U.S. patent application number 16/227398 was filed with the patent office on 2020-06-25 for compounds and their use for reducing uric acid levels.
The applicant listed for this patent is Acquist LLC. Invention is credited to Alexandre Larivee, John J. Piwinski, Arshad Siddiqui, Karen Thai, Raymond P. Warrell, JR..
Application Number | 20200197393 16/227398 |
Document ID | / |
Family ID | 60913101 |
Filed Date | 2020-06-25 |
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United States Patent
Application |
20200197393 |
Kind Code |
A9 |
Warrell, JR.; Raymond P. ;
et al. |
June 25, 2020 |
Compounds and Their Use for Reducing Uric Acid Levels
Abstract
Bifunctional compounds that increase uric acid excretion and
reduce uric acid production, and monofunctional compounds that
either increase uric acid excretion or reduce uric acid production.
Methods of using these compounds for reducing uric acid levels in
blood or serum, for treating disorders associated with excess uric
acid, and for maintaining normal uric acid levels in blood or
serum, or the whole body, are also provided. Pharmaceutical
compositions comprising the bifunctional and monofunctional
compounds are also provided.
Inventors: |
Warrell, JR.; Raymond P.;
(Westfield, NJ) ; Piwinski; John J.; (Lebanon,
NJ) ; Larivee; Alexandre; (Montreal, CA) ;
Siddiqui; Arshad; (Newton, MA) ; Thai; Karen;
(Ottawa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Acquist LLC |
Westfield |
NJ |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20190117654 A1 |
April 25, 2019 |
|
|
Family ID: |
60913101 |
Appl. No.: |
16/227398 |
Filed: |
December 20, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2017/040836 |
Jul 6, 2017 |
|
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16227398 |
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62358669 |
Jul 6, 2016 |
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62732737 |
Sep 18, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 19/06 20180101;
C07D 403/14 20130101; C07D 401/14 20130101; A61K 31/513 20130101;
C07D 417/14 20130101; C07D 403/12 20130101 |
International
Class: |
A61K 31/513 20060101
A61K031/513; A61P 19/06 20060101 A61P019/06; C07D 417/14 20060101
C07D417/14; C07D 403/12 20060101 C07D403/12; C07D 401/14 20060101
C07D401/14 |
Claims
1. A compound selected from the group consisting of a) compounds
having a structure represented by Formula (II): ##STR00048##
wherein W, X, and Y are each independently O, S, NR.sup.2 or
N(R.sup.2).sub.2; A is phenyl, heteroaryl, C5-C10 branched or
unbranched cycloalkyl, C6-C10 bicycloalkyl or C5-C10
spirocycloalkyl; each Z is independently present or absent and, if
present, is independently selected from one or more halogen atoms,
--CN, --CF.sub.3, --OCF.sub.3, --OCHF.sub.2, --OR.sup.2,
--C(O)R.sup.2, SR.sup.2, --S(O).sub.gR.sup.3 where g is 1 or 2,
--N(R.sup.2).sub.2, --NO.sub.2, --CO.sub.2R.sup.2,
--OCO.sub.2R.sup.3, OC(O)R.sup.2, --CON(R.sup.2).sub.2,
--NR.sup.2C(O)R.sup.2, --SO.sub.2N(R.sup.2).sub.2,
--NR.sup.2SO.sub.2R.sup.3, --NR.sup.2SO.sub.2N(R.sup.2).sub.2 or
--NR.sup.2C(O)N(R.sup.2).sub.2, --C(O)NHOR.sup.2, alkyl, aryl,
alkenyl, alkynyl and cycloalkyl; wherein each R.sup.1 is C1-C8
branched or unbranched alkyl, optionally substituted with Z;
wherein each R.sup.2 is independently H, alkyl or aryl; wherein
each R.sup.3 is independently alkyl or aryl, optionally substituted
with one or more halogen atoms or OR.sup.2; and wherein a, b, c, d,
and e are each independently carbon or nitrogen, or four of a, b,
c, d, and e are each independently carbon or nitrogen and one of a,
b, c, d, and e is O, with the proviso that at least one of a, b, c,
d and e is nitrogen, and Z is not connected directly to nitrogen or
oxygen; and b) tautomers of any of the foregoing compounds.
2. The compound according to claim 1, wherein three of a, b, c, d,
and e are nitrogen.
3. The compound according to claim 1, wherein R.sup.1 is
--CH.sub.3.
4. The compound according to claim 3, wherein --XR.sup.1 is
--SCH.sub.3 or --OCH.sub.3.
5. The compound according to claim 4, wherein A is substituted
phenyl.
6. The compound according to claim 5, wherein A is --CF.sub.3
substituted phenyl
7. The compound according to claim 6, which has a structure
represented by Formula (II.sub.dd).
8. The compound according to claim 1, which is selected from the
group consisting of compounds having a structure represented by
Formula (II.sub.i) Formula (II.sub.j), Formula (II.sub.k), Formula
(II.sub.l), Formula (II.sub.m), Formula (II.sub.n), Formula
(II.sub.o), Formula (II.sub.p), Formula (II.sub.q), Formula
(II.sub.r), Formula (II.sub.s), Formula (II.sub.t), Formula
(II.sub.u), Formula (II.sub.v), Formula (II.sub.w), Formula
(II.sub.x), Formula (II.sub.y), Formula (II.sub.z), Formula Formula
(II.sub.aa), Formula (II.sub.bb), Formula (II.sub.cc), Formula
(II.sub.dd), Formula (II.sub.ee), Formula (II.sub.ff), Formula
(II.sub.gg), Formula (II.sub.hh), Formula (II.sub.ii), Formula
(II.sub.jj), Formula (II.sub.kk), Formula (II.sub.ll), Formula
(II.sub.mm), Formula (II.sub.2), Formula (II.sub.3), Formula
(II.sub.4), Formula (II.sub.5), Formula (II.sub.6), Formula
(II.sub.7), Formula (II.sub.8), Formula (II.sub.9), Formula
(II.sub.10), Formula (II.sub.11), and tautomers thereof.
9. A compound selected from the group consisting of a) compounds
having a structure represented by Formula (I): ##STR00049## wherein
W, X, and Y are each independently O, S, NR.sup.2 or
N(R.sup.2).sub.2; T is --CONR.sup.2--, --C(NR.sup.2)NH--,
--C(NOR.sup.2)NH--, --C(N--NR.sup.2)NH--, --C(SR.sup.2)N--, or
--NHC(O)--; A is phenyl, heteroaryl, C5-C10 branched or unbranched
cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl; each Z
is independently present or absent and, if present, is
independently selected from one or more halogen atoms, --CN,
--CF.sub.3, --OR.sup.2, --C(O)R.sup.2, SR.sup.2,
--S(O).sub.gR.sup.3 where g is 1 or 2, --N(R.sup.2).sub.2,
--NO.sub.2, --CO.sub.2R.sup.2, --OCO.sub.2R.sup.3, OC(O)R.sup.2,
--CON(R.sup.2).sub.2, --NR.sup.2C(O)R.sup.2,
--SO.sub.2N(R.sup.2).sub.2, --NR.sup.2SO.sub.2R.sup.3,
--NR.sup.2SO.sub.2N(R.sup.2).sub.2 or
--NR.sup.2C(O)N(R.sup.2).sub.2, --C(O)NHOR.sup.2, alkyl, aryl,
alkenyl and alkynyl; wherein each R.sup.2 is independently H, alkyl
or aryl; wherein each R.sup.3 is independently alkyl or aryl,
optionally substituted with one or more halogen atoms or OR.sup.2;
and wherein a, b, c, d, and e are each independently carbon or
nitrogen, or four of a, b, c, d, and e are each independently
carbon or nitrogen and one of a, b, c, d, and e is O, with the
proviso that at least one of a, b, c, d and e is nitrogen, and Z is
not connected directly to nitrogen or oxygen; and b) tautomers of
any of the foregoing compounds.
10. The compound according to claim 9, wherein A is thiazole or
isothiazole.
11. The compound according to claim 10, wherein three of a, b, c,
d, and e are nitrogen.
12. The compound according to claim 11, which has a structure
represented by Formula (I.sub.o).
13. A pharmaceutical composition comprising a compound having a
structure represented by Formula (I) or Formula (II); a tautomer of
Formula (I) of Formula (II), or a combination thereof, and a
pharmaceutically acceptable carrier.
14. The pharmaceutical composition according to claim 13, which
comprises a compound having a structure represented by Formula
(II.sub.d) or a tautomer thereof.
15. A method for reducing uric acid levels in blood, serum or a
whole body of a subject, or for preventing elevation of uric acid
levels in blood, serum or a whole body of a subject, or for
treating or preventing a disorder associated with excess uric acid,
comprising administering to a subject in need thereof a compound
having a structure represented by Formulae (I) or Formula (II); a
tautomer of Formula (I) or Formula (II), or a combination thereof,
in an amount effective to reduce blood or serum uric acid
levels.
16. The method according to claim 15, wherein the disorder
associated with excess uric acid is selected from gout,
hyperuricemia, tumor lysis syndrome, kidney disease, arthritis,
kidney stones, kidney failure, urolithiasis, plumbism,
hyperparathyroidism, psoriasis, inborn genetic errors of metabolism
such as Lesch-Nyhan syndrome, sarcoidosis, nonalcoholic fatty liver
disease (NAFLD), nonalcoholic steatohepatitis (NASH),
cardiovascular disease, atherosclerosis, hypertension, obesity,
diabetes, insulin resistance, metabolic syndrome, and
transplantation of blood, bone marrow or solid organs.
17. The method according to claim 15, which comprises
administration of a compound having a structure represented by
Formula (II.sub.dd).
18. The method according to claim 15, wherein the compound,
tautomer or combination thereof is administered parenterally,
intraperitoneally, intravenously, intranasally, intrarectally, or
orally.
19. The method according to claim 18, wherein the compound,
tautomer or combination thereof is administered by injection,
infusion, or oral administration.
20. The method according to claim 15, wherein the compound,
tautomer or combination thereof is administered intermittently.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Appln. No. PCT/US2017/040836, filed Jul. 6, 2017, which claims
priority to U.S. Provisional Appln. No. 62/358,669, filed Jul. 6,
2016, the disclosures of which are incorporated by reference in
their entireties.
TECHNICAL FIELD
[0002] This invention relates to pharmaceutical compositions and
methods for reducing uric acid in blood or serum of a subject, or
in the whole body of a subject, employing bifunctional and
monofunctional compounds as active agents.
BACKGROUND
[0003] Uric acid (UA) has emerged as a central regulator of
inflammation and fat formation in various human illnesses. Gout
afflicts more than 8 million U.S. subjects, and is associated with
chronic elevation of uric acid (UA) in blood. The incidence of this
condition has doubled in the past ten years. When UA exceeds
solubility limits, it forms crystals that settle into joints and
kidney, causing severe pain, destructive arthritis, and kidney
failure. Uric acid itself is known to be directly toxic to a number
of other tissues, and the generation of uric acid by xanthine
oxidase is also known to be toxic due to oxidative stress induced
by release of oxygen free radicals. In addition, nonalcoholic
steatohepatitis (NASH) I disease associated by excess production
liver fat that triggers hepatitis and leads to cirrhosis--is
expected to become the most common global contributor to requisite
liver transplantation. Treatment for disorders associated with
excess uric acid such as chronic gout and NASH entails extended--if
not lifelong--therapy focused on reducing UA production or
increasing its excretion. For example, the standard-of-care for
initial therapy of gout is allopurinol, a drug that inhibits
xanthine oxidase (XO), a key production enzyme. Launched in 2009,
Uloric.RTM. (febuxostat; Takeda), has similar activity as an XO
inhibitor with somewhat higher efficacy and improved safety.
Xanthine oxidase inhibitors are used as initial therapy in more
than 90% of gout patients; nonetheless, the therapeutic target is
achieved in less than one-third of patients, the drugs have
multiple side effects, and hypersensitivity (especially to
allopurinol) is common. Given that most patients do not actually
respond, the continued use of ineffective treatment administered
over many months in order to determine the low percentage of
patients who might respond represents an important burden to
patients as well as substantial costs to global healthcare systems,
Moreover, the high proportion of failures causes many patients to
become non-compliant with therapy and thus at increased risk for
development of chronic complications of gout, especially
destructive arthritis and renal insufficiency. Lastly, the U.S. FDA
has issued a warning letter to physicians that Uloric.RTM. is
associated with an increase in cardiac and all-cause mortality.
[0004] Since 2000, rapid advances in the biology of proteins known
as transporters have presented an array of new drug targets. The
enzyme URAT1 is a high capacity renal transporter that reabsorbs
most of the UA that is initially filtered into the urine from the
blood by the kidney. Inhibitors of certain urate transporters may
prevent such reabsorption and thereby increase UA excretion.
Several drugs are now known to inhibit URAT1, including
benzbromarone (approved but withdrawn in the US by Sanofi in 2003),
and lesinurad (Zurampic.RTM., AstraZeneca), which was approved in
the U.S. and EU in 2016.
[0005] These drugs are all mono-functional. That is, they inhibit
only one of the two equilibrium paths that reduce the levels of UA
in blood (i.e., decreased production or increased excretion).
Allopurinol is an example of a drug that decreases UA production by
inhibiting xanthine oxidase, but it has no effect on renal
excretion. As expected, allopurinol does not affect the activity of
URAT1 or other renal urate transporters. Benzbromarone and
lesinurad increase UA excretion (i.e., they promote uricosuria)
primarily via inhibition of URAT1, but these agents have no effect
on UA production, since they have no substantial effect on xanthine
oxidase. Since xanthine oxidase inhibition is the principal,
preferred, and primary 1.sup.st-line form of treatment for
hyperuricemia, agents that promote uricosuria are used second-line
and are commonly employed only in combination with xanthine oxidase
inhibitors rather than as single-agents. However, because of
structural similarities, other transporter enzymes may also be
inhibited by URAT1 inhibitors, including but not limited to organic
anion transporters such as OAT1, OAT3 and GLUT9a/b.
[0006] Non-sedating 5-carboxanilide derivatives of barbiturates,
including merbarone (5-(N-phenylcarboxamido)-2-thio-barbituric
acid), have been evaluated as potential cytotoxic anticancer drugs.
Subsequently, it was discovered that clinical treatment with
merbarone was associated with a marked reduction of UA levels in
blood. Despite these discoveries, the cytotoxic activity of
merbarone completely precluded its use as a treatment for a chronic
disorder associated with excess uric acid, since the safety of such
use (primarily its genotoxicity) posed a serious risk to other
aspects of human health. Such clinical utility would only be
possible if the genotoxic activity could be chemically dissociated
and eliminated from the hypouricemic activity. The inventors have
since described a number of non-genotoxic hypouricemic derivatives
of merbarone.
[0007] There exists a compelling need for new drugs than can reduce
UA levels in blood or the whole body and provide better treatment
for patients afflicted by gout. Reduction in UA is universally
acknowledged as beneficial for patients with gout, as well as other
disorders associated with excess uric acid, and such reduction is
directly linked to patient benefit. More specifically, reduction of
serum uric acid below a "target" level is accepted by international
drug regulatory agencies (e.g., the U.S. Food and Drug
Administration [FDA], the European Medicines Agency [EMA], etc.) as
an endpoint for commercial drug approval in gout. As previously
noted, drugs that can overcome the limited clinical activity of
xanthine oxidase inhibitors are available or are currently being
investigated, but only as "add-ons" for combination use. The
approval of lesinurad [Zurampic.RTM.] is the most recent example.
The present invention relates to new compounds that can provide
alternatives to current therapy for elevated UA levels and
treatment of other disorders associated with excess uric acid such
as gout. Certain of these compounds have the particular advantage
of bifunctional activity (i.e., decreasing UA production by
inhibiting xanthine oxidase and increasing UA excretion by
inhibiting a renal urate transporter), making them suitable for use
as initial therapy and as single agents rather than "add-on"
therapies. In addition, certain of the compounds have reduced
toxicity compared to prior art drugs such as merbarone.
SUMMARY
[0008] In a first aspect, compounds having a structure represented
by Formula (I) are provided:
##STR00001##
wherein [0009] W, X, and Y are each independently O, S, NR.sup.2 or
N(R.sup.2).sub.2; [0010] T is --CONR.sup.2--, --C(NR.sup.2)NH--,
--C(NOR.sup.2)NH--, --C(N--NR.sup.2)NH--, --C(SR.sup.2)N--, or
--NHC(O)--; A is phenyl, heteroaryl, C5-C10 branched or unbranched
cycloalkyl, C6-C10 bicycloalkyl or C5-C10 spirocycloalkyl; [0011]
each Z is independently present or absent and, if present, is
independently selected from one or more halogen atoms, --CN,
--CF.sub.3, --OR.sup.2, --C(O)R.sup.2, SR.sup.2,
--S(O).sub.gR.sup.3 where g is 1 or 2, --N(R.sup.2).sub.2,
--NO.sub.2, --CO.sub.2R.sup.2, --OCO.sub.2R.sup.3, OC(O)R.sup.2,
--CON(R.sup.2).sub.2, --NR.sup.2C(O)R.sup.2,
--SO.sub.2N(R.sup.2).sub.2, --NR.sup.2SO.sub.2R.sup.3,
--NR.sup.2SO.sub.2N(R.sup.2).sub.2 or
--NR.sup.2C(O)N(R.sup.2).sub.2, --C(O)NHOR.sup.2, alkyl, aryl,
alkenyl and alkynyl; wherein each R.sup.2 is independently H, alkyl
or aryl; wherein each R.sup.3 is independently alkyl or aryl,
optionally substituted with one or more halogen atoms or OR.sup.2;
and wherein a, b, c, d, and e are each independently carbon or
nitrogen, or four of a, b, c, d, and e are each independently
carbon or nitrogen and one of a, b, c, d, and e is O, with the
proviso that at least one of a, b, c, d and e is nitrogen, and Z is
not connected directly to nitrogen or oxygen.
[0012] In a second aspect, compounds having a structure represented
by Formula (II) are provided:
##STR00002##
wherein [0013] W, X, and Y are each independently O, S, NR.sup.2 or
N(R.sup.2).sub.2; [0014] A is phenyl, heteroaryl, C5-C10 branched
or unbranched cycloalkyl, C6-C10 bicycloalkyl or C5-C10
spirocycloalkyl; [0015] each Z is independently present or absent
and, if present, is independently selected from one or more halogen
atoms, --CN, --CF.sub.3, --OCF.sub.3, --OCHF.sub.2, --OR.sup.2,
--C(O)R.sup.2, SR.sup.2, --S(O).sub.gR.sup.3 where g is 1 or 2,
--N(R.sup.2).sub.2, --NO.sub.2, --CO.sub.2R.sup.2,
--OCO.sub.2R.sup.3, OC(O)R.sup.2, --CON(R.sup.2).sub.2,
--NR.sup.2C(O)R.sup.2, --SO.sub.2N(R.sup.2).sub.2,
--NR.sup.2SO.sub.2R.sup.3, --NR.sup.2SO.sub.2N(R.sup.2).sub.2 or
--NR.sup.2C(O)N(R.sup.2).sub.2, --C(O)NHOR.sup.2, alkyl, aryl,
alkenyl, alkynyl and cycloalkyl; wherein each R.sup.1 is C1-C8
branched or unbranched alkyl, optionally substituted with Z;
wherein each R.sup.2 is independently H, alkyl or aryl; wherein
each R.sup.3 is independently alkyl or aryl, optionally substituted
with one or more halogen atoms or OR.sup.2; and wherein a, b, c, d,
and e are each independently carbon or nitrogen, or four of a, b,
c, d, and e are each independently carbon or nitrogen and one of a,
b, c, d, and e is O, with the proviso that at least one of a, b, c,
d and e is nitrogen, and Z is not connected directly to nitrogen or
oxygen.
[0016] A further aspect relates to methods for reducing uric acid
levels in blood or serum of a subject, or preventing elevation of
uric acid levels in blood or serum of a subject, comprising
administering a compound having a structure represented by Formula
(I), Formula (II), or a combination thereof, to a subject in need
thereof in an amount effective to reduce blood or serum uric acid
levels or prevent elevation of blood or serum uric acid levels. In
a modification of this embodiment, the methods comprise
administering a compound according to a specific embodiment of the
compounds of Formula (I), Formula (II), or a combination thereof,
as described above, to a subject in need thereof in an amount
effective to reduce blood or serum uric acid levels or prevent
elevation of blood or serum uric acid levels.
[0017] In certain embodiments of these methods, a compound having a
structure represented by Formula (I), Formula (II), or a
combination thereof, is administered to a subject with a disorder
associated with excess uric acid such as gout, hyperuricemia,
kidney disease, arthritis, kidney stones, kidney failure,
urolithiasis, plumbism, hyperparathyroidism, psoriasis, inborn
genetic errors of metabolism (including but not limited to
Lesch-Nyhan syndrome), sarcoidosis, cardiovascular disease
(including but not limited to atherosclerosis and hypertension),
diabetes or insulin resistance, obesity, metabolic syndrome, or
transplantation of blood, bone marrow or solid organs, to reduce
uric acid levels.
[0018] A further aspect relates to methods for treating a disorder
associated with excess uric acid associated with or caused by
elevated uric acid in blood or serum comprising administering to a
subject in need thereof a compound having a structure represented
by Formula (I), Formula (II), or a combination thereof, in an
amount effective to reduce blood or serum uric acid levels or
prevent elevation of blood or serum uric acid levels, thereby
treating the disorder associated with excess uric acid. One such
embodiment relates to methods for treating a disorder of excess
uric acid associated with or caused by elevated uric acid in blood
or serum comprising administering to the subject a compound
according to a specific embodiment of the compounds of Formula (I),
Formula (II), or a combination, as described above.
[0019] A further aspect of the invention provides pharmaceutical
compositions comprising a compound having a structure represented
by Formula (I), Formula (II), or a combination thereof, and a
pharmaceutically acceptable carrier. In a specific embodiment, the
pharmaceutical composition comprises a compound according to a
specific embodiment of the compounds of Formula (I), Formula (II),
or a combination thereof, as described above.
[0020] A further aspect provides methods for synthesizing the
compounds discussed above, as discussed in more detail below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 illustrates a general scheme for synthesis of
compounds according to Formula (I).
[0022] FIG. 2 illustrates a general scheme for synthesis of
compounds according to Formula (II).
[0023] FIG. 3 illustrates an alternative general scheme for
synthesis of compounds according to Formula (II).
[0024] FIG. 4 illustrates a general scheme for synthesis of
compounds containing a substituent on X of the barbiturate
ring.
[0025] FIG. 5 illustrates an alternative general scheme for
synthesis of compounds containing a substituent on X of the
barbiturate ring.
[0026] FIG. 6 illustrates a further alternative general scheme for
synthesis of compounds containing a substituent on X of the
barbiturate ring.
[0027] FIG. 7 illustrates a method for forming the triazole
heterocyclic ring when it is not known in the art.
[0028] FIG. 8 illustrates alternative general schemes for synthesis
of the A ring of the compounds.
[0029] FIG. 9 illustrates the synthesis scheme described in Example
1.
[0030] FIG. 10 illustrates the synthesis scheme described in
Example 2.
[0031] FIG. 11 illustrates the synthesis scheme described in
Example 3.
[0032] FIG. 12 illustrates the synthesis scheme described in
Example 4.
[0033] FIG. 13 illustrates the synthesis scheme described in
Example 5.
[0034] FIG. 14 illustrates the synthesis scheme described in
Example 6.
[0035] FIG. 15 illustrates the synthesis scheme described in
Example 7.
[0036] FIG. 16 illustrates the synthesis scheme described in
Example 8.
[0037] FIG. 17 illustrates the synthesis scheme described in
Example 9.
[0038] FIG. 18 illustrates the synthesis scheme described in
Example 10.
[0039] FIG. 19 illustrates the synthesis scheme described in
Example 11.
[0040] FIG. 20 illustrates the synthesis scheme described in
Example 12.
[0041] FIG. 21 illustrates the synthesis scheme described in
Example 13.
[0042] FIG. 22 illustrates the synthesis scheme described in
Example 14.
[0043] FIG. 23 illustrates the synthesis scheme described in
Example 15.
[0044] FIG. 24 illustrates the synthesis scheme described in
Example 16.
[0045] FIG. 25 illustrates the synthesis scheme described in
Example 17.
[0046] FIG. 26 illustrates the synthesis scheme described in
Example 18.
[0047] FIG. 27 illustrates the synthesis scheme described in
Example 19.
[0048] FIG. 28 illustrates the synthesis scheme described in
Example 20.
[0049] FIG. 29 illustrates the synthesis scheme described in
Example 21.
[0050] FIG. 30 illustrates the synthesis scheme described in
Example 22.
[0051] FIG. 31 illustrates the synthesis scheme described in
Example 23.
[0052] FIG. 32 illustrates the synthesis scheme described in
Example 24.
[0053] FIG. 33 illustrates the synthesis scheme described in
Example 25.
[0054] FIG. 34 illustrates the synthesis scheme described in
Example 26.
[0055] FIG. 35 illustrates the synthesis scheme described in
Example 27.
[0056] FIG. 36 illustrates the synthesis scheme described in
Example 28.
[0057] FIG. 37 illustrates the synthesis scheme described in
Example 29.
[0058] FIG. 38 illustrates the synthesis scheme described in
Example 30.
[0059] FIG. 39 illustrates the synthesis scheme described in
Example 31.
[0060] FIG. 40 illustrates the synthesis scheme described in
Example 32.
[0061] FIG. 41 illustrates an alternative general scheme for
synthesis of compounds according to Formula (II).
[0062] FIG. 42 illustrates an alternative general scheme for
synthesis of compounds according to Formula (I).
[0063] FIG. 43A and FIG. 43B illustrate general schemes for
synthesis of compounds according to Formula (II).
[0064] FIG. 44A and FIG. 44B illustrate additional general schemes
for synthesis of compounds according to Formula (II).
[0065] FIG. 45 illustrates the synthesis scheme described in
Example 33.
[0066] FIG. 46 illustrates the synthesis scheme described in
Example 34.
[0067] FIG. 47 illustrates the synthesis scheme described in
Example 35.
[0068] FIG. 48 illustrates the synthesis scheme described in
Example 36.
[0069] FIG. 49 illustrates the synthesis scheme described in
Example 37.
[0070] FIG. 50 illustrates the synthesis scheme described in
Example 38.
[0071] FIG. 51 illustrates the synthesis scheme described in
Example 39.
[0072] FIG. 52 illustrates the synthesis scheme described in
Example 40.
[0073] FIG. 53 illustrates the synthesis scheme described in
Example 41.
[0074] FIG. 54 illustrates the synthesis scheme described in
Example 42.
DETAILED DESCRIPTION
[0075] Before describing several exemplary embodiments provided
herein, it is to be understood that the invention is not limited to
the details of construction or process steps set forth in the
following description. The invention is capable of other
embodiments and of being practiced or being carried out in various
ways.
[0076] Reference throughout this specification to "one embodiment,"
"certain embodiments," "one or more embodiments" or "an embodiment"
means that a particular feature, structure, material, or
characteristic described in connection with the embodiment is
included in at least one embodiment of the invention. Thus, the
appearances of the phrases such as "in one or more embodiments,"
"in certain embodiments," "in one embodiment" or "in an embodiment"
in various places throughout this specification are not necessarily
referring to the same embodiment of the invention. Furthermore, the
particular features, structures, materials, or characteristics may
be combined in any suitable manner in one or more embodiments.
[0077] As used herein, the term "bifunctional" with respect to
disclosed compounds means that the compound inhibits both a renal
transporter, including but not limited to URAT1, and xanthine
oxidase. The potency of inhibition of either target may vary, but
in general an IC50 of less than about 100 .mu.M for both xanthine
oxidase and a renal transporter such as URAT1 is considered
bifunctional. An IC50 of less than about 50 .mu.M for both xanthine
oxidase and URAT1 is considered a particularly active bifunctional
compound, and an IC50 of less than 10 .mu.M is considered a highly
potent bifunctional compound.
[0078] As used herein, the term "monofunctional" with respect to
disclosed compounds means that the compound inhibits an enzyme in
the uric acid metabolic pathway involved in uric acid excretion
that is either a renal transporter, including but not limited to
URAT1, or an enzyme involved in uric acid production, including but
not limited to xanthine oxidase, but not both. The potency of
inhibition of single target may vary, but in general an IC50 of
greater than about 100 .mu.M for one of xanthine oxidase or URAT1,
and an IC50 of less than about 100 NM for the other of xanthine
oxidase or URAT1, is considered monofunctional. An IC50 of less
than about 50 .mu.M for one of xanthine oxidase or URAT1, and an
IC50 of greater than about 100 .mu.M for the other of xanthine
oxidase or URAT1, is considered a particularly active
monofunctional compound. An IC50 of less than about 10 .mu.M for
one of xanthine oxidase or URAT1, and an IC50 of greater than about
100 .mu.M for the other of xanthine oxidase or URAT1, is considered
a highly potent monofunctional compound. As previously noted, other
transporter enzymes may also be inhibited by "bifunctional"
inhibitors or by "monofunctional" URAT1 inhibitors, including but
not limited to organic anion transporters such as OAT1, OAT3 and
GLUT9a/b.
[0079] As used herein, the term "treatment" refers to reducing
elevated uric acid levels in blood or serum, preferably by reducing
levels to the normal, low-normal or sub-normal range, with an
overall goal of relieving symptoms and/or preventing recurrences of
active disease. For example, a typical "therapeutic target" for
treatment of elevated serum uric acid is a level .ltoreq.6.0 mg/dL.
"Elevated" uric acid levels generally refer above-normal uric acid
levels, as long-term elevated levels can result in conditions that
require additional treatment.
[0080] As used herein, the term "preventing" elevation of uric acid
levels in blood or serum refers to maintaining normal or
therapeutically acceptable uric acid levels in blood or serum in a
subject who would otherwise experience an increase in uric acid
levels, with an overall goal of preventing development or
recurrence of symptoms and/or preventing recurrences of active
disease. It will be appreciated that prevention of elevation of
uric acid levels, or achievement of a sustained reduction in uric
acid, is a goal of the long-term maintenance therapy discussed
below, as well as certain short-term conditions.
[0081] The numbering of the positions on the barbiturate ring used
herein follows the convention of Warrell (U.S. Pat. No. 4,880,811).
It is also to be understood that although the compounds disclosed
herein are generally illustrated by specific chemical structures,
the disclosure of the compounds is intended to include their
tautomers. Representative examples of tautomers in the barbiturate
ring include the structures depicted below, as well as any
additional tautomers on the substituents of Formula (I) or Formula
(II):
##STR00003##
[0082] The compounds described herein meet certain needs in the
therapeutic field of reduction of uric acid levels in blood and
treatment of disorders associated with excess uric acid in blood or
serum, or in the whole body. Certain of the compounds are potent
monofunctional inhibitors of URAT1 or xanthine oxidase. Certain of
the compounds are bifunctional inhibitors of both URAT1 and
xanthine oxidase.
[0083] The improved biological activity profile of the compounds of
the invention and their potency make these compounds useful new
drugs for reducing uric acid levels in blood or the whole body, and
for treating disorders associated with, or caused by, excess uric
acid levels in blood or serum or the whole body, including gout. Of
particular significance is the advantage that the bifunctional
compounds can be used effectively as monotherapy for reducing uric
acid levels in blood, for treating or preventing disorders
associated with excess uric acid uric acid, and specifically for
treating gout. In certain embodiments, the bifunctional compounds
can be used effectively for treating or preventing non-alcoholic
fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH),
and metabolic syndrome, atherosclerosis or other forms of
carciovascular disease, hypertension, chronic kidney disease,
obesity, diabetes or insulin resistance, and metabolic
syndrome.
[0084] In a first aspect, compounds having a structure represented
by Formula (I) are provided:
##STR00004##
wherein [0085] W, X, and Y are each independently O, S, NR.sup.2 or
N(R.sup.2).sub.2; [0086] T is --CONR.sup.2--, --C(NR.sup.2)NH--,
--C(NOR.sup.2)NH--, --C(N--NR.sup.2)NH--, --C(SR.sup.2)N--, or
--NHC(O)--; [0087] A is phenyl, heteroaryl, C5-C10 branched or
unbranched cycloalkyl, C6-C10 bicycloalkyl or C5-C10
spirocycloalkyl; [0088] each Z is independently present or absent
and, if present, is independently selected from one or more halogen
atoms, --CN, --CF.sub.3, --OR.sup.2, --C(O)R.sup.2, SR.sup.2,
--S(O).sub.gR.sup.3 where g is 1 or 2, --N(R.sup.2).sub.2,
--NO.sub.2, --CO.sub.2R.sup.2, --OCO.sub.2R.sup.3, OC(O)R.sup.2,
--CON(R.sup.2).sub.2, --NR.sup.2C(O)R.sup.2,
--SO.sub.2N(R.sup.2).sub.2, --NR.sup.2SO.sub.2R.sup.3,
--NR.sup.2SO.sub.2N(R.sup.2).sub.2 or
--NR.sup.2C(O)N(R.sup.2).sub.2, --C(O)NHOR.sup.2, alkyl, aryl,
alkenyl and alkynyl; wherein each R.sup.2 is independently H, alkyl
or aryl; wherein each R.sup.3 is independently alkyl or aryl,
optionally substituted with one or more halogen atoms or OR.sup.2;
and wherein a, b, c, d, and e are each independently carbon or
nitrogen, or four of a, b, c, d, and e are each independently
carbon or nitrogen and one of a, b, c, d, and e is O, with the
proviso that at least one of a, b, c, d and e is nitrogen, and Z is
not connected directly to nitrogen or oxygen.
[0089] In one or more embodiments, the compound having a structure
represented by Formula (I) is a compound wherein T is
--CONR.sup.2--.
[0090] In one or more embodiments, the compound having a structure
represented by Formula (I) is a compound wherein the 5-member
heterocyclic ring is a substituted or unsubstituted triazole, or a
substituted or unsubstituted tetrazole (i.e., a, b, c, d, and e are
each independently carbon or nitrogen, with the proviso that three
or four of a, b, c, d and e are nitrogen, and Z is not connected
directly to nitrogen).
[0091] In one or more embodiments, the compound having a structure
represented by Formula (I) is a compound wherein A is heteroaryl
having two heteroatoms, for example thiazole or isothiazole.
[0092] In a specific non-limiting embodiment, the compound having a
structure represented by Formula (I) is a compound wherein X is O
or S; Y and W are O; A is substituted or unsubstituted thiazole or
isothiazole; each Z is independently present or absent; each
R.sup.2 is H, and; the 5-member heterocyclic ring is substituted,
or unsubstituted triazole or substituted or unsubstituted tetrazole
(i.e., a, b, c, d, and e are each independently carbon or nitrogen,
with the proviso that three or four of a, b, c, d and e are
nitrogen, and Z is not connected directly to nitrogen).
[0093] In a further specific non-limiting embodiment, the compound
having a structure represented by Formula (I) is a compound wherein
W, X and Y are each independently O or S; T is CONR.sup.2; A is
heteroaryl; Z is absent; R.sup.2 is H, and; the 5-member
heterocyclic ring is triazole. In one or more of these embodiments,
the heteroaryl A is thiazole or isothiazole.
[0094] Specific examples of compounds having a structure
represented by Formula (I) include the following:
##STR00005##
[0095] In a second aspect, compounds having a structure represented
by Formula (II) are provided:
##STR00006##
wherein [0096] W, X, and Y are each independently O, S, NR.sup.2 or
N(R.sup.2).sub.2; [0097] A is phenyl, heteroaryl, C5-C10 branched
or unbranched cycloalkyl, C6-C10 bicycloalkyl or C5-C10
spirocycloalkyl; [0098] each Z is independently present or absent
and, if present, is independently selected from one or more halogen
atoms, --CN, --CF.sub.3, --OCF.sub.3, --OCHF.sub.2, --OR.sup.2,
--C(O)R.sup.2, SR.sup.2, --S(O).sub.gR.sup.3 where g is 1 or 2,
--N(R.sup.2).sub.2, --NO.sub.2, --CO.sub.2R.sup.2,
--OCO.sub.2R.sup.3, OC(O)R.sup.2, --CON(R.sup.2).sub.2,
--NR.sup.2C(O)R.sup.2, --SO.sub.2N(R.sup.2).sub.2,
--NR.sup.2SO.sub.2R.sup.3, --NR.sup.2SO.sub.2N(R.sup.2).sub.2 or
--NR.sup.2C(O)N(R.sup.2).sub.2, --C(O)NHOR.sup.2, alkyl, aryl,
alkenyl, alkynyl and cycloalkyl; wherein each R.sup.1 is C1-C8
branched or unbranched alkyl, optionally substituted with Z;
wherein each R.sup.2 is independently H, alkyl or aryl; wherein
each R.sup.3 is independently alkyl or aryl, optionally substituted
with one or more halogen atoms or OR.sup.2; and wherein a, b, c, d,
and e are each independently carbon or nitrogen, or four of a, b,
c, d, and e are each independently carbon or nitrogen and one of a,
b, c, d, and e is O, with the proviso that at least one of a, b, c,
d and e is nitrogen, and Z is not connected directly to nitrogen or
oxygen.
[0099] In one or more embodiments, the 5-member heterocyclic ring
of the compound having a structure represented by Formula (II) is a
substituted or unsubstituted triazole, or substituted or
unsubstituted tetrazole (i.e., a, b, c, d, and e are each
independently carbon or nitrogen, with the proviso that three or
four of a, b, c, d and e are nitrogen, and Z is not connected
directly to nitrogen).
[0100] In one or more embodiments, the compound having a structure
represented by Formula (II) is a compound wherein R.sup.1 is
--CH.sub.3.
[0101] In a one or more embodimenst, the compound having a
structure represented by Formula (II) is a compound wherein
--XR.sup.1 is --SCH.sub.3, --SC(CH.sub.3) or --OCH.sub.3.
[0102] In a specific non-limiting embodiment, the compound having a
structure represented by Formula (II) is a compound wherein X is O,
S or N(R.sup.2).sub.2; Y and W are each independently O or S; A is
substituted or unsubstituted phenyl, bicycloalkyl, spirocycloalkyl,
pyridine or diazine; Z is alkyl, cycloalkyl, halogen, CF.sub.3, or
N(R.sup.2).sub.2; each R.sup.1 is C1-C3 branched or unbranched
alkyl, optionally substituted with Z; each R.sup.2 is H, and; the
5-member heterocyclic ring is substituted or unsubstituted
triazole, or substituted or unsubstituted tetrazole (i.e., a, b, c,
d, and e are each independently carbon or nitrogen, with the
proviso that three or four of a, b, c, d and e are nitrogen, and Z
is not connected directly to nitrogen).
[0103] Specific examples of compounds having a structure
represented by Formula (II) include the following:
[0104] 1. A compound wherein the 5-member heterocyclic ring is an
unsubstituted triazole or an unsubstituted tetrazole.
Representative examples of such compounds include: [0105] The
compound wherein A is substituted or unsubstituted phenyl; and X is
S; including certain exemplary compounds wherein R.sup.1 is
--CH.sub.3 or --C(CH.sub.3).sub.2; and tautomers thereof, such as a
structure represented by Formula (II.sub.i):
##STR00007##
[0106] a structure represented by Formula (II.sub.j):
##STR00008##
[0107] a structure represented by Formula (II.sub.k):
##STR00009##
[0108] a structure represented by Formula (II.sub.l):
##STR00010##
[0109] a structure represented by Formula (II.sub.m):
##STR00011##
[0110] a structure represented by Formula (II.sub.n):
##STR00012##
[0111] a structure represented by Formula (II.sub.o):
##STR00013##
[0112] a structure represented by Formula (II.sub.p):
##STR00014##
[0113] a structure represented by Formula (II.sub.q):
##STR00015##
[0114] a structure represented by Formula (II.sub.r):
##STR00016##
[0115] a structure represented by Formula (II.sub.s):
##STR00017##
[0116] a structure represented by Formula (II.sub.t):
##STR00018##
[0117] a structure represented by Formula (II.sub.u):
##STR00019##
[0118] a structure represented by Formula (II.sub.u):
##STR00020##
[0119] a structure represented by Formula (II.sub.w):
##STR00021##
[0120] a structure represented by Formula (II.sub.x):
##STR00022##
[0121] a structure represented by Formula (II.sub.y):
##STR00023##
[0122] a structure represented by Formula (II.sub.z):
##STR00024##
[0123] a structure represented by Formula (II.sub.aa):
##STR00025##
[0124] a structure represented by Formula (II.sub.bb):
##STR00026##
[0125] a structure represented by Formula (II.sub.cc):
##STR00027##
[0126] a structure represented by Formula (II.sub.dd):
##STR00028##
[0127] a structure represented by Formula (II.sub.ee):
##STR00029##
[0128] a structure represented by Formula (II.sub.2):
##STR00030##
[0129] a structure represented by Formula (II.sub.3):
##STR00031##
[0130] a structure represented by Formula (II.sub.4):
##STR00032##
[0131] a structure represented by Formula (II.sub.5):
##STR00033##
[0132] a structure represented by Formula (II.sub.6):
##STR00034##
and
[0133] a structure represented by Formula (II.sub.7):
##STR00035##
[0134] Among the foregoing class of compounds, particularly useful
compounds are the compounds wherein A is CF.sub.3-substituted
phenyl, X is S and R.sup.1 is --C(CH.sub.3).sub.2, and tautomers
thereof. [0135] The compound wherein A is unsubstituted phenyl; and
X is NR.sup.2 or N(R.sup.2).sub.2; and tautomers thereof, such as a
structure represented by Formula (II.sub.ll):
##STR00036##
[0135] and
[0136] a structure represented by Formula (II.sub.mm):
##STR00037## [0137] The compound wherein A is heteroaryl such as
substituted or unsubstituted pyridine; and X is S; including
certain exemplary compounds wherein R.sup.1 is --CH.sub.3; and
tautomers thereof, such as a structure represented by Formula
(II.sub.8):
##STR00038##
[0137] and
[0138] a structure represented by Formula (II.sub.9):
##STR00039## [0139] The compound wherein A is heteroaryl such as
substituted or unsubstituted quinoline; and X is S; including
certain exemplary compounds wherein R.sup.1 is --CH.sub.3; and
tautomers thereof, such as a structure represented by Formula
(II.sub.10):
##STR00040##
[0139] and
[0140] a structure represented by Formula (II.sub.11):
##STR00041##
[0141] 2. A compound wherein the 5-member heterocyclic ring is a
substituted triazole. Representative examples of such compounds
include: [0142] The compound wherein A is phenyl; X is O or S; and
R.sup.1 is methyl (CH.sub.3); and tautomers thereof, such as a
structure represented by Formula (II.sub.ff):
##STR00042##
[0142] and
[0143] a structure represented by Formula (II.sub.gg):
##STR00043##
[0144] 3. A compound wherein A is spirocycloalkyl. Representative
examples of such compounds include structures represented by
Formula (II.sub.hh):
##STR00044##
[0145] 4. A compound wherein A is pyridine or diazine.
Representative examples of such compounds include a structure
represented by Formula (II.sub.ii):
##STR00045##
[0146] a structure represented by Formula (II.sub.jj):
##STR00046##
and
[0147] a structure represented by Formula (II.sub.kk):
##STR00047##
[0148] As disclosed herein, reference to compounds having a
structure represented by Formula (I), Formula II, or a combination
thereof, is intended to include all compounds falling within the
generic structure, as well as the specific embodiments described
and their tautomers.
[0149] The compounds disclosed herein can be synthesized various
general procedures, for example as depicted in FIGS. 1-8, FIGS. 43A
and B, and FIGS. 44A and B. In general the various synthetic routes
center on the coupling of a substituted phenyl, heterocyclic,
cycloalkyl or spirocyclic A ring with an appropriately substituted
barbiturate ring. Several different coupling agents can be employed
in this process. Many of the compounds of Formula (I) can be made
as illustrated in FIG. 1 if the appropriately substituted nitro or
amino ring A is known in the art.
[0150] As shown in FIG. 2, for synthesis of Formula (II) compounds
4,6-dichloro-2-thiopyrimidine-5-carbonyl chloride with the
appropriate R.sup.1 group already incorporated can be protected
with various protecting groups, including with a para-methoxy
benzyl group. Other protecting groups are also possible, although
para-methoxy benzyl is preferred. This can then be reacted with the
appropriate amino derivative attached to ring A containing the
desired heterocycle, which is usually a triazine or tetrazole.
However, in many cases this moiety needs to be synthesized. This is
accomplished by employing a Sonogashira cross-coupling reaction
between the appropriately substituted halogen (e.g., bromine)
containing amino derivative with an acetylene. This acetylene is
best protected with a trimethylsilyl group at one end. The
trimethyl silyl group is then removed by base and the resulting
acetylene is reacted with the appropriate azide to produce the
intermediate that is to be coupled to the
4,6-dichloro-2-alkylthiopyrimidine-5-carbonyl chloride. The azide
may or may not be substituted with a protecting group. One such
group is azidomethyl pivalate, which can be subsequently removed by
base. The resultant coupled product is then deprotected by the
appropriate reagents. For example, the PMB groups can be removed by
acid and the pivalate group by base to produce the desired final
product. However, those skilled in the art will realize that other
protecting groups can be employed.
[0151] As shown in FIG. 3, for synthesis of Formula (II) compounds,
4,6-dichloro-2-(methylthio)pyrimidine-5-carbonyl chloride can be
protected with various protecting groups, including with a
para-methoxy benzyl group. Other protecting groups are also
possible, although para-methoxy benzyl is preferred. This can then
be reacted with the appropriate amino derivative attached to ring A
containing the desired heterocycle, which is usually a triazine or
tetrazole. This moiety can be protected with a protecting group or
not. In some cases the reactions work without a protecting group.
Once the two moieties are coupled together, the thiomethyl group
can be oxidized by a variety of agents including mCPBA. This
produces the methyl sulfone, which can be displaced by a variety
alkylated sulfhydryl agents. Then the protecting groups are
removed. The PMB group is usually removed by acid, such as triflic
acid. If there is a protecting group on the triazine or similar
nitrogen heterocycle, this group is removed by other agents
depending on the nature of the group. For example, methyl pivalate
is removed by base, trimethoxybenzyl by acid, and
[2-(Trimethylsilyl)ethoxy]methyl by fluoride. Those skilled in the
art will realize that other protecting groups are possible.
[0152] FIG. 41 illustrates a synthesis scheme that is similar to
the schemes described in FIG. 2 and FIG. 3 except that
4,6-dichloro-2-thiopyrimidine-5-carboxylic acid is used in place of
the acid chloride. In this case an appropriate coupling agent is
employed to form the bond between the amino heterocycle and the
bibratuate ring. Such agents include T.sub.3P/Et.sub.3N, EDC, DCC
and carbonyl di-miidazole, although there are many others that can
be employed, as one skilled in the art would know. If other
substituents are desired in addition to the methyl group on sulfur,
one skilled in the art would employ the scheme described in FIG.
13.
[0153] FIG. 42 illustrates synthesis of a compound of Formula (I),
wherein barbiturate acid is activated with an appropriate coupling
agent as described in other figures. This can then be reacted with
the appropriate amino derivative that contains a halogen, usually
bromine. Subsequent Sonogashira cross-coupling reaction with a
substituted acetylene then produces the intermediate that can be
reacted with the appropriately protected azide. In many cases
different protecting groups can be employed, but azidomethyl
pivalate is often preferred, in which the pivolate group can often
be easily removed by base.
[0154] FIGS. 4-6 depict methods of synthesis that result in
compounds containing a substituent on X of the barbiturate ring.
FIG. 4 is the most straightforward way of making such compounds, as
it generally follows the sequence depicted in FIG. 1. However, this
method involves a subsequent last step, which involves alkylation
of the X group. This is only possible if R.sup.2 is an alkyl group,
and separation of the various possible isomers obtained may be
necessary. The synthesis outlined in FIG. 5 is more direct in that
it ensures that the R.sup.2 group is attached to the barbiturate
ring. This process involves the introduction of the R.sup.2 group
early in the synthesis, first by condensation of malonate to an
appropriately substituted urea, followed by alkylation, which again
is conducted with an alkyl halide (i.e., R.sup.2 is alkyl). In
certain cases, a protecting group may be necessary depending on the
nature of the substituents. In another case the R.sup.2 group on
the X can be introduced in the earliest part of the synthesis. It
can be intact on the substituted urea or thiourea. This would allow
for compounds that contain an aryl or heterocyclic aryl group on X.
This is depicted in FIG. 6 and involves the condensation of the
R.sup.2 substituted urea or thiourea with malonate. The subsequent
barbiturate ring is then coupled to the appropriate amino A ring
compound to produce the desired product.
[0155] FIG. 7 depicts methods for forming the triazole heterocyclic
ring when it is not known in the art. Addition of azide to an amino
containing A ring can be accomplished via a variety of methods
which all involve the addition of azide to the acetylene.
Protecting groups as illustrated in the figure may be necessary.
Sometimes the acetylene containing A ring may not be known in the
art, so it needs to be synthesized. This can be accomplished via a
variety of methods as illustrated in FIG. 8. Treatment of the
corresponding aldehyde of the ring A compound with
1-diazo-1-((dimethylperoxy)(oxo)-.lamda..sup.4-phosphanyl)propan-2-one,
which is known in the art, or Sonogashira reaction on the halide of
a ring A compound would produce the corresponding acetylenic A ring
containing compound. Subsequent addition of azide to the acetylene
would produce the triazole. This can then be coupled by the methods
described above to produce the desired targeted compound.
[0156] As shown in FIG. 43A, for synthesis of Formula (II)
compounds 4,6-dichloro-2-mercaptopyrimidine-5-carboxylic acid with
the appropriate R.sup.1 group already incorporated can be protected
with various protecting groups, including with a para-methoxy
benzyl group. Other protecting groups are also possible, although
para-methoxy benzyl is preferred. This can then be reacted with the
appropriate amino derivative attached to ring A containing the
desired heterocycle, which is usually a triazine. However, in many
cases this moiety needs to be synthesized. This is accomplished by
employing a Sonogashira cross-coupling reaction between the
appropriately substituted halogen (e.g., bromine) containing amino
derivative with an acetylene. This acetylene is best protected with
a trimethylsilyl group at one end. The trimethyl silyl group is
then removed by base and the resulting acetylene is reacted with
the appropriate azide to produce the intermediate that is to be
coupled to the 4,6-PMB-protected carboxylic acid, described above.
The azide may or may not be substituted with a protecting group.
One such group is azidomethyl pivalate, which can be subsequently
removed by base or a SEM group which can be removed by fluoride or
acid. The resultant coupled product is then deprotected by the
appropriate reagents. For example, the PMB groups can be removed by
acid and the pivalate or SEM group by base or fluoride (or acid),
respectively, to produce the desired final product. However, those
skilled in the art will realize that other protecting groups can be
employed.
[0157] An alternative procedure involves the coupling of the
appropriately substituted barbituric acid with an isocyanate
attached to ring A containing the heterocycle, in most cases a
protected triazine. This sequence is depicted in FIG. 43B. The
isocyanate can be synthesized by employing a Sonogashira
cross-coupling reaction between the appropriately substituted
halogen (e.g., bromine) containing amino derivative with an
acetylene. This acetylene is best protected with a trimethylsilyl
group at one end. The trimethyl silyl group is then removed by base
and the resulting acetylene is reacted with the appropriate azide
to produce the intermediate isocyanate. Usually a base such as
sodium t-butoxide is employed in the coupling. The resultant
coupled product is then deprotected on the azide by the appropriate
reagents. For example, the pivalate or SEM groups by base or
fluoride (or acid), respectively, to produce the desired final
product. However, those skilled in the art will realize that other
protecting groups can be employed.
[0158] Alternatively, the thio-substituted barbiturate ring can be
constructed to allow different R1 groups on the sulfur of the ring,
as depicted in FIG. 44A. An appropriately protected amine is
reacted with di(1H-imidazol-1-yl)methanethione, followed by ammonia
to produce the protected thiourea. This is then condensed with
diethyl malonate with base to produce the barbiturate ring. The
sulfur atom is then alkylated with the appropriate alkyl halide to
provide the R1 group on the sulfur. This is then reacted with the
appropriate isocyanate, which is prepared as described in FIG. 43B.
The resultant coupled product is then deprotected on the azide by
the appropriate reagents. For example, the pivalate or SEM groups
by base or fluoride (or acid), respectively, to produce the desired
final product. However, those skilled in the art will realize that
other protecting groups can be employed.
[0159] Another sequence that can be utilized to prepare compounds
of the invention is depicted in FIG. 44B. The amino derivative
containing the A ring substituted with the heterocycle, which is
usually a protected triazine is prepared as described in FIG. 43A.
This is reacted with the di-substituted PMB-bartiturate, which
contains an acid halide or other appropriate leaving group like
penta-fluorophenol at the C-5 position. This results in the coupled
product protected by PMB groups and a protecting group on the
triazine like a pivalate or SEM group. The resultant coupled
product is then deprotected by the appropriate reagents. For
example, the PMB groups can be removed by acid and the pivalate or
SEM group by base or fluoride (or acid), respectively, to produce
the desired final product. However, those skilled in the art will
realize that other protecting groups can be employed.
[0160] In one aspect, the invention provides methods for reducing
uric acid levels in the blood or serum, or in the whole body, of a
subject comprising administering a compound having a structure
represented by Formula (I), Formula (II), or a combination thereof,
to the subject in an amount effective to reduce blood or serum uric
acid levels. It is to be understood that all such methods for
reducing uric acid levels correspond to a compound having a
structure represented by any of Formula (I) or Formula (II), or a
combination thereof, for use in medicine as well as a compound
having a structure represented by Formula (I), Formula (II), or a
combination thereof, for use in the treatment of elevated uric acid
levels. Typically, the compound having a structure represented by
Formula (I), Formula (II), or a combination thereof, will be
administered when the level of uric acid in the blood of the
subject is elevated, i.e., in the upper range of normal or above
normal levels. One skilled in the art would further recognize that
continued administration after normal uric acid levels are achieved
is also contemplated in order to maintain uric acid levels within
the normal range or to reduce the overall body burden of excess
uric acid that may have occurred due to a period of previous
excess. Accordingly, methods for preventing elevation of uric acid
levels in blood or serum, or the whole body, are also an aspect of
the invention. It is to be understood that all such methods for
preventing elevation of uric acid correspond to a compound having a
structure represented by Formula (I), Formula (II), or a
combination thereof, for therapeutic use as well as a compound
having a structure represented by Formula (I), Formula (II), or a
combination thereof, for prevention of elevated uric acid
levels.
[0161] Normal uric acid levels in serum are generally in the range
of 4.3 mg/dL to 8.0 mg/dL. In certain embodiments, a compound
having a structure represented by any of Formulae (I), Formula
(II), or a combination thereof, is administered to a subject with a
serum uric acid level of at least about 6 mg/dL. Administration may
continue until a serum uric acid level of about 6.0 mg/dL or less
is reached; however, it is generally considered to be beneficial to
maintain uric acid levels below this target in patients with
disorders associated with excess uric acid.
[0162] In certain embodiments, the invention provides methods of
treating a disorder associated with excess uric acid in blood or
serum or the whole body. The method of treating such disorders
comprises administering a compound having a structure represented
by Formula (I), Formula (II), or a combination thereof, to a
subject in need thereof in an amount effective to reduce serum uric
acid levels, thereby treating the disorder associated with excess
uric acid in the subject. These disorders are associated with, or
caused by, elevated uric acid levels in blood or serum or the whole
body which are in the upper range of normal or above normal, and
include gout; hyperuricemia; kidney disease; arthritis; kidney
stones; kidney failure; urolithiasis; plumbism;
hyperparathyroidism; psoriasis; inborn genetic errors of metabolism
(such as Lesch-Nyhan syndrome) and sarcoidosis. In certain
embodiments, the bifunctional compounds can be used effectively for
treating or preventing other disorders associated with excess uric
acid including NAFLD, NASH, atherosclerosis or other forms of
cardiovascular disease, hypertension, chronic kidney disease,
obesity, diabetes, insulin resistance, and metabolic syndrome,
and/or transplantation of blood, bone marrow or solid organs.
[0163] These drugs are particularly useful for treating gout and
kidney disease (including acute uric acid nephropathy, chronic
urate nephropathy, uric acid nephrolithiasis, and chronic kidney
disease). In addition, treatment of some cancers with chemotherapy
leads to the release of large amounts of uric acid into the blood,
which can damage the kidneys. Chemotherapy-induced hyperuricemia,
particularly the disorder known as "tumor lysis syndrome," may also
be treated, prevented or ameliorated according to the methods of
the invention. Administration of a compound having a structure
represented by Formula (I), Formula (II), or a combination thereof,
to a subject with excess uric acid, such as a subject suffering
from gout, kidney disease, or a risk of inducing elevated uric acid
levels due to chemotherapy, treats, prevents or ameliorates these
disorders by reducing uric acid levels in blood, or preventing or
controlling their level of increase. In specific embodiments, the
disorder associated with excess uric acid treated by administration
of a compound having a structure represented by Formula (I),
Formula (II), or a combination thereof, is gout. It is to be
understood that all such methods for treating disorders associated
with excess uric acid or elevated uric acid levels in serum
(hyperuricemia) correspond to a compound having a structure
represented by Formula (I), Formula (II), or a combination thereof,
for therapeutic use as well as a compound having a structure
represented by any of Formulae (I), Formula (II), or a combination
thereof, for treatment of disorders associated with excess uric
acid in blood or serum or the whole body.
[0164] The dose of a compound having a structure represented by
Formula (I), Formula (II), or a combination thereof, administered
to the subject may be any dose sufficient to achieve a desired
reduction in uric acid levels in blood or serum over the
time-course of administration. In certain embodiments, a daily dose
of about 20 to about 1,500 mg/m.sup.2/day is administered. In other
embodiments, a daily dose of about 20 to about 500 mg/m.sup.2/day,
about 20 to about 250 mg/m.sup.2/day, about 20 to about 150
mg/m.sup.2/day or about 20 to about 100 mg/m.sup.2/day is
administered. In other embodiments, a daily dose of about 50 to
about 1,500 mg/m.sup.2/day is administered. In other embodiments, a
daily dose of about 50 to about 500 mg/m.sup.2/day, about 50 to
about 150 mg/m.sup.2/day, about 50 to about 100 mg/m.sup.2/day, or
about 20 to about 100 mg/m.sup.2/day is administered.
[0165] In certain embodiments of any of the foregoing methods, a
compound having a structure represented by Formula (I), Formula
(II), or a combination thereof, is administered to the subject
parenterally, intraperitoneally, intravenously, intranasally,
intrarectally, or orally. Particularly useful routes of
administration include injection, infusion, or oral administration.
The amount of the drug administered per dose is an amount
sufficient to achieve a reduction in uric acid levels in blood or
serum, or the whole body, to prevent elevation of uric acid levels
in blood or serum or the whole body, or to treat or prevent
disorders associated with excess uric acid over the course of
therapy. One skilled in the art will recognize that
individualization of dosage based on a patient's body composition
or his/her hypouricemic response to treatment may be medically
necessary or desirable.
[0166] The drug(s) may be administered to the subject either
intermittently or continuously over a period of time in order to
achieve the desired reduction in uric acid levels in blood or serum
or the whole body, or to treat a disorder associated with excess
uric acid. For example, doses may be administered intermittently
several times per day, daily, once, twice or three times per week,
or at monthly intervals. In a specific example, a compound having a
structure represented by Formula (I), Formula (II), or a
combination thereof, may be administered to the subject by
continuous intravenous infusion over 24 hours for about five days.
Alternatively, a compound having a structure represented by Formula
(I), Formula (II), or a combination thereof, may be administered to
the subject by intravenous infusion over about 1 hour to about 5
hours for about five consecutive days. In a specific example, a
compound having a structure represented by Formula (I), Formula
(II), or a combination thereof, may be administered to the subject
by intramuscular injection or by intravenous infusion over about 10
minutes for about five consecutive days. In further specific
embodiments, a compound having a structure represented by Formula
(I), Formula (II), or a combination thereof, may be administered to
the subject by daily bolus injections for about five days. The
period of time of administration in any of the foregoing protocols
may be modified to achieve the desired reduction in uric acid
levels and UA body burden, including about 2 days, about 3 days,
about 4 days, about one week or about two weeks of administration,
or for longer periods in repeated treatment cycles, and these
treatments may be repeated at intervals of every two to every 10
weeks.
[0167] In addition to continuous intravenous infusion or bolus
intravenous or subcutaneous injection, a compound having a
structure represented by Formula (I), Formula (II), or a
combination thereof, may be administered to the subject orally. In
this embodiment, an oral dose in amounts as described above may be
administered in one, two, three or four administrations per day for
1, 2, 3, 4, or 5 days to achieve the desired reduction in uric acid
levels. In further embodiments, the oral dose as described above
may be administered once per day, or in one, two, three or four
administrations per day for one week or two weeks, to achieve the
desired reduction in uric acid levels.
[0168] It will be appreciated that a subject in need of reduced
levels of uric acid in blood or serum or the whole body, or in need
of treatment of a disorder associated with excess uric acid, will
be treated more aggressively initially to achieve the desired
reduction in uric acid. Following initial therapy and reduction of
uric acid to normal or sub-normal levels, the subject may be
further treated over a period of time, or over a lifetime, to
maintain normal or sub-normal levels of uric acid in blood or serum
and prevent elevation of uric acid levels subsequent to the initial
treatment. The maintenance or preventive protocol may comprise
reduced dosages and/or less frequent administration of a compound
having a structure represented by Formula (I), Formula (II), or a
combination thereof, as necessary or desired to maintain normal or
sub-normal uric acid levels in blood or serum or the whole body.
For example, in a maintenance protocol the drug(s) may be
administered daily, weekly, monthly, or intermittently as uric acid
levels rise between treatment periods. Such maintenance protocols
will serve to maintain normal or sub-normal uric acid levels for a
prolonged period of time and reduce the subject's lifetime risk of
developing a disorder associated with excess uric acid. The initial
reduction of uric acid levels from above normal or high normal to
normal or sub-normal, and maintenance of normal or sub-normal uric
acid levels are both features included in treatment of a disorder
associated with excess uric acid. It is anticipated that in certain
embodiments, a typical patient will require daily treatment of
varying duration, and that such daily treatment may be provided
intermittently for life or for extended periods.
[0169] In certain embodiments of any of the foregoing methods,
blood or serum uric acid levels of the subject are reduced by at
least 25% compared to uric acid levels prior to administration of a
compound having a structure represented by Formula (I), Formula
(II), or a combination thereof. In certain further embodiments,
blood or serum uric acid levels of the subject are reduced by 50%
or more compared to levels prior to administration. In a specific
embodiment, uric acid levels are reduced by about 75% even at daily
doses of 500 mg/m.sup.2/day or less.
[0170] In a second aspect of the invention methods are provided for
treating a disorder associated with excess uric acid in blood or
serum or the whole body comprising administering to a subject in
need thereof a compound having a structure represented by Formula
(I), Formula (II), or a combination thereof, in an amount effective
to reduce blood or serum uric acid levels, thereby treating the
disorder associated with excess uric acid. Specific embodiments of
the methods for treating a disorder associated with excess uric
acid metabolism relating to dosing, routes of administration,
initial therapy and maintenance therapy are as described above for
reducing uric acid levels in blood or serum. The initial reduction
in uric acid levels is typically rapid, and often occurs within 1-3
days. Upon reduction in uric acid levels to normal or sub-normal
levels, continued maintenance or preventive therapy may also result
in a detectable improvement in at least one symptom of excess uric
acid, such as reduced inflammation, reduced pain, slowing of
deformity development, reduction of kidney stones, improvement in
kidney function, prevention of tumor lysis syndrome, improved
cognition, improvement in (or reduction of actual or risk for)
cardiovascular disease and hypertension, reversal of insulin
resistance, or improvement in parameters of liver function. One
skilled in the art will recognize that prevention of recurrent
symptoms or complications of disease due to recurrence of excess
uric acid that may necessitate extended treatment, would be highly
desirable to maximize patient benefit.
[0171] In embodiments corresponding to the foregoing methods, the
invention relates to use of a compound disclosed herein, or a
combination thereof, for reducing uric acid levels in blood or
serum or the whole body of a subject in need thereof, preventing
elevation of uric acid levels in blood or serum or the whole body
of a subject, or treating a disorder associated with excess uric
acid. Each of the methods of treatment or prevention disclosed,
including routes of administration, dosage and compounds
administered, are also applicable to such uses of the
compounds.
[0172] A further aspect of the invention provides a pharmaceutical
composition comprising a compound having a structure represented by
Formula (I), Formula (II), or a combination thereof, and a
pharmaceutically acceptable carrier. In certain embodiments of the
pharmaceutical compositions, the composition is formulated as a
solution or tablet. Solutions or dispersions of the drug(s) can be
prepared in water or saline. In certain embodiments of the
pharmaceutical compositions, the pharmaceutically acceptable
carrier is one or more component selected from the group consisting
of one or more of a solvent, a dispersing agent, a coating (e.g.,
lecithin), a surfactant (e.g., hydroxypropylcellulose), a
preservative (e.g., paraben, phenol, thimerosal, sorbic acid,
chlorobutanol), an emulsion, an alcohol (e.g., ethanol), a polyol
(e.g., glycerol, propylene glycol), and an isotonic agent (e.g.,
sugars, sodium chloride).
[0173] In certain embodiments of the foregoing pharmaceutical
compositions, the composition is formulated for controlled release
of the compound having a structure represented by Formula (I),
Formula (II), or a combination thereof. In certain embodiments of
the foregoing methods, a compound having a structure represented by
Formula (I), Formula (II), or a combination thereof, is
administered in a form for controlled release. The controlled
release compositions may include pharmaceutically acceptable
carriers or excipients which cause release of the active ingredient
more slowly or which extend the duration of its action within the
body. Examples of controlled release compositions include
pharmaceutically acceptable carriers or excipients which delay
absorption of the active ingredient (e.g., aluminum monostearate,
gelatin, natural or synthetic hydrophilic gums). Alternatively,
controlled release of the pharmaceutical composition may employ a
device such as a pump, implant or transdermal patch.
[0174] In certain embodiments of the foregoing pharmaceutical
compositions, the composition is formulated for improved oral
bioavailability or extended release in the body. For example,
microemulsions, particle size reduction and complexation
technologies may be used to improve dissolution rates or
equilibrium solubilities of the compounds. Other suitable chemical
and physical means for improving oral bioavailability or extended
release will also be known to those skilled in the art.
EXAMPLES
[0175] General Procedure for Triphosgene coupling:
2-(Methylthio)pyrimidine-4,6-diol (2 eq) was added to a stirring
solution of sodium tert-butoxide (2.0 eq) dissolved in DMSO (0.2 M)
at rt for 5 min. In a separate flask, the appropriately substituted
amine was dissolved in 1,4-dioxane (0.8 M), to this solution was
added triphosgene (0.33 eq) in one-portion. The suspension was
stirred vigorously for 2 min at rt, then iPr.sub.2NEt (2 eq) was
added. The suspension was stirred vigorously at rt for 2 min.
Freshly prepared solution of sodium
6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added to the
suspension in one-portion. The reaction was stirred at 90.degree.
C. for 30 min, until complete consumption of starting material
observed via LCMS. The reaction mixture was loaded directly on C18
column and purified via reverse-phase chromatography.
Example 1: Preparation of
4-hydroxy-N-(4-(4-methyl-1H-1,2,3-triazol-5-yl)phenyl)-2-(methylthio)-6-o-
xo-1,6-dihydropyrimidine-5-carboxamide (101, Formula (II.sub.ff),
with Reference to FIG. 9)
[0176] Step One. 1-Nitro-4-(prop-1-yn-1-yl)benzene: A round bottom
flask containing 1-bromo-4-nitrobenzene (1.00 g, 4.95 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (174 mg, 0.248 mmol), and CuI (47 mg,
0.248 mmol) was purged with nitrogen for 15 min. Anhydrous
acetonitrile (2.5 mL) was added, followed by propyne in heptane
(13.2 mL, 99.0 mmol, 3% in heptane) and Et.sub.3N (1.4 mL, 9.90
mmol). The reaction mixture was sealed and allow to stir at rt for
20 h. The reaction mixture was then concentrated, diethyl ether was
added, then filtered through a small pad of Celite. The filtrate
was concentrated then purified via ISCO (SiO.sub.2, gradient eluent
from 0 to 25% ethyl acetate in hexanes over 20 CV) to yield the
product as a yellow solid (645 mg, >99% purity, 81% yield).
[0177] R.sub.f: 0.79 (25% ethyl acetate in hexanes).
[0178] LCMS: R.sub.T=1.73 min; purity=>99%.
[0179] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0180] Step Two. 5-Methyl-4-(4-nitrophenyl)-1H-1,2,3-triazole
(101-B): Sodium azide (111 mg, 1.71 mmol) was added to 101-A (229
mg, 1.42 mmol) dissolved in anhydrous DMF (7.1 mL) at rt. The
reaction was sealed in a pressure vessel and heated to 120.degree.
C. for 18 h. The reaction mixture was then allowed to warm up to
rt, dichloromethane was added, followed by water. The aqueous layer
was extracted with dichloromethane (3.times.20 mL), the combined
organic extract was washed with brine, dried over MgSO.sub.4, then
concentrated under reduced pressure to yield the product as a brown
solid (180 mg, >99% purity, 62% yield), without further
purification.
[0181] LCMS: m/z [M+1].sup.+=205.29; R.sub.T=1.29 min;
purity=>99%.
[0182] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0183] Step Three. 4-(5-Methyl-1H-1,2,3-triazol-4-yl)aniline
(101-C): Tin (II) chloride (938 mg, 4.51 mmol) was added to 101-B
(230 mg, 1.13 mmol) in EtOH (3.8 mL) and conc. HCl (710 .mu.L) at
rt, the resulting reaction mixture was heated to reflux for 1 h.
After complete consumption of the starting material was observed
via LCMS, the reaction was allowed to cool to rt, before pouring
into a solution of K.sub.3PO.sub.4 (.about.1.0 g) in MeOH (10 mL)
at rt. The resulting reaction was stirred at rt for 30 min until
the pH is not longer acidic. The precipitate was filtered, washed
with additional methanol. The filtrate was collected and
concentrated under reduced pressure. The crude product was purified
via ISCO (SiO.sub.2, gradient eluent from 0 to 15% methanol in
dichloromethane over 12 CV) to yield the product as a brown oil (69
mg, 35% yield).
[0184] LCMS: m/z [M+I].sup.+=175.42; R.sub.T=0.83 min.
[0185] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0186] Step Four.
4-hydroxy-N-(4-(4-methyl-1H-1,2,3-triazol-5-yl)phenyl)-2-(methylthio)-6-o-
xo-1,6-dihydropyrimidine-5-carboxamide (101): 101 was synthesized
following general procedure 2. 2-(Methylthio)pyrimidine-4,6-diol
(171 mg, 1.08 mmol) was added to a stirring solution of sodium
tert-butoxide (104 mg, 1.08 mmol) dissolved in DMSO (2.7 mL) at rt
for 5 min. In a separate flask, aniline 101-C was dissolved in
1,4-dioxane (680 mL), to this solution was added triphosgene (53
mg, 0.178 mmol) in one-portion. The suspension was stirred
vigorously for 2 min at rt, then iPr.sub.2NEt (190 .mu.L) was
added. The suspension was stirred vigorously at rt for 2 min.
Freshly prepared solution of sodium
6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added to the
suspension in one-portion. The reaction was stirred at 90.degree.
C. for 30 min, until complete consumption of starting material
observed via LCMS. The reaction mixture was loaded directly on C18
column and purified via reverse-phase chromatography (gradient
eluent from 0 to 100% acetonitrile in water with an ammonium
formate buffer 10 mM over 20 CV) to yield the product as brown
solid (29.2 mg, 97.7% purity, 15% yield), after lyophilization.
[0187] .sup.1H NMR (400 MHz, DMSO-d6) .delta. 7.69 (br s, 4H), 2.44
(s, 3H), 2.37 (s, 3H).
[0188] LCMS: m/z [M+1].sup.+=359.0; R.sub.T=1.37 min;
purity=97.7%.
[0189] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 2: Preparation of
N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-
-dihydropyrimidine-5-carboxamide (102). (102, Formula (II.sub.i),
with Reference to FIG. 10)
[0190] Step One. 1-(3,4,5-trimethoxybenzyl)thiourea (102-A):
(3,4,5-Trimethoxyphenyl)methanamine (2.5 mL, 14.6 mmol) was added
dropwise to a solution of 1,1'-thiocarbonyl diimidazole (3.91 g,
22.0 mmol) dissolved in dichloromethane (36.5 mL) at 0.degree. C.
The reaction mixture was then allowed to warm up to rt over 2 h.
After complete consumption of the starting material was observed
via LCMS, a solution of ammonia in methanol (7.5 mL, 52.6 mmol, 7.0
M in MeOH) was added, then stirred for an additional 20 h. The
reaction mixture was concentrated under reduced pressure,
dichloromethane was added, the precipitate was isolated and washed
with additional CH.sub.2Cl.sub.2, then dried under high vacuum to
yield the product as a light pink solid (2.83 g, 76% yield).
[0191] LCMS: m/z [M+1].sup.+=257.07; R.sub.T=1.06 min.
[0192] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0193] Step Two.
6-hydroxy-2-(isopropylthio)-3-(3,4,5-trimethoxybenzyl)pyrimidin-4(3H)-one
(102-B): A mixture of 102-A (781 mg, 3.05 mmol), diethyl malonate
(465 .mu.L, 3.05 mmol), and NaOMe (1.4 mL, 6.10 mmol, 4.4 M in
MeOH) in methanol (2.4 mL) was heated to reflux for 3 h. The
reaction was then cooled to .about.50.degree. C., isopropyl iodide
(3.5 mL, 30.5 mmol) was then added in one-portion. The reaction was
stirred for an additional 30 min at 50.degree. C. The reaction
mixture was then cooled to rt, then concentrated under reduced
pressure. The crude product was purified via reverse-phase
chromatography (gradient eluent from 0 to 100% acetonitrile in
water with an ammonium formate buffer 10 mM over 15 CV) to yield
the product as a white solid (433 mg, 97.7% purity, 38% yield),
after lyophilization.
[0194] LCMS: m/z [M+1].sup.+=367.02; R.sub.T=1.41 min;
purity=97.7%.
[0195] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0196] Step Three.
N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1-(-
3,4,5-trimethoxybenzyl)-1,6-dihydropyrimidine-5-carboxamide
(102-C): 102-C was synthesized following general procedure 2. 102-B
(97 mg, 0.265 mmol) was added to a stirring solution of sodium
tert-butoxide (25 mg, 0.265 mmol) dissolved in DMSO (870 .mu.L) at
rt for 5 min. In a separate flask, aniline XX was dissolved in
1,4-dioxane (220 .mu.L), to this solution was added triphosgene (17
mg, 0.0578 mmol) in one-portion. The suspension was stirred
vigorously for 2 min at rt, then iPr.sub.2NEt (60 .mu.L) was added.
The suspension was stirred vigorously at rt for 2 min. Freshly
prepared solution of sodium
6-hydroxy-2-(isopropylthio)-3-(3,4,5-trimethoxybenzyl)pyrimidin-4(3H)-ola-
te in DMSO was added to the suspension in one-portion. The reaction
was stirred at 90.degree. C. for 30 min, until complete consumption
of starting material observed via LCMS. The reaction mixture was
loaded directly on C18 column and purified via reverse-phase
chromatography (gradient eluent from 30 to 100% acetonitrile in
water with an ammonium formate buffer 10 mM over 15 CV) to yield
the product as brown solid (31.2 mg, 80.2% purity, 26% yield),
after lyophilization.
[0197] LCMS: m/z [M+1].sup.+=552.9; R.sub.T=1.80 min;
purity=80.2%.
[0198] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0199] Step Four.
N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-
-dihydropyrimidine-5-carboxamide (102): A solution of 102-C (31.2
mg, 0.0452 mmol, 80% purity) in dichloromethane (1.5 mL) was added
trifluoroacetic acid (270 .mu.L). The resulting reaction mixture
was sealed in a pressure vessel then heated to 60.degree. C. for 20
h. The reaction mixture was allowed to cool to rt, then
concentrated under reduced pressure. The crude product was
co-evaporated several times with methanol (3.times.), then purified
via reverse-phase chromatography (C18, gradient eluent from 30 to
100% acetonitrile in water with an ammonium formate buffer 10 mM
over 20 CV) to yield the product as an off-white solid (6.0 mg,
98.6% purity, 35% yield), after lyophilization.
[0200] .sup.1H NMR (400 MHz, DMSO-d6+AcOD) .delta. 8.23 (s, 1H),
7.87 (d, J=8.2 Hz, 2H), 7.68 (d, J=8.2 Hz, 2H), 3.92 (dt, J=13.7,
6.9 Hz, 1H), 1.36 (d, J=6.9 Hz, 6H).
[0201] LCMS: m/z [M+1].sup.+=373.1; R.sub.T=1.49 min;
purity=98.6%.
[0202] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 3: Preparation of
N-(5-(1H-1,2,3-triazol-5-yl)pyridin-2-yl)-4,6-dihydroxy-2-(methylthio)pyr-
imidine-5-carboxamide (103, Formula (II.sub.jj), with Reference to
FIG. 11
[0203] Step One. 5-((Trimethylsilyl)ethynyl)pyridin-2-amine
(103-A): To a sealed tube was added 2-amino-5-bromopyridine (1.00
g, 5.8 mmol), Pd(dba).sub.2Cl.sub.2 (202 mg, 0.29 mmol), PPh.sub.3
(151 mg, 0.58 mmol), CuI (110 mg, 0.578 mmol), Et.sub.3N (10 mL)
and TMS-acetylene (963 mg, 9.8 mmol) sequentially. The mixture was
degassed and heated at 85.degree. C. for 2 h. After complete
consumption of starting material was observed via LCMS, the solvent
was removed in vacuo and the crude was purified over silica
(gradient eluent from 0 to 100% ethyl acetate in hexanes). 103-A
was obtained as beige solid (812 mg, 74% yield).
[0204] LCMS: m/z [M+1].sup.+=191.3; R.sub.T=1.55 min
[0205] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0206] Step Two. 5-Ethynylpyridin-2-amine (103-B): 103-A (500 mg,
2.6 mmol) was dissolved in THF (5 mL) and to this solution was
added TBAF (5 mL, 1 M in THF). The reaction was stirred at rt for
10 min and THF was removed in vacuo. The crude was dissolved in
EtOAc and this solution was passed through a pad of silica and
washed with EtOAc. The filtrate was concentrated to yield 103-B as
a beige solid (256 mg, 82% yield).
[0207] LCMS: m/z [M+1].sup.+=118.8; R.sub.T=0.41 min.
[0208] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0209] Step Three.
N-(5-Ethynylpyridin-2-yl)-4,6-dihydroxy-2-(methylthio)pyrimidine-5-carbox-
amide (103-C): t-BuONa (136 mg, 1.4 mmol) was dissolved in DMSO (2
mL) and to this solution was added
2-(methylthio)pyrimidine-4,6-diol (224 mg, 1.4 mmol). The solution
was stirred at rt for 5 min and left aside for the second step. At
the same time, 103-B (84 mg, 0.71 mmol) was dissolved in DCE (1 mL)
and to the solution was added CDI (115 mg, 0.71 mmol) in
one-portion. The suspension was stirred vigorously for 2 min at rt
and iPr.sub.2NEt (250 uL, 1.4 mmol) was added. The solution was
stirred at rt vigorously for 2 min. Freshly prepared solution of
sodium 6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added
to the suspension. The reaction was stirred at 90.degree. C. for 30
min. DCE solvent was removed in vacuo and the product was isolated
by ISCO (120 g C18 column, gradient eluent from 0 to 50%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV). Product elutes at 35% MeCN in water. The product was
isolated as a beige solid (62 mg, 29% yield), after
lyophilization.
[0210] LCMS: m/z [M+1].sup.+=303.0; R.sub.T=1.59 min
[0211] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0212] Step Four.
N-(5-(1H-1,2,3-triazol-5-yl)pyridin-2-yl)-4,6-dihydroxy-2-(methylthio)pyr-
imidine-5-carboxamide (103). 103-C (62 mg, 0.21 mmol) was dissolved
in DMSO (2 mL) and to this solution was added NaN.sub.3 (67 mg, 1.0
mmol). The mixture was stirred at 180.degree. C. for 30 min. The
product was purified by ISCO (60 g C18 column, gradient eluent from
0 to 50% acetonitrile in water with an ammonium bicarbonate buffer
10 mM over 20 CV, product elutes at 22% MeCN in water). The product
was isolated as an off-white solid (25 mg, 35% yield), after
lyophilization.
[0213] 1HNMR (500 MHz, DMSO-d6, DCl in D.sub.2O) .delta. 12.11 (s,
1H), 8.88 (dd, J=2.4, 0.8 Hz, 1H), 8.45 (s, 1H), 8.33 (dd, J=8.6,
2.4 Hz, 1H), 8.22 (dd, J=8.7, 0.8 Hz, 1H), 2.56 (s, 3H).
[0214] LCMS: m/z [M-1].sup.-=346.0; R.sub.T=1.29 min;
purity=94.4%.
[0215] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 4: Preparation of
4-hydroxy-2-methoxy-N-(4-(4-methyl-1H-1,2,3-triazol-5-yl)phenyl)-6-oxo-1,-
6-dihydropyrimidine-5-carboxamide (104, Formula (II.sub.gg), with
Reference to FIG. 12)
[0216] 104 was synthesized following general procedure 1. To a
stirring solution of 101-C (23 mg, 0.132 mmol) in anhydrous DMSO
(130 .mu.L) was added 1,1'carbonyldiimidazole (33 mg, 0.198 mmol)
was added at rt, under inert atmosphere. The resulting solution was
stirred for 20 min at rt. In a separate flask containing
2-methoxypyrimidine-4,6-diol (21 mg, 0.145 mmol) was added
anhydrous 1,4-dioxane (440 .mu.L), then heated to 50.degree. C.
Et.sub.3N (29 .mu.L, 0.211 mmol) was added and stirred for 15 min
at 50.degree. C. The isocyanate generated from the amine in DMSO
was added to the stirring suspension, then heated to 80.degree. C.
until complete consumption of the starting materials were observed
via LCMS (30 min). The reaction mixture was cooled to rt, then
acidified with 6M HCl (aq), the reaction mixture was directly
loaded onto a C18 column and purified via ISCO (gradient eluent
from 0 to 50% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 20 CV) to yield the product as an off-white solid
(1.6 mg, 97.0% purity, 4% yield), after lyophilization.
[0217] .sup.1H NMR (400 MHz, DMSO-d6) .delta. 8.14 (s, 1H), 7.69
(s, 4H), 3.79 (s, 3H), 2.44 (s, 3H).
[0218] LCMS: m/z [M+1].sup.+=342.7; R.sub.T=1.24 min;
purity=97.0%.
[0219] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 5: Preparation of
N-(3-(1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]pentan-1-yl)-2,4-dihydroxy-6-ox-
o-1,6-dihydropyrimidine-5-carboxamide (105, Formula (II.sub.hh),
with Reference to FIG. 13)
[0220] Step One. tert-Butyl
(3-(hydroxymethyl)bicyclo[1.1.1]pentan-1-yl)carbamate (105-A).
3-((Tert-butoxycarbonyl)amino)bicyclo[1.1.1]pentane-1-carboxylic
acid (600 mg, 2.6 mmol) was added to THF (25 mL). The solution was
cooled to 0.degree. C. under N.sub.2. To the solution was added
LiAH.sub.4 (401 mg, 10.6 mmol) under N.sub.2. The reaction was
warmed up to rt and stirred for 1 h. Na.sub.2SO.sub.4 decahydrate
(500 mg) was added slowly to the reaction and the reaction was
diluted with EtOAc (30 mL). The precipitate was filtered and the
filtrate was concentrated in vacuo to yield crude PA67 (420 mg, 75%
yield), which was used without purification.
[0221] 1HNMR (500 MHz, CDCl.sub.3) .delta. 3.7 (s, 2H), 1.94 (s,
6H), 1.44 (s, 9H).
[0222] Step Two. tert-Butyl
(3-formylbicyclo[1.1.1]pentan-1-yl)carbamate (105-B).
SO.sub.3--Pyridine (500 mg, 3.1 mmol) was added portion wise (small
exotherm) to a solution of DMSO (1.2 g, 15 mmol), 105-A (335 mg,
1.6 mmol) and iPr.sub.2Net (811 mg, 6.3 mmol) in CH.sub.2Cl.sub.2
(6 mL) at rt. The reaction was stirred for 20 min at rt. The
reaction mixture was diluted with CH.sub.2Cl.sub.2 (30 mL). The
reaction was then washed with sat. NaHCO.sub.3 (10 mL), brine (10
mL) and dried over MgSO.sub.4 and concentrated in vacuo. The crude
105-B was used in the next step, without further purification.
[0223] 1HNMR (500 MHz, CDCl.sub.3) .delta. 9.66 (s, 1H), 2.29 (s,
6H), 1.44 (s, 9H).
[0224] Step Three. tert-butyl
(3-ethynylbicyclo[1.1.1]pentan-1-yl)carbamate (105-C). 105-B (80
mg, 0.38 mmol) was dissolved in dry MeOH/THF (2 mL, 1:1 v/v, dried
over MgSO.sub.4 overnight). To the solution was added
K.sub.2CO.sub.3 (105 mg, 0.76 mmol) and dimethyl
diazo-2-oxopropylphosphonate (95 mg, 0.49 mmol). The mixture was
stirred overnight. ISCO purification was performed (dry loading
with silica, gradient 1HNMR (500 MHz, CDCl.sub.3) .delta. 2.29 (s,
6H), 2.10 (s, 1H), 1.25 (s, 9H). t eluent from 0 to 50% ethyl
acetate in hexanes) to yield the desired product, 105-C (42 mg, 53%
yield).
[0225] Step Four. tert-butyl
(3-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]pentan-1-yl)c-
arbamate (105-D). CuSO.sub.4 (263 mg in 3 mL water, 1.6 mmol) was
added to sodium ascorbate (390 mg in 3 mL water, 2.0 mmol). The
solution was stirred at rt for 1 min and DMSO (8 mL) was added to
the mixture. The suspension was added to 105-C (70 mg, 0.33 mmol)
and PMB-N.sub.3 (161 mg, 0.99 mmol) mixture in 4 mL MeOH. The
resulting mixture was stirred at rt for 30 min. The precipitate was
filtered by Celite and washed with methanol. The filtrate was
concentrated to remove MeOH and the product was extracted with
EtOAc/H.sub.2O (40 mL/20 mL). The organic layer was washed with
brine and dried over MgSO.sub.4, then concentrated in vacuo to give
the crude product. The crude product was purified by silica pad,
(30% ethyl acetate in hexanes to remove the excess azide. The
product was flushed out by 1/1 MeOH/DCM) to yield the desired
product (115 mg, 95% yield).
[0226] LCMS: m/z [M+1].sup.+=371.1; R.sub.T=1.61 min
[0227] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0228] Step Five.
3-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]pentan-1-amine
(105-E). 105-D (80 mg, 0.22 mol) was dissolved in TFA (4 mL) and
the reaction was stirred at rt for 30 min. Removal of the solvent
in vacuo and the crude was dissolved in EtOAc (30 mL). The organic
layer was washed with sat. aqueous Na.sub.2CO.sub.3 (10 mL) and
brine (10 mL). The organic layer was then dried over MgSO.sub.4 and
the solvent was removed in vacuo to yield the crude product (58 mg,
99% yield).
[0229] LCMS: m/z [M+1].sup.+=271.0; R.sub.T=1.08 min
[0230] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0231] Step Six.
4-Hydroxy-N-(3-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)bicyclo[1.1.1]p-
entan-1-yl)-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide
(105-F). 105-E (57 mg, 0.21 mmol) was dissolved in dioxane (0.3
mL). To this solution was added CDI (45 mg, 0.27 mmol), the
reaction was stirred at rt for 3 min and iPr.sub.2NEt (82 mg, 0.63
mmol) was added. The solution was stirred at rt for 10 min. To this
solution was added freshly prepared
2-(methylthio)pyrimidine-4,6-diol (100 mg, 0.63 mmol) with NaOt-Bu
(61 mg, 0.63 mmol) in DMSO (1 mL). The mixture was heated up to
90.degree. C. for 1 h until complete consumption of the starting
material was observed by LCMS. The crude product was then purified
by ISCO (gradient eluent from 0 to 100% acetonitrile in water with
an ammonium bicarbonate buffer 10 mM, product eluted at 35% MeCN in
water) to yield the product (42 mg, 44% yield), after
lyophilization.
[0232] LCMS: m/z [M+1].sup.+=455.1; R.sub.T=1.54 min
[0233] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0234] Step Seven.
N-(3-(1H-1,2,3-Triazol-4-yl)bicyclo[1.1.1]pentan-1-yl)-2,4-dihydroxy-6-ox-
o-1,6-dihydropyrimidine-5-carboxamide (105). 106-F (20 mg, 0.044
mmol) was dissolved in TFA (4 mL) and TfOH (0.2 mL) was added. The
reaction mixture was heated at 85.degree. C. for 4 h. To the
reaction was added 0.3 mL iPr.sub.2NEt (To prevent decomposition
caused by TfOH) and the reaction was concentrated in vacuo. The
crude product was purified by ISCO (gradient eluent from 0 to 100%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 15 CV) to yield the product as a white solid (27 mg, 87%
yield).
[0235] 1HNMR (500 MHz, DMSO-d6+TFA) .delta. 9.91 (s, 1H), 7.74 (s,
1H), 2.55 (s, 3H), 2.45 (s, 6H).
[0236] LCMS: m/z [M+1].sup.+=334.9; R.sub.T=1.23 min;
purity=97.0%.
[0237] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 6: Preparation of
N-(6-(1H-1,2,3-triazol-4-yl)pyridin-3-yl)-4-hydroxy-2-(methylthio)-6-oxo--
1,6-dihydropyrimidine-5-carboxamide (106, Formula (II.sub.ii), with
Reference to FIG. 14)
[0238] Step One. 6-Bromopyridin-3-amine (106-A). To a solution of
2-bromo-5-nitropyridine (502 mg, 2.47 mmol) in a mixture of
EtOH/THF/H.sub.2O/NH.sub.4Cl (sat.) (5.0 mL, 4:4:1:1 v/v) was added
Fe powder (1.40 g, 25.1 mmol) and the mixture was heated to
80.degree. C. overnight. The reaction mixture was filtered through
a small pad of Celite/MgSO.sub.4 mixture (1:1) using EtOAc. The
crude product was concentrated and subjected to purification via
ISCO (SiO.sub.2, 0-50% % ethyl acetate in hexanes) to afford the
product as a brown solid (411 mg, 96.1% yield).
[0239] 1H NMR (400 MHz, CDCl.sub.3) .delta. 7.84 (dd, J=3.1, 0.5
Hz, 1H), 7.21 (dd, J=8.5, 0.6 Hz, 1H), 6.87 (dd, J=8.5, 3.1 Hz,
1H), 3.73 (br s, 2H). 13C NMR (101 MHz, CDCl.sub.3) .delta. 142.1,
137.1, 129.6, 127.8, 124.7.
[0240] LCMS: m/z [M+2H].sup.+=175.2, R.sub.T=0.92 min.
[0241] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0242] Step Two. 6-((Trimethylsilyl)ethynyl)pyridin-3-amine
(106-B). To a dry 15 mL round bottom flask was added
PdCl.sub.2(PPh.sub.3).sub.2 (25 mg, 0.0360 mmol), CuI (4.9 mg,
0.0260 mmol) and 6-bromopyridin-3-amine 106-A (200 mg, 1.16 mmol)
was added THF (4 mL) and the solution was degassed by bubbling with
N.sub.2. The solution mixture was treated with 2-ethanolamine (140
.mu.L, 2.32 mmol) and ethynyltrimethylsilane (200 .mu.L, 1.42 mmol)
and the reaction was stirred overnight at 60.degree. C. The
reaction mixture was filtered through a pad of Celite using EtOAc.
The crude product was concentrated and subjected to purification
via ISCO (SiO.sub.2, 0-25% ethyl acetate in hexanes) to afford a
brown solid (179 mg, 81.4% yield).
[0243] 1H NMR (400 MHz, CDCl.sub.3) .delta. 8.02 (dd, J=2.9, 0.5
Hz, 1H), 7.28-7.22 (m, 1H), 6.87 (dd, J=8.4, 2.8 Hz, 1H), 3.86 (s,
2H), 0.24 (s, 9H).
[0244] LCMS: m/z [M+H].sup.+=191.3, R.sub.T=1.52 min.
[0245] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0246] Step Three. 6-Ethynylpyridin-3-amine (106-C). To a solution
of 106-B (529 mg, 2.78 mmol) in THF (14 mL) was added TBAF (3.0 mL
3.00 mmol, 1 M in THF) and the resulting black solution was stirred
at room temperature. After 30 min., the reaction mixture was washed
with water (20 mL) and extracted with CH.sub.2Cl.sub.2 (3.times.10
mL). The organic phase was washed with brine (10 mL), dried over
MgSO.sub.4 and then concentrated. The crude solid was subjected to
purification via ISCO (10-100% ethyl acetate in hexanes) to afford
106-C as a brown solid (310 mg, 94.5% yield).
[0247] 1H NMR (400 MHz, CDCl.sub.3) .delta. 8.04 (dd, J=2.9, 0.7
Hz, 1H), 7.28 (dd, J=8.4, 0.7 Hz, 1H), 6.90 (dd, J=8.4, 2.9 Hz,
1H), 3.88 (brs, 2H), 3.02 (s, 1H).
[0248] LCMS: m/z [M+H].sup.+=119.4, R.sub.T=0.50 min; 98%
purity.
[0249] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0250] Step Four.
N-(6-Ethynylpyridin-3-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrim-
idine-5-carboxamide (106-D). To a solution of t-BuONa (163 mg, 1.69
mmol) in DMSO (2.4 mL) was added
4,6-dihydroxy-2-methylmercaptopyrimidine (268 mg, 1.69 mmol) and
the solution was stirred at rt for 5 min. At the same time, the
106-C (100 mg, 0.846 mmol) was dissolved in DCE (1.2 mL) and to the
solution was added CDI (251 mg, 0.846 mmol). The suspension was
stirred vigorously for 2 min at rt and iPr.sub.2NEt (300 .mu.L) was
added. The solution was stirred vigorously for 2 min. Then the
prepared DMSO solution was added to the suspension at once and the
reaction was stirred at 90.degree. C. for 30 min. Afterwards, DCE
was removed in vacuo and the crude product was subject to
purification by ISCO (C18 column, 0-50% acetonitrile in water with
an ammonium formate buffer 10 mM). Product precipitated in column,
column was flushed with 100% DMSO. The solvent was removed to yield
the 106-D as a red solid (67.2 mg, 13.1% yield).
[0251] LCMS: m/z [M+H].sup.+=303.0, R.sub.T=1.36 min; 94%
purity.
[0252] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0253] Step Five.
4-Hydroxy-N-(4-(5-hydroxy-1H-pyrazol-3-yl)phenyl)-2-(methylthio)-6-oxo-1,-
6-dihydropyrimidine-5-carboxamide (106). The 106-D (67.2 mg, 0.220
mmol) and sodium azide (72.3 mg, 1.11 mmol) were dissolved in DMSO
(2.2 mL). The solution was stirred at 180.degree. C. and the
consumption of the starting material was monitored via LCMS. After
2 h, the crude product was subjected to purification by ISCO (C18
column, 0-40% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM) and was lyophilized to afford a white solid (7.4 mg,
9.7% yield).
[0254] 1H NMR (400 MHz, DMSO-d6, DCl in D.sub.2O) .delta. 9.18 (d,
J=2.2 Hz, 1H), 9.04 (s, 1H), 8.63 (dd, J=8.9, 2.3 Hz, 1H), 8.47
(app d, J=8.9 Hz, 1H), 2.54-2.53 (m, J=4.7 Hz, 3H).
[0255] 1H NMR (400 MHz, CD.sub.3OD, DCl in D.sub.2O) .delta. 9.44
(d, J=2.1 Hz, 1H), 8.84 (s, 1H), 8.69 (dd, J=8.9, 2.1 Hz, 1H), 8.56
(d, J=8.9 Hz, 1H), 2.64 (s, 3H).
[0256] LCMS: m/z [M+H].sup.+=346.1, R.sub.T=1.19 min; 99%
purity.
[0257] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 7: Preparation of
4-hydroxy-N-(4-(5-hydroxy-1H-pyrazol-3-yl)phenyl)-2-(methylthio)-6-oxo-1,-
6-dihydropyrimidine-5-carboxamide (107, Formula (II.sub.j), with
Reference to FIG. 15)
[0258] NaH (2.4 mg, 0.0615 mmol) was added to XY (19.6 mg, 0.056
mmol) in THF (280 .mu.L) at 0.degree. C., and stirred for 30 min at
0.degree. C. 5-(Trifluoromethyl)-5H-dibenzo[b,d]thiophen-5-ium
trifluoromethanesulfonate (27 mg, 0.067 mmol) was then added in
one-portion, and the resulting reaction mixture was warmed up to rt
over 72 h. The reaction was quenched with methanol (5 mL) then
concentrated under reduced pressure. The crude product was purified
via reverse-phase chromatography (C18, gradient eluent from 30 to
100% acetonitrile in water with an ammonium formate buffer 10 mM
over 15 CV) to yield the product 107, after lyophillization, as a
white solid (2.0 mg, 9% yield).
[0259] .sup.1H NMR (400 MHz, DMSO-d6+AcOD) .delta. 8.09 (s,
5H).
[0260] LCMS: m/z [M+1].sup.+=399.1; R.sub.T=2.01 min;
purity=99.2%.
[0261] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 8: Preparation of
N-(4-(1H-1,2,4-Triazol-3-yl)phenyl)-2-(methylthio)-4,6-dioxo-1,4,5,6-tetr-
ahydropyrimidine-5-carboxamide (108, Formula (II.sub.k), with
Reference to FIG. 16)
[0262] 2-(Methylthio)pyrimidine-4,6-diol (300 mg, 1.90 mmol) was
added to a stirring solution of sodium tert-butoxide (182 mg, 1.90
mmol) dissolved in DMSO (4.8 mL) at rt for 5 min. In a separate
flask, 4-(4H-1,2,4-triazol-3-yl)aniline (160 mg, 0.95 mmol, 95%
purity) was dissolved in 1,4-dioxane (1.2 mL), to this solution was
added triphosgene (93 mg, 0.314 mmol) in one-portion. The
suspension was stirred vigorously for 2 min at rt, then
iPr.sub.2NEt (330 .mu.L, 1.90) was added. The suspension was
stirred vigorously at rt for 2 min. Freshly prepared solution of
sodium 6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added
to the suspension in one-portion. The reaction was stirred at
90.degree. C. for 30 min, until complete consumption of starting
material observed via LCMS. The reaction mixture was loaded
directly on C18 column and purified via reverse-phase
chromatography (C18, gradient eluent from 5 to 100% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 20 CV) to
yield the product 108, after lyophillization, as a beige solid
(23.5 mg, 7.0% yield).
[0263] .sup.1H NMR (400 MHz, DMSO-d6+AcOD) .delta. 8.07 (s, 1H),
7.99 (d, J=8.8 Hz, 2H), 7.69 (d, J=8.7 Hz, 2H), 2.50 (s, 3H).
[0264] LCMS: m/z [M+1].sup.+=345.2; R.sub.T=1.21 min;
purity=95.2%.
[0265] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 9: Preparation of
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2-(ethylthio)-4-hydroxy-6-oxo-1,6-dih-
ydropyrimidine-5-carboxamide (109, Formula (II), with Reference to
FIG. 17)
[0266] Step One. 2-(Ethylthio)-6-hydroxypyrimidin-4(3H)-one
(109-A). A mixture of 6-hydroxy-2-mercaptopyrimidin-4(3H)-one (250
mg, 1.73 mmol) and NaOMe (400 .mu.L, 1.76 mmol, 4.4 M in MeOH) in
methanol (1.5 mL) was heated to 50.degree. C. for 30 min.
Iodoethane (140 .mu.L, 1.74 mmol) was added dropwise to the
reaction mixture and the reaction was stirred overnight at
50.degree. C. Afterwards, the reaction mixture was filtered, and
then washed with methanol. The filtrate was concentrated to afford
the crude product as a white solid. The crude product was
triturated with ethyl acetate to afford 109-A as a white solid (270
mg, 90.5% yield).
[0267] LCMS: m/z [M+1].sup.+=173.4; R.sub.T=0.78 min;
purity=92.5%.
[0268] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0269] Step Two.
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2-(ethylthio)-4-hydroxy-6-oxo-1,6-dih-
ydropyrimidine-5-carboxamide (109). Compound 109 was synthesized in
accordance in General Procedure 1. The reaction mixture was loaded
directly on C18 column and purified via reverse-phase
chromatography (C18, gradient eluent from 5 to 75% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 20 CV) to
yield impure product 109 after concentration. The impure product
was purified via reverse-phase chromatography (C18, gradient eluent
from 0 to 50% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 20 CV) to partially purified product 109. Further
purification via trituration was performed using the centrifuge.
The solid was washed with water, the supernatant was removed, the
sequence was repeated three additional times, and then washed with
acetonitrile two times. The resulting solid was collected and
lyophilized to yield the pure product 109 as an off-white solid
(11.2 mg, 2% yield).
[0270] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.58 (s, 1H),
7.87 (d, J=8.7 Hz, 2H), 7.81 (d, J=8.7 Hz, 2H), 3.47 (q, J=7.5 Hz,
2H), 1.53 (t, J=7.4 Hz, 3H).
[0271] LCMS: m/z [M+1].sup.+=359.2; R.sub.T=1.42 min;
purity=95.2%.
[0272] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
Example 10: Preparation of
N-(3-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-di-
hydropyrimidine-5-carboxamide (110, Formula (II.sub.m), with
Reference to FIG. 18)
[0273] Step One. 3-((Trimethylsilyl)ethynyl)aniline (110-A). To a
solution of PdCl.sub.2(PPh.sub.3).sub.2 (124 mg, 0.176 mmol), CuI
(22 mg, 0.118 mmol) and 3-bromoaniline (320 .mu.L, 2.94 mmol) was
added THF (10 mL) and the solution was degassed by bubbling with
N.sub.2. The solution mixture was treated with 2-ethanolamine (360
.mu.L, 5.88 mmol) and ethynyltrimethylsilane (620 .mu.L, 4.41 mmol)
and the reaction was stirred for 2 days at 65.degree. C. The
reaction mixture was filtered through a pad of Celite using ethyl
acetate. The crude product was concentrated and carried onto the
subsequent reaction without further purification.
[0274] LCMS: m/z [M+1].sup.+=190.3; R.sub.T=1.82 min;
purity=66%.
[0275] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0276] Step Two. 3-Ethynylaniline (110-B). To a solution of 110-A
(556 g, 2.94 mmol) in THF (15 mL) was added TBAF (1 M in THF, 4.40
mL, 4.40 mmol) and the resulting black solution was stirred at room
temperature. After 1 h, the reaction mixture was washed with water
(40 mL) and extracted with CH.sub.2Cl.sub.2 (3.times.10 mL). The
organic phase was washed with brine (10 mL), dried over MgSO.sub.4,
and then concentrated. The crude product was subjected to
purification via ISCO (SiO.sub.2, 10 to 50% ethyl acetate in
hexanes) to afford 110-B as a dark brown oil (320 mg, 93.2%
yield).
[0277] 1H NMR (400 MHz, CDCl.sub.3) .delta. 7.10 (ddd, J=8.1, 7.6,
0.5 Hz, 1H), 6.92-6.88 (m, 1H), 6.83-6.79 (m, 1H), 6.67 (ddd,
J=8.1, 2.4, 1.0 Hz, 1H), 3.68 (brs, 2H), 3.01 (s, 1H).
[0278] LCMS: m/z [M+1].sup.+=118.4; R.sub.T=1.22 min;
purity=97%.
[0279] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0280] Step Three. 3-(1H-1,2,3-Triazol-5-yl)aniline (110-C). A
solution of 3-ethynylaniline 110-B (300 mg, 2.56 mmol), CuI (24.4
mg, 0.128 mmol) and trimethylsilyl azide (510 .mu.L, 3.84 mmol) in
anhydrous MeOH/DMF solution (12.0 mL, 1:23 v/v) was heated
overnight at 100.degree. C. The reaction mixture was washed with
water (20 mL) and extracted with EtOAc (3.times.10 mL). The organic
phase was washed with saturated NH.sub.4Cl (10 mL), brine (10 mL),
dried over MgSO.sub.4 and then concentrated. The crude product was
subjected to purification via ISCO (10-100% ethyl acetate in
hexanes) to afford the product as a pink solid (205 mg, 50%
yield).
[0281] LCMS: m/z [M+1].sup.+=161.4; R.sub.T=0.79 min;
purity=98%.
[0282] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0283] Step Four.
N-(3-(1H-1,2,3-triazol-5-yl)phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-di-
hydropyrimidine-5-carboxamide (110). To a solution of t-BuONa (120
mg, 1.25 mmol) in DMSO (3.1 mL) was added
4,6-dihydroxy-2-methylmercaptopyrimidine (198 mg, 1.25 mmol) and
the solution was stirred at rt for 5 min. At the same time, 110-C
(100 mg, 0.624 mmol) was dissolved in 1,4-dioxane (0.800 mL) and to
the solution was added triphosgene (61.0 mg, 0.206 mmol). The
suspension was stirred vigorously for 2 min at rt and iPr.sub.2NEt
(220 .mu.L, 1.25 mmol) was added. The solution was stirred
vigorously for 2 min. Then the prepared DMSO solution was added to
the suspension at once and the reaction was stirred at 90.degree.
C. for 30 min. Afterwards, water (2 mL) was added to the reaction
mixture and the crude solution was transferred into a flask using
MeOH. The crude mixture was concentrated and the crude product was
subject to purification by ISCO (30 g, C18 column, 10 mM AmF in
water/MeCN). The C18 column was flushed with DMSO and was
evaporated with air drying to afford the crude product. The product
was subject to purification by ISCO (30 g, C18 column, 0 to 50%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM) to
afford the product (22.8 mg, 5.3% yield).
[0284] 1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.59 (s, 1H), 8.11
(s, 1H), 7.68 (s, 3H), 2.85 (s, 3H).
[0285] LCMS: m/z [M+1].sup.+=345.1; R.sub.T=1.33 min;
purity=>99%.
[0286] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 11: Preparation of
N-(4-(1H-1,2,4-triazol-3-yl)phenyl)-6-hydroxy-2-(isopropylthio)-4-oxo-1,4-
-dihydropyrimidine-5-carboxamide (112, Formula (II.sub.n), with
Reference to FIG. 19)
[0287] Step One.
6-Hydroxy-2-(isopropylthio)-3-(3,4,5-trimethoxybenzyl)pyrimidin-4(3H)-one
(112-A). A mixture of 102-A (1.00 g, 3.91 mmol), diethyl malonate
(600 .mu.L, 3.91 mmol), and NaOMe (1.8 mL, 7.82 mmol, 4.4 M in
MeOH) in methanol (4.4 mL) was heated to reflux for 3 h. The
reaction was then cooled to .about.50.degree. C., isopropyl iodide
(4.4 mL, 39.1 mmol) was then added in one-portion. The reaction was
stirred for an additional 30 min at 50.degree. C. The reaction
mixture was then cooled to rt, then concentrated under reduced
pressure. The crude product was purified via reverse-phase
chromatography (gradient eluent from 0 to 100% acetonitrile in
water with an ammonium formate buffer 10 mM over 15 CV) to yield
the product as a white solid (695 mg, 49% yield), after
lyophilization.
[0288] LCMS: m/z [M+1].sup.+=367.02; R.sub.T=1.42 min;
purity=>99%.
[0289] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0290] Step Two.
N-(4-(1H-1,2,4-Triazol-3-yl)phenyl)-2-(isopropylthio)-4,6-dioxo-1-(3,4,5--
trimethoxybenzyl)-1,4,5,6-tetrahydropyrimidine-5-carboxamide
(112-B). 112-A (354 mg, 0.924 mmol) was added to a stirring
solution of sodium tert-butoxide (87 mg, 0.924 mmol) dissolved in
DMSO (4.2 mL) at rt for 5 min. In a separate flask,
4-(1H-1,2,4-triazol-5-yl)aniline (141 mg 0.840 mmol, 95% purity)
was dissolved in 1,4-dioxane (1.1 mL), to this solution was added
triphosgene (82 mg, 0.277 mmol) in one-portion. The suspension was
stirred vigorously for 2 min at rt, then iPr.sub.2NEt (290 .mu.L,
1.68) was added. The suspension was stirred vigorously at rt for 2
min. Freshly prepared solution of sodium
6-hydroxy-2-(methylthio)pyrimidin-4-olate in DMSO was added to the
suspension in one-portion. The reaction was stirred at 90.degree.
C. for 30 min, until complete consumption of starting material
observed via LCMS. The reaction mixture was loaded directly on C18
column and purified via reverse-phase chromatography (C18, gradient
eluent from 10 to 75% acetonitrile in water with an ammonium
bicarbonate buffer 10 mM over 20 CV) to yield the product 112-C,
after lyophillization, as a white solid (34 mg, 6% yield).
[0291] LCMS: m/z [M+1].sup.+=553.4; R.sub.T=1.73 min;
purity=85.3%.
[0292] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0293] Step Three.
N-(4-(1H-1,2,4-triazol-3-yl)phenyl)-6-hydroxy-2-(isopropylthio)-4-oxo-1,4-
-dihydropyrimidine-5-carboxamide (112). Solution of 112-B (31.2 mg,
0.0452 mmol) in dichloromethane (1.5 mL) was added trifluoroacetic
acid (270 .mu.L), the reaction was sealed in a high-pressure vessel
and heated to 60.degree. C. After 20 h, the reaction mixture was
concentrated and co-evaporated with methanol (3.times.). The crude
product was purified via reverse-phase chromatography (C18,
gradient eluent from 0 to 60% acetonitrile in water with an
ammonium bicarbonate buffer 10 mM over 20 CV) to yield the product
7, after lyophillization, as a white solid. Further purification
via trituration was performed using the centrifuge. The solid was
washed with water, the supernatant was removed, the sequence was
repeated three additional times, and then washed with acetonitrile
two times. The resulting solid was collected and lyophilized to
yield the pure product 112 as an off-white solid (13.0 mg, 64%
yield).
[0294] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 9.49 (s, 1H),
8.15-8.08 (m, 2H), 7.88-7.80 (m, 2H), 4.28-4.16 (m, 1H), 1.59 (d,
J=6.5 Hz, 6H).
[0295] LCMS: m/z [M+1].sup.+=373.1; R.sub.T=1.07 min;
purity=98.9%.
[0296] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
Example 12: Preparation of
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2-((cyclopropylmethyl)thio)-4,6-dioxo-
-1,4,5,6-tetrahydropyrimidine-5-carboxamide (113, Formula
(II.sub.o), with Reference to FIG. 20)
[0297] Step One.
2-((Cyclopropylmethyl)thio)-1-(3,4,5-trimethoxybenzyl)pyrimidine-4,6(1H,5-
H)-dione (113-A). To a mixture of 102-A (697 mg, 2.71 mmol, 99.4%
purity), diethyl malonate (940 mL, 6.13 mmol, and NaOMe (2.8 mL,
12.3 mmol, 4.4 M in MeOH) in methanol (4.9 mL). The reaction was
heated to reflux for 2 h, the reaction was then cooled to
50.degree. C., (bromomethyl)cyclopropane (290 .mu.L, 2.98 mmol) was
added stirred for an additional 12 h at 50.degree. C., incomplete
conversion was observed. An additional NaOMe solution (0.5 mL, 2.71
mmol, 4.4 M in MeOH) was added to the reaction mixture, followed by
the addition of (bromomethyl)cyclopropane (300 .mu.L, 3.09 mmol).
The resulting reaction mixture was stirred for 30 min, complete
conversion was observed via LCMS, the reaction mixture was then
concentrated. Ethyl acetate (.about.10 mL) was added, the
precipitate was filtered and washed with isopropanol (.about.10
mL), then washed with ethyl acetate. The product was isolated as a
white solid (715 mg, 67% yield).
[0298] LCMS: m/z [M+1].sup.+=379.4; m/z [M-1].sup.-=377.5;
R.sub.T=1.45 min; purity=96.5%.
[0299] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0300] Step Two.
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2-((cyclopropylmethyl)thio)-4,6-dioxo-
-1-(3,4,5-trimethoxybenzyl)-1,4,5,6-tetrahydropyrimidine-5-carboxamide
(113B). Compound 113B was synthesized following General Procedure
1. Crude 113A was purified via reverse-phase chromatography (C18,
gradient eluent from 0 to 70% acetonitrile in water with an
ammonium bicarbonate buffer 10 mM over 20 CV) to yield the
partially purified product 113A, after lyophillization, as a yellow
solid (255 mg, 16% yield).
[0301] LCMS: m/z [M+1].sup.+=565.5; R.sub.T=1.82 min;
purity=50.0%.
[0302] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0303] Step Three.
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2-((cyclopropylmethyl)thio)-4,6-dioxo-
-1,4,5,6-tetrahydropyrimidine-5-carboxamide (113). Solution of
113-B (225 mg, 0.199 mmol) in dichloromethane (6.6 mL) was added
trifluoroacetic acid (1.2 mL), the reaction was sealed in a
high-pressure vessel and heated to 60.degree. C. After 20 h, the
reaction mixture was concentrated and co-evaporated with methanol
(3.times.). The crude product 113 was purified via reverse-phase
chromatography (C18, gradient eluent from 0 to 40% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 20 CV);
fractions containing the product was collected, and then
concentrated under reduced pressure. The residue was added water
with ammonium bicarbonate buffer 10 mM (.about.20 mL), then
sonicated. The precipitate was filtered off and the filtrate was
concentrated. The residue was purified via reverse-phase
chromatography (C18, gradient eluent from 0 to 50% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 20 CV) to
yield partially pure product, which was purified again via
reverse-phase chromatography (C18, gradient eluent from 0 to 30%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV) to yield partially pure product as a white solid, after
concentration. The white solid was triturated with deionized water,
then the filtrate was lyophilized. Further purification via
trituration was performed using the centrifuge. The solid was
washed with water, the supernatant was removed, the sequence was
repeated three additional times, and then washed with acetonitrile
two times. The resulting solid was collected and dried under high
vacuum to yield the pure product 113 as an off-white solid (3.0 mg,
4% yield).
[0304] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.61-8.58 (m,
1H), 7.89 (d, J=5.6 Hz, 2H), 7.83-7.78 (m, 2H), 2.40-2.19 (m, 2H),
1.32 (br s, 4H), 0.49 (dd, J=10.3, 4.6 Hz, 1H).
[0305] LCMS: m/z [M+1].sup.+=385.2; R.sub.T=1.54 min;
purity=96.4%.
[0306] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
Example 13: Preparation of
N-(4-(1H-tetrazol-5-yl)phenyl)-2-(ethylthio)-4-hydroxy-6-oxo-1,6-dihydrop-
yrimidine-5-carboxamide (114, Formula (II.sub.p), with Reference to
FIG. 21)
[0307] Step One. 2-(4-methoxybenzyl)-5-(4-nitrophenyl)-2H-tetrazole
(114-A). To a solution of 5-(4-nitrophenyl)-1H-tetrazole (1.00 g,
5.23 mmol) and K.sub.2CO.sub.3 (1.33 g, 5.76 mmol) in DMF (3.5 mL)
was added p-methoxybenzyl chloride (901 mg, 5.76 mmol). The
reaction mixture was stirred at 55.degree. C. for 18 h. The mixture
cooled to rt and was diluted with sat. aq. NH.sub.4Cl (50 mL) and
extracted with EtOAc (3.times.25 mL). The combined organic extracts
were pooled together and washed with brine (50 mL), dried over
MgSO.sub.4, and then concentrated. The crude product was
precipitated upon addition of ethyl acetate followed by filtration.
The filtrate was concentrated and this method was repeated until
the newly formed precipitate was greater than 90% purity. No
further purification was performed.
[0308] 1H NMR (400 MHz, CDCl.sub.3) .delta. 8.31 (s, 4H), 7.43-7.38
(m, 2H), 6.94-6.88 (m, 2H), 5.76 (s, 2H), 3.80 (s, 3H).
[0309] LCMS: m/z [M+1].sup.+=312.2; R.sub.T=1.79 min;
purity=>99%.
[0310] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
[0311] Step Two. 4-(2-(4-methoxybenzyl)-2H-tetrazol-5-yl)aniline
(114-B). To a solution of the 114-A (1.09 g, 3.52 mmol) in a
mixture of EtOH/THF/H.sub.2O/NH.sub.4Cl (sat.) (7 mL, 4:4:1:1 v/v)
was added Fe powder (1.98 g, 35.5 mmol) and the mixture was heated
to 80.degree. C. overnight. The reaction mixture was filtered
through a small pad of Celite/MgSO.sub.4 mixture (1:1) using EtOAc.
The crude product obtained after filtration was concentrated in
vacuo. The product was not purified any further to afford an
off-white solid (1.04 g, quant. yield).
[0312] LCMS: m/z [M+1].sup.+=282.3; R.sub.T=1.51 min;
purity=97.1%.
[0313] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
[0314] Step Three.
N-(4-(2-(4-methoxybenzyl)-2H-tetrazol-5-yl)phenyl)-4,6-bis((4-methoxybenz-
yl)oxy)-2-(methylthio)pyrimidine-5-carboxamide (114-C). To a cooled
solution (0.degree. C.) of 2-(methylthio)pyrimidine-5-carbonyl
chloride (183 mg, 0.711 mmol) in THF (2.5 mL) was added Et.sub.3N
(109 .mu.L, 0.782 mmol), followed by the corresponding aniline
intermediate 114-B (200 mg, 0.711 mmol) dissolved in THF (2.5 mL).
The reaction mixture was warmed to rt and stirred for 1.5 h. Once
the starting material was fully consumed, the solution was cooled
to 0.degree. C. and treated with PMBOH (333 .mu.L, 2.84 mmol)
followed by NaH (114 mg, 2.84 mmol, 60% dispersed in oil). The
solution was stirred at 0.degree. C. for 10 min and then warmed to
rt. After 1.5 h, the solution was cooled to 0.degree. C. and
treated with water. The solution was concentrated then washed with
water and filtered. The precipitate was then washed with MTBE to
afford the desired product as a white solid (362 mg, 72% yield).
The product was used without additional purification.
[0315] LCMS: m/z [M+1].sup.+=706.5; R.sub.T=2.11 min;
purity=94.1%.
[0316] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
[0317] Step Four.
N-(4-(2-(4-methoxybenzyl)-2H-tetrazol-5-yl)phenyl)-4,6-bis((4-methoxybenz-
yl)oxy)-2-(methylsulfonyl)pyrimidine-5-carboxamide (114-D). To a
solution of the methythioether substrate (362 mg, 0.512 mmol) in
CH.sub.2Cl.sub.2 (10.2 mL) was added mCPBA (241 mg, 1.08 mmol) at
0.degree. C. and open to air. After 10 min, the reaction was warmed
to rt and stirred for 3.5 h. The reaction mixture was treated with
NaHCO.sub.3 (sat) (50 mL) and stirred for 30 min. The organic
fraction was washed with NaHCO.sub.3 (sat) (3.times.10 mL), brine
(10 mL), dried (MgSO.sub.4), filtered and then concentrated. Based
on LC-MS, traces of mCBA was in the crude product and so the sample
was dissolved in CH.sub.2Cl.sub.2 (20 mL) and once more subjected
to the previous workup to afford a yellow solid (320 mg, 85%
yield), which was used without any additional purification.
[0318] LCMS: m/z [M+1].sup.+=738.4; R.sub.T=1.91 min;
purity=88.6%.
[0319] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
[0320] Step Five.
2-(Ethylthio)-N-(4-(2-(4-methoxybenzyl)-2H-tetrazol-5-yl)phenyl)-4,6-bis(-
(4-methoxybenzyl)oxy)pyrimidine-5-carboxamide (114-E). To a
solution of the 114-D (320 mg, 0.434 mmol) in THF (8.7 mL) at
0.degree. C. was added ethanethiol (160 .mu.L, 2.17 mmol) followed
by dropwise addition of KOtBu (2.60 mL, 2.60 mmol, 1 M in THF). The
resulting solution was stirred at 0.degree. C. for 10 min.
Afterwards, the reaction mixture was warmed to rt and stirred for 2
h. The solution was treated with H.sub.2O (20 mL) and extracted
with EtOAc (3.times.10 mL). The organic fraction was washed with
brine (20 mL), dried (MgSO.sub.4), filtered and concentrated to
afford the product as an off-white solid (257 mg, 82% yield), which
was used without any additional purification.
[0321] LCMS: m/z [M+1].sup.+=720.1; R.sub.T=2.14 min;
purity=81.1%.
[0322] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
[0323] Step Six.
N-(4-(2H-Tetrazol-5-yl)phenyl)-2-(ethylthio)-4-hydroxy-6-oxo-1,6-dihydrop-
yrimidine-5-carboxamide (114). To a solution of the 114-E (257 mg,
0.356 mmol) in CH.sub.2Cl.sub.2 (11.9 mL) at rt was added TFA (2.1
mL). The reaction vessel was sealed and heated at 60.degree. C.
After 48 h, the mixture was concentrated, using MeOH to
co-evaporate any residual TFA. The crude product was subjected to
purification via to ISCO (60 g, C18 column, from 0 to 50%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM) to
afford the product as a white solid (87 mg, 68% yield).
[0324] 1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.08 (d, J=8.6 Hz,
2H), 7.83 (d, J=8.7 Hz, 2H), 3.43 (q, J=7.4 Hz, 2H), 1.52 (t, J=7.4
Hz, 3H).
[0325] LCMS: m/z [M+1].sup.+=360.3; R.sub.T=1.35 min;
purity=>99%.
[0326] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
Example 14: Preparation of
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(isobutylthio)-6-oxo-1,6--
dihydropyrimidine-5-carboxamide (115, Formula (II.sub.q), with
Reference to FIG. 22)
[0327] Step One.
N-(4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4,6-bis((4-methox-
ybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxamide (115-A). To a
stirring solution of
4,6-dichloro-2-(methylthio)-5-pyrimidinecarbonyl chloride (227 mg,
0.881 mmol) and triethylamine (98 mg, 0.97 mmol) in anhydrous THF
(6.3 mL) was added amine XX (247 mg, 280 mmol) at 0.degree. C. The
reaction was warmed up to rt over 1 h, then cooled to 0.degree. C.
again. para-Methoxylbenzylamine (220 .mu.L, 1.76 mmol) was added,
followed by portion-wise addition of sodium hydride (109 mg, 2.73
mmol, 60% dispersed in mineral oil). The reaction was allowed to
stir at 0.degree. C. for 5 min, and then warmed up to rt overnight.
Additional sodium hydride (50 mg) was added, then stirred for an
additional 1 h; when complete conversion was observed via LCMS, the
reaction was quenched with methanol (.about.10 mL). tert-Butyl
methyl ether (.about.20 mL) was added, and then sonicated. The
precipitate was filtered and washed with MeOH and TBME, the
precipitate was collected. The product was isolated as an off-white
solid (550 mg, 80% yield).
[0328] LCMS: m/z [M+1].sup.+=705.5; R.sub.T=2.02 min;
purity=90.6%.
[0329] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0330] Step Two.
N-(4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4,6-bis((4-methox-
ybenzyl)oxy)-2-(methylsulfonyl)pyrimidine-5-carboxamide (115-B).
115-A (550 mg, 0.707 mmol) was dissolved in dichloromethane (3.5
mL) and cooled to 0.degree. C. meta-Chloroperoxybenzoic acid (332
mg, 1.48 mmol, 77% in water) was added and the resulting reaction
mixture was warmed up to rt overnight. The reaction was quenched
with sat. NaHCO.sub.3 aq. solution and stirred vigorously for 30
min. The organic phase was extracted, and washed with sat.
NaHCO.sub.3 aq. solution (3.times.), and then brine (1.times.). The
resulting organic extract was dried over MgSO.sub.4 then
concentrated to yield the crude product as an orange solid (440 mg,
70% yield). No further purification required.
[0331] LCMS: m/z [M+1].sup.+=737.5; R.sub.T=1.84 min;
purity=83.4%.
[0332] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0333] Step Three.
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(isobutylthio)-6-oxo-1,6--
dihydropyrimidine-5-carboxamide (115). To a stirring solution of
115-B (105 mg, 0.119 mmol) and 2-methylpropane-1-thiol (64 .mu.L,
0.594 mmol) in anhydrous THF (2.4 mL) at 0.degree. C., was added
KOtBu (714 mL, 0.714 mmol, 1.0 M in THF), under inert atmosphere.
The resulting reaction mixture was warmed up to rt over 30 min,
after complete consumption of the starting material was observed
via LCMS, the reaction was quenched with deionized water (.about.5
mL). The reaction mixture was extracted with EtOAc (3.times.), the
combined organic extracts was dried over MgSO.sub.4, and then
concentrated. The crude product 115-C was used without further
purification. The crude product 115-C was isolated as an off-white
solid.
[0334] Crude 115-C was suspended in dichloromethane (2.4 mL), TFA
(700 .mu.L) was added, followed by TfOH (100 .mu.L), the resulting
reaction mixture was sealed in a high pressure vessel and heated to
80.degree. C. After 20 h, the reaction mixture was added methanol,
and then co-evaporated with methanol (3.times.) under reduced
pressure. The crude product was purified via reverse-phase
chromatography (C18, gradient eluent from 5 to 25% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 20 CV) to
yield impure product 115 after lyophilization. Further purification
via trituration was performed using the centrifuge. The solid was
washed with water, the supernatant was removed, the sequence was
repeated three additional times, and then washed with acetonitrile
two times. The resulting solid was collected and lyophilized to
yield the pure product 115 as an off-white solid (25.4 mg, 55%
yield).
[0335] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.61 (s, 1H),
7.90 (d, J=7.6 Hz, 2H), 7.81 (d, J=8.5 Hz, 2H), 3.37 (d, J=6.7 Hz,
2H), 2.14 (d, J=6.9 Hz, 1H), 1.17 (d, J=6.6 Hz, 6H).
[0336] LCMS: m/z [M+1].sup.+=387.0; R.sub.T=1.60 min;
purity=98.6%.
[0337] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
Example 15: Preparation
N-(3-Fluoro-4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(methylthio)-6-o-
xo-1,6-dihydropyrimidine-5-carboxamide (116, Formula (II.sub.r),
with Reference to FIG. 23)
[0338] Step One. 3-Fluoro-4-((trimethylsilyl)ethynyl)aniline
(116-A). A round bottom flask containing 4-bromo-2-fluoroaniline
(380 mg, 2.0 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (84 mg, 0.12 mmol),
and CuI (15 mg, 0.08 mmol) was purged with nitrogen for 15 min.
Anhydrous THF (6.7 mL) was added, followed by
trimethylsilylacetylene (560 .mu.L, 4.0 mmol) and ethanolamine (240
.mu.L, 4.0 mmol). The reaction mixture was sealed and heated to
65.degree. C. for 20 h. The reaction mixture was cooled to rt,
filtered through a small pad of Celite. The filtrate was
concentrated and used without further purification.
[0339] LCMS: R.sub.T=1.86 min.
[0340] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0341] Step Two. 4-Ethynyl-3-fluoroaniline (116-B). To a solution
of crude 116-A (2.0 mmol) dissolved in methanol (2.0 mL) was added
potassium carbonate (553 mg, 4.00 mmol) at rt. The resulting
reaction mixture was stirred at rt for 2 h, or until complete
consumption of the starting material was observed. The reaction
mixture was filtered, and the filtrate was concentrated. The crude
product 116-B was used without further purification.
[0342] LCMS: m/z [M+1].sup.+=136.1; R.sub.T=1.34 min.
[0343] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0344] Step Three.
3-Fluoro-4-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)aniline
(116-C). To a solution of crude 116-B (2.0 mmol) was dissolved in a
mixture of anhydrous MeOH/DMF (10.0 mL, 1:9, v/v), under inert
atmosphere. At room temperature, CuI (38 mg, 0.20 mmol) was added,
followed by the addition of 5-(azidomethyl)-1,2,3-trimethoxybenzene
(487 mg, 2.14 mmol), the reaction mixture was sealed in a high
pressure vessel and heated to 100.degree. C. for 2 h, the reaction
was then cooled to rt, then concentration. The crude product was
purified via ISCO (SiO.sub.2, gradient eluent from 0 to 40% ethyl
acetate in hexanes over 20 CV) to yield the product as a brown oil
(374 mg, 37% yield over 3 steps).
[0345] LCMS: m/z [M+1].sup.+=359.3; R.sub.T=1.39 min;
purity=70%.
[0346] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0347] Step Four.
N-(3-Fluoro-4-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4-
,6-bis((4-methoxybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxamide
(116-D). To a stirring solution of 116-C (374 mg, 0.731 mmol, 70%
purity) in anhydrous THF (5.2 mL) at 0.degree. C., under inert
atmosphere was added Et.sub.3N (110 .mu.L, 0.804 mmol), followed by
the addition of 4,6-dichloro-2-(methylthio)pyrimidine-5-carbonyl
chloride (188 mg, 0.731 mmol). The resulting reaction mixture was
warmed up to rt over 60 min, until complete consumption of the
starting materials were observed via LCMS. The reaction mixture was
then cooled to 0.degree. C., then p-methoxylbenzyl alcohol (180
.mu.L, 1.46 mmol) was added, followed by careful addition of NaH
(91 mg, 2.27 mmol, 60% dispersed in oil). The reaction was kept at
0.degree. C. for 5 min, then warmed up to rt over 20 h; the
progress of the reaction was monitored by LCMS. Methanol (.about.1
mL) was added to quench the reaction, then TBME (20 mL) was added
and the resulting suspension was sonicated for 10 min. The
suspension was then filtered, and the solid was washed with TBME,
the brown solid (308 mg, 54% yield) was collected.
[0348] LCMS: m/z [M+1].sup.+=784.3; R.sub.T=2.03 min;
purity=92.9%.
[0349] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0350] Step Five.
N-(3-Fluoro-4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(methylthio)-6-o-
xo-1,6-dihydropyrimidine-5-carboxamide (116). 116-D (308 mg, 0.365
mmol) dissolved in CH.sub.2Cl.sub.2 (7.3 mL) was added TFA (2.1 mL)
and sealed in a pressure vessel, heated to 60.degree. C. for 20 h.
Reaction was cooled to rt, then TfOH (50 .mu.L) was added, sealed
and heated to 60.degree. C. for 20 h. The reaction mixture was
cooled to rt, then co-evaporated with MeOH (3.times.). TBME and
deionized water (.about.10 mL, 1:1 v/v) was added; the suspension
was sonicated. The solid was filtered and washed with water and
washed with TBME. The green solid was collected (302 mg,
quantitative yield, 69.6% purity) as the TMB-protected
intermediate
[0351] The TMB-protected intermediate (288 mg, 0.256 mmol) was
added CH.sub.2Cl.sub.2 (1.3 mL), TfOH (640 .mu.L), the resulting
reaction mixture was sealed in a microwave vessel and heated to
80.degree. C. for 20 h. The reaction was then cooled to rt, then
co-evaporated with methanol (3.times.). The crude product was
purified via reverse-phase chromatography (C18, gradient eluent
from 0 to 30% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 20 CV) to yield impure product 116 after
lyophilization. Further purification via trituration was performed
using the centrifuge. The solid was washed with water, the
supernatant was removed, the sequence was repeated three additional
times, and then washed with acetonitrile two times. The resulting
solid was collected and lyophilized to yield the pure product 116
as an off-white solid (12.9 mg, 14% yield).
[0352] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.66 (s, 1H),
7.92 (t, J=8.2 Hz, 1H), 7.84 (dd, J=12.3, 2.0 Hz, 1H), 7.52 (dd,
J=8.6, 1.9 Hz, 1H), 2.86 (s, 3H).
[0353] LCMS: m/z [M+1].sup.+=363.1; m/z [M-1].sup.-=361.3;
R.sub.T=1.38 min; purity=98.6%.
[0354] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 16: Preparation of
N-(2-Fluoro-4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(methylthio)-6-o-
xo-1,6-dihydropyrimidine-5-carboxamide (117, Formula (II.sub.s),
with Reference to FIG. 24)
[0355] Step One. 2-Fluoro-4-((trimethylsilyl)ethynyl)aniline
(117-A). A round bottom flask containing 4-bromo-2-fluoroaniline
(380 mg, 2.0 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (84 mg, 0.12 mmol),
and CuI (15 mg, 0.08 mmol) was purged with nitrogen for 15 min.
Anhydrous THF (6.7 mL) was added, followed by
trimethylsilylacetylene (560 .mu.L, 4.0 mmol) and ethanolamine (240
.mu.L, 4.0 mmol). The reaction mixture was sealed and heated to
65.degree. C. for 20 h. The reaction mixture was cooled to rt,
filtered through a small pad of Celite. The filtrate was
concentrated and used without further purification.
[0356] LCMS: R.sub.T=1.91 min.
[0357] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0358] Step Two. 4-Ethynyl-2-fluoroaniline (117-B). To a solution
of crude 116-A (2.0 mmol) dissolved in methanol (2.0 mL) was added
potassium carbonate (553 mg, 4.00 mmol) at rt. The resulting
reaction mixture was stirred at rt for 2 h, or until complete
consumption of the starting material was observed. The reaction
mixture was filtered, and the filtrate was concentrated. The crude
product 117-B was used without further purification.
[0359] LCMS: R.sub.T=1.37 min.
[0360] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0361] Step Three.
2-Fluoro-4-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)aniline
(117-C). To a solution of crude 117-B (2.0 mmol) was dissolved in a
mixture of anhydrous MeOH/DMF (10.0 mL, 1:9, v/v), under inert
atmosphere. At room temperature, CuI (38 mg, 0.20 mmol) was added,
followed by the addition of 5-(azidomethyl)-1,2,3-trimethoxybenzene
(487 mg, 2.14 mmol), the reaction mixture was sealed in a high
pressure vessel and heated to 100.degree. C. for 2 h, the reaction
was then cooled to rt, then concentration. The crude product was
purified via ISCO (SiO.sub.2, gradient eluent from 0 to 35% ethyl
acetate in hexanes over 20 CV) to yield the product as a brown oil
(566 mg, 70% yield over 3 steps).
[0362] LCMS: m/z [M+1].sup.+=359.2; R.sub.T=1.36 min;
purity=89.9%.
[0363] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0364] Step Four.
N-(2-Fluoro-4-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4-
,6-bis((4-methoxybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxamide
(117-D). To a stirring solution of 117-C (285 mg, 0.715 mmol, 89.8%
purity) in anhydrous THF (5.1 mL) at 0.degree. C., under inert
atmosphere was added Et.sub.3N (110 .mu.L, 0.804 mmol), followed by
the addition of 4,6-dichloro-2-(methylthio)pyrimidine-5-carbonyl
chloride (184 mg, 0.731 mmol). The resulting reaction mixture was
warmed up to rt over 60 min, until complete consumption of the
starting materials were observed via LCMS. The reaction mixture was
then cooled to 0.degree. C., then p-methoxylbenzyl alcohol (180
.mu.L, 1.43 mmol) was added, followed by careful addition of NaH
(89 mg, 2.22 mmol, 60% dispersed in oil). The reaction was kept at
0.degree. C. for 5 min, then warmed up to rt over 20 h; the
progress of the reaction was monitored by LCMS. Methanol (.about.1
mL) was added to quench the reaction, then TBME (20 mL) was added
and the resulting suspension was sonicated for 10 min. The
suspension was then filtered, and the solid was washed with TBME,
the brown solid (461 mg, 74% yield) was collected.
[0365] LCMS: m/z [M+1].sup.+=783.6; R.sub.T=2.05 min;
purity=90.0%.
[0366] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0367] Step Five.
N-(2-Fluoro-4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(methylthio)-6-o-
xo-1,6-dihydropyrimidine-5-carboxamide (117). 117-D (461 mg, 0.530
mmol, 90.0% purity) dissolved in CH.sub.2Cl.sub.2 (5.3 mL) was
added TFA (3.8 mL) and sealed in a pressure vessel, heated to
60.degree. C. for 20 h. Reaction was cooled to rt, then TfOH (50
.mu.L) was added, sealed and heated to 60.degree. C. for 20 h. The
reaction mixture was cooled to rt, then co-evaporated with MeOH
(3.times.). TBME and deionized water (.about.10 mL, 1:1 v/v) was
added; the suspension was sonicated. The solid was filtered and
washed with water and washed with TBME. The green solid was
collected (100 mg, 27% yield, 77.4% purity) as the TMB-protected
intermediate.
[0368] The TMB-protected intermediate (100 mg, 0.0989 mmol, 77.4%
purity) was added CH.sub.2Cl.sub.2 (1.3 mL), TfOH (640 .mu.L), the
resulting reaction mixture was sealed in a microwave vessel and
heated to 80.degree. C. for 20 h. The reaction was then cooled to
rt, then co-evaporated with methanol (3.times.). The crude product
was purified via reverse-phase chromatography (C18, gradient eluent
from 0 to 30% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 20 CV) to yield impure product 117 after
lyophilization. Further purification via trituration was performed
using the centrifuge. The solid was washed with water, the
supernatant was removed, the sequence was repeated three additional
times, and then washed with acetonitrile two times. The resulting
solid was collected and lyophilized to yield the pure product 117
as an off-white solid (13.0 mg, 35% yield).
[0369] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.61 (s, 1H),
8.41-8.32 (m, 1H), 7.70 (t, J=7.9 Hz, 2H), 2.90 (s, 3H).
[0370] LCMS: m/z [M+1].sup.+=362.8; R.sub.T=0.92 min;
purity=97.2%.
[0371] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate; Eluent B: Acetonitrile.
Example 17: Preparation of
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2-(ethylamino)-4-hydroxy-6-oxo-1,6-di-
hydropyrimidine-5-carboxamide (118, Formula (II.sub.ll), with
Reference to FIG. 25)
[0372] Step One.
2-(Ethylamino)-N-(4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4,-
6-bis((4-methoxybenzyl)oxy)pyrimidine-5-carboxamide (118-A). 115-C
(138 mg, 0.167 mmol, 89% purity) dissolved in anhydrous THF (3.3
mL) at rt, under inert atmosphere was added N-ethylamine (330
.mu.L, 0.668 mmol, 2.0 M in THF). The reaction was allowed to stir
at rt for 30 min, until complete consumption of starting material
was observed. The reaction mixture was concentrated under reduced
pressure. The crude product was used without further
purification.
[0373] LCMS: m/z [M+1].sup.+=702.6; R.sub.T=1.96 min;
purity=88.1%.
[0374] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0375] Step Two.
N-(4-(1H-1,2,3-triazol-4-yl)phenyl)-2-(ethylamino)-4-hydroxy-6-oxo-1,6-di-
hydropyrimidine-5-carboxamide (118). TfOH (50 .mu.L) was added to a
solution of 118-A (0.167 mmol) in TFA (980 .mu.L) and
CH.sub.2Cl.sub.2 (3.3 mL). The reaction was heated at 80.degree. C.
for 72 h. After complete consumption of starting material was
observed via LCMS, the reaction was then cooled to rt, then
co-evaporated with methanol (3.times.). The crude product was
purified via reverse-phase chromatography (C18, gradient eluent
from 0 to 25% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 30 CV) to yield product 118 after lyophilization
as a white solid (22.8 mg, 40% yield).
[0376] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.57 (s, 1H),
7.88 (d, J=8.8 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 3.66-3.58 (m, 2H),
1.41 (t, J=7.3 Hz, 3H).
[0377] LCMS: m/z [M+1].sup.+=342.2; R.sub.T=1.24 min;
purity=>99%.
[0378] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
Example 18: Preparation of
N-(4-(1H-1,2,3-Triazol-4-yl)phenyl)-2-(ethyl(methyl)amino)-4-hydroxy-6-ox-
o-1,6-dihydropyrimidine-5-carboxamide (119, Formula (II.sub.mm),
with Reference to FIG. 26)
[0379] 115-C (146 mg, 0.188 mmol) dissolved in anhydrous THF (3.8
mL) at rt, under inert atmosphere was added N-ethylmethylamine (64
.mu.L, 0.750 mmol). The reaction was allowed to stir at rt for 30
min, until complete consumption of starting material was observed.
The reaction mixture was concentrated under reduced pressure. The
crude product was used without further purification. The crude
product was dissolved in CH.sub.2Cl.sub.2 (3.8 mL), TFA (1.1 mL)
was added at rt, followed by the addition of TfOH (70 .mu.L). The
resulting reaction was sealed in a pressure vessel and heated to
80.degree. C. for 72 h. The reaction was cooled, and then
co-evaporated with methanol (3.times.) concentrated under reduced
pressure. The crude product was purified via reverse-phase
chromatography (C18, gradient eluent from 0 to 25% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 30 CV) to
yield impure product 119 after lyophilization. Further purification
via trituration was performed using the centrifuge. The solid was
washed with water, the supernatant was removed, the sequence was
repeated three additional times, then washed with acetonitrile two
times. The resulting solid was collected and lyophilized to yield
the pure product 119 as a white solid (28.5 mg, 42% yield).
[0380] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.64 (s, 1H),
7.92 (d, J=8.7 Hz, 2H), 7.80 (d, J=8.8 Hz, 2H), 3.83 (d, J=7.5 Hz,
2H), 3.49 (s, 3H), 1.46 (t, J=7.3 Hz, 3H).
[0381] LCMS: m/z [M+1].sup.+=356.2; R.sub.T=1.33 min;
purity=99.5%.
[0382] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 19: Preparation of
N-(4-(1H-1,2,3-triazol-5-yl)phenyl)-2-((cyclopropylmethyl)thio)-4-hydroxy-
-6-oxo-1,6-dihydropyrimidine-5-carboxamide (121, Formula
(II.sub.t), with Reference to FIG. 27)
[0383] Step One.
4,6-Bis((4-methoxybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxylic
acid (XZ). A flask containing
4,6-dichloro-2-(methylthio)pyrimidine-5-carboxylic acid (1.099 g,
4.46 mmol) was dissolved in anhydrous THF (32 mL, under inert
atmosphere. The solution was cooled to 0.degree. C., then
p-methoxylbenzyl alcohol (1.10 mL, 8.92 mmol) was added, followed
by careful addition of NaH (535 mg, 13.4 mmol, 60% dispersed in
oil). The reaction was kept at 0.degree. C. for 5 min, then warmed
up to rt over 20 h; the progress of the reaction was monitored by
LCMS. Methanol (.about.1 mL) was added to quench the reaction; the
reaction mixture was concentrated under reduced pressure. The
residue was added CH.sub.3CN and sonicated. The suspension was then
filtered, and the solid was washed with CH.sub.3CN, the off-white
solid (1.234 g, 58% yield) was collected.
[0384] LCMS: m/z [M+1].sup.+=442.9; R.sub.T=1.86 min;
purity=90.0%.
[0385] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0386] Step Two. 2,6-Difluoro-4-((trimethylsilyl)ethynyl)aniline
(121-A). A round bottom flask containing
4-bromo-2,6-difluoroaniline (1.00 g, 4.80 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (202 mg, 0.288 mmol), and CuI (37 mg,
0.1.92 mmol) was purged with nitrogen for 15 min. Anhydrous THF
(16.0 mL) was added, followed by trimethylsilylacetylene (1.40 mL,
9.62 mmol) and ethanolamine (580 .mu.L, 9.62 mmol). The reaction
mixture was sealed and heated to 65.degree. C. for 20 h. The
reaction mixture was cooled to rt, filtered through a small pad of
Celite. The filtrate was concentrated and used without further
purification.
[0387] LCMS: m/z [M+1].sup.+=226.0; R.sub.T=2.01 min.
[0388] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0389] Step Three. 4-Ethynyl-2,6-difluoroaniline (121-B). To a
solution of crude 121-A (4.80 mmol) dissolved in methanol (4.8 mL)
was added potassium carbonate (1.33 mg, 9.60 mmol) at rt. The
resulting reaction mixture was stirred at rt for 2 h, or until
complete consumption of the starting material was observed. The
reaction mixture was filtered, and the filtrate was concentrated.
The crude product 121-B was used without further purification.
[0390] LCMS: R.sub.T=1.49 min.
[0391] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0392] Step Four.
2,6-Difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)aniline
(121-C). To a solution of crude 121-B (4.80 mmol) was dissolved in
a mixture of anhydrous MeOH/DMF (2.4 mL, 1:9, v/v), under inert
atmosphere. At room temperature, CuI (91 mg, 0.480 mmol) was added,
followed by the addition of p-methoxybenzyl azide (838 mg, 5.14
mmol), the reaction mixture was sealed in a high pressure vessel
and heated to 100.degree. C. for 48 h, the reaction was then cooled
to rt, then concentration. The crude product was purified via ISCO
(SiO.sub.2, gradient eluent from 0 to 50% ethyl acetate in hexanes
over 20 CV) to yield the product as a brown solid (1.057 g, 61%
yield over 3 steps).
[0393] LCMS: m/z [M+1].sup.+=317.1; R.sub.T=1.55 min;
purity=87.3%.
[0394] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0395] Step Five.
N-(2,6-Difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4,6--
bis((4-methoxybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxamide
(121-D). T.sub.3P (920 .mu.L, 1.55 mmol, 50% in EtOAc) was added to
a mixture of 121-C (93 mg, 0.258 mmol), triethyl amine (490 .mu.L,
3.48 mmol) and XZ (178 mg, 0.387 mmol) in EtOAc (1.3 mL). The
reaction was heated at 80.degree. C. overnight. After complete
consumption of starting material was observed via LCMS, the mixture
was concentrated. MeOH/H.sub.2O (.about.10 mL, 1:1 v/v) was added
to the residue, and sonicated to yield the title compound (108 mg,
23% yield) as a brown solid.
[0396] LCMS: m/z [M+1].sup.+=741.4; R.sub.T=2.07 min;
purity=60.7%.
[0397] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0398] Step Six.
N-(2,6-Difluoro-4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(methylthio)-
-6-oxo-1,6-dihydropyrimidine-5-carboxamide (121). TfOH (35 .mu.L)
was added to a solution of 121-C (114 mg, 0.0935 mmol) in TFA (550
.mu.L) and CH.sub.2Cl.sub.2 (1.9 mL). The reaction was heated at
80.degree. C. for 20 h. After complete consumption of starting
material was observed via LCMS, MeOH 10 mL was added to the
mixture. The resulting mixture was concentrated to remove
CH.sub.2Cl.sub.2 and TFA. Add MeOH 10 mL two more times to
co-evaporate with CH.sub.2Cl.sub.2 and TFA. The crude product was
purified via reverse-phase chromatography (C18, gradient eluent
from 0 to 25% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 30 CV) to yield impure product 121 after
lyophilization. Further purification via trituration was performed
using the centrifuge. The solid was washed with water, the
supernatant was removed, the sequence was repeated, then washed
with acetonitrile. The resulting solid was collected and
lyophilized to yield the pure product 121 as a white solid (6.4 mg,
18% yield).
[0399] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.54 (s, 1H),
7.52 (s, 2H), 2.79 (s, 3H).
[0400] LCMS: m/z [M+1].sup.+=381.1; R.sub.T=1.36 min;
purity=98.6%.
[0401] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
Example 20: Preparation of
N-(3,5-difluoro-4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(methylthio)-
-6-oxo-1,6-dihydropyrimidine-5-carboxamide (122, Formula
(II.sub.u), with Reference to FIG. 28)
[0402] Step One. 3,5-difluoro-4-((trimethylsilyl)ethynyl)aniline.
To a mixture of PdCl.sub.2(PPh.sub.3).sub.2 (168 mg, 0.24 mmol),
CuI (30 mg, 0.16 mmol) and 3,5-difluoro-4-iodoaniline (1.02 g, 4.0
mmol), was added THF 14.0 mL, followed by ethanolamine (489 mg, 8.0
mmol) and TMS-acetylene (786 mg, 8.0 mmol). The reaction was heated
at 65.degree. C. for 20 h. After complete consumption of starting
material was observed via LCMS, the mixture was filtered through
Celite, washed by ethyl acetate (3.times.10 mL). The filtrate was
concentrated to yield the desired product, which was used as crude
without further purification.
[0403] LCMS: m/z [M+1].sup.+=226.1; R.sub.T=1.81 min;
purity=>87%.
[0404] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0405] Step Two. 4-ethynyl-3,5-difluoroaniline. To a solution of
3,5-difluoro-4-((trimethylsilyl)ethynyl)aniline (1.1 g, 4.0 mmol)
in MeOH (4.0 mL), was added K.sub.2CO.sub.3 (1.11 g, 8.0 mmol). The
reaction was stirred at rt for 2 h. After complete consumption of
starting material was observed via LCMS, the mixture was filtered
through Celite, washed by MeOH (3.times.5 mL). The filtrate was
concentrated to yield the desired product, which was used without
further purification (product were to be used right away to avoid
decomposition).
[0406] LCMS: m/z [M+1].sup.+=154.3; R.sub.T=1.43 min;
purity=>90%.
[0407] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0408] Step Three.
3,5-difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)aniline.
To a suspension of 4-ethynyl-3,5-difluoroaniline (306 mg, 2.0 mmol)
and CuI (38 mg, 0.20 mmol) in MeOH/DMF (1.0 mL, 1:9, v/v), was
added PMB-N.sub.3 (359 mg, 2.2 mmol). The reaction tube was sealed
and heated at 100.degree. C. for 2 h. After complete consumption of
starting material was observed via LCMS, the mixture was
concentrated, and the residue was purified by ISCO (25 g SiO.sub.2,
0-80% ethyl acetate in hexanes), yielding the title compound as a
yellow solid (265 mg, 42% yield).
[0409] LCMS: m/z [M+1].sup.+=317.0; R.sub.T=1.46 min;
purity=>99%.
[0410] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0411] Step Four.
N-(3,5-difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4,6--
bis((4-methoxybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxamide.
T.sub.3P (636.36 mg, 2 mmol) was added to a mixture of
3,5-difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)aniline
(158 mg, 0.50 mmol), triethylamine (455 mg, 4.5 mmol) and
4,6-bis((4-methoxybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxylic
acid (331 mg, 0.75 mmol) in EtOAc 2.5 mL. The reaction was heated
at 100.degree. C. overnight. After complete consumption of starting
material was observed via LCMS, the mixture was concentrated. MeOH
(10 mL) was added to the residue, and sonicated to yield the title
compound as a light yellow solid (406 mg, quantitative yield).
[0412] LCMS: m/z [M+1].sup.+=741.2; R.sub.T=2.06 min;
purity=>90%.
[0413] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0414] Step Five.
N-(3,5-difluoro-4-(1H-1,2,3-triazol-4-yl)phenyl)-4-hydroxy-2-(methylthio)-
-6-oxo-1,6-dihydropyrimidine-5-carboxamide (122). TfOH (100 .mu.L)
was added to a solution of
N-(3,5-difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl)-4,6--
bis((4-methoxybenzyl)oxy)-2-(methylthio)pyrimidine-5-carboxamide
(200 mg, 0.27 mmol) in TFA 1.6 mL and CH.sub.2Cl.sub.2 (5.0 mL).
The reaction was heated at 80.degree. C. for 2 days. After complete
consumption of starting material was observed via LCMS, MeOH (10
mL) was added to the mixture. The resulting mixture was
concentrated to remove CH.sub.2Cl.sub.2 and TFA. MeOH (10 mL) was
added two more times to co-evaporate with TFA. MeOH (5 mL) was
added, and the mixture was filtered, the precipitate collected was
washed with acetonitrile (2.times.5 mL), water (1.times.5 mL), and
acetone (1.times.5 mL) to yield the title compound as light yellow
solid (74.1 mg, 72% yield).
[0415] LCMS: m/z [M+1].sup.+=380.9; R.sub.T=1.39 min;
purity=>96.3%.
[0416] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0417] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.68 (s, 1H),
7.59 (d, J=11.0 Hz, 2H), 2.77 (s, 3H).
Example 21: Preparation of N-(4-(1H-1,2,3-triazol-4-yl)
benzyl)-4,6-dihydroxy-2-(methylthio) pyrimidine-5-carboxamide
(1102, Formula (II.sub.v), with Reference to FIG. 29)
[0418] Step One. 2-Fluoro-4-((trimethylsilyl)ethynyl) aniline
(1102-A). A round bottom flask containing 4-bromo-2-fluoroaniline
(1.00 g, 5.26 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (222 mg, 0.316
mmol), and CuI (40 mg, 0.21 mmol) was purged with nitrogen for 15
min. Anhydrous THF (17.5 mL) was added, followed by trimethylsilyl
acetylene (1.5 mL, 10.5 mmol) and ethanolamine (634 .mu.L, 10.5
mmol). The reaction mixture was sealed and heated to 65.degree. C.
for 20 h. The reaction mixture was cooled to rt, filtered through a
small pad of Celite. The filtrate was concentrated and the crude
product was used without further purification.
[0419] LCMS: R.sub.T=1.91 min.
[0420] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0421] Step Two. 4-Ethynyl-2-fluoroaniline (1102-B). To a solution
of crude 1102-A (5.26 mmol) dissolved in methanol (5.3 mL) was
added potassium carbonate (1.45 g, 10.5 mmol) at rt. The resulting
reaction mixture was stirred at rt until complete consumption of
the starting material was observed. The reaction mixture was
filtered, and the filtrate was concentrated. The crude product was
purified via ISCO (0 to 30% ethyl acetate in hexanes, over 20 CV)
to yield the product as a yellow oil (543 mg, 72% yield over 2
steps, 93.9% purity).
[0422] LCMS: R.sub.T=1.37 min; purity=93.9%.
[0423] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0424] Step Three.
(4-(4-Amino-3-fluorophenyl)-1H-1,2,3-triazol-1-yl) methyl pivalate
(1102-C). To a solution of 1102-B (543 mg, 3.78 mmol) was dissolved
in anhydrous 1,4-dioxane (7.6 mL). CuI (72 mg, 0.378 mmol) was
added, followed by the addition of iPr.sub.2NEt (660 .mu.L, 3.78
mmol) and azidomethyl pivalate (7.6 mL, 3.78 mmol, 0.5 M in
2-methoxypropane). The resulting reaction mixture was heated to
60.degree. C. for 8 h. The reaction mixture was then cooled to rt
and then concentrated under reduced pressure. The crude product was
purified via ISCO (0 to 60% ethyl acetate in hexanes, over 15 CV)
to yield the product as a yellow solid (823 mg, 72% yield, 97.3%
purity).
[0425] LCMS: m/z [M+1].sup.+=293.2; R.sub.T=1.51 min;
purity=97.3%
[0426] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0427] Step Four. N-(2-Fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methylthio)
pyrimidine-5-carboxamide (1102-D). To a solution of 1102-C (412 mg,
1.37 mmol) dissolved in anhydrous THF (9.8 mL), under inert
atmosphere was cooled to 0.degree. C. Et.sub.3N (210 .mu.L, 1.51
mmol) was added followed by the addition of
4,6-dichloro-2-(methylthio) pyrimidine-5-carbonyl chloride X (352
mg, 1.37 mmol). The resulting reaction mixture was warmed up to rt
over 18 h. The reaction mixture was cooled to 0.degree. C.,
p-methoxybenzyl alcohol (510 .mu.L, 4.11 mmol), followed by the
addition of NaH (219 mg, 5.48 mmol, 60% dispersed in oil). The
reaction was stirred at 0.degree. C. for 5 min, then warmed up to
rt over 20 h. Methanol (.about.10 mL) was added, followed by TBME
(.about.10 mL), the resulting suspension was sonicated and the
precipitate was filtered and collected to yield the product as a
brown solid (722 mg, 63% yield, 72.0% purity).
[0428] LCMS: m/z [M+1].sup.+=603.2; R.sub.T=1.97 min;
purity=72.0%
[0429] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0430] Step Five. N-(2-Fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methyl sulfonyl)
pyrimidine-5-carboxamide (1102-E). To a solution of 1102-D (364 mg,
0.435 mmol, 72.0% purity) dissolved in CH.sub.2Cl.sub.2 (2.2 mL)
and CHCl.sub.3 (2.0 mL) was added mCPBA (158 mg, 0.914 mmol, 77% in
H.sub.2O) at 0.degree. C. The reaction was warmed up to rt over 20
h with vigorous stirring. After complete conversion was observed
via LCMS, the reaction mixture was loaded onto a 6 g Si-TMA acetate
column. Acid impurities were retained on the column and the product
was eluted with methanol. The crude product was used without
further purification.
[0431] LCMS: m/z [M+1].sup.+=635.4; R.sub.T=1.71 min.
[0432] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0433] Step Six. N-(2-Fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-2-(isopropylthio)-4,6-bis((4-methoxybenzyl) oxy)
pyrimidine-5-carboxamide (1102-F). To a solution of crude 1102-D
(0.435 mmol) and iPrSH (200 .mu.L, 2.18 mmol) in anhydrous THF (8.7
mL) was added KOtBu (2.6 mL, 0.261 mmol, 1.0 M in THF) at 0.degree.
C. The reaction mixture was then warmed up to rt over 30 min,
H.sub.2O (10 mL) was added then extracted with EtOAc
(3.times..about.10 mL). The combined organic extracts were dried
over MgSO.sub.4, and then concentrated under reduced pressure to
yield the crude product as a brown solid, used without further
purification.
[0434] LCMS: m/z [M+1].sup.+=631.2; R.sub.T=2.11 min.
[0435] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0436] Step Seven. N-(2-Fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxa-
mide (1102). Crude 1102-F (0.435 mmol) in dichloromethane (8.7 mL)
was added trifluoroacetic acid (2.6 mL) at rt. The reaction was
stirred for 1 h, and then co-evaporated with MeOH
(3.times..about.20 mL). The crude product was purified via
reverse-phase chromatography (C18, gradient eluent from 0 to 30%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV) to yield 1102 as an off-white solid (12.3 mg, 3% yield
over 3 steps, 97.2% purity), after lyophilization.
[0437] .sup.1H NMR (400 MHz, DMSO-d6+Acetone+TFA) .delta. 8.70 (s,
1H), 8.56 (s, 1H), 7.78 (dd, J=17.0, 10.3 Hz, 2H), 4.11-4.00 (m,
1H), 1.41 (d, J=6.9 Hz, 6H).
[0438] LCMS: m/z [M+1].sup.+=391.2; R.sub.T=1.55 min;
purity=97.2%.
[0439] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 22: Preparation of N-(3-Fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxa-
mide (1104, Formula (II.sub.w), with Reference to FIG. 30)
[0440] Step One. 3-Fluoro-4-((trimethylsilyl)ethynyl) aniline
(1104-A). A round bottom flask containing 4-bromo-3-fluoroaniline
(2.00 g, 10.5 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (442 mg, 0.63
mmol), and CuI (80 mg, 0.42 mmol) was purged with nitrogen for 15
min. Anhydrous THF (35 mL) was added, followed by trimethylsilyl
acetylene (2.9 mL, 21.0 mmol) and ethanolamine (1.3 mL, 21.0 mmol).
The reaction mixture was sealed and heated to 65.degree. C. for 20
h. The reaction mixture was cooled to rt, filtered through a small
pad of Celite. The filtrate was concentrated and the crude product
was used without further purification.
[0441] LCMS: R.sub.T=1.86 min.
[0442] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0443] Step Two. 4-Ethynyl-3-fluoroaniline (1104-B). To a solution
of crude 1104-A (10.5 mmol) dissolved in methanol (21 mL) was added
potassium carbonate (2.90 g, 21.0 mmol) at rt. The resulting
reaction mixture was stirred at rt until complete consumption of
the starting material was observed. The reaction mixture was
filtered, and the filtrate was concentrated. The crude product was
purified via ISCO (0 to 30% ethyl acetate in hexanes, over 20 CV)
to yield the product as a brown oil (1.70 g, 90% yield, 75.2%
purity).
[0444] LCMS: R.sub.T=1.43 min (75.2% purity).
[0445] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0446] Step Three.
(4-(4-Amino-3-fluorophenyl)-1H-1,2,3-triazol-1-yl) methyl pivalate
(1104-C). To a solution of 1104-B (1.033 g, 5.75 mmol, 75.2%
purity) was dissolved in anhydrous 1,4-dioxane (11.5 mL). CuI (110
mg, 0.575 mmol) was added, followed by the addition of iPr.sub.2NEt
(1.0 mL, 5.75 mmol) and azidomethyl pivalate (995 mg, 6.33 mmol).
The resulting reaction mixture was heated to 60.degree. C. for 20
h. The reaction mixture was then cooled to rt and then concentrated
under reduced pressure. The crude product was purified via ISCO (0
to 60% ethyl acetate in hexanes, over 15 CV) to yield the product
as a orangish-yellow solid (605 mg, 35% yield, 95.9% purity).
[0447] LCMS: m/z [M+1].sup.+=293.0; R.sub.T=1.54 min (95.9%
purity)
[0448] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0449] Step Four. N-(3-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methylthio)
pyrimidine-5-carboxamide (1104-D). To a solution of 1104-C (512 mg,
1.99 mmol) dissolved in anhydrous THF (14.2 mL), under inert
atmosphere was cooled to 0.degree. C. Et.sub.3N (305 .mu.L, 2.19
mmol) was added followed by the addition of 4,6-dichloro-2-(methyl
thio) pyrimidine-5-carbonyl chloride X (512 mg, 1.99 mmol). The
resulting reaction mixture was warmed up to rt over 20 h. The
reaction mixture was cooled to 0.degree. C., p-methoxybenzyl
alcohol (740 V L, 5.97 mmol), followed by the addition of NaH (318
mg, 7.96 mmol, 60% dispersed in oil). The reaction was stirred at
0.degree. C. for 5 min, then warmed up to rt over 20 h, then
concentrated under reduced pressure. The crude product was purified
via reverse-phase chromatography (C18, gradient eluent from 20 to
100% acetonitrile in water with an ammonium formate buffer 10 mM
over 20 CV) to yield the product as an off-white solid (351 mg, 25%
yield, 89.8% purity), after lyophilization.
[0450] LCMS: m/z [M+1].sup.+=603.1; R.sub.T=1.88 min (89.8%
purity)
[0451] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0452] Step Five. N-(3-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methyl sulfonyl)
pyrimidine-5-carboxamide (1104-E). To a solution of 1104-D (250 mg,
0.373 mmol, 89.8% purity) dissolved in CH.sub.2Cl.sub.2 (1.9 mL)
and CHCl.sub.3 (1.9 mL) was added mCPBA (176 mg, 0.783 mmol, 77% in
H.sub.2O) at 0.degree. C. The reaction was warmed up to rt over 20
h with vigorous stirring. After complete conversion was observed
via LCMS, the reaction mixture was loaded onto a 6 g Si-TMA acetate
column. Acid impurities were retained on the column and the product
was eluted with methanol. The crude product was used without
further purification.
[0453] LCMS: m/z [M+1].sup.+=635.1; R.sub.T=1.67 min (75.5%
purity).
[0454] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0455] Step Six. N-(3-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-2-(isopropylthio)-4,6-bis((4-methoxybenzyl) oxy)
pyrimidine-5-carboxamide (1104-F). To a solution of crude 1104-D
(0.373 mmol) and iPrSH (170 .mu.L, 1.87 mmol) in anhydrous THF (7.5
mL) was added KOtBu (2.2 mL, 2.24 mmol, 1.0 M in THF) at 0.degree.
C. The reaction mixture was then warmed up to rt over 30 min,
H.sub.2O (10 mL) was added then extracted with EtOAc
(3.times..about.10 mL). The combined organic extracts were dried
over MgSO.sub.4, and then concentrated under reduced pressure; the
crude product was used without further purification.
[0456] LCMS: m/z [M+1].sup.+=631.2; R.sub.T=2.02 min.
[0457] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0458] Step Seven. N-(3-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxa-
mide (1104). Crude 1104-F (0.373 mmol) in dichloromethane (7.5 mL)
was added trifluoroacetic acid (2.2 mL) at rt. The reaction was
stirred for 1 h, and then co-evaporated with MeOH
(3.times..about.20 mL). The crude product was purified via
reverse-phase chromatography (C18, gradient eluent from 0 to 25%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV) to yield the product 1104 as a white solid (25.8 mg,
18% yield over 3 steps, 99.1% purity), after lyophilization.
[0459] .sup.1H NMR (400 MHz, CHCl.sub.3+TFA) .delta. 8.66 (s, 1H),
7.92 (s, 1H), 7.85 (d, J=12.9 Hz, 1H), 7.53 (s, 1H), 4.22 (d, J=6.4
Hz, 1H), 1.55 (d, J=6.8 Hz, 6H).
[0460] LCMS: m/z [M+1].sup.+=391.0; R.sub.T=1.60 min (99.1%
purity).
[0461] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 23: Preparation of
N-(2,6-Difluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxa-
mide (1105, Formula (II.sub.x), with Reference to FIG. 31
[0462] Step One. 4,6-Bis((4-methoxybenzyl) oxy)-2-(methylthio)
pyrimidine-5-carboxylic acid (XZ). A flask containing
4,6-dichloro-2-(methylthio) pyrimidine-5-carboxylic acid (1.099 g,
4.46 mmol) was dissolved in anhydrous THF (32 mL, under inert
atmosphere. The solution was cooled to 0.degree. C., then
p-methoxylbenzyl alcohol (1.10 mL, 8.92 mmol) was added, followed
by careful addition of NaH (535 mg, 13.4 mmol, 60% dispersed in
oil). The reaction was kept at 0.degree. C. for 5 min, then warmed
up to rt over 20 h; the progress of the reaction was monitored by
LCMS. Methanol (.about.1 mL) was added to quench the reaction; the
reaction mixture was concentrated under reduced pressure. The
residue was added CH.sub.3CN and sonicated. The suspension was then
filtered, and the solid was washed with CH.sub.3CN, the off-white
solid (1.234 g, 58% yield) was collected.
[0463] LCMS: m/z [M+1].sup.+=442.9; R.sub.T=1.86 min; (90.0%
purity).
[0464] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0465] Step Two. 2,6-Difluoro-4-((trimethylsilyl)ethynyl) aniline
(1105-A). A round bottom flask containing
4-bromo-2,6-difluoroaniline (3.94 g, 18.9 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (798 mg, 1.14 mmol), and CuI (144 mg,
0.0758 mmol) was purged with nitrogen for 15 min. Anhydrous THF (38
mL) was added, followed by trimethylsilyl acetylene (5.3 mL, 37.9
mmol) and ethanolamine (2.3 mL, 37.9 mmol). The reaction mixture
was sealed and heated to 65.degree. C. for 20 h. The reaction
mixture was cooled to rt, filtered through a small pad of Celite.
The filtrate was concentrated and used without further
purification.
[0466] LCMS: R.sub.T=1.98 min.
[0467] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0468] Step Three. 4-Ethynyl-2,6-difluoroaniline (1105-B). To a
solution of crude 1105-A (9.47 mmol) dissolved in methanol (9.5 mL)
was added potassium carbonate (2.61 g, 18.9 mmol) at rt. The
resulting reaction mixture was stirred at rt for 20 h, or until
complete consumption of the starting material was observed. The
reaction mixture was filtered, and the filtrate was concentrated.
The crude product was purified via ISCO (0 to 10% ethyl acetate in
hexanes, over 20 CV to yield the product as a yellow solid (508 mg,
35% yield over 2 steps) LCMS: R.sub.T=1.46 min.
[0469] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0470] Step Four.
(4-(4-Amino-3,5-difluorophenyl)-1H-1,2,3-triazol-1-yl) methyl
pivalate (1105-C). To a solution of 1104-B (508 mg, 3.32 mmol) was
dissolved in anhydrous 1,4-dioxane (6.6 mL). CuI (63 mg, 0.332
mmol) was added, followed by the addition of iPr.sub.2NEt (580
.mu.L, 3.32 mmol) and azidomethyl pivalate (6.6 mL, 3.32 mmol, 0.5
M in 2-methoxypropane). The resulting reaction mixture was heated
to 60.degree. C. for 20 h. The reaction mixture was then cooled to
rt and then concentrated. The crude reaction mixture was sonicated
with a mixture of H.sub.2O/MeOH (1:1 v/v), the precipitate was
filtered and washed with H.sub.2O, MeOH, and then TBME to yield as
a solid (817 mg, 78% yield, 97.7% purity).
[0471] LCMS: m/z [M+1].sup.+=311.0; R.sub.T=1.68 min (97.7%
purity).
[0472] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0473] Step Five. (4-(4-(4,6-bis((4-methoxybenzyl)
oxy)-2-(methylthio)
pyrimidine-5-carboxamido)-3,5-difluorophenyl)-1H-1,2,3-triazol-1-yl)
methyl pivalate (1105-D). Fluoro-N, N, N', N'-bis(tetramethylene)
formamidinium hexafluorophosphate, BTFFH (270 mg, 0.853 mmol) and
XY (330 mg, 0.711 mmol) in anhydrous dichloromethane (2.4 mL) was
added iPr.sub.2Net (370 .mu.L, 2.13 mmol), the resulting reaction
was stirred for 30 min at rt. 1105-C (150 mg, 0.474 mmol, 97.7%
purity) was added then sealed in a pressure vessel heated to
80.degree. C. for 24 h. The reaction mixture was cooled to rt, then
extracted with EtOAc (3.times.), the organic extracts were combined
and washed with brine, then dried over MgSO.sub.4 and concentrated
under reduced pressure. The crude product was purified via ISCO (0
to 60% ethyl acetate in hexanes, over 20 CV) to yield the product
as an off-white solid (176 mg, 51% yield, >99% purity).
[0474] LCMS: m/z [M+1].sup.+=735.3; R.sub.T=2.08 min (99%
purity).
[0475] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0476] Step Six. (4-(4-(4,6-Bis((4-methoxybenzyl) oxy)-2-(methyl
sulfonyl)
pyrimidine-5-carboxamido)-3,5-difluorophenyl)-1H-1,2,3-triazol-1-yl)
methyl pivalate (1105-E). To a solution of 1105-D (176 mg, 0.239
mmol) dissolved in CH.sub.2Cl.sub.2 (1.2 mL) was added mCPBA (113
mg, 0.503 mmol, 77% in H.sub.2O) at 0.degree. C. The reaction was
warmed up to rt over 20 h with vigorous stirring. After complete
conversion was observed via LCMS, the reaction mixture was quenched
with sat. NaHCO.sub.3, then stirred vigorously for 30 min, the
organic layer was separated and washed with sat. NaHCO.sub.3 (aq)
(3.times.), until all mCBA and mCPBA impurities were removed from
organic phase. The resulting organic extract was dried over
MgSO.sub.4, and then concentrated under reduced pressure. The crude
product was used without further purification.
[0477] LCMS: m/z [M+1].sup.+=767.4; R.sub.T=1.89 min(67.5%
purity).
[0478] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0479] Step Seven. N-(2,6-difluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-2-(isopropylthio)-4,6-bis((4-methoxybenzyl) oxy)
pyrimidine-5-carboxamide (1105-F). To a solution of crude 1105-E
(120 mg, 0.106 mmol, 67.5% purity) and iPrSH (50 .mu.L, 0.530 mmol)
in anhydrous THF (2.1 mL) was added KOtBu (740 .mu.L, 0.742 mmol,
1.0 M in THF) at 0.degree. C. The reaction mixture was then warmed
up to rt over 30 min, H.sub.2O (10 mL) was added then extracted
with EtOAc (3.times..about.10 mL). The combined organic extracts
were dried over MgSO.sub.4, and then concentrated under reduced
pressure; the crude product was used without further
purification.
[0480] LCMS: m/z [M+1].sup.+=769.3; R.sub.T=1.99 min.
[0481] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0482] Step Eight. N-(2,6-Difluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(isopropylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxa-
mide (1105). TFA (630 .mu.L) was added to a solution of 1105-F
(0.1.06 mmol) CH.sub.2Cl.sub.2 (2.1 mL). The reaction was stirred
at rt for 0.5 h. After complete consumption of starting material
was observed via LCMS, MeOH 10 mL was added to the mixture. The
resulting mixture was concentrated to remove CH.sub.2Cl.sub.2 and
TFA. Add MeOH 10 mL two more times to co-evaporate with
CH.sub.2Cl.sub.2 and TFA. The crude product was purified via
reverse-phase chromatography (C18, gradient eluent from 0 to 35%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 30 CV) to yield impure product 1105 after lyophilization.
Further purification via trituration was performed using the
centrifuge. The solid was washed with water, the supernatant was
removed, the sequence was repeated, then washed with acetonitrile.
The resulting solid was collected and lyophilized to yield the pure
product 1105 as a white solid (1.1 mg, 2% yield over 3 steps,
>99% purity).
[0483] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.63 (s, 1H),
7.53 (s, 2H), 4.28-4.15 (m, 1H), 1.55 (d, J=6.7 Hz, 6H).
[0484] LCMS: m/z [M+1].sup.+=409.2; R.sub.T=1.51 min (99.1%
purity).
[0485] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 24: Preparation of
N-(2,6-dichloro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-dihydroxy-2-(methylthio) pyrimidine-5-carboxamide
(1106, Formula (II.sub.y), with Reference to FIG. 32)
[0486] Step One. 2,6-dichloro-4-((trimethylsilyl)ethynyl) aniline
(1106-A). A round bottom flask containing
4-bromo-2,6-dichloroaniline (1.2 g, 5.0 mmol), Pd(PPh.sub.3).sub.4
(115 mg, 0.1 mmol), and CuI (38 mg, 0.2 mmol) was purged with
nitrogen for 15 min. Anhydrous CH.sub.3CN (10.0 mL) was added,
followed by trimethylsilyl acetylene (0.8 mL, 5.5 mmol) and
triethylamine (2.1 mL, 15.0 mmol). The reaction mixture was heated
to 65.degree. C. for 12 h. After completion reaction mixture was
cooled to rt, filtered through a small pad of Celite. The filtrate
was concentrated and used in next step without further
purification.
[0487] LCMS: R.sub.T=2.18 min (98% Purity).
[0488] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0489] Step Two. 2,6-dichloro-4-ethynylaniline (1106-B). To a
solution of crude 1106-A (1.28 g, 5.0 mmol) dissolved in methanol
(20.0 mL) was added potassium carbonate (1.38 g, 10.0 mmol) at rt.
The resulting reaction mixture was stirred at rt until complete
consumption of the starting material was observed. The reaction
mixture was filtered, and the filtrate was concentrated. The crude
product was purified via ISCO (0 to 30% ethyl acetate in hexanes,
over 20 CV) to yield the product as a yellow solid (721 mg, 78%
yield over 2 steps).
[0490] LCMS: R.sub.T=1.72 min (99.5% purity).
[0491] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0492] Step Three.
(4-(4-amino-3,5-dichlorophenyl)-1H-1,2,3-triazol-1-yl) methyl
pivalate (1106-C). To a solution of 1106-B (372 mg, 2.0 mmol)
dissolved in anhydrous 1,4-dioxane (5.0 mL). CuI (38 mg, 0.2 mmol)
was added, followed by the addition of DIPEA (0.7 mL, 4.0 mmol) and
azidomethyl pivalate (377 mg, 2.4 mmol). The resulting reaction
mixture was heated to 60.degree. C. for 12 h. The reaction mixture
was then cooled to rt and concentrated. The crude product was
purified via ISCO (SiO2, 0 to 50% ethyl acetate in hexanes over 20
CV) to yield 1106-C as an off-white solid (610 mg, 89% yield).
[0493] LCMS: m/z [M+1].sup.+=343.0; R.sub.T=1.76 min (98.8%
purity)
[0494] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0495] Step Four. (4-(4-(4,6-bis((4-methoxybenzyl)
oxy)-2-(methylthio)
pyrimidine-5-carboxamido)-3,5-dichlorophenyl)-1H-1,2,3-triazol-1-yl)
methyl pivalate (1106-D). Fluoro-N, N, N', N'-bis (tetramethylene)
formamidinium hexafluorophosphate, BTFFH (427 mg, 1.35 mmol) and XY
(403 mg, 1.5 mmol) in anhydrous 1,2 dichloroethane (4.0 mL) was
added DIPEA (0.42 mL, 2.4 mmol), the resulting reaction was stirred
for 30 min at rt. 1106-C (200 mg, 0.6 mmol, 87.7% purity) was added
then sealed tube and heated to 80.degree. C. for 16 h. The reaction
mixture was cooled to rt, and then concentrated under reduced
pressure. The crude product was purified via ISCO (0 to 40% ethyl
acetate in hexanes, over 20 CV) to yield the product as white solid
(55 mg, 12% yield).
[0496] LCMS: m/z [M+1].sup.+=768.1; R.sub.T=2.13 min (99.5%
purity)
[0497] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0498] Step Five. N-(2,6-dichloro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-dihydroxy-2-(methylthio) pyrimidine-5-carboxamide
(1106). 2.0 M aqueous solution of NaOH (1.0 mL) was added to 1106-D
(50 mg, 0.07 mmol) in THF (1.0 mL) at rt. The reaction mixture was
stirred for 1 h, and then concentrated and evaporated with MeOH to
obtained intermediate crude product which was dissolved in THF (2.0
mL), followed by the addition of TFA (2.0 mL) at rt. The reaction
was stirred at rt for 2 h. After complete consumption of starting
material was observed via LCMS, MeOH 10 mL was added to the
mixture. The resulting mixture was concentrated to get crude
product. The crude product was purified via reverse-phase
chromatography (C18, gradient eluent from 0 to 35% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 30 CV)
followed by lypholization to yield ammonium salt of 1106 as white
solid (8.0 mg, 30% yield).
[0499] .sup.1H NMR (400 MHz, CDCl3+TFA): .delta. 10.86 (s, 1H),
8.45 (s, 1H), 7.85 (s, 2H), 6.81-6.39 (m, 2H), 2.92 (s, 1H), 2.74
(s, 3H).
[0500] LCMS: m/z [M+1].sup.+=414.9; R.sub.T=1.37 min (97.3%
purity).
[0501] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 25: Preparation of N-(4-(1H-1,2,3-triazol-4-yl)
phenyl)-2-((2-(dimethyl amino) ethyl)
thio)-4-hydroxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide (1107,
Formula (II.sub.z), with Reference to FIG. 33)
[0502] Step one. N-(4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methyl sulfonyl)
pyrimidine-5-carboxamide (1107-A). To a solution of
N-(4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl) phenyl)-4,6-bis
((4-methoxy benzyl) oxy)-2-(methylthio) pyrimidine-5-carboxamide
(71 mg, 0.1 mmol) dissolved in CH.sub.2Cl.sub.2 (10.0 mL) was added
mCPBA (67 mg, 0.22 mmol, 77% in H.sub.2O) at 0.degree. C. The
reaction was warmed up to rt over 2 h with vigorous stirring. After
complete conversion was observed via LCMS, the reaction mixture was
quenched with sat. NaHCO.sub.3, then stirred vigorously for 30 min,
the organic layer was separated and washed with sat. NaHCO.sub.3
(aq) (3.times.), until all mCPBA and mCPBA impurities were removed
from organic phase. The resulting organic extract was dried over
MgSO4, and then concentrated under reduced pressure. The crude
product 1107-A (68 mg, 92% yield) was used in next step without
further purification.
[0503] LCMS: m/z [M+1].sup.+=737.2; R.sub.T=1.80 min (92.7%
purity)
[0504] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0505] Step two. 2-((2-(dimethylamino) ethyl)
thio)-N-(4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy) pyrimidine-5-carboxamide
(1107-B). To a solution of crude 1107-A (68 mg, 0.092 mmol, 92.7%
purity) and dimethylamino ethane thiol (65 mg, 0.46 mmol) in
anhydrous THF (3.0 mL) was added KOtBu (0.65 mL, 0.65 mmol, 1.0 M
in THF) at 0.degree. C. The reaction mixture was then warmed up to
rt over 30 min, H.sub.2O (10 mL) was added then extracted with
EtOAc (3.times..about.10 mL). The combined organic extracts were
dried over MgSO4, and concentrated under reduced pressure; the
crude product 1107-B was used in next step without further
purification.
[0506] LCMS: m/z [M+1].sup.+=762.3; R.sub.T=1.72 min (68%
purity).
[0507] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0508] Step Three. N-(4-(1H-1,2,3-triazol-4-yl)
phenyl)-2-((2-(dimethylamino) ethyl)
thio)-4-hydroxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide (1107).
To a microwave vial TFA (2.0 mL) was added to a solution of 1107-B
(0.092 mmol) in CH.sub.2Cl.sub.2 (2.0 mL) followed by addition of
TfOH (0.1 mL). The reaction was sealed and heated at 80.degree. C.
for 48 h. After complete consumption of starting material was
observed via LCMS, it was cooled to rt, MeOH 10 mL was added to the
reaction mixture and concentrated to remove excess of TFA. Add MeOH
10 mL two more times to co-evaporate with CH.sub.2Cl.sub.2 and TFA.
The crude product was purified via reverse-phase chromatography
(C18, gradient eluent from 0 to 35% acetonitrile in water with an
ammonium bicarbonate buffer 10 mM over 30 CV) to yield impure
product 1107 after lyophilization. Further purification via
trituration was performed using the centrifuge. The solid was
washed with water, the supernatant was removed, the sequence was
repeated, then washed with acetonitrile. The resulting solid was
collected and lyophilized to yield the pure product 1107 as a white
solid (5.5 mg, 15% yield over 2 steps).
[0509] .sup.1H NMR (400 MHz, DMSO+DCl in D.sub.2O) .delta. 8.63 (s,
1H), 7.79 (d, J=8.2 Hz, 2H), 7.55 (d, J=8.3 Hz, 2H) 3.32 (dd, J=8.0
Hz, 4H), 2.70 (s, 6H).
[0510] LCMS: m/z [M+1].sup.+=402.1; R.sub.T=1.04 min (98.2%
purity)
[0511] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 26: Preparation of N-(5-(1H-1,2,3-Triazol-4-yl)
thiazol-2-yl)-2,4,6-trihydroxypyrimidine-5-carboxamide (1108,
Formula (I.sub.o), with Reference to FIG. 34)
[0512] Step One.
N-(5-Bromothiazol-2-yl)-2,4,6-trihydroxypyrimidine-5-carboxamide
(1108-A). To a solution of 2-amino-5-bromothiazole monohydrobromide
(938 mg, 3.61 mmol) in dry DMSO (3.6 mL), under inert atmosphere
was added triethylamine (1.0 mL, 7.22 mmol) and CDI (1.17 g, 7.22
mmol) in one portion at room temperature. The resulting reaction
mixture was stirred for 20 h to generate the corresponding
isocyanate.
[0513] In a separate flask, to a suspension of barbituric acid (462
mg, 3.61 mmole) in anhydrous 1,4-dioxane (12.0 mL) at 55.degree. C.
was added triethylamine (810 .mu.L, 7.22 mmol) and reaction mixture
stirred at 55.degree. C. for 30 minutes, then the isocyanate
generated from the previous step was added. The resulting reaction
mixture was heated at 80.degree. C. for 4 h 30 min. Reaction
mixture cooled to rt, then acidified with 6 M HCl, the precipitate
formed was filtered and washed with H.sub.2O, and MeOH to yield the
product as a brown solid (899 mg, 75% yield, >99% purity).
[0514] LCMS: m/z [M+1].sup.+=333.1/335.1; R.sub.T=0.99 min
[0515] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0516] Step Two. 2, 4, 6-Trihydroxy-N-(5-((trimethylsilyl) ethynyl)
thiazol-2-yl) pyrimidine-5-carboxamide (1108-B). A round bottom
flask containing 1108-A (899 mg, 2.71 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (114 mg, 0.163 mmol), and CuI (21 mg,
0.108 mmol) was purged with nitrogen for 15 min. Anhydrous DMF (5.4
mL) was added, followed by trimethylsilylacetylene (760 .mu.L, 5.42
mmol) and triethylamine (760 .mu.L, 5.42 mmol). The reaction
mixture was sealed and heated to 65.degree. C. for 20 h. The
reaction mixture was cooled to rt, filtered through a small pad of
Celite. The filtrate was concentrated and purified via
reverse-phase chromatography (C18, gradient eluent from 0 to 100%
acetonitrile in water with an ammonium formate buffer 10 mM over 20
CV) to yield impure 1108-B as a brown solid, after lyophilization.
Further purification via trituration was performed, the solid was
washed with ethyl acetate and methanol. The resulting solid was
collected and lyophilized to yield the pure product 1108-B as a
brown solid (182 mg, 20% yield, 87.9% purity).
[0517] LCMS: m/z [M+1].sup.+=351.2; R.sub.T=1.33 min (87.9%
purity).
[0518] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0519] Step Three.
N-(5-Ethynylthiazol-2-yl)-2,4,6-trihydroxypyrimidine-5-carboxamide
(1108-C). To a solution of crude 1108-B (182 mg, 0.457 mmol)
dissolved in methanol (2.3 mL) was added potassium carbonate (12.6
mg, 0.914 mmol) at rt. The resulting reaction mixture was stirred
at rt for 20 h, or until complete consumption of the starting
material was observed. The reaction mixture was filtered, and the
filtrate was concentrated. The crude product was used without
further purification.
[0520] LCMS: m/z [M+1].sup.+=278.8; R.sub.T=0.96 min (97.7%
purity).
[0521] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0522] Step Four. (4-(2-(2,4,6-trihydroxypyrimidine-5-carboxamido)
thiazol-5-yl)-1H-1,2,3-triazol-1-yl) methyl pivalate (1108-D). To a
solution of crude 1108-C (125 mg, 0.450 mmol) was dissolved in
anhydrous DMSO (900 .mu.L). CuI (9 mg, 0.045 mmol) was added,
followed by the addition of iPr.sub.2NEt (80 .mu.L, 457 mmol) and
azidomethyl pivalate (78 mg, 0.503 mmol). The resulting reaction
mixture was heated to 60.degree. C. for 20 h. The reaction mixture
was then cooled to rt and then concentrated. The crude reaction
mixture was triturated with ethyl acetate, the precipitate was
filtered and washed with H.sub.2O, MeOH, and then TBME to yield as
a solid (148 mg, 67% yield over 2 steps, 88.9% purity).
[0523] LCMS: m/z [M+1].sup.+=436.0; R.sub.T=1.18 min (88.9%
purity).
[0524] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0525] Step Five. N-(5-(1H-1,2,3-Triazol-4-yl)
thiazol-2-yl)-2,4,6-trihydroxypyrimidine-5-carboxamide (1108). 2.5
M aqueous solution of NaOH (610 .mu.L) was added to 1108-D (148 mg,
0.302 mmol, 88.9% purity) in MeOH (6.1 mL) at rt. The reaction was
stirred for 10 min, and then acidified with 6 M HCl, then
concentrated under reduced pressure. The crude product was purified
via reverse-phase chromatography (C18, gradient eluent from 0 to
30% acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV) to yield the product 1108 as a brown solid, after
lyophilization (6.5 mg, 6% yield, 96.0% purity).
[0526] .sup.1H NMR (400 MHz, DMSO+TFA) .delta. 8.85 (s, 1H), 8.60
(s, 1H).
[0527] LCMS: m/z [M+1].sup.+=321.8; R.sub.T=0.82 min (96.0%
purity).
[0528] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
Example 27: Preparation of
N-(2,3-difluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-dihydroxy-2-(methylthio) pyrimidine-5-carboxamide
(1109, Formula (II.sub.aa), with Reference to FIG. 35)
[0529] Step One. 2,3-difluoro-4-((trimethylsilyl)ethynyl) aniline
(1109-A). A round bottom flask containing
4-bromo-2,3-difluoroaniline (1.04 g, 5.0 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (210 mg, 0.3 mmol), and CuI (38 mg, 0.2
mmol) was purged with nitrogen for 15 min. Anhydrous THF (10.0 mL)
was added, followed by trimethylsilyl acetylene (1.4 mL, 10.0 mmol)
and ethanolamine (0.6 mL, 10.0 mmol). The reaction mixture was
heated to 65.degree. C. for 12 h. After completion reaction mixture
was cooled to rt, filtered through a small pad of Celite. The
filtrate was concentrated and used in next step without further
purification.
[0530] LCMS: R.sub.T=1.90 min (90% Purity).
[0531] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0532] Step Two. 4-ethynyl-2,3-difluoroaniline (1109-B). To a
solution of crude 1109-A (1.1 g, 4.9 mmol) dissolved in methanol
(20.0 mL) was added potassium carbonate (1.35 g, 9.8 mmol) at rt.
The resulting reaction mixture was stirred at rt until complete
consumption of the starting material was observed. The reaction
mixture was filtered, and the filtrate was concentrated. The crude
product was purified via ISCO (0 to 30% ethyl acetate in hexanes,
over 20 CV) to yield the product as a yellow solid (207 mg, 77%
purity).
[0533] LCMS: R.sub.T=1.43 min (77.0% purity).
[0534] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0535] Step Three.
(4-(4-amino-2,3-difluorophenyl)-1H-1,2,3-triazol-1-yl) methyl
pivalate (1109-C). To a solution of crude 1109-B (200 mg, 1.3 mmol)
dissolved in anhydrous 1,4-dioxane (3.0 mL). CuI (25 mg, 0.13 mmol)
was added, followed by the addition of DIPEA (0.5 mL, 2.6 mmol) and
azidomethyl pivalate (250 mg, 1.56 mmol). The resulting reaction
mixture was heated to 60.degree. C. for 12 h. The reaction mixture
was then cooled to rt and concentrated. The crude product was
purified via ISCO (SiO2, 0 to 50% ethyl acetate in hexanes over 20
CV) to yield 1109-C as an off-white solid (208 mg, 14% yield over
three steps).
[0536] LCMS: m/z [M+1].sup.+=311.1; R.sub.T=1.61 min (98.2%
purity)
[0537] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0538] Step Four. (4-(4-(4,6-bis((4-methoxybenzyl)
oxy)-2-(methylthio)
pyrimidine-5-carboxamido)-2,3-difluorophenyl)-1H-1,2,3-triazol-1-yl)
methyl pivalate (1109-D). Fluoro-N, N, N', N'-bis (tetramethylene)
formamidinium hexafluorophosphate, BTFFH (435 mg, 1.4 mmol) and XY
(383 mg, 0.825 mmol) in anhydrous 1,2 dichloroethane (5.0 mL) was
added DIPEA (0.5 mL, 2.75 mmol), the resulting reaction was stirred
for 30 min at rt. 1109-C (170 mg, 0.55 mmol, 98.2% purity) was
added and reaction was heated to 80.degree. C. for 16 h. After
completeion it was cooled to rt, and then concentrated under
reduced pressure. The crude product was purified via ISCO (0 to 40%
ethyl acetate in hexanes, over 20 CV) to yield the product as white
solid (290 mg, 74.2% yield).
[0539] LCMS: m/z [M+1].sup.+=735.4; RT=2.18 min (98.5% purity)
[0540] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0541] Step Five. N-(2,3-difluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-dihydroxy-2-(methylthio) pyrimidine-5-carboxamide
(1109). 2.0 M aqueous solution of NaOH (3.0 mL) was added to 1109-D
(290 mg, 0.4 mmol) in THF: MeOH (6.0 mL) at rt. The reaction
mixture was stirred for 1 h, and then concentrated and
co-evaporated with MeOH to obtained intermediate crude product
which was re-dissolved in THF (2.0 mL), followed by the addition of
TFA (2.0 mL) at rt and allowed to stir for 2 h. After complete
consumption of starting material was observed via LCMS, MeOH 10 mL
was added to the mixture. The resulting mixture was concentrated to
get crude product. The crude product was purified via reverse-phase
chromatography (C18, gradient eluent from 0 to 35% acetonitrile in
water with an ammonium bicarbonate buffer 10 mM over 30 CV)
followed by lypholization to yield 1109 as white solid (21.0 mg,
14% yield).
[0542] .sup.1H NMR (400 MHz, CDCl3+TFA): .delta. 8.63 (s, 1H), 8.28
(t, J=7.3 Hz, 1H), 7.69 (t, J=8.4 Hz, 1H), 2.80 (s, 3H).
[0543] LCMS: m/z [M+1]+=381.0; RT=0.89 min (99.07% purity).
[0544] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium bicarbonate;
Eluent B: Acetonitrile.
Example 28: Preparation of 2-((2-(dimethylamino) ethyl)
thio)-N-(2-fluoro-4-(1H-1,2,3-triazol-4-yl) phenyl)
4-hydroxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide (1110, Formula
(II.sub.bb), with Reference to FIG. 36)
[0545] Step one. N-(2-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methylsulfonyl)
pyrimidine-5-carboxamide (1110-A). To a solution of
N-(2-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methylthio)
pyrimidine-5-carboxamide (182 mg, 0.217 mmol, 67% purity) dissolved
in CHCl.sub.3 (7.0 mL) was added mCPBA (158 mg, 0.912 mmol, 77% in
H2O) at 0.degree. C. The reaction was warmed up to rt over 20 h
with vigorous stirring. After complete conversion was observed via
LCMS, the reaction mixture was quenched with sat. NaHCO.sub.3, then
stirred vigorously for 30 min, the organic layer was separated and
washed with sat. NaHCO.sub.3(aq) (3.times.), until all mCPBA and
mCPBA impurities were removed from organic phase. The resulting
organic extract was dried over MgSO4, and then concentrated under
reduced pressure. The crude product 1110-A was used in next step
without further purification.
[0546] LCMS: m/z [M+1]+=635.2; R.sub.T=1.64 min (62% purity)
[0547] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0548] Step two. 2-((2-(dimethylamino) ethyl)
thio)-N-(2-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy) pyrimidine-5-carboxamide
(1110-B). To a solution of crude 1110-A (258 mg, 0.4 mmol, 60%
purity) and dimethylamino ethane thiol (283 mg, 2.0 mmol) in
anhydrous THF (5.0 mL) was added KOtBu (2.8 mL, 2.8 mmol, 1.0 M in
THF) at 0.degree. C. The reaction mixture was then warmed up to rt
over 40 min, H.sub.2O (10 mL) was added then extracted with EtOAc
(3.times..about.10 mL). The combined organic extracts were dried
over MgSO4, and concentrated under reduced pressure; the crude
product 1110-B was used in next step without further
purification.
[0549] LCMS: m/z [M+1].sup.+=660.2; R.sub.T=1.46 min (68%
purity).
[0550] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0551] Step Three. 2-((2-(dimethylamino) ethyl)
thio)-N-(2-fluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-6-oxo-1,6-dihydropyrimidine-5-carboxamide (1110).
TFA (2.0 mL) was added to a solution of 1110-B (0.4 mmol) in
CH.sub.2Cl.sub.2 (2.0 mL) and the reaction was stirred at rt for 3
h. After complete consumption of starting material was observed via
LCMS, MeOH 10 mL was added to the reaction mixture and concentrated
to remove excess of TFA. Add MeOH 10 mL two more times to
co-evaporate CH.sub.2Cl.sub.2 and TFA. The crude product was
purified via reverse-phase chromatography (C18, gradient eluent
from 0 to 35% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 30 CV) to yield impure product 1110 after
lyophilization. Further purification via trituration was performed
using the centrifuge. The solid was washed with water, the
supernatant was removed, the sequence was repeated, then washed
with acetonitrile. The resulting solid was collected and
lyophilized to yield the pure product 1110 as a white solid (12.0
mg, 12% yield over 3 steps).
[0552] 1H NMR (400 MHz, DMSO+TFA): .delta. 9.56 (s, 1H), 8.37 (s,
1H) 8.31 (t, J=8.5 Hz, 1H), 7.80 (d, J=11.6 Hz, 1H), 7.73 (d, J=8.5
Hz, 1H) 3.47 (m, 4H), 2.84 (s, 6H).
[0553] LCMS: m/z [M+1].sup.+=420.0; R.sub.T=1.09 min (95.6%
purity).
[0554] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
Example 29: Preparation of
N-(2,5-Difluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (1111, Formula (II.sub.cc), with Reference to FIG. 37)
[0555] Step One. 2,5-Difluoro-4-((trimethylsilyl)ethynyl) aniline
(1111-A). A round bottom flask containing
2,5-difluoro-4-iodoaniline (1.20 g, 4.61 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (194 mg, 0.277 mmol), and CuI (35 mg,
0.184 mmol) was purged with nitrogen for 15 min. Anhydrous THF (9.2
mL) was added, followed by trimethylsilyl acetylene (1.3 mL, 9.22
mmol) and ethanolamine (556 .mu.L, 9.22 mmol). The reaction mixture
was sealed and heated to 65.degree. C. for 2 h. The reaction
mixture was cooled to rt, filtered through a small pad of Celite.
The filtrate was concentrated and used without further
purification.
[0556] LCMS: m/z [M+1].sup.+=226.2; R.sub.T=1.87 min.
[0557] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0558] Step Two. 4-Ethynyl-2,5-difluoroaniline (1111-B). Crude
1111-A (2.31 mmol) was dissolved in THF (4.6 mL) and to this
solution was added TBAF (4.6 mL, 1 M in THF). The reaction was
stirred at rt for 10 min and THF was removed in vacuo. The crude
was dissolved in EtOAc and this solution was passed through a pad
of silica and washed with EtOAc. The filtrate was concentrated and
the crude product was used without further purification.
[0559] LCMS: R.sub.T=1.43 min.
[0560] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0561] Step Three.
2,5-difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl) aniline
(1111-C). To a solution of crude 1111-B (2.31 mmol) was dissolved
in anhydrous 1,4-dioxane (4.6 mL). CuI (44 mg, 0.231 mmol) was
added, followed by the addition of iPr.sub.2NEt (400 .mu.L, 2.31
mmol) and p-methoxylbenzyl pivalate (377 mg, 2.31 mmol). The
resulting reaction mixture was heated to 60.degree. C. for 20 h.
The reaction mixture was then cooled to rt and then concentrated.
The crude reaction mixture was purified via ISCO (SiO2, 0 to 50%
ethyl acetate in hexanes, over 20 CV) to yield the product as an
orange foam (352 mg, 43% yield over 3 steps, 89.3% purity).
[0562] LCMS: m/z [M+1].sup.+=317.3; R.sub.T=1.50 min (89.3%
purity).
[0563] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0564] Step Four.
N-(2,5-Difluoro-4-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)
phenyl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methylthio)
pyrimidine-5-carboxamide (1111-D). T.sub.3P (3.4 mL, 5.70 mmol, 50%
in EtOAc) was added to a mixture of 1111-C (336 mg, 0.950 mmol),
triethylamine (1.2 mL, 8.55 mmol) and XZ (594 mg, 1.24 mmol) in
EtOAc (4.8 mL). The reaction was heated at 80.degree. C. overnight.
After complete consumption of starting material was observed via
LCMS, the mixture was concentrated. TBME was added to the residue,
and sonicated, the solid was filtered and washed with H.sub.2O and
TBME to yield the title compound as a light brown solid (575 mg,
40% yield, 82.1% purity).
[0565] LCMS: m/z [M+1].sup.+=741.1; R.sub.T=2.13 min (82.1%
purity).
[0566] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
[0567] Step Five. N-(2,5-difluoro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (1111). TfOH (90 .mu.L) was added to a solution of 1111-D (210
mg, 0.252 mmol) in TFA (1.5 mL) and 1,2-dichloroethane (5.0 mL).
The reaction was heated at 80.degree. C. for 20 h in a sealed
pressure vessel. After complete consumption of starting material
was observed via LCMS, MeOH 10 mL was added to the mixture. The
resulting mixture was concentrated to remove CH.sub.2Cl.sub.2 and
TFA. Add MeOH 10 mL two more times to co-evaporate with
CH.sub.2Cl.sub.2 and TFA. The crude product was purified via
reverse-phase chromatography (C18, gradient eluent from 0 to 30%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 30 CV) to yield the product 1111 as a white solid (25.4 mg,
26% yield, 96.7% purity) after lyophilization.
[0568] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.63 (s, 1H),
8.50-8.41 (m, 1H), 7.72 (s, 1H), 2.77 (s, 3H).
[0569] LCMS: m/z [M+1].sup.+=381.0; R.sub.T=1.46 min (96.7%
purity).
[0570] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 30: Preparation of
N-(4-(1H-1,2,3-triazol-4-yl)-2-(trifluoromethyl)
phenyl)-4-hydroxy-2-(methyl
thio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide (1112, Formula
(II.sub.dd), with Reference to FIG. 38
[0571] Step One.
2-(Trifluoromethyl)-4-((trimethylsilyl)ethynyl)aniline (1112-A). A
round bottom flask containing 4-iodo-2-(trifluoromethyl) aniline
(280 mg, 2.02 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (84 mg, 0.12
mmol), and CuI (30 mg, 0.16 mmol) was purged with nitrogen for 15
min. Anhydrous THF (4.0 mL) was added, followed by trimethylsilyl
acetylene (570 .mu.L, 4.04 mmol) and triethylamine (560 .mu.L, 4.04
mmol). The reaction mixture was sealed and heated to 65.degree. C.
for 20 h. The reaction mixture was cooled to rt, filtered through a
small pad of Celite. The filtrate was concentrated and used without
further purification.
[0572] LCMS: R.sub.T=2.08 min.
[0573] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0574] Step Two. 4-Ethynyl-2-(trifluoromethyl) aniline (1112-B). To
a solution of crude 1112-A (2.02 mmol) dissolved in methanol (4.0
mL) was added potassium carbonate (558 mg, 4.04 mmol) at rt. The
resulting reaction mixture was stirred at rt until complete
consumption of the starting material was observed. The reaction
mixture was filtered, and the filtrate was concentrated. The crude
product was purified via ISCO (0 to 30% ethyl acetate in hexanes,
over 20 CV) to yield the product as a yellow solid (280 mg, 74%
yield over 2 steps, 98.6% purity).
[0575] LCMS: R.sub.T=1.64 min(98.6% Purity).
[0576] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0577] Step Three. (4-(4-Amino-3-(trifluoromethyl)
phenyl)-1H-1,2,3-triazol-1-yl) methyl pivalate (1112-C). To a
solution of 1111-B (280 mg, 1.51 mmol) was dissolved in anhydrous
1,4-dioxane (3.0 mL). CuI (29 mg, 0.151 mmol) was added, followed
by the addition of iPr.sub.2NEt (260 .mu.L, 1.51 mmol) and
azidomethyl pivalate (261 mg, 1.66 mmol). The resulting reaction
mixture was heated to 60.degree. C. for 20 h. The reaction mixture
was then cooled to rt and then concentrated. The crude product was
purified via ISCO (SiO.sub.2, 0 to 50% ethyl acetate in hexanes
over 20 CV) to yield the product as an off-white solid (370 mg, 70%
yield, 97.0% purity).
[0578] LCMS: m/z [M+1].sup.+=343.2; R.sub.T=1.70 min (97.0%
purity).
[0579] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0580] Step Four. (4-(4-(4,6-Bis ((4-methoxy benzyl)
oxy)-2-(methylthio) pyrimidine-5-carboxamido)-3-(trifluoromethyl)
phenyl)-1H-1,2,3-triazol-1-yl) methyl pivalate (1112-D). Fluoro-N,
N, N', N'-bis (tetramethylene) formamidinium hexafluorophosphate,
BTFFH (85 mg, 0.270 mmol) and XY (111 mg, 0.230 mmol) in anhydrous
dichloromethane (1.5 mL) was added iPr.sub.2Net (120 .mu.L, 0.88
mmol), the resulting reaction was stirred for 30 min at rt. 1112-C
(50 mg, 0.150 mmol, 97.0% purity) was added then sealed in a
pressure vessel heated to 80.degree. C. for 24 h. The reaction
mixture was cooled to rt, and then concentrated under reduced
pressure. The crude product was purified via ISCO (0 to 40% ethyl
acetate in hexanes, over 20 CV) to yield the product as an
off-white solid (62 mg, 46% yield, 84.8% purity).
[0581] LCMS: m/z [M+1].sup.+=767.3; R.sub.T=2.19 min(84.8%
purity).
[0582] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0583] Step Five. (4-(4-(4,6-Bis ((4-methoxy benzyl)
oxy)-2-(methylthio) pyrimidine-5-carboxamido)-3-(trifluoromethyl)
phenyl)-1H-1,2,3-triazol-1-yl) methyl pivalate (1112). 2.5 M
Aqueous solution of NaOH (690 .mu.L) was added to 1112-D (62 mg,
0.0686 mmol, 84.8% purity) in THF (1.4 mL) at rt. The reaction was
stirred for 20 h, and then co-evaporated with ethyl acetate. The
crude product was sonicated with ethyl acetate, and then filtered,
the precipitate was washed with EtOAc, then collected.
[0584] The intermediate was added CH.sub.2Cl.sub.2 (1.4 mL),
followed by the addition of TFA (400 .mu.L) at rt. The reaction was
stirred at rt for 15 min. After complete consumption of starting
material was observed via LCMS, MeOH 10 mL was added to the
mixture. The resulting mixture was concentrated to remove
CH.sub.2Cl.sub.2 and TFA. Add MeOH 10 mL two more times to
co-evaporate with CH.sub.2Cl.sub.2 and TFA. The crude product was
purified via trituration was performed using the centrifuge. The
solid was washed with water, the supernatant was removed, and the
sequence was repeated, and then washed with acetonitrile. The
resulting solid was collected and lyophilized to yield the pure
product 1112 as an off-white solid (18.4 mg, 63% yield over 2
steps, 95.4% purity).
[0585] .sup.1H NMR (400 MHz, CDCl.sub.3+TFA) .delta. 8.65 (d, J=1.1
Hz, 1H), 8.36 (d, J=8.6 Hz, 1H), 8.17 (s, 1H), 8.07 (d, J=8.9 Hz,
1H), 2.83 (s, 3H).
[0586] LCMS: m/z [M+1].sup.+=413.2; R.sub.T=1.41 min(95.4%
purity).
[0587] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 31: Preparation of N-(6-(1H-1,2,3-triazol-4-yl)
pyridazin-3-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-ca-
rboxamide (1113, Formula (II.sub.kk), with Reference to FIG.
39)
[0588] Step One. 6-((trimethylsilyl)ethynyl) pyridazin-3-amine
(1113-A). A round bottom flask containing 6-iodopyridazin-3-amine
(1.1 g, 5.0 mmol), trimethylsilyl acetylene (3.58 mL, 25.0 mmol)
and CuI (380 mg, 2.0 mmol) was dissolved in EtOAc (30.0 mL) and
purged with nitrogen for 15 min. Reaction mixture was then cooled
at -20.degree. C. and PdCl.sub.2 (dppf) (732 mg, 1 mmol), DIPEA
(1.75 mL, 10.0 mmol) was added. After 10 min The reaction mixture
was brought to rt and stirred for 12 h. After completion reaction
mixture was filtered through a small pad of Celite. The filtrate
was concentrated and Purified via silica gel column chromatography
using 0 to 5% MeOH in DCM as eluent to obtained 1113-A as
off--white solid (680 mg, 70% Yield).
[0589] LCMS: m/z [M+1]+=192.0; R.sub.T=1.41 min (95.1% Purity).
[0590] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0591] Step Two. 6-ethynylpyridazin-3-amine (1113-B). To a solution
of 1113-A (680 mg, 3.6 mmol) dissolved in THF (15.0 mL) was added
TBAF (7.2 mL, 7.2 mmol, 1M In THF) at rt. The resulting reaction
mixture was stirred at rt until complete consumption of the
starting material was observed. The reaction mixture was
concentrated. The crude product was purified via ISCO (0 to 30%
MeOH in DCM, over 20 CV) to yield the product as a brown solid (380
mg, 89.8% yield).
[0592] LCMS: m/z [M+1]+=120.4; R.sub.T=0.31 min (99.2% purity).
[0593] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0594] Step Three.
(4-(6-aminopyridazin-3-yl)-1H-1,2,3-triazol-1-yl) methyl pivalate
(1113-C). To a solution of 1113-B (198 mg, 1.7 mmol) dissolved in
anhydrous 1,4-dioxane (5.0 mL). CuI (33 mg, 0.17 mmol) was added,
followed by the addition of DIPEA (0.6 mL, 3.4 mmol) and
azidomethyl pivalate (321 mg, 2.0 mmol). The resulting reaction
mixture was heated to 60.degree. C. for 12 h. The reaction mixture
was then cooled to rt and concentrated. The crude product was
purified via ISCO (SiO2, 0 to 50% ethyl acetate in hexanes over 20
CV) to yield 1113-C as brown solid (328 mg, 71% yield).
[0595] LCMS: m/z [M+1]+=277.3; R.sub.T=1.18 min (95.2% purity)
[0596] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0597] Step Four. (4-(6-(4,6-bis((4-methoxybenzyl)
oxy)-2-(methylthio) pyrimidine-5-carboxamido)
pyridazin-3-yl)-1H-1,2,3-triazol-1-yl) methyl pivalate (1113-D). To
a stirred solution of HBTU (380 mg, 1.0 mmol) and XY (443 mg, 1.0
mmol) dissolved in anhydrous DMF (5.0 mL) was added DIPEA (0.49 mL,
2.8 mmol), the resulting reaction was stirred for 30 min at rt.
1113-C (138 mg, 0.5 mmol,) was added then reaction mixture was
heated to 60.degree. C. for 16 h. reaction mixture was cooled to
rt, and then concentrated under reduced pressure. The crude product
was purified via ISCO (0 to 40% ethyl acetate in hexanes, over 20
CV) to yield the product as white solid (60 mg, 78% purity).
[0598] LCMS: m/z [M+1].sup.+=701.3; R.sub.T=2.07 min(78.0%
purity).
[0599] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate pH:
3.8; Eluent B: Acetonitrile.
[0600] Step Five. N-(6-(1H-1,2,3-triazol-4-yl)
pyridazin-3-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-ca-
rboxamide (1113). 2.0 M Aqueous solution of NaOH (2.0 mL) was added
to 1113-D (60 mg, 0.06 mmol, 78.0% purity) in THF: MeOH (3.0 mL,
1:1) at rt. The reaction mixture was stirred for 1 h, and then
concentrated and evaporated with MeOH to obtained intermediate
crude product which was dissolved in CH.sub.2Cl.sub.2 (3.0 mL),
followed by the addition of TFA (2.0 mL) at rt. The reaction was
stirred at rt for 30 min. After complete consumption of starting
material was observed via LCMS, MeOH 10 mL was added to the
mixture. The resulting mixture was concentrated to remove
CH.sub.2Cl.sub.2 and TFA. Add MeOH 10 mL two more times to
co-evaporate CH.sub.2Cl.sub.2 and TFA. The crude product was
purified via trituration using the centrifuge. The solid was washed
with water, the supernatant was removed, and the sequence was
repeated, and then washed with acetonitrile. The resulting solid
was collected and lyophilized to yield the pure product 1113 as an
off-white solid (4.8 mg, 22% yield over 2 steps).
[0601] 1H NMR (400 MHz, 1M K.sub.2CO.sub.3 in D.sub.2O): .delta.
8.28 (d, J=9.6 Hz, 1H), 8.01 (s, 1H), 7.91 (d, J=9.0 Hz, 1H), 2.18
(s, 3H).
[0602] LCMS: m/z [M+1].sup.+=347.0; R.sub.T=1.20 min (97.8%
purity).
[0603] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 32: Preparation of N-(2-chloro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (1114, Formula (II.sub.ee), with Reference to FIG. 40)
[0604] Step One. 2-chloro-4-((trimethylsilyl)ethynyl) aniline
(1114-A). A round bottom flask containing 2-chloro-4-iodoaniline
(1.27 g, 5.0 mmol), Pd(PPh.sub.3).sub.4 (115 mg, 0.1 mmol), and CuI
(38 mg, 0.2 mmol) was purged with nitrogen for 15 min. Anhydrous
CH.sub.3CN (10.0 mL) was added, followed by trimethylsilyl
acetylene (0.8 mL, 5.5 mmol) and triethylamine (2.1 mL, 15.0 mmol).
The reaction mixture was heated to 65.degree. C. for 12 h. After
completion reaction mixture was cooled to rt, filtered through a
small pad of Celite. The filtrate was concentrated and used in next
step without further purification.
[0605] LCMS: R.sub.T=2.02 min (97.7% Purity).
[0606] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0607] Step Two. 2-chloro-4-ethynylaniline (1114-B). To a solution
of crude 1114-A (1.32 g, 5.9 mmol) dissolved in methanol (20.0 mL)
was added potassium carbonate (1.63 g, 11.8 mmol) at rt. The
resulting reaction mixture was stirred at rt until complete
consumption of the starting material was observed. The reaction
mixture was filtered, and the filtrate was concentrated. The crude
product was purified via ISCO (0 to 30% ethyl acetate in hexanes,
over 20 CV) to yield the product as a yellow solid (580 mg, 77%
yield over 2 steps).
[0608] LCMS: R.sub.T=1.55 min (97.9% purity).
[0609] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0610] Step Three.
(4-(4-amino-3-chlorophenyl)-1H-1,2,3-triazol-1-yl) methyl pivalate
(1114-C). To a solution of 1114-B (303 mg, 2.0 mmol) dissolved in
anhydrous 1,4-dioxane (5.0 mL). CuI (38 mg, 0.2 mmol) was added,
followed by the addition of DIPEA (0.7 mL, 4.0 mmol) and
azidomethyl pivalate (377 mg, 2.4 mmol). The resulting reaction
mixture was heated to 60.degree. C. for 12 h. The reaction mixture
was then cooled to rt and concentrated. The crude product was
purified via ISCO (SiO2, 0 to 50% ethyl acetate in hexanes over 20
CV) to yield 1114-C as an off-white solid (521 mg, 84% yield).
[0611] LCMS: m/z [M+1].sup.+=309.2; R.sub.T=1.62 min (87.7%
purity).
[0612] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0613] Step Four. (4-(4-(4,6-bis((4-methoxybenzyl) oxy)-2-(methyl
thio)
pyrimidine-5-carboxamido)-3-chlorophenyl)-1H-1,2,3-triazol-1-yl)
methyl pivalate (1114-D). Fluoro-N, N, N', N'-bis (tetramethylene)
formamidinium hexafluorophosphate, BTFFH (316 mg, 1.0 mmol) and XY
(443 mg, 1.0 mmol) in anhydrous 1,2 dichloroethane (4.0 mL) was
added DIPEA (0.49 mL, 2.8 mmol), the resulting reaction was stirred
for 30 min at rt. 1114-C (206 mg, 0.7 mmol, 87.7% purity) was added
then sealed tube and heated to 80.degree. C. for 16 h. The reaction
mixture was cooled to rt, and then concentrated under reduced
pressure. The crude product was purified via ISCO (0 to 40% ethyl
acetate in hexanes, over 20 CV) to yield the product as an
off-white solid (100 mg, 21% yield, 85% purity).
[0614] LCMS: m/z [M+1].sup.+=734.3; R.sub.T=2.19 min(85.0%
purity).
[0615] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0616] Step Five. N-(2-chloro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (1114). 2.0 M Aqueous solution of NaOH (2.0 mL) was added to
1114-D (100 mg, 0.12 mmol, 85.0% purity) in THF: MeOH (3.0 mL, 1:1)
at rt. The reaction mixture was stirred for 1 h, and then
concentrated and evaporated with MeOH to obtained intermediate
crude product which was dissolved in CH.sub.2Cl.sub.2 (3.0 mL),
followed by the addition of TFA (2.0 mL) at rt. The reaction was
stirred at rt for 30 min. After complete consumption of starting
material was observed via LCMS, MeOH 10 mL was added to the
mixture. The resulting mixture was concentrated to remove
CH.sub.2Cl.sub.2 and TFA. Add MeOH 10 mL two more times to
co-evaporate CH.sub.2Cl.sub.2 and TFA. The crude product was
purified via trituration using the centrifuge. The solid was washed
with water, the supernatant was removed, and the sequence was
repeated, and then washed with acetonitrile. The resulting solid
was collected and lyophilized to yield the pure product 1114 as an
off-white solid (7.0 mg, 16% yield over 2 steps, 95.2% purity).
[0617] 1H NMR (400 MHz, 1M K.sub.2CO.sub.3 in D.sub.2O): .delta.
7.97 (d, J=8.8 Hz, 1H), 7.75 (s, 1H), 7.66 (s, 1H), 7.47 (d, J=9.1
Hz, 1H), 2.18 (s, 3H).
[0618] LCMS: m/z [M+1].sup.+=379.2; R.sub.T=1.20 min (95.2%
purity).
[0619] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
Example 33: Preparation of N-(3-chloro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (101) (a Compound Having a Structure Represented by Formula
II.sub.2, with Reference to Synthesis Illustrated in FIG. 45)
[0620] Step One. 3-chloro-4-((trimethylsilyl)ethynyl) aniline
(101-A): A round bottom flask containing 3-chloro-4-iodoaniline
(1.27 g, 5.0 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (175 mg, 0.1 mmol),
and CuI (38 mg, 0.2 mmol) was purged with nitrogen for 15 min.
Anhydrous THF (10.0 mL) was added, followed by trimethylsilyl
acetylene (1.4 mL, 10.0 mmol) and triethylamine (1.4 mL, 10.0
mmol). The reaction mixture was heated to 65.degree. C. for 12 h.
After completion reaction mixture was cooled to rt, filtered
through a small pad of Celite. The filtrate was concentrated to get
crude product which was purified by using column chromatography
using hexane: EtOAc (0 to 100% gradient) to yield (1.01 g, 90%) of
product 101-A.
[0621] LCMS: R.sub.T=1.95 min (98% Purity). HPLC conditions:
Column: XBridge C18, 3.5 .mu.m, 4.6.times.30 mm; Gradient: 5% B for
0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min.
Eluent A: Milli-Q H2O+10 mM ammonium formate; Eluent B:
Acetonitrile.
[0622] Step Two. 3-chloro-4-ethynylaniline (101-B): To a solution
of 101-A (0.446 g, 2.0 mmol) dissolved in THF (3.0 mL) was added 1M
solution of TBAF (3.0 ml, 3.0 mmol) at rt. The resulting reaction
mixture was stirred at rt until complete consumption of the
starting material was observed. The reaction mixture was quenched
with water and extracted with EtOAc and then concentrated. The
crude product was used in next step without any purification.
[0623] LCMS: m/z [M+1].sup.+=152.1; R.sub.T=1.40 min (97.9%
purity).
[0624] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0625] Step Three.
(4-(4-amino-2-chlorophenyl)-1H-1,2,3-triazol-1-yl) methyl pivalate
(101-C): To a solution of 101-B (303 mg, 2.0 mmol) dissolved in
anhydrous 1,4-dioxane (5.0 mL). CuI (38 mg, 0.2 mmol) was added,
followed by the addition of DIPEA (0.7 mL, 4.0 mmol) and
azidomethyl pivalate (377 mg, 2.4 mmol). The resulting reaction
mixture was heated to 60.degree. C. for 12 h. The reaction mixture
was then cooled to rt and concentrated. The crude product was
purified via ISCO (SiO2, 0 to 50% ethyl acetate in hexanes over 20
CV) to yield 101-C as an off-white solid (210 mg, 34% yield over
two steps).
[0626] LCMS: m/z [M+1].sup.+=309.2; R.sub.T=1.60 min (91%
purity).
[0627] HPLC conditions: Column: XBridge C18, 3.5.mu.m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0628] Step Four. (4-(4-(4,6-bis((4-methoxybenzyl)
oxy)-2-(methylthio)
pyrimidine-5-carboxamido)-2-chlorophenyl)-1H-1,2,3-triazol-1-yl)
methyl pivalate (101-D): Fluoro-N, N, N', N'-bis (tetramethylene)
formamidinium hexafluorophosphate, BTFFH (400 mg, 1.25 mmol) and XY
(350 mg, 0.75 mmol) in anhydrous 1,2 dichloromethane (3.0 mL) was
added DIPEA (0.4 mL, 2.5 mmol), the resulting reaction was stirred
for 30 min at rt. 101-C (160 mg, 0.5 mmol, 91% purity) was added in
a sealed tube and heated to 80.degree. C. for 16 h. The reaction
mixture was cooled to rt, and then concentrated under reduced
pressure. The crude product was purified via ISCO (0 to 40% ethyl
acetate in hexanes, over 20 CV) to yield 101-D as an off-white
solid (138 mg, 35% yield).
[0629] LCMS: m/z [M].sup.+=733.5; R.sub.T=2.16 min (95.2%
purity).
[0630] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate pH:
3.8; Eluent B: Acetonitrile.
[0631] Step Five. N-(2-chloro-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (101): 2.0 M Aqueous solution of NaOH (0.75 mL) was added to
101-D (108 mg, 0.15 mmol, 91.0% purity) in MeOH (2.0 mL) at rt. The
reaction mixture was stirred for 1 h, and then concentrated and
evaporated with MeOH to obtained intermediate crude product which
was dissolved in CH.sub.2Cl.sub.2 (3.0 mL), followed by the
addition of TFA (2.0 mL) at rt. The reaction was stirred at rt for
30 min. After complete consumption of starting material was
observed via LCMS, MeOH 10 mL was added to the mixture. The
resulting mixture was concentrated to remove CH.sub.2Cl.sub.2 and
TFA. Add MeOH 10 mL two more times to co-evaporate CH.sub.2Cl.sub.2
and TFA. The crude product was purified via trituration using the
centrifuge. The solid was washed with water, the supernatant was
removed, and the sequence was repeated, and then washed with
acetonitrile. The resulting solid was collected and lyophilized to
yield the pure product 101 as an off-white solid (7.0 mg, 12.5%
yield, 96.8% purity).
[0632] 1H NMR (400 MHz, CDCl.sub.3+TFA): .delta. 8.67 (s, 1H), 8.02
(d, J=1.4 Hz, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.67 (dd, J=8.8, 1.7 Hz,
1H), 2.79 (s, 3H).
[0633] LCMS: m/z [M+1].sup.+=379.2; R.sub.T=1.44 min (96.8%
purity).
[0634] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
Example 34: Preparation of
N-(4-(1H-1,2,3-triazol-4-yl)-2-(trifluoromethoxy)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (102) (a Compound Having a Structure Represented by Formula
II.sub.3, with Reference to Synthesis Illustrated in FIG. 46)
[0635] Step One. 2-(trifluoromethoxy)-4-((trimethylsilyl)ethynyl)
aniline (102-A): A round bottom flask containing
4-iodo-2-(trifluoromethoxy) aniline (1.52 g, 5.0 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (175 mg, 0.1 mmol), and CuI (38 mg, 0.2
mmol) was purged with nitrogen for 15 min. Anhydrous THF (10.0 mL)
was added, followed by trimethylsilyl acetylene (1.4 mL, 10.0 mmol)
and triethylamine (1.4 mL, 10.0 mmol). The reaction mixture was
heated to 65.degree. C. for 12 h. After completion reaction mixture
was cooled to rt, filtered through a small pad of Celite. The
filtrate was concentrated to get crude product which was purified
by using column chromatography using hexane: EtOAc (0 to 100%
gradient) to yield (1.21 g, 88%) of product 102-A.
[0636] LCMS: R.sub.T=2.09 min (99.1% Purity).
[0637] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0638] Step Two. 4-ethynyl-2-(trifluoromethoxy) aniline (102-B): To
a solution of 102-A (0.546 g, 2.0 mmol) dissolved in THF (3.0 mL)
was added 1M solution of TBAF (3.0 ml, 3.0 mmol) at rt. The
resulting reaction mixture was stirred at rt until complete
consumption of the starting material was observed. The reaction
mixture was quenched with water and extracted with EtOAc and then
concentrated. The crude product was used in next step without any
purification.
[0639] LCMS: R.sub.T=1.65 min (98% purity).
[0640] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0641] Step Three. 2-(trifluoromethoxy)-4-(1-((2-(trimethylsilyl)
ethoxy) methyl)-1H-1,2,3-triazol-4-yl) aniline (102-C): To a
solution of 102-B (402 mg, 2.0 mmol) dissolved in anhydrous
1,4-dioxane: MeOH (1:1, 5.0 mL). CuI (38 mg, 0.2 mmol) was added,
followed by the addition of DIPEA (0.7 mL, 4.0 mmol) and SEM-azide
(416 mg, 2.4 mmol). The resulting reaction mixture was heated to
60.degree. C. for 12 h. The reaction mixture was then cooled to rt
and concentrated. The crude product was purified via ISCO (SiO2, 0
to 50% ethyl acetate in hexanes over 20 CV) to yield 102-C as an
off-white solid (520 mg, 70% yield over two steps).
[0642] LCMS: m/z [M+1].sup.+=375.2; R.sub.T=1.89 min (97%
purity).
[0643] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0644] Step four.
4-hydroxy-2-(methylthio)-6-oxo-N-(2-(trifluoromethoxy)-4-(1-((2-(trimethy-
lsilyl) ethoxy) methyl)-1H-1,2,3-triazol-4-yl)
phenyl)-1,6-dihydropyrimidine-5-carboxamide (102-D):
6-hydroxy-2-(methylthio) pyrimidin-4(3H)-one (316 mg, 2.0 mmol) was
added to a stirring solution of sodium tert-butoxide (192 mg, 2.0
mmol) dissolved in DMSO (2.0 mL) at rt for 5 min. In a separate
flask, aniline 102-C (374 mg, 1.0 mmol) was dissolved in
1,4-dioxane (2.0 mL), to this solution was added triphosgene (98
mg, 0.33 mmol) in one-portion. The suspension was stirred
vigorously for 2 min at rt, then iPr.sub.2NEt (0.35 mL) was added.
The suspension was stirred vigorously at rt for 2 min. Freshly
prepared solution of sodium
6-hydroxy-2-(methylthio)-3-(3,4,5-trimethoxybenzyl)
pyrimidin-4(3H)-olate in DMSO was added to the suspension in
one-portion. The reaction was stirred at 90.degree. C. for 30 min,
until complete consumption of starting material observed via LCMS.
Reaction mixture was then cooled at rt; Methanol was added to get
clear solution followed by addition of 1M HCl to precipitate; which
was filtered and dried and used in next step without any further
purification.
[0645] LCMS: m/z [M+1].sup.+=559.2; R.sub.T=2.01 min (90%
purity).
[0646] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0647] Step five. N-(4-(1H-1,2,3-triazol-4-yl)-2-(trifluoromethoxy)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (102): A solution of crude 102-D (90% purity) in dichloromethane
(2.0 mL) was added trifluoroacetic acid (2.0 mL). The resulting
reaction mixture was stirred at rt for 20 h. The reaction mixture
was then concentrated under reduced pressure. The crude product was
co-evaporated several times with methanol (3.times.), then purified
via reverse-phase chromatography (C18, gradient eluent from 0 to
20% acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV) to yield the product as an off-white solid (107 mg, 25%
yield over two steps), after lyophilization.
[0648] .sup.1H NMR (400 MHz, 1M K.sub.2CO.sub.3 in D.sub.2O)
.delta. 8.07 (d, J=8.2 Hz, 1H), 7.79 (s, 1H) 7.58 (s, 1H), 7.53 (d,
J=8.9 Hz, 1H), 2.21 (s, 3H).
[0649] LCMS: m/z [M+1].sup.+=429.7; R.sub.T=1.50 min (98.9%
purity).
[0650] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 35: Preparation of
N-(2-(difluoromethoxy)-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (103) (a Compound Having a Structure Represented by Formula
(II.sub.4), with Reference to Synthesis Illustrated in FIG. 47)
[0651] Step One. 2-(difluoromethoxy)-4-((trimethylsilyl)ethynyl)
aniline (103-A): A microwave vial containing
4-bromo-2-(difluoromethoxy) aniline (1.19 g, 5.0 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (702 mg, 1.0 mmol), and CuI (190 mg, 1
mmol) was purged with nitrogen for 15 min. diethyl amine (20.0 mL)
was added, followed by trimethylsilyl acetylene (1.4 mL, 10.0 mmol)
and vial was sealed. The reaction mixture was heated to 120.degree.
C. for 30 min in microwave. After completion reaction mixture was
cooled to rt, filtered through a small pad of Celite. The filtrate
was washed with EtOAc and concentrated to get crude product which
was purified by using column chromatography using hexane: EtOAc (0
to 100% gradient) to yield (0.825 g, 65%) of product 103-A
[0652] LCMS: R.sub.T=1.96 min (98% Purity). HPLC conditions:
Column: XBridge C18, 3.5 .mu.m, 4.6.times.30 mm; Gradient: 5% B for
0.2 min, 5% to 100% B in 1.8 min; 100% B for 1 min; 3 mL/min.
Eluent A: Milli-Q H2O+10 mM ammonium formate; Eluent B:
Acetonitrile.
[0653] Step Two. 2-(difluoromethoxy)-4-ethynylaniline (103-B): To a
solution of 103-A (0.77 g, 3.0 mmol) dissolved in THF (4.0 mL) was
added 1M solution of TBAF (6.0 ml, 6.0 mmol) at rt. The resulting
reaction mixture was stirred at rt until complete consumption of
the starting material was observed. The reaction mixture was
quenched with water and extracted with EtOAc and then concentrated.
The crude product was used in next step without any
purification
[0654] LCMS: R.sub.T=1.51 min (97% purity).
[0655] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0656] Step Three. 2-(difluoromethoxy)-4-(1-((2-(trimethylsilyl)
ethoxy) methyl)-1H-1,2,3-triazol-4-yl) aniline (103-C): To a
solution of 103-B (550 mg, 3.0 mmol) dissolved in anhydrous
1,4-dioxane (5.0 mL). CuI (57 mg, 0.3 mmol) was added, followed by
the addition of DIPEA (1.05 mL, 6.0 mmol) and SEM-azide (630 mg,
3.6 mmol). The resulting reaction mixture was heated to 60.degree.
C. for 12 h. The reaction mixture was then cooled to rt and
concentrated. The crude product was purified via ISCO (SiO2, 0 to
50% ethyl acetate in hexanes over 20 CV) to yield 103-C as an
off-white solid (720 mg, 67% yield over two steps).
[0657] LCMS: m/z [M+1].sup.+=357.2; R.sub.T=1.80 min (96%
purity).
[0658] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0659] Step four. N-(2-(difluoromethoxy)-4-(1-((2-(trimethylsilyl)
ethoxy) methyl)-1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (103-D): 6-hydroxy-2-(methylthio) pyrimidin-4(3H)-one (95 mg, 0.6
mmol) was added to a stirring solution of sodium tert-butoxide (58
mg, 0.6 mmol) dissolved in DMSO (1.0 mL) at rt for 5 min. In a
separate flask, aniline 103-C (107 mg, 0.3 mmol) was dissolved in
1,4-dioxane (0.5 mL), to this solution was added triphosgene (30
mg, 0.1 mmol) in one-portion. The suspension was stirred vigorously
for 2 min at rt, then iPr.sub.2NEt (0.1 mL) was added. The
suspension was stirred vigorously at rt for 2 min. Freshly prepared
solution of sodium
6-hydroxy-2-(methylthio)-3-(3,4,5-trimethoxybenzyl)
pyrimidin-4(3H)-olate in DMSO was added to the suspension in
one-portion. The reaction was stirred at 90.degree. C. for 30 min,
until complete consumption of starting material observed via LCMS.
Reaction mixture was then cooled at rt; Methanol was added to get
clear solution followed by addition of 1M HCl to precipitate; which
was filtered and dried and used in next step without any further
purification.
[0660] LCMS: m/z [M+1].sup.+=541.3; R.sub.T=1.94 min (88%
purity).
[0661] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0662] Step five. N-(4-(1H-1,2,3-triazol-4-yl)-2-(trifluoromethoxy)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (103): A solution of crude 103-D (88% purity) in dichloromethane
(1.0 mL) was added trifluoroacetic acid (10 mL). The resulting
reaction mixture was stirred at rt for 20 h. The reaction mixture
was then concentrated under reduced pressure. The crude product was
co-evaporated several times with methanol (3.times.), then purified
via reverse-phase chromatography (C18, gradient eluent from 0 to
20% acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV) to yield the product 103 as white solid (14 mg, 13%
yield over two steps), after lyophilization as an ammonium
salt.
[0663] .sup.1H NMR (500 MHz, DMSO+TFA) .delta. 12.15 (s, 1H), 8.43
(d, J=8.6 Hz, 1H), 8.32 (s, 1H) 7.78 (s, 1H), 7.75 (dd, J=8.5, 1.8
Hz, 1H), 7.20 (dt, J=91.7, 62.3 Hz, 3H) 2.54 (s, 1H), 2.53 (s,
3H).
[0664] LCMS: m/z [M-1].sup.+=409.1; R.sub.T=1.42 min (97.8%
purity).
[0665] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 36: Preparation of N-(2-cyano-4-(1H-1,2,3-triazol-4-yl)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (104) (a Compound Having a Structure Represented by Formula
(II.sub.5), with Reference to the Synthesis Illustrated in FIG.
48)
[0666] Step One. 2-amino-5-((trimethylsilyl)ethynyl) benzonitrile
(104-A): A round bottom flask containing 2-amino-5-iodobenzonitrile
(1.22 g, 5.0 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (175 mg, 0.1 mmol),
and CuI (38 mg, 0.2 mmol) was purged with nitrogen for 15 min.
Anhydrous THF (10.0 mL) was added, followed by trimethylsilyl
acetylene (1.4 mL, 10.0 mmol) and triethylamine (1.4 mL, 10.0
mmol). The reaction mixture was heated to 65.degree. C. for 12 h.
After completion reaction mixture was cooled to rt, filtered
through a small pad of Celite. The filtrate was concentrated to get
crude product which was purified by using column chromatography
using hexane: EtOAc (0 to 100% gradient) to yield (0.98 g, 92%) of
product 104-A.
[0667] LCMS: R.sub.T=1.88 min (97% Purity).
[0668] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0669] Step Two. 2-amino-5-ethynylbenzonitrile (104-B): To a
solution of 104-A (0.856 g, 4.0 mmol) dissolved in THF (2.0 mL) was
added 1M solution of TBAF (8.0 ml, 8.0 mmol) at rt. The resulting
reaction mixture was stirred at rt until complete consumption of
the starting material was observed. The reaction mixture was
quenched with water and extracted with EtOAc and then concentrated.
The crude product was used in next step without any
purification.
[0670] LCMS: R.sub.T=1.38 min (96% purity).
[0671] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0672] Step Three. 2-amino-5-(1-((2-(trimethylsilyl) ethoxy)
methyl)-1H-1,2,3-triazol-4-yl) benzonitrile (104-C): To a solution
of 104-B (426 mg, 3.0 mmol) dissolved in anhydrous 1,4-dioxane (5.0
mL). CuI (38 mg, 0.3 mmol) was added, followed by the addition of
DIPEA (1.05 mL, 6.0 mmol) and SEM-azide 630 mg, 3.6 mmol). The
resulting reaction mixture was heated to 60.degree. C. for 12 h.
The reaction mixture was then cooled to rt and concentrated. The
crude product was purified via ISCO (SiO2, 0 to 50% ethyl acetate
in hexanes over 20 CV) to yield 104-C as an off-white solid (520
mg, 70% yield over two steps).
[0673] LCMS: m/z [M+1].sup.+=316.2; R.sub.T=1.72 min (95%
purity).
[0674] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile
[0675] Step four.
4-hydroxy-2-(methylthio)-6-oxo-N-(2-(trifluoromethoxy)-4-(1-((2-(trimethy-
lsilyl) ethoxy) methyl)-1H-1,2,3-triazol-4-yl)
phenyl)-1,6-dihydropyrimidine-5-carboxamide (104-D):
6-hydroxy-2-(methylthio) pyrimidin-4(3H)-one (95 mg, 0.6 mmol) was
added to a stirring solution of sodium tert-butoxide (58 mg, 0.6
mmol) dissolved in DMSO (1.0 mL) at rt for 5 min. In a separate
flask, aniline 104-C (95 mg, 0.3 mmol) was dissolved in 1,4-dioxane
(2.0 mL), to this solution was added triphosgene (30 mg, 0.1 mmol)
in one-portion. The suspension was stirred vigorously for 2 min at
rt, then iPr.sub.2NEt (0.1 mL) was added. The suspension was
stirred vigorously at rt for 2 min. Freshly prepared solution of
sodium 6-hydroxy-2-(methylthio)-3-(3,4,5-trimethoxybenzyl)
pyrimidin-4(3H)-olate in DMSO was added to the suspension in
one-portion. The reaction was stirred at 90.degree. C. for 30 min,
until complete consumption of starting material observed via LCMS.
Reaction mixture was then cooled at rt; Methanol was added to get
clear solution followed by addition of 1M HCl to precipitate; which
was filtered and dried and used in next step without any further
purification.
[0676] LCMS: m/z [M+1].sup.+=500.3; R.sub.T=1.91 min (80%
purity).
[0677] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0678] Step five. N-(4-(1H-1,2,3-triazol-4-yl)-2-(trifluoromethoxy)
phenyl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamid-
e (104): A solution of crude 104-D (80% purity) in dichloromethane
(1.0 mL) was added trifluoroacetic acid (1.0 mL). The resulting
reaction mixture was stirred at rt for 20 h. The reaction mixture
was then concentrated under reduced pressure. The crude product was
co-evaporated several times with methanol (3.times.), then purified
via reverse-phase chromatography (C18, gradient eluent from 0 to
20% acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 20 CV) to yield the product 104 as an off-white solid (11 mg,
10% yield over two steps), after lyophilization as an ammonium
salt.
[0679] .sup.1H NMR (500 MHz, DMSO+TFA) .delta. 12.28 (s, 1H), 8.39
(s, 1H), 8.34 (d, J=8.8 Hz, 1H), 8.27 (d, J=2.0 Hz, 1H), 8.16 (dd,
J=8.8, 2.0 Hz, 1H), 7.20-6.90 (t, 1.5H ammonia peak), 2.51 (s,
3H).
[0680] LCMS: m/z [M-1].sup.+=368.3; R.sub.T=1.35 min (96.5%
purity).
[0681] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 37: Preparation of
N-(4-(1H-1,2,3-Triazol-4-yl)-3-(trifluoromethyl)phenyl)-4-hydroxy-2-(meth-
yl-thio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide (105) (a
Compound Having a Structure Represented by Formula (II.sub.6), with
Reference to the Synthesis Illustrated in FIG. 49)
[0682] Step One.
3-(Trifluoromethyl)-4-((trimethylsilyl)ethynyl)aniline (105-A): A
round bottom flask containing 4-bromo-3-(trifluoromethyl)aniline
(2.17 g, 9.04 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (381 mg, 0.543
mmol), and CuI (69 mg, 0.36 mmol) was purged with nitrogen for 15
min. Anhydrous THF (40 mL) was added, followed by trimethylsilyl
acetylene (2.60 mL, 18.4 mmol) and ethanolamine (1.09 mL, 18.1
mmol). The reaction mixture was sealed and heated to 65.degree. C.
for 16 h. The reaction mixture was cooled to rt, filtered through a
small pad of Celite. The filtrate was concentrated and the crude
product was used without further purification.
[0683] LCMS: R.sub.T=1.97 min.
[0684] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0685] Step Two. 4-Ethynyl-3-(trifluoromethyl)aniline (105-B): To a
solution of crude 105-A (9.04 mmol) dissolved in methanol (18.0 mL)
was added potassium carbonate (2.50 g, 18.1 mmol) at rt. The
resulting reaction mixture was stirred at rt for 2 h. The reaction
mixture was filtered, and the filtrate was concentrated. The crude
product was purified via ISCO (0 to 50% ethyl acetate in hexanes,
over 20 CV) to yield the product as a brown oil (1.46 g,
purity=18%; contaminated with 4-bromo-3-(trifluoromethyl)aniline
(82%)).
[0686] LCMS: R.sub.T=1.55 min; purity=18%.
[0687] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0688] Step Three.
3-(Trifluoromethyl)-4-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,3-tria-
zol-4-yl)aniline (105-C): 105-B (1.26 g, 6.81 mmol, purity=18%) was
dissolved in anhydrous 1,4-dioxane (13.5 mL). CuI (65 mg, 0.34
mmol) was added, followed by the addition of iPr.sub.2NEt (590
.mu.L, 3.39 mmol) and (2-(2-azidoethoxy)ethyl)trimethylsilane (590
mg, 3.40 mmol). The resulting reaction mixture was heated to
60.degree. C. for 16 h. The reaction mixture was then cooled to rt
and then concentrated under reduced pressure. The crude product was
purified via ISCO (0 to 40% ethyl acetate in hexanes, over 20 CV)
to yield the product as orange oil (118 mg, 5% yield over three
steps, >99% purity).
[0689] Step Three.
3-(Trifluoromethyl)-4-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-1,2,3-tria-
zol-4-yl)aniline (105-C): 105-B (1.26 g, 6.81 mmol, purity=18%) was
dissolved in anhydrous 1,4-dioxane (13.5 mL). CuI (65 mg, 0.34
mmol) was added, followed by the addition of iPr.sub.2NEt (590
.mu.L, 3.39 mmol) and (2-(2-azidoethoxy)ethyl)trimethylsilane (590
mg, 3.40 mmol). The resulting reaction mixture was heated to
60.degree. C. for 16 h. The reaction mixture was then cooled to rt
and then concentrated under reduced pressure. The crude product was
purified via ISCO (0 to 40% ethyl acetate in hexanes, over 20 CV)
to yield the product as orange oil (118 mg, 5% yield over three
steps, >99% purity).
[0690] LCMS: m/z [M+1].sup.+=359.3; R.sub.T=1.79 min;
purity=>99%.
[0691] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0692] Step Four.
4-Hydroxy-2-(methylthio)-6-oxo-N-(3-(trifluoromethyl)-4-(1-((2-(trimethyl-
silyl)-ethoxy)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1,6-dihydropyrimidine--
5-carboxamide (105-D): 6-Hydroxy-2-(methylthio)pyrimidin-4(3H)-one
(84 mg, 0.53 mmol) was added to a stirring solution of sodium
tert-butoxide (51 mg, 0.53 mmol) dissolved in DMSO (0.90 mL) at
room temperature for 5 min. In a separate flask, aniline 105-C (95
mg, 0.265 mmol) was dissolved in 1,4-dioxane (0.65 mL). To this
solution was added triphosgene (26 mg, 0.088 mmol) in one-portion.
The suspension was stirred vigorously for 2 min at room
temperature, then iPr.sub.2NEt (92 .mu.L, 0.53 mmol) was added. The
suspension was stirred vigorously at room temperature for 2 min.
The previous solution prepared in DMSO was added to this suspension
in one-portion. The reaction was then stirred at 80.degree. C. for
45 min. Reaction mixture was then cooled to room temperature and
diluted with CH.sub.2Cl.sub.2. The solution was washed with water
(1.times.) and with brine (1.times.) then dried over MgSO.sub.4,
filtered, and concentrated under reduced pressure. The crude
product was purified via ISCO (0 to 10% methanol in
dichloromethane, over 30 CV) to yield the product as orange oil (39
mg, 27% yield, 84% purity).
[0693] LCMS: m/z [M+1].sup.+=543.2; R.sub.T=1.95 min (84%
purity).
[0694] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0695] Step Five.
N-(4-(1H-1,2,3-Triazol-4-yl)-3-(trifluoromethyl)phenyl)-4-hydroxy-2-(meth-
ylthio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide (105):
Trifluoroacetic acid (0.70 mL) was added to 105-D (37 mg, 0.068
mmol) in dichloromethane (0.70 mL). The reaction mixture was
stirred at room temperature for 3 h, and then co-evaporated with
MeOH (3.times..about.5 mL). The crude product was purified via
reverse-phase chromatography (C18, gradient eluent from 0 to 30%
acetonitrile in water with an ammonium bicarbonate buffer 10 mM
over 30 CV) to yield 105 as a white solid (8.2 mg, 29% yield,
>99.9% purity), after lyophilization.
[0696] .sup.1H NMR (500 MHz, CDCl.sub.3: TFA (10:1)) .delta. 8.34
(s, 1H), 8.22 (d, J=2.1 Hz, 1H), 7.97 (dd, J=8.3, 2.2 Hz, 1H), 7.66
(d, J=8.5 Hz, 1H), 2.74 (s, 3H).
[0697] LCMS: m/z [M+1].sup.+=413.2; R.sub.T=1.44 min;
purity=>99.9%.
[0698] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 38: Preparation of N-(2-(1H-1,2,3-triazol-4-yl)
quinolin-6-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-car-
boxamide (106) (a Compound Having a Structure Represented by
Formula (II.sub.10), with Reference to the Synthesis Illustrated in
FIG. 50)
[0699] Step One. 2-Chloroquinolin-6-amine (106-A): To
2-chloro-6-nitroquinoline (418 mg, 2.0 mmol) and NH.sub.4Cl (535
mg, 10.0 mmol) was added EtOH (20 mL) and water (0.4 mL). The
reaction mixture was heated to 60.degree. C. and Fe (335 mg, 6.0
mmol) was added in several portions. The reaction mixture was
stirred for 2 h maintaining the temperature at 60.degree. C. The
mixture was cooled to room temperature and the ethanol was removed
under reduced pressure. The aqueous mixture was diluted with 100 mL
of EtOAc and solids were removed by filtration. The filtrate was
concentrated under reduced pressure to yield the desired product,
339 mg (95%), as a yellow solid.
[0700] LCMS: m/z [M+1].sup.+=179.2; R.sub.T=1.26 min (99%
purity).
[0701] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0702] Step Two. 2-((trimethylsilyl)ethynyl)quinolin-6-amine
(106-B): A round bottom flask containing 2-chloro-6-aminoquinoline
(356 mg, 2.0 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (281 mg, 0.4 mmol),
and CuI (152 mg, 0.8 mmol) was purged with nitrogen for 15 min.
triethylamine (10.0 ml) was added, followed by trimethylsilyl
acetylene (1.4 mL, 10.0 mmol). The reaction mixture was heated to
65.degree. C. for 16 h. After completion reaction mixture was
cooled to rt, filtered through a small pad of Celite. The filtrate
was concentrated to get crude product which was purified by using
column chromatography using hexane: EtOAc (0 to 100% gradient) to
yield (425 mg, 88%) of product 106-B.
[0703] LCMS: m/z [M+1].sup.+=241.2; R.sub.T=1.75 min (96%
Purity).
[0704] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0705] Step Three. 2-ethynylquinolin-6-amine (106-C): To a solution
of 106-B (480 mg, 2.0 mmol) dissolved in THF (2.0 mL) was added 1M
solution of TBAF (4.0 ml, 4.0 mmol) at rt. The resulting reaction
mixture was stirred at rt until complete consumption of the
starting material was observed. The reaction mixture was quenched
with water and extracted with EtOAc and then concentrated. The
crude product 106-C was used in next step without any
purification.
[0706] LCMS: m/z [M+1].sup.+=169.3; R.sub.T=1.43 min (95%
purity).
[0707] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0708] Step Four. 2-(1-((2-(trimethylsilyl) ethoxy)
methyl)-1H-1,2,3-triazol-4-yl) quinolin-6-amine (106-D): To a
solution of 106-C (336 mg, 2.0 mmol) dissolved in methanol (3.0
mL). CuI (38 mg, 0.3 mmol) was added, followed by the addition of
DIPEA (0.7 mL, 4.0 mmol) and SEM-azide (416 mg, 2.4 mmol). The
resulting reaction mixture was heated to 60.degree. C. for 12 h.
The reaction mixture was then cooled to rt and concentrated. The
crude product was purified via ISCO (SiO2, 0 to 50% ethyl acetate
in hexanes over 20 CV) to yield 106-D as an off-white solid (550
mg, 81% yield over two steps).
[0709] LCMS: m/z [M+1].sup.+=342.6; R.sub.T=1.68 min (99.5%
purity).
[0710] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0711] Step Five.
4-hydroxy-2-(methylthio)-6-oxo-N-(2-(1-((2-(trimethylsilyl) ethoxy)
methyl)-1H-1,2,3-triazol-4-yl)
quinolin-6-yl)-1,6-dihydropyrimidine-5-carboxamide (106-E):
6-hydroxy-2-(methylthio) pyrimidin-4(3H)-one (158 mg, 1.0 mmol) was
added to a stirring solution of sodium tert-butoxide (96 mg, 1.0
mmol) dissolved in DMSO (1.0 mL) at rt for 5 min. In a separate
flask, aniline 106-D (171 mg, 0.5 mmol) was dissolved in
1,4-dioxane (2.0 mL), to this solution was added triphosgene (82
mg, 0.275 mmol) in one-portion. The suspension was stirred
vigorously for 2 min at rt, then iPr.sub.2NEt (0.2 mL) was added.
The suspension was stirred vigorously at rt for 2 min. Freshly
prepared solution of sodium
6-hydroxy-2-(methylthio)-3-(3,4,5-trimethoxybenzyl)
pyrimidin-4(3H)-olate in DMSO was added to the suspension in
one-portion. The reaction was stirred at 90.degree. C. for 30 min,
until complete consumption of starting material observed via LCMS.
Reaction mixture was then cooled at rt; concentrated under vacuum.
Purification was done by reverse phase column chromatography using
10 mM ammonium bicarbonate buffer (PH=10.0) and acetonitrile. Pure
fraction was lyophilized to get desired product 106-E
[0712] LCMS: m/z [M+1].sup.+=526.3; R.sub.T=2.10 min (98%
purity).
[0713] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0714] Step Six. N-(2-(1H-1,2,3-triazol-4-yl)
quinolin-6-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-car-
boxamide (106): A solution of 106-E (80 mg, 0.15 mmol) in
dichloromethane (2.0 mL) was added trifluoroacetic acid (2.0 mL).
The resulting reaction mixture was stirred at rt for 2 h. The
reaction mixture was then concentrated under reduced pressure. The
crude product was co-evaporated several times with methanol
(3.times.), then purified via reverse-phase chromatography (C18,
gradient eluent from 0 to 20% acetonitrile in water with an
ammonium bicarbonate buffer 10 mM over 20 CV) to yield the product
106 as white solid (36 mg, 60% yield), after lyophilization.
[0715] .sup.1H NMR (500 MHz, DMSO+TFA) .delta. 12.02 (s, 1H), 8.78
(s, 1H), 8.72 (d, J=8.7 Hz, 1H), 8.47 (d, J=1.9 Hz, 1H), 8.29 (d,
J=8.7 Hz, 1H), 8.21 (d, J=9.0 Hz, 1H), 8.04 (dd, J=9.1, 2.2 Hz,
1H), 2.55 (s, 3H).
[0716] LCMS: m/z [M+1].sup.+=396.1; R.sub.T=0.93 min (99.0%
purity).
[0717] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate pH: 10.0; Eluent B: Acetonitrile.
Example 39: Preparation of
N-(4-(1H-1,2,3-Triazol-5-yl)-2-(trifluoromethyl)phenyl)-4-hydroxy-2-(isop-
ropyl-thio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide (107) (a
Compound Having a Structure Represented by Formula (II.sub.7), with
Reference to the Synthesis Illustrated in FIG. 51)
[0718] Step One. 1-(3,4,5-Trimethoxybenzyl)thiourea (107-A):
(3,4,5-Trimethoxyphenyl)methanamine (2.5 mL, 14.6 mmol) was added
dropwise to a solution of 1,1'-thiocarbonyl diimidazole (3.91 g,
22.0 mmol) dissolved in dichloromethane (36.5 mL) at 0.degree. C.
The reaction mixture was then allowed to warm up to rt over 2 h.
After complete consumption of the starting material was observed
via LCMS, a solution of ammonia in methanol (7.5 mL, 52.6 mmol, 7.0
M in MeOH) was added, then stirred for an additional 20 h. The
reaction mixture was concentrated under reduced pressure,
dichloromethane was added, the precipitate was isolated and washed
with additional CH.sub.2Cl.sub.2, then dried under high vacuum to
yield the product as a light pink solid (2.83 g, 76% yield).
[0719] LCMS: m/z [M+1].sup.+=257.07; R.sub.T=1.06 min.
[0720] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0721] Step Two.
6-Hydroxy-2-(isopropylthio)-3-(3,4,5-trimethoxybenzyl)
pyrimidin-4(3H)-one (107-B): A mixture of 107-A (781 mg, 3.05
mmol), diethyl malonate (465 .mu.L, 3.05 mmol), and NaOMe (1.4 mL,
6.10 mmol, 4.4 M in MeOH) in methanol (2.4 mL) was heated to reflux
for 3 h. The reaction was then cooled to .about.50.degree. C.,
isopropyl iodide (3.5 mL, 30.5 mmol) was then added in one-portion.
The reaction was stirred for an additional 30 min at 50.degree. C.
The reaction mixture was then cooled to rt, then concentrated under
reduced pressure. The crude product was purified via reverse-phase
chromatography (gradient eluent from 0 to 100% acetonitrile in
water with an ammonium formate buffer 10 mM over 15 CV) to yield
the product as a white solid (433 mg, 97.7% purity, 38% yield),
after lyophilization.
[0722] LCMS: m/z [M+1].sup.+=367.02; R.sub.T=1.41 min;
purity=97.7%.
[0723] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0724] Step Three.
4-Hydroxy-2-(isopropylthio)-6-oxo-N-(2-(trifluoromethyl)-4-(1-((2-(trimet-
hylsilyl)-ethoxy)methyl)-1H-1,2,3-triazol-4-yl)phenyl)-1-(3,4,5-trimethoxy-
benzyl)-1,6-dihydropyrimi-dine-5-carboxamide (107-C): 107-B (159
mg, 0.434 mmol) was added to a stirring solution of sodium
tert-butoxide (42 mg, 0.437 mmol) dissolved in DMSO (1.50 mL) at
room temperature for 5 min. In a separate flask, aniline 100-C (78
mg, 0.22 mmol) was dissolved in 1,4-dioxane (1.10 mL). To this
solution was added triphosgene (21 mg, 0.071 mmol) in one-portion.
The suspension was stirred vigorously for 2 min at room
temperature, then iPr.sub.2NEt (76 .mu.L, 0.44 mmol) was added. The
suspension was stirred vigorously at room temperature for 2 min.
The previous solution prepared in DMSO was added to this suspension
in one-portion. The reaction was then stirred at 80.degree. C. for
45 min. Reaction mixture was then cooled to room temperature and
water was added. The mixture was extracted with 2-methyl-THF
(3.times.). Combined organic layers were washed with brine
(1.times.) then dried over MgSO.sub.4, filtered, and concentrated
under reduced pressure. The crude product was purified via ISCO (0
to 5% methanol in dichloromethane, over 25 CV) to yield the product
as an off-white solid (146 mg, 90% yield, 81% purity).
[0725] LCMS: m/z [M+1].sup.+=751.6; R.sub.T=2.29 min;
purity=81%.
[0726] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
[0727] Step Four.
N-(4-(1H-1,2,3-Triazol-5-yl)-2-(trifluoromethyl)phenyl)-4-hydroxy-2-(isop-
ropyl-thio)-6-oxo-1,6-dihydropyrimidine-5-carboxamide (107):
Trifluoroacetic acid (1.20 mL) was added to a solution of 107-C
(146 mg, 0.194 mmol) in dichloromethane (6.5 mL). The resulting
reaction mixture was sealed in a pressure vessel then heated to
60.degree. C. for 16 h. The reaction mixture was allowed to cool to
rt, then concentrated under reduced pressure. The crude product was
co-evaporated several times with methanol (3.times.5 mL), then
purified via reverse-phase chromatography (C18, gradient eluent
from 0 to 30% acetonitrile in water with an ammonium bicarbonate
buffer 10 mM over 25 CV). A second purification via reverse-phase
chromatography (C18, gradient eluent from 20 to 60% acetonitrile in
water with an ammonium formate buffer 10 mM over 24 CV) was done to
yield the product 107 as a white solid (9.2 mg, >99.9% purity,
11% yield), after lyophilization.
[0728] .sup.1H NMR (500 MHz, CDCl.sub.3: TFA (10:1)) .delta. 8.54
(s, 1H), 8.43 (d, J=8.6 Hz, 1H), 8.10 (s, 1H), 7.99 (d, J=8.5 Hz,
1H), 4.19-4.13 (m, 1H), 1.51 (d, J=6.8 Hz, 6H).
[0729] LCMS: m/z [M+1].sup.+=441.1; R.sub.T=1.60 min;
purity=>99.9%. HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 40: Preparation of
N-(5-(1H-1,2,3-triazol-4-yl)-3-(trifluoromethyl)
pyridin-2-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carb-
oxamide (108) (a Compound Having a Structure Represented by Formula
(II.sub.8), with Reference to the Synthesis Illustrated in FIG.
52)
[0730] Step One. 3-(trifluoromethyl)-5-((trimethylsilyl)ethynyl)
pyridin-2-amine (108-A): A round bottom flask containing
5-iodo-3-(trifluoromethyl) pyridin-2-amine (1.0 g, 3.5 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (25 mg, 0.035 mmol), and CuI (7 mg,
0.035 mmol) was purged with nitrogen for 15 min. Anhydrous THF
(15.0 mL) was added, followed by trimethylsilyl acetylene (0.6 mL,
4.2 mmol) and triethylamine (4.9 mL, 35.0 mmol). The reaction
mixture was heated to 65.degree. C. for 3 h. After completion
reaction mixture was cooled to rt, filtered through a small pad of
Celite. The filtrate was concentrated to get crude product which
was purified by using column chromatography using hexane: EtOAc (0
to 100% gradient) to yield (0.452 g, 50%) of product 108-A.
[0731] LCMS: m/z [M+1].sup.+=259.2; R.sub.T=1.95 min (94%
Purity).
[0732] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0733] Step Two. 5-ethynyl-3-(trifluoromethyl) pyridin-2-amine
(108-B): To a solution of 108-A (0.452 g, 1.75 mmol) dissolved in
THF (2.0 mL) was added 1M solution of TBAF (3.5 ml, 3.5 mmol) at
rt. The resulting reaction mixture was stirred at rt until complete
consumption of the starting material was observed. The reaction
mixture was quenched with water and extracted with EtOAc and then
concentrated. The crude product was purified by using column
chromatography with hexane: EtOAc (0 to 100% gradient) to yield
(0.228 g, 70%) of product 108-B.
[0734] LCMS: m/z [M+1].sup.+=187.2; R.sub.T=1.43 min (98%
purity).
[0735] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0736] Step Three. 3-(trifluoromethyl)-5-(1-((2-(trimethylsilyl)
ethoxy) methyl)-1H-1, 2, 3-triazol-4-yl) pyridin-2-amine (108-C):
To a solution of 108-B (228 mg, 1.23 mmol) dissolved in MeOH (5.0
mL). CuI (24 mg, 0.123 mmol) was added, followed by the addition of
DIPEA (0.65 mL, 3.7 mmol) and SEM-azide (255 mg, 1.47 mmol). The
resulting reaction mixture was heated to 60.degree. C. for 12 h.
The reaction mixture was then cooled to rt and concentrated. The
crude product was purified via ISCO (SiO2, 0 to 50% ethyl acetate
in hexanes over 20 CV) to yield 108-C as an off-white solid (243
mg, 55% yield).
[0737] LCMS: m/z [M+1].sup.+=360.3; R.sub.T=1.78 min (95%
purity).
[0738] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0739] Step four. 4,6-bis((4-methoxybenzyl)
oxy)-2-(methylthio)-N-(3-(trifluoromethyl)-5-(1-((2-(trimethylsilyl)
ethoxy) methyl)-1H-1,2,3-triazol-4-yl) pyridin-2-yl)
pyrimidine-5-carboxamide (108-D):
[0740] To a stirred solution of 108-C (180 mg, 0.5 mmol) in THF
under N.sub.2 atmosphere at rt was added 1M LiHMDS solution in THF
(0.5 ml, 0.5 mmol). The reaction mixture was allowed to stir for 30
min at rt. After 30 min, perfluorophenyl 4,6-bis((4-methoxybenzyl)
oxy)-2-(methylthio) pyrimidine-5-carboxylate (300 mg, 0.5 mmol)
dissolved in THF (2.0 ml) was added slowly to the reaction mixture.
After stirring reaction mixture for another 30 min at rt it was
quenched with water; extracted with ethyl acetate. The combined
ethyl acetate layer was washed with brine, dried over sodium
sulfate and concentrated under vacuum to get crude product (312 mg;
80% Yield) which was used in next step without any further
purification.
[0741] LCMS: m/z [M+1].sup.+=784.5; R.sub.T=2.22 min (98%
purity).
[0742] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0743] Step five. N-(5-(1H-1,2,3-triazol-4-yl)-3-(trifluoromethyl)
pyridin-2-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carb-
oxamide (108):
[0744] A solution of crude 108-D (156 mg, 0.2 mmol) in
dichloromethane (2.0 mL) was added trifluoroacetic acid (1.0 mL).
The resulting reaction mixture was stirred at rt for 1 h then
heated at 80.degree. C. for 6 h. Reaction mixture was cooled at rt
and concentrated to get crude product which was co-evaporated 2-3
times with methanol. The resulting reside was re-dissolved in MeOH
(2.0 ml) and ethylenediamine (0.4 ml, 6 mmol, 30 eq) and reaction
mixture was stirred for another 12 h it was then concentrated under
vacuum to get crude product. Finally, crude product was purified
via reverse-phase chromatography (C18, gradient eluent from 0 to
100% acetonitrile in water with an ammonium formate buffer 10 mM
over 22 CV) to yield the product 108 as an off-white solid (18 mg,
23% yield), after lyophilization as an ammonium salt.
[0745] .sup.1H NMR (500 MHz, DMSO+DCl in D.sub.2O) .delta. 9.30 (d,
J=2.1 Hz, 1H), 8.81 (s, 1H), 8.76 (d, J=2.1 Hz, 1H), 8.23 (s, 1H,
--HCOOH peak), 2.60 (s, 3H).
[0746] LCMS: m/z [M-1].sup.+=412.3; R.sub.T=1.26 min (99.2%
purity).
[0747] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium formate pH: 3.8; Eluent B: Acetonitrile.
Example 41: Preparation of N-(3-fluoro-5-(1H-1,2,3-triazol-4-yl)
pyridin-2-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carb-
oxamide (109) (a Compound Having a Structure Represented by Formula
(II.sub.9), with Reference to the Synthesis Illustrated in FIG.
53)
[0748] Step One. 3-(fluoro)-5-((trimethylsilyl)ethynyl)
pyridin-2-amine (109-A): In a microwave vial containing
5-bromo-3-fluoropyridin-2-amine (0.955 g, 5.0 mmol),
PdCl.sub.2(PPh.sub.3).sub.2 (702 mg, 1.0 mmol), and CuI (380 mg,
2.0 mmol) was purged with nitrogen for 15 min. Diethylamine (20.0
mL) was added, followed by trimethylsilyl acetylene (3.5 mL, 25.0
mmol). Microwave vial was sealed and heated to 120.degree. C. for 2
h in microwave. After completion reaction mixture was cooled to rt,
filtered through a small pad of Celite. The filtrate was
concentrated to get crude product which was purified by using
column chromatography in hexane: EtOAc (0 to 100% gradient) to
yield (0.75 g, 72%) of product 109-A.
[0749] LCMS: m/z [M+1].sup.+=209.3; R.sub.T=1.77 min (95%
purity).
[0750] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0751] Step Two. 5-ethynyl-3-fluoropyridin-2-amine (109-B): To a
solution of 109-A (0.75 g, 3.6 mmol) dissolved in THF (5.0 mL) was
added 1M solution of TBAF (7.2 ml, 7.2 mmol) at rt. The resulting
reaction mixture was stirred at rt until complete consumption of
the starting material was observed. The reaction mixture was
quenched with water and extracted with EtOAc and then concentrated.
The crude product was used in next step without any
purification.
[0752] LCMS: m/z [M+1].sup.+=137.2; R.sub.T=1.11 min (96%
purity).
[0753] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0754] Step Three. 3-fluoro-5-(1-((2-(trimethylsilyl) ethoxy)
methyl)-1H-1,2,3-triazol-4-yl) pyridin-2-amine (109-C): To a
solution of 109-B (490 mg, 3.6 mmol) dissolved in MeOH (5.0 mL).
CuI (69 mg, 0.36 mmol) was added, followed by the addition of DIPEA
(1.25 mL, 7.2 mmol) and SEM-azide (748 mg, 4.32 mmol). The
resulting reaction mixture was heated to 60.degree. C. for 12 h
then cooled to rt and concentrated. The crude product was purified
via ISCO (SiO2, 0 to 50% ethyl acetate in hexanes over 20 CV) to
yield 109-C as an off-white solid (835 mg, 75% yield).
[0755] LCMS: m/z [M+1].sup.+=310.2; R.sub.T=1.61 min (97%
purity).
[0756] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0757] Step four. N-(3-fluoro-5-(1-((2-(trimethylsilyl) ethoxy)
methyl)-1H-1,2,3-triazol-4-yl)
pyridin-2-yl)-4,6-bis((4-methoxybenzyl) oxy)-2-(methylthio)
pyrimidine-5-carboxamide (109-D): To a stirred solution of 109-C
(150 mg, 0.5 mmol) in THF under N.sub.2 atmosphere at rt was added
1M LiHMDS solution in THF (0.5 ml, 0.5 mmol). The reaction mixture
was allowed to stir for 30 min at rt. After 30 min, perfluorophenyl
4,6-bis((4-methoxybenzyl) oxy)-2-(methylthio)
pyrimidine-5-carboxylate (300 mg, 0.5 mmol) dissolved in THF (2.0
ml) was added slowly to the reaction mixture. After stirring
reaction mixture for another 30 min at rt it was quenched with
water; extracted with ethyl acetate. The combined ethyl acetate
layer was washed with brine, dried over sodium sulfate and
concentrated under vacuum to get crude product (265 mg; 72% Yield)
which was used in next step without any further purification.
[0758] LCMS: m/z [M+1].sup.+=734.6; R.sub.T=2.15 min (97%
purity).
[0759] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0760] Step five. N-(3-fluoro-5-(1H-1,2,3-triazol-4-yl)
pyridin-2-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carb-
oxamide (109): A solution of crude 109-D (265 mg, 0.36 mmol) in
dichloromethane (2.0 mL) was added trifluoroacetic acid (1.0 mL).
The resulting reaction mixture was stirred at rt for 1 h then
heated at 80.degree. C. for 6 h. Reaction mixture was cooled at rt
and concentrated to get crude product which was co-evaporated 2-3
times with methanol. The resulting reside was re-dissolved in MeOH
(2.0 ml) and ethylenediamine (0.7 ml, 10.8 mmol, 30 eq) and
reaction mixture was stirred for another 12 h it was then
concentrated under vacuum to get crude product. Finally, crude
product was purified via reverse-phase chromatography (C18,
gradient eluent from 0 to 100% acetonitrile in water with an
ammonium formate buffer 10 mM over 22 CV) to yield the product 109
as an off-white solid (28 mg, 22% yield), after lyophilization.
[0761] .sup.1H NMR (500 MHz, DMSO+TFA) .delta. 8.68 (d, J=1.8 Hz,
1H), 8.56 (s, 1H), 8.51 (d, J=10.5 Hz, 1H), 2.41 (s, 3H).
[0762] LCMS: m/z [M+I].sup.+=364.1; R.sub.T=1.17 min (96.0%
purity).
[0763] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM ammonium formate
pH: 3.8; Eluent B: Acetonitrile.
Example 42: Preparation of
N-(2-(1H-1,2,3-triazol-4-yl)-5-(trifluoromethyl)
quinolin-6-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-car-
boxamide (110) (a Compound Having a Structure Represented by
Formula (II.sub.11), with Reference to the Synthesis Illustrated in
FIG. 54)
[0764] Step One. 2-Chloroquinolin-6-amine (110-A): To
2-chloro-6-nitroquinoline (2.08 g, 10.0 mmol) and NH.sub.4Cl (2.68
g, 50.0 mmol) was added EtOH (100 mL) and water (2.0 mL). The
reaction mixture was heated to 60.degree. C. and Fe (1.68 g, 30.0
mmol) was added in several portions. The reaction mixture was
stirred for 2 h maintaining the temperature at 60.degree. C. The
mixture was cooled to room temperature and the ethanol was removed
under reduced pressure. The aqueous mixture was diluted with 500 mL
of EtOAc and solids were removed by filtration. The filtrate was
concentrated under reduced pressure to yield the desired product,
1.64 g (92%), as a yellow solid.
[0765] LCMS: m/z [M+1].sup.+=179.2; R.sub.T=1.25 min (98%
Purity).
[0766] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0767] Step Two. 2-chloro-5-(trifluoromethyl) quinolin-6-amine
(110-B): To a solution of 110-A (890 mg, 5.0 mmol, 1.0 equiv) and
sodium trifluoromethylsulfinate (2.34 g, 15.0 mmol, 3.0 equiv) in
dichloromethane (18.0 mL) and water (7.0 mL) at 0.degree. C. was
slowly added tert-butylhydroperoxide (70% solution in water, 2.26
g, 25 mmol, 5.0 equiv) with vigorous stirring. The reaction was
allowed to warm to room temperature and monitored by LCMS until
completion. Reaction does not go to completion in 20 h, a second
addition of sodium trifluoromethylsulfinate (3.0 equiv) and
tert-butylhydroperoxide (5.0 equiv) was added to drive the reaction
towards completion. Upon consumption of starting material, the
reaction was partitioned between dichloromethane (100 mL) and
saturated sodium bicarbonate (50 mL). The organic layer was
separated, and the aqueous layer was extracted with dichloromethane
(3.times.50 mL). The organic layers were dried with sodium sulfate,
concentrated, and purified by column chromatography on silica gel
to get 110-B (308 mg, 25% Yield).
[0768] .sup.1H NMR (500 MHz, DMSO) .delta. 8.21-8.10 (m, 1H), 7.81
(d, J=9.3 Hz, 1H), 7.53 (d, J=9.1 Hz, 1H), 7.35 (d, J=9.4 Hz, 1H),
6.49 (s, 2H).
[0769] LCMS: m/z [M+1].sup.+=247.1; R.sub.T=1.68 min (99.5%
purity).
[0770] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0771] Step Three. 5-(trifluoromethyl)-2-((trimethylsilyl)ethynyl)
quinolin-6-amine (110-C): A round bottom flask containing 110-B
(330 mg, 1.34 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (190 mg, 0.27
mmol), and CuI (103 mg, 0.54 mmol) was purged with nitrogen for 15
min. triethylamine (4.5 ml) was added, followed by trimethylsilyl
acetylene (0.94 mL, 6.7 mmol). The reaction mixture was heated to
65.degree. C. for 16 h. After completion reaction mixture was
cooled to rt, filtered through a small pad of Celite. The filtrate
was concentrated to get crude product which was purified by using
column chromatography using hexane: EtOAc (0 to 100% gradient) to
yield (315 mg, 76%) of product 110-C.
[0772] LCMS: m/z [M+1].sup.+=309.1; R.sub.T=2.04 min (95%
purity).
[0773] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0774] Step Four. 2-ethynyl-5-(trifluoromethyl) quinolin-6-amine
(110-D): To a solution of 110-C (308 mg, 1.0 mmol) dissolved in THF
(2.0 mL) was added 1M solution of TBAF (2.0 ml, 2.0 mmol) at rt.
The resulting reaction mixture was stirred at rt until complete
consumption of the starting material was observed. The reaction
mixture was quenched with water and extracted with EtOAc and then
concentrated. The crude product 110-D was used in next step without
any purification.
[0775] LCMS: m/z [M+1].sup.+=237.1; R.sub.T=1.54 min (90%
purity).
[0776] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0777] Step Five. 5-(trifluoromethyl)-2-(1-((2-(trimethylsilyl)
ethoxy) methyl)-1H-1,2,3-triazol-4-yl) quinolin-6-amine (110-E): To
a solution of 110-D (236 mg, 1.0 mmol) dissolved in methanol (5.0
mL). CuI (20 mg, 0.1 mmol) was added, followed by the addition of
DIPEA (0.35 mL, 2.0 mmol) and SEM-azide (208 mg, 1.2 mmol). The
resulting reaction mixture was heated to 65.degree. C. for 12 h.
The reaction mixture was then cooled to rt and concentrated. The
crude product was purified via ISCO (SiO.sub.2, 0 to 50% ethyl
acetate in hexanes over 20 CV) to yield 110-E as an off-white solid
(180 mg, 45% yield over two steps).
[0778] LCMS: m/z [M+1].sup.+=410.3; R.sub.T=1.94 min (99.0%
purity).
[0779] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM
ammonium formate; Eluent B: Acetonitrile.
[0780] Step Six.
4-hydroxy-2-(methylthio)-6-oxo-N-(5-(trifluoromethyl)-2-(1-((2-(trimethyl-
silyl) ethoxy) methyl)-1H-1,2,3-triazol-4-yl)
quinolin-6-yl)-1,6-dihydropyrimidine-5-carboxamide (110-F):
6-hydroxy-2-(methylthio) pyrimidin-4(3H)-one (127 mg, 0.8 mmol) was
added to a stirring solution of sodium tert-butoxide (77 mg, 0.8
mmol) dissolved in DMSO (2.0 mL) at rt for 5 min. In a separate
flask, aniline 110-E (164 mg, 0.4 mmol) was dissolved in
1,4-dioxane (2.0 mL), to this solution was added triphosgene (40
mg, 0.132 mmol) in one-portion. The suspension was stirred
vigorously for 2 min at rt, then iPr.sub.2NEt (0.14 mL) was added.
The suspension was stirred vigorously at rt for 2 min. Freshly
prepared solution of sodium
6-hydroxy-2-(methylthio)-3-(3,4,5-trimethoxybenzyl)
pyrimidin-4(3H)-olate in DMSO was added to the suspension in
one-portion. The reaction was stirred at rt for 30 min, until
complete consumption of starting material observed via LCMS.
Reaction mixture was then cooled at rt; concentrated under vacuum.
Purification was done by reverse phase column chromatography using
10 mM ammonium bicarbonate buffer (PH=10.0) and acetonitrile. Pure
fraction was lyophilized to get desired product 110-F (170 mg,
70%).
[0781] LCMS: m/z [M+1].sup.+=594.3; R.sub.T=2.01 min (98%
purity).
[0782] HPLC conditions: Column: XBridge C18, 3.5 m, 4.6.times.30
mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8 min; 100% B for
1 min; 3 mL/min. Eluent A: Milli-Q H2O+10 mM ammonium formate;
Eluent B: Acetonitrile.
[0783] Step Seven. N-(2-(1H-1,2,3-triazol-4-yl)-5-(trifluoromethyl)
quinolin-6-yl)-4-hydroxy-2-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-car-
boxamide (110): A solution of crude 110-F (170 mg, 0.29 mmol) in
dichloromethane (1.5 mL) was added trifluoroacetic acid (0.5 mL).
The resulting reaction mixture was stirred at rt for 6 h. Reaction
mixture was concentrated to get crude product which was
co-evaporated 2-3 times with methanol. The resulting reside was
re-dissolved in MeOH (2.0 ml) and ethylenediamine (1.0 ml, 14.5
mmol, 50 eq) and reaction mixture was stirred for another 12 h it
was then concentrated under vacuum to get crude product. Finally,
crude product was purified via reverse-phase chromatography (C18,
gradient eluent from 0 to 100% acetonitrile in water with an
ammonium formate buffer 10 mM over 22 CV) to yield the product 110
as an off-white solid (80 mg, 60% yield), after lyophilisation.
[0784] .sup.1H NMR (500 MHz, DMSO+TFA) .delta. 12.00 (s, 1H),
8.70-8.65 (m, 1H), 8.64 (s, 1H), 8.35 (d, J=9.2 Hz, 1H), 8.32 (d,
J=9.3 Hz, 1H), 8.20 (d, J=9.3 Hz, 1H), 2.56 (s, 3H).
[0785] LCMS: m/z [M+1].sup.+=464.1; R.sub.T=1.11 min (99.0%
purity).
[0786] HPLC conditions: Column: XBridge C18, 3.5 .mu.m,
4.6.times.30 mm; Gradient: 5% B for 0.2 min, 5% to 100% B in 1.8
min; 100% B for 1 min; 3 mL/min. Eluent A: Milli-Q H.sub.2O+10 mM
ammonium bicarbonate pH: 10.0; Eluent B: Acetonitrile.
Example 43: Bioactivity Assays
[0787] The biological activities of compounds having structures
represented by Formulae (I) and Formula (II) were evaluated in two
assays: xanthine oxidase activity and URAT1 activity.
[0788] Xanthine oxidase inhibition was determined using a standard
fluorescence-based assay for xanthine oxidase activity (McHale A,
Grimes H, Coughlan M P: Int J Biochem. 10:317-9, 1979) with minor
variations. The procedure was internally standardized using
allopurinol and DPI as controls for all experiments after
determination of their optimal inhibitory concentrations.
Experiments on test compounds were performed in triplicate in
multi-well plates using 10 concentrations of each compound that
ranged over a 3-fold dilution.
[0789] URAT1 (SLC22A12) activity was evaluated in a cellular uptake
assay using a 96-well plate with stably transfected URAT-1/CHO
cells. .sup.3H-orotate was used as the test transport agent, which
was measured in a liquid scintillation counter, using benzbromarone
as a positive control, and DMSO and non-transfected CHO cells as
negative controls (Solvo Biotechnology, Boston, Mass.). Generally
determined over 7 concentrations (range, 0.01 to 150 .mu.M), a
semi-log plot (percent relative transport of oratate vs. time) was
generated to determine the concentration at which 50% inhibition
was observed (i.e., the IC50).
[0790] The results of these assays for the exemplary compounds
according to Formula (I) and Formula (II) are shown in Table 1:
TABLE-US-00001 TABLE 1 URAT1 Xanthine Oxidase Compound IC50 (.mu.M)
IC50 (.mu.M) Formula (II.sub.ff) 2.92 Formula (II.sub.i) 1.077
<0.02 Formula (II.sub.jj) >9.4 0.02 Formula (II.sub.gg) 1.73
Formula (II.sub.hh) 211.45 Formula (II.sub.ii) >40 0.377 Formula
(II.sub.j) >300 Formula (II.sub.k) 10.2 0.28 Formula (II.sub.l)
2.43 <0.02 Formula (II.sub.m) 8.64 Formula (II.sub.n) 3.57
Formula (II.sub.o) 1.14 Formula (II.sub.p) >50 0.20 Formula
(II.sub.q) 14.16 0.04 Formula (II.sub.r) 1.85 0.21 Formula
(II.sub.s) >3.5 0.12 Formula (II.sub.ll) 1.02 Formula
(II.sub.mm) 2.86 Formula (II.sub.t) 19.88 0.18 Formula (II.sub.u)
4.30 Formula (II.sub.v) 18.53 0.10 Formula (II.sub.w) >30 0.66
Formula (II.sub.x) .gtoreq.30 Formula (II.sub.y) 0.33 Formula
(II.sub.z) 0.015 Formula (I.sub.o) 5.297 Formula (II.sub.aa) >90
0.103 Formula (II.sub.bb) >90 0.206 Formula (II.sub.cc) 0.519
Formula (II.sub.dd) 0.064 Formula (II.sub.kk) 0.315 Formula
(II.sub.ee) 0.029 Allopurinol >300.sup..dagger. 2.0 to 5.0
Lesinurad 18.61* >300.sup..dagger. 52.5 .+-. 5.9.sup..dagger.*
.sup..dagger.Presentation estimate; Proc. EULAR Abstract #THU0357,
2008 *URAT1 assay as described herein
[0791] The results of bioactivity assays for additional exemplary
compounds according to Formula (II) are shown in Table 2:
TABLE-US-00002 TABLE 2 URAT1 Xanthine Oxidase Compound IC50 (.mu.M)
IC50 (.mu.M) Formula (II.sub.2) -- 0.53 Formula (II.sub.3) 4.9 0.06
Formula (II.sub.4) 2.3 0.06 Formula (II.sub.5) 7.5 0.04 Formula
(II.sub.6) -- >10.0 Formula (II.sub.7) >100 0.07 Formula
(II.sub.8) 19.9 0.06 Formula (II.sub.9) -- 0.52 Formula (II.sub.10)
>100 0.19 Formula (II.sub.11) -- 0.22 Allopurinol
>300.sup..dagger. 2.0 to 5.0 Lesinurad 18.61*
>300.sup..dagger. 52.5 .+-. 5.9.sup..dagger.*
.sup..dagger.Presentation estimate; Proc. EULAR Abstract #THU0357,
2008 *URAT1 assay as described herein
[0792] Among the compounds listed in Table 1, Formulae (II.sub.i),
(II.sub.jj), (II.sub.ii), (II.sub.k), (II.sub.l), (II.sub.p),
(II.sub.q), (II.sub.r), (II.sub.s), and (II.sub.t) are particularly
potent inhibitors of xanthine oxidase compared to allopurinol.
Several of the compounds also effectively inhibit URAT1 (e.g.,
Formulae (II.sub.i), (II.sub.k), (II.sub.l), (II.sub.q),
(II.sub.r), (II.sub.s)), although not all of them were tested.
These are the most promising bifunctional inhibitors. Two
representative examples of a particularly effective bifunctional
inhibitors are Formulae (II.sub.r) and (II.sub.s).
[0793] Of the compounds listed in Table 2, all are particularly
potent inhibitors of xanthine oxidase compared to allopurinol, with
the exception of Formula (II.sub.6). Formula (II.sub.3), Formula
(II.sub.4), and Formula (II.sub.5) were also found to be
particularly potent inhibitors of URAT1 compared to lesinurad.
[0794] While many compounds were potent inhibitors, the extent of
inhibition of each enzyme/channel was different. Such variability
allows the intelligent selection of a pharmaceutically acceptable
product that exhibits greater or lesser inhibition of one or the
other enzyme target. For example, greater inhibition of XO might be
deemed preferable for a patient whose primary metabolic defect was
over-production of uric acid. Conversely, greater inhibition of
URAT1 might be deemed preferable for a patient whose primary
metabolic defect was under-excretion of uric acid. However, it
should be noted that almost all patients with hyperuricemia or a
disorder associated with excess uric acid will benefit from
reduction in serum uric acid, and bifunctional compounds can be
expected to exert a beneficial effect in such patients. The
practitioner, guided by the present disclosure, will be able to
select particular compounds as appropriate for a specific use based
on the level of skill in the art.
[0795] By way of comparison, allopurinol has an IC50 for XO ranging
from about 2.0 to about 5.0 .mu.M and an IC50 for URAT1 of >300
.mu.M. Lesinurad has an IC50 for XO of >300 M and an IC50 for
URAT1 ranging from 18 to 53 .mu.M. Thus, neither of these compounds
is considered bifunctional, since both are selective inhibitors of
only one enzyme that affects either production or excretion of uric
acid. In contrast, certain of the compounds described herein are
not only bifunctional, several are substantially more potent
inhibitors of either or both XO and URAT1.
[0796] While in many clinical situations it is desirable to treat
hyperuricemia with a drug that is highly potent against both XO and
URAT1, it is also contemplated that selection of a particular
compound of the invention for treatment of hyperuricemia or a
disorder associated with excess uric acid may be based on the
phenotype of the patient being treated (i.e., the relative
contributions of over-production of uric acid and under-excretion
of uric acid to the patient's specific disease). Where
over-production of uric acid predominates, use of compounds
according to the invention that are substantially more potent
against XO than URAT1 may be appropriate. Where under-excretion of
uric acid predominates, use of compounds according to the invention
that are substantially more potent against URAT1 than XO may be
appropriate. Although the genetics of these two pathways are not
completely understood, chemical testing to determine the extent to
which each contributes to the hyperuricemia of a particular patient
has been published, and may be useful to clarify the patient's
disease phenotype for selection of an appropriate drug that
balances these respective activities, as can be appropriately
determined by those skilled in the art.
[0797] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It will be apparent to those
skilled in the art that various modifications and variations can be
made to the method and apparatus of the present invention without
departing from the spirit and scope of the invention. Thus, it is
intended that the present invention include modifications and
variations that are within the scope of the appended claims and
their equivalents.
* * * * *