U.S. patent application number 16/185258 was filed with the patent office on 2021-05-13 for cyclic dinucleotide.
The applicant listed for this patent is TAKEDA PHARMACEUTICAL COMPANY LIMITED. Invention is credited to Kosuke Hidaka, Taisuke Kato, Michiyo Mochizuki, Yoshihisa Nakada, Yasuo Nakagawa, Morihisa Saitoh, Tomohiro Seki, Masaki Seto, Akito Shibuya, Zenyu Shiokawa, Yusuke Tominari, Masato Yoshikawa, Yayoi Yoshitomi.
Application Number | 20210137962 16/185258 |
Document ID | / |
Family ID | 1000005550533 |
Filed Date | 2021-05-13 |
United States Patent
Application |
20210137962 |
Kind Code |
A9 |
Yoshikawa; Masato ; et
al. |
May 13, 2021 |
CYCLIC DINUCLEOTIDE
Abstract
The present disclosure 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
present disclosure relates to a compound represented by the formula
(I): ##STR00001## wherein each symbol is as defined in the
description, or a salt thereof.
Inventors: |
Yoshikawa; Masato; (Fujisawa
Kanagawa, JP) ; Saitoh; Morihisa; (Fujisawa Kanagawa,
JP) ; Kato; Taisuke; (Fujisawa Kanagawa, JP) ;
Yoshitomi; Yayoi; (Fujisawa Kanagawa, JP) ; Seki;
Tomohiro; (Fujisawa Kanagawa, JP) ; Nakagawa;
Yasuo; (Fujisawa Kanagawa, JP) ; Tominari;
Yusuke; (Fujisawa Kanagawa, JP) ; Seto; Masaki;
(Fujisawa Kanagawa, JP) ; Shibuya; Akito;
(Fujisawa Kanagawa, JP) ; Hidaka; Kosuke;
(Fujisawa Kanagawa, JP) ; Shiokawa; Zenyu;
(Fujisawa Kanagawa, JP) ; Nakada; Yoshihisa;
(Fujisawa Kanagawa, JP) ; Mochizuki; Michiyo;
(Fujisawa Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TAKEDA PHARMACEUTICAL COMPANY LIMITED |
Osaka-Shi Osaka |
|
JP |
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Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20190192549 A1 |
June 27, 2019 |
|
|
Family ID: |
1000005550533 |
Appl. No.: |
16/185258 |
Filed: |
November 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/IB2017/057588 |
Dec 1, 2017 |
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16185258 |
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62589300 |
Nov 21, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/7076 20130101;
A61K 47/6807 20170801; A61P 35/00 20180101; A61K 31/7084
20130101 |
International
Class: |
A61K 31/7084 20060101
A61K031/7084; A61P 35/00 20060101 A61P035/00; A61K 47/68 20060101
A61K047/68; A61K 31/7076 20060101 A61K031/7076 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 1, 2016 |
JP |
2016-234553 |
May 30, 2017 |
JP |
2017-107216 |
Claims
1. A compound having Formula (X): ##STR00447## wherein: R.sup.1 and
R.sup.2 are each independently a hydroxy group or a halogen atom;
B.sup.1 is: ##STR00448## R.sup.18 is hydrogen or C.sub.1-6 alkyl,
R.sup.19 a halogen atom; B.sup.2 is: ##STR00449## and and Q.sup.2
and Q.sup.4 are each independently an oxygen atom or a sulfur atom,
or a pharmaceutically acceptable salt thereof.
2-26. (canceled)
27. The compound of claim 1 selected from the group consisting of:
##STR00450## ##STR00451## or a pharmaceutically acceptable salt
thereof.
28-200. (canceled)
201. The compound of claim 27 that is: ##STR00452## or a
pharmaceutically acceptable salt thereof.
202. The compound of claim 27 that is: ##STR00453##
203. The compound of claim 27 that is: ##STR00454## or a
pharmaceutically acceptable salt thereof.
204. The compound of claim 27 that is: ##STR00455## or a
pharmaceutically acceptable salt thereof.
205. The compound of claim 27 that is: ##STR00456## or a
pharmaceutically acceptable salt thereof.
206. The compound of claim 27 that is: ##STR00457## or a
pharmaceutically acceptable salt thereof.
207. The compound of claim 201 that is the triethylamine salt.
208. The compound of claim 202 that is the triethylamine salt.
209. The compound of claim 203 that is the triethylamine salt.
210. The compound of claim 204 that is the triethylamine salt.
211. The compound of claim 205 that is the triethylamine salt.
212. The compound of claim 206 that is the triethylamine salt.
213. The compound of claim 201 that is the sodium salt.
214. The compound of claim 202 that is the sodium salt.
215. The compound of claim 203 that is the sodium salt.
216. The compound of claim 204 that is the sodium salt.
217. The compound of claim 205 that is the sodium salt.
218. The compound of claim 206 that is the sodium salt.
219. A pharmaceutical composition comprising the compound of claim
1, or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable excipient.
220. A method of treating a patient comprising administering to the
patient a therapeutically effective amount of the compound of claim
1, or a pharmaceutically acceptable salt thereof, wherein the
patient has cancer.
Description
TECHNICAL FIELD
[0001] The present disclosure provides a cyclic dinucleotide having
a STING (stimulator of interferon genes) agonistic activity, which
may be useful as an agent for the prophylaxis or treatment of
cancer and other diseases.
BACKGROUND OF THE INVENTION
[0002] STING is a receptor recognizing nucleic acid different from
TLR (toll-like receptor). Examples of the natural ligand to be
recognized include bacteria/protozoa-derived cyclic dinucleotides
(CDNs), 2',3'-cGAMP synthesized by the 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 natural
ligands, is decomposed by ENPPI
(ecto-nucleotide-pyrophosphatase/phosphodiesterase), which is one
of pyrophosphatases/phosphodiesterases, and that the other CDNs are
decomposed by phosphodiesterase (Nat Chem Biol 10:1043-1048 (2014);
Cell Res 25:539-550 (2015); Biochemistry 55:837-849 (2016)). STING
is activated by these natural ligands, and induces the
phosphorylation of TBK1 (TANK binding kinase 1) in the downstream,
and activates IRF3 (Interferon regulatory factor 3) signal and NFkB
signal in the further downstream, and thereby type-I interferon
(IFN) response is induced (Trends in Immunology 35:88-93 (2014)).
The importance of STING signal on cancer are indicated by a test
using a knockout mouse. It is reported that in tumor-allografted
mice using knockout mice for STING and its downstream signal, IRF3,
the cancer cells grow by suppression of cancer immune system,
compared with in wild-type mouse (Immunity 41: 830-842 (2014)). In
addition, it is also reported that the cancer cell growth in a
tumor-allografted mouse is suppressed by radiation therapy, but in
knockout mice for STING and IFNAR1 (interferon (alpha and beta)
receptor 1, receptor of type-I IFN produced by the downstream
signal), the effect by the radiation therapy is reduced (Immunity
41:843-852 (2014)). For those reasons, it is considered that STING
plays an important role on suppression of cancer cell growth, and
the activation of the immune signal, which is induced by the
activation of STING, leads to anticancer activity. Therefore, STING
agonist may be used as an anticancer agent targeting cancer
immunity. In addition, the activation of STING is considered to
plays an important role on immune effect of vaccine, since the
activation activates natural immunity (Ther Adv Vaccines 1:131-143
(2013)). Therefore, STING agonist may be used as an adjuvant for
various vaccines.
[0003] The following cyclicdinucleotides are known.
[0004] Patent Document 1 (WO 2014/093936) discloses a compound
represented by the following formula:
##STR00002##
wherein each symbol is as defined in Patent Document 1, which
according to Patent Document 1 is a STING-dependent TBK1 activator,
and is useful for the treatment of cancer (particularly solid
cancer) and the like, and also useful as an adjuvant.
[0005] Patent Document 2 (WO 2014/189805) discloses a compound
represented by the following formula:
##STR00003##
wherein each symbol is as defined in Patent Document 2, which
according to Patent Document 2 is an immune stimulator via STING,
and is useful for the treatment of cancer and the like.
[0006] Patent Document 3 (WO 2015/077354) and Non-Patent Document 8
(Cell reports 11, 1018-1030 (2015)) disclose a compound represented
by the following formula:
##STR00004##
wherein each symbol is as defined in Patent Document 3, which
according to Patent Document 3 is a STING agonist, and is useful
for the treatment of cancer and the like.
[0007] Patent Document 4 (WO 2013/185052) and Non-Patent Document 9
(Sci. Transl. Med. 283, 283ra52 (2015)) disclose c-di-AMP,
c-di-GMP, c-di-IMP, c-AMP-GMP, c-AMP-IMP and c-GMP-IMP, which
according to Patent Document 4 are STING-dependent TBK1
activators.
[0008] Patent Document 5 (WO 2014/189806) discloses a compound
represented by the following formula:
##STR00005##
wherein each symbol is as defined in Patent Document 5, which
according to Patent Document 5 inhibits STING-dependent signal
transduction, and is useful for the treatment of autoimmune disease
and the like.
[0009] Patent Document 6 (WO 2015/185565) discloses a compound
represented by the following formula:
##STR00006##
wherein each symbol is as defined in Patent Document 6, which
according to Patent Document 6 is a STING modulator, and is useful
for the treatment of inflammation, allergic autoimmune disease,
cancer and the like, and also useful as a vaccine adjuvant.
[0010] Patent Document 7 (WO 2014/179760) discloses a compound
represented by the following formula:
##STR00007##
wherein each symbol is as defined in Patent Document 7, which
according to Patent Document 7 can increase Type I interferon
production, and is useful for the treatment of cancer, autoimmune
disease, allergic reaction and the like, and also useful as an
adjuvant.
[0011] Patent Document 8 (WO 2014/179335) and Non-Patent Document
10 (Mol. Cell 154, 748-762 (2013)) disclose a compound represented
by the following formula:
##STR00008##
wherein each symbol is as defined in Patent Document 8, which
according to Patent Document 8 can increases Type I interferon
production, and is useful for the treatment of diseases
characterized by inflammation, autoimmune disease, Sjogren's
syndrome and the like.
[0012] Patent Document 9 (WO 2015/017652) discloses a compound
represented by the following formula:
##STR00009##
which according to Patent Document 9 is a STING modulator, and is
useful for the treatment of cancer, autoimmune disease and the
like, and also useful as a vaccine.
[0013] Patent Document 10 (WO 2016/096577) discloses a compound
represented by the following formula:
##STR00010##
which according to Patent Document 10 is a STING agonist, and is
useful for the treatment of cancer (particularly solid pancreatic
cancer) and the like.
[0014] Patent Document 11 (WO 2011/003025) discloses a compound
represented by the following formula:
##STR00011##
Which according to Patent Document 11 is a STING agonist.
[0015] Patent Document 12 (WO 2016/096174) discloses a compound
represented by the following formula:
##STR00012##
which according to Patent Document 12 is a STING agonist.
[0016] Patent Document 13 (WO 2016/120305) discloses a compound
represented by the following formula:
##STR00013##
which according to Patent Document 13 is a STING agonist.
[0017] Patent Document 14 (WO 2016/145102) discloses a compound
represented by the following formula:
##STR00014##
wherein each symbol is as defined in Patent Document 14, which
according to Patent Document 14 is a STING agonist.
[0018] Patent Document 15 (WO 2017/027646) discloses a compound
represented by the following formula:
##STR00015##
wherein each symbol is as defined in Patent Document 15, which
according to Patent Document 15 is a STING agonist.
[0019] Patent Document 16 WO 2017/075477) discloses a compound
represented by the following formula:
##STR00016##
wherein each symbol is as defined in Patent Document 16, which
according to Patent Document 16 is a STING agonist.
[0020] Patent Document 17 (WO 2017/027645) discloses a compound
represented by the following formula:
##STR00017##
wherein each symbol is as defined in Patent Document 17, which
according to Patent Document 17 is a STING agonist.
BRIEF SUMMARY OF THE INVENTION
[0021] In one aspect, the disclosure provides a compound
represented by the formula (I):
##STR00018##
wherein:
[0022] the partial structure represented by formula (A-1):
##STR00019##
[0023] is a partial structure represented by formula (IIA):
##STR00020##
[0024] a partial structure represented by formula (IIB):
##STR00021##
[0025] R.sup.1 and R.sup.2 are each independently a hydroxy group
or a halogen atom;
[0026] B.sup.1 is a group represented by
##STR00022##
[0027] R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each
independently a hydrogen atom or a substituent;
[0028] Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14, Y.sup.15 and
Y.sup.16 are each independently N or CR.sup.1a;
[0029] Z.sup.11, Z.sup.12, Z.sup.13, Z.sup.14, Z.sup.15 and
Z.sup.16 are each independently N or C;
[0030] R.sup.1a is a hydrogen atom or a substituent;
[0031] B.sup.2 is a group represented by
##STR00023##
[0032] R.sup.23, R.sup.24, R.sup.25, R.sup.26 and R.sup.27 are each
independently a hydrogen atom or a substituent;
[0033] Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24, Y.sup.2 and Y.sup.26
are each independently N or CR.sup.2a;
[0034] Z.sup.21, Z.sup.22, Z.sup.23, Z.sup.24, Z.sup.25 and
Z.sup.26 are each independently N or C;
[0035] R.sup.2a is a hydrogen atom or a substituent;
provided that
[0036] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14l,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0037] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0038] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C; X.sup.1 and X.sup.2 are each independently an oxygen
atom or a sulfur atom; and
[0039] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom, or a salt thereof (hereinafter
sometimes to be referred to as compound (I)).
[0040] In another aspect, the disclosure provides a compound
represented by the formula (I), wherein at least one of B.sup.1 and
B.sup.2 is
##STR00024##
[0041] R.sup.18 is hydrogen or C1-6 alkyl; and
[0042] R.sup.19 is a halogen atom, or a salt thereof.
[0043] In another aspect, X.sup.1 and X.sup.2 are O.
[0044] In another aspect, the disclosure provides a compound
represented by the formula (I), wherein
[0045] B.sup.1 is
##STR00025##
and
[0046] B.sup.2 is
##STR00026##
or a salt thereof. In another aspect, X.sup.1 and X.sup.2 are
O.
[0047] In another aspect, the disclosure provides
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[3,2-1][1,3,6,-
9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-p-
yrrolo[2,3-d]pyrimidin-4-one, or a salt thereof.
[0048] In another aspect, the disclosure provides
7-((2R,5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluo-
ro-10,16-dihydroxy-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-d-
ihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, or a salt thereof.
[0049] In another aspect, the disclosure provides
2-amino-9-((5R,7R,8R,12aR,14S,15S,15aS,16R)-14-(4-aminopyrazolo[1,5-a][1,-
3,5]triazin-8-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-me-
thanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-1,-
9-dihydro-6H-purin-6-one, or a salt thereof.
[0050] In another aspect, the disclosure provides
7-((2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15--
fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofur-
o[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, or a salt thereof.
[0051] In another aspect, the disclosure provides a compound having
Formula (XIV):
(CD-L).sub.n-A (XIV)
or a pharmaceutically acceptable salt thereof, wherein:
[0052] CD is a group represented by any one of Formula (XX)-(XXIX),
see below;
[0053] L is a linker;
[0054] A is an antibody, antibody fragment, or antigen-binding
fragment;
[0055] n is 1-10.
[0056] In another aspect, the disclosure provides a compound having
Formula (XIV), or a pharmaceutically acceptable salt thereof,
wherein A is an antibody.
[0057] In another aspect, the disclosure provides a compound having
Formula (XIV), or a pharmaceutically acceptable salt thereof,
wherein A is an antigen-binding fragment.
[0058] In another aspect, the disclosure provides medicament
comprising the compound or salt of formula (I). In another
embodiment, the medicament is a STING agonist. In another
embodiment, the medicament is an agent for the prophylaxis or
treatment of cancer.
[0059] In another aspect, the disclosure provides a method of
activating a STING in a mammal, which comprises administering an
effective amount of a compound having formula (I), or a salt
thereof, to the mammal.
[0060] In another aspect, the disclosure provides a method for the
prophylaxis or treatment of cancer in a mammal, which comprises
administering an effective amount of the compound having formula
(I), or a salt thereof, to the mammal.
[0061] In another aspect, the disclosure provides a compound having
formula (I), or a salt thereof, for use in prevention or treatment
of cancer.
[0062] In another aspect, the disclosure provides the use of the
compound having formula (I), or a salt thereof, for the production
of an agent for the prophylaxis or treatment of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] FIG. 1 is a line graph showing Payload 1 release from
ADC1.
[0064] FIG. 2 is a line graph showing Payload 1 release from
ADC2.
[0065] FIG. 3 is a line graph showing Payload 2 release from
ADC3.
[0066] FIG. 4 is a bar graph showing that HEK293 cells that express
the surface receptor targets for ADC1 exhibit the expected increase
in the STING pathway marker phospho-IRF3 (pIRF3) when treated with
the ADC1.
[0067] FIG. 5 is a bar graph showing that HEK293 cells that express
the surface receptor targets for ADC2 exhibit the expected increase
in the STING pathway marker phospho-IRF3 (pIRF3) when treated with
the ADC2.
[0068] FIG. 6 is a bar graph showing that HEK293 cells that express
the surface receptor targets for ADC3 exhibit the expected increase
in the STING pathway marker phospho-IRF3 (pIRF3) when treated with
the ADC3.
[0069] FIG. 7 is a bar graph showing the Western blot analyses of
downstream signaling pathway activation (TBK1 and IRF3) for
compound Ex. 14.
[0070] FIG. 8 is line graph showing the anti-tumor activity of
compound Ex. 3a in the colon carcinoma CT-26 syngeneic mice
model.
[0071] FIG. 9 is line graph showing the anti-tumor activity of
compound Ex. 14 in the colon carcinoma CT-26 syngeneic mice
model.
[0072] FIG. 10 is line graph showing the anti-tumor activity of
compound Ex. 3a in the colon carcinoma B16F10 syngeneic mice
model.
[0073] FIG. 11 is line graph showing the anti-tumor activity of
compound Ex. 14 in the colon carcinoma B16F10 syngeneic mice
model.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The present disclosure is explained in detail below.
[0075] The definition of each substituent used in the present
specification is described in detail in the following. Unless
otherwise specified, each substituent has the following
definition.
[0076] In the present specification, examples of the "halogen atom"
include fluorine, chlorine, bromine and iodine.
[0077] In the present specification, examples of the "C.sub.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.
[0078] In the present specification, examples of the "optionally
halogenated C.sub.1-6 alkyl group" include a C.sub.1-6 alkyl group
optionally having 1 to 7, preferably 1 to 5, halogen atoms.
Specific examples thereof include methyl, chloromethyl,
difluoromethyl, trichloromethyl, trifluoromethyl, ethyl,
2-bromoethyl, 2,2,2-trifluoroethyl, tetrafluoroethyl,
pentafluoroethyl, propyl, 2,2-difluoropropyl,
3,3,3-trifluoropropyl, isopropyl, butyl, 4,4,4-trifluorobutyl,
isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl,
5,5,5-trifluoropentyl, hexyl and 6,6,6-trifluorohexyl.
[0079] In the present specification, examples of the "C.sub.2-6
alkenyl group" include ethenyl, 1-propenyl, 2-propenyl,
2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,
3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,
4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl and 5-hexenyl.
[0080] In the present specification, examples of the "C.sub.2-6
alkynyl group" include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,
2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,
4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl
and 4-methyl-2-pentynyl.
[0081] In the present specification, examples of the "C.sub.3-10
cycloalkyl group" include cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl,
bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl and adamantyl.
[0082] In the present specification, examples of the "optionally
halogenated C.sub.3-10 cycloalkyl group" include a C.sub.3-10
cycloalkyl group optionally having 1 to 7, preferably 1 to 5,
halogen atoms. Specific examples thereof include cyclopropyl,
2,2-difluorocyclopropyl, 2,3-difluorocyclopropyl, cyclobutyl,
difluorocyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and
cyclooctyl.
[0083] In the present specification, examples of the "C.sub.3-10
cycloalkenyl group" include cyclopropenyl, cyclobutenyl,
cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl.
[0084] In the present specification, examples of the "C.sub.6-14
aryl group" include phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl,
2-anthryl and 9-anthryl.
[0085] In the present specification, examples of the "C.sub.7-16
aralkyl group" include benzyl, phenethyl, naphthylmethyl and
phenylpropyl.
[0086] In the present specification, examples of the "C.sub.1-6
alkoxy group" include methoxy, ethoxy, propoxy, isopropoxy, butoxy,
isobutoxy, sec-butoxy, tert-butoxy, pentyloxy and hexyloxy.
[0087] In the present specification, examples of the "optionally
halogenated C.sub.1-6 alkoxy group" include a C.sub.1-6 alkoxy
group optionally having 1 to 7, preferably 1 to 5, halogen atoms.
Specific examples thereof include methoxy, difluoromethoxy,
trifluoromethoxy, ethoxy, 2,2,2-trifluoroethoxy, propoxy,
isopropoxy, butoxy, 4,4,4-trifluorobutoxy, isobutoxy, sec-butoxy,
pentyloxy and hexyloxy.
[0088] In the present specification, examples of the "C.sub.3-10
cycloalkyloxy group" include cyclopropyloxy, cyclobutyloxy,
cyclopentyloxy, cyclohexyloxy, cycloheptyloxy and
cyclooctyloxy.
[0089] In the present specification, examples of the "C.sub.1-6
alkylthio group" include methylthio, ethylthio, propylthio,
isopropylthio, butylthio, sec-butylthio, tert-butylthio, pentylthio
and hexylthio.
[0090] In the present specification, examples of the "optionally
halogenated C.sub.1-6 alkylthio group" include a C.sub.1-6
alkylthio group optionally having 1 to 7, preferably 1 to 5,
halogen atoms. Specific examples thereof include methylthio,
difluoromethylthio, trifluoromethylthio, ethylthio, propylthio,
isopropylthio, butylthio, 4,4,4-trifluorobutylthio, pentylthio and
hexylthio.
[0091] In the present specification, examples of the "C.sub.1-6
alkyl-carbonyl group" include acetyl, propanoyl, butanoyl,
2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl,
2,2-dimethylpropanoyl, hexanoyl and heptanoyl.
[0092] In the present specification, examples of the "optionally
halogenated C.sub.1-6 alkyl-carbonyl group" include a C.sub.1-6
alkyl-carbonyl group optionally having 1 to 7, preferably 1 to 5,
halogen atoms. Specific examples thereof include acetyl,
chloroacetyl, trifluoroacetyl, trichloroacetyl, propanoyl,
butanoyl, pentanoyl and hexanoyl.
[0093] In the present specification, examples of the "C.sub.1-6
alkoxy-carbonyl group" include methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,
isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl,
pentyloxycarbonyl and hexyloxycarbonyl.
[0094] In the present specification, examples of the "C.sub.6-14
aryl-carbonyl group" include benzoyl, 1-naphthoyl and
2-naphthoyl.
[0095] In the present specification, examples of the "C.sub.7-16
aralkyl-carbonyl group" include phenylacetyl and
phenylpropionyl.
[0096] In the present specification, examples of the "5- to
14-membered aromatic heterocyclylcarbonyl group" include
nicotinoyl, isonicotinoyl, thenoyl and furoyl.
[0097] In the present specification, examples of the "3- to
14-membered non-aromatic heterocyclylcarbonyl group" include
morpholinylcarbonyl, piperidinylcarbonyl and
pyrrolidinylcarbonyl.
[0098] In the present specification, examples of the "mono- or
di-C.sub.1-6 alkyl-carbamoyl group" include methylcarbamoyl,
ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl and
N-ethyl-N-methylcarbamoyl.
[0099] In the present specification, examples of the "mono- or
di-C.sub.7-16 aralkyl-carbamoyl group" include benzylcarbamoyl and
phenethylcarbamoyl.
[0100] In the present specification, examples of the "C.sub.1-6
alkylsulfonyl group" include methylsulfonyl, ethylsulfonyl,
propylsulfonyl, isopropylsulfonyl, butylsulfonyl, sec-butylsulfonyl
and tert-butylsulfonyl.
[0101] In the present specification, examples of the "optionally
halogenated C.sub.1-6 alkylsulfonyl group" include a C.sub.1-6
alkylsulfonyl group optionally having 1 to 7, preferably 1 to 5,
halogen atoms. Specific examples thereof include methylsulfonyl,
difluoromethylsulfonyl, trifluoromethylsulfonyl, ethylsulfonyl,
propylsulfonyl, isopropylsulfonyl, butylsulfonyl,
4,4,4-trifluorobutylsulfonyl, pentylsulfonyl and hexylsulfonyl.
[0102] In the present specification, examples of the "C.sub.6-14
arylsulfonyl group" include phenylsulfonyl, 1-naphthylsulfonyl and
2-naphthylsulfonyl.
[0103] In the present specification, examples of the "substituent"
include a halogen atom, a cyano group, a nitro group, an optionally
substituted hydrocarbon group, an optionally substituted
heterocyclic group, an acyl group, an optionally substituted amino
group, an optionally substituted carbamoyl group, an optionally
substituted thiocarbamoyl group, an optionally substituted
sulfamoyl group, an optionally substituted hydroxy group, an
optionally substituted sulfanyl (SH) group and an optionally
substituted silyl group.
[0104] In the present specification, examples of the "hydrocarbon
group" (including "hydrocarbon group" of "optionally substituted
hydrocarbon group") include a C.sub.1-6 alkyl group, a C.sub.2-6
alkenyl group, a C.sub.2-6 alkynyl group, a C.sub.3-10 cycloalkyl
group, a C.sub.3-10 cycloalkenyl group, a C.sub.6-14 aryl group and
a C.sub.7-16 aralkyl group.
[0105] In the present specification, examples of the "optionally
substituted hydrocarbon group" include a hydrocarbon group
optionally having substituent(s) selected from the following
Substituent group A.
"Substituent group A:"
[0106] (1) a halogen atom,
[0107] (2) a nitro group,
[0108] (3) a cyano group,
[0109] (4) an oxo group,
[0110] (5) a hydroxy group,
[0111] (6) an optionally halogenated C.sub.1-6 alkoxy group,
[0112] (7) a C.sub.6-14 aryloxy group (e.g., phenoxy,
naphthoxy),
[0113] (8) a C.sub.7-16 aralkyloxy group (e.g., benzyloxy),
[0114] (9) a 5- to 14-membered aromatic heterocyclyloxy group
(e.g., pyridyloxy),
[0115] (10) a 3- to 14-membered non-aromatic heterocyclyloxy group
(e.g., morpholinyloxy, piperidinyloxy),
[0116] (11) a C.sub.1-6 alkyl-carbonyloxy group (e.g., acetoxy,
propanoyloxy),
[0117] (12) a C.sub.6-14 aryl-carbonyloxy group (e.g., benzoyloxy,
1-naphthoyloxy, 2-naphthoyloxy),
[0118] (13) a C.sub.1-6 alkoxy-carbonyloxy group (e.g.,
methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy,
butoxycarbonyloxy),
[0119] (14) a mono- or di-C.sub.1-6 alkyl-carbamoyloxy group (e.g.,
methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy,
diethylcarbamoyloxy),
[0120] (15) a C.sub.6-14 aryl-carbamoyloxy group (e.g.,
phenylcarbamoyloxy, naphthylcarbamoyloxy),
[0121] (16) a 5- to 14-membered aromatic heterocyclylcarbonyloxy
group (e.g., nicotinoyloxy),
[0122] (17) a 3- to 14-membered non-aromatic
heterocyclylcarbonyloxy group (e.g., morpholinylcarbonyloxy,
piperidinylcarbonyloxy),
[0123] (18) an optionally halogenated C.sub.1-6 alkylsulfonyloxy
group (e.g., methylsulfonyloxy, trifluoromethylsulfonyloxy),
[0124] (19) a C.sub.6-14 arylsulfonyloxy group optionally
substituted by a C.sub.1-6 alkyl group (e.g., phenylsulfonyloxy,
toluenesulfonyloxy),
[0125] (20) an optionally halogenated C.sub.1-6 alkylthio
group,
[0126] (21) a 5- to 14-membered aromatic heterocyclic group,
[0127] (22) a 3- to 14-membered non-aromatic heterocyclic
group,
[0128] (23) a formyl group,
[0129] (24) a carboxy group,
[0130] (25) an optionally halogenated C.sub.1-6 alkyl-carbonyl
group,
[0131] (26) a C.sub.6-14 aryl-carbonyl group,
[0132] (27) a 5- to 14-membered aromatic heterocyclylcarbonyl
group,
[0133] (28) a 3- to 14-membered non-aromatic heterocyclylcarbonyl
group,
[0134] (29) a C.sub.1-6 alkoxy-carbonyl group,
[0135] (30) a C.sub.6-14 aryloxy-carbonyl group (e.g.,
phenyloxycarbonyl, 1-naphthyloxycarbonyl,
2-naphthyloxycarbonyl),
[0136] (31) a C.sub.7-16 aralkyloxy-carbonyl group (e.g.,
benzyloxycarbonyl, phenethyloxycarbonyl),
[0137] (32) a carbamoyl group,
[0138] (33) a thiocarbamoyl group,
[0139] (34) a mono- or di-C.sub.1-6 alkyl-carbamoyl group,
[0140] (35) a C.sub.6-14 aryl-carbamoyl group (e.g.,
phenylcarbamoyl),
[0141] (36) a 5- to 14-membered aromatic heterocyclylcarbamoyl
group (e.g., pyridylcarbamoyl, thienylcarbamoyl),
[0142] (37) a 3- to 14-membered non-aromatic heterocyclylcarbamoyl
group (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl),
[0143] (38) an optionally halogenated C.sub.1-6 alkylsulfonyl
group,
[0144] (39) a C.sub.6-14 arylsulfonyl group,
[0145] (40) a 5- to 14-membered aromatic heterocyclylsulfonyl group
(e.g., pyridylsulfonyl, thienylsulfonyl),
[0146] (41) an optionally halogenated C.sub.1-6 alkylsulfinyl
group,
[0147] (42) a C.sub.6-14 arylsulfinyl group (e.g., phenylsulfinyl,
1-naphthylsulfinyl, 2-naphthylsulfinyl),
[0148] (43) a 5- to 14-membered aromatic heterocyclylsulfinyl group
(e.g., pyridylsulfinyl, thienylsulfinyl),
[0149] (44) an amino group,
[0150] (45) a mono- or di-C.sub.1-6 alkylamino group (e.g.,
methylamino, ethylamino, propylamino, isopropylamino, butylamino,
dimethylamino, diethylamino, dipropylamino, dibutylamino,
N-ethyl-N-methylamino),
[0151] (46) a mono- or di-C.sub.6-14 arylamino group (e.g.,
phenylamino),
[0152] (47) a 5- to 14-membered aromatic heterocyclylamino group
(e.g., pyridylamino),
[0153] (48) a C.sub.7-16 aralkylamino group (e.g.,
benzylamino),
[0154] (49) a formylamino group,
[0155] (50) a C.sub.1-6 alkyl-carbonylamino group (e.g.,
acetylamino, propanoylamino, butanoylamino),
[0156] (51) a (C.sub.1-6 alkyl)(C.sub.1-6 alkyl-carbonyl) amino
group (e.g., N-acetyl-N-methylamino),
[0157] (52) a C.sub.6-14 aryl-carbonylamino group (e.g.,
phenylcarbonylamino, naphthylcarbonylamino),
[0158] (53) a C.sub.1-6 alkoxy-carbonylamino group (e.g.,
methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino,
butoxycarbonylamino, tert-butoxycarbonylamino),
[0159] (54) a C.sub.7-16 aralkyloxy-carbonylamino group (e.g.,
benzyloxycarbonylamino),
[0160] (55) a C.sub.1-6 alkylsulfonylamino group (e.g.,
methylsulfonylamino, ethyl sulfonylamino),
[0161] (56) a C.sub.6-14 arylsulfonylamino group optionally
substituted by a C.sub.1-6 alkyl group (e.g., phenylsulfonylamino,
toluenesulfonylamino),
[0162] (57) an optionally halogenated C.sub.1-6 alkyl group,
[0163] (58) a C.sub.2-6 alkenyl group,
[0164] (59) a C.sub.2-6 alkynyl group,
[0165] (60) a C.sub.3-10 cycloalkyl group,
[0166] (61) a C.sub.3-10 cycloalkenyl group, and
[0167] (62) a C.sub.6-14 aryl group.
[0168] The number of the above-mentioned substituents in the
"optionally substituted hydrocarbon group" is, for example, 1 to 5,
preferably 1 to 3. When the number of the substituents is two or
more, the respective substituents may be the same or different.
[0169] In the present specification, examples of the "heterocyclic
group" (including "heterocyclic group" of "optionally substituted
heterocyclic group") include (i) an aromatic heterocyclic group,
(ii) a non-aromatic heterocyclic group and (iii) a 7- to
10-membered bridged heterocyclic group, each containing, as a
ring-constituting atom besides carbon atom, 1 to 4 heteroatoms
selected from a nitrogen atom, a sulfur atom and an oxygen
atom.
[0170] In the present specification, examples of the "aromatic
heterocyclic group" (including "5- to 14-membered aromatic
heterocyclic group") include a 5- to 14-membered (preferably 5- to
10-membered) aromatic heterocyclic group containing, as a
ring-constituting atom besides carbon atom, 1 to 4 heteroatoms
selected from a nitrogen atom, a sulfur atom and an oxygen
atom.
[0171] Preferable examples of the "aromatic heterocyclic group"
include 5- or 6-membered monocyclic aromatic heterocyclic groups
such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl,
isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl,
pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl,
1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl,
triazinyl and the like; and
[0172] 8- to 14-membered fused polycyclic (preferably bi or
tricyclic) aromatic heterocyclic groups such as benzothiophenyl,
benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,
benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl,
thienopyridinyl, furopyridinyl, pyrrolopyridinyl,
pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl,
imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl,
furopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl,
oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl,
naphtho[2,3-b]thienyl, phenoxathiinyl, indolyl, isoindolyl,
1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl,
naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, phenazinyl,
phenothiazinyl, phenoxazinyl and the like.
[0173] In the present specification, examples of the "non-aromatic
heterocyclic group" (including "3- to 14-membered non-aromatic
heterocyclic group") include a 3- to 14-membered (preferably 4- to
10-membered) non-aromatic heterocyclic group containing, as a
ring-constituting atom besides carbon atom, 1 to 4 heteroatoms
selected from a nitrogen atom, a sulfur atom and an oxygen
atom.
[0174] Preferable examples of the "non-aromatic heterocyclic group"
include 3- to 8-membered monocyclic non-aromatic heterocyclic
groups such as aziridinyl, oxiranyl, thiiranyl, azetidinyl,
oxetanyl, thietanyl, tetrahydrothienyl, tetrahydrofuranyl,
pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl,
oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl,
thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl,
tetrahydroisooxazolyl, piperidinyl, piperazinyl,
tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl,
tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl,
tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl,
thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanyl,
azocanyl, diazocanyl and the like; and
[0175] 9- to 14-membered fused polycyclic (preferably bi or
tricyclic) non-aromatic heterocyclic groups such as
dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl,
dihydrobenzothiazolyl, dihydrobenzisothiazolyl,
dihydronaphtho[2,3-b]thienyl, tetrahydroisoquinolyl,
tetrahydroquinolyl, 4H-quinolizinyl, indolinyl, isoindolinyl,
tetrahydrothieno[2,3-c]pyridinyl, tetrahydrobenzazepinyl,
tetrahydroquinoxalinyl, tetrahydrophenanthridinyl,
hexahydrophenothiazinyl, hexahydrophenoxazinyl,
tetrahydrophthalazinyl, tetrahydronaphthyridinyl,
tetrahydroquinazolinyl, tetrahydrocinnolinyl, tetrahydrocarbazolyl,
tetrahydro-1-carbolinyl, tetrahydroacrydinyl, tetrahydrophenazinyl,
tetrahydrothioxanthenyl, octahydroisoquinolyl and the like.
[0176] In the present specification, preferable examples of the "7-
to 10-membered bridged heterocyclic group" include quinuclidinyl
and 7-azabicyclo[2.2.1]heptanyl.
[0177] In the present specification, examples of the
"nitrogen-containing heterocyclic group" include a "heterocyclic
group" containing at least one nitrogen atom as a ring-constituting
atom.
[0178] In the present specification, examples of the "optionally
substituted heterocyclic group" include a heterocyclic group
optionally having substituent(s) selected from the above-mentioned
"Substituent group A.
[0179] The number of the substituents in the "optionally
substituted heterocyclic group" is, for example, 1 to 3. When the
number of the substituents is two or more, the respective
substituents may be the same or different.
[0180] In the present specification, examples of the "acyl group"
include a formyl group, a carboxy group, a carbamoyl group, a
thiocarbamoyl group, a sulfino group, a sulfo group, a sulfamoyl
group and a phosphono group, each optionally having "1 or 2
substituents selected from a C.sub.1-6 alkyl group, a C.sub.2-6
alkenyl group, a C.sub.3-10 cycloalkyl group, a C.sub.3-10
cycloalkenyl group, a C.sub.6-14 aryl group, a C.sub.7-16 aralkyl
group, a 5- to 14-membered aromatic heterocyclic group and a 3- to
14-membered non-aromatic heterocyclic group, each of which
optionally has 1 to 3 substituents selected from a halogen atom, an
optionally halogenated C.sub.1-6 alkoxy group, a hydroxy group, a
nitro group, a cyano group, an amino group and a carbamoyl
group".
[0181] Examples of the "acyl group" also include a
hydrocarbon-sulfonyl group, a heterocyclylsulfonyl group, a
hydrocarbon-sulfinyl group and a heterocyclylsulfinyl group.
[0182] Here, the hydrocarbon-sulfonyl group means a hydrocarbon
group-bonded sulfonyl group, the heterocyclylsulfonyl group means a
heterocyclic group-bonded sulfonyl group, the hydrocarbon-sulfinyl
group means a hydrocarbon group-bonded sulfinyl group and the
heterocyclylsulfinyl group means a heterocyclic group-bonded
sulfinyl group.
[0183] Preferable examples of the "acyl group" include a formyl
group, a carboxy group, a C.sub.1-6 alkyl-carbonyl group, a
C.sub.2-6 alkenyl-carbonyl group (e.g., crotonoyl), a C.sub.3-10
cycloalkyl-carbonyl group (e.g., cyclobutanecarbonyl,
cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl), a
C.sub.3-10 cycloalkenyl-carbonyl group (e.g.,
2-cyclohexenecarbonyl), a C.sub.6-14 aryl-carbonyl group, a
C.sub.7-16 aralkyl-carbonyl group, a 5- to 14-membered aromatic
heterocyclylcarbonyl group, a 3- to 14-membered non-aromatic
heterocyclylcarbonyl group, a C.sub.1-6 alkoxy-carbonyl group, a
C.sub.6-14 aryloxy-carbonyl group (e.g., phenyloxycarbonyl,
naphthyloxycarbonyl), a C.sub.7-16 aralkyloxy-carbonyl group (e.g.,
benzyloxycarbonyl, phenethyloxycarbonyl), a carbamoyl group, a
mono- or di-C.sub.1-6 alkyl-carbamoyl group, a mono- or
di-C.sub.2-6 alkenyl-carbamoyl group (e.g., diallylcarbamoyl), a
mono- or di-C.sub.3-10 cycloalkyl-carbamoyl group (e.g.,
cyclopropylcarbamoyl), a mono- or di-C.sub.6-14 aryl-carbamoyl
group (e.g., phenylcarbamoyl), a mono- or di-C.sub.7-16
aralkyl-carbamoyl group, a 5- to 14-membered aromatic
heterocyclylcarbamoyl group (e.g., pyridylcarbamoyl), a
thiocarbamoyl group, a mono- or di-C.sub.1-6 alkyl-thiocarbamoyl
group (e.g., methylthiocarbamoyl, N-ethyl-N-methylthiocarbamoyl), a
mono- or di-C.sub.2-6 alkenyl-thiocarbamoyl group (e.g.,
diallylthiocarbamoyl), a mono- or di-C.sub.3-10
cycloalkyl-thiocarbamoyl group (e.g., cyclopropylthiocarbamoyl,
cyclohexylthiocarbamoyl), a mono- or di-C.sub.6-14
aryl-thiocarbamoyl group (e.g., phenylthiocarbamoyl), a mono- or
di-C.sub.7-16 aralkyl-thiocarbamoyl group (e.g.,
benzylthiocarbamoyl, phenethylthiocarbamoyl), a 5- to 14-membered
aromatic heterocyclylthiocarbamoyl group (e.g.,
pyridylthiocarbamoyl), a sulfino group, a C.sub.1-6 alkylsulfinyl
group (e.g., methylsulfinyl, ethylsulfinyl), a sulfo group, a
C.sub.1-6 alkylsulfonyl group, a C.sub.6-14 arylsulfonyl group, a
phosphono group and a mono- or di-C.sub.1-6 alkylphosphono group
(e.g., dimethylphosphono, diethylphosphono, diisopropylphosphono,
dibutylphosphono).
[0184] In the present specification, examples of the "optionally
substituted amino group" include an amino group optionally having
"1 or 2 substituents selected from a C.sub.1-6 alkyl group, a
C.sub.2-6 alkenyl group, a C.sub.3-10 cycloalkyl group, a
C.sub.6-14 aryl group, a C.sub.7-16 aralkyl group, a C.sub.1-6
alkyl-carbonyl group, a C.sub.6-14 aryl-carbonyl group, a
C.sub.7-16 aralkyl-carbonyl group, a 5- to 14-membered aromatic
heterocyclylcarbonyl group, a 3- to 14-membered non-aromatic
heterocyclylcarbonyl group, a C.sub.1-6 alkoxy-carbonyl group, a 5-
to 14-membered aromatic heterocyclic group, a carbamoyl group, a
mono- or di-C.sub.1-6 alkyl-carbamoyl group, a mono- or
di-C.sub.7-16 aralkyl-carbamoyl group, a C.sub.1-6 alkylsulfonyl
group and a C.sub.6-14 arylsulfonyl group, each of which optionally
has 1 to 3 substituents selected from "Substituent group A".
[0185] Preferable examples of the optionally substituted amino
group include an amino group, a mono- or di-(optionally halogenated
C.sub.1-6 alkyl) amino group (e.g., methylamino,
trifluoromethylamino, dimethylamino, ethylamino, diethylamino,
propylamino, dibutylamino), a mono- or di-C.sub.2-6 alkenylamino
group (e.g., diallylamino), a mono- or di-C.sub.3-10
cycloalkylamino group (e.g., cyclopropylamino, cyclohexylamino), a
mono- or di-C.sub.6-14 arylamino group (e.g., phenylamino), a mono-
or di-C.sub.7-16 aralkylamino group (e.g., benzylamino,
dibenzylamino), a mono- or di-(optionally halogenated C.sub.1-6
alkyl)-carbonylamino group (e.g., acetylamino, propionylamino), a
mono- or di-C.sub.6-14 aryl-carbonylamino group (e.g.,
benzoylamino), a mono- or di-C.sub.7-16 aralkyl-carbonylamino group
(e.g., benzylcarbonylamino), a mono- or di-5- to 14-membered
aromatic heterocyclylcarbonylamino group (e.g., nicotinoylamino,
isonicotinoylamino), a mono- or di-3- to 14-membered non-aromatic
heterocyclylcarbonylamino group (e.g., piperidinylcarbonylamino), a
mono- or di-C.sub.1-6 alkoxy-carbonylamino group (e.g.,
tert-butoxycarbonylamino), a 5- to 14-membered aromatic
heterocyclylamino group (e.g., pyridylamino), a carbamoylamino
group, a (mono- or di-C.sub.7-16 alkyl-carbamoyl) amino group
(e.g., methylcarbamoylamino), a (mono- or di-C.sub.7-16
aralkyl-carbamoyl) amino group (e.g., benzylcarbamoylamino), a
C.sub.1-6 alkylsulfonylamino group (e.g., methylsulfonylamino,
ethylsulfonylamino), a C.sub.6-14 arylsulfonylamino group (e.g.,
phenylsulfonylamino), a (C.sub.1-6 alkyl)(C.sub.1-6 alkyl-carbonyl)
amino group (e.g., N-acetyl-N-methylamino) and a (C.sub.1-6
alkyl)(C.sub.6-14 aryl-carbonyl) amino group (e.g.,
N-benzoyl-N-methylamino).
[0186] In the present specification, examples of the "optionally
substituted carbamoyl group" include a carbamoyl group optionally
having "1 or 2 substituents selected from a C.sub.1-6 alkyl group,
a C.sub.2-6 alkenyl group, a C.sub.3-10 cycloalkyl group, a
C.sub.6-14 aryl group, a C.sub.7-16 aralkyl group, a C.sub.1-6
alkyl-carbonyl group, a C.sub.6-14 aryl-carbonyl group, a
C.sub.7-16 aralkyl-carbonyl group, a 5- to 14-membered aromatic
heterocyclylcarbonyl group, a 3- to 14-membered non-aromatic
heterocyclylcarbonyl group, a C.sub.1-6 alkoxy-carbonyl group, a 5-
to 14-membered aromatic heterocyclic group, a carbamoyl group, a
mono- or di-C.sub.1-6 alkyl-carbamoyl group and a mono- or
di-C.sub.7-16 aralkyl-carbamoyl group, each of which optionally has
1 to 3 substituents selected from "Substituent group A".
[0187] Preferable examples of the optionally substituted carbamoyl
group include a carbamoyl group, a mono- or di-C.sub.1-6
alkyl-carbamoyl group, a mono- or di-C.sub.2-6 alkenyl-carbamoyl
group (e.g., diallylcarbamoyl), a mono- or di-C.sub.3-10
cycloalkyl-carbamoyl group (e.g., cyclopropylcarbamoyl,
cyclohexylcarbamoyl), a mono- or di-C.sub.6-14 aryl-carbamoyl group
(e.g., phenylcarbamoyl), a mono- or di-C.sub.7-16 aralkyl-carbamoyl
group, a mono- or di-C.sub.1-6 alkyl-carbonyl-carbamoyl group
(e.g., acetylcarbamoyl, propionylcarbamoyl), a mono- or
di-C.sub.6-14 aryl-carbonyl-carbamoyl group (e.g.,
benzoylcarbamoyl) and a 5- to 14-membered aromatic
heterocyclylcarbamoyl group (e.g., pyridylcarbamoyl).
[0188] In the present specification, examples of the "optionally
substituted thiocarbamoyl group" include a thiocarbamoyl group
optionally having "1 or 2 substituents selected from a C.sub.1-6
alkyl group, a C.sub.2-6 alkenyl group, a C.sub.3-10 cycloalkyl
group, a C.sub.6-14 aryl group, a C.sub.7-16 aralkyl group, a
C.sub.1-6 alkyl-carbonyl group, a C.sub.6-14 aryl-carbonyl group, a
C.sub.7-16 aralkyl-carbonyl group, a 5- to 14-membered aromatic
heterocyclylcarbonyl group, a 3- to 14-membered non-aromatic
heterocyclylcarbonyl group, a C.sub.1-6 alkoxy-carbonyl group, a 5-
to 14-membered aromatic heterocyclic group, a carbamoyl group, a
mono- or di-C.sub.1-6 alkyl-carbamoyl group and a mono- or
di-C.sub.7-16 aralkyl-carbamoyl group, each of which optionally has
1 to 3 substituents selected from "Substituent group A".
[0189] Preferable examples of the optionally substituted
thiocarbamoyl group include a thiocarbamoyl group, a mono- or
di-C.sub.1-6 alkyl-thiocarbamoyl group (e.g., methylthiocarbamoyl,
ethylthiocarbamoyl, dimethylthiocarbamoyl, diethylthiocarbamoyl,
N-ethyl-N-methylthiocarbamoyl), a mono- or di-C.sub.2-6
alkenyl-thiocarbamoyl group (e.g., diallylthiocarbamoyl), a mono-
or di-C.sub.3-10 cycloalkyl-thiocarbamoyl group (e.g.,
cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), a mono- or
di-C.sub.6-14 aryl-thiocarbamoyl group (e.g., phenylthiocarbamoyl),
a mono- or di-C.sub.1-6 aralkyl-thiocarbamoyl group (e.g.,
benzylthiocarbamoyl, phenethylthiocarbamoyl), a mono- or
di-C.sub.1-6 alkyl-carbonyl-thiocarbamoyl group (e.g.,
acetylthiocarbamoyl, propionylthiocarbamoyl), a mono- or
di-C.sub.6-14 aryl-carbonyl-thiocarbamoyl group (e.g.,
benzoylthiocarbamoyl) and a 5- to 14-membered aromatic
heterocyclylthiocarbamoyl group (e.g., pyridylthiocarbamoyl).
[0190] In the present specification, examples of the "optionally
substituted sulfamoyl group" include a sulfamoyl group optionally
having "1 or 2 substituents selected from a C.sub.1-6 alkyl group,
a C.sub.2-6 alkenyl group, a C.sub.3-10 cycloalkyl group, a
C.sub.6-14 aryl group, a C.sub.7-16 aralkyl group, a C.sub.1-6
alkyl-carbonyl group, a C.sub.6-14 aryl-carbonyl group, a
C.sub.7-16 aralkyl-carbonyl group, a 5- to 14-membered aromatic
heterocyclylcarbonyl group, a 3- to 14-membered non-aromatic
heterocyclylcarbonyl group, a C.sub.1-6 alkoxy-carbonyl group, a 5-
to 14-membered aromatic heterocyclic group, a carbamoyl group, a
mono- or di-C.sub.1-6 alkyl-carbamoyl group and a mono- or
di-C.sub.7-16 aralkyl-carbamoyl group, each of which optionally has
1 to 3 substituents selected from "Substituent group A".
[0191] Preferable examples of the optionally substituted sulfamoyl
group include a sulfamoyl group, a mono- or di-C.sub.1-6
alkyl-sulfamoyl group (e.g., methylsulfamoyl, ethylsulfamoyl,
dimethylsulfamoyl, diethylsulfamoyl, N-ethyl-N-methylsulfamoyl), a
mono- or di-C.sub.2-6 alkenyl-sulfamoyl group (e.g.,
diallylsulfamoyl), a mono- or di-C.sub.3-10 cycloalkyl-sulfamoyl
group (e.g., cyclopropylsulfamoyl, cyclohexylsulfamoyl), a mono- or
di-C.sub.6-14 aryl-sulfamoyl group (e.g., phenylsulfamoyl), a mono-
or di-C.sub.7-16 aralkyl-sulfamoyl group (e.g., benzylsulfamoyl,
phenethylsulfamoyl), a mono- or di-C.sub.1-6
alkyl-carbonyl-sulfamoyl group (e.g., acetylsulfamoyl,
propionylsulfamoyl), a mono- or di-C.sub.6-14
aryl-carbonyl-sulfamoyl group (e.g., benzoylsulfamoyl) and a 5- to
14-membered aromatic heterocyclylsulfamoyl group (e.g.,
pyridylsulfamoyl).
[0192] In the present specification, examples of the "optionally
substituted hydroxy group" include a hydroxy group optionally
having "a substituent selected from a C.sub.1-6 alkyl group, a
C.sub.2-6 alkenyl group, a C.sub.3-10 cycloalkyl group, a
C.sub.6-14 aryl group, a C.sub.7-16 aralkyl group, a C.sub.1-6
alkyl-carbonyl group, a C.sub.6-14 aryl-carbonyl group, a
C.sub.7-16 aralkyl-carbonyl group, a 5- to 14-membered aromatic
heterocyclylcarbonyl group, a 3- to 14-membered non-aromatic
heterocyclylcarbonyl group, a C.sub.1-6 alkoxy-carbonyl group, a 5-
to 14-membered aromatic heterocyclic group, a carbamoyl group, a
mono- or di-C.sub.1-6 alkyl-carbamoyl group, a mono- or
di-C.sub.1-16 aralkyl-carbamoyl group, a C.sub.1-6 alkylsulfonyl
group and a C.sub.6-14 arylsulfonyl group, each of which optionally
has 1 to 3 substituents selected from "Substituent group A".
[0193] Preferable examples of the optionally substituted hydroxy
group include a hydroxy group, a C.sub.1-6 alkoxy group, a
C.sub.2-6 alkenyloxy group (e.g., allyloxy, 2-butenyloxy,
2-pentenyloxy, 3-hexenyloxy), a C.sub.3-10 cycloalkyloxy group
(e.g., cyclohexyloxy), a C.sub.6-14 aryloxy group (e.g., phenoxy,
naphthyloxy), a C.sub.7-16 aralkyloxy group (e.g., benzyloxy,
phenethyloxy), a C.sub.1-6 alkyl-carbonyloxy group (e.g.,
acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, pivaloyloxy), a
C.sub.6-14 aryl-carbonyloxy group (e.g., benzoyloxy), a C.sub.7-16
aralkyl-carbonyloxy group (e.g., benzylcarbonyloxy), a 5- to
14-membered aromatic heterocyclylcarbonyloxy group (e.g.,
nicotinoyloxy), a 3- to 14-membered non-aromatic
heterocyclylcarbonyloxy group (e.g., piperidinylcarbonyloxy), a
C.sub.1-6 alkoxy-carbonyloxy group (e.g., tert-butoxycarbonyloxy),
a 5- to 14-membered aromatic heterocyclyloxy group (e.g.,
pyridyloxy), a carbamoyloxy group, a C.sub.1-6 alkyl-carbamoyloxy
group (e.g., methylcarbamoyloxy), a C.sub.7-16 aralkyl-carbamoyloxy
group (e.g., benzylcarbamoyloxy), a C.sub.1-6 alkylsulfonyloxy
group (e.g., methylsulfonyloxy, ethylsulfonyloxy) and a C.sub.6-14
arylsulfonyloxy group (e.g., phenylsulfonyloxy).
[0194] In the present specification, examples of the "optionally
substituted sulfanyl group" include a sulfanyl group optionally
having "a substituent selected from a C.sub.1-6 alkyl group, a
C.sub.2-6 alkenyl group, a C.sub.3-10 cycloalkyl group, a
C.sub.6-14 aryl group, a C.sub.7-16 aralkyl group, a C.sub.1-6
alkyl-carbonyl group, a C.sub.6-14 aryl-carbonyl group and a 5- to
14-membered aromatic heterocyclic group, each of which optionally
has 1 to 3 substituents selected from "Substituent group A" and a
halogenated sulfanyl group.
[0195] Preferable examples of the optionally substituted sulfanyl
group include a sulfanyl (--SH) group, a C.sub.1-6 alkylthio group,
a C.sub.2-6 alkenylthio group (e.g., allylthio, 2-butenylthio,
2-pentenylthio, 3-hexenylthio), a C.sub.3-10 cycloalkylthio group
(e.g., cyclohexylthio), a C.sub.6-14 arylthio group (e.g.,
phenylthio, naphthylthio), a C.sub.7-16 aralkylthio group (e.g.,
benzylthio, phenethylthio), a C.sub.1-6 alkyl-carbonylthio group
(e.g., acetylthio, propionylthio, butyrylthio, isobutyrylthio,
pivaloylthio), a C.sub.6-14 aryl-carbonylthio group (e.g.,
benzoylthio), a 5- to 14-membered aromatic heterocyclylthio group
(e.g., pyridylthio) and a halogenated thio group (e.g.,
pentafluorothio).
[0196] In the present specification, examples of the "optionally
substituted silyl group" include a silyl group optionally having "1
to 3 substituents selected from a C.sub.1-6 alkyl group, a
C.sub.2-6 alkenyl group, a C.sub.3-10 cycloalkyl group, a
C.sub.6-14 aryl group and a C.sub.7-16 aralkyl group, each of which
optionally has 1 to 3 substituents selected from "Substituent group
A". Examples of the optionally substituted silyl group include a
tri-C.sub.1-6 alkylsilyl group (e.g., trimethylsilyl,
tert-butyl(dimethyl)silyl).
[0197] The term "antibody-drug conjugate" or "ADC" as used herein
refers to a compound that is linked to an antibody and is defined
by the formula (CD-L).sub.n-A, wherein CD is a group represented by
any one of Formula (XX)-(XXIX), see below, L is a linker, A is a
protein, e.g., an antibody, and n is 1-10. In one embodiment, the
linker is represented by --X.sup.3-T-Z-Q-, wherein X.sup.3 is a
divalent radical that connects CD to the rest of linker, or is
absent, T is a peptide, or is absent, Z is a spacer, and Q is
heterobifunctional group or a heterotrifunctional group. By way of
illustration, the following generic formula shows an ADC of the
disclosure having a para-aminobenzyl-based connector, an
alanine-alanine-based dipeptide, a propanone-based spacer, and a
succinimide thioether-based heterobifunctional group:
##STR00027##
[0198] In the present disclosure, the term "linker" refers to any
chemical moiety capable of linking a protein, e.g., antibody,
antibody fragment (e.g., antigen binding fragments) or functional
equivalent to a cyclic dinucleotide. Linkers may be susceptible to
cleavage (a "cleavable linker") thereby facilitating release of the
cyclic dinucleotide. For example, such cleavable linkers may be
susceptible to acid-induced cleavage, photo-induced cleavage,
peptidase-induced cleavage, esterase-induced cleavage, and
disulfide bond cleavage, at conditions under which the cyclic
dinucleotide and/or the antibody remains active. Alternatively,
linkers may be substantially resistant to cleavage (a "noncleavable
linker").
[0199] In the present disclosure, non-cleavable linkers are any
chemical moiety capable of linking a cyclic dinucleotide to an
antibody in a stable, covalent manner and does not fall off under
the categories listed above for cleavable linkers. Thus,
non-cleavable linkers are substantially resistant to acid-induced
cleavage, photo-induced cleavage, peptidase-induced cleavage,
esterase-induced cleavage and disulfide bond cleavage. Furthermore,
non-cleavable refers to the ability of the chemical bond in the
linker or adjoining to the linker to withstand cleavage induced by
an acid, photolabile-cleaving agent, a peptidase, an esterase, or a
chemical or physiological compound that cleaves a disulfide bond,
at conditions under which a cyclic dinucleotide and/or the antibody
does not lose its activity.
[0200] Some cleavable linkers are cleaved by peptidases ("peptidase
cleavable linkers"). Only certain peptides are readily cleaved
inside or outside cells, see e.g. Trout et al., 79 Proc. Natl.
Acad. Sci. USA, 626-629 (1982) and Umemoto et al. 43 Int. J.
Cancer, 677-684 (1989). Furthermore, peptides are composed of
.alpha.-amino acid units and peptidic bonds, which chemically are
amide bonds between the carboxylate of one amino acid and the amino
group of a second amino acid. Other amide bonds, such as the bond
between a carboxylate and the .alpha.-amino acid group of lysine,
are understood not to be peptidic bonds and are considered
non-cleavable.
[0201] Some linkers are cleaved by esterases ("esterase cleavable
linkers"). Only certain esters can be cleaved by esterases present
inside or outside of cells. Esters are formed by the condensation
of a carboxylic acid and an alcohol. Simple esters are esters
produced with simple alcohols, such as aliphatic alcohols, and
small cyclic and small aromatic alcohols.
[0202] In some embodiments, the cleavable linker component
comprises a peptide comprising one to ten amino acid residues. In
these embodiments, the peptide allows for cleavage of the linker by
a protease, thereby facilitating release of the cyclic dinucleotide
upon exposure to intracellular proteases, such as lysosomal enzymes
(Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Exemplary
peptides include, but are not limited to, dipeptides, tripeptides,
tetrapeptides, and pentapeptides. Exemplary dipeptides include, but
are not limited to, alanine-alanine (ala-ala), valine-citrulline
(vc or val-cit), alanine-phenylalanine (af or ala-phe);
phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine
(phe-homolys); and N-methyl-valine-citrulline (Me-val-cit).
Exemplary tripeptides include, but are not limited to,
glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine
(gly-gly-gly).
[0203] A peptide may comprise naturally-occurring and/or
non-natural amino acid residues. The term "naturally-occurring
amino acid" refer to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys,
Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr.
"Non-natural amino acids" (i.e., amino acids do not occur
naturally) include, by way of non-limiting example, homoserine,
homoarginine, citrulline, phenylglycine, taurine, iodotyrosine,
seleno-cysteine, norleucine ("Nle"), norvaline ("Nva"),
beta-alanine, L- or D-naphthalanine, ornithine ("Orn"), and the
like. Peptides can be designed and optimized for enzymatic cleavage
by a particular enzyme, for example, a tumor-associated protease,
cathepsin B, C and D, or a plasmin protease.
[0204] Amino acids also include the D-forms of natural and
non-natural amino acids. "D-" designates an amino acid having the
"D" (dextrorotary) configuration, as opposed to the configuration
in the naturally occurring ("L-") amino acids. Natural and
non-natural amino acids can be purchased commercially (Sigma
Chemical Co., Advanced Chemtech) or synthesized using methods known
in the art.
[0205] In the present disclosure, the term "heterobifunctional
group" or the term "heterotrifunctional group" refers to a chemical
moiety that connects a linker and another therapeutically active
molecule, e.g., protein, e.g., an antibody. See, e.g., WO
2017/191579. Heterobi- and tri-functional groups are characterized
as having different reactive groups at either end of the chemical
moiety. Non-limiting exemplary heterobifunctional groups
include:
##STR00028##
wherein the "*" indicates the attachment point to any available
carbon atom, nitrogen atom, oxygen atom, or sulfur atom attached to
the antibody. In embodiment, the heterobifunctional group is
##STR00029##
and is attached to a sulfur atom attached to the antibody.
[0206] A non-limiting exemplary heterotrifunctional group is:
##STR00030##
[0207] In the present disclosure, the term "spacer" refers to
chemical moiety that connects a heterobi- and tri-functional group
to the rest of the linker, e.g., a peptide, or, if a heterobi- or
tri-functional group is absent, connects the rest of the linker or
the cyclic dinucleotide to any available carbon atom, nitrogen
atom, oxygen atom, or sulfur atom attached to the antibody.
Non-limiting exemplary spacers include --NH--, --S--, --O--,
--NHC(.dbd.O)CH.sub.2CH.sub.2--,
--S(.dbd.O).sub.2--CH.sub.2CH.sub.2--, --C(.dbd.O)NHNH,
--C(.dbd.O)O--, --C(.dbd.O)NH--, --CH.sub.2--,
--CH.sub.2CH.sub.2--, --CH.sub.2CH.sub.2CH.sub.2--,
--CH.sub.2.dbd.CH.sub.2--, --C.ident.C--, --CH.dbd.N--O--,
polyethylene glycol (PEG),
##STR00031##
[0208] The term "drug antibody ratio" or "DAR" refers to the number
of CDs linked to A (i.e., a protein, e.g., an antibody or
antigen-binding fragment thereof). Thus, in the ADC having the
generic formula (CD-L).sub.n-A, the DAR is defined by the variable
"n."
[0209] When referring to a compound having formula (CD-L).sub.n-A
representing an individual ADC, the DAR refers to the number of CDs
linked to the individual A, e.g., n is an integer of 1 to 10.
[0210] When referring to a compound having formula (CD-L).sub.n-A
representing a plurality of ADCs, the DAR refers to the average
number of cyclic dinucleotides linked to the As, e.g., n is an
integer or fraction of 1 to 10). Thus, by way of an example, a
compound having formula (CD-L).sub.n-A comprising a first ADC with
3 cyclic dinucleotides per A and a second ADC with 4 cyclic
dinucleotides per A would have a DAR, i.e., an "n," of 3.5
[0211] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer can be linear or branched, it can comprise
modified amino acids, and it can be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified naturally or by intervention, for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included within the definition are, for
example, polypeptides containing one or more analogs of an amino
acid (including, for example, unnatural amino acids, etc.), as well
as other modifications known in the art. It is understood that,
because the polypeptides of this disclosure are based upon
antibodies, in certain embodiments, the polypeptides can occur as
single chains or associated chains.
[0212] A "monoclonal" antibody or antigen-binding fragment thereof
refers to a homogeneous antibody or antigen-binding fragment
population involved in the highly specific recognition and binding
of a single antigenic determinant, or epitope. This is in contrast
to polyclonal antibodies that typically include different
antibodies directed against different antigenic determinants. The
term "monoclonal" antibody or antigen-binding fragment thereof
encompasses both intact and full-length monoclonal antibodies as
well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single
chain (scFv) mutants, fusion proteins comprising an antibody
portion, and any other modified immunoglobulin molecule comprising
an antigen recognition site. Furthermore, "monoclonal" antibody or
antigen-binding fragment thereof refers to such antibodies and
antigen-binding fragments thereof made in any number of manners
including but not limited to by hybridoma, phage selection,
recombinant expression, and transgenic animals.
[0213] The term "humanized" antibody or antigen-binding fragment
thereof refers to forms of non-human (e.g. murine) antibodies or
antigen-binding fragments that are specific immunoglobulin chains,
chimeric immunoglobulins, or fragments thereof that contain minimal
non-human (e.g., murine) sequences. Typically, humanized antibodies
or antigen-binding fragments thereof are human immunoglobulins in
which residues from the complementary determining region (CDR) are
replaced by residues from the CDR of a non-human species (e.g.
mouse, rat, rabbit, hamster) that have the desired specificity,
affinity, and capability ("CDR grafted") (Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988);
Verhoeyen et al., Science 239:1534-1536 (1988)). In some instances,
the Fv framework region (FR) residues of a human immunoglobulin are
replaced with the corresponding residues in an antibody or fragment
from a non-human species that has the desired specificity,
affinity, and capability. The humanized antibody or antigen-binding
fragment thereof can be further modified by the substitution of
additional residues either in the Fv framework region and/or within
the replaced non-human residues to refine and optimize antibody or
antigen-binding fragment thereof specificity, affinity, and/or
capability. In general, the humanized antibody or antigen-binding
fragment thereof will comprise substantially all of at least one,
and typically two or three, variable domains containing all or
substantially all of the CDR regions that correspond to the
non-human immunoglobulin whereas all or substantially all of the FR
regions are those of a human immunoglobulin consensus sequence. The
humanized antibody or antigen-binding fragment thereof can also
comprise at least a portion of an immunoglobulin constant region or
domain (Fc), typically that of a human immunoglobulin. Examples of
methods used to generate humanized antibodies are described in U.S.
Pat. No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA,
91(3):969-973 (1994), and Roguska et al., Protein Eng.
9(10):895-904 (1996). In some embodiments, a "humanized antibody"
is a resurfaced antibody.
[0214] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. The variable
regions of the heavy and light chain each consist of four framework
regions (FR) connected by three complementarity determining regions
(CDRs) also known as hypervariable regions. The CDRs in each chain
are held together in close proximity by the FRs and, with the CDRs
from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al. Sequences of
Proteins of Immunological Interest, (5th ed., 1991, National
Institutes of Health, Bethesda Md.)); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Al-lazikani
et al (1997) J. Molec. Biol. 273:927-948)). In addition,
combinations of these two approaches are sometimes used in the art
to determine CDRs.
[0215] The Kabat numbering system is generally used when referring
to a residue in the variable domain (approximately residues 1-107
of the light chain and residues 1-113 of the heavy chain) (e.g.,
Kabat et al., Sequences of Immunological Interest. 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)). Unless explicitly indicated otherwise, the numbering
system used herein is the Kabat numbering system.
[0216] The amino acid position numbering as in Kabat, refers to the
numbering system used for heavy chain variable domains or light
chain variable domains of the compilation of antibodies in Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991). Using this numbering system, the actual linear amino acid
sequence can contain fewer or additional amino acids corresponding
to a shortening of, or insertion into, a FR or CDR of the variable
domain. For example, a heavy chain variable domain can include a
single amino acid insert (residue 52a according to Kabat) after
residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and
82c, etc. according to Kabat) after heavy chain FR residue 82. The
Kabat numbering of residues can be determined for a given antibody
by alignment at regions of homology of the sequence of the antibody
with a "standard" Kabat numbered sequence. Chothia refers instead
to the location of the structural loops (Chothia and Lesk J. Mol.
Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when
numbered using the Kabat numbering convention varies between H32
and H34 depending on the length of the loop (this is because the
Kabat numbering scheme places the insertions at H35A and H35B; if
neither 35A nor 35B is present, the loop ends at 32; if only 35A is
present, the loop ends at 33; if both 35A and 35B are present, the
loop ends at 34). The AbM hypervariable regions represent a
compromise between the Kabat CDRs and Chothia structural loops, and
are used by Oxford Molecular's AbM antibody modeling software.
TABLE-US-00001 Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2
L50-L56 L50-L56 L50-56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B
H26-H35B H26-H32_34 (Kabat Numbering) H1 H31-35 H26-H35 H26-32
(Chothia Numbering) H2 H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102
H95-H102
[0217] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
Chothia numbering scheme, which refers to the location of
immunoglobulin structural loops (see, e.g., Chothia C & Lesk A
M, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J
Mol Biol 273: 927-948; Chothia C et al., (1992) J Mol Biol 227:
799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and
U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering
convention, the Chothia CDR-H1 loop is present at heavy chain amino
acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at
heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is
present at heavy chain amino acids 95 to 102, while the Chothia
CDR-L1 loop is present at light chain amino acids 24 to 34, the
Chothia CDR-L2 loop is present at light chain amino acids 50 to 56,
and the Chothia CDR-L3 loop is present at light chain amino acids
89 to 97. The end of the Chothia CDR-H1 loop when numbered using
the Kabat numbering convention varies between H32 and H34 depending
on the length of the loop (this is because the Kabat numbering
scheme places the insertions at H35A and H35B; if neither 35A nor
35B is present, the loop ends at 32; if only 35A is present, the
loop ends at 33; if both 35A and 35B are present, the loop ends at
34).
[0218] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
IMGT numbering system as described in Lefranc M-P, (1999) The
Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic
Acids Res 27: 209-212. According to the IMGT numbering scheme,
VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57,
VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to
32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions
89 to 97.
[0219] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to
MacCallum R M et al., (1996) J Mol Biol 262: 732-745. See also,
e.g., Martin A. "Protein Sequence and Structure Analysis of
Antibody Variable Domains," in Antibody Engineering, Kontermann and
Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin
(2001).
[0220] In certain aspects, the CDRs of an antibody or
antigen-binding fragment thereof can be determined according to the
AbM numbering scheme, which refers AbM hypervariable regions which
represent a compromise between the Kabat CDRs and Chothia
structural loops, and are used by Oxford Molecular's AbM antibody
modeling software (Oxford Molecular Group, Inc.).
[0221] The term "human" antibody means an antibody produced by a
human or an antibody having an amino acid sequence corresponding to
an antibody produced by a human made using any technique known in
the art. This definition of a human antibody includes intact or
full-length antibodies, fragments thereof, and/or antibodies
comprising at least one human heavy and/or light chain polypeptide
such as, for example, an antibody comprising murine light chain and
human heavy chain polypeptides.
[0222] The term "chimeric" antibodies refers to antibodies wherein
the amino acid sequence of the immunoglobulin molecule is derived
from two or more species. Typically, the variable region of both
light and heavy chains corresponds to the variable region of
antibodies derived from one species of mammals (e.g. mouse, rat,
rabbit, etc.) with the desired specificity, affinity, and
capability while the constant regions are homologous to the
sequences in antibodies derived from another (usually human) to
avoid eliciting an immune response in that species.
[0223] The term "epitope" or "antigenic determinant" are used
interchangeably herein and refer to that portion of an antigen
capable of being recognized and specifically bound by a particular
antibody. When the antigen is a polypeptide, epitopes can be formed
both from contiguous amino acids and noncontiguous amino acids
juxtaposed by tertiary folding of a protein. Epitopes formed from
contiguous amino acids are typically retained upon protein
denaturing, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation.
[0224] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present disclosure. Specific illustrative
embodiments are described in the following.
[0225] "Or better" when used herein to refer to binding affinity
refers to a stronger binding between a molecule and its binding
partner. "Or better" when used herein refers to a stronger binding,
represented by a smaller numerical Kd value. For example, an
antibody which has an affinity for an antigen of "0.6 nM or
better", the antibody's affinity for the antigen is <0.6 nM,
i.e. 0.59 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6
nM.
[0226] By "specifically binds," it is generally meant that an
antibody binds to an epitope via its antigen binding domain, and
that the binding entails some complementarity between the antigen
binding domain and the epitope. According to this definition, an
antibody is said to "specifically bind" to an epitope when it binds
to that epitope, via its antigen binding domain more readily than
it would bind to a random, unrelated epitope. The term
"specificity" is used herein to qualify the relative affinity by
which a certain antibody binds to a certain epitope. For example,
antibody "A" may be deemed to have a higher specificity for a given
epitope than antibody "B," or antibody "A" may be said to bind to
epitope "C" with a higher specificity than it has for related
epitope "D."
[0227] By "preferentially binds," it is meant that the antibody
specifically binds to an epitope more readily than it would bind to
a related, similar, homologous, or analogous epitope. Thus, an
antibody which "preferentially binds" to a given epitope would more
likely bind to that epitope than to a related epitope, even though
such an antibody may cross-react with the related epitope.
[0228] An antibody is said to "competitively inhibit" binding of a
reference antibody to a given epitope if the antibody
preferentially binds to that epitope or an overlapping epitope to
the extent that it blocks, to some degree, binding of the reference
antibody to the epitope. Competitive inhibition may be determined
by any method known in the art, for example, competition ELISA
assays. An antibody may be said to competitively inhibit binding of
the reference antibody to a given epitope by at least 90%, at least
80%, at least 70%, at least 60%, or at least 50%.
[0229] The phrase "substantially similar," or "substantially the
same", as used herein, denotes a sufficiently high degree of
similarity between two numeric values (generally one associated
with an antibody of the disclosure and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values can be
less than about 50%, less than about 40%, less than about 30%, less
than about 20%, or less than about 10% as a function of the value
for the reference/comparator antibody.
[0230] A polypeptide, antibody, polynucleotide, vector, cell, or
composition which is "isolated" is a polypeptide, antibody,
polynucleotide, vector, cell, or composition which is in a form not
found in nature. Isolated polypeptides, antibodies,
polynucleotides, vectors, cell or compositions include those which
have been purified to a degree that they are no longer in a form in
which they are found in nature. In some embodiments, an antibody,
polynucleotide, vector, cell, or composition which is isolated is
substantially pure.
[0231] As used herein, "substantially pure" refers to material
which is at least 50%/o pure (i.e., free from contaminants), at
least 90% pure, at least 95% pure, at least 98% pure, or at least
99% pure.
[0232] The term "antibody" refers an immunoglobulin molecule that
recognizes and specifically binds to a target, such as a protein,
polypeptide, peptide, carbohydrate, polynucleotide, lipid, or
combinations of the foregoing through at least one antigen
recognition site within the variable region of the immunoglobulin
molecule. As used herein, the term "antibody" encompasses intact
polyclonal antibodies, intact monoclonal antibodies, chimeric
antibodies, humanized antibodies, human antibodies, fusion proteins
comprising an antibody, and any other modified immunoglobulin
molecule so long as the antibodies exhibit the desired biological
activity. An antibody can be of any the five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses
(isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2),
based on the identity of their heavy-chain constant domains
referred to as alpha, delta, epsilon, gamma, and mu, respectively.
The different classes of immunoglobulins have different and well
known subunit structures and three-dimensional configurations.
Antibodies can be naked or conjugated to other molecules such as
toxins, radioisotopes, etc. As used herein, the term "antibody"
encompasses bispecific and multispecific antibodies.
[0233] The term "antibody fragment" refers to a portion of an
intact antibody. An "antigen-binding fragment" refers to a portion
of an intact antibody that binds to an antigen. An antigen-binding
fragment can contain the antigenic determining variable regions of
an intact antibody. Examples of antibody fragments include, but are
not limited to Fab, Fab', F(ab')2, and Fv fragments, linear
antibodies, and single chain antibodies. An "antigen-binding
fragment" can be a bispecific or multispecific antigen-binding
fragment.
[0234] The term "drug delivery agent" as used herein refers to a
chemical moiety that alters the distribution, targeting, or
lifetime of the molecule into which it is incorporated. In some
embodiments a drug delivery agent provides an enhanced affinity for
a selected target, e.g., molecule, cell or cell type, compartment,
receptor e.g., a cellular or organ compartment, tissue, organ or
region of the body, as compared to a species absent such a drug
delivery agent. Drug delivery agents providing enhanced affinity
for a selected target are termed targeting drug delivery agents.
See, e.g., WO 2013/075035 A1. All patents, patent applications, and
publications cited herein are fully incorporated by reference in
their entirety.
[0235] The term "drug delivery conjugate" as used herein refers to
a compound comprising any one of Formula (XX)-(XXIX), a drug
delivery agent and, optionally, a covalent linker.
[0236] Some drug delivery agents have endosomolytic properties.
Endosomolytic drug delivery agents promote the lysis of the
endosome and/or transport of any one of Formula (XX)-(XXIX), from
the endosome to the cytoplasm of the cell. The endosomolytic drug
delivery agent may be a polyanionic peptide or peptidomimetic which
shows pH-dependent membrane activity and fusogenicity. In one
embodiment, the endosomolytic drug delivery agent assumes its
active conformation at endosomal pH. The "active" conformation is
that conformation in which the endosomolytic drug delivery agent
promotes lysis of the endosome and/or transport of Formula
(XX)-(XXIX) from the endosome to the cytoplasm of the cell.
Exemplary endosomolytic drug delivery agents include the GALA
peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the
EALA peptide (Vogel et al., J. Am. Chem. Soc, 1996, 118:
1581-1586), and their derivatives (Turk et al., Biochem. Biophys.
Acta, 2002, 1559: 56-68). In one embodiment, the endosomolytic
component may contain a chemical group. e.g., an amino acid, which
will undergo a change in charge or protonation in response to a
change in pH. The endosomolytic component may be linear or
branched.
[0237] Drug delivery agents can improve transport, hybridization,
and specificity properties of the resultant drug delivery
conjugate.
[0238] Drug delivery agents can include therapeutic modifiers,
e.g., for enhancing uptake; diagnostic compounds or reporter groups
e.g., for monitoring distribution; and cross-linking agents.
General examples include lipids, steroids, vitamins, carbohydrates,
proteins, peptides, polyamines, synthetic polymers or oligomers
(such as PEG's), peptidomimetics, or any combinations thereof.
[0239] Drug delivery agents can include a naturally occurring
substance, such as a protein, e.g., human serum albumin (HSA),
low-density lipoprotein (LDL), high-density lipoprotein (HDL), or
globulin, a carbohydrate, e.g., a dextran, pullulan, chitin,
chitosan, inulin, cyclodextrin or hyaluronic acid; or a lipid. The
drug delivery agent may also be a recombinant or synthetic
molecule, such as a synthetic polymer or oligomer, e.g., a
synthetic polyamino acid, an oligonucleotide, e.g., an aptamer.
Examples of polyamino acids include a polylysine (PLL), poly
L-aspartic acid, poly L-glutamic acid. Other examples of synthetic
polymers or oligomers include styrene-maleic acid anhydride
copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl
ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide
copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol
(PVA), polyurethane, poly(2-ethylacrylic acid),
N-isopropylacrylamide polymers, or polyphosphazine. Example of
polyamines include: polyethylenimine, polylysine (PLL), spermine,
spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic
polyamine, dendrimer polyamine, arginine, amidine, protamine,
cationic lipid, cationic porphyrin, quaternary salt of a polyamine,
or an alpha helical peptide.
[0240] Drug delivery agents can also include targeting groups,
e.g., a cell or tissue targeting agent, e.g., a lectin,
glycoprotein, lipid or protein, e.g., an antibody, that binds to a
specified cell type. A targeting group can be a thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin
carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent
galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,
vitamin B 12, biotin, an RGD peptide, an RGD peptide mimetic or an
aptamer.
[0241] Other examples of drug delivery agents include dyes,
intercalating agents (e.g., acridines), cross-linkers (e.g.,
psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin),
polycyclic aromatic hydrocarbons (e.g., phenazine,
dihydrophenazine), artificial endonucleases or a chelator (e.g.,
EDTA), lipophilic molecules, e.g., cholesterol, cholic acid,
adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-0(hexadecyl)glycerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid,
03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and
peptide conjugates (e.g., antennapedia peptide, Tat peptide),
alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),
MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled
markers, enzymes, haptens (e.g., biotin), transport/absorption
facilitators (e.g., aspirin, vitamin E, folic acid), synthetic
ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole
clusters, acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, or AP.
[0242] Drug delivery agents may also include hormones and hormone
receptors. They can also include non-peptidic species, such as
lipids, lectins, carbohydrates, vitamins, cofactors, multivalent
lactose, multivalent galactose, N-acetyl-galactosamine,
N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or
aptamers. The drug delivery agent can be, for example, a
lipopolysaccharide, an activator of p38 MAP kinase, or an activator
of NF-KB.
[0243] The drug delivery agent can be a substance, which can
increase the uptake of any one of Formula (XX)-(XXIX) into the
cell, for example, by disrupting the cell's cytoskeleton, e.g., by
disrupting the cell's microtubules, microfilaments, and/or
intermediate filaments.
[0244] The drug delivery agent can increase the uptake of any one
of Formula (XX)-(XXIX) into the cell by, for example, activating an
inflammatory response. Exemplary drug delivery agents that would
have such an effect include tumor necrosis factor alpha (TNFalpha),
interleukin-1 beta, or gamma interferon.
[0245] In one embodiment, the drug delivery agent is a lipid or
lipid-based molecule. Such a lipid or lipid-based molecule can bind
a serum protein, e.g., human serum albumin (HSA). An HSA binding
drug delivery agent allows for distribution of the drug delivery
conjugate to a target tissue, e.g., a non-kidney target tissue of
the body. Other molecules that can bind HSA can also be used as
drug delivery agents. For example, naproxen or aspirin can be used.
A lipid or lipid-based drug delivery agent can (a) increase
resistance to degradation of the drug delivery conjugate, (b)
increase targeting or transport into a target cell or cell
membrane, and/or (c) can be used to adjust binding to a serum
protein, e.g., HSA. A lipid or lipid-based drug delivery agent can
also enable the formation of nanoparticles, such as micelles, which
can impact the biodistribution of any one of Formula
(XX)-(XXIX).
[0246] A lipid based drug delivery agent can be used to modulate,
e.g., control the binding of the drug delivery conjugate to a
target tissue. For example, a lipid or lipid-based drug delivery
agent that binds to HSA more strongly will be less likely to be
targeted to the kidney and therefore less likely to be cleared from
the body. A lipid or lipid-based drug delivery agent that binds to
HSA less strongly can be used to target the conjugate to the
kidney.
[0247] In one embodiment, the lipid based drug delivery agent binds
HSA with a sufficient affinity such that the drug delivery
conjugate is distributed to a non-kidney tissue. In one embodiment,
the affinity is not so strong that the HSA-drug delivery agent
binding cannot be reversed.
[0248] In another embodiment, the lipid based drug delivery agent
binds HSA weakly or not at all, such that the drug delivery
conjugate is distributed to the kidney. Other moieties that target
to kidney cells can also be used in place of or in addition to the
lipid based drug delivery agent.
[0249] In another embodiment, the drug delivery agent is a moiety,
e.g., a vitamin, which is taken up by a target cell, e.g., a
proliferating cell. These are particularly useful for treating
disorders characterized by unwanted cell proliferation, e.g., of
the malignant or non-malignant type, e.g., cancer cells. Exemplary
vitamins include vitamin A, E, and K. Other exemplary vitamins
include B vitamins, e.g., folic acid, B 12, riboflavin, biotin,
pyridoxal or other vitamins or nutrients taken up by cancer cells.
Also included are HAS, low density lipoprotein (LDL) and
high-density lipoprotein (HDL).
[0250] In another embodiment, the drug delivery agent is a
cell-permeation agent, e.g., a helical cell-permeation agent. In
one embodiment, the agent is amphipathic. Exemplary amphipathic
agents include a peptide such as tat or antennopedia, and a
lipid-PEG conjugate. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. In one
embodiment, the helical agent is an alpha-helical agent having a
lipophilic and a lipophobic phase.
[0251] The drug delivery agent can be a peptide or peptidomimetic.
A peptidomimetic (also referred to herein as an
oligopeptidomimetic) is a molecule capable of folding into a
defined three-dimensional structure similar to a natural peptide.
The peptide or peptidomimetic moiety can be about 5-50 amino acids
long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino
acids long. A peptide or peptidomimetic can be, for example, a cell
permeation peptide, cationic peptide, amphipathic peptide, or
hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or
Phe). The peptide moiety can be a dendrimer peptide, constrained
peptide or crosslinked peptide. In another embodiment, the peptide
moiety can include a hydrophobic membrane translocation sequence
(MTS). An exemplary hydrophobic MTS-containing peptide is RFGF. An
RFGF analogue containing a hydrophobic MTS can also be a targeting
moiety. The peptide moiety can be a "delivery" peptide, which can
carry large polar molecules including peptides, oligonucleotides,
and protein across cell membranes. For example, certain sequences
from the HIV Tat protein and the Drosophila Antennapedia have been
found to be capable of functioning as delivery peptides. A peptide
or peptidomimetic can be encoded by a random sequence of DNA, such
as a peptide identified from a phage-display library, or
one-bead-one-compound (OBOC) combinatorial library (Lam et ah,
Nature, 354:82-84, 1991). In one embodiment, the peptide or
peptidomimetic is a cell targeting peptide such as an
arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A
peptide moiety can range in length from about 5 amino acids to
about 40 amino acids. The peptide moieties can have a structural
modification, such as to increase stability or direct
conformational properties. Any of the structural modifications
described below can be utilized. An RGD peptide moiety can be used
to target a tumor cell, such as an endothelial tumor cell or a
breast cancer tumor cell (Zitzmann et ah, Cancer Res., 62:5139-43,
2002). An RGD peptide can facilitate targeting of any one of
Formula (XX)-(XXIX) to tumors of a variety of other tissues,
including the lung, kidney, spleen, or liver (Aoki et ah, Cancer
Gene Therapy 8:783-787, 2001).
[0252] In some embodiments, the RGD peptide will facilitate
targeting of a drug delivery conjugate to the kidney. The RGD
peptide can be linear or cyclic, and can be modified, e.g.,
glycosylated or methylated to facilitate targeting to specific
tissues. For example, a glycosylated RGD peptide can deliver a drug
delivery conjugate to a tumor cell expressing civB3 (Haubner et ah,
Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers
enriched in proliferating cells can be used. For example, RGD
containing peptides and peptidomimetic s can target cancer cells,
in particular cells that exhibit an integrin. Thus, one could use
RGD peptides, cyclic peptides containing RGD, RGD peptides that
include D-amino acids, as well as synthetic RGD mimics. In addition
to RGD, one can use other moieties that target the integrin drug
delivery agent. Generally, such drug delivery agents can be used to
control proliferating cells and angiogenesis. Preferred conjugates
of this type of drug delivery agent target PECAM-1, VEGF, or other
cancer gene, e.g., a cancer gene described herein.
[0253] A "cell permeation peptide" is capable of permeating a cell,
e.g., a mammalian cell, such as a human cell. A cell permeation
peptide can include a RALA peptide. (Molecular Therapy: Nucleic
Acids, 2017, 6, 249-258; J Control Release. 2014, 189:141-9; and
Nanomedicine (Lond). 2015, 10(19): 2989-3001). A cell permeation
peptide can also include a nuclear localization signal (NLS). For
example, a cell permeation peptide can be a bipartite amphipathic
peptide, such as MPG, which is derived from the fusion peptide
domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni
et al, Nucl. Acids Res. 31:2717-2724, 2003). A cell permeation
peptide may form a nano particle.
[0254] In one embodiment, a targeting peptide can be an amphipathic
a-helical peptide. Exemplary amphipathic a-helical peptides
include, but are not limited to, cecropins, lycotoxins, paradaxins,
buforin, CPF, bombinin-like peptide (BLP), cathelicidins,
ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial
peptides (HFIAPs), magainines, brevinins-2, dermaseptins,
melittins, pleurocidin, H2A peptides, Xenopus peptides,
esculentinis-1, and caerins. A number of factors will preferably be
considered to maintain the integrity of helix stability. For
example, a maximum number of helix stabilization residues will be
utilized (e.g., leu, ala, or lys), and a minimum number helix
destabilization residues will be utilized (e.g., proline, or cyclic
monomeric units. The capping residue will be considered (for
example Gly is an exemplary N-capping residue and/or C-terminal
amidation can be used to provide an extra H-bond to stabilize the
helix. Formation of salt bridges between residues with opposite
charges, separated by i+3, or i+4 positions can provide stability.
For example, cationic residues such as lysine, arginine,
homoarginine, omithine or histidine can form salt bridges with the
anionic residues glutamate or aspartate.
[0255] Peptide and peptidomimetic drug delivery agents include
those having naturally occurring or modified peptides, e.g., D or L
peptides; a, 3, or y peptides; N-methyl peptides; azapeptides;
peptides having one or more amide, i.e., peptide, linkages replaced
with one or more urea, thiourea, carbamate, or sulfonyl urea
linkages; or cyclic peptides.
[0256] The targeting drug delivery agent can be any drug delivery
agent that is capable of targeting a specific receptor. Examples
are: folate, GalNAc, galactose, mannose, mannose-6P, clusters of
sugars such as GalNAc cluster, mannose cluster, galactose cluster,
or an aptamer. A cluster is a combination of two or more sugar
units. The targeting drug delivery agents also include integrin
receptor drug delivery agents, Chemokine receptor drug delivery
agents, transferrin, biotin, serotonin receptor drug delivery
agents, PSMA, endothelin, GCPII, somatostatin, LDL and HDL drug
delivery agents. The drug delivery agents can also be based on
nucleic acid, e.g., an aptamer. The aptamer can be unmodified or
have any combination of modifications disclosed herein.
[0257] Endosomal release agents include imidazoles, poly or
oligoimidazoles, PEIs, peptides, fusogenic peptides,
polycaboxylates, polyacations, masked oligo or poly cations or
anions, acetals, polyacetals, ketals/polyketyals, orthoesters,
polymers with masked or unmasked cationic or anionic charges,
dendrimers with masked or unmasked cationic or anionic charges.
[0258] Drug delivery agent can be a PK modulator (pharmacokinetic
modulator). PK modulators include lipophiles, bile acids, steroids,
phospholipid analogues, peptides, protein binding agents, PEG,
vitamins etc. Exemplary PK modulators include, but are not limited
to, cholesterol, fatty acids, cholic acid, lithocholic acid,
dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,
naproxen, ibuprofen, vitamin E, biotin etc.
[0259] Oligonucleotides that comprise a number of phosphorothioate
linkages are also known to bind to serum protein, thus short
oligonucleotides, e.g., oligonucleotides of about 5 bases, 10
bases, 15 bases or 20 bases, comprising multiple phosphorothioate
linkages in the backbone are also amenable to the present invention
as drug delivery agents (e.g., as PK modulating drug delivery
agents).
[0260] In addition, aptamers that bind serum components (e.g.,
serum proteins) are also amenable to the present disclosure as PK
modulating drug delivery agents.
[0261] When the drug delivery agent comprises a combination of two
or more moieties, e.g., a lipid and a carbohydrate, e.g., a lipid
and a peptide, the two or more moieties can all have the same
properties, all have different properties, or some moieties have
the same properties while others have different properties. For
example, a drug delivery agent can have targeting properties,
endosomolytic activity, and/or PK modulating properties. For
example, one moiety of a drug delivery agent can be hydrophilic,
and another can be hydrophobic. In some embodiments, the moieties
have different properties.
[0262] Drug delivery agents and linkers are described in U.S. Pat.
Nos. 7,745,608; 7,626,014; 8,034,921; US 2005/0164235; and WO
2013/075035. All patents, patent applications, and publications
cited herein are fully incorporated by reference in their
entirety.
[0263] The definition of each symbol in the formula (I) is
explained in detail in the following embodiments.
[0264] In one embodiment, the partial structure:
##STR00032##
is a partial structure represented by formula (IIA):
##STR00033##
a partial structure represented by formula (IIB):
##STR00034##
[0265] In another embodiment, the partial structure:
##STR00035##
is a partial structure represented by the formula (IIA):
##STR00036##
[0266] In another embodiment, the partial structure represented by
the formula (IIA):
##STR00037##
is a partial structure represented by the formula (IIAa):
##STR00038##
[0267] In another embodiment, the partial structure represented by
the formula (IIA):
##STR00039##
is a partial structure represented by the formula (IIAb):
##STR00040##
[0268] In another embodiment, the partial structure represented by
the formula (IIB):
##STR00041##
is a partial structure represented by the formula (IIBa):
##STR00042##
[0269] In another embodiment, the partial structure represented by
the formula (IIB):
##STR00043##
is a partial structure represented by the formula (IIBb):
##STR00044##
[0270] In another embodiment, the partial structure:
##STR00045##
[0271] In another embodiment, the partial structure:
##STR00046##
[0272] In another embodiment, R.sup.1 and R.sup.2 are each
independently a hydroxy group or a halogen atom.
[0273] In another embodiment, R.sup.1 and R.sup.2 are each
independently a hydroxy group or a fluorine atom.
[0274] In another embodiment, R.sup.1 is a hydroxy group.
[0275] In another embodiment, R.sup.2 is a hydroxy group or a
fluorine atom.
[0276] In another embodiment B.sup.1 is a group represented by
##STR00047##
[0277] In another embodiment, R.sup.13, R.sup.14, R.sup.15,
R.sup.16 and R.sup.17 are each independently a hydrogen atom or a
substituent.
[0278] In another embodiment, Y.sup.11, Y.sup.12, Y.sup.13,
Y.sup.14, Y.sup.15 and Y.sup.16 are each independently N or
CR.sup.1a.
[0279] In another embodiment, Z.sup.11, Z.sup.12, Z.sup.13,
Z.sup.14, Z.sup.15 and Z.sup.16 are each independently N or C.
[0280] In another embodiment, R.sup.1a is a hydrogen atom or a
substituent.
[0281] In another embodiment, R.sup.13 is a hydrogen atom.
[0282] In another embodiment, R.sup.14 is a hydrogen atom or an
optionally substituted amino group,
[0283] In another embodiment, R.sup.14 is a hydrogen atom or an
amino group.
[0284] In another embodiment, R.sup.14 is a hydrogen atom.
[0285] In another embodiment, R.sup.15 is preferably a hydrogen
atom.
[0286] In another embodiment, R.sup.13 and R.sup.14 are both
hydrogen atoms, and R.sup.14 is a hydrogen atom or an optionally
substituted amino group (particularly a hydrogen atom or an amino
group).
[0287] In another embodiment, R.sup.13, R.sup.14 and R.sup.15 are
all hydrogen atoms.
[0288] In another embodiment, R.sup.16 is a hydrogen atom.
[0289] In another embodiment, R.sup.17 is a hydrogen atom.
[0290] In another embodiment, Y.sup.11 is N.
[0291] In another embodiment, Y.sup.12 is N or CH,
[0292] In another embodiment, Y.sup.12 is N.
[0293] In another embodiment, Y.sup.13 is N or CR.sup.1a wherein
R.sup.1a is a halogen atom (e.g., a fluorine atom).
[0294] In another embodiment, Y.sup.13 is N or CF.
[0295] In another embodiment, Y.sup.13 is CF.
[0296] In another embodiment, Y.sup.11 is N, Y.sup.12 is N or CH,
and Y.sup.13 is N or CR.sup.1a wherein R.sup.1a is a halogen atom
(e.g., a fluorine atom).
[0297] In another embodiment, Y.sup.11 is N, Y.sup.12 is N or CH,
and Y.sup.13 is N or CF.
[0298] In another embodiment, Y.sup.11 is N, Y.sup.12 is N, and
Y.sup.13 is CF, Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is N, or
Y.sup.11 is N, Y.sup.12 is CH, and Y.sup.13 is N.
[0299] In another embodiment, Y.sup.11 is N, Y.sup.12 is N, and
Y.sup.13 is CF.
[0300] In another embodiment, Y.sup.14 is N.
[0301] In another embodiment, Y.sup.15 is N.
[0302] In another embodiment, Y.sup.16 is N or CR.sup.1a wherein
R.sup.1a is a halogen atom (e.g., a fluorine atom).
[0303] In another embodiment, Y.sup.16 is N or CF.
[0304] In another embodiment, Y.sup.16 is N.
[0305] In another embodiment, Y.sup.14 is N, Y.sup.15 is N, and
Y.sup.16 is N or CR.sup.1a wherein R.sup.1a is a halogen atom
(e.g., a fluorine atom).
[0306] In another embodiment, Y.sup.14 is N, Y.sup.15 is N, and
Y.sup.16 is N or CF.
[0307] In another embodiment, Y.sup.14 is N, Y.sup.15 is N, and
Y.sup.16 is N, or Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is
CF.
[0308] In another embodiment, Y.sup.14 is N, Y.sup.15 is N, and
Y.sup.16 is N.
[0309] In another embodiment, Z.sup.11 is C.
[0310] In another embodiment, Z.sup.12 is C.
[0311] In another embodiment, Z.sup.13 is N.
[0312] In another embodiment, Z.sup.11 is C, Z.sup.12 is C, and
Z.sup.13 is N.
[0313] In another embodiment, Z.sup.14 is preferably C.
[0314] In another embodiment, Z.sup.15 is preferably C.
[0315] In another embodiment, Z.sup.16 is preferably N.
[0316] In another embodiment, Z.sup.14 is C, Z.sup.15 is C, and
Z.sup.16 is N.
[0317] In another embodiment, B.sup.2 is a group represented by
##STR00048##
[0318] In another embodiment, R.sup.23, R.sup.24, R.sup.25,
R.sup.26 and R.sup.27 are each independently a hydrogen atom or a
substituent.
[0319] In another embodiment, Y.sup.21, Y.sup.22, Y.sup.23,
Y.sup.24, Y.sup.25 and Y.sup.26 are each independently N or
CR.sup.2a.
[0320] In another embodiment, Z.sup.21, Z.sup.22, Z.sup.23,
Z.sup.24, Z.sup.25 and Z.sup.26 are each independently N or C.
[0321] In another embodiment, R.sup.2a is a hydrogen atom or a
substituent.
[0322] In another embodiment, R.sup.23 is a hydrogen atom.
[0323] In another embodiment, R.sup.24 is a hydrogen atom or an
optionally substituted amino group.
[0324] In another embodiment, R.sup.24 is a hydrogen atom or an
amino group.
[0325] In another embodiment, R.sup.24 is a hydrogen atom.
[0326] In another embodiment, R.sup.25 is a hydrogen atom.
[0327] In another embodiment, R.sup.26 is a hydrogen atom.
[0328] In another embodiment, R.sup.27 is a hydrogen atom.
[0329] In another embodiment, Y.sup.21 is N.
[0330] In another embodiment, Y.sup.22 is N.
[0331] In another embodiment, Y.sup.23 is N or CR.sup.2a wherein
R.sup.2a is a halogen atom (e.g., a fluorine atom).
[0332] In another embodiment, Y.sup.23 is N or CF.
[0333] In another embodiment, Y.sup.23 is CF.
[0334] In another embodiment, Y.sup.21 is N, Y.sup.22 is N, and
Y.sup.23 is N or CR.sup.2a wherein R.sup.2a is a halogen atom
(e.g., a fluorine atom).
[0335] In another embodiment, Y.sup.21 is N, Y.sup.22 is N, and
Y.sup.23 is N or CF.
[0336] In another embodiment, Y.sup.21 is N, Y.sup.22 is N, and
Y.sup.23 is CF, or Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is
N.
[0337] In another embodiment, Y.sup.21 is N, Y.sup.22 is N, and
Y.sup.23 is CF.
[0338] In another embodiment, Y.sup.24 is N.
[0339] In another embodiment, Y.sup.25 is N or CH.
[0340] In another embodiment, Y.sup.25 is N.
[0341] In another embodiment, Y.sup.26 is N or CR.sup.2a wherein
R.sup.2a is a halogen atom (e.g., a fluorine atom).
[0342] In another embodiment, Y.sup.26 is N or CF.
[0343] In another embodiment, Y.sup.26 is N.
[0344] In another embodiment, Y.sup.24 is N, Y.sup.25 is N or CH,
and Y.sup.26 is N or CR.sup.2a wherein R.sup.2a is a halogen atom
(e.g., a fluorine atom).
[0345] In another embodiment, Y.sup.24 is N, Y.sup.25 is N or CH,
and Y.sup.26 is N or CF.
[0346] In another embodiment, Y.sup.24 is N, Y.sup.25 is N, and
Y.sup.26 is N; Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is CF; or
Y.sup.24 is N, Y.sup.25 is CH, and Y.sup.26 is N.
[0347] In another embodiment, Y.sup.24 is N, Y.sup.25 is N, and
Y.sup.26 is N.
[0348] In another embodiment, Z.sup.21 is N or C
[0349] In another embodiment, Z.sup.21 is C.
[0350] In another embodiment, Z.sup.22 is C.
[0351] In another embodiment, Z.sup.23 is N or C.
[0352] In another embodiment, Z.sup.23 is N.
[0353] In another embodiment, Z.sup.21 is N or C, Z.sup.22 is C,
and Z.sup.23 is N or C.
[0354] In another embodiment, Z.sup.21 is C, Z.sup.22 is C, and
Z.sup.23 is N, or Z.sup.21 is N, Z.sup.22 is C, and Z.sup.23 is
C.
[0355] In another embodiment, Z.sup.21 is C, Z.sup.22 is C, and
Z.sup.23 is N.
[0356] In another embodiment, Z.sup.24 is N or C.
[0357] In another embodiment, Z.sup.24 is C.
[0358] In another embodiment, Z.sup.25 is C.
[0359] In another embodiment, Z.sup.26 is N or C.
[0360] In another embodiment, Z.sup.26 is N.
[0361] In another embodiment, Z.sup.24 is N or C, Z.sup.25 is C,
and Z.sup.26 is N or C.
[0362] In another embodiment, Z.sup.24 is C, Z.sup.25 is C, and
Z.sup.26 is N, or Z.sup.24 is N, Z.sup.25 is C, and Z.sup.26 is
C.
[0363] In another embodiment, Z.sup.24 is C, Z.sup.25 is C, and
Z.sup.26 is N.
[0364] In another embodiment, B.sup.1 and B.sup.2 are each
independently
##STR00049##
[0365] In another embodiment, at least one of B.sup.1 and B.sup.2
is
##STR00050##
and in this case, the other is
##STR00051##
[0366] In another embodiment, B.sup.1 is
##STR00052##
[0367] B.sup.2 is
##STR00053##
[0368] In another embodiment,
[0369] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0370] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0371] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C.
[0372] In another embodiment, X.sup.1 and X.sup.2 are each
independently an oxygen atom or a sulfur atom.
[0373] In another embodiment, X.sup.1 and X.sup.2 are both oxygen
atoms.
[0374] In another embodiment, Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4
are each independently an oxygen atom or a sulfur atom.
[0375] In another embodiment, Q.sup.1 is an oxygen atom.
[0376] In another embodiment, Q.sup.3 is an oxygen atom.
[0377] In another embodiment, Q.sup.1 and Q.sup.3 are both oxygen
atoms, and Q.sup.2 and Q.sup.4 are each independently an oxygen
atom or a sulfur atom.
[0378] In another embodiment, Q.sup.1 is an oxygen atom, Q.sup.2 is
an oxygen atom or a sulfur atom, Q.sup.3 is an oxygen atom, and
Q.sup.4 is an oxygen atom or a sulfur atom.
[0379] In another embodiment, Q.sup.1 is an oxygen atom, Q.sup.2 is
a sulfur atom, Q.sup.3 is an oxygen atom, and Q.sup.4 is a sulfur
atom.
[0380] In another embodiment, Compound (1) is a compound
represented by the formula (Ia):
##STR00054##
wherein the partial structure:
##STR00055##
is a partial structure represented by the formula (IIAa):
##STR00056##
or a partial structure represented by the formula (IIBa):
##STR00057##
or a salt thereof (hereinafter sometimes to be referred to as
compound (Ia)).
[0381] In another embodiment, Compound (I) is a compound
represented by the formula (Ib):
##STR00058##
wherein the partial structure:
##STR00059##
##STR00060##
or a partial structure represented by the formula (IIBb):
##STR00061##
or a salt thereof (hereinafter sometimes to be referred to as
compound (Ib)).
[0382] Examples of compound (I) include the following
compounds.
Compound A1
[0383] Compound A1 is a compound having formula (I), wherein the
partial structure:
##STR00062##
[0384] is a partial structure represented by the formula (IIA):
##STR00063##
[0385] R.sup.1 is a hydroxy group;
[0386] R.sup.2 is a hydroxy group or a halogen atom (e.g., a
fluorine atom);
[0387] B.sup.1 is a group represented by
##STR00064##
[0388] R.sup.13 is a hydrogen atom;
[0389] R.sup.14 is a hydrogen atom or an optionally substituted
amino group;
[0390] R.sup.15 is a hydrogen atom;
[0391] R.sup.16 is a hydrogen atom;
[0392] R.sup.17 is a hydrogen atom;
[0393] Y.sup.11 is N;
[0394] Y.sup.12 is N or CH;
[0395] Y.sup.13 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0396] Y.sup.14 is N;
[0397] Y.sup.15 is N;
[0398] Y.sup.16 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0399] Z.sup.11 is C;
[0400] Z.sup.12 is C;
[0401] Z.sup.13 is N;
[0402] Z.sup.14 is C;
[0403] Z.sup.15 is C;
[0404] Z.sup.16 is N;
[0405] B.sup.2 is a group represented by
##STR00065##
[0406] R.sup.23 is a hydrogen atom;
[0407] R.sup.24 is a hydrogen atom;
[0408] R.sup.25 is a hydrogen atom;
[0409] R.sup.26 is a hydrogen atom;
[0410] R.sup.27 is a hydrogen atom;
[0411] Y.sup.21 is N;
[0412] Y.sup.22 is N;
[0413] Y.sup.23 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0414] Y.sup.24 is N;
[0415] Y.sup.25 is N or CH;
[0416] Y.sup.26 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0417] Z.sup.21 is N or C;
[0418] Z.sup.22 is C;
[0419] Z.sup.23 is N or C;
[0420] Z.sup.24 is N or C;
[0421] Z.sup.25 is C;
[0422] Z.sup.26 is N or C;
provided that
[0423] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0424] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0425] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0426] X.sup.1 and X.sup.2 are each independently an oxygen atom or
a sulfur atom; and
[0427] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom.
Compound B1
[0428] Compound B1 is a compound having formula (I), wherein
the partial structure:
##STR00066##
[0429] is a partial structure represented by the formula (IIA):
##STR00067##
[0430] R.sup.1 is a hydroxy group;
[0431] R.sup.2 is a hydroxy group or a fluorine atom;
[0432] B.sup.1 is a group represented by
##STR00068##
[0433] R.sup.13 is a hydrogen atom;
[0434] R.sup.14 is a hydrogen atom or an amino group;
[0435] R.sup.15 is a hydrogen atom;
[0436] R.sup.16 is a hydrogen atom;
[0437] R.sup.17 is a hydrogen atom;
[0438] Y.sup.11 is N;
[0439] Y.sup.12 is N or CH;
[0440] Y.sup.13 is N or CF;
[0441] (e.g., Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is CF;
Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is N; or Y.sup.11 is N,
Y.sup.12 is CH, and Y.sup.13 is N);
[0442] Y.sup.14 is N;
[0443] Y.sup.15 is N;
[0444] Y.sup.16 is N or CF;
[0445] (e.g., Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is N; or
Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is CF);
[0446] Z.sup.11 is C;
[0447] Z.sup.12 is C;
[0448] Z.sup.13 is N;
[0449] Z.sup.14 is C;
[0450] Z.sup.15 is C;
[0451] Z.sup.16 is N;
[0452] B.sup.2 is a group represented by
##STR00069##
[0453] R.sup.23 is a hydrogen atom;
[0454] R.sup.24 is a hydrogen atom;
[0455] R.sup.25 is a hydrogen atom;
[0456] R.sup.26 is a hydrogen atom;
[0457] R.sup.27 is a hydrogen atom;
[0458] Y.sup.21 is N;
[0459] Y.sup.22 is N;
[0460] Y.sup.23 is N or CF;
[0461] (e.g., Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is CF; or
Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is N);
[0462] Y.sup.24 is N;
[0463] Y.sup.25 is N or CH;
[0464] Y.sup.26 is N or CF;
[0465] (e.g., Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is N;
Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is CF; or Y.sup.24 is N,
Y.sup.25 is CH, and Y.sup.26 is N);
[0466] Z.sup.21 is N or C;
[0467] Z.sup.22 is C;
[0468] Z.sup.23 is N or C;
[0469] (e.g., Z.sup.21 is C, Z.sup.22 is C, and Z.sup.23 is N; or
Z.sup.21 is N, Z.sup.22 is C, and Z.sup.23 is C);
[0470] Z.sup.24 is N or C;
[0471] Z.sup.25 is C;
[0472] Z.sup.26 is N or C;
[0473] (e.g., Z.sup.24 is C, Z.sup.25 is C, and Z.sup.26 is N; or
Z.sup.24 is N, Z.sup.25 is C, and Z.sup.26 is C) provided that
[0474] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.5 and Y.sup.16 is CR.sup.1a,
[0475] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0476] iii) at least one of Z.sup.1, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0477] X.sup.1 and X.sup.2 are both oxygen atoms;
[0478] Q.sup.1 is an oxygen atom;
[0479] Q.sup.2 is an oxygen atom or a sulfur atom;
[0480] Q.sup.3 is an oxygen atom; and
[0481] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound A1-a
[0482] Compound A1-a is a compound having formula (Ia) wherein the
partial structure:
##STR00070##
[0483] is a partial structure represented by the formula
(IIAa):
##STR00071##
[0484] R.sup.1 is a hydroxy group;
[0485] R.sup.2 is a hydroxy group or a halogen atom (preferably a
fluorine atom);
[0486] B.sup.1 is a group represented by
##STR00072##
[0487] R.sup.13 is a hydrogen atom;
[0488] R.sup.14 is a hydrogen atom or an optionally substituted
amino group;
[0489] R.sup.15 is a hydrogen atom;
[0490] R.sup.16 is a hydrogen atom;
[0491] R.sup.17 is a hydrogen atom;
[0492] Y.sup.11 is N;
[0493] Y.sup.12 is N or CH;
[0494] Y.sup.13 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0495] Y.sup.14 is N;
[0496] Y.sup.15 is N;
[0497] Y.sup.16 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0498] Z.sup.11 is C;
[0499] Z.sup.12 is C;
[0500] Z.sup.13 is N;
[0501] Z.sup.14 is C;
[0502] Z.sup.15 is C;
[0503] Z.sup.16 is N;
[0504] B.sup.2 is a group represented by
##STR00073##
[0505] R.sup.23 is a hydrogen atom;
[0506] R.sup.24 is a hydrogen atom;
[0507] R.sup.25 is a hydrogen atom;
[0508] R.sup.26 is a hydrogen atom;
[0509] R.sup.27 is a hydrogen atom;
[0510] Y.sup.21 is N;
[0511] Y.sup.22 is N;
[0512] Y.sup.23 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0513] Y.sup.24 is N;
[0514] Y.sup.25 is N or CH;
[0515] Y.sup.26 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0516] Z.sup.21 is N or C;
[0517] Z.sup.22 is C;
[0518] Z.sup.23 is N or C;
[0519] Z.sup.24 is N or C;
[0520] Z.sup.25 is C;
[0521] Z.sup.26 is N or C;
provided that
[0522] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0523] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0524] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0525] X.sup.1 and X.sup.2 are each independently an oxygen atom or
a sulfur atom; and
[0526] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom.
Compound B1-a
[0527] Compound B1-a is a compound having formula (Ia) wherein the
partial structure:
##STR00074##
[0528] is a partial structure represented by the formula
(IIAa):
##STR00075##
[0529] R.sup.1 is a hydroxy group;
[0530] R.sup.2 is a hydroxy group or a fluorine atom;
[0531] B.sup.1 is a group represented by
##STR00076##
[0532] R.sup.13 is a hydrogen atom;
[0533] R.sup.14 is a hydrogen atom or an amino group;
[0534] R.sup.15 is a hydrogen atom;
[0535] R.sup.16 is a hydrogen atom;
[0536] R.sup.17 is a hydrogen atom;
[0537] Y.sup.11 is N;
[0538] Y.sup.12 is N or CH;
[0539] Y.sup.13 is N or CF;
[0540] (e.g., Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is CF;
Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is N; or Y.sup.11 is N,
Y.sup.12 is CH, and Y.sup.13 is N);
[0541] Y.sup.14 is N;
[0542] Y.sup.13 is N;
[0543] Y.sup.16 is N or CF;
[0544] (e.g., Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is N; or
Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is CF);
[0545] Z.sup.11 is C;
[0546] Z.sup.12 is C;
[0547] Z.sup.13 is N;
[0548] Z.sup.14 is C;
[0549] Z.sup.15 is C;
[0550] Z.sup.16 is N;
[0551] B.sup.2 is a group represented by
##STR00077##
[0552] R.sup.23 is a hydrogen atom;
[0553] R.sup.24 is a hydrogen atom;
[0554] R.sup.25 is a hydrogen atom;
[0555] R.sup.26 is a hydrogen atom;
[0556] R.sup.27 is a hydrogen atom;
[0557] Y.sup.21 is N;
[0558] Y.sup.22 is N;
[0559] Y.sup.23 is N or CF;
[0560] (e.g., Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is CF; or
Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is N)
[0561] Y.sup.24 is N;
[0562] Y.sup.25 is N or CH;
[0563] Y.sup.26 is N or CF;
[0564] (e.g., Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is N;
Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is CF; or Y.sup.24 is N,
Y.sup.25 is CH, and Y.sup.26 is N);
[0565] Z.sup.21 is N or C;
[0566] Z.sup.22 is C;
[0567] Z.sup.23 is N or C;
[0568] (e.g., Z.sup.21 is C, Z.sup.22 is C, and Z.sup.23 is N; or
Z.sup.21 is N, Z.sup.22 is C, and Z.sup.23 is C);
[0569] Z.sup.24 is N or C;
[0570] Z.sup.25 is C;
[0571] Z.sup.26 is N or C;
[0572] (e.g., Z.sup.24 is C, Z.sup.25 is C, and Z.sup.26 is N, or
Z.sup.24 is N, Z.sup.25 is C, and Z.sup.26 is C);
provided that
[0573] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0574] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0575] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0576] X.sup.1 and X.sup.2 are both oxygen atoms;
[0577] Q.sup.1 is an oxygen atom;
[0578] Q.sup.2 is an oxygen atom or a sulfur atom;
[0579] Q.sup.3 is an oxygen atom; and
[0580] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound A1-b
[0581] Compound A1-b is a compound having formula (Ib) wherein the
partial structure:
##STR00078##
[0582] is a partial structure represented by the formula
(IIAb):
##STR00079##
[0583] R.sup.1 is a hydroxy group;
[0584] R.sup.2 is a hydroxy group or a halogen atom (e.g., a
fluorine atom);
[0585] B.sup.1 is a group represented by
##STR00080##
[0586] R.sup.13 is a hydrogen atom;
[0587] R.sup.14 is a hydrogen atom or an optionally substituted
amino group;
[0588] R.sup.15 is a hydrogen atom;
[0589] R.sup.16 is a hydrogen atom;
[0590] R.sup.17 is a hydrogen atom;
[0591] Y.sup.11 is N;
[0592] Y.sup.12 is N or CH;
[0593] Y.sup.13 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0594] Y.sup.14 is N;
[0595] Y.sup.15 is N;
[0596] Y.sup.16 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0597] Z.sup.11 is C;
[0598] Z.sup.12 is C;
[0599] Z.sup.13 is N;
[0600] Z.sup.14 is C;
[0601] Z.sup.15 is C;
[0602] Z.sup.16 is N;
[0603] B.sup.2 is a group represented by
##STR00081##
[0604] R.sup.23 is a hydrogen atom;
[0605] R.sup.24 is a hydrogen atom;
[0606] R.sup.25 is a hydrogen atom;
[0607] R.sup.26 is a hydrogen atom;
[0608] R.sup.27 is a hydrogen atom;
[0609] Y.sup.21 is N;
[0610] Y.sup.22 is N;
[0611] Y.sup.23 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0612] Y.sup.24 is N;
[0613] Y.sup.25 is N or CH;
[0614] Y.sup.26 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0615] Z.sup.21 is N or C;
[0616] Z.sup.22 is C;
[0617] Z.sup.23 is N or C;
[0618] Z.sup.24 is N or C;
[0619] Z.sup.25 is C;
[0620] Z.sup.26 is N or C;
provided that
[0621] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0622] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0623] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0624] X.sup.1 and X.sup.2 are each independently an oxygen atom or
a sulfur atom; and
[0625] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom.
Compound B1-b
[0626] Compound B1-b is a compound having formula (Ib) wherein the
partial structure:
##STR00082##
[0627] is a partial structure represented by the formula
(IIAb):
##STR00083##
[0628] R.sup.1 is a hydroxy group;
[0629] R.sup.2 is a hydroxy group or a fluorine atom;
[0630] B.sup.1 is a group represented by
##STR00084##
[0631] R.sup.13 is a hydrogen atom;
[0632] R.sup.14 is a hydrogen atom or an amino group;
[0633] R.sup.15 is a hydrogen atom;
[0634] R.sup.16 is a hydrogen atom;
[0635] R.sup.17 is a hydrogen atom;
[0636] Y.sup.11 is N;
[0637] Y.sup.12 is N or CH;
[0638] Y.sup.13 is N or CF;
[0639] (e.g., Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is CF;
Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is N; or Y.sup.11 is N,
Y.sup.12 is CH, and Y.sup.13 is N);
[0640] Y.sup.14 is N;
[0641] Y.sup.13 is N;
[0642] Y.sup.16 is N or CF;
[0643] (e.g., Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is N; or
Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is CF);
[0644] Z.sup.11 is C;
[0645] Z.sup.12 is C;
[0646] Z.sup.13 is N;
[0647] Z.sup.14 is C;
[0648] Z.sup.15 is C;
[0649] Z.sup.16 is N;
[0650] B.sup.2 is a group represented by
##STR00085##
[0651] R.sup.23 is a hydrogen atom;
[0652] R.sup.26 is a hydrogen atom;
[0653] R.sup.27 is a hydrogen atom;
[0654] Y.sup.21 is N;
[0655] Y.sup.22 is N;
[0656] Y.sup.23 is N or CF;
[0657] (e.g., Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is CF; or
Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is N);
[0658] Y.sup.24 is N;
[0659] Y.sup.25 is N or CH;
[0660] Y.sup.26 is N or CF;
[0661] (e.g., Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is N;
Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is CF, or Y.sup.24 is N,
Y.sup.25 is CH, and Y.sup.26 is N);
[0662] Z.sup.21 is N or C;
[0663] Z.sup.22 is C;
[0664] Z.sup.23 is N or C;
[0665] (e.g., Z.sup.21 is C, Z.sup.22 is C, and Z.sup.23 is N; or
Z.sup.21 is N, Z.sup.22 is C, and Z.sup.23 is C);
[0666] Z.sup.24 is N or C;
[0667] Z.sup.25 is C;
[0668] Z.sup.26 is N or C;
[0669] (e.g., Z.sup.24 is C, Z.sup.25 is C, and Z.sup.26 is N; or
Z.sup.24 is N, Z.sup.25 is C, and Z.sup.26 is C); provided that
[0670] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0671] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0672] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0673] X.sup.1 and X.sup.2 are both oxygen atoms;
[0674] Q.sup.1 is an oxygen atom;
[0675] Q.sup.2 is an oxygen atom or a sulfur atom;
[0676] Q.sup.3 is an oxygen atom; and
[0677] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound A2
[0678] Compound A2 is a compound having formula (I) wherein the
partial structure:
##STR00086##
[0679] is a partial structure represented by the formula (IIA):
##STR00087##
or
[0680] a partial structure represented by the formula (IIB):
##STR00088##
[0681] R.sup.1 is a hydroxy group;
[0682] R.sup.2 is a hydroxy group or a halogen atom (e.g., a
fluorine atom);
[0683] B.sup.1 is a group represented by
##STR00089##
[0684] R.sup.13 is a hydrogen atom;
[0685] R.sup.14 is a hydrogen atom or an optionally substituted
amino group;
[0686] R.sup.15 is a hydrogen atom;
[0687] R.sup.16 is a hydrogen atom;
[0688] R.sup.17 is a hydrogen atom;
[0689] Y.sup.11 is N;
[0690] Y.sup.12 is N or CH;
[0691] Y.sup.13 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0692] Y.sup.14 is N;
[0693] Y.sup.15 is N;
[0694] Y.sup.16 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0695] Z.sup.11 is C;
[0696] Z.sup.12 is C;
[0697] Z.sup.13 is N;
[0698] Z.sup.14 is C;
[0699] Z.sup.15 is C;
[0700] Z.sup.16 is N;
[0701] B.sup.2 is a group represented by
##STR00090##
[0702] R.sup.23 is a hydrogen atom;
[0703] R.sup.24 is a hydrogen atom or an optionally substituted
amino group;
[0704] R.sup.25 is a hydrogen atom;
[0705] R.sup.26 is a hydrogen atom;
[0706] R.sup.27 is a hydrogen atom;
[0707] Y.sup.21 is N;
[0708] Y.sup.22 is N;
[0709] Y.sup.23 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0710] Y.sup.24 is N;
[0711] Y.sup.25 is N or CH;
[0712] Y.sup.26 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0713] Z.sup.21 is N or C;
[0714] Z.sup.22 is C;
[0715] Z.sup.23 is N or C;
[0716] Z.sup.24 is N or C;
[0717] Z.sup.25 is C;
[0718] Z.sup.26 is N or C;
provided that
[0719] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0720] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.21 and Y.sup.26 is CR.sup.2a, or
[0721] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0722] X.sup.1 and X.sup.2 are each independently an oxygen atom or
a sulfur atom; and
[0723] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom.
Compound B2
[0724] Compound B2 is a compound having formula (I) wherein the
partial structure:
##STR00091##
[0725] is a partial structure represented by the formula (IIA):
##STR00092##
or
[0726] a partial structure represented by the formula (IIB):
##STR00093##
[0727] R.sup.1 is a hydroxy group;
[0728] R.sup.2 is a hydroxy group or a fluorine atom;
[0729] B.sup.1 is a group represented by
##STR00094##
[0730] R.sup.13 is a hydrogen atom;
[0731] R.sup.14 is a hydrogen atom or an amino group;
[0732] R.sup.15 is a hydrogen atom;
[0733] R.sup.16 is a hydrogen atom;
[0734] R.sup.17 is a hydrogen atom;
[0735] Y.sup.11 is N;
[0736] Y.sup.12 is N or CH;
[0737] Y.sup.13 is N or CF;
[0738] (e.g., Y.sup.11 is N, Y.sup.12 is N, and Y.sup.11 is CF;
Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is N; or Y.sup.11 is N,
Y.sup.12 is CH, and Y.sup.13 is N);
[0739] Y.sup.14 is N;
[0740] Y.sup.15 is N;
[0741] Y.sup.16 is N or CF;
[0742] (e.g., Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is N; or
Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is CF);
[0743] Z.sup.11 is C;
[0744] Z.sup.12 is C;
[0745] Z.sup.13 is N;
[0746] Z.sup.14 is C;
[0747] Z.sup.15 is C;
[0748] Z.sup.16 is N;
[0749] B.sup.2 is a group represented by
##STR00095##
[0750] R.sup.23 is a hydrogen atom;
[0751] R.sup.24 is a hydrogen atom or an amino group;
[0752] R.sup.25 is a hydrogen atom;
[0753] R.sup.26 is a hydrogen atom;
[0754] R.sup.27 is a hydrogen atom;
[0755] Y.sup.21 is N;
[0756] Y.sup.22 is N;
[0757] Y.sup.23 is N or CF;
[0758] (e.g., Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is CF; or
Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is N);
[0759] Y.sup.24 is N;
[0760] Y.sup.25 is N or CH;
[0761] Y.sup.26 is N or CF;
[0762] (e.g., Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is N;
Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is CF, or Y.sup.24 is N,
Y.sup.25 is CH, and Y.sup.26 is N);
[0763] Z.sup.21 is N or C;
[0764] Z.sup.22 is C;
[0765] Z.sup.23 is N or C;
[0766] (e.g., Z.sup.21 is C, Z.sup.22 is C, and Z.sup.23 is N; or
Z.sup.21 is N, Z.sup.22 is C, and Z.sup.23 is C);
[0767] Z.sup.24 is N or C;
[0768] Z.sup.25 is C;
[0769] Z.sup.26 is N or C;
[0770] (e.g., Z.sup.24 is C, Z.sup.25 is C, and Z.sup.26 is N; or
Z.sup.24 is N, Z.sup.25 is C, and Z.sup.26 is C)
provided that
[0771] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0772] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0773] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0774] X.sup.1 and X.sup.2 are both oxygen atoms;
[0775] Q.sup.1 is an oxygen atom;
[0776] Q.sup.2 is an oxygen atom or a sulfur atom;
[0777] Q.sup.3 is an oxygen atom; and
[0778] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound A2-a
[0779] Compound A2-a is a compound having formula (Ia) wherein the
partial structure:
##STR00096##
[0780] is a partial structure represented by the formula
(IIAa):
##STR00097##
[0781] a partial structure represented by the formula (IIBa):
##STR00098##
[0782] R.sup.1 is a hydroxy group;
[0783] R.sup.2 is a hydroxy group or a halogen atom (e.g., a
fluorine atom);
[0784] B.sup.1 is a group represented by
##STR00099##
[0785] R.sup.13 is a hydrogen atom;
[0786] R.sup.14 is a hydrogen atom or an optionally substituted
amino group;
[0787] R.sup.15 is a hydrogen atom;
[0788] R.sup.16 is a hydrogen atom;
[0789] R.sup.17 is a hydrogen atom;
[0790] Y.sup.11 is N;
[0791] Y.sup.12 is N or CH;
[0792] Y.sup.13 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0793] Y.sup.14 is N;
[0794] Y.sup.15 is N;
[0795] Y.sup.16 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0796] Z.sup.11 is C;
[0797] Z.sup.12 is C;
[0798] Z.sup.13 is N;
[0799] Z.sup.14 is C;
[0800] Z.sup.15 is C;
[0801] Z.sup.16 is N;
[0802] B.sup.2 is a group represented by
##STR00100##
[0803] R.sup.23 is a hydrogen atom;
[0804] R.sup.24 is a hydrogen atom or an optionally substituted
amino group;
[0805] R.sup.25 is a hydrogen atom;
[0806] R.sup.26 is a hydrogen atom;
[0807] R.sup.21 is a hydrogen atom;
[0808] Y.sup.21 is N;
[0809] Y.sup.22 is N;
[0810] Y.sup.23 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0811] Y.sup.24 is N;
[0812] Y.sup.25 is N or CH;
[0813] Y.sup.26 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0814] Z.sup.21 is N or C;
[0815] Z.sup.22 is C;
[0816] Z.sup.23 is N or C;
[0817] Z.sup.24 is N or C;
[0818] Z.sup.25 is C;
[0819] Z.sup.26 is N or C;
provided that
[0820] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0821] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0822] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0823] X.sup.1 and X.sup.2 are each independently an oxygen atom or
a sulfur atom; and
[0824] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom.
Compound B2-a
[0825] Compound B2-a is a compound having formula (I) wherein the
partial structure:
##STR00101##
[0826] is a partial structure represented by the formula
(IIAa):
##STR00102##
[0827] a partial structure re represented by the formula
(IIBa):
##STR00103##
[0828] R.sup.1 is a hydroxy group;
[0829] R.sup.2 is a hydroxy group or a fluorine atom;
[0830] B.sup.1 is a group represented by
##STR00104##
[0831] R.sup.13 is a hydrogen atom;
[0832] R.sup.14 is a hydrogen atom or an amino group;
[0833] R.sup.15 is a hydrogen atom;
[0834] R.sup.16 is a hydrogen atom;
[0835] R.sup.17 is a hydrogen atom;
[0836] Y.sup.11 is N;
[0837] Y.sup.12 is N or CH;
[0838] Y.sup.13 is N or CF;
[0839] (e.g., Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is CF;
Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is N; or Y.sup.11 is N,
Y.sup.12 is CH, and Y.sup.13 is N);
[0840] Y.sup.14 is N;
[0841] Y.sup.15 is N;
[0842] Y.sup.16 is N or CF;
[0843] (e.g., Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is N; or
Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is CF);
[0844] Z.sup.11 is C;
[0845] Z.sup.12 is C;
[0846] Z.sup.13 is N;
[0847] Z.sup.14 is C;
[0848] Z.sup.15 is C;
[0849] Z.sup.16 is N;
[0850] B.sup.2 is a group represented by
##STR00105##
[0851] R.sup.23 is a hydrogen atom;
[0852] R.sup.24 is a hydrogen atom or an amino group;
[0853] R.sup.25 is a hydrogen atom;
[0854] R.sup.26 is a hydrogen atom;
[0855] R.sup.27 is a hydrogen atom;
[0856] Y.sup.21 is N;
[0857] Y.sup.22 is N;
[0858] Y.sup.23 is N or CF;
[0859] (e.g., Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is CF; or
Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is N);
[0860] Y.sup.24 is N;
[0861] Y.sup.25 is N or CH;
[0862] Y.sup.26 is N or CF;
[0863] (e.g., Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is N;
Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is CF; or Y.sup.24 is N,
Y.sup.25 is CH, and Y.sup.26 is N);
[0864] Z.sup.21 is N or C;
[0865] Z.sup.22 is C;
[0866] Z.sup.23 is N or C;
[0867] (e.g., Z.sup.21 is C, Z.sup.22 is C, and Z.sup.23 is N; or
Z.sup.21 is N, Z.sup.22 is C, and Z.sup.23 is C);
[0868] Z.sup.24 is N or C;
[0869] Z.sup.25 is C;
[0870] Z.sup.26 is N or C;
[0871] (e.g., Z.sup.24 is C, Z.sup.25 is C, and Z.sup.26 is N; or
Z.sup.24 is N, Z.sup.25 is C, and Z.sup.26 is C);
provided that
[0872] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0873] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0874] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0875] X.sup.1 and X.sup.2 are both oxygen atoms;
[0876] Q.sup.1 is an oxygen atom;
[0877] Q.sup.2 is an oxygen atom or a sulfur atom;
[0878] Q.sup.3 is an oxygen atom; and
[0879] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound A2-b
[0880] Compound A2-b is a compound having formula (Ib) wherein the
partial structure:
##STR00106##
[0881] is a partial structure represented by the formula
(IIAb):
##STR00107##
or
[0882] a partial structure represented by the formula (IIBb):
##STR00108##
[0883] R.sup.1 is a hydroxy group;
[0884] R.sup.2 is a hydroxy group or a halogen atom (e.g., a
fluorine atom);
[0885] B.sup.1 is a group represented by
##STR00109##
[0886] R.sup.13 is a hydrogen atom;
[0887] R.sup.14 is a hydrogen atom or an optionally substituted
amino group;
[0888] R.sup.15 is a hydrogen atom;
[0889] R.sup.16 is a hydrogen atom;
[0890] R.sup.17 is a hydrogen atom;
[0891] Y.sup.11 is N;
[0892] Y.sup.12 is N or CH;
[0893] Y.sup.13 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0894] Y.sup.14 is N;
[0895] Y.sup.15 is N;
[0896] Y.sup.16 is N or CR.sup.1a wherein R.sup.1a is a halogen
atom (e.g., a fluorine atom);
[0897] Z.sup.11 is C;
[0898] Z.sup.12 is C;
[0899] Z.sup.13 is N;
[0900] Z.sup.14 is C;
[0901] Z.sup.15 is C;
[0902] Z.sup.16 is N;
[0903] B.sup.2 is a group represented by
##STR00110##
[0904] R.sup.23 is a hydrogen atom;
[0905] R.sup.24 is a hydrogen atom or an optionally substituted
amino group;
[0906] R.sup.25 is a hydrogen atom;
[0907] R.sup.26 is a hydrogen atom;
[0908] R.sup.27 is a hydrogen atom;
[0909] Y.sup.21 is N;
[0910] Y.sup.22 is N;
[0911] Y.sup.23 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0912] Y.sup.24 is N;
[0913] Y.sup.25 is N or CH;
[0914] Y.sup.26 is N or CR.sup.2a wherein R.sup.2a is a halogen
atom (e.g., a fluorine atom);
[0915] Z.sup.21 is N or C;
[0916] Z.sup.22 is C;
[0917] Z.sup.23 is N or C;
[0918] Z.sup.24 is N or C;
[0919] Z.sup.25 is C;
[0920] Z.sup.26 is N or C;
provided that
[0921] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0922] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0923] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0924] X.sup.1 and X.sup.2 are each independently an oxygen atom or
a sulfur atom; and
[0925] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom.
Compound B2-b
[0926] Compound B2-b is a compound having formula (Ib) wherein the
partial structure:
##STR00111##
[0927] is a partial structure represented by the formula
(IIAb):
##STR00112##
or
[0928] a partial structure represented by the formula (IIBb):
##STR00113##
[0929] R.sup.1 is a hydroxy group;
[0930] R.sup.2 is a hydroxy group or a fluorine atom;
[0931] B.sup.1 is a group represented by
##STR00114##
[0932] R.sup.13 is a hydrogen atom;
[0933] R.sup.14 is a hydrogen atom or an amino group;
[0934] R.sup.15 is a hydrogen atom;
[0935] R.sup.16 is a hydrogen atom;
[0936] R.sup.17 is a hydrogen atom;
[0937] Y.sup.11 is N;
[0938] Y.sup.12 is N or CH;
[0939] Y.sup.13 is N or CF;
[0940] (e.g., Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is CF;
Y.sup.11 is N, Y.sup.12 is N, and Y.sup.13 is N; or Y.sup.11 is N,
Y.sup.12 is CH, and Y.sup.13 is N);
[0941] Y.sup.14 is N;
[0942] Y.sup.15 is N;
[0943] Y.sup.16 is N or CF;
[0944] (e.g., Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is N; or
Y.sup.14 is N, Y.sup.15 is N, and Y.sup.16 is CF);
[0945] Z.sup.11 is C;
[0946] Z.sup.12 is C;
[0947] Z.sup.13 is N;
[0948] Z.sup.14 is C;
[0949] Z.sup.15 is C;
[0950] Z.sup.16 is N;
[0951] B.sup.2 is a group represented by
##STR00115##
or
[0952] R.sup.23 is a hydrogen atom;
[0953] R.sup.24 is a hydrogen atom or an amino group;
[0954] R.sup.25 is a hydrogen atom;
[0955] R.sup.26 is a hydrogen atom;
[0956] R.sup.27 is a hydrogen atom;
[0957] Y.sup.21 is N;
[0958] Y.sup.22 is N;
[0959] Y.sup.23 is N or CF;
[0960] (e.g., Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is CF; or
Y.sup.21 is N, Y.sup.22 is N, and Y.sup.23 is N);
[0961] Y.sup.24 is N;
[0962] Y.sup.25 is N or CH;
[0963] Y.sup.26 is N or CF;
[0964] (e.g., Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is N;
Y.sup.24 is N, Y.sup.25 is N, and Y.sup.26 is CF, or Y.sup.24 is N,
Y.sup.25 is CH, and Y.sup.26 is N);
[0965] Z.sup.21 is N or C;
[0966] Z.sup.22 is C;
[0967] Z.sup.23 is N or C;
[0968] (e.g., Z.sup.21 is C, Z.sup.22 is C, and Z.sup.23 is N; or
Z.sup.21 is N, Z.sup.22 is C, and Z.sup.23 is C)
[0969] Z.sup.24 is N or C;
[0970] Z.sup.25 is C;
[0971] Z.sup.26 is N or C;
[0972] (e.g., Z.sup.24 is C, Z.sup.25 is C, and Z.sup.26 is N; or
Z.sup.24 is N, Z.sup.25 is C, and Z.sup.26 is C)
provided that
[0973] i) at least one of Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14,
Y.sup.15 and Y.sup.16 is CR.sup.1a,
[0974] ii) at least one of Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24,
Y.sup.25 and Y.sup.26 is CR.sup.2a, or
[0975] iii) at least one of Z.sup.13, Z.sup.16, Z.sup.23 and
Z.sup.26 is C;
[0976] X.sup.1 and X.sup.2 are both oxygen atoms;
[0977] Q.sup.1 is an oxygen atom;
[0978] Q.sup.2 is an oxygen atom or a sulfur atom;
[0979] Q.sup.3 is an oxygen atom; and
[0980] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound C2
[0981] Compound C2 is a compound having formula (I) wherein the
partial structure:
##STR00116##
[0982] is a partial structure represented by the formula (IIA):
##STR00117##
or
[0983] a partial structure represented by the formula (IIB):
##STR00118##
[0984] R.sup.1 is a hydroxy group;
[0985] R.sup.2 is a hydroxy group or a fluorine atom;
[0986] B.sup.1 and B.sup.2 are each independently
##STR00119##
and
[0987] at least one of B.sup.1 and B.sup.2 is
##STR00120##
[0988] and e.g., the other is
##STR00121##
[0989] X.sup.1 and X.sup.2 are both oxygen atoms;
[0990] Q.sup.1 is an oxygen atom;
[0991] Q.sup.2 is an oxygen atom or a sulfur atom;
[0992] Q.sup.3 is an oxygen atom; and
[0993] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound C2-a
[0994] Compound C2-a is a compound having formula (Ia) wherein the
partial structure:
##STR00122##
[0995] is a partial structure represented by the formula
(IIAa):
##STR00123##
or
[0996] a partial structure represented by the formula (IIBa):
##STR00124##
[0997] R.sup.1 is a hydroxy group;
[0998] R.sup.2 is a hydroxy group or a fluorine atom;
[0999] B.sup.1 and B.sup.2 are each independently
##STR00125##
and
[1000] at least one of B.sup.1 and B.sup.2 is
##STR00126##
[1001] and, e.g., the other is
##STR00127##
[1002] X.sup.1 and X.sup.2 are both oxygen atoms;
[1003] Q.sup.1 is an oxygen atom;
[1004] Q.sup.2 is an oxygen atom or a sulfur atom;
[1005] Q.sup.3 is an oxygen atom; and
[1006] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound C2-b
[1007] Compound C2-b is a compound having formula (Ib) wherein the
partial structure:
##STR00128##
[1008] is a partial structure represented by the formula
(IIAb):
##STR00129##
[1009] a partial structure represented by the formula (IIBb):
##STR00130##
[1010] R.sup.1 is a hydroxy group;
[1011] R.sup.2 is a hydroxy group or a fluorine atom;
[1012] B.sup.1 and B.sup.2 are each independently
##STR00131##
[1013] at least one of B.sup.1 and B.sup.2 is
##STR00132##
[1014] and, e.g., the other is
##STR00133##
[1015] X.sup.1 and X.sup.2 are both oxygen atoms;
[1016] Q.sup.1 is an oxygen atom;
[1017] Q.sup.2 is an oxygen atom or a sulfur atom;
[1018] Q.sup.3 is an oxygen atom; and
[1019] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound D2
[1020] Compound D2 is a compound having formula (I) wherein the
partial structure:
##STR00134##
[1021] is a partial structure represented by the formula (IIA):
##STR00135##
or
[1022] a partial structure represented by the formula (IIB):
##STR00136##
[1023] R.sup.1 is a hydroxy group;
[1024] R.sup.2 is a hydroxy group or a fluorine atom;
[1025] B.sup.1 is a group represented by
##STR00137##
[1026] B.sup.2 is a group represented by
##STR00138##
[1027] X.sup.1 and X.sup.2 are both oxygen atoms;
[1028] Q.sup.1 is an oxygen atom;
[1029] Q.sup.2 is an oxygen atom or a sulfur atom;
[1030] Q.sup.3 is an oxygen atom; and
[1031] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound D2-a
[1032] Compound D2-a is a compound having formula (Ia) wherein the
partial structure:
##STR00139##
[1033] is a partial structure represented by the formula
(IIAa):
##STR00140##
or
[1034] a partial structure re resented by the formula (IIBa):
##STR00141##
[1035] R.sup.1 is a hydroxy group;
[1036] R.sup.2 is a hydroxy group or a fluorine atom;
[1037] B.sup.1 is a group represented by
##STR00142##
[1038] B.sup.2 is a group represented by
##STR00143##
[1039] X.sup.1 and X.sup.2 are both oxygen atoms;
[1040] Q.sup.1 is an oxygen atom;
[1041] Q.sup.2 is an oxygen atom or a sulfur atom;
[1042] Q.sup.3 is an oxygen atom; and
[1043] Q.sup.4 is an oxygen atom or a sulfur atom.
Compound D2-b
[1044] Compound D2-b is a compound having formula (Ib) wherein the
partial structure:
##STR00144##
[1045] is a partial structure represented by the formula
(IIAb):
##STR00145##
or
[1046] a partial structure represented by the formula (IIBb):
##STR00146##
[1047] R.sup.1 is a hydroxy group;
[1048] R.sup.2 is a hydroxy group or a fluorine atom;
[1049] B.sup.1 is a group represented by
##STR00147##
[1050] B.sup.2 is a group represented by
##STR00148##
[1051] X.sup.1 and X.sup.2 are both oxygen atoms;
[1052] Q.sup.1 is an oxygen atom;
[1053] Q.sup.2 is an oxygen atom or a sulfur atom;
[1054] Q.sup.3 is an oxygen atom; and
[1055] Q.sup.4 is an oxygen atom or a sulfur atom.
[1056] Specific examples of compound (I) include the compounds of
Examples 1 to 20 and 3a.
[1057] When compound (I) is in a form of a salt, the salt is
preferably a pharmacologically acceptable salt. Examples include
salts with inorganic base, salts with organic base, salts with
inorganic acid, salts with organic acid, and salts with basic or
acidic amino acid.
[1058] Preferable examples of the salt with inorganic base include
alkali metal salts such as sodium salt, potassium salt and the
like; alkaline-earth metal salts such as calcium salt, magnesium
salt and the like; aluminium salt and ammonium salt.
[1059] Preferable examples of the salt with organic base include
salts with trimethylamine, triethylamine, pyridine, picoline,
ethanolamine, diethanolamine, triethanolamine, tromethamine
[tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine,
benzylamine, dicyclohexylamine and N,N-dibenzyl ethylene
diamine.
[1060] Preferable examples of the salt with inorganic acid include
salts with hydrochloric acid, hydrobromic acid, nitric acid,
sulfuric acid and phosphoric acid.
[1061] Preferable examples of the salt with organic acid include
salts with formic acid, acetic acid, trifluoroacetic acid, phthalic
acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric
acid, succinic acid, malic acid, methanesulfonic acid,
benzenesulfonic acid and p-toluenesulfonic acid.
[1062] Preferable examples of the salt with basic amino acid
include salts with arginine, lysine and ornithine.
[1063] Preferable examples of the salt with acidic amino acid
include salts with aspartic acid and glutamic acid.
[1064] When compound (1) is in a form of a salt, the salt is
preferably a salt with triethylamine or sodium, more preferably a
salt with triethylamine.
[1065] The production method of the compound of the present
invention is explained in the followings.
[1066] The raw material compound and reagent used and the compound
obtained in each step in the following production method may be
each in a form of a salt, and examples of such salt include those
similar to the salts of the compound of the present disclosure.
[1067] When the compound obtained in each step is a free form, it
can be converted to the objective salt according to a method known
per se. When the compound obtained in each step is a salt, it can
be converted to the objective free form or the other salt according
to a method known per se.
[1068] The compound obtained in each step can be used directly as
the reaction mixture or as a crude product for the next reaction.
Alternatively, the compound obtained in each step can be isolated
and purified from a reaction mixture according to a method known
per se, for example, a separation means such as concentration,
crystallization, recrystallization, distillation, solvent
extraction, fractional distillation, column chromatography and the
like.
[1069] When the raw material compound and reagent used in each step
are commercially available, the commercially available product can
also be used directly.
[1070] In the reaction in each step, while the reaction time varies
depending on the kind of the reagent and solvent to be used, it is
generally 1 min-48 hr, preferably 10 min-8 hr, unless otherwise
specified.
[1071] In the reaction in each step, while the reaction temperature
varies depending on the kind of the reagent and solvent to be used,
it is generally -78.degree. C.-300.degree. C., preferably
-78.degree. C.-150.degree. C., unless otherwise specified.
[1072] In the reaction in each step, while the pressure varies
depending on the kind of the reagent and solvent to be used, it is
generally 1 atm-20 atm, preferably 1 atm-3 atm, unless otherwise
specified.
[1073] Microwave synthesizer such as Initiator manufactured by
Biotage and the like may be used for the reaction in each step.
While the reaction temperature varies depending on the kind of the
reagent and solvent to be used, it is generally room
temperature-300.degree. C., preferably 50.degree. C.-250.degree.
C., unless otherwise specified. While the reaction time varies
depending on the kind of the reagent and solvent to be used, it is
generally 1 min-48 hr, preferably 1 min-8 hr, unless otherwise
specified.
[1074] In the reaction in each step, the reagent is used in an
amount of 0.5 equivalents-20 equivalents, preferably 0.8
equivalents-5 equivalents, relative to the substrate, unless
otherwise specified. When the reagent is used as a catalyst, the
reagent is used in an amount of 0.001 equivalent-1 equivalent,
preferably 0.01 equivalent-0.2 equivalent, relative to the
substrate. When the reagent is used as a reaction solvent, the
reagent is used in a solvent amount.
[1075] Unless otherwise specified, the reaction in each step is
carried out without solvent, or by dissolving or suspending the raw
material compound in a suitable solvent. Examples of the solvent
include those described in Examples and the following solvents.
[1076] alcohols: methanol, ethanol, tert-butyl alcohol,
2-methoxyethanol and the like;
[1077] ethers: diethyl ether, diphenyl ether, tetrahydrofuran,
1,2-dimethoxyethane and the like;
[1078] aromatic hydrocarbons: chlorobenzene, toluene, xylene and
the like;
[1079] saturated hydrocarbons: cyclohexane, hexane and the
like;
[1080] amides: N,N-dimethylformamide, N-methylpyrrolidone and the
like;
[1081] halogenated hydrocarbons: dichloromethane, carbon
tetrachloride and the like;
[1082] nitriles: acetonitrile and the like;
[1083] sulfoxides: dimethyl sulfoxide and the like;
[1084] aromatic organic bases: pyridine and the like;
[1085] anhydrides: acetic anhydride and the like;
[1086] organic acids: formic acid, acetic acid, trifluoroacetic
acid and the like;
[1087] inorganic acids: hydrochloric acid, sulfuric acid and the
like;
[1088] esters: ethyl acetate and the like;
[1089] ketones: acetone, methyl ethyl ketone and the like;
[1090] water.
[1091] The above-mentioned solvent can be used in a mixture of two
or more kinds thereof in an appropriate ratio.
[1092] When a base is used for the reaction in each step, examples
thereof include those described in Examples and the following
bases.
inorganic bases: sodium hydroxide, magnesium hydroxide, sodium
carbonate, calcium carbonate, sodium hydrogencarbonate and the
like; organic bases: triethylamine, diethylamine, pyridine,
4-dimethylaminopyridine, N,N-dimethylaniline,
1,4-diazabicyclo[2.2.2]octane, 1,8-diazabicyclo[5.4.0]-7-undecene,
imidazole, piperidine and the like; metal alkoxides: sodium
ethoxide, potassium tert-butoxide and the like; alkali metal
hydrides: sodium hydride and the like; metal amides: sodium amide,
lithium diisopropylamide, lithium hexamethyldisilazide and the
like; organic lithiums: n-butyllithium and the like.
[1093] When an acid or an acid catalyst is used for the reaction in
each step, examples thereof include those described in Examples and
the following acids and acid catalysts. inorganic acids:
hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid,
phosphoric acid and the like;
organic acids: acetic acid, trifluoroacetic acid, citric acid,
p-toluenesulfonic acid, 10-camphorsulfonic acid and the like; Lewis
acid: boron trifluoride diethyl ether complex, zinc iodide,
anhydrous aluminium chloride, anhydrous zinc chloride, anhydrous
iron chloride and the like.
[1094] Unless otherwise specified, the reaction in each step is
carried out according to a method known per se, for example, the
method described in Jikken Kagaku Kouza, 5th Edition, vol. 13-19
(the Chemical Society of Japan ed.); Shin Jikken Kagaku Kouza, vol.
14-15 (the Chemical Society of Japan ed.); Fine Organic Chemistry,
Revised 2nd Edition (L. F. Tietze, Th. Eicher, Nankodo); Organic
Name Reactions, the Reaction Mechanism and Essence, Revised Edition
(Hideo Togo, Kodansha); ORGANIC SYNTHESES Collective Volume I-VII
(John Wiley & Sons Inc); Modern Organic Synthesis in the
Laboratory A Collection of Standard Experimental Procedures (Jie
Jack Li, OXFORD UNIVERSITY); Comprehensive Heterocyclic Chemistry
III, Vol. 1-Vol. 14 (Elsevier Japan); Strategic Applications of
Named Reactions in Organic Synthesis (translated by Kiyoshi
Tomioka, Kagakudojin); Comprehensive Organic Transformations (VCH
Publishers Inc.), 1989, or the like, or the method described in
Examples.
[1095] In each step, the protection or deprotection reaction of an
functional group is carried out according to a method known per se,
for example, the method described in "Protective Groups in Organic
Synthesis, 4th Ed", Wiley-Interscience, Inc., 2007 (Theodora W.
Greene, Peter G. M. Wuts); "Protecting Groups 3rd Ed." Thieme, 2004
(P. J. Kocienski), or the like, or the method described in
Examples.
[1096] Examples of the protecting group for a hydroxy group (e.g.,
a hydroxy group of an alcohol and the like, a phenolic hydroxy
group) include ether-type protecting groups such as methoxymethyl
ether, benzyl ether, tert-butyldimethylsilyl ether,
tetrahydropyranyl ether and the like; carboxylate ester-type
protecting groups such as acetate ester and the like; sulfonate
ester-type protecting groups such as methanesulfonate ester and the
like; and carbonate ester-type protecting groups such as
tert-butylcarbonate and the like.
[1097] Examples of the protecting group for a carbonyl group of an
aldehyde include acetal-type protecting groups such as
dimethylacetal and the like; and cyclic acetal-type protecting
groups such as 1,3-dioxane and the like.
[1098] Examples of the protecting group for a carbonyl group of a
ketone include ketal-type protecting groups such as dimethylketal
and the like; cyclic ketal-type protecting groups such as
1,3-dioxane and the like; oxime-type protecting groups such as
O-methyloxime and the like; and hydrazone-type protecting groups
such as N,N-dimethylhydrazone and the like.
[1099] Examples of the protecting group for a carboxyl group
include ester-type protecting groups such as methyl ester and the
like; and amide-type protecting groups such as N,N-dimethylamide
and the like.
[1100] Examples of the protecting group for a thiol include
ether-type protecting groups such as benzyl thioether and the like;
and ester-type protecting groups such as thioacetate ester,
thiocarbonate, thiocarbamate and the like.
[1101] Examples of the protecting group for an amino group and an
aromatic heterocycle such as imidazole, pyrrole, indole and the
like include carbamate-type protecting groups such as benzyl
carbamate and the like; amide-type protecting groups such as
acetamide, benzamide, isobutylamide and the like; alkyl amine-type
protecting groups such as N-triphenylmethylamine and the like; and
sulfonamide-type protecting groups such as methanesulfonamide and
the like.
[1102] The protecting groups can be removed according to a method
known per se (e.g., a method using acid, base, ultraviolet rays,
hydrazine, phenylhydrazine, sodium N-methyldithiocarbamate,
tetrabutylammonium fluoride, palladium acetate or trialkylsilyl
halide (e.g., trimethylsilyl iodide, trimethylsilyl bromide), a
reduction method).
[1103] When reduction reaction is carried out in each step,
examples of the reducing agent to be used include metal hydrides
such as lithium aluminium hydride, sodium triacetoxyborohydride,
sodium cyanoborohydride, diisobutylaluminium hydride (DIBAL-H),
sodium borohydride, tetramethylammonium triacetoxyborohydride and
the like; boranes such as borane tetrahydrofuran complex and the
like; Raney nickel; Raney cobalt; hydrogen; formic acid;
triethylsilane and the like. When carbon-carbon double bond or
triple bond is reduced, a method using a catalyst such as
palladium-carbon, Lindlar's catalyst and the like may be
employed.
[1104] When oxidation reaction is carried out in each step,
examples of the oxidizing agent to be used include peroxides such
as m-chloroperbenzoic acid (mCPBA), hydrogen peroxide, tert-butyl
hydroperoxide and the like; perchlorates such as tetrabutylammonium
perchlorate and the like; chlorates such as sodium chlorate and the
like; chlorites such as sodium chlorite and the like; periodic
acids such as sodium periodate and the like; hypervalent iodine
reagents such as iodosylbenzene and the like; reagents containing
manganese such as manganese dioxide, potassium permanganate and the
like; leads such as lead tetraacetate and the like, reagents
containing chromium such as pyridinium chlorochromate (PCC),
pyridinium dichromate (PDC), Jones reagent and the like; halogen
compounds such as N-bromosuccinimide (NBS) and the like; oxygen;
ozone; sulfur trioxido-pyridine complex; osmium tetroxide; selenium
dioxide; 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and the
like.
[1105] When radical cyclization reaction is carried out in each
step, examples of the radical initiator to be used include azo
compounds such as azobisisobutyronitrile (AIBN) and the like;
water-soluble radical initiators such as
4-4'-azobis-4-cyanopentanoic acid (ACPA) and the like;
triethylboron in the presence of air or oxygen; benzoyl peroxide
and the like. Examples of the radical reagent to be used include
tributylstannane, tristrimethylsilylsilane,
1,1,2,2-tetraphenyldisilane, diphenylsilane, samarium iodide and
the like.
[1106] When Wittig reaction is carried out in each step, examples
of the Wittig reagent to be used include alkylidene phosphoranes
and the like. The alkylidene phosphoranes can be prepared according
to a method known per se, for example, by reacting a phosphonium
salt with a strong base.
[1107] When Horner-Emmons reaction is carried out in each step,
examples of the reagent to be used include phosphonoacetates such
as methyl dimethylphosphonoacetate, ethyl diethylphosphonoacetate
and the like; and bases such as alkali metal hydrides, organic
lithiums and the like.
[1108] When Friedel-Crafts reaction is carried out in each step,
examples of the reagent to be used include a combination of a Lewis
acid and an acid chloride or a combination of a Lewis acid and an
alkylating agent (e.g., an alkyl halide, an alcohol, an olefin).
Alternatively, an organic acid or an inorganic acid can also be
used instead of a Lewis acid, and an anhydride such as acetic
anhydride and the like can also be used instead of an acid
chloride.
[1109] When aromatic nucleophilic substitution reaction is carried
out in each step, a nucleophile (e.g., an amine, imidazole) and a
base (e.g., an organic base) are used as a reagent.
[1110] When nucleophilic addition reaction by a carbo anion,
nucleophilic 1,4-addition reaction (Michael addition reaction) by a
carbo anion or nucleophilic substitution reaction by a carbo anion
is carried out in each step, examples of the base to be used for
generation of the carbo anion include organic lithiums, metal
alkoxides, inorganic bases, organic bases and the like.
[1111] When Grignard reagent is carried out in each step, examples
of the Grignard reagent to be used include arylmagnesium halides
such as phenylmagnesium bromide and the like; alkylmagnesium
halides such as methylmagnesium bromide and the like, and the like.
The Grignard reagent can be prepared according to a method known
per se, for example, by reacting an alkyl halide or an aryl halide
with metal magnesium in an ether or tetrahydrofuran as a
solvent.
[1112] When Knoevenagel condensation reaction is carried out in
each step, a compound having an activated methylene group with two
electron withdrawing groups (e.g., malonic acid, diethyl malonate,
malononitrile etc.) and a base (e.g., an organic base, a metal
alkoxide, an inorganic base) are used as a reagent.
[1113] When Vilsmeier-Haack reaction is carried out in each step,
phosphoryl chloride and an amide derivative (e.g.,
N,N-dimethylformamide etc.) are used as a reagent.
[1114] When azidation reaction of an alcohol, an alkyl halide or a
sulfonate is carried out in each step, examples of the azidating
agent to be used include diphenylphosphorylazide (DPPA),
trimethylsilylazide, sodium azide and the like. For example, for
the azidation reaction of an alcohol, a method using
diphenylphosphorylazide and 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU), a method using trimethylsilylazide and a Lewis acid, and the
like are employed.
[1115] When reductive amination reaction is carried out in each
step, examples of the reducing agent to be used include sodium
triacetoxyborohydride, sodium cyanoborohydride, hydrogen and formic
acid. When the substrate is an amine compound, examples of the
carbonyl compound to be used include paraformaldehyde, aldehydes
such as acetaldehyde and the like, and ketones such as
cyclohexanone and the like. When the substrate is a carbonyl
compound, examples of the amine to be used include ammonia, primary
amines such as methylamine and the like; secondary amines such as
dimethylamine and the like, and the like.
[1116] When Mitsunobu reaction is carried out in each step, an
azodicarboxylate (e.g., diethyl azodicarboxylate (DEAD),
diisopropyl azodicarboxylate (DIAD) etc.) and triphenylphosphine
are used as a reagent.
[1117] When esterification reaction, amidation reaction or ureation
reaction is carried out in each step, examples of the reagent to be
used include acyl halides such as acid chlorides, acid bromides and
the like; activated carboxylic acids such as anhydrides, activated
esters, sulfates and the like. Examples of the activating agent of
the carboxylic acid include carbodiimide condensing agents such as
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD)
and the like; triazine condensing agents such as
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
n-hydrate (DMT-MM) and the like; carbonate condensing agents such
as 1,1-carbonyldiimidazole (CDI) and the like; diphenylphosphoryl
azide (DPPA); benzotriazol-1-yloxy-trisdimethylaminophosphonium
salt (BOP reagent); 2-chloro-1-methyl-pyridinium iodide (Mukaiyama
reagent); thionyl chloride; lower alkyl haloformates such as ethyl
chloroformate and the like;
O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphorate (HATU); sulfuric acid; combinations thereof
and the like. When carbodiimide condensing agent is used, an
additive such as 1-hydroxybenzotriazole (HOBt),
N-hydroxysuccinimide (HOSu), dimethylaminopyridine (DMAP) and the
like may be added to the reaction system.
[1118] When coupling reaction is carried out in each step, examples
of the metal catalyst to be used include palladium compounds such
as palladium(II) acetate, tetrakis(triphenylphosphine)palladium(O),
dichlorobis(triphenylphosphine)palladium(II),
dichlorobis(triethylphosphine)palladium(II),
tris(dibenzylideneacetone)dipalladium(0),
1,1'-bis(diphenylphosphino)ferrocene palladium(II) chloride and the
like; nickel compounds such as
tetrakis(triphenylphosphine)nickel(0) and the like; rhodium
compounds such as tris(triphenylphosphine)rhodium(III) chloride and
the like; cobalt compounds; copper compounds such as copper oxide,
copper(I) iodide and the like; platinum compounds and the like. In
addition, a base can be added to the reaction system, and examples
thereof include inorganic bases and the like.
[1119] When thiocarbonylation reaction is carried out in each step,
phosphorus pentasulfide is typically used as the thiocarbonylating
agent. Alternatively, a reagent having a
1,3,2,4-dithiadiphosphetane-2,4-disulfide structure (e.g.,
2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide
(Lawesson reagent) etc.) can also be used instead of phosphorus
pentasulfide.
[1120] When Wohl-Ziegler reaction is carried out in each step,
examples of the halogenating agent to be used include
N-iodosuccinimide, N-bromosuccinimide (NBS), N-chlorosuccinimide
(NCS), bromine, sulfuryl chloride and the like. In addition, the
reaction can be accelerated by subjecting a radical initiator such
as heat, light, benzoyl peroxide, azobisisobutyronitrile and the
like to the reaction system reaction.
[1121] When halogenation reaction of a hydroxy group is carried out
in each step, examples of the halogenating agent to be used include
hydrohalic acids and acid halides of inorganic acids, specifically,
hydrochloric acid, thionyl chloride, phosphorus oxychloride and the
like for chlorination, 48% hydrobromic acid and the like for
bromination. In addition, a method of producing an alkyl halide by
reacting an alcohol with triphenylphosphine and carbon
tetrachloride or carbon tetrabromide or the like can be employed.
Alternatively, a method of producing an alkyl halide via two step
comprising converting an alcohol to the corresponding sulfonate,
and then reacting the sulfonate with lithium bromide, lithium
chloride or sodium iodide can also be employed.
[1122] When Arbuzov reaction is carried out in each step, examples
of the reagent to be used include alkyl halides such as ethyl
bromoacetate and the like; phosphites such as triethyl phosphite,
tri(isopropyl) phosphite and the like, and the like.
[1123] When sulfonate esterification reaction is carried out in
each step, examples of the sulfonating agent to be used include
methanesulfonyl chloride, p-toluenesulfonyl chloride,
methanesulfonic anhydride, p-toluenesulfonic anhydride and the
like.
[1124] When hydrolysis reaction is carried out in each step, an
acid or a base is used as a reagent. Examples of the acid include
pyridine 2,2,2-trifluoroacetate. For acid hydrolysis reaction of
t-butyl ester, formic acid, triethylsilane and the like may be
added to reductively-trap t-butyl cation which is by-produced.
[1125] When dehydration reaction is carried out in each step,
examples of the dehydrating agent to be used include sulfuric acid,
diphosphorus pentaoxide, phosphorus oxychloride,
N,N'-dicyclohexylcarbodiimide, alumina, polyphosphoric acid and the
like.
[1126] Examples of the protecting group for a hydroxy group to be
used in each step include ether-type protecting groups such as
bis(4-methoxyphenyl)(phenyl)methyl ether and the like, in addition
to those exemplified above.
[1127] Examples of the protecting group for an amino group to be
used in each step include imidamide-type protecting groups such as
N,N-dimethylformimidamide and the like, in addition to those
exemplified above.
[1128] The deprotection reaction in each step can also be carried
out using acetic acid, 1,1,1,3,3,3-hexafluoropropan-2-ol,
triethylamine trihydrofluoride, methylamine, 2-methylpropan-2-amine
or hydrogen fluoride-pyridine, trifluoroacetic acid, instead of
those exemplified above.
[1129] When rearrangement reaction is carried out in each step,
examples of the reagent to be used include bases such as
triethylamine and the like.
[1130] When phosphoramidite reaction is carried out in each step, a
phosphoramiditing agent (e.g., phosphordiamidites such as
3-((bis(diisopropylamino)phosphino)oxy)propanenitrile and the like;
chlorophosphoramidites such as 2-cyanoethyl
diisopropylchlorophosphoramidite and the like) and a base (e.g.,
organic bases) are used as a reagent.
[1131] When thiophosphoramidite reaction is carried out in each
step, a thiophosphoramiditing agent (e.g., 2-cyanoethyl
dipropan-2-ylphosphoramidochloridothioite) and a base (e.g.,
organic bases) are used as a reagent.
[1132] When H-phosphonation reaction is carried out in each step,
examples of the H-phosphonating agent to be used include diphenyl
phosphite and the like.
[1133] When H-thiophosphonation reaction is carried out in each
step, examples of the H-thiophosphonating agent to be used include
a combination of an activator such as diphenyl phosphite and the
like and a sulfur atom source such as lithium sulfide and the like,
and the like.
[1134] When condensation reaction is carried out in each step,
examples of the activator to be used include pyridine
2,2,2-trifluoroacetate, 1H-tetrazole,
5-(ethylsulfanyl)-1H-tetrazole (hereinafter sometimes to be
referred to as 5-(ethylsulfanyl)-2H-tetrazole) and the like.
[1135] When oxidation reaction is carried out in each step,
examples of the oxidizing agent to be used include halogens such as
iodine and the like generally used for synthesis of nucleic acid,
and the like, in addition to those exemplified above.
[1136] When sulfurization reaction is carried out in each step,
examples of the sulfurizing agent to be used include
3H-benzo[c][1,2]dithiol-3-one, 3H-benzo[c][1,2]dithiol-3-one
1,1-dioxide,
((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione
and the like. In addition, for example, sulfur-2,6-lutidine
suspension, sulfur-carbon disulfide solution, tetramethylthiuram
disulfide (TETD) (H. Vu et al., Tetrahedron Lett., 32, 3005-3008
(1991)), Beauge's reagent (R. P. Lyer et al., J. Am. Chem. Soc.,
112, 1253-1254 (1990)) and Lawesson's reagent can also be used for
sulfurization reaction. Moreover, as a method of formation of
phosphorodithioate structure, the document by Marshall et al
(Science 259: 1564-1570, 1993) and the document by Caruthers and
Nielsen et al (WO 1989/011486) can be used as a reference.
[1137] When cyclization reaction is carried out in each step,
examples of the activator to be used include
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide, pivaloyl
chloride,
2-(benzoyltriazol-1-yloxy)-1,3-dimethyl-2-pyrrolidin-1-yl-1,3,2-diazaphos-
pholidinium hexafluorate (BOMP), N,N-bis(2-oxazolidinyl)phosphonic
chloride (BopCl), pyrophosphoric acid and the like. When an
activator is not used, the reaction may be carried out under
heating. In addition, a base may be added to the reaction system.
Examples of the base include organic bases and the like.
[1138] When bond-forming reaction between nucleobase and ribose is
carried out in each step, examples of the activator to be used
include trimethylsilyl N-(trimethylsilyl)acetimidate,
trimethylsilyl trifluoromethanesulfonate and the like.
[1139] When the partial structure:
##STR00149##
[1140] is a partial structure represented by the formula (IIA):
##STR00150##
compound (IA) can be produced from compound (1a) or (1b) and
compound (2a) or (2b) according to the below method.
##STR00151## ##STR00152##
wherein PG.sup.a is a hydroxy-protecting group, and the other
symbols are as defined above.
[1141] Alternatively, when the partial structure:
##STR00153##
is the partial structure represented by the formula (IIA), compound
(IA) can also be produced from compound (8a) or (8b) and compound
(9a) or (9b) according to the below method.
##STR00154## ##STR00155##
wherein PG.sup.b is a hydroxy-protecting group, and the other
symbols are as defined above.
[1142] When the partial structure:
##STR00156##
[1143] is a partial structure represented by the formula (IIB):
##STR00157##
compound (IB) can be produced from compound (15a) or (15b) and
compound (2a) or (2b) according to the below method.
##STR00158## ##STR00159##
wherein each symbol is as defined above.
[1144] Alternatively, when the partial structure:
##STR00160##
is the partial structure represented by the formula (IB), compound
(IB) can also be produced from compound (8a) or (8b) and compound
(21a) or (2 Ib) according to the below method.
##STR00161## ##STR00162##
wherein PG.sup.c is a hydroxy-protecting group, and the other
symbols are as defined above.
[1145] Preferable examples of the hydroxy-protecting group for
PG.sup.a, PG.sup.b or PG.sup.c include ether-type protecting groups
such as bis(4-methoxyphenyl)(phenyl)methyl ether,
3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl ether and the like.
[1146] When R.sup.1 and R.sup.2 are each a hydroxy group, the
hydroxy group may be protected. Preferable examples of the
hydroxy-protecting group include ether-type protecting groups such
as tert-butyldimethylsilyl ether,
3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl ether and the like.
[1147] When R.sup.1 is a hydroxyl group, the hydroxyl group and the
hydroxyl group represented by PG.sup.a may be protected by a
1,1,3,3-tetraisopropyldisiloxanyl group:
##STR00163##
[1148] In this case, a compound wherein the R.sup.1 hydroxyl group
is protected by a 3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl
group:
##STR00164##
is produced in the deprotection reaction for removal of
PG.sup.a.
[1149] When B.sup.1 has a functional group, the functional group
may be protected. For example, when B.sup.1 is a group represented
by
##STR00165##
and
[1150] R.sup.14 is --NH.sub.2 group, the --NH.sub.2 group may be
protected. In addition, for example, when B.sup.1 is a group
represented by
##STR00166##
the --NH.sub.2 group may be protected.
[1151] When B.sup.2 has a functional group, the functional group
may be protected. For example, when B.sup.2 is a group represented
by
##STR00167##
and
[1152] R.sup.24 is --NH.sub.2 group, the --NH.sub.2 group may be
protected. In addition, for example, when B.sup.2 is a group
represented by
##STR00168##
the --NH.sub.2 group may be protected.
[1153] Preferable examples of the protecting group for the
--NH.sub.2 group include amide-type protecting groups such as
benzamide, isobutylamide and the like, and imidamide-type
protecting groups such as N,N-dimethylformimidamide and the
like.
[1154] The above-mentioned compounds (1a), (1b), (9a) and (9b) can
be produced from compound (27) according to the below method.
##STR00169##
wherein each symbol is as defined above.
[1155] The above-mentioned compounds (2a), (2b), (8a) and (8b) can
be produced from compound (29) according to the below method.
##STR00170##
wherein each symbol is as defined above.
[1156] The above-mentioned compounds (15a), (15b), (21a) and (21b)
can be produced from compound (31) according to the below
method.
##STR00171##
wherein each symbol is as defined above.
[1157] The substituent of the thus-obtained compound (I) can be
subjected to modification (i.e., substituent introduction,
functional group transformation) according to a means known per se
to give the other compound or a salt thereof encompassed in
Compound (I). Known general methods can be employed for the
substituent introduction and functional group transformation, and
examples thereof include conversion of a halogen atom (e.g.,
fluorine, chlorine, bromine, iodine) or an optionally halogenated
C.sub.1-6 alkylsulfonyl-oxy group [e.g., methanesulfonyloxy,
ethanesulfonyloxy, trichloromethanesulfonyloxy,
trifluoromethanesulfonyloxy (triflate)] to a methyl group, a
cyclopropyl group, a vinyl group, a cyano group, a formyl group, a
carbonyl group, a carboxyl group, a hydroxy group, an amino group,
a boryl group and the like; conversion of a formyl group to an
ethynyl group by Seyferth-Gilbert homologation reaction; conversion
of an ester to a carboxy group by hydrolysis; conversion of a
carboxy group to a carbamoyl group by amidation; conversion of a
carboxy group to a hydroxymethyl group by reduction; conversion of
a carbonyl group to an alcohol by reduction or alkylation;
reductive amination of a carbonyl group; oximation of a carbonyl
group; acylation of an amino group; ureation of an amino group;
sulfonylation of an amino group; alkylation of an amino group;
replacement or amination of an activated halogen by an amine;
alkylation of a hydroxy group; and replacement or amination of a
hydroxy group.
[1158] In case of the substituent introduction and functional group
transformation, when the compound has a reactive moiety in which
undesirable reaction may occur, a protecting group may be
introduced into the reactive moiety in advance according to a
method known per se as necessary. By removing the protecting group
according to a method known per se after the objective reaction,
the compound encompassed in the present disclosure can be
produced.
[1159] For Example, when the raw material compound or intermediate
has an amino group, a carboxyl group or a hydroxy group, these
groups may be protected by a protecting group generally used in
peptide chemistry and the like. By removing the protecting group as
necessary after the reaction, the objective compound can be
obtained.
[1160] When compound (I) contains an isomer such as an optical
isomer, a stereoisomer, a regioisomer, a rotamer and the like, any
isomer and a mixture thereof are also encompassed in compound (I)
wherein. For example, when compound (I) contains an optical isomer,
an optical isomer resolved from racemate compound is also
encompassed in compound (I) wherein. These isomers can be obtained
as a single product according to a synthetic method known per se
and a separation method known per se (e.g., concentration, solvent
extraction, column chromatography and recrystallization).
[1161] Compound (I) may be a crystal, and a single crystal form and
a mixture of crystal forms are both encompassed in compound (I)
wherein. The crystal can be produced by crystallizing according to
a crystallization method known per se.
[1162] Compound (I) may be a pharmaceutically acceptable cocrystal
or a salt thereof. The cocrystal or a salt thereof means a
crystalline substance constituted with two or more special solids
at room temperature, each having different physical properties
(e.g., structure, melting point, melting heat, hygroscopicity and
stability). The cocrystal or a salt thereof can be produced
according to a cocrystallization method known per se.
[1163] Compound (I) may be a hydrate, a non-hydrate, a solvate or a
non-solvate, and they are all encompassed in compound (I).
[1164] Compound (I) also encompasses a compound labeled or
substituted with an isotope (e.g., .sup.2H, .sup.3H, .sup.11C,
.sup.14C, .sup.18F, .sup.35S, .sup.125I) and the like. The compound
labeled or substituted with an isotope may be used, for example, as
a tracer (PET tracer) used in positron emission tomography (PET),
and is useful in the field of medical diagnosis and the like.
[1165] Compound (I) may be a prodrug.
[1166] The prodrug of compound (I) means a compound which is
converted to compound (I) with a reaction due to an enzyme, gastric
acid and the like under the physiological condition in the living
body, that is, a compound which is converted to compound (I) by
enzymatic oxidation, reduction, hydrolysis and the like; a compound
which is converted to compound (I) by hydrolysis and the like due
to gastric acid, and the like.
[1167] Examples of the prodrug for compound (I) include
[1168] (1) a compound obtained by subjecting the amino in compound
(I) to acylation, alkylation or phosphorylation (e.g., a compound
obtained by subjecting the amino in compound (I) to
eicosanoylation, alanylation, pentylaminocarbonylation,
(5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxycarbonylation,
tetrahydrofuranylation, pyrrolidylmethylation,
pivaloyloxymethylation, tert-butylation, ethoxycarbonylation,
tert-butoxycarbonylation, acetylation or
cyclopropylcarbonylation);
[1169] (2) a compound obtained by subjecting the hydroxy in
compound (I) to acylation, alkylation, phosphorylation or boration
(e.g., a compound obtained by subjecting the hydroxy in compound
(I) to acetylation, palmitoylation, propanoylation, pivaloylation,
succinylation, fumarylation, alanylation or
dimethylaminomethylcarbonylation);
[1170] (3) a compound obtained by subjecting the carboxy in
compound (I) to esterification or amidation (e.g., a compound
obtained by subjecting the carboxy in compound (I) to ethyl
esterification, phenyl esterification, carboxymethyl
esterification, dimethylaminomethyl esterification,
pivaloyloxymethyl esterification, ethoxycarbonyloxyethyl
esterification, phthalidyl esterification,
(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl esterification,
cyclohexyloxycarbonylethyl esterification or methylamidation).
These compounds can be produced from compound (I) according to a
method known per se.
[1171] The prodrug of compound (I) may also be one which is
converted to compound (1) under physiological conditions as
described in "IYAKUHIN no KAIHATSU (Development of
Pharmaceuticals)", Vol. 7, Design of Molecules, p. 163-198,
Published by HIROKAWA SHOTEN (1990).
[1172] In the present specification, the prodrug may be in a form
of a salt, and examples of such salt include those similar to the
salts of the compound represented by the formula (I).
[1173] Compound (I) may also be used as a payload in an antibody
(or peptidic antigen recognition sequence)-drug conjugate (the
payload is the moiety corresponding to the above-mentioned drug).
When compound (I) is used as a payload, compound (I) may be bonded
to an antibody (or a peptidic antigen recognition sequence) via a
linker.
[1174] Examples of the above-mentioned payload include
(1) compound (I) wherein B.sup.1 is a group represented by
##STR00172##
and (2) compound (I) wherein B.sup.2 is a group represented by
##STR00173##
[1175] When compound (I) is used as a payload, compound (1) can be
converted an antibody (or peptidic antigen recognition
sequence)-drug conjugate, as follows.
(1) B.sup.1 in the formula (I) is converted to a group represented
by
##STR00174##
or
[1176] B.sup.2 in the formula (I) is converted to a group
represented by
##STR00175##
wherein
[1177] R.sup.a and R.sup.b are each independently
[1178] (i) a C.sub.1-6 alkyl group,
[1179] (ii) an acyl group, or
[1180] (iii) a group represented by the formula:
##STR00176##
wherein
[1181] R is a linker bonding to an antibody or a peptidic antigen
recognition sequence (the bond is a covalent bond between the
linker and the functional group in the side chain of the antibody
or peptidic antigen recognition sequence), and
[1182] each R.sup.1 is a hydrogen atom or a substituent.
[1183] (2) R.sup.1 and R.sup.2 in the formula (I) are each
independently converted to a group represented by the formula:
--OR.sup.3 wherein R.sup.3 is
[1184] (i) a C.sub.1-6 alkyl group,
[1185] (ii) an acyl group (preferably a C.sub.1-6 alkoxy-carbonyl
group),
[1186] (iii) a group represented by the formula:
##STR00177##
[1187] (iv) a group represented by the formula:
##STR00178##
[1188] (v) a group represented by the formula:
##STR00179##
[1189] (vi) a group represented by the formula:
##STR00180##
[1190] (vii) a group represented by the formula:
##STR00181##
[1191] (viii) a group represented by the formula:
##STR00182##
or
[1192] (ix) a group represented by the formula:
##STR00183##
[1193] wherein each R and each R' in the formulas are each as
defined above.
[1194] (3) Q.sup.2H and Q.sup.4H in the formula (I) are each
independently converted to
[1195] (i) a group represented by the formula: --SR.sup.4 wherein
R.sup.4 is
[1196] (a) a C.sub.1-6 alkyl group,
[1197] (b) an acyl group,
[1198] (c) a group represented by the formula: --SR,
[1199] (d) a group represented by the formula:
##STR00184##
[1200] (e) a group represented by the formula:
##STR00185##
[1201] (f) a group represented by the formula:
##STR00186##
[1202] (g) a group represented by the formula:
##STR00187##
[1203] (h) a group represented by the formula:
##STR00188##
or
[1204] (i) a group represented by the formula:
##STR00189##
[1205] (ii) a group represented by the formula: --OR.sup.5 wherein
R is
[1206] (a) a C.sub.1-6 alkyl group,
[1207] (b) an acyl group,
[1208] (c) a group represented by the formula:
##STR00190##
[1209] (d) a group represented by the formula:
##STR00191##
[1210] (e) a group represented by the formula:
##STR00192##
[1211] (f) a group represented by the formula:
##STR00193##
or
[1212] (iii) a group represented by the formula: --NHR.sup.6
wherein R.sup.6 is
[1213] (a) a C.sub.1-6 alkyl group,
[1214] (b) an acyl group,
[1215] (c) a group represented by the formula:
##STR00194##
[1216] (d) a group represented by the formula:
##STR00195##
or
[1217] (e) a group represented by the formula:
##STR00196##
[1218] wherein each R and each R' in the formulas are each as
defined above.
[1219] In addition, when compound (I) is used as a payload, the
linker described in Chem. Rev., 114, 9154-9218 (2014), Pharma. Res.
32, 3526-3540 (2015), Bioconjugate Chem. 21, 5-13 (2010), The AAPS
journal, 17, 339-351 (2015), WO 2011/005761 and the like may be
used.
[1220] The disclosure also provides the following particular
embodiments.
Embodiment 1
[1221] A compound having Formula (I):
##STR00197##
or a pharmaceutically acceptable salt thereof, wherein:
[1222] the partial structure represented by formula (A-1):
##STR00198##
[1223] is a partial structure represented by formula (IIA), or
formula (IIB):
##STR00199##
[1224] R.sup.1 and R.sup.2 are each independently a hydroxy group
or a halogen atom;
[1225] B.sup.1 is a group represented by formula (B.sup.1-A) or
formula (B.sup.1-B):
##STR00200##
[1226] R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each
independently a hydrogen atom or a substituent;
[1227] Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14, Y.sup.15 and
Y.sup.16 are each independently N or CR.sup.1a;
[1228] Z.sup.11, Z.sup.12, Z.sup.13, Z.sup.14, Z.sup.15 and
Z.sup.16 are each independently N or C;
[1229] R.sup.1a is a hydrogen atom or a substituent;
[1230] B.sup.2 is a group represented by formula (B.sup.2-A) or
formula (B.sup.2-B):
##STR00201##
[1231] R.sup.23, R.sup.24, R.sup.25, R.sup.26 and R.sup.27 are each
independently a hydrogen atom or a substituent;
[1232] Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24, Y.sup.25 and
Y.sup.26 are each independently N or CR.sup.2a;
[1233] Z.sup.21, Z.sup.22, Z.sup.23, Z.sup.24, Z.sup.25 and
Z.sup.26 are each independently N or C;
[1234] R.sup.2a is a hydrogen atom or a substituent;
[1235] X.sup.1 and X.sup.2 are each independently an oxygen atom or
a sulfur atom; and
[1236] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom.
provided that:
[1237] at least one of B.sup.1 or B.sup.2 is:
##STR00202##
wherein:
[1238] R.sup.18 is hydrogen or C.sub.1-6 alkyl; and
[1239] R.sup.19 a halogen atom.
Embodiment 2
[1240] The compound of Embodiment 1, or a pharmaceutically
acceptable salt thereof, wherein R.sup.19 is fluoro or chloro.
Embodiment 3
[1241] The compound of Embodiments 1 or 2, or a pharmaceutically
acceptable salt thereof, having formula (X):
##STR00203##
[1242] The compound of Embodiments 1 or 2, or a pharmaceutically
acceptable salt thereof, having formula (XI).
##STR00204##
Embodiment 5
[1243] The compound of any one of Embodiments 1-4, or a
pharmaceutically acceptable salt thereof, wherein R.sup.1 is a
hydroxy group.
Embodiment 6
[1244] The compound of any one of Embodiments 1-4, or a
pharmaceutically acceptable salt thereof, wherein R.sup.1 is fluoro
atom.
Embodiment 7
[1245] The compound of any one of Embodiments 1-3, 5, or 6, or a
pharmaceutically acceptable salt thereof, wherein R.sup.2 is a
hydroxyl group.
Embodiment 8
[1246] The compound of any one of Embodiments 1-3, 5, or 6, or a
pharmaceutically acceptable salt thereof, wherein R.sup.2 is a
fluoro atom.
Embodiment 9
[1247] The compound of any one of Embodiments 1-8, or a
pharmaceutically acceptable salt thereof, wherein Q.sup.4 is a
sulfur atom.
Embodiment 10
[1248] The compound of any one of Embodiments 1-9, or a
pharmaceutically acceptable salt thereof, wherein Q.sup.2 is an
oxygen atom.
Embodiment 11
[1249] The compound of any one of Embodiments 1-9, or a
pharmaceutically acceptable salt thereof, wherein Q.sup.2 is a
sulfur atom.
Embodiment 12
[1250] The compound of any one of Embodiments 1-3 or 5-11, or a
pharmaceutically acceptable salt thereof, having the formula
(XII):
##STR00205##
Embodiment 13
[1251] The compound of any one of Embodiments 1, 2, 4-6, or 9-11,
or a pharmaceutically acceptable salt thereof, having the formula
(XIII):
##STR00206##
Embodiment 14
[1252] The compound of any one of Embodiments 1-13, or a
pharmaceutically acceptable salt thereof, wherein B.sup.1 is:
##STR00207##
Embodiment 15
[1253] The compound of any one of Embodiments 1-13, or a
pharmaceutically acceptable salt thereof, wherein B.sup.2 is:
##STR00208##
Embodiment 16
[1254] The compound of any one of Embodiments 2-15, or a
pharmaceutically acceptable salt thereof, wherein R.sup.19 is a
fluoro atom.
Embodiment 17
[1255] The compound of any one of Embodiments 1-16, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
hydrogen.
Embodiment 18
[1256] The compound of any one of Embodiments 1-16, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
methyl.
Embodiment 19
[1257] The compound of any one of Embodiments 14 or 16-18, or a
pharmaceutically acceptable salt thereof, wherein B.sup.2 is
selected from the group consisting of:
##STR00209## ##STR00210##
[1258] each of which is optionally and independently substituted
at:
[1259] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1260] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 20
[1261] The compound of Embodiment 19, or a pharmaceutically
acceptable salt thereof, wherein B.sup.2 is selected from the group
consisting of:
##STR00211## ##STR00212## ##STR00213## ##STR00214##
Embodiment 21
[1262] The compound of Embodiment 20, or a pharmaceutically
acceptable salt thereof, wherein B.sup.2 is selected from the group
consisting of:
##STR00215##
Embodiment 22
[1263] The compound of Embodiment 21, or a pharmaceutically
acceptable salt thereof, wherein B.sup.2 is selected from the group
consisting of:
##STR00216##
Embodiment 23
[1264] The compound of any one of Embodiments 15-18, or a
pharmaceutically acceptable salt thereof, wherein B.sup.1 is
selected from the group consisting of:
##STR00217## ##STR00218##
each of which is optionally and independently substituted at:
[1265] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1266] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 24
[1267] The compound of Embodiment 23, or a pharmaceutically
acceptable salt thereof, wherein B.sup.1 is selected from the group
consisting of:
##STR00219## ##STR00220## ##STR00221## ##STR00222##
Embodiment 25
[1268] The compound of Embodiment 24, or a pharmaceutically
acceptable salt thereof, wherein B.sup.1 is selected from the group
consisting of:
##STR00223##
Embodiment 26
[1269] The compound of Embodiment 25, or a pharmaceutically
acceptable salt thereof, wherein B.sup.1 is selected from the group
consisting of:
##STR00224##
Embodiment 27
[1270] The compound of Embodiment 1, or a pharmaceutically
acceptable salt thereof, selected from the group consisting of:
##STR00225## ##STR00226## ##STR00227## ##STR00228##
Embodiment 28
[1271] The compound of Embodiment 27, or a pharmaceutically
acceptable salt thereof, selected from the group consisting of:
##STR00229## ##STR00230## ##STR00231##
Embodiment 29
[1272] The compound of Embodiment 27, or a pharmaceutically
acceptable salt thereof, selected from the group consisting of:
##STR00232##
Embodiment 30
[1273] The compound of any one of Embodiments 1-29, wherein the
pharmaceutically acceptable salt is the triethylamine salt or the
sodium salt e.g., the di-triethylamine salt or di-sodium salt.
Embodiment 31
[1274] A pharmaceutical composition comprising the compound of any
one of Embodiments 1-30, or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable excipient.
Embodiment 32
[1275] A compound having Formula (XIV):
(CD-L).sub.n-A (XIV)
or a pharmaceutically acceptable salt thereof, wherein:
[1276] CD is a group represented by any one of Formula
(XX)-(XXIX):
##STR00233## ##STR00234## ##STR00235##
[1277] R.sup.1 and R.sup.2 are each independently a hydroxy group,
hydrogen, amino group, or a halogen atom;
[1278] B.sup.3 and B.sup.4 are independently an optionally
substituted 5- to 14-membered aromatic heterocyclic group;
[1279] X.sup.1 and X.sup.2 are each independently an oxygen atom,
CH.sub.2, or a sulfur atom;
[1280] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom;
[1281] L is a linker;
[1282] A is an antibody, antibody fragment, or antigen-binding
fragment; and
[1283] n is 1-10.
Embodiment 33
[1284] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein R.sup.1 and R.sup.2 are each
independently a hydroxy group or a halogen atom.
Embodiment 34
[1285] The compound of Embodiments 32 or 33, or a pharmaceutically
acceptable salt thereof, wherein X.sup.1 and X.sup.2 are each
independently an oxygen atom or a sulfur atom.
Embodiment 35
[1286] The compound of any one of Embodiments 31-34, or a
pharmaceutically acceptable salt thereof, wherein Q.sup.1 and
Q.sup.3 are an oxygen atom.
Embodiment 36
[1287] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XX-A):
##STR00236##
Embodiment 37
[1288] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXI-A):
##STR00237##
Embodiment 38
[1289] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXII-A):
##STR00238##
Embodiment 39
[1290] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIII-A):
##STR00239##
Embodiment 40
[1291] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIV-A):
##STR00240##
Embodiment 41
[1292] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXV-A):
##STR00241##
Embodiment 42
[1293] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVI-A):
##STR00242##
Embodiment 43
[1294] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVII-A):
##STR00243##
Embodiment 44
[1295] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVIII-A):
##STR00244##
Embodiment 45
[1296] The compound of Embodiment 32, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIX-A):
##STR00245##
Embodiment 46
[1297] The compound of any one of Embodiments 32-45, or a
pharmaceutically acceptable salt thereof, wherein:
[1298] L is --X.sup.3-T-Z-Q-;
[1299] X.sup.3 is --(CH.sub.2).sub.o--,
##STR00246##
[1300] o is 1, 2, or 3; or
[1301] X.sup.3 is absent;
[1302] T is a peptide, or is absent;
[1303] Z is a spacer;
[1304] Q is a heterobifunctional group or heterotrifunctional
group, or is absent.
Embodiment 47
[1305] The compound of Embodiment 46, or a pharmaceutically
acceptable salt thereof, having the formula (XXX):
##STR00247##
Embodiment 48
[1306] The compound of Embodiment 46, or a pharmaceutically
acceptable salt thereof, having the formula (XXXI):
##STR00248##
Embodiment 49
[1307] The compound of any one of Embodiments 46-48, or a
pharmaceutically acceptable salt thereof, wherein:
[1308] X.sup.3 is
##STR00249##
[1309] T is
##STR00250##
and
[1310] R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl.
Embodiment 50
[1311] The compound of Embodiment 49, or a pharmaceutically
acceptable salt thereof, wherein:
[1312] X.sup.3 is
##STR00251##
Embodiment 51
[1313] The compound of any one of Embodiments 46-50, or a
pharmaceutically acceptable salt thereof, wherein:
[1314] Z is
##STR00252##
or --(CH.sub.2CH.sub.2O).sub.s--;
[1315] m is 1, 2, 3, 4, 5, or 6; and
[1316] s is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Embodiment 52
[1317] The compound of any one of Embodiments 46-48, or a
pharmaceutically acceptable salt thereof, wherein:
[1318] X.sup.3 is --CH.sub.2--;
[1319] Z is
##STR00253##
and
[1320] p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Embodiment 53
[1321] The compound of any one of Embodiments 46-48, or a
pharmaceutically acceptable salt thereof, wherein:
[1322] X.sup.3 is --CH.sub.2CH.sub.2--;
[1323] Z is
##STR00254##
[1324] q is 1, 2, 3, 4, 5, or 6; and
[1325] r is 1, 2, 3, 4, 5, or 6.
Embodiment 54
[1326] The compound of any one of Embodiments 46-53, or a
pharmaceutically acceptable salt thereof, wherein:
[1327] Q is a heterobifunctional group selected from the group
consisting of:
##STR00255##
[1328] R.sup.29 is hydrogen or C.sub.1-6 alkyl;
[1329] R.sup.30a and R.sup.30b are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, halo,
--C(.dbd.O)OR.sup.29, --NH.sub.2, C.sub.1-6 alkoxy, --CN,
--NO.sub.2, and --OH;
[1330] R.sup.31a and R.sup.31b are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, halo,
--C(.dbd.O)OR.sup.29, --NH.sub.2, --N(CH.sub.3).sub.2, C.sub.1-6
alkoxy, --CN, --NO.sub.2, and --OH, and
[1331] * indicates the attachment point to any available carbon
atom, nitrogen atom, oxygen atom, or sulfur atom attached to the
antibody, antibody fragment, or antigen-binding fragment.
Embodiment 55
[1332] The compound of Embodiment 51, or a pharmaceutically
acceptable salt thereof, having formula (XXXII):
##STR00256##
[1333] wherein:
[1334] R.sup.10a and R.sup.10b are independently C.sub.1-3 alkyl;
and
[1335] m is 2, 3, 4, or 5.
Embodiment 56
[1336] The compound of Embodiment 55, or a pharmaceutically
acceptable salt thereof, having formula (XXXIII):
##STR00257##
[1337] wherein p is 4, 5, or 6.
Embodiment 57
[1338] The compound of Embodiment 56, or a pharmaceutically
acceptable salt thereof, having formula (XXXIV):
##STR00258##
[1339] wherein:
[1340] q is 1, 2, or 3; and
[1341] r is 1, 2, or 3.
Embodiment 58
[1342] The compound of any one of Embodiments 32-57, or a
pharmaceutically acceptable salt thereof, wherein n is 2-8.
Embodiment 59
[1343] The compound of any one of Embodiments 32-58, or a
pharmaceutically acceptable salt thereof, wherein B.sup.3 and
B.sup.4 are independently an optionally substituted 8- to
14-membered fused bicyclic aromatic heterocyclic.
Embodiment 60
[1344] The compound of Embodiment 59, or a pharmaceutically
acceptable salt thereof, wherein:
[1345] B.sup.3 is a group represented by formula (B.sup.3-A) or
formula (B.sup.3-B):
##STR00259##
[1346] R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each
independently a hydrogen atom or a substituent;
[1347] Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14, Y.sup.15 and
Y.sup.16 are each independently N or CR.sup.1a;
[1348] Z.sup.11, Z.sup.12, Z.sup.13, Z.sup.14, Z.sup.15 and
Z.sup.16 are each independently N or C;
[1349] R.sup.1a is a hydrogen atom or a substituent;
[1350] B.sup.4 is a group represented by formula (B.sup.4-A) or
formula (B.sup.4-B):
##STR00260##
[1351] R.sup.23, R.sup.24, R.sup.25, R.sup.26 and R.sup.27 are each
independently a hydrogen atom or a substituent;
[1352] Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24, Y.sup.25 and
Y.sup.26 are each independently N or CR.sup.2a;
[1353] Z.sup.21, Z.sup.22, Z.sup.23, Z.sup.24, Z and Z.sup.26 are
each independently N or C;
[1354] R.sup.2a is a hydrogen atom or a substituent.
Embodiment 61
[1355] The compound of any one of Embodiments 32-60, or a
pharmaceutically acceptable salt thereof, wherein at least one of
B.sup.3 or B.sup.4 is:
##STR00261##
[1356] R.sup.18 is hydrogen or C.sub.1-6 alkyl; and
[1357] R.sup.19 is a halogen atom.
Embodiment 62
[1358] The compound of Embodiment 61, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is:
##STR00262##
Embodiment 63
[1359] The compound of Embodiment 61, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is:
##STR00263##
Embodiment 64
[1360] The compound of any one of Embodiments 61-63, or a
pharmaceutically acceptable salt thereof, wherein R.sup.19 is a
fluoro atom.
Embodiment 65
[1361] The compound of any one of Embodiments 61-64, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
hydrogen.
Embodiment 66
[1362] The compound of any one of Embodiments 61-64, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
methyl.
Embodiment 67
[1363] The compound of any one of Embodiments 62 or 64-66, or a
pharmaceutically acceptable salt thereof, wherein B.sup.4 is
selected from the group consisting of:
##STR00264## ##STR00265##
[1364] each of which is optionally and independently substituted
at:
[1365] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1366] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 68
[1367] The compound of Embodiment 67, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00266## ##STR00267## ##STR00268## ##STR00269##
Embodiment 69
[1368] The compound of Embodiment 68, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00270##
Embodiment 70
[1369] The compound of Embodiment 69, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00271##
Embodiment 71
[1370] The compound of any one of Embodiments 63-66, or a
pharmaceutically acceptable salt thereof, wherein B; is selected
from the group consisting of:
##STR00272## ##STR00273##
[1371] each of which is optionally and independently substituted
at:
[1372] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1373] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 72
[1374] The compound of Embodiment 71, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00274## ##STR00275## ##STR00276## ##STR00277##
Embodiment 73
[1375] The compound of Embodiment 72, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00278##
Embodiment 74
[1376] The compound of Embodiment 73, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00279##
Embodiment 75
[1377] The compound of any one of Embodiments 32-39, 44, or 46-74,
or a pharmaceutically acceptable salt thereof, wherein Q.sup.2 is
an oxygen atom.
Embodiment 76
[1378] The compound of any one of Embodiments 32-39, 44, or 46-74,
or a pharmaceutically acceptable salt thereof, wherein Q.sup.2 is a
sulfur atom.
Embodiment 77
[1379] The compound of any one of Embodiments 32-35, 40-43, 45, 46,
49-54, or 58-74, or a pharmaceutically acceptable salt thereof,
wherein Q.sup.4 is an oxygen atom.
Embodiment 78
[1380] The compound of any one of Embodiments 32-35, 40-43, 45, 46,
49-54, or 58-74, or a pharmaceutically acceptable salt thereof,
wherein Q.sup.4 is a sulfur atom.
Embodiment 79
[1381] The compound of any one of Embodiments 32-78, or a
pharmaceutically acceptable salt thereof, wherein X.sup.1 and
X.sup.2 are an oxygen atom and R.sup.1 and R.sup.2 are
independently a hydroxy group, a fluoro atom, or a chloro atom.
Embodiment 80
[1382] The compound of any one of Embodiments 32-79, or a
pharmaceutically acceptable salt thereof, wherein the antibody is
an anti-GCC antibody. Antibodies that bind to human guanylyl
cyclase C (GCC) are disclosed, e.g., in US 20130315923 and WO
2011050242.
Embodiment 81
[1383] The compound of Embodiment 80, or a pharmaceutically
acceptable salt thereof, wherein the antibody is an anti-GCC
antibody comprising a heavy chain region comprising amino acid
sequence SEQ. ID. No. 1.
Embodiment 82
[1384] The compound of Embodiments 80 or 81, or a pharmaceutically
acceptable salt thereof, wherein the antibody is an anti-GCC
antibody comprising a light chain region comprising amino acid
sequence SEQ. ID. No. 2.
Embodiment 83
[1385] A pharmaceutical composition comprising the compound of any
one of Embodiments 32-82, or a pharmaceutically acceptable salt
thereof, and a pharmaceutically acceptable excipient.
Embodiment 84
[1386] A method of treating a patient comprising administering to
the patient a therapeutically effective amount of the compound of
any one of Embodiments 1-30 or 32-82, or a pharmaceutically
acceptable salt thereof, wherein the patient has cancer.
Embodiment 85
[1387] The method of Embodiment 84, wherein the cancer is any one
or more of the cancers of Table 7.
Embodiment 86
[1388] The method of Embodiment 85, wherein the cancer is selected
from the group consisting of solid tumor and lymphoma.
Embodiment 87
[1389] The method of any one of Embodiments 84-86 further
comprising administering a therapeutically effective amount of a
second therapeutic agent useful in the treatment of cancer.
Embodiment 88
[1390] The pharmaceutical composition of Embodiments 31 or 83 for
use in treating cancer.
Embodiment 89
[1391] The pharmaceutical composition of Embodiment 88, wherein the
cancer is any one or more of the cancers of Table 7.
Embodiment 90
[1392] The pharmaceutical composition of Embodiment 88, wherein the
cancer is selected from the group consisting of solid tumor and
lymphoma.
Embodiment 91
[1393] A compound of any one of Embodiments 1-30 or 32-82, or a
pharmaceutically acceptable salt thereof, for use in treatment of
cancer.
Embodiment 92
[1394] The compound of Embodiment 91, wherein the cancer is any one
or more of the cancers of Table 7.
Embodiment 93
[1395] The compound of Embodiment 91, wherein the cancer is
selected from the group consisting of solid tumor and lymphoma.
Embodiment 94
[1396] Use of a compound of any one of Embodiments 1-30 or 32-82,
or a pharmaceutically acceptable salt thereof, for the manufacture
of a medicament for treatment of cancer.
Embodiment 95
[1397] The use of Embodiment 94, wherein the cancer is any one or
more of the cancers of Table 7.
Embodiment 96
[1398] The use of Embodiment 94, wherein the cancer is selected
from the group consisting of solid tumor and lymphoma.
Embodiment 97
[1399] A kit comprising the compound of any one of Embodiments 1-30
or 32-82, or a pharmaceutically acceptable salt, and instructions
for administering the compound, or a pharmaceutically acceptable
salt thereof, to a patient having cancer.
Embodiment 98
[1400] The kit of Embodiment 97, wherein the cancer is any one or
more of the cancers of Table 7.
Embodiment 99
[1401] The kit of Embodiment 97, wherein the cancer is selected
from the group consisting of solid tumor and lymphoma.
Embodiment 100
[1402] The kit of any one of Embodiments 97-99 further comprising
one or more additional therapeutic agents.
Embodiment 101
[1403] A compound having Formula (XL):
CD-L.sup.1-R.sup.28 (XL)
or a pharmaceutically acceptable salt thereof, wherein:
[1404] CD is a group represented by any one of Formula
(XX)-(XXIX):
##STR00280## ##STR00281##
[1405] R.sup.1 and R.sup.2 are each independently a hydroxy group,
hydrogen, amino group, or a halogen atom;
[1406] B.sup.3 and B.sup.4 are independently an optionally
substituted 5- to 14-membered aromatic heterocyclic group;
[1407] X.sup.1 and X.sup.2 are each independently an oxygen atom,
CH.sub.2, or a sulfur atom;
[1408] Q.sup.1, Q.sup.2, Q.sup.3 and Q.sup.4 are each independently
an oxygen atom or a sulfur atom;
[1409] L.sup.1 is a linker;
[1410] R.sup.28 is selected from the group consisting of --N.sub.3,
--ONH.sub.2, --OH, --CN, --NO.sub.2, --CHO,
--NR.sup.29C(.dbd.O)CH.dbd.CH.sub.2, --SH,
--S(.dbd.O).sub.2(CH.dbd.CH.sub.2), --NR.sup.29C(.dbd.O)CH.sub.2Br,
--NR.sup.29C(.dbd.O)CH.sub.2I, --C(.dbd.O)NHNH.sub.2, --CO.sub.2H,
--NH.sub.2, --C.dbd.CH,
##STR00282##
[1411] R.sup.29 is hydrogen or C.sub.1-6 alkyl;
[1412] R.sup.30a and R.sup.30b are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, halo,
--C(.dbd.O)OR.sup.29, --NH.sub.2, C.sub.1-6 alkoxy, --CN,
--NO.sub.2, and --OH;
[1413] R.sup.31a and R.sup.31b are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, halo,
--C(.dbd.O)OR.sup.29, --NH.sub.2, --N(CH.sub.3).sub.2, C.sub.1-6
alkoxy, --CN, --NO.sub.2, and --OH; and
[1414] the linker is attached to cyclic dinucleotide through any
available carbon, nitrogen, oxygen, or sulfur atom.
Embodiment 102
[1415] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein R.sup.1 and R.sup.2 are each
independently a hydroxy group or a halogen atom.
Embodiment 103
[1416] The compound of Embodiments 101 or 102, or a
pharmaceutically acceptable salt thereof, wherein X.sup.1 and
X.sup.2 are each independently an oxygen atom or a sulfur atom
Embodiment 104
[1417] The compound of any one of Embodiments 101-103, or a
pharmaceutically acceptable salt thereof, wherein Q.sup.1 and
Q.sup.3 are an oxygen atom.
Embodiment 105
[1418] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XX-A):
##STR00283##
Embodiment 106
[1419] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXI-A):
##STR00284##
Embodiment 107
[1420] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXII-A):
##STR00285##
Embodiment 108
[1421] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIII-A):
##STR00286##
Embodiment 109
[1422] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIV-A):
##STR00287##
Embodiment 110
[1423] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXV-A):
##STR00288##
Embodiment 111
[1424] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVI-A):
##STR00289##
Embodiment 112
[1425] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVII-A):
##STR00290##
Embodiment 113
[1426] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVIII-A):
##STR00291##
Embodiment 114
[1427] The compound of Embodiment 101, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIX-A):
##STR00292##
Embodiment 115
[1428] The compound of any one of Embodiments 101-114, or a
pharmaceutically acceptable salt thereof, wherein:
[1429] L.sup.1 is --X.sup.3-T-Z--;
[1430] X.sup.3 is --(CH.sub.2).sub.o--,
##STR00293##
[1431] o is 1, 2, or 3; or
[1432] X.sup.3 is absent;
[1433] T is a peptide, or is absent; and
[1434] Z is a spacer.
Embodiment 116
[1435] The compound of Embodiment 115, or a pharmaceutically
acceptable salt thereof, wherein:
[1436] X.sup.3 is
##STR00294##
[1437] T is
##STR00295##
and
[1438] R.sup.10a and R.sup.10b are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl.
Embodiment 117
[1439] The compound of Embodiment 116, or a pharmaceutically
acceptable salt thereof, wherein:
[1440] X.sup.3 is
##STR00296##
Embodiment 118
[1441] The compound of any one of Embodiments 115-117, or a
pharmaceutically acceptable salt thereof, wherein:
[1442] Z is
##STR00297##
or --(CH.sub.2CH.sub.2O).sub.s--;
[1443] m is 1, 2, 3, 4, 5, or 6; and
[1444] s is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Embodiment 119
[1445] The compound of Embodiment 115, or a pharmaceutically
acceptable salt thereof, wherein: X.sup.3 is --CH.sub.2--;
[1446] Z is
##STR00298##
and
[1447] p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Embodiment 120
[1448] The compound of Embodiment 115, or a pharmaceutically
acceptable salt thereof, wherein:
[1449] X.sup.3 is --CH.sub.2CH.sub.2--;
[1450] Z is
##STR00299##
[1451] q is 1, 2, 3, 4, 5, or 6; and
[1452] r is 1, 2, 3, 4, 5, or 6.
Embodiment 121
[1453] The compound of any one of Embodiments 101-120, or a
pharmaceutically acceptable salt thereof, wherein B.sup.3 and
B.sup.4 are independently an optionally substituted 8- to
14-membered fused bicyclic aromatic heterocyclic.
Embodiment 122
[1454] The compound of Embodiment 121, or a pharmaceutically
acceptable salt thereof, wherein:
[1455] B.sup.3 is a group represented by formula (B.sup.3-A) or
formula (B.sup.3-B):
##STR00300##
[1456] R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each
independently a hydrogen atom or a substituent;
[1457] Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14, Y.sup.15 and
Y.sup.16 are each independently N or CR.sup.1a;
[1458] Z.sup.11, Z.sup.12, Z.sup.13, Z.sup.14, Z.sup.15 and
Z.sup.16 are each independently N or C;
[1459] R.sup.1a is a hydrogen atom or a substituent;
[1460] B.sup.4 is a group represented by formula (B.sup.4-A) or
formula (B.sup.4-B):
##STR00301##
[1461] R.sup.23, R.sup.24, R.sup.25, R.sup.26 and R.sup.27 are each
independently a hydrogen atom or a substituent;
[1462] Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24, Y.sup.25 and
Y.sup.26 are each independently N or CR.sup.2a;
[1463] Z.sup.21, Z.sup.22, Z.sup.23, Z.sup.24, Z.sup.25 and
Z.sup.26 are each independently N or C;
[1464] R.sup.2a is a hydrogen atom or a substituent.
Embodiment 123
[1465] The compound of any one of Embodiments 101-122, or a
pharmaceutically acceptable salt thereof, wherein at least one of
B.sup.3 or B.sup.4 is:
##STR00302##
[1466] R.sup.18 is hydrogen or C.sub.1-6 alkyl; and
[1467] R.sup.19 is a halogen atom.
Embodiment 124
[1468] The compound of Embodiment 123, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is:
##STR00303##
Embodiment 125
[1469] The compound of Embodiment 123, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is:
##STR00304##
Embodiment 126
[1470] The compound of any one of Embodiments 123-125, or a
pharmaceutically acceptable salt thereof, wherein R.sup.19 is a
fluoro atom.
Embodiment 127
[1471] The compound of any one of Embodiments 123-126, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
hydrogen.
Embodiment 128
[1472] The compound of any one of Embodiments 123-126, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
methyl.
Embodiment 129
[1473] The compound of any one of Embodiments 124 or 126-128, or a
pharmaceutically acceptable salt thereof, wherein B.sup.4 is
selected from the group consisting of:
##STR00305## ##STR00306##
each of which is optionally and independently substituted at:
[1474] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1475] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 130
[1476] The compound of Embodiment 129, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00307## ##STR00308## ##STR00309## ##STR00310##
Embodiment 131
[1477] The compound of Embodiment 130, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00311##
Embodiment 132
[1478] The compound of Embodiment 131, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00312##
Embodiment 133
[1479] The compound of any one of Embodiments 125-128, or a
pharmaceutically acceptable salt thereof, wherein B.sup.3 is
selected from the group consisting of:
##STR00313## ##STR00314##
each of which is optionally and independently substituted at:
[1480] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1481] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 134
[1482] The compound of Embodiment 133, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00315## ##STR00316## ##STR00317## ##STR00318##
Embodiment 135
[1483] The compound of Embodiment 134, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00319##
Embodiment 136
[1484] The compound of Embodiment 135, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00320##
Embodiment 137
[1485] The compound of any one of Embodiments 101-108, 113, or
115-136, or a pharmaceutically acceptable salt thereof, wherein
Q.sup.2 is an oxygen atom.
Embodiment 138
[1486] The compound of any one of Embodiments 101-108, 113, or
115-136, or a pharmaceutically acceptable salt thereof, wherein
Q.sup.2 is a sulfur atom.
Embodiment 139
[1487] The compound of any one of Embodiments 101-104, 109-112,
114, or 115-136, or a pharmaceutically acceptable salt thereof,
wherein Q.sup.4 is an oxygen atom.
Embodiment 140
[1488] The compound of any one of Embodiments 101-04, 109-112, 114,
or 115-136, or a pharmaceutically acceptable salt thereof, wherein
Q.sup.4 is a sulfur atom.
Embodiment 141
[1489] The compound of any one of Embodiments 101-142, or a
pharmaceutically acceptable salt thereof, wherein X.sup.1 and
X.sup.2 are an oxygen atom and R.sup.1 and R.sup.2 are
independently a hydroxy group or a halogen atom.
Embodiment 142
[1490] The compound of any one of Embodiments 101-141, wherein
R.sup.2 is
##STR00321##
Embodiment 143
[1491] The compound of Embodiment 142, selected from the group
consisting of:
##STR00322##
Embodiment 144
[1492] A compound having Formula (XLI):
(CD-L.sup.2).sub.u-DA (XLI)
[1493] or a pharmaceutically acceptable salt thereof, wherein:
[1494] CD is a group represented by any one of Formula
(XX)-(XXIX):
##STR00323## ##STR00324## ##STR00325##
[1495] R.sup.1 and R.sup.2 are each independently a hydroxy group,
hydrogen, amino group, or a halogen atom;
[1496] B.sup.3 and B.sup.4 are independently an optionally
substituted 5- to 14-membered aromatic heterocyclic group;
[1497] X.sup.1 and X.sup.2 are each independently an oxygen atom,
CH.sub.2, or a sulfur atom;
[1498] Q.sup.1, Q.sup.2, Q.sup.3, and Q.sup.4 are each
independently an oxygen atom or a sulfur atom;
[1499] L.sup.2 is a linker; or
[1500] L.sup.2 is absent;
[1501] DA is a drug delivery agent; and
[1502] u is 1-1000.
Embodiment 145
[1503] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein R.sup.1 and R.sup.2 are each
independently a hydroxy group or a halogen atom.
Embodiment 146
[1504] The compound of Embodiments 144 or 145, or a
pharmaceutically acceptable salt thereof, wherein X.sup.1 and
X.sup.2 are each independently an oxygen atom or a sulfur atom.
Embodiment 147
[1505] The compound of any one of Embodiments 144-146, or a
pharmaceutically acceptable salt thereof, wherein Q.sup.1 and
Q.sup.3 are an oxygen atom.
Embodiment 148
[1506] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XX-A):
##STR00326##
Embodiment 149
[1507] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXI-A):
##STR00327##
Embodiment 150
[1508] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXII-A):
##STR00328##
Embodiment 151
[1509] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIII-A):
##STR00329##
Embodiment 152
[1510] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIV-A):
##STR00330##
Embodiment 152
[1511] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXV-A):
##STR00331##
Embodiment 153
[1512] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVI-A):
##STR00332##
Embodiment 154
[1513] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVII-A):
##STR00333##
Embodiment 155
[1514] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXVIII-A):
##STR00334##
Embodiment 156
[1515] The compound of Embodiment 144, or a pharmaceutically
acceptable salt thereof, wherein CD is group represented by Formula
(XXIX-A):
##STR00335##
Embodiment 157
[1516] The compound of any one of Embodiments 144-156, or a
pharmaceutically acceptable salt thereof, wherein B.sup.3 and
B.sup.4 are independently an optionally substituted 8- to
14-membered fused bicyclic aromatic heterocyclic.
Embodiment 158
[1517] The compound of Embodiment 157, or a pharmaceutically
acceptable salt thereof, wherein:
[1518] B.sup.3 is a group represented by formula (B.sup.3-A) or
formula (B.sup.3-B):
##STR00336##
[1519] R.sup.13, R.sup.14, R.sup.15, R.sup.16 and R.sup.17 are each
independently a hydrogen atom or a substituent;
[1520] Y.sup.11, Y.sup.12, Y.sup.13, Y.sup.14, Y.sup.15 and
Y.sup.16 are each independently N or CR.sup.1a;
[1521] Z.sup.11, Z.sup.12, Z.sup.13, Z.sup.14, Z.sup.15 and
Z.sup.16 are each independently N or C;
[1522] R.sup.1a is a hydrogen atom or a substituent;
[1523] B.sup.4 is a group represented by formula (B.sup.4-A) or
formula (B.sup.4-B):
##STR00337##
[1524] R.sup.23, R.sup.24, R.sup.25, R.sup.26 and R.sup.21 are each
independently a hydrogen atom or a substituent;
[1525] Y.sup.21, Y.sup.22, Y.sup.23, Y.sup.24, Y.sup.25 and
Y.sup.26 are each independently N or CR.sup.2a;
[1526] Z.sup.21, Z.sup.22, Z.sup.23, Z.sup.24, Z.sup.25 and
Z.sup.26 are each independently N or C;
[1527] R.sup.2a is a hydrogen atom or a substituent.
Embodiment 159
[1528] The compound of any one of Embodiments 144-158, or a
pharmaceutically acceptable salt thereof, wherein at least one of
B.sup.3 or B.sup.4 is:
##STR00338##
[1529] R.sup.18 is hydrogen or C.sub.1-6 alkyl; and
[1530] R.sup.19 is a halogen atom.
Embodiment 160
[1531] The compound of Embodiment 159, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is:
##STR00339##
Embodiment 161
[1532] The compound of Embodiment 159, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is:
##STR00340##
Embodiment 162
[1533] The compound of any one of Embodiments 159-161, or a
pharmaceutically acceptable salt thereof, wherein R.sup.19 is a
fluoro atom.
Embodiment 163
[1534] The compound of any one of Embodiments 159-162, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
hydrogen.
Embodiment 164
[1535] The compound of any one of Embodiments 159-162, or a
pharmaceutically acceptable salt thereof, wherein R.sup.18 is
methyl.
Embodiment 165
[1536] The compound of any one of Embodiments 159 or 162-164, or a
pharmaceutically acceptable salt thereof, wherein B.sup.4 is
selected from the group consisting of:
##STR00341## ##STR00342##
[1537] each of which is optionally and independently substituted
at:
[1538] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1539] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 166
[1540] The compound of Embodiment 165, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00343## ##STR00344## ##STR00345## ##STR00346##
Embodiment 167
[1541] The compound of Embodiment 166, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00347##
Embodiment 168
[1542] The compound of Embodiment 167, or a pharmaceutically
acceptable salt thereof, wherein B.sup.4 is selected from the group
consisting of:
##STR00348##
Embodiment 169
[1543] The compound of any one of Embodiments 161-164, or a
pharmaceutically acceptable salt thereof, wherein B is selected
from the group consisting of:
##STR00349## ##STR00350##
[1544] each of which is optionally and independently substituted
at:
[1545] (i) any available carbon atom with a halo, C.sub.1-6 alkyl,
C.sub.1-6 alkoxy, C.sub.1-6 alkylthio, or amino group; and/or
[1546] (ii) any available nitrogen atom with a C.sub.1-6 alkyl
group.
Embodiment 170
[1547] The compound of Embodiment 169, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00351## ##STR00352## ##STR00353## ##STR00354##
Embodiment 171
[1548] The compound of Embodiment 170, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00355##
Embodiment 172
[1549] The compound of Embodiment 171, or a pharmaceutically
acceptable salt thereof, wherein B.sup.3 is selected from the group
consisting of:
##STR00356##
Embodiment 173
[1550] The compound of any one of Embodiments 144-151, 155, or
157-172, or a pharmaceutically acceptable salt thereof, wherein
Q.sup.2 is an oxygen atom.
Embodiment 174
[1551] The compound of any one of Embodiments 144-151, 155, or
157-172, or a pharmaceutically acceptable salt thereof, wherein
Q.sup.2 is a sulfur atom.
Embodiment 175
[1552] The compound of any one of Embodiments 144-147, 152-154,
156, or 157-172, or a pharmaceutically acceptable salt thereof,
wherein Q.sup.4 is an oxygen atom.
Embodiment 176
[1553] The compound of any one of Embodiments 144-147, 152-154,
156, or 157-172, or a pharmaceutically acceptable salt thereof,
wherein Q.sup.4 is a sulfur atom.
Embodiment 177
[1554] The compound of any one of Embodiments 144-176, or a
pharmaceutically acceptable salt thereof, wherein X.sup.1 and
X.sup.2 are an oxygen atom and R.sup.1 and R.sup.2 are
independently a hydroxy group, a fluoro atom, or a chloro atom.
Embodiment 178
[1555] The compound of any one of Embodiments 144-177, or a
pharmaceutically acceptable salt thereof, wherein u is 1-100.
Embodiment 179
[1556] The compound of Embodiment 178, or a pharmaceutically
acceptable salt thereof, wherein u is 1-10.
Embodiment 180
[1557] The compound of Embodiment 179, or a pharmaceutically
acceptable salt thereof, wherein u is 1-5.
Embodiment 181
[1558] The compound of Embodiment 180, or a pharmaceutically
acceptable salt thereof, wherein u is 1.
Embodiment 182
[1559] The compound of any one of Embodiments 144-181, or a
pharmaceutically acceptable salt thereof, wherein:
[1560] L.sup.2 is --X.sup.4-T.sup.1-Z.sup.1-Q.sup.A-;
[1561] X.sup.4 is --(CH.sub.2).sub.v--,
##STR00357##
[1562] v is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or
[1563] X.sup.4 is absent;
[1564] T.sup.1 is a peptide, or is absent;
[1565] Z is a spacer;
[1566] Q.sup.A is a heterobifunctional group or heterotrifunctional
group, or is absent.
Embodiment 183
[1567] The compound of Embodiment 182, or a pharmaceutically
acceptable salt thereof, wherein:
[1568] X.sup.4 is
##STR00358##
[1569] T.sup.1 is
##STR00359##
and
[1570] R.sup.10c and R.sup.10d are independently selected from the
group consisting of hydrogen and optionally substituted C.sub.1-6
alkyl.
Embodiment 184
[1571] The compound of Embodiment 183, or a pharmaceutically
acceptable salt thereof, wherein:
[1572] X.sup.4 is
##STR00360##
Embodiment 185
[1573] The compound of any one of Embodiments 182-184, or a
pharmaceutically acceptable salt thereof, wherein:
[1574] Z.sup.1 is
##STR00361##
or --(CH.sub.2CH.sub.2O).sub.s1--;
[1575] m.sub.1 is 1, 2, 3, 4, 5, or 6; and
[1576] s.sub.1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Embodiment 186
[1577] The compound of Embodiment 182, or a pharmaceutically
acceptable salt thereof, wherein:
[1578] X.sup.4 is --CH.sub.2--;
[1579] Z1 is
##STR00362##
and
[1580] p1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
Embodiment 187
[1581] The compound of Embodiment 182, or a pharmaceutically
acceptable salt thereof, wherein:
[1582] X.sup.4 is --CH.sub.2CH.sub.2--;
[1583] Z.sup.1 is
##STR00363##
[1584] q.sub.1 is 1, 2, 3, 4, 5, or 6; and
[1585] r.sub.1 is 1, 2, 3, 4, 5, or 6.
Embodiment 188
[1586] The compound of any one of Embodiments 182, 186, or 187, or
a pharmaceutically acceptable salt thereof, wherein T.sup.1 is
absent.
Embodiment 189
[1587] The compound of any one of Embodiments 182-188, or a
pharmaceutically acceptable salt thereof, wherein Q.sup.A is a
heterobifunctional group selected from the group consisting of:
##STR00364##
[1588] R.sup.29 is hydrogen or C.sub.1-6 alkyl;
[1589] R.sup.30a and R.sup.30b are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, halo,
--C(.dbd.O)OR.sup.29, --NH.sub.2, C.sub.1-6 alkoxy, --CN,
--NO.sub.2, and --OH;
[1590] R.sup.31a and R.sup.31b are independently selected from the
group consisting of hydrogen, C.sub.1-6 alkyl, halo,
--C(.dbd.O)OR.sup.29, --NH.sub.2, --N(CH.sub.3).sub.2, C.sub.1-6
alkoxy, --CN, --NO.sub.2, and --OH; and
[1591] * indicates the attachment point to any available carbon
atom, nitrogen atom, oxygen atom, or sulfur atom attached to the
drug delivery agent.
Embodiment 190
[1592] The compound of Embodiment 182, or a pharmaceutically
acceptable salt thereof, wherein X.sup.4, T.sup.1, and Q.sup.A are
absent.
Embodiment 191
[1593] The compound of any one of Embodiments 144-190, or a
pharmaceutically acceptable salt thereof, wherein the drug delivery
agent comprises lipids, steroids, vitamins, carbohydrates,
proteins, peptides, polyamines, polyethylene glycols, or
peptidomimetics, or any combinations thereof.
Embodiment 192
[1594] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
lipids.
Embodiment 193
[1595] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
steroids.
Embodiment 194
[1596] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
vitamins.
Embodiment 195
[1597] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
carbohydrates.
Embodiment 196
[1598] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
proteins.
Embodiment 197
[1599] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
peptides.
Embodiment 198
[1600] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
polyamines.
Embodiment 199
[1601] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
polyethylene glycols.
Embodiment 200
[1602] The compound of Embodiment 191, or a pharmaceutically
acceptable salt thereof, wherein the drug delivery agent comprises
peptidomimetics
[1603] Since compound (I) or a prodrug thereof (in the present
specification, sometimes to be collectively abbreviated as "the
compound of the present disclosure") has a STING agonistic
activity, it may be useful as an agent for the prophylaxis or
treatment of cancer, a cancer growth inhibitor or a cancer
metastasis inhibitor.
[1604] Since the compound of the present disclosure shows a STING
agonistic activity, and is superior in terms of efficacy
expression, pharmacokinetics (e.g., absorption, distribution,
metabolism, excretion), solubility (e.g., water solubility),
interaction with other medicaments (e.g., drug-metabolizing enzyme
inhibitory action), safety (e.g., acute toxicity, chronic toxicity,
genetic toxicity, reproductive toxicity, cardiotoxicity,
carcinogenicity, central toxicity) and stability (e.g., chemical
stability, stability to an enzyme), it may be useful as a
medicament.
[1605] Therefore, the compound of the present disclosure may be
used for increased STING activity in a mammal (e.g., mouse, rat,
hamster, rabbit, cat, dog, cow, sheep, monkey, human).
[1606] The compound of the present disclosure may be used as a
medicament such as an agent for the prophylaxis or treatment of
diseases possibly influenced by STING (in the present
specification, sometimes to be abbreviated as "STING-related
diseases"), for example, cancers [e.g., colorectal cancers (e.g.,
colorectal cancer, rectal cancer, anus cancer, familial colorectal
cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal
stromal tumor), lung cancers (e.g., non-small-cell lung cancer,
small-cell lung cancer, malignant mesothelioma), mesothelioma,
pancreatic cancers (e.g., pancreatic ductal carcinoma, pancreatic
endocrine tumor), pharynx cancer, larynx cancer, esophageal cancer,
stomach cancers (e.g., papillary adenocarcinoma, mucinous
adenocarcinoma, adenosquamous carcinoma), duodenal cancer, small
intestinal cancer, breast cancers (e.g., invasive ductal carcinoma,
non-invasive ductal carcinoma, inflammatory breast cancer), ovarian
cancers (e.g., ovarian epithelial cancer, extragonadal germ cell
tumor, ovarian germ cell tumor, ovarian low-malignant potential
tumor), testis tumor, prostate cancers (e.g., hormone-dependent
prostate cancer, non-hormone dependent prostate cancer,
castration-resistant prostate cancer), liver cancers (e.g.,
hepatocellular cancer, primary liver cancer, extrahepatic bile duct
cancer), thyroid cancers (e.g., medullary thyroid carcinoma), renal
cancers (e.g., renal cell cancers (e.g., clear cell renal cell
cancer), transitional cell cancer of renal pelvis and ureter),
uterine cancers (e.g., cervical cancer, uterine body cancer, uterus
sarcoma), gestational choriocarcinoma, brain tumors (e.g.,
medulloblastoma, glioma, pineal astrocytic tumors, pilocytic
astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, pituitary
adenoma), retinoblastoma, skin cancers (e.g., basalioma, malignant
melanoma), sarcomas (e.g., rhabdomyosarcoma, leiomyosarcoma, soft
tissue sarcoma, spindle cell sarcoma), malignant bone tumor,
bladder cancer, blood cancers (e.g., multiple myeloma, leukemias
(e.g., acute myelogenous leukemia), malignant lymphoma, Hodgkin's
disease, chronic myeloproliferative disease), cancer of unknown
primary], a cancer growth inhibitor, a cancer metastasis inhibitor,
an apoptosis promoter, an agent for the treatment of precancerous
lesions (e.g., myelodysplastic syndromes), and the like.
[1607] In another embodiment, the cancer is selected from any one
or more of the cancers of Table 7.
TABLE-US-00002 TABLE 7 adrenal cancer acinic cell carcinoma
acoustic neuroma acral lentigious melanoma acrospiroma acute
eosinophilic leukemia acute erythroid leukemia acute lymphoblastic
leukemia acute megakaryoblastic leukemia acute monocytic leukemia
acute promyelocytic leukemia adenocarcinoma adenoid cystic
carcinoma adenoma adenomatoid odontogenic tumor adenosquamous
carcinoma adipose tissue neoplasm adrenocortical carcinoma adult
T-cell leukemia/lymphoma aggressive NK-cell leukemia AIDS-related
lymphoma alveolar rhabdomyosarcoma alveolar soft part sarcoma
ameloblastic fibroma anaplastic large cell lymphoma anaplastic
thyroid cancer angioimmunoblastic T-cell lymphoma, angiomyolipoma
angiosarcoma astrocytoma atypical teratoid rhabdoid tumor B-cell
chronic lymphocytic leukemia B-cell prolymphocytic leukemia B-cell
lymphoma basal cell carcinoma biliary tract cancer bladder cancer
blastoma bone cancer Brenner tumor Brown tumor Burkitt's lymphoma
breast cancer brain cancer carcinoma carcinoma in situ
carcinosarcoma cartilage tumor cementoma myeloid sarcoma chondroma
chordoma choriocarcinoma choroid plexus papilloma clear-cell
sarcoma of the kidney crani opharyngioma cutaneous T-cell lymphoma
cervical cancer colorectal cancer Degos disease desmoplastic small
round cell tumor diffuse large B-cell lymphoma dysembryoplastic
neuroepithelial tumor, dysgerminoma embiyonal carcinoma endocrine
gland neoplasm endodermal sinus tumor enteropathy-associated T-cell
lymphoma esophageal cancer fetus in fetu fibroma fibrosarcoma
follicular lymphoma follicular thyroid cancer ganglioneuroma
gastrointestinal cancer germ cell tumor gestational choriocarcinoma
giant cell fibroblastoma giant cell tumor of the bone glial tumor
glioblastoma multiforme glioma gliomatosis cerebri glucagonoma
gonadoblastoma granulosa cell tumor gynandroblastoma gallbladder
cancer gastric cancer hairy cell leukemia hemangioblastoma head and
neck cancer hemangiopericytoma hematological malignancy
hepatoblastoma hepatosplenic T-cell lymphoma Hodgkin's lymphoma
non-Hodgkin's lymphoma invasive lobular carcinoma intestinal cancer
kidney cancer laryngeal cancer lentigo maligna. lethal midline
carcinoma leukemia leydig cell tumor liposarcoma lung cancer
lymphangioma lymphangiosarcoma lymphoepithelioma lymphoma acute
lymphocytic leukemia acute myelogeous leukemia chronic lymphocytic
leukemia liver cancer small cell lung cancer non-small cell lung
cancer MALT lymphoma malignant fibrous histiocytoma malignant
peripheral nerve sheath tumor malignant triton tumor mantle cell
lymphoma marginal zone B-cell lymphoma mast cell leukemia
mediastinal germ cell tumor medullary carcinoma of the breast
medullary thyroid cancer, medulloblastoma melanoma, meningioma,
merkel cell cancer mesothelioma metastatic urothelial carcinoma
mixed Mullerian tumor mucinous tumor multiple myeloma muscle tissue
neoplasm mycosis fungoides myxoid liposarcoma myxoma myxosarcoma
nasopharyngeal carcinoma neurinoma neuroblastoma neurofibroma
neuroma nodular melanoma ocular cancer oligoastrocytoma
oligodendroglioma oncocytoma optic nerve sheath meningioma optic
nerve tumor oral cancer osteosarcoma ovarian cancer Pancoast tumor
papillary thyroid cancer paraganglioma pinealoblastoma pineocytoma
pituicytoma pituitary adenoma pituitary tumor plasmacytoma polyet
bryoma precursor T-lymphoblastic lymphoma primary central nervous
system lymphoma primary effusion lymphoma preimary peritoneal
cancer prostate cancer pancreatic cancer pharyngeal cancer
pseudomyxoma periotonei renal cell carcinoma renal medullary
carcinoma retinoblastoma rhabdomyoma rhabdomyosarcoma Richter's
transformation rectal cancer sarcoma Schwannomatosis seminoma
Sertoli cell tumor sex cord-gonadal stromal tumor signet ring cell
carcinoma skin cancer small blue round cell tumors small cell
carcinoma soft tissue sarcoma somatostatinoma soot wart spinal
tumor splenic marginal zone lymphoma squamous cell carcinoma
synovial sarcoma Sezary's disease small intestine cancer squamous
carcinoma stomach cancer T-cell lymphoma testicular cancer thecoma
thyroid cancer transitional cell carcinoma throat cancer urachal
cancer urogenital cancer urothelial carcinoma uveal melanoma
uterine cancer verrucous carcinoma visual pathway glioma vulvar
cancer vaginal cancer Waldenstrom's macroglobulinemia Warthin's
tumor Wilms' tumor
[1608] In another embodiment, the cancer is a solid tumor or
lymphoma.
[1609] Particularly, the compound of the present disclosure may be
used as a medicament for colorectal cancer, breast cancer, skin
cancer, malignant lymphoma or lung cancer.
[1610] The compound of the present disclosure may be administered
orally or parenterally as it is or in a mixture with a
pharmacologically acceptable carrier as a medicament, to a mammal
(preferably humans).
[1611] The medicament containing the compound of the present
disclosure (hereinafter sometimes to be abbreviated as "the
medicament of the present disclosure") is explained in detail
below. Examples of the dosage form of the medicament of the present
disclosure include oral preparations such as tablet (e.g.,
sugar-coated tablet, film-coated tablet, sublingual tablet, buccal,
orally disintegrating tablet), pill, granule, powder, capsule
(e.g., soft capsule, microcapsule), syrup, emulsion, suspension,
films (e.g., orally disintegrable films, oral mucosa-adhesive film)
and the like. In addition, examples of the dosage form of the
medicament of the present disclosure include parenteral
preparations such as injection, drip infusion, transdermal
absorption type preparation (e.g., iontophoresis transdermal
absorption type preparation), suppository, ointment, nasal
preparation, pulmonary preparation, eye drop and the like.
Moreover, the medicament of the present disclosure may be a release
control preparation such as an immediate-release preparation, a
sustained-release preparation (e.g., a sustained-release
microcapsule) and the like.
[1612] As the dosage form of the medicament of the present
disclosure, a nanoparticle preparation and a preparation using a
bacteria-derived membrane can also be used.
[1613] The medicament of the present disclosure may be prepared
according to a method known per se (e.g., the method described in
the Japanese Pharmacopoeia etc.) generally used in the field of
preparation. In addition, the medicament of the present disclosure
may contain a suitable amount of an additive such as a excipient, a
binder, a disintegrant, a lubricant, a sweetening agent, a
surfactant, a suspending agent, an emulsifier, a colorant, a
preservative, an aromatic, a corrigent, a stabilizer, a thickening
agent and the like generally used in the field of preparation as
necessary. Examples of the pharmacologically acceptable carrier
include these additives.
[1614] For example, tablet may be prepared using an excipient, a
binder, a disintegrant, a lubricant and the like, and pill or
granule may be prepared using an excipient, a binder and a
disintegrant. Powder or capsule may be prepared using an excipient
and the like, syrup may be prepared using a sweetening agent and
the like, and emulsion or suspension may be prepared using a
suspending agent, a surfactant, an emulsifier and the like.
[1615] Examples of the excipient include lactose, sucrose, glucose,
starch, sucrose, crystalline cellulose, powdered glycyrrhiza,
mannitol, sodium hydrogencarbonate, calcium phosphate and calcium
sulfate.
[1616] Examples of the binder include 5 to 10 wt % starch liquid
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.
[1617] Examples of the disintegrant include starch and calcium
carbonate.
[1618] Examples of the lubricant include magnesium stearate,
stearic acid, calcium stearate and purified talc.
[1619] Examples of the sweetener include glucose, fructose, invert
sugar, sorbitol, xylitol, glycerin and simple syrup.
[1620] Examples of the surfactant include sodium lauryl sulfate,
polysorbate 80, sorbitan monofatty acid ester and polyoxyl 40
stearate.
[1621] Examples of the suspending agent include gum arabic, sodium
alginate, sodium carboxymethyl cellulose, methyl cellulose and
bentonite.
[1622] Examples of the emulsifier include gum arabic, tragacanth,
gelatin and polysorbate 80.
[1623] For example, when the medicament of present disclosure is a
tablet, for example, an excipient (e.g., lactose, sucrose, starch),
a disintegrant (e.g., starch, calcium carbonate), a binder (e.g.,
starch, gum arabic, carboxymethyl cellulose, polyvinyl pyrrolidone,
hydroxypropyl cellulose) or a lubricant (e.g., talc, magnesium
stearate, polyethylene glycol 6000) is added to the compound of the
present disclosure, and the mixture is compression-molded,
according to a method known per se, and then where necessary, the
molded product is coated according to a method known per se for the
purpose of masking of taste, enteric property or durability, to
give a tablet. As the coating agent for the coating, for example,
hydroxypropylmethyl cellulose, ethyl cellulose, hydroxymethyl
cellulose, hydroxypropyl cellulose, polyoxyethylene glycol, Tween
80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethyl
cellulose phthalate, hydroxymethyl cellulose acetate succinate,
Eudragit (methacrylic acid-acrylic acid copolymer, manufactured by
Rohm, DE) and pigment (e.g., iron oxide red, titanium dioxide) may
be used.
[1624] Examples of the injection include intravenous injection as
well as subcutaneous injection, intracutaneous injection,
intramuscular injection, intraperitoneal injection, drip injection
and the like.
[1625] Such injections are prepared according to a method known per
se, or by dissolving, suspending or emulsifying the compound of the
present disclosure in a sterilized aqueous or oily liquid. Examples
of the aqueous liquid include physiological saline, isotonic
solutions containing glucose or other auxiliary drugs (e.g.,
D-sorbitol, D-mannitol, sodium chloride) and the like. The aqueous
liquid may contain a suitable solubilizing agent such as an alcohol
(e.g., ethanol), a polyalcohol (e.g., propylene glycol,
polyethylene glycol), a nonionic surfactant (e.g., polysorbate 80,
HCO-50) and the like. Examples of the oily 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 may be blended with a buffer (e.g., phosphate buffer,
sodium acetate buffer), a soothing agent (e.g., benzalkonium
chloride, procaine hydrochloride), a stabilizer (e.g., human serum
albumin, polyethylene glycol), a preservative (e.g., benzyl
alcohol, phenol) and the like. A prepared injection may be
generally filled in an ampoule.
[1626] While the content of the compound of the present disclosure
in the medicament of the present disclosure varies depending on the
form of the pharmaceutical preparation, it is generally about 0.01
to about 100 wt %, preferably about 2 to about 85 wt %, more
preferably about 5 to about 70 wt/o, relative to the entire
preparation.
[1627] While the content of the additive in the medicament of the
present disclosure varies depending on the form of the
pharmaceutical preparation, it is generally about 1 to about 99.9
wt %, preferably about 10 to about 90 wt %, relative to the entire
preparation.
[1628] The compound of the present disclosure is stable and low
toxic, and may be used safely. While the daily dose of the compound
of the present disclosure varies depending on the condition and
body weight of patients, the kind of compound, administration route
and the like, in the case of, for example, oral administration to
patients for the treatment of cancer, the daily dose to an adult
(body weight about 60 kg) is about 1 to about 1000 mg, preferably
about 3 to about 300 mg, more preferably about 10 to about 200 mg,
as the compound of the present disclosure, which may be given in a
single administration or administered in 2 or 3 portions a day.
[1629] When the compound of the present disclosure is administered
parenterally, it is generally administered in the form of a liquid
(e.g., injection). While the dose of the compound of the present
disclosure varies depending on the subject of administration,
target organ, symptom, administration method and the like, it is,
for example, about 0.01 mg to about 100 mg, preferably about 0.01
to about 50 mg, more preferably about 0.01 to about 20 mg, relative
to 1 kg body weight, which is preferably given by intravenous
injection.
[1630] The compound of the present disclosure may be used
concurrently with other drugs. To be specific, the compound of the
present disclosure may be used together with medicaments such as
hormonal therapeutic agents, chemotherapeutic agents,
immunotherapeutic agents, medicaments inhibiting the action of cell
growth factors or cell growth factor receptors and the like. In the
following, the drugs that can be used in combination with the
compound of the present disclosure are abbreviated as concomitant
drugs.
[1631] Examples of the "hormonal therapeutic agents" include
fosfestrol, diethylstylbestrol, chlorotrianisene,
medroxyprogesterone acetate, megestrol acetate, chlormadinone
acetate, cyproterone acetate, danazol, allylestrenol, gestrinone,
mepartricin, raloxifene, ormeloxifene, levormeloxifene,
anti-estrogens (e.g., tamoxifen citrate, toremifene citrate), pill
preparations, mepitiostane, testrolactone, aminoglutethimide, LH-RH
agonists (e.g., goserelin acetate, buserelin, leuprorelin,
leuprorelin acetate), droloxifene, epitiostanol, ethinylestradiol
sulfonate, aromatase inhibitors (e.g., fadrozole hydrochloride,
anastrozole, retrozole, exemestane, vorozole, formestane),
anti-androgens (e.g., flutamide, bicartamide, nilutamide,
enzalutamide), 5.alpha.-reductase inhibitors (e.g., finasteride,
epristeride, dutasteride), aderenal cortex hormone drugs (e.g.,
dexamethasone, prednisolone, betamethasone, triamcinolone),
androgen synthesis inhibitors (e.g., abiraterone), retinoid and
drugs that retard retinoid metabolism (e.g., liarozole), thyroid
hormone, and DDS (Drug Delivery System) preparations thereof.
[1632] Examples of the "chemotherapeutic agents" include alkylating
agents, antimetabolites, anticancer antibiotics and plant-derived
anticancer agents.
[1633] Examples of the "alkylating agents" include nitrogen
mustard, nitrogen mustard-N-oxide hydrochloride, chlorambutyl,
cyclophosphamide, ifosfamide, thiotepa, carboquone, improsulfan
tosylate, busulfan, nimustine hydrochloride, mitobronitol,
melphalan, dacarbazine, ranimustine, sodium estramustine phosphate,
triethylenemelamine, carmustine, lomustine, streptozocin,
pipobroman, etoglucid, carboplatin, cisplatin, miboplatin,
nedaplatin, oxaliplatin, altretamine, ambamustine, dibrospidium
hydrochloride, fotemustine, prednimustine, pumitepa, ribomustin,
temozolomide, treosulphan, trophosphamide, zinostatin stimalamer,
adozelesin, cystemustine, bizelesin, and DDS preparations
thereof.
[1634] Examples of the "antimetabolites" include mercaptopurine,
6-mercaptopurine riboside, thioinosine, methotrexate, pemetrexed,
enocitabine, cytarabine, cytarabine ocfosfate, ancitabine
hydrochloride, 5-FU drugs (e.g., fluorouracil, tegafur, UFT,
doxifluridine, carmofur, gallocitabine, emitefur, capecitabine),
aminopterine, nelzarabine, leucovorin calcium, tabloid, butocine,
calcium folinate, levofolinate calcium, cladribine, emitefur,
fludarabine, gemcitabine, hydroxycarbamide, pentostatin,
piritrexim, idoxuridine, mitoguazone, thiazophrine, ambamustine,
bendamustine, and DDS preparations thereof.
[1635] Examples of the "anticancer antibiotics" include
actinomycin-D, actinomycin-C, mitomycin-C, chromomycin-A3,
bleomycin hydrochloride, bleomycin sulfate, peplomycin sulfate,
daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin
hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride,
neocarzinostatin, mithramycin, sarcomycin, carzinophilin, mitotane,
zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin
hydrochloride, and DDS preparations thereof (e.g.,
doxorubicin-including PEG liposome).
[1636] Examples of the "plant-derived anticancer agents" include
etoposide, etoposide phosphate, vinblastine sulfate, vincristine
sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel,
cabazitaxel, vinorelbine, and DDS preparations thereof.
[1637] Examples of the "immunotherapeutic agents (BRM)" include
picibanil, krestin, sizofiran, lentinan, ubenimex, interferons,
interleukins, macrophage colony-stimulating factor, granulocyte
colony-stimulating factor, erythropoietin, lymphotoxin, BCG
vaccine, Corynebacterium parvum, levamisole, polysaccharide K,
procodazole, anti-CTLA4 antibodies (e.g., ipilimumab,
tremelimumab), anti-PD-1 antibodies (e.g., nivolumab,
pembrolizumab), and anti-PD-L1 antibody.
[1638] Example of the "cell growth factors" in the "medicaments
inhibiting the action of cell growth factors or cell growth factor
receptors" include any substances that promote cell proliferation,
which are normally peptides having not more than 20,000 molecular
weight that are capable of exhibiting their activity at low
concentrations by binding to a receptor, including (1) EGF
(epidermal growth factor) or substances possessing substantially
the same activity as EGF [e.g., TGF.alpha.], (2) insulin or
substances possessing substantially the same activity as insulin
[e.g., insulin, IGF (insulin-like growth factor)-1, IGF-2], (3) FGF
(fibroblast growth factor) or substances possessing substantially
the same activity as FGF [e.g., 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 (nerve growth factor),
PDGF (platelet-derived growth factor), TGF.beta. (transforming
growth factor .beta.), HGF (hepatocyte growth factor), VEGF
(vascular endothelial growth factor), heregulin, angiopoietin].
[1639] Examples of the "cell growth factor receptors" include any
receptors capable of binding to the aforementioned cell growth
factors, including EGF receptor, heregulin receptor (e.g., HER3),
insulin receptor, IGF receptor-1, IGF receptor-2, FGF receptor-1 or
FGF receptor-2, VEGF receptor, angiopoietin receptor (e.g., Tie2),
PDGF receptor and the like.
[1640] Examples of the "medicaments inhibiting the action of cell
growth factors or cell growth factor receptors" include EGF
inhibitor, TGF.alpha. inhibitor, heregulin inhibitor, insulin
inhibitor, IGF inhibitor, FGF inhibitor, KGF inhibitor, CSF
inhibitor, EPO inhibitor, IL-2 inhibitor, NGF inhibitor, PDGF
inhibitor, TGF.beta. inhibitor, HGF inhibitor, VEGF inhibitor,
angiopoietin inhibitor, EGF receptor inhibitor, HER2 inhibitor,
HER4 inhibitor, insulin receptor, 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, ALK inhibitor, RORI
inhibitor, Trk inhibitor, Ret inhibitor, mTOR inhibitor, Aurora
inhibitor, PLK inhibitor, MEK (MEK1/2) inhibitor, MET inhibitor,
CDK inhibitor, Akt inhibitor, ERK inhibitor, PI3K inhibitor and the
like. More specifically, anti-VEGF antibody (e.g., Bevacizumab,
Ramucurumab), anti-HER2 antibody (e.g., Trastuzumab, Pertuzumab),
anti-EGFR antibody (e.g., Cetuximab, Panitumumab, Matuzumab,
Nimotuzumab), anti-HGF antibody, Imatinib, Erlotinib, Gefitinib,
Sorafenib, Sunitinib, Dasatinib, Lapatinib, Vatalanib, Ibrutinib,
Bosutinib, Cabozantinib, Crizotinib, Alectinib, Vismodegib,
Axitinib, Motesanib, Nilotinib,
6-[4-(4-ethylpiperazin-1-ylmethyl)phenyl]-N-[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]qu-
inazolin-7-yloxy]propyl]-N-ethylamino]ethyl phosphate (AZD-1152),
4-[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-ylam-
ino]benzoic acid,
N-[2-methoxy-5-[(E)-2-(2,4,6-trimethoxyphenyl)vinylsulfonylmethyl]phenyl]-
glycine sodium salt (ON-1910Na), Volasertib, Selumetinib,
Trametinib,
N-[2(R),3-dihydroxypropoxy]-3,4-difluoro-2-(2-fluoro-4-iodophenylamino)be-
nzamide (PD-0325901), Bosutinib, Regorafenib, Afatinib, Idelalisib,
Ceritinib, Dabrafenib and the like may be used.
[1641] In addition to the aforementioned drugs, L-asparaginase,
L-arginase, arginine deiminase, aceglatone, procarbazine
hydrochloride, protoporphyrin-cobalt complex salt, mercuric
hematoporphyrin-sodium, topoisomerase I inhibitors (e.g.,
irinotecan, topotecan, indotecan, Indimitecan), topoisomerase II
inhibitors (e.g., sobuzoxane), differentiation inducers (e.g.,
retinoid, vitamin D), other angiogenesis inhibitors (e.g.,
humagillin, shark extract, COX-2 inhibitor), .alpha.-blockers
(e.g., tamsulosin hydrochloride), bisphosphonic acids (e.g.,
pamidronate, zoledronate), thalidomide, lenalidomide, pomalidomide,
azacytidine, decitabine, proteasome inhibitors (e.g., bortezomib,
carfilzomib, ixazomib), NEDD8 inhibitors (e.g., Pevonedistat), UAE
inhibitors, PARP inhibitors (e.g., Olaparib, Niraparib, Veliparib),
antitumor antibodies such as anti-CD20 antibodies (e.g., Rituximab,
Obinutuzumab), anti-CCR4 antibodies (e.g., Mogamulizumab) and the
like, antibody-drug conjugates (e.g., trastuzumab emtansine,
Brentuximab vedotin) and the like may also be used as a concomitant
drug.
[1642] By combining the compound of the present disclosure and a
concomitant drug, a superior effect such as (1) the dose may be
reduced as compared to single administration of the compound of the
present disclosure or a concomitant drug, (2) the drug to be
combined with the compound of the present disclosure may be
selected according to the condition of patients (mild case, severe
case and the like), (3) the period of treatment may be set longer,
(4) a sustained treatment effect may be designed, (5) a synergistic
effect may be afforded by a combined use of the compound of the
present disclosure and a concomitant drug, and the like, may be
achieved.
[1643] In the present specification, the compound of the present
disclosure and a concomitant drug used in combination are referred
to as the "combination agent of the present disclosure".
[1644] For use of the combination agent of the present disclosure,
the administration time of the compound of the present disclosure
and the concomitant drug is not restricted, and the compound of the
present disclosure and the concomitant drug can be administered to
an administration subject simultaneously, or may be administered at
different times. When administered at a time interval, the interval
differs depending on the effective ingredient to be administered,
dosage form and administration method, and for example, when the
concomitant drug is administered first, the compound of the present
disclosure may be administered within time range of from 1 min to 3
days, preferably from 10 min to 1 day, more preferably from 15 min
to 1 hr after administration of the concomitant drug. When the
compound of the present disclosure is administered first, the
concomitant drug is administered within time range of from 1 min to
1 day, preferably from 10 min to 6 hrs, more preferably from 15 min
to 1 hr after administration of the compound of the present
disclosure. The dosage of the concomitant drug may be determined
according to the dose clinically set, and may be appropriately
selected depending on the administration subject, administration
route, disease, combination and the like.
[1645] Examples of the administration mode of the combined use of
the compound of the present disclosure and the concomitant drug
include the following methods: (1) The compound of the present
disclosure and the concomitant drug are simultaneously produced to
give a single preparation, which is then administered. (2) The
compound of the present disclosure and the concomitant drug are
separately produced to give two kinds of preparations which are
administered simultaneously by the same administration route. (3)
The compound of the present disclosure and the concomitant drug are
separately produced to give two kinds of preparations which are
administered by the same administration route at different times.
(4) The compound of the present disclosure and the concomitant drug
are separately produced to give two kinds of preparations which are
administered simultaneously by different administration routes. (5)
The compound of the present disclosure and the concomitant drug are
separately produced to give two kinds of preparations which are
administered by different administration routes at different times
(e.g., the compound of the present disclosure and the concomitant
drug are administered in this order, or in the reverse order).
[1646] The dose of the concomitant drug may be appropriately
determined in accordance with its clinical dose, and the ratio of
the compound of the present disclosure and the concomitant drug may
be appropriately determined depending on the administration
subject, administration route, target disease, symptom,
combination, and the like. For example, when the administration
subject is human, the concomitant drug is used in 0.01 to 100
(parts by weight), relative to 1 part by weight of the compound of
the present disclosure.
[1647] Furthermore, the compound of the present disclosure or the
combination agent of the present disclosure may be used
concurrently with a non-drug therapy. To be precise, the compound
of the present disclosure or the combination agent of the present
disclosure may be combined with a non-drug therapy such as (1)
surgery, (2) hypertensive chemotherapy using angiotensin II etc.,
(3) gene therapy, (4) thermotherapy, (5) cryotherapy, (6) laser
cauterization and (7) radiotherapy.
[1648] For example, by using the compound of the present disclosure
or the combination agent of the present disclosure before or after
the above-mentioned surgery and the like, or before or after a
combined treatment of two or three kinds thereof, effects such as
prevention of emergence of resistance, prolongation of Disease-Free
Survival, suppression of cancer metastasis or recurrence,
prolongation of life and the like may be afforded.
[1649] In addition, it is possible to combine a treatment with the
compound of the present disclosure or the combination agent of the
present disclosure with a supportive therapy [(i) administration of
antibiotic (e.g., .beta.-lactam type such as pansporin and the
like, macrolide type such as clarithromycin and the like) for the
complication with various infectious diseases, (ii) administration
of high-calorie transfusion, amino acid preparation or general
vitamin preparation for the improvement of malnutrition, (iii)
administration of morphine for pain mitigation, (iv) administration
of a pharmaceutical agent for ameliorating side effects such as
nausea, vomiting, anorexia, diarrhea, leucopenia, thrombocytopenia,
decreased hemoglobin concentration, hair loss, hepatopathy,
renopathy, DIC, fever and the like and (v) administration of a
pharmaceutical agent for suppressing multiple drug resistance of
cancer and the like].
EXAMPLES
[1650] The present disclosure is explained in detail by referring
to the following Examples, Experimental Examples and Formulation
Examples, which are not to be construed as limitative, and the
disclosure may be changed within the scope of the present
disclosure.
[1651] In the following Examples, the "room temperature" generally
means about 10.degree. C. to about 35.degree. C. The ratios
indicated for mixed solvents are volume mixing ratios, unless
otherwise specified. % means wt %, unless otherwise specified.
[1652] In silica gel column chromatography, "Diol" means use of
3-(2,3-dihydroxypropoxy)propylsilane-bound silica gel. In silica
gel column chromatography and HPLC (high-performance liquid
chromatography), "C18" means use of octadecyl-bound silica gel. The
ratios of elution solvents are volume mixing ratios, unless
otherwise specified.
[1653] The measurement by LC/MS was performed under the following
conditions [column: L-Column2 ODS, 3.0 mmI.D..times.50 mm, mobile
phase: acetonitrile/5 mM ammonium acetate buffer
solution=900/100)].
[1654] In Examples, the following abbreviations are used.
[1655] mp: melting point [1656] MS: mass spectrum [1657]
[M+H].sup.+, [M-H].sup.-: molecular ion peak [1658] M: mol
concentration [1659] N: normal [1660] CDCl.sub.3: deuterated
chloroform [1661] DMSO-d.sub.6: deuterated dimethyl sulfoxide
[1662] D.sub.2O: deuterated water [1663] .sup.1H NMR: proton
nuclear magnetic resonance [1664] .sup.31P NMR: phosphorus nuclear
magnetic resonance [1665] LC/MS: liquid chromatograph mass
spectrometer [1666] ESI: ElectroSpray Ionization [1667] APCI:
Atmospheric Pressure Chemical Ionization [1668] THF:
tetrahydrofuran [1669] DMF: N,N-dimethylformamide [1670] DCM:
dichloromethane
[1671] .sup.1H NMR and .sup.31P NMR was measured by
Fourier-transform type NMR. For the analysis, ACD/SpecManager
(trade name) and the like were used. Peaks with very mild protons
such as a hydroxy group, an amino group and the like are not
described.
[1672] MS was measured by LC/MS. As ionization method, ESI method
or APCI method was used. The data indicates actual measured value
(found). Generally, a molecular ion peak is observed. In the case
of a compound having a tert-butoxycarbonyl group, a peak after
elimination of a tert-butoxycarbonyl group or a tert-butyl group
may be observed as a fragment ion. In the case of a compound having
a hydroxy group, a peak after elimination of H.sub.2O may be
observed as a fragment ion. In the case of a salt, a molecular ion
peak or fragment ion peak of free form is generally observed.
Example 1
Synthesis of
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[3,2-1][1,3,6,-
9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-p-
yrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
##STR00365## ##STR00366## ##STR00367##
[1673] 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
[1674] To a solution of
7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-f-
luoro-3H-pyrrolo[2,3-d]pyrimidin-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
atmosphere at room temperature. To the reaction mixture was added
saturated aqueous sodium hydrogencarbonate solution, and the
mixture was extracted with ethyl acetate. The organic layer was
washed successively with saturated aqueous sodium hydrogencarbonate
solution and saturated brine, 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:
[M-H].sup.- 586.0.
B)
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3-O-(tert-butyl(dimethyl)si-
lyl)-beta-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-
-4-one
[1675] To a solution of
7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihy-
droxytetrahydrofuran-2-yl)-5-fluoro-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one
(2.65 g) in DMF (20 mL) were added imidazole (0.614 g) and
tert-butyldimethylchlorosilane (0.816 g), and the mixture was
stirred overnight at room temperature. The reaction mixture was
diluted with water, and extracted with ethyl acetate. The extract
was washed with saturated brine, 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: [M-H].sup.- 700.2.
C)
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3-O-(tert-butyl(dimethyl)si-
lyl)-2-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-ribofuranosyl-
)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[1676]
7-(5-O-(Bis(4-methoxyphenyl)(phenyl)methyl)-3-O-(tert-butyl(dimethy-
l)silyl)-beta-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrim-
idin-4-one (5.43 g) was subjected to azeotropic dehydration 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),
1H-tetrazole (0.542 g) and 1-methyl-1H-imidazole (0.306 mL), and
the mixture was stirred overnight under argon atmosphere at room
temperature, poured into saturated aqueous sodium hydrogencarbonate
solution, and extracted with ethyl acetate. The organic layer was
washed successively with saturated aqueous sodium hydrogencarbonate
solution and saturated brine, and 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 obtained 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: [M-H].sup.- 901.2.
D)
(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((((2R,3R,4R,5R)-4-((te-
rt-butyldimethylsilyl)oxy)-2-(5-fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(-
4H)-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphory-
l)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate
[1677]
N-Benzoyl-2'-deoxy-2'-fluoro-3'-O-(hydroxy(oxido)phosphoranyl)adeno-
sine (1.2 g) and
7-(5-O-([bis(4-methoxyphenyl)(phenyl)methyl)]-3-O-([tert-butyl(dimethyl)s-
ilyl)]-2-O-({(2-cyanoethoxy)([diisopropylamino)]phosphino)}-beta-D-ribofur-
anosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (3.4
g) were subjected to azeotropic dehydration with anhydrous
acetonitrile, and anhydrous acetonitrile (15 mL) and anhydrous THF
(5 mL) were added thereto. To the mixture was added a mixture of
5-(ethylsulfanyl)-2H-tetrazole (1.07 g) (which was subjected to
azeotropic dehydration with anhydrous acetonitrile) and anhydrous
acetonitrile (10 mL), and the mixture was stirred under argon
atmosphere at 55.degree. C. for 2 hr. 70% tert-Butyl hydroperoxide
aqueous solution (1.12 mL) was added thereto, and the mixture was
stirred at room temperature for 20 min. 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 min. 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].sup.+ 952.2
E)
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-16-((tert-butyl(dimethyl)silyl)o-
xy)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-y-
l)-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9-
,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6-yl)benzamide
[1678]
(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]pyrimidi-
n-7(4H)-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosp-
horyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate
(350 mg) was subjected to azeotropic dehydration 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 atmosphere at room temperature
for 1 hr. Water (232 .mu.L) and iodine (121 mg) were added thereto,
and the mixture was stirred at room temperature for additional 20
min. 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 min. Toluene was added
thereto, and the mixture was concentrated under reduced pressure.
To the residue were added anhydrous acetonitrile (15 mL) and
2-methylpropan-2-amine (5.26 mL), and the mixture was stirred at
room temperature for 2 hr, and 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].sup.+ 897.1
F)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-16-((ter-
t-butyl(dimethyl)silyl)oxy)-15-fluoro-2,10-dihydroxy-2,10-dioxidooctahydro-
-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradec-
in-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[1679] To
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-16-((tert-butyl(dimethyl)-
silyl)oxy)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimi-
din-7-yl)-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-1][-
1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6-yl)benz-
amide (332 mg) was added 40% methylamine ethanol solution (7.6 mL),
and the mixture was stirred under argon atmosphere at room
temperature for 1 hr, and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
(methanol/ethyl acetate), and the obtained residue was purified by
HPLC (L-column2 ODS, 50'150 mm, mobile phase: 5 mM aqueous ammonium
acetate solution/acetonitrile) to give the title compound (106.4
mg). MS: [M+H].sup.+ 793.1
G)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluor-
o-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 di-triethylamine salt
[1680] To
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
6-((tert-butyl(dimethyl)silyl)oxy)-15-fluoro-2,10-dihydroxy-2,10-dioxidooc-
tahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclot-
etradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(106.4 mg) was added triethylamine trihydrofluoride (1094 .mu.L).
The reaction mixture was stirred at 50.degree. C. for 1 hr, and
cooled to room temperature, ethoxy(trimethyl)silane (6266 .mu.L)
was added thereto, and the mixture was stirred at room temperature
for 1 hr. The reaction mixture was concentrated under reduced
pressure, and to the residue were added ethoxy(trimethyl)silane
(6266 .mu.L) and methanol (1 mL), and the mixture was stirred at
room temperature for 1 hr. The reaction mixture was concentrated
under reduced pressure, and the residue was purified by C18 column
chromatography (acetonitrile/10 mM triethylammonium acetate buffer
solution) to give the title compound (82 mg). .sup.1H NMR (400 MHz,
D.sub.2O) .delta. 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=3.4 Hz),
4.42-4.50 (1H, m), 4.54 (1H, d, J=4.2 Hz), 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). .sup.31P NMR (162 MHz,
D.sub.2O) .delta. -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-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-p-
yrrolo[2,3-d]pyrimidin-4-one disodium salt
##STR00368##
[1682] Deionized water (480 mL) was passed through a column
prepared by packing AG (trade name) 50W-X8 cation-exchange resin
(100-200 mesh, 30 g) in an empty column. Then, 1 M aqueous sodium
hydroxide solution (288 mL) and deionized water (540 mL) were
passed through the resin. Deionized water (54 mL) containing
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[3,2-1][1,3,6,-
9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-p-
yrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (1.53 g) was
passed through the resin after the above-mentioned pre-treatment,
and deionized water (72 mL) was passed through the resin, and the
obtained aqueous solution was freeze-dried to give the title
compound (1.28 g). .sup.1H NMR (300 MHz, D.sub.2O) .delta.
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=2.0 Hz), 7.91 (1H, s), 8.07
(1H, s), 8.18 (1H, s). .sup.31P NMR (121 MHz, D.sub.2O) .delta.
-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-fluoro--
10,16-dihydroxy-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-dihy-
dro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (Optical
Isomer)
##STR00369##
[1683] A)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
6-((tert-butyl(dimethyl)silyl)oxy)-15-fluoro-10-hydroxy-2,10-dioxido-2-sul-
fanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosph-
acyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-o-
ne (Optical Isomer)
[1684]
(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]pyrimidi-
n-7(4H)-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosp-
horyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate
(700 mg) was subjected to azeotropic dehydration with anhydrous
acetonitrile and anhydrous pyridine, and anhydrous pyridine (50 mL)
was added thereto. To the mixture was added
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (475 mg) at
room temperature, and the mixture was stirred under argon
atmosphere at room temperature for 1 hr. Water (464 .mu.L) and
3H-benzo[c][1,2]dithiol-3-one (186 mg) were added thereto, and the
mixture was stirred at room temperature for 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. To the residue were added anhydrous acetonitrile
(30 mL) and 2-methylpropan-2-amine (10 mL), and the mixture was
stirred at room temperature for 1 hr, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (methanol/ethyl acetate), and to the obtained
residue was added 40% methylamine ethanol solution (7.3 mL). The
mixture was stirred under argon atmosphere at room temperature for
30 min, and the reaction mixture was concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (methanol/ethyl acetate). The obtained residue was
resolved into two diastereomers (tR1 and tR2, retention times of
which by LC/MS are from shorter to longer in this order) by HPLC
(L-column2 ODS, 50.times.150 mm, mobile phase: 5 mM aqueous
ammonium acetate solution/acetonitrile) to give the title compound
(70 mg, tR1) and the title compound (150 mg, tR2). MS (tR1):
[M+H].sup.+ 809.1. MS (tR2): [M+H].sup.+ 809.1
B)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluor-
o-10,16-dihydroxy-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-di-
hydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
(Optical Isomer)
[1685] To
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
6-((tert-butyl(dimethyl)silyl)oxy)-15-fluoro-10-hydroxy-2,10-dioxido-2-sul-
fanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosph-
acyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-o-
ne (optical isomer) (70 mg, derived from tR1) were added methanol
(3.0 mL) and triethylamine trihydrofluoride (1.41 mL). The reaction
mixture was concentrated to remove the methanol, and the residue
was stirred at 55.degree. C. for 1 hr. 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 hr.
The reaction mixture was concentrated under reduced pressure, and
the residue was purified by C18 column chromatography
(acetonitrile/10 mM triethylammonium acetate buffer solution) to
give the title compound (56 mg). .sup.1H NMR (400 MHz, D2O) .delta.
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). .sup.31P NMR (162 MHz, D2O)
5-2.43, 54.03.
Example 3
Synthesis of
7-((2R,5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluo-
ro-10,16-dihydroxy-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-d-
ihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 1.7 triethylamine salt
##STR00370##
[1687] To
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
6-((tert-butyl(dimethyl)silyl)oxy)-15-fluoro-10-hydroxy-2,10-dioxido-2-sul-
fanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosph-
acyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-o-
ne (optical isomer) (150 mg, derived from tR2) were added methanol
(3.0 mL) and triethylamine trihydrofluoride (3.02 mL). The reaction
mixture was concentrated to remove the methanol, and the residue
was stirred at 55.degree. C. for 1 hr. 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 hr.
The reaction mixture was concentrated under reduced pressure, and
the residue was purified by C18 column chromatography
(acetonitrile/10 mM triethylammonium acetate buffer solution) to
give the title compound (155 mg). .sup.1H NMR (400 MHz, D2O)
.delta. 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). .sup.31P NMR (162 MHz,
D2O) 6-2.41, 55.33.
Example 3a
Synthesis of
7-((2R,5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluo-
ro-10,16-dihydroxy-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-d-
ihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one disodium salt
##STR00371##
[1689] Deionized water (400 mL) was passed through a column
prepared by packing AG (trade name) 50W-X8 cation-exchange resin
(100-200 mesh, 25.3 g) in an empty column. Then, 1 M aqueous sodium
hydroxide solution (240 mL) and deionized water (450 mL) were
passed through the resin. Deionized water (45 mL) containing
7-((2R,5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluo-
ro-10,16-dihydroxy-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-d-
ihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (1.30
g) was passed through the resin after the above-mentioned
pre-treatment, and deionized water (60 mL) was passed through the
resin to give an aqueous solution containing the title
compound.
[1690] Deionized water (400 mL) was passed through a column
prepared by packing AG (trade name) 50W-X8 cation-exchange resin
(100-200 mesh, 26.3 g) in an empty column. Then, 1 M aqueous sodium
hydroxide solution (270 mL) and deionized water (540 mL) were
passed through the resin. Deionized water (54 mL) containing
7-((2R,5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluo-
ro-10,16-dihydroxy-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-d-
ihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (1.18
g) was passed through the resin after the above-mentioned
pre-treatment, and deionized water (63 mL) was passed through the
resin to give an aqueous solution containing the title compound.
The two aqueous solutions containing the title compound were
combined, and freeze-dried to give the title compound (2.0 g).
.sup.1H NMR (300 MHz, D.sub.2O) .delta. 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). .sup.31P NMR (121 MHz, D.sub.2O) .delta. -2.38, 55.3.
Example 4
Synthesis of
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[3,2-1][1,3,6,9,11,-
2,10]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrol-
o[2,3-d]pyrimidin-4-one di-triethylamine salt
##STR00372## ##STR00373##
[1691] A)
(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimeth-
ylsilyl)-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)tetrahydrofur-
an-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1692]
N-Benzoyl-2'-O-(tert-butyl(dimethyl)silyl)-3'-O-(hydroxy(oxido)phos-
phoranyl)adenosine (369 mg) and
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3-O-(tert-butyl(dimethyl)sily-
l)-2-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-ribofuranosyl)--
5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (817 mg) were
subjected to azeotropic dehydration 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 subjected to
azeotropic dehydration with anhydrous acetonitrile) and anhydrous
acetonitrile (2.4 mL), and the mixture was stirred under argon
atmosphere at room temperature for 1.5 hr. To the reaction solution
was added 70/o tert-butyl hydroperoxide aqueous solution (0.276
mL), and the mixture was stirred at room temperature for additional
40 min. 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 hr.
The reaction mixture was concentrated under reduced pressure, and
the residue was subjected to azeotropic dehydration 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].sup.+ 1064.3.
B)
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis((tert-butyl(dimethyl)s-
ilyl)oxy)-10-(2-cyanoethoxy)-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--
1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6-yl)b-
enzamide
[1693]
(2R,3R,4R,5R)-5-(6-Benzamido-9H-purin-9-yl)-4-((tert-butyldimethyls-
ilyl)oxy)-2-((((((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(5-fluor-
o-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)-5-(hydroxymethyl)tetrahydrofu-
ran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl
hydrogen phosphonate (538 mg) was subjected to azeotropic
dehydration with anhydrous acetonitrile and anhydrous pyridine, and
suspended in anhydrous pyridine (12 mL).
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (327 mg) was
added thereto, and the mixture was stirred at room temperature for
1 hr. Water (0.319 mL) and iodine (167 mg) were added thereto, and
the mixture was stirred at room temperature for additional 30 min.
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].sup.+ 1062.3.
C)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-bi-
s((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
[1694]
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-Bis((tert-butyl(dimeth-
yl)silyl)oxy)-10-(2-cyanoethoxy)-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[-
3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6--
yl)benzamide (509 mg) was dissolved in 33% methylamine ethanol
solution (10 mL), the solution was stirred under argon atmosphere
at room temperature for 1 hr, and the solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (methanol/ethyl acetate). The obtained residue was
purified by HPLC (L-column2 ODS, 50.times.150 mm, mobile phase: 5
mM aqueous ammonium acetate solution/acetonitrile) to give the
title compound (227 mg). MS: [M+H].sup.+ 905.2.
D)
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[3,2-1][1,3,6,9,1-
1,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrr-
olo[2,3-d]pyrimidin-4-one di-triethylamine salt
[1695] To 7-((5R,7R,8R
12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-bis((tert-butyl(d-
imethyl)silyl)oxy)-2,10-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofur-
o[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 (227 mg) was added
triethylamine trihydrofluoride (0.818 mL), and the mixture was
stirred at 50.degree. C. for 5 hr. The solvent was evaporated under
reduced pressure, the residue was neutralized with 1 M aqueous
triethylammonium bicarbonate solution, and the solvent was again
evaporated under reduced pressure. The residue was purified by C18
silica gel column chromatography (10 mM triethylammonium acetate
buffer solution/acetonitrile), and the obtained solid was
freeze-dried to give the title compound (103 mg). .sup.1H NMR (300
MHz, D.sub.2O) .delta. 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=1.5 Hz), 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, s). .sup.31P NMR (121 MHz,
D.sub.2O) .delta. -1.96, -1.21.
Example 5
7-((5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-15,16-dihyd-
roxy-2,10-dioxido-2,10-disulfanyloctahydro-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 di-triethylamine salt (Optical
Isomer)
##STR00374## ##STR00375##
[1696] A)
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3-O-(tert-butyl(dime-
thyl)silyl)-2-O-(hydroxy(oxido)phosphoranyl)-beta-D-ribofuranosyl)-5-fluor-
o-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[1697]
7-(5-O-(Bis(4-methoxyphenyl)(phenyl)methyl)-3-O-(tert-butyl(dimethy-
l)silyl)-beta-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrim-
idin-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 hr. Water (1 mL) and hydroxide ammonium (2
mL) were added thereto. The mixture was stirred at room temperature
for 30 min, and the reaction mixture was diluted with water, and
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: [M-H].sup.- 764.1.
B)
7-(3-O-(tert-butyl(dimethyl)silyl)-2-O-(hydroxy(oxido)phosphoranyl)-bet-
a-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[1698] A mixture of
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3-O-(tert-butyl(dimethyl)sily-
l)-2-O-(hydroxy(oxido)phosphoranyl)-beta-D-ribofuranosyl)-5-fluoro-3,7-dih-
ydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (611 mg) and 80% acetic acid
(4 mL) was stirred at room temperature for 2 hr. 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].sup.+ 464.1.
C)
(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((te-
rt-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cy-
anoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(-
5-fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1699] A mixture of
7-(3-O-(tert-butyl(dimethyl)silyl)-2-O-(hydroxy(oxido)phosphoranyl)-beta--
D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(358 mg) and
N-benzoyl-5'-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2'-O-(tert-butyl(dime-
thyl)silyl)-3'-O-((2-cyanoethoxy)(diisopropylamino)phosphino)adenosine
(1.02 g) was subjected to azeotropic dehydration 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 atmosphere at room temperature for
1 hr.
((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione
(199 mg) was added thereto, and the mixture was stirred at room
temperature for additional 30 min. The reaction mixture was
quenched with a 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, 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 (328 mg). MS: [M+H].sup.+
1080.3.
D)
N-(9-((5R,78R,12aR,14R,15R,15aR,16R)-15,16-bis((tert-butyl(dimethyl)sil-
yl)oxy)-2-(2-cyanoethoxy)-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]p-
yrimidin-7-yl)-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methanofuro-
[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6-
-yl)benzamide (Optical Isomer)
[1700] To a mixture of
(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-cyan-
oethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(5--
fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)tetrahydrofuran-3-yl
hydrogen phosphonate (328 mg) and anhydrous pyridine (10 mL) was
added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (196
mg). The mixture was stirred under argon atmosphere at room
temperature for 30 min, water (0.5 mL) and
3H-benzo[c][1,2]dithiol-3-one (61 mg) were added thereto, and the
mixture was stirred at room temperature for 1 hr. The reaction
mixture was diluted with saturated aqueous sodium bicarbonate
solution, and 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
buffer solution/acetonitrile) to give the title compound (optical
isomer) (92 mg, tR4). MS: [M+H].sup.+ 1094.3.
E)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-bi-
s((tert-butyl(dimethyl)silyl)oxy)-2,10-dioxido-2,10-disulfanyloctahydro-12-
H-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)
[1701] 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-p-
yrrolo[2,3-d]pyrimidin-7-yl)-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5-
,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14--
yl)-9H-purin-6-yl)benzamide (optical isomer) (90 mg) was dissolved
in 40% methylamine-methanol solution (3.0 mL), the solution was
stirred under argon atmosphere at room temperature for 1 hr, 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].sup.+
937.1.
F)
7-((5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-15,16-di-
hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-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 di-triethylamine salt (Optical
Isomer)
[1702] 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-dioxido-2,10-disulfanyloctahydro-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) (32 mg) and triethylamine trihydrofluoride (0.12 mL) was
stirred at 50.degree. C. for 3 hr. The mixture was cooled to room
temperature, and neutralized with 1 M aqueous triethylammonium
bicarbonate solution, and the solvent was evaporated under reduced
pressure. The residue was purified by C18 silica gel column
chromatography (10 mM triethylammonium acetate buffer
solution/acetonitrile). The obtained solid was freeze-dried to give
the title compound (21 mg). .sup.1H NMR (300 MHz, D.sub.2O) .delta.
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=1.1 Hz), 6.36 (1H, d, J=7.4 Hz), 7.31 (1H, d, J=1.9 Hz), 7.95
(1H, s), 8.11 (1H, s), 8.22 (1H, s). .sup.31P NMR (121 MHz,
D.sub.2O) .delta. 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-dihyd-
roxy-2,10-dioxido-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-4-
H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (Optical
Isomer)
##STR00376## ##STR00377##
[1703] A)
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(dime-
thyl)silyl)-beta-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]py-
rimidin-4-one
[1704] To a solution of
7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihy-
droxytetrahydrofuran-2-yl)-5-fluoro-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one
(2.65 g) in DMF (20 mL) were added imidazole (0.614 g) and
tert-butyldimethylchlorosilane (0.816 g), and the mixture was
stirred overnight at room temperature. The reaction mixture was
diluted with water, and extracted with ethyl acetate. The extract
was washed with saturated brine, 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: [M-H].sup.- 700.3.
B)
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(dimethyl)si-
lyl)-3-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-ribofuranosyl-
)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[1705]
7-(5-O-(Bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(dimethy-
l)silyl)-beta-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrim-
idin-4-one (2.37 g) was dissolved in DMF (6.75 mL), and to the
solution were added
3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (2.15 mL),
1H-tetrazole (0.237 g) and 1-methyl-1H-imidazole (0.134 mL), and
the mixture was stirred under argon atmosphere at room temperature
for 20.5 hr. To the reaction solution was added saturated aqueous
sodium bicarbonate solution, and the mixture was extracted with
ethyl acetate. The extract was washed with saturated brine, 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:
[M-H].sup.- 900.2.
C)
7-(2-O-(tert-butyl(dimethyl)silyl)-3-O-(hydroxy(oxido)phosphoranyl)-bet-
a-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[1706]
7-(5-O-(Bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(dimethy-
l)silyl)-3-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-ribofuran-
osyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (1.54
g) was dissolved in acetonitrile (15 mL), pyridine
2,2,2-trifluoroacetate (0.396 g) and water (0.062 mL) were added
thereto at room temperature, and the mixture was stirred at room
temperature for 50 min. 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 min. The reaction
mixture was concentrated under reduced pressure, to the residue was
added 80% acetic acid (8.54 mL), and the mixture was stirred at
room temperature for 1 hr.
[1707] Similarly,
7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(di
methyl)silyl)-3-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-rib-
ofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(1.13 g) was dissolved in acetonitrile (10 mL), and pyridine
2,2,2-trifluoroacetate (0.290 g) and water (0.045 mL) were added
thereto at room temperature, and the mixture was stirred at room
temperature for 50 min. 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 min. The reaction
mixture was concentrated under reduced pressure, to the residue was
added 80% acetic acid (6.27 mL), and the mixture was stirred at
room temperature for 1 hr.
[1708] The reaction mixtures were combined, and concentrated under
reduced pressure, and the residue was purified by silica gel column
chromatography (methanol/ethyl acetate) to give the title compound
(961 mg). MS: [M+H].sup.+ 464.0.
D)
(2R,3R,4R,5R)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((te-
rt-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cy-
anoethoxy)phosphorothioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(-
5-fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1709]
7-(2-O-(tert-Butyl(dimethyl)silyl)-3-O-(hydroxy(oxido)phosphoranyl)-
-beta-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-o-
ne (400 mg) and
N-benzoyl-5'-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3'-O-(tert-butyl(dime-
thyl)silyl)-2'-O-((2-cyanoethoxy)(diisopropylamino)phosphino)adenosine
(1.02 g) were subjected to azeotropic dehydration with anhydrous
acetonitrile (three times), and was suspended in anhydrous
acetonitrile (6 mL). Pyridine 2,2,2-trifluoroacetate (333 mg) was
added thereto, and the mixture was stirred under argon atmosphere
at room temperature for 1 hr.
(Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione
(195 mg) was added thereto, and the mixture was stirred at room
temperature for additional 30 min. 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 1,1,1,3,3,3-hexafluoropropan-2-ol (5 mL), and the
solution was stirred at room temperature for 1 hr. The reaction
mixture was concentrated under reduced pressure, and the residue
was subjected to azeotropic dehydration 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].sup.+ 1080.3.
E)
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis((tert-butyl(dimethyl)s-
ilyl)oxy)-10-(2-cyanoethoxy)-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-
,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-9H-purin-6-yl-
)benzamide (Optical Isomer)
[1710]
((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 hydrogen phosphonate (556 mg) was subjected to azeotropic
dehydration with anhydrous acetonitrile and anhydrous pyridine, and
dissolved in anhydrous pyridine (15 mL). To the solution was added
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (332 mg) at
room temperature, and the mixture was stirred under argon
atmosphere at room temperature for 30 min. Water (1 mL) and
3H-1,2-benzodithiol-3-one (108 mg) were added thereto, and the
mixture was stirred at room temperature for additional 1 hr. To the
reaction mixture was added saturated aqueous sodium bicarbonate
solution, and the mixture was extracted with ethyl acetate. The
extract was washed with saturated brine, 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/methanol). The obtained residue was
resolved into four diastereomers (tR1, tR2, tR3 and tR4, retention
times of which by LC/MS are from shorter to longer in this order)
by HPLC (L-column2 ODS, 50.times.150 mm, mobile phase: 5 mM aqueous
ammonium acetate solution/acetonitrile) to give the title compound
(120 mg, tR2) and the title compound (183 mg, tR4). tR2 MS:
[M+H].sup.+ 1094.3. tR4 MS: [M+H].sup.+ 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-dioxido-2,10-di sulfanyl
octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyc-
lotetradecin-14-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(Optical Isomer)
[1711]
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-Bis((tert-butyl(dimeth-
yl)silyl)oxy)-10-(2-cyanoethoxy)-14-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo-
[2,3-d]pyrimidin-7-yl)-2-oxido-2-sulfanyl-10-sulfidooctahydro-12H-5,8-meth-
anofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-9H-p-
urin-6-yl)benzamide (optical isomer, derived from tR2) (120 mg) was
dissolved in 33% methylamine ethanol solution (5.0 mL), the
solution was stirred under argon atmosphere at room temperature for
1.5 hr, 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].sup.+ 937.2.
G) 7-((5R,7R,8R
12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dio-
xido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]p-
entaoxadiphosphacyclotetradecin-14-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-
-d]pyrimidin-4-one di-triethylamine salt (Optical Isomer)
[1712] To
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-dioxido-2,10-disulfanyloctahy-
dro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra-
decin-4-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(optical isomer, derived from tR2) (84.4 mg) was added
triethylamine trihydrofluoride (0.294 mL), and the mixture was
stirred at 50.degree. C. for 2 hr. The reaction solution was cooled
to room temperature, and neutralized with 1 M aqueous
triethylammonium bicarbonate solution, and the solvent was
evaporated under reduced pressure. The residue was purified by C18
silica gel column chromatography (10 mM triethylammonium acetate
buffer solution/acetonitrile), and the obtained solid was
freeze-dried to give the title compound (58.4 mg). .sup.1H NMR (300
MHz, D.sub.2O) .delta. 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, brs), 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=2.3 Hz), 8.05 (1H,
s), 8.20 (1H, s), 8.66 (1H, s). .sup.31P NMR (121 MHz, D.sub.2O)
.delta. 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-dihyd-
roxy-2,10-dioxido-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-4-
H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (Optical
Isomer)
##STR00378##
[1713] 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-dioxido-2,10-disulfanyloctahy-
dro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra-
decin-14-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(Optical Isomer)
[1714]
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-Bis((tert-butyl(dimeth-
yl)silyl)oxy)-10-(2-cyanoethoxy)-14-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo-
[2,3-d]pyrimidin-7-yl)-2-oxido-2-sulfanyl-10-sulfidooctahydro-12H-5,8-meth-
anofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-9H-p-
urin-6-yl)benzamide (optical isomer, derived from tR4) (183 mg) was
dissolved in 33% methylamine ethanol solution (5.0 mL), the
solution was stirred under argon atmosphere at room temperature for
1 hr, 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].sup.+ 937.1.
B)
7-((5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dih-
ydroxy-2,10-dioxido-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 di-triethylamine salt (Optical
Isomer)
[1715] To
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-dioxido-2,10-disulfanyloctahy-
dro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetra-
decin-14-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(optical isomer, derived from tR4) (107 mg) was added triethylamine
trihydrofluoride (0.372 mL), and the mixture was stirred at
50.degree. C. for 2.5 hr. The reaction solution was cooled to room
temperature, and neutralized with 1 M aqueous triethylammonium
bicarbonate solution, and the solvent was evaporated under reduced
pressure. The residue was purified by C18 silica gel column
chromatography (10 mM triethylammonium acetate buffer
solution/acetonitrile), and the obtained solid was freeze-dried to
give the title compound (78 mg). .sup.1H NMR (300 MHz, D.sub.2O)
.delta. 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, brs),
4.65-4.71 (1H, m), 4.89 (1H, d, J=4.2 Hz), 4.99-5.11 (1H, m),
5.29-5.42 (1H, m), 6.23-6.31 (2H, m), 7.00 (1H, d, J=1.9 Hz), 8.03
(1H, s), 8.17 (1H, s), 8.56 (1H, s). .sup.31P NMR (121 MHz,
D.sub.2O) .delta. 52.8, 55.1.
Example 8
Synthesis of
2-amino-9-((5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrro-
lo[2,3-d]pyrimidin-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 di-triethylamine salt
##STR00379## ##STR00380## ##STR00381##
[1716] A)
N-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)meth-
yl)-3,4-dihydroxytetrahydrofuran-2-yl)-5-fluoro-H-pyrrolo[2,3-d]pyrimidin--
4-yl)benzamide
[1717]
(2R,3R,4R,5R)-2-((Benzoyloxy)methyl)-5-(4-chloro-5-fluoro-7H-pyrrol-
o[23-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl dibenzoate (14.09 g)
was charged into 17 seal tube containers in 17 parts, and 2 M
ammonia isopropanol solution (20 mL) was added thereto,
respectively. Each mixture was stirred with microwave irradiation
at 130.degree. C. for 5 hr. The obtained mixtures were combined,
and concentrated under reduced pressure. The obtained residue was
subjected to azeotropic evaporation twice with toluene to remove
the solvent, pyridine (100 mL) was added thereto, and the mixture
was cooled to 0.degree. C. Benzoyl chloride (26.6 mL) was added
thereto at 0.degree. C., and the mixture was stirred overnight at
room temperature. To the reaction mixture was added water, 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). To the
obtained compound were added pyridine (350 mL) and ethanol (100
mL), and the mixture was cooled to 0.degree. C. 1 M Aqueous sodium
hydroxide solution (103 mL) was added thereto, and the mixture was
stirred at 0.degree. C. for 1 hr. 1 M Aqueous sodium hydroxide
solution (45.7 mL) was added again thereto, and the mixture was
stirred at 0.degree. C. for 1 hr. To the reaction mixture was added
strong acidic cation-exchange resin DOWEX.TM. 50Wx4 100-200 (95 g)
at room temperature, the solid was removed by filtration, and the
filtrate was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (methanol/ethyl
acetate). To the obtained 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 hr. 4,4'-Dimethoxytrityl chloride
(5.98 g) was added again thereto, and the mixture was stirred at
room temperature for 1 hr. 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
saturated brine, 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].sup.+ 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-p-
yrrolo[2,3-d]pyrimidin-4-yl)benzamide
[1718] To a mixture of
N-(7-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-d-
ihydroxytetrahydrofuran-2-yl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)ben-
zamide (7.51 g) and DMF (30 mL) were added imidazole (1.924 g) and
tert-butyldimethylchlorosilane (2.13 g), and the mixture was
stirred overnight at room temperature. To the reaction mixture was
added water, and the mixture was extracted with ethyl acetate. The
extract was washed with water and saturated brine, 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
(2.94 g). MS, found: 501.2.
C)
N-benzoyl-7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(d-
imethyl)silyl)-3-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-rib-
ofuranosyl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-amine
[1719]
N-(7-((2R,3R,4R,5R)-5-((Bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-
-3-((tert-butyldimethylsilyl)oxy)-4-hydroxytetrahydrofuran-2-yl)-5-fluoro--
7H-pyrrol o[2,3-d]pyrimidin-4-yl)benzamide (1.03 g) was subjected
to azeotropic dehydration with anhydrous toluene, and anhydrous DMF
(5 mL) was added thereto under argon atmosphere.
3-((Bis(diisopropylamino)phosphino)oxy)propanenitrile (0.771 g),
1H-tetrazole (0.090 g) and 1-methyl-1H-imidazole (0.051 mL) were
added thereto, and the mixture was stirred for 6 hr. To the
reaction mixture was added saturated aqueous sodium
hydrogencarbonate solution, and the mixture was extracted with
ethyl acetate. The extract was washed with saturated aqueous sodium
hydrogencarbonate solution and saturated brine, 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). .sup.1H NMR (300 MHz,
CDCl.sub.3) .delta. -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
hydrogen phosphonate
[1720] To
N-benzoyl-7-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert--
butyl(dimethyl)silyl)-3-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-bet-
a-D-ribofuranosyl)-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-4-amine
(1.12 g) were added acetonitrile (5 mL), water (40 .mu.L) and
pyridine 2,2,2-trifluoroacetate (258 mg). The mixture was stirred
at room temperature for 10 min, tert-butylamine (5.43 mL) was added
thereto, and the mixture was stirred at room temperature for 1 hr.
The reaction mixture was concentrated under reduced pressure, 80%
acetic acid (5.5 mL) was added thereto, and the mixture was stirred
at room temperature for additional 1 hr. 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 (527 mg). MS: [M+H].sup.+ 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-butyldimeth-
ylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)-
tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahydrofu-
ran-3-yl hydrogen phosphonate
[1721]
(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
hydrogen phosphonate (527 mg) and
3'-O-(tert-butyl(dimethyl)silyl)-2'-O-((2-cyanoethoxy)(diisopropylamino)p-
hosphino)-N-isobutyrylguanosine (1.083 g) were subjected to
azeotropic dehydration 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 min. 70% tert-Butyl
hydroperoxide aqueous solution (382 .mu.L) was added thereto, and
the mixture was stirred at room temperature for additional 20 min.
To the reaction mixture were added sodium thiosulfate (693 mg) and
water (1 mL), 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 hr. The reaction
mixture was concentrated under reduced pressure, and the residue
was subjected to azeotropic dehydration 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].sup.+ 1149.4.
F)
N-(7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis((tert-butyl(dimethyl)s-
ilyl)oxy)-10-(2-cyanoethoxy)-2-hydroxy-7-(2-((2-methylpropanoyl)amino)-6-o-
xo-1,6-dihydro-9H-purin-9-yl)-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,-
2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-5-fluoro-7H-p-
yrrolo[2,3-d]pyrimidin-4-yl)benzamide
[1722]
(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-butyldi-
methylsilyl)oxy)-5-(hydroxymethyl)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-
-yl)tetrahydrofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahyd-
rofuran-3-yl hydrogen phosphonate (175 mg) was subjected to
azeotropic dehydration three times with anhydrous acetonitrile, and
anhydrous pyridine (3 mL) and
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (98 mg) were
added thereto. The mixture was stirred under argon atmosphere at
room temperature for 10 min, water (96 .mu.L) and iodine (50 mg)
were added thereto, and the mixture was stirred at room temperature
for additional 20 min. 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 min, and 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].sup.+ 1147.6.
G)
2-amino-9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(4-amino-5-fluoro-7H-pyr-
rolo[2,3-d]pyrimidin-7-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)-1,9-dihydro-6H-purin-6-one
[1723]
N-(7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-Bis((tert-butyl(dimeth-
yl)silyl)oxy)-10-(2-cyanoethoxy)-2-hydroxy-7-(2-((2-methylpropanoyl)amino)-
-6-oxo-1,6-dihydro-9H-purin-9-yl)-2,10-dioxidooctahydro-12H-5,8-methanofur-
o[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-5-fluoro--
7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzamide (48 mg) was subjected to
azeotropic dehydration twice with anhydrous acetonitrile, 33%
methylamine ethanol solution (2 mL) was added thereto, and the
mixture was stirred overnight under argon atmosphere at room
temperature. The obtained mixture was concentrated under reduced
pressure, and the residue was purified by silica gel column
chromatography (methanol/ethyl acetate). The obtained residue was
purified by HPLC (L-column2 ODS, 50.times.150 mm, mobile phase: 5
mM aqueous ammonium acetate solution/acetonitrile), the obtained
fraction was concentrated under reduced pressure, and the obtained
product was freeze-dried to give the title compound (5 mg). MS:
[M+H].sup.+ 920.3.
H) 2-amino-9-((5R,7R,8R
12aR,14R,15R,15aS,16R)-14-(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-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-p-
urin-6-one di-triethylamine salt
[1724] A mixture of
2-amino-9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(4-amino-5-fluoro-7H-pyrro-
lo[2,3-d]pyrimidin-7-yl)-15,16-bis((tert-butyl(dimethyl)silyl)oxy)-2,10-di-
hydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]p-
entaoxadiphosphacyclotetradecin-7-yl)-1,9-dihydro-6H-purin-6-one (5
mg) and triethylamine trihydrofluoride (200 .mu.L) was stirred at
50.degree. C. for 1 hr. The reaction mixture was cooled to room
temperature, and neutralized with 1 M aqueous triethylammonium
hydrogen carbonate solution. The reaction mixture was purified by
C18 column chromatography (acetonitrile/10 mM triethylammonium
acetate buffer solution), the objective fraction was concentrated
under reduced pressure, and the obtained product was freeze-dried
to give the title compound (3.2 mg). .sup.1H NMR (600 MHz,
D.sub.2O) .delta. 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 (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, 4.3 Hz),
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, s), 7.14 (1H,
d, J=1.6 Hz), 7.87 (1H, s), 8.10 (1H, s).
Example 10
Synthesis of
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[3,2-1][1,3,6,9,11,-
2,10]pentaoxadiphosphacyclotetradecin-7-yl)-1,5-dihydro-4H-imidazo[4,5-c]p-
yridin-4-one
##STR00382## ##STR00383##
[1725] A)
(2R,3R,4R,5R)-2-((benzoyloxy)methyl)-5-(4-oxo-4,5-dihydro-1H-imi-
dazo[4,5-c]pyridin-1-yl)tetrahydrofuran-3,4-diyl dibenzoate
[1726] To 4-chloro-111-imidazo[4,5-c]pyridine (20 g) were added
formic acid (123 mL) and water (30 mL) at room temperature, and the
mixture was stirred at 100.degree. C. for 2 hr. The mixture was
cooled to room temperature, and concentrated under reduced
pressure. To the residue was added conc. hydrochloric acid (100
mL), and the mixture was stirred at 100.degree. C. for 1 hr. 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).
Trimethylsilyl N-(trimethyl silyl)acetimidate (38.2 mL) was added
thereto at room temperature, and the mixture was stirred for 10
min. To the reaction mixture was added
(2S,3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl
dibenzoate (79.0 g) at room temperature, and the mixture was heated
to 80.degree. C. To the reaction solution was added trimethylsilyl
trifluoromethanesulfonate (28.2 mL), and the mixture was stirred
under argon atmosphere overnight at 80.degree. 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), .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 4.65-4.96 (31, m), 5.91 (2H, brs), 6.23-6.42
(11, m), 6.64 (1H, brs), 7.15 (1H, brs), 7.32-7.56 (9H, m),
7.92-8.15 (7H, m), 12.11-12.50 (1H, m). MS: [M+H].sup.+ 580.1
B)
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1-
H-imidazo[4,5-c]pyridin-4(5H)-one
[1727] To
(2R,3R,4R,5R)-2-((benzoyloxy)methyl)-5-(4-oxo-4,5-dihydro-1H-imi-
dazo[4,5-c]pyridin-1-yl)tetrahydrofuran-3,4-diyl dibenzoate (30.0
g) was added 40% methylamine methanol solution (200 mL), and the
mixture was stirred under argon atmosphere at room temperature for
1 hr, and concentrated under reduced pressure. The residue was
recrystallized from hexane-ethyl acetate to give the title compound
(11.2 g). MS: [M+H].sup.+ 268.1
C)
1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)phenyl)methoxy)methyl)-3,4-dih-
ydroxytetrahydrofuran-2-yl)-1H-imidazo[4,5-c]pyridin-4(5H)-one
[1728] To a solution of
1-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H--
imidazo[4,5-c]pyridin-4(5H)-one (18.0 g) in pyridine (140 mL) was
added 4,4'-dimethoxytrityl chloride (18.46 g) at room temperature,
and the mixture was stirred at room temperature for 1 hr. The
reaction mixture was concentrated under reduced pressure, to the
residue was added water, and the mixture was extracted with ethyl
acetate. The obtained extract was washed with saturated brine,
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].sup.+ 570.2
D)
1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((te-
rt-butyldimethylsilyl)oxy)-3-hydroxytetrahydrofuran-2-yl)-1H-imidazo[4,5-c-
]pyridin-4(5H)-one
[1729] To a solution of
1-((2R,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihy-
droxytetrahydrofuran-2-yl)-1H-imidazo[4,5-c]pyridin-4(5H)-one
(18.02 g) in DMF (158 mL) were added imidazole (4.30 g) and
tert-butyldimethylchlorosilane (5.72 g), and the mixture was
stirred at room temperature for 3 hr.
tert-Butyldimethylchlorosilane (2.38 g) was added again thereto,
and the mixture was stirred for 3 hr. The reaction mixture was
diluted with water, and extracted with ethyl acetate. The extract
was washed with saturated brine, 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-H-imidazo[4,5-c]pyridin-1-yl)-
tetrahydrofuran-3-yl hydrogen phosphonate
[1730] 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]p-
yridin-4(5H)-one (8.0 g) and pyridine (117 mL) was added diphenyl
phosphite (4.5 mL). The mixture was stirred at room temperature for
1 hr. 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, and extracted with ethyl acetate.
The extract was washed with saturated brine, 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-o-
xo-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1731] A mixture of
(2R,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-bu-
tyldimethylsilyl)oxy)-2-(4-oxo-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)t-
etrahydrofuran-3-yl hydrogen phosphonate (8.2 g) and
1,1,1,3,3,3-hexafluoropropan-2-ol (30 mL) was stirred at room
temperature for 2 hr. Methanol (10 mL) was added thereto, the
mixture was stirred at 60.degree. C. for 1 hr, 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].sup.+ 446.1
G)
(2R,3R,4R,5R)-5-((((((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((te-
rt-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cy-
anoethoxy)phosphoryl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(4-oxo-
-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1732]
(2R,3R,4R,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(hydroxymethyl)-2--
(4-oxo-4,5-dihydro-1H-imidazo[4,5-c]pyridin-1-yl)tetrahydrofuran-3-yl
hydrogen phosphonate (250 mg) and
N-benzoyl-5'-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2'-O-(tert-butyl(dime-
thyl)silyl)-3'-O-((2-cyanoethoxy)(diisopropylamino)phosphino)adenosine
(776 mg) were subjected to azeotropic dehydration with anhydrous
acetonitrile, and anhydrous acetonitrile (5.61 mL) was added
thereto. To the mixture was added pyridine 2,2,2-trifluoroacetate
(271 mg), and the mixture was stirred at room temperature for 40
min. 70% tert-Butyl hydroperoxide aqueous solution (231 .mu.L) was
added thereto, and the mixture was stirred at room temperature for
20 min. 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 hr. 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].sup.+ 1046.3
H)
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis((tert-butyl(dimethyl)s-
ilyl)oxy)-2-(2-cyanoethoxy)-10-hydroxy-2,10-dioxido-7-(4-oxo-4,5-dihydro-1-
H-imidazo[4,5-c]pyridin-1-yl)octahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,-
11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6-yl)benzamide
[1733]
(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
hydrogen phosphonate (160 mg) was subjected to azeotropic
dehydration with anhydrous pyridine, and anhydrous pyridine (3.0
mL) was added thereto. To the mixture was added
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (99 mg) at
room temperature, and the mixture was stirred at room temperature
for 1 hr. Water (96 .mu.L) and iodine (50.5 mg) were added thereto,
and the mixture was stirred at room temperature for additional 20
min. 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].sup.+ 1044.3
I)
1-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15,16-bi-
s((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)-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one
[1734] To
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis((tert-butyl(dim-
ethyl)silyl)oxy)-2-(2-cyanoethoxy)-10-hydroxy-2,10-dioxido-7-(4-oxo-4,5-di-
hydro-1H-imidazo[4,5-c]pyridin-1-yl)octahydro-12H-5,8-methanofuro[3,2-1][1-
,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6-yl)benza-
mide (10.6 mg) was added 40% methylamine ethanol solution (5.0 mL),
and the mixture was stirred under argon atmosphere at room
temperature for 1 hr, and 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].sup.+ 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[3,2-1][1,3,6,9,1-
1,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-1,5-dihydro-4H-imidazo[4,5-c-
]pyridin-4-one
[1735] To 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[3,2-1][1,3,-
6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-1,5-dihydro-4H-imidazo[-
4,5-c]pyridin-4-one (9.0 mg) were added methanol (1.0 mL) and
triethylamine trihydrofluoride (165 .mu.L). The reaction mixture
was concentrated to remove the methanol, and the residue was
stirred at 55.degree. C. for 1 hr. 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 hr. The
reaction mixture was concentrated under reduced pressure, and the
residue was purified by C18 column chromatography (acetonitrile/10
mM triethylammonium acetate buffer solution) to give the title
compound (0.3 mg). .sup.1H NMR (400 MHz, D.sub.2O) a 4.15 (2H, d,
J=11.5 Hz), 4.46 (4H, brs), 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). .sup.31P NMR (162 MHz,
D.sub.2O) .delta. -2.36, -1.94.
Example 11
Synthesis of
2-amino-9-((5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(4-amino-1H-imidazo[4,5-c]-
pyridin-1-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-methan-
ofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-1,9-di-
hydro-6H-purin-6-one di-triethylamine salt
##STR00384## ##STR00385## ##STR00386##
[1736] A)
N-benzoyl-1-(2-O-(tert-butyl(dimethyl)silyl)-35-O-(di-tert-butyl-
silylene)-beta-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine
[1737]
N-Benzoyl-1-(Beta-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine
(2.41 g) was dissolved in DMF (25 mL), di-tert-butylsilanediyl
bis(trifluoromethanesulfonate) (23 mL) was added thereto at
0.degree. C., and the mixture was stirred at 0.degree. C. for 75
min. Di-tert-butylsilanediyl bis(trifluoromethanesulfonate) (0.63
mL) was added thereto at 0.degree. C., and the mixture was stirred
at 0.degree. C. for additional 30 min. Di-tert-butylsilanediyl
bis(trifluoromethanesulfonate) (0.63 mL) was added thereto at
0.degree. C., and the mixture was stirred at 0.degree. C. for
additional 35 min. To the reaction mixture was added 1H-imidazole
(2.22 g), and the mixture was stirred at room temperature for 10
min. tert-Butyldimethylchlorosilane (1.18 g) was added thereto, and
the mixture was stirred at 60.degree. C. for 1.5 hr.
tert-Butyldimethylchlorosilane (0.294 g) was added thereto, and the
mixture was stirred at 60.degree. C. for 14.5 hr. The reaction
mixture was diluted with water, and extracted with ethyl acetate.
The extract was washed with saturated brine, 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].sup.+ 625.2.
B)
N-benzoyl-1-(2-O-(tert-butyl(dimethyl)silyl)-beta-D-ribofuranosyl)-1H-i-
midazo[4,5-c]pyridin-4-amine
[1738] Pyridinium poly(hydrogen fluoride) (1.86 mL) was dissolved
in pyridine (10 mL) at 0.degree. C., the solution was added to a
solution of
N-benzoyl-1-(2-O-(tert-butyl(dimethyl)silyl)-3,5-O-(di-tert-butylsilylene-
)-beta-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine (1.64 g)
in THF (13 mL) at 0.degree. C., and the mixture was stirred at
0.degree. C. for 8 min. This reaction was repeated twice. The
reaction solutions were combined, diluted with water, and extracted
with ethyl acetate. The extract was washed with saturated brine,
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].sup.+ 485.1.
C)
N-benzoyl-1-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(d-
imethyl)silyl)-beta-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine
[1739]
N-Benzoyl-1-(2-O-(tert-butyl(dimethyl)silyl)-beta-D-ribofuranosyl)--
1H-imidazo[4,5-c]pyridin-4-amine (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 hr. The reaction mixture was concentrated under
reduced pressure, to the residue was added water, and the mixture
was extracted with ethyl acetate. The extract was washed with
saturated brine, 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].sup.+ 787.3.
D)
N-benzoyl-1-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-butyl(d-
imethyl)silyl)-3-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-rib-
ofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine
[1740]
N-Benzoyl-1-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-but-
yl(dimethyl)silyl)-beta-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine
(3.29 g) was dissolved in anhydrous DMF (9.5 mL), to the solution
were added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile
(2.98 mL), 1H-tetrazole (0.328 g) and 1-methyl-1H-imidazole (0.185
mL), and the mixture was stirred under argon atmosphere at room
temperature for 2.5 hr. To the reaction solution was added
saturated aqueous sodium bicarbonate solution, and the mixture was
extracted with ethyl acetate. The extract was washed with saturated
brine, 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].sup.+ 987.4.
E)
N-benzoyl-1-(2-O-(tert-butyl(dimethyl)silyl)-3-O-(hydroxy(oxido)phospho-
ranyl)-beta-D-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine
[1741]
N-Benzoyl-1-(5-O-(bis(4-methoxyphenyl)(phenyl)methyl)-2-O-(tert-but-
yl(dimethyl)silyl)-3-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-beta-D-
-ribofuranosyl)-1H-imidazo[4,5-c]pyridin-4-amine (3.85 g) was
dissolved in acetonitrile (30 mL), pyridine 2,2,2-trifluoroacetate
(0.904 g) and water (0.141 mL) were added thereto at room
temperature, and the mixture was stirred at room temperature for 30
min. 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 min. The reaction mixture was concentrated under
reduced pressure, and to the residue was added
1,1,1,3,3,3-hexafluoropropan-2-ol (25 mL), and the mixture was
stirred at room temperature for 3.5 hr. The reaction mixture was
concentrated under reduced pressure, acetic acid (20 mL) and water
(5 mL) were added thereto, and the mixture was stirred at room
temperature for 1 hr. 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
(1.85 g). MS: [M+H].sup.+ 549.1.
F)
(2R,3R,4R,5R)-5-(4-benzamido-1H-imidazo[4,5-c]pyridin-1-yl)-4-((tert-bu-
tyldimethylsilyl)oxy)-2-((((((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy-
)-5-(hydroxymethyl)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydrof-
uran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1742]
N-Benzoyl-1-(2-O-(tert-butyl(dimethyl)silyl)-3-O-(hydroxy(oxido)pho-
sphoranyl)-beta-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)s-
ilyl)-2'-O-((2-cyanoethoxy)(diisopropylamino)phosphino)-N-isobutyrylguanos-
ine (637 mg) were subjected to azeotropic dehydration 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 argon atmosphere
at room temperature for 1 hr. 70% tert-Butyl hydroperoxide aqueous
solution (0.225 mL) was added thereto, and the mixture was stirred
at room temperature for additional 1 hr. 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), and the solution was stirred
at room temperature for 45 min. The reaction mixture was
concentrated under reduced pressure, and the residue was subjected
to azeotropic dehydration 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].sup.+ 1131.4.
G) N-(1-((5R
7R,8R,12aR,14R,15R,15aR,16R)-15,16-bis((tert-butyl(dimethyl)silyl)oxy)-10-
-(2-cyanoethoxy)-2-hydroxy-7-(2-(isobutyrylamino)-6-oxo-1,6-dihydro-9H-pur-
in-9-yl)-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]-
pentaoxadiphosphacyclotetradecin-14-yl)-1H-imidazo[4,5-c]pyridin-4-yl)benz-
amide
[1743]
(2R,3R,4R,5R)-5-(4-Benzamido-1H-imidazo[4,5-c]pyridin-1-yl)-4-((ter-
t-butyldimethylsilyl)oxy)-2-((((((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl-
)oxy)-5-(hydroxymethyl)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahy-
drofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-y-
l hydrogen phosphonate (469 mg) was subjected to azeotropic
dehydration with anhydrous acetonitrile and anhydrous pyridine, and
suspended in anhydrous pyridine (10 mL).
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (268 mg) was
added thereto, and the mixture was stirred under argon atmosphere
at room temperature for 15 min. Water (1 mL) and iodine (158 mg)
were added thereto, and the mixture was stirred at room temperature
for additional 13 min. 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 subjected to
azeotropic dehydration with toluene. The residue was purified by
silica gel column chromatography (methanol/ethyl acetate) to give
the title compound (419 mg). MS: [M+H].sup.+ 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[3,2-1][1,3,6,9,11,2,10]pentaoxadi-
phosphacyclotetradecin-7-yl)-1,9-dihydro-6H-purin-6-one
[1744]
N-(1-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15,16-Bis((tert-butyl(dimeth-
yl)silyl)oxy)-10-(2-cyanoethoxy)-2-hydroxy-7-(2-(isobutyrylamino)-6-oxo-1,-
6-dihydro-9H-purin-9-yl)-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-1][-
1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-1H-imidazo[4,5-c]p-
yridin-4-yl)benzamide (419 mg) was dissolved in 33% methylamine
ethanol solution (10.0 mL), and the solution was stirred under
argon atmosphere at room temperature for 18.5 hr. 33% Methylamine
ethanol solution (5 mL) was added thereto, the mixture was stirred
for additional 3 hr, and the solvent was evaporated under reduced
pressure. The residue was dissolved in 33% methylamine ethanol
solution (10.0 mL), the solution was stirred at room temperature
for 1 hr, 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].sup.+ 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-meth-
anofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-1,9--
dihydro-6H-purin-6-one di-triethylamine salt
[1745] To
2-amino-9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(4-amino-1H-imida-
zo[4,5-c]pyridin-1-yl)-15,16-bis((tert-butyl(dimethyl)silyl)oxy)-2,10-dihy-
droxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pen-
taoxadiphosphacyclotetradecin-7-yl)-1,9-dihydro-6H-purin-6-one (173
mg) was added triethylamine trihydrofluoride (0.625 mL), and the
mixture was stirred at 50.degree. C. for 3 hr. The mixture was
cooled to room temperature, and neutralized with 1 M aqueous
triethylammonium bicarbonate solution, and the solvent was
evaporated under reduced pressure. The residue was purified by C18
silica gel column chromatography (10 mM triethylammonium acetate
buffer solution/acetonitrile), and the obtained solid was
freeze-dried to give the title compound (123 mg). .sup.1H NMR (300
MHz, D.sub.2O) .delta. 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=3.8 Hz), 4.65 (1H, d, J=4.2 Hz), 4.97-5.10 (1H, m), 5.79
(1H, brs), 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). .sup.31P NMR
(121 MHz, D.sub.2O) .delta. -1.36.
Example 12
Synthesis of
8-((5R,7R,8R,12aR,14S,15S,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihyd-
roxy-2,10-dioxido-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]t-
riazin-4(3H)-one di-triethylamine salt (Optical Isomer)
##STR00387## ##STR00388##
[1746] A)
8-((2S,3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahy-
drofuran-2-yl)pyrazolo[1,5-a][1,3,5]triazin-4(3H)-one
[1747] To a solution of
4-((2S,3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran--
2-yl)-1H-pyrazol-5-amine (17.3 g) and potassium carbonate (24.6 g)
in DMF (200 mL) was added ethyl formimidate hydrochloride (19.5 g)
at room temperature. The reaction mixture was stirred overnight at
room temperature. To the reaction mixture was added diethyl
carbonate (126 g) at room temperature, and the reaction mixture was
stirred at 100.degree. C. for 1 hr. To the reaction mixture was
added 20% sodium ethoxide ethanol solution (60.6 g) at room
temperature. The reaction mixture was stirred at 100.degree. 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 saturated brine, 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].sup.+ 539.1.
B)
8-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)py-
razolo[1,5-a][1,3,5]triazin-4(3H)-one
[1748] A solution of
8-((2S,3S,4R,5R)-3,4-bis(benzyloxy)-5-((benzyloxy)methyl)tetrahydrofuran--
2-yl)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) was stirred under
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].sup.+ 269.0.
C)
8-((2S,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-di-
hydroxytetrahydrofuran-2-yl)pyrazolo[1,5-a][1,3,5]triazin-4(3H)-one
[1749] To a solution of
8-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyra-
zolo[1,5-a][1,3,5]triazin-4(3H)-one (1.72 g) in dehydrated pyridine
(30 mL) was added 4,4'-dimethoxytrityl chloride (2.39 g) under
ice-cooling. The reaction mixture was stirred under argon
atmosphere at room temperature for 2 hr. To the reaction mixture
was added 4,4'-dimethoxytrityl chloride (0.217 g) under
ice-cooling. The reaction mixture was stirred under argon
atmosphere at room temperature for 3 hr. To the reaction mixture
was added 4,4'-dimethoxytrityl chloride (0.217 g) at room
temperature. The reaction mixture was stirred under argon
atmosphere at room temperature for 30 min. To the reaction mixture
was added saturated aqueous sodium hydrogencarbonate solution at
room temperature, and the mixture was extracted with ethyl acetate.
The extract was washed with water and saturated brine, 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:
[M-H].sup.- 569.1.
D)
8-((2S,3R,4R,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((te-
rt-butyldimethylsilyl)oxy)-4-hydroxytetrahydrofuran-2-yl)pyrazolo[1,5-a][1-
,3,5]triazin-4(3H)-one
[1750] tert-Butyldimethylchlorosilane (536 mg) and silver(I)
nitrate (604 mg) were added to a solution of
8-((2S,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-dihy-
droxytetrahydrofuran-2-yl)pyrazolo[1,5-a][1,3,5]triazin-4(3H)-one
(1.69 g) and dehydrated pyridine (1.20 mL) in dehydrated THF (20
mL) at room temperature under argon atmosphere. The reaction
mixture was stirred at room temperature for 8 hr, and to the
reaction mixture were added tert-butyldimethylchlorosilane (89 mg)
and silver(I) nitrate (101 mg) at room temperature. The reaction
mixture was stirred overnight at room temperature. The insoluble
substance was removed by filtration, and washed with ethyl acetate.
To the filtrate was added saturated aqueous sodium
hydrogencarbonate solution, and the mixture was extracted with
ethyl acetate. The extract was washed with saturated brine, 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: [M-H].sup.- 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 hydrogen phosphonate
[1751] Diphenyl phosphite (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) at room
temperature. The reaction mixture was stirred under argon
atmosphere at room temperature for 1 hr. To the reaction mixture
was added water (20 mL), and the mixture was stirred for 1 hr. The
reaction mixture was poured into water at room temperature, and
extracted with ethyl acetate. The extract was washed with water and
saturated brine, 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: [M-H].sup.- 747.1.
F)
(2R,3R,4S,5S)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(4-o-
xo-3,4-dihydropyrazolo[1,5-a][1,3,5]triazin-8-yl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1752] To
(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]tr-
iazin-8-yl)tetrahydrofuran-3-yl hydrogen phosphonate (300 mg) was
added 80% aqueous acetic acid solution (10 mL), and the mixture was
stirred at room temperature for 1 hr. 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].sup.+ 447.0.
G)
(2R,3R,4S,5S)-2-((((((2R,3R,4R,5R)-2-(6-benzamido-9H-purin-9-yl)-4-((te-
rt-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)(2-cy-
anoethoxy)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
hydrogen phosphonate
[1753]
(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
hydrogen phosphonate (190 mg) and
N-benzoyl-5'-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3'-O-(tert-butyl(dime-
thyl)silyl)-2'-O-((2-cyanoethyl)(diisopropylamino)phosphino)adenosine
(505 mg) were subjected to azeotropic process three times with
dehydrated acetonitrile. To the residue were added dehydrated
acetonitrile (5 mL) and pyridine 2,2,2-trifluoroacetate (164 mg).
The reaction mixture was stirred under argon atmosphere at room
temperature for 10 min,
((dimethylamino-methylidene)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 min. 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 hr, and the reaction
mixture was subjected to azeotropic process twice with
acetonitrile. The residue was purified by silica gel column
chromatography (methanol/ethyl acetate) to give the title compound
(260 mg). MS: [M+H].sup.+ 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-dioxido-2,10-disulfanyloctahydro-12H-
-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-1-
4-yl)pyrazolo[15-a][1,3,5]triazin-4(3H)-one (Optical Isomer)
[1754]
(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 hydrogen phosphonate (260 mg) was subjected to azeotropic
process twice with dehydrated acetonitrile. The residue was
subjected to azeotropic process once with dehydrated pyridine. To a
solution of the residue in dehydrated pyridine (5 mL) was added
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (158 mg), and
the mixture was stirred under argon atmosphere at room temperature
for 20 min. To the reaction mixture were added water (0.15 mL) and
3H-benzo[c][1,2]dithiol-3-one (49.4 mg) at room temperature. The
reaction mixture was stirred at room temperature for 1 hr. To the
reaction mixture was added saturated aqueous sodium
hydrogencarbonate solution at room temperature, and the mixture was
extracted with ethyl acetate. The extract was washed with saturated
aqueous sodium thiosulfate solution and saturated brine, dried over
anhydrous sodium sulfate, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(methanol/ethyl acetate), and then purified by HPLC (L-column2 ODS,
50.times.150 mm, mobile phase: 5 mM aqueous ammonium acetate
solution/acetonitrile) to give fractions, and the fraction having
the longest retention time was concentrated under reduced pressure.
To the residue was added 33% methylamine ethanol solution (5 mL),
and the mixture was stirred at room temperature for 1 hr. 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].sup.+ 920.2.
I) 8-((5R,7R,8R
12aR,14S,15S,15aS,16R)-7-(6-amino-9H-purin-9-yl)-15,16-dihydroxy-2,10-dio-
xido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]p-
entaoxadiphosphacyclotetradecin-14-yl)pyrazolo[1,5-a][1,3,5]triazin-4(3H)--
one di-triethylamine salt (Optical Isomer)
[1755] 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-dioxido-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) was added triethylamine trihydrofluoride (0.177
mL) at room temperature, and the mixture was stirred at 50.degree.
C. for 2 hr. To the reaction mixture was added
ethoxytrimethylsilane (5 mL) at room temperature, 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 buffer
solution/acetonitrile), and the obtained product was freeze-dried
to give the title compound (13 mg). .sup.1H NMR (300 MHz, D.sub.2O)
.delta. 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-amino-9-((5R,7R,8R,12aR,14S,15S,15aS,16R)-14-(4-aminopyrazolo[1,5-a][1,-
3,5]triazin-8-yl)-2,10,15,16-tetrahydroxy-2,10-dioxidooctahydro-12H-5,8-me-
thanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-1,-
9-dihydro-6H-purin-6-one di-triethylamine salt
##STR00389## ##STR00390##
[1756] A)
(2S,3S,4R,5R)-2-(4-(N-benzoylbenzamido)pyrazolo[1,5-a][1,3,5]tri-
azin-8-yl)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl
dibenzoate
[1757] To a solution of
(2S,3R,4S,5R)-2-(4-aminopyrazolo[1,5-a][1,3,5]triazin-8-yl)-5-(hydroxymet-
hyl)tetrahydrofuran-3,4-diol (2.98 g) and
N,N-dimethyl-4-aminopyridine (1.36 g) in dehydrated pyridine (50
mL) was added benzoyl chloride (12.5 g) under ice-cooling. The
reaction mixture was stirred under argon atmosphere for 1 hr. To
the reaction mixture was added benzoyl chloride (3.13 g) under
ice-cooling. The reaction mixture was stirred under argon
atmosphere at room temperature for 2 hr. To the reaction mixture
was added water, and the mixture was extracted with ethyl acetate.
The extract was washed with water and saturated brine, 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].sup.+ 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
[1758] To a solution of
(2S,3S,4R,5R)-2-(4-(N-benzoylbenzamido)pyrazolo[1,5-a][1,3,5]triazin-8-yl-
)-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyl dibenzoate (6.51
g) in a mixed solvent of pyridine (50 mL) and ethanol (25 mL) was
added IM aqueous sodium hydroxide solution (49.6 mL) under
ice-cooling, and the mixture was stirred at room temperature for 2
hr. To the reaction mixture was added strong acidic cation-exchange
resin DOWEX.TM. 50Wx4 100-200 (40 g) at room temperature, and the
mixture was stirred at room temperature for 15 min. The solid was
removed by filtration, and the filtrate was concentrated under
reduced pressure. To the residue was added methanol, and the
obtained solid was collected by filtration to give the title
compound (1.82 g). MS: [M+H].sup.+ 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)benzamid-
e
[1759] To a solution of
N-(8-((2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)p-
yrazolo[1,5-a][1,3,5]triazin-4-yl)benzamide (1.89 g) in dehydrated
pyridine (30 mL) was added 4,4'-dimethoxytrityl chloride (517 mg)
under ice-cooling. The reaction mixture was stirred under argon
atmosphere at room temperature for 3 hr. To the reaction mixture
was added 4,4'-dimethoxytrityl chloride (517 mg) at room
temperature, and the mixture was stirred overnight under argon
atmosphere at room temperature. To the reaction mixture was added
4,4'-dimethoxytrityl chloride (1035 mg) at room temperature, and
the mixture was stirred under argon atmosphere at room temperature
for 1 hr. To the reaction mixture was added saturated aqueous
sodium hydrogencarbonate solution at room temperature, and the
mixture was extracted with ethyl acetate. The extract was washed
with saturated brine, 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: [M-H].sup.- 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
[1760] To a solution of
N-(8-((2S,3R,4S,5R)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3,4-d-
ihydroxytetrahydrofuran-2-yl)pyrazolo[1,5-a][1,3,5]triazin-4-yl)benzamide
(2.16 g) in dehydrated THF (30 mL) were added silver(I) nitrate
(708 mg) and dehydrated pyridine (1.22 g). The reaction mixture was
stirred under argon atmosphere for 15 min, and
tert-butyldimethylchlorosilane (628 mg) was added thereto. The
reaction mixture was stirred under argon atmosphere at room
temperature for 2 hr. To the reaction mixture were added silver(I)
nitrate (163 mg), tert-butyldimethylchlorosilane (145 mg) and
dehydrated pyridine (507 mg) at room temperature. The reaction
mixture was stirred overnight under argon atmosphere at room
temperature. To the reaction mixture were added silver(I) nitrate
(436 mg), tert-butyldimethylchlorosilane (387 mg) and dehydrated
pyridine (761 mg) at room temperature. The reaction mixture was
stirred under argon atmosphere at room temperature for 2 hr. To the
reaction mixture was added saturated aqueous sodium
hydrogencarbonate solution at room temperature, and the mixture was
extracted with ethyl acetate. The extract was washed with saturated
brine, 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: [M-H].sup.- 786.2.
E)
(2R,3R,4S,5S)-5-(4-benzamidopyrazolo[1,5-a][1,3,5]triazin-8-yl)-4-((ter-
t-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1761]
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 subjected to
azeotropic process twice with dehydrated toluene, and dissolved in
dehydrated DMF (10 mL). To the reaction mixture were added
3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (487 mg),
1H-tetrazole (87 mg) and 1-methyl-1H-imidazole (51 mg). The
reaction mixture was stirred under argon atmosphere at room
temperature for 5 hr. To the reaction mixture was added saturated
aqueous sodium hydrogencarbonate solution at room temperature, and
the mixture was extracted with ethyl acetate. The extract was
washed with saturated brine, 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 obtained mixture in
acetonitrile (10 mL) were added water (0.04 mL) and pyridine
2,2,2-trifluoroacetate (256 mg) at room temperature. The reaction
mixture was stirred at room temperature for 30 min, tert-butylamine
(5.38 mL) was added thereto, and the mixture was stirred at room
temperature for 45 min. The solvent was evaporated 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 hr, 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].sup.+
550.2.
F)
(2R,3R,4S,5S)-5-(4-benzamidopyrazolo[1,5-a][1,3,5]triazin-8-yl)-4-((ter-
t-butyldimethylsilyl)oxy)-2-((((((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl-
)oxy)-5-(hydroxymethyl)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahy-
drofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-y-
l hydrogen phosphonate
[1762] 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
hydrogen phosphonate (240 mg) and
5'-O-(bis(4-methoxyphenyl)(phenyl)methyl)-3'-O-(tert-butyl(dimethyl)silyl-
)-2'-O-((2-cyanoethyl)(diisopropylamino)phosphino)-N-isobutyrylguanosine
(635 mg) was subjected to azeotropic process three times with
dehydrated acetonitrile. To the residue was added a solution of
dehydrated acetonitrile (5 mL), dehydrated THF (2.5 mL) and
5-(ethylthio)-2H-tetrazole (171 mg) (which was in advance subjected
to azeotropic process with dehydrated acetonitrile) in dehydrated
acetonitrile (2.5 mL). The reaction mixture was stirred under argon
atmosphere at room temperature for 2 hr. 70% Aqueous tert-butyl
hydroperoxide solution (0.179 mL) was added thereto, and the
mixture was stirred at room temperature for 20 min. 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 hr. The residue was subjected to azeotropic
process 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].sup.+
1132.3.
G)
N-(8-((5R,7R,8R,12aR,14S,15S,15aR,16R)-15,16-bis((tert-butyl(dimethyl)s-
ilyl)oxy)-10-(2-cyanoethoxy)-2-hydroxy-7-(2-((2-methylpropanoyl)amino)-6-o-
xo-1,6-dihydro-9H-purin-9-yl)-2,10-dioxidooctahydro-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-yl)benzamide
[1763] The crude product (430 mg) containing
(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)o-
xy)-5-(hydroxymethyl)-2-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydr-
ofuran-3-yl)oxy)(2-cyanoethoxy)phosphoryl)oxy)methyl)tetrahydrofuran-3-yl
hydrogen phosphonate was subjected to azeotropic process twice with
dehydrated acetonitrile. The obtained residue was subjected to
azeotropic process once with dehydrated pyridine. To a solution of
the obtained residue in dehydrated pyridine (3 mL) was added
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (245 mg), and
the mixture was stirred at room temperature under argon atmosphere
for 10 min. To the reaction mixture were added water (0.239 mL) and
iodine (125 mg), and the mixture was stirred at room temperature
for 20 min. 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 min. 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].sup.+ 1130.4.
H)
2-amino-9-((5R,7R,8R,12aR,14S,15S,15aS,16R)-14-(4-aminopyrazolo[1,5-a][-
1,3,5]triazin-8-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 di-triethylamine salt
[1764] A mixture of
N-(8-((5R,7R,8R,12aR,14S,15S,15aR,16R)-15,16-bis((tert-butyl(dimethyl)sil-
yl)oxy)-10-(2-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][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)pyrazolo[1,5-a][-
1,3,5]triazin-4-yl)benzamide (553 mg) and 33% methylamine ethanol
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
give a fraction, and the obtained fraction was concentrated under
reduced pressure. To a solution of the obtained residue in methanol
(3 mL) was added triethylamine trihydrofluoride (0.072 mL) at room
temperature, and the mixture was stirred at 50.degree. C. for 4 hr.
To the reaction mixture was added triethylamine trihydrofluoride
(0.181 mL) at room temperature, and the mixture was stirred
overnight at 50.degree. C. To the reaction mixture was added
triethylamine trihydrofluoride (0.181 mL) at room temperature, and
the mixture was stirred at 50.degree. C. for 5 hr. To the reaction
mixture was added ethoxytrimethylsilane (1.034 mL) at room
temperature, and the mixture was stirred for 5 min. The reaction
mixture was concentrated under reduced pressure, the residue was
purified by C18 silica gel column chromatography (10 mM
triethylammonium acetate buffer solution/acetonitrile), and the
obtained product was freeze-dried to give the title compound (10
mg). .sup.1H NMR (300 MHz, D.sub.2O) .delta. 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-f-
luoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-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
##STR00391## ##STR00392##
[1765] 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)phospho-
rothioyl)oxy)methy)-4-((tert-butyldimethylsilyloxy)-2-(5-fluoro-4-oxo-3H-p-
yrrolo[2,3-d]pyrimidin-7(4H)-yl)tetrahydrofuran-3-yl hydrogen
phosphonate
[1766]
7-(3-O-(tert-Butyl(dimethyl)silyl)-2-O-(hydroxy(oxido)phosphoranyl)-
-beta-D-ribofuranosyl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-o-
ne (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)
were subjected to azeotropic process three times with dehydrated
acetonitrile. To the residue were added anhydrous acetonitrile (40
mL), anhydrous THF (20 mL) and an anhydrous acetonitrile solution
(40 mL) of 5-(ethylsulfanyl)-2H-tetrazole (7.49 g). The reaction
mixture was stirred at room temperature for 30 min under argon
atmosphere,
((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione
(4.33 g) was added to the reaction mixture, and the mixture was
stirred at room temperature for 30 min. 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.
To the residue was added 80% aqueous acetic acid solution (100 mL),
the mixture was stirred at room temperature for 1 hr, and the
reaction mixture was subjected to azeotropic process 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].sup.+ 968.3.
B)
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-16-((tert-butyl(dimethyl)silyl)o-
xy)-2-(2-cyanoethoxy)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2-
,3-d]pyrimidin-7-yl)-10-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-metha-
nofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-p-
urin-6-yl)benzamide (Optical Isomer)
[1767]
(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)phosphorot-
hioyl)oxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-2-(5-fluoro-4-oxo-3H-py-
rrolo[2,3-d]pyrimidin-7(4H)-yl)tetrahydrofuran-3-yl hydrogen
phosphonate (1.07 g) was subjected to azeotropic process three
times with dehydration pyridine. To a solution of the residue in
anhydrous pyridine (150 mL) was added
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (4.66 g), and
the mixture was stirred at room temperature for 30 min under argon
atmosphere. To the reaction mixture were added
3H-benzo[c][1,2]dithiol-3-one (1.46 g) and water (4.55 mL) at room
temperature, and the reaction mixture was stirred for 30 min. LCMS
analysis of the reaction mixture showed four peaks with the same
mass ([M+H]+ 971.1), indicating four diastereomers being formed.
The reaction mixture was concentrated under reduced pressure, and
the residue was purified by silica gel column chromatography
(methanol/ethyl acetate), and then purified by HPLC (L-column2 ODS,
50.times.150 mm, mobile phase: 5 mM aqueous ammonium acetate
solution/acetonitrile). The fraction having the longest retention
time was concentrated under reduced pressure to give the title
compound (379 mg). MS: [M+H].sup.+ 982.1.
C)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-16-((ter-
t-butyl(dimethyl)silyl)oxy)-15-fluoro-2,10-dioxido-2,10-disulfanyloctahydr-
o-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetrade-
cin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(Optical Isomer)
[1768] 40% Methylamine methanol solution (10 mL) of
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-oxido-10-sulfanyl-2-sulfidooctahydro-12H-5,8-methano-
furo[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-pur-
in-6-yl)benzamide (optical isomer) (379 mg) was stirred at room
temperature for 3 hr under argon atmosphere. 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].sup.+
825.1.
D)
7-((2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
5-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanof-
uro[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 di-triethylamine
salt
[1769] To a solution of
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-16-((tert--
butyl(di
methyl)silyl)oxy)-15-fluoro-2,10-dioxido-2,10-disulfanyloctahydro-
-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradec-
in-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(optical isomer) (319 mg) in methanol (1 mL) was added
triethylamine trihydrofluoride (2.52 mL) at room temperature, and
the mixture was stirred at 50.degree. C. for 3 hr, and diluted with
methanol (10 mL). To the reaction mixture was added
ethoxytrimethylsilane (12 mL) at room temperature, and the mixture
was stirred for 10 min. The reaction mixture was concentrated under
reduced pressure, the residue was diluted with methanol, and the
mixture was concentrated again under reduced pressure. To the
residue was added triethylammonium acetate buffer solution, and the
solid was collected by filtration, and washed with acetonitrile to
give a white solid. The obtained white solid was purified by silica
gel column chromatography (ODS, 10 mM triethylammonium acetate
buffer solution/acetonitrile). 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 buffer solution/acetonitrile). The fraction containing the
title compound obtained by silica gel column chromatography (ODS,
10 mM triethylammonium acetate buffer solution/acetonitrile) was
concentrated under reduced pressure, and the residue was
freeze-dried to give the title compound (195 mg). MS: [M+H].sup.+
711.0.
E)
7-((2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
5-fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanof-
uro[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
[1770] Deionized water (60 mL) was passed through a column prepared
by packing AG (trade name) 50W-X8 cation-exchange resin (100-200
mesh, 3.9 g) in an empty column. Then, 1 M 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-(6-amino-9H-purin-9-yl)-15--
fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofur-
o[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 di-triethylamine salt
(195 mg) was passed through the resin after the above-mentioned
pre-treatment, and deionized water (19 mL) was passed through the
resin, and the obtained aqueous solution was freeze-dried to give
the title compound (165 mg). .sup.1H NMR (300 MHz, D.sub.2O)
.delta. 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). .sup.31P NMR (121 MHz, D.sub.2O) .delta. 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-fluoro--
2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1-
][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihy-
dro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (Optical
Isomer)
##STR00393## ##STR00394##
[1771] A)
5-fluoro-7-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetr-
ahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one
[1772] To a solution of
7-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-5-f-
luoro-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (37.9 g) in pyridine
(760 mL) was added 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane
(44.4 mL), and the mixture was stirred at room temperature for 3 hr
under argon atmosphere. The solvent was evaporated under reduced
pressure, and to the residue were added ethyl acetate and water.
The organic layer was washed with water and saturated brine, and
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].sup.+ 528.2.
B) 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]trioxadisilocin-9--
yl diisopropylphosphoramidite
[1773]
5-Fluoro-7-((6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetraisopropyltetrahy-
dro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-3,7-dihydro-4H-pyrrolo[-
2,3-d]pyrimidin-4-one (20 g) was subjected to azeotropic
dehydration three times with anhydrous acetonitrile, and dissolved
in anhydrous DMF (100 mL). To this solution were added 1H-tetrazole
(2.79 g), 1-methyl-1H-imidazole (1.65 mL), and
3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (24.1 mL),
and the mixture was stirred overnight at room temperature under
argon atmosphere, poured into saturated aqueous sodium
hydrogencarbonate solution, and extracted with ethyl acetate. The
organic layer was washed successively with saturated aqueous sodium
hydrogencarbonate solution and saturated brine, 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 (20.8 g) as a mixture of
two diastereomers. .sup.1H NMR (300 MHz, DMSO-d.sub.6) .delta.
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-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]trioxadisilocin-9-yl)oxy-
)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl hydrogen
phosphonate
[1774] A mixture of
(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)tet-
rahydrofuran-3-yl hydrogen phosphonate (3.48 g) 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]trioxadisilocin-9--
yl diisopropylphosphoramidite (7.82 g) was subjected to azeotropic
dehydration with anhydrous acetonitrile, and suspended in anhydrous
acetonitrile (25 mL) and anhydrous THF (15 mL).
5-(Ethylsulfanyl)-2H-tetrazole (3.11 g), which was subjected in
advance to azeotropic dehydration with anhydrous acetonitrile, was
dissolved in anhydrous acetonitrile (6 mL), the solution was added
to the above-mentioned suspension, and the mixture was stirred at
room temperature for 1 hr under argon atmosphere.
((Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione
(3.27 g) was added thereto, and the mixture was stirred at room
temperature for additional 30 min. 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
(6.73 g). MS: [M+H].sup.+ 1096.2.
D)
(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-((((2-cyanoethoxy)(((2R,3-
R,4R,5R)-2-(5-fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)-4-((3-hydr-
oxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-5-(hydroxymethyl)tetrahydrofura-
n-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl
hydrogen phosphonate
[1775]
(2R,3R,4R,5R)-5-(6-Benzamido-9H-purin-9-yl)-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-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl-
)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl
hydrogen phosphonate (6.73 g) was dissolved in a mixed solvent of
THF (74.8 mL) and water (16.6 mL), and the solution was ice-cooled.
Trifluoroacetic acid (16.6 mL) was added thereto, and the mixture
was stirred 0.degree. C. for 2 hr. The reaction mixture was
quenched with an aqueous solution of sodium bicarbonate (25.8 g),
and extracted with ethyl acetate. The organic layer was washed with
saturated brine, 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].sup.+
1114.2.
E)
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-tetra(propan-2-yl)disiloxanyl)oxy)-2-oxido-10-sulfidooc-
tahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclot-
etradecin-14-yl)-9H-purin-6-yl)benzamide
[1776]
(2R,3R,4R,5R)-5-(6-Benzamido-9H-purin-9-yl)-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)tetrahydro-
furan-3-yl)oxy)phosphorothioyl)oxy)methyl)-4-fluorotetrahydrofuran-3-yl
hydrogen phosphonate (6.43 g) was subjected to azeotropic
dehydration with anhydrous acetonitrile and anhydrous pyridine, and
suspended in anhydrous pyridine (130 mL).
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (3.73 g) was
added thereto, and the mixture was stirred at room temperature for
1 hr under argon atmosphere. Water (5.17 mL) and iodine (1.90 g)
were added thereto, and the mixture was stirred at room temperature
for additional 1 hr. 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 saturated
brine, 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].sup.+ 1112.2.
F)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluor-
o-2-hydroxy-16-((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2,10-dio-
xido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]penta-
oxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]py-
rimidin-4-one (Optical Isomer)
[1777] To
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-10-(2-cyanoethoxy)-15-flu-
oro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydro-
xy-16-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl)oxy)-2-oxido-10-su-
lfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosph-
acyclotetradecin-14-yl)-9H-purin-6-yl)benzamide (6.42 g) was added
40% methylamine methanol solution (30 mL), and the mixture was
stirred at room temperature for 1 hr under argon atmosphere. The
obtained mixture was concentrated under reduced pressure, and the
residue was purified by silica gel column chromatography
(methanol/ethyl acetate). The obtained residue was purified by HPLC
(L-column2 ODS, 50.times.150 mm, mobile phase: 5 mM aqueous
ammonium acetate solution/acetonitrile), the obtained fraction was
concentrated under reduced pressure, and the residue was
freeze-dried to give the title compound (420 mg). MS: [M+H].sup.+
955.2.
G)
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluor-
o-2,16-dihydroxy-2,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-
-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-di-
hydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-trimethylamine salt
(Optical Isomer)
[1778] To
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
5-fluoro-2-hydroxy-16-((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2-
,10-dioxido-10-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,1-
0]pentaoxadiphosphacyclotetradecin-7-yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2-
,3-d]pyrimidin-4-one (optical isomer, derived from tR2) (420 mg)
was added triethylamine trihydrofluoride (9.68 mL), and the mixture
was stirred at 50.degree. C. for 7.5 hr. To the reaction mixture
was added ethoxytrimethylsilane (36.3 mL) at room temperature, and
the mixture was stirred for 30 min. The reaction mixture was
concentrated under reduced pressure, and the residue was purified
by C18 silica gel column chromatography (10 mM triethylammonium
acetate buffer solution/acetonitrile), and freeze-dried to give the
title compound (298 mg). .sup.1H NMR (300 MHz, D.sub.2O) .delta.
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). .sup.31P NMR (121 MHz,
D.sub.2O) .delta. 52.2, 1.63.
Example 16
Synthesis of
7-((5R,7R,8R,12aR,14R,15R,5aS,18R)-14-(6-amino-9H-purin-9-yl)-2,10,18-tri-
hydroxy-2,10-dioxidohexahydro-14H-15,12a-(epoxymethano)-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 di-triethylamine salt
(Optical Isomer)
##STR00395## ##STR00396##
[1779] A)
(1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((((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-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-
-yl)oxy)phosphoryl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate
[1780]
(1S,3R,4R,7S)-3-(6-Benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-d-
ioxabicyclo[2.2.1]heptan-7-yl hydrogen phosphonate (2.00 g) 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]trioxadisilocin-9--
yl diisopropylphosphoramidite (4.23 g) were subjected to azeotropic
dehydration with anhydrous acetonitrile (about 50 mL, three times),
and suspended in anhydrous THF (16 mL).
5-(Ethylsulfanyl)-2H-tetrazole (1.75 g), which was subjected in
advance to azeotropic dehydration with anhydrous acetonitrile
(about 30 mL, three times), was dissolved in anhydrous acetonitrile
(16 mL), the solution was added to the above-mentioned suspension,
and the mixture was stirred at room temperature for 15 hr under
argon stream. 70% tert-Butyl hydroperoxide aqueous solution (1.86
mL) was added thereto, and the mixture was stirred at room
temperature for additional 30 min. The reaction mixture was
quenched with 10% aqueous sodium thiosulfate solution (13 mL), and
concentrated under reduced pressure. The residue was subjected
successively to azeotropic dehydration with acetonitrile (about 80
mL) and toluene (about 80 mL), the residue was subjected to silica
gel column chromatography (methanol/ethyl acetate), and the
objective fraction was concentrated under reduced pressure to give
the title compound (5.36 g) as a white amorphous solid (a mixture
of two diastereomers). MS: [M+H].sup.+ 1090.2.
B)
(1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((((2-cyanoethoxy)(((2R,3-
R,4R,5R)-2-(5-fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)-4-((3-hydr-
oxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-5-(hydroxymethyl)tetrahydrofura-
n-3-yl)oxy)phosphoryl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate
[1781]
(1R,3R,4R,7S)-3-(6-Benzamido-9H-purin-9-yl)-1-((((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-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl-
)oxy)phosphoryl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate (5.36 g) was dissolved in a mixed solvent of
THF (60 mL) and water (13 mL), and the solution was ice-cooled.
Trifluoroacetic acid (13.18 mL) was added thereto, and the mixture
was stirred at 0.degree. C. for 4 hr. The reaction mixture was
quenched with a solution of sodium bicarbonate (20.65 g) in water
(250 mL) little by little, saturated with NaCl, and extracted with
ethyl acetate-THF (3:1). The organic layer was washed with
saturated brine, 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 objective fraction was concentrated under reduced
pressure to give the title compound (3.06 g) as a white amorphous
solid (a mixture of two diastereomers). MS: [M+H].sup.+ 1108.3.
C) N-(9-((5R,7R,8R,12aR,14R,15R
15aS,18R)-10-(2-cyanoethoxy)-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-
-d]pyrimidin-7-yl)-2-hydroxy-18-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disi-
loxanyl)oxy)-2,10-dioxidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofu-
ro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14(12H)-yl)-9H--
purin-6-yl)benzamide
[1782]
(1R,3R,4R,7S)-3-(6-Benzamido-9H-purin-9-yl)-1-((((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)tetrahydro-
furan-3-yl)oxy)phosphoryl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate (3.06 g) was subjected successively to
azeotropic dehydration 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-dioxaphosphinane
2-oxide (1.78 g) was added thereto, and the mixture was stirred at
room temperature for 1 hr under argon stream. Water (1.74 mL) and
iodine (911 mg) were added thereto, and the mixture was stirred at
room temperature for additional 15 min. 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 saturated
brine, 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 objective fraction
was concentrated under reduced pressure to give the title compound
(2.38 g) as a white amorphous solid (a mixture of two
diastereomers). MS: [M+H].sup.+ 1106.3.
D)
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2,10-dih-
ydroxy-18-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl)oxy)-2,10-diox-
idohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2-
,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl)-5-fluoro-3,7-dihydro-4H-py-
rrolo[2,3-d]pyrimidin-4-one
[1783]
N-(9-((5R,7R,8R,12aR,14R,15R,15aS,18R)-10-(2-Cyanoethoxy)-7-(5-fluo-
ro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxy-18-((3-hyd-
roxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl)oxy)-2,10-dioxidohexahydro-14H--
15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadipho-
sphacyclotetradecin-14(12H)-yl)-9H-purin-6-yl)benzamide (2.38 g)
was dissolved in 40% methylamine methanol solution (40 mL), the
solution was stirred at room temperature for 1 hr, 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 objective fraction
was concentrated under reduced pressure to give 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-2,10-dioxidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro-
[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl)-5-fluo-
ro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine
salt (Optical Isomer)
[1784]
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-Amino-9H-purin-9-yl)-2,10-
-dihydroxy-18-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl)oxy)-2,10--
dioxidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,-
11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl)-5-fluoro-3,7-dihydro-4-
H-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.degree. C. for 2 hr. The reaction mixture was allowed to cool to
room temperature, neutralized with 1 M aqueous triethylammonium
hydrogencarbonate solution (110 mL), and stirred at room
temperature for 15 min. The reaction mixture was concentrated under
reduced pressure, and the residue was subjected to ODS column
chromatography (acetonitrile/10 mM triethylammonium acetate buffer
solution). The objective fraction was concentrated under reduced
pressure, and the residue was freeze-dried to give the title
compound (504 mg) as a white solid. .sup.1H NMR (300 MHz, D.sub.2O)
.delta. 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=4.5 Hz), 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). .sup.31P NMR (121
MHz, D.sub.2O) .delta. -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-dihy-
droxy-2,10-dioxido-2-sulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-metha-
nofuro[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
di-trimethylamine salt (Optical Isomer)
##STR00397##
[1785] A)
N-(9-((5R,7R,8R,12aR,14R,15R,15aS,18R)-10-(2-cyanoethoxy)-7-(5-f-
luoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-18-((3-hydroxy-1,-
1,3,3-tetraisopropyldisiloxanyl)oxy)-2,10-dioxido-2-sulfanylhexahydro-14H--
15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadipho-
sphacyclotetradecin-14(12H)-yl)-9H-purin-6-yl)benzamide
[1786]
(1R,3R,4R,7S)-3-(6-Benzamido-9H-purin-9-yl)-1-((((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)tetrahydro-
furan-3-yl)oxy)phosphoryl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate (3.71 g) was subjected to azeotropic
dehydration with anhydrous acetonitrile and anhydrous pyridine, and
suspended in anhydrous pyridine (70 mL).
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (2.15 g) was
added thereto, and the mixture was stirred at room temperature for
1 hr under argon atmosphere. 3H-Benzo[c][1,2]dithiol-3-one (674 mg)
was added thereto, and the mixture was stirred at room temperature
for additional 1 hr. To the reaction mixture was added saturated
aqueous sodium hydrogencarbonate solution, and the mixture was
extracted with ethyl acetate. The organic layer was washed with
saturated brine, 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].sup.+ 1122.2
B)
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-10-hydro-
xy-18-((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2,10-dioxido-2-su-
lfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,1-
1,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl)-5-fluoro-3,7-dihydro-4H-
-pyrrolo[2,3-d]pyrimidin-4-one (Optical Isomer)
[1787]
N-(9-((5R,7R,8R,12aR,14R,15R,15aS,18R)-10-(2-Cyanoethoxy)-7-(5-fluo-
ro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-18-((3-hydroxy-1,1,3-
,3-tetraisopropyldisiloxanyl)oxy)-2,10-dioxido-2-sulfanylhexahydro-14H-15,-
12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosph-
acyclotetradecin-14(12H)-yl)-9H-purin-6-yl)benzamide (2.78 g) was
dissolved in 40% methylamine methanol solution (50 mL), the
solution was stirred at room temperature for 1 hr under argon
atmosphere, and the solvent was evaporated under reduced pressure.
The residue was purified by silica gel column chromatography
(methanol/ethyl acetate). The obtained residue was purified by HPLC
(L-column2 ODS, 50.times.150 mm, mobile phase: 5 mM aqueous
ammonium acetate solution/acetonitrile) 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-di-
hydroxy-2,10-dioxido-2-sulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-met-
hanofuro[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
di-triethylamine salt (Optical Isomer)
[1788] To
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-1-
0-hydroxy-18-((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2,10-dioxi-
do-2-sulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,-
3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl)-5-fluoro-3,7-dih-
ydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (optical isomer) (30 mg) were
added triethylamine trihydrofluoride (0.70 mL) and methanol (1 mL),
and the mixture was stirred at 50.degree. C. for 3 hr. To the
reaction solution was added ethoxy(trimethyl)silane (2.5 mL), 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 buffer solution/acetonitrile), and the obtained solid was
freeze-dried to give the title compound (25 mg). .sup.1H NMR (300
MHz, D.sub.2O) .delta. 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). .sup.31P NMR (121
MHz, D.sub.2O) .delta. 55.76, -1.56.
Example 18
Synthesis of
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2,18-dihyd-
roxy-2,10-dioxido-10-sulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-metha-
nofuro[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
di-triethylamine salt (Optical Isomer)
##STR00398## ##STR00399##
[1789] A)
(1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((((2-cyanoethoxy)-
(((6aR,8R,9R,9aR)-8-(5-fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)-2-
,3,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-
-yl)oxy)phosphorothioyl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate
[1790] A mixture of
(1S,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-dioxabi-
cyclo[2.2.1]heptan-7-yl hydrogen phosphonate (3.0 g) 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]trioxadisilocin-9--
yl diisopropylphosphoramidite (5.86 g) was subjected to azeotropic
dehydration with anhydrous acetonitrile, and suspended in anhydrous
THF (25 mL). 5-(Ethylsulfanyl)-2H-tetrazole (2.62 g), which was
subjected in advance to azeotropic dehydration with anhydrous
acetonitrile, was dissolved in anhydrous acetonitrile (25 mL), the
solution was added to the above-mentioned suspension, and the
mixture was stirred overnight at room temperature under argon
atmosphere. ((Dimethylaminomethyl idene)amino)-3H-1,2,4-di
thiazoline-3-thione (2.75 g) was added thereto, and the mixture was
stirred at room temperature for additional 30 min. 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].sup.+ 1106.3.
B)
(1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((((2-cyanoethoxy)(((2R,3-
R,4R,5R)-2-(5-fluoro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-7(4H)-yl)-4-((3-hydr-
oxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-5-(hydroxymethyl)tetrahydrofura-
n-3-yl)oxy)phosphorothioyl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate
[1791]
(1R,3R,4R,7S)-3-(6-Benzamido-9H-purin-9-yl)-1-((((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-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl-
)oxy)phosphorothioyl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl
hydrogen phosphonate (4.96 g) was dissolved in a mixed solvent of
THF (60 mL) and water (12 mL), and the solution was ice-cooled.
Trifluoroacetic acid (12.1 mL) was added thereto, and the mixture
was stirred at 0.degree. C. for 3 hr. 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].sup.+ 1124.2.
C)
N-(9-((5R,7R,8R,12aR,14R,15R,15aS,18R)-10-(2-cyanoethoxy)-7-(5-fluoro-4-
-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxy-18-((3-hydroxy-
-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2-oxido-10-sulfidohexahydro-14H-15-
,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosp-
hacyclotetradecin-14(12H)-yl)-9H-purin-6-yl)benzamide
[1792]
(1R,3R,4R,7S)-3-(6-Benzamido-9H-purin-9-yl)-1-((((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)tetrahydro-
furan-3-yl)oxy)phosphorothioyl)oxy)methyl)-2,5-dioxabicyclo[2.2.1]heptan-7-
-yl hydrogen phosphonate (3.77 g) was subjected to azeotropic
dehydration with anhydrous acetonitrile and anhydrous pyridine, and
suspended in anhydrous pyridine (70 mL).
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (2.17 g) was
added thereto, and the mixture was stirred at room temperature for
1 hr under argon atmosphere. Water (2.12 mL) and iodine (1.11 g)
were added thereto, and the mixture was stirred at room temperature
for additional 1 hr. 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].sup.+
1122.2.
D)
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2-hydrox-
y-18-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl)oxy)-2,10-dioxido-1-
0-sulfanyl-hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,-
6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl)-5-fluoro-3,7-dihyd-
ro-4H-pyrrolo[2,3-d]pyrimidin-4-one (Optical Isomer)
[1793] To
N-(9-((5R,7R,8R,12aR,14R,15R,15aS,18R)-10-(2-cyanoethoxy)-7-(5-f-
luoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydroxy-18-((3--
hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2-oxido-10-sulfidohexahydro-
-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxa-
diphosphacyclotetradecin-14(12H)-yl)-9H-purin-6-yl)benzamide (2.38
g) was added 40% methylamine methanol solution (40 mL), and the
mixture was stirred at room temperature for 1 hr under argon
atmosphere. The obtained mixture was concentrated under reduced
pressure, and the residue was purified by silica gel column
chromatography (methanol/ethyl acetate). The obtained residue was
purified by HPLC (L-column2 ODS, 50.times.150 mm, mobile phase: 5
mM aqueous ammonium acetate solution/acetonitrile), the obtained
fraction was concentrated under reduced pressure, and the residue
was freeze-dried to give the title compound (710 mg). MS:
[M+H].sup.+ 965.2.
E)
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2,18-dih-
ydroxy-2,10-dioxido-10-sulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8-met-
hanofuro[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
di-triethylamine salt (Optical Isomer)
[1794] To a solution of
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-2-hydroxy--
18-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl)oxy)-2,10-dioxido-10--
sulfanylhexahydro-14H-15,12a-(epoxymethano)-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, derived from tR2)
(710 mg) in methanol (20 mL) was added triethylamine
trihydrofluoride (4.80 mL), and the mixture was stirred at
50.degree. C. for 3 hr. To the reaction mixture was added
ethoxytrimethylsilane (2.29 mL) at room temperature, and the
mixture was stirred for 30 min. The reaction mixture was
concentrated under reduced pressure, and the residue was purified
by C18 silica gel column chromatography (10 mM triethylammonium
acetate buffer solution/acetonitrile), and freeze-dried to give the
title compound (530 mg). .sup.1H NMR (300 MHz, D.sub.2O) .delta.
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, 4.3 Hz), 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). 3P NMR (121 MHz, D.sub.2O) .delta.
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-pyrrolo[2,3-d]pyrimidin-7-yl)-2,10,16-trihydroxy-2,10-dioxid-
ooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyc-
lotetradecin-14-yl)-1,9-dihydro-6H-purin-6-one di-triethylamine
salt (Optical Isomer)
##STR00400## ##STR00401##
[1795] A)
(2R,3R,4R,5R)-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-tetraisopropyltetrahydr-
o-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl)oxy)phosphoryl)oxy)methyl)-
-4-fluoro-5-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl
hydrogen phosphonate
[1796]
2'-Deoxy-2'-fluoro-3'-O-(hydroxy(oxido)phosphoranyl)-N-isobutyrylgu-
anosine (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]trioxadisilocin-9--
yl diisopropylphosphoramidite (1535 mg) were subjected to
azeotropic dehydration 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 subjected to azeotropic dehydration with anhydrous
acetonitrile) and anhydrous acetonitrile (10 ml), and the mixture
was stirred at room temperature for 7 hr under argon atmosphere. To
the mixture were 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]trioxadisilocin-9--
yl diisopropylphosphoramidite (1181 mg, 1.62 mmol) (which was
subjected to azeotropic dehydration with anhydrous acetonitrile)
and anhydrous acetonitrile (3 ml), and a mixture of
5-(ethylsulfanyl)-2H-tetrazole (633 mg) (which was subjected to
azeotropic dehydration with anhydrous acetonitrile) and anhydrous
acetonitrile (3 ml), and the mixture was stirred overnight at room
temperature under argon atmosphere. To the reaction mixture was
added 700% tert-butyl hydroperoxide aqueous solution (0.674 mL),
and the mixture was stirred at room temperature for additional 30
min. To the reaction mixture was added a mixture of sodium
thiosulfate pentahydrate (2817 mg) and water (4 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 (1260 mg). MS: [M+H].sup.+
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-tetraisopropyldisi-
loxanyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphoryl)oxy)meth-
yl)-4-fluoro-5-(2-isobutylamido-6-oxo-H-purin-9(6H)-yl)tetrahydrofuran-3-y-
l hydrogen phosphonate
[1797] To a mixture of
(2R,3R,4R,5R)-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-6H-furo-
[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl)oxy)phosphoryl)oxy)methyl)-4-fluoro-
-5-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl
hydrogen phosphonate (1.26 g), THF (16 ml) and water (4 ml) was
added trifluoroacetic acid (3.18 ml) at 0.degree. C., and the
mixture was stirred for 4 hr. To the mixture was added an aqueous
solution of sodium hydrogencarbonate (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].sup.+ 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-12-
H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin--
14-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-2-methylpropanamide
[1798]
(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-tetraisopropyl-
disiloxanyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphoryl)oxy)-
methyl)-4-fluoro-5-(2-isobutylamido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofura-
n-3-yl hydrogen phosphonate (1 g) was subjected to azeotropic
dehydration with anhydrous acetonitrile and anhydrous pyridine, and
suspended in anhydrous pyridine (24 mL).
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (598 mg) was
added thereto, and the mixture was stirred at room temperature for
15 min under argon atmosphere. Water (0.584 mL) and iodine (305 mg)
were added thereto, and the mixture was stirred at room temperature
for additional 25 min. 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].sup.+ 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-hydrox-
y-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2,10-dioxidooctahydro-12H-5,8-met-
hanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl)-1,-
9-dihydro-6H-purin-6-one (Optical Isomer)
[1799] To
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-10-(2-cyanoethoxy)-15-flu-
oro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydro-
xy-16-((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy)-2,10-dioxidooctah-
ydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetr-
adecin-14-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-2-methylpropanamide
(800 mg, 0.74 mmol) was added 33% methylamine ethanol solution (30
mL), the mixture was stirred overnight at room temperature under
argon atmosphere, and the solvent was evaporated under reduced
pressure. The residue was purified by silica gel column
chromatography (methanol/ethyl acetate). The obtained residue was
purified by HPLC (L-column2 ODS, 50.times.150 mm, mobile phase: 5
mM aqueous ammonium acetate solution/acetonitrile) to give the
title compound (163.4 mg). MS: [M+H].sup.+ 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-diox-
idooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphac-
yclotetradecin-14-yl)-1,9-dihydro-6H-purin-6-one di-triethylamine
salt (Optical Isomer)
[1800] A mixture of
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-metha-
nofuro[3,2-1][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) was stirred at
50.degree. C. for 1 hr under argon atmosphere. To the reaction
mixture was added ethoxy(trimethyl)silane (1.046 mL), the mixture
was stirred at room temperature for additional 1 hr, and the
solvent was evaporated under reduced pressure. The residue was
purified by C18 silica gel column chromatography (10 mM
triethylammonium acetate buffer solution/acetonitrile), and
freeze-dried to give the title compound (4.6 mg). .sup.1H NMR (300
MHz, D.sub.2O) .delta. 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, brs), 7.95
(1H, s). .sup.31P NMR (121 MHz, D.sub.2O) .delta. -1.61, -1.52.
Example 20
Synthesis of
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[3,2-1][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)
##STR00402## ##STR00403##
[1801] A)
(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimeth-
ylsilyl)oxy)-2-((((2-cyanoethoxy)(((6aR,8R,9R,9aR)-8-(5-fluoro-4-oxo-3H-py-
rrolo[2,3-d]pyrimidin-7(4H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3-
,2-f][1,3,5,2,4]trioxadisilocin-9-yl)oxy)phosphorothioyl)oxy)methyl)tetrah-
ydrofuran-3-yl hydrogen phosphonate
[1802] A mixture of
(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)o-
xy)-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen phosphonate (3.0
g) 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]trioxadisilocin-9--
yl diisopropylphosphoramidite (5.36 g) was subjected to azeotropic
dehydration with anhydrous acetonitrile, and suspended in anhydrous
acetonitrile (25 mL) and anhydrous THF (15 mL).
5-(Ethylsulfanyl)-2H-tetrazole (2.13 g), which was subjected in
advance to azeotropic dehydration with anhydrous acetonitrile, was
dissolved in anhydrous acetonitrile (15 mL), the solution was added
to the above-mentioned suspension, and the mixture was stirred
overnight at room temperature under argon atmosphere.
((Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazoline-3-thione
(2.24 g) was added thereto, and the mixture was stirred at room
temperature for additional 1 hr. 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
(5.15 g). MS: [M+H].sup.+ 1208.3.
B)
(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]pyrimidin-7(4H)-yl)-4-((3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy-
)-5-(hydroxymethyl)tetrahydrofuran-3-yl)oxy)phosphorothioyl)oxy)methyl)tet-
rahydrofuran-3-yl hydrogen phosphonate
[1803]
(2R,3R,4R,5R)-5-(6-Benzamido-9H-purin-9-yl)-4-((tert-butyldimethyls-
ilyl)oxy)-2-((((2-cyanoethoxy)(((6aR,8R,9R,9aR)-8-(5-fluoro-4-oxo-3H-pyrro-
lo[2,3-d]pyrimidin-7(4H)-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2--
f][1,3,5,2,4]trioxadisilocin-9-yl)oxy)phosphorothioyl)oxy)methyl)tetrahydr-
ofuran-3-yl hydrogen phosphonate (5.15 g) was dissolved in a mixed
solvent of THF (48 mL) and water (10 mL), and the solution was
ice-cooled. Trifluoroacetic acid (11.5 mL) was added thereto, and
the mixture was stirred at 0.degree. C. for 3 hr. 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 saturated brine, 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].sup.+ 1226.4.
C)
N-(9-((5R,7R,8R,12aR,14R,15R,15aR,16R)-15-((tert-butyl(dimethyl)silyl)o-
xy)-10-(2-cyanoethoxy)-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyri-
midin-7-yl)-2-hydroxy-16-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl-
)oxy)-2-oxido-10-sulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,-
10]pentaoxadiphosphacyclotetradecin-14-yl)-9H-purin-6-yl)benzamide
[1804]
(2R,3R,4R,5R)-5-(6-Benzamido-9H-purin-9-yl)-4-((tert-butyldimethyls-
ilyl)oxy)-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)phosphorothioyl)oxy)methyl-
)tetrahydrofuran-3-yl hydrogen phosphonate (5.13 g) was subjected
to azeotropic dehydration with anhydrous acetonitrile and anhydrous
pyridine, and suspended in anhydrous pyridine (100 mL).
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (2.70 g) was
added thereto, and the mixture was stirred at room temperature for
1 hr under argon atmosphere. Water (2.64 g) and iodine (1.38 g)
were added thereto, and the mixture was stirred at room temperature
for additional 1 hr. The reaction mixture was quenched with sodium
thiosulfate (5.19 g) and water (2.64 g), and extracted with ethyl
acetate. The organic layer was washed with water and saturated
brine, 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].sup.+ 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-oxido-10-sulfanyl-5,8-me-
thano-12H-furo[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-y-
l)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (Optical
Isomer)
[1805] To N-(9-((5R,7R,8R
12aR,14R,15R,15aR,16R)-15-((tert-butyl(dimethyl)silyl)oxy)-10-(2-cyanoeth-
oxy)-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydr-
oxy-16-((3-hydroxy-1,1,3,3-tetra(propan-2-yl)disiloxanyl)oxy)-2-oxido-10-s-
ulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosp-
hacyclotetradecin-14-yl)-9H-purin-6-yl)benzamide (3.5 g) was added
40% methylamine methanol solution (50 mL), and the mixture was
stirred at room temperature for 1 hr under argon atmosphere. The
obtained mixture was concentrated under reduced pressure, and the
residue was purified by silica gel column chromatography
(methanol/ethyl acetate). The obtained residue was purified by HPLC
(L-column2 ODS, 50.times.150 mm, mobile phase: 5 mM aqueous
ammonium acetate solution/acetonitrile), the obtained fraction was
concentrated under reduced pressure, and the residue was
freeze-dried to give the title compound (210 mg). MS: [M+H].sup.+
1067.4.
E)
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[3,2-1][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)
[1806] 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-oxido-10-sulfanyl-5,8-meth-
ano-12H-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, derived from tR2) (210 mg) in methanol (1.0
mL)-triethylamine (0.4 mL) was added triethylamine trihydrofluoride
(0.962 mL), and the mixture was stirred at 50.degree. C. for 2.5
hr. To the reaction mixture was added ethoxytrimethylsilane (4.59
mL) at room temperature, and the mixture was stirred for 30 min.
The reaction mixture was concentrated under reduced pressure, and
the residue was purified by C18 silica gel column chromatography
(10 mM triethylammonium acetate buffer solution/acetonitrile), and
freeze-dried to give the title compound (125 mg).
[1807] .sup.1H NMR (300 MHz, D.sub.2O) .delta. 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, 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). .sup.31P NMR (121 MHz, D.sub.2O)
.delta. 52.4, 1.21.
Example 23
Synthesis of
7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-10,15,16-t-
rihydroxy-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-methyl-3,7-d-
ihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
(Optical Isomer 1 and 2)
##STR00404## ##STR00405## ##STR00406##
[1808] 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-me-
thyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2,4,4-tetraisopr-
opyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl]oxy}phosphory-
l]oxy}methyl)tetrahydrofuran-3-yl hydrogen phosphonate
[1809]
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-4-{[tert-butyl(dim-
ethyl)silyl]oxy}-2-(hydroxymethyl)tetrahydrofuran-3-yl hydrogen
phosphonate (250 mg) and 2-cyanoethyl
(6aR,8R,9R,9aR)-8-(5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]p-
yrimidin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]t-
rioxadisilocin-9-yl diisopropylphosphoramidoite (359 mg)(Example 24
step C) were subjected to azeotropic dehydration with anhydrous
acetonitrile, and anhydrous acetonitrile (1.1 mL) and anhydrous
tetrahydrofuran (0.7 mL) were added thereto. To the mixture was
added a solution of 5-(ethylsulfanyl)-2H-tetrazole (175 mg) (which
was subjected to azeotropic dehydration with anhydrous
acetonitrile) in anhydrous acetonitrile (0.75 mL), and the mixture
was stirred under argon atmosphere at room temperature for 2 hr.
Tert-butyl hydroperoxide (5.5 mol/L) in nonane (0.25 mL) was added
thereto, and the mixture was stirred at room temperature for 40
min. The reaction mixture was cooled in ice bath and a solution of
sodium thiosulfate (345 mg) in water (0.275 mL) was added. The
mixture was allowed to stir at room temperature for 15 min and was
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].sup.+ 1206.3
B)
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-4-{[tert-butyl(dimethy-
l)silyl]oxy}-2-({[(2-cyanoethoxy)({(2R,3R,4R,5R)-2-(5-fluoro-3-methyl-4-ox-
o-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-5-(hydroxymethyl)-4-[(3-hyd-
roxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]tetrahydrofuran-3-yl}oxy)phosph-
oryl]oxy}methyl)tetrahydrofuran-3-yl hydrogen phosphonate
[1810]
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-4-{[tert-butyl(dim-
ethyl)silyl]oxy}-2-({[(2-cyanoethoxy){[(6aR,8R,9R,9aR)-8-(5-fluoro-3-methy-
l-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2,2,4,4-tetraisopropy-
ltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl]oxy}phosphoryl]o-
xy}methyl)tetrahydrofuran-3-yl hydrogen phosphonate (315 mg) was
dissolved in a mixture of tetrahydrofuran (2.8 mL) and water (0.71
mL) and the solution was allowed to stir and cooled in ice bath.
Trifluoroacetic acid (0.71 mL) was added drop wise and the mixture
was allowed to stir at 0-5.degree. C. for 2 hr 15 min. Sodium
bicarbonate (1.14 g) was added gradually to the reaction mixture
while maintaining good stirring followed by addition of water (3
mL) and ethyl acetate (10 mL). Separated aqueous layer was
extracted with ethyl acetate (10 mL). Combined organics was washed
with brine (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].sup.+ 1224.3
C)
N-(9-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-15-{[tert-butyl(dimethyl)silyl]o-
xy}-10-(2-cyanoethoxy)-7-(5-fluoro-3-methyl-4-oxo-3,4-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[3,2-1][1,3,6,9,11,2,-
10]pentaoxadiphosphacyclotetradecin-14-yl}-9H-purin-6-yl)benzamide
[1811]
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-4-{[tert-butyl(dim-
ethyl)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)ph-
osphoryl]oxy}methyl)tetrahydrofuran-3-yl hydrogen phosphonate (143
mg) was subjected to azeotropic dehydration with anhydrous
acetonitrile and anhydrous pyridine (2.3 mL) was added thereto. To
the mixture was added 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane
2-oxide (75 mg), and the mixture was stirred under argon atmosphere
at room temperature for 45 min. Water (75 .mu.L) and
3H-1,2-benzodithiol-3-one 1,1-dioxide (29 mg) were added thereto,
and the mixture was stirred at room temperature for additional 40
min. 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 min. The mixture was
concentrated, toluene was added thereto, and the mixture was
concentrated under reduced pressure (repeat 4.times.). 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].sup.+ 1238.3
D)
7-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-{[ter-
t-butyl(dimethyl)silyl]oxy}-10-hydroxy-16-[(3-hydroxy-1,1,3,3-tetraisoprop-
yldisiloxanyl)oxy]-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-met-
hyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
[1812] 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-py-
rrolo[2,3-d]pyrimidin-7-yl)-16-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxan-
yl)oxy]-2,10-dioxido-2-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,-
9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl}-9H-purin-6-yl)benzamide
(83 mg) was added 33% methylamine ethanol solution (2.0 mL), and
the mixture was stirred under argon atmosphere at room temperature
for 1 hr, and concentrated under reduced pressure. The crude
product was purified by reverse phase chromatography (ISCO
RediSepRf Gold HP C18 15.5 g column) eluted with 0 to 70% ACN in 10
mM aq NH.sub.4OAc to give two isolated single isomers of the title
compounds: peak 1 (early fraction, 11 mg) and peak 2 (late
fraction, 44.5 mg). MS: [M+H].sup.+ 1081.3
E)
7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-10,15,16-
-trihydroxy-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-methyl-3,7-
-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
(Optical Isomer 1)
[1813] To
7-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-1-
5-{[tert-butyl(dimethyl)silyl]oxy}-10-hydroxy-16-[(3-hydroxy-1,1,3,3-tetra-
isopropyldisiloxanyl)oxy]-2,10-dioxido-2-sulfanyloctahydro-12H-5,8-methano-
furo[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl}-5-fluor-
o-3-methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (324 mg,
peak 2/late fraction from step D) were added pyridine (6.0 mL) and
triethylamine trihydrofluoride (0.303 mL). The reaction mixture was
stirred at 55.degree. C. for 16 hr. LCMS analysis showed some of
monoprotected intermediate. Methanol (2.0 mL) was added and the
reaction mixture was stirred at 55.degree. C. for 3 hr. LCMS then
showed completed reaction. 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 hr and
was filtered on a pad of Celite.RTM.. The filtrate was concentrated
under reduce pressure, and the residue was purified by reverse
phase chromatography (ISCO RediSepRf Gold HP C18Aq column) eluted
with 0 to 15% ACN in 10 mM aq triethylammonium acetate to give the
title compound (195 mg). MS: [M+H].sup.+ 707.1 .sup.1H NMR (400
MHz, D.sub.2O) .delta. 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
(11H, 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).
.sup.31P NMR (162 MHz, D.sub.2O) .delta. -2.22, 54.80. .sup.19F NMR
(376 MHz, D.sub.2O) .delta. -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-dioxido-2-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][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 di-triethylamine salt
(Optical Isomer 2)
[1814] Optical isomer 2 was prepared by using the same procedure as
for optical isomer 1.
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-tetraisopropyl-
disiloxanyl)oxy]-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-methy-
l-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (24.5 mg, peak
1/early fraction from step D) give the title compound (11.8 mg).
MS: [M+H].sup.+ 707.1. .sup.1H NMR (400 MHz, D.sub.2O) .delta. ppm
1.25 (18H, t, 0.1=7.22 Hz), 3.17 (q, J=7.24 Hz, 12H), 3.35 (3H, s),
4.12-4.22 (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).
.sup.31P NMR (162 MHz, D.sub.2O) .delta. -2.42, 54.11. .sup.19F NMR
(376 MHz, D.sub.2O) .delta. -165.54
Example 24
Synthesis of
7-[(5R,7R,8R,12aR,14R,15R,15aR,6R)-14-(6-amino-9H-purin-9-yl)-15-fluoro-1-
0,16-dihydroxy-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-methyl--
3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
(Optical Isomer 1 and 2)
##STR00407## ##STR00408## ##STR00409## ##STR00410##
[1815] A)
7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran--
2-yl]-5-fluoro-3-methyl-pyrrolo[2,3-d]pyrimidin-4-one
[1816]
7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-y-
l]-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (300 mg)
was dissolved in N,N-dimethylformamide (3.30 mL) and potassium
carbonate (192 mg) was added. The suspension was cooled in 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 IM HCl to pH .about.6. The resulted 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[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl]-3-methyl-3,7-dihydro-4H-pyr-
rolo[2,3-d]pyrimidin-4-one
[1817]
7-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-y-
l]-5-fluoro-3-methyl-pyrrolo[2,3-d]pyrimidin-4-one (985 mg)
(pre-azeotroped with acetonitrile 3 times and dried in vacuum for 5
hours) was dissolved in pyridine (9.75 mL) and
N,N-dimethylformamide (4.80 mL). After stirring for 15 min
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (1.02 mL) was added
drop wise. 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). Separated organic layer was washed with water
(2.times.50 mL) and brine (50 mL), dried over Na.sub.2SO.sub.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]p-
yrimidin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]t-
rioxadisilocin-9-yl diisopropylphosphoramidoite
[1818] 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]trioxadisilocin-8-yl]-3-methyl-3,7-dihydro-4H-pyrro-
lo[2,3-d]pyrimidin-4-one (1.41 g) was azeotroped with dry
acetonitrile (3.times.5 mL), dissolved in dry N,N-dimethylformamide
(6.17 mL) under Argon. 1H-tetrazole 0.45M in acetonitrile (7.05
mL), 1-methyl-1H-imidazole (123 .mu.L) and 2-cyanoethyl
N,N,N',N'-tetraisopropylphosphorodiamidite (1.86 mL) were added.
The mixture was stirred at room temperature for 1 hour. The mixture
was diluted with 70 ml of EtOAc, washed with saturated NaHCO.sub.3
(.times.2) and brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated under reduced pressure. The crude residue was purified
by silica gel chromatography using EtOAc/hexane (0.5% Et.sub.3N) 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-(5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyr-
imidin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]tri-
oxadisilocin-9-yl]oxy}phosphoryl]oxy)methyl)-4-fluorotetrahydrofuran-3-yl
hydrogen phosphonate
[1819] In a 100 mL round bottom flask 2-cyanoethyl
(6aR,8R,9R,9aR)-8-(5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]p-
yrimidin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]t-
rioxadisilocin-9-yl diisopropylphosphoramidoite (937 mg) and
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-4-fluoro-2-(hydroxymethy-
l)tetrahydrofuran-3-yl hydrogen phosphonate (425 mg) were
azeotroped with dry CH.sub.3CN (3.times.5 mL), suspended in dry
tetrahydrofuran (1.80 mL) and acetonitrile (2.95 mL) under Argon. A
solution of 5-(ethylthio)-1H-tetrazole (380 mg) (pre-azeotroped
with acetonitrile 3.times.5 mL) in dry acetonitrile (1.80 mL) was
added. The mixture was allowed to stir at room temperature for 1
hour. Tert-butyl hydroperoxide (5.5 mol/L) in nonane (425 .mu.L)
was added. The mixture was stirred for 40 min at room temperature
then cooled in an ice bath. A solution of sodium thiosulfate (500.0
mg) in water (700 .mu.L as added and the mixture was stirred for 10
min. 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) as a mixture of two
optical isomers was afforded. 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-(5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyri-
midin-7-yl)-5-(hydroxymethyl)-4-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxa-
nyl)oxy]tetrahydrofuran-3-yl}oxy)phosphoryl]oxy}methyl)-4-fluorotetrahydro-
furan-3-yl hydrogen phosphonate
[1820] 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]pyr-
imidin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]tri-
oxadisilocin-9-yl]oxy}phosphoryl]oxy}methyl)-4-fluorotetrahydrofuran-3-yl
hydrogen phosphonate (1.10 g) in tetrahydrofuran (11.0 mL) and
water (2.50 mL) cooled with an ice bath was slowly added
trifluoroacetic acid (2.50 mL). The mixture was stirred with
cooling for 4 hours. Sodium bicarbonate (3800 mg) was added,
followed by 8 ml of water. The mixture was stirred for 5 min,
brought to room temperature and extracted with EtOAc twice. The
combined organic layer was washed with brine, dried over
Na.sub.2SO.sub.4, filtered and the filtrate was evaporated under
reduced pressure to give a crude product. Chromatography using
silica gel column (methanol/DCM) afforded 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-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-[(-
3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dioxido-2-sulfanyloc-
tahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclot-
etradecin-14-yl}-9H-purin-6-yl)benzamide
[1821] In a 100 mL round bottom flask
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-2-({[(2-cyanoethoxy)({(2-
R,3R,4R,5R)-2-(5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimi-
din-7-yl)-5-(hydroxymethyl)-4-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxany-
l)oxy]tetrahydrofuran-3-yl}oxy)phosphoryl]oxy}methyl)-4-fluorotetrahydrofu-
ran-3-yl hydrogen phosphonate (149 mg) was azeotroped with
acetonitrile (3.times.4 mL) and dried under vacuum, dissolved in
dry pyridine (2.35 mL) and cooled in an ice bath.
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (180 mg) in
pyridine (0.120 ml) was added. The mixture was allowed to stir at
room temperature for 1.5 hour. Water (70.3 .mu.L) was added,
followed by 3H-1,2-benzodithiol-3-one (56.2 mg). The mixture was
allowed to stir at room temperature for 40 min. Sodium thiosulfate
(135 mg) in 1 mL of water was added. The mixture was stirred for 10
min, concentrated under reduced pressure, azeotroped with toluene
to remove the 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-fluor-
o-10-hydroxy-16-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-di-
oxido-2-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]penta-
oxadiphosphacyclotetradecin-7-yl}-5-fluoro-3-methyl-3,7-dihydro-4H-pyrrolo-
[2,3-d]pyrimidin-4-one
[1822] The mixture of two isomers
N-(9-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-10-(2-cyanoethoxy)-15-fluoro-7-(5--
fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-[(3--
hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dioxido-2-sulfanylocta-
hydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotet-
radecin-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 substance was evaporated off under reduced
pressure. The crude product was purified by reverse phase
chromatography (ISCO RediSepRf Gold HP C18 column) eluted with 0 to
50% ACN in 10 mM aq NH.sub.4OAc to give two isolated single isomers
of the title compounds: peak 1 (early fraction, 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-fluor-
o-10,16-dihydroxy-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-meth-
yl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine
salt (Optical Isomer 1)
[1823] 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-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dio-
xido-2-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentao-
xadiphosphacyclotetradecin-7-yl}-5-fluoro-3-methyl-3,7-dihydro-4H-pyrrolo[-
2,3-d]pyrimidin-4-one (35.6 mg, peak 2/late fraction from step G).
Pyridine (273 .mu.L) was added, followed by triethylamine
trihydrofluoride (27.3 .mu.L) and triethylamine (414 .mu.L). The
mixture was allowed to stir at room temperature overnight and at
50.degree. C. for 2 hours. The reaction mixture was cooled in 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 plug of Celite.RTM..
The filtrate was concentrated under reduced pressure and the
residue was purified by reverse phase chromatography (ISCO
RediSepRf Gold HP C18Aq column) eluted with 0 to 10% ACN in 10 mM
aq triethylammonium acetate to give the title compound (23.9 mg).
MS: [M+H]+ 709.2. .sup.1H NMR (400 MHz, METHANOL-d4) .delta.
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=12 Hz), 6.37 (d, 1H, J=16 Hz), 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).
.sup.31P NMR (162 MHz, D.sub.2O) .delta. -2.45, 55.32. .sup.19F NMR
(376.5 MHz, D.sub.2O) .delta. -164.60, -200.71.
I)
7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluor-
o-10,16-dihydroxy-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-meth-
yl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine
salt (Optical Isomer 2)
[1824] 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-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dio-
xido-2-sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentao-
xadiphosphacyclotetradecin-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 step G).
Pyridine (92 .mu.L) was added, followed by triethylamine
trihydrofluoride (9.2 .mu.L) and triethylamine (140 .mu.L). The
mixture was allowed to stir at 50.degree. C. for 2 hours. The
reaction mixture was cooled with 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 plug of Celite.RTM.. The filtrate was
concentrated under reduced pressure and the residue was purified by
reverse phase chromatography (ISCO RediSepRf Gold HP C18Aq column)
eluted with 0 to 10% ACN in 10 mM aq triethylammonium acetate to
give the title compound (1.94 mg). MS: [M+H]+ 709.3. .sup.1H NMR
(400 MHz, DEUTERIUM OXIDE) .delta. 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 (br
d, 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). .sup.31P NMR (162 MHz, D.sub.2O) .delta. -2.52, 53.94.
.sup.19F NMR (376.5 MHz, D.sub.2O) .delta. -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-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-methyl-3,7-dih-
ydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
##STR00411## ##STR00412##
[1825] A)
N-(9-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-10-(2-cyanoethoxy)-15-flu-
oro-7-(5-fluoro-3-methyl-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-dio-
xidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphospha-
cyclotetradecin-14-yl}-9H-purin-6-yl)benzamide
[1826] In a 100 mL round bottom flask
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-2-({[(2-cyanoethoxy)((2R-
,3R,4R,5R)-2-(5-fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimid-
in-7-yl)-5-(hydroxymethyl)-4-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl-
)oxy]tetrahydrofuran-3-yl}oxy)phosphoryl]oxy)
methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate (1.14 g)
was azeotroped with acetonitrile (3.times.4 mL) and dried under
vacuum, dissolved in pyridine (18.0 mL) and cooled in an ice bath.
2-Chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane 2-oxide (586 mg) was
added in 2 portions. The mixture was allowed to stir at room
temperature for 40 min. Water (0.554 mL) was added, followed by
3H-1,2-benzodithiol-3-one 1,1-dioxide (251 mg). The mixture was
allowed to stir at room temperature for 40 min. 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 the 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,12R,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[3,2-1][1,3,6,9,11,2,10]pentaoxadipho-
sphacyclotetradecin-7-yl}-5-fluoro-3-methyl-3,7-dihydro-4H-pyrrol
o[2,3-d]pyrimidin-4-one
[1827] To a 25 mL round-bottom flask was added
N-(9-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-10-(2-cyanoethoxy)-15-fluoro-7-(5--
fluoro-3-methyl-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-hydro-
xy-16-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dioxidooctah-
ydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetr-
adecin-14-yl}-9H-purin-6-yl)benzamide (650 mg) and methylamine (33
mass %) in absolute ethanol (22.7 mL). The mixture was stirred at
room temperature for 3 hours. The reaction mixture was concentrated
under reduced pressure and the residue was purified by reverse
phase chromatography (ISCO RediSepRf Gold HP C18 column) eluted
with 0 to 60% ACN in 10 mM aq NH.sub.4OAc 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-fluor-
o-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-methyl-3,7-d-
ihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
[1828] 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-[(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]pentaoxadiphos-
phacyclotetradecin-7-yl}-5-fluoro-3-methyl-3,7-dihydro-4H-pyrrolo[2,3-d]py-
rimidin-4-one (32.0 mg) and pyridine (246 .mu.L) was added. To the
suspension was added triethylamine trihydrofluoride (24.6 .mu.L),
followed by triethylamine (372 .mu.L). The mixture was allowed to
stir at room temperature overnight. The reaction mixture was cooled
in 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 plug of Celite.RTM..
The filtrate was concentrated under reduced pressure and the
residue was purified by reverse phase chromatography (ISCO
RediSepRf Gold HP C18Aq column) eluted with 0 to 5% ACN in 10 mM aq
triethylammonium acetate to give the title compound (8.3 mg). MS:
[M+H]+ 693.2. .sup.1H NMR (400 MHz, METHANOL-d4) .delta. 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=4 Hz),
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). .sup.31P NMR (162 MHz,
D.sub.2O) .delta. -1.66, -2.26. .sup.19F NMR (376.5 MHz, D.sub.2O)
.delta. -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--
16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1-
][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-5-chloro-3,7-dihy-
dro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt (Optical
Isomer 1 and Optical Isomer 2)
##STR00413## ##STR00414## ##STR00415##
[1829] A)
(6aR,8R,9R,9aR)-8-(5-chloro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]p-
yrimidin-7-yl)-2,2,4,4-tetraisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]t-
rioxadisilocin-9-yl-cyanoethyl diisopropylphosphoramidoite
[1830] 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]trioxadisilocin-8-yl]-3,7-dihydro-4H-pyrrolo[2,3-d]-
pyrimidin-4-one (1.469 g) (synthesized in the same manner as in the
method of Example 15 A)) was azeotroped with toluene (3.times.) and
dried in vacuum. The round bottom flask was evacuated and back
filled with argon (3.times.). Anhydrous N,N-dimethylformamide (5.00
mL) was added to dissolve the starting material with sonication for
a minute, followed by 2-cyanoethyl
N,N,N',N'-tetraisopropylphosphorodiamidite (2.50 mL),
1-methylimidazole (0.13 m) and then 1H-tetrazole (0.45M in
acetonitrile, 6.70 mL). The mixture was stirred under atmosphere of
argon at room temperature for 3 hours. The mixture was diluted with
EtOAc (200 mL). Saturated NaHCO.sub.3 solution was added. The
aqueous layer was extracted with EtOAc twice. The combined EtOAc
layer was washed with water then brine, dried over anhydrous
Na.sub.2SO.sub.4, filtered. The filtrate was concentrated under
reduced pressure to give a crude oily product. The crude material
was purified by column chromatography (ISCO RediSepRf DIOL HP Gold
100 g column) eluted with EtOAc:hexanes (1:9 to 1:1) to give the
title compound (1.77 g). MS: [M+H]+ 661.3 for the hydrolyzed
fragment of the product.
B)
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-4-fluoro-2-(hydroxymet-
hyl)tetrahydrofuran-3-yl hydrogen phosphonate
[1831]
N-(9-[(2R,3R,4R,5R)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl)-
-3-fluoro-4-hydroxytetrahydrofuran-2-yl]-9H-purin-6-yl}benzamide
(2.64 g) was dissolved in anhydrous pyridine (18.0 mL) under argon,
cooled with an ice bath. Diphenyl phosphite (1.60 mL) was added
slowly over 1 min. The reaction solution was allowed to warm to
room temperature and stirred for 1 hour. The second portion of
diphenyl phosphite (0.35 mL) was added slowly and the mixture was
kept stirring for 30 more min. The mixture was cooled with an ice
bath. Water (2.0 mL) was added and the mixture was stirred at room
temperature for 30 min. The mixture was concentrated under reduced
pressure and azeotroped with toluene to further remove pyridine and
water. The residue was dried on vacuum pump. To the residue was
added acetic acid (10.0 mL) and water (2.0 mL). The mixture was
stirred at room temperature for 30 min. The mixture was
concentrated under reduced pressure and azeotroped with toluene,
then dried on vacuum pump to give a crude oil, which was
chromatographed on a silica column using MeOH/DCM (0/100 to 50/50)
to afford 1.33 g of 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-te-
traisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl]oxy}(-
2-cyanoethoxy)phosphorothioyl]oxy}methyl)-4-fluorotetrahydrofuran-3-yl
hydrogen phosphonate
[1832] In a 250 mL round bottom flask a mixture of
(6aR,89R,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]trioxadisilo-
cin-9-yl 2-cyanoethyl diisopropylphosphoramidoite (2.23 g) and
(2R,3R,4R,5R)-5-[6-(benzoylamino)-9H-purin-9-yl]-4-fluoro-2-(hydroxymethy-
l)tetrahydrofuran-3-yl hydrogen phosphonate (1.17 g) was azeotroped
with dry acetonitrile (25 mL.times.3), dried on vacuum pump for 1
hour. To the mixture was added anhydrous acetonitrile (20.0 mL)
under argon, followed by a solution of 5-(ethylthio)-1H-tetrazole
(1.00 g) (pre-azeotroped with dry acetonitrile, 10 mL.times.3 and
dried on vacuum pump) in anhydrous acetonitrile (10.0 mL). The
mixture was stirred at room temperature for 1 hour 20 min.
((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazoline-3-thione
(655 mg) was added and the mixture was stirred at room temperature
for 40 min. The mixture was concentrated under reduced pressure.
Chromatography on silica column using MeOH/DCM (0/100 to 40/60)
afforded 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-(hydroxyme-
thyl)-4-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]tetrahydrofuran--
3-yl}oxy)(2-cyanoethoxy)phosphorothioyl]oxy}methyl)-4-fluorotetrahydrofura-
n-3-yl hydrogen phosphonate
[1833] In a round bottom flask
(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-tetr-
aisopropyltetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yl]oxy}(2--
cyanoethoxy)phosphorothioyl]oxy}methyl)-4-fluorotetrahydrofuran-3-yl
hydrogen phosphonate (1.73 g) was dissolved in tetrahydrofuran (16
mL) and water (4.0 mL). The solution was cooled with an ice bath.
Triflouroacetic acid (4.0 mL) was dropwise added. The mixture was
stirred with cooling for 4 hours. At 0.degree. C. sodium
bicarbonate (7.00 g) was portion wise added, followed by water and
EtOAc. The mixture was stirred for 3 min then brought to room
temperature. The layers were separated. The aqueous layer was
extracted with EtOAc (3.times.). The combined EtOAc solution was
washed with brine, dried over anhydrous Na.sub.2SO.sub.4, filtered.
The filtrate was concentrated under reduce pressure and dried on
vacuum pump to give the crude product. The crude material was
purified on a silica 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[2,3-d]pyrimidin-7-yl)-10-(2-cyanoethoxy)-15-fluoro-16-[(3-hydroxy-
-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2-oxido-2-sulfanyl-10-sulfidooctah-
ydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetr-
adecin-14-yl}-9H-purin-6-yl)benzamide (Mixture of Two Major Optical
Isomers)
[1834] 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-(hydroxymethy-
l)-4-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]tetrahydrofuran-3-y-
l}oxy)(2-cyanoethoxy)phosphorothioyl]oxy)
methyl)-4-fluorotetrahydrofuran-3-yl hydrogen phosphonate (1.20 g)
was azeotroped with dry pyridine (fresh bottle)/acetonitrile
(.times.2) then dry acetonitrile. The resulting white solid was
dried under vacuum. Under an atmosphere of argon anhydrous pyridine
(20.0 mL) (fresh bottle) was added, followed by
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (0.700 g). The
clear solution was allowed to stir at room temperature for 1 hour
10 min. Water (0.70 mL) was added, followed by
3H-1,2-benzodithiol-3-one 1,1-dioxide (0.300 g). The mixture was
stirred at room temperature for 20 min. The mixture was
concentrated under reduced pressure, azeotroped with toluene
(.times.2), dried on vacuum pump to give a crude residue. The crude
residue was chromatographed on a silica column using MeOH/DCM
(0/100 to 20/80). The title products (590 mg) from the early
fraction was afforded and identified as the mixture of major
isomers of peaks 2 and 4 from LCMS; while the second crop of
products (389 mg) from the late fraction was 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-fluor-
o-16-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dioxido-2,10--
disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadip-
hosphacyclotetradecin-7-yl}-5-chloro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidi-
n-4-one (Single Optical Isomers 1 and 2)
[1835] Under N.sub.2 atmosphere
N-(9-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-7-(5-chloro-4-oxo-3,4-dihydro-7H-p-
yrrolo[2,3-d]pyrimidin-7-yl)-10-(2-cyanoethoxy)-15-fluoro-16-[(3-hydroxy-1-
,1,3,3-tetraisopropyldisiloxanyl)oxy]-2-oxido-2-sulfanyl-10-sulfidooctahyd-
ro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetrad-
ecin-14-yl}-9H-purin-6-yl)benzamide (0.584 g, mixture of 2 major
isomers, early fraction of column chromatograph) was stirred in
methylamine (33 mass %) in absolute ethanol (15.0 mL) at room
temperature for 2 hours. The mixture was concentrated under reduced
pressure and dried on vacuum pump to give crude solid. Two
subsequent purifications on silica gel columns, using MeOH/EtOAc
(0/100 to 40/60) afforded 2 single optical isomers of the title
compounds: isomer 1 (early fraction/peak 1, 73 mg) and isomer 2
(late fraction/peak 2, 304 mg), and a mix fraction of peak 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-fluor-
o-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-
-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-5-chloro-3,7-di-
hydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
(Optical Isomer 1)
[1836] To a polypropylene tube was added
7-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro--
16-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dioxido-2,10-di-
sulfanyloctahydro-2H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphos-
phacyclotetradecin-7-yl}-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 triethylamine
trihydrofluoride (0.060 mL), then triethylamine (0.90 mL). The
reaction mixture in the sealed propylene tube was stirred at
50.degree. 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.RTM. using a plastic filter funnel, washed
with water (1 mL.times.4). The clear aqueous filtrate was
concentrated under reduced pressure, azeotroped with toluene for
multiple times, dried on vacuum for 10 min to give a crude residue.
The crude residue was purified by C18 column chromatography
(water-acetonitrile/10 mM triethylammonium acetate buffer
solution). The product fraction was concentrated under reduced
pressure, azeotroped with toluene (.times.4), then with
endotoxin-free water (.times.5). The final residue was dissolved in
endotoxin-free water and lyophilized to afford the title product
(optical isomer 1, 29.0 mg). MS [M+H]+ 727.0. .sup.1H NMR (400 MHz,
deuterium oxide) 6=8.19 (s, 1H), 8.16 (s, 1H), 7.96 (s, 1H), 7.63
(s, 1H), 6.39 (br d, J=15.7 Hz, 1H), 6.34 (br d, 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 (br
d, J=3.8 Hz, 1H), 4.51 (br d, J=8.2 Hz, 1H), 4.42 (br s, 1H), 4.40
(br s, 1H), 4.35-4.28 (m, 1H), 4.16-4.10 (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). .sup.31P NMR (162 MHz, deuterium oxide) .delta. 55.29, 54.54.
.sup.19F NMR (376 MHz, deuterium oxide) .delta. -200.90.
I)
7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluor-
o-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-
-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-5-chloro-3,7-di-
hydro-4H-pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt
(Optical Isomer 2)
[1837] To a polypropylene tube was added
7-{(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-9H-purin-9-yl)-15-fluoro--
16-[(3-hydroxy-1,1,3,3-tetraisopropyldisiloxanyl)oxy]-2,10-dioxido-2,10-di-
sulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadipho-
sphacyclotetradecin-7-yl}-5-chloro-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin--
4-one (optical isomer 2, 186 mg, late fraction from chromatograph).
Pyridine (1.1 mL) was added, followed by triethylamine
trihydrofluoride (0.161 mL), then triethylamine (2.4 mL). The
reaction mixture in the sealed propylene tube was stirred at
50.degree. 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.RTM. using a plastic filter funnel, washed
with water (3 mL.times.4). The clear aqueous filtrate was
concentrated under reduced pressure, azeotroped with toluene
multiple times, dried on vacuum for 10 min to give a crude residue.
The crude residue was purified by C18 column chromatography
(water-acetonitrile/10 mM triethylammonium acetate buffer
solution). The product fraction was concentrated under reduced
pressure, azeotroped with toluene (.times.4), then with
endotoxin-free water (.times.5). The final residue was dissolved in
endotoxin-free water and lyophilized to afford the title product
(optical isomer 2, 109 mg). MS [M+H]+ 727.0.
[1838] .sup.1H NMR (400 MHz, deuterium oxide) .delta. 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 (br d, J=9.0 Hz, 1H), 4.33 (br d, J=14.7
Hz, 2H), 4.28-4.16 (m, 2H), 3.98 (br dd, J=5.0, 11.8 Hz, 1H), 3.04
(q, J=7.4 Hz, 12H), 1.12 (t, J=7.3 Hz, 18H). .sup.31P NMR (162 MHz,
deuterium oxide) .delta. 55.12, 52.04. .sup.19F NMR (376 MHz,
deuterium oxide) .delta. -200.43.
Example 27
Linker-Payload 1
##STR00416##
[1840] To a mixture of
6-(2,5-dioxopyrrol-1-yl)-N-[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1--
methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]hexanamide
(mc-Val-Ala-PAB-OH, Synchem, Elk Grove Village, Ill., 0.0822 mmol,
40.0 mg) in anhydrous acetonitrile (19.1 mmol, 1.00 mL, 783 mg) was
added cesium iodide (0.0986 mmol, 25.6 mg) and boron trifluoride
diethyl etherate (0.0986 mmol, 0.0125 mL, 14.0 mg). The reaction
was stirred at room temperature overnight. After 18 h of stirring
the mixture was diluted with -10 mL of DCM and filtered over a
Celite.RTM. pad. The solid was washed with DCM. The filtrate was
concentrated to provide an orange solid. The crude product
(mc-Val-Ala-PAB-I) was used without further purification.
[1841] A vial charged with a solution of disodium
(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-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2-s-
ulfidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosp-
hacyclotetradecin-10-olate 2,10-dioxide (Payload 1, also "compound
Ex. 3a", 0.0106 mmol, 7.80 mg) and
6-(2,5-dioxopyrrol-1-yl)-N-[(1S)-1-[[(1S)-2-[4-(iodomethyl)anilino]-1-met-
hyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]hexanamide
(mc-Val-Ala-PAB-I, 0.0208 mmol, 12.4 mg, crude) in
N,N-dimethylformamide (5.17 mmol, 0.400 mL, 378 mg) was sealed and
heated to 60.degree. C. In 2 h the reaction was cooled to room
temperature and purified by chromatography on C18 column
(continuous gradient from 20-60% CH3CN/water with 0.1% formic acid)
to provide
N-[(2S)-1-{[(2S)-1-{[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-7H-pyrrolo[2,3-d]pyri-
midin-7-yl)-10,16-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2--
1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-yl]sulfanyl}methyl)p-
henyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-(2,5-diox-
o-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (5.5 mg, 45% yield) as a
white solid. .sup.1H NMR (400 MHz, METHANOL-d4) .delta. 8.23-8.35
(m, 2H), 8.10 (s, 1H), 7.89-8.02 (m, 1H), 7.84 (s, 1H), 7.52-7.64
(m, 2H), 7.41 (br d, 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 t, 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
##STR00417##
[1843] Linker-payload 2 was generated according to the procedure in
Example 27, using 2-amino-9-[(5R,7R,8R,12aR,14R
15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-10,18-dihydroxy-2,10-dioxido-2-s-
ulfanyl
hexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9-
,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin--
6-one sesqui-triethylamine salt (Payload 2) in place of Payload
1.
[1844] Payload 2 was prepared according to the method below.
2-amino-9-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)-10,-
18-dihydroxy-2,10-oxido-2-sulfanylhexahydro-14H-15,12a-(epoxymethano)-5,8--
methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(12H)-
-yl)-1,9-dihydro-6H-purin-6-one sesqui-triethylamine salt (Optical
Isomer)
A) (1R,3R,4R,7S)-3-(6-benzamido-9H-purin-9-yl)-1-((((((2R,3R,4R,
R)-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)-2,5-dioxabicyclo[2.2.1]heptan-7-yl hydrogen
phosphonate
[1845]
(1S,3R,4R,7S)-3-(6-Benzamido-9H-purin-9-yl)-1-(hydroxymethyl)-2,5-d-
ioxabicyclo[2.2.1]heptan-7-yl hydrogen phosphonate (700 mg) and
5'-O-[bis(4-methoxyphenyl)(phenyl)methyl]-3'-O-[tert-butyl(dimethyl)silyl-
]-2'-O-{(2-cyanoethoxy)[diisopropylamino]phosphanyl}-N-(2-methylpropanoyl)-
guanosine (2280 mg) were subjected to azeotropic dehydration with
anhydrous acetonitrile, and anhydrous acetonitrile (15 mL) and
anhydrous tetrahydrofuran (5 mL) were added thereto. To the mixture
was added a mixture of 5-(ethylsulfanyl)-2H-tetrazole (611 mg)
(which was subjected to azeotropic dehydration with anhydrous
acetonitrile) and anhydrous acetonitrile (10 mL), and the mixture
was stirred overnight under argon atmosphere at room temperature.
70% tert-Butyl hydroperoxide (643 .mu.L) was added thereto, and the
mixture was stirred at room temperature for 20 min. 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 min. 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].sup.+
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-oxido-2-sulfidohexah-
ydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pent-
aoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one
(Optical Isomer)
[1846]
(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)(2-cyanoethoxy)phosphoryl)oxy)m-
ethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-yl hydrogen phosphonate (980
mg) was subjected to azeotropic dehydration with anhydrous
acetonitrile and anhydrous pyridine, and anhydrous pyridine (50 mL)
was added thereto. To the mixture was added
2-chloro-5,5-dimethyl-1,3,2-dioxaphosphinane 2-oxide (615 mg) at
room temperature, and the mixture was stirred under argon
atmosphere at room temperature for 1 hr. Water (600 .mu.L) and
3H-benzo[c][1,2]dithiol-3-one (240 mg) were added thereto, and the
mixture was stirred at room temperature for additional 30 min. 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. To the residue were added anhydrous acetonitrile
(15 mL) and 2-methylpropan-2-amine (5.0 mL), and the mixture was
stirred at room temperature for 1.5 hr, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (methanol/ethyl acetate), and to the obtained
residue was added 40% methylamine ethanol solution (30 mL). The
mixture was stirred overnight under argon atmosphere at room
temperature, and the reaction mixture was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (methanol/ethyl acetate). The obtained residue was
resolved into two diastereomers (tR1 and tR2, retention times of
which by LC/MS are from shorter to longer in this order) by HPLC
(L-column2 ODS, 50.times.150 mm, mobile phase: 5 mM aqueous
ammonium acetate solution/acetonitrile) to give the title compound
(38 mg, tR1) and the title compound (322 mg, tR2). MS (tR1):
[M+H].sup.+ 817.1. MS (tR2): [M+H].sup.+ 817.1
C)
2-amino-9-[(5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-9H-purin-9-yl)--
2,10,18-trihydroxy-10-oxido-2-sulfidohexahydro-14H-15,12a-(epoxymethano)-5-
,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(1-
2H)-yl]-1,9-dihydro-6H-purin-6-one sesqui-triethylamine salt
(Optical Isomer)
[1847] To
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-oxido-2-sulfi-
dohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,-
10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one
(optical isomer) (38 mg, tR1) were added methanol (3.0 mL) and
triethylamine trihydrofluoride (0.76 mL). The reaction mixture was
concentrated to remove the methanol, and the residue was stirred at
55.degree. C. for 1 hr. 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 hr. The reaction mixture was
concentrated under reduced pressure, and the residue was purified
by C18 column chromatography (acetonitrile/10 mM triethylammonium
acetate buffer solution) to give the title compound (27 mg).
.sup.1H NMR (400 MHz, D.sub.2O) .delta. 1.23 (13H, t, J=7.3 Hz),
3.15 (9H, q, J=7.3 Hz), 4.04 (1H, d, J=8.3 Hz), 4.08-4.19 (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). .sup.31P NMR (162 MHz, D.sub.2O) .delta. -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-
,18-dihydroxy-2,10-dioxido-2-sulfanylhexahydro-14H-15,12a-(epoxymethano)-5-
,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7(1-
2H)-yl]-1,9-dihydro-6H-purin-6-one sesqui-triethylamine salt
(Optical Isomer; Payload 2)
[1848] To
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-oxido-2-sulfi-
dohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,6,9,11,2,-
10]pentaoxadiphosphacyclotetradecin-7(12H)-yl]-1,9-dihydro-6H-purin-6-one
(optical isomer) (322 mg, tR2) were added methanol (3.0 mL) and
triethylamine trihydrofluoride (3.2 mL). The reaction mixture was
concentrated to remove the methanol, and the residue was stirred at
55.degree. C. for 1 hr. 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 hr. The reaction mixture was
concentrated under reduced pressure, and the residue was purified
by C8 column chromatography (acetonitrile/10 mM triethylammonium
acetate buffer solution) to give the title compound (266 mg).
.sup.1H NMR (400 MHz, D.sub.2O) .delta. 1.23 (14H, t, J=7.3 Hz),
3.15 (10H, q, J=7.3 Hz), 4.02 (1H, d, J=8.1 Hz), 4.13-4.24 (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=4.2 Hz), 5.61-5.73 (1H, m), 5.95 (1H, d, J=8.3 Hz), 6.15 (1H,
s), 7.87 (1H, s), 8.00 (1H, s), 8.25 (1H, s). .sup.31P NMR (162
MHz, D.sub.2O) .delta. -1.93, 55.44.
Example 29
General ADC Conjugation Procedure
[1849] A solution of antibody was diluted to the desired reaction
concentration with an appropriate buffer. The pH was adjusted to 7
by addition of 0.5 M tris, 25 mM EDTA solution in water (pH 8.0).
TCEP (2.2 equiv, 5 mM in water) was added with stirring. After 1
hour of incubation at room temperature, 5 equiv of linker-payload
(5 mM in DMA) was added. After 2 additional hours of gentle
stirring at room temperature the mixture was purified twice over 3
mL 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), and then buffer exchanged to
Dulbecco's PBS and concentrated to 1-3 mg/mL (as determined by UV
absorption using a standard IgG1 extinction coefficient) using a
Vivaspin6 column (GE Healthcare). Sample was then characterized for
drug-antibody-ratio (DAR) by QTOF mass spectrometry. The monomer
composition of the ADC was determined by size exclusion
chromatography (SEC).
Size Exclusion Chromatography (SEC) Protocol
Sample Preparation
[1850] Prepare a water blank by adding HPLC water to an HPLC vial.
Naked antibody controls and ADC samples are obtained from the
chemists. The controls and samples are diluted with 1.times.PBS if
the concentration is above 5 mg/mL, or injected neat if the
concentration is 5 mg/mL or below.
HPLC System Setup
[1851] An Agilent 1100 HPLC system is used for analysis. The system
is set up with system wash (5% Acetonitrile in HPLC water) on one
pump channel, and the mobile phase (as described above) on a
different pump channel. The column used is Tosoh Biosep TSK Gel,
G3000SWx1; P/N 8541; 250A; 5 um; 7.8 mm.times.300 mm. The flow rate
is set to 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 minutes. The DAD is set to 280 nm. The
injection volume for each control, sample, and blank is typically
10 uL, but can be adjusted depending on UV absorbance. Data
Analysis: All peaks at 280 nm within appropriate time window
(typically 2-10 minutes) are integrated.
Example 30
Preparation of ADC1
[1852] The synthesis was performed according to the General ADC
Conjugation Procedure.
[1853] Antibody: Antibody 1, an anti-GCC antibody (5F9), 100 uL of
a 60 mg/mL solution in a buffer of 50 mM histidine and 100 mM
arginine at pH 6.0, receptor target guanylyl cyclase C (GCC)
TABLE-US-00003 Antibody 1 heavy chain: (SEQ. ID No. 1)
QVQLQQWGAGLLKPSETLSLTCAVFGGSFSGYYWSWIRQPPGKGLEWIGE
INHRGNTNDNPSLKSRVTISVDTSKNQFALKLSSVTAADTAVYYCARERG
YTYGNFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFOPKPK
DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody 1 light
chain: (SEQ. ID No. 2)
EIVMTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPGQAPRLLIYG
ASTRATGIPARFSGSGSGTEFTLTIGSLQSEDFAVYYCQQYKTWPRTFGQ
GTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
[1854] Buffer: 50 mM histidine and 100 mM arginine at pH 6.0, 200
uL, final concentration 20 mg/mL
[1855] Linker-payload:
N-[(2S)-1-{[(2S)-1-{[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-7H-pyrrolo[2,3-d]pyri-
midin-7-yl)-10,16-dihydroxy-2,10-dioxidooctahydro-12H-5,8-methanofuro[3,2--
1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2-yl]sulfanyl}methyl)p-
henyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-(2,5-diox-
o-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (Linker-payload 1)
[1856] Product ADC: ADC1, yield=1.9 ml at 2.5 mg/mL (79%), DAR 3.5,
99% monomer (i.e., not aggregated)
Example 31
Preparation of ADC2
[1857] The synthesis was performed according to the General ADC
Conjugation Procedure.
[1858] Antibody: Antibody 2, a non-GCC targeting antibody directed
to a membrane-anchored protein over expressed in certain solid
tumors including lung and breast, 100 uL at 11.8 mg/mL in 25 mM
sodium acetate, pH 5.5.
[1859] Buffer: Dulbecco's PBS, pH 7.4, 200 uL, final concentration
4 mg/mL
[1860] Linker-payload: N-[(2S)-1-{[(2S)-1-{[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-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-10,16-dihydroxy-2,10-dio-
xidooctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphospha-
cyclotetradecin-2-yl]sulfanyl}methyl)phenyl]amino}-1-oxopropan-2-yl]amino)-
-3-methyl-1-oxobutan-2-yl]-6-(2,5-dioxo-2,5-dihydro-H-pyrrol-1-yl)hexanami-
de (Linker-payload 1)
[1861] Product ADC: ADC2, yield=0.6 ml at 1.4 mg/mL (70%), DAR 2.6,
99% monomer
Example 32
Preparation of ADC3
[1862] The synthesis was performed according to the General ADC
Conjugation Procedure.
[1863] Antibody: Antibody 1, 33 uL at 60 mg/mL in a buffer of 50 mM
histidine and 100 mM arginine at pH 6.0
[1864] Buffer: 50 mM histidine and 100 mM arginine at pH 6.0, 66 uL
final concentration of antibody solution 20 mg/mL
[1865] Linker-payload:
N-[(2S)-1-{[(2S)-1-{[4-({[(5R,7R,8R,12aR,14R,15R,15aS,18R)-7-(2-amino-6-o-
xo-1,6-dihydro-9H-purin-9-yl)-14-(6-amino-9H-purin-9-yl)-10,18-dihydroxy-2-
,10-dioxidohexahydro-14H-15,12a-(epoxymethano)-5,8-methanofuro[3,2-1][1,3,-
6,9,11,2,10]pentaoxadiphosphacyclotetradecin-2(12H)-yl]sulfanyl}methyl)phe-
nyl]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 2)
[1866] Product ADC: 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 Systems or ADCs in an In Vitro Lysosome
Model
Materials:
[1867] Buffer: 47.9 mg/ml of Potassium Phosphate Monobasic
(Sigma-Aldrich Product Number P5379); 6.8 mg/ml of Sodium Phosphate
Dibasic (Sigma-Aldrich Product Number S0876), 1.7 mg/ml of
Ethylenediaminetetraacetic Acid (Sigma-Aldrich Product Number
ED4SS) in purified water. pH was adjusted to 6.0 using IN HCl or IN
KOH.
[1868] Rat Liver Tritosomes purchased from XenoTech
[1869] Linker-payload (10 mM DMSO)
[1870] ADC (1-40 uM in the storage buffer used for production).
Procedure:
[1871] For evaluation of payload release from linker-payload
compounds, a 120.95 uM DMSO working solution was prepared from 10
mM stock solution of linker-payload compounds. Then, the 120.95 uM
DMSO working solution was diluted to 1 uM in 225 uL tritosome
buffer solution containing 22.4% rat liver tritosomes.
[1872] For evaluation of ADC molecules, 1.86 uL of ADC solution was
diluted with 223.1 uL of mixture of rat liver tritosomes in
tritosome buffer.
[1873] The solutions thus prepared were incubated for 24 hours at
37.degree. C. 40 uL aliquots were removed at 0.5, 1, 3, 5, and 24
hours and diluted with 160 uL of 0.1% formic acid in methanol
solution in a 96-well plate, which was stored in -80.degree. C.
freezer until the completion of the experiment. After collecting
the last time point, a fresh 200 uL of 0.1% formic acid in methanol
solution and spiked with 150 nM carbutamide which is an internal
standard solution was added into the samples. The samples were
mixed well and centrifuged at 4000 g for 10 minute, and the 96-well
plate was submitted for analysis of payload concentration by
LC/MS/MS.
LC/Ms/Ms System:
[1874] 5 uL of samples were injection into the LC/MS/MS with a
Waters Xselect C18 CSH 3.5 u 2.1 mmID.times.30 mm length column.
Mobile phase aqueous solvent contained 0.1% formic acid in water,
and mobile phase organic solvent contained 0.1% formic acid in 5%
water and 95% acetonitrile. The samples were running in 3 minutes
gradient at 1.5 mL/min flow rate. Initially, the instrument was
running 100% aqueous mobile phase solvent for 0.5 minutes, and then
it was increasing to 100% organic solvent in next 1.5 minutes. The
system will hold at 100% organic solvent for another 0.5 minutes,
and then it would change to 100% aqueous phase solvent in next 0.5
minutes.
Analysis:
[1875] To monitor and calculate the release of linker-payload and
payload from ADC, a peak area or concentration versus time curve
was plotted. The data would be analyzed by using Excel-Fit program
to calculate linear range rate and t1/2 of formation payload from
the tested molecules.
Tritosomal Payload Release Data:
[1876] As shown in FIG. 1, FIG. 2, and FIG. 3, when subjected to
the above protocol ADC1, ADC2, and ADC3 release the expected
payloads.
Phospho-IRF3 Assay Protocol
[1877] HEK293 cells engineered to express the tumor-associated cell
surface targets for mAbs Antibody 1 (ADC1 and ADC3) and Antibody 2
(ADC2), as well as target non-expressing HEK293 cells
(HEK293-Vect), were seeded at 1.5.times.10E5 cells/well (500
uL/well) in Poly-D-Lysine-coated 24-well plates (Corning) and were
cultured overnight. After overnight serum deprivation, the cells
were treated with various concentrations of ADCs for 6 hours and
then lysed in RIPA buffer (ThermoFisher Scientific) with protease
and phosphatase inhibitors (ThermoFisher Scientific). The cell
lysates were subjected to Western blotting analysis for
phospho-IRF3 (CST) and IRF3 (BD Biosciences). The level of
phospho-IRF3 was normalized to its total IRF3.
Phospho-IRF3 Assay Results
[1878] As shown in FIG. 4, FIG. 5, and FIG. 6, HEK293 cells that
express the surface receptor targets for ADC1, ADC2, and ADC3
exhibit the expected increase in the STING pathway marker
phospho-IRF3 (pIRF3) when treated with the ADC1, ADC2, and ADC3,
respectively. Conversely, the pIRF3 response is not upregulated
upon ADC treatment in vector HEK293 cells that do not express the
corresponding surface receptor targets.
Example 34
Pharmacological Profiles of Selected Cyclic Dinucleotides
[1879] The pharmacological profiles of selected cyclic
dinucleotides are shown in Table 6.
TABLE-US-00004 STING-293T STING- cell 293T cell EC.sub.50 (.mu.M)
EC.sub.50 (.mu.M) Reporter Reporter Cellular THP-1 Dual Human STING
without with permeability cell plasma binding digitonin digitonin
Ratio EC.sub.50s EC.sub.50 (.mu.M) stability Ex (.mu.M) (A) (B)
(A)/(B) Reporter (/h) Ex3a 0.015 2.9 0.13 22.3 5.6 0.58 Ex5 0.050
3.6 0.19 18.9 N.D. <0.01 Ex14 0.013 0.34 0.13 2.6 2.3 <0.01
cGAMP 0.015 21 0.039 539 18 1.39
Protocol of THP-1 Dual Lucia Reporter Gene Assay
[1880] THP1-Dual.TM. cells (InvivoGen #thpd-nfis) were derived from
the human THP-1 monocyte cell line by stable integration of the
Lucia luciferase gene, a secreted luciferase reporter gene, under
the control of an ISG54 (interferon-stimulated gene) minimal
promoter in conjunction with five interferon (IFN)-stimulated
response elements. On the day of experiment, the cells were plated
to a black, 384-well plate (Corning 356697) at 7500 cells/25 .mu.L
per well density in growth media (RPMI 1640, 2 mM L-glutamine, 25
mM HEPES, 10% heat-inactivated fetal bovine serum, 100 .mu.g/mL
Normocin.TM., 100 U/mL-100 pig/mL Pen-Strep, 10 .mu.g/mL of
blasticidin, and 100 .mu.g/mL of Zeocin). The cell plates were
dosed with 62.5 nL of the testing compounds, and then incubated at
37.degree. C. for 20 hours. At the end of the incubation, 15
.mu.L/well of the QUANTI-Luc.TM. (InvivoGen # rep-qlcl) were added,
and luminescence was measured immediately using the LeadSeeker.
Protocol of Western Blot Analyses of Downstream Signaling Pathway
Activation (TBK and IRF3)
[1881] 1.5.times.10.sup.6 THP-1-Dual Cells (Invivogen catalog #
thpd-nfis) were treated with DMSO or the indicated concentrations
of Ex14 for 3 hours. After stimulation, cells were collected on
ice, centrifuged at 800 RCF for 5 minutes, and washed once in
ice-cold PBS.
[1882] Cell pellets were lysed in 1% Triton X-100 whole cell lysis
buffer (100/% glycerol, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 .mu.M
EDTA, and 1 .mu.M DTT) containing protease and phosphatase
inhibitor cocktails (Sigma # P8340 and CalBiochem #524629,
respectively). Cell lysates were cleared of insoluble debris by
centrifuging at 16,000 RCF for 10 minutes. The protein
concentrations of the lysates were determined by Bradford assay
using BSA standards.
[1883] Lysates were denatured in NuPAGE.TM. LDS Sample Buffer
(4.times., catalog # NP0008) containing DTT as a reducing agent.
Denatured lysates were resolved on NuPAGE.TM. 4-12% Bis-Tris gels
in MES/SDS running buffer and transferred to PVDF membranes using a
semi-dry blotting method. Membranes were probed overnight at
4.degree. C. for phospho-TBK1 S172, phospho-IRF3 S396, and GAPDH
using antibodies from Cell Signaling Technology (#5483, #4947, and
#5174, respectively).
[1884] After an overnight primary probe, membranes were washed
extensively, then probed with secondary antibody (Alexa Fluor.RTM.
680 goat anti-rabbit IgG, Life Technologies #A21109) in
Odyssey.RTM. Blocking Buffer (catalog #927-400000) at room
temperature. After extensive washing, membranes were developed
using a LI-COR ODYSSEY CLx.
[1885] The relative intensities of the phospho-TBK1 and
phospho-IRF3 bands were quantified and normalized to the levels of
GAPDH. GAPDH-normalized phospho-TBK1 and phospho-IRF3 values were
graphed as fold intensity over the non-stimulated (DMSO) control.
See FIG. 7.
Protocol of Human Plasma Stability Assay
[1886] The compound was incubated in heparinized human plasma at
37.degree. C. for 0, 1, 2, 4, 8 and 24 hours, and the change in
compound concentration was measured by LC/MS/MS method. The
elimination rate constant (=k(/h)) was calculated based on the
fitted curve assuming the exponential decrease of concentration as
follows; concentration at time x=initial
concentration*exp(-k*x).
Syngeneic Tumor Model Testing
[1887] Ex14 and Ex3a were tested in syngeneic tumor models in mice.
When Ex14 and Ex3a were administered intravenously (every 3 days, 3
times), anti-tumor effects were observed. See FIG. 8 and FIG. 9 for
the anti-tumor effect colon carcinoma CT-26 syngeneic mice model,
and FIG. 10 and FIG. 11 for the anti-tumor effect in the colon
carcinoma B16F10 syngeneic mice model.
The Protocol for the Anti-Tumor Effect Evaluation
[1888] 8.times.10.sup.4 B16F10 and 2.times.10.sup.5 CT-26 mouse
tumor cells were subcutaneously implanted in the flank of female
C57BL/6 (B16F10) and Balb/C (CT-26) mice (n=7 per group). Vehicle
(1.times.PBS), compound Ex3a (30 mg/kg) or Ex14 (1.0 mg/kg) were
intravenously administered (final volume of 100 .mu.L) to
tumor-bearing mice once the tumor volume has reached 100 mm.sup.3
(Day 0). Vehicle, Ex3a or Ex14 were given every 3 days for a total
of 3 doses (q3dx3). Tumor and body weight measurements were taken
three times per week using vernier calipers and mettler scale,
respectively. Tumor volumes were determined by multiplying the
square of the width (`W`), measured along the short axis of the
tumor, by one-half the length (`L`) measured along the short axis
of the tumor (V=W.sup.2.times.0.5L)
[1889] The compounds of the Examples are shown in the following
tables. MS in the tables means actual measured value. The compound
example 9 was synthesized in the same manner as in the method of
Example 5.
TABLE-US-00005 TABLE 1-1 Ex. No. IUPAC NAME Structure Additive MS 1
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[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-
yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d] pyrimidin-4-one
##STR00418## 2Et3N 677.1 1a
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[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-
yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d] pyrimidin-4-one
##STR00419## 2Na 678.9 2
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-
9H-purin-9-yl)-15-fluoro-10,16-dihydroxy-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) ##STR00420## 2Et3N 695.0 3
7-((2R,5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-
amino-9H-purin-9-yl)-15-fluoro-10,16-dihydroxy-
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) ##STR00421## 1.7Et3N 693.1 3a
7-((2R,5R,7R,8R,12aR,14R,15R,15aR,16R)-14-6-
amino-9H-purin-9-yl)15-fluoro-10,16-dihydroxy-
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
##STR00422## 2Na 695.0 4
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[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-
yl)-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3-d] pyrimidin-4-one
##STR00423## 2Et3N 677.1 5
7-((5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-amino-
9H-purin-9-yl)-15,16-dihydroxy-2,10-dioxido-2,10-
disulfanyloctahydro-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) ##STR00424## 2Et3N 707.1
TABLE-US-00006 TABLE 1-2 Ex. No. IUPAC NAME Structure Additive MS 6
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[3,2-1]
[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) ##STR00425## 2Et3N 707.1 7
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[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-
14-yl)-5-fluoro-3,7-dyhydro-4H-pyrrolo[2,3-d] pyrimidin-4-one
(optical isomer) ##STR00426## 2Et3N 709.0 8
2-amino-9-((5R,7R,8R,12aR,14R,15R,15aS,16R)-14-
(4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyridimin-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 ##STR00427## 2Et3N 692.2 9
9-((5R,7R,8R,12aR,14R,15R,15aS,16R)-7-(4-amino-5-
fluoro-7H-pyrrolo[2,3-d]pyrimidine-7-yl)-15,16-
dihydro-2,10-dioxine-2,10-disulfanyloctahydro-12H-
5,8-methanofuro[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-
14-yl)-1,9-dihydro-6H-purine-6-one ##STR00428## 2Et3N 707.1 10
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[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-
yl-1,5-dihydro-4H-imidazo[4,5-c]pyridin-4-one ##STR00429##
659.1
TABLE-US-00007 TABLE 1-3 Ex. No. IUPAC NAME Structure Additive MS
11 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[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-
yl)-1,9-dihydro-6H-purin-6-one ##STR00430## 2Et3N 674.2 12
8-((5R,7R,8R,12aR,14S,15S,15aS,16R)-7-(6-amino-
9H-purin-yl)-15,16-dihydroxy-2,10-dioxido-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)
##STR00431## 2Et3N 692.2 13
2-amino-9-((5R,7R,8R,12aR,14S,15S,15aS,16)-14-(4-
aminopyrazolo[1,5-a][1,3,5]triazin-8-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 ##STR00432## 2Et3N 675.1 14
7-((2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-14-(6-
amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-2,10-
dioxido-2,10-disulfanyloctahydro-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
##STR00433## 2Na 711.0 15
7-((5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-amino-
9H-purin-9-yl)-15-fluoro-2,16-dihydroxy-2,10-
dioxido-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 (optical
isomer) ##STR00434## 2Et3N 695.0
TABLE-US-00008 TABLE 1-4 Ex. No. IUPAC NAME Structure Additive MS
16 7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-
9H-purin-9-yl)-2,10,18-trihydroxy-2,10-
dioxidohexahydro-14H-15,12a-(epoxymethano)-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) ##STR00435## 2Et3N 687.0 17
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-15,12a-(epoxymethano)-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) ##STR00436## 2Et3N 702.9 18
7-((5R,7R,8R,12aR,14R,15R,15aS,18R)-14-(6-amino-
9H-purin-9-yl)-2,18-dihydroxy-2,10-dioxido-10-
sulfanylhexahydro-14H-15,12a-(epoxymethano)-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) ##STR00437## 2Et3N 702.9 19
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[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-
14-yl)-1,9-dihydro-6H-purin-6-one (optical isomer) ##STR00438##
2Et3N 692.9 20 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[3,2-1]
[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) ##STR00439## 2Et3N 693.9
TABLE-US-00009 TABLE 1-5 Ex. No. IUPAC NAME Structure Additive MS
23 (optical isomer 1) 7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-
amino-9H-purin-9-yl)-10,15,16-trihydroxy-
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-methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt ##STR00440## 2
Et.sub.3N 707.1 23 (optical isomer 2)
7-[(5R,7R,8R,12aR,14R,15R,15aS,16R)-14-(6-
amino-9H-purin-9-yl)-10,15,16-trihydroxy-
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-methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt ##STR00441## 2
Et.sub.3N 707.1 24 (optical isomer 1)
7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-
amino-9H-purin-9-yl)-15-fluoro-10,16-
dihydroxy-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-methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt ##STR00442## 2
Et.sub.3N 709.2 24 (optical isomer 2)
7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-
amino-9H-purin-9-yl)-15-fluoro-10,16-
dihydroxy-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-methyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one di-triethylamine salt ##STR00443## 2
Et.sub.3N 709.3 25 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-l2H-5,8- methanofuro[3,2-1]
[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 di-triethylamine salt ##STR00444## 2
Et.sub.3N 693.2 26 (optical isomer 1)
7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-
amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-
2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofura[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-
7-yl]-5-chloro-3,7-dihydro-4H-pyrrolo[2,3-d] pyrimidin-4-one
di-triethylamine salt ##STR00445## 2 Et.sub.3N 727.0 26 (optical
isomer 2) 7-[(5R,7R,8R,12aR,14R,15R,15aR,16R)-14-(6-
amino-9H-purin-9-yl)-15-fluoro-16-hydroxy-
2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8- methanofuro[3,2-1]
[1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-
7-yl]-5-chloro-3,7-dihydro-4H-pyrrolo[2,3-d] pyrimidin-4-one
di-triethylamine salt ##STR00446## 2 Et.sub.3N 727.0
Example 35
STING-Binding Test
[1890] Test compound (2 .mu.L), Streptavidin-Terbium (4 .mu.L,
Cisbio), Fluorescein-labeled 2',3'-cGAMP
(c[G(2',5')p-2'-Fluo-AHC-A(3',5')p]) (Biolog, Germany) and
biotinylated STING protein (2 .mu.L, wild-type, WT) were mixed
using assay buffer (Dulbecco's Phosphate-Buffered Saline (Wako Pure
Chemical Industries, Ltd.) containing 0.01% bovine serum albumin
free of fatty acid (Wako Pure Chemical Industries, Ltd.)), and the
mixture was left to stand at room temperature for 3 hr (The final
concentration: Streptavidin-Terbium; diluted by 1/1000, FITC-cGAMP;
1 .mu.M, biotinylated STING protein; 100 nM). The time-resolved
fluorescence resonance energy transfer (TR-FRET) was measured at
the wavelength of 520 nm and 486 nm by EnVision (PerkinElmer,
Waltham, Mass., US). 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 count at 520 nm divided by the
count at 486 nm. The results are shown in Tables 2 and 2A.
[1891] The above-mentioned biotinylated STING protein (wild-type
(WT)) was prepared by the following method.
Preparation Method of Biotinylated Wild-Type STING Protein
[1892] ECOS (trade name) Competent E. coli BL21(DE3) was purchased
from Nippon Gene Co., Ltd. Ampicillin, kanamycin, NaCl, glycerol,
isopropylthiogalactoside, (+)-biotin, imidazole, SEM nuclease
recombinant and BCA protein assay kit were purchased from Wako Pure
Chemical Industries, Ltd. Tryptone, Bacto, and Yeast Extract, Bacto
were purchased from Difco Laboratories, tris buffered saline (TBS)
tablets, pH7.6 was purchased from Takara Bio Inc., Lysozyme (Egg
White), 6.times.Cryst was purchased from Seikagaku Corporation, and
cOmplet (trade name) and EDTA-free protease inhibitor cocktail were
purchased from Roche. Ni-NTA Superflow Cartridge manufactured by
QIAGEN was used, and HiLoad 26/60 Superdex 200 .mu.g manufactured
by GE Healthcare was used.
[1893] Into pRSF1b (Novagen) having altered multiple cloning site
was inserted Escherichia coli BirA, and transfected to ECOS JM109,
whereby pRH8/FLAG-BirA was constructed.
pET21HH/His-Avi-SUMO-FLAG-hTMEM 173(139-379)(H232R) (which was
constructed by the method mentioned in the EXAMPLE 36) and
pRH8/FLAG-BirA for Avi tag biotinylation were simultaneously
transformed to ECO (trade name) Competent E. coli BL21(DE3) to
prepare His-Avi-SUMO-FLAG-hSTING (139-379, H232R)-expressing cell
line. The expressing cell line was added to LB medium (10 g/L
Tryptone, 5 g/L Yeast Extract, 5 g/L NaCl) containing ampicillin
(100 .mu.g/L) and kanamycin (50 .mu.g/L), and the mixture was
pre-cultured at 30.degree. C., and expanded to TB medium (12 g/L
Tryptone, 24 g/L Yeast Extract, 4 mL/L Glycerol, 2.3 g/L
KH.sub.2PO.sub.4, 12.5 g/L K.sub.2HPO.sub.4) containing the same
antibiotics, and the mixture was cultured at 37.degree. C. When the
turbidity of the culture solution reached 500 KU, the culture
temperature was reduced to 16.degree. C., 0.1 mM
isopropylthiogalactoside and 50 LM (+)-biotin were added thereto,
and the mixture was cultured for additional 16 hr.
[1894] The culture solution was centrifuged, the obtained fungus
bodies were suspended in Lysis Buffer (50 mM TrisHCl, 150 mM NaCl,
20 mM Imidazole, 1 mg/mL Lysozyme, 5 U/mL SEM Nuclease,
recombinant, Complete EDTA-free, pH7.6), and the protein was
extracted by ultrasonic fragmentation. The reagent was added
thereto so that the salt concentration of the extract was adjusted
to 300 mM NaCl, and the supernatant was collected by
centrifugation. The obtained supernatant was passed through NiNTA
superflow Cartridge equilibrated with Wash Buffer (50 mM TrisHCl,
300 mM NaCl, 20 mM Imidazole, pH7.6), and the Cartridge was washed
with Wash Buffer, and eluted with Elution Buffer (50 mM TrisHCl,
300 mM NaCl, 250 mM Imidazole, pH7.6). The eluate was passed
through HiLoad 26/60 Superdex 200 .mu.g column equilibrated with
Storage Buffer (50 mM TrisHCl, 150 mM NaCl, pH7.6), and the eluted
fraction was collected as biotinylated His-Avi-SUMO-FLAG-hSTING
(139-379, H232R). The protein concentration was measured using BCA
protein assay kit, and the fraction was cryopreserved at
-80.degree. C. until used.
TABLE-US-00010 TABLE 2 STING-binding test Example binding
inhibition rate of No. test compound (30 .mu.M) 1 76% 2 79% 3 64%
3a 93% 5 71% 8 90% 13 68% 14 89% 15 89% 16 91% 17 86% 18 94% 19 93%
20 98%
TABLE-US-00011 TABLE 2 ASTING-binding test Example No. IC.sub.50
(.mu.M) 23 optical isomer 1) 0.17 23 optical isomer 2) 20 24
(optical isomer 1) 0.02.9 24 (optical isomer 2) 0.29 25 0.083 26
(optical isomer 1) 0.53 26 (optical isomer 2) 0.075
[1895] As is clear from the above-mentioned results, the compound
of the present disclosure inhibits the binding of wild-type STING
protein and the natural ligand 2',3'-cGAMP, that is, the compound
of the present disclosure binds to wild-type STING protein.
Example 36
Reporter Gene Assay
Preparation of Various Plasmids
(i) Expression Plasmid Construction of Human TMEM173 and the
Like
[1896] Expression plasmid of human STING (in the present
specification, sometimes to be referred to as human TMEM173) in E.
coli was obtained by overlap extension PCR for introduction of
mutation using human TMEM 173 cDNA Clone (GeneCopoeia) as a
template. First, PCRs were performed using two kinds of Primer
(5'-CCTGGCCCCAGCTGAGATCTCTG-3' (C-hTMEM(139aa)-F) (SEQ. ID No. 3)
and 5'-GTAAACCCGATCCTTGATGCCAGCACGGTCACCGGTC-3' (hTMEM
173(H232R)-R)) (SEQ. ID No. 4) and two kinds of Primer
(5'-CTGCCCCAGCAGACCGGTGACCGTGCTGGCATCAAG-3' (hTMEM 173(H232R)-F)
(SEQ. ID No. 5) and 5'-ATAATAGCGGCCGCTCAAGAGAAATCCGTGCGGAGAGG-3'
(hTMEM173-st-Not-R) (SEQ. ID No. 6). The PCR was each performed
using PrimeStar MAX DNA Polymerase (Takara Bio Inc.) successively
(1) at 98.degree. C. for 1 min, (2) 25-times repetitions of at
98.degree. C. for 10 sec and at 72.degree. C. for 10 sec, and (3)
at 72.degree. C. for 1 min. Then, the PCR was performed using the
obtained segment as a template, and using two kinds of Primer
(C-hTMEM(139aa)-F and hTMEM173-st-Not-R). The PCR was performed
using PrimeStar GXL DNA Polymerase (Takara Bio Inc.) successively
(1) at 98.degree. C. for 1 min, (2) 35-times repetitions of at
98.degree. C. for 10 sec and at 72.degree. C. for 1.5 min, and (3)
at 72.degree. C. for 1 min. The obtained segment was cut with Not I
(Takara Bio Inc.), inserted into the Stu I/Not I site of vector
wherein His-Avi-SUMO Tag was attached to pET21a (Novagen), using
Ligation High (Toyobo Co., Ltd), and transfected to ECOS JM109
(Nippon Gene Co., Ltd.), whereby pET21HH/His-Avi-SUMO-hTMEM
173(139-379)(H232R) was constructed.
[1897] Second, the PCR was performed using this plasmid as a
template, and using two kinds of Primer
(5'-CGACTACAAGGACGACGATGACAAGGGATCCCTGGCCCCAGCTGAGATCTCTG-3'
(C-FLAG-Bam-hTMEM173(139aa)-F) (SEQ. ID No. 7) and
hTMEM173-st-Not-R). The PCR was performed using PrimeStar MAX DNA
Polymerase successively (1) at 98.degree. C. for 1 min, (2)
25-times repetitions of at 98.degree. C. for 10 sec and at
72.degree. C. for 10 sec, and (3) at 72.degree. C. for 1 min. The
obtained segment was cut with Not I, inserted into the Stu I/Not I
site of pET21a to which His-Avi-SUMO Tag was attached, using
Ligation High, as mentioned above, and transfected to ECOS JM109,
whereby pET21HH/His-Avi-SUMO-FLAG-hTMEM 173(139-379)(H232R) was
constructed.
[1898] Expression plasmid for Reporter Assay was obtained by
overlap extension PCR for introduction of mutation using human TMEM
173 cDNA Clone as a template, as mentioned above. First, PCRs were
performed using two kinds of Primer
(5'-GTACCCATACGATGTTCCAGATTACGCTGGATCCGCCACCATGCCCCACTCCAGC
CTGCATC-3' (HA-Bam-ko-hTMEM173-F) (SEQ. ID No. 8) and
hTMEM173(H232R)-R) and two kinds of Primer (hTMEM173(H232R)-F and
hTMEM173-st-Not-R). The PCR was each performed using PrimeStar MAX
DNA Polymerase successively (1) at 98.degree. C. for 1 min, (2)
25-times repetitions of at 98.degree. C. for 10 sec and at
72.degree. C. for 10 sec, and (3) at 72.degree. C. for 1 min. Then,
the PCR was performed using the obtained segment as a template, and
using two kinds of Primer
(5'-ATAATATCTAGAATTCGCCACCATGTACCCATACGATGTTCCAGATTACGC-3'
(Xba-Eco-ko-HA-F) (SEQ. ID No. 9) and hTMEM173-st-Not-R). The PCR
was performed using PrimeStar GXL DNA Polymerase successively (1)
at 98.degree. C. for 1 min, (2) 35-times repetitions of at
98.degree. C. for 10 sec, at 65.degree. C. for 5 sec and at
72.degree. C. for 1.5 min, and (3) at 72.degree. C. for 1 min. The
obtained segment was cut with Xba I (Takara Bio Inc.) and Not I,
inserted into the Nhe I/Not I site of Zeocin-resistance vector
wherein multiple cloning site was inserted into pcDNA3.3
(Invitrogen), using Ligation High, and transfected to ECOS JM109,
whereby pcDNA3.3zeo/HA-hTMEM173(H232R) was constructed.
[1899] Plasmid expressing human mutated-type TMEM173 (R232H) was
constructed by PCR using human TMEM173 cDNA Clone (GeneCopoeia) as
a template, and using two kinds of Primer
(5'-TTCCAGATTACGCTGGATCCGCCACCATGCCCCACTCCAGCCTGCATC-3'
(Bam-ko-hTEME173v1-F) (SEQ. ID No. 10) and
5'-CCTCTAGACTCGAGCGGCCGCTCAAGAGAAATCCGTGCGGAGAGG-3' (hTMEM
173-st-Not-R2) (SEQ. ID No. 11). The PCR was performed using
PrimeStar MAX DNA Polymerase (Takara Bio Inc.) successively (1) at
98.degree. C. for 1 min, (2) 30-times repetitions of at 98.degree.
C. for 10 sec and at 68.degree. C. for 10 sec, and (3) at
72.degree. C. for 1 min. The obtained segment was inserted into the
Bam HI/Not I site of vector wherein HA-Tag was attached to
pcDNA3.1(+) (ThermoFischer), using Gibson Assembly (NEB), and
transfected to ECOS JM109 (Nippon Gene Co., Ltd.), whereby
pcDNA3.1HA/HA-hTMEM173v1 was constructed.
[1900] Plasmid expressing human wild-type TMEM173 (H232R) was
constructed by PCR using pcDNA3.3zeo/HA-hTMEM173(H232R) plasmid as
a template, and using two kinds of Primer
(5'-GGAGACCCAAGCTGGCTAGCGCCACCATGTACCCATACGATG-3' (Nhe-ko-HA-F)
(SEQ. ID No. 12) and
5'-CCTCTAGACTCGAGCGGCCGCTCAAGAGAAATCCGTGCGGAGAGG-3 (hTMEM
173-st-Not-R.sup.2) (SEQ. ID No. 11). The PCR was performed using
PrimeStar MAX DNA Polymerase successively (1) at 98.degree. C. for
1 min, (2) 30-times repetitions of at 98.degree. C. for 10 sec and
at 72.degree. C. for 35 sec, and (3) at 72.degree. C. for 1 min.
The obtained segment was inserted into the Nhe I/Not I site of
pcDNA3.1zeo (ThermoFischer) using Gibson Assembly, and transfected
to ECOS JM109, whereby pcDNA3.1zeo/HA-hTMEM173(H232R) was
constructed.
(ii) Firefly Luciferase Expression Plasmid Construction
[1901] Firefly luciferase expression plasmid was constructed by
inserting CMV Promoter into Reporter vector. The PCR was performed
using pcDNA3.1(+) vector (Invitrogen) as a template, and using two
kinds of Primer (5'-ATAATAAGATCTGTTGACATTGATTATTGACTAGTTATTAATAG-3'
(CMVPro-BglII-F) (SEQ. ID No. 13) and
5'-ATAATAAAGCTTGAGCTCTGCTTATATAGACCTCCC-3' (CMVPro-HindIII-R) (SEQ.
ID No. 14). The PCR was performed using PrimeStar MAX DNA
Polymerase successively (1) at 98.degree. C. for 1 min, (2)
25-times repetitions of at 98.degree. C. for 10 sec and at
68.degree. C. for 3 sec, and (3) at 72.degree. C. for 1 min. The
obtained segment was cut with Bgl II and Hind III (Takara Bio
Inc.), inserted into the Bgl II/Hind III site of pGL4.17 (Promega
Corporation) using Ligation High (Toyobo Co., Ltd), and transfected
to ECOS JM109, whereby pGL4.17/CMV Pro was constructed.
Reporter Gene Assay (1)
[1902] A stable expressing 293T cell line transfected with
pNL[NLucP/ISRE/Hygro]vector (Promega, Fitchburg, Wis., US) was
constructed. pcDNA3.3zeo/HA-hTMEM 173(H232R) was transfected to the
cells using FugeneHD (Promega, Fitchburg, Wis., US), 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. The cells were collected, and cryopreserved by
CELLBANKER 1 (Nippon Zenyaku Kogyo Co., Ltd.).
[1903] On the day of the assay, the test compound diluted with
assay buffer (DMEM containing 0.1% bovine serum albumin free of
fatty acid) was added to a white 384-well plate (Corning, NY, US)
by 10 .mu.L/well. The cryopreserved cells were thawed, and the
cells suspended in assay buffer were seeded thereto by 10
.mu.L/well (10000 cells/well). The cells were cultured at
37.degree. C. under 5% CO.sub.2 condition for 20 hr, and NanoGlo
reagent (Promega, Fitchburg, Wis., US) solution (20 .mu.L) was
added thereto. After incubated for 5 min, the luminescence level
was measured using EnVision (PerkinElmer, Waltham, Mass., US). The
activity level of each test compound was calculated when the count
in the cells treated with 2',3'-cGAMP (30 .mu.M) was considered as
100%, and the count in the cells treated with the solvent was
considered as 0%. The results are shown in Table 3.
TABLE-US-00012 TABLE 3 STING agonistic activity test Example
activity level of No. test compound (30 .mu.M) 1 77% 2 77% 3 76% 3a
107% 5 100% 14 71% 15 81% 16 123% 17 99% 18 81% 20 125%
[1904] As is clear from the above-mentioned results, the compound
of the present disclosure has an agonist activity against wild-type
STING.
Reporter Gene Assay (2)
[1905] A stable expressing 293T cell line transfected with
pNL[NLucP/ISRE/Hygro]vector (Promega, Fitchburg, Wis., US) was
constructed. A suspension, which was prepared by transfecting
pcDNA3. 1 zeo/HA-hTMEM173(H232R) or pcDNA3.1HA/HA-hTMEM173v1
together with firefly luciferase expression plasmid to the cells
using FugeneHD (Promega, Fitchburg, Wis., US), was added to a
384-well plate (Corning, NY, US), and the cells were cultured for
two days. The culture solution was removed, and the test compound
diluted with assay buffer (50 mM HEPES pH 7.0, 100 mM KCl, 3 mM
MgCl.sub.2, 85 mM Sucrose, 0.1 mM DTT, 0.2% BSA, 1 mM ATP, 0.1 mM
GTP, 10 g/ml Digitonin) was added thereto by 15 .mu.L/well. The
cells were cultured at 37.degree. C. under 5% CO.sub.2 condition
for 30 min, and the assay buffer was removed. Dulbecco's Modified
Eagle's Medium (DMEM) (Wako Pure Chemical Industries, Ltd.)
containing 10.sup.0' fetal bovine serum was added thereto by 20
.mu.L/well, and the cells were cultured at 37.degree. C. under 5%
CO.sub.2 condition for 4 hr. Luminescence signals derived from
firefly luciferase and NanoLuc Luciferase were each measured using
EnVision (PerkinElmer, Waltham, Mass., US) according to Nano-Glo
Dual-Luciferase Reporter Assay System (Promega, Fitchburg, Wis.,
US) protocol. The ratio of the count of NanoLuc Luciferase divided
by the count of firefly luciferase was used for calculation. The
activity level of each test compound was calculated when the ratio
in the cells treated with 2',3'-cGAMP (30 .mu.M) was considered as
100%, and the ratio in the cells treated with the solvent was
considered as 0%. The results are shown in Table 4.
TABLE-US-00013 TABLE 4 STING agonistic activity test activity level
against activity level against Example WT STING of test
mutated-type STING (R232H) No. compound (30 .mu.M) of test compound
(30 .mu.M) 1 97% 70% 1a 99% 92% 2 112% 77% 3 97% 73% 5 107% 95% 13
99% 85% 14 99% 92% 15 91% 100% 16 114% 108% 17 110% 85% 18 98% 96%
19 101% 62% 20 113% 85%
[1906] As is clear from the above-mentioned results, the compound
of the present disclosure has an agonist activity against wild-type
STING and mutated-type STING (R232H).
Example 37
Phosphorylated IRF3 Protein Detection in FaDu Cell
[1907] Human larynx cancer cell line FaDu cell (ATCC) was seeded,
the medium was replaced with serum free medium one day after the
seeding. After the replacement, the cell was cultured for one day,
and to the cell were added the natural ligand 2',3'-cGAMP (the
final concentration; 30 .mu.M) and test compound (the final
concentration; 15 .mu.M or 30 .mu.M). 6 hr after the addition, the
cell was washed with PBS, the cell extract was prepared, and the
phosphorylated IRF3 protein was detected by ELISA method or Western
blotting method. The IRF3 protein phosphorylation activity of the
test compound was calculated when the value in the sample with
adding the natural ligand 2',3'-cGAMP at the final concentration of
30 .mu.M was considered as 100%. The results are shown in Table
5.
TABLE-US-00014 TABLE 5 IRF3 protein phosphorylation test activity
level of Example No. test compound 1 468.8% at 30 .mu.M 2 88.7% at
30 .mu.M 3 464.6% at 30 .mu.M 5 117.4% at 30 .mu.M
[1908] As is clear from the above-mentioned results, the compound
of the present disclosure promotes the phosphorylation of IRF3,
which is the downstream signal of STING, as in natural ligand
2',3'-cGAMP. That is to say, the compound of the present disclosure
activates the downstream signal of STING, as a STING agonist.
Example 38
Formulation Example
[1909] A medicament containing the compound of the present
disclosure as an active ingredient can be produced, for example,
based on the following composition.
1. Capsule
TABLE-US-00015 [1910] (1) the compound obtained in Example 1 10 mg
(2) lactose 90 mg (3) crystalline cellulose 70 mg (4) magnesium
stearate 10 mg 1 capsule 180 mg
[1911] (1), (2), (3) and 5 mg of (4) are blended and granulated.
Thereto is added the remaining 5 mg of (4), and the total amount is
filled in a gelatin capsule.
2. Tablet
TABLE-US-00016 [1912] (1) the compound obtained in Example 1 10 mg
(2) lactose 35 mg (3) cornstarch 150 mg (4) crystalline cellulose
30 mg (5) magnesium stearate 5 mg 1 tablet 230 mg
[1913] (1), (2), (3), 20 mg of (4) and 2.5 mg of (5) are blended
and granulated. Thereto is added the remaining 10 mg of (4) and the
remaining 2.5 mg of (5), and the mixture is compression formed to
give a tablet.
INDUSTRIAL APPLICABILITY
[1914] The compound of the present disclosure may have a STING
agonistic activity. Therefore, the compound of the present
disclosure may be used as a STING agonist, and may be useful as an
agent for the prophylaxis or treatment of STING-related diseases
including cancer and the like.
[1915] It is to be understood that the foregoing described
embodiments and exemplifications are not intended to be limiting in
any respect to the scope of the disclosure, and that the claims
presented herein are intended to encompass all embodiments and
exemplifications whether or not explicitly presented herein
[1916] All patents, patent applications, and publications cited
herein are fully incorporated by reference in their entirety.
Sequence CWU 1
1
141449PRTArtificial SequenceAntibody 1 heavy chain 1Gln Val Gln Leu
Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Ala Val Phe Gly Gly Ser Phe Ser Gly Tyr 20 25 30Tyr Trp
Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45Gly
Glu Ile Asn His Arg Gly Asn Thr Asn Asp Asn Pro Ser Leu Lys 50 55
60Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ala Leu65
70 75 80Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala 85 90 95Arg Glu Arg Gly Tyr Thr Tyr Gly Asn Phe Asp His Trp Gly
Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe 115 120 125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu 130 135 140Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp145 150 155 160Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200
205Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
210 215 220Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys2214PRTArtificial SequenceAntibody 1 light chain 2Glu Ile Val
Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Asn 20 25 30Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Gly Ser Leu Gln
Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Lys Thr
Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Asn Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205Phe Asn Arg Gly Glu Cys 210323DNAHomo sapiens
3cctggcccca gctgagatct ctg 23437DNAHomo sapiens 4gtaaacccga
tccttgatgc cagcacggtc accggtc 37536DNAHomo sapiens 5ctgccccagc
agaccggtga ccgtgctggc atcaag 36638DNAHomo sapiens 6ataatagcgg
ccgctcaaga gaaatccgtg cggagagg 38753DNAHomo sapiens 7cgactacaag
gacgacgatg acaagggatc cctggcccca gctgagatct ctg 53862DNAHomo
sapiens 8gtacccatac gatgttccag attacgctgg atccgccacc atgccccact
ccagcctgca 60tc 62951DNAHomo sapiens 9ataatatcta gaattcgcca
ccatgtaccc atacgatgtt ccagattacg c 511048DNAHomo sapiens
10ttccagatta cgctggatcc gccaccatgc cccactccag cctgcatc
481145DNAHomo sapiens 11cctctagact cgagcggccg ctcaagagaa atccgtgcgg
agagg 451242DNAHomo sapiens 12ggagacccaa gctggctagc gccaccatgt
acccatacga tg 421344DNAArtificial SequenceCMVPro-BglII-F
13ataataagat ctgttgacat tgattattga ctagttatta atag
441436DNAArtificial SequenceCMVPro-HindIII-R 14ataataaagc
ttgagctctg cttatataga cctccc 36
* * * * *