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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

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


Astellas Pharma (Originator), LAUNCHED 1993


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.


Canada 2037408 2002-12-17 EXPIRY 2011-03-01
Canada 1338491 1996-07-30            2013-07-30
United States 5665727 1994-09-09            2014-09-09
United States 5260301 1994-02-28            2011-02-28

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 Link out

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:

Figure imgf000002_0001

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.”


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:

PROTOPIC® (tacrolimus) Structural Formula Illustration

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 PrografAdvagrafProtopic) 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.

Figure imgf000003_0001

(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….



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.


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).



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

Figure imgb0071

Synthetic Processes


Process 1

         (Introduction of common Hydroxy-Protective Group)

        Figure imgb0009


Process 2

         (Introduction of common Hydroxy-Protective Group)

        Figure imgb0010


Process 3

         (Formation of Double Bond)

        Figure imgb0011


Process 4

         (Oxidation of Hydroxyethylene Group)

        Figure imgb0012


Process 5

     (Reduction of Allyl Group)

    Figure imgb0013

    in which
    R¹, R², R³, n and the symbol of a line and dotted line are each as defined above,
    R 1 a

    Figure imgb0014

    and R 2 a

    Figure imgb0015

    are each commonly protected hydroxy, and
    R 2 b

    Figure imgb0016

    is a common leaving group.

    • The microorganism which can be used for the production of the FR-900506, FR-900520 and/or FR-900525 substances is FR-900506 FR-900520 and/or FR-900525 substance(s)-producing strain belonging to the genusStreptomyces, among which Streptomyces tsukubaensis No. 9993 has been newly isolated from a soil sample collected at Toyosato-cho, Tsukuba-gun, Ibaraki Prefecture, Japan.
    • A lyophilized sample of the newly isolated Streptomyces tsukubaensis No. 9993 has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology (No. 1-3, Higashi 1-chome, Yatabemachi Tsukuba-gun, Ibaraki Prefecture, Japan) under the deposit number of FERM P-7886 (deposited date: October 5th, 1984), and then converted to Budapest Treaty route of the same depository on October 19, 1985 under the new deposit number of FERM BP-927.
    • The Streptomyces tsukubaensis No. 9993 has the following morphological, cultural, biological and physiological characteristics.
  • This white powder of the FR-900506 substance could be transformed into a form of crystals by recrystallization thereof from acetonitrile, which possess the following physical and chemical properties.
    (1) Form and Color:
    colorless prisms

    Elemental Analysis:
    C: 64.30 %, H: 8.92 %, N: 1.77 %
    64.20 %, 8.86 %, 1.72 %,

    (3) Melting Point:
    127 – 129 °C
    (4) Specific Rotation:
    [α] 23 D

    Figure imgb0025

    : -84.4° (c = 1.02, CHCl₃)
    (5) ¹³C Nuclear Magnetic Resonance Spectrum:

    Figure imgb0026

    the chart of which being shown in Figure 3,
    (6) ¹H Nuclear Magnetic Resonance Spectrum:
    the chart of which being shown in Figure 4.

  • Other physical and chemical properties, that is, the color reaction, solubility, ultraviolet absorption spectrum, infrared absorption spectrum, thin layer chromatography and property of the substance of the colorless prisms of the FR-900506 substance were the same as those for the white powder of the same under the identical conditions.
  • From the above physical and chemical properties and the analysis of the X ray diffraction, the FR-900506 substance could be determined to have the following chemical structure.

    Figure imgb0027



    The total synthesis of FK-506 is described: This synthesis was performed by previously constructing three building fragments (XX), (XXXII) and (XLVI), which later were coupled sequentially. First the synthesis of these fragments will be presented, and afterwards their sequential coupling will be described. 1) (2RS,4R,6S,7R,8S,10R)-2-(Bis(dimethylamino)phosphono)-7-(tert-butyldimethylsilyloxy)-6,8-dimethoxy-10-(1,3-dithian-2-yl)-4-methylundecane (XX). The reaction of L-arabitol (I) with 2-acetoxyisobutyryl chloride in acetonitrile gives the diacetoxycompound (II), which by treatment with sodium methoxide in THF yields (2S,4S)-1,2:4,5-diepoxy-3-pentanol (III). The protection of (III) with TBS-Cl in THF affords the protected compound (IV), which is condensed with ethoxyacetylene (V) by means of butyllithium and boron trifluoride ethearate in THF giving the diacetylenic alcohol (VI). Cyclization of (VI) by means of HgCl2 and p-toluenesulfonic acid in refluxing ethanol yields the dilactone (VII), which is methylated by means of methyl iodide and lithium diisopropylamide in THF affording the methylated dilactone (VIII). The deprotection of (VIII) with HF in acetonitrile gives the hydroxydilactone (IX), which is benzylated with benzyl trichloroacetimidate and trifluoromethanesulfonic acid in dichloromethane-cyclohexane yielding the benzyl protected dilactone (X). The methanolysis of (X), followed by methylation with NaH and methyl iodide in DMF affords the nonanedioic ester (XI), which is debenzylated by hydrogenolysis with H2 over Pd/C in ethyl acetate giving the hydroxy diester (XII). The lactonization of (XII) with pyridinium p-toluenesulfonate in dichloromethane yields the lactone-methyl ester (XIII), which is selectively reduced with L-Selectride in THF affording the lactol-methyl ester (XIV). The reaction of (XIV) with propane-1,3-dithiol and boron trifluoride ethearate in dichloromethane gives the 1,3-dithiane derivative (XV), which by reduction of its lactone group with LiAlH4 in THF yields (2R,4S,5R,6S,8R)-8-(1,3-dithian-2-yl)-4,6-dimethoxy-2-methylnonane-1,5-diol (XVI). The reaction of (XVI) with I2, pyridine and triphenylphosphine in benzene affords the 1-iodo derivative (XVII), which is protected with TBS trifluoromethanesulfonate and triethylamine in dichloromethane giving the protected iodide (XVIII). Finally, this compound is condensed with ethylphosphonic acid bis(dimethylamide) (XIX) by means of butyllithium in THF to afford the first building fragment (XX).




    Isolation and Purification

    The cultured broth thus obtained was filtered with an aid of diatomaseous earth (5 kg). The mycelial cake was extracted with acetone (50 liters), yielding 50 liters of the extract. The acetone extract from mycelium and the filtrate (135 liters) were combined and passed through a column of a non-ionic adsorption resin “Diaion HP-20” (Trade Mark, maker Mitsubishi Chemical Industries Ltd.) (10 liters). After washing with water (30 liters) and 50% aqueous acetone (30 liters), elution was carried out with 75% aqueous acetone. The eluate (30 liters) was evaporated under reduced pressure to give residual water (2 liters). This residue was extracted with ethyl acetate (2 liters) three times. The ethyl acetate extract was concentrated under reduced pressure to give an oily residue. The oily residue was mixed with twice weight of acidic silica gel (special silica gel grade 12, maker Fuji Devision Co.), and this mixture was slurried in ethyl acetate. After evaporating the solvent, the resultant dry powder was subjected to column chromatography of the same acidic silica gel (800 ml) which was packed with n-hexane. The column was developed with n-hexane (3 liters), a mixture of n-hexane and ethyl acetate (4:1 v/v, 3 liters) and ethyl acetate (3 liters). The fractions containing the object compound were collected and concentrated under reduced pressure to give an oily residue. The oily residue was dissolved in a mixture of n-hexane and ethyl acetate (1:1 v/v, 30 ml) and subjected to column chromatography of silica gel (maker Merck Co., Ltd. 230-400 mesh) (500 ml) packed with the same solvents system. Elution was carried out with a mixture of n-hexane and ethyl acetate (1:1 v/v, 2 liters and 1:2 v/v, 1.5 liters) and ethyl acetate (1.5 liters).

    Fractions containing the first object compound were collected and concentrated under reduced pressure to give crude FR-900506 substance (3 g) in the form of yellowish powder.




Synthesis pathway

Synthesis a)

Trade Names

Country Trade name Manufacturer
Germany Advagraf Astellas
Prograf – “-
Protopic – “-
France Prograf – “-
Protopic – “-
United Kingdom – “- – “-
Italy Prograf Fujisawa
Japan – “- Astellas
USA – “- – “-
Ukraine Prograf Astellas Ireland Co.., Ltd., Ireland;
Fujisawa Ireland Ltd., Ireland
Protopic Astellas Ireland Co.., Ltd.. (Issue series and packaging), Ireland;
Astellas Toyama Co., Ltd.., Plant Toyama, Japan
Advagraf Astellas Ireland Co.., Ltd., Ireland


  • ampoules of 5 mg / 1 ml;
  • Capsules 0.5 mg, 1 mg, 5 mg;
  • granules 0.2%;
  • Ointment 0.1%


  1. Manufacturing; selection:
    • EP 184 162 (Fujisawa Pharmaceutical; appl. 11/6/1986; GB -prior. 05.02.1985, 1/4/1985).
  2. synthesis of FK-506:
    • EP 378 318 (Fujisawa Pharmaceutical; appl. 18.7.1990; USA-prior. 11.1.1989, 30.6.1989).
    • Ireland, R. et al .: J. Org. Chem. (JOCEAH) 61, 6856 (1996).
  3. Synthesis of Intermediates:
    • Danishefsky, SJ et al .: J. Org. Chem. (JOCEAH) 55 (9) 2786 (1990).
    • Schreiber, SL et al .: J. Am. Chem. Soc. (JACSAT) 112 (4), 5583 (1990).
    • US 4,940,797 (Fujisawa Pharmaceutical; 10.7.1990; USA-prior. 23.3.1989).
  4. Alternative synthesis :
    • Shinkai, I. et al .: J. Am. Chem. Soc. (JACSAT) 111 (3) 1157 (1989).
    • Shinkai, I. et al .: Tetrahedron Lett. (TELEAY) 29 (3), 281 (1988).




  1.  Kino T, Hatanaka H, Hashimoto M, Nishiyama M, Goto T, Okuhara M, Kohsaka M, Aoki H, Imanaka H (1987). “FK-506, a novel immunosuppressant isolated from a Streptomyces. I. Fermentation, isolation, and physico-chemical and biological characteristics.”. J Antibiot (Tokyo) 40 (9): 1249–55. PMID 2445721.
  2. Pritchard D (2005). “Sourcing a chemical succession for cyclosporin from parasites and human pathogens.”. Drug Discov Today 10 (10): 688–91. doi:10.1016/S1359-6446(05)03395-7.PMID 15896681. Supports source organism, but not team information
  3.  Ponner, B, Cvach, B (Fujisawa Pharmaceutical Co.): Protopic Update 2005
  4.  Healthy Ontario: Tacrolimus topical ointment
  5.  Alloway RR, Germain M, Osama Gaber, A, Bodziak KA, Mulgaonkar SP, Gohh RY, Kaplan B, Katz E, Beckert M, Gordon RD, A Phase II Open-Label, Multi-Center Prospective, Conversion Study in Stable Kidney Transplant Patients to Compare the Pharmacokinetics of LCP-Tacro Tablets Once-A-Day to Prograf Capsules Twice-A-Day. American Transplant Congress, 2008
  7. identifier: NCT01187953
  8.  William F. Ganong. Review of medical physiology (22nd ed.). Lange medical books. p. 530. ISBN 0-07-144040-2.
  9.  Liu J, Farmer J, Lane W, Friedman J, Weissman I, Schreiber S (1991). “Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes.”. Cell 66 (4): 807–15.doi:10.1016/0092-8674(91)90124-HPMID 1715244.
  10.  McCauley, Jerry (2004-05-19). “Long-Term Graft Survival In Kidney Transplant Recipients”Slide Set Series on Analyses of Immunosuppressive TherapiesMedscape. Retrieved 2006-06-06.
  11.  M.M. Abou-Jaoude, R. Naim, J. Shaheen, N. Naufal, S. Abboud, M. AlHabash, M. Darwish, A. Mulhem, A. Ojjeh, and W.Y. Almawi (2005). “Tacrolimus (FK506) versus cyclosporin microemulsion (Neoral) as maintenance immunosuppresion therapy in kidney transplant recipients.”. Transplantation Proceedings 37 (7): 3025–3028. doi:10.1016/j.transproceed.2005.08.040PMID 16213293.
  12.  Elizabeth Haddad, Vivian McAlister, Elizabeth Renouf, Richard Malthaner, Mette S. Kjaer, and Lise Lotte Gluud (2006). “Cyclosporin versus Tacrolimus for Liver Transplanted Patients”. In McAlister, Vivian. Cochrane Database of Systematic Reviews 4 (CD005161): CD005161. doi:10.1002/14651858.CD005161.pub2PMID 17054241.
  13.  J.G. O’Grady, A. Burroughs, P. Hardy, D. Elbourne, A. Truesdale, and The UK and Ireland Liver Transplant Study Group (2002). “Tacrolimus versus emulsified cyclosporin in liver transplantation: the TMC randomised controlled trial”. Lancet 360 (9340): 1119–1125. doi:10.1016/S0140-6736(02)11196-2PMID 12387959.
  14.  Baumgart DC, Pintoffl JP, Sturm A, Wiedenmann B, Dignass AU (2006). “Tacrolimus is safe and effective in patients with severe steroid-refractory or steroid-dependent inflammatory bowel disease–a long-term follow-up”. Am J Gastroenterol 101 (5): 1048–1056. doi:10.1111/j.1572-0241.2006.00524.xPMID 16573777.
  15.  Baumgart DC, MacDonald JK, Feagan BG (2008). “Tacrolimus (FK506) for induction of remission in refractory ulcerative colitis”. In Baumgart, Daniel C. Cochrane Database Syst Rev 16 (3): CD007216. doi:10.1002/14651858.CD007216PMID 18646177.
  16.  Silverberg, NB; Lin, P; Travis, L; Farley-Li, J; Mancini, AJ; Wagner, AM; Chamlin, SL; Paller, AS (2004). “Tacrolimus ointment promotes repigmentation of vitiligo in children: a review of 57 cases.”.Journal of the American Academy of Dermatology 51 (5): 760–6. doi:10.1016/j.jaad.2004.05.036PMID 15523355.
  17.  Naesens M, Kuypers DR, Sarwal M (2009). “Calcineurin inhibitor nephrotoxicity”. Clin. J. Am. Soc. Nephrol. 4 (2): 481–509. doi:10.2215/CJN.04800908PMID 19218475.
  18.  Miwa Y, Isozaki T, Wakabayashi K, et al. (2008). “Tacrolimus-induced lung injury in a rheumatoid arthritis patient with interstitial pneumonitis”. Mod Rheumatol 18 (2): 208–11. doi:10.1007/s10165-008-0034-3PMID 18306979.
  19.  O’Donnell MM, Williams JP, Weinrieb R, Denysenko L (2007). “Catatonic mutism after liver transplant rapidly reversed with lorazepam”Gen Hosp Psychiatry 29 (3): 280–1.doi:10.1016/j.genhosppsych.2007.01.004PMID 17484951.
  20.  Hanifin JM, Paller AS, Eichenfield L, Clark RA, Korman N, Weinstein G, Caro I, Jaracz E, Rico MJ; US Tacrolimus Ointment Study Group (2005). “Efficacy and safety of tacrolimus ointment treatment for up to 4 years in patients with atopic dermatitis”. J Am Acad Derm 53 (2 suppl 2): S186–94. doi:10.1016/j.jaad.2005.04.062PMID 16021174.
  21.  N H Cox and Catherine H Smith (December 2002). “Advice to dermatologists re topical tacrolimus” (PDF). Therapy Guidelines Committee. British Association of Dermatologists.
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  24. 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
  25. and U.S. Pat. No. 4,894,366 issued on Jan. 16, 1990!.
  26. Total synthesis of FK506 and an FKBP probe reagent, (C8,C9-13C2)-FK506
    J Am Chem Soc 1990, 112(14): 5583
  27. A diastereospecific, non-racemic synthesis of the C.10-C.18 segment of FK-506
    Tetrahedron Lett 1988, 29(3): 277



  1. Est-il possible de prendre deux trois phrases pour mon blog perso ?

  2. Vօus publiez continuellement des postes intéressants

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DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 30 year tenure till date Dec 2017, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 50 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 19 lakh plus views on New Drug Approvals Blog in 216 countries...... , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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