Prodrug platform for delivery of amines, amides, and phenols

Abstract

The present invention discloses a prodrug platform that can be used to deliver pharmaceutically active amines, amides and phenols and its use in diagnosing, preventing and / or treating various diseases. Compared with the parent drug (e.g., gemcitabine), the prodrug of the present invention shows a significant overall safety improvement (therapeutic index (TI) improvement) in vivo.

Description

Technical Field

[0001] The present invention relates to a prodrug platform that can be used to deliver pharmaceutically active amines, amides and phenols and its use in diagnosing, preventing and / or treating various diseases. In addition, the present invention relates to a phosphorus-containing prodrug of gemcitabine and its use in treating cancer and viral infectious diseases. Background of the Invention

[0003] Oral administration of drugs such as gemcitabine may be limited by their poor oral bioavailability. Shipley LA. et al., "Metabolism and disposition of gemcitabine, and oncolytic deoxycytidine analog, in mice, mice, and dogs". Drug Metabolism & Disposition, 1992, 20(6): 849-55. In addition, when administered orally, drugs such as gemcitabine may be involved in causing adverse dose-limiting intestinal lesions characterized by moderate to significant loss of mucosal epithelium throughout the length of the intestine (atrophic enteropathy). Horton ND et al., "Toxicity of single-dose oral gemcitabine in mice," American Association for Cancer Research, Poster Presentation, Orlando, FL, March 27-31, 2004.

[0004] To improve the therapeutic window, prodrugs are often used to address the ADMET (absorption, distribution, metabolism, excretion and toxicity) properties and risks of preclinical drug candidates or drugs already on the market. Recent developments in the field of drug discovery have seen prodrugs being used to selectively deliver active drugs to desired tissues where biological targets are located, which can result in enhanced efficacy in desired tissues or reduced toxicity in undesired tissues.

[0005] As is well known in the art, the liver is an important target organ for many drugs and prodrugs, and there remains a need to develop liver-targeted drugs or prodrugs that can allow oral administration, pass intact through the intestinal tract without significant degradation, and deliver pharmaceutically active agents to diseased areas in the liver with acceptable safety and efficacy.

[0006] Gemcitabine hydrochloride (2',2'-difluoro-2'-deoxycytidine hydrochloride) is an antineoplastic agent. Selling, for treating various cancers, such as pancreatic cancer, breast cancer and non-small cell lung cancer (NSCLC), and being evaluated for ovarian cancer. In addition, gemcitabine can also be used to treat HCV and as a regulator of immune function (see U.S. Patent number 6,555,518). In another aspect, the combination of gemcitabine and decitabine (decitabine) shows that HIV-1 is effectively suppressed by a mechanism different from the drug mechanism currently used to treat HIV-1 infection (Antimicrobial Agents and Chemotherapy, 2012, 56, 1942-1948). Gemcitabine also shows antiviral activity for other viruses, such as poliovirus (ACS Infect. Dis., 2017, 3 (1), 45-53), human rhinovirus (Antiviral Research, 2017, 145, 6-13) and influenza A virus (J. Biol. Chem., 2012, 287 (42), 35324-35332).

[0007]

[0008] It is currently administered by intravenous infusion at a dose of approximately 1000 to 1250 mg / m2 over 30 minutes once a week for up to 7 weeks, followed by a week of rest after treatment.

[0009] Various prodrugs and sustained-release formulations of gemcitabine have been explored to find improvements. Examples of such prodrugs and sustained-release formulations can be found in the following documents: WO 04 / 0412303, "Gemcitabine Prodrugs, Pharmaceutical Compositions and Uses Thereof", Gallop et al.; WO 98 / 32762, "Gemcitabine Derivatives", Myhren, Finn et al.; WO 02 / 09768, "Therapeutic polyesters and polyamides", Uhrich, Kathryn E.; WO 02 / 76476, "Prodrugs of anticanceragents based on substituted aromatic acids", Greenwald, Richard m et al.; WO 02 / 65988, "Terminally branched polymeric lilüers and polymeric conjugates as prodrugs prodrug)”, Choe, Yun Hwang et al.

[0010] Gemcitabine amide derivatives have been described in the art as useful intermediates for the synthesis of gemcitabine (see, e.g., Britton et al., U.S. Pat. No. 5,420,266, and Grindey et al., U.S. Pat. No. 5,464,826), and may also be used as prodrug moieties for the administration of gemcitabine. See, e.g., Gallop et al., WO 04 / 041203.

[0011] It is reported that LY2334737 (amide prodrug of gemcitabine) is an oral administration agent (J. Med. Chem., 2009, 52, 6958-6961). More importantly, it has shown clinical benefits in Phase I human clinical trials (Invest. New Drugs, 2015, 33, 1206-1216, and references cited therein). However, hepatotoxicity has been observed in some patients and is believed to be related to the genetic polymorphism of the cytidine deaminase gene (rs818202).

[0012] Hepatocellular carcinoma (HCC) is a difficult-to-treat cancer. The combination of gemcitabine and other anticancer agents (e.g., doxorubicin and oxaliplatin) has shown promising results in improving overall survival (Am. J. Clin. Oncol., 2012, 35 (5), 418-423, and references cited therein), and overall survival remains the most important endpoint of HCC. In another report, gemcitabine and docetaxel showed significant anticancer activity in patients with advanced liver cancer (Cancer Research on Prevention and Treatment, 2012, 39 (11), 1369-1372). In addition, gemcitabine chemotherapy has also shown improvement in immune function in patients with advanced liver cancer (Journal of Hainan Medical University, 2016, 22(17), 2029-2031), which should make gemcitabine a potential partner in combination therapy with immuno-oncology products (such as checkpoint inhibitors PD-1, PD-L1, CTLA4 antibodies).

[0013] Liver-targeted prodrugs of gemcitabine will selectively deliver gemcitabine active metabolites to the liver and thus be useful in treating liver cancer. Therefore, there is a need to develop liver-targeted prodrugs of gemcitabine that can allow oral administration, pass intact through the intestinal tract without significant degradation, and deliver the pharmaceutically active agent to diseased areas in the liver with acceptable safety and efficacy.

[0014] Oral delivery of liver-targeted prodrugs of gemcitabine can also be used as a monotherapy or as part of a combination therapy to treat other solid tumors (such as lung cancer, pancreatic cancer, colon cancer, prostate cancer, breast cancer, etc.). For example, the combination of gemcitabine and albumin-bound paclitaxel as first-line therapy for patients with advanced pancreatic cancer showed significant therapeutic benefit, with a DCR of 77.8%; PFS and OS were 5 months and 8 months, respectively (Chinese Clinical Oncology, 2016, 21(7), 642-645). SUMMARY OF THE INVENTION

[0016] After a lot of work, we surprisingly found that cyclic phosphates / amides according to the present invention can deliver pharmaceutically active aromatic amines, amides and phenols (including gemcitabine) in vivo, which can achieve oral delivery of intravenous (iv) drugs or selectively deliver active drugs containing amines, amides or phenols to the liver. Such prodrugs can protect active drugs through the gastrointestinal tract (GI) and reach the liver, and once activated in the liver, the active drug can reach other tissues where the biological target is located.

[0017] These prodrugs pass through the gastrointestinal tract essentially intact and are converted to active drugs in the liver, thereby minimizing the formation of potentially undesirable metabolites in the gastrointestinal tract, liver, and plasma. For example, it was found that the N-phosphoramide prodrug of gemcitabine delivered dFdC to the liver after oral administration, but generated significantly lower levels of dFdU (in both the liver and plasma) than gemcitabine itself (intravenous or oral administration), which should be understood to be much less toxic than gemcitabine while maintaining appropriate efficacy and safety when administered orally or intravenously.

[0018] Therefore, the present invention aims to provide a new prodrug platform, to deliver the medicine containing aromatic amine, amide or phenol in vivo, it can be given orally or intravenously and has potential tissue selectivity (such as liver selectivity).When the prodrug is given orally, they pass the intestinal tract intactly and enter the portal vein blood flow, have less gastrointestinal toxicity and better bioavailability compared with the parent drug, and keep the effect of the parent drug at a lower dose simultaneously.Therefore, these prodrugs can not only deliver the active drug to the liver, but also can reach other organs, because the blood leaving the liver can bring the active drug into other organs.It is expected that these phosphorus-containing prodrugs can also be used for treating the disease of other organs / tissues except treating liver-related diseases.

[0019] According to a first aspect of the present invention, there is provided a compound having formula (I):

[0020]

[0021] Its stereoisomers, salts, hydrates, solvates or crystalline forms thereof;

[0022] in,

[0023] Ar is independently selected from the group consisting of optionally substituted aryl and heteroaryl;

[0024] X is independently selected from -NR'-, -O-, -S-, optionally substituted heterocyclyl and optionally substituted heteroaryl, preferably with the proviso that when X is -O-, Q must be an aromatic or heteroaromatic group;

[0025] R' is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and

[0026] Q is a group such that HXQ together represent a pharmaceutically active agent (drug) or a prodrug thereof, and preferably the pharmaceutically active agent is a compound having at least one free (aromatic) amine, amide or phenol group (HX-) which can be reacted to obtain a compound of formula (I), in particular to form the X moiety.

[0027] In a preferred embodiment of the first aspect of the invention, Ar is optionally substituted phenyl, preferably 3-chlorophenyl, or optionally substituted pyridyl, preferably 3-pyridyl and 4-pyridyl.

[0028] In another preferred embodiment of the first aspect of the invention, X is -NR'-, and R' is hydrogen, optionally substituted alkyl or optionally substituted aryl.

[0029] In another preferred embodiment of the first aspect of the present invention, X is -O- or -S-.

[0030] In another preferred embodiment of the first aspect of the invention, Q is a group such that HXQ together represent a pharmaceutically active agent for the diagnosis, prevention and / or treatment of a disease selected from the group consisting of cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases, and preferably HXQ together represent gemcitabine, tenofovir disoproxil, metformin, topotecan, entacapone, nebicapone, PARP inhibitors (such as rucaparib, pamiparib, talazoparib, olaprib, veliparib, HY-14478 and HY-102035), HIF-PHD inhibitors (such as roxadustat and HY-101023), pyridoxine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, dapoxetine, pioglitazone, sitagliptin, lenvatinib, TLR7 agonists (such as HY-103039 and GS-9620), STING agonists (such as HY-19711, HY-103665 and HY-123943), EP4 antagonists (such as HY-42794, grapiprant, HY-111539, HY-112152, HY-10413, HY-50901, HY-10797 and HY-103088) or apatinib.

[0031] In another preferred embodiment of the first aspect of the present invention, the compound according to formula (I) is selected from the group consisting of:

[0032] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0033] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0034] 4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;

[0035] 4-((4-(3-bromophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;

[0036] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((4-(3-fluorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0037] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((4-(4-methoxyphenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one; and

[0038] ((2R,3R,5R)-5-(4-((4-(3-chloro-4-methoxyphenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl pivalate;

[0039] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate;

[0040] ((2R,3R,5R)-3-acetoxy-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluorotetrahydrofuran-2-yl)methyl acetate;

[0041] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((propionyloxy)methyl)tetrahydrofuran-3-yl propanoate;

[0042] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((2-methoxyacetoxy)methyl)tetrahydrofuran-3-yl 2-methoxyacetate;

[0043] 1-(4-argio-2-oxo-1,3,2-dioxaphosphinane-2-yl)-5-fluoropyrimidine-2,4(1H,3H)-dione;

[0044] 4-((4-argio-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2RS,3R,4SR,5RS)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;

[0045] Valine ((2RS,3SR,4R,5RS)-5-(4-((4-argio-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl ester;

[0046] (Z)-3-((1-(4-argio-2-oxo-1,3,2-dioxaphosphinane-2-yl)-5-(tert-butyl)-1H-imidazol-4-yl)methylene)-6-((Z)-benzylidene)piperazine-2,5-dione;

[0047] (Z)-1-(4-argio-2-oxo-1,3,2-dioxaphosphinane-2-yl)-3-((Z)-benzylidene)-6-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione;

[0048] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((pivaloyloxy)methyl)tetrahydrofuran-3-yl pivalate; and

[0049] Isopropyl (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isopropoxycarbonyloxy)methyl)tetrahydrofuran-3-yl carbonate,

[0050] A compound selected from the group consisting of:

[0051]

[0052]

[0053]

[0054]

[0055] as well as,

[0056]

[0057]

[0058]

[0059] wherein Ar is as defined herein.

[0060] Another aspect of the present invention provides a method for diagnosing, preventing and / or treating a disease in a mammal, the method comprising administering an effective amount of a compound according to formula (I) to a mammal in need thereof. Preferably, the disease is selected from the group consisting of cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases, and the compound is administered orally or intravenously, and preferably the mammal is selected from the group consisting of humans, mice, rats, rabbits, hamsters, guinea pigs, monkeys, dogs, cats, pigs, sheep, goats, cattle and horses. Most preferably, the pharmaceutically active agent is gemcitabine, tenofovir disoproxil, metformin, topotecan, entacapone, nitrobenzylcapone, PARP inhibitors (such as rucaparib, pamiparib, talazopanib, olaparib, veliparib, HY-14478 and HY-102035), HIF-PHD inhibitors (such as roxadustat and HY-101023), pioglitazone, sitagliptin, lenvatinib, TLR7 stimulant HY-103039 and GS-9620), STING agonists (such as HY-19711, HY-103665 and HY-123943), EP4 antagonists (such as HY-42794, glapiron, HY-111539, HY-112152, HY-10413, HY-50901, HY-10797 and HY-103088), or apatinib.

[0061] The present invention also provides the use of a compound according to formula (I) in the preparation of a medicament for diagnosing, preventing and / or treating a disease in a mammal. Preferably, the disease is selected from the group consisting of cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases, and the compound is administered orally or intravenously, and preferably the mammal is selected from the group consisting of humans, mice, rats, rabbits, hamsters, guinea pigs, monkeys, dogs, cats, pigs, sheep, goats, cattle and horses. Most preferably, the pharmaceutically active agent is gemcitabine, tenofovir disoproxil, metformin, topotecan, entacapone, nitrobenzylcapone, PARP inhibitors (such as rucaparib, pamiparib, talazopanib, olaparib, veliparib, HY-14478 and HY-102035), HIF-PHD inhibitors (such as roxadustat and HY-101023), pioglitazone, sitagliptin, lenvatinib, TLR7 stimulant HY-103039 and GS-9620), STING agonists (such as HY-19711, HY-103665 and HY-123943), EP4 antagonists (such as HY-42794, glapiron, HY-111539, HY-112152, HY-10413, HY-50901, HY-10797 and HY-103088), or apatinib.

[0062] The present invention further provides the use of a compound according to formula (I) in the preparation of a medicament for diagnosing, preventing and / or treating a disease in a mammal. Preferably, the disease is selected from the group consisting of cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases, and the compound is administered orally or intravenously, and preferably the mammal is selected from the group consisting of humans, mice, rats, rabbits, hamsters, guinea pigs, monkeys, dogs, cats, pigs, sheep, goats, cattle and horses. Most preferably, the pharmaceutically active agent is gemcitabine, tenofovir disoproxil, metformin, topotecan, entacapone, nitrobenzylcapone, PARP inhibitors (such as rucaparib, pamiparib, talazopanib, olaparib, veliparib, HY-14478 and HY-102035), HIF-PHD inhibitors (such as roxadustat and HY-101023), pioglitazone, sitagliptin, lenvatinib, TLR7 stimulant HY-103039 and GS-9620), STING agonists (such as HY-19711, HY-103665 and HY-123943), EP4 antagonists (such as HY-42794, glapiron, HY-111539, HY-112152, HY-10413, HY-50901, HY-10797 and HY-103088), or apatinib.

[0063] The present invention further provides a compound according to formula (I) for diagnosing, preventing and / or treating a disease in a mammal. Preferably, the disease is selected from the group consisting of cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases, and the compound is administered orally or intravenously, and preferably the mammal is selected from the group consisting of humans, mice, rats, rabbits, hamsters, guinea pigs, monkeys, dogs, cats, pigs, sheep, goats, cattle and horses. Most preferably, the pharmaceutically active agent is gemcitabine, tenofovir disoproxil, metformin, topotecan, entacapone, nitrobenzylcapone, PARP inhibitors (such as rucaparib, pamiparib, talazopanib, olaparib, veliparib, HY-14478 and HY-102035), HIF-PHD inhibitors (such as roxadustat and HY-101023), pioglitazone, sitagliptin, lenvatinib, TLR7 stimulant HY-103039 and GS-9620), STING agonists (such as HY-19711, HY-103665 and HY-123943), EP4 antagonists (such as HY-42794, glapiron, HY-111539, HY-112152, HY-10413, HY-50901, HY-10797 and HY-103088), or apatinib.

[0064] The present invention further provides a pharmaceutical composition comprising a compound according to formula (I) in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0065] Another aspect of the present invention provides a method for preparing a pharmaceutical composition, the method comprising the step of combining a compound according to formula (I) with a pharmaceutically acceptable carrier, diluent or excipient.

[0066] In addition, the present invention also provides a method for preparing a compound of formula (I), which comprises coupling a compound of formula HXQ with a compound of formula Y in the presence of a base and a catalyst as necessary to obtain a compound of formula (I).

[0067]

[0068] wherein Ar, X and Q are as defined herein.

[0069] Alternatively, the compound of formula (I) can be prepared by a method comprising reacting a compound of formula HXQ with phosphoryl trichloride in a solvent (e.g., pyridine) in the presence of a base and a catalyst as necessary, followed by adding a 1,3-propanediol derivative of formula Z to react to obtain a compound of formula (I),

[0070]

[0071] Another aspect of the invention provides a method for delivering a pharmaceutically active agent to a mammal, preferably the liver of the mammal, wherein the pharmaceutically active agent is as defined herein, and is preferably a compound having at least one free (aromatic) amine, amide or phenolic group; and the method comprises the steps of preparing a compound according to formula (I) herein, and administering the compound according to any one of claims 1 to 6 to a mammal in need thereof.

[0072] Preferably, the pharmaceutically active agent as defined above is suitable for treating a liver disease, is directed against a liver disease or is targeted to the liver.

[0073] Based on the platform technology of the present invention, we have found various novel phosphoramide derivatives of gemcitabine as prodrugs. These prodrugs pass through the gastrointestinal (GI) tract intact and are converted into gemcitabine in the liver, thereby minimizing the formation of deoxydifluorouridine (dFdU) (the main metabolite of gemcitabine) in the gastrointestinal tract, liver and blood plasma. Compared with gemcitabine itself (intravenous or oral administration), the significantly lower levels of dFdU (in both liver and blood plasma) observed using these phosphorus-containing prodrugs should be understood as being much less toxic than gemcitabine, while maintaining appropriate efficacy and safety when administered orally or intravenously.

[0074] Therefore, the present invention aims to provide the novel phosphorus-containing prodrug of gemcitabine, which can be given orally or intravenously and selectively gemcitabine active metabolites are delivered to the liver, while minimizing the formation of dFdU. When the prodrug is given orally, they pass the intestinal tract intactly and enter the portal vein blood flow, have less gastrointestinal toxicity and better bioavailability compared with the parent drug (i.e. gemcitabine, also known as dFdC), and keep the effect of the parent drug at a lower dose. Therefore, these prodrugs can not only deliver gemcitabine to the liver, but also can reach other organs, because the blood leaving the liver can bring gemcitabine into other organs. It is expected that, except for treating liver cancer, these phosphorus-containing gemcitabine prodrugs can also be used for treating the cancer of other organs (such as pancreas, lung, prostate etc.).

[0075] According to a preferred aspect of the present invention, there is provided a compound of formula (I):

[0076]

[0077] Its stereoisomers, salts, hydrates, solvates or crystalline forms thereof;

[0078] in,

[0079] Ar is independently selected from the group consisting of optionally substituted aryl and heteroaryl;

[0080] R 3 independently selected from the group consisting of: hydrogen, acyl, and alkoxycarbonyl;

[0081] R 5 are independently selected from the group consisting of hydrogen, acyl, alkoxycarbonyl, and -PO(OAr')(NH-CR 1 R 2 -CO 2 R 4 ),

[0082] or R 3 and R 5 Together they form a cyclic phosphate group as shown below:

[0083]

[0084] in

[0085] R 1 and R 2 are independently selected from the group consisting of: H, alkyl and alkylaryl, or R 1 and R 2 Together they form an alkylene chain to provide a ring system together with the C atoms to which they are attached;

[0086] R 4 is selected from the group consisting of: alkyl, aryl, and alkylaryl; and

[0087] Ar' is a monocyclic aromatic ring moiety or a fused bicyclic aromatic ring moiety, either of which is carbocyclic or heterocyclic and is optionally substituted.

[0088] In a preferred embodiment of the preferred aspects of the invention, Ar is optionally substituted phenyl, or optionally substituted pyridyl, preferably 3-pyridyl and 4-pyridyl.

[0089] In another preferred embodiment of the preferred aspects of the present invention, R 3 is hydrogen or acyl, preferably isobutyryl.

[0090] In another preferred embodiment of the preferred aspects of the present invention, R 5 is -PO(OAr')(NH-CR 1 R 2 -CO 2 R 4 ), R 1 and R 2 Independently selected from the group consisting of: H, C 1-6 Alkyl, C 1-3 Alkyl C 5-7 Aryl groups, or when they are taken together to form an alkylene chain, together with the C atoms to which they are attached, provide a C 3-8 Carbocyclic aliphatic ring; R 4 Selected from the group consisting of: C 1-16 Primary or secondary alkyl, C 5-7 Carbocyclic aromatic groups and C 1-6 Alkyl C 5-11 Aryl, preferably methyl, ethyl or benzyl; and Ar' is optionally substituted phenyl, preferably phenyl, pCF 3 C 6 H 4 -, pFC 6 H 4 -, pNO 2 C 6 H 4 -、pClC 6 H 4 - and oClC 6 H 4 -.

[0091] In another preferred embodiment of the preferred aspects of the present invention, R 1 and R 2 are independently selected from the group consisting of: H, methyl, benzyl and -CH 2 CH(CH3 ) 2 , or R 1 and R 2 Together with the C atoms to which they are attached, they provide C 5-6 ring, preferably a pentyl ring.

[0092] In another preferred embodiment of the preferred aspects of the present invention, R 5 is hydrogen, acyl (preferably isobutyryl) or -PO(OPh)(NH-CR 1 R 2 -CO 2 R 4 ), wherein Ph represents phenyl, R 1 Yes H, R 2 is a methyl group, and R 4 It is ethyl.

[0093] In another preferred embodiment of the preferred aspects of the present invention, Ar is optionally substituted phenyl or optionally substituted 3-pyridyl or 4-pyridyl, R 3 is hydrogen or isobutyryl, and R 5 is hydrogen or isobutyryl, or R 3 and R 5 Together they form a cyclic group as shown below:

[0094]

[0095] In another preferred embodiment of the preferred aspects of the invention, Ar is 3-chlorophenyl, 3-pyridyl or 4-pyridyl.

[0096] In another preferred embodiment of the preferred aspects of the present invention, the compound according to formula (A) is selected from the group consisting of:

[0097] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0098] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0099] 4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;

[0100] 4-((4-(3-bromophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;

[0101] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((4-(3-fluorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0102] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((4-(4-methoxyphenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one; and

[0103] ((2R,3R,5R)-5-(4-((4-(3-chloro-4-methoxyphenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl pivalate;

[0104] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate;

[0105] ((2R,3R,5R)-3-acetoxy-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluorotetrahydrofuran-2-yl)methyl acetate;

[0106] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((propionyloxy)methyl)tetrahydrofuran-3-yl propanoate;

[0107] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((2-methoxyacetoxy)methyl)tetrahydrofuran-3-yl 2-methoxyacetate;

[0108] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((pivaloyloxy)methyl)tetrahydrofuran-3-yl pivalate; and

[0109] Isopropyl (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinan-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isopropoxycarbonyloxy)methyl)tetrahydrofuran-3-yl carbonate.

[0110] Another aspect of the invention provides a method for treating a susceptible tumor in a mammal, the method comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to formula (A). Preferably, the susceptible tumor is selected from the group consisting of: T-cell lymphoma, soft tissue sarcoma, pancreatic cancer, breast cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, bladder cancer and hepatocellular carcinoma (HCC).

[0111] The present invention also provides the use of a compound according to formula (A) in the preparation of a medicament for treating a sensitive tumor. Preferably, the sensitive tumor is selected from the group consisting of: T-cell lymphoma, soft tissue sarcoma, pancreatic cancer, breast cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, bladder cancer and hepatocellular carcinoma (HCC).

[0112] The present invention further provides a compound according to formula (A) for use in treating a sensitive tumor. Preferably, the sensitive tumor is selected from the group consisting of: T-cell lymphoma, soft tissue sarcoma, pancreatic cancer, breast cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, bladder cancer and hepatocellular carcinoma (HCC).

[0113] The present invention further provides a pharmaceutical composition comprising a compound according to formula (A) in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0114] Another aspect of the present invention provides a method for preparing a pharmaceutical composition, the method comprising the step of combining a compound according to formula (A) with a pharmaceutically acceptable carrier, diluent or excipient.

[0115] In addition, the present invention also provides a method for preparing a compound of formula (A), the method comprising the following steps:

[0116] (i) protecting the 5'-position (5'-OH) of gemcitabine with a suitable protecting group to obtain a compound of formula (IV), wherein PG represents a suitable protecting group for the -OH group;

[0117]

[0118] (ii) modifying a compound of formula (IV) (e.g. by reacting a compound of formula (IV) with a compound of formula (V)) to obtain a compound of formula (III) (i.e. preparing an N-prodrug of gemcitabine), wherein Ar is as defined herein;

[0119]

[0120]

[0121] (iii) removing the protecting group (PG) at the 5'-position (5'-OH) of gemcitabine to obtain a compound of formula (II);

[0122]

[0123] And, optionally further

[0124] (iv) modifying the compound of formula (II) at the 3'-position (3'-OH) and / or the 5'-position (5'-OH) to obtain a compound of formula (A) wherein Ar, R 3 and R 5 As defined in this article

[0125]

[0126] Furthermore, the present invention provides a method for treating sensitive tumors in mammals, said method comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to formula (A) in combination with at least one (preferably one or two) oncolytic and / or immuno-oncological agents.

[0127] Preferably, the oncolytic agent is selected from the group consisting of: 5-fluorouracil, chloroquine, S-1 (combination drug tegafur / gimeracil / oteracil, Liu TW et al. Lancet Oncol. 2016, 17: 12-4), vinorelbine, sorafenib, elpamotide, capecitabine, carboplatin, cisplatin, oxaliplatin, Aurora kinase inhibitors (e.g. MSC1992371A, Investigational New Drugs, 2014, 32(1), 94-103), EGFR inhibitors (e.g., erlotinib, gefitinib), tyrosine kinase inhibitors (e.g., lapatinib, vandetanib), topoisomerase inhibitors (e.g., irinotecan, exatecan, indotecan (LMP400) and indimitecan (LMP776), albumin-bound paclitaxel (nab-paclitaxel), paclitaxel, docetaxel, pemetrexed, curcumin, and radiation therapy.

[0128] Preferably, the immuno-oncology agent is selected from the group consisting of: checkpoint inhibitors, PD-1, PD-L1, CTLA-4 and VEGF-A antibodies.

[0129] Another aspect of the present invention provides a method of treating a susceptible viral infection in a mammal, the method comprising administering to a mammal in need thereof a therapeutically effective amount of a compound according to formula (A).

[0130] Another aspect of the present invention provides a method for diagnosing, preventing and / or treating a disease in a mammal, the method comprising administering to a mammal in need thereof an effective amount of a compound according to the present invention in combination with other therapeutic agents, such as immune checkpoint inhibitors, microtubule inhibitors, PARP inhibitors, BTK inhibitors, EGFR TK inhibitors, cytotoxic platinum agents. Preferably, the disease is selected from the group consisting of: cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] Figure 1 Plasma exposure of Compound 4 and gemcitabine (also known as dFdC) following intravenous (equivalent dose of 15 mg / kg gemcitabine) or oral (80 mg / kg) administration of Compound 4;

[0132] Figure 2 . Liver concentrations of dFdC and dFdU at 2 hours after administration of compound 4 (intravenous, equivalent to 15 mg / kg gemcitabine; oral, 80 mg / kg) and gemcitabine (intravenous, 15 mg / kg);

[0133] Figure 3 . Tissue distribution of compound 27 relative to dFdC after administration of compound 27 and dFdC ("mpk" in the figure represents mg / kg);

[0134] Figure 4 . Tissue distribution of the metabolite dFdU after administration of compound 27 and dFdC;

[0135] Figure 5 . Tissue distribution of compound 28 relative to dFdC after administration of compound 28 and dFdC;

[0136] Figure 6 . Tissue distribution of the metabolite dFdU after administration of compound 28 and dFdC;

[0137] Figure 7 . Liver levels of dFdC and dFdU in mice after intravenous administration of dFdC (3 mg / kg) or oral administration of compound 29 at 30 mg / kg (A: liver dFdC and dFdU levels 2 hours after intravenous administration of dFdC (3 mg / kg) or oral administration of compound 29 (30 mg / kg) in mice (n=3); B: therapeutic index (TI) of liver dFdC level relative to liver dFdU level);

[0138] Figure 8 . Plasma concentration of topotecan in mice: Topotecan administered intravenously at 2 mg / kg relative to compound 10 administered intravenously at 3 mg / kg in mice (A: plasma topotecan exposure after intravenous administration of 2 mg / kg topotecan to mice (n=3); B: plasma compound 10 exposure after intravenous administration of 3 mg / kg compound 10 (equivalent to 1 mol of topotecan) to mice (n=3);

[0139] Fig. 9. Plasma concentration of topotecan in mice: Oral administration of topotecan at 10 mg / kg relative to intravenous administration of compound 10 at 3 mg / kg in mice (A: plasma topotecan exposure after oral administration of 10 mg / kg topotecan to mice (n=3); B: plasma compound 10 exposure after intravenous administration of 3 mg / kg compound 10 (equivalent to 1 mol of topotecan) to mice (n=3));

[0140] Fig.10 . tissue distribution of compound 10 relative to topotecan after administration of compound 10 and topotecan; and

[0141] Fig.11 . Tissue distribution of the metabolite topotecan after administration of compound 10 and topotecan. DETAILED DESCRIPTION OF THE INVENTION

[0143] definition

[0144] The terms used herein are to be interpreted as having their ordinary and typical meanings to those of ordinary skill in the art. However, the following terms are given specific definitions as defined below.

[0145] The term "alkyl" means a branched or straight chain, cyclic or acyclic, saturated or unsaturated (eg, alkenyl or alkynyl) hydrocarbon group. When cyclic, the alkylene group is preferably C 3 To C 12 More preferably C 5 To C 10 More preferably C 5 To C 7 When cyclic, the alkyl group is preferably C 1 To C 16 More preferably C 1 To C 6 Saturated alkyl groups such as methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0146] The term "aryl" or "aromatic group" refers to an aromatic group containing 5 to 14 ring atoms, for example phenyl or naphthyl.

[0147] The term "heteroaryl" or "heteroaromatic group" refers to an aryl group containing one, two, three or four (preferably one) heteroatoms independently selected from the group consisting of O, N and S. Examples of such heteroaryl groups include pyridyl, pyrrolyl, furanyl and thienyl.

[0148] The term "heterocyclyl" refers to a cyclic group containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, and may be selected from the group consisting of pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidinyl The number of ring atoms in the heterocyclic group can be in the range of 3 to 20, such as 4 to 6, 5 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0149] Aryl, heteroaryl and heterocyclic groups may be substituted or unsubstituted. When substituted, there are usually one to three substituents, preferably one substituent. Substituents may include halogen atoms (which means F, Cl, Br and I atoms) and halomethyl (such as CF 3 and CCl 3 ); oxygen-containing groups such as oxo, hydroxyl, carboxyl, carboxyl C 1-6 Alkyl, alkoxy, alkanoyl, alkanoyloxy, aryloxy, aroyl and aroyloxy groups; nitrogen-containing groups such as amino, C 1-6 alkylamino, cyano, azido and nitro groups; sulfur-containing groups such as thio, C 1-6 Alkylthio, sulfonyl and sulfoxide; alkyl as defined above, which may itself be substituted; and aryl as defined above, which may itself be substituted, such as phenyl and substituted phenyl. The substituents on the alkyl and aryl groups are as defined above.

[0150] The term "acyl" refers to a group RCO-, wherein R represents an alkyl group, which is derived from an organic acid, usually by removing the hydroxyl group from all the acid groups. Preferred examples of acyl groups include C 1-6 Acyl groups, such as formyl, acetyl, propionyl, butyryl (eg isobutyryl).

[0151] The terms "alkoxy" and "aryloxy" refer to alkyl-O- (e.g., wherein the alkyl group is C 1 To C 16 Alkyl, preferably C 1 To C 6alkyl) and aryl-O- (e.g. wherein aryl is a 5 to 14 membered aromatic monocyclic moiety or a bifused ring moiety, optionally containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, preferably aryl is phenyl).

[0152] The term "alkoxycarbonyl" refers to an alkoxy-C(O)-, preferably C 1-16 Alkoxycarbonyl, and more preferably C 1-6 Alkoxycarbonyl groups such as methylcarbonyl, ethylcarbonyl, propylcarbonyl and butylcarbonyl.

[0153] The terms "alkanoyl" and "aroyl" refer to alkyl-CO- (e.g., wherein the alkyl group is C 1 To C 16 Alkyl, preferably C 1 To C 6 alkyl) and aryl-CO- (e.g. wherein aryl is a 5 to 14 membered aromatic monocyclic moiety or a bifused ring moiety, optionally containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, preferably aryl is phenyl).

[0154] The terms "alkanoyloxy" and "aroyloxy" refer to alkyl-CO-O (e.g., wherein the alkyl group is C 1 To C 16 Alkyl, preferably C 1 To C 6 alkyl) and aryl-CO-O (e.g. wherein aryl is a 5 to 14 membered single aromatic ring system or a difused aromatic ring system, optionally containing 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, preferably aryl is phenyl).

[0155] The term "free" in the expression "the pharmaceutically active agent is a compound having at least one free (aromatic) amine, amide or phenolic moiety" means herein that the pharmaceutically active agent has an (aromatic) amine, amide or phenolic group, which can be used to carry out a suitable reaction or reactions to obtain the compound of formula (I) according to the present invention.

[0156] Compounds of formula (I)

[0157] One aspect of the present invention relates to a compound represented by formula (I), a stereoisomer, a pharmaceutically acceptable salt, a hydrate, a solvate or a crystalline form thereof,

[0158]

[0159] in,

[0160] Ar is independently selected from the group consisting of optionally substituted aryl and heteroaryl;

[0161] X is independently selected from -NR'-, -O-, -S-, optionally substituted heterocyclyl and optionally substituted heteroaryl; preferably, provided that when X is -O-, Q must be an aromatic or heteroaromatic group;

[0162] R' is independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted aryl, and optionally substituted heteroaryl; and

[0163] Q is a group such that HXQ together represent a pharmaceutically active agent (drug) or a prodrug thereof, and preferably the pharmaceutically active agent is a compound having at least one free (aromatic) amine, amide or phenolic group.

[0164] Preferably, the group Ar includes substituted or unsubstituted aryl, wherein the term "aryl" and the possible substitution of the group are as defined herein. Preferably, Ar is substituted or unsubstituted phenyl. Particularly preferred substituents are electron withdrawing groups, such as halogen (preferably chlorine or fluorine), trihalomethyl (preferably trifluoromethyl), cyano and nitro. For example, Ar can be phenyl, 3-chloro-phenyl, 3,5-dichloro-phenyl, p-trifluoromethyl-phenyl, p-cyano-phenyl or p-nitro-phenyl. When Ar is heteroaryl, preferably it is an optionally substituted pyridyl.

[0165] Suitably, Ar is a 5 to 14 membered aromatic ring moiety.One or both rings may include 1, 2, 3 or 4 heteroatoms, preferably 1 heteroatom, independently selected from O, S and N.

[0166] Preferably, Ar is a carbon monocyclic aromatic ring moiety. More preferably, Ar is C 6 A monocyclic aromatic ring moiety, ie an optionally substituted phenyl group.

[0167] One, two, three or four substituents, which may be the same or different, may be present on Ar and are selected from the group consisting of: halogen, which may be -F, -CI, -Br or -I; -NO; -NH 2 ; optionally substituted -C 1-3 Alkyl; optionally substituted -C 1-3 Alkoxy, preferably methoxy (-OCH 3 ); optionally substituted -SC 1-3 Alkyl; -CN; optionally substituted -COC 1-3 alkyl; and optionally substituted -CO 2 C 1-3 The optional substituents are one or more (up to six, preferably three) members selected from the group consisting of halogen (which may be F, CI, Br and I) and NO 2 Preferred substituents on Ar include F, Cl, CF3 and NO 2 .

[0168] Most preferably, Ar is optionally substituted phenyl, preferably 3-chlorophenyl, or optionally substituted pyridyl, preferably 3-pyridyl and 4-pyridyl.

[0169] Preferably, the group X is -NR'-, and R' is hydrogen, optionally substituted alkyl or optionally substituted aryl. In another preferred embodiment, X is -O- or -S-.

[0170] As long as HXQ together represent a pharmaceutically active agent or a prodrug thereof, there is no particular restriction on the nature and identity of the chemical group Q. However, for forming compounds of formula (I), it is preferred that the pharmaceutically active agent is a compound having at least one free (aromatic) amine, amide or phenol.

[0171] Most preferably, the compound of formula (I) is selected from the group consisting of:

[0172] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0173] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0174] 4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;

[0175] 4-((4-(3-bromophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;

[0176] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((4-(3-fluorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0177] 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((4-(4-methoxyphenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one;

[0178] ((2R,3R,5R)-5-(4-((4-(3-chloro-4-methoxyphenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl pivalate;

[0179] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate;

[0180] ((2R,3R,5R)-3-acetoxy-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluorotetrahydrofuran-2-yl)methyl acetate;

[0181] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((propionyloxy)methyl)tetrahydrofuran-3-yl propanoate;

[0182] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((2-methoxyacetoxy)methyl)tetrahydrofuran-3-yl 2-methoxyacetate;

[0183] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((pivaloyloxy)methyl)tetrahydrofuran-3-yl pivalate; and

[0184] Isopropyl (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isopropoxycarbonyloxy)methyl)tetrahydrofuran-3-yl carbonate,

[0185] A compound selected from the group consisting of:

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] as well as,

[0192]

[0193] wherein Ar is as defined herein.

[0194] One aspect of the present invention relates to a compound represented by formula (A), a stereoisomer, a pharmaceutically acceptable salt, a hydrate, a solvate or a crystalline form thereof,

[0195]

[0196] in,

[0197] Ar is independently selected from the group consisting of optionally substituted aryl and heteroaryl;

[0198] R 3 independently selected from the group consisting of: hydrogen, acyl, and alkoxycarbonyl;

[0199] R 5 are independently selected from the group consisting of hydrogen, acyl, alkoxycarbonyl, and -PO(OAr')(NH-CR 1 R 2 -CO 2 R 4 ),

[0200] or R 3 and R 5 Together they form a cyclic phosphate group as shown below:

[0201]

[0202] in

[0203] R 1 and R 2 are independently selected from the group consisting of: H, alkyl and alkylaryl, or R 1 and R 2 Together they form an alkylene chain to provide a ring system together with the C atoms to which they are attached;

[0204] R 4 is selected from the group consisting of: alkyl, aryl, and alkylaryl; and

[0205] Ar' is a monocyclic aromatic ring moiety or a fused bicyclic aromatic ring moiety, either of which is carbocyclic or heterocyclic and is optionally substituted.

[0206] Preferably, the group Ar' includes substituted or unsubstituted aryl, wherein the term "aryl" and the possible substitutions of the group are as defined herein. Preferably, Ar' is substituted or unsubstituted phenyl. Particularly preferred substituents are electron withdrawing groups, such as halogen (preferably chlorine or fluorine), trihalomethyl (preferably trifluoromethyl), cyano and nitro. For example, Ar' can be phenyl, 3-chloro-phenyl, 3,5-dichloro-phenyl, p-trifluoromethyl-phenyl, p-cyano-phenyl or p-nitro-phenyl. When Ar' is heteroaryl, preferably it is an optionally substituted pyridyl.

[0207] Suitably, Ar' is a 5 to 14 membered aromatic ring moiety.One or both rings may include 1, 2, 3 or 4 heteroatoms, preferably 1 heteroatom, independently selected from O, S and N.

[0208] Preferably, Ar' is a carbon monocyclic aromatic ring moiety. More preferably, Ar' is C 6 A monocyclic aromatic ring moiety, ie an optionally substituted phenyl group.

[0209] One, two, three or four substituents, which may be the same or different, may be present on Ar' and are selected from the group consisting of: halogen, which may be -F, -CI, -Br or -I; -NO 2 ;-NH 2 ; optionally substituted -C 1-3 Alkyl; optionally substituted -C 1-3 Alkoxy, preferably methoxy (-OCH 3 ); optionally substituted -SC 1-3 Alkyl; -CN; optionally substituted -COC 1-3 alkyl; and optionally substituted -CO 2 C 1-3 The optional substituents are one or more (up to six, preferably three) members selected from the group consisting of halogen (which may be F, CI, Br and I) and NO2 Preferred substituents on Ar' include F, Cl, CF 3 and NO 2 .

[0210] Substituents may be in any position of the ring moiety. When the ring moiety is phenyl, a single substituent at the 2 position (ortho position) or the 4 position (para position) is preferred. When Ar' is phenyl, a single substituent at the 4 position is more preferred.

[0211] Preferably, R 4 It is C 1-16 Primary or secondary alkyl, C 5-7 Carbocyclic aromatic or C 1-6 Alkyl C 5-11 More preferably, R 4 It is C 1-10 Alkyl (most preferably C 1-6 Alkyl), phenyl or C 1-3 Alkyl C 5-7 Preferably, R 4 is unsubstituted.

[0212] Preferably, R 4 It is a methyl group (-CH 3 ), ethyl (-C 2 H 5 ), n-propyl or isopropyl (-C 3 H 7 ), n-butyl or isobutyl (-C 4 H 9 ) or benzyl (-CH 2 C 6 H 5 ).

[0213] Suitably, R 1 and R 2 Each independently selected from the group consisting of: H, C 1-6 Primary, secondary or tertiary alkyl, C 1-3 Alkyl C 5-7 Aryl groups, or when they are taken together to form an alkylene chain, together with the C atoms to which they are attached, provide a C 3-8 Carbocyclic aliphatic ring.

[0214] Alternatively, preferably, R 1 and R 2 are independently H, methyl (-CH 3 ), sec-butyl (-CH 2 -CH-(CH 3 ) 2 ), benzyl (-CH 2 C 6 H 5), or together with the C atoms to which they are attached provide C 5-6 ring.

[0215] Preferably, the group Ar includes substituted or unsubstituted aryl, wherein the term "aryl" and the possible substitution of the group are as defined herein. Preferably, Ar is substituted or unsubstituted phenyl. Particularly preferred substituents are electron withdrawing groups, such as halogen (preferably chlorine or fluorine), trihalomethyl (preferably trifluoromethyl), cyano and nitro. For example, Ar can be phenyl, 3-chloro-phenyl, 3,5-dichloro-phenyl, p-trifluoromethyl-phenyl, p-cyano-phenyl or p-nitro-phenyl. When Ar is heteroaryl, preferably it is an optionally substituted pyridyl, such as 3-pyridyl or 4-pyridyl.

[0216] Preferably, R 3 It is hydrogen, C 1-6 Alkyl, C 1-6 Acyl and C 1-6 Alkoxycarbonyl, more preferably hydrogen or isobutyryl.

[0217] Synthesis of the compounds of the present invention

[0218] Compounds in the present invention can be prepared by the methods described herein and the related open literature procedures used by those skilled in the art. All starting materials and reagents are well known in the art and / or easily commercially available, or prepared by the methods described herein. For example, the method for preparing gemcitabine (2', 2'-difluoro-2'-deoxycytidine) is disclosed in U.S. Patent No. 6,555,518. It should be understood that the following methods are provided only for illustrative purposes, and should not limit the present invention defined by the claims. The protection and deprotection in the scheme can be carried out according to procedures well known in the art (e.g., "Protecting Groups in Organic Synthesis", 3rd edition, Wiley, 1999) at each stage of synthesis.

[0219] In general, the synthesis of compounds of formula (I) can be accomplished as described in the following sections.

[0220]

[0221] Scheme 1. Direct coupling of active drug with phosphorus-containing prodrug.

[0222] If possible, it is preferred to directly couple the active drug HXQ with a suitable key intermediate of the desired phosphorus-containing prodrug moiety in the presence of a suitable base to obtain compounds of formula (I), as described in Scheme 1. The phosphorylation reagent Y is readily prepared using literature procedures, and the reaction will produce the final prodrug in a single step, and is generally preferred as the last step.

[0223] In particular, the synthesis of compounds of formula (I) wherein HXQ is gemcitabine comprises the following general steps:

[0224] (1) Protection of the 5'-position of gemcitabine, which can be performed according to procedures known in the art (e.g., "Protecting Groups in Organic Synthesis", 3rd edition, Wiley, 1999). For example, selective protection of 5'-OH can be achieved using TBDPSCl under appropriate conditions;

[0225] (2) Preparation of 4-amino prodrug of gemcitabine;

[0226] (3) removal of protecting groups; and

[0227] (4) Modification of existing N-prodrugs of gemcitabine.

[0228] The following is an example of a method for preparing a specific compound of the present invention (i.e., 4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one).

[0229] (1) Protection of the 5'-position of gemcitabine

[0230] Selective protection of the 3',5'-OH group of a nucleoside can generally be achieved under appropriate reaction conditions. For example, treatment of gemcitabine with tert-butyldimethylsilyl chloride (TBSCl) in the presence of a suitable base and appropriate reaction conditions yields a 3',5'-OTBS derivative of gemcitabine, as shown below.

[0231]

[0232] (2) Preparation of N-prodrug of gemcitabine

[0233] Prodrugs can be introduced at different stages of the synthesis. Most commonly, these prodrugs are introduced late in the synthesis due to the instability of various prodrugs, but prodrugs can also be introduced early in the synthesis due to other considerations. For example, treatment of 3',5'-TBS-protected gemcitabine with p-nitrophenol 1,3-propanediol cyclic phosphate (Q) in the presence of a suitable base (such as sodium hydride) produces an N-phosphate prodrug derivative, as shown below. Alternatively, the racemic form of compound Q can also be used to produce compound 3 (as a diastereomer, with the benzyl group as the chiral center), and then the two diastereomers are separated.

[0234]

[0235] Other phosphorylation reagents and methods can also be used to attach phosphorus-containing groups directly to the 4-amino group of gemcitabine, either in a suitably protected form or to gemcitabine itself.

[0236] (3) Removal of protecting groups

[0237] Once the desired N-prodrug group is attached, the molecule can be further modified at other positions or subjected to a deprotection reaction to remove the protecting group. For example, the 3',5'-TBS protecting group was removed by treating compound 5 with ammonium fluoride under appropriate reaction conditions.

[0238]

[0239] Other prodrugs of formula (I) may also be prepared and some examples are provided in the following section.

[0240] For example, prodrugs of metformin can be prepared using the following reaction scheme:

[0241]

[0242] The preparation of topotecan prodrug is illustrated as follows:

[0243]

[0244] Another amine prodrug is exemplified via the preparation of a sitagliptin prodrug as described below:

[0245]

[0246] Alternatively, the coupling reactions can be performed in a sequential manner as described in Scheme 2 below.

[0247]

[0248] Scheme 2. Sequential reactions to obtain phosphorus-containing prodrugs.

[0249] Reaction of the active drug HXQ with phosphoryl trichloride in the presence of a suitable base (usually at low temperature) followed by addition of the desired 1,3-propanediol derivative will also produce the desired prodrug.

[0250] The preparation of tenofovir disoproxil prodrug was accomplished using the strategy outlined in Scheme 2:

[0251]

[0252] Of note, the following prodrugs were also prepared using the synthetic chemistry described above or appropriate variations thereon.

[0253] A compound selected from the group consisting of:

[0254]

[0255]

[0256]

[0257] as well as,

[0258] as well as,

[0259] wherein Ar is as defined herein.

[0260] (4) Modification of existing N-prodrugs of gemcitabine

[0261] If necessary, other modifications may also be made to other positions of gemcitabine. Alternatively, another phosphate prodrug group may be selectively attached to the 5'-position. For example, compound 4a is treated with phenyl dichlorophosphate under suitable reaction conditions in the presence of a suitable base and then treated with ethyl alanine to obtain compound 53, wherein both the 5'-position and the N4-position have a phosphate prodrug.

[0262]

[0263] In another aspect, the 3' and 5'-OH groups are linked to form a cyclic group. For example, treating compound 4a with phosphoryl trichloride in the presence of a suitable base and under suitable reaction conditions, followed by treating with ethyl alanine, produces a compound of formula (A), wherein R 3 and R 5 Together they form a cyclic phosphate group.

[0264]

[0265] Preparation of key precursors

[0266] Various precursors can be prepared according to the procedures reported in the prior art literature. For example, nitrophenyl 1,3-propanediol cyclic phosphate can be prepared according to the procedures disclosed in J. Am. Chem. Soc., 2004, 126, 5154-5163.

[0267]

[0268] Alternatively, propane-1,3-diol can be prepared stereoselectively to give a chiral compound, and chiral compound Q can be obtained after reaction with dichlorophosphoryl p-nitrophenolate.

[0269] Other precursors of formula (V) can be prepared in a similar manner to that described above.

[0270] All stereoisomers of the compounds of the present invention are included, as mixtures or in pure or substantially pure form. The compounds of the present invention may have a stereocenter at the phosphorus atom and at any carbon including any R substituents. Therefore, the compounds of formula (I) / formula (A) may exist in enantiomeric or diastereomeric form or in a mixture thereof. The preparation method may utilize racemate, enantiomer or diastereoisomer as raw material. When preparing enantiomers or diastereoisomer products, they may be separated by conventional methods. For example, chromatography or fractional crystallization may be used to separate diastereomeric mixtures, and the derivatives of enantiomers may be separated by chromatography.

[0271] One aspect of the invention provides a method for synthesizing and isolating a single isomer of a compound of formula (I) / formula (A). Since phosphorus is a stereogenic atom, forming a prodrug with racemic substituted 1,3-propanediol will produce a mixture of isomers. In another aspect, using an enantiomerically enriched substituted 1,3-propanediol with an R configuration yields an enantiomerically enriched R cis and R trans prodrug. These compounds can be separated by a combination of column chromatography and / or fractional crystallization.

[0272] Prodrugs are typically introduced late in the synthesis, although some prodrugs may be introduced early in the synthesis due to other considerations.

[0273] Alternatively, compounds of formula (I) / formula (A) can be prepared in the following manner: using suitable protecting groups to block 3' and 5' hydroxyl functional groups, followed by functionalization of the N4-amino group. Typical protecting groups are well known and summarized in the art (Protecting Groups in Organic Synthesis, 3rd edition, Theodora Greene, Peter Wuts (Wiley-Interscience) 1999). On the other hand, compounds of formula (I) / formula (A) can be prepared without using protecting groups.

[0274] Preparation, dosage and use

[0275] The compounds of the invention are administered in a total daily dose of 0.01 to 2500 mg. In one aspect, the range is from about 5 mg to about 500 mg. The dose may be administered in multiple divided doses as convenient.

[0276] The compounds of the present invention can be administered as a daily dose or an appropriate portion of a daily dose (e.g., twice a day (bid)) when used in combination with other agents. The compounds of the present invention can be used in a multi-drug regimen, also referred to as combination therapy or "cocktail" therapy, in which multiple agents can be administered together, can be administered separately at the same time or at different intervals, or can be administered sequentially. The compounds of the present invention can be administered after a course of treatment with another agent, during a course of treatment with another agent, as part of a treatment regimen, or can be administered before another agent treatment in a treatment program.

[0277] For purposes of the present invention, the compound can be administered in a variety of ways, including oral, parenteral, inhalation spray, topical or rectal, in a formulation containing a pharmaceutical carrier, adjuvant and vehicle. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular and intraarterial injection using various infusion techniques. Intraarterial and intravenous injections as used herein include administration through a catheter. Intravenous administration is generally preferred.

[0278] Pharmaceutically acceptable salts include acetate, adipate, benzenesulfonate, bromide, camphorsulfonate, chloride, citrate, edisylate, estolate, fumarate, glucoheptonate, gluconate, glucoranate, hippurate, hyclate, hydrobromide, hydrochloride, iodide, isethionate, lactate, lactobionate, maleate, methanesulfonate, methylbromide, methylsulfate, naphthylsulfonate, nitrate, oleate, palmoate, phosphate, polygalacturonate, stearate, succinate, sulfate, sulfosalicylate, tannate, tartrate, terephthalate, toluenesulfonate and triethyl iodide.

[0279] The pharmaceutical composition containing the active ingredient can be in any form suitable for the intended method of administration. For example, when used for oral use, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs can be prepared. Compositions intended for oral use can be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions can contain one or more agents (including sweeteners, flavoring agents, coloring agents and preservatives) to provide a palatable preparation. Tablets containing active ingredients mixed with non-toxic pharmaceutical excipients suitable for preparing tablets are acceptable. These excipients can be, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants such as corn starch or alginic acid; adhesives such as starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate, alone or with a wax, may be employed.

[0280] Formulations for oral use may also be presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, such as calcium phosphate or kaolin, or as soft gelatin capsules where the active ingredient is mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil.

[0281] The aqueous suspension of the present invention contains an active substance mixed with an excipient suitable for preparing an aqueous suspension. Such excipients include: suspending agents, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic, and dispersants or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecanethenoxycetanol), condensation products of ethylene oxide and partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives (e.g., ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate), one or more coloring agents, one or more flavoring agents, and one or more sweeteners (e.g., sucrose or saccharin).

[0282] Oily suspensions can be prepared by suspending the active ingredient in a vegetable oil (such as peanut oil, olive oil, sesame oil or coconut oil) or a mineral oil (such as liquid paraffin). Oral suspensions can contain thickeners such as beeswax, hard paraffin or acetyl alcohol. Sweeteners (such as those described above) and flavorings can be added to provide a palatable oral preparation. These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0283] Dispersible powders and granules of the present invention suitable for preparing aqueous suspensions by adding water provide the active ingredient mixed with a dispersant or wetting agent, a suspending agent and one or more preservatives. Suitable dispersants or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients such as sweeteners, flavoring agents and coloring agents may also be present.

[0284] The pharmaceutical composition of the present invention can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil (such as olive oil or peanut oil), a mineral oil (such as liquid paraffin) or a mixture of these. Suitable emulsifiers include naturally occurring gums (such as gum arabic and gum tragacanth), naturally occurring phospholipids (such as soybean lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (such as sorbitan monooleate) and condensation products of these partial esters with ethylene oxide (such as polyoxyethylene sorbitan monooleate). The emulsion can also contain sweeteners and flavorings.

[0285] Syrups and elixirs may be formulated with sweeteners such as glycerol, sorbitol or sucrose. Such preparations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

[0286] The pharmaceutical composition of the present invention can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. The suspension can be prepared according to known techniques using those suitable dispersants or wetting agents and suspending agents mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol, or prepared as a lyophilized powder. Among them, acceptable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils can generally be used as solvents or suspension media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can also be used for the preparation of injections.

[0287] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary according to the treated host and the specific mode of administration. For example, a time-release formulation intended for oral administration to a person can contain 20 to 2000 μmol (approximately 10 to 1000 mg) of active substance mixed with a suitable and convenient amount of carrier material, and the suitable and convenient amount can vary in about 5 to about 95% of the total composition. It is preferred that a pharmaceutical composition that provides an easily measurable amount of administration can be prepared. For example, an aqueous solution intended for intravenous infusion can contain about 0.05 to about 50 μmol (approximately 0.025 to 25 mg) of active ingredient / ml solution so that an infusion of a suitable volume can be performed at a rate of about 30 mL / hour.

[0288] As noted above, formulations of the invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.

[0289] As noted above, formulations of the invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.

[0290] Tablets can be prepared by compression or molding optionally with one or more auxiliary ingredients. Compressed tablets can be prepared by compressing in a suitable machine an active ingredient in a free-flowing form (such as a powder or granule) optionally mixed with a binder (e.g., polyvinylpyrrolidone, gelatin, hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethylcellulose), a surfactant, or a dispersant. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored, and can be formulated to provide a sustained or controlled release of the active ingredient therefrom, wherein hydroxypropylmethylcellulose is used, for example, in different proportions to provide the desired release characteristics. Tablets can optionally be enteric coated to provide release in the intestinal tract portion other than the stomach. When such compounds are sensitive to acid hydrolysis, it is particularly advantageous to use compounds of formula (I) / formula (A).

[0291] Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0292] Formulations for rectal administration may be presented as a suppository with a suitable base containing for example cocoa butter or a salicylate.

[0293] Formulations suitable for vaginal administration may also be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0294] Preparations suitable for parenteral administration include: aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The preparation can be presented in unit dose or multi-dose sealed containers, such as ampoules and vials, and can be stored under freeze-dried (lyophilized) conditions, requiring only the addition of a sterile liquid carrier (e.g., water) for injection immediately before use. Injection solutions and suspensions are prepared from sterile powders, granules, and tablets of the aforementioned types.

[0295] Preparations suitable for parenteral administration can be administered by continuous infusion via an indwelling pump or via a medical bag. Continuous infusion includes infusion via an external pump. Infusion can be performed by Hickman or PICC or any other suitable means of parenteral or intravenous administration of the preparation.

[0296] Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, or an appropriate fraction thereof, of the drug.

[0297] However, it should be understood that the specific dosage level for any particular patient will depend on a variety of factors, including the activity of the specific compound employed; the age, weight, general health, sex, and diet of the individual to be treated; the time and route of administration; rate of excretion; other drugs previously administered; and the severity of the specific disease being treated, as is well known to those skilled in the art.

[0298] The phosphorus-containing prodrug of formula (I) according to the present invention can be converted into the corresponding pharmaceutically active agent HXQ in vivo, and therefore can be used to treat a variety of diseases, including but not limited to cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases, such as T-cell lymphoma, soft tissue sarcoma, pancreatic cancer, breast cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, bladder cancer and hepatocellular carcinoma (HCC). In this regard, the prodrug of the present invention can also be used in combination with one or more other active agents / methods for treating cancer, such as antiproliferative drugs / antitumor drugs, cell growth inhibitors, anti-invasive agents, growth factor inhibitors, anti-angiogenic agents, gene therapy methods, immunotherapy methods, cytotoxic agents and targeted therapies (e.g., PI3Kd inhibitors). Example

[0299] The compounds used in the present invention and their preparation can be further understood by examples, which illustrate some methods for preparing these compounds. However, these examples should not be construed as particularly limiting the present invention, and variants of compounds currently known or developed later are considered to fall within the scope of the present invention claimed below. Unless otherwise stated, the starting materials and reagents used in the examples are commercially available (such as from Aldrich) or prepared according to known methods described in the prior art documents.

[0300] The following abbreviations are used in this manual:

[0301] DMAP-Dimethylaminopyridine

[0302] DMF-Dimethylformamide

[0303] DIEA-Diisopropylethylamine

[0304] TEA-Triethylamine

[0305] TBSCl-tert-butyldimethylsilyl chloride

[0306] EtOAc-ethyl acetate

[0307] THF-Tetrahydrofuran

[0308] TBAF-Tetrabutylammonium fluoride

[0309] NMR - Nuclear Magnetic Resonance

[0310] LC-MS - Liquid Chromatography Mass Spectrometry

[0311] TMS-Tetramethylsilane

[0312] RT - Room temperature

[0313] iv - intravenous administration

[0314] PO - oral administration

[0315] THU-Tetrahydrouridine

[0316] PK-pharmacokinetics

[0317] MRT 0-t - the average residence time from zero time to observation time

[0318] MRT 0-inf - Average residence time from zero time to infinity

[0319] AUC 0-t - Area under the curve from time zero to observation time

[0320] AUC 0-inf -The area under the curve from zero time to infinity

[0321] TI - Therapeutic Index

[0322] General experimental methods

[0323] 1 H NMR spectra were recorded on a Bruker Avance III and Bruker Avance Neo (400 MHz), and TMS was used as an internal standard.

[0324] LC-MS was performed on a quadrupole mass spectrometer on an Agilent LC / MSD 1200 series (column: Ultimate XB-C18 (50×4.6 mm, 5 μm), operating in ES (+) or (-) ionization mode; T=30° C.; flow rate=1.5 mL / min; detection wavelength: 214 nm.

[0325] Example 1

[0326] Preparation of 4-(((2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound 4) & 4-(((2S,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound 5)

[0327]

[0328] (i) Preparation of 1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-4-(((4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (Compound 3)

[0329] To a solution of 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)-oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound 2, 1.0 g, 2.04 mmol) in THF (15.0 mL) was added NaH (122 mg, 3.18 mmol, 60% in mineral oil) portionwise at 0° C. After the addition, the mixture was stirred at the same temperature for 1 hour. Then (4S)-4-(3-chlorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane 2-oxide (Q, 752 mg, 2.04 mmol; prepared according to the procedure of Journal of Medicinal Chemistry, 2018, Vol. 61, p. 4904) in THF (5.0 mL) was added dropwise to the above mixture at 0°C. The reaction mixture was then stirred at 0°C for another 2 hours. The reaction was heated with H 2 O (30mL) quenched, and then extracted with EtOAc (50mL * 2). The combined organic layer was washed with brine (50mL) and washed with Na 2 SO 4 The residue was purified by column chromatography on silica gel (DCM / CH 3 OH=80 / 1) to give the desired product (compound 3, 620 mg, 42%) as a yellow solid.

[0330] LC-MS: Rt = 1.93 min, [M+H] + =722.

[0331] (ii) Preparation of 4-(((2R,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound 4) & 4-(((2S,4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (Compound 5)

[0332] To 1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)-oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-4-(((4S)-4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (Compound 3, 620 mg, 0.86 mol, 5) in CH 3 NH OH (10 mL) was added to the solution 4 F (318 mg, 8.6 mmol) was added, and the mixture was stirred at 25° C. for 24 hours. The mixture was purified by preparative HPLC to give the product compound 4 (112 mg) and compound 5 (33 mg) as a white solid.

[0333] Compound 4: 1H NMR (400 MHz, DMSO-d6): δ11.0 (brs, 1H), 7.91 (d, J = 8.8 Hz, 1H), 7.50 (s, 1H), 7.43-7.38 (m, 3H), 6.33-6.27 (m, 2H), 6.12 (t, J = 7.6 Hz, 1H), 5.65 (d, J = 10.8 Hz, 1H), 5.25 (s ,1H),4.56-4.50(m,1H),4.40-4.35(m,1H),4.19-4.15(m,1H),3.88-3.84(m,1H),3 .78(d,J=12.4Hz,1H),3.63(d,J=12.4Hz,1H),2.48-2.31(m,1H),2.08-2.03(m,1H).

[0334] LC-MS: Rt = 2.863 min, [M+H] + =494.

[0335] Compound 5: 1H NMR (400MHz, DMSO-d6): δ11.7(brs,1H),7.89(d,J=7.6Hz,1H),7.46-7.36( m,4H),6.31(d,J=5.6Hz,1H),6.24(d,J=7.6Hz,1H),6.10(t,J=8.0Hz,1H),5 .56(brs,1H),5.24(s,1H),4.48-4.33(m,2H),4.19-4.15(m,1H),3.87-3.8 4(m,1H),3.77(d,J=12.0Hz,1H),3.63(d,J=12.0Hz,1H),2.10-2.00(m,2H).

[0336] LC-MS: Rt = 2.954 min, [M+H] + =494.

[0337] Alternatively, when the racemic reagent Compound 7 was used, the following racemic prodrug was also prepared in a similar manner:

[0338]

[0339] Example 2

[0340] Synthesis of 2-[({[(dimethylamino)iminomethyl]amino}iminomethyl)amino]-4-(3-chlorophenyl)-1,3,2-dioxaphosphinane-2-one (8)

[0341]

[0342] Amino[(dimethylamino)iminomethyl]formamidine hydrochloride (6, 676 mg, 4.08 mmol), 4-(3-chlorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane-2-oxide (7, 500 mg, 1.36 mmol, prepared according to the procedure described in J. Am. Chem. Soc., 2004, 126, 5154-5163 (with slight modifications as needed)) and Cs 2 CO 3 A mixture of (2.7 g, 8.16 mmol) in DMF (10 mL) was stirred at room temperature for 5 hours. The reaction mixture was filtered. The filtrate was concentrated. The residue was purified by preparative HPLC to give the product (8, 111.7 mg, 23%) as a white solid.

[0343] 1 HNMR (400MHz, CD 3OD): δ7.44-7.27(m,4H),5.56-5.51(m,1H),4.62-4.55(m,1H),4.36-4.26(m,1H),3.10-3.01(m,6H),2.20-2.11(m,1H),2.05-1.93(m,1H).

[0344] LC-MS: Column: Waters Xbridge C18 5um, 50*4.6mm; Mobile phase: B (ACN): A (0.02% NH 4 Ac); gradient (B%), 6 min. Rt 1 =3.392, Rt 2 =3.536, Rt 3 =3.572, Rt=4.055; MS calculated value: 359, MS found value: 360 [M+H] + .

[0345] Example 3

[0346] Synthesis of (4R)-9-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)oxy)-10-((dimethylamino)methyl)-4-ethyl-4-hydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione (10)

[0347]

[0348] To (R)-10-((dimethylamino)methyl)-4-ethyl-4,9-dihydroxy-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14(4H,12H)-dione hydrochloride (9, 300 mg, 0.655 mmol) and K 2 CO 3 4-(3-chlorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane 2-oxide (7, 242 mg, 0.655 mmol, prepared according to the procedure described in J.Am.Chem.Soc., 2004, 126, 5154-5163 (slightly modified when necessary)) was added to the mixture in DMF (3.0 mL). After addition, the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was filtered. The filtrate was diluted with water (30 mL) and extracted with EtOAc (60 mL*2). The combined organic layers were washed with brine (30 mL*2) and washed with Na 2 SO 4Dry, filter, and concentrate in vacuo.The residue was purified by preparative TLC (DCM / MeOH=10 / 1) to give the title compound 10 (94 mg, 22%) as a yellow solid.

[0349] 1 H NMR (400 MHz, DMSO-d 6 ): δ8.99(s,1H),8.20-8.17(m,1H),7.83-7.78(m,1H),7.59(s,1H),7.53-7.42(m,3H),7.26(s,1H),6.54(brs,1H),5.94-5.91(m,1H) ,5.42(s,2H),5.31(s,2H),4.79-4.66(m,2H),3.94(s,2H),2.51-2.40(m,2H),2.35(s,6H),1.92-1.82(m,2H),0.88(t,J=7.2Hz,3H).

[0350] LC-MS: Column: Waters Xbridge C18 5um, 50*4.6mm; Mobile phase: B (ACN): A (0.02% NH 4 Ac); gradient (B%), 6 min. Rt 1 =2.865, Rt 2 =2.946, Rt 3 =3.104; MS calculated value: 651, MS found value: 652 [M+H] + .

[0351] Example 4

[0352] Synthesis of (3R)-3-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (12)

[0353]

[0354] To a mixture of (R)-3-amino-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (11, 250 mg, 0.61 mmol) and KOH (35 mg, 0.61 mmol) in 5 mL of DMF was added a solution of 4-(3-chlorophenyl)-2-(4-nitrophenoxy)-1,3,2-dioxaphosphinane 2-oxide (44, 490 mg, 1.84 mmol, prepared according to the procedure described in J. Am. Chem. Soc., 2004, 126, 5154-5163 (with slight modifications as needed)) in 2 mL of DMF. After stirring at room temperature overnight, the reaction mixture was diluted with 20 mL of water and extracted with DCM (20 mL*3). The separated organic layer was concentrated, and the residue was purified twice by preparative HPLC to give compound 12 (20 mg, yield 5%) as a white solid.

[0355] 1 HNMR (400MHz, CD 3 OD): δ7.48-7.19(m,5H),6.94-6.89(m,1H),5.45-4.42(m,1H),5.13-4.91(m,2 H),4.51-4.12(m,6H),3.97-3.83(m,1H),2.98-2.67(m,4H),2.15-1.94(m,2H).

[0356] LC-MS: Column: Waters Xbridge C18 5um, 50*4.6mm; Mobile phase: B (ACN): A (0.02% NH 4 Ac); gradient (B%), 6 min. Rt 1 =3.349, Rt=4.055; MS calculated value: 637, MS found value: 638 [M+H] + .

[0357] Example 5

[0358] Synthesis of diisopropyl ((((((2R)-1-(6-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(oxy))bis(methylene)dicarbonate (15)

[0359]

[0360] At 0°C in N 2To a mixture of (R)-diisopropyl(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(oxy))bis(methylene)dicarbonate (13, 688 mg, 1.33 mmol) in anhydrous pyridine (33 mL) was added POCl 3 4-(4-(4-chlorophenyl)propane-1-yl)-2-nitropropane-2-yl)-4 ... 4 Cl (100mL * 2) washing. The organic phase is concentrated in vacuo. The residue is purified by preparative HPLC (eluted with water / acetonitrile = 1% to 60%) to give a crude white solid. The solid is then purified again by preparative HPLC to give the title compound ((((((2R)-1-(6-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphorinane-2-yl)amino)-9H-purine-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(oxy))bis(methylene)dicarbonate diisopropyl ester (15, 104mg, 10%) as a white solid.

[0361] 1 H NMR (400 MHz, DMSO-d 6 ): δ8.61-8.28(m,1H),8.20-8.15(m,1H),7.60-7.45(m,1H),7.33-7.29(m,3H),6.14-5.70(m,1H),5.67-5.58(m,4H),5.10-4.39(m,5H) ,4.23-4.21(m,1H),3.97-3.91(m,2H),3.93-3.73(m,1H),2.95-2.35(m,1H),2.19-2.05(m,1H),1.32-1.28(m,12H),1.25-1.21(m,3H).

[0362] LC-MS: Column: Waters Xbridge C18 5um, 50*4.6mm; Mobile phase: B (ACN): A (0.02% NH 4 Ac); gradient (B%), 6 min. Rt 1 =3.439, Rt 2=3.650; MS calculated value: 749, MS found value: 750 [M+H] + .

[0363] Example 6

[0364] Synthesis of diisopropyl (((((((2R)-1-(6-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(oxy))bis(methylene))dicarbonate (20)

[0365]

[0366] At 0°C in N 2 To a mixture of (R)-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)-bis(oxy))bis(methylene)dicarbonate (13, 800 mg, 1.54 mmol) in pyridine (40 mL) was added POCl 3 4-(4-(pyridin-3-yl)propane-1,3-diol (43,707mg, 4.62mmol, prepared according to the procedure described in J.Am.Chem.Soc., 2004,126,5154-5163 (slightly modified when necessary)) was added to the mixture at 0 ° C. After addition, the reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was concentrated in a vacuum. The residue was purified by C18 (eluted with water / acetonitrile=1% to 70%) to give a yellow solid. The solid was then purified again by preparative TLC (DCM / MeOH=13 / 1) to give the title compound ((((((2R)-1-(6-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(oxy))bis(methylene))dicarbonate (20, 25.4 mg, 2%) as a white solid.

[0367] 1 H NMR (400 MHz, DMSO-d 6): δ8.71-8.20(m,3H),8.16(s,1H),7.86-7.82(m,1H),7.37-7.29(m,1H),5.84 -5.82(m,1H),5.84-5.64(m,4H),4.94-4.90(m,2H),4.87-4.76(m,1H),4.67-4. 64(m,1H),4.43-4.38(m,1H),4.23-4.21(m,1H),3.97-3.77(m,2H),3.73-3.69 (m,1H),3.06-2.99(m,1H),2.40-2.10(m,1H),1.32(s,12H),1.29-1.25(m,3H).

[0368] LC-MS: Column: Waters Xbridge C18 5um, 50*4.6mm; Mobile phase: B (ACN): A (0.02% NH 4 Ac); gradient (B%), 6 min. Rt = 3.324; MS calculated value: 716, MS found value: 717 [M+H] + .

[0369] Example 7

[0370] Synthesis of diisopropyl (((((((2R)-1-(6-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(oxy))bis(methylene))dicarbonate (21)

[0371]

[0372] At 0°C in N 2 To a mixture of (R)-(((((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)-bis(oxy))bis(methylene)dicarbonate (13, 519 mg, 1.0 mmol) in pyridine (30 mL) was added POCl 34-(pyridin-4-yl)propane-1,3-diol (38,459mg, 3.0mmol, prepared according to the procedure described in J.Am.Chem.Soc., 2004,126,5154-5163 (slightly modified when necessary)) was added to the mixture at 0 ° C. After addition, the reaction mixture was slowly warmed to room temperature and stirred for 16 hours. The reaction mixture was concentrated in a vacuum. The residue was purified by C18 (eluted with water / acetonitrile=1% to 70%) to give a yellow solid. The solid was then purified again by preparative TLC (DCM / MeOH=13 / 1) to give the title compound ((((((2R)-1-(6-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphoryl)bis(oxy))bis(methylene))dicarbonate (21, 35.8 mg, 5%) as a white solid.

[0373] 1 H NMR (400 MHz, DMSO-d 6 ): δ8.68-8.44(m,3H),8.16(s,1H),7.43-7.38(m,2H),6.18-6.08(m,1H), 5.77-5.52(m,4H),5.17-5.08(m,1H),5.02-4.85(m,2H),4.77-4.61(m,1H) ,4.61-4.56(m,1H),4.47-4.43(m,1H),4.34-4.15(m,2H),3.99-3.91(m,1 H),2.42-2.34(m,1H),2.28-2.20(m,1H),1.20(s,12H),1.29-1.22(m,3H).

[0374] LC-MS: Column: Waters Xbridge C18 5um, 50*4.6mm; Mobile phase: B (ACN): A (0.02% NH 4 Ac); gradient (B%), 6 min. Rt = 3.279; MS calculated value: 716, MS found value: 717 [M+H] + .

[0375] Example 8

[0376] Synthesis of 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (27)

[0377]

[0378] Synthesis of 1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (41)

[0379] To 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (1.22 g, 2.5 mmol) and Cs 2 CO 3 2-(4-nitrophenoxy)-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane 2-oxide (840 mg, 2.5 mmol, 42, prepared according to the procedure described in J. Am. Chem. Soc., 2004, 126, 5154-5163 (1.63 g, 5.0 mmol) was added to a mixture in THF (20 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with water (50 mL) and extracted with EtOAc (50 mL*3). The combined organic phases were concentrated in vacuo. The residue was purified by preparative HPLC (water / acetonitrile = 30% to 100%) to give the title compound (500 mg, 29%, 41) as a yellow solid.

[0380] LC-MS: Rt = 1.837 min, [M+H] + =689.

[0381] Synthesis of 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (27)

[0382] To a solution of 1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-4-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (500 mg, 0.73 mmol, 41) in THF (5.0 mL) was added TBAF (1.46 mL, 1.46 mmol, 1 M in THF) dropwise at 0°C. After addition, the reaction mixture was stirred at the same temperature for 30 hours. The reaction mixture was purified by preparative HPLC (water / acetonitrile = 10% to 70%) to give compound 27 (120 mg, 36%) as a white solid.

[0383] 1 H NMR (400 MHz, DMSO-d 6 ): δ8.58-8.57(m,2H),7.95(brs,1H),7.43(s,2H),7.34-7.32(m,2H),6.10-6.09(m,1H),5.71-5.66(m,1H),5.27-5.25(m,1H),4.56-4.53( m,1H),4.47-4.43(m,1H),4.20-4.15(m,1H),3.88-3.83(m,1H),3.80- 3.76(m,1H),3.66-3.60(m,1H),2.49-2.48(m,1H),2.15-2.11(m,1H).

[0384] LC-MS: Rt 1 =2.800, [M+H] + =461.

[0385] Example 9

[0386] Synthesis of 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (28)

[0387]

[0388] Synthesis of 1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (50)

[0389] To 4-amino-1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)pyrimidin-2(1H)-one (982 mg, 2.0 mmol) and Cs 2 CO 3 2-(4-nitrophenoxy)-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane 2-oxide (672 mg, 2.0 mmol, 49, prepared according to the procedure described in J. Am. Chem. Soc., 2004, 126, 5154-5163 (slightly modified when necessary)) was added to a mixture in THF (10 mL). After addition, the reaction mixture was stirred at 30 ° C for 2 hours. The reaction mixture was then quenched with water (50 mL) and extracted with EtOAc (50 mL*3). The combined organic phases were concentrated in vacuo. The residue was purified by preparative HPLC (water / acetonitrile=40% to 100%) to give the target compound (500 mg, 36%, 50) as a yellow solid.

[0390] LC-MS: Rt = 1.99 min, [M+H] + =689.

[0391] Synthesis of 1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (28)

[0392] To a solution of 1-((2R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)-3,3-difluorotetrahydrofuran-2-yl)-4-((2-oxo-4-(pyridin-3-yl)-1,3,2-dioxaphosphinane-2-yl)amino)pyrimidin-2(1H)-one (500 mg, 0.73 mmol) in THF (5.0 mL) was added TBAF (1.46 mL, 1.46 mmol, 1 M in THF) dropwise at 0°C. After addition, the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by preparative HPLC to give compound 28 (110 mg, 33%) as a white solid.

[0393] 1 H NMR (400 MHz, DMSO-d 6 ): δ8.65(s,1H),8.55(d,J=4.0Hz,1H),7.91-7.89(m,2H),7.43-7.38(m ,1H),6.31-6.29(m,2H),6.12-6.08(m,1H),5.72-5.69(m,1H),5.26-5. 23(m,1H),4.58-4.52(m,1H),4.41-4.39(m,1H),4.20-4.15(m,1H),3.8 7-3.76(m,2H),3.66-3.60(m,1H),2.49-2.47(m,1H),2.08-2.04(m,1H).

[0394] LC-MS: Rt=2.811, [M+H] + =461.

[0395] Example 10

[0396] Synthesis of (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isobutyryloxy)methyl)tetrahydrofuran-3-yl isobutyrate (Compound 29)

[0397]

[0398] To a mixture of 4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (150 mg, 0.304 mmol, compound 4a, prepared according to Example 1) in DCM / DMF (3 mL / 0.3 mL) was added TEA (154 mg, 1.52 mmol) dropwise at room temperature followed by isobutyryl chloride (324 mg, 3.04 mmol). After addition, the reaction mixture was stirred at room temperature for 16 hours. The mixture was quenched with water (50 mL) and extracted with EtOAc (20 mL*2). The combined organic phases were concentrated. The residue was purified by C18 (water / acetonitrile = 10% to 60%) to give the title compound (41 mg, compound 29) as a white solid. The white solids (Y1430-12483-022 (49 mg) and Y1430-12483-034 (41 mg)) were combined (90 mg, total yield 21%, compound 29, as a mixture of diastereomers).

[0399] 1 H NMR (400 MHz, DMSO-d 6 ): δ8.01-7.98(m,1H),7.54-7.34(m,5H),6.36-6.34(m,1H),5.73-5.64(m,1H),5.19-5. 15(m,1H),4.72-4.46(m,5H),2.72-2.57(m,2H),2.36-2.16(m,2H),1.23-1.09(m,12H).

[0400] LC-MS: R t1 =2.976min, R t2 =3.165min, R t3 =3.322min, [M+H] + =634.

[0401] The following compounds were prepared in a similar manner:

[0402] ((2R,3R,5R)-3-acetoxy-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluorotetrahydrofuran-2-yl)methyl acetate;

[0403] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((propionyloxy)methyl)tetrahydrofuran-3-yl propanoate;

[0404] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((2-methoxyacetoxy)methyl)tetrahydrofuran-3-yl 2-methoxyacetate;

[0405] (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinane-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((pivaloyloxy)methyl)tetrahydrofuran-3-yl pivalate;

[0406] Isopropyl (2R,3R,5R)-5-(4-((4-(3-chlorophenyl)-2-oxo-1,3,2-dioxaphosphinan-2-yl)amino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-2-((isopropoxycarbonyloxy)methyl)tetrahydrofuran-3-yl carbonate.

[0407] Mouse pharmacokinetic study 1

[0408] 1. Experimental Design

[0409] 1.1 Experimental animals

[0410] The C57BL / 6 mice used in this experiment were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the administration test was performed on male mice with a body weight range of 16 to 19 g. The animals were housed under appropriate air conditions (temperature: 20 to 25°C; humidity: 40% to 70%; light / dark cycle: 12 hours), and food and water were provided ad libitum.

[0411] 1.2 Preparation

[0412] 1.2.1 Administration by intravenous injection

[0413] The solution medium for intravenous administration was DMSO: 30% solutol HS-15: saline = 10: 10: 80 (v / v / v). The test substance was dissolved in the medium to obtain a clear solution, and filtered through PTFE to obtain a colorless clear solution for intravenous administration.

[0414] 1.2.2 Oral administration

[0415] The solution medium for oral administration was DMSO: 30% solutol HS-15: saline = 10: 10: 80 (v / v / v). The test substance was dissolved in the medium to obtain a clear solution.

[0416] 1.3 Administration

[0417] Animals were randomly divided into groups. All animals were fasted overnight (12-15 hours) before dosing (but water was provided ad libitum), and food was provided 4 hours after dosing. For intravenous administration of gemcitabine, the formulation was administered to mice by tail vein injection (dosage was 15 mg / kg).

[0418] The pharmacokinetic (PK) characteristics of the liver-targeted gemcitabine (dFdC) prodrug according to Formula (I) / Formula (A) (i.e., Compound 4) were evaluated in male C57BL / C mice after intravenous administration (iv) at an equivalent dose of 15 mg / kg gemcitabine or after oral administration (PO or ig) at a dose of 80 mg / kg.

[0419] 1.4 Sample collection, processing and analysis

[0420] Blood samples were collected from designated animals at 0.083, 0.25, 0.5, 1, 2, 4, and 8 hours after intravenous administration and at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after oral administration (n=3 / time point / compound). Systemic blood samples were collected into EDTA-K containing tetrahydrouridine (THU, 0.5 mM). 2Treatment tubes to inhibit further metabolism of gemcitabine (dFdC) to dFdU. In addition, 2 hours after intravenous or oral administration, 3 animals were sacrificed to collect liver tissue. The plasma was separated by centrifugation and frozen before analysis. The liver tissue was treated by homogenization in X5 volumes of 20% methanol, and the homogenate was centrifuged to collect the supernatant. The plasma and tissue concentrations of the prodrug, the parent drug (i.e., gemcitabine, also known as dFdC) and dFdU (the main metabolite of dFdC) were determined by LC / MS / MS analysis (instrument: Agilent 6470. MS: positive ion, AJS ESI, MRM detection; HPLC conditions: column: Agilent ZORBAX XDB-C18, 5μm, 2.1×50mm (column number: 50-241), flow rate: 0.30mL·min -1 Pharmacokinetic parameters were calculated using WinNonlin software (Pharsight Corp., Mountain View, CA).

[0421] 2. Results

[0422] from Figure 1 As can be seen, intravenous or oral administration of a prodrug compound according to formula (I) / formula (A) (e.g., compound 4) results in significant exposure of the prodrug in the systemic circulation, indicating that the prodrug is well absorbed in the intestine. In addition, the detection of gemcitabine (also known as dFdC) in the circulation further demonstrates that the prodrug approach is feasible: dFdC is released from the prodrug in vivo ( Figure 1 ).

[0423] The prodrug compounds according to the present invention can enhance the exposure of dFdC by intravenous or oral administration of the prodrug, which is based on the half-life (T 1 / 2 ) were compared: T for intravenous administration of gemcitabine, intravenous administration of compound 4 and oral administration of compound 4, respectively. 1 / 2 =1.1 hours, 2.1 hours and 3.3 hours. After intravenous administration of gemcitabine at 15 mg / kg, or intravenous administration of compound 4 (equivalent dose of 15 mg / kg gemcitabine), or oral administration (80 mg / kg) of compound 4, other dFdC PK parameters are shown in Table 1, which demonstrates the extended half-life of compound 4 compared to gemcitabine.

[0424] Table 1. Plasma PK parameters of dFdC after intravenous administration of 15 mg / kg gemcitabine or intravenous administration of compound 4 (equivalent dose of 15 mg / kg gemcitabine) or oral administration of compound 4 (80 mg / kg)

[0425]

[0426] Hepatic drug exposure of dFdC and dFdU was measured 2 hours after intravenous (equivalent dose of 15 mg / kg gemcitabine) or oral administration (80 mg / kg) of Compound 4 compared to intravenous gemcitabine (15 mg / kg). Figure 2 As can be seen in the results, there is no detectable dFdC level in the liver, while significant levels of dFdU are detected in the liver after intravenous administration of gemcitabine. In contrast, intravenous and oral administration of compound 4 obtain significant levels of dFdC in the liver. Therefore, compared with the intravenous administration group of gemcitabine at the same dose, 1331 times high dFdC concentrations were produced after intravenous administration of compound 4. Similarly, oral administration of compound 4 also produces significantly higher concentrations of dFdC. On the contrary, the dFdU level produced by compound 4 administration (intravenous or oral) is much lower than that of the intravenous administration group of gemcitabine. For intravenous and oral administration of compound 4, the overall safety improvement (therapeutic index improvement) of compound 4 relative to gemcitabine is greater than 3500 times and 2035 times respectively (Table 2, showing the therapeutic index improvement of compound 4 relative to gemcitabine).

[0427] Table 2. Improvement of the therapeutic index (TI) of prodrugs relative to gemcitabine in the liver

[0428]

[0429]

[0430] TI = [dFdC] - improvement factor x [dFdU] - improvement factor

[0431] Mouse pharmacokinetic study 2

[0432] According to the procedure of the above mouse pharmacokinetic study 1, the PK parameters of compound 27, compound 28, compound 29 and compound 10 were tested, and the tissue distribution of the above compounds in multiple tissues (such as liver, lung, pancreas and kidney) was also determined. The experimental results are respectively Figures 3 to 11 Shown in.

[0433] Similar to compound 4, intravenous or oral administration of each of the above prodrug compounds resulted in significant exposure of the prodrug in the systemic circulation, and the detection of the corresponding active drug (e.g., dFdC) in the circulation further demonstrated that the prodrug approach is feasible: the corresponding active drug (e.g., dFdC) is released from the prodrug in vivo.

[0434] Furthermore, the above-mentioned prodrug compounds can enhance the exposure of the corresponding active drug (eg, dFdC) by intravenous or oral administration of the prodrug and show an increased half-life relative to the corresponding active drug.

[0435] Furthermore, intravenous and oral administration of the above-mentioned prodrugs respectively resulted in significant levels of the corresponding active drug (eg, dFdC) in the liver and showed an overall improved safety profile (improved therapeutic index) relative to the corresponding active drug.

[0436] Pharmacological research

[0437] Human clinical studies of gemcitabine have shown potential as part of a combination therapy for hepatocellular carcinoma, cholangiocarcinoma, and pancreatic cancer. In a Phase II study, gemcitabine combined with oxaliplatin and erlotinib as a first-line therapy for hepatocellular carcinoma showed a progression-free survival of 41% of patients at 26 weeks (Cancer Medicine, 2017, 6, 2042-2051). In another Phase II study of Chinese patients with metastatic pancreatic adenocarcinoma, gemcitabine plus albumin-bound paclitaxel treatment showed an overall response rate of 35%, which met the pre-set primary endpoint.

[0438] 14-Day Safety Study in Mice

[0439] A 14-day mouse safety study was conducted to evaluate whether compounds of Formula (I) / Formula (A) (e.g., Compound 4) have adverse effects in mice after 14 days of oral gavage administration, and to determine the reproducible PK properties of the prodrug (e.g., Compound 4) and its metabolites dFdC and dFdU.

[0440] Male (M) and female (F) CD-1 mice (body weight 18-22g) provided by Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. were randomly divided into four groups, 5 animals of each sex. Compound 4 was administered to animals by oral gavage. A series of doses were selected to determine the maximum tolerated dose and dose-limiting toxicity. A dosing volume of 5mL / kg was used and applied daily.

[0441]

[0442] Endpoints were evaluated including clinical symptoms, body weight, food consumption, hematology, clinical chemistry, plasma concentrations of compound 4 and its metabolites (dFdC, dFdU), macroscopic pathology, organ weights.

Claims

1. A compound having formula (I): (I)， Stereoisomers or salts thereof; in, Ar is pyridyl or phenyl substituted by halogen; as well as X is -NH- or -O-, and X and Q are groups such that HXQ together represent tenofovir disoproxil having structural formula 13, topotecan having structural formula 9, or sitagliptin having structural formula 11, respectively: 。 2. A compound selected from the group consisting of: or 。 3. A method for preparing a compound of formula (I) according to claim 1, comprising: If necessary, the compound of formula HXQ is coupled with the compound of formula Y in the presence of a base and a catalyst to obtain the compound of formula (I), wherein Ar, X and Q are as defined in claim 1.

4. A method for preparing a compound of formula (I) according to claim 1, comprising: The compound of formula HXQ is reacted with phosphoryl trichloride in the presence of a base and a catalyst as necessary, and then a 1,3-propanediol derivative of formula Z is added to react to obtain the compound of formula (I), wherein Ar, X and Q are as defined in claim 1.

5. Use of a compound according to claim 1 or 2 in the preparation of a medicament for diagnosing, preventing and / or treating a disease in a mammal.

6. The use according to claim 5, wherein the disease is selected from the group consisting of cancer, metabolic diseases, viral infections, immune diseases, nervous system diseases and blood diseases.

7. The use according to claim 5, wherein the compound is administered orally or intravenously.

8. The use according to claim 5, wherein the mammal is selected from the group consisting of humans, mice, rats, rabbits, hamsters, guinea pigs, monkeys, dogs, cats, pigs, sheep, goats, cows and horses.

9. The use according to claim 5, wherein the agent is used in combination with other therapeutic agents, and the other therapeutic agents are selected from immune checkpoint inhibitors, microtubule inhibitors, PARP inhibitors, BTK inhibitors, EGFR TK inhibitors and cytotoxic platinum agents.

10. A pharmaceutical composition comprising: a combination of the compound according to claim 1 or 2 and a pharmaceutically acceptable carrier, diluent or excipient.

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