Rational design, synthesis, and potency of N-substituted indoles, pyrroles, and triarylpyrazoles as potential fructose 1,6-bisphosphatase inhibitors

Article abstract of DOI:10.1002/cmdc.200900493

By using computer modeling and lead structures from our earlier SAR results, a broad variety of pyrrole-, indole-, and pyrazole-based compounds were evaluated as potential fructose 1,6-bisphosphatase (FBPase) inhibitors. The docking studies yielded promising structures, and several were selected for synthesis and FBPase inhibition assays: 1-[4-(trifluoromethyl)-benzoyl]-1H- indole-5-carboxamide, 1-(α-naphthalen-1-ylsulfonyl)-7-nitro-1H-indole, 5-(4-carboxyphenyl)-3-phenyl-1-[3-(trifluoromethyl) phenyl]-1H-pyrazole, 1-(4-carboxyphenylsulfonyl)-1H-pyrrole, and 1-(4-carbomethoxyphenylsulfonyl)-1H- pyrrole were synthesized and tested for inhibition of FBPase. The IC ₅₀ values were determined to be 0.991 and 1.34 mm, and 575, 135, and 32 nm, respectively. The tested compounds were significantly more potent than the natural inhibitor AMP (4.0 μm) by an order of magnitude; indeed, the best inhibitor showed an IC₅₀ value toward FBPase more than two orders of magnitude better than that of AMP. This level of activity is virtually the same as that of the best currently known FBPase inhibitors. This work shows that such indole derivatives are promising candidates for drug development in the treatment of type II diabetes.

Full text of DOI:10.1002/cmdc.200900493

MED
DOI: 10.1002/cmdc.200900493
Rational Design, Synthesis, and Potency of N-Substituted
Indoles, Pyrroles, and Triarylpyrazoles as Potential
Fructose 1,6-Bisphosphatase Inhibitors
Aleksandra Rudnitskaya, Dmitry A. Borkin, Ken Huynh, Bꢀla Tçrçk,* and Kimberly Stieglitz*[a]
By using computer modeling and lead structures from our ear-
lier SAR results, a broad variety of pyrrole-, indole-, and pyra-
zole-based compounds were evaluated as potential fructose
1,6-bisphosphatase (FBPase) inhibitors. The docking studies
yielded promising structures, and several were selected for
synthesis and FBPase inhibition assays: 1-[4-(trifluoromethyl)-
benzoyl]-1H-indole-5-carboxamide, 1-(a-naphthalen-1-ylsulfon-
values were determined to be 0.991 and 1.34 mm, and 575,
135, and 32 nm, respectively. The tested compounds were sig-
nificantly more potent than the natural inhibitor AMP (4.0 mm)
by an order of magnitude; indeed, the best inhibitor showed
an IC50 value toward FBPase more than two orders of magni-
tude better than that of AMP. This level of activity is virtually
the same as that of the best currently known FBPase inhibitors.
This work shows that such indole derivatives are promising
candidates for drug development in the treatment of type II
diabetes.
yl)-7-nitro-1H-indole,
5-(4-carboxyphenyl)-3-phenyl-1-[3-(tri-
fluoromethyl)phenyl]-1H-pyrazole, 1-(4-carboxyphenylsulfonyl)-
1H-pyrrole, and 1-(4-carbomethoxyphenylsulfonyl)-1H-pyrrole
were synthesized and tested for inhibition of FBPase. The IC50
Introduction
Owing to the increasing rate of obesity worldwide, the so-
called type II or non-insulin-dependent diabetes is becoming
one of the most frequently diagnosed diseases. As the number
of individuals with impaired glucose tolerance in the pre-dia-
betic state is steadily rising, the inhibition of fructose 1,6-bi-
sphosphatase (FBPase), as one of the principal treatment op-
tions, continues to attract significant attention.[1] In recent
years many new potential therapeutics have been screened
and identified to inhibit gluconeogenesis by blocking FBPase
activity. Some of these compounds have excellent IC50 values,
such as MB05032 (IC50 =16 nm),[2] benzimidazole phosphonic
acid (IC50 =90 nm),[3] and 10A (IC50 =16 nm).[4] Most drug candi-
dates that reached early clinical trials were later determined to
have serious toxic side effects or problems in delivery.[2–4] The
therapeutic use of most drugs currently on the market (such
as metformin and glibenclamide), is limited due to toxic side
effects.[5–7] Therefore, the search for the ideal drug to amelio-
rate high blood glucose levels continues. Chronically elevated
blood glucose levels in patients with type II diabetes is caused
by excessive glucose production by the liver coupled with de-
creased glucose uptake and metabolism by muscle, fat, and
liver. The gluconeogenesis pathway and rate-limiting enzymes
within this pathway such as FBPase have been the focus of ef-
forts to treat diabetes by decreasing glucose production. The
prevention of hyperglycemia through control of gluconeogen-
esis is widely considered to be a viable strategy for the treat-
ment of type II diabetes. FBPase converts fructose 1,6-bisphos-
phate (FBP) into fructose 6-phosphate and inorganic phos-
phate; inhibition of this step along the gluconeogenesis path-
way has been found to be effective in the control of blood
sugar levels in rat models of the pre-diabetic state.[4,8]
In a recent study[5] we synthesized and evaluated a broad
group of compounds including substituted pyrazoles, pyrroles,
indoles, and carbazoles for inhibition of FBPase in order to
identify potential lead compounds that modulate the activity
of this enzyme. Based on enzyme inhibition assays and dock-
ing studies we identified a small group of leads. These com-
pounds showed similar or better IC50 values (3.1, 4.8, 6.1, and
11.9 mm) than that of adenosine monophosphate (AMP), the
natural inhibitor of murine FBPase (IC50 =4.0 mm). Docking pro-
grams were used to interpret the experimental results.[5]
In this study we built upon our previous SAR data, and by
the use of extended targeted molecular modeling and docking
studies, we designed and evaluated a broad variety of new in-
hibitor candidates. Based on the in silico results, we identified
and synthesized five new inhibitor compounds for synthesis.
Herein we report two new improved lead compounds for the
inhibition of FBPase. We used three of the most commonly ap-
plied biochemical modeling programs to carry out parallel
docking studies with a broad range of derivatives of the origi-
nal lead compounds. Although there are three potential bind-
ing sites in the FBPase structure to target,[9] according to the
results from our previous study as well as other studies, we in-
[a] A. Rudnitskaya, D. A. Borkin, K. Huynh, Prof. B. Tçrçk, Prof. K. Stieglitz
Department of Chemistry
University of Massachusetts Boston
100 Morrissey Blvd., Boston, MA 02125 (USA)
Fax: (+1)617-287-6030
E-mail: bela.torok@umb.edu
kstieglitz@rcc.mass.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.200900493.
384
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ChemMedChem 2010, 5, 384 – 389

FBPase Inhibitors
vestigated small-molecule binding at the allosteric regulatory
site that binds AMP (Figure 1).[5,10]
After analysis of the results and comparison of the docking
data with those of the natural inhibitor AMP, as well as taking
Figure 1. A close-up view of the AMP binding pocket of FBPase (PDB ID:
1KZ8[9]). The image was produced by using POVScript+.[33] The actual posi-
tion of AMP in the crystallographic structure is shown. The docking pro-
grams were tested for accuracy using AMP as previously described.[5]
synthetic approaches into consideration (difficulty, cost, and
stability), five compounds were synthesized. The compounds
prepared were then tested in FBPase inhibition assays to deter-
mine their potency; the results were then compared with that
of AMP.
Figure 2. The library of pyrrole-, pyrazole-, and indole-based potential
FBPase inhibitor candidates used in this study (see the Supporting Informa-
tion for individual structures).
followed by insertion of the ligand in the known allosteric
pocket of the enzyme. The complete series of docking results
are summarized in table S1 (Supporting Information). Based on
the calculated binding energies and binding constants (pre-
dicted Ki values in AutoDock) of the small-molecule structures
in the three docking programs, we selected the best 13 com-
pounds. For each docking program used, the docking scores
of the potential inhibitors (small molecules) were compared
with that of AMP. The compounds predicted to bind FBPase
with greater than twofold higher affinity than AMP were se-
lected for viewing interactions in the AMP binding pocket by
the molecular graphics program Chimera, and were modified
as described above. The program X-Score[12,13] was used as a
common measurement tool for comparison of performance of
the small molecules in the docking programs. The binding pa-
rameters (energies, Ki values) of these compounds in the inhib-
itor–FBPase interaction are listed in Table 1.
Results and Discussion
The small-molecule structures used in this study were generat-
ed based on our earlier investigations.[5] The major groups ana-
lyzed were indoles, pyrroles, and pyrazoles. The compounds
were evaluated virtually by using the most commonly applied
biomolecular docking programs such as AutoDock4, Dock6,
and Surflex2. Based on the position of chosen ligands in the
protein pocket and on the evaluation of functional groups of
our protein located next to the ligand, we visually determined
where interactions might occur and what types of functional
groups would likely improve both theoretical docking scores
and experimentally determined IC50 values. The most reasona-
ble functional groups were NH2, CH2NH2, COOH, COOCH3, and
CONH2. The three lead compounds were systematically modi-
fied by retaining their original scaffold and adding hydrophilic
or lipophilic substituents of various sizes. We designed and
constructed a library of inhibitor candidates (207) to provide a
systematic variation of several substituents. A schematic repre-
sentation of the compounds used in the docking studies is
shown in Figure 2, and the complete list is provided in the
Supporting Information (figure S1).
As summarized in Table 1, through docking, we identified 13
compounds as candidates for chemical synthesis. The com-
pounds in Table 1 are organized with docking scores from Sur-
flex2 greater than that of AMP (142, (R)-95, 100, 106, 147, and
176). Molecules are also organized with Dock6 docking scores
lower than AMP, including 21, 84E, 64, 84, 160, and 152. The
compounds selected for synthesis were: 1-[4-(trifluoromethyl)-
benzoyl]-1H-indole-5-carboxamide (142), 1-(a-naphthalen-1-yl-
sulfonyl)-7-nitro-1H-indole (176), 5-(4-carboxyphenyl)-3-phenyl-
The new modified structures were drawn in ChemDraw
Ultra 9.0, optimized in Gaussian 98,[11] and tested with the
three docking programs mentioned above. The FBPase crystal
structure was modified by removing the natural inhibitor AMP,
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385

K. Stieglitz, B. Tçrçk, et al.
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than those of the indole derivatives 142 and 176. We were
therefore able to narrow down the basic scaffold in our
second-generation lead compounds to the triarylpyrazoles
and, most importantly, to the substituted N-sulfonylpyrroles
(Scheme 1). Importantly, the activity of our new inhibitors, es-
pecially 84E (IC50 =32 nm) is virtually the same as that of the
best currently known inhibitor compounds.[2–4]
Table 1. Docking data for AMP and the 13 best inhibitor candidates.
Compd
Surflex2
Energy
Score
Dock6
Energy
Score
AutoDock4
Final Docked
Energy
X-Score
Energy
Score
AMP
142
(R)-95
100
106
(S)-120
147
21
176
84E
164
84
4.00
5.33
5.09
4.82
4.70
4.60
4.27
4.13
4.12
4.02
2.91
4.43
4.15
3.47
À25.66
À25.19
À19.32
À21.85
À26.85
À21.54
À21.41
À35.68
À25.76
À34.22
À33.53
À31.18
À29.48
À29.46
À15.23
16.03
À14.26
À9.17
À23.38
À23.47
À16.81
À22.68
À22.90
À16.57
À23.52
À15.43
À22.52
À25.20
À20.26
À23.84
À16.04
À24.32
Docking studies and visual examination of the results using
Chimera suggested that similar interactions of the molecules
occur in the AMP binding pocket relative to the natural inhibi-
tor AMP, as reported previously.[5] More specifically, both 142
and 176 showed hydrogen bonding and electrostatic interac-
tions with Lys112, Tyr113, and Arg140, as well as enhanced
(short-distance) hydrophobic interactions with Leu30 and
Val160 compared with AMP. Compound 100 also showed
shorter distances than the phosphate group of AMP in hydro-
gen bonding of its carboxylic acid moiety with Tyr113, Lys112,
Thr27, and Arg140. The fluorine atoms are buried in the bind-
ing pocket and interact with Thr31 and the backbone of Gly21
(Figure 3A). The sulfate group oxygen atoms of compound 84
interact with Arg140 and Tyr113 and the backbone of Thr31
(Figure 3B). Compound 84E exhibits enhanced hydrophobic
interactions with Val160, Leu30, and Leu34 of FBPase; its sul-
fate group oxygen atoms interact with Arg140 and Tyr113, as
well as the backbone of Thr31 (Figure 3C). The most striking
difference from AMP was the number of new interactions es-
tablished in the hydrophobic interior of the AMP binding
pocket (Figure 3).
À15.70
À12.02
À11.77
À15.59
À15.36
À18.50
À15.97
À15.25
À9.42
160
152
À9.22
1-[3-(trifluoromethyl)phenyl]-1H-pyrazole (100), 1-(4-carbome-
thoxyphenylsulfonyl)-1H-pyrrole (84E), and 1-(4-carboxyphenyl-
sulfonyl)-1H-pyrrole (84). Compounds 142 and 176 could be
synthesized by a base-assisted N acylation of the correspond-
ing commercially available indole derivatives with the appro-
priate acid chlorides, and the two were produced by using es-
sentially the same procedure. The pyrazole derivative 100 was
synthesized by a domino cyclization-aromatization process
with 100E, and then it was hydrolyzed by a solution of potas-
sium hydroxide in ethanol. Compound 84E was prepared by a
method we reported earlier, a K-10 montmorillonite-catalyzed
cyclization process;[14] the ester was hydrolyzed to furnish 84
by the same method mentioned above for the conversion of
100E into 100. Despite moderate yields in certain cases, we
were able to obtain the products, and tested their activity in
FBPase inhibition. Notably, we attempted the synthesis of
other compounds that were also identified as reasonable can-
didates. In some cases, despite successful synthesis and proper
identification by mass spectrometry, the stability of the prod-
uct was not satisfactory; in most cases the half-life of these
compounds was less than 2–3 h. These stability problems limit-
ed the number of inhibitor candidates for biological assays to
five.
Conclusion
In summary, we applied computer-based drug design using
docking programs and graphics-display programs to systemati-
cally select molecules for synthesis, testing, and analysis in
FBPase inhibition. The synthesized compounds were tested for
their biological potency by an in vitro enzyme inhibition assay.
The biological response to the new compounds is approxi-
mately two orders of magnitude better than that generated by
the natural inhibitor AMP. These results highlight two N-sulfo-
nylpyrroles and a triarylpyrazole as our second-generation lead
compounds, with potency similar to that of the best currently
known compounds.
Because AMP is the natural inhibitor of FBPase, any new pro-
posed inhibitor for this enzyme was not only docked into the
allosteric AMP binding site, but was also compared with AMP
by determination of its IC50 value. Based on earlier studies, we
determined the biological activity of the selected compounds
in a murine FBPase inhibition assay. For direct comparison, the
potency of AMP was also determined under identical condi-
tions, and the IC50 value of AMP was determined to be 4.0 mm.
For 84E, 84, 100, 142, and 176, the respective IC50 values were
found to be 32 nm, 135 nm, 575 nm, 991 nm, and 1.34 mm. It
appears that the computer-based systematic inhibitor design
resulted in compounds that are more potent than AMP by ap-
proximately two orders of magnitude. Our data clearly indicate
that not only the N-sulfonylpyrroles 84 and 84E, but also the
pyrazole derivative 100 gave significantly lower IC50 values
Experimental Section
Design of inhibitor candidates
The small-molecule structures used in this study were generated as
described below. Three lead structures were systematically
changed by retaining their original scaffold and adding substitu-
ents of various character. These major groups were indoles, pyr-
roles, and pyrazoles. Thus a wide range of derivatives was con-
structed to provide a systematic variation of the substituents for
docking studies. The following method was used to propose the
substituents: the FBPase crystallographic structure was modified
by removing the natural inhibitor AMP, and then replacing it with
the ligand in question by docking to the allosteric AMP binding
site.
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FBPase Inhibitors
Docking studies
The 3D crystal structure coordinates were downloaded
from the RCSB Protein Data Bank (PDB).[15] The ligand
was removed from the binding pocket of interest. The
energy-minimized ligand with full charges was loaded
into the program PRODRG.[16] The format used was
mol2 for Dock6[17] and Surflex2,[18] whereas PDBQ
format was used for AutoDock4.[19] The molecules were
drawn in GaussView 2.1,[11] energy-minimized (HF/STO-
3G), and converted into PDBQ then mol2 format (Chem-
Draw Ultra 9.0 and Dundee PRODRG). The structure of
FBPase (PDB ID: 1FTA) was obtained from the RCSB
PDB.
Using Surflex-Dock 2.0, a conformational search of rotat-
able bonds was performed, and these conformations
were aligned to the protein (protomol); the binding
energy was then determined in log format. AutoDock4
was used to dock the ligand into a set of grids describ-
ing the target protein, and this provided estimated in-
hibition constants in log format. Dock6 was used to
generate a set of overlapping spheres that fill the active
site. To orient a ligand within the active site, some of
the sphere centers were “matched” with ligand atoms.
Approximations were made to the usual molecular me-
chanics attractive and dispersive terms for use on a
grid. The program presented energy scores in log
format. X-Score[12,13] was used to compare binding ener-
gies by analyzing binding strength between the protein
and ligand, as determined by electrostatic, hydrophilic,
and hydrophobic interactions.
Synthesis of inhibitor candidates
All chemicals and solvents were purchased from Aldrich.
The inhibitor candidates were synthesized based on
published methods.[20–24] In all cases, compounds were
purified by preparative thin-layer chromatography (TLC).
Identification and determination of purity were carried
out by gas chromatography–mass spectrometry with an
Agilent 6850-5973N GC–MS system (70 eV electron
impact ionization, 30 m long DB-5 type column) and
NMR spectroscopy (1H, 13C, and 19F when applicable)
using a 300 MHz superconducting Varian NMR spec-
Scheme 1. Synthesis and biological potency of the second-generation lead compounds.
Figure 3. AMP binding pocket of human FBPase (PDB ID: 1FTA)[34] with the docking results of the three best inhibitors: A) 100, B) 84, and C) 84E. This image
was generated with the program Chimera.[35]
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trometer, with [D6]DMSO or CDCl3 as solvents and (CH3)4Si as inter-
nal standard. Coupling constants are given in Hz. The purity of the
compounds exceeded 95% (GC).
1H), 7.59 (m, 1H), 7.38–7.49 (m, 7H), 6.93 ppm (s, 1H); 13C NMR
(74.467 MHz, CDCl3): d=215.85, 171.40, 152.88, 143.31, 140.09,
135.19, 132.23, 130.54, 129.56, 129.24, 128.78, 128.70, 128.51,
128.08, 125.86, 124.30, 122.08, 106.64 ppm; 19F NMR (300 MHz,
CDCl3): d=À63.07 ppm.
1-(4-Carbomethoxyphenylsulfonyl)-1H-pyrrole (84E): 4-Carbome-
thoxyphenylsulfonamide (1 mmol) and 2,5-dimethoxytetrahydrofur-
an (1.5 mmol) were mixed in Et2O (3 mL) in a round-bottom flask.
K-10 montmorillonite (500 mg) was then added. After stirring for
5 min, the solvent was evaporated to produce a dry mixture of re-
actants adsorbed at the catalyst surface. The dry mixture was trans-
ferred to a reaction tube and irradiated in a focused microwave re-
actor (CEM Discover Benchmate) at 1008C. The reaction tempera-
ture was determined and maintained by a built-in infrared temper-
ature detector–controller. After satisfactory conversion, Et2O
(10 mL) was added to the cold mixture, and the product was sepa-
rated from the catalyst by filtration. The product was isolated as
colorless crystals and purified by flash chromatography to give
198 mg 84E (75% yield): mp: 115–1178C (dec.); 1H NMR
(300.126 MHz, CDCl3): d=8.12 (d, J=8.1 Hz, 2H), 7.91 (d, J=8.1 Hz,
2H), 7.16 (d, J=4.8 Hz, 2H), 6.31 ppm (d, J=4.8 Hz, 2H); 13C NMR
(74.467 MHz, CDCl3): d=165.15, 142.59, 134.76, 130.34, 127.73,
127.37, 120.81, 114.18, 52.72 ppm.
1-[4-(trifluoromethyl)benzoyl]-1H-indole-5-carboxamide
(142):
Indole-5-carboxamide (1 mmol, 160 mg) was dissolved in CH2Cl2
(1 mL), and tBuOK (1.1 mmol, 123 mg) was carefully added at room
temperature. The bright-orange solution was stirred for 30 min,
and then 4-trifluoromethylbenzoyl chloride (1.2 mmol, 178 mg)
was added. The reaction mixture was allowed to react under con-
tinuous stirring for 12 h. The reaction was then poured into dis-
tilled H2O (2 mL), and the product was extracted with CH2Cl2. The
combined extracts were dried over Na2SO4, and the solvent was re-
moved under vacuum. The crude product was purified by prepara-
tive TLC to give 22 mg 142 (7% yield). 1H NMR (300.126 MHz,
[D6]DMSO); d=8.68 (d, J=8.4 Hz, 1H), 8.53 (m, 3H), 8.26 (d, J=
8.7 Hz, 2H), 7.78 (d, J=3.9 Hz, 1H), 7.74 (s, 2H, NH2), 7.22 ppm (d,
J=3.3 Hz, 1H); 13C NMR (74.467 MHz, [D6]DMSO): d=169.51,
168.09, 138.20, 138.02, 137.62; 131.14, 130.76, 130.43, 129.84,
127.37, 126.48, 126.21, 125.01, 121.58, 121.41, 121.13, 116.14,
112.37, 111.66, 110.15 ppm; 19F NMR (282.4 MHz, [D6]DMSO): d=
À60.87 ppm; MS (70 eV) for C17H11N2O2F3: m/z (%): 332 [M]+ (100),
313 (17), 173 (85), 145 (40).
1-(4-Carboxyphenylsulfonyl)-1H-pyrrole (84): 4-Carbomethoxy-
phenylsulfonamide (120 mg) was dissolved in EtOH (0.1 mL). KOH
(8m, in 0.1 mL EtOH) was then added to the solution, which was
stirred for 12 h at room temperature. After satisfactory conversion,
the solution was acidified to pH 1 with concd HCl. A white precipi-
tate formed which was washed (H2O, 15 mL) and dried. The crude
product was purified by flash chromatography to give 98 mg 84
(89% yield). Colorless crystals: mp: 225–2278C (dec.); 1H NMR
(300.126 MHz, [D6]DMSO): d=8.14 (d, J=8.7 Hz, 2H), 8.08 (d, J=
8.7 Hz, 2H), 7.39 (t, J=2.1 Hz, 2H), 6.40 (d, J=2.1 Hz, 2H),
3.39 ppm (brs, 1H); 13C NMR (74.467 MHz, [D6]DMSO): d=166.38,
142.04, 136.56, 131.17, 127.69, 121.82, 115.02 ppm.
1-(a-naphthalen-1-ylsulfonyl)-7-nitro-1H-indole (176): tBuOK
(1.1 mmol, 123 mg) was carefully added to a solution of 7-nitroin-
dole (1 mmol, 162 mg) in CH2Cl2 at room temperature. The bright-
orange solution was stirred for 30 min, and a-naphthylsulfonyl
chloride (1.2 mmol, 272 mg) was then added. The reaction mixture
was allowed to react under continuous stirring for 12 h. The reac-
tion was then poured into distilled H2O (2 mL), and the product
was extracted with CH2Cl2. The combined extracts were dried over
Na2SO4, and the solvent was removed under vacuum. The crude
product was purified by preparative TLC to give 215 mg 176 (60%
1
yield). H NMR (300.126 MHz, CDCl3): d=8.42 (m, 2H), 8.13 (d, J=
5-(4-Carbomethoxyphenyl)-3-phenyl-1-[3-(trifluoromethyl)-
phenyl]-1H-pyrazole (100E): (E)-methyl-4-(3-oxo-3-phenylprop-1-
enyl)benzoate (266 mg, 1 mmol) and 3-trifluoromethyphenylhydra-
zine (157 mL, 1.2 mmol) were dissolved in AcOH (5 mL), followed
by the addition of I2 (253 mg). The mixture was stirred for 24 h at
room temperature and then quenched with a saturated solution of
Na2CO3 (5 mL). The product was extracted with Et2O (3ꢂ15 mL).
The combined organic extracts were washed with a saturated solu-
tion of Na2S2O3 (10 mL), dried over anhydrous Na2SO4, and the sol-
vent was removed under vacuum. The crude product was purified
by flash chromatography to yield 320 mg 100E (76% yield). Color-
less crystals: mp: 189–1928C (dec.); 1H NMR (300.126 MHz,
[D6]DMSO): d=8.03 (d, J=8.4 Hz, 2H), 7.92 (d, J=8.4 Hz, 2H), 7.78
(brs, 1H), 7.59 (m, 1H), 7.35–7.46 (m, 7H), 6.92 (s, 1H), 3.94 ppm (s,
3H); 13C NMR (74.467 MHz, CDCl3): d=172.30, 171.10, 152.82,
143.50, 134.35, 132.31, 130.15, 129.94, 129.51, 128.77, 128.62,
128.46, 128.05, 125.85, 124.21, 122.06, 117.09, 105.59 ppm; 19F NMR
(300 MHz, CDCl3): d=À63.10 ppm; MS (70 eV) m/z (Irel): 422 (100%)
[M]+, 423 (26%) [M]+, 391 (23%), 392 (6%).
8.1H, 1H), 7.97 (m, 2H), 7.84 (d, J=7.8 Hz, 1H), 7.75 (d, J=8.1H,
1H), 7.68 (d, J=3.9 Hz, 1H), 7.61 (m, 2H), 7.57 (dd, J=7.5, 8.1 Hz,
1H), 7.38 (dd, J=8.1, 7.8 Hz, 1H), 6.82 ppm (d, J=3.9 Hz, 1H);
13C NMR (74.467 MHz, CDCl3): d=135.12, 134.08, 130.71, 129.06,
128.80, 127.88, 127.63, 127.28, 126.43, 125.85, 123.94, 123.81,
123.36, 120.88, 116.75, 108.92 ppm; MS (70 eV) for C18H12N2O4S: m/
z (%): 352 [M]+ (92), 241 (18), 191 (55), 127 (100).
FBPase assays
Acetone-precipitated murine liver homogenates were resuspended
in deionized H2O, sterile filtered, and then purified and dialyzed
against 50 mm Tris buffer (pH 7.5) for three changes over 48 h at
48C. The crude protein extract was then run over a gel filtration
column (G25–150), and the fractions were analyzed by Bradford
assay and SDS-PAGE. Fractions containing FBPase activity were
pooled and re-dialyzed in 50 mm Tris (pH 7.5), loaded on a Matrix
Gel Blue Affinity column (Cibacron Blue 3GA dye coupled to cross-
linked 5% agarose), and eluted in the presence of 1 mm FBP. Frac-
tions were tested for activity, pooled, and re-dialyzed in 50 mm Tris
5-(4-Carboxyphenyl)-3-phenyl-1-[3-(trifluoromethyl)phenyl]-1H-
pyrazole (100): Compound 100E (175 mg) was dissolved in EtOH
(0.2 mL). KOH (8m, 76 mL in EtOH) was then added to the solution.
The mixture was stirred for 12 h at room temperature and then
acidified to pH 1 with concd HCl. The precipitate was filtered off,
washed with H2O (5 mL), and dried for 12 h. The crude product
was purified by preparative TLC to give 145 mg 100 (87% yield).
White crystals: mp: 215–2178C (dec.); 1H NMR (300.126 MHz,
CDCl3): d=8.09 (d, J=8.4 Hz, 2H), 7.93 (d, J=8.4 Hz, 2H), 7.78 (brs,
(pH 7.5). The final concentration of wild-type enzyme was deter-
[25]
mined by absorption at l 280 nm (A280
)
and compared with the
Bio-Rad version of the Bradford dye-binding assay.[26] Enzyme
purity was verified by SDS-PAGE[27] and non-denaturing PAGE.[28,29]
The FBPase high-throughput screen is based on measurement of
phosphatase activity by colorimetric assay using an ammonium
molybdate Malachite Green reagent.[30,31] The reagent was pre-
388
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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 384 – 389

FBPase Inhibitors
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order to minimize experimental error. The amount of enzyme was
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Support of our work by the University of Massachusetts Boston is
greatly appreciated.
Keywords: docking · heterocycles · indoles · inhibitors ·
N substitution
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Received: December 1, 2009
Revised: December 16, 2009
Published online on January 12, 2010
ChemMedChem 2010, 5, 384 – 389
ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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