Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 00/61566 PCT/US00/09965
Process for Preparing MKC-442
This application claims priority to U.S. provisional application no.
60/128,925, filed
on April 13, 1999.
This application is in the area of synthetic pharmaceutical chemistry, and in
particular,
includes an efficient method for preparing 6-benzyl-1-(ethoxymethyl)-5-
isopropyluracil,
which is also known as MKC-442.
In 1983, the etiological cause of Acquired Immune Deficiency Syndrome (AIDS)
was
determined to be the human immunodeficiency virus (HIV). It is currently
believed that a
key factor in whether a person infected with HIV develops AIDS is the amount
of HIV in the
body at a given time (i.e., "the viral load"). Researchers have focused on
halting HIV
replication and reducing viral load by blocking one or both of two key enzymes
required for
viral replication, reverse transcriptase and protease.
The first enzyme, reverse transcriptase, is active early in the replication
cycle of HIV
and allows the virus, which is made of RNA, to produce DNA necessary for
continued
replication. This enzyme can be inhibited by two general classes of drugs
defined by their
structure as well as their mechanism of action. The first general class,
nucleoside analogue
reverse transcriptase inhibitors such as 3'-azido-3'-deoxythymidine (AZT),
2',3'-
dideoxyinosine (ddI), 2',3'-dideoxycytidine (ddC), and (-)-cis-2-hydroxymethyl-
5-(cytosin-1-
yl)-1,3-oxathiolane (3TC), bear a strong chemical resemblance to the natural
building blocks
(nucleosides) of DNA and interfere with the function of the enzyme by
displacing the natural
nucleosides used by the enzyme. The second general class, non-nucleoside
reverse
transcriptase inhibitors, such as MKC-442 and nevirapine, is composed of an
extremely
diverse group of chemicals that act by binding to the enzyme and modifying it
so that it
functions less efficiently.
Since 1985, a number of nucleoside reverse transcriptase inhibitors, including
AZT,
ddI, ddC, 3TC, 2',3'-dideoxy-2',3'-didehydrothymidine (D4T), and cis-2-
hydroxymethyl-S-
(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC), have been proven to be effective
against HIV.
After cellular phosphorylation to the 5'-triphosphate by cellular kinases,
these synthetic
nucleosides are also incorporated into a growing strand of viral DNA, causing
chain
termination due to the absence of the 3'-hydroxyl group.
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MKC-442 functions as a non-nucleoside reverse transcriptase inhibitor. MKC-442
is
considered an allosteric inhibitor because it appears to exert its activity by
binding to an
"allosteric position", i.e., one other than the binding site, of the enzyme.
Preclinical tests
suggest that MKC-442 may possess characteristics that address several of the
therapeutic
challenges of HIV. When tested in cell culture assay systems against wild-type
(drug-
sensitive) and several mutant strains of HIV known to be resistant to
established non-
nucleoside reverse transcriptase inhibitors, MKC-442 retained much of its
ability to inhibit
HIV replication. In these studies, MKC-442 displayed greater potency than
nevirapine
against wild-type and mutant strains of HIV. Preclinical studies of MKC-442 in
two drug
combinations with AZT or with ddI and in three drug combinations with AZT and
saquinavir
have demonstrated synergistic inhibition of HIV replication.
Studies in animals suggest a favorable safety and pharmacokinetic profile for
MKC-
442. Animal pharmacokinetic analyses showed good oral bioavailability and
excellent
penetration into the central nervous system, a significant site of HIV
replication that is poorly
penetrated by many currently marketed anti-HIV drugs. In rats, for example,
the
concentration of MKC-442 in the brain was 100% of that seen in the plasma.
A Phase I study evaluated the pharmacokinetics and tolerance of single
escalating
doses of MKC-442 in HIV-infected volunteers. The compound was generally well
tolerated,
with only a few participants experiencing minor adverse effects at the higher
dose levels. In
the groups receiving higher doses, concentrations of the drug in the plasma
reached levels
much higher than the levels required to suppress 90% of the virus in culture.
Preliminary data from a Phase I/II double-blind, placebo controlled trial
designed to
evaluate the safety and e~cacy of repeated multiple oral doses of MKC-442 in
HIV-infected
patients has now also been evaluated. A total of 49 patients were treated with
MKC-442 for
up to two months. Doses ranging from 100 mg to 1000 mg twice a day were given
to groups
of six to eight patients at each dosage level. At the highest doses tested
(750 mg and 1000
mg twice a day), the viral load was reduced by an average of 96% in all
patients after one
week. This reduction was mostly sustained at two weeks whereafter it was
followed by a
gradual increase in viral load toward baseline levels. A single point mutation
at position 13
of the reverse transcriptase that may be associated with resistance was found
in the virus
obtained from some patients. In over 308 patient-weeks of drug exposure, MKC-
442 was
well tolerated.
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MKC-442 is described, for example, in U.S. Patent No. 5,461,060, which is
incorporated herein by reference in its entirety.
U.S. Patent No. 5,604,209, issued on February 18, 1997 to Ubasawa et al., and
assigned to Mitsubishi Chemical Corporation, discloses that certain 6-benzyl-1-
ethoxymethyl-5-substituted uracil derivatives, including MKC-442, and certain
2',3'-
dideoxyribonucleosides, including 2',3'-dideoxyinosine (ddI), 3'-azido-3'-
deoxythymidine
(AZT), AZT triphosphate, and 2',3'-dideoxycytidine (ddC), exhibit a
synergistic effect
against HIV.
Because of the pharmaceutical activity of MKC-442 alone and in combination
with
other antiviral agents, there is growing interest in efficient methods for its
manufacture.
Since MKC-442 is a 5,6-substituted uracil derivative that has an alkoxyalkyl
group in
the 1 position (see Figure 1 ), methods for its synthesis must include steps
to add these three
1,5,6-substituents. Typical synthetic routes to MKC-442 have included
condensation of the
alkoxyalkyl moiety with a 5-substituted thiouracil, followed by
desulfurization of the
thiouracil, lithiation at the 6-position, reaction with benzaldehyde,
subsequent acetylation and
eventual reduction of the hydroxy group using hydrogenolysis to yield MKC-442
.
Rosowsky, et al., J. Med Chem., 1981, 24, 1177; Tanaka, et al., J. Med Chem.
1992, 35,
4713. See also Baba, et al., Nucleosides and Nucleotides, 14(3-5), 575-583
(1995). Baba et
al. noted that it is well known that glycosidation of 6-substituted uracils
almost invariably
results in the formation of N-3-glycosylated products due to steric hindrance
by the
substituents, and for that reason the 6-substituent should be introduced after
the acyclic
portion has been introduced.
Danel, et al. reported a synthesis of 5,6-disubstituted acyluridine
derivatives that
includes inserting the ethoxymethyl moiety of the molecule at the N-1 position
in the last step
ofthe synthesis (J. Med. Chem., 1996, 39, 2427-2431; Synthesis, August 1995,
934-936).
Danel, et al. first reacts an arylacetonitrile with a 2-bromoester in a
Reformatsky reaction to
form an ethyl-2-alkyl-4-aryl-3-oxobutyrate (see Scheme 1 ), i.e., a ~i-keto
ester. The (3-keto
ester is then condensed with thiourea in the presence of sodium ethoxide to
form a 2-
thiouracil which is refluxed with chloroacetic acid in aqueous acetic acid
overnight to give 6-
benzyl-5-ethyluracil. Silylation of the uracil is required prior to
condensation with an
alkylating agent of choice. While reasonable for small scale, the Danel method
has
substantial drawbacks for the manufacturing scale preparation of MKC-442, as
it requires a
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WO 00/61566 PCT/US00/09965
large excess of Zn and the use of sodium metal to generate the base in situ,
which is a serious
safety concern. Also, a 20-fold excess of the ethoxide was specified in the
article, which is
not reasonable for industrial scale-up. Further, the Danel method required the
use of fifteen
equivalents of thiourea, which prevents a clean crystallization of the
thiouracil derivative.
Further, the thiouracil intermediate was not at all soluble in the
chloroacetic acid solution
used, and when the material was heated to reflux, a sticky mass formed and
coated the
agitator and flask walls. Finally, when the desulfurization reaction was
homogenized using
either alcohols or tetrahydrofuran, the thiouracil was converted to an
undetermined impurity.
On scale up of the Danel method, the yield of MKC-442 dropped dramatically,
providing
only 25-50% product having only 90% purity.
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WO 00/61566 PCT/US00/09965
Scheme 1
I. CH3CHZCHBrCO2Et, Zn,
CHZCN THF, reflux, 2.5 h
2. HCI (aq), OS h
90%
CHzCOCHEtCOZEt
1. MeCN/MgCl2 /Et3N
Et CpzEt 10-15° C,OS h
2. PhCHZCOCI/Et3N I
0° C, OSh; 20-25°C,12h
COZK
85 %
1. NaOEt, HZNCSNH2,
EtOH, reflux, 8h O
2. CICHZCOZH(aq)
reflux, 24 h HN Et
68 %
O NH
2
1. HMDS/(IVH~S04, reflux, 2 h O
2. MeCN, TMS ttiflate, RZOCHR'ORZ
O HN Et
or Cn~~R' -45°C, 1-
89-95 % O N
2 ~ R'
R O 3a~
R' Rz
3a H Et
3b Me Me
3c Me Et
3d H CH2CH20H
3e Me CH2CH20H
5
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WO 00/61566 PCT/US00/09965
Orr and Musso in 1996 reported the synthesis of a 5-substituted uracil
derivative (not
a 5,6-disubstituted uracil) by the reaction of ethyl phenylpropanoate with
thiourea. They
reported that, while the conventional literature precedent involves the use of
sodium metal as
the base in the preparation of the 2-thiouracil from thiourea, it can cause
problems in the
synthesis and result in a low yield. Synthetic Communications, 26(1), 179-189
(1996). Orr et
al. compared the preparations of 2-thiouracil through the reactions of ethyl
phenyl propanoate
with (i) sodium, ethylfortnate, diethyl ether, and then thiourea and ethanol
with reflux; (ii)
lithium diisopropylamide, tetrahydrofuran, ethyl formate, low temperature,
then thiourea and
ethanol in reflux; (iii) potassium tert-butoxide in tetrahydrofuran, ethyl
formate, diethyl ether
at ambient temperature, then thiourea and 2-propanol at reflux. Orr et al.
reported that the
use of lithium diisopropylamide or potassium tert-butoxide instead of sodium
metal in the
preparation of 5-benzyl-2-thiouracil improves the yield of reaction, and that
substituting 2-
propanol for ethanol as the solvent for condensing the enolate with thiourea
results in
improved yields. Using ethyl orthoformate, the Orr reaction cannot be used to
provide a 5,6-
disubstituted uracil, but at best, a 5-substituted uracil or a 5,5-
dihydrouracil (see Scheme 2).
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WO 00/61566 PCT/US00/09965
Scheme 2
R'OzCCH=C ~ RZ ii EtO,CCH2CH, ~ R2
1. R'=H 3
i
2. R'=CHZCH3
iii
RZ=OH
O ~ Ra ~ Rs
CHp iv, v or vi EtO2CCHZCH2
HN
/ \NH 4a: R3~CHg
4b: R3~CHZCHzCHg
4c: R3~(CHZ)3CH3
4d: R3~CH2-c-C3H5
4e ~t3 ~CHzPh
a Reagents: (i) EtOH, Et20, HC1, reflux; (ii) EtOH, HZ, Pt02; (iii)
MeZCO, R3Br, K2C03; (iv) Na, HCOZEt, Et20 then thiourea, EtOH,
reflux; (v) LDA, THF, HCOZEt, -60° - -78° then thiourea, EtOH,
reflux; (vi) tent-BuOK in THF, HCOZEt, Et20, ambient temperature;
then thiourea, 2-PrOH, reflux.
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WO 00/61566 PCT/US00/09965
Baba et al. reported a method to produce MKC-442 by reacting a silylated 5-
isopropyl-2-thiouracil with carcinogenic CH3CHZOCHZC1 (KI/CHZC12) to yield the
N-1-
ethoxymethyl product (Nucleosides and Nucleotides, 14(3-5), 575-583 (1995)).
Lithiation
with lithium diisopropylamide (LDA) resulted in a 6-substituted product.
Oxidative
hydrolysis of the 6-substituted product followed by conventional
hydrogenolysis led to
MKC-442.
In light of the fact that MKC-442 is useful in the treatment of HIV-infected
patients,
there is a need to develop cost effective and scalable methods for
manufacturing pure MKC-
442 in large quantity, high purity, and high yield. It is therefore an object
of the present
invention to provide an improved synthetic process for preparing 6-benzyl-1-
(ethoxymethyl)-
5-isopropyluracil (MKC-442) in high yield and greater purity.
Summary of the Invention
It has been discovered that a 5,6-disubstituted uracil can be produced in good
yield by
the reaction of an appropriate ~-keto ester with thiourea in the presence of
potassium t-
butoxide. When the ~-keto ester is ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate,
the resulting
product is 6-benzyl-1-(ethoxymethyl)-S-isopropyluracil, also known as MKC-442.
Therefore, in one embodiment, a method for producing a 5,6-disubstituted
uracil is
provided that includes the use of two bases simultaneously to effectively
synthesize the 5,6-
disubstituted uracil in high yield from a ~-keto ester. Preferred bases
include potassium
carbonate and potassium t-butoxide as the co-bases in acetonitrile. This
embodiment is
depicted in Scheme 3.
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WO 00/61566 PCT/US00/09965
Scheme 3
t-BuOK
KZCO3 O
CH3CN H,N
w
---~ ~ ~ ~ ,
p 82 C S H
/ O
10% CIACOH (aq.) I ACOH
c.105°C I 8 h
1. HMDS I (NH4~S04 (cat.) O
ii-N 125°C / ~ h H~N \
/ .
O N
H
2. /~O~O~
Sulfuric acid (0.5X) I CH3CN
C.85°C I 2-5 h
3. Reuystallization: EthanoIIVllater
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WO 00/61566 PCT/US00/09965
As a nonlimiting example for the synthesis of MKC-442, an isopropyl (3-keto
ester
such as ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate can be synthesized using any
known
method, including from benzyl cyanide and ethyl-2-bromo-3,3-dimethyl-
propionate with
activated zinc in a Reformatsky reaction. The (3-keto ester is then reacted
with a two-base
system, including but not limited to potassium carbonate and potassium t-
butoxide, in a
solvent, for example acetonitrile.
The process provides several advantages over the prior art. First, the
reaction is a
single-step process that proceeds to form a product in greater than 80% yield
. Additionally,
the two-base system results in products of greater purity. For example, the
use of potassium
carbonate and potassium t-butoxide in acetonitrile results in a product having
a purity of 90-
95%.
In synthesizing the substituted thiouracil ring, bases of high pKb alone
destroy the
starting (3-keto ester and have higher levels of impurities. As a result, use
of bases with high
pKb alone leads to products of lower yield. Bases of lower pKbs tend to have
longer reaction
1 S times and tend to contain residual starting material. It is therefore
beneficial to use a two-
base system to counteract these tendencies.
Bases useful in the two-base system of the present invention include, but are
not
limited to potassium carbonate, potassium t-butoxide, sodium carbonate, di-
isopropyl amine,
triethyl amine, 2,6-dimethyl pyridine, sodium acetate, potassium acetate,
ammonia,
potassium ethoxide, and potassium cyanide. The bases are used in combination
with a
solvent.
Solvents useful in the two-base system of the present invention include, but
are not
limited to acetonitrile, butyronitrile, isobutyronitrile, chloroacetonitrile
and other alkyl nitrite
solvents, isopropyl alcohol, methanol, ethanol, propanol, butanol, and other
alcohol solvents.
In another embodiment of the present invention, a method for producing a 5,6-
disubstituted uracil is provided that includes reacting a ~i-keto ester with
thiourea in the
presence of potassium t-butoxide, preferably in iso-propyl alcohol. This
embodiment of this
reaction is depicted in Scheme 4.
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WO 00/61566 PCT/US00/09965
Scheme 4
S
H2N~NH2
tBuOK I iPA O
C.85°C / 3 h H,
\ O~/
II II S N
O O
H
10% CIAcOH (aq.) I AcOH
c.105°C I 8 h
1. HMDS I (NH4~S04 (cat.) O
H'N 125°C / 4 h H'N
w \
O' O' _N
i
2. /~O~O~\ H
Sulfuric acid (0.5X) I CH3CN
c.85°C / 2-5 h
3. Recrystallization: EthanoI/Vllater
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As a nonlimiting example for the synthesis of MKC-442, an isopropyl (3-
ketoester
such as ethyl-2-isopropyl-3-oxo-4-phenyl-butyrate can be synthesized using any
known
method, including from benzyl cyanide and ethyl-2-bromo-3,3-dimethyl-
propionate with
activated zinc in a Reformatsky reaction, and then the ethyl-2-isopropyl-3-oxo-
4-phenyl-
butyrate is condensed with thiourea in the presence of potassium t-butoxide in
process step
(A) of the present invention to form a benzyl isopropyl thiouracil, which is
desulfurized in
process step (B), and then the benzyl isopropyl uracil is alkoxyalkylated in
process step (C) to
form MKC-442. The improved process of the present invention provides several
unexpected
advantages over the prior art while producing MKC-442 in greater yield and
purity.
The process provides several advantages over the prior art. The substitution
of t-
BuOK for NaOEt in the cyclocondensation reaction of step (A) affords a
dramatically purer
product. The increased yields gained with t-BuOK have precedent in the
literature, however
the increased purity of the product obtained is heretofore unknown and
unexpected.
Additionally, the substitution of t-BuOK for NaOEt afforded a tractable, crude
thiouracil,
making the workup both manageable and scalable. It is also surprising that
this reaction of
step (A) occurs with a sub-stoichiometric amount of t-BuOK. Finally, the use
of diethoxy
methane in the presence of an acid catalyst such as sulfuric acid of step (C),
rather than the
very toxic TMS triflate or chloromethyl ether used in the prior art, offers a
cost advantage as
well as a safety benefit.
The slow and controlled evolution of hydrogen sulfide in the desulfurization
reaction
of step (B) is also unexpected. A rapid and uncontrollable gas evolution would
prevent scale
up and commercialization of the process.
The alkylation reaction of step (C) has two unexpected aspects associated with
it.
First, it is surprising that this reaction proceeds so efficiently in the
presence of sub-
stoichiometric amounts of sulfuric acid. Secondly, it is extremely convenient
that the
removal of HMDS is not necessary. The alkylation proceeds in an efficient
fashion with sub-
stoichiometric amounts of sulfuric acid, even in the presence of HMDS.
Detailed Description of the Invention
An improved process for preparing 5,6-disubstituted uracils is provided. This
improved process offers several advantages over the prior art. Most
significantly, the process
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of the present invention produces MKC-442 in higher yields, without
sacrificing purity of the
product. In fact, the process can yield a product having 95% or greater
purity.
The invention includes at least the following embodiments:
A first process for preparing a 5,6-disubstituted uracil, preferably MKC-442,
via the
reaction of a (3-keto ester with thiourea in a two-base system. The process
includes the use of
two bases simultaneously to effectively synthesize the 5,6-disubstituted
uracil. The process
includes reacting a (3-keto ester with thiourea in the presence of a two-base
system and a
solvent at temperatures ranging from 80-85 ° C.
The one-step process of this first process of the present invention comprises
reacting a
2,4-disubstituted-3-oxo-butyrate, preferably ethyl-2-isopropyl-3-oxo-4-
phenylbutyrate, with
thiourea in a two-base system and a solvent. Preferred bases for the two-base
system include,
but are not limited to, potassium t-butoxide and potassium carbonate. A
preferred solvent for
the reaction includes, but is not limited to, acetonitrile. The reaction
proceeds at a
temperature from 80 °C to 85 °C. A preferred reaction
temperature is about 85 °C.
As used herein, the term "two-base system" refers to the use of two bases
simultaneously in the presence of a solvent. Nonlimiting examples of bases
included in the
two-base system are potassium carbonate, potassium t-butoxide, sodium
carbonate, di-
isopropyl amine, triethyl amine, 2,6-dimethyl pyridine, sodium acetate,
potassium acetate,
ammonia, potassium ethoxide, and potassium cyanide. The bases are used in
combination
with a solvent.
Solvents useful in the two-base system of the present invention include, but
are not
limited to acetonitrile, butyronitrile, isobutyronitrile, chloroacetonitrile
and other alkyl nitrite
solvents, isopropyl alcohol, methanol, ethanol, propanol, butanol, and other
alcohol solvents.
A second process for preparing a 5,6-disubstituted uracil, preferably MKC-442,
that
includes:
(i) a step (A) that includes the preparation of a 5,6-disubstituted thiouracil
via the
reaction of a 2,4-disubstituted-3-oxo-butyrate ester with thiourea in the
presence of potassium
t-butoxide in an organic solvent, wherein the intermediate is a 5,6-
disubstituted-2-thio-uracil;
(ii) the process of step (A) wherein the solvent is an alcohol, preferably
isopropyl
alcohol;
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(iii) the process of step (A) wherein molar ratio of thiourea to 2,4-
disubstituted-3-
keto-butyrate is not greater than approximately 10% excess, and more
preferably no more
than 5% excess;
(iv) the process of step (A) wherein the reaction is quenched with water and
acetic
acid;
(v) a step (B) that includes the process of step (A) further including
desulfurizing the
5,6-disubstituted-2-thiouracil to a 5,6-disubstituted-uracil, preferably using
10% chloroacetic
acid (aq) in acetic acid;
(vi) the process of step (B) further including isolating the thiouracil prior
to
desulfurization;
(vii) the process of step (B) wherein the acetic acid is between approximately
25 and
40% of the total solvent volume used, and preferably, approximately 35%;
(viii) the process of step (B) wherein the reaction is heated to between
85° C and 105°
C to form a solution;
(ix) a step (C) that includes the silylation of the 5,6-disubstituted uracil
of step (v)
with any suitable reagent according to known methods;
(x) the process of step (C) wherein the reagent is a silylating agent such as
hexamethyldisilizane;
(xi) the process of step (x) wherein the hexamethyldisilizane is reacted in
the presence
of a catalytic amount of ammonium sulfate;
(xii) reacting the product of step C with an alkylating or alkoxyalkylating
agent, for
example, diethoxymethane;
(xiii) the process of step (xii) wherein the reaction is accomplished in
sulfuric acid or
methanesulfonic acid;
(xiv) the process of step (xiii), wherein sub-stoichiometric equivalent
amounts of
sulfuric acid are used, and preferably, between 0.25 and 0.5 equivalent
amounts; and
(xv) the process of step (xiv), wherein the reaction is run in refluxing
acetonitrile (or
other alkyl nitrites), preferably for 2-5 hours.
The two-stage process of the second process of the present invention comprises
steps
(A) and (B) in Stage I. Step (A) comprises cyclizing isopropyl-(3-ketoester
with thiourea and
potassium tert-butoxide in isopropyl alcohol at reflux to produce benzyl
isopropyl thiouracil.
The benzyl thiouracil is then desulfurized under reflux in step (B) to produce
benzyl
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isopropyl uracil (BIU) using chloroacetic solution, 10% CIAcOH (aq), AcOH. The
reaction
work up involves a quench with water and acetic acid, then filtration of a
crystalline solid in
70-80% yield.
The Stage I process differs from Danel's process (J. Med. Chem. 1996, 39,2427-
2431 ) by eliminating the use of sodium metal, used by Danel to generate the
base in situ.
Elimination of the sodium resolved a serious safety concerns during scale up.
Danel also
specified a 20-fold excess of the ethoxide while only a 5%-20% excess is
required by the
process of the present invention. Additionally, the Danel process produces a
thiouracil
intermediate which, when heated to reflux, forms a sticky mass which coats the
agitator and
flask walls. In contrast, using discretely isolated thiouracil produced by the
process of step
(A) of the present invention, conversion and purity are increased. The
addition of acetic acid
to the reaction in step (B) (approximately 35% of the total solvent volume)
accomplished two
tasks: it allowed for the formation of a solution when heated to 95°C;
it served as a
recrystallization cosolvent when the reaction was scaled. The process of the
present
invention eliminates the sticky mass formed by the Danel process, making the
improved
process scalable to production sized equipment. In addition, the BIU was
recovered in
approximately 90% yield of >98% pure material.
In Stage II of the process of the present invention, BIU is silylated with
1,1,1,3,3,3
hexamethyldisilazane (HMDS) in the presence of a catalytic amount of ammonium
sulfate,
followed by alkylation with diethoxymethane in acetonitrile containing
sulfuric acid. This
process is referred to herein as step (C). Optionally, one can also use
methanesulfonic acid as
the acid catalyst. The conditions were optimized to employ sub-stoichiometric
amounts (0.25
- 0.5 equivalents) of concentrated sulfuric acid at reflux in acetonitrile for
2-5 hours in order
to effect alkylation. Typical reaction profiles showed MKC-442 at 90-95%
yields possessing
<3% BIU as an impurity.
The two-stage process of the present invention provides several improvements
over
the prior art processes. First, the substitution of t-BuOK for NaOEt in the
cyclocondensation
reaction of step (A) affords a dramatically purer product. The increased
yields gained with t-
BuOK has literature precedence, but in this case, the increased purity of the
product obtained
was unexpected. Moreover, the substitution of t-BuOK for the NaOEt
unexpectedly afforded
a tractable, crude thiouracil, making the workup both manageable and scalable.
With the
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NaOEt, an intractable, sticky mass was obtained, making scale-up difficult.
Also, it is
surprising that this reaction occurs with a sub-stoichiometric amount of t-
BuOK.
Another advantage to the process of the present invention is the slow and
controlled
evolution of hydrogen sulfide in the desulfurization reaction of step (B). A
rapid and
difficult-to-control gas evolution would have severely limited the scalability
of the process.
Finally, the alkylation reaction of step (C) has several unexpected aspects
associated
with it. First, it is surprising that this reaction proceeds so efficiently in
the presence of sub-
stoichiometric amounts of sulfuric acid. Second, it is unexpected and quite
convenient that
the removal of the HMDS is unnecessary. The alkylation proceeds in an
efficient fashion,
with sub-stoichiometric amounts of sulfuric acid, even in the presence of
HMDS. Finally, the
use of diethoxymethane as an alkylating agent offers a significant advantage
over the prior art
processes which employ trimethylsilyl triflate or chloromethyl ether. Not only
is the
diethoxymethane less expensive, it is also a significantly less toxic reagent,
making its use
safer than the reagents of the prior art.
Example 1
Stage I: Synthesis of the i-propyl ~i-ketoester
=N
1. Zn~.THF
2. Et-Br-isovaletate
3. 10 ~ H,S04
A 12 liter reaction flask fitted with a mechanical stirrer and twin condensers
was
purged with argon and charged with -100 mesh zinc metal (301 g, 4.6 mol) and
5000 ml
tetrahydrofuran (THF). A catalytic amount of iodine (3.0 g) was added and the
suspension
was stirred until the color faded. The activated zinc suspension was then
treated with benzyl
cyanide (179 g, 1.53 mol) and the mix was heated to 60°C.
The reaction was then treated with ethyl 2-bromoisovalerate (481 g, 2.30 mol).
Approximately 50 g of the bromo ester were added, and the reaction was allowed
to initiate,
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raising the batch temperature to 69°C after five minutes. The remaining
ester was then added
over 35 minutes, so that a minimal reflux was maintained. Following the
addition, the
reaction was left to stir at 65°C for one hour.
An in-process sample was obtained and quenched with 1.8 M sulfuric acid. Gas
chromatography showed 90% isopropyl ~3-ketoester, and 1 % benzyl cyanide. The
batch was
then cooled to 35°C and 4L THF were removed by evaporation under
reduced pressure. The
reaction was then diluted with 3L EtOAc and was carefully quenched with 2.OL
1.8M
HZS04(aq). After one hour, the layers were separated. The organics were then
washed with
1L saturated NaHC03 and 500 ml brine. The aqueous layers were then back
extracted with
250 ml EtOAc each. The organics were combined and concentrated to an oil. 341
g 90%
yield, 90% AUC (area under the curve) by gas chromatography. The material is
typically
used as is, however it can be distilled at 5 mm Hg, 110-120°C to give
240 g 96% (AUC) by
gas chromatography.
Example 2
Synthesis of Isopropyl Benzyl Uracil Using a Two-Base System
1. thiourea,t-BuOK,K,COg/CH3CN
2. 10 %CIAcOH(aq), AcOH
A 1000 mL round bottom flask fitted with a mechanical stirrer and condensor
was
charged with isopropyl - (3 -ketoester (74.Og, 289 mmol), thiourea (45.6g, 599
mmoL), and
acetonitrile (500 mL). The reaction mixture was then charged with potassium t-
butoxide
(37.Og, 330 mmoL), potassium carbonate (61.9g, 448 mmoL), and heated to
82°C. After 3
hours, the mix was cooled to c. 40°C and quenched with water (300 mL).
The volume of the
entire mixture was reduced by half, the pH was adjusted to c. 3 (via HCl), and
the
precipitated solids were collected via filtration. The solids were dried at
45°C for 18 h
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affording 62.7g (81%) of a white solid having a purity of 95% (AUC).
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Synthesis of Isopropyl Benzyl Uracil
HN
O
1. 'Ihiourea,tBOK,IPA
2. 10'7oCIAcOH (aq).AcOH
A 3000 ml 4 neck round bottom flask fitted with a mechanical stirrer and
condenser
was charged with iso-propyl beta ketoester (259 g, 1.04 mol), thiourea (83 g,
1.1 mol) and
1150 ml isopropanol. The reaction was then treated with potassium t-butoxide
(140 g, 1.25
mol) added in 20 g portions, and the mix was heated to 85°C. After 2
hours, the mix was
cooled to 60°C and quenched with water (600 ml). The 2-propanol was
removed by
evaporation under reduced pressure and the remaining solution was cooled to
30°C and
further diluted with 600 ml water and 450 ml acetic acid (pH=4). The solids
were collected
by vacuum filtration and were washed with 2 x 250 ml water. Dried to leave 200
g, >99.5%
AUC by HPLC, 78% yield.
166 g of the thiouracil were resuspended in a solution containing water (450
ml),
glacial acetic acid (200 ml), and chloracetic acid (250 g). The heterogeneous
reaction was
heated at 95°C. After about one hour, a solution formed, and after one
additional hour, a
white solid precipitated. The reaction was heated for six more hours then was
cooled to room
temperature and the product was collected by vacuum filtration. The product, a
white
crystalline solid, was washed with water (2 x 150 ml) and dried in a vacuum
oven (60°C, 5
mm Hg) overnight. The product was isolated in a 70% overall yield from the
ketoester and
had a 99.5% AUC purity by HPLC.
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Stage 2: Alkylation of Isopropyl Benzyl Uracil, Synthesis of MKC-442
U
HN
1. HMDS,(NHQ)oSOp
O
2. DEM.H,S04,ACN
A 1 L 3 neck round bottom flask was charged with 6-benzyl-5-i-propyl uracil
(BIU)
(50 g, 0.20 mol), ammonium sulfate (0.5 g, catalytic) and 1,1,1,3,3,3-
hexamethyldisilazane
(258 g, 1.6 mol). The slurry was heated to reflux (125-130°C) under
argon for four hours.
The resulting solution was concentrated to a thick oil (about 125 ml) under
reduced pressure
(85°C, 30 mm Hg). The oil was resuspended in 350 ml acetonitrile.
Sulfuric acid (4.9 g, 0.05
mol) followed by diethoxymethane (36 g, 0.30 mol) were added at 80°C. A
precipitate
formed after the H2S04 was added, but a solution reformed after about 30
minutes. The
solution was stirred for 5 hours. An additional portion of HZS04 was added
(2.5 g, 0.025
mol) and the mix was heated for an additional 2.5 hours.
After 10 hours, HPLC showed <3% remaining starting material and >90% MKC-442.
The reaction was quenched with 175 ml 0.5 N KOH at 50-60°C (pH=10-11 )
and the
acetonitrile was removed by evaporation under reduced pressure (65-
70°C, 30 m Hg). The
remaining slurry was diluted with 175 ml ethyl acetate and the layers were
split. The
aqueous layer was washed with ethyl acetate (50 ml) and the organic fractions
combined.
The organic fractions were concentrated to a volume of about 75 ml, then were
chased with
75 ml ethanol to a volume of about 75 ml. The crude MKC-442 was then taken up
in an
additional 100 ml EtOH at 85°C and was diluted with 60 ml HZO, cooled
to ambient and
crystallized. After 2 hours, the crude material was filtered and washed with
25 ml 25% EtOH
in water. 52 g, 94% HPLC AUC.
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The material was then taken up in 115 ml EtOH at 85°C, diluted with 50
ml HZO, and
the product was left to cool to ambient temperature and crystallize. The
product was filtered
and washed twice with 25 ml 25% EtOH in water to leave 45 g, 99.8% AUC, with
0.10%
(AUC) BIU.
This invention has been described with reference to its preferred embodiments.
Variations and modifications of the invention, will be obvious to those
skilled in the art from
the foregoing detailed description of the invention. It is intended that all
of these variations
and modifications be included within the scope of this invention.
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