2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors of bacterial type IV secretion

Article abstract of DOI:10.1016/j.bmc.2014.09.036

A novel series of 8-amino imidazo[1,2-a]pyrazine derivatives has been developed as inhibitors of the VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imidazo[1,2-a]pyrazines has been used to probe the structure-activity relationships of these inhibitors, which show potential as antibacterial agents.

Full text of DOI:10.1016/j.bmc.2014.09.036

Bioorganic & Medicinal Chemistry 22 (2014) 6459–6470
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2- and 3-substituted imidazo[1,2-a]pyrazines as inhibitors
of bacterial type IV secretion
James R. Sayer a, , Karin Walldén b,à, Thomas Pesnot a,§, Frederick Campbell a,–, Paul J. Gane c,k
,
Michela Simone c, Hans Koss a, Floris Buelens b, , Timothy P. Boyle a,àà, David L. Selwood c,
Gabriel Waksman b, Alethea B. Tabor a,
⇑
a Department of Chemistry, UCL, 20, Gordon Street, London WC1H 0AJ, UK
b Institute of Structural and Molecular Biology, UCL and Birkbeck, Malet Street, London WC1E 7HX, UK
c Wolfson Institute for Biomedical Research, UCL, The Cruciform Building, Gower Street, London WC1E 6BT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 June 2014
Revised 15 September 2014
Accepted 16 September 2014
Available online 28 September 2014
A novel series of 8-amino imidazo[1,2-a]pyrazine derivatives has been developed as inhibitors of the
VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic
route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imi-
dazo[1,2-a]pyrazines has been used to probe the structure–activity relationships of these inhibitors,
which show potential as antibacterial agents.
Ó 2014 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/3.0/).
Keywords:
8-Amino imidazo[1,2-a]pyrazine
Bacterial type IV secretion system
HP0525
ATPase inhibitor
1. Introduction
secretion of proteins toxic to host cells; and the secretion of effec-
tor molecules required for the propagation of the microorganism
Microorganisms have evolved a number of macromolecular
secretion machineries to translocate proteins and nucleoprotein
complexes from the bacterial cytosol to the host cell. Seven types
of secretion systems (I–VII) have so far been identified, with a
diverse range of functions including: the transfer of plasmid DNA
from one cell to another (the major mechanism for the spread of
antibiotic resistance genes between pathogenic bacteria); the
within the host cell.1
Bacterial secretion systems represent attractive targets for the
development of novel antibacterial agents.2 As these systems are
not required for bacterial growth, it is believed that bacteria would
be slow to develop resistance to drugs targeting these system. Sev-
eral groups have developed promising small molecule inhibitors
that are effective against the type III secretion systems (T3SS)
found in Gram-negative pathogens such as Yersinia, Salmonella
and Chlamydia.2
⇑
Corresponding author. Tel.: +44 (0) 20 7679 4695; fax: +44 (0) 20 7679 7463.
E-mail address: a.b.tabor@ucl.ac.uk (A.B. Tabor).
Type IV secretion systems (T4SS) are vital for the pathogenicity
of
a number of important Gram-negative bacteria, such as
Present address: MedPharm Ltd, R&D Centre, Unit 3/Chancellor Court, 50 Occam
Road, Surrey Research Park, Guildford GU2 7AB, UK.
Helicobacter pylori, Legionella pneumophilia and Bordetella pertussis,
which cause serious infections in both animals and plants.3,4 H.
pylori utilizes the type IV secretion system to translocate the toxic
protein CagA into gastric epithelial cells, and in doing so induces a
number of changes in the host cell.5,6 To date, little attention has
been paid to T4SS as targets for antibacterial agents, although a
VirB8 dimerisation inhibitor has recently been described as a
T4SS inhibitor.7 Type IV secretion systems require ATP as an energy
source to drive this transport and therefore require a class of
ATPases known as VirB11 ATPases, which are associated with the
inner membrane. The crystal structure of the VirB11 ATPase
à
Present address: Dept. Cell and Molecular Biology, Karolinska Institutet, Berzelius
väg 35, SE-171 77 Stockholm, Sweden.
§
Present address: Redx Oncology Ltd, Duncan Building, Royal Liverpool University
Hospital, Crown Street, Liverpool L69 3GA, UK.
–
Present address: Leiden Institute of Chemistry, Gorlaeus Laboratories, Einstein-
weg 55, 2333 CC Leiden, Netherlands.
k
Present address: The EMBL-European Bioinformatics Institute, Wellcome Trust
Genome Campus, Hinxton, Cambridge CB10 1SD, UK.
Present address: Department of Theoretical and Computational Biophysics, Max
Planck Institute for Biophysical Chemistry, Am Faßberg 11, D-37077 Göttingen,
Germany.
àà
Present address: NewSouth Innovations Pty Limited, Rupert Myers Building,
HP0525 has been solved, with the apo-,8 ADP bound9 and ATP
cS
UNSW, Sydney, NSW 2052, Australia.
http://dx.doi.org/10.1016/j.bmc.2014.09.036
0968-0896/Ó 2014 The Authors. Published by Elsevier Ltd.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

6460
J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
bound8 forms of HP0525 being studied. The HP0525 forms double
hexameric ring structures where each subunit monomer consists
of 328 amino acid residues comprising the N-terminal domain
(NTD) and C-terminal domain (CTD). Each domain forms a hexa-
meric ring with the CTDs forming a closed ring mounted onto
the dynamic open hexameric ring formed by the NTDs. The result
is a dome-like structure, open at the NTD end and closed at the
CTD end, which is large enough to accommodate a macromolecule
such as CagA. The nucleotide-free (apo-HP0525) form exists as an
asymmetric hexamer with the NTDs displaying mobility and the
CTDs maintaining the scaffold. When three molecules of ATP bind,
three of the subunits are locked into rigid conformations. Hydroly-
sis of these three ATP molecules to give ADP, together with binding
of a further three ATP molecules to the remaining three nucleotide-
free subunits results in a perfect hexameric rigid form. When all
ATP molecules are hydrolysed and released the symmetric hexa-
meric ring returns to its asymmetric form. The structure of
HP1451, an inhibitory factor of HP0525 which regulates Cag-med-
iated secretion, bound to HP0525, has also been studied.10
Whilst targeting the ATPase activity of HP0525 would repre-
sent an attractive approach to generating novel antibacterial
agents,11 so far only one group have previously published a
series of inhibitors of this enzyme.12 In this paper, we describe
a novel series of imidazo[1,2-a]pyrazine derivatives which act
as inhibitors of the HP0525 ATPase from H. pylori. In the course
of this work we have developed a flexible synthetic route to
the core heterocycle, which can deliver either 2-aryl or 3-aryl
imidazo[1,2-a]pyrazines.
2.2. Synthesis of first generation imidazo[1,2-a]pyrazine
inhibitors
The classical synthetic route to substituted imidazo[1,2-a]pyra-
zines involves a simple condensation between 2-amino pyrazine
and chloroacetaldehyde. Previously reported methods14 using
DMF as a solvent proved to be low yielding, but with methanol
as solvent, the product was isolated in 98% yield. However, all sub-
sequent attempts to functionalise the imidazo[1,2-a]pyrazine core
via bromination14,15 gave extremely poor yields and inseparable
mixtures of dibrominated regioisomers. Furthermore, these could
not be further transformed using telesubstitution with ammo-
nia,15,16 to give 8-amino imidazo[1,2-a]pyrazines as planned.
We therefore planned to prepare the core heterocycle with
functional groups already installed at the 2- or 3-positions, and
with a leaving group at the 8-position to allow amino or other
functionality to be added. In order to access the 3-substituted het-
erocycles 1–6, we adapted the procedure of MacCoss et al.17 to give
3-aryl substituted 8-chloroimidazo[1,2-a]pyrazines from 2-aryl-2-
hydroxy amines 14a–e (Scheme 1). In each case the synthesis of
the amino alcohol was achieved via
ketone with pyridinium tribromide to give 15a–e. This was fol-
lowed by substitution of the
-bromine with an azide,19 giving
a
-bromination18 of the aryl
a
16a–e, which were then reduced20 to give alcohols 17a–e. Hydro-
genation21 to give amino alcohols 14a–e was performed at atmo-
spheric pressure, with the exception of the thiophene analogue
which required 3 bar pressure19 to go to completion. Coupling with
2,3-dichloropyrazine in 1,4-dioxane afforded the pyrazinyl-amino
alcohols 18a–e in good yields. Swern oxidation22 to the ketones
19a–e was followed by acid-induced cyclisation to form the 3-
aryl-8-chloroimidazo[1,2-a]pyrazines 20a–e.
2. Results and discussion
2.1. Lead identification
3-substituted imidazo[1,2-a]pyrazine derivatives have previ-
ously been reported as kinase inhibitors,23–25 and a range of strat-
egies is available for synthesis of the key heterocyclic core.
However, 2-aryl imidazo[1,2-a]pyrazine derivatives have been less
frequently explored,26 and consequently fewer synthetic
approaches to these compounds have been reported. Using the
Structures from the kinase-directed SoftFocus library (BioFocus)
were modeled into ATP-binding site of HP0525 ATPase7 (1NLY) and
scored using DOCK 6.13 A group of 3-aryl 8-amino imidazo[1,2-
a]pyrazines 1–6 (Fig. 1) was identified among the best-scoring
compounds. These were selected for experimental study, along
with the regioisomeric 2-aryl 8-amino imidazo[1,2-a]pyrazines
7–13 (Fig. 1).
a-bromo aryl ketones 15a–e, the key 2-aryl-8-chloroimidazo[1,2-
a]pyrazine intermediates 21a–e were readily prepared by conden-
sation with 2-amino-3-chloropyrazine (Scheme 2).
O
O
S
NH
OMe
OMe
O
1, Ar =
3, Ar =
4, Ar =
2, Ar =
N
N
N
Ar
H
N
S
O
O
NH
5, Ar =
6, Ar =
N
N
S
N
Ar
O
O
S
N
NH
7, Ar =
9, Ar =
10, Ar =
OMe
OMe
O
8, Ar =
N
Ar
N
H
N
S
O
O
NH
O
11, Ar =
13, Ar =
12,Ar =
N
N
Ar
S
N
Figure 1. Initial lead structures of 2- and 3-substituted 8-amino imidazo[1,2-a]pyrazines.

J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
6461
NaN3
Cl
Cl
N
N
DMSO
0oC - RT,
1-5 h
(COCl)2, DMSO,
Et3N, CH2Cl2, 3 h,
H2, Pd/C
MeOH, RT
3-16 h
Et3N, Dioxane
Reflux, 16-20 h
NaBH4, MeOH
0oC 1 h
OH
O
O
O
OH
OH
-78 o
64-90%
C
H
N
H
N
N
N
Br
N3
N
N
NH2
N3
Ar
18a-e
Ar
15a-e
Ar
16a-e
Ar
Ar
Ar
95-99%
86-99%
58-64%
63-99%
Cl
Cl
14a-e
17a-e
19a-e
TFA/TFAA,
PhMe, RT
72 h
18 - 99%
Cl
N
N
20a-e
N
Ar
Scheme 1. Synthesis of 3-aryl-8-chloroimidazo[1,2-a]pyrazines 20a–e.
inorganic phosphate (Pi) using an in vitro ATPase assay (see
Section 4 and SI). Initially, the inhibitory effects of the compounds
were evaluated by performing the ATPase assay with and without
H2N
N
N
NaHCO3, tBuOH,
reflux, 20 - 40 h
Cl
Cl
Br
O
N
N
Ar
compound present at concentrations of 500
lM (or 250 lM),
N
Ar
15a-e
16 - 61%
50 M and 5 M (data not shown). The compounds with inhibitory
l
l
21a-e
activities were further analyzed for dose-dependency by estimat-
ing their IC50s (Fig. 2). Those with the highest inhibitory effect,
11, 5 and 6 with IC50s of 6, 20 and 48 lM, respectively, showed
Scheme 2. Synthesis of 2-aryl-8-chloroimidazo[1,2-a]pyrazines 21a–e.
similarities in their chemical structures, see below.
To test our hypothesis that the inhibitors bind in the substrate
pocket, as suggested by the molecular docking, we tested the mode
of inhibition of 11. Steady-state kinetic data displayed Michaelis–
Menten behavior, and 11 unambiguously behaved as a competitive
inhibitor (Fig. 2).
We verified that our analogues displayed suitable physicochem-
ical profiles by calculating logP and logS using AlogP2.1 applet.34
The results presented in Table 2 suggest that our first generation
of compounds display limited solubility (0.1 to 10 mg/L) and logP
values that are high but within limits described by the Lipinski’s
‘rule of five’ (i.e., 65).35
To install the sulfonamido and sulfonamidoaniline groups,
Buchwald–Hartwig coupling chemistry was employed.27 tBu-
XPhos28 has been previously used successfully in the Pd-catalysed
amidation of aryl bromides with tert-butyl carbamate.29 The combi-
nation of tBu-XPhos/Pd(dba)2/K2CO3/tBuOH gave low to moderate
yields for all couplings with p-toluene sulfonamide 22 and
N-(4-aminophenyl)-4-methylbenzenesulfonamide 23 (Table 1),
with the exception of 21d, for which Pd(dppf)Cl2 had to be used30
as an alternative. As the coupling of sulfonamides with aryl chlo-
rides had been reported31 to give high yields using DavePhos32
a
complete range of precatalyst, base, solvent and temperature was
screened for the conversion of 21a to 7 and 11 (Table S1, Support-
ing information). The combination of Pd2(dba)3/DavePhos/NaOtBu/
toluene gave 7 in excellent yield, however this reaction is clearly
highly substrate-dependent and these optimised coupling condi-
tions did not give such good results with other 2- and 3-aryl-8-
chloroimidazo[1,2-a]pyrazines. Likewise, conditions for micro-
wave coupling were explored, which gave excellent yields of 7
but which were less successful for the synthesis of 11 (Table 1).
Methyl sulfones have previously been used to attach a variety of
nucleophiles onto imidazo[1,2-a]pyrazine rings.24,33 As an alterna-
tive to the Buchwald–Hartwig coupling, 21a was converted to
methyl sulfone 24 (Scheme 3). Treatment with 4-toluene sulfon-
amide/NaH/DMF gave 7 in good yield, however for this substrate
no improvement over the optimised Buchwald–Hartwig conditions
could be effected via this route.
The regiochemistry of the 2- and 3-substituted imidazo[1,2-
a]pyrazines was confirmed by 2D NMR. For the 2-substituted imi-
dazo[1,2-a]pyrazine 21a, HMBC (Fig. S1, Supporting information)
showed correlation between the protons and carbons in the 3-
and 5-positions of the imidazo[1,2-a]pyrazine rings. Conversely,
with the 3-substituted imidazo[1,2-a]pyrazines no correlation
was observed between the protons in the corresponding 2- and
5-positions.
2.4. Second generation imidazo[1,2-a]pyrazine inhibitors
The promising IC50 values observed for these compounds, and
especially for 11, prompted a further investigation of related struc-
tures. Table 2 shows that compounds with substituents in the 8-
position where the sulfonamide moiety was remote from the core
heterocycle showed the greatest potency. In particular, 11 showed
the most promise as a lead for further investigation. However, the
physicochemical properties (partition coefficient and solubility) of
this series of compounds, were poor, and we therefore sought to
explore structural modifications that would give candidates suit-
able for preclinical drug development with improved solubility as
well as potency. As the sulfonamide could potentially act as a bioiso-
stere of one of the phosphate groups of ATP,36 we aimed to evaluate
analogues with different spacing and flexible or rigid linkers
between the core heterocycle and the sulfonamide, and also ana-
logues with the sulfonamide group absent. Analogues with hetero-
cyclic groups at position 2, or lacking the aryl group altogether, were
also evaluated, in a further effort to improve the solubility of this
series. Finally, substitution at position 6 was also investigated.
A series of analogues of 11, differing at the 8-position, were pre-
pared (Table 3) via either Buchwald–Hartwig coupling from 21a or
nucleophilic substitution of the methyl sulfone 24, in moderate to
good yields. Deleting the arylsulfonamide group completely (25)
resulted in complete loss of activity, further suggesting that this
region of the 8-substituted imidazo[1,2-a]pyrazines is important
for binding to the active site. The majority of variants of
2.3. Biochemical evaluation of first generation imidazo[1,2-a]
pyrazine inhibitors
The HP0525 protein was produced recombinantly in Escherichia
coli and purified to high purity as described previously.10 The ATP-
ase activity of HP0525 was measured by monitoring the release of
the
N-(4-aminophenyl)-4-methylbenzenesulfonamido
group
(26–30) showed a decrease in potency compared to 11 when the

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J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
Table 1
Synthesis of 2- and 3-substituted 8-amino imidazo[1,2-a]pyrazines from 20a–e and 21a–e
H
N
S
O
O
O
H
O
S
N
NH
Cl
S
NH
23
22
NH2
O
O
S
O
N
N
N
N
N
NH2
N
Ar1
Ar1
Ar2 Buchwald-Hartwig coupling
Ar1
O
N
N
N
Ar2
Ar2
Buchwald-Hartwig coupling
20a-e,
21a-e
5, 6, 11, 12, 13
Ar2
1-4, 7-10
Starting
Material
Ar1
Ar3NH2
Conditions
Product
(Yield)
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 40 h
20a
20b
H
H
22
22
1 (41%)
2 (26%)
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 40 h
O
OMe
OMe
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 40 h
20c
H
22
3 (30%)
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 40 h
20d
20a
20e
H
H
H
22
23
23
4 (67%)
5 (35%)
6 (30%)
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 46 h
DavePhos (3 mol %), Pd2(dba)3 (1 mol %) NaOtBu (1.4 equiv) toluene,
reflux, 24 h
S
DavePhos (3 mol %), Pd2(dba)3 (1 mol %) NaOtBu (1.4 equiv) toluene,
reflux, 24 h
21a
21a
24
H
H
H
22
22
22
7 (93%)
7 (46%)
7 (72%)
DavePhos (3 mol %), Pd2(dba)3 (1 mol %) NaOtBu (1.4 equiv) toluene,
160 °C (lW), 10 min
NaH (2 equiv), DMF, 100 °C, 20 h
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 48 h
O
21b
21c
H
H
22
22
8 (13%)
9 (2%)
OMe
OMe
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 48 h
21d
H
22
Pd(dppf)2Cl2 (2 mol %), K2CO3 (1.2 equiv), tBuOH, reflux, 21 h
10 (29%)
DavePhos (3 mol %), Pd2(dba)3 (1 mol %) NaOtBu (1.4 equiv) toluene,
reflux, 24 h
21a
21a
H
H
23
23
11 (22%)
11 (47%)
DavePhos (3 mol %), Pd2(dba)3 (1 mol %) NaOtBu (1.4 equiv) toluene,
160 °C (lW), 10 min
tBu-XPhos (5 mol %) Pd(dba)2 (1 mol %) K2CO3 (1.2 equiv) tBuOH,
reflux, 48 h
21e
21b
H
H
23
23
12 (8%)
13 (9%)
S
DavePhos (3 mol %), Pd2(dba)3 (1 mol %) NaOtBu (1.4 equiv) toluene,
reflux, 24 h
O
sulfonamido group was placed at a greater distance from the imi-
dazo[1,2-a]pyrazine ring, modified, or deleted completely. How-
ever, when the aniline group was replaced by an ethyl linker (31)
comparable inhibition with better solubility and logP were
obtained. Replacing the p-toluene sulfonamidyl group with
quinoline-8-sulfonamide (33, 34) also resulted in compounds of high
potency. Surprisingly, when the N-(4-aminophenyl)-4-meth-
ylbenzenesulfonamido group was replaced by 3-(pyridin-3-yl)aniline
(32) a further slight improvement in potency was seen (Table 3).
Unfortunately, the most active compounds in this series (32, 33
and 34) all exhibited comparable solubility and logP to 11.
The importance of the aryl substituent in position 2 was further
reinforced by compound 35; deleting the aryl substituent resulted
in a complete loss of activity (Table 4). As the naphthalene substi-
tuent is highly lipophilic and is a major contributor to the insolu-
bility of this series of inhibitors, we replaced this with
a

J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
6463
O
O
O
S
O
22
NH
Cl
S
(i) NaSMe, DMSO,
100 oC, 48 h, 90%
O
N
S
H2N
O
N
N
N
N
N
N
N
N
NaH, DMF, 100 oC, 72%
(ii) mCPBA, CH2Cl2,
r.t., 2 h, 56%
7
21a
24
Scheme 3. Alternative route to 7 via methylsulfone 24.
Figure 2. Dose-dependent and steady-state inhibition of HP0525. Dose–response curves used for IC50 estimations of compounds 11 (A) and 32 (B). Michaelis–Menten (C) and
Lineweaver–Burk (D) plots, corresponding to z without inhibitor, and z with 11. Error bars represent standard deviations using triplicate data.
quinoxaline (36). However, this gave no appreciable improvement
in either potency or solubility.
representative structure. However, the resolution of 1G6O is
2.50 Å compared to 2.80 Å for 1NLY, and a Ramachandran38 analy-
sis gives 4% of residues in more favourable regions for 1G6O. The
crystal structure of the ADP-bound HP0525, 1G6O, with heteroat-
oms and ADP removed, was therefore used for the docking studies.
Examination of the ADP/ATP binding pocket shows that it adopts a
conical topology in which the entrance is wide open and the
bottom of the cavity very narrow and very likely to tolerate small
groups only. In accordance with kinases and phosphorylases
topologies the entrance to the active side is highly lipophilic and
the end of the cavity highly hydrophilic. In the entrance to the
active site (adenosine binding region), lipophilicity is governed
by three aromatic residues (Tyr140, Phe144 and Phe145).
Hydrophilicity within the cavity (triphosphate binding region) is
created by a tetrad Gly181/Ser182/Gly183/Lys184 along with
Arg133. A third binding region, not exploited by ADP/ATP, is
located in the direction of the 20-ribose hydroxyl and is likely to
tolerate small aliphatic moieties (Fig. 3a).
In order to explore the effects of substitution at the 6-position
of the imidazo[1,2-a]pyrazine, 37 and 38 were synthesized. The
key 6,8-dibromoimidazo[1,2-a]pyrazine 39 was prepared by con-
densation of 4,6-dibromo-2-aminopyrazine with 2-bromoacetyl
naphthalene (Scheme 4). Nucleophilic reaction with the appropri-
ate monotosylated diamine proceeded smoothly and exclusively at
the 8-position to afford 37 or 38 in good yield. However, 38
showed poorer physicochemical properties and IC50 compared to
the analogue 31. Likewise, 37 showed poorer solubility and logP
compared with 11, which lacks the 6-substitutent, but showed
comparable potency (Table 4). Overall, this suggests that the bro-
mide substituent at the 5-position does not improve potency and
leads to a poorer physicochemical profile.
2.5. Docking studies
In order to further understand the binding of these imidazo[1,2-
a]pyrazines to HP0525, and to direct the design of more potent
inhibitors, molecular docking studies were carried out using Auto-
Dock Vina.37 Structural studies showed that the conformation of
the apo form of each unit is variable, but the two structures
Docking of the lead compound 11 (Fig. 3b), and comparison
with the binding of ADP, showed a binding mode in which the
inhibitor is deeply buried within the enzymatic cavity. In this ori-
entation, the naphthalene group occupies the purine-binding
region of the active site, possibly making
p-stacking interactions
1G6O9 and 1NLY8 which possess ADP, or the ATP mimic ATP-
cS,
with Phe145. The core imidazo[1,2-a]pyrazine ring sits in place
of the ribose moiety of ADP, with the sulfonamide occupying the
phosphate binding region and making polar contacts with
Gly181, Lys184 and Thr185. Examining the surface of the ADP
binding site with the docked 11 (Fig. 3c) suggested that these
two binding regions could be explored to increase the potency of
this series of inhibitors. For example, in order to optimize the pre-
dicted interaction of the sulfonamide moiety with the phosphate
binding site, a series of analogues (30, 31, 33, 34) in which different
spacer lengths and orientations between the sulfonamide and the
respectively, are structurally highly similar with an RMS of
0.50 Å for CA atoms and 0.78 Å for all atoms. Both of these struc-
tures contain two identical chains, A and B. For drug design pur-
poses any of the four chains from the two crystal structures
above are acceptable for use in modelling/screening. Investigation
of the ligand–protein interactions of each, in particular the
hydrogen bonding, reveals a more extensive network of interac-
tions in 1NLY and the A chain also includes the active site metal
(Mg). This might therefore be taken to be the more physiological

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J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
Table 2
IC50 values for first generation compounds
R
N
N
Ar1
N
Ar2
Compound
R
Ar1
H
Ar2
IC50
/
l
M
AlogsP
AlogpS
ꢀ5.7
O
O
O
S
S
NH
1
77 16
96 42
3.7
O
O
NH
2
3
4
H
H
H
4.3
3.1
3.3
ꢀ5.8
ꢀ4.7
ꢀ5.4
O
O
O
OMe
OMe
S
S
NH
144 14
O
NH
154 22
H
N
S
5
6
H
H
20
48
3
7
4.6
3.5
ꢀ6.0
ꢀ5.1
O
O
O
NH
NH
H
N
S
O
O
S
O
S
S
NH
7
8
H
H
H
H
88 14
82 14
4.3
4.5
3.5
3.5
ꢀ5.7
ꢀ5.8
ꢀ4.7
ꢀ5.4
O
O
O
NH
O
O
O
OMe
OMe
S
S
NH
9
167 43
133 16
O
NH
10
H
N
S
11
12
13
H
H
H
6
1
5.0
3.9
5.5
ꢀ6.0
ꢀ5.1
ꢀ6.2
O
O
O
O
NH
NH
NH
H
N
S
O
99 18
S
H
N
O
S
O
18
4
8-position were synthesized and tested. Docking studies on 30
(IC50 58 M), 31 (IC50 M), and 34 (IC50 M) suggest that the
the phosphate binding region. This may indicate that the sulfon-
amide group is not always necessary for binding if other H-bond
donor or acceptor groups are present in the correct orientation.
l
7
l
7 l
propyl chain of 30 may position the sulfonamide moiety too far
from the phosphate binding region (Fig. 3d). Intriguingly, the most
potent lead compound in this series, 32, which is also the most
ligand efficient, lacks the sulfonamide group completely. Here
the docking (Fig. 3e) suggests a similar orientation, with the naph-
thalene group occupying the purine-binding region, and the imi-
dazo[1,2-a]pyrazine ring occupying the ribose-binding area.
However, the 3-(pyridin-3-yl)aniline moiety in this case occupies
3. Conclusions
Following a virtual high throughput screen, a novel series of 8-
amino imidazo[1,2-a]pyrazine derivatives have been developed,
using a flexible synthetic route to deliver 2- and 3-aryl regioisom-
ers. Biochemical evaluation showed moderate to good potency

J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
6465
Table 3
Structures and IC50 values for 2nd generation compounds, varying at 8-position
R
N
N
N
Compound
R
Synthesis method
—
IC50
/
l
M
AlogsP
AlogpS
25
NH2
H2N
>1000
3.2
ꢀ4.3
26
27
A
A
18
5
4.4
4.0
ꢀ5.3
NH
H
N
H3C
O
S
61 28
ꢀ5.4
O
NH
H
N
S
28
29
A
A
29
9
5.0
5.3
ꢀ6.0
ꢀ6.0
O
O
O
O
NH
O
S
S
75 35
58 38
N
H
O
O
O
N
H
NH
30
31
B
B
4.3
4.1
ꢀ5.7
ꢀ5.6
H
N
7
4
2
1
NH
S
O
NH
32
A
5.0
ꢀ5.8
N
H
N
S
S
33
34
A
A
7
7
2
2
5.1
5.0
ꢀ6.0
ꢀ6.0
O
O
O
N
N
NH
NH
O
N
H
Method A: Buchwald–Hartwig coupling from 21a. Method B: NaH, 24. Section 4 are given in the Supporting information.
highlighting this class of compound as competitive inhibitors of
the HP0525 ATPase from H. pylori, with potential as antibacterial
agents. The structure–activity relationships of these 8-amino imi-
dazo[1,2-a]pyrazines has been explored through docking studies,
however co-crystallisation of these inhibitors with HP0525 is
imperative to fully understand the interaction within the nucleo-
tide binding site and aid in the development of more potent inhib-
itors. Furthermore, development of these compounds will require
improvements in their aqueous solubility to enable a more suitable
physicochemical profile.
the General Amber Force Field.41 The resulting binding poses were
scored using the Hawkins GB/SA function of DOCK 6.13
Further molecular docking studies were carried out using Auto-
Dock Vina.37 The crystal structure of ADP-HP0525 (PDB entry
1G60) was used to define a docking grid around the nucleotide
binding site, with a size of 14 ꢁ 16 ꢁ 24 and a grid center of
ꢀ12.034, 24.627 and 22.363 in the x, y, and z coordinates, respec-
tively. An ‘exhaustiveness’ parameter of 8 was used. Ligand
structures were generated using chem3D pro and further
prepared using AutoDock Tools (ADT)42 as recommended in the
documentation.
4. Experimental section
4.2. General chemistry
4.1. Lead identification and molecular docking
Melting points (Mp) were recorded on a Gallenkamp Melting
Point Apparatus and are uncorrected. 1H and 13C NMR were
recorded using Bruker AV400 (400 and 100 MHz, respectively),
AV500 (500 and 125 MHz, respectively) and AV600 (600 and
150 MHz, respectively) spectrometers as indicated. Chemical shifts
are quoted on the d scale in units of ppm using TMS as an internal
standard. Spectra were obtained using CDCl3, CD3OD, CD2Cl2 and
DMSO-d6 as solvents and coupling constants (J) are reported in
The initial lead compounds were identified through screening of
the SoftFocus kinase-targeted compound library (BioFocus). The
pharmacophore alignment function of MOE39 was used to produce
a rough initial alignment to ATP of the diverse input set based on
common chemical features. The aligned structures were then
energy-minimised in the context of the nucleotide binding site of
HP0525 (PDB ID 1NLY8), with the atoms of the protein frozen, using
NAMD.40 Ligand force field parameters were assigned according to
Hz with the following splitting abbreviations:
s (singlet), d

6466
J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
Table 4
Variants of the lead compound 11 at the 2- and 6-positions
Compound
IC50
/
l
M
AlogsP
AlogpS
ꢀ4.8
35
>1000
2.7
H
N
S
O
O
NH
N
N
N
36
37
38
28
6
4.2
6.6
5.0
ꢀ5.3
ꢀ6.3
ꢀ5.8
H
N
S
O
O
NH
N
N
N
N
N
6
2
H
N
S
O
O
NH
N
N
N
Br
77 20
H
N
S
O
O
NH
N
N
N
Br
O
O
O
O
Br
S
R
N
S
R
N
NH
Br
N
NH2
Br
H
H
O
NH2
N
N
N
N
N
N
N
H2O, THF, cat. HCl
80 oC, 3 days, 25%
Br
Br
Hunig's base
Br
39
37 R = (CH2)2, 82%
38, R = p-C6H4, 79%
Scheme 4. Synthesis of 37 and 38 via 6,8-dibromoimidazo[1,2-a]pyrazine 39.
(doublet), t (triplet), dd (doublet of doublets), bs (broad singlet).
Infra-Red (IR) spectroscopy was carried out using a PerkinElmer
Spectrum 100 FT-IR Spectrometer using thin films. Absorption
tribromide (7.50 g, 23.6 mmol) was added and the reaction was
stirred at 50 °C for 16 h. The reaction mixture was cooled to room
temperature and the solvents removed in vacuo. The resulting
orange slurry was suspended in H2O (30 mL) and extracted with
EtOAc (4 ꢁ 30 mL). The combined organic extracts were washed
with H2O (2 ꢁ 20 mL) and brine (1 ꢁ 20 mL), dried (Na2SO4), fil-
tered and concentrated in vacuo to give a yellow oil. Flash
chromatography was carried out (applied in petroleum ether;
eluted 0% to 10% to 33% CH2Cl2) to afford the title compound as
a pale yellow oil (2.30 g, 7.90 mmol, 84%). Rf = 0.68 (CH2Cl2); IR
maxima (m
max) are reported in wavenumbers (cmꢀ1).
Solvents and reagents were obtained from commercial sources
and were used as received unless otherwise stated. Petroleum
ether refers to the fraction of light petroleum ether boiling in the
range 40–60 °C.
Representative examples of each of the synthetic routes shown
in Schemes 1–3 are given. Full experimental for the preparation of
the remaining compounds, and full compound characterisation, is
given in the Supplementary data.
(m
max/cmꢀ1, thin film): 1677, 1598, 1574; 1H NMR (500 MHz,
CDCl3): dH = 4.65 (s, 2H), 6.86 (d, J = 8.4 Hz, 1H), 7.09 (d,
J = 7.7 Hz, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.22 (t, J = 7.3 Hz, 1H),
7.40–7.47 (m, 3H), 7.92 (dd, J = 7.6, 1.5 Hz, 1H); 13C NMR
(125 MHz, CDCl3): dC = 36.8, 117.6, 119.5, 123.0, 124.4, 126.2,
129.9, 131.3, 134.2, 155.0, 156.5, 191.6; LRMS m/z (EI+): 292 [M
4.3. General method for synthesis of a-bromo aryl ketones,
illustrated for the preparation of 2-bromo-1-(2-phenoxyphe-
nyl)ethanone, 15b
(
81Br)]+, 290 [M (79Br)]+, 212 [MꢀBr]+, 197 [MꢀCH2Br]+; HRMS
1-(2-Phenoxyphenyl)ethanone (2.00 g, 9.42 mmol) was dis-
solved in chloroform (60 mL) and ethanol (60 mL). Pyridinium
m/z (EI+): Found 289.99403 [M(79Br)]+; C14H11BrO2 requires
289.99369.

J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
6467
Figure 3. (a) Binding of ADP to HP0525 (PDB 1G6O). Key residues are labelled. (b) Comparison of the binding of 11 and ADP (Yellow). (c) Binding of 11 to HP0525, showing
regions that could be explored to increase the potency of the series of inhibitors. (d) Compounds 11 (white), 30 (magenta), 31 (violet-brown) and 34 (turquoise) are overlaid,
when docked in HP0525. Polar bonds are indicated corresponding to interactions involving compound 11. (e) Comparison of the docking of 32 (white/blue) and ADP in
HP0525.
4.4. General method for synthesis of
a-azido aryl ketones,
4.5. General method for synthesis of a-azido aryl alcohols,
illustrated for the preparation of 2-azido-1-(2-naphthyl)-
illustrated for the preparation of 2-azido-1-(2-naphthyl)ethanol
ethanone 16a
17a
2-(Bromoacetyl)naphthalene (2.00 g, 8.03 mmol) was dissolved
in DMSO (10 mL) and the mixture was cooled on ice such that the
temperature was kept below 10 °C. Sodium azide (0.630 g,
9.64 mmol) was added in one portion and the reaction was stirred
under argon at room temperature for 90 min. The reaction was
quenched with H2O (20 mL), and extracted with EtOAc
(3 ꢁ 30 mL). The organic layers were combined, washed with
H2O, dried (Na2SO4) and filtered. The solvent was removed in
vacuo to give the title compound as a brown/orange oil (1.69 g,
8.01 mmol, 100%) with NMR consistent with literature values.43
Azidoketone 16a (2.11 g, 10.0 mmol) was dissolved in anhy-
drous MeOH (100 mL) and cooled on ice. Sodium borohydride
(568 mg, 15.0 mmol) was added portion wise and the mixture
was stirred on ice under argon for 1 h until the reaction had gone
to completion by TLC. The solvent was removed and the resulting
residue was taken up in CH2Cl2 (100 mL) and carefully washed
with H2O (2 ꢁ 60 mL) followed by brine (60 mL). The organic
extracts were dried over Na2SO4, filtered and concentrated in
vacuo to give the title compound as a brown oil (2.14 g, 10.0 mmol,
100%). Spectroscopic data (for the racemic material) was consistent
with that previously reported44 (for the (S)-enantiomer): Rf = 0.65
Rf = 0.63 (5:1 petroleum ether/EtOAc); IR (m
max/cmꢀ1, thin film):
2105, 1690; 1H NMR (600 MHz, CDCl3): dH = 4.73 (s, 2H), 7.59–
7.62 (m, 1H), 7.65–7.68 (m, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.95 (d,
J = 8.6 Hz, 1H), 7.99–8.01 (m 2H), 8.42 (s, 1H); 13C NMR
(150 MHz, CDCl3): dC = 55.0, 123.3, 127.2, 127.9 129.0, 129.1,
129.6, 129.8, 131.7, 132.4, 136.0, 193.2; LRMS m/z (EI+): 211
[M]+, 155 [MꢀCH2N3]+, 127 [Naphthalene]+.
(3:1 petroleum ether/EtOAc); IR (m
max/cmꢀ1, thin film): 3398,
2100; 1H NMR (500 MHz, CDCl3): dH = 2.70 (br s, 1H), 3.46–3.58
(m, 2H), 5.02 (dd, J = 8.1, 3.9 Hz, 1H), 7.44 (dd J = 8.4, 1.6 Hz, 1H),
7.49–7.52 (m, 2H), 7.83–7.86 (m, 4H); 13C NMR (125 MHz, CDCl3):
dC = 58.1, 73.6, 123.7, 125.1, 126.4, 126.5, 127.8, 128.1, 128.6, 133.3
(2 signals), 138.0; LRMS m/z (EI+): 221, 157 [MꢀCH2N3]+, 147, 129.

6468
J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
4.6. General method for synthesis of
a-amino aryl alcohols,
eluted 100:1 to 30:1 CH2Cl2/EtOAc) afforded the title compound
illustrated for the preparation of 2-amino-1-(2-naphthyl)-
ethanol 14a
as a yellow solid (903 mg, 3.03 mmol, 57%). Mp: 160 °C; Rf = 0.30
(30:1 CH2Cl2/EtOAc); IR (m
max/cmꢀ1, thin film): 1680; 1H NMR
(500 MHz, CDCl3): dH = 5.10 (d, J = 4.3, 2H), 6.54 (s, 1H), 7.58–
7.62 (m, 1H), 7.64–7.67 (m, 1H), 7.68 (d, J = 6.1 Hz, 1H), 7.91 (d,
J = 8.0 Hz, 1H), 7.96 (d, J = 8.6 Hz, 1H), 8.00–8.02 (m, 2H), 8.10
(dd, J = 8.6, 1.8 Hz, 1H), 8.62 (s, 1H); 13C NMR (125 MHz, CDCl3):
dC = 48.4, 123.4, 127.3, 128.0, 129.0, 129.2, 129.8, 130.1, 131.3,
131.8, 132.6, 136.2, 139.7, 193.8; LRMS m/z (ESI+): 300
[M(37Cl)+H]+, 298 [M(35Cl)+H]+, 282 [M(37Cl)ꢀOH]+, 280
[M(35Cl)ꢀOH]+; HRMS m/z (ESIꢀ): Found 296.0591 [M(35Cl)ꢀH]ꢀ;
Azidoalcohol 17a (2.18 g, 10.2 mmol) was dissolved in anhy-
drous MeOH (50 mL) and 10% palladium on carbon (218 mg, 10%
w/w) was added. The vessel was evacuated and purged with Ar
(3ꢁ) and under static vacuum a balloon of hydrogen was added.
The reaction mixture was stirred under hydrogen atmosphere until
completion as determined by TLC and disappearance of N3 peak by
IR. After 3½ h, the hydrogen was carefully released, the vessel
evacuated and purged argon (3ꢁ), and the reaction mixture was fil-
tered through Celite (pre-washed with MeOH). Solvent removal in
vacuo gave the crude compound as a orange oil (1.91 g, 10.2 mmol,
100%). Spectroscopic data was consistent with that previously
C16H11ClN3O requires 296.0591.
4.9. General method for synthesis of 3-aryl-8-chloro-
imidazo[1,2-a]pyrazines, illustrated for the preparation of 8-
chloro-3-(2-naphthyl)imidazo[1,2-a]pyrazine 20a
reported.44 Rf = 0.06 (5:1 EtOAc/MeOH); IR ( max/cmꢀ1, thin film):
m
3290, 3054, 2916, 1599; 1H NMR (500 MHz, CDCl3): dH = 2.82 (m,
2H), 4.59–4.75 (m, 1H), 7.36–7.40 (m, 3H), 7.74–7.76 (m, 4H);
13C NMR (125 MHz, CDCl3): dC = 49.8, 74.3, 123.8, 124.5, 125.7,
126.0, 127.5, 128.1, 132.8, 133.1 (2 signals), 139.7; LRMS m/z
(ESI+): 229.2 [M+MeCN]+, 211.2 [M+Na]+, 188.1 [M+H]+, 170
[MꢀOH]+.
Compound 19a (903 mg, 3.03 mmol) was dissolved in anhy-
drous toluene (40 mL) and the mixture was cooled on ice. Trifluo-
roacetic acid (1.64 mL, 21.2 mmol) was added and the reaction was
allowed to stir on ice for 30 min, followed by the addition of triflu-
oroacetic anhydride (2.95 mL, 21.2 mmol). The reaction mixture
was then stirred on ice for a further 30 min and then at room tem-
perature for 65 h. The reaction was then diluted with toluene
(20 mL) and washed with NaHCO3 solution (10% w/v, 3 ꢁ 20 mL)
and brine (20 mL). The organics were dried (MgSO4), filtered and
concentrated to give the crude product as an amber oil. Purification
was carried out via flash chromatography (applied in CH2Cl2;
eluted 80:1 to 10:1 CH2Cl2/EtOAc) to afford the title compound
as an off white solid (386 mg, 1.38 mmol, 45%). Mp: 166 °C;
4.7. General method for synthesis of 2-[(3-chloropyrazin-2-
yl)amino]-1-(2-aryl)ethanol, illustrated for the preparation of
2-[(3-chloropyrazin-2-yl)amino]-1-(2-naphthyl)ethanol 18a
Amino alcohol 14a (289 mg, 1.55 mmol), 2,3-dichloropyrazine
(177 lL, 1.70 mmol) and Et3N (301 lL, 2.16 mmol) were dissolved
in 1,4-dioxane (3 mL) and the reaction was stirred under reflux,
under argon. After 19 h, the reaction was cooled to room tempera-
ture and the solvent removed in vacuo. The residue was taken up in
CH2Cl2 and washed with H2O (3 ꢁ 20 mL) and brine (1 ꢁ 20 mL).
The organic extracts were dried (Na2SO4), filtered and concen-
trated to give the crude product as an amber oil. Purification was
carried out via flash chromatography (applied in CH2Cl2; eluted
0% to 33% EtOAc) to afford the title compound as a yellow oil
Rf = 0.21 (10:1 CH2Cl2/EtOAc); IR (m
max/cmꢀ1, thin film): 3102,
3052; 1H NMR (500 MHz, CDCl3): dH = 7.58–7.62 (m, 2H), 7.64
(dd, J = 8.6, 1.7 Hz, 1H), 7.73 (d, J = 4.6 Hz, 1H), 7.92–7.95 (m, 2H),
8.02 (s, 1H), 8.04–8.05 (m, 1H), 8.04 (s, 1H), 8.30 (d, J = 4.6 Hz,
1H); 13C NMR (125 MHz, CDCl3): dC = 116.4, 124.7, 125.2, 127.3,
127.4, 127.6, 128.0, 128.2, 128.6, 129.4, 129.7, 133.4, 133.5,
134.8, 138.4, 144.5; LRMS m/z (ESI+): 282 [M(37Cl)+H]+, 280
[M(35Cl)+H]+; HRMS m/z (ESI+): Found 280.0646 [M(35Cl)+H]+;
(295 mg, 0.983 mmol, 63%). Rf = 0.64 (2:1 CH2Cl2/EtOAc); IR (mmax
/
cmꢀ1, thin film): 3419, 3054, 2922, 1523; 1H NMR (500 MHz,
CDCl3): dH = 3.65–3.70 (m, 1H), 3.88 (br s, 1H), 3.92–3.97 (m, 1H),
5.12 (dd, J = 7.5, 2.8 Hz), 5.67 (t, J = 5.3 Hz), 7.47–7.50 (m, 3H),
7.59 (d, J = 2.7 Hz, 1H), 7.81–7.84 (m, 3H), 7.86 (s, 1H), 7.91 (d,
J = 2.7 Hz, 1H); 13C NMR (125 MHz, CDCl3): dC = 49.4, 73.9, 123.9,
124.8, 126.1, 126.4, 127.8, 128.0, 128.4, 131.3, 133.2, 133.3,
135.1, 139.4, 140.2, 151.5; LRMS m/z (ESI+): 300.1 [M(35Cl)+H]+,
284.2 [M(37Cl)ꢀOH]+, 282.2 [M(35Cl)ꢀOH]+; HRMS m/z (ESIꢀ):
Found 298.0731 [M(35Cl)ꢀH]ꢀ; C16H13ClN3O requires 298.0747.
C16H11ClN3 requires 280.0642.
4.10. General method for the synthesis of 4-methyl-N-[4-[3-
arylimidazo[1,2-a]pyrazine-8-yl]]-sulfonamides, illustrated for
the preparation of 4-methyl-N-[4-[3-(2-naphthyl)imidazo[1,2-
a]pyrazine-8-yl]aminophenyl]benzenesulfonamide 5
All glassware was evacuated and flushed with argon prior to
use. Compound 20a (283 mg, 1.01 mmol), N-(4-aminophenyl)-4-
methylbenzenesulfonamide 23 (318 mg, 1.21 mmol), K2CO3
(167 mg, 1.21 mmol), Pd(dba)2 (5.80 mg, 1 mol %) and tert-butyl
4.8. General method for synthesis of 2-[(3-chloropyrazin-2-
yl)amino]-1-(2-aryl)ethanones, illustrated for the preparation
of 2-[(3-chloropyrazin-2-yl)amino]-1-(2-naphthyl)ethanone
19a
t
XPhos (21.5 mg, 5 mol %) were taken up in BuOH (6 mL) and the
reaction was stirred under reflux under Ar for 46 h. The reaction
mixture was cooled to room temperature, diluted with MeOH
(100 mL) and filtered through Celite (pre-washed with MeOH).
Flash chromatography (applied in CH2Cl2; eluted 100:1 to 50:1 to
8:1 CH2Cl2/EtOAc) was carried out to give the title compound as
a yellow solid (181 mg, 0.355 mmol, 35%). Mp: >200 °C; Rf = 0.12
DMSO (982
(60 mL) and the reaction mixture was cooled to and maintained
at ꢀ78 °C. Oxalyl chloride (586 L, 6.93 mmol) was added drop
lL, 13.9 mmol) was dissolved in anhydrous CH2Cl2
l
wise and the mixture was stirred for 20 min. 17a (1.60 g,
5.33 mmol), dissolved in anhydrous CH2Cl2 (40 mL) was added
dropwise, and stirred for 20 min. Et3N (3.54 mL, 26.6 mmol) was
added dropwise and the reaction mixture was allowed to warm
to room temperature over a period of 2.5 h. The reaction was then
quenched with H2O (50 mL) and organics extracted, which were
then washed with 2.0 M HCl (2 ꢁ 40 mL), NaHCO3 (satd aq
40 mL), H2O (40 mL) and brine (40 mL). The organic layer was
dried (MgSO4), filtered and the solvent removed in vacuo to give
a yellow/orange solid. Flash chromatography (applied in CH2Cl2;
(10:1 CH2Cl2/EtOAc); IR (m
max/cmꢀ1, thin film): 3240, 3057, 1623,
1500, 1330, 1154; 1H NMR (600 MHz, CDCl3): dH = 2.37 (s, 3H),
7.08–7.10 (m, 2H), 7.17 (s, 1H), 7.21 (d, J = 8.3 Hz, 2H), 7.52 (d,
J = 4.7 Hz, 1H), 7.57–7.58 (m, 2H), 7.64 (dd, J = 8.5, 1.6 Hz, 1H),
7.65 (d, J = 8.3 Hz, 2H), 7.73 (s, 1H), 7.79–7.80 (m, 3H), 7.91–7.92
(m, 2H), 8.00 (d, J = 8.5 Hz, 1H), 8.03 (s, 1H), 8.39 (br s, 1H); 13C
NMR (150 MHz, CDCl3): dC = 21.6, 109.4, 120.3, 123.7, 125.2,
125.4, 127.1, 127.3, 127.9, 128.1, 128.9, 129.0, 129.3, 129.7,
130.4, 131.2, 133.1, 133.4, 136.1, 137.2, 143.7, 146.2; LRMS m/z

J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
6469
(ESIꢀ): 504 [MꢀH]ꢀ; HRMS m/z (ESIꢀ): Found 504.1503 [MꢀH]ꢀ;
4.13. 8-(Methylsulfonyl)-2-(naphthalen-2-yl)imidazo[1,2-a]-
pyrazine 24
C
29H22N5O2S requires 504.1494.
4.11. General method for synthesis of 2-aryl-8-chloro-
imidazo[1,2-a]pyrazines, illustrated for the preparation of 8-
chloro-2-(2-naphthyl)imidazo[1,2-a]pyrazine 21a
Compound 21a (1.12 g, 4.01 mmol) was dissolved in anhydrous
DMSO (16 mL). NaSMe (337 mg, 4.81 mmol) was added portion-
wise and the reaction was stirred at 100 °C for 16 h. The mixture
was then cooled to room temperature, diluted with brine (50 mL)
and extracted with CH2Cl2 (100 mL). The organic layer was washed
with H2O (5 ꢁ 30 mL) and brine (30 mL), dried (MgSO4), filtered
and solvent removed in vacuo. Flash chromatography (applied in
CH2Cl2; eluted 0% to 1% to 2% EtOAc) afforded 8-(methylthio)-2-
(naphthalen-2-yl)imidazo[1,2-a]pyrazine as an off white/yellow
solid (989 mg, 3.40 mmol, 85%). Mp: 168 °C; Rf = 0.47 (5% EtOAc/
2-(Bromoacetyl)naphthalene 15a (3.14 g, 12.6 mmol), 2-amino-
3-chloropyrazine (1.63 g, 12.6 mmol), NaHCO3 (1.32 g, 16.7 mmol)
and tBuOH (60 mL) were stirred under reflux for 40 h. The reaction
mixture was cooled to room temperature and the solvent removed
in vacuo. The resulting orange solid was taken up in H2O (100 mL)
and extracted with CH2Cl2 (3 ꢁ 150 mL). The combined organic
layers were washed with H2O (75 mL) and brine (75 mL), dried
(MgSO4), filtered and concentrated in vacuo to give crude orange
solid. On addition of CH2Cl2 and MeOH (ꢂ1:1), insoluble material
filtered off to give the title compound as a cream fluffy solid
(1.05 g). Purification of the remaining filtrate via flash chromatog-
raphy (applied in petroleum ether; eluted 10% to 20% to 33%
EtOAc) afforded the title compound as a pale orange/brown solid
(1.38 g, 4.95 mmol, 39%). Mp: Decomposed before melting;
CH2Cl2); IR (m
max/cmꢀ1, thin film): 3055; 1H NMR (600 MHz,
CDCl3): dH = 2.71 (s, 3H), 7.47–7.52 (m, 2H), 7.72 (d, J = 4.5 Hz,
1H), 7.82 (d, J = 4.5 Hz, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.90 (d,
J = 8.5 Hz, 1H), 7.94 (d, J = 7.4 Hz, 1H), 7.98 (s, 1H), 8.04 (dd,
J = 8.5, 1.6 Hz, 1H), 8.55 (s, 1H); 13C NMR (150 MHz, CDCl3):
dC = 12.3, 110.3, 115.1, 124.3, 125.6, 126.4, 126.5, 127.9, 128.5,
128.6, 129.0, 130.2, 133.5 (2 signals), 138.9, 146.4, 154.4; LRMS
m/z (ES+): 292 [M+H]+; HRMS m/z (ES+): Found 292.0909 [M+H]+;
Rf = 0.34 (1:1 petroleum ether/EtOAc); IR (
m
max/cmꢀ1, thin film):
C17H14N3S requires 292.0908.
3026, 2921, 1495; 1H NMR (600 MHz, (CD3)2SO): dH = 7.55–7.58
(m, 2H), 7.76 (d, J = 4.4 Hz, 1H), 7.95–7.97 (m, 1H), 8.04 (d,
J = 8.6 Hz, 1H) 8.07–8.09 (m, 1H), 8.16 (dd, J = 8.6, 1.7 Hz, 1H),
8.64 (s, 1H), 8.67 (d, J = 4.4 Hz, 1H), 8.87 (s, 1H,); 13C NMR
(150 MHz, CDCl3): dC = 113.7, 120.6, 124.1, 124.9, 126.6, 126.7,
127.7 (2 signals), 128.4, 128.6, 129.9, 133.1, 133.2, 137.5, 141.2,
146.4; LRMS m/z (ESI+): 282 [M(37Cl)+H]+, 280 [M(35Cl)+H]+; HRMS
m/z (EI+): Found 279.05574 [M(35Cl)]+; C16H10N3Cl requires
279.05578.
8-(Methylthio)-2-(naphthalen-2-yl)imidazo[1,2-a]pyrazine
(1.77 g, 6.07 mmol) was dissolved in anhydrous CH2Cl2 (50 mL)
and the mixture was cooled on ice. mCPBA (5.23 g, 30.3 mmol)
was added in one portion and the reaction continued to stir at
room temperature for 5 h. The reaction was partitioned with
NaHCO3 (40 mL) and extracted with CH2Cl2 (3 ꢁ 30 mL); the com-
bined organic extracts were then washed with brine (30 mL), dried
(MgSO4), filtered and concentrated in vacuo. Flash chromatography
(applied in CH2Cl2; eluted 2–5% EtOAc) afforded the title com-
pound as a yellow solid (1.10 g, 3.40 mmol, 56.0%). Mp: >200 °C;
4.12. General method for the synthesis of 4-methyl-N-[4-[2-
arylimidazo[1,2-a]pyrazine-8-yl]]-sulfonamides, illustrated for
the preparation of 4-methyl-N-[4-[2-(2-naphthyl)imidazo[1,2-
a]pyrazine-8-yl]aminophenyl]benzenesulfonamide 11
Rf = 0.22 (10% EtOAc/CH2Cl2); IR (m
max/cmꢀ1, thin film): 3121,
3010, 1312, 1138; 1H NMR (600 MHz, CDCl3): dH = 3.84 (s, 3H),
7.51–7.54 (m, 2H), 7.86–7.87 (m, 1H), 7.92 (d, J = 8.5 Hz, 1H),
7.95–7.96 (m, 1H), 8.02 (d, J = 4.3 Hz, 1H), 8.08 (dd, J = 8.5, 1.4 Hz,
1H), 8.26 (s, 1H), 8.32 (d, J = 4.3 Hz, 1H), 8.59 (s, 1H); 13C NMR
(150 MHz, CDCl3): dC = 41.7, 110.9, 122.1, 124.3, 126.7, 126.8,
127.0, 127.9, 128.1, 128.7, 128.8, 129.1, 133.5, 134.0, 136.3,
148.6, 149.8; LRMS m/z (EI+): 323 [M]+; HRMS m/z (EI+): Found
323.07266 [M]+; C17H13N3O2S requires 323.07230.
All glassware was evacuated and flushed with argon prior to
use. 21a (50.0 mg, 0.178 mmol), N-(4-aminophenyl)-4-methylben-
zenesulfonamide 23 (56.3 mg, 0.215 mmol), NaOtBu (24.1 mg,
0.250 mmol, 1.4 equiv), 1 mol % Pd2(dba)3 (1.6 mg) and 3 mol %
DavePhos (2.1 mg) were weighed into a 25 mL round bottom flask.
Toluene (2 mL) was added and the reaction was stirred under
reflux for 24 h. The reaction mixture was cooled to room tempera-
ture and the solvents removed in vacuo. The residue was taken up
in CH2Cl2 (30 mL) and washed with water (3 ꢁ 30 mL). The com-
bined aqueous extracts were washed with CH2Cl2 (30 mL). The
CH2Cl2 layers were combined, washed with brine (30 mL), dried
over MgSO4 and the solvents removed in vacuo to give an off-white
solid. Flash chromatography (applied in toluene; eluted 3:1 tolu-
ene/EtOAc) gave the title compound as an off-white solid (20 mg,
0.039 mmol, 22%). Mp: decomposed before melting; Rf = 0.50 (1:1
4.14. Conversion of 24 to 4-methyl-N-[2-(2-naphthyl)-
imidazo[1,2-a]pyrazine-8-yl]benzenesulfonamide 7
NaH (7.1 mg of a 60% suspension in mineral oil, 4.6 mg,
0.186 mmol, 2.0 equiv) was washed by stirring in anhydrous hex-
ane (3 mL), syringing out the solvent and drying. Anhydrous DMF
(0.5 mL) was added to the flask under Ar, followed by 4-toluene
sulfonamide 22 (31.8 mg, 0.186 mmol, 2.0 equiv) in anhydrous
DMF (0.5 mL). The contents were then stirred at rt for 20 min
before 24 (30 mg, 0.093 mmol) in DMF (2 mL) was added dropwise,
and the reaction was stirred at 100 °C for 20 h. The reaction was
cooled to room temperature and quenched with satd aq NH4Cl
(30 mL) and extracted with ethyl acetate (3 ꢁ 30 mL). The com-
bined organic layers were washed with H2O (5 ꢁ 30 mL) and brine,
dried (MgSO4), filtered and solvent removed. Flash chromatogra-
phy (applied in petroleum ether; gradient 20% to 66% EtOAc) affor-
ded 7 (27.8 mg, 0.669 mmol, 72%). Mp: >200 °C; Rf = 0.32 (1:1
petroleum ether/EtOAc); IR (m
max/cmꢀ1, thin film): 3126, 2923,
2853, 1507, 1325, 1143; 1H NMR (600 MHz, CD3OD): dH = 2.42
(s, 3H), 7.20 (d, J = 5.2 Hz, 1H), 7.29 (d, J = 8.9 Hz, 2H), 7.36 (d,
J = 8.2 Hz, 2H), 7.52–7.56 (m, 2H), 7.58 (d, J = 8.9 Hz, 2H), 7.75
(d, J = 8.2 Hz, 2H), 7.92 (d, J = 7.1, 1H), 7.96–7.99 (m, 3H), 8.13
(dd, J = 8.5, 1.7 Hz, 1H), 8.53 (s, 1H), 8.56 (s, 1H); 13C NMR
(150 MHz, CD3OD): dC = 21.4, 113.9, 115.9 (2 signals), 122.9,
124.9, 125.8, 126.1, 127.6, 127.7, 128.4, 128.9, 129.3, 129.7,
130.7, 131.0, 133.5 (2 signals) 135.0 (2 signals), 137.7, 138.2,
145.2, 146.1, 148.1; LRMS m/z (ESI+): 506 [M+H]+, (ESIꢀ): 504
[MꢀH]ꢀ; HRMS m/z (ESI+): Found 506.1651 [M+H]+; C29H24N5O2S
requires 506.1651.
CH2Cl2/EtOAc); IR
(m ,
max/cmꢀ1 thin film): 3253; 1H NMR
(600 MHz, DMSO-d6): dH = 2.37 (s, 3H), 7.16 (br d, J = 5.2 Hz, 1H),
7.39 (d, J = 8.2 Hz, 2H), 7.51–7.54 (m, 2H), 7.86 (br d, J = 5.2 Hz,
1H), 7.89 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 7.7 Hz, 1H), 7.98 (d,
J = 8.6 Hz, 1H), 8.01–8.05 (m, 2H), 8.52 (s, 1H), 8.59 (s, 1H), 11.69

6470
J.R. Sayer et al. / Bioorg. Med. Chem. 22 (2014) 6459–6470
(s, 1H, NH); 13C NMR (150 MHz, DMSO-d6): dC = 21.0, 111.0, 115.3,
116.8, 123.8, 124.2, 126.2, 126.3, 126.6, 127.7, 128.3, 128.4, 129.5,
130.0, 132.8, 133.2, 135.6, 140.0, 142.7, 144.5, 145.3; LRMS m/z
(ESI+): 415 [M+H]+, (ESIꢀ): 413 [MꢀH]ꢀ, HRMS m/z (ESI+): Found
415.1219 [M+H]+; C23H19N4O2S requires 415.1229.
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4.15. Enzyme activity measurements
Assays for the activity of the HP0525 inhibitors were performed
using a colorimetric ATPase assay (Innova Biosciences), see SI for
details. The enzymatic reactions were performed in 96-well format
for 30 min at 37 °C followed by measuring the absorbance at
620 nm, detecting the presence of inorganic phosphate product.
For the IC50 measurements, each reaction contained 100 mM
Tris–HCl (pH 7.5), 2.5 mM MgCl2, 125
NaCl, 0.5 mM DTT 0.053 M HP0525 and various concentrations
of inhibitors (0–50 M or 0–250 M). Michaelis–Menten kinetics
were performed under the same conditions as above but with var-
ious concentrations of ATP ranging from 0 to 500 M, with and
without 10 M of compound 11. Both IC50 and Michaelis–Menten
lM ATP, 5% DMSO, 25 mM
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l
kinetics measurements were made in triplicate.
Acknowledgments
We thank the BBSRC for a PhD studentship (BBS/SE/2006/1326/
9) to JS, a PhD studentship (BBS/K/2005/12145) to F.B., and a
Research Grant (BB/D005469/1) to T.P.B. The Wellcome Trust are
also thanked for a Research Grant (082227) to K.W. and for a
PhD studentship (096617/Z/11/Z) to H.K. We would also like to
thank UCLB for HEIF Proof of Concept Funding (T.P., F.C., P.G. and
M.S.) and BioFocus for access to their SoftFocus library.
Supplementary data
34. Tetko, I. V.; Gasteiger, J.; Todeschini, R.; Mauri, A.; Livingstone, D.; Ertl, P.;
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Supplementary data (experimental details for the synthesis of
compounds 1–4, 6–10, 12, 13, 25–38, details of the optimization
of the conversion of 21a to 7, NMR data confirming the regiochem-
istry of 21a, 1H NMR spectra for all novel compounds, full descrip-
tion of the ATPase assay, IC50 curves for all compounds) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2014.09.036.
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