Design, synthesis, and antiviral activity of entry inhibitors that target the CD4-binding site of HIV-1

Article abstract of DOI:10.1021/jm3002247

The CD4 binding site on HIV-1 gp120 has been validated as a drug target to prevent HIV-1 entry to cells. Previously, we identified two small molecule inhibitors consisting of a 2,2,6,6-tetramethylpiperidine ring linked by an oxalamide to a p-halide-substituted phenyl group, which target this site, specifically, a cavity termed "Phe43 cavity". Here we use synthetic chemistry, functional assessment, and structure-based analysis to explore variants of each region of these inhibitors for improved antiviral properties. Alterations of the phenyl group and of the oxalamide linker indicated that these regions were close to optimal in the original lead compounds. Design of a series of compounds, where the tetramethylpiperidine ring was replaced with new scaffolds, led to improved antiviral activity. These new scaffolds provide insight into the surface chemistry at the entrance of the cavity and offer additional opportunities by which to optimize further these potential-next- generation therapeutics and microbicides against HIV-1.

Full text of DOI:10.1021/jm3002247

Article
pubs.acs.org/jmc
Design, Synthesis, and Antiviral Activity of Entry Inhibitors That
Target the CD4-Binding Site of HIV-1
Francesca Curreli,† Spreeha Choudhury,† Ilya Pyatkin,‡ Victor P. Zagorodnikov,‡ Anna Khulianova Bulay,‡
Andrea Altieri,‡ Young Do Kwon,§ Peter D. Kwong,§ and Asim K. Debnath*,†
†Lindsley F. Kimball Research Institute, New York Blood Center, 310 E. 67th Street, New York, New York 10065, United States
‡Asinex Ltd., 20 Geroev Panfilovtzev, Building 1, Moscow 125480, Russia
§Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland
20892, United States
S
* Supporting Information
ABSTRACT: The CD4 binding site on HIV-1 gp120 has been validated as a
drug target to prevent HIV-1 entry to cells. Previously, we identified two small
molecule inhibitors consisting of a 2,2,6,6-tetramethylpiperidine ring linked by an
oxalamide to a p-halide-substituted phenyl group, which target this site,
specifically, a cavity termed “Phe43 cavity”. Here we use synthetic chemistry,
functional assessment, and structure-based analysis to explore variants of each
region of these inhibitors for improved antiviral properties. Alterations of the
phenyl group and of the oxalamide linker indicated that these regions were close
to optimal in the original lead compounds. Design of a series of compounds,
where the tetramethylpiperidine ring was replaced with new scaffolds, led to
improved antiviral activity. These new scaffolds provide insight into the surface
chemistry at the entrance of the cavity and offer additional opportunities by which to optimize further these potential-next-
generation therapeutics and microbicides against HIV-1.
residues; however, another residue, arginine 59 (Arg59) of
CD4, also makes critical interaction by forming a salt bridge
with aspartic acid residue at position 368 (Asp368) of gp120. In
addition, gp120 residues in the span of 365−371 and 425−430
contribute to about 58% of the interactions.6 Several hydro-
phobic residues in those two regions are critical and make
hydrophobic contacts with the hydrophobic residues in CD4.
Insertions into this cavity were found to enhance the affinity of
CD4 and of CD4-mimetics,7 and we were the first to identify
small molecule inhibitors, NBD-556 (1) and NBD-557 (2)8
(Figure 1), which appear to insert into the “Phe43 cavity”.9,10
These two compounds show low micromolar potency against
several laboratory strains and primary isolates. In addition,
these molecules induce conformational changes in gp120
similar to CD4.11 Recently, we cocrystallized 1 with the clade C
strain C1086 version of gp120 coree.12 The structure reveals
that 1 binds within the “Phe43 cavity” of gp120 with its 4-
chlorophenyl group extended deep inside the cavity surrounded
by hydrophobic residues, such as Trp112, Val255, Trp427, and
Met475. The distal NH of the oxalamide group from 4-
chlorophenyl ring forms an H-bond with Gly473. However, no
apparent interaction of the tetramethylpiperidine ring with any
residues in the cavity was observed; rather it was exposed
outside of the pocket. The structural insight led us (this
INTRODUCTION
■
Human immunodeficiency virus type-1 (HIV-1) is the
etiological agent responsible for the AIDS pandemic. According
to 2011 UNAIDS report, 34 million people are living with HIV
worldwide, a rise of 17% compared to 2001. However, the
availability of low cost highly active antiretroviral therapies
(HAART) has helped to lower the rate of new infection and
death. Despite the success, emergence of resistance to the
available drugs is a major problem, which justifies continuous
search for novel drugs against this virus.
The first step in HIV life cycle is the entry/fusion of the virus
to the target cells. The envelope glycoprotein gp120 initiates
this process by binding to the cellular receptor CD4 on cell
surface. This binding induces substantial structural rearrange-
ment in gp120 which is required for its subsequent binding to
cellular chemokine receptor CXCR4 or CCR5.1,2 This
interaction helps to expose the fusion domain of gp41 that
initiates fusion and the entry of the virus to the host cells.
Although all these steps are legitimate targets for developing
entry inhibitors, only drugs targeting gp413,4 and CCR55 have
been successfully developed and clinically available. The X-ray
structure of gp120 bound to CD4 and the fragment of an
antibody (Fab) 17b was solved in 1998.6 The structure reveals
that phenylalanine at position 43 (Phe43) binds to the vestibule
of a cavity of gp120, termed “Phe43 cavity”, and makes major
hydrophobic contacts. This hydrophobic contribution accounts
for 28% of the contact interactions between CD4 and gp120
Received: February 20, 2012
© XXXX American Chemical Society
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analogue of 1.14 In addition, we (this report) and Lalonde et
al. observed that variations of the oxalamide chain resulted in
either poor activity or no antiviral activity at all.14 Therefore, we
turned our attention to modify the 2,2,6,6-tetramethylpiper-
idine ring. The structural insights described before motivated us
to replace the 2,2,6,6-tetramethylpiperidine ring with other
scaffolds that may provide a similar interaction as was observed
between Arg59 and Asp368 as well as interact with other
available binding residues. Since 1 was prepared by simple
coupling reaction between 2-(4-chlorophenylamino)-2-oxoace-
tyl chloride and 4-amino-2,2,6,6-tetramethylpiperidine, we first
identified 208 commercially available amines with diverse
structures to replace the tetramethylpiperidine part and created
a virtual library with 33 anilines consisting of 6864 compounds.
Besides 4-chloroaniline, we also used a variety of substituted
anilines to investigate how they fit in the “Phe43 cavity”. The
structure of the compounds in the virtual library was in 2D
format; therefore, we used the LigPrep module within Maestro
(Schrodinger, San Diego, CA) to generate 3D structures. The
LigPrep module not only converts 2D structure to 3D but also
considers tautomeric, stereochemical, and ionization states of
the structure and generates conformations and performs energy
minimization. The LigPrep application generated 20 688
structures representing all tautomeric, stereochemical, and
ionization states of the molecules. We then performed a
GLIDE-based docking15,16 of these structures using the
standard precision (SP) mode and selected 500 best scored
structures to dock again using the extra precision (XP) scoring
mode. We selected 50 top scored structures from this
simulation. However, it is worth mentioning that within the
top 50 structures the same molecule appeared multiple times
because of different stereochemical, tautomeric, and ionization
states. We noticed that the top XP scored molecule (−8.8) and
several others within the top 50 molecules contained (2-
(amino)piperidine-2-yl)methyl)-4-methylthiazolyl-5-yl)-
methanol scaffold with variable substitutions in the phenyl-
amine moiety. Another major observation was that the
positively charged piperidine ring of the scaffold formed a salt
bridge with Asp368 of gp120 (Figure 2). This interaction was
Figure 1. Structures of 1 and 2 identifying different pharmacophoric
regions.
manuscript) and others to believe that the tetramethylpiper-
idine moiety can be replaced with other groups that may be
able to take advantage of additional interactions, especially with
Asp368, near the entrance of the cavity and yield more potent
antivirals.9,13,14
In this report, we present the systematic modification of 1 to
more potent inhibitors using structure-based design, synthesis,
antiviral screening, and structure−activity relationship analyses.
Our data indicate that structure-guided modifications, especially
of region III with new scaffolds never reported before,
improved the activity of several analogues by ∼6-fold compared
to 1.
RESULTS AND DISCUSSION
■
Virtual Screening To Identify New Scaffolds To
Replace 2,2,6,6-Tetramethylpiperidine Ring. We initiated
a strategy for designing next generation entry inhibitors
targeted to “Phe43 cavity” based on our earlier identified
small molecule CD4-mimetics 18 (Figure 1) and the X-ray
structure of 1 bound to HIV-1 gp120 core. In our initial
attempt for a limited structure−activity relationship (SAR)
study on 1 we wanted to extend the hydrophobic portion of the
oxalamide chain in region II (Figure 1) as defined by Lalonde
et al.14 and designed several succinamide containing com-
pounds (Supporting Information). However, these compounds
failed to show any antiviral activity in both single-cycle and
multicycle inhibition assay. Recently, Lalonde et al also
reported similar observation when testing a succinamide
Figure 2. GLIDE-based docking of compound 6 in the “Phe43 cavity”. (a) Compound 6 is shown as docked inside the cavity. The 4-chlorophenyl
moiety is located deep inside the cavity. The protonated “N” of piperidine ring is within salt-bridge (H-bond interaction) distance from Asp368. (b)
Shown are the interactions of 6 with the residues in the “Phe43 cavity” of HIV-1 gp120 as mapped by the Maestro software in Schrodinger Suite
̈
2011.
B
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a
Scheme 1
a
Reagents: (a) CICOCOOEt, NEt3, DCM; (b) NaOH, EtOH, H2O; (c) TBTU, NEt3, amine; (d) HCI/dioxane (this step is only required to
deprotect N-Boc amines).
absent in most of the other top scored compounds. Since we
wanted to capture this interaction in our newly designed
compounds, we decided to use this scaffold and its structural
variations as the main target for synthesis.
in antiviral activity in a single-cycle pseudovirus based assay. In
addition, the toxicity in TZM-bl cells also decreased
remarkably. Unfortunately, both compounds were much less
active in MT-2 cell based multicycle infectivity assay. A similar
study was done with our follow-up most active compound
NBD-10007 (9). Again we observed remarkable improvement
in antiviral potency as well as toxicity for both 10 and 11 in the
TZM-bl based pseudovirus assay. However, both compounds
again showed much lower potency in MT-2 based infectivity
assay. Since these two assay methods are different and different
cells were used, some variations in activity were expected.
However, no clear SAR could be discerned from the multicycle
infectivity based antiviral data. Recently, a multicenter
comparison study of HIV neutralization assay indicated that
pseudovirus assays are generally more sensitive than multicycle
virus inhibition assay; however, it also depends on the virus and
inhibitor used.17 The presence of either hydroxymethanol or
hydroxyethanol on the thiazole ring did not make any
appreciable differences in antiviral activity. These groups most
likely work as the solubilizing unit of these molecules instead of
conferring any direct binding. Next, we have substituted H of
the NH of the piperidine ring with methyl (12) and acetyl
(13). The antiviral activity of methyl substituted piperidine
(12) improved compared to its unsubstituted counterpart (9),
but the toxicity level remained the same. The acetyl substituted
piperidine analogue (13) showed much worse activity, but the
toxicity improved substantially. In both cases, the antiviral
activity in MT-2 cells was poor, as was observed with
unsubstituted analogues. Complete removal of the piperidine
ring with the methylaminoethyl (−CH2NHCH3) moiety also
showed potent antiviral activity in single-cycle pseudovirus-
based assay but showed poor activity in MT-2 cell based
multicycle infectivity assay. Again, the toxicity of this analogue
was high. We also tested three structurally diverse analogues
(17−19), but they either had poor activity or no activity at all
in both infectivity assays.
Since 6 and 9 showed much better activity profile in both
assays, we restricted our synthesis effort in designing analogues
based only on these two active inhibitors. This time, we focused
our attention mostly to include substitutions to the phenyl ring
(Table 2). Substituting 4-Cl with CF3 (20) resulted in
somewhat reduced antiviral activity. When the acetyl group
was introduced at the para position of the phenyl ring (23), the
compound became totally inactive. The introduction of a
smaller substituent (F) at the ortho position of the phenyl ring
was found to be detrimental to the antiviral activity (21).
However, interestingly, 4-Cl and 4-CH3 groups with 3-F (27,
28, 38, and 39) in both −CH2OH and −CH2CH2OH
substituted thaizoles showed much improved activity. This is
consistent with the observation reported with 2,2,6,6-
tetramethylpiperidine containing compounds9 and recently
with a large number of compounds where 4-amino-2,2,6,6-
Initially, we restricted our synthesis effort using 4-
chlorophenylamine in region I (Figure 1) because of the fact
that we observed earlier that the phenyl moiety could tolerate
limited substitutions and 4-chloro was the most active so far.
We also selected a few other top scored compounds for
synthesis that did not satisfy the salt-bridge formation to
compare their activity; however, we needed to make some
minor changes because of the lack of availability of the exact
reagents that were used to create the virtual library (Table 2).
All these compounds with the variations were docked using the
same method and inspected for their fit in the Phe43 cavity. All
of them fit very well as the original scaffold with high score
(>−7.0).
Chemistry. The oxalamide derivatives were prepared by
adopting the general procedure in Scheme 1. This allows
probing different haloaryl derivatives in region I and virtually
any kind of available amine in region III (Figure 1). A series of
commercially available arylamines (Tables 1 and 2 and Table
S1) have been coupled with the ethyl 2-chloro-2-oxoacetate (a).
The resulting intermediate was hydrolyzed (b) and coupled
with commercially available amines (c) (Tables 1 and 2 and
Table S1). When Boc-protected amines were used, an
additional step of cleaving was necessary (d). The resulting
1
coupling product was HPLC purified and characterized by H
NMR and LC−MS. Some of the reported compounds were
isolated as a diastereoisomeric mixture with variable purity. No
further attempt was made to separate the individual isomer at
this stage.
Structure−Activity Relationship (SAR). In our initial
attempt to understand the SAR we decided to explore few
compounds by replacing the oxalamide moiety in region II of
our earlier reported compounds, 1, with succinamide (Table S1
in Supporting Information). No docking-based selection
method was used. Rather those compounds were synthesized
based on reagent availability. However, none of the compounds
showed any noticeable antiviral activity either in single-cycle or
in multicycle assay. On the basis of the docking simulation, the
best XP-scored compound had the 4-bromo-3-fluorophenyl
moiety (Figure S1). However, we decided initially to explore
the 4-chloroaniline based on our earlier reported compound 1.
Therefore, we synthesized the compound, NBD-09027 (6), and
it showed low micromolar potency with good selectivity index
(SI) in both single-cycle (in TZM-bl cells) and multicycle (in
MT-2 cells) assays. Therefore, we concentrated our attention to
understand the SAR of the piperidine ring of our initial best hit,
6. Then we synthesized two additional compounds by moving
the nitrogen from position 2 in the initial hit to positions 3 (7)
and 4 (8). Both compounds showed substantial improvement
C
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Table 1. Structure and Antiviral Activity of Oxalamide Series Compounds in Single-Cycle (TZM-bl) and Multicycle (MT-2)
Inhibition Assays
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Table 1. continued
a
The number in parentheses indicates % toxicity at that dose.
tetramethylpiperidine was replaced by diverse amines.14 In a
more drastic move we have replaced the phenyl ring with a
seven-membered alicyclic ring, and those compounds (29 and
40) showed no antiviral activity. This confirms our earlier
observation that the phenyl ring can only accommodate limited
substitutions because of the narrow hydrophobic pocket in the
“Phe43 cavity”.
interaction. One of its NH attached to the phenyl ring formed
an H-bond with Asn425. This is somewhat different from what
we observed with 6 (Figure 2) where in addition to the same
H-bond the distal NH of the oxalamide linker also formed an
H-bond with Met426. This predicted binding information is
expected to guide future optimization of this class of inhibitors.
To understand the expected binding mode of two of the
most active compounds that contained 4-Cl with 3-F
substituents in the phenyl rings NBD-11009 (27) and NBD-
11018 (39), we performed GLIDE-based docking simulations
in XP mode as described before. The top scoring
conformations of these two inhibitors indicated two possible
binding modes (Figure 3). In both cases, the 4-Cl-3-F-phenyl
ring was surrounded by hydrophobic residues similar to what
we observed with 6; however, there was considerable difference
between two binding modes of the piperidinethiazolyl moiety
of 27 (Figure 3 a,b). Surprisingly, the positively charged
piperidine nitrogen of the top scored (−8.22) 27 conformation
did not form any H-bond/salt-bridge with Asp368, whereas in
the next best scored (−7.89) conformation it indeed formed
the H-bond/salt-bridge with Asp368. In both cases the
−CH2OH formed H-bond with Trp427. On the contrary, the
positively charged piperidine nitrogen in both top scored
(−7.96) and the next best scored (−7.81) conformations of 39
formed H-bond/salt-bridge with Asp368 (Figure 3c,d). This
difference in the binding mode may be attributed to the larger
size of the ethanol moiety on the thiazole ring. However, the
−CH2CH2OH group had different binding modes in two
different conformations. One noticeable feature was that in all
these conformations the oxalamide moiety had the same
CONCLUSIONS
■
After our first discovery of two small molecule inhibitors that
target the “Phe43 cavity” in 2005, it has been conclusively
shown that these compounds bind to the cavity with
remarkable ability to alter the conformation of gp120 similar
to that observed when CD4 bound to gp120. This
extraordinary ability of these small molecules resulted in
solving the crystal structures of these molecules with gp120,
which provided details of the binding mode of these
compounds in the cavity. Despite many attempts by us and
others using traditional medicinal chemistry, the 2,2,6,6-
tetramethylpiperidine ring could not be replaced successfully
by any other chemical scaffold until this report to improve the
antiviral activity. In fact, all attempts reported until recently14
indicate failure to obtain compounds with activity better than 1
and 2 . In this study, we have successfully utilized the structural
insights of the X-ray crystal structure of 1 bound to gp120 to
design a large number of molecules with new scaffold replacing
the 2,2,6,6-tertamethylpiperidine ring. Several of these new
compounds showed improved potency in single-cycle as well as
multicycle HIV-1 inhibition assays. The most notable achieve-
ment is the identification of new scaffolds which should provide
opportunities to optimize these compounds further. Since these
compounds prevent HIV-1 from entering cells, they may serve
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Table 2. Structure−Activity Relationship (SAR) Analysis of Oxalamide Compounds in Single-Cycle (TZM-bl) and Multicycle
(MT-2) Assays
a
The number in parentheses indicates % toxicity at that dose.
F
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Figure 3. Binding mode of two of the most active compounds, 27 and 39. (a, b) The docking-based top scoring two conformations of 27 showed
distinct differences in binding of the piperidinethiazolyl moiety. The piperidine NH formed an H-bond/salt-bridge with Asp368 in the second best
scored conformation but not in the top scored conformation. (c, d) Both top scored two conformations of 39, on the other hand, formed the H-
bond/salt-bridge with Asp368. However, the ethanol moiety in the thiazolyl ring showed two distinct binding modes. The symbol notations are same
as in Figure-2.
a
Scheme 2
a
Reagents: (a) CICOCOOEt, NEt3, DCM; (b) NaOH, EtOH, H2O; (c) TBTU, NEt3, amine; (d) HCI/dioxane.
compounds were ≥95% unless otherwise mentioned. HPLC analyses
were performed on a Water XTerra MS C18 analytical column (2.1
mm × 30 mm, particle size 3.5 μm) using DMSO/acetonitrile (50:50),
at a flow rate of 15 mL/min at 25 °C. The oxalamide compounds were
isolated as diastereoisomers with variable isomer ratios.
MT-2 cells (human T-cell leukemia cells, obtained through the
AIDS Research and Reference Reagent Program from Dr. D.
Richman.) were grown in RPMI 1640 medium (Gibco) supplemented
with 10% fetal bovine serum (FBS), penicillin, and streptomycin (100
U/μL each). TZM-bl cells (a HeLa cell line that expresses CD4,
CXCR4, and CCR5 and expresses luciferase and β-galactosidase under
control of the HIV-1 promoter, obtained from Dr. John C. Kappes, Dr.
Xiaoyun Wu, and Tranzyme Inc.) and 293T cells (ATCC) were grown
in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supple-
mented with 10% FBS, penicillin, and streptomycin. pNL4-3.Luc.R-E-
DNA was obtained through the AIDS Research and Reference
as potential leads to develop next-generation therapeutics and
microbicides against HIV-1.
EXPERIMENTAL SECTION
■
All reagents were purchased from commercial suppliers or obtained in-
house (Asinex) and were used without further purification. The
melting points were measured on a Sanyo Gallenkamp melting point
apparatus (Sanyo Gallenkamp, U.K.). The LC−MS analyses for the
compounds were done at Surveyor MSQ (Thermo Finnigan, U.S.)
with APCI ionization. The 1H NMR spectra were recorded on
Mercury Plus 400 MHz spectrometer (Varian, Germany). Chemical
shift values are given in ppm relative to tetramethylsilane (TMS), with
the residual solvent proton resonance as internal standard. High-
resolution mass spectra were recorded on a Bruker APEX II ICR-
FTMS. Purity of compounds was determined by HPLC. Purity of
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Reagent Program from Dr. N. Landau and env expression vector
pHXB2-env (X4) DNA from Dr. K. Page and Dr. D. Littman.18
Virtual Screening Using GLIDE-Based Docking. We used the
do) (5-(hydroxymethyl)-4-methylthiazol-2-yl)methyl)piperidine-1-car-
boxylate (4′) (0.250 g, 0.462 mmol) was dissolved in dioxane (20 mL),
and 15% solution HCl in dioxane (20 mL) was added. The resulting
mixture was stirred at room temperature for 6 h. Solvent was distilled
off and residue was triturated with acetone/ether to give N1-(4-chloro-
3-fluorophenyl)-N2-((5-(hydroxymethyl)-4-methylthiazol-2-yl)-
(piperidin-2-yl)methyl)oxalamide hydrochloride (27) (0.114 g, 52%)
(diastereoisomeric mixture, 1:5). LC−MS (APCI+) m/z: calcd for
C19H22ClFN4O3S, 440.11; found, 441 (M + H+), 443.15 (M+ + 2),
automated docking software GLIDE 5.7 (Schrodinger, Portland, OR)
̈
within Schrodinger Suite 2011 that applies a two-stage scoring process
̈
to sort out the best conformations and orientations of the ligand
(defined as pose) based on its interactions with the receptor. GLIDE
has been used successfully in drug design.19−25 We used our recently
solved X-ray crystal structure of 1 with the clade C strain C1086
version of gp120 coree at 2.7 Å resolution for docking simulations
(PDB code 3TGS).12 Three-dimensional coordinates of the ligands
and their isomeric, ionization, and tautomeric states were generated
1
444.15. HPLC: >92%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH,
1.32−1.75 (m, 6H, CH2), 2.27 (s, 3H, -CH3), 2.75−3.00 (m, 1H,
CH2-N), 3.27 (m, 1H, CH2-N), 3.80 (m, 1H, CH-N), 4.56 (s, 2H,
-CH2-OH), 5.38 (t, 1H for one isomer, CH-), 5.51 (t, 1H for the
other, CH-), 6.00−6.30 (H2O + H+ + -OH signals), 7.51 (t, 1H, ArH-
), 7.59 (d, 1H, ArH-), 7.87 (d, 1H, ArHt-), 8.50−9.50 (m br, 2H,
using the LigPrep (including Ionizer) module within the Schrodinger
̈
Suite 2011 programs. The protein was prepared using the “protein
preparation tool”, and the structures were minimized with Macro-
+
model software within Schrodinger Suite 2011. A grid file
̈
NH2 ), 9.53 (br, 1H, CONH), 11.00 (s, 1H for one isomer, CONH),
encompassing the area in the cavity that contains information on
the properties of the associated receptor was created. Conformational
flexibility of the ligands was handled via an exhaustive conformational
11.09 (s, 1H for the other, CONH).
N1-(4-Chlorophenyl)-N2-((5-(hydroxymethyl)-4-methylthia-
zol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (6). White solid.
Yield: 42% (diastereoisomeric mixture, 1:1). LC−MS (APCI+) m/z:
calcd for C19H23ClN4O3S, 422.12; found, 422.98 (M + H+). HPLC:
search. Initially, we used Schrodinger’s proprietary GlideScore scoring
̈
function in standard precision (SP) mode. We selected 500 top-scored
compounds to dock again in extra precision (XP) mode to score the
optimized poses. The 50 top-scored ligands were selected from this
simulation for further study.
1
95.8%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.32−1.90 (m,
6H), 2.27 (s, 3H, -CH3), 2.75−3.00 (m, 1H), 3.33, (m, 1H), 3.80 (m,
1H), 4.56 (s, 2H, -CH2-OH), 5.38 (t, 1H for one isomer), 5.51 (t, 1H
for the other), 7.40 (d, 2H), 7.84 (d, 2H), 8.50−9.30 (m br, 2H,
Synthesis of Oxalamide Series, Compounds 6−40. Since the
compounds from the oxalamide series were synthesized according to
general Scheme 1, we presented a representative synthesis (Scheme 2)
of one of the most active compounds (27) from Table 2, and all other
compounds in this series were synthesized by following this method.
The deprotection step (d) was only used when N-Boc protected
amines were used as described below.
+
NH2 ), 9.61 (d, 1H for one isomer, NH), 9.68 (d, 1H for the other,
NH), 11.80 (s, 1H for one isomer, CONH), 10.91 (s, 1H for the
other, CONH).
N1-(4-Chlorophenyl)-N2-((5-(hydroxymethyl)-4-methylthia-
zol-2-yl)(piperidin-3-yl)methyl)oxalamide·2HCl (7). White solid.
Yield: 50% (diastereoisomeric mixture, 1:1). LC−MS (APCI+) m/z:
calcd for C19H23ClN4O3S, 422.12; found, 423.26 (M + H+). HPLC: >
97.5%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.34 (m, 1H), 1.65
(br, 2H), 1.82 (m, 2H), 2.27 (s, 3H, -CH3), 2.55 (m, 1H), 2.75 (m,
2H, -CH2-NH), 3,15 (m, 2H, -CH2-NH), 4.56 (s, 2H, -CH2-OH),
5.08 (m, 1H), 7.40 (d, 2H, Ar-H), 7.83 (d, 2H, Ar-H), 8.80 (br, 1H,
Ethyl 2-(4-Chloro-3-fluorophenylamino)-2-oxoacetate (2′).
TEA (2.28 g, 0.023 mol) was added at once to a solution of 3 g
(0.0206 mol) of 4-chloro-3-fluoroaniline in 50 mL of DCM, and then
ethyl 2-chloro-2-oxoacetate (2.81 g, 0.0206 mol) was added dropwise
at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and then at
room temperature for 6 h. The mixture was washed with 25% aqueous
solution of K2CO3 (2 × 50 mL) and water (50 mL). The product was
dried over Na2SO4 and evaporated. The residue was washed with ether
and dried in air to give ethyl 2-(4-chloro-3-fluorophenylamino)-2-
oxoacetate (2′) (3.97 g, 78.6%) as a white powder. LC−MS (APCI+)
m/z: calcd for C10H9ClFNO3, 245.03; found, 245 (M + H+).
2-(4-Chloro-3-fluorophenylamino)-2-oxoacetic Acid (3′).
Ethyl 2-(4-chloro-3-fluorophenylamino)-2-oxoacetate (4.18 g, 0.017
mol) was added to a solution of NaOH (1.361 g, 0.0340 mol) in a
mixture 50 mL of EtOH and 50 mL of water, and the resulting mixture
was stirred at room temperature for 6 h. The mixture was acidified
with 2 N HCl to pH 4−5 at 0 °C. The precipitate was filtered, washed
with water, and dried in air to afford 2-(4-chloro-3-fluorophenylami-
no)-2-oxoacetic acid (3′) (2 g, 55.2%) as a white solid. LC−MS
(APCI+) m/z: calcd for C8H5ClFNO3, 216.99; found, 217 (M + H+).
tert-Butyl 2-((2-(4-Chloro-3-fluorophenylamino)-2-
oxoacetamido)(5-(hydroxymethyl)-4-methylthiazol-2-yl)-
methyl)piperidine-1-carboxylate (4′). A mixture of 2-(4-chloro-3-
fluorophenylamino)-2-oxoacetic acid (3′) (0.3 g, 1.378 mmol), TBTU
(0.530 g 1.65 mmol), and TEA (0.166 g, 1.65 mmol) in DCM (20
mL) was stirred at room temperature for 1 h. Then 2-[amino-(5-
hydroxymethyl-4-methylthiazol-2-yl)methyl]piperidine-1-carboxylic
acid tert-butyl ester (0.470 g, 1.378 mmol) was added, and stirring was
continued for 6 h. The reaction mixture was washed with 25% aqueous
solution of K2CO3 (2 × 50 mL) and water (50 mL). The product was
dried over Na2SO4 and evaporated. The residue was purified by
column chromatography on silica gel (EtOAc/hexane, 1/1) to afford
tert-butyl 2-((2-(4-chloro-3-fluorophenylamino)-2-oxoacetamido)(5-
(hydroxymethyl)-4-methylthiazol-2-yl)methyl)piperidine-1-carboxy-
late (4′) (0.53 g, 72%) as a white solid. LC−MS (APCI+) m/z: calcd
for C24H30ClFN4O5S, 540.16; found, 541 (M + H+).
+
+
NH2 ), 9.30 (br d and d, 1H, NH2 ), 9.50 (d and d, 1H, CONH),
10.75 (s, 1H, CONH).
N1-(4-Chlorophenyl)-N2-((5-(hydroxymethyl)-4-methylthia-
zol-2-yl)(piperidin-4-yl)methyl)oxalamide·2HCl (8). White solid.
Yield: 46% (single diastereoisomer). LC−MS (APCI+) m/z: calcd for
C19H23ClN4O3S, 422.12; found, 423.26 (M + H+). HPLC: 95.0%. H
1
NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.49 (br m, 2H), 1.68 (br d,
1H), 1.93 (br d, 1H), 2.26 (s, 3H, -CH3), 2,35 (br m, 1H), 2.80 (br m,
2H), 3.25 (br m, 2H), 3.55 (m, 1H), 4.56 (s, 2H, -CH2-OH), 4.68 (br
t, 1H), 4.95 (m, 1H), 7.39 (d, 2H, Ar-H), 7.83 (d, 2H, Ar-H), 8.68 (br,
+
+
1H, NH2 ), 9.08 (br, 1H, NH2 ), 9.41 (d, 1H, CONH), 10.78 (s, 1H,
CONH).
N1-(4-Chlorophenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCOOH (9). White
solid. Yield: 55% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C20H25ClN4O3S, 436.13; found, 437.30 (M + H+).
1
HPLC: >95.0%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.10−
1.80 (m, 6H), 2.26 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.00−3.80
(12H + NH + H2O signal), 5.00 (br, 1H for one isomer), 5.10 (br, 1H
for the other), 7.41 (d, 2H), 7.82 (d, 2H), 9.10 (br 1H for one isomer,
+
+
NH2 ), 9.38 (br, 1H for the other, NH2 ), 10.85 (s, 1H, NH).
N1-(4-Chlorophenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(piperidin-3-yl)methyl)oxalamide·2HCl (10). White
solid. Yield: 37% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C20H25ClN4O3S, 436.13; found, 437.27 (M + H+).
1
HPLC: 98.2%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.40 (m,
1H), 1.65 (br, 2H), 1.82 (m, 2H), 2.27 (s, 3H, -CH3), 2.55 (br, 1H),
2.65−2.88 (m, 4H), 3,13 (m, 2H), 3,55 (m, 2H, -CH2-CH2-OH), 5.07
+
(m, 1H), 7.39 (d, 1H, Ar-H), 7.82 (d, 1H, Ar-H), 8.75 (br, 1H, NH2+),
+
9.11 (br, 1H for one isomer, NH2 ), 9.24 (br, 1H for the other, NH2 ),
9.48 (d, 1H for one isomer, CO-NH), 9.51 (d, 1H for the other, CO-
NH), 10.75 (s, 1H, CO-NH).
N1-(4-Chloro-3-fluorophenyl)-N2-((5-(hydroxymethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·2HCl
(27). tert-Butyl 2-((2-(4-chloro-3-fluorophenylamino)-2-oxoacetami-
N1-(4-Chlorophenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(piperidin-4-yl)methyl)oxalamide·2HCl (11). White
H
dx.doi.org/10.1021/jm3002247 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
solid. Yield: 44% (single diastereoisomer). LC−MS (APCI+) m/z:
N1-(4-Chlorophenyl)-N2-((1S,2R)-1-morpholino-1-phenylpro-
pan-2-yl)oxalamide (19). White solid. Yield: 30%. LC−MS (APCI+)
m/z: calcd for C21H24ClN3O3: 401.15; found, 402.28 (M + H+).
calcd for C20H25ClN4O3S, 436.13; found, 437.21 (M + H+). HPLC:
1
95%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.45 (m, 2H), 1.68
1
HPLC: 95%. H NMR (CDCl3, 45 °C, 400 MHz) δH, 1.10 (d, 3H,
(d, 1H), 1.95 (d, 1H), 2.25 (s, 3H, CH3), 2.35 (m, 1H), 2.80 (m, 4H),
-CH3), 2.45 (br, 2H, -N-CH2-), 2.56 (br, 2H, -N-CH2-), 3.31 (d, 1H,
Ph-CH-N), 3.79 (m br, 4H, -O-CH2-), 4.65 (m, 1H, N-CH-CH3),
7.28−7.45 (m, 5H, Ar-H), 7.52 (br d, 2H, Ar-H), 7.57 (br d, 2H, Ar-
H).
3.24 (m, 2H), 3,40−3.70(m, 3H), 4,95 (t, 1H,), 7.39 (d, 2H, Ar-H),
+
+
7.82 (d, 2H, Ar-H), 8.65 (br, 1H, NH2 ), 8.88 (br, 1H, NH2 ), 9.35 (d,
1H, CO-NH), 10.75 (s, 1H, CO-NH).
N1-(4-Chlorophenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(1-methylpiperidin-2-yl)methyl)oxalamide (12). White
solid. Yield: 38% (single diastereoisomer). LC−MS (APCI+) m/z:
calcd for C21H27ClN4O3S, 450.15; found, 451.12 (M + H+). HPLC:
N1-((5-(Hydroxymethyl)-4-methylthiazol-2-yl)(piperidin-2-
yl)methyl)-N2-(4-(trifluoromethyl)phenyl)oxalamide·2HCl (20).
White solid. Yield: 56% (diastereoisomeric mixture, 1:20). LC−MS
(APCI+) m/z: calcd for C20H23F3N4O3S, 456.14; found, 457.90 (M +
H+). HPLC: 97.7%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−
1.76 (m, 6H), 2.27 (s, 3H, -CH3), 2.62 (br, 1H, CH), 3.30, (br d, 1H),
3.82 (br, 1H), 4.56 (s, 2H, -CH2-O), 5.40 (t, 1H for one isomer), 5.51
(t, 1H for the other), 7.65 (d, 2H), 8.20 (d, 2H), 8.61−9.50 (m, 2H,
1
93.0%. H NMR (CDCl3, 45 °C, 400 MHz) δH, 1.20−2.20 (m, 8H)
2.35(s, 3H, -CH3), 2.38 (s, 3H, -CH3), 2.71 (br, 1H, -CH-NMe), 2.95
(m, 4H, -CH2-CH2-OH), 3.80 (m, 2H, -CH2-CH2-OH), 5.10 (br, 1H,
CONH-CH), 7.31 (d, 2H ArH), 7.53 (d, 2H ArH,), 8.40 (br, 1H,
CONH-CH) 9.28 (s, 1H, Ar-NHCO-).
+
N1-((1-Acetylpiperidin-2-yl)(5-(2-hydroxyethyl)-4-methyl-
thiazol-2-yl)methyl)-N2-(4-chlorophenyl)oxalamide (13). White
solid. Yield: 36% (single diastereoisomer). LC−MS (APCI+) m/z:
calcd for C22H27ClN4O4S, 478.14; found, 479.12 (M + H+). HPLC:
NH2 ), 9.65 (br d, 1H, CONH), 11.00 (s, 1H for one isomer,
CONH), 11.08 (s, 1H for the other, CONH).
N1-(2,4-Difluorophenyl)-N2-((5-(hydroxymethyl)-4-methyl-
thiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (21). White
solid. Yield: 58% (diastereoisomeric mixture, 1:5). LC−MS (APCI+)
m/z: calcd for C19H22F2N4O3S, 424.14; found, 425.86 (M + H+).
HPLC: 95.6%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80
(m, 6H), 2.27 (s, 3H, -CH3), 2.80−4.00 (m, 3H + H2O signal), 4.56
(s, 2H, -CH2-O), 5.35 (t, 1H for one isomer), 5.41 (t, 1H for the
1
90.0%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.10−2.20 (m,
9H), 2.27 (s, 3H, -CH3), 2.80 (m, 2H,), 3.56 (m, 2H, -CH2-OH), 4.75,
(m, 1H,) 5.12 (m, 1H), 5.48 (m, 1H,), 7.41 (d, 2H), 7.82 (d, 2H),
8.35 (br 1H, CO-NH), 10.75 (s, 1H, CO-NH).
N1-(4-Chlorophenyl)-N2-((5-(hydroxymethyl)-4-methylthia-
zol-2-yl)(pyrrolidin-2-yl)methyl)oxalamide·HCOOH (14). White
solid. Yield: 39% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C18H21ClN4O3S, 408.10; found, 409.28 (M + H+).
HPLC: 93.2%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.40−1.90
(m, 4H, -CH2-), 2.27 (s, 3H, -CH3), 2.87 (m, 2H), 3.10−3.70 (NH +
H2O signal), (3.80 (m, 1H), 4.56 (s, 2, -CH2-OH), 4.98 (br, 1H for
one isomer), 5.08 (br, 1H for the other), 7.40 (d, 2H), 7.82 (d, 2H),
+
other), 7.11 (t, 1H), 7.34 (t, 3H), 7.61 (m, 3H), 8.38 (br 1H, NH2 ),
+
8.70 (br d, 1H, NH2 ), 9.51 (d, 1H for one isomer, NH), 9.70 (d, 1H
for the other, NH), 10.25 (s, 1H for one isomer, Ar-CONH), 10.30 (s,
1H for the other, Ar-CONH).
N1-(3,4-Difluorophenyl)-N2-((5-(hydroxymethyl)-4-methyl-
thiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (22). White
solid. Yield: 45% (diastereoisomeric mixture, 3:2). LC−MS (APCI+)
m/z: calcd for C19H22F2N4O3S, 424.14; found, 425.90 (M + H+).
HPLC: 98.3%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80
(m, 6H), 2.27 (s, 3H -CH3), 2.82 (br, 1H), 3.30, (br, 1H), 3.82 (m,
1H), 4.56 (s, 2H, -CH2-O), 5.40 (t, 1H for one isomer), 5.55 (t, 1H
for the other), 7.40 (m, 1H), 7.67 (d, 1H) 7.91 (m, 1H), 8.60 (br, 1H
+
+
9.08 (br 1H for one isomer, NH2 ), 9.40 (br, 1H for the other, NH2 ),
10.85 (s, 1H, NH).
N1-(4-Chlorophenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(pyrrolidin-2-yl)methyl)oxalamide·HCOOH (15). White
solid. Yield: 53% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C19H23ClN4O3S, 422.12; found, 423.27 (M + H+).
+
+
for one isomer, NH2+), 8.90 (br, 1H for the other, NH2+), 9.20 (br, 1H
for one isomer, NH2 ), 9.35 (br, 1H for the other, NH2 ), 9.68 (br d +
d, 1H, CONH), 10.80 (s, 1H, CONH-Ar), 11.00 (s, 1H, CONH-Ar).
N1-(4-Acetylphenyl)-N2-((5-(hydroxymethyl)-4-methylthia-
zol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (23). White solid.
Yield: 42% (diastereoisomeric mixture, 3:2). LC−MS (APCI+) m/z:
calcd for C21H26N4O4S, 430.17; found, 431.19 (M + H+). HPLC:
1
HPLC: 95.0%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.52 (m,
1H, -CH2-), 1.69 (m, 2H, -CH2-), 1.85 (m, 1H, -CH2-) 2.27 (s, 3H,
-CH3), 2.80 (t, 2H, -CH2-CH2-OH), 2.87 (m, 2H, CH2-N), 3.53 (m,
2H, -CH2-CH2-OH), 3.85 (m, 1H, CH-N), 4.98 (br, 1H for one
isomer), 5.08 (br, 1H for the other), 7.40 (d, 2H), 7.82 (d, 2H), 9.08
+
+
1
(br 1H for one isomer, NH2 ), 9.46 (br, 1H for the other, NH2 ),
10.85 (s, 1H, NH).
96.5%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80 (m,
6H), 2.27 (s, 3H, -CH3), 2.52 (s, 3H, -CH3), 2.82 (br, 1H), 3.30, (br,
1H), 3.82 (m, 1H), 4.56 (s, 2H, -CH2-O), 5.40 (t, 1H for one isomer),
5.55 (t, 1H for the other), 7.90 (s, 4H, ArH), 8.60 (br, 1H for one
N1-(4-Chlorophenyl)-N2-(1-(5-(2-hydroxyethyl)-4-methyl-
thiazol-2-yl)-2-(methylamino)ethyl)oxalamide·2HCl (16). White
solid. Yield: 41%. LC−MS (APCI+) m/z: calcd for C17H21ClN4O3S,
396.10; found, 397.08 (M + H+). HPLC: 94.2%. 1H NMR (DMSO-d6,
50 °C, 400 MHz) δH, 2.27 (s, 3H, -CH3), 2.59 (br, 3H, NH-CH3),
2.82 (m, 2H, -CH2-CH2-OH), 3.55 (m, 3H, -NCH3), 3.60 (m, 2H,
-CH2-OH), 5.30 (m, 2H), 5.55 (m, 1H, CH-HetAr), 7.41 (d, 2H,
ArH), 7.82 (d, 2H, ArH), 9.05 (br 1H, NH), 9.28 (br 1H, NH), 9.75
(br d, 1H, NH), 10.85 (s, 1H, NH).
+
+
isomer, NH2+), 8.95 (br, 1H for the other, NH2 +), 9.10 (br, 1H for one
isomer, NH2 ), 9.25 (br, 1H for the other, NH2 ), 9.65 (d, 1H for one
isomer, CONH), 9.71 (d, 1H for the other, CONH), 10.80 (s, 1H,
CONH-Ar), 11.00 (s, 1H, CONH-Ar).
N1-(3-Chloro-4-fluorophenyl)-N2-((5-(hydroxymethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·2HCl
(24). White solid. Yield: 33% (diastereoisomeric mixture, 1:1). LC−
MS (APCI+) m/z: calcd for C19H22ClFN4O3S, 440.11; found, 441.13
(M + H+). HPLC: 92.7%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH,
1.30−1.80 (m, 6H), 2.27 (s, 3H, -CH3), 2.82 (br, 1H), 3.30, (br, 1H),
3.82 (br, 1H), 4.56 (s, 2H, -CH2-O), 5.40 (t, 1H for one isomer), 5.55
(t, 1H for the other), 7.40 (t, 1H, ArH), 7.82 (m, 1H, ArH), 8.08 (m,
N1-(4-Chlorophenyl)-N2-(1-ethyl-1H-pyrazol-4-yl)oxalamide
(17). White solid. Yield: 35%. Mp: 232−233. LC−MS (APCI+) m/z:
calcd for C13H13ClN4O2: 292.07; found, 293.04 (M + H+). HPLC:
1
98.8%. H NMR (DMSO-d6, 400 MHz) δH, 1.38 (t, 3H, -CH3), 4.11
(q, 2H, -CH2-), 7.41 (d, 2H, Ar-H), 7.70 (s, 1H, HetAr-H), 7.86 (d,
2H, Ar-H), 8.10 (s, 1H, HetAr-H), 10.75 (s, 1H, NH), 11.10 (s, 1H,
NH).
+
1H ArH), 8.60 (br, 1H for one isomer, NH2 ), 8.95 (br, 1H for the
+
+
other, NH2 +), 9.19 (br, 1H for one isomer, NH2 ), 9.40 (br, 1H for the
other, NH2 ), 9.68 (d + d, 1H, CONH), 10.80 (s, 1H, CONH-Ar),
11.00 (s, 1H, CONH-Ar).
(2S,4R,5S)-Methyl 4-(2-(4-Chlorophenylamino)-2-oxoaceta-
mido)-5-phenylpyrrolidine-2-carboxylate (18). White solid.
Yield: 39%. LC−MS (APCI+) m/z: calcd for C20H20ClN3O4:
N1-(4-Fluorophenyl)-N2-((5-(hydroxymethyl)-4-methylthia-
zol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (25). White solid.
Yield: 50% (diastereoisomeric mixture, 1:1). LC−MS (APCI+) m/z:
calcd for C19H23FN4O3S, 406.15; found, 407.16 (M + H+). HPLC:
1
401.11; found, 402.14 (M + H+). HPLC: 95%. H NMR (DMSO-
d6, 50 °C, 400 MHz) δH, 2.18 (m, 2H, -CH2-), 3.30 (br, 1H, -NH-),
3.69 (s, 3H, O-CH3), 4.05 (m, 1H, -N-CH-Ph), 4.17 (m, 1H, -CH-),
4.27 (m, 1H, -CH-), 7.20 (t, 1H, Ar-H), 7.28 (t, 2H, Ar-H meta), 7.38
(d, 2H, Ar-H), 7.44 (d, 2H, Ar-H), 7.81 (d, 2H, Ar-H), 9.12 (br 1H,
NH), 10.58 (s, 1H, Ar-NH).
1
94.7%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80 (m,
6H), 2.27 (s, 3H, -CH3), 2.80−3.00 (br, 1H), 3.30 (br, 1H), 3.82 (m,
1H), 4.51(s, 2H, -CH2-O), 5.40 (t, 1H for one isomer), 5.51 (t, 1H for
I
dx.doi.org/10.1021/jm3002247 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
the other), 7.12 (m, 2H, ArH), 7.75 (m, 2H, ArH), 8.60 (br, 1H for
5.50 (t, 1H for the other), 7.07 (m, 1H, ArH), 7.28 (m, 1H, ArH),
+
+
+
one isomer, NH2 ), 8.95 (br, 1H for the other, NH2 ), 9.19 (br, 1H for
7.60 (m, 1H, ArH), 8.50 (br, 1H for one isomer, NH2 +), 8.74 (br, 1H
+
+
+
one isomer, NH2 ), 9.40 (br, 1H for the other, NH2 ), 9.65 (d + d,
1H, CONH), 10.70 (s, 1H for one isomer, CONH-Ar), 10.75 (s, 1H
for the other, CONH-Ar).
for the other, NH2 ), 9.09 (br, 1H for one isomer, NH2 ), 9.21 (br, 1H
+
for the other, NH2 ), 9.57 (d, 1H for one isomer, CONH), 9.70 (d,
1H for the other, CONH), 11.01 (s, 1H for one isomer, CONH-Ar),
11.32 (s, 1H for the other, CONH-Ar).
N1-(2-Fluoro-4-methylphenyl)-N2-((5-(hydroxymethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (26).
White solid. Yield: 39% (diastereoisomeric mixture, 1:9). LC−MS
(APCI+) m/z: calcd for C20H25FN4O3S:420.16; found, 421.19 (M +
H+). HPLC: 95.1%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−
1.80 (m, 6H), 2.27 (s, 3H, -HetAr-CH3), 2.31 (s, 3H, Ar-CH3), 2.75−
3.00 (br, 1H), 3.30 (br, 1H), 3.82 (m, 1H), 4.51(s, 2H, -CH2-O), 5.40
(t, 1H for one isomer), 5.51 (t, 1H for the other), 7.02 (d, 1H, ArH),
7.10 (d, 1H, ArH), 7.50 (t, 1H, ArH), 8.51 (br, 1H for one isomer,
N1-(3,4-Difluorophenyl)-N2-((5-(2-hydroxyethyl)-4-methyl-
thiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·2HCl (33). White
solid. Yield: 32% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C20H24F2N4O3S, 438.15; found, 439.19 (M + H+).
1
HPLC: 97.9%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 11.30−
1.80 (m, 6H), 2.25 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.30, (br, 1H),
3,55 (m, 2H, -CH2-CH2-OH), 3.80 (br, 1H), 5.36 (t, 1H for one
isomer), 5.50 (t, 1H for the other), 7.40 (m, 1H, ArH), 7.65 (m, 1H,
+
+
+
NH2 ), 8.61 (br, 1H for the other, NH2 ), 9.10 (br d, 1H for one
ArH), 7.91 (m, 1H, ArH), 8.56 (br, 1H for one isomer, NH2 ), 8.90
+
+
+
+
isomer, NH2 ), 9.25 (br, 1H for the other, NH2 ), 9.60 (d, 1H for one
isomer, CONH), 9.67 (d, 1H for the other, CONH), 10.10 (s, 1H for
one isomer, CONH-Ar), 10.20 (s, 1H for the other, CONH-Ar).
N1-(3-Fluoro-4-methylphenyl)-N2-((5-(hydroxymethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (28).
White solid. Yield: 34% (diastereoisomeric mixture, 2:3). LC−MS
(APCI+) m/z: calcd for C20H25FN4O3S, 420.16; found, 421.19 (M +
H+). HPLC: 95.8%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−
1.80 (m, 6H), 2.26 (s, 3H, -CH3), 2.80 (m, 1H), 3.30, (br, 1H), 3.80
(m, 1H,), 4.56 (s, 2H, CH2-OH), 5.40 (t, 1H for one isomer), 5.51 (t,
1H for the other), 7.24 (m, 1H, ArH), 7.55 (d, 1H, ArH), 7.66 (d, 1H,
(br, 1H for the other, NH2 ), 9.11 (br d, 1H for one isomer, NH2 ),
+
9.29 (br, 1H for the other, NH2 ), 9.61 (d, 1H for one isomer,
CONH), 9.69 (d, 1H for the other, CONH), 10.85 (s, 1H for one
isomer, CONH-Ar), 11.00 (s, 1H for the other, CONH-Ar).
N1-(4-Acetylphenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(piperidin-2-yl)methyl)oxalamide·2HCl (34). White
solid. Yield: 44% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C22H28N4O4S, 444.18; found, 445.20 (M + H+). HPLC:
1
96.6%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80 (m,
6H), 2.25 (s, 3H, -CH3), 2.52 (s, 3H, CO-CH3), 2.75−3.00 (br, 3H),
3.30, (br, 1H), 3.55 (m, 2H, -CH2-CH2-OH), 3.80 (br, 1H), 5.36 (t,
1H for one isomer), 5.50 (t, 1H for the other), 7.86 (s, 4H, ArH), 8.56
+
ArH), 8.51 (br, 1H for one isomer, NH2 ), 8.65 (br, 1H for the other,
+
+
+
+
NH2+), 9.11 (br, 1H for one isomer, NH2 ), 9.24 (br, 1H for the other,
NH2 ), 9.61 (d, 1H for one isomer, CONH), 9.68 (d, 1H for the
other, CONH), 10.75 (s, 1H for one isomer, CONH-Ar), 10.82 (s, 1H
for the other, CONH-Ar).
(br, 1H for one isomer, NH2+), 8.90 (br, 1H for the other, NH2+), 9.10
(br, 1H for one isomer, NH2 ), 9.25 (br, 1H for the other, NH2 ), 9.61
(d, 1H for one isomer, CONH), 9.69 (d, 1H for the other, CONH),
10.86 (s, 1H for one isomer, CONH-Ar), 11.00 (s, 1H for the other,
CONH-Ar).
N1-Cycloheptyl-N2-((5-(hydroxymethyl)-4-methylthiazol-2-
yl)(piperidin-2-yl)methyl)oxalamide·HCl (29). White solid. Yield:
28% (diastereoisomeric mixture, 2:3). LC−MS (APCI+) m/z: calcd for
N1-(3-Chloro-4-fluorophenyl)-N2-((5-(2-hydroxyethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·2HCl
(35). White solid. Yield: 35% (diastereoisomeric mixture, 1:1). LC−
MS (APCI+) m/z: calcd for C20H24ClFN4O3S, 454.12; found, 455.14
(M + H+). HPLC: 97.0%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH,
1.30−1.80 (m, 6H), 2.25 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.30, (br,
1H), 3.55 (m, 2H), 3.80 (br, 1H), 5.39 (t, 1H for one isomer), 5.51 (t,
1H for the other), 7.40 (t, 1H, ArH), 7.80 (m, 1H, ArH), 8.15 (m, 1H,
1
C20H32N4O3S, 408.22; found, 409.57 (M + H+). HPLC: 97.5%. H
NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80 (m, 18H), 2.26 (s,
3H, -CH3), 2.75−3.00 (m, 1H), 3.27, (m, 1H), 3.40, (m, 1H), 3.76
(m, 2H), 4.56 (s, 2H, CH2-OH), 5.33 (t, 1H for one isomer), 5.50 (t,
1H for the other), 8.30−8,55 (br m, 1H, NH), 8.53 (br, 1H for one
isomer, NH), 8.77 (br, 1H for the other, NH), 9.11 (br, 1H for one
isomer, NH), 9.25 (br, 1H for the other, NH), 9.40 (br, 1H, CO-NH).
N1-(4-Chlorophenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCOOH (30). White
solid. Yield: 55% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C20H25ClN4O3S:436.13; found, 437.30 (M + H+).
+
ArH), 8.58 (br, 1H for one isomer, NH2 ), 8.90 (br, 1H for the other,
+
+
NH2+), 9.15 (br, 1H for one isomer, NH2 ), 9.30 (br, 1H for the other,
NH2 ), 9.61 (d, 1H for one isomer, CONH), 9.69 (d, 1H for the
other, CONH), 10.86 (s, 1H for one isomer, CONH-Ar), 11.00 (s, 1H
for the other, CONH-Ar).
1
N1-(4-Fluorophenyl)-N2-((5-(2-hydroxyethyl)-4-methylthia-
zol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (36). White solid.
Yield: 47% (diastereoisomeric mixture, 1:1.2). LC−MS (APCI+) m/z:
calcd for C20H25FN4O3S, 420.16; found, 421.18 (M + H+). HPLC:
HPLC: >95.0%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.10−
1.80 (m, 6H), 2.26 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.00−3.80
(12H + NH + H2O signal), 5.00 (br, 1H for one isomer), 5.10 (br, 1H
for the other), 7.41 (d, 2H), 7.82 (d, 2H), 9.10 (br 1H for one isomer,
1
+
+
95.3%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80 (m,
NH2 ), 9.38 (br, 1H for the other, NH2 ), 10.85 (s, 1H, NH).
N1-((5-(2-Hydroxyethyl)-4-methylthiazol-2-yl)(piperidin-2-
yl)methyl)-N2-(4-(trifluoromethyl)phenyl)oxalamide·2HCl (31).
White solid. Yield: 39% (diastereoisomeric mixture, 1:1). LC−MS
(APCI+) m/z: calcd for C21H25F3N4O3S, 470.16; found, 471.21 (M +
H+). HPLC: 95.5%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−
1.80 (m, 6H), 2.25 (s, 3H, -CH3) 2.75−3.00 (m, 3H), 3.30, (br, 1H),
3,55 (m, 2H, -CH2-CH2-OH), 3.80 (br, 1H), 5.38 (t, 1H for one
isomer), 5.55 (t, 1H for the other), 7.77 (d, 2H, ArH), 8.02 (d, 2H,
6H), 2.26 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.30 (br, 1H), 3.55 (m,
2H), 3.80 (br, 1H), 5.39 (t, 1H for one isomer), 5.50 (t, 1H for the
other), 7.17 (m, 2H, ArH), 7.82 (m, 2H, ArH), 8.51 (br, 1H for one
+
+
isomer, NH2+), 8.90 (br, 1H for the other, NH2 +), 9.12 (br, 1H for one
isomer, NH2 ), 9.28 (br, 1H for the other, NH2 ), 9.51 (d, 1H for one
isomer, CONH), 9.60 (d, 1H for the other, CONH), 10.70 (s, 1H for
one isomer, CONH-Ar), 10.76 (s, 1H for the other, CONH-Ar).
N1-(2-Fluoro-4-methylphenyl)-N2-((5-(2-hydroxyethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·2HCl
(37). White solid. Yield: 65% (diastereoisomeric mixture, 1:1). LC−
MS (APCI+) m/z: calcd for C21H27FN4O3S, 434.18; found, 435.21 (M
+
ArH), 8.60 (br, 1H for one isomer, NH2 ), 8.91 (br, 1H for the other,
+
+
NH2+), 9.12 (br, 1H for one isomer, NH2 ), 9.26 (br, 1H for the other,
NH2 ), 9.63 (d, 1H for one isomer, CONH), 9.70 (d, 1H for the
other, CONH), 10.98 (s, 1H for one isomer, CONH-Ar), 11.20 (s, 1H
for the other, CONH-Ar).
1
+ H+). HPLC: 93.4%. H NMR (DMSO-d6, 50 °C, 400 MHz) δH,
1.30−1.80 (m, 6H), 2.26 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.30, (br,
1H), 3.60 (m, 2H), 3.80 (br, 1H), 5.35 (t, 1H for one isomer), 5.50 (t,
1H for the other), 7.01 (d, 1H, ArH), 7.10 (d, 1H, ArH), 7.51 (m, 1H,
N1-(2,4-Difluorophenyl)-N2-((5-(2-hydroxyethyl)-4-methyl-
thiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (32). White
solid. Yield: 43% (diastereoisomeric mixture, 1:1). LC−MS (APCI+)
m/z: calcd for C20H24F2N4O3S, 438.15; found 438.50 (M + H+).
HPLC: 97.5%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80
(m, 6H), 2.25 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.30, (br, 1H), 3.55
(m, 2H, -CH2-CH2-OH), 3.80 (br, 1H), 5.39 (t, 1H for one isomer),
+
ArH), 8.51 (br, 1H for one isomer, NH2 ), 8.65 (br, 1H for the other,
+
+
NH2+), 9.11 (br, 1H for one isomer, NH2 ), 9.24 (br, 1H for the other,
NH2 ), 9.53 (d, 1H for one isomer, CONH), 9.70 (d, 1H for the
other, CONH), 10.15 (s, 1H for one isomer, CONH-Ar), 10.24 (s, 1H
for the other, CONH-Ar).
J
dx.doi.org/10.1021/jm3002247 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
N1-(3-Fluoro-4-methylphenyl)-N2-((5-(2-hydroxyethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (38).
White solid. Yield: 47% (diastereoisomeric mixture, 1:1). LC−MS
(APCI+) m/z: calcd for C21H27FN4O3S, 434.18; found, 435.24 (M +
H+). HPLC: 96.1%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−
1.80 (m, 6H), 2.20 (s, 3H, Ar-CH3), 2.26 (s, 3H, HetAr-CH3), 2.75−
3.00 (m, 3H), 3.30 (br, 1H), 3.55 (m, 2H, -CH2-CH2-OH), 3.80 (s,
1H), 5.39 (t, 1H for one isomer), 5.57 (t, 1H for the other), 7.24 (t,
1H, ArH), 7.55 (d, 1H, ArH), 7.70 (d, 1H, ArH), 8.55 (br, 1H for one
determined as previously described.26 In brief, 1 × 104 MT-2 cells
were infected with HIV-1 at 100 TCID50 (50% tissue culture infective
dose) (0.01 MOI) in 200 μL of medium in the presence or absence of
small molecules at graded concentrations and incubated overnight.
The culture supernatants were then removed and replaced with fresh
medium. On the fourth day postinfection, an amount of 100 μL of
culture supernatants was collected from each well, mixed with an equal
volume of 5% Triton X-100, and tested for p24 antigen by sandwich-
ELISA. The percent inhibition of p24 production and IC50 values were
calculated by the GraphPad Prism software (GraphPad Software Inc.).
Determination of Cytotoxicity. In TZM-bl Cells. The cytotoxicity
of small molecules in TZM-bl cells was measured by a colorimetric
method using XTT (sodium 3′-(1-(phenylamino)carbonyl)-3,4-
tetrazolium-bis(4-methoxy-6-nitro)bezenesulfonic acid hydrate), a
light yellowish tetrazolium dye, as previously described.27 Briefly,
100 μL of a compound at graded concentrations was added to equal
volume of cells (105/mL) in wells of 96-well plates followed by
incubation at 37 °C for 3 days and addition of XTT (PolySciences,
Inc., Warrington, PA). The soluble intracellular formazan was
quantitated colorimetrically at 450 nm 4 h later. The percent of
cytotoxicity and the CC50 (the concentration for 50% cytotoxicity)
values were calculated by the GraphPad Prism software (GraphPad
Software Inc.).
+
+
isomer, NH2 ), 8.91 (br, 1H for the other, NH2 ), 9.12 (br d, 1H for
+
+
one isomer, NH2 ), 9.26 (br, 1H for the other, NH2 ), 9.58 (d, 1H for
one isomer, CONH), 9.65 (d, 1H for the other, CONH,), 10.75 (s,
1H for one isomer, CONH-Ar), 10.80 (s, 1H for the other, CONH-
Ar).
N1-(4-Chloro-3-fluorophenyl)-N2-((5-(2-hydroxyethyl)-4-
methylthiazol-2-yl)(piperidin-2-yl)methyl)oxalamide·HCl (39).
White solid. Yield: 50% (diastereoisomeric mixture, 4:5). LC−MS
(APCI+) m/z: calcd for C20H24ClFN4O3S, 454.12; found, 455.18 (M +
H+). HPLC: 96.2%. 1H NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−
1.80 (m, 6H), 2.26 (s, 3H, -CH3), 2.75−3.00 (m, 3H), 3.30 (br, 1H),
3.60 (m, 2H, -CH2-CH2-OH), 3.80 (m, 1H,), 5.35 (t, 1H for one
isomer), 5.50 (t, 1H for the other), 7.55 (t, 1H, ArH), 7.65 (d, 1H,
+
ArH), 7.90 (d, 1H, ArH), 8.59 (br, 1H for one isomer, NH2 +), 8.90
+
(br, 1H for the other, NH2 ), 9.11 (br, 1H for one isomer, NH2 ), 9.24
In MT-2 Cells. Cytotoxicity of small molecules in MT-2 cells was
measured as previously described.26 Briefly, 100 μL of a small molecule
at graded concentrations was added to an equal volume of MT-2 cells
(105 cells/mL) in 96-well plates followed by incubation at 37 °C for 4
days. After addition of XTT, the soluble intracellular formazan was
quantitated colorimetrically and the percent of cytotoxicity and the
CC50 values were calculated as above.
+
(br, 1H for the other, NH2 ), 9.60 (d, 1H for one isomer, CONH),
9.68 (d, 1H for the other, CONH), 10.92 (s, 1H for one isomer,
CONH-Ar), 11.09 (s, 1H for the other, CONH-Ar).
N1-Cycloheptyl-N2-((5-(2-hydroxyethyl)-4-methylthiazol-2-
yl)(piperidin-2-yl)methyl)oxalamide·HCl (40). Yellow oil. Yield:
57% (diastereoisomeric mixture, 1:1). LC−MS (APCI+) m/z: calcd for
1
C21H34N4O3S, 422.24; found, 423.60 (M + H+). HPLC: 98.5%. H
NMR (DMSO-d6, 50 °C, 400 MHz) δH, 1.30−1.80 (m, 18H), 2.26 (s,
3H, -CH3), 2.75−3.00 (m, 3H), 3.30 (br, 1H), 3.40 (m, 1H), 3.58 (m,
2H, -CH2-CH2-OH), 3.80 (m, 1H), 5.35 (t, 1H for one isomer), 5.50
(t, 1H for the other), 8.30−8.55 (br m, 1Hσ, NH), 8.53 (br, 1H for
one isomer, NH), 8.77 (br, 1H for the other, NH), 9.11 (br, 1H for
one isomer, NH), 9.25 (br, 1H for the other, NH), 9.40 (br, 1H, CO-
NH).
ASSOCIATED CONTENT
* Supporting Information
Synthesis procedures (Scheme S1) of succinamide series
compounds (41−51), listing of their structures and antiviral
activity (Table S1), and listing of the 50 top GLIDE XP-scored
poses (Figure S1). This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
Measurement of Antiviral Activity. Pseudoviruses Preparation.
To prepare the X4-tropic pseudovirus NL4-3-HXB2-Luc, 5 × 106
293T cells were seeded in a T75 flask and transfected 24 h later in 20
mL of medium with a mixture of 10 μg of pNL4-3.Luc.R-E- DNA and
10 μg of env expression vector pHXB2-env (X4) DNA using FuGENE
6 (Roche). Pseudovirus-containing supernatant was collected 2 days
after transfection and stored in aliquots at −80 °C. Pseudovirus was
titered by infecting the TZM-bl cells to calculate the 50% tissue culture
infectious dose (TCID50). TZM-bl cells were plated in 96-well plates
at 104 cells/well 24 h before infection. On the day of the infection 100
μL of serial 2-fold dilutions of pseudovirus were added to the cells.
After 3 days of incubation the cells were washed 2 times with PBS and
lysed with 50 μL of cell culture lysis reagent (Promega). An amount of
20 μL of lysates was transferred to a white 96-well plate (Costar) and
mixed with 100 μL of luciferase assay reagent (luciferase assay system,
Promega). The luciferase activity was immediately measured with a
Tecan infinite M1000 reader (Tecan). Wells producing relative
luminescence units (RLU) 4 times the background were scored as
positive, and the TCID50 was calculated by the Spearman−Karber
statistical method.
Single-Cycle Neutralization Assay. The inhibitory activity of small
molecules was tested on NL4-3-HXB2-Luc pseudotyped virus
expressing Env of the HIV-1HXB2 (X4). Briefly, 100 μL of TZM-bl
cells at 1 × 105 cells/mL was added to the wells of a 96-well tissue
culture plate and cultured at 37 °C overnight. Then 50 μL of a test
compound at graded concentrations was mixed with 50 μL of the
NL4-3-HXB2-Luc virus at about 100 TCID50. After incubation at 37
°C for 30 min, the mixtures were added to the cells and incubated at
37 °C for 3 days. The cells were then harvested and lysed for
measuring luciferase activity as described above.
AUTHOR INFORMATION
Corresponding Author
*Phone: (212) 570-3373. E-mail: adebnath@nybloodcenter.
■
org.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by an intramural fund from the New
York Blood Center (A.K.D.), the Intramural AIDS Targeted
Antiviral Program (IATAP), and intramural funding from the
National Institute of Allergy and Infectious Diseases.
ABBREVIATIONS USED
■
SAR, structure−activity relationship; LC−MS, liquid chroma-
tography−mass spectrometry; NMR, nuclear magnetic reso-
nance; DCM, dichloromethane; TBTU, N,N,N′,N′-tetramethyl-
O-(benzotriazol-1-yl)uranium tetrafluoroborate
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