Synthesis, in vitro evaluation and cocrystal structure of 4-oxo-[1]benzopyrano[4,3-c]pyrazole cryptosporidium parvum inosine 5′-monophosphate dehydrogenase (Cp IMPDH) inhibitors

Article abstract of DOI:10.1021/jm501527z

Cryptosporidium inosine 5′-monophosphate dehydrogenase (CpIMPDH) has emerged as a therapeutic target for treating Cryptosporidium parasites because it catalyzes a critical step in guanine nucleotide biosynthesis. A 4-oxo-[1]benzopyrano[4,3-c]pyrazole derivative was identified as a moderately potent (IC₅₀ = 1.5 μM) inhibitor of CpIMPDH. We report a SAR study for this compound series resulting in 8k (IC₅₀ = 20 ± 4 nM). In addition, an X-ray crystal structure of CpIMPDH·IMP·8k is also presented.

Full text of DOI:10.1021/jm501527z

Brief Article
pubs.acs.org/jmc
Synthesis, in Vitro Evaluation and Cocrystal Structure of 4‑Oxo-
[1]benzopyrano[4,3‑c]pyrazole Cryptosporidium parvum Inosine 5′-
Monophosphate Dehydrogenase (CpIMPDH) Inhibitors
Zhuming Sun,† Jihan Khan,§ Magdalena Makowska-Grzyska,∥ Minjia Zhang,‡ Joon Hyung Cho,†
Chalada Suebsuwong,† Pascal Vo,† Deviprasad R. Gollapalli,‡ Youngchang Kim,∥,⊥ Andrzej Joachimiak,∥,⊥
Lizbeth Hedstrom,‡,§ and Gregory D. Cuny*,†
†Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Science and Research
Building 2, Room 549A, Houston, Texas 77204, United States
§
‡Departments of Biology and Chemistry, Brandeis University, 415 South Street, Waltham, Massachusetts 02454, United States
∥Center for Structural Genomics of Infectious Diseases, Computational Institute, University of Chicago, 5735 South Ellis Avenue,
Chicago, Illinois 60637, United States
⊥Structural Biology Center, Biosciences, Argonne National Laboratory, 9700 South Cass Avenue Argonne, Illinois 60439, United
States
S
* Supporting Information
ABSTRACT: Cryptosporidium inosine 5′-monophosphate de-
hydrogenase (CpIMPDH) has emerged as a therapeutic target
for treating Cryptosporidium parasites because it catalyzes a
critical step in guanine nucleotide biosynthesis. A 4-oxo-
[1]benzopyrano[4,3-c]pyrazole derivative was identified as a
moderately potent (IC50 = 1.5 μM) inhibitor of CpIMPDH.
We report a SAR study for this compound series resulting in 8k
(IC50 = 20 4 nM). In addition, an X-ray crystal structure of
CpIMPDH·IMP·8k is also presented.
novo.6−8 Instead, the parasite converts adenosine salvaged from
the host into guanine nucleotides via a linear pathway
INTRODUCTION
■
Cryptosporidium parvum and Cryptosporidium hominis are
dependent on IMPDH activity. Interestingly, these parasites
intracellular protozoan parasites that invade the brush border
appear to have obtained their IMPDH gene by lateral gene
epithelial cells of the small intestine. Cryptosporidiosis is
transfer from bacteria. Consequently, CpIMPDH is structurally
prevalent in the developing world where it results in life-
distinct from mammalian IMPDH enzymes9 and is poorly
threatening diarrhea and severe malnutrition in children.1
inhibited by the prototypical human IMPDH inhibitor
Cryptosporidium oocysts are water-transmitted and highly
mycophenolic acid (CpIMPDH IC50 ∼ 10 μM; hIMPDH1 Ki
resistant to water purification methods, also leading to
= 33 nM; hIMPDH2 Ki ∼ 7 nM).10,11 These structural and
mechanistic differences also provide an opportunity to design
selective CpIMPDH inhibitors as therapeutic agents for treating
cryptosporidiosis.12 CpIMPDH inhibitors may also be effective
against bacterial infections.13,14
significant disease burden in the developed world.2 Infections
resolve in immunocompetent hosts but can be chronic and fatal
in immunocompromised patients. Furthermore, because
oocysts can readily be obtained and water supplies are relatively
easily accessed, these organisms represent a credible bioterror-
ism threat.3 Currently, vaccine therapies against C. parvum and
C. hominis are not available and the only approved drug,
nitazoxanide, has an ill-defined mechanism of action and is not
particularly effective.4 Thus, new chemotherapeutic agents are
needed for the treatment of cryptosporidiosis.
One emerging molecular target for the treatment of
cryptosporidiosis is the oxidoreductase inosine 5′-mono-
phosphate dehydrogenase (IMPDH), which catalyzes the
conversion of inosine-5′-monophosphate (IMP) into xantho-
sine-5′-monophosphate (XMP) as the rate-determining step in
guanine nucleotide biosynthesis.5 Genomic analysis revealed
that Cryptosporidium cannot synthesize purine nucleotides de
Previously, we have reported the optimization of several
structurally distinct compound series, including C64 and
Q21,15−18 as well as the first demonstration of in vivo efficacy
of a CpIMPDH inhibitor (e.g., P131) in a mouse model of
cryptosporidiosis (Figure 1).19 This later study also revealed
several additional hurdles required in the development of
efficacious compounds, including preferential compound
distribution to gastrointestinal enterocytes (as opposed to
systemic distribution) and minimizing the impact of IMPDH
Received: July 3, 2014
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
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The presence of the acid proved crucial for these reactions.20
The regioisomeric [1]benzopyrano[4,3-c]pyrazol-4(2H)-one
derivative 9c was prepared using the methodology outlined in
Scheme 2 (method B). 4-Hydroxycoumarin (9a) was treated
a
Scheme 2. Synthesis of Regioisomers 9c (Method B)
Figure 1. Structures of previously described inhibitors C64 and Q21
that have been cocrystallized with CpIMPDH, P131 that demon-
strated in vivo efficacy in a cryptosporidiosis animal model, and a new
inhibitor 8a identified by HTS.
a
Reagents and conditions: (a) POCl3, DMF, 1,2-dichloroethane, rt, 12
h, then saturated aqueous Na2CO3; (b) t-butyl carbazate, KHCO3,
ethyl acetate, 85 °C, 5 h, then TFA in CH2Cl2 (1:4), 2 h, rt; (c)
DIPEA, EtOH, rt, 12 h.
inhibition on gut microbiome populations. The study reported
herein is a continuation of our effort to identify and optimize
structurally distinct CpIMPDH inhibitors and to develop a
common pharmacophore as a guide for the future design of
additional CpIMPDH inhibitors.
Our current structure−activity relationship (SAR) study was
initiated based on 4-oxo-N-(3-methoxyphenyl)-[1]-
benzopyrano[4,3-c]pyrazole-1(4H)-acetamide (8a, Figure 1),
identified by high throughput screening, as a moderately potent
CpIMPDH inhibitor (IC50 = 1.5 0.2 μM).
with POCl3 and DMF, similar to standard Vilsmeier−Haack
conditions, but at room temperature. The reaction was
terminated by the addition of aqueous Na2CO3, which
generated product 9b. Upon reaction with 6a in ethanol in
the presence of DIPEA, the regioisomeric pyrazole 9c was
obtained. Presumably, the terminal NH2 of hydrazine 6a
condensed with the carbonyl of the vinylogous amide of 9b,
which was followed by cyclization via an addition−elimination
reaction to generate the isolated product.21
RESULTS AND DISCUSSION
■
The preparation of 8l−n, as analogues of 8k with additional
substituents on the acetamide and [1]benzopyrano[4,3-c]-
pyrazole, is outlined in Scheme 3 (method C). Anilines 2l or 2k
Chemistry. 4-Oxo-[1]benzopyrano[4,3-c]pyrazole ana-
logues (8a−n and 13a−f) were prepared using four general
synthetic methods. The synthesis of analogues 8a−k used the
methodology shown in Scheme 1 (method A). Anilines 2a−k
Scheme 3. Synthesis of 4-Oxo-[1]benzopyrano[4,3-
c]pyrazole Analogue 8l−n (Method C)
a
Scheme 1. Synthesis of 4-Oxo-[1]benzopyrano[4,3-
a
c]pyrazole Derivatives 8a−k (Method A)
a
Reagents and conditions: (a) bromoacetyl chloride (3), K2CO3,
a
CH2Cl2, 0 °C to rt; (b) t-butyl carbazate, KHCO3, EtOAc/H2O (1:2),
85 °C, 5 h; (c) TFA in CH2Cl2 (1:4), 2 h; (d) 4-chloro-3-
formylcoumarin (7a), AcOH (cat), EtOH, 105 °C, 20 min.
Reagents and conditions: (a) 21 (4-Cl-3-OMePhNHMe) or 2k (4-
Cl-3-OMePhNH2), K2CO3, CH2Cl2, 0 °C to rt; (b) for 4l, t-butyl
carbazate, K2CO3, KI, acetone, 65 °C, 18 h or for 4m, t-butyl
carbazate, DIPEA, toluene 105 °C, 16 h; (c) TFA in CH2Cl2 (1:4), 2
h, then 7a (or 4-chloro-3-formyl-7-methylcoumarin, 7b), AcOH (cat.),
EtOH, 105 °C, 20 min.
were treated with bromoacetyl chloride, 3, in CH2Cl2 in the
presence of K2CO3 to afford aryl amides 4a−k, which were
treated with t-butyl carbazate in aqueous KHCO3 to provide
the N-Boc-protected hydrazines 5a−k via an SN2 reaction. In
the next step, trifluoroacetic acid was used to remove the t-butyl
carbamate protecting group in 5a−k to give 6a−k, which were
used without purification. The hydrazines 6a−k were refluxed
in ethanol with 4-chloro-3-formylcoumarin (7a) in the presence
of a catalytic amount of acetic acid to provide analogues 8a−k.
were treated with 3a or 3b to afford aryl amides 4l or 4m. A
stronger base (e.g., K2CO3), organic solvent (e.g., acetone), and
the presence of potassium iodide were required to displace the
primary chloride of 4l to furnish 5l. In the case of 5m, DIPEA
in toluene proved effective. The required intermediate 7b was
synthesized from the corresponding 4-hydroxycoumarin
B
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Journal of Medicinal Chemistry
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following typical Vilsmeier−Haack conditions (see Experimen-
tal Section). Analogues 8l−n were synthesized from acid-
catalyzed cyclization of hydrazines (5l−m des-Boc intermedi-
ates) and 7a or 7b using the same method described in Scheme
1.
Initial attempts to synthesize analogues 13a−f following the
methodology outlined in Scheme 1 (method A) proved
problematic. Thus, an alternate method was developed that is
shown in Scheme 4 (method D). Aldehydes 7a and 10a−b
a
Scheme 4. Synthesis of Derivatives 13a−f (Method D)
Figure 2. Predicted binding mode of 8a (green) in complex with
CpIMPDH·IMP. The red dotted line indicates an ionic−dipole
interaction between the amide of 8a and the side chain of E329. The
phenyl ring of 8a is stacked above Y358′.
production.12 IC50 values were determined by averaging the
results of three independent experiments unless otherwise
noted.
The regioisomeric derivative 9c did not inhibit CpIMPDH,
indicating that the relative orientation of the anilide on the
fused pyrazole was crucial for inhibitory activity (Table 1).
Table 1. Monosubstituted N-Phenyl 4-Oxo-[1]benzo
Pyrano[4,3-c]pyrazole-1(4H)-acetamide Derivatives for
CpIMPDH Inhibition
a
Reagents and conditions: (a) ethyl hydrazinoacetate hydrochloride
(14), AcOH (cat), EtOH, 105 °C, 0.5−2 h; (b) 12a, LiOH (aq), THF,
rt, 12 h then EDC, TEA, DMF, 12b−c, aqueous LiOH, THF, rt, 12 h;
(c) NH2R, HBTU, DIPEA, DMF, rt, 12 h.
were refluxed in the presence of ethyl hydrazinoacetate
hydrochloride and a catalytic amount of acetic acid in ethanol
to afford pyrazoles 11a−c. Hydrolysis of the ester using 2 M
aqueous LiOH in THF afforded acids 12b−c. In the case of
12a, the lactone ring also opened during this step and required
relactonization using EDC and TEA in DMF. The acids 12a−c
were treated with either 4-chloro-3-methoxyaniline or hetero-
cyclic anilines in the presence of HBTU and DIPEA in DMF to
afford analogues 13a−f. Intermediates 10a−b were again
prepared using Vilsmeier−Haack reactions (see Experimental
Section).
Predicted Binding Mode of Inhibitor 8a with
CpIMPDH·IMP. Inhibitor 8a was docked into the binding
site observed in one of our previously reported crystal
structures of the catalytic domain of CpIMPDH (PDB code:
4IXH)15 using AutoDock Tools 1.5.6. The top 10 binding
conformations were examined, and the two best conformations
(binding energies of −7.86 and −7.76 kcal/mol, respectively)
were selected based on similarity of their binding modes with
Q21,15 including π-interactions between the 4-oxo-[1]-
benzopyrano[4,3-c]pyrazole with the hypoxanthine of IMP
and the 3-methoxyphenyl with Y358′ (where prime denotes a
residue from the adjacent subunit). However, the hydrogen
atom of the amide for these two conformations formed ionic−
dipole interactions with two different oxygen atoms in the side
chain of E329. Therefore, the conformation that formed an
interaction similar to Q21 was selected as the predicted binding
mode for the N-series and is shown in Figure 2.
ID
method
R1
IC50 (nM)
9c
8a
8b
8c
8d
8e
B
A
A
A
A
A
>5000
1500 200
>5000
3-OMe
3-Cl
2-Cl
>5000
4-F
460 95
580 130
4-CF3
Next, the SAR study focused on the monosubstituted aniline
moiety of 8a. Analogues with a 2-chloro substituent (8c)
showed no inhibitory activity. Replacement of the 3-methoxy
with a chlorine (8b) likewise led to loss of inhibition. However,
the 4-fluoro and 4-trifluoromethyl derivatives (8d and 8e)
displayed a 3-fold increase in activity, indicating that
monosubstitution on the amide phenyl moiety could provide
only moderate increases in potency, similar to our observation
with other inhibitor series.15,16,18 Therefore, disubstituted and
fused anilines were examined. The 2,4-dichloro substituted
analogue 8g showed no inhibition activity (Table 2). In light of
this finding, combined with the result of 8c, it appeared that an
ortho-chloro was not well tolerated in the binding pocket,
possibly due to a clash with Y358′. However, the 3,4-dichloro
analogue 8f displayed significantly improved potency. A further
increase in potency was achieved by replacing the 3-chloro
substituent with a methoxy (8k, CpIMPDH IC50 = 20 4 nM).
However, replacing the remaining chloro of 8k with another
methoxy (8i) resulted in the loss of enzyme inhibition.
Tethering the ethers into a dioxane ring (8h) also resulted in
a significantly lower IC50 value compared to 8k. The loss of
activity for these two derivatives containing electron donating
Evaluation of CpIMPDH Inhibition. Biological character-
ization of the 4-oxo-[1]benzopyrano[4,3-c]pyrazole derivatives
was performed following our published procedures.15,16
CpIMPDH was expressed and purified as previously
reported.22−24 Enzymatic activity was monitored by NADH
C
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methylene position (8m) resulted in a 3-fold loss of potency,
likely due to a steric clash with the side chain of E329. The
addition of a methyl to the 7-position of the 4-oxo-
[1]benzopyrano[4,3-c]pyrazole ring system (8n) was also
detrimental, revealing the steric limitation of this region of
the molecule, likely the result of clashes with either M302 or
the ribose of IMP. Replacing the lactone ring with an ether
bridge (13e) resulted in a 3-fold loss of inhibition, indicating
the carbonyl contributes to binding. Replacement of the lactone
with an ethylene bridge (13f) resulted in a further decrease of
potency. Therefore, the lactone appears essential for
CpIMPDH inhibition, although the rationale for this
observation was not obvious from the docking model.
Table 2. Disubstituted N-Phenyl and N-Heteroaryl 4-Oxo-
[1]benzopyrano[4,3-c]pyrazole-1(4H)-acetamide
Derivatives for CpIMPDH Inhibition
The original screening hit (8a) in addition to 11 other
CpIMPDH inhibitors (e.g., 8d−f, 8h, 8j, 8k, 8m, 13b, and
13d−f) failed to inhibit hIMPDH2 (<20% inhibition at 5 μM),
which also has high sequence identity (85%) to hIMPDH1.
These results demonstrated that CpIMPDH inhibitory potency
could be increased, while preserving selectivity against a human
orthologue.
Crystal Structure of CpIMPDH·8k·IMP. The structure of a
CpIMPDH complex with IMP and 8k was solved at 2.40 Å
resolution using molecular replacement with the structure of
apo CpIMPDH (PDB code: 3FFS)25 as the search model. Like
the structures of CpIMPDH·IMP in complex with other
inhibitors,15,25 the 4-oxo-[1]benzopyrano[4,3-c]pyrazole-based
inhibitor interacts with residues from two adjacent subunits.
One aromatic moiety of 8k, the 4-oxo[1]benzopyrano[4,3-
c]pyrazole, π-stacks with the hydroxanthine of IMP (Figure 3A)
in an interaction similar to that observed previously for two
other CpIMPDH complexes and as predicted in the docking of
8a.15,26 The pyrazole portion interacts with the side chain of
M308 and forms n−π* contacts between the carbonyl group of
the 4-oxo[1]benzopyrano-[4,3-c]pyrazole moiety and the main
chain carbonyl of M302.26 This latter interaction was not
predicted in the docking model but provides an explanation for
the importance of the lactone. The remaining portion of the
inhibitor circumvents A165 and extends into the pocket formed
at the subunit interface. The amide NH of 8k forms a H-bond
with a side chain oxygen atom of E329 and is part of the
extensive H-bonding network involving T221, S354′, and
Y358′. Another common feature observed in all CpIMPDH
inhibitor complexes is the interaction of the second aromatic
moiety of the inhibitor with the side chain of Y358′. In the case
of 8k, this interaction is observed for the 4-chloro-3-
methoxyphenyl. In addition, this moiety is involved in contacts
with H166 and P26′ via polar and van der Waals interactions.
Inhibitor 8k does not extend as deep into the cavity formed at
the subunit interface as does the 2-(4-pyridyl)benzoxazole
derivative Q21 (Figure 3B).15 However, similarly to the bromo
substituent of inhibitor C64,25 the chloro substituent of 8k is
contacting the main chain carbonyl oxygen atom of G357′
(Figure 3C).
groups in the para-position of the anilide is potentially due to
weakening of the H-bond donating ability of the amide NH,
which is a critical interaction with E329 observed in cocrystal
structures of other CpIMPDH inhibitors.15,25 However, the
naphthyl substituted analogue 8j demonstrated an IC50 value of
67 nM.
On the basis of these results, bioisosteres of the naphthyl
were investigated. Benzo[d]imidazole 13a and 1H-indol-5-yl
13c had only weak or no inhibitory activity. However, the
(1H)-indole 13b and quinolin-6-yl 13d showed moderate
inhibition (CpIMPDH IC50 = 120
respectively), albeit less than 8j.
30 and 80
40 nM,
Further modifications were performed on 8k that retained
the 3-methoxy-4-chloro phenyl moiety (Table 3). Addition of a
methyl group on the amide nitrogen (8l) resulted in loss of
activity, again indicating the importance of the H-bond donor
function of the amide NH. Addition of a methyl group on the
Table 3. Modifications of the Acetamide and 4-Oxo-
[1]benzopyrano[4,3-c]pyrazole Regions of 8k for CpIMPDH
Inhibition
CONCLUSION
■
An SAR study of CpIMPDH inhibitor 8a was conducted with
guidance from an in silico docking model based on a previously
crystallized CpIMPDH inhibitor complex. The orientation of
the anilide on the fused pyrazole was crucial, a 4-chloro-3-
methoxy substitution on the anilide (e.g., 8k) achieved the
greatest potency among this series of derivatives, and the
secondary amide and the lactone of the 4-oxo-[1]benzopyrano-
[4,3-c]pyrazole were vital for binding. Overall, this study
ID
method
R1
R2
R3
X
Y
IC50(nM)
20
8k
A
H
H
H
O
CO
CO
CO
CO
CH2
4
8l
C
C
C
D
D
Me
H
H
H
O
>5000
63 11
2000
8m
8n
13e
13f
Me
H
H
O
H
Me
H
O
H
H
O
70 27
100 30
H
H
H
CH2
CH2
D
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Journal of Medicinal Chemistry
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maintained at 0 °C for 20 min and then allowed to warm to rt.
Saturated aqueous NaHCO3 was added. The reaction mixture was
extracted with CH2Cl2, and the organic layer was washed by brine,
dried over anhydrous Na2SO4, filtered, and evaporated in vacuo. The
product was purified by column chromatography (0−50% EtOAc in
hexane) to afford 4a−m.
General Procedure for 5a−k. Compound 4a−k (5.6 mmol), t-
butyl carbazate (11.2 mmol), and KHCO3 (16.8 mmol) were
suspended in 30 mL of EtOAC and H2O (1:2). The mixture was
refluxed at 85 °C for 5 h. The mixture was allowed to cool to rt, and
the crude product was extracted with EtOAC, dried with anhydrous
MgSO4, filtered, and concentrated. The product was purified by
column chromatography (10−70% EtOAc in hexane).
t-Butyl 2-(2-((4-Chloro-3-methoxyphenyl)(methyl)amino)-2-
oxoethyl)hydrazinecarboxylate (5l). Compound 4l (1 mmol), t-
butyl carbazate (2 mmol), K2CO3 (3 mmol), and KI (2 mmol) were
suspended in 20 mL of acetone. The mixture was heated at 65 °C for
18 h, allowed to cool to rt, and then evaporated to dryness. The
residue was diluted with aqueous NH4Cl and extracted with EtOAc
(20 mL × 3). The organic layer was dried over anhydrous MgSO4,
filtered, and concentrated. The product was purified by silica gel
column chromatography (5% EtOAc in CH2Cl2) to give 5l as an oil
(66% yield).
t-Butyl 2-(1-((4-Chloro-3-methoxyphenyl)amino)-1-oxopropan-
2-yl)hydrazinecarboxylate (5m). Compound 4m (1.7 mmol), t-
butyl carbazate (3.3 mmol), and DIPEA (3.3 mmol) were suspended
in 10 mL of toluene. The mixture was refluxed at 105 °C for 16 h,
allowed to cool to rt, and then evaporated to dryness. The residue was
diluted with aqueous NH4Cl and extracted with EtOAc (20 mL × 3).
The organic layer was dried over anhydrous MgSO4, filtered, and
concentrated. The crude product was purified by silica gel
chromatography (0−50% EtOAc in hexane) to give 5m as a solid
(83% yield).
General Procedures for 8a−n. Compounds 5a−m (0.8 mmol)
were treated with 2.5 mL of TFA in CH2Cl2 (1:4) at rt for 2 h. The
mixture was evaporated in vacuo to afford 6a−m as colorless oils. The
intermediates 6a−m were dissolved in 2 mL of EtOH, which was then
added to a suspension of 7a−b (0.8 mmol) in 2 mL of EtOH. Acetic
acid (20 μL) was added. The mixture was refluxed at 105 °C for 20
min then allowed to cool to rt. The mixture was evaporated in vacuo,
partitioned between aqueous NaHCO3 and EtOAc. The organic layer
was dried over anhydrous MgSO4, filtered, and concentrated. The
products 8a−n was collected after silica gel chromatography using 20−
85% EtOAc in hexane as eluent.
3-[(Dimethylamino)methylene]chroman-2,4-dione (9b). 4-Hy-
droxycoumarin (9a, 9.2 mmol) in 1.5 mL of DMF added to 10 mL
of 1,2-dichloroethane. To this solution was added 1.1 mL of POCl3.
The mixture was stirred at rt for 12 h. Next, saturated aqueous
Na2CO3 was added. The reaction mixture was extracted with EtOAc,
dried over anhydrous MgSO4, filtered, and concentrated. The crude
product was purified by silica gel chromatography (0−25% EtOAc in
CH2Cl2) to give 9b (48% yield) as a yellow solid.
N-(3-Methoxyphenyl)-[1]benzopyrano[4,3-c]pyrazol-4(2H)-one
(9c). Intermediate 6a (2.1 mmol, prepared from 5a following the
procedure described above) was dissolved in 3 mL of EtOH then was
added to 9b (2.1 mmol) suspended in 3 mL of EtOH. To this mixture
was added DIPEA (8.4 mmol). The reaction mixture was stirred at rt
for 12 h and then evaporated in vacuo. The crude product was
partitioned between aqueous NH4Cl and EtOAc. The organic layer
was dried over anhydrous MgSO4, filtered, and concentrated. The
material was purified by silica gel chromatography using 0−20%
EtOAc in CH2Cl2 as eluent to give 9c as a white solid (56% yield).
General Procedure for 11a−c. Compounds 7a or 10a−b (8.6
mmol) were suspended in 15 mL of EtOH with ethyl hydrazinoacetate
hydrochloride (9.5 mmol). Acetic acid (30 μL) was added. The
mixture was refluxed at 105 °C for 30 min (for 11a) or 2 h (for 11b−
c) and then allowed to cool to rt. The reaction mixture was evaporated
in vacuo, partitioned between aqueous NaHCO3 and EtOAc. The
organic layer was dried over anhydrous MgSO4, filtered, and
concentrated. The crude product was purified by silica gel
Figure 3. (A) Structure of CpIMPDH·IMP·8k. (B) Overlay of
CpIMPDH structures with 8k and Q21. (C) Overlay of CpIMPDH
structures with 8k and C64. In all panels, chains A (slate) and D
(violet) are shown in cartoon representations. Residues involved in
IMP and inhibitor binding are shown as sticks. IMP (light gray), 8k
(yellow), C64 (green), and Q21 (teal) are shown as sticks. Hydrogen
and halogen bonds are depicted as red dashed lines and a water
molecule as a red sphere. Prime indicates residues from an adjacent
monomer.
provides a structurally distinct inhibitor series that will further
assist in the continuing development of CpIMPDH inhibitors
for the treatment of cryptosporidiosis and possibly other
infectious diseases.13,14 Finally, a crystal structure of
CpIMPDH·IMP and 8k (e.g., N109) provides further support
for a general binding mode of CpIMPDH inhibitors featuring
three key interactions: (i) π-interaction between an aryl/
heteroaryl moiety and the hydroxanthine of IMP, (ii) H-bond
with E329, and (iii) extension of an aryl/herteroaryl group into
an adjacent subunit forming interactions with Y358′. This
pharmacophore provides a template that can be extended to the
discovery of other structurally distinct chemical scaffolds of
selective CpIMPDH inhibitors.
EXPERIMENTAL SECTION
Chemistry. All test compounds had a purity ≥95% as determined
by HPLC analyses.
■
General Procedure for 4a−m. To a suspension of 2a−l (6.5
mmol) in 15 mL of CH2Cl2 at 0 °C, acyl halides (3 or 3a−b, 8.5
mmol) and K2CO3 (1.25 g, 9.1 mmol) were added. The reaction was
E
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Journal of Medicinal Chemistry
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chromatography using 20−75% EtOAc in hexane as eluent to give
products 11a−c.
tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophos-
phate; EDC, 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide;
rt, room temperature; TEA, triethylamine; TFA, trifluoroacetic
acid; XMP, xanthosine 5′-monophosphate
[1]Benzopyrano[4,3-c]pyrazole-1(4H)acetic Acid (12a). Com-
pound 11a (7.3 mmol) was dissolved in 30 mL of THF, and then
18 mL of aqueous LiOH (2M) was added. The mixture was stirred at
rt for 12 h, and then 5% aqueous HCl was added until the pH = 2. The
mixture was evaporated to dryness, and then the residue was dissolved
in EtOAc, concentrated to dryness, and used without further
purification. The intermediate was dissolved in 8 mL of DMF, and
then 3.6 mL of TEA and 1.23 g of EDC were added in order to reform
the lactone. The mixture was stirred at rt for 18 h, and then 5%
aqueous HCl was added until the pH = 2. The mixture was evaporated
in vacuo, partitioned between EtOAc and aqueous NH4Cl. The
organic layer was evaporated in vacuo to give 12a, which was used
without further purification (75% yield).
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ASSOCIATED CONTENT
* Supporting Information
Procedures for 2l, 7b, and 10a−b, compound characterization,
IC50 determinations, gene cloning, protein expression, crystal-
lization, and statistics for data collection/refinement of the X-
ray crystal structure. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
Accession Codes
PDB ID code: 4QJ1.
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-713-743-1274. E-mail: gdcuny@central.uh.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institute of Allergy
and Infectious Diseases (U01AI075466 and R01AI93459 to
L.H., R56AI106743 to G.D.C. and Contracts
HHSN272200700058C and HHSN272201200026C to the
Center for Structural Genomics of Infectious Diseases,
University of Chicago, IL) and the New England Regional
Center of Excellence for Biodefense and Emerging Infectious
Diseases. We also thank Minyi Gu for help with gene cloning.
The use of the 19-ID beamline at the Structural Biology Center
at the Advanced Photon Source was supported by the U.S.
Department of Energy, Office of Biological and Environmental
Research, under contract DE-AC02-06CH11357.
ABBREVIATIONS USED
■
Cp, Cryptosporidium parvum; DIPEA, N,N-diisopropylethyl-
amine; IMP, inosine 5′-monophosphate; IMPDH, IMP
dehydrogenase; ND, not determined; HBTU, N,N,N,N-
F
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Journal of Medicinal Chemistry
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