Tadalafil

GF-196960, IC-351, Cialis

6R-trans)-6-(1,3-benzodioxol-5-yl)- 2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino [1', 2':1,6] pyrido[3,4-b]indole-1,4-dione

Pyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione,6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-, (6R-trans)-; (6R,12aR)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-ethylpyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione; GF 196960;  Adcirca;

171596-29-5  ^casno

Molecular Weight:
389.40

Molecular Formula:C₂₂H₁₉N₃O₄

GlaxoSmithKline (Originator), Lilly Icos (Marketer), Lilly (Licensee), Lilly Icos (Licensee)

Launched-2003

Tadalafil is currently marketed as Cialis. Cialis was developed by Eli Lilly as a treatment for impotence. In this capacity, it is reported that tadalafil functions by inhibiting the formation of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5). The inhibition of PDE5 presumably lessens impotence by increasing the amount ot c(iMP, resulting in smooth muscle relaxation and increased blood flow.

Tadalafil is a PDE5 inhibitor marketed in pill form for treating erectile dysfunction (ED) under the name Cialis, and under the name Adcirca for the treatment of pulmonary arterial hypertension. In October 2011 the U.S. Food and Drug Administration (FDA) approved Cialis for treating the signs and symptoms of benign prostatic hyperplasia (BPH) as well as a combination of BPH and erectile dysfunction (ED) when the conditions coincide. It initially was developed by the biotechnology company ICOS, and then again developed and marketed world-wide by Lilly ICOS, LLC, the joint venture of ICOS Corporation and Eli Lilly and Company. Cialis tablets, in 2.5 mg, 5 mg, 10 mg, and 20 mg doses, are yellow, film-coated, and almond-shaped. The approved dose for pulmonary arterial hypertension is 40 mg (two 20-mg tablets) once daily.

Tadalafil can be prepared via a series of intermediates. One synthesis scheme is illustrated in Scheme 1: Scheme 1

U.S. Patent No. 5,859,006 describes the synthesis of the tadalafil intermediate (Compound III) from D-tryptophan methyl ester (Compound II) and piperonal (Compound I) using trifluoroacetic acid and dichloromethane, a halogenated solvent. Compound III is then reacted with chloroacetyl chloride (Compound IV) and chloroform, providing another intermediate of tadalafil (Compound V). WO 04/011463 describes a process of preparing tadalafil intermediates from D-tryptophan methyl ester HCl salt and piperonal by refluxing the reagents in isopropyl alcohol; the obtained intermediate is reacted with chloroacetyl chloride and THF, resulting in another intermediate of tadalafil.

Tadalafil is also manufactured and sold under the name of Tadacip by the Indian pharmaceutical company Cipla in doses of 10 mg and 20 mg.

On November 21, 2003 the FDA approved tadalafil (as Cialis) for sale in the United States as the third ED prescription drug pill (after sildenafil citrate(Viagra) and vardenafil (Levitra)). Like sildenafil and vardenafil, tadalafil is recommended as an ‘as needed’ medication. Cialis is the only one of the three that is also offered as a once-daily medication.

Moreover, tadalafil was approved in May 2009 in the United States for the treatment of pulmonary arterial hypertension and is under regulatory review in other regions for this condition. In late November 2008, Eli Lilly sold the exclusive rights to commercialize tadalafil for pulmonary arterial hypertension in the United States to United Therapeutics for an upfront payment of $150 million.

The FDA’s approval of Viagra (Sildenafil) on March 27, 1998 was a ground-breaking commercial event for the treatment of ED, with sales exceedingUS$1 billion. Subsequently, the FDA approved Levitra (vardenafil) on August 19, 2003, and Cialis (tadalafil) on November 21, 2003.

Cialis was discovered by Glaxo Wellcome (now GlaxoSmithKline) under a partnership between Glaxo and ICOS to develop new drugs that began in August 1991. ^[1][2] In 1993, the Bothell, Washington biotechnology company ICOS Corporation began studying compound IC351, a phosphodiesterase type 5 (PDE5) enzyme inhibitor. In 1994, Pfizer scientists discovered that sildenafil, which also inhibits the PDE5 enzyme, caused penile erection in men participating in a clinical study of a heart medicine. Although ICOS scientists were not testing compound IC351 for treating ED, they recognized its potential usefulness for treating that disorder. Soon, in 1994, ICOS received a patent for compound IC351 (structurally unlike sildenafil and vardenafil), and Phase 1 clinical trials began in 1995. In 1997, the Phase 2 clinical studies were initiated for men experiencing ED, then progressed to the Phase 3 trials that supported the drug’s FDA approval. Although Glaxo had an agreement with ICOS to share profits 50/50 for drugs resulting from the partnership, Glaxo let the agreement lapse in 1996 as the drugs developed were not in the company’s core markets.^[3]

In 1998, ICOS Corporation and Eli Lilly and Company formed the Lilly ICOS, LLC, joint venture company to further develop and commercialize tadalafil as a treatment for ED. Two years later, Lilly ICOS, LLC, filed a new drug application with the FDA for compound IC351 (under the tadalafil generic name, and the Cialis brand name). In May 2002, Lilly ICOS reported to the American Urological Association that clinical trial testing demonstrated that tadalafil was effective for up to 36 hours, and one year later, the FDA approved tadalafil. One advantage Cialis has over Viagra and Levitra is its 17.5-hour half-life (thus Cialis is advertised to work for up to 36 hours, after which time there remains approximately 25 percent of the absorbed dose in the body) when compared to the four-hour half–life of sildenafil (Viagra).

In 2007, Eli Lilly and Company bought the ICOS Corporation for $2.3 billion. As a result, Eli Lilly owned Cialis and then closed the ICOS operations, ending the joint venture and firing most of ICOS’s approximately 500 employees, except for 127 employees of the ICOS biologics facility, which subsequently was bought by CMC Biopharmaceuticals A/S (CMC).

Tadalafil Molecule

Persons surnamed “Cialis” objected to Eli Lilly and Company’s so naming the drug, but the company has maintained that the drug’s trade name is unrelated to the surname.^[4]

On October 6, 2011, the U.S. FDA approved tadalafil ^[5] to treat the signs and symptoms of benign prostatic hyperplasia (BPH). BPH is a condition in males in which the prostate gland becomes enlarged, obstructing the free flow of urine. Symptoms may include sudden urges to urinate (urgency), difficulty in starting urination (hesitancy), a weak urine stream, and more frequent urination- especially at night. The FDA has also approved tadalafil for treatment of both BPH and erectile dysfunction (ED) where the two conditions co-exist.

Although available since 2003 in 5, 10, 20 mg dosage, in late 2008/early 2009, the U.S. FDA approved the commercial sale of Cialis in 2.5 mg dosage as a one-a-day treatment for ED. The 2.5 mg dose avoids earlier dispensing restrictions on higher dosages. The price of the 5 mg and 2.5 mg are often similar, so some people score and split the pill.^[6] The manufacturer does not recommend splitting.

Moreover, tadalafil (Adcirca) 40 mg was approved in 2009 in the United States and Europe (and 2010 in Canada and Japan) as a once-daily therapy to improve exercise ability in patients withpulmonary arterial hypertension. In patients with pulmonary arterial hypertension, the pulmonary vascular lumen is decreased as a result of vasoconstriction and vascular remodeling, resulting in increased pulmonary artery pressure and pulmonary vascular resistance. Tadalafil is believed to increase pulmonary artery vasodilation, and inhibit vascular remodeling, thus lowering pulmonary arterial pressure and pulmonary vascular resistance. Right heart failure is the principal consequence of pulmonary arterial hypertension.

On October 6, 2011, the U.S. FDA approved tadalafil [6] to treat the signs and symptoms of benign prostatic hyperplasia (BPH). BPH is a condition in males in which the prostate gland becomes enlarged, obstructing the free flow of urine. Symptoms may include sudden urges to urinate (urgency), difficulty in starting urination (hesitancy), a weak urine stream, and more frequent urination- especially at night. The FDA has also approved tadalafil for treatment of both BPH and erectile dysfunction (ED) where the two conditions co-exist.

Tadalafil has been used in approximately 15,000 men participating in clinical trials, and over eight million men worldwide (primarily in the post-approval/post-marketing setting). The most commonside effects when using tadalafil are headache, stomach discomfort or pain, indigestion, burping, acid reflux. back pain, muscle aches, flushing, and stuffy or runny nose. These side effects reflect the ability of PDE5 inhibition to cause vasodilation (cause blood vessels to widen), and usually go away after a few hours. Back pain and muscle aches can occur 12 to 24 hours after taking the drug, and the symptom usually disappears after 48 hours.
In May 2005, the U.S. Food and Drug Administration found that tadalafil (along with other PDE5 inhibitors) was associated with vision impairment related to NAION (nonarteritic anterior ischemic optic neuropathy) in certain patients taking these drugs in the post-marketing (outside of clinical trials) setting. Most, but not all, of these patients had underlying anatomic or vascular risk factors for development of NAION unrelated to PDE5 use, including: low cup to disc ratio (“crowded disc”), age over 50, diabetes, hypertension, coronary artery disease, hyperlipidemia and smoking. Given the small number of NAION events with PDE5 use (fewer than one in one million), the large number of users of PDE5 inhibitors (millions) and the fact that this event occurs in a similar population to those who do not take these medicines, the FDA concluded that they were not able to draw a cause and effect relationship, given these patients underlying vascular risk factors or anatomical defects. However, the label of all three PDE5 inhibitors was changed to alert clinicians to a possible association.

In October 2007, the FDA announced that the labeling for all PDE5 inhibitors, including tadalafil, requires a more prominent warning of the potential risk of sudden hearing loss as the result of postmarketing reports of deafness associated with use of PDE5 inhibitors.^[7]

Selectivity compared with other PDE5 inhibitors

Tadalafil, sildenafil, and vardenafil all act by inhibiting the PDE5 enzyme. These drugs also inhibit other PDE enzymes. Sildenafil and vardenafil inhibit PDE6, an enzyme found in the eye, more than tadalafil.^[9] Some sildenafil users see a bluish tinge and have a heightened sensitivity to light because of PDE6 inhibition.^[3] Sildenafil and vardenafil also inhibit PDE1 more than tadalafil.^[9]PDE1 is found in the brain, heart, and vascular smooth muscle.^[9] It is thought that the inhibition of PDE1 by sildenafil and vardenafil leads to vasodilation, flushing, and tachycardia.^[9] Tadalafil inhibits PDE11 more than sildenafil or vardenafil.^[9] PDE11 is expressed in skeletal muscle, the prostate, the liver, the kidney, the pituitary gland, and the testes.^[9] The effects on the body of inhibiting PDE11 are not known.^[9]

20 mg Cialis tablet

In the United States, the FDA relaxed rules on prescription drug marketing in 1997, allowing advertisements targeted directly to consumers.^[10] Lilly-ICOS hired the Grey Worldwide Agency in New York, part of the Grey Global Group, to run the Cialis advertising campaign.^[11] Marketers for Cialis has taken advantage of its greater duration compared to its competitors in advertisements for the drug; Stuart Elliot of The New York Times opined: “The continuous presence of women in Cialis ads is a subtle signal that the drug makes it easier for them to set the pace with their men, in contrast to the primarily male-driven imagery for Levitra and Viagra.”^[11] Iconic themes in Cialis ads include couples in bathtubs and the slogan “When the moment is right, will you be ready?”^[11] Cialis ads were unique among the ED drugs in mentioning specifics of the drug.^[12] As a result, Cialis ads were also the first to describe the side effects in an advertisement, as the FDA requires advertisements with specifics to mention side effects. One of the first Cialis ads aired at the 2004 Super Bowl.^[12] Just weeks before the Super Bowl, the FDA required more possible side effects to be listed in the advertisement, including priapism.^[12] Although many parents objected to the Cialis ad being aired during the Super Bowl, Janet Jackson‘s halftime “wardrobe malfunction” overshadowed Cialis.^[12] In January 2006, the Cialis ads were tweaked, adding a doctor on screen to describe side effects and only running ads where more than 90 percent of the audience are adults, effectively ending Super Bowl ads.^[10] In 2004, Lilly-ICOS, Pfizer, and GlaxoSmithKline spent a combined $373.1 million to advertise Cialis, Viagra, and Levitra respectively.^[12] Cialis has sponsored many golf events, including the America’s Cup and the PGA Tour, once being title sponsor of the PGA Tour Western Open tournament.^[13]

CIALIS (tadalafil) is a selective inhibitor of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterase type 5 (PDE5). Tadalafil has the empirical formula C22H19N3O4 representing a molecular weight of 389.41. The structural formula is:

The chemical designation is pyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione, 6-(1,3-benzodioxol-5-yl)2,3,6,7,12,12a-hexahydro-2-methyl-, (6R,12aR)-. It is a crystalline solid that is practically insoluble in water and very slightly soluble in ethanol.

CIALIS is available as almond-shaped tablets for oral administration. Each tablet contains 2.5, 5, 10, or 20 mg of tadalafil and the following inactive ingredients: croscarmellose sodium, hydroxypropyl cellulose, hypromellose, iron oxide, lactose monohydrate, magnesium stearate, microcrystalline cellulose, sodium lauryl sulfate, talc, titanium dioxide, and triacetin.

Tadalafil, (6R-trans)-6-(l,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2- methyl-pyrazino[r,2':l,6]pyrido[3,4-b]indole-l,4-dione, with the structural formula shown below, is a white crystalline powder. (CAS# 171596-29-5). Tadalafil is a potent and selective inhibitor of the cyclic guanosine monophosphate (cGMP) – specific phosphodiesterase enzyme, PDE5. The inhibition of PDE5 increases the amount of cGMP, resulting in smooth muscle relaxation and increased blood flow. Tadalafil is therefore currently used in the treatment of male erectile dysfunction, and is commercially available as CIALIS ^®.

Tadalafil U.S. Patent No. 5,859,006 describes the synthesis of tadalafil via the cyclization of TDCL (i.e., cis-methyl l,2,3,4-tetrahydro-2-chloroacetyl-l-(3,4- methylenedioxyphenyl)-9H-pyrido[3,4-b]mdole-3-carboxylate) using methylamine by purification by flash chromatography, followed by subsequent crystallization from methanol. Crude tadalafil typically requires additional purification steps, such as multiple extractions, crystallization, and/or flash chromatography, to remove the impurities present in the compound after synthesis is complete. Such purification processes increase the cost of producing tadalafil. Also, when repeating the US ’006 process, about 250 volumes of methanol were necessary for the crystallization step

Tadalafil can be prepared via a series of intermediates. One synthesis for preparing tadalafil is illustrated below in Scheme I:

SCHEME I

U.S. Patent No. 5,859,006 discloses the synthesis of a tadalafil intermediate

(Compound III) from D-tryptophan methyl ester (Compound II) and piperonal (Compound

I) using trifluoroacetic acid and dichloromethane, a halogenated solvent. Compound III is then reacted with chloroacetyl chloride (Compound IV) and chloroform to provide another intermediate of tadalafil (Compound V).

WO 2004/011463 discloses a process of preparing tadalafil intermediates from D-tryptophan methyl ester HCl salt and piperonal by refluxing the reagents in isopropyl alcohol, reacting the intermediate thus obtained with chloroacetyl chloride and tetrahydrofuran (THF) to provide another intermediate of tadalafil.

WO 2006/110893 discloses a process for the preparation of methyl ester intermediate (Compound III), and tadalafil using the methyl ester intermediate (CompoundII).

U.S. Patent Application Publication No. 2006/0258865 Al discloses a synthesis of the tadalafil intermediate (Compound III) from D-tryptophan methyl ester

(Compound II) and piperonal (Compound I) using a dehydrating agent selected from Na₂SO₄, K₂SO₄, MgSO₄, CaSO₄, CaCl_2, molecular sieve or mixtures thereof and a high boiling solvent such as N,N-Dimethyl acetamide. Compound III is then reacted with chloroacetyl chloride (Compound IV) in the presence of a base such as NaHCO₃ and an organic solvent such as dichloromethane, providing another intermediate of tadalafil (Compound V), which is further reacted with aqueous methyl amine solution to provide tadalafil.

………………………………………….

EP2004644A1

WO2007110734A1

Scheme II and III.

……………………………………………………………………………………………………a compound of .Formula I

SCHEME III

SCHEME IV

EXAMPLE l

The reaction scheme of this example is generally shown below in SchemeIV.

SCHEME IV

Compound – 1                                           Compound – II

Into a clean dry glass flask charged with ethanol (250 ml) under a nitrogen atmosphere was added Compound 1 (25 g) under stirring. The reaction mass was cooled to 0 to 5⁰C and monomethylamine gas was purged into the reaction mixture for about 2 hours while maintaining the temperature between 0 to 5⁰C. The temperature was raised to 75 to ⁰C and the reaction mixture was stirred under reflux for 2 hours. The reaction mixture was then cooled to 0 to 5°C and monomethylamine gas was again purged into the reaction mixture at 0 to 5°C. The temperature was again raised to 75 to 80⁰C and stirred for about 1 hour. The reaction mixture was concentrated under vacuum to about 1/3 its original volume, cooled to 5 to 1O⁰C and stirred for 1 hour at this temperature. The solids were filtered and washed with chilled ethanol (50 ml). The wet solids were dried under vacuum for 6 hours.

Yield: 25g; Mp: 202-206.7⁰C

Specific rotation (25°C) :+44.0 ( C=l% in DMSO)

¹³C NMR, DMSO-D6 : 25.78, 25.92, 57.89, 57.98, 101.17, 108.09, 108.32,

109.08, 111.48, 117.82, 118.62, 122.23, 122.97, 126.97, 135.97, 136.22, 136.55, 146.99,

147.48, 173.13

¹H NMR, DMSO-D6, 300 MHz, Delta values: 2.6(m,lH), 2.7(m,3H),

2.8(d,lH), 3.0(d,lH), 3.6(bs,lH), 5.1(m,lH), 6.0(s,3H), 6.9-7.1(m, 5H), 7.2(d,lH),

7.4(d,lH), 7.8(bs, IH), 10.3(s, IH)

EXAMPLE 2

The reaction scheme of this example is generally shown below in SchemeV.

SCHEME V

Formula III                                                                                     Formula II

Into a clean dry flask charged with dichloromethane (200 ml) was added

Compound II (25 g) obtained in Example 1 under stirring at 25 to 30⁰C. Next, triethylamine (16.11 g) was added to the reaction mixture and stirred for 30 minutes at 20 to 30⁰C. The reaction mixture was cooled to 0 to 5°C and a solution of chloroacetyl chloride (12.93 g) in chloroform (50 ml) was added to the reaction mixture while maintaining temperature between -5 to 5⁰C. The reaction mixture was stirred at -5 to 5°C for about 2 hours. Saturated aqueous sodium bicarbonate solution (50 ml) was added to the reaction mass slowly and the temperature of the reaction mixture was raised to 25 to 30⁰C. The lower organic layer was separated and washed twice with water (75 ml). The chloroform extract was dried over anhydrous sodium sulfate. The organic layer was concentrated under vacuum until a thick yellow slurry was obtained. The slurry was cooled to 0 to5°C. The solids obtained were filtered and washed with 50 ml chilled chloroform. The wet product was dried at 75⁰C under vacuum for 6 hours.

Yield: 22.5 g; HPLC Purity: 97%; Mp: 180-182⁰C

Specific rotation(25°C): -154.3(C=1% in DMSO)

¹³C. NMR(DMSO-Do, 300 MHZ)= 21.11, 25.88, 44.207, 51.60, 53.95,

101.16,107.66 109.56, 111.38, 118.36, 118.75, 121.58,122.74, 126.30, 130.31, 134.13,

136.57, 146.66, 147.03,167.43, 168.45

¹H. NMR (CDC13, 300 MHZ):2.4(bs,3H), 3.1(m,lH), 3.8(m,lH),

4.3(bs,2H), 4.9(m,lH), 5.4(m,lH), 5.9(s,2H), 6.6-6.8(m,3H), 6.9(bs,lH), 7.1-7.3(m,3H),

7.6(d, IH), 7.7(bs,lH)

¹H. NMR (DMSO-D6, 300 MHZ): 2.0 (bs,3H), 2.9(m,lH), 3.4(m,lH),

4.5(m,lH), 4.8(m,lH), 4.9(m,lH), 6.0(m,2H), 6.4-6.8(m,4H), 6.9-7.2(m,2H), 7.3(d, IH),

7.4(bs,lH), 7.5(d,lH), 10.8(s,lH)

EXAMPLE 3

The reaction scheme of this example is generally shown below in SchemeVI.

SCHEME VI

Formula II                                                                         Formula I

Into a clean dry round bottom (RB) flask was charged tetrahydrofuran

(THF) (175 ml) under a nitrogen blanket and then cooled to -35 to -40⁰C. Next 92 ml n- butyllithium (1.6 m solution in hexane) was added while maintaining the temperature between -35 to -40⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -40⁰C for 15 minutes. A solution of compound of formula II (22.5 g) obtained in Example 2 in THF (75 ml) was prepared and slowly added to the reaction mixture while maintaining the temperature between -35 to -40⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -40⁰C for 2.5 hours. Saturated aqueous ammonium chloride solution (25 ml) and 50 ml ethyl acetate was added to the reaction mixture at -35 to -40⁰C. The temperature was raised to 25 to 30⁰C and the two layers formed were separated. The upper organic layer was collected. The lower aqueous layer was thrice extracted with ethyl acetate (25 ml). The organic layers were combined together and washed with water (50 ml). The organic extract was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain crude tadalafil as a dark brown solid. [0058] Yield: 22 g; HPLC Purity: 50%.

EXAMPLE 4

Purification of crude tadalafil

The crude tadalafil (22 g) obtained in Example 3 was suspended in 110 ml methanol and stirred for 1 hour at 25 to 30⁰C. The solids obtained were filtered and washed with 25 ml chilled methanol. The wet product was dried at 60⁰C under vacuum for 6 hours. This was further purified by using isopropyl alcohol. Yield: 9 g; HPLC Purity: >99.5%.

EXAMPLE 5

The reaction scheme of this example is generally shown below in SchemeVII.

Scheme VII

Formula VI where R = -OCH3 Formula VIA [0062] Into a clean dry RB flask charged with methanol (1900 ml) was added D- tryptophan methyl ester (190 g) under stirring at 25 to 30⁰C. The reaction mixture was cooled to 0 to 5⁰C. Monomethylamine gas was purged into the reaction mixture at 0 to 5°C for about 5-7 hours under stirring. The temperature of the reaction mixture was slowly raised to about 25 to 3O⁰C and stirred at this temperature for 5-7 hours. The reaction mixture was concentrated under vacuum to distill out the solvent. Diisopropyl ether (950 ml) was added and cooled to 25 to 3O⁰C under stirring for 1-2 hrs. The solids obtained were filtered, washed with Diisopropyl ether and dried under vacuum. [0063] MP: 122.4-124⁰C; Yield: 150 g (78.9 % w/w).

Specific rotation(25°C): +12.5 (C=I % in DMSO)

13 C NMR (300 MHZ,DMSO-D6): 25.71, 31.40, 55.67, 110.93, 111.55,

118.42, 118.73, 121.09, 123.95, 127.66,136.44, 175.39.

¹H NMR (300 MHZ,DMSO-D6): 1.6(bs,2H), 2.5(m,3H), 2.8(m,lH),

3.1(m,lH), 3.4(m, IH), 6.9-7.2(m,3H), 7.3(d,lH), 7.5(d,lH), 7.8(bs,lH), 10.8(bs,lH)

EXAMPLE 6

The reaction scheme of this example is generally shown below in Scheme VIII.

SCHEME VIII

Formula VIA                                Formula VII

Into a clean, dry flask charged with methylene dichloride (MDC) (1000 ml) was added D-tryptophan methyl amide, the compound of Formula VIA (50 g), and piperonal, the compound of Formula VII (31.09 g), under stirring at 25 to 30⁰C. The reaction mixture was cooled to 0 to 5°Cunder nitrogen atmosphere. Trifluoroacetic acid (85.3 g) was dissolved in MDC (250 ml) and the solution was slowly added to the reaction mixture at 0 to 5°C. The temperature of the reaction mixture was raised to 20 to 30⁰C and stirred at this temperature for 14-16 hours. The reaction was monitored by TLC, workup was done as follows, the pH of the reaction mixture was adjusted to 8-9 using sodium carbonate solution under stirring, the two layers were settled, separated and the lower MDC layer was washed with water. The MDC layer was then dried over anhydrous sodium sulfate. The reaction mass was concentrated under vacuum at 40 to 5O⁰C to remove the solvent. The compound was precipitated using ethyl acetate, the solids were filtered, washed with ethyl acetate and dried.

Yield: 52.5 g; Yield: 105% w/w, HPLC Purity: 71% cis and 27% trans isomer (HPLC).

EXAMPLE 7

The reaction scheme of this example is generally shown below in Scheme IX.

SCHEME IX

1]CICOCH2C1 2]crystn

Formula H

Into a clean dry flask charge with dichloromethane (400 ml) under a nitrogen atmosphere was added the compound of Formula III obtained in Example 6 and triethylamine (28.96 g) under stirring at 20 to 3O⁰C. The reaction mixture was then cooled to 0 to 5⁰C. A mixture of chloroacetyl chloride (25.85 g) in dichloromethane (100 ml) was prepared and slowly added to the reaction mixture while maintaining the temperature between -5 to 5⁰C in 1-2 hrs. The reaction mixture was stirred at 0 to 5⁰C for 30 min and then saturated sodium bicarbonate solution (100 ml) was added at 5 to 10⁰C under stirring. The temperature of the reaction mixture was raised to 25 to 30⁰C and stirred at this temperature for 15 minutes. The layers were then separated. The lower MDC layer was collected, washed twice with 100 ml water and dried over anhydrous sodium sulfate. The

MDC layer was concentrated to distill out MDC until a stirrable mass was left behind. The mass was cooled to 25-3O⁰C and filtered, washed, to yield off-white to light yellow colored solids. The resulted product was the cis isomer, the trans isomer left behind in the mother liquor.

Yield = 25.5 g (50%w/w); HPLC Purity: > 97%.

The physical and spectral data was similar to that obtained in Example 2.

EXAMPLE 8

The reaction scheme of this example is generally shown below in SchemeX.

SCHEME X

Formula II

Into a clean dry round bottom (RB) flask was charged THF (1625 ml) under a nitrogen blanket and then cooled to -35 to -40⁰C. Next, 505 ml n-butyllithium (1.6 m solution in hexane) was added while maintaining the temperature between -35 to -4O⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -4O⁰C for 15 minutes. 72 ml diisopropyl amine was then added at -35 to -40⁰C and then stirred at 0-5⁰C for 1 hr. A solution of Compound of formula II (125 g) obtained in Example 7 in THF (625 ml) was prepared and slowly added to the reaction mixture while maintaining the temperature between -40 to -5O⁰C. After the addition was complete, the reaction mixture was stirred at -35 to -40⁰C for 2-6 hours. Saturated aqueous ammonium chloride solution (250 ml) and ethyl acetate (125 ml) was added to the reaction mixture at -35 to -40⁰C. The temperature was raised to 25 to 30⁰C and the two layers formed were separated. The upper organic layer was collected. The lower aqueous layer was extracted with ethyl acetate (65 ml). The organic layers were combined together and distilled. Isopropyl alcohol (1250 ml) was added and the distillation was continued. A mixture of methanol (250 ml) and isopropanol (375 ml) were added and crude tadalafϊl was obtained upon cooling. The crude product was filtered, washed with water and dried. [0076] Yield: 60 g; (48% w/w); HPLC Purity: >99%.

EXAMPLE 9

Purification of crude Tadalafil

The crude tadalafil obtained in Example 8 was suspended in methanol (600 ml) and stirred for 1 hour at reflux. The mixture was cooled and the solids obtained were filtered and washed with chilled methanol (60 ml). The wet product was dried at under vacuum.

Yield: 56 g; HPLC Purity: 99.8%.

……………………………………………………………………………………

Beilstein J. Org. Chem. 2011, 7, 442–495.

http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-7-57#S28

A different approach was used in the synthesis of the phosphodiesterase inhibitor tadalafil (132, Cialis) starting from commercially available (D)-tryptophan methyl ester to form the indolopiperidine motif 135 via a Pictet–Spengler reaction followed by a double condensation to install the additional diketopiperazine ring (Scheme 28) [38,39].

To achieve the high levels of cis selectivity required from the Pictet–Spengler reaction, an extensive investigation of solvents and the influence of additives was undertaken [40]. It was identified that the use of a specific 23 mol % of benzoic acid significantly increased the cis/trans ratio from a base level of 55:45 to 92:8 (16 h reaction time at ambient temperature) in an overall yield of 86%. It was also determined that more polar solvents such as acetonitrile and nitromethane preferentially solvated the trans product and thereby allowed the isolation of the ciscompound by precipitation. It was also shown that by heating the reaction mixture under reflux the product distribution could be driven to the thermodynamically more favoured cis isomer having both the ester and the piperonyl moiety in equatorial positions. Hence, after heating under reflux for 8 h the cis/trans ratio was found to be 99:1 and the product could be isolated in an overall yield of 91%. This work represents an impressive example of a well considered and executed process optimisation study.

………………………………

The process disclosed in the patent US 5 859 006 (Scheme 1) involves condensation of D-tryptophan methyl ester with a piperonal derivative to yield a compound of formula (II). After conversion into a thioamide derivative of formula (III), cyclization occurs in presence of both an alkylating and reducing agents to provide a tetrahydro-β-carboline derivative of formula (IV), which on treatment with chloroacetyl chloride and methyl amine, gives Tadalafil. The compound of formula (IV) can also be obtained in one step, after separation of the other diastereoisomer, by a Pictet Spengler reaction between D-tryptophan methyl ester and piperonal in presence of an acid, such as trifluoroacetic acid.

• The patent application WO2007/10038 discloses the reaction of D-tryptophan with piperonal to provide a tetrahydro-β-carboline acid that was cyclised to Tadalafil in presence of a sarcosine derivative.
  The patent application WO2007/1107 discloses the reaction of D-tryptophan methyl amide with piperonal, to provide an intermediate that after reaction with chloroacetyl chloride cyclises to Tadalafil in presence of butyllithium.
  Thus, the active substance prepared by the processes known up till now can only be obtained in a satisfactory quality after running through a large number of process steps. Moreover a toxic alkylating agent, such as methylamine, is often used.

EP2181997A1

Example 1

• (1R,3R)-methyl-1,2,3,4-tetrahydro-2-(2-(benzyl(methyl)amino)acetyl)-1-(3,4-methylenedioxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (VII)
•  
  A 50 mL three-necked flask fitted with thermometer and reflux condenser was charged with (1R,3R)-methyl 1,2,3,4-tetrahydro-2-chloroacetyl-1-(3,4-methylenedioxyphenyl)-9H-pyrido [3,4-b] indole-3-carboxylate (VI) (1.39 g, 3.26 mmol), DMA (5.33 mL), K₂CO₃ (0.5 g, 3.6 mmol) and N-benzylmethylamine (0.41 mL, 3.26 mmol). The resultant solution was stirred at room temperature. After 2 hours, the mixture was poured in brine (20 mL) and extracted with isopropyl acetate. The combined organic phases were washed with brine (3 x 5 mL), dried over sodium sulfate and concentrated to a residue under reduced pressure, affording 1.5 g of the desired product (VII), as a white solid. Yield: 70%.
  ¹H NMR (d₆-DMSO 300 MHz, 298K) 2.24 (s, 3H), 2.94-3.00 (m, 5H), 3.44-3.68 (m, 3H), 5.56 (bd, J = 6.4, 1H), 5.95 (s, 1H), 5.96 (s, 1H), 6.55 (bd, J = 7.4, 1H), 6.75 (bs, 1H), 6.77 (d, J = 8.0, 1H), 6.84 (bs, 1H), 7.05 (td, J = 7.4, 0.9, 1H), 7.12 (td, J = 7.5, 1.2, 1H), 7.17-7.32 (m, 6H), 7.56 (d, J = 7.7, 1H), 10.76 (bs, 1H)
  ¹³C NMR (d₆-DMSO 75.4 MHz, 298K) 21.9, 42.5, 51.3, 51.9, 52.4, 61.0, 61.7, 101.5, 107.0, 108.0, 109.8, 111.8, 118.5, 119.2, 122.0, 123.0, 126.7, 127.7, 128.7, 129.6, 131.1, 134.7, 137.1, 138.6, 147.1, 147.5, 170.6, 171.5

Example 2

• (6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-pyrazino [1',2':1,6] pyrido [3,4-b] indole-1,4-dione (Tadalafil) (I)

  Under H₂ atmosphere (3 atm) and magnetic stirring, Raney^® Ni (2800 slurry in water, 0.0276 g, 0.47 mmol), previously washed with methanol (3 times), was added to a solution of (1R,3R)-methyl-1,2,3,4-tetrahydro-2-(2-(benzyl(methyl)amino)acetyl)-1-(3,4-methylenedioxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (VII) (3.00 g, 4.70 mmol) in DMA (21.3 mL). The mixture was heated at 90°C for 17 hours and then cooled to room temperature. The suspension was filtered over a pad of Celite^® and the resulting solutionand the resulting solution was concentrated until 6 mL. Methanol (12 mL) was added and the solid which was so obtained was filtered over Buchner, washed with methanol (4 mL) and oven-dried under reduced pressure for 2 hours, affording 1.3 g of the title compound, as a white solid. Yield: 70%
  ¹H NMR (d₆-DMSO 300 MHz, 298K): 2.91-3.00 (m, 4H), 3.32 (s, 1H), 3.47-3.54 (dd, J = 4.6, 11.3, 1H), 3.93 (d, J = 17.1, 1H), 4.17 (d, J = 17.1, 1H), 4.35-4.40 (dd, J = 4.27, 11.6, 1H), 5.91 (s, 2H), 6.11 (s, 1H), 6.76 (s, 2H), 6.85 (s, 1H), 6.98-7.06 (m, 2H), 7.28 (d, J = 7.9, 1H), 7.52 (d, J = 7.3, 1H), 11.0 (s, 1H)
  ¹³C NMR (d₆-DMSO 75.4 MHz, 298K) 23.8, 33.4, 52.0, 55.9, 56.1, 101.5, 105.3, 107.6, 108.6, 111.9, 118.7, 119.5, 119.9, 121.8, 126.4, 134.5, 136.8, 137.6, 146.7, 147.6, 167.1, 167.5

References

DAUGAN A ET AL: “THE DISCOVERY OF TADALAFIL: A NOVEL AND HIGHLY SELECTIVE PDE5 INHIBITOR. 2: 2,3,6,7,12,12A-HEXAHYDROPYRAZINO[1′,2′:1,6 ÜPYRIDO[3,4-B ÜINDOLE-1,4-DIONE” JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 46, no. 21, 2003, pages 4533-4542, XP008052656 ISSN: 0022-2623

• Tadalafil bound to proteins in the PDB
• National Institutes of Health – Medlineplus
• Material Safety Data Sheet PDF file
• Official Cialis (Tadalafil) Website
• U.S. National Library of Medicine: Drug Information Portal – Tadalafil

STRUCTURAL FORMULA
Monomer
APQPPNILLL LMDDMGWGDL GVYGEPSRET PNLDRMAAEG LLFPNFYSAN 50
PLCSPSRAAL LTGRLPIRNG FYTTNAHARN AYTPQEIVGG IPDSEQLLPE 100
LLKKAGYVSK IVGKWHLGHR PQFHPLKHGF DEWFGSPNCH FGPYDNKARP 150
NIPVYRDWEM VGRYYEEFPI NLKTGEANLT QIYLQEALDF IKRQARHHPF 200
FLYWAVDATH APVYASKPFL GTSQRGRYGD AVREIDDSIG KILELLQDLH 250
VADNTFVFFT SDNGAALISA PEQGGSNGPF LCGKQTTFEG GMREPALAWW 300
PGHVTAGQVS HQLGSIMDLF TTSLALAGLT PPSDRAIDGL NLLPTLLQGR 350
LMDRPIFYYR GDTLMAATLG QHKAHFWTWT NSWENFRQGI DFCPGQNVSG 400
VTTHNLEDHT KLPLIFHLGR DPGERFPLSF ASAEYQEALS RITSVVQQHQ 450
EALVPAQPQL NVCNWAVMNW APPGCEKLGK CLTPPESIPK KCLWSH 496
Disulfide bridges
139-139′ 282-393 282′-393′ 463-492 463′-492′ 475-481 475′-481′
Modified residues
C
53 , 53′
3-oxoAla
O
CO2H
H NH2

Glycosylation sites (N)
Asn-178 Asn-178′ Asn-397 Asn-397′

Vimizim (elosufase alfa)

Elosulfase alfa nonproprietary drug name  GET STRUCTURE

MOLECULAR FORMULA C5020H7588N1364O1418S34

MOLECULAR WEIGHT 110.8 kDa (peptide)

SPONSOR BioMarin Pharmaceutical Inc.

CODE DESIGNATION BMN 110, rhGALNS

CAS REGISTRY NUMBER 9025-60-9

THERAPEUTIC CLAIM Treatment of Morquio Syndrome

CHEMICAL NAMES

1. Sulfatase, chondroitin

2. Human N-acetylgalactosamine-6-sulfatase (chondroitinsulfatase, galactose-6-sulfate
sulfatase, EC=3.1.6.4) dimer (139-139′)-disulfide glycosylated (produced by CHO cells)

Company: BioMarin Pharmaceutical Inc.
Date of FDA Approval: February 14, 2014
Treatment for: Mucopolysaccharidosis Type IVA

• BMN 110
• Chondroitin 6-sulfatase
• Chondroitin sulfatase
• Chondroitin sulfate sulfatase
• Chondroitinase
• Chondrosulfatase
• E.C. 3.1.6.4
• Elosulfase alfa
• rhGALNS
• UNII-ODJ69JZG85
• Vimizim

CLINICAL….http://clinicaltrials.gov/search/intervention=Elosulfase%20alfa%20OR%20bmn%20110

Vimizim (elosufase alfa) is an enzyme replacement therapy for patients with Mucopolysaccharidosis Type IVA (Morquio A syndrome).

• FDA Advisory Committee Recommends Approval for BioMarin’s Vimizim for the Treatment of Patients With Morquio A Syndrome - November 20, 2013

Feb 16, 2014 Approval FDA Approves Vimizim to Treat Mucopolysaccharidosis Type IVA

The U.S. Food and Drug Administration today approved Vimizim (elosulfase alfa), the first FDA-approved treatment for Mucopolysaccharidosis Type IVA (Morquio A syndrome). Morquio A syndrome is a rare, autosomal recessive lysosomal storage disease caused by a deficiency in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Vimizim is intended to replace the missing GALNS enzyme involved in an important metabolic pathway. Absence of this enzyme leads to problems with bone development, growth and mobility. There are approximately 800 patients with Morquio A syndrome in the United States.

Vimizim was granted priority review. An FDA priority review provides for an expedited review of drugs for serious diseases or conditions that may offer major advances in treatment. Vimizim is also the first drug to receive the Rare Pediatric Disease Priority Review Voucher – a provision that aims to encourage development of new drugs and biologics for the prevention and treatment of rare pediatric diseases.

“This approval and rare pediatric disease priority review voucher underscores the agency’s commitment to making treatments available to patients with rare diseases,” said Andrew E. Mulberg, M.D., deputy director, Division of Gastroenterology and Inborn Errors Products in the FDA’s Center for Drug Evaluation and Research (CDER). “Prior to today’s approval, patients with this rare disease have had no approved drug treatment options.”

The safety and effectiveness of Vimizim were established in a clinical trial involving 176 participants with Morquio A syndrome, ranging in age from 5 to 57 years. Participants treated with Vimizim showed greater improvement in a 6-minute walk test than participants treated with placebo. On average, patients treated with Vimizim in the trial walked 22.5 meters farther in 6 minutes compared to the patients who received placebo.

The most common side effects in patients treated with Vimizim during clinical trials included fever, vomiting, headache, nausea, abdominal pain, chills and fatigue. The safety and effectiveness of Vimizim have not been established in pediatric patients less than 5 years of age. Vimizim is being approved with a boxed warning to include the risk of anaphylaxis. During clinical trials, life-threatening anaphylactic reactions occurred in some patients during Vimizim infusions.

Vimizim is marketed by Novato, Calif.-based BioMarin Pharmaceutical Inc.

Elosulfase alfa (GALNS), a proposed treatment for Morqio A syndrome. Morquio A syndrome is an inherited, autosomal recessive disease caused by a deficiency of a particular lysosomal enzyme, N- acetylgalactosamine- 6 sulfatase. BioMarin’s experimental drug for Morquio A syndrome is an enzyme replacement of elosulfase alfa (called BMN 110), which is designed to clear keratan sulfate from the lysosome. BMN 110 is being studied to determine if it is safe, if it will slow the progression of the disease and if it will improve some of the symptoms.

BioMarin started BMN 110 clinical studies in humans in 2009 to evaluate safety and efficacy. In a phase III Multicenter, Multinational, Extension Studythe Long-Term Efficacy and Safety of BMN 110 in Patients With Mucopolysaccharidosis IVA (Morquio A Syndrome) MOR-005 was evaluated. Participants will receive 2 mg/kg weekly or every other weekly dosing of study drug via infusion until the MOR- 004 study is unblinded and the optimal dose is selected. All subjects will then be treated with the optimal dose for up to approximately 5 years or until the drug is approved.

Omacetaxine mepesuccinate 高三尖杉酯碱

Alkaloid from Cephalotaxus harringtonia; FDA approved orphan drug status for Ceflatonin in the treatment of chronic myeloid leukemia due to being an inducer of apoptosis in myeloid cells and inhibitor of angiogenesis.
26833-87-4 CAS NO

1-((1S,3aR,14bS)-2-Methoxy-1,5,6,8,9,14b-hexahydro-4H-cyclopenta(a)(1,3)dioxolo(4,5-h)pyrrolo(2,1-b)(3)benzazepin-1-yl) 4-methyl (2R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl)butanedioate

1-((11bS,12S,14aR)-13-methoxy-2,3,5,6,11b,12-hexahydro-1H-[1,3]dioxolo[4',5':4,5]benzo[1,2-d]cyclopenta[b]pyrrolo[1,2-a]azepin-12-yl) 4-methyl 2-hydroxy-2-(4-hydroxy-4-methylpentyl)succinate

Also known as:  NSC-141633,

• BRN 5687925
• Ceflatonin
• CGX-635
• Homoharringtonine
• Myelostat
• NSC 141633
• Omacetaxine mepesuccinate
• Omapro
• Synribo
• UNII-6FG8041S5B
• 高三尖杉酯碱

CGX-635-14 (formulation), CGX-635, HHT, ZJ-C, Myelostat, Ceflatonin

 USFDA on 26th October 2012  APPROVED

US FDA:    link

The US Food and Drug Administration has now issued full approval for Israeli drugmaker Teva’s Synribo (omacetaxine mepesuccinate) for chronic myeloid leukaemia (CML).

Synribo is indicated for adult patients with chronic phase (CP) or accelerated phase (AP) CML with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs).

Read more at: http://www.pharmatimes.com/Article/14-02-17/US_green_light_for_Teva_s_CML_drug_Synribo.aspx#ixzz2tdkbGFcw

Homoharringtonine is an angiogenesis-inhibiting and apoptosis-inducing alkaloid which was approved in October 2012 by the FDA for the treatment of adult patients with chronic or accelerated phase chronic myeloid leukemia (CML) with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKI). In November 2012, the product was commercialized as Synribo(R) on the U.S. market by Teva.

The original developer, ChemGenex, selected homoharringtonine for the combination trials due to its complementary mechanism of action that can reduce Bcr-Abl protein expression associated with resistance to imatinib mesylate.

In 2004, the compound received orphan drug designation from the EMEA for the treatment of AML and CML. Orphan drug designation was granted by the FDA for the treatment of CML in 2006 and for the treatment of myelodysplasia in 2009. Fast track designation was assigned to homoharringtonine for CML in 2006. In 2009, the product was licensed to Hospira by ChemGenex Pharmaceuticals for development and marketing in Europe, the Middle East and parts of Africa.

Homoharringtonine, AKA HHT or omacetaxine mepesuccinate, is a cephalotaxine ester and protein synthesis inhibitor with established clinical activity as a single agent in hematological malignancies. Homoharringtonine is synthesized from cephalotaxine, which is an extract from the leaves of the plant, Cephalotaxus species. In October 2005, homoharringtonine received Orphan Drug designation from the EMEA for the treatment of chronic myeloid leukemia (CML). Then in March 2006, homoharringtonine received Orphan Drug status from the FDA for the treatment of CML. In November 2006, homoharringtonine, for the treatment of CML, was granted Fast Track designation by the FDA. Most recently, in October 2012, homoharringtonine was marketed under the brand name Synribo” and FDA approved for patients who are intolerant and/or resistant to two or more tyrosine kinase inhibitors used to treat accelerated or chronic phase CML

Omacetaxine mepesuccinate is administered subcutaneously and acts differently from TKIs. It may have a therapeutic advantage for patients who have failed TKIs. Omacetaxine is currently in global phase 2/3 clinical trials for CML and has been granted Orphan Drug designations by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMEA) as well as Fast Track status by the FDA. In vitro and animal model trails are promising and recent results showed that omacetaxine has potential to treat resistant leukemia mainly CML and ALL.

Tetrahedron Letters,Vo1.23,No.34,pp 3431-3434 … - Brock University

Omacetaxine mepesuccinate

Omacetaxine mepesuccinate (INN, trade name Synribo) is a semi-synthetic analogue of an alkaloid from Cephalotaxus harringtonia that is indicated for treatment of chronic myelogenous leukemia (CML). It was approved by the US FDA in October 2012 for the treatment of adult patients with CML with resistance and/or intolerance to two or more tyrosine kinase inhibitors (TKIs).^[1]

Omacetaxine is indicated for use as a treatment for patients with chronic myeloid leukaemia who are intolerant of tyrosine kinase inhibitors.^[2][3]

In June 2009, results of a long-term open label Phase II study were published, which investigated the use of omacetaxine infusions in CML patients. After twelve months of treatment, about one third of patients showed a cytogenetic response.^[4] A study in patients who had failed imatinib and who had the drug resistant T315I mutation achieved cytogenetic response in 28% of patients and haematological response in 80% of patients, according to preliminary data.^[5]

Phase I studies including a small number of patients have shown benefit in treating myelodysplastic syndrome (MDS, 25 patients)^[6] and acute myelogenous leukaemia (AML, 76 patients).^[7] Patients with solid tumors did not benefit from omacetaxine.^[8]

Omacetaxine is a protein translation inhibitor. It inhibits protein translation by preventing the initial elongation step of protein synthesis. It interacts with the ribosomal A-site and prevents the correct positioning of amino acid side chains of incoming aminoacyl-tRNAs. Omacetaxine acts only on the initial step of protein translation and does not inhibit protein synthesis from mRNAs that have already commenced translation.^[9]

Omacetaxine mepesuccinate

SYNRIBO contains the active ingredient omacetaxine mepesuccinate, a cephalotaxine ester. It is a protein synthesis inhibitor. Omacetaxine mepesuccinate is prepared by a semi-synthetic process from cephalotaxine, an extract from the leaves of Cephalotaxus sp. The chemical name of omacetaxine mepesuccinate is cephalotaxine, 4-methyl (2R)-hydroxyl-2-(4-hydroxyl-4-methylpentyl) butanedioate (ester).

Omacetaxine mepesuccinate has the following chemical structure:

The molecular formula is C₂₉H₃₉NO₉ with a molecular weight of 545.6 g/mol. SYNRIBO for injection is a sterile, preservative-free, white to off-white, lyophilized powder in a single-use vial. Each vial contains 3.5 mg omacetaxine mepesuccinate and mannitol.

SYNRIBO is intended for subcutaneous administration after reconstitution with 1.0 mL of 0.9% Sodium Chloride Injection, USP. The pH of the reconstituted solution is between 5.5 and 7.0.

…………………………………..

INTRODUCTION

Harringtonines 3 are particular cephalotaxanes formed by attachement of a branched hydroxyacyloxy side-chain at the 3-position of various cephalotaxines moieties. Harringtoriines are natural esters of cephalotaxines exhibiting generally a strong cytotoxic activity. However the lost only one atom of this minimal structure lead to a dramatic lost of activity (see below). Some example of harringtonines are harringtonine

3a, homoharringtonine 3b, drupangtonine 3c, anhydroharringtonine 3d and neoharringtonine 3e.

SCHEME 1 DEFINITION NOMENCLATURE AND NUMBERING OF CEPHALOTAXANES

Examples of harringtonines

Examples of cephalotaxines

Harringtonine 3a (n = 2) Anhydroharringtonine 3d Homoharringtonine 3b (n = 3)

(-)-Cephalotaxine 2a

Drupacine 2b Drupangtonine 3c Neoharringtonine 3e (n = 2)

…………………………………

The term “cephalotaxanes” refers to compounds or salts thereof which have a basic skeleton of formula

where p is equal to 1 or 2 (it being possible for the two units to be identical or different and linked via a single bond or an oxygen atom), which can contain various oxygenated substituents (aliphatic or aromatic ethers, free or esterified alcohols, substituted or free enols and/or phenols, bridged ethers, and more generally any substituent usually encountered in the natural state on compounds of this type).

Harringtonines are alkaloids which are of high interest in anticancer chemotherapy, in particular on certain haematosarcomas which are multi-resistant to the existing therapies. The selectivity of harringtonines, which is based on a novel mechanism of action relating to protein synthesis, is such that this series is favoured with a great future in anticancer therapy.

Several literature compilations give a seemingly exhaustive review of all of the knowledge relating to cephalotaxanes, these compilations being, chronologically: [C. R. Smith, Jr, R. G. Powell and K. L. Mikolajczack, Cancer Treat. Rep., Vol. 60, 1157 (1976); C. R. Smith, Jr, L. Kenneth, K. L. Mikolajczack and R. G. Powell in “Anticancer Agent Based on Natural Product Model”, 391 (1980); Liang Huang and Zhi Xue in “The Alkaloids”, Vol. XXIII (A. Brossi Ed.), 157 (1984); M. Suffness and G. A. Cordell in “The Alkaloids, Chemistry and Pharmacology” (A. Brossi Ed.), Vol. 25, 57-69, 295-298 (1'987); P. J. O'Dwyer, S. A. King, D. F. Hoth, M. Suffness and B. Leyland-Jones, Journal of Clinical Oncology, 1563 (1986); T. Hudlicky, L. D. Kwart and J. W. Reed, in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, 199 (1998)].

Antiparasitic activities, in particular on the haematozoon of malaria, have also been recognized [J. M. Whaun and N. D. Brown, Ann Trop. Med. Par., Vol. 84, 229 (1990)].

Homo-harringtonine (HHT), the most active member of the series, is active at and above daily doses of 2.5 mg/m² of body area per 24 hours, i.e., as a guide, at doses twenty times lower than that for Taxol. HHT has already undergone fourteen phase I and II clinical trials and it is the only known product capable of a 70% reinduction of full haematological remissions in patients suffering from chronic myeloid leukaemias that have become resistant to alpha-interferon [S. O'Brien, H. Kantarjian, M. Keating, M. Beran, C. Koler, L. E. Robertson, J. Hester, M. Rios, M. Andreeff and M. Talpaz, Blood, 332 (1995); Leukemia Insights, Vol. 3, No. 1 (1998)].

Harringtonines were extracted over 35 years ago from an exclusively Asiatic cephalotaxacea known as Cephalotaxus harringtonia, following the programme of research into novel anticancer agents in the plant kingdom developed by the National Cancer Institute. In fact, the Cephalotaxus alkaloids consist essentially (at least 50%) of cephalotaxine, a biosynthetic precursor of the harringtonines, the latter individually representing only a few percent of the total alkaloids.

Besides their low concentration in the natural state in plant starting material, harringtonines are mixed with many congeners which have very similar chemical structures. Thus, in a high resolution high performance liquid chromatography (HPLC) chromatogram of a semi-purified alkaloid extract, no less than several tens of cephalotaxine esters are counted.

Numerous antileukemia drugs have been investigated but so far, there is no single drug that is effective and safe. As discussed in U.S. 3,497,593, an alkaloid from Tylophora plant is said to have antitumor activity against mouse leukemia (L-1210). U.S. 3,928,584 discloses an organic composition derived from tree sap and is said to have activity against mouse leukemia P-388. Also U.S. 4,431,639 discloses that an extract of Rhisoma Stractylis promotes the production of lymphocytes in the circulating blood, consequently eliminating cancer growth

• Harringtonine or Homoharringtonine, hereinafter referred to as HH, has been known to be effective against acute chronic granulocytic and monocytic leukemia (Journal of Chinese Internal Medicine 3:162-164, 1978). However, it is highly toxic and causes damage to heart and hematopoietic organs. The results of experiments in animals, such as mice, rabbits and dogs, indicate that most of them die from cardiotoxicity after receiving the drug. Therefore, there is a need to improve the HH drug for safe use against leukemia. This drug is of special importance in that all known antileukemia drugs are effective against lymphatic leukemia and there are no effective drugs for treating nonlymphatic leukemia

All the literature from 1972 to the present date [Mikolajczack et al., Tetrahedron, 1995 (1972); T. Hudlicky, L. D. Kwart and J. W. Reed in “Alkaloid: Chemical and Biological Perspectives” (S. W. Pelletier Ed.), Vol. 5, 639 (1987); M. A. Miah, T. Hudlicky and J. Reed in “The Alkaloids”, Vol. 51, p. 236 (1998)] mention the impossibility hitherto of esterifying the highly sterically hindered secondary hydroxyl of cephalotaxane 2a with the tertiary carboxyl of the alkanoyl chain of harringtonic acid 3 totally preformed to give a harringtonine 4b, i.e. the conversion 2a+3e(4b as described in the example featured in the scheme below

• ……………………………………………………..

SYNTHESIS

Tetrahedron Lett 1982,23(34),3431,  J Org Chem 1983,48(26),5321

The oxidation of 2-methyl-1-cyclopentene-1-carbaldehyde (I) with O3 and Ag2O gives 2,6-dioxoheptanoic acid (II), which is esterified with cephalotaxine (III) by means of (COCl)2, yielding the ester (IV). Reformatsky reaction of (IV) with methyl bromoacetate (V) and Zn affords the adduct (VI), which is treated with an excess of methylmagnesium iodide to provide the target homoharringtonine (as a single diastereomer), along with some starting cephalotaxine that is separated by chromatography.

………………………………

SYNTHESIS

EP 1064285; FR 2776292; WO 9948894, Tetrahedron Lett 1999,402931

The intermediate (racemic)-2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid (VIII) has been obtained by several related methods: 1. The Grignard condensation of 4-methyl-3-pentenyl bromide (I) with diethyl oxalate (II) in HF gives the 2-oxoheptenoate (III), which is condensed with methyl acetate (IV) by means of LiHMDS in THF to yield 3-(ethoxycarbonyl)-3-hydroxy-7-methyl-6-octenoic acid methyl ester (V).

The cyclization of (V) by means of Ts-OH in hot toluene or by means of hot aqueous formic acid affords 2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid ethyl ester (VI), which is hydrolyzed with KOH in boiling water to provide the corresponding dicarboxylic acid (VII). Finally, this compound is regioselectively monoesterified by means of BF3/MeOH in methanol to furnish the intermediate (racemic)-2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid (VIII). 2.

The reaction of 3-(ethoxycarbonyl)-3-hydroxy-7-methyl-6-octenoic acid methyl ester (V) with HCl in hot methanol gives 3-(ethoxycarbonyl)-3,7-dihydroxy-7-methyloctanoic acid methyl ester (IX), which is then cyclized by means of ZnCl2 in hot dichloroethane to yield the previously described intermediate (VIII). 3. The hydrolysis of 3-(ethoxycarbonyl)-3-hydroxy-7-methyl-6-octenoic acid methyl ester (V) with KOH in refluxing methanol/water gives the corresponding diacid (X), which is regioselectively monoesterified by means of BF3/MeOH in methanol to yield 3-carboxy-3-hydroxy-7-methyl-6-octenoic acid methyl ester (XI).

Finally, this compound is cyclized by means of Ts-OH in hot toluene to afford the previously described carboxylic intermediate (VIII). The racemic acid (VIII) is submitted to optical resolution by esterification with quinine (XII) by means of 2,4,6-trichlorobenzoyl chloride and TEA or DCC to give a diastereomeric mixture of esters (XIII) that is separated by preparative HPLC to obtain the desired diastereomer (XIV).

The hydrolysis of (XIV) with KOH in refluxing ethanol/water gives the corresponding chiral dicarboxylic acid (XV), which is regioselectively monoesterified with BF3/MeOH in methanol to yield the chiral (R)-2-(methoxycarbonylmethyl)-6,6-dimethyltetrahydropyran-2-carboxylic acid (XVI).

The esterification of (XVI) with cephalotaxine (XVII) by means of 2,4,6-trichlorobenzoyl chloride and TEA in toluene affords the corresponding ester (XVIII), which is treated with HBr in dichloromethane/HOAc, providing the bromoester (XIX). Finally, this compound is treated with NaHCO3, CaCO3 or BaCO3 in acetone/water to give the target hydroxyester.

………………………………………….

EXTRACTION

EP0203386B1

• Throughout the specification, the concentration of the solvent is the same as first given unless stated otherwise. Redeuced pressure means about 2,27 kPa (17 mm Hg. abs), l is liter, kg is kilogram. ml is milliliter. Yield in weight %.
  Example 1. HH is extracted from the skins, stems, leaves and seeds of Cephalotaxus fortunel Hook and other related species, such as Cephalotaxus sinensis Li, C. hainanensis, and C. wilsoniana, including C.oliveri mast and C.harringtonia.
• 1 kg of finely ground Cephalotaxus fortunel Hook is extracted with 8 l of 90% ethanol at room temperature for 24 hrs. The solution is filtered to yield a filtrate A and filtercake. The filtercake is percolated with ethanol and filtered again to yield filtrate B. A and B are combined and distilled under reduced pressure to recover ethanol and an aqueous residue. To this residue, 2% HCl is added to adjust the pH to 2.5. The solids are separated from the solution by filtration to yield a filtrate C. The solids are washed once with 2% HCl and filtered to yield a filtrate D. C and D are combined and the pH adjusted to 9.5 by adding saturated sodium carbonate solution. The alkaline filtrate is extracted with chloroform and the chloroform layer separated from the aqueous layer. This extration process is repeated five times. All the chloroform extracts are combined and distilled at reduced pressure to recover chloroform and alkaloid as a solid residue respectively.
• The solid alkaloid is then dissolved in 20 ml. of 6% citric acid in water. The solution is divided into three equal portions. These are adjusted to pH 7,8 and 9 by adding saturated sodium carbonate solution.
• The portions having pH 8 and 9 are combined and extracted with chloroform. The chloroform extracts are distilled under reduced pressure, whereby chloroform is removed and recovered and a solid residue of crude Harringtonine is obtained.
• The crude Harringtonine is dissolved in pure ethanol i.e. alkaloid : anhydrous ethanol 1:10 , and crystallized. The crystals are refined by recrystalliation in diethyl ether. Overall yield of Harringtonine is about 0.1% including yield from mixed HH from the subsequent process.
  Harringtonine has the following chemical structure:

  wherein R is

  melting point:

  135° – 137°C

  crystal:

  colorless

  infrared spectrum:

  3750, 1660, 1505, 1490, 1050, and 945 cm⁻¹.
• The portion having a pH of 7 and the mother liquors from the foregoing crystallization of Harringtonine are combined and passed through a liquid chromatographic column of diameter to height ratio 1:50 packed with alumina. The column is finally flushed with chloroform and followed by chloroform-methanol of 9:1 mixture. The resulting alkaloids are mixture of HH. The mixed HH is then separated from each other by countercurrent distribution employing chloroform and pH 5 buffer. The first fraction of the countercurrent distribution is Homoharringtonine and the last fraction of the countercurrent distribution is Harringtonine. Homoharringtonine is purified by crystallization in methyl alcohol.
  Homoharringtonine has the following chemical structure:

  wherein R is

  yield:

  0.02%

  melting point:

  144° – 146°C

  infrared spectrum:

  3500∼3400, 1750, 1665, 1030 and 940 cm⁻¹.

…………………………………………………………………………..

EXTRACTION

EP1064285B1

All the literature from 1972 to the present date [Mikolajczack et al.,Tetrahedron, 1995 (1972); T. Hudlicky, L.D. Kwart and J.W. Reed in "Alkaloid: Chemical and Biological Perspectives" (S.W. Pelletier Ed.), Vol. 5, 639 (1987); M.A. Miah, T. Hudlicky and J. Reed in "The Alkaloids", Vol. 51, p. 236 (1998)] mention the impossibility hitherto of esterifying the highly sterically hindered secondary hydroxyl of cephalotaxine 2a with the tertiary carboxyl of the alkanoyl chain of harringtonic acid 3e totally preformed to give a harringtonine 4b , i.e. the conversion 2a + 3e ( 4b as described in the example featured in the scheme below

Example 46

Preparation of purified (-) cephalotaxine from total alkaloidic extract of Cephalotaxus sp

• [0319]
• Partially racemized cephalotaxine [H. Wenkui; L. Yulin; P. Xinfu, Scientia Sinica,; 23; 7; 835 (1980)]
• ¹H NMR of two batches of cephalotaxine (extracted in the same conditions as above) with the optically active NMR shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorate (1 éq) showed the following results:

  • Batch A: ¹H NMR 400 MHz (CDCl₃)(δ ppm): 6.06 (1H, OCH₂O (+)-cephalotaxine) and 5.82 (1H, OCH₂O (+)-cephalotaxine) ; 5.99 (1H, OCH₂O (-)-cephalotaxine) and 5.76 (1H, OCH₂O (-)-cephalotaxine).
    Presence of 11 ± 5 % de (+)-cephalotaxine.
    [α]²² = -134,0° (c = 0,214; CHCl₃) : calculated rate 25 ± 5 %
  • Batch B: slightly racemized (1%)
    [α]¹⁹ = -173,3° (c = 0,208; CHCl₃)

Enantiomeric enrichment of the natural cephalotaxine:

• Crude chromatographied cephalotaxine (20g) was dissolved at 55°C in dry methanol (100 ml). Crystallization occurs by cooling with rotary evaporator and after filtration the product thus obtained showed 99.9% of HPLC purity.
  [α]²⁰ _D =-130° (C1, CHD₃) corresponding to 10 % of racemization. The crystallized product thus obtained (20g) was dissolved again in hot methanol (100 ml).
  Slowly cooling the solution allows translucent prisms composed of pure enantiomeric (-)-cephalotaxine [α]²⁰ _D= -185°(C1,CHCl₃).
  After filtration, the mother liquors was allowed to slowly evaporate at room temperature and crystals in the form of macled needles exclusively composed of racemic cephalotaxine [α]^D ₂₀ = 0,5° (C1 ; CHCl₃) were obtained.
  After filtration, the second mother liquors allowed prisms composed of (-)-cephalotaxine identical to this obtained at the first crystallization.
  After filtration, the third mother liquors still allowed macled needles (urchins) composed of (±)-cephalotaxine.
  The cycle is repeated three times. The combined prismatic crystals was recrystallized once to give enantiomerically pure (-)-cephalotaxine, while the combined macled needles treated in the same way gives 100% racemic cephalotaxine.

Chemical evaluation of the enantiomeric purity of natural cephalotaxine:

• A sample of partially racemized natural cephalotaxine was inserted in the process, which sequence is described in the Examples 1,2,3,4,5,6,15,19 and 21, by using a pure (2R)-homoharrintonic acid resulting from Example 19.
  The HPLC analysis of the diastereomeric mixture of anhydro-homoharrintonine thus obtained showed a significant enantio-epi-homoharringtonine rate (11% ± 3%) corresponding to the (+)-cephalotaxine content in the racemic mixture of origin (it has been demonstrated that the two antipodes of the homoharringtonic acid react in a stoechiometric way comparable to the pure enantiomeric cephalotaxine).

Example 47Preparation of homoharringtonine, from anhydro-homoharringtonine:

1)° Method A

• A commercial solution of hydrobromic acid in acetic acid (17.4 ml, 86.6 mmol, HBr 30% w/w) was added to a stirred solution of anhydrohomoharringtonine resulting from Example 21 (50.8 g, 9.63 mmol) in anhydrous dichloromethane (25.6 ml) at -10°C. After stirring at -10°C for 3 hours was added water (240 ml) and the reaction mixture was become viscous. The temperature was allowed to rise to room temperature and after stirring for 2.5 hours was added sodium carbonate 0.76M (406 ml) to pH 8. The resulting aqueous layer was saturated with sodium chloride, then was extracted with dichloromethane (3 × 230 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a foam. After phase reverse chromatography below-mentioned were obtained 4.03g of homoharringtonine (77%). The product thus obtained showed identical characteristics to this resulting from Example 25.

2°) Method B

• To a stirred solution of anhydrohomoharringtonine resulting from Example 21 (214 mg, 0.406 mmol) in anhydrous dichloromethane (1.1 ml) was added at -10°C a commercial solution of hydrobromic acid in acetic acid (0.728 ml, 3.6 mmol, HBr 30% w/w). After stirring at -10°C for 3 hours, was added water (13 ml) and then the temperature was raised to 20°C. After stirring at 20°C for 3 hours, was added a sodium carbonate solution (0.76M; 31.5 ml) up to pH 8. The resulting aqueous layer, after saturation with sodium chloride, was extracted with dichloromethane (3 × 20 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product was purified by phase reverse chromatography below-mentioned to provide homoharringtonine (166 mg, 75%). The product thus obtained showed identical characteristics to this resulting from Example 25.

……………………

SEMISYNTHESIS

US6831180

EXAMPLE 27 Preparation of homoharringtonine as a pharmaceutical use from crude semi-synthetic homoharringtonine resulting from example 25 by preparative high-performance liquid chromatography

1°) Method A

Crude homoharringtonine (35 g) is dissolved in buffer (triethylamine (1.55/1000) in deionised water and orthophosphoric acid to adjust pH to 3. The solution was filtered then injected on a preparative high-performance liquid chromatograph equipped with axial compression and high pressure pump (stationary phase: n-octadecylsilane, 15 μm, porosity 100, 1 kg; mobile phase; buffer/tetrahydrofurane 85/15). Elution was performed at a flow rate of 0.2 l/min. Fractions contain was monitored by U.V. detector and TLC. Retained fraction were finally checked by HPLC then combined, alkalinised with 2.5% aqueous ammonia and extracted with dichloromethane (4×400 ml). After concentration under reduced pressure homoharringtonine is obtained as a pale yellow resin which on trituration in a 8/2 water-methanol mixture gave pure homoharringtonine as a white crystalline solid (mp=127° C.), HPLC purity was higher than 99.8%.

2°) Method B

Same procedure of purification as method A was performed but mobile phase buffer/methanol (68/32) was used instead buffer/tetrahydrofurane.

3°) Method C

Same procedure of purification as method A was performed but mobile phase buffer/acetonitrile (85/15) was used instead buffer/tetrahydrofurane.

EXAMPLE 28 Preparation of homoharringtonine as a pharmaceutical use from semi-purified natural cephalotaxine

Crude homoharringtonine, prepared according to Example 25 from a partially racemized natural cephalotaxine and purified by chromatography and crystallisation according to the method A of Example 27, gave an homoharringtonine showing a non natural enantiomeric epi-homoharringtonine content less than 0.05%.

EXAMPLE 46 Preparation of purified (−) cephalotaxine from total alkaloidic extract of cephatotaxus sp

Partially racemized cephalotaxine [H. Wenkui; L. Yulin; P. Xinfu, Scientia Sinica; 23; 7; 835 (1980)]

¹H NMR of two batches of cephalotaxine (extracted in the same conditions as above) with the optically active NMR shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorate (1éq) showed the following results:

Batch A: ¹H NMR 400 MHz (CDCl₃)(δ ppm): 6.06 (1H, OCH₂O (+)-cephalotaxine) and 5.82 (1H, OCH₂O (+)-cephalotaxine); 5.99 (1H, OCH₂O (−)-cephalotaxine) and 5.76 (1H, OCH₂O (−)-cephalotaxine). Presence of 11±5% de (+)-cephalotaxine. [α]²²=−134,0°(c=0,214; CHCl₃): calculated rate 25±5%

Batch B: slightly racemized (1%) [α]¹⁹=−173,3°(c=0,208; CHCl₃)

Enantiomeric Enrichment of the Natural Cephalotaxine:

Crude chromatographied cephalotaxine (20 g) was dissolved at 55° C. in dry methanol (100 ml). Crystallization occurs by cooling with rotary evaporator and after filtration the product thus obtained showed 99.9% of HPLC purity, [α]²⁰ _D=−130°(C1, CHD₃) corresponding to 10% of racemization. The crystallized product thus obtained (20 g) was dissolyed again in hot methanol (100 ml).

Slowly cooling the solution allows translucent prisms composed of pure enantiomeric (-−)-cephalotaxine [α]²⁰ _D=−185°(C1, CHCl₃).

After filtration, the mother liquors was allowed to slowly evaporate at room temperature and crystals in the form of macled needles exclusively composed of racemic cephalotaxine [α]^D ₂₀=0,5°(C1; CHCl₃) were obtained.

After filtration, the second mother liquors allowed prisms composed of (−)-cephalotaxine identical to this obtained at the first crystallization.

After filtration, the third mother liquors still allowed macled needles (urchins) composed of (±)-cephalotaxine.

The cycle is repeated three times. The combined prismatic crystals was recrystallized once to give enantiomerically pure (−)-cephalotaxine, while the combined macled needles treated in the same way gives 100% racemic cephalotaxine.

Chemical Evaluation of the Enantiomeric Purity of Natural Cephalotaxine:

A sample of partially racemized natural cephalotaxine was inserted in the process, which sequence is described in the Examples 1,2,3,4,5,6,15,19 and 21, by using a pure (2R)-homoharrintonic acid resulting from Example 19. The HPLC analysis of the diastereomeric mixture of anhydro-homoharrintonine thus obtained showed a significant enantio-epi-homoharringtonine rate (11%±3%) corresponding to the (+)-cephalotaxine content in the racemic mixture of origin (it has been demonstrated that the two antipodes of the homoharringtonic acid react in a stoechiometric way comparable to the pure enantiomeric cephalotaxine).

EXAMPLE 47

Preparation of homoharringtonine, from anhydro-homoharringtonine

1°) Method A

A commercial solution of hydrobromic acid in acetic acid (17.4 ml, 86.6 mmol, HBr 30% w/w) was added to a stirred solution of anhydrohomoharringtonine resulting from Example 21 (50.8 g, 9.63 mmol) in anhydrous dichloromethane (25.6 ml) at −10° C. After stirring at −10° C. for 3 hours was added water (240 ml) and the reaction mixture was become viscous. The temperature was allowed to rise to room temperature and after stirring for 2.5 hours was added sodium carbonate 0.76M (406 ml) to pH 8. The resulting aqueous layer was saturated with sodium chloride, then was extracted with dichloromethane (3×230 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a foam. After phase reverse chromatography below-mentioned were obtained 4.03 g of homoharringtonine (77%). The product thus obtained showed identical characteristics to this resulting from Example 25.

2°) Method B

To a stirred solution of anhydrohomoharringtonine resulting from Example 21 (21.4 mg, 0.406 mmol) in anhydrous dichloromethane (1.1 ml) was added at −10° C. a commercial solution of hydrobromic acid in acetic acid (0.728 ml, 3.6 mmol, HBr 30% w/w). After stirring at −10° C. for 3 hours, was added water (13 ml) and then the temperature was raised to 20° C. After stirring at 20° C. for 3 hours, was added a sodium carbonate solution (0.76M; 31.5 ml) up to pH 8. The resulting aqueous layer, after saturation with sodium chloride, was extracted with dichloromethane (3×20 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product was purified by phase reverse chromatography below-mentioned to provide homoharringtonine (166 mg, 75%). The product thus obtained showed identical characteristics to this resulting from Example 25.

…………………………………

EXTRACTION

US20100240887

The remarkable clinical efficacy of Homoharringtonine (HHT) resulting in lot of observations of complete remission of leukemia and other solid cancer in human being since 1988. Recently, research articles reported that the HHT efficacy in glaucoma, inhibition of Hepatities B virus replication and using in bone marrow transplantation. For example, the University of Texas M.D. Anderson Cancer Center and National Cancer Institute reported that “Ninety-two percent of patients achieved CHR with HHT.” [Susan O'Brien, at al.; Sequential homoharringtonine and interferon-α in the treatment of early chronic phase chronic myelogenous leukemia; Blood, Vol 93, No 12 (June 15), 1999: pp 4149-4153]. Another article reported that “the median number of days on HHT per month was 2 days with a median follow-up of 26 months; the estimated 2-year survival rate was 90%.” (Susan O’Brien, at al.; Simultaneous homoharringtonine and interferon-α in the treatment of patients with chronic-phase chronic myelogenous leukemia; American Cancer Society; Apr. 1, 2002, Vol 94, No. 7).

On Nov. 8, 1988, U.S. Pat. No. 4,783,454 titled Process for producing harringtonine and homoharringtonine disclosed the technique of isolation of a purified HHT from bark of Cephalotaxus. However, the natural source ofCephalotaxus is very limited. Trees of Cephalotaxus grow slowly. Bark ofCephalotaxus has very low content of HHT. Extracting HHT from bark ofCephalotaxus the yield was about 0.02% only. More important to harvest bark ofCephalotaxus will kill and destroy trees. Supply of HHT is very short now. Therefore, it is necessary to find a new manufacturing method.

DETAILED DESCRIPTION

Great progress has been made in research on Homoharringtonine (HHT) production and on future generation HHT drug since 1988. For example, the University of Texas M.D. Anderson Cancer Center and National Cancer Institute reported that “Ninety-two percent of patients achieved CHR with HHT.” Another article reported that “the median number of days on HHT per month was 2 days with a median follow-up of 26 months; the estimated 2-year survival rate was 90%.”

The good clinical results of HHT in treating cancer brought to the major problem, which is the supply of HHT both short term and long term. It is apparent that a huge amount of bark of Cephalotaxus is needed for collection, extraction and purification of HHT. It is clear that due to the slow growth of the trees ofCephalotaxus, which is a nature source of HHT, and the killing of trees by harvesting bark is not a sustainable resource for HHT production.

Present invention disclosed new methods for producing HHT. The new methods of producing HHT are shown as follows.

1. Tissue Culture (Plant Cell Culture):

Culture manipulation to promote secretion of HHT is a new way for an extracellular product HHT. The biosynthetic methods can yield more HHT through precursor of HHT feeding. The production of HHT increased significantly after the addition of the precursors and special biochemical agents. Content of precursor of HHT abounds in tree and it is very cheap. The present methods include several significant developments in technique of culture plant tissues that are

• • (a) yields of HHT selected from rapid growth, resistance to infections organisms; and
  • (b) HHT can excrete into media.

Traditional method of plant culture is very difficult to overcome the problem of high cost. Therefore, traditional method appears too long to have commercial value. HHT is secondary metabolite of Cephalotaxus. Secondary compound acts in defense against the harmful effects of toxins, carcinogens or mutagens found in the plant. In fact, traditional method is very difficult to increase HHT contenting in plant tissues. The present new method uses a special biochemical agent for increasing content of HHT and more easily to purify HHT from other metabolites.

More important is that the key of the present new technique for producing high content of HHT in plant cell culture is to increase production of HHT by directed fermentation through precursor of HHT feeding. The present new methods are used special metabolite of Cephalotaxus for markedly enhance production of HHT. Therefore, the present invention disclosed a new source for the long term of producing HHT.

2. Using Precursor of HHT:

Recent research’s results have established that direct production of HHT from its precursor and advances in biosynthetic understanding for HHT metabolism. Biosynthesis or semisynthesis of HHT from major nonactivity ingredients is well established through great advances in special biochemistry reactions. Using precursor of HHT for semisynthesis and increase of production in plant cell culture are new developing methods for producing HHT.

3. Using Leaves:

Our new method use leaves of tree of Cephalotaxus not use the bark. So far, the extraction of HHT is used bark. The leaves are harvested from the trees ofCephalotaxus, which grow in mountains of South China. The natural source of leaves is very abundance. The new methods do not use bark. Therefore, it can avoid destroy trees. The natural source of Cephalotaxus tree is very limited and slow growing. In fact, bark of Cephalotaxus has very low yield of HHT. The yield of HHT from Cephalotaxus bark is about 50-100 mg/kg of dried bark. The present new method, therefore, has a great economic and environmental value.

4. Semisynthesis:

HHT has received important chemical studies particularly in regard to structure and anticancer activity relationship and semisynthesis.

A great progress in biochemistry allows semisynthesis to use precursor of HHT from leaves of Cephalotaxus and to produce HHT. The total chemical synthesis of HHT appears too long to have commercial value too. Semisynthesis method can yield a high efficient conversion of precursor to HHT. It is other better biological source for manufacturing HHT. This new method uses closing chemical analogues to convert to HHT. This analogue is produced from leaves or other organ of Cephalotaxus. The present invention disclosed that new methods and techniques of manufacturing HHT could avoid chopping down Cephalotaxus trees which governmental environmentalists are trying to have declared a threatened species.

5. Using Taxol Residual

The anticancer drug Taxol is the most promising new chemotherapeutic agents that developed for cancer treatment in the past twenty years. Taxol has a unique mechanism of action. It has been shown to promote tubulin polymerization and stabilize microtubules against depolymerization. The FDA approved the clinical use of Taxol for several types of cancer. So far, annual sales of Taxol are more than $2 billion in market. Taxol is extracted from bark or leaves of an evergreen tree named Taxus species including Taxus brevifolia (or called Pacific yew). After Taxol has been extracted from bark or leaves, all residual materials of Taxus brecifolia named Taxus residual, which are waste.

Both taxol and HHT can be extracted from yew tree. The content of taxol is less than 0.01% in yew tree. The content of HHT in yew tree is about 0.01% -0.22%. The content of HHT is much higher than content of Taxol. Taxol extracted from bark of yew is difficult and expensive. One reason is that the presences of closely related congeners are similar to Taxol. A major congener is Cephalomannine (CPM), which is a waster of process in manufacturing of Taxol.

The chemical and physical characters are very close between Taxol and Cephalomannine (CPM).

CPM characterized by the same ring structure as Taxol and distinguishes from them only in C-13 ester structure. The present invention disclosed that CPM and related derivative are used to produce HHT.

The following specific examples will provide detailed illustrations of methods of producing relative drugs, according to the present invention and pharmaceutical dosage units containing demonstrates its effectiveness in treatment of cancer cells. These examples are not intended, however, to limit or restrict the scope of the invention in any way, and should not be construed as providing conditions, parameters, reagents, or

EXAMPLE 1

Production of HHT by Culture Cells

So far, HHT is extracted from bark and skins of Cephalotaxus species. However, growth of Cephalotaxus species is very slow and concentration of HHT in plant is extremely low. Furthermore, it is difficult to harvest the plants because of their low propagation rate and the danger of drastic reduced in plant availability. Also, cost of total chemical synthesis of HHT is very expensive and is not available for commerce now. For the reasons given above it is more difficult to obtainCephalotaxus on a large scale for long time. Therefore, Cephalotaxus cell cultures are one of best methods for obtaining HHT. In this present invention, special elicitation is disclosed and it will significantly increase production of HHT.

The methods of cell and tissue culture are disclosed as below.

Parts of bark, stems, leaves, or roots of Cephalotaxus species were surface disinfected by treatment in 70% ethanol for 10 minutes and followed by 0.1 HgCl₂for 3 minutes. Plant materials were washed five times for 10 minutes each by sterilized water. Parts of plant were cut into small pieces (0.5-1 mm) and put pieces to Murashige and Skoog’s (MS) medium and supplemented with derivative of new active ingredient of phylum mycota (IPM), precursor of HHT which is a derivative of Cephalotaxus (CEP), tyrosine (TYR) naphthaleneacetic acid (NAA), Kinetin (3 mg/L), and 3% sucrose (w/v). PH of medium was adjusted to 5.7˜5.8. Agar (10 g/L) added to medium. Callus tissues are collected from agar media and suspension cultured cells were harvested by filtration and cultured in MS medium.

The cultures were kept in a culture room at 26° C.±1° C. Friable callus tissues were obtained. The callu was inoculated into 4 L of MS liquid medium containing sucrose, derivative of CEP, PHE, TYR, NAA and Kinetin. Then callus tissues were cultivated 26° C. for 35 days on rotary shaker operated at 120 rpm in the dark. Cells were subcultured into fresh medium of same composition every 2 weeks and maintained at 120 rpm at 26°±1° C. Packed cell volume (PCV), fresh weight (FW), dry weight (DW), concentration of HHT and concentration of sugar were determined every 5th day. The cells were harvested and dried.

In general, callus and suspension cultures of cephalotaxus species grow very slow and no production of free or esterified HHT. However, according to the present invention, addition of IPM to cultures cause a drastic increasing in HHT after 30 days of incubation. For example, in control group (no IPM), HHT in cultured cells is 0.020 mg/g dry weight, but in treatment group (addition of IPM) HHT is about 0.050 mg/g dry weight. Therefore, IPM can increase 250% of content of HHT. It has resulted in plant cell culture systems that producing HHT at concentration higher than those produced by the mother plant. The production of HHT increases significantly after the addition of precursors (CEP). Addition of CEP can increase HHT. Obviously, the present invention provided a new commercial and economic method for producing HHT. The IPM and precursors (CEP) play key role in cultured cells.

EXAMPLE 2

Semi-Synthesis of HHT

HHT shows a significant inhibitory activity against leukemia and other cancer. Concentration of HHT, however, has only 0.01% in natural sources. Cephalotazine (CEP) is major alkaloids present in plant extracts and the concentration ofCephalotaxus has about 1%. Therefore, concentration of CEP is about 100 times higher then HHT in nature plant sources. But CEP is inactive. For the reason given above, semisynthesis of HHT from CEP will increase huge natural sources of HHT.

• • (1) Extraction of CEP

10 kg of dried stems or leaves or roots of Cephalotaxus species were milled, placed in a percolator, along 80 L of 95% of ethanol, and allowed to stand 24 hours. The ethanol was recovered under reduced pressure (below 40° C.). 20 L of 5% tartaric acid was added to concentrated ethanol solution. The ammonia water was added to the acidic solution and adjusted pH to 9. The solution of pH 9 was filtered and yielded a filtrate. The filtrate was extracted with CHCl₃. CHCl₃ was recovered under reduced pressure and residue was obtained. The residue was chromatographed packed with alumna and eluted by CHCl₃-MeOH (9:1). Eluate was concentrated under reduced pressure. Residue was dried under vacuum. The product is CEP.

• • (2) Semisynthesized HHT from CEP

Materials and Methods

Melting points were determined on a Fisher-Johns apparatus. Infrared spectra were obtained on a Perkin-Elmer 567 infrared spectrophotometer or on a Beckman 4230 IR spectrophotometer. Peak positions were given in cm⁻¹. The IR spectra of solid samples were measured as potassium bromide dispersions, and the spectra of liquids were determined in chloroform or carbon tetrachloride solutions. NMR spectra were measured on a Varian A-60, Perkin-Elmer R-32, Varian EM-390, or Brüker WH-90 NMR spectrometer. Chemical-shift values were given in parts per million downfield from Me₄Si as an internal standard. Mass spectra were run on an AE1 MS-12 Finnigan 3300, or CEC21-110B mass spectrometer.

Preparative thin-layer chromatography was accomplished using 750-μm layers of aluminum oxide HF-254 (type E), aluminum oxide 60 PF-254 (type E), silica gel HF-254 (type 60 PF-254), or silica gel GF-254. Visualization was by short-wave ultraviolet light. Grace silica gel, Grade 923, and Woelm neutral aluminum oxide, activity III, were used for column chromatography. Analytical thin-layer chromatography was run on plastic sheets precoated with aluminum oxide F-254 neutral (type T), 200-μm thick, and on Polygram Sil G/UV254 (silica gel), 250 μm on plastic sheets. Visualization was usually by short-wave ultraviolet light, phosphomolybdic acid, or iodoplatinate.

Preparation of α-Ketoester-Harringtonine

1 g of Benzene-α-acetone Na was put into 10 L of benzene. Mixture was stirred at room temperature then was dissolved in 10 L of pyridine and stirred at 0° C. Oxalic chloride was added from a dropping funnel to solution of pyridine. Stirring was continued while the solution warmed to room temperature and stand overnight. Excess reagent was removed. This solution was dissolved in CH₂Cl₂and cooled to near 0° C. in an ice water bath. 5 g of CEP, 2.5 L of CH₂Cl₂ and 2.5 L of pyridine were added to cold CH₂Cl₂ solution. Manipulations were done in a dry N₂ atmosphere and all glassware heat-dried just before use. The suspension was stirred at room temperature and overnight. The mixture was washed with 10% Na₂CO₃ and saturated aqueous NaCl, then dried with auhydrous magenesium sulfate, and filtered and the solvents were removed in vacuo. Evaporation provided as an amorphous solid α-ketoester-harringtonine (mp 143˜145° C.).

Semi-Synthesis of HHT

10 L of CH₃CHBrCOOEt and activated zin dust and THF were added to the α-ketoester-harringtonine (at −78° C.) for 6 hours followed by slow warming to room temperature with stirred. The reaction mixture was diluted with 10 L CHCl₃ and 10 L H₂O and solid Na₂CO₃ was added. CHCl₃ was evaporated under reduced pressure and residue was obtained.

The residue was purified by chromatography on alumina. The column was flushed with chloroform and followed by chloroform-methanol (9:1). The solvents were recovered under reduced pressure to provide as a solid. Solid was dissolved in pure ethanol and crystallized. The crystals were refined by recrystalization in diethyl ether. The crystals dried under vacuum. The product is HHT, which has the following characters:

[α]_D −119° (C=0.96),

MSm/e (%): 689 (M⁺, 3), 314 (3), 299 (20), 298 (100), 282 (3), 266 (4), 20 (3), 150 (8), 131 (12), 73 (18)

EXAMPLE 3

HHT Extracted from Plant Tissue

Extraction of HHT has several major methods which including extraction by organic solvent, chromatograph and adjust pH.

HHT was extracted from plant tissue culture, plant cells or leaves of Cephalotaxusspecies.

1 kg of ground Cephalotaxus fortunei Hook was extracted with 10 liters of water at room temperature for 24 hrs. To filtered the solution to yield a filtrate. Ten liters of 90% ethanol added to filtrate. The mixture was Centrifugalized to yield a sediment. Percolated the sediment with ethanol and filter again to yield filtrate, combined filtrates, and distilled under reduced pressure to recover ethanol and an aqueous residue. To this residue, added 10% of HCl to adjust the pH to 2.5. To separated the solids from the solution by filtration to yield a filtrate (1). Washed the solids once with 2% HCl and filtered to yield a filtrate (2). Combined (1) and (2) and adjusted the pH to 9.5 by adding saturated sodium carbonate solution. Extracted the alkaline filtrate with chloroform and separated the chloroform layer from the aqueous layer. To repeated this extraction process five times. Combined all the chloroform extracts and distilled at reduced pressure to recover chloroform and alkaloid as a solid residue obtained. The solid alkaloid was then dissolved in 6% citric acid in water. The solution was divided into three equal portions. These were adjusted to pH 7, 8 and 9 by adding saturated sodium carbonate solution. The portions having pH 8 and 9 were combined and extracted with chloroform. The chloroform extracts were distilled under reduced pressure, whereby chloroform was removed and recovered and crude HHT was obtained. The crude HHT was dissolved in pure ethanol and crystallized. The crystals were refined by recrystallization in diethyl ether. The crude HHT obtained.

The portion having a pH of 7 passed through a liquid chromatographic column packed with alumina of diameter to height 1:50. The column was finally flushed with chloroform and followed by chloroform-methanol of 9:1 mixture. The resulting alkaloids were mixture crude of HHT. Combined crude HHT and then separated from each other by countercurrent distribution employing chloroform and pH 5 buffers. The first fraction of the countercurrent distribution was HHT. HHT was purified by crystallization in methyl alcohol. The crystallization was purified by recrystallization in methyl alcohol and dried under vacuum.

…………………….

EP1373275A2

Example 1 : Preparation of harringtonine drug substance by purification of commercial natural harringtonine

A. Analytical profile of starting product

By combination of HPLC analysis with UV detection (see Figure 6) and mass spectrometry detection (see figure 7 and 8) a total of 6.5% of related compound (identified as b,c: position isomer of harringtonine = 3.4%; d: homoharringtonine = 3%; e: 4′-demethyl harringtonine = 0.01%; f: drupacine derivative: 0.05%) are found in the starting product.

B. Chromatography of natural harringtonine

Natural harringtonine (5 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 80 mm; length: 1000 mm) containing 1000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi). Unwanted fractions are discarded based upon in-line UV spectrophotometric detection. Kept fractions are collected in 16 separate containers which each are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase). During the development phase, a dual in-line UV-MS detection is used. After discarding of the fractions representing more than 0.5 % of the total content of harringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure. Then crude concentrated solution of harringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 1.5 %. C. Crystallization of raw harringtonine

Under a laminar flow hood, the above raw harringtonine (4.1 grams) is dissolved in methanol (5ml), at 30°C. The resulting alcoholic solution was filtered on a 0.25 μ sterile Millipore filter to remove microparticules and germs and collected in a sterilized rotary flask. Then, desionized water (50mL) is added and methanol is completely removed under vacuum at 30°C in using a decontaminated rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of harringtonine is kept under vacuum and rotation is continued during appearance of white crystals of pure harringtonine. The stirring is continued until no more crystal occurs. Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water (10 mL x 2). The white translucent crystals are then dried using high vacuum at 40°C for 24 hours. Overall yield is 76%. All operations were documented prior to start the process and full current Good Manufacturing Practices were applied. This clinical batch corresponds to 400 therapeutic units dosed at 10mg.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. Figure 7 and 9 compare HPLC chromatogram before and after purification in using this process. Table II shows the comparison of the corresponding related compound content.

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC) thermogravimetry, 2D NMR, solid NMR and X-ray powder diffractometry.

Infrared Spectrometry:

Identical IR spectra were obtained by either the KBr pellet and/or mineral oil mull preparation technique. Figure 5 shows typical infrared spectrum (KBr) for unambiguous identification at the solid state of the crystalline harringtonine obtained by this process. A series of sharp absorption bands are noted at 615, 654, 674, 689, 709, 722, 750, 761 805, 850, 928, 989, 1022, 1033, 1062, 1083, 1112, 1162, 1205, 1224, 1262, 1277, 1308, 1340, 1364, 1382, 1438 1486, 1508, 1625, 1656, 1725, 1745, 2883, 2936, 2972, 3079, 3353, 3552 and 3647 cm^“1

Differential Scanning Calorimetry (DSC) And Thermogravimetry (TG) Measurement of DSC and TG were obtained on a Mettler Toledo STAR System. Approximately 12 mg of harringtonine drug substance were accurately weighed (12.4471 mg) into a DSC pan. The sample was heated from 25°C to 200°C at a rate of 10°C/min. The DSC data were obtained following a standard method in the art. The DSC curve of crystalline harringtonine drug substance ((Figure 4), exhibits a melting endotherm at 79.5 °C . No subsequent decomposition occurred under the upper tested temperature 200°C. Simultaneous TG measurement, indicated a loss on drying of 1.3 % which did not correspond to a lost of structural molecule of solvent or water.

Example 2: Preparation of homoharringtonine drug substance by purification of raw semi- synthetic (hemi-synthetic) homoharringtonine

A. Analytical profile of starting product

Crude reaction mixture of raw homoharringtonine contains a potential of 250 grams of homoharringtonine DS together with process impurities such as catalyst, unchanged starting product (anhydro-homo-harringtonine), and some related side product. HPLC analysis with UV detection (see left-side chromatogram on Figure 10) indicated a total of 9 % of related impurities. B. Chromatography of semi-synthetic homoharringtonine

Raw semi-synthetic homoharringtonine (550 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 450 mm; length: 1000 mm) containing 48,000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi, flow-rate 540 L/hour). Unwanted fractions are discarded based upon by- passed in-line UV spectrophotometric detector. Kept fractions are collected in 30 separate stainless steel containers (20 or 50 L each) which are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase) and equipped with a diode array detector. After discarding of the fractions representing more than 0.5 % of the total content of homoharringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure in using a mechanically stirred thin film evaporator. Then crude concentrated solution of homoharringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 0.5 % (see rigth-side chromatogram on Figure 10)

C. Crystallization of homoharringtonine DS

In a controlled clean room, under a laminar flow hood, the above raw homoharringtonine DS (210 grams) is dissolved in methanol (240 mL), at 30°C. The resulting alcoholic solution is filtered on a 0.25 μ sterile Millipore filter to remove microparticules and germs and collected in a sterilized pilot rotary flask. Then, desionized water (2400mL) is added and methanol is completely removed under vacuum at 30°C in using a decontaminated pilot rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of homoharringtonine DS is kept under vacuum and rotation is continued during appearance of white crystals of pure homoharringtonine. The stirring is continued until no more crystal occurs. Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water (450 mL x 2). The white cryitals are then dried using high vacuum at 60°C for 48 hours. Overall yield is 88% from potential content of homoharringtonine in raw semi-synthetic homoharringtonine. All operations were documented prior to start the process and full current Good Manufacturing Practices were applied. This clinical batch corresponds to 40,000 therapeutic units dosed at 5mg.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. Figure 11 shows HPLC chromatogram before and after crystallization. Total of related impurities of homoharringtonine DS is 0.03%.

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC), thermogravimetry (TD), 2D NMR, solid NMR and X-ray powder diffractometry.

Infrared Spectrometry:

Identical IR spectra were obtained by either the KBr pellet and/or mineral oil mull preparation technique. Figure 3 shows typical infrared spectrum (KBr) for unambiguous identification at the solid state of the crystalline homoharringtonine obtained by this process. A series of sharp absorption bands are noted at 612, 703, 771 , 804, 826, 855, 879, 932, 1029, 1082, 1119,

1135, 1161 , 1191 , 1229, 1274, 1344, 1367, 1436, 1457, 1488, 1505, 1653, 1743, 2814, 2911 ,

2958, 3420, and 3552 cm^“1

Differential Scanning Calorimetry (DSC) And Thermogravimetry (TG)

Measurement of DSC and TG were obtained on a Mettler Toledo STAR System. Approximately 11 mg of homoharringtonine drug substance were accurately weighed (10.6251 mg) into a DSC pan. The sample was heated from 25°C to 250°C at a rate of 5°C/min. The

DSC data were obtained following a standard method in the art. The DSC curve of crystalline homoharringtonine drug substance (Figure 1), exhibits a melting endotherm at 145.6 °C.

Melting range performed by the capillary method (Bucchi Apparatus) gave 143-145°C. Literature indicated 144-146°C [Anonymous, Acta Bot. Sin. 22, 156 (1980) cited by L. Huang and Z. Xue, Cephalotaxus Alkaloids, in "The Alkaloids", vol. XXIII, pp157, (1988).

Crystallization medium was not published. This is the only literature reference regarding melting point of a crystalline form of HHT] X-Ray Powder Diffraction

X-ray powder diffraction pattern was collected on a INEL microdiffractomer, model

DIFFRACTINEL. Powdered homoharringtonine DS was packed in a glass capillary tube and was analyzed according to a standard method in the art. The X-ray generator was opered at 45 kV and 40 mA, using the copper Kalpha line as the radiation source. The sample was rotated along the chi axis and data was collected between 0 and 120 deg 2-theta. A collection time of 1200 sec was used. As showed on Figure 2, the x-ray powder diffraction for this crystalline form of homoharringtonine shows a typical pattern including major reflection peaks at approximately 7.9, 9.2, 10.9, 14.9 16.0, 17.7, 19.5, 19.7, 21.78, 23.1 , 25.3, 25.4 and 25.7 deg 2-theta.

Example 3: Preparation of homoharringtonine drug substance by purification of a commercial sample of impure homoharringtonine from Chinese source

A. Analytical profile of starting product

Analytical HPLC chromatogram of natural homoharringtonine (China National Pharmaceutical) is displayed on Figure 12 (bottom left).

B. Chromatography of Natural Homoharringtonine

Natural homoharringtonine (25 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 200 mm; length: 1000 mm) containing 12,000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi, flow-rate 120 IJhour). Unwanted fractions are discarded based upon bypassed in-line UV spectrophotometric detector. Kept fractions are collected in 22 separate stainless steel containers which are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase) and equipped with a diode array detector. After discarding of the fractions representing more than 0.5 % of the total content of homoharringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure in using a mechanically stirred thin film evaporator. Then crude concentrated solution of homoharringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 0.5 %.

C. Crystallization of homoharringtonine DS

In a controlled clean room, under a laminar flow hood, the above chromatographied homoharringtonine DS (18 grams) is dissolved in methanol (35 mL), at 30°C. The resulting alcoholic solution is filtered on a 0.25 μ sterile Millipore filter to remove microparticules and germs and collected in a sterilized pilot rotary flask. Then, desionized water (300 mL) is added and methanol is completely removed under vacuum at 30°C in using a decontaminated pilot rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of homoharringtonine DS is kept under vacuum and rotation is continued during appearance of white crystals of pure homoharringtonine. The stirring is continued until no more crystal occurs.

Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water

(50 mL x 2). The white crystals are then dried using high vacuum at 60°C for 48 hours. Overall yield is 84% from potential content of homoharringtonine in raw semi-synthetic homoharringtonine. All operations were documented prior to start the process and full current

Good Manufacturing Practices were applied.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. Figure 12 (bottom right) shows HPLC chromatogram after crystallization. Total of related impurities of homoharringtonine DS is 0.05%.

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC), thermogravimetry (TD), 2D NMR, solid NMR and X-ray powder diffractometry. Infrared Spectra, Differential Scanning Calorimetry (DSC) and X-Ray Powder Diffraction gave patterns strictly superimposable to the one of example 2 obtained from semi-synthetic homoharringtonine (Figure 3, 1 , and 2, respectively).

………………………………….

KOREAN PAPER.. LINK

Title: 한국산 개비자(Cephalotaxus koreans)에서의 Harringtonine과 Homoharringtonine의 확인 및 함량 분석
Author: 박호일 ; 이연 (한국생물공학회)
Source: 한국생물공학회지 = Korean journal of biotechnology and bioengineering; ISSN:1225-7117 @ 1225-7117 @ ; VOL.11; NO.6; PAGE.689-695; (1996)
Pub.Country: Korea
Language: Korean
Abstract: Harringtonine and homoharringtonine known as anti-cancer agents were isolated from Korean native plumyew(Cephalotaxus koreana) using column chromatography(CHCl3:MeOH=19:1, Rf=0.28). The structure of the mixture of two compounds was characterized by 1H-NMR. Comparison of our spectra of harringtonine and homoharringtonine with previously reported ones indicated that the two are identical. The contents of harringtonine and homoharringtonine in the needles, stems, and roots of Korean native plumyew were determined by high performance liquid chromatography(HPLC). The contents of both compounds varied with the site of location and the part of plant. The content of harringtonine was higher in needles and roots than in stems, whereas the content of homoharringtonlne was lower than harringtonine. Homoharringtonine contents in needles at Mt. Palgong, Mt. Dukyu, Mt. Baekyang, Mt. Jiri, and Namhae were higher than in stems and roots. But homoharringtonine contents in needles al Mt. Jokye and Jindo were lower than in stems and roots.

http://img.kisti.re.kr/originalView/originalView.jsp

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References

MOXIFLOXACIN

Bay-12-8039

US FDA:link

CAS 354812-41-2

186826-86-8 HYDROCHLORIDE

Moxifloxacin is a fourth-generation synthetic fluoroquinolone antibacterial agent developed by Bayer AG (initially called BAY 12-8039). It is marketed worldwide (as the hydrochloride) under the brand names Avelox, Avalox, and Avelon for oral treatment. In most countries, the drug is also available in parenteral form for intravenous infusion. Moxifloxacin is also sold in an ophthalmic solution (eye drops) under the brand names Vigamox, Moxezafor the treatment of conjunctivitis (pink eye).

A United States patent application was submitted on 30 June 1989, for Avelox (moxifloxacin hydrochloride).^[1] In 1999 Avelox was approved by theU.S. Food and Drug Administration (FDA) for use in the United States.^[2]

In the United States, moxifloxacin is licensed for the treatment of acute bacterial sinusitis, acute bacterial exacerbation of chronic bronchitis, community acquired pneumonia, complicated and uncomplicated skin and skin structure infections, and complicated intra-abdominal infections.^[3]

In the European Union, it is licensed for acute bacterial exacerbations of chronic bronchitis, non-severe community-acquired pneumonia, and acute bacterial sinusitis. Based on its investigation into reports of rare but severe cases of liver toxicity and skin reactions, the European Medicines Agency recommended in 2008 that the use of the oral (but not the IV) form of moxifloxacin be restricted to infections in which other antibacterial agents cannot be used or have failed.^[4] In the US, the marketing approval does not contain these restrictions, though the label contains prominent warnings against skin reactions.

MOXIFLOXACIN

Avelox (moxifloxacin) was launched in the United States in 1999 and is currently marketed in more than 80 countries worldwide. In the United States, Avelox is marketed by Bayer’s partner Merck.

In 2011 the FDA added two boxed warnings for this drug in reference to spontaneous tendon ruptures and the fact that moxifloxacin may cause worsening of myasthenia gravis symptoms, including muscle weakness and life-threatening breathing problems.^[5]

Moxifloxacin is used to treat a number of infections including: respiratory tract infections, cellulitis, anthrax, intraabdominal infections, endocarditis,meningitis, and tuberculosis.^[6]

The initial approval by the FDA (December 1999)^[7] encompassed the following indications:

• Acute Exacerbations of Chronic Bronchitis (AECB)

• Acute Bacterial Sinusitis (ABS)

• Community Acquired Pneumonia (CAP)

Additional indications were approved by the FDA as follows:

• April 2001: Uncomplicated Skin and Skin Structure Infections (uSSSI)^[8]

• May 2004: Community Acquired Pneumonia caused by multi-drug resistant Streptococcus pneumoniae.^[9]

• June 2005: Complicated Skin and Skin Structure Infections (cSSSI)^[10]

• November 2005: Complicated Intra-Abdominal Infections (cIAI).^[11]

The European Union requires that moxifloxacin only be prescribed when other antibiotics that have been initially recommended for treatment cannot be used or have failed.^[12][13]

At the current time,^[when?] there are no approved uses within the pediatric population for Oral and I.V. moxifloxacin. A significant number of drugs found within this class, including moxifloxacin, are not licensed by the FDA for use in children due to the risk of permanent injury to the musculoskeletal system.^[14][15][16]

In ophthalmology, moxifloxacin is approved for the treatment of conjunctival infections caused by susceptible bacteria.^[17]

Note: Moxifloxacin may be licensed for other uses, or restricted, by the various regulatory agencies worldwide

Marketing authorisations for the tablet and injectable forms of Moxifloxacin are held by Bayer, while Alcon (now a subsidiary of Novartis) produces ophthalmic solutions for treating conjunctivitis under the brand names of Moxeza, Vigamox, and Moxivig. Avelox generated sales of USD320 million in the first 9 months of 2013.

Moxifloxacin is available in three distinct administration forms. Formulated as a salt, Moxifloxacin hydrochloride is sold as an oral 400 mg film-coated tablet and as an injectable solution for infusion by Bayer; although in the US it is distributed by Merck Sharp and Dohme under license from Bayer.

Alcon has formulated Moxifloxacin hydrochloride as a 0.5% ophthalmic solution under license from Bayer. Moxifloxacin was first discovered in 1988 and received the first market authorisation eleven years later in 1999 in the US.

Moxifloxacin hydrochloride is a synthetic broad-spectrum antibacterial agent. The active moiety, moxifloxacin has been shown to be clinically active against most strains of microorganisms such as aerobic gram-positive microorganisms including staphylococcus aureus, streptococcus pneumonia (penicillin-susceptible strains) and streptococcus pyogenes, aerobic gram-negative microorganisms including haemophilus influenza hemophilus parainfluenzae, klebisiella pneumonia. Moxifloxacin is commercially available under the brand name of AVELOX® marketed by Bayer pharms.

VIGAMOX® (moxifloxacin hydrochloride ophthalmic solution) 0.5% is a sterile solution for topical ophthalmic use. Moxifloxacin hydrochloride is an 8-methoxy fluoroquinolone anti-infective, with a diazabicyclononyl ring at the C7 position.

http://images.rxlist.com/images/rxlist/vigamox1.gif” width=”286″ height=”171″ />

C₂₁H₂₄FN₃0₄•HC1       Mol Wt 437.9

Chemical Name: l-Cyclopropyl-6-fluoro-l,4-dihydro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolol[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarboxylic acid, monohydrochloride. Moxifloxacin hydrochloride is a slightly yellow to yellow crystalline powder. Each mL of VIGAMOX® solution contains 5.45 mg moxifloxacin hydrochloride, equivalent to 5 mg moxifloxacin base.

Contains: Active: Moxifloxacin 0.5% (5 mg/mL); Inactives: Boric acid, sodium chloride, and purified water. May also contain hydrochloric acid/sodium hydroxide to adjust pH to approximately 6.8.

VIGAMOX® solution is an isotonic solution with an osmolality of approximately 290 mOsm/kg.

Market Considerations

Amongst the US approvals, Dr. Reddy’s, Teva, Torrent, and Aurobindo have received tentative approvals for the 400 mg oral tablet formulation. Akorn, Teva and Apotex have received tentative approvals for a Moxifloxacin hydrochloride ophthalmic solution. No 180 day period of exclusivity has been awarded since all patents were found to be valid.

In the UK, Teva, Rivopharm, and Double-E Pharma have received marketing authorisations for the 400 mg Moxifloxacin tablets, while Noridem has received a market authorisation for the equivalent 400mg/250ml solution for infusion. A similar trend of generic competition, for tablets and infusions, following molecule patent expiry is expected throughout Europe. Currently no generic market authorisations for ophthalmic formulations have been granted in major European countries. However, Sandoz and Hexal have gained market authorisations in some European markets for the ophthalmic dosage form following Novartis’ acquisition of Alcon.

In Canada the only generic manufacturer holding a marketing authorisation is Sandoz, however, this was granted as a New Drug Submission rather than as an ANDS. Following patent expiries from mid-2014, the European and North American markets are likely to have significant competition if the numbers of companies filing litigation, ANDS, ANDAs and the like in the northern hemisphere is anything to go by.

MOXIFLOXACIN

History

Moxifloxacin was first patented (United States patent) in 1991 by Bayer A.G., and again in 1997.^[47] Avelox was subsequently approved by the U.S. Food and Drug Administration (FDA) for use in the United States in 1999 to treat specific bacterial infections.^[2] Ranking 140th within the top 200 prescribed drugs in the United States for 2007^[48] moxifloxacin, in the same manner asciprofloxacin, has proven to be a blockbuster drug for Bayer A. G., generating billions of dollars in additional revenue. In 2007 alone, Avelox generated sales of $697.3 million dollars worldwide.^[26]

Moxifloxacin is also manufactured by Alcon as Vigamox.^{[citation needed]}

Patent

A United States patent application was made on 30 June 1989, for Avelox (moxifloxacin hydrochloride),(Bayer A.G. being the assignee), which was subsequently approved on 5 February 1991. This patent was scheduled to expire on 30 June 2009. However, this patent was extended for an additional two and one half years on 16 September 2004, and as such is not expected to expire until 2012.^[49] Moxifloxacin was subsequently (ten years later) approved by the U.S. Food and Drug Administration (FDA) for use in the United States in 1999. There have been at least four additional United States patents filed regarding moxifloxacin hydrochloride since the 1989 United States application,^[47][50] as well as patents outside of the USA.

Additional regulatory history

6/12/2002 Changes made to minimize the impact of warnings concerning adverse reactions.^[51]

26 June 2003 New Zealand Pharmacovigilance warns of moxifloxacin induced respiratory insufficiency.^[52]

10/6/2003 Changes made to minimize the impact of post marketing reports as well as the risk of tendon injuries.^[53]

29 December 2008 Addition of numerous adverse reactions associated with the use of moxifloxacin.^[54]

27 April 2009 Issuance of a Medication Guide and revisions to include new safety information including the addition of the Black Box Warning to the Medication Guide. The FDA had determined that Moxifloxacin poses a serious and significant public health concern, requiring the distribution of a Medication Guide.^[55]

24 June 2009 Updating of the carton and container labels to include a statement to let dispensers know that a Medication Guide must be dispensed with the product.(emphasis added)^[56]

Patent related

As indicated by the Key Patent Indicator (Fig. 2), patents in the families with priority DE3824072A, 15/07/1988 (‘072), and DE4200414A, 10/01/1992, (‘414) provide protection for the Moxifloxacin molecule and are considered to be the main constraint to generic entry. As patents in the ‘414 family have expired or were never granted, the only remaining constraint to generic entry is the ‘072 family. The term of the Australian patent of this family have been extended to 19 June 2014 while the Canadian member will enjoy the longer term of 17 years from grant, expiring in November 2015.

Supplementary protection certificates (SPCs) have been granted in France, Germany, Spain and the UK, and will expire in June 2014. Given that there are less than 2 years until these SPCs expire, and that as yet no applications for paediatric extension of the SPCs have been published in Europe, they are unlikely to be extended by 6 months on the basis of the approved Paediatric Investigation Plans.

The US member, 4,990,517 (‘517), protecting the general structure of the Moxifloxacin molecule, expired in June 2012, after enjoying 6 month paediatric extension on top of a 901 day s156 extension. However, the absence of generics on the US market is due to Bayer securing a divisional patent, 5,607,942 (‘942). This patent claims the Moxifloxacin molecule specifically and is due to expire in September 2014, after being awarded a 6 month paediatric extension.

Members of the ‘072 family from both Canada and the US have been the subject of litigation after generic manufacturers identified these patents in paragraph IV filings, and the equivalent in Canada, as early as 2006. After filing an infringement suit in the US against Teva in relation to US ‘517, US ‘942, Bayer enjoyed a satisfying validification of their patents when Teva agreed that it would be infringing two of the patents, while the third was decided by the court to be equally valid.

In Canada, Novopharm, Cobalt, Apotex, Mylan and Apotex have also all tested the litigation waters relating to the equivalent patent with no success noted so far.

A third patent family that promises to be a constraint for generic ophthalmic formulations is Alcon’s 1998 patent, US10250498P (Fig. 2), which identifies an ophthalmic formulation of Moxifloxacin and its use in the treatment and prevention of eye infections. Patents in this family are set to expire in August 2019. US members 6,716,830 (‘830) and 7,671,070 (‘070) have been awarded a 6 month paediatric extension, extending their expiration until March 2020. The validity of US ‘830 was upheld following Teva filing paragraph IV certifications to manufacture generic Vigamox. Teva has since appealed this ruling.

In addition, Alcon has filed patent infringement suits against Watson, Lupin and Apotex in relation to US ‘070 after they submitted Abbreviated New Drug Applications (ANDAs)with paragraph IV filings in preparation for commercialisation of a Moxeza/Vigamox generic equivalent. There has been no outcome from these suits to date. In addition, applications for Orders of Prohibition against Cobalt, Apotex and Teva have also been noted for the equivalent Canadian patent following the filing of Abbreviated New Drug Submissions (ANDS) by these companies.

The equivalent European patent 1,117,401 was revoked following opposition by Teva filed in the European patent office. Its divisional patents, 1,384,478 (granted) and 2,301,541 (accepted) have restricted claims to the use of Moxifloxacin in the topical treatment of ophthalmic infections caused by P. aeruginosa and H. influenza, respectively. This may provide a prepared generic competitor an opportunity to launch their Moxifloxacin ophthalmic equivalent in Europe soon after the expiry of patents protecting the molecule, subject to legal review of the remaining claims of the patent.

Alcon have secured additional protection for their Moxeza ophthalmic formulation by way of patents in the family with priority US5987708P (09/06/2008). Patent claims specify ratios of Moxifloxacin to inactive ingredients and additional inactive ingredients and therefore generic competitors are likely to circumvent the patent by reformulation. Lupin has filed paragraph IV certifications to US8450311, which is currently subject of a patent infringement suit.

Families with priorities DE19546249A (12/12/1995), DE19751948A (24/11/1997), DE19855758A (10/11/1998) and US36433499A (30/07/1999) are not considered to be a constraint to generic entry because the protected technologies are likely to be circumvented.

Generic equivalents

In 2007, the U.S. District Court for the District of Delaware held that two Bayer patents on Avelox (moxifloxacin hydrochloride) are valid and enforceable, and infringed by Dr. Reddy’s ANDA for a generic version of Avelox.^[70][71] The district court sided with Bayer, citing the Federal Circuit’s prior decision in Takeda v. Alphapharm^[72] as “affirming the district court’s finding that defendant failed to prove a prima facie case of obviousness where the prior art disclosed a broad selection of compounds, any one of which could have been selected as a lead compound for further investigation, and defendant did not prove that the prior art would have led to the selection of the particular compound singled out by defendant.” According to Bayer’s press release^[70] announcing the court’s decision, it was noted that Teva had also challenged the validity of the same Bayer patents at issue in the Dr. Reddy’s case. Within Bayer’s first quarter 2008 stockholder’s newsletter^[73]

Bayer stated that they had reached an agreement with Teva Pharmaceuticals USA, Inc., the adverse party, to settle their patent litigation with regard to the two Bayer patents. Under the settlement terms agreed upon, Teva would obtain a license to sell its generic moxifloxacin tablet product in the U.S. shortly before the second of the two Bayer patents expires in March 2014.

• Economic impact: adverse reactions:

The advocacy group Public Citizen has lobbied for increasing safety warnings and for the removal of some fluoroquinolone drugs from clinical practice.^{[74][75][76][77][78][79][80][81]}

DESCRIPTION

• Moxifloxacin is a therapeutic agent that shows a broad spectrum antibacterial action. Moxifloxacin is the international non-proprietary name (INN) for 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-oxo-3-quinolinecarboxylic acid.
• The structure of moxifloxacin corresponds to formula (I):
• Racemic moxifloxacin was firstly described in EP-A-350733 and, particularly, moxifloxacin having a (S,S)-configuration is described inEP-A-550903 .
• In experimental example 19 of EP-A-550903 and example Z19 of EP-A-591808 , a method for preparing and isolating moxifloxacin base is described. The same method is described in patent document EP-A-592868 ). These published patent applications neither describe nor suggest the possible existence of a crystalline form of moxifloxacin base. In WO-A-2008059521 it is disclosed that by performing the mentioned examples an acetonitrile solvated form of moxifloxacin with low purity is obtained, and so the obtained solvate form can not be used as such in pharmaceutical formulations. In the above mentioned examples, the obtained moxifloxacin base crude is purified and isolated by chromatography using methylene chloride/methanol/17% aqueous ammonia as the solvent system. The purification process disclosed in said documents has been reproduced by the authors of the present invention, but only an amorphous form of moxifloxacin was obtained. This process for the purification and isolation of moxifloxacin base is complex and difficult to perform on industrial scale due to the need for purifying the product by column chromatography.
• WO-A-9926940 discloses a process for the preparation of moxifloxacin from a difluoro precursor comprising the step of adjusting the pH to 6.8- 7.0. However, the reproduction of this example shows that at this pH moxifloxacin hydrochloride or a mixture of moxifloxacin hydrochloride and moxifloxacin base is obtained, since the X-ray diffractogram of the isolated compound corresponds with the hydrochloride moxifloxacin. This fact has also been confirmed by the reproduction of the subsequent crystallization described in the international patent application of the previous compound in ethanol/water. The behaviour of this solid was very different regarding its solubility in respect what was described in the international patent application.
• WO-A-2004091619 and WO-A-2007010555 disclose the preparation of moxifloxacin base as a solid. Nevertheless, they do not describe nor suggest the preparation of a crystalline form of moxifloxacin base. The authors of the present invention have proved that the X-ray diffractogram of the product obtained by reproducing the reference example disclosed in WO-A-2004091619 , based on a pH adjustment to 7.0-7.2, corresponds to the X-ray diffractogram of the monohydrate of moxifloxacin hydrochloride disclosed in US 5849752 . Similarly, by reproducing the reference example of WO-A-2007010555 , also for obtaining moxifloxacin base but based on a pH adjustment to 5.0-6.0, it is neither expected to obtain moxifloxacin base due to the adjustment of the solution within a range of acidic pH values.
• [0008]
  WO-A-2008059521 discloses a process for the preparation of a crystalline form of moxifloxacin base, designated as Form I, by recrystallization in a ketosolvent, such as acetone. This form is characterized by its X-ray diffraction pattern corresponding to a hemihydrate. The best yield obtained is a 78.2% starting from moxifloxacin hydrochloride in example 18. This crystalline form has a tendency to occlude solvent molecules within the crystalline network in amounts very superiors to the allowed ones by for Guidelines Residual Solvents (CPMP/ICH/283/95) and can be difficult to impossible to remove by drying, what forces to carry out laborious treatments, either physical, or chemical to reach allowed solvent levels. The presence of any non-aqueous solvents in amounts over the allowed ones would not make this crystalline form suitable for the preparation of pharmaceutical formulations.
• The existence of polymorphs is unpredictable and there is no a priori established procedure to prepare an unknown polymorph. The difference in the physical properties of different morphological forms results from the orientation and intermolecular interactions of adjacent molecules are complexes in the bulk solid. Furthermore, the different solid forms of a pharmaceutically active ingredient can have different characteristics, and offer certain advantages in methods of manufacture and also in pharmacology. Thus, the discovery of new solid forms can contribute to clear improvements in the efficiency of methods of production and/or improvements in the characteristics of the pharmaceutical formulations of the active ingredients, since some forms are more adequate for one type of formulation, and other forms for other different formulations.

Moxifloxacin Hydrochloride namely (4aS-Cis) -l-cyclopropyl-7- (2, 8- diazabicyclo [4.3.0] non-8-yl) -6-fluoro-8-methoxy-4-oxo-l, 4-dihydro-3- quinoline carboxylic acidhydrochloride has the formula

Moxifloxacin Hydrochloride

Moxifloxacin is a fluoroquinolone broad spectrum antibacterial particularly against Gram-positive bacteria significantly better than those of Sparfloxacin and Ciprofloxacin that was disclosed in EP No 350,733 and EP No 550,903. Moxifloxacin has activity against Gram- negative and Gram-positive organisms, including Streptococcus pneumonia, Staphylococcus aureus, Pseudomonas aeruginosa, particularly against the respiratory disease-causing pathogens like Mycoplasma pneumonia, Mycobacterium tuberculosis, Chlamydia pneumoniae and the activity shown to be unaffected by B-lactamases .

US Patent No 5,157,117 discloses (l-cyclopropyl-6, 7-difluoro-8-methoxy- 4-oxo-l, 4-dihydro-3-quinoline carboxylic acid-O³, 0⁴)bis (acyloxy-O) borate and process for its preparation by reacting ethyl-1- cyclopropyl-6, 7-difluoro-8-methoxy-4-oxo-l, -dihydro-3-quinoline carboxylate with Boric acid and acetic anhydride in presence of zinc chloride and its conversion to Gatifloxacin hydrochloride.

WO 2005/012285 discloses the process for the preparation of moxifloxacin hydrochloride using a novel intermediate namely (4aS-Cis)-(1-cyclopropyl-7-(2,8-diazabicyclo[4,3,0]non-8-yl)-6-fluoro-8-methoxy-4-oxo-1,4-dihydro-3-quinoline carboxylic acid-O³,O⁴)bis(acycloxy-O)borate.

Hydrates of Moxifloxacin hydrochloride known are the anhydrous and monohydrate. US Patent No. 5,849,752 discloses the monohydrate of Moxifloxacin hydrochloride and its preparation by treating the anhydrous crystalline form with ethanol/ water mixtures.

The prior art disclosed in European Patent No’s EP 350,733, EP 550,903 and EP 657,448 discloses the preparation of Moxifloxacin hydrochloride involving the condensation of l-cyclopropyl-6, 7-difluoro-8-methoxy-4- oxo-1, 4-dihydro-3-quinoline carboxylic acid or its esters with (S,S) 2,8-Diaza bicyclo [4.3.0] nonane in presence of a base and its conversion to hydrochloride at higher temperatures leading to the desired Moxifloxacin along with its positional isomer namely (4aS-Cis)-l- cyclopropyl-6- (2, 8-diazabicyclo [4.3.0] non-8-yl) -7-fluoro-8-methoxy-4- oxo-1, 4-dihydro-3-quinoline carboxylic acid as a major impurity. As the impurity and the Moxifloxacin are positional isomers they are difficult to separate. Purification of Moxifloxacin to remove this isomer results in lower yields thereby increasing the product cost. Similarly methods described in the prior art involves the preparation of Moxifloxacin and then its conversion to its hydrochloride thereby incorporating an additional step in the manufacturing process also leading to lowering of yields.

Moxifloxacin and its pharmacologically acceptable salts are disclosed in European patents EP 350733, EP 550903 and EP 657,448. The disclosed process for the preparation of moxifloxacin hydrochloride comprises of condensing l-cyclopropyl-6,7- difluoro-8-methoxy-4-oxo-l,4-dihydro-3-quinoline carboxylic acid or its esters with (S,S)2,8-diazobicyclo[4.3.0]nonane, in presence of a base at high temperature followed by conversion into hydrochloride salt .

This process not only produces desired moxifloxacin hydrochloride but also its positional isomer namely l-cyclopropyl-7-fluoro- 1,4- dihydro -8- methoxy -6- (4aS,7aS)- octahydro- 6H -pyrrolo [3,4-b] pyridine-6-yl] -4- oxo-quinolinecarboxylic acid as a major impurity which is difficult to separate. The purification of moxifloaxcin to remove this isomer results in lower yields thereby increasing the product cost.

The International publication WO 2005/012285 discloses an improved process for the preparation of moxifloxacin hydrochloride incorporated herein by reference. The disclosed process involves the preparation of moxifloxacin hydrochloride from the ethyl 1 -cyclopropyl-6,7-difluoro-8-methoxy-4-oxo- 1 ,4-dihydro-3-quinolme carboxylate through a novel intermediate (4aS-cis)-l-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8- yl)-6-fluoro-8-methoxy-4-oxo- 1 ,4-dihydro-3 -quinolinecarboxylicacid-0³,0⁴)bis(acyloxy -0)-borate.

US patent application 6897315 discloses a process for the preparation of 8-methoxy-3-quinoline carboxylic acid especially moxifloxacin incorporated herein by reference. The disclosed process involves the preparation of moxifloxacin from 8-halo moxifloxacin derivative using methanol and potassium tertiary butoxide. US patent 5639886 discloses one-pot process for the preparation of 3-quinoline carboxylic acid derivatives including moxifloxacin.

WO 2004 091619 claims anhydrous crystalline form-Ill of moxifloxacin hydrochloride and WO 2004/039804 claims amorphous form of moxifloxacin hydrochloride. US Pat.No.5, 849,752 discloses specific crystalline forms of anhydrous moxifloxacin mono hydrochloride and monohydrated moxifloxacin mono hydrochloride. Anhydrous moxifloxacin mono hydrochloride disclosed in US Pat. No.5, 849,752 has been designated as “Form-I” and the hydrated form as “Form-II” in US Pat. No.7,230,006. It also discloses a novel crystalline Form-Ill of anhydrous moxifloxacin mono hydrochloride.

US patent US 5,480,879 discloses the melting range of moxifloxacin in example part as 203-208°C and does not speaks about polymorphism of moxifloxacin. Experiment executed as per the procedure given in example Zl 9 of US 5,480,879 and resulted in acetonitrile solvated form of moxifloxacin with low purity and the obtained solvated form can not used for formulations

U.S. Pat. No. 5,849, 752 (“the ‘752 patent”), incorporated by reference, described two crystalline forms of moxifloxacin hydrochloride namely, anhydrous moxifloxacin hydrochloride and monohydrated moxifloxacin hydrochloride. For convenience, the anhydrous crystalline form described in the 752 patent is designated as “Form I”, and the hydrated form as “Form II”. According to U.S. Pat. No. ‘752’, moxifloxacin hydrochloride monohydrate Form II was obtained by stirring a suspension of the anhydrous moxifloxacin hydrochloride in aqueous media until hydration. Moxifloxacin hydrochloride monohydrate of ‘752’ was also prepared by crystallizing moxifloxacin hydrochloride from a media having a water content which is stoichiometrically sufficient but limited to 10%.

WO patent application publication No. 04/091619 disclosed anhydrous Form III of moxifloxacin hydrochloride.

WO patent application publication No. 04/039804 disclosed amorphous form of moxifloxacin hydrochloride.

WO 2005/054240 disclosed two novel crystalline forms which were designated as Form A and Form B of moxifloxacin hydrochloride.

WO patent application publication No. 07/010555 disclosed two crystalline forms which were Form X and Form Y of moxifloxacin hydrochloride. According to WO Publication No. 2007/010555, Form Y was obtained by crystallization of moxifloxacin hydrochloride from the mixture of methanol and water in the ratio of about 8:1 by volume.

WO patent application publication No. 07/148137 disclosed hydrate form of moxifloxacin hydrochloride. According to WO Publication No. 2007/148137, moxifloxacin hydrochloride monohydrate was obtained by crystallization moxifloxacin hydrochloride by humidification of moxifloxacin hydrochloride at 50-90% relative humidity at 25-60° C. for 8 to 24 hours.

WO patent application publication No. 08/028959 disclosed crystalline form of moxifloxacin hydrochloride. According to WO Publication No. 2008/028959, moxifloxacin hydrochloride was obtained by dissolving moxifloxacin hydrochloride in a mixture of methanol and water and adding acetone and recovering moxifloxacin hydrochloride crystalline form.

WO patent application publication No. 08/059521 disclosed process for the preparation of anhydrous crystalline form I of moxifloxacin hydrochloride.

WO patent application publication No. 08/095964 disclosed crystalline form of moxifloxacin base.

……………………

SYNTHESIS

Drugs Fut 1997,22(2),109

36th Intersci Conf Antimicrob Agents Chemother (Sept 15-18, New Orleans) 1996,Abst. F1.

The anhydrization of pyridine-2,3-dicarboxylic acid (I) with acetic anhydride gives the corresponding anhydride (II), which by treatment with benzylamine (III) is converted into the benzylimide (IV). The hydrogenation of (IV) with H2 over Pd/C yields 8-benzyl-2,8-diazabicyclo[4.3.0]nonane-7,9-dione (V), which is further hydrogenated with LiAlH4, affording (?-cis-8-benzyl-2,8-diazabicyclo[4.3.0]nonane (VI) (1). The optical resolution of (VI) by separation of the cis-(R,R)-isomer as crystalline L-(+)-tartrate and further purification of the cis-(S,S)-isomer (VII) as the D-(-)-tartrate affords enantiomerically pure (S,S)-8-benzyl-2,8-diazabicyclo[4.3.0]nonane (VII). The debenzylation of (VII) by hydrogenolysis with H2 over Pd/C gives (S,S)-2,8-diazabicyclo[4.3.0]nonane (VIII), which is condensed with 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (IX) in basic medium and finally salified with HCl. The 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (IX) has been obtained as follows: The reaction of 2,4,5-trifluoro-3-methoxybenzoyl chloride (X) with malonic acid monoethyl ester monopotassium salt (XI) by means of triethylamine gives 2-(2,4,5-trifluoro-3-methoxybenzoyl)acetic acid ethyl ester (XII), which is condensed with triethyl orthoformate yielding the corresponding ethoxymethylene derivative (XIII). The reaction of (XIII) with cyclopropylamine affords the cyclopropylaminomethylene derivative (XIV), which is finally cyclized to (IX) by means of NaF in DMF.

………………………..

J Label Compd Radiopharm 2000,43(8),795

The condensation of 2,4,5-trifluoro-3-methoxybenzoyl chloride (I) with 14C-labeled diethyl malonate (II) by means of MgCl2 and TEA gives the benzoylmalonate (III), which is monodecarboxylated with TsOH in refluxing water, yielding the benzoylacetate (IV). The reaction of (IV) with triethyl orthoformate and Ac2O at 140 C affords the benzoylacrylate (V), which is treated with cyclopropylamine (VI) in cyclohexane to provide ethyl 3-(cyclopropylamino)-2-(2,4,5-trifluoro-3-methoxybenzoyl)acrylate (VII). The cyclization of (VII) by means of K2CO3 in hot N-methylpyrrolidone gives the quinolone carboxylate (VIII), which is hydrolyzed with NaOH in hot methanol, affording the carboxylic acid (IX). Finally, this compound is condensed with (S,S)-2,8-diazabicyclo[4.3.0]octane (X) by means of 1,4-diazabicyclo[2.2.2]octane (DABCO) in refluxing acetonitrile.

…………………….

……………………

EP1651630A1

The reaction scheme is given below: Stage-I

Acetic anhydride

Ethyl-l-cyclopropyl-6, 7- ( l-cyclopropyl-6, 7-difluoro-l, 4- difluoro-1, 4-dihydro-8- dihydro-8- methoxy-4-oxo-3- methoxy-4-oxo-3-quinoline quinoline carboxylic acid-0³, 0⁴) carboxylate . Bis ( acetate-O) -borate (Borate complex)

Stage-II

Triet yl amine

Acetonitrile

l-cyclo propyl-6, 7-difluoro- [S, S] -2, 8-diazabicyclo- (1- cyclo propyl-6, fluoro-7 (2, 8- 1, 4-dihydro-8- methoxy-4-oxo [4 ,3.0]nonane Diazabicyclo-nonane) 1,4- -3-quinoline carboxylic acid- dihydro-8-methoxy-4-oxo-3 0³,0⁴)Bis ( acetate-O) -borate- quinoline carboxylic acid- (Borate complex) (0³,0⁴) bis (acetate-O) -borate itage-III

(1- cyclo propyl-6, fluoro- (2, 8- Moxifloxacin HCI pseudohydrate Diazabicyclo-nonane) 1,4- dihydro-8-methoxy-4-oxo-3 -quinolinecarboxylicacid- (O^O⁴) bis (acetate-O) -borate

Stage-TV

Moxifloxacin HCI pseudohydrate Moxifloxacin HCI monohydrate

EXAMPLE – I

Stage-1: Preparation of l-cyclopropyl-6, 7-difluoro-8-methoxy-4-oxo- l,4-dihydro-3-quinoline carboxylic acid-O³,O*)bis (acyloxy-O)borate

Acetic anhydride (175 g) is heated to 70°C and boric acid (30 g) is slowly added lot wise in a temperature range of 70°C to 90°C. The temperature is then raised, maintained under reflux for 1 hr followed by cooling to about 70°C. Ethyl-l-cyclopropyl-6, 7-difluoro-8-methoxy-4- oxo-1, 4-dihydro-3-quinoline carboxylate (100 g) is added under stirring. The temperature is then raised and maintained for 1 hr in the range of 100°C to 105°C. The reaction mass is cooled to 0°C, chilled water (400 ml) is added slowly followed by cold water (600 ml) at temperature 0°C to 5°C and maintained for 2 hrs at 0°C to 5°C. The product which is a boron acetate complex is filtered, washed with water (500 ml) and dried at 55°C to 60°C under vacuum to constant weight. The dry wt is 130.0 g corresponding to yield of 95.2%.

Stage-2: Preparation of (4aS-Cis) -l-Cyclopropyl-7- (2, 8-diazabicyclo [4.3.0]non-8-yl) -6-fluoro-8-methoxy-4-oxo-l , 4-dihydro-3-quinoline carboxylicacid-0³,0*)bis (acyloxy-O)borate

The boron acetate complex (130 g) prepared in stage 1 is suspended in acetonitrile (650 ml), and [S, S] -2, 8-diazabicyclo [4.3.0] nonane (47 g) and triethyl amine (72.9 g) are added. The temperature is raised to reflux and maintained for 1 hr. at reflux, followed by cooling to about 40°C. The solvent is removed under vacuum at temperature below 40°C, and n-hexane (200 ml) is added. After maintaining the reaction mass for 1 hr at room temperature the product is isolated by filtration followed by washing of the wet cake with n-hexane . The product is dried at about 45°C to about 50°C to constant weight.

Dry wt of the novel intermediate is 117.0 g corresponding to yield of 71.5%.

Elemental analysis: C: 56.42%, H: 5.62%, N: 7.76% and the calculated values for the intermediate, formula C₂₅H₂₉BFN₃0₈C: 56.6%, H: 5.47%, N: 7.92%

IR Spectrum (KBr, cm-¹) : 3415, 3332, 2936, 1718, 1630, 1573, 1526, 1445, 1273, 1042, 935, 860, 798, 682

^ NMR (200 MHz, CDC1₃, ppm) : 9.00 (1H), 7.82 (1H), 4.12 (4H), 3.57 (3H), 3.43 (4H), 3.07 (2H) , 2.75 (2H), 2.4 (1H),’ 2.1 (6H), 1.84 (2H) , 1.6 (1H), 1.31 (2H)

Mass Spectrum (MJ : 530.3 [M⁺H] , 470.2 [M⁺ - CH₃COOH] , 428.2 [M⁺- (CH₃CO)₂0, 100%], 402.2, 388.2

Stage -3: Preparation of Moxifloxacin Hydrochloride pseudohydrate

The intermediate (117 g) prepared stage-2 is dissolved in ethanol (600 ml) by stirring for about 30 min. at room temperature and the insolubles if any are filtered off. pH of the filtrate is adjusted to about 0.5 by addition of hydrochloric acid at room temperature and maintained for 2 hrs. The reaction mass is cooled, and maintained for two hrs, at about 0°C to about 5°C. The product is filtered, washed with chilled ethanol (50 ml) and dried at about 50°C to about 55°C till constant weight.

The dry weight of the Moxifloxacin hydrochloride pseudohydrate is 87.5g corresponding to yield of 91.0%. Water content of the product by KF is 0.64% w/w.

X-ray diffraction pattern data are given in Table-1 EXAMPLE – II

Stage- 2 : Preparation of Moxifloxacin pseudohydrate with out isolating (4aS-Cis) -l-Cyclopropyl-7- (2 , 8-diazabicyclo [4.3.0] on-8-yl) -6-fluoro- 8-methoxy-4-oxo-l,4-dihydro-3-quinolinecarboxylicacid-0³,0⁴)bis (acyloxy-O) borate

The boron acetate complex (130 g) prepared in stage-1 of Example-1 is suspended in acetonitrile (650ml) and [S, S] -2, 8-Diazabicyclo [4.3.0]nonane (47 g) & triethyl amine (72.9 g) are added. Temperature of the reaction mass is raised to reflux, maintained for 1 hr. at reflux and cooled to room temperature. Methanol (600 ml) is added and maintained for 30 min at room temperature to obtain a clear solution. The solution is filtered to remove insolubles if any and pH of the filtrate is adjusted to about 0.5 with hydrochloric acid (57.5 g) . The reaction mass is maintained for 2 hrs at temperature in the range of about 20°C to about 25°C, cooled to 0°C followed by maintaining the reaction mass at about 0°C to about 5°C for 2 hrs. The product is filtered, washed with methanol (50 ml) and dried at about 50°C to 55°C until constant weight.

Dry wt of the Moxifloxacin hydrochloride pseudohydrate is 88g corresponding to yield of 68.7%.

EXAMPLE – III : Preparation of Moxifloxacin Hydrochloride monohydrate

Moxifloxacin hydrochloride (50 g) prepared as above is suspended in a mixture of ethanol (250 ml) and hydrochloric acid (25 ml) . Raised the temperature, maintained for two hrs at 40°C to 45°C followed by cooling to about 25°C. The product is filtered and dried under vacuum at 50-55°C until become constant weight.

Dry wt of Moxifloxacin hydrochloride monohydrate is 46 g corresponding to yield of 90.5%.

The IR spectral data and XRD pattern are identical with available Moxifloxacin hydrochloride monohydrate.

WO2008059521A2

The preferred embodiments of the present invention is represented as follows

Formula-2a Formula-2b

Formula-3a Formula-3b Formula-3c Formula-3d

Formula-4b

Formula-4c

SCHEME-5:

SCHEME-6:

 Example-1: Preparation of (S,S)-2-benzyl-8-trityI-2,8-diazabicyclo (4.3.0) nonane:

A mixture of (S₅S)-2-benzyl-2,8-diazabicyclo-(4.3.0) nonane 50 grams, dichloromethane 300 ml and triethylamine 28 ml was stirred at 25-35°C. Trityl chloride 71 grams was added to the reaction mixture. The reaction mixture was stirred at 25-35⁰C for 5 hrs. The organic layer was washed with 5% sodium bicarbonate solution followed by water. The organic layer was distilled off completely to provide the title compound as a residue.

Yield: 96 grams

Example-2:

Preparation of (S,S)-8-trityI-2,8-diazabicyclo (4.3.0) nonane:

A mixture of (S,S)-2-benzyl-8-trityl-2,8-diazabicyclo (4.3.0) nonane 130 grams,

2- butanol 1600 ml and 5% Pd/C was taken in an autoclave and heated to 45-50°C at 4.0-4.5 Kg/cm² of hydrogen pressure. The reaction mixture was stirred at this condition for 45-50 hrs. The reaction mixture was cooled to 25-30⁰C and filtered through hyflow bed. The solvent was distilled off completely to provide the title compound as a residue.

Yield: 95 grams.

Example-3:

Preparation of condensed compouad of formula-4a:

A mixture of 20 grams of l-cyclopropyl-NjN-diethyl-όjT-difluoro-l^-dihydro-δ- methoxy-4-oxoquinoline-3-carboxamide, 11.8 grams of (S,S)2,8-diazabicyclo (4.3.0) nonane, 100 ml of acetonitrile and 2 grams of l,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) was heated to 80⁰C. The reaction mixture was stirred for 35 hours at ⁰C. The reaction mixture was cooled to 32⁰C and stirred for 45 minutes at 32°C. The solvent was distilled off under reduced pressure. Water (100 ml) was added to the obtained residue. The reaction mixture was cooled to 25-35⁰C. The reaction mixture was extracted with ethyl acetate. The solvent was distilled off to get the title compound as a residue. Yield: 12 grams.

Example-4:

Preparation of condensed compound of formula-4b:

A mixture of 5 grams of l-cyclopropyl-NjN-diethyl-όJ-difluoro-M-dihydro-δ- methoxy-4-oxoquinoline-3-carboxamide, 2.9 grams of (S,S)-8-phenyloxy carbonyl-2,8- diazabicyclo (4.3.0) nonane, 25 ml of acetonitrile and 0.5 grams of diazabicyclo[5.4.0]undec-7-ene (DBU) was heated to 80⁰C. The reaction mixture was stirred for 35 hours at 75-8O⁰C. The reaction mixture was cooled to 25-35°C. The reaction mixture was stirred for 45 minutes at 25-35°C. The solvent was distilled off under reduced pressure. Water (100 ml) was added to the obtained residue. The reaction mixture was cooled to 25-35°C. The reaction mixture was extracted with ethyl acetate. The solvent was distilled off to get the title compound as a residue. Yield: 1 gram.

Example-5: Preparation of condensed compound of formula-4c:

A mixture of 5 grams of l-cyclopropyl-N_jN-diethyl-βJ-difluoro-l^-dihydro-S- methoxy-4-oxoquinoline-3-carboxamide, 2.9 grams of (S,S)-8-tertiary butyloxycarbonyl- 2,8-diazabicyclo (4.3.0) nonane, 25 ml of acetonitrile and 0.5 grams of diazabicyclo[5.4.0]undec-7-ene (DBU) was heated to 80⁰C. The reaction mixture was stirred for 35 hours at 75-80⁰C. The reaction mixture was cooled to 25-35⁰C and stirred for 45 minutes at 25-35°C. The solvent was distilled off under reduced pressure. Water (100 ml) was added to the obtained residue. The reaction mixture was cooled to 25-35⁰C. The reaction mixture was extracted with ethyl acetate. The solvent was distilled off to get the title compound as a residue Yield: lgram. Example-6:

Preparation of condensed compound of formula-4d:

A mixture of 20 grams of l-cyclopropyl-6,7-difluoro-8-methoxy-l,4-dihydro-4- oxoquinoline-3-carboxamide, 2.9 grams of (S,S)-2,8-diazabicyclo (4.3.0) nonane , 100 ml of acetonitrile and 2 grams of diazabicyclo[5.4.0]undec-7-ene (DBU) was heated to 75-80°C. The reaction mixture was stirred for 35 hours at 75-80°C. The reaction mixture was cooled to 25-35⁰C. The reaction mixture was stirred for 45 minutes at 25-35⁰C. The solid obtained was filtered and washed with acetonitrile. The material was dried at 40-45⁰C to get the title compound. Yield: 12 grams; M.R: 210-212⁰C.

Example-7:

Preparation of condensed compound of formula-4e:

A mixture of (S,S)-8-trityl-2,8-diazabicyclo (4.3.0) nonane (42 grams), l-cyclopropyl-N,N-diethyl-6,7-difluoro-l,4-dihydro-8-methoxy-4-oxoquinoline-3- carboxamide (10 grams), potassium carbonate ( 7.9 grams) and dimethyl formamide (80 ml) was heated to 110-120⁰C and stirred for 20 hours at 110-120⁰C. The reaction mixture was cooled to 80⁰C. The solvent was distilled off under reduced pressure. Water (100 ml) was added to the obtained residue. The reaction mixture was cooled to 25-35°C. The reaction mixture was extracted with ethyl acetate. The solvent was distilled off to obtain the title compound as a residue. Yield: 14.5 grams

Example-8: Preparation of moxifloxacin from 4d:

A mixture of 165 ml of water, 35 grams of sodium hydroxide, 150 ml of ethylene glycol and 12 grams of condensed compound of formula-4d was heated to 115°C. The reaction mixture was stirred for 15 hours at 115°C. The reaction mixture was cooled to 5°C. The pH of the reaction mixture was adjusted to 5.5 using hydrochloric acid and stirred for 30 minutes at 5°C. The obtained solid was filtered and washed with water. The solid was dried at 45°C to get the title compound. Yield: 8 grams; M.R: 203-205⁰C Example-9:

Preparation of moxifloxacin from 4b:

Moxifloxacin can be prepared from 4b (3grams), by a method which is analogous to the method illustrated in Example-8 Yield: 0.85 grams; M.R: 203-205°C

Example-10:

Preparation of moxifloxacin from 4c:

Moxifloxacin can be prepared from 4c (3 grams), by a method which is analogous to the method illustrated in Example-8 Yield: 1.0 grams; M.R: 203-205°C

Example-11:

Preparation of moxifloxacin from 4a: Moxifloxacin can be prepared from 4a (10 grams), by a method which is analogous to the method illustrated in Example-8 Yield: 7.5 grams; M.R: 203-205⁰C

Example-12: Preparation of moxifloxacin from 4e:

The condensed compound of formula-4e (14.5 grams) was dissolved in ethyl acetate 100ml. Aqueous hydrochloric acid (5 ml in 20 ml of water) was added to the above reaction mixture. The reaction mixture was stirred at 25-30°C for 30-45 min. 20ml of water was added to the reaction mixture. The two layers were separated. The aqueous layer was washed twice with ethyl acetate. The pH of the aqueous layer was adjusted to 10.8 using sodium hydroxide solution. The reaction mixture was extracted with methylene chloride. The solvent distilled off to get a residue. The residue was suspended in 165 ml of water, 35 grams of sodium hydroxide; 150 ml of ethylene glycol was heated to 115°C. The reaction mixture was stirred for 15 hours at 115°C. The reaction mixture was cooled to 5°C. The pH of the reaction mixture was adjusted to 5.5 using hydrochloric acid and stirred for 30 minutes at 5°C.The obtained solid was filtered and washed with water and dried at 45⁰C to get the title compound. Yield: 4.5 grams; M.R: 203-205⁰C

Example-13:

Preparation of moxifloxacin hydrochloride compound of formula-1:

A mixture of 8 grams of moxifloxacin, 16 ml of water and 64 ml of methanol was heated to reflux temperature of 70⁰C. The reaction mixture was filtered to remove undissolved material. Filtrate was cooled to 35°C. The pH of the filtrate was adjusted to 1.6 using hydrochloric acid. The reaction mixture was cooled to 15⁰C. The reaction mixture was stirred for 45 minutes at 15⁰C. The solid obtained was filtered and washed with methanol and dried at 40-45⁰C to get crystalline Form-Y of moxifloxacin hydrochloride monohydrate. Yield: 5.5 grams

Example- 14:

Preparation of moxifloxacin hydrochloride monohydrate:

A mixture of 25 grams of moxifloxacin, 16 ml of water and 64 ml of methanol was heated to reflux temperature of 70⁰C. The reaction mixture was filtered to remove undissolved material. Filtrate was cooled to 35°C. The pH of the filtrate was adjusted to 1.6 using hydrochloric acid. The reaction mixture was cooled to 15⁰C. The reaction mixture was stirred for 45 minutes at 15⁰C. The obtained solid was taken into a mixture of 22 ml of water and 0.5ml of hydrochloric acid and stirred 30 min at 5°C. The compound was filtered and washed with water, dried at 40-45⁰C to get moxifloxacin hydrochloride monohydrate particles having oval shape. Yield: 20 grams

Example-15:

Preparation of anhydrous crystalline Form-I of moxifloxacin hydrochloride: Moxifloxacin hydrochloride (Form-Y, 10 grams) suspended in 50 ml of methanol and the reaction mixture was heated to 45-5O⁰C. 18ml of dichloromethane was added slowly to the reaction mixture. The reaction mixture was stirred at 45-50°C for 15-20 minutes. The reaction mixture was cooled slowly to around 0-5°C and stirred for 1-1.5 hours. The precipitated solid was filtered under nitrogen atmosphere and washed with 5 ml of methanol. The solid obtained was dried at 110-120°C till the moisture content reached below 0.5%. Yield: 8.0 grams.

Bulk density: 0.28 g/ml; Tapped density: 0.52 g/ml Particle size distribution : D( v,0.1) :1.6 μm ; D( v_s0.5) : 5.5 μm ; D( v,0.9) :25.0 μm

 Preparation of Moxifloxacin

A solution of lOOg of 1-Cyclopropyl -6,7difluoro-l,4-dihydro-4-oxo-methoxyquinolone- 3-carboxilic acid), 52gr of (S,S)2,8 diazabicyclo[4,3,0]nonane, lOgr of 1,8- diazabycyclo[5,4,0]undec-7-ene and 300ml of acetonitrile is heated to 65 -70°C temperature and stirred till the reaction get completed and evaporated the solvent. Added 5 %aqueous isopropylalcohol to the reaction mass and adjusted the pH to basic with caustic solution and then filtered the reaction mass. Combined the total filtrate and adjusted the pH to 5.0 to 6.0 with aqueous HCl.and isolated the separated solid at a temperature of 10-15⁰C to afford Moxifloxacin.

………………….

SYNTHESIS REVIEW

http://www.beilstein-journals.org/bjoc/single/articleFullText.htm?publicId=1860-5397-9-265

Beilstein J. Org. Chem. 2013, 9, 2265–2319.

While isolated pyridines are a vital component in numerous drugs the related quinoline/quinolone scaffolds are becoming increasingly common. One such example is nalidixic acid (1.101, in fact a naphthyridone, Figure 2). Nalidixic acid is a prototype quinolone antibiotic that has been used extensively as an effective treatment against both gram positive and gram negative bacteria. In general, quinolone antibiotics act by interfering with the enzymes DNA-gyrase and/or topoisomerase of bacteria [55]. Moxifloxacin (1.102, Avelox) and levofloxacin (1.103, Levaquin) are two third-generation fluoroquinolone antibiotics which also appear amongst the top selling drugs. These compounds display similar SAR data [56]. For instance, in the case of moxifloxacin the fluorine atom in the C6 position enhances microbial activity while a methoxy group in the C8 position is reported to increase potency and decrease toxicity. Furthermore, it was found that a cyclopropyl group was beneficial for the enzyme–DNA binding complex while the bulky nitrogen-based appendage at C7 helps to bind to DNA gyrase and hinders the efflux of the drug from the bacterial cell.

Figure 2: Structures of nalidixic acid, levofloxacin and moxifloxacin.

Due to the elaborate substitution pattern of the parent quinolone ring systems these compounds are usually prepared via a linear consecutive sequence. In the case of moxifloxacin, an intramolecular base catalysed nucleophilic aromatic substitution is used to prepare the bicyclic ring system of the highly substituted aromatic1.104 (Scheme 19). A SNAr reaction is then used to introduce the saturated piperidinopyrrolidine appendage 1.105to furnish the desired structure [57-60]. In order to obtain a high yield for the substitution reaction a one-pot procedure was developed, initial masking of the acid (1.104) is achieved by silylation with subsequent borane chelate formation. Addition of the amine nucleophile 1.105 under basic conditions then renders the desired product in high yield. The available patent literature however does not comment on regioselectivity issues of the SNAr reaction due to the presence of the second fluoride substituent in the substrate, although not necessarily as electronically favourable for displacement it is certainly more accessible.

Scheme 19: Synthesis of moxifloxacin.

The saturated (S,S)-2,8-diazabicyclo[4.3.0]nonane (1.105) used in the final step can be prepared by a double nucleophilic substitution between tosylamine and 2,3-bis-chloromethylpyridine (1.112) followed by catalytic reduction of the resulting bicycle using palladium on carbon in acetic acid (Scheme 20). As the corresponding sulfonamide 1.113 was found to be a crystalline solid a resolution using (D)-(+)-O,O-dibenzoyltartaric acid was reported to separate the enantiomers [Petersen, U.; Schenke, T.; Krebs, A.; Schenke, T.; Philipps, T.; Grohe, K.; Bremm, K.-D.; Endermann, R.; Metzger, K. G. New Quinoline and Naphthyridinonecarboxylic Acid Derivatives. Ger. Patent DE 4 208 792 A1, March 23, 1993.].

Scheme 20: Synthesis of (S,S)-2,8-diazabicyclo[4.3.0]nonane 1.105.

…………………………………

PAPER

First way of enantioselective synthesis of moxifloxacin intermediate LI GuangXun, WU Lei, FU QingQuan, TANG Zhuo, ZHANG XiaoMei A new method of enantioselective synthesis of (S,S)-2,8-diazobicyclo [4.3.0] nonane was found by using (R)-2-amino-2-phenyl-ethanol as chiral induction reagent. The entire synthetic process included 8 steps which were easy to operate with high yield. The purification method was only simple recrystallization or even used directly in the next step without further purification. The total yield was 29%.

2013 Vol. 56 (3): 307-311 [Abstract] ( 22 ) [ PDF (518 KB)   ] ( 92 )  [Supporting Information]  DOI: 10.1007/s11426-012-4803-7

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Sonidegib/Erismodegib

CODE DESIGNATION ..LDE225, NVP-LDE-225

Treatment of medulloblastoma PHASE3 2014 FDA FILING

Treatment of advanced basal cell carcinoma PHASE3 2014 FDA FILING

Treatment of SOLID TUMORS..PHASE1 2017 FDA FILING

READMalignant Solid Tumors of Childhood

THERAPEUTIC CLAIM Oncology, Antineoplastics & Adjunctive Therapies

CHEMICAL NAMES

1. [1,1'-Biphenyl]-3-carboxamide, N-[6-[(2R,6S)-2,6-dimethyl-4-morpholinyl]-3-pyridinyl]-2-
methyl-4′-(trifluoromethoxy)-, rel-

2. N-{6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl}-2-methyl-4′-
(trifluoromethoxy)biphenyl-3-carboxamide

N-[6-[(2S,6R)-2,6-dimethylmorpholin-4-yl]pyridin-3-yl]-2-methyl-3-[4-(trifluoromethoxy)phenyl]benzamide

N-(6-((2S,6R)-2,6-dimethylmorpholino)pyridin-3-yl)-2-methyl-4′-(trifluoromethoxy)biphenyl-3-carboxamide

MOLECULAR FORMULA C26H26F3N3O3

MOLECULAR WEIGHT 485.5

SPONSOR Novartis Pharma AG

CAS REGISTRY NUMBER 956697-53-3  free form

NOTE… DIPHOSPHATE SALT IS THE DRUG WITH CAS 1218778-77-8

sonidegib - European Medicines Agency READ THIS..

Summary EudraCT Number: 2012-004022-21 Sponsor’s Protocol … READ THIS

Sonidegib (INN) or Erismodegib (USAN), also known as LDE225 is a Hedgehog signalling pathway inhibitor (via smoothened antagonism) being developed as an anticancer agent by Novartis.^[1][2] It has been investigated as a potential treatment for:

• Pancreatic cancer^[3][4][5][6]
• Breast cancer^[7][8]
• Basal cell carcinoma of the skin^[9][10][11]
• Small cell lung cancer^[12]
• Medulloblastoma^[13][14]
• Advanced solid tumours (including ovarian, breast, pancreatic, stomach, oesophageal cancers and glioblastoma multiforme)^[15][16][17]
• Acute leukaemia^[18]
• Chronic myeloid leukaemia^[19]
• Myelofibrosis and Essential thrombocythaemia^[20]

NVP-LDE-225, a product candidate developed by Novartis, is in phase III clinical trials for the treatment of medulloblastoma and basal cell carcinoma. Phase II trials are in progress for the treatment of adult patients with relapsed or refractory or untreated elderly patients with acute leukemia.

Early clinical trials are ongoing for the oral treatment of advanced solid tumors, for the treatment of myelofibrosis in combination with ruxolitinib and for the treatment of small cell lung cancer. A phase II clinical trial for the treatment of basal cell carcinomas in Gorlin’s syndrome patients with a cream formulation of NVP-LDE-225 was discontinued in 2011 since the formulation did not demonstrate tumor clearance rate sufficient to support further development.

Dana-Farber Cancer Institute and the Massachusetts General Hospital are conducting phase I clinical trials for the treatment of locally advanced or metastatic pancreatic cancer in combination with chemotherapy. In 2009, orphan drug designation was assigned in the E.U. for the treatment of Gorlin syndrome.

It has demonstrated significant efficacy against melanoma in vitro and in vivo.^[21] It also demonstrated efficacy in a mouse model of pancreatic cancer.^[22]

NVP-LDE225 Diphosphate salt (Erismodegib, Sonidegib) 

Synonym:Erismodegib, Sonidegib

CAS Number:1218778-77-8

Mol. Formula:C₂₆H₂₆F₃N₃O₃ ∙ 2H₃PO₄

MW:681.5

nmr.http://www.chemietek.com/Files/Line2/Chemietek,%20NVP-LDE225%20[02],%20NMR.pdf

hplc–http://www.chemietek.com/Files/Line3/Chemietek,%20NVP-LDE225%20[02],%20HPLC.pdf

Brief Description:

The following Examples serve to illustrate the invention without limiting the scope thereof, it is understood that the invention is not limited to the embodiments set forth herein, but embraces ali such forms thereof as come within the scope of the disclosure,

Step 1:

To a solution of 2-chloro-5-nitro-pyridine 1 (5.58 g, 35.2 mmoL) and c/s-2,6- dimethylmorpholine (4.05 g, 35.2 mmoL) in anhydrous DMF (30 mi.) was added K₂CO₃ (9.71 g, 70.4 mnrtoL). The mixture was heated at 50ºC overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over anhydrous Na₂SO₄ and concentrated to give crude product 3 as a yellow solid, after purification by silica gel chromatography, obtained pure product (7.80 g, 93.2%). LC-MS m/z: 238.2 (M+ 1).

Step 2:

The above material 3 (7.3Og. 30.8 mmoL) was hydrogenated in the presence of 10% Pd-C (1.0 g) in MeOH (120 ml) under hydrogen overnight. The suspension was filtered through celite and the filtrate was concentrated to give the crude product 4 (5.92 g) as a dark brown oil which was used directly in the next step without further purification. LC-MS m/z. 208.2 (M+1).

Step 3:

To a solution of 3-bromo-2-methyl benzoic acid (2.71 g, 12.6 mmoL), 6-((2S,6R)-2,6- dimethylmorpholino)pyridin-3-arnine 4 (2.61 g, 12.6 mmoL), and HATU (4.80 g, 12.6 mmoL) in anhydrous DMF (30 mL) was added diisopropylethylamine (6.58 mL, 37.8 mmoL) dropwise. The resulting mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (50 mL), and then extracted with EtOAc (3×120 mL). The organic layer was dried and concentrated to give the crude product. This crude product was then purified by flash column chromatography using 30% EtOAc in hexane as eiuent to give 5 as a white solid (4.23 g, 83.0%). LC-MS m/z: 404.1 (M+1).

Step 4:

A mixture of 4-(trif!uoromethoxy)phenylboronic acid (254 mg, 1.24 mmol), 3-bromo- N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-ylJ-4-methyl-benzamide 5 (250 mg, 0.62mmol), Pd(PPh₃)₄ (36 mg, 0.03 mmol), Na₂CO₃ (2.0M aqueous solution, 1.23 mL, 2.4 mmol) and DME (4.5 mL) in a sealed tube was heated at 130ºC overnight. The reaction mixture was diluted with EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine and concentrated to give the crude product which was then purified by preparative mass triggered HPLC (C₁₈ column, etuted with CH₃CN-H₂O containing 0.05% TFA) to give N-(6-((2S,6R)-2,6-dimethyfmorpholino)pyridin-3-yl)-2-rnethyl- 4'-(trifluoromethoxy)biphenyi-3-carboxamide (183.5 mg, 61.1% yield). LC-MS m/z: 486.2 (M+1).

The resultant crystalline product (Form A) was converted to the amorphous form by dissolving in 3% w/w aqueous ethanol, and the resultant solution spray dried at about 150ºC.

Form B was prepared by heating the amorphous form in an oven at 110ºC for 2 hours. In a further embodiment, the invention relates to a process step or steps, or an intermediate as described herein.

EXAMPLESThe present invention is further exemplified, but not limited, by the following example that illustrates the preparation of compounds of Formula I according to the invention.Example 1 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide

Step 1: To a solution of 3-iodo-4-methyl-benzoic acid (10.0 g, 38.2 mmol) in methanol (70 ml) is added concentrated sulfuric acid (0.5 ml). The reaction mixture is heated at 70° C. for 48 hours, cooled to room ambient temperature and then concentrated. After that, ethyl acetate (100 ml) and aqueous NaHCO₃ (saturated, 100 ml) solution are added to the residue. The organic layer is separated and washed again with aqueous NaHCO₃ (saturated, 100 ml) solution. The organic layer is separated, dried over anhydrous Na₂SO₄ and concentrated to yield 3-iodo-4-methyl-benzoic acid methyl ester 1. It is used without further purification in the next step. ¹H NMR (400 MHz, DMSO-d₆) δ 8.31 (s, 1H), 7.87 (d, 1H, J=8.4 Hz), 7.48 (d, 1H, J=8.4 Hz), 3.85 (s, 3H), 3.35 (s, 3H); LC-MS m/z: 277.0 (M+1).

Step 2: To a round-bottom flask containing 3-iodo-4-methyl-benzoic acid methyl ester (1.38 g, 5.00 mmol), 4-cyanophenylboronic acid (1.10 g, 7.48 mmol), palladium acetate (168 mg, 0.748 mmol), 2-(dicyclohexylphosphino)biphenyl (0.526 g, 1.50 mmol) and potassium fluoride (0.870 g, 15.0 mmol) is added anhydrous 1,4-dioxane (15 ml). The flask is purged with argon and sealed. The mixture is stirred at 130° C. for 18 hours, cooled to ambient temperature and then water (20 ml) and ethyl acetate (20 ml) are added. Solid is removed under vacuum filtration. The filtrate is extracted with EtOAc (20 ml×2). The organic layers are combined, washed with aqueous HCl (5%, 20 ml) and saturated NaHCO₃ (20 ml). It is dried over MgSO₄, and concentrated. The residue is purified by silica gel column chromatography (EtOAc/Hexane, gradient) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid methyl ester 2; LC-MS m/z: 252.1 (M+1).

Step 3: To a solution of 4′-cyano-6-methyl-biphenyl-3-carboxylic acid methyl ester 2 (2.56 g, 10.3 mmol) in 1,4-dioxane-H₂O (1:1 mixture, 20 ml) is added NaOH (1.22 g, 30.2 mmol)). The reaction is stirred at ambient temperature for 24 hours. To this mixture is added aqueous HCl (1 N, 36 ml) and it is then extracted with ethyl acetate (40 ml×3). The organic layers are combined, dried over anhydrous Na₂SO₄. The solver is removed. The solid obtained is washed with small amount of acetonitrile and air dried to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid 3: ¹H NMR (DMSO-d₆) δ 7.94 (d, 2H, J=8.0 Hz), 7.84 (dd, 1H, J₁=8.4 Hz, J₂=1.2 Hz), 7.75 (d, 1H, J=1.2 Hz), 7.61 (d, 2H, J=8.0 Hz), 7.48 (d, 1H, J=8.4 Hz), 2.29 (s, 3 H); LC-MS m/z 238.1 (M+1).

Step 4: To a suspension of 4′-cyano-6-methyl-biphenyl-3-carboxylic acid 3 (40 mg, 0.17 mmol) in anhydrous methylene chloride (5 ml) is added 2 drops of DMF. Then oxalyl chloride (32 mg, 22 μl, 0.25 mmol) is added. The mixture is stirred at ambient temperature until it turns clear. After that, it is concentrated, re-dissolved in anhydrous methylene chloride (3 ml), and added to a solution of 4-(morpholine-4-sulfonyl)-phenylamine (61 mg, 0.25 mmol) and triethylamine (34 mg, 47 μl, 0.33 mmol) in methylene chloride (2 ml). The mixture is stirred for 2 hours, concentrated and the residue is purified by preparative mass triggered HPLC (C₁₈ column, eluted with CH₃CN—H₂O containing 0.05% TFA) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [4-(morpholine-4-sulfonyl)-phenyl]-amide: ¹H NMR (DMSO-d₆) δ 10.64 (s, 1H), 8.07 (d, 2H, J=8.8 Hz), 7.97 (d, 2H, J=8.4 Hz), 7.95 (d, 1H, J=8.8 Hz), 7.89 (s, 1H), 7.43 (d, 2H, J=8.4 Hz), 7.67 (d, 2H, J=8.8 Hz), 7.53 (d, 2H, J=8.8 Hz), 3.63 (m, 4H), 2.84 (m, 4H) 2.32 (s, 3H); LC-MS m/z: 462.1 (M+1).

Example 2 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide

Step 1: To a solution of 2-chloro-5-nitro-pyridine 4 (2.38 g, 15 mmol.) and cis-2,6-dimethylmorpholine (1.73 g, 15 mmol.) is added K₂CO₃ (4.14 g, 30 mmol.). The mixture was heated at 50° C. overnight. After concentration, the residue is partitioned between EtOAc and water. The EtOAc layer is dried over anhydrous Na₂SO₄ and concentrated to give crude product 6 as a yellow solid. The crude product is used directly in next step without further purification. LC-MS m/z: 238.1 (M+1).

Step 2: The above crude material 6 is hydrogenated in the presence of Pd—C (0.2 g) in MeOH (100 mL) under hydrogen over 10 h. The suspension is filtered through celite and the filtrate is concentrated to give the crude product 7 as a dark brown oil which is used directly in the next step without further purification. LC-MS m/z: 208.1 (M+1).

Step 3: To a solution of 3-bromo-4-methyl benzoic acid (108 mg, 0.5 mmol.), 6-(2,6-Dimethyl-morpholin-4-yl)-pyridin-3-ylamine 7 (104 mg, 0.5 mmol.), amd HATU (190 mg, 0.5 mmol.) in dry DMF (5 mL) is added triethylamine (139 uL, 1.0 mmol.) dropwise. The resulting mixture is stirred at room temperature for 2 h. After concentration, the residue is partitioned between EtOAc and water. The organic layer is dried and concentrated to give the crude product. The final compound is purified by flash column chromatography using 50% EtOAc in hexane as eluent to give 8 as a white solid. LC-MS m/z: 404.1 (M+1).

Step 4: A mixture of 4-cyanophenyl boronic acid (18 mg, 0.12 mmol), 3-bromo-N-[6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-4-methyl-benzamide 8 (40 mg, 0.1 mmol), Pd(PPh₃)₄ (11 mg, 0.01 mmol), and Na₂CO₃ (42 mg, 0.4 mmol) in a combined solvent system of toluene (0.2 mL) and water (0.2 mL) and ethanol (0.05 mL) is heated at 140° C. under microwave irradiation for 30 min. The reaction mixture is diluted with EtOAc and water. The aqueous layer is extracted with EtOAc. The combined organic layer is washed with brine and concentrated to give the crude product which is purified by preparative mass triggered HPLC (C₁₈ column, eluted with CH₃CN—H₂O containing 0.05% TFA) to give 4′-cyano-6-methyl-biphenyl-3-carboxylic acid [6-(2,6-dimethyl-morpholin-4-yl)-pyridin-3-yl]-amide. LC-MS m/z: 427.2 (M+1).

Reference

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Oritavancin
(4R)-22-O-(3-Amino-2,3,6-trideoxy-3-C-methyl-alpha-L-arabinohexopyranosyl)-N3-(p-(p-chlorophenyl)benzyl)vancomycin

(3S, 6R, 7R, 22R, 23S, 26S, 36R, 38aR) -22 – (3-Amino-2 ,3,6-trideoxy-3-C-methyl-alpha-L-mannopyranosyloxy) -3 – (carbamoylmethyl ) -10,19-dichloro-44-[2-O-[3 - (4'-chlorobiphenyl-4-ylmethylamino) -2,3,6-trideoxy-3-C-methyl-alpha-L-mannopyranosyl] – beta-D-glucopyranosyloxy] -

Also known as N^DISACC-(4-(4-chlorophenyl)benzyl)A82846B and LY333328,N-(4-(4-chlorophenyl)benzyl)A82846B

Abbott (Supplier), Lilly (Originator), InterMune (Licensee)

The medicines company—

1. the Oritavancin Program Results.pdf

   phx.corporate-ir.net/External.File?item…t=1‎

   Jul 2, 2013 - Inhibits two key steps of cell wall synthesis: – Transglycosylation. – Transpeptidation. • Disrupts bacterial membrane integrity. Differentiated from  …

FDA Accepts Filing of NDA for IV Antibiotic Oritavancin with Priority Review

PARSIPPANY, NJ — (Marketwired) — 02/19/14 — The Medicines Company (NASDAQ: MDCO) today announced that the U.S. Food and Drug Administration (FDA) has accepted the filing of a new drug application (NDA) for oritavancin, an investigational intravenous antibiotic, with priority review. The Medicines Company is seeking approval of oritavancin for the treatment of acute bacterial skin and skin structure infections (ABSSSI) caused by susceptible gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), administered as a single dose.

In December 2013, the FDA designated oritavancin as a Qualified Infectious Disease Product (QIDP). The QIDP designation provides oritavancin priority review, and an additional five years of exclusivity upon approval of the product for the treatment of ABSSSI. Priority review means the FDA’s goal is to take action on the application within six months, compared to 10 months under standard review. The FDA action date (PDUFA date) for oritavancin is August 6, 2014.
Oritavancin (INN, also known as LY333328) is a novel semi-synthetic glycopeptide antibiotic being developed for the treatment of serious Gram-positive infections. Originally discovered and developed by Eli Lilly, oritavancin was acquired by InterMune in 2001 and then by Targanta Therapeuticsin late 2005.^[1]

In Dec 2008 the FDA declined to approve it, and an EU application was withdrawn.

In 2009 the development rights were acquired by The Medicine Co. who are running clinical trials for a possible new FDA application in 2013.^[2]

Its structure is similar to vancomycin^[3] It is a lipoglycopeptide

About Oritavancin

Oritavancin is an investigational intravenous antibiotic for which The Medicines Company is seeking approval in the treatment of ABSSSI caused by susceptible gram-positive bacteria, including MRSA. In clinical trials, the most frequently reported adverse events associated with oritavancin were nausea, headache, vomiting and diarrhea. Hypersensitivity reactions have been reported with the use of antibacterial agents including oritavancin.

Oritavancin shares certain properties with other members of the glycopeptide class of antibiotics, which includes vancomycin, the current standard of care for serious Gram-positive infections in the United States and Europe.^[4] Data presented at the 47th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in September 2007 demonstrated that oritavancin possesses potent and rapid bactericidal activity in vitro against a broad spectrum of both resistant and susceptible Gram positive bacteria, including Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Enterococci, and Streptococci.^[5] Two posters presented at the meeting also demonstrated that oritavancin was more active than either metronidazole or vancomycin against strains of Clostridium difficile tested.^[6]

Anthrax : Research presented at the American Society for Microbiology (ASM) 107th Annual General Meeting in May 2007, suggested oritavancin’s potential utility as a therapy for exposure to Bacillus anthracis, the gram-positive bacterium that causes anthrax, having demonstrated efficacy in a mouse model both pre- and post-exposure to the bacterium^[7]

oritavancin

The 4′-chlorobiphenylmethyl group disrupts the cell membrane of gram positive bacteria.^[8] It also acts by inhibition of transglycosylation and inhibition of transpeptidation.^[9]

Results have been presented (in 2003) but possibly not yet published from two pivotal Phase 3 clinical trials testing the efficacy of daily intravenous oritavancin for the treatment of complicated skin and skin-structure infections (cSSSI) caused by Gram-positive bacteria. The primary endpoints of both studies were successfully met, with oritavancin achieving efficacy with fewer days of therapy than the comparator agents (vancomycin followed by cephalexin). In addition, oritavancin showed a significantly improved safety profile with a 19.2 percent relative reduction in the overall incidence of adverse events versus vancomycin/cephalexin (p<0.001) in the second and larger pivotal trial.^[10]

A Phase 2 clinical study was planned to run until May 2008 entitled “Single or Infrequent Doses for the Treatment of Complicated Skin and Skin Structure Infections (SIMPLIFI),” evaluating the efficacy and safety of either a single dose of oritavancin or an infrequent dose of oritavancin compared to the previously studied dosing regimen of 200 mg oritavancin given once daily for 3 to 7 days.^[11] Results published May 2011.^[12]

Regulatory submissions

USA

On February 11, 2008, Targanta submitted a New Drug Application (NDA) to the US FDA seeking approval of oritavancin;^[13] in April 2008, the FDA accepted the NDA submission for standard review.^[14] On 9 Dec 2008 the FDA said insufficient data for approval of oritavancin had been provided and they requested a further phase 3 clinical study to include more patients with MRSA.^[15]

Europe

June 2008, Targanta’s Marketing Authorization Application (MAA) for oritavancin was submitted and accepted for review by the European Medicines Agency (EMEA),^[16] but the company later withdrew the application in Aug 2009.^[17]

About The Medicines Company

The Medicines Company’s purpose is to save lives, alleviate suffering, and contribute to the economics of healthcare by focusing on 3,000 leading acute/intensive care hospitals worldwide. Its vision is to be a leading provider of solutions in three areas: acute cardiovascular care, surgery and perioperative care, and serious infectious disease care. The company operates in the Americas, Europe and the Middle East, and Asia Pacific regions with global centers today in Parsippany, NJ, USA and Zurich, Switzerland.

“We look forward to working with the FDA during the review process, and sharing the knowledge we have gained in our studies of oritavancin,” said Matthew Wikler, MD, Vice President and Medical Director, Infectious Disease Care for The Medicines Company. “We believe that upon approval, oritavancin, administered as a single dose for the treatment of ABSSSI, will offer new options for both physicians and their patients for the treatment of these infections.”

The oritavancin NDA is based on data from two Phase 3 clinical trials, SOLO I and SOLO II, which were conducted under a Special Protocol Assessment (SPA) agreement with the FDA. These Phase 3 trials evaluated the efficacy and safety of a single 1200mg dose of oritavancin compared to 7 to 10 days of twice-daily vancomycin in adults with ABSSSI, including infections caused by MRSA. The combined SOLO studies were conducted in 1,959 patients (modified intent-to -treat population, or mITT), with 405 of the patients suffering from an ABSSSI with a documented MRSA infection.

oritavancin

Drug substance

Oritavancin diphosphate

CLINICAL TRIALS..http://clinicaltrials.gov/search/intervention=oritavancin

• LY 333328 diphosphate
• LY333328 diphosphate
• Oritavancin diphosphate
• UNII-VL1P93MKZN
• 192564-14-0 CAS NO

INTRODUCTION

Oritavancin

Oritavancin inhibits cell wall synthesis by complexing with the terminal D-Ala-D-Ala of a nascent peptidoglycan chain and also to the pentaglycine bridge, thus inhibiting transglyco- sylation and transpeptidation. Unlike other glycopeptides, oritavancin is able to bind to depsipeptides including D-Ala-D-Lac, which fa- cilitates its inhibition of cell wall synthesis even in organisms exhibiting VanA-type resistance. Oritavancin forms homodimers prior to binding to D-Ala-D-Ala or D-Ala-D-Lac, which increases its binding affinity for the target site.The p-chloro-phenylbenzyl side chain of oritavancin interacts with the cell membrane, exerting two beneficial effects. This binding acts to main- tain the antibacterial in a prime position for peptidoglycan interactions and it also imparts oritavancin with the ability to disrupt the bac- terial membrane potential and thus increase membrane permeability.[22,23] Oritavancin has been shown to dissipate membrane potential in both stationary and exponential phase growing bacteria, which is rare and may carry clinical implications in terms of its activity against slowly growing organisms and biofilms. The dual mechanism of action could also theoretically increase effectiveness and reduce the risk of resist- ance selection. In addition to the aforemen- tioned mechanisms, it has also been hypothesized that oritavancin inhibits RNA synthesis.

vancomycin, desmethylvancomycin, eremomycin, teicoplanin (complex of five compounds), dalbavancin, oritavancin, telavancin, and A82846B (LY264826) having structures A, B, C, D, E, F, G and H:

R = B-2-Acetylamido-glucopyraπosyl- Attorney Docket No 33746-704 602

Dalbavancin, oritavancin and telavancin are semisynthetic lipoglycopeptides that demonstrate promise for the treatment of patients with infections caused by multi-drug-resistant Gram-positive pathogens. Each of these agents contains a heptapeptide core, common to all glycopeptides, which enables them to inhibit transglycosylation and transpeptidation (cell wall synthesis). Modifications to the heptapeptide core result in different in vitro activities for the three semisynthetic lipoglycopeptides. All three lipoglycopeptides contain lipophilic side chains, which prolong their half-life, help to anchor the agents to the cell membrane and increase their activity against Gram-positive cocci. In addition to inhibiting cell wall synthesis, telavancin and oritavancin are also able to disrupt bacterial membrane integrity and increase membrane permeability; oritavancin also inhibits RNA synthesis. Enterococci exhibiting the VanA phenotype (resistance to both vancomycin and teicoplanin) are resistant to both dalbavancin and telavancin, whileoritavancin retains activity. Dalbavancin, oritavancin and telavancin exhibit activity against VanB vancomycin-resistant enterococci.

All three lipoglycopeptides demonstrate potent in vitro activity against Staphylococcus aureus and Staphylococcus epidermidis regardless of their susceptibility to meticillin, as well as Streptococcus spp. Both dalbavancin and telavancin are active against vancomycin-intermediate S. aureus (VISA), but display poor activity versus vancomycin-resistant S. aureus (VRSA). Oritavancin is active against both VISA and VRSA. Telavancin displays greater activity against Clostridium spp. than dalbavancin, oritavancin or vancomycin. The half-life of dalbavancin ranges from 147 to 258 hours, which allows for once-weekly dosing, the half-life of oritavancin of 393 hours may allow for one dose per treatment course, while telavancin requires daily administration. Dalbavancin and telavancin exhibit concentration-dependent activity and AUC/MIC (area under the concentration-time curve to minimum inhibitory concentration ratio) is the pharmacodynamic parameter that best describes their activities.

Oritavancin’s activity is also considered concentration-dependent in vitro, while in vivo its activity has been described by both concentration and time-dependent models; however, AUC/MIC is the pharmacodynamic parameter that best describes its activity. Clinical trials involving patients with complicated skin and skin structure infections (cSSSIs) have demonstrated that all three agents are as efficacious as comparators. The most common adverse effects reported with dalbavancin use included nausea, diarrhoea and constipation, while injection site reactions, fever and diarrhoea were commonly observed withoritavancin therapy. Patients administered telavancin frequently reported nausea, taste disturbance and insomnia. To date, no drug-drug interactions have been identified for dalbavancin, oritavancin or telavancin. All three of these agents are promising alternatives for the treatment of cSSSIs in cases where more economical options such as vancomycin have been ineffective, in cases of reduced vancomycin susceptibility or resistance, or where vancomycin use has been associated with adverse events.

Oritavancin diphosphate (oritavancin) is a semi-synthetic lipoglycopeptide derivative of a naturally occurring glycopeptide. Its structure confers potent antibacterial activity against gram-positive bacteria, including vancomycin-resistant enterococci (VRE), methicillin- and vancomycin-resistant staphylococci, and penicillin-resistant streptococci. The rapidity of its bactericidal activity against exponentially-growing S. aureus (≧3-log reduction within 15 minutes to 2 hours against MSSA, MRSA, and VRSA) is one of the features that distinguishes it from the prototypic glycopeptide vancomycin (McKay et al., J Antimicrob Chemother. 63(6):1191-9 (2009), Epub 2009 Apr. 15).

Oritavancin inhibits the synthesis of peptidoglycan, the major structural component of the bacterial cell wall by a mechanism that is shared with glycopeptides, such as vancomycin (Allen et al., Antimicrob Agents Chemother 41(1):66-71 (1997); Cegelski et al., J Mol Biol 357:1253-1262 (2006); Arhin et al., Poster C1-1471: Mechanisms of action of oritavancin in Staphylococcus aureus [poster]. 47th Intersci Conf Antimicro Agents Chemo, Sep. 17-20, 2007; Chicago, Ill.). Oritavancin, like vancomycin, binds to the Acyl-D-Alanyl-D-Alanine terminus of the peptidoglycan precursor, lipid-bound N-acetyl-glucosamine-N-acetyl-muramic acid-pentapeptide (Reynolds, Eur J Clin Microbiol Infect Dis 8(11):943-950 (1989); Nicas and Allen, Resistance and mechanism of action.

In: Nagarajan R, editor. Glycopeptide antibiotics. New York: Marcel Dekker 195-215 (1994); Allen et al., Antimicrob Agents Chemother 40(10):2356-2362 (1996); Allen and Nicas, FEMS Microbiology Reviews 26:511-532 (2003); Kim et al., Biochemistry 45:5235-5250 (2006)). However, oritavancin inhibits cell wall biosynthesis even when the substrate is the altered peptidoglycan precursor that is present in VRE and vancomycin-resistant S. aureus (VRSA). Thus, the spectrum of oritavancin antibacterial activity extends beyond that of vancomycin to include glycopeptide-resistant enterococci and staphylococci (Ward et al., Expert Opin Investig Drugs 15:417-429 (2006); Scheinfeld, J Drugs Dermatol 6:97-103 (2007)). Oritavancin may inhibit resistant bacteria by interacting directly with bacterial proteins in the transglycosylation step of cell wall biosynthesis (Goldman and Gange, Curr Med Chem 7(8):801-820 (2000); Halliday et al., Biochem Pharmacol 71(7):957-967 (2006); Wang et al., Poster C1-1474: Probing the mechanism of inhibition of bacterial peptidoglycan glycotransferases by glycopeptide analogs. 47th Intersci Conf Antimicro Agents Chemo, Sep. 17-20, 2007). Oritavancin also collapses transmembrane potential in gram positive bacteria, leading to rapid killing (McKay et al., Poster C1-682: Oritavancin disrupts transmembrane potential and membrane integrity concomitantly with cell killing in Staphylococcus aureus and vancomycin-resistant Enterococci. 46th Intersci Conf Antimicro Agents Chemo, San Francisco, Calif., Sep. 27-30, 2006). These multiple effects contribute to the rapid bactericidal activity of oritavancin.

Vancomycin (U.S. Patent 3,067,099); A82846A, A82846B, and A82846C (U.S. Patent 5,312,738, European Patent Publication 256,071 A1); PA-42867 factors A, C, and D (U.S. Patent4,946,941 and European Patent Publication 231,111 A2); A83850 (U.S. Patent No. 5,187,082); avoparcm (U.S. Patent 3,338,786 and U.S. Patent 4,322,343); actmoidin, also known as K288 (J. Antibiotics Series A 14:141 (1961); helevecardin (Chem. Abstracts 110:17188 (1989) and Japanese Patent Application 86/157,397); galacardin (Chem. Abstracts 110:17188 (1989) and Japanese Patent Application 89/221,320); and M47767 (European Patent Publication 339,982).

Oritavancin is in clinical development against serious gram-positive infections, where administration of the drug is via intravenous infusion using several dosages administered over a series of days. The development of alternative dosing regimens for the drug could expand treatment options available to physicians. The present invention is directed to novel dosing regimens.

Means for the preparation of the glycopeptide antibiotics, including oritavancin and analogs thereof, may be found, for example, in U.S. Pat. No. 5,840,684,

ORITAVANCIN DIPHOSPHATE

SYNTHESIS

LY-333328 was synthesized by reductocondensation of the glycopeptide antibiotic A82846B (I) with 4′-chlorobiphenyl-4-carboxaldehyde (II) by means of sodium cyanoborohydride in refluxing methanol.

J Antibiot1996, 49, (6) :575-81

(3S,6R,7R,22R,23S,26S,36R,38aR)-3-(Carbamoylmethyl)-10,19-dichloro-7,28,30,32-tetrahydroxy-6-(N-methyl-D-leucylamido)-2,5,24,38,39-pentaoxo-22-(L-vancosaminyloxy)-44-[2-O-(L-vancosaminyl)-beta-D-glucopyranosyloxy]-2,3,4,5,6,7,23,24,25,26,36,37,38,38a-tetradecahydro-1H,22H-8,11:18,21-dietheno-23,36-(iminomethano)-13,16:31,36-dimetheno-[1,6,9]oxadiazacyclohexadecino[4,5-m][10,2,16]benzoxadiazacyclotetracosine-26-carboxylic acid; A82846B (I)
4′-chloro[1,1'-biphenyl]-4-carbaldehyde (II)

LY-333328 was synthesized by reductocondensation of the glycopeptide antibiotic A82846B (I) with 4′-chlorobiphenyl-4-carboxaldehyde (II) by means of sodium cyanoborohydride in refluxing methanol.

…………………..

WO1996030401A1

EXAMPLE 4

Preparation of Compound 229

A three liter 3-necked flask was fitted with a

condenser, nitrogen inlet and overhead mechanical stirring apparatus. The flask was charged with pulverized A82846B acetate salt (20.0 g, 1.21 × 10^-3 mol) and methanol (1000 mL) under a nitrogen atmosphere. 4′-chlorobiphenylcarboxaldehyde (2.88 g, 1.33 × 10^-2 mol, 1.1 eq.) was added to this stirred mixture, followed by methanol (500 mL). Finally, sodium cyanoborohydride (0.84 g, 1.33 × 10^-2 mol, 1.1 eq.) was added followed by methanol (500 mL). The resulting mixture was heated to reflux (about 65°C).

After 1 hour at reflux, the reaction mixture attained homogeneity. After 25 hours ac reflux, the heat source was removed and the clear reaction mixture was measured with a pH meter (6.97 at 58.0°C). 1 N NaOH (22.8 mL) was added

dropwise to adjust the pH to 9.0 (at 54.7°C). The flask was equipped with a distillation head and the mixture was concentrated under partial vacuum to a weight of 322.3 grams while maintaining the pot temperature between 40-45°C.

The distillation head was replaced with an addition funnel containing 500 mL of isopropanol (IPA). The IPA was added dropwise to the room temperature solution over 1 hour. After approximately 1/3 of the IPA was added, a granular precipitate formed. The remaining IPA was added at a faster rate after precipitation had commenced. The flask was weighed and found to hold 714.4 grams of the IPA/methanol slurry.

The flask was re-equipped with a still-head and

distilled under partial vacuum to remove the remaining methanol. The resulting slurry (377.8 g) was allowed to chill in the freezer overnight. The crude product was filtered through a polypropylene pad and rinsed twice with 25 mL of cold IPA. After pulling dry on the funnel for 5 minutes, the material was placed in the vacuum oven to dry at 40°C. A light pink solid (22.87 g (theory = 22.43 g) ) was recovered. HPLC analysis versus a standard indicated 68.0% weight percent of Compound 229 (4- [4-chlorophenyl] benzyl-A82846B] in the crude solid, which translated into a

corrected crude yield of 69.3%.

The products of the reaction were analyzed by reverse-phase HPLC utilizing a Zorbax SB-C18 column with ultraviolet light (UV; 230 nm) detection. A 20 minute gradient solvent system consisting of 95% aqueous buffer/5% CH₃CN at time=0 minutes to 40% aqueous buffer/60% CH₃CN at time=20 minutes was used, where the aqueous buffer was TEAP (5 ml CH₃CN, 3 ml phosphoric acid in 1000 ml water).

………………….

WO2008097364A2

Oritavancin (also termed N-(4-(4-chlorophenyl)benzyl)A82846B and LY333328) has the following Formula III:

References

KW 4490

cis-4-Cyano-4-(2,3-dihydro-8-methoxy-1,4-benzodioxin-5-yl)cyclohexanecarboxylic Acid 

Cyclohexanecarboxyli​c acid, 4-​cyano-​4-​(2,​3-​dihydro-​8-​methoxy-​1,​4-​benzodioxin-​5-​yl)​-​, cis-

cis-​4-​Cyano-​4-​(2,​3-​dihydro-​8-​methoxy-​1,​4-​benzodioxin-​5-​yl)​cyclohexane-​1-​carboxylic acid;

cis-​4-​Cyano-​4-​(8-​methoxy-​1,​4-​benzodioxan-​5-​yl)​cyclohexanecarboxyli​c acid

KF 66490; KW 4490;

MF C₁₇ H₁₉ N O₅

A phosphodiesterase type 4 inhibitor, commonly referred to as a PDE4 inhibitor, is a drug used to block the degradative action ofphosphodiesterase 4 (PDE4) on cyclic adenosine monophosphate (cAMP). It is a member of the larger family of PDE inhibitors. The PDE4 family of enzymes are the most prevalent PDE in immune cells. They are predominantly responsible for hydrolyzing cAMP within both immune cells and cells in the central nervous system

PDE4 hydrolyzes cyclic adenosine monophosphate (cAMP) to inactive adenosine monophosphate (AMP). Inhibition of PDE4 blocks hydrolysis of cAMP, thereby increasing levels of cAMP within cells.

Practical synthesis of the PDE4 inhibitor, KW-4490

ORGN 699

Arata Yanagisawa, arata.yanagisawa@kyowa.co.jp1, Koichiro Nishimura2, Tetsuya Nezu2, Kyoji Ando2, Ayako Maki2, Eiichiro Imai2, and Shin-ichiro Mohri2. (1) Pharmaceutical Research Center, Medicinal Chemistry Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka, Japan, (2) Pharmaceutical Research Center, Sakai Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, 1-1-53 Takasu-cyo, Sakai-ku, Sakai, Osaka, Japan

A practical and scaleable synthesis of the PDE4 inhibitor, KW-4490 (1), was developed for the multi-kilogram preparation. This improved synthesis features construction of the 1-arylcyclohexene by Diels-Alder reaction, followed by a newly established acid-mediated hydrocyanation. The synthesis was achieved in 7 steps in 38% overall yield. Efforts toward increasing the regioselectivity in the Diels-Alder reaction, optimization of crystallization-induced dynamic resolution of the hydrocyanation product, and investigation of other synthetic routes will be presented.

New Reactions and Methodology, Metal-Mediated Reactions, Physical Organic Chemistry, Molecular Recognition and Self-Assembly 7:00 PM-9:00 PM, Wednesday, August 20, 2008 Pennsylvania Convention Center — Blrm A/B, PosterDivision of Organic ChemistryThe 236th ACS National Meeting, Philadelphia, PA, August 17-21, 2008

A team at Kyowa Hakko Kirin in Japan has used a crystallisation-induced dynamic resolution in the synthesis of KW-4490, a PDE-4 inhibitor being developed for asthma and chronic obstructive pulmonary disease.6 Towards the end of the synthesis, they were faced with a mixture of cis and trans diastereomers of an intermediate derived from a hydrocyanation reaction, which was about 62:38 cis:trans; altering the conditions of the reaction did not give a selective process. The desired isomer was the cis, so they wanted to convert the unwanted trans isomer to cis to improve the yield (Scheme 2).

They first tried using a base-induced isomerisation using a base such as potassium t-butoxide, but although this worked to a degree the best ratio of products obtained was 75:25. The same result was obtained when they tested the system on both pure cis and trans isomers, indicating that this ratio represented the thermodynamic equilibrium. However, they realised that the cis isomer was less soluble in ethanol, so they thought the answer might lie in crystallisation-induced dynamic resolution.

They therefore suspended a crude mixture of the two isomers in ethanol and added a catalytic amount of potassium t-butoxide to effect the isomerisation. It was stirred and warmed, and hexane added portion-wise to crash the cis isomer out of solution. The group managed to increase the ratio of isomers to 99:1 by continuous isomerisation, with a 90% isolated yield.

Scheme 2: Kyowa Hakko Kirin found a way to improve the yield of the cis isomer

Compound (XIII) is disclosed in WO00/14085 as being useful as a PDE-IV inhibitor. A method for the preparation of a typical compound among compounds (XIII) disclosed in WO00/14085 is as follows:

However, this method is not practically satisfactory as a industrially applicable preparation method, because of (1) requiring multiple steps, (2) low overall yield, (3) requiring purification by silica-gel column chromatography, and the like.

REFERENCE EXAMPLE 1

Synthesis of cis-4-cyano-4-(2,3-dihydro-8-methoxy-1,4-benzodioxine-5-yl)cyclohexanecarboxylic acid

(1) Synthesis of cis-4-cyano-4-(2,3-dihydro-8-methoxy-1,4-benzodioxine-5-yl)cyclohexanecarboxylic acid ethyl ester

Under a nitrogen atmosphere, trifluoromethanesulfonic acid (2.25 g) and trimethylsilylcyanide (1.57 mL) were dissolved in benzotrifluoride (10 mL), and a solution of 4-(2,3-dihydro-8-methoxy-1,4-benzodioxine-5-yl)-3-cyclohexenecarboxylic acid ethyl ester (0.79 g) prepared according to the method described in EXAMPLE 1 in benzotrifluoride (10 mL) was added dropwise at −25° C. After being stirred for for one hour at −20° C., an aqueous saturated sodium hydrogen carbonate was added and the mixture was extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was crystallized from ethanol (1 mL) to give a solid substance (0.64 g). The solid substance (0.030 g) was crystallized from a mixed solvent of diisopropyl ether and ethyl acetate (0.36 mL, diisopropyl ether/ethyl acetate=4/1) to give cis-4-cyano-4-(2,3-dihydro-8-methoxy-1,4-benzodioxine-5-yl)cyclohexanecarboxylic acid ethyl ester (0.019 g, 47.3%) as a solid.

Melting point 131° C.

¹H-NMR (CDCl₃, δ ppm) 6.84 (d, J=8.9 Hz, 1H), 6.49 (d, J=8.9 Hz, 1H), 4.39−4.33 (m, 4H), 4.17 (q, J=7.1 Hz, 2H), 3.88 (s, 3H), 2.44 (brd, J=12.6 Hz, 2H), 2.32 (tt, J=11.8, 3.8 Hz, 1H), 2.18−1.95 (m, 4H), 1.86 (dt, J=3.6, 12.6 Hz, 2H), 1.28 (t, J=7.1 Hz, 3H).

[0184] IR (KBr, cm⁻¹) 2953, 2228, 1722, 1607, 1504, 1460, 1381, 1325, 1281, 1117, 1043, 953, 787.

MS (m/z) 346(M+H)⁺.

Table 1.

•  Yanagisawa, Arata; Organic Process Research & Development 2010, 14(5), P1182-1187 
•  Yanagisawa, Arata; Organic Letters 2009, 11(22), P5286-5289 
• US 20010056117
• WO 2002059105
• WO 2000014085

RAMELTEON

(5)-N-[2-(l,6,7,8-tetrahydro-2H-indeno-[5,4-ό]furan-8- yl)ethyl]propionamide

EP885210B1 , EP1792899A1 and J. Med Chem. 2002, 45, 4222-4239

read all at

http://www.allfordrugs.com/2014/02/23/ramelteon-tak-375-melatonin-mt1mt2-receptor-agonist/

Bhasma : The ancient Indian nanomedicine

Dilipkumar pal

Department of Pharmaceutical Sciences, Guru Ghasidash Vishwavidyalya (A Central University), Koni, Bilaspur – 495 009, Chhattisgarh 

India

Ayurveda is the science made up of Veda (knowledge) and Ayush (life) i.e. knowledge of life. An Ayurvedic system adopts a holistic approach towards health care by balancing the physical, mental and spiritual functions of the human body. Rasa-Shastra (vedic-chemistry) is one of the parts of Ayurveda, which deals with herbo-mineral/metals/non-metals preparations called Bhasmas. Rasayana (immunomodulation and anti-aging quality) and yogavahi (ability to target drugs to the site) are characteristics of a properly made herbo-mineral/metals/non-metals preparation, which is also nontoxic, gently absorbable, adaptable and digestible in the body

read at http://www.japtr.org/article.asp?issn=2231-4040;year=2014;volume=5;issue=1;spage=4;epage=12;aulast=Pal

Pal D, Sahu CK, Haldar A. Bhasma : The ancient Indian nanomedicine. J Adv Pharm Technol Res [serial online] 2014 [cited 2014 Feb 26];5:4-12. Available from: http://www.japtr.org/text.asp?2014/5/1/4/126980

Bhasma^[1] in Ayurveda has been defined as a substance obtained by calcination.

Use of both bhasma (Residue after incineration – calcined preparation) as well as in pishti (powdered gem or metal) form along with appropriate herbs for treatment of critical ailments is a medicinal preparation in Ayurveda and to some extent Unani (both Indian branches of medical science using natural curative methods. The procedures for preparing these medicines are time-consuming and complicated.

Bhasma is a calcined preparation in which the gem or metal is converted into ash. Gems or metals are purified to remove impurities and treated by triturating and macerating in herbal extracts. The dough so obtained is calcinated to obtain the ashes.^[2]^

Bhasma or vibhooti is the sacred ash from the dhuni or fire of a yogi or avadhoota, or from the sacrificial fire or yajna, where special wood, ghee, herbs, grains and other auspicious and purifying items are offered in worship along with mantras. It is believed that bhasma destroys sins (paap), and that it links us with the divine. It is called ‘bhasma’ because it has the power to consume all evils. Any matter, broken up through the process of fire is reduced to its ‘bhasmic’ form, which is infinitely more refined and pure than the original matter, devoid as it is of all impurities niranjan. The grossness of matter obscures the subtle essence inherent within it, just as wood hides fire and milk conceals butter and cheese, but when it is burnt (or churned in the case of milk) only the pure essence remains. Similarly, the great heat of tapasya and the churning of the mind in meditation reveals the underlying subtle spirit or atman.

Vibhuti

In certain circumstances Bhasma, ‘Vibhuti‘ (Sanskrit) and ‘Thiruneeru’ (Tamil) are synonymous.

Bhasmikaran

Bhasmikaran is a process by which a substance which is otherwise bioincompatible is made biocompatible by certain samskaras or processes (Puranik and Dhamankar, 1964e). The objectives of samskara are :- a) elimination of harmful matters from the drug b) modification of undesirable physical properties of the drug c)conversion of some of the characteristics of the drug d) enhancement of the therapeutic action(Puranik and Dhamankar, 1964e). Various steps involved in the preparation of bhasma(or bhasmikaran) are:- 1) Shodhan -Purification, 2) Maran – Powdering, 3) Chalan- Stirring, 4) Dhavan – Washing, 5) Galan- Filtering, 6) Putan- Heating, 7) Mardan- Triturating, 8) Bhavan- Coating with herbal extract, 9) Amrutikaran – Detoxification and 10) Sandharan- Preservation (Puranik and Dhamankar, 1964e). Selection of these steps depends on the specific metal. Sometimes there is an overlapping of the steps e.g. maran is achieved by puttan. Since the present thesis work is on bhasma, Bhasmikaran process is elaborated in details in the following paragraphs.

Steps of bhasmikaran

1. Shodhan: The principle objective of shodhan is to remove unwanted part from the raw material and separate out impurities( Vaiday and Dole 1996b). Metals obtained from ores may contain several impurities, which are removed by subjecting them to Shodhan process. In context of bhasma, shodhan means purifying and making the product suitable for the next step i.e. Maran. Ayurveda classifies shodhan into a) General process and b) Specific process.

General process for shodhan:

“The sheets of metals are heated till red hot and are successively dipped into liquids like oil, buttermilk, cow’s urine etc. The procedure is repeated seven times”.

b. Specific process for shodhan For some metals a specific process is described for shodhan e.g. for purification of Jasad, the molten mass is poured in cow’s milk 21 times (Shastri K,1979b).

2. Maran : Maran literally means killing. As the name suggests in maran process, a change is brought about in the chemical form or state of the metal. This makes it to lose its metallic characteristics and physical nature. In short, after maran, metal can be converted into powder or other form suitable for administration. To convert various metals into a form appropriate for human consumption, several techniques have been employed which ultimately gave birth to concept: “Bhasma prepared by using Rasa i.e. mercury is the best, whereas the one prepared using herbs are of better quality and those prepared using Gandhak (sulfur) are of inferior quality. Thus there are 3 methods given for maran. It is carried out by heating the metal in presence of 1) mercury 2) plants and 3 ) sulfur.

When various maran procedures for different metals were reviewed, it was found that mercury is mainly used. The unique property of mercury to amalgamate with many metals must have been the reason behind its maximum use in the process of Bhasmikaran. Ancient practitioners might have found it as the most suitable chemical and therefore probably have mentioned that bhasmas using mercury are superior. Plants used in maran process may be serving as catalyst in the process or the minerals in the plants may be forming complexes with the metals. However, no such explanation can be obtained for the use of sulfur.

3. Chalan: Process of stirring during heating the metal is chalan. Stirring is carried out either with iron rod or stick made from a specific plant. As we know today, iron serves as catalyst in many chemical reactions. The phytoconstituents of plant stick may be enhancing the therapeutic effect. For example, stick of Neem is used for chalan process of Jasad bhasma, which is used topically for ophthalmic diseases. We can interpret the significance of this process now. Neem is an antiseptic (Puranik and Dhamankar, 1964h). Zinc is antiseptic, astringent and has ulcer healing property (Block et al., 1982b). These effects of both the constituents may impart the final product better therapeutic activity.

4. Dhavan: In this process, several water washes are given to the product obtained in the previous stage. Perhaps this is to remove the excess amounts of agents used in shodhan or maran stage. Such agents may adversely affect the quality of final product. Hence intermediates are washed with water, thereby water soluble constituents are removed (Puranik and Dhamankar, 1964h).

5. Galan: The product is then sifted either through a fine cloth or through sieves of suitable mesh so as to separate residual material larger in size (Puranik and Dhamankar, 1964h).

6. Puttan: The term puttan means ignition. The general term used for heating in the process of Bhasmikaran is Puta. A special earthen pot, Sharav is generally used for the process. It has two parts, each having a shape of soccer. Sharav is used for direct heating of the material. Its shallowness is useful in heating the material faster and uniformly. After keeping the material on the shallow surface, other part is used as a lid, by placing it in an inverted position. This Puttan process can be looked upon as the key step in manufacturing of bhasma. The classification of putta is primarily done on the basic nature of the process and is as under :- (Puranik and Dhamankar, 1964f) 1)Chandraputta 2) Dhanyarashiputta 3) Suryaputta 4)Bhugarbhaputta 5) Agniptuta.

Toxicity

Modern medical science finds that mercury is inherently toxic, and that its toxicity is not due to the presence of impurities. While mercury does have anti-microbial properties, and formerly waswidely used in Western medicine, its toxicity does not warrant the risk of using it as a health product in most circumstances.^[3][4] The Centers for Disease Control and Prevention have also reported a number of cases of lead and mercury poisoning associated with rasa shastra containing Ayurvedic medicines.^[5]

Literal and symbolic meaning of bhasma

The Sanskrit word bhasma literally means ‘disintegration’. Bha implies bhartsanam (to destroy), while sma implies smaranam (to remember). Bhasma is thus a reminder to us of the ephemeral nature of life. Also, if we wish to unite with God (or the ‘supreme self’) and remember him constantly, our ego or ‘little self’ has first to be disintegrated or burnt to ashes. Bhasma is a symbol of this process. It is also called raksha because it protects one from all fears. When applied to the forehead before sleep, it is said to keep away spirits or ghosts, whether external or those which manifest from the depths of the mind in the form of nightmares.

Bhasma symbolises the burning of our false identification with the mortal body, and freedom from the limitations of the painfully illusive cycle of birth and death. It also reminds us of the perishable quality of the body, which will one day be reduced to mere ashes. As it says in the Bible, “Ashes to ashes; soul to soul” – the body will return to dust but the soul will continue its journey until it unites with God. All the saints and sages beseech us to remember the ephemeral nature of our earthly existence. In the Rubayyat of Omar Khyyam the poet tells us to, “ . . . make the most of what we yet may spend, before we too into the dust descend, dust into dust, and under dust to lie.” Here he calls for us to seek the eternal, not the temporal. Ash or dust, on the other hand, can be said to represent permanency (or the soul itself), because the ash, just like imperishable truth, does not itself decay. The realised soul is said to rise from the ashes (of the individual self) as the mythical phoenix. The Sufis say, “To reach the goal we have to be burned with the fire of love, so that nothing remains but ashes, and from the ashes will resurrect the new being. Only then can there be real creation!”

The power of bhasma

Bhasma is also called ‘vibhooti’, because it gives spiritual power. The Sanskrit word, vibhooti means ‘glory’, as it gives glory to one who applies it, protection (raksha) from ill health and negative forces, and attracts the higher forces of nature. Another meaning of vibhooti is ‘healing power’, and it is widely used as a medicinal treatment in both Ayurveda and Chinese and Tibetan medicine, which are all ancient and profound systems for the rejuvenation of life. Gold, silver, copper, pearls, mica and other precious stones and metals have curative properties which can quite safely and most effectively be taken into the body after being reduced to ash using great heat.

In Indian villages you will find tantric healers called ojhas who say certain mantras over the ash, which the sick person then applies to the body or eats. These healers can take some earth in their hands, hold it up to the sun, repeat some mantras, and the earth turns into the most beautifully scented ash for curative purposes. Vibhooti is also the name given to siddhis (perfections or psychic powers), as it acts as a vehicle for them. Patanjali’s Yoga Sutras devotes an entire chapter to yogic siddhis. Vibhooti also means ‘dominion’, and is the subtle power lying behind creation, from which all things manifest. From vibhooti or bhasma, anything can be created by a tantric and aghora, because the potential of creation lies within it, and he has penetrated the law and controlled the elements.

Maha Yogi Shiva, father of tantra, is usually depicted naked in sadhana, his whole body covered in bhasma. The first verse of the Shiva Panchakshara Stotram gives the following description: Naagendrahaaraaya trilochanaaya, bhasmaangaraagaaya maheshwaraaya. Nityaaya shuddhaaya digambaraaya – ‘Salutations to the mighty three-eyed Shiva, eternal and pure, wearing the king of snakes as his garland, naked and besmeared with sacred ash.’ Some other names given to Lord Shiva are Bhasmashayaaya (abode of bhasma) and Bhasmabhootaaya (covered with bhasma). Covering the body with ash is considered to be an auspicious act for discovering one’s Shiva nature. Shiva is said to be responsible for mahapralaya, the dissolution of the universe at the end of each kalpa. At this time he dances his tandava nritya, the dance of destruction.

The great tantric siddha Avadhoota Dattatreya was referred to as Bhasma Nishta – one who loves bhasma. Bhasma is generally applied on the forehead, while many sadhus also apply it on the arms, chest and stomach. Some ascetics, especially nagas (naked ascetics) rub it all over the body. While applying it, many devotees also consume a pinch. Shaivites use only bhasma from cremated bodies, which is believed to be very powerful. Bhasma has the power of fire. Agni, the inner fire, scorches and reduces all impurities in the body. It is said that one who smears ash on the body is purified as if bathed in fire. This is known as ‘the bath of fire’. After smearing the body with ash, one should reflect on and realise the highest truth.

Tripundra

Sannyasins wear three lines of bhasma on the forehead. These three lines (tripundra), with a red dot of kumkum underneath, between the eyebrows, symbolise Shiva-Shakti (the unity of energy and matter that creates the entire seen and unseen universe). The lower line represents tamoguna (the state of inertia and darkness), the middle line represents rajas (activity and dynamism) and the top line represents sattwa (balance and illumination). The red dot or tika represents the power of shakti through sadhana, which can take the sadhaka beyond the three gunas or qualities to the state of turiya, the fourth dimension of existence. This is the state of trigunatita – beyond the three gunas.

Swami Niranjanananda says, “The three stripes represent the tradition of the paramahamsas. Jignasus are one stripe sannyasins, representing the drive and motivation to overcome the tamasic tendency. Karma sannyasins are given two stripes, representing their drive to overcome the rajasic along with the tamasic tendencies. Poorna sannyasins are given three stripes, which represent their motivation to transcend the three gunas and attain inner sublimation. The red dot represents the spiritual power or energy that gives us the strength to control the three gunas. It is the awakening of that shakti which is the real aim of sannyasa.”

Bhasma and tattwa shuddhi

Consciousness manifests as energy, which then condenses into matter. In the tantric practice of tattwa shuddhi, in order to experience consciousness free from matter, we reverse the process of evolution back through more and more subtle dimensions to its original cause. Bhasma is an integral part of tattwa shuddhi sadhana, as a symbol of purification on the physical, subtle and causal realms of consciousness. The process of disintegration undergone in tattwa shuddhi is the breaking down of conscious awareness. Just as we reduce matter to its bhasmic form, the ‘fire’ of this practice leads us to the realisation of our essential essence. The stages of pratyahara (sense withdrawal) and dharana (concentration) take us through the more subtle states of consciousness, culminating in samadhi, the ultimate experience or ‘Shiva consciousness’. The journey is from gross matter to pure consciousness.

At the end of the practice of tattwa shuddhi, bhasma is applied to the forehead with the repetition of mantras. It is taken on the middle and ring fingers and wiped slowly on the forehead from left to right, repeating the mantra Om Hraum Namah Shivaya. Sannyasins use the index, middle and ring fingers, and repeat the mantra Om Hamsa. The bhasma used in tattwa shuddhi is prepared from gobar or cow dung. The word gobar literally means ‘gift from the cow’; it is also known as go-maya. The cow is a pure and sacred animal, full of auspicious qualities. It is even said to contain all the devas and devatas within it. Not only does gobar have mystical qualities, but it also contains useful hormones with germicidal properties. The word go also means ‘senses’. So bhasma is also symbolic of the disintegration of the senses which keep us trapped and bound in the gross material world. The transformation of gobar to bhasma is parallel to the transformation from the material world to cosmic consciousness that we find in tattwa shuddhi.

Panchagni bhasma

During the Sat Chandi Mahayajna, and on other auspicious occasions at Rikhia Dham, devotees receive the precious prasadam of panchagni bhasma. This is much prized by sadhakas, because as it has the power of Swami Satyananda’s sadhana behind it, it quickly helps to raise the consciousness at the time of mantra japa and other sadhana when applied to the forehead. Just keeping it in the pooja room is auspicious. This bhasma given is from the Maha Kaal Chita Dhuni, where the previously fierce fires of Sri Swamiji’s panchagni tapasya now lie smouldering quietly under ashes in their shanta roopa or peaceful form. Dhuni is the yogi’s fire, which is the witness or sakshi to his sadhana. It is also where he cooks, takes warmth, and chants the name of God. (Maha means ‘great’, kaal is ‘time’ and chita is ‘consciousness’).

This akhanda dhuni, eternal fire, has been burning in Sri Swamiji’s pooja area ever since he first came to Rikhia in 1989 and devotees come daily for its darshan. It was the centre and support of his life during his austerity, and is the very heart of the Rikhia Ashram (next to Sri Swamiji himself). Although Sri Swamiji no longer goes to this area, the fire is still tended daily. The ashes are moved to the side and the burning embers taken out. Balls of dried cow dung mixed with purifying herbs (vanaspati) are then placed inside along with fresh wood. The embers are then replaced, and the whole area is covered over once more with the ashes. From time to time the ash is removed, carefully sieved through fine cloth, and given as prasadam (that which has been blessed by a divine power.

For the panchagni sadhana itself, Sri Swamiji prepared his own bhasma to protect his body from the great heat, according to the formula prescribed in the Devi Bhagavat Purana. This special bhasma is called mahabhasma and is made from pure cow dung cakes, reeds and ghee. It is treated eleven times with many herbs, honey and other ingredients, being re-burnt each time. Bhasma is smeared on the body only during the first few days of the panchagni sadhana, and is applied in the morning. Sri Swamiji’s dog-cum-companion Bholenath, in whom he manifested the spirit of Bhairava, also took part in the tapasya. “Alsatian dogs can’t bear the heat,” commented Sri Swamiji. “I would put bhasma on him in the morning and he would sit with me.”

Tantric siddhas like Maha Yogi Shiva, Avadhoota Dattatreya and Sri Swamiji are extremely rare beings, and a gift to us beyond our understanding. They belong to a great tradition and leave behind for us a great spiritual legacy. The parampara, the line of avadhootas (those who have become immortal), continues, just as the Mahakaal Chita Dhuni continues to smoulder, unseen beneath the symbol of their glory, their bhasma – the sacred ash.

 

Abhrak Bhasma

Strengthens body, ligaments & Saptadhatu, effective in raktapitta, vat diseases and joint pain. Relieves problems related to chronic hyperacidity like stomache, headache etc.

 

Details

The Indian ayurved philosophy is founded on three basic classifications of human body known as doshasand its well-being:

Vat - related to the central nervous system and gastric tract,

Pitta -digestion and metabolism, governs movement of heat in the body and

Kapha - concerned with structure, stability and fluid balance in the body.

Indian herbal medicines are designed for the specific maintenance of these respective doshas or the aspects of the human body.

Abhrak Bhasma or Sahastraputi is an ancient Indian ayurvedic medicine which is trusted worldwide for healing Vat related issues of the human body. It is also found extremely effective for treating heart related issues.

Abhrak is known to have a high penetrative property which spreads in the body at a faster rate and impacts micro tissues quickly.That is why this herbal medicine is supremely effective in cell regeneration. When used as an alterative, it strengthens ligaments. It is known for rapidly increasing the production of T-cell phagocytes, antibacterial components, which are responsible for a strong immune system.

Kesar Herbals has been in the ayurvedic medicine manufacturing business for about 25 years. This company is one of the foremost and trusted ayurvedic medicine suppliers in India.The concept was let entire mankind benefit from the ancient herbal wealth of India.

Composition:

Purified Mica, Magnesium, Iron, Potassium, Calcium and Aluminium.

Major Benefits of Abhrak Bhasma

• It finds its application in curing diseases like Hepatitis, Tuberculosis, Asthma and Plague.
• Abhrak Bhasma is also recommended for breathlessness due to chronic Bronchitis, Asthma, and even Chest Congestion. When taken in the initial stage of TB, it can completely cure it.
• It is beneficial to all the seven dhatus (energy elements) in the body.
• It rejuvenates the human mind.
• It improves blood circulation, improves the general tone of tissues and conductivity.
• It cures erectile dysfunction and impotency in men.
• Abhrak is hematinic, which means increases the count of red blood cells. This also increases oxygen carrying ability of the cells.
• It helps in curing jaundice and anemia.
• It restores damaged tissues and maintains their health.
• It is highly effective in cases of bone marrow depletion and hepatic dysfunction.
• It is effective in curing respiratory tract infections.
• It is beneficial for curing Bells Palsy and dysentery.
• It is beneficial in curing anemia, pernicious and azoospermia.
• Also, helps in controlling many types of veneral diseases like EBV, HIV, Lupus bronchitis and pneumonia.
• It cures breast cancer.
• It is also known to cure chronic hyperacidity like stomach-ache and vomiting with blood.

 

Suvarna Bhasma

Boosts immunity, Acts as a nervine tonic , Indicated in old age debility, urinal disorders, anemia, Shwas, Helps to improve complexion, Kas & chronic fever etc.

 

Details

Swarna Bhasma is an ancient Indian ayurvedic medicine that enjoys wide popularity and application.Gold has been accepted as most useful ingredient in Indian ayurvedic medicine system. SwarnaBhasma is used for the effective treatment of Gonorrhoea and Syphilis.

Fortified with potent ayurvedic herbs it is effective for maintaining a healthy life-style and relieves stress.Bhasma denotes the metal based medicine prepared from metals after scientific process to harness the hidden richness of raw metals for therapeutic effects. Swarna Bhasmaor Gold Ash is a therapeutic form of gold metal of nano-sized particles that is one of the most treasured ayurvedic medicines in the world.

It has multi-purpose ayurvedic medicine: it can be used as an antacid, haematinic, and alterative. It is highly effective as a cardiac tonic, maintaining the vitality of the human heart. It is a general vitality booster.

Kesar Herbals firmly believes in the power of nature in healing various ailments. Ayurvedic medicines from India are a gift from nature itself, without any side-effects, and hence they are trusted world-wide.Through scientific processes, nature’s best healing powers from herbs are obtained and used for medicinal purposes, helping million people worldwide. Kesar Herbals is involved in consulting, manufacturing and supplying FDA approved ayurvedic medicines for over three decades.

Major Benefits of Swarna Bhasma

• Swarna Bhasma improves resistance power in human body.
• It is responsible for boosting immunity and strengthening the human body.
• It can be used on a regular basis as an alterative and acts as a nerve tonic.
• Swarna Bhasma is excellent in treating Anemia, and chronic fever.
• Due to its gold base, it is helpful in improving complexion.
• Swarna Bhasma eradicates all chronic disorders, when used consistently.
• It is indicated in chronic fever, cough, Asthma, urinary disorders, sleeplessness and weak digestion.
• It strengthens weak muscles, debilities associated with old-age.
• It is excellent for treating Tuberculosis.
• It increases sexual power.
• Swarna Bhasma slows down the process of degeneration of tissues thus helps to prevent premature ageing.
• It helps prevent symptoms of ageing like wrinkles, dullness of skin, dark circles around the eyes, debility, fatigue, etc.
• Swarna Bhasma strengthens tissues and ligaments of the human body.
• It is highly effective in maintaining vigor, vitality and stamina.

Hirak Bhasma

effective against immunity disorders, Bone marrow depression and arthritis, increases stamina, vitality and strength

Ayurvedic medicinal science has been at the forefront of solving most diseases and disorders of the human body because of the purity of its ingredients- this forms the core of the ancient Indian healing system. Ayurvedic medicines and herbal products are effective because of the composition or the formulation of herbs. One such effective ayurvedic medicine is the Hirak Bhasma, which contains diamond ash.

According to Ayurveda, diamond ash is very useful in cancers, immunity disorders, Crippling Rheumatoid arthritis, and even bone marrow depression. Hirak Bhasma is known for toning heart muscles and avoiding cardiovascular syndromes and diseases. It is highly effective as a tonic and alterative, which means, it can be used regularly to strengthen immunity, aid stamina and overall vitality or the functionality of the human body.

Hirak Bhasma is made with diamond- the most precious as well as the hardest gemstone known to mankind. In its healing formulation, diamond is known since ages to make the human body stronger. It is also known for sharpening the human mind by strengthening memory power.

The effectiveness of ayurvedic medicines depends on the company. So be careful in selecting the brand for these medicines and products. Kesar Herbals has been in the ayurvedic consulting space and even manufacturing and supplying of ayurvedic medicines and herbal products for over 25 years. The trust and good will it represents in the field of Ayurveda is affirmed by its customers the world over. It is important to ascertain the brand equity of the company before purchasing these medicines.

Composition:

Hirak Bhasma (diamond ash), Kumari Rasa (Aloe Vera Juice), Gulab Jala (Rose Water), Sunthi Kwath (Dry Ginger Dicoction)

Benefits:

• Hirak Bhasma is known the world over to make the human body stronger and the mind sharper.
• It is also recommended for the well-being of the human heart.
• It helps in toning the muscles of the heart.
• Its regular consumption as a tonic or an alterative, under medical prescription or supervision is known for toning the heart muscles.
• Hirak Bhasma also increases stamina, vitality and strength.
• It is highly effective against immunity disorders.
• It is known for curing Crippling Rheumatoid
• It is effective against Bone marrow depression and arthritis.
• Hirak Bhasma is also known to cure one of the most dangerous and lethal diseases known to mankind: cancer. When used in the initial stages, under medical supervision, it can cure cancerous cells.

1.  Ayurvedic formulary of India , 1978a
2.  Major Herbs of Ayurveda, Elizabeth M. Williamson. ISBN 0-443-07203-5
3.  http://www.atsdr.cdc.gov/toxprofiles/tp46.html
4.  http://toxnet.nlm.nih.gov/cgi-bin/sis/search/f?./temp/~jcaJ1Z:2
5.  CDC Morbidity and Mortality Weekly Report: Lead Poisoning Associated with Ayurvedic Medications — Five States, 2000–2003

Healthline just published an interesting infograph that gives a visualization of what your daily value of cholesterol looks like.  In the graphic, you can see what 300 mg of cholesterol looks like for 20 high cholesterol foods: http://www.healthline.com/health/high-cholesterol/daily-value

This is a very informative resource as it helps us visualize what their cholesterol intake look like

Fried Chicken:

4 pieces=300mg cholesterol

Croissants:

6 2/3 rolls=300mg cholesterol

Cheddar Cheese:

12 3/4 slices=300mg cholesterol

Prosciutto:

28 slices=300mg cholesterol

Corned Beef:

14 thin slices=300mg cholesterol

Butter:

1 1/5 sticks=300mg cholesterol

read at

http://www.healthline.com/health/high-cholesterol/daily-value