Fragment-Based Approach to Targeting Inosine-5′-monophosphate Dehydrogenase (IMPDH) from Mycobacterium tuberculosis

Article abstract of DOI:10.1021/acs.jmedchem.7b01622

Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5′-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotid

Full text of DOI:10.1021/acs.jmedchem.7b01622

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A fragment-based approach to targeting inosine-5´-monophosphate
dehydrogenase (IMPDH) from Mycobacterium tuberculosis
Ana Trapero, Angela Pacitto, Vinayak Singh, Mohamad Sabbah, Anthony G
Coyne, Valerie Mizrahi, Tom L. Blundell, David B. Ascher, and Chris Abell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01622 • Publication Date (Web): 16 Mar 2018
Downloaded from http://pubs.acs.org on March 17, 2018
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A fragmentꢀbased approach to targeting inosineꢀ5´ꢀ
monophosphate dehydrogenase (IMPDH) from
Mycobacterium tuberculosis
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a
b
c#
a
a
Ana Trapero, Angela Pacitto, Vinayak Singh, Mohamad Sabbah, Anthony G. Coyne,
c
*,b
*,b,d
*,a
Valerie Mizrahi, Tom L. Blundell, David B. Ascher,
Chris Abell
a
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW,
United Kingdom.
b
Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge
CB2 1GA, United Kingdom.
c
MRC/NHLS/UCT Molecular Mycobacteriology Research Unit & DST/NRF Centre of
Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine
and Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town,
Rondebosch 7701, Cape Town, South Africa
d
Department of Biochemistry and Molecular Biology, Bio21 Institute, University of Melbourne,
30 Flemington Road, Parkville, Victoria 3052, Australia.
#
Present address: H3D Drug Discovery and Development Centre, Department of Chemistry and
Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch
7701, Cape Town, South Africa
ABSTRACT
Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are
needed to combat emergence of drug resistance. Inosine 5´ꢀmonophosphate dehydrogenase
(IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an
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attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors
using fragmentꢀbased screening and structureꢀbased design techniques. Screening of a fragment
library for Mycobacterium thermoresistible (Mth) IMPDH (ꢁCBS) inhibitors identified a low
affinity phenylimidazole derivative. Xꢀray crystallography of the Mth IMPDH ꢁCBS
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IMP−inhibitor complex revealed that two molecules of the fragment were bound in the NAD
binding pocket of IMPDH. The linking the two molecules of the fragment afforded compounds
with more than 1000ꢀfold improvement in IMPDH affinity over the initial fragment hit.
KEYWORDS: IMPDH, Mycobacterium tuberculosis, fragmentꢀbased drug discovery, 2ꢀ
acylaminoimidazole derivatives, Xꢀray crystallography.
INTRODUCTION
Tuberculosis (TB) is an contagious infectious disease caused by Mycobacterium tuberculosis
(
Mtb), which can be transmitted through the air as droplets. The infection predominantly affects
the lungs, but can spread to other parts of the body, especially in patients with a suppressed
immune system.
The World Health Organization (WHO) has estimated that nearly oneꢀthird of the world’s
1
population is infected with Mtb, leading to 1.8 million TB deaths in 2015. Although there has
been a slow decline in new TB cases and TBꢀrelated deaths in recent years, the emergence and
spread of multidrug resistant (MDR) and extensively drug resistant (XDR) strains of Mtb has
increased the threat that this disease poses for global public health. According to the WHO
approximately 480,000 cases of MDRꢀTB emerged in 2015, and the cure rate of those patients
1
was only 50%.
Current TB treatments require combinations of the four of the firstꢀline drugs isoniazid,
rifampicin, ethambutol, pyrazinamide and streptomycin, which must be taken for six months or
2
longer. Resistant strains are not susceptible to the standard drugs, and although MDRꢀTB is
3
treatable using secondꢀline drugs, such treatments have a number severe side effects.
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Consequently, there is an urgent need for the development of novel and more effective drugs for
the treatment of drug resistant TB.
Inosineꢀ5´ꢀmonophosphate dehydrogenase (IMPDH, E.C. 1.1.1.205) has received considerable
4
5, 6
interest in recent years as an important target enzyme for immunosuppressive, anticancer,
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and antiviral drugs. Most recently, IMPDH has emerged as a promising antimicrobial drug
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ꢀ11
target.
IMPDH catalyzes the first unique step in the de novo synthesis of guanine nucleotides, the
oxidation of inosine 5´ꢀmonophosphate (IMP) to xanthosine 5´ꢀmonophosphate (XMP) with the
+
concomitant reduction of the cofactor nicotinamide adenine dinucleotide (NAD ) to NADH
12
(
Figure 1). XMP is then subsequently converted to guanosine 5´ꢀmonophosphate (GMP) by a
1
3
GMP synthetase.
IMPDH has been deemed essential in every pathogen analyzed to date, including Mtb,
Staphylococcus aureus, and Pseudomonas aeruginosa, which are three of the most serious
1
4ꢀ16
17
bacterial threats.
However this has been somewhat contradictory, in comparing cell versus
animal work. IMPDH is an ubiquitous enzyme present in several eukaryotes, bacteria and
18
protozoa. The IMPDH reaction involves two chemical transformations. The first step of the
IMPDH catalysed reaction involves the attack of catalytic Cys on substrate IMP followed by
+
hydride transfer to NAD , forming the covalent enzyme intermediate EꢀXMP*. In the second
11
step, EꢀXMP* is hydrolysed to XMP. The enzyme exists in two different conformations, an
open form that accommodates both the substrate and cofactor during the first step, and a closed
+
19
form where the active site flap moves into the NAD ꢀbinding site for the EꢀXMP* hydrolysis.
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Figure 1. Purine nucleotide biosynthesis. The commonly occurring guanine nucleotide
biosynthetic and salvage reactions are shown, as is the adenine nucleotide biosynthetic pathway.
The IMPDH reaction is boxed. NK, nucleoside kinase; HPRT, hypoxanthine phosphoribosyl
transferase; XPRT, xanthine phosphoribosyl transferase; GPRT, guanine phosphoribosyl
transferase; GMPS, guanosine 5′ꢀmonophosphate synthetase; GMPR, guanosine 5′ꢀ
monophosphate reductase; ADSS, adenylosuccinate synthetase; ADSL, adenylosuccinate lyase.
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In recent years, there has been considerable effort aimed at identifying small molecule inhibitors
of IMPDH as potential antitubercular agents. Xꢀray crystal structures of a truncated form of the
20ꢀ26
Mtb enzyme in complex with some of these compounds have been reported.
In antibacterial drug discovery, and especially in TB drug discovery, highꢀthroughput screening
HTS) typically identifies a number of leads that show high potency in vitro, but most did not
(
show any translation to an in vivo effect. It is also inevitable that the HTS libraries represent
only a small fraction of possible chemical space and so limit confidence in finding a good
starting point for subsequent development. Phenotypic screens can potentially lead to the
identification of a molecule that modifies a disease phenotype by acting on a previously
27
undescribed target or by acting simultaneously on more than one target. However, for many of
these hits the relevant target or targets has not yet been identified, thus preventing further targetꢀ
28, 29
based optimization of the compounds.
The previously reported IMPDH inhibitors, such as compounds 7759844 (1), MAD1, P41,
VCC234718 and DDD00079282 (Figure 2), were identified by phenotypic screening or target
21, 23ꢀ25
based HTS of compound libraries.
We have sought to develop IMPDH inhibitors using a fragmentꢀbased approach. Fragmentꢀ
based drug discovery (FBDD) is now established in both industry and academia as an
alternative approach to highꢀthroughput screening for the generation of hits or chemical tools
30
for drug targets.
We have previously reported the discovery of several series of novel and potent inhibitors using
FBDD to target Mtb. Previously we have reported the fragment elaboration strategies that we
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have applied which have included fragment growing, merging and linking. Although, fragment
linking is conceptually the most appealing strategy for fragment elaboration, in practice, this
3
1, 32
strategy can be challenging where the choice of the optimal fragment linker can be crucial.
In the fragmentꢀbased approach, biophysical techniques are usually used to identify small
chemical compounds (fragments) that bind with low affinity to the drug target. Xꢀray
crystallography is then usually employed to establish the binding mode of the fragment and to
facilitate the design of elaborated fragments. The availability of highꢀresolution Xꢀray crystal
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structures of a truncated form of the IMPDH,
in both the substrateꢀfree and
substrate/ligandꢀbound forms, make this enzyme attractive to a fragmentꢀbased approach.
In this work, the discovery of a new class of potent nM inhibitors of IMPDH using a FBDD
approach is reported. A library of 960 fragments was screened against Mth IMPDH ꢁCBS using
a biochemical assay. The fragment hits from this assay were examined using Xꢀray
crystallography and an Xꢀray crystal structure of one of the fragment complexes was solved to a
resolution of 1.45 Å. Examination of the Xꢀray crystal structure suggested a strategy of
fragmentꢀlinking for optimization of this fragment hit.
i
Figure 2. Structures of previously reported IMPDH inhibitors (αK values against IMP). All
compounds showed uncompetitive inhibition with respect to IMP.
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RESULTS AND DISCUSSION
Fragment Screening.
An inꢀhouse fragment library composed of 960 fragments was screened using a biochemical
assay against Mth IMPDH ꢁCBS. Mth IMPDH, which shares 85% sequence identity with Mtb
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IMPDH and is 100% identical in the active site,
was chosen for the fragment screening and
structural studies because it gave higher protein expression yields and better diffracting crystals
than the Mtb orthologue. IMPDH activity was monitored spectrophotometrically by measuring
the formation of NADH at 340 nm. The biochemical assay was performed at a fragment
concentration of 1 mM and hits were reꢀtested in triplicate. Compound 1 (7759844) previously
23
reported as IMPDH inhibitor was used as a positive control in assays (Table 1).
The screen resulted in 18 hits (1.9% hit rate), where a hit was defined as a compound that gave
greater than 50% inhibition at a concentration of 1 mM. A complete list of fragments hits
identified is included in Table 1. A number of common scaffolds were observed, in particular
phenylimidazole (fragments 2−4), aminopyrazole (fragments 5−6) and naphthol (fragments
7
−11), and the remaining compounds contained a substituted phenyl or a heterocyclic five
membered ring (fragments 12−19).
Table 1. Structures and activities for compound 1 (7759844) and the most potent fragment
hits found in the screen against Mth IMPDH ꢀCBS
%
Inhibition
% Inhibition
at 1 mM
Compd
Structure
Compd
Structure
at 1 mM
a
1
92 ± 1
11
77 ± 1
64 ± 6
2
3
65 ± 2
52 ± 4
12
13
72 ± 1
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59 ± 1
53 ± 3
14
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86 ± 6
51 ± 1
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50 ± 3
16
88 ± 10
7
8
68 ± 3
57 ± 1
17
18
53 ± 2
79 ± 4
9
98 ± 2
66 ± 1
19
99 ± 1
10
a
% Inhibition at 10 µM.
The IC50 values of six of the most active fragments were measured and the IC50 and ligand
efficiency (LE) values of these fragments are summarized in Table 2. The fragment screen
provided an array of hits with IC50 ranging from 325 ꢂM to 674 ꢂM and ligand efficiencies from
+
0
.31 to 0.42. The inhibition constant [K
i
] with respect to both substrates IMP and NAD were
determined by assaying various concentrations of each inhibitor with five different
concentrations of substrate and a fixed saturating concentration of the coꢀsubstrate. The
inhibition data for these fragments are summarized in Table 2. All compounds yielded an
+
uncompetitive inhibition pattern with respect to NAD with K
i
values ranging from 262 to 525
µM. Fragments 14, 16 and 19 yielded a mixed inhibition with respect to IMP with K values
i
ranging from 126 to 398 µM and compounds 1, 2, 11 and 18 yielded an uncompetitive
inhibition with K
i
values ranging from 361 to 609 µM.
Inhibition constants of compound 2 towards fullꢀlength Mtb IMPDH were also determined.
i
Compound 2 inhibited fullꢀlength Mtb IMPDH enzyme with a K value of 572 ± 14 µM with
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IMP as the substrate and a K
similar to the K
i
value of 534 ± 18 µM with NAD as the substrate, which are
i
values observed for the Mth IMPDH ꢁCBS enzyme.
i
Table 2. IC50, Ligand Efficiency, and alpha K values of fragment hits and compound 1
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(
7759844) against Mth IMPDH ꢀCBS
IC50 (µM)
Compd
a
IMP αK
i
(µM)
i
NAD αK (µM)
(
LE)
0.77 ± 0.06
b
1
2
0.86 ± 0.03 (UC) 0.55 ± 0.02 (UC)
(
0.40)
6
74 ± 53
0.36)
609 ±3 (UC)
361 ± 53 (UC)
238 ± 6 (Mixed)
398 ± 81 (Mixed)
554 ± 70 (UC)
126 ± 34 (Mixed)
512 ± 23 (UC)
310 ± 30 (UC)
262 ± 39 (UC)
525 ± 64 (UC)
452 ± 26 (UC)
355 ± 47 (UC)
(
3
36 ± 6
0.39)
11
(
4
00 ± 7
0.42)
14
16
(
4
33 ± 60
0.31)
(
5
12 ± 37
0.37)
1
8
9
(
3
25 ± 9
0.37)
1
(
a
Ligand efficiency was calculated using the equation LE = (1.37 × pIC )/HA, where HA means
50
b
heavy atom, i.e., a nonꢀhydrogen atom. UC: Uncompetitive inhibition.
Xꢁray Structure of compounds 1 (7759844) and 2
Compound 1 and the six fragment hits (2, 11, 14, 16, 18 and 19) were selected for structural
characterization using Xꢀray crystallography by soaking into preformed crystals of Mth IMPDH
20
ꢁ
CBS as previously described. After molecular replacement, clear electron density was
observed in the 2F – F difference map (σ = 3.0) for IMP, and in addition density was observed
0
c
for one molecule of compound 1 (Figure 3A) and two molecules of compound 2 (Figure 3B),
+
which were partially occupying the NAD binding site. None of the other fragments (11,14, 16,
1
8 and 19) showed any electron density in the Xꢀray crystal structures. Although the kinetic
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studies of compounds 1 and 2 suggested that these two compounds are uncompetitive with
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respect to NAD , the binding mode of compounds 1 and 2 closely resemble that of other
2
1, 24, 25
previously reported uncompetitive inhibitors of Mtb IMPDH.
It has been proposed that
+
the uncompetitive mode of inhibition of IMPDH inhibitors with respect to NAD is consistent
1
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with their binding preferentially to the covalent IMPDHꢀXMP* intermediate after NADH has
2
4, 25, 33
been released.
The structure of Mth IMPDH ꢁCBS with compound 1 showed that the inhibitor binds in the
24, 25
NAD pocket in a near identical manner to our recently described IMPDH inhibitors.
Compound 1 formed strong π interactions with the hypoxanthine group of IMP, P45′, Y471′,
and polar interactions with G409, E442, P45′ and G470′. The electron density revealed two
molecules of compound 2 within the NAD binding pocket of IMPDH. One molecule of
compound 2 stacked with IMP, forming extensive π interactions with the hypoxanthine group of
IMP. This fragment was further stabilised through polar interactions, hydrogen bonds and π
interactions to surrounding residues in the active site pocket, including A269, G318 and E442.
The other molecule of compound 2 sits closer to the opening of the active site, making polar
interactions with N273 and E442, and π interactions with H270 and Y471′.
A comparison of the structure of Mth IMPDH ꢁCBS with compound 1 with the fragment 2
structure shows that the two molecules of 2 mimic the position of the larger inhibitor 1 (Figure
3C.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A
B
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
Figure 3. Xꢀray crystal structure of compounds 1 and 2 bound to IMPDH. Ligand interactions
are represented as dotted lines; hydrogen bonds are represented in red, polar interactions in
orange, ionic interactions in yellow and aromatic and π interactions in green dotted lines.
Protein–ligand interactions were analyzed using Arpeggio. (a) Interactions made by 1 (green) in
the Xꢀray crystal structure of the complex of IMPDH with IMP (orange). (b) Interactions made
by 2 (yellow) in the Xꢀray crystal structure of the complex of IMPDH with IMP. (c) Structural
alignment of the IMPDH crystal structures of 1 (green) and 2 (yellow).
Fragment elaboration
Fragment 2 was selected as the starting point for exploration because of the ease of synthetic
modification and the availability of a Xꢀray crystal structure to guide the optimization. For
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chemical elaboration of 2, fragment linking as well as fragment growing were considered. As
the two molecules of fragment 2 are found to bind in adjacent regions of the target protein, the
fragmentꢀlinking approach was the more attractive option. However, before fragment linking,
fragment 2 was further optimised with the aim of improving the binding affinity. The structures
and inhibitory activities of these compounds against Mth IMPDH ꢁCBS are summarized in
Tables 3 and 4. The corresponding data for fragment 2 have also been included for comparative
purposes.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All compounds were evaluated at a concentration of 100 ꢀM with Mth IMPDH ꢁCBS.
Fragment growing.
The fragmentꢀgrowing strategy involves using structureꢀbased drug design to form additional
interactions by growing out from the starting fragment. Fragment 2 was modified at the 2ꢀ
position of the imidazole ring to explore the introduction of various aromatic rings linked by a
thioacetamide (20−22) to form π interactions with the hypoxanthine group of IMP (Figure S1).
Such modifications gave compounds with improved Mth IMPDH ꢁCBS inhibition. The phenyl
and benzofuran derivatives (20 and 21), showed 13 and 31% inhibition, respectively. Mth
IMPDH ꢁCBS inhibition was shown to be sensitive to minor modifications of the phenyl
substituent groups e.g. the 4ꢀiodo substituted 22 showed 30% inhibition at 100 µM, whereas the
nonꢀsubstituted compound, 20, showed 13% inhibition at the same concentration. The effect of
the removal of the 4ꢀbromo group was investigated and compounds 23−25 were synthesized.
Removal of the bromo substituent in compounds 20−22 (13−31% inhibition at 100 µM) was
tolerated (23−25, 3−46% inhibition at 100 µM). The importance of the aromatic amide linked
by a thioacetamide was subsequently examined. Replacing the phenyl with isopropyl (26)
resulted in complete loss of activity. Substitution of the thioacetamide by a thioacetic acid also
led to a complete loss in activity (compounds 27 and 28).
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. Structures and activities for fragment 2 and compounds 20−28 against Mth
IMPDH ꢀCBS
%
Inhibition
Compd
Structure
at 100 µM
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
< 5%
2
2
0
13 ± 3
31 ± 6
21
2
2
3
3
0 ± 3
2
< 5%
24
36 ± 1
2
5
6
4
6 ± 3
2
<
5%
5%
5%
27
<
28
<
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Fragment linking and SAR.
Examination of the Xꢀray crystal structure of the previously reported inhibitor 1 when overlaid
with fragment 2 revealed that the distance between the 4ꢀposition of the phenyl ring of fragment
2
and the 2ꢀposition of the imidazole ring represents the closest approach of the molecules
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Figure S1). On the basis of this structural information, three different linkers were designed to
connect the two copies of the fragment 2 at these positions (compounds 29−31). Initially, a
thioacetamide and urea linker moieties were examined. Compounds 29 and 30 showed 20%
and 56% of Mth IMPDH ꢁCBS inhibition respectively, at 100 µM (Table 4). Interestingly,
compound 30 showed a 12ꢀfold improvement in Mth IMPDH ꢁCBS inhibitory activity with an
IC of 58 µM, compared to the fragment 2.
5
0
The lactate linker was then used but all attempts to link 4ꢀphenylimidazole with 4ꢀ(4ꢀ
bromophenyl)ꢀ1Hꢀimidazole were unsuccessful. We therefore decided to synthesize compound
3
1, which contains 1ꢀmethylꢀ4ꢀphenylꢀ1Hꢀimidazole and 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazole
linked with a lactate analogue, as in compound 1. Compound 31 (Table 4) showed markedly
improved Mth IMPDH ꢁCBS inhibition with a LE of 0.29 and IC50 of 0.52 µM, which is 1300ꢀ
fold more potent compared to the fragment 2. Although compound 31 binds in the cofactor site,
the mechanism of inhibition can vary depending on its relative affinities for the E•IMP and Eꢀ
8
, 11
XMP* complexes.
Kinetic evaluation of compound 31 showed the mode of Mth IMPDH
+
ꢁ
CBS inhibition was uncompetitive with respect to both IMP and the NAD cofactor (see
Figure S2, Supporting Information) with a K
and a K value of 0.20 ± 0.01 with NAD as the substrate.
Inhibition constants of compound 31 towards fullꢀlength Mtb IMPDH were also determined.
Compound 31 inhibited fullꢀlength Mtb IMPDH enzyme with a K value of 0.61 ± 0.05 µM with
i
value of 0.30 ± 0.02 µM with IMP as the substrate
i
i
IMP as the substrate and a K value of 0.39 ± 0.02 µM with NAD as the substrate. The inhibition
i
constants were consistent with the data using the Mth IMPDH ꢁCBS enzyme.
Removal of the bromo substituent in compound 29 to give compound 32 was well tolerated
(
Table 4). The importance of the imidazole group for the inhibitory activity against Mth IMPDH
ꢁ
CBS was confirmed by replacing of the 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazole substituent of
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
compound 31 with 4ꢀ(4ꢀbromophenyl)oxazole (33) which resulted in a 4ꢀfold loss of activity
(
Table 4). Replacing the 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazole of 31 by a phenyl (34) resulted in
complete loss of activity (Table 4). However, the 4ꢀiodophenyl derivative 35 demonstrated
slightly improved activity (IC50 = 0.47 µM, LE = 0.34) compared to compounds 31 (IC50 = 0.52
µM, LE = 0.29) and 1 (IC50 = 0.77 µM, LE = 0.40). It is noteworthy that LE of compound 35
was comparable to that of the original fragment hit 2 (LE=0.36) and other reported IMPDH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
34
inhibitors.
Table 4. Structures and activities for fragment 2 and compounds 29−35 against Mth
IMPDH ꢀCBS
%
Inhibition
IC50 (µM)
(LE)
Compd
Structure
a
at 100 µM
2
< 5%
674 ± 53
(
0.36)
2
9
0
b
2
0 ± 5
ND
3
56 ± 11
58 ± 8
0.21)
(
c
9
9 ± 11
0.52 ± 0.004
(0.29)
31
32
24 ± 1
ND
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c
3
3
83 ± 2
2.21 ± 0.08
(
0.26)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
5
c
4
± 1
ND
c
3
97 ± 1
0.47 ± 0.03
0.34)
(
a
Ligand efficiency was calculated using the equation LE = (1.37 × pIC )/HA, where HA means
heavy atom, i.e., a nonꢀhydrogen atom. ND: not determined. IC50 values were determined for
50
b
c
compounds that showed >50% inhibition. %Inhibition at 10 µM.
The importance of the 4ꢀbromo substituent on phenyl ring in compound 31 was also explored
(
compounds 36−39, Table 5). Removal of the bromo substituent (36) resulted in a 5ꢀfold loss in
activity. Whereas replacing this group with an iodine (39) or a morpholine ring (37) resulted in
less than 1.3ꢀfold loss in activity. The electronic nature of the substituent in this position had
little effect on inhibitory activity. For example, an electronꢀdonating methoxy (38) retained
activity comparable to the bromine derivative (31). Among them, imidazoles 31, 37−38, and 39
were found to be potent inhibitors of Mth IMPDH ꢁCBS with IC50 values ranging from 520 to
6
90 nM.
The (S)ꢀisomer of 31 was found to bind preferentially (Table 5), the racemate 31 having
approximately half the Mth IMPDH ꢁCBS inhibition of the (S)ꢀisomer 31. This observation
21, 35
accords with the results previously reported for other series of IMPDH inhibitors.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5. Structures and activities for compounds 31, 36−39, and (S)ꢁ31 against Mth
IMPDH ꢀCBS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
%
Inhibition
IC50 (µM)
(LE)
Compd
R
1
a
at 10 µM
99 ± 1
77 ± 3
94 ± 1
95 ± 1
96 ± 1
99 ± 1
31
36
37
Br
H
0.52 ± 0.004
(
0.29)
2.76 ± 0.21
0.26)
(
0.52 ± 0.01
(0.24)
38
39
OMe
I
0.64 ± 0.03
(0.27)
0.69 ± 0.06
(0.28)
(S)ꢁ31
Br
0.27 ± 0.02
(0.30)
a
Ligand efficiency was calculated using the equation LE = (1.37 × pIC50)/HA, where HA means
heavy atom, i.e., a nonꢀhydrogen atom.
Xꢁray structure of compound 31
From crystals soaked with compound 31, the 2F – F difference map (σ = 3.0) revealed strong
0
c
density for the inhibitor. The structure of compound 31 (Figure 4A) showed that it bound in a
nearly identical manner to compounds 1 and 2 in the NAD binding pocking (Figure 4B),
stacking with IMP, and maintaining the interactions with H270, N273, E442, and P45 and Y471
from the neighbouring subunit. Compound 31 made additional interactions in the binding
pocket, including polar interactions with D267 and N297.
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A
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
B
Figure 4. Xꢀray crystal structure of compound 31 bound to IMPDH. Protein–ligand interactions
were analyzed using Arpeggio. (a) Interactions made by 31 in the Xꢀray crystal structure of the
complex of IMPDH with IMP (orange). Ligand interactions are represented as dotted lines;
hydrogen bonds are represented in red, polar interactions in orange, ionic interactions in yellow
and aromatic and π interactions in green dotted lines. (b) Structural alignment of the IMPDH
crystal structures of 1 (lilac), 2 (yellow) and 31 (green).
Wholeꢁcell activity against Mtb.
The wholeꢀcell activity of the most potent analogues 31, 33−39, and (S)ꢀ31 in vitro was
determined against Mtb H37Rv (see Table S1, Supporting Information). No significant
inhibition of bacterial growth was detected for any of the compounds (MIC90 ≥ 50 ꢀM) over the
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
tested concentration range (0−100 ꢀM). There are currently onꢀgoing efforts to explain the lack
of efficacy of the potent Mth IMPDH ꢁCBS inhibitors described which could be caused by low
membrane permeability, poor metabolic stability and/or drugꢀefflux mechanisms.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthetic Chemistry.
Compounds 20−29 and 32 were synthesized from 4ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol or 4ꢀ(4ꢀ
bromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol according to the sequence described in Scheme 1.
Thioacetic derivatives 27 and 28 were prepared by treatment of 4ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol
or 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol with 2ꢀchloroacetic acid in the presence of NaOH
followed by neutralization with hydrochloric acid.
Thioacetamide 26 was synthesized by amide coupling between thioacetic derivative 28 and
isopropylamine. Similarly, thioacetamides 20−25, 29 and 32 were prepared by crossꢀlinking 4ꢀ
phenylꢀ1Hꢀimidazoleꢀ2ꢀthiol
or
4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol
with
αꢀ
chloroacetamides 40−43, which were obtained by acylation of anilines with various substituents
with chloroacetyl chloride.
The synthesis of urea 30 was achieved as shown in Scheme 2 by coupling amine 44 and Nꢀ(4ꢀ
(
1Hꢀimidazolꢀ4ꢀyl)phenyl)ꢀ1Hꢀimidazoleꢀ1ꢀcarboxamide,
which
was
obtained
from
commercially available 4ꢀ(1Hꢀimidazolꢀ4ꢀyl)aniline as a crude intermediated, in the presence of
N,Nꢀdiisopropylethylamine. Amine 44 was synthesized from benzyl ((5ꢀ(4ꢀbromophenyl)ꢀ1Hꢀ
3
6
imidazolꢀ2ꢀyl)methyl)carbamate, followed by the deprotection of the benzyloxycarbonyl
group under acidic condition.
Imidazole derivatives 31, 36−38, and 39 were prepared following the synthetic procedure
outlined in Scheme 3. 2ꢀAminoimidazoles 50−54 were synthesized according to a published
3
7
microwaveꢀassisted protocol. In brief, 2ꢀaminoimidazoles 50−54 were prepared by reaction of
the commercially available αꢀhaloketones and Nꢀacetylguanidine, followed by deacetylation
(
Scheme 3). Acid 59 was synthesized starting with imidazole 55, which was prepared by
3
8
reaction of 2ꢀbromoꢀ4'ꢀhydroxyacetophenone with formamide as reported previously. The
phenol 57 was synthesized by alkylation of imidazole 55, followed by deprotection of the
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methyl ether with BBr
3
. Substituted phenol 57 was converted to the ether 58 upon treatment
2 3
with methyl 2ꢀbromopropionate in the presence of Cs CO . Enantiomerically pure phenyl ethers
were synthesized by using Mitsunobu reaction conditions with ethyl Dꢀlactate (Scheme 4). After
the hydrolysis of the ester group, the resulting carboxylic acid 59 was treated with thionyl
chloride to give the acid chloride 60, which was reacted with 2ꢀaminoimidazoles 50−54 to
afford imidazole derivatives 31, 36−38, and 39.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The synthesis of 2ꢀacylaminooxazole 33 and amides 34−35 were achieved as shown in Scheme
5
. 2ꢀacylaminooxazole derivative 33 was obtained by coupling the acid chloride derivative 60
with 2ꢀaminooxazole derivative 61, which was prepared by reaction of 2,4′ꢀ
dibromoacetophenone with urea. Similarly, amides 34−35 were prepared by coupling the
corresponding anilines with the acid chloride derivative 60.
a
Scheme 1. Synthesis of thioacetamide and thioacetic derivatives 20−29 and 32
a
Reagent and conditions: (a) 2ꢀchloroacetic acid, NaOH, EtOH, 80 ºC, 69−72%; (b)
isopropylamine, HATU, DIPEA, EtOAc, rt, 73%; (c) chloroacetyl chloride, TEA, DCM, rt, 89
−
99%; (d) NaOH, MeOH, H
2
O, 70 ºC, 69−96%.
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 2. Synthesis of urea 30
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagent and conditions: (a) CDI, THF, rt, quant.; (b) HCl (4M), dioxane, 100 ºC, quant.; (c)
DIPEA, DMF, rt, 26%.
a
Scheme 3. Synthesis of 2ꢁacylaminoimidazole derivatives 31, 36−38, and 39
a
Reagent and conditions: (a) acetylguanidine
SO , MeOH:H O (1:1 v/v), ꢀW, 100 ºC; 2. aq. Na
9%; (d) CH I, Cs CO , DMF, rt, 88%; (e) BBr (1M in DCM), ꢀ78ºC−rt, DCM, 96%; (f)
Cs CO , methyl 2ꢀbromopropionate, DMF, 60 ºC, 92%; (g) NaOH, THF:H O (2:1 v/v), 80 ºC,
5%; (h) SOCl
,
CH
3
CN, ꢀW, 100 ºC, 71−88%; (b) 1. 20%
CO , 79−95%; (c) formamide, 150 ºC,
H
2
4
2
2
3
9
3
2
3
3
2
3
2
6
2
, 80 ºC, quant.; (i) TEA, DCM, 40 ºC, 61−72%.
a
Scheme 4. Synthesis of the Sꢁenantiomer of 31
a
3 2
Reagent and conditions: (a) 1. Ethyl Dꢀlactate, PPh , DEAD, THF, rt; 2. NaOH, THF:H O (2:1
v/v), rt, 56%; (b) SOCl , 80 ºC, quant.; (c) 50, TEA, DCM, 40 ºC, 69%.
2
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a
Scheme 5. Synthesis of 2ꢁacylaminooxazole derivative 33 and amides 34−35
1
1
1
1
1
1
1
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1
2
2
2
2
2
2
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0
a
Reagent and conditions: (a) urea, CH CN, 80 ºC, 78%; (b) TEA, DCM, rt (for 34 and 35) or 40
3
ºC (for 33), 64−76%.
CONCLUSIONS
FBDD has emerged as a robust approach to identify small molecules that bind to a wide range
of therapeutic targets. Fragment elaboration strategies have resulted in the development of a
number of compounds that have progressed into clinical trials. Within the area of TB drug
discovery, a number of HTS and phenotypic screens have been performed during the past
decade. Although HTS identified a number of leads that show high potency in vitro, the
translation to an in vivo effect has proven challenging.
This study illustrates the successful application of a fragmentꢀbased approach followed by
fragment optimization to obtain nanomolar affinity ligands of IMPDH. A library of 960
fragments were screened against Mth IMPDH ꢁCBS, and from the screen the phenylimidazole
fragment hit 2 (IC50 = 674 µM) was identified. Kinetic experiments showed that 2 was an
+
uncompetitive inhibitor of Mth IMPDH ꢁCBS with respect to NAD and IMP. Two molecules
of the fragment 2 were shown to bind at the NAD binding site of the enzyme. The Xꢀray crystal
structure also revealed that one molecule of fragment 2 makes
π interactions with IMP and the
other molecule sits closer to the opening of the active site, making polar interactions with N273
and E442, and π interactions with H270 and Y471′. This provides potential for further
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optimization of fragment 2. To explore better the possibilities given by fragment 2, fragmentꢀ
linking and fragmentꢀgrowing strategies were employed, resulting in low micromolar to
nanomolar affinity compounds. Among them, compounds 31, 35, 37− 39 were the most potent
IMDPH inhibitors of the series described in this work with IC50 values between 0.47 and 0.69
ꢂM, which represent >1000ꢀfold improvement in Mth IMPDH ꢁCBS potency over the initial
fragment hit. Compound 31 was shown to bind at the NAD binding site of the enzyme and the
0
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Xꢀray crystal structure also revealed that it makes
π interactions with IMP, and maintaining the
interactions with H270, N273, E442, and P45 and Y471 from the neighbouring subunit.
Moreover, compound 31 made additional interactions in the binding pocket, including polar
interactions with D267 and N297. A comparison of this structure with the fragment 2 structure
shows that the two molecules of 2 mimic the position of the larger inhibitor 31. This is the first
example of utilizing the fragmentꢀbased approach specifically to identify new potent inhibitors
of IMPDH. Further structural optimization to improve the cellular activity of these analogues is
ongoing with the aim of developing novel classes of antiꢀTB agents.
EXPERIMENTAL SECTION
1
1
. Chemistry
.1 General Experimental Methods. Solvents were distilled prior to use and dried by standard
1
13
methods. Unless otherwise stated, H and C NMR spectra were obtained in CDCl
DMSOꢀd solutions using either a Bruker 400 MHz AVANCE III HD Smart Probe, 400 MHz
QNP cryoprobe, or 500 MHz DCH cryoprobe spectrometer. Chemical shifts (δ) are given in
3
, MeOD, or
6
1
13
ppm relative to the residual solvent peak (CDCl
3
: H, δ = 7.26 ppm; C, δ = 77.16 ppm), and
the coupling constants (J) are reported in hertz (Hz). Optical rotations were measured on a
−1
PerkinꢀElmer Polarimeter 343 at 589 nm (Na Dꢀline), and specific rotations are reported in 10
2
−1
deg cm g . Microwave reactions were performed using a Biotage Initiator system under
reaction conditions as indicated for each individual reaction.
Reactions were monitored by TLC and LCMS to determine consumption of starting materials.
Flash column chromatography was performed using an Isolera Spektra One/Four purification
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system and the appropriately sized Biotage SNAP column containing KPꢀsilica gel (50 ꢂm).
Solvents are reported as volume/volume eluent mixture where applicable.
High resolution mass spectra (HRMS) were recorded using a Waters LCT Premier Time of
Flight (TOF) mass spectrometer or a Micromass QuadrapoleꢀTime of Flight (QꢀTOF)
spectrometer.
1
1
1
1
1
1
1
1
1
1
2
2
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0
Liquid chromatography mass spectrometry (LCMS) was carried out using an Ultra Performance
Liquid Chromatographic system (UPLC) Waters Acquity Hꢀclass coupled to a Waters SQ Mass
Spectrometer detector. Samples were detected using a Waters Acquity TUV detector at 2
wavelengths (254 and 280 nm). Samples were run using an Acquity UPLC HSS column and a
flow rate of 0.8 mL/min. The eluent consisted of 0.1% formic acid in water (A) and acetonitrile
(
B); gradient, from 95% A to 5% A over a period of 4 or 7 min.
All final compounds had a purity greater than 95% by LCMS analysis.
General Method A: Synthesis of thioacetamides 20–25, 29, and 32.
To a solution of the 2ꢀchloroacetamide derivative (0.17 mmol) in MeOH (8 mL), 4ꢀ(4ꢀ
bromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol or 4ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol (0.17 mmol) was added,
followed by a solution of NaOH (0.67 mmol) in H
at 70 ºC for 3h. After cooling to rt, the solvents were removed in vacuo and the resulting residue
was taken up in 30 mL of EtOAc and washed with H O (15 mL). The aqueous layer was
extracted with EtOAc (3 × 30 mL) and the combined organic layers were washed with brine and
2
O (2.5 mL). The reaction mixture was stirred
2
4
dried over MgSO . Filtration and evaporation afforded crude products, which were purified as
indicated below.
General Method B: Synthesis of thioacetic derivatives 27 and 28.
A solution of 4ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol or 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol (2.80
mmol) and NaOH (5.0 mmol) in EtOH (5 mL) was reflux for 1 h. After cooling to rt, a solution
of 2ꢀchloroacetic acid (2.80 mmol) in EtOH (2 mL) was added. The reaction was stirred at
reflux for an additional 3 h, and then cooled to 0 ºC. The reaction mixture was diluted with cold
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water (5 mL) and acidified with 1M HCl. The precipitated product was collected by filtration
and washed with DCM (2 x 2 mL).
General Method C: Synthesis of compounds 31, 33, and 36–39.
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2
A mixture of acid 59 (0.20 mmol) and SOCl (2 ml) were heated at 80 ºC for 2 h. The solvent
was removed under reduced pressure to give the acid chloride 60 as a white solid, which was
immediately dissolved in anhydrous DCM and it was added slowly dropwise at 0 ºC to a
solution of the corresponding substituted 2ꢀAminoimidazoles (0.20 mmol) and TEA (0.80
mmol) in anhydrous DCM (5 mL). The reaction mixture was stirred at 40 ºC for 36 h, and then
diluted with DCM (20 mL) and washed with saturated aqueous NaHCO
layer was then extracted with DCM (2 x 20 mL), and the combined organic layers were dried
over MgSO , filtered and the solvent was removed under reduced pressure to afford a yellow
3
. The aqueous phase
4
oil, which was purified by flash chromatography eluting with the solvent system specified.
General Method D: Synthesis of amides 34–35.
2
A mixture of acid 59 (0.20 mmol) and SOCl (2 mL) were heated at 80 ºC for 2 h. The solvent
was removed under reduced pressure to give 60 as a white solid, which was immediately
dissolved in anhydrous DCM and it was added slowly dropwise at 0 ºC to a solution of the
corresponding aniline (0.20 mmol) and triethylamine (0.80 mmol) in anhydrous DCM (5 mL).
The reaction mixture was stirred at rt for 4 h, and then diluted with DCM (20 mL) and washed
with saturated aqueous NaHCO
3
. The aqueous phase layer was then extracted with DCM (2 x
, filtered ant the solvent was
2
0 mL), and the combined organic layers were dried over MgSO
4
removed under reduced pressure to afford a yellow oil, which was purified by flash
chromatography eluting with the solvent system specified.
General Method E: Synthesis of αꢁchloroacetamides 40–43.
3
Et N (4.77 mmol) followed by a solution of chloroacetyl chloride (4.77 mmol) in DCM (3 mL)
were added to a stirred solution of the corresponding aniline (4.38 mmol) in DCM (5 mL) at rt.
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The reaction mixture was stirred at rt for 2 – 4 h. The reaction was then diluted with DCM (20
3
mL) and washed with saturated aqueous NaHCO , 1M HCl and brine, dried over anhydrous
MgSO , and the solvent was removed under reduced pressure. Compound 43 was purified by
4
flash chromatography eluting with the solvent system specified, although other analogues were
used in subsequent reactions without further purification.
1
1
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2
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2
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General Method F: Synthesis of Substituted Nꢁ(1Hꢁimidazolꢁ2ꢁyl)acetamides 45–49.
A mixture of the corresponding 2ꢀbromoacetophenone derivative (0.38 mmol) and
acetylguanidine (1.13 mmol) in anhydrous acetonitrile (3 mL) was heated at 100 ºC using
microwave irradiation for 15 min. The solvent was removed, and the residue was taken in H
2
O
(
3 mL), filtered and the solid was washed with H O (2 mL x 2) and DCM (2 mL). The solid
2
obtained was used in the next step without further purification.
General Method G: Synthesis of Substituted 2ꢁAminoimidazoles 50–54.
To a solution of the corresponding substituted Nꢀ(1Hꢀimidazolꢀ2ꢀyl)acetamides (0.31 mmol) in
a 1:1 v/v mixture of MeOH and H O (2.4 mL) was added concentrated H SO (0.6 mL) and the
reaction mixture was heated at 100 °C under microwave irradiation for 15–30 min. The reaction
mixture was concentrated, and the resulting residue was resuspended in H O (5 mL) and a
saturated aqueous Na CO was added until pH 8. The product was extracted into EtOAc (3 x 40
2
2
4
2
2
3
mL). The combined organic fractions were dried over MgSO , and the solvent was removed
4
under reduced pressure. The resulting solid was used in the next reaction without further
purification.
2
ꢁ((4ꢁ(4ꢁbromophenyl)ꢁ1Hꢁimidazolꢁ2ꢁyl)thio)ꢁNꢁphenylacetamide (20). Compound 40 (29
mg, 0.17 mmol) was reacted with 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol (43 mg, 0.17 mmol)
according to the general method A. Purification by flash chromatography (1–20% v/v MeOH in
1
DCM) afforded 20 (59 mg, 0.15 mmol, 89% yield) as a white solid. H NMR (400 MHz,
MeOD) δ 7.62 (br s, 2H), 7.56 – 7.39 (m, 5H), 7.36 – 7.17 (m, 2H), 7.13 – 7.00 (m, 1H), 3.82
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2
2
2
2
2
2
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3
(
s, 2H) ppm. C NMR (100 MHz, MeOD) δ 169.6, 141.3, 139.6, 132.8, 129.8, 129.8, 127.5,
–
1
25.5, 121.4, 121.2, 121.2, 116.7, 39.6 ppm. LCMS (ESI–) m/z 388.0 [M – H] , retention time
+
2
.20 min (100%). HRMS (ESI+): m/z calculated for C H BrN OS [M + H] : 388.0119.
1
7
15
3
Found: 388.0121.
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Nꢁ(benzofuranꢁ5ꢁyl)ꢁ2ꢁ((4ꢁ(4ꢁbromophenyl)ꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetamide
(21).
Compound 41 (36 mg, 0.17 mmol) was reacted with 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol
(
(
43 mg, 0.17 mmol) according to the general method A. Purification by flash chromatography
1
1–20% v/v MeOH in DCM) afforded 21 (70 mg, 0.16 mmol, 96% yield) as a white solid. H
NMR (400 MHz, DMSOꢀd
6
) δ 12.50 (br s, 1H), 10.35 (br s, 1H), 7.94 (dd, J = 10.6, 2.0 Hz,
2
1
1
H), 7.73 – 7.64 (m, 2H), 7.52 – 7.44 (m, 4H), 7.34 (dd, J = 8.8, 2.2 Hz, 1H), 6.91 (dd, J = 2.2,
1
3
6
.0 Hz, 1H), 3.99 (s, 2H) ppm. C NMR (100 MHz, DMSOꢀd ) δ 166.5, 150.8, 146.7, 140.2,
39.9, 134.3, 133.6, 131.4, 127.4, 126.2, 119.0, 116.9, 115.7, 111.6, 111.3, 107.0, 37.9. LCMS
–
(
ESI–) m/z 428.0 [M – H] , retention time 2.22 min (100%). HRMS (ESI+): m/z calculated for
+
C
19
H15BrN
3
O
2
S [M + H] : 428.0068. Found: 428.0065.
2
4
ꢁ((4ꢁ(4ꢁbromophenyl)ꢁ1Hꢁimidazolꢁ2ꢁyl)thio)ꢁNꢁ(4ꢁiodophenyl)acetamide (22). Compound
2 (50 mg, 0.17 mmol) was reacted with 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol (43 mg, 0.17
mmol) according to the general method A. Purification by flash chromatography (1–20% v/v
1
MeOH in DCM) afforded 22 (60 mg, 0.11 mmol, 69% yield) as a white solid. H NMR (500
MHz, MeOD) δ 7.63 – 7.58 (m, 4H), 7.56 – 7.45 (m, 3H), 7.38 – 7.31 (m, 2H), 3.81 (s, 2H)
13
ppm. C NMR (125 MHz, MeOD) δ 169.6, 139.6, 138.9, 132.7, 127.5, 127.1, 123.0, 116.6,
–
1
11.4, 88.1, 39.7 ppm. LCMS (ESI–) m/z 511.7 [M – H] , retention time 2.30 min (100%).
+
HRMS (ESI+): m/z calculated for C17
H14BrIN
3
OS [M + H] : 513.9086. Found: 513.9087.
Nꢁphenylꢁ2ꢁ((4ꢁphenylꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetamide (23). Compound 40 (42 mg, 0.25
mmol) was reacted with 4ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol (44 mg, 0.25 mmol) according to the
general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM) afforded
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1
3 (61 mg, 0.20 mmol, 82% yield) as a white solid. H NMR (500 MHz, MeOD) δ 7.71 (brs,
H), 7.54 – 7.49 (m, 2H), 7.47 (brs, 1H), 7.35 (t, J = 7.4 Hz, 2H), 7.31 – 7.20 (m, 3H), 7.11 –
1
3
.03 (m, 1H), 3.83 (s, 2H) ppm. C NMR (125 MHz, MeOD) δ 168.3, 142.7, 139.5, 138.5,
38.2, 128.4, 128.3, 126.7, 124.4, 124.1, 123.7, 119.8, 38.3 ppm. LCMS (ESI–) m/z 308.0 [M –
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
–
+
H] , retention time 1.82 min (95%). HRMS (ESI+): m/z calculated for C17
16 3
H N OS [M + H] :
3
10.1014. Found: 310.1004.
Nꢁ(benzofuranꢁ5ꢁyl)ꢁ2ꢁ((4ꢁphenylꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetamide (24). Compound 41 (36
mg, 0.17 mmol) was reacted with 4ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol (30 mg, 0.17 mmol) according
to the general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM)
1
afforded 24 (42 mg, 0.12 mmol, 70% yield) as a white solid. H NMR (500 MHz, MeOD) δ
7
.87 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 2.2 Hz, 1H), 7.68 (d, J = 7.7 Hz, 2H), 7.46 (br s, 1H), 7.40
(
d, J = 8.8 Hz, 1H), 7.37 – 7.29 (m, 3H), 7.23 (t, J = 7.4 Hz, 1H), 6.76 (dd, J = 2.2, 0.9 Hz, 1H),
13
3
1
.84 (s, 2H) ppm. C NMR (125 MHz, MeOD) δ 169.6, 153.5, 147.4, 140.9, 134.7, 129.8,
–
29.1, 128.1, 125.8, 118.9, 114.2, 112.1, 107.7, 39.7 ppm. LCMS (ESI–) m/z 348.0 [M – H] ,
+
retention time 1.81 min (100 %). HRMS (ESI+): m/z calculated for C19
50.0963. Found: 350.0971.
H
16
N O
3 2
S [M + H] :
3
Nꢁ(4ꢁiodophenyl)ꢁ2ꢁ((4ꢁphenylꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetamide (25). Compound 42 (74 mg,
.25 mmol) was reacted with 4ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol (44 mg, 0.25 mmol) according to
0
the general method A. Purification by flash chromatography (1–20% v/v MeOH in DCM)
1
afforded 25 (70 mg, 0.16 mmol, 70% yield) as a white solid. H NMR (500 MHz, MeOD) δ
7
6
1
2
4
.66 (d, J = 7.7 Hz, 2H), 7.61 – 7.54 (m, 2H), 7.44 (br s, 1H), 7.38 – 7.31 (m, 4H), 7.23 (t, J =
13
.7 Hz, 1H), 3.80 (s, 2H) ppm. C NMR (125 MHz, MeOD) δ 169.7, 140.8, 139.6, 138.9,
–
29.8, 128.2, 125.8, 123.0, 88.1, 39.8 ppm. LCMS (ESI–) m/z 433.9 [M – H] , retention time
+
.13 min (98%). HRMS (ESI+): m/z calculated for C H IN OS [M + H] : 435.9981. Found:
1
7
15
3
35.9989.
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Journal of Medicinal Chemistry
Page 28 of 52
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Nꢁisopropylꢁ2ꢁ((4ꢁphenylꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetamide (26). A solution of propanꢀ2ꢀ
amine (22 µL, 0.25 mmol), 2ꢀ((4ꢀphenylꢀ1Hꢀimidazolꢀ2ꢀyl)thio)acetic acid (60 mg, 0.25 mmol),
HATU (194 mg, 0.50 mmol), DIPEA (90 µL, 0.50 mmol) in EtOAc (10 mL) was stirred for 5 h
at rt. The reaction mixture was diluted with EtOAc (40 mL) and washed with saturated aqueous
0
1
2
3
4
5
6
7
8
9
0
1
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0
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0
3 4
NaHCO (20 mL), water (20 mL) and brine (20 mL). The organic layer was dried over MgSO
and filtered. The solvent was removed under reduced pressure to give an oil residue, which was
purified by flash chromatography (5–100% v/v EtOAc in petroleum ether) to give the desired
1
product as a white solid (50 mg, 0.18 mmol, 73% yield). H NMR (500 MHz, MeOD) δ 7.37 (d,
J = 7.8 Hz, 2H), 7.14 (s, 1H), 7.05 (t, J = 7.7 Hz, 2H), 6.93 (t, J = 7.4 Hz, 1H), 3.62 (dt, J =
1
3
1
1
2
3.1, 6.4 Hz, 1H), 3.00 (s, 2H), 0.78 (d, J = 6.6 Hz, 6H) ppm. C NMR (125 MHz, MeOD) δ
70.3, 141.1, 140.8, 133.7, 129.7, 128.1, 125.7, 119.7, 43.0, 38.8, 22.4 ppm. LCMS (ESI–) m/z
–
73.9 [M – H] , retention time 1.52 min (100%). HRMS (ESI+): m/z calculated for C14
H
18
N
3
OS
+
[
M + H] : 276.1171. Found: 276.1172.
2
ꢁ((4ꢁ(4ꢁbromophenyl)ꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetic acid (27). Following the general
procedure B, from 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀthiol (250 mg, 0.98 mmol) was obtained
1
2
7 (220 mg, 0.70 mmol, 72% yield) as a white solid. H NMR (400 MHz, MeOD) δ 7.65 – 7.56
13
(
m, 2H), 7.57 – 7.39 (m, 3H), 3.80 (s, 2H) ppm. C NMR (100 MHz, MeOD) δ 172.9, 141.9,
–
1
40.2, 132.9, 132.6, 127.5, 121.8, 118.9, 37.4 ppm. LCMS (ESI–) m/z 311.0[M – H] , retention
+
time 1.59 min (98%). HRMS (ESI+): m/z calculated for C H BrN O S [M + H] : 312.9646.
1
1
10
2
2
Found: 312.9654.
2
4
6
1
ꢁ((4ꢁphenylꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetic acid (28). Following the general procedure B, from
ꢀphenylꢀ1Hꢀimidazoleꢀ2ꢀthiol (500 mg, 2.80 mmol) was obtained 28 (450 mg, 1.92 mmol,
1
9% yield) as a white solid. H NMR (500 MHz, MeOD) δ 7.68 (d, J = 7.2 Hz, 2H), 7.59 (s,
13
H), 7.41 (t, J = 7.7 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H), 3.86 (s, 2H) ppm. C NMR (125 MHz,
MeOD) δ 172.7, 141.7, 139.6, 131.6, 130.0, 129.1, 126.0, 118.9, 37.5 ppm. LCMS (ESI+) m/z
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Journal of Medicinal Chemistry
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+
2
35.1 [M + H] , retention time 1.22 min (100%). HRMS (ESI+): m/z calculated for
+
C
11
H
11
N
2
O
2
S [M + H] : 235.0541. Found: 235.0536.
Nꢁ(4ꢁ(1Hꢁimidazolꢁ4ꢁyl)phenyl)ꢁ2ꢁ((4ꢁ(4ꢁbromophenyl)ꢁ1Hꢁimidazolꢁ2ꢁyl)thio)acetamide
29). Compound 43 (40 mg, 0.17 mmol) was reacted with 4ꢀ(4ꢀbromophenyl)ꢀ1Hꢀimidazoleꢀ2ꢀ
1
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(
thiol (43 mg, 0.17 mmol) according to the general method A. Purification by flash
chromatography (1–20% v/v MeOH in DCM) afforded 29 (51 mg, 0.12 mmol, 69% yield) as a
1
white solid. H NMR (400 MHz, MeOD) δ 7.72 (d, J = 1.1 Hz, 1H), 7.66 (d, J = 8.5 Hz, 4H),
1
3
7
1
4
.59 – 7.47 (m, 5H), 7.40 (br s, 1H), 3.86 (s, 2H) ppm. C NMR (125 MHz, MeOD) δ 169.4,
41.2, 138.4, 137.0, 132.8, 127.5, 126.2, 121.4, 116.7, 111.4, 39.7 ppm. LCMS (ESI–) m/z
–
51.9 [M – H] , retention time 1.65 min (100%). HRMS (ESI+): m/z calculated for
+
C
20
H17BrN
5
OS [M + H] : 454.0337. Found: 454.0334.
1
ꢁ(4ꢁ(1Hꢁimidazolꢁ4ꢁyl)phenyl)ꢁ3ꢁ((5ꢁ(4ꢁbromophenyl)ꢁ1Hꢁimidazolꢁ2ꢁyl)methyl)urea (30).
To a solution of 4ꢀ(1Hꢀimidazolꢀ4ꢀyl)aniline (50 mg, 0.31 mmol) in THF (5 mL) was added 1,1ꢀ
carbonyldiimidazole (76 mg, 0.47 mmol). The mixture was stirred at rt overnight, and then it
was filtered. The filter was washed with THF (2 x 3 mL) and the resulting solid (30 mg, 0.12
mmol) was dissolved in DMF (3 mL), 44 (42 mg, 0.12 mmol) and N,Nꢀdiisopropylethylamine
(
74 µL, 0.48 mmol) were added. The reaction mixture was stirred at rt for 14 h, and then it was
poured into water, extracted with EtOAc, dried over Na SO , and concentrated. After cooling to
2
4
0
ºC, a 1:5 v/v mixture of MeOH and DCM (12 mL) was added and the suspended solid was
collected by filtration and dried at vacuum to yield 30 (35 mg, 0.08 mmol, 26% yield) as a white
1
solid. H NMR (500 MHz, DMSOꢀd ) δ 12.03 (br s, 2H), 8.64 (br s, 1H), 7.73 – 7.68 (m, 2H),
6
7
7
1
1
.64 – 7.59 (m, 2H), 7.57 (d, J = 1.9 Hz, 1H), 7.52 – 7.47 (m, 2H), 7.47 – 7.39 (m, 2H), 7.39 –
13
.32 (m, 2H), 6.58 (br s, 1H), 4.33 (d, J = 5.5 Hz, 2H) ppm. C NMR (125 MHz, DMSOꢀd
6
) δ
55.5, 146.9, 140.6, 138.9, 138.9, 135.9, 134.6, 131.7, 128.8, 126.6, 125.1, 125.0, 119.0, 118.4,
–
18.2, 113.7, 111.7, 37.6 ppm. LCMS (ESI–) m/z 437.0 [M – H] , retention time 5.07 min
+
(
97%). HRMS (ESI+): m/z calculated for C20
H18BrN
6
O [M + H] : 437.0725. Found: 437.0725.
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