U.S. patent application number 17/166512 was filed with the patent office on 2021-05-27 for 4'-substituted nucleoside reverse transcriptase inhibitors and preparations thereof.
This patent application is currently assigned to Merck Sharp & Dohme Corp.. The applicant listed for this patent is Merck Sharp & Dohme Corp., Merck Sharp & Dohme Limited. Invention is credited to Kevin M. Belyk, Edward Cleator, Andrew William Gibson, Stephen Philip Keen, Jongrock Kong, David R. Lieberman, Mark McLaughlin, Erika M. Milczek, Jeffrey C. Moore, Feng Peng, Zhiguo Jake Song, Alejandro Diequez Vazquez, Michael J. Williams.
Application Number | 20210154221 17/166512 |
Document ID | / |
Family ID | 1000005380919 |
Filed Date | 2021-05-27 |
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United States Patent
Application |
20210154221 |
Kind Code |
A1 |
McLaughlin; Mark ; et
al. |
May 27, 2021 |
4'-SUBSTITUTED NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS AND
PREPARATIONS THEREOF
Abstract
The present invention is directed to 4'-substituted nucleoside
derivatives of Formula I ##STR00001## and their use in the
inhibition of HIV reverse transcriptase, the prophylaxis of
infection by HIV, the treatment of infection by HIV, and the
prophylaxis, treatment, and delay in the onset or progression of
AIDS and/or ARC. The present invention also provides processes for
the preparation of 4'-substituted nucleoside derivatives of Formula
I and derivatives thereof.
Inventors: |
McLaughlin; Mark; (Summit,
NJ) ; Cleator; Edward; (Whittlesford, GB) ;
Kong; Jongrock; (Princeton, NJ) ; Gibson; Andrew
William; (Welwyn Garden City, GB) ; Lieberman; David
R.; (London, GB) ; Vazquez; Alejandro Diequez;
(Toledo, ES) ; Keen; Stephen Philip; (Ware,
GB) ; Williams; Michael J.; (Morris Plains, NJ)
; Moore; Jeffrey C.; (Westfield, NJ) ; Milczek;
Erika M.; (New York, NY) ; Peng; Feng;
(Dayton, NJ) ; Belyk; Kevin M.; (Metuchen, NJ)
; Song; Zhiguo Jake; (Edison, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Merck Sharp & Dohme Corp.
Merck Sharp & Dohme Limited |
Rahway
Hertfordshire |
NJ |
US
GB |
|
|
Assignee: |
Merck Sharp & Dohme
Corp.
Rahway
NJ
Merck Sharp & Dohme Limited
Hertfordshire
|
Family ID: |
1000005380919 |
Appl. No.: |
17/166512 |
Filed: |
February 3, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15761742 |
Mar 20, 2018 |
10953029 |
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PCT/US2016/052409 |
Sep 19, 2016 |
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17166512 |
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62302452 |
Mar 2, 2016 |
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62222304 |
Sep 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/18 20180101;
C07H 19/14 20130101; A61K 31/7064 20130101; C07H 19/16 20130101;
C07D 307/33 20130101; C07H 23/00 20130101; C07H 1/00 20130101; A61K
45/06 20130101 |
International
Class: |
A61K 31/7064 20060101
A61K031/7064; C07D 307/33 20060101 C07D307/33; A61K 45/06 20060101
A61K045/06; C07H 1/00 20060101 C07H001/00; C07H 19/14 20060101
C07H019/14; C07H 23/00 20060101 C07H023/00; C07H 19/16 20060101
C07H019/16; A61P 31/18 20060101 A61P031/18 |
Claims
1-12. (canceled)
13. A process for preparing a compound of Formula (IA) ##STR00075##
wherein X is H, F, Cl, or Br; Y is N, C(H), C(F), C(Cl), C(Br),
C(CH.sub.3); Z is NH.sub.2; comprising: (a.) coupling sugar
##STR00076## with nucleobase ##STR00077## to provide protected
nucleoside ##STR00078## and (b.) converting the protected
nucleoside (A) to the compound of Formula (IA); wherein Z.sup.1 is
Cl, N(H)PG, or N(PG).sub.2, R.sup.d is (i) a group of the formula
##STR00079## wherein R.sup.p is C.sub.1-C.sub.3 alkyl,
C.sub.1-C.sub.3 alkoxy, halo, CF.sub.3, or N(CH.sub.3).sub.2; (ii)
--Si(R.sup.s).sub.3; (iii) --C(O)C.sub.1-C.sub.3 alkyl; or (iv)
--C(O)CH.sub.2N(H)C(O)CH.sub.3; each R.sup.s is independently
C.sub.1-C.sub.6 alkyl; unsubstituted phenyl; phenyl substituted by
on to three C.sub.1-C.sub.3 alkyl, halo, or C.sub.1-C.sub.3 alkoxy;
LG is OAc, OBz, halo, or --O--C.sub.2-C.sub.8 alkenyl; and PG is an
amino protecting group.
14. The process of claim 13, wherein R.sup.d is
4-methylbenzoyl.
15. The process of claim 14, wherein in the compound of Formula
(IA), X is F, Y is N, and Z is NH.sub.2.
16. The process of claim 15, wherein LG is --OAc in the sugar (C);
and Z.sup.1 is N(H)Si(R.sup.s).sub.3 in the protected
nucleoside.
17. The process of claim 16, wherein in step (a) said coupling is
conducted in the presence of a Lewis or Brcnsted acid in an aprotic
solvent to provide nucleoside (A).
18. The process of claim 16, wherein Z.sup.1 is
--N(H)Si(CH.sub.3).sub.3.
19. The process of claim 16, wherein converting in step (b)
comprises reacting the protected nucleoside (A) with an alkali
metal C.sub.1-C.sub.3 alkoxide.
20. The process of claim 16, wherein the sugar (C) is prepared by
reacting lactol ##STR00080## with acetic anhydride.
21. The process of claim 15, wherein LG is
--O(CH.sub.2).sub.3C(H).dbd.CH.sub.2 in the sugar (C); and Z.sup.1
is N(H)C(O)--OR.sup.s and X is F in the nucleobase (B) and in the
protected nucleoside
22. The process of claim 21, wherein in step (a) said coupling is
conducted by treating sugar (C) with an activating agent selected
from iodine, bromine, N-iodosuccinimide, N-bromosuccinimide,
Balarenga's reagent (Py.sub.2I), or diiodo-dimethylhydantoin; in
the presence of nucleobase (B) in an aprotic solvent.
23. The process of claim 21, wherein Z is
N(H)C(O)--OC(CH.sub.3).sub.3.
24. The process of claim 21, wherein converting in step (b)
comprises treating the protected nucleoside (A) with an alkali
metal C.sub.1-C.sub.3 alkoxide and then with a strong acid.
25. The process of claim 21, wherein the sugar (C) is prepared by
reacting lactol ##STR00081## with pent-4-en-1-ol to provide the
sugar ##STR00082##
26. The process of claim 13, wherein the sugar (C) is prepared by
reducing lactone ##STR00083## with a selective reducing agent to
provide lactol ##STR00084## and converting lactol (10A) to the
sugar (C).
27. The process of claim 26, wherein the selective reducing agent
is sodium bis(2-methoxyethoxy)aluminum hydride.
28. The process of claim 26, wherein the lactone (10) is prepared
by: reacting dioxolane ##STR00085## with an acid to provide a
deprotected intermediate; and acylating the deprotected
intermediate with a p-methylbenzoylating agent to provide the
lactone (10).
29. The process of claim 28, wherein the p-methylbenzoylating agent
is p-methylbenzoyl chloride.
30. The process of claim 28, wherein the process further comprises
isolating the deprotected intermediate, wherein the deprotected
intermediate is ##STR00086##
31. The process of claim 28, wherein the dioxolane (9) is prepared
by reacting TIPS intermediate ##STR00087## with a fluoride agent to
provide the dioxolane (91.
32. The process of claim 31, wherein the TIPS intermediate (8) is
prepared by reducing ketone ester ##STR00088##
33. The process of claim 32, wherein the reducing comprises
asymmetric transfer hydrogenation of the ketone ester (7) with
formic acid/triethylamine in the presence of a chiral catalyst.
34. The process of claim 32, wherein the chiral catalyst is
RuCl--(S,S)-Ts-DENEB catalyst.
35. A compound of the formula ##STR00089##
36-38. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] The retrovirus designated human immunodeficiency virus
(HIV), particularly the strains known as HIV type-1 (HIV-1) and
type-2 (HIV-2), have been etiologically linked to the
immunosuppressive disease known as acquired immunodeficiency
syndrome (AIDS). HIV seropositive individuals are initially
asymptomatic but typically develop AIDS related complex (ARC)
followed by AIDS. Affected individuals exhibit severe
immunosuppression which makes them highly susceptible to
debilitating and ultimately fatal opportunistic infections.
Replication of HIV by a host cell requires integration of the viral
genome into the host cell's DNA. Since HIV is a retrovirus, the HIV
replication cycle requires transcription of the viral RNA genome
into DNA via an enzyme known as reverse transcriptase (RT).
[0002] Reverse transcriptase has three known enzymatic functions:
The enzyme acts as an RNA-dependent DNA polymerase, as a
ribonuclease, and as a DNA-dependent DNA polymerase. In its role as
an RNA-dependent DNA polymerase, RT transcribes a single-stranded
DNA copy of the viral RNA. As a ribonuclease, RT destroys the
original viral RNA and frees the DNA just produced from the
original RNA. And as a DNA-dependent DNA polymerase, RT makes a
second, complementary DNA strand using the first DNA strand as a
template. The two strands form double-stranded DNA, which is
integrated into the host cell's genome by the integrase enzyme.
[0003] It is known that compounds that inhibit enzymatic functions
of HIV RT will inhibit HIV replication in infected cells. These
compounds are useful in the prophylaxis or treatment of HIV
infection in humans. Among the compounds approved for use in
treating HIV infection and AIDS are nucleoside RT inhibitors (NRTI)
such as 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine
(ddI), 2',3'-dideoxycytidine (ddC), d4T, 3TC, abacavir,
emtricitabine, and tenofovir disoproxil fumarate, as well as
non-nucleoside RT inhibitors (nNRTI) such as nevirapine,
delavirdine, and efavirenz.
[0004] While each of the foregoing drugs is effective in treating
HIV infection and AIDS, there remains a need to develop additional
HIV antiviral drugs including additional RT inhibitors. A
particular problem is the development of mutant HIV strains that
are resistant to the known inhibitors. The use of anti-retrovirals
to treat AIDS often leads to viruses that are less sensitive to the
inhibitors. This resistance is typically the result of mutations
that occur in the reverse transcriptase segment of the pol gene.
The continued use of antiviral compounds to prevent HIV infection
will inevitably result in the emergence of new resistant strains of
HIV. Accordingly, there is a continuing need for new RT inhibitors
that are effective against mutant HIV strains.
[0005] In addition, new routes for preparing nucleoside RT
inhibitors are needed, particularly for preparing multi-kilogram
quantities of drug substance required to support animal toxicology
studies and subsequent human clinical trials. For instance, several
routes to the 4'-substituted nucleoside derivative,
4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA)
##STR00002##
have appeared in the literature, including two reports in Organic
Letters published in 2011 and 2015 (Kuwahara et al., Org. Lett.
2011, 13, 5264 and Kuwahara/Ohrui et al., Org. Lett. 2015, 17,
828). EFdA has been reported to provide potent antiviral activity
against wild-type and multi-drug resistant HIV-1 strains. The
published routes to EFdA have drawbacks with respect to production
of multi-kilogram quantities of drug substance required for further
studies. In particular, some of the published routes use the chiral
starting material (R)-glyceraldehyde acetonide, which is not
readily available at large scale and is also prone to
stereochemical erosion. In addition, the published routes lack a
sufficient number of crystalline intermediates to enable purity
control without resort to chromatographic purification. The
published routes also use hazardous or impractical reagents, and
synthetic methods that are not optimized for large-scale
implementation due to the reagents' toxicities or the methods'
hazards.
SUMMARY OF THE INVENTION
[0006] The present invention is directed to 4'-substituted
nucleoside derivatives and their use in the inhibition of HIV
reverse transcriptase, the prophylaxis of infection by HIV, the
treatment of infection by HIV, and the prophylaxis, treatment, and
delay in the onset or progression of AIDS and/or ARC.
[0007] The present invention also provides a process for the
preparation of 4'-ethynyl-2'-deoxyribonucleosides, such as EFdA and
the compounds having structural Formula I. In addition, the
invention provides certain synthetic intermediates which are useful
in preparing 4'-ethynyl-2'-deoxyribonucleosides.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention is directed to compounds having
structural Formula I:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 is
--H, --C(O)R.sup.3, --C(O)OR.sup.3, --C(O)N(R.sup.3).sub.2,
##STR00004##
or a pro-drug modification of the mono-, di- or triphosphate; and
R.sup.2 is --H, --C(O)R.sup.4, --C(O)OR.sup.4 or
--C(O)N(R.sup.4).sub.2; R.sup.3 and R.sup.4 are each independently
selected at each occurrence from --H, --C.sub.1-C.sub.6 alkyl,
--C.sub.1-C.sub.6 haloalkyl, --(C.sub.1-C.sub.3
alkylene).sub.m-(C.sub.3-C.sub.7 cycloalkyl), --(C.sub.1-C.sub.3
alkylene).sub.m-(aryl), --(C.sub.1-C.sub.3 alkylene).sub.m-(4 to
7-membered heterocycloalkyl), --(C.sub.1-C.sub.3
alkylene).sub.m-(5- or 6-membered monocyclic heteroaryl) or
--(C.sub.1-C.sub.3 alkylene).sub.m-(9- or 10-membered bicyclic
heteroaryl), [0009] wherein each of said --C.sub.1-C.sub.6 alkyl,
said --C.sub.3-C.sub.7 cycloalkyl group, said aryl group, said 4 to
7-membered heterocycloalkyl group, said 5- or 6-membered monocyclic
heteroaryl group or said 9- or 10-membered bicyclic heteroaryl
group is unsubstituted or substituted with R.sup.5; m is an integer
selected from 0 (zero) or 1; and R.sup.5 represents from one to
five substituent groups, each independently selected from
--C.sub.1-C.sub.6alkyl, --C.sub.2-C.sub.6 alkenyl,
--C.sub.2-C.sub.6 alkynyl, --C.sub.1-C.sub.6 haloalkyl, aryl or a
5-6-member heteroaryl.
[0010] In Embodiment A of this invention are compounds of Formula
I, or a pharmaceutically acceptable salt thereof, wherein R.sup.1
is --H, --C(O)R.sup.3, --C(O)OR.sup.3, --C(O)N(R.sup.3).sub.2, or a
pro-drug modification of one of the following mono-, di- or
triphosphate moieties:
##STR00005##
R.sup.2 is --H, --C(O)R.sup.4, --C(O)OR.sup.4 or
--C(O)N(R.sup.4).sub.2.
[0011] In Embodiment B of this invention are compounds of Formula
I, or a pharmaceutically acceptable salt thereof, wherein R.sup.1
is --H, --C(O)R.sup.3, --C(O)OR.sup.3, or --C(O)N(R.sup.3).sub.2;
and R.sup.2 is --H, --C(O)R.sup.4, --C(O)OR.sup.4 or
--C(O)N(R.sup.4).sub.2.
[0012] In Embodiment C of this invention are compounds of Formula I
or Embodiment A or B, or a pharmaceutically acceptable salt
thereof, wherein at least one of R.sup.1 or R.sup.2 is --H.
[0013] In Embodiment D of this invention are compounds of Formula I
wherein R.sup.1 is
##STR00006##
R.sup.2 is --H.
[0014] Examples of compounds of Formula I of this invention are the
following Compound 1 or Compound 2, or a pharmaceutically
acceptable salt thereof:
##STR00007##
[0015] Reference to the compounds of this invention as those of a
specific formula or embodiment, e.g., Formula I, or Embodiments A,
B or C thereof, or any other generic structural formula or specific
compound described or claimed herein, is intended to encompass the
specific compound or compounds falling within the scope of the
Formula or embodiment, including salts thereof, particularly
pharmaceutically acceptable salts, solvates (including hydrates) of
such compounds and solvated salt forms thereof, where such forms
are possible, unless specified otherwise. The present invention
includes each of the Examples described herein, and
pharmaceutically acceptable salts thereof. The invention also
encompasses pharmaceutical compositions comprising an effective
amount of a compound of the invention or a pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable
carrier.
[0016] As used herein, the term "alkyl" refers to a monovalent
straight or branched chain, saturated aliphatic hydrocarbon radical
having a number of carbon atoms in the specified range. Thus, for
example, "C.sub.1-6 alkyl" (or "C.sub.1-C.sub.6 alkyl") refers to
any of the hexyl alkyl and pentyl alkyl isomers as well as n-,
iso-, sec- and t-butyl, n- and iso-propyl, ethyl and methyl. As
another example, "C.sub.1-4 alkyl" (or "C.sub.1-C.sub.4 alkyl")
refers to n-, iso-, sec- and t-butyl, n- and isopropyl, ethyl and
methyl.
[0017] The term "alkenyl" refers to a monovalent straight or
branched chain aliphatic hydrocarbon radical containing one
carbon-carbon double bond and having a number of carbon atoms in
the specified range. Thus, for example, "C.sub.2-6 alkenyl" (or
"C.sub.2-C.sub.6 alkenyl") refers to all of the hexenyl and
pentenyl isomers as well as 1-butenyl, 2-butenyl, 3-butenyl,
isobutenyl, 1-propenyl, 2-propenyl, and ethenyl (or vinyl).
[0018] The term "alkynyl" refers to a monovalent straight or
branched chain aliphatic hydrocarbon radical containing one
carbon-carbon triple bond and having a number of carbon atoms in
the specified range. Thus, for example, "C.sub.2-6 alkynyl" (or
"C.sub.2-C.sub.6 alkynyl") refers to all of the hexynyl and
pentynyl isomers as well as 1-butynyl, 2-butynyl, 3-butynyl,
1-propynyl, 2-propynyl, and ethynyl.
[0019] The term "alkylene" refers to any divalent linear or
branched chain aliphatic hydrocarbon radical having a number of
carbon atoms in the specified range. Thus, for example, "--C.sub.1.
C.sub.3 alkylene-" refers to any of the C.sub.1 to C.sub.3 linear
or branched alkylenes. A particular class of alkylenes includes
--(CH.sub.2).sub.1-3--, --(CH.sub.2).sub.2-3--,
--(CH.sub.2).sub.1-2--, --CH.sub.2--, --CH(CH.sub.3)--, and
--C(CH.sub.3).sub.2--.
[0020] The term "cycloalkyl" refers to any monocyclic ring of an
alkane having a number of carbon atoms in the specified range.
Thus, for example, "C.sub.3-C.sub.7 cycloalkyl" (or
"C.sub.3-C.sub.7 cycloalkyl") refers to cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and cycloheptyl. A particular class of
interest for compounds of Formula I and embodiments thereof is
C.sub.3-C.sub.6 cycloalkyl. "Heterocycloalkyl" refers to a
cycloalkyl ring wherein one or two of the carbon atoms in the ring
are replaced by a heteroatom independently selected from N, O and
S.
[0021] The term "halogen" (or "halo") refers to fluorine, chlorine,
bromine and iodine (alternatively referred to as fluoro, chloro,
bromo, and iodo). A particular class of interest for compounds of
Formula I and embodiments thereof is each of fluoro of chloro.
[0022] The term "haloalkyl" refers to an alkyl group as defined
above in which one or more of the hydrogen atoms have been replaced
with halo (i.e., --F, --Cl, --Br and/or --I). Thus, for example,
"C.sub.1-C.sub.6 haloalkyl" refers to a C.sub.1 to C.sub.6 linear
or branched alkyl group as defined above with one or more halo
substituents.
[0023] The term "C(O)" refers to carbonyl.
[0024] The term "aryl" (or "C.sub.6-C.sub.10 aryl") refers to (i)
phenyl, or (ii) 9- or 10-membered bicyclic, fused carbocylic ring
systems in which at least one ring is aromatic. Suitable aryls
include, for example, phenyl, naphthyl, tetrahydronaphthyl
(tetralinyl), or indenyl. In a particular class of compounds of
Formula I and embodiments thereof, aryl is phenyl or naphthyl, and
more particularly aryl is phenyl.
[0025] The term "heteroaryl" refers to (i) a 5- or 6-membered
heteroaromatic ring containing from 1 to 4 heteroatoms
independently selected from N, O and S, wherein each N is
optionally in the form of an oxide to the extent chemically
possible, (ii) a 9- or 10-membered bicyclic fused ring system,
wherein the fused ring system contains from 1 to 6 heteroatoms
independently selected from N, O and S, wherein each ring in the
fused ring system contains zero, one, or more than one heteroatom,
and at least one ring is aromatic, and each N is optionally in the
form of an oxide to the extent chemically possible, and each S in a
ring which is not aromatic is optionally S(O) or S(O).sub.2.
Suitable 5- and 6-membered heteroaromatic rings include, for
example, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl,
triazinyl, thienyl, furanyl, imidazolyl, pyrazolyl, triazolyl
triazolyl (i.e., 1,2,3-triazolyl or 1,2,4-triazolyl), tetrazolyl,
oxazolyl, isooxazolyl, oxadiazolyl (i.e., the 1,2,3-, 1,2,4-,
1,2,5-(furazanyl), or 1,3,4-isomer), oxatriazolyl, thiazolyl,
isothiazolyl, and thiadiazolyl. Suitable 9- and 10-membered
heterobicyclic, fused ring systems include, for example,
benzofuranyl, indolyl, indazolyl, naphthyridinyl, isobenzofuranyl,
benzopiperidinyl, benzisoxazolyl, benzoxazolyl, chromenyl,
quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, isoindolyl,
benzodioxolyl (e.g., benzo-1,3-dioxolyl:
##STR00008##
benzopiperidinyl, benzisoxazolyl, benzoxazolyl, chromanyl,
isochromanyl, benzothienyl, benzofuranyl, imidazo[1,2-a]pyridinyl,
benzotriazolyl, dihydroindolyl, dihydroisoindolyl, indazolyl,
indolinyl, isoindolinyl, quinoxalinyl, quinazolinyl,
2,3-dihydrobenzofuranyl, and 2,3-dihydrobenzo-1,4-dioxinyl
(i.e.,
##STR00009##
[0026] It is understood that the specific rings and ring systems
suitable for use in the present invention are not limited to those
listed in the preceding paragraphs. These rings and ring systems
are merely representative. Unless expressly stated to the contrary
in a particular context, any of the various cyclic rings and ring
systems described herein may be attached to the rest of the
compound at any ring atom (i.e., any carbon atom or any heteroatom)
provided that the attachment is chemically allowed and a stable
compound results.
[0027] When any variable occurs more than one time in any
constituent or in Formula I or in any other formula depicting and
describing compounds of the present invention, its definition on
each occurrence is independent of its definition at every other
occurrence. Also, combinations of substituents and/or variables are
permissible only if such combinations result in stable
compounds.
[0028] Unless expressly stated to the contrary, substitution by a
named substituent is permitted on any atom in a ring (e.g.,
cycloalkyl, aryl, or heteroaryl) provided such ring substitution is
chemically allowed and results in a stable compound.
[0029] Unless expressly stated to the contrary, all ranges cited
herein are inclusive. For example, a heteroaromatic ring described
as containing from "1 to 4 heteroatoms" means the ring can contain
1, 2, 3 or 4 heteroatoms. It is also to be understood that any
range cited herein includes within its scope all of the sub-ranges
within that range. Thus, for example, a heteroaryl group described
as containing from "1 to 4 heteroatoms" is intended to include as
aspects thereof, heteroaromatic rings containing 2 to 4
heteroatoms, 3 or 4 heteroatoms, 1 to 3 heteroatoms, 2 or 3
heteroatoms, 1 or 2 heteroatoms, 1 heteroatom, 2 heteroatoms, 3
heteroatoms, or 4 heteroatoms. "Optionally" substituted means a
chemical moeity can be unsubstituted or substituted with the noted
substituents.
[0030] As would be recognized by one of ordinary skill in the art,
certain of the compounds of the present invention can exist as
tautomers. All tautomeric forms of these compounds, whether
isolated individually or in mixtures, are within the scope of the
present invention. For example, in instances where an --OH
substituent is permitted on a heteroaromatic ring and keto-enol
tautomerism is possible, it is understood that the substituent
might in fact be present, in whole or in part, in the oxo (.dbd.O)
form.
[0031] A "stable" compound is a compound which can be prepared and
isolated and whose structure and properties remain or can be caused
to remain essentially unchanged for a period of time sufficient to
allow use of the compound for the purposes described herein (e.g.,
therapeutic or prophylactic administration to a subject). The
compounds of the present invention are limited to stable compounds
embraced by Formula I and its embodiments.
[0032] The compounds of Formula I may have one or more chiral
(asymmetric) centers. Centers of asymmetry that are present in the
compounds of Formula I can all independently of one another have
(R) or (S) configuration, except for the chiral centers depicted in
structural Formula I as having a specific stereoconfiguration. The
present invention encompasses all such stereoisomeric forms of the
compounds of Formula I.
[0033] The invention includes all possible enantiomers and
diastereomers and mixtures of two or more stereoisomers of
compounds of Formula I when chiral centers are possible in R.sup.3,
R.sup.4 and/or R.sup.5, for example mixtures of enantiomers and/or
diastereomers, in all ratios. Thus, enantiomers are a subject of
the invention in enantiomerically pure form, both as levorotatory
and as dextrorotatory antipodes, in the form of racemates and in
the form of mixtures of the two enantiomers in all ratios. In the
case of a cis/trans isomerism the invention includes both the cis
form and the trans form as well as mixtures of these forms in all
ratios. The preparation of individual stereoisomers can be carried
out, if desired, by separation of a mixture by customary methods,
for example by chromatography or crystallization, by the use of
stereochemically uniform starting materials for the synthesis or by
stereoselective synthesis. Optionally a derivatization can be
carried out before a separation of stereoisomers. The separation of
a mixture of stereoisomers can be carried out at an intermediate
step during the synthesis of a compound of Formula I or it can be
done on a final racemic product. Absolute stereochemistry may be
determined by X-ray crystallography of crystalline products or
crystalline intermediates which are derivatized, if necessary, with
a reagent containing a stereogenic center of known configuration.
Alternatively, absolute stereochemistry may be determined by
Vibrational Circular Dichroism (VCD) spectroscopy analysis. The
present invention includes all such isomers, as well as salts,
solvates (which includes hydrates) and solvated salts of such
racemates, enantiomers, diastereomers and tautomers and mixtures
thereof.
[0034] The atoms in a compound of Formula I may exhibit their
natural isotopic abundances, or one or more of the atoms may be
artificially enriched in a particular isotope having the same
atomic number, but an atomic mass or mass number different from the
atomic mass or mass number predominantly found in nature. The
present invention is meant to include all suitable isotopic
variations of the compounds of generic Formula I. For example,
different isotopic forms of hydrogen (H) include protium (.sup.1H)
and deuterium (.sup.2H). Protium is the predominant hydrogen
isotope found in nature. Enriching for deuterium may afford certain
therapeutic advantages, such as increasing in vivo half-life or
reducing dosage requirements, or may provide a compound useful as a
standard for characterization of biological samples.
Isotopically-enriched compounds within generic Formula I can be
prepared without undue experimentation by conventional techniques
well known to those skilled in the art or by processes analogous to
those described in the Examples and descriptions herein using
appropriate isotopically-enriched reagents and/or
intermediates.
[0035] The compounds can be administered in the form of
pharmaceutically acceptable salts. The term "pharmaceutically
acceptable salt" refers to a salt which is not biologically or
otherwise undesirable (e.g., is neither toxic nor otherwise
deleterious to the recipient thereof). When the compounds of
Formula I contain one or more acidic or basic groups the invention
also includes the corresponding pharmaceutically acceptable salts.
Thus, the compounds of Formula I which contain acidic groups (e.g.,
--COOH) can be used according to the invention as, for example but
not limited to, alkali metal salts, alkaline earth metal salts or
as ammonium salts. Examples of such salts include but are not
limited to sodium salts, potassium salts, calcium salts, magnesium
salts or salts with ammonia or organic amines such as, for example,
ethylamine, ethanolamine, triethanolamine or amino acids. Compounds
of Formula I which contain one or more basic groups, i.e. groups
which can be protonated, can be used according to the invention in
the form of their acid addition salts with inorganic or organic
acids as, for example but not limited to, salts with hydrogen
chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric
acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic
acid, naphthalenedisulfonic acids, oxalic acid, acetic acid,
trifluoroacetic acid, tartaric acid, lactic acid, salicylic acid,
benzoic acid, formic acid, propionic acid, pivalic acid,
diethylacetic acid, malonic acid, succinic acid, pimelic acid,
fumaric acid, maleic acid, malic acid, sulfaminic acid,
phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic
acid, citric acid, adipic acid, etc. If the compounds of Formula I
simultaneously contain acidic and basic groups in the molecule the
invention also includes, in addition to the salt forms mentioned,
inner salts or betaines (zwitterions). Salts can be obtained from
the compounds of Formula I by customary methods which are known to
the person skilled in the art, for example by combination with an
organic or inorganic acid or base in a solvent or dispersant, or by
anion exchange or cation exchange from other salts. The present
invention also includes all salts of the compounds of Formula I
which, owing to low physiological compatibility, are not directly
suitable for use in pharmaceuticals but which can be used, for
example, as intermediates for chemical reactions or for the
preparation of pharmaceutically acceptable salts.
[0036] Another embodiment of the present invention is a compound of
Formula I wherein the compound or its salt is in a substantially
pure form. As used herein "substantially pure" means suitably at
least about 60 wt. %, typically at least about 70 wt. %, preferably
at least about 80 wt. %, more preferably at least about 90 wt. %
(e.g., from about 90 wt. % to about 99 wt. %), even more preferably
at least about 95 wt. % (e.g., from about 95 wt. % to about 99 wt.
%, or from about 98 wt. % to 100 wt. %), and most preferably at
least about 99 wt. % (e.g., 100 wt. %) of a product containing a
compound of Formula I or its salt (e.g., the product isolated from
a reaction mixture affording the compound or salt) consists of the
compound or salt. The level of purity of the compounds and salts
can be determined using a standard method of analysis such as thin
layer chromatography, gel electrophoresis, high performance liquid
chromatography, and/or mass spectrometry. If more than one method
of analysis is employed and the methods provide experimentally
significant differences in the level of purity determined, then the
method providing the highest purity level governs. A compound or
salt of 100% purity is one which is free of detectable impurities
as determined by a standard method of analysis. A substantially
pure compound can be either a substantially pure mixture of
stereoisomers or a substantially pure individual diastereomer or
enantiomer.
[0037] Furthermore, compounds of the present invention may exist in
amorphous form and/or one or more crystalline forms, and as such
all amorphous and crystalline forms and mixtures thereof of the
compounds of Formula I are intended to be included within the scope
of the present invention. In addition, some of the compounds of the
instant invention may form solvates with water (i.e., a hydrate) or
common organic solvents. Such solvates and hydrates, particularly
the pharmaceutically acceptable solvates and hydrates, of the
instant compounds are likewise encompassed within the scope of this
invention, along with un-solvated and anhydrous forms.
[0038] It is understood that a compound of Formula I (or any
embodiment thereof and pharmaceutically acceptable salts thereof)
may be converted intracellularly/in vivo by one or more mechanisms
(e.g., enzyme-catalyzed chemical reactions) to the corresponding
nucleoside 5' triphosphate, i.e., wherein R.sup.1 is
--P(O)(OH)--O--P(O)(OH)--O--P(O)(OH).sub.2) and R.sup.2 is H. While
not wishing to be bound by any particular theory, the nucleoside
5'triphosphate is generally understood to be responsible for
inhibiting the HIV RT enzyme and for the resulting antiviral
activity after administration of the compound of Formula I to a
subject. For example, Compound 2 described herein is a nucleoside
5'triphosphate analog of Compound 1.
[0039] Accordingly, prodrugs of the compounds of the invention are
contemplated herein. A discussion of prodrugs is provided in T.
Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987)
14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in
Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical
Association and Pergamon Press. The term "prodrug" herein means a
compound (e.g., a drug precursor), which may be in the form of a
pharmaceutically acceptable salt, that is transformed
intracellularly/in vivo to provide a 4'-substituted Nucleoside
Derivative which is an inhibitor of HIV reverse transcriptase. A
nucleoside 5'triphosphate is an example of a 4'-substituted
Nucleoside Derivative. The in vivo transformation may occur by
various mechanisms, e.g., an enzyme-catalyzed chemical reaction, a
metabolic chemical reaction, and/or a spontaneous chemical reaction
(e.g., solvolysis), such as, for example, through hydrolysis in
blood. This invention encompasses any prodrugs which convert, due
to intracellular/in vivo conversion, to a 4'-substituted Nucleoside
Derivative of a compound of Formula I which is an inhibitor of HIV
reverse transcriptase. For example, 4'-substituted Nucleoside
Derivatives of Formula I include, but are not limited to, compounds
of Formula I wherein:
a) R.sup.1 is --P(O)(OH)--O--P(O)(OH)--O--P(O)(OH).sub.2; or b)
R.sup.1 is --P(O)(OH)--O--P(O)(OH)--O--P(O)(OH).sub.2 and R.sup.2
is --H.
[0040] Prodrugs of compounds of Formula I can exhibit enhanced
solubility, absorption, and/or lipophilicity compared to the
compounds per se, thereby resulting in increased bioavailability
and efficacy. When the compound of Formula I contains a hydroxy
group at the 5' and/or 3' positions, (i.e., when R.sup.1 is --H,
and/or R.sup.2 is --H), the prodrug can be a derivative of the
hydroxy group such as when R.sup.1=--C(O)R.sup.3, --C(O)OR.sup.3 or
--C(O)N(R.sup.3).sub.2, and/or R.sup.2=--C(O)R.sup.4,
--C(O)OR.sup.4 or --C(O)N(R.sup.4).sub.2.
[0041] In Formula I, R.sup.1 also includes pro-drug modification of
the mono-, di- or triphosphate. This prodrug modification can be a
derivative of one or more of the hydroxy groups on a mono-, di- or
triphosphate moiety, such as an ester (--OC(O)R), a carbonate ester
(--OC(O)OR), an ether (--OR), a phosphate ester
(--O--P(.dbd.O)(OC(O)R).sub.2), or a mono-phosphate prodrug such as
a phosphoramidate (which can be converted in vivo to the
corresponding nucleoside monophosphate). The pro-drug modification
of the mono-, di- or triphosphate also includes, but is not limited
to, 5'-alcohol-derived prodrugs such as
--P(O)(--O--C.sub.1-C.sub.6alkyl).sub.2;
--P(O)(--NH--(.alpha.-aminoacyl group))(--O-aryl) known as
"McGuigan" type prodrugs; --P(O)(--O--(C.sub.1-C.sub.6
alkylene)-(S-acyl)(--NH-arylalkyl); S-acyl-2-thioethyl (SATE)
prodrugs; or a cyclic phosphate ester that forms a bridge between
two ribose hydroxyl groups, such as:
##STR00010##
e.g.,
##STR00011##
wherein the cyclic phosphate ester forms a bridge between the
3'--OH group and 5'--OH groups; and those described in U.S. Pat.
No. 7,879,815; International Publication Nos. WO2005/003047,
WO2008/082602, WO2010/0081628, WO2010/075517 and WO2010/075549;
Mehellou, Chem. Med. Chem., 5:1841-1842 (2005); Bobeck et al.,
Antiviral Therapy 15:935-950 (2010); Furman et al., Future
Medicinal Chemistry, 1:1429-1452 (2009); and Erion, Microsomes and
Drug Oxidations, Proceedings of the International Symposium, 17th,
Saratoga Springs, N.Y., United States, Jul. 6-10, 2008, 7-12
(2008).
[0042] As further examples of prodrugs, if a compound of Formula I
contains an alcohol functional group at any of the above-described
positions in the compound, a prodrug can be formed by the
replacement of one or more of the hydrogen atoms of the alcohol
groups with a group such as, for example,
(C.sub.1-C.sub.6)alkanoyloxymethyl,
1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl,
1-methyl-1-((C.sub.1-C.sub.6)alkanoyloxy)ethyl,
(C.sub.1-C.sub.6)alkoxycarbonyloxymethyl,
N--(C.sub.1-C.sub.6)alkoxycarbonylaminomethyl, succinoyl,
(C.sub.1-C.sub.6)alkanoyl, .alpha.-amino(C.sub.1-C.sub.4)alkyl,
.alpha.-amino(C.sub.1-C.sub.4)alkylene-aryl, arylacyl and
.alpha.-aminoacyl, or .alpha.-aminoacyl-.alpha.-aminoacyl, where
each .alpha.-aminoacyl group is independently selected from the
naturally occurring L-amino acids, or glycosyl (the radical
resulting from the removal of a hydroxyl group of the hemiacetal
form of a carbohydrate).
[0043] The compounds of Formula I also contains an amine functional
group. A prodrug of a compound of Formula I can be formed by the
replacement of a hydrogen atom in the amine group with a group such
as, for example, R-carbonyl-, RO-carbonyl-, NRR'-carbonyl- wherein
R and R' are each independently (C.sub.1-C.sub.10)alkyl,
(C.sub.3-C.sub.7) cycloalkyl, benzyl, a natural .alpha.-aminoacyl,
--C(OH)C(O)OY.sup.1 wherein Y.sup.1 is H, (C.sub.1-C.sub.6)alkyl or
benzyl, --C(OY.sup.2)Y.sub.3 wherein Y.sup.2 is (C.sub.1-C.sub.4)
alkyl and Y.sup.3 is (C.sub.1-C.sub.6)alkyl; carboxy
(C.sub.1-C.sub.6)alkyl; amino(C.sub.1-C.sub.4)alkyl or mono-N- or
di-N,N--(C.sub.1-C.sub.6)alkylaminoalkyl; --C(Y.sup.4)Y.sup.5
wherein Y.sup.4 is H or methyl and Y.sup.5 is mono-N- or
di-N,N--(C.sub.1-C.sub.6)alkylamino morpholino; piperidin-1-yl or
pyrrolidin-1-yl, and the like.
[0044] Pharmaceutically acceptable esters of the present compounds
include the following groups: (1) carboxylic acid esters obtained
by esterification of the hydroxy group of a hydroxyl compound, in
which the non-carbonyl moiety of the carboxylic acid portion of the
ester grouping is selected from straight or branched chain alkyl
(e.g., methyl, ethyl, n-propyl, isopropyl, t-butyl, sec-butyl or
n-butyl), alkoxyalkyl (e.g., methoxymethyl), aryl (e.g., benzyl),
aryloxyalkyl (for example, phenoxymethyl), aryl (e.g., phenyl
optionally substituted with, for example, halogen, C.sub.1-4alkyl,
--O--(C.sub.1-4alkyl) or amino); (2) sulfonate esters, such as
alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino
acid esters (e.g., L-valyl or L-isoleucyl); (4) phosphonate esters
and (5) mono-, di- or triphosphate esters. The phosphate esters may
be further esterified by, for example, a C.sub.1-20 alcohol or
reactive derivative thereof, or by a 2,3-di (C.sub.6-24)acyl
glycerol.
[0045] If a 4'-substituted deoxyribose derivative contains a
carboxylic acid functional group, a prodrug can comprise an ester
formed by the replacement of the hydrogen atom of the acid group
with a group such as, for example, (C.sub.1-C.sub.8)alkyl,
(C.sub.2-C.sub.12)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having
from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having
from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to
6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7
carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to
8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9
carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10
carbon atoms, 3-phthalidyl, 4-crotonolactonyl,
gamma-butyrolacton-4-yl,
di-N,N--(C.sub.1-C.sub.2)alkylamino(C.sub.2-C.sub.3)alkyl (such as
.beta.-dimethylaminoethyl), carbamoyl-(C.sub.1-C.sub.2)alkyl,
N,N-di (C.sub.1-C.sub.2)alkylcarbamoyl-(C.sub.1-C.sub.2)alkyl and
piperidino-, pyrrolidino- or morpholino(C.sub.2-C.sub.3)alkyl, and
the like.
[0046] Other examples include the following: when the compound of
Formula I contains a carboxylic acid group, the prodrug can be an
ester or an amide, and when the compound of Formula I contains a
primary amino group or another suitable nitrogen that can be
derivatized, the prodrug can be an amide, carbamate, urea, imine,
or a Mannich base. Conventional procedures for the selection and
preparation of suitable prodrug derivatives are described, for
example, in Design of Prodrugs, edited by H. Bundgaard, Elsevier,
1985; J. J. Hale et al., J. Med. Chem. 2000, vol. 43, pp.
1234-1241; C. S. Larsen and J. Ostergaard, "Design and application
of prodrugs" in: Textbook of Drug Design and Discovery, 3rd
edition, edited by C. S. Larsen, 2002, pp. 410-458; and Beaumont et
al., Current Drug Metabolism 2003, vol. 4, pp. 461-458; the
disclosures of each of which are incorporated herein by reference
in their entireties.
[0047] Accordingly, the compounds within the structural Formula I,
embodiments and specific compounds described and claimed herein
encompass salts, any possible stereoisomers and tautomers, physical
forms (e.g., amorphous and crystalline forms), solvate and hydrate
forms thereof and any combination of these forms, as well as the
salts thereof, pro-drug forms thereof which include any combination
of stereoisomer, tautomer, solvate, hydrate, salt and/or physical
forms of said pro-drugs, where such forms are possible unless
specified otherwise.
[0048] The invention also encompasses methods for the treatment or
prophylaxis of infection by HIV, for the inhibition of HIV reverse
transcriptase, or for the treatment, prophylaxis, or delay in the
onset of AIDS in a subject in need thereof, which comprises
administering to the subject an effective amount of a compound of
the invention or a pharmaceutically acceptable salt thereof.
[0049] The invention also encompasses a compound of the invention,
or a pharmaceutically acceptable salt thereof, for use in the
preparation of a medicament for the treatment or prophylaxis of
infection by HIV, for the inhibition of HIV reverse transcriptase,
or for the treatment, prophylaxis, or delay in the onset of AIDS in
a subject in need thereof.
[0050] The invention also encompasses a pharmaceutical composition
comprising an effective amount of a compound of the invention, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier and further comprising an effective amount of an
additional anti-HIV agent selected from the group consisting of HIV
antiviral agents, immunomodulators, and anti-infective agents.
Within this embodiment, the anti-HIV agent is an antiviral selected
from the group consisting of HIV protease inhibitors, HIV reverse
transcriptase inhibitors, HIV integrase inhibitors, HIV fusion
inhibitors, HIV entry inhibitors, and HIV maturation
inhibitors.
[0051] Compounds of Embodiment A, B or C each form a subset of the
compounds included in Formula I. Any description above or which
follows that refers to a compound of Formula I also applies to a
compound of each of Embodiment A, B or C and any embodiments
thereof.
[0052] Other embodiments of the present invention include the
following:
[0053] (a) A pharmaceutical composition comprising an effective
amount of a compound of Formula I as defined above, or a prodrug or
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
[0054] (b) A pharmaceutical composition which comprises the product
prepared by combining (e.g., mixing) an effective amount of a
compound of Formula I as defined above, or a prodrug or
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
[0055] (c) The pharmaceutical composition of (a) or (b), further
comprising an effective amount of one or more an anti-HIV agents
selected from the group consisting of HIV antiviral agents,
immunomodulators, and anti-infective agents.
[0056] (d) The pharmaceutical composition of (c), wherein the
anti-HIV agent is selected from one or more of an antiviral
selected from the group consisting of HIV protease inhibitors,
nucleoside HIV reverse transcriptase inhibitors, non-nucleoside HIV
reverse transcriptase inhibitors, HIV integrase inhibitors, HIV
fusion inhibitors, HIV entry inhibitors and HIV maturation
inhibitors.
[0057] (e) A combination which is (i) a compound of Formula I as
defined above, or a prodrug or pharmaceutically acceptable salt
thereof, and (ii) an anti-HIV agent selected from the group
consisting of HIV antiviral agents, immunomodulators, and
anti-infective agents; wherein the compound and the anti-HIV agent
are each employed in an amount that renders the combination
effective for inhibition of HIV reverse transcriptase, for
treatment or prophylaxis of infection by HIV, or for treatment,
prophylaxis of, or delay in the onset or progression of AIDS.
[0058] (f) The combination of (e), wherein the anti-HIV agent is an
antiviral selected from the group consisting of HIV protease
inhibitors, nucleoside HIV reverse transcriptase inhibitors,
non-nucleoside HIV reverse transcriptase inhibitors, HIV integrase
inhibitors, HIV fusion inhibitors, HIV entry inhibitors and HIV
maturation inhibitors.
[0059] (g) A method for the inhibition of HIV reverse transcriptase
in a subject in need thereof which comprises administering to the
subject an effective amount of a compound of Formula I or a prodrug
or pharmaceutically acceptable salt thereof.
[0060] (h) A method for the prophylaxis or treatment of infection
by HIV (e.g., HIV-1) in a subject in need thereof which comprises
administering to the subject an effective amount of a compound of
Formula I or a prodrug or pharmaceutically acceptable salt
thereof.
[0061] (i) The method of (h), wherein the compound of Formula I is
administered in combination with an effective amount of at least
one other HIV antiviral selected from the group consisting of HIV
protease inhibitors, HIV integrase inhibitors, non-nucleoside HIV
reverse transcriptase inhibitors, nucleoside HIV reverse
transcriptase inhibitors, HIV fusion inhibitors, HIV entry
inhibitors and HIV maturation inhibitors.
[0062] (j) A method for the prophylaxis, treatment or delay in the
onset or progression of AIDS in a subject in need thereof which
comprises administering to the subject an effective amount of a
compound of Formula I or a prodrug or pharmaceutically acceptable
salt thereof.
[0063] (k) The method of (j), wherein the compound is administered
in combination with an effective amount of at least one other HIV
antiviral selected from the group consisting of HIV protease
inhibitors, HIV integrase inhibitors, non-nucleoside HIV reverse
transcriptase inhibitors, nucleoside HIV reverse transcriptase
inhibitors, HIV fusion inhibitors, HIV entry inhibitors and HIV
maturation inhibitors.
[0064] (l) A method for the inhibition of HIV reverse transcriptase
in a subject in need thereof which comprises administering to the
subject the pharmaceutical composition of (a), (b), (c) or (d) or
the combination of (e) or (f).
[0065] (m) A method for the prophylaxis or treatment of infection
by HIV (e.g., HIV-1) in a subject in need thereof which comprises
administering to the subject the pharmaceutical composition of (a),
(b), (c) or (d) or the combination of (e) or (f).
[0066] (n) A method for the prophylaxis, treatment, or delay in the
onset or progression of AIDS in a subject in need thereof which
comprises administering to the subject the pharmaceutical
composition of (a), (b), (c) or (d) or the combination of (e) or
(f).
[0067] The present invention also includes a compound of Formula I
or pharmaceutically acceptable salt thereof, (i) for use in, (ii)
for use as a medicament for, or (iii) for use in the preparation of
a medicament for: (a) therapy (e.g., of the human body), (b)
medicine, (c) inhibition of HIV reverse transcriptase, (d)
treatment or prophylaxis of infection by HIV, or (e) treatment,
prophylaxis of, or delay in the onset or progression of AIDS. In
these uses, the compounds of the present invention can optionally
be employed in combination with one or more anti-HIV agents
selected from HIV antiviral agents, anti-infective agents, and
immunomodulators.
[0068] Additional embodiments of the invention include the
pharmaceutical compositions, combinations and methods set forth in
(a)-(n) above and the uses (i)(a)-(e) through (iii)(a)-(e) set
forth in the preceding paragraph, wherein the compound of the
present invention employed therein is a compound of one of the
embodiments described above. In all of these embodiments, the
compound may optionally be used in the form of a prodrug or
pharmaceutically acceptable salt or pharmaceutically acceptable
salt of a prodrug.
[0069] Additional embodiments of the present invention include each
of the pharmaceutical compositions, combinations, methods and uses
set forth in the preceding paragraphs, wherein the compound of the
present invention or its salt employed therein is substantially
pure. With respect to a pharmaceutical composition comprising a
compound of Formula I or its prodrug or salt and a pharmaceutically
acceptable carrier and optionally one or more excipients, it is
understood that the term "substantially pure" is in reference to a
compound of Formula I or its prodrug and/or salt per se.
[0070] Still additional embodiments of the present invention
include the pharmaceutical compositions, combinations and methods
set forth in (a)-(n) above and the uses (i)(a)-(e) through
(iii)(a)-(e) set forth above, wherein the HIV of interest is HIV-1.
Thus, for example, in the pharmaceutical composition (d), the
compound of Formula I is employed in an amount effective against
HIV-1 and the anti-HIV agent is an HIV-1 antiviral selected from
the group consisting of HIV-1 protease inhibitors, HIV-1 reverse
transcriptase inhibitors, HIV-1 integrase inhibitors, HIV-1 fusion
inhibitors and HIV-1 entry inhibitors. The compounds of Formula I
may also be useful agents against HIV-2.
[0071] The term "administration" and variants thereof (e.g.,
"administering" a compound) in reference to a compound of Formula I
means providing the compound to the individual in need of treatment
or prophylaxis and includes both self-administration and
administration to the patient by another person. When a compound or
a prodrug thereof is provided in combination with one or more other
active agents (e.g., antiviral agents useful for treating or
prophylaxis of HIV infection or AIDS), "administration" and its
variants are each understood to include provision of the compound
or prodrug and other agents at the same time or at different times.
When the agents of a combination are administered at the same time,
they can be administered together in a single composition or they
can be administered separately.
[0072] As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients, as well
as any product which results from combining the specified
ingredients. Ingredients suitable for inclusion in a pharmaceutical
composition are "pharmaceutically acceptable" ingredients, which
means the ingredients must be compatible with each other and not
deleterious to the recipient thereof.
[0073] The term "subject" as used herein refers to an animal,
preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment.
[0074] The term "effective amount" as used herein means an amount
sufficient to inhibit HIV reverse transcriptase, inhibit HIV
replication, exert a prophylactic effect, and/or a exert a
therapeutic effect after administration. One embodiment of
"effective amount" is a "therapeutically effective amount" which is
an amount of a compound that is effective for inhibiting HIV
reverse transcriptase, inhibiting HIV replication (either of the
foregoing which may also be referred to herein as an "inhibition
effective amount"), treating HIV infection, treating AIDS, delaying
the onset of AIDS, and/or slowing progression of AIDS in a patient.
Another embodiment of "effective amount" is a "prophylactically
effective amount" which is an amount of the compound that is
effective for prophylaxis of HIV infection or prophylaxis of AIDS
in a patient. It is understood that an effective amount can
simultaneously be both a therapeutically effective amount, e.g.,
for treatment of HIV infection, and a prophylactically effective
amount, e.g., for prevention or reduction of risk for developing
AIDS. When the compound of Formula I is administered as a salt,
reference to an amount of the compound is to the free form (i.e.,
the non-salt form) of the compound.
[0075] In the method of the present invention (i.e., inhibiting HIV
reverse transcriptase, treating or prophylaxis of HIV infection,
inhibiting HIV replication, treating or prophylaxis of AIDS,
delaying the onset of AIDS, or delaying or slowing progression of
AIDS), the compounds of this invention, optionally in the form of a
salt, can be administered by means that produce contact of the
active agent with the agent's site of action. They can be
administered by conventional means available for use in conjunction
with pharmaceuticals, either as individual therapeutic agents or in
a combination of therapeutic agents. They can be administered
alone, but typically are administered with a pharmaceutical carrier
selected on the basis of the chosen route of administration and
standard pharmaceutical practice. The compounds of the invention
can, for example, be administered orally, parenterally (including
subcutaneous injections, intravenous, intramuscular, intrasternal
injection or infusion techniques, or by implantable device), by
inhalation spray, or rectally, in the form of a unit dosage of a
pharmaceutical composition containing an effective amount of the
compound and conventional non-toxic pharmaceutically acceptable
carriers, adjuvants and vehicles, any of which administration
methods can be provided as a single dose, once-daily, or less
frequently such as once weekly or once monthly, twice yearly or
once yearly in, for example but not limited to, the dosage ranges
and amounts described below. Liquid preparations suitable for oral
administration (e.g., suspensions, syrups, elixirs and the like)
can be prepared according to techniques known in the art and can
employ any of the usual media such as water, glycols, oils,
alcohols and the like. Solid preparations suitable for oral
administration (e.g., powders, pills, capsules and tablets) can be
prepared according to techniques known in the art and can employ
such solid excipients as starches, sugars, kaolin, lubricants,
binders, disintegrating agents and the like. Parenteral
compositions can be prepared according to techniques known in the
art and typically employ sterile water as a carrier and optionally
other ingredients, such as a solubility aid. Injectable solutions
can be prepared according to methods known in the art wherein the
carrier comprises a saline solution, a glucose solution or a
solution containing a mixture of saline and glucose. Further
description of methods suitable for use in preparing pharmaceutical
compositions for use in the present invention and of ingredients
suitable for use in said compositions is provided in Remington's
Pharmaceutical Sciences, 18th edition, edited by A. R. Gennaro,
Mack Publishing Co., 1990 and in Remington--The Science and
Practice of Pharmacy, 22nd Edition, published by Pharmaceutical
Press and Philadelphia College of Pharmacy at University of the
Sciences, 2012, ISBN 978 0 85711-062-6 and prior editions.
[0076] Formulations of compounds described by Formula I that result
in drug supersaturation and/or rapid dissolution may be utilized to
facilitate oral drug absorption. Formulation approaches to cause
drug supersaturation and/or rapid dissolution include, but are not
limited to, nanoparticulate systems, amorphous systems, solid
solutions, solid dispersions, and lipid systems. Such formulation
approaches and techniques for preparing them are well known in the
art. For example, solid dispersions can be prepared using
excipients and processes as described in reviews (e.g., A. T. M.
Serajuddin, J Pharm Sci, 88:10, pp. 1058-1066 (1999)).
Nanoparticulate systems based on both attrition and direct
synthesis have also been described in reviews such as Wu et al (F.
Kesisoglou, S. Panmai, Y. Wu, Advanced Drug Delivery Reviews, 59:7
pp. 631-644 (2007)).
[0077] The compounds of Formula I, and pharmaceutically acceptable
salts thereof, can be useful for inhibiting HIV reverse
transcriptase and for inhibiting HIV replication in vitro and in
vivo. More particularly, the compounds of Formula I can be useful
for inhibiting the polymerase function of HIV-1 reverse
transcriptase. The testing of Compound 1 in the assay set forth in
the RT Polymerase Assay below, illustrates the ability of compounds
of the invention to inhibit the RNA-dependent DNA polymerase
activity of HIV-1 reverse transcriptase. The compounds of Formula I
may also be useful agents against HIV-2.
[0078] The compounds of Formula I can be administered in a dosage
range of 0.001 to 1000 mg/kg of mammal (e.g., human) body weight
per day, or at other time intervals as appropriate, in a single
dose or in divided doses. One example of a dosage range is 0.01 to
500 mg/kg body weight per day, or at other time intervals as
appropriate, administered orally or via other routes of
administration in a single dose or in divided doses. Another
example of a dosage range is 0.1 to 100 mg/kg body weight per day,
or at other time intervals as appropriate, administered orally or
via other routes of administration in single or divided doses. For
oral (e.g., tablets or capsules) or other routes of administration,
the compositions can be provided containing 1.0 to 500 milligrams
of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50,
75, 100, 150, 200, 250, 300, 400, and 500 milligrams of the active
ingredient for the symptomatic adjustment of the dosage to the
patient to be treated. The specific dose level and frequency of
dosage for any particular patient may be varied and will depend
upon a variety of factors including the activity of the specific
compound employed, the metabolic stability and length of action of
that compound, the age, body weight, general health, sex, diet,
mode and time of administration, rate of excretion, drug
combination, the severity of the particular condition, and the host
undergoing therapy. In some cases, depending on the potency of the
compound or the individual response, it may be necessary to deviate
upwards or downwards from the given dose. Compounds of the
invention can be administered as a single dose, once-daily, or less
frequently such as once weekly, once monthly, twice yearly or once
yearly in, for example but not limited to, the dosage ranges and
amounts noted above. Furthermore, the compound may be formulated
for immediate or modified release such as extended or controlled
release.
[0079] As noted above, the present invention is also directed to
use of a compound of Formula I with one or more anti-HIV agents. An
"anti-HIV agent" is any agent which is directly or indirectly
effective in the inhibition of HIV, the treatment or prophylaxis of
HIV infection, and/or the treatment, prophylaxis or delay in the
onset or progression of AIDS. It is understood that an anti-HIV
agent is effective in treating, preventing, or delaying the onset
or progression of HIV infection or AIDS and/or diseases or
conditions arising therefrom or associated therewith. For example,
the compounds of this invention may be effectively administered,
whether at periods of pre-exposure and/or post-exposure, in
combination with effective amounts of one or more anti-HIV agents
selected from HIV antiviral agents, imunomodulators,
antiinfectives, or vaccines useful for treating HIV infection or
AIDS. Suitable HIV antivirals for use in combination with the
compounds of the present invention include, for example, those
listed in Table A as follows:
TABLE-US-00001 TABLE A Antiviral Agents for Treating HIV infection
or AIDS Name Type abacavir, ABC, Ziagen .RTM. nRTI abacavir +
lamivudine, Epzicom .RTM. nRTI abacavir + lamivudine + zidovudine,
Trizivir .RTM. nRTI amprenavir, Agenerase .RTM. PI atazanavir,
Reyataz .RTM. PI AZT, zidovudine, azidothymidine, Retrovir .RTM.
nRTI capravirine nnRTI darunavir, Prezista .RTM. PI ddC,
zalcitabine, dideoxycytidine, Hivid .RTM. nRTI ddI, didanosine,
dideoxyinosine, Videx .RTM. nRTI ddI (enteric coated), Videx EC
.RTM. nRTI delavirdine, DLV, Rescriptor .RTM. nnRTI dolutegravir,
Tivicay .RTM. InI doravirine, MK-1439 nnRTI efavirenz, EFV, Sustiva
.RTM., Stocrin .RTM. nnRTI efavirenz + emtricitabine + tenofovir
DF, Atripla .RTM. nnRTI + nRTI EFdA
(4'-ethynyl-2-fluoro-2'-deoxyadenosine) nRTI Elvitegravir InI
emtricitabine, FTC, Emtriva .RTM. nRTI emtricitabine + tenofovir
DF, Truvada .RTM. nRTI emvirine, Coactinon .RTM. nnRTI enfuvirtide,
Fuzeon .RTM. FI enteric coated didanosine, Videx EC .RTM. nRTI
etravirine, TMC-125 nnRTI fosamprenavir calcium, Lexiva .RTM. PI
indinavir, Crixivan .RTM. PI lamivudine, 3TC, Epivir .RTM. nRTI
lamivudine + zidovudine, Combivir .RTM. nRTI lopinavir PI lopinavir
+ ritonavir, Kaletra .RTM. PI maraviroc, Selzentry .RTM. EI
nelfinavir, Viracept .RTM. PI nevirapine, NVP, Viramune .RTM. nnRTI
PPL-100 (also known as PL-462) (Ambrilia) PI raltegravir, MK-0518,
Isentress .TM. InI Rilpivirine nnRTI ritonavir, Norvir .RTM. PI
saquinavir, Invirase .RTM., Fortovase .RTM. PI stavudine, d4T,
didehydrodeoxythymidine, Zerit .RTM. nRTI tenofovir DF (DF =
disoproxil fumarate), TDF, Viread .RTM. nRTI Tenofovir,
hexadecyloxypropyl (CMX-157) nRTI Tenofovir alafenamide fumarate
(GS-7340) nRTI tipranavir, Aptivus .RTM. PI vicriviroc EI EI =
entry inhibitor; FI = fusion inhibitor; InI = integrase inhibitor;
PI = protease inhibitor; nRTI = nucleoside reverse transcriptase
inhibitor; nnRTI = non-nucleoside reverse transcriptase inhibitor.
Some of the drugs listed in the table are used in a salt form;
e.g., abacavir sulfate, delavirdine mesylate, indinavir sulfate,
atazanavir sulfate, nelfinavir mesylate, saquinavir mesylate.
[0080] It is understood that the scope of combinations of the
compounds of this invention with anti-HIV agents is not limited to
the HIV antivirals listed in Table A, but includes in principle any
combination with any pharmaceutical composition useful for the
treatment or prophylaxis of AIDS. The HIV antiviral agents and
other agents will typically be employed in these combinations in
their conventional dosage ranges and regimens as reported in the
art, including, for example, the dosages described in the
Physicians' Desk Reference, Thomson PDR, Thomson PDR, 57th edition
(2003), the 58th edition (2004), or the 59th edition (2005) and the
current Physicians' Desk Reference (68th ed.). (2014), Montvale,
N.J.: PDR Network. The dosage ranges for a compound of the
invention in these combinations can be the same as those set forth
above.
[0081] The compounds of this invention are also useful in the
preparation and execution of screening assays for antiviral
compounds. For example, the compounds of this invention are useful
for isolating enzyme mutants, which are excellent screening tools
for more powerful antiviral compounds. Furthermore, the compounds
of this invention are useful in establishing or determining the
binding site of other antivirals to HIV reverse transcriptase,
e.g., by competitive inhibition.
[0082] In another aspect, the present invention provides a process
for preparation of the compound of Formula (IA).
##STR00012##
wherein X is H, F, Cl, or Br;
Y is N, C(H), C(F), C(Cl), C(Br), or C(CH.sub.3); and
[0083] cZ is NH2. Thus, in embodiment in no. 1, the present
invention provides a process comprising:
[0084] (a.) coupling sugar
##STR00013##
with nucleobase H
##STR00014##
to provide protected nucleoside
##STR00015##
and
[0085] (b.) converting the protected nucleoside (A) to the compound
of Formula (IA); wherein
Z.sup.1 is Cl, N(H)PG, or N(PG).sub.2,
R.sup.d is
[0086] (i) a group of the formula
##STR00016##
[0086] wherein R.sup.p is C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3
alkoxy, halo, CF.sub.3, or N(CH.sub.3).sub.2; [0087] (ii)
--Si(R.sup.s).sub.3; or [0088] (iii) --C(O)C.sub.1-C.sub.3 alkyl;
or [0089] (iv) --C(O)CH.sub.2N(H)C(O)CH.sub.3; each R.sup.s is
independently C.sub.1-C.sub.6 alkyl; unsubstituted phenyl; phenyl
substituted by one to three C.sub.1-C.sub.3 alkyl, halo, or
C.sub.1-C.sub.3 alkoxy; LG is OAc, OBz, halo, or
--O--C.sub.2-C.sub.8 alkenyl; and PG is an amino protecting
group.
[0090] The amino protecting group can be a carbamate-based
protecting group such as --C(O)OR.sup.s (e.g., Boc, CBz); a silyl
protecting group such as --Si(R.sup.s).sub.3, (e.g.,
(--Si(CH.sub.3).sub.3, --Si(CH.sub.3).sub.2(C(CH.sub.3).sub.3)); or
an amide protecting group such as --C(O)R.sup.s (e.g.,
--C(O)CH.sub.3, --C(O)Ph).
[0091] In embodiment no. 2, the present invention provides a
process as set forth in embodiment no. 1, wherein R.sup.d is
4-methylbenzoyl (Tol):
##STR00017##
[0092] In embodiment no. 3, the present invention provides a
process as set forth in embodiment no. 2, wherein in the compound
of Formula (IA), X is F, Y is N, and Z is NH.sub.2.
[0093] In embodiment no. 4, the present invention provides a
process as set forth in embodiment no. 3, wherein in step (a.),
[0094] in the sugar (C), LG is --OAc or
--O(CH.sub.2).sub.3C(H).dbd.CH.sub.2; and
[0095] in the protected nucleoside (A), X is F, Y is N, and Z.sup.1
is N(H)PG.
[0096] In embodiment no. 5, the present invention provides a
process as set forth in embodiment no. 4, wherein LG is --OAc in
the sugar (C); and
Z.sup.1 is N(H)Si(R.sup.s).sub.3 in the protected nucleoside.
[0097] In embodiment no. 6, the present invention provides a
process as set forth in embodiment no. 5, wherein in step (a) said
coupling is conducted in the presence of a Lewis or Bronsted acid
in an aprotic solvent to provide nucleoside (A). Suitable Lewis
acids for the coupling include TMSOTf, TiCl.sub.4, and SnCl.sub.4.
Suitable Brcnsted acids include, for example,
trifluoromethanesulfonic acid.
[0098] In embodiment no. 7, the present invention provides a
process as set forth in embodiment no. 6, wherein the Lewis or
Bronsted acid is TMSOTf.
[0099] In embodiment no. 8, the present invention provides a
process as set forth in embodiment no. 6 or 7, wherein the aprotic
solvent is acetonitrile.
[0100] In embodiment no. 9, the present invention provides a
process as set forth in any one of embodiment nos. 6-8, wherein the
coupling is conducted at a temperature from 60 to 85.degree. C.
[0101] In embodiment no. 10, the present invention provides a
process as set forth in embodiment no. 9, wherein the volume ratio
of the aprotic solvent to sugar (C) is at least 10. For instance,
volume ratio of the aprotic solvent to sugar (C) is 20 to 40.
[0102] In embodiment no. 11, the present invention provides a
process as set forth in any one of embodiment nos. 6-10, wherein
step (a.) further comprises isolating the protected nucleoside (A)
by crystallizing the protected nucleoside (A) from a mixture
containing the aprotic solvent and the nucleobase (B); and
separating the protected nucleoside (A) from the mixture.
[0103] In embodiment no. 12, the present invention provides a
process as set forth in any one of embodiment nos. 4-11, wherein
Z.sup.1 is --N(H)Si(CH.sub.3).sub.3.
[0104] In embodiment no. 13, the present invention provides a
process as set forth in embodiment no. 12, wherein the nucleobase
(B) is prepared by reacting N,O-bis(trimethylsilyl)acetamide with
2-fluoroadenine.
[0105] In embodiment no. 14, the present invention provides a
process as set forth in any one of embodiment nos. 5-13, wherein
converting in step (b) comprises reacting the protected nucleoside
(A) with an alkali metal C.sub.1-C.sub.3 alkoxide.
[0106] In embodiment no. 15, the present invention provides a
process as set forth in embodiment no. 14, wherein the alkali metal
C.sub.1-C.sub.3 alkoxide is sodium methoxide. In specific
embodiments, the conversion is conducted with 3 to 10 mol. % NaOMe
to the protected nucleoside (A).
[0107] In embodiment no. 16, the present invention provides a
process as set forth in any one of embodiment nos. 5-15, wherein
the sugar (C) is prepared by reacting lactol
##STR00018##
with acetic anhydride.
[0108] In embodiment no. 17, the present invention provides a
process as set forth in embodiment no. 4, wherein
[0109] LG is --O(CH.sub.2).sub.3C(H).dbd.CH.sub.2 in the sugar (C);
and
[0110] Z.sup.1 is N(H)C(O)--OR.sup.s and X is F or Cl in the
nucleobase (B) and in the protected nucleoside (A). In specific
embodiments, Z.sup.1 is N(H)C(O)--OR.sup.s and X is F in the
nucleobase (B) and in the protected nucleoside (A).
[0111] In embodiment no. 18, the present invention provides a
process as set forth in embodiment no. 17, wherein in step (a) said
coupling is conducted by treating sugar (C) with a activating agent
in the presence of nucleobase (B) in an aprotic solvent. Typically,
the activating agent is iodine, bromine, N-iodosuccinimide,
N-bromosuccinimide, Balarenga's reagent (Py.sub.2I), or
diiodo-dimethylhydantoin. For instance in one embodiment, the
activating agent is iodine.
[0112] In embodiment no. 19, the present invention provides a
process as set forth in embodiment no. 18, wherein the aprotic
solvent is acetonitrile, propionitrile, ethyl acetate,
dichloromethane, THF, toluene, 1,4-dioxane, an acetonitrile-THF
mixture, an acetonitrile-dichloromethane mixture, an
acetonitrile-toluene mixture or an acetonitrile-N-methylpyrrolidone
mixture.
[0113] In embodiment no. 20, the present invention provides a
process as set forth in embodiment no. 18 or 19, wherein the
coupling is conducted at a temperature from -78 to 45.degree. C.
For instance, the coupling can be conducted at -40 to 10.degree.
C.
[0114] In embodiment no. 21, the present invention provides a
process as set forth in any one of embodiment nos. 17-20, wherein
Z.sup.1 is N(H)C(O)--OC(CH.sub.3).sub.3.
[0115] In embodiment no. 22, the present invention provides a
process as set forth in any one of embodiment nos. 17-21, wherein
converting in step (b) comprises treating the protected nucleoside
(A) with an alkali metal C.sub.1-C.sub.3 alkoxide (e.g., sodium
methoxide) and with a strong acid, in separate steps. The strong
acid can be, for instance, trifluoroacetic acid or a mineral acid.
The term "mineral acid" refers to a common inorganic acid, such as
hydrochloric acid or sulphuric acid. In some embodiments, the
protected nucleoside (A) is treated with the strong acid first and
then with the alkali metal C.sub.1-C.sub.3 alkoxide. In other
embodiments, the protected nucleoside (A) is treated first with the
alkali metal C.sub.1-C.sub.3 alkoxide, and then with the strong
acid.
[0116] In embodiment no. 23, the present invention provides a
process as set forth in any one of embodiment nos. 17-22, wherein
the sugar (C) is prepared by
[0117] reacting lactol
##STR00019##
with pent-4-en-1-ol to provide the sugar
##STR00020##
wherein LG is --O--(CH.sub.2).sub.3CH.dbd.CH.sub.3 (herein referred
as sugar (C3).
[0118] In embodiment no. 24, the present invention provides a
process as set forth in embodiment no. 23, wherein the reacting
further comprises reacting lactol (10A) with acetic anhydride and
pent-4-en-1-ol to provide the sugar (C).
[0119] In embodiment no. 25, the present invention provides a
process as set forth in embodiment no. 2, wherein in the compound
of Formula (IA), X is Cl, Y is C(F) or C(H), and Z is NH.sub.2.
[0120] In embodiment no. 26, the present invention provides a
process as set forth in embodiment no. 25, wherein in step
(a.),
[0121] in the sugar (C), LG is --Cl (referred to as sugar (C2));
and
[0122] X is Cl, Y is C(F), and Z is Cl in the nucleobase (B) and in
the protected nucleoside (A).
[0123] In embodiment no. 27, the present invention provides a
process as set forth in embodiment no. 25, wherein in step (a.)
said coupling is conducted by
[0124] treating nucleobase (B) with an alkali metal base in an
aprotic solvent to provide a alkali metal salt of the nucleobase
(B); and
[0125] reacting the alkali metal salt of the nucleobase (B) with
sugar (C) to provide the protected nucleoside (A).
[0126] Suitable alkali metal bases for treatment of the nucleobase
(B) include, for example, NaHMDS, NaH, KHMDS, and LDA. In
embodiment no. 28, the alkali metal base is NaHMDS.
[0127] In embodiment no. 29, the present invention provides a
process as set forth in any one of embodiment nos. 25-28, wherein
converting in step (b) comprises reacting the protected nucleoside
(A) with ammonia. For example, the conversion can be conducted in a
solution of ammonia in a C.sub.1-C.sub.3 alcohol, such as
methanol.
[0128] In embodiment no. 30, the present invention provides a
process as set forth in any one of embodiment nos. 26-29, wherein
the sugar (C2) is prepared by reacting lactol
##STR00021##
with acetic anhydride to provide the sugar
##STR00022##
and reacting the sugar (C1) with hydrochloric acid to provide the
sugar (C2)
[0129] In embodiment no. 31, the present invention provides a
process as set forth in any one of embodiment nos. 2-30, wherein
the sugar (C) is prepared by
[0130] reducing lactone
##STR00023##
with a selective reducing agent to provide lactol
##STR00024##
and converting lactol (10A) to the sugar (C).
[0131] In embodiment no. 32, the present invention provides a
process as set forth in embodiment no. 31, wherein the selective
reducing agent is sodium bis(2-methoxyethoxy)aluminum hydride.
[0132] In embodiment no. 33, the present invention provides a
process as set forth in embodiment no. 31, wherein the lactone (10)
is prepared by:
[0133] reacting dioxolane
##STR00025##
with an acid to provide a deprotected intermediate; and
[0134] acylating the deprotected intermediate with a
p-methylbenzoylating agent to provide the lactone (10).
[0135] The acylation of the deprotected intermediate described in
embodiment no. 33 can be performed using a number of methods known
to those skilled in the art of organic chemistry, for instance, by
using p-methylbenzoylating agents such as anhydrides of
p-methylbenzoic acid or p-methylbenzoyl chloride. In embodiment no.
34, the p-methylbenzoylating agent is p-methylbenzoyl chloride.
[0136] In embodiment no. 35, the present invention provides a
process as set forth in embodiment no. 33, wherein the process
further comprises isolating lactone (10) by crystallizing lactone
(10). Suitable solvents for crystallizing and isolating lactone
(10) include a mixture of pyridine:water and a mixture of isopropyl
acetate:heptane.
[0137] In embodiment no. 36, the present invention provides a
process as set forth in embodiment no. 33, wherein the deprotected
intermediate is
##STR00026##
[0138] In embodiment no. 37, the present invention provides a
process as set forth in any one of embodiment nos. 33-36, wherein
the dioxolane (9) is prepared by
[0139] reacting TIPS intermediate
##STR00027##
with a fluoride agent to provide the dioxolane (9). Suitable
fluoride agents include, for example, tetraalkylammonium fluoride,
potassium fluoride, and hydrofluoric acid. In embodiment no. 38,
the fluoride agent is tetrabutylammonium fluoride.
[0140] In embodiment no. 39, the present invention provides a
process as set forth in embodiment no. 37, wherein the TIPS
intermediate (8) is prepared by reducing ketone ester
##STR00028##
[0141] In embodiment no. 40, the reduction set forth in embodiment
no. 39 is performed by asymmetric transfer hydrogenation of the
ketone ester (7) with formic acid/triethylamine in the presence of
a chiral catalyst. Suitable chiral catalysts for the hydrogenation
include, for example, ruthenium-based catalysts containing chiral
ligands, such as DENEB.TM., available from Takasago International
Corporation, Tokyo, Japan. In embodiment no. 41, the chiral
catalyst is RuCl--(S,S)-Ts-DENEB.TM..
[0142] In embodiment no. 42, the present invention provides a
process as set forth in any one of embodiment nos. 39-41, wherein
the ketone ester (7) is prepared by converting alcohol
##STR00029##
to the ketone ester (7).
[0143] In embodiment no. 43, the present invention provides a
process as set forth in embodiment no. 42, wherein the conversion
of alcohol (4) to ketone ester (7) comprises:
[0144] (i.) oxidizing alcohol (4) to provide carboxylic acid
##STR00030##
[0145] (ii.) esterifying the carboxylic acid (5) provide methyl
ester
##STR00031##
[0146] (iii.) reacting an alkali metal enolate of tert-butyl
acetate with methyl ester (6) to provide ketone ester (7).
[0147] Suitable oxidation conditions to oxidize alcohol (4) and
provide carboxylic acid (5) include a two-step oxidation process or
a direct oxidation process. Two-step oxidation processes include
oxidation of the alcohol moiety to an aldehyde, such as by using
Dess-Martin periodinane or Parikh-Doering oxidation (DMSO, SO.sub.3
pyr, triethylamine), followed by oxidation of the aldehyde to the
carboxylic acid, using for example, Pinnick oxidation (NaClO.sub.2,
tert-butanol, NaH.sub.2PO.sub.4). Direct oxidation is a one-step
oxidation process of the alcohol (4) to the carboxylic acid (5).
Typically this one-step oxidation process includes treatment of
alcohol (4) with 2,2,6,6-tetrmethylpiperidin-1-oxyl (TEMPO), NaOCl,
NaOCl.sub.2 at a pH of about 4.
[0148] The esterification of the carboxylic acid (5) to provide the
methyl ester (6) can be performed in a number of ways. The
carboxylic acid (5) can be treated with a base which is suitable
for forming the carboxylate anion (e.g., DBU), and a methylating
agent such as methyl iodide or dimethylsulfate. Alternatively, the
the carboxylic acid (5) can be activated with a carbodiimide agent
such as N,N'-carbonyldiimidazole, and then quenched with methanol
to provide the methyl ester (6).
[0149] Formation of the alkali metal enolate of tert-butyl acetate
can be conducted with with an alkali metal base such as LDA or
NaHMDS. The enolate the alkali metal enolate is reacted with the
methyl ester (6) to form the ketone ester (7).
[0150] In some embodiments, further improvement of the purity of
the acid (5) may be desired. Accordingly, in embodiment no. 44, the
present invention provides a process as set forth in embodiment no.
43, wherein the process further comprises:
[0151] (i.) treating carboxylic acid (5) with an amine to form a
salt of (5) with the amine
##STR00032##
[0152] (ii.) isolating the salt (amine-5); and
[0153] (iii.) reacting the salt (amine-5) with an acid (e.g.,
citric acid) to provide purified carboxylic acid (5).
[0154] In some embodiments, the amines used to treat carboxylic
acid (5) can be an achiral amine such as tert-butylamine. In other
embodiments, the acid (5) is treated with a chiral amine such as
phenylmethylamine, phenylethylamine, 1-amino-2-indanol,
1-(1-napthyl)ethylamine, 1-(2-napthyl)ethylamine, cinchonine, or
norephredine. In embodiment no. 45, the carboxylic acid (5) is
treated with (1R,2S)-(+)-cis-1-amino-2-indanol.
[0155] In embodiment no. 46, the present invention provides a
process as set forth in any one of embodiment nos. 43-45, wherein
the alcohol (4) is prepared by:
[0156] (i.) reacting diol
##STR00033##
with an acetonide-forming agent and an acid to form acetonide
##STR00034##
and
[0157] (ii.) treating acetonide (3a) with an alkali metal
C.sub.1-C.sub.3 alkoxide to form the alcohol (4).
[0158] In embodiment no. 47, the acetonide-forming reacted with
diol (3) in embodiment no. 46 is 2,2-dimethoxypropane,
2-methoxypropene or acetone. In embodiment no. 48, the
acetonide-forming agent is 2,2-dimethoxypropane.
[0159] In embodiment no. 49, the alkali metal C.sub.1-C.sub.3
alkoxide used to form the alcohol (4) in embodiment no. 46, 47 or
48 is sodium methoxide.
[0160] In embodiment no. 50, the present invention provides a
process as set forth in any one of embodiment nos. 46-49, wherein
the diol (3) is prepared by contacting diacetoxy alcohol
##STR00035##
with a lipase.
[0161] In embodiment no. 51, the lipase contacted with diacetoxy
alcohol (I-2) is Candida antarctica lipase A.
[0162] In embodiment no. 52, the present invention provides a
process as set forth in any one of embodiment nos. 50 and 51,
wherein the diacetoxy alcohol (I-2) is prepared by adding lithiated
alkyne adduct
##STR00036##
to diacetoxyacetone
##STR00037##
[0163] In another aspect, the present invention provides processes
for the preparation of certain synthetic intermediates useful in
the preparation of the compound of the Formula (IA). Thus, in
embodiment no. 53, the present invention provides a process for the
preparation of the lactone
##STR00038##
using the conditions set forth in any one of embodiment nos. 33-52.
In embodiment no. 54 the present invention provides a process for
the preparation of the lactone
##STR00039##
using the conditions set forth in any one of embodiment nos. 33 and
36-52.
[0164] In embodiment no. 55, the present invention provides a
process for preparation of the protected nucleoside
##STR00040##
wherein X is H, F, Cl or Br, using the conditions set forth in any
one of embodiment nos. 5-13, 16 and 31-52. In a class of this
process embodiment, the protected nucleoside (A2aa) is
##STR00041##
[0165] In embodiment no. 56, the present invention provides a
process for preparation of the protected nucleoside
##STR00042##
wherein X is H, F, Cl or Br, using the conditions set forth in any
one of embodiment nos. 17-24 and 31-52. In a class of this process
embodiment, the protected nucleoside (A2bb) is
##STR00043##
[0166] In embodiment no. 57, the present invention provides a
process for the preparation of the protected nucleoside
##STR00044##
wherein X is H, F, Cl, or Br and R.sup.S7 is H, F, Cl, Br, or
CH.sub.3 using the conditions set forth in any one of embodiment
nos. 25-52. In a particular class of this process embodiment, X is
Cl and R.sup.S7 is F. In another particular class of this process
embodiment, X is Cl and R.sup.S7 is H.
[0167] In another aspect, the presention invention provides
synthetic intermediates useful in the preparation of the compound
of Formula (IA). Thus, in embodiment no. 58, the present invention
provides the protected nucleoside (A2aa), (A2bb), (A2a), (A2b), or
(A3a).
[0168] In embodiment no. 59, the present invention provides the
protected nucleoside (A2aa).
[0169] In embodiment no. 60, the present invention provides the
protected nucleoside (A2b).
[0170] In embodiment no. 61, the present invention provides the
protected nucleoside (A3a) wherein X is H, F, Cl, or Br and
R.sup.S7 is H, F, Cl, Br, or CH.sub.3. In a class of this
embodiment, X is Cl and R.sup.S7 is F in the protected nucleoside
(A3a). In another class of this embodiment, In a class of this
embodiment, X is Cl and R.sup.S7 is H.
[0171] In embodiment no. 62, the present invention provides the
lactone
##STR00045##
[0172] In embodiment no. 63, the present invention provides the
lactone
##STR00046##
[0173] In embodiment no. 64, the present invention provides the
sugar
##STR00047##
[0174] In embodiment no. 65, the present invention provides the
sugar
##STR00048##
[0175] Abbreviations and acronyms employed herein include the
following:
TABLE-US-00002 ACN = acetonitrile NHS = normal human serum AcOH =
acetic acid; Ac = acyl; NovoCor AD L = Lipase from Candida sp.
Ac.sub.2O = acetic anhydride recombinant, expressed in Aspergillus
oryzae ADA = adenosine deaminase NMR = nuclear magnetic resonance
Boc = tert-butyloxycarbonyl QTof = Quadrupole time of flight BTMSA
= bis-trimethylsilylacetamide NTP = nucleoside triphosphate DMF =
N,N-dimethylformamide dNTP = 2'-deoxy nucleoside triphosphate BzCl
= benzoyl chloride pet. ether = petroleum ether DCM =
dichloromethane Ppm, ppm = parts per million DME =
1,2-dimethoxyethane PPTS = 4-toluenesulfonic acid DMSO =
dimethylsulfoxide r.t. = room temperature EtOAc = ethyl acetate RT
= reverse transcriptase Ecosorb C-941 = activated carbon TBAF =
tetrabutylammonium fluoride EGTA = ethylene glycol tetraacetic acid
TEAB = triethylammonium bromide Et.sub.3N = triethylamine TEMPO =
2,2,6,6-tetramethyl- L = liter 1-piperidinyloxy LDA = lithium
diisopropylamide THF = tetrahydrofuran Me = methyl, Et = Ethyl, Pr
= propyl, TIPS = triisopropylsilyl Bu = Butyl, Bz = benzoyl TFA =
trifluoroacetic acid MeOH = methanol TLC = thin layer
chromatography mL or ml = milliliter TMS = trimethysilyl mol =
moles; M = molar TMSOTf = trimethylsilyl mmol = millimoles
trifluoromethanesulfonate MTBE = methyl tert-butyl ether Tol =
p-methylbenzoyl NaHMDS = sodium hexamethyldisilazide TS-DENEB =
N-[(1S,2S)-1,2-Diphenyl-2-(2- KHMDS--potassium hexamethyldisilazide
(4-methylbenzyloxy)ethylamino)-ethyl]-4- CBz = benzyloxy carbonyl
methylbenzene sulfonamide(chloro)ruthenium(II) DBU =
1,8-Diazabicyclo[5.4.0]undec-7-ene
[0176] Compounds of the invention can be prepared by methods well
known in the art of organic chemistry. See, for example, J. March,
`Advanced Organic Chemistry` 6th Edition, John Wiley and Sons.
During synthetic sequences it may be necessary and/or desirable to
protect sensitive or reactive groups on any of the molecules
concerned. This is achieved by means of conventional protecting
groups, such as those described in T. W. Greene and P. G. M. Wutts
`Protective Groups in Organic Synthesis` 4th Edition, John Wiley
and Sons. The protective groups are optionally removed at a
convenient subsequent stage using methods well known in the
art.
[0177] The compounds of Formula I can be readily prepared according
to the following reaction schemes and examples, or modifications
thereof, using readily available starting materials and reagents.
In these reactions, it is also possible to make use of variants
which are themselves known to those of ordinary skill in this art,
but are not mentioned in greater detail. Furthermore, other methods
for preparing compounds of the invention will be readily apparent
to the person of ordinary skill in the art in light of the
following reaction schemes and examples. Unless otherwise
indicated, all variables are as defined above.
##STR00049##
[0178] Compound 1 can be selectively reacted at the 5'-OH when
treated with chlorides of type R.sup.1--Cl in the presence of a
base such as triethylamine to produce compounds of type 1-2.
##STR00050##
[0179] Compound 1 can be selectively protected with a 5'-OH
protecting group (PG) to provide intermediate 2-2. Reaction of 2-2
with chlorides of type R.sup.2--Cl in the presence of a base such
as triethylamine to produce intermediate 2-3. Removal of the
protecting group at the 5'-OH can provide compounds of type
2-4.
##STR00051##
[0180] Compound 1 can be treated with chlorides of type R.sup.1--Cl
in the presence of a base such as triethylamine to produce
intermediate 3-2. Intermediate 3-2 can be treated with chlorides of
type R.sup.2--Cl in presence of a base such as triethylamine to
produce compounds of type 3-3.
[0181] Scheme 4 shows one embodiment of the process of the present
invention, wherein the lactol 10A is prepared. As compared to the
published procedures which disclose preparation of
4-ethynyl-2-deoxysugar coupling partner suitable for preparing
EFdA, such as M. Kageyama et al., Biosci. Biotechnol. Biochem.,
76(6) 1219-1225 (2012), the process depicted in Scheme 4 utilizes a
more readily available starting material, and is able to be
performed at larger scales without chromatography.
##STR00052## ##STR00053##
[0182] As shown in Scheme 4, dihydroxyacetone is acetylated with
acetic anhydride to provide diacetoxyacetone (I-1). A lithium
adduct of ethynyltriisopropylsilane is added to diacetoxy acetone
(I-1) to yield the diacetoxy alcohol (1-2). Treatment of the
diacetoxy alcohol with a lipase (such as Candida antartica lipase
A) provides enantiomerically enriched diol (3). The enzymatic
hydrolysis selectively yields the R-diol with a percent
enantiomeric excess (% ee) of greater than 90%, such as 92-96%
ee.
[0183] Conversion of diol (3) to the the alcohol (4) occurs by
first treating diol (3) with an acetonide forming agent such as
2,2-dimethoxypropane, acetone, or 2-methoxypropene and an acid such
as pyridinium p-toluenesulfonate; followed by treating the
intermediate acetonide with an alkali metal C.sub.1-C.sub.3
alkoxide such as sodium methoxide to remove the acetate group.
Those of skill in the art will recognize that alternative ketal
forming agents, such as diethyl ketal, cyclopentyl ketal and
cyclohexyl ketal can be used in place of acetonide forming agents,
to form similarly ketal-protected synthetic intermediates.
[0184] Formation of the ketone ester (7) from alcohol (4) occurs by
oxidation of the alcohol (4) with an oxidation agent followed by
esterification of the resultant carboxylic acid (5); and then
condensation of the ester with an alkali metal enolate of
tert-butyl acetate. In one embodiment, the oxidation conditions
used include the reagents 2,2,6,6-tetramethylpiperidin-1-yl)oxyl
(TEMPO), NaOCl, NaOCl.sub.2 with a buffer system having a pH of
about 4. Other suitable oxidizing systems include Dess-Martin
periodinane, (diacetoxy)iodobenzene (DAIB), and Parikh-Doering
oxidation (DMSO, SO.sub.3 pyridine, triethylamine). The carboxylic
acid (5) is esterified to prepare the methyl ester (6) by using
dimethylsulfate and a base, or by using methanol and a
carbodiimide-coupling agent such as N,N'-carbonyldiimidazole.
[0185] Optionally, the purity of the carboxylic acid (5) can be
improved by forming a salt of the carboxylic acid with an amine,
such as a chiral amine, isolation of the amine salt, followed by
treatment with an acid to break the salt.
[0186] The ketone ester (7) is reduced, such as under asymmetric
transfer hydrogenation conditions, to provide the TIPS intermediate
(8). Suitable chiral catalysts include ruthenium-based catalysts
with chiral ligands such as DENEB.TM. from Takasago International
Corporation, Tokyo, Japan with a reducing agent such as formic
acid/triethylamine. The triisopropylsilane group is removed from
the TIPS intermediate (8) by treatment with a fluoride agent, such
as potassium fluoride or tetrabutylammonium fluoride, to provide
the dioxolane (9).
[0187] Deprotection and lactonization under acidic conditions
(e.g., HCl) provides the lactone (10D) which can be isolated as a
solid. The lactone (10D) is acylated with p-methylbenzoylating
agent (p-methyl-Ph-C(O)X.sup.1 in Scheme 4, wherein X.sup.1 is a
leaving group) such as p-methylbenzoyl chloride to provide lactone
(10). The lactone (10) is reduced with a selective reducing agent,
such as sodium bis(2-methoxyethoxy)aluminum hydride, to provide the
lactol (10A).
##STR00054##
[0188] Scheme 5 shows embodiments of the process, wherein the
lactol is converted to the coupling precursors (C1), (C2), and
(C3). Sugar (C1) is prepared by reacting lactol 10A with acetic
anhydride. Sugar (C2) is prepared by treatment of sugar (C1) with
hydrochloric acid. Sugar (C3) is prepared by treatment of either
lactol (10A) or sugar (C1).
[0189] Scheme 6 shows one embodiment of the process for preparing
the compounds of Formula (IA), wherein X, Y, Z, Z.sup.1 and LG are
as set forth above.
##STR00055##
[0190] As shown in Scheme 6, sugar (C) is coupled with nucleobase
(B) to provide the protected nucleoside (A). Protected nucleoside
(A) is converted to the compound of Formula (IA).
[0191] Scheme 7 shows one embodiment of a process for preparing
EFdA (wherein in the compound of Formula IA, X is F, Y is N, and Z
is NH.sub.2).
##STR00056##
[0192] The amino group of 2-fluoroadenine is protected with a
protecting group. In specific embodiments, the amino protecting
group is a trimethylsilyl group. The reaction mixture containing
the nucleobase is combined with the sugar (C1) to provide the
protected nucleoside (A1) in the presence of a Lewis acid.
Conversion of the protected nucleoside (A1) to yield EFdA can be
effected by treatment with an alkali metal C.sub.1-C.sub.3 alkoxide
which (e.g., NaOCH.sub.3) cleaves both the p-toluoyl and the TMS
groups. In alternative embodiments, the conversion can be affected
by treatment with NH.sub.3 in a C.sub.1-C.sub.3 alcohol, such as
methanol.
[0193] In specific aspects of this embodiment, 2-fluoroadenine is
treated with an excess of BTMSA and a Lewis acid such as TMS-OTf.
The reaction mixture containing the nucleobase is combined with the
sugar (C1) to provide the protected nucleoside (A1) wherein the
amino group is protected with a TMS group, such that Z.sup.1 is
--N(H)Si(CH.sub.3).sub.3. Isolation of the protected nucleoside
(A1) containing the TMS-protected amino group is advantageous
because the desired .beta.-anomer selectively crystallizes from the
reaction mixture after concentration of the reaction mixture and
cooling. In some embodiments, the isolated protected nucleoside
(A1) shows over a 99:1 ratio favoring the desired .beta.-anomer.
This feature obviates the need for column chromatography
purification, and thus reduces the environmental- and cost-burdens
associated with using large volumes of organic solvent for eluting
the column.
[0194] Scheme 8 shows another embodiment of a process for preparing
EFdA, wherein R.sup.s is as set forth above.
##STR00057##
[0195] As shown in Scheme 8, sugar (C3) is activated with an
activating agent, such as iodine, and then coupled with a
carbamate-protected 2-fluoradenine to provide the protected
nucleoside (A2). Conversion of the protected nucleoside to EFdA is
effected by removal of the carbamate and p-toluoyl protecting
groups.
[0196] In specific aspects of this embodiment, the amino group in
the nucleobase and the protected nucleoside is protected with a Boc
group. Treatment of the Boc-protected nucleoside (A2) with an
alkali metal C.sub.1-C.sub.3 alkoxide (e.g., NaOCH.sub.3) cleaves
the p-toluoyl groups on the sugar moiety, and treatment with a
strong acid (e.g., TFA) removes the carbamate protecting group to
provide EFdA. In some embodiments, the Boc-protected nucleoside
(A2) is treated with the strong acid before treatment with an
alkali metal C.sub.1-C.sub.3 alkoxide. In other embodiments,
treatment with the alkali metal C.sub.1-C.sub.3 alkoxide occurs
before treatment with the strong acid.
[0197] Scheme 9 shows a general method suitable for preparing
4'-ethynyl-2'-deoxyribonucleosides having substituted 7-deaza
adenine moieties, wherein R.sup.S7 is H, F, Cl, or Br.
##STR00058##
[0198] As shown in Scheme 9, protected nucleoside (A3) is formed
from the coupling of sugar (C2) to sodium salt of nucleobase (B2).
Treatment of protected nucleoside (A3) with ammonia, such as a
solution of ammonia in a C.sub.1-C.sub.3 alcohol) results in the
compound of Formula (IA-2).
[0199] General Chemical Procedures: All reagents were either
purchased from common commercial sources or synthesized according
to literature procedures beginning from commercial reagents.
Commercial reagents were used without further purification. Unless
otherwise indicated, percent is percent by weight given the
component and the total weight of the composition, temperature is
in .degree. C. or is at ambient temperature and pressure is at or
near atmospheric. 1H NMR spectra were obtained on a Varian VNMR
System 400 (400 MHz) and are reported as ppm downfield from
Me.sub.4Si with number of protons, multiplicities, and coupling
constants in Hertz indicated parenthetically. Where LC/MS data are
presented, analyses were performed using an Agilent 6110A MSD or an
Applied Biosystems API-100 mass spectrometer. The parent ion is
given. Preparative HPLC was performed on a Waters preparative HPLC
system fitted with a Waters Xselect.C18 column, typically using
gradient elution with water/acetonitrile containing 0.075%
trifluoro acetic acid. Flash column chromatography was performed
using pre-packed normal phase silica from Biotage, Inc. or bulk
silica from Fisher Scientific. Unless otherwise indicated, column
chromatography was performed using a gradient elution of petroleum
ether/ethyl acetate, from petroleum ether 100% to 100% ethyl
acetate. The term "room temperature" in the examples refers to the
ambient temperature which was typically in the range of about
20.degree. C. to about 26.degree. C.
Example 1
Synthesis of
(2R,3S,5R)-5-(4-amino-2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)--
2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (1)
##STR00059## ##STR00060##
[0200] Step 1: Synthesis of
2,4-dichloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine
[0201] 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (1 g, 5.32 mmol)
was massed into a 250 mL round-bottom flask and dried over
P.sub.2O.sub.5 under vacuum overnight. To this were injected
acetonitrile (60 mL) and acetic acid (12 mL) at room temperature,
followed by the addition of
1-(chloromethyl)-4-fluoro-1,4-diazabicyclo[2.2.2]octane-1,4-diium
tetrafluoroborate (2.64 g, 7.45 mmol) under argon atmosphere. The
mixture was heated to 70.degree. C. and stirred for 36 hours. The
resulting mixture was cooled to 25.degree. C., diluted with DCM
(150 mL), washed with water (2.times.50 mL) and brine (2.times.50
mL) successively. The organic layer was collected, dried over
anhydrous Na.sub.2SO.sub.4, filtered and the filtrate was
concentrated under reduced pressure. silica gel column using ethyl
acetate/petroleum ether (0% to 20% EtOAc in Pet. ether) to give a
crude solid, which was further purified by C18 gel column
reverse-phase-chromatography with the following conditions: Column,
60 A, 120 g; mobile phase, water (with 0.05% TFA) and acetonitrile
(5% acetonitrile up to 40% in 15 min, 40% up to 47% in 5 min, hold
47% 5 min, up to 95% in 3 min, down to 5% in 5 min); Detector, UV
254 nm. The product-containing fractions were collected, extracted
with EtOAc (2.times.50 mL). The organic layer was collected, washed
with brine (2.times.30 mL), dried over anhydrous Na.sub.2SO.sub.4,
filtered and the filtrate was concentrated under reduced pressure
to give 2,4-dichloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine. LC-MS:
(ES, m/z): 206.05 [M+H].sup.+. .sup.1H-NMR: (300 MHz, d.sub.6-DMSO,
ppm): .delta. 12.71 (brs, 1H), 7.77 (d, J=2.4 Hz, 1H).
.sup.19F-NMR:(282 MHz, d.sub.6-DMSO, ppm): .delta.-169.79 (s,
1F).
Step 2: Synthesis of 2-oxopropane-1,3-diyl Diacetate
[0202] To a solution of 1,3-dihydroxypropan-2-one (90 g, 999 mmol)
in pyridine (400 ml) was added acetic anhydride (408 g, 3997 mmol)
at 0.degree. C. After the resulting mixture was stirred at
20.degree. C. for 16 hours, it was concentrated under reduced
pressure. The residue was diluted with DCM (1000 mL), washed with
2N HCl (2.times.1000 mL), NaHCO.sub.3(3.times.1000 mL). The
combined organic layers was dried with anhydrous Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The residue was
poured into stirring petroleum ether (1500 mL), the product was
precipitated out and filtered to give 2-oxopropane-1,3-diyl
diacetate. H-NMR: (300 MHz, CDCl.sub.3, ppm): .delta. 4.76 (s, 4H),
2.18 (s, 6H).
Step 3: Synthesis of
2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl
Diacetate
[0203] To a solution of ethynyltriisopropylsilane (58.6 g, 322
mmol) in THF (500 ml) under argon was added n-butyllithium (127 mL,
318 mmol) dropwise with stirring at -78.degree. C. The resulting
solution was stirred for 1 hour at -60.degree. C. To this solution
was added a solution of 2-oxopropane-1,3-diyl diacetate (56 g, 322
mmol) in THF (100 mL) dropwise with stirring at -78.degree. C. The
resulting solution was allowed to react, with stirring, for an
additional 1 hour at -60.degree. C. The reaction was then quenched
by the addition of 25 mL of AcOH at -78.degree. C. The resulting
solution was diluted with 500 mL of MTBE. The resulting mixture was
washed with 1.times.450 mL of citric acid (10%). The combined
organic extracts were washed with 2.times.300 mL of brine, dried
over anhydrous sodium sulfate and concentrated under vacuum to give
2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl diacetate
as a liquid. H-NMR: (300 MHz, CDCl.sub.3, ppm): .delta. 4.26 (d,d,
J=11.4 Hz, 13.2 Hz, 4H), 3.22-3.05 (bs, 1H), 2.11 (s, 6H),
1.09-0.99 (m, 21 H).
Step 4: Synthesis of
(R)-2-hydroxy-2-(hydroxymethyl)-4-(triisopropylsilyl)but-3-yn-1-yl
Acetate
[0204] To a mixture of KH.sub.2PO.sub.4/KOH (240 mL, 0.1 M, pH=7.5)
and NovoCor AD L (60 g, 140 mmol) was added a solution of
2-hydroxy-2-((triisopropylsilyl)ethynyl)propane-1,3-diyl diacetate
(50 g, 140 mmol) in methanol (240 ml) under argon at ambient
temperature. The resulting solution was stirred for 16 hours at
30.degree. C. The reaction progress was monitored by TLC. The
resulting solution was diluted with 1000 ml of brine and MTBE (1000
mL), CELITE.RTM. 545 (50 g) was added, and stirred at 30.degree. C.
for 30 minutes. Then the mixture was filtered, and the filtrate was
extracted with 3.times.1000 ml of MTBE. The organic layers combined
washed with brine (3.times.500 ml), dried over anhydrous magnesium
sulfate and filtered. The filtrate was concentrated under reduced
pressure to afford
(R)-2-hydroxy-2-(hydroxymethyl)-4-(triisopropylsilyl)but-3-yn-1-yl
acetate. H-NMR: (400 MHz, CDCl.sub.3, ppm): .delta.4.35 (d, J=11.2
Hz, 1H), 4.23 (d, J=11.2 Hz, 1H) 3.75-3.65 (d,d, J=11.2 Hz, 26.0
Hz, 2H), 2.70-2.50 (bs, 1H), 2.13 (s, 3H), 1.09-1.00 (m, 18H),
0.92-0.89 (m, 3H).
Step 5: Synthesis of
(S)-(2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)methan-
ol
[0205] To a solution of
(R)-2-hydroxy-2-(hydroxymethyl)-4-(triisopropylsilyl)but-3-yn-1-yl
acetate (70 g, 223 mmol) in MTBE (350 ml) and acetonitrile (280 ml)
were added PPTS (8.39 g, 33.4 mmol) and 2,2-dimethoxypropane (70 g,
672 mmol). The resulting solution was stirred for 4 hours at
50.degree. C. This was followed by the addition of sodium
methanolate (111 ml, 111 mmol) dropwise with stirring at 0.degree.
C. The resulting solution was stirred for an additional 30 minutes
at 25.degree. C., then the pH value was adjusted to 7 by 10% citric
acid. The resulting mixture was diluted with 600 ml MTBE and washed
with 2.times.200 mL of sodium bicarbonate. The combined organic
layers were washed with brine (3.times.300 ml), dried over
anhydrous sodium sulfate and concentrated under vacuum to give
(S)-(2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)methan-
ol. H-NMR: (300 MHz, CDCl.sub.3, ppm): .delta. 5.30 (s 1H),
4.14-4.13 (m, 2H), 3.75-3.70 (m, 1H), 3.65-3.55 (m, 1H), 1.85-1.95
(m, 1H), 1.54 (s, 3H), 1.44 (s, 3H), 1.08-1.01 (m, 21H).
Step 6: Synthesis of
(R)-2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxyli-
c Acid
[0206] To a mixture of
(S)-(2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)methan-
ol (52 g, 166 mmol) in MTBE (200 ml) and acetone (165 ml) under
argon were added TEMPO (2.86 g, 18.30 mmol), NaOCl (36.1 g, 399
mmol) and NaClO.sub.2 (124 g, 166 mmol) in water,
NaH.sub.2PO.sub.4H2(120 g) in water (600 ml) at ambient
temperature. The resulting mixture was warmed to 35.degree. C. and
stirred for 4 hours under argon. The reaction progress was
monitored by TLC. The reaction mixture was cooled down to ambient
temperature. The organic layer was washed with 200 ml of
NaHSO.sub.3 and 2.times.200 ml of water, dried over anhydrous
sodium sulfate. The filtrate was concentrated under reduced
pressure to give
(R)-2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxyli-
c acid. H-NMR: (300 MHz, d.sub.6-DMSO, ppm): .delta. 13.50 (s, 1H),
4.34 (d, J=8.7 Hz, 1H), 4.09 (d, J=8.7 Hz, 1H), 3.08 (s, 1H), 1.41
(s, 3H), 1.36 (s, 3H), 1.03-0.96 (m, 21H).
Step 7: Synthesis of
(1R,2S)-2-hydroxy-2,3-dihydro-1H-inden-1-aminium(R)-2,2-dimethyl-4-((trii-
sopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxylate
[0207] To a mixture of
(R)-2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxyli-
c acid (120 g, 368 mmol)) in MTBE (1000 ml) under argon was added
(1R,2S)-1-amino-2,3-dihydro-1H-inden-2-ol (49.3 g, 331 mmol) at
ambient temperature. The resulting mixture was warmed to 50.degree.
C. and stirred for 4 hours under argon. After 4 hours, the solution
was slowly cooled to 25.degree. C. and stirred for 24 hours under
argon. The solids were collected by filtration and washed with MTBE
(3.times.300 ml) to give
(1R,2S)-2-hydroxy-2,3-dihydro-1H-inden-1-aminium(R)-2,2-dimethyl-4-(-
(triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxylate. H-NMR:
(300 MHz, d.sub.6-DMSO, ppm): .delta. 7.66-7364 (m, 1H), 7.26-7.17
(m, 3H), 4.73-4.72 (m, 1H), 4.51-4.49 (m, 1H), 4.26-4.18 (m, 2H),
3.16-3.12 (m, 2H), 1.50 (s, 3H), 1.3 7 (m, 3H), 1.30-1.22 (m, 2H),
0.97 (s, 18H), 0.88-0.86 (m, 1H).
Step 8: Synthesis of (R)-methyl
2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxylate
[0208] To a mixture of
(1R,2S)-2-hydroxy-2,3-dihydro-1H-inden-1-aminium
(R)-2,2-dimethyl-4((triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxylat-
e (160 g, 336 mmol) in MTBE (2000 ml) was added 1000 ml of citric
acid (10%) at 0.degree. C. The mixture was stirred for 10 minutes.
The organic layer was collected, washed with 3.times.1000 ml of
brine, dried over anhydrous sodium sulfate and filtered. The
filtrate was concentrated under reduced pressure to give 100 g of
(4R)-2,2-dimethyl-4-[2-[tris(propan-2-yl)silyl]ethynyl]-1,3-dioxolane-4-c-
arboxylic acid as a liquid. To 100 g of
(4R)-2,2-dimethyl-4-[2-[tris(propan-2-yl)silyl]ethynyl]-1,3-dioxolane-4-c-
arboxylic acid in DMF (1000 ml) under argon were added
Cs.sub.2CO.sub.3 (329 g, 1009 mmol) and Mel (52.6 ml, 841 mmol) at
ambient temperature. The resulting solution was stirred overnight
at ambient temperature. The solids were filtered out. The resulting
filtrate was diluted with 2500 ml of EA. The resulting mixture was
washed with 3.times.1000 ml of brine. The combined organic layers
was dried over anhydrous sodium sulfate and filtered. The filtrate
was concentrated under reduced pressure to give (R)-methyl
2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxylate.
H-NMR: (400 MHz, CDCl.sub.3, ppm): .delta. 4.48 (d, J=8.8 Hz, 1H),
4.22 (d, J=8.8 Hz, 1H), 3.83 (s, 3H), 1.53 (s, 3H), 1.49 (s, 3H),
1.10-1.02 (m, 21H).
Step 9: Synthesis of (R)-tert-butyl
3-(2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)-3-oxopr-
opanoate
[0209] To a solution diisopropyl amine (53.7 g 532 mmol) in THF
(500 ml) was added n-BuLi (212 ml of 2.5 mol in hexane) over 40
minutes with internal temperature maintained below -68.degree. C.
To the LDA solution was added tert-butyl acetate (61.4 g, 529 mmol)
in THF (1000 ml) under argon at -78.degree. C. The resulting
mixture was stirred for 1 hour under argon (-60.degree. C.). To the
mixture was added a solution of (R)-methyl
2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolane-4-carboxylate
(90 g, 264 mmol) in THF (200 ml) at -78.degree. C. The reaction
progress was monitored by TLC. The resulting solution was allowed
to react, with stirring, for an additional 1 hour at -78.degree. C.
The reaction was then quenched by the addition of 15 ml AcOH. The
resulting solution was diluted with 2500 mL of MTBE The combined
organic layers were washed with brine (4.times.1000 mL), dried over
anhydrous sodium sulfate and filtered. The filtrate was
concentrated under reduced pressure to give (R)-tert-butyl
3-(2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)-3-oxopr-
opanoate. H-NMR: (300 MHz, CDCl.sub.3, ppm): .delta. 4.52 (d, J=8.7
Hz, 1H), 4.43 (d, J=8.7 Hz, 1H), 3.68 (dd, J=16.5 Hz, 36.6 Hz, 2H),
1.49-1.45 (m, 15H), 1.09-0.98 (m, 21H).
Step 10: Synthesis of (S)-tert-butyl
3-((R)-2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)-3-h-
ydroxypropanoate
[0210] Under argon, to a stirred and cooled 0.degree. C. solution
of Et.sub.3N (32.8 ml, 235 mmol) in THF (50 ml) was added formic
acid (27.1 g, 589 mmol). The mixture was stirred at ambient
temperature for 10 minutes (flask 1). A second flask with a stirred
solution of (R)-tert-butyl
3-(2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)-3-oxopr-
opanoate (100 g, 235 mmol) in THF (1000 ml) and MTBE (250 ml) was
added (((S,S)-TS-DENEB (0.345 g, 0.530 mmol). The prepared formic
acid/Et.sub.3N mixture was added from flask 1 to flask 2. The
reaction mixture was stirred at 38.degree. C. under Ar. The
reaction was monitored by TLC. After 11 hours, the reaction mixture
was cooled to 22.degree. C., then charged with 10% citric acid
solution (500 mL). The mixture was stirred and the layers were
split. The organic solution was treated with Ecosorb C-941(25 g).
The slurry was stirred for 1 hour and filtered. The filtrate was
concentrated under reduced pressure to give (S)-tert-butyl
3-((R)-2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)-3-h-
ydroxypropanoate. The product was used in the next step directly
without further purification. H-NMR: (400 MHz, CDCl.sub.3, ppm):
.delta. 4.22 (d, J=8.4 Hz, 1H), 4.11 (d, J=8.4 Hz, 1H), 4.03-4.02
(m, 1H), 2.78-2.70 (m, 2H), 2.48-2.39 (m, 1H), 1.50-1.41 (m, 15H),
1.08-1.04 (m, 21H).
Step 11: Synthesis of (S)-tert-butyl
3-((R)-4-ethynyl-2,2-dimethyl-1,3-dioxolan-4-yl)-3-hydroxypropanoate
[0211] To a mixture of (S)-tert-butyl
3-((R)-2,2-dimethyl-4-((triisopropylsilyl)ethynyl)-1,3-dioxolan-4-yl)-3-h-
ydroxypropanoate (40 g, 94 mmol) in THF (300 ml) under argon was
added 1 M TBAF in THF (94 ml) dropwise with stirring at 0.degree.
C. After the reaction mixture was stirred for 20 minutes at
25.degree. C., it was quenched by saturated brine (100 mL) at
0.degree. C. The resulting mixture was diluted with MTBE (2500 mL).
The organic layers were washed with brine (4.times.1000 mL), dried
over anhydrous sodium sulfate and filtered. The filtrate was
concentrated under reduced pressure to give (S)-tert-butyl
3-((R)-4-ethynyl-2,2-dimethyl-1,3-dioxolan-4-yl)-3-hydroxypropanoate.
H-NMR:(400 MHz, CDCl.sub.3, ppm): .delta. 4.21 (d, d, J=8.4 Hz,
40.0 Hz, 2H), 4.17-4.15 (m, 1H), 3.30-3.20 (brs, 1H), 2.72-2.71 (m,
1H), 2.56-2.54 (m, 2H), 1.50-1.47 (m, 15H).
Step 12: Synthesis of
(4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one
[0212] To a mixture of (S)-tert-butyl
3-((R)-4-ethynyl-2,2-dimethyl-1,3-dioxolan-4-yl)-3-hydroxypropanoate
(40 g, 148 mmol) in 1,2-DME (200 ml) was added HCl (36.5 ml, 444
mmol) dropwised with stirring at 0.degree. C. over 2 minutes. After
the mixture was stirred at 45.degree. C. for 1 hour, it was
concentrated under reduced pressure. Isopropyl acetate (50 ml) was
added to the residue dropwise with stirring at ambient temperature
for 16 h. The solid was collected by filtration to give
(4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one.
H-NMR: (400 MHz, DMSO, ppm): .delta. 5.82 (d, J=5.6 Hz, 1H), 5.56
(t, J=6.0 Hz, 12.0 Hz, 1H), 4.75 (s, 1H), 4.38-4.34 (m, 2H),
2.94-2.87 (m, 1H), 2.39-2.34 (m, 1H).
Step 13: Synthesis of
(2R,3S)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)-5-oxotetrahydrofuran-3-
-yl 4-methylbenzoate
[0213] To a mixture of
(4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one
(8.5 g, 54.4 mmol) in pyridine (90 mL) under argon was added
4-methylbenzoyl chloride (15.11 mL, 114 mmol) at 0.degree. C. The
resulting mixture was stirred for 2 hours at 0.degree. C. under an
atmosphere of argon. The reaction mixture was poured into ice water
(300 mL) and stirred for 10 minutes. The mixture was filtered, the
filter cake was washed with ice water (10.times.100 mL), and then
dried at 25.degree. C. for 24 hours to afford (2R,
3S)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)-5-oxotetrahydrofuran-3-yl
4-methylbenzoate. LC-MS: (ES, m/z): 393.20 [M+H].sup.+1. H-NMR (300
MHz, CDCl.sub.3, ppm): .delta. 8.00 (d, J=8.1 Hz, 2H), 7.91 (d,
J=8.1 Hz, 2H), 7.30-7.25 (m, 4H), 5.85-5.82 (m, 1H), 4.77 (dd,
J=9.0 Hz, 2H), 3.27-3.18 (m, 1H), 2.94-2.87 (m, 1H), 2.73 (s, 1H),
2.44 (d, J=3.9 Hz, 6H).
Step 14: Synthesis of
(2R,3S)-2-ethynyl-5-hydroxy-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate
[0214] To a stirred solution of methyl
(2R,3S)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)-5-oxotetrahydrofuran-3-
-yl 4-methylbenzoate (1160 mg, 2.96 mmol) in anhydrous toluene (30
mL) and DCM (6 mL) under argon in a 100-mL 3-necked round-bottom
flask, was added bis(2-methoxyethoxy)aluminum(III) sodium hydride
toluene solution (70% w/w, 0.598 g, 2.96 mmol) dropwise with
stirring at -78.degree. C. in 3 minutes. The resulting solution was
stirred at the same temperature for 90 minutes. The reaction
progress was monitored by TLC. The reaction was quenched by the
addition of acetic acid (1.7 mL), then hydrochloric acid (1 N, 30
mL) was added and the mixture was extracted with ethyl acetate
(3.times.30 mL). The combined organic fractions were washed with
brine (saturated, 2.times.30 mL), dried over anhydrous sodium
sulfate, filtered and concentrated under vacuum. The residue was
purified by silica gel column chromatography with ethyl
acetate/petrol ether (15/85) to give
(2R,3S)-2-ethynyl-5-hydroxy-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate. H-NMR: (300 MHz, CDCl.sub.3, ppm):
.delta. 7.92-8.00 (m, 4H), 7.19-7.27 (m, 4H), 5.74-5.84 (m, 1H),
5.65-5.70 (m, 1H), 4.68(s, 1H), 4.54 (dd, J=11.4 Hz, 35.4 Hz, 1H),
2.49-2.61 (m, 2.5H), 2.36-2.42 (m, 6.5H).
[0215] An alternative preparation is described below which omits
the chromatographic purification.
[0216] To a stirred solution of methyl
(2R,3S)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)-5-oxotetrahydrofuran-3-
-yl 4-methylbenzoate (4.5 g, 22.94 mmol) in toluene (67.5 mL) and
DCM (9 mL) was added bis(2-methoxyethoxy)aluminum(III) sodium
hydride toluene solution (65% w/w, 0.3.57 g, 11.47 mmol) dropwise
with stirring at -60.degree. C. over 2 hours. The resulting
solution was stirred at the same temperature for 2 hours. The
reaction progress was monitored by LC. The reaction was quenched by
the addition of acetic acid (1.31 mL, 22.94 mmol) over 20 minutes,
keeping the temperature below -60.degree. C. MTBE (22.50 mL) was
added, followed by aqueous citric acid solution (10 wt %, 22.5 mL)
and hydrochloric acid (1 N; 90.0 mL). The organic phase was
separated, dried over anhydrous magnesium sulfate, filtered and
concentrated under vacuum to give
(2R,3S)-2-ethynyl-5-hydroxy-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate. The product was used in the next
transformation without further purification.
Step 15: Synthesis of
(2R,3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate
[0217] To a stirred solution of methyl
(2R,3S)-2-ethynyl-5-hydroxy-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate (925 mg, 2.345 mmol) in dried DCM (30 mL)
under argon in a 100-mL 3-necked round-bottom flask, was added
4-dimethylaminopyridine (430 mg, 3.52 mmol), followed by the
addition of a solution of acetic anhydride (0.332 ml, 3.52 mmol) in
dichloromethane (3 mL) dropwise with stirring at 0.degree. C. The
resulting solution was stirred at 0.degree. C. for 1 hour. The
reaction was quenched with water (30 mL) and extracted with
dichloromethane (3.times.30 mL). The combined organic layer was
washed with brine (2.times.30 mL), dried over anhydrous sodium
sulfate, filtered and concentrated under vacuum. The residue was
purified by silica gel column chromatography with ethyl
acetate/petrol ether (10/90) to give
(2R,3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate. H-NMR: (300 MHz, CDCl.sub.3, ppm):
.delta. 7.90-8.10 (m, 4H), 7.19-7.27 (m, 4H), 6.45-6.50 (m, 1H),
5.84 (t, J=7.5 Hz, 1H), 4.72-4.75 (d, J=11.7 Hz, 1H), 4.50-4.61 (m,
1H), 2.64-2.78 (m, 3H), 2.40-2.43 (m, 6H), 1.90 (s, 3H).
[0218] An alternative preparation is described below which omits
the chromatographic purification.
[0219] To a stirred suspension of methyl (2R,
3S)-2-ethynyl-5-hydroxy-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-
-yl 4-methylbenzoate (26.0 g, 65.9 mmol) in DCM (130 mL) at
0.degree. C. was added acetic anhydride (9.35 mL, 99 mmol),
triethylamine (11.91 mL, 86 mmol), and 4-dimethylaminopyridine
(1.61 g, 13.18 mmol). The resulting solution was stirred at
0.degree. C. for 2.5 hours then quenched with MTBE (650 mL) and
aqueous citric acid (10 wt %; 130 mL). The organic layer was washed
with aqueous citric acid (10 wt %; 130 mL) and water (3.times.130
mL), then concentrated under vacuum to give (2R,
3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-
-yl 4-methylbenzoate. The product was used in the next
transformation without further purification.
Step 16: Synthesis of
(2R,3S)-5-chloro-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofura-
n-3-yl 4-methylbenzoate
[0220] Methyl (2R,
3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-
-yl 4-methylbenzoate (7 g, 16.04 mmol) was dried over P205 under
vacuum overnight and then added into an oven dried 250 ml
round-bottom flask, followed by the addition of dry DCM (140 mL)
under argon atmosphere at room temperature. The mixture was stirred
until it became clear, then cooled to 0.degree. C. HCl gas was
bubbled into the mixture while maintaining the temperature below
5.degree. C. The reaction progress was monitored by LCMS. After
bubbling was continued for 30 minutes then argon was bubbled into
the mixture for 10 minutes to remove the dissolved residual HCl.
The resulting DCM solution was partitioned between cold biphasic
mixture of MTBE (210 mL) and aqueous NaHCO.sub.3(saturated, 105
mL). The organic layer was collected, washed with cold aqueous
NaHCO.sub.3(saturated, 2.times.105 mL), dried over anhydrous
MgSO.sub.4 and filtered. The filtrate was concentrated under
reduced pressure to give
(2R,3S)-5-chloro-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydr-
ofuran-3-yl 4-methylbenzoate (.alpha./.beta.=2/1), which was used
to next reaction step directly without further purification. LC-MS:
(ES, m/z): 1) Cl converted to OH, 417.25 [M+Na]+, 377.36 [M-OH]+.
2) Cl converted to OMe, 431.34 [M+Na].sup.+, 377.36 [M-OMe]+.
H-NMR: (400 MHz, CD.sub.3CN, ppm): .delta. 8.02 (d, J=8.0 Hz, 41H),
7.96-7.93 (m, 3H), 7.36-7.26 (m, 4H), 6.50-6.48 (m, 1H), 5.94 (t,
J=8.0 Hz, 0.3H), 5.79 (dd, J=1.2 Hz, J=7.2 Hz, 0.6H), 4.78 (d,
J=11.6 Hz, 0.32H), 4.57 (d, J=12.0 Hz, 0.32H), 4.50 (dd, J=11.2 Hz,
J=14.4 Hz, 1.21H), 4.51-4.47 (m, 1H), 3.06-3.04 (m, 1H), 2.98-2.91
(m, 1.4H), 2.74 (d, J=15.6 Hz, 0.6H), 2.42-2.39 (m, 6H).
Step 17: Synthesis of
(2R,3S,5R)-5-(2,4-dichloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-et-
hynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate
[0221] To a stirred solution of
2,4-dichloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine (550 mg, 2.67
mmol) in anhydrous THF (18 mL) under argon atmosphere was injected
1M NaHMDS in THF (2.67 mL, 2.67 mmol) at -20.degree. C. in 5
minutes. The resulting mixture was maintained at -20.degree. C. for
30 minutes and then gradually warmed to 20.degree. C. To the above
was injected a solution of
(2R,3S)-5-chloro-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofura-
n-3-yl 4-methylbenzoate (918 mg, 2.223 mmol) in anhydrous THF (18
mL) in 5 minutes. The resulting mixture was stirred at 20.degree.
C. for 6 hours. The reaction was quenched by the addition of
diluted aqueous HCl (0.5 N, 5 mL) and extracted with MTBE
(3.times.50 mL). The organic layer was collected, washed with brine
(2.times.30 mL), dried over anhydrous MgSO.sub.4 and filtered. The
filtrate was concentrated under reduced pressure, the residue was
purified by silica gel column using ethyl acetate/petroleum ether
(0% to 10% EtOAc in pet. ether) to give a crude product. The crude
product (.alpha.+.beta. mixture) was treated with MTBE (4 mL)
stirred at room temperature for 15 hours. A solid was precipitated
from the medium and was collected by filtration, washed with cold
MTBE (2 mL), and dried under vacuum to give
(2R,3S,5R)-5-(2,4-dichloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-et-
hynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate. .sup.1H-NMR- (.beta. isomer): (300 MHz,
CDCl.sub.3, ppm): .delta. 8.02 (d, J=8.1 Hz, 2H), 7.90 (d, J=8.1
Hz, 2H), 7.29 (d, J=8.1 Hz, 2H), 7.25 (d, J=8.1 Hz, 2H), 7.09 (d,
J=2.7 Hz, 1H), 6.83 (t, J=6.0 Hz, 1H), 5.90 (t, J=6.3 Hz, 1H), 4.83
(d, J=12.0 Hz, 1H), 4.61 (d, J=12.0 Hz, 1H), 2.87 (t, J=6.3 Hz,
2H), 2.71 (s, 1H), 2.45, 2.43 (2s, 6H). .sup.19F-NMR (isomer): (282
MHz, CDCl.sub.3, ppm): .delta. -165.25 (s, 1F). LC-MS: (ES, m/z):
582.40 [M+H].sup.+.
Step 18: Synthesis of
(2R,3S,5R)-5-(4-amino-2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)--
2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (1)
[0222]
(2R,3S,5R)-5-(2,4-dichloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl-
)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate (240 mg, 0.412 mmol) was massed into an oven-dried
80 ml steel bomb and cooled to -40.degree. C. To the above was
added isopropanolic ammonia (i-PrOH/liquid NH.sub.3=1/1, v/v, mixed
at -40.degree. C., 30 mL). After the resulting mixture was heated
to 80.degree. C. and stirred for 16 hours, it was cooled to room
temperature and then concentrated under reduced pressure. The
residue was purified by silica gel column chromatography using
DCM/MeOH (94/6) to give a crude solid. The crude was triturated
with DCM/MeOH (20/1, v/v, 4 mL) and stirred at room temperature for
2 hours. A precipitate was formed and then collected by filtration,
washed with DCM/MeOH (v/v, 20/1, 2.times.2 mL) and lyophilized
overnight to give
(2R,3S,5R)-5-(4-amino-2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)--
2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol. .sup.1H-NMR: (300
MHz, d.sub.6-DMSO, ppm): .delta. 7.68 (brs, 2H), 7.32 (d, J=2.0 Hz,
1H), 6.44 (t, J=5.8 Hz, 1H), 5.52 (d, J=5.6 Hz, 1H), 5.27 (t, J=6.0
Hz, 1H), 4.44 (q, J=6.4 Hz, 1H), 3.60 (q, J=6.0 Hz, 1H), 3.53 (q,
J=6.4 Hz, 1H), 3.48 (s, 1H), 2.48-2.41 (m, 1H), 2.37-2.30 (m, 1H).
.sup.19F-NMR: (282 MHz, d.sub.6-DMSO, ppm): .delta. -166.67 (s,
1F). LC-MS: (ES, m/z): 327.00 [M+H].sup.+.
Example 2
Synthesis of Ammonium
((2R,3S,5R)-5-(4-amino-2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
-2-ethynyl-3-hydroxytetrahydrofuran-2-yl)methyl Triphosphate
(2)
##STR00061##
[0223] Step 1: Synthesis of
2-((4,4,6,6-tetraoxido-1,3,5,2,4,6-trioxatriphosphinan-2-yl)oxy)benzoate
[0224] In a glove box under an argon atmosphere, dry tributylamine
(0.4 ml, 1.679 mmol) was added to the flask containing
tributylammonium pyrophosphate (112 mg, 0.205 mmol) dissolved in
0.4 mL dimethylformamide (DMF) to give a clear solution. The clear
solution was then injected into the flask containing dry
2-chloro-4H-benzo[d][1,3,2]dioxaphosphinin-4-one (27.6 mg, 0.136
mmol) in dimethylformamide (0.4 mL) under vigorous stirring. The
resulting mixture was stirred at 30.degree. C. for 30 min to give
2-((4,4,6,6-tetraoxido-1,3,5,2,4,6-trioxatriphosphinan-2-yl)oxy)b-
enzoate which was used to the next reaction directly without any
work-up.
Step 2: Synthesis of Ammonium
((2R,3S,5R)-5-(4-amino-2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
-2-ethynyl-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate
(2)
[0225]
(2R,3S,5R)-5-(4-amino-2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin--
7-yl)-2-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (20 mg, 0.061
mmol) was massed into a 10 mL over-dried round-bottom flask and
then dried over P.sub.205 under high vacuum overnight. To this
flask was added activated molecular sieves 4A (300 mg). Under argon
atmosphere, a solution of
2-((4,4,6,6-tetraoxido-1,3,5,2,4,6-trioxatriphosphinan-2-yl)oxy)benzoate
(1.8 eq, freshly prepared) in dry DMF (0.6 ml) was transferred into
the above flask by syringes and the mixture was stirred at
30.degree. C. for 3 h. The reaction progress was monitored by TLC
(acetonitrile: 0.1 M ammonium chloride=7:3). When most of started
nucleoside was consumed, the mixture was cooled to 0.degree. C.,
followed by the injection of iodine solution [3% iodine in
pyridine/water (9/1), 0.5 mL]. As the iodine was consumed,
drop-wise addition of the iodine solution was continued until a
permanent brown color of iodine was maintained. After 15 min,
triethylammonium bicarbonate buffer (1.0 M, 1.0 mL) was added with
stirring at 10.degree. C. and the mixture was stirred for 15 min.
The volatile was removed under reduced pressure (inner temperature
was not over 25.degree. C.). The residue was re-dissolved in water
(2 mL) and extracted with chloroform (2.times.2 mL). The collected
aqueous layer which containing crude product was then purified by
preparative-HPLC with the following conditions (1
#-Pre-HPLC-011(Waters)): Column: X Bridge Prep Amide, 19*150 mm, 5
um; Mobile phase: water with 50 mmol ammonium bicarbonate and
acetonitrile (90% acetonitrile to 55% in 10 min, up to 30% in 2
min); Detector, UV 254 & 220 nm. The product-containing
fractions were collected and concentrated to an approximate volume
of 10 mL. Then ACN (1 mL) and TEAB buffer (2 M, 0.05 mL) were added
to the solution, and then the mixture was lyophilized for 40 h to
give ammonium
((2R,3S,5R)-5-(4-amino-2-chloro-5-fluoro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-
-2-ethynyl-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate.
LC-MS: (ES, m/z): 564.95 [M-H-4NH.sub.3].sup.-. H-NMR: (400 MHz,
D.sub.2O, ppm): .delta. 7.07 (s, 1H), 6.46 (s, 1H), 4.15-4.05 (m,
2H), 2.56-2.49 (m, 2H). P-NMR: (161 MHz, D.sub.2O, ppm): .delta.
-5.81 (s, 1P), -11.49-11.41 (d, J=13.20 Hz, 1P), -19.28 (s,
1P).
Example 3
Synthesis of
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)-
tetrahydrofuran-3-ol (EFdA) from
(2R,3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate
##STR00062##
[0226] Step 1: Synthesis of
(2R,3S,5R)-2-ethynyl-5-(2-fluoro-6-((trimethylsilyl)amino)-9H-purin-9-yl)-
-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate
[0227] To a stirred solution of 2-fluoro-7H-purin-6-amine (4.79 g,
31.3 mmol) in MeCN (210 mL) was added N,
O-bis(trimethylsilyl)acetamide (35.3 mL, 144 mmol). The reaction
mixture was heated to 81.degree. C. and stirred for 1 hour. The
resulting solution was cooled to room temperature and
trimethylsilyl trifluoromethanesulfonate (7.84 mL, 43.3 mmol) was
added, followed by acetonitrile (105 mL). To the above was added a
solution of
(2R,3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate (10.5 g, 24.06 mmol) in MeCN (100 mL) over
2 hours. The resulting mixture was stirred at 81.degree. C. for 14
hours, then concentrated to 150 mL volume by simple distillation.
The reaction mixture was seeded with product (0.1 wt %) and slowly
cooled to room temperature to give a slurry. The solid was
collected by filtration to give
(2R,3S,5R)-2-ethynyl-5-(2-fluoro-6-((trimethylsilyl)amino)-9H-purin--
9-yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate (6.73 g). .sup.1H-NMR- (.beta. isomer): (400 MHz,
CDCl.sub.3, ppm): .delta. 8.04 (d, J=8.0 Hz, 2H), 7.94 (d, J=8.0
Hz, 2H), 7.87 (s, 1H), 7.20 (d, J=8.0 Hz, 2H), 7.24 (d, J=8.0 Hz,
2H), 6.52 (dd, J=8.0, 4.0 Hz, 1H), 6.07 (dd, J=8.0, 4.0 Hz, 1H),
5.50 (s, 1H), 4.84 (d, J=12.0 Hz, 1H), 4.69 (d, J=12.0 Hz, 1H),
3.25-3.18 (m, 1H), 2.91-2.85 (m, 1H), 2.70 (s, 1H), 2.46 (s, 3H),
2.42 (s, 3H), 0.41 (s, 9H).
Step 2: Synthesis of
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)-
tetrahydrofuran-3-ol (EFdA)
[0228]
(2R,3S,5R)-2-ethynyl-5-(2-fluoro-6-((trimethylsilyl)amino)-9H-purin-
-9-yl)-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate (6.0 g, 9.97 mmol) was dissolved in THF (60 mL)
and cooled to -25.degree. C. Sodium methoxide in methanol (30 wt %;
1.80 g, 9.97 mmol) was slowly added, keeping the internal
temperature below -20.degree. C. The reaction mixture was stirred
at -25.degree. C. for 16 hours then quenched with acetic acid (1.14
mL, 19.94 mmol). The resulting solution was heated to 45.degree. C.
and concentrated to 60 mL. Water was added (0.90 g, 49.9 mmol) and
the solvent was switched to ACN. The resulting slurry was
concentrated to 50 mL, cooled to room temperature and aged for 30
minutes. The solid was collected by filtration, washed with ACN
(3.times.30 mL) and water (2.times.6 mL), and dried to give
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)-
tetrahydrofuran-3-ol (2.93 g). .sup.1H-NMR: (600 MHz, d.sub.6-DMSO,
ppm): .delta. 8.29 (s, 1H), 7.82 (br s, 2H), 6.24 (dd, J=7.2, 5.0
Hz, 1H), 5.55 (d, J=5.5, 1H), 5.27 (dd, J=6.8, 5.7 Hz, 1H), 4.57
(m, 1H), 3.65 (dd, J=11.9, 5.7 Hz, 1H), 3.56 (dd, J=11.9, 6.8 Hz,
1H), 3.49 (s, 1H), 2.70 (m, 1H), 2.43 (m, 1H). .sup.19F-NMR: (282
MHz, d.sub.6-DMSO, ppm): LC-MS: (ES, m/z): 316.0818
[M+Na].sup.+.
Example 4
Synthesis of
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)-
tetrahydrofuran-3-ol (EfdA) from
(2R,3S)-2-Ethynyl-2-[4-methylbenzoyl)oxymethyl]-5-(pent-4-en-1-yloxy)tetr-
ahydrofuran-3-yl 4-methylbenzoate
##STR00063##
[0229] Step 1:Synthesis of
(2R,3S)-2-Ethynyl-2-[4-methylbenzoyl)oxymethyl]-5-(pent-4-en-1-yloxy)tetr-
ahydrofuran-3-yl 4-methylbenzoate
[0230] To a stirred solution of p-toluenesulfonic acid monohydrate
(0.76 g, 4 mmol) and 4-penten-1-ol (0.38 g, 4.4 mmol) in toluene
(20 mL) at 0.degree. C. was added
(2R,3S)-2-ethynyl-5-hydroxy-2-[(4-methylbenzoyl)oxymethyl]tetrahydrofuran-
-3-yl 4-methylbenzoate (11.15 wt % in toluene, 14.15 g, 4 mmol).
The reaction mixture was stirred for 1 hour and quenched with water
(30 mL). The organic layer was washed with saturated aqueous sodium
bicarbonate (30 mL), water (30 mL) and saturated aqueous sodium
chloride (30 mL). The resulting toluene solution was assayed by
HPLC against a standard to give
(2R,3S)-2-ethynyl-2-[4-methylbenzoyl)oxymethyl]-5-(pent-4-en-1-yloxy)tetr-
ahydrofuran-3-yl 4-methylbenzoate, (1.6 g). HRMS
(QTof).sub.m/z.:[M+Na].sup.+ calcd for C.sub.28H.sub.30O.sub.6Na
485.1940, found 485.1926.
Step 2: Synthesis of
(2R,3S)-2-ethynyl-2-[4-methylbenzoyl)oxymethyl]-5-(pent-4-en-1-yloxy)
tetrahydrofuran-3-yl 4-methylbenzoate
[0231] To a stirred solution of
(2R,3S)-5-acetoxy-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofur-
an-3-yl 4-methylbenzoate (2.0 g, 4.58 mmol) and 4-penten-1-ol
(0.395 g, 4.58 mmol) in acetonitrile (20 mL) at 0.degree. C. was
added trimethylsilyl trifluoromethanesulfonate (0.083 ml, 0.458
mmol). The reaction mixture was stirred for 45 minutes and quenched
with saturated sodium hydrogen carbonate solution (20 mL). The
organic layer was washed 5% brine solution (20 mL), and the
organics were concentrated to give
(2R,3S)-2-ethynyl-2-[4-methylbenzoyl)oxymethyl]-5-(pent-4-en-1-yloxy)tetr-
ahydrofuran-3-yl
4-methylbenzoate.(2R,3S)-2-ethynyl-2-[4-methylbenzoyl)oxymethyl]-5-(pent--
4-en-1-yloxy) tetrahydrofuran-3-yl 4-methylbenzoate (2.1 g).
.sup.1H-NMR: (400 MHz, DMSO, ppm, mixture of anomers): .delta.
8.01-7.88 (m, 8H), 7.40-7.32 (m, 83), 5.92-5.58 (m, 4H), 5.40-5.27
(m, 2H), 5.09-4.87 (m, 4H), 4.64-4.35 (m, 4H), 3.78-3.32 (m, 6H),
2.70-2.41 (m, 4H), 2.40-2.35 (m, 12H), 2.20-2.12 (m, 2H), 2.04-1.93
(m, 2H), 1.69-1.61 (m, 2H), 1.56-1.42 (m, 2H).
Step 3: Synthesis of Tert-Butyl
(2-fluoro-9H-purin-6-yl)carbamate
[0232] To a stirred suspension of 2-fluoro-9H-purin-6-amine (20 g,
131 mmol) and 4-(dimethylamino)pyridine (1.6 g, 13 mmol) in THF
(200 mL) at 0.degree. C. was added di-tert-butyldicarbonate (100 g,
457 mmol) previously dissolved in THF (100 mL). The resulting
suspension was stirred at 0.degree. C. for 12 hours then diluted
with MTBE (200 mL) and quenched with water (200 mL). The organic
layer was washed with aqueous citric acid (10 wt %; 100 mL), water
(2.times.100 mL) and saturated aqueous sodium chloride (100 mL).
The resulting solution was then concentrated under reduced pressure
to a 100 mL volume and diluted with ethanol (absolute, 400 mL).
Aqueous sodium hydroxide (2.5 M, 313 mL, 784 mmol) was then added
at 20.degree. C. over 1 hour and the batch aged for 48 hours at
this temperature. Solvents were distilled under reduced pressure
and the aqueous solution was cooled to 0.degree. C. and neutralized
with hydrochloric acid (1N, 705 mL) to give a slurry. The solid was
collected by filtration, washed with water (2.times.50 mL) and
dried to give tert-butyl (2-fluoro-9H-purin-6-yl)carbamate (20 g,
79 mmol). 1H-NMR: (400 MHz, DMSO, ppm): .delta. 8.42 (s, 1H), 1.954
(s, 9H). HRMS (QTof) m/z: [M+H].sup.+ calcd for
C.sub.10H.sub.13FN.sub.5O.sub.2 254.1052, found 254.1053.
Step 4: Synthesis of
(2R,3S,5R)-5-(6-((Tert-butoxycarbonyl)amino)-2-fluoro-9H-purin-9-yl)-2-et-
hynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate
[0233] A dry stirred mixture of
(2R,3S)-2-ethynyl-2-(((4-methylbenzoyl)oxy)methyl)-5-(pent-4-en-1-yloxy)t-
etrahydrofuran-3-yl 4-methylbenzoate (3.63 g, 7.85 mmol) and
tert-butyl (2-fluoro-9H-purin-6-yl)carbamate (2.29 g, 9.04 mmol) in
acetonitrile (80 mL) was cooled to -25.degree. C. Iodine (6.30 g,
24.8 mmol) was added and the resulting mixture was stirred for 17
h, under a nitrogen atmosphere, at -25.degree. C. The reaction
mixture was then warmed to 0.degree. C. and stirred at this
temperature for a further 6 h. The reaction was quenched with
aqueous sodium sulfite (10 wt %; 30 mL), diluted with water (40
mL), and then extracted with methyl tert-butyl ether (80 mL). The
resulting organic layer was washed with aqueous sodium
hydrogencarbonate (7 wt %; 40 mL) and then with aqueous sodium
chloride (2 wt %; 42 mL). The organic layer obtained was
concentrated to a volume of 35 mL, to form a slurry. To this
stirred slurry, at ambient temperature, was slowly added water (12
mL). The resulting slurry was aged at ambient temperature and then
filtered, washing the solid with 1:1 acetonitrile/water (2.times.10
mL). The resulting solid was dried to give
(2R,3S,5R)-5-(6-((tert-butoxycarbonyl)amino)-2-fluoro-9H-purin-9-yl)-2-et-
hynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate (3.00 g). .sup.1H-NMR: (400 MHz, CDCl.sub.3, ppm):
.delta. 8.06-8.02 (m, 4H), 7.92-7.89 (m, 2H), 7.33-7.29 (m, 2H),
7.26-7.22 (m, 2H), 6.56 (t, J=6.5 Hz, 1H), 6.08 (dd, J=7.2, 5.5 Hz,
1H), 4.89 (d, J=12.0 Hz, 1H), 4.67 (d, J=12.0 Hz, 1H), 3.24 (ddd,
J=13.9, 7.4, 6.3 Hz, 1H), 2.95 (ddd, J=14.0, 6.9, 5.4 Hz, 1H), 2.73
(s, 1H), 2.47 (s, 3H), 2.43 (s, 3H), 1.59 (s, 9H). HRMS (QTof) m/z:
[M+H].sup.+ calcd for C.sub.33H.sub.33FN.sub.5O.sub.7 630.2364,
found 630.2295.
Step 5: Synthesis of Tert-Butyl
(9-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-
-2-fluoro-9H-purin-6-yl)carbamate
[0234] A stirred solution of
(2R,3S,5R)-5-(6-((tert-butoxycarbonyl)amino)-2-fluoro-9H-purin-9-yl)-2-et-
hynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate (1.5 g, 2.382 mmol) in a mixture of
tetrahydrofuran (10 ml) and methanol (5 ml) was cooled to
-20.degree. C. Sodium methoxide (1.634 ml of a 25 wt % solution in
methanol, 7.15 mmol) was added and the reaction was stirred for 4 h
whilst monitoring the reaction progress by HPLC. The reaction was
quenched by the addition of phosphoric acid (0.489 ml, 7.15 mmol)
and the reaction was warmed to room temperature and the solid was
filtered washing the cake with methanol. The filtrate was
concentrated to provide tert-butyl
(9-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-
-2-fluoro-9H-purin-6-yl)carbamate, (0.47 g). .sup.1H NMR (400 MHz,
CDCl.sub.3, ppm). 8.16 (s, 1H), 7.97 (s, 1H), 6.42-6.39 (dd,
J=8.68, 5.78 Hz, 1H), 5.05-5.03 (m, 1H), 4.74-4.73 (m, 1H),
4.13-4.06 (m, 1H), 3.91-3.85 (m, 1H), 3.13-3.07 (m, 1H), 2.82 (S,
1H) 2.61 (bs, 1H), 2.54-2.50 (m, 1H), 1.57 (s, 9H) HRMS (QTof) m/z:
[M+H].sup.+ calcd for C.sub.17H.sub.21FN.sub.5O.sub.5 394.1527,
found 394.1526.
Step 6: Synthesis of
(2R,3S,5R)-5-(6-Amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)
tetrahydrofuran-3-ol (EFdA)
[0235] To a stirred solution of tert-butyl
(9-((2R,4S,5R)-5-ethynyl-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-
-2-fluoro-9H-purin-6-yl)carbamate (0.05 g, 0.127 mmol) in
dichloromethane (0.5 ml) was added trifluoroacetic acid (0.1 ml).
The reaction mixture was aged at 20.degree. C. for 16 h then
concentrated to give
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)
tetrahydrofuran-3-ol (1), (0.012 g), which was confirmed by HPLC
assay to a known standard.
Step 7: Synthesis of
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(((4-methylbenz-
oyl)oxy)methyl) tetrahydrofuran-3-yl 4-methylbenzoate
[0236] To a stirred solution of
(2R,3S,5R)-5-(6-((tert-butoxycarbonyl)amino)-2-fluoro-9H-purin-9-yl)-2-et-
hynyl-2-(((4-methylbenzoyl)oxy)methyl)tetrahydrofuran-3-yl
4-methylbenzoate (0.5 g, 0.794 mmol) in toluene (5 ml) at
20.degree. C., was added trifluro acetic acid (0.5 ml). The
reaction was aged for 24 h whilst monitoring the reaction progress
by HPLC. The reaction was quenched by the addition of saturated
sodium hydrogen carbonate (10 ml) and ethyl acetate was added (10
ml). The organics were washed with water (10 ml) and concentrated
to give
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(((4-methylbenz-
oyl)oxy)methyl) tetrahydrofuran-3-yl 4-methylbenzoate (0.40 g). 1H
NMR (400 MHz, CDCl.sub.3, ppm). 8.3-8.1 (d, J=8.29 Hz, 2H),
7.93-7.91 (d, J=8.29 Hz, 2H), 7.89 (s, 1H), 7.30-7.28 (d, J=8.29
Hz, 2H), 7.23-7.21 (d, J=8.29 Hz, 2H), 6.52-6.49 (t, J=6.68 Hz,
1H), 6.07-6.04 (dd, J=7.22, 5.35 Hz, 1H), 5.89 (bs, 2H), 4.86-4.83
(d, J=11.76 Hz, 1H), 4.67-4.64 (d, J=11.76 Hz, 1H), 3.22-3.18 (m,
1H), 2.90-2.87 (m, 1H), 2.69 (s, 1H), 2.45 (s, 3H). 2.44-2.43 (m,
1H), 2.45 (s, 1H). HRMS (QTof) m/z: [M+H].sup.+ calcd for
C.sub.28H.sub.25FN.sub.5O.sub.5 530.1840, found 530.1885.
Step 8: Synthesis of
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)
tetrahydrofuran-3-ol (EfdA)
[0237] A stirred solution
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(((4-methylbenz-
oyl)oxy)methyl) tetrahydrofuran-3-yl 4-methylbenzoate (0.084 g,
0.159 mmol) in a mixture of tetrahydrofuran (0.84 ml) and methanol
(0.42 ml) was cooled to -20.degree. C. Sodium methoxide (0.109 ml
of a 25 wt % solution in methanol, 0.476 mmol) was added and the
reaction was stirred for 16 h whilst monitoring the reaction
progress by HPLC. The reaction was quenched by the addition of
phosphoric acid (0.47 g, 0.476 mmol) and the reaction was warmed to
room temperature and the solid was filtered washing the cake with
methanol. The filtrate was concentrated to to give
(2R,3S,5R)-5-(6-amino-2-fluoro-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)
tetrahydrofuran-3-ol (EfdA), (0.039 g), which was confirmed by HPLC
assay to a known standard.
RT Polymerase Assay
[0238] Full-length wild-type and 2 mutant RT proteins were
expressed in Escherichia coli BL21(DE3) cells and purified.
Briefly, the heterodimeric nucleic acid substrate used in the HIV-1
RT polymerase reactions was generated by annealing biotinylated DNA
primer to a 500 nucleotide RNA template. The HIV-1 RT enzyme (final
concentration of 50 pM) was combined with an inhibitor compound or
DMSO (10% DMSO in the final reaction mixture) in assay buffer (62.5
mM Tris-HCl, pH 7.8, 1.25 mM dithiothreitol, 7.5 mM MgCl.sub.2, 100
mM KCl, 0.03% CHAPS, 0.125 mM EGTA). This mixture was pre-incubated
for 30 minutes at room temperature in microtiter plates. The
polymerization reaction was initiated by the addition of
template/primer substrate (final concentration: 16.6 nM) and dNTPs
(final concentration: 2 .mu.M dCTP, dGTP, dATP, and 66.6 nM
Ru-dUTP). After 90 min of incubation at 37.degree. C., reactions
were quenched by the addition of EDTA (25 mM). The resulting
mixture was incubated for an additional 5 minutes at room
temperature followed by transferring the solution (50 .mu.L) to
blocked avidin plate from Meso Scale Discovery (MSD). The mixtures
were incubated at room temperature for 60 min prior to the
quantification of the reaction product via an ECL 6000 imager
instrument. The resulting data is shown in Table 1.
TABLE-US-00003 TABLE 1 Example No. Structure dNTP IC.sub.50 (nM) 2
##STR00064## 490
Viking Assay:
[0239] Assessing antiviral potency in a multiple round HIV-1
infection assay. HIV-1 replication was monitored using MT4-gag-GFP
clone D3 (hereafter designate MT4-GFP), which are MT-4 cells
modified to harbor a GFP reporter gene, the expression of which is
dependent on the HIV-1 expressed proteins tat and rev. Productive
infection of an MT4-GFP cell with HIV-1 results in GFP expression
approximately 24 h post-infection.
[0240] MT4-GFP cells were maintained at 37.degree. C./5%
CO.sub.2/90% relative humidity in RPMI 1640 supplemented with 10%
fetal bovine serum, 100 U/mL penicillin/streptomycin, and 400
.mu.g/mL G418 to maintain the reporter gene. For infections,
MT4-GFP cells were placed in the same medium lacking G418 and
infected overnight with H9IIIB virus at an approximate multiplicity
of infection of 0.01 in the same incubation conditions. Cells were
then washed and re-suspended in either RPMI 1640 containing 10% or
50% normal human serum at 1.6.times.10.sup.5 cells/mL (10% or 50%
NHS conditions) or in 100% normal human serum at 2.times.10.sup.5
cells/mL (100% NHS conditions). Compound plates were prepared by
dispensing compounds dissolved in DMSO into wells of 384 well poly
D lysine-coated plates (0.21/well) using an ECHO acoustic
dispenser. Each compound was tested in a 10 point serial 3-fold
dilution (typical final concentrations: 8.4 .mu.M-0.43 nM).
Controls included no inhibitor (DMSO only) and a combination of
three antiviral agents (efavirenz, indinavir, and an integrase
strand transfer inhibitor at final concentrations of 4 .mu.M each).
Cells were added (50 .mu.L/well) to compound plates and the
infected cells were maintained at 37.degree. C./5% CO.sub.2/90%
relative humidity.
[0241] Infected cells were quantified at two time points, .about.48
h and .about.72 h post-infection, by counting the number of green
cells in each well using an Acumen eX3 scanner. The increase in the
number of green cells over -24 h period gives the reproductive
ratio, R.sub.0, which is typically 5- and has been shown
experimentally to be in logarithmic phase (data not shown).
Inhibition of R.sub.0 is calculated for each well, and EC.sub.50s
determined by non-linear 4-parameter curve fitting.
CTG Assay:
Assessing Cytotoxicity in CellTiter-Glo Luminescent Cell Viability
Assay (CTG).
[0242] MT4-GFP cells were seeded in RPMI 1640 supplemented with 10%
fetal bovine serum, 100 U/mL penicillin/streptomycin overnight at
37.degree. C./5% CO.sub.2/90% relative humidity. Cells were then
washed and resuspended in RPMI 1640 containing 10% normal human
serum at a density of 0.8.times.10.sup.5 cells/mL. Compound plates
were prepared by dispensing compounds dissolved in DMSO into wells
of 384 well solid black plates (Corning 3571) using an ECHO
acoustic dispenser (0.2 .mu.l/well). Each compound was tested in a
10 point serial 3-fold dilution (final concentrations: 8.4
.mu.M-0.43 nM). Controls included DMSO. Cells were added (50
.mu.L/well) to compound plates and were maintained at 37.degree.
C./5% CO.sub.2/90% relative humidity. CTG reagent (Promega, G7573)
was added to the cell plates after 48 h incubation according to the
manufacturer's description. Luminescence signals were recorded on
EnVision plate reader (PerkinElmer). LD.sub.50s were determined by
non-linear 4-parameter curve fitting. The resulting data is shown
in Table 2 with the marketed HIV nucleoside reverse transcriptase
inhibitor AZT (azidothymidine, zidovudine) included as a
control.
TABLE-US-00004 TABLE 2 Viking, EC.sub.50 (10% NHS) CTG Structure
(nM) (.mu.M) AZT ##STR00065## 37 >8.4 Ex- Viking, EC.sub.50
ample (10% NHS) CTG No. Structure (nM) (.mu.M) 1 ##STR00066## 1.2
>8.4
[0243] Antiviral Persistence in Human Peripheral Blood Mononuclear
Cells (PBMC)
[0244] PBMCs were obtained from Biological Specialty Corporation,
activated with 5 .mu.g/ml PHA-P (Sigma L1668) for 72 hrs, and then
washed with fresh medium. Following activation, PBMCs were cultured
in media containing 10 IU/ml IL-2 (Roche 11011456001). Cells were
treated with a titration of compound for 6 or 24 hrs and washed
with fresh medium. To evaluate persistence of antiviral activity
following wash-out, PBMCs were maintained for either 24 h or 72 h
and then infected by adding wild-type HIV-1-GFP virus (17
.mu.L/well) to the plates and incubating at 37.degree. C./5%
CO.sub.2/90% relative humidity. Infected cells were quantified at
24 h post-infection by counting the number of green cells in each
well using an Acumen eX3 scanner. EC50 values in Table 3 were
calculated by non-linear 4-parameter curve fitting.
[0245] The antiviral persistence assay is meant to assess for the
persistence of antiviral activity upon removal of the nucleoside.
The data in Table 3 demonstrates the antiviral persistence of
compounds of this invention in comparison to marketed nucleoside
AZT. The publication AIDS Research and Therapy, 2009, 6:5,
highlights the value of antiviral persistence.
TABLE-US-00005 TABLE 3 EC.sub.50 EC.sub.50 24 h 72 h Structure (nM)
(nM) AZT ##STR00067## 19 68 EX- EC.sub.50 EC.sub.50 AMPLE 24 h 72 h
No. Structure (nM) (nM) 1 ##STR00068## 0.38 3.8
Adenosine Deaminase (ADA) Half-Life
[0246] The data in Table 4 was generated by testing the reactivity
of the substrate compound with human ADA type 1 in the presence of
Tris-HCl buffer (pH 7.5) at 40.degree. C. and monitoring by LCMS
for consumption of starting material. The time necessary for 50%
conversion to the corresponding inosine product is noted as the
T.sub.1 in Table 4. It is known that deamination by adenosine
deaminase decreases the therapeutic potential of adenosine-like
nucleoside inhibitors especially in vivo (see references below).
The compounds shown in Table 4 have been shown to have varying
degrees of stability to adenosine deaminase when compared to EDA
((2R,3S,5R)-5-(6-amino-9H-purin-9-yl)-2-ethynyl-2-(hydroxymethyl)tetrahyd-
rofuran-3-ol) and natural deoxyadenosine. It is possible that
compounds that are more resistant to ADA will have better
pharmacokinetic properties.
References
[0247] Journal of Medicinal Chemistry 1996, 39, 19, 3847; Chemical
Pharmaceutical Bulletin. 1994, 42, 8, 1688-1690; Antimicrobial
Agents and Chemotherapy (2013), 57(12), 6254-6264; [0248]
Collection of Czechoslovak Chemical Communications (2006), 71(6),
769-787; [0249] Microbiologica (1995), 18(4), 359-70; J Antivir
Antiretrovir S10.doi:10.4172/jaa.S0-002.
TABLE-US-00006 [0249] TABLE 4 Structure (Substrate) Inosine Product
T.sub.1/2 Deoxy- adenosine ##STR00069## ##STR00070## <10 min EDA
##STR00071## ##STR00072## <60 min Example No Structure
(Substrate) Inosine Product T.sub.1/2 1 ##STR00073## ##STR00074##
NA NA: Substrate insensitive to Adenosine Deaminase. 100% of the
substrate was seen after 10 days of incubation with ADA.
[0250] While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, the practice of the invention encompasses all of the
usual variations, adaptations and/or modifications that come within
the scope of the following claims. Recitation or depiction of a
specific compound in the claims (i.e., a species) without a
specific stereoconfiguration designation, or with such a
designation for less than all chiral centers, is intended to
encompass the racemate, racemic mixtures, each individual
enantiomer, a diastereoisomeric mixture and each individual
diastereomer of the compound where such forms are possible due to
the presence of one or more asymmetric centers. All publications,
patents and patent applications cited herein are incorporated by
reference in their entirety into the disclosure.
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