Patent Grant
9890198
The invention relates to novel cyclosporine derivatives, their pharmaceutical compositions comprising the same, and methods for treating or preventing viral infections, inflammation, dry eye, central nervous disorders, cardiovascular diseases, cancer, obesity, diabetes, muscular dystrophy, and hair loss.
Cyclosporins in nature are poly-N-methyl, cyclic undecapeptides, isolated from fungi. Cyclosporin A has an immunosuppressive activity and has been used for almost 30 years to prevent rejection in kidney, heart and liver transplant recipients. It possesses anti-inflammatory properties and has been used for treating severe rheumatoid arthritis, severe psoriasis, Behget's uveitis, and dry eye disease. In addition, it is useful for treating severe ulcerative colitis, Crohn's disease, alopecia greata, aplastic anemia, HSV-1 stromal keratitis, systemic lupus erythematosus, and severe lupus nephritis. However, its strong immunosuppressive activity limits its applications in many diseases.
The anti-HIV activity of cyclosporin A was first discovered in 1986 and has been continually studied since then (Klatzmann, D., et al., 1986, C R Acad. Sci. III, 303(9):343-8; Wainberg, M. A., et al., 1988, Blood, 72, 1904-10; Luban, J., et al., 1993, Cell, 73, 1067-1078; each of which is incorporated herein by reference). Its non-immunosuppressive derivative, NIM-811, was reported to have potent anti HIV activity due to its ability to inhibit cyclophilin A (Franke, E. K., et al., 1994, Nature, 372, 359-362; Thali, M., et al., 1994, Nature, 372, 363-365; Gamble, T. R., et al., 1996, Cell, 87, 1157-1159; Rosenwirth B., et al., 1994, Antimicrob. Agents Chemother., 38, 1763-1772; each of which is incorporated herein by reference).
Cyclosporin A and its non-immunosuppressive derivatives, as such as NIM-811 (N-MeIle-4-Cyclosporin), Debio-025, and SCY-635, bind and inhibit cyclophilins; cyclophilins interact with HCV protein NS5A and NS5B and stimulate its RNA-binding activity. As a result, these compounds have an effective anti-HCV activity (Watashi, K., et al., 2007, Rev. Med. Virol., 17:245-252.37; Inoue, K., et al., 2001, Nippon Rinsho., 59, 1326-30; Inoue, K., et al., 2003, J. Gastroenterol., 38, 567-72; Watashi, K., et al., 2003, Hepatology, 38, 1282-8; Gaither, L. A., et al., 2010, Virology, 397, 43-55; each of which is incorporated herein by reference). Currently, NIM-811, Debio-025, and SCY-635 are undergoing clinical trials for treating HCV.
NIM-811 and Debio-025 have a chemical structure similar to cyclosporine A and possess a poor pharmacokinetic profile. In addition, they are metabolized by P450 for inducing drug interactions (Lill, J., et al., 2000, Am J Health-Syst Pharm 57, 1579; incorporated herein by reference).
SCY-635 has an improved pharmacokinetic profile and low blood serum binding. In addition, it has a low potential for drug-drug interactions. SCY-635's in vitro anti-HCV activity (EC50) was reported to be 0.10 μM (Hopkins, S. et al., 2010, Antimicrob. Agents Chemother., 54, 660-672, incorporated herein by reference). However, SCY-635 is not chemically stable, as it is easily converted to its diastereoisomer by epimerization. Its diastereoisomer is expected to have poor binding activity with cyclophilins, and as a result, its anti-viral activity in vivo may be affected (See, e.g., WO2012/009715, WO2012/021796, and WO2012/075494, each of which incorporated herein by reference in its entirety).
Cyclosporin A and its non-immunosuppressive derivatives were also found to possess anti-HBV activity through the inhibition of cyclophilins (Chokshi, S., et al., 2012, Gut 61:A11; Chokshi, S., et al., 2012, Poster Presentations, 47th Annual Meeting of the European Association for the Study of the Liver (EASL 2012), Barcelona, Spain; Chokshi, S., et al., 2011, Abstract 190 (Poster Presentations), 46th Annual Meeting of the European Association for the Study of the Liver (EASL 2011), Berlin, March 30-April 3; Tian, X. C., et al., 2010, J. Virol., 84, 3373-3381; Xia, W. L., et al., 2004, Hepatobiliary Pancreat Dis Int., 4, 18-22; Michael, J., et al., 2003, J. Virol., 77, 7713-7719; each of which is incorporated herein by reference).
Furthermore, cyclophilins were reported to regulate the life cycle and pathogenesis of several viruses, including severe acute respiratory syndrome coronavirus, vaccinia virus, and herpes simplex virus (Castro, A. P., et al., 2003, J. Virol., 77, 9052-9068; Chen, Z., L., et al., 2005, J. Infect. Dis. 191(5):755-760; Arai, C., et al., Nihon Rinsho Meneki Gakkai Kaishi., 35(1), 87-91; Labetoulle, M., 2012, J Fr Ophtalmol., 35(4), 292-307; De Clercq, E., 2008, Expert Opin Emerg Drugs., 13(3):393-416; Vahlne, A., 1992, Arch Virol., 122(1-2):61-75; each of which is incorporated herein by reference). Cyclosporin A and its non-immunosuppressive derivatives also possess such anti viral-activities.
N-MeVal-4-Cyclosporin (SDZ 220-384), another non-immunosuppressive cyclosporine derivative, was reported to have similar biological activities to that of NIM-811 (Fliri, H., et al., 1993, Ann. N Y Acad Sci. 696, 47-53; Zenke, G., et al., 1993, Ann N Y Acad Sci. 23; 685:330-5).
Hepatitis C virus (HCV) is a small (55-65 nm in size), enveloped, positive sense single strand RNA virus in the Flaviviridae family. HCV has a high rate of replication and an exceptionally high mutation rate. About 80% of people infected with HCV develop chronic, persistent infection. More than 4 million Americans have been infected with HCV and more than 200 million people are estimated to be infected chronically worldwide. About 35,000 new cases of hepatitis C are estimated to occur in the United States each year. HCV infection is responsible for about 50% of all chronic liver disease, 30% of all liver transplants, and 30% of all cirrhosis, end-stage liver disease, and liver cancer in the U.S. The peg-interferon and ribavirin combination is the standard treatment for chronic hepatitis C, but it has low efficacy against HCV infection. Recently, the FDA has approved Vertex's Incivek (telaprevir) and Merck's Victrelis (boceprevir) as an add-on to the current interferon/ribavirin therapy for treating HCV. Both drugs are HCV protease inhibitors that target the virus to prevent its replication. However, due to HCV's fast mutation rate, drug resistance can be developed in a short period of time. Thus, there exists a need for an effective therapeutic for HCV treatment.
Hepatitis B virus (HBV) is a 42 nm partially double stranded DNA virus composed of a 27 nm nucleocapsid core (HBcAg) that is surrounded by an outer lipoprotein envelope containing the surface antigen (HBsAg). More than 2 billion people have been infected, and there are 350 million chronic carriers of the virus. The disease has caused epidemics in parts of Asia and Africa. Chronic hepatitis B will cause liver cirrhosis and liver cancer, a fatal disease with a very poor response to current chemotherapies. The infection is preventable by vaccination, and HBV load and replication can be reduced by current antiviral drugs, such as lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread), telbivudine (Tyzeka), entecavir (Baraclude), and the two immune system modulators interferon alpha-2a and PEGylated interferon alpha-2a (Pegasys). However, none of the available drugs can clear the infection. There remains a need for an effective therapeutic to treat HBV infection.
The non-immunosuppressive cyclosporin derivatives bind to cyclophilins, a family of host proteins that catalyze cis-trans peptidyl-prolyl isomerization in protein folding and regulation, which are crucial for the processing and maturation of the viral proteins for viral replication. HIV and HCV are viruses with a high mutation rate. All current anti-viral drugs target the virus itself; when the virus mutates, it leads to the development of drug resistance. Instead of directly targeting the virus, targeting host cofactors (cyclophilins) will be slow down the development of drug resistance due to a higher genetic barrier (Rosenwirth, B., et al., 1994, Antimicrob. Agents Chemother., 38, 1763-1772; Tang, H. L. et al., 2010, Viruses, 2, 1621-1634; Hopkins, S. et al., 2010, Oral Presentation, Scynexis's SCY-635 Demonstrates Impressive Barrier to Resistance in HCV Treatment, the 45th Annual Meeting of the European Association for the Study of the Liver (EASL 2010), Vienna, Austria, April 14-18; each of which is incorporated herein by reference). Cyclosporine derivatives affect a new target, cyclophilins, and therefore represent a new mechanism of action against viruses.
There are 17 cyclophilins in the human genome, but the functions of these cyclophilin isoforms are still unclear (Davis, T. L., et al., 2010, PLoS Biol. 8(7):e1000439; incorporated herein by reference). Cyclophilin A, B, C, D, and other such isoforms play an important role in the pathophysiology of a number of serious diseases, such as cancer (Campa, M J., et al., 2003, Cancer Res., 63(7), 1652-6; Li, M., et al., 2006, Cancer, 106: 2284-94; Yang, H., et al., 2007, Biochem Biophys Res Commun., 361(3):763-7; Obchoei, S., et al., 2009, Med Sci Monit., 15(11), RA221-32; Andersson, Y., et al., 2009, Br J Cancer, 101, 1307-1315; Lee, J., 2010, Arch Pharm Res., 33(2), 181-7; Lee, J., et al., 2010, J Exp Clin Cancer Res., 29:97; Obchoei, S., 2011, Molecular Cancer, 10:102; Takahashi, M., et al., 2012, Oncol Rep., 27(1):198-203; Qian, Z., et al., 2010, BMC Cancer, 12:442; each of which is incorporated herein by reference), inflammations (the result of interactions between a secreted extracellular cyclophilin and CD-147, a surface protein; Yurchenko V., 2005, Immunology, 117(3):301-9; Yurchenko, V., 2010, Clin Exp Immunol., 160(3):305-17; Malesevie, M., 2010, Angew Chem Int Ed Engl., 49(1):213-5; each of which is incorporated herein by reference), rheumatoid arthritis (Wells, G., et al., 2000, Cochrane Database Syst Rev., (2):CD001083; Kim, H., et al., 2005, Clin Immunol., 116(3):217-24; Yang, Y., Rheumatology (Oxford), 47(9):1299-310; Yurchenko, V., et al., 2006, Immunology, 117(3):301-9; Damsker, J. M., 2009, Immunology, 126(1):55-62; Wang, L., et al., 2010, J Clin Immunol., 30(1):24-33; Billich A., et al., 1997, J Exp Med., 185:975-80; De Ceuninck F., et al., 2003, Arthritis Rheum., 48:2197-206; each of which is incorporated herein by reference), respiratory inflammation (Foda, H. D., et al., 2001, Am J Respir Cell Mol Biol., 25:717-24; Hasaneen, N. A., et al., FASEB J., 19:1507-9. Yurchenko, V., et al., 2006, Immunology, 117(3):301-9; Gwinn, W. M., 2006, J Immunol., 177(7):4870-9; Onoue, S., 2009, J Control Release., 138(1):16-23; Balsley, M. A., et al., 2010, J Immunol., 185(12):7663-70; Balsley, M., et al., 2010, Am. J. Respir. Crit. Care Med., 181(1): A6821; Stemmy, E. J., et al., 2011, J. Asthma, 48(10):986-993; Stemmy, E. J., et al., 2011, Am J Respir Cell Mol Biol., 45(5):991-8; Amin, K., 2012, Respir Med., 106(1):9-14; Onoue, S., 2012, Eur J Pharm Biopharm., 80(1):54-60; each of which is incorporated herein by reference), lupus (Caccavo, D., et al., 1997, Arthritis & Rheumatism, 40(1):27-35; Dostál, C., et al., 1998, Lupus, 7(1):1 29-36; Tam, L S., et al., 1998, Q J Med., 91(8):573-580; Fu, L W., et al., 1998, Rheumatology 37 (2): 217-221; Hallegua, D., et al., 2009, Lupus, 9: 241-251; each of which is incorporated herein by reference), psoriasis (Ellis, C. N., 1991, N Engl J Med., 324, 277-284; Lebwohl, M., et al., 1998, J Am Acad Dermatol., 39(3):464-75; Rosmarin, D M., et al., 2010, J Am Acad Dermatol., 62(5):838-53; each of which is incorporated herein by reference), atopic dermatitis (Naeyaert, J. M., et al., 1999, Dermatology, 198:145-152; Pacor, M L., et al., 2001, Recenti Prog Med., 92(6):390-1; Ricci, G., et al., 2009, Drugs, 69(3):297-306; Simon, D., 2011, Curr Probl Dermatol., 41:156-64; each of which is incorporated herein by reference), dry eye disease (Pflugfelder, S. C., 2004, Am J Ophthalmol., 137(2), 337-42; Kymionis, G. D., et al 2008, Clin Ophthalmol., 2, 829-836; Kunert, K. S., et al., 2002, Arch Ophthalmol., 120, 330-7; Yavuz, B., et al., 2012, Scientific World Journal. 2012:194848; each of which is incorporated herein by reference), severe Graves' ophthalmopathy (Prummel, M. F., 1989, N Engl J Med., 321(20), 1353-9; incorporated herein by reference), endogenous uveitis (Nussenblatt, R. B., et al., 1991, Am J Ophthalmol., 112(2), 138-46; which is incorporated herein by reference), Wegener's granulomatosis (Georganas, C., et al., 1996, Clin Rheumatol., 15(2), 189-92; incorporated herein by reference), vernal keratoconjutivitis (Pucci, N., et al., 2002, Ann Allergy Asthma Immunol., 89, 298-303; incorporated herein by reference), atopic keratoconjutivitis (Akpek, E. K., et al., 2004, Ophthalmology, 111, 476-82; incorporated herein by reference), ligneous conjutivitis (Rubin, B. I., et al., 1991, Am J Ophthalmol., 112, 95-96; incorporated herein by reference), conjuctival linchen planus (Levell, N. J., et al., 1992, Br J Dermatol., 127, 66-7; incorporated herein by reference), and superior limbic keratoconjutivitis (Perry, H. D., et al., 2003, Ophthalmology, 110, 1578-81; incorporated herein by reference), inflammatory bowel disease-Crohn's Disease and Ulcerative Colitis (Sandborn, W. J., 1995, Inflamm Bowel Dis. 1:48-63; Shibolet, O., et al., 2005, Cochrane Database Syst Rev., (1):CD004277; Rufo, P. A., et al., 2006, Paediatr Drugs, 8(5):279-302; Reindl, W., et al., 2007, Gut., 56(7):1019; Hart, A. L., et al., 2010, Aliment Pharmacol Ther., 32(5):615-27; Cheifetz, A. S., et al., 2011, J Clin Gastroenterol., 45(2):107-12; Sharkey, L., 2011, J Crohns Colitis., 5(2):91-4; Fabro, M., et al., 2011, Curr Drug Targets., 12(10):1448-53; Van Assche, G., et al., 2011, Gut., 60(1):130-3; each of which is incorporated herein by reference), NSAID-induced enteropathy (LoGuidice, A., at al., 2010, Toxicol. Sci., 118, 276-285; which is incorporated herein by reference), cardiovascular diseases (including vascular stenosis, atherosclerosis, abdominal aortic aneurysms, aortic rupture, cardiac hypertrophy, pulmonary arterial hypertension, myocarditis and myocardial fibrosis, and ischaemic heart diseases; Jin, Z. G., et al., 2000, Circ Res., 87(9):789-96; Yurchenko, V., et al., 2005, Immunology, 117, 301-309; Suzuki, J., et al., 2006, Circ Res., 98(6):811-7; Satoh, K., et al., 2008, Circulation., 117(24):3088-98; Nishihara, M., et al., 2008, J Mol Cell Cardiol., 44(2):441-442; Satoh, K., et al., 2010, Circ J., 74(11):2249-56; Satoh, K., et al., 2010, Antioxid Redox Signal., 12(5):675-82; Hausenloy, D. J., et al., 2012, Br J Pharmacol. 165(5):1235-45; Coppinger, J. A., et al., 2004, Blood, 103(6):2096-104; Satoh, K., et al., 2010, Antioxid Redox Signal., 1:12(5), 675-682; Nigro, P., et al., 2010, J Exp Med., 208(1):53-66; Wang, W. L., et al., 2011, Med Hypotheses, 77(5):734-8; Hattori, F., 2012, J Mol Cell Cardiol., 53(1):1-2; Seizer P., 2012, J Mol Cell Cardiol., 53(1):6-14; each of which is incorporated herein by reference), and ischaemic brain diseases (Boulos, S., et al., 2007, Neurobiol Dis., 25:54-64; incorporated herein by reference).
Due to cyclophilin inhibition, cyclosporin derivatives also possess the following biological activities: anti-fungal (Kirkland, T. N., et al., 1983, Antimicrob Agents Chemother., 24(6): 921-924; Mody, C. H., et al., 1988, Infect Immun., 56(1): 7-12; Roilides, E., et al., 1994, Antimicrob Agents Chemother., 38(12): 2883-2888; Moussaff, M., et al., 1997, Appl Environ Microbiol., 63(5):1739-43; Cruz, M. C., et al., 2000, Antimicrob Agents Chemother., 44(1):143-9; each of which is incorporated herein by reference), anti-malarial (Nickell, S. P., et al., 1982, Infect Immun., 37(3):1093-100; Murphy, J. R., et al, 1988, Antimicrob Agents Chemother., 32(4):462-6; Marín-Menéndez, A., et al., 2012, Mol Biochem Parasitol., 184(1):44-7; each of which is incorporated herein by reference), and anti-parasitic (including Leishmania donovani, Cryptosporidium parvum, Hymenolepis nana, Toxoplasma, Trypanosoma cruzi, and Schistosome; Chappell, L. H., et al., 1992, Parasitology, 105 Suppl:S25-40; Bell, A., et al., 1996, Gen Pharmacol., 27(6):963-71; Yau, W. L., et al., 2010, PLoS Negl Trop Dis., 4(6):e729; Yurchenko, V., et al., 2008, Int J Parasitol., 38(6):633-9; Perkins, M. E., et al., 1998, Antimicrob Agents Chemother., 42(4):843-8; Matsuzawa, K., et al., 1998, Int J Parasitol., 28(4):579-88; Silverman, J. A., et al., 1997, Antimicrob Agents Chemother., 41(9):1859-66; Búa, J., et al., 2008, Parasitology, 135(2):217-28; Búa, J., et al., 2004, Bioorg Med Chem Lett., 14(18):4633-7; Bout, D. T, et al., 1984, Am J Trop Med. Hyg., 33(1):185-6; Bout, D., et al., 1986, Infect Immun., 52(3):823-7; Munro, G. H., et al., 1991, Parasitology, 102 Pt 1:57-63; each of which is incorporated herein by reference). In addition, cyclosporin derivatives can promote hair growth (Watanabe, S., et al., 1991, J Dermatol., (12):714-9; Paus R., et al., 1994, J Invest Dermatol., 103:2, 143-7; Hozumi, Y., et al., 1994, J Dermatol Sci., 7 Suppl: S33-8; Takahashi, T., et al., 2001, J Invest Dermatol., 117(3):605-11; Taylor M., et al., 1993, J Invest Dermatol., 100:3, 237-9; Gafter-Gvili, A., et al., 2004, Arch Dermatol Res., 296(6):265-9; each of which is incorporated herein by reference).
Recent research for Alzheimer's disease indicated that Cyclophilin A is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration (Bell, R. D., et al., 2012, Nature, 485(7399):512-6; Bell, R. D., et al., 2009, Acta Neuropathol., 118(1):103-13; each of which is incorporated herein by reference).
Due to the function of extracellular cyclophilins, it is necessary to emphasize that the special target of a secreted extracellular cyclophilin using a cell-impermeable derivative of cyclosporine will be very effective in reducing inflammation for diseases such as respiratory inflammation and cardiovascular diseases (Yurchenko V., 2005, Immunology, 117(3):301-9; Yurchenko, V., 2010, Clin Exp Immunol., 160(3):305-17; Malesević, M., 2010, Angew Chem Int Ed Engl., 49(1):213-5; Balsley, M. A., et al., 2010, J Immunol., 185(12):7663-70; Balsley, M., et al., 2010, Am. J. Respir. Crit. Care Med., 181(1): A6821; Satoh, K., et al., 2010, Circ J., 74(11):2249-56; each of which is incorporated herein by reference).
Cyclophilin D (CypD) is very important for mitochondrial related neuro and cardiovascular functions because it is an integral part of the mitochondrial permeability transition pore (mPTP). Unregulated opening of the mPTP can lead to mitochondrial swelling and cell death. Thus, the CypD-mediated mPTP is directly linked to a new pharmacologic treatment strategy for many neuro and cardiovascular diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, ALS, aging, heart failure, traumatic brain injury, spinal cord injury, epilepticus, stroke, ischemia-reperfusion injury in the brain, heart, kidney, and particularly in myocardial infarction. The CypD-mediated mPTP is also linked to a new treatment strategy for cancer, obesity, diabetes, and muscular dystrophy (Henry-Mowatt, J., 2004, Oncogene, 23, 2850-60; Galluzzi, L., 2006, Oncogene, 25, 4812-4830; Hirai, K., et al., 2001, J Neurosci., 21, 3017-3023; Friberg, H., et al., 2002, Biochimie, 84, 241-250; Waldmeier, P. C., et al., 2003, Curr Med Chem., 10, 1485-506; Hansson, M. J., et al., 2004, J Bioenerg Biomembr., 36, 407-13; Sullivan, P. G., et al., 2005, J Neurosci Res., 79, 231-9; Baines, C. P., et al, 2005, Nature 434, 658-662; Shanmuganathan, S., et al, 2005, Am J Physiol Heart Circ Physiol., 289, H237-H242; McBride, H. M., et al., 2006, Curr Biol., 16, R551-560; Mandemakers, W., et al., 2007, J Cell Sci., 120, 1707-1716; Kroemer, G., et al., 2007, Physiol Rev., 87, 99-163; Ibarra, A., et al., 2007, Brain Res., 1149, 200-209; Michelakis, E. D., et al, 2008, Circulation, 117, 2431-2434; Du, H., et al, 2008, Nature Medicine, 14, 1097-1105; Piot C., et al., 2008, N Engl J Med., 359, 473-81; Hatton, J., et al., 2008, J Neurosurg., 109, 699-707; Tatsuta, T., et al., 2008, EMBO J, 27, 306-314; Reutenauer, J., et al., 2008, Br J Pharmacol., 155, 574-84; Mazzeo, A. T., et al., 2009, Exp Neurol., 218, 363-370; Galluzzi, L., et al, 2009, Nature Rev Neurosci., 10, 481-494; Halestrap, A. P., et al., 2009, Biochim Biophys Acta., 1787, 1402-15; Arnett, A. L. H., et al., 2009, Curr. Opin. Genet. Dev., 19, 290-297; Tiepolo, T., et al., 2009, Br J Pharmacol., 157, 1045-1052; Wissing, E. R., et al., 2010, Neuromuscul Disord., 20, 753-60; Halestrap, A. P., et al., 2010, Biochem Soc Trans., 38, 841-860; Cernak, I., et al., 2010, J Cereb Blood Flow Metab., 30, 255-66; Elrod, J. W., et al., 2010, J Clin Invest., 120, 3680-3687; Duchen, M. R., et al., 2010, Essays Biochem., 47, 115-37; Schapira, A. H. V., et al., 2011, Parkinson's Disease, Volume 2011, 1-7 Article ID 159160; Osman, M. M., et al., 2011, Neuropeptides, 45, 359-368; Devalaraja-Narashimha K., et al., 2011, FEES Lett., 585, 677-82; Fujimoto, K., et al., 2010, Proc Natl Acad Sci USA. 107, 10214-9; Irwin, W. A., et al., 2003, Nat Genet., 35, 267-271; Angelin, A., et al., 2007, Proc Natl Acad Sci USA, 104, 991-6; Merlini, L., et al., 2008, Proc Natl Acad Sci USA, 105, 5225-9; Millay, D. P., 2008, Nat Med., 14, 442-7; each of which is incorporated herein by reference). Cyclosporine A and its derivatives can block CypD to prevent mitochondrial swelling and cell death, and therefore could be useful for treatment of the aforementioned diseases, for example, as a neuro and cardiovascular protective agent or as a novel mitochondrial medicine.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
In one aspect, the present invention provides a compound of Formula (I):
or pharmaceutically acceptable salt thereof, wherein:
and
In another aspect, the present invention provides a compound of Formula (I) as shown above, or pharmaceutically acceptable salt thereof, wherein:
In another aspect, the present invention provides a compound of Formula (I) as shown above, or pharmaceutically acceptable salt thereof, wherein:
In another aspect, the present invention provides a compound of Formulae (II) through (VI):
or pharmaceutically acceptable salt thereof, wherein: represents a single bond or double bond;
each W is independently O, S, CH2, or NR1;
each R1 is independently H, alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, aryl or substituted aryl, or heterocycle or substituted heterocycle; or R1 and R3 together with the nitrogen atom to which they are attached, form a saturated or unsaturated heterocyclic ring containing from three to seven ring atoms, which ring may optionally contain another heteroatom selected from the group consisting of nitrogen, oxygen and sulfur and may be optionally substituted by from one to four groups which may be the same or different selected from the group consisting of alkyl, phenyl and benzyl; and
each occurrence of R3 and R5 is independently H, alkyl or substituted alkyl, alkenyl or substituted alkenyl, alkynyl or substituted alkynyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, or aryl or substituted aryl.
In another aspect, the present invention provides a compound of Formulae (II) through (VI): as shown above, or pharmaceutically acceptable salt thereof, wherein:
In another aspect, the present invention provides a compound of Formulae (II) through (VI) as shown above, or pharmaceutically acceptable salt thereof, wherein:
In yet another aspect, the present invention provides a pharmaceutical composition comprising at least one compound as described herein and a pharmaceutically-acceptable carrier.
In a further aspect, the present invention provides a method for treating or preventing a viral infection in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein.
In another aspect, the present invention provides a method for treating or preventing hepatitis C virus infection in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein.
In yet another aspect, the present invention provides a method for inhibiting a cyclophilin in a subject in need thereof, which comprises administrating to said subject an effective cyclophilin-inhibiting amount of at least one compound as described herein.
In yet another aspect, the present invention provides a method for treating or preventing diseases that are mediated by cyclophilins in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein.
In yet another aspect, the present invention provides a method for treating or preventing diseases in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases are selected from inflammation, respiratory inflammation, rheumatoid arthritis, and dry eye.
In yet another aspect, the present invention provides a method for treating or preventing diseases in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases are selected from neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's Diseases, and ALS; traumatic brain injury; stroke; and ischemia-reperfusion injury in the brain, heart, and kidney.
In yet another aspect, the present invention provides a method for treating or preventing diseases in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases are selected from cardiovascular diseases, vascular stenosis, atherosclerosis, abdominal aortic aneurysms, cardiac hypertrophy, aortic rupture, pulmonary arterial hypertension, myocarditis and myocardial fibrosis, and ischaemic heart diseases.
In yet another aspect, the present invention provides a method for treating or preventing diseases or conditions in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases or conditions are selected from cancer, obesity, diabetes, muscular dystrophy, and hair loss.
In yet another aspect, the present invention provides a method for treating or preventing diseases or conditions in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases or conditions are selected from allergic conjunctivitis, atopic and vernal keratoconjunctivitis, atopic keratoconjunctivitis, anterior uveitis, Behcet's disease, blepharitis, chronic ocular surface inflammation caused by viral infection, corneal transplant rejection, corneal sensitivity impaired due to surgery on the cornea or other surface of the eye, meibomian gland disease, ptyregia, ocular symptoms of graft versus host disease, ocular allergy, ocular cicatricial pemphigoid, Steven Johnson syndrome, vernal keratoconjunctivitis, uveitis, herpes simplex keratitis, ocular rosacea, and Pinguecula.
The following are definitions of terms used in the present specification. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.
The terms “alkyl” and “alk” refer to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. Exemplary “alkyl” groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. The term “(C1-C4)alkyl” refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and isobutyl. The term “(C1-C6)alkyl” refers to a straight or branched chain alkane (hydrocarbon) radical containing from 1 to 6 carbon atoms, such as n-hexyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, 2,2-dimethylbutyl, in addition to those exemplified for “(C1-C4)alkyl.” “Substituted alkyl” refers to an alkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substitutents forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, oxo (i.e., ═O), CF3, OCF3, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORe, P(═O)2ORe, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORd, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NRbC(═O)ORe, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein each occurrence of Ra is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; each occurrence of Rb, Rc and Rd is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc together with the N to which they are bonded optionally form a heterocycle; and each occurrence of Re is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. In the aforementioned exemplary substitutents, groups such as alkyl, cycloalkyl, alkenyl, alkynyl, cycloalkenyl, heterocycle and aryl can themselves be optionally substituted.
The term “alkenyl” refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplaries of such groups include ethenyl or allyl. The term “C2-C6 alkenyl” refers to a straight or branched chain hydrocarbon radical containing from 2 to 6 carbon atoms and at least one carbon-carbon double bond, such as ethylenyl, propenyl, 2-propenyl, (E)-but-2-enyl, (Z)-but-2-enyl, 2-methy(E)-but-2-enyl, 2-methy(Z)-but-2-enyl, 2,3-dimethyl-but-2-enyl, (Z)-pent-2-enyl, (E)-pent-1-enyl, (Z)-hex-1-enyl, (E)-pent-2-enyl, (Z)-hex-2-enyl, (E)-hex-2-enyl, (Z)-hex-1-enyl, (E)-hex-1-enyl, (Z)-hex-3-enyl, (E)-hex-3-enyl, and (E)-hex-1,3-dienyl. “Substituted alkenyl” refers to an alkenyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substitutents forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, oxo (i.e., ═O), CF3, OCF3, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORe, P(═O)2ORe, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORd, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NRbC(═O)ORe, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein each occurrence of Ra is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; each occurrence of Rb, Rc and Rd is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc together with the N to which they are bonded optionally form a heterocycle; and each occurrence of Re is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. The exemplary substitutents can themselves be optionally substituted.
The term “alkynyl” refers to a straight or branched chain hydrocarbon radical containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond. An exemplary of such groups includes ethynyl. The term “C2-C6 alkynyl” refers to a straight or branched chain hydrocarbon radical containing from 2 to 6 carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl. “Substituted alkynyl” refers to an alkynyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substitutents forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, oxo (i.e., ═O), CF3, OCF3, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORe, P(═O)2ORe, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORd, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NRbC(═O)ORe, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein each occurrence of Ra is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; each occurrence of Rb, Rc and Rd is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc together with the N to which they are bonded optionally form a heterocycle; and each occurrence of Re is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. The exemplary substitutents can themselves be optionally substituted.
The term “cycloalkyl” refers to a fully saturated cyclic hydrocarbon group containing from 1 to 4 rings and 3 to 8 carbons per ring. “C3-C7 cycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. “Substituted cycloalkyl” refers to a cycloalkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substitutents forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, oxo (i.e., ═O), CF3, OCF3, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORe, P(═O)2ORe, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORd, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NRbC(═O)ORe, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein each occurrence of Ra is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; each occurrence of Rb, Rc and Rd is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc, together with the N to which they are bonded optionally form a heterocycle; and each occurrence of Re is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. The exemplary substitutents can themselves be optionally substituted. Exemplary substituents also include spiro-attached or fused cyclic substituents, especially spiro-attached cycloalkyl, spiro-attached cycloalkenyl, spiro-attached heterocycle (excluding heteroaryl), fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substitutents can themselves be optionally substituted.
The term “cycloalkenyl” refers to a partially unsaturated cyclic hydrocarbon group containing 1 to 4 rings and 3 to 8 carbons per ring. Exemplaries of such groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, etc. “Substituted cycloalkenyl” refers to a cycloalkenyl group substituted with one more substituents, preferably 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substitutents forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, oxo (i.e., ═O), CF3, OCF3, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORe, P(═O)2ORe, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORd, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NRbC(═O)ORe, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein each occurrence of Ra is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; each occurrence of Rb, Rc and Rd is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc, together with the N to which they are bonded optionally form a heterocycle; and each occurrence of Re is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. The exemplary substitutents can themselves be optionally substituted. Exemplary substituents also include spiro-attached or fused cyclic substituents, especially spiro-attached cycloalkyl, spiro-attached cycloalkenyl, spiro-attached heterocycle (excluding heteroaryl), fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substituents can themselves be optionally substituted.
The term “aryl” refers to cyclic, aromatic hydrocarbon groups that have 1 to 5 aromatic rings, especially monocyclic or bicyclic groups such as phenyl, biphenyl or naphthyl. Where containing two or more aromatic rings (bicyclic, etc.), the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl, phenanthrenyl and the like). “Substituted aryl” refers to an aryl group substituted by one or more substituents, preferably 1 to 3 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substitutents forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, oxo (i.e., ═O), CF3, OCF3, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORe, P(═O)2ORe, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORd, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NRbC(═O)ORe, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein each occurrence of Ra is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; each occurrence of Rb, Rc and Rd is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc, together with the N to which they are bonded optionally form a heterocycle; and each occurrence of Re is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. The exemplary substitutents can themselves be optionally substituted. Exemplary substituents also include fused cyclic groups, especially fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substituents can themselves be optionally substituted.
The terms “heterocycle” and “heterocyclic” refer to fully saturated, or partially or fully unsaturated, including aromatic (i.e., “heteroaryl”) cyclic groups (for example, 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 8 to 16 membered tricyclic ring systems) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3, or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. (The term “heteroarylium” refers to a heteroaryl group bearing a quaternary nitrogen atom and thus a positive charge.) The heterocyclic group may be attached to the remainder of the molecule at any heteroatom or carbon atom of the ring or ring system. Exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, hexahydrodiazepinyl, 4-piperidonyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane and tetrahydro-1,1-dioxothienyl, and the like. Exemplary bicyclic heterocyclic groups include indolyl, isoindolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzo[d][1,3]dioxolyl, 2,3-dihydrobenzo[b][1,4]dioxinyl, quinuclidinyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofurazanyl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), triazinylazepinyl, tetrahydroquinolinyl and the like. Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like.
“Substituted heterocycle” and “substituted heterocyclic” (such as “substituted heteroaryl”) refer to heterocycle or heterocyclic groups substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment. Exemplary substituents include but are not limited to one or more of the following groups: hydrogen, halogen (e.g., a single halogen substituent or multiple halo substitutents forming, in the latter case, groups such as CF3 or an alkyl group bearing Cl3), cyano, nitro, oxo (i.e., ═O), CF3, OCF3, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, aryl, ORa, SRa, S(═O)Re, S(═O)2Re, P(═O)2Re, S(═O)2ORe, P(═O)2ORe, NRbRc, NRbS(═O)2Re, NRbP(═O)2Re, S(═O)2NRbRc, P(═O)2NRbRc, C(═O)ORd, C(═O)Ra, C(═O)NRbRc, OC(═O)Ra, OC(═O)NRbRc, NRbC(═O)ORe, NRdC(═O)NRbRc, NRdS(═O)2NRbRc, NRdP(═O)2NRbRc, NRbC(═O)Ra, or NRbP(═O)2Re, wherein each occurrence of Ra is independently hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl; each occurrence of Rb, Rc and Rd is independently hydrogen, alkyl, cycloalkyl, heterocycle, aryl, or said Rb and Rc together with the N to which they are bonded optionally form a heterocycle; and each occurrence of Re is independently alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, heterocycle, or aryl. The exemplary substitutents can themselves be optionally substituted. Exemplary substituents also include spiro-attached or fused cyclic substituents at any available point or points of attachment, especially spiro-attached cycloalkyl, spiro-attached cycloalkenyl, spiro-attached heterocycle (excluding heteroaryl), fused cycloalkyl, fused cycloalkenyl, fused heterocycle, or fused aryl, where the aforementioned cycloalkyl, cycloalkenyl, heterocycle and aryl substituents can themselves be optionally substituted.
The term “alkylamino” refers to a group having the structure —NHR′, wherein R′ is hydrogen, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, as defined herein. Examples of alkylamino groups include, but are not limited to, methylamino, ethylamino, n-propylamino, iso-propylamino, cyclopropylamino, n-butylamino, tert-butylamino, neopentylamino, n-pentylamino, hexylamino, cyclohexylamino, and the like.
The term “dialkylamino” refers to a group having the structure —NRR′, wherein R′ and R′ are each independently alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, aryl or substituted aryl, heterocyclyl or substituted heterocyclyl, as defined herein. R and R′ may be the same or different in an dialkylamino moiety. Examples of dialkylamino groups include, but are not limited to, dimethylamino, methyl ethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(iso-propyl)amino, di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino, di(neopentyl)amino, di(n-pentyl)amino, di(hexyl)amino, di(cyclohexyl)amino, and the like. In certain embodiments, R and R′ are linked to form a cyclic structure. The resulting cyclic structure may be aromatic or nonaromatic. Examples of cyclic diaminoalkyl groups include, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolyl, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.
The terms “halogen” or “halo” refer to chlorine, bromine, fluorine or iodine.
Unless otherwise indicated, any heteroatom with unsatisfied valences is assumed to have hydrogen atoms sufficient to satisfy the valences.
The compounds of the present invention may form salts which are also within the scope of this invention. Reference to a compound of the present invention is understood to include reference to salts thereof, unless otherwise indicated. The term “salt(s)”, as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when a compound of the present invention contains both a basic moiety, such as but not limited to a pyridine or imidazole, and an acidic moiety such as but not limited to a carboxylic acid, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of a compound of the present invention may be formed, for example, by reacting a compound I with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
The compounds of the present invention which contain a basic moiety, such as but not limited to an amine or a pyridine or imidazole ring, may form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, hydroxyethanesulfonates (e.g., 2-hydroxyethanesulfonates), lactates, maleates, methanesulfonates, naphthalenesulfonates (e.g., 2-naphthalenesulfonates), nicotinates, nitrates, oxalates, pectinates, persulfates, phenylpropionates (e.g., 3-phenylpropionates), phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates, tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.
Compounds of the present invention which contain an acidic moiety, such but not limited to a carboxylic acid, may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glycamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.
Prodrugs and solvates of the compounds of the invention are also contemplated herein. The term “prodrug” as employed herein denotes a compound that, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield a compound of the present invention, or a salt and/or solvate thereof. Solvates of the compounds of the present invention include, for example, hydrates.
Compounds of the present invention, and salts or solvates thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.
All stereoisomers of the present compounds (for example, those which may exist due to asymmetric carbons on various substituents), including enantiomeric forms and diastereomeric forms, are contemplated within the scope of this invention. Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers (e.g., as a pure or substantially pure optical isomer having a specified activity), or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the present invention may have the S or R configuration as defined by the International Union of Pure and Applied Chemistry (IUPAC) 1974 Recommendations. The racemic forms can be resolved by physical methods, such as, for example, fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by any suitable method, including without limitation, conventional methods, such as, for example, salt formation with an optically active acid followed by crystallization.
Compounds of the present invention are, subsequent to their preparation, preferably isolated and purified to obtain a composition containing an amount by weight equal to or greater than 90%, for example, equal to greater than 95%, equal to or greater than 99% pure (“substantially pure” compound I), which is then used or formulated as described herein. Such “substantially pure” compounds of the present invention are also contemplated herein as part of the present invention.
All configurational isomers of the compounds of the present invention are contemplated, either in admixture or in pure or substantially pure form. The definition of compounds of the present invention embraces both cis (Z) and trans (E) alkene isomers, as well as cis and trans isomers of cyclic hydrocarbon or heterocyclic rings.
Throughout the specifications, groups and substituents thereof may be chosen to provide stable moieties and compounds.
Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, the entire contents of which are incorporated herein by reference.
Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (
Isomeric mixtures containing any of a variety of isomer ratios may be utilized in accordance with the present invention. For example, where only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those of ordinary skill in the art will readily appreciate that analogous ratios are contemplated for more complex isomer mixtures.
The present invention also includes isotopically labeled compounds, which are identical to the compounds disclosed herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine and chlorine, such as 2H, 3H, 13C, 11C, 14C, 15N, 18O, 17O, 31P, 32P, 35S, 18F, and 36Cl, respectively. Compounds of the present invention, or an enantiomer, diastereomer, tautomer, or pharmaceutically acceptable salt or solvate thereof, which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds can generally be prepared by carrying out the procedures disclosed in the Schemes and/or in the Examples below, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.
If, for instance, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties. In general, the term “substituted” whether preceded by the term “optionally” or not, and substituents contained in formulas of this invention, refer to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For purposes of this invention, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, this invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Combinations of substituents and variables envisioned by this invention are preferably those that result in the formation of stable compounds useful in the treatment, for example, of infectious diseases or proliferative disorders. The term “stable”, as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.
Compounds
The novel cyclosporin derivatives of the present invention are potent inhibitors of cyclophilins and are useful for inhibiting viruses such as HCV, HBV, and HIV.
In one aspect, the present invention provides a compound Formula (I):
or pharmaceutically acceptable salt thereof, wherein:
In one aspect, the present invention provides a compound Formula (I):
or pharmaceutically acceptable salt thereof, wherein:
In certain embodiments, R3 is (C7-C10)alkyl. In certain other embodiments, R3 is (C7-C8)alkyl. In yet other embodiments, R3 is (C7-C12) linear alkyl. In yet other embodiments, R3 is (C7-C10) linear alkyl. In yet other embodiments, R3 is (C7-C8) linear alkyl.
In certain embodiments, R4 is hydroxyl. In certain other embodiments, R4 is C(═O)ORA.
In certain embodiments, R8 is n-butyl. In certain other embodiments, R8 is (E)-but-2-enyl. In certain other embodiments, R8 is
In yet other embodiments, R8 is —(CH2)4—SR9. In yet other embodiments, R8 is —(CH2)4—(C═O)OR9. In yet other embodiments, R8 is —(CH2)3—(C═O)OR9. In certain embodiments, each occurrence of R9 is independently hydrogen. In certain other embodiments, each occurrence of R9 is independently (C1-C6)alkyl. In certain embodiments, R2 is ethyl.
In certain embodiments, the compound of Formula I has the structure of Formulae (II) through (VI):
or pharmaceutically acceptable salt thereof, wherein:
In certain embodiments, the compound of Formula I has the structure of Formulae (II) through (VI):
or pharmaceutically acceptable salt thereof, wherein:
In certain embodiments, R3 is (C7-C10)alkyl. In certain other embodiments, R3 is (C7-C8)alkyl. In yet other embodiments, R3 is (C7-C12) linear alkyl. In yet other embodiments, R3 is (C7-C10) linear alkyl. In yet other embodiments, R3 is (C7-C8) linear alkyl.
In certain embodiments, R4 is hydroxyl. In certain other embodiments, R4 is C(═O)ORA.
In certain embodiments, W is O. In certain other embodiments, W is S. In yet other embodiments, W is NH. In yet other embodiments, W is CH2, yet other embodiments, W is NR1. In yet other embodiments, W is N—(C1-C4)alkyl.
In certain embodiments, m is 1. In certain other embodiments, m is 2. In yet other embodiments, m is 3. In yet other embodiments, m is 4 or 5.
In certain embodiments, p is 0. In certain other embodiments, p is 1. In yet other embodiments, m is 2. In yet other embodiments, m is 3, 4 or 5.
In certain embodiments, R3 is H, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, CH2CMe3, phenyl, CH2-phenyl,
In certain embodiments, R3 is —(CH2)nNRARB, wherein n is an integer of 2, 3, 4, 5 or 6, or integer of 7, 8, 9, 10, 11 or 12; and wherein each occurrence of RA and RB is independently hydrogen; (C1-C4)alkyl, optionally substituted by one or more groups RD which may be the same or different, in which each occurrence of RD is independently halogen, hydroxy, O(C1-C4)alkyl, C(═O)(C1-C4)alkyl, C(═O)O(C1-C4)alkyl; or RA and RB, together with the nitrogen atom to which they are attached, form a saturated or unsaturated heterocyclic ring containing from three to seven ring atoms, which ring may optionally contain another heteroatom selected from the group consisting of nitrogen, oxygen and sulfur and may be optionally substituted by from one to four groups which may be the same or different selected from (C1-C4)alkyl, phenyl and benzyl.
In certain embodiments, R3 is —(CH2)nNRARB, wherein n is an integer of 2, 3, 4, 5 or 6, or integer of 7, 8, 9, 10, 11 or 12; and wherein RA and RB, together with the nitrogen atom to which they are attached, form a saturated or unsaturated heterocyclic ring containing from three to seven ring atoms, which ring may optionally contain another heteroatom selected from nitrogen, oxygen and sulfur and may be optionally substituted by from one to four groups which may be the same or different selected from (C1-C4)alkyl, phenyl and benzyl.
In certain embodiments, n is 2. In certain other embodiments, n is 3. In yet other embodiments, n is 4, 5, or 6. In yet other embodiments, n is 7 or 8. In yet other embodiments, n is 9 or 10. In yet other embodiments, n is 11 or 12.
In certain embodiments, R3 is —(CH2)nNRARB, wherein n is an integer of 7, 8, 9, 10, 11 or 12; and wherein each occurrence of RA and RB is independently hydrogen; (C1-C4)alkyl, optionally substituted by one or more groups RD which may be the same or different, in which each occurrence of RD is independently halogen, hydroxy, O(C1-C4)alkyl, C(═O)(C1-C4)alkyl, C(═O)O(C1-C4)alkyl; or RA and RB, together with the nitrogen atom to which they are attached, form a saturated or unsaturated heterocyclic ring containing from three to seven ring atoms, which ring may optionally contain another heteroatom selected from the group consisting of nitrogen, oxygen and sulfur and may be optionally substituted by from one to four groups which may be the same or different selected from (C1-C4)alkyl, phenyl and benzyl.
In certain embodiments, n is 7. In certain other embodiments, n is 8. In yet other embodiments, n is 9, 10, 11 or 12.
In certain embodiments, R3 is 2-aminoethyl, 2-aminopropyl, 3-aminopropyl, 2-monoalkylaminoethyl, 2-monoalkylaminopropyl, 3-monoalkylaminopropyl, 2-dialkylaminoethyl, 2-dialkylaminopropyl, or 3-dialkylaminopropyl, wherein said alkyl is (C1-C4)alkyl.
In certain embodiments, R3 is 2-aminoethyl, 2-aminopropyl, 3-aminopropyl, 2-monoalkylaminoethyl, 2-monoalkylaminopropyl, 3-monoalkylaminopropyl, 2-dialkylaminoethyl, 2-dialkylaminopropyl, or 3-dialkylaminopropyl, wherein said alkyl is (C1-C4)alkyl, wherein R3 is dimethylaminoethyl, diethylaminoethyl, methylethylaminoethyl, methyl-iso-butylaminoethyl, ethyl-iso-butylaminoethyl, methyl-tert-butylaminoethyl, or ethyl-tert-butylaminoethyl.
In certain embodiments, R3 is
in which n is an integer of 2, 3, 4, 5, or 6, and m is an integer of 2, 3, or 4. In certain embodiments, n is 2. In certain other embodiments, n is 3. In yet other embodiments, n is 4, or 5, or 6. In certain embodiments, m is 2. In certain other embodiments, m is 3. In certain other embodiments, m is 4. In certain embodiments, n is 7. In certain other embodiments, n is 8. In yet other embodiments, n is 9, 10, 11 or 12.
In certain embodiments, W is NR1, and R1 and R3 together with the nitrogen atom to which they are attached, form a heterocycle selected from
in which RC is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, CH2CMe3, Ph, CH2Ph, CH2CH2OH, or CH2CH2O(C1-C4)alkyl.
In certain embodiments, R5 is H, (C1-C6)alkyl, (C2-C6)alkenyl, phenyl, benzyl, CH2—S—(C1-C6)alkyl, CH2—O—(C1-C6)alkyl, (C2-C6)ORA, (C1-C6)-monoalkyl amine, (C1-C6)-dialkyl amine, or (C1-C6)-cyclic amine, in which said phenyl or benzyl is optionally substituted by one to three substitutents selected from (C1-C4)alkyl, (C1-C4)alkoxy, and halogen; and RA is H, (C1-C6)alkyl, phenyl, CH2-phenyl, (C1-C6)alkylOH, (CH2)pO(CH2)mOH, (CH2)pO(CH2)mO(CH2)mOH, (C1-C6)alkylO(C1-C4)alkyl, (CH2)pO(CH2)mO(C1-C4)alkyl, or (CH2)pO(CH2)mO(CH2)mO(C1-C4)alkyl; p is an integer of 0, 1, 2, 3, 4, or 5; and m is an integer of 1, 2, 3, 4 or 5.
In certain embodiments, R5 is H. In certain other embodiments, R5 is methyl. In yet other embodiments, R5 is CH2—S—(C1-C6)alkyl, e.g., CH2—S—CH3. In yet other embodiments, R5 is CH2—O—(C1-C6)alkyl, e.g., CH2—O—CH2—CH3. In yet other embodiments, R5 is (C2-C6)alkenyl, e.g., CH2—CH═CH2. In yet other embodiments, R5 is benzyl. In yet other embodiments, R5 is (C2-C6)OH. In yet other embodiments, R5 is (C1-C6)-monoalkyl amine, e.g., CH2—NH-Me. In yet other embodiments, R5 is (C1-C6)-dialkyl amine, e.g., CH2—CH2—N(Et)2. In yet other embodiments, R5 is (C1-C6)-cyclic amine, e.g., CH2—CH2-morpholine.
In certain embodiments, each occurrence RA and RB is independently H, (C1-C6)alkyl, phenyl, CH2-phenyl, (C1-C6)alkylOH, (CH2)pO(CH2)mOH, or (CH2)pO(CH2)mO(CH2)mOH, (C1-C6)alkylO(C1-C4)alkyl, (CH2)pO(CH2)mO(C1-C4)alkyl, or (CH2)pO(CH2)mO(CH2)mO(C1-C4)alkyl. In certain other embodiments, RA and RB, together with the nitrogen atom to which they are attached, form a heterocycle selected from
in which RC is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, CH2CMe3, Ph, CH2Ph, CH2CH2OH, or CH2CH2O(C1-C4)alkyl.
In certain embodiments, wherein represents a single bond. In certain other embodiments, wherein
represents a double bond.
In another aspect, the present invention provides a compound of Formulae (IIa)-(VIa):
or a pharmaceutically acceptable salt thereof, wherein:
In another aspect, the present invention provides a compound of Formulae (IIa)-(VIa):
or a pharmaceutically acceptable salt thereof, wherein:
In another aspect, the present invention provides a compound of Formulae (IIb)-(VIb):
or a pharmaceutically acceptable salt thereof, wherein:
In another aspect, the present invention provides a compound of Formulae (IIb)-(VIb):
or a pharmaceutically acceptable salt thereof, wherein:
In certain embodiments, R3 is (C7-C10)alkyl. In certain other embodiments, R3 is (C7-C8)alkyl. In yet other embodiments, R3 is (C7-C12) linear alkyl. In yet other embodiments, R3 is (C7-C10) linear alkyl. In yet other embodiments, R3 is (C7-C8) linear alkyl.
In certain embodiments, R4 is hydroxyl. In certain other embodiments, R4 is C(═O)ORA.
In certain embodiments, the compound of Formula I has the structure of Formulae (II) through (VI):
or a pharmaceutically acceptable salt thereof, wherein:
In certain embodiments, q is 1. In certain other embodiments, q is 2. In certain other embodiments, n is independently an integer of 6, 7, 8, 9, 10 or 11.
In certain embodiments, W is S. In certain other embodiments, W is O. In yet other embodiments, W is NH. In yet other embodiments, W is N—(C1-C4)alkyl.
In certain embodiments, R1 is hydrogen. In certain other embodiments, R1 is (C1-C6)alkyl. In certain embodiments, R3 is (C1-C6)alkyl. In certain other embodiments, R3 is NRCCH2(CH2)pNRARB.
In certain embodiments, R1 and R3 together with the nitrogen atom to which they are attached, form a heterocycle selected from
in which RC is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, CH2CMe3, Ph, CH2Ph, CH2CH2OH, or CH2CH2O(C1-C4)alkyl.
In certain embodiments, R5 is H, (C1-C6)alkyl, (C2-C6)alkenyl, phenyl, benzyl, CH2—S—(C1-C6)alkyl, CH2—O—(C1-C6)alkyl, (C2-C6)ORA, (C1-C6)-monoalkyl amine, (C1-C6)-dialkyl amine, or (C1-C6)-cyclic amine, in which said phenyl or benzyl is optionally substituted by one to three substitutents selected from (C1-C4)alkyl, (C1-C4)alkoxy, and halogen; and RA is H, (C1-C6)alkyl, phenyl, CH2-phenyl, (C1-C6)alkylOH, (CH2)pO(CH2)mOH, (CH2)pO(CH2)mO(CH2)mOH, (C1-C6)alkylO(C1-C4)alkyl, (CH2)pO(CH2)mO(C1-C4)alkyl, or (CH2)pO(CH2)mO(CH2)mO(C1-C4)alkyl; p is an integer of 0, 1, 2, 3, 4, or 5; and m is an integer of 1, 2, 3, 4 or 5.
In certain other embodiments, R5 is H, (C1-C4)alkyl, (C2-C4)alkenyl, phenyl, benzyl, CH2—S—(C1-C4)alkyl, CH2—O—(C1-C4)alkyl, (CH2)2OH, or (CH2)2O(C1-C4)alkyl. In certain embodiments, R5 is H. In certain other embodiments, R5 is methyl.
In certain embodiments, each occurrence RA and RB is independently H, (C1-C6)alkyl, phenyl, CH2-phenyl, (C1-C6)alkylOH, (CH2)pO(CH2)mOH, or (CH2)pO(CH2)mO(CH2)mOH, (C1-C6)alkylO(C1-C4)alkyl, (CH2)pO(CH2)mO(C1-C4)alkyl, or (CH2)pO(CH2)mO(CH2)mO(C1-C4)alkyl. In certain other embodiments, each occurrence RA and RB is independently H or (C1-C6)alkyl. In yet other embodiments, RA and RB, together with the nitrogen atom to which they are attached, form a heterocycle selected from
in which RC is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, t-Bu, CH2CMe3, Ph, CH2Ph, or CH2CH2OH and CH2CH2ORd.
In one aspect, the present invention provides a compound selected from the following:
or a pharmaceutically acceptable salt thereof.
In a further aspect, the present invention provides a compound as described in the Examples.
In certain embodiments, the compounds are selected from:
In another aspect, the present invention provides a pharmaceutical composition comprising at least one compound described herein and a pharmaceutically-acceptable carrier or diluent.
In a further aspect, the present invention provides a method for treating or preventing a viral infection in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound described herein. In certain embodiments, the viral infection is HIV infection. In certain other embodiments, the viral infection is HBV infection. In yet other embodiments, the viral infection is HCV infection. In yet other embodiments, the viral infection is influenza A virus infection, severe acute respiratory syndrome coronavirus infection or vaccinia virus infection.
In another aspect, the present invention provides a method for treating or preventing hepatitis C virus infection in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound described herein.
In yet another aspect, the present invention provides a method for inhibiting a cyclophilin in a subject in need thereof, which comprises administrating to said subject an effective cyclophilin-inhibiting amount of at least one compound as described herein.
In yet another aspect, the present invention provides a method for treating or preventing diseases that are mediated by cyclophilins in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein.
In yet another aspect, the present invention provides a method for treating or preventing diseases in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases are selected from inflammation, respiratory inflammation, rheumatoid arthritis, and dry eye.
In yet another aspect, the present invention provides a method for treating or preventing diseases in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases are selected from neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's Diseases, and ALS; traumatic brain injury; stroke; and ischemia-reperfusion injury in the brain, heart, and kidney.
In yet another aspect, the present invention provides a method for treating or preventing diseases in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases are selected from cardiovascular diseases, vascular stenosis, atherosclerosis, abdominal aortic aneurysms, cardiac hypertrophy, aortic rupture, pulmonary arterial hypertension, myocarditis and myocardial fibrosis, and ischaemic heart diseases.
In yet another aspect, the present invention provides a method for treating or preventing diseases or conditions in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases or conditions are selected from cancer, obesity, diabetes, muscular dystrophy, and hair loss.
In yet another aspect, the present invention provides a method for treating or preventing diseases or conditions in a mammalian species in need thereof, the method comprising administering to the mammalian species a therapeutically effective amount of at least one compound as described herein, wherein the diseases or conditions are selected from allergic conjunctivitis, atopic and vernal keratoconjunctivitis, atopic keratoconjunctivitis, anterior uveitis, Behcet's disease, blepharitis, chronic ocular surface inflammation caused by viral infection, corneal transplant rejection, corneal sensitivity impaired due to surgery on the cornea or other surface of the eye, meibomian gland disease, ptyregia, ocular symptoms of graft versus host disease, ocular allergy, ocular cicatricial pemphigoid, Steven Johnson syndrome, vernal keratoconjunctivitis, uveitis, herpes simplex keratitis, ocular rosacea, and Pinguecula.
Methods of Preparation
In certain embodiments, the compound of formulae (I) and (II) can be prepared by treating cyclosporin A or an analog thereof with a base (e.g., LDA) to form a sarcosine enolate at 3-position, and then CO2 gas is introduced to yield carboxylic acid-3-cyclosporin, after formation of its corresponding methyl ester and reduction of the methyl ester side chain to alcohol, its mesylate, tosylate or chloride can be formed by treatment with MsCl or TsCl in dichloromethane solution, and they can be converted to the methylene on the sarcosine by treatment with a base (e.g., NaH), when sulfur nuclectrophile is used for 1,4-addition reaction on the methylene group, the methylene sulfur side chain with S-conformation can be formed on the sarcosine of position 3 as novel cyclosporine derivatives. For example:
[α-Methylene-Sar]-3-cyclosporin can also be prepared using a method analogous to the procedure described in Reference Example 2 or WO2012/051194A1 (which is incorporated herein by reference).
In certain embodiments, the above resulting methylene-3-cyclosporin can be converted to its methylene amine side chain to form novel cyclosporine derivatives. For example:
Therefore, the following derivatives can be prepared by this procedure.
In certain embodiments, the above resulting alcohol can be converted to its methylene oxygen ether side chain to form novel cyclosporine derivatives too. For example:
In Schemes 1-4 above, the symbols have the same meaning as defined in the claims and throughout the specification, unless otherwise noted.
In certain other embodiments, the compound of formula (IIa) or (IIIa) can be obtained according to the procedures described herein.
Pharmaceutical Compositions
This invention also provides a pharmaceutical composition comprising at least one of the compounds as described herein or a pharmaceutically-acceptable salt or solvate thereof, and a pharmaceutically-acceptable carrier.
The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as butylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.
As set out above, certain embodiments of the present pharmaceutical agents may be provided in the form of pharmaceutically-acceptable salts. The term “pharmaceutically-acceptable salt”, in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al., (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).
The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, butionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.
In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term “pharmaceutically-acceptable salts” in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polybutylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.
Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium carbonate, and sodium starch glycolate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and polyethylene oxide-polybutylene oxide copolymer; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxybutylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets, may be, made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxybutylmethyl cellulose in varying butortions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples are embedding compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if apbutriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isobutyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, butylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Additionally, cyclodextrins, e.g., hydroxybutyl-.beta.-cyclodextrin, may be used to solubilize compounds.
Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active pharmaceutical agents of the invention.
Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be apbutriate.
Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or butellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary butellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and butane.
Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving, or dispersing the pharmaceutical agents in the buter medium. Absorption enhancers can also be used to increase the flux of the pharmaceutical agents of the invention across the skin. The rate of such flux can be controlled, by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. One strategy for depot injections includes the use of polyethylene oxide-polybutylene oxide copolymers wherein the vehicle is fluid at room temperature and solidifies at body temperature.
Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
The compounds and pharmaceutical compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, the compound of the present invention may be administered concurrently with another anti-HCV agent), or they may achieve different effects (e.g., control of any adverse effects).
The compounds of the invention may be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, topically, orally, or by other acceptable means. The compounds may be used to treat arthritic conditions in mammals (i.e., humans, livestock, and domestic animals), birds, lizards, and any other organism, which can tolerate the compounds.
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
The representative examples which follow are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. It should further be appreciated that the contents of those cited references are incorporated herein by reference to help illustrate the state of the art. The following examples contain important additional information, exemplification and guidance which can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
[α-Carboxy-sar]-3-cyclosporin (5.00 g, 4.01 mmol) was dissolved in N,N-dimethylformamide (30 ml). Iodomethane (2.85 g, 20.10 mmol) and potassium carbonate (1.38 g, 10.00 mmol) were added. The mixture was stirred at room temperature for 2 hours. Then ethyl acetate (60 ml) and water (60 ml) were added and the mixture was separated. The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 5.32 g of crude product, which was directly used for the next step without purification (yield: ˜100%) [Molecular Formula: C64H113N11O14; Exact Mass: 1259.85; MS (m/z): 1260.7 (M+1)+, 1282.7 (M+Na)+; TLC Rf: 0.55 (dichloromethane/methanol=9/1)].
[α-Methoxycarbonyl-Sar]-3-cyclosporin (2.00 g, 1.59 mmol) was dissolved in tetrahydrofuran (30 ml). Cesium chloride (1.33 g, 7.90 mmol) and sodium borohydride (0.60 g, 15.89 mmol) were added in portions. Then methanol (30 ml) was added dropwise to the mixture over 2 hours. After addition, the mixture was stirred at room temperature overnight. Most of solvent was then evaporated under reduced pressure. Ethyl acetate (50 ml) and water (50 ml) were added. The ethyl acetate layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 1.99 g of crude product, which was purified by on silica gel column with dichloromethane/methanol (from 100:0 to 95:5) to give the 1.50 g of pure product (yield: 76%) [Molecular Formula: C63H113N11O13; Exact Mass: 1231.85; MS (m/z): 1232.7 (M+1)+, 1254.7 (M+Na)+].
To a solution of [α-hydroxymethyl-Sar]-3-cyclosporin (30 mg, 0.024 mmol) in dichloromethane (2 ml) at 0° C. were added triethylamine (52.8 μl, 0.38 mmol), and methanesulfonyl chloride (23 mg, 0.20 mmol). The mixture was stirred at room temperature for two hours. Then reaction mixture was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 33 mg of crude product, which was directly used in next step reaction without further purification [Molecular Formula: C64H115N11O15S; Exact Mass: 1309.83; MS (m/z): 1310.7 (M+1)+].
To a solution of [α-hydroxymethyl-Sar]-3-cyclosporin (30 mg, 0.024 mmol) in dichloromethane (2 ml) at 0° C. were added triethylamine (52.8 μl, 0.384 mmol, 16 equivalents) and methanesulfonyl chloride (23 mg, 0.20 mmol). The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 30 mg of crude product, which was directly used in next step reaction without further purification [Molecular Formula: C63H112ClN11O12; Exact Mass: 1249.82; MS (m/z): 1250.7 (M+1)+, 1272.9 (M+Na)+].
To a solution of either [α-methanesulfonatemethyl-Sar]-3-cyclosporin (33 mg, 0.025 mmol) or [α-chloromethyl-Sar]-3-cyclosporin (30 mg, 0.025 mmol) in tetrahydrofuran (3 ml) was added sodium hydride (15.3 mg, 60% in oil, 0.38 mmol, 10 equivalents) at 0° C. The mixture was stirred at 0° C. for one hour and then warmed up to room temperature for 30 minutes. After removal of solvent, the residue was dissolved in dichloromethane (20 ml). The dichloromethane layer was washed with 1 N hydrochloric acid, saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethylene/methanol (20/1) to give 16 mg of product (yield: 54%) [Molecular Formula: C63H111N11O12; Exact Mass: 1213.84; MS (m/z): 1214.7 (M+1)+, 1236.7 (M+Na)+; TLC Rf: 0.55 (ethyl acetate/methanol=20/1); HPLC RT: 7.0 min (C8 reverse phase column: 150 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-cyclosporin can also be prepared by a method analogous to the procedure described in WO2012/051194A1 (which is incorporated herein by reference).
To a solution of [α-methylene-Sar]-3-cyclosporine (0.60 g, 0.50 mmol) and 2-(dimethylamino)ethanethiol (0.63 g, 6.00 mmol) in methanol (20 ml) was added triethylamine (0.82 ml, 6.0 mmol). The reaction mixture was stirred overnight at room temperature. After removal of solvent, the residue was subjected to chromatography using dichloromethane/methanol as eluent to give 0.35 g of (R)-2-(N,N-dimethylamino)ethylthiomethyl-Sar]-3-cyclosporin (isomer A) and 0.20 g of [(S)-2-(N,N-dimethylamino)ethylthiomethyl-Sar]-3-cyclosporin (isomer B) [Molecular Formula: C67H122N12O12S; Exact Mass: 1218.9; MS (m/z): 1319.80 (M+1)+; TLC Rf: 0.20 (ethyl acetate/methanol=5/1); HPLC RT: 12.55 min (isomer A) and 13.22 min (isomer B) (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-cyclosporin (0.31 g, 0.25 mmol) and 2-diethylaminoethanethiol (0.40 g, 3.00 mmol) in methanol (10 ml) was added triethylamine (0.41 ml, 3.00 mmol, 12 equivalents). The reaction mixture was stirred overnight at room temperature. After removal of solvent, the residue was subjected to chromatography using dichloromethane/methanol as eluent to yield 0.20 g of [(R)-2-(N,N-Diethylamino)ethylthiomethyl-Sar]-3-cyclosporin (isomer A) and 0.08 g of [(S)-2-(N,N-Diethylamino)ethylthiomethyl-Sar]-3-cyclosporin (isomer B) [Molecular Formula: C69H126N12O12S; Exact Mass: 1346.93; MS (m/z): 1347.80 (M+1)+; TLC Rf: 0.23 (ethyl acetate/methanol=5/1); HPLC RT: 13.37 min (isomer A) and 13.91 min (isomer B) (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (0.36 g, 0.29 mmol) in benzene (30 ml) were added a solution of sodium hydroxide (1.20 g, 30 mmol) in water (2 ml), 2-bromo-N,N-diethylethylamine hydrobromide (3.80 g, 14.56 mmol) and tetra-n-butylammonium bromide (0.20 g, 0.62 mmol). The reaction mixture was stirred at 30° C. for 20 hours. After diluted with ice water, the mixture was separated. The aqueous layer was extracted with dichloromethane (30 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 210 mg of product [Molecular Formula: C69H126N12O13; Exact Mass: 1330.96; MS (m/z): 1331.71 (M+1)+; TLC Rf: 0.38 (dichloromethane/methanol=95/5); HPLC RT: 14.12 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (0.50 g, 0.41 mmol) in benzene (30 ml) were added a solution of sodium hydroxide (1.00 g, 25.00 mmol) in water (1 ml), t-butyl bromoacetate (3.20 g, 16.41 mmol) and tetra-n-butylammonium bromide (0.40 g, 1.24 mmol). The mixture was stirred at room temperature for 10 hours. After diluted with ice water, the mixture was separated. The aqueous layer was extracted with dichloromethane (30 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (hexane/acetone=2/1) to give 0.41 g product [Molecular Formula: C69H123N11O15; Exact Mass: 1345.92; MS (m/z): 1346.61 (M+1)+; TLC Rf: 0.60 (dichloromethane/methanol=95/5); HPLC RT: 18.29 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (0.35 g, 0.28 mmol) in benzene (15 ml) were added a solution of sodium hydroxide (0.60 g, 15.00 mmol) in water (1 ml), ethyl bromoacetate (1.60 g, 9.58 mmol) and tetra-n-butylammonium bromide (0.20 g, 0.62 mmol). The mixture was stirred at room temperature for 10 hours. After diluted with ice water, the mixture was separated. The aqueous layer was extracted with dichloromethane (15 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (hexane/acetone=2/1) to give 0.31 g of product [Molecular Formula: C67H119N11O15; Exact Mass: 1317.89; MS (m/z): 1318.46 (M+1)+; TLC Rf: 0.55 (dichloromethane/methanol=95/5); HPLC RT: 17.40 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-((tert-butoxycarbonyl)methoxy)methyl-Sar]-3-cyclosporin (0.18 g, 0.13 mmol) in dichloromethane (5 ml) were added trifluoroacetic acid (1 ml) and triethylsilane (10 drops). The mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. Then dichloromethane (10 ml) and water (10 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by C-18 chromatography (acetonitrile/water) to give 75 mg of product [Molecular Formula: C65H115N11O15; Exact Mass: 1289.86; MS (m/z): 1290.56 (M+1)+; HPLC RT: 11.03 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-(carboxymethoxy)methyl-Sar]-3-cyclosporin (30 mg, 0.02 mmol) in methanol (1 ml) was added a solution of sodium hydroxide (1.00 mg, 0.02 mmol) in water (0.5 ml). The mixture was stirred at room temperature 1 hour and dried in high vacuum to give 28 mg of product [Molecular Formula: C65H114N11NaO15; Exact Mass: 1311.84; MS (m/z): 1290.56 (M+1−Na)+; HPLC RT: 10.98 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-(ethoxycarbonylmethoxy)methyl-Sar]-3-cyclosporin (0.25 g, 0.19 mmol) in methanol (30 ml) were added lithium chloride (0.33 g, 7.85 mmol) and sodium borohydride (0.60 g, 15.89 mmol) in portions. After addition, the mixture was stirred at room temperature overnight. Most of solvent was then evaporated under reduced pressure. Ethyl acetate (50 ml) and water (50 ml) were added. The ethyl acetate layer was separated and washed with brine (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified on silica gel column with (dichloromethane/methanol=95/5) to give the product [Molecular formula: C65H117N11O14; Exact Mass: 1275.88; MS (m/z): 1276.55 (M+1)+; TLC Rf: 0.39 (dichloromethane/methanol=9/1); HPLC RT: 15.31 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (1.03 g, 0.84 mmol) in benzene (50 ml) were added a solution of sodium hydroxide (1.34 g, 33.47 mmol) in water (1.34 ml), tetramethylammonium hydroxide pentahydrate (3.04 g, 16.73 mmol) and 2-dimethylaminoethyl chloride hydrochloride (2.41 g, 16.73 mmol). The mixture was stirred at room temperature for 5 days. Sodium bicarbonate saturated solution (100 ml) was added and the mixture was separated. Then the aqueous layer was extracted with ethyl acetate (50 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 303 mg of product was obtained [Molecular Formula: C67H122N12O13; Exact Mass: 1302.93; MS (m/z): 1303.70 (M+1)+, 1325.85 (M+Na)+; TLC Rf: 0.36 (dichloromethane/methanol=9/1); HPLC RT: 18.19 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (0.27 g, 0.22 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (0.70 g, 17.55 mmol) in water (0.70 ml), tetramethylammonium hydroxide pentahydrate (0.80 g, 4.39 mmol) and 2-(4-morpholinyl)ethyl chloride hydrochloride (0.82 g, 4.39 mmol). The mixture was stirred at 30 to 40° C. for a week. Sodium bicarbonate saturated solution (30 ml) was added and then the mixture was separated. The aqueous layer was extracted with ethyl acetate (25 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 56 mg of product was obtained [Molecular Formula: C69H124N12O14; Exact Mass: 1344.94; MS (m/z): LCMS: 1345.72 (M+1)+, 1367.83 (M+Na)+; TLC Rf: 0.50 (dichloromethane/methanol=9/1); HPLC RT: 16.64 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (0.320 g, 0.26 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (0.83 g, 20.80 mmol) in water (0.85 ml), tetramethylammonium hydroxide pentahydrate (0.95 g, 5.20 mmol) and 1-(2-chloroethyl)pyrrolidine hydrochloride (0.88 g, 5.20 mmol). The mixture was stirred at room temperature for a weekend. Sodium bicarbonate saturated solution (30 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (25 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 103 mg of product was obtained [Molecular Formula: C69H124N12O13; Exact Mass: 1328.94; MS (m/z): 1329.75 (M+1)+, 1351.82 (M+Na)+; TLC Rf: 0.37 (dichloromethane/methanol=9/1); HPLC RT: 18.94 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (0.28 g, 0.22 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (0.36 g, 9.07 mmol) in water (0.36 ml), tetramethylammonium hydroxide pentahydrate (0.82 mg, 4.53 mmol) and 1-(2-chloroethyl)piperidine hydrochloride (0.83 g, 4.53 mmol). The mixture was stirred at 30 to 40° C. for 20 hours. Sodium bicarbonate saturated solution (30 ml) was added and the mixture was separated. Then the aqueous layer was extracted with ethyl acetate (25 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 121 mg of product was obtained [Molecular Formula: C70H126N12O13; Exact Mass: 1342.96; MS (m/z): 1343.76 (M+1)+, 1365.83 (M+Na)+; TLC Rf: 0.44 (dichloromethane/methanol=9/1); HPLC RT: 19.26 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-hydroxymethyl-Sar]-3-cyclosporin (0.50 g, 0.41 mmol) in benzene (30 ml) were added a solution of sodium hydroxide (1.00 g, 25.00 mmol) in water (1 ml), 3-bromopropionaldehyde dimethyl acetal (1.80 g, 10.00 mmol) and tetra-n-butylammonium bromide (0.20 g, 0.62 mmol). After stirred at room temperature for 10 hours, the mixture was diluted with ice water and the mixture was separated. The aqueous layer was extracted with dichloromethane (20 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 0.48 g of crude product, which was used for next step [Molecular Formula: C68H123N11O15; Exact Mass: 1333.92; MS (m/z): 1334.50 (M+1)+].
To a solution of crude [(R)-(3,3-dimethoxypropoxy)methyl-Sar]-3-cyclosporin (0.48 g, 0.36 mmol) in dichloromethane (30 ml) were added trifluoroacetic acid (5 ml) and water (4 ml) at 0° C. Then the mixture was allowed to warm to room temperature and stirred for 3 hours. After the mixture was separated, the dichloromethane layer was washed with saturated sodium bicarbonate solution (20 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (hexane/acetone=2/1) to give 0.31 g of product [Molecular Formula: C66H117N11O14; Exact Mass: 1287.88; MS (m/z): 1288.63 (M+1)+].
To a solution of [(R)-(2-formylethoxy)methyl-Sar]-3-cyclosporin (0.13 g, 0.10 mmol) in chloroform (5 ml) were added dimethylamine hydrochloride (0.10 g, 1.22 mmol) and acetic acid (5 drops). After the mixture was stirred at room temperature for 5 minutes, tetramethylammonium triacetoxyborohydride (65 mg, 0.25 mmol) was added in portions and stirring was continued for one hour. Then dichloromethane (10 ml) and saturated sodium bicarbonate solution (10 ml) were added and separated. The organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 89 mg of product [Molecular Formula: C68H124N12O13; Exact Mass: 1316.94; MS (m/z): 1317.64 (M+1)+; TLC Rf: 0.39 (dichloromethane/methanol=95/5); HPLC RT: 13.92 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-(2-formylethoxy)methyl-Sar]-3-cyclosporin (100 mg, 0.08 mmol) in chloroform (4 ml) were added diethylamine (100 mg, 1.37 mmol) and acetic acid (4 drops). After the mixture was stirred at room temperature for 5 minutes, tetramethylammonium triacetoxyborohydride (50 mg, 0.19 mmol) was added in portions and stirring was continued for 1 hour. Then dichloromethane (10 ml) and saturated sodium bicarbonate solution (10 ml) were added and the mixture was separated. The organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 56 mg of product [Molecular Formula: C70H128N12O13; Exact Mass: 1344.97; MS (m/z): 1345.71 (M+1)+; TLC Rf: 0.40 (dichloromethane/methanol=95/5); HPLC RT: 14.59 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-(2-formylethoxy)methyl-Sar]-3-cyclosporin (300 mg, 0.23 mmol) in dichloromethane (15 ml) were added morpholine (101 mg, 1.16 mmol) and tetramethylammonium triacetoxyborohydride (306 mg, 1.16 mmol). The reaction mixture was stirred at room temperature for two hours. Then sodium bicarbonate saturated solution (30 ml) and dichloromethane (15 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 124 mg of product was obtained [Molecular Formula: C70H126N12O14; Exact Mass: 1358.95; MS (m/z): 1359.71 (M+1)+, 1381.79 (M+Na)+; TLC Rf: 0.40 (dichloromethane/methanol=9/1); HPLC RT: 14.2 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-cyclosporin (315 mg, 0.24 mmol) in dichloromethane (15 ml) were added pyrrolidine (87 mg, 1.22 mmol) and tetramethylammonium triacetoxyborohydride (322 mg, 1.22 mmol). The reaction mixture was stirred at room temperature for two hours. Then sodium bicarbonate saturated solution (30 ml) and dichloromethane (15 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 22 mg of product was obtained [Molecular Formula: C70H126N12O13; Exact Mass: 1342.96; MS (m/z): 1343.75 (M+1)+, 1365.82 (M+Na)+; TLC Rf: 0.33 (dichloromethane/methanol=9/1); HPLC RT: 14.3 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-(2-formylethoxy)methyl-Sar]-3-cyclosporine (350 mg, 0.27 mmol) in dichloromethane (20 ml) were added piperidine (115 mg, 1.34 mmol) and tetramethylammonium triacetoxyborohydride (352 mg, 1.34 mmol). The reaction mixture was stirred overnight at room temperature. Then sodium bicarbonate saturated solution (30 ml) and dichloromethane (15 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 35 mg of product was obtained [Molecular Formula: C71H128N12O13; Exact Mass: 1356.97; MS (m/z): 1357.76 (M+1)+, 1379.83 (M+Na)+; TLC Rf: 0.36 (dichloromethane/methanol=9/1); HPLC RT: 14.4 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of LDA (2.0 M in tetrahydrofuran, 23 ml, 46 mmol) in tetrahydrofuran (80 ml) at −78° C. under nitrogen, [(γ-hydroxy)-N-MeLeu]-4-cyclosporin (4.40 g, 3.61 mmol) in tetrahydrofuran (15 ml) was added over 3 min. After the mixture was stirred at −78° C. for 3 hours, carbon dioxide gas was bubbled into the reaction mixture for 1 hour. Then the mixture was allowed to warm to room temperature slowly and kept stirring for 3 hours. Most of tetrahydrofuran was evaporated. Dichloromethane (100 ml) and water (50 ml) were added. The PH of the mixture was adjusted to around 5 by adding aqueous citric acid solution. The mixture was separated and the organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 3.20 g of crude product, which was used for next step without purification [Molecular Formula: C63H111N11O15; Exact Mass: 1261.83; MS (m/z): 1262.49 (M+1)+].
[(γ-Hydroxy)-N-MeLeu]-4-cyclosporin was prepared by Sebekia benihana biotransformation according to a method described by Kuhnt M. et al., 1996, Microbial Biotransformation Products of Cyclosporin A, J. Antibiotics, 49 (8), 781.
To a mixture of [α-carboxy-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (3.20 g 2.53 mmol) and potassium carbonate (1.30 g, 9.40 mmol) in N,N-dimethylformamide (20 ml) was added iodomethane (1.80 g, 12.70 mmol). The mixture was stirred overnight at room temperature. Dichloromethane (80 ml) and water (50 ml) were added and the mixture was separated. The dichloromethane layer was washed with water (25 ml) and brine (25 ml), dried over magnesium sulfate and evaporated under reduced pressure to give crude 3.00 g of product [Molecular Formula: C64H113N11O15; Exact Mass: 1275.84; MS (m/z): 1276.75 (M+1)+].
To a suspension of [α-methoxycarbonyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (3.00 g, 2.35 mmol) and lithium chloride (1.50 g, 35.30 mmol) in methanol (100 ml) was added sodium borohydride (2.50 g, 66.10 mmol) in portions. The mixture was stirred overnight at room temperature. Most of solvent was evaporated under reduced pressure. Dichloromethane (80 ml) and water (50 ml) were added and the mixture was separated. The dichloromethane layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 1.30 g of product [Molecular Formula: C63H113N11O14; Exact Mass: 1247.85; MS (m/z): 1248.48 (M+1)+; 1H NMR spectrum (600 MHz, CDCl3, δ in ppm): 0.68 (d, J=5.4 Hz, 3H), 0.80-1.00 (m, 30H), 1.07 (d, J=6.0 Hz, 3H), 1.16-1.29 (m, 10H), 1.32 (d, J=7.2 Hz, 3H), 1.39-1.46 (m, 2H), 1.59-1.63 (m, 6H), 1.68-1.83 (m, 7H), 2.02-2.11 (m, 4H), 2.31-2.33 (m, 1H), 2.37-2.42 (m, 2H), 2.67 (s, 6H), 3.09 (s, 3H), 3.19 (s, 3H), 3.20 (s, 3H), 3.22 (s, 3H), 3.47 (s, 3H), 3.72-3.75 (m, 1H), 3.82 (br, 1H), 3.97-3.99 (m, 1H), 4.07-4.10 (m, 1H), 4.50-4.52 (m, 1H), 4.65-4.67 (t, J=8.4 Hz, 1H), 4.79-4.81 (m, 1H), 4.90-4.95 (m, 2H), 5.00-5.05 (m, 2H), 5.09 (d, J=10.8 Hz, 1H), 5.30-5.35 (m, 2H), 5.46 (d, J=6.0 Hz, 1H), 5.52-5.53 (m, 1H), 5.66-5.68 (m, 1H), 7.12 (d, J=7.8 Hz, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.60 (d, J=7.2 Hz, 1H), 7.87-7.89 (d, J=9.6 Hz, 1H)].
To a solution of [α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (250 mg, 0.20 mmol) in dichloromethane (10 mL) at room temperature were added triethylamine (0.33 mL, d 0.726, 2.40 mmol) and triethylamine hydrochloride (95.6 mg, 1.00 mmol), followed by adding p-toluenesulfonyl chloride (0.23 g, 1.20 mmol) under stirring. The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with brine, dried over magnesium sulfate and the solvent was evaporated under reduced pressure. The reaction mixture of [α-chloromethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular formula: C63H112ClN11O13; Exact Mass: 1265.81; MS (m/z): 1266.32 (M+1)+, 1288.43 (M+Na)+] and [α-p-toluenesulfonylmethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular formula: C70H119N11O16S; Exact Mass: 1401.856; MS (m/z): 1402.34 (M+1)+, 1424.62 (M+Na)+] was directly used in next step reaction without further purification. To a solution of the above mixture in tetrahydrofuran (20 ml) was added sodium hydride (320 mg, 60% in oil, 8 mmol) at 0° C. The mixture was stirred at 0° C. for one hour and then warmed up to room temperature for 30 minutes. The reaction was quenched with a saturated ammonia chloride solution. After removing tetrahydrofuran, the crude product was extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using ethyl acetate/methanol (20/1) to give 45 mg of product (yield: 18%) [Molecular formula: C63H111N11O13; Exact Mass: 1229.84; MS (m/z): 1230.6 (M+1)+, 1252.82 (M+Na)+; TLC Rf: 0.50 (ethyl acetate/methanol=10/1); HPLC RT: 15.38 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm); 1H NMR spectrum (600 MHz, CDCl3, δ in ppm): 0.72 (d, J=5.4 Hz, 3H), 0.84-1.00 (m, 30H), 1.17-1.26 (m, 15H), 1.34 (d, J=6.0 Hz, 3H), 1.44-1.47 (m, 2H), 1.59-1.62 (m, 6H), 1.69-1.76 (m, 4H), 1.94-1.99 (m, 1H), 2.09-2.13 (m, 3H), 2.34-2.37 (m, 3H), 2.65 (s, 3H), 2.67 (s, 3H), 3.09 (s, 3H)), 3.10 (s, 3H), 3.19 (s, 3H), 3.44 (s, 3H), 3.46 (s, 3H), 3.80 (m, 1H), 3.91 (m, 1H), 4.47-4.50 (m, 1H), 4.68-4.71 (t, J=9.0 Hz, 1H), 4.78-4.81 (m, 1H), 4.98-5.02 (m, 2H), 5.06-5.11 (m, 3H), 5.24 (s, 1H), 5.32 (m, 2H), 5.41-5.43 (m, 2H), 5.64-5.66 (m, 1H), 7.11 (d, J=7.2 Hz, 1H), 7.49 (d, J=7.2 Hz, 1H), 7.74 (d, J=8.4 Hz, 1H), 7.84 (d, J=9.6 Hz, 1H)].
[(R)-α-Hydroxymethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (crude, 2.00 g), carbon tetrabromide (2.66 g, 8.02 mmol) and triphenylphosphine (2.11 g, 8.02 mmol) were dissolved in dichloromethane (30 ml). The mixture was stirred under nitrogen at room temperature for two hours. Then the mixture was added into a suspension of sodium hydride (60% dispersion in mineral oil) (0.77 g, 19.25 mmol) in tetrahydrofuran (30 ml) under nitrogen at 0° C. The mixture was stirred at 0° C. for one hour. Most of solvents then were evaporated under reduced pressure. The residue was treated with water (10 ml) slowly at 0° C. Ethyl acetate (30 ml) and water (30 ml) were added and the mixture was separated. The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (hexane/acetone from 90/10 to 70/30) to give 0.68 g product of [α-methylene-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C63H111N11O13; Exact Mass: 1229.84; MS (m/z): 1230.50 (M+1)+, 1252.68 (M+Na)+; TLC Rf: 0.50 (ethyl acetate/methanol=10/1); HPLC RT: 15.36 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (250 mg, 0.20 mmol) in methylene chloride (10 mL) was added dropwise 1-chloro-N,N,2-trimethyl-1-propenylamine (131 μl, d 1.01, 1.0 mmol) at 0° C. under nitrogen atmosphere. After stirred for 30 minutes at 0° C., the mixture was allowed to warm to room temperature and stirred for another hour. The reaction mixture was washed with sodium bicarbonate solution, brine, dried over magnesium sulfate and evaporated under reduced pressure. The crude product containing [α-chloromethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular formula: C63H112ClN11O13; Exact Mass: 1265.81; MS (m/z): 1266.32 (M+1)+, 1288.43 (M+Na)] was used in next step reaction without further purification. To a solution of the above crude product in tetrahydrofuran (20 ml) was added sodium hydride (320 mg, 60% in oil, 8 mmol) at 0° C. under stirring. The mixture was stirred at 0° C. for one hour and then warmed up to room temperature for another 30 minutes. The reaction was then quenched with a saturated ammonia chloride solution. After removing tetrahydrofuran, the residue was extracted with ethyl acetate. The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using ethyl acetate/methanol (20/1) to give 33 mg of product (yield: 13%) [Molecular formula: C63H111N11O13; Exact Mass: 1229.84; MS (m/z): 1230.45 (M+1)+, 1252.65 (M+Na)+; TLC Rf: 0.50 (ethyl acetate/methanol=10/1); HPLC RT: 15.36 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
n-Buli (2.2 M, 49.30 ml, 108.46 mmol) was added into a solution of diisopropylamine (15.39 ml, 108.46 mmol) in tetrahydrofuran (150 ml) at −78° C. under nitrogen. After the reaction mixture was stirred for an hour, a solution of [(γ-hydroxy)-NMeLeu]-4-cyclosporin (12.00 g, 9.86 mmol) in tetrahydrofuran (30 ml) was added over 10 min. The stirring was continued at −78° C. for two hours. Carbon dioxide gas was bubbled through the reaction mixture for two hour and the mixture was stirred at −78° C. for another hour. Then the cooling bath was removed and the reaction mixture was allowed to warm up to room temperature slowly with bubbling out of unreacted carbon dioxide. The mixture was cooled to about 0-5° C. by ice bath and chloromethyl chloroformate (13.98 g, 108.46 mmol) was added. The mixture was allowed to warm to room temperature and stirred for overnight. Water (30 ml) was added to quench the reaction. Most of solvent was then evaporated under reduced pressure. Ethyl acetate (100 ml) and water (80 ml) were added. The ethyl acetate layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography with hexane/acetone (from 90:10 to 70:30) as eluent to give 4.74 g of pure product of [α-Methylene-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin* [Molecular Formula: C63H111N11O13; Exact Mass: 1229.84; MS (m/z): 1230.39 (M+1)+, 152.59 (M+Na)+; TLC Rf: 0.50 (ethyl acetate/methanol=10/1); HPLC RT: 15.38 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
Synthesis of [α-Methylene-Sar]-3-cyclosporin can also be prepared using a method analogous to that described in WO2012/051194A1.
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) and methylmercaptoacetate (0.24 g, 2.00 mmol) were dissolved in acetonitrile (15 ml), followed by adding 20 equivalents of potassium carbonate (0.55 g, 4.0 mmol). The mixture was stirred overnight at room temperature. After removal of solvents, the residue was dissolved in dichloromethane (20 ml). The dichloromethane solution was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was subject to the flash chromatography using ethyl acetate/methanol as eluent to give product. [Molecular formula: C66H117N11O15S; Exact Mass: 1335.84; MS (m/z): 1336.50 (M+1)+, 1358.80 (M+Na)+; TLC Rf: 0.30 (ethyl acetate/methanol=20/1); HPLC RT: 14.33 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.86 g, 0.70 mmol) and 2-aminoethanethiol hydrochloride (0.80 g, 7.00 mmol) were dissolved in methanol (80 ml), followed by adding 20 equivalents of lithium hydroxide (0.17 g, 7.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was subjected to the flash chromatography using dichloromethane/methanol as eluent to give 0.60 g of product [Molecular Formula: C65H118N12O13S; Exact Mass: 1306.87; MS (m/z): 1307.56 (M+1)+, 1329.73 (M+Na)+, TLC Rf: 0.025 (dichloromethane/methanol=5/1); HPLC RT: 10.97 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.62 g, 0.50 mmol) and 2-(dimethylamino)ethanethiol (0.49 g, 6.00 mmol) were dissolved in methanol (30 ml) followed by adding triethylamine (0.82 ml, 6.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was subjected to chromatography using dichloromethane/methanol as eluent to yield 0.41 g of [(R)-(2-(N,N-dimethylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (isomer A) and 0.18 g of [(S)-(2-(N,N-dimethylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (isomer B) [Molecular Formula: C67H122N12O13S; Exact Mass: 1334.9; MS (m/z): 1335.7 (M+1)+; TLC Rf: 0.05 (ethyl acetate/methanol=5/1); HPLC RT: 10.88 min (isomer A) and 11.30 min (isomer B) (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.31 g, 0.25 mmol) and 2-diethylaminoethanethiol (0.40 g, 3.00 mmol) were dissolved in methanol (30 ml), followed by adding triethylamine (0.41 ml, 3.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was subjected to chromatography using dichloromethane/methanol as eluent to yield 0.15 g of [(R)-(2-(N,N-diethylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy-N-MeLeu]-4-cyclosporin (isomer A) and 0.10 g of [(S)-(2-(N,N-diethylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy-N-MeLeu]-4-cyclosporin (isomer B) [Molecular Formula: C69H126N12O13S; Exact Mass: 1362.93; MS (m/z): 1363.75 (M+1)+; TLC Rf: 0.1 (ethyl acetate/methanol=5/1); HPLC RT: 11.64 min (isomer A) and 11.85 min (isomer B) (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(Amino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.31 g, 0.25 mmol) and acetone (0.40 ml, 5.44 mmol) were dissolved in chloroform (30 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (164 mg, 0.63 mmol) in portions and a few drops of acetic acid. The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using methylene/methanol as eluent to give 0.25 g of pure product [Molecular Formula: C68H124N12O13S; Exact Mass: 1348.91; MS (m/z): 1349.59 (M+1)+; TLC Rf: 0.1 (ethyl acetate/methanol=5/1); HPLC RT: 11.97 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(N-Isopropylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (49 mg, 0.034 mmol) and formaldehyde (100 μl, 37% in water) were mixed with chloroform (10 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (22 mg, 0.085 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 30 mg of pure product [Molecular Formula: C69H126N12O13S; Exact Mass: 1362.93; MS (m/z): 1363.72 (M+1)+, 1385.81 (M+Na)+; TLC Rf: 0.15 (ethyl acetate/methanol=5:1); HPLC RT: 12.26 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(N-Isopropylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (49 mg, 0.034 mmol) and acetaldehyde (100 μl, 37% in water) were mixed with chloroform (10 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (22 mg, 0.085 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 37 mg of pure product [Molecular Formula: C70H128N12O13S; Exact Mass: 1376.94; MS (m/z): 1377.84 (M+1)+; TLC Rf: 0.15 (ethyl acetate/methanol=5/1); HPLC RT: 12.36 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(N-isopropylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) and isobutyraldehyde (91 μl, 10 mmol) were dissolved in chloroform (30 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (0.13 g, 0.50 mmol) in portion. The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 19 mg of pure product [Molecular Formula: C72H132N12O13S; Exact Mass: 1404.98; MS (m/z): 1405.89 (M+1)+, 1427.94 (M+Na)+; TLC Rf: 0.25 (ethyl acetate/methanol=5/1); HPLC RT: 14.46 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-Aminoethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (42 mg, 0.032 mmol) and isobutyraldehyde (15 μl, 0.165 mmol) were dissolved in chloroform (10 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (21 mg, 0.080 mmol) in portions. The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 23 mg of pure product [Molecular Formula: C73H134N12O13S; Exact Mass: 1418.99; MS (m/z): 1419.73 (M+1)+, 1441.87 (M+Na)+; TLC Rf: 0.36 (ethyl acetate/methanol=5:1); HPLC RT: 14.46 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(Amino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.45 g, 0.34 mmol) and pivalaldehyde (100 μl, 37% in water) were mixed with chloroform (50 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (0.22 g, 0.85 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 11 mg of pure product [Molecular Formula: C70H128N12O13S; Exact Mass: 1376.94; MS (m/z): 1377.72 (M+1)+, 1399.82 (M+Na)+; TLC Rf: 0.15 (ethyl acetate/methanol=5/1); HPLC RT: 12.36 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(N-Neopentylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (49 mg, 0.034 mmol) and formaldehyde (100 μl, 37% in water) were mixed with chloroform (10 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (22 mg, 0.085 mmol. The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 31 mg of pure product [Molecular Formula: C71H130N12O13S; Exact Mass: 1390.96; MS (m/z): 1391.71 (M+1)+, 1413.86 (M+Na)+; TLC Rf: 0.25 (ethyl acetate/methanol=5/1); HPLC RT: 13.28 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(N-Neopentylamino)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (46 mg, 0.034 mmol) and acetaldehyde (10 μl, 0.17 mmol) were dissolved in chloroform/methanol, followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (22 mg, 0.085 mmol) in portions. The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 28 mg of product [Molecular Formula: C72H132N12O13S; Exact Mass: 1404.98; MS (m/z): 1405.75 (M+1)+, 1427.95 (M+Na)+; TLC Rf: 0.25 (ethyl acetate/methanol=5/1); HPLC RT: 13.65 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (260 mg, 0.21 mmol) and 2-morpholinoethanethiol (300 mg, 2.04 mmol) in methanol (30 ml) was added lithium hydroxide (140 mg, 5.83 mmol). The reaction mixture was stirred at room temperature overnight. Most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 102 mg of product [Molecular Formula: C69H124N12O14S; Exact Mass: 1376.91; MS (m/z): 1399.85 (M+Na)+; TLC Rf: 0.30 (dichloromethane/methanol=9/1); HPLC RT: 11.03 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm); 1H NMR spectrum (600 MHz, CDCl3, δ in ppm): 0.68 (d, J=6.6 Hz, 3H), 0.79 (d, J=6.6 Hz, 3H), 0.82 (m, 6H,), 0.85 (d, J=6.6 Hz, 3H), 0.88 (d, J=7.2 Hz, 3H), 0.90 (d, J=6.6 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H), 0.97-1.00 (m, 9H), 1.08 (d, J=6.6 Hz, 3H), 1.21-1.25 (m, 11H), 1.31 (d, J=7.2 Hz, 3H), 1.39-1.47 (m, 2H), 1.54-1.61 (m, 8H), 1.66-1.70 (m, 2H), 1.75 (m, 1H), 2.01-2.11 (m, 4H), 2.36-2.43 (m, 7H), 2.55-2.59 (m, 2H), 2.67 (m, 8H), 2.93-3.04 (m, 2H), 3.10 (s, 3H), 3.24 (s, 6H), 3.26 (s, 3H), 3.48 (s, 3H), 3.52 (br, 1H), 3.67 (m, 6H), 4.51 (m, 1H), 4.59 (t, J=8.4 Hz, 1H), 4.81 (m, 1H), 4.94-5.00 (m, 2H), 5.04 (t, J=6.6 Hz, 1H), 5.08 (d, J=10.8 Hz, 1H), 5.27-5.31 (m, 1H), 5.33-5.37 (m, 1H), 5.48 (m, 2H), 5.67 (m, 1H), 7.14 (d, J=7.8 Hz, 1H), 7.49 (d, J=7.8 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 8.11 (d, J=9.6 Hz, 1H)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.37 g, 0.30 mmol) and 2-(N-piperidino)ethylthiol (0.44 g, 3.00 mmol) were dissolved in methanol (30 ml), followed by adding 10 equivalents of lithium hydroxide. The mixture was stirred overnight at room temperature. After removal of solvent, the residue was dissolved in dichloromethane (30 ml). The dichloromethane solution was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography using dichloromethane/methanol as eluent to give 0.20 g of product [Molecular Formula: C70H126N12O13S; Exact Mass: 1374.93; MS (m/z): 1375.65 (M+1)+, 1397.80 (M+Na)+; TLC Rf: 0.18 (ethyl acetate/methanol=5/1); HPLC RT: 12.09 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (1.37 g, 1.11 mmol) and 2-mercaptoethylpiperazine (0.49 g, 3.33 mmol) were dissolved in methanol (25 ml), followed by adding lithium hydroxide (0.26 g, 11.10 mmol). The mixture was stirred at room temperature overnight. Most of solvent was evaporated under reduced pressure. The residue was mixed with ethyl acetate (60 ml) and saturated sodium bicarbonate solution (60 ml) and separated. The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C69H125N13O13S; Exact Mass: 1375.92; MS (m/z): 1376.55 (M+1)+, 1398.69 (M+Na)+; TLC Rf: 0.11 (dichloromethane/methanol=9:1); HPLC RT: 8.06 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.30 g, 0.24 mmol) and 2-(4-methylpiperazino)ethylthiol (0.42 g, 2.62 mmol) were dissolved in methanol (30 ml), followed by adding 10 equivalents of lithium hydroxide. The mixture was stirred overnight at room temperature. After removal of solvent, the residue was dissolved in methylene chloride (30 ml). The dichloromethane solution was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography using dichloromethane/methanol as eluent to give 0.22 g of product [Molecular Formula: C70H127N13O13S; Exact Mass: 1389.94; MS (m/z): 1390.9 (M+1)+; TLC Rf: 0.08 (ethyl acetate/methanol=5/1); HPLC RT: 10.07 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.30 g, 0.24 mmol) and 3-(1-ethyl-4-piperazino)ethylthiol (0.30 g, 1.72 mmol) were dissolved in methanol (15 ml), followed by adding lithium hydroxide (58 mg, 2.41 mmol). The mixture was stirred overnight at room temperature. Then most of the solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=94/6) to give product [Molecular formula: C71H129N13O13S; Exact Mass: 1403.96; MS (m/z): 1404.55 (M+1)+; TLC Rf: 0.30 (dichloromethane/methanol=85/15); HPLC RT: 8.83 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(S)-(2-(N-piperazinyl)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (80 mg, 0.058 mmol) and propionaldehyde (MW 58.08, d 0.81, 42 μl, 0.580 mmol) were dissolved in dichloromethane (25 ml), followed by adding tetramethylammonium triacetoxyborohydride (153 mg, 0.580 mmol) in portions and acetic acid (3 drops). The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane and methanol as eluent to give product [Molecular Formula: C72H131N13O13S; Exact Mass: 1417.97; MS (m/z): 1418.60 (M+1)+, 1440.79 (M+Na)+; TLC Rf: 0.37 (dichloromethane/methanol=9:1); HPLC RT: 9.61 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(N-piperazinyl)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (80 mg, 0.058 mmol) and isobutyraldehyde (MW 72.11, d 0.794, 53 μl, 0.58 mmol) were dissolved in dichloromethane (25 ml), followed by adding tetramethylammonium triacetoxyborohydride (153 mg, 0.58 mmol) in portions and acetic acid (3 drops). The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane and methanol as eluent to give product [Molecular Formula: C73H133N13O13S; Exact Mass: 1431.99; MS (m/z): 1432.63 (M+1)+, 1454.78 (M+Na)+; TLC Rf: 0.44 (dichloromethane/methanol=9:1); HPLC RT: 10.08 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(2-(N-piperazinyl)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (80 mg, 0.058 mmol) and 2-bromoethanol (FW 124.97, d 1.762, 41 μl, 0.58 mmol) were dissolved in dichloromethane (15 ml), followed by adding sodium carbonate (15.4 mg, 0.15 mmol). The mixture was stirred at room temperature for a weekend. Then the reaction mixture was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane and methanol as eluent to give 20.8 mg of product [Molecular Formula: C71H129N13O14S; Exact Mass: 1419.95; MS (m/z): 1420.52 (M+1)+, 1442.72 (M+Na)+; TLC Rf: 0.20 (dichloromethane/methanol=9:1); HPLC RT: 8.79 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (280 mg, 0.23 mmol) and 2-(N-pyrrolidinyl)ethanethiol (280 mg, 2.14 mmol) in methanol (30 ml) was added lithium hydroxide (114 mg, 4.75 mmol). The reaction mixture was stirred overnight at room temperature. Most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 126 mg of product [Molecular Formula: C69H124N12O13S; Exact Mass: 1360.91; MS (m/z): 1361.80 (M+1)+; TLC Rf: 0.23 (dichloromethane/methanol=95/5); HPLC RT: 11.59 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (200 mg, 0.16 mmol) and 2-mercaptoethanol (MW 78.13, d 1.114, 112 μl, 1.60 mmol) were dissolved in methanol (10 ml), followed by adding lithium hydroxide (23 mg, 0.96 mmol). The mixture was stirred at room temperature overnight. Most of solvent was evaporated under reduced pressure. The residue was mixed with ethyl acetate (20 ml) and saturated sodium bicarbonate solution (20 ml) and separated. The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C65H117N11O14S; Exact Mass: 1307.85; MS (m/z): 1308.44 (M+1)+, 1330.68 (M+Na)+; TLC Rf: 0.41 (dichloromethane/methanol=9:1); HPLC RT: 12.61 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.12 mmol) and 2-ethoxyethanethiol (160 mg, 1.51 mmol) in methanol (10 ml) was added lithium hydroxide (50 mg, 2.08 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate/methanol=97/3) to give a pure product [Molecular formula: C67H121N11O14S; Exact Mass: 1335.88; MS (m/z): 1336.49 (M+1)+; TLC Rf: 0.43 (dichloromethane/methanol=97/3); HPLC RT: 15.51 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(S)-(2-hydroxyethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.28 g, 0.21 mmol) in benzene (15 ml) were added a solution of sodium hydroxide (0.60 g, 15.00 mmol) in water (1 ml), ethyl bromoacetate (1.50 g, 8.98 mmol) and tetra-n-butylammonium bromide (0.20 g, 0.62 mmol). The mixture was stirred at room temperature for 10 hours. After diluted with ice water, the mixture was separated. The aqueous layer was extracted with dichloromethane (15 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate/methanol=97/3) to give a pure product [Molecular Formula: C69H123N11O16S; Exact Mass: 1393.89; MS (m/z): 1394.48 (M+1)+; TLC Rf: 0.45 (dichloromethane/methanol=95/5); HPLC RT: 15.28 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a suspension of [(S)-((ethoxycarbonylmethoxy)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (100 mg, 0.07 mmol) and lithium chloride (15 mg, 0.35 mmol) in methanol (10 ml) was added sodium borohydride (125 mg, 3.29 mmol) in portions. The mixture was stirred overnight at room temperature for 6 hours. Most of solvent was evaporated under reduced pressure. Dichloromethane (50 ml) and water (200 ml) were added and separated. The dichloromethane layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give the product [Molecular formula: C67H121N11O15S; Exact Mass: 1351.88; MS (m/z): 1352.46 (M+1)+; TLC Rf: 0.32 (dichloromethane/methanol=95/5); HPLC RT: 12.87 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (300 mg, 0.24 mmol) and 2-(2-methoxyethoxy)ethanethiol (330 mg, 2.42 mmol) in methanol (20 ml) was added lithium hydroxide (60 mg, 2.50 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate/methanol=97/3) to give a pure product [Molecular formula: C68H123N11O15S; Exact Mass: 1365.89; MS (m/z): 1366.49 (M+1)+; TLC Rf: 0.37 (dichloromethane/methanol=97/3); HPLC RT: 14.72 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (3.00 g, 2.44 mmol) and 3-aminopropanethiol (1.33 g, 14.59 mmol) were dissolved in methanol (80 ml), followed by adding lithium hydroxide (0.35 g, 14.65 mmol). The mixture was stirred at room temperature for a weekend. After removal of solvent, the residue was purified by chromatography using dichloromethane/methanol as eluent to give product [Molecular Formula: C66H120N12O13S; Exact Mass: 1320.88; MS (m/z): 1321.52 (M+1)+, 1343.67 (M+Na)+, TLC Rf: 0.028 (dichloromethane/methanol=5/1); HPLC RT: 10.24 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.30 g, 0.24 mmol) and 3-(N,N-dimethyl)propylthiol (0.36 g, 2.40 mmol) were dissolved in methanol (25 ml), followed by adding lithium hydroxide (59 mg, 2.44 mmol). The mixture was stirred at room temperature overnight. After removal of solvent, the residue was purified by flash chromatography using dichloromethane/methanol as eluent to give 0.18 g of pure product [Molecular Formula: C68H124N12O13S; Exact Mass: 1348.91; MS (m/z): 1349.70 (M+1)+, 1371.83 (M+Na); TLC Rf: 0.15 (ethyl acetate/methanol=5/1); HPLC RT: 11.53 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.30 g, 0.24 mmol) and 3-(N,N-dimethyl)propylthiol (0.36 g, 2.44 mmol) were dissolved in methanol (25 ml), followed by adding lithium chloride (59 mg, 2.4 mmol). The mixture was stirred at room temperature overnight. After removal of solvent, the residue was purified by flash chromatography using dichloromethane/methanol as eluent to give 0.30 g of product [Molecular Formula: C70H128N12O13S; Exact Mass: 1376.94; MS (m/z): 1377.90 (M+1)+, 1399.76 (M+Na)+; TLC Rf: 0.17 (ethyl acetate/methanol=5/1); HPLC RT: 12.06 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(3-Aminopropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.11 mmol) and acetone (MW 58.08, d 0.791, 42 μl, 0.57 mmol) were dissolved in 10 ml of dichloromethane, followed by adding tetramethylammonium triacetoxyborohydride (74.7 mg, 0.28 mmol) in portions and a few drops of acetic acid. The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C69H126N12O13S; Exact Mass: 1362.93; MS (m/z): 1363.60 (M+1)+; TLC Rf: 0.38 (dichloromethane/methanol=9/1); HPLC RT: 10.89 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (200 mg, 0.16 mmol) and 3-(N-ethyl-N-isopropylamino)propylthiol (200 mg, 1.25 mmol) in methanol (25 ml) was added lithium hydroxide (89 mg, 3.71 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 88 mg of product [Molecular Formula: C71H130N12O13S; Exact Mass: 1390.96; MS (m/z): 1413.81 (M+Na)+; TLC Rf: 0.40 (dichloromethane/methanol=9/1); HPLC RT: 12.49 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
[(S)-(3-Aminopropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.11 mmol) and isobutyraldehyde (MW 72.11, d 0.794, 15 μl, 0.17 mmol) were dissolved in dichloromethane (25 ml), followed by adding a few drops of acetic acid and tetramethylammonium triacetoxyborohydride (30 mg, 0.11 mmol) in portions. The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with saturated sodium bicarbonate solution (25 ml) and brine (25 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C70H128N12O13S; Exact Mass: 1376.94; MS (m/z): 1477.56 (M+1)+, 1399.71 (M+Na)+; TLC Rf: 0.18 (dichloromethane/methanol=9:1); HPLC RT: 11.70 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(3-Aminopropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.11 mmol) and isobutyraldehyde (MW 72.11, d 0.794, 15 μl, 0.17 mmol) were dissolved in dichloromethane (25 ml), followed by adding a few drops of acetic acid and tetramethylammonium triacetoxyborohydride (30 mg, 0.11 mmol in portions. The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with saturated sodium bicarbonate solution (25 ml) and brine (25 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C73H134N12O13S; Exact Mass: 1433.01; MS (m/z): 1433.64 (M+1)+, 1455.78 (M+Na)+; TLC Rf: 0.24 (dichloromethane/methanol=95:5); HPLC RT: 14.45 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(3-Aminopropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (50 mg, 0.04 mmol) and pivalaldehyde (MW 86.13, d 0.793, 21 μl, 0.19 mmol) were mixed with 20 ml of dichloromethane, followed by adding tetramethylammonium triacetoxyborohydride (24.9 mg, 0.10 mmol) and a few drops of acetic acid. The mixture was stirred at room temperature for 6 hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution (30 ml) and brine (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C71H130N12O13S; Exact Mass: 1390.96; MS (m/z): 1391.54 (M+1)+, 1413.69 (M+Na)+; TLC Rf: 0.24 (dichloromethane/methanol=5/1); HPLC RT: 13.21 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.20 mmol) and 3-(N-piperidinyl)propanethiol (318 mg, 2.00 mmol) in methanol (30 ml) was added lithium hydroxide (96 mg, 4.00 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 135 mg of product [Molecular Formula: C71H128N12O13S; Exact Mass: 1388.94; MS (m/z): 1389.84 (M+1)+; TLC Rf: 0.30 (dichloromethane/methanol=95/5); HPLC RT: 12.19 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm); 1H NMR spectrum (600 MHz, CDCl3, δ in ppm): 0.68 (d, J=6.0 Hz, 3H), 0.79 (d, J=6.0 Hz, 3H), 0.82-0.86 (m, 9H), 0.88 (d, J=6.6 Hz, 3H), 0.91 (d, J=6.6 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H), 0.97-1.00 (m, 9H), 1.09 (d, J=6.6 Hz, 3H), 1.21-1.24 (m, 11H), 1.31-1.46 (m, 8H), 1.53 (m, 5H), 1.61 (m, 11H), 1.67-1.70 (m, 2H), 1.74-1.76 (m, 2H), 1.99-2.11 (m, 4H), 2.31-2.35 (m, 4H), 2.37-2.41 (m, 2H), 2.53-2.60 (m, 2H), 2.67 (s, 6H), 2.91-2.98 (m. 2H), 3.09 (s, 3H), 3.24 (s, 6H), 3.26 (s, 3H), 3.48 (s, 3H), 3.56 (m, 1H), 3.65 (m, 1H), 4.51 (m, 1H), 4.58 (t, J=8.4 Hz, 1H), 4.81 (m, 1H), 4.94-5.02 (m, 2H), 5.04 (t, J=6.6 Hz, 1H), 5.08 (d, J=10.8 Hz, 1H), 5.28-5.32 (m, 1H), 5.33-5.37 (m, 1H), 5.49 (m, 2H), 5.67 (dd, J=10.8 Hz and 3.6 Hz, 1H), 7.14 (d, J=8.4 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 8.09 (d, J=10.2 Hz, 1H)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (213 mg, 0.17 mmol) and 3-(N-pyrrolidinyl)propanethiol (280 mg, 1.93 mmol) in methanol (25 ml) was added lithium hydroxide (94 mg, 3.92 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give 57 mg of product [Molecular Formula: C70H126N12O13S; Exact Mass: 1374.93; MS (m/z): 1375.75 (M+1)+; TLC Rf: 0.23 (dichloromethane/methanol=95/5); HPLC RT: 11.83 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (210 mg, 0.17 mmol) and 3-morpholinopropanethiol (300 mg, 1.86 mmol) in methanol (25 ml) was added lithium hydroxide (140 mg, 5.83 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 78 mg of product [Molecular Formula: C70H126N12O14S; Exact Mass: 1390.92; MS (m/z): 1413.77 (M+Na)+; TLC Rf: 0.33 (dichloromethane/methanol=9/1); HPLC RT: 11.35 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm); 1H NMR spectrum (600 MHz, CDCl3, δ in ppm): 0.68 (d, J=6.0 Hz, 3H), 0.79 (d, J=5.4 Hz, 3H), 0.82-0.86 (m, 9H), 0.88 (d, J=6.6 Hz, 3H), 0.91 (d, J=6.6 Hz, 3H), 0.93 (d, J=6.6 Hz, 3H), 0.97-1.00 (m, 9H), 1.09 (d, J=6.6 Hz, 3H), 1.21-1.24 (m, 11H), 1.31 (d, J=7.2 Hz, 3H), 1.38-1.46 (m, 2H), 1.61 (m, 11H), 1.67-1.70 (m, 2H), 1.74-1.76 (m, 2H), 2.03-2.11 (m, 4H), 2.35-2.43 (m, 8H), 2.55-2.63 (m, 2H), 2.67 (s, 6H), 2.91-2.98 (m. 2H), 3.10 (s, 3H), 3.24 (3, 6H), 3.26 (s, 3H), 3.49 (s, 3H), 3.52 (m, 1H), 3.65-3.67 (m, 5H), 4.51 (m, 1H), 4.59 (t, J=8.4 Hz, 1H), 4.81 (m, 1H), 4.94-5.01 (m, 2H), 5.04 (t, J=6.6 Hz, 1H), 5.08 (d, J=12 Hz, 1H), 5.28-5.30 (m, 1H), 5.33-5.37 (m, 1H), 5.49 (m, 2H), 5.67 (m, 1H), 7.14 (d, J=7.8 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.65 (d, J=7.2 Hz, 1H), 8.12 (d, J=9.6 Hz, 1H)].
To a solution of [(S)-(3-(N-Morpholino)propylthio)methyl-Sar]-3-[(γ-hydroxyl)-N-MeLeu]-4-cyclosporin (27 mg, 0.02 mmol) in methanol (5 ml) was added oxone (25 mg, 0.04 mmol). The reaction mixture was stirred at room temperature for one hour. Then most of the solvent was evaporated under reduced pressure. Dichloromethane (20 ml) and saturated sodium bicarbonate solution (5 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give product [Molecular formula: C70H126N12O15S; Exact Mass: 1406.92; MS (m/z): 1407.47 (M+1)+; TLC Rf: 0.23 (dichloromethane/methanol=9/1); HPLC RT: 9.35 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(S)-(3-(N-Morpholino)propylthio)methyl-Sar]-3-[(γ-hydroxyl)-N-MeLeu]-4-cyclosporin (30 mg, 0.02 mmol) in methanol (5 ml) were added oxone (40 mg, 0.07 mmol) and water (0.3 ml) at 0° C. The reaction mixture was stirred at room temperature 1 hour. Dichloromethane (30 ml) and cold saturated sodium bicarbonate solution (5 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give product [Molecular formula: C70H126N12O16S; Exact Mass: 1422.91; MS (m/z): 1423.54 (M+1)+; TLC Rf: 0.28 (dichloromethane/methanol=9/1); HPLC RT: 9.38 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (280 mg, 0.23 mmol) and 4-(3-thiopropyl)thiomorpholine (280 mg, 1.58 mmol) in methanol (15 ml) was added lithium hydroxide (80 mg, 3.33 mmol). The reaction mixture was stirred at room temperature overnight. Then most of the solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give product [Molecular formula: C70H126N12O13S2; Exact Mass: 1406.90; MS (m/z): 1407.51 (M+1)+; TLC Rf: 0.35 (dichloromethane/methanol=9/1); HPLC RT: 11.18 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (2.95 g, 2.40 mmol) and 1-Boc-4-(3-mercaptopropanyl)piperazine (MW: 260.4, 3.18 g, 5.10 mmol) were dissolved in methanol (75 ml), followed by adding lithium hydroxide (MW: 23.95, 0.35 g, 14.4 mmol). The mixture was stirred at room temperature overnight. After removal of solvent, the residue was purified by chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C75H135N13O15S; Exact Mass: 1489.99; MS (m/z): 1490.54 (M+1)+, 1512.63 (M+Na)+; HPLC RT: 12.51 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(R)-(3-(4-N-Boc-N-piperazinyl)propylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (1.59 g, 1.07 mmol) was dissolved in methanol (20 ml). Then 4 M hydrochloric acid in dioxane (4 ml) was added. The mixture was stirred at room temperature for one hour. Most of solvent was evaporated under reduced pressure. The residue was mixed ethyl acetate (60 ml) and saturated sodium bicarbonate solution (60 ml) and separated. The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The crude product was purified by chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular Formula: C70H127N13O13S; Exact Mass: 1389.94; MS (m/z): 1390.56 (M+1)+, 1412.70 (M+Na)+; TLC Rf: 0.37 (dichloromethane/methanol=5:1); HPLC RT: 8.20 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.30 g, 0.24 mmol) and 3-(4-methylpiperazino)propylthiol (MW: 174.31, 0.42 g, 2.44 mmol) were dissolved in methanol (25 ml), followed by adding 10 equivalents of lithium hydroxide (58 mg, 2.40 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by flash chromatography using dichloromethane/methanol as eluent to give 0.20 g of product [Molecular Formula: C71H129N13O13S; Exact Mass: 1403.96; MS (m/z): 1404.9 (M+1)+, 1426.9 (M+Na)+; TLC Rf: 0.10 (ethyl acetate/methanol=5/1); HPLC RT: 10.07 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) and 3-(1-ethyl-4-piperazino)propylthiol (MW: 188.33, 0.20 g, 1.06 mmol) were dissolved in methanol (15 ml), followed by adding lithium hydroxide (MW: 23.95, 48 mg, 20 mmol). The mixture was stirred overnight at room temperature. After removal of solvents, the residue was subject to chromatography using dichloromethane/methanol as eluent to give product [Molecular formula: C72H131N13O13S; Exact Mass: 1417.97; MS (m/z): 1418.58 (M+1)+; TLC Rf: 0.13 (ethyl acetate/methanol=5/1)]. HPLC RT: 8.70 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(S)-(3-(N-piperazinyl)propylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.11 mmol) and propionaldehyde (MW 58.08, d 0.81, 80 μl, 1.11 mmol) were dissolved in dichloromethane (20 ml), followed by adding acetic acid (5 drops) and tetramethylammonium triacetoxyborohydride (MW: 263.09, 72 mg, 0.28 mmol) in portions. The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane and methanol as eluent to give product [Molecular Formula: C73H133N13O13S; Exact Mass: 1431.99; MS (m/z): 1432.61 (M+1)+, 1454.69 (M+Na)+; TLC Rf: 0.24 (dichloromethane/methanol=9:1); HPLC RT: 9.07 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-3-(N-piperazinylpropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.11 mmol) and 2-bromopropane (MW 123.00, d 1.310, 102 μl, 1.08 mmol) were dissolved in dichloromethane (15 ml), followed by adding sodium carbonate (MW: 105.99, 28.6 mg, 0.27 mmol). The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane and methanol as eluent to give product [Molecular Formula: C73H135N13O13S; Exact Mass: 1431.99; MS (m/z): 1432.58 (M+1)+, 1454.72 (M+Na)+; TLC Rf: 0.14 (dichloromethane/methanol=9:1); HPLC RT: 8.74 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-3-(N-piperazinylpropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (168 mg, 0.12 mmol) and isobutyraldehyde (MW 72.11, d 0.794, 110 μl, 1.21 mmol) were dissolved in dichloromethane (25 ml), followed by adding acetic acid (5 drops) and tetramethylammonium triacetoxyborohydride (79.5 mg, 0.30 mmol) in portions. The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane and methanol as eluent to give product [Molecular Formula: C74H135N13O13S; Exact Mass: 1446.00; MS (m/z): 1446.58 (M+1)+, 1468.69 (M+Na)+; TLC Rf: 0.43 (dichloromethane/methanol=9:1); HPLC RT: 9.59 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-3-(N-piperazinylpropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.11 mmol) and neopentyl bromide (MW 151.05, d 1.195, 136 μl, 1.08 mmol) were dissolved in dichloromethane (20 ml), followed by adding sodium carbonate (MW: 105.99, 28.6 mg, 0.27 mmol). The mixture was stirred at room temperature overnight. Then the reaction mixture was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using dichloromethane and methanol as eluent to give product [Molecular Formula: C75H137N13O13S; Exact Mass: 1460.02; MS (m/z): 1460.64 (M+1)+, 1482.72 (M+Na)+; TLC Rf: 0.43 (dichloromethane/methanol=9:1); HPLC RT: 11.25 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (600 mg, 0.49 mmol) and methyl 3-mercaptopropionate (0.54 ml, d 1.085, 4.88 mmol) in methanol (15 ml) was added lithium hydroxide (94 mg, 3.90 mmol). The reaction mixture was stirred at room temperature for 4 hours. Most of solvent was evaporated under reduced pressure. Ethyl acetate (30 ml) and brine (30 ml) were added and the mixture was separated. The organic layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (hexane/acetone) to give 270 mg of [(S)-(2-(methoxycarbonyl)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C67H119N11O15S; Exact Mass: 1349.86. MS (m/z): 1350.43 (M+1)+, 1372.62 (M+Na)] and 260 mg of [(R)-(2-(methoxycarbonyl)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C67H119N11O15S; Exact Mass: 1349.86. MS (m/z): 1350.42 (M+1)+, 1372.63 (M+Na)+].
[(S)-(2-(Methoxycarbonyl)ethylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (170 mg, 0.13 mmol) was dissolved in tetrahydrofuran (30 ml), followed by adding cesium chloride (1.00 g, 5.94 mmol) and sodium borohydride (1.00 g, 26.43 mmol). Then 30 ml of methanol was added dropwise to the mixture over 30 minutes. After addition, the mixture was stirred at room temperature one hour. Most solvent was then evaporated under reduced pressure. Ethyl acetate (30 ml) and water (30 ml) were added. The ethyl acetate layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography with dichloromethane/methanol (from 100:0 to 96:4) as eluent to give the 55 mg of pure product [Molecular Formula: C66H119N11O14S; Exact Mass: 1321.87; MS (m/z): 1322.45 (M+1)+, 1344.67 (M+Na)+; TLC Rf: 0.54 (dichloromethane/methanol=9:1); HPLC RT: 13.06 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
According this method, 28 mg of pure [(R)-(3-Hydroxypropylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C66H119N11O14S; Exact Mass: 1321.87; MS (m/z): 1322.44 (M+1)+, 1344.69 (M+Na)+; TLC Rf: 0.54 (dichloromethane/methanol=9:1); HPLC RT: 13.02 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)] was obtained.
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (200 mg, 0.16 mmol) and 3-mercapto-1-propanol (125 mg, 1.36 mmol) in methanol (15 ml) was added lithium hydroxide (80 mg, 3.33 mmol). The reaction mixture was stirred at room temperature overnight. Then most of the solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give product of [(R)-(3-Hydroxypropylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular formula: C66H119N11O14S; Exact Mass: 1321.87; MS (m/z): 1322.53 (M+1)+; TLC Rf: 0.54 (dichloromethane/methanol=9/1); HPLC RT: 13.02 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.31 g, 0.25 mmol) and 1-mercapto-3-methoxypropane (265 mg, 10 mmol) were dissolved in methanol (10 ml), followed by adding 10 equivalents of lithium hydroxide (60 mg). The mixture was stirred overnight at room temperature. After removal of solvents, the residue was dissolved in ethyl acetate (15 ml). The ethyl acetate solution was washed with brine, dried over magnesium sulfite and evaporated under reduced pressure. The residue was subject to a flash chromatography using ethyl acetate/methanol as eluent to give 35 mg of pure product [Molecular formula: C67H121N11O14S; Exact Mass: 1335.88; MS (m/z): 1336.43 (M+1)+, 1358.74 (M+Na)+; TLC Rf: 0.31 (ethyl acetate/methanol=20/1); HPLC RT: 15.21 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.12 mmol) and 3-methyl-3-methoxybutanethiol (165 mg, 1.23 mmol) in methanol (10 ml) was added lithium hydroxide (50 mg, 2.08 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate/methanol=98/2) to give a pure product [Molecular formula: C69H125N11O14S; Exact Mass: 1363.91; MS (m/z): 1364.53 (M+1)+; TLC Rf: 0.33 (ethyl acetate/methanol=98/2); HPLC RT: 16.10 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (1.23 g, 1.0 mmol) and 4-aminobutylthiol (1.06 g, 10.00 mmol) were dissolved in methanol (80 ml), followed by adding 10 equivalents of lithium hydroxide (0.24 g, 10.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvents, the residue was subjected to the flash chromatography using dichloromethane/methanol as eluents to give 0.40 g of product [Molecular formula: C67H122N12O13S; Exact Mass: 1334.89; MS (m/z): 1335.55 (M+1)+, 1357.64 (M+Na)+, TLC Rf: 0.05 (dichloromethane/methanol=5/1); HPLC RT: 10.53 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(S)-(4-Aminobuylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (70 mg, 0.05 mmol) and acetaldehyde (MW 44.05, d 0.78, 100 μl, 0.18 mmol)) were dissolved in chloroform (5 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (32.87 mg, 0.125 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular formula: C71H130N12O13S; Exact Mass: 1390.96; MS (m/z): 1391.63 (M+1)+, 1413.79 (M+Na)+; TLC Rf: 0.17 (ethyl acetate/methanol=5/1); HPLC RT: 12.55 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(S)-(4-Aminobuylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (60 mg, 0.05 mmol) and acetone (100 μl) were dissolved in chloroform (5 ml), followed by adding tetramethylammonium triacetoxyborohydride (29.5 mg, 0.11 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product. [Molecular formula: C70H128N12O13S; Exact Mass: 1376.94; MS (m/z): 1377.58 (M+1)+, 1399.79 (M+Na)+; TLC Rf: 0.15 (ethyl acetate/methanol=5/1); HPLC RT: 11.21 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(S)-(4-Aminobuylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (80 mg, 0.060 mmol) and isobutyraldehyde (MW 72.11, d 0.794, 27 μl, 0.3 mmol) were dissolved in chloroform (5.0 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (39.4 mg, 0.15 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular formula: C71H130N12O13S; Exact Mass: 1390.96; MS (m/z): 1391.58 (M+1)+, 1413.74 (M+Na)+; TLC Rf: 0.20 (ethyl acetate/methanol=5/1); HPLC RT: 11.99 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(S)-(4-Aminobuylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (80 mg, 0.060 mmol) and isobutyraldehyde (MW 72.11, d 0.794, 27 μl, 0.3 mmol) were dissolved in chloroform (5.0 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (39.4 mg, 0.15 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give product [Molecular formula: C75H138N12O13S; Exact Mass: 1447.023; MS (m/z): 1447.63 (M+1)+, 1470.84 (M+Na)+; TLC Rf: 0.25 (ethyl acetate/methanol=5/1); HPLC RT: 13.84 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(S)-(4-Aminobuylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (67 mg, 0.05 mmol) and trimethylacetaldehyde (MW 86.13, d 0.781, 27.6 μl, 0.25 mmol) were dissolved in chloroform (5 ml), followed by adding 2.5 equivalents of tetramethylammonium triacetoxyborohydride (32.87 mg, 0.125 mmol). The mixture was stirred at room temperature for two hours. Then the reaction mixture was washed with saturated sodium bicarbonate solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel using dichloromethane/methanol as eluent to give 25 mg of pure product [Molecular formula: C72H132N12O13S; Exact Mass: 1404.98; MS (m/z): 1405.54 (M+1)+, 1427.72 (M+Na)+; TLC Rf: 0.17 (ethyl acetate/methanol=5/1); HPLC RT: 12.66 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (213 mg, 0.17 mmol) and 4-mercapto-1-butanol (156 mg, 1.44 mmol) in methanol (25 ml) was added lithium hydroxide (94 mg, 3.92 mmol). The reaction mixture was stirred at room temperature overnight. Then most of the solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was subject to a flash chromatography using ethyl acetate/methanol as eluent to give product of isomer A as [(S)-(4-hydroxylbutylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin and product of isomer B as [(R)-(4-hydroxylbutylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin [Molecular formula: C67H121N11O14S; Exact Mass: 1335.88; MS (m/z): 1336.44 (M+1)+, 1358.67 (M+Na)+; TLC Rf: 0.38 (dichloromethane/methanol=9/1); TLC Rf (isomer A): 0.25 (ethyl acetate/methanol=20/1, twice development), and TLC Rf (isomer B): 0.20 (ethyl acetate/methanol=20/1, twice development); HPLC RT: 13.57 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (1.28 g, 1.04 mmol) and methyl 4-mercaptobutanoate (0.84 g, 6.24 mmol) in methanol (25 ml) was added lithium hydroxide (0.15 g, 6.24 mmol). The reaction mixture was stirred at room temperature overnight. Most of solvent was evaporated under reduced pressure. Ethyl acetate (30 ml) and brine (30 ml) were added and the mixture was separated. The organic layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (hexane/acetone) to give 300 mg of [(S)-(4-(methoxycarbonyl)propylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C68H128N11O15S; Exact Mass: 1363.88; MS (m/z): 1364.43 (M+1)+, 1386.64 (M+Na)+; HPLC RT: 15.26 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)] and 220 mg of [(R)-(4-(methoxycarbonyl)propylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C68H128N11O15S; Exact Mass: 1363.88; MS (m/z): 1364.43 (M+1)+, 1386.64 (M+Na)+; HPLC RT: 15.13 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(4-(Methoxycarbonyl)propylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (200 mg, 0.15 mmol) was dissolved in tetrahydrofuran (15 ml), followed by adding cesium chloride (200 mg, 1.18 mmol) and sodium borohydride (300 mg, 7.93 mmol). Then 10 ml of methanol was added dropwise to the mixture over 30 minutes. After addition, the mixture was stirred at room temperature one hour. Most solvent was then evaporated under reduced pressure. Ethyl acetate (30 ml) and water (30 ml) were added. The ethyl acetate layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography with dichloromethane/methanol (from 100:0 to 96:4) as eluent to give 13 mg of pure product [Molecular Formula: C67H121N11O14S; Exact Mass: 1335.88; MS (m/z): 1336.50 (M+1)+, 1358.70 (M+Na)+; TLC Rf: 0.39 (dichloromethane/methanol=9:1); HPLC RT: 13.25 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
According to this method, 11 mg of pure [(R)-(4-hydroxybutylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin was obtained [Molecular Formula: C67H121N11O14S; Exact Mass: 1335.88; MS (m/z): 1336.50 (M+1)+, 1358.70 (M+Na)+; TLC Rf: 0.39(dichloromethane/methanol=9:1); HPLC RT: 13.28 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (300 mg, 0.244 mmol) and 1-mercapto-4-methoxybutane (MW: 120.21, 292 mg, 10 mmol) were dissolved in methanol (10 ml), followed by adding six equivalents of lithium hydroxide (35 mg). The mixture was stirred overnight at room temperature. After removal of solvents, the residue was dissolved in ethyl acetate (15 ml). The ethyl acetate solution was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was subject to the flash chromatography using methylene chloride/methanol as eluent to give 57 mg of pure product [Molecular formula: C66H123N11O14S; Exact Mass: 1349.90; MS (m/z): 1350.45 (M+1)+, 1372.58 (M+Na)+; TLC Rf: 0.30 (methylene chloride/methanol=20/1); HPLC RT: 15.54 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(S)-(4-hydroxylbutylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.32 g, 0.24 mmol) in benzene (30 ml) were added a solution of sodium hydroxide (1.20 g, 30 mmol) in water (2 ml), 2-bromo-N,N-diethylethylamine hydrobromide (MW: 261, 3.80 g, 14.56 mmol) and tetra-n-butylammonium bromide (0.20 g, 0.62 mmol). The reaction mixture was stirred at 30° C. for 48 hours. After diluted with ice water, the mixture was separated. The aqueous layer was extracted with dichloromethane (30 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give product [Molecular formula: C73H134N12O14S; Exact Mass: 1434.99; MS (m/z): 1435.64 (M+1)+; TLC Rf: 0.30 (dichloromethane/methanol=9/1); HPLC RT: 12.06 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (480 mg, 0.39 mmol) and 5-mercapto-2-pentanone (MW: 118.19, 500 mg, 4.24 mmol) in methanol (30 ml) was added lithium hydroxide (120 mg, 5.00 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (80 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate) to give a pure product [Molecular formula: C68H121N11O14S; Exact Mass: 1347.88; MS (m/z): 1348.49 (M+1)+; TLC Rf: 0.46 (dichloromethane/methanol=97/3); HPLC RT: 14.86 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(S)-4-oxopentylthiomethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (80 mg, 0.06 mmol) in methanol (5 ml) was added sodium borohydride (36 mg, 0.95 mmol) in portions. After addition, the mixture was stirred at room temperature for one hour. Most of solvent was then evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added. The dichloromethane layer was separated and washed with brine (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified on silica gel column with (ethyl acetate/methanol=99/1) to give a pure product [Molecular formula: C68H123N11O14S; Exact Mass: 1349.90; MS (m/z): 1350.49 (M+1)+; TLC Rf: 0.35 (dichloromethane/methanol=97/3); HPLC RT: 14.25 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(S)-4-oxopentylthiomethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (100 mg, 0.07 mmol) in tetrahydrofuran (25 ml) at 0° C. was added a solution of methylmagnesium bromide in ether (1 ml, 3 M, 3.00 mmol). After addition, the mixture was slowly warmed to room temperature and stirred at room temperature for one and half hour. Then the reaction was quenched by adding aqueous ammonium chloride solution. Dichloromethane (30 ml) and water (30 ml) were added. The dichloromethane layer was separated and washed with brine (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified on silica gel column with (ethyl acetate/methanol=99/1) to give a pure product [Molecular formula: C69H125N11O14S; Exact Mass: 1363.91; MS (m/z): 1364.48 (M+1)+; TLC Rf: 0.42 (dichloromethane/methanol=97/3); HPLC RT: 14.74 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (1.00 g, 0.81 mmol) and methyl 5-mercaptopentanoate (0.72 g, 4.88 mmol) in methanol (30 ml) was added lithium hydroxide (195 mg, 8.13 mmol). The reaction mixture was stirred at room temperature for 3 hours. Most of solvent was evaporated under reduced pressure. Ethyl acetate (30 ml) and brine (30 ml) were added and the mixture was separated. The organic layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (hexane/acetone) to give 210 mg of [(S)-(4-(methoxycarbonyl)butylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C69H123N11O15S; Exact Mass: 1377.89; MS (m/z): 1378.49 (M+1)+, 1400.67 (M+Na)+; HPLC RT: 15.72 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)] and 270 mg of [(R)-(4-(methoxycarbonyl)butylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C69H123N11O15S; Exact Mass: 1377.89; MS (m/z): 1378.50 (M+1)+, 1400.68 (M+Na)+; HPLC RT: 15.53 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(4-(M ethoxycarbonyl)butylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (170 mg, 0.13 mmol) was dissolved in tetrahydrofuran (30 ml), followed by adding cesium chloride (1.00 g, 5.94 mmol) and sodium borohydride (1.00 g, 26.43 mmol). Then 30 ml of methanol was added dropwise to the mixture over 30 minutes. After addition, the mixture was stirred at room temperature one hour. Most solvent was then evaporated under reduced pressure. Ethyl acetate (30 ml) and water (30 ml) were added. The ethyl acetate layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography with dichloromethane/methanol (from 100:0 to 96:4) as eluent to give 47 mg of pure product of [(S)-(5-hydroxypentylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin [Molecular Formula: C68H123N11O14S; Exact Mass: 1349.90; MS (m/z): 1350.52 (M+1)+, 1372.72 (M+Na)+; TLC Rf: 0.54 (dichloromethane/methanol=9:1); HPLC RT: 14.19 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
According this method, 48 mg of pure [(R)-(5-hydroxypentylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin was obtained [Molecular Formula: C68H123N11O14S; Exact Mass: 1349.90; MS (m/z): 1350.47 (M+1)+, 1372.71 (M+Na)+; TLC Rf: 0.54 (dichloromethane/methanol=9:1); HPLC RT: 14.14 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(S)-(4-(Methoxycarbonyl)butylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (340 mg, 0.25 mmol) was dissolved in tetrahydrofuran (10 ml) and put into an ice bath. Then 0.63 ml of 3 M methylmagnesium bromide in ether (1.89 mmol) was added dropwise to the mixture over one hour. After the mixture was stirred 0° C. for two hour, 3 ml of brine was added dropwise to quench the reaction. Most solvent was then evaporated under reduced pressure. Ethyl acetate (10 ml) and water (10 ml) were added. The ethyl acetate layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography with dichloromethane/methanol as eluent to give 17 mg of pure product [Molecular Formula: C70H127N11O14S; Exact Mass: 1377.93; MS (m/z): 1378.55 (M+1)+, 1400.79 (M+Na)+; TLC Rf: 0.48 (dichloromethane/methanol=9:1); HPLC RT: 15.22 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
According to this method, 48 mg of pure [(R)-(5-hydroxy-5-methylhexylthio)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin was obtained [Molecular Formula: C70H127N11O14S; Exact Mass: 1377.93; MS (m/z): 1378.45 (M+1)+, 1400.70 (M+Na)+; TLC Rf: 0.41 (dichloromethane/methanol=9:1); HPLC RT: 15.11 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (123 mg, 0.1 mmol) and (R)-1-mercapto-5-hexanol (110 mg, 10 mmol) were dissolved in methanol (10 ml), followed by adding 10 equivalents of lithium hydroxide (14.4 mg). The mixture was stirred overnight at room temperature. After removal of solvents, the residue was dissolved in ethyl acetate (15 ml). The ethyl acetate solution was washed with brine, dried over magnesium sulfite and evaporated under reduced pressure. The residue was subject to a flash chromatography using ethyl acetate/methanol as eluent to give 25 mg of pure product [Molecular formula: C69H125N11O14S; Exact Mass: 1363.91; MS (m/z): 1364.68 (M+1)+, 1386.87 (M+Na)+; TLC Rf: 0.31 (methylene chloride/methanol=20/1); HPLC RT: 14.72 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) and 6-mercapto-1-hexanol (MW: 134.24, 0.27 g, 2.00 mmol) were dissolved in methanol (30 ml), followed by adding 10 equivalents of lithium hydroxide (48 mg, 2.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvents, the residue was dissolved in dichloromethane (30 ml). The dichloromethane solution was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was subject to the flash chromatography using ethyl acetate/methanol as eluent to give 58 mg of product. [Molecular formula: C69H125N11O14S; Exact Mass: 1363.91; MS (m/z): 1364.59 (M+1)+, 1386.76 (M+Na)+; TLC Rf: 0.28 (ethyl acetate/methanol=20/1); HPLC RT: 14.84 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (500 mg, 0.41 mmol) and dimethyl 4-mercaptobutylmalonate (650 mg, 2.95 mmol) in methanol (80 ml) was added lithium hydroxide (110 mg, 4.58 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (100 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was used for next step. [Molecular formula: C72H127N11O17S; Exact Mass: 1449.91; MS (m/z): 1450.37 (M+1)+.
To a solution of [(S)-(6-methoxy-5-methoxycarbonyl)-6-oxohexylthiomethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.17 mmol) in methanol (40 ml) was added sodium borohydride (350 mg, 9.26 mmol) in portions. The reaction mixture was stirred at room temperature 3 hours. Then most of solvent was evaporated under reduced pressure. Dichloromethane (100 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give a pure product [Molecular formula: C70H127N11O15S; Exact Mass: 1393.92; MS (m/z): 1394.45 (M+1)+; TLC Rf: 0.25 (dichloromethane/methanol=95/5); HPLC RT: 12.62 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.246 g, 0.2 mmol) and 1-mercapto-2-propanol (0.184 g, 2 mmol) were dissolved in methanol (10 ml), followed by adding 10 equivalents of lithium hydroxide (48 mg). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was dissolved in ethyl acetate (15 ml). The ethyl acetate solution was washed with brine, dried over magnesium sulfite and evaporated under reduced pressure. The residue was subject to a flash chromatography using ethyl acetate/methanol as eluent to give the product of isomer A as [(S)-(2-Hydroxypropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin and the product of isomer B as [(R)-(2-Hydroxypropylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin [Molecular formula: C66H119N11O14S; Exact Mass: 1321.87; MS (m/z): 1322.50 (M+1)+, 1344.76 (M+Na)+; TLC Rf (isomer A): 0.29 (methylene chloride/methanol=20/1, twice development); TLC Rf (isomer B): 0.26 (methylene chloride/methanol=20/1, twice development); HPLC RT: 13.62 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (500 mg, 0.41 mmol) and 4-mercapto-4-methyl-2-pentanone (680 mg, 5.15 mmol) in methanol (25 ml) was added lithium hydroxide (160 mg, 6.66 mmol). The reaction mixture was stirred at room temperature for 3 days. Then most of the solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate) to give the product [Molecular formula: C69H123N11O14S; Exact Mass: 1361.90; MS (m/z): 1362.50 (M+1)+; TLC Rf: 0.47 (dichloromethane/methanol=97/3); HPLC RT: 15.51 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(S)-(2-methyl-4-oxypentan-2-ylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (68 mg, 0.03 mmol) in methanol (5 ml) were added sodium borohydride (36 mg, 0.95 mmol) in portions. After addition, the mixture was stirred at room temperature one hour. Most solvent was then evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The dichloromethane layer was washed with brine (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified on silica gel column with dichloromethane/methanol (95/5) as eluent to give the product [Molecular formula: C69H125N11O14S; Exact Mass: 1363.91; MS (m/z): 1364.44 (M+1)+; TLC Rf: 0.38 (dichloromethane/methanol=97/3); HPLC RT: 15.03 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (580 mg, 0.47 mmol) and dimethyl 3-mercaptopropylmalonate (MW: 206.26, 600 mg, 2.91 mmol) in methanol (30 ml) was added lithium hydroxide (110 mg, 4.58 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (100 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was used for next step. [Molecular formula: C71H125N11O17S; Exact Mass: 1435.90; MS (m/z): 1436.45 (M+1)+.
To a solution of [(S)-((5-methoxy-4-methoxycarbonyl)-5-oxopentylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (280 mg, 0.19 mmol) in methanol (30 ml) was added sodium borohydride (360 mg, 9.52 mmol) in portions. The reaction mixture was stirred at room temperature 3 hours. Then most of the solvent was evaporated under reduced pressure. Dichloromethane (100 ml) and water (30 ml) were added and separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give a pure product [Molecular formula: C69H125N11O15S; Exact Mass: 1379.91; MS (m/z): 1380.49 (M+1)+; TLC Rf: 0.23 (dichloromethane/methanol=95/5); HPLC RT: 12.05 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) in benzene (30 ml) were added a solution of sodium hydroxide (1.00 g, 25 mmol) in water (2 ml), 2-bromo-N,N-diethylethylamine hydrobromide (MW: 261, 2.80 g, 10.72 mmol) and tetra-n-butylammonium bromide (0.2 g, 0.62 mmol). The mixture was stirred at 30° C. for 20 hours. Then ice water (30 ml) was added and the mixture was separated. The aqueous layer was extracted with dichloromethane (25 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 240 mg of product [Molecular Formula: C69H126N12O14; Exact Mass: 1346.95; MS (m/z): 1347.59 (M+1)+; TLC Rf: 0.41 (dichloromethane/methanol=9/1); HPLC RT: 12.20 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (300 mg, 0.24 mmol) in benzene (15 ml) were added sodium hydroxide (0.38 g. 9.60 mmol), tetramethylammonium hydroxide pentahydrate (0.44 g, 2.40 mmol) and 1-(2-chloroethyl)piperidine hydrochloride (MW: 184.10, 0.44 g, 2.40 mmol). The mixture was stirred at 30° C. for 36 hours. Then ice water (20 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (20 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel with dichloromethane/methanol (95/5) as eluent to give 100 mg of pure product [Molecular Formula: C70H126N12O14; Exact Mass: 1358.95; MS (m/z): 1359.69 (M+1)+, 1381.75 (M+Na)+; TLC Rf: 0.05 (dichloromethane/methanol=20/1); HPLC RT: 12.43 min (C8 reverse phase column: 150 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.50 g, 0.40 mmol) in benzene (20 ml) were added sodium hydroxide (0.64 g, 16.00 mmol), tetramethylammonium hydroxide pentahydrate (0.72 g, 4.00 mmol) and 4-(2-chloroethyl)morpholine hydrochloride (MW: 186.08, 0.74 g, 4.00 mmol). The mixture was stirred at 30° C. for a week. Then ice water (20 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (20 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel with dichloromethane/methanol (95/5) as eluent to give 60 mg of product [Molecular Formula: C69H124N12O15; Exact Mass: 1360.93; MS (m/z): 1361.63 (M+1)+, 1383.75 (M+Na)+; TLC Rf: 0.10 (dichloromethane/methanol=5:1); HPLC RT: 11.49 min (C8 reverse phase column: 150 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.37 g, 0.30 mmol) in benzene (15 ml) were added sodium hydroxide (0.48 g. 12.00 mmol), tetramethylammonium hydroxide pentahydrate (0.54 g, 3.00 mmol) and 3-dimethylaminoethyl chloride hydrochloride (0.43 g, 3.00 mmol). The mixture was stirred at 30° C. for 36 hours. Then ice water (20 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (20 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel with dichloromethane/methanol (95/5) as eluent to give 90 mg of pure product [Molecular Formula: C67H122N12O14; Exact Mass: 1318.92; MS (m/z): 1319.70 (M+1)+, 1341.80 (M+Na)+); TLC Rf: 0.05 (dichloromethane/methanol=5:1); HPLC RT: 11.43 min (C8 reverse phase column: 150 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.38 g, 0.30 mmol) in benzene (15 ml) were added sodium hydroxide (0.48 g, 12.00 mmol), tetramethylammonium hydroxide pentahydrate (0.54 g, 3.00 mmol) and 1-(2-chloroethyl)pyrrolidine hydrochloride (0.44 g, 3.00 mmol). The mixture was stirred at 30° C. for 36 hours. Then ice water (20 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (20 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel with dichloromethane/methanol (95/5) as eluent to give to give 120 mg of the expected isomer [Molecular Formula: C69H124N12O14; Exact Mass: 1344.94; MS (m/z): 1345.62 (M+1)+, 1367.76 (M+Na)+; TLC Rf: 0.05 (dichloromethane/methanol=10/1); HPLC RT: 12.09 min (C8 reverse phase column: 150 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.36 g, 0.29 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (0.60 g, 15.00 mmol) in water (1 ml), ethyl bromoacetate (1.60 g, 9.58 mmol) and tetra-n-butylammonium bromide (0.20 g, 0.62 mmol). The mixture was stirred at room temperature for 10 hours. After diluted with ice water, the mixture was separated. The aqueous layer was extracted with dichloromethane (15 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give the product [Molecular formula: C67H119N11O16; Exact Mass: 1333.88; MS (m/z): 1334.50 (M+1)+; TLC Rf: 0.35 (dichloromethane/methanol=95/5); HPLC RT: 15.16 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% NH4OAc in water; operation temperature: 64° C.; Detector: 210 nm)].
To a solution of [(R)-(ethoxycarbonylmethoxy)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.23 g, 0.17 mmol) in methanol (30 ml) were added lithium chloride (0.30 g, 7.14 mmol) and sodium borohydride (0.66 g, 17.46 mmol) in portions. After addition, the mixture was stirred at room temperature overnight. Most solvent was then evaporated under reduced pressure. Ethyl acetate (50 ml) and water (50 ml) were added. The ethyl acetate layer was separated and washed with brine (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified on silica gel column with (dichloromethane/methanol=95/5) to give the product [Molecular formula: C65H117N11O15; Exact Mass: 1291.87; MS (m/z): 1292.51 (M+1)+; TLC Rf: 0.28 (dichloromethane/methanol=9/1); HPLC RT: 12.55 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
[(R)-Hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (5.00 g, 4.01 mmol) was dissolved in benzene (100 ml). 2-(2-Bromoethyl)-1,3-dioxane (7.82 g, 40.10 mmol), tetra-n-butylammonium bromide (0.99 g, 3.09 mmol), sodium hydroxide (3.21 g, 8.02 mmol) and water (3.3 ml) were added. The reaction mixture was stirred at 35° C. for nine hours. And the stirring was continued overnight at room temperature. Then 50 ml of brine was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (25 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel with hexane/acetone as eluent, 1.50 g of product obtained [Molecular Formula: C69H123N11O16; Exact Mass: 1361.91; (m/z): 1362.64 (M+1)+, 1384.85 (M+Na)+].
[(R)-(2-(1,3-Dioxan-2-yl)ethoxy)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (1.29 g, 0.95 mmol) was dissolved in dioxane (25 ml), followed by adding hydrochloric acid solution (1 N, 25 ml). The reaction mixture was stirred overnight at room temperature. Most of dioxane was evaporated under reduced pressure. Then the aqueous layer was extracted with ethyl acetate (25 ml×2). The combined ethyl acetate layers were dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified on silica gel with hexane/acetone as eluent to give 600 mg of product [Molecular Formula: C66H117N11O15; Exact Mass: 1303.87; MS (m/z): 1304.59 (M+1)+, 1326.78 (M+Na)+; HPLC RT: 14.2 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(R)-(2-Formylethoxy)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (300 mg, 0.23 mmol) was dissolved in dichloromethane (15 ml). Morpholine (100 mg, 1.15 mmol) and tetramethylammonium triacetoxyborohydride (302 mg, 1.15 mmol) were added. The reaction mixture was stirred overnight at room temperature. Then sodium bicarbonate saturated solution (30 ml) and dichloromethane (15 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 105 mg of pure product was obtained [Molecular Formula: C70H126N12O15; Exact Mass: 1374.95; MS (m/z): 1375.70 (M+1)+, 1397.80 (M+Na)+; TLC Rf: 0.37 (dichloromethane/methanol=9/1); HPLC RT: 12.2 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
[(R)-(2-Formylethoxy)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (200 mg, 0.15 mmol) was dissolved in dichloromethane (15 ml). Pyrrolidine (95 mg, 1.34 mmol) and tetramethylammonium triacetoxyborohydride (353 mg, 1.34 mmol) were added. The reaction mixture was stirred overnight at room temperature. Then sodium bicarbonate saturated solution (30 ml) and dichloromethane (15 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 50 mg of pure product was obtained [Molecular Formula: C70H126N12O14; Exact Mass: 1358.95; MS (m/z): 1359.74 (M+1)+, 1381.79 (M+Na)+; TLC Rf: 0.40 (dichloromethane/methanol=9/1); HPLC RT: 12.7 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
[(R)-(2-Formylethoxy)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (200 mg, 0.15 mmol) was dissolved in dichloromethane (15 ml). Piperidine (114 mg, 1.34 mmol) and tetramethylammonium triacetoxyborohydride (353 mg, 1.34 mmol) were added. The reaction mixture was stirred overnight at room temperature. Then sodium bicarbonate saturated solution (30 ml) and dichloromethane (15 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 43 mg of product was obtained [Molecular Formula: C71H128N12O14; Exact Mass: 1372.97; (m/z): MS (m/z): 1373.79 (M+1)+, 1395.86 (M+Na)+; TLC Rf: 0.27 (dichloromethane/methanol=9/1); HPLC RT: 17.8 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
[(R)-(2-Formylethoxy)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (100 mg, 0.08 mmol) was dissolved in dichloromethane (15 ml). 3-Amino-2,2-dimethyl-1-propanol (40 mg, 0.39 mmol) and tetramethylammonium triacetoxyborohydride (100 mg, 0.39 mmol) were added. The reaction mixture was stirred at room temperature for 6 hours. Then sodium bicarbonate saturated solution (30 ml) and dichloromethane (15 ml) were added and the mixture was separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 10 mg of pure product was obtained [Molecular Formula: C71H130N12O15; Exact Mass: 1390.98; MS (m/z): 1391.64 (M+1)+, 1413.77 (M+Na)+; TLC Rf: 0.37 (dichloromethane/methanol=9/1); HPLC RT: 11.46 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.38 g, 0.30 mmol) in benzene (15 ml) were added sodium hydroxide (0.48 g, 12.00 mmol), tetramethylammonia hydroxide (0.54 g, 3.0 mmol) and 3-dimethylaminoethyl chloride hydrochloride (0.43 g, 3.00 mmol). The mixture was stirred at 30° C. for 36 hours. Then ice water (20 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (20 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel with dichloromethane/methanol (95/5) as eluent to give to give 70 mg of pure product [Molecular Formula: C68H124N12O14; Exact Mass: 1332.94. MS (m/z): 1333.64 (M+1)+, 1355.73 (M+Na)+; TLC Rf: 0.04 (dichloromethane/methanol=5/1); HPLC RT: 11.78 min (C8 reverse phase column: 150 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.20 mmol) in benzene (15 ml) were added a solution of sodium hydroxide (400 mg, 10.00 mmol) in water (0.5 ml), 3-diethylaminepropyl chloride hydrochloride (500 mg, 2.69 mmol) and tetramethylammonium hydroxide pentahydrate (430 mg, 2.41 mmol). The mixture was stirred at 32° C. for 4 days. Then ice water (30 ml) was added and the mixture was separated. The aqueous layer was extracted with dichloromethane (50 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give 120 mg of product [Molecular Formula: C70H128N12O14; Exact Mass: 1360.97; MS (m/z): 1361.72 (M+1)+; TLC Rf: 0.38 (dichloromethane/methanol=9/1); HPLC RT: 16.71 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) in benzene (30 ml) were added a solution of sodium hydroxide (1.00 g, 25 mmol) in water (2 ml), 2-(3-bromopropoxy)tetrahydro-2H-pyran (2.50 g, 11.21 mmol) and tetra-n-butylammonium bromide (0.2 g, 0.62 mmol). The mixture was stirred at 30° C. for 4 hours. Then ice water (30 ml) was added and the mixture was separated. The aqueous layer was extracted with dichloromethane (25 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give the product [Molecular Formula: C66H119N11O15; Exact Mass: 1305.89; MS (m/z): 1306.46 (M+1)+; TLC Rf: 0.38 (dichloromethane/methanol=9/1); HPLC RT: 12.94 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (0.34 g, 0.27 mmol) in benzene (10.0 ml) were added 4-bromobutyl acetate (1.56 ml, d 1.348, 10.81 mmol), tetra-n-butylammonium bromide (0.30 g, 0.94 mmol) and sodium hydroxide (0.70 g, 17.6 mmol). The mixture was stirred at room temperature for two hours and then washed with brine, dried over magnesium sulfate. After removal of solvent under reduced pressure, the residue was purified by chromatography on silica gel to give pure product [Molecular formula: C69H123N11O16; Exact Mass: 1361.91; MS (m/z): 1362.51 (M+1)+, 1385.77 (M+Na)+].
[(R)-(4-Acetoxybutyloxy)methyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (49 mg, 0.036 mmol) was dissolved in methanol (5 ml). Water (3 ml) and potassium carbonate (99 mg, 0.72 mmol) were added and the mixture was stirred for two hours. After removal of methanol, the residue was dissolved in ethyl acetate (6 ml). The ethyl acetate layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel with ethyl acetate/methanol as eluent to give 22 mg of the pure product [Molecular formula: C67H121N11O15; Exact Mass: 1319.90; MS (m/z): 1320.58 (M+1)+, 1342.78 (M+Na)+; TLC Rf: 0.20 (methylene/methanol=25/1); HPLC RT: 13.62 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(S)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.624 g, 0.50 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (1.40 g, 35 mmol) in water (2 ml), 1-bromo-4-methoxybutane (0.84 g, 5.0 mmol) and tetra-n-butylammonium bromide (0.563 g, 1.75 mmol). The mixture was stirred at room temperature overnight and then at 30° C. for 4 hours. Then ice water (30 ml) was added and separated. The aqueous layer was extracted with dichloromethane (25 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=20:1) to give 50 mg of product [Molecular formula: C68H123N11O15; Exact Mass: 1333.92; MS (m/z): 1334.44 (M+1)+, 1356.67 (M+Na); TLC Rf: 0.33 (dichloromethane/methanol=20/1); HPLC RT: 15.52 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% NH4OAc in water; operation temperature: 64° C.; Detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) in benzene (30 ml) were added a solution of sodium hydroxide (1.00 g, 25 mmol) in water (2 ml), 5-bromopentyl acetate (2.20 g, 10.53 mmol) and tetra-n-butylammonium bromide (0.2 g, 0.62 mmol). The mixture was stirred at 30° C. for 10 hours. Then ice water (30 ml) was added and the mixture was separated. The aqueous layer was extracted with dichloromethane (25 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give the product [Molecular Formula: C68H123N11O15; Exact Mass: 1333.92; MS (m/z): 1334.46 (M+1)+; TLC Rf: 0.30 (dichloromethane/methanol=95/5); HPLC RT: 14.22 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(γ-hydroxy)-N-MeLeu]-4-cyclosporin (4.50 g, 3.70 mmol) in anhydrous dimethyl sulfoxide (25 ml) was added acetic anhydride (15 ml). The reaction mixture was stirred at room temperature for 17 hours. After diluted with ethyl acetate (75 ml), the mixture was washed with saturated sodium bicarbonate water solution and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified on silica gel chromatography with dichloromethane/methanol (98/2) as eluant to give 2.35 g of [(γ-methylthio)methoxy-N-MeLeu]-4-cyclosporin [Molecular Formula: C64H115N11O13S; Exact Mass: 1277.84; MS (m/z): 1300.70 (M+Na)+; TLC Rf: 0.30 (dichloromethane/methanol=95/5); HPLC RT: 19.57 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [γ-(Methylthio)methoxy-N-MeLeu]-4-cyclosporin (1.20 g, 0.94 mmol) in anhydrous tetrahydrofuran (40 ml) was added Raney Ni (˜2 g). The resulting suspension was stirred and heated to 60° C. for 30 minutes and the reaction was monitored by LC-MS. The reaction mixture was filtered and the filter cake was washed with tetrahydrofuran. The filtrate was collected and evaporated under reduced pressure. The residue was purified by chromatography using eluant of ethyl acetate/methanol (97.5/2.5) to give 0.60 g of product [Molecular Formula: C63H113N11O13; Exact Mass: 1231.85; MS (m/z): 1232.70 (M+1)+; TLC Rf: 0.46 (dichloromethane/methanol=95/5); HPLC RT: 20.63 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
n-Butyllithium (2.8 M in tetrahydrofuran/hexane, 5.00 ml, 14.00 mmol) was added to a solution of diisopropylamine (1.44 g, 14.30 mmol) in tetrahydrofuran (30 ml) at −78° C. under nitrogen atmosphere. After the mixture was stirred for one and half hour, a solution of [γ-(methoxy)-N-MeLeu]-4-cyclosporin (1.20 g, 0.97 mmol) in tetrahydrofuran (6 ml) was added slowly. The stirring was continued at −78° C. for 2 hours. Then carbon dioxide gas was bubbled into the reaction mixture for one hour. The mixture was allowed to warm to room temperature slowly and stirred for another 3 hours. After most of solvent was evaporated under reduced pressure, dichloromethane (30 ml) and water (30 ml) were added. The PH of the mixture was adjusted to around 5 by adding aqueous citric acid. The mixture was separated, and the dichloromethane layer was washed with brine, dried over magnesium sulfate and concentrated under reduced pressure to give 1.20 g of crude product used for next step [Molecular Formula: C64H113N11O15; Exact Mass: 1275.84; MS (m/z): 1298.53 (M+Na)+].
To a mixture of [α-carboxy]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (1.20 g. 0.94 mmol) and potassium carbonate (0.80 g, 5.79 mmol) in N,N-dimethylformamide (25 ml) was added iodomethane (0.80 g, 5.63 mmol). The mixture was stirred at room temperature overnight. Dichloromethane (75 ml) and water (30 ml) were added and the mixture was separated. The dichloromethane layer was washed with water (25 ml) and brine (25 ml), dried magnesium sulfate and concentrated under reduced pressure to give 1.10 g of crude product [Molecular Formula: C65H115N11O15; Exact Mass: 1289.86; MS (m/z): 1312.72 (M+Na)+].
To a suspension of [α-methoxycarbonyl-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (1.10 g, 0.85 mmol) and lithium chloride (1.00 g, 23.53 mmol) in methanol (80 ml) was added sodium borohydride (2.00 g, 52.91 mmol) in portions. The mixture was stirred at room temperature overnight and concentrated under reduced pressure. Dichloromethane (50 ml) and water (30 ml) were added and the mixture was separated. The dichloromethane layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 310 mg of product [Molecular Formula: C64H115N11O14; Exact Mass: 1261.86; MS (m/z): 1262.68 (M+1)+].
[α-Methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin was prepared according to the method described in Example 28 [Molecular Formula: C64H113N11O13; Exact Mass: 1243.85; MS (m/z): 1244.57 (M+1)+; TLC Rf: 0.34 (hexane/acetone=6/1); HPLC RT: 17.10 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid), operation temperature: 64° C.; detector: 210 nm].
[α-Methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (0.24 g, 0.19 mmol) and 2-(N,N-dimethylamino)ethylthiol hydrochloride (0.27 g, 1.91 mmol) was dissolved in methanol (30 ml), followed by adding 20 equivalents of lithium hydroxide (46 mg, 1.90 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by flash chromatography using methylene chloride/methanol (96/4) as eluent to give 0.11 g of pure product [Molecular Formula: C68H124N12O13S; Exact Mass: 1348.93; MS (m/e): 1349.85 (M+1)+, 1371.81 (M+Na)+; TLC Rf: 0.20 (ethyl acetate/methanol (5:1); HPLC RT: 12.42 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (280 mg, 0.23 mmol) and 2-diethylaminoethanethiol hydrochloride (570 mg, 3.37 mmol) in methanol (15 ml) was added lithium hydroxide (142 mg, 5.92 mmol). The reaction mixture was stirred overnight at room temperature. Most of solvent was evaporated under reduced pressure. Dichloromethane (80 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 110 mg of product [Molecular Formula: C70H128N12O13S; Exact Mass: 1376.94; MS (m/z): 1377.67 (M+1)+; TLC Rf: 0.35 (dichloromethane/methanol=95/5); HPLC RT: 13.17 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (230 mg, 0.18 mmol) and 2-(N-pyrrolidinyl)ethanethiol (340 mg, 2.59 mmol) in methanol (15 ml) was added lithium hydroxide (120 mg, 5.00 mmol). The reaction mixture was stirred overnight at room temperature. Most of solvent was evaporated under reduced pressure. Dichloromethane (30 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 55 mg of product [Molecular Formula: C70H126N12O13S; Exact Mass: 1374.93; MS (m/z): 1375.57 (M+1)+; TLC Rf: 0.29 (dichloromethane/methanol=95/5); HPLC RT: 12.90 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.20 mmol) and 2-morpholinoethanethiol (260 mg, 1.76 mmol) in methanol (15 ml) was added lithium hydroxide (120 mg, 5.00 mmol). The reaction mixture was stirred at room temperature overnight. Most of solvent was evaporated under reduced pressure. Dichloromethane (60 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 70 mg of product [Molecular Formula: C70H126N12O14S; Exact Mass: 1390.92; MS (m/z): 1391.58 (M+1)+; TLC Rf: 0.38 (dichloromethane/methanol=9/1); HPLC RT: 12.48 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) and 3-(N,N-dimethylamino)propylthiol (0.24 g, 2.00 mmol) were dissolved in methanol (20 ml), followed by adding 10 equivalents of lithium hydroxide (48 mg, 2.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by flash chromatography using methylene chloride/methanol as eluent to give 80 mg of pure product [Molecular Formula: C69H126N12O13S; Exact Mass: 1362.93; MS (m/e): 1363.70 (M+1)+. TLC Rf: 0.20 (ethyl acetate/methanol=10/1); HPLC RT: 12.82 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (0.25 g, 0.20 mmol) and 3-(N,N-diethylamino)propylthiol (0.30 g, 2.00 mmol) were dissolved in methanol (20 ml), followed by adding 10 equivalents of lithium hydroxide (48 mg, 2.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by flash chromatography using methylene chloride/methanol as eluent to give 120 mg of pure product [Molecular Formula: C71H130N12S; Exact Mass: 1390.96; MS (m/e): 1391.64 (M+1)+, 1413.79 (M+Na)+; TLC Rf: 0.25 (ethyl acetate/methanol=10/1); HPLC RT: 13.57 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (0.37 g, 0.30 mmol) and 3-(N-piperidino)propylthiol (0.48 g, 3.00 mmol) were dissolved in methanol (30 ml), followed by adding 10 equivalents of lithium hydroxide (72 mg, 3.00 mmol). The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by flash chromatography using methylene chloride/methanol as eluent to give 60 mg of pure product [Molecular Formula: C72H130N12O13S; Exact Mass: 1402.96; MS (m/e): 1403.69 (M+1)+, 1425 (M+Na)+; TLC: Rf: 0.3 (ethyl acetate/methanol=10/1); HPLC RT: 13.59 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (280 mg, 0.23 mmol) and 3-(N-pyrrolidinyl)propanethiol (350 mg, 2.41 mmol) in methanol (15 ml) was added lithium hydroxide (120 mg, 5.00 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (80 ml) and water (25 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=94/6) to give 47 mg of product [Molecular Formula: C71H128N12O13S; Exact Mass: 1388.94; MS (m/z): 1389.68 (M+1)+; TLC Rf: 0.30 (dichloromethane/methanol=95/5); HPLC RT: 13.25 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-Methylene-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (320 mg, 0.26 mmol) and 3-morpholinopropanethiol (600 mg, 3.73 mmol) in methanol (25 ml) was added lithium hydroxide (140 mg, 5.83 mmol). The reaction mixture was stirred at room temperature overnight. Then most of solvent was evaporated under reduced pressure. Dichloromethane (60 ml) and water (25 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 58 mg of product [Molecular Formula: C71H128N12O14S; Exact Mass: 1404.94; MS (m/z): 1405.52 (M+1)+; TLC Rf: 0.39 (dichloromethane/methanol=9/1); HPLC RT: 15.96 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
[(S)-(4-Hydroxylbutylthio)methyl-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (100 mg, 0.075 mmol) was dissolved in of benzene (3 ml), followed by adding 3.5 equivalents of tetrabutylammonium bromide, 75 equivalents of 50% of sodium hydroxide and iodomethene (0.425 g. 3.00 mol). The mixture was stirred overnight at room temperature and diluted with benzene (10 ml). The organic mixture was washed with brine, dried over magnesium sulfite and evaporated under reduced pressure. The residue was subject to a flash chromatography using ethyl acetate/methanol as eluent to give 25 mg of product [Molecular formula: C69H125N11O14S; Exact Mass: 1363.91; MS (m/z): 1364.35 (M+1)+, 1386.70 (M+Na)+; TLC Rf: 0.38 (ethyl acetate/methanol=20/1); HPLC RT: 16.72 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temp: 64° C.; Detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (0.20 g, 0.16 mmol) in benzene (10 ml) were added a solution of sodium hydroxide (0.48 g, 12.00 mmol) in water (1 ml), 2-bromo-N,N-diethylethylamine hydrobromide (1.10 g, 4.21 mmol) and tetra-n-butylammonium bromide (0.10 g, 0.31 mmol). The mixture was stirred at 35° C. for 40 hours. Ice water (10 ml) was added and the mixture was separated. The aqueous layer was extracted with dichloromethane (20 ml). The combined organic layers was washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 36 mg of product [Molecular Formula: C70H128N12O14; Exact Mass: 1360.97; MS (m/z): 1383.74 (M+Na)+; TLC Rf: 0.32 (dichloromethane/methanol=95/5); HPLC RT: 13.66 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm); 1H NMR spectrum (600 MHz, CDCl3, δ in ppm): 0.65 (d, J=4.8 Hz, 3H), 0.82 (m, 9H), 0.87 (m, 6H), 0.91 (d, J=6. Hz, 3H), 0.93 (d, J=6.0 Hz, 3H), 0.98-1.01 (m, 15H), 1.07 (d, J=6.6 Hz, 3H), 1.11 (s, 6H), 1.23 (m, 6H), 1.33 (d, J=7.2 Hz, 3H), 1.39-1.47 (m, 2H), 1.53-1.58 (m, 4H), 1.6 (m, 3H), 1.67-1.75 (m, 3H), 1.98-2.12 (m, 4H), 2.43-2.48 (m, 3H), 2.50-2.54 (m, 4H), 2.60 (t, J=6.0 Hz, 2H), 2.67 (s, 3H), 2.68 (s, 3H), 3.07 (s, 3H), 3.10 (s, 3H), 3.12 (s, 3H), 3.24 (s, 3H), 3.26 (s, 3H), 3.48 (m, 4H), 3.52-3.56 (m, 1H), 3.60-3.62 (m, 1H), 3.67-3.70 (m, 1H), 3.80 (m, 1H), 4.06 (t, J=9.6 Hz, 1H), 4.52 (m, 1H), 4.57 (m, 1H), 4.80 (m, 1H), 4.91 (t, J=7.8 Hz, 1H), 5.04 (m, 3H), 5.11 (d, J=11.4 Hz, 1H), 5.28-5.34 (m, 2H), 5.50 (d, J=7.2 Hz, 1H), 5.67 (m, 1H), 7.10 (d, J=7.8 Hz 1H), 7.48 (d, J=7.80 Hz, 1H), 7.58 (d, J=7.2 Hz, 1H), 7.91 (d, J=10.2 Hz, 1H)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.20 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 2-dimethylaminoethyl chloride hydrochloride (570 mg, 3.96 mmol). The mixture was stirred at 40 to 50° C. for two days. Sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 2-dimethylaminoethyl chloride hydrochloride (570 mg, 3.96 mmol) were added and the mixture was kept stirring at 40 to 50° C. for another two days. Another portion of 2-dimethylaminoethyl chloride hydrochloride (1.14 g, 7.91 mmol) was added and the stirring was continued at 40 to 50° C. for one more day. Sodium bicarbonate saturated solution (30 ml) was added and the mixture was separated. Then the aqueous layer was extracted with ethyl acetate (25 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. The residue was dissolved in ethyl acetate (25 ml). The resulting ethyl acetate phase was washed with acetic acid solution (5 ml in 10 ml water) and sodium bicarbonate saturated solution (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 21 mg product was obtained [Molecular Formula: C68H124N12O14; Exact Mass: 1332.94; MS (m/z): 1333.75 (M+1)+, 1355.87 (M+Na)+; TLC Rf: 0.22 (dichloromethane/methanol=9/1); HPLC RT: 17.3 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)]
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.20 mmol) in benzene (20 ml) was added a solution of sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), followed by tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 2-(4-morpholinyl)ethyl chloride hydrochloride (737 mg, 3.96 mmol). The mixture was stirred at 40 to 50° C. for two days. Sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 2-(4-morpholinyl)ethyl chloride hydrochloride (737 mg, 3.96 mmol) were added and the mixture was kept stirring at 40 to 50° C. for another two days. Another portion of 2-(4-morpholinyl)ethyl chloride hydrochloride (1.47 g, 7.91 mmol) was added and the stirring was continued at 40 to 50° C. for two more days. Sodium bicarbonate saturated solution (30 ml) was added and the mixture was separated. Then the aqueous layer was extracted with ethyl acetate (25 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. The residue was dissolved in ethyl acetate (25 ml). And the resulting ethyl acetate phase was washed with acetic acid solution (5 ml in 10 ml water) and sodium bicarbonate saturated solution (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 45 mg product was obtained [Molecular Formula: C70H126N12O15; Exact Mass: 1374.95; MS (m/z): 1375.63 (M+1)+, 1397.79 (M+Na)+; TLC Rf: 0.42 (dichloromethane/methanol=9/1); HPLC RT: 12.9 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)]
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.20 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 3-dimethylaminopropyl chloride hydrochloride (626 mg, 3.96 mmol). The mixture was stirred at 40 to 50° C. for two days. Sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 3-dimethylaminopropyl chloride hydrochloride (626 mg, 3.96 mmol) were added and the mixture was kept stirring at 40 to 50° C. for another two days. Another portion of 3-dimethylaminopropyl chloride hydrochloride (1.25 g, 7.91 mmol) was added and the stirring was continued at 40 to 50° C. for one more day. Sodium bicarbonate saturated solution (30 ml) was added and the mixture was separated. Then the aqueous layer was extracted with ethyl acetate (25 ml×2). The combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. The residue was dissolved in ethyl acetate (25 ml). And the resulting ethyl acetate phase was washed with acetic acid solution (5 ml in 10 ml water) and sodium bicarbonate saturated solution (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 36 mg product was obtained [Molecular Formula: C69H126N12O14; Exact Mass: 1346.95; MS (m/z): 1347.65 (M+1)+, 1369.74 (M+Na)+; TLC Rf: 0.21 (dichloromethane/methanol=9/1). HPLC RT: 18.8 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[(γ-methoxy)-N-MeLeu]-4-cyclosporin (250 mg, 0.20 mmol) in benzene (20 ml) was added a solution of sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), followed by adding tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 3-diethylaminopropyl chloride hydrochloride (737 mg, 3.96 mmol). The mixture was stirred at 40 to 50° C. for two days. Sodium hydroxide (633 mg, 15.85 mmol) in water (0.70 ml), tetramethylammonium hydroxide pentahydrate (720 mg, 3.96 mmol) and 3-diethylaminopropyl chloride hydrochloride (737 mg, 3.96 mmol) were added and the mixture was kept stirring at 40 to 50° C. for another two days. Another portion of 3-diethylaminopropyl chloride hydrochloride (1.47 g, 7.91 mmol) was added and the stirring was continued at 40 to 50° C. for two more days. Sodium bicarbonate saturated solution (30 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (25 ml×2). Then the combined organic layers were dried over magnesium sulfate and evaporated under reduced pressure. The residue was dissolved in ethyl acetate (25 ml). And the resulting ethyl acetate phase was washed with acetic acid solution (5 ml in 10 ml water) and sodium bicarbonate saturated solution (30 ml), dried over magnesium sulfate and evaporated under reduced pressure. After purified on silica gel, 38 mg product was obtained [Molecular Formula: C71H130N12O14; Exact Mass: 1374.98; MS (m/z): 1375.70 (M+1)+, 1397.80 (M+Na)+; TLC Rf: 0.24 (dichloromethane/methanol=9/1); HPLC RT: 19.6 min (C8 reverse phase column: 250 mm; acetonitrile/0.077% ammonium acetate in water; operation temperature: 64° C.; detector: 210 nm)]
[α-Methylene-Sar]-3-[(γ-methylthiomethoxy)-N-MeLeu]-4-cyclosporin was prepared according to the method described in Example 28. The product was purified by chromatography on silica gel (ethyl acetate/methanol) [Molecular Formula: C65H115N11O13S; Exact Mass: 1289.84; MS (m/z): 1290.70 (M+1)+, 1312.67 (M+Na)+].
[α-Methylene-Sar]-3-[(γ-methylthiomethoxy)-N-MeLeu]-4-cyclosporin (0.32 g, 0.25 mmol) and 2-(N,N-dimethyl)ethanethiol (0.26 g, 2.50 mmol) were dissolved in methanol (20 ml), followed by adding 24 equivalents of triethylamine. The mixture was stirred overnight. After removal of solvent, the residue was subject to chromatography using dichloromethane/methanol as eluent to give 0.14 g of pure product [Molecular Formula, C69H126N12O13S2; Exact Mass: 1394.90; MS (m/z): 1395.70 (M+1)+, 1417.68 (M+Na)+; TLC Rf: 0.10 (ethyl acetate/methanol=10:1); HPLC RT: 13.30 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-methylthiomethoxy)-N-MeLeu]-4-cyclosporin (0.27 g, 0.21 mmol) and 2-(N,N-diethyl)ethanethiol (0.28 g, 2.10 mmol) were dissolved in methanol (20 ml), followed by adding 24 equivalents of triethylamine. The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by chromatography on silica gel using dichloromethane/methanol as eluent to give 0.17 g of pure product [Molecular Formula, C71H130N12O13S2; Exact Mass: 1422.93; MS (m/z): 1423.70 (M+1)+, 1445.67 (M+Na)+; TLC Rf: 0.35 (ethyl acetate/methanol=10:1); HPLC RT: 13.95 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(γ-hydroxy)-N-MeLeu]-4-cyclosporin (1.20 g, 0.99 mmol) in dichloromethane (80 ml) was added diisopropylethylamine (FW 129.25, d 0.742, 1.32 ml, 0.98 g, 7.60 mmol), followed by adding chloromethyl ethyl ether (FW 94.54, d 1.02, 2.22 ml, 2.27 g, 24 mmol) dropwise. The mixture was stirred overnight at room temperature and TLC was used to monitor the completion of the reaction. The reaction mixture was washed with 1 N hydrochloric acid, saturated sodium bicarbonate water solution and brine. After dried over magnesium sulfate, the mixture was evaporated under reduced pressure to give a yellowish oil, which was further purified by flash chromatography using dichloromethane/methanol as eluent to give 0.95 g of the product [Molecular Formula: C65H117N11O14; Exact Mass: 1275.88; MS (m/z): 1276.70 (M+H)+, 1298.70 (M+Na)+; TLC Rf: 0.37 (ethyl acetate)].
[α-Methylene-Sar]-3-[(γ-ethoxymethoxy)-N-MeLeu]-4-cyclosporin was prepared according to the method described in Example 28. The product was purified by chromatography on silica gel with ethyl acetate/methanol as eluent [Molecular Formula: C66H117N11O14; Exact Mass: 1287.68; MS (m/z): 1288.72 (M+1)+, 1310.70 (M+Na)+].
[α-Methylene-Sar]-3-[(γ-ethoxymethoxy)-N-MeLeu]-4-cyclosporin (0.27 g, 0.21 mmol) and 2-(N,N-diethyl)ethanethiol (0.28 g, 2.1 mmol) were dissolved in methanol (20 ml), followed by adding 12 equivalents of triethylamine. The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by chromatography on silica gel using dichloromethane/methanol as eluent to give 90 mg of pure product [Molecular Formula: C72H132N12O14S; Exact Mass: 1420.97; MS (m/z): 1421.75 (M+1)+, 1443.72 (M+Na)+; TLC: Rf: 0.40 (ethyl acetate/methanol=10/1); HPLC RT: 13.58 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of LDA (2.0 M in tetrahydrofuran, 5 ml, 10 mmol) in tetrahydrofuran (15 ml) at −78° C. under nitrogen atmosphere was added [N-MeIle]-4-cyclosporin (1.20 g, 1.00 mmol) in tetrahydrofuran (15 ml) over 3 min After the mixture was stirred at −78° C. for 3 hours, carbon dioxide gas was bubbled into the reaction mixture for 1 hour. Then the mixture was allowed to warm to room temperature slowly and kept stirring for another 3 hours. Most of tetrahydrofuran was evaporated under reduced pressure. Dichloromethane (100 ml) and water (50 ml) were added. The PH of the mixture was adjusted to around 5 by adding aqueous citric acid solution. The mixture was then separated and the organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 1.10 g of crude product, which was used for next step without purification [Molecular Formula: C63H111N11O14; Exact Mass: 1245.83; MS (m/z): 1246.68 (M+1)+].
To a mixture of [α-carboxy-Sar]-3-[N-MeIle]-4-cyclosporin (1.00 g, 0.80 mmol) and potassium carbonate (0.70 g, 5.07 mmol) in N,N-dimethylformamide (10 ml) was added iodomethane (1.50 g, 10.56 mmol). The mixture was stirred overnight at room temperature. Dichloromethane (80 ml) and water (50 ml) were added and the mixture was separated. The dichloromethane layer was washed with water (25 ml) and brine (25 ml), dried over magnesium sulfate and evaporated under reduced pressure to give crude 1.00 g of product [Molecular Formula: C64H113N11O14; Exact Mass: 1259.85; MS (m/z): 1260.51 (M+1)+].
To a suspension of [α-methoxycarbonyl-Sar]-3-[N-MeIle]-4-cyclosporin (1.00 g, 0.79 mmol) and lithium chloride (0.60 g, 14.11 mmol) in methanol (80 ml) was added sodium borohydride (3.00 g, 79.26 mmol) in portions. The mixture was stirred overnight at room temperature. Most of solvent was evaporated under reduced pressure. Dichloromethane (100 ml) and water (50 ml) were added and the mixture was separated. The dichloromethane layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give 420 mg of product [Molecular Formula: C63H113N11O13; Exact Mass: 1231.85; MS (m/z): 1232.59 (M+1)+; TLC Rf: 0.32 (dichloromethane/methanol=95/5); HPLC RT: 14.32 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[N-MeIle]-4-cyclosporin was prepared according to the method described in Example 28 [Molecular Formula: C63H111N11O12; Exact Mass: 1213.84; MS (m/z): 1214.59 (M+1)+; TLC Rf: 0.34 (hexane/acetone=6/1); HPLC RT: 17.47 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid), operation temperature: 64° C.; detector: 210 nm].
To a solution of [α-Methylene-Sar]-3-[N-MeIle]-4-cyclosporin (300 mg, 0.25 mmol) and 2-diethylaminoethanethiol hydrochloride (408 mg, 2.41 mmol) in methanol (20 ml) was added lithium hydroxide (116 mg, 4.83 mmol). The reaction mixture was stirred overnight at room temperature. Most of solvent was evaporated under reduced pressure. Dichloromethane (80 ml) and water (30 ml) were added and the mixture was separated. The organic layer was washed with water and brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 170 mg of product [Molecular Formula: C69H126N12O12S; Exact Mass: 1346.93; MS (m/z): 1347.68 (M+1)+; TLC Rf: 0.32 (dichloromethane/methanol=95/5); HPLC RT: 13.54 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[N-MeIle]-4-cyclosporin (0.39 g, 0.32 mmol) in benzene (20 ml) were added a solution of sodium hydroxide (0.80 g, 20 mmol) in water (1 ml), 2-bromo-N,N-diethylethylamine hydrobromide (2.40 g, 9.20 mmol) and tetra-n-butylammonium bromide (0.10 g, 3.10 mmol). The mixture was stirred at 30° C. for 40 hours. Ice water (10 ml) was added and the mixture was separated. The aqueous layer was extracted with dichloromethane (50 ml). The combined organic layers was washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give 270 mg of product [Molecular Formula: C69H126N12O13; Exact Mass: 1330.96; MS (m/z): 1331.73 (M+1)+; TLC Rf: 0.34 (dichloromethane/methanol=95/5); HPLC RT: 13.42 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene]-3-[N-MeVal]-4-cyclosporin was prepared according to the method described in Example 28. The product was purified by chromatography on silica gel (ethyl acetate/methanol). [Molecular Formula: C62H109N11O12; Exact Mass: 1199.83; MS (m/z): 1200.56 (M+1)+, 1222.72 (M+Na)+].
[α-Methylene-Sar]-3-[N-MeVal]-4-cyclosporin (88 mg, 0.07 mmol) and 2-(N,N-dimethyl)ethanethiol hydrochloride (0.10 g, 7.30 mmol) were dissolved in methanol (20 ml), followed by adding 20 equivalents of lithium hydroxide. The mixture was stirred overnight at room temperature. After removal of solvent, the residue was purified by flash chromatography using dichloromethane/methanol as eluent to give 30 mg of pure product [Molecular Formula: C66H120N12O12S; Exact Mass: 1304.89; MS (m/z): 1305.68 (M+1)+, 1327.83 (M+Na)+; TLC Rf: 0.05 (ethyl acetate/methanol=5/1); HPLC RT: 12.23 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[N-MeVal]-4-cyclosporin (0.20 g, 0.16 mmol) and 2-(N,N-9diethyl)ethanethiol hydrochloride (0.28 g, 1.70 mmol) were dissolved in methanol (20 ml), followed by adding 20 equivalents of lithium hydroxide (77 mg, 3.20 mmol). The mixture was stirred overnight. After removal of solvent, the residue was purified by chromatography on silica gel using dichloromethane/methanol as eluent to give 100 mg of pure product [Molecular Formula: C68H124N12O12S; Exact Mass: 1332.92; MS (m/e): 1333.58 (M+1)+, 1355.79 (M+Na)+; TLC Rf: 0.08 (ethyl acetate/methanol=5/1); HPLC RT: 12.77 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[(R)-α-Hydroxymethyl-Sar]-3-[N-MeVal]-4-cyclosporin was prepared according to the method described in Example 2. The product was purified by chromatography on silica gel (ethyl acetate/methanol) [Molecular Formula: C62H111N11O13; Exact Mass: 1217.84; MS (m/z): 1218.56 (M+1)+, 1240.75 (M+Na)+].
To a solution of [(R)-α-hydroxymethyl-Sar]-3-[N-MeVal]-4-cyclosporin (0.12 g, 0.10 mmol) in benzene (15 ml) were added sodium hydroxide (0.20 g. 5.00 mmol), tetramethylammonium hydroxide pentahydrate (0.18 g, 1.00 mmol) and 3-dimethylaminoethyl chloride hydrochloride (0.14 g, 1.00 mmol). The mixture was stirred at 30° C. overnight. Ice water (20 ml) was added and the mixture was separated. The aqueous layer was extracted with ethyl acetate (20 ml). The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was subjected to chromatography on silica gel (dichloromethane/methanol=95/5) to give the 30 mg of pure product [Molecular Formula: C66H120N12O13; Exact Mass: 1288.91; MS (m/z): 1289.73 (M+1)+, 1311.71 (M+Na)+; TLC Rf: 0.14 (dichloromethane/methanol=10/1); HPLC RT: 12.00 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
[α-Methylene-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (0.37 g, 0.30 mmol) and piperidine (0.26 g, 3.00 mmol) were dissolved in acetonitrile/water (20 ml) in the presence of the catalytic amount of copper (II) acetate. The mixture was stirred overnight at room temperature. After removal of solvent, the residue was dissolved in dichloromethane (30 ml). The dichloromethane phase was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was further purified by chromatography on silica gel (dichloromethane/methanol, 96/4) to give 0.17 g of product [Molecular Formula: C68H122N12O13; Exact Mass: 1314.93; MS (m/z): 1315.74 (M+1)+, 1337.86 (M+Na)+; TLC Rf: 0.10 (ethyl acetate/methanol=5/1); HPLC RT: 11.70 min (C8 reverse phase column: 150 mm; acetonitrile/water (0.05% TFA); operation temperature: 64° C.; detector: 210 nm)].
Under nitrogen atmosphere, to a mixture of [(γ-Hydroxy)-NMeLeu]-4-cyclosporin (FW 1218.61, 800 mg, 0.66 mmol) and ally 2,2,2-trichloroacetimidate (FW 202.47, 930 mg, 4.6 mmol) in 150 ml of DCM was added trimethylsilyl trifluoromethanesulfonate (FW 222.26, d 1.228, 250 mg, 1.12 mmol) at 0° C. The resulting mixture was allowed to warm to room temperature and stirred for overnight. Then the mixture was washed with saturated NaHCO3 water solution and brine. The organic layer was separated, dried over MgSO4 and evaporated under vacuum. The residue was purified by column chromatography using DCM/MeOH (98/2) to give product [Molecular formula: C65H115N11O13; Exact Mass: 1257.87; MS (m/z): 1280.7 (M+Na)+; TLC Rf: 0.46 (DCM/MeOH=95/5); HPLC RT: 16.45 minutes (C8 reverse phase column, 250 mm, acetonitrile/0.05% TFA in water, operation temperature: 64° C.; Detector: 210 nm)].
To a solution of [α-methylene-Sar]-3-cyclosporin (FW 1214.62, 1.0 g, 0.82 mmol) in nitromethane (15 ml) was added 1,8-diazabicyclo[5,4,0]undec-7-ene (FW 152.24, 1.0 g, 6.6 mmol). After stirred at room temperature for 2 days, the reaction mixture was concentrated under reduced pressure. The residue was mixed with water dichloromethane. The dichloromethane layer was washed with aqueous citric acid solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (hexane/acetone=3/1) to give the product [Molecular formula: C64H114N12O14; Exact Mass: 1274.86; MS (m/z): 1275.54 (M+1)+].
To a mixture of [(R)-2-nitroethyl-Sar]-3-cyclosporin (FW 1275.66, 210 mg, 0.16 mmol) and zinc (FW 65.38, 1 g, 15.3 mmol) in ethanol (20 ml) was added 10% aqueous hydrochloric acid (10 ml). The reaction mixture was stirred at room temperature overnight (monitored by LC-MS) and filtered. The filter cake was washed with ethanol. The filtrate was concentrated and diluted with dichloromethane. The dichloromethane layer was washed with aqueous saturated sodium bicarbonate and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give the product [Molecular formula: C64H116N12O12; Exact Mass: 1244.88; MS (m/z): 1245.54 (M+1)+].
To a solution of [(R)-2-aminoethyl-Sar]-3-Cyclosporin (FW 1245.68, 122 mg, 0.1 mmol) in chloroform (6 ml) were added formaldehyde aqueous 37% solution (0.6 ml) and acetic acid (6 drops). The reaction mixture was stirred at room temperature for 5 min. Then tetramethylammonium triacetoxyborohydride (FW 263.10, 131 mg, 0.5 mmol) was added and the reaction mixture was continued to stir for one hour. The mixture was diluted with dichloromethane. The organic layer was washed with aqueous saturated sodium bicarbonate and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give the product [Molecular formula: C66H120N12O12; Exact Mass: 1272.91; MS (m/z): 1273.70 (M+1)+; TLC Rf: 0.27 (dichloromethane/methanol); HPLC RT: 12.42 minutes].
To a solution of 3-amino cyclosporine (FW 1245.68, 124 mg, 0.1 mmol) in chloroform (6 ml) were added acetaldehyde (FW 44.06, 78 mg, 1.77 mmol) and acetic acid (drops). The reaction mixture was stirred at room temperature for 5 min. Then tetramethylammonium triacetoxyborohydride (FW 263.10, 126 mg, 0.48 mmol) was added and the reaction mixture was continued to stir for 1 hour. The mixture was diluted with dichloromethane. The organic layer was washed with aqueous saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give product [Molecular formula: C68H124N12O12; Exact Mass: 1300.95; MS (m/z): 1301.72 (M+1)+; TLC Rf: 0.33 (dichloromethane/methanol=95/5); HPLC RT: 13.28 minutes (C8 reverse phase column, 250 mm, acetonitril-water/(0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
n-Butyllithium (15.84 ml, 2.89 M, 45.79 mmol) was added to a solution of diisopropylamine (6.50 ml, 45.79 mmol) in tetrahydrofuran (80 ml) at −78° C. under nitrogen and the reaction mixture was stirred for an hour. A solution of cyclosporine A (5.00 g, 4.16 mmol) in tetrahydrofuran (20 ml) was added over 10 minutes and the mixture was stirred at −78° C. for another two hours. Then methyl bromoacetate (7.00 g, 45.79 mmol) and the reaction mixture was stirred at −78° C. for one hour. The reaction mixture was allowed to warm up to room temperature slowly and stirred overnight. Most of tetrahydrofuran was removed under vacuum at room temperature. Ethyl acetate (50 ml) and 50 ml brine were added and separated. The aqueous layer was extracted with ethyl acetate (20 ml×3). The combined ethyl acetate layers were dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by column to give to give 2.11 g of [(2-methoxy-2-oxoethyl)-Sar]-3-cyclosporin [Molecular Formula: C65H115N11O14; Exact Mass: 1273.86; MS (m/z): 1274.44 (M+1)+, 1296.63 (M+Na)+].
[(2-(t-Butoxy)-2-oxoethyl)-Sar]-3-cyclosporin was synthesized using a method analogous to a procedure described by Seebach D, et al., 1993, Helv Chim Acta, 76, 1564-1590.
[(2-Methoxy-2-oxoethyl)-Sar]-3-cyclosporin (1.00 g, 0.79 mmol) was dissolved in tetrahydrofuran (30 ml), followed by adding cesium chloride (1.00 g, 5.94 mmol) and sodium borohydride (1.00 g, 26.43 mmol). Then 30 ml of methanol was added dropwise to the mixture over one hour. After addition, the mixture was stirred at room temperature for another hour. Most solvent was then evaporated under reduced pressure. Ethyl acetate (30 ml) and water (30 ml) were added. The ethyl acetate layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography to give the product of isomer A as [(R)-(2-hydroxyethyl)-Sar]-3-cyclosporin [Molecular Formula: C64H115N11O13; Exact Mass: 1245.87; MS (m/z): 1246.49.52 (M+1)+, 1268.72 (M+Na)+; TLC Rf: 0.46 (dichloromethane/methanol=9:1); HPLC RT: 16.06 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)] and the product of isomer B as [(S)-(2-hydroxyethyl)-Sar]-3-cyclosporin [Molecular Formula: C64H115N11O13; Exact Mass: 1245.87; MS (m/z): 1246.49 (M+1)+, 1268.68 (M+Na)+; TLC Rf: 0.46 (dichloromethane/methanol=9:1); HPLC RT: 15.15 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% trifluoroacetic acid); operation temperature: 64° C.; detector: 210 nm)].
To a solution of [α-Methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (FW 1230.62, 1.6 g, 1.3 mmol) in 20 ml of nitromethane was added 1,8-diazabicyclo[5,4,0]undec-7-ene (4 ml). The reaction mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was mixed with water and dichloromethane and separated. The organic layer was washed with aqueous citric acid solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give the product (R-isomer) 600 mg [Molecular formula: C64H114N12O15; Exact Mass: 1290.85; MS (m/z): 1291.72 (M+1)+. HPLC RT: 14.95 minutes]. (S-isomer) 360 mg [Molecular formula: C64H114N12O15; Exact Mass: 1290.85; MS (m/z): 1291.72 (M+1)+; HPLC RT: 14.43 minutes].
To a mixture of [(R)-2-nitroethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (FW 1291.66, 400 mg, 0.31 mmol) and zinc (1.5 g) in ethanol (30 ml) was added 10% aqueous hydrochloric acid (30 ml). The reaction mixture was stirred at room temperature overnight (monitored by LC-MS) and filtered. The filter cake was washed with ethanol. The combined filtrate was concentrated and diluted with dichloromethane. The organic layer was washed with aqueous saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give the product [Molecular formula: C64H116N12O13; Exact Mass: 1260.88; MS (m/z): 1261.70 (M+1)+].
To a solution of [(R)-2-aminoethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (FW 1261.68, 180 mg, 0.14 mmol) in chloroform (6 ml) were added formaldehyde aqueous 37% solution (0.8 ml) and acetic acid (8 drops). The reaction mixture was stirred at room temperature for 5 min. Then tetramethylammonium triacetoxyborohydride (263.10, 200 mg, 0.76 mmol) was added and the reaction mixture was continued to stir for one hour. The mixture was diluted with dichloromethane. The organic layer was washed with aqueous saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give the product [Molecular formula: C66H120N12O13; Exact Mass: 1288.91; MS (m/z): 1289.76 (M+1)+; TLC Rf: 0.32 (dichloromethane/methanol=9/1); HPLC RT: 11.14 minutes].
To a solution of [(R)-2-aminoethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (FW 1261.68, 129 mg, 0.10 mmol) in chloroform (10 ml) were added acetaldehyde (FW 44.06, 80 mg, 1.8 mmol) and acetic acid (7 drops). The reaction mixture was stirred at room temperature for 5 min. Then tetramethylammonium triacetoxyborohydride (FW 263.10, 200 mg, 0.76 mmol) was added and the reaction mixture was continued to stir for one hour. The mixture was diluted with dichloromethane. The organic layer was washed with aqueous saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give the product [Molecular formula: C68H124N12O13; Exact Mass: 1316.94; MS (m/z): 1317.70 (M+1)+; TLC Rf: 0.39 (Dichloromethane/methanol=9/1); HPLC RT: 12.06 minutes].
To a mixture of [(S)-2-nitroethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (FW 1291.66, 350 mg, 0.27 mmol) and zinc (1.5 g) in ethanol (25 ml) was added 10% aqueous hydrochloric acid (15 ml). The reaction mixture was stirred at room temperature overnight (monitored by LC-MS) and filtered. The filter cake was washed with ethanol. The combined filtrate was concentrated and diluted with dichloromethane. The organic solution was washed with aqueous saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=96/4) to give the product [Molecular formula: C64H116N12O13; Exact Mass: 1260.88; MS (m/z): 1261.64 (M+1)+].
To a solution of [(S)-2-aminoethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (FW 1261.68, 13 0 mg, 0.10 mmol) in chloroform (10 ml) were added formaldehyde aqueous 37% solution (0.7 ml) and acetic acid (7 drops). The reaction mixture was stirred at room temperature for 5 min. Then tetramethylammonium triacetoxyborohydride (263.10, 200 mg, 0.76 mmol) was added and the reaction mixture was continued to stir for one hour. The mixture was diluted with dichloromethane. The organic layer was washed with aqueous saturated sodium bicarbonate solution and brine, dried over Magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give the product [Molecular formula: C66H120N12O13; Exact Mass: 1288.91; MS (m/z): 1289.70 (M+1)+; TLC Rf: 0.35 (Dichloromethane/methanol=9/1); HPLC RT: 11.02 minutes].
To a solution of [(S)-2-aminoethyl-Sar]-3-[(γ-hydroxy)-NMeLeu]-4-cyclosporin (FW 1261.68, 129 mg, 0.10 mmol) in chloroform (10 ml) were added acetaldehyde (FW 44.06, 80 mg, 1.8 mmol) and acetic acid (7 drops). The reaction mixture was stirred at room temperature for 5 minutes. Then tetramethylammonium triacetoxyborohydride (FW 263.10, 200 mg, 0.76 mmol) was added and the reaction mixture was continued to stir for one hour. The mixture was diluted with dichloromethane. The organic layer was washed with aqueous saturated sodium bicarbonate solution and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=95/5) to give the product [Molecular formula: C68H124N12O13; Exact Mass: 1316.94; MS (m/z): 1317.70 (M+1)+; TLC Rf: 0.41 (Dichloromethane/methanol=9/1); HPLC RT: 11.96 minutes].
To a mixture of [(R)-2-nitroethyl-Sar]-3-cyclosporin (FW 1275.66, 360 mg, 0.28 mmol) and dimethymethyleneammonium iodide (FW 185.01, 550 mg, 15.4 mmol) acetonitrile (in 25 ml) was added triethylamine (10 drops). The reaction mixture was stirred at room temperature overnight (monitored by LC-MS). The mixture was concentrated and diluted with dichloromethane. The organic layer was washed with aqueous water and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude product was used for next step.
To a mixture of cylcosporine (crude form previous step FW 1332.76, 0.28 mmol) and tri-n-butyltin hydride (FW 291.07, 0.41 g, 1.4 mmol) in benzene (25 ml) was added AIBN (FW 164.21, 46 mg, 0.28 mmol) under nitrogen atmosphere. The mixture was heated to reflux with stirring for 8 hours. After the reaction was completed (monitored by LC-MS), the reaction mixture was washed with aqueous saturated sodium bicarbonate solution and brine followed by evaporated under vacuum. The residue was purified by chromatography (Dichloromethane/methanol=95/5) to give the product [Molecular formula: C67H122N12O12; Exact Mass: 1286.93; MS (m/z): 1287.71 (M+1)+; TLC Rf: 0.36 (dichloromethane/methanol=95/5); HPLC RT: 12.57 minutes].
To a solution of [α-methylene-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (0.61 g, 0.50 mmol) and diethyl malonate (1.00 g, 6.25 mmol) in ethanol anhydrous (30 ml) was added sodium ethoxide (0.82 g, 12.05 mmol). The reaction mixture was stirred at room temperature overnight and concentrated under reduced pressure. The residue was mixed with water (30 ml) and dichloromethane (80 ml) and separated. The organic layer was washed with aqueous citric acid and brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by chromatography (hexane/acetone=2/1) to give the product. [Molecular formula: C70H123N11O17; Exact Mass: 1389.91; MS (m/z): 1390.56 (M+1)+; TLC Rf: 0.30 (dichloromethane/methanol=95/5); HPLC RT: 15.32 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% TFA); operation temperature: 64° C.; Detector: 210 nm)].
To a suspension [(R)-3-ethoxy-2-(ethoxycarbonyl)-3-oxopropyl)-Sar]-3-[(γ-hydroxy)-N-MeLeu]-4-cyclosporin (150 mg, 0.11 mmol) and lithium chloride (580 mg, 13.81 mmol) in methanol (50 ml) was added sodium borohydride (1.20 g, 31.75 mmol) in portions. The mixture was stirred overnight at room temperature. Most of solvent was evaporated under reduced pressure. Dichloromethane (100 ml) and water (50 ml) were added and separated. The dichloromethane layer was washed with brine, dried over Magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography (dichloromethane/methanol=97/3) to give product. [Molecular formula: C66H119N11O15; Exact Mass: 1305.89; MS (m/z): 1306.48 (M+1)+; TLC Rf: 0.22 (dichloromethane/methanol=9/1); HPLC RT: 10.65 min (C8 reverse phase column: 250 mm; acetonitrile/water (0.05% TFA); operation temperature: 64° C.; Detector: 210 nm)].
n-Butyllithium (2.87 M, 27 mmol, 9.4 ml, 10 eq) was added to a solution of diisopropylamine (3.8 ml, 27 mmol, 10 eq) in tetrahydrofuran (80 ml) at −78° C. under nitrogen. After the reaction mixture was stirred for an hour, a solution of cyclosporine A (3.2 g, 2.66 mmol) in tetrahydrofuran (15 ml) was added over 10 min. The stirring was continued at −78° C. for 2 hours. Carbon dioxide gas was bubbled through the reaction mixture for 20-25 minutes and stirred at −78° C. for another hour. Then the cooling bath was removed and the reaction mixture was allowed to warm up to 0° C. slowly. Most of tetrahydrofuran was removed under vacuum at room temperature. The residue was quenched by the addition of saturated citric acid solution and the pH of the mixture was adjusted to around 7-8. The unreacted cyclosporin was extracted with ether (40 ml×2). The PH of the aqueous layer was adjusted to 3˜4 with 1 N hydrochloric acid and the precipitated oil was extracted with ethyl acetate (100 ml). The aqueous layer was extracted with ethyl acetate (100 ml×3). The combined ethyl acetate layers were washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give semi-solid product (2.61 g, yield: 78%) [Molecular Formula: C63H111N11O14; Exact Mass: 1245.83; MS (m/z): 1246.7 (M+1)+, 1268.7 (M+Na)+].
[α-Carboxy-Sar]-3-cyclosporin was synthesized according to a procedure described by Seebach D, et al., 1993, Helv Chim Acta, 76, 1564-1590.
[α-Methylene-Sar]-3-cyclosporin can also be prepared using a method analogous to the procedure described in WO2012/051194A1.
[N-MeVal]-4-Cyclosporin (SDZ 220-384) was prepared according to procedures described by Papageorgiou C, et al., 1994, Bioorg & Med Chem Lett, 4, 267-272 and its key cyclosporine ring-opening between position 3 and 4 cited as reference 14: Su Z and Wenger R, Unpublished results; Papageorgiou C, et al., 1994, J. Med. Chem., 37, 3674-3676 and its key cyclosporine ring-opening between position 3 and 4 cited as reference 11: Su Z and Wenger R, Unpublished results.
To a solution of cyclosporin A (1) (12.00 g, 19.98 mmol) in acetic anhydride (MW: 102.09, d 1.082, 40 ml) were added pyridine (MW: 79.01, d 0.978, 40 ml) and 4-N,N-dimethylaminopyridine (MW: 122.17, 0.40 g). This mixture was stirred for overnight at room temperature, and then the mixture was diluted with 600 ml of ethyl acetate. The mixture was washed with brine, saturated ammonium chloride solution and 15% of sodium bicarbonate solution. The organic phase was dried over sodium sulphate, filtered and evaporated under the reduced pressure. Then all of pyridine was azeotropically evaporated out under the reduced pressure by adding toluene to the mixture to give a pale yellow solid residue, which was purified by flash chromatography on a silica gel column (100-200 mesh) with eluent of ethyl acetate/hexane (1/3) to give the 11.80 g (9.48 mmol, 95%) of cyclosporin A-acetate (2).
To a suspension of trimethyloxonium-fluoroborate (MW: 147.91, 2.96 g, 20 mmol, 2.50 equiv.) in dichloromethane (80 ml) was added cyclosporine A-acetate (2) (10.00 g, 8.00 mmol). The suspension was stirred for 18 hours at room temperature, and then a solution of sodium methoxide (9.90 mmol) in methanol (40 ml) was added. After the mixture was stirred for another half hour, 2 N solution of sulfuric acid in methanol (40 ml) was added. The mixture was stirred for 15-30 minutes at room temperature and neutralized with 15% potassium bicarbonate solution. Then the mixture was extracted twice with 700 ml of ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulphate and evaporated under reduced pressure. The residue was purified by flash chromatography on a silica column (100-200 mesh) with eluent of methanol/methyl t-butyl ether to give the 7.15 g (5.60 mmol, 70%) of linear undecapeptide peptide (1).
To a solution of linear undecapeptide peptide (3) (7.00 g, 5.50 mmol) in tetrahydrofuran (80 ml) was added phenyl isothiocyanate (MW: 135.19, d 1.130, 0.86 ml, 7.15 mmol, 1.30 equiv.). The mixture was stirred for 3 hours at room temperature and evaporated under reduced pressure. The residue was purified by flash chromatography on a silica gel column (100-200 mesh) with eluent of acetone in hexane (1/5) to give the 6.99 g (4.95 mmol, 90%) of linear phenylthiourea undecapeptide (4) [Exact Mass: 1410.89; MS m/z: 1433.88 (M+Na)+].
To a solution of linear phenylthiourea undecapeptide (4) (6.80 g, 4.82 mmol) in toluene (300 ml) was added trifluoroacetic acid (MW: 114.02, d 1.480, 8.00 ml) at room temperature. The mixture was stirred for 1.5 to 2 hours and quenched by a slurry of sodium bicarbonate in water. Then the mixture was separated and the water phase was extracted with toluene (100 ml) and ethyl acetate (100 ml) subsequently. The combined organic layers were dried over sodium sulphate and evaporated under reduced pressure. The residue was purified by flash chromatography on a silica column (100-200 mesh) with eluent of acetone/hexane (3/1) to give the 3.88 g (3.37 mmol, 70%) of linear decapeptide peptide (5) [Exact Mass: 1148.78; MS m/z: 1149.78 (M+1)+].
This Edman degradation was carried according to the similar method described by Edman P, et al, 1967, Eur. J. Biochem., 1, 80.
To a solution of linear decapeptide peptide (5) (3.80 g, 3.30 mmol) in dichloromethane (150 ml) were added Boc-MeVal (6) (MW: 231.29, 0.92 g, 3.96 mmol, 1.2 equiv.), 1-propanephosphonic acid cyclic anhydride (MW: 318.18, 2.10 ml, 50 wt. % solution in ethyl acetate) and triethylamine (MW: 101.19, d 0.726, 0.46 ml, 3.30 mmol) at 0° C. The resulting mixture was stirred at room temperature for 5 hours. Then the mixture was washed with brine. The aqueous layer was extracted with ethyl acetate (100 ml). The combined organic layers were dried over sodium sulphate. Removal of the solvent under reduced pressure gave the residue, which was purified by flash chromatography on a silica column (100-200 mesh) with eluent of acetone/hexane (1/2.5) to give the 4.05 g (2.97 mmol, 90%) of linear Boc-N-MeVal-decapeptide peptide (7) [Exact Mass: 1361.91; MS m/z: 1384.91 (M+Na)+].
To a solution of linear [Boc-N-MeVal]-4-decapeptide peptide (7) (4.00 g, 2.94 mmol) in ethyl alcohol (150 ml) at 0° C. was added 0.5 N sodium hydroxide solution (7.1 ml, 1.20 equiv.). The mixture was stirred and kept at 0° C. for 16 hours. Then the PH of the mixture was adjusted to around 3 by adding 0.5 N hydrochloric acid. Most of solvent was evaporated under the reduced pressure and the residue was dissolved in 200 ml of ethyl acetate. The mixture was washed with a pH 3 buffer, dried over sodium sulphate, filtered and evaporated under the reduced pressure. The residue was purified by flash chromatography on a silica column (100-200 mesh) with eluent of methanol/ethyl acetate (1/8) to yield 2.55 g (1.89 mmol, 64.3%) of the free acid (8).
To a solution of free acid (8) (2.55 g, 1.89 mmol) in dichloromethane (25 ml) was slowly added 5 ml of trifluoroacetic acid (MW: 114.02, d 1.480) at 0° C. The solution was stirred at room for 2 hours. Then ethyl acetate (300 ml) was added and the solvent was removed under reduced pressure. Another ethyl acetate (300 ml) was added and the solvent was removed under reduced pressure again. The residue was purified by flash chromatography on a silica gel column (100-200 mesh) with eluent of methanol/acetone (1/3) to give the 2.01 g (1.61 mmol, 85%) of linear [N-MeVal]-4-decapeptide peptide free acid (9) [Exact Mass: 1247.85; MS m/z: 1248.85 (M+1)+].
To a solution of linear [N-MeVal]-4-decapeptide peptide free acid (9) (1.03 g, 0.83 mmol) in dichloromethane (250 ml) were added 1-propanephosphonic acid cyclic anhydride (MW: 318.18, 0.53 ml, 50 wt. % solution in ethyl acetate), 2,4,6-collidine (MW: 121.18, d 0.917, 0.11 ml, 0.83 mmol)) at 0° C. The mixture was stirred at room temperature for 24 hours. Then the mixture was passed through a thin layer of silica gel and washed twice by 40 ml of ethyl acetate. The collected organic solution was evaporated under the reduced pressure. The residue was purified by flash chromatography on a silica gel column (230-400 mesh) with eluent of methanol/acetone (1/6) to give the 611 mg (0.50 mmol, 60%) of (N-Methyl-Val)-4-Cyclosporin acetate (10) [Exact Mass: 1229.84; MS m/z: 1252.82 (M+Na)+].
To a solution of [N-MeVal]-4-Cyclosporin acetate (10) (0.60 g, 0.49 mmol) in methanol (40 ml) was added a solution of sodium methoxide in methanol (0.5 M, 1.9 ml, 2.0 equiv.). The mixture was stirred for 0.5 hour at 0° C. and 24 hours at room temperature. The PH of the mixture was adjusted to around 6 by adding 0.5 N hydrochloric acid. After the solvent was evaporated under reduced pressure, the residue was dissolved in 200 ml of ethyl acetate. The organic solution was washed by aqueous sodium bicarbonate and brine, dried over sodium sulphate and filtered. After removal of solvent, the residue was purified by flash chromatography on a silica gel column (230-400 mesh) with eluent of acetone/hexane (1/2) to give the 406 mg (0.34 mmol, 70%) of [N-MeVal]-4-Cyclosporin (11) [Exact Mass: 1187.83; 84; MS m/z: 1210.81 (M+Na].
[N-MeIle]-4-Cyclosporin (NIM-811) was prepared according to the procedure used for the synthesis of (N-MeVal)-4-cyclosporin (SDZ 220-384).
[N-MeThr]-4-Cyclosporin can be prepared according to the procedure used for the synthesis of (N-MeVal)-4-cyclosporin (SDZ 220-384).
The side chain intermediates were synthesized according to procedures described by Urquhart G G, 1994, Org Synth, Coll. Vol III, 363
A mixture of 4-(2-chloroethyl)morpholine (7.00 g, 37 mmol) and thiourea (2.90 g, 38 mmol) in 95% ethanol (55 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (3.40 g, 85 mmol) in water (20 ml) was added, and the mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with benzene. The benzene layer was washed with brine, dried over magnesium sulfate and evaporated to provide 3.80 g of crude product, which was used for the addition reaction.
The mixture of 1-(2-chloroethyl)piperidine hydrochloride (7.00 g, 38 mmol) and thiourea (4.60 g, 61 mmol) in 95% ethanol (30 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (2.40 g) in water (20 ml) was added. The mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with ether. The organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 3.20 g of crude product, which was used for the addition reaction without purification.
The mixture of 1-(2-chloroethyl)piperidine hydrochloride (7.0 g, 41 mmol) and thiourea (3.20 g, 40 mmol) in 95% ethanol (30 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (3.40 g, 85 mmol) in water (20 ml) was added. The mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with benzene. The organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 3.80 g of crude product, which was used for the addition reaction without purification.
To a suspension of 1-bromoro-3-chloropropane (30.00 g, 191 mmol) and potassium carbonate (17.00 g, 123 mmol) in dichloromethane (160 ml) was added pyrrolidine (3.50 g, 49 mmol) portions. The mixture was stirred at room temperature overnight. Then the mixture was filtered and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate/methanol=95/5) to give 6.00 g of product.
A mixture of 1-(3-chloropropyl)pyrrolidine (3.4 g, 23 mmol) and thiourea (1.8 g, 23 mmol) in 95% ethanol (55 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (1.20 g, 30 mmol) in water (10 ml) was added, and the mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with benzene. The organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 1.80 g of crude product, which was used for the addition reaction.
The mixture of 1-(3-chloropropyl)piperidine hydrochloride (7.50 g, 38 mmol) and thiourea (4.60 g, 61 mmol) in 95% ethanol (30 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (2.40 g) in water (20 ml) was added. The mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with ether. The organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 3.50 g of crude product, which was used for the addition reaction without purification.
To a suspension of 1-bromoro-3-chloropropane (30.00 g, 191 mmol) and potassium carbonate (14.00 g, 101 mmol) in dichloromethane (160 ml) was added morpholine (4.00 g, 46 mmol) in portions. Then the mixture was stirred at room temperature overnight. The mixture was filtered and evaporated under reduced pressure. The residue was purified by chromatography (ethyl acetate) to give 5.60 g of product.
A mixture of 1-(3-chloropropyl)morpholine (4.20 g, 25.76 mmol) and thiourea (2.00 g, 26.27 mmol) in 95% ethanol (55 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (1.3 g, 32.50 mmol) in water (10 ml) was added, and the mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with benzene. The benzene layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 2.20 g of crude product, which was used for the addition reaction.
A mixture of 2-(4-methylpiperazino)ethyl chloride (8.00 g, 40 mmol) and thiourea (4.87 g, 64 mmol) in 95% ethanol (55 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (2.60 g) in water (20 ml) was added, and the mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with benzene. The benzene layer was washed with brine, dried magnesium sulfate and evaporated to provide 3.0 g of crude product, which was used for the addition reaction.
A mixture of 3-(4-methylpiperazino)propyl chloride (8.5 g, 40 mmol) and thiourea (4.87 g, 64 mmol) in 95% ethanol (70 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (2.6 g) in water (20 ml) was added, and the mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with ether. The organic layer was washed with brine, dried over magnesium sulfate and evaporated to provide 2.5 g of crude product, which was used for the addition reaction.
To a suspension of 1-bromoro-3-chloropropane (11.00 g, 70 mmol) and potassium carbonate (13.00 g, 94 mmol) in dichloromethane (100 ml) was added ethylisopropylamine (4.10 g, 47 mmol) in portions. The mixture was stirred at room temperature overnight. The mixture was filtered and concentrated under reduced pressure. The residue was purified by chromatography (ethyl acetate/methanol=95/5) to give 6.10 g of product.
A mixture of 3-chloropropylethylisopropylamine (4.20 g, 25.66 mmol) and thiourea (2.00 g, 26.32 mmol) in 95% ethanol (55 ml) was heated to reflux for 24 hours. A solution of sodium hydroxide (1.30 g, 32.50 mmol) in water (10 ml) was added, and the mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with benzene. The benzene layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure to give 1.20 g of crude product, which was used for the addition reaction.
A mixture of N-(3-bromopropyl)phthalimide (20.00 g, 74.63 mmol) and thiourea (9.98 g, 131.34 mmol) in 95% ethanol (80 ml) was heated to reflux for 3 hours. A solution of sodium hydroxide (4.48 g, 111.94 mmol) in water (30 ml) was added, and the mixture was continued to reflux for another 3 hours. After cooled to room temperature, the mixture was evaporated under reduced pressure. The residue was mixed with ethyl acetate (50 ml) and brine (50 ml). The organic layer was separated and washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The residue was mixed with toluene (100 ml) and heated to reflux with removal of water azeotropically for two hours. After removal of toluene, the residue was purified by chromatography on silica gel with hexane and ethyl acetate as solvent to give 9.10 g of N-(3-mercaptopropyl)phthalimide. To a solution of N-(3-mercaptopropyl)phthalimide in methanol (50 ml) was added ethanolamine. The mixture was stirred and heated to reflux for two hours. After cooled to room temperature, the mixture was used for the addition reaction without further purification.
To a suspension of piperizaine (30.00 g, 348.27 mmol) and sodium carbonate (106 g, 348.27 mmol) in dichloromethane (200 ml) was added dropwise a solution of Di-tert-butyl dicarbonate (18.98 g, 87.07 mmol) in dichloromethane (30 ml) at room temperature for one hour. Then the mixture was stirred at room temperature overnight. The mixture was mixed with water (100 ml) and separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was dissolved in dichloromethane (150 ml). Sodium carbonate (15.55 g, 146.77 mmol) and 1-bromo-2-chloroethane (21.05 g, 146.77 mmol) were added. The mixture was stirred at room temperature for a weekend. The mixture was mixed with water (100 ml) and separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using hexane and ethyl acetate as eluent to give 6.85 g of 1-Boc-4-(2-chloroethyl)piperazine.
To a solution of 1-Boc-4-(2-chloroethyl)piperazine (6.85 g, 27.62 mmol) in methanol (50 ml) was added thiourea (4.12 g, 55.24 mmol). The mixture was heated to reflux for 2 hours. A solution of sodium hydroxide (1.66 g, 41.43 mmol) in water (10 ml) was added, and the mixture was continued to reflux for another hour. Then most solvent was evaporated under reduced pressure. The residue was mixed with ethyl acetate (50 ml) and brine (30 ml) and separated. The ethyl acetate was dried over magnesium sulfate and evaporated under reduced pressure. The residue was dissolved in methanol (20 ml). 4 M hydrochloric acid in dioxane (10 ml) was added. The mixture was stirred at room temperature overnight and most of solvent was evaporated under reduced pressure. The residue was used for the addition reaction without further purification.
To a suspension of piperizaine (30.00 g, 348.27 mmol) and sodium carbonate (106 g, 348.27 mmol) in dichloromethane (200 ml) was added dropwise a solution of Di-tert-butyl dicarbonate (18.98 g, 87.07 mmol) in dichloromethane (30 ml) at room temperature for one hour. Then the mixture was stirred at room temperature overnight. The mixture was mixed with water (100 ml) and separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was dissolved in dichloromethane (100 ml). Sodium carbonate (15.44 g, 145.70 mmol) and 1-bromo-3-chloropropane (15.29 g, 97.13 mmol) were added. The mixture was stirred at room temperature overnight. The mixture was mixed with water (80 ml) and separated. The dichloromethane layer was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using hexane and ethyl acetate as eluent to give 11.77 g of 1-Boc-4-(3-chloropropanyl)piperazine.
To a solution of 1-Boc-4-(3-chloropropanyl)piperazine (11.77 g, 44.90 mmol) in ethanol (100 ml) was added thiourea (6.82 g, 89.80 mmol). The mixture was heated to reflux for 4 hours. A solution of sodium hydroxide (2.69 g, 67.35 mmol) in water (40 ml) was added, and the mixture was continued to reflux for another 2 hours. Then most solvent was evaporated under reduced pressure. The residue was mixed with ethyl acetate (100 ml) and brine (50 ml) and separated. The ethyl acetate was dried over magnesium sulfate and evaporated under reduced pressure. The residue was purified by chromatography on silica gel using hexane and acetone as eluent to give 7.5 g of tert-butyl 4-N-Boc-1-(3-mercaptopropanyl)piperazine.
The following compounds can be prepared according to a method analogous to those described herein.
Stability of Cyclosporin derivatives was evaluated in methanol at 65° C. and 50° C., and HPLC was used to monitor possible isomerization of these compounds. It was found that SCY-635 is not stable and can easily convert to its corresponding epimer, which is expected to have low or no anti-viral activity.
SCY-635
When the epimer was treated with MeOH at 65° C., it also was found that it partially transformed to SCY-635. At the endpoint of the equilibrium, this solution contained about 58% of SCY-635 and about 42% of epimer.
Based on the isomerization data, the inventors suggest that the epimerization of SCY-635 occurs through the following reaction mechanism:
Thus, the two carbon side chain at position 3 of the sarcosine of cyclosporine contributes to the instability, because it can form a six-membered ring transition state, and stimulate the epimerization. Additionally, the epimerization is accelerated by the γ-hydroxyl group at the 4-position of leucine.
Accordingly, the inventors envisioned novel cyclosporine derivatives having enhanced stability while maintaining good cyclophilin binding activity. In particular, the inventors have surprisingly found that the masking the γ-hydroxyl group on leucine at position 4, elongating side carbon chain (e.g., with 3 carbons or higher), and/or substituting the amine terminal at position 3 with a bulky side chain can prevent or minimize the epimerization. Specially, when the methylene substituents are introduced on position-3, those analogs are very stable, and can prevent the epimerization.
Anti-HCV activity of cyclosporine derivatives were evaluated in the HCV subgenomic replicon assay. The assay use the cell line ET (luc-ubi-neo/ET), which is a Huh7 human hepatoma cell line harboring an HCV replicon with a stable luciferase (Luc) reporter. HCV RNA replication was assessed by quantifying HCV replicon-derived luciferase activity. The antiviral activity of cyclosporine analogs were evaluated after drug treatment to derive EC50 and EC90 values by using the luciferase end point (Krieger, N., et al., 2001, J. Virol. 75, 4614-4624; Pietschmann, T., et al., 2002, J. Virol. 76, 4008-4021; each of which is incorporated herein by reference). Cytotoxicity was assessed in parallel.
Anti-HIV activity of cyclosporine derivatives were evaluated by cytoprotection assay in an acute infection model using CEM-SS cells and either HIV-1IIIB or HIV-1 RF. Antiviral activity was determined as a reduction in virus-caused cytopathic effects when compounds prevent virus replication. Cytoprotection and compound cytotoxicity were evaluated using the tetrazollium dye MTS (Promega) to calculate cell viability following virus infection after 6-day incubation (Zhou G., et al., 2011, J. Med. Chem. 27, 7220-31; which is incorporated herein by reference).
The effect of cyclosporine analogs on mitochondrial permeability transition (MPT) was determined by a modified mitochondrial swelling assay measured as an influx of Ca2+, published by J. Blattner et al., 2001, Analytical Biochem, 295, 220-226. Briefly, rat neuronal mitochondria in an ice-cold sucrose buffer were obtained after rat whole-body perfusion and a series of centrifugation. The total protein concentration in each sample batch was determined for standardization between assays. The mitochondrial swelling was induced by 100 micromolar of Calcium Chloride. Each compound (100 nM) was added to mitochondria 5 minutes before the addition of Ca.2+. The value of absorbance at the certain wavelength (620 nm) reflected the degree of mitochondrial swelling. The percentage of the swelling was calculated by comparing the absorbance in the present or absent compound of the interests.
In above assay, the results in mitochondrial swelling strongly indicate that cyclosporine analogs can penetrate the mitochondrial membrane and inhibit mitochondrial swelling.
This patent application is a continuation-in-part of International Application No. PCT/US2011/063295, filed Dec. 5, 2011, which claims priority to U.S. Application Ser. No. 61/419,326, filed Dec. 3, 2010; this patent application also claims priority to U.S. Application Ser. Nos. 61/655,236, filed Jun. 4, 2012; 61/684,928, filed Aug. 20, 2012; and the entire contents of each of the foregoing applications are hereby incorporated by reference herein.
| Number | Name | Date | Kind |
|---|---|---|---|
| 3869448 | Earley et al. | Mar 1975 | A |
| 4108985 | Ruegger et al. | Aug 1978 | A |
| 4703033 | Seebach | Oct 1987 | A |
| 5767069 | Ko et al. | Jun 1998 | A |
| 5965527 | Barriere et al. | Oct 1999 | A |
| 5981479 | Ko et al. | Nov 1999 | A |
| 5994299 | Barriere et al. | Nov 1999 | A |
| 6583265 | Ellmerer-Muller et al. | Jun 2003 | B1 |
| 6927208 | Wenger et al. | Aug 2005 | B1 |
| 7439227 | Scalfaro et al. | Oct 2008 | B2 |
| 7511013 | Molino et al. | Mar 2009 | B2 |
| 7696166 | Molino | Apr 2010 | B2 |
| 7718767 | Fliri et al. | May 2010 | B2 |
| 20060069015 | Molino et al. | Mar 2006 | A1 |
| 20060160727 | Fliri et al. | Jul 2006 | A1 |
| 20100167996 | Fliri et al. | Jul 2010 | A1 |
| 20100173836 | Li et al. | Jul 2010 | A1 |
| 20100173837 | Hopkins | Jul 2010 | A1 |
| 20100196316 | Or | Aug 2010 | A1 |
| 20120088734 | Frydrych | Apr 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| 101068829 | Nov 2007 | CN |
| 61-212599 | Sep 1986 | JP |
| 2000-502320 | Feb 2000 | JP |
| 2011127080 | Feb 2013 | RU |
| WO-0001715 | Jan 2000 | WO |
| WO-2006038088 | Apr 2006 | WO |
| WO-2006038119 | Apr 2006 | WO |
| WO-2006039164 | Apr 2006 | WO |
| WO-2006039668 | Apr 2006 | WO |
| WO-2007015824 | Feb 2007 | WO |
| WO-2008143996 | Nov 2008 | WO |
| WO-2012009715 | Jan 2012 | WO |
| WO-2012021796 | Feb 2012 | WO |
| WO-2012051194 | Apr 2012 | WO |
| WO-2012075494 | Jun 2012 | WO |
| Entry |
|---|
| Evers, 2003, Bioorganic & Medicinal Chemistry Letters, 13, 4415-4419. |
| Papageorgiou, Bioorganic & Medicinal Chemistry, Letters, vol. 6, No, 4, p. 497, 1996. |
| Berge, S.M. et al., “Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, vol. 66, No. 1, pp. 1-19 (Jan. 1977). |
| Bouchard, M.J. et al., “Activation and Inhibition of Cellular Calcium and Tyrosine Kinase Signaling Pathways Identify Targets of the HBx Protein Involved in Hepatitis B Virus Replication,” Journal of Virology, vol. 77, No. 14, pp. 7713-7719, 8 pages. (Jul. 2003). |
| Castro, A.P.V. et al., “Redistribution of Cyclophilin A to Viral Factories during Vaccinia Virus Infection and Its Incorporation into Mature Particles,” Journal of Virology, vol. 77, No. 16, pp. 9052-9068, 18 pages (Aug. 2003). |
| Chen, Z. et al., “Function of HAb18G/CD147 in Invasion of Host Cells by Severe Acute Respiratory Syndrome Coronavirus,” The Journal of Infectious Diseases, vol. 191, Issue 5, pp. 755-760 (Mar. 1, 2005). |
| Chokshi, S. et al., “1104: Characterization of Antiviral Activities of Cyclophilin Inhibitors DEB025 (Alisporivir) and NIM811 on Hepatitis B Virus (HBV) Replication and HBSAG Secretiona In Vitro,” 07a: Viral Hepatitis B&D: Experimental, Journal of Hepataology, vol. 54, pp. S437-S438 (2011). |
| Office Action issued by the State Intellectual Property Office of China for Application No. 201180035147.9 mailed on Feb. 20, 2014 (16 pages—includes English translation). |
| Evers, et al., “Synthesis of Non-Immunosuppressive Cyclophilin-Binding Cyclosporin A Derivatives as Potential Anti HIV-1 Drugs,” Bioorganic & Medicinal Chemistry Letters, vol. 13, pp. 4415-4419 (2003). |
| Extended European Search Report issued by the European Patent Office for European Patent Application No. 11845460.2 dated Apr. 30, 2014 (11 pgs.). |
| Fliri, H. et al., “Cyclosporins. Structure-activity relationships,” Ann N.Y. Acad. Sci., vol. 696, pp. 47-53 (Nov. 1993). |
| Franke, E.A. et al., “Specific incorporation of cyclophilin A into HIV-1 virions,” Nature, vol. 372, pp. 359-362 (Nov. 24, 1994). |
| Hopkins, S. et al., “SCY-635, a Novel Nonimmunosuppressive Analog of Cyclosporine That Exhibits Potent Inhibition of Hepatitis C Virus RNA Replication In Vitro,” Antimicrobial Agents and Chemotherapy, vol. 54, No. 2, pp. 660-672, 14 total pages (Feb. 2010). |
| Hopkins, S. et al., “SCYNEXIS's SCY-635 Demonstrates Impressive Barrier to Resistance in HCV Treatment,” The 45th Annual Meeting of the European Association for the Study of the Liver, Vienna, Austria, 2 pages (Apr. 15, 2010). |
| Inoue, K. et al., “Combined interferon α2b and cyclosporin A in the treatment of chronic hepatitis C: controlled trial,” Journal of Gastroenterology, vol. 38, No. 6, pp. 567-572 (Jun. 2003). |
| Inoue, K. et al., “IFN combined cyclosporin A therapy,” Nihon Rinsho, vol. 59, No. 7, pp. 1326-1330 (Jul. 2001). |
| International Search Report and Written Opinion issued by the U.S. Patent and Trademark Office as International Searching Authority for International Application No. PCT/US11/047571 mailed Feb. 29, 2012 (13 pgs.). |
| International Search Report and Written Opinion issued by the U.S. Patent and Trademark Office as International Searching Authority for International Application No. PCT/US11/063295 mailed Apr. 23, 2012 (9 pgs.). |
| International Search Report and Written Opinion issued by the U.S. Patent and Trademark Office as International Searching Authority for International Application No. PCT/US12/51572 mailed Oct. 16, 2012 (11 pgs.). |
| International Search Report and Written Opinion issued by the U.S. Patent and Trademark Office as the International Searching Authority for International Application No. PCT/US11/44362 mailed on Feb. 2, 2012 (9 pages). |
| International Search Report and Written Opinion issued by the U.S. Patent and Trademark Office as the International Searching Authority for International Application No. PCT/US14/030491 mailed on Nov. 5, 2014 (11 pages). |
| Klatzmann, D. et al., “Functional inhibition by cyclosporin A of the lymphocyte receptor for the AIDS virus (HIV),” C.R. Acad. Sci. III, vol. 303, No. 9, pp. 343-348, 7 total pages(1986) (abstract included). |
| Krieger, N. et al., “Enhancement of Hepatitis C Virus RNA Replication by Cell Culture-Adaptive Mutations,” Journal of Virology, vol. 75, No. 10, pp. 4614-4624, 12 pages (May 2001). |
| Kuhnt, M. et al., “Microbial biotransformation products of cyclosporin A,” The Journal of Antibiotics, vol. 49, No. 8, pp. 781-787 (1996). |
| Lill, J. et al., “Cyclosporine-Drug Interactions and the Influence of Patient Age,” Am. J. Health-Syst. Pharm., vol. 57, pp. 1579-1584 (Sep. 1, 2000). |
| Liu, X. et al., “Cyclophilin A interacts with influenza A virus M1 protein and impairs the early stage of the viral replication,” Cellular Microbiology, vol. 11, No. 5, pp. 730-741 (Feb. 6, 2009). |
| Luban, Jeremy, “Absconding with the Chaperone: Essential Cyclophilin-Gag Interaction in HIV-1 Virions,” Cell, vol. 87, pp. 1157-1159 (Dec. 27, 1996). |
| Luban,J. et al., “Human immunodeficiency virus type 1 Gag protein binds to cyclophilins A and B,” Cell, vol. 73, Issue 6, pp. p1067-p1078 (Jun. 18, 1993). |
| Gilden, D., “Sandoz Axes Cyclosporin Research,” GMHC Treatment Issues, vol. 9, No. 12, pp. 1 & 7 (Dec. 1995). |
| Office Action issued by the State Intellectual Property Office of China for Application No. 201280051142.X dated Feb. 25, 2015 (17 total pages). |
| Papageorgiou, C. et al., “Calcineurin has a very tight-binding pocket for the side chain of residue 4 of cyclosporin,” Bioorganic & Medicinal Chemistry Letters, vol. 4, No. 2, pp. 267-272 (Jan. 1994). |
| Papageorgiou, C. et al., “Improved Binding Affinity for Cyclophilin A by a Cyclosporin Derivative Singly Modified at Its effector Domain,” Journal of Medicinal Chemistry, vol. 37, No. 22, pp. 3674-3676 (1994). |
| Pietschmann, T. et al., “Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture,” Journal of Virology, vol. 76, No. 8, pp. 4008-4021, 15 page (Apr. 2002). |
| Rosenwirth, B. et al., “Inhibition of human immunodeficiency virus type 1 replication by SDZ NIM 811, a nonummunosuppressive cyclosporine analog,” Antimicrobial Agents and Chemotherapy, vol. 38, No. 8, pp. 1763-1772, 11 pages (Aug. 1994). |
| Seebach, D. et al., “Modification of Cyclosporin A (CS): Generation of an enolate at the sarcosine residue and reactions with electrophiles,” Helvetica Chimica Acta, vol. 76, Issue 4, pp. 1564-1590 (Jun. 30, 1993). |
| Tang, Hengli, “Cyclophilin Inhibitors as a Novel HCV Therapy,” Viruses, vol. 2, No. 8, pp. 1621-1634 (Aug. 5, 2010). |
| Tian, X. et al., “Hepatitis B Virus (HBV) Surface Antigen Interacts with and Promotes Cyclophilin A Secretion: Possible Link to Pathogenesis of HBV Infection,” Journal of Virology, vol. 84, No. 7, pp. 3373-3381, 10 pages (Apr. 2010). |
| Wainberg, M.A. et al., “The effect of cyclosporine A on infection of susceptible cells by human immunodeficiency virus type 1,” Blood, vol. 72, No. 6, pp. 1904-1910, 8 pages (Dec. 1988). |
| Watashi, K. et al., “Chemical genetics approach to hepatitis C virus replication: cyclophilin as a target for anti-hepatitis C virus strategy,” Reviews in Medical Virology, vol. 17, Issue 4, pp. 245-252 (Jul./Aug. 2007). |
| Watashi, K. et al., “Cyclosporin A Suppresses Replication of Hepatitis C Virus Genome in Cultured Hepatocytes,” Hepatology, vol. 38, No. 5, pp. 1282-1288 (2003). |
| Zenke, G. et al., “Molecular mechanisms of Immunosuppression by Cyclosporins,” Annals of the New York Academy of Sciences, vol. 685, pp. 330-335 (Jun. 1993). |
| Thali, et al., “Functional Association of Cyclophilin A with HIV-1 Virions,” Nature, vol. 372, pp. 363-365 (1994). |
| Carry, et al., “Semisynthetic Di- and Tri-Functionalized Non-Immunosuppressive Cyclosporin A Derivatives as Potential Anti-HIV 1 Drugs,” Synlett, vol. 2, pp. 316-320 (2004). |
| Edman, P., “Preparation of Phenyl Thiohydantoins from Some Natural Amino Acids,” ACTA Chem. Scand., vol. 4, pp. 277-282 (1950). |
| Edman, P. and Begg, G., “A Protein Sequenator,” Eur. J. Biochem., vol. 1, pp. 80-91 (1967). |
| Xia, et al., “Inhibitory Effect of Cyclosporine A on Hepatitis B Virus Replication in vitro and its Possible Mechanisms,” Hepatobiliary Pancreat Dis Int, vol. 4, pp. 18-22 (2005). |
| Papageorgiou, C., et al., “Anti HIV-1 Activity of a Hydrophilic Cyclosporin Derivative with Improved Binding Affinity to Cyclophilin A”, Bioorganic & Medicinal Chemistry Letters, 6(1):23-26; including Corrected Figure, published in Bioorganic & Medicinal Chemistry Letters, Additions and Corrections, 6(4):497, 1996 (5 pages). |
| Number | Date | Country | |
|---|---|---|---|
| 20130210704 A1 | Aug 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61419326 | Dec 2010 | US | |
| 61655236 | Jun 2012 | US | |
| 61684928 | Aug 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2011/063295 | Dec 2011 | US |
| Child | 13840088 | US | |
| Parent | 13840088 | Mar 2013 | US |
| Child | 13840088 | US |
