Synthesis and Biological Characterization of Aryl Uracil Inhibitors of Hepatitis C Virus NS5B Polymerase: Discovery of ABT-072, a trans-Stilbene Analog with Good Oral Bioavailability

Article abstract of DOI:10.1021/acs.jmedchem.7b01630

ABT-072 is a non-nucleoside HCV NS5B polymerase inhibitor that was discovered as part of a program to identify new direct-acting antivirals (DAAs) for the treatment of HCV infection. This compound was identified during a medicinal chemistry effort to impr

Full text of DOI:10.1021/acs.jmedchem.7b01630

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Article
Synthesis and Biological Characterization of Aryl Uracil
Inhibitors of Hepatitis C Virus NS5B Polymerase: Discovery of
ABT-072, a Trans-Stilbene Analog with Good Oral Bioavailability
John Randolph, A. Chris Krueger, Pamela Donner, John K. Pratt, Dachun Liu, Christopher E Motter,
Todd W Rockway, Mike D Tufano, Rolf Wagner, Hock B Lim, Jill M Beyer, Rubina Mondal, Neeta S
Panchal, Lynn Colletti, Yaya Liu, Gennadiy Koev, Warren M Kati, Lisa E Hernandez, David W.A. Beno,
Kenton L. Longenecker, Kent D. Stewart, Emily O. Dumas, Akhteruzzaman Molla, and Clarence Maring
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01630 • Publication Date (Web): 17 Jan 2018
Downloaded from http://pubs.acs.org on January 17, 2018
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Synthesis and Biological Characterization of Aryl Uracil Inhibitors of
Hepatitis C Virus NS5B Polymerase: Discovery of ABTꢀ072, a TransꢀStilbene
Analog with Good Oral Bioavailability
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John T. Randolph*, A. Chris Krueger*, Pamela L. Donner, John K. Pratt, Dachun Liu,
Christopher E. Motter, Todd W. Rockway, Michael D. Tufano, Rolf Wagner, Hock B. Lim, Jill
M. Beyer, Rubina Mondal, Neeta S. Panchal, Lynn Colletti, Yaya Liu, Gennadiy Koev, Warren
M. Kati, Lisa E. Hernandez, David W. A. Beno, Kenton L. Longenecker, Kent D. Stewart, Emily
O. Dumas, Akhteruzzaman Molla, Clarence J. Maring
Research and Development, AbbVie Inc., North Chicago, IL 60064, USA
KEYWORDS. Hepatitis C, antiviral, NS5B, ABTꢀ072
abstract
ABTꢀ072 is a nonꢀnucleoside HCV NS5B polymerase inhibitor that was discovered as part of a
program to identify new directꢀacting antivirals (DAAs) for the treatment of HCV infection.
This compound was identified during a medicinal chemistry effort to improve on an original
lead, inhibitor 1, which we described in a previous publication. Replacement of the amide
linkage in 1 with a transꢀolefin resulted in improved compound permeability and solubility, and
provided much better pharmacokinetic properties in preclinical species. Replacement of the
dihydrouracil in 1 with an Nꢀlinked uracil provided better potency in the genotype 1 replicon
assay. Results from Phase 1 clinical studies supported onceꢀdaily oral dosing with ABTꢀ072 in
HCV infected patients. A Phase 2 clinical study that combined ABTꢀ072 with the HCV protease
inhibitor ABTꢀ450 provided a sustained virologic response at 24 weeks post dosing (SVR24) in
10 of 11 patients who received treatment.
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Introduction
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Hepatitis C virus (HCV) infection is a global health problem, with over 185 million
infected individuals at risk of serious and potentially lifeꢀthreatening liver disease.1 The majority
of those currently infected are asymptomatic and thus likely unaware of their dilemma. Yet, 75ꢀ
85% will eventually develop a chronic infection, and 10ꢀ20% will develop cirrhosis, a very
serious condition that greatly increases the odds of developing hepatocellular carcinoma. HCVꢀ
related deaths are on the rise in the US, with close to 20,000 US death certificates listing HCV as
the cause of death in 2014, according to recent CDC mortality statistics. In the US, the total
number of combined deaths from 60 other infectious diseases, including HIV, tuberculosis, and
pneumococcal disease is surpassed by the number of HCV deaths alone, and this is reported to
be only a fraction of total deaths that can be attributed to complications from liver disease caused
by HCV.2
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Recent advances in HCV therapy have been realized through the use of drug
combinations with orally administered directꢀacting antivirals (DAAs).3 Highly effective HCV
combination therapy utilizes two or more DAAs targeting multiple HCV drug target proteins that
play different roles in the viral life cycle.4 DAAs that inhibit HCV NS5B polymerase are among
the most clinically useful agents for HCV treatment.5 These antiviral agents interfere with
replication of the viral RNA genome, and are divided into two inhibitor classes. Nucleoside
inhibitors bind to the catalytic site of NS5B as their triphosphate metabolites and function by
interrupting the genetic message as RNA chain terminators. Nonꢀnucleoside inhibitors (NNIs)
bind to one of four different allosteric sites in NS5B, commonly referred to as NNI sites I, II, III,
and IV. NNI site binders inhibit NS5B by affecting the conformation of the enzyme, thereby
interfering with its catalytic function.6
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Research at AbbVie targeting HCV polymerase was first to discover a series of Nꢀaryl
uracil analogs as a novel structural class of NS5B NNI.7 Our original report described
dihydrouracil analog 1, which is a potent inhibitor of genotypes 1a and 1b polymerase and is
active in genotype 1 (GT1) replicon assays (1a EC50 = 51 nM and 1b EC50 = 19 nM). Replicon
activity is maintained when the assay is conducted in the presence of 40% human plasma (1a
EC50 = 61 nM and 1b EC50 = 22 nM), indicating a negligible effect of plasma protein binding on
cell potency. However, compound 1 has poor pharmacokinetic properties in rat, with high
plasma clearance and poor oral bioavailability (F = 1.4%), providing low plasma concentrations
of drug following oral dosing. Physical properties of this compound that can be associated with
poor oral exposures include low aqueous solubility (< 1.5 ꢀM at pH 7.2) and poor membrane
permeability. The solubility problem is likely related to the compound’s high melting point
(>300 oC). Based on an excellent antiviral activity profile, compound 1 became the primary lead
for our medicinal chemistry team, which launched an effort to design inhibitors with an
improved pharmacokinetic profile.
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Figure 1. A novel inhibitor of HCV NS5B.7
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Chemistry
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Synthesis of dihydrouracil analogs was accomplished as shown in Schemes 1 and 2.
Ipsoꢀnitration of diꢀtertꢀbutyl salicylate 2 resulted in replacement of the alkyl group para to the
hydroxyl substituent. Methylation followed by nitro reduction provided compound 3, which was
Nꢀalkylated with acrylic acid in refluxing toluene to form the propionic acid derivative. Heating
this compound at 120 oC with urea in acetic acid formed the dihydrouracil, which was subjected
to ester hydrolysis to provide 4. Synthesis of amide analogs, such as 1 and 5, proceeded via the
acid chloride, formed by heating 4 in thionyl chloride. Alternatively, the acid chloride
intermediate formed from 4 could be reduced to aldehyde 6 by the action of lithium triꢀtertꢀ
butoxyaluminum hydride. Aldehyde 6 proved to be a versatile intermediate for making multiple
analogs, including benzylamine 7, formed by reductive amination with Nꢀ(4ꢀ
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aminophenyl)methanesulfonamide, and benzylether 8, accessed through the benzyl bromide
intermediate derived from the corresponding benzyl alcohol. Olefination of 6 using
phosphonates 9 and 10 provided stilbene derivatives 11 and 12, which were converted to 13 and
14 by nitro reduction and sulfonylation. Compound 13 was subjected to hydrogenation to
provide 15, or cyclopropanation to give 16.
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Other dihydrouracil derivatives were synthesized as shown in Scheme 2. Selective
nitration para to the hydroxyl group in phenol 17, followed by methylation and nitro reduction,
provided aniline intermediate 18. The dihydrouracil group was assembled by the 2ꢀstep process
of heating with acrylic acid in toluene to affect Nꢀalkylation to the propionic acid derivative,
followed by heating with urea in acetic acid. Demethylation with boron tribromide gave 19, a
step which proved necessary in order to achieve satisfactory yields in the subsequent nitration.
Methylation with TMSꢀdiazomethane, followed by hydrogenolysis to reduce the nitro group,
provided aniline 20. Acylation with 4ꢀnitrobenzoyl chloride, and 2ꢀstep conversion of the nitro
group to methanesulfonamide, gave 21. The nitration product of 17 was iodinated with
iodochloride, followed by methylation and nitro reduction to give 22. This compound was
converted to dihydrouracil 23 and subjected to Sonogashira coupling with Nꢀ(4ꢀ
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ethynylphenyl)methanesulfonamide to provide acetylene analog 24.
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Uracil derivatives were synthesized as shown in Scheme 3. The uracil group was
installed on compound 3 via a 2ꢀstep procedure that involved reaction with (E)ꢀ3ꢀ
methoxyacryloyl isocyanate to give the corresponding Nꢀ(phenylcarbamoyl)acrylamide
derivative, followed by heating with sulfuric acid in aqueous ethanol. Uracil intermediate 25
was converted to 5ꢀfluoro derivative 26. Ester hydrolysis, followed by conversion to the acid
chloride and reduction with lithium triꢀtertꢀbutoxyaluminum hydride gave aldehyde
intermediates 27 and 28. Olefination with phosphonate 9, followed by nitro reduction and
methanesulfonylation provided stilbene analogs 29 and 30. Compound 31 was prepared from 27
using the same sequence of steps, but replacing 9 with diethyl (3ꢀfluoroꢀ4ꢀ
nitrobenzyl)phosphonate.
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Results and Discussion
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Our medicinal chemistry team investigated replacement of the amide linker between the
two phenyl groups in 1. Suitable alternative linkers that eliminated one or both of the two
hydrogenꢀbonding groups were expected to provide compounds with better membrane
permeability. Improvements in aqueous solubility might also be provided, if the resulting
compounds were less crystalline than 1.
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Allꢀcarbon linkers were found to be particularly well suited for our needs, providing good
passive membrane permeability as demonstrated in a PAMPA assay, as well as better aqueous
solubility (Table 1). In addition to these improvements, the stilbene (13) and acetylene (24)
analogs were found to be more potent in GT1 enzyme and replicon inhibitory assays. Compound
13 is a subꢀnanomolar inhibitor of both 1a and 1b polymerase as the result of a > 30ꢀfold
improvement in enzyme activity relative to amideꢀlinked 1. It is also a potent inhibitor in the
GT1b replicon (EC50 = 1.1 nM) due to a nearly 20ꢀfold improvement in cellꢀculture activity.
Activity improvements for acetylene 24 relative to 1 were 10ꢀfold in the enzyme assays and 5ꢀ
fold in the replicon. The triꢀsubstituted olefin 14 was also more active than 1, although it was
substantially less active and less soluble than stilbene analog 13.
Both 13 and 24 have linker groups that impart constraints on compound conformation, as
does the amideꢀlinked analog 1. The more flexible ethylꢀlinked compound 15 was considerably
less active, illustrating that the degree of conformational control provided by 1, 13, and 24 is
important to the activity of these inhibitors. The poor activity of cyclopropylꢀlinked analog 16 is
likely the result of this particular linking group stabilizing a conformation that does not bind well
to the enzyme.
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Other alternative linking strategies investigated were unproductive. Reversing the amide
group gave compound 21, which had improved aqueous solubility, but similar activity and
PAMPA permeability relative to 1. Unfortunately, the increased solubility of 21 resulted in no
significant improvement in oral PK properties in rat (F = 4.3%). Alkylation of the amide
nitrogen was detrimental to activity. Tertiary amide analog 5 suffered a > 10ꢀfold loss in
potency in the enzyme assays, as well as a nearly 100ꢀfold loss in replicon potency.
Benzylamineꢀlinked 7 had the best aqueous solubility of compounds made in this series,
although it had poor PAMPA permeability and was substantially less active than 1. The loss in
activity for 7, as well as benzyl ether 8, is likely due in part to greater linker flexibility in these
analogs relative to more active compounds, such as 1, 13, and 24.
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Table 1. In vitro properties of dihydrouracil HCV polymerase inhibitors.
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H
O
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N
R
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O
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Stilbene analog 13 and acetylene analog 24 have similar metabolic stability in vitro, with
34ꢀ50% of parent remaining following 30 minute incubation in human and rat liver microsomes.
Substantial improvements in both PAMPA permeability and aqueous solubility for these two
compounds in comparison to 1 made them good candidates for pharmacokinetic study in rat.
Both compounds gave higher plasma exposures and better oral bioavailability following a 5
mg/kg oral dose (Figure 2 and Table 2). Stilbene 13 provided the best overall results, with
relatively low IV clearance (0.64 L/hr/kg) and higher plasma levels with oral dosing (30ꢀfold and
7ꢀfold higher AUC relative to 1 and 24, respectively). Plasma concentrations of 13 were
maintained above 100 ng/mL (> 200 nM) out to 8 hours post dosing, exceeding the plasmaꢀ
adjusted 1b replicon EC50 value for 13 (3.1 nM) by over 60ꢀfold. By contrast, acetylene 24
achieved a maximum concentration of 60 ng/mL (128 nM), an exposure level that is only a few
fold above its plasmaꢀadjusted 1b replicon EC50 of 30 nM.
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Figure 2. Plasma concentrations for 13 (a) and 24 (b) following a 5 mg/kg dose in rat.
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Table 2. Pharmacokinetic properties for dihydrouracil analogs.
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IV CLp
(L/hr/kg)
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F%
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1.0
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Uracil analogs of stilbene inhibitors have improved properties in comparison to
dihydrouracil analogs (Table 3). Uracil 29 is 2ꢀfold to 3ꢀfold more active than 13 in both 1a and
1b replicon assays, both with and without the addition of 40% human plasma. In addition, 29
provided a longer plasma halfꢀlife and better plasma levels following a 5 mg/kg oral dose in rat,
as well as a substantial increase in oral bioavailability from 18% to 44%. Further improvement
in pharmacokinetic properties was provided with fluorinated analogs. 5ꢀFluorouracil analog 30
provided a higher plasma concentration and better oral bioavailability. However, the replicon
activity for this compound was relatively poor. Fluorophenyl analog 31 provided even greater
plasma exposures and relatively low clearance. While the replicon activity for 31 was better than
fluorouracil 30, it was still weak in comparison to the nonꢀfluorinated analog 29.
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Table 3. Comparison of uracil and dihydrouracil analogs.
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Due to a superior replicon activity profile and good PK properties in rat, compound 29
was selected for further in vivo characterization (Figure 3 and Table 4). Pharmacokinetic studies
in dog revealed low plasma clearance and high oral bioavailability. Plasma exposures increased
in a dose proportional manner following oral dosing in both rat and dog. Concentrations in liver
tissue were high relative to plasma levels in both rat and dog. A 2.5 mg/kg oral dose in dog
provided a 24ꢀhour liver concentration of over 1 ꢀg/g (liver to plasma ratio = 10).
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Figure 3. Plasma and liver concentrations for 29 in rat (a) and dog (b).
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0.1
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IV; 5 mg/kg
PO; 5 mg/kg w/
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Liver
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Time (Hours Post Dose)
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Table 4. Pharmacokinetic properties of 29 in rat and dog.
Cmax
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CLp
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Species
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12 hr
24 hr
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5 mg/kg (IV)
5 mg/kg (PO)
30 mg/kg (PO)
2.5 mg/kg (IV)
2.5 mg/kg (PO)
30 mg/kg (PO)
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2.00
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The NS5B inhibitory potency of 29 is far greater against GT1 proteins than against other
genotypes (Figure 4). IC50 values obtained in polymerase assays for other HCV genotypes
ranged from 3 ꢀM for GT3a to 23 ꢀM for GT2a. For the GT1a polymerase, the activity of 29 is
highly sensitive to singleꢀpoint amino acid variations. The greatest loss of inhibitory activity is
seen with cysteine variants at position 316. Loss of inhibitory activity to the M414T variant is
observed in both GT1a and GT1b. In GT1b, the Y448H variant imparts the greatest loss of
inhibitory activity. Replicons containing these amino acid variants were selected during in vitro
resistance selection studies (data not shown) and have clinical relevance as they were observed in
human HCV infected subjects treated with dasabuvir, a compound that is structurally similar to
29.8,9 The overall enzyme inhibitory activity profile for 29, shown in Figure 4, is consistent with
binding to the NNI III (Palm 1) binding site of NS5B10 based on analogies to two structurally
similar compounds11ꢀ13 that are highly resistant to the M414T variant, and whose Palm 1 binding
sites were confirmed via Xꢀray crystallography.
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Figure 4. Inhibitory activity of compound 29 in polymerase enzyme assays. (a) Activity against
different HCV genotypes. (b) Activity against GT1 polymerase with singleꢀpoint amino acid
variations.
(a)
(b)
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Human Pharmacokinetics
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Based on these and other preclinical results, compound 29 was selected for clinical study
and designated ABTꢀ072. Human pharmacokinetic results are shown in Figure 5 and Table 5.14
Singleꢀdose administration of ABTꢀ072 at 80, 160, and 320 mg in healthy subjects resulted in
doseꢀproportional increases in plasma exposure. Maximum concentrations were achieved within
5ꢀ9 hours of dosing with a mean terminal phase halfꢀlife of 7ꢀ9 hours. Following dosing with
ABTꢀ072 at 160 mg once daily for 2 days in HCV GT1ꢀinfected patients (N=4), the HCV RNA
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least squares mean maximum decrease from baseline was 1.18 log10 IU/mL. The mean Cmax
,
AUC, and t1/2 values in HCV infected patients were similar to those obtained in healthy subjects.
These results supported further development of ABTꢀ072 as a onceꢀdaily nonꢀnucleoside NS5B
inhibitor for the treatment of HCV infection.
Figure 5. Mean (+SD) ABTꢀ072 (29) plasma concentrationꢀtime profiles in healthy subjects.
600
80 mg (n=8)
160 mg (n=8)
320 mg (n=8)
500
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0
0
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Time (hr)
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Table 5. Mean ± SD pharmacokinetics parameters of ABTꢀ072 in healthy subjects and HCV
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GT1ꢀinfected patients.
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HCV GT1ꢀ
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Healthy Subjects
Infected Patients
ABTꢀ072 160 mg
(N = 4)
Pharmacokinetic
Parameter
ABTꢀ072 80 mg ABTꢀ072 160 mg
ABTꢀ072 320 mg
(N = 8)
(N = 8)
(N = 8)
303 ± 89.1
6.8 ± 1.8
Cmax (ng/mL)
Tmax (h)
180 ± 37.6
5.1 ± 1.6
486 ± 154
415 ± 142
8.8 ± 2.6
3.6 ± 0.5
t
† (h)
8.17 ± 1.34
2442 ± 885
7.63 ± 2.32
4639 ± 1446
7.01 ± 0.87
7197 ± 2328
8.56 ± 1.21
4175 ± 1669
½
AUC∞ (ng•h/mL) $
†
Presented as harmonic mean and pseudo SD.
AUC24 presented for HCVꢀinfected patients.
$
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Conclusions
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Research on a novel series of Nꢀarylꢀdihydrouracil HCV NS5B inhibitors has identified
stilbene analogs with better antiviral potency and improved ADME properties in comparison to
early lead molecule 1. Replacement of the amide linker between the two aryl groups in 1
provided several analogs with much better permeability, and improved aqueous solubility. A
transꢀsubstituted olefin provided the most potent inhibitor, stilbene 13, which gave > 30ꢀfold
improvement in IC50 against both GT1a and GT1b polymerase, and 20ꢀfold improvement in
GT1b replicon EC50 compared to 1. Pharmacokinetic properties for 13 were also improved, with
reduced clearance and increased plasma compound levels upon oral dosing in rat. The
pharmacokinetic properties of stilbene analog 13 were further improved by replacing the
dihydrouracil group with uracil to give 29. Compound 29 is a potent inhibitor of HCV GT1
replicons, with an EC50 of 1 nM against GT1a and 0.3 nM against GT1b. Replicon potency is
attenuated 8ꢀfold to 17ꢀfold when activity is measured in the presence of 40% human plasma.
The resistance profile for 29 is consistent with binding to the NNI III (Palm 1) binding site of
NS5B. Compound 29 was selected for clinical evaluation and designated ABTꢀ072.
Pharmacokinetic studies in human support development of ABTꢀ072 as a onceꢀdaily nonꢀ
nucleoside NS5B inhibitor for the treatment of HCV infection.14,15 Phase 2 studies have
demonstrated the effectiveness of ABTꢀ072 for treating genotype 1 infected HCV patients, when
used in combination with HCV protease inhibitor ABTꢀ450, coꢀadministered with a low dose of
ritonavir (ABTꢀ450/r), and ribavirin.16ꢀ18 ABTꢀ072, administered once daily as a 400 mg dose in
combination with ABTꢀ450/r and ribavirin, demonstrated a sustained virologic response 24
weeks postꢀtreatment (SVR24) of 91% (N=10/11) in HCV genotype 1 infected adults.16
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Experimental Section
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Biological Evaluation. Biochemical activity of compounds was determined by
measuring their ability to inhibit the formation of RNA products in a standard polymerase
inhibition assay.9 Inhibition of genotypes 1a (H77) and 1b (BK) HCV NS5B was determined by
measuring the amount of 3HꢀUTP incorporated into RNA by scintillation counting. IC50 values
for inhibitors were calculated using a standard IC50 equation.
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Cellꢀculture activity of compounds was determined using subgenomic replicons
transfected into Huhꢀ7 cells.9 The ability of compounds to inhibit replication of genotype 1a
(H77) and 1b (Con1) replicons expressing the firefly luciferase gene was determined by
measuring the level of luciferase in the cell lysate using a luminometer. Replicon inhibition
activity, reported as EC50, was measured both in the absence of human plasma (HP) and in the
presence of 40% HP in order to determine the extent to which inhibitorꢀprotein interactions
affect compound potency.
Pharmacokinetic properties of compounds were determined using SpragueꢀDawley rats
(n = 3 for each experiment) via both intravenous and oral administration of a 5 mg/kg dose.
Compounds were formulated using DMSO: PEG400 (1:9, v/v) and were dosed as a solution.19
Chemistry. General Procedures. Reagents and solvents, including anhydrous solvents,
were obtained from commercial sources and used as supplied. Column chromatography was
carried out on silica gel. All final compounds were purified to >95% purity as determined by
reverse phase HPLC performed on a Waters 2695 Separation Module / Waters 2489 UV/Visible
Detector equipped with a YMC ODSꢀA, 5.5 ꢁm, 120Å, 4.6 X 150 mm column using a solvent
gradient of 10ꢀ100% acetonitrile in water (0.1% TFA). 1H NMR spectra were measured using
either a Brucker AMX 300 MHz or a Varian Inova 400 MHz spectrometer. Chemical shifts are
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reported in ppm (
δ
) and referenced to an internal standard of tetramethylsilane (δ 0.00 ppm). H
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– 1H couplings are assumed to be firstꢀorder, and peak multiplicities are reported in the usual
manner. MS analysis was conducted using a Finnigan SSQ7000 (ESI) mass spectrometer.
Methyl 5ꢀaminoꢀ3ꢀtertꢀbutylꢀ2ꢀmethoxybenzoate (3). Methyl 3,5ꢀdiꢀtertꢀbutylꢀ2ꢀ
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hydroxybenzoate (2) (28.66 g, 108.4 mmol) was dissolved with stirring in 430 mL glacial acetic
acid and the resulting mixture was treated dropꢀwise with fuming nitric acid (90%,179.26 mL).
When the addition was complete, the resulting mixture was stirred for 2.5 h. The reaction
mixture was poured onto 2.0 L of crushed ice and allowed to stand for 0.5 h.
Afterwards, 1.0 L of water was added and the ice water mixture was allowed to melt. The
mixture was then filtered, and the collected solid product was washed with water and dried to
provide methyl 3ꢀtertꢀbutylꢀ2ꢀhydroxyꢀ5ꢀnitrobenzoate (24.57 g, 89%). This compound (11.41
g, 45.0 mmol), potassium carbonate (9.34 g, 67.6 mmol), acetone (200 mL), and dimethyl sulfate
(6.46 g, 67.6 mmol) were added together, and the resultant mixture was heated to reflux for 16 h.
After cooling, the mixture was filtered, the solid was washed with ethyl acetate, and the filtrate
was concentrated under vacuum. The oily product was dissolved in ethyl acetate (600 mL),
washed with water, dried over sodium sulfate, and filtered. The filtrate was concentrated under
vacuum to give a crude product that was purified by column chromatography on silica gel,
eluting with a solvent gradient of 5ꢀ40% ethyl acetate in hexanes to yield methyl 3ꢀtertꢀbutylꢀ2ꢀ
methoxyꢀ5ꢀnitrobenzoate (10.42 g, 87%). This compound was combined with iron powder
(10.89 g, 195 mmol), ammonium chloride (3.13 g, 58.5 mmol), water (30 mL), and methanol
(150 mL), and the resulting mixture was refluxed for 1 h. The mixture was cooled to rt, filtered
through Celite, and the Celite was washed with methanol. The filtrate was concentrated under
vacuum, and the residue was dissolved in ethyl acetate (600 mL) and washed with water and
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brine. The organic extract was dried over sodium sulfate, filtered and concentrated under
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vacuum to provide 3 as an oil (9.25g, 100%): H NMR (300 MHz, DMSOꢀd6) δ ppm 1.29 (s,
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9
9H), 3.56 (s, 3H), 3.80 (s, 3H), 4.98 (s, 2H), 6.68ꢀ6.74 (m, 2H); MS (ESI) m/z 238 (M+H)+.
3ꢀtertꢀbutylꢀ5ꢀ(2,4ꢀdioxotetrahydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀmethoxybenzoic acid (4).
A solution of 3 (16.44g, 69.3 mmol) in toluene (200 mL) was heated to reflux and acrylic acid
was added slowly (1 mL of acrylic acid added every 3 h, 5.23 mL total, 76.2 mmol). The
mixture was then refluxed for 24 h. The resulting mixture was cooled to rt and concentrated
under vacuum to give 3ꢀ(3ꢀtertꢀbutylꢀ4ꢀmethoxyꢀ5ꢀ(methoxycarbonyl)phenylamino)propanoic
acid as an oil (21.4 g, 100%): 1H NMR (300 MHz, DMSOꢀd6) δ ppm 1.30 (s, 9H), 2.47 (t, J =
7.17 Hz, 2H), 3.19 (t, J = 6.80 Hz, 2H), 3.57 (s, 3H), 3.82 (s, 3H), 6.61 (d, J = 2.94 Hz, 1H), 6.72
(d, J = 2.94 Hz, 1H), 12.26 (s, 1H); MS (ESI) m/z 310 (M+H)+.
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A solution of 3ꢀ(3ꢀtertꢀbutylꢀ4ꢀmethoxyꢀ5ꢀ(methoxycarbonyl)phenylamino)propanoic
acid (21.4 g, 69.3 mmol) and urea (10.4 g, 173 mmol) in glacial acetic acid (200 mL) was heated
at 120oC for 18.5 h, and the resulting mixture was concentrated under vacuum to give an oil.
Methanol (13 mL) and ethyl acetate (350 mL) were added, and the resulting mixture was allowed
to stand for 24ꢀ48 h, during which time a precipitate formed. The resulting solid was collected
by filtration, washed with methanol (10 mL), and air dried to yield methyl 3ꢀtertꢀbutylꢀ5ꢀ(2,4ꢀ
1
dioxotetrahydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀmethoxybenzoate as a solid (15.26 g, 66%): H NMR
(300 MHz, DMSOꢀd6) δ ppm 1.35 (s, 9H), 2.70 (t, J = 6.62 Hz, 2H), 3.72 (s, 3H), 3.76 (t, J =
6.80 Hz, 2H), 3.87 (s, 3H), 7.40 (d, J = 2.57 Hz, 1H), 7.46 (d, J = 2.57 Hz, 1H), 10.36 (s, 1H);
MS (ESI) m/z 335 (M+H)+.
To a solution of methyl 3ꢀtertꢀbutylꢀ5ꢀ(2,4ꢀdioxotetrahydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀ
methoxybenzoate (4.52g, 13.52 mmol) in methanol (70 mL) and tetrahydrofuran (70 mL) was
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added a solution of aqueous sodium hydroxide (1.0 M, 68 mL). The mixture was stirred for 12 h
and concentrated under vacuum to remove the organic solvents. Aqueous hydrochloric acid (1.0
M, 80 mL) was added, resulting in the formation of a solid, and the mixture was concentrated
under vacuum. Concentrated hydrochloric acid (100 mL) was added, and the mixture was heated
to 100oC for 1.5 h. The mixture was cooled to rt, water was added (100 mL), and the resulting
solid was collected by filtration, washed with water, and dried to provide 4 as a solid (3.55 g,
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82%): H NMR (300 MHz, DMSOꢀd6) δ ppm 1.35 (s, 9H), 2.70 (t, J = 6.62 Hz, 2H), 3.77 (m,
5H), 7.36 (d, J = 2.94 Hz, 1H), 7.44 (d, J = 2.94 Hz, 1H), 10.35 (s, 1H), 13.13 (s, 1H); MS (ESI)
m/z 321 (M+H)+.
3ꢀtertꢀbutylꢀ5ꢀ(2,4ꢀdioxotetrahydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀmethoxybenzaldehyde (6).
A solution of 4 (4.07g, 12.71 mmol) in thionyl chloride (40.82 mL, 559 mmol) was refluxed for
2 h and was concentrated under vacuum to provide a light yellow solid. The solid was dissolved
in tetrahydrofuran (125 mL), and the solution was cooled to –78oC with stirring. To the cooled
mixture was added lithium triꢀtertꢀbutoxyaluminum hydride (1.0 M in THF, 14 mL, 14.0 mmol)
slowly over 10 min. The mixture was stirred at –78oC for 2 h, and was then quenched with
aqueous hydrochloric acid (1.0 M, 25 mL) and allowed to warm to rt. Ethyl acetate (100 mL)
was added, the organic layer was separated, and the aqueous layer was washed with ethyl
acetate. The organic extracts were combined and washed with saturated sodium bicarbonate
solution. The organic layer was dried over sodium sulfate, filtered and concentrated under
1
vacuum to provide 6 as a solid (3.73 g, 96%): H NMR (300 MHz, DMSOꢀd6) δ ppm 1.38 (s,
9H), 2.71 (t, J = 6.62 Hz, 2H), 3.80 (t, J = 6.80, 2H), 3.93 (s, 3H), 7.56 (q, J = 2.70 Hz, 2H),
10.26 (s, 1H), 10.40 (s, 1H); MS (ESI) m/z 305 (M+H)+.
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1ꢀ(3ꢀtertꢀbutylꢀ4ꢀmethoxyꢀ5ꢀ(4ꢀnitrostyryl)phenyl)dihydropyrimidineꢀ2,4(1H,3H)ꢀ
dione (11). To a solution of 6 (1.00 g, 3.29 mmol) and diethyl 4ꢀnitrobenzylphosphonate (0.853
g, 3.12 mmol) in dichloromethane (50 mL) was added solid potassium tertꢀbutoxide (0.737 g,
6.57 mmol) portionwise at rt. The resultant dark red solution was stirred at rt for 1.5 h, followed
by the addition of aqueous hydrochloric acid solution (1.0 M, 50 mL). The mixture was stirred
for 30 min, followed by the addition of dichloromethane (50 mL). The organic layer was
separated and dried over sodium sulfate, filtered and concentrated to give a crude product that
was purified by column chromatography on silica gel using 1% methanol in dichloromethane as
eluent to provide 11 as a solid (1.12g, 80%). Note: the material obtained is a mixture of approx.
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9/1 trans/cis olefin isomers. H NMR (300 MHz, DMSOꢀd6) δ ppm 1.39 (s, 9H), 2.72 (t, J =
6.60 Hz, 2H), 3.78 (s, 3H), 3.81 (m, 2H), 7.24 (d, J = 2.5 Hz, 1H), 7.38 (d, J = 16.20 Hz, 1H),
7.57 (m, 1H), 7.61 (d, J = 2.2 Hz, 1H), 7.94 (d, J = 8.8 Hz, 2H), 8.24 (d, J = 8.8 Hz, 2H), 10.38
(s, 1H); MS (ESI) m/z 424 (M+H)+.
(E)ꢀNꢀ(4ꢀ(3ꢀtertꢀbutylꢀ5ꢀ(2,4ꢀdioxotetrahydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀ
methoxystyryl)phenyl)methanesulfonamide (13). A mixture of 11 (1.1 g, 2.60 mmol), iron
powder (0.725 g, 12.99 mmol), and ammonium chloride (0.208 g , 3.90 mmol) in
tetrahydrofuran (40 mL), ethanol (40 mL), and water (12 mL) was heated to 90oC for 45 min.
The mixture was cooled to rt, filtered through Celite and washed with ethanol (20 mL), and the
filtrate was concentrated under vacuum to give a solid. The solid was dissolved in ethyl acetate
(100 mL), washed with water, dried over sodium sulfate, filtered and concentrated under
vacuum. The resultant yellow solid (830 mg, 2.11 mmol) was dissolved in dichloromethane (50
mL) and pyridine (0.512 mL, 6.33 mmol) was added, followed by methanesulfonyl chloride
(0.181 mL, 2.32 mmol). The resultant solution was stirred at rt for 16 h. Dichloromethane (100
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mL) was added, and the solution was extracted with a 1N aqueous hydrochloric acid solution (2
x 50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under
vacuum. The crude product was purified by column chromatography on silica gel using 2%
9
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methanol in dichloromethane to provide 13 as a solid (480 mg, 39%): H NMR (500 MHz,
DMSOꢀd6) δ ppm 1.37 (s, 9H), 2.71 (t, J = 6.7 Hz, 2H), 3.01 (s, 3H), 3.75 (s, 3H), 3.79 (t, J = 6.6
Hz, 2H), 7.13 (d, J = 16.5 Hz, 1H), 7.15 (d, J = 2.4 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 7.25 (d, J =
16.5 Hz, 1H), 7.51 (d, J = 2.4 Hz, 1H), 7.61 (d, J =8.6 Hz, 2H), 9.80 (bs, 1H), 10.30 (s, 1H); MS
(ESI) m/z 472 (M+H)+.
3ꢀtertꢀbutylꢀ5ꢀiodoꢀ4ꢀmethoxyaniline (22). To a vigorously stirred solution of 2ꢀtertꢀ
butylphenol (10.0 g, 66.6 mmol) in heptane (67 mL) was added at a fast drip a solution of 70%
nitric acid (4.25 mL, 66.6 mmol) diluted with water (4.25 mL). The resulting dark red/brown
mixture was stirred vigorously for 2 h. The suspended solid was collected by filtration then
washed with hexane (300 mL), water (200 mL) and once again with hexane (200 mL) to provide
1
2ꢀtertꢀbutylꢀ4ꢀnitrophenol as a solid (4.65 g, 36%): H NMR (300 MHz, CDCl3): δ ppm 1.44 (s,
9H), 5.85 (s, 1H), 6.76 (d, J = 8.82 Hz, 1H), 8.01 (dd, J = 8.64, 2.76 Hz, 1H), 8.22 (d, J = 2.94
Hz, 1H).
To a solution of 2ꢀtertꢀbutylꢀ4ꢀnitrophenol (4.5 g, 23.05 mmol) in methanol (120 mL) and
water (30 mL) was added iodine monochloride (1.16 mL, 23.05 mmol) dropꢀwise over a period
of 10 min. The mixture was stirred for 2 h, and was poured into water (1.0 L) and allowed to
stand overnight. The resulting solid was collected by filtration, washed with water (3 x 50 mL),
and dried under vacuum to provide 2ꢀ(tertꢀbutyl)ꢀ6ꢀiodoꢀ4ꢀnitrophenol as a tan solid (7.14 g,
96%): 1H NMR (300 MHz, CDCl3): δ ppm 1.42 (s, 9H), 6.17 (s, 1H), 8.20 (d, J = 2.3 Hz, 1H),
8.47 (d, J = 2.7 Hz, 1H).
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A solution of 2ꢀtertꢀbutylꢀ6ꢀiodoꢀ4ꢀnitrophenol (5.5 g, 17.13 mmol) in methyl tertꢀbutyl
ether (15 mL) was placed in a 50 mL pressure vessel and cooled in an ice bath. A solution of
trimethylsilyldiazomethane (2.0 M in diethyl ether, 12.85 mL, 25.7 mmol) was added, followed
by dropꢀwise addition of methanol (1.0 mL). The vessel was sealed and stirred at rt for 16 h, and
the vessel was cooled in an ice bath before being opened. The resulting solution was partitioned
between ethyl acetate and water, and the organic layer was washed with 1.0M aqueous
hydrochloric acid, saturated aqueous potassium carbonate solution, and brine. The organic layer
was dried over sodium sulfate, filtered and concentrated to provide 1ꢀtertꢀbutylꢀ3ꢀiodoꢀ2ꢀ
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1
methoxyꢀ5ꢀnitrobenzene as a red oil (5.4 g, 84%): H NMR (300 MHz, CDCl3): δ ppm 1.42 (s,
9H), 3.98 (s, 3H), 8.24 (d, J = 2.7 Hz, 1H), 8.56 (d, J = 2.7 Hz, 1H).
A mixture of 1ꢀtertꢀbutylꢀ3ꢀiodoꢀ2ꢀmethoxyꢀ5ꢀnitrobenzene (5.80 g, 17.31 mmol),
ammonium chloride (1.39 g, 26.0 mmol), and iron powder (4.83 g, 87 mmol) in a 2/2/1 mixture
of tetrahydrofuran/ethanol/water (200 mL) was refluxed for 2 h, cooled and filtered through
Celite. The filtrate was concentrated under vacuum, and the residue was partitioned between
water and ethyl acetate. The organic layer was washed with brine, dried with sodium sulfate,
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filtered and concentrated to provide 22 as a brown oil (5.28 g, 100% yield): H NMR (300 MHz,
DMSOꢀd6): δ ppm 1.42 (s, 9H), 3.63 (s, 3H), 4.89 (s, 2H), 6.51 (d, J = 2.7 Hz, 1H), 6.86 (d, J =
2.7 Hz, 1H); MS (ESI) m/z 306 (M+H)+.
1ꢀ(3ꢀtertꢀbutylꢀ5ꢀiodoꢀ4ꢀmethoxyphenyl)dihydropyrimidineꢀ2,4(1H,3H)ꢀdione (23).
A solution of 22 (8.2 g, 26.9 mmol) was treated with acrylic acid (5.53 mL, 81 mmol) and stirred
at rt overnight to give a thick, viscous mixture. To the mixture was added acetic acid (60 mL)
and urea (7.3 g, 120 mmol), and the mixture was heated at 120°C for 24 h. The mixture was
cooled to rt and concentrated under vacuum, and the residue was dissolved in toluene and
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concentrated under vacuum to give a brownish solid. The solid was suspended in a mixture of
ethyl acetate (50 mL) and saturated aqueous sodium bicarbonate (100 mL) and stirred for 0.5 h
to neutralize any remaining acetic acid. The solid was collected by filtration and washed
repeatedly with 50 mL portions of water and finally with 3/1 hexane/ethyl acetate (50 mL) to
provide 23 as an offꢀwhite solid (7.1 g, 66%): 1H NMR (300 MHz, DMSOꢀd6) δ ppm 1.37 (s,
9H), 2.69 (q, J = 6.74 Hz, 2H), 3.74 (t, J = 6.62 Hz, 2H), 3.82 (s, 3H), 7.26 (d, J = 2.57 Hz, 1H),
7.68 (d, J = 2.21 Hz, 1H), 10.34 (s, 1H); MS (ESI) m/z 403 (M+H)+.
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Nꢀ(4ꢀ((3ꢀ(tertꢀbutyl)ꢀ5ꢀ(2,4ꢀdioxotetrahydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀ
methoxyphenyl)ethynyl)phenyl)methanesulfonamide (24). To a solution of 4ꢀethynylaniline
(3.64 g, 31.1 mmol) in dichloromethane (62 mL) was added pyridine (9.83 g, 124 mmol)
followed by dropꢀwise addition of methanesulfonyl chloride (4.27 mL, 37.3 mmol). The mixture
was stirred at 25°C for 2 h, and concentrated under vacuum. The residue was dissolved in ethyl
acetate (350 mL) and washed with 1N aqueous hydrochloric acid, water, and saturated sodium
chloride solution. The organic layer was dried over sodium sulfate, filtered, and concentrated
under vacuum to give a crude product that was purified by column chromatography on silica gel,
eluting with 0ꢀ1% methanol in dichloromethane to provide Nꢀ(4ꢀ
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ethynylphenyl)methanesulfonamide as a colorless solid (5.09 g, 84%): H NMR (300 MHz,
DMSOꢀd6) δ ppm 3.03 (s, 3H), 4.11 (s, 1H), 7.19 (d, J = 8.82 Hz, 2H), 7.44 (d, J = 8.46 Hz, 2H),
10.03 (s, 1H); MS (ESI) m/z 196 (M+H)+.
A mixture of 23 (1.77 g, 4.40 mmol), Nꢀ(4ꢀethynylphenyl)methanesulfonamide (0.945 g,
4.84 mmol), copper(I) iodide (0.029 g, 0.154 mmol), triethylamine (3.07 mL, 22.0 mmol), and
dichlorobis(triphenylphosphine)palladium(II) (0.154 g, 0.22 mmol) in acetonitrile (60 mL) was
purged by bubbling with nitrogen for 5 min, and then heated at 80°C for 30 min. The mixture
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was cooled to rt and diluted with ethyl acetate (400 mL). The mixture was extracted with 1N
aqueous hydrochloric acid, water, and brine. The organic layer was dried over sodium sulfate,
filtered, and concentrated under vacuum to give a crude product that was purified by column
chromatography on silica gel, eluting with 0ꢀ5% methanol in dichloromethane to provide 24 as a
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colorless solid (0.98 g, 47%): H NMR (300 MHz, DMSOꢀd6) δ ppm 1.35 (s, 9 H) 2.70 (t,
J=6.62 Hz, 2 H) 3.06 (s, 3 H) 3.77 (t, J = 6.62 Hz, 2 H) 4.05 (s, 3 H) 7.25 (dd, J = 5.52, 2.94 Hz,
3 H) 7.35 (d, J = 2.57 Hz, 1 H) 7.55 (d, J = 8.46 Hz, 2 H) 10.09 (s, 1 H) 10.37 (s, 1 H); MS (ESI)
m/z 470 (M+H)+.
Methyl 3ꢀ(tertꢀbutyl)ꢀ5ꢀ(2,4ꢀdioxoꢀ3,4ꢀdihydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀ
methoxybenzoate (25). A solution of 3 (2.0 g, 8.43 mmol) in N,Nꢀdimethylacetamide (30 mL)
was cooled to ꢀ25°C. A 0.5 M solution of Eꢀ3ꢀmethoxyacryloyl isocyanate in benzene (21.9 mL,
10.96 mmol) was added dropꢀwise, and the resulting solution was stirred at rt for 4 h, and then
poured into water. The product was extracted into dichloromethane, washed with brine, dried
over sodium sulfate, filtered and concentrated under vacuum to provide a solid. This material
(3.1 g, 8.51 mmol) was dissolved in ethanol (60 mL). To this solution was added a mixture of
concentrated sulfuric acid (6 mL) and water (60 mL), and the heterogeneous mixture was heated
at 100°C for 3 h. The ethanol solvent was removed under vacuum, and the aqueous mixture was
extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, and
concentrated under vacuum to give a crude product that was purified by column chromatography
on silica gel, eluting with 1% methanol in dichloromethane to provide 25 as a solid (1.23 g,
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44%): H NMR (300 MHz, DMSOꢀd6) δ ppm 1.32 (s, 9H), 3.76 (s, 3H), 3.88 (s, 3H), 5.64 (d, J
= 8.09 Hz, 1H), 7.46 (d, J = 2.94 Hz, 1H), 7.56 (d, J = 2.57 Hz, 1H), 7.72 (d, J = 7.72 Hz, 1H),
11.41 (s, 1H); MS (ESI) m/z 333 (M+H)+.
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3ꢀtertꢀbutylꢀ5ꢀ(2,4ꢀdioxoꢀ3,4ꢀdihydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀmethoxybenzaldehyde
(27). A solution of 25 (1.23 g, 3.7 mmol) in ethanol (5 mL) and 1M sodium hydroxide solution
(10 mL) was stirred at rt for 18 h. The solution was acidified with 1M aqueous hydrochloric acid
and the resulting solid was collected by filtration and dried (0.945 g, 80%). This material (0.945
g, 2.97 mmol) was mixed with thionyl chloride (4.5 mL), and the mixture was heated at 80°C for
40 min. After evaporating to dryness, the resulting acid chloride intermediate was dissolved in
dry tetrahydrofuran (8 mL) and cooled to ꢀ78°C. A 1.0 M solution of lithium triꢀtertꢀ
butoxyaluminum hydride in tetrahydrofuran (3.0 mL, 3.0 mmol) was added dropꢀwise. The
mixture was stirred at ꢀ78°C for 45 min, and was quenched with 1M aqueous hydrochloric acid
(5 mL). The mixture was extracted into ethyl acetate, and the organic layer was dried over
sodium sulfate, filtered, and concentrated under vacuum. The crude product was purified by
column chromatography on silica gel using a solvent gradient of 0ꢀ1% methanol in
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dichloromethane to provide 27 (0.635 g, 71%): H NMR (300 MHz, DMSOꢀd6) δ ppm 1.39 (s,
9H), 3.97 (s, 3H), 5.67 (dd, J = 7.9, 2.0 Hz, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.67 (d, J = 2.8 Hz,
1H), 7.75 (d, J = 7.9 Hz, 1H), 10.27 (s, 1H), 11.44 (s, 1H); MS (ESI) m/z 303 (M+H)+.
(E)ꢀNꢀ(4ꢀ(3ꢀtertꢀbutylꢀ5ꢀ(2,4ꢀdioxoꢀ3,4ꢀdihydropyrimidinꢀ1(2H)ꢀyl)ꢀ2ꢀ
methoxystyryl)phenyl)methanesulfonamide (29). To a solution of 27 (0.634 g, 2.1 mmol) and
diethyl 4ꢀnitrobenzylphosphonate (0.573 g, 2.1 mmol) in anhydrous dichloromethane (25 mL)
was added potassium tertꢀbutoxide (0.494 g, 4.4 mmol) portionꢀwise, and the resulting
red/brown heterogeneous mixture was stirred for 1.5 h. The reaction was quenched with 1M
aqueous hydrochloric acid (15 mL) and was partitioned between water and ethyl acetate. The
organic layer was dried over sodium sulfate, filtered and concentrated under vacuum to give a
crude product that was purified by column chromatography on silica gel, eluting with 1%
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methanol in dichloromethane to provide a solid (0.735 g, 83%). This solid (0.735 g, 1.74 mmol)
was mixed with ammonium chloride (0.14 g, 2.62 mmol) and iron powder (0.487 g, 8.72 mmol)
in ethanol (10 mL), water (5 mL), and tetrahydrofuran (10 mL). The mixture was stirred and
heated at 75° C for 1 h. The cooled mixture was filtered through Celite, and the filtrate was
concentrated under vacuum. The resulting crude aniline intermediate (0.683 g, 1.75 mmol) was
dissolved in pyridine (0.564 mL, 6.98 mmol) and dichloromethane (15 mL). Methanesulfonyl
chloride (0.163 mL, 2.1 mmol) was added dropꢀwise and the solution was stirred at rt for 18 h.
The mixture was partitioned between 1M aqueous hydrochloric acid and dichloromethane, and
the organic layer was dried over sodium sulfate, filtered and concentrated under vacuum. The
crude product was purified by column chromatography on silica gel using a solvent gradient of
1ꢀ2% methanol in dichloromethane to provide 29 as a colorless solid (0.465 g, 57%): 1H NMR
(300 MHz, DMSOꢀd6) δ ppm 1.38 (s, 9H), 3.01 (s, 3H), 3.79 (s, 3H) 5.65 (d, J = 7.7 Hz, 1H),
7.17ꢀ7.28 (m, 5H), 7.58ꢀ7.70 (m, 3H), 7.75 (d, J = 7.7 Hz, 1H), 9.86 (s, 1H), 11.42 (s, 1H); MS
(ESI) m/z 470 (M+H)+.
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