Discovery of a novel class of potent HCV NS4B inhibitors: SAR studies on piperazinone derivatives

Article abstract of DOI:10.1021/jm4012643

HTS screening identified compound 2a (piperazinone derivative) as a low micromolar HCV genotype 1 (GT-1) inhibitor. Resistance mapping studies suggested that this piperazinone chemotype targets the HCV nonstructural protein NS4B. Extensive SAR studies were performed around 2a and the amide function and the C-3/C-6 cis stereochemistry of the piperazinone core were essential for HCV activity. A 10-fold increase in GT-1 potency was observed when the chiral phenylcyclopropyl amide side chain of 2a was replaced with p- fluorophenylisoxazole-carbonyl moiety (67). Replacing the C-6 nonpolar hydrophobic moiety of 67 with a phenyl moiety (95) did not diminish the GT-1 potency. A heterocyclic thiophene moiety (103) and an isoxazole moiety (108) were incorporated as isosteric replacements for the C-6 phenyl moiety (95), resulting in significant improvement in GT-1b and 1a potency. However, the piperazonone class of compounds lacks GT-2 activity and, consequently, were not pursued further into development.

Full text of DOI:10.1021/jm4012643

Article
pubs.acs.org/jmc
Discovery of a Novel Class of Potent HCV NS4B Inhibitors: SAR
Studies on Piperazinone Derivatives
Ramesh Kakarla,* Jian Liu,† Devan Naduthambi,‡ Wonsuk Chang,§ Ralph T. Mosley,∥ Donghui Bao,⊥
Holly M. Micolochick Steuer,# Meg Keilman,▽ Shalini Bansal,○ Angela M. Lam,◆ William Seibel,¶
Sandra Neilson,¶ Phillip A. Furman,+ and Michael J. Sofia□
Pharmasset, Inc. 303A College Road East, Princeton, New Jersey 08540, United States
ABSTRACT: HTS screening identified compound 2a (piper-
azinone derivative) as a low micromolar HCV genotype 1
(GT-1) inhibitor. Resistance mapping studies suggested that
this piperazinone chemotype targets the HCV nonstructural
protein NS4B. Extensive SAR studies were performed around
2a and the amide function and the C-3/C-6 cis stereo-
chemistry of the piperazinone core were essential for HCV
activity. A 10-fold increase in GT-1 potency was observed
when the chiral phenylcyclopropyl amide side chain of 2a was
replaced with p-fluorophenylisoxazole-carbonyl moiety (67).
Replacing the C-6 nonpolar hydrophobic moiety of 67 with a
phenyl moiety (95) did not diminish the GT-1 potency. A
heterocyclic thiophene moiety (103) and an isoxazole moiety
(108) were incorporated as isosteric replacements for the C-6
phenyl moiety (95), resulting in significant improvement in
GT-1b and 1a potency. However, the piperazonone class of
compounds lacks GT-2 activity and, consequently, were not
pursued further into development.
The HCV virion is an enveloped positive-strand RNA virus
of the family flaviviridae with a single oligoribonucleotide
genomic sequence of about 9600 bases, which encodes a
polyprotein of about 3010 amino acids.5 The protein products
of the HCV gene consist of the structural proteins C, E1, and
E2 and the nonstructural proteins NS2, NS3, NS4A, NS4B,
NS5A, and NS5B. The nonstructural (NS) proteins are
believed to provide the catalytic machinery for viral replication.
A number of molecular targets have been pursued in an effort
to identify direct acting antivirals as anti-HCV therapeutics.6
These include, but are not limited to, the NS3/4A7 protease,
NS5A,8 and the NS5B9 polymerase. Agents targeting these
nonstructural proteins have proven to be clinically effective at
reducing HCV viral load in infected patients. However, the
emergence of resistant virus, and the observation that
combining direct acting antivirals appears to be necessary to
INTRODUCTION
■
Hepatitis C virus (HCV) infection is a major global health
problem that affects more than 200 million individuals
worldwide and an estimated 4.5 million people in the United
States.1 Ten to twenty percent of chronically infected
individuals eventually develop liver-destroying cirrhosis or
hepatocellular carcinoma.2 Recently approved standard of care
(SOC) for genotype 1 patients involves the combination of a
NS3/4A protease inhibitor, pegylated α-interferon (IFN), and
the oral nucleoside ribavirin. The sustained virologic response
(SVR or undetectable HCV RNA in serum post treatment) rate
for most-difficult-to-treat genotype-1 HCV patients is about
70% with approved drugs boceprevir and telaprevir in
combination with ribavirin and IFN.3 The current SOC
provides limited clinical benefit for HCV genotype 2, 3, 4, 5,
and 6 infected patients.4 Moreover, there is no established
vaccine for HCV. Consequently, there is an urgent need for
improved oral drugs or drug combination therapies that
effectively combat chronic HCV infection, are pan-genotypic,
have a high barrier to resistance, and are well tolerated.
Special Issue: HCV Therapies
Received: August 15, 2013
© XXXX American Chemical Society
A
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Figure 1. HCV NS4B inhibitors with sub-μM GT1b potency.
a
Scheme 1. Synthesis of Piperazinone Core 7 and Compounds 2a, 2b, and 67
a
Reagents and conditions: (a) MeNHOMe·HCl, TBTU, DIPEA, CH3CN, 0 °C to RT (98%); (b) LiAlH4, THF, −78 °C (98%); (c) NH2-Leu-
OMe, NaBH(OAc)3, DCE (90%); (d) 20% Et2NH in DCM, DCM (95%); (e) ( )-(1R,2R)-2-phenylcyclopropanecarboxylic acid, TBTU, DIPEA,
DCM (90%); (f) 5-(4-fluorophenyl)isoxazole-3-carboxylic acid, TBTU, DIPEA, DCM (91%).
achieve sustained viral suppression in the absence of IFN, has
necessitated the search for novel and safe direct acting antivirals
that can be combined with other anti-HCV agents in
development.10
One of the HCV auxiliary NS proteins is NS4B. This protein
is a relatively poorly characterized 27 kDa protein with at least
four predicted transmembrane (TM) domains.11 It is believed
that as a consequence of polyprotein processing by the NS3/4A
protease, the N- and C-terminal parts of NS4B are oriented
toward the cytosolic side of the endoplasmic reticulum (ER)
membrane. Furthermore, it is believed that HCV NS4B
associates with a number of additional NS proteins and permits
formation of the so-called “membranous web” structure that
facilitates HCV replication.11 Because NS4B plays a key role in
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HCV replication, disrupting NS4B function represents an
attractive new anti-HCV strategy.
Efforts to find inhibitors of HCV that act on the NS4B
protein utilized either the purified NS4B protein binding assay
or the HCV GT1 replicon luciferase reporter-based in vitro
assay and resulted in the identificatioin of low μM inhibitors.12a
The first reported potent sub-μM (HCV 1b) NS4B inhibitor
was anguizole12b (1a) (Figure 1), which showed potency in
both GT1b and GT1a replicon assays (EC50) of 0.31 and 0.56
μM, respectively. Lead optimization studies based on the
anguzole core resulted in a novel imidazo[1,2-a]pyridines12c
(1b) series (Figure 1) with low nM HCV GT1a and 1b
replicon activity. Further PK profile optimization studies on 1b
resulted in pyrazolo[1,5-a]pyridine12d analogue 1c (Figure 1)
with improved viral resistance profile when compared to
compound 1b. Recently, a new potent HCV GT1b Inhibitor
(1d) based on the 2-(pyridine-2-yl)-indole12e−g (1d) core with
1.7 nM (EC50)1b replicon activity was also disclosed (Figure
1). Anguizole and compounds 1b, 1c, and 1d all showed similar
resistance profiles at amino acid residues (H94, F98, V105) of
TM1 domain. Resistant profile data of 1a−1d appears to
indicate that each of these molecules are interacting at the same
region of NS4B, however, due to the lack of cocrystal data, their
specific mode of binding and identification of key molecular
interactions with the NS4B protein have not been determined.
Although the current clinical status of compounds 1c and 1d is
not available, it is known that anguizole (1a) was evaluated in a
phase 1 clinical trial, but no further data has been reported.
Herein, we report the identification and SAR development of a
novel class of highly potent HCV NS4B inhibitors that is
structurally distinct from those that have been previously
disclosed.
A high-throughput screen (HTS) was carried out on 277000
compounds using a HCV 1b replicon luciferase reporter-based
assay resulted in identification of the active compound 2a.
Compound 2a contained a piperazinone core and a cyclo-
propylphenyl side chain (Figure 1). The activity of 2a was
confirmed after resynthesis (Scheme 1) from commercially
available L-leucine derivatives. Compound 2a (trans-R,R)
exhibited HCV replicon genotype 1b (GT-1b) potency of 0.8
μM and genotype 1a (GT-1a) potency of 1.0 μM. trans-S,S
isomer 2b did not show any HCV replicon activity (GT-1b or
GT-1a) when tested up to a 20 μM concentration. Compound
2a was inactive against HIV and inactive as a HCV NS5B
inhibitor. Cytotoxicity assessment against a panel of cell lines
(Huh7, HepG2, BxPC3, and CEM) showed that 2a’s replicon
activity could not be attributed to a cytotoxic effect (CC50 range
= 40−76 μM). Compound 2a with its unique diisobutylpiper-
azin-2-one core was further investigated as a novel compound
class targeting HCV.
Figure 2. HTS hit compound (2a) and its inactive isomer (2b).
sensitivity was determined using the luciferase reporter assay.
Compound 2a and 95 showed a 17-fold reduction and >20-fold
reduction in activity against these mutant replicons, respec-
tively, thus indicating that NS4B was the target for this class of
molecules. The known NS4B inhibitor anguizole showed ∼10-
fold reduction in activity against residue 94 and 98 mutant
replicons. Despite submicromolar potency of 2a and 95 against
GT-1a and GT-1b replicons, no appreciable activity was
observed using a GT-2a replicon. JFH-1 GT-2a WT naturally
contains a Leu at position 98. When GT-2a L98 was mutated to
Phe, some inhibition was recovered for compound 2a and 95.
SYNTHESIS OF HCV NS4B INHIBITORS
■
The general synthesis of piperazinone core 7 (Scheme 1) was
achieved by employing a modified literature procedure.13 The
aldehyde 5 was prepared via Weinreb amide intermediate 4,
which was in turn synthesized from commercially available
Fmoc-L-leucine 3. Reductive amination of methyl ester of L-
leucine with aldehyde 5 furnished 6, which on subsequent
internal cyclization afforded the core 3,6-disiobutylpiperazinone
7. Compound 7 was coupled to commercially available
( )-(R,R)-phenyl-cyclopropane carboxylic acid followed by
chiral column (SFC) separation to furnish active inhibitor 2a
and its inactive isomer 2b (Scheme 1). TBTU mediated
coupling of 7 with 5-(4-fluorophenyl)isoxazole-3-carboxylic
acid furnished inhibitor 67 (Scheme 1).
Synthesis of core 12 bearing a furanyl moiety began by
treating furanyl-2-boronic acid (8) with glyoxylic acid
monohydrate and bis(4-methoxyphenyl)methanamine in di-
chloromethane using the Petasis14 multicomponent coupling
reaction to afford the acid 9 (Scheme 2). The acid compound 9
was converted to the corresponding Weinreb amide compound
10. Weinreb amide 10 was reduced with LiAlH4 and then
underwent a reductive amination with the methyl ester of L-
leucine to furnish the corresponding ester 11. Ester 11 was
cyclized using aqueous acetic acid followed by SFC (chiral)
separation providing the cores 12 and 13. Compound 12 was
then coupled to the 5-(4-fluorophenyl)isoxazole-3-carboxylic
acid to furnish the inhibitor 107.
Synthesis of oxazole core 16 began with coupling of
commercially available FMOC-L-Ser-OtBu and the hydro-
chloride salt of L-leucine methyl ester by the method described
for compound 7 (Scheme 1) to afford intermediate 14
(Scheme 3). Acid hydrolysis followed by the nitrogen
protection with a Boc group furnished alcohol 15. Oxidation
of hydroxy group in 15 with Dess−Martin’s reagent followed
by treatment with tosylmethyl isocyanide and SFC (chiral)
separation furnished the desired oxazole core 16. Oxazole core
16 was subsequently treated with HCl to deprotect the Boc
Because 2a did not inhibit HCV polymerase (data not
shown), resistant replicons were generated in an effort to
determine the viral target for this class of compounds. The
generation of a resistant replicon resulted in the identification
of a replicon with decreased sensitivity to 2a. Genotypic and
phenotypic analyses suggested that the NS4B protein (NS4B)
was the target for 2a. When known inhibitors of NS3/4A and
NS5A were tested against the compound 2a-resistant replicon,
no significant shift in HCV EC50 values was observed. Three
separate rounds of resistance selection generated high
frequency mutations at residues 90 and 98 of NS4B. High
frequency mutations found in NS4B were constructed in the
GT-1b ET:PVI replicon by site-directed mutagenesis, and the
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a
Scheme 1. Piperazinone 20 was coupled with 5-(4-fluorophen-
yl)-isoxazole-3-carboxylic acid giving 21, and subsequent
oxidation of the vinyl goup with OsO4 afforded the acid 22.
Acid 22 was treated with 2,2-dimethoxy-ethylamine to give the
acetal product 23, which was cyclized to furnish the inhibitor
109.
Scheme 2. Synthesis of Furanyl Derivative 107
Synthesis of pyrrolidine 26 began with a Petasis14 multi-
component coupling reaction utilizing (1-(tert-butoxycarbon-
yl)-1H-pyrrol-2-yl)boronic acid (24) followed by treatment
with N-methylmethoxyamine to afford the corresponding
Weinreb amide derivative 25 (Scheme 5). Amide 25 was
subjected to reductive amination, deprotection, and acylation as
described for compound 107 (Scheme 2) to furnish inhibitor
111 (trans diastereomers).
RESULTS AND DISCUSSION
■
A study was first undertaken to understand which structural
elements of the piperazinone core were critical to its HCV
inhibitory activity. This was accomplished by preparing
analogues of key structural motifs found in 2a (Table 1).
Compounds derived from combination of core 2a with trans-
S,S, side chain or core 2b with trans-R,R side chain were shown
to be inactive, indicating that the stereochemistry of the core as
well as the side chain were essential for activity. Alkylation of
the N-1 amide nitrogen (27) resulted in a 10-fold loss in GT-1b
potency. Removal of the core carbonyl group was also shown to
be detrimental to GT-1b activity as exemplified by compound
28. Together, these results seemed to imply that the
piperazinone N-1 nitrogen and C-2 carbonyl groups might be
providing an important hydrogen bond donor and acceptor
interaction with the NS4B protein. The cis stereochemical
relationship between C-3 and C-6 of the piperazinone core was
also shown to be important because changing the stereo-
chemistry at C-3 or C-6 to the R-stereochemistry as shown in
compound 29 and 30 resulted in complete loss of activity.
Molecular modeling of 2a indicated that the lowest energy
conformers of 2a have the i-Bu at C-3 above plane and the i-Bu
at C-6 in plane with the piperazinone ring. In the case of 29 and
30, the i-Bu substituents at C-3 and C-6 are in opposite
orientation to each other and also out of plane to the
piperazinone ring. On the basis of these molecular modeling
and activity results, we concluded that the cis stereochemical
relationship between C-3 and C-6 substituents of the
piperazinone core confers the preferred orientation for tight
binding.
a
Reagents and conditions: (a) glyoxylic acid monohydrate, bis(4-
methoxyphenyl)methanamine, DCM (94%); (b) MeNHOMe·HCl,
TBTU, DIPEA, CH3CN (93%); (c) LiAlH4,THF, −78 °C, then NH2-
Leu-OMe, NaBH(OAc)3, DCM (49%); (d) 70% aqueous AcOH,
reflux (75%); (e) 5-(4-fluorophenyl)-isoxazole-3-carboxylic acid,
TBTU, DIPEA, DCM (50%).
a
Scheme 3. Synthesis of Oxazole Derivative 108
Futher investigation of the C-3 and C-6 core substitutents
showed that substituting an ethyl moiety for the i-Bu at C-3
(31) or C-6 (32) resulted in substantial loss of activity.
Replacing the i-Bu moiety with a sec-Bu (33) group at C-6 also
led to a loss of GT-1b potency. The only modification to 2a
that maintained GT-1b activity was replacement of n-Pr (34)
for i-Bu at C-6. The observed results for modifications at C-3
and C-6 suggest that these positions are sensitive to the nature
of the hydrophobic groups. On the basis of the piperazinone
core SAR, it is apparent that the amide function and the cis
stereochemistry (S,S) of the piperazinone core are essential for
GT1b activity.
A series of replacements for the cyclopropyl moiety were
investigated (Table 2). Replacement of the cyclopropyl amide
moiety of 2a with propiolamide (35) produced a 3-fold loss in
activity. Cinnamide (trans-E) 36 showed 2-fold loss in activity,
while the cis-(Z)-cinnamide (37) showed a complete loss of
activity. These findings indicated that the trans geometry of the
a
Reagents and conditions: (a) HCl in dioxane, 70 °C; (b) (BOC)2O,
DIPEA, DCM, DMF (2 steps 87%); (c) DMP, DCM, 0 °C to RT; (d)
tosylmethyl isocyanide, K2CO3, MeOH, 70 °C (2 steps 15%); (e) HCl
in dioxane, DCM, 0 °C to RT; (f) HATU, DIPEA, DMF at RT (2
steps, 70%).
group and coupled with 5-(4-fluorophenyl)isoxazole-3-carbox-
ylic acid to furnish inhibitor 108.
Synthesis of piperazinone core derivative 109 bearing a
oxazole moiety began with Fmoc protection of (S)-2-amino-
but-3-en-1-ol (17) to give Fmoc protected alcohol 18 (Scheme
4). Oxidation of 18 with Dess−Martin’s reagent followed by
reductive amination with (S)-2-amino-4-methylpentanoic acid
methyl ester furnished 19. Compound 19 was converted to the
piperazinone core 20 via the reaction sequence outlined in
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a
Scheme 4. Syntheses of Inhibitors 21, 22, and 109
a
Reagents and conditions: (a) Fmoc-Cl, aq dioxane, 0 °C to RT (quantitative); (b) DMP, DCM; (c) (S)-2-amino-4-methylpentanoic acid methyl
ester HCl salt, NaBH(OAc)3, THF (2 steps 42%); (d) 40% Et2NH in EtOH, 60 °C (42%); (e) 5-(4-fluorophenyl)isoxazole-3-carboxylic acid,
HOBT hydrate, EDC, DIPEA, CH3CN (30%); (f) OsO4, oxone, DMF (60%); (g) 2,2-dimethoxy-ethylamine, HOBT hydrate, EDC, DIPEA,
CH3CN (50%); (h), TFA, DCM (75%).
a
Scheme 5. Synthesis of Pyrrolidine Derivative 111
a
Reagents and conditions: (a) glyoxylic acid monohydrate, bis(4-methoxyphenyl)methanamine, DCM; (b) MeNHOMe·HCl, TBTU, DIPEA,
CH3CN (2 steps 98%); (c) LiAlH4,THF, −78 °C, then NH2-Leu-OMe, NaBH(OAc)3, DCM, (67%); (d) 5-(4-fluorophenyl)-isoxazole-3-carboxylic
acid, TBTU, DIPEA, CH3CN; (e) 70% aq AcOH, reflux (2 steps 12%).
E
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Table 1. SAR of Piperazinone Core
Table 2. SAR of Cyclopropyl Amide Side Chain
Replacements
a
For description of the assay, see ref 15. Replicon data is the average of
n = 2 with 0.2 μM SD.
involved in π−π interactions with the target protein.
Replacement of cyclopropyl moiety with a bulky pheny moiety
(40) led to significant loss in GT-1b activity. On the basis of
the observed SAR, we decided to utilize the trans-cinnamide
side chain for further SAR studies.
In an effort to improve the potency of the nonchiral
cinnamide side chain, investigation of phenyl group substituent
effects was undertaken. Several analogues16 were prepared by
coupling the piperazinone core (7) with commercially available
cinnamic acid derivatives (Scheme 1). For selected compounds,
GT1b inhibitory activity is shown in Table 3. Methyl (41),
trifluoromethyl (42), and methoxy (43) phenyl substituents
were not tolerated, as indicated by the significant decrease in
GT1b activity. Loss of activity was also observed for
compounds having a nitro (44), dimethylamino (45), methyl
sulfone (46), or carboxylate (47) phenyl substituent. These
results indicate that strong electron withdrawing and polar
functionalities were not tolerated on the phenyl moiety of the
cinnamide side chain. Improvement in potency was seen for p-
chloro (49) and p-fluoro (52) phenyl-substituted cinnamide
derivatives, however, these derivatives only showed comparable
potency to 2a. The p-Br, m-Cl, and o-Cl analogues 48, 50, and
51 showed 4-fold loss in activity. Interestingly, the disubstituted
compounds 53 and 54 did demonstrate GT-1b potency similar
to the mono substituted analogue 52. For the cinnamide series,
the SAR showed that substituents at the para-position
a
For description of the assays, see ref 15. Replicon data is the average
of n = 2 with 0.2 mM SD.
side chain is important for potency. Further analysis indicated
that the side chain carbonyl group may not be required for
potency as shown by the N-alkylated analogue 38. Removal of
side chain phenyl group (39) led to complete loss of activity,
indicating that the phenyl group is essential and might be
F
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substituents. The replicon potency of compounds 49, 52, and
53 which lack the chiral cyclopropyl unit was shown to be
comparable to that observed for compound 2a and,
consequently, served as the basis for further lead optimization
by exploring cinnamide double-bond replacements.
Table 3. SAR of the trans-Cinnamide Side Chain Analogues
To further improve the potency relative to compounds
having the cinnamide side chain (52), we investigated amide
side chains having 5-membered heterocyclic ring systems in
place of the cinnamide double bond. The hope was that such a
replacement might provide additional electrostatic interactions
while still maintaining a favorable geometry. A commercially
available diverse set of heterocyclic acid derivatives capable of
forming H-bond acceptor and/or donor interactions were
coupled to the piperazinone core16 by the method shown in
Scheme 1, and the activity of selected compounds is shown in
Table 4. Compound 56 exhibiting H-acceptor characteristics
Table 4. HCV Replicon Activity of Heterocyclic Amide Side
Chain
a
For description of the assay, see ref 15. Replicon data is the average of
n = 2 determinations with 0.2 μM SD.
a
For description of the assay, see ref 15. Replicon data is the average of
led to a several-fold decrease in GT-1b activity. Similarly, the
oxazole 57 and imidazole 60 did not show GT-1b activity up to
10 μM. Pyrazole 61 and imidazole 62 having H-donor ability
were also shown to be inactive up to 10 μM. However, the
isoxazole 58 and oxadiazole 59 showed a 2-fold increase in GT-
1b activity. We surmise that both 67 and 68 provide favorable
n = 2 determinations with 0.2 μM SD.
consistently produced compounds with improved potency
relative to compounds having a meta- or ortho-substituted
phenyl moiety and that fluoro and chloro are optimal
G
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substituent orientation and electronic character to result in the
observed 2-fold increase in GT-1b potency when compared to
the corresponding cinnamide 36. Replacing the side chain
phenyl group of 58 with other heterocyclic moieties that can
provide similar π−π interaction with protein did not pan out.
Isoxazole 58 became the basis for further lead optimization by
studying substituent effects on the phenyl moiety.
Table 5. Effect of Phenyl Ring Substitution on the GT-1b
Potency of Isoxazole Derivatives
The next step in our attempt to improve the GT-1b potency
beyond that of 58 investigated the effect of substituents on the
phenyl moiety while maintaining intact the piperazinone core
and isoxazole amide side chain (Table 5). Synthesis of these
compounds was achieved by the method in Scheme 1. As
expected (see Table 3), the para-substituted methyl (63),
methoxy (64), cyano (70), and nitro (72) derivatives showed
no improvement in GT-1b activity, while the GT-1b potency of
para-F (67), para-Cl (65), and para-Br (66) derivatives
increased by 8-fold, 4-fold, and 2-fold, respectively. The 3,4-
and 2,4-disubstituted compounds (68 and 69) showed a 5-fold
decrease in potency when compared to the para-F substituted
compound (67). After optimizing the GT-1b potency of the
piperazinone side chain, we decided to focus on modification of
the core in an attempt to further improve the GT-1b potency.
Piperazinone core C-6 substituent effects were probed using
hydrophobic moieties (acyclic, branched, and cyclic) to study
space constraints and substituents having H-acceptor and/or
H-donor moieties to evaluate potential electrostatic interactions
(Table 6).16 The neopentyl (75), sec-butyl (76), and tert-butyl
(84) derivatives produced a significant reduction in GT-1b
activity compared to 67. The n-propyl analogue 73 retained
potency, while the ethyl analogue 82 and n-Bu analogue 80
showed a 6−4-fold decrease in potency, respectively. The
propenyl compound 74 exhibited 2-fold improvement in GT-
1b potency, yet the allyl moiety (77) and vinyl moiety (21)
containing compounds showed 3-fold and 8-fold decrease in
GT-1b potency, respectively. The cyclopropyl methyl com-
pound 78 showed 3-fold decrease in potency, and the
cyclopropyl analogue 85 showed 8-fold loss of potency. The
i-propyl analogue 83 retains the GT-1b potency of parent
compound 67. The thio-ether analogues 79 and 81 showed 2-
and 3-fold decrease in GT-1b potency, respectively. Among the
cycloalkyl analogues, cyclobutyl derivative 86 showed 6-fold
loss of potency, but the cyclopentyl analogue 87 proved to be a
slightly more potent inhibitor than 67. Among the 6-membered
cycloalkyl analogues, cyclohexyl compound 88 proved to be
slightly less potent, while the tetrahydropyran analogue 93
showed a 4-fold loss of potency, and thio-pyran 94 showed 8-
fold decrease in potency. The phenyl analogue 95 showed
potency similar to that seen for 74 and 87 but demonstrated
better metabolic stability17 and was chosen for further SAR
optimization.
a
The substituent effects on the C-6 phenyl group of 95 were
investigated but did not lead to any improvement in potency
(Table 7). A nearly 10-fold decrease in GT-1b potency was
observed when a methyl (96) or fluoro (99) substituent was
introduced at the 4-position of the phenyl moiety. Significant
loss of potency also occurred with introduction of chloro (98)
and trifluoromethyl (102) substitution, indicating that the
substitution at the 4-position of the phenyl moiety was not well
tolerated. In the case of fluoro substituted compounds,
substitution at the meta position (100) resulted in 4-fold loss
of potency, while the ortho-F compound (101) showed only a
modest loss of potency. Because we were not able to improve
the GT-1b potency of inhibitor 95 through the addition of
For description of the assay, see ref 15. Replicon data is the average of
n = 2 determinations with 0.20 μM SD.
substituents on the phenyl moiety, our next step was to
investigate heterocyclic moieties as replacements for the C-6
phenyl group.
We envisioned that introduction of a heterocyclic moiety at
the C-6 position of the piperazinone ring should result in
retention of any favorable (π−π) interactions provided by a C-6
phenyl group and may introduce additional interactions that
could further improve the GT1 potency. Consequently, we
synthesized a series of C-6 heterocyclic derivatives (103−113)
H
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Table 6. Piperazinone C-6 Substituent SAR
Table 7. Substitution Effects on the GT-1b Potency of
Piperazinone C-6 Phenyl Analogues
a
For description of the assay, see ref 15. Replicon data is the average of
n = 2 determinations with 0.20 μM SD.
unsubstituted 2-pyrrole compound 111 showed a 11-fold loss
of activity, but the 3-pyrrole compound 112 showed similar
potency to the phenyl analogue 98.
a
For description of the assay, see ref 15. Replicon data is the average of
An attempt to further optimize the potency of 3-thiophene
compound (104) led to the 2-chloro thiophene derivative
(113) that showed a 6-fold improvement in the GT-1b potency
(Table 8). Further evaluation of inhibitors 103 and 108 in both
GT-1a and 1b replicons showed that the GT-1b potency was
superior to GT-1a potency (Table 9). The thiophene derivative
103 exhibited a preferable metabolic stability, yet the oxazole
108 had superior solubility characteristics and none of these
compounds showed any CYP (1A2, 2C9, 2D6, and 3A4)
inhibition up to 10 μM. Neither 103 nor 108 demonstrated any
appreciable GT-2 activity when tested in a replicon assay.
Unfortunately, due to their lack of broad genotype coverage,
the piperazinone class of NS4B inhibitors did not progress
further into preclinical development.
n = 2 determinations with 0.20 μM SD and for 95 it was 0.02 μM
SD.
using synthetic methods depicted in Schemes 2−5 (Table 8).
The 2-thiophene compound 103 showed a dramatic 10-fold
increase in potency, while the 3-thiophene compound 104
showed a less dramatic 3-fold increase the GT1b potency when
compared to the corresponding phenyl analogue 95. The 3-
methyl-thiophene compound 105 was 110-fold less active, and
5-methyl-thiophene 106 was 30-fold less active than the
unsubstituted 2-thiophene analogue 103. The furan derivative
107 was 3-fold less active than 95 and 30-fold less active than
the 2-thiophene derivative 103. The 2-oxazole 109 and 2-
thiazole 110 were inactive, but the 4-oxazole 108 retained the
activity of the phenyl derivative 95. The results for the oxazoles
108 and 109 suggested that the position of the nitrogen atom
in the heterocyclic ring was critical to the GT-1b potency. The
CONCLUSION
■
HCV infection is a disease that afflicts a large patient
population with negative long-term consequences. Research
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which HCV nonstructural protein was the target for
piperazinone 2a revealed that mutations at residues 90 and
98 in the TM1 domain of NS4B resulted in a significant
reduction in the HCV inhibitory activity of compound 2a, thus
supporting NS4B was the primary target. An extensive
chemistry-driven SAR development effort looking at multiple
positions of the piperazinone core and amide side chain was
performed on compound 2a. Although we did not have
structural information to guide the inhibitor design, we were
able to effectively develop a robust SAR picture that led to
potent HCV GT1 inhibitors. Initial studies kept the
piperazinone core constant, explored the side chain cyclopropyl
replacements as well as the substituents on the side chain
phenyl moiety to optimize compound 2a potency. These
studies resulted in the p-fluorophenylisoxazole amide as the
optimized side chain as shown in compound 67. The core C-6
SAR investigation demonstrated that the i-Bu moiety could be
replaced with a phenyl moiety and resulted in molecule 95 with
double-digit nanomolar GT-1b potency. Further optimization
of the lead compounds GT-1b potency led to the single-digit
nanomolar GT-1b and low double-digit nanomolar GT-1a
HCV inhibitors 103 and 108 having acceptable metabolic
stability and solubility parameters. Through this effort, we have
discovered a useful novel prototype inhibitor of HCV NS4B
that has good genotype 1b and 1a potency with suitable
stability, solubility parameters, and no CYP liabilities but lacked
broad genotype coverage suitable for further development.
The identification and optimization of the piperazinone class
of NS4B inhibitors did however introduce a new structural class
of NS4B inhibitors into the repertoire of HCV replication
inhibitors. Each of the previously identified NS4B inhibitors
1a−d incorporates a critical 6,5-bicyclic ring system containing
similar patterns of substitution. In fact, compounds 1b and 1c
are based on the original anguizole motif where each are
substituted similarly and include an essential amide side chain.
Even though indole inhibitor 1d does not display an amide side
chain directly attached to the 5-membered ring of the bicycle, it
shares with 1b and 1c a common amide-type functionality on
its side chain at a similar distance from the 5-membered ring
point of attachment. The clear structural similarities among
inhibitors 1a−d and the existence of identical resistance profiles
supports the hypothesis that they all share a common binding
site on the NS4B protein. In stark contrast to the known NS4B
inhibitors 1a−d, the piperazinone class of inhibitors exemplified
by lead 2a and optimized compound 103 do not incorporate a
bicyclic nucleus, however, this class does incorporate an amide
side chain. Any attempts to incorporate structural features, such
as bicyclic systems, similar to those noted in 1a−d, resulted in
loss of activity. In addition, efforts to draw structural
comparisons between the piperazinones 2a and 103 with
inhibitors 1a−d did not lead to any understanding of common
topographical motifs. However, irrespective of the apparent lack
of significant structural similarity between the piperazinone
class of NS4B inhibitors and the previously reported inhibitors
1a−d, they all share a F98L resistant phenotype and lose
substantial activity when assayed against the GT2a replicon
where the wild-type phenotype is L98. It can therefore be
surmised that because of this common resistant amino acid
substitution and activity loss against the GT2a replicon, there
must be some overlap in binding sites between the structurally
similar inhibitors 1a−d and the piperazinone class of NS4B
inhibitors. Therefore, it may be reasonable to speculate that
overlapping binding sites occur in the region of the NS4B
Table 8. Optimization of GT-1b Potency with C-6
Heterocyclic Moieties
a
For description of the assay, see ref 15. Replicon data is the average of
n = 2 determinations with 0.025 μM SD, and for 113 it was 0.002
μM SD.
has shown that the combination of potent direct acting
antivirals can provide a cure for this debilitating and often fatal
disease. Therefore, novel direct acting antiviral agents acting by
new mechanisms of action have the potential to become part of
future therapeutic regimens. With this desire in mind, an HTS
campaign was undertaken that led to the identification of
piperazinone 2a as a novel and specific HCV inhibitor.
Compound 2a was shown to be an effective inhibitor of
HCV genotype 1b and 1a replicons, with a 50-fold activity/
toxicity window in whole cells and excellent selectivity over
other viruses. Resistance mapping studies aimed at determining
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a
Table 9. Lead Compounds Profiling Data
a
For description of the assays, see ref 15. GT 1a Replicon data is the average of n = 2 determinations with 0.010 μM SD.
× 30 mm × 2 mm column was used. The mobile phase was HPLC
grade water (A) and HPLC grade acetonitrile (B) system. SFC
separation conditions were as follows: Berger Multi-Gram SFC from
Mettler Toledo Co, Ltd.; column, AD 250 mm × 30 mm (5 μm), 38
°C; mobile phase, supercritical CO2 (A), MeOH (B), A/B = 75/25 at
60 mL/min; nozzle pressure (100 Bar), nozzle temperature (60 °C);
evaporator temperature (20 °C); trimmer temperature (25 °C);
wavelength (220 nm). LC/MS was conducted on Shimadzu LCMS
2010EV using electrospray positive [ES + ve to give MH+] equipped
with a Shim-pack XR-ODS 2.2 μm column (3.0 mm × 30 mm, 3.0 mm
i.d.), eluting with 0.0375% TFA in water (solvent A) and 0.01875%
TFA in acetonitrile (solvent B). Unless specified otherwise, Column
chromatography was performed on Intelli Flash 280 (AnaLogix) using
silica flash columns.
protein where the amide side chains of 1a−d and that of the
piperazinones bind. However, definitive proof of this common
binding site will only occur when cocrytal structures of these
different inhibitors with the NS4B protein are obtained. The
possibility exists that with the identification of the structurally
novel and potent piperazinone class of NS4B inhibitors in
conjunction with the information reported for the other classes
of NS4B inhibitors, further work may lead to the identification
of NS4B inhibitors that can overcome the genotype coverage
limitations that currently plague the search for clinically viable
NS4B inhibitors.
EXPERIMENTAL SECTION
■
General Procedures for the Synthesis of Piperazinone Core.
(3S,6S)-3,6-Diisobutylpiperazin-2-one (7). A. (S)-(9H-Fluoren-9-yl)-
methyl-(1-methoxy-(methyl)-amino)-4-methyl-1-oxopentan-2-yl)-
carbamate (4). A mixture of FMOC-Leu-OH (3, 0.5 g, 1.41 mmol),
TBTU (0.68 g, 2.12 mmol), and N,O-dimethylhydroxylamine
hydrochloride (0.207 mg, 2.12 mmol) in MeCN (5 mL) were stirred
at 0 °C under nitrogen for 10 min. DIPEA (0.736 mL, 4.23 mmol) was
added dropwise (5 min) and stirred for 30 min. The reaction mixture
was allowed to warm to rt and stirred for 2.5 h. The reaction mixture
concentrated in vacuo, and the residue was dissolved in EtOAc (100
mL) and washed sequentially with 1 M aqueous HCl (15 mL × 2), 1
M aqueous NaHCO3 (15 mL × 2), and brine (20 mL). The solution
was dried over Na2SO4 and concentrated in vacuo to dryness to afford
4 (574 mg, 98% yield), which was used for the next step without
General Methods. Unless specified otherwise, starting materials
were available from commercial sources. Dry solvents and reagents
were of commercial quality and were used as purchased. Nuclear
magnetic resonance (NMR) spectra were recorded on Bruker Advance
II 400 MHz and Varian AS 400 MHz spectrometers at room
temperature with tetramethylsilane as an internal standard. Chemical
shifts (δ) are reported in parts per million (ppm), and peak
multiplicity are reported as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), or br s (broad singlet). Purities of the final
compounds were determined by HPLC and UPLC-MS and were
≥95% purity. HPLC conditions to assess ≥95% purity of final
compounds were as follows: Shimadzu HPLC 20AB Sepax HP-C18
4.6 mm × 50 mm (5 μm); flow rate, 3.0 mL/min; acquisition time, 6
min; wavelength, UV 230 nm; oven temperature, 40 °C. UPLC-MS
conditions to assess ≥95% purity for final compounds were as follows:
Waters Acquity UPLCMS UPLC BEH C18 2.1 mm × 50 mm (1.7
μm); flow rate, 0.8 mL/min; wavelength, UV 254 nm; oven
temperature, 50 °C. Chiral HPLC was conducted on Shimadzu
HPLC 20A. The preparative HPLC system includes two sets of Gilson
306 pumps, a Gilson 156 UV/vis detector, and a Gilson 215 injector
and fraction collector, with Unipoint control software. A YMC 25 mm
1
further purification. H NMR (400 MHz, CDCl3) δ 7.76−7.58 (m, 4
H), 7.41−7.29 (m, 4 H), 5.42 (d, J = 9.2 Hz, 1 H), 4.82−4.80 (m, 1
H), 4.37−4.32 (m, 2 H), 4.23−4.20 (t, J = 14.0 Hz, 1 H), 3.79 (s, 3
H), 3.21 (s, 3 H), 1.74−1.70 (m, 1 H), 1.52−1.48 (m, 1 H), 0.99−0.94
(m, 6 H). MS (ESI): m/z 413.2 (M + 1).
B. (S)-(9H-Fluoren-9-yl)methyl (4-methyl-1-oxopentan-2-yl)-
carbamate (5). To a solution of 4 (0.4 g, 0.97 mmol) in dry THF
(10 mL) at −78 °C was added LiAlH4 (80 mg, 1.94 mmol), and the
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reaction was stirred under argon for 30 min. The reaction was
quenched with 6% aqueous HCl (10 mL, pH 4−5) at −78 °C, and the
solution was allowed to warm to 0 °C and stirred for 5 min. This
mixture was partitioned between EtOAc/brine (1:2, 100 mL) and
extracted with EtOAc (50 mL). The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo to afford 5 (320
mg, 98% yield), which was used for the next step without further
concentrated in vacuo to afford compound 9 (3.5 g, 94% yield) as
pale-yellow foam. The crude product was used for the next step
without further purification. 1H NMR (400 MHz, CDCl3) δ 7.31 (s, 1
H), 7.27−7.24 (m, 4 H), 6.81−6.79 (m, 4 H), 6.28 (br s, 1 H), 6.24
(br s, 1 H), 4.80 (s, 1 H), 4.44 (s, 1 H) 3.76 (s, 3 H), 3.75 (s, 3 H)
3.70 (s, 1 H). MS (ESI): m/z 368.0 (M + 1).
B. 2-((Bis(4-methoxyphenyl)methyl)amino)-2-(furan-2-yl)-N-me-
thoxy-N-methyl-acetamide (10). To a mixture of 9 (0.5 g, 1.36
mmol), TBTU (0.66 g, 2.04 mmol), and N,O-dimethylhydroxylamine
hydrochloride (0.2 g, 2.04 mmol) in dry MeCN (6 mL), DIPEA (710
uL, 4.08 mmol) was added and stirred at rt for 4 h. The reaction
mixture was concentrated in vacuo, and the crude product was purified
by column chromatography (0−40% EtOAc in hexanes) to afford
1
purification. H NMR (400 MHz, CDCl3) δ 9.58 (s, 1 H), 7.77−7.59
(m, 4 H), 7.42−7.30 (m, 4 H), 5.18 (d, J = 7.2 Hz, 1 H), 4.45 (d, J =
6.8 Hz, 2 H), 4.34−4.32 (m, 1 H), 4.24−4.21 (t, J = 6.8 Hz, 1 H),
1.75−1.68 (m, 2 H), 1.48−1.39 (m, 1 H), 0.98−0.96 (m, 6 H). MS
(ESI): m/z 438.1 (M + 1).
C. (S)-Methyl 2-(((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)-
amino)-4-methylpentyl)-amino)-4-methylpentanoate (6). To a
well dried mixture of aldehyde 5 (290 mg, 0.86 mmol) and Leu-
OMe hydrochloride (160 mg, 0.86 mmol) were added DCM (3 mL)
and NaBH(OAc)3 (260 mg, 1.2 mmol) and stirred at rt under
nitrogen for 6 h. The reaction was quenched with saturated aqueous
NaHCO3 (50 mL) and was extracted with EtOAc (50 mL) and dried
over Na2SO4. The solvent was concentrated in vacuo and the crude
product purified by column chromatography (0−25% EtOAc in
1
compound 10 (520 mg, 93% yield) as a white foam. H NMR (400
MHz, CDCl3) δ 7.37 (s, 1 H), 7.34−7.25 (m, 4 H), 6.85−6.79 (m, 4
H), 6.33−6.32 (m, 1 H), 6.22 (d, J = 3.2 Hz, 1 H), 4.83 (s, 1 H), 4.68
(s, 1 H) 3.77 (s, 3 H), 3.75 (s, 3 H) 3.28 (s, 3 H), 3.20 (s, 3 H). MS
(ESI): m/z 411.1 (M +1).
C. (2S)-Methyl 2-((2-((Bis(4-methoxyphenyl)methyl)amino)-2-
(furan-2-yl)ethyl)amino)-4-methyl-pentanoate (11). To a solution
of compound 10 (0.2 g, 1.22 mmol) in dry THF (3 mL) at −78 °C,
LiAlH4 (40 mg, 0.97 mmol) was added and stirred at −78 °C under
argon for 3 h. The reaction mixture was quenched with saturated
aqueous NH4Cl solution (2 mL) by dropwise addition. This mixture
was partitioned between EtOAc and brine (1:1, 50 mL) and extracted
with EtOAc (50 mL). The organic layers were combined and washed
with brine, dried over MgSO4, and concentrated in vacuo. The crude
aldehyde intermediate was used for the next step without further
purification. To a solution of aldehyde (0.4 g, 1.14 mmol) and HCl salt
of Leu-OMe (210 mg, 1.14 mmol) in DCM (8 mL), NaBH(OAc)3
(340 mg, 1.59 mmol) was added and the mixture was stirred at rt
under argon for 6 h. The reaction mixture was quenched with
saturated aqueous NaHCO3 solution (30 mL), and the aqueous
solution was extracted with EtOAc (30 mL), dried over Na2SO4, and
concentrated in vacuo. The crude product was purified by column
chromatography (0−40% EtOAc in hexanes) to afford compound 11
(0.27 g, 49% yield) as a pale-yellow oil. 1H NMR (400 MHz, CDCl3)
δ 7.37−7.36 (m, 1 H), 7.30−7.17 (m, 4 H), 6.85−6.76 (m, 4 H),
6.32−6.31 (m, 1 H), 6.16−6.11 (m, 1 H), 4.64 (d, J = 5.2 Hz, 1 H),
3.78−3.67 (m, 7 H) 3.28−3.20 (m, 1 H), 2.94−2.88 (m, 1 H) 2.75−
2.66 (m, 1 H), 1.96 (s, 1 H), 1.70−1.64 (m, 1 H), 1.48−1.40 (m, 2 H),
0.94−0.86 (m, 6 H). MS (ESI): m/z 481.1 (M + 1).
1
hexanes) to afford 6 (360 mg, 90% yield) as a white solid. H NMR
(400 MHz, CDCl3) δ 7.77−7.59 (m, 4 H), 7.41−7.29 (m, 4 H), 4.82
(d, J = 5.6 Hz, 1 H), 4.40 (d, J = 7.2 Hz, 2 H), 4.24 (t, J = 6.8 Hz, 1
H), 3.71 (s, 3 H), 3.27−3.24 (m, 1 H), 2.71−2.42 (m, 2 H), 1.76−1.60
(m, 2 H), 1.46−1.30 (m, 4 H), 0.92−0.87 (m, 12 H). MS (ESI): m/z
467.2 (M + 1).
D. (3S,6S)-3,6-Diisobutylpiperazin-2-one (7). To a solution of 6
(120 mg, 0.26 mmol) in DCM (1.6 mL) was added diethyl amine (0.4
mL, 3.86 mmol 20% in DCM) at rt under nitrogen and stirred
overnight. The reaction mixture was concentrated in vacuo, and the
crude product was purified by column chromatography (0−25%
EtOAc in hexanes) to afford 7 (52 mg, 95% yield) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 5.83 (s, 1 H), 3.47−3.39 (m, 2 H), 3.02−
2.98 (m, 1 H), 2.79−2.74 (m, 1 H), 1.80−1.54 (m, 4 H), 1.45−1.31
(m, 2 H), 0.96−0.90 (m, 12 H). MS (ESI): m/z 213.1 (M + 1).
( 3 S , 6 S ) - 3 , 6 - D i i s o b u t y l - 4 - ( ( 1 R , 2 R ) - 2 -
phenylcyclopropanecarbonyl)piperazin-2-one (2a) and (3S,6S)-3,6-
Diisobutyl-4-((1S,2S)-2-phenylcyclopropanecarbonyl)piperazin-2-
one (2b). To a well dried ( )-(1R,2R)-2-phenylcyclopropanecarbox-
ylic acid (53.5 mg, 0.33 mmol), TBTU (127.5 mg, 0.396 mmol), and 7
(70 mg, 0.33 mmol) was added MeCN (3 mL) followed by DIPEA
(0.17 mL, 0.99 mmol) dropwise (5 min) and stirred at rt for 3 h. The
reaction mixture was concentrated in vacuo, and the crude residue was
dissolved in EtOAc (25 mL) and washed sequentially with 1 M
aqueous HCl (15 mL × 2), 1 M aqueous NaHCO3 (15 mL × 2), and
brine (20 mL). The solution was dried over Na2SO4 and concentrated
in vacuo, and the crude product was purified by column
chromatography (0−60% EtOAc in hexanes) to afford a mixture of
products 2a and 2b (105.8 mg, 90% yield). The product mixture (2a
and 2b) was separated by SFC (chiral) column to furnish 2a (30 mg,
25% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.50 (s,
1 H), 3.23−3.26 (m, 1 H), 3.07 (dd, J = 9.6 Hz, 3.6 Hz,1 H), 2.80 (dd,
J = 12.8 Hz, 4.4 Hz, 1 H), 2.58 (dd, J = 13.2 Hz, 5.2 Hz, 1 H), 2.36 (s,
1 H), 1.74−1.78 (m, 1 H), 1.59−1.65 (m, 1 H),1.28−1.48 (m, 4 H),
0.80−0.89 (m, 12 H). MS (ESI): m/z 213.1 (M + 1). Compound 2b
D. (3S,6R)-6-(Furan-2-yl)-3-isobutylpiperazin-2-one (12) and
(3S,6S)-6-(Furan-2-yl)-3-isobutylpiperazin-2-one (13). Compound
11 (0.4 g, 0.84 mmol) was dissolved in 70% aqueous AcOH (5
mL) and heated under reflux for 3 h. The reaction mixture was
concentrated in vacuo, and the crude product was purified by column
chromatography (20−60% EtOAc in hexanes) to afford compounds
12 (85 mg, 40% yield) and 13 (75 mg, 35% yield). Compound 12: 1H
NMR (400 MHz, CDCl3) δ 7.39−7.38 (m, 1H), 6.40 (s, 1H), 6.45−
6.34 (m, 1H), 6.26−6.25 (m, 1H), 4.63−4.61 (m, 1H), 3.46−3.43 (m,
1H), 3.24−3.23 (m, 2H), 1.91 (s, 1H), 1.89−1.79 (m, 2H), 1.53−1.47
(m, 1H) 0.95−0.82 (m, 6H). MS (ESI): m/z 223.0 (M + 1).
1
Compound 13: H NMR (400 MHz, CDCl3) δ 7.38−7.37 (m, 1H),
6.34−6.33 (m, 1H), 6.26−6.25 (m, 1H), 6.07 (s, 1H), 4.68 (m, 1H),
3.75 (d, J = 7.2 Hz, 1H), 3.47−3.43 (m, 1H), 3.40−3.35 (m, 2H),
3.06−3.01 (m, 1H), 1.89−1.78 (m, 2H), 1.60−1.54 (m, 1H) 0.97−
0.92 (m, 6H). MS (ESI): m/z 223.0 (M + 1).
1
(29 mg, 25% yield, white solid): H NMR (400 MHz, DMSO-d6) δ
7.50 (s, 1 H), 3.23−3.26 (m, 1 H), 3.07 (dd, J = 9.6 Hz, 3.6 Hz,1 H),
2.80 (dd, J = 12.8 Hz, 4.4 Hz, 1 H), 2.58 (dd, J = 13.2 Hz, 5.2 Hz, 1
H), 2.36 (s, 1 H), 1.74−1.78 (m, 1 H), 1.59−1.65(m, 1 H),1.28−1.48
(m, 4 H), 0.80−0.89 (m, 12 H). MS (ESI): m/z 213.1 (M + 1).
(3S,6R)-6-(Furan-2-yl)-3-isobutylpiperazin-2-one (12) and
(3S,6S)-6-(Furan-2-yl)-3-isobutylpipera-zin-2-one (13). A. 2-((Bis(4-
methoxyphenyl)methyl)amino)-2-(furan-2-yl)acetic Acid (9). To a
stirred solution of glyoxylic acid monohydrate (920 mg, 10.11 mmol)
in DCM (70 mL) were added bis(4-methoxyphenyl)methanamine
(2.46 g, 10.11 mmol) and 2-furanboronic acid (1.13 g, 10.11 mmol),
and the reaction mixture was stirred at rt for 2 min to form clear
solution. The solution was purged with argon (2 min), and the sealed
reaction mixture was stirred for overnight. The reaction mixture was
(3S,6R)-6-(tert-Butoxymethyl)-3-isobutylpiperazin-2-one (14).
Synthesized from FMOC-L-Ser-OtBu (42.0 g, 0.114 mol) and HCl
salt of Leu-OMe (21.0 g, 0.115 mol) by the method described for the
compound 7 (Scheme 1) to afford 14 (4.2 g, 5.7% yield) as a colorless
1
gum. H NMR (400 MHz, DMSO-d6) δ 7.38−7.37 (m, 1 H), 3.35−
3.39 (m, 1 H), 3.21−3.32 (m, 2 H), 3.03−3.06 (m, 1 H), 2.77−2.84
(m, 2 H), 1.72−174 (m, 1 H), 1.56 −1.57 (m, 1 H), 1.34−1.33 (m, 1
H), 1.121−1.126 (m, 9 H) and 0.82−0.88 (m, 6 H). MS (ESI): m/z
243.2 (M + 1).
(2S,5R)-tert-Butyl 5-(Hydroxymethyl)-2-isobutyl-3-oxopiperazine-
1-carboxylate (15). Compound 14 (2.00 g, 8.25 mmol) and HCl in
dioxane (4 N, 20 mL) were stirred at 70 °C for 1.5 h. The reaction
L
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mixture was concentrated in vacuo, and the crude compound was
dissolved in DMF/DCM (2:1, 30 mL) and DIPEA (3.5 mL) was
added followed by BOC-anhydride (1.60 mL, 6.96 mmol) in DCM (5
mL). After 5 h stirring, the reaction mixture was concentrated in
vacuo, and the oily residue was partioned in EtOAc (15 mL) and water
(15 mL). The organic layer was separated and washed with brine,
dried (MgSO4), concentrated in vacuo, and purified by column
chromatography (0−90% EtOAc in hexanes) to give 15 (2.06 g, 87%
yield) as a white solid. 1H NMR (DMSO-d6) δ 7.69 (s, 1 H), 4.88 (t, J
= 5.6 Hz, 1 H), 4.40−4.22 (m, 1 H), 4.11−3.90 (m, 1 H), 3.44−3.24
(m, 2 H), 2.90−2.76 (m, 2 H), 1.65−1.56 (m, 2 H), 1.51−1.46 (m, 1
H), 1.41 (s, 9 H), 0.91 (m, 6 H). MS (ESI): m/z 287.2 (M + 1).
(2S,5R)-tert-Butyl 2-Isobutyl-5-(oxazol-5-yl)-3-oxopiperazine-1-
carboxylate (16). To a solution of the alcohol 15 (0.50 g, 1.75
mmol) in DCM (25 mL) at 0 °C was added Dess−Martin reagent
(0.965 g, 4.55 mmol) and water (0.5 mL). Reaction mixture was
warmed to rt and stirred for 6 h. Then, i-PrOH (0.3 mL) was added
and the mixture was stirred for 15 min followed by saturated aqueous
sodium thiosulfate (10 mL) and saturated aqueous NaHCO3 (10 mL)
were added. Organic layer was separated, and the aqueous layer was
extracted with EtOAc (2 × 5 mL). Combined organic layers were
washed with brine, dried (MgSO4), and concentrated to give the
aldehyde product. To the crude aldehyde in MeOH (20 mL) was
added tosylmethyl isocyanide (480 mg, 2.46 mmol) and K2CO3 (483
mg, 3.50 mmol) and was stirred at 70 °C for 1 h. The reaction mixture
was concentrated in vacuo, and EtOAc (30 mL) and water (20 mL)
were added. Organic layer was separated, and the aqueous layer was
extracted with EtOAc (20 mL × 2). Combined organic layers were
washed with brine, dried (MgSO4), concentrated in vacuo, and
purified by column chromatography (0−95% EtOAc in hexanes) to
give 16 (78 mg, 14% yield) as a white solid. 1H NMR (CDCl3) δ 7.90
(s, 1 H), 7.08 (s, 1 H), 5.93 (br s, 1 H), 4.88−4.84 (m, 1 H), 4.69−
4.64 (m, 1 H), 4.50−4.44 (m, 1 H), 3.18 (t, J = 12.4 Hz, 1 H), 1.78−
1.62 (m, 3 H), 1.02 (d, J = 5.6 Hz, 3 H), 0.97 (d, J = 6.0 Hz, 3 H). MS
(ESI): m/z 324.1 (M + 1).
(2R,5S)-N-(2,2-Dimethoxyethyl)-4-(5-(4-fluorophenyl)isoxazole-3-
carbonyl)-5-isobutyl-6-oxopiperazine-2-carboxamide (23). A. (S)-
(9H-Fluoren-9-yl)methyl (1-Hydroxybut-3-en-2-yl)carbamate (18).
To a mixture of 17 (HCl salt, 1g, 8.1 mmol) and a solution of K2CO3
(3.4 g, 24.3 mmol) in water (50 mL) was added a solution of Fmoc-Cl
(2.3g, 8.91 mmol) in dioxane (50 mL) at 0 °C and stirred at rt for 3 h.
The reaction mixture was extracted with DCM (100 mL × 3) and
washed with saturated aqueous NH4Cl (50 mL) and brine (50 mL).
The organic layer was dried over Na2SO4 and concentrated in vacuo,
and the residue was purified by column chromatography (0−30%
EtOAC in hexanes) to give 18 (2.5 g, quantitative) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2 H), 7.60 (d, J = 7.6
Hz, 2 H), 7.41 (t, J = 7.6 Hz, 2 H), 7.32 (t, J = 7.6 Hz, 2 H), 5.89−5.74
(m, 1 H), 5.25 (d, J = 12.4 Hz, 2 H), 5.12 (s, 1 H), 4.45 (d, J = 6.8 Hz,
2 H), 4.40−4.26 (m, 1 H), 4.22 (t, J = 6.8 Hz, 1 H), 3.78−3.61(m, 2
H), 1.93(s, 1 H). MS (ESI): m/z 310.0 (M + 1).
was purified by column chromatography (10−50% EtOAC in
hexanes) to give 19 (1.1 g, 42% yield) as a pale-brown solid. 1H
NMR (400 MHz, CDCl3) δ 9.25(s, 1 H), 7.78−7.75 (m, 2 H), 7.62−
7.60 (m, 2 H), 7.41(t, J = 7.6 Hz, 2 H), 7.43−7.38 (m, 2 H), 7.34−
7.29 (m, 2 H), 5.81−5.74 (m, 1 H), 5.24−5.17 (m, 2 H), 4.46−4.39
(m, 3 H), 4.27−4.21 (m, 2 H), 3.71 (s, 3 H), 3.30 (t, J = 7 Hz, 1 H),
1.87−1.67 (m, 3 H), 0.98−0.87 (m, 6 H). MS (ESI): m/z 437.3 (M +
1).
C. (3S,6S)-3-Isobutyl-6-vinylpiperazin-2-one (20). To a solution of
19 (0.55 g, 1.26 mmol) in EtOH (6 mL) was added diethylamine (4
mL) at rt and heated at 60 °C overnight. The reaction mixture
concentrated in vacuo, and the residue was purified by column
chromatography (10−40% EtOAC in hexanes) to give 20 (92 mg,
42% yield) as a colorless solid. 1H NMR (400 MHz, CD3OD) δ 5.93−
5.85 (m, 1 H), 5.26−5.21 (m, 2 H), 4.03−3.95 (m, 1 H), 3.34−3.29
(m, 1 H), 3.02 (dd, J = 13.4 Hz, 4.8 Hz, 1 H), 2.85 (dd, J = 13.4 Hz,
4.8 Hz, 1 H), 1.88−1.80 (m, 1 H), 1.72−1.64 (m, 1 H), 1.55−1.48 (m,
1 H), 0.95(d, J = 6.4 Hz, 3 H), 0.92 (d, J = 6.4 Hz, 3 H). MS (ESI):
m/z 183.0 (M + 1).
D. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-vinylpiperazin-2-one (21). A mixture of 20 (61.9 mg, 0.34 mmol),
5-(4-fluorophenyl)-isoxazole-3-carboxylic acid (84.4 mg, 0.41 mmol),
HOBT hydrate (65.1 mg, 0.425 mmol), EDC (78.2 mg, 0.41 mmol),
and DIPEA (0.12 mL, 0.68 mmol) in MeCN (5 mL) were stirred at rt
overnight. The reaction mixture was concentrated in vacuo, and the
residue was purified by column chromatography (0−60% EtOAC in
1
hexanes) to give 21 (37.5 mg, 30% yield) as a brown solid. H NMR
(400 MHz, CDCl3) δ 7.80−7.77 (m, 2 H), 7.21−7.15 (m, 2 H), 6.87
(s, 1 H), 6.20 (s, 1 H), 5.76−5.68 (m, 1 H), 5.43 (d, J = 17.2 Hz, 1 H),
5.32 (d, J = 10.0 Hz, 1 H), 5.36−5.27 (m, 1 H), 4.88−4.82 (m, 1 H),
4.28−4.21 (m, 1 H), 3.21(dd, J = 14.0 Hz, 11.0 Hz, 1H), 1.92−1.67
(m, 3 H), 1.07 (d, J = 6.4 Hz, 3 H), 0.98 (d, J = 6.4 Hz, 3 H). MS
(ESI): m/z 372.1 (M + 1).
E. (2R,5S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-5-isobutyl-
6-oxopipera-zine-2-carboxylic Acid (22). To a solution of 21 (92.9
mg, 0.25 mmol) in DMF (5 mL) was added OsO4 (2.5% in t-BuOH,
0.03 mL, 0.0025 mmol) and stirred at rt for 5 min ,and then oxone
(614.8 mg, 1 mmol) was added and stirred at rt for 3 h. Na2SO3 (189
mg, 1.5 mmol) was added to the reaction mixture and stirred for 1 h,
and then EtOAc (20 mL) and 1N aqueous HCl (20 mL) were added.
The organic layer was separated, washed with 1N aqueous HCl (10
mL × 2) and brine (20 mL), dried over Na2SO4, and concentrated in
vacuo. The residue was purified by column chromatography (0−10%
MeOH in DCM) to give 22 (58.4 mg, 60% yield) as a brown solid. 1H
NMR (400 MHz, CD3OD) δ 7.95−7.91 (m, 2 H), 7.30−7.25 (m, 2
H), 7.05(s, 1 H), 5.16 (dd, J = 10.0 Hz, 4.0 Hz, 1 H), 4.78−4.70 (m, 1
H), 4.18−4.10 (m, 1 H), 3.49−3.43 (m, 1 H), 1.89−1.68 (m, 3 H),
1.06 (d, J = 6.0 Hz, 3 H), 0.99 (d, J = 6.0 Hz, 3 H). MS (ESI): m/z
390.0 (M + 1).
F. (2R,5S)-N-(2,2-Dimethoxyethyl)-4-(5-(4-fluorophenyl)-
isoxazole-3-carbonyl)-5-isobutyl-6-oxopiperazine-2-carboxamide
(23). Compound 22 (96 mg, 0.25 mmol) and 2,2-dimethoxy-
ethylamine (0.03 mL, 0.3 mmol) were coupled (96 mg, 0.25 mmol)
according to the method described for the preparation of compound
21 to give 23 (60 mg, 50% yield) as a pale-yellow gum. 1H NMR (400
MHz, CDCl3) δ 7.81−7.77 (m, 2 H), 7.22−7.17 (m, 2 H), 6.91 (s, 1
H), 6.60 (s, 1 H), 6.32 (t, J = 5.6 Hz, 1 H), 5.29−5.26 (m, 1 H), 5.11
(dd, J = 14.0 Hz, 4.0 Hz, 1 H), 4.43−4.38 (m, 3 H), 3.50−3.36 (m, 8
H), 1.93−1.65 (m, 3 H), 1.08 (d, J = 6.4 Hz, 3 H), 0.98 (d, J = 6.4 Hz,
3 H). MS (ESI): m/z 477.1 (M + 1).
B. (S)-Methyl 2-(((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)-
amino)but-3-en-1-yl)amino)-4-methyl-pentanoate (19). To a sol-
ution of 18 (1.85 g, 6 mmol) in anhydrous DCM (30 mL) was added
Dess−Martin’s reagent (5.33 g, 12.6 mmol) and stirred at rt for 1 h.
The reaction mixture was diluted with ether (21 mL), and a solution of
sodium thiosulfate (10.43 g, 66 mmol) in saturated aqueous NaHCO3
(20 mL) was added. The reaction mixture was stirred for 10 min, the
layers were separated, and the aqueous layer was extracted with ether
(50 mL × 3). The combined organic layers were washed with
saturated aqueous NaHCO3 (10 mL), water (10 mL), and brine (10
mL) and then dried over Na2SO4 and concentrated in vacuo to furnish
the crude aldehyde (1.8g, quantitative), which was used for the next
step without further purification. MS (ESI): m/z 308.0 (M + 1). To a
solution of the crude aldehyde (1.842 g, 6 mmol) in THF (20 mL)
was added HCl salt of Leu-OMe (1.2 g, 6.6 mmol) and NaBH(OAc)3
(1.9 g, 9 mmol) and stirred at rt overnight. The reaction mixture was
quenched with saturated aqueous NaHCO3 (20 mL) and extracted
with EtOAc (100 mL × 3). The combined organic layers were dried
over anhydrous Na2SO4 and concentrated in vacuo, and the residue
( 3 S , 6 S ) - 3 , 6 - D i i s o b u t y l - 1 - m e t h y l - 4 - ( ( 1 R , 2 R ) - 2 -
phenylcyclopropanecarbonyl)piperazin-2-one (27). To a mixture of
2a (100 mg, 0.281 mmol) and BMAP (0.5 mL, 1.800 mmol) in DCM
(25 mL) were added iodomethane (2700 mg, 19.005 mmol) stirred at
rt for 36 h. The reaction mixture was washed with water (15 mL × 2)
and dried over MgSO4, and the organic layers were concentrated in
vacuo. The residue was purified by pre-HPLC under basic conditions
to afford 27 (12 mg, 12% yield) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 7.09 −7.21 (m, 3 H), 6.96−7.00 (m, 2 H), 4.50−4.77 (m, 2
H), 3.00−3.50 (m, 1 H), 2.63−2.80 (m, 1 H), 2.30−2.43 (m, 1 H),
M
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1.81−1.96 (m, 1 H), 1.07−1.75 (m, 10 H), 0.70−0.97 (m, 9 H), 0.47−
0.55 (m, 3 H). MS (ESI): m/z 371.2 (M + 1).
Leu-OMe according to the reaction sequence outlined in Scheme 1
for compound 2a. H NMR (400 MHz, CDCl3) δ 7.32−7.05 (m, 5
H), 6.29 (m, 1 H), 5.21−4.70 (m, 1 H), 4.57−4.04 (m, 1 H), 3.56−
3.48 (m, 1 H), 3.12−2.60 (m, 1 H), 2.56−2.51 (m, 1 H), 2.12−2.05
(m, 1 H), 1.94−1.58 (m, 5 H), 1.49−1.28 (m, 4 H), 1.01−0.92 (m, 9
H). MS (ESI): m/z 343.1 (M + 1).
1
((2S, 5S)-2, 5-Diisobutylpiperazin-1-yl)((1R, 2R)-2-
phenylcyclopropyl)methanone (28). To a solution of compound 7
(300 mg, 1.41 mmol) in THF (30 mL) was added LiAlH4 (107 mg,
2.82 mmol) at −40 °C and the mixture was stirred for 6 h. The
reaction mixture was quenched with water, filtered, extracted withed
EtOAc (50 mL), dried over Na2SO4, and concentrated in vacuo to give
the decarbonylated compound (200 mg, 72% yield), which was used
for the next reaction without further purification. The decarbonylated
compound (200 mg, 1.01 mmol) was coupled to (1R,2R)-2-
phenylcyclopropanecarboxylic acid (164 mg, 1.01 mmol) according
to the method described for compound 2a to give compound 28 (110
mg, 28% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.24−
7.30 (m, 2 H), 7.16−7.22 (m, 1 H), 7.06−7.10 (m, 2 H), 4.54−4.58
(m, 1 H), 3.84−3.88 (m, 1 H), 3.03−3.14 (m, 3 H), 2.78−2.86 (m, 1
H), 2.36−2.45 (m, 1 H), 1.87−1.95 (m, 1 H), 1.71−1.74 (m, 2 H),
1.27−1.51 (m, 6 H), 0.91−0.96 (m, 6 H), 0.63−0.82 (m, 6 H). MS
(ESI): m/z 343.1 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-(3-phenylpropioloyl)piperazin-2-one
(35). Compound 7 (400 mg, 1.88 mmol) was coupled with 3-
phenylpropiolic acid (275 mg, 1.88 mmol) according to the procedure
described for the preparation of compound 2a (Scheme 1) to give 35
(195 mg, 30% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6)
δ 7.51−7.55 (m, 2 H), 7.34−7.45 (m, 3 H), 6.10−6.14 (m, 1 H),
5.08−5.18 (m, 0.5 H), 4.92−5.15 (m, 0.5 H), 4.47−4.70 (m, 1 H),
3.59−3.67 (m, 1 H), 3.11−3.14 (m, 0.5 H), 2.67−2.73 (m, 0.5 H),
1.78−1.86 (m, 2 H), 1.65−1.73 (m, 2 H), 1.34−1.40 (m, 2 H), 0.90−
1.06 (m, 12 H). MS (ESI): m/z 341.1 (M + 1).
(3S,6S)-4-Cinnamoyl-3,6-diisobutylpiperazin-2-one (36). Com-
pound 7 (200 mg, 0.941 mmol) was coupled with cinnamic acid
(139 mg, 0.939 mmol) according to the procedure described for the
preparation of compound 2a (Scheme 1) to give 36 (39 mg, 13%
yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.72−7.76 (m,
1 H), 7.46−7.53 (m, 2 H), 7.35−7.42 (m, 3 H), 6.81−6.87 (m, 1 H),
5.82 (s, 1 H), 4.57−4.82 (m, 2 H), 3.59−3.64 (m, 1 H), 2.66−3.16
(m, 1 H), 1.69−1.89 (m, 4 H), 1.32−1.42 (m, 2 H), 1.05−1.10 (m, 3
H), 0.90−1.03 (m, 9H). MS (ESI): m/z 343.1 (M + 1) and 365.1 (M
+ 23).
(3S,6S)-3,6-Diisobutyl-4-((Z)-3-phenylacryloyl)piperazin-2-one
(37). Compound 35 (140 mg, 0.41 mmol) in MeOH (15 mL) was
added Lindlar-Pd (30 mg), and the mixture was stirred at rt under H2
(40 psi) for 24 h. The reaction mixture was filtered, concentrated in
vacuo, and purified by prep HPLC under basic conditions to afford 37
(41 mg, 29% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
7.23−7.36 (m, 5 H), 6.67−6.74 (m, 1 H), 6.02(d, J = 2.4 Hz, 1 H),
5.46−5.72 (m, 1 H), 4.65−5.19 (m, 1 H), 3.85−4.26 (m, 1 H), 2.85−
3.41 (m, 1 H), 2.50−2.80 (m, 1 H), 1.45−1.72 (m, 4 H), 1.21−1.25
(m, 1 H), 1.05−1.11 (m, 1 H), 0.81−0.90 (m, 9 H), 0.70−0.78 (m, 3
H). MS (ESI): m/z 343.3 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-((E)-3-phenylallyl)piperazin-2-one(38).
To a solution of compound 7 (150 mg, 0.706 mmol) in DMF (10 mL)
was added (E)-(3-chloroprop-en-1-yl)benzene (210 mg, 0.85 mmol)
and K2CO3 (97 mg, 0.706 mmol) and stirred at 80 °C for 12 h. The
reaction mixture was diluted with water (10 mL) and extracted with
EtOAc (15 mL × 3), and the combined organic layers were washed
with brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by prep HPLC under acidic conditions to give 38 (63 mg,
27% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.58 (s,
1 H), 7.37−7.39 (m, 2 H), 7.27−7.30 (m, 2 H), 7.20−7.21 (m, 1 H),
6.51−6.55 (m, 1 H), 6.22 (m, 1 H), 3.45−3.49 (m, 1 H), 2.87 (m, 1
H), 2.65−2.67 (m, 2 H), 1.80−1.78 (m, 1 H), 1.58−1.60 (m, 2 H),
1.34−1.37 (m, 2 H), 1.19 (s, 3 H) 0.78−0.84 (m, 12 H). MS (ESI):
m/z 329.3 (M + 1).
( 3 R , 6 S ) - 3 , 6 - D i i s o b u t y l - 4 - ( ( 1 R , 2 R ) - 2 -
phenylcyclopropanecarbonyl)piperazin-2-one (29). Compound 29
was synthesized from Fmoc-Leu and D-Leu-OMe according to the
1
reaction sequence outlined in Scheme 1 for compound 2a. H NMR
(400 MHz, DMSO-d6) δ 7.25−7.29 (m, 2 H), 7.19−7.21 (m, 1 H),
7.05−7.08 (m, 2 H), 6.48−6.63 (m, 1 H), 5.17−5.21 (m, 0.5 H),
4.54−4.57 (m, 0.5 H), 4.39−4.42 (m, 0.5 H), 3.79−3.82 (m, 0.5 H),
3.65−3.69 (m, 0.5 H), 3.41−3.50 (m, 1 H), 3.22−3.27 (m, 0.5 H),
2.43−2.50 (m, 3 H), 1.89−1.92 (m, 1 H), 1.63−1.75 (m, 4 H), 1.33−
1.49 (m, 3 H), 0.90−1.02 (m, 6 H), 0.58−0.80 (m, 6 H). MS (ESI):
m/z 357.1 (M + 1).
( 3 S , 6 R ) - 3 , 6 - D i i s o b u t y l - 4 - ( ( 1 R , 2 R ) - 2 -
phenylcyclopropanecarbonyl)piperazin-2-one (30). Compound 30
was synthesized from Fmoc-D-leu and Leu-OMe according to the
1
reaction sequence outlined in Scheme 1 for compound 2a. H NMR
(400 MHz, DMSO-d6) δ 7.26−7.30 (m, 2 H), 7.20−7.23 (m, 1 H),
7.06−7.12 (m, 2 H), 6.44−6.60 (m, 0.5 H), 5.13−5.17 (m, 0.5 H),
4.53−4.56 (m, 0.5 H), 4.39−4.42 (m, 0.5 H), 3.82−3.85 (m, 0.5 H),
3.61−3.66 (m, 0.5 H), 3.48−3.49 (m, 1 H), 3.19−3.23 (m, 0.5 H),
2.55−2.57 (m, 1 H), 1.94−1.97 (m, 0.5 H), 1.83−1.91 (m, 2.5 H),
1.63−1.71 (m, 4 H), 1.30−1.52 (m, 3 H), 0.92−1.01 (m, 12 H). MS
(ESI): m/z 357.1 (M + 1).
( 3 S , 6 S ) - 6 - E t h y l - 3 - i s o b u t y l - 4 - ( ( 1 R , 2 R ) - 2 -
phenylcyclopropanecarbonyl)piperazin-2-one (31). Compound 31
was synthesized from Fmoc-Ala and Leu-OMe according to the
1
reaction sequence outlined in Scheme 1 for compound 2a. H NMR
(400 MHz, CDCl3) δ 7.32−7.05 (m, 5 H), 5.97 (s, 1 H), 5.22−4.71
(m, 1 H), 4.58−4.06 (m, 1 H), 3.50−3.39 (m, 1 H), 3.12−2.60 (m, 1
H), 2.56−2.51 (m, 1 H), 1.95−1.91 (m, 1 H), 1.86−1.72 (m, 2 H),
1.70−1.58 9m, 2 H), 1.56−1.45 (m, 2 H), 1.33−1.28 (m, 1 H), 1.02−
0.93 (m, 9 H). MS (ESI): m/z 329.20 (M − 1).
( 3 S , 6 S ) - 3 - E t h y l - 6 - i s o b u t y l - 4 - ( ( 1 R , 2 R ) - 2 -
phenylcyclopropanecarbonyl)piperazin-2-one (32). Compound 32
was synthesized from Leu-OMe and Fmoc-Ala according to the
(3S,6S)-4-(Cyclopropanecarbonyl)-3,6-diisobutylpiperazin-2-one
(39). Compound 7 (200 mg, 0.941 mmol), cyclopropanecarboxylic
acid (86 mg, 1.0 mmol), HATU (360 mg, 0.947 mmol), and DIPEA
(129 mg, 1.000 mmol) in DCM (15 mL) were stirred at rt for 2 h. The
reaction mixture was washed with water (15 mL × 2), dried over
MgSO4, and concentrated in vacuo. The residue was purified by prep
HPLC under basic conditions to afford 39 (128 mg, 49% yield) as a
1
reaction sequence outlined in Scheme 1 for compound 2a. H NMR
(400 MHz, CDCl3) δ 7.10−7.40 (m, 5 H), 5.90 (s, 1 H), 5.05−5.15
(m, 0.3 H), 4.70−4.80 (m, 0.7 H), 4.45−4.55 (m, 0.7 H), 4.05−4.15
(m, 0.3 H), 3.50−3.65 (m, 1 H), 2.45−2.55 (m, 2 H), 0.90−2.20 (m,
17 H). MS(ESI): m/z 328.9 (M + 1)].
1
colorless syrup. H NMR (400 MHz, DMSO-d6) δ 6.63−6.91 (m, 1
(3S, 6S)-6-((S)-sec-Butyl)-3-isobutyl-4-((1R, 2R)-2-
phenylcyclopropanecarbonyl)piperazin-2-one (33). Compound 33
was synthesized from (2S,3R)-2((((9H-fluoren-9-yl)methoxy)-
carbonyl)amino)-3-methylpentanoic acid and Leu-OMe according to
H), 4.17−5.21 (m, 2 H), 3.52−3.65 (m, 1 H), 2.57−3.15 (m, 1 H),
1.59−1.89 (m, 5 H), 1.32−1.41 (m, 2 H), 0.91−1.06 (m, 14 H), 0.82−
0.88 (m, 2 H). MS (ESI): m/z 281.2 (M + 1).
1
the reaction sequence outlined in Scheme 1 for compound 2a. H
(3S,6S)-4-([1,1′-Biphenyl]-3-carbonyl)-3,6-diisobutylpiperazin-2-
one (40). Compound 7 (100 mg, 0.47 mmol) was coupled with [1,1′-
biphenyl]-3-carboxylic acid (112 mg, 0.564 mmol) according to the
procedure described for the preparation of compound 2a (Scheme 1)
NMR (400 MHz, CDCl3) δ 7.05−7.30 (m, 5 H), 5.90−6.05 (m, 1H),
4.40−4.65 (m, 1 H), 4.05−4.15 (m, 0.5 H), 3.55−3.65 (m, 0.5 H),
3.15−3.25 (m, 2 H), 2.50−2.65 (m, 1 H), 0.85−1.95 (m, 20 H). MS
(ESI): m/z 357.1 (M + 1).
1
to give 40 (37 mg, 20% yield) as a white solid. H NMR (400 MHz,
CD3OD) δ 7.77 (d, J = 8.0 Hz, 1 H), 7.67−7.56 (m, 4 H), 7.45−7.36
(m, 4 H), 5.20 (d, J = 8.4 Hz, 1 H), 3.79−3.73 (m, 1 H), 3.62−3.52
(m, 1 H), 3.20−3.14 (m, 1 H), 1.92−1.70 (m, 3 H), 1.45−1.19 (m, 4
(3S,6S)-3-Isobutyl-4-((1R,2R)-2-phenylcyclopropanecarbonyl)-6-
propylpiperazin-2-one (34). Compound 34 was synthesized from (S)-
2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pentanoic acid and
N
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H), 1.10−0.96 (m, 6 H), 0.79−0.65 (m, 5 H). MS (ESI): m/z 393.2
(M + 1).
J = 15.2 Hz, 8.0 Hz, 1 H), 6.30 (brs, 1 H), 5.30 (dd, J = 8.0 Hz, 4.0 Hz,
1 H), 4.78 (dd, J = 13.2 Hz, 4.0 Hz, 1 H), 4.53 (t, J = 8.0 Hz, 1 H),
3.91 (s, 1 H), 3.65−3.55 (m, 1 H), 2.67 (t, J = 12.0 Hz,1 H), 1.89−
1.63 (m, 4 H), 1.38−1.34 (m, 2 H), 1.04−0.93 (m, 12 H). MS (ESI):
m/z 401.5 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-((E)-3-(p-tolyl)acryloyl)piperazin-2-one
(41). Compound 7 (60 mg, 0.29 mmol) and 4-methylcinnamic acid
(50 mg, 0.3 mmol) were coupled according to the procedure described
for the preparation of compound 2a to furnish 41 (95 mg, 95% yield)
as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.72 (dd, J = 14.0 Hz,
4.5 Hz, 1 H), 7.41 (t, J = 6.5 Hz, 1 H), 7.19 (d, J = 8.0 Hz, 1 H), 6.82−
6.76 (dd, J = 15.0 Hz, 7.2 Hz, 1 H) (s, 1H), 5.93 (br s, 1 H), 5.35 (br
s, 1 H), 4.81 (dd, J = 12.5 Hz, 6.5 Hz, 1 H), 4.57 (br s, 1 H), 3.60 (m,
1 H), 3.14 (m, 1 H), 2.67 (m, 1 H), 2.37 (m, 1 H), 1.75 (m, 4 H), 1.36
(m, 2 H), 1.06−0 (m, 12 H). MS (ESI): m/z 357.2 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-((E)-3-(4-(trifluoromethyl)phenyl)-
acryloyl)piperazin-2-one (42). Compound 7 (60 mg, 0.29 mmol) and
4-trifluorocinnamic acid (65 mg, 0.3 mmol) were coupled according to
the procedure described for the preparation of compound 2a to
(3S,6S)-4-((E)-3-(4-Bromophenyl)acryloyl)-3,6-diisobutylpipera-
zin-2-one (48). Compound 7 (60 mg, 0.29 mmol) and 4-
bromocinnamic acid (55 mg, 0.3 mmol) were coupled according to
the procedure described for the preparation of compound 2a to
1
furnish 48 (70 mg, 65% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 8.80−8.60 (m, 2 H), 7.85−7.70 (m, 2 H), 7.4 (br s, 1 H),
6.95 (br s, 1 H), 5.95 (s, 1 H), 5.38 (br s, 1 H), 5.06−4.99 (m, 1 H),
4.80 (br s, 1 H), 4.55 (br s, 1 H), 4.05 (m, 1 H), 3.10 (m, 1 H), 2.70
(m, 1 H), 1.90−1.10 (m, 5 H), 1.05−0.85 (m, 12 H). MS (ESI): m/z
377.2 (M + 1).
(3S,6S)-4-((E)-3-(4-Chlorophenyl)acryloyl)-3,6-diisobutylpipera-
zin-2-one (49). Compound 7 (60 mg, 0.29 mmol) and 4-
chlorocinnamic acid (55 mg, 0.3 mmol) were coupled according to
the procedure described for the preparation of compound 2a to
1
furnish 42 (44 mg, 38% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 7.77−7.58 (m, 5 H), 6.91 (dd, J = 8.0 Hz, 7.2 Hz, 1 H), 5.87
(s, 1 H), 5.33 (d, J = 9.6 Hz, 1 H), 4.81 (d, J = 13.6 Hz, 1 H), 4.56−
4.53 (m, 1 H), 4.00 (d, J = 12.4 Hz, 1 H), 3.62 (m, 2 H), 2.70 (m, 1
H), 1.90−1.69 (m, 2 H), 1.44−0.93 (m, 14 H). MS (ESI): m/z 411.2
(M + 1).
1
furnish 49 (70 mg, 65% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 8.80−8.60 (m, 2 H), 7.85−7.70 (m, 2 H), 7.4 (br s, 1 H),
6.95 (br s, 1 H), 5.95 (s, 1 H), 5.38 (br s, 1 H), 5.06−4.99 (m, 1 H),
4.80 (d, 1 H), 4.55 (br s, 1 H), 4.05 (m, 1 H), 3.10 (m, 1 H), 2.70 (m,
1 H), 1.90−1.10 (m, 5 H), 1.05−0.85 (m, 12 H). MS (ESI): m/z
377.2 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-((E)-3-(4-methoxyphenyl)acryloyl)-
piperazin-2-one (43). Compound 7 (60 mg, 0.29 mmol) and (E)-3-
(4-methoxyphenyl) acrylic acid (60 mg, 0.34 mmol) were coupled
according to the procedure described for the preparation of compound
2a to give 43 (84 mg, 96% yield) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 7.71 (m, 1 H), 7.46−7.43 (br s, 2 H), 6.92−6.90 (br s, 2 H),
6.69 (m, 1 H), 5.93 (br s, 1 H), 4.79 (m, 1 H), 4.56 (m, 1 H), 3.83 (s,
3 H) 3.66−3.52 (m, 1 H), 2.67 (m, 1 H), 1.87−1.60 (m, 5 H), 1.38−
1.28 (m, 2 H), 1.05−0.93 (m, 12 H). MS (ESI): m/z 373.2 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-((E)-3-(4-nitrophenyl)acryloyl)piperazin-
2-one (44). Compound 7 (99.8 mg, 0.47 mmol) and (E)-3-(4-
nitrophenyl) acrylic acid (109 mg, 0.564 mmol) were coupled
according to the procedure described for the preparation of compound
2a to give 44 (82 mg, 45% yield) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 8.21 (d, J = 8.4 Hz, 2 H), 7.73 (d, J = 15.2 Hz, 1 H), 7.63 (d,
J = 8.4 Hz, 2 H), 6.95 (d, J = 15.2 Hz, 1 H), 6.50 (s, 1 H), 4.77 (d, J =
13.2 Hz, 1 H), 4.55−4.49 (m, 1 H), 3.64−3.52 (m, 1 H), 2.72−2.66
(m, 1 H), 1.91−1.58 (m, 4 H), 1.42−1.32 (m, 2 H), 1.04−0.89 (m, 12
H). MS (ESI): m/z 388.1 (M + 1).
(3S,6S)-4-((E)-3-(3-Chlorophenyl)acryloyl)-3,6-diisobutylpipera-
zin-2-one (50). Compound 7 (60 mg, 0.29 mmol) and 3-
chlorocinnamic acid (60 mg, 0.33 mmol) were coupled according to
the procedure described for the preparation of compound 2a to
1
furnish 50 (88 mg, 98% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 7.67 (dd, J = 15.6 Hz, 5.6 Hz, 1 H), 7.50 (d, J = 15.6 Hz, 1
H); 7.38−7.29 (m, 2 H), 6. 83 (dd, J = 15.6 Hz, 5.6 Hz, 1 H), 5.95 (br
s, 1 H), 5.33 (dd, J = 9.6 Hz, 4.0 Hz, 1 H), 4.79 (dd, J = 13.2 Hz, 4.0
Hz, 1H), 4.56−4.53 (m, 1 H), 3.65−3.56 (m, 2 H), 2.68 (dd, J = 13.2
Hz, 11.2 Hz, 1 H), 1.88−1.63 (m, 4 H), 1.39−1.34 (m, 2 H), 1.05−
0.93 (m, 12 H). MS (ESI): m/z 379 (M + 1).
(3S,6S)-4-((E)-3-(2-Chlorophenyl)acryloyl)-3,6-diisobutylpipera-
zin-2-one (51). Compound 7 (60 mg, 0.29 mmol) and 2-
chlorocinnamic acid (60 mg, 0.33 mmol) were coupled according to
the procedure described for the preparation of compound 2a to
1
furnish 51 (50 mg, 56% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 7.84 (d, J = 15.6 Hz, 1 H), 7.50 (d, J = 15.6 Hz, 1 H); 7.35
(dd, J = 8.4 Hz, 2.0 Hz, 1 H), 7.20−7.16 (m, 1 H), 6.99 (d, J = 15.6
Hz, 1 H), 5.89 (br s, 1 H), 5.33 (dd, J = 8.0 Hz, 4.0 Hz, 1 H), 4.79 (dd,
J = 13.2 Hz, 4.0 Hz, 1 H), 4.46 (dd, J = 8.0 Hz, 4.0 Hz, 1 H), 3.69−
3.56 (m, 2 H), 2.71 (dd, J = 13.2 Hz, 11.2 Hz, 1 H), 1.84−1.64 (m, 4
H), 1.39−1.32 (m, 2 H), 1.07−0.92 (m, 12 H). MS (ESI): m/z 379
(M + 1).
(3S,6S)-4-((E)-3-(4-(Dimethylamino)phenyl)acryloyl)-3,6-diisobu-
tylpiperazin-2-one (45). Compound 7 (60 mg, 0.28 mmol) and (E)-
3-[4-(dimethylamino)phenyl]acrylic acid (60 mg, 0.3 mmol) were
coupled according to the procedure described for the preparation of
compound 2a to give the product 45 (100 mg, 96% yield) as an yellow
1
syrup. H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 16.0 Hz, 1 H),
7.41−7.39 (br s, 2 H), 6.68−6.66 (br s, 2 H), 6.64−6.58 (m, 1 H),
5.83 (br s, 1 H), 4.83−4.80 (m, 1 H), 4.61−4.59 (m,1 H), 3.66−3.57
(m, 1 H), 3.01 (s, 6 H); 2.68−2.62 (m,1 H), 1.86−1.66 (m, 4 H),
1.38−1.32 (m, 2 H), 1.05−0.92 (m, 12 H). MS (ESI): m/z 387.4 (M
+ 1).
(3S,6S)-3,6-Diisobutyl-4-((E)-3-(4-(methylsulfonyl)phenyl)-
acryloyl)piperazin-2-one (46). Compound 7 (99.8 mg, 0.47 mmol)
and (E)-3-(4-(methylsulfonyl)phenyl)acrylic acid (127.6 mg, 0.564
mmol) were coupled according to the procedure described for the
preparation of compound 2a to give the product 46 (151 mg, 76%
yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.4
Hz, 2 H), 7.73 (d, J = 15.6 Hz, 1 H), 7.66 (d, J = 8.4 Hz, 2 H), 6.94 (d,
J = 15.6 Hz, 1 H), 6.33 (s, 1 H), 4.78 (d, J = 13.2 Hz, 3.8 Hz, 1 H),
4.55−4.51 (m, 1 H), 3.65−3.52 (m, 1 H), 3.06 (s, 3 H), 2.69 (d, J =
13.2 Hz, 11.2 Hz, 1 H), 1.91−1.62 (m, 4 H), 1.43−1.33 (m, 2 H),
1.06−0.91 (m, 12 H). MS (ESI): m/z 421.4 (M + 1).
(3S,6S)-4-((E)-3-(4-Fluorophenyl)acryloyl)-3,6-diisobutylpipera-
zin-2-one (52). Compound 7 (53 mg, 0.25 mmol) and 4-
fluorocinnamic acid (50 mg, 0.3 mmol) were coupled according to
the procedure described for the preparation of compound 2a to
1
furnish 52 (80 mg, 94% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 7.71−7.57 (m, 3 H), 7.19−7.11 (m, 3 H), 5.19 (dd, J = 10.5
Hz, 3.2 Hz, 1 H) 4.72 (m, 1 H), 4.36 (dd, J = 14.4 Hz, 3.2 Hz, 1 H),
3.58−3.52 (m, 1 H), 3.19 (dd, J = 12.0 Hz, 10.8 Hz, 1 H), 2.79−2.74
(m, 1 H), 2.37 (m, 1 H), 1.85−1.60 (m, 4 H), 1.41 (t, J = 7.2 Hz, 2
H), 1.03−0.94 (m, 12 H). MS (ESI): m/z 361.5 (M + 1).
(3S,6S)-4-((E)-3-(3,4-Difluorophenyl)acryloyl)-3,6-diisobutylpiper-
azin-2-one (53). Compound 7 (53 mg, 0.25 mmol) and 3,4-
difluorocinnamic acid (60 mg, 0.33 mmol) were coupled according
to the procedure described for the preparation of compound 2a to
1
furnish 53 (90 mg, 97% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 7.66−7.60 (m, 1 H), 7.35−7.13 (m, 3 H), 6. 73 (dd, J = 15.2
Hz, 4.8 Hz, 1 H), 6.23 (d, J = 15.2, 1 H), 5.31 (dd, J = 9.6 Hz, 3.6 Hz,
1 H), 4.78 (dd, J = 13.2 Hz, 4.0 Hz, 1 H), 4.53 (dd, J = 8.8 Hz, 4 Hz, 1
H), 3.64−3.55 (m, 1 H), 3.59 (m, 1 H), 3.15 (dd, J = 14.0 Hz, 11.2
Hz, 1 H), 2.68 (dd, J = 14.0 Hz, 10.8 Hz, 1 H), 1.87−1.65 (m, 4 H),
1.38−1.34 (m, 2 H), 1.04−0.92 (m, 12 H). MS (ESI): m/z 379.1 (M
+ 1).
Methyl 4-((E)-3-((2S,5S)-2,5-Diisobutyl-3-oxopiperazin-1-yl)-3-ox-
oprop-1-en-1-yl)benzoate (47). Compound 7 (55 mg, 0.26 mmol)
and (E)-3-[4-(methoxycarbonyl)phenyl] acrylic acid (60 mg, 0.29
mmol) were coupled according to the procedure described for the
preparation of compound 2a to furnish 47 (94 mg, 90% yield) as a
white solid. 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 8.0 Hz, 2 H),
7.72 (dd, J = 15.2 Hz, 8.0 Hz, 1 H), 7.55 (t, J = 8.0 Hz, 2 H), 6.91 (dd,
O
dx.doi.org/10.1021/jm4012643 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(3S,6S)-4-((E)-3-(2,4-Difluorophenyl)acryloyl)-3,6-diisobutylpiper-
azin-2-one (54). Compound 7 (60 mg, 0.29 mmol) and 2,4-
difluorocinnamic acid (60 mg, 0.33 mmol) were coupled according
to the procedure described for the preparation of compound 2a to
furnish 54 (104 mg, 95% yield) as a white solid. 1H NMR (400 MHz,
CDCl3) δ 7.66−7.73 (m, 1 H), 7.41−7.47 (m, 1 H), 6.81−6.97 (m, 3
H), 6.43 (d, J = 13.8 Hz, 1 H), 5.29 (dd, J = 10.0 Hz, 4.0 Hz, 1 H),
4.01 (d, J = 11.2 Hz, 1 H), 3.57−3.60 (m, 1 H), 3.08−3.14 (m, 1 H),
1.72−1.86 (m, 4 H), 1.34−1.38 (m, 2 H), 0.91−1.02 (m, 12 H). MS
(ESI): m/z 379.1 (M + 1).
coupled according to the procedure described for the preparation of
compound 2a to give 61 (33 mg, 18% yield) as a white solid. 1H NMR
(400 MHz, CDCl3) δ 11.7 (br s, 1 H), 7.69−7.64 (m, 2 H), 7.45−7.35
(m, 3 H), 6.94 (s, 1 H), 6.05 (s, 1 H), 5.38−5.31 (m, 1 H), 4.88−4.79
(m, 1 H), 3.76−3.63 (m, 1 H), 3.15−3.05 (m, 1 H), 1.92−1.55 (m, 4
H), 1.43−1.28 (m, 2 H), 1.12−0.78 (m, 12 H). MS (ESI): m/z 383.4
(M + 1).
(3S,6S)-3,6-Diisobutyl-4-(2-phenyl-1H-imidazole-4-carbonyl)-
piperazin-2-one (62). Compound 7 (100 mg, 0.47 mmol) and 2-
phenyl-1H-imidazole-4-carboxylic acid (106 mg, 0.56 mmol) were
coupled according to the procedure described for the preparation of
compound 2a to give 62 (45 mg, 25% yield) as a white solid. 1H NMR
(400 MHz, CD3OD) δ 7.91 (d, J = 6.8 Hz, 2 H), 7.74 (s, 1 H), 7.48−
7.39 (m, 3 H), 5.86 (d, J = 12.0 Hz, 1 H), 5.20 (d, J = 9.2 Hz, 1 H),
3.78−3.62 (m, 1 H), 3.17−3.11 (m, 1 H), 1.89−1.73 (m, 4 H), 1.44−
1.39 (m, 2 H), 1.07−0.91 (m, 12 H). MS (ESI): m/z 383.2 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-(5-(p-tolyl)isoxazole-3-carbonyl)-
piperazin-2-one (63). Compound 7 (100 mg, 0.47 mmol) and 5-(p-
tolyl)isoxazole-3-carboxylic acid (114 mg, 0.56 mmol) were coupled
according to the procedure described for the preparation of compound
(3S,6S)-3,6-Diisobutyl-4-(5-phenylfuran-2-carbonyl)piperazin-2-
one (55). Compound 7 (100 mg, 0.47 mmol) and 5-phenylfuran-2-
carboxylic acid (106 mg, 0.564 mmol) were coupled according to the
procedure described for the preparation of compound 2a to give 55
1
(52 mg, 29% yield) as a white solid. H NMR (400 MHz, CDCl3) δ
7.67 (d, J = 7.6 Hz, 2 H), 7.44−7.39 (m, 2 H), 7.36−7.33 (m, 1 H),
7.21(d, J = 3.6 Hz, 1 H), 6.75 (d, J = 3.6 Hz, 1 H), 6.04 (br s, 1 H),
5.35−5.26 (s, 1 H), 4.77 (dd, J = 20.0 Hz, 4 Hz, 1H), 3.82−3.73 (m, 1
H), 3.25−3.12 (m, 1 H), 1.92−1.65 (m, 4 H), 1.41−1.37 (m, 2 H),
1.00−0.89 (m, 12 H). MS (ESI): m/z 383.1 (M + 1).
1
(3S,6S)-3,6-Diisobutyl-4-(2-phenylthiazole-4-carbonyl)piperazin-
2-one (56). Compound 7 (100 mg, 0.47 mmol) and 2-phenylthiazole-
4-carboxylic acid (116 mg, 0.564 mmol) were coupled according to the
procedure described for the preparation of compound 2a to give 56
(58 mg, 31% yield) as a colorless solid. 1H NMR (400 MHz, CDCl3) δ
8.07 (s, 1 H), 7.94−7.89 (m, 2 H), 7.45−7.43 (m, 3 H), 6.48 (s, 1 H),
5.31−5.29 (m, 1 H), 5.13 (d, J = 14.0 Hz, 1 H), 3.92−3.84 (m, 1 H),
3.08 (dd, J = 14.0 Hz, 10.8 Hz, 1 H), 1.90−1.66 (m, 4 H), 1.41−1.24
(m, 2 H), 1.09−0.82 (m, 12 H). MS (ESI): m/z 400.1 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-(2-phenyloxazole-4-carbonyl)piperazin-
2-one (57). Compound 7 (99.8 mg, 0.47 mmol) and 2-phenyloxazole-
4-carboxylic acid (106.7 mg, 0.564 mmol) were coupled according to
the procedure described for the preparation of compound 2a to give
57 (55 mg, 30% yield) as a white solid. 1H NMR (400 MHz, CD3OD)
δ 8.47 (s, 1 H), 8.08−8.03 (m, 2 H), 7.56−7.50 (m, 3 H), 5.39 (dd, J =
14.2 Hz, 3.6 Hz, 1 H), 5.16−5.13 (m, 1 H), 3.84−3.74 (m, 1 H), 3.18
(dd, J = 14.0 Hz, 11.2 Hz, 1 H), 1.92−1.66 (m, 4 H), 1.50−1.38 (m, 2
H), 1.08−0.92 (m, 12 H). MS (ESI): m/z 384.4 (M + 1).
2a to give 63 (107 mg, 57% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 7.67 (d, J = 8.0 Hz, 2 H), 7.27 (d, J = 8.0 Hz, 2 H),
6.85 (s, 1 H), 6.51 (s, 1H), 5.26 (dd, J = 9.4 Hz, 4 Hz, 1 H), 4.86 (dd,
J = 14.2 Hz, 4.0 Hz, 1 H), 3.77−3.67 (m, 1 H), 3.10 (dd, J = 14.2 Hz,
11.0 Hz, 1 H), 2.40 (s, 3 H), 1.91−1.67 (m, 4 H), 1.41−1.33 (m, 2 H),
1.08−0.91 (m, 12 H). MS (ESI): m/z 398.2 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-(5-(4-methoxyphenyl)isoxazole-3-
carbonyl)piperazin-2-one (64). Compound 7 (100 mg, 0.47 mmol)
and 5-(4-methoxyphenyl)isoxazole-3-carboxylic acid (123 mg, 0.56
mmol) were coupled according to the procedure described for the
preparation of compound 2a to give 64 (106 mg, 55% yield) as a white
1
solid. H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.8 Hz, 2 H), 6.98
(d, J = 8.8 Hz, 2 H), 6.78 (s, 1 H), 6.51 (s, 1 H), 5.26 (dd, J = 9.4 Hz,
4.4 Hz, 1 H), 4.85 (dd, J = 14.0 Hz, 4.4 Hz, 1 H), 3.85 (s, 3 H), 3.77−
3.66 (m, 1 H), 3.09 (dd, J = 13.8 Hz, 10.8 Hz, 1 H), 1.91−1.67 (m, 4
H), 1.41−1.33 (m, 2 H), 1.07−0.90 (m, 12 H). MS (ESI): m/z 414.1
(M + 1).
(3S,6S)-4-(5-(4-Chlorophenyl)isoxazole-3-carbonyl)-3,6-diisobu-
tylpiperazin-2-one (65). Compound 7 (100 mg, 0.47 mmol) and 5-
(4-chlorophenyl)isoxazole-3-carboxylic acid (126 mg, 0.56 mmol)
were coupled according to the procedure described for the preparation
(3S,6S)-3,6-Diisobutyl-4-(5-phenylisoxazole-3-carbonyl)-
piperazin-2-one (58). Compound 7 (100 mg, 0.47 mmol) and 5-
phenylisoxazole-3-carboxylic acid (107 mg, 0.56 mmol) were coupled
according to the procedure described for the preparation of compound
1
1
of compound 2a to give 65 (109 mg, 56% yield) as a white solid. H
2a to give 58 (120 mg, 67% yield) as a white solid. H NMR (400
NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.4 Hz, 2 H), 7.46 (d, J = 8.4
Hz, 2 H), 6.91 (s, 1 H), 6.49 (s, 1 H), 5.26 (dd, J = 9.4 Hz, 4.0 Hz, 1
H), 4.85 (dd, J = 14.2 Hz, 4.0 Hz, 1 H), 3.77−3.67 (m, 1 H), 3.10 (dd,
J = 14.2 Hz, 11.0 Hz, 1 H), 1.91−1.67 (m, 4 H), 1.41−1.34 (m, 2 H),
1.07−0.91 (m, 12 H). MS (ESI): m/z 418.4 (M + 1).
MHz, CDCl3) δ 7.82−7.78 (m, 2 H), 7.51−7.45 (m, 3 H), 6.92 (s, 1
H), 6.36 (s, 1 H), 5.29−5.26 (m, 1 H), 4.87 (dd, J = 14.2 Hz, 4.0 Hz, 1
H), 3.78−3.67 (m, 1 H), 3.11 (dd, J = 14.0 Hz, 10.8 Hz, 1 H), 1.92−
1.82 (m, 1 H), 1.81−1.67 (m, 3 H), 1.42−1.34 (m, 2 H), 1.08 (d, J =
6.4 Hz, 3 H), 0.99−0.93 (m, 9 H). MS (ESI): m/z 384.2 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-(5-phenyl-1,2,4-oxadiazole-3-carbonyl)-
piperazin-2-one (59). Compound 7 (50 mg, 0.24 mmol) and 5-
phenyl-1,2,4-oxadiazole-3-carboxylic acid (54 mg, 0.28 mmol) were
coupled according to the procedure described for the preparation of
(3S,6S)-4-(5-(4-Bromophenyl)isoxazole-3-carbonyl)-3,6-diisobu-
tylpiperazin-2-one (66). Compound 7 (100 mg, 0.47 mmol) and 5-
(4-bromophenyl)isoxazole-3-carboxylic acid (151 mg, 0.56 mmol)
were coupled according to the procedure described for the preparation
1
1
of compound 2a to give 66 (130 mg, 60% yield) as a white solid. H
compound 2a to give 59 (30 mg, 33% yield) as a colorless gum. H
NMR (400 MHz, CDCl3) δ 7.66−7.60 (m, 4 H), 6.92 (s, 1 H), 6.54
(s, 1 H), 5.27−5.24 (m, 1 H), 4.86−4.76 (m, 1 H), 3.77−3.66 (m, 1
H), 3.10 (dd, J = 13.6 Hz, 11.2 Hz, 1 H), 1.91−1.69 (m, 4 H), 1.41−
1.34 (m, 2 H), 1.07−0.91 (m, 12 H). MS (ESI): m/z 462.1 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3,6-diisobu-
tylpiperazin-2-one (67). Compound 7 (100 mg, 0.47 mmol) and 5-
(4-fluorophenyl)isoxazole-3-carboxylic acid (117 mg, 0.564 mmol)
were coupled according to the procedure described for the preparation
NMR (400 MHz, CDCl3) δ 8.21−8.18 (m, 2 H), 7.67−7.54 (m, 3 H),
5.93 (s, 1 H), 5.32−5.29 (m, 1 H), 4.89−4.82 (m, 1 H), 3.87−3.74
(m, 1 H), 3.14 (dd, J = 13.8 Hz, 11.0 Hz, 1 H), 1.94−1.63 (m, 4 H),
1.43−1.32 (m, 2 H), 1.11−0.89 (m, 12 H). MS (ESI): m/z 385.2 (M
+ 1).
(3S,6S)-3,6-Diisobutyl-4-(1-phenyl-1H-imidazole-4-carbonyl)-
piperazin-2-one (60). Compound 7 (100 mg, 0.47 mmol) and 1-
phenyl-1H-imidazole-4-carboxylic acid (106 mg, 0.56 mmol) were
coupled according to the procedure described for the preparation of
1
of compound 2a to give 67 (172 mg, 91% yield) as a white solid. H
1
NMR (400 MHz, CD3OD) δ 7.96−7.92 (m, 2 H), 7.30−7.26 (m, 2
H), 7.06 (s, 1 H), 5.16−5.12 (m, 1 H), 4.53 (dd, J = 14.2 Hz, 4.4 Hz, 1
H), 3.79−3.71 (m, 1 H), 3.21 (dd, J = 14.2 Hz, 11.0 Hz, 1 H), 1.92−
1.62 (m, 4 H), 1.48−1.32 (m, 2 H), 1.12−0.88 (m, 12 H). MS (ESI):
m/z 402.2 (M + 1).
(3S,6S)-4-(5-(3,4-Difluorophenyl)isoxazole-3-carbonyl)-3,6-diiso-
butylpiperazin-2-one (68). Compound 7 (45 mg, 0.21 mmol) and 5-
(3,4-difluorophenyl)isoxazole-3-carboxylic acid (56 mg, 0.25 mmol)
were coupled according to the procedure described for the preparation
compound 2a to give 60 (83 mg, 46% yield) as a colorless oil. H
NMR (400 MHz, CDCl3) δ 7.99 (s, 1 H), 7.77 (s, 1 H), 7.53−7.49
(m, 2 H), 7.43−7.39 (m, 3 H), 5.94 (s, 1 H), 5.84−5.76 (m, 1 H),
5.36−5.32 (m, 1 H), 3.76−3.64 (m, 1 H), 3.10−3.04 (m, 1 H), 1.87−
1.71 (m, 4 H), 1.42−1.27 (m, 2 H), 1.08−0.93 (m, 12 H). MS (ESI):
m/z 383.3 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-(5-phenyl-1H-pyrazole-3-carbonyl)-
piperazin-2-one (61). Compound 7 (100 mg, 0.47 mmol) and 5-
phenyl-1H-pyrazole-3-carboxylic acid (106 mg, 0.56 mmol) were
P
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1
of compound 2a to give 68 (57 mg, 65% yield) as a white solid. H
NMR (400 MHz, CD3OD) δ 7.65−7.60 (m, 1 H), 7.55−7.53 (m, 1
H), 7.33−7.26 (m, 1 H), 6.89 (s, 1 H), 5.93 (s, 1 H), 5.28 (dd, J = 9.8
Hz, 3.8 Hz, 1H), 4.86 (dd, J = 14.0 Hz, 3.8 Hz, 1 H), 3.79−3.69 (m, 1
H), 3.12 (dd, J = 14.0 Hz, 10.8 Hz, 1 H), 1.92−1.85 (m, 1 H), 1.79−
1.65 (m, 3 H), 1.42−1.32 (m, 2 H), 1.08−0.92 (m, 12 H). MS (ESI):
m/z 420.2 (M + 1).
mmol) by the method described for the preparation of compound 7
(Scheme 1) to afford (3S,6S)-3-Isobutyl-6-propenyl-piperazin-2-one
(1.623 g, 8% yield) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ
7.56 (s, 1 H), 5.43−5.57(m, 2 H), 3.74 (s, 1 H), 3.07 (dd, J = 10.8 Hz,
3.2 Hz, 1 H), 2.82 (dd, J = 12.8 Hz, 4.8 Hz, 1 H), 2.63 (dd, J = 12.8
Hz, 5.6 Hz, 1 H), 1.74−1.82 (m, 1 H), 1.64 (d, J = 7.2 Hz, 3 H), 1.47−
1.54 (m, 1 H), 1.35−1.42 (m, 1 H), 0.82−0.88 (m, 7 H). MS (ESI):
m/z 197.2 (M + 1).
(3S,6S)-4-(5-(2,4-Difluorophenyl)isoxazole-3-carbonyl)-3,6-diiso-
butylpiperazin-2-one (69). Compound 7 (50 mg, 0.24 mmol) and 5-
(2,4-difluorophenyl)isoxazole-3-carboxylic acid (53 mg, 0.24 mmol)
were coupled according to the procedure described for the preparation
of compound 2a to furnish 69 (72 mg, 73% yield) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 8.00−7.93 (m, 1 H), 7.07−6.95 (m, 3 H),
5.88 (s, 1 H), 5.30−5.22 (m, 1 H), 4.81−4.76 (m, 1 H), 3.78−3.70
(m, 1 H), 3.15−2.79 (m, 1 H), 1.89−1.61 (m, 4 H), 1.42−1.31 (m, 2
H) 1.09−0.76 (m, 12 H). MS (ESI): m/z 420.1 (M + 1).
4-(3-((2S,5S)-2,5-Diisobutyl-3-oxopiperazine-1-carbonyl)isoxazol-
5-yl)benzonitrile (70). Compound 7 (100 mg, 0.47 mmol) and 5-(4-
cyanophenyl)isoxazole-3-carboxylic acid (121 mg, 0.56 mmol) were
coupled according to the procedure described for the preparation of
compound 2a to give 70 (61 mg, 32% yield) as a white solid. 1H NMR
(400 MHz, CDCl3) δ 7.91 (d, J = 8.4 Hz, 2 H), 7.79 (d, J = 8.4 Hz, 2
H), 7.07 (s, 1 H), 6.32 (s, 1 H), 5.30−5.24 (m, 1 H), 4.87−4.83 (m, 1
H), 3.79−3.67 (m, 1 H), 3.12 (dd, J = 13.6 Hz, 11.2 Hz, 1 H), 1.92−
1.67 (m, 4 H), 1.42−1.35 (m, 2 H), 1.08−0.90 (m, 12 H). MS (ESI):
m/z 409.2 (M + 1).
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-((E)-prop-1-en-1-yl)pipera-zin-2-one (74). (3S,6S)-3-Isobutyl-6-
propenyl-piperazin-2-one (40 mg, 0.20 mmol) and 5-(4-fluorophenyl)-
isoxazole-3-carboxylic acid (45 mg, 0.22 mmol) were coupled
according to the procedure described for the preparation of compound
1
2a to furnish 74 (32 mg, 41% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 7.80−7.76 (m, 2 H), 7.20−7.15 (m, 2 H), 6.86 (s, 1
H), 5.92−5.81 (m, 2 H), 5,34−5.25 (m, 2 H), 4.80−4.72 (m, 1 H),
4.20−4.13 (m, 1 H), 3.19 (dd, J = 14.0 Hz, 11.2 Hz, 1 H), 1.92−1.68
(m, 6 H) and 1.08−0.77 (m, 6 H). MS (ESI): m/z 386.2 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
neopentylpiperazin-2-one (75). A. (3S,6S)-6-(2,2-Dimethyl-propyl)-
3-isobutyl-piperazin-2-one. Synthesized from (S)-2-((((9H-fluoren-
9-yl)methoxy)carbonyl)amino)-4,4-dimethylpentanoic acid (15.0 g, 40
mmol) and HCl salt of Leu-OMe (3.5 g, 19.35 mmol) by the method
described for the preparation of compound 7 (Scheme 1) to afford
(3S,6S)-6-(2,2-dimethyl-propyl)-3-isobutyl-piperazin-2-one (1.6 g,
1
35% yield) as a white solid. H NMR (400 MHz, DMSO-d6) δ 7.37
(s, 1 H), 3.30 (dd, J = 5.6 Hz, 4.0 Hz, 1 H), 3.07 (dd, J = 9.6 Hz, 4.0
Hz, 1 H), 2.83 (dd, J = 12.8 Hz, 4.0 Hz, 1 H), 2.56 (dd, J = 13.2 Hz,
6.0 Hz, 1 H), 1.90 (s, 1 H), 1.74−1.79 (m, 1 H), 1.33−1.49 (m, 4 H),
0.80−0.90 (m, 15 H). MS (ESI): m/z 227.0 (M + 1).
(3S,6S)-4-(5-(4-(Dimethylamino)phenyl)isoxazole-3-carbonyl)-
3,6-diisobutylpiperazin-2-one (71). Compound 7 (100 mg, 0.47
mmol) and 5-(4-(dimethylamino)phenyl)isoxazole-3-carboxylic acid
(131 mg, 0.56 mmol) were coupled according to the procedure
described for the preparation of compound 2a to give 71 (52 mg, 26%
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-neopentylpiperazin-2-one (75). (3S,6S)-6-(2,2-Dimethyl-propyl)-
3-isobutyl-piperazin-2-one (56 mg, 0.25 mmol) and 5-(4-
fluorophenyl)isoxazole-3-carboxylic acid (53 mg, 0.25 mmol) were
coupled according to the procedure described for the preparation of
compound 2a to give 75 (94 mg, 90% yield) as a white solid. 1H NMR
(400 MHz, CD3OD) δ 7.86−7.82 (m, 2 H), 7.29−7.26 (m, 2 H), 7.06
(s, 1 H), 5.16−5.12 (m, 1 H), 4.53 (dd, J = 14.2 Hz, 4.4 Hz, 1 H),
3.79−3.71 (m, 1 H), 3.21 (dd, J = 14.2 Hz, 11.0 Hz, 1 H), 1.92−1.62
(m, 4 H), 1.48−1.32 (m, 2 H), 1.12−0.88 (m, 15 H). MS (ESI): m/z
416.1 (M + 1).
(3S,6S)-6-((R)-sec-Butyl)-4-(5-(4-fluorophenyl)isoxazole-3-carbon-
yl)-3-isobutylpiperazin-2-one (76). A. (3S,6S)-6-((R)-sec-Butyl)-3-
isobutylpiperazin-2-one. Synthesized from (2S,3R)-2-(Fmoc)amino-
3-methylpentanoic acid (14.5 g, 40 mmol) and HCl salt of Leu-OMe
(7.8 g, 42.95 mmol) by the method described for the preparation of
compound 7 (Scheme 1) to afford (3S,6S)-6-((R)-sec-butyl)-3-
isobutylpiperazin-2-one (4.26 g, 50% yield) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 5.64 (s, 1 H), 3.10−3.43 (m, 3 H), 2.68−
2.94 (m, 1 H), 1.05−1.94 (m, 6 H), 0.89−0.96 (m, 12 H). MS (ESI):
m/z 213.1 (M + 1).
1
yield) as white solid. H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 8.8
Hz, 2 H), 6.72 (d, J = 8.8 Hz, 2 H), 6.67 (s, 1 H), 6.43 (s, 1 H), 5.29−
5.25 (m, 1 H), 4.87−4.81 (m, 1 H), 3.78−3.65 (m, 1 H), 3.08 (dd, J =
13.6 Hz, 10.8 Hz, 1 H), 3.03 (s, 6 H), 1.90−1.67 (m, 4 H), 1.41−1.32
(m, 2 H), 1.08−0.90 (m, 12 H). MS (ESI): m/z 427.2 (M + 1).
(3S,6S)-3,6-Diisobutyl-4-(5-(4-nitrophenyl)isoxazole-3-carbonyl)-
piperazin-2-one (72). Compound 7 (60 mg, 0.28 mmol) and 5-(4-
nitrophenyl)-isoxazole-3-carboxylic acid (60 mg, 0.28 mmol) were
coupled according to the procedure described for the preparation of
compound 2a to furnish 72 (90 mg, 74% yield) as a pale-yellow solid.
1H NMR (400 MHz, CDCl3) δ 8.37−7.34 (m, 2 H), 7.99−7.96 (m, 2
H), 7.12 (s, 1 H), 6.20 (s, 1 H), 5.29−5.25 (m, 1 H), 4.88−4.76 (m, 1
H), 3.79−3.69 (m, 1 H), 3.18 (dd, J = 14.0 Hz, 10.8 Hz, 1 H), 1.91−
1.69 (m, 4 H), 1.42−1.35 (m, 2 H) and 1.08−0.92 (m, 12 H). MS
(ESI): m/z 429.1 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
propylpiperazin-2-one (73). A. (3S,6S)-3-Isobutyl-6-propyl-pipera-
zin-2-one. Synthesized from Fmoc-L-norvaline (19.82 g, 58 mmol)
and HCl salt of Leu-OMe (4.7 g, 26.25 mmol) by the method
described for the preparation of compound 7 (Scheme 1) to afford
(3S,6S)-3-isobutyl-6-propyl-piperazin-2-one (3.4 g, 25% yield) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.53 (s, 1 H), 3.06 (dd,
J = 14.0 Hz, 4.0 Hz, 1 H), 2.78 (dd, J = 12.8 Hz, 4.0 Hz, 1 H), 2.59
(dd, J = 12.8 Hz, 5.2 Hz, 1 H), 2.49 (dd, J = 3.6 Hz, 2.0 Hz, 1 H), 2.35
(s, 1 H), 1.73−1.80 (m, 1 H), 1.36−1.51 (m, 6 H), 0.80−0.90 (m, 9
H). MS (ESI): m/z 198.9 (M + 1).
B. (3S,6S)-6-((R)-sec-Butyl)-4-(5-(4-fluorophenyl)isoxazole-3-car-
bonyl)-3-isobutylpiperazin-2-one (76). (3S,6S)-6-((R)-sec-Butyl)-3-
isobutylpiperazin-2-one (50 mg, 0.24 mmol) and 5-(4-fluorophenyl)-
isoxazole-3-carboxylic acid (53 mg, 0.25 mmol) were coupled
according to the procedure described for the preparation of compound
2a to furnish 76 (79 mg, 83% yield) as a colorless solid. 1H NMR (400
MHz, CDCl3) δ 7.83−7.76 (m, 2 H), 7.22−7.16 (m, 2 H), 6.86 (s, 1
H), 6.24 (s, 1 H), 5.33−5.25 (m, 1 H), 5.00−4.70 (m, 1 H), 3.76−3.36
(m, 1 H), 3.34−3.11 (m, 1H), 1.91−1.71 (m, 3 H), 1.48−1.16 (m, 2
H), 1.05−0.71 (m, 12 H). MS (ESI): m/z 402.20 (M + 1).
(3S,6S)-6-Allyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-3-iso-
butylpiperazin-2-one (77). A. (3S,6S)-6-Allyl-3-isobutylpiperazin-2-
one. Synthesized from (S)-2-((((9H-fluoren-9-yl)methoxy)-carbonyl)-
amino)pent-4-enoic acid (12.5 g, 37 mmmol) and HCl salt of Leu-
OMe (7.8 g, 42.95 mmol) by the method described for the preparation
of compound 7 (Scheme 1) to afford (3S,6S)-6-allyl-3-isobutylpiper-
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-propylpiperazin-2-one (73). (3S,6S)-3-Isobutyl-6-propylpiperazin-
2-one (50 mg, 0.25 mmol) and 5-(4-fluorophenyl)isoxazole-3-
carboxylic acid (53 mg, 0.25 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 73
1
(93 mg, 95% yield) as a white solid. H NMR (400 MHz, CDCl3) δ
7.80−7.77 (m, 2 H), 7.21−7.17 (m, 2 H), 6.86 (s, 1 H), 6.08 (s, 1 H),
5.29−5.28 (m, 1 H), 4.89−4.78 (m, 1 H), 3.71−3.63 (m, 1 H), 3.17−
2.83 (m, 1 H), 1.921.65 (m, 3 H), 1.58−1.37 (m, 4 H), 1.08−0.77 (m,
9 H). MS (ESI): m/z 388.1 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
((E)-prop-1-en-1-yl)pipe-razin-2-one (74). A. (3S,6S)-3-Isobutyl-6-
propenyl-piperazin-2-one. Synthesized from 2-amino-pent-3-enoic
acid (11.93 g, 104 mmol) and HCl salt of Leu-OMe (7.8 g, 42.95
1
azin-2-one (2.6 g, 36% yield) as a white solid. H NMR (400 MHz,
CDCl3) δ 6.01 (s, 1 H), 5.66−5.74 (m, 1 H), 5.15−5.17 (m, 1 H),
5.12−5.15 (m, 1 H), 3.38−3.43 (m, 2 H), 2.97−3.01 (m, 1 H), 2.79−
2.84 (m, 1 H), 2.28−2.33 (m, 1 H), 2.16−2.20 (m, 1 H), 1.70−1.79
Q
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(m, 2 H), 1.66−1.69 (m, 1 H), 1.53−1.59 (m, 1 H), 0.90−0.96 (m, 6
H). MS (ESI): m/z 197.2 (M + 1).
described for the preparation of compound 2a to furnish 80 (60 mg,
79% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.80−7.76
(m, 2 H), 7.20−7.15 (m, 2 H), 6.86 (s, 1 H), 6.43 (br s, 1 H), 5.28−
5.25 (m, 1 H), 4.87−4.77 (m, 1 H), 3.69−3.59 (m, 1 H), 3.13 (dd, J =
14.0 Hz, 11.2 Hz, 0.5H), 2.84 (dd, J = 14.0 Hz, 11.2 Hz, 0.5H), 2.80−
2.74 (m, 2 H), 1.92−1.68 (m, 4 H), 1.58−1.24 (m, 7 H), 1.07−0.76
(m, 9 H). MS (ESI): m/z 402.2 (M + 1).
B. (3S,6S)-6-Allyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-3-
isobutylpiperazin-2-one (77). (3S,6S)-6-Allyl-3-isobutylpiperazin-2-
one (40 mg, 0.20 mmol) and 5-(4-fluorophenyl)isoxazole-3-carboxylic
acid (45 mg, 0.22 mmol) were coupled according to the procedure
described for the preparation of compound 2a to furnish 77 (60 mg,
79% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.81−7.76
(m, 2 H), 7.22−7.16 (m, 2 H), 6.86 (m, 1 H), 5.99 (br s, 1 H), 5.76−
5.67 (m, 1 H), 5.32−5.20 (m, 2 H), 4.88−4.78 (m, 1 H), 3.76−3.65
(m, 1 H), 3.18 (dd, J = 14.0 Hz, 11.2 Hz, 0.5 H), 2.89 (dd, J = 14.0 Hz,
11.2 Hz, 0.5 H), 2.42−2.36 (m, 1 H), 2.16−2.05 (m, 1 H), 1.92−1.63
(m, 4 H), 1.08−0.77 (m, 6 H). MS (ESI): m/z 386.1 (M + 1).
(3S,6S)-6-(Cyclopropylmethyl)-4-(5-(4-fluorophenyl)isoxazole-3-
carbonyl)-3-isobutyl-pipe-razin-2-one (78). A. (3S,6S)-6-(Cyclopro-
pylmethyl)-3-isobutylpiperazin-2-one. Synthesized from Fmoc-cyclo-
propylalanine (23.6 g, 67.3 mmol) and HCl salt of Leu-OMe (6.6 g,
37.0 mmol) by the method described for the preparation of compound
7 (Scheme 1) to afford (3S,6S)-6-(cyclopropylmethyl)-3-isobutylpi-
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(2-(methylthio)ethyl)-piperazin-2-one (81). A. (3S,6S)-3-Isobutyl-6-
(2-(methylthio)ethyl)piperazin-2-one. Synthesized from (S)-2-
(Fmoc)amino-4-(methylthio)butanoic acid (24.9 g, 67 mmol) and
HCl salt of Leu-OMe (6.6 g, 37.0 mmol) by the method described for
the preparation of compound 7 to afford (3S,6S)-3-isobutyl-6-(2-
1
(methylthio)ethyl)piperazin-2-one (5.0 g, 49% yield). H NMR (400
MHz, DMSO-d6) δ 6.14 (s, 1 H), 3.54 (s, 1 H), 3.39−3.42 (m, 1 H),
3.03−3.07 (m, 1 H), 2.83−2.87 (m, 1 H), 2.78 (s, 1 H), 2.53−2.58
(m, 2 H), 2.09−2.15 (m, 3 H), 1.86−1.90 (m, 1 H), 1.71−1.82 (m, 2
H), 1.54−1.57 (m, 1 H), 0.90−0.96 (m, 6 H). MS (ESI): m/z 231.1
(M + 1).
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(2-(methylthio)ethyl)-piperazin-2-one (81). (3S,6S)-3-Isobutyl-6-
(2-(methylthio)ethyl)piperazin-2-one (50 mg, 0.22 mmol) and 5-(4-
fluorophenyl)isoxazole-3-carboxylic acid (45 mg, 0.22 mmol) were
coupled according to the procedure described for the preparation of
compound 2a to furnish 81 (71 mg, 78% yield) as a colorless solid. 1H
NMR (400 MHz, CDCl3) δ 7.81−7.76 (m, 2 H), 7.22−7.16 (m, 2 H),
6.87 (s, 1 H), 6.41 (s, 1 H), 5.33−5.26 (m, 1 H), 4.93−4.79 (m, 1 H),
3.86−3.77 (m, 1 H), 3.20−2.91 (m, 1 H), 2.63−2.58 (m, 2 H), 2.13
(s, 3 H), 1.92−1.70 (m, 5 H), 1.08−0.78 (m, 6 H). MS (ESI): m/z
420.10 (M + 1).
(3S,6S)-6-Ethyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-3-iso-
butylpiperazin-2-one (82). A. (3S,6S)-3-Isobutyl-6-ethylpiperazin-2-
one. Synthesized from (S)-2-(Fmoc)amino-butanoic acid (21.8 g, 67
mmol) and HCl salt of Leu-OMe (6.6 g, 37.0 mmol) by the method
described for the compound 7 to afford (3S,6S)-3-isobutyl-6-
ethylpiperazin-2-one (2.1 g, 65% yield). 1H NMR (400 MHz,
CDCl3) δ 6.03 (s, 1 H), 3.21−3.37 (m, 2 H), 2.91−2.97 (m, 1 H),
2.72−2.76 (m, 1 H), 1.63−1.67 (m, 1 H), 1.44−1.52 (m, 3 H), 0.85−
0.91 (m, 9 H). MS (ESI): m/z 185.1 (M + 1).
1
perazin-2-one (1.6 g, 11.5% yield) as a white solid. H NMR (400
MHz, DMSO-d6) δ 7.49 (s, 1 H), 3.24−3.27 (m, 1 H), 3.05−3.08 (dd,
J = 9.6 Hz, 4.0 Hz, 1 H), 2.82−2.86 (dd, J = 13.2 Hz, 4.4 Hz, 1 H),
2.69−2.74 (dd, J = 12.8 Hz, 5.2 Hz, 1 H), 1.72−1.78 (m, 1 H), 1.39−
1.49 (m, 2 H), 1.31−1.36 (m, 2 H), 0.81−0.89 (m, 6 H), 0.62−0.67
(m, 1 H), 0.38−0.40 (m, 2 H), 0.01−0.08 (m, 2 H). MS (ESI): m/z
210.9 (M + 1).
B. (3S,6S)-6-(Cyclopropylmethyl)-4-(5-(4-fluorophenyl)isoxazole-
3-carbonyl)-3-isobutylpipera-zin-2-one (78). (3S,6S)-6-(Cyclopro-
pylmethyl)-3-isobutylpiperazin-2-one (50 mg, 0.24 mmol) and 5-(4-
fluorophenyl)isoxazole-3-carboxylic acid (53 mg, 0.25 mmol) were
coupled according to the procedure described for the preparation of
1
compound 2a to furnish 78 (53 mg, 54% yield) as a white solid. H
NMR (400 MHz, CDCl3) δ 7.79−7.61 (m, 2 H), 7.19−7.15 (m, 2 H),
6.86 (s, 1 H), 6.47 (s, 1 H), 5.29−5.25 (m, 1 H), 4.93−4.88 (m, 1 H),
3.78−3.74 (m, 1 H), 3.21 (dd, J = 14.0 Hz, 11.0 Hz, 1 H), 1.88−1.52
(m, 4 H), 1.30−1.22 (m, 1 H), 1.08−0.96 (m, 6 H), 0.74−0.52 (m, 3
H), 0.21−0.08 (m, 2 H). MS (ESI): m/z 400.1 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
((methylthio)methyl)-piperazin-2-one (79). A. (3S,6R)-3-Isobutyl-6-
((methylthio)methyl)piperazin-2-one. Synthesized from (R)-2-
(Fmoc)amino-3-(methylthio)propanoic acid (23.9 g, 67 mmol) and
HCl salt of Leu-OMe (6.6 g, 37.0 mmol) by the method described for
the preparation of compound 7 to afford (3S,6R)-3-isobutyl-6-
((methylthio)methyl)piperazin-2-one (1.7 g, 12% yield). 1H NMR
(400 MHz, DMSO-d6) δ 7.49 (s, 1 H), 3.31−3.33 (m, 1 H), 3.06−
3.09 (m, 1 H), 2.84−2.89 (m, 2 H), 2.68−2.71 (m, 1 H), 2.53−2.54
(m,1 H), 2.05−2.06 (m, 3 H), 1.74−1.78 (m, 1 H), 1.51−1.54 (m, 1
H), 1.36−1.39 (m, 1 H), 0.82−0.88 (m, 6 H). MS (ESI): m/z 217.14
(M +1).
B. (3S,6S)-6-Ethyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-3-
isobutylpiperazin-2-one (82). (3S,6S)-3-Isobutyl-6-ethylpiperazin-2-
one (50 mg, 0.27 mmol) and 5-(4-fluorophenyl)isoxazole-3-carboxylic
acid (56 mg, 0.27 mmol) were coupled according to the procedure
described for the preparation of compound 2a to furnish 82 (50 mg,
1
50% yield) as a colorless solid. H NMR (400 MHz, CDCl3) δ 7.80−
7.76 (m, 2 H), 7.26−7.16 (m, 2 H), 6.86 (s, 1 H), 5.97 (s, 1 H), 5.32−
5.27 (m, 1 H), 4.91−4.80 (m, 1 H), 3.64−3.55 (m, 1 H), 3.18−2.83
(m, 1 H), 1.92−1.69 (m, 2 H), 1.63−1.48 (m, 3 H), 1.09−0.77 (m, 9
H). MS (ESI): m/z 374.10 (M + 1).
B. (3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-((methylthio)methyl)-piperazin-2-one (78). (3S,6R)-3-Isobutyl-6-
((methylthio)methyl)piperazin-2-one (50 mg, 0.23 mmol) and 5-(4-
fluorophenyl)isoxazole-3-carboxylic acid (50 mg, 0.24 mmol) were
coupled according to the procedure described for the preparation of
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
isopropylpiperazin-2-one (83). A. (3S,6S)-3-Isobutyl-6-isopropylpi-
perazin-2-one. Synthesized from Fmoc-L-valine (27.4 g, 80 mmol)
and HCL salt of Leu-OMe (3.4 g, 23 mmol) by the method described
for the preparation of compound 7 (Scheme 1) to afford the (3S,6S)-
3-isobutyl-6-isopropylpiperazin-2-one (2.5 g, 22% yield) as a white
solid. 1H NMR (400 MHz, CDCl3) δ 5.88 (s, 1 H), 3.40 (dd, J = 10.4
Hz, 4.0 Hz, 1H), 3.09−3.06 (m, 1 H), 2.94−2.86 (m, 2 H), 1.77−1.72
(m, 2 H), 1.59−1.55 (dd, J = 10.4 Hz, 4.4 Hz, 1 H), 1.31−1.23 (m, 1
H), 0.95−0.90 (m, 12 H). MS (ESI): m/z 199.0 (M + 1).
1
compound 2a to furnish 79 (30 mg, 30% yield) as a white solid. H
NMR (CDCl3) δ 7.82−7.77 (m, 2 H), 7.25−7.17 (m, 2 H), 6.88 (s,
0.5 H), 6.78 (s, 0.5 H), 6.52 (br s, 1 H), 5.39−5.28 (m, 1 H), 5.04−
4.82 (m, 1 H), 4.50−4.39 (m, 1 H), 3.85- 3.74 (m, 1 H), 3.42−2.36
(m, 2 H), 2.15 (s, 2 H), 2.13 (s, 1 H), 1.94−1.86 (m, 1 H), 1.81−1.72
(m, 2 H), 1.10−0.78 (m, 6 H). MS (ESI): m/z 406.2 (M + 1).
(3S,6S)-6-Butyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-3-iso-
butylpiperazin-2-one (80). A. (3S,6S)-3-Isobutyl-6-butyl-piperazin-2-
one. Synthesized from Fmoc-(S)-2-aminohexanoic acid (9.91 g, 28.5
mmol) and HCl salt of Leu-OMe (4.7 g, 26.25 mmol) by the method
described for the preparation of compound 7 (Scheme 1) to afford
(3S,6S)-3-isobutyl-6-butyl-piperazin-2-one (3.4 g, 25% yield) as a
white solid. MS (ESI): m/z 213.3 (M + 1).
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-isopropylpiperazin-2-one (83). (3S,6S)-3-Isobutyl-6-isopropylpi-
perazin-2-one (50 mg, 0.25 mmol) and 5-(4-fluorophenyl)isoxazole-
3-carboxylic acid (52 mg, 0.25 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 83
1
(43 mg, 44% yield) as a white solid. H NMR (400 MHz, CDCl3) δ
7.80−7.76 (m, 2 H), 7.21−7.16 (m, 2 H), 6.87 (s, 1 H), 5.97 (s, 1 H),
5.31−5.27 (m, 1 H), 4.92−4.77 (m, 1 H), 3.53−3.44 (m, 1 H), 3.23−
2.90 (m, 1 H), 1.90−1.69 (m, 4 H), 1.08−0.77 (m, 12 H). MS (ESI):
m/z 388.1 (M + 1).
(3S,6S)-6-(tert-Butyl)-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-
3-isobutylpiperazin-2-one (84). A. (3S,6S)-6-(tert-Butyl)-3-isobutyl-
B. (3S,6S)-6-Butyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-3-
isobutylpiperazin-2-one (80). (3S,6S)-3-Isobutyl-6-butyl-piperazin-2-
one (40 mg, 0.19 mmol) and 5-(4-fluorophenyl)isoxazole-3-carboxylic
acid (50 mg, 0.24 mmol) were coupled according to the procedure
R
dx.doi.org/10.1021/jm4012643 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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piperazin-2-one. Synthesized from Fmoc-L-tert-leucine (23.0 g, 16
mmol) and HCL salt of Leu-OMe (5.0 g, 27.0 mmol) by the method
described for the preparation of compound 7 (Scheme 1) to afford
(3S,6S)-6-(tert-butyl)-3-isobutylpiperazin-2-one (1.0 g, 16% yield) as a
H), 2.77 (dd, J = 12.8 Hz, 4.0 Hz, 1 H), 2.68 (dd, J = 12.8 Hz, 4.8 Hz,
1 H), 1.97 (s, 1 H), 1.38−1.55 (m, 9 H), 1.08−1.22 (m, 3 H), 0.80−
0.90 (m, 6 H). MS (ESI): m/z 225.1 (M + 1).
B. (3S,6S)-6-Cyclopentyl-4-(5-(4-fluorophenyl)isoxazole-3-car-
bonyl)-3-isobutylpiperazin-2-one (87). (3S,6S)-6-Cyclopentyl-3-iso-
butylpiperazin-2-one (50 mg, 0.22 mmol) and 5-(4-fluorophenyl)-
isoxazole-3-carboxylic acid (46 mg, 0.22 mmol) were coupled
according to the procedure described for the preparation of compound
1
white solid. H NMR (400 MHz, DMSO-d6) δ 7.18 (s, 1 H), 3.05−
3.09 (m, 2 H), 2.97−2.99 (m, 1 H), 2.66−2.69 (m, 1 H), 2.60−2.64
(m, 1 H), 1.76−1.89 (m, 1 H), 1.38−1.44 (m, 2 H), 0.86−0.89 (m, 15
H). MS (ESI): m/z 213.0 (M + 1).
1
2a to furnish 87 (56 mg, 61% yield) as a white solid. H NMR (400
B. (3S,6S)-6-(tert-Butyl)-4-(5-(4-fluorophenyl)isoxazole-3-carbon-
yl)-3-isobutylpiperazin-2-one (84). (3S,6S)-3-Isobutyl-6-isopropylpi-
perazin-2-one (50 mg, 0.24 mmol) and 5-(4-fluorophenyl)-isoxazole-
3-carboxylic acid (50 mg, 0.24 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 84
(78 mg, 82% yield) as a colorless solid. 1H NMR (400 MHz, CDCl3) δ
7.80−7.77 (m, 2 H), 7.21−7.16 (m, 2 H), 6.87 (s, 1 H), 5.85 (s, 1 H),
5.30−5.27 (m, 1 H), 4.95−4.79 (m, 1 H), 3.48−3.37 (m, 1 H), 3.25−
2.92 (m, 1 H), 1.90−1.81 (m, 1 H), 1.77−1.71 (m, 2 H), 1.09−0.79
(m, 15 H). MS (ESI): m/z 402.10 (M − 1).
MHz, CDCl3) δ 7.81−7.76 (m, 2 H), 7.21−7.16 (m, 2 H), 6.87 (s, 1
H), 5.94 (s, 1 H), 5.29−5.26 (m, 1 H), 4.93−4.79 (m, 1 H), 3.53−3.41
(m, 1 H), 3.19−2.86 (m, 1 H), 1.91−1.58 (m, 10 H), 1.32−1.23 (m, 2
H), 1.09−0.77 (m, 6 H). MS (ESI): m/z 414.1 (M + 1).
(3S,6S)-6-Cyclohexyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-
3-isobutylpiperazin-2-one (88). A. (3S,6S)-6-Cyclohexyl-3-isobutyl-
piperazin-2-one. Synthesized from Fmoc-L-cyclohexylglycine (22.78
g, 60 mmol) and HCl salt of Leu-OMe (6.0 g, 32.8 mmol) by the
method described for the preparation of compound 7 (Scheme 1) to
afford the product (3S,6S)-6-cyclohexyl-3-isobutylpiperazin-2-one (1.2
(3S,6S)-6-Cyclopropyl-4-(5-(4-fluorophenyl)isoxazole-3-carbon-
yl)-3-isobutylpiperazin-2-one (85). A. (3S,6S)-6-Cyclopropyl-3-iso-
butylpiperazin-2-one. Synthesized from Fmoc-L-cyclopropylglycine
(13.78 g, 43 mmol) and HCl salt of Leu-OMe (6.0 g, 32.9 mmol) by
the method described for the compound 7 (Scheme 1) to afford
(3S,6S)-6-cyclopropyl-3-isobutylpiperazin-2-one (1.7 g, 20% yield) as
1
g, 8% yield) as a white solid. H NMR (400 MHz, DMSO-d6) δ 7.45
(s, 1 H), 3.08 (dd, J = 10.0 Hz, 4.4 Hz, 1 H), 2.95 (d, J = 5.6 Hz, 1 H),
2.76 (dd, J = 13.6 Hz, 6.0 Hz, 1 H), 2.68 (dd, J = 13.2 Hz, 4.4 Hz, 1
H), 1.59−1.77 (m, 6 H), 1.38−1.45 (m, 3 H), 1.09−1.16 (m, 3 H),
0.89−0.95 (m, 5 H), 0.72−0.88 (m, 3 H). MS (ESI): m/z 239.0 (M +
1).
1
a white solid. H NMR (400 MHz, DMSO-d6) δ 7.575 (s, 1 H), 3.43
(t, J = 4.0 Hz, 1 H), 2.81−2.82 (m, 1 H), 2.78−2.79 (m, 2 H), 2.38−
2.39 (m, 2 H), 1.72−1.80 (m, 1 H), 1.49−1.52 (m, 1 H), 1.45−1.47
(m, 1 H), 0.95−1.05 (m, 1 H), 0.87 (d, J = 3.2 Hz, 3 H), 0.82 (d, J =
3.2 Hz, 3 H), 0.42−0.45 (m, 1 H), 0.32−0.36 (m, 1 H), 0.24−0.26 (m,
1 H), 0.08−0.12 (m, 1 H). MS (ESI): m/z 197.1 (M + 1).
B. (3S,6S)-6-Cyclohexyl-4-(5-(4-fluorophenyl)isoxazole-3-carbon-
yl)-3-isobutylpiperazin-2-one (88). (3S,6S)-6-Cyclohexyl-3-isobutyl-
piperazin-2-one (50 mg, 0.24 mmol) and 5-(4-fluorophenyl)-isoxazole-
3-carboxylic acid (43 mg, 0.21 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 88
1
(46 mg, 51% yield) as a white solid. H NMR (400 MHz, CDCl3) δ
B. (3S,6S)-6-Cyclopropyl-4-(5-(4-fluorophenyl)isoxazole-3-car-
bonyl)-3-isobutylpiperazin-2-one (85). (3S,6S)-6-Cyclopropyl-3-iso-
butylpiperazin-2-one (50 mg, 0.25 mmol) and 5-(4-fluorophenyl)-
isoxazole-3-carboxylic acid (52 mg, 0.25 mmol) were coupled
according to the procedure described for the preparation of compound
7.81−7.76 (m, 2 H), 7.21−7.16 (m, 2 H), 6.86 (s, 1 H), 5.86 (s, 1 H),
5.30−5.27 (m, 1 H), 4.92−4.78 (m, 1 H), 3.53−3.44 (m, 1 H), 3.26−
2.93 (m, 1 H), 1.88−1.70 (m, 8 H), 1.45−1.37 (m, 1 H), 1.29−1.11
(m, 5 H), 1.09−0.77 (m, 6 H). MS (ESI): m/z 428.1 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(3,3,3-trifluoro-2-(trifluoromethyl)propyl)piperazin-2-one (89).
A. (3S,6S)-3-Isobutyl-6-(3,3,3-trifluoro-2-(trifluoromethyl)-propyl)-
piperazin-2-one. Synthesized from (S)-2-((((9H-fluoren-9-yl)-
methoxy)carbonyl)amino)-5,5,5-trifluoro-4-(trifluoro-methyl)-
pentanoic acid (9.22 g, 20 mmol) and HCl salt of Leu-OMe (3.0 g,
16.4 mmol) by the method described for the compound 7 (Scheme 1)
to afford (3S,6S)-3-isobutyl-6-(3,3,3-trifluoro-2-(trifluoromethyl)-
propyl)piperazin-2-one (321 mg, 5% yield). 1H NMR (400 MHz,
CDCl3) δ 6.83 (s, 1 H), 3.87−3.89 (m, 0.5 H), 3.72−3.75 (m, 0.5 H),
3.52−3.56 (m, 1 H), 2.89−2.92 (m, 1 H), 3.11−3.15 (m, 1 H), 2.86−
2.90 (m, 1 H), 2.28−2.35 (m, 1 H), 1.97−2.01 (m, 1 H), 1.66−1.80
(m, 2 H), 1.49−1.52 (m, 1 H), 0.85−0.89 (m, 6 H). MS (ESI): m/z
321.1 (M + 1).
1
2a to furnish 85 (78 mg, 79% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 7.80−7.77 (m, 2 H), 7.20−7.16 (m, 2 H), 6.85 (s, 1
H), 6.05 (s, 1 H), 5.29−5.24 (m, 1 H), 4.94−4.86 (m, 1 H), 3.35−3.02
(m, 1 H), 2.88−2.77 (m, 1 H), 1.92−1.88 (m, 1 H), 1.83−1.71 (m, 2
H), 1.09−0.92 (m, 6 H), 0.79−0.75 (m, 2 H), 0.64−0.60 (m, 2 H),
0.43−0.27 (m, 1 H). MS (ESI): m/z 386.1 (M + 1).
(3S,6S)-6-Cyclobutyl-4-(5-(4-fluorophenyl)isoxazole-3-carbonyl)-
3-isobutylpiperazin-2-one (86). A. (3S,6S)-6-Cyclobutyl-3-isobutyl-
piperazin-2-one. Synthesized from Fmoc-(S)-amino-cyclobutyl-acetic
acid (11.3 g, 32.19 mmol) and HCl salt of Leu-OMe (3.67 g, 20.3
mmol) by the method described for the compound 7 (Scheme 1) to
afford (3S,6S)-6-cyclobutyl-3-isobutylpiperazin-2-one (1.55 g, 26%
1
yield) as a white solid. H NMR (400 MHz, CDCl3) δ 5.74 (s, 1 H),
3.35−3.38 (m, 1 H), 3.24−3.26 (m, 1 H), 2.89−2.94 (dd, J = 13.2 Hz,
4.4 Hz, 1 H), 2.68−2.73 (m, 1 H), 2.41−2.45 (m, 1 H), 1.90−2.04 (m,
3 H), 1.63−1.81 (m, 5 H), 1.51−1.54 (m, 2 H), 0.85−0.94 (m, 6 H).
MS (ESI): m/z 211.1 (M + 1).
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(3,3,3-trifluoro-2-(trifluoromethyl)propyl)piperazin-2-one (89).
(3S,6S)-3-Isobutyl-6-(3,3,3-trifluoro-2-(trifluoromethyl)propyl)-piper-
azin-2-one (40 m g, 0.14 mmol) and 5-(4-fluorophenyl)-isoxazole-3-
carboxylic acid (30 mg, 0.14 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish
B. (3S,6S)-6-Cyclobutyl-4-(5-(4-fluorophenyl)isoxazole-3-carbon-
yl)-3-isobutylpiperazin-2-one (86). (3S,6S)-6-Cyclobutyl-3-isobutyl-
piperazin-2-one (42 mg, 0.20 mmol) and 5-(4-fluorophenyl)isoxa-zole-
3-carboxylic acid (42 mg, 0.20 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 98
1
compound 89 (40 mg, 48% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 7.82−7.79 (m, 2 H), 7.22−7.17 (m, 2 H), 6.91 (s, 1
H), 5.39 (t, J = 6.0 Hz, 0.5 H), 5.28 (dd, J = 7.2 Hz, 4.0 Hz, 1 H), 5.06
(d, J = 13.2 Hz, 1 H), 4.89 (d, J = 7.2 Hz, 0.5 H), 3.99−3.85 (m, 1 H),
3.40−3.34 (m, 1 H), 3.17 (dd, J = 14.8 Hz, 10.2 Hz, 1 H), 2.07−1.83
(m, 3 H), 1.77−1.71 (m, 2 H), 1.08 (d, J = 6.4 Hz, 2 H), 0.99 (d, J =
6.4 Hz, 2 H), 0.94 (d, J = 6.0 Hz, 1 H), 0.81 (d, J = 6.0 Hz, 1 H). MS
(ESI): m/z 510.2 (M + 1).
1
(55 mg, 69% yield) as a white solid. H NMR (400 MHz, CDCl3) δ
7.80−7.77 (m, 2 H), 7.26−7.16 (m, 2 H), 6.87 (s, 1 H), 5.91 (s, 1 H),
5.30−5.25 (m, 1 H), 4.84−5.73 (m, 1 H), 3.69−3.55 (m, 1 H), 3.09
(dd, J = 14.0 Hz, 11.2 Hz, 0.5 H), 2.73 (dd, J = 14.0 Hz, 11.2 Hz, 0.5
H), 2.38−2.29 (m, 1 H), 2.14−1.66 (m, 9 H), 1.08−0.76 (m, 6 H).
MS (ESI): m/z 400.0 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-6-(hydroxy-
methyl)-3-isobutylpipera-zin-2-one (90). Compound 14 (2.00 g, 8.25
mmol) and HCl in dioxane (4 N, 20 mL) were stirred at 70 °C for 1.5
h and then concentrated under vacuo to give the crude (3S,6R)-6-
(hydroxymethyl)-3-isobutylpiperazin-2-one, which was used for the
next step without further purification. Crude compound of (3S,6R)-6-
(hydroxymethyl)-3-isobutylpiperazin-2-one (0.20 g, 0.90 mmol) and
5-(4-fluorophenyl)-isoxazole-3-carboxylic acid (0.19 g, 0.90 mmol)
(3S,6S)-6-Cyclopentyl-4-(5-(4-fluorophenyl)isoxazole-3-carbon-
yl)-3-isobutylpiperazin-2-one (87). A. (3S,6S)-6-Cyclopentyl-3-iso-
butylpiperazin-2-one. Synthesized from Fmoc-L-cyclopentyl-glycine
(10.23 g, 28 mmol) and HCl salt of Leu-OMe (6.7 g, 36.8 mmol) by
the method described for the preparation of compound 7 (Scheme 1)
to afford (3S,6S)-6-cyclopentyl-3-isobutyl-piperazin-2-one (1.1 g, 16%
1
yield) as a white solid. H NMR (400 MHz, DMSO-d6) δ 7.51 (s, 1
H), 3.08 (dd, J = 10.0 Hz, 3.6 Hz, 1 H), 2.93 (dd, J = 7.6 Hz, 3.6 Hz, 1
S
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were coupled according to the procedure described for the preparation
of compound 2a to furnish 90 (0.294 g, 87% yield) as a white solid. 1H
NMR (400 MHz, DMSO-d6) δ 8.03−7.99 (m, 2 H), 7.93 (s, 0.3 H),
7.90 (s, 0.7 H), 7.45−7.41 (m, 2 H), 7.37 (s, 1 H), 5.01 (m, 0.3 H),
4.92−4.89 (m, 1.7 H), 4.69−4.60 (m, 0.3 H), 4.21 (dd, J = 14.4 Hz,
4.4 Hz, 0.7 H), 3.53−3.48 (m, 1 H), 3.46−3.41 (m, 1 H), 3.31−3.27
(m, 1.7 H), 3.05 (m, 0.3 H), 1.81 (t, J = 9.6 Hz, 1 H), 1.68−1.61 (m, 2
H), 0.98 (d, J = 6.0 Hz, 2.4 H), 0.94 (d, J = 6.0 Hz, 2.4 H), 0.81 (d, J =
6.4 Hz, 0.6 H), 0.61 (d, J = 6.4 Hz, 0.6 H). MS (ESI): m/z 376.2 (M +
1).
(3S,6S)-3-isobutyl-6-(tetrahydro-2H-thiopyran-4-yl)piperazin-2-one
(0.77 g, 37% yield) as a brown solid. H NMR (400 MHz, CDCl3) δ
6.09 (s, 1 H), 3.31−3.37 (m, 2 H), 3.10−3.14 (m, 1 H), 2.62−2.74
(m, 5 H), 1.90−2.03 (m, 3 H), 1.84−1.88 (m, 1 H), 1.71−1.80 (m, 1
H), 1.37−1.54 (m, 4 H), 0.87−0.95 (m, 6 H). MS (ESI): m/z 257.17
(M + 1).
1
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(tetrahydro-2H-thiopyran-4-yl)piperazin-2-one (94). (3S,6S)-3-
Isobutyl-6-(tetrahydro-2H-thiopyran-4-yl)piperazin-2-one (50 mg,
0.27 mmol) and 5-(4-fluorophenyl)isoxazole-3-carboxylic acid (44
mg, 0.27 mmol) were coupled according to the procedure described
for the preparation of compound 2a to furnish compound 94 (37 mg,
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(methoxymethyl)pipera-zin-2-one (91). To a soluton of alcohol 90
(50 mg, 0.13 mmol) in MeCN (3 mL) under argon was added MeI
(83 uL, 1.33 mmol) and Ag2O (154 mg, 0.66 mmol) and stirred at rt
for 3 days. The reaction mixture was filtered through a short pad of
Celite, concentrated in vacuo, and purified by column chromatography
(0−80% EtOAc in hexanes) to give 91 (19 mg, 37% yield) as a
1
42% yield) as a colorless gum. H NMR (400 MHz, CDCl3) δ 7.81−
7.77 (m, 2 H), 7.22−7.16 (m, 2 H), 6.87 (s, 1 H), 6.32 (s, 1 H), 5.32−
5.26 (m, 1 H), 4.92−4.71 (m, 1 H), 3.65−3.54 (m, 1 H), 3.29−2.95
(m, 1 H), 2.73−2.66 (m, 4 H), 2.09−2.06 (m, 2 H), 1.90−1.82 (m, 1
H), 1.76−1.66 (m, 2 H), 1.60−1.54 (m, 2 H), 1.08−0.0.78 (m, 6 H).
MS (ESI): m/z 445.97 (M + 1).
1
colorless solid. H NMR (400 MHz, DMSO-d6) δ 8.06−8.00 (m, 3
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
phenylpiperazin-2-one (95). A. (3S,6S)-3-Isobutyl-6-phenylpipera-
zin-2-one. Synthesized from Fmoc-L-2-phenylglycine (23.41 g, 63
mmol) and HCl salt of Leu-OMe (8 g, 56 mmol) by the method
described for the compound 7 (Scheme 1) to afford the (3S,6S)-3-
isobutyl-6-phenylpiperazin-2-one (2.0 g, 9% yield) as a white solid. 1H
NMR (400 MHz, DMSO-d6) δ 7.35−7.40 (m, 2 H), 7.24−7.32 (m, 2
H), δ 6.12 (s, 1 H), 4.62 (q, J = 7.2 Hz, 1 H), 3.49 (dd, J = 10.4 Hz,
3.2 Hz, 1 H), 3.22−3.27 (m, 1 H), 2.98 (dd, J = 13.6 Hz, 5.2 Hz, 1 H),
1.68−1.87 (m, 2 H), 1.54−1.61 (m, 1 H), 0.94 (d, J = 3.2 Hz, 3 H),
0.91 (d, J = 3.2 Hz, 3 H). MS (ESI): m/z 233.2 (M + 1).
H), 7.45−7.40 (m, 2 H), 7.38 (s, 1 H), 4.90 (m, 1 H), 4.70−4.56 (m, 1
H), 4.21 (m, 1 H), 3.68 (m, 1 H), 3.44−3.37 (m, 1 H), 3.23 (s, 2 H),
3.18−3.05 (m, 1 H), 2.69 (s, 1 H), 1.81 (t, J = 10.0 Hz, 1 H), 1.69−
1.59 (m, 2 H), 0.98 (d, J = 5.6 Hz, 3 H), 0.94 (d, J = 6.0 Hz, 3 H). MS
(ESI): m/z 390.2 (M +1).
(3S,6S)-6-((Dimethylamino)methyl)-4-(5-(4-fluorophenyl)-
isoxazole-3-carbonyl)-3-isobutyl-piperazin-2-one (92). To a solution
of the alcohol 90 (0.20 g, 0.53 mmol) in DMF (3 mL) was added
Et3N (0.23 mL, 1.65 mmol) and MsCl (70 uL, 0.90 mmol) and stirred
at rt for 16 h and then concentrated in vacuo. The residue was purified
by column chromatography (0−75% EtOAc in hexanes) to give the
crude mesylate product (0.132 g, 55% yield), which was used for the
next step without further purification. MS (ESI): m/z 454.2 (M + 1).
To a solution of the mesylate (30 mg, 0.07 mmol) in DMF (2 mL)
was added dimethyamine in THF (2 M, 0.17 mL, 0.34 mmol) and
heated at 60 °C for 16 h. The reaction mixture was concentrated in
vacuo and purified by column chromatography (0−90% EtOAc in
hexanes) to give 92 (19 mg, 71% yield) as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 8.01 (m, 2 H), 7.76 (s, 1 H), 7.68 (s, 3 H), 7.45−
7.38 (m, 3 H), 4.91−4.89 (m, 1 H), 4.70−4.64 (m, 1 H), 4.25−4.18
(m, 1 H), 3.74−3.64 (m, 1 H), 3.22 (m, 1 H), 2.36−2.25 (m, 2 H),
2.09 (s, 6 H), 1.84 (t, J = 10.8 Hz, 1 H), 1.67−1.63 (m, 1 H), 0.98 (d, J
= 5.6 Hz, 2.5 H), 0.94 (d, J = 6.0 Hz, 2.5 H), 0.81 (d, J = 6.4 Hz, 0.5
H), 0.61 (d, J = 6.4 Hz, 0.5 H). MS (ESI): m/z 403.2 (M + 1).
(3S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(tetrahydro-2H-pyran-4-yl)-piperazin-2-one (93). A. (3S)-3-Isobutyl-
6-(tetrahydro-2H-pyran-4-yl)piperazin-2-one. Synthesized from
Fmoc-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (11.98 g, 31.4
mmol) and hydrochloride salt of Leu-OMe (3.0 g, 16.4 mmol) by the
method described for the compound 7 to afford (3S)-3-isobutyl-6-
(tetrahydro-2H-pyran-4-yl)piperazin-2-one (0.50 g, 6.70% yield) as a
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-phenylpiperazin-2-one (95). (3S,6S)-3-Isobutyl-6-phenylpiperazin-
2-one (50 mg, 0.22 mmol) and 5-(4-fluorophenyl)isoxazole-3-
carboxylic acid (45 mg, 0.22 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 95
1
(69 mg, 76% yield) as a white solid. H NMR (400 MHz, CDCl3) δ
7.82−7.78 (m, 2 H), 7.44−7.36 (m, 5 H), 7.22−7.16 (m, 2 H), 6.89
(s, 1 H), 6.04 (s, 1 H), 5.43−5.36 (m, 1 H), 4.97−4.88 (m, 2 H),
3.37−3.06 (m, 1 H), 2.04−1.94 (m, 1 H), 1.93−1.84 (m, 1 H), 1.80−
1.70 (m, 1 H), 1.11−0.81 (m, 6 H). MS (ESI): m/z 422.1 (M +1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(p-tolyl)piperazin-2-one (96). A. (3S,6S)-3-Isobutyl-6-p-tolyl-piper-
azin-2-one. Synthesized from (S)-2-amino-2-(p-tolyl)acetic acid (5.0g,
30 mmol) and HCl salt of Leu-OMe (4.37 g, 24.3 mmol) by the
method described for the preparation of compound 7 (Scheme 1) to
afford (3S,6S)-3-Isobutyl-6-p-tolyl-piperazin-2-one (1.44 g, 19% yield)
as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.87 (s, 1 H), 7.15
(s, 4 H), 4.41−4.44 (m, 1 H), 3.17−3.20 (m, 1 H), 3.00−3.03 (m, 1
H), 2.68−2.72 (m, 1 H), 2.31 (s, 1 H), 2.28 (s, 3 H), 1.72−1.80 (m, 1
H), 1.51−1.62 (m, 1 H), 1.44−1.49 (m, 1 H), 0.89 (d, J = 5.6 Hz, 3
H), 0.85 (d, J = 6.4 Hz, 3 H). MS (ESI): m/z 247.1 (M + 1).
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(p-tolyl)piperazin-2-one (96). (3S,6S)-3-Isobutyl-6-p-tolyl-pipera-
zin-2-one (50 mg, 0.20 mmol) and 5-(4-fluorophenyl)-isoxazole-3-
carboxylic acid (41 mg, 0.20 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 96
1
white solid. H NMR (400 MHz, CDCl3) δ 6.12−6.35 (m, 1 H),
3.94−4.09 (m, 2 H), 2.94−3.38 (m, 5 H), 2.00−2.01 (m, 1 H), 1.20−
1.86 (m, 9 H), 0.87−0.93 (m, 6 H). MS (ESI): m/z 241.17 (M + 1).
B. (3S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(tetrahydro-2H-pyran-4-yl)-piperazin-2-one (93). (3S)-3-Isobutyl-6-
(tetrahydro-2H-pyran-4-yl)piperazin-2-one (36 mg, 0.15 mmol) and
5-(4-fluorophenyl)isoxazole-3-carboxylic acid (35 mg, 0.17 mmol)
were coupled according to the procedure described for the preparation
of compound 2a to furnish 93 (10 mg, 15% yield) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 7.80−7.77 (m, 2 H), 7.21−7.10 (m, 2 H),
6.88 (s, 1 H), 6.17 (br s, 1 H), 5.34−5.27 (m, 1 H), 4.99 and 4.82 (dd,
J = 13.2 Hz, 4.4 Hz, 1 H), 4.06−4.00 (m, 2 H), 3.59−3.47 (m, 1 H),
3.43−3.34 (m, 2 H), 3.23 and 2.95 (dd, J = 13.6 Hz, 11.2 Hz, 1 H),
1.90−1.42 (m, 8 H) and 1.08−0.78 (m, 6 H). MS (ESI): m/z 430.1
(M + 1).
1
(77 mg, 88% yield) as a white solid. H NMR (400 MHz, CDCl3) δ
7.82−7.78 (m, 2 H), 7.28−7.18 (m, 6 H), 6.89 (s, 0.7 H), 6.81 (s, 0.3
H), 6.01 (br s, 0.7 H), 5.97 (br s, 0.3 H), 5.45−5.36 (m, 1 H), 4.94−
4.73 (m, 2 H), 3.33 (dd, J = 14.0 Hz, 10.4 Hz, 1 H), 2.38 (s, 1 H), 2.37
(s, 2 H), 2.05−1.68 (m, 3 H), 1.17−0.81 (m, 6 H). MS (ESI): m/z
436.2 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(o-tolyl)piperazin-2-one (97). A. (3S,6S)-3-Isobutyl-6-o-tolyl-piper-
azin-2-one. Synthesized from (S)-amino-o-tolyl-acetic acid (4 g, 24.2
mmol) and HCl salt of Leu-OMe (3.3 g, 22.9 mmol) by the method
described for the preparation of compound 7 (Scheme 1) to afford
(3S,6S)-3-Isobutyl-6-o-tolyl-piperazin-2-one (1.2 g, 20% yield) as a
white solid. 1H NMR (400 MHz, DMSO-d6) δ 7.67 (s, 1 H), 7.18 (dd,
J = 4.0 Hz, 2.0 Hz, 2 H), 7.11 (dd, J = 4.8 Hz, 3.2 Hz, 2 H), 4.66 (m, 1
H), 3.17 (dd, J = 10.0 Hz, 3.6 Hz, 1 H), 3.01 (dd, J = 13.2 Hz, 4.4 Hz,
1 H), 2.63 (dd, J = 13.2 Hz, 5.6 Hz, 1 H), 2.29−2.31 (m, 1 H), 2.24 (s,
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(tetrahydro-2H-thiopyran-4-yl)piperazin-2-one (94). A. (3S,6S)-3-
Isobutyl-6-(tetrahydro-2H-thiopyran-4-yl)piperazin-2-one. Synthe-
sized from Fmoc-2-amino-2-(tetrahydro-2H-thiopyran-4-yl)acetic
acid (12 g, 31 mmol) and HCl salt of Leu-OMe (3.0 g, 16.4 mmol)
by the method described for the preparation of compound 7 to afford
T
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Journal of Medicinal Chemistry
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3 H), 1.71−1.77 (m, 1 H), 1.43−1.62 (m, 2 H), 0.86 (d, J = 6.8 Hz, 3
H), 0.82 (d, J = 6.8 Hz, 3 H). MS (ESI): m/z 247.1 (M + 1).
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(o-tolyl)piperazin-2-one (97). (3S,6S)-3-Isobutyl-6-(o-tolyl)-
piperazin-2-one (50 mg, 0.2 mmol) and 5-(4-fluorophenyl)isoxazole-
3-carboxylic acid (41 mg, 0.2 mmol) were coupled according to the
procedure described for the preparation of compound 2a to furnish 97
(77 mg, 87% yield) as a colorless solid. 1H NMR (400 MHz, CDCl3) δ
7.82−7.77 (m, 2 H), 7.42−7.39 (m, 1 H), 7.30−7.17 (m, 5 H), 6.89
(s, 1 H), 5.90 (s, 1 H), 5.46−5.37 (m, 1 H), 5.17−5.02 (m, 1 H),
4.98−4.87 (m, 1 H), 3.30−2.96 (m, 1 H), 2.45 (s, 3 H), 2.04−1.71
(m, 3 H), 1.12−0.83 (m, 6 H). MS (ESI): m/z 436.1 (M + 1).
(3S,6S)-6-(4-Chlorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-car-
bonyl)-3-isobutyl-piperazin-2-one (98). A. (3S,6S)-6-(4-Chloro-phe-
nyl)-3-isobutyl-piperazin-2-one. Synthesized from (S)-2-amino-2-(4-
chlorophenyl)acetic acid (10.0 g, 53.88 mmol) and HCl salt of Leu-
OMe (13.9 g, 76.51 mmol) by the method described for the
preparation of compound 7 (Scheme 1) to afford (3S,6S)-6-(4-
chlorophenyl)-3-isobutyl-piperazin-2-one (445 mg, 3% yield) as a
1.60 (m, 2 H), 0.95 (dd, J = 12.8 Hz, 6.4 Hz, 6 H). MS (ESI): m/z
251.0 (M + 1).
B. (3S,6S)-6-(3-Fluorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-
carbonyl)-3-isobutyl-piperazin-2-one (100). (3S,6S)-6-(3-Fluoro-
phenyl)-3-isobutyl-piperazin-2-one (50 mg, 0.2 mmol) and 5-(4-
fluorophenyl)isoxazole-3-carboxylic acid (41 mg, 0.2 mmol) were
coupled according to the procedure described for the preparation of
compound 2a to furnish 100 (67 mg, 76% yield) as a colorless solid.
1H NMR (400 MHz, CDCl3) δ 7.82−7.78 (m, 2 H), 7.44−7.37 (m, 1
H), 7.26−7.18 (m, 3 H), 7.13−7.05 (m, 2 H), 6.90−6.81 (m, 1 H),
6.12−6.10 (m, 1 H), 5.45−5.35 (m, 1 H), 5.01−4.77 (m, 2 H), 3.35−
3.04 (m, 1 H), 1.99−1.92 (m, 1 H), 1.89−1.08 (m, 1 H), 1.78−1.71
(m, 1 H), 1.11−0.81 (m, 6 H). MS (ESI): m/z 440.0 (M + 1).
(3S,6S)-6-(2-Fluorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-car-
bonyl)-3-isobutyl-piperazin-2-one (101). A. (3S,6S)-6-(3-Fluoro-
phenyl)-3-isobutyl-piperazin-2-one. Synthesized from (S)-amino-(2-
fluorophenyl)-acetic acid (5.0 g, 29.58 mmol) and HCl salt of Leu-
OMe (7.8 g, 42.95 mmol) by the method described for the preparation
of compound 7 (Scheme 1) to afford (3S,6S)-6-(2-fluorophenyl)-3-
1
1
isobutyl-piperazin-2-one (660 mg, 9% yield). H NMR (400 MHz,
white solid. H NMR (400 MHz, CDCl3) δ 7.34−7.37 (m, 2 H),
CDCl3) δ 7.28−7.36 (m, 2 H), 7.17−7.21 (m, 1 H), 7.04−7.09 (m, 1
H), 6.14 (s, 1 H), 4.96 (dd, J = 7.8 Hz, 4.0 Hz, 1 H), 3.50 (dd, J = 10.8
Hz, 3.6 Hz, 1 H), 3.32 (dd, J = 10.8 Hz, 3.6 Hz, 1 H), 3.07 (dd, J =
13.2 Hz, 3.6 Hz, 1 H), 1.85−1.91 (m, 1 H), 1.75−1.81 (m, 1 H),
1.51−1.58 (m, 2 H), 0.91−0.96 (m, 6 H). MS (ESI): m/z 251.1 (M +
1).
7.20−7.25 (m, 2 H), 5.94 (s, 1 H), 4.60 (dd, J = 7.8 Hz, 4.8 Hz, 1 H),
3.51(dd, J = 10.4 Hz, 3.2 Hz, 1 H), 3.26 (dd, J = 13.6 Hz, 4.8 Hz,1 H),
2.94−2.99 (m, 1 H), 1.75−1.88 (m, 3 H), 1.55−1.61 (m, 1 H), 0.94
(dd, J = 12.8 Hz, 6.4 Hz, 6H). MS (ESI): m/z 267.0 (M + 1).
B. (3S,6S)-6-(4-Chlorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-
carbonyl)-3-isobutyl-piperazin-2-one (98). (3S,6S)-6-(4-Chloro-phe-
nyl)-3-isobutyl-piperazin-2-one (53 mg, 0.20 mmol) and 5-(4-
fluorophenyl)-isoxazole-3-carboxylic acid (41 mg, 0.20 mmol) were
coupled according to the procedure described for the preparation of
B. (3S,6S)-6-(2-Fluorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-
carbonyl)-3-isobutyl-piperazin-2-one (101). (3S,6S)-6-(2-Fluoro-
phenyl)-3-isobutyl-piperazin-2-one (50 mg, 0.20 mmol) and 5-(4-
fluorophenyl)-isoxazole-3-carboxylic acid (41 mg, 0.20 mmol) were
coupled according to the procedure described for the preparation of
1
compound 2a to furnish 98 (73 mg, 85% yield) as a white solid. H
NMR (400 MHz, CDCl3) δ 7.82−7.78 (m, 2 H), 7.42−7.34 (m, 4 H),
7.20 (m, 2 H), 6.90 (s, 1 H), 5.93 (br s, 1 H), 5.39 (dd, J = 14.4 Hz,
4.4 Hz, 1 H), 4.99−4.76 (m, 2 H), 3.30 (dd, J = 14.0 Hz, 10.8 Hz, 1
H), 2.01−1.70 (m, 3 H), 1.02 (d, J = 6.4 Hz, 2 H), 1.00 (d, J = 6.8 Hz,
2 H), 0.97−0.82 (m, 2 H). MS (ESI): m/z 456.2 (M + 1).
(3S,6S)-6-(4-Fluorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-car-
bonyl)-3-isobutyl-piperazin-2-one (99). A. (3S,6S)-6-(4-Fluorophen-
yl)-3-isobutyl-piperazin-2-one. Synthesized from (S)-amino-(4-fluo-
rophenyl)-acetic acid (10 g, 60 mmol) and HCl salt of Leu-OMe (1.56
g, 10.67 mmol) by the method described for the preparation of
compound 7 (Scheme 1) to afford (3S,6S)-6-(4-fluorophenyl)-3-
isobutyl-piperazin-2-one (420 mg, 3% yield) as a white solid. 1H NMR
(400 MHz, DMSO-d6) δ 7.96 (s, 1 H), 7.37 (dd, J = 6.4 Hz, 2.8 Hz, 2
H),7.23 (dd, J = 11.2 Hz, 9.2 Hz, 2 H), 4.54 (d, J = 2.4 Hz, 1 H), 3.26
(dd, J = 10 Hz, 3.6 Hz, 1 H), 3.10 (dd, J = 13.2 Hz, 4.4 Hz, 1 H), 2.79
(dd, J = 12.8 Hz, 4.8 Hz, 1 H), 2.45 (s, 1 H), 1.80−1.84 (m, 1 H),
1.53−1.70 (m, 2 H), 0.95 (d, J = 6.8 Hz, 3 H), 0.91 (d, J = 6.4 Hz, 3
H). MS (ESI): m/z 251.1 (M +1).
1
compound 2a to furnish 101 (69 mg, 86% yield) as a white solid. H
NMR (CDCl3) δ 7.83−7.78 (m, 2 H), 7.43−7.34 (m, 2 H), 7.25−7.09
(m, 4 H), 6.89 (s, 1 H), 5.90 (br s, 1 H), 5.41 (dd, J = 10.0 Hz, 4.8 Hz,
1 H), 5.17−5.02 (m, 2 H), 3.49 (dd, J = 14.4 Hz, 11.2 Hz, 1 H), 2.01−
1.86 (m, 2 H), 1.82−1.70 (m, 1 H), 1.11 (d, J = 6.4 Hz, 3 H), 1.01 (d,
J = 6.8 Hz, 3 H). MS (ESI): m/z 440.2 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(4-(trifluoromethyl)-phenyl)piperazin-2-one (102). A. (3S,6S)-3-
Isobutyl-6-(4-trifluoromethyl-phenyl)-piperazin-2-one. Synthesized
from (S)-amino-(4-trifluoromethyl-phenyl)-acetic acid (3.0 g, 13.70
mmol) and HCl salt of Leu-OMe (3.5 g, 19.20 mmol) by the method
described for the preparation of compound 7 (Scheme 1) to afford
(3S,6S)-3-isobutyl-6-(4-trifluoromethyl-phenyl)-piperazin-2-one (217
1
mg, 5% yield) as a white solid. H NMR (400 MHz, CDCl3) δ 7.65
(d, J = 8.0 Hz, 2 H), 7.42 (d, J = 8.0 Hz, 2 H), 6.07 (s, 1 H), 4.70−4.71
(m, 1 H), 3.54 (dd, J = 10.4 Hz, 3.6 Hz, 1 H), 3.33 (dd, J = 13.2 Hz,
4.8 Hz, 1 H), 3.02 (dd, J = 13.2 Hz, 4.4 Hz, 1 H), 1.75−1.88 (m, 3 H),
1.58−1.62 (m, 1 H), 0.97 (d, J = 6.4 Hz, 3 H), 0.94 (d, J = 6.4 Hz, 3
H). MS (ESI): m/z 301.0 (M + 1).
B. (3S,6S)-6-(4-Fluorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-
carbonyl)-3-isobutyl-piperazin-2-one (99). (3S,6S)-6-(4-Fluorophen-
yl)-3-isobutyl-piperazin-2-one (50 mg, 0.20 mmol) and 5-(4-
fluorophenyl)-isoxazole-3-carboxylic acid (41 mg, 0.20 mmol) were
coupled according to the procedure described for the preparation of
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(4-(trifluoromethyl)-phenyl)piperazin-2-one (102). (3S,6S)-3-Iso-
butyl-6-(4-trifluoromethyl-phenyl)-piperazin-2-one (50 mg, 0.17
mmol) and 5-(4-fluorophenyl)isoxazole-3-carboxylic acid (35 mg,
0.17 mmol) were coupled according to the procedure described for the
preparation of compound 2a to furnish compound 102 (43 mg, 53%
1
compound 2a to furnish 99 (68 mg, 78% yield) as a white solid. H
NMR (400 MHz, CDCl3) δ 7.82−7.78 (m, 2 H), 7.40−7.37 (m, 2 H),
7.22−7.18 (m, 2 H), 7.14−7.10 (m, 2 H), 6.90 (s, 1 H), 6.06 (br s, 1
H), 5.39−5.36 (m, 1 H), 4.98−4.76 (m, 2 H), 3.31 (dd, J = 13.6 Hz,
10.0 Hz, 1 H), 2.01−1.92 (m, 1 H), 1.89−1.73 (m, 2 H), 1.11 (d, J =
6.4 Hz, 2 H), 1.00 (d, J = 6.8 Hz, 2 H), 0.96−0.82 (m, 2 H). MS
(ESI): m/z 440.2 (M +1).
1
yield) as a colorless solid. H NMR (400 MHz, CDCl3) δ 7.69−7.67
(m, 2 H), 7.51−7.47 (m, 2 H), 7.32−7.20 (m, 3 H), 7.12−7.08 (m, 1
H), 6.03 (s, 1 H), 5.35−4.80 (m, 1 H), 4.78−4.72 (m, 1 H), 4.69−4.17
(m, 1 H), 3.37−2.81 (m, 1 H), 2.60−2.55 (m, 1 H), 1.98−1.91 (m, 2
H), 1.88−1.82 (m, 1 H) 1.79−1.60 (m, 2 H), 1.40−1.33 (m, 1 H),
1.04−0.91 (m, 6 H). MS (ESI): m/z 445.1 (M + 1).
(3S,6S)-6-(3-Fluorophenyl)-4-(5-(4-fluorophenyl)isoxazole-3-car-
bonyl)-3-isobutylpiperazin-2-one (100). A. (3S,6S)-6-(3-Fluorophen-
yl)-3-isobutyl-piperazin-2-one. Synthesized from (S)-amino-(3-fluo-
rophenyl)-acetic acid (9 g, 53 mmol) and HCl salt of Leu-OMe (6.9 g,
38 mmol) by the method described for the preparation of compound
7 (Scheme 1) to afford (3S,6S)-6-(3-fluorophenyl)-3-isobutyl-
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(thiophen-2-yl)piperazin-2-one (103). A. (3S,6R)-3-Isobutyl-6-thio-
phen-2-yl-piperazin-2-one. Synthesized from (R)-amino-(thiophen-
2-yl)-acetic acid (5.0 g, 31.85 mmol) and HCl salt of Leu-OMe (38.6
g, 47.78 mmol) by the method described for the preparation of
compound 7 (Scheme 1) to afford (3S,6R)-3-isobutyl-6-thiophen-2-yl-
1
piperazin-2-one (478 mg, 4% yield) as a white solid. H NMR (400
1
MHz, CDCl3) δ 7.32−7.38 (m, 1 H), 6.99−7.07 (m, 2 H), 6.10 (s, 1
H), 4.62 (dd, J = 7.6 Hz, 4.4 Hz, 1 H), 3.50 (dd, J = 13.2 Hz, 4.4 Hz, 1
H), 3.00 (dd, J = 13.2 Hz, 4.8 Hz, 1 H), 1.76−1.88 (m, 2 H), 1.55−
piperazin-2-one (610 mg, 8.0% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 7.26−7.28 (m, 1 H), 6.97−7.01 (m, 2 H), 5.96 (s, 1
H), 4.88 (dd, J = 9.2 Hz, 4.4 Hz, 1 H), 3.49 (dd, J = 10.0 Hz, 3.6 Hz, 1
U
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Journal of Medicinal Chemistry
Article
H), 3.36−3.41 (m, 1 H), 2.93−2.99 (m, 1 H), 1.88−1.94 (m, 1 H),
1.75−1.84 (m, 1 H), 1.63−1.71 (m, 1 H), 1.54−1.60 (m, 1 H), 0.94−
0.99 (m, 6 H). MS (ESI): m/z 239.1 (M + 1).
(m, 1 H), 1.68−1.84 (m, 1 H), 1.53−1.59 (m, 1 H), 0.95−1.01 (m, 6
H). MS (ESI): m/z 253.1 (M + 1).
B. (3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(3-methylthiophen-2-yl)-piperazin-2-one (106). (3S,6R)-3-Isobu-
tyl-6-(3-methyl-thiophen-2-yl)-piperazin-2-one (50 mg, 0.20 mmol)
and 5-(4-fluorophenyl)-isoxazole-3-carboxylic acid (45 mg, 0.22
mmol) were coupled according to the method described for the
preparation of compound 2a to furnish product 106 (40 mg, 46%
yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.21−8.07 (m,
2 H), 7.26−7.19 (m, 2 H), 7.13−7.08 (m, 1 H), 6.89−6.79 (m, 1 H),
6.64−6.52 (m, 1 H), 6.08 (br s, 1 H), 5.50−5.14 (m, 1 H), 5.06−4.91
(m, 1 H), 4.80−4.74 (m, 1 H), 4.00−3.46 (m, 1 H), 1.95 (s, 3 H),
1.93−1.71 (m, 3 H), 1.16−0.66 (m, 6 H). MS (ESI): m/z 442.1 (M +
1).
B. (3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(thiophen-2-yl)piperazin-2-one (103). (3S,6R)-3-Isobutyl-6-thio-
phen-2-yl-piperazin-2-one (112 mg, 0.46 mmol) and 5-(4-fluorophen-
yl)-isoxazole-3-carboxylic acid (117.2 mg, 0.57 mmol) were coupled
according to the method described for the preparation of compound
2a to give 103 (100 mg, 50% yield) as an yellow solid. 1H NMR (400
MHz, CDCl3) δ 7.81−7.77 (m, 2 H), 7.35−7.32 (m, 1 H), 7.21−7.12
(m, 3 H), 7.04−7.00 (m, 1 H), 6.89 (s, 1 H), 6.45(s, 1 H), 5.35−5.31
(m, 1 H), 5.18−5.15 (m, 1 H), 5.04−4.99 (m, 1 H), 3.51−3.45 (m, 1
H), 1.95−1.71 (m, 3 H), 1.09 (d, J = 6.4 Hz, 3 H), 1.00 (d, J = 6.4 Hz,
3 H). MS (ESI): m/z 428.0 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-6-(furan-2-
yl)-3-isobutylpiperazin-2-one (107). Compound 12 (40 mg, 0.18
mmol) and 5-(4-fluorophenyl)isoxazole-3-carboxylic acid (55 mg, 0.3
mmol) were coupled according to the procedure described for the
preparation of compound 2a to afford 107 (17 mg, 24% yield) as a
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(thiophen-3-yl)piperazin-2-one (104). A. (3S,6S)-3-Isobutyl-6-thio-
phen-3-yl-piperazin-2-one. Synthesized from (S)-2-amino-2-(thio-
phen-3-yl)acetic acid (4 g, 25 mmol) and HCl salt of Leu-OMe (3.73
g, 20.6 mmol) by the method described for the preparation of
compound 7 (Scheme 1) to afford (3S,6S)-3-isobutyl-6-thiophen-3-yl-
1
colorless gum. H NMR (400 MHz, CDCl3) δ 7.72−7.69 (m, 2 H),
1
piperazin-2-one (1.1 g, 18% yield) as a white solid. H NMR (400
7.19−7.15 (m, 2 H), 7.11−7.10 (m, 1 H), 6.71−6.60 (m, 1 H), 6.30
(s, 1 H), 6.26−6.22 (m, 2 H), 5.45−5.40 (m, 1 H), 5.01−4.95 (m, 1
H), 4.76−4.64 (m, 1 H), 3.93−3.54 (m, 1 H), 1.90−1.86 (m, 2 H),
1.75−1.69 (m, 1 H) 1.05−0.74 (m, 6 H). MS (ESI): m/z 412.1 (M +
1).
MHz, CDCl3) δ 7.35−7.37 (m, 1 H), 7.17−7.18 (m, 1 H), 6.99−7.00
(m, 1 H), 6.12 (s, 1 H), 4.71−4.74 (m, 1 H), 3.48−3.51 (m, 1 H),
3.23−3.27 (m, 1 H), 3.03−3.07 (m, 1 H), 1.77−1.90 (m, 3 H), 1.52−
1.59 (m, 1 H), 0.94−0.99 (m, 6 H). MS (ESI): m/z 239.1 (M + 1).
B. (3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(thiophen-3-yl)piperazin-2-one (104). (3S,6S)-3-Isobutyl-6-thio-
phen-3-yl-piperazin-2-one (48 mg, 0.20 mmol) and 5-(4-fluorophen-
yl)-isoxazole-3-carboxylic acid (45 mg, 0.22 mmol) were coupled
according to the method described for the preparation of compound
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(oxazol-5-yl)piperazin-2-one (108). To a solution 16 (26 mg, 0.078
mmol) in DCM (1.5 mL) at 0 °C was added 4 N HCl in dioxane (0.5
mL) and then stirred at rt for 1 h. The reaction mixture was
concentrated in vacuo to give the Boc deprotected compound, which
was used for the next step without further purification. To a stirred
solution of 5-(4-fluorophenyl)-isoxazole-3-carboxylic acid (16 mg,
0.078 mmol) and HATU (22.9 mg, 0.079 mmol) in DMF (1.5 mL),
BOC deprotected compound in DMF (0.5 mL) and DIPEA (0.1 mL)
was added and stirred for 1 h. The reaction mixture was concentrated
in vacuo, and the crude product was purified by column
chromatography (0−95% EtOAc in hexanes) to furnish 108 (24.6
1
2a to furnish 104 (45 mg, 52% yield) as a white solid. H NMR (400
MHz, CDCl3) δ 7.82−7.77 (m, 2 H), 7.42−7.10 (m, 5 H), 6.89 (br s,
1 H), 6.04 (br s, 1 H), 5.44−5.34 (m, 1 H), 5.02−4.89 (m, 2 H), 3.41
(dd, J = 15.2 Hz, 12.0 Hz, 0.5 H), 3.13 (dd, J = 15.2 Hz, 12.0 Hz, 0.5
H), 2.02−1.72 (m, 3 H) and 1.11−0.80 (m, 6 H). MS (ESI): m/z
428.0 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(5-methylthiophen-2-yl)piperazin-2-one (105). A. (3S,6R)-3-Isobu-
tyl-6-(5-methyl-thiophen-2-yl)-piperazin-2-one. Synthesized from
(R)-2-amino-(5-methyl-thiophen-2-yl)-acetic acid (9 g, 52.6 mmol)
and HCl salt of Leu-OMe (6.37 g, 35.2 mmol) by the method
described for the preparation of compound 7 (Scheme 1) to afford
(3S,6R)-3-isobutyl-6-(5-methyl-thiophen-2-yl)-piperazin-2-one (0.57
1
mg, 71% yield) as a white solid. H NMR (400 MHz, CDCl3) δ 8.01
(s, 1 H), 7.80−7.77 (m, 2 H), 7.21−7.16 (m, 2 H), 7.02 (br s, 1 H),
6.90 (s, 1 H), 6.86 (s, 0.5 H), 6.83 (s, 0.5 H), 5.52−5.44 (m, 0.5 H),
5.37−5.29 (m, 0.5 H), 5.18−4.88 (m, 2 H), 3.65−3.56 (m, 0.5 H),
3.38−3.29 (m, 0.5 H), 1.93−1.86 (m, 1 H), 1.76−1.71 (m, 1 H),
1.10−0.80 (m, 6 H). MS (ESI): m/z 413.2 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(oxazol-2-yl)piperazin-2-one (109). To a solution of 23 (11.2 mg,
0.0235 mmol) in DCM (2 mL) was added TFA (0.6 mL) and stirred
at rt for 5 h. The reaction mixture was concentrated in vacuo, and the
residue was purified by column chromatography (10−60% EtOAC in
hexanes) to give 109 (7.3 mg, 75% yield) as a colorless solid. 1H NMR
(400 MHz, CDCl3) δ 7.80−7.77 (m, 2 H), 7.21−7.17 (m, 2 H), 6.92
(s, 1 H), 6.83 (s, 1 H), 6.69 (d, J = 5.6 Hz, 1 H), 5.92 (d, J = 5.6 Hz, 1
H), 5.42−5.38 (m, 1 H), 5.34−5.28 (m, 1 H), 4.51 (dd, J = 11.0 Hz,
4.6 Hz, 1 H), 3.73 (dd, J = 15.0 Hz, 11.0 Hz, 1 H), 1.91−1.68 (m, 3
H), 1.09 (d, J = 6.4 Hz, 3 H), 1.00 (d, J = 6.4 Hz, 3 H). MS (ESI): m/z
413.0 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(thiazol-2-yl)piperazin-2-one (110). To a solution of the alcohol 90
(0.30 g, 0.80 mmol) in DCM (10 mL) at 0 °C was added Dess−
Martin’s reagent (441 mg, 1.04 mmol) and water (0.05 mL). The
reaction mixture was stirred at rt for 6 h and then i-PrOH (0.2 mL),
saturated aqueous sodium thiosulfate (5 mL), and saturated aqueous
NaCO3 (5 mL) were added. The aqueous layer was extracted with
EtOAc (5 mL × 3), the combined organic layers were dried over
MgSO4 and concentrated in vacuo, and the crude aldehyde product
was used for the next step without further purification. To a solution of
crude aldehyde (0.20 g, 0.53 mmol) in EtOH (5 mL) was added HCl
salt of 2-aminoethanethiol (60.5 mg, 0.533 mmol) and KOAc (62.8
mg, 0.640 mmol) and stirred at rt for 2 h. The reaction mixture was
concentrated in vacuo, and EtOAc (4 mL) and water (3 mL) were
added. The aqueous layer was extracted with EtOAc (5 mL × 3). The
1
g, 4% yield) as a white solid. H NMR (400 MHz, CDCl3) δ 7.34−
7.35 (m, 1 H), 6.77−6.78 (m, 1 H), 6.73−6.74 (m, 1 H), 6.60−6.61
(s, 1 H), 6.02 (s, 1 H), 5.79 (s, 2 H), 4.76−4.79 (m, 1 H), 3.45−3.48
(m, 1 H), 3.33−3.37 (m, 1 H), 2.90−2.95 (m, 1 H), 2.51 (s, 3 H), 2.45
(s, 3 H), 1.78−1.90 (m, 4 H), 1.52−1.58(m, 1 H), 0.94−0.99 (m, 6
H). MS (ESI): m/z 253.15 (M + 1).
B. (3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(5-methylthiophen-2-yl)-piperazin-2-one (105). (3S,6R)-3-Isobu-
tyl-6-(5-methyl-thiophen-2-yl)-piperazin-2-one (50 mg, 0.20 mmol)
and 5-(4-fluorophenyl)-isoxazole-3-carboxylic acid (45 mg, 0.22
mmol) were coupled according to the method described for the
preparation of compound 2a to furnish 105 (40 mg, 46% yield) as a
1
white solid. H NMR (400 MHz, CDCl3) δ 7.81−7.77 (m, 2 H),
7.21−7.16 (m, 2 H), 6.90−6.87 (m, 2 H), 6.66−6.64 (m, 1 H), 6.08
(br s, 1 H), 5.41−5.29 (m, 1 H), 5.06−4.91 (m, 2 H), 3.46 (dd, J =
14.0 Hz, 9.8 Hz, 1 H), 2.47 (s, 3 H), 1.98−1.70 (m, 3 H) and 1.10−
0.80 (m, 6 H). MS (ESI): m/z 442.2 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(3-methylthiophen-2-yl)-piperazin-2-one (106). A. (3S,6R)-3-Isobu-
tyl-6-(3-methyl-thiophen-2-yl)-piperazin-2-one. Synthesized from
(R)-2-amino-(3-methyl-thiophen-2-yl)acetic acid (9 g, 52.6 mmol)
and HCl salt of Leu-OMe (3.6 g, 19.6 mmol) by the method described
for the preparation of compound 7 (Scheme 1) to afford (3S,6R)-3-
isobutyl-6-(3-methyl-thiophen-2-yl)-piperazin-2-one (268 mg, 2%
1
yield). H NMR (400 MHz, CDCl3) δ 7.17−7.18 (m, 1 H), 6.81−
6.82 (m, 1 H), 5.86 (s, 1 H), 4.93−4.97 (m, 1 H), 3.35−3.52 (m, 1 H),
3.30−3.34 (m, 1 H), 2.92−2.98 (m, 1 H), 2.22 (s, 3 H), 1.92−1.98
V
dx.doi.org/10.1021/jm4012643 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
combined organic layers were dried over MgSO4 and concentrated in
vacuo, and the crude thiazolidine product was used for the next step
without further purification. To a solution of the thiazolidine (60 mg,
0.14 mmol) in dioxane (2 mL) was added activated MnO2 (100 mg)
and stirred at 55 °C for 6 h. The reaction mixture was filtered, and the
filtrate was concentrated and purified by column chromatopraphy (0−
80% EtOAc in hexanes) to give the product 110 (14 mg, 24% yield).
1H NMR (DMSO-d6) δ 9.91 (s, 1 H), 8.06 (m, 2 H), 7.53−7.37 (m, 4
fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-(1H-pyrrol-2-yl)-
piperazin-2-one (12 mg, 14% yield). Compound 111 (brown solid):
1H NMR (400 MHz, CDCl3) δ 9.23−9.09 (d, J = 53 Hz, 1 H), 7.81−
7.76 (m, 2 H), 7.21−7.16 (m, 2 H), 6.94−6.79 (m, 3 H), 6.18−6.16
(m, 1 H), 5.43−5.29 (m, 1 H), 4.97−4.85 (m, 1 H), 3.77 (br s, 1 H),
3.51−3.16 (m, 1 H), 1.90−1.84 (m, 2 H), 1.74−1.60 (m, 1 H), 1.09−
0.77 (m, 6 H). MS (ESI): m/z 411.1 (M + 1). (3S,6S)-4-(5-(4-
Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-(1H-pyrrol-2-yl)-
piperazin-2-one (brown solid): 1H NMR (400 MHz, CDCl3) δ 9.05−
8.81 (m, 1 H), 7.79−7.65 (m, 2 H), 7.19−7.08 (m, 2 H), 7.08−6.61
(m, 3 H), 6.05−5.91 (m, 1 H), 5.45−5.29 (m, 1 H), 4.83−4.58 (m, 1
H), 3.93−3.48 (m, 2 H), 1.96−1.81 (m, 2 H), 1.78−1.67 (m, 1 H),
1.04−0.75 (m, 6 H). MS (ESI): m/z 411.1 (M + 1).
(3S,6S)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(1H-pyrrol-3-yl)piperazin-2-one (112). Synthesized from (1-(tert-
butoxycarbonyl)-1H-pyrrol-2-yl)boronic acid (2.13 g, 10.11 mmol)
by the procedures described for the preparation of 26 (Scheme 5) to
furnish 112 (15 mg, 17% yield) as a brown solid. 1H NMR (400 MHz,
CDCl3) δ 8.42 (br s, 1 H), 7.81−7.76 (m, 2 H), 7.20−7.16 (m, 2 H),
6.86−6.79 (m, 3 H), 6.24−6.21 (m, 1 H), 5.97 (br s, 1 H), 5.35−5.32
(m, 1 H), 4.89−4.75 (m, 2 H), 3.45−3.11 (m, 1 H), 1.99−1.71 (m, 3
H), 1.10−0.0.78 (m, 6 H). MS (ESI): m/z 409.07 (M − 1).
H), 4.95 (t, J = 7.6 Hz, 1 H), 4.33−4.27 (m, 2 H), 3.83 (m, 1 H), 1.63
(m, 1 H), 1.52−1.48 (m, 2 H), 0.95 (d, J = 6.4 Hz, 6 H). MS (ESI):
m/z 429.2 (M + 1).
(3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-6-
(1H-pyrrol-2-yl)piperazin-2-one (111). A. tert-Butyl 2-(1-((Bis(4-
methoxyphenyl)methyl)amino)-2-(methoxy-(methyl)-amino)-2-
oxo-ethyl)-1H-pyrrole-1-carboxylate (25). To a stirred solution of
glyoxylic acid monohydrate (0.92 g, 10.11 mmol) in DCM (70 mL),
bis(4-methoxyphenyl)methanamine (2.46 g, 10.11 mmol) and 24
(2.13 g, 10.11 mmol) were added and stirred at rt for 2 min to form a
clear solution. The solution was purged with argon (2 min), and the
sealed reaction mixture was stirred overnight. Reaction mixture was
concentrated in vacuo to yield crude acid product (4.5 g, 95% yield) as
pale-yellow foam. Crude acid product (4.5 g, 10 mmol), TBTU (4.87
g, 15 mmol), and N,O-dimethylhydroxylamine hydrochloride (1.47 g,
15 mmol) were dissolved in dry MeCN (50 mL), and DIPEA (5.3 mL,
30.22 mmol) was added and stirred at rt for 12 h. The reaction mixture
was concentrated in vacuo, and the crude product was purified by
column chromatography (5 to 40% EtOAc in hexanes) to afford 25
(5.1 g, 98% yield) as a pale-yellow foam. 1H NMR (400 MHz, CDCl3)
δ 7.36−7.25 (m, 4 H), 7.15−7.14 (m, 1 H), 6.82−6.76 (m, 4 H), 6.18
(s, 1 H), 6.07 (t, J = 3.2 Hz, 1 H), 5.72 (br s, 1 H), 4.82 (s, 1 H) 3.76
(s, 3 H), 3.74 (s, 3 H) 3.24 (s, 3 H), 3.13 (s, 3 H). 2.58 (br s, 1 H),
1.53 (s, 9 H). MS (ESI): m/z 510.1 (M + 1).
(3S,6S)-6-(2-Chlorothiophen-3-yl)-4-(5-(4-fluorophenyl)isoxazole-
3-carbonyl)-3-isobutyl-piperazin-2-one (113). A. (3S,6S)-6-(2-Chlor-
othiophen-3-yl)-3-isobutylpiperazin-2-one and (3S,6R)-6-(2-Chlor-
othiophen-3-yl)-3-isobutylpiperazin-2-one. Synthesized from 2-
chlorothiophen-3-ylboronic acid (4.15 g, 56.12 mmol) and (S)-methyl
2-amino-4-methylpentanoate hydrochloride (3.15 g, 17.39 mmol) by
the method described for the preparation of compound 12 (Scheme
2) to afford (3S,6S)-6-(2-chlorothiophen-3-yl)-3-isobutylpiperazin-2-
one (237 mg, 1.6% yield) and (3S,6R)-6-(2-chlorothiophen-3-yl)-3-
isobutylpiperazin-2-one (385 mg, 2.6% yield). (3S,6S)-6-(2-Chlor-
othiophen-3-yl)-3-isobutylpiperazin-2-one (white solid): 1H NMR
(400 MHz, DMSO-d6) δ 7.13 (d, J = 6.0 Hz, 1 H), 6.99 (d, J = 6.0
Hz, 1 H), 5.91 (s, 1 H), 4.78−4.75 (m, 1 H), 3.82−3.74 (m, 1 H),
3.54−3.50 (m, 1 H), 3.26−3.21 (m, 1 H), 3.10−3.06 (m, 1 H), 1.88−
1.80 (m, 2 H), 1.64−1.54 (m, 1 H), 0.99−0.94 (m, 6 H). MS (ESI):
m/z 272.83 (M + 1). (3S,6R)-6-(2-Chlorothiophen-3-yl)-3-isobutylpi-
B. tert-Butyl 2-(1-((Bis(4-methoxyphenyl)methyl)amino)-2-(((S)-1-
methoxy-4-methyl-1-oxo-pentan-2-yl)amino)ethyl)-1H-pyrrole-1-
carboxylate (26). To a solution of 25 (1.5 g, 2.94 mmol) in dry THF
(30 mL) at −78 °C, LiAlH4 (220 mg, 5.89 mmol) was added and
stirred under argon for 3 h. The reaction mixture was quenched with
saturated aqueous NH4Cl solution (5 mL) by dropwise addition. The
reaction mixture was partitioned between EtOAc and brine and
extracted with EtOAc (20 mL × 2). The organic layers were
combined, washed with brine, and dried over MgSO4 and
concentrated in vacuo to afford the crude aldehyde (unstable, slowly
decomposes at rt), which was used for the next step without further
purification. To a mixture of aldehyde compound (1.2 g, 2.66 mmol)
and HCl salt of Leu-OMe (0.58 g, 2.66 mmol) in DCM (24 mL),
NaBH(OAc)3 (790 mg, 3.73 mmol) was added and stirred at rt under
argon for 6 h. The reaction mixture was quenched with saturated
aqueous NaHCO3 solution (25 mL), extracted with EtOAc (30 mL),
dried over Na2SO4, and concentrated in vacuo. The crude product was
purified by column chromatography (0−40% EtOAc in hexanes) to
1
perazin-2-one (white solid): H NMR (400 MHz, DMSO-d6) δ 7.14
(d, J = 4.8 Hz, 1 H), 6.96 (d, J = 4.8 Hz, 1 H), 5.82 (s, 1 H), 4.86−4.82
(m, 1 H), 3.77−3.75 (m, 1 H), 3.52−3.48 (m, 1 H), 3.34−3.30 (m, 1
H), 2.89−2.84 (m, 1 H), 1.99−1.92 (m, 1 H), 1.86−1.78 (m, 1 H),
1.62−1.53 (m, 1 H), 1.00−0.94 (m, 6 H). MS (ESI): m/z 272.84 (M
+ 1).
B. (3S,6S)-6-(2-Chlorothiophen-3-yl)-4-(5-(4-fluorophenyl)-
isoxazole-3-carbonyl)-3-isobutyl-piperazin-2-one (113). (3S,6S)-6-
(2-Chlorothiophen-3-yl)-3-isobutylpiperazin-2-one (55 mg, 0.20
mmol) and 5-(4-fluorophenyl)-isoxazole-3-carboxylic acid (45 mg,
0.22 mmol) were coupled according to the method described for the
preparation of compound 2a to furnish 113 (42 mg, 45% yield) as a
1
1
afford 26 (1.54 g, 67% yield) as a pale-yellow foam. H NMR (400
white solid. H NMR (400 MHz, CDCl3) δ 7.85−7.77 (m, 2 H),
MHz, CDCl3) δ 7.24−7.21 (m, 4 H), 6.80−6.78 (m, 5 H), 6.15−6.14
(m, 2 H), 4.72 (s, 1 H), 4.30 (br s, 1 H), 3.79−3.75 (m, 4 H), 3.64 (s,
3 H) 3.26−3.90 (m, 2 H), 2.68−2.65 (m, 1 H) 2.05 (br s, 1 H), 1.70−
1.65 (m, 1 H), 1.48−1.41 (m, 11 H), 0.91−0.85 (m, 6 H). MS (ESI):
m/z 580.1 (M + 1).
7.21−7.16 (m, 3 H), 6.99−6.95 (m, 1 H), 6.88 (s, 1 H), 6.03 (br s, 1
H), 5.46 and 5.36 (dd, J = 10.0 Hz, 4.4 Hz, 1 H), 5.07−4.89 (m, 2 H),
3.43 and 3.16 (dd, J = 14.4 Hz, 11.2 Hz, 1 H), 1.99−1.70 (m, 3 H) and
1.10−0.81 (m, 6 H). MS (ESI): m/z 463.92 (M + 2).
C. (3S,6R)-4-(5-(4-Fluorophenyl)isoxazole-3-carbonyl)-3-isobutyl-
6-(1H-pyrrol-2-yl)pipera-zin-2-one (111). To a stirred solution of 26
(225 mg, 0.39 mmol), TBTU (161 mg, 0.5 mmol), and 5-(4-
fluorophenyl)-isoxazole-3-carboxylic acid (116 mg, 0.56 mmol) in
anhydrous MeCN (6 mL), DIPEA (0.41 mL, 2.33 mmol) was added
and stirred at rt for 24 h. The reaction mixture was concentrated in
vacuo, and the crude product was purified by column chromatography
(0−50% EtOAc in hexanes) to afford crude amide compound (250
mg, 83% yield) as pale-yellow foam. The crude amide compound (250
mg, 0.32 mmol) was dissolved in 70% aqueous AcOH (6 mL) and
heated under reflux (80 °C) for 24 h. The reaction mixture was
concentrated under vacuo, and the crude product was purified by
column chromatography (0−4% MeOH in CH2Cl2) to afford 111 (13
mg, 15% yield) and it is trans core isomer (3S,6S)-4-(5-(4-
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 860-460-3706. E-mail: rameshkakarla@yahoo.com.
Address: 9 Chamberlain Lane, South Glastonbury, Connecticut
06073, United States.
Present Addresses
†For J.L.: Department of Chemistry & Chemical Biology,
Rutgers University, 610 Taylor Road, Piscataway, New Jersey
08854, United States.
‡For D.N.: Department of Medicinal Chemistry, Gilead
Sciences, Foster City, California 94404, United States.
W
dx.doi.org/10.1021/jm4012643 | J. Med. Chem. XXXX, XXX, XXX−XXX

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Article
§For W.C.: Department of Medicinal Chemistry, Merck
Research Laboratories, West Point, Pennsylvania 19486, United
States.
inhibitor for treating genotype 1 chronic hepatitis C virus. Nature
Biotechnol. 2011, 29, 993−1003. (b) Venkatraman, S. Discovery of
boceprevir, a direct-acting NS3/4A protease inhibitor for treatment of
chronic hepatitis C infections. Trends Pharmacol. Sci. 2012, 33, 289−
294. (c) Chen, K. X.; Njoroge, F. G. The journey to the discovery of
boceprevir: an NS3-NS4 HCV protease inhibitor for the treatment of
chronic hepatitis C. Prog. Med. Chem. 2010, 49, 1−36.
(4) (a) Sheridan, C. W. New Merck and Vertex drugs raise standard
of care in hepatitis C. Nature Biotechnol. 2011, 29, 553−554. (b) Fried,
M. W.; Russo, M. W.; Fried, M. W. Side effects of therapy for chronic
hepatitis C. Gastroenterology 2003, 124, 1711−1719. (c) Manns, M. P.;
Wedemeyer, H.; Cornberg, M. Treating viral hepatitis C: efficacy, side
effects, and complications. Gut 2006, 55, 1350−1359.
∥For R.M.: 247 East Fourth Avenue, Roselle, New Jersey
07203, United States.
⊥For D.B.: Cumbre Inc., 1502 Viceroy Drive, Dallas, Texas
75235, United States.
#For H.S.: Department of Biology, Gilead Sciences, Foster City,
California 94404, United States.
▽For M.K.: 66 North Edgewater Avenue, Yardley, Pennsylva-
nia 19067, United States.
○For S. B.: Xeme Biopharma, 5 Parkway Avenue, Plainsboro,
New Jersey 08536, United States.
(5) (a) Kato, N. Genome of human hepatitis C virus (HCV): gene
organization, sequence diversity, and variation. Microb. Comp.
Genomics 2000, 5 (3), 129−151. (b) Dubuisson, J. Hepatitis C virus
proteins. World J. Gastroenterol. 2007, 13 (17), 2406−2415.
◆For A.L.: Novira Therapeutics, 3805 Old Easton Road,
Doylestown, Pennsylvania 18902, United States.
¶For W.S. and S.N.: University of Cincinnati, Cincinnati, Ohio
45221, United States.
(6) (a) Asselah, T.; Marcellin, P. Direct acting antivirals for the
treatment of chronic hepatitis C: One pill a day for tomorrow. Liver
Int. 2012, 32 (Suppl 1), 88−102. (b) Welsch, C.; Jesudian, A.;
Zeuzem, S.; Jacobson, I. New direct-acting antiviral agents for the
treatment of hepatitis C virus infection and perspectives. Gut Liver
2012, 61, i36−i46. (c) Pockros, P. J. Drugs in development for chronic
hepatitis C: a promising future. Expert Opin. Biol. Ther. 2011, 11,
1611−1622. (d) Fusco, D. N.; Chung, R. T. Novel therapies for
hepatitis C: insights from the structure of the virus. Annu. Rev. Med.
2012, 63, 373−387. (e) Lemon, S. M.; McKeating, J. A.; Pietschmann,
T.; Frick, D. N.; Glenn, J. S.; Tellinghuisen, T. L.; Symons, J.; Furman,
P. A. Development of novel therapies for hepatitis C. Antiviral Res.
2010, 86, 79−92.
+For P.F.: 194 Parkside Drive, Saint Augustine, Florida 32095,
United States.
□For M.J.S.: Oncore Biopharma, Inc., 3805 Old Easton Road,
Doylestown, Pennsylvania 18902, United States.
Notes
The authors declare the following competing financial
interest(s): All of the authors except W.S. and S.N. were
employees of Pharmasset where this work was completed.
Gilead Sciences acquired Pharmasset in January 2012.
Pharmasset was acquired by Gilead Sciences in January 2012
(7) (a) Mani, N.; Rao, B. G.; Kieffer, T. L.; Kwong, A. D. Recent
progress in the development of HCV protease inhibitors. Methods
Principals Med. Chem. 2011, 50, 307−328. (b) Chary, A.; Holodniy, M.
Recent advances in hepatitis C virus treatment: review of HCV
protease inhibitor clinical trials. Rev. Recent Clin. Trials 2010, 5, 158−
173. (c) Kwo, P. Y.; Vinayek, R. The therapeutic approaches for
hepatitis C virus: protease inhibitors and polymerase inhibitors. Gut
2011, 5, 406−417.
ACKNOWLEDGMENTS
■
We are grateful to the scientists at Wuxi-Aptec for synthesizing
valuable intermediates involved in the synthesis of HCV
inhibitors and providing SFC (Chiral) separation service for
some of the compounds.
ABBREVIATIONS USED
(8) Reviriego, C. Daclatasvir dihydrochloride: treatment of hepatitis
C virus HCV NS5A inhibitor. Drugs Future 2011, 36, 735−739.
(9) (a) Kwo, P. Y.; Vinayek, R. The therapeutic approaches for
hepatitis C virus: protease inhibitors and polymerase inhibitors. Gut
2011, 5, 406−417. (b) Sofia, M. J.; Chang, W.; Furman, P. A.; Mosley,
R. T.; Ross, B. S. Nucleoside, nucleotide, and non-nucleoside
inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymer-
ase. J. Med. Chem. 2012, 55, 2481−2531. (c) Li, H.; Shi, S. T. Non-
nucleoside inhibitors of hepatitis C virus polymerase: current progress
and future challenges. Future Med. Chem. 2010, 2, 121−141.
(d) Watkins, W. J.; Ray, A. S.; Chong, L. S. HCV NS5B polymerase
inhibitors. Curr. Opin. Drug Discovery Dev. 2010, 13, 441−465.
(10) (a) Rong, L.; Dahari, H.; Ribeiro, R. M.; Perelson, A. S. Rapid
emergence of protease inhibitor resistance in hepatitis C virus. Sci.
Transl. Med. 2010, 2, 1−9. (b) Wang, C.; Sun, J. H.; O’Boyle, D. R.;
Nower, P.; Valera, L.; Roberts, S.; Fridell, R. A.; Gao, M. Persistence of
variants in hepatitis C virus-infected patients treated with the NS5A
replication complex inhibitor daclatasvir. Antimicrob. Agents Chemother.
2013, 57 (5), 2054−2065. (c) Delang, L.; Vliegen, I.; Leyssen, P.;
Neyts, J. In vitro selection and characterization of HCV replicons
resistent to multiple non-nucleoside polymerase inhibitors. J. Hepatol.
2012, 56 (1), 41−48.
(11) Gouttenoire, J.; Roingeard, P.; Penin, F.; Moradpour, D.
Hepatitis C virus nonstructural protein 4B: a journey into unexplored
territory. Rev. Med. Virol. 2010, 20 (2), 117−129.
(12) (a) Meanwell, N. A.; Belema, M. Hepatitis C virusProgress
towards inhibiting the nonenzymatic viral proteins. Annu. Rep. Med.
Chem. 2011, 46, 268−270. (b) Brysona, P. D.; Choa, N. J.; Einava, S.;
Leea, C.; Taid, V.; Bechteld, J.; Sivarajad, M.; Robertsd, C.; Schmitzd,
U.; Glenn, J. S. A small molecule inhibits HCV replication and alters
NS4B’s subcellular distribution. Antiviral Res. 2010, 87, 1−8.
■
AcOH, acetic acid; BuOH, butanol; Boc, tert-butyloxycarbonyl;
CyP, cytochrome P450; DCM, dichloromethane; DIPEA, N,N-
diisopropylethylamine; DMAP, 4-(dimethylamino)pyridine;
DMF, dimethylformamide; EDC, N-(3-dimethylaminoprop-
yl)-N′-ethylcarbodiimide hydrochloride; EtOAc, ethyl acetate;
Fmoc, 9-fluorenylmethyloxycarbonyl; HATU, (O-(7-azabenzo-
triazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-fluorophos-
phate); HLM, human liver microsomal; HOBt, N-hydrox-
ybenzotriazole; Leu, L-leucine; LiAlH4, lithium aluminum
hydride; MeCN, acetonitrile; MeOH, methanol; Phe, phenyl-
alanine; i-PrOH, 2-propanol; SFC, supercritical fluid chroma-
tography; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethy-
luronium tetrafluoroborate; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; WT, wild type
REFERENCES
■
(1) (a) Te, H. S.; Jensen, D. M. Epidemiology of hepatitis B and C
viruses: a global overview. Clin. Liver Dis. 2010, 14, 1−21. (b) Shepard,
C. W.; Finelli, L.; Alter, M. J. Global epidemiology of hepatitis C virus
infection. Lancet Infect. Dis. 2005, 5, 558−567.
(2) (a) El-Serag, H. B. Epidemiology of viral hepatitis and
hepatocellular carcinoma. Gastroenterology 2012, 142, 1264−1273.
(b) Perz, J. F.; Armstrong, G. L.; Farrington, L. A.; Hutin, Y. J. F.; Bell,
B. P. The contributions of hepatitis B virus and hepatitis C virus
infections to cirrhosis and primary liver cancer worldwide. J. Hepatol.
2006, 45, 529−538.
(3) (a) Kwong, A. D.; Kauffman, R. S.; Hurter, P.; Mueller, P.
Discovery and development of telaprevir: an NS3-4A protease
X
dx.doi.org/10.1021/jm4012643 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(c) Shotwell, J. B.; Baskaran, S.; Chong, P.; Creech, K. L.; Crosby, R.
M.; Dicks, H.; Fang, J.; Garrido, D.; Mathis, A.; Maung, J.; Parks, D. J.;
Pouliot, J. J.; Price, D. J.; Rai, R.; Seal, J. W.; Schmitz, U.; Tai, V. W. F.;
Thomson, M.; Xie, M.; Xiong, J. J.; Peat, A. J. Imidazo[1,2-a]pyridines
that directly interact with hepatitis C NS4B: initial preclinical
characterization. ACS Med. Chem. Lett. 2012, 3, 565−569. (d) Miller,
J. F.; Chong, P. Y.; Shotwell, J. B.; Catalano, J. G.; Tai, V. W.; Fang, J.;
Banka, A.; Roberts, C. D.; Youngman, M.; Zhang, H.; Xiong, Z.;
Mathis, A.; Pouliot, J. J.; Hamatake, R. K.; Price, D. J.; Seal, J. W.;
Stroup, L. L.; Creech, K. L.; Carballo, L. H.; Todd, D.; Spaltenstein,
A.; Furst, S.; Hong, Z.; Peat, A. J. Hepatitis C replication inhibitors
that target the viral NS4B protein. J. Med. Chem. 2013, 10.1021/
jm400125h. (e) Gu, Z.; Graci, J. D.; Lahser, F. C.; Breslin, J.; Jung, S.
P.; Crona, J. H.; McMonagle, P.; Xia, E.; Liu, S.; Karp, G.; Zhu, J.;
Huang, S.; Nomeir, A.; Weetall, M.; Almstead, N. G.; Peltz, S. W.;
Tong, X.; Ralston, R.; Colacino, J. M. Identification of PTC725: an
orally bioavailable small molecule that selectively targets the hepatitis
C virus NS4B protein. Antimicrob. Agents Chemother. 2013, 57 (5),
2054−2065. (f) Zhang, X.; Zhang, N.; Chen, G.; Turpoff, A.; Ren, H.;
Takasugi, J.; Morrill, C.; Zhu, J.; Li, C.; Lennox, W.; Paget, S.; Liu, Y.;
Almstead, N.; George Njoroge, F.; Gu, Z.; Komatsu, T.; Clausen, V.;
Espiritu, C.; Graci, J.; Colacino, J.; Lahser, F.; Risher, N.; Weetall, M.;
Nomeir, A.; Karp, G. M. Discovery of novel HCV inhibitors: synthesis
and biological activity of 6-(indol-2-yl)pyridine-3-sulfonamides target-
ting hepatitis C virus NS4B. Bioorg. Med. Chem. Lett. 2013, 23 (13),
3947−3953. (g) Chen, G.; Ren, H.; Turpoff, A.; Arefolov, A.; Wilde,
R.; Takasugi, J.; Khan, A.; Almstead, N.; Gu, Z.; Komatsu, T.; Freund,
C.; Breslin, J.; Colacino, J.; Hedrick, J.; Weetall, M.; Karp, G. M.
Discovery of of N-(41-(indol-2-yl)phenyl)-sulfonamides as novel
inhibitors of HCV replication. Bioorg. Med. Chem. Lett. 2013, 23
(13), 3942−3946.
(13) Masaki, V. M.; Ohta, M. Synthese von 3,6-disubstituierten 2-
oxopiperazinen. Bull. Chem. Soc. Jpn. 1963, 36 (8), 922−925.
(14) (a) Petasis, N. A.; Goodman, A.; Zavialov, T. A new synthesis of
α-arylglycines from aryl boronic acids. Tetrahedron 1997, 53 (48),
16463−16470. (b) Haurena, C.; Le Gall, E.; Sengmany, S.; Martens,
T.; Troupel, M. A straightforward three-component synthesis of α-
amino esters containing a phenylalanine or a phenylglycine scaffold. J.
Org. Chem. 2010, 75 (8), 2645−2650.
(15) Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.;
Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang, H.-R.;
Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Steuer, H. M. M.;
Niu, C.; Otto, M. J.; Furman, P. A. Discovery of a β-D-21-deoxy-21-α-
fluoro-20-β-C-methyl-uridine nucleotide prodrug (PSI-7977) for the
treatment of hepatitis C virus. J. Med. Chem. 2010, 53, 7202−7218.
(16) (a) Sofia, M. J.; Kakarla, R.; Liu, J.; Naduthambi, D.; Mosley, R.;
Steuer, H. M. Preparation of piperazine derivatives and their uses to
treat viral infections, including hepatitis C. U.S. Patent
US20120202794A1, September 8, 2012. (b) Sofia, M. J.; Kakarla, R.;
Liu, J.; Naduthambi, D.; Mosley, R.; Steuer, H. M. Preparation of
pyrazine and imidazolidine derivatives and their uses to treat viral
infections, including hepatitis C. WO 2012103113A1, February 8,
2012.
(17) HLM of compound 95 is 54 min (half-life) and for the
compounds, 74 and 87 is 17 and 29 min, respectively.
Y
dx.doi.org/10.1021/jm4012643 | J. Med. Chem. XXXX, XXX, XXX−XXX