Parallel synthesis of potent, pyrazole-based inhibitors of Helicobacter pylori dihydroorotate dehydrogenase

Article abstract of DOI:10.1021/jm020112w

The identification of several potent pyrazole-based inhibitors of bacterial dihydroorotate dehydrogenase (DHODase) via a directed parallel synthetic approach is described below. The initial pyrazole-containing lead compounds were optimized for potency against Helicobacter pylori DHODase. Using three successive focused libraries, inhibitors were rapidly identified with the following characteristics: K_i < 10 nM against H. pylori DHODase, sub-μg/mL H. pylori minimum inhibitory concentration activity, low molecular weight, and > 10 000-fold selectivity over human DHODase.

Full text of DOI:10.1021/jm020112w

J . Med. Chem. 2002, 45, 4669-4678
4669
P a r a llel Syn th esis of P oten t, P yr a zole-Ba sed In h ibitor s of Helicoba cter pylor i
Dih yd r oor ota te Deh yd r ogen a se
Tasir S. Haque,*,† Seifu Tadesse,†,‡ J ovita Marcinkeviciene,§ M. J ohn Rogers,|, Christine Sizemore,|,
Lisa M. Kopcho,§ Karen Amsler,|,# Lisa D. Ecret,| Dong Liang Zhan,† Frank Hobbs,† Andrew Slee,|,#
George L. Trainor,† Andrew M. Stern,§ Robert A. Copeland,§ and Andrew P. Combs†
Department of Medicinal Chemistry, Department of Chemical Enzymology, and Antimicrobials Group, Bristol-Myers Squibb
Company, Experimental Station, Route 141 and Henry Clay Road, Wilmington, Delaware 19880
Received March 11, 2002
The identification of several potent pyrazole-based inhibitors of bacterial dihydroorotate
dehydrogenase (DHODase) via a directed parallel synthetic approach is described below. The
initial pyrazole-containing lead compounds were optimized for potency against Helicobacter
pylori DHODase. Using three successive focused libraries, inhibitors were rapidly identified
with the following characteristics: Ki < 10 nM against H. pylori DHODase, sub-µg/mL H. pylori
minimum inhibitory concentration activity, low molecular weight, and >10 000-fold selectivity
over human DHODase.
In tr od u ction
Helicobacter pylori is a Gram-negative microaero-
philic bacterium that infects up to 50% of the world’s
human population.1 H. pylori resides in the acidic
surroundings of the stomach, utilizing a high urease
enzyme activity to provide a locally alkaline environ-
ment. First reported in 1982, H. pylori has been
implicated in numerous gastrointestinal disorders and
is associated with gastric ulcers, gastritis, and gastric
cancer.2 The current treatment of H. pylori infections
typically utilizes a multiple drug therapy involving at
least one broad spectrum antibiotic (antimicrobial
therapy) and a proton pump inhibitor (antisecretory
therapy). However, a H. pylori specific antimicrobial
would be very desirable; a specific agent should avoid
many of the negative gastrointestinal side effects as-
sociated with a broad spectrum antibacterial resulting
from eradication of the normal gastrointestinal flora.
Dihydroorotate dehydrogenase (DHODase) is involved
in the de novo pyrimidine synthesis pathway in most
prokaryotic and eukaryotic cells. DHODase catalyzes
the oxidation of dihydroorotate to orotate (Figure 1), the
fourth step in de novo pyrimidine biosynthesis.3 Two
major families of DHODases have been described.4 A
low degree of similarity exists between the sequences
of the two families (usually <20%).5 The enzymes may
be distinguished by their location and cofactor utiliza-
tion. Family 1 is found in the cytosol, while family 2 is
membrane-associated. H. pylori DHODase belongs to
family 2, as do all other DHODase enzymes from Gram-
negative bacteria and mammals. All family 2 enzymes
identified to date make use of an endogenous flavin
mononucleotide (FMN) redox cofactor and exogenous
F igu r e 1. DHODase involvement in the pyrimidine biosyn-
thetic pathway.
coenzyme Q6 electron acceptor in the FMN oxidation
half-reaction.6
As pyrimidines are crucial to the survival of the
bacteria, it is not surprising to find that many organ-
isms have pyrimidine salvage pathways, whereby py-
rimidines may be generated by catabolism of nucleic
acids and via salvage from exogenous sources of nucle-
otides. The genome sequences of two strains of H. pylori
have been reported.7,8 Examination of the two genomes
suggests that while the pathway for purine salvage is
present in H. pylori, the pathway for pyrimidine salvage
is, to a large extent, missing.6 If this salvage pathway
is in fact absent, then H. pylori would be critically
dependent on biosynthesis of pyrimidines; therefore, the
pyrimidine pathway would be an attractive target for
antibacterial therapy. DHODase is also present in
mammalian cells. On the basis of previous research
demonstrating that human DHODase could be inhibited
with high selectivity over bacterial DHODase,9 we
hypothesized that it would be possible to identify
compounds that would selectively inhibit bacterial
DHODase over the corresponding human enzyme.10 It
should be noted that a high degree of selectivity over
human DHODase is required, as inhibition of human
* To whom correspondence should be addressed. Tel: (302)467-6028.
Fax: (302)467-6951. E-mail: Tasir.haque@bms.com.
† Department of Medicinal Chemistry.
‡ Current address: Amgen, Inc., Thousand Oaks, CA 91320.
§ Department of Chemical Enzymology.
| Antimicrobials Group.
Current address: National Institute of Allergy and Infectious
Diseases, Bethesda, MD 20892.
# Current address: Enanta Pharmaceuticals, Watertown, MA 02472.
10.1021/jm020112w CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/06/2002

4670 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Haque et al.
commercial suppliers or an in-house collection). Com-
putational resources for this project were limited, so no
structure-based or diversity selection computational
methods were applied. Instead, the side chains were
chosen to explore specific hypotheses regarding inhibitor
binding (e.g., benzylamines in RB set of library 2) or to
maximize diversity at a position using readily available
and chemically compatible reagents (e.g., RA amine set
of library 1).
F igu r e 2. Pyrazole DHODase inhibitors 1 and 2.
The synthesis of the first library was carried out as
shown in Scheme 1. The first amine side chain (RA) was
attached to backbone amide linker (BAL)-linked poly-
styrene resin via a reductive amination. The monoacid/
monoester pyrazole core containing RC was then coupled
to the amine, after which the ester was saponified with
LiOH in tetrahydrofuran (THF)/H2O/MeOH. Finally,
the second amine side chain (RB) was coupled to the
pyrazole acid, and the resulting product was cleaved
from the polymer support using trifluoroacetic acid.
DHODase has been shown to have an immunosuppres-
sive effect.11,12 Such an effect is clearly undesirable when
combatting a bacterial infection.
We have previously reported the selective inhibition
of bacterial DHODase by pyrazoles6 and thiadiazo-
lidinediones.10 Pyrazole compounds 1 and 2 (Figure 2)
were demonstrated to be potent inhibitors of H. pylori
DHODase (Ki values against H. pylori DHODase of 26
and 50 nM, respectively, and minimum inhibitory
concentration (MIC) of 3 µg/mL vs H. pylori for com-
pound 1). These compounds are competitive with coen-
zyme Q6 and were highly selective for H. pylori over
human DHODase. In this paper, we discuss our efforts
to improve upon the enzyme inhibition and antibacterial
potency of the pyrazole series of compounds, beginning
from lead compounds 1 and 2.
All compounds were examined by analytical high-
performance liquid chromatography (HPLC) chroma-
tography (detecting at 220 and 254 nm) and flow
injection mass spectrometry following cleavage from
solid support, whereupon it was discovered that 43 of
the desired 120 compounds were not obtained in amounts
sufficient for biological assay. Comparison of the prod-
ucts obtained to those that failed showed that the poor
yields were correlated to the use of anilines at RA. For
example, 4-aminopyridine (A6, shown in Figure 4) failed
with all RB combinations. After several test reactions
were conducted with anilines, we noted that while the
reductive amination of anilines to the aldehyde linker
proceeded smoothly, subsequent coupling of the pyrazole
to the secondary aniline (using standard O-(7-azaben-
zotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (HATU) coupling conditions) gave poor
yields of the desired amide. We chose to purify this first
library, obtain as many of the desired products as
possible, and then revisit our approach to the synthesis
of solid phase pyrazole libraries.
Ch em istr y
After compounds 1 and 2 were identified as H. pylori
DHODase inhibitors, we began to investigate the chem-
istry required for synthesis of analogues in a parallel
format. The two amides off the central pyrazole in the
lead compounds suggested natural starting places for
disconnections. The core pyrazole could be accessed with
one carboxylic acid protected as the ethyl ester, thus
allowing the two coupling sites to be differentiated
(Scheme 1). We chose to synthesize small, focused
libraries so that information gained in the assay of a
particular library could be incorporated into the plan-
ning of the subsequent library. An iterative approach
also allowed the incorporation of a larger number of side
chains at each position, without the exponential increase
in the total number of compounds expected in a “true”
combinatorial library. Additionally, we elected to use a
pyrazole core that had been synthesized by “traditional”
solution phase methods rather than assembling the
pyrazole on a solid support. Synthesis of the core was
accomplished with high selectivity for the desired re-
gioisomer, which was then separated from any of the
minor regioisomer by column chromatography. By using
the isomerically pure pyrazole core and synthesizing the
libraries in a spatially separate format, it was possible
to unambiguously assign both the identity and the
regiochemistry of all of our products. In contrast, most
on-support synthetic approaches to the desired pyra-
zoles would have required a lengthy development time
to obtain regiospecifically substituted products or would
have resulted in a mixture of two regioisomers (requir-
ing postassay analysis to determine the identity of the
active regioisomer).
The pyrazole synthesis on solid support was optimized
to allow for incorporation of anilines at the RA position
in library 2. After a variety of coupling reagents and
conditions were tried, double coupling of the resin-bound
amine at 50 °C using PyBrOP as the coupling reagent
was found to provide a high yield (75-90%) of the
support-bound pyrazole product (Figure 3). In both the
second and the third libraries, PyBrOP was used
whenever a pyrazole acid was coupled to a support-
bound aniline, and HATU was used for coupling a
pyrazole acid to a support-bound alkylamine.
We concurrently developed an alternate synthetic
approach to aniline-substituted pyrazole amides, whereby
the furan-protected pyrazole acid could be coupled onto
support and then oxidized to the carboxylic acid to
liberate the second coupling site (Figure 3). In this route,
the RB side chains are initially reductively aminated
onto the support, and the RA side chain is coupled in
the final step before cleavage. Coupling of the primary
aniline to the support-bound carboxylic acid would allow
for the incorporation of anilines at RA, as the difficulties
with the aniline coupling were only observed when the
aniline was attached to the linker. However, a small
loss in yield of the final products was observed in this
The side chain sets for the libraries were selected
based on a combination of knowledge of medicinal
chemistry and availability of reagents (from either

Helicobacter pylori Dihydroorotate Dehydrogenase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4671
Sch em e 1. Support-Bound Synthesis of Pyrazoles
Resu lts a n d Discu ssion
Library 1 (Figure 4) was built around the 4-methox-
yphenyl-pyrazole core identified from lead compounds
1 and 2. This library was designed to provide a rapid,
coarse exploration of the structure-activity relation-
ships (SAR) around both of the amine substituents. The
77 compounds isolated after purification were assayed
for activity against H. pylori DHODase (Figure 5). Two
new inhibitors were identified (3 and 4, Figure 6).
However, neither of the new inhibitors had better than
1 µM IC50 values vs H. pylori DHODase. Compound 3
illustrates the importance of the orientation of the
pyrazole aryl substituent for inhibitory activity, as 3 was
50-fold less potent than the corresponding pyrazole
regioisomer 1 (Figure 2).
The second pyrazole library (library 2, Figure 7) was
designed to specifically explore the RB site, with a
variety of different side chains at RB, two RA side chains,
and two pyrazole cores (RC). Addition of a new side chain
at RA and a new core (i.e., new side chain at RC) allowed
for a preliminary exploration of potential additive
interactions between side chains. The RB side chains
were selected in order to probe the binding pocket of
the benzyl side chain. Synthesis of library 2 was high
yielding (70-95%) and quite clean, with product purities
uniformly in excess of 80% (as determined by HPLC/
F igu r e 3. Two synthetic routes for accessing aniline-contain-
ing RA side chains. In the left route, PyBrOP is used to couple
the pyrazole acid to the support-bound aniline; in the right
route, the aniline is coupled to the support-bound pyrazole
after RuCl3-catalyzed oxidation of the furan to the carboxylic
acid.
1
UV detection at 254 nm and examination of product H
NMR spectra). Therefore, the library was assayed for
inhibitory activity without further purification, and
selected active inhibitors were resynthesized, purified,
and reevaluated. A very good correlation between Ki
values obtained with unpurified and purified material
was observed for this series of compounds.
route, possibly due to cleavage during the oxidation step.
A mild reduction in purity was also observed in several
cases where oxidation of the furan to the carboxylic acid
was incomplete.
The first method described above (use of PyBrOP as
coupling reagent) permits the inclusion of anilines at
both RA and RB positions without changing the core
pyrazole used in the synthesis. We also found complete
hydrolysis of the support-bound ester to the carboxylic
acid to be more reliable than complete oxidation of the
support-bound furan to the carboxylic acid. For these
two reasons, the more versatile PyBrOP coupling re-
agent route was chosen for libraries 2 and 3.
A number of compounds from this second library were
inhibitors of H. pylori DHODase; 18 compounds had
inhibitory activity at concentrations under 11 µM, with
7 of the 18 having Ki values between 10 and 100 nM.
When RB was 4-fluorobenzyl (B17), little change in
enzyme inhibition (vs compound 1) was observed, but
as the RB para substitutent increased in size (chloro to
methoxy to cyclohexyl), there was a corresponding

4672 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Haque et al.
F igu r e 4. Side chains for pyrazole library 1.
F igu r e 5. Graph of library 1, % inhibition (at 10 µM
compound) of H. pylori DHODase vs side chains RA and RB.
F igu r e 6. Compounds 3 (RA ) A8, RB ) B9) and 4 (RA ) A8,
RB ) B4) from library 1. Note that 1 (Figure 1) and 3 are
regioisomers around the pyrazole core.
decrease in potency. Analogues with aromatic side
chains at RB were considerably more potent DHODase
inhibitors than analogues with RB ) alkyl side chains.
For example, compare the activity of the benzyl RB side
chain in 1 (A13:B15:C1, 26 nM) with the two most
potent alkyl RB replacements, isobutyl (compound 5,
A13:B11:C1, 138 nM) or tetrahydrofuryl (racemic com-
pound 6, A13:B13:C1, 331 nM), Table 1.
The RC ) 4-methoxyphenyl (C1) inhibitors in general
were more potent than the corresponding RC ) phenyl
(C2) inhibitors, especially for mildly potent compounds
(e.g., 331 nM for A13:B13:C1 vs 4400 nM for A13:B13:
C2). At the RA position, the 4-(pyrrolidine-1-carbonyl)-
phenyl substituent A13 was clearly superior to the
cyclopentyl substituent A3, with none of the compounds
containing the latter RA substituent having inhibitory
activity better than 10 µM vs H. pylori DHODase.
All compounds from library 2 were evaluated for
whole cell activity vs H. pylori. MIC was determined
against a reference American Type Culture Collection

Helicobacter pylori Dihydroorotate Dehydrogenase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4673
significant decrease in inhibition (Ki ) 91 nM for 10,
A9:B15:C3).
At the RB position, the (3-aminomethyl)pyridine (B22,
Table 3, 11) or unsubstituted benzylamine (B15, Table
3, 8) side chains were preferred over the 3- or 4-methyl-
substituted benzylamine side chains (B16 and B21,
Table 3, 12). At the RC position, the 4-chlorophenyl
substituent (C3) was consistently favored over the
4-methoxyphenyl (C1) or unsubstituted phenyl (C2)
substituent. For example, compare Table 3, compounds
11 and 13. Finally, all of the compounds containing a
methyl RC substituent (C4) were inactive against H.
pylori DHODase. For the pyrazole substituent (RC), the
general order of activity was 4-chlorophenyl > 4-meth-
oxyphenyl > phenyl . methyl.
Several compounds with good whole cell activity vs
H. pylori were identified from library 3. Compounds 8
and 11 (MIC values of 0.125 and 0.250 µg/mL, respec-
tively) were the most potent compounds identified both
in terms of DHODase inhibition and activity in the MIC
assay. These compounds were of sufficient antibacterial
potency that they could be carried forward into a proof
of principle study. Preliminary data suggest that H.
pylori growth cannot recover after treatment of the
organism with these inhibitors and subsequent com-
pound removal. These results suggest that the com-
pounds act as bactericidal agents; however, this con-
clusion must be viewed with some caution because of
the very slow growth rates of H. pylori in cell culture.
F igu r e 7. Side chains for pyrazole library 2.
(ATCC) strain of H. pylori under standard conditions.
As discussed above, numerous inhibitors with Ki values
lower than 1 µM against the H. pylori DHODase enzyme
were identified. However, as compared to lead com-
pound 1, none of the library 2 compounds had an
improved activity (<3 µg/mL) vs H. pylori bacteria in
the whole cell assay.
To define further the whole cell activity of the pyra-
zole-based compounds, spontaneous resistant mutants
of H. pylori were obtained at 4× and 8× MIC levels of
compound 1. These resistant mutants were obtained at
a frequency of approximately (1-2) × 10-7 viable H.
pylori cells plated for both the ATCC strain 43629 and
the SS1 strain,13 implying that a single target is the
site of action for compound 1.14 The resistance was
determined to be at high levels of 1 (>64 µg/mL).
Genomic DNA was prepared, and the pyrD gene was
amplified from several of these resistant mutants. In
multiple isolates, a common mutation CfA was deter-
mined, changing codon 18 from GCG fGAG, corre-
sponding to an AlafGlu mutant enzyme (A18E). To
confirm this, the CfA change was introduced by in vitro
mutagenesis of the wild-type enzyme. The mutant and
wild-type H. pylori enzymes were cloned and overpro-
duced in a pyrD mutant of Escherichia coli, so there was
no contamination of endogenous E. coli DHODase
activity.15 Purification and biochemical analysis of the
mutant A18E DHODase revealed that while the kinetic
parameters for the dihydroorotate substrate and coen-
zyme Q0 were unchanged, the Ki for compound 1 was
elevated 6-fold. The increase in Ki is less than the
increase in MIC (at least 8-fold) for the resistant
mutants. However, the reported toxicity of upstream
intermediates in the pathway16 and the overall effect
of inhibition of the pathway for pyrimidine biosynthesis
on cell growth were not considered in the assay of the
single enzyme. These factors may account for the
observed differences between the changes in Ki and MIC
values.
For our final directed library (Figure 8), we focused
our attention on the RA side chain. It was believed that
reduction of the molecular weight of the inhibitors and
incorporation of pyridine side chains at RB might result
in compounds with more desirable physical character-
istics. By decreasing the size of the molecules and
adding aromatic or tertiary amines, aqueous solubility
and cell permeability of the compounds may be im-
proved, potentially resulting in greater antimicrobial
potency. Several side chains were selected to determine
which characteristics of the somewhat large A13 side
chain were necessary for tight binding to H. pylori
DHODase. We were also able to examine several other
side chains at RA and RB, while still focusing on benzyl
derivatives at RC. This allowed us to probe for additive
effects between sites, while synthesizing a relatively
small number of compounds (160 members in library
3). All products from the third library were purified by
preparative liquid chromatography/mass spectrometry
(LC/MS) prior to biological assay.
There were a number of potent (Ki < 100 nM)
compounds identified in library 3 (see Table 2). It is
clear that the A13 side chain can be truncated all the
way down to the unsubstituted aniline side chain (A14)
at the RA position with no loss of inhibitory potency
(Table 3, compounds 7 and 8). The structural constraint
introduced by the amide in the A13 side chain is not
necessary for activity, as demonstrated by compound 9
with the extended A18 side chain (Ki ) 8 nM, A18:B22:
C3). However, replacement of the aniline with an alkyl
side chain at RA (e.g., A9, A15, or A16) resulted in a
DHODase has also been identified as the target of an
antifungal compound in Aspergillus nidulans.17 In this
case, moderate and high level resistant mutants were

4674 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Ta ble 1. Selected Actives from Libraries 1 and 2
Haque et al.
Ta ble 2. Comparison of Number of Active Compounds (within
Three Potency Ranges) in the Three Pyrazole Libraries
size (no. of
compds)
1 µM <
Ki < 10 µM
100 nM <
Ki < 1 µM
Ki <
100 nM
library
1
2
3
77
44
160
2
7
5
0
4
6
0
7
31
binding in the human DHODase enzyme.15 These data
support the N-terminal portion of the enzyme in cofactor
binding and specificity in interactions with inhibitors,
supported by the recent description of the X-ray crystal-
lographic structure of human DHODase with inhibi-
tors.18
A spontaneously resistant mutant of H. pylori was
obtained with compound 13 using a method analogous
to that described above. Each mutant was then tested
for cross-resistance to the pyrazole not used to develop
the strain. (For example, the MIC of compound 1 was
determined for the mutant developed using compound
13.) As shown in Table 4, a high level of cross-resistance
between the strains derived from the two pyrazoles was
observed. This result suggests that bacterial isolates
resistant to one pyrazole inhibitor will be resistant to
the other pyrazole inhibitors identified above. The onset
of spontaneous resistance in vitro to compound 1 was
rapid, and cross-resistance to other members of the
inhibitor class is high, suggesting a liability to the
pyrazoles as potential H. pylori therapeutic agents.
F igu r e 8. Side chains for pyrazole library 3.
obtained from several mutations; the high level resis-
tant enzyme was about 2500 times less sensitive to the
inhibitor as measured by IC50 determination in crude
enzyme preparations. An alignment of DHODase en-
zymes shows that the A18E mutation in the H. pylori
enzyme is close to a corresponding histidine 26 muta-
tion, defined by alanine scanning as affecting brequinar
Con clu sion
A number of potent H. pylori DHODase inhibitors
based upon a pyrazole core were identified. Directed
parallel synthesis enabled the rapid optimization of lead
compounds via three focused libraries. All of the librar-
ies discussed above were developed, synthesized, and

Helicobacter pylori Dihydroorotate Dehydrogenase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4675
Ta ble 3. Selected Inhibitors from Library 3
Ta ble 4. MIC of Compounds 1 and 13 vs Wild Type and
Resistant H. pylori Isolates
The synthesis of the first two small libraries allowed
us to rapidly identify motifs important to enzyme
inhibitor potency. This knowledge was incorporated into
the design of the third library. The third library was
devised with the goal of finding multiple potent DHO-
Dase inhibitors. These could then be screened for whole
cell activity against H. pylori. We favored the approach
of finding many potent inhibitors and screening for the
whole cell activity in our third library, rather than
trying to optimize whole cell activity in an iterative,
stepwise fashion. As shown in Table 2, library 3
demonstrates the utility of this approach: library 3
contained 31 compounds with Ki values under 100 nM,
several of which possessed potent whole cell activity.
We believe that a similar strategy could be successful
for a variety of medicinal targets, where the generation
of large numbers of potent compounds in later libraries
would allow for selection of compounds having desirable
biological and pharmacological characteristics.
wild type
MIC (µg/mL)
1 resistant isolate
MIC (µg/mL)
13 resistant
isolate MIC
compd
1
13
3.00
0.50
>64
32
>64
24
purified by two chemists in 9 months. The most potent
DHODase inhibitors are very selective (>10 000-fold)
for H. pylori over human DHODase enzymes. The
inhibitors display low µg/mL to sub-µg/mL antibacterial
activity against H. pylori. However, no antibacterial
activity is seen against a variety of other Gram-positive
and Gram-negative organisms (Staphylococcus aureus,
Enterococcus faecalis, Streptococcus pyogenes, E. coli,
Haemophilus influenzae, Moraxella catarrhalis, and
Psuedomonas aeruginosa). The authors speculate that
this selectivity is due to the absence of a pyrimidine
salvage pathway in H. pylori and the presence of an
active pathway in the other organisms. Compounds 8
and 11 are the most potent DHODase enzyme inhibitors
identified (Ki ) 4 nM for both). Both compounds were
also very active against H. pylori in a whole cell
antimicrobial assay (MIC ) 0.125 µg/mL for 8, 0.250
µg/mL for 11). The pyrazoles have a high level of
spontaneous resistance (on the order of (1-2) × 10-7
viable H. pylori cells) as well as cross-resistance within
the class, suggesting a single target as the site of action
resulting in whole cell activity.
Exp er im en ta l Section
En zym ology/Biology. P r otein Exp r ession a n d P u r ifi-
ca tion . H. pylori and human DHODase were expressed and
purified as previously described.6 A full description is provided
in the Supporting Information accompanying this paper.
En zym a tic Activity Assa ys, In h ibition Stu d ies, a n d
Cellu la r Stu d ies. Human and H. pylori enzymatic activity
assays and inhibition studies and H. pylori cellular studies
were performed as previously described.6
Ch em istr y. Intermediates were monitored by thin-layer
chromatography (silica gel plates), flow injection positive ion

4676 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Sch em e 2. Synthesis of Pyrazole Cores
Haque et al.
electrospray mass spectrometry using a Micromass ZMD
quadrapole mass spectrometer, and HPLC on a Varian Prostar
instrument (eluting with acetonitrile/water through a 4.6 mm
× 50 mm C-18 column and monitoring at 220 and 254 nm).
Final products of support-bound syntheses were characterized
by flow injection positive ion electrospray mass spectrometry,
by HPLC (254 nm detection), and by 1H LC NMR with a
Varian Inova 400 MHz NMR spectrometer equipped with a
60 µL active volume flow probe and a Gilson 215 liquids
handler, using 3:1 DMSO-d6/CDCl3 as solvent. NMR spectra
of compounds synthesized in solution were obtained on a
Varian VXR/Unity 300 MHz spectrometer. Elemental analysis
of products was performed by Quantitative Technologies, Inc.
Pyrazole cores and library compounds were synthesized from
commercially available starting materials and solvents.
Solu tion P h a se Syn th esis of P yr a zole Cor es. The
pyrazole monoacid/monofuran cores were synthesized as previ-
ously described (as shown in Scheme 2).19 Reaction of the
appropriate hydrazine and 2-furanpropanoic acid, â-oxo-, ethyl
ester in the presence of acetic acid resulted in the desired
pyrazole ester in good to high regioselectivity.20 The product
was then further purified by crystallization or silica gel flash
chromatography to provide the desired pyrazole as a single
regioisomer. Finally, the furan was oxidized to the carboxylic
acid as previously reported, with sodium periodate and ru-
thenium(III) chloride21 or potassium permanganate to provide
(after purification) the desired pyrazole acid/ester in 55-85%
yield.
Su p p or t-Bou n d Syn th esis of P yr a zoles. The pyrazoles
were synthesized on polystyrene beads using Robbins filtration
blocks.22 Amines were reductively aminated onto a BAL linker,
resulting in a support-bound secondary amine. Monoacid/
monoester pyrazole cores were then coupled to the secondary
amines using PyBrOP. The resulting support-bound ethyl ester
was hydrolyzed with LiOH in THF/H2O/MeOH. Finally, the
second amine was coupled to the pyrazole acid, resulting in
the desired products. Pyrazoles were cleaved into 2 mL 96 well
plates using TFA/CH2Cl2. A representative procedure (used
for synthesis of the second library, including both resin-bound
amines and anilines) is given below.
Step 2A. Cou p lin g of P yr a zole Ca r boxylic Acid Cor es
on to Resin -Bou n d Secon d a r y Am in es. The resin-bound
amine from step 1 was distributed into two fritted tubes (one
for each of the two pyrazole cores), 0.500 g/tube. One of the
pyrazole cores was dissolved in DMF to afford a 0.45 M
solution. To this solution was added 2 equiv of DIEA, followed
by 1 equiv of HATU. The resulting solution was added to the
tubes containing resin-bound amine. The tubes were capped
and agitated on an orbital shaker for 4 h (running the reaction
overnight afforded no advantage). The solution was drained
from the resin, and the resin was washed with DMF (5×),
methanol (5×), and CH2Cl2 (7×). Reaction completion was
determined by ninhydrin test and by cleaving a small portion
(20-30 mg) of resin with 1:1 TFA/CH2Cl2 for 1 h and then
checking by ES-MS and HPLC. Acylation was typically
complete after one coupling reaction, but if necessary, a second
coupling reaction was performed to fully acylate the resin-
bound amine.
Step 2B. Cou p lin g of P yr a zole Ca r boxylic Acid Cor es
on to Resin -Bou n d Secon d a r y An ilin es. The resin-bound
amine from step 1 was distributed into two vials (one for each
of the two pyrazole cores), 0.500 g/vial. Each of the pyrazole
cores was dissolved in DMF to afford two 0.45 M solutions.
To each solution was added two equiv of DIEA, followed by 1
equiv of PyBrOP (bromo-tris-pyrrolidino-phosphonium hexaflu-
orophosphate). The resulting solution was added to one of the
vials containing resin-bound amine. The vial was capped,
placed in a 50 °C sand bath, and agitated on an orbital shaker
for 2 h. The suspension containing the resin was transferred
to a fritted tube, drained, and rinsed with DMF (3×). The resin
was transferred back into a glass vial, and the coupling
reaction was then repeated using the same conditions as above
(PyBrOP, DIEA in DMF, 50 °C, 2 h). The suspension contain-
ing the resin was transferred to a fritted tube and drained,
and the resin was washed with DMF (5×), methanol (5×), and
CH2Cl2 (7×). Reaction completion was determined by ninhy-
drin test and by cleaving a small portion (20-30 mg) of resin
with 1:1 TFA/CH2Cl2 for 1 h and then checking for product
formation by ES-MS and HPLC.
Step 3. Hyd r olysis of Su p p or t-Bou n d Eth yl Ester .
Lithium hydroxide (5 equiv relative to resin loading) was
dissolved in a THF/MeOH/H2O mixture (5:2:2 ratio, 0.1 M
LiOH final concentration). The resulting solution was added
to the resin-bound ester in the Robbins blocks. The blocks were
then sealed and agitated for 4 h at room temperature. The
wells were then drained, and the resin was washed with THF
(4×), H2O (5×), MeOH (5×), and CH2Cl2 (7×). A small portion
of resin was cleaved, and the reaction progress was checked
by mass spectroscopy and HPLC. Hydrolysis of resin-bound
ester was typically complete after one treatment with LiOH.
Step 4. Cou p lin g of Am in es or An ilin es on to Resin -
Bou n d P yr a zole Ca r boxylic Acid Cor es. A 9 equiv amount
of DIEA and 4.5 equiv of HATU (for amine nucleophiles) or
4.5 equiv of PyBrOP (for aniline nucleophiles) were dissolved
in enough DMF to yield a solution that was 0.1-0.2 M in
coupling reagent. The resulting solution was added to the
Step 1. Red u ctive Am in a tion of Am in e on to P S-BAL
Resin . The primary amine (or aniline) was dissolved in a
mixture of 1% acetic acid in DMF to afford a solution that was
0.2 M in amine. To this solution was added sodium triacetoxy-
borohydride (0.2 M final concentration). The solution was
immediately added to 1.00 g of PS-PEG-BAL (polystyrene-
poly(ethylene glycol)-(4-formyl-3,5-dimethoxyphenyl)) resin
(initial loading ) 0.55 mmol/g) in a fritted tube. The reaction
was mixed several times over 1 h, and then, the tube was
capped, placed on an orbital shaker, and allowed to stir
overnight (15 h). The solution was then drained from the resin,
and the resin was washed with methanol (1×), 10% N,N-
diisopropylethylamine (DIEA) in DMF (2×), DMF (7×), CH2-
Cl2 (7×), methanol (3×), and CH2Cl2 (3×). The resin was then
dried in vacuo. Completion of reaction was determined by solid
phase 1H NMR of the resin.

Helicobacter pylori Dihydroorotate Dehydrogenase
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4677
Sch em e 3. Solution Phase Synthesis of Compound 11 (Standard Solution Phase Scale-Up Method)
Robbins block wells containing resin-bound carboxylic acid.
The acid was preactivated with the coupling reagent for 10-
15 min. The desired amine or aniline was then added to the
appropriate wells (in a minimal amount of DMF, if needed).
The block was then sealed and agitated on an orbital shaker
overnight. The solution was drained from the resin, and the
resin was washed with DMF (7×), methanol (5×), and CH2-
Cl2 (7×). Reaction completion was determined by cleaving a
small portion (20-30 mg) of resin with 1:1 TFA/CH2Cl2 for 1
h and then checking by ES-MS and HPLC. Coupling to the
resin-bound acid was typically complete after one reaction, but
if necessary, a second coupling reaction was performed to fully
react the resin-bound acid to the desired amide.
Step 5. Clea va ge of P r od u ct fr om Solid Su p p or t.
Products were cleaved from Robbins blocks directly into 2 mL
96 well plates. To each well was added 0.70 mL of 1:1
trifluoroacetic acid/CH2Cl2. After 1 h, the cleavage solution was
drained into a 2 mL 96 well plate, and the resin was washed
with 0.5 mL of CH2Cl2. The wash solution was combined with
the cleavage solution, and the samples were evaporated to
dryness.
Step 6. P u r ifica tion of Libr a r y P r od u cts. When neces-
sary, compounds were purified by LC/MS on a Waters Micro-
mass instrument, collecting on the expected product molecular
weight. Compounds purified by LC/MS were collected via
mass-directed fractionation using a Micromass ZMD mass
spectrometer and Gilson 306 LC pumps, with fraction collec-
tion controlled by Micromass Fractionlynx software. Fractions
containing product were evaporated to dryness, and products
were characterized by analytical LC/MS and 1H NMR.
Solu tion P h a se Sca le-Up of P yr a zole P r od u cts. In
nearly all cases, products were either scaled up on support
using a larger quantity of resin or in solution using analogous
techniques (for example, see synthesis of compound 13 in
Scheme 3). In solution, anilines were coupled via generation
of the appropriate acid chloride, while amines were coupled
via the acid chloride or the 1-hydroxybenzotriazole-activated
ester (generated with EDC‚HCl). One exception was compound
7, which was scaled up in solution using 4-aminobenzonitrile
(coupling via the mixed anhydride generated with POCl323) and
then hydrolyzing the nitrile to the primary amide with sodium
bicarbonate24 (see Scheme 4 in Supporting Information).
H), 8.02 (bt, 1 H), 9.16 (s, 1 H). Anal. (C30H29N5O4‚0.6H2O) C,
H, N.
1
Com p ou n d 4. H NMR (300 MHz, CDCl3): δ 3.84 (s, 3 H),
4.47 (d, J ) 5.9 Hz, 2 H), 6.38 (d, J ) 8.4 Hz, 1 H), 6.58 (bt, J
) 5.7 Hz, 1 H), 6.94 (d, J ) 9.1 Hz, 2 H), 7.20-7.40 (m, 18 H),
7.53 (d, J ) 8.4 Hz, 1 H). Anal. (C32H28N4O3‚0.2H2O) C, H, N.
1
Com p ou n d 5. H NMR (300 MHz, acetone-d6): δ 0.90 (d,
J ) 6.6 Hz, 6 H), 1.82-2.00 (m, 5 H), 2.86 (bd, 2 H), 3.47 (bq,
4 H), 3.83 (s, 3 H), 7.00 (d, J ) 8.8 Hz, 2 H), 7.38 (s, 1 H), 7.42
(d, J ) 8.8 Hz, 2 H), 7.51 (d, J ) 8.8 Hz, 2 H), 7.75 (bt, 1 H),
7.77 (d, J ) 8.4 Hz, 2 H), 9.95 (s, 1 H). Anal. (C27H31N5O4‚
0.4H2O) C, H, N.
Com p ou n d 6. 1H NMR (300 MHz, CD2Cl2): δ 1.46 (m, 1
H), 1.78-1.95 (m, 8 H), 3.28 (m, 1 H), 3.37-3.61 (bm, 5 H),
3.70 (dd, J ) 6.6, 7.0 Hz, 1 H), 3.79 (m, 1 H), 3.82 (s, 3 H),
3.96 (m, 1 H), 6.93 (d, J ) 9.2 Hz, 2 H), 7.31 (bt, J ) 5.7 Hz,
1 H), 7.35 (d, J ) 9.2 Hz, 2 H), 7.42 (d, J ) 8.8 Hz, 2 H), 7.48
(s, 1 H), 7.65 (d, J ) 8.4 Hz, 2 H), 9.59 (s, 1 H). Anal.
(C28H31N5O5‚0.6H2O) C, H, N.
1
Com p ou n d 7. H NMR (300 MHz, DMSO-d6): δ 4.40 (d, J
) 6.3 Hz, 2 H), 7.17-7.27 (m, 6 H), 7.53 (s, 4 H), 7.55 (s, 1 H),
7.67 (d, J ) 8.8 Hz, 2 H), 7.79 (d, J ) 8.8 Hz, 2 H), 7.83 (bs,
1 H), 9.01 (bt, J ) 5.0 Hz, 1 H), 10.70 (s, 1 H). Anal.
(C25H20N5O3Cl‚0.2H2O) C, H, N.
1
Com p ou n d 8. H NMR (300 MHz, acetone-d6): δ 4.59 (d,
J ) 6.2 Hz, 2 H), 7.11 (tt, J ) 1.7, 7.3 Hz, 1 H), 7.19-7.38 (m,
7 H), 7.45 (s, 1 H), 7.48-7.59 (m, 4 H), 7.72 (d, J ) 8.1 Hz, 2
H), 8.32 (bt, 1 H), 9.78 (s, 1 H). Anal. (C29H19N4O2Cl‚0.6H2O)
C, H, N.
Com p ou n d 9. 1H NMR (300 MHz, CD2Cl2): δ 1.55 (m, 2
H), 1.68 (m, 3 H), 2.35 (s, 2 H), 3.09 (m, 3 H), 4.59 (d, J ) 6.3
Hz, 2 H), 6.89 (d, J ) 8.8 Hz, 2 H), 7.29 (dd, J ) 4.7, 7.7 Hz,
1 H), 7.35 (s, 1 H), 7.42-7.47 (m, 6 H), 7.71 (dt, J ) 8.1, 1.8
Hz, 1 H), 7.76 (t, J ) 6.2 Hz, 1 H), 8.45 (dd, J ) 1.5, 4.7 Hz,
1 H), 8.53 (s, 1 H), 9.45 (s, 1 H). Anal. (C28H27N6O2Cl) C, H, N.
Com p ou n d 10. 1H NMR (300 MHz, CDCl3): δ 0.89 (d, J )
6.6 Hz, 6 H), 1.84 (m, 1 H), 3.15 (t, J ) 6.6 Hz, 2 H), 4.61 (d,
J ) 6.2 Hz, 2 H), 6.65 (bt, 1 H), 7.24-7.41 (m, 11 H). Anal.
(C22H23N4O2Cl‚0.3H2O) C, H, N.
Com p ou n d 11. 1H NMR (300 MHz, 10:1 CDCl3/CD3OD):
δ 4.55 (d, J ) 6.2 Hz, 2 H), 7.12 (t, J ) 7.3 Hz, 1 H), 7.20-
7.30 (m, 3 H), 7.41 (s, 4 H), 7.51 (t, J ) 5.2 Hz, 1 H), 7.58 (m,
3 H), 7.65 (s, 1 H), 8.49 (bs, 2 H), 9.16 (s, 1 H). Anal.
(C23H18N5O2Cl‚1.0H2O) C, H, N.
Com p ou n d 1. 1H NMR (300 MHz, CDCl3): δ 1.83-1.96 (m,
4 H), 3.37 (t, J ) 6.3 Hz, 2 H), 3.62 (t, J ) 6.8 Hz, 2 H), 3.82
(s, 3 H), 4.57 (d, J ) 6.2 Hz, 2 H), 6.94 (d, J ) 9.1 Hz, 2 H),
7.23-7.41 (m, 10 H), 7.64 (d, J ) 8.4 Hz, 2 H), 7.70 (s, 1 H),
9.57 (s, 1 H). Anal. (C30H29N5O4‚0.3H2O) C, H, N.
1
Com p ou n d 12. H NMR (300 MHz, acetone-d6): δ 2.26 (s,
3 H), 4.54 (d, J ) 4.0 Hz, 2 H), 7.08-7.13 (m, 3 H), 7.24 (d, J
) 8.1 Hz, 2 H), 7.32 (m, 2 H), 7.45 (s, 1 H), 7.46-7.59 (m, 4
H), 7.72 (d, J ) 8.4 Hz, 2 H), 8.20 (bt, 1 H), 9.78 (s, 1 H). Anal.
(C25H21N4O2Cl) C, H, N.
Com p ou n d 3. 1H NMR (300 MHz, 10:1 CD2Cl2/CD3OD): δ
1.88 (m, 4 H), 3.43 (t, J ) 6.4 Hz, 2 H), 3.54 (t, J ) 6.3 Hz, 2
H), 3.83 (s, 3 H), 4.44 (d, J ) 5.9 Hz, 2 H), 6.94 (dt, J ) 9.1,
2.2 Hz, 2 H), 7.22-7.34 (m, 6 H), 7.36 (dt, J ) 9.2, 2.2 Hz, 2
H), 7.48 (dt, J ) 8.8, 2.0 Hz, 2 H), 7.71 (dt, J ) 8.8, 1.9 Hz, 2
1
Com p ou n d 13. H NMR (300 MHz, acetone-d6): δ 3.81 (s,
3 H), 4.59 (d, J ) 6.6 Hz, 2 H), 6.98 (d, J ) 9.2 Hz, 2 H), 7.07
(t, J ) 7.3 Hz, 1 H), 7.24-7.31 (m, 3 H), 7.35 (s, 1 H), 7.41 (d,

4678 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Haque et al.
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Inhibition of Dihydroorotate Dehydrogenase Activity by Brequi-
nar Sodium. Cancer Res. 1992, 52, 3521-3527.
(10) Marcinkeviciene, J .; Rogers, M. J .; Kopcho, L.; J iang, W.; Wang,
K.; Murphy, D. J .; Lippy, J .; Link, S.; Chung, T. D. Y.; Hobbs,
F.; Haque, T.; Trainor, G. L.; Slee, A.; Stern, A. M.; Copeland,
R. A. Selective Inhibition of Bacterial Dihydroorotate Dehydro-
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J ) 9.1 Hz, 2 H), 7.68 (d, J ) 8.4 Hz, 2 H), 7.75 (d, J ) 8.4 Hz,
1 H), 8.35 (bt, 1 H), 8.41 (dd, J ) 1.8, 4.7 Hz, 1 H), 8.58 (s, 1
H), 9.64 (bs, 1 H). Anal. (C24H21N5O3‚0.4H2O) C, H, N.
Ack n ow led gm en t. We thank the Wilmington ana-
lytical chemistry group for their invaluable support in
both the purification and the analysis of the libraries
discussed above.
(11) Cherwinski, H. M.; Cohn, R. G.; Cheung, P.; Webster, D. J .; Xu,
Y.-Z.; Caulfield, J . P.; Young, J . M.; Nakano, G.; Ransom, J . T.
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Su p p or tin g In for m a tion Ava ila ble: Charts containing
graphs of percent inhibition (at 10 µM compound concentra-
tion) vs RARB side chains for libraries 2 and 3, Scheme 4
showing the solution phase synthesis of compound 7, and a
detailed description of methods used for protein expression and
purification. This material is available free of charge via the
Internet at http://pubs.acs.org.
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