EA038805B1 - Cyclic dinucleotides as sting (stimulator of interferon genes) agonists - Google Patents

Abstract

The invention provides a compound having a STING agonistic activity, which may be expected to be useful as an agent for the prophylaxis or treatment of STING-related diseases. The invention relates to a compound represented by the formula (X)wherein each symbol is as defined in the description, or a salt thereof, to a pharmaceutical composition comprising a compound of the formula (X) and to a method of treating cancer comprising administering a compound of the formula (X).

Description

The technical field to which the invention relates

Provided herein is a cyclic dinucleotide having agonist activity

STING (genes stimulated by interferon), which may be useful as an agent for the prevention or treatment of cancer and other diseases.

State of the art

STING is a receptor that recognizes a nucleic acid other than a TLR (toll-like receptor). Examples of the recognizable natural ligand include cyclic dinucleotides (CDN) derived from bacteria / protozoa, 2 ', 3'-cGAMP, synthesized upstream cGAS (cyclic GMP-AMP synthase), and the like. (Trends in Immunology 35: 88-93 (2014)). It is reported that 2 ', 3'-cGAMP, which is one of the natural ligands, is degraded by ENPP1 (ectonucleotide pyrophosphatase / phosphodiesterase), which is one of the pyrophosphatases / phosphodiesterases, and that other CDNs are degraded by phosphodiesterase (Nat. Chem. Biol. 10 : 10431048 (2014); Cell Res 25: 539550 (2015); Biochemistry 55: 837-849 (2016)). STING is activated by these natural ligands and induces downstream phosphorylation of TBK1 (TANK-binding kinase 1) and activates the IRF3 (interferon regulatory factor 3) signal and NFkB signal downstream, and thus induces an interferon I-type response (IFN) response (Trends in Immunology 35: 88-93 (2014)). The importance of the STING signal in cancer is indicated by a knockout mouse test. It is reported that in tumor allograft mice, when using knockout mice for STING and its downstream signal, IRF3, cancer cells grow by suppressing the cancerous immune system compared to wild-type mice. (Immunity 41: 830-842 (2014)). In addition, it is also reported that the growth of cancer cells in mice with allogeneic tumor graft is suppressed by radiation therapy, but in knockout mice for STING and IFNAR1 (interferon (α and β) receptor 1, IFN type I receptor produced by the downstream signal), the effect of radiation therapy decreases (Immunity 41: 843-852 (2014)). For these reasons, STING is believed to play an important role in suppressing the growth of cancer cells, and the activation of the immune signal that is induced by the activation of STING leads to anti-cancer activity. Therefore, the STING agonist can be used as an antineoplastic agent targeting cancer immunity. In addition, STING activation is believed to play an important role in the immune action of the vaccine, as the activation activates natural immunity (Ther Adv Vaccines 1: 131-143 (2013)). Therefore, the STING agonist can be used as an adjuvant for various vaccines.

The following cyclic nucleotides are known.

Patent Document 1 (WO 2014/093936) describes a compound represented by the following formula:

each symbol is as defined in Patent 1, which according to Patent Document 1 is a STING-dependent TBK1 activator and is useful for treating cancer (especially solid cancer) and the like. and is also useful as an adjuvant.

Patent Document 2 (WO 2014/189805) describes a compound represented by the following formula:

Oh

wherein each character is as defined in Patent Document 2, which according to Patent Document 2 is an immunostimulant via STING and is useful for treating cancer and the like.

- 1 038805

Patent Document 3 (WO 2015/077354) and Non-Patent Document 8 (Cell reports 11, 1018-1030 (2015)) describe a compound represented by the following formula:

wherein each character is as defined in Patent Document 3, which according to Patent Document 3 is a STING agonist and is useful for treating cancer and the like.

Patent Document 4 (WO 2013/185052) and Non-Patent Document 9 (Sci. Transl. Med. 283, 283ra52 (2015)) describe c-di-AMP, c-di-HMP, c-di-IMP, c-AMP -GMP, c-AMP-IMP and c-GMP-IMP, which according to Patent Document 4 are STING-dependent activators of TBK1.

Patent Document 5 (WO 2014/189806) describes a compound represented by the following formula:

where each character is as defined in Patent Document 5, which according to Patent Document 5 inhibits STING-dependent signal transduction and is useful for treating an autoimmune disease and the like.

Patent Document 6 (WO 2015/185565) describes a compound represented by the following formula:

where each symbol is as defined in Patent Document 6, which according to Patent Document 6 is a STING modulator and is useful for treating inflammation, allergic autoimmune disease, cancer and the like, and is also useful as a vaccine adjuvant.

Patent Document 7 (WO 2014/179760) describes a compound represented by the following formula:

where each character is as defined in Patent Document 7, which according to Patent Document 7 can increase the production of Type I interferon and is useful for treating cancer, autoimmune disease, allergic reaction and the like, and is also useful as an adjuvant.

Patent Document 8 (WO 2014/179335) and Non-Patent Document 10 (Mol. Cell 154, 748-762 (2013)) describe a compound represented by the following formula:

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wherein each symbol is as defined in Patent Document 8, which according to Patent Document 8 can increase the production of Type I interferon and is useful for treating diseases characterized by inflammation, autoimmune disease, Sjogren's syndrome and the like.

Patent Document 9 (WO 2015/017652) describes a compound represented by the following formula:

nh _2, which according to Patent Document 9 is a STING modulator, is useful for the treatment of cancer, autoimmune disease and the like, and is also useful as a vaccine.

Patent Document 10 (WO 2016/096577) describes a compound represented by the following formula:

NHj

Which, according to Patent Document 10, is a STING agonist and is useful in the treatment of cancer (especially solid pancreatic cancer) and the like.

Patent Document 11 (WO 2011/003025) describes a compound represented by the following formula:

which according to Patent Document 11 is a STING agonist.

Patent Document 12 (WO 2016/096174) describes a compound represented by the following formula:

- 3 038805

which according to Patent Document 12 is a STING agonist.

Patent Document 13 (WO 2016/120305) describes a compound represented by the following formula:

Formula (I)

which according to Patent Document 13 is a STING agonist.

Patent Document 14 (WO 2016/145102) describes a compound represented by the following formula:

wherein each character is as defined in Patent Document 14, which according to Patent Document 14 is a STING agonist.

Patent Document 15 (WO 2017/027646) describes a compound represented by the following formula:

each character is as defined in Patent Document 15, which according to Patent Document 15 is a STING agonist.

Patent Document 16 (WO 2017/075477) describes a compound represented by the following formula:

- 4,038805, each character being as defined in Patent Document 16, which according to Patent Document 16 is a STING agonist.

Patent Document 17 (WO 2017/027645) describes a compound represented by the following formula:

each character is as defined in Patent Document 17, which according to Patent Document 17 is a STING agonist.

Brief description of the essence of the invention

In one aspect, provided herein is a compound represented by formula (X) d -Ί ' ^P ' P

R ² (U _B 1

-ov ²

7- ^ _(x) where Q ² and Q ⁴ each independently represent an oxygen atom or a sulfur atom,

B ¹ is

R ¹⁸ is hydrogen or C _1-6 alkyl; and

R ¹⁹ represents a halogen atom or a salt thereof.

In another aspect, provided herein is a compound represented by Formula (I), wherein B ¹ is

B ² is

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In another aspect, provided herein is a medicament comprising a compound or salt of formula (X).

In another embodiment, the drug is a STING agonist. In another embodiment of the invention, the medicament is an agent for the prevention or treatment of cancer.

In another aspect, provided herein is a method for activating STING in a mammal, which comprises administering to the mammal an effective amount of a compound of formula (X), or a salt thereof.

In another aspect, provided herein is a method for preventing or treating cancer in a mammal, which comprises administering to the mammal an effective amount of a compound of formula (X), or a salt thereof.

In another aspect, provided herein is a compound of formula (X), or a salt thereof, for use in the prevention or treatment of cancer.

In another aspect, provided herein is the use of a compound of formula (X), or a salt thereof, for the manufacture of an agent for the prevention or treatment of cancer.

Brief description of graphic materials

FIG. 1 is a line graph illustrating the release of Payload 1 from ADC1;

fig. 2 is a line graph illustrating the release of Payload 1 from ADC2;

fig. 3 is a line graph illustrating the release of Payload 2 from ADC3;

fig. 4 is a bar graph illustrating that HEK293 cells that express surface receptor targets for ADC1 show the expected increase in the STING pathway phospho-IRF3 marker (pIRF3) upon treatment with ADC1;

fig. 5 is a bar graph illustrating that HEK293 cells that express surface receptor targets for ADC2 exhibit the expected increase in the STING pathway phospho-IRF3 marker (pIRF3) upon treatment with ADC2;

fig. 6 is a bar graph illustrating that HEK293 cells that express surface receptor targets for ADC3 show the expected increase in the STING pathway phospho-IRF3 marker (pIRF3) upon treatment with ADC3;

fig. 7 is a bar graph illustrating Western blot analysis of downstream signaling pathway activation (TBK1 and IRF3) for the compound of Example 14;

fig. 8 is a line graph illustrating the antitumor activity of the compound of Example 3a in a CT-26 colon carcinoma model of syngeneic mice;

fig. 9 is a line graph illustrating the antitumor activity of the compound of Example 14 in a CT-26 colon carcinoma model of syngeneic mice;

fig. 10 is a line graph illustrating the antitumor activity of the compound of Example 3 a in the B16F10 colon carcinoma model of syngeneic mice;

fig. 11 is a line graph illustrating the antitumor activity of the compound of Example 14 in the B16F10 syngeneic mouse colon carcinoma model.

Detailed description of the essence of the invention

This description is explained in detail below.

The definition of each substituent used in this description is detailed below.

Unless otherwise indicated, each substituent has the following definition.

In this specification, examples of the halogen atom include fluorine, chlorine, bromine, and iodine.

In this specification, examples of the C _1-6 alkyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl , 2,2-dimethylbutyl, 3,3-dimethylbutyl and 2-ethylbutyl.

A compound or a pharmaceutically acceptable salt thereof of formula (X) o H ⁴ H 'p

R ² θ 'B ¹ ν θ

В ^{2 R} \, 0 * ™ <X>.

Embodiment of the invention 1. A compound or a pharmaceutically acceptable salt thereof, wherein R ¹ is a hydroxy group.

Embodiment of the invention 2. A compound or a pharmaceutically acceptable salt thereof, wherein R ¹ is a fluorine atom.

Embodiment 3. A compound according to any one of Embodiments 1, 2, or a pharmaceutically acceptable salt thereof, wherein R ² is a hydroxyl group.

Embodiment of the Invention 4. A compound according to any one of Embodiments of the Invention 1,

- 6 038805 or a pharmaceutically acceptable salt thereof, wherein R ² is a fluorine atom.

Embodiment 5. A compound according to any one of Embodiments 14, or a pharmaceutically acceptable salt thereof, wherein Q ⁴ is a sulfur atom.

Embodiment 6. A compound according to any one of Embodiments 15, or a pharmaceutically acceptable salt thereof, wherein Q ² is an oxygen atom.

Embodiment 7. A compound according to any one of Embodiments 15, or a pharmaceutically acceptable salt thereof, wherein Q ² is a sulfur atom.

Embodiment 8. A compound according to any one of Embodiments 17, or a pharmaceutically acceptable salt thereof, of formula (XII)

Embodiment 9. A compound according to any one of Embodiments 18, or a pharmaceutically acceptable salt thereof, wherein B ¹ is

Embodiment 10. A compound according to any one of Embodiments 1-9, or a pharmaceutically acceptable salt thereof, wherein R ¹⁹ is a fluorine atom.

Embodiment 11. A compound according to any one of Embodiments 1-10, or a pharmaceutically acceptable salt thereof, wherein R ¹⁸ is hydrogen.

Embodiment 12. A compound according to any one of Embodiments 1-10, or a pharmaceutically acceptable salt thereof, wherein R ¹⁸ is methyl.

Embodiment 13. A compound according to any one of Embodiments 1-12, or a pharmaceutically acceptable salt thereof, wherein B ^{2 is} selected from the group consisting of

Nil, Oh ϋ

nJ \ J J.

* 'Ν' _and ,

Embodiment 14. The compound of Embodiment 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of

- 7 038805

- 8 038805

Embodiment 28. A compound of Embodiment 27, or a pharmaceutically acceptable salt thereof, selected from the group consisting of

- 9 038805

- 10 038805

Embodiment 29. A compound of Embodiment 27, or a pharmaceutically acceptable salt thereof, selected from the group consisting of

Embodiment 30. A compound according to any one of Embodiments 1-29, wherein the pharmaceutically acceptable salt is a triethylamine salt or a sodium salt, for example a diethylamine salt or a disodium salt.

Embodiment 31. A pharmaceutical composition comprising a compound according to any one of Embodiments 1-30, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

Embodiment 32. A method of treating a patient, comprising administering to the patient a therapeutically effective amount of a compound according to any one of Embodiments 1-30, or a pharmaceutically acceptable salt thereof, wherein the patient has cancer.

Embodiment of the invention 33. The method according to embodiment 32, characterized in that the cancer is any one or more types of cancer from table. 7.

Embodiment 34. The method of embodiment 32, wherein the cancer is selected from the group consisting of a solid tumor and lymphoma.

Embodiment 35. A method according to any one of Embodiments 3234, further comprising administering a therapeutically effective amount of a second therapeutic agent useful for treating cancer.

Embodiment 36. Pharmaceutical composition according to Embodiments 31 for use in the treatment of cancer.

Embodiment 37. Pharmaceutical composition according to embodiment 36, characterized in that the cancer is any one or more types of cancer from table. 7.

Embodiment 38. A pharmaceutical composition according to embodiment 37, wherein the cancer is selected from the group consisting of a solid tumor and a lymphoma.

Embodiment 39. A compound according to any one of Embodiments 1-30, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer.

Embodiment 40. The compound of Embodiment 39, differing in that the cancer is any one or more of the cancers from Table. 7.

Embodiment 41. A compound of embodiment 40, wherein the cancer is selected from the group consisting of solid tumor and lymphoma.

Embodiment 42. Use of a compound according to any one of Embodiments 1-30, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of cancer.

An embodiment of the invention 43. Use according to an embodiment of the invention 42, characterized in that the cancer is any one or more types of cancer from table. 7.

Embodiment 44. Use in Embodiment 43, wherein the cancer is selected from the group consisting of a solid tumor and a lymphoma.

Embodiment 45. A kit further comprising a compound according to any one of Embodiments 1-30 or a pharmaceutically acceptable salt and instructions for administering the compound or a pharmaceutically acceptable salt thereof to a patient with cancer.

An embodiment of the invention 46. A kit according to an embodiment of the invention 45, characterized in that the cancer is any one or more types of cancer from the table. 7.

An embodiment of the invention 47. A kit according to an embodiment of the invention 46, characterized in that the cancer is selected from the group consisting of a solid tumor and a lymphoma.

Embodiment 48. A kit according to any one of Embodiments 45-47, further comprising one or more additional therapeutic agents.

Since Compound (X) or a prodrug thereof (sometimes collectively referred to herein as a compound of this invention) has STING agonist activity, it may be useful as an agent for the prevention or treatment of cancer, a cancer growth inhibitor or an inhibitor of cancer metastasis.

Since the compound of this invention exhibits STING agonist activity and surpasses it in terms of expression of efficacy, pharmacokinetics (e.g. absorption, distribution, metabolism, excretion), solubility (e.g. water solubility), interactions with other drugs (e.g. , inhibitory effect on drug metabolism), safety (e.g. acute toxicity, chronic toxicity, genetic toxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity) and stability (e.g. chemical stability, enzyme stability), it can be useful in as a medicine.

Therefore, the compound of this invention can be used to increase STING activity in a mammal (eg, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human).

The compound of this invention can be used as a medicament, such as an agent for the prevention or treatment of diseases possibly affected by STING (sometimes abbreviated as STING-related diseases herein), for example, cancer [eg, various types of cancer colon and rectum (eg, colon and rectal cancer, rectal cancer, anus cancer, familial colon and rectal cancer, hereditary non-polyposis colon and rectal cancer, gastrointestinal stromal tumor), lung cancer (eg, non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (eg, pancreatic duct carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, stomach cancer (eg, papillary adenocarcinoma, mucinous glandular carcinoma ), duodenal cancer, small bowel cancer, breast cancer (e.g. p, invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (eg, ovarian epithelial cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian tumor with low malignant potential), tumor (testis, prostate cancer for example, hormone-dependent prostate cancer, non-hormone-dependent prostate cancer, castration-resistant prostate cancer), liver cancer (eg, hepatocellular cancer, primary liver cancer, extrahepatic bile duct cancer), thyroid cancer (eg, medullary thyroid carcinoma), kidney cancer (eg, renal cell carcinoma (eg, clear cell renal cell carcinoma), transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (eg, cervical cancer, uterine body cancer, uterine sarcoma), gestational choriocarcinoma, brain tumors (eg, medulloblastoma, glioma, pineal tumors, n Ilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary adenoma), retinoblastoma, skin cancer (eg, basalioma, malignant melanoma), sarcomas (eg, rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, malignant cell carcinoma) , blood cancer (eg, multiple myeloma, leukemia (eg, acute myelogenous leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disease), primary cancer of unknown origin], cancer growth inhibitor, cancer metastasis inhibitor, apoptosis stimulant, precancerous lesions (for example, myelodis-12 038805 plastic syndromes), etc.

In another embodiment of the invention, the cancer is selected from any one or more cancers from table. 7.

Table 7

adrenal cancer lymphoepithelioma adenocarcinoma lymphoma acoustic schwannoma acute lymphoblastic leukemia acral lentiginous melanoma acute myeloid leukemia acrospyrome chronic lymphocytic leukemia acute eosinophilic leukemia liver cancer acute erythroid leukemia small cell lung cancer acute lymphoblastic leukemia non-small cell lung cancer acute megakaryoblastic leukemia MALT lymphoma acute monocytic leukemia malignant fibrous histiocytoma acute promyelocytic leukemia malignant tumor of the membranes of the peripheral nerves adenocarcinoma malignant newt tumor adenocystic carcinoma mantle cell lymphoma zones adenoma B-cell lymphoma of the marginal zone adenomatoid odontogenic tumor basophilic leukemia glandular squamous cell carcinoma mediastinal embryonic cell tumor neoplasm of adipose tissue medullary breast cancer, adrenocortical carcinoma medullary thyroid cancer Adult cell leukemia / lymphoma medulloblastoma aggressive NK cell leukemia Melanoma, AIDS-related lymphoma Meningioma alveolar rhabdomyosarcoma Merkel cell cancer soft tissue alveolar sarcoma mesothelioma ameloblastic fibroma metastatic urothelial carcinoma anaplastic large cell lymphoma Muller mixed tumor anaplastic thyroid cancer swelling of the mucosa

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glands angioimmunoblastic T-cell lymphoma. multiple myeloma angiomyolipoma muscle growth angiosarcoma fungal mycosis astrocytoma myxoid liposarcoma atypical teratoid rhabdoid tumor myxoma B-cell chronic lymphocytic leukemia myxosarcoma B-cell prolymphocytic leukemia nasopharyngeal carcinoma B cell lymphoma neuroma basal cell carcinoma neuroblastoma bile duct cancer neurofibroma bladder cancer neuroma blastoma nodular melanoma bone cancer ocular cancer Brenner's tumor oligoastrocytoma Brown's tumor oligodendroglioma Burkitt's lymphoma bronchiogenic adenoma mammary cancer meningioma of the optic nerve sheath brain cancer optic nerve tumor carcinoma oral cancer carcinoma in situ osteosarcoma carcinosarcoma ovarian cancer cartilage swelling Pancost's tumor cementoma papillary thyroid cancer myeloid tumor paraganglioma chondroma pineoblastoma chordoma pineocytoma choriocarcinoma pituicitis

- 14038805

choroid plexus papilloma pituitary adenoma clear cell renal sarcoma pituitary tumor craniopharyngeoma plasmacytoma cutaneous T cell lymphoma polyembryoma cervical cancer precursor of T-lymphoblastic lymphoma colon and rectal cancer primary central nervous system lymphoma Degos syndrome primary effusion lymphoma desmoplastic small round cell tumor. primary peritoneal cancer diffuse large B cell lymphoma prostate cancer dysembryoplastic neuroepithelial tumor pancreas cancer dysgerminoma pharyngeal cancer embryonic cancer peritoneal pseudomyxoma endocrine neoplasms glands renal cell carcinoma endodermal sinus tumor renal medullary carcinoma T-cell lymphoma associated with enteropathy retinoblastoma esophageal carcinoma rhabdomyoma included fetus rhabdomyosarcoma fibroma Richter's transformation fibrosarcoma rectal cancer follicular lymphoma sarcoma follicular thyroid cancer Schwannomatosis Ganglioneuroma seminoma gastrointestinal cancer Sertoli cell tumor embryonic cell tumor germ cord tumor gonadal tumor choriocarcinoma of the uterus cricoid cell carcinoma

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giant cell fibroblastoma skin cancer giant cell bone tumor small round cell blue tumors glial tumor small cell carcinoma glioblastoma multiforme sarcoma of soft tissues glioma somatostatinoma brain gliomatosis chimney sweep cancer glucagonoma spinal cord tumor dysgerminoma lymphoma of the marginal zone of the spleen folliculoma squamous carcinoma gynandroblastoma synovial sarcoma carcinoma of the gallbladder Cesari's disease gastrointestinal cancer small bowel cancer hairy cell leukemia squamous cell carcinoma hemangioblastoma stomach cancer head and neck cancer T cell lymphoma hemangiopericytoma testicular cancer hematological malignancy tekoma hepatoblastoma thyroid cancer T-cell lymphoma of the liver and spleen transitional cell carcinoma Hodgkin's lymphoma throat cancer non-Hodgkin lymphoma urinary duct cancer invasive lobular carcinoma cancer of the genitourinary system bowel cancer urothelial carcinoma kidney cancer uveal melanoma laryngeal cancer uterine cancer malignant lentigo verrucous carcinoma fatal midline carcinoma optic pathway glioma leukemia vulvar cancer Leydig cell tumor vaginal cancer liposarcoma Waldenstrom's macroglobulinemia lung cancer bronchiogenic lymphoma lymphangioma Wilms tumor lymphangiosarcoma

In another embodiment, the cancer is a solid tumor or lymphoma.

In particular, the compound of this invention can be used as a medicament for the treatment of colon and rectal cancer, breast cancer, skin cancer, malignant lymphoma, or lung cancer.

The compound of this invention can be administered to a mammal (preferably humans) orally or parenterally as it is, or in admixture with a pharmacologically acceptable carrier as a drug.

A drug containing a compound of the present invention (hereinafter sometimes abbreviated as a drug of the present invention) is explained in detail below. Examples of the dosage form of the medicament of the present invention include oral preparations such as a tablet (e.g., sugar-coated tablet, film-coated tablet).

- 16 038805 coated, sublingual tablet, buccal, disintegrating tablet), pill, granule, powder, capsule (e.g. soft capsule, microcapsule), syrup, emulsion, suspension, films (e.g. orally disintegrating films, film binding to oral mucosa), etc. In addition, examples of the dosage form of the medicament of the present invention include parenteral preparations such as injection, drip infusion, transdermal absorption type preparation (for example, transdermal absorption type preparation for iontophoresis), suppository, ointment, nasal preparation, pulmonary preparation, ophthalmic drops, etc.

In addition, the drug of the present invention may be a controlled release preparation such as an immediate release preparation, a sustained release preparation (for example, a sustained release microcapsule), and the like.

As the dosage form of the medicament according to the present invention, a nanoparticle preparation and a preparation using a membrane obtained from bacteria can also be used.

The medicament of the present invention can be prepared according to a method known per se (for example, the method described in the Japanese Pharmacopoeia, etc.) commonly used in the field of preparation. In addition, the medicament of the present invention may contain a suitable amount of an additive such as an excipient, binder, disintegrant, lubricant, sweetener, surfactant, suspending agent, emulsifier, color, preservative, flavor, flavoring agent, stabilizer, thickener and the like, commonly used in the field of preparation as needed. Examples of a pharmacologically acceptable carrier include these additives.

For example, a tablet can be prepared using an excipient, binder, disintegrant, lubricant, and the like, and a tablet or granule can be prepared using an excipient, binder, and disintegrant. A powder or capsule can be prepared using an excipient and the like, a syrup can be prepared using a sweetener and the like, and an emulsion or suspension can be prepared using a suspending agent, surfactant, emulsifier and the like. ...

Examples of the excipient include lactose, sucrose, glucose, starch, sucrose, crystalline cellulose, powdered licorice root, mannitol, sodium bicarbonate, calcium phosphate, and calcium sulfate.

Examples of the binder contain 5 to 10 wt% liquid starch paste, 10 to 20 wt% gum arabic solution or gelatin solution, 1 to 5 wt% tragacanth solution, carboxymethyl cellulose solution, sodium alginate solution and glycerin.

Examples of the disintegrant include starch and calcium carbonate.

Examples of the lubricant include magnesium stearate, stearic acid, calcium stearate, and refined talc.

Examples of the sweetener include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, and simple syrup.

Examples of the surfactant include sodium lauryl sulfate, polysorbate 80, sorbitan mono fatty acid ester, and polyoxyl stearate 40.

Examples of the suspending agent include gum arabic, sodium alginate, sodium carboxymethyl cellulose, methyl cellulose, and bentonite.

Examples of the emulsifier include gum arabic, tragacanth, gelatin, and polysorbate 80.

For example, when the medicament of this invention is a tablet, for example, an excipient (for example, lactose, sucrose, starch), a disintegrant (for example, starch, calcium carbonate), a binder (for example, starch, gum arabic, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose) or a lubricant (e.g. talc, magnesium stearate, polyethylene glycol 6000) is added to the compound of this invention, and the mixture is compression molded according to a method known per se, and then, if necessary, the molded product is coated according to the method known to mask taste, entericity or longevity to form a tablet.

As the coating agent for coating, for example, hydroxypropyl methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate ) and a pigment (eg red iron oxide, titanium dioxide).

Examples of injections include intravenous injection as well as subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, drip injection, and the like.

Such injections are obtained according to a method known per se, or by dissolving, suspending or emulsifying a compound of this invention in a sterilized aqueous or oily liquid. Examples of aqueous fluid include saline, isotonic

- 17 038805 solutions containing glucose or other auxiliary drugs (for example, D-sorbitol, D-mannitol, sodium chloride), etc. The aqueous liquid may contain a suitable solubilizing agent such as alcohol (eg ethanol), polyhydric alcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant (eg polysorbate 80, HCO-50), and the like. Examples of the oil liquid include sesame oil, soybean oil, and the like.

The oily liquid may contain a solubilizing agent. Examples of the solubilizing agent include benzyl benzoate, benzyl alcohol and the like. In addition, the injection can be mixed with a buffer (eg, phosphate buffer, sodium acetate buffer), a sedative (eg, benzalkonium chloride, procaine hydrochloride), a stabilizer (eg, human serum albumin, polyethylene glycol), a preservative (eg, benzyl alcohol, phenol ) etc. The resulting injection can usually be filled into an ampoule.

Although the content of the compound of this invention in the medicament of this invention varies depending on the form of the pharmaceutical preparation, it is usually from about 0.01 to about 100 wt%, preferably from about 2 to about 85 wt%, more preferably from about 5 to about 70 wt.% Based on the entire preparation.

Although the content of the additive in the medicament of this invention varies depending on the form of the pharmaceutical preparation, it is usually from about 1 to about 99.9 wt%, preferably from about 10 to about 90 wt%, based on the total formulation.

The compound of this invention is stable and low toxic and can be used safely. Although the daily dose of the compound of this invention varies depending on the condition and body weight of the patients, the type of compound, the route of administration, and the like, in the case of, for example, oral administration to patients for the treatment of cancer, the daily dose for an adult (body weight about 60 kg) is from about 1 to about 1000 mg, preferably from about 3 to about 300 mg, more preferably from about 10 to about 200 mg of a compound of this invention, which may be administered in a single dose or 2 or 3 servings per day.

When a compound of this invention is administered parenterally, it is usually administered in the form of a liquid (eg, injection). Although the dose of a compound of this invention varies depending on the subject of administration, target organ, symptom, route of administration, and the like, it is, for example, from about 0.01 to about 100 mg, preferably from about 0.01 to about 50 mg, more preferably from about 0.01 to about 20 mg, based on 1 kg of body weight, which is administered by intravenous injection.

The compound of this invention can be used concurrently with other drugs.

Specifically, the compound of this invention can be used in conjunction with drugs such as hormone therapeutic agents, chemotherapeutic agents, immunotherapeutic agents, drugs that inhibit the action of cell growth factors or cell growth factor receptors, and the like.

In the following, drugs that can be used in combination with a compound of this invention are abbreviated as concomitant drugs.

Examples of hormonal therapeutic agents include phospestrol, diethylstilbestrol, chlorotrianisene, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone, mepartricin, raloxifene, ormeloxifene pill form, mepitiostane, testrolactone, amiglutethimide, LH-RH agonists (eg goserelin acetate, buserelin, leuprorelin, leuprorelin acetate), droloxifene, epitiostanol, ethinylestradiol retrosolate, aromatase inhibitors, vorosetol, eczemastrozol formestane), antiandrogens (for example, flutamide, bikartamide, nilutamide, enzalutamide), 5a-reductase inhibitors (for example, finasteride, episteride, dutasteride), adrenal hormone preparations (for example, dexamethasone, prednisolone, betamethasone synthesis), triamitcinors for example, abiraterone), a retinoid and drugs that slow down metabolism of retinoids (eg liarozol), thyroid hormone and their DDS (drug delivery system) preparations.

Examples of chemotherapeutic agents include alkylating agents, antimetabolites, anticancer antibiotics, and plant derived anticancer agents.

Examples of alkylating agents include nitrogen mustard, nitrogen mustard-K-oxide hydrochloride, chlorambutyl, cyclophosphamide, ifosfamide, thiotepa, carboquone, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melphalan, dnimacustinustin sodium, fosfamine , streptozocin, pipobroman, etoglucid, carboplatin, cisplatin, miboplatin, nedaplatin, oxaliplatin, altretamin, ambamustin, dibrosidium hydrochloride, fotemustin, prednimustin, pumitepa, ribomustin, temozolomide DS, trosulfamidin ...

Examples of antimetabolites include mercaptopurine, 6-mercaptopurine riboside, thioinosine, methotrexate, pemetrexed, enocitabine, cytarabine, cytarabine octophosphate, antcitabine hydrochloride, drug-18 038805 veins 5-FU, emitfur, capecitabine), aminopterin, nelzarabine, leucovorin-calcium, tabloid, butocin, calcium folinate, calcium levofolinate, cladribine, emitfur, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, pyritrexime, idoxazuridamine, thiustazoguidine

DDS.

Examples of antineoplastic antibiotics include actinomycin-D, actinomycin-C, mitomycin-C, chromomycin-A3, bleomycin hydrochloride, bleomycin sulfate, peplomycin sulfate, daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin pyrochloride hydrochloride, mitomycin hydrochloride carcinophilin, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride, and their DDS preparations (eg, PEG liposome containing doxorubicin).

Examples of herbal anticancer agents include etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, cabazitaxel, vinorelbine, and their DDS preparations.

Examples of immunotherapeutic agents (BRM) include picibanil, krestin, sizofiran, lentinan, ubenimex, interferons, interleukins, macrophage colony-stimulating factor, granulocyte colony-stimulating factor, erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium antibacterial -CTLA4 (e.g. ipilimumab, tremelimumab), anti-PD-1 antibodies (e.g. nivolumab, pembrolizumab) and anti-PD-L1 antibody.

Examples of cell growth factors in drugs that inhibit the action of cell growth factors or cell growth factor receptors include any substances that promote cell proliferation, which are usually peptides having molecular weights of no more than 20,000, which are able to exert their activity at low concentrations by binding with a receptor, including (1) EGF (epidermal growth factor) or substances having substantially the same activity as EGF [e.g. TGFa], (2) insulin or substances having substantially the same activity as insulin [e.g. insulin, IGF (insulin-like growth factor) -1, IGF-2], (3) FGF (fibroblast growth factor) or substances having essentially the same activity as FGF [for example , acidic FGF, basic FGF, KGF (keratinocyte growth factor), FGF-10], and (4) other cell growth factors (e.g. CSF (colony stimulating factor), EPO (erythropoietin), IL-2 (interleukin-2), NGF (growth factor is not moats), PDGF (platelet growth factor), TGFe (transforming growth factor β), HGF (hepatocyte growth factor), VEGF (vascular endothelial growth factor), heregulin, angiopoietin].

Examples of cell growth factor receptors include any receptor capable of binding to the aforementioned cell growth factors, including an EGF receptor, a heregulin receptor (e.g., HER3), an insulin receptor, an IGF-1 receptor, an IGF-2 receptor, an FGF-1 receptor, or an FGF-β receptor. 2, VEGF receptor, angiopoietin receptor (e.g., Tie2), PDGF receptor and the like.

Examples of drugs that inhibit the action of cell growth factors or cell growth factor receptors include EGF inhibitor, TGFa inhibitor, heregulin inhibitor, insulin inhibitor, IGF inhibitor, FGF inhibitor, KGF inhibitor, CSF inhibitor, EPO inhibitor, IL-2 inhibitor, NGF inhibitor, inhibitor PDGF, TGFe inhibitor, HGF inhibitor, VEGF inhibitor, angiopoietin inhibitor, EGF receptor inhibitor, HER2 inhibitor, HER4 inhibitor, insulin receptor inhibitor, IGF-1 receptor inhibitor, IGF-2 receptor inhibitor, FGF receptor-1 inhibitor, FGF receptor-2 inhibitor , FGF receptor-3 inhibitor, FGF receptor-4 inhibitor, VEGF receptor inhibitor, Tie-2 inhibitor, PDGF receptor inhibitor, Abl inhibitor, Raf inhibitor, FLT3 inhibitor, c-Kit inhibitor, Src inhibitor, PKC inhibitor, Smo inhibitor, inhibitor ALK, ROR1 inhibitor, Trk inhibitor, Ret inhibitor, mTOR inhibitor, Aurora inhibitor, PLK inhibitor, MEK inhibitor (MEK1 / 2), MET inhibitor, CDK inhibitor, Akt inhibitor, ERK inhibitor, PI3K inhibitor, and etc.

More specifically, anti-VEGF antibody (eg, bevacizumab, ramucirumab), anti-HER2 antibody (eg, trastuzumab, pertuzumab), anti-EGFR antibody (eg, cetuximab, panitumumab, matuzumab, nimotuzumab), anti-HGF antibody, imlotinitinab gefitinib, sorafenib, sunitinib, dasatinib, lapatinib, vatalanib, ibrutinib, bosutinib, cabozantinib, crizotinib, alectinib, vismodegib, axitinib, motesanib, nilotinib, 6- [4-iperazyl-4-phylline-ethyl 1 (R) -phenylethyl] 7H-pyrrolo [2,3-d] pyrimidin-4-amine (AEE-788), vandetanib, temsirolimus, everolimus, enzastaurin, tozasertib, 2- [N- [3- [4- [ 5- [N- (3-fluorophenyl) carbamoylmethyl] -1H-pyrazol-3-ylamino] quinazolin-7yloxy] propyl] -N-ethylamino] ethyl phosphate (AZD-1152), 4- [9-chloro-7- ( 2,6-difluorophenyl) -5H-pyrimido [5,4-d] [2] benzazepin-2-ylamino] benzoic acid, sodium salt N- [2-methoxyu-5 - [(E) -2- (2, 4,6trimethoxyphenyl) vinylsulfonylmethyl] phenyl] glycine (ON-1910Na), volasertib, selumetinib, trametinib, N- [2 (R), 3-dihydroxypropoxy] -3,4-difluoro-2- (2-fluoro-4 -iodophenylamino) benzamide (PD-0325901), bosutinib, regorafenib, afatinib, idealisib, Ceritinib, dabrafenib, etc.

In addition to the above drugs, L-asparaginase, L-arginase, arginine deiminase,

- 19 038805 aceglatone, procarbazine hydrochloride, protoporphyrin-cobalt complex salt, sodium mercury hematoporphyrin, topoisomerase I inhibitors (eg irinotecan, topotecan, indotecan, indimitecan), topoisomerase D inhibitors, retuserase II ), other inhibitors of angiogenesis (eg humagillin, shark extract), COX-2 inhibitor), α-blockers (eg, tamsulosin hydrochloride), bisphosphonic acids (eg pamidronate, zoledronate), thalidomide, lenalidomide, pomalidomide, azacitidine, decitabine proteasome inhibitors (e.g. bortezomib, carfilzomib, ixazomib), NEDD8 inhibitors (e.g. pevoneedistat), UAE inhibitors, PARP inhibitors (e.g. olaparib, niraparib, veliparib), anti-tumor antibodies such as anti-CD20 antibodies (e.g. rituxime obinutuzumab), anti-CCR4 antibodies (e.g. mogamulizumab), etc., antibody-drug conjugates (e.g. trastuzumab emtansine, brentuximab vedotin) etc. can also be used as a concomitant medication.

By combining a compound of this invention and a concomitant drug, an improved effect can be achieved, such as (1) the dose can be reduced compared to a single administration of a compound of this invention or a concomitant drug, (2) a drug to be combined with the compound of this invention can be selected according to the condition of the patients (mild case, severe case, etc.), (3) the treatment period can be set longer, (4) long-term treatment, the effect can be planned, (5) a synergistic effect can be achieved by the combined use of a compound of this invention and a concomitant preparation.

As used herein, a compound of this invention and a concomitant drug used in combination are referred to as a combination agent of this invention.

For the use of the combination agent of this invention, the time of administration of the compound of this invention and the concomitant drug is not limited, and the compound of this invention and the concomitant drug may be administered to the subject of administration at the same time or may be administered at different times. When administered at a time interval, the interval differs depending on the effective component to be administered, dosage form and route of administration, and, for example, when the concomitant drug is administered first, the compound of this invention can be administered within a time range of 1 min to 3 days, preferably 10 minutes to 1 day, more preferably 15 minutes to 1 hour after taking the concomitant preparation. When the compound of this invention is administered first, the concomitant drug is administered at a time interval of 1 minute to 1 day, preferably 10 minutes to 6 hours, more preferably 15 minutes to 1 hour, after administration of the compound of this invention. The dosage of the concomitant drug can be determined in accordance with the clinically determined dose, and can be appropriately selected depending on the subject of administration, route of administration, disease, combination, and the like.

Examples of a route of administration for the combined use of a compound of this invention and a concomitant drug include the following routes.

(1) A compound of the present invention and a concomitant drug are simultaneously prepared to produce a single drug, which is then administered.

(2) The compound of the present invention and the concomitant drug are prepared separately to obtain two kinds of drugs that are administered simultaneously by the same administration route.

(3) The compound of the present invention and the concomitant drug are prepared separately to obtain two kinds of drugs that are administered at different times by the same administration route.

(4) The compound of the present invention and the concomitant drug are prepared separately to obtain two kinds of drugs that are administered simultaneously by different routes of administration.

(5) The compound of the present invention and the concomitant drug are prepared separately to prepare two kinds of drugs that are administered by different routes of administration at different times (for example, the compound of the present invention and the concomitant drug are administered in this order or in reverse order).

The dose of the concomitant drug can be appropriately determined according to its clinical dose, and the ratio of the compound of this invention to the concomitant drug can be appropriately determined depending on the subject of administration, route of administration, target disease, symptom, combination, and the like. ... For example, when the subject of administration is a human, the concomitant drug is administered in an amount of 0.01 to 100 wt. relative to 1 wt.d. compounds of this invention.

In addition, the compound of the present invention or the combination agent of the present invention can be used concurrently with non-drug therapy. To be precise, a compound of this invention or a combination agent of this invention can be combined with non-drug therapy such as (1) surgery, (2) hypertensive

- 20 038805 chemotherapy using angiotensin II, etc., (3) gene therapy, (4) thermotherapy, (5) cryotherapy, (6) laser moxibustion and (7) radiation therapy.

For example, using a compound of this invention or a combination agent of this invention before or after the above operation, and the like. or before or after combined treatment of two or three of its types, effects such as prevention of the emergence of resistance, prolongation of disease-free survival, suppression of metastasis or recurrence of cancer, prolongation of life, and the like are observed.

In addition, it is possible to combine treatment with a compound of this invention or a combination agent of this invention with maintenance therapy [(i) administration of an antibiotic (e.g., β-lactam type such as pansporin and the like, macrolide type such as clarithromycin and the like) for complications of various infectious diseases, (ii) the administration of a high-calorie transfusion, an acid preparation or a general vitamin preparation to improve malnutrition, (iii) the administration of morphine to reduce pain, (iv) the administration of a pharmaceutical agent to alleviate side effects such as nausea, vomiting, anorexia, diarrhea, leukopenia, thrombocytopenia, decreased hemoglobin concentration, hair loss, hepatopathy, renopathy, disseminated intravascular coagulation, fever, etc., (v) administration of a pharmaceutical agent to suppress multidrug resistance of cancer, and etc.].

Examples of

This description is explained in detail with reference to the following examples, experimental examples and examples of drugs, which should not be construed as limiting, and the description can be changed within the scope of this description.

In the following examples, room temperature usually means about 10 to about 35 ° C. The ratios shown for mixed solvents are by volume, unless otherwise indicated. % means wt.%, unless otherwise indicated.

In silica gel column chromatography, Diol means the use of silica gel coupled with 3- (2,3-dihydroxypropoxy) propylsilane. In silica gel column chromatography and HPLC (high performance liquid chromatography), C18 means using octadecyl-linked silica gel.

Elution solvent ratios are volume ratios unless otherwise indicated.

LC / MS measurement was carried out under the following conditions [column: L-column 2 ODS, 3.0 mm I.D. χ 50 mm, mobile phase: acetonitrile / 5 mM ammonium acetate buffer solution = 900/100)].

The examples use the following abbreviations: so pl. - melting point,

MS - mass spectrum,

[M + H] +, [MH] ^- - molecular ion peak,

M - molar concentration,

N - normal,

CDC13 - deuterated chloroform,

DMSO-d ₆ - deuterated dimethyl sulfoxide,

D ₂ O - deuterated water,

1H NMR - proton nuclear magnetic resonance, ³¹ P NMR - phosphorus nuclear magnetic resonance,

LC / MS - liquid chromatography-mass spectrometry,

IER - electrospray ionization,

APCI - atmospheric pressure chemical ionization,

THF - tetrahydrofuran,

DMF - Ν, Ν-dimethylformamide,

DXM stands for dichloromethane.

1H NMR and ³¹ P-NMR was measured using NMR Fourier transform. ACD / SpecManager (trade name), etc. were used for the analysis. Peaks with highly mobile protons such as hydroxy, amino and the like are not described.

MS was measured by LC / MS. The ionization method used was the IES method or the APCI method. The data indicates the actual measured value (found). A molecular ion peak is usually observed. In the case of a compound having a tert-butoxycarbonyl group, a peak after removal of the tert-butoxycarbonyl group or the tert-butyl group can be observed as a fragmentary ion. In the case of a compound having a hydroxy group, a peak after removal of H ₂ O can be observed as a fragmented ion. In the case of a salt, a molecular ion peak or a free-form fragment ion peak is usually observed.

Example 1. Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro2,10,16-trihydroxy -2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadi-21 038805 phosphacyclotetradecin-7-yl) -5-fluoro -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt

A) 7 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) -5-fluoro-3H- pyrrolo [2,3-d] pyrimidin-4 (7H) -one.

To a solution of 7 - ((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -5-fluoro-3Hpyrrolo [2,3-d] pyrimidine-4 (7H ) -one (6.26 g) in pyridine (100 ml) was added 4,4'-dimethoxytrityl chloride (8.92 g) at room temperature and the mixture was stirred overnight under argon at room temperature. To the reaction mixture was added a saturated aqueous sodium hydrogen carbonate solution, and the mixture was extracted with ethyl acetate. The organic layer was sequentially washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (8.65 g). MS: [MH] ^- 586,0.

B) 7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3-O- (tert-butyl (dimethyl) silyl) -β-D-ribofuranosyl) -5-fluoro-3 , 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

To a solution of 7 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) -5-fluoro-3H- pyrrolo [2,3-d] pyrimidin-4 (7H) -one (2.65 g) in DMF (20 ml), imidazole (0.614 g) and tert-butyldimethylchlorosilane (0.816 g) were added, the mixture was stirred overnight

- 22 038805 at room temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (1.1 g).

MS: [MH] 700.2.

C) 7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3-O- (tert-butyl (dimethyl) silyl) -2-O - ((2-cyanoethoxy) (diisopropylamino) phosphino ) -β-D-ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3-O- (tert-butyl (dimethyl) silyl) -β-D-ribofuranosyl) 5-fluoro-3,7-dihydro- 4H-pyrrolo [2,3-d] pyrimidin-4-one (5.43 g) was azeotroped three times with anhydrous toluene and dissolved in anhydrous DMF (15 ml). To the solution were added 3 ((bis (diisopropylamino) phosphino) oxy) propanenitrile (3.19 ml), III-tetrazole (0.542 g) and 1-methyl-Shimidazole (0.306 ml), the mixture was stirred overnight in an argon atmosphere at room temperature. temperature, poured into saturated aqueous sodium hydrogen carbonate solution and extracted with ethyl acetate.

The organic layer was sequentially washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (DIOL, ethyl acetate / hexane). The resulting crude product was purified again by silica gel column chromatography (ethyl acetate / hexane containing 0.5% triethylamine) to give the title compound (4.22 g) as a mixture of two diastereomers. MS: [MH] ^- 901.2.

D) (2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -2 - ((((((2R, 3R, 4R, 5R) -4 - ((tert -butyldimethylsilyl) oxy) -2- (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -5- (hydroxymethyl) tetrahydrofuran-3yl) oxy) (2 -cyanoethoxy) phosphoryl) oxy) methyl) -4-fluorotetrahydrofuran-3-yl hydrophosphonate.

N-Benzoyl-2'-deoxy-2'-fluoro-3'-O- (hydroxy (oxide) phosphoranyl) adenosine (1.2 g) and 7- (5-O - ([6is (4methoxyphenyl) (phenyl) methyl)] - 3-O - ([tert-butyl (dimethyl) silyl)] - 2-O - ({(2-cyanoethoxy) ([diisopropylamino)] phosphino)} - β-D-ribofuranosyl) -5-fluoro -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (3.4 g) was azeotropically dried with anhydrous acetonitrile and anhydrous acetonitrile (15 ml) and anhydrous THF (5 ml ). To the mixture was added a mixture of 5- (ethylsulfanyl) -2H-tetrazole (1.07 g) (which was azeotropically dried with anhydrous acetonitrile) and anhydrous acetonitrile (10 ml), and the mixture was stirred under argon at 55 ° C for 2 h. To this was added a 70% aqueous solution of tert-butyl hydroperoxide (1.12 ml) and the mixture was stirred at room temperature for 20 minutes. To the reaction mixture was added a mixture of sodium thiosulfate (5920 mg) and water (3 ml), and the mixture was concentrated under reduced pressure. To the residue was added 80% acetic acid (30 ml) and the mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (1.1 g). MS: [M + H] + 952.2.

E) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16 - ((tert-butyl (dimethyl) silyl) oxy) -15-fluoro-7- (5fluoro- 4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2,10-dihydroxy-2,10-dioxidooctahydro12H-5,8-methanofuro [3,2-l ] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purine-6yl) benzamide.

(2R, 3R, 4R, 5R) -5- (6-Benzαmido-9H-purin-9-yl) -2 - ((((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl ) oxy) -2- (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -5- (hydroxymethyl) tetrahydrofuran-3yl) oxy) (2-cyanoethoxy ) phosphoryl) oxy) methyl) -4-fluorotetrahydrofuran-3-yl hydrophosphonate (350 mg) was subjected to azeotropic drying with anhydrous acetonitrile and anhydrous pyridine, and anhydrous pyridine (15 ml) was added thereto. To the mixture was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide (238 mg) and the mixture was stirred under argon at room temperature for 1 hour. Water (232 μL) was added thereto and iodine (121 mg) and the mixture was stirred at room temperature for an additional 20 minutes. To the reaction mixture was added a mixture of sodium thiosulfate pentahydrate (91 mg) and water (1 ml), and the mixture was stirred at room temperature for 5 minutes. Toluene was added thereto, and the mixture was concentrated under reduced pressure. Anhydrous acetonitrile (15 ml) and 2-methylpropan-2-amine (5.26 ml) were added to the residue and the mixture was stirred at room temperature for 2 hours, concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (332 mg). MS: [M + H] + 897.1.

F) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -16 - ((tert-butyl (dimethyl) silyl ) oxy) -15-fluoro-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin -7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

To N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16 - ((tert-butyl (dimethyl) silyl) oxy) -15-fluoro-7- (5fluoro-4 -oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2,10-dihydroxy-2,10-dioxidooctahydro12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purine-6yl) benzamide (332 mg) was added 40% ethanolic methylamine solution (7.6 ml) and the mixture was stirred

- 23 038805 argon atmosphere at room temperature for 1 hour, concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) and the resulting residue was purified by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution) to give the title compound ( 106.4 mg). MS: [M + H] + 793.1.

G) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) - 15-fluoro-2,10,16-tri - hydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3 , 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt.

K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -16 - ((tert-butyl (dimethyl) silyl) oxy) -15-fluoro-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (106.4 mg) was added triethylamine trihydrofluoride (1094 μl). The reaction mixture was stirred at 50 ° C for 1 h and cooled to room temperature, ethoxy (trimethyl) silane (6266 μL) was added to it and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was added ethoxy (trimethyl) silane (6266 μl) and methanol (1 ml), stir the mixture at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by C18 column chromatography (acetonitrile / 10 mM acetate buffer triethylammonium) to give the title compound (82 mg).

¹ H NMR (400 MHz, D ₂ O) δ 1.18 (18H, t, J = 7.3 Hz), 3.11 (12H, q, J = 7.4 Hz), 4.01-4, 14 (2H, m), 4.17-4.26 (1H, m), 4.34 (2H, d, J = 3.4Hz), 4.42-4.50 (1H, m), 4 , 54 (1H, d, J = 4.2Hz), 4.86-4.99 (2H, m), 5.38-5.60 (1H, m), 6.30-6.45 (2H , m), 7.24 (1H, d, J = 2.0 Hz), 7.91 (1H, s), 8.05 (1H, s), 8.17 (1H, s).

³¹ P NMR (162 MHz, D2O) δ -2,16, -1,66.

Example 1a. Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro2,10,16-trihydroxy-2, 10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro- 4H-pyrrolo [2,3-d] pyrimidin-4-one disodium salt

Deionized water (480 ml) was passed through a column prepared by loading 50W-X8 AG (trade name) cation exchange resin (100-200 mesh, 30 g) into an empty column. A 1M aqueous sodium hydroxide solution (288 ml) and deionized water (540 ml) were then passed through the resin. Deionized water (54 ml) containing 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) - 15-fluoro-2.10 , 16-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (1.53 g) after the above pretreatment was passed through the resin, and deionized water (72 ml) was passed through the resin, obtained the aqueous solution was freeze-dried to give the title compound (1.28 g).

1H NMR (300 MHz, D2O) δ 4.05-4.17 (2H, m), 4.20-4.28 (1H, m), 4.33-4.40 (2H, m), 4, 44-4.51 (1H, m), 4.55 (1H, d, J = 4.2 Hz), 4.84-5.01 (2H, m), 5.39-5.61 (1H, m), 6.31-6.40 (2H, m), 7.25 (1H, d, J = 2.0Hz), 7.91 (1H, s), 8.07 (1H, s), 8.18 (1H, s).

³¹ P NMR (121 MHz, D2O) δ -2,2, -1,6.

Example 2. Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro10,16-dihydroxy-2 , 10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3 , 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt (optical isomer)

- 24 038805

О 5) MeNH ₂

A) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -16 - ((tert-butyl (dimethyl) silyl ) oxy) -15-fluoro-10-hydroxy-2, 10-dioxido-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10 ] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer) (2R, 3R, 4R, 5R) -5- ( 6-benzamido-9H-purin-9-yl) -2 - (((((2R, 3R, 4R, 5R) -4 ((tert-butyldimethylsilyl) oxy) -2- (5-fluoro-4-oxo -3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) -4-fluorotetrahydrofuran- 3-yl hydrogenphosphonate (700 mg) was azeotropically dried with anhydrous acetonitrile and anhydrous pyridine, and anhydrous pyridine (50 ml) was added thereto. To the mixture at room temperature was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide (475 mg) and the mixture was stirred under argon at room temperature for 1 hour. Water (464 μl) and 3H-benzo [c] [1,2] dithiol-3-one (186 mg), the mixture was stirred at room temperature for an additional 30 min. To the reaction mixture was added a mixture of sodium thiosulfate (913 mg) and water (3 ml), and the mixture was concentrated under reduced pressure. Anhydrous acetonitrile (30 ml) and 2-methylpropan-2-amine (10 ml) were added to the residue, the mixture was stirred at room temperature for 1 hour, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate), and a 40% ethanolic methylamine solution (7.3 ml) was added to the resulting residue. The mixture was stirred under argon at room temperature for 30 minutes and the reaction mixture was concentrated under reduced pressure. The residue was purified using silica gel column chromatography (methanol / ethyl acetate). The resulting residue was divided into two diastereomers (tR1 and tR2, the retention times of which from LC / MS from shorter to longer in this order) by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution) to give the title compound (70 mg, tR1) and the title compound (150 mg, tR2). MS (tR1): [M + H] + 809.1. MS (tR2): [M + H] + 809.1.

B) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-10,16-dihydroxy2,10 -dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -16 - ((tert-butyl (dimethyl) silyl) oxy) -15-fluoro-10-hydroxy-2, 10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer) (70 mg, obtained from tR1) methanol (3.0 ml) and triethylamine trihydrofluoride (1.41 ml). The reaction mixture was concentrated to remove methanol and the residue was stirred at 55 ° C for 1 hour. The residue was cooled to room temperature, ethoxy (trimethyl) silane (7.8 ml) was added thereto and the mixture was stirred at room temperature for 2 hours. the mixture was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (acetonitrile / 10 mM triethylammonium acetate buffer) to give the title compound (56 mg).

1H NMR (400 MHz, D2O) δ 1.23 (18H, t, J = 7.3 Hz), 3.15 (12H, q, J = 7.3 Hz), 4.15-4.23 (3H , m), 4.37-4.50 (2H, m), 4.54 (1H, d, J = 10.3 Hz), 4.61 (1H, d, J = 3.9 Hz), 4 , 96 (2H, dt, J = 7.9, 3.9 Hz), 5.77-5.95 (1H, m), 6.36 (1H, d, J = 8.1 Hz), 6, 40 (1H, d, J = 15.4 Hz), 7.51 (1H, d, J = 1.7 Hz), 7.99 (2H, d, J = 13.7 Hz), 8.23 ( 1H, s).

³¹ P NMR (162 MHz, D2O) δ -2,43, 54,03.

Example 3. Synthesis of 7 - ((2R, 5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro10,16-dihydroxy -2,10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one-1,7 triethylamine salt

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K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -16 - ((tert-butyl (dimethyl) silyl) oxy) - 15-fluoro-10-hydroxy-2,10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer) (150 mg, obtained from tR2) methanol (3.0 ml) and triethylamine trihydrofluoride (3.02 ml). The reaction mixture was concentrated to remove methanol and the residue was stirred at 55 ° C for 1 hour. The residue was cooled to room temperature, ethoxy (trimethyl) silane (16.4 ml) was added thereto and the mixture was stirred at room temperature for 2 hours. the mixture was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (acetonitrile / 10 mM triethylammonium acetate buffer) to give the title compound (155 mg).

1H NMR (400 MHz, D ₂ O) δ 1.23 (15H, t, J = 7.3 Hz), 3.15 (10H, q, J = 7.3 Hz), 4.06 (1H, dd , J = 11.7, 4.9 Hz), 4.18 (1H, dd, J = 11.6, 2.8 Hz), 4.29-4.47 (3H, m), 4.51 ( 1H, d, J = 8.8 Hz), 4.58 (1H, d, J = 3.9 Hz), 4.93-5.14 (2H, m), 5.44-5.64 (1H , m), 6.33-6.45 (2H, m), 7.32 (1H, d, J = 1.5 Hz), 7.95 (1H, s), 8.06 (1H, s) , 8.24 (1H, s).

³¹ P NMR (162 MHz, D ₂ O) δ -2.41, 55.33.

Example 3a. Synthesis 7 - ((2R, 5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro10,16-dihydroxy-2, 10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3, 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one disodium salt

Deionized water (400 ml) was passed through a column prepared by loading 50W-X8 AG (trade name) cation exchange resin (100-200 mesh, 25.3 g) into an empty column. A 1M aqueous sodium hydroxide solution (240 ml) and deionized water (450 ml) were then passed through the resin. Deionized water (45 ml) containing 7 - ((2R, 5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) - 15-fluoro-10 , 16-dihydroxy-2,10-dioxido-2-sulfanyloctahydro-12H-5,8methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5 -Fluoro-3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (1.30 g) after the above pretreatment was passed through the resin and deionized water (60 ml) was passed through the resin to obtain an aqueous solution containing the title compound.

Deionized water (400 ml) was passed through a column prepared by loading 50W-X8 AG (trade name) cation exchange resin (100-200 mesh, 26.3 g) into an empty column. A 1M aqueous sodium hydroxide solution (270 ml) and deionized water (540 ml) were then passed through the resin. Deionized water (54 ml) containing 7 - ((2R, 5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) - 15-fluoro-10 , 16-dihydroxy-2,10-dioxido-2-sulfanyloctahydro-12H-5,8methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5 -fluoro-3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (1.18 g) after the above pretreatment was passed through the resin and deionized water (63 ml) was passed through the resin to obtain an aqueous solution containing the title compound. Two aqueous solutions containing the title compound were combined and freeze-dried to give the title compound (2.0 g).

1H NMR (300 MHz, D ₂ O) δ 3.97-4.07 (1H, m), 4.11-4.20 (1H, m), 4.27-4.42 (3H, m), 4.43-4.51 (1H, m), 4.55 (1H, d, J = 3.8 Hz), 4.88-5.12 (2H, m), 5.34-5.59 ( 1H, m), 6.30-6.40 (2H, m), 7.28 (1H, d, J = 1.9 Hz), 7.91 (1H, s), 8.01 (1H, s ), 8.18 (1H, s).

³¹ P NMR (121 MHz, D ₂ O) δ -2.38, 55.3.

Example 4. Synthesis 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -2,10,15,16 tetrahydroxy-2 , 10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro -4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt

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A) (2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 ((((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -2- (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin7 (4H) -yl) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate.

N-Benzoyl-2'-O- (tert-butyl (dimethyl) silyl) -3'-O- (hydroxy (oxide) phosphoranyl) adenosine (369 mg) and 7- (5-O- (bis (4-methoxyphenyl ) (phenyl) methyl) -3-O- (tert-butyl (dimethyl) silyl) -2-O - ((2-cyanoethoxy) (diisopropylamino) phosphino) -β-D-ribofuranosyl) -5-fluoro-3, 7-dihydro-4H-pyrrolo [2,3-d] pyrimidine-4one (817 mg) was azeotropically dried with anhydrous acetonitrile (three times) and suspended in anhydrous acetonitrile (3.6 ml) and anhydrous THF (1.2 ml) ... To the suspension was added a mixture of 5 (ethylsulfanyl) -2H-tetrazole (262 mg) (which was azeotropically dried with anhydrous acetonitrile) and anhydrous acetonitrile (2.4 ml), and the mixture was stirred under argon at room temperature for 1.5 h. To the reaction solution was added a 70% aqueous solution of tert-butyl hydroperoxide (0.276 ml), and the mixture was stirred at room temperature for an additional 40 minutes. The reaction mixture was quenched with sodium thiosulfate (636 mg) and water (2 ml) and the solvent was evaporated under reduced pressure. The residue was dissolved in 80% acetic acid (5 ml) and the solution was stirred at room temperature for 2.5 hours. The reaction mixture was concentrated under reduced pressure and the residue was dried azeotropically with anhydrous acetonitrile and toluene. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (538 mg). MS: [M + H] + 1064.3.

B) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) - 7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [ 3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14yl) -9H-purin-6-yl) benzamide.

(2R, 3R, 4R, 5R) -5- (6-Benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 ((((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -2- (5-Fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin7 (4H) -yl) -5- (hydroxymethyl) tetrahydrofuran- 3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate (538 mg) was azeotropically dried with anhydrous acetonitrile and anhydrous pyridine and suspended in anhydrous pyridine (12 ml). To it was added 2-chloro-5,5dimethyl-1,3,2-dioxaphosphinane-2-oxide (327 mg) and the mixture was stirred at room temperature for 1 hour. Water (0.319 ml) and iodine (167 mg ) and the mixture was stirred at room temperature for an additional 30 minutes. The reaction mixture was quenched with sodium thiosulfate (208 mg) and water (2 ml) and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (509 mg). MS: [M + H] ⁺ 1062.3.

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C) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl ( dimethyl) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-2,10 dioxidooctahydro-12Н-5,8-methanofuro [3, 2-1] [1,3,6,9, P, 2,10] pentaoxadiphosphacyclotetradecin-14yl) -9Н-purin-6-yl) benzamide (509 mg) was dissolved in 33% ethanol solution of methylamine (10 ml), solution stirred under argon at room temperature for 1 hour and the solvent was evaporated off under reduced pressure. The residue was purified using silica gel column chromatography (methanol / ethyl acetate). The resulting residue was purified by HPLC (L-2 ODS column, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution) to give the title compound (227 mg). MS: [M + H] + 905.2.

D) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -2.10,15,16-tetrahydroxy2,10 -dioxide octahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt.

K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl (dimethyl ) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 -yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (227 mg) was added triethylamine trihydrofluoride (0.818 ml) and the mixture was stirred at 50 ° C for 5 h. The solvent was evaporated under reduced pressure, the residue was neutralized with 1M aqueous solution of triethylammonium hydrogencarbonate and the solvent was again evaporated under reduced pressure. The residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and the resulting solid was freeze-dried to give the title compound (103 mg).

1H NMR (300 MHz, D ₂ O) δ 1.26 (18H, t, J = 7.4 Hz), 3.18 (12H, q, J = 7.4 Hz), 4.06-4.29 (3H, m), 4.34-4.52 (3H, m), 4.62 (1H, d, J = 3.8 Hz), 4.69-4.83 (1H, m), 4, 84-4.95 (1H, m), 4.98-5.09 (1H, m), 6.14 (1H, d, J = 1.5Hz), 6.41 (1H, dd, J = 8.3, 1.5 Hz), 7.33 (1H, d, J = 1.9 Hz), 7.99 (1H, s), 8.16 (1H, s), 8.25 (1H, With).

³¹ P NMR (121 MHz, D ₂ O) δ -1.96, -1.21.

Example 5.7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -15,16-dihydroxy2,10-dioxido 2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7- dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one di-triethylamine salt (optical isomer)

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A) 7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3-O- (tert-butyl (dimethyl) silyl) -2-O- (hydroxy (oxide) phosphoranyl) -β- D-ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3-O- (tert-butyl (dimethyl) silyl) -β-D-ribofuranosyl) 5-fluoro-3,7-dihydro- 4H-pyrrolo [2,3-d] pyrimidin-4-one (835 mg) was dissolved in pyridine (4 ml) and diphenyl phosphite (0.456 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. To this were added water (1 ml) and ammonium hydroxide (2 ml). The mixture was stirred at room temperature for 30 min and the reaction mixture was diluted with water, extracted with ethyl acetate. The extract was dried over anhydrous magnesium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (611 mg). MS: [MH] ^- 764.1.

B) 7- (3-O- (tert-butyl (dimethyl) silyl) -2-O- (hydroxy (oxide) phosphoranyl) -β-D-ribofuranosyl) -5fluoro-3,7-dihydro-4H-pyrrolo [ 2,3-d] pyrimidin-4-one.

A mixture of 7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3-O- (tert-butyl (dimethyl) silyl) -2-O- (hydroxy (oxide) phosphoranyl) -β-D -ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] nyrimidin-4-one (611 mg) and 80% acetic acid (4 ml) were stirred at room temperature for 2 h The reaction mixture was diluted with methanol and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (358 mg). MS: [M + H] + 464.1.

C) (2R, 3R, 4R, 5R) -5 - (((((2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert -butyldimethylsilyl) oxy) -2- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 ((tert-butyldimethylsilyl) oxy) -2- (5-fluoro-4- oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) tetrahydrofuran-3-yl hydrophosphonate.

A mixture of 7- (3-O- (tert-butyl (dimethyl) silyl) -2-O- (hydroxy (oxide) phosphoranyl) -β-D-ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (358 mg) and N-benzoyl-5'-O- (bis (4methoxyphenyl) (phenyl) methyl) -2'-O- (tert-butyl (dimethyl) silyl ) -3'-O - ((2-cyanoethoxy) (diisopropylamino) phosphino) adenosine (1.02 g) was azeotropically dried with anhydrous acetonitrile and suspended in anhydrous acetonitrile (10 ml). Pyridine 2,2,2-trifluoroacetate (386 mg) was added thereto and the mixture was stirred under argon at room temperature for 1 hour.

- 29 038805 ((dimethylaminomethylidene) amino) -3H-1,2,4-dithiazoline-3-thione (199 mg) and the mixture was stirred at room temperature for an additional 30 minutes. The reaction mixture was quenched with an aqueous solution (2.5 ml) of sodium thiosulfate (250 mg) and the solvent was evaporated under reduced pressure. The residue was dissolved in 1,1,1,3,3,3-hexafluoropropan-2-ol (10 ml), the solution was stirred at room temperature for 30 min, the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (328 mg). MS: [M + H] + 1080.3.

D) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2- (2cyanoethoxy) - 7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -10-oxide-10-sulfanyl-2-sulfidooctahydro-12H-5, 8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin14-yl) -9H-purin-6-yl) benzamide (optical isomer).

To a mixture (2R, 3R, 4R, 5R) -5 - (((((2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert -butyldimethylsilyl) oxy) -2- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -2- (5-fluoro-4 -oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) tetrahydrofuran-3-yl hydrophosphonate (328 mg) and anhydrous pyridine (10 ml) were added 2-chloro-5,5dimethyl-1, 3,2-dioxaphosphinane 2-oxide (196 mg). The mixture was stirred under argon at room temperature for 30 min, water (0.5 ml) and 3H-benzo [c] [1,2] dithiol-3-one (61 mg) were added to it, the mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with saturated aqueous sodium hydrogen carbonate solution and was extracted with ethyl acetate. The extract was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) and then purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) to give the title compound (optical isomer) (92 mg, tR4). MS: [M + H] + 1094.3.

E) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl ( dimethyl) silyl) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer).

N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2- (2-cyanoethoxy) - 7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -10-oxide-10-sulfanyl-2sulfidooctahydro-12H-5,8- methanofuro [3,2-U1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14yl) -9H-purin-6-yl) benzamide (optical isomer) (90 mg) was dissolved in a 40% solution of methylamine methanol (3 , 0 ml), the solution was stirred in an argon atmosphere at room temperature for 1 h, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (32 mg). MS: [M + H] + 937.1.

F) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -15,16-dihydroxy-2,10dioxide-2 , 10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro -4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

A mixture of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl (dimethyl) silyl ) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl ) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer) (32 mg) and triethylamine trihydrofluoride (0.12 ml) were stirred at 50 ° C for for 3 hours. The mixture was cooled to room temperature and neutralized with 1M aqueous solution of triethylammonium hydrogencarbonate, and the solvent was evaporated under reduced pressure. The residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer).

The resulting solid was freeze-dried to give the title compound (21 mg).

1H NMR (300 MHz, D2O) δ 1.23 (18H, t, J = 7.3 Hz), 3.15 (12H, q, J = 7.4 Hz), 3.99-4.09 (1H , m), 4.23-4.30 (2H, m), 4.30-4.41 (2H, m), 4.41-4.45 (1H, m), 4.47-4.54 (1H, m), 4.83-4.88 (2H, m), 4.96-5.13 (3H, m), 6.11 (1H, d, J = 1.1Hz), 6, 36 (1H, d, J = 7.4Hz), 7.31 (1H, d, J = 1.9Hz), 7.95 (1H, s), 8.11 (1H, s), 8, 22 (1H, s).

³¹ P NMR (121 MHz, D2O) δ 52,25, 54,88.

Example 6. Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15,16 dihydroxy-2,10-dioxido -2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -5-fluoro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt (optical isomer)

- 30 038805

A) 7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -v-O-ribofuranosyl) -5-fluoro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

To a solution of 7 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyiphenyl) (phenyl) methoxy) methyl) -3,4-dihydrocetetrahydrofuran-2-yl) -5-fluoro-3H- pyrrolo [2,3-d] pyrimidin-4 (7H) -one (2.65 g) in DMF (20 ml), imidazole (0.614 g) and tert-butyldimethylchlororeilane (0.816 g) were added and the mixture was stirred overnight at room temperature. temperature. The reaction mixture was diluted with water and extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (1.2 g). MS: [MH] ^- 700,3.

B) 7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -3-O - ((2-cyanoethokei) (diisopropylamino) foefino ) -β-D-ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

7- (5-O- (bie (4-methokeiphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) eilyl) -β-D-ribofuranosyl) 5-fluoro-3,7-dihydro- 4H-pyrrolo [2,3-d] pyrimidin-4-one (2.37 g) was dissolved in DMF (6.75 ml) and 3 - ((bis (diisopropylamino) phosphino) oxy) propanenitrile (2, 15 ml), 1H-tetrazole (0.237 g) and 1-methyl-1H-imidazole (0.134 ml), the mixture was stirred under an argon atmosphere at room temperature for 20.5 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution and extracted mixture with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane containing 0.5% triethylamine) to give the title compound (2.67 g). MS: [MH] ^- 900,2.

C) 7- (2-O- (tert-butyl (dimethyl) silyl) -3-O- (hydrokei (oceanido) phosphoranyl) -β-D-ribofuranosyl) -5fluoro-3,7-dihydro-4H-pyrrolo [ 2,3-d] pyrimidin-4-one.

- 31 038805

7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -3-O - ((2-cyanoethoxy) (diisopropylamino) phosphino) - β-D-ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4one (1.54 g) was dissolved in acetonitrile (15 ml), added to it at room temperature pyridine2,2,2-trifluoroacetate (0.396 g) and water (0.062 ml) and the mixture was stirred at room temperature for 50 minutes. To the reaction solution was added 2-methylpropan-2-amine (8.07 ml) at room temperature, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, 80% acetic acid (8.54 ml) was added to the residue, and the mixture was stirred at room temperature for 1 h.

Similarly, 7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -3-O - ((2cyanoethoxy) (diisopropylamino) phosphino) - β-D-ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3d] pyrimidin-4-one (1.13 g) was dissolved in acetonitrile (10 ml) and added to it at room temperature pyridine-2,2,2-trifluoroacetate (0.290 g) and water (0.045 ml), the mixture was stirred at room temperature for 50 minutes. To the reaction solution was added 2-methylpropan-2-amine (5.92 ml) at room temperature, and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was concentrated under reduced pressure, 80% acetic acid (6.27 ml) was added to the residue, and the mixture was stirred at room temperature for 1 hour.

The reaction mixtures were combined and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (961 mg). MS: [M + H] + 464.0.

D) (2R, 3R, 4R, 5R) -2 - (((((2R, 3R, 4R, 5R) -2- (6-benzamido-9H-purin-9-yl) -4 - ((tert -butyldimethylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 ((tert-butyldimethylsilyl) oxy) -5- (5-fluoro-4- oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) tetrahydrofuran-3-yl hydrophosphonate.

7- (2-O- (tert-butyl (dimethyl) silyl) -3-O- (hydroxy (oxide) phosphoranyl) -β-D-ribofuranosyl) -5fluoro-3,7-dihydro-4H-pyrrolo [2, 3-d] pyrimidin-4-one (400 mg) and X-benzoyl-5'-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3'-O- (tert-butyl (dimethyl) silyl) -2'-O - ((2-cyanoethoxy) (diisopropylamino) phosphino) adenosine (1.02 g) (three times) was azeotropically dried with anhydrous acetonitrile and suspended in anhydrous acetonitrile (6 ml). Pyridine-2,2,2-trifluoroacetate (333 mg) was added thereto and the mixture was stirred under argon at room temperature for 1 hour. (Dimethylaminomethylidene) amino) -3H-1,2,4-dithiazoline-3 β-thione (195 mg) and the mixture was stirred at room temperature for an additional 30 minutes. The reaction mixture was quenched with sodium thiosulfate (316 mg) and water (0.5 ml) and the solvent was evaporated under reduced pressure. The residue was dissolved in 1,1,1,3,3,3hexafluoropropan-2-ol (5 ml) and the solution was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was dried azeotropically with anhydrous acetonitrile and toluene. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (556 mg). MS: [M + H] + 1080.3.

E) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) - 14- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-2,10 disulfidooctahydro-12H-5,8-methanofuro [ 3,2-Al 1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7yl) -9H-purin-6-yl) benzamide (optical isomer).

((2R, 3R, 4R, 5R) -2 - (((((2R, 3R, 4R, 5R) -2- (6-benzamido-9H-purin-9-yl) -4 - ((tert- butyldimethylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 ((tert-butyldimethylsilyl) oxy) -5- (5-fluoro-4-oxo -3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) tetrahydrofuran-3-yl hydrophosphonate (556 mg) was subjected to azeotropic drying with anhydrous acetonitrile and anhydrous pyridine, dissolved in anhydrous pyridine (15 ml). 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinine 2-oxide (332 mg) was added to the solution at room temperature and the mixture was stirred in an argon atmosphere at room temperature for 30 minutes. Water (1 ml ) and 3H-1,2benzodithiol-3-one (108 mg) and the mixture was stirred at room temperature for an additional 1 hour. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture and the mixture was extracted with ethyl acetate The extract was washed with saturated aqueous sodium chloride solution and dried. over anhydrous sodium sulfate, the solvent was evaporated off Under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / methanol). The resulting residue was divided into four diastereomers (tR1, tR2, tR3 and tR4, the retention times of which from LC / MS from shorter to longer in this order) by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM aqueous solution of acetate ammonium / acetonitrile) to give the title compound (120 mg, tR2) and the title compound (183 mg, tR4). tR2 MS: [M + H] + 1094.3. tR4 MS: [M + H] + 1094.3.

F) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl ( dimethyl) silyl) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (op

- 32 038805 tic isomer).

N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) -14- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-oxide-2-sulfanyl10-sulfidooctahydro-12H-5,8-methanofuro [ 3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7yl) -9H-purin-6-yl) benzamide (optical isomer obtained from tR2) (120 mg) was dissolved in 33 % ethanolic solution of methylamine (5.0 ml), the solution was stirred in an argon atmosphere at room temperature for 1.5 hours and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (84.4 mg). MS: [M + H] + 937.2.

G) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-dihydroxy-2,10-dioxide -2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -5-fluoro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl (dimethyl ) silyl) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14 -yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer obtained from tR2) (84.4 mg) triethylamine trihydrofluoride (0.294 ml) was added and the mixture was stirred at 50 ° C. for 2 hours. The reaction solution was cooled to room temperature and neutralized with 1M triethylammonium hydrogencarbonate aqueous solution, and the solvent was evaporated under reduced pressure. The residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and the resulting solid was freeze-dried to give the title compound (58.4 mg).

1H NMR (300 MHz, D2O) δ 1.26 (18H, t, J = 7.4 Hz), 3.18 (12H, q, J = 7.3 Hz), 3.94-4.05 (1H , m), 4.10-4.20 (1H, m), 4.28-4.45 (2H, m), 4.50 (2H, br.s), 4.59 (1H, d, J = 4.2 Hz), 4.71-4.81 (1H, m), 5.32 (2H, d, J = 10.6 Hz), 6.23-6.32 (2H, m), 7 , 17 (1H, d, J = 2.3Hz), 8.05 (1H, s), 8.20 (1H, s), 8.66 (1H, s).

³¹ P NMR (121 MHz, D ₂ O) δ 55.8, 59.2.

Example 7. Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15,16 dihydroxy-2,10-dioxido -2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -5-fluoro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt (optical isomer)

A) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl ( dimethyl) silyl) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 14-yl) -5-fluoro-3, 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer).

N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) -14- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-oxide-2-sulfanyl10-sulfidooctahydro-12H-5,8-methanofuro [ 3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7yl) -9H-purin-6-yl) benzamide (optical isomer obtained from tR4) (183 mg) was dissolved in 33 % ethanol solution of methylamine (5.0 ml), the solution was stirred in an argon atmosphere at room temperature for 1 hour and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (107 mg). MS: [M + H] + 937.1.

B) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-dihydroxy-2,10-dioxide -2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -5 -fluoro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl (dime

- 33 038805 tyl) silyl) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10 ] pentaoxadiphosphacyclotetradecin-14-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer obtained from tR4) (107 mg) triethylamine trihydrofluoride (0.372 ml) and the mixture was stirred at 50 ° C for 2.5 hours. The reaction solution was cooled to room temperature and neutralized with 1M aqueous triethylammonium hydrogencarbonate solution, the solvent was evaporated under reduced pressure. The residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and the resulting solid was freeze-dried to give the title compound (78 mg).

1H NMR (300 MHz, D ₂ O) δ 1.26 (18H, t, J = 7.4 Hz), 3.18 (12H, q, J = 7.2 Hz), 4.09-4.18 (1H, m), 4.21-4.29 (2H, m), 4.33-4.43 (1H, m), 4.51 (2H, br.s), 4.65-4.71 (1H, m), 4.89 (1H, d, J = 4.2Hz), 4.99-5.11 (1H, m), 5.295.42 (1H, m), 6.23-6, 31 (2H, m), 7.00 (1H, d, J = 1.9Hz), 8.03 (1H, s), 8.17 (1H, s), 8.56 (1H, s).

³¹ P NMR (121 MHz, D ₂ O) δ 52.8, 55.1.

Example 8. Synthesis of 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (4-amino-5-fluoro-7Hpyrrolo [2,3-d] pyrimidine- 7-yl) -2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -1,9-dihydro-6H-purin-6-one diethylamine salt

νη ₂

A) N- (7 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) -5-fluoro -7H-pyrrolo [2,3-d] pyrimidin-4-yl) benzamide.

(2R, 3R, 4R, 5R) -2 - ((Benzoyloxy) methyl) -5- (4-chloro-5-fluoro-7H-pyrrolo [2,3-d] pyrimidine-7

- 34 038805 yl) tetrahydrofuran-3,4-diyldibenzoate (14.09 g) was charged into 17 hermetically sealed containers in 17 parts, a 2M solution of ammonia in isopropanol (20 ml) was added to it, respectively. Each mixture was stirred under microwave irradiation at 130 ° C for 5 hours. The resulting mixtures were combined and concentrated under reduced pressure. The resulting residue was azeotroped twice with toluene to remove the solvent, pyridine (100 ml) was added thereto, and the mixture was cooled to 0 ° C. Benzoyl chloride (26.6 ml) was added thereto at 0 ° C and the mixture was stirred overnight at room temperature. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The extract was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane). Pyridine (350 ml) and ethanol (100 ml) were added to the resulting compound, and the mixture was cooled to 0 ° C. To this was added 1M aqueous sodium hydroxide solution (103 ml) and the mixture was stirred at 0 ° C for 1 h. To it was added 1 M aqueous sodium hydroxide solution (45.7 ml) and the mixture was stirred at 0 ° C for 1 h DOWEX ™ 50Wx4 100-200 strong acidic cation exchange resin (95 g) was added to the reaction mixture at room temperature, the solid was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified using silica gel column chromatography (methanol / ethyl acetate). To the resulting product (5.71 g) were added pyridine (60 ml) and 4,4'-dimethoxytrityl chloride (5.98 g) and the mixture was stirred at room temperature for 4 hours. To this was added 4,4'-dimethoxytrityl chloride (5 , 98 g), and the mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure, water was added thereto and the mixture was extracted with ethyl acetate. The extract was washed with water and a saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (8.50 g). MS: [M + H] + 691.2.

B) N- (7 - ((2R, 3R, 4R, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3 - ((tert-butyldimethylsilyl) oxy) -4-hydroxytetrahydrofuran -2-yl) -5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-4-yl) benzamide.

To a mixture of N- (7 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) -5-fluoro -7H-pyrrolo [2,3-d] pyrimidin-4-yl) benzamide (7.51 g) and DMF (30 ml) were added imidazole (1.924 g) and tert-butyldimethylchlorosilane (2.13 g), the mixture was stirred in overnight at room temperature. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The extract was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (2.94 g). MS found: 501.2.

C) N-benzoyl-7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -3-O - ((2cyanoethoxy) (diisopropylamino ) phosphino) -β-D-ribofuranosyl) -5-fluoro-7H-pyrrolo [2,3-d] pyrimidin4-amine.

N- (7 - ((2R, 3R, 4R, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3 - ((tert-butyldimethylsilyl) oxy) -4-hydroxytetrahydrofuran-2 -yl) -5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-4-yl) benzamide (1.03 g) was azeotropically dried with anhydrous toluene and anhydrous DMF (5 ml) was added under argon ... To this was added 3 - ((bis (diisopropylamino) phosphino) oxy) propanenitrile (0.771 g), 1H tetrazole (0.090 g) and 1-methyl-1H-imidazole (0.051 ml), the mixture was stirred for 6 hours. saturated aqueous sodium hydrogen carbonate solution and the mixture was extracted with ethyl acetate. The extract was washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate containing 0.5% triethylamine / hexane) to give the title compound (1.12 g).

1H NMR (300 MHz, CDCl ₃ ) δ -0.17 (3H, d, J = 2.6 Hz), -0.04-0.00 (3H, m), 0.78 (9H, s), 1.04 (3H, d, J = 6.8 Hz), 1.13-1.23 (9H, m), 2.32 (1H, t, J = 6.6 Hz), 2.68 (1H , td, J = 6.4, 1.9 Hz), 3.21-3.36 (1H, m), 3.42-3.71 (4H, m), 3.79 (6H, d, J = 1.1 Hz), 3.85-4.05 (1H, m), 4.25-4.45 (2H, m), 4.64-4.82 (1H, m), 6.39 ( 1H, dd, J = 14.2, 5.9 Hz), 6.74-6.89 (4H, m), 7.19-7.41 (8H, m), 7.43-7.67 ( 5H, m), 7.98 (2H, d, J = 7.6 Hz), 8.50 (1H, s), 8.64 (1H, d, J = 5.3 Hz).

D) (2R, 3R, 4R, 5R) -5- (4-benzamido-5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2- (hydroxymethyl) tetrahydrofuran-3-yl hydrophosphonate.

To N-benzoyl-7- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -3-O - ((2cyanoethoxy) (diisopropylamino) phosphino) -β-D-ribofuranosyl) -5-fluoro-7H-pyrrolo [2,3-d] pyrimidine4-amine (1.12 g) was added acetonitrile (5 ml), water (40 μl) and pyridine-2, 2,2-trifluoroacetate (258 mg). The mixture was stirred at room temperature for 10 minutes, tert-butylamine (5.43 ml) was added and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, to which was added 80% acetic acid (5.5 ml ) and the mixture was stirred at room temperature for an additional 1 h. The solvent was evaporated under reduced pressure and the residue was purified by column chromatography on silica gel (methanol / ethyl acetate) with

- 35 038805 obtaining the title compound (527 mg). MS: [M + H] + 567.2.

E) (2R, 3R, 4R, 5R) -5- (4-benzamido-5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - (((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) -2- (2isobutylamido-6-oxo-1H-purine-9 ( 6H) -yl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate.

(2R, 3R, 4R, 5R) -5- (4-Benzamido-5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - (hydroxymethyl) tetrahydrofuran-3-yl hydrophosphonate (527 mg) and 3'-O- (tert-butyl (dimethyl) silyl) -2'-O - ((2-cyanoethoxy) (diisopropylamino) phosphino) -N-isobutyrylguanosine (1.083 g) was subjected to azeotropic drying three times with anhydrous acetonitrile and anhydrous acetonitrile (10 ml) was added thereto. Pyridine-2,2,2-trifluoroacetate (359 mg) was added thereto, and the mixture was stirred at room temperature for 30 minutes. A 70% aqueous solution of tert-butyl hydroperoxide (382 μl) was added thereto and the mixture was stirred at room temperature for an additional 20 minutes. Sodium thiosulfate (693 mg) and water (1 ml) were added to the reaction mixture, and the mixture was concentrated under reduced pressure. To the residue was added 80% acetic acid (5 ml) and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was azeotroped three times with toluene. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (175 mg). MS: [M + H] + 1149.4.

F) N- (7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) - 2-hydroxy-7- (2 - ((2-methylpropanoyl) amino) -6-oxo-1,6-dihydro-9H-purin-9-yl) -2,10dioxidooctahydro-12H-5,8-methanofuro [3 , 2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14yl) -5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-4-yl) benzamide.

(2R, 3R, 4R, 5R) -5- (4-Benzamido-5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - ((((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) -2- (2-isobutylamido-6-oxo-1H-purine-9 ( 6H) -yl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate (175 mg) was azeotropically dried three times with anhydrous acetonitrile and anhydrous pyridine (3 ml) was added to it ) and 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (98 mg). The mixture was stirred under argon at room temperature for 10 minutes, water (96 μl) and iodine (50 mg) were added thereto, and the mixture was stirred at room temperature for an additional 20 minutes. The reaction mixture was added to a mixture of sodium thiosulfate (98 mg) and water (0.4 ml) and the mixture was stirred for 5 minutes, concentrated under reduced pressure. The residue was purified twice by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (48 mg). MS: [M + H] + 1147.6.

G) 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (4-amino-5-fluoro-7H-pyrrolo [2,3-d] pyrimidine- 7-yl) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H5,8-methanofuro [3,2-l] [1, 3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -1,9-dihydro-6H-purine-6one.

N- (7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) -2- hydroxy-7- (2 - ((2-methylpropanoyl) amino) -6-oxo-1,6-dihydro-9H-purin-9-yl) -2,10 dioxidooctahydro-12H-5,8-methanofuro [3,2 -l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14yl) -5-fluoro-7H-pyrrolo [2,3-d] pyrimidin-4-yl) benzamide (48 mg) was subjected to twice azeotropic drying with anhydrous acetonitrile, a 33% ethanolic solution of methylamine (2 ml) was added thereto, and the mixture was stirred overnight under argon at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate). The resulting residue was purified by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution), the resulting fraction was concentrated under reduced pressure and the resulting product was lyophilized to give the title compound (5 mg ). MS: [M + H] + 920.3.

H) 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (4-amino-5-fluoro-7H-pyrrolo [2,3-d] pyrimidine- 7-yl) -2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -1,9-dihydro-6H-purin-6-one diethylamine salt.

A mixture of 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (4-amino-5-Fluoro-7H-pyrrolo [2,3d] pyrimidin-7-yl ) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3 , 6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -1,9-dihydro6H-purin-6-one (5 mg) and triethylamine trihydrofluoride (200 μl) were stirred at 50 ° C for 1 h. The reaction mixture was cooled to room temperature and neutralized with 1M aqueous triethylammonium hydrogen carbonate solution. The reaction mixture was purified by C18 column chromatography (acetonitrile / 10 mM triethylammonium acetate buffer), the target fraction was concentrated under reduced pressure, and the resulting product was lyophilized to give the title compound (3.2 mg).

1H NMR (600 MHz, D ₂ O) δ 1.23 (18H, t, J = 7.3 Hz), 3.15 (12H, q, J = 7.3 Hz), 4.06-4.11 (1H, m), 4.14-4.19

- 36 038805 (1H, m), 4.21-4.26 (1H, m), 4.31-4.37 (2H, m), 4.40 (1H, d, J = 1.5 Hz) , 4.56 (1H, dd, J = 15.2, 4.3Hz), 4.61-4.68 (1H, m), 5.00 (1H, ddd, J = 8.5, 6, 6, 4.5 Hz), 5.60 (1H, td, J = 7.9, 4.1 Hz), 5.93 (1H, d, J = 8.5 Hz), 6.26 (1H, With),

7.14 (1H, d, J = 1.6Hz), 7.87 (1H, s), 8.10 (1H, s).

Example 10. Synthesis 1 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -2,10,15,16 tetrahydroxy- 2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -1,5-dihydro-4H- imidazo [4,5-c] pyridin-4-one

BzO OBz

- 37 038805

one)

NHBz

NC

A) (2R, 3R, 4R, 5R) -2 - ((benzoyloxy) methyl) -5- (4-oxo-4,5-dihydro-1H-imidazo [4,5-c] pyridin-1yl) tetrahydrofuran- 3,4-diyldibenzoate.

Formic acid (123 ml) and water (30 ml) were added to 4-chloro-1H-imidazo [4,5-c] pyridine (20 g) at room temperature and the mixture was stirred at 100 ° C for 2 h. The mixture was cooled to room temperature and concentrated under reduced pressure. Conc. Was added to the remainder. hydrochloric acid (100 ml) and the mixture was stirred at 100 ° C for 1 h. The mixture was concentrated under reduced pressure, the residue was suspended in MeOH (100 ml) and diisopropyl ether (100 ml) and triethylamine (50 ml) was added thereto. The resulting solid was collected by filtration and dissolved in acetonitrile (600 ml). To this was added trimethylsilyl-N (trimethylsilyl) acetimidate (38.2 ml) at room temperature and the mixture was stirred for 10 minutes. To the reaction mixture at room temperature was added (2S, 3R, 4R, 5R) -2-acetoxy-5 - ((benzoyloxy) methyl) tetrahydrofuran3,4-diyldibenzoate (79.0 g) and the mixture was heated to 80 ° C. To the reaction solution was added trimethylsilyl trifluoromethanesulfonate (28.2 ml), and the mixture was stirred under an argon atmosphere overnight at 80 ° C. The mixture was cooled to room temperature, water (500 ml) was added thereto, and the resulting solid was collected by filtration. The solid was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (25.9 g).

1H NMR (400 MHz, CDCl ₃ ) δ 4.65-4.96 (3H, m), 5.91 (2H, br.s), 6.23-6.42 (1H, m), 6, 64 (1H, br.s), 7.15 (1H, br.s), 7.32-7.56 (9H, m), 7.92-8.15 (7H, m), 12.11- 12.50 (1H, m). MS: [M + H] + 580.1.

B) 1 - ((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) -1H-imidazo [4,5c] pyRidin-4 (5H) -one.

К (2R, 3R, 4R, 5R) -2 - ((benzoyloxy) methyl) -5- (4-oxo-4,5-dihydro-1H-imidazo [4,5-c] pyridin-1yl) tetrahydrofuran-3 , 4-diyldibenzoate (30.0 g) was added 40% methanol solution of methylamine (200 ml) and the mixture was stirred under argon atmosphere at room temperature for 1 hour, concentrated under reduced pressure. The residue was recrystallized from hexane-ethyl acetate to give the title compound (11.2 g). MS: [M + H] ⁺ 268, 1.

C) 1 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) -1H-imidazo [4, 5-c] pyridin-4 (5H) -one.

To a solution of 1 - ((2R, 3R, 4S, 5R) -3,4-dihydroxy-5 - (hydroxymethyl) tetrahydrofuran-2-yl) -1Himidazo [4,5-c] pyridin-4 (5H) -oneα ( 18.0 g) in pyridine (140 ml) was added at room temperature

- 38 038805

4,4'-dimethoxytrityl chloride (18.46 g) and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, water was added to the residue, and the mixture was extracted with ethyl acetate. The resulting extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (18.2 g). MS: [M + H] + 570.2.

D) 1 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -3-hydroxytetrahydrofuran-2- yl) -1H-imidazo [4,5-c] pyridin-4 (5H) -one.

To a solution of 1 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) -1H-imidazo [4, 5-c] pyridin-4 (5H) -one (18.02 g) in DMF (158 ml), imidazole (4.30 g) and tert-butyldimethylchlorosilane (5.72 g) were added, the mixture was stirred at room temperature for 3 hours tert-Butyldimethylchlorosilane (2.38 g) was added again and the mixture was stirred for 3 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane). The isolated regioisomer of the title compound was dissolved in MeOH and triethylamine and the solution was stirred overnight at room temperature. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (11.5 g). MS: [M + H] + 684.2.

E) (2R, 3R, 4R, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -2- (4-oxo-4, 5-dihydro-1H-imidazo [4,5-c] pyridin-1-yl) tetrahydrofuran-3-yl hydrophosphonate.

To a mixture of 1 - ((2R, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -3-hydroxytetrahydrofuran-2- yl) -1H-imidazo [4,5-c] pyridin-4 (5H) -one (8.0 g) and pyridine (117 ml) diphenylphosphite (4.5 ml) was added. The mixture was stirred at room temperature for 1 hour. Water (20 ml) was added to the reaction mixture. The mixture was stirred at room temperature for 30 min and the reaction mixture was diluted with water, extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (8.5 g). MS: [M + H] + 748.3.

F) (2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) -2- (4-oxo-4,5-dihydro-1Himidazo [4,5-c] pyridin-1-yl) tetrahydrofuran-3-yl hydrophosphonate.

A mixture of (2R, 3R, 4R, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -2- (4-oxo-4,5 -dihydro-1H-imidazo [4,5-c] pyridin-1-yl) tetrahydrofuran-3-yl hydrophosphonate (8.2 g) and 1,1,1,3,3,3-hexafluoropropan-2-ol ( 30 ml) was stirred at room temperature for 2 hours. Methanol (10 ml) was added thereto, the mixture was stirred at 60 ° C for 1 hour, and the solvent was evaporated under reduced pressure. The residue was recrystallized from ethyl acetate and diisopropyl ether to give the title compound (4.3 g). MS: [M + H] + 446.1.

G) (2R, 3R, 4R, 5R) -5 - (((((2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert -butyldimethylsilyl) oxy) -2- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -2- (4-oxo-4,5 -dihydro-1H-imidazo [4,5-c] pyridin-1-yl) tetrahydrofuran-3-yl hydrophosphonate (2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl ) -2- (4-oxo-4,5dihydro-1H-imidazo [4,5-c] pyridin-1-yl) tetrahydrofuran-3-yl hydrophosphonate (250 mg) and N-benzoyl-5'O- (bis (4-methoxyphenyl) (phenyl) methyl) -2'-O- (tert-butyl (dimethyl) silyl) -3'-O - ((2-cyanoethoxy) (diisopropylamino) phosphino) adenosine (776 mg) was subjected to azeotropic drying with anhydrous acetonitrile, added to them anhydrous acetonitrile (5.61 ml). Pyridine-2,2,2-trifluoroacetate (271 mg) was added to the mixture, and the mixture was stirred at room temperature for 40 minutes. To this was added a 70% aqueous solution of tert-butyl hydroperoxide (231 μl) and the mixture was stirred at room temperature for 20 minutes. To the reaction mixture was added a mixture of sodium thiosulfate (1.3 g) and water (3 ml), and the mixture was concentrated under reduced pressure. To the residue was added 1,1,1,3,3,3-hexafluoropropan-2-ol (15 ml) and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (ethyl acetate / hexane and methanol / ethyl acetate) to give the title compound (170 mg). MS: [M + H] + 1046.3.

H) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2- (2-cyanoethoxy ) -10-hydroxy-2,10-dioxido-7- (4-oxo-4,5-dihydro-1H-imidazo [4,5-c] pyridin-1-yl) octahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purine-6yl) benzamide.

(2R, 3R, 4R, 5R) -5 - (((((2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl ) oxy) -2- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -2- (4-oxo-4,5-dihydro -1H-imidazo [4,5-c] pyridin-1-yl) tetrahydrofuran-3-yl hydrophosphonate (160 mg) was azeotropically dried with anhydrous pyridine and added to it without

- 39 038805 aqueous pyridine (3.0 ml). To the mixture at room temperature was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide (99 mg) and the mixture was stirred at room temperature for 1 hour. Water (96 μL) and iodine were added (50.5 mg) and the mixture was stirred at room temperature for an additional 20 minutes. To the reaction mixture was added a mixture of sodium thiosulfate (190 mg) and water (0.4 ml), and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (10.6 mg). MS: [M + H] + 1044.3.

I) 1 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl ( dimethyl) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl) -1,5-dihydro-4H-imidazo [4,5-c] pyridin-4-one.

To N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2- (2cyanoethoxy) -10 -hydroxy-2,10-dioxido-7- (4-oxo-4,5-dihydro-1H-imidazo [4,5-c] pyridin-1-yl) octahydro-12H-5,8-methanofuro [3, 2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purine-6yl) benzamide (10.6 mg) was added 40% ethanol solution of methylamine (5.0 ml ) and the mixture was stirred in an argon atmosphere at room temperature for 1 h, concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (9.0 mg). MS: [M + H] + 887.2.

J) 1 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -2,10,15,16-tetrahydroxy-

2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacylotetradecin-7yl) -1,5-dihydro-4H-imidazo [ 4,5-c] pyridin-4-one.

K 1 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl (dimethyl ) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 -yl) -1,5-dihydro-4H-imidazo [4,5-c] pyridin-4-one (9.0 mg) methanol (1.0 ml) and triethylamine trihydrofluoride (165 μl) were added. The reaction mixture was concentrated to remove methanol and the residue was stirred at 55 ° C for 1 h. The mixture was cooled to room temperature, ethoxy (trimethyl) silane (0.90 ml) was added thereto and the mixture was stirred at room temperature for 1 h. the mixture was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (acetonitrile / 10 mM triethylammonium acetate buffer) to give the title compound (0.3 mg).

¹ H NMR (400 MHz, D ₂ O) δ 4.15 (2H, d, J = 11.5 Hz), 4.46 (4H, br.s), 4.56-4.62 (1H, m ), 4.98-4.99 (2H, m), 6.05-6.20 (2H, m), 6.79-6.87 (1H, m), 7.06-7.12 (1H , m), 8.00 (1H, s), 7.96-8.10 (1H, m), 8.23 (2H, s).

³¹ P NMR (162 MHz, D ₂ O) δ -2.36, -1.94.

Example 11. Synthesis of 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (4-amino-1H-imidazo [4,5c] pyridin-1-yl) -2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7- yl) -1, 9-dihydro-6H-purin-6-one diethylamine salt

- 40 038805

A) N-benzoyl-1- (2-O- (tert-butyl (dimethyl) silyl) -3,5-O- (di-tert-butylsilylene) -β-D-ribofuranosyl) -1H-imidazo [4, 5-c] pyridin-4-amine.

N-Benzoyl-1- (β-D-ribofuranosyl) -1H-imidazo [4,5-c] pyridin-4-amine (2.41 g) was dissolved in DMF (25 ml), to which was added at 0 ° C di-tert-butylsilandiyl bis (trifluoromethanesulfonate) (2.32 ml) and the mixture was stirred at 0 ° C for 75 minutes. To this was added at 0 ° C di-tert-butylsilanediyl bis (trifluoromethanesulfonate) (0.63 ml) and the mixture was stirred at 0 ° C for an additional 30 minutes. To this was added di-tert-butylsilanediyl bis (trifluoromethanesulfonate) (0.63 ml) at 0 ° C and the mixture was stirred at 0 ° C for an additional 35 minutes. To the reaction mixture was added 1Himidazole (2.22 g) and the mixture was stirred at room temperature for 10 minutes. To this was added tert-butyldimethylchlorosilane (1.18 g) and the mixture was stirred at 60 ° C for 1.5 hours. Tert-butyldimethylchlorosilane (0.294 g) was added thereto and the mixture was stirred at 60 ° C for 14.5 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (3.28 g). MS: [M + H] + 625.2.

B) N-benzoyl-1- (2-O- (tert-butyl (dimethyl) silyl) -β-D-ribofuranosyl) -1H-imidazo [4,5-c] pyridin4-amine.

- 41 038805

Poly (hydrofluoride) pyridinium (1.86 ml) was dissolved in pyridine (10 ml) at 0 ° C, the solution was added to a solution of N-benzoyl-1- (2-O- (tert-butyl (dimethyl) silyl) -3, 5-O- (di-tert-butylsilylene) -β-Dribofuranosyl) -1H-imidazo [4,5-c] pyridin-4-amine (1.64 g) in THF (13 ml) at 0 ° C and stirred mix at 0 ° C for 8 min.

This reaction was repeated twice. The reaction solutions were mixed, diluted with water and extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane and methanol / ethyl acetate) to give the title compound (2.24 g). MS: [M + H] + 485.1.

C) N-benzoyl-1- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -β-D-ribofuranosyl) -1H-imidazo [4,5-c] pyridin-4-amine.

N-Benzoyl-1- (2-O- (tert-butyl (dimethyl) silyl) -β-D-ribofuranosyl) -1H-imidazo [4,5-c] pyridine-4amine (2.24 g) was dissolved in pyridine (25 ml), 4.4'dimethoxytrityl chloride (2.04 g) was added thereto at room temperature and the mixture was stirred at room temperature for 4.5 hours. The reaction mixture was concentrated under reduced pressure, water was added to the residue and the mixture was extracted with ethyl acetate ... The extract was washed with a saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (3.29 g). MS: [M + H] + 787.3.

D) N-benzoyl-1- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -3-O - ((2cyanoethoxy) (diisopropylamino ) phosphino) -β-D-ribofuranosyl) -1H-imidazo [4,5-c] pyridin-4-amine.

N-Benzoyl-1- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -β-Dribofuranosyl) -1H-imidazo [4,5 -c] pyridin-4-amine (3.29 g) was dissolved in anhydrous DMF (9.5 ml) and 3 - ((bis (diisopropylamino) phosphino) oxy) propanenitrile (2.98 ml), 1H- tetrazole (0.328 g) and 1-methyl-1H-imidazole (0.185 ml), the mixture was stirred under argon at room temperature for 2.5 hours. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction solution, and the mixture was extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane containing 0.5% triethylamine) to give the title compound (3.85 g). MS: [M + H] + 987.4.

E) N-benzoyl-1- (2-O- (tert-butyl (dimethyl) silyl) -3-O- (hydroxy (oxide) phosphoranyl) -β-D-ribofuranosyl) -1H-imidazo [4, 5-c] pyridin-4-amine.

N-Benzoyl-1- (5-O- (bis (4-methoxyphenyl) (phenyl) methyl) -2-O- (tert-butyl (dimethyl) silyl) -3-O - ((2cyanoethoxy) (diisopropylamino) phosphino ) -β-D-ribofuranosyl) -1H-imidazo [4,5-c] pyridin-4-amine (3.85 g) was dissolved in acetonitrile (30 ml), pyridine 2,2,2 trifluoroacetate was added at room temperature (0.904 g) and water (0.141 ml) and the mixture was stirred at room temperature for 30 minutes. To the reaction solution was added 2-methylpropan-2-amine (9 ml) at room temperature, and the mixture was stirred at room temperature for 40 minutes. The reaction mixture was concentrated under reduced pressure and 1,1,1,3,3,3-hexafluoropropan-2-ol (25 ml) was added to the residue, the mixture was stirred at room temperature for 3.5 hours. The reaction mixture was concentrated under reduced pressure. was added to it acetic acid (20 ml) and water (5 ml) and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (methanol / ethyl acetate) to obtain the specified in the title compound (1.85 g). MS: [M + H] + 549.1.

F) (2R, 3R, 4R, 5R) -5- (4-benzamido-1H-imidazo [4,5-c] pyridin-1-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2- ( (((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) -2- (2-isobutylamido-6oxo-1H-purin-9 (6H) -yl ) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate.

N-Benzoyl-1- (2-O- (tert-butyl (dimethyl) silyl) -3-O- (hydroxy (oxide) phosphoranyl) -β-D-ribofuranosyl) -1H-imidazo [4,5-c] pyridin-4-amine (300 mg) and 5'-O- (bis (4-methoxyphenyl) (phenyl) methyl) -3'-O- (tert-butyl (dimethyl) silyl) -2'-O - ((2- cyanoethoxy) (diisopropylamino) phosphino) -N-isobutyrylguanosine (637 mg) was subjected to azeotropic drying with anhydrous acetonitrile (three times) and suspended in anhydrous acetonitrile (6 ml). Pyridine 2,2,2-trifluoroacetate (264 mg) was added thereto and the mixture was stirred under an argon atmosphere at room temperature for 1 hour. A 70% aqueous solution of tert-butyl hydroperoxide (0.225 ml) was added thereto and the mixture was stirred at room temperature for for an additional 1 hour. The reaction mixture was quenched with sodium thiosulfate (400 mg) and water (1.5 ml) and the solvent was evaporated under reduced pressure. The residue was dissolved in 80% acetic acid (5 ml), the solution was stirred at room temperature for 45 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to azeotropic drying with anhydrous acetonitrile and toluene. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (469 mg). MS: [M + H] + 1131.4.

G) N- (1 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) - 2-hydroxy-7- (2- (isobutyrylamino) -6-oxo-1,6-dihydro-9H-purin-9-yl) -2,10-dioxidooktαguidro-12H-5,8-meta-nofuro [3,2- l] [1,3,6,9,11,2,10] nentaoksaduphosphosphatsuclotetrαdecin-14-yl) -1Himidazo [4,5-c] pyridin-4-yl) benzamide.

(2R, 3R, 4R, 5R) -5- (4-Benzamido-1H-imidazo [4,5-c] pyridin-1-yl) -4 - ((tert-butyldimethylsilyl) oxy) 2 - (((( ((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) -2- (2-isobutylamido-6-oxo1H-purin-9 (6H) -yl) tetrahydrofuran- 3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran 3-yl hydrophosphonate (469 mg) was dried azeotropically with anhydrous acetonitrile and anhydrous pyridine and suspended in anhydrous pyridine (10 ml). 2-Chloro-5,5-dimethyl1,3,2-dioxaphosphine-2-oxide (268 mg) was added thereto and the mixture was stirred under argon at room temperature for 15 minutes. Water (1 ml) and iodine (158 mg) were added thereto and the mixture was stirred at room temperature for an additional 13 minutes. The reaction mixture was quenched with sodium thiosulfate (170 mg) and water (0.5 ml), the solvent was evaporated under reduced pressure and the residue was azeotroped with toluene. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (419 mg). MS: [M + H] + 1129.3.

H) 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (4-amino-1H-imidazo [4,5-c] pyridin-1-yl) 15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6 , 9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -1,9-dihydro-6H-purin-6-one.

N- (1 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) -2- hydroxy-7- (2- (isobutyrylamino) -6-oxo-1,6-dihydro-9H-purin-9-yl) -2,10-dioxidooktagudro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] nentaoksaduphosphaclotetradecin-14-ul) -1Himidazo [4,5-c] pyridin-4-yl) benzamide (419 mg) was dissolved in a 33% ethanol solution of methylamine (10 , 0 ml), and the solution was stirred in an argon atmosphere at room temperature for 18.5 h. A 33% ethanolic solution of methylamine (5 ml) was added thereto, the mixture was stirred for an additional 3 h, and the solvent was evaporated under reduced pressure. The residue was dissolved in 33% ethanolic methylamine solution (10.0 ml), the solution was stirred at room temperature for 1 hour, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (173 mg). MS: [M + H] + 902.3.

I) 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (4-amino-1H-imidazo [4,5-c] pyridin-1-yl) 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl ) -1,9-dihydro-6H-purin-6-one diethylamine salt.

K 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (4-amino-1H-imidazo [4,5-c] pyridin-1-yl) 15 , 16-bis ((tert-butyl (dimethyl) silyl) oxy) -2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6, 9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -1,9-dihydro-6H-purin-6-one (173 mg), triethylamine trihydrofluoride (0.625 ml) was added and the mixture was stirred at 50 ° C for 3 h The mixture was cooled to room temperature and neutralized with 1M aqueous triethylammonium hydrogen carbonate solution, and the solvent was evaporated under reduced pressure. The residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and the resulting solid was freeze-dried to give the title compound (123 mg).

1H NMR (300 MHz, D ₂ O) δ 1.25 (18H, t, J = 7.4 Hz), 3.17 (12H, q, J = 7.2 Hz), 3.87-4.10 (1H, m), 4.11-4.30 (3H, m), 4.32-4.45 (2H, m), 4.52 (1H, d, J = 3.8Hz), 4, 65 (1H, d, J = 4.2 Hz), 4.97-5.10 (1H, m), 5.79 (1H, br.s), 5.94 (2H, d, J = 8, 3 Hz), 7.10 (1H, d, J = 6.8 Hz), 7.68-7.78 (1H, m), 7.83 (1H, s), 7.94-8.26 ( 1H, m).

³¹ P NMR (121 MHz, D2O) δ -1,36.

Example 12. Synthesis of 8 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15,16 dihydroxy-2,10-dioxido -2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) pyrazolo [1,5-a] [1,3,5] triazin-4 (3H) -one diethylamine salt (optical isomer)

- 43 038805

1) HN ^ OEtHCI K ₂ CO ₃ DMF

2) EtOCOOEt

DMTrCI

3) NaOEt, EtOH pyridine

A) 8 - ((2S, 3S, 4R, 5R) -3,4-bis (benzyloxy) -5 - ((benzyloxy) methyl) tetrahydrofuran-2-yl) pyrazolo [1,5a] [1,3,5 ] triazin-4 (3H) -one.

To a solution of 4 - ((2S, 3S, 4R, 5R) -3,4-bis (benzyloxy) -5 - ((benzyloxy) methyl) tetrahydrofuran-2-yl) -1Hpyrazol-5-amine (17.3 g) and potassium carbonate (24.6 g) in DMF (200 ml), ethyl formimidate hydrochloride (19.5 g) was added at room temperature. The reaction mixture was stirred overnight at room temperature. Diethyl carbonate (126 g) was added to the reaction mixture at room temperature and the reaction mixture was stirred at 100 ° C for 1 hour. To the reaction mixture was added 20% ethanolic sodium ethoxide solution (60.6 g) at room temperature. The reaction mixture was stirred at 100 ° C for 30 min. The reaction mixture was neutralized with acetic acid at room temperature and extracted with ethyl acetate. The extract was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (5.79 g). MS: [M + H] + 539.1.

B) 8 - ((2S, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) pyrazolo [1,5a] [1,3,5] triazine-4 (3H )-he.

Solution 8 - ((2S, 3S, 4R, 5R) -3,4-bis (benzyloxy) -5 - ((benzyloxy) methyl) tetrahydrofuran-2yl) pyrazolo [1,5-a] [1,3,5] triazin-4 (3H) -one (3.49 g) and palladium hydroxide (1.37 g, 10% Pd) in methanol (30 ml) were stirred under a hydrogen atmosphere overnight at room temperature. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure to give the title compound (1.72 g). MS: [M + H] + 269.0.

C) 8 - ((2S, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) pyrazolo [1,5-a ] [1,3,5] triazin-4 (3H) -one.

- 44 038805

To a solution of 8 - ((2S, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) pyrazolo [1.5а] [1,3,5] triazin-4 (3Н ) -one (1.72 g) in anhydrous pyridine (30 ml) was added 4,4'-dimethoxytrityl chloride (2.39 g) while cooling in an ice bath. The reaction mixture was stirred under argon at room temperature for 2 hours. To the reaction mixture was added 4,4'-dimethoxytrityl chloride (0.217 g) while cooling in an ice bath. The reaction mixture was stirred under argon at room temperature for 3 hours. To the reaction mixture was added 4,4'-dimethoxytrityl chloride (0.217 g) at room temperature. The reaction mixture was stirred under argon at room temperature for 30 minutes. A saturated sodium hydrogencarbonate aqueous solution was added to the reaction mixture at room temperature, and the mixture was extracted with ethyl acetate. The extract was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (1.62 g). MS: [MH] ^- 569,1.

D) 8 - ((2S, 3R, 4R, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3 - ((tert-butyldimethylsilyl) oxy) -4-hydroxytetrahydrofuran-2- yl) pyrazolo [1,5-a] [1,3,5] triazin-4 (3H) -one.

tert-Butyldimethylchlorosilane (536 mg) and silver nitrateC) (604 mg) at room temperature in an argon atmosphere were added to a solution of 8 - ((2S, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) pyrazolo [1,5-a] [1,3,5] triazin-4 (3H) -one (1.69 g) and anhydrous pyridine (1, 20 ml) in anhydrous THF (20 ml). The reaction mixture was stirred at room temperature for 8 hours and tert-butyldimethylchlorosilane (89 mg) and silver (I) nitrate (101 mg) were added to the reaction mixture at room temperature. The reaction mixture was stirred overnight at room temperature. The insoluble matter was removed by filtration and washed with ethyl acetate. A saturated aqueous sodium hydrogen carbonate solution was added to the filtrate, and the mixture was extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (740 mg). MS: [MH] ^- 683.1.

E) (2R, 3R, 4S, 5S) -2 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -5- (4-oxo-3, 4-dihydropyrazolo [1,5-a] [1,3,5] triazin-8-yl) tetrahydrofuran-3-yl hydrophosphonate.

At room temperature, diphenylphosphite (0.41 ml) was added to a solution of 8 - ((2S, 3R, 4R, 5R) -5 ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3 - ((tert-butyldimethylsilyl ) oxy) -4-hydroxytetrahydrofuran-2-yl) pyrazolo [1,5-a] [1,3,5] triazin-4 (3H) -one (740 mg) in pyridine (10 ml). The reaction mixture was stirred under an argon atmosphere at room temperature for 1 hour. Water (20 ml) was added to the reaction mixture and the mixture was stirred for 1 hour. The reaction mixture was poured into injection at room temperature and extracted with ethyl acetate. The extract was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (300 mg). MS: [MH] ^- 747.1.

F) (2R, 3R, 4S, 5S) -4 - ((tert-butyldimethylsilyl) oxy) -2- (hydroxymethyl) -5- (4-oxo-3,4-dihydropyrazolo [1,5-a] [1 , 3,5] triazin-8-yl) tetrahydrofuran-3-yl hydrophosphonate.

K (2R, 3R, 4S, 5S) -2 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -5- (4-oxo-3,4 -dihydropyrazolo [1,5-a] [1,3,5] triazin-8-yl) tetrahydrofuran-3-yl hydrophosphonate (300 mg) was added 80% aqueous acetic acid solution (10 ml) and the mixture was stirred at room temperature in for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (170 mg). MS: [M + H] + 447.0.

G) (2R, 3R, 4S, 5S) -2 - (((((2R, 3R, 4R, 5R) -2- (6-benzamido-9H-purin-9-yl) -4 - ((tert -butyldimethylsilyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 ((tert-butyldimethylsilyl) oxy) -5- (4-oxo-3, 4-dihydropyrazolo [1,5-a] [1,3,5] triazin-8-yl) tetrahydrofuran-3-yl hydrophosphonate.

(2R, 3R, 4S, 5S) -4 - ((tert-Butyldimethylsilyl) oxy) -2- (hydroxymethyl) -5- (4-oxo-3,4-dihydropyrazolo [1,5-a] [1,3 , 5] triazin-8-yl) tetrahydrofuran-3-yl hydrophosphonate (190 mg) and N-benzoyl-5'-O- (bis (4methoxyphenyl) (phenyl) methyl) -3'-O- (tert-butyl ( dimethyl) silyl) -2'-O - ((2-cyanoethyl) (diisopropylamino) phosphino) adenosine (505 mg) was subjected to azeotropic distillation process three times with anhydrous acetonitrile. Anhydrous acetonitrile (5 ml) and pyridine-2,2,2-trifluoroacetate (164 mg) were added to the residue. The reaction mixture was stirred under argon at room temperature for 10 min, ((dimethylaminomethylidene) amino) -3H-1,2,4-dithiazoline-3-thione (96 mg) was added to the reaction mixture, and the mixture was stirred at room temperature for 20 minutes. To the reaction mixture was added an aqueous solution (0.2 ml) of sodium thiosulfate (0.2 g), and the mixture was concentrated under reduced pressure. To the residue was added 80% aqueous acetic acid solution (5 ml), the mixture was stirred at room temperature for 1 hour, and the reaction mixture was subjected to azeotropic distillation process with acetonitrile twice. The residue was purified by silica gel column chromatography (meta- 45 038805 nol / ethyl acetate) to give the title compound (260 mg). MS: [M + H] + 1063.2.

H) 8 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl ( dimethyl) silyl) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 14-yl) pyrazolo [1,5-a] [1,3,5] triazin-4 (3H) -one (optical isomer).

(2R, 3R, 4S, 5S) -2 - (((((2R, 3R, 4R, 5R) -2- (6-Benzαmudo-9H-purun-9-yl) -4 - ((tert-butyldumethylsilyl ) oxy) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 ((tert-butyldimethylsilyl) oxy) -5- (4-oxo-3,4- dihydropyrazolo [1,5-a] [1,3,5] triazin-8-yl) tetrahydrofuran-3-yl hydrophosphonate (260 mg) was azeotroped twice with anhydrous acetonitrile. The residue was azeotroped once with anhydrous pyridine. To a solution of the residue in anhydrous pyridine (5 ml) was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2 oxide (158 mg) and the mixture was stirred under argon at room temperature for 20 minutes. Water (0.15 ml) and 3H-benzo [c] [1,2] dithiol-3one (49.4 mg) were added to the reaction mixture at room temperature. The reaction mixture was stirred at room temperature for 1 hour. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture at room temperature, and the mixture was extracted with ethyl acetate. The extract was washed with a saturated aqueous sodium thiosulfate solution and a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (methanol / ethyl acetate), then purified by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution) to obtain fractions, the fraction with the longest retention time concentrated under reduced pressure. A 33% ethanolic solution of methylamine (5 ml) was added to the residue, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (25 mg). MS: [M + H] + 920.2.

I) 8 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aS, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-dihydroxy-2,10dioxide-2 , 10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) pyrazolo [1,5-a] [1 , 3,5] triazin-4 (3H) -one diethylamine salt (optical isomer).

To a solution of 8 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aR, 16R) -7- (6-amino-9H-purin-9-yl) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14- yl) pyrazolo [1,5-a] [1,3,5] triazin-4 (3H) -one (optical isomer) (25 mg) in methanol (2 ml), triethylamine trihydrofluoride (0.177 ml) was added at room temperature and stir the mixture at 50 ° C for 2 h. To the reaction mixture at room temperature was added ethoxytrimethylsilane (5 ml) and the mixture was stirred for 10 min. The reaction mixture was concentrated under reduced pressure, the residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and the resulting product was lyophilized to give the title compound (13 mg).

1H NMR (300 MHz, D ₂ O) δ 4.02-4.19 (3H, m), 4.32-4.42 (2H, m), 4.44-4.49 (1H, m), 4.59-4.64 (1H, m), 4.76-4.80 (1H, m), 4.95-5.04 (1H, m), 5.16 (1H, d, J = 4 , 9 Hz), 5.31 (1H, ddd, J = 9.8, 8.7, 4.2 Hz), 6.22 (1H, d, J = 8.3 Hz), 7.99 (1H , s), 8.00 (1H, s), 8.14 (1H, s), 8.49 (1H, s).

Example 13. Synthesis of 2-αmuno-9 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aS, 16R) -14- (4-αmunopyrαzolo [1,5-a] [1,3,5] triazine -8-yl) -2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10 ] pentaoxadiphosphacyclotetradecin-7-yl) -1,9-dihydro-6H-purin-6-one diethylamine salt

- 46 038805

A) (2S, 3S, 4R, 5R) -2- (4- (N-benzoylbenzamido) pyrazolo [1,5-a] [1,3,5] triazin-8-yl) -5 - ((benzoyloxy) methyl) tetrahydrofuran-3,4-diyldibenzoate.

To a solution of (2S, 3R, 4S, 5R) -2- (4-aminopyrazolo [1,5-a] [1,3,5] triazin-8-yl) -5- (hydroxymethyl) tetrahydrofuran-3,4- diol (2.98 g) and K, N-dimethyl-4-aminopyridine (1.36 g) in anhydrous pyridine (50 ml) was added benzoyl chloride (12.5 g) while cooling in an ice bath. The reaction mixture was stirred under argon for 1 hour. Benzoyl chloride (3.13 g) was added to the reaction mixture while cooling in an ice bath. The reaction mixture was stirred under argon at room temperature for 2 hours. Water was added to the reaction mixture and the mixture was extracted with ethyl acetate. The extract was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (6.51 g). MS: [M + H] + 788.2.

B) N- (8 - ((2S, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) pyrazolo [1,5-a]

[1,3,5] triazin-4-yl) benzamide.

To a solution of (2S, 3S, 4R, 5R) -2- (4- (N-benzoylbenzamido) purazolo [1,5-a] [1,3,5] triazin-8-yl) -5 ((benzoyloxy) methyl ) tetrahydrofuran-3,4-diyldibenzoate (6.51 g) in a solvent of mixed pyridine (50 ml) and ethanol (25 ml) was added 1M aqueous sodium hydroxide solution (49.6 ml) while cooling in an ice bath and the mixture was stirred at DOWEX ™ 50Wx4 100-200 strong acidic cation exchange resin (40 g) was added to the reaction mixture at room temperature for 2 hours. The mixture was stirred at room temperature for 15 minutes. The solid was removed by filtration, and the filtrate was concentrated under reduced pressure. Methanol was added to the residue, and the resulting solid was collected by filtration to give the title compound (1.82 g). MS: [M + H] ⁺ 372.1.

C) N- (8 - ((2S, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) pyrazolo [1, 5-a] [1,3,5] triazin-4-yl) benzamide.

To a solution of N- (8 - ((2S, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl) pyrazolo [1,5-a] [1,3,5] triazin-4-yl) benzamide (1.89 g) in anhydrous pyridine (30 ml) was added 4,4'-dimethoxytrityl chloride (517 mg) with cooling in an ice bath. The reaction mixture was stirred under argon at room temperature for 3 hours. To the reaction mixture at room temperature was added 4,4'-dimethoxytrityl chloride (517 mg) and the mixture was stirred overnight under argon at room temperature. To the reaction mixture at room temperature was added 4,4'dimethoxytrityl chloride (1035 mg) and the mixture was stirred under argon at room temperature for 1 hour. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture at room temperature and the mixture was extracted with ethyl acetate ... The extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (2160 mg). MS: [MH] ^- 672.1.

D) N- (8 - ((2S, 3R, 4R, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3 - ((tert-butyldimethylsilyl) oxy) -4-hydroxytetrahydrofuran -2-yl) pyrazolo [1,5-a] [1,3,5] triazin-4-yl) benzamide.

To a solution of N- (8 - ((2S, 3R, 4S, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3,4-dihydroxytetrahydrofuran-2-yl) pyrazolo [1, 5-a] [1,3,5] triazin-4-yl) benzamide (2.16 g) in anhydrous THF (30 ml) was added silver nitrate (708 mg) and anhydrous pyridine (1.22 g). The reaction mixture was stirred under argon atmosphere for 15 minutes and tert-butyldimethylchlorosilane (628 mg) was added thereto. The reaction mixture was stirred under argon at room temperature for 2 hours. Silver nitrate (163 mg), tert-butyldimethylchlorosilane (145 mg) and anhydrous pyridine (507 mg) were added to the reaction mixture at room temperature. The reaction mixture was stirred overnight under argon atmosphere at room temperature. Silver nitrate (436 mg), tert-butyldimethylchlorosilane (387 mg) and anhydrous pyridine (761 mg) were added to the reaction mixture at room temperature. The reaction mixture was stirred under argon at room temperature for 2 hours. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture at room temperature, and the mixture was extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give the title compound (980 mg). MS: [MH] ^- 786.2.

E) (2R, 3R, 4S, 5S) -5- (4-benzamidopyrazolo [1,5-a] [1,3,5] triazin-8-yl) -4 - ((tert-butyldimethylsilyl) oxy) - 2- (hydroxymethyl) tetrahydrofuran-3-yl hydrophosphonate.

N- (8 - ((2S, 3R, 4R, 5R) -5 - ((bis (4-methoxyphenyl) (phenyl) methoxy) methyl) -3 - ((tert-butyldimethylsilyl) oxy) -4-hydroxytetrahydrofuran-2 -yl) pyrazolo [1,5-a] [1,3,5] triazin-4-yl) benzamide (980 mg) was azeotroped twice with anhydrous toluene and dissolved in anhydrous DMF (10 ml). To the reaction mixture were added 3 - ((bis (diisopropylamino) phosphino) oxy) propanenitrile (487 mg), III-tetrazole (87 mg) and 1-methyl-1H-imidazole (51 mg). The reaction mixture was stirred under argon at room temperature for 5 hours. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture at room temperature, and the mixture was extracted with ethyl acetate. The extract was washed with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate containing 0.5% triethylamine / hexane). To a solution of the resulting mixture at room temperature in acetonitrile (10 ml) were added water (0.04 ml) and pyridine-2,2,2 trifluoroacetate (256 mg). The reaction mixture was stirred at room temperature for 30 minutes, tert-butylamine (5.38 ml) was added thereto and the mixture was stirred at room temperature for 45 minutes. The solvent was evaporated off under reduced pressure. To the residue was added 80% aqueous acetic acid solution (5.4 ml), the mixture was stirred at room temperature for 1 hour, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (530 mg). MS: [M + H] + 550.2.

F) (2R, 3R, 4S, 5S) -5- (4-benzamidopyrazolo [1,5-a] [1,3,5] triazin-8-yl) -4 - ((tert-butyldimethylsilyl) oxy) - 2 - ((((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5- (hydroxymethyl) -2- (2-isobutylamido-6oxo-1H-purine-9 (6H ) -yl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate.

A mixture of (2R, 3R, 4S, 5S) -5- (4-benzamidopyrazolo [1,5-a] [1,3,5] triazin-8-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - (hydroxymethyl) tetrahydrofuran-3-yl hydrophosphonate (240 mg) and 5'-O- (6is (4methoxyphenyl) (phenyl) methyl) -3'-O- (tert-butyl (dimethyl) silyl) -2'-O - ((2-cyanoethyl) (diisopropylamino) phosphino) -K-isobutyrylguanosine (635 mg) was subjected to azeotropic distillation process three times with anhydrous acetonitrile. To the residue was added a solution of anhydrous acetonitrile (5 ml), anhydrous THF (2.5 ml) and 5- (ethylthio) -2H-tetrazole (171 mg) (which was previously subjected to azeotropic distillation with anhydrous acetonitrile) in anhydrous acetonitrile (2 , 5 ml). The reaction mixture was stirred under argon at room temperature for 2 hours. A 70% aqueous solution of tert-butyl hydroperoxide (0.179 ml) was added thereto, and the mixture was stirred at room temperature for 20 minutes. To the reaction mixture was added an aqueous solution (0.12 ml) of sodium thiosulfate pentahydrate (0.12 g), and the reaction mixture was concentrated under reduced pressure. To the residue was added 80% aqueous acetic acid solution (3 ml) and the mixture was stirred at room temperature for 1 hour. The residue was azeotroped twice with acetonitrile. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give a crude product (420 mg) containing the title compound. MS: [M + H] + 1132.3.

G) N- (8 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2- 48 038805 cyanoethoxy) -2-hydroxy-7- (2 - ((2-methylpropanoyl) amino) -6-oxo-1,6-dihydro-9H-purin-9-yl) -2.10 dioxidooctahydro-12H-5.8 -methanofuro [3,2-1] [1,3,6,9, P, 2,10] pentaoxadiphosphacyclotetradecin-14yl) pyrazolo [1,5-a] [1,3,5] triazin-4-yl) benzamide ...

Crude product (430 mg) containing (2R, 3R, 4S, 5S) -5- (4-benzamidopyrazolo [1,5-a] [1,3,5] triazin-8yl) -4 - ((tert-butyldimethylsilyl ) oxy) -2 - ((((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl) oxy) -5 (hydroxymethyl) -2- (2-isobutylamido-6-oxo-1H- purin-9 (6H) -yl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphoryl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate was azeotroped twice with anhydrous acetonitrile. The resulting residue was subjected once to azeotropic distillation process with anhydrous pyridine. To a solution of the resulting residue in anhydrous pyridine (3 ml) was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine-2-oxide (245 mg) and the mixture was stirred at room temperature under argon atmosphere for 10 min. ... Water (0.239 ml) and iodine (125 mg) were added to the reaction mixture, and the mixture was stirred at room temperature for 20 minutes. To the reaction mixture was added an aqueous solution (0.4 ml) of sodium thiosulfate pentahydrate (245 mg), and the mixture was stirred at room temperature for 5 minutes. Toluene was added thereto, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give a crude product (553 mg) containing the title compound. MS: [M + H] + 1130.4.

H) 2-Amino-9 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aS, 16R) -14- (4-αmunnuрαzolo [1,5-a] [1,3,5] truαзun-8 -ul) 2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 7-yl) -1,9-dihydro-6H-purin-6-one diethylamine salt.

A mixture of N- (8 - ((5R, 7R, 8R, 12aR, 14S, 15S, 15aR, 16R) -15,16-bis ((tert-butyl (dimethyl) silyl) oxy) -10- (2cyanoethoxy) -2 -hydroxy-7- (2 - ((2-methylpropanoyl) amino) -6-oxo-1,6-dihydro-9H-purin-9-yl) -2,10 dioxidooctahydro-12H-5,8-methanofuro [3, 2-Th1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14yl) pyrazolo [1,5-a] [1,3,5] triazin-4-yl) benzamide (553 mg) and 33% ethanol methylamine solution (20 ml) was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (methanol / ethyl acetate) and then purified by HPLC (ODS, mobile phase: water / acetonitrile (containing 5 mM ammonium acetate)) to obtain a fraction, the resulting fraction was concentrated under reduced pressure. To a solution of the resulting residue in methanol (3 ml) at room temperature was added triethylamine trihydrofluoride (0.072 ml) and the mixture was stirred at 50 ° C for 4 hours. To the reaction mixture at room temperature was added triethylamine trihydrofluoride (0.181 ml) and the mixture was stirred for nights at 50 ° C. Triethylamine trihydrofluoride (0.181 ml) was added to the reaction mixture at room temperature and the mixture was stirred at 50 ° C for 5 hours. Ethoxytrimethylsilane (1.034 ml) was added to the reaction mixture at room temperature and the mixture was stirred for 5 minutes. The reaction mixture was concentrated under reduced pressure, the residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and the resulting product was lyophilized to give the title compound (10 mg).

¹ H NMR (300 MHz, D ₂ O) δ 3.99-4.08 (1H, m), 4.11-4.31 (4H, m), 4.34-4.42 (1H, m) , 4.52-4.60 (2H, m), 4.94-5.05 (1H, m), 5.27-5.35 (1H, m), 5.51-5.62 (1H, m), 5.94 (1H, d, J = 8.3 Hz), 7.89 (1H, s), 8.07 (1H, s), 8.08 (1H, s).

Example 14 7 - ((2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) - 15-fluoro-16hydroxy- 2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-U1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3 , 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one disodium salt

- 49 038805

A) (2R, 3R, 4R, 5R) -5 - (((((2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4-fluoro-2 - (hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -2- (5-fluoro-4-oxo-3H-pyrrolo [ 2,3-d] pyrimidin-7 (4H) -yl) tetrahydrofuran-3-yl hydrophosphonate 7 (3-O- (tert-butyl (dimethyl) silyl) -2-O- (hydroxy (oxide) phosphoranyl) -β -D-ribofuranosyl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (8.89 g) and N-benzoyl-5'-O- (bis (4- methoxyphenyl) (phenyl) methyl) -3'-O - ((2-cyanoethoxy) (diisopropylamino] phosphino) -2'-deoxy-2'-fluoroadenosine (18.5 g) was subjected to azeotropic distillation process three times with anhydrous acetonitrile. the residue was added anhydrous acetonitrile (40 ml), a solution of anhydrous THF (20 ml) and anhydrous acetonitrile (40 ml) 5 (ethylsulfanyl) -2H-tetrazole (7.49 g). The reaction mixture was stirred at room temperature for 30 min under atmosphere argon, ((dimethylaminomethylidene) amino) 3H-1,2,4-dithiazoline-3-thione (4.33 g) was added to the reaction mixture, the mixture was stirred at room temperature for 30 minutes. To the reaction mixture was added an aqueous solution (5 ml) containing sodium thiosulfate (5.24 g), and the mixture was concentrated under reduced pressure. An 80% aqueous solution of acetic acid (100 ml) was added to the residue, the mixture was stirred at room temperature for 1 h, and the reaction mixture was subjected to azeotropic distillation with toluene. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (14.3 g). MS: [M + H] + 968.3.

B) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16 - ((tert-butyl (dimethyl) silyl) oxy) -2- (2-cyanoethoxy) -15 -fluoro-7- (5-fluoro-4-oxo-3, 4-dihydro-7H-pyrrolo [2,3-b] pyrimidin-7-yl) -10-oxide-10-sulfanyl-2-sulfidooctahydro -12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purin-6-yl) benzamide (optical isomer) ...

(2R, 3R, 4R, 5R) -5 - (((((2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4-fluoro-2- ( hydroxymethyl) tetrahydrofuran-3-yl) oxy) (2-cyanoethoxy) phosphorothioyl) oxy) methyl) -4 - ((tert-butyldimethylsilyl) oxy) -2 (5-fluoro-4-oxo-3H-pyrrolo [2,3 -d] pyrimidin-7 (4H) -yl) tetrahydrofuran-3-yl hydrophosphonate (1.07 g) was subjected to azeotropic distillation process three times with anhydrous pyridine. To a solution of the residue in anhydrous pyridine (150 ml) was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide (4.66 g) and the mixture was stirred at room temperature for 30 min under argon atmosphere ... To the reaction mixture at room temperature were added 3H-benzo [c] [1,2] dithiol-3-one (1.46 g) and water (4.55 ml), the reaction mixture was stirred for 30 min. LCMS analysis of the reaction mixture showed four peaks of equal mass ([M + H] ⁺ 971.1), indicating the formation of four diastereomers. The reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography on silica gel (methanol / ethyl acetate), then purified by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM aqueous ammonium acetate / acetonitrile solution). The fraction with the longest retention time was concentrated under reduced pressure to give the title compound (379 mg). MS: [M + H] ⁺ 982.1.

C) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aR, 16R) -14- (6-amino-9H-purin-9-yl) -16 - ((tert-butyl (dime- tyl) silyl) oxy) -15-fluoro-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2, 10] πentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4H-πyrrolo [2,3-d] πiri- 50 038805 midin-4-one (optical isomer).

40% Methanol solution of methylamine (10 ml) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -16 - ((tert-butyl (dimethyl) silyl) oxy) -2- (2 -cyanoethoxy) -15-fluoro-7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -10-oxide-10-sulfanyl- 2-sulfidooctahydro-12H-5,8-methanofuro [3,2-l]

[1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purin-6-yl) benzamide (optical isomer) (379 mg) was stirred at room temperature for 3 hours under argon ... The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (319 mg). MS: [M + H] + 825.1.

D) 7 - ((2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) - 15-fluoro-16-hydroxy -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5 -fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt.

To a solution of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -16 - ((tert-butyl (dimethyl) silyl ) oxy) -15-fluoro-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin -7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer) (319 mg) in methanol (1 ml) trihydrofluoride was added at room temperature triethylamine (2.52 ml) and the mixture was stirred at 50 ° C for 3 h, diluted with methanol (10 ml). Ethoxytrimethylsilane (12 ml) was added to the reaction mixture at room temperature and the mixture was stirred for 10 minutes. The reaction mixture was concentrated under reduced pressure, the residue was diluted with methanol, and the mixture was concentrated again under reduced pressure. Triethylammonium acetate buffer was added to the residue, and the solid was collected by filtration and washed with acetonitrile to give a white solid. The resulting white solid was purified by silica gel column chromatography (ODS, 10 mM triethylammonium acetate / acetonitrile buffer). On the other hand, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) and silica gel column chromatography (ODS, 10 mM triethylammonium acetate / acetonitrile buffer). The fraction containing the title compound obtained by silica gel column chromatography (ODS, 10 mM triethylammonium acetate / acetonitrile buffer) was concentrated under reduced pressure, and the residue was lyophilized to give the title compound (195 mg). MS: [M + H] + 711.0.

E) 7 - ((2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) -15-fluoro-16-hydroxy2 , 10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one disodium salt.

Deionized water (60 ml) was passed through a column prepared by loading 50W-X8 AG (trade name) cation exchange resin (100-200 mesh, 3.9 g) into an empty column. Then, a 1M aqueous sodium hydroxide solution (36 ml) and deionized water (68 ml) were passed through the resin. Deionized water (15 ml) containing 7 - ((2R, 5R, 7R, 8R, 10R, 12aR, 14R, 15R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) - 15-fluorine -16-hydroxy-2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-Fluoro-3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (195 mg) after the above pretreatment was passed through the resin and deionized water (19 ml) was passed through the resin, the resulting aqueous the solution was freeze-dried to give the title compound (165 mg).

¹ H NMR (300 MHz, D ₂ O) δ 4.00-4.08 (1H, m), 4.22-4.45 (4H, m), 4.50-4.57 (1H, m) , 4.81-4.84 (1H, m), 4.98-5.14 (2H, m), 5.40-5.61 (1H, m), 6.33-6.43 (2H, m), 7.28 (1H, d, J = 1.9 Hz), 7.94 (1H, s), 8.05 (1H, s), 8.21 (1H, s).

³¹ P NMR (121 MHz, D ₂ O) δ 52.1, 55.3.

Example 15. Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro2,16-dihydroxy-2 , 10-dioxide-10-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3 , 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt (optical isomer)

- 51 038805

A) 5-Fluoro-7 - ((6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f]

[1,3,5,2,4] trioxasilocin-8-yl) -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

To a solution of 7 - ((2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-ul) -5-fluoro-3Hpyrrolo [2,3-d] pyrimidine-4 (7H ) -one (37.9 g) in pyridine (760 ml) was added 1,3-dichloro-1,1,3,3 tetraisopropyldisiloxane (44.4 ml), the mixture was stirred at room temperature for 3 hours under argon. The solvent was evaporated away under reduced pressure, and ethyl acetate and water were added to the residue. The organic layer was washed with water and a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was diluted with isopropyl ether (IPE) and the mixture was stirred overnight at room temperature. The solid was collected by filtration to give the title compound (32.3 g). The mother liquor was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give the title compound (13.3 g). MS: [M + H] + 528.2.

B) 2-Cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3H-purrolo [2,3-d] purumidin-7 (4H) -yl) -2,2 , 4,4 tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasulotsin-9-yl diisopropylphosphoramidite.

5-Fluoro-7 - ((6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2, 4] trioxasilocin-8-yl) -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (20 g) was subjected to azeotropic drying three times with anhydrous acetonitrile and dissolved in anhydrous DMF (100 ml) ... To this solution was added 1H-tetrazole (2.79 g), 1-methyl-1H-imidazole (1.65 ml) and 3 - ((bis (diisopropylamino) phosphino) oxy) propanenitrile (24.1 ml), the mixture was stirred overnight at room temperature under argon atmosphere, poured into saturated aqueous sodium hydrogen carbonate solution, and extracted with ethyl acetate.

The organic layer was sequentially washed with a saturated aqueous sodium hydrogen carbonate solution and a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane containing 0.5% triethylamine) to give the title compound (20.8 g) as a mixture of two diastereomers.

- 52 038805 ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 0.91-1.19 (40H, m), 2.69-2.82 (2H, m), 3.46-3.73 ( 2H, m), 3.78-4.12 (5H,

m), 4.47-4.61 (2H, m), 6.02-6.12 (1H, m), 7.14-7.23 (1H, m), 7.84-7.91 ( 1H, m), 12.00-12.40 (1H, br).

C) (2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -2 - ((((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) - 8- (5-fluoro4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [ 1,3,5,2,4] trioxasilocin-9-yl) oxy) phosphorothioyl) oxy) methyl) -4-fluorotetrahydrofuran-3-yl hydrophosphonate.

A mixture of (2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4-fluoro-2- (hydroxymethyl) tetrahydrofuran-3yl hydrophosphonate (3.48 g) and 2-cyanoethyl - (6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3H-pyrrolo [2,3d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro- 6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl diisopropylphosphoramidite (7.82 g) was azeotropically dried with anhydrous acetonitrile and suspended in anhydrous acetonitrile (25 ml) and anhydrous THF (15 ml). 5- (Ethylsulfanyl) -2Htetrazole (3.11 g), which was previously subjected to azeotropic distillation with anhydrous acetonitrile, was dissolved in anhydrous acetonitrile (6 ml), the solution was added to the above suspension and the mixture was stirred at room temperature for 1 h in an atmosphere argon. To this was added ((dimethylaminomethyl-ide) amino) -3H-1,2,4-dithiazoline-3-thione (3.27 g) and the mixture was stirred at room temperature for an additional 30 minutes. The solvent was evaporated away under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (6.73 g). MS: [M + H] + 1096.2.

D) (2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -2 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) - 2- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 5 - (hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphorothioyl) oxy) methyl) -4-fluorotetrahydrofuran-3-yl hydrophosphonate.

(2R, 3R, 4R, 5R) -5- (6-Benzamido-9H-purin-9-yl) -2 - ((((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) -8- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1, 3,5,2,4] trioxasilocin-9-yl) oxy) phosphorothioyl) oxy) methyl) -4-fluorotetrahydrofuran-3-yl hydrophosphonate (6.73 g) was dissolved in a mixture of THF solvents (74.8 ml) and water (16.6 ml), the solution was cooled in an ice bath. Trifluoroacetic acid (16.6 ml) was added thereto and the mixture was stirred at 0 ° C for 2 hours. The reaction mixture was quenched with aqueous sodium hydrogen carbonate solution (25.8 g) and extracted with ethyl acetate. The organic layer was washed with a saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (6.43 g). MS: [M + H] + 1114.2.

E) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2-cyanoethoxy) -15-fluoro-7- (5-fluoro-4-oxo-3 , 4dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-16 - ((3-hydroxy-1,1,3,3-tetra (propan-2yl) disiloxanyl) oxy) -2-oxide-10-sulfidooctahydro-12H-5,8-methanofuro [3,2-Sh1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purin-6-yl) benzamide.

(2R, 3R, 4R, 5R) -5- (6-Benzamido-9H-purin-9-yl) -2 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) -2- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 5- ( hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphorothioyl) oxy) methyl) -4-fluorotetrahydrofuran-3-yl hydrophosphonate (6.43 g) was subjected to azeotropic drying with anhydrous acetonitrile and anhydrous pyridine, suspended in anhydrous pyridine (130 ml) 2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide (3.73 g) was added thereto, and the mixture was stirred at room temperature for 1 hour under argon. Water (5.17 ml) and iodine (1.90 g) were added thereto and the mixture was stirred at room temperature for an additional 1 h. The reaction mixture was quenched with sodium thiosulfate (7.16 g) and water (3.6 ml) and extracted with ethyl acetate. The organic layer was washed with water and a saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (4.60 g). MS: [M + H] + 1112.2.

F) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) -15-fluoro-2-hydroxy-16- ( (3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxido-10-sulfanyloctahydro-12H-5,8methanofuro [3,2-l] [1,3,6,9,11 , 2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one (optical isomer).

To N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2-cyanoethoxy) -15-fluoro-7- (5-fluoro-4-oxo-3, 4dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-16 - ((3-hydroxy-1,1,3,3-tetra (propan-2-yl) disiloxanyl) oxy ) -2-oxide-10-sulfidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purine- 6-yl) benzamide (6.42 g) was added 40% methanol solution of methylamine (30 ml) and the mixture was stirred at room temperature for 1 hour under argon atmosphere. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate). The resulting residue was purified by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution), the resulting fraction was concentrated under reduced pressure and the residue was lyophilized to give the title compound (420 mg). MS: [M + H] + 955.2.

G) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-2,16-dihydroxy2, 10-dioxide-10-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3, 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-2-hydroxy-16 - (( 3hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxide-10-sulfanyloctahydro-12H-5,8methanofuro [3,2-l] [1,3,6,9,11,2, 10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one (optical isomer obtained from tR2) (420 mg) triethylamine trihydrofluoride was added (9, 68 ml) and the mixture was stirred at 50 ° C for 7.5 hours. Ethoxytrimethylsilane (36.3 ml) was added to the reaction mixture at room temperature and the mixture was stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and freeze-dried to give the title compound (298 mg).

1H NMR (300 MHz, D ₂ O) δ 1.13-1.29 (16H, m), 3.12 (11H, q, J = 7.2 Hz), 3.99-4.29 (4H, m), 4.31-4.43 (2H, m), 4.50 (1H, d, J = 9.1 Hz), 4.81-5.08 (3H, m), 5.39-5 , 61 (1H, m), 6.30-6.42 (2H, m), 7.24 (1H, d, J = 1.9 Hz), 7.92 (1H, s), 8.09 ( 1H, s), 8.19 (1H, s).

³¹ P NMR (121 MHz, D2O) δ 52,2, 1,63.

Example 16. Synthesis 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -2.10.18trihydroxy-2.10 -dioxydhexahydro-14H-15,12- (enoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl) - 5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer)

Me me

- 54 038805

A) (1R, 3R, 4R, 7S) -3- (6-benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) - 8- (5-fluoro4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f]

[1,3,5,2,4] trioxasilocin-9-yl) oxy) phosphoryl) oxy) methyl) -2,5-dioxabicyclo [2,2,1] heptan-7-ylhydrogenphosphonate.

(1S, 3R, 4R, 7S) -3- (6-Benzamido-9H-purin-9-yl) -1- (hydroxymethyl) -2,5-dioxabicyclo [2,2,1] heptan7-yl-hydrophosphonate ( 2.00 g) and 2-cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3H-pyrrolo [2,3d] pyrimidin-7 (4H) -yl) -2 , 2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yldiisopropylphosphoramidite (4.23 g) was subjected to azeotropic drying with anhydrous acetonitrile (about 50 ml, three times) and suspended in anhydrous THF (16 ml). 5- (Ethylsulfanyl) -2H-tetrazole (1.75 g), which was previously subjected to azeotropic distillation with anhydrous acetonitrile (about 30 ml, three times), was dissolved in anhydrous acetonitrile (16 ml), the solution was added to the above suspension and the mixture was stirred at room temperature for 15 h in a flow of argon. A 70% aqueous solution of tert-butyl hydroperoxide (1.86 ml) was added thereto and the mixture was stirred at room temperature for an additional 30 minutes. The reaction mixture was quenched with 10% aqueous sodium thiosulfate solution (13 ml) and concentrated under reduced pressure. The residue was sequentially azeotropically dried with acetonitrile (about 80 ml) and toluene (about 80 ml), the residue was purified by silica gel column chromatography (methanol / ethyl acetate), the target fraction was concentrated under reduced pressure to give the title compound (5.36 d) as a white amorphous solid (mixture of two diastereomers). MS: [M + H] + 1090.2.

B) (1R, 3R, 4R, 7S) -3- (6-benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) - 2- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 5 - (hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphoryl) oxy) methyl) -2,5-dioxabicyclo [2.2.1] heptane-7yl-hydrophosphonate.

(1R, 3R, 4R, 7S) -3- (6-Benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) -8- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1, 3,5,2,4] trioxasilocin-9-yl) oxy) phosphoryl) oxy) methyl) -2,5-dioxabicyclo [2,2,1] heptan-7-ylhydrogenphosphonate (5.36 g) was dissolved in a mixture of solvents THF (60 ml) and water (13 ml), the solution was cooled in an ice bath. To this was added trifluoroacetic acid (13.18 ml) and the mixture was stirred at 0 ° C for 4 h. The reaction mixture was carefully quenched with a solution of sodium bicarbonate (20.65 g) in water (250 ml), saturated NaCl and extracted with ethyl acetate-THF (3: 1). The organic layer was washed with a saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography (methanol / ethyl acetate), and the target fraction was concentrated under reduced pressure to obtain the title compound (3.06 g) as a white amorphous solid (mixture of two diastereomers). MS: [M + H] + 1108.3.

C) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -10- (2-cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro -7Hpyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-18 - ((3-hydroxy-1,1,3,3-tetra (propan-2-yl) disiloxanyl) oxy) -2 , 10-dioxidohexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14 (12H) -yl ) -9H-purin-6-yl) benzamide.

(1R, 3R, 4R, 7S) -3- (6-Benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) -2- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 5- ( hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphoryl) oxy) methyl) -2,5-dioxabicyclo [2.2.1] heptane-7yl-hydrophosphonate (3.06 g) was successively azeotropically dried with anhydrous acetonitrile (about 100 ml) and anhydrous pyridine (about 100 ml) and dissolved in anhydrous pyridine (75 ml). 2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide (1.78 g) was added thereto and the mixture was stirred at room temperature for 1 hour under argon flow. Water (1.74 ml) and iodine (911 mg) were added thereto and the mixture was stirred at room temperature for an additional 15 minutes. The reaction mixture was quenched with 10% aqueous sodium thiosulfate solution (7.5 ml) and concentrated under reduced pressure. The residue was diluted with water (100 ml) and the mixture was extracted with ethyl acetate-THF (3: 1). The organic layer was washed with a saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. Toluene (about 100 ml) was added thereto and the mixture was concentrated again under reduced pressure. The residue was subjected to silica gel column chromatography (methanol / ethyl acetate), and the target fraction was concentrated under reduced pressure to obtain the title compound (2.38 g) as a white amorphous solid (mixture of two diastereomers). MS: [M + H] + 1106.3.

D) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -2,10-dihydroxy-18 - ((3hydroxy -1,1,3,3-tetra (propan-2-yl) disiloxanyl) oxy) -2,10-dioxidohexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -10- (2-cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro-7Hpyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-18 - ((3-hydroxy-1,1,3,3-tetra (propan-2-yl) disiloxanyl) oxy) -2.10 -dioxydhexahydro-14H-15,12- (epoxymethano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] penta-55 038805 oxadiphosphacyclotetradecin-14 (12H) -yl) -9H-purin-6-yl) benzamide (2.38 g) was dissolved in 40% methanol solution of methylamine (40 ml), the solution was stirred at room temperature for 1 h and the reaction mixture was concentrated under reduced pressure. Toluene (about 80 ml) was added thereto and the mixture was concentrated again under reduced pressure. The residue was subjected to silica gel column chromatography (methanol / ethyl acetate), and the target fraction was concentrated under reduced pressure to obtain the title compound (756 mg) as a white solid. MS: [M + H] + 949.2.

E) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -2,10,18-trihydroxy-2,10dioxydohexahydro -14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl) -5- fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -1 4- (6-Amino-9H-purin-9-yl) -2,1 0-dihydroxy-18 - ((3 hydroxy-1,1,3,3-tetra (propan-2-yl) disiloxanyl) oxy) -2,10-dioxidohexahydro-14H-15,12а (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl) -5fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (756 mg) was dissolved in methanol (2 ml) and triethylamine (0.8 ml), triethylamine trihydrofluoride (3.90 ml) was added thereto and the mixture was stirred at 50 ° C for 2 h. The reaction mixture was left to cool to room temperature, neutralized 1M aqueous triethylammonium hydrogen carbonate solution (110 ml) and stirred at room temperature for 15 minutes. The reaction mixture was concentrated under reduced pressure and the residue was purified by ODS column chromatography (acetonitrile / 10 mM triethylammonium acetate buffer). The target fraction was concentrated under reduced pressure, and the residue was lyophilized to give the title compound (504 mg) as a white solid.

1H NMR (300 MHz, D ₂ O) δ 1.20 (18H, t, J = 7.4 Hz), 3.12 (12H, q, J = 7.2 Hz), 4.02-4.16 (4H, m), 4.23-4.37 (3H, m), 4.55 (1H, d, J = 4.5Hz), 4.82-4.93 (3H, m), 6, 12 (1H, s), 6.39 (1H, dd, J = 8.3, 1.5 Hz), 7.19 (1H, d, J = 1.9 Hz), 7.96 (1H, s ), 8.11 (1H, s), 8.17 (1H, s).

³¹ P NMR (121 MHz, D2 O) δ -1.91, -1.80.

Example 17 Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -10,18 dihydroxy-2,10-dioxido -2-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl ) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3d] pyrimidin-4-one diethylamine salt (optical isomer)

A) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -10- (2-cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro -7Hpyrrolo [2,3-0] pyrimidin-7-yl) -18 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10dioxide-2-sulfanylhexahydro-14H-15,12а - (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14 (12H) -yl) -9H-purin-6-yl) benzamide.

(1 R, 3R, 4R, 7S) -3 - (6-Benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) -2 - (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphoryl) oxy) methyl) -2,5-dioxabicyclo [2.2.1] heptane-7yl hydrogenphosphonate (3.71 g) was subjected to azeotropic drying with anhydrous acetonitrile and anhydrous pyridine, suspended in anhydrous pyridine (70 ml). To this was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide (2.15 g) and the mixture was stirred at room temperature for 1 hour under argon. To this was added 3H-benzo [c] [1,2] dithiol-3-one (674 mg) and the mixture was stirred at room temperature for an additional 1 hour. Saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture and the mixture was extracted with ethyl acetate. The organic layer was washed with a saturated sodium chloride aqueous solution and dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (2.78 g). MS: [M + H] + 1122.2.

- 56 038805

B) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -10-hydroxy-18 - ((3-hydroxy1 , 1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxide-2-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-1] [1,3,6 , 9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer).

N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -10- (2-Cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro-7Hpyrrolo [2,3-b] pyrimidin-7-yl) -18 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10dioxide-2-sulfanylhexahydro-14H-15,12- ( epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyklotetradecin-14 (12H) -yl) -9H-purin-6-yl) benzamide ( 2.78 g) was dissolved in 40% methanol solution of methylamine (50 ml), the solution was stirred at room temperature for 1 hour under argon and the solvent was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (methanol / ethyl acetate). The resulting residue was purified by HPLC (L-2 ODS column, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution) to give the title compound (30 mg). MS: [M + H] + 965.3.

C) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -10,18-dihydroxy-2,10dioxy-2 -sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyklotetradecin-7 (12H) -yl) - 5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

K 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -10-hydroxy-18 - ((3-hydroxy1, 1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxide-2-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6, 9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer) (30 mg) was added triethylamine trihydrofluoride (0.70 ml) and methanol (1 ml), the mixture was stirred at 50 ° C for 3 h. Ethoxy (trimethyl) silane (2.5 ml) was added to the reaction solution, the mixture was stirred at room temperature for 30 min and the solvent was evaporated under reduced pressure. The residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and the resulting solid was lyophilized to give the title compound (25 mg).

¹ H NMR (300 MHz, D2O) δ 1.19 (18H, t, J = 7.4 Hz), 3.11 (12H, q, J = 7.2 Hz), 3.99-4.09 ( 3H, m), 4.09-4.17 (2H, m), 4.22-4.29 (1H, m), 4.34-4.38 (1H, m), 4.50-4, 54 (2H, m), 4.90-4.96 (1H, m), 5.06 (1H, s), 6.11 (1H, s), 6.35 (1H, dd, J = 8, 3, 1.1 Hz), 7.19 (1H, d, J = 2.3 Hz), 7.94 (1H, s), 8.08 (1H, s), 8.16 (1H, s) ...

³¹ P NMR (121 MHz, D ₂ O) δ 55.76, -1.56.

Example 18 Synthesis of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -2.18dihydroxy-2,10-dioxido -10-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl ) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer)

- 57 038805

A) (1R, 3R, 4R, 7S) -3- (6-benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) - 8- (5-fluoro4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f]

[1,3,5,2,4] trioxasilocin-9-yl) oxy) phosphorothioyl) oxy) methyl) -2,5-dioxabicyclo [2,2,1] heptan-7-ylhydrogenphosphonate.

A mixture of (1S, 3R, 4R, 7S) -3- (6-benzamido-9H-purin-9-yl) -1- (hydroxymethyl) -2,5-dioxabicyclo [2,2,1] heptan-7-yl -hydrophosphonate (3.0 g) and 2-cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3Hpyrrolo [2,3-d] pyrimidin-7 (4H) -yl ) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl-diisopropylphosphoramidite (5.86 g) was subjected to azeotropic drying with anhydrous acetonitrile, suspended in anhydrous THF (25 ml). 5- (Ethylsulfanyl) -2H-tetrazole (2.62 g), which was previously subjected to azeotropic distillation with anhydrous acetonitrile, was dissolved in anhydrous acetonitrile (25 ml), the solution was added to the above suspension and the mixture was stirred at room temperature overnight in argon atmosphere. To this was added ((dimethylaminomethylidene) amino) -3H-1,2,4-dithiazoline-3-thione (2.75 g) and the mixture was stirred at room temperature for an additional 30 minutes. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (4.96 g). MS: [M + H] + 1106.3.

B) (1R, 3R, 4R, 7S) -3- (6-benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) - 2- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 5 - (hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphorothioyl) oxy) methyl) -2,5-dioxabicyclo [2,2,1] heptan-7-yl-hydrophosphonate.

(1R, 3R, 4R, 7S) -3- (6-Benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) -8- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1, 3,5,2,4] trioxasilocin-9-yl) oxy) phosphorothioyl) oxy) methyl) -2,5-dioxabicyclo [2,2,1] heptan-7-yl-hydrophosphonate (4.96 g) was dissolved in a mixture of solvents THF (60 ml) and water (12 ml), the solution was cooled in an ice bath. To this was added trifluoroacetic acid (12.1 ml) and the mixture was stirred at 0 ° C for 3 hours.The reaction mixture was quenched with a solution of sodium bicarbonate (18.8 g) in water (100 ml) and extracted with ethyl acetate-THF (3: 1 ). The organic layer was dried over sodium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (3.77 g). MS: [M + H] + 1124.2.

C) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -10- (2-cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro -7Hpyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-18 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 2-oxide-10-sulfidohexahydro-14H- 15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14 (12H) -yl) -9H-purine-6 -yl) benzamide.

(1R, 3R, 4R, 7S) -3- (6-Benzamido-9H-purin-9-yl) -1 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) -2- (5-fluoro-4oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 5- ( hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphorothioyl) oxy) methyl) -2,5-dioxabicyclo [2.2.1] heptan-7-yl-hydrophosphonate (3.77 g) was subjected to azeotropic drying with anhydrous acetonitrile and anhydrous pyridine, suspended in anhydrous pyridine (70 ml). To this was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine-2-oxide (2.17 g) and the mixture was stirred at room temperature for 1 hour under argon. Water (2.12 ml) and iodine (1.11 g) were added thereto and the mixture was stirred at room temperature for an additional 1 hour. The reaction mixture was quenched with 10% aqueous sodium thiosulfate solution (15 ml) and the solvent was evaporated under reduced pressure. The residue was diluted with water (100 ml) and the mixture was extracted with ethyl acetate-THF (3: 1). The organic layer was dried over sodium sulfate and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (2.21 g). MS: [M + H] + 1122.2.

D) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) - 14- (6-amino-9H-purin-9-yl) -2-hydroxy-18 - ((3-hydroxy1 , 1,3,3-tetra (propan-2-yl) disiloxanyl) oxy) -2,10-dioxide-10-sulfanyl-hexahydro-14H-15,12а (epoxymetano) -5,8-methanofuro [3,2 -l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl) -5fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidine-4- he (optical isomer).

To N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -10- (2-cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro- 7Hpyrrolo [2, 3-d] pyrimidin-7-yl) -2-hydroxy-18 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) 2-oxide-10-sulfidohexahydro-14H-15 , 12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14 (12H) -yl) -9H-purine-6- yl) benzamide (2.38 g) was added 40% methanol solution of methylamine (40 ml) and the mixture was stirred at room temperature for 1 hour under argon. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate). The resulting residue was purified by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution), the resulting fraction was concentrated under reduced pressure, and the residue was lyophilized to give the title compound (710 mg) ... MS: [M + H] +965.2.

E) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) - 14- (6-amino-9H-purin-9-yl) -2,18-dihydroxy-2,10-dioxide -10-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10]

- 58 038805 pentaoxadiphosphacyclotetradecin-7 (12H) -yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

To a solution of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -2-hydroxy-18 - ((3hydroxy-1 , 1,3,3-tetra (propan-2-yl) disiloxanyl) oxy) -2,10-dioxido-10-sulfanylhexahydro-14H15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ( optical isomer obtained from tR2) (710 mg) in methanol (20 ml) was added triethylamine trihydrofluoride (4.80 ml) and the mixture was stirred at 50 ° C for 3 h. Ethoxytrimethylsilane (2.29 ml) and the mixture was stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer), freeze-dried to give the title compound (530 mg).

¹ H NMR (300 MHz, D2O) δ 1.22 (18H, t, J = 7.2 Hz), 3.14 (12H, q, J = 7.6 Hz), 4.03-4.19 ( 4H, m), 4.25-4.41 (3H, m), 4.65 (1H, d, J = 4.5 Hz), 4.84 (1H, d, J = 4.5 Hz), 4.91 (1H, s), 4.99 (1H, ddd, J = 10.1, 8.2, 4.3Hz), 6.14 (1H, s), 6.39 (1H, d, J = 7.9 Hz), 7.17 (1H, d, J = 2.3 Hz), 7.97 (1H, s), 8.11 (1H, s), 8.19 (1H, s) ...

³¹ P NMR (121 MHz, D ₂ O) δ 52.5, 1.74.

Example 19 Synthesis of 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15-fluoro-7- (5-fluoro-4-oxo-3,4-dihydro-7H- nirrolo [2,3-d] pyrimidin-7-yl) -2,10,16-trihydroxy-2,10-dioxidooctahydro-12H-5,8methanofuro [3,2-l] [1,3,6,9, 11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -1, 9-dihydro-6H-purin-6-one diethylamine salt (optical isomer)

Me me

A) (2R, 3R, 4R, 5R) -2 - (((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3H-pyrrolo [2 , 3d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9yl) oxy) phosphoryl) oxy) methyl) -4-fluoro-5- (2-isobutylamido-6-oxo-1H-purin-9 (6H) -yl) tetrahydrofuran 3-yl-hydrophosphonate.

2'-Deoxy-2'-fluoro-3'-O- (hydroxy (oxide) phosphoranyl) -N-isobutyrylguanosine (680 mg) and 2 cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro- 4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4 tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2 , 4] trioxasilocin-9-yl-diisopropylphosphoramidite (1535 mg) was subjected to azeotropic drying with anhydrous acetonitrile (three times) and suspended in anhydrous THF (10.00 ml). To the suspension was added a mixture of 5- (ethylsulfanyl) -2H-tetrazole (633 mg) (which was azeotropically dried with anhydrous acetonitrile) and anhydrous acetonitrile (10 ml), and the mixture was stirred at room temperature for 7 hours under argon. To the mixture was added a mixture of 2-cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2 , 2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl-diisopropylphosphoramidite (1181 mg, 1.62 mmol) (which was subjected to azeotropic drying with anhydrous acetonitrile) and anhydrous acetonitrile (3 ml), a mixture of 5- (ethylsulfanyl) -2H-tetrazole (633 mg) (which was azeotropically dried with anhydrous acetonitrile) and anhydrous acetonitrile (3 ml), the mixture was stirred overnight at room temperature in an argon atmosphere. To the reaction mixture was added a 70% aqueous solution of tert-butyl hydroperoxide (0.674 ml) and the mixture was stirred at room temperature for an additional 30 minutes. To the reaction mixture was added a mixture of sodium thiosulfate pentahydrate (2817

- 59 038805 mg) and water (4 ml), the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (1260 mg). MS: [M + H] + 1062.3.

B) (2R, 3R, 4R, 5R) -2 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) -2- (5-fluoro-4-oxo-3H-pyrrolo [2 , 3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphoryl ) oxy) methyl) -4-fluoro-5- (2-isobutylamido-6-oxo-1H-purin-9 (6H) -yl) tetrahydrofuran-3-yl-hydrophosphonate.

To a mixture (2R, 3R, 4R, 5R) -2 - ((((2-cyanoethoxy) (((6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3H-pyrrolo [2 , 3d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9yl) oxy) phosphoryl) oxy) methyl) -4-fluoro-5- (2-isobutylamido-6-oxo-III-purin-9 (6H) -yl) tetrahydrofuran 3-yl-hydrophosphonate (1.26 g), THF (16 ml) and water (4 ml) was added trifluoroacetic acid (3.18 ml) at 0 ° C and the mixture was stirred for 4 hours. To the mixture was added an aqueous solution of sodium hydrogen carbonate (4.98 g) and the mixture was saturated with sodium chloride. The mixture was extracted with ethyl acetate / THF and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (1000 mg). MS: [M + H] + 1080.3.

C) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2-cyanoethoxy) -15-fluoro-7- (5-fluoro-4-oxo-3 , 4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-16 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxidooctahydro -12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -6-oxo-6,9-dihydro-1H-purine -2-yl) -2-methylpropanamide (2R, 3R, 4R, 5R) -2 - ((((2cyanoethoxy) (((2R, 3R, 4R, 5R) -2- (5-fluoro-4-oxo 3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy1,1,3,3-tetraisopropyldisiloxanyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-3 -yl) oxy) phosphoryl) oxy) methyl) -4-fluoro-5- (2-isobutylamido-6-oxo-1H-purin-9 (6H) -yl) tetrahydrofuran-3-yl-hydrophosphonate (1 g) was subjected to azeotropic drying with anhydrous acetonitrile and anhydrous pyridine and suspended in anhydrous pyridine (24 ml). 2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinine 2-oxide (598 mg) was added thereto and the mixture was stirred at room temperature for 15 minutes under argon. Water (0.584 ml) and iodine (305 mg) were added thereto and the mixture was stirred at room temperature for an additional 25 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (800 mg). MS: [M + H] + 1078.2.

D) 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 15-fluoro-7- (5-fluoro-4-oxo-3,4-dihydro-7H- pyrrolo [2,3-d] pyrimidin-7-yl) -2,10-dihydroxy-16 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxidooctahydro-12H- 5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -1,9-dihydro-6H-purin-6-one (optical isomer ).

To N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2-cyanoethoxy) -15-fluoro-7- (5-fluoro-4-oxo-3, 4dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-16 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxidooctahydro- 12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -6-oxo-6,9-dihydro-1H-purine- 2-yl) -2-methylpropanamide (800 mg, 0.74 mmol) was added 33% ethanolic solution of methylamine (30 ml), the mixture was stirred overnight at room temperature under argon atmosphere, the solvent was evaporated under reduced pressure. The residue was purified using silica gel column chromatography (methanol / ethyl acetate). The resulting residue was purified by HPLC (L-2 ODS column, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution) to give the title compound (163.4 mg). MS: [M + H] + 955.3.

E) 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 15-fluoro-7- (5-fluoro-4-oxo-3,4-dihydro-7H- pyrrolo [2,3-d] pyrimidin-7-yl) -2,10,16-trihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6, 9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -1,9-dihydro-6H-purin-6-one diethylamine salt (optical isomer).

A mixture of 2-amino-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 15-fluoro-7- (5-fluoro-4-oxo-3,4-dihydro-7Hpyrrolo [2 , 3-d] pyrimidin-7-yl) -2,10-dihydroxy-16 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -2,10-dioxidooctahydro-12H-5,8 -methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl) -1,9-dihydro-6H-purin-6-one (optical isomer) (21 , 4 mg), triethylamine trihydrofluoride (0.183 ml) and methanol (0.07 ml) were stirred at 50 ° C for 1 h under argon atmosphere. To the reaction mixture was added ethoxy (trimethyl) silane (1.046 ml), the mixture was stirred at room temperature for an additional 1 hour, and the solvent was evaporated under reduced pressure. The residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer) and freeze-dried to give the title compound (4.6 mg).

1H NMR (300 MHz, D ₂ O) δ 1.20 (18H, t, J = 7.3 Hz), 3.13 (12H, q, J = 7.3 Hz), 4.07 (2H, d , J = 11.1 Hz), 4.25 (2H, d, J = 7.6 Hz), 4.33-4.99 (4H, m), 5.04-5.25 (1H, m) , 5.41-5.70 (1H, m), 6.17 (1H, d, J = 19.0 Hz), 6.38 (1H, d, J = 9.2 Hz), 7.32 ( 1H, s), 7.79 (1H, br s), 7.95 (1H, s).

- 60 038805 ³¹ P NMR (121 MHz, D ₂ O) δ -1.61, -1.52.

Example 20. Synthesis 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -2,10,15,16 tetrahydroxy-2 -oxido-10-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt (optical isomer) νη ₂

A) cyanoethoxy)

(2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - ((((2 (((6aR, 8R , 9R, 9aR) -8- (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4 teraisopropylterahydro-6H-furo [ 3,2-f] [1,3,5,2,4] trioxasilocin-9-yl) oxy) phosphorothioyl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate.

A mixture of (2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 (hydroxymethyl) tetrahydrofuran-3-yl hydrophosphonate (3 , 0 d) and 2-cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2, 2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f]

[1,3,5,2,4] trioxasilocin-9-yl-diisopropylphosphoramidite (5.36 g) was azeotropically dried with anhydrous acetonitrile and suspended in anhydrous acetonitrile (25 ml) and anhydrous THF (15 ml). 5 (Ethylsulfanyl) -2H-tetrazole (2.13 g), which was previously subjected to azeotropic distillation with anhydrous acetonitrile, was dissolved in anhydrous acetonitrile (15 ml), the solution was added to the above suspension and the mixture was stirred overnight at room temperature in an atmosphere argon. To this was added ((dimethylaminomethylidene) amino) -3H-1,2,4-dithiazoline-3-thione (2.24 g), the mixture was stirred at room temperature for an additional 1 hour. The solvent was evaporated under reduced pressure, the residue was purified from by column chromatography on silica gel (meta- 61 038805 nol / ethyl acetate) to give the title compound (5.15 g). MS: [M + H] + 1208.3.

B) (2R, 3R, 4R, 5R) -5- (6-benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - ((((2cyanoethoxy) ((( 2R, 3R, 4R, 5R) -2- (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -4 - ((3-hydroxy1,1, 3,3-tetraisopropyldisiloxanyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphorothioyl) oxy) methyl) tetrahydrofuran-3-yl hydrophosphonate.

(2R, 3R, 4R, 5R) -5- (6-Benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - ((((2-cyanoethoxy) ((( 6aR, 8R, 9R, 9aR) -8- (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidin-7 (4H) -yl) -2,2,4,4-tetraisopropyltetrahydro- 6Н-furo [3,2-1] [1,3,5,2,4] trioxasilocin-9-yl) oxy) phosphorothioyl) oxy) methyl) tetrahydrofuran-3-yl-hydrophosphonate (5.15 g) was dissolved in a mixture of solvents THF (48 ml) and water (10 ml), the solution was cooled in an ice bath. To this was added trifluoroacetic acid (11.5 ml) and the mixture was stirred at 0 ° C for 3 hours. The reaction mixture was quenched with a solution of sodium bicarbonate (12.5 g) in water (100 ml) and extracted with ethyl acetate-THF. The organic layer was washed with a saturated sodium chloride aqueous solution and dried over sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (5.13 g). MS: [M + H] + 1226.4.

C) N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 15 - ((tert-butyl (dimethyl) silyl) oxy) -10- (2-cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-16 - ((3-hydroxy1,1,3 , 3-tetra (propan-2-yl) disiloxanyl) oxy) -2-oxide-10-sulfidooctahydro-12H-5,8-methanofuro [3,2l] [1,3,6,9,11,2,10 ] pentaoxadiphosphacyclotetradecin-14-yl) -9H-purin-6-yl) benzamide (2R, 3R, 4R, 5R) -5 (6-Benzamido-9H-purin-9-yl) -4 - ((tert-butyldimethylsilyl) oxy) -2 - ((((2-cyanoethoxy) (((2R, 3R, 4R, 5R) -2 (5-fluoro-4-oxo-3H-pyrrolo [2,3-d] pyrimidine-7 (4H ) -yl) -4 - ((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy) -5- (hydroxymethyl) tetrahydrofuran-3-yl) oxy) phosphorothioyl) oxy) methyl) tetrahydrofuran 3-yl-hydrophosphonate (5.13 g) was subjected to azeotropic drying with anhydrous acetonitrile and anhydrous pyridine and suspended in anhydrous pyridine (100 ml). To this was added 2-chloro-5,5-dimethyl1,3,2-dioxaphosphine 2-oxide (2.70 g) and the mixture was stirred at room temperature for 1 hour under argon. To this were added water (2.64 g) and iodine (1.38 g) and the mixture was stirred at room temperature for an additional 1 hour.The reaction mixture was quenched with sodium thiosulfate (5.19 g) and water (2.64 g) and was extracted with ethyl acetate. The organic layer was washed with water and a saturated sodium chloride aqueous solution and dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (3.53 g). MS: [M + H] + 1224.4.

D) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) -15 - (((1,1-dimethylethyl) dimethylsilyl) oxy) octahydro-2,10-dihydroxy-16 - ((3-hydroxy-1,1,3,3-tetrakis (1-methylethyl) disiloxanyl) oxy) -2-oxide-10-sulfanyl-5,8 -methano-12H-furo [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2 , 3-d] pyrimidin-4-one (optical isomer).

To N- (9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 15 - ((tert-butyl (dimethyl) silyl) oxy) -10- (2-cyanoethoxy) -7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-16 - ((3-hydroxy1,1,3,3- tetra (propan-2-yl) disiloxanyl) oxy) -2-oxide-10-sulfidooctahydro-12H-5,8-methanofuro [3,2l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin- 14-yl) -9H-purin-6-yl) benzamide (3.5 g) was added 40% methanolic methylamine solution (50 ml) and the mixture was stirred at room temperature for 1 hour under argon. The resulting mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate). The resulting residue was purified by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution), the resulting fraction was concentrated under reduced pressure and the residue was lyophilized to give the title compound (210 mg) ... MS: [M + H] +1067.4.

E) 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -2,10,15,16-tetrahydroxy-2oxido -10-sulfanyloctahydro-12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro -4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer).

To a solution of 7 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15 - (((1,1-dimethylethyl) dimethylsilyl) oxy) octahydro-2,10-dihydroxy-16 - ((3-hydroxy-1,1,3,3-tetrakis (1-methylethyl) disiloxanyl) oxy) -2-oxide-10-sulfanyl-5,8 -methano-12Н-furo [3,2-1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl) -5-fluoro-3,7-dihydro-4H-pyrrolo [2 , 3-d] pyrimidin-4-one (optical isomer obtained from tR2) (210 mg) in methanol (1.0 ml) - triethylamine (0.4 ml), triethylamine trihydrofluoride (0.962 ml) was added and the mixture was stirred at 50 ° C for 2.5 hours. Ethoxytrimethylsilane (4.59 ml) was added to the reaction mixture at room temperature and the mixture was stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by C18 silica gel column chromatography (10 mM triethylammonium acetate / acetonitrile buffer), freeze-dried to give the title compound (125 mg).

1H NMR (300 MHz, D ₂ O) δ 1.13-1.28 (18H, m), 3.12 (12H, q, J = 7.2 Hz), 4.02-4.11 (1H, m), 4.13-4.25 (2H,

- 62 038805

m), 4.27-4.40 (2H, m), 4.46 (1H, d, J = 6.4 Hz), 4.73-4.79 (2H, m), 4.80-4 , 91 (1H, m), 5.02 (1H, td, J = 8.8, 4.2 Hz),

6.08 (1H, d, J = 1.7 Hz), 6.35 (1H, d, J = 8.1 Hz), 7.27 (1H, d, J = 1.7 Hz), 7, 93 (1H, s), 8.14 (1H, s), 8.18 (1H, s).

³¹ P NMR (121 MHz, D2O) δ 52,4, 1,21.

Example 23. Synthesis of 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -10,15,16trihydroxy-2,10 -dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-fluoro-3-methyl -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt (optical isomer 1 and 2)

ΝΗΒζ ΝΗΒζ

A) (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -4 - {[tert-butyl (dimethyl) silyl] oxy} -2 ({[( 2-cyanoethoxy) {[(6aR, 8R, 9R, 9aR) -8- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidine-7 -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl] oxy} phosphoryl] oxy} methyl) tetrahydrofuran -3-yl-hydrophosphonate.

(2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -4 - {[tert-butyl (dimethyl) silyl] oxy} -2 (hydroxymethyl) tetrahydrofuran-3 -yl-hydrophosphonate (250 mg) and 2-cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3- d] pyrimidin-7-yl) -2,2,4,4-tetraisopropyltetrahydro6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl-diisopropylphosphoramidite (359 mg) ( stage C, example 24) was subjected to azeotropic drying with anhydrous acetonitrile and to them were added anhydrous acetonitrile (1.1 ml) and anhydrous tetrahydrofuran (0.7 ml). To the mixture was added a solution of 5- (ethylsulfanyl) -2Htetrazole (175 mg) (which was subjected to azeotropic drying with anhydrous acetonitrile) in anhydrous acetonitrile (0.75 ml), and the mixture was stirred in an argon atmosphere at room temperature for 2 hours. tert-butyl hydroperoxide (5.5 mol / L) in nonane (0.25 ml) was added and the mixture was stirred at room temperature for 40 minutes. The reaction mixture was cooled in an ice bath and a solution of sodium thiosulfate (345 mg) in water (0.275 ml) was added. The mixture was left stirring at room temperature for 15 minutes and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (methanol / dichloromethane) to give the title compound (318 mg) as a mixture of diastereomers. MS: [M + H] + 1206.3.

B) (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -4 - {[tert-butyl (dimethyl) silyl] oxy} -2 - ({[ (2cyanoethoxy) ({(2R, 3R, 4R, 5R) -2- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidine-7- yl) 5- (hydroxymethyl) -4 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] tetrahydrofuran-3-yl} oxy) phosphoryl] oxy} methyl) tetrahydrofuran-3-yl hydrophosphonate.

(2R, 3R, 4R, 5R) -5- [6- (Benzoylamino) -9H-purin-9-yl] -4 - {[tert-butyl (dimethyl) silyl] oxy} -2 - ({[(2cyanoethoxy ) {[(6aR, 8R, 9R, 9aR) -8- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidine-7yl) -2 , 2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl] oxy} phosphoryl] oxy} methyl) tetrahydrofuran-3-yl- hydrophosphonate (315 mg) was dissolved in a mixture of tetrahydrofuran (2.8 ml) and water (0.71 ml) and the solution was left stirring, cooled in an ice bath. Trifluoroacetic acid (0.71 ml) was added dropwise and the mixture was left stirring at 0-5 ° C for 2 h 15

- 63 038805 min. Sodium hydrogen carbonate (1.14 g) was gradually added to the reaction mixture while maintaining good stirring, followed by the addition of water (3 ml) and ethyl acetate (10 ml). The separated aqueous layer was extracted with ethyl acetate (10 ml). The combined organic phases were washed with saturated aqueous sodium chloride solution (3 ml), dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / methylene chloride) to give the title compound (145 mg). MS: [M + H] + 1224.3.

C) N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15 - {[tert-butyl (dimethyl) silyl] oxy} -10- (2-cyanoethoxy) -7 - (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -16 - [(3-hydroxy1,1,3,3 -tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl } -9H-purin-6-yl) benzamide (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -4 - {[tert-butyl (dimethyl) silyl ] oxy} -2 - ({[(2-cyanoethoxy) ({(2R, 3R, 4R, 5R) -2- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -5 (hydroxymethyl) -4 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] tetrahydrofuran-3-yl} oxy) phosphoryl] oxy } methyl) tetrahydrofuran-3-yl-hydrophosphonate (143 mg) was dried azeotropically with anhydrous acetonitrile, and anhydrous pyridine (2.3 ml) was added thereto. To the mixture was added 2-chloro5,5-dimethyl-1,3,2-dioxaphosphine-2-oxide (75 mg), and the mixture was stirred under argon at room temperature for 45 minutes. To this was added water (75 μl) and 3H-1,2-benzodithiol-3-one-1,1-dioxide (29 mg) and the mixture was stirred at room temperature for an additional 40 minutes. To the reaction mixture was added a solution of sodium thiosulfate pentahydrate (150 mg) in water (0.35 ml) and the mixture was stirred at room temperature for 5 minutes. The mixture was concentrated, toluene was added thereto and the mixture was concentrated under reduced pressure (repeated 4X). The crude product was purified by silica gel column chromatography (methanol / dichloromethane) to give the title compound (84 mg) as a mixture of diastereomers. MS: [M + H] + 1238.3.

D) 7 - {(5R, 7R, 8r, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) -15- {[tert-butyl (dimethyl) silyl ] oxy} - 10-hydroxy-16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2sulfanyloctahydro-12H-5,8-methanofuro [3,2-l ] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7yl} -5-fluoro-3-methyl-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ...

To N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -15 - {[tert-butyl (dimethyl) silyl] oxy} -10- (2-cyanoethoxy) -7- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -16 - [(3-hydroxy1,1,3,3- tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl} -9H-purin-6-yl) benzamide (83 mg) was added 33% ethanolic methylamine solution (2.0 ml) and the mixture was stirred under argon at room temperature for 1 hour, concentrated under reduced pressure. The crude product was purified by reverse phase chromatography (ISCO RediSepRf Gold HP C18 15.5 g column) eluting with 0 to 70% ACN in 10 mM aq. NH ₄ OAc to give two isolated individual isomers of the title compounds: peak 1 (early cut, 11 mg) and peak 2 (late cut, 44.5 mg). MS: [M + H] + 1081.3.

E) 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -10,15,16-trihydroxy-2,10dioxide -2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-fluoro-3-methyl-3 , 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer 1).

K 7 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15 - {[tert-butyl (dimethyl) silyl] oxy} - 10-hydroxy-16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7yl} -5-fluoro-3-methyl-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ( 324 mg, peak 2 / last fraction from step D) pyridine (6.0 ml) and triethylamine trihydrofluoride (0.303 ml) were added. The reaction mixture was stirred at 55 ° C for 16 hours. LC-MS analysis indicated the presence of some of the monoprotected intermediates. Methanol (2.0 ml) was added and the reaction mixture was stirred at 55 ° C for 3 h. LCMS then indicated the reaction was complete. The reaction mixture was concentrated under reduced pressure, and water (6.7 ml) and calcium chloride (700 mg) were added. The resulting suspension was stirred for 1 hour and filtered through a Celite® pad. The filtrate was concentrated under reduced pressure and the residue was purified by reverse phase chromatography (ISCO RediSepRf Gold HP C18Aq column) eluting with 0 to 15% ACN in 10 mM aq. triethylammonium acetate to give the title compound (195 mg). MS: [M + H] + 707.1.

¹ H NMR (400 MHz, D ₂ O) δ 1.27 (18H, t, J = 7.3 Hz), 3.19 (12H, q, J = 7.3 Hz), 3.35 (3H, s), 4.06-4.10 (1H, m), 4.18-4.26 (1H, m), 4.32-4.37 (1H, m), 4.39-4.54 ( 3H, m), 4.63 (1H, d, J = 4.0 Hz), 4.80-4.82 (1H, m), 5.03-5.12 (2H, m), 6.15 -6.17 (1H, m), 6.35-6.39 (1H, m), 7.33-7.37 (1H, m), 8.13 (1H, s), 8.15 (1H , s), 8.27 (1H, s).

³¹ P NMR (162 MHz, D ₂ O) δ -2.22, 54.80.

¹⁹ F NMR (376 MHz, D2O) δ -164.6.

F) 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9H-purin-9-yl) -10,15,16-trihydroxy-2,10 - 64 038805 dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-fluoro-3 -methyl-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer 2).

Optical isomer 2 was obtained using the same procedure as for optical isomer 1. 7 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purine- 9-yl) -15- {[tert-butyl (dimethyl) silyl] oxy} -10-hydroxy-16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxido2 -sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7yl} -5-fluoro-3-methyl-3,7-dihydro -4H-pyrrolo [2,3-d] pyrimidin-4-one (24.5 mg, peak 1 / first fraction from Step D) gives the title compound (11.8 mg). MS: [M + H] + 707.1.

¹ H NMR (400 MHz, D2O) δ ppm. 1.25 (18H, t, J = 7.22 Hz), 3.17 (q, J = 7.24 Hz, 12H), 3.35 (3H, s), 4.124.22 (3H, m), 4.40-4.53 (3H, m), 4.62 (1H, m), 4.83-4.90 (1H, m), 4.93-4.98 (1H, m), 5, 09-5.15 (1H, m), 6.16 (1H, s), 6.28-6.36 (1H, m), 7.50 (1H, s), 8.12 (1H, s) , 8.15 (1H, s), 8.31 (1H, s).

³¹ P NMR (162 MHz, D2O) δ -2,42, 54,11.

¹⁹ F NMR (376 MHz, D ₂ O) δ -165.54.

Example 24. Synthesis of 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro10,16-dihydroxy-2 , 10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-fluoro-3 -methyl-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt (optical isomer 1 and 2)

- 65 038805

A) 7 - [(2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl] -5-fluoro-3-methylpyrrolo [2,3-d] pyrimidine-4 -he.

7 - [(2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl] -5-fluoro-3,7-dihydro-4H pyrrolo [2,3-d] pyrimidine -4-one (300 mg) was dissolved in, Ν-dimethylformamide (3.30 ml) and potassium carbonate (192 mg) was added. The suspension was cooled in an ice bath. Iodomethane (0.080 ml) was added. The mixture was stirred with cooling for 5 min and then stirred at room temperature overnight. The mixture was concentrated and water was added, neutralized with 1M HCl to pH ~ 6. The resulting solid was collected by filtration and dried under vacuum overnight to give the title compound (266 mg). MS: [M + H] + 300.1.

B) 5-Fluoro-7 - [(6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2f] [1,3,5,2, 4] trioxasilocin-8-yl] -3-methyl-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one.

7 - [(2R, 3R, 4S, 5R) -3,4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-yl] -5-fluoro-3-methylpyrrolo [2,3-d] pyrimidin-4-one (985 mg) (previously azeotroped 3 times with acetonitrile and dried in vacuo for 5 h) was dissolved in pyridine (9.75 ml) and N, N-dimethylformamide (4.80 ml). After stirring for 15 minutes, 1,3-dichloro1,1,3,3-tetraisopropyldisiloxane (1.02 ml) was added dropwise. The mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and azeotroped with toluene. The residue was partitioned between EtOAc (120 ml) and water (50 ml). The separated organic layer was washed with water (2x50 ml) and saturated aqueous sodium chloride solution (50 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by chromatography using EtOAc / hexane to give the title compound (1.41 g). MS: [M + H] + 542.3.

C) 2-Cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidine-7 -yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl-diisopropylphosphoramidite.

In a 15 ml round bottom flask 5-fluoro-7 - [(6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3 , 5,2,4] trioxasilocin-8-yl] -3-methyl-3,7-dihydro-4H-pyrrolo [2,3d] pyrimidin-4-one (1.41 g) was subjected to azeotropic distillation with dry acetonitrile ( 3x5 ml), dissolved in dry N, N-dimethylformamide (6.17 ml) under argon. Added 1H-tetrazole 0.45M in acetonitrile (7.05 ml), 1-methyl-1H-imidazole (123 μl) and 2-cyanoethyl-N, N, N ', N'-tetraisopropylphosphorodiamidite (1.86 ml). The reaction mixture was stirred at room temperature for 1 h. The mixture was diluted with 70 ml EtOAc, washed with saturated NaHCO ₃ (x 2) and saturated aqueous sodium chloride solution, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude residue was purified by column chromatography using EtOAc / hexane (0.5% Et ₃ N) to give the title compound (1.87 g). MS: [M + H] + 742.4.

D) (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[(2-cyanoethoxy) {[(6aR, 8R, 9R, 9aR ) -8- (5fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2,2,4,4-tetraisopropyltetrahydro6H-furo [ 3,2-f] [1,3,5,2,4] trioxasilocin-9-yl] oxy} phosphoryl] oxy} methyl) -4-fluorotetrahydrofuran-3yl-hydrophosphonate.

In a 100 ml round-bottom flask, 2-cyanoethyl- (6aR, 8R, 9R, 9aR) -8- (5-fluoro-3-methyl-4-oxo-3,4-dihydro7H-pyrrolo [2,3-d] pyrimidine- 7-yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl-diisopropylphosphoramidite (937 mg) and (2R , 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] 4-fluoro-2- (hydroxymethyl) tetrahydrofuran-3-yl-hydrophosphonate (425 mg) was azeotroped with dry CH3CN (3x5 ml), suspended in dry tetrahydrofuran (1.80 ml) and acetonitrile (2.95 ml) under argon. A solution of 5- (ethylthio) -1H-tetrazole (380 mg) (previously subjected to azeotropic distillation with acetonitrile 3x5 ml) in dry acetonitrile (1.80 ml) was added. The mixture was stirred at room temperature for 1 hour. Tert-butyl hydroperoxide (5.5 mol / L) in nonane (425 μL) was added. The mixture was stirred for 40 minutes at room temperature, then cooled in an ice bath. A solution of sodium thiosulfate (500.0 mg) in water (700 μl) was added and the mixture was stirred for 10 minutes. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography (methanol / DCM). The title compound (1.10 g) was obtained as a mixture of two optical isomers. MS: [M + H] + 1094.3.

E) (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[(2-cyanoethoxy) ({(2R, 3R, 4R, 5R ) -2- (5fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -5 - (hydroxymethyl) -4 - [(3 - hydroxy1,1,3,3-tetraisopropyldisiloxanyl) oxy] tetrahydrofuran-3-yl} oxy) phosphoryl] oxy} methyl) -4-fluorotetrahydrofuran-3-yl-hydrophosphonate.

To a solution of (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[(2-cyanoethoxy) {[(6aR, 8R, 9R, 9aR ) -8- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2,2,4,4 tetraisopropyltetrahydro-6H- furo [3,2-1] [1,3,5,2,4] trioxasilocin-9-yl] oxy} phosphoryl] oxy} methyl) -4fluorotetrahydrofuran-3-yl-hydrophosphonate (1.10 g) in tetrahydrofuran ( 11.0 ml) and water (2.50 ml) cooled in an ice bath, trifluoroacetic acid (2.50 ml) was added slowly. The mixture was stirred and cooled for 4 hours. Sodium hydrogen carbonate (3800 mg) was added, followed by the addition of 8 ml of water. The mixture was stirred for 5 min, brought to room temperature and extracted twice with EtOAc. The combined organic layer was washed with saturated aqueous sodium chloride solution, dried over Na2SO _4, filtered and the filtrate was evaporated under reduced pressure to give crude product. Silica gel column chromatography (methanol / DCM) gave the title compound (1.14 g). MS: [M + H] + 1112.3.

F) N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2-cyanoethoxy) -15-fluoro-7- (5-fluoro-3-methyl-4 -oxo3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxido -2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl} -9H-purin-6-yl) benzamide ...

In a 100 ml round bottom flask (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[(2cyanoethoxy) ({(2R, 3R, 4R, 5R) -2- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) 5- (hydroxymethyl) -4 - [( 3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] tetrahydrofuran-3yl} oxy) phosphoryl] oxy} methyl) -4-fluorotetrahydrofuran-3-yl-hydrophosphonate (149 mg) was azeotroped with acetonitrile (3x4 ml ) and dried in vacuo, dissolved in dry pyridine (2.35 ml) and cooled in an ice bath. Added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (180 mg) in pyridine (0.120 ml). The mixture was stirred at room temperature for 1.5 hours. Water (70.3 μl) was added followed by the addition of 3H-1,2-benzodithiol-3-one (56.2 mg). The mixture was left stirring at room temperature for 40 minutes. Sodium thiosulfate (135 mg) was added in 1 ml of water. The mixture was stirred for 10 minutes, concentrated under reduced pressure, azeotroped with toluene to remove pyridine. The residue was purified by silica gel column chromatography (methanol / DCM) to give the title compound as a mixture of two optical isomers (63.6 mg). MS: [M + H] + 1126.3.

G) 7 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-10-hydroxy-16- [ (3hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2-sulfanyloctahydro-12H-5.8methanofuro [3,2-l] [1,3,6,9,11,2 , 10] pentaoxadiphosphacyclotetradecin-7-yl} -5-fluoro-3-methyl-3,7-dihydro4H-pyrrolo [2,3-d] pyrimidin-4-one.

A mixture of two isomers N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2-cyanoethoxy) -15-fluoro-7- (5fluoro-3-methyl-4- oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -16 - [(3-hydroxy-1,1,3,3tetraisopropyldisiloxanyl) oxy] -2,10-dioxide 2-sulfanyloctahydro-12H-5,8-methanofuro [3,2l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl} -9H-purin-6-yl) benzamide (60, 7 mg) was stirred in methylamine (33 wt%) in ethanol (3.00 ml) at room temperature for 1 hour. The volatile matter was evaporated under reduced pressure. The crude product was purified by reverse phase chromatography (ISCO RediSepRf Gold HP C18 column) eluting with 0 to 50% ACN in 10 mM aqueous NH4OAc to give two isolated individual isomers of the title compounds: peak 1 (early cut, 12 mg ) and peak 2 (late fraction, 35.6 mg). MS: [M + H] + 969.3.

H) 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-10,16-dihydroxy2,10 -dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-fluoro-3-methyl -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer 1).

A 15 ml polypropylene conical tube was loaded with 7 {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) - 15-fluoro-10-hydroxy -16 - [(3-hydroxy1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6 , 9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl} -5-fluoro-3-methyl-3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one (35.6 mg, peak 2 / last fraction from stage G). Pyridine (273 μl) was added, followed by the addition of triethylamine trihydrofluoride (27.3 μl) and triethylamine (414 μl). The mixture was left stirring at room temperature overnight and at 50 ° C for 2 hours. The reaction mixture was cooled in an ice bath and diluted with water (1.3 ml). A solution of calcium chloride (146.0 mg) in water (1.0 ml) was added slowly. The mixture was stirred for 30 min then filtered through a Celite® pad. The filtrate was concentrated under reduced pressure and the residue was purified by reverse phase chromatography (ISCO RediSepRf Gold HP C18Aq column) eluting with 0 to 10% ACN in 10 mM aq. triethylammonium acetate to give the title compound (23.9 mg). MS: [M + H] + 709.2.

1H NMR (400 MHz, methanol- ^) δ 8.29-8.24 (m, 1H), 8.22 (s, 1H), 8.08-8.04 (m, 1H), 7.46- 7.40 (m, 1H), 6.48 (d, 1H, J = 12Hz), 6.37 (d, 1H, J = 16Hz), 5.80-5.57 (m, 1H), 5.33-5.12 (m, 1H), 5.05-4.95 (m, 1H), 4.50-4.42 (m, 2H), 4.41-4.34 (m, 1H ), 4.34-4.24 (m, 2H), 4.08-3.96 (m, 1H), 3.57 (s, 3H), 3.52-3.48 (m, 1H), 3.25-3.11 (m, 12H), 1.39-1.24 (m, 18H).

³¹ P NMR (162 MHz, D2O) δ -2,45, 55,32.

¹⁹ F NMR (376.5 MHz, D ₂ O) δ -164.60, -200.71.

I) 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-10,16-dihydroxy -dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphospha- 67 038805 cyclotetradecin-7-yl] -5-fluoro -3-methyl-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer 2).

A 15 ml polypropylene conical tube was loaded with 7 {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) - 15-fluoro-10-hydroxy -16 - [(3-hydroxy1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2-sulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6 , 9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl} -5-fluoro-3-methyl-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (12.0 mg , peak 1 / early fraction from stage G). Pyridine (92 μl) was added, followed by the addition of triethylamine trihydrofluoride (9.2 μl) and triethylamine (140 μl). The mixture was left stirring at 50 ° C for 2 hours. The reaction mixture was cooled in an ice bath and diluted with water (1.0 ml). A solution of calcium chloride (6.98 mg) in water (1.0 ml) was added slowly. The mixture was stirred for 30 min, then filtered through a Celite® pad. The filtrate was concentrated under reduced pressure and the residue was purified by reverse phase chromatography (ISCO RediSepRf Gold HP C18Aq column) eluting with 0 to 10% ACN in 10 mM aq. triethylammonium acetate to give the title compound (1.94 mg). MS: [M + H] + 709.3.

1H NMR (400 MHz, deuterium oxide) δ 8.20 (s, 1H), 8.06 (s, 1H), 7.99 (s, 1H), 7.46-7.40 (m, 1H), 6.37 (d, 1H, J = 16 Hz), 6.27 (d, 1H, J = 8 Hz), 5.83 (dd, 1H, J1 = 52 Hz, J2 = 4 Hz), 4.98 -4.84 (m, 2H), 4.57 (brd, J = 3.9 Hz, 2H), 4.54-4.48 (m, 2H), 4.46-4.36 ( m, 3H), 4.20-4.11 (m, 3H), 3.55-3.47 (m, 3H), 3.20-3.06 (dd, 12H), 1.28-1, 12 (t, 18H).

³¹ P NMR (162 MHz, D2O) δ -2,52, 53,94.

¹⁹ F NMR (376.5 MHz, D ₂ O) δ -165.55, -202.45.

Example 25. Synthesis of 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro2,10,16-trihydroxy -2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-fluoro-3-methyl -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one ditriethylamine salt

A) N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2-cyanoethoxy) - 15-fluoro-7- (5-fluoro-3-methyl-4 -oxo3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2-hydroxy-16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2 , 10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl} -9H-purin-6-yl) benzamide ...

In a 100 ml round bottom flask (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[(2cyanoethoxy) ({(2R, 3R, 4R, 5R) -2- (5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) 5- (hydroxymethyl) -4 - [( 3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] tetrahydrofuran-3-yl} oxy) phosphoryl] oxy} methyl) -4-fluorotetrahydrofuran-3-yl-hydrophosphonate (1.14 g) was subjected to azeotropic distillation with acetonitrile (3x4 ml) and dried in vacuo, dissolved in pyridine (18.0 ml) and cooled in an ice bath. Added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide (586 mg) in 2 portions. The mixture was left stirring at room temperature for 40 minutes. Water (0.554 ml) was added followed by the addition of 3H-1,2-benzodithiol-3-one-1,1-dioxide (251 mg). The mixture was left stirring at room temperature for 40 minutes. A solution of sodium thiosulfate (810 mg) in 2 ml of water was added. The mixture was concentrated under reduced pressure and azeotroped with toluene to remove pyridine. The residue was purified by silica gel column chromatography (methanol / DCM) to give the title compound (650 mg). MS: [M + H] 1111.3.

B) 7 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) - 15-fluoro-2,10-dihydroxy-16 [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxidooctahydro-12H-5,8-methanofuro

- 68 038805

[3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl} -5-fluoro-3-methyl-3,7-dihydro-4Hpyrrolo [2,3-d ] pyrimidin-4-one.

N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -10- (2cyanoethoxy) -15-fluoro-7- (5-fluoro-3-methyl -4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2hydroxy-16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxidooctahydro-12H-5,8methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl} -9H-purin-6-yl) benzamide (650 mg) and methylamine (33 wt%) in absolute ethanol (22.7 ml). The mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by reverse phase chromatography (ISCO RediSepRf Gold HP C18 column) eluting with 0 to 60% ACN in 10 mM aq. NH ₄ OAc to give the title compound (32.0 mg). MS: [M + H] + 953.3.

C) 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) - 15-fluoro-2,10,16-trihydroxy -2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-U1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-fluoro-3-methyl-3, 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one-ditriethylamine α salt.

A 15 ml polypropylene conical tube was loaded with 7 {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) - 15-fluoro-2,10 -dihydroxy-16 - [(3hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2l] [1,3,6,9,11 , 2,10] pentaoxadiphosphacyclotetradecin-7-yl} -5-fluoro-3-methyl-3,7-dihydro-4H-pyrrolo [2,3d] pyrimidin-4-one (32.0 mg) and pyridine (246 μl). Triethylamine trihydrofluoride (24.6 μl) was added to the suspension, followed by the addition of triethylamine (372 μl). The mixture was left stirring at room temperature overnight. The reaction mixture was cooled in an ice bath and diluted with water (1 ml). A solution of calcium chloride (18.6 mg) in water (1 ml) was added slowly. The mixture was stirred for 30 min, then filtered through a Celite® pad. The filtrate was concentrated under reduced pressure and the residue was purified by reverse phase chromatography (ISCO RediSepRf Gold HP C18Aq column) eluting with 0 to 5% ACN in 10 mM aq. triethylammonium acetate to give the title compound (8.3 mg). MS: [M + H] + 693.2.

¹ H NMR (400 MHz, methanol-d ₄ ) δ 8.36 (br., 1H), 8.22 (s, 1H), 8.05 (s, 1H), 7.38 (d, 1H, J = 2), 6.48 (d, 1H, J = 8), 6.37 (d, 1H, J = 16 Hz), 5.70 (dd, 1H, J1 = 52 Hz, J2 = 4 Hz), 5.20-5.07 (m, 1H), 4.98-4.92 (m, 1H), 4.53 (d, 1H, J = 4Hz), 4.45-4.33 (m, 2H), 4.33-4.19 (m, 3H), 4.09-4.03 (m, 1H), 3.45 (s, 3H), 3.17 (qt, 12H), 1.28 (t, 18H).

³¹ P NMR (162 MHz, D ₂ O) δ -1.66, -2.26.

¹⁹ F NMR (376.5 MHz, D ₂ O) δ -164.5, -201.7.

Example 26. Synthesis of 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-16hydroxy-2,10 -dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-Sh1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-chloro-3,7- dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer 1 and optical isomer 2)

- 69 038805

A) (6aR, 8R, 9R, 9aR) -8- (5-chloro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2.2, 4,4 tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl-2-cyanoetyl diisopropylphosphoramidite.

In a 100 ml round-bottom flask 5-chloro-7 - [(6aR, 8R, 9R, 9aS) -9-hydroxy-2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3 , 5,2,4] trioxasilocin-8-yl] -3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4one (1.469 g) (synthesized in the same manner as in Example 15A)) , subjected to azeotropic distillation with toluene (3x) and dried in vacuo. The round bottom flask was evacuated and back filled with argon (3x). Anhydrous N, N-dimethylformamide (5.00 ml) was added to dissolve the starting material by sonication for a minute, followed by the addition of 2-cyanoethylN, N, N ', N'-tetraisopropylphosphorodiamidite (2.50 ml), 1- methylimidazole (0.13 ml) and then 3-tetrazole (0.45 M in acetonitrile, 6.70 ml). The mixture was stirred under argon at room temperature for 3 hours. The mixture was diluted with EtOAc (200 ml). _{Saturated NaHCO 3} solution was added. The aqueous layer was extracted twice with EtOAc. The combined EtOAc layer was washed with water, then with saturated aqueous sodium chloride solution, dried over anhydrous Na ₂ SO ₄ , filtered. The filtrate was concentrated under reduced pressure to obtain a crude oily product. The crude material was purified by column chromatography (ISCO RediSepRf DIOL HP Gold 100 g column) eluting with EtOAc: hexanes (1: 9 to 1: 1) to give the title compound (1.77 g). MS: [M + H] + 661.3 for hydrolyzed product fragment.

B) (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -4-fluoro-2- (hydroxymethyl) tetrahydrofuran 3-yl hydrophosphonate.

N- {9 - [(2R, 3R, 4R, 5R) -5 - {[bis (4-methoxyphenyl) (phenyl) methoxy] methyl} -3-fluoro-4hydroxytetrahydrofuran-2-yl] -9H-purine-6 -yl} benzamide (2.64 g) under argon was dissolved in anhydrous pyridine (18.0 ml), cooled in an ice bath. Diphenylphosphite (1.60 ml) was added slowly over 1 min. The reaction solution was allowed to warm to room temperature and stirred for 1 hour. A second portion of diphenylphosphite (0.35 ml) was added slowly and the mixture was continued to stir for an additional 30 minutes. The mixture was cooled in an ice bath. Added water (2.0 ml)

- 70 038805 and the mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure and azeotroped with toluene to further remove pyridine and water. The residue was dried under a vacuum pump. Acetic acid (10.0 ml) and water (2.0 ml) were added to the residue. The mixture was stirred at room temperature for 30 minutes. The mixture was concentrated under reduced pressure and azeotroped with toluene, then dried under a vacuum pump to give a crude oil which was chromatographed on a silica gel column using MeOH / DCM (0/100 to 50/50) to give 1.33 g of the indicated in the title product as a white solid. MS: [M + H] + 438.1.

C) (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[{[(6aR, 8R, 9R, 9aR) -8- ( 5-chloro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2,2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [ 1,3,5,2,4] trioxasilocin-9-yl] oxy} (2-cyanoethoxy) phosphorothioyl] oxy} methyl) -4-fluorotetrahydrofuran-3-yl hydrophosphonate.

In a 250 ml round-bottom flask, a mixture of (6aR, 8R, 9R, 9aR) -8- (5-chloro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3d] pyrimidin-7-yl) -2, 2,4,4-tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl-2cyanoethyl diisopropylphosphoramidite (2.23 g) and (2R, 3R, 4R , 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -4fluoro-2- (hydroxymethyl) tetrahydrofuran-3-yl-hydrophosphonate (1.17 g) was subjected to azeotropic distillation with dry acetonitrile (25 ml χ 3), dried under a vacuum pump for 1 h. Anhydrous acetonitrile (20.0 ml) was added to the mixture in an argon atmosphere, followed by the addition of a solution of 5 (ethylthio) -1H-tetrazole (1.00 g) distillation with dry acetonitrile, 10 ml χ 3 and dried under a vacuum pump) in anhydrous acetonitrile (10.0 ml). The mixture was stirred at room temperature for 1 hour 20 minutes. ((Dimethylaminomethylidene) amino) -3H-1,2,4-dithiazoline-3-thione (655 mg) was added and the mixture was stirred at room temperature for 40 minutes. The mixture was concentrated under reduced pressure.

Column chromatography on silica gel using MeOH / DCM (0/100 to 40/60) gave the title compound (1.78 g) as a mixture of 2 optical isomers. MS: [M + H] + 1112.4.

E) (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[({(2R, 3R, 4R, 5R) -2- ( 5-chloro-4-oxo-3,4 dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -5- (hydroxymethyl) -4 - [(3-hydroxy-1,1,3,3 -tetraisopropyldisiloxanyl) oxy] tetrahydrofuran-3-yl} oxy) (2-cyanoethoxy) phosphorothioyl] oxy} methyl) -4fluorotetrahydrofuran-3-yl hydrophosphonate.

(2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 ({[{[(6aR, 8R, 9R, 9aR) -8- (5-chloro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -2,2,4,4 tetraisopropyltetrahydro-6H-furo [3,2-f] [1,3,5,2,4] trioxasilocin-9-yl] oxy} (2-cyanoethoxy) phosphorothioyl] oxy} methyl) -4-fluorotetrahydrofuran-3-yl-hydrophosphonate (1.73 g) in tetrahydrofuran (16 ml) and water (4.0 ml). The solution was cooled in an ice bath. Trifluoroacetic acid (4.0 ml) was added dropwise. The mixture was stirred with cooling for 4 h. Sodium hydrogen carbonate (7.00 g) was added portionwise at 0 ° C followed by the addition of water and EtOAc. The mixture was stirred for 3 minutes, then brought to room temperature. The layers were separated. The aqueous layer was extracted with EtOAc (3x). The combined phases of the EtOAc solution were washed with saturated aqueous sodium chloride solution, dried over anhydrous Na ₂ SO ₄ , filtered. The filtrate was concentrated under reduced pressure and dried under a vacuum pump to give a crude product. The crude material was purified on a silica gel column using MeOH / DCM (0/100 to 40/60) to afford the title product (1.43 g) as a mixture of 2 optical isomers. MS: [M + H] + 1130.3.

F) N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (5-chloro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3d ] pyrimidin-7-yl) -10- (2-cyanoethoxy) -15-fluoro-16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2-oxide-2-sulfanyl-10 -sulfidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl} -9H-purin-6-yl) benzamide (mixture two main optical isomers).

In a round bottom flask (2R, 3R, 4R, 5R) -5- [6- (benzoylamino) -9H-purin-9-yl] -2 - ({[({(2R, 3R, 4R, 5R) -2 ( 5-chloro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -5- (hydroxymethyl) -4 - [(3-hydroxy1,1,3,3 -tetraisopropyldisiloxanyl) oxy] tetrahydrofuran-3-yl} oxy) (2-cyanoethoxy) phosphorothioyl] oxy} methyl) -4-fluorotetrahydrofuran-3-yl-hydrophosphonate (1.20 g) was azeotropically distilled with dry pyridine (freshly distilled) acetonitrile (χ2), then with dry acetonitrile. The resulting white solid was dried under vacuum. Under argon, anhydrous pyridine (20.0 ml) (from a freshly distilled bottle) was added, followed by the addition of 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide (0.700 g). The clear solution was left stirring at room temperature for 1 hour 10 minutes. Water (0.70 ml) was added followed by the addition of 3H-1,2benzodithiol-3-one-1,1-dioxide (0.300 g). The mixture was stirred at room temperature for 20 minutes. The mixture was concentrated under reduced pressure, azeotroped with toluene (χ2), dried under a vacuum pump to give a crude residue. The crude residue was chromatographed on a silica gel column using MeOH / DCM (0/100 to 20/80). The title products (590 mg) from the early fractions were obtained and identified as a mixture of the main isomers of peaks 2 and 4

- 71,038805 from LC-MS; while the products of the second batch (389 mg) from the late cut were a mixture of all 4 isomers. MS: [M + H] + 1144.2.

G) 7 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) -15-fluoro-16 - [(3-hydroxy1 , 1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2,10-disulfanyloctahydro-12H-5.8methanofuro [3,2-l] [1,3,6,9,11,2,10 ] pentaoxadiphosphacyclotetradecin-7-yl} -5-chloro-3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one (single optical isomers 1 and 2).

In an atmosphere of N ₂ N- (9 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -7- (5-chloro-4-oxo-3,4-dihydro-7Hpyrrolo [2,3 -d] pyrimidin-7-yl) -10- (2-cyanoethoxy) -15-fluoro-16 - [(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2-oxide-2-sulfanyl -10-sulfidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-14-yl} -9H-purin-6-yl) benzamide (0.584 g, mixture of 2 main isomers, early fraction from column chromatography) was stirred in methylamine (33 wt%) in absolute ethanol (15.0 ml) at room temperature for 2 h. The mixture was concentrated under reduced pressure and dried under vacuum. pump to get a crude solid. Two subsequent purifications on silica gel columns using MeOH / EtOAc (0/100 to 40/60) gave 2 individual optical isomers of the title compounds: isomer 1 (early cut / peak 1, 73 mg) and isomer 2 (late cut / peak 2, 304 mg), fraction of the mixture of peaks 1 and 2 (0.30 g). MS (optical isomer 1): [M + H] + 987.2. MS (optical isomer 2): [M + H] + 987.2.

H) 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) - 15-fluoro-16-hydroxy-2,10 - dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-chloro-3 , 7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer 1).

7 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9Hpurin-9-yl) - 15-fluoro-16 - [(3-hydroxy -1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2 , 10] pentaoxadiphosphacyclotetradecin-7yl} -5-chloro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer 1, 69.2 mg, early fraction from column chromatography), pyridine (0.40 ml), followed by the addition of triethylamine trihydrofluoride (0.060 ml) followed by triethylamine (0.90 ml). The reaction mixture was stirred in a sealed polypropylene tube at 50 ° C overnight. The reaction mixture was cooled to room temperature, diluted with water (1.30 ml). A solution of calcium chloride (92.5 mg) in water (1.30 ml) was added. The cloudy white mixture was stirred at room temperature for 1 hour and then filtered through Celite® using a plastic filter funnel, washed with water (1 ml x 4). The clear aqueous filtrate was concentrated under reduced pressure, azeotroped several times with toluene, and dried in vacuo for 10 minutes to give a crude residue. The crude residue was purified by C18 column chromatography (water-acetonitrile / 10 mM triethylammonium acetate buffer). The product fraction was concentrated under reduced pressure, subjected to azeotropic distillation with toluene (x4), then with water without endotoxin (x5). The final residue was dissolved in water without endotoxin and lyophilized to give the title product (optical isomer 1, 29.0 mg). MS [M + H] + 727.0.

1H NMR (400 MHz, deuterium oxide) δ 8.19 (s, 1H), 8.16 (s, 1H), 7.96 (s, 1H), 7.63 (s, 1H), 6.39 ( brd, J = 15.7 Hz, 1H), 6.34 (brd, J = 8.0 Hz, 1H), 5.60-5.41 (m, 1H), 5.22 -5.14 (m, 1H), 5.14-5.02 (m, 1H), 4.55 (brd, J = 3.8Hz, 1H), 4.51 (brd , J = 8.2 Hz, 1H), 4.42 (br.s, 1H), 4.40 (br.s, 1h), 4.35-4.28 (m, 1H), 4.164.10 ( m, 1H), 4.06 (br.dd, J = 4.3, 11.5 Hz, 1H), 3.13 (q, J = 7.3 Hz, 12H), 1.21 (t, J = 7.3 Hz, 18H).

³¹ P NMR (162 MHz, deuterium oxide) δ 55.29, 54.54.

¹⁹ F NMR (376 MHz, deuterium oxide) δ -200.90.

I) 7 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) - 14- (6-amino-9H-purin-9-yl) - 15-fluoro-16-hydroxy-2,10 - dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7-yl] -5-chloro-3,7 -dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one diethylamine salt (optical isomer 2).

7 - {(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9Hpurin-9-yl) - 15-fluoro-16 - [(3-hydroxy -1,1,3,3-tetraisopropyldisiloxanyl) oxy] -2,10-dioxide-2,10-disulfanyloctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2 , 10] pentaoxadiphosphacylotetradecin-7yl} -5-chloro-3,7-dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one (optical isomer 2, 186 mg, late fraction from the chromatograph). Pyridine (1.1 ml) was added, followed by triethylamine trihydrofluoride (0.161 ml) followed by triethylamine (2.4 ml). The reaction mixture was stirred in a sealed polypropylene tube at 50 ° C overnight. The reaction mixture was cooled to room temperature, diluted with water (3.50 ml). A solution of calcium chloride (250 mg) in water (3.50 ml) was added. The cloudy white mixture was stirred at room temperature for 1 hour. The mixture was filtered through Celite® using a plastic filter funnel, washed with water (3 ml x 4). The clear aqueous filtrate was concentrated under reduced pressure, azeotroped multiple times with toluene, and dried in vacuo for 10 minutes to obtain a crude residue. The crude residue was purified by C18 column chromatography (water-acetonitrile / 10

- 72 038805 mM triethylammonium acetate buffer solution). The product fraction was concentrated under reduced pressure, subjected to azeotropic distillation with toluene (x4), then with water without endotoxin (x5). The final residue was dissolved in water without endotoxins and lyophilized to give the title product (optical isomer 2, 109 mg). MS [M + H] + 727.0.

1H NMR (400 MHz, deuterium oxide) δ 8.10 (s, 1H), 7.89 (s, 1H), 7.86 (s, 1H), 7.55 (s, 1H), 6.30 ( d, J = 15.9 Hz, 1H), 6.24 (d, J = 8.2 Hz, 1H), 5.49-5.27 (m, 1H), 5.07-4.86 (m , 2H), 4.85 (d, J = 3.9 Hz, 1H), 4.44 (brd, J = 9.0 Hz, 1H), 4.33 (brd, J = 14.7 Hz, 2H), 4.28-4.16 (m, 2H), 3.98 (dd br, J = 5.0, 11.8 Hz, 1H), 3.04 (q, J = 7.4 Hz, 12H), 1.12 (t, J = 7.3 Hz, 18H).

³¹ P NMR (162 MHz, deuterium oxide) δ 55.12, 52.04.

¹⁹ F NMR (376 MHz, deuterium oxide) δ -200.43.

Example 27. Linker Payload 1

To a mixture of 6- (2,5-dioxopyrrol-1-yl) -N - [(1S) -1 - [[(1 S) -2- [4- (hydroxymethyl) anilino] -1-methyl-2-oxoethyl ] carbamoyl] -2-methyl-propyl] hexanamide (mc-Val-Ala-PAB-OH, Synchem, Elk Grove Village, IL, 0.0822 mmol, 40.0 mg) in anhydrous acetonitrile (19.1 mmol, 1 , 00 ml, 783 mg), cesium iodide (0.0986 mmol, 25.6 mg) and boron tehfluoride etherate (0.0986 mmol, 0.0125 ml, 14.0 mg) were added. The reaction mixture was stirred at room temperature overnight. After stirring for 18 hours, the mixture was diluted with ~ 10 ml DCM and filtered through a Celite® pad. The solid was washed with DCM. The filtrate was concentrated to give an orange solid. The crude product (mc-Val-Ala-PAB-I) was used without further purification.

Disodium solution (5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin9-yl) -15-fluoro-7- (5-fluoro-4- oxo-3,4-dihydro-7H-pyrrolo [2,3-th] pyrimidin-7-yl) -16-hydroxy-2sulfido-octahydro-12H-5,8-methanofuro [3,2-l] [1,3, 6,9,11,2,10] pentaoxadiphosphacyclotetradecin-10olate-2,10-dioxide (payload 1, also the compound from example 3a, 0.0106 mmol, 7.80 mg) and 6 (2,5-dioxopyrrole-1 -yl) -N - [(1S) -1 - [[(1S) -2- [4- (submethyl) anilino] -1-methyl-2-oxoethyl] carbamoyl] -2methylpropyl] hexanamide (mc-Val Α-Ala-PAB-I, 0.0208 mmol, 12.4 mg, crude) in K, K-dimethylformamide (5.17 mmol, 0.400 ml, 378 mg) was sealed and heated to 60 ° C. After 2 h, the reaction was cooled to room temperature and purified by C18 column chromatography (continuous gradient 20-60% CH3CN / water with 0.1% formic acid) to give N - [(2S) -1 - {[( 2S) -l - {[4 ({[(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9H-purin-9-yl) - 15-fluoro-7 - (5-fluoro-4-oxo-3,4-dihydro-7Н-pyrrolo [2,3-th] pyrimidin-7-yl) -10,16-dihydroxy-2,10-dioxidooctahydro-12Н-5,8methanofuro [3 , 2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-2-yl] sulfanyl} methyl) phenyl] amino} -1-oxopropan-2-yl] amino} -3-methyl- 1-oxobutan-2-yl] -6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl) hexanamide (5.5 mg, 45% yield) as a white solid.

¹ H NMR (400 MHz, methanol-J4) δ 8.23-8.35 (m, 2H), 8.10 (s, 1H), 7.89-8.02 (m, 1H), 7.84 (s, 1H), 7.527.64 (m, 2H), 7.41 (brd, J = 8.53 Hz, 2H), 7.22-7.38 (m, 2H), 6.75 -6.83 (m, 2H), 6.32-6.43 (m, 2H), 5.71-5.90 (m, 2H), 4.54-4.59 (m, 2H), 4 , 42-4.51 (m, 2H), 4.07-4.35 (m, 8H), 3.46 (br, J = 7.03 Hz, 2H), 2.27 (br. t., J = 7.34 Hz, 2H), 2.03-2.15 (m, 2H), 1.53-1.70 (m, 4H), 1.39-1.47 (m, 3H ), 1.23-1.37 (m, 2H), 0.92-1.02 (m, 6H).

LCMS: m / z 1163,1164 [M + H].

Example 28. Linker Payload 2

- 73 038805

Linker payload 2 was generated according to the procedure in Example 27 using 2amino-9 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purine-9 -yl) -10,18-dihydroxy-2,10dioxide-2-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11 , 2,10] pentaoxadiphosphacyclotetradecin-7 (1 2H) -yl] - 1,9-dihydro-6H-purin-6-one sesquitriethylamine salt (payload 2) instead of payload 1.

Payload 2 was obtained according to the method below.

2-Amuho-9 - ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) - 14- (6-amino-9H-purin-9-yl) -10,18-dihydroxy2,10-oxide -2-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl ) -1,9-dihydro-6H-purin-6-one sesquitriethylamine salt (optical isomer).

A) (1R, 3R, 4R, 7S) -3- (6-benzamido-9H-purin-9-yl) -1 - ((((((2R, 3R, 4R, 5R) -4 - ((tert -butyldimethylsilyl) oxy) -5- (hydroxymethyl) -2- (2-isobutylamido-6-oxo-1H-purin-9 (6H) -yl) tetrahydrofuran-3-yl) oxy) (2cyanoethoxy) phosphoryl) oxy) methyl ) -2,5-dioxabicyclo [2,2,1] heptan-7-yl-hydrophosphonate.

(1S, 3R, 4R, 7S) -3- (6-Benzamido-9H-purin-9-yl) -1- (hydroxymethyl) -2,5-dioxabicyclo [2,2,1] heptan7-yl-hydrophosphonate ( 700 mg) and 5'-O- [bis (4-methoxyphenyl) (phenyl) methyl] -3'-O- [tert-butyl (dimethyl) silyl] -2'-O- {(2-cyanoethoxy) [diisopropylamino ] phosphanyl} -K- (2-methylpropanoyl) guanosine (2280 mg) was subjected to azeotropic drying with anhydrous acetonitrile, and anhydrous acetonitrile (15 ml) and anhydrous tetrahydrofuran (5 ml) were added. To the mixture was added a mixture of 5- (ethylsulfanyl) -2H-tetrazole (611 mg) (which was azeotropically dried with anhydrous acetonitrile) and anhydrous acetonitrile (10 ml), and the mixture was stirred overnight under argon at room temperature. 70% tert-butyl hydroperoxide (643 μl) was added thereto and the mixture was stirred at room temperature for 20 minutes. To the reaction mixture was added a mixture of sodium thiosulfate (5920 mg) and water (3 ml), and the mixture was concentrated under reduced pressure. To the residue was added 80% acetic acid (30 ml) and the mixture was stirred at room temperature for 20 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol / ethyl acetate) to give the title compound (980 mg). MS: [M + H] + 1030.2.

B) 2-amino-9 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -14- (6-amino-9H-purin-9-yl) -18 - {[tert-butyl (dimethyl) silyl] oxy} -2,10-dihydroxy-10-oxide-2-sulfidohexahydro-14H-15,12a- (epoxymetano) 5,8-methanofuro [3,2-l] [1,3,6, 9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (12H) -yl] -1,9-dihydro-6Hpurin-6-one (optical isomer).

(1R, 3R, 4R, 7S) -3- (6-Benzαmido-9H-nurin-9-yl) -1 - ((((((2R, 3R, 4R, 5R) -4 - ((tert-butyldimethylsilyl ) oxy) -5- (hydroxymethyl) -2- (2-isobutylamido-6-oxo-1H-purin-9 (6H) -yl) tetrahydrofuran-3-yl) oxy) (2cyanoethoxy) phosphoryl) oxy) methyl) - 2,5-dioxabicyclo [2,2,1] heptane-7-yl-hydrophosphonate (980 mg) was azeotropically dried with anhydrous acetonitrile and anhydrous pyridine, with the addition of anhydrous pyridine (50 ml). To the mixture at room temperature was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphine 2-oxide (615 mg) and the mixture was stirred under argon at room temperature for 1 hour. Water (600 μL) was added thereto and 3H-benzo [c] [1,2] dithiol-3-one (240 mg) and the mixture was stirred at room temperature for an additional 30 minutes. To the reaction mixture was added a mixture of sodium thiosulfate (1180 mg) and water (3 ml), and the mixture was concentrated under reduced pressure. Anhydrous acetonitrile (15 ml) and 2-methylpropan-2-amine (5.0 ml) were added to the residue and the mixture was stirred at room temperature for 1.5 h, concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / ethyl acetate), and a 40% ethanolic methylamine solution (30 ml) was added to the resulting residue. The mixture was stirred overnight under argon atmosphere at room temperature and the reaction mixture was concentrated under reduced pressure. The residue was purified using silica gel column chromatography (methanol / ethyl acetate). The resulting residue was divided into two diastereomers (tR1 and tR2, the retention times of which from LC / MS from shorter to longer in the order indicated) by HPLC (L-column 2 ODS, 50x150 mm, mobile phase: 5 mM ammonium acetate / acetonitrile aqueous solution) to give the title compound (38 mg, tR1) and the title compound (322 mg, tR2). MS (tR1): [M + H] + 817.1. MS (tR2): [M + H] + 817.1.

C) 2-Amuho-9 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) - 14- (6-amino-9H-purin-9-yl) -2,10,18-trihydroxy -10-oxide-2-sulfidohexahydro-14H-15,12- (epoxymethano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 ( 1 2H) -yl] - 1,9-dihydro-6H-purin-6-one sesquitriethylamine salt (optical isomer).

K 2-amino-9 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) - 14- (6-amino-9H-purin-9-yl) -18- {[tert-butyl ( dimethyl) silyl] oxy} -2,10-dihydroxy-10-oxide-2-sulfidohexahydro-14H-15,12- (epoxymetano) -5,8methanofuro [3,2-l] [1,3,6,9, 11,2,10] pentaoxadiphosphacyclotetradecin-7 (1 2H) -yl] - 1,9-dihydro-6H-purin6-one (optical isomer) (38 mg, tR1) methanol (3.0 ml) and triethylamine trihydrofluoride ( 0.76 ml). The reaction mixture was concentrated to remove methanol and the residue was stirred at 55 ° C for 1 h. The mixture was cooled to room temperature, ethoxy (trimethyl) silane (4.2 ml) was added thereto and the mixture was stirred at room temperature for 2 h. the mixture was concentrated under reduced pressure, and the residue was purified by C18 column chromatography (acetonitrile / 10 mM triethylammonium acetate buffer) to give the title compound (27 mg).

1H NMR (400 MHz, D2O) δ 1.23 (13H, t, J = 7.3 Hz), 3.15 (9H, q, J = 7.3 Hz), 4.04 (1H, d, J = 8.3 Hz), 4.084.19 (3H, m), 4.28 (1H, d, J = 12.2 Hz), 4.37-4.52 (2H, m), 4.65 (1H , d, J = 4.2 Hz), 4.90 (1H, d, J = 4.6 Hz), 5.36 (1H, s), 5.55 (1H, td, J = 8.5, 4.0 Hz), 5.98 (1H, d, J = 8.3 Hz), 6.16 (1H, s), 7.94 (1H, s), 8.21 (1H, s), 8 , 25 (1H, s).

³¹ P NMR (162 MHz, D ₂ O) δ -1.45, 53.78.

Synthesis of 2-amino-9 - [(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) - 14- (6-amino-9H-purin-9-yl) -10,18dihydroxy-2,10- dioxido-2-sulfanylhexahydro-14H-15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-7 (1 2H) -yl] -1,9-dihydro-6H-purin-6-one sesquitriethylamine salt (optical isomer; payload 2).

K 2-amino-9 - [(5R, 7R, 8R, 12aR, 1 4r, 15R, 15aS, 18R) - 14- (6-amino-9H-purin-9-yl) -18- {[tert-butyl (dimethyl) silyl] oxy} -2,10-dihydroxy-10-oxide-2-sulfidohexahydro-14H-15,12a- (epoxymetano) -5,8methanofuro [3,2-l] [1,3,6,9 , 11,2,10] pentaoxadiphosphacyclotetradecin-7 (1 2H) -yl] - 1,9-dihydro-6H-purin6-one (optical isomer) (322 mg, tR2) methanol (3.0 ml) and triethylamine trihydrofluoride were added (3.2 ml). The reaction mixture was concentrated to remove methanol and the residue was stirred at 55 ° C for 1 hour. The mixture was cooled to room temperature, ethoxy (trimethyl) silane (14 ml) was added thereto and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated. under reduced pressure, and the residue was purified by C18 column chromatography (acetonitrile / 10 mM triethylammonium acetate buffer) to give the title compound (266 mg).

1H NMR (400 MHz, D ₂ O) δ 1.23 (14H, t, J = 7.3 Hz), 3.15 (10H, q, J = 7.3 Hz), 4.02 (1H, d , J = 8.1 Hz), 4.134.24 (2H, m), 4.27-4.42 (4H, m), 4.59 (1H, d, J = 4.4 Hz), 5.01 (1H, s), 5.11 (1H, d, J = 4.2Hz), 5.61-5.73 (1H, m), 5.95 (1H, d, J = 8.3Hz) , 6.15 (1H, s), 7.87 (1H, s), 8.00 (1H, s), 8.25 (1H, s).

³¹ P NMR (162 MHz, D2 O) δ -1.93, 55.44.

Example 29 General Procedure for ADC Conjugation.

The antibody solution was diluted to the desired reaction concentration with an appropriate buffer. The pH was adjusted to 7 by adding a 0.5M solution of Tris, 25 mM EDTA in water (pH 8.0). TCEP (2.2 eq., 5 mM in water) was added with stirring. After 1 h incubation at room temperature, 5 equiv. linker payload (5 mM in DMA). After 2 additional hours of gentle stirring at room temperature, the mixture was purified twice through 3 ml of spinOUT desalting columns (G-Bioscience, pre-equilibrated with a buffer containing 10 mM histidine, 7.5% sucrose (w / v) and 0.08% polysorbate 20 (w / v) at pH 5.2), then the buffer was changed to Dulbecco's PBS and concentrated to 1-3 mg / ml (as determined by UV absorbance using a standard IgG1 extinction coefficient) using a Vivaspin6 column (GE Healthcare). The sample was then characterized for drug-antibody ratio (DAR) using QTOF mass spectrometry. The monomeric composition of the ADC was determined by size exclusion chromatography (SEC).

Size exclusion chromatography (SEC) protocol.

Sample preparation.

Prepare pure water by adding HPLC water to the HPLC vial. Naked control antibodies and ADC samples were obtained from chemists. Control samples and samples are diluted with 1XPBS if the concentration is higher than 5 mg / ml, or injected in pure form if the concentration is 5 mg / ml or lower.

HPLC system setup.

An Agilent 1100 HPLC system was used for analysis. The system was installed by flushing the system (5% acetonitrile in HPLC water) in one pump channel and mobile phase (as described above) in the other pump channel. The column used is Tosoh Biosep TSK gel, G3000SWxl; P / N 8541; 250A; 5 microns; 7.8 mm χ 300 mm. The flow rate is set at 1 ml / min and each run is isocratic with 100% mobile phase [100 mM sodium phosphate, 300 mM sodium chloride, pH 6.8, 10% acetonitrile (v / v)] for 20 min. ... DAD is set at 280 nm. The injection volume for each control, sample and pure water is usually 10 μl, but can be adjusted based on UV absorption. Analysis data: all peaks at 280 nm within the appropriate time window (usually 2-10 min) are integrated.

Example 30. Obtaining ADC1.

Synthesis was performed according to the general ADC conjugation procedure.

Antibody: Antibody 1, anti-GCC antibody (5F9), 100 μl of a 60 mg / ml solution in 50 mM histidine and 100 mM arginine buffer at pH 6.0, guanylyl cyclase C (GCC) receptor target.

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Heavy chain antibody 1:

QVQLQQWGAGLLKPSETLSLTCAVFGGSFSGYYWSWIRQPPGKGLEWIGEINHRGNTND

NPSLKSRVTISVDTSKNQFALKLSSVTAADTAVYYCARERGYTYGNFDHWGQGTLVTVSSASTK

GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV

VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (SEQ. ID No. 1)

Antibody light chain 1:

EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYGASTRATGIP

IARFSGSGSGTEFTLTIGSLQSEDFAVYYCQQYKTWPRTFGQGTNVEIKRTVAAPSVFIFPPSDE

QLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE

KHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ. ID No. 2)

Buffer: 50 mM histidine solution and 100 mM arginine solution at pH 6.0, 200 μl, final concentration 20 mg / ml.

Linker payload: N - [(2S) -1 - {[(2S) -1- {[4- ({[(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- ( 6amino-9H-purin-9-yl) -15-fluoro-7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -10 , 16dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-2-yl] sulfanyl} methyl) phenyl] amino} -1-oxopropan-2-yl] amino} -3-methyl-1-oxobutan2-yl] -6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamide ( linker payload 1).

ADC product: ADC1, yield = 1.9 ml at 2.5 mg / ml (79%), DAR 3.5, 99% monomer (i.e. not aggregated).

Example 31. Obtaining ADC2.

Synthesis was performed according to the general ADC conjugation procedure.

Antibody: Antibody 2, a non-GCC targeting antibody directed to a membrane-bound protein overexpressed in some solid tumors, including lung and mammary gland, 100 μl at 11.8 mg / ml in 25 mM sodium acetate, pH 5.5.

Buffer: Dulbecco's PBS, pH 7.4, 200 μl, final concentration 4 mg / ml.

Linker payload: N - [(2S) -1 - {[(2S) -1- {[4- ({[(5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- ( 6amino-9H-purin-9-yl) -15-fluoro-7- (5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3-d] pyrimidin-7-yl) -10 , 16dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-2-yl] sulfanyl} methyl) phenyl] amino} -1-oxopropan-2-yl] amino} -3 -methyl-1-oxobutan-2-yl] 6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamide (linker-payload 1).

ADC product: ADC2, yield = 0.6 ml at 1.4 mg / ml (70%), DAR 2.6, 99% monomer.

Example 32. Getting ADC3.

Synthesis was performed according to the general ADC conjugation procedure.

Antibody: Antibody 1, 33 μl at 60 mg / ml in 50 mM histidine and 100 mM arginine buffer at pH 6.0.

Buffer: 50 mM histidine solution and 100 mM arginine solution at pH 6.0, 66 μl, final concentration of antibody solution 20 mg / ml.

Linker payload: N - [(2S) - 1 - {[(2S) -1- {[4 - ({[(5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 18R) -7- ( 2-amino6-oxo-1, 6-dihydro-9H-purin-9-yl) - 14- (6-amino-9H-purin-9-yl) -10,18-dihydroxy-2,10-dioxidohexahydro-14H -15,12- (epoxymetano) -5,8-methanofuro [3,2-l] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetradecin-2 (12H) -yl] sulfanyl} methyl) phenyl ] amino} -1-oxopropan-2-yl] amino} -3-methyl-1-oxobutane-2ul] -6- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanamide (linker-payload 2).

ADC product: ADC3, yield = 1.9 ml at 1.0 mg / ml (95%), DAR 4.3, 98% monomer.

Example 33. Tritosomal Payload Release Assay — Measurement of payload release from linker-payload or ADC systems in an in vitro lysosome model.

Materials.

Buffer: 47.9 mg / ml monobasic potassium phosphate (Sigma-Aldrich, product number P5379); 6.8 mg / ml sodium phosphate dibasic (Sigma-Aldrich, product number S0876), 1.7 mg / ml ethylenediamine tetraacetic acid (Sigma-Aldrich, product number ED4SS) in purified water. The pH was adjusted to 6.0 using 1N HCl or 1N KOH.

Rat liver tritosomes were purchased from XenoTech.

Linker payload (10 mM DMSO).

ADC (1-40 μm in storage buffer used for manufacturing).

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Methodology.

To evaluate the release of the payload from the linker-payload compounds, a 120.95 μM DMSO working solution was prepared from a stock solution of 10 mM linker-payload compounds.

Then the working solution of DMSO 120.95 μM was diluted to 1 μM in 225 μl of tritosomal buffer solution containing 22.4% rat liver tritosomes.

To evaluate ADC molecules, 1.86 μl of ADC solution was diluted with 223.1 μl of a mixture of rat liver tritosomes in tritosomal buffer.

The solutions prepared in this way were incubated for 24 h at 37 ° C. Aliquots of 40 μl were removed after 0.5, 1, 3, 5 and 24 h and diluted with 160 μl of 0.1% formic acid solution in methanol in a 96-well plate, which was stored in a freezer at -80 ° C until the end of the experiment. ... After collecting the last time point, fresh 200 μl of 0.1% formic acid solution in methanol was added to the samples and 150 nM carbutamide, which is an internal standard solution, was added. The samples were mixed well and centrifuged at 4000 g for 10 min and the 96 well plate was presented for analysis of payload concentration by LC / MS / MS.

LC / MS / MS system.

μl of samples were injected into LC / MS / MS onto a Waters Xselect C18 CSH 3.5u 2.1 mm ID x 30 mm column. The aqueous solvent of the mobile phase contained 0.1% formic acid in water, and the organic solvent of the mobile phase contained 0.1% formic acid in 5% water and 95% acetonitrile. The samples were run with a gradient for 3 minutes at a flow rate of 1.5 ml / min. Initially, the instrument was operated for 0.5 min with 100% aqueous solvent of the mobile phase, followed by increasing to 100% organic solvent over the next 1.5 min. The system will be held at 100% organic solvent for another 0.5 min, and then it will be replaced with 100% aqueous phase solvent for the next 0.5 min.

Analysis.

To monitor and calculate the release of the linker payload and payload from the ADC, a peak area or concentration versus time curve was plotted. The data will be analyzed using Excel-Fit to calculate the linear velocity of the range and t1 / 2 of the payload release from the tested molecules.

Tritosomal payload release data.

As illustrated in FIG. 1, 2, and 3, when exposed to the above protocols ADC1, ADC2, and ADC3 release the expected payloads.

Phospho-IRF3 assay protocol.

HEK293 cells designed to express tumor associated targets on the cell surface for mAbs 1 (ADC1 and ADC3) and antibody 2 (ADC2), as well as non-expressing HEK293 target cells (HEK293-Vect), were plated at 1.5x10E5 cells / well ( 500 μl / well) in poly-D-lysine coated 24 well plates (Corning) and cultured overnight. After overnight serum deprivation, cells were treated with various concentrations of ADC for 6 h and then lysed in RIPA buffer (ThermoFisher Scientific) with protease and phosphatase inhibitors (ThermoFisher Scientific). Cell lysates were subjected to Western blotting for phospho-1IR3 (CST) and IRF3 (BD Biosciences). The phospho-1IR3 level was normalized to its total IRF3.

Analysis results of phospho-1IR3.

As illustrated in FIG. 4, 5 and 6, HEK293 cells that express surface receptor targets for ADC1, ADC2 and ADC3 show the expected increase in the STING pathway marker phospho-IG3 (pIRF3) when treated with ADC1, ADC2 and ADC3, respectively. In contrast, the pIRF3 response is not upregulated by ADC treatment in HEK293 vector cells that do not express their respective surface target receptors.

Example 34 Pharmacological profiles of selected cyclic dinucleotides.

Pharmacological profiles of the selected cyclic dinucleotides are shown in table. 6.

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Table 6

Example Bind STING (μM) STINGSOOT EC ₅₀ cells (μM) Reporter without digitonin a (A) STING293T cells ec ₅₀ (μM) Report P with DIGITON (B) Cellular permeability of the spine Ratio EC5Q (A) / (B) Double cells THP-1 EC ₅₀ (μM) Reporter Stability of human plasma (/ h) Example For 0.015 2.9 0, 13 22.3 5, 6 0.58 Example 5 0.050 3.6 0, 19 18.9 There is no data <0.01 Example 14 0.013 0.34 0, 13 2.6 2,3 <0.01 cGAMP 0.015 21 0.039 539 eighteen 1.39

Protocol for the analysis of immunoluminescence analysis of the gene-reporter of double THP-1.

THP1-Dual ™ cells (InvivoGen cat # thpd-nfis) were obtained from the human THP-1 monocyte cell line by stably integrating the luciferase gene in an immunoluminescence assay, a secreted luciferase reporter gene, under the control of the minimal promoter ISG54 (interferon-stimulated gene) in combined with five interferons (IFN) -stimulated response elements. On the day of the experiment, cells were plated on a black 384-well plate (Corning 356697) at a density of 7,500 cells / 25 μl per well in culture medium (RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg / ml Normocin ™, 100 U / ml-100 μg / ml Pen-Strep, 10 μg / ml Blasticidin and 100 μg / ml Zeocin). Cell plates were dosed with 62.5 nl of test compounds and then incubated at 37 ° C for 20 hours. At the end of incubation, 15 μl / well of QUANTI-Luc ™ (InvivoGen cat # rep-qlc1) was added and luminescence was immediately measured using a LeadSeeker.

Western blotting protocol for downstream signaling pathway activation (TBK1 and IRF3).

, 5x106 double THP-1 cells (Invivogen cat # thpd-nfis) were treated with DMSO or the indicated concentrations of Ex14 for 3 h. After stimulation, cells were harvested on ice, centrifuged at 800 RCF for 5 min and washed once in ice cold PBS ...

Cell pellets were lysed in 1% Triton X-100 whole cell lysis buffer (10% glycerol, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 μM EDTA, and 1 μM DTT) containing protease and phosphatase inhibitor cocktails ( Sigma Cat # P8340 and CalBiochem Cat # 524629 respectively). Cell lysates were purified from insoluble debris by centrifugation at 16,000 RCF for 10 min. Protein concentration in lysates was determined by the Bradford method using BSA standards.

Lysates were denatured in NuPAGE ™ LDS sample buffer (4X, cat # NP0008) containing DTT as a reducing agent. Denatured lysates were separated on 4-12% NuPAGE ™ bis-Trisgels in MES / SDS buffer and transferred to PVDF membranes by semi-dry blotting. Membranes were tested overnight at 4 ° C for PhosPho-TBKI S172, Phospho-IRF3 S396 and GAPDH using antibodies from Cell Signaling Technology (Cat # 5483, Cat # 4947 and Cat # 5174, respectively).

After the primary probe overnight, the membranes were washed thoroughly, then subjected to secondary antibody testing (goat anti-rabbit IgG Alexa Fluor® 680, Life Technologies cat # A21109) in Odyssey® blocking buffer (cat # 927-400000) at room temperature. After intensive flushing, the membranes were developed using the LI-COR ODYSSEY CLx.

The relative intensities of the phospho-TBKl and phospho-IRF3 bands were quantified and normalized to GAPDH levels. The GAPDH-normalized values of Phospho-TBKI and phospho-IRF3 were presented as a fold intensity versus unstimulated (DMSO) control. See fig. 7.

Human plasma stability analysis protocol.

The compound was incubated in heparinized human plasma at 37 ° C for 0, 1, 2, 4, 8, and 24 h, the change in the concentration of the compound was measured by LC / MS / MS. The elimination rate constant (k (h)) was calculated based on the fitted curve, assuming an exponential decrease

- 78 038805 concentration as follows: concentration at time x = initial concentration * exp (-k * x).

Syngeneic tumor model testing.

Example 14 and Example 3a were tested in mouse syngeneic tumor models. When Example 14 and Example 3a were administered intravenously (every 3 days, 3 times), antitumor effects were observed. See fig. 8 and 9, which illustrate the antitumor effect of colon carcinoma in a CT-26 syngeneic mouse model, and FIG. 10 and 11 - the antitumor effect of colon carcinoma of the B16F10 syngeneic mouse model.

Protocol for the assessment of the antitumor effect.

8x104 B16F10 and 2x105 CT-26 mouse tumor cells were implanted subcutaneously in the flank of female C57BL / 6 (B16F10) and Balb / C (CT-26) mice (n = 7 per group). Carrier medium (1 x PBS), the compound of Example 3a (30 mg / kg) or Example 14 (1.0 mg / kg) was administered intravenously (final volume 100 μl) to tumor-bearing mice as soon as the tumor volume reached 100 mm ³ (day 0 ). The carrier medium of Example 3a or Example 14 was given every 3 days for a total of 3 doses (q3dx3). Tumor and body weight measurements were performed three times a week using a caliper and mettler scale, respectively. Tumor volumes were determined by multiplying the square of the width (W), measured along the short axis of the tumor, half the length (L), measured along the short axis of the tumor (V = W x ^{0 2, 5} L).

The compounds of the examples are shown in the following tables.

MC in the tables means the actual measured value. The compound of example 9 was synthesized in the same way as in the method of example 5.

Table 1-1 | Example No.

IUPAC title

Structure

Doba

MC

7 ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aR, 16R) -14- (6-amino-9Hpurin-9-yl) -15-fluoro2,10,16-trihydroxy-2,10dioxidooctahydro-12H -5.8methanofuro [3.2—

1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3-

d] pyrimidin-4-one ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aR, 16R) -14- (6-amino-9Hpurin-9-yl) -15-fluoro2,10,16-trihydroxy -2.10dioxidooctahydro-12H-5.8methanofuro [3.2—

1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3-

d] pyrimidin-4-one ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aR, 16R) -14- (6-amino-9Hpurin-9-yl) -15-fluoro-10,16 dihydroxy-2 , 10-dioxido2-sulfanyloctahydro-12H5,8-methanofuro [3,2-

1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3-

d] pyrimidin-4-one ((2R, 5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9Hpurin-9-yl) -15-fluoro-10,16 dihydroxy- 2,10-dioxido2-sulfanyloctahydro-12H5,8-methanofuro [3,2-

1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3-

d] pyrimidin-4-one ___________

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Per 7- ((2R, 5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino-9Hpurin-9-yl) -15-fluoro-10, 16dihydroxy-2,10-dioxido2-sulfanyloctahydro -12H5,8-methanofuro [3,2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3- d] pyrimidin-4-one O. Λ " T 2Na 695, ο 4 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 16R) -14- (6-amino-9Hpurin-9-yl) -2,10,15,16 tetrahydroxy-2,10- dioxidooctahydro-12H-5, 8methanofuro [3,2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin- 7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3- d] pyrimidin-4-one F ⁰ s he ν А Т О '' ОН nh ₂ 2Et3 Ν 677 , one 5 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 16R) -14- (6-amino-9Hpurin-9-yl) -15,16- dihydroxy-2,10-dioxido2,10-disulfanyloctahydro12H- 5,8-methanofuro [3,21] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3-d] pyrimidine -4-one F ® o sh ί K r Chgrmn ONO O. X, JI l l / 7 'y? H * T ° 'SH νη ₂ 2Et3 Ν 707 , one

Table 1-2

Example No. Name no IUPAC Structure Add avk a MC 6 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 16R) -7- (6-amino-9Hpurin-9-yl) -15,16dihydroxy-2,10-dioxido2,10-disulfanyloctahydro12H-5, 8-methanofuro [3,2-1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin14-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3— d] pyrimidine -4- - ^zn mA 5 F О '' SH 2Et 3N 70 7, one

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he (optical isomer) 7 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 16R) -7- (6-amino-9Hpurin-9-yl) -15,16dihydroxy-2,10-dioxido2,10-disulfanyloctahydro12H-5, 8-methanofuro [3,21] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin14-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3d] pyrimidine-4- he (optical isomer) ΝΗ _? O- ^SH m 1 'D // Ίι ^N AA π y ό- -sh 2Et 3Ν 70 9, 0 eight 2-amino-9- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 16R) -14- (4-amino-5fluoro-7H-pyrrolo [2,3d] pyrimidin-7-yl) - 2,10,15,16-tetrahydroxy- 2,10-dioxidooctahydro12H-5,8-methanofuro [3,21] [1,3,6,9,11,2,10] pentaok sadifosfacyclotetradecin7-yl) -1,9-dihydro-6Npurin-6-one О О ОН II ОНО О ('ll Л Aw nh ₂ F & ОН 2Et 3Ν 69 2, 2 9 9- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 16R) -7- (4-amino-5-fluoro7H-pyrrolo [2,3- d] pyrimidin-7-yl) -15,16 dihydroxy-2,10-dioxide- 2.10- disulfanylooctahydro-12 H5,8-methanofuro [3,2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin- 14-yl) -1,9-dihydro-6Hpurin-6-one ... θ ν G okAo <Ni * H M ^N g HS '-b 2Et 3Ν 70 7, one 10 one- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 16R) -14- (6-amino-9Hpurin-9-yl) -2,10,15,16 tetrahydroxy-2,10- dioxidooctahydro-12H-5.8methanofuro [3,2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin- 7-yl) -1,5-dihydro-4Nimidazo [4,5-c] pyridin-4-one О Γ ϊ “> X ⁰ ? ³ Τ ή 'ΌΗ ΝΗ ^υ 65 9, one

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Table 1-3

Example No. eleven 12 thirteen 14 IUPAC title 2-amino-9- ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (4-amino1H-imidazo [4,5с] pyridin-1-yl) 2,10,15,16 tetrahydroxy-2,10 dioxidooctahydro- 12 H5,8-methanofuro [3.2— 1] [1,3,6,9,11,2,10] penta oxadiphosphacyclotetradets in-7-yl) -1,9-dihydro-6Нpurin-6-one eight- ((5R, 7R, 8R, 12aR, 14S, 15S, 15aS, 16R) -7- (6-amino-9Hpurin-9-yl) -15,16- dihydroxy-2,10dioxide-2,10-disulfanyloctahydro12H-5 , 8-methanofuro [3,2-1] [1,3,6,9,11,2,10] penta oxadiphosphacyclotetradec in-14-yl) pyrazolo [1,5-a] [1,3,5] triazine -4 (ZN) he 2-amino-9- ((5R, 7R, 8R, 12aR, 14S, 15S, 15aS, 16R) -14- (4aminopyrazolo [1,5 - a] [1,3,5] triazin-8-yl) 2,10,15,16 tetrahydroxy -2.10 dioxidooctahydro- 12 H5, 8-methanofuro [3.2 - 1] [1,3,6,9,11,2,10] penta oxadiphosphacyclotetradets in-7-yl) -1,9-dihydro-6Нpurin-6-one 7- ((2R, 5R, 7R, 8R, 10R, 12aR, 1 4R, 15R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) -15fluoro-16-hydroxy-2,10 dioxido-2, 10 - disulfanyloctahydro12H-5,8-methanofuro [3,2-1] [1,3,6,9,11,2,10] penta oxadiphosphacyclotetradets in-7-yl) -5-fluoro-3, 7dihydro-4H- pyrrolo [2,3d] pyrimidin-4-one Structure 0 p. P ^N N A • p // ΊΊ ^νη IT νΠ O u b w C - T 0 '' ^OH ΝΠ2 νη ₂ hs - ° ... Λ 1 is a C JI Λ g L »" HN N. _n VV π hs '' 6 o ™ m i 7 h ΝΗ it o. V, JI А nh ₂ ° '° hL f ιτ> V °; ° JO' SH N4 ₂ Doba vka 2Et3 N 2Et3 N 2Et3 N 2Na MC | 67 4, 2 69 2, 2 67 5, one 71 one, 0 15 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15aR, 16R) -14- (6-amino9H-purin-9-yl) -15-fluoro2,16-dihydroxy-2,10dioxide-10- sulfanyloctahydro-12H5,8-methanofuro [3,2 - 1] [1,3,6,9,11,2,10] penta oxadiphosphacyclotetradec in-7-yl) -5-fluoro-3, 7-dihydro-4H-pyrrolo [2,3— d] pyrimidin-4-one F 0 ο- 7 ^H v JI nh ₂ 2Et3 N 69 50

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Table 1-4

sixteen 17 eighteen 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 18R) -14- (6-amino-9Hpurin-9-yl) -2.10.18trihydroxy-2.10- dioxidohexahydro-14H15,12- (epoxymetano) -5.8methanofuro [3,2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin- 7 (12H) -yl) -5-fluoro-3, 7dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 18R) -14- (6-amino-9Hpurin-9-yl) -10,18dihydroxy-2,10-dioxido2-sulfanylhexahydro-14H15,12- (epoxymethane) -5.8methanofuro [3.2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin- 7 (12H) -yl) -5-fluoro-3, 7dihydro-4H-pyrrolo [2,3-d] pyrimidin-4-one 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aS, 18R) -14- (6-amino-9Hpurin-9-yl) -2.18dihydroxy-2,10-dioxido10-sulfanylhexahydro14H-15,12- (epoxymethane) 5,8-methanofuro [3,2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin- 7 (12H) -yl) -5-fluoro-3, 7dihydro-4H-pyrrolo [2,3d] pyrimidin-4-one F θ ОН II phase I О '' he nh ₂ F ° n T О 'ОН nh ₂ F θ ° -L L А. a'Z S ^Ch o ° but Tn ₂ ° '' ^SH 2Et 3Ν 2Et 3Ν 2Et 3N 68 7, 0 70 2, 9 70 2, 9 nineteen 2-amino-9- ((5R, 7R, 8R, 12aR, 14R, 15R, 15 aR, 16R) -15-fluoro-7- (5-fluoro4-oxo-3, 4-dihydro-7Hpyrrolo [2, 3-d] pyrimidine-7yl ) -2,10,16-trihydroxy2,10-dioxidooctahydro12H-5,8-methanofuro [3.21] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin14-yl) -1,9-dihydro -6Npurin-6-one ϊ / o = (2 su .ρ O. °, <Α % τ 2Et 3N 69 2, 9 twenty 7- ((5R, 7R, 8R, 12aR, 14R, 15R, 15aS, 16R) -14- (6-amino-9Hpurin-9-yl) -2,10,15,16tetrahydroxy-2-oxide-10sulfanyloctahydro-12H5, 8-methanofuro [3,2- 1] [1,3,6,9,11,2,10] pentaok sadiphosphacyclotetradecin7-yl) -5-fluoro-3, 7-dihydro4H-pyrrolo [2,3- d] pyrimidin-4-one F θ o. OH \ Η, l ъ Ϊη ₂ ° '' ^sh 2Et 3N 69 3, 3

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Table 1-5

Example no. IUPAC title Structure Doba vka MC 23 (optical isomer one) 7- [(5R, 7R, 8R, 12aR, 14R, 15 R, 15aS, 16R) -14- (6amino-9H-purin-9-yl) 10,15,16-trihydroxy2,10-dioxido-2sulfanyloctahydro12Н-5,8 - methanofuro [3,2- 1] [1,3, 6, 9, 11,2,10] pentaoxadiphosphacyclotetra adecin-7-yl] -5-fluoro-Zmethyl-3,7-dihydro-4Hpyrrolo [2,3 - d] pyrimidin-4-one diethylamine salt 1 about V A o N ' but oh x / / 1 ns o ° o Nil, 2 Et ₃ N 707, 1 23 (optical isomer 2) 7- [(5R, 7R, 8R, 12aR, 14R, 15 R, 15aS, 16R) -14- (6amino-9H-purin-9-yl) 10,15,16-trihydroxy2,10-dioxido-2sulfanyloctahydro12Н-5,8 - methanofuro [3,2- 1] [1,3, 6, 9,11,2,10] pentaoxadiphosphacyclotetra adecin-7-yl] -5-fluoro-Zmethyl-3,7-dihydro-4Hpyrrolo [2,3 - d] pyrimidin-4-one diethylamine salt \ И, С ¹ | (N ^Н К- ° 1 ^J Ν 'HQ О \ __>N' - | ^С .ΙΑ Ά ^0Η NH. 2 Et ₃ N 707, 1 24 (optical isomer one) 7- [(5R, 7R, 8R, 12aR, 14R, 15 R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) 15-fluoro-10,16- dihydroxy-2,10-dioxido-2sulfanyloctahydro12H -5.8methanofuro [3.2- 1] [1,3,6,9,11,2,10] pentaoxadiphosphacycloteter adecin-7-yl] -5-fluoro-Zmethyl-3,7-dihydro-4Hpyrrolo [2,3d] pyrimidin-4-one ditriethylamine salt 1 o 0 ^{SH L} Ν., J ° y, Y to N._ N ° 1 HO 0 Ν - Λ O ' ^CH NH, 2 Et ₃ N 709, 2

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24 (optical isomer 2) 7- [(5R, 7R, 8R, 12aR, 14R, 15 R, 15aR, 16R) -14- (6- amino-9H-purin-9-yl) 15-fluoro-10.16 - dihydroxy-2.10 - dioxido-2sulfanyloctahydro12H-5.8methanofuro [3,2-1] [1,3,6,9,11,2 , 10] pen taoxadiphosphacycloteter adecin-7-yl] -5-fluoro-Zmethyl-3,7-dihydro-4Hpyrrolo [2,3 - d] pyrimidin-4-one diethylamine salt ... _m ) I ..% s? 1, U uD -N / \ \ ____ / N. ' ^N ° ⁰ 14 ---. oh ^{he is} NN. 2 Et ₃ N 709, 3 25 7- [(5R, 7R, 8R, 12aR, 14R, 15 R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) 15-fluoro-2,10,16— trihydroxy-2,10dioxidooctahydro-12 H5,8-methanofuro [3,21] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetra adecin-7-yl] -5-fluoro-Zmethyl-3,7-dihydro-4Hpyrrolo [2, 3 - d] pyrimidin-4-one diethylamine salt I C Ί 1 f o _N 1 \ ' ^η -Ρ O' ^0Η NH, 2 Et ₃ N 693, 2 26 (optical isomer one) 7- [(5R, 7R, 8R, 12aR, 14R, 15 R, 15aR, 16R) -14- (6- amino-9H-purin-9-yl) 15-fluoro-16-hydroxy2,10-dioxide-2,10disulfanyloctahydro12H-5.8methanofuro [3,2-1] [1,3,6,9,11,2,10 ] pen taoxadiphosphacycloteter adecin-7-yl] -5-chloro3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one diethylamine salt ^c (θ o · \ ί νη Fo D JJ \ ° 34 Fl nA Ν'-d O ' ^SH NH; 2 Et ₃ N 727, 0 26 (optical isomer 2) 7- [(5R, 7R, 8R, 12aR, 14R, 15 R, 15aR, 16R) -14- (6amino-9H-purin-9-yl) 15-fluoro-16-hydroxy2,10-dioxide-2,10disulfanyloctahydro12H-5 , 8methanofuro [3.2— 1] [1,3,6,9,11,2,10] pentaoxadiphosphacyclotetra adecin-7-yl] -5-chloro- 3,7-dihydro-4Hpyrrolo [2,3-d] pyrimidin-4-one diethylamine salt Cl and O% | NH _N 5'1 ^Hu °: / n ° -₽ N-c 0 ^Sl NH; 2 Et ₃ N 727, 0

Example 35. STING binding test.

Test compound (2 μl), streptavidin-terbium (4 μl, Cisbio), labeled with fluorescein 2 ', 3'-cGAMP (c [G (2', 5 ') p-2'-Fluo-AHC-A (3' , 5 ') p]) (Biolog, Germany) and biotinylated STING protein (2 μl, wild type, WT) were mixed using assay buffer (Dulbecco's phosphate buffered saline (Wako Pure Chemical Industries, Ltd.) containing 0, 01% fatty acid-free bovine serum albumin (Wako Pure Chemical Industries, Ltd.)) and the mixture was left to stand at room temperature for 3 hours (final concentration: streptavidin-terbium; diluted to 1/1000, FITC-cGAMP; 1 μM biotinylated STING protein; 100 nM). Time-resolved fluorescence inductive resonance energy transfer (TR-FRET) was measured at 520 nm and 486 nm using Envision (PerkinElmer, Waltham, MA, USA). The inhibition rate of the binding of wild-type STING protein and 2 ', 3'-cGAMP of the test compound was calculated using the ratio of the amount at 520 nm divided by the number at 486 nm. The results are illustrated in table. 2 and 2A.

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The above biotinylated STING protein (wild type (WT)) was obtained by the following method.

A method of obtaining biotinylated wild-type STING protein.

ECOS (trade name) competent E. coli BL21 (DE3) were purchased from Nippon Gene Co., Ltd. Ampicillin, kanamycin, NaCl, glycerin, isopropylthiogalactoside, (+) - biotin, imidazole, SEM recombinant nucleotide, and BCA protein kit were purchased from Wako Pure Chemical Industries, Ltd. Tryptone, Bacto and Yeast Extract, Bacto were purchased from Difco Laboratories, Tris Buffered Salt (TBS) tablets, pH7.6 were purchased from Takara Bio Inc., Lysozyme (egg white), 6x Cryst was purchased from Seikagaku Corporation, a cOmplet (trade name) and protease inhibitor cocktail without EDTA were purchased from Roche. Ni-NTA Superflow Cartridge from QIAGEN and HiLoad 26/60 Superdex 200 pg from GE Healthcare were used.

Escherichia coli BirA was inserted into pRSF1b (Novagen), which has an altered multiple cloning site and transfected into ECOS JM109, resulting in pRH8 / FLAGBirA.pET21HH / His-Avi-SUMO-FLAG-hTMEM173 (139-379) (H232R) (which was created according to the method indicated in example 36) and pRH8 / FLAG-BirA for biotinylation Avi-tags were simultaneously transformed into ECO (trade name), competent E. coli BL21 (DE3) for the preparation of His-AviSUMO-FLAG-hSTING ( 139-379, H232R) -expressing cell line. The expression cell line was added to LB medium (10 g / L Tryptone, 5 g / L yeast extract, 5 g / L NaCl) containing ampicillin (100 μg / L) and kanamycin (50 μg / L), and the mixture previously cultured at 30 ° C, expanded to TB medium (12 g / L Trypton, 24 g / L yeast extract, 4 ml / L glycerol, 2.3 g / L KH ₂ PO ₄ , 12.5 g / L KH2PO4) containing the same antibiotics, and the mixture was cultured at 37 ° C. When the turbidity of the culture solution reached 500 KU, the culture temperature was lowered to 16 ° C, 0.1 mM isopropylthiogalactoside and 50 μM (+) - biotin were added to it, and the mixture was cultured for an additional 16 h.

The culture solution was centrifuged, the resulting fungal bodies were suspended in lysis buffer (50 mM Tris HCl, 150 mM NaCl, 20 mM imidazole, 1 mg / ml lysozyme, 5 U / ml SEM nuclease, recombinant, completely without EDTA, pH 7, 6), the protein was recovered by ultrasonic fragmentation. The reagent was added to it so that the salt concentration in the extract was brought to 300 mM NaCl, and the supernatant was collected by centrifugation. The resulting supernatant was passed through an overflow NiNTA cartridge equilibrated with wash buffer (50 mM Tris HCl, 300 mM NaCl, 20 mM imidazole, pH 7.6), and the cartridge was washed with wash buffer and eluted with elution buffer (50 mM Tris HCl, 300 mM NaCl, 250 mM imidazole, pH 7.6). The eluate was passed through a HiLoad 26/60 Superdex 200 pg column equilibrated with storage buffer (50 mM Tris HCl, 150 mM NaCl, pH 7.6) and the eluted fraction was collected as biotinylated His-Avi-SUMO-FLAG-hSTING (139 -379, H232R). Protein concentration was measured using a BCA protein assay kit, and the fraction was cryopreserved at -80 ° C prior to use.

table 2

STING binding test

Example no. the rate of inhibition of binding of the test compound (30 μM) one 76% 2 79% 3 64% Per 93% 5 71% eight 90% thirteen 68% 14 89% 15 89% sixteen 91% 17 86% eighteen 94% nineteen 93% twenty 98%

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Table 2A

Example no.

STING binding test

1C ₅₀ (μM) (optical isomer 1)

0.17 (optical isomer 2) (optical isomer 1)

0.029 (optical isomer 2)

0.29

0.083 (optical isomer 1)

0.53 (optical isomer 2)

0.075

As is clear from the above results, the compound of this invention inhibits the binding of the wild-type STING protein and the natural ligand 2 ', 3'-cGAMP, i. E. the compound of this invention binds to the wild type STING protein.

Example 36. Analysis of the reporter gene.

Obtaining various plasmids.

(i) Construction of expression plasmid human TMEM173 and the like.

The human STING expression plasmid (sometimes referred to herein as human TMEM173) in E. coli was prepared by overlapping primer PCR to introduce mutation using the human TMEM173 cDNA clone (GeneCopoeia) as template. First, PCR was performed using two types of primer (5'-CCTGGCCCCAGCTGAGATCTCTG-3 '(C-hTMEM (139aa) -F) (SEQ ID NO. 3) and 5'-GTAAACCCGATCCTTGATGCCAGCACGGTCACCGGTC-3' (hTMREM173) (H232 ) (SEQ ID NO. 4) and two kinds of primer (5'-CTGCCCCAGCAGACCGGTGACCGTGCTGGCATCAAG-3 '(hTMEM173 (H232R) -F) (SEQ ID NO. 5) and 5'-ATAATAGCGGCCGCTCAAGAGAGATCCGTG-3 R) (SEQ ID NO. 6) Each PCR was performed using PrimeStar MAX DNA Polymerase (Takara Bio Inc.) sequentially (1) at 98 ° C for 1 min, (2) 25 times at 98 ° C for 10 s and at 72 ° C for 10 s, and (3) at 72 ° C for 1 min.Then PCR was performed using the obtained segment as a template using two types of primers (C-hTMEM (139aa) - F and hTMEM173-st-NotR) PCR was performed using PrimeStar GXL DNA Polymerase (Takara Bio Inc.) sequentially (1) at 98 ° C for 1 min, (2) 35 times at 98 ° C for 10 s and at 72 ° C for 1.5 min, and (3) at 72 ° C for 1 min. The resulting segment was cut with Not I (Takara Bio Inc.), inserted into the Stu I / Not I site of the vector in which the His-Avi-SUMO tag was attached to pET21a (Novagen) using Ligation High (Toyobo Co., Ltd). ) and ported to ECOS JM109 (Nippon Gene Co., Ltd.), resulting in pET21HH / His-Avi-SUMO-hTMEM173 (139-379) (H232R).

Second, PCR was carried out using this plasmid as a template and using two types of primer (5'-CGACTACAAGGACGACGATGACAAGGGATCCCTGGCCCCAGCTGAGATCTCTG-3 '(C-FLAGBam-hTMEM173 (139aa) -F) (SEQTM ID NO173. -R). PCR was performed using PrimeStar MAX DNA polymerase sequentially (1) at 98 ° C for 1 min, (2) 25 times at 98 ° C for 10 s and at 72 ° C for 10 s, and (3) at 72 ° C for 1 min. The resulting segment was cut with Not I, inserted into the Stu I / Not I site of pET21a, to which the His-Avi-SUMO tag was attached, using Ligation High as mentioned above, and transferred into ECOS JM109, resulting in the creation of pET21HH / His -Avi-SUMO-FLAG-hTMEM173 (139-379) (H232R).

An expression plasmid for reporter assay was obtained by overlapping primer PCR to introduce mutation using the human TMEM173 cDNA clone as template as mentioned above. First, PCR was carried out using two types of primer (5'-GTACCCATACGATGTTCCAGATTACGCTGGATCCGCCACCATGCCCCACTCCAGCCTGCATC-3 '(HA-Bam-ko-hTMEM173-F) (SEQ ID NO. 8) and hTMEM173 (h232R H232R) -F and hTMEM173-st-Not-R). Each PCR was performed using PrimeStar MAX DNA polymerase sequentially (1) at 98 ° C for 1 min, (2) 25 times at 98 ° C for 10 s and 72 ° C for 10 s, and ( 3) at 72 ° C for 1 min. Then, PCR was performed using the obtained segment as a template using two types of primer (5'ATAATATCTAGAATTCGCCACCATGTACCCATACGATGTTCCAGATTACGC-3 '(Xba-Eco-ko-HA-F) (SEQ ID NO. 9) and hTMEM173-st-Not-R). PCR was performed using PrimeStar GXL DNA polymerase (1) at 98 ° C for 1 min, (2) 35 times at 98 ° C for 10 s, at 65 ° C for 5 s and 72 ° C for 1.5 min, and (3) at 72 ° C for 1 min. The resulting segment was cut with Xba I (Takara Bio Inc.) and Not I, inserted into the Nhe I / Not I site of the zeocin resistance vector, where the multiple cloning site was inserted into pcDNA3,3 (Invitrogen) using Ligation High, and transferred into ECOS JM109 whereby DNA3,3zeo / HA-hTMEM173 (H232R) was created.

A plasmid expressing the human mutant type TMEM173 (R232H) was created using

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PCR using the human cDNA clone TMEM173 (GeneCopoeia) as template and using two kinds of primers (5'-TTCCAGATTACGCTGGATCCGCCACCATGCCCCACTCCAGCCTGCATC-3 '(Bam-ko-hTEME 173v1F) (SEQGACGATC. (hTMEM173-st-Not-R2) (SEQ ID NO. 11) PCR was performed using PrimeStar MAX DNA polymerase (Takara Bio Inc.) sequentially (1) at 98 ° C for 1 min, (2) 30- a single repeat at 98 ° C for 10 s and at 68 ° C for 10 s, and (3) at 72 ° C for 1 min The resulting segment was inserted into the Bam HI / Not I site of the vector, where the HA-Tag was attached to pcDNA3,1 (+) (ThermoFischer) using Gibson Assembly (NEB) and ported to ECOS JM109 (Nippon Gene Co., Ltd.), whereby pcDNA3,1HA / HA-hTMEM173v1 was created.

Plasmid expressing human wild-type TMEM173 (H232R) was constructed by PCR using pcDNA3,3zeo / HA-hTMEM173 (H232R) plasmid as template and using two primer species (5'-GGAGACCCAAGCTGGCTAGCGCCATACCAT NGTACCC 3 ' HA-F) (SEQ ID NO. 12) and 5'-CCTCTAGACTCGAGCGGCCGCTCAAGAGAAATCCGTGCGGAGAGG-3 '(hTMEM 173-st-Not-R2) (SEQ ID NO. 11) PCR was performed using PrimeStar MAX DNA polymerase sequentially (1) at 98 ° C for 1 min, (2) 30 repetitions at 98 ° C for 10 s and at 72 ° C for 35 s and (3) at 72 ° C for 1 min.The resulting segment was inserted into pcDNA3,1zeo Nhe I / Not I site (ThermoFischer) using Gibson Assembly and transfected into ECOS JM109, whereby pcDNA3,1 zeo / HA-hTMEM 173 (H232R) was generated.

(ii) construction of a firefly luciferase expression plasmid.

A firefly luciferase expression plasmid was created by inserting the CMV promoter into a reporter vector. PCR was performed using the vector pcDNA3,1 (+) (Invitrogen) as a template and using two types of primer (5'-ATAATAAGATCTGTTGACATTGATTATTGACTAGTTATTAATAG-3 '(CMVPro-BgIII-F) (SEQ ID NO. 13) and 5'-ATAATAATCAGCTT -3 '(CMVPro-HindIII-R) (SEQ ID NO. 14) PCR was performed using PrimeStar MAX DNA polymerase sequentially (1) at 98 ° C for 1 min, (2) 25 times at 98 ° C for 10 s and at 68 ° C for 3 s and (3) at 72 ° C for 1 min The resulting segment was cut with Bgl II and Hind III (Takara Bio Inc.), inserted into the Bgl II site / Hind III pGL4,17 (Promega Corporation) using Ligation High (Toyobo Co., Ltd) and transferred to ECOS JM109, whereby pGL4,17 / CMV Pro was generated.

Reporter gene analysis (1).

Was created stably expressing cell line 293T, transfected with the vector pNL [NLucP / ISRE / Hygro] (Promega, Fitchburg, WI, USA). pcDNA3,3zeo / HA-hTMEM173 (H232R) were transfected into cells using FugeneHD (Promega, Fitchburg, Wisconsin, USA) and the cells were cultured for one day using Dulbecco's Modified Eagle's Medium (DMEM) (Wako Pure Chemical Industries, Ltd ) containing 10% fetal bovine serum. Cells were harvested and cryopreserved with CELLBANKER 1 (Nippon Zenyaku Kogyo Co., Ltd.).

On the day of assay, test compound was diluted with assay buffer (DMEM containing 0.1% fatty acid free bovine serum albumin) was added to a white 384 well plate (Corning, NY, USA) at 10 μl / well. The cryopreserved cells were thawed, and the cells were suspended in assay buffer and seeded with 10 μl / well (10,000 cells / well). The cells were cultured at 37 ° C under 5% CO2 for 20 h, and a NanoGlo reagent solution (Promega, Fitchburg, Wisconsin, USA) (20 μL) was added to them. After incubation for 5 min, the luminescence level was measured using Envision (PerkinElmer, Waltham, MA, USA). The activity level of each test compound was calculated when the number of cells treated with 2 ', 3'-cGAMP (30 μM) was taken as 100% and the number of cells treated with vehicle was taken as 0%. The results are illustrated in table. 3.

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Table 3

Study of the agonist activity of STING

Example no. the level of activity of the test compound (30 μM) one 77% 2 77% 3 76% Per 107% 5 one hundred% 14 71% 15 81% sixteen 123% 17 99% eighteen 81% twenty 125%

As is clear from the above results, the compound of this invention has wild-type STING agonist activity.

Reporter gene analysis (2).

Was constructed stably expressing cell line 293T, transfected with the vector pNL [NLucP / ISRE / Hygro] (Promega, Fitchburg, WI, USA). The suspension, which was obtained by transfection of pcDNA3,1zeo / HA-hTMEM173 (H232R) or pcDNA3,1HA / HA-hTMEM173v1 together with the plasmid for expression of firefly luciferase to cells using FugeneHD (Promega, Fitchburg, Wis., USA), was added to 384- plate (Corning, NY, USA) and cells were cultured for two days. The culture solution was removed and the test compound was diluted with analytical buffer (50 mM HEPES, pH 7.0, 100 mM KCl, 3 mM MgCl ₂ , 85 mM sucrose, 0.1 mM DTT, 0.2% BSA, 1 mM APT, 0, 1 mM GTP, 10 μg / ml digitonin), 15 μl / well was added to it. The cells were cultured at 37 ° C under 5% CO2 for 30 min and the assay buffer was removed. Dulbecco's Modified Eagle's Medium (DMEM) (Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum, to which was added 20 μl / well, and the cells were cultured at 37 ° C under 5% CO2 for 4 h. Luminescence signals obtained from firefly luciferase and NanoLuc luciferase were each measured using Envision (PerkinElmer, Waltham, MA, USA) according to the protocol of the Nano-Glo-Glo-Luciferase Reporter Assay System (Promega, Fitchburg , Wisconsin, USA). The ratio of the amount of NanoLuc luciferase divided by the amount of firefly luciferase was used for the calculation. The activity level of each test compound was calculated when the ratio in cells treated with 2 ', 3'-cGAMP (30 μM) was taken as 100%, and the ratio in cells treated with vehicle was taken as 0%. The results are illustrated in table. 4.

Table 4

Study of the agonist activity of STING

Example no. the level of activity against WT STING of the test compound (30 μM) the level of activity against the STING mutant type (R232H) of the test compound (30 μM) one 97% 70% 1a 99% 92% 2 112% 77% 3 97% 73% 5 102% 95% thirteen 99% 85% 14 99% 92% 15 91% one hundred% sixteen 114% 108% 17 110% 85% eighteen 98% 96% nineteen 101% 62% twenty 113% 85%

As is clear from the above results, the compound of this invention has agonist activity against wild type STING and mutated type STING (R232H).

- 89 038805

Example 37. Detection of phosphorylated IRF3 protein in FaDu cell.

FaDu cells of the human laryngeal cancer cell line (ATCC) were seeded, the medium was replaced with serum-free medium one day after seeding. After replacement, the cell was cultured for one day, and natural ligand 2 ', 3'-cGAMP (final concentration; 30 μM) and test compound (final concentration; 15 μM or 30 μM) were added to the cells. 6 h after the addition, the cells were washed with PBS, a cell extract was prepared, and the phosphorylated IRF3 protein was determined by ELISA or Western blotting. The phosphorylation activity of the IRF3 protein of the test compound was calculated when the value in the sample with the addition of the natural ligand 2 ', 3'-cGAMP at a final concentration of 30 μM was considered equal to 100%. The results are illustrated in table. 5.

Table 5

IRF3 protein phosphorylation test

Example no. activity level Test compound one 4 6 8.8% at 30 μM 2 8 8.7% at 30 μM 3 4 64.6% at 30 μM 5 117.4% at 30 μM

As is clear from the above results, the compound of this invention promotes phosphorylation of IRF3, which is the downstream STING signal, as in the natural ligand 2 ', 3'cGAMP. That is, the compound of the present invention activates the downstream STING signal as a STING agonist.

Example 38. Example of a preparation.

A medicament containing a compound of the present invention as an active ingredient can be obtained, for example, based on the following composition.

1. Capsule.

(1) The compound obtained in Example 1 is 10 mg.

(2) Lactose 90 mg.

(3) Crystalline Cellulose 70 mg

(4) Magnesium stearate 10 mg.

capsule - 180 mg.

(1), (2), (3) and 5 mg (4) are mixed and granulated. To these are added the remaining 5 mg (4) and the entire amount is filled into a gelatin capsule.

2. Tablet.

(1) The compound obtained in Example 1 is 10 mg.

(2) Lactose 35 mg

(3) Cornstarch 150 mg

(4) Crystalline Cellulose 30 mg

(5) Magnesium stearate 5 mg.

tablet - 230 mg.

(1), (2), (3), 20 mg (4) and 2.5 mg (5) are mixed and granulated. To these are added the remaining 10 mg (4) and the remaining 2.5 mg (5), and the mixture is compressed to obtain a tablet.

Industrial applicability

The compound of this invention may have STING agonist activity. Therefore, the compound of the present invention can be used as a STING agonist and can be useful as an agent for the prevention or treatment of STING-associated diseases, including cancer and the like.

It should be understood that the foregoing described embodiments and examples are not intended to limit in any way the scope of the description, and that the claims presented herein are intended to cover all embodiments and examples, whether or not they are presented. in this document, explicitly or not.

All patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

Claims (17)

CLAIM 1. The compound having the formula (X) where R ¹ and R ² each independently represents a hydroxy group or a halogen atom; B ¹ represents E'- ¹ R ¹⁸ is hydrogen or C _1-6 alkyl; R ¹⁹ represents a halogen atom; B ² is Q ² and Q ⁴ each independently represent an oxygen atom or a sulfur atom, or a pharmaceutically acceptable salt thereof. 2. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R ¹⁹ is fluoro or chloro. 3. A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R ¹ is hydroxy. 4. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein R ² is a hydroxyl group or a fluorine atom. ^{5. A compound according to any one of claims 1 to 4} , or a pharmaceutically acceptable salt thereof, wherein Q 4 is a sulfur atom. 6. A compound according to any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein Q ² is an oxygen atom. 7. A compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, wherein Q ² is a sulfur atom. 8. A compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, having the formula (XII) 9. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, wherein R ¹⁸ is hydrogen or methyl. 10. The compound according to claim 1, selected from the group consisting of - 91 038805 or a pharmaceutically acceptable salt thereof. 11. A compound according to claim 10, which is - 92 038805 or a pharmaceutically acceptable salt thereof. 12. A compound according to claim 10 which is or a pharmaceutically acceptable salt thereof. 13. A compound according to claim 10, which is or a pharmaceutically acceptable salt thereof. 14. A compound according to any one of claims 1 to 13, wherein the pharmaceutically acceptable salt is a triethylamine salt. 15. A compound according to any one of claims 1-13, wherein the pharmaceutically acceptable salt is sodium salt. 16. A pharmaceutical composition for activating an interferon gene stimulator comprising a therapeutically effective amount of a compound according to any one of claims 1 to 15 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. 17. A method of treating cancer in a patient, comprising administering to the patient a therapeutically effective amount of a compound according to any one of claims 1-15, or a pharmaceutically acceptable salt thereof.