FDA Secure Supply Chain Pilot Program: 13 companies prequalified

In August 2013, the FDA initiated the so called Secure Supply Chain Pilot Program (SSCPP) to enhance the security of imported drugs. Now, the first companies have been listed. Read more.

In August 2013, the U.S. Food and Drug Administration (FDA) initiated the so called Secure Supply Chain Pilot Program (SSCPP) to enhance the security of imported drugs.

The goal was to enable qualified firms to expedite the importation of active pharmaceutical ingredients and finished drug products into the United States.

With this program, FDA wants to better focus its imports surveillance resources on preventing the entry of high-risk drugs that are the most likely to compromise the quality and safety of the U.S. drug supply.

The SSCPP is a voluntary program. Each firm accepted to participate in the program will be allowed to have up to five drugs subject to expedited import entry review. The SSCPP will be jointly administered by FDA’s Center for Drug Evaluation and Research (CDER) and Office of Regulatory Affairs (ORA).

Currently, the following companies have been accepted into the program:

• AbbVie Inc.
• Allergan, Inc.
• Astellas U.S. Technologies, Inc.
• Bristol-Myers Squibb Company
• Celgene Corporation
• GE Healthcare Inc.
• GlaxoSmithKline LLC
• Merck Sharp & Dohme Corporation
• Mylan Pharmaceuticals Inc.
• Novartis Pharmaceuticals Corporation
• Pfizer, Inc.
• Teva Pharmaceutcials USA, Inc.
• Watson Laboratories, Inc.

Source: FDA press release

Pimecrolimus

137071-32-0 ^cas 

(3S,4R,5S,8R,9E,12S,14S,15R,16S,18R,19R,26aS)- 3-{(E)-2-[(1R,3R,4S)-4-Chloro-3-methoxycyclohexyl]- 1-methylvinyl}-8-ethyl-5,6,8,11,12,13,14,15,16,17,

18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy- 14,16-dimethoxy-4,10,12, 18-tetramethyl-15,19-epoxy- 3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1, 7,20,21(4H,23H)-tetrone

The systematic name of pimecrolimus is (lR,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(lE)-2- {(1 R,3R,4S)-4-chloro-3-methoxycyclohexyl} - 1 -methylvinyl] – 17-ethyl- 1,14- dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-ll,28-dioxa-4-aza- tricyclo[22.3.1.0⁴'⁹]octacos-18-ene-2,3,10,16-tetraone.

Pimecrolimus is the 32 epichloro derivative of ascomycin.

Pimecrolimus is an immunomodulating agent used in the treatment of atopic dermatitis (eczema). It is currently available as a topical cream, once marketed by Novartis, (however Galderma will be promoting the molecule in Canada in early 2007) under the trade name Elidel.

NMR…http://file.selleckchem.com/downloads/nmr/S500401-Pimecrolimus-NMR-Selleck.pdf

HPLC…….http://file.selleckchem.com/downloads/hplc/S500401-Pimecrolimus-HPLC-Selleck.pdf

http://file.selleckchem.com/downloads/hplc/S500401-Pimecrolimus-HPLC-Selleck.pdf

Pimecrolimus is an immunomodulating agent used in the treatment of atopic dermatitis (eczema). It is available as a topical cream, once marketed by Novartis (however, Galderma has been promoting the compound in Canada since early 2007) under the trade name Elidel.

Pimecrolimus is an ascomycin macrolactam derivative. It has been shown in vitro that pimecrolimus binds to macrophilin-12(also referred to as FKBP-12) and inhibits calcineurin. Thus pimecrolimus inhibits T-cell activation by inhibiting the synthesis and release of cytokines from T-cells. Pimecrolimus also prevents the release of inflammatory cytokines and mediators from mast cells.

Pimecrolimus is a chemical that is used to treat atopic dermatitis (eczema). Atopic dermatitis is a skin condition characterized by redness, itching, scaling and inflammation of the skin. The cause of atopic dermatitis is not known; however, scientists believe that it may be due to activation of the immune system by various environmental or emotional triggers. Scientists do not know exactly how pimecrolimus reduces the manifestations of atopic dermatitis, but pimecrolimus reduces the action of T-cells and mast cells which are part of the immune system and contribute to responses of the immune system. Pimecrolimus prevents the activation of T-cells by blocking the effects of chemicals (cytokines) released by the body that stimulate T-cells. Pimecrolimus also reduces the ability of mast cells to release chemicals that promote inflammation.

Pimecrolimus, like tacrolimus, belongs to the ascomycin class of macrolactam immunosuppressives, acting by the inhibition of T-cell activation by the calcineurin pathway and inhibition of the release of numerous inflammatory cytokines, thereby preventing the cascade of immune and inflammatory signals.^[1] Pimecrolimus has a similar mode of action to that of tacrolimus but is more selective, with no effect on dendritic (Langerhans) cells.^[2] It has lower permeation through the skin than topical steroids or topical tacrolimus^[3] although they have not been compared with each other for their permeation ability through mucosa. In addition, in contrast with topical steroids, pimecrolimus does not produce skin atrophy.^[4] It has been proven to be effective in various inflammatory skin diseases, e.g., seborrheic dermatitis,^[5] cutaneous lupus erythematosus,^[6]oral lichen planus,^[7] vitiligo,^[8] and psoriasis.^[9][10] Tacrolimus and pimecrolimus are both calcineurin inhibitors and function as immunosuppressants.^[11]

Ascomycin macrolactams belong to a new group of immunosuppressive, immunomodulatory and anti-inflammatory agents and include, e.g., ascomycin (FK520), tacrolimus (FK506) and pimecrolimus (ASM 981). The main biological effect of ascomycin macrolactams appears to be the inhibition of the synthesis of both Th1 and Th2-type cytokines in target cells.

As used herein, the term “ascomycin macrolactam” means ascomycin, a derivative of ascomycin, such as, e.g., tacrolimus and pimecrolimus, or a prodrug or metabolite of ascomycin or a derivative thereof.

Ascomycin, also called immunomycin, is a structurally complex macrolide produced by Streptomyces hygroscopicus. Ascomycin acts by binding to immunophilins, especially macrophilin-12. It appears that ascomycin inhibits the production of Th1 (interferon- and IL-2) and Th2 (IL-4 and IL-10) cytokines. Additionally, ascomycin preferentially inhibits the activation of mast cells, an important cellular component of the atopic response. Ascomycin produces a more selective immunomodulatory effect in that it inhibits the elicitation phase of allergic contact dermatitis but does not impair the primary immune response when administered systemically. The chemical structure of ascomycin is depicted below.

Tacrolimus (FK506) is a synthetic derivatives of ascomycin. As a calcineurin inhibitor, it works through the FK-binding protein and inhibits the dephosphorylation of nuclear factor of activated T cells (NFAT), thereby preventing the transport of the cytoplasmic component of NFAT to the cell nucleus. This leads to transcriptional inhibition of proinflammatory cytokine genes such as, e.g., interleukin 2, which are dependent on the nuclear factor of activated NFAT. The chemical structure of tacrolimus is depicted below.

Pimecrolimus, an ascomycin derivative, is a calcineurin inhibitor that binds with high affinity to the cytosolic receptor macrophilin-12, inhibiting the calcium-dependent phosphatase calcineurin, an enzyme required for the dephosphorylation of the cytosolic form of the nuclear factor of the activated T cell (NF-AT). It thus targets T cell activation and proliferation by blocking the release of both TH1 and TH2 cytokines such as IF-g, IL-2, -4, -5, and -10.3 It also prevents the production of TNF-a and the release of proinflammatory mediators such as histamine, hexosaminidase, and tryptase from activated mast cells.3 It does not have general antiproliferative activity on keratinocytes, endothelial cells, and fibroblasts, and in contrast to corticosteroids, it does not affect the differentiation, maturation, functions, and viability of human dendritic cells. The chemical structure of pimecrolimus is depicted below.

Pimecrolimus is an anti-inflammatory compound derived from the macrolactam natural product ascomycin, produced by certain strains of Streptomyces.

In January 2006, the United States Food and Drug Administration (FDA) announced that Elidel packaging would be required to carry a black box warning regarding the potential increased risk of lymph node or skin cancer, as for the similar drug tacrolimus. Whereas current practice by UKdermatologists is not to consider this a significant real concern and they are increasingly recommending the use of such new drugs.^[12]

Importantly, although the FDA has approved updated black-box warning for tacrolimus and pimecrolimus, the recent report of the American Academy of Dermatology Association Task Force finds that there is no causal proof that topical immunomodulators cause lymphoma or nonmelanoma skin cancer, and systemic immunosuppression after short-term or intermittent long-term topical application seems an unlikely mechanism.^[13] Another recent review of evidence concluded that postmarketing surveillance shows no evidence for this systemic immunosuppression or increased risk for any malignancy.^[14] However, there are still some strong debates and controversies regarding the exact indications of immunomodulators and their duration of use in the absence of active controlled trials.^[15] Dermatologists’ and Allergists’ professional societies, the American Academy of Dermatology[1], and the American Academy of Allergy, Asthma, and Immunology, have protested the inclusion of the black box warning. The AAAAI states “None of the information provided for the cases of lymphoma associated with the use of topical pimecrolimus or tacrolimus in AD indicate or suggest a causal relationship.”[2].

ELIDEL® (pimecrolimus) Cream 1% contains the compound pimecrolimus, the immunosuppressant 33-epi-chloro-derivative of the macrolactam ascomycin.

Chemically, pimecrolimus is (1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-12-[(1E)-2{(1R,3R,4S)-4-chloro-3-methoxycyclohexyl}-1-methylvinyl]-17-ethyl-1,14-dihydroxy-23,25 dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0 4,9]octacos-18-ene2,3,10,16-tetraone.

The compound has the empirical formula C₄₃H₆₈CINO₁₁ and the molecular weight of 810.47. The structural formula is

Pimecrolimus is a white to off-white fine crystalline powder. It is soluble in methanol and ethanol and insoluble in water.

Each gram of ELIDEL Cream 1% contains 10 mg of pimecrolimus in a whitish cream base of benzyl alcohol, cetyl alcohol, citric acid, mono- and di-glycerides, oleyl alcohol, propylene glycol, sodium cetostearyl sulphate, sodium hydroxide, stearyl alcohol, triglycerides, and water.

The second representative of the immunosuppressive macrolides for topical application – after tacrolimus (Protopic ®) – has 21 October in the trade. Pimecrolimus is approved for short-term and intermittent long-term treatment for patients aged two years who suffer from mild to moderate atopic dermatitis.

Pimecrolimus is a lipophilic derivative of macrolactam Ascomycin. The macrolides inhibit the production and release of pro-inflammatory cytokines by blocking the phosphatase calcineurin.The anti-inflammatory effect unfolds the drug in the skin. Since he is only minimally absorbed to not measurable, it hardly affects the local or systemic immune response. Therefore, the authorization neither restricts nor a maximum daily dose treatable area or duration of therapy.The cream can also be applied on the face, head and neck, and in skin folds, but not simultaneously with other anti-inflammatory topical agents such as glucocorticoids.

In studies in phases II and III patients aged three months and treated a maximum of one year.In two six-week trials involving 186 infants and young children as well as 403 children and adolescents, the verum symptoms and itching decreased significantly better than the cream base. Already in the first week of itching in 44 percent of children and 70 percent of the infants improved significantly. In adults, pimecrolimus was less effective than 0.1 percent betamethasone 17-valerate.

In the long-term treatment the verum significantly reduced the incidence of flares, revealed two studies with 713 and 251 patients. About a half and one year each about twice as many of the small patients were free of acute disease exacerbations than with the cream base (example: 61 versus 34 per cent of children, 70 versus 33 percent of infants older than six months). Moreover, the use of topical corticosteroids decreased significantly.

In a study of 192 adults with moderate to severe eczema half suffered six months no relapses more (24 percent with placebo). In the long-term therapy pimecrolimus was less effective than 0.1 percent triamcinolone acetonide cream and 1 percent hydrocortisone cream in adults.

The new topicum is-apart from burning and irritation at the application site – relatively well tolerated. It is neither kontaktsensibilisierend still phototoxic or sensitizing and does not cause skin atrophy. As in atopic Ekzen but usually a long-term therapy is necessary studies can reveal long-term adverse effects of the immunosuppressant on the skin only beyond one year.Also available from direct comparative studies between tacrolimus and pimecrolimus. They could help to delineate the importance of the two immunosuppressants.

Pimecrolimus (registry number 137071-32-0; Figure 1) is a macro lide having anti-inflammatory, antiproliferative and immunosuppressive properties. This substance is present as an active ingredient in the Elidel ® drug recently approved in Europe and in the USA for topical treatment of inflammatory conditions of the skin such as atopic dermatitis.

Figure 1: structural formula of pimecrolimus

19^th Ed., vol. π, pg. 1627, spray-drying consists of bringing together a highly dispersed liquid and a sufficient volume of hot air to produce evaporation and drying of the liquid droplets. Spray-drying however is often limited to aqueous solutions unless special expensive safety measures are taken. Also, in spite of the short contact time, certain undesirable physical and chemical characteristics of the emerging solids are in particular cases unavoidable. The turbulence present in a spray-drier as a result of the moving air may alter the product in an undesirable manner. Modifications to the spray-drying technique are disclosed in WO 03/063821 and WO 03/063822. [00012] European Patent EP 427 680 Bl discloses a method of synthesizing amorphous pimecrolimus (Example 66a). The method yields amorphous pimecrolimus as a colorless foamy resin.

U.S. Patent No. US 6,423,722 discloses crystalline forms of pimecrolimus, such as form A, form B, etc. US 722 also contend that by performing example 66a from the European Patent EP 427 680 Bl, amorphous pimecrolimus is obtained.

The preparation of pimecrolimus was described for the first time in the patent application EP427680 on behalf of Sandoz. Used as raw material in such document is ascomycin (compound identified by registry number 11011-38-4), a natural product obtained through fermentation from Streptomyces strains (such as for example Streptomyces hygroscopicus var ascomyceticus, or Streptomyces hygroscopicus tsukubaensis N°9993). Pimecrolimus is obtained from the ascomycin through a sequence of four steps of synthesis (scheme 1)

Scheme 1 : synthesis process described in EP427680

From a structural point of view, pimecrolimus is the 33-epi-chloro derivative of ascomycin. As described in EP427680, the simultaneous presence – in the structure of ascomycin – of two secondary hydroxyl groups in position 24 and in position 33, requires the protection of the hydroxyl in position 24 before substituting the second hydroxyl in position 33 with an atom of chlorine.

In order to obtain the monoprotection of the hydroxyl in position 24 of ascomycin, such synthesis process provides for the preparation of 24,33-disilyl derivative and the subsequent selective removal of the silyl ester in position 33.

The high ratio between the silylating agent and the substrate and the non-complete selectivity of the subsequent step of deprotection requires carrying out two chromatographic purifications on the column of silica gel (Baumann K., Bacher M., Damont A., Hogenauer K., Steck A. Tetrahedron, (2003), 59, 1075-1087). The general yields of such synthesis process are not indicated in literature; an experiment by the applicant revealed that such yields amount to about 16% molar starting from ascomycin.

Other synthesis processes were recently proposed as alternatives to the synthesis of EP427680.

In particular, the International patent application WO2006040111 on behalf of Novartis provides for the direct substitution of the hydroxyl in position 33 of ascomycin with an atom of chlorine and a second alternative, described in the international patent application WO2006060614 on behalf of Teva, uses – as a synthetic intermediate – a sulfonate derivative in position 33 of ascomycin. Both the proposed synthetic alternatives are not entirely satisfactory in that in WO2006040111 the proposed halogenating agents (chlorophosphorane and N- chlorosuccinimide) are not capable, according to the same authors, of regioselectively substituting the hydroxyl function in position 33, while in WO2006060614 the quality characteristics of the obtained product are, even after chromatographic purification and/or crystallisation, low for a product to be used for pharmaceutical purposes (i.e. purity of 96% as described in the experimental part).

Generally, purified enzymatic systems may be used for the organic synthesis of polyfunctional molecules (Wang Y-F, Wong C-H. J Org Chem (1988) 53, 3127- 3129; Santaniello E., Ferraboschi P., Grisenti P., Manzocchi A. Chem. Rev. (1992), 92(5), 1071-140; Ferraboschi P., Casati S., De Grandi S., Grisenti P., Santaniello E. Biocatalysis (1994), 10(1-4), 279-88); WO2006024582). WO2007103348 and WO2005105811 describe the acylation of rapamycin in position 42 in the presence of lipase from Candida antartica.

…………………….

EP2432791A1

Scheme 2: synthesis of pimecrolimus for enzymatic transesterification of ascomycin.

Scheme 3. Synthesis of pimecrolimus for enzyme-catalyzed alcoholysis from 33,24- diacetate of ascomycin

Example 1

Preparation of the 33-acetyl derivative of ascomvcin (compound I of scheme II)

Lipase from Candida antarctica (CAL B, Novozym 435) [0.140 g (2 U/mg)

FLUKA] was added to a solution of ascomycin (100 mg; 0.126 mmol) in toluene (8 ml) and vinyl acetate (4.5 eq; 0.473 g). The reaction is kept under stirring at the temperature of 30° C for 80 hrs then the enzyme is taken away for filtration and the filtrate is concentrated at low pressure to obtain 105 mg of 33-acetyl ascomycin.

A sample of such intermediate was purified for analytical purposes by chromatography on silica gel (n-hexane/acetone = 8/2 v/v as eluents) and thus crystallised by acetone/water.

The following analysis were carried out on such sample: ¹H-NMR (500MHz) δ:

2.10 (CH₃CO), 3.92 and 4.70 (24CH and 33CH); IR (cm-¹): 3484.245, 2935.287,

1735.331, 1649.741, 1450.039,

1372.278; DSC: endotherm at 134.25° C; [α]_D=-74,0° (c=0.5 CHCl₃).

Spectrum of MS (ESI +): m/z: 856.4 (M+23; 100.0%)

Elementary analysis calculated for C₄₅H₇iNO₁₃: C 64.80%; H, 8.58%; N, 1.68%;

O, 24.94%

Elementary analysis found: C 64.78%; H, 8.54%; N, 1.59%; O, 24.89%

Preparation of the 24-tgrt-butyldimethylsilylether-33 -acetyl derivative of ascomvcin (intermediate 24-silyl-33-Oac; compound II of scheme 2)

2,6-lutidine (0.29Og; 2.7 mmolels) and tert-butyldimethylsilyl triflate (0.238g; 0.9 mmoles) are added to a solution of 33-acetyl derivative of ascomycin (150 mg;

0.18 mmoles) in dichloromethane (5ml). The reaction is left under stirring at ambient temperature for 30 minutes. After this period the reaction mixture is washed with a solution saturated with sodium bicarbonate (5 ml) and organic phase obtained is washed in sequence with HCl 0.1N (5 ml 3 times) and with a solution at 30% of NaCl (5ml). The organic phase is anhydrified on sodium sulphate, filtered and concentrated to residue under vacuum to obtain 128 mg of product.

Spectrum of MS (ESI +): m/z: 970.5 (M+23; 100.0%)

¹H-NMR (500 MHz) δ: 0.05 and 0.06 ((CHs)₂Si), 0.90 ((CH₃)₃C-Si), 2.10

(CH₃CO), 4.70 (33CH)

IR (cm-’): 3462.948, 2934.450, 1739.236, 1649.937

Elementary analysis calculated for C₅₁H₈₅NOi₃Si: C 64.59%; H, 9.03%; N, 1.48%; O, 21.93%

Elementary analysis found: C 64.50%; H, 9.05%; N, 1.41%; O, 21.88%

DSC= endoderma a 236,43° C. [α]_D=-81,4° (c=0.5 CHCl₃).

Preparation of 24-tert-butyldimethylsilylether of ascomycin (intermediate 24- silyl-33-OH; compound III of scheme 2) n-octan-1-ol (0.035g; 0.265 mmoles) and CAL B (Novozym 435) [0.100 g (2

U/mg) FLUKA] are added to a solution of 24-tert-butyldimethylsilylether-33- acetyl derivative of ascomycin (50 mg; 0.053 mmoles) in tert-butylmethylether (4 ml). The reaction is kept under stirring at the temperature of 40° C for 120 hours.

After this period the reaction mixture is filtered and the filtrate is evaporated to residue under vacuum to obtain a reaction raw product which is purified by chromatography on silica gel: 44 mg of product (0.048 mmoles) are recovered through elution with petroleum ether/acetone 7/3.

The chemical/physical properties of the obtained product match those of a reference sample obtained according to patent EP427680.

Preparation of 24-tert-butyldimethylsilylether-33-epi-chloro ascomycin

(intermediate 24-silyl-33-chloro; compound IV of scheme 2)

A solution of 24-silyl FR520, i.e. 24-silyl ascomycin (165 g; 0.18 moles) in anhydrous toluene (1.4 litres) and pyridine (50 ml) is added to a suspension of dichlorotriphenylphosphorane (99.95g) in anhydrous toluene (1.1 litres), under stirring at ambient temperature (20-25 °C) in inert atmosphere.

After adding, the reaction mixture is heated at the temperature of 60° C for 1 hour.

After this period the temperature of the reaction mixture is taken to 25° C and thus the organic phase is washed in sequence with water (1 time with 1 L) and with an aqueous solution of NaCl at 10% (4 times with 1 L each time), then it is anhydrified on sodium sulphate, filtered and concentrated under vacuum to obtain about 250 g of a moist solid of toluene. Such residue product is retaken with n- hexane (500 ml) and then evaporated to dryness (in order to remove the toluene present). The residue product is diluted in n-hexane (500 ml) under stirring at ambient temperature for about 45 minutes and then the undissolved solid taken away for filtration on buckner (it is the sub-product of dichlorophosphorane).

The filtrate is concentrated at low pressure to obtain 148.6 g of a solid which is subsequently purified by chromatography on silica gel (elution with n- heptane/acetone = 9/1) to obtain 123 g (0.13 moles) of product.

The chemical/physical properties of the obtained product match those described in literature (EP427680).

Preparation of the pimecrolimus from 24-fert-butyldimethylsilylether-33-epi- chloro ascomycin

The intermediate 24-silyl-33 chloro (123g; 0.13 Moles; compound IV of scheme

2) is dissolved under stirring at ambient temperature in a dichloromethane/methanol mixture=l/l=v/v (1.1 litres) then p-toluenesulfonic acid monohydrate (10.11 g) is added.

The reaction is kept under stirring at the temperature of 20-25° C for 72 hours, thus a solution of water (600 ml) and sodium bicarbonate (4.46 g) is added to the reaction mixture. The reaction mixture is kept under stirring at ambient temperature for 10 minutes, the organic phase is then prepared and washed with an aqueous solution at 10% of sodium chloride (600 ml).

The organic phase is anhydrified on sodium sulphate, filtered and concentrated under vacuum to obtain 119 g of raw pimecrolimus. Such raw product is purified by chromatography on silica gel (n-hexane/acetone as eluents) and thus crystallised by ethyl acetate, cyclohexane/water to obtain 66 g (81.5 mmoles) of purified pimecrolimus.

The chemical/physical data obtained matches the data indicated in literature.

Example 2

Preparation of ascomvcin 24.33-diacetate (intermediate 24, 33-diacetate; compound V of scheme 3)

DMAP (4.5 eq; 0.136 g) and acetic anhydride (4.5 eq; 0.114 g) are added to a solution of ascomycin (200 mg; 0.25 mmoles) in pyridine (2.5 ml), under stirring at the temperature of 0° C.

The reaction is kept under stirring for 1.5 hours at the temperature of 0° C then it is diluted with water and it is extracted with ethyl acetate (3 times with 5 ml). The organic extracts are washed with HCl 0.5 N (5 times with 10 ml), anhydrified on

Na₂SO₄ concentrated under vacuum.

The residue product was purified by chromatography on silica gel (n- hexane/acetone 8/2 v/v as eluent) to obtain ascomycin 24,32-diacetate (210 mg;

0.24 mmoles).

We carried out the following analysis on such purified sample:

¹H-NMR (500 MHz) δ: 2.02 and 2.06 (2 CH₃CO), 5.20 and 4.70 (24CH and

33CH);

IR (Cm-¹): 3462.749, 2935.824, 1734.403, 1650.739, 1449.091, 1371.079.

DSC: endothermic peak at 234.10° C ; [α]_D=- 100.0° (C=0.5 CHCl₃).

Spectrum of MS (ESI+): m/z: 898.4 (100.0%; m+23)_.

Elementary analysis calculated for C₄₇H₇₃NO₁₄: C 64.44%; H 8.40%; N 1.60%; O

25.57%

Elementary analysis found: C 64.55%; H 8.44%; N 1.61%; O 25.40%

Preparation of the 24-acetyl ascomycin (intermediate 24-acetate-33-OH; compound VI of scheme 3)

Lipase from Candida antartica (CAL B Novozym 435) [1.1 g (2 U/mg) FLUKA] is added to a solution of ascomycin 33,24-diacetate (500 mg; 0.57 mmol) in

TBDME (25 ml) and n-octan-1-ol (4.5 eq; 0.371 g). The reaction is kept under stirring at 30° C for 100 hours, then the enzyme is taken away for filtration and the obtained filtrate is concentrated under low pressure to obtain 425 mg (0.51 mmoles) of product.

A sample was purified for analytical purposes by chromatography on silica gel (n- hexane/acetone = 7:3 v/v as eluents) and thus crystallised by acetone/water.

We carried out the following analysis on such purified sample: ¹H-NMR

(500MHz) δ: 2.05 (CH3CO); IR (an ¹): 3491.528, 2935.860, 1744.728, 1710.227,

1652.310, 1448.662, 1371.335. DSC: endothermic peak at 134.68° C; [α]_D=-

102.7° (c=0.5 CHCl₃)

Spectrum of MS (ESI +): m/z: 856.4 (M+23; 100.0%)

Elementary analysis calculated for C₄₅H₇₁NO₁₃: C 64.80%; H, 8.58%; N, 1.68%;

0, 24.94%

Elementary analysis found: C 64.71%; H, 8.49%; N, 1.60%; O, 24.97%

Preparation of the 24-acetyl-33epi-chloro ascomycin (intermediate 24-Acetate-33- chloro; compound VII of scheme 3) Supported triphenylphosphine (0.335 g; 1.1 mmoles) is added to a solution of 24- acetyl ascomycin (400 mg; 0.48 mmoles) in carbon tetrachloride (5 ml). The reaction mixture is kept under reflux for 3 hours then it is cooled at ambient temperature. The obtained suspension is filtered and the filtrate is concentrated to residue under vacuum to obtain 0.45g of reaction raw product which is purified by chromatography on silica gel: 163mg (0.19 mmoles) of product are obtained by elution with petroleum ether/acetone = 90/10.

¹H-NMR δ: 2.08 (CH₃CO); 4.60 (33CH); IR (Cm^“1)= 3464.941, 2934.360,

1738.993, 1650.366, 1450.424, 1371.557; DSC: endothermic peak at 231.67° C

[α]_D=-75.2° (c=0.5 CHCl₃)

Spectrum of MS (ESI +): m/z: 874.3 (M+23; 100.0%)

Elementary analysis calculated for C₄₅H₇₀ClNO₁₂: C 63.40%; H, 8.28%; Cl,

4.16%; N, 1.64%; O, 22.52%

Elementary analysis found: C 63.31%; H, 8.30%; Cl, 4.05%; N, 1.58%; O,

22.42%.

Preparation of pimecrolimus from 24-acetyl-33-epi-chloro ascomycin

A solution of 24-acetyl-33-epi-chloro ascomycin (200 mg; 0.23 mmoles; compound VII) in methanol (2 ml) and HCl 3N (1 ml) is stirred at ambient temperature for 40 hours. After this period, the reaction is neutralised with an aqueous bicarbonate solution, the methanol evaporated under vacuum. The mixture is extracted with dichloromethane (3 times with 5 ml), anhydrified on sodium sulphate, filtered and concentrated to residue to obtain a residue product which is purified by chromatography on silica gel (n-hexane/acetone as eluents) and thus crystallised by ethyl acetate, cyclohexane/water to obtain 78 mg of purified pimecrolimus (0.096 mmoles).

The chemical/physical characteristics of the obtained product matches the data indicated in literature for pimecrolimus.

Example 4 (comparative^*)

Verification of the method of synthesis of pimecrolimus described in EP427680 Imidazole (508 mg) and tert-Butyldimethylsilylchloride (1.125 g) are added in portions to a solution of 2g (2.53 mmoles) of ascomycin in anhydrous N,N- dimethylformamide (40 ml). The reaction mixture is kept under stirring at ambient temperature for 4.5 days. The reaction is thus processed diluting it with ethyl acetate (200 ml) and processing it using water (5 x 100 ml). The organic phase is separated, anhydrified on sodium sulphate, filtered and evaporated to residue under vacuum to obtain a foamy raw product which is subsequently purified by chromatography on silica gel (1:30 p/p): 2.1 g (2.05 mmoles; yields 81% molars) of ascomycin 24,33 disilyl intermediate are obtained by elution with n- hexane/ethyl acetate 3/1. The chemical/physical data of such intermediate matches that indicated in EP427680.

2.1 g (2.05 mmoles) of ascomycin 24,33 disilyl intermediate are dissolved in a solution under stirring at the temperature of 0°C composed of acetonitrile (42 ml) and aqueous HF 40% (23.1 ml). The reaction mixture is kept under stirring at the temperature of 0°C for 2 hours then it is diluted with dichloromethane (30 ml). Then the reaction is washed in sequence with a saturated aqueous solution using sodium bicarbonate (30 ml) and water (30 ml). The separated organic phase is anhydrified on sodium sulphate, filtered and evaporated to residue under vacuum to obtain a foamy residue which is subsequently purified by chromatography on silica gel (1:30 p/p): 839 mg (0.92 mmoles; yields 45% molars) of ascomycin 24 monosilyl intermediate are obtained by elution with dichloromethane/methanol 9/1. The chemical/physical data of such intermediate matches that obtained on the compound III scheme 2 and matches the data of literature indicated in EP427680. A mixture of 839 mg (0.92 mmoles; yields 45% molars) of ascomycin 24 monosilyl intermediate, triphenylphosphine (337 mg) in carbon tetrachloride (36.4 ml) is heated under stirring under reflux for 15 hours. After this period the reaction mixture is evaporated to residue under vacuum to obtain a solid product purified by chromatography on silica gel (1:30 p/p): 535 mg (0.57 mmoles; yields 63% molars) of ascomycin 24 monosilyl intermediate, 33-chloro derivative are obtained by elution with n-hexane/ethyl acetate 2/1. The chemical/physical data of such intermediate matches those we obtained on compound IV scheme 2 and matches the data of literature indicated in EP427680.

535 mg (0.57 mmoles) of ascomycin 24 monosilyl intermediate, 33-chloro derivative are dissolved under stirring at ambient temperature in acetonitrile (16.4 ml) and aqueous HF 40% (0.44 ml). The reaction mixture is kept under stirring at ambient temperature for 45′ and then it is diluted with ethyl acetate (100 ml). The organic phase is thus washed in sequence with an aqueous solution of sodium bicarbonate (70 ml) with water (2 x 70 ml) and thus it is anhydrified on sodium sulphate, filtered and evaporated under vacuum to obtain a solid which is subsequently purified by chromatography on silica gel (1 :30 p/p): 323 mg (0.399 mmoles; yields 70% molars) of pimecrolimus is obtained by elution with n- hexane/ethyl acetate 2/3. The chemical/physical characteristics of the obtained product matches the data indicated in literature regarding pimecrolimus; the overall yield of the process is 16%.

………………………..

POLYMORPHS…….WO2006060615A1

Example 7: Preparation of amorphous pimecrolimus by precipitation [00094] 19,5 g purified pimecrolimus (colorless resin) was dissolved in 217 ml acetone at 4O⁰C and concentrated. Residue: 38,76 g. The residue was diluted with 6 ml distilled water with stirring. Finally 1 ml acetone was added. This solution was added slowly to 2 L chilled distilled water that was stirred efficiently. After the addition had been completed, the suspension was stirred 20 min at O⁰C. Then the solid was filtered and dried at 45⁰C in vacuum oven overnight. Product: 15,65 g yellowish solid. Amorphous (XRD, DSC).

Example 8: Preparation of amorphous pimecrolimus by grinding

[00095] Procedure of grinding: 200 mg of Pimecrolimus sample was ground gently in an agate mortar using a pestle for half a minute. ,

References

• Elidel official homepage
• FDA News
• NPS RADAR
• Article about American Academy of Dermatology speaking out against black box warning
• Report of the Calcineurin Task Force of the ACAAI and AAAAI

Umirolimus, Biolimus

Biosensors (Originator)

40 -O-[(2′-ethoxy) ethyl]rapamycin

(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-12-{(2R)-1-[(1S,3R,4R)-4-(2-Ethoxyethoxy)-3-methoxycyclohexyl]-2-propanyl}-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36 -dioxa-4-azatricyclo[30.3.1.04,9]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentone

Umirolimus inhibits T cell and smooth muscle cell proliferation, and was designed for use in drug eluting stents. This analog has a chemical modification at position 40 of the rapamycin ring. It has potent immunosuppressive properties that are similar to those of sirolimus, but the drug is more rapidly absorbed by the vessel wall, readily attaches and enters smooth muscle cell membranes causing cell cycle arrest at G0, and is comparable to sirolimus in terms of potency.

The key biologic event associated with the restenotic process is clearly the proliferation of smooth muscle cells in response to the expansion of a foreign body against the vessel wall. This proliferative response is initiated by the early expression of growth factors such as PDGF isoforms, bFGF, thrombin, which bind to cellular receptors.

However, the key to understanding the mechanism by which compounds like umirolimus inhibit cell proliferation is based on events which occur downstream of this growth factor binding. The signal transduction events which culminate in cell cycle arrest in the G1 phase are initiated as a result of ligand binding to an immunophilin known as FK binding protein-12. The FK designation was based on early studies conducted with tacrolimus, formerly known as FK-506, which binds this cytoplasmic protein with high affinity.

Subsequent investigations showed that rapamycin also binds to this intracellular target, forming an FKBP12–rapamycin complex which is not in itself inhibitory, but does have the capacity to block an integral protein kinase known as target of rapamycin (TOR). TOR was first discovered in yeast J.N. Heitman, N.R. Movva and M.N. Hall, Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast, Science 253 (1991), pp. 905–909. View Record in Scopus | Cited By in Scopus (434)and later identified in eukaryotic cells, where it was designated as mTOR, the mammalian target of rapamycin. The importance of mTOR is based on its ability to phosphorylate a number of key proteins, including those associated with protein synthesis (p70s6kinase) and initiation of translation (4E-BP1).

Of particular significance is the role that mTOR plays in the regulation of p27kip1, an inhibitor of cyclin-dependent kinases such as cdk2. The binding of agents like rapamycin and umirolimus to mTOR is thought to block mTOR’s crucial role in these cellular events, resulting in arrest of the cell cycle, and ultimately, cell proliferation.

Introduction

It is known that Biolimus A9, a rapamycin derivative, is an immunosuppressant, and is also proven to have anti-tumor and anti-fungal effect.

Several prior arts had disclosed the improvements of the product yield of rapamycin derivatives. U.S. Pat. No. 7,193,078 to Isozaki et al. disclosed a process for producing Biolimus A9, giving an example to obtain a yield of 46% by reacting rapamycin with 2-ethoxyethyl trifluoromethane sulfonate (or 2-ethoxyethyl triflate) in an organic solvent.

However, the Isozaki’s prior art still has the following drawbacks:

• 1. Even one example ever showed a 46% yield of Biolimus A9, it however just revealed a small-scale laboratory experiment with only one gram (1.09 mmol) of rapamycin and 1.95g (8.78 mmol) of 2-ethoxyethyl triflate. After amplifying or expanding the process to be larger scale, the yield will be remarkably reduced to thereby decrease the commercial or industrial value of this prior art (Note: The low yield after simulated process amplification will be hereinafter discussed in Examples 3, 4 of this application).
• 2. Even the reactant of 2-ethoxyethyl triflate is a compound with high activity, it is unstable and will be decomposed such as after being stored for one week at room temperature. Also, the triflate is not UV-absorbable and is therefore unsuitable for process tracking when proceeding the reaction. Such poor properties will affect the material storage, production scheduling and process tracking for commercially making the Biolimus A9.

sirolimus 42 – ether derivatives are a class of sirolimus derivative, is a new generation macrolide immunosuppressant and anticancer drugs. The compounds discovered by the Swiss company Sandoz, mainly applicable to organ transplant recipient’s immune suppression and cancer. The synthesis of such substances currently on the patent literature have W09409010, CN102127092A and CN102268015A.

Patent Document W09409010 on Synthesis of this type of structure are used sirolimus protected materials in acidic or neutral reaction conditions, and then removing the protective group to obtain the target product. Such as 42-0 – (4 – hydroxymethyl) benzyl – sirolimus, the first synthesis of the formula V, and then removal of silyl ether protecting groups have the formula VI.

This synthetic method has several drawbacks: 1, the reaction reagent relatively difficult to obtain; 2, the intermediate prepared in the reaction yield is low;

3, sirolimus, structural part to participate in a two-step reaction, reduction reaction yield, costs.

CN102127092A mention a synthetic sirolimus 42 – ether derivative everolimus one way. This synthetic route similar to the W09409010 (route of reaction formula 1), but with silane reagents and reaction conditions are different.First reaction toluene as solvent, 50 ° C _60 ° C between the reaction and after-treatment of the intermediate after the first column chromatography, yield 32%.The second step in tetrahydrofuran as a solvent, the reaction overnight at 0 ° C, after treatment by a column chromatography to give the product, the yield was 66%, with a total yield of 21.1%.

Reaction Scheme I:

This method has the defects include: 1, the reaction reagent relatively difficult to obtain. 2, the structure part of sirolimus to attend two-step reaction, reduction reaction yield, costs. 3, the use of highly toxic solvents, are not suitable for practical application. 4, the reaction temperature is relatively harsh, difficult to control.

CN102268015A discloses a method for synthesizing everolimus. The first step to sirolimus or sirolimus derivatives as raw materials in -20 ° C was added dropwise trifluoromethanesulfonic anhydride and incubated for 3 hours, was isolated intermediates 02, the yield was 87.4% or 95.32%. The second step of the intermediate 02 with ethylene glycol mono-protected in 50 ° C reaction intermediate 03 was isolated in a yield of 79.0% or 76.78%. The third step of dilute hydrochloric acid was added dropwise at room temperature intermediate 03 was deprotected product everolimus. The total yield was 48.4% or 52.5%. See Reaction Scheme 2 synthetic route.

Reaction Scheme 2:

The method of the defects as follows: 1, to protection and deprotection of ethylene glycol. 2, the reaction steps excessive structural part to participate in the sirolimus-step reaction. 3, the reaction yield improved, but also greatly increased operating costs.

………………..

.

CN102731527A

Examples 5,42-0-(2_ ethoxy) ethyl – Synthesis of sirolimus

[0044] In the IOOml three-necked flask was added 2g of sirolimus, 2. 78g of 4_ dimethylaminopyridine, 5. 34g of chlorine acid glycol ester and 20ml of acetonitrile, 35 ° C The reaction was stirred 36 hours ended. The reaction solution was poured into an equal volume of saturated sodium bicarbonate solution and extracted with 5% potassium bisulfate solution was washed twice with a saturated sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and concentrated through the column. Silica gel column chromatography (EA: PE = I :20-2: 1), obtained by rotary evaporation 42-0 – (2 – ethoxy) ethyl – sirolimus I. 57g (yield: 73.3 %). HPLC analysis showed that: a purity of 88.2%.

…………

US7872122

A process for making Biolimus A9 represented by formula (1),

comprising reacting sirolimus of formula (2),

with

2-ethoxyethyl pentafluorobenzene sulfonate

under catalyzing by an organic base,

and in the presence of an organic solvent,

to undergo a nucleophilic substitution reaction to obtain Biolimus A9 of formula (1).

the Biolimus A9 of the present invention will be presented as below-mentioned:
Biolimus A9

Reaction Parameters

Quantity of Alkylbenzene Sulfonate: 1˜20 equivalents, preferably being 5˜10 equivalents, per equivalent of sirolimus.

Reaction Temperature: 40˜80° C., preferably being 55˜65° C. Reaction Time: 12˜72 hours, preferably being 16˜30 hours.

After the reaction is completed, the rough product is collected, washed, dried and purified to obtain the Biolimus A9 of the present invention with high yield of 45%.

Since the product Biolimus A9 is a polyene macrolide, which is easily oxidized and decomposed during the storage or material handling.

Accordingly, a proper antioxidant may be homogeneously mixed with the Biolimus A9 to enhance the stability when stored or handled.

The proper antioxidants may be selected from: Butylatd hydroxytoluene (BHT), DL-α-tocopherol, propyl gallate, ascorbyl palmitate, 3-tert-butyl-4-hydroxyanisole, 2-tert-butyl-4-hydroxyanisole, and fumaric acid.

The Butylated hydroxytoluene (BHT) is the most preferable antioxidant adapted for use in the present invention.

The process for making Biolimus A9 in accordance with the present invention will be described in detail in view of the following examples:

EXAMPLE 1

A. Synthesis of 2-ethoxyethyl pentafluorobenzene sulfonate

In a reaction flask, 25 grams (93.8 mmol) of pentafluorobenzene sulfonyl chloride (or pentafluorobenzene sulfochloride) and 86 ml of tetrahydrofuran were added and nitrogen gas was filled into the flask.

The flask is then cooled to 0° C. and is dripped therein with 2-ethoxyethanol (8.5g, 94.5 mmol) and triethyl amine (15 g, 148.5 mmol). After dripping, the reaction solution is stirred for 30 minutes, and then filtered, concentrated and the residue is separated from the solution and further purified by silica gel column chromatography to obtain a colorless oily product of 2-ethoxyethyl pentafluorobenzene sulfonate (26.6 g, 83.1 mmol) having a yield of 88.6%.

B. Synthesis of Biolimus A9

In a reaction flask, 1 g (1.1 mmol) of sirolimus, 7.8 g (60.3 mmol) of ethyl di-isopropyl amine, 3.5 ml of methylene chloride and 2.8 g (8.7 mmol) of 2-ethoxyethyl pentrafluorobenzene sulfonate as previously obtained were added therein.

The reaction mixture in the flask was heated to 60° C. and agitated for 23 hours. It is cooled, and further added therein with ethyl acetate (100 ml) and aqueous solution of hydrochloric acid (1N, 100 ml ) under agitation.

Then, it is settled for separating the organic and aqueous layers. The organic layer is collected, and washed with pure water (100 ml) and saturated saline (100 ml). The washed organic liquid is then dried and concentrated. The residue is then separated from the liquid and further purified by silica gel column chromatography to obtain white solid product of Biolimus A9(0.49 g, 0.5 mmol) with a yield of 45.4%.

EXAMPLE 2 Process Amplification of Example 1B

In a reaction flask, 10 g (10.9 mmol) of sirolimus, 78 g (603.5 mmol) of ethyl di-isopropyl amine, 35 ml of methylene chloride and 28 g (87.4 mmol) of 2-ethoxyethyl pentrafluorobenzene sulfonate were added therein.

The reaction mixture in the flask was heated to 60° C. and agitated for 24 fours. It is cooled, and further added therein with ethyl acetate (500 ml) and aqueous solution of hydrochloric acid (1N, 500 ml ) under agitation.

Then, it is settled for separating the organic and aqueous layers. The organic layer is collected, and washed with pure water (500 ml) and saturated saline (400 ml). The washed organic liquid is then dried and concentrated. The residue is then separated from the liquid and further purified by silica gel column chromatography to obtain white solid product of Biolimus A9(4.8 g, 4.9 mmol) with a yield of 44.5%.

This example is a process amplification of the previous Example 1, Step B, by amplifying or expanding the quantity of each reactant for about 10 times of that of the Example 1 (of small scale).

By the way, the production yield (44.5%) of this Example is still as high as that of the previous Example 1 of small scale. It indicates that the reproducibility of high yield can still be obtained in accordance with the present invention even after process amplification, proving that the present invention is suitable for commercialization or mass production. The product may be further purified to obtain a high-purity final product of Biolimus A9 such as by middle-performance liquid chromatography or the like. The Biolimus A9 thus obtained is identified by the X-ray powder diffractogramm as shown in the single drawing FIGURE as attached herewith.

EXAMPLE 3 Comparative Example for Simulating the Process of the Prior Art of U.S. Pat. No. 7,193,078

A. Synthesis of 2-ethoxyethyl trifluoromethane sulfonate

In a reaction flask, 2-ethoxyethanol (10 g, 111 mmol), methylene chloride (177 ml) and 2,6-dimethyl pyridine (23.8 g, 222.3 mmol) were added into the flask, which is filled therein with nitrogen gas. It is cooled to 0° C. and added dropwise with trifluoromethane sulfonic acid anhydride (37.6 g, 133.4 mmol). After completing the dripping of said sulfonic acid anhydride, the reaction mixture is agitated for one hour and a saturated aqueous solution of ammonium chloride (20 ml) is added and further agitated for 10 minutes.

It is then settled for separating the layers. The organic layer is collected, and is respectively washed with aqueous solution of hydrochloric acid (1N, 100 ml), pure water (100 ml), saturated aqueous solution of sodium bicarbonate (100 ml) and saturated saline (100 ml). The washed organic layer is dried, concentrated and the residue is then separated and further purified with silica gel column chromatography to obtain the oily product of 22.5 g (101.3 mmol) of 2-ethoxyethyl trifluoromethane sulfonate (or 2-ethoxyethyl triflate), with a yield of 91.3%.

B. Synthesis of Biolimus A9

In a reaction flask, sirolimus (1 g, 1.1 mmol), ethyl di-isopropyl amine (7.8 g, 60.3 mmol), methylene chloride (3.5 ml) and 2-ethoxyethyl triflate (2.0 g, 8.8 mmol) as previously made in Example 3A were added into the flask, which is filled with nitrogen gas. The reaction mixture is heated to 60° C. and is agitated for one hour and twenty minutes. Then, it is cooled, added with ethyl acetate (100 ml) and aqueous solution of hydrochloric acid (1N, 100 ml) and is further agitated. After agitation, it is settled for separating the layers. The organic layer is collected and respectively washed with pure water (100 ml), saturated saline (80 ml). The washed organic layer is dried and concentrated. The residue is then separated and purified by silica gel column chromatography to obtain white product of Biolimus A9 (0.48 g, 0.49 mmol), with a yield of 44.5%.

EXAMPLE 4 Comparative Example for Simulative Process Amplification of Example 3B

In a reaction flask, sirolimus (10 g, 10.9 mmol), ethyl di-isopropyl amine (78 g, 603.5 mmol), methylene chloride (35 ml) and 2-ethoxyethyl triflate (20 g, 88 mmol), each having a quantity about 10 times of that used in Example 3B, were added into the flask, which is filled with nitrogen gas. The reaction mixture is heated to 60° C. and is agitated for one hour and twenty minutes. Then, it is cooled, added with ethyl acetate (500 ml) and aqueous solution of hydrochloric acid (1N, 500 ml) and is further agitated. After agitation, it is settled for separating the layers. The organic layer is collected and respectively washed with pure water (500 ml), saturated saline (400 ml). The washed organic layer is dried and concentrated. The residue is then separated and purified by silica gel column chromatography to obtain white product of Biolimus A9 (2.9 g, 2.9 mmol), having a yield of 26.8% only.

Comparatively, via this process amplification, the yield of Biolimus A9 of the prior art is remarkably reduced in comparison with its small-scale production (Example 3B). Therefore, the prior art of U.S. Pat. No. 7,193,078 may be considered as a process especially suitable for small-scale production, such as a laboratory experiment, rather than a large-scale commercial or industrial production, which is thus inferior to this application, when compared with this application which has shown the high yields both in small-scale process (Example 1) and large-scale process (Example 2).

Accordingly, this application is more suitable for commercialization for mass production.

Moreover, the essential reactant of 2-ethoxyethyl triflate of the prior art (U.S. Pat. No. 7,193,078), even having high activity, is unstable because it will be decomposed into unknown compounds after one-week storage (by NMR spectrographic detection) as accompanied with physical change from its original colorless transparent liquid to a black viscous oily product, to thereby be inferior to this application because the 2-ethoxyethyl pentafluoro benzene sulfonate (which is obviously different from the 2-ethoxyethyl triflate as used in the prior art) of this application is still stable after one-week storage as aforementioned.

Furthermore, the 2-ethoxyethyl pentafluorobenzene sulfonate of this application may absorb ultra-violet rays to have a better tractability during the process proceeding than that of the 2-ethoxyethyl triflate (which is not UV-absorbable) of the prior art. So, this application is also beneficial for better production scheduling, reliable process tracking and efficient production management than the prior art.

So, this application is more suitable for commercial production even when considering the stability of product storage and improvement of process monitoring, control and management.

EXAMPLE 5

The Biolimus A9, as obtained from Example 2, is respectively added with anti-oxidant, namely Butylated Hydroxytoluene (or BHT), for 0.1%, 0.2, 0.5%, and 1% (w/w) based on 100% (wt) of Biolimus to enhance its stability at 40° C. by revealing a high yield of more than 99.4% even after six-week storage. Comparatively, a control test is provided by adding 0% of anti-oxidant (BHT) into Biolimus A9, resulting in a reduction of yield to be 69.7% after six-week storage. The yield data of different amounts of anti-oxidant as added into Biolimus A9 with respect to time lapse of weeks are summarized in Table 1 as below-mentioned.

………………………….

US7193078

The O-(2-ethoxyethyl)-rapamycin can be produced by reaction between rapamycin and 2-ethoxyethyl triflate in the presence of N,N-diisopropylethylamine in methylene chloride.

An example of the O-alkylrapamycin derivative (with R=hydroxyalkyl) is O-(2-hydroxyethyl)-rapamycin represented by the general formula 3 below.

The O-(2-hydroxyethyl)-rapamycin can be produced by reaction between rapamycin and t-butyldimethylsilyloxyethyl triflate in the presence of N,N-diisopropylethylamine in methylene chloride, followed by deprotecting of t-butyldimethylsilyl group.

EXAMPLES

The invention will be described with reference to the following examples, which demonstrate the efficient production of O-alkylrapamycin derivatives by the process of the present invention.

Example 1

• (1) Synthesis of 2-ethoxyethyl Triflate

In a round bottom flask containing a stirring bar was placed 9.0 g (100 mmol) of ethoxyethanol. The atmosphere in the flask was replaced with nitrogen by using a nitrogen bubbler. The flask was given 160 mL of methylene chloride and 23.3 mL (120 mmol) of 2,6-lutidine. The flask cooled with ice was given dropwise 20.2 mL (120 mmol) of trifluoromethanesulfonic acid anhydride over 20 minutes. After stirring for 1 hour, the reaction liquid was mixed with 20 mL of saturated solution of ammonium chloride. The resulting mixture was washed sequentially with 1N hydrochloric acid (100 mL), deionized water (100 mL), saturated solution of sodium hydrogen carbonate (100 mL), and saturated aqueous solution of sodium chloride (100 mL). The organic layer was separated and dried with anhydrous sodium sulfate. With the sodium sulfate filtered off, the solution was concentrated under reduced pressure. The residue underwent silica gel chromatography. Thus there was obtained 15.03 g (67.6% yields) of 2-ethoxyethyl triflate from the fraction in eluate of 20% ethyl acetate-hexane.

• (2) Synthesis of 40-O-[(2′-ethoxy)ethyl]rapamycin

In a round bottom flask containing a stirring bar was placed 1.0 g (1.09 mmol) of rapamycin. With the flask connected to a condenser, the atmosphere in the flask was replaced with nitrogen by using a nitrogen bubbler. To the flask was added 3.5 mL of methylene chloride for dissolution. To the flask was further added 10 mL (57.5 mmol) of N,N-diisopropylethylamine and 1.95 g (8.78 mmol) of the previously synthesized 2-ethoxyethyl triflate with vigorous stirring. With the flask kept at 60° C. in an oil bath, the content was stirred for 1 hour and 20 minutes. The resulting mixture was diluted with 100 mL of ethyl acetate and washed sequentially with 100 mL of 1N hydrochloric acid, 100 mL of deionized water, and 80 mL of saturated aqueous solution of sodium chloride. The ethyl acetate phase was separated and then stirred with 5 g of anhydrous sodium sulfate for 20 minutes. With the sodium sulfate filtered off, the solution was concentrated by using a rotary evaporator. The concentrated solution was purified using a column chromatograph, with a silica gel bed measuring 4 cm in diameter and 26 cm high. Elution was accomplished by flowing sequentially 300 mL of ethyl acetate/n-hexane (1:1 v/v), 1000 mL of ethyl acetate/n-hexane (3:2, v/v), and 300 mL of ethyl acetate/n-hexane (7:3, v/v). The desired fraction was collected and concentrated, and the concentrate was vacuum dried in a desiccator. Thus there was obtained 494 mg (0.501 mmol) of the desired product (46% yields).

Example 2

In a round bottom flask containing a stirring bar was placed 1.0 g (1.09 mmol) of rapamycin. With the flask connected to a condenser, the atmosphere in the flask was replaced with nitrogen by using a nitrogen bubbler. To the flask was added 3.5 mL of chloroform for dissolution. To the flask was further added 10 mL (57.5 mmol) of N,N-diisopropylethylamine and 1.95 g (8.78 mmol) of the 2-ethoxyethyl triflate previously synthesized in Example 1 with vigorous stirring. With the flask kept at 60° C. in an oil bath, the content was stirred for 1 hour and 20 minutes. The resulting mixture was diluted with 100 mL of ethyl acetate and washed sequentially with 100 mL of 1N hydrochloric acid, 100 mL of deionized water, and 80 mL of saturated aqueous solution of sodium chloride. The ethyl acetate phase was separated and then stirred with 5 g of anhydrous sodium sulfate for 20 minutes. With the sodium sulfate filtered off, the solution was concentrated using a rotary evaporator. The concentrated solution was purified using column chromatograph, with a silica gel bed measuring 4 cm in diameter and 26 cm high. Elution was accomplished by flowing sequentially 300 mL of ethyl acetate/n-hexane (1:1, v/v), 1000 mL of ethyl acetate/n-hexane (3:2, v/v), and 300 mL of ethyl acetate/n-hexane (7:3, v/v). The desired fraction was collected and concentrated, and the concentrate was vacuum dried in a desiccator. Thus there was obtained 451 mg (0.458 mmol) of the desired product (42% yields).

Example 3

In a round bottom flask containing a stirring bar was placed 1.0 g (1.09 mmol) of rapamycin. With the flask connected to a condenser, the atmosphere in the flask was replaced with nitrogen by using a nitrogen bubbler. To the flask was added 3.5 mL of methylene chloride for dissolution. To the flask was further added 8 mL (57.4 mmol) of triethylamine and 1.95 g (8.78 mmol) of the 2-ethoxyethyl triflate previously synthesized in Example 1 with vigorous stirring. With the flask kept at 60° C. in an oil bath, the content was stirred for 1 hour and 20 minutes. The resulting mixture was diluted with 100 mL of ethyl acetate and washed sequentially with 100 mL of 1N hydrochloric acid, 100 mL of deionized water, and 80 mL of saturated aqueous solution of sodium chloride. The ethyl acetate phase was separated and then stirred with 5 g of anhydrous sodium sulfate for 20 minutes. With the sodium sulfate filtered off, the solution was concentrated using a rotary evaporator. The concentrated solution was purified using column chromatograph, with a silica-gel bed measuring 4 cm in diameter and 26 cm high. Elution was accomplished by flowing sequentially 300 mL of ethyl acetate/n-hexane (1:1, v/v), 1000 mL of ethyl acetate/n-hexane (3:2, v/v), and 300 mL of ethyl acetate/n-hexane (7:3, v/v). The desired fraction was collected and concentrated, and the concentrate was vacuum dried in a desiccator. Thus there was obtained 344 mg (0.349 mmol) of the desired product (32% yields).

Example 4

In 2 mL of methanol was dissolved 500 mg of the 40 -O-[(2′-ethoxy)ethyl]rapamycin which had been obtained in Example 1. The resulting solution was added dropwise to 20 mL of deionized water with stirring. The solids which had precipitated out were filtered off and washed with a small amount of water and finally dried under reduced pressure at 40° C. for more than 10 hours. Thus there was obtained 483 mg of white powder.

This product gave an NMR chart as shown in FIG. 1. This NMR chart indicates the structure of 40 -O-[(2′-ethoxy) ethyl]rapamycin represented by the general formula 4.

Comparative Example

A sample of 40-O-[(2′-ethoxy)ethyl]rapamycin was synthesized by the process disclosed in WO94/09010 official gazette so as to evaluate yields.

In a round bottom flask containing a stirring bar was placed 1.0 g (1.09 mmol) of rapamycin. With the flask connected to a condenser, the atmosphere in the flask was replaced with nitrogen by using a nitrogen bubbler. To the flask was added 3.5 mL of toluene for dissolution. To the flask was further added 467 mg (4.36 mmol) of 2,6-lutidine and 1.95 g (8.78 mmol) of the 2-ethoxyethyl triflate previously synthesized in Example 1 with vigorous stirring. With the flask kept at 60° C. in an oil bath, the content was stirred for 1 hour and 20 minutes. The resulting mixture was diluted with 100 mL of ethyl acetate and washed sequentially with 100 mL of 1N hydrochloric acid, 100 mL of deionized water, and 80 mL of saturated aqueous solution of sodium chloride. The ethyl acetate phase was separated and then stirred with 5 g of anhydrous sodium sulfate for 20 minutes. With the sodium sulfate filtered off, the solution was concentrated using a rotary evaporator. The concentrated solution was purified using column chromatograph, with a silica gel bed measuring 4 cm in diameter and 26 cm high. Elution was accomplished by flowing sequentially 300 mL of ethyl acetate/n-hexane (1:1, v/v), 1000 mL of ethyl acetate/n-hexane (3:2, v/v), and 300 mL of ethyl acetate/n-hexane (7:3, v/v). The desired fraction was collected and concentrated, and the concentrate was vacuum dried in a desiccator. Thus there was obtained 247 mg (0.251 mmol) of the desired product (23% yields).

NMR

http://www.google.com/patents/US20050192311

This product gave an NMR chart as shown in FIG. 1. This NMR chart indicates the structure of 40-O-[(2′-ethoxy)ethyl]rapamycin represented by the general formula 4.

Tacrolimus, Fujimycin

104987-11-3  CAS, 804.0182, C44H69NO12

• Astagraf XL
• FK 506
• FR 900506
• FR900506
• LCP-Tacro
• Prograf
• Protopic
• Tacrolimus
• Tacrolimus hydrate
• Tsukubaenolide hydrate
• UNII-WM0HAQ4WNM

3S-[3R^*[E(1S^*,3S^*,4S^*)],4S^*,5R^*,8S^*,9E,12R^*,14R^*,15S^*,16R^*,18S^*,19S^*,26aR^*-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5, 19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c] [1,4] oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone, monohydrate

17-Allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0^4,9]octacos-18-ene-2,3,10,16-tetraone

Astellas Pharma (Originator), LAUNCHED 1993

CTK8E6891, 109581-93-3 MONOHYDRATE TACROLIMUS

Tacrolimus (also FK-506 or Fujimycin) is an immunosuppressive drug whose main use is after organ transplant to reduce the activity of the patient’s immune system and so the risk of organ rejection. It is also used in a topical preparation in the treatment of severe atopic dermatitis, severe refractory uveitis after bone marrow transplants, and the skin condition vitiligo. It was discovered in 1984 from the fermentation broth of a Japanese soil sample that contained the bacteria Streptomyces tsukubaensis. Tacrolimus is chemically known as a macrolide. It reduces peptidyl-prolyl isomerase activity by binding to the immunophilin FKBP-12 (FK506 binding protein) creating a new complex. This FKBP12-FK506 complex interacts with and inhibits calcineurin thus inhibiting both T-lymphocyte signal transduction and IL-2 transcription.

PATENT

Pan Sup Chang, Hoon Cho, “Water soluble polymer-tacrolimus conjugated compounds and process for preparing the same.” U.S. Patent US5922729, issued April, 1997.

US5922729 

Tacrolimus is a naturally-occurring macrolide isolated from the fermentation broth of Streptomyces tsukubaensis that was originally discovered by Fujisawa (now Astellas Pharma) in 1984. Tacrolimus possesses immunosuppressive properties and suppresses IL-2 production from helper T-cells, resulting in inhibition of the activation and proliferation of cytotoxic T-cells. In the cell, tacrolimus binds to an immunophilin called FKBP-12 and forms a tacro-immunophilin complex that, in turn, binds to calcineurin and prevents the dephosphorylation of cytoplasmic NF-AT thus disallowing it from reaching the nucleus, thereby strongly inhibiting IL-2 gene transcription. As a result, T-cell activation and proliferation is inhibited.

In 1993, Prograf(R) (tacrolimus capsules and injection) received clearance from the Japanese Ministry of Health and Welfare and was introduced in Japan the same year for the treatment of kidney and liver transplant rejection. Based on two large phase III comparative clinical trials, the product received clearance from the FDA in April 1994, and was made available two months later for commercial use in the U.S. The product is available extensively for transplant rejection. Prograf(R) was also launched in Japan for the treatment of myasthenia gravis and for the treatment of heart transplant rejection; the latter indication was approved in the U.S. in 2006 and launched in 2007. In 2008, Astellas Pharma preregistered the compound in Japan for the oral treatment of all cases of myasthenia gravis. The same year, Senju launched the product in Japan for the treatment of vernal and perennial allergic conjunctivitis in patients unresponsive to anti-allergic drugs. In 2009, the product was approved and commercialized in Japan for the treatment of ulcerative colitis. In 1999, Astellas Pharma launched Protopic(R) (tacrolimus ointment) in Japan for the treatment of atopic dermatitis and in 2001, Protopic(R) was commercialized in the U.S. and Europe. In April 2005, tacrolimus (capsules) was commercialized again by Astellas Pharma in Japan for the treatment of rheumatoid arthritis (RA) in patients who respond insufficiently to current therapies. The following year, Senju received approval in Japan for the use of tacrolimus for the treatment of vernal conjunctivitis and perennial allergic conjunctivitis. A once-daily capsule was approved in the E.U. in 2006. The compound was launched in 2007 in Japan for lupus nephritis. In 2009, the product was approved in US for the prophylaxis of organ rejection in allogeneic kidney transplantation in combination with mycophenolate mofetil and, in the E.U., for the prophylaxis of transplant rejection in adult and pediatric, kidney, liver or heart allograft recipients. In 2011, the compound was launched in Japan for the prophylaxis of organ rejection in patients receiving allogeneic small bowel transplants. In 2013, the indication for interstitial pneumonia associated with polymyositis/dermatomyositis was approved in Japan and an extended release formulation was approved in the U.S. for the prophylaxis of organ rejection in adult patients receiving kidney transplants. This extended release formulation was launched in the U.S. in August 2013. Veloxis Pharmaceuticals (formerly LifeCycle Pharma) is developing a once-daily tablet formulation of tacrolimus (Envarsus®) with improved bioavailability and reduced variability compared with the modified-release version of the compound. Envarsus® has been pre-registered in E.U. and the U.S. for the prevention of transplant rejection in kidney transplant patients. The company is also evaluating the compound in phase II trials for the treatment of autoimmune hepatitis.

In terms of clinical development, the National Cancer Institute (NCI) is developing tacrolimus in phase III for the treatment of graft-versus-host disease (GVHD). Phase III trials are also underway at Astellas Pharma for the treatment of psoriasis, ulcerative colitis and chronic focal encephalitis (Rasmussen’s encephalitis), while early clinical trials are ongoing for asthma. In 2009, Astellas Pharma withdrew an NDA seeking approval in the U.S. based on potential clinical challenges that would result from FDA requirements to conduct additional clinical studies. Kyoto University had been conducting phase II clinical studies for the treatment of Crohn’s disease; however, no recent development has been reported for this research.

In 2003, Sucampo Pharmaceuticals obtained a license from Astellas Pharma to develop and market tacrolimus for ophthalmic indications in the U.S. and Europe, however, in June 2005, the company voluntarily discontinued its tacrolimus eye drops development program due to FDA safety concerns. In 2005, Senju and Astellas Pharma established an agreement to codevelop an eye drop formulation of tacrolimus in Japan. Also, Astellas Pharma granted Senju exclusive manufacturing and marketing rights of the compound. In 2003, Astellas Pharma and GlaxoSmithKline signed an agreement for the copromotion of Protopic(R) in the U.S for atopic dermatitis. An additional agreement for the copromotion of Protopic(R) in South America for the same indication was signed in 2004 between Astellas Pharma and Roche. Tacrolimus was designated orphan drug status in Japan in 1993 and in 2005 for the suppression of organ rejection in allogenic kidney transplantation and for the treatment of vernal conjunctivitis, respectively, in patients unresponsive to anti-allergic drugs. In the E.U., the latter indication was assigned orphan drug designation in 2004. The product was withdrawn from the community register of designated orphan medicinal products in the E.U. in April 2010 on request of the sponsor. In 1998 and 2005, the FDA assigned orphan drug designation for the prophylaxis of GVHD and for the prophylaxis of organ rejection in patients receiving heart transplants. Finally, in 2008, orphan designation was received in Japan for the treatment of myasthenia gravis. In 2012, an additional orphan drug designation was assigned in the U.S. for the treatment of hemorrhagic cystitis. This designation was granted in Japan in 2012 for the treatment of interstitial pneumonia accompanied with polymyositis/dermatomyositis complex. In 2012, orphan drug designation was assigned in Japan for the treatment of interstitial pneumonia accompanied with polymyositis/dermatomyositis complex. In 2012, the product was licensed by Veloxis Pharmaceuticals to Chiesi on an exclusive basis for the commercialization and distribution in Europe, Turkey and CIS countries for the prevention of rejection in kidney transplant recipients. In 2013, an additional orphan drug designation was assigned in the U.S. for the prophylaxis of organ rejection in patients receiving allogeneic kidney transplant.

Tacrolimus, also known as FK-506 or FR-900506, has the chemical tricyclic structure shown below:

corresponding to C44H69NO-|2- Tacrolimus appears in the form of white crystals or crystalline powder. It is practically insoluble in water, freely soluble in ethanol and very soluble in methanol and chloroform. The preparation of tacrolimus is described in EP-A-0 184 162 and analogues of tacrolimus are disclosed e.g. in EP-A-0444659 and US 6,387,918

Tacrolimus is an immunosuppressive agent produced by Streptomyces tsukubaensis No. 9993 and is the compound of formula (I) wherein R.sub.1 and R.sub.2 are both hydrogen. Tacrolimus, which is also called FK-506, has first discovered by Tanaka, Kuroda and their colleague in Japan see, J. Am. Chem. Soc., 1987, 109, 5031 and U.S. Pat. No. 4,894,366 issued on Jan. 16, 1990!.

July 19, 2013 /PRNewswire/ — Astellas Pharma US, Inc. (“A.stellas”), a U.S. subsidiary of Tokyo-based Astellas Pharma Inc., announced today that the U.S. Food and Drug Administration (FDA) has approved Astagraf XL (tacrolimus extended-release capsules) for the prophylaxis of organ rejection in patients receiving a kidney transplant with mycophenolate mofetil (MMF) and corticosteroids, with or without basiliximab induction.

“Each transplant recipient is different and requires a personalized treatment approach. The approval of Astagraf XL marks an important milestone in post-transplant care as it provides physicians with a new treatment option for kidney t recipients,” said Sef Kurstjens, M.D., PhD., chief medical officer, Astellas Pharma, Inc. “Astellas is pleased to continue our more than 20-year commitment to the field of transplant immunology.”

http://www.drugs.com/newdrugs/astellas-announces-fda-approval-astagraf-xl-tacrolimus-extended-release-capsules-prophylaxis-organ-3855.html

Read more at http://www.drugs.com/newdrugs/astellas-announces-fda-approval-astagraf-xl-tacrolimus-extended-release-capsules-prophylaxis-organ-3855.html#43KX8fBakpHoz5Kh.99

PROTOPIC (tacrolimus) Ointment contains tacrolimus, a macrolide immunosuppressant produced by Streptomyces tsukubaensis. It is for topical dermatologic use only. Chemically, tacrolimus is designated as [3S[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10, 12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4] oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone,monohydrate. It has the following structural formula:

Tacrolimus has an empirical formula of C₄₄H₆₉NO₁₂•H₂O and a formula weight of 822.03. Each gram of PROTOPIC Ointment contains (w/w) either 0.03% or 0.1% of tacrolimus in a base of mineral oil, paraffin, propylene carbonate, white petrolatum and white wax.

FK-506 (also Tacrolimus or fujimycin) is a potent calcineurin (protein phosphatase 2B) inhibitor that requires FK 506-binding protein 12 (FKBP12) for activity (IC50 = 3 nM). FK-506 inhibits secretion of IL-1, IL-2 (IC50 = 1 nM), IL-3, IL-4, IL-6 (IC50 = 35 nM), GM-CSF, TNFα (IC50 = 10 nM), IFNγ and Myc from activated T-cells in vitro. FK-506 exhibits potent immunosuppressive, neuroprotective and anticonvulsant activity in vivo. The physiological effects of FK-506 also include regulation of nitric oxide neurotoxicity, neurotransmitter release, and regulation of Ca2+ release via the ryanodine and inositol-(1,4,5)-trisphosphate (IP3) receptors. Furthermore, it has become clear that, predominantly as a result of CaN inhibition, FK506 alters multiple biochemical processes in a variety of cells besides lymphocytes. FK506 and ascomycin inhibit signaling pathways in astrocytes and change the pattern of cytokine and neurotrophin gene expression.

Tacrolimus (also FK-506 or fujimycin, trade names Prograf, Advagraf, Protopic) is an immunosuppressive drug that is mainly used after allogeneic organ transplant to reduce the activity of the patient’s immune system and so lower the risk of organ rejection. It is also used in a topical preparation in the treatment of atopic dermatitis (eczema), severe refractory uveitis after bone marrow transplants, exacerbations of minimal change disease, and the skin condition vitiligo.

It is a 23-membered macrolide lactone discovered in 1984 from the fermentation broth of a Japanese soil sample that contained the bacteria Streptomyces tsukubaensis. It reduces interleukin-2 (IL-2) production by T-cells.

Tacrolimus was discovered in 1984; it was among the first macrolide immunosuppressants discovered, preceded by the discovery of rapamycin (sirolimus) on Rapa Nui (Easter Island) in 1975.It is produced by a type of soil bacterium, Streptomyces tsukubaensis. The name tacrolimus is derived from ‘Tsukuba macrolide immunosuppressant’.

Tacrolimus 0.1%

Indication	For use after allogenic organ transplant to reduce the activity of the patient’s immune system and so the risk of organ rejection. It was first approved by the FDA in 1994 for use in liver transplantation, this has been extended to include kidney, heart, small bowel, pancreas, lung, trachea, skin, cornea, and limb transplants. It has also been used in a topical preparation in the treatment of severe atopic dermatitis.
Pharmacodynamics	Tacrolimus is a macrolide antibiotic. It acts by reducing peptidyl-prolyl isomerase activity by binding to the immunophilin FKBP-12 (FK506 binding protein) creating a new complex. This inhibits both T-lymphocyte signal transduction and IL-2 transcription. Although this activity is similar to cyclosporine studies have shown that the incidence of acute rejection is reduced by tacrolimus use over cyclosporine. Tacrolimus has also been shown to be effective in the topical treatment of eczema, particularly atopic eczema. It suppresses inflammation in a similar way to steroids, but is not as powerful. An important dermatological advantage of tacrolimus is that it can be used directly on the face; topical steroids cannot be used on the face, as they thin the skin dramatically there. On other parts of the body, topical steroid are generally a better treatment.
Mechanism of action	The mechanism of action of tacrolimus in atopic dermatitis is not known. While the following have been observed, the clinical significance of these observations in atopic dermatitis is not known. It has been demonstrated that tacrolimus inhibits T-lymphocyte activation by first binding to an intracellular protein, FKBP-12. A complex of tacrolimus-FKBP-12, calcium, calmodulin, and calcineurin is then formed and the phosphatase activity of calcineurin is inhibited. This prevents the dephosphorylation and translocation of nuclear factor of activated T-cells (NF-AT), a nuclear component thought to initiate gene transcription for the formation of lymphokines. Tacrolimus also inhibits the transcription for genes which encode IL-3, IL-4, IL-5, GM-CSF, and TNF-, all of which are involved in the early stages of T-cell activation. Additionally, tacrolimus has been shown to inhibit the release of pre-formed mediators from skin mast cells and basophils, and to downregulate the expression of FceRI on Langerhans cells.

Tacrolimus was first approved by the Food and Drug Administration (FDA) in 1994 for use in liver transplantation; this has been extended to include kidney, heart, small bowel, pancreas, lung, trachea, skin, cornea, bone marrow, and limb transplants.

The branded version of the drug is owned by Astellas Pharma, and is sold under the trade names Prograf given twice daily, Advagraf, a sustained release formulation allowing once daily dosing, and Protopic (Eczemus in Pakistan by Brookes Pharma), the topical formulation. Advagraf is available in 0.5, 1, 3 and 5 mg capsules, the ointment is concentrations of 0.1% and 0.03%.

A second once-daily formulation of tacrolimus is in Phase 3 clinical trials in the U.S. and Europe. This formulation also has a smoother pharmacokinetic profile that reduces the peak-to-trough range in blood levels compared to twice-daily tacrolimus.Data from the first Phase 3 trial in stable kidney transplant patients showed that this once-daily formulation was non-inferior in efficacy and safety compared to twice-daily tacrolimus. A second Phase 3 trial in de novo patients is ongoing.

Tacrolimus, which is also referred to as FK-506 (Fermentek catalogue number 506), is a 23-membered macrolide lactone and belongs to the group of polyketides. Tacrolimus was first isolated in the 1980′s from the fermentation broth of the soil bacteria Streptomyces tsukubaensis. The antibiotic macrolide compound tacrolimus was e.g. reported in 1984 by Kino et al. (J. Antibiotics 40, 1249-1255, 1984). Later on tacrolimus was prepared as a microbial natural product by using different microorganisms, i.e. soil bacteria such as Streptomyces sp. MA6858 (US 5,116,756) ATCC 55098, Streptomyces tsukubaensis NRRL 18488 (EP-B 0 356 399 and US 5,200,41 1 ), Streptomyces clavuligerus CKD 1119 (KR-B 100485877) or Streptomyces glaucescens MTCC 5115 (US 2007191415).

The product tacrolimus exhibits immunosuppressive activities which are due to its effect to reduce the activity of the enzyme peptidyl-propyl isomerase and to the binding to the protein immunophilin FKBP12 (FK506 binding protein). Tacrolimus and the structurally similar polyketides ascomycin and rapamycin require initial binding to the highly conserved protein cyclophilin FKBP12 in order to be physiologically active. The rapamycin/FKBP12 complex binds to mTOR (mammalian target of rapamycin), a serine- threonine kinase that appears to act as a central controller for sensing the cellular environment and regulating translation initiation (see e.g. Easton J. B. and Houghton P.J., 2004, Expert Opin Ther Targets; 8(6):551-64). However, the tacrolimus/FKBP12 complex was found to bind to a different cellular target and inhibits the phosphatase activity of calcineurin, in analogy to cyclosporine (see Allison A.C., 2000, Immunopharmacology; 47(2-3):63-83).

Tacrolimus is often used for immunosuppression following e.g. organ transplantation. Furthermore, tacrolimus and its derivatives have been shown to be effective in treating a number of diseases such as asthma, inflammatory diseases and hyperproliferative skin disease. Tacrolimus and other immunosuppressant such as rapamycin, cyclosporine, or a combination thereof are also useful in the treatment of various auto-immmune diseases. For many years calcineurin inhibitors (e.g. cyclosporine and tacrolimus) have been the mainstay of immunosuppressive therapy. These two compounds are potent suppressors of cellular immune response and have significantly improved the outcome of organ transplants during the past two decades (see Allison A.C., 2000, Immunopharmacology; 47(2-3):63-83). Gene clusters encoding the biosynthetic pathways of a great number of medically important drugs of microbial origin have already been cloned and sequenced, including the gene cluster of macrolides rapamycin, ascomycin and tacrolimus. With respect to cloning of the tacrolimus gene cluster, a partial sequence, mostly encompassing genes encoding polyketide synthase (PKS), was reported in the literature (see Motamedi H. and Shafiee A. 1998, Eur J Biochem; 256(3):528-34). On the other hand, scientists reported cloning of the ascomycin gene cluster in 2000 (see Wu K et al. 2000, Gene; 251(1 ):81- 90, US 6,503,737). Tacrolimus structurally and by the biosynthetic origin resembles ascomycin (FK520) and rapamycin (see Reynolds et al.; Drugs and the Pharmaceutical Sciences, 1997, 82, 497-520. They all can be synthesised by combined polyketide (PKS) and non-ribosomal peptide biosynthetic pathways (NRPS) (see McDaniel R et al. 2005, Chem Rev; 105(2):543-58).

Tacrolimus and ascomycin are structurally similar. As only structural difference, the allyl side chain at carbon 21 of tacrolimus is replaced by an ethyl side chain in ascomycin. The structures of tacrolimus (FK506) and ascomycin (FK520) compounds are shown as formulae (Ia) and (Ib). The structures of ascomycin and tacrolimus already suggest complex biosynthetic pathways which can be divided into four steps considering the biosynthetic mechanism:

1. chain initiation using the unusual shikimate derived starter,

2. chain elongation common to most PKS derived compounds,

3. chain termination and cyclization by incorporation of L-pipecolic acid and

4. post-PKS processing.

During the tacrolimus fermentation process, undesired ascomycin (FK520) product is also produced as an impurity, thus lowering the final yield of tacrolimus and causing significant additional costs to the downstream isolation processes of tacrolimus.

(Ia) FK506, R = -CH₂-CH = CH₂

(Ib) FK520, R = CH₂-CH₃

For oral administration, tacrolimus is currently formulated and marketed as soft gelatine capsules comprising the equivalent of 0.5, 1 or 5 mg anhydrous tacrolimus and marketed under the trade name Prograf® and Protropic®. The recommended initial oral dose is from about 0.1 to 0.2 mg/kg/day in patients. The dose aims at a certain trough plasma level from about 5 to about 20 ng/ml. Prograf^® is indicated for the prophylaxis of organ rejection in patients receiving allogeneic liver or kidney transplants. There remains a need for novel pharmaceutical compositions and/or dosage forms comprising tacrolimus exhibiting enhanced bioavailability. An increased bioavailability may allow a reduction in the dosage units taken by a patient, e.g. down to a single dose daily, and may also reduce or negate the need for food to be takes simultaneously with the dosage form thereby allowing patients more freedom on when the drug is taken. Furthermore, it is contemplated that fluctuations in the plasma concentration versus time profile may be significantly reduced. Further, enhanced bioavailability may also result in a more reproducible (i.e. less variable compared to that of Prograf®) release profile….

………………….

EP2451955A1

h) Determination of tacrolimus and ascomycin production with HPLC of thiostrepton resistant ccr disrupted mutants derived by secondary homologous recombination using pKC1 139-ccrTs.:

Method for tacrolimus and ascomycin determination: The analysis for determination of tacrolimus or ascomycin production thereof was carried out by isocratic reversed phase HPLC using an appropriate column and running conditions: column Nucleosil-100 C18 (150×4.0 mm, particle size 3 μm), flow 1.5 ml/min, T°C=60°C, mobile phase: 560 ml water, 335 ml acetonitrile, 70 ml MTBE and 0.2 ml 85% H₃PO₄, detection 210 nm, sample injection 20 μl.

The tacrolimus and ascomycin content in samples quantification was performed by using external standards of tacrolimus and ascomycin, where tacrolimus was eluted at 12.5 min and ascomycin at 11.5 min. Results are expressed as % of ascomycin production compared to tacrolimus production in samples.

……………………….

http://www.drugfuture.com/synth/syndata.aspx?ID=124071

A new total synthesis of FK-506 is described: This synthesis has been performed by previous construction of two building fragments (XXIV) and (LI), which later were coupled and cyclized. (Schemes 1-3): 1) (1R*S*,3R,5S,6R,7S,9R)-6-(tert-butyldimethylsilyloxy)-9-(1,3-dithian-2-yl)-5,7-dimethoxy-1-methyldecyl diphenyl phosphine oxide (XXIV). The Sharpless asymetric epoxidation of 1,4-pentadien-3-ol (I) with (-)-diisopropyltartrate and tert-butylhydroperoxide gives the epoxy alcohol (II) with high optical purity, which is benzylated in the usual way to (III). The reaction of (III) with lithioacetonitrile and then HCl yields lactone (IV), which is methylated with lithium diisopropylamide and methyl iodide to lactone (V) as major isomer (separated by chromatography on SiO2). The reduction of (V) with LiAlH4 affords the diol (VI), which is converted into the bis(tert-butyl carbonate) (VII) with 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (BOC-N). The reaction of (VII) with Br2 and K2CO3 in dichloromethane gives the bromocarbonate (VIII), which by selective saponification of the cyclic carbonate with NaOCH3 in methanol yields the epoxy alcohol (IX). Methylation of (IX) with NaH and methyl iodide affords the methyl ether (X), which is converted into the butyrolactone (XI) with lithioacetonitrile as before. The protection of the OH group of (XI) with TBS-Cl gives the silyl ether (XII), which by trans-selective methylation with lithium diisopropylamide and methyl iodide yields lactone (XIII). The reduction of (XIII) with LiAlH4 affords diol (XIV) as major isomer (separated by column chromatography). The selective esterification of the primary OH group of (XIV) with pivaloyl chloride gives the hydroxy ester (XV), which is methylated with NaH and methyl iodide as usual to the methoxy derivative (XVI). Debenzylation of (XVI) by hydrogenolysis with H2 over Pd/C yields the hydroxy ester (XVII), which is silylated with TBS-SO3CF3 to the fully protected compound (XVIII).

Selective deprotection of (XVIII) with trifluoroacetic acid in THF – water affords the primary alcohol (XIX), which is oxidized with oxalyl chloride and DMSO in dichloromethane to the aldehyde (XX). The protection of the aldehyde group of (XX) with propane-1,3-dithiol and BF3 gives the dithiane derivative (XXI), which is resilylated with TBS-SO3CF3 as before to the dithiane (XXII). The pivaloyl group of (XXII) is eliminated with LiAlH4 in THF yielding the alcohol (XXIII), which is finally treated with benzenesulfonyl chloride and then with ethyl diphenylphosphine oxide and butyllithium in THF to obtain the first building group, the phosphine derivative (XXIV).

2) [2S,3S,5S,6R,7S,8E,9(1'R,3'R,4'R)]-2-Allyl-3-(tert-butyldimethylsilylox y)-6,8-dimethyl-7-(triethylsilyloxy)-5-(triisopropylsilyloxy)-9-[3-meth oxy-4-(triisopropylsilyloxy)cyclohexyl]-8-nonenal (LI). Quinic acid (XXV) is converted into the lactone (XXVI) by known methods. Then this lactone is treated with thiocarbonyldiimidazole in refluxing dichloroethane yielding the bis(thiocarbonyl)lactone (XXVII), which by reaction with tributyltin hydride and AIBN in refluxing xylene is converted into the lactone (XXIX), either directly or through the intermediate thiocarbonyl-lactone (XXVIII). The silylation of (XXIX) with TIPS-SO3CF3 as usual affords the protected lactone (XXX). Opening of the lactone ring with methylchloroaluminum N-methoxy-N-methylamide gives the methoxyamide (XXXI), which is methylated with methyl trifluoromethylsulfonate to the methoxy-N-methoxyamide (XXXII). The reduction of (XXXII) with diisobutylaluminum hydride gives the aldehyde (XXXIII), which is condensed with 2-lithio-2-(triethylsilyl)propanal (XXXIV), yielding unsaturated aldehyde (XXXV). The condensation of (XXXV) with the boron enolate of oxazolidone (XXVI) affords the oxazolidone derivative (XXXVII), which is treated with methylchloroaluminum N-methoxy-N-methylamide to give the methoxyamide (XXXVIII). The silylation of (XXXVIII) with TES-SO3CF3 as usual yields the silylated amide (XXXIX), which is reduced with diisobutylaluminum hydride to the aldehyde (XL). The condensation of (XL) with chiral acetate (XLI) by means of lithium diisopropylamide in THF affords the hydroxy ester (XLII). Transesterification of (XLII) with NaOCH3 and methanol gives methyl ester (XLIII).

http://www.drugfuture.com/synth/syndata.aspx?ID=124071

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EP0184162B1

Use of the microorganism streptomyces tsukubaensis No. 9993 for the production of the FR-900506 substance of the formula:

References

• Tacrolimus levels in Liver Transplants-Indian Study by Dr.Pradeep Naik,Dr.Dharmesh Kapoor, Dr.DCS Reddy
• Prograf prescribing information at Fujisawa
• Pimecrolimus (Elidel Cream) FDA adivisory page (for eczema treatment)
• Tacrolimus (FK506) product page from Fermentek
• U.S. National Library of Medicine: Drug Information Portal – Tacrolimus

Octreotide

(D)-Phe-Cys-Phe-(D)-Trp-Lys-Thr-Cys-Thr-ol.

(4R,7S,10S,13R,16S,19R)-10-(4-aminobutyl)-19-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-16-benzyl-N-[(2R,3R)-1,3-dihydroxybutan-2-yl]-7-(1-hydroxyethyl)-13-(1H-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-
pentazacycloicosane-4-carboxamide

L-cysteinamide, D-phenylalanyl-L-cysteiny-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-,cyclic (2→7)-disulfide; [R-(R*,R*)].

Octreotide is the acetate salt of a cyclic octapeptide. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin.

Sandostatin LAR Depot
L-Cysteinamide, D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-(2-hydroxy-1-(hydroxymethyl)propyl)-, cyclic(2-7)-disulfide, (R-(R*,R*))-, acetate (salt)
Octreotide Acetate Depot
AC1L1GVR
AC1Q2BPN
CCRIS 8708
Octreotide acetate [USAN:JAN]
UNII-75R0U2568I
83150-76-9 (Parent)
AC-663

Octreotide (brand name Sandostatin,^[1] Novartis Pharmaceuticals) is an octapeptide that mimics natural somatostatin pharmacologically, though it is a more potent inhibitor of growth hormone, glucagon, and insulin than the natural hormone. It was first synthesized in 1979 by the chemist Wilfried Bauer.

Since octreotide resembles somatostatin in physiological activities, it can:

• inhibit secretion of many hormones, such as gastrin, cholecystokinin, glucagon, growth hormone, insulin, secretin, pancreatic polypeptide, TSH, and vasoactive intestinal peptide,
• reduce secretion of fluids by the intestine and pancreas,
• reduce gastrointestinal motility and inhibit contraction of the gallbladder,
• inhibit the action of certain hormones from the anterior pituitary,
• cause vasoconstriction in the blood vessels, and
• reduce portal vessel pressures in bleeding varices.

It has also been shown to produce analgesic effects, most probably acting as a partial agonist at the mu opioid receptor.^[2][3]

Acromegaly is a hormonal disorder that results when the pituitary gland produces excess growth hormone (GH). It most commonly affects middle-aged adults and can result in serious illness and premature death. Once recognized, acromegaly is treatable in most patients, but because of its slow and often insidious onset, it frequently is not diagnosed correctly.

Octreotide is one drug used to treat acromegaly. Octreotide exerts pharmacologic actions similar to those of the natural hormone somatostatin. Octreotide decreases GH and IGF-1 levels, as well as glucagons and insulin. Octreotide also suppresses luteinizing hormone (LH) response to gonadotropin releasing hormone (GnRH), decreases splanchnic blood flow, and inhibits the release of serotonin, gastrin, vasoactive intestinal peptide, secretin, motilin, and pancreatic polypeptide. In many patients, GH levels fall within one hour and headaches improve within minutes after the injection of octreotide. Several studies have shown that octreotide is effective for long-term treatment. Octreotide also has been used successfully to treat patients with acromegaly caused by non-pituitary tumors. In some acromegaly patients who already have diabetes, octreotide can reduce the need for insulin and improve blood sugar control.

Octreotide is currently available as Sandostatin LAR® Depot, which is, upon reconstitution, a suspension of microspheres containing octreotide acetate. Sandostatin LAR® Depot is the only medication indicated for the long-term maintenance therapy in acromegalic patients. It is also indicated for the long-term treatment of severe diarrhea and flushing episodes associated with metastatic carcinoid tumors and profuse water diarrhea associated with VIP-secreting tumors. Sandostatin LAR® T Depot is administered via intramuscular injection every four weeks, following a titration period. Octreotide acetate has also been available in an immediate-release formulation, Sandostatin® Injection solution, which was required to be administered by injection three times daily.

Octreotide is an octapeptide with the following amino acid sequence: L-cysteinamide, D-phenylalanyl-L-cysteiny-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-,cyclic (2→7)-disulfide; [R-(R*,R*)]. The structure of octreotide is shown below.

The chemical formula is C₄₉H₆₆N₁₀O₁₀S₂ and its molecular weight is 1019.3 Da. Its therapeutic category is gastric antisecretory agent.

The Food and Drug Administration (FDA) has approved the usage of a salt form of this peptide, octreotide acetate, as an injectable depot formulation for the treatment of growth hormone producing tumors (acromegaly and gigantism), pituitary tumors that secrete thyroid stimulating hormone(thyrotropinoma), diarrhea and flushing episodes associated with carcinoid syndrome, and diarrhea in patients with vasoactive intestinal peptide-secreting tumors (VIPomas).

Octreotide is used in nuclear medicine imaging by labelling with indium-111 (Octreoscan) to noninvasively image neuroendocrine and other tumours expressing somatostatin receptors.^[4] More recently, it has been radiolabelled with carbon-11^[5] as well as gallium-68, enabling imaging with positron emission tomography (PET), which provides higher resolution and sensitivity.

Octreotide can also be labelled with a variety of radionuclides, such as yttrium-90 or lutetium-177, to enable peptide receptor radionuclide therapy(PRRT) for the treatment of unresectable neuroendocrine tumours.

Octreotide is the acetate salt of a cyclic octapeptide. It is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin. Octreotide is known chemically as L-Cysteinamide, D-phenylalanyl-L-cysteinyl-L-phenylalanyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1- (hydroxy-methyl) propyl]-, cyclic (2→7)-disulfide; [R-(R*,R*)].

Sandostatin LAR Depot is available in a vial containing the sterile drug product, which when mixed with diluent, becomes a suspension that is given as a monthly intragluteal injection. The octreotide is uniformly distributed within the microspheres which are made of a biodegradable glucose star polymer, D,L-lactic and glycolic acids copolymer. Sterile mannitol is added to the microspheres to improve suspendability.

Sandostatin LAR Depot is available as: sterile 5-mL vials in 3 strengths delivering 10 mg, 20 mg, or 30 mg octreotide-free peptide. Each vial of Sandostatin LAR Depot delivers:

Each syringe of diluent contains:

The molecular weight of octreotide is 1019.3 (free peptide, C₄₉H₆₆N₁₀O₁₀S₂) and its amino acid sequence is

Octreotide has also been used off-label for the treatment of severe, refractory diarrhea from other causes. It is used in toxicology for the treatment of prolonged recurrent hypoglycemia after sulfonylurea and possibly meglitinides overdose. It has also been used with varying degrees of success in infants with nesidioblastosis to help decrease insulin hypersecretion.

Octreotide has been used experimentally to treat obesity, particularly obesity caused by lesions in the hunger and satiety centers of thehypothalamus, a region of the brain central to the regulation of food intake and energy expenditure.^[6] The circuit begins with an area of the hypothalamus, the arcuate nucleus, that has outputs to the lateral hypothalamus (LH) and ventromedial hypothalamus (VMH), the brain’s feeding and satiety centers, respectively.^[7][8] The VMH is sometimes injured by ongoing treatment for acute lymphoblastic leukemia (ALL) or surgery or radiation to treat posterior cranial fossa tumors.^[6] With the VMH disabled and no longer responding to peripheral energy balance signals,

Octreotide has also been investigated for patients with pain from chronic pancreatitis,^[11] and it may be useful in the treatment of thymic neoplasms.

The drug has been used off-label, injected subcutaneously, in the management of hypertrophic pulmonary osteoarthropathy (HPOA) secondary to non-small cell lung carcinoma. Although its mechanism is not known, it appears to reduce the pain associated with HPOA.^{[citation needed]}

It has been used in the treatment of malignant bowel obstruction.^[12]

Octreotide may be used in conjunction with midodrine to partially reverse peripheral vasodilation in the hepatorenal syndrome. By increasing systemic vascular resistance, these drugs reduce shunting and improve renal perfusion, prolonging survival until definitive treatment with liver transplant.^[13] Similarly, octreotide can be used to treat refractory chronic hypotension.^[14]

While successful treatment has been demonstrated in case reports,^[15][16] larger studies have failed to demonstrate efficacy in treating chylothorax.^[17]

Octreotide is often give as an infusion for management of acute haemorrhage from esophageal varices in liver cirrhosis on the basis that it reduces portal venous pressure, though current evidence suggests that this effect is transient and does not improve survival.^[18]

A small study has shown that octreotide may be effective in the treatment of idiopathic intracranial hypertension.^[19][20]

Octreotide has not been adequately studied for the treatment of children, pregnant and lactating women. The drug is given to these groups of patients only if a risk-benefit analysis is positive.^[21][22]

Acetate

C₅₃H₇₄N₁₀O₁₄S₂   ,  1139.34326

The most frequent adverse effects (more than 10% of patients) are headache, hypothyroidism, cardiac conduction changes, gastrointestinal reactions (including cramps, nausea/vomiting and diarrhoea or constipation), gallstones, reduction of insulin release, hyperglycemia^[23] or hypoglycemia, and (usually transient) injection site reactions. Slow heart rate, skin reactions such aspruritus, hyperbilirubinemia, hypothyroidism, dizziness and dyspnoea are also fairly common (more than 1%). Rare side effects include acute anaphylactic reactions, pancreatitis andhepatitis.^[21][22] One study reported a possible association with rheumatoid arthritis.^[24]

Some studies reported alopecia in patients who were treated by octreotide.^[25] Rats which were treated by octreotide experienced erectile dysfunction in a 1998 study.^[26]

A prolonged QT interval has been observed in patients, but it is uncertain whether this is a reaction to the drug or part of the patients’ illnesses.^[21]

Octreotide is absorbed quickly and completely after subcutaneous application. Maximal plasma concentration is reached after 30 minutes. The elimination half-life is 100 minutes (1.7 hours) on average when applied subcutaneously; after intravenous injection, the substance is eliminated in two phases with half-lives of 10 and 90 minutes, respectively.^[21][22]

Conventional synthesis of octreotide may be divided into two main approaches, liquid-phase synthesis and solid-phase synthesis. · Octreotide first disclosed in US4395403, in which Octreotide is prepared by solution phase peptide synthesis. The process comprises; removing protected group from peptide;^‘ linking together by an amide bond to two peptide unit; converting a function group at the N- or C- terminal; oxidizing a straight chain polypeptide by boron tristrifluoroacetate.

Since all the synthesis steps are carried out in liquid phase, US’403 process is a time- consuming, multi-step synthesis and it is difficult to separate octreotide from the reaction mixtures. Another solution phase approach described in US6987167 and WO2007110765A2, in which the cyclization of partially deprotected octreotide is carried out in the solution phase using iodine under specific conditions in presence of alcoholic solvents.

US6346601 B1 , WO2005087794A1 and WO2010089757A2 disclose a process for the preparation of octreotide by hybrid approach i. e synthesis of fragments on solid phase and condensing the obtained fragments in a liquid phase.

US6476186 describes the solid phase synthesis, in which the synthesis of octreotide using Thr(ol)(tBu)-2CI-trityl resin as starting material, followed by the cleavage of the straight chain peptide from the resin using a strong acid and the formation of the intra-molecular disulfide bond on the completely deprotected octreotide by oxidation using charcoal catalyst.

US20040039161A1 provides a solid phase .peptide synthetic method for the preparation of C-terminal alcohols using trichloroacetimidate activated linker, making the required peptide chain on the resin support, cleaving the attached peptide; air oxidation to form said C- terminal amino alcohol containing peptide and a 36.3% yield of octreotide after HPLC purification.

Charcoal oxidation or air oxidation needs longer reaction time and results in low yield. Further, in large scale, the conversion of dithiol to disulfide bond ends in unconverted starting material.

Another solid phase approach describes in Bioconjugate chem. 2009, 20, 1323-1331. This article discloses the process of somatostatin and octreotide analogues using solid phase peptide synthesis with CTC resin.

Journal of Harbin Institute of Technology, 2008, Vol 40 (2), 292-295, discloses the process for the preparation of octreotide using CTC resin. According to this process the obtained octreotide has the purity 70.26% by HPLC. During the process of peptide bond formation which is mediated by a coupling agent, the carboxylic group of amino acid interacts with the coupling agent to form an activated intermediate, which in turn interacts with the amino group of the next amino acid.

Racemization is a side-reaction that occurs during the preparation of a peptide. In large scale production, the formations of small amounts of epimers are possible. Detection and removal of these impurities are very difficult. This constitutes one of the most serious drawbacks for the implementation of peptides in commercial scale production.

WO2005087794A1

Conventional syntheses of OCT may be divided two main approaches, direct solid-phase synthesis and liquid-phase synthesis. Direct solid-phase synthesis comprises attachment of a C-terminal amino acid to a resin, and step-by step elongation of the peptide chain, with pre- activated amino acids.

This route is expensive because it requires large excesses of starting amino acids and additionally is quite labor consuming as the peptide size increases, necessitating complex purification procedures to separate the product from the impurities since they are very similar to the final product. These shortcomings are especially important for large scale industrial production of the product. For example, see Canadian Patent Application 2,309,312 and U.S. Patent No. 6,476,186. With each successive condensation reaction required to add an amino acid, waste of starting materials increases, and purification steps are repeated. Liquid-phase synthesis comprises condensation of amino acids in solution. Several blocks, containing from 2 to 5 amino acids may be synthesized independently, followed by condensation of these synthons to each other in the required sequence.

For example, see WO 03/097668; U.S. Patent No. 4,395,403; and RU 2196144 C1. The advantage of this kind of processes is that it is less expensive than the previous one and the product is easier to purify. This method is also more effective for scale-up. However, liquid phase synthesis of lengthy peptide blocks, for example having more than 3 amino acids, is inefficient. Liquid-phase octreotide synthesis has the drawback is that the method is extremely labor-intensive and time consuming.

U.S. Patent No. 6,346,601 describes a method for octreotide synthesis where a solid-phase method is used to obtain a 7-mer, followed by condensation in solution with the modified amino acid threoninol. However, by using solid- phase synthesis to produce a 7-mer, only one less condensation is required compared to the solid-phase process for forming octreotide itself. Thus, only a marginal efficiency is introduced.

Summary of the invention According to an embodiment of the invention, there is provided a process for obtaining octreotide or a pharmaceutically acceptable salt thereof by hybrid solid-phase – liquid-phase synthesis. The synthesis comprises the steps of condensing two or three peptide blocks using liquid phase condensation to form a condensation product followed by cyclizing the product.

Each peptide block contains two or more amino acid residues, and at least one of the blocks is synthesized by solid-phase synthesis. The condensation product comprises in sequence the amino acids residues of octreotide. In the step of cyclizing, the condensation product is cyclized to form a disulfide bridge between the two cysteine residues, thereby forming octreotide. Further, according to another embodiment of the invention, a process is provided for obtaining an intermediate in octreotide synthesis by hybrid solid- phase – liquid-phase synthesis.

The synthesis of the intermediate comprises the steps of obtaining two or three peptide blocks, each peptide block containing two or more amino acid residues, and at least one of the blocks is synthesized by solid-phase synthesis. Subsequently, the peptide blocks are condensed using liquid phase condensation to form a condensation product, wherein the condensation product comprises in sequence the amino acids residues of octreotide.

This invention provides a more cost-effective and labor-saving method for obtaining OCT and its pharmaceutically acceptable salts by means of hybrid solid-phase – liquid-phase synthesis. The invention involves liquid phase condensation of two peptide blocks, at least one of which is obtained by solid- phase synthesis, the blocks containing more two or more amino acid residue in every block, followed by formation of a disulfide bridge from the two cysteine groups. Optionally, three blocks may be condensed. This hybrid solid phase-liquid phase method involves formation of one or more blocks of the octreotide amino acid sequence by solid-phase synthesis, followed by liquid phase condensation of the block(s) with required supplementary amino acids or other block(s) of amino acids.

This method is a blend of solid-phase and liquid-phase synthesis methods, combining the efficiencies of preparing shorter (6-mer or less) peptides using a solid-phase method with relative cheapness and easiness of purification of the product, characteristic of the liquid-phase method. Generally, the methods of invention comprise synthesizing specific side- chain protected peptide fragment intermediates of OCT on a solid support or in solution, coupling of the protected fragments in solution to form a protected OCT, followed by deprotection of the side chains and oxidation to yield the final OCT. The present invention further relates to individual peptide fragments which act as intermediates in the synthesis of the OCT

………………

WO2013046233A2

Stage-I: Preparation of protected octreotide anchored to 2-CTC Resin

Method -1:

Octreotide was synthesized manually on 2-chlorotrityl chloride resin (substitution 0.90 mmol/g) by standard Fmoc solid phase synthesis strategy. The resin was soaked in the mixture of DC and DMF for the swelling. Fmoc-Thr(tBu)-OL was treated with the swelled 2- CTC resin in DCM in the presence of DIEA and substitution level was determined by weight gain measurements and also by UV Method. After the coupling of the first amino acid onto the resin, the un-reacted linkers on the resin (polymer) are protected, to avoid the undesired peptide chain formation, with a solution of 5% DIEA and 10% methanol in DCM. This process of capping is performed after anchoring the first protected amino acid to the resin. The complete synthesis was achieved by stepwise coupling of Fmoc-Amino acids to the growing peptide chain on the resin. All the couplings were carried out in DMF. The N- terminal Fmoc group was removed with 20 %( V/V) piperidine in DMF. The couplings were performed by dissolving the Fmoc-Amino acid (2 eq.) and HOBt (2 eq.) in DMF. The solution was cooled on ice and then DIC (2 eq.) was added. The reaction mixture was added to the resin and allowed to react for 2 hrs. The efficiency of the coupling was monitored using the Kaiser Ninhydrin test. The coupling step was repeated if Kaiser test was found positive. The sequence of addition for the synthesis of Octeriotide was Fmoc-Cys(Trt), Fmo-Thr(tBu), Fmoc-Lys(Boc), Fmoc-Trp(Boc), Fmoc-Phe, Fmoc-Cys(Trt), Boc-D-Phe.

Method -2:

Octreotide was synthesized manually on 2-chlorotrityl chloride resin (substitution 0.90 mmol/g) by standard Fmoc solid phase synthesis strategy. The resin was soaked in the mixture of MDC and DMF for the swelling. Fmoc-Thr-OL was treated with the swelled 2-CTC resin in DCM in the presence of DIEA and substitution level was determined by weight gain measurements and also by UV Method. After the coupling of the first amino acid onto the resin, the un-reacted linkers on the resin (polymer) are protected, to avoid the undesired peptide chain formation, with a solution of 5% DIEA and 10% methanol in DCM. This process of capping is performed after anchoring the first protected amino acid to the resin. The complete synthesis was achieved by stepwise coupling of Fmoc-Amino acids to the growing peptide chain on the resin. All the couplings were carried out in DMF. The N- terminal Fmoc group was removed with 20 %( V7V) piperidine in DMF. The couplings were performed by dissolving the Fmoc-Amino acid (2 eq.) and HOBt (2 eq.) in DMF. The solution was cooled on ice and then DIC (2 eq.) was added. The reaction mixture was added to the resin and allowed to react for 2 hrs. The efficiency of the coupling was monitored using the Kaiser Ninhydrin test. The coupling step was repeated if Kaiser test was found positive. The sequence of addition for the synthesis of Octeriotide was Fmoc-Cys(Trt), Fmo-Thr(tBu), Fmoc-Lys(Boc), Fmoc-Trp(Boc), Fmoc-Phe, Fmoc-Cys(Trt), Boc-D-Phe.

Stage-ll: Cleavage of peptide from resin along with global deprotection

The peptide resin (200 g, obtained in stage I) was swelled in DCM (500 mL) for 15 to 20 minutes under nitrogen at 25-30° C. The cocktail mixture (2.0 L – TFA (1.8 L), water (80 mL) DCM (80mL) and TIPS (80 mL)) was charged to the resin at 25-30° C. and the obtained reaction mixture was stirred for 2.5 hours at 25-30°C under nitrogen atmosphere. The reaction mixture was filtered and washed the resin with TFA (250 mL). The obtained filtrate was charged into cold MTBE (4 L, pre-cooled to a temperature of 0 -5° C) under stirring and allowing the temperature to rise more than 5° C. The reaction mixture was stirred for 45-75 minutes at 0-5°C. The obtained suspension was filtered, washed the solid with MTBE (5 L) and dried the solid under nitrogen. The product was stir with 5%ethanol in ethyl acetate at 25-30°C. Filtered the product, wash ith ethyl acetate and dried under vacuum to obtain a desired product

Stage-Ill: Disulphide bridge formation

The free thiol (100 g) obtained above is dissolved in methanol (22.0 L) with small amount of acetic acid and water (4.5 L) and stirred. Iodine solution (20gm iodine in 500 mL methanol) was added to the reaction mass slowly up to yellow color persists. The reaction was maintained for another 2 hrs, and the excess iodine quenched with Indion 830-S Resin (900 g) and filtered the resin. The filtrate was evaporated and precipitated using TBE or directly taken the solution for purification using preparative HPLC.

Stage -IV: Preparative HPLC Purification

Method-1 :

The crude disulphide bridge peptide was purified on a preparative reverse phase HPLC system using Kromasil C-18, 10 micron (50 x 250 mm). and eluting with a solvent system of 0.2% acetic acid in water(A) and 0.2% acetic acid in methanol(B). A linear gradient of 20- 60% B was used at a flow rate of 80mlJmin and detection at 220 nm.

The octreotide was eluted at around 25% methanol. The fractions were collected at regular intervals and assayed by HPLC to determine the purity of fractions. The desired purities fractions were pooled together and evaporated using Rota evaporator. The aqueous layer was lyophilized to isolate octreotide acetate

Method-2:

The crude disulphide bridge peptide was purified on a preparative reverse phase HPLC system using Kromasil C-18, 10 micron (50 x 250 mm) and eluting with a solvent system of 0.4% acetic acid in water(A) and methanol(B). A linear gradient of 25-60% B was used at a flow rate of 80mL/min and detection at 220 nm.

The octreotide was eluted at around 25% methanol. The fractions are collected at regular intervals and are assayed by HPLC to determine the purity and fractions. The desired purities may be pooled together and were evaporated using Rota evaporator. The aqueous layer was lyophilized to isolate octreotide acetate >

……………………….

WO2010089757A2

Octreotide is a highly potent and pharmacologically selective analog of somatostatin. It inhibits growth hormone for long duration and is thereof indicated for acromegaly to control and reduce the plasma level of growth hormone. The presence of D-Phe at the N-terminal and an amino alcohol at the C-terminal, along with D-Tryptophan and a cyclic structure makes it very resistant to metabolic degradation.

Octreotide comprises 8 amino acids which has the following structural formula:

(D)Phe-Cys-Phe-{D)Trp-Lys-Thr-Cys-Thr-OL

Formula(l) wherein sulphur atoms of the Cys at the position 2 and of the Cys at the position 7 are mono-cyclic to form an -S-S- bridge.

A considerable number of known, naturally occurring small and medium-sized cyclic peptides as well as some of their artificial derivatives and analogs possessing desirable pharmacological properties have been synthesized. However, wider medical use is often hampered due to complexity of their synthesis and purification. Therefore, improved methods for making these compounds in simple, lesser steps and at lesser cost are desirable and this is the felt need of the industry and the mankind.

Conventional synthesis of octreotide may be divided into two main approaches, direct solid-phase synthesis and liquid-phase synthesis. Solution phase synthesis has been described by Bauer et al., (Sandoz) (Eur. Pat. Appl. 29,579 and U.S. Pat. No. 4,395,403). The process comprises: removing protected group from peptide; linking together by an amide bond two peptide unit; converting a function group at the N- or C-terminal; oxidizing a straight chain polypeptide by boron tristrifluoroacetate. This process involves a time-consuming, multi-step synthesis, and it is difficult to separate octreotide from the reaction mixtures since all the synthesis steps are carried out in liquid phase.Another solution phase approach described by Chaturvedi, et al., (Wockhardt) in U.S. Pat. No. 6,987,167 and EP 1506219 A, claims the cyclization of partially deprotected octreotide in the solution phase using iodine under conditions and for a time sufficient to form the octreotide.

Synthesis in solid phase have been described subsequently (Mergler et al., Alsina et al., Neugebauer). The above prior art for solid phase peptide synthesis cites the octapeptide formation, by starting the synthesis from the threoninol residue which makes it mandatory to protect this residue. Mergler et al., (Peptides: Chemistry and Biology. Proceedings of the 12* American Peptide Symposium. Smith, J.A. And Rivier J.E. Eds ESCOM, Leiden, Poster 292 Presentation, (1991) ) describes a synthetic process, using an aminoethyl resin upon which the Threoninol residue is incorporated with the two alcohol functions protected in acetal form The synthesis is carried out following an Fmoc/tBu protection scheme, forming the disulphide bridge on resin by oxidation of the thiol groups of the previously deprotected cysteine residues and releasing and deprotecting the peptide with a 20% mixture of TFA/DCM.

In early 1997, Alsina J. et al. ( Alsina J., Chiva C, Ortiz M., Rabanal F., Giralt E., and Albericio F., Tetrahedron Letters, 38, 883-886, 1997) described the incorporation, on active carbonate resins, of a Threoninol residue with the amino group protected by the Boc group and the side chain protected by a BzI group. The synthesis was then continued by Boc/Bzl strategy. Formation of the disulfide bridge was carried out directly on resin using iodine and the peptide was cleaved from the resin and its side chain protecting groups were simultaneously removed with HF/anisole 9/1. At the final stage the formyl group was removed with a piperidine/DMF solution.

Neugebauer (Neugebauer W., Lefevre M.R., Laprise R, Escher E., Peptides: Chemistry, Structure and Biology, p 1017, Marshal G.R. And Rivier J.E. Eds. ESCOM.Leiden (1990) described a linear synthesis with a yield of only 7%.

Edwards et al., (Edwards B.W., Fields C.G., Anderson CJ., Pajeau T.S., Welch M.J., Fields G.B., J.Med.Chem. 37, 3749-3757 (1994) carried out another another solid- phase type approximation; they synthesized step-by-step on the resin, the peptide D- Phe-Cys(Acm)-Phe-D-Tφ(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-HMP-Resin. Next they proceeded to form the disulfide on resin and then release the peptide from the resin by means of aminolysis with threoninol, with obtaining a total yield of only 14%.

The solid phase synthesis described by Yao-Tsung Hsieh et. al., in U.S. Pat. No. 6,476,186 involves the synthesis of octreotide by using Thr(ol)(tBu)-2Cl-trityl resin as starting material followed by the cleavage of the straight chain peptide from the resin by using a strong acid and the formation of the intra-molecular disulfide bond on the completely deprotected octreotide by oxidation using charcoal catalyst and a higher yield of >70%.

Another solid phase synthesis described by Berta Ponsati et.al (Lipotec) in U.S. Pat No. 6,346,601 and EP 0953577 B involve the coupling of threoninol on the protected heptapeptide in solution, after a selective acid cleavage from the chlorotrityl resin without affecting the peptide side-chain protecting groups.

A hybrid solid phase-liquid phase method for synthesis of octreotide described by Iarov et al., (Dalton Chemical Laboratories) in WO 2005087794 wherein the method comprises liquid phase condensation of two or three peptide blocks in which at least one peptide block is synthesized by solid-phase method.

EP 1511761 Bl involves cyclization on the semi-protected linear peptide wherein one of the cysteine residue is protected with an orthogonal protecting group. The radioactive isotope labeling of octreotide by the coupling of bifunctional chelating agents like DTPA or DOTA to the peptide was described by Te- Wei Lee et al., in U.S. Pat. No. 5,889,146 (Inst, of Nuclear Energy Research)

The method for cyclization of linear vapreotide by means of intramolecular cysteine formation has been described by Quattrini et. al., (Lonza AG) in WO 2006048144, wherein the process involves the synthesis of linear vapreotide peptide on Sieber-resin (from Novabiochem) by Fmoc standard groups, wherein the side chain protecting groups are D or L-Trp(Boc), Cys(Trt), Lys(Boc), Tyr(tBu). The protected peptide is cleaved off in 5% TFA in dichloromethane and then globally deprotected by acidolysis in a cleavage mix of 300 equivalents of concentrated TFA, 12 equivalents of Dithiothreitol, 12 equivalents of Dichloromethane, 50 equivalents of water forl hour at room temperature. The Boc groups are removed. The product was subjected to charcoal method using trace amounts of activated, powdered charcoal wherein a concentration of the linear cysteinyl peptide of 50 mg/ml (1 eq.) in DMF in the presence of 1 eq. Diisopropyl-ethyl-amine and that additionally air was sparged at low pressure into the liquid under stirring. After 15-20 hrs, 100% conversion was achieved with 84% (w/w) analytical yield of 79% vapreotide.

The formation of intramolecular disulphide formation in a polypeptide by reacting with hydrogen peroxide has been described by Mineo Niwa et al. (Fujisawa Pharmaceutical Co.) in U.S. Pat. No.5, 102,985 wherein the reaction is to be carried out at a pH of about 6 tol 1, wherein the molar ratio of H₂O₂ to polypeptide is within the range of 1:1 to 100:1. The above cited prior art mainly carries out the cyclization of the peptide on the resin or on partially protected or protected peptides. The use of partial or minimal protecting group strategies and improvement in the activation methods have considerable effect on limitations of poor solubility and possible danger of racemization due to the overactivation of carboxyl groups. However, these approaches do not overcome the problem of the poor coupling efficiency between large peptide segments, because of the intrinsic difficulty of obtaining effective molar concentrations for high molecular weight molecules.

Example 8:

Oxidation of S-H peptide with DMSO-HCl to get S-S peptide:

(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-OL

Formula (1)

S-H peptide ( 9g) was dissolved in 6.5L DMSO and under ice-cooling 6.5L IM HCl was added slowly so that temperature is below 26°C. Stirring was continued for 6 hours. At room temperature after six hours reaction mixture was diluted with 13L of water and filtered through Whatman no. 41 through Celite bed. The filtrate was loaded on C- 18 column for concentration. The compound was eluted with 100% acetonitrile. The eluant was concentrated on rotavap and then the concentrated solution was centri-evaporated to dryness. The RP-HPLC profile of crude octreotide is depicted in Figure 1.

Weight of crude peptide =3.9g.(45%)

Purity: 44.25%

Example 9:

Purification of crude octreotide:

The crude octreotide was loaded on to cation ion exchange column and eluted using a salt gradient using a Akta Purifier (by Amersham, Sweden) low pressure chromatography system. The IEX fractions of purity >70% were further loaded for RP-HPLC purification on Kromacil C-18 column of (250x50mm,100A°.) The peptide was purified by using aqueous TF A(O-0.5%) and methanol/ethanol and/or Acetonitrile in a gradient program on a Shimadzu preparative HPLC System consisting of a controller, 2 LC8A pumps, and UV-Vis detector. The purified peptide was analysed by analytical RP-HPLC (Figure 5). Fractions of > 99% purity were subjected either by RP-HPLC or IEX to salt exchange and concentrated to remove organic solvent either by rota or reverse osmosis and subsequently lyophilized to get final API with purification step yield of 70% or above.The MS spectrum of octreotide is depicted in Figure 6.

References

Ann-Teresa Cusenza

It is a great pleasure to write about ANN…. I read her blog everyday……………………….

Ann-Teresa Cusenza, MLS, MBA

 She is Managing Editor, Orphan Druganaut Blog

read at

http://orphandruganaut.wordpress.com/this is all about orphan drugs, great work ANN

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TUESDAY March 11, 2014, 2014 — The U.S. Food and Drug Administration on Tuesday approved the first device aimed at preventing migraines.

The device, called Cefaly, is a headband-like device that runs on a battery and sits across the forehead and over the ears, the FDA said in a statement.

“The user positions the device in the center of the forehead, just above the eyes, using a self-adhesive electrode,” the agency explained. “The device applies an electric current to the skin and underlying body tissues to stimulate branches of the trigeminal nerve, which has been associated with migraine headaches.”

Cefaly is made by Belgium-based Cefaly Technology and is available by prescription only. The device is only indicated for use by adults and should only be used for 20 minutes per day, the FDA said. The agency also noted that “the user may feel a tingling or massaging sensation where the electrode is applied.”

http://www.drugs.com/news/fda-approves-first-device-prevent-migraines-50727.html?utm_source=ddc&utm_medium=email&utm_campaign=Today%27s+news+summary+-+March+13%2C+2014

CEFALY DRUG-FREE MIGRAINE PAIN RELIEF

Millions of Cefaly treatments studied have proven that it is an effective, drug free, migraine pain reliever – Join the tens of thousands of people around the world who have tried Cefaly and are free of migraine pain and pain relieving medication.

Cefaly is a CE and ISO certified medical device designed to treat and prevent migraine headaches. Cefaly can considerably reduce or replace the consumption of side effect producing medications. Cefaly is the first cranial analgesic electrotherapeutic device to acquire ISO medical certification proven effective on migraine pain with no side effects.

Cefaly’s patented design uses TENS technology which has been researched in medical circles for over 40 years. TENS technology is known for its safety and dramatic absence of side effects, making Cefaly unique to all other headache and migraine pain treatments.

Cefaly is designed and developed in accordance with the strictest quality standards eliminating all possible short-term and long-term dangers.

Cefaly is an innovative, lightweight and extremely cost effective pain relieving solution. Its self adhesive electrode is placed directly on to the forehead. Worn conveniently like a pair of eyeglasses, Cefaly connects to the electrode and begins its subtle treatment.

read

http://www.cefaly.ca/#howitworks

The first non-inhaled treatment for a difficult-to-treat form of severe asthma is getting closer to market after GlaxoSmithKline said it would initiate global filings for the drug at the end of this year, on the back of strong late-stage clinical data.

Mepolizumab – a monoclonal antibody that inhibits interleukin 5 – is being investigated as a treatment for severe eosinophilic asthma in patients who experience exacerbations despite high-dose oral or inhaled corticosteroids (ICS) and an additional controller such as long-acting beta-2 agonist.

Read more at: http://www.pharmatimes.com/Article/14-03-13/GSK_to_file_severe_asthma_drug_by_year_end.aspx#ixzz2vuANtYaK
Follow us: @PharmaTimes on Twitter

Mepolizumab (proposed trade name Bosatria) is a humanized monoclonal antibody that recognizes interleukin-5 (IL-5), and is used to treat certain kinds of asthma and white blood cell diseases.

IL 5

Recent studies have concluded that mepolizumab may improve exacerbations in patients with severe eosinophilic asthma, an adult-onset asthma which represents less than 5% of all asthma.

IL-5 is a chemical messenger in the immune system that stimulates the growth of eosinophils. In eosinophilic asthma, eosinophils are present in the lungs. When mepolizumab was given to people with eosinophilic asthma, it eliminated eosinophils from the bloodstream,and reduced eosinophils in the lungs and bone marrow. Mepolizumab also reduced the number of asthma exacerbations, and reduced the need for corticosteroids.^[1]Mepolizumab improved the quality of life, but the improvement was “not clinically meaningful,” according to a reviewer.^[2] [3]

In a recent multi-centre, double-blinded, randomised, controlled trial study of Mepolizumab in severe eosinophilic asthma, Mepolizumab reduced the number of clinically significant exacerbations compared to a placebo. Additionally Mepolizumab reduced sputum and blood eosinophil counts and was shown to be safe for up to 12 months.^[4]

Mepolizumab is also in development for the management of hypereosinophilic syndrome by GlaxoSmithKline (GSK) and has received orphan drug designation by the FDA.^[5] Mepolizumab has been shown to reduce the need for corticosteroids and improve symptoms in FIP1L1/PDGFRA negative hypereosinophilic syndrome.^[6]

UK pharma giant GlaxoSmithKline (LSE: GSK) says that a pivotal Phase III study of mepolizumab, an investigational IL-5 antagonist monoclonal antibody, met its primary endpoint of reduction in the frequency of exacerbations, in patients with severe eosinophilic asthma.

Mepolizumab could add £400 million ($668 million) to GSK’s revenue by 2021, according to estimates from Barclays reported by The Wall Street Journal. Analysts from Deutsche Bank forecast £300 million in mepolizumab sales by 2018 for the company, already a leader in the asthma treatment sector.

The study (MEA115588) evaluated the efficacy of two-dose regimens of mepolizumab in the treatment of patients with severe eosinophilic asthma. Patients remained on their current asthma maintenance therapy throughout the study and were randomized to receive either mepolizumab 75mg intravenous (IV), 100mg subcutaneous (SC), or placebo every four weeks.

For the primary end point, both mepolizumab treatment arms showed statistically significant reductions in the frequency of clinically significant exacerbations of asthma compared to placebo (75mg IV, 47%, p<0.001; 100mg SC, 53%, p<0.001).

Adverse events reported in the study were similar across all treatment groups. The most common reported adverse events across all treatment groups were nasopharyngitis, headache, upper respiratory tract infection and asthma. The frequency of adverse events was 83% in the placebo group, 84% in the mepolizumab 75mg IV and 78% in the mepolizumab 100mg SC group. The frequency of serious adverse events was 14% in the placebo group, 7% in the mepolizumab 75mg IV and 8% in the mepolizumab 100mg SC group.

Backs up earlier studies; regulatory filing mooted at year end

Dave Allen, head of GSK Respiratory Therapy Area Unit, R&D, said: “We are really pleased to have generated further positive data on mepolizumab, consistent with the findings from our earlier exacerbation study. We now have two studies showing a reduction in exacerbations in a specific group of patients with a severe form of asthma who continue to exacerbate despite treatment with high doses of their current maintenance therapies. This is very positive news for patients. For GSK it is exciting that this is the first non-inhaled treatment for severe asthma and we will be progressing towards global filings at the end of the year.”

In addition, a second Phase III study (MEA115575) designed to evaluate the use of mepolizumab 100mg SC, every four weeks in comparison to placebo in reducing daily oral corticosteroid use while maintaining asthma control also met its primary endpoint. The study showed that patients on mepolizumab 100mg SC were able to achieve greater reductions in their maintenance oral corticosteroid dose during weeks 20-24 compared to patients on placebo (p =0.008), while maintaining asthma control.

In this study adverse events were similar across treatment groups. The most common reported adverse events in the two treatment groups were headache, nasopharyngitis, bronchitis, sinusitis, fatigue and asthma. The frequency of adverse events was 92% in the placebo and 84% in the mepolizumab treatment group. Frequency of serious adverse events was 18% in the placebo group and 1% in the mepolizumab group.

Mepolizumab Useful in Refractory Eosinophilic Asthma, a Rare Subtype of Asthma

Eosinophilic inflammation, which may be a consequence of interleukin-5 action, is a characteristic feature of some forms of asthma.A study examined the prednisone-sparing effect of mepolizumab (proposed trade name Bosatria), a monoclonal antibody against interleukin-5, in a rare subgroup of patients who have sputum eosinophilia and airway symptoms despite continued treatment with prednisone.
Eosinophil.
Eosinophil.Eeosinophilic form of asthma represents less than 5% of cases of adult-onset asthma and is difficult to treat.

Crystal structure of human IL-5. .

Mepolizumab reduced the number of blood and sputum eosinophils and allowed prednisone sparing in patients who had asthma with sputum eosinophilia despite prednisone treatment.

Mepolizumab therapy reduced exacerbations by 43% and improved Asthma Quality of Life Questionnaire (AQLQ) scores in patients with refractory eosinophilic asthma.

Eosinophils may have a role as important effector cells in the pathogenesis of severe exacerbations of asthma in patients with eosinophilic asthma.

Cytokine targets for immunomodulators for allergic disorders.

Mediators from Eosinophils

References

EMA publishes New Process Validation Guideline

After the publication of the Annex 15 draft at the beginning of February 2014, the EMA made a move towards the revision of its process validation guideline. The final document was published on 27 February 2014. For a long time now, the EMA had already announced this revision in a concept paper. What’s new?  click here

After the publication of the Annex 15 draft at the beginning of February 2014, the EMA made a move towards the revision of its process validation guideline. The final document was published on 27 February 2014. For a long time now, the EMA had already announced this revision in a concept paper. The objective of the revision was to integrate modern GMP aspects:

• Integration of the ICH Q8, Q9 and Q10 Guidelines
• Incorporation of Process Analytical Technology (PAT), Quality by Design (QbD) and Real-Time Release Testing (RTRT).
• Extension with regard to an “enhanced approach” and integration of “continuous process verification”
• Integration of the Annexes to the current Note for Guidance
• Harmonisation with the current FDA Guidance on Process Validation 

The deadline for comments on the draft for the revision of the process validation guideline ended in October 2012 already. Now, elements in accordance with the Annex 15 have also flowed into the final document. In the following, you will read a short evaluation of the document with regard to the original draft from March 2012, the (still) applicable Note for Guidance on Process Validation and FDA’s Guidance on Process Validation. The GMP relevant aspects of the documents will also be addressed.

The original 7-page long Note for Guidance on Process Validation has more than doubled and now contains 15 pages. Even the original revision draft had only 11 pages. The change in the title to “Guideline on process validation for finished products- information and data to be provided in regulatory submissions” is noticeable. The title itself gives indication about the content of the document, namely marketing authorisation matters.

Like in the draft, the document is composed of 8 numerated chapters, a summary, definitions, references, an Annex I (Process validation scheme) and an Annex II (Standard/non-standard processes) which is a new part compared to the draft. A sub section on “Design space verification” has been newly added to the chapter on process validation.

There haven’t been big changes to the draft document released in 2012. Only the chapter “Design space verification” is brand new, all other parts have been mostly updated. The chapter on ongoing process validation has been removed. Compared to the draft, indications about standard/ non-standard processes are now available in the Annex II – like in the currently applicable Note for Guidance.

What are the changes to the currently applicable Note for Guidance on Process Validation?

Compared to the current Note for Guidance, the revision remains in its final version pretty difficult to read and rather general. This is a marketing authorisation document, which is clearly addressed in the title and only applies to finished dosage forms of chemical medicinal products for human and veterinary use but not for old ones, which are already authorised and on the market. The introduction of a validation life cycle and the integration of continued process verification (CPV) are completely new although this approach is already acquainted from ICH Q8. The “traditional approach” remains accepted. Like in the Annex 15 draft the hybrid approach remains here in the final document “nebulous”. The idea to integrate modern elements from ICH Q8, Q10 (and Q11) into the document is clearly noticeable. Yet, far less concrete references are made to ICH Q9.

A stronger overlap of the FDA Guidance would have been desirable. FDA’s Guidance also deals with APIs and biologicals, and the process validation life cycle runs like a thread through the whole FDA document. FDA’s Guidance also contains GMP aspects. The FDA Guidance explicitly addresses old products which should be integrated to stage 3 of the life cycle. Yet, there is another big difference. The revised document doesn’t highlight statistical methods like the FDA Guidance.

Before the finalisation, a comparison with the Annex 15 has been made which is a nice thing. This explains the long period between the publication of the draft (March 2012) and that of the finalisation (February 2014).

What is significant for the GMP world? On the one hand almost nothing, on the other hand quite a lot:  one may wonder why? Direct references to the Annex 15 can be found with regard to the “ongoing process verification” and “concurrent validation”,  which is almost nothing looking at the whole document. Moreover, validation in general is required to be executed according to the GMP regarding “continuous process verification” and “change control”; these are the essential parts of the document, and (almost) the complete document should therefore be seen from a GMP perspective.

The new EMA guideline on process validation will apply by the end of August 2014.

(NaturalNews) Researchers have just discovered that flavonoids extracted from common onions slow the rate of colon cancer growth in mice just as effectively as a chemotherapy drug. And while the mice on chemo saw their LDL cholesterol go up (a possible side effect of the drug), the mice on onion extract actually saw their LDL levels drop.

Onion flavonoids slow colon tumor growth by 67% in vivo

Learn more: http://www.naturalnews.com/044318_onion_extract_colon_cancer_chemotherapy_drug.html##ixzz2wD3udzfF

http://www.naturalnews.com/044318_onion_extract_colon_cancer_chemotherapy_drug.html#

Nemonoxacin 奈诺沙星

378746-64-6 CAS

TG-873870

WARNER CHILCOTT ORIGINATOR

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

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid

7-[3(S)-Amino-5(S)-methylpiperidin-1-yl]-1-cyclopropyl-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
Taigexyn has been approved in Taiwan IN 2014

Nemonoxacin is a novel non-fluorinated quinolone antibiotic undergoing clinical trials.

Taigexyn Granted QIDP and Fast Track Designations

TaiGen Biotechnology announced that the FDA has granted nemonoxacin (Taigexyn) Qualified Infectious Disease Product (QIDP) and Fast Track designations for community-acquired bacterial pneumonia (CAP) and acute bacterial skin and skin structure infections (ABSSSI).

Safety and clinical pharmacokinetics of nemonoxacin, a novel non-fluorinated quinolone, in healthy Chinese volunteers following single and multiple oral doses

Nemonoxacin is a novel non-fluorinated quinolone broad spectrum antibiotic available in both oral and intravenous formulations. Nemonoxacin demonstrates activity against gram-positive and gram-negative bacteria and atypical pathogens. Nemonoxacin also possesses activities against methicillin-­resistant Staphylococcus aureus (MRSA) and vancomycin-resistant pathogens.

Nemonoxacin is a novel non-flourinated quinolone antibiotic registered in Taiwan for the oral treatment of community-acquired pneumonia. Clinical trials are in development at TaiGen Biotechnology for the treatment of diabetic foot infections and for the treatment of moderate to severe community-acquired pneumonia with an intravenous formulation. The drug is thought to accomplish its antibacterial action through topoisomerase inhibition.

Originally developed at Procter & Gamble, nemonoxacin was the subject of a strategic alliance formed in January 2005 between P&G and TaiGen to further the development and commercialization of nemonoxacin. In 2012, the product was licensed by TaiGen Biotechnology to Zhejiang Medicine in China for manufacturing, sales and marketing. In 2014, TaiGen out-licensed the exclusive rights of the product in Russian Federation, Commonwealth Independent States and Turkey to R-Pharm.

TaiGen has completed two Phase 2 clinical studies, one in CAP and the other in diabetic foot infections with demonstrated efficacy and safety. In the clinical trials conducted to date, nemonoxacin has shown activity against drug-resistant bacteria such as MRSA, quinolone-resistant MRSA, as well as quinolone-resistant Streptococcus pneumoniae.

Malate salt

Nemonoxacin malate anhydrous
951163-60-3 CAS NO, MW: 505.5209

Nemonoxacin malate hemihydrate
951313-26-1, MW: 1029.0566

Chemical structure of nemonoxacin as a malate salt (C20H25N3O4·C4H6O5·H2O). Nemonoxacin is the free base, and its molecular mass is 371.44 g/mol. The molecular mass of the salt, nemonoxacin malate, is 514.53 g/mol.

……………………..

isomeric compounds are:

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD1…….DESIRED

(3S,5R)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD 1′….NOT DESIRED

EP2303271A1

Example 1

Malate salts of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1) and (3S,5R)-7- [3-ammo-5-methyl-piperidinyl]- 1 -cyclopropyl- 1 ,4-dihydro-8-methoxy-4-oxo-3- quinolinecarboxylic acid (Compound 1′) were synthesized as follows:

(A) Synthesis of (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (Compound 9) and (3S,5R)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (Compound 9′): Compound 9′ was synthesized as shown in Scheme 1 below:

Scheme 1

3 4 Boc

A 50-L reactor was charged with Compound 2 (5.50 kg, 42.60 mol), methanol (27 L) and cooled to 10-15⁰C. Thionyl chloride (10.11 kg, 2.0 equiv.) was added via an addition funnel over a period of 65 min, with external cooling to keep temperature below 30°. The resulting solution was stirred at 25⁰C for 1.0 hour, after which methanol was removed under reduced pressure. The oily residue was azeotroped with ethyl acetate (3 x 2.5 L) to remove residual methanol, dissolved in ethyl acetate (27.4 L), charged into a 50 L reactor, and neutralized by slow addition of triethylamine (3.6 kg) below 3O⁰C. The resulting suspension was filtered to remove triethylamine hydrochloride.

The filtrate was charged to a 50 L reactor, along with DMAP (0.53 kg). Di- fert-butyl dicarbonate (8.43 kg) was added via hot water heated addition funnel, over a period of 30 min at a temperature of 20-30⁰C. The reaction was complete after 1 hour as determined by TLC analysis. The organic phase was washed with ice cold IN HCl (2 x 7.5 L), saturated sodium bicarbonate solution (1 x 7.5 L), dried over magnesium sulfate, and filtered. After ethyl acetate was removed under reduced pressure, crystalline slurry was obtained, triturated with MTBE (10.0 L), and filtered to afford Compound 3 as a white solid (5.45 kg, 52.4%).

Anal. Calcd for C_HH_I7NO₅ : C, 54.3; H, 7.04; N, 5.76. Found: C, 54.5; H, 6.96; N, 5.80. HRMS (ESI⁺) Expected for C_HH_I8NO₅, [M+H] 244.1185. Found

244.1174; ¹H NMR (CDCl₃, 500 MHz):δ=4.54 (dd, J= 3.1, 9.5 Hz, IH), 3.7 (s, 3H), 2.58-2.50 (m, IH), 2.41 (ddd, IH, J= 17.6, 9.5, 3.7), 2.30-2.23 (m, IH), 1.98-1.93 (m, IH), 1.40 (s, 9H); ¹³C NMR (CDCl₃, 125.70 MHz) δ 173.3, 171.9, 149.2, 83.5, 58.8, 52.5, 31.1, 27.9, 21.5. Mp 70.2⁰C.

A 50-L reactor was charged with Compound 3 (7.25 kg, 28.8 mol), DME (6.31 kg), and Bredereck’s Reagent (7.7 kg, 44.2 mole). The solution was agitated and heated to 75⁰C + 5⁰C for three hours. The reaction was cooled to O⁰C over an hour, during which time a precipitate formed. The mixture was kept at O⁰C for an hour, filtered, and dried in a vacuum oven for at least 30 hours at 3O⁰C + 5⁰C to give compound 4 as a white crystalline solid (6.93 kg, 77.9%).

Anal. Calcd for Ci₄H₂₂N₂O₅: C, 56.4; H, 7.43; N, 9.39. Found C, 56.4; H, 7.32; N, 9.48; HRMS (ESI⁺) Expected for Ci₄H₂₂N₂O₅, [M+H] 299.1607. Found 299.1613; ¹H NMR (CDCl₃, 499.8 MHz) δ = 7.11 (s, IH), 4.54 (dd, IH, J= 10.8, 3.6), 3.74 (s, 3H), 3.28-3.19 (m, IH), 3.00 (s, 6H), 2.97-2.85 (m,lH), 1.48 (s, 9H); ¹³C NMR (CDCl₃, 125.7 MHz) δ = 172.6, 169.5, 150.5, 146.5, 90.8, 82.2, 56.0, 52.3, 42.0, 28.1, 26.3. MP 127.9⁰C. A 10-gallon Pfaudler reactor was charged with ESCAT 142 (Engelhard Corp.

N.J, US) 5% palladium powder on carbon (50% wet, 0.58 kg wet wt), Compound 4 (1.89 kg, 6.33 mol), and isopropanol (22.4 Kg). After agitated under a 45-psi hydrogen atmosphere at 45⁰C for 18 hrs, the reaction mixture was cooled to room temperature and filtered though a bed of Celite (0.51 kg). The filtrate was evaporated under reduced pressure to give a thick oil, which was solidified on standing to afford Compound 5 (1.69 kg, 100%) as a 93:7 diastereomeric mixture.

A sample of product mixture was purified by preparative HPLC to give material for analytical data. Anal. Calcd for Ci₂Hi₉NO₅: C, 56.0; H, 7.44; N, 5.44. Found C, 55.8; H, 7.31; N, 5.44; MS (ESI⁺) Expected for Ci₂Hi₉NO₅, [M+H] 258.1342. Found 258.1321; ¹H NMR (CDCl₃, 499.8 MHz) δ = 4.44 (m, IH), 3.72 (s, 3H), 2.60-2.48 (m, 2H), 1.59-1.54 (m, IH), 1.43 (s, 9H), 1.20 (d, j = 6.8 Hz,3H); ¹³C NMR (CDCl₃, 125.7 MHz) δ = 175.7, 172.1, 149.5, 83.6, 57.4, 52.5, 37.5, 29.8, 27.9, 16.2. Mp 89.9⁰C.

A 50-L reactor was charged with Compound 5 (3.02 kg, 11.7 mol), absolute ethanol (8.22 kg), and MTBE (14.81 kg). Sodium borohydride (1.36 kg, 35.9 mol) was added in small portions at 0⁰C + 5⁰C. A small amount of effervescence was observed. The reaction mixture was warmed to 1O⁰C + 5⁰C and calcium chloride dihydrate (2.65 kg) was added in portions at 1O⁰C + 5⁰C over an hour. The reaction was allowed to warm to 2O⁰C + 5⁰C over one hour and agitated for an additional 12 hours at 20⁰C + 5⁰C. After the reaction was cooled to -5⁰C + 5⁰C, ice-cold 2N HCl (26.9 kg) was added slowly at of O⁰C + 5⁰C. Agitation was stopped. The lower aqueous phase was removed. The reactor was charged with aqueous saturated sodium bicarbonate (15.6 kg) over five minutes under agitation. Agitation was stopped again and the lower aqueous phase was removed. The reactor was charged with magnesium sulfate (2.5 kg) and agitated for at leastlO minutes. The mixture was filtered though a nutsche filter, and concentrated under reduced pressure to afford Compound 6 (1.80 kg, 66%). Anal. Calcd for CnH₂₃NO₄: C, 56.6 H, 9.94; N, 6.00. Found C, 56.0; H, 9.68;

N, 5.96; HRMS (ESI⁺) Expected for CnH₂₄NO₄, [M+H] 234.1705. Found 234.1703; ¹H NMR (CDCl₃, 500 MHz) δ = 6.34 (d, J= 8.9 Hz, IH, NH), 4.51 (t, J= 5.8, 5.3 Hz, IH, NHCHCH₂OH), 4.34 (t, J= 5.3, 5.3 Hz, IH, OBCHCH₂OH), 3.46-3.45, (m, IH, NHCH), 3.28 (dd, J= 10.6, 5.3 Hz, NHCHCHHOH), 3.21 (dd, J= 10.2, 5.8 Hz , IH, CH₃CHCHHOH), 3.16 (dd, J = 10.2, 6.2 Hz, IH, NHCHCHHOH), 3.12 (dd, J= 10.6, 7.1 Hz , IH, CH₃CHCHHOH), 1.53-1.50 (m, IH, CH₃CHCHHOH), 1.35 (s, 9H, 0(CH_B)₃, 1.30 (ddd, J = 13.9, 10.2, 3.7 Hz, IH, NHCHCHHCH), 1.14 (ddd, J= 13.6, 10.2, 3.4 Hz, IH, NHCHCHHCH), 0.80 (d, J= 6.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125.7 MHz) δ 156.1, 77.9, 50.8, 65.1, 67.6, 65.1, 35.6, 32.8, 29.0, 17.1. Mp 92.1⁰C. A 50 L reactor was charged with a solution of Compound 6 (5.1 kg) in isopropyl acetate (19.7 kg). The reaction was cooled to 15⁰C + 5°C and triethylamine (7.8 kg) was added at that temperature. The reactor was further cooled to O⁰C + 5⁰C and methanesulfonyl chloride (MsCl) (6.6 kg) was added. The reaction was stirred for a few hours and monitored for completion by HPLC or TLC. The reaction was quenched by saturated aqueous bicarbonate solution. The organic phase was isolated and washed successively with cold 10% aqueous triethylamine solution, cold aqueous HCl solution, cold saturated aqueous bicarbonate solution, and finally saturated aqueous brine solution. The organic phase was dried, filtered, and concentrated in vacuo below 55⁰C + 5⁰C to afford compound 7 as a solid/liquid slurry, which was used in the subsequent reaction without further purification.

After charged with 9.1 kg of neat benzylamine, a 50 L reactor was warmed to 55⁰C, at which temperature, a solution of compound 7 (8.2 kg) in 1,2- dimethoxyethane (14.1 kg) was added. After the addition, the reaction was stirred at 6O⁰C + 5⁰C for several hours and monitored for completion by TLC or HPLC. The reaction was cooled to ambient temperature and the solvent was removed under vacuum. The residue was diluted with 11.7 kg of 15% (v/v) ethyl acetate/hexanes solution and treated, while agitating, with 18.7 kg of 20% (wt) aqueous potassium carbonate solution. A triphasic mixture was obtained upon standing. The upper organic layer was collected. The isolated middle layer was extracted twice again with 11.7 kg portions of 15% (v/v) ethyl acetate/hexanes solution. The combined organic layers were concentrated under vacuum to give an oily residue. The residue was then purified by chromatography to afford Compound 8 as an oil. A 40 L pressure vessel was charged with 0.6 kg 50% wet, solid palladium on carbon (ElOl, 10 wt. %) under flow of nitrogen. A solution of Compound 8 (3.2 kg) in 13.7 kg of absolute ethanol was then added to the reactor under nitrogen. The reactor was purged with nitrogen and then pressurized with hydrogen at 45 psi. The reaction was then heated to 45°C. It was monitored by TLC or LC. Upon completion, the reaction was cooled to ambient temperature, vented, and purged with nitrogen. The mixture was filtered through a bed of Celite and the solid was washed with 2.8 kg of absolute ethanol. The filtrate was concentrated under vacuum to afford Compound 9 as a waxy solid.

TLC R/(Silica F₂₅₄, 70:30 v/v ethyl acetate-hexanes, KMnO₄ stain) = 0.12; ¹H NMR (300 MHz, CDCl₃) δ 5.31 (br s, IH), 3.80-3.68 (m, IH), 2.92 (d, J=I 1.4 Hz,

IH), 2.77 (AB quart, J_AB=12.0 Hz, v=50.2 Hz, 2H), 2.19 (t, J=10.7 Hz, IH), 1.82-1.68 (m, 2H), 1.54 (br s, IH), 1.43 (s, 9H), 1.25-1.15 (m, IH), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ: 155.3, 78.9, 54.3, 50.8, 45.3, 37.9, 28.4, 27.1, 19.2; MS (ESI+) m/z 215 (M+H), 429 (2M+H). Similarly, (3S,5R)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester

(Compound 9′) was synthesized as shown in Scheme 2.

Scheme 2

_HN Boc HN Boc

NaBH₄,EtOH _w - ^“ MsCI₁TEA . „ _. – - _. „ Benzyl Amine

THF EA₁CoId

(B) Synthesis of l-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-l,4-dihydro-quinoline-3- carboxylic acid (Compound 10): Compound 10 was prepared according to the method described in U.S. Patent

6,329,391.

(C) Synthesis of borone ester chelate of l-Cyclopropyl-7-fluoro-8-methoxy-4-oxo- l,4-dihydro-quinoline-3-carboxylic acid (Compound 11):

Scheme 3

Toluene, tert-Butylmethyl ether 20-50⁰C, filter

A reactor was charged with boron oxide (2.0 kg, 29 mol), glacial acetic acid (8.1 L, 142 mol), and acetic anhydride (16.2 L, 171 mol). The resulting mixture was refluxed at least 2 hours, and then cooled to 40⁰C, at which temperature, 7- fluoroquinolone acid compound 10 (14.2 kg, 51 mol) was added. The mixture was refluxed for at least 6 hours, and then cooled to about 90⁰C. Toluene (45 L) was added to the reaction. At 5O⁰C, terϊ-butylmethyl ether (19 L) was added to introduce precipitation. The mixture was then cooled to 20⁰C and filtered to isolate the precipitation. The isolated solid was then washed with teτt-butylmethyl ether (26 L) prior to drying in a vacuum oven at 4O⁰C (50 torr) to afford Compound 11 in a yield of 86.4%. Raman (cm ¹): 3084.7, 3022.3, 2930.8, 1709.2, 1620.8, 1548.5, 1468.0, 1397.7, 1368.3, 1338.5, 1201.5, 955.3, 653.9, 580.7, 552.8, 384.0, 305.8. NMR (CDCl₃, 300 MHz) δ (ppm): 9.22 (s, IH), 8.38-8.33 (m, IH), 7.54 (t, J=9.8 Hz, IH), 4.38-4.35 (m, IH), 4.13 (s, 3H), 2.04 (s, 6H), 1.42-1.38 (m, 2H), 1.34-1.29 (m, 2H). TLC (Whatman MKC18F Silica, 6θA, 200 μm), Mobile Phase: 1 :1 (v/v) CH₃CN : 0.5N NaCl (aq), UV (254/366 nm) visualization; R^O.4-0.5. (D) Synthesis of malate salt of (3S,5S)-7-[3-amino-5-methyl-piperidmyl]-l- cyclopropyl-l,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1) and malate salt of (3S,5R)-7-[3-amino-5-methyl-piperidmyl]-l-cyclopropyl-l,4- dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (Compound 1′)

Compound 1 was synthesized from compound 9 as shown in Scheme 4 below:

Scheme 4

5O⁰C 3 d

a 6 0 N HCI (aq) CH₂CI₂ 35°40°C 12 h t> Extract pH ad]ust to ~7-8 50″-65″C filter

A reactor was charged with Compound 11 (4.4 kg, 10.9 mol), Compound 9 (2.1 kg, 9.8 mol), triethylamine (TEA) (2.1 L, 14.8 mol), and acetonitrile (33.5 L, 15.7 L/kg). The resulting mixture was stirred at approximately 50⁰C till completion of the reaction, as monitored by HPLC or reverse phase TLC. It was cooled to approximately 35°C and the reaction volume was reduced to approximately half by distillation of acetonitrile under vacuum between 0-400 torr. After 28.2 kg of 3.0 N NaOH (aq) solution was added, the reaction mixture was warmed to approximately 4O⁰C, distilled under vacuum until no further distillates were observed, and hydro lyzed at room temperature. Upon completion of hydrolysis, which was monitored by HPLC or reverse phase TLC, 4-5 kg of glacial acetic acid was added to neutralize the reaction mixture.

The resulting solution was extracted 3 times with 12.7 kg (9.6 L) of dichloromethane. The organic layers were combined and transferred to another reactor. The reaction volume was reduced to approximately a half by evaporation at 40⁰C. After 20.2 Kg 6.0N HCl (aq) solution was added, the reaction mixture was stirred for at least 12 hours at 35°C. After the reaction was completed as monitored by HPLC or reverse phase TLC, agitation was discontinued to allow phase separation. The organic phase was removed and the aqueous layer was extracted with 12.7 kg (9.6 L) of dichloromethane. The aqueous layer was diluted with 18.3 kg distilled water and warmed to approximately 50⁰C. Dichloromethane was further removed by distillation under vacuum (100-400 torr).

The pH of the aqueous solution was then adjusted to 7.8-8.1 by adding about 9.42 kg of 3.0 N NaOH (aq) below 65°C. The reaction mixture was stirred at 50⁰C for at least an hour and then cooled to room temperature. The precipitate was isolated by suction filtration, washed twice with 5.2 kg of distilled water, and dried with suction for at least 12 hours and then in a convection oven at 55°C for additional 12 hours. Compound 12 (3.2 kg, 79%) was obtained as a solid.

A reactor was charged with 3.2 kg of Compound 12 and 25.6 kg of 95% ethanol. To the reactor was added 1.1 kg of solid D,L-malic acid. The mixture was refluxed temperature (~80°C). Distilled water (-5.7 L) was added to dissolve the precipice and 0.2 kg of activated charcoal was added. The reaction mixture was passed through a filter. The clear filtrate was cooled to 45°C and allowed to sit for at least 2 hours to allow crystallization. After the reaction mixture was further cooled to 5°C, the precipitate was isolated by suction filtration, washed with 6.6 kg of 95% ethanol, and dried with suction for at least 4 hours. The solid was further dried in a convection oven at 45⁰C for at least 12 hours to afford 3.1 kg of Compound 1 (yield: 70%). NEMONOXACIN

NMR (D₂O, 300 MHz) δ (ppm): 8.54 (s, IH), 7.37 (d, J=9.0 Hz, IH), 7.05 (d, J=9.0 Hz, IH), 4.23-4.18 (m, IH), 4.10-3.89 (m, IH), 3.66 (br s, IH), 3.58 (s, 3H), 3.45 (d, J=9.0 Hz, IH), 3.34 (d, J=9.3 Hz, IH), 3.16 (d, J=12.9 Hz, IH), 2.65 (dd, J=16.1, 4.1 Hz, IH), 2.64-2.53 (m, IH), 2.46 (dd, J=16.1, 8.0 Hz, IH), 2.06 (br s, IH), 1.87 (d, J=14.4 Hz, IH), 1.58-1.45 (m, IH), 1.15-0.95 (m, 2H), 0.91 (d, J=6.3 Hz, 3H), 0.85-0.78 (m, 2H).

Similarly, Compound 1′ was synthesized from Compound 9′ as shown in Scheme 5 below:

Scheme 5

(3S,5R)-7-[3-amino-5-methyl-piperidinyl]-l-cyclopropyl-l,4-dihydro-8- methoxy-4-oxo-3 -quinolinecarboxylic acid

COMPD 1′….NOT DESIRED

…………………

US20070232650

US2007/232650 A1,

malate salts of

(3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (hereinafter Compound I, see also intermediate (23) in Section D, of Detailed Description of the Invention).

EXAMPLES Example 1 Synthesis of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid and malate salt thereof A. Synthesis of (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8)

(2S)-1-(1,1-Dimethylethyl)-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester, (2). A 50-L reactor is charged with compound (1) (5.50 Kg, 42.60 mol), methanol (27 L) and cooled to 10-15° C. Thionyl chloride (10.11 Kg, 2.0 equiv.) is added via addition funnel over a period of 65 min, with external cooling to maintain temperature at <30°. The resulting solution is stirred at 25° C.+5° C. for 1.0 hour, after which the methanol is distilled off under reduced pressure. The resulting thick oil is azeotroped with ethyl acetate (3×2.5 L) to remove residual methanol. The residue is dissolved in ethyl acetate (27.4 L), charged into a 50 L reactor, and neutralized by the addition of triethylamine (3.6 Kg) from an addition funnel over 30 minutes. The temperature of the neutralization is maintained below 30° C. via external cooling. The resulting suspension of triethylamine hydrochloride is removed by filtration, and the clarified mother liquor solution is charged to a 50 L reactor, along with DMAP (0.53 Kg). Di-tert-butyl dicarbonate (8.43 Kg) is added via hot water heated addition funnel, over a period of 30 min with external cooling to maintain temperature at about 20-30° C. The reaction is complete after 1 hour as determined by TLC analysis. The organic phase is washed with ice cold 1N HCl (2×7.5 L), saturated sodium bicarbonate solution (1×7.5 L), and dried over magnesium sulfate. The mixture is filtered through a nutsche filter and ethyl acetate is removed under reduced pressure to yield a crystalline slurry that is triturated with MTBE (10.0 L) and filtered to afford intermediate (2) as a white solid (5.45 Kg, 52.4%). Anal. Calcd for C₁₁H₁₇NO₅: C, 54.3; H, 7.04; N, 5.76. Found: C, 54.5; H, 6.96; N, 5.80. HRMS (ESI⁺) Expected for C₁₁H₁₈NO₅, [M+H] 244.1185. Found 244.1174; ¹H NMR (CDCl₃, 500 MHz): δ=4.54 (dd, J=3.1, 9.5 Hz, 1H), 3.7 (s, 3H), 2.58-2.50 (m, 1H), 2.41 (ddd, 1H, J=17.6, 9.5, 3.7), 2.30-2.23 (m, 1H), 1.98-1.93 (m, 1H), 1.40 (s, 9H); ¹³C NMR (CDCl₃, 125.70 MHz) δ 173.3, 171.9, 149.2, 83.5, 58.8, 52.5, 31.1, 27.9, 21.5; Mp 70.2° C.

(2S,4E)-1-(1,1-Dimethylethyl)-4-[(dimethylamino)methylene]-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester (3). A 50-L reactor is charged with intermediate (2) (7.25 Kg, 28.8 mol), DME (6.31 Kg), and Bredereck’s Reagent (7.7 Kg, 44.2 mole). The solution is agitated and heated to 75° C.±5° C. for at least three hours. The progress of the reaction is monitored by HPLC. The reaction is cooled to 0° C.±5° C. over on hour during which time a precipitate forms. The mixture is held at 0° C.±5° C. for one hour and filtered though a nutsche filter and the product dried in a vacuum oven for at least 30 hours at 30° C.±5° C. to give intermediate (3) as a white crystalline solid (6.93 Kg, 77.9%). Anal. Calcd for C₁₄H₂₂N₂O₅: C, 56.4; H, 7.43; N, 9.39. Found C, 56.4; H, 7.32; N, 9.48; HRMS (ESI⁺) Expected for C₁₄H₂₂N₂O₅, [M+H] 299.1607. Found 299.1613; ¹H NMR(CDCl₃, 499.8 MHz)δ=7.11 (s, 1H), 4.54 (dd, 1H, J=10.8, 3.6), 3.74 (s, 3H), 3.28-3.19 (m, 1H), 3.00 (s, 6H), 2.97-2.85 (m, 1H), 1.48 (s, 9H); ¹³C NMR (CDCl₃, 125.7 MHz) δ=172.6, 169.5, 150.5, 146.5, 90.8, 82.2, 56.0, 52.3, 42.0, 28.1, 26.3. Mp 127.9° C.

(2S,4S)-1-(1,1-Dimethylethyl)-4-methyl-5-oxo-1,2-pyrrolidinedicarboxylic acid-2-methyl ester (4). A 10-gallon Pfaudler reactor is inerted with nitrogen and charged with ESCAT 142 5% palladium powder on carbon (50% wet, 0.58 Kg wet wt.), intermediate (3) (1.89 Kg, 6.33 mol) and isopropanol (22.4 Kg). The reaction mixture is agitated under a 45-psi hydrogen atmosphere at 45° C. for 18 hrs. The reaction mixture is then cooled to room temperature and filtered though a bed of Celite (0.51 Kg) in a nutsche filter to remove catalyst. The mother liquor is evaporated under reduced pressure to give a thick oil that crystallizes on standing to afford 4 (1.69 Kg, 100%) as a 93:7 diastereomeric mixture. A sample of product mixture is purified by preparative HPLC to give material for analytical data. Anal. Calcd for C₁₂H₁₉NO₅: C, 56.0; H, 7.44; N, 5.44. Found C, 55.8; H, 7.31; N, 5.44; MS (ESI⁺) Expected for C₁₂H₁₉NO₅, [M+H] 258.1342. Found 258.1321; ¹H NMR (CDCl₃, 499.8 MHz) δ=4.44 (m, 1H), 3.72 (s, 3H), 2.60-2.48 (m, 2H), 1.59-1.54 (m, 1H), 1.43 (s, 9H), 1.20 (d, j=6.8 Hz,3H); ¹³C NMR (CDCl₃, 125.7 MHz) δ=175.7, 172.1, 149.5, 83.6, 57.4, 52.5, 37.5, 29.8, 27.9, 16.2. Mp 89.9° C.

(1S,3S)-(4-Hydroxyl-1-hydroxymethyl-3-methyl-butyl)-carbamic acid tert-butyl ester (5). A 50-L reactor is charged with intermediate (4) (3.02 Kg, 11.7 mol), absolute ethanol (8.22 Kg), and MTBE (14.81 Kg). The solution is agitated and cooled to 0° C.±5° C. and sodium borohydride (1.36 Kg, 35.9 mol) is added in small portions so as to maintain reaction temperature at 0° C.±5° C. A small amount of effervescence is observed. The reaction mixture is warmed to 10° C.±5° C. and calcium chloride dihydrate (2.65 Kg) is added portion wise at a slow rate over an hour so as to maintain a reaction temperature of 10° C.±5° C. The reaction is allowed to warm to 20° C.±5° C. over one hour and agitated for an additional 12 hours at 20° C.±5° C. The reaction is cooled to −5° C.±5° C., ice-cold 2N HCl (26.9 Kg) is added at a rate to maintain a reaction temperature of 0° C.±5° C. Agitation is stopped to allow phases to separate. The lower aqueous phase (pH=1) is removed. The reactor is charged with aqueous saturated sodium bicarbonate (15.6 Kg) over five minutes. Agitation is stopped to allow phases to separate. The lower aqueous phase (pH=8) is removed. The reactor is charged with magnesium sulfate (2.5 Kg) and agitated for at least 10 minutes. The mixture is filtered though a nutsche filter, and condensed under reduced pressure to afford intermediate (5) (1.80 Kg, 66%). Anal. Calcd for C₁₁H₂₃NO₄: C, 56.6; H, 9.94; N, 6.00. Found C, 56.0; H, 9.68; N, 5.96; HRMS (ESI⁺) Expected for C₁₁H₂₄NO₄, [M+H] 234.1705. Found 234.1703; ¹H NMR (CDCl₃, 500 MHz)δ=6.34(d, J=8.9 Hz, 1H, NH), 4.51 (t, J=5.8, 5.3 Hz, 1H, NHCHCH₂OH), 4.34 (t, J=5.3, 5.3 Hz, 1H, CH3CHCH₂OH), 3.46-3.45, (m, 1H, NHCH), 3.28 (dd, J=10.6, 5.3 Hz, NHCHCHHOH), 3.21 (dd, J=10.2, 5.8 Hz, 1H, CH₃CHCHHOH), 3.16 (dd, J=10.2, 6.2 Hz, 1H, NHCHCHHOH), 3.12 (dd, J=10.6, 7.1 Hz, 1H, CH₃CHCHHOH), 1.53-1.50 (m, 1H, CH₃CHCHHOH), 1.35 (s, 9H, O(CH ₃)₃, 1.30 (ddd, J=13.9, 10.2, 3.7 Hz, 1H, NHCHCHHCH), 1.14 (ddd, J=13.6, 10.2, 3.4 Hz, 1H, NHCHCHHCH), 0.80 (d, J=6.6 Hz, 3H, CH₃); ¹³C NMR (CDCl₃, 125.7 MHz) δ 156.1, 77.9, 50.8, 65.1, 67.6, 65.1, 35.6, 32.8, 29.0, 17.1. Mp 92.1° C.

(2S,4S)-Methanesulfonic acid 2-tert-butoxycarbonylamino-5-methanesulfonyloxy-4-methyl-pentyl ester (6). A 50 L reactor is charged with a solution of intermediate (5) (5.1 Kg) in isopropyl acetate (i-PrOAc) 11.8 Kg followed by a rinse with an additional 7.9 Kg i-PrOAc. The reaction is cooled to 15° C.±5° C. and triethylamine (TEA) (7.8 Kg) is added while maintaining the set temperature. The reactor is further cooled to 0° C.±5° C. and methanesulfonyl chloride (MsCl) (6.6 Kg) is added to the reaction solution while maintaining the set temperature. The reaction is stirred for a few hours and monitored for completion by HPLC or TLC. The reaction is quenched by the addition of a saturated aqueous bicarbonate solution and the resulting isolated organic phase is washed successively with cold 10% aqueous triethylamine solution, cold aqueous HCl solution, cold saturated aqueous bicarbonate solution, and finally saturated aqueous brine solution. The organic phase is dried, filtered, and concentrated in vacuo below 55° C.±5° C. until a solid/liquid slurry containing intermediate (6) is obtained. The slurry is used crude in subsequent reaction without further characterization.

(3S,5S)-(1-Benzyl-5-methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (7). A 50 L reactor is charged with 9.1 Kg of neat benzylamine. The reactor is brought to 55° C. and a solution of intermediate (6) (8.2 Kg) in 1,2-dimethoxyethane (DME) (14.1 Kg) is added to the reactor while maintaining a temperature of 60° C.±5° C. After complete addition of this solution, the reaction is stirred at 60° C.±5° C. for several hours and monitored for completion by TLC or HPLC. The reaction is cooled to ambient temperature and volatiles (DME) are removed by rotary evaporation under vacuum. The residue is diluted with 11.7 Kg of 15% (v/v) ethyl acetate/hexanes solution and treated, while agitating, with 18.7 Kg of 20% (wt) aqueous potassium carbonate solution. A triphasic mixture is obtained upon settling. The bottom aqueous phase is removed and the middle phase is set aside. The upper organic phase is collected and held for combination with extracts from additional extractions. The isolated middle phase is extracted twice again with 11.7 Kg portions of 15% (v/v) ethyl acetate/hexanes solution, each time combining the extracts with original organic phase. The combined organic extracts are transferred into a rotary evaporator and solvent is removed under vacuum until an oily residue remains. The residue is then purified via large-scale preparative chromatography to afford purified intermediate (7) as an oil.

(3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8). A 40 L pressure vessel is charged with 0.6 Kg 50% wet, solid palladium on carbon (E101, 10 wt. %) under flow of nitrogen. A solution of 3.2 Kg intermediate (7) in 13.7 Kg of absolute ethanol is then charged to the reactor under nitrogen. The reactor is purged with nitrogen and is then pressurized with hydrogen at 45 psi. The reaction is then heated to 45° C. while maintaining a hydrogen pressure of 45 psi. The reaction is monitored by TLC or LC until complete. The reaction is cooled to ambient temperature, vented, and purged with nitrogen. The reactor contents are filtered through a bed of Celite and the solids are washed with 2.8 Kg of absolute ethanol. The filtrate is concentrated by rotary evaporation under vacuum until a waxy solid is obtained to afford intermediate (8): TLC R_f (Silica F₂₅₄, 70:30 v/v ethyl acetate-hexanes, KMnO₄ stain)=0.12; ¹H NMR (300 MHz, CDCl₃) δ 5.31 (br s, 1H), 3.80-3.68 (m, 1H), 2.92 (d, J=11.4 Hz, 1H), 2.77 (AB quart, J_AB=12.0 Hz, Δν=50.2 Hz, 2H), 2.19 (t, J=10.7 Hz, 1H), 1.82-1.68 (m, 2H), 1.54 (br s, 1H), 1.43 (s, 9H), 1.25-1.15 (m, 1H), 0.83 (d, J=6.6 Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 155.3, 78.9, 54.3, 50.8, 45.3, 37.9, 28.4, 27.1, 19.2; MS (ESI⁺) m/z 215 (M+H), 429 (2M+H).

B. Synthesis of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (19)

Intermediate (12): A reactor is charged with a solution of intermediate (11) (1.2 Kg, 7.7 mol, 1.0 eq) in anhydrous toluene (12 L) followed by ethylene glycol (1.8 L, 15.7 mol, 4.2 eq) and solid p-toluenesulfonic acid (120 g, 10 wt. %). The reaction mixture is stirred at ambient temperature for at least 30 minutes and then heated to reflux, collecting the water/toluene azeotrope in a Dean Stark type trap apparatus until the reaction is complete as determined by TLC analysis (15% EtOAc/Hexanes v/v). Upon completion, the reaction is cooled to ambient temperature and poured into an aqueous solution of sodium bicarbonate (6 L). The organic toluene phase was removed and washed with saturated sodium bicarbonate solution (6 L), distilled water (2×6 L), and saturated aqueous brine (6 L). The organic phase was removed and dried over MgSO₄, filtered, and evaporated under reduced pressure to afford intermediate (12) as an oil (1.3 Kg, 86%). The material is used without further purification in subsequent reaction steps.

Intermediate (13): A reactor is charged with a solution of intermediate (12) (1.2 Kg, 6.0 mol, 1.0 eq) in anhydrous tetrahydrofuran (12 L) and n-butyllithium (2.5M in hexanes, 2.6 L, 6.6 mol, 1.1 eq) is added at −40° C., while maintaining this temperature throughout the addition. The reaction is stirred for at least one hour at −40° C. and trimethylborate (0.9 L, 7.8 mol, 1.3 eq) is added to the mixture while maintaining the temperature at or below −40° C. The reaction mixture is stirred for at least one hour at −40° C. until complete as determined by TLC analysis (30% EtOAc/Hexanes v/v). The reaction is warmed slightly to −30° C. and acetic acid (3 L) is added slowly. Upon complete addition, water is added (0.5 L) to the reaction and the mixture is allowed to quickly warm to ambient temperature while stirring overnight. Organic solvent is removed from the reaction by distillation under reduced pressure at 45° C. To the reaction residue is added 3-4 volumes of water (6 L) and 30% hydrogen peroxide (0.7 L, 1.0 eq) slowly at ambient temperature with cooling provided to control the exotherm. The reaction is stirred for at least an hour at ambient temperature until complete as determined by TLC (15% EtOAc/Hexanes v/v). The reaction mixture is cooled to 0-5° C. and excess peroxide is quenched with the addition of 10% aqueous sodium bisulfite solution (2 L). The mixture is tested to ensure a negative peroxide result and the reaction is acidified by the addition of 6N HCl (aq) (1.2 L). The reaction is stirred until the hydrolysis reaction is complete as determined by TLC or NMR analysis. The resulting solids are collected by suction filtration to afford intermediate (13) as a yellow solid (1.0 Kg, 79%).

Intermediate (14): A reactor is charged with intermediate (13) (0.53 Kg, 3.0 mol, 1.0 eq) and dissolved in dry toluene (2.7 Kg, 3.1 L). To this solution is added dimethylsulfate (0.49 Kg, 3.9 mol, 1.30 eq) followed by solid potassium carbonate (0.58 Kg, 4.2 mol, 1.4 eq). The reaction mixture is heated to reflux and held for at least 1 hour until complete as determined by HPLC. During this time, vigorous gas evolution is observed. The reaction is then cooled to ambient temperature and diluted with distilled water (3.2 L) along with 30% NaOH (aq) (0.13 Kg, 0.33 eq). The aqueous phase is separated and the remaining toluene phase is extracted twice more with distilled water (3.2 L) combined with 30% NaOH (aq) (0.13 Kg, 0.33 eq), removing the aqueous phase each time. The organic upper phase is concentrated by distillation in vacuo (<100 mbar) at approximately 40° C. until a concentrated toluene solution is achieved. The resulting solution is cooled to ambient temperature, checked for quality and yield by HPLC, and carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (14) assumed, 0.56 Kg).

Intermediate (15a,b): A reactor is charged with 1.8 Kg (2.1 L) anhydrous toluene along with sodium hydride (0.26 Kg, 6.6 mol, 2.20 eq) as a 60 wt. % dispersion in mineral oil. To this mixture is added (0.85 Kg, 7.2 mol, 2.4 eq) diethylcarbonate as the reaction mixture is heated to 90° C. over 1 hour. A solution of intermediate (14) (˜1.0 eq) in toluene from the previous step is added to the reaction while maintaining a temperature of 90° C.±5° C. Gas evolution can be observed during this addition. After complete addition, the reaction is stirred for at least 30 minutes or until complete as determined by HPLC analysis. Upon completion, the mixture is cooled to ambient temperature and diluted with 10 wt. % aqueous sulfuric acid (3.8 Kg, 3.9 mol, 1.3 eq) with agitation. The phases are allowed to separate and the lower aqueous phase is removed. The remaining organic phase is concentrated in vacuo (<100 mbar) at approximately 40° C. until a concentrated toluene solution is achieved. The resulting solution is cooled to ambient temperature and carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (15a,b) assumed, 0.85 Kg).

Intermediate (16a,b; 17a,b): A reactor is charged with a solution of intermediate (15a,b) (0.85 Kg, ˜3.0 mol, ˜1.0 eq) in toluene from the previous step. To the reactor is then added dimethylformamide-dimethylacetal (0.54 Kg, 4.5 mol, 1.5 eq) and the resulting solution is heated to reflux temperature (˜95-105° C.). The lower boiling solvent (methanol from reaction) is allowed to distill off while the temperature is maintained at ≧90° C. Heating is continued for at least 1 hour or until complete as determined by HPLC analysis. Upon completion, the reaction containing the mixture of intermediate (16a,b), is cooled to ambient temperature and toluene (1.8 Kg, 2.1 L) along with cyclopropylamine (0.21 Kg, 3.6 mol, 1.2 eq) are added to the reaction. The reaction is stirred at ambient temperature for at least 30 minutes until complete as determined by HPLC. Upon completion, the reaction is diluted with 10 wt. % aqueous sulfuric acid (2.9 Kg, 3.0 mol, 1.0 eq) with agitation, and the phases are then allowed to separate. The aqueous phase is removed and the organic phase is concentrated under reduced pressure (<100 mbar) at approximately 40° C. by distillation. When the desired concentration is achieved, the solution is cooled to ambient temperature and the toluene solution containing the mixture of intermediate (17a,b) is carried forward to the next step in the synthesis without further purification (theoretical yield for intermediate (17a,b) assumed, ˜1.1 Kg).

Intermediate (18): A reactor is charged with a solution of the mixture of intermediate (17a,b) (˜4.7 Kg, ˜3.0 mol) at ambient temperature. To the reactor is added N,O-bis(trimethylsilyl)acetamide (0.61 Kg, 3.0 mol, 1.0 eq) and the reaction is heated to reflux temperature (˜105-115° C.) for at least 30 minutes or until complete as determined by HPLC analysis. If not complete, an additional amount of N,O-bis(trimethylsilyl)acetamide (0.18 Kg, 0.9 mol, 0.3 eq) is added to the reaction to achieve completion. Upon completion, the reaction is cooled to below 40° C. and organic solvent is removed under reduced pressure (<100 mbar) at approximately 40° C. by distillation until a precipitate is formed. The reaction is cooled to ambient temperature and the precipitated solids are isolated by suction filtration and washed with distilled water twice (1×1.8 L, 1×0.9 L). The solid is dried to afford intermediate (18) as a white solid (0.76 Kg, 82%). The material is used without further purification in the next reaction step.

Intermediate (19): A reactor is charged with solid intermediate (18) (0.76 Kg, ˜2.5 mol, ˜1.0 eq) at ambient temperature followed by ethanol (5.3 Kg, 6.8 L) and 32 wt. % aqueous hydrochloric acid (1.1 Kg, 10 mol). The reaction mixture is brought to reflux temperature (76-80° C.) during which time the mixture first becomes homogeneous and later becomes heterogeneous. The mixture is heated at reflux for at least 5 hours or until complete as determined by TLC analysis (15% EtOAc/Hexanes v/v). Upon completion, the reaction is cooled to 0° C.±5° C. and the precipitated solid is isolated by filtration and washed with distilled water (1.7 Kg) followed by ethanol (1.7 Kg). The isolated solid is dried to afford intermediate (19) as a white solid (0.65 Kg, ˜95%). ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 14.58 (s, 1H), 8.9 (s, 1H), 8.25 (m, 1H), 7.35 (m, 1H), 4.35 (m, 1H), 4.08 (s, 3H), 1.3 (m, 2H), 1.1 (m, 2H) ¹⁹F NMR (CDCl₃+CFCl₃, 292 MHz) δ (ppm): −119. HPLC: 99.5% by area.

C. Synthesis of borone ester chelate of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (20)

A reactor is charged with boron oxide (2.0 Kg, 29 mol) followed by dilution with glacial acetic acid (8.1 L, 142 mol) and acetic anhydride (16.2 L, 171 mol). The resulting mixture is heated to reflux temperature for at least 2 hours. The reaction contents are cooled to 40° C. and the solid 7-fluoroquinolone acid intermediate (19) (14.2 Kg, 51 mol) is added to the reaction mixture. The mixture is again heated to reflux temperature for at least 6 hours. Reaction progress is monitored by HPLC and NMR. The mixture is cooled to approximately 90° C. and toluene (45 L) is added to the reaction. The reaction is further cooled to 50° C. and tert-butylmethyl ether (19 L) is added to the reaction mixture to bring about precipitation of the product. The mixture is then cooled to 20° C. and the solid product 19 is isolated by filtration. The isolated solids are then washed with tert-butylmethyl ether (26 L) prior to drying in a vacuum oven at 40° C. (50 torr). The product yield obtained for intermediate (20) in this reaction is 86.4%. Raman (cm⁻¹): 3084.7, 3022.3, 2930.8, 1709.2, 1620.8, 1548.5, 1468.0, 1397.7, 1368.3, 1338.5, 1201.5, 955.3, 653.9, 580.7, 552.8, 384.0, 305.8. NMR (CDCl₃, 300 MHz) δ (ppm): 9.22 (s, 1H), 8.38-8.33 (m, 1H), 7.54 (t, J=9.8 Hz, 1H), 4.38-4.35 (m, 1H), 4.13 (s, 3H), 2.04 (s, 6H), 1.42-1.38 (m, 2H), 1.34-1.29 (m, 2H). TLC (Whatman MKC18F Silica, 60 Å, 200 μm), Mobile Phase: 1:1 (v/v) CH₃CN:0.5N NaCl (aq), UV (254/366 nm) visualization; R_f=0.4-0.5.

D. Coupling of 1-Cyclopropyl-7-fluoro-8-methoxy-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (20) to (3S,5S)-(5-Methyl-piperidin-3-yl)-carbamic acid tert-butyl ester (8), and synthesis of malate salt of (3S,5S)-7-[3-amino-5-methyl-piperidinyl]-1-cyclopropyl-1,4-dihydro-8-methoxy-4-oxo-3-quinolinecarboxylic acid (25)

A reactor is charged with solid intermediate (20) (4.4 Kg, 10.9 mol) followed by dilution with a solution of triethylamine (TEA) (2.1 L, 14.8 mol) and piperidine side chain intermediate (8) (2.1 Kg, 9.8 mol) in acetonitrile (33.5 L, 15.7 L/Kg) at room temperature. The resulting mixture is warmed to approximately 50° C. until reaction is judged complete. Reaction progress is monitored by HPLC or reverse phase TLC. When complete, the reaction is cooled to approximately 35° C. and reaction volume is reduced to approximately half by distillation of acetonitrile under vacuum between 0-400 torr. The reactor is then charged with 28.2 Kg of 3.0N NaOH (aq) solution and the temperature is raised to approximately 40° C. Distillation under vacuum is continued between 1-4 hours or until no further distillates are observed. The reaction is then cooled to room temperature and the hydrolysis reaction is monitored by HPLC or reverse phase TLC. Upon completion, the reaction mixture is neutralized to a pH of between 6-8 by adding ˜4-5 Kg of glacial acetic acid. The reactor is then charged with 12.7 Kg (9.6 L) of dichloromethane as an extraction solvent, the mixture is agitated, phases are allowed to separate, and the organic dichloromethane phase is removed. The extraction process is repeated two additional times using 12.7 Kg (9.6 L) of dichloromethane, collecting the lower, organic phase each time. The aqueous phase is discarded and the organic extracts are combined in a single reactor. The reactor contents are heated to 40° C. and the reaction volume is reduced to approximately one half by distillation. The reactor is then charged with 20.2 Kg 6.0N HCl (aq) solution, the temperature is adjusted to 35° C., and agitation is allowed for at least 12 hours to permit the Boc deprotection reaction to occur. The reaction is monitored by HPLC or reverse phase TLC. When complete, agitation is discontinued and the phases are allowed to separate. The lower, organic phase is removed and set aside. The reactor is then charged with 12.7 Kg (9.6 L) of dichloromethane as an extraction solvent, the mixture is agitated, phases are allowed to separate, and the organic dichloromethane phase is removed. The organic extracts are combined and discarded. The remaining aqueous phase is diluted with 18.3 Kg distilled water and the temperature is raised to approximately 50° C. Distillation under vacuum (100-400 torr) is performed to remove residual dichloromethane from the reaction. The pH of the reaction is then adjusted to between 7.8-8.1 using about 9.42 Kg of 3.0N NaOH (aq) solution while keeping the temperature of the reaction below 65° C. The reaction is cooled to 50° C. and the precipitated solids are aged for at least an hour prior to cooling the mixture to room temperature. The solids are isolated by suction filtration and washed twice with 5.2 Kg portions of distilled water. The solids are dried for at least 12 hours with suction and then for an additional 12 hours in a convection oven at 55° C. The yield achieved for intermediate (23) in this example is 3.2 Kg (79%). A reactor is charged with 3.2 Kg solid intermediate (23) and the solids are suspended in 25.6 Kg of 95% ethanol as solvent. To the reactor is then added 1.1 Kg of solid D,L-malic acid (24), and the mixture is heated to reflux temperature (˜80° C.). Distilled water (˜5.7 L) is added to the reaction until a complete solution is achieved and 0.2 Kg of activated charcoal is added. The reaction mixture is passed through a filter to achieve clarification, cooled to 45° C. and held for a period of at least 2 hours to allow crystallization to occur. The reaction mixture is further cooled to 5° C. and the suspended solids are isolated by suction filtration. The solids are then washed with 6.6 KG of 95% ethanol and dried for at least 4 hours with suction under vacuum. The solids are then further dried in a convection oven for at least 12 hours at 45° C. to afford 3.1 Kg of intermediate (24) (70%). NMR (D₂O, 300 MHz) δ (ppm): 8.54 (s, 1H), 7.37 (d, J=9.0 Hz, 1H), 7.05 (d, J=9.0 Hz, 1H), 4.23-4.18 (m, 1H), 4.10-3.89 (m, 1H), 3.66 (br s, 1H), 3.58 (s, 3H), 3.45 (d, J=9.0 Hz, 1H), 3.34 (d, J=9.3 Hz, 1H), 3.16 (d, J=12.9 Hz, 1H), 2.65 (dd, J=16.1, 4.1 Hz, 1H), 2.64-2.53 (m, 1H), 2.46 (dd, J=16.1, 8.0 Hz, 1H), 2.06 (br s, 1H), 1.87 (d, J=14.4 Hz, 1H), 1.58-1.45 (m, 1H), 1.15-0.95 (m, 2H), 0.91 (d, J=6.3 Hz, 3H); 0.85-0.78 (m, 2H). TLC (Whatman MKC18F Silica, 60 Å, 200 μm), Mobile Phase: 1:1 (v/v) CH₃CN:0.5N NaCl (aq), UV (254/366 nm) visualization. HPLC: Mobile Phase H₂O with 0.1% formic acid/Acetonitrile with 0.1% formic acid, gradient elution with 88% H₂O/formic acid to 20% H₂O/formic acid, Zorbax SB-C8 4.6 mm×150 mm column, Part No. 883975.906, 1.5 ml/min rate, 20 min run time, 292 nm, Detector Model G1314A, S/N JP72003849, Quat Pump Model G1311A, S/N US72102299, Auto Sampler Model G1313A, S/N DE14918139, Degasser Model G1322A, S/N JP73007229; approximate retention time for intermediate (19): 13.0 min; approximate retention time for intermediate (20): 11.6 min; approximate retention time for intermediate (21): 16.3 min; approximate retention time for intermediate (22): 18.2 min; approximate retention time for intermediate (23): 8.6 min; approximate retention time for compound (25): 8.6 min.

………………..

REF

A. ARJONA ET AL: “Nemonoxacin“, DRUGS OF THE FUTURE, vol. 34, no. 3, 1 January 2009 (2009-01-01), page 196, XP55014485, ISSN: 0377-8282, DOI: 10.1358/dof.2009.034.03.1350294

WO 2007110834

WO 2007110835

WO 2007110836

WO 1999014214

WO 2010077798

1, nemonoxacin; 2, delafloxacin; 3, finafloxacin; 4, zabofloxacin; 5, JNJ-Q2; 6, DS-8587; 7, KPI-10; 8, ozenoxacin; 9, chinfloxacin; 10, ACH-702.

NEW DRUG APPROVALS WILL TOUCH 2 LAKH VIEWS THIS MONTH

THANKS FOR YOUR SUPPORT

http://newdrugapprovals.org/ 2 LAKH VIEWS SOON

DR ANTHONY MELVIN CRASTO

Vatiquinone

1213269-98-7 CAS ,

MW440.6636, C29 H44 O3

Reverses visual loss due to retinal ganglion degeneration.

• 2,5-Cyclohexadiene-1,4-dione, 2-((3R,6e,10E)-3-hydroxy-3,7,11,15-tetramethyl-6,10,14-hexadecatrien-1-yl)-3,5,6-trimethyl-

• alpha-Tocotrienol quinone
• EPI 743
• UNII-6O85FK9I0X
• Vatiquinone
• ATQ-3

Biogen Idec, Atlas Venture Pump $17M into Ataxion

• Biogen Idec and Atlas Venture have agreed to invest a combined $17 million of Series A financing in a nearly-year-old drug developer focused on hereditary ataxias. Biogen Idec is separately providing R&D and other funding to the company, called Ataxion. The biotech giant has the option to acquire Ataxion to continue development of the program upon completion of a Phase I multiple ascending dose (MAD) study at pre-negotiated terms, including undisclosed upfront and milestone payments. Earlier this month, Edison Pharmaceuticals won FDA “fast-track” designation for its own Fredrich’s ataxia drug, the company’s lead drug candidate EPI-743, now in Phase II trials. And on February 12, the developer of a preclinical gene therapy for Friedrich’s ataxia, Voyager Therapeutics, was launched by Third Rock Ventures with $45 million in Series A financing. read at http://www.genengnews.com/gen-news-highlights/biogen-idec-atlas-venture-pump-17m-into-ataxion/81249632/
• EPI-743 is being developed at Edison Pharmaceuticals in phase II clinical trials for several indications; Leigh syndrome, Friedreich’s ataxia, Parkinson’s disease, Pearson syndrome, cobalamin C deficiency syndrome and Rett’s syndrome. The licensee, Dainippon Sumitomo is developing the product in phase II/III study for the treatment of Leigh syndrome in children. Preclinical studies are also underway for the treatment of Huntington’s disease. In 2011, an orphan drug designation was assigned by the FDA for the treatment of inherited mitochondrial respiratory chain diseases and by the EMA for the treatment of Leigh syndrome, and in 2014, the FDA assigned another orphan drug for the treatment of Friedreich’s ataxia. In 2014, the product was granted fast track designation for this indication. In 2013, the compound was licensed to Dainippon Sumitomo Pharma by Edison Pharmaceuticals in Japan for development and commercialization for the treatment of pediatric orphan inherited mitochondrial and adult central nervous system diseases.
• OLD ARTICLE

19 February 2013 EPI-743 Vatiquinone  is a new drug that is based on vitamin E. Tests have shown that it can help improve the function of cells with mitochondrial problems. It may be able to treat people with genetic disorders that affect metabolism and mitochondria Edison Pharmaceuticals and Bambino Gesu Children’s Hospital have announced the commencement of EPI-743 Phase 2 cobalamin C deficiency syndrome trial. EPI-743 is an orally bioavailable small molecule and a member of the para-benzoquinone class of drugs. The trial’s principal investigator, Bambino Gesu Children’s Hospital, division of metabolism Professor Carlo Dionisi-Vici said, “Given the central role of glutathione in cellular redox balance and antioxidant defense systems, we are eager to explore whether a therapeutic that increases glutathione such as EPI-743 will provide clinical benefit.” Improvement in visual function is the primary endpoint of the placebo-controlled study while secondary outcome measurements assess neurologic and neuromuscular function, glutathione biomarkers, quality of life, in addition to safety parameters. The investigation is aimed at assessing the efficacy of EPI-743 in disorders of intermediary metabolism that also result in redox disturbances. EPI-743 is an orally absorbed small molecule that readily crosses into the central nervous system. It works by targeting the enzyme NADPH quinone oxidoreductase 1 (NQO1). Its mode of action is to synchronize energy generation in mitochondria with the need to counter cellular redox stress Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative and cardiodegenerative disorder caused by decreased levels of the protein frataxin. The disease causes the progressive loss of voluntary motor coordination (ataxia) and cardiac complications. Symptoms typically begin in childhood, and the disease progressively worsens as the patient grows older; patients eventually become wheelchair-bound due to motor disabilities. Patients with Friedreich’s ataxia develop loss of visual acuity or changes in color vision. Most have jerky eye movements (nystagmus), but these movements by themselves do not necessarily interfere with vision. ……………… Bioorg Med Chem Lett 2011, 21(12): 3693 http://www.sciencedirect.com/science/article/pii/S0960894X11005440 We report that α-tocotrienol quinone (ATQ3) is a metabolite of α-tocotrienol, and that ATQ3 is a potent cellular protectant against oxidative stress and aging. ATQ3 is orally bioavailable, crosses the blood–brain barrier, and has demonstrated clinical response in inherited mitochondrial disease in open label studies. ATQ3 activity is dependent upon reversible 2e-redox-cycling. ATQ3 may represent a broader class of unappreciated dietary-derived phytomolecular redox motifs that digitally encode biochemical data using redox state as a means to sense and transfer information essential for cellular function.

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WO2010126909 The active component of the formulation of the present invention is selected from alpha- tocotrienol quinone, beta-tocotrienol quinone, gamma-tocotrienol quinone, delta-tocotrienol quinone, and mixtures thereof. In one embodiment, the formulation of the present invention comprises alpha-tocotrienol quinone as the active component. In other embodiments, the formulations of the present invention comprise one or more tocotrienol quinones of Formula I or mixtures thereof, in a pharmaceutically acceptable vehicle, and in other embodiments, the formulations of the present invention comprise alpha-tocotrienol quinone in a pharmaceutically acceptable vehicle. In other particular embodiments, the formulations are administered orally. In other embodiments, the formulations of the present invention comprise one or more tocotrienol quinones of Formula I or mixtures thereof, in an ophthalmically acceptable vehicle for topical, periocular, or intraocular administration, and in other embodiments, the formulations of the present invention comprise alpha-tocotrienol quinone in an ophthalmically acceptable vehicle.

[0120] The formulations of the present invention comprise tocotrienol quinones which can be produced synthetically from the respective tocotrienol by oxidation with suitable oxidizing agents, as for example eerie ammonium nitrate (CAN). Particularly, the formulations of the present invention comprise alpha-tocotrienol quinone (CAS Reg. No. 1401-66-7) produced by oxidation of alpha-tocotrienol. A preferred process for the production of alpha-tocotrienol has been described in co-owned US provisional application USAN 61/197,585 titled “Process for Enrichment and Isolation of alpha-Tocotrienol from Natural Extracts”.

[0121] Syntheses of various members of the tocotrienol family in the d,l- or (RS)-form have been published, see for example Schudel et al, HeIv. Chim. Acta (1963) 46, 2517-2526; H. Mayer et al, HeIv. Chim. Acta (1967) 50, 1376-11393; H.-J. Kabbe et al, Synthesis (1978), 888-889; M. Kajiwara et al, Heterocycles (1980) 14, 1995-1998; S. Urano et al, Chem. Pharm. Bull. (1983) 31, 4341-4345, Pearce et al, J. Med Chem. (1992), 35, 3595-3606 and Pearce et al, J. Med. Chem. (1994). 37, 526-541. None of these reported processes lead to the natural form of the tocotrienols, but rather produces racemic mixtures. Syntheses of natural form d-tocotrienols have been published. See for example. J. Scott et al, HeIv. CMm. Acta (1976) 59, 290-306, Sato et al. (Japanese Patent 63063674); Sato et al. (Japanese Patent NoJP 01233278) and Couladouros et al. (US Patent No. 7,038,067).

[0122] While synthetic and natural tocopherols are readily available in the market, the natural tocotrienol supply is limited, and generally comprises a mixture of tocotrienols. Crude palm oil which is rich in tocotrienols (800-1500 ppm) offers a potential source of natural tocotrienols. Carotech, Malaysia is able to extract and concentrate tocotrienols from crude palm oil, by a process patented in U.S. Pat. No. 5,157,132. Tocomin®-50 typically comprises about 25.32% mixed tocotrienols (7.00% alpha-tocotrienol, 14.42% gamma-tocotrienol, 3.30% delta-tocotrienol and 0.6% beta-tocotrienol ), 6.90% alpha-tocopherol and other phytonutrients such as plant squalene, phytosterols, co-enzyme QlO and mixed carotenoids.

[0123] Other methods for isolation or enrichment of tocotrienol from certain plant oils and plant oil by-products have been described in the literature. For some examples of such isolation and purification processes, see for instance Top A. G. et al, U.S. Pat. No. 5,190,618; Lane R et al, U.S. Pat No. 6,239,171; Bellafiore, L. et al. U.S. Pat. No.6,395,915; May, CY et al, U.S. Pat. No.6,656,358; Jacobs, L et al, U.S. Pat. No. 6,838,104; Sumner, C et al. Int. Pat. Pub. WO 99/38860, or Jacobs, L, Int. Pat. Pub. WO 02/500054. The compounds for use in the present invention and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions for use in the present invention may be varied so as to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient. When administered in combination with other therapeutic agents, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.