A library of novel allosteric inhibitors against fructose 1,6-bisphosphatase

Article abstract of DOI:10.1016/j.bmc.2009.04.030

The identification of a proper lead compound for fructose 1,6-bisphosphatase (FBPase) is a critical step in the process of developing novel therapeutics against type-2 diabetes. Herein, we have successfully generated a library of allosteric inhibitors against FBPase as potential anti-diabetic drugs, of which, the lead compound 1b was identified through utilizing a virtual high-throughput screening (vHTS) system, which we have developed. The thiazole-based core structure was synthesized via the condensation of α-bromo-ketones with thioureas and substituents on the two aryl rings were varied. 4c was found to inhibit pig kidney FBPase approximately fivefold better than 1b. In addition, we have also identified 10b, a tight binding fragment, which can be use for fragment-based drug design purposes.

Full text of DOI:10.1016/j.bmc.2009.04.030

Bioorganic & Medicinal Chemistry 17 (2009) 3916–3922
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A library of novel allosteric inhibitors against fructose 1,6-bisphosphatase
*
Sabrina Heng, Kimberly R. Gryncel, Evan R. Kantrowitz
Boston College, Department of Chemistry, Merkert Chemistry Center, Chestnut Hill, MA 02467, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 February 2009
Revised 7 April 2009
Accepted 9 April 2009
Available online 19 April 2009
The identification of a proper lead compound for fructose 1,6-bisphosphatase (FBPase) is a critical step in
the process of developing novel therapeutics against type-2 diabetes. Herein, we have successfully gen-
erated a library of allosteric inhibitors against FBPase as potential anti-diabetic drugs, of which, the lead
compound 1b was identified through utilizing a virtual high-throughput screening (vHTS) system, which
we have developed. The thiazole-based core structure was synthesized via the condensation of a-bromo-
ketones with thioureas and substituents on the two aryl rings were varied. 4c was found to inhibit pig
kidney FBPase approximately fivefold better than 1b. In addition, we have also identified 10b, a tight
binding fragment, which can be use for fragment-based drug design purposes.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Fructose 1,6-bisphosphatase
Type-2 diabetes
ZINC database
Enzyme inhibition
Thiazole derivatives
Virtual high-throughput screening
1. Introduction
tified at the center of the molecule where the four subunits con-
verge.9,10 The enzyme does not exhibit substrate cooperativity
In 2007, it was estimated that 7.8% of the population in the Uni-
ted States (23.6 million people) have diabetes,1 while the world-
wide frequency of diabetes is expected to continue to grow by
6% annually, potentially reaching a total of 200–300 million cases
by 2010.2 In adults, type-2 diabetes, also known as non-insulin-
dependent diabetes mellitus accounts for about 90–95% of all diag-
nosed cases of diabetes. The liver has a critical role in regulating
endogenous glucose production from de novo synthesis (gluconeo-
genesis) or the catabolism of glycogen (glycogenolysis).3 Increased
rates of hepatic glucose production are largely responsible for the
development of overt hyperglycemia, in particular fasting hyper-
glycemia, in patients with diabetes.4 Therefore enzymes that regu-
late rate-controlling steps in the gluconeogenic or glycogenolytic
pathways are obvious molecular targets for therapeutic interven-
tions.5 As a rate-limiting and highly regulated enzyme in the gluco-
neogenesis pathway, fructose-1,6-bisphosphatase (FBPase) is an
attractive target in the development of new anti-diabetic
pharmaceuticals.
but is cooperative with respect to the binding of AMP and metal
cofactors.11 Without effectors the enzyme exists in the R-quater-
nary structure. AMP induces the transition from the active R-state
to the inactive T-state.12
Targeting the AMP binding site has historically been challeng-
ing due to the abundance of AMP-binding enzymes controlling
other key biosynthetic pathways resulting in issues with specific-
ity. Other difficulties that need to be overcome include the hydro-
philic nature of AMP sites and their reliance on the negatively
charged phosphate group of AMP for binding affinity.13 Target-
based virtual database screening has become a useful tool for
the identification of inhibitors for protein–ligand and protein–
protein interactions.14 In light of the abovementioned challenges,
virtual screening, in the use of high-performance computing to
analyze chemical databases and prioritize compounds for synthe-
sis and assay,15 then provides a more cost-effective approach to
discovering allosteric inhibitors that bind to the desired allosteric
site and yet are structurally distinct from the traditional AMP
analogs.
In the present work, we have successfully generated a library
of allosteric inhibitors against FBPase of which, the lead com-
pound was identified utilizing a virtual high-throughput screen-
ing (vHTS) system, which we have developed. In this paper, the
synthesis and the ability of the compounds in this library to inhi-
bit FBPase in vitro are also described, thus demonstrating how
vHTS can be utilized to find and develop novel inhibitors against
FBPase.
FBPase is a tetramer of four identical polypeptide chains (Mr
34,000/chain) and exists as a dimer of dimers.6 The enzyme exists
in at least two distinct quaternary conformations called R and T.7
The enzyme is subject to competitive substrate inhibition by fruc-
tose-2,6-bisphosphate8 and to allosteric inhibition by adenosine
monophosphate (AMP). A ‘novel allosteric site’ has also been iden-
* Corresponding author. Tel.: +1 617 552 4558.
E-mail address: evan.kantrowitz@bc.edu (E.R. Kantrowitz).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.030

S. Heng et al. / Bioorg. Med. Chem. 17 (2009) 3916–3922
3917
spectrum of inhibitory activities, ranging from 10 mM to 20 lM.
2. Results
Of these, we decided to pursue a more detailed structure activity
relationship (SAR) study of 1. Compound 1, contains a thiazole ring,
a functionality that has been found to be associated with a plethora
of biological activities,28 several of which have been developed into
potent inhibitors of FBPase.9,29 More importantly, 1 was chosen be-
cause it has reasonable activity (IC50 of 31.6 1.6 lM) and pos-
sesses two aromatic rings that would allow for a variety of
functional groups to be investigated.
2.1. In silico screening
The identification of a proper lead compound for FBPase is a
critical step in the process of developing novel therapeutics against
diabetes. To this end, target-based virtual database screening has
become a useful tool for the identification of inhibitors for pro-
tein–ligand and protein–protein interactions.15,16 In our labora-
tory, we have in-place, a vHTS system that is set-up to screen
millions of compounds against a desired target. The two essential
components for a successful screen are the docking software and
the database of small molecules. In an effort to make virtual
screening more accessible to a broader community, Irwin et al.17
developed ZINC, a free database of structures of small molecules,
many of them ‘drug-like’ or ‘lead-like’. Virtual screening using
the ZINC database has now been used for the development of
inhibitors for a variety of targets including cyclooxygenase-2,18 an-
thrax edema factor19 and the H5N1 avian influenza virus.20
2.3. Chemistry
The synthesis of the inhibitors shown in Table 1 was carried out
by modifying reported procedures and is illustrated in Scheme 1.
Where the primary thiourea could not be purchased commercially,
it was easily synthesized by reacting the respective amine 20–24
with excess potassium thiocynate in the presence of 1 equiv con-
centrated HCl. In most cases, the pure primary thioureas could
be isolated by precipitation and purified in good yield by crystalli-
The virtual high-throughput screening system we have devel-
oped consists of four parts: (1) a MySQL database containing en-
tries of the molecules in the ZINC6 database in mol2, pdbq and
mae format, (2) a set of unix tar files containing the executable
zation.30 The condensation of
a-bromoketones 25–29 with the N-
monosubstituted thioureas 11–18 via the Hantzsch reaction were
originally accomplished by heating the reaction mixture under re-
flux, with varying reaction times from 7 h to 24 h.28,31 Although
combinatorial chemistry has been widely adopted in drug discov-
ery in recent years, it can be both labor and time intensive. Gener-
ating a small library of 29 compounds via a two-step synthetic
pathway as described, would require setting up 29 reaction vessels,
each used twice. This was made more challenging when problems
arose in the second synthetic step. Even after 24 h, overall yields
were low (<30%) and purifications were tedious.
In order to enhance the capabilities of combinatorial chemistry,
we modified the second synthetic step according to the recent
work by Kabalka and Mereddy,32 who demonstrated the use of
microwave irradiation to promote the rapid one-pot synthesis of
a series of 2-aminothiazoles. Using the same methodology, we
were able to reduce the reaction times from 24 h to a mere 45 s
and increased the yields from less than 30% to greater than 90%
after recrystallization.
21
22
SUFLEX
program and associated auxiliary files for AUTODOCK
,
24,25
DOCK5,23 and GLIDE
(Schrödinger, Inc.), (3) a MySQL database
for storage of the results of the docking calculations, and (4) a UNIX
shell script that automates the process and provides the ability to
distribute the computations over multiple computers.
Validation of AUTODOCK and DOCK5 were performed utilizing the
structure of human FBPase with AMP bound (PDB entry 1FTA).
AMP was first removed from its site, and its coordinates changed
so that AMP was in a different spatial position, distant from the ac-
tual binding site. AMP was then docked into the allosteric site of
human FBPase using the respective docking programs. Both pro-
grams performed well in matching the docked conformation with
the one observed in the crystal structure.
We then screened ꢀ3 million compounds, which are character-
ized as purchasable in the ZINC6 database, against the AMP-bind-
ing site of human FBPase using a combination of the above
mentioned programs. We then examined, in detail, a representa-
tive sample of the compounds that showed high-binding affinity.
The eventual selection of the compounds for in vitro biochemical
assays was based on the following rationales: (i) not all the in silico
hits could be obtained commercially, thus limiting the number of
compounds that could be directly tested in vitro, (ii) hits that were
analogs of or were structurally similar to AMP were eliminated,
(iii) only one member of a group of similar compounds was char-
acterized thus maximizing chemical diversity, and (iv) the com-
pounds had to be considered drug-like as defined by Lipinski’s
rule of 5.26
Having designed an efficient route to the compounds in groups
1–8, we were able to expand the library further by synthesizing the
2-aminothiazole fragments according to Scheme 2. Compounds
10a–10c (Table 2), were synthesized from thiourea 19 according
to the method described above.
3. Discussion
The ability of compounds 1a–10c to inhibit the activity of pig
kidney FBPase was determined using a coupled-enzyme assay27
with AMP as the control, and the results obtained are shown in Ta-
bles 1 and 2. To validate our results, all 29 inhibitors were docked
against the allosteric site of a pig kidney T-state crystal structure
(PDB entry 1FRP) using the high precision mode (XP) mode in Glide
(SchrÖdinger).25 A graph of IC50 values was plotted against the
resultant docking score obtained from Glide. As showed in Figure
2, there is good agreement between the actual inhibitory activity
against the pig kidney enzyme and the binding affinity as predicted
by Glide.
2.2. Experimental assays
The selected compounds were assayed for inhibitory activity
using purified pig kidney FBPase, employing a coupled-enzyme as-
say with AMP as the control.27 The degree of amino acid sequence
conservation within the FBPase coding region among mammalian
species is very high, with 90% of the residues identical and another
6% similar. Thus the pig kidney enzyme is an excellent model sys-
tem to study enzyme inhibition with respect to human FBPase. In
addition, the use of pig kidney enzyme was conducted to support
our ongoing crystallographic work using this enzyme. Figure 1
illustrates some of the compounds from this screen that were
found to inhibit pig kidney FBPase. As shown, the vHTS system suc-
cessfully identified inhibitors of pig kidney FBPase with a broad
The results displayed in Tables 1 and 2 revealed that substitut-
ing a hydroxyl at R5 is essential for inhibition. This trend is consis-
tent regardless of the substitution on ring 1, as inhibitors with
hydroxyl functionality at R5 resulted in improved inhibition over
those without. Remarkably, while 7a, even at 300
significant inhibitory activity against the enzyme, the R5 hydroxyl
substituted 7b inhibited the enzyme at an IC50 of 48 M.
Likewise, comparison of 5a (IC50 = 270 14 M) with 5b
lM, showed no
3
l
a
l

3918
S. Heng et al. / Bioorg. Med. Chem. 17 (2009) 3916–3922
Figure 1. Structures and IC50 of AMP and various inhibitors identified by the vHTS system.
when the hydroxyl is replaced by nitro at R5 (3d). Similarly, a
nitro group at R5 in 8e eliminates inhibition compared to 8a
Table 1
Inactivation of pig kidney FBPase by inhibitors 1a–9b
(IC50 = 2.5 0.1 mM) and 8b (IC50 = 320
4 lM).
Given the high concentration of positively charged residues
present at the AMP binding site as shown in Figure 3, it is reason-
able to assume that by adding a second hydroxyl at R4, that the
binding affinity of the inhibitors for the allosteric site might in-
crease. This is true for groups 3 and 4 where the additional hydro-
xyl at R4 improved enzyme inhibition. However, with the rest, the
differences in inhibitory activity between the single and double
hydroxyls were negligible.
Ring 1
Ring 3
R3
R2
S
R5
N
N
H
R1
R4
1a - 9b
Manipulation of the substituent positions on Ring 1 within the
same functional group seemed to have little effect on activity. This
is demonstrated by the relatively small changes in inhibitory activ-
Compound
R1
R2
R3
R4
R5
FBPase, IC50
145 11
(lM)
1a
1b
1c
2a
2b
3a
3b
3c
3d
4a
4b
4c
5a
5b
5c
6a
6b
7a
7b
8a
8b
8c
8d
8e
9ac
9bc
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
SO2NH2
SO2NH2
SO2NH2
H
H
OH
OH
OH
OH
H
H
H
OH
H
H
H
H
OH
H
H
H
OH
H
H
OH
H
H
H
H
H
H
OH
OH
H
OH
H
OH
OH
NO2
H
OH
OH
H
OH
OH
H
OH
H
OH
H
OH
OH
H
32
2
ity between 1a (IC50 = 145 11
lM) and 2a (IC50 = 104
7 lM)
55 0.5
where the sulfonamide was moved from R3 to R2. Similar observa-
tions can be made for the hydroxyl and nitro substitutions at R1, R2
an R3. Given the relative size differences between these functional
groups, this may suggest that the area where Ring 1 binds is not
restrictive. However, between the different functional groups,
there is a marked effect on the activity of the inhibitor. Overall, it
would seem that hydroxyls are better for activity as opposed to
sulfonamides and nitros. Inhibitors with no substitution on Ring
1 seemed to fare the worst as a group.
In an effort to develop potent inhibitors against FBPase, part of
our strategy is to use the fragment based approach to enhance
inhibitor affinity. Thus, we investigated the effect that the phenyl-
thiazole fragment 10a–10c have on the enzyme activity. To our
surprise, the fragment 4-(2-aminothiazol-5-yl)phenol 10b, inhib-
SO2NH2
SO2NH2
H
H
H
104
50
7
6
124 6.0
48 3.0
11 0.5
NDa
H
OH
OH
OH
H
H
H
H
H
NO2
NO2
H
H
H
H
H
COOH
COOH
35
13 0.5
0.5
270 14
4
H
H
H
H
6
15
1
2
H
20
NO2
NO2
H
H
H
H
H
H
H
NDb
246 12
NDb
48
3
2500 110
ited the enzyme with an IC50 of 1.1 0.1 lM, having better inhibi-
H
318
343
119
NDb
4
4
4
tory activity than all the diphenylthiazole-based inhibitors found
in Table 1. In addition, the binding of 10b at the allosteric site
was observed in a low resolution X-ray crystallography data of
the enzymeꢁ10b complex that we had obtained.
OH
OH
H
H
H
NO2
H
OH
H
H
571 5.0
48
4
In order to confirm that the FBPase inhibition by the achyrofuran
analogs was due to binding at the allosteric site and not the active
site, a competition experiment was performed using the analog of
AMP, 20,30-O-(2,4,6-trinitrophenyl)adenosine 5-monophosphate
(TNP-AMP). This analog has been shown to bind at the allosteric site
of FBPase and exhibits fluorescence only when bound to the enzyme.
TNP-AMP was added to the FBPase at a concentration equal to 0.5
times their respective Kd. Increasing concentrations of the respective
inhibitors were added which resulted in a substantial diminution of
the TNP-AMP fluorescence, indicating that these inhibitors may be
competing with TNP-AMP at the allosteric site. Binding constants
for the inhibitors could not be determined by this method due to
the relatively low solubility of these compounds.
Assays were performed in 0.2 M Tris, 4 mM MgCl2, 4 mM (NH4)2SO4, 0.1 EDTA, pH
7.5.
a
Approximately 87% inhibition at 100
No detectable inhibition observed.
Commercially available compounds.
lM.
b
c
(IC50 = 15
1 lM) showed that the R5 hydroxyl substitution (5b)
resulted in an ꢀ18-fold improved inhibition. On the other hand,
functionalizing R5 with nitro instead of hydroxyl, eliminated
inhibition. For example, a comparison of 3a (IC50 = 124
6 lM)
with 3b (IC50 = 48 M) and 3d showed that the inhibitory activ-
3
l
ity of the inhibitor with an R5 hydroxyl substitution (3b) improved
threefold from 3a while having its inhibitory activity eliminated

S. Heng et al. / Bioorg. Med. Chem. 17 (2009) 3916–3922
Scheme 1. Reagents and conditions: (i) concd HCl, potassium thiocynate, EtOH, reflux, 18 h (ii) EtOH, MW (71 °C, 400 W), 45 s.
Scheme 2. Reagents and conditions: (i) EtOH, MW (71 °C, 400 W), 45 s.
3919
Table 2
Inactivation of pig kidney FBPase by inhibitors 10a–10c
Figure 3. X-ray crystal structure of AMP bound to pig kidney FBPase at the
allosteric site. (PDB entry 1RDZ). The electrostatic potentials calculated using the
Adaptive Poisson–Boltzmann Solver36 were map on a surface representation of the
Compounds
R4
R5
FBPase, IC50 (lM)
37
protein created using PYMOL
.
10a
10b
10c
H
H
OH
H
OH
OH
1392 68
1.1 0.1
93
5
structure of pig kidney FBPaseꢁ1b demonstrated how a vHTS sys-
tem was used to identify and develop novel inhibitors against
FBPase. By eliminating the sulfonamide on the original molecule
(1b) as identified by the vHTS system, and adding a hydroxyl at
R2 to ring 1 and a second hydroxyl at R4 to ring 3, we have designed
Assays were performed in 0.2 M Tris, 4 mM MgCl2, 4 mM (NH4)2SO4, 0.1 EDTA, pH
7.5.
and synthesized 4c (IC50 = 6 0.5
in potency over 1b (IC50 = 32
l
M) which has a fivefold increase
2
lM) against pig kidney FBPase.
Currently, one of the most potent allosteric inhibitor of FBPase is
MB05032, which inhibits human liver FBPase at an EC50 of
16 nM, a value that is approximately 370-fold better than 4c. How-
ever, in line with the fragment-based drug design strategy, a future
goal is to obtain higher resolution X-ray data of the enzymeꢁ10b
complex so that we can use the resultant structure, with 10b
bound, in our vHTS system to screen for fragments that have a high
(micromolar or better) affinity for the allosteric site. Thus, by syn-
thetically linking the appropriate fragment with 10b, we will be
able to generate a more potent and specific inhibitor of FBPase.
And although we only focused on developing a library based upon
compound 1 in this work, our vHTS yielded many other potential
hits, which we are currently in the process of further evaluating
as inhibitors against FBPase.
Figure 2. Graph of the Glide25 docking Score (high-precision XP mode) of
compounds 1a–10c versus their experimentally determined IC50 values. A more
negative Glide docking score corresponds to tighter binding and therefore predicts
better inhibition. This graph was prepared using Kaleidagraph with two outlaying
points removed (3d and 10b).
5. Experimental
5.1. Isolation and purification of pig kidney FBPase
Plasmid pEK28433 was transformed into Escherichia coli strain
EK1601.34 Bacteria were cultured with vigorous agitation at 37 °C
4. Conclusion
in YT35 media containing ampicillin at 100
l
g per mL. Induction
of T7 RNA polymerase was initiated by addition of 0.4 mM isopro-
pyl-b- -thiogalactopyranoside. After further growth for 16–22 h at
Compounds 1a–9b represent a novel class of inhibitors against
FBPase that are not analogs of AMP. The in vitro data in Table 1 and
the observation of inhibitor electron density in a low-resolution
D
37 °C, the cells were harvested by centrifugation, broken open by
sonication, and purified as described previously34 with the modifi-

3920
S. Heng et al. / Bioorg. Med. Chem. 17 (2009) 3916–3922
cation of eluting from the Dyematrex Blue A column with a 50 mM
KH2PO4 buffer at pH 7.5 containing 5 mM ATP instead of AMP. Pure
fractions were pooled and dialyzed extensively against a buffer
containing 50 mM KH2PO4 and 1 mM MgCl2 at pH 7.5 before per-
forming enzyme activity assays.
(37.4%, 2.14 mL) was added dropwise. The suspension that formed
was heated to reflux and when all suspension had dissolved, a
solution of potassium thiocynate (2.6 g, 0.0255 mol) in 5 mL of eth-
anol was added to the suspension. The reaction mixture was stir-
red at reflux for 18 h. The precipitate formed upon cooling was
dried under vacuum and recrystallized from ethanol to yield 3 g
(76.3%) of 11 as white solid; 1H (DMSO-d6): 7.28 (br, 2H), 7.70–
7.75 (m, 4H), 10.21 (br, 1H).
5.2. Measurement of FBPase activity
FBPase activity was measured spectrophotometrically by
employing the coupling enzymes phosphoglucose isomerase and
glucose-6-phosphate dehydrogenase.27 The reduction of NADP+
to NADPH was monitored directly at 340 nm. Specifically, buffer
(0.2 M Tris, 4 mM MgCl2, 4 mM (NH4)2SO4, 0.1 EDTA, pH 7.5),
0.2 mM of NADP+, 1.4 units of phosphoglucose isomerase and 0.5
5.4.2.2. 4-(4-(4-Hydroxyphenyl)thiazol-2-ylamino)benzenesul-
fonamide (1b). A solution of 11 (200 mg, 0.86 mmol) and 2-bro-
mo-40-hydroxyacetophenone 25 (188 mg, 0.87 mmol) in 5 mL of
ethanol were placed in the microwave cavity and subjected to
MW irradiation at 71 °C (400 W). The reaction mixture was cooled
to room temperature and solvent was removed under vacuo. The
resultant solid was washed with ethanol and crystallized from eth-
anol/n-pentane to yield 280 mg (94%) of 1b as a pale yellow solid;
1H (DMSO-d6): 6.73 (d, J = 8.8 Hz, 2H), 7.07 (s, 1H), 7.65–7.70 (m,
4H), 7.78 (d, J = 8.8 Hz, 2H,), 10.57 (br, 1H). HRMS (ESI) [M+H]+
calcd for C15H14N3O3S2, 348.0477; found 348.0486.
units glucose-6-phosphate dehydrogenase, 0–300
and 9 ng of FBPase, were mixed in a cuvette and equilibrated at
30 °C. FBP (70 M) was then added to initiate the reaction. A340
lM of inhibitor
l
data were collected as a function of time using a JASCO V-630 spec-
trophotometer. After a lag phase, due to coupling, a straight line
was fit to the progress curve and the slope was determined as
D
A340 per min. The amount of NADPH produced per min. was cal-
culated using the 340 nm millimolar extinction coefficient of 6.22.
At each inhibitor concentration, reactions were performed in dupli-
cate. Inhibition curves were obtained for each compound by plot-
ting the relative activity versus inhibitor concentration.
5.4.2.3. 4-(4-Phenylthiazol-2-ylamino)benzenesulfonamide
(1a). Compound 1a (275 mg, 97%, white solid) was prepared in a
similar manner as described for the synthesis of 1b except using
2-bromoacetophenone 26. (DMSO-d6): 7.21 (br, 2H), 7.34 (m,
1H), 7.45 (d, J = 9.6 Hz, 2H), 7.46 (s, 1H), 7.80–7.90 (m, 4H), 7.96
(d, J = 8.0 Hz, 2H), 10.74 (bs, 1H). HRMS (ESI) [M+H]+ calcd for
C15H14N3O2S2, 332.0527; found 332.0522.
5.3. Fluorescence measurements
Fluorescence data were collected using a JASCO FP-6300 spec-
trofluorometer. Excitation and emission wavelengths of 410 and
535 nm, respectively, were used for TNP-AMP (Molecular Probes).
5.4.2.4. 4-(4-(2,4-Dihydroxyphenyl)thiazol-2-ylamino)benzene-
sulfonamide (1c). Compound 1c (300 mg, 96%, brown solid) was
prepared in a similar manner as described for the synthesis of 1b
except using 2-bromo-20-40-dihydroxyoacetophenone 27. 1H
(DMSO-d6): 6.36 (d, J = 8.8 Hz, 1H), 6.40 (br, 1H), 7.27 (s, 1H),
7.74–7.82 (m, 5H), 10.84 (br, 1H). HRMS (ESI) [M+H]+ calcd for
C15H14N3O2S2, 332.0527; found 332.0517. HRMS (ESI) [M+H]+
calcd for C15H14N3O4S2, 364.0426; found 364.0443.
0.15 mg of the respective FBPase, 0.08 mM MgCl2 and 20
tose 1,6-bisphosphate in 50 mM Tris-acetate buffer, pH 7.5, was
added to a 2 mL cuvette and stirred. TNP-AMP (3.5 M) was added
lM fruc-
l
to the cuvette and the emission data was obtained. Microliter vol-
umes of each inhibitor were subsequently added to the cuvette and
resultant emission data was obtained after each addition. The final
concentration of each inhibitor in the enzyme solution was be-
tween 5 and 50
5.4. Chemistry
5.4.1. General
l
M.
5.4.2.5. 3-Thioureidobenzenesulfonamide (12). Compound 12
(800 mg, 60%, white solid) was prepared in a similar manner as de-
scribed for the synthesis of 11 and by substituting sulfanilamide
with 3-aminobenzenesulfonamide 21. 1H (DMSO-d6): 7.37 (s,
1H), 7.48–7.56 (overlapped, m, 2H), 7.70 (d, J = 7.6 Hz, 1H), 7.93
(bs, 2H), 9.95 (br, 2H).
All materials were reagent grade and used without further puri-
fication. Thin-layer chromatography (TLC) was performed on Silica
Gel 60 aluminum backed pates (250
l
m) from Sorbent Technolo-
5.4.2.6. 3-(4-Phenylthiazol-2-ylamino)benzenesulfonamide
(2a). Compound 2a (560 mg, 90%, yellow solid) was prepared from
12 and according to the manner described for 1a. 1H (DMSO-d6):
6.82 (d, J = 8.8 Hz, 2H), 7.17 (s, 1H), 7.18 (d, J = 9.6 Hz, 1H), 7.76
(d, J = 8.8 Hz, 2H), 7.78 (d, J = 7.6 Hz, 2H), 7.85 (s, 1H), 7.86 (d,
J = 9.2 Hz, 1H), 9.58 (s, 1H), 10.5 (s, 1H). HRMS (ESI) [M+H]+ calcd
for C15H14N3O2S2, 332.0527; found 332.0517.
gies and visualized by Abs254 irradiation. Flash chromatography
was conducted with Silica Gel 60 (230 mesh) from AK Scientific
Inc. All final products were characterized by 1H NMR and HRMS.
Purity of all final compounds were determined by HPLC. 1H NMR
spectra were recorded on a Varian 400 spectrometer. Proton chem-
ical shifts are reported in ppm (d) relative to internal tetramethyl-
silane (TMS, d 0.0) or with the solvent reference relative to TMS
employed as the internal standard (CDCl3, d 7.24 ppm; DMSO-d6,
d 2.50). Standard abbreviations indicating multiplicity were used
as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = mul-
tiplet and br = broad. Mass spectra were obtained at the Mass Spec-
trometry Facilities at Boston College. HPLC separations were
performed on a Biologic Duo-Flow chromatrography system (Bio-
Rad). Microwave reactions were performed using a General Electric
Spacemaker II, operating at 400 W.
5.4.2.7. 3-(4-(4-Hydroxyphenyl)thiazol-2-ylamino)benzenesul-
fonamide (2b). Compound 2b (600 mg, 93%, brown solid) was
prepared from 12 and according to the manner described for 1b.
1H (DMSO-d6): 6.52 (s, 1H), 6.78–6.86 (overlapped, m, 4H), 7.47
(d, J = 7.2 Hz, 2H), 7.80 (d, J = 7.6 Hz, 2H), 9.80 (br, 1H), 10.40 (br,
1H), 11.61 (br, 1H). HRMS (ESI) [M+H]+ calcd for C15H14N3O3S2,
348.0477; found 348.0492.
5.4.2.8. 4-(4-Phenylthiazol-2-ylamino)phenol (3a). Compound
3a (158 mg, 93%, white solid) was prepared from commercially
available 1-(4-hydroxyphenyl)thiourea 13 and according to the
manner described for 1a. 1H (DMSO-d6): 6.76 (d, J = 8.8 Hz, 2H),
7.23 (s, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.48
5.4.2. Preparation of analogs
5.4.2.1. 4-Thioureidobenzenesulfonamide (11). A solution of
sulfanilamide 20 (3 g, 0.017 mol) in 15 mL of ethanol was stirred
at room temperature while concentrated hydrochloric acid

S. Heng et al. / Bioorg. Med. Chem. 17 (2009) 3916–3922
3921
(d, J = 6.8 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H), 9.95 (br, 1H). HRMS (ESI)
to the manner described for 1a. 1H (DMSO-d6): 6.37–6.40 (m, 1H),
6.82 (d, J = 8.8 Hz, 2H), 7.00–7.02 (m, 1H), 7.04 (s, 1H), 7.10 (t,
J = 8 Hz, 1H), 7.31 (t, J = 2.2 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 10.13
(br, 1H). HRMS (ESI) [M+H]+ calcd for C15H13N2OS, 269.0749; found
269.0751.
[M+H]+ calcd for C15H13N2OS, 269.0749; found 269.0740.
5.4.2.9. 4-(4-(4-Hydroxyphenyl)thiazol-2-ylamino)phenol
(3b). Compound 3b (154 mg, 91%, white solid) was prepared
from 13 and according to the manner described for 1b. 1H
(DMSO-d6): 6.76 (d, J = 8.8 Hz, 2H), 6.80 (d, J = 8.8 Hz, 2H),
6.95 (s, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H),
9.97 (br, 1H). HRMS (ESI) [M+H]+ calcd for C15H13N2O2S,
285.0698; found 285.0698.
5.4.2.18. 2-(4-(4-Hydroxyphenyl)thiazol-2-ylamino)phenol
(5b). Compound 5b (300 mg, 94%, white solid) was prepared from
15 and according to the manner described for 1b. 1H (DMSO-d6):
6.37–6.39 (m, 1H), 6.82 (d, J = 8.8 Hz, 2H), 7.00–7.02 (m, 1H),
7.11 (t, J = 8 Hz, 1H), 7.30 (t, J = 2.2 Hz, 2H), 7.74 (d, J = 8.8 Hz,
2H), 10.13 (br, 1H). HRMS (ESI) [M+H]+ calcd for C15H13N2O2S,
285.0698; found 285.0704.
5.4.2.10. 4-(2-(4-Hydroxyphenylamino)thiazol-4-yl)benzene-1,3-
diol (3c). Compound 3c (40 mg, 94%, brown solid) was prepared
from 13 and according to the manner described for 1c. 1H
(DMSO-d6): 6.31 (d, J = 8.4 Hz, 1H), 6.32 (s, 1H), 6.81 (d,
J = 8.8 Hz, 2H), 7.01 (s, 1H), 7.27 (d, J = 8.8 Hz, 2H), 7.53 (d,
J = 8.0 Hz, 1H), 10.29 (br, 1H). HRMS (ESI) [M+H]+ calcd for
C15H13N2O3S, 301.0647; found 301.0652.
5.4.2.19. 4-(2-(2-Hydroxyphenylamino)thiazol-4-yl)benzene-
1,3-diol (5c). Compound 5c (330 mg, 92%, dark brown solid) was
prepared from 15 and according to the manner described for 1c.
1H (DMSO-d6): 6.30 (d, J = 2.4 Hz, 1H), 6.32 (br, 1H), 6.86 (t,
J = 7.3 Hz, 1H), 6.93–7.00 (m, 2H), 7.05 (s, 1H), 7.53 (d, J = 9.0 Hz,
1H), 7.65 (d, J = 7.8 Hz, 1H), 9.93 (br, 1H). HRMS (ESI) [M+H]+ calcd
for C15H13N2O3S, 301.0647; found 301.0634.
5.4.2.11. 4-(4-(4-Nitrophenyl)thiazol-2-ylamino)phenol (3d). Com-
pound 3d (176 mg, 95%, yellow solid) was prepared from 13 and
according to the manner described for 1b except using 2-bromo-
40-nitroacetophenone 28. 1H (DMSO-d6): 6.77 (d, J = 8.8 Hz, 2H),
7.48 (d, J = 8.8 Hz, 2H), 7.63 (s, 1H), 8.15 (d, J = 9.2 Hz, 2H), 8.29
(d, J = 9.2 Hz, 2H) 10.06 (br, 1H). HRMS (ESI) [M+H]+ calcd for
C15H12N3O3S, 314.0599; found 314.0612.
5.4.2.20. N-(4-Nitrophenyl)-4-phenylthiazol-2-amine (6a). Com-
pound 6a (700 mg, 93%, orange solid) was prepared from commer-
cially available 1-(4-nitrophenyl)-2-thiourea 16, according to the
manner described for 1a. 1H (DMSO-d6): 7.35 (t, J = 6.6 Hz, 1H),
7.46 (t, J = 7.8 Hz, 2H), 7.57 (s, 1H), 7.85 (d, J = 9.2 Hz, 2H), 8.19 (d,
J = 11.6 Hz, 2H), 8.28 (d, J = 9.2 Hz, 2H), 10.29 (br, 1H). HRMS (ESI)
[M+H]+ calcd for C15H12N3O2S, 298.0650; found 298.0650.
5.4.2.12. 1-(3-Hydroxyphenyl)thiourea (14). Compound 14 (480
mg, 44%, white solid) was prepared in a similar manner as de-
scribed for the synthesis of 11 and by substituting sulfanilamide
with 3-aminophenol 22. 1H (DMSO-d6): 6.52 (d. J = 8.2 Hz, 1H),
6.85 (d, J = 8.6 Hz, 1H), 7.00 (s, 1H), 7.10 (t, J = 8.0 Hz, 1H), 9.44
(s, 1H), 9.65 (s, 1H), 9.64 (s, 1H).
5.4.2.21. 4-(2-(4-Nitrophenylamino)thiazol-4-yl)phenol (6b). Com-
pound 6b (755 mg, 95%, orange solid) was prepared from 16 and
according to the manner described for 1b. 1H (DMSO-d6): 6.83 (d,
J = 8.8 Hz, 2H), 7.28 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.94 (d,
J = 9.2 Hz, 2H), 8.27 (d, J = 9.6 Hz, 2H), 11.05 (br, 1H). HRMS (ESI)
[M+H]+ calcd for C15H12N3O3S, 314.0599; found 314.0600.
5.4.2.13. 3-(4-Phenylthiazol-2-ylamino)phenol (4a). Compound
4a (290 mg, 91%, white solid) was prepared from 14 and according
to the manner described for 1a. 1H (DMSO-d6): 6.50 (d, J = 8.4 Hz,
1H), 6.71 (s, 1H), 6.74–6.77 (m, 2H), 7.08 (t, J = 8.0 Hz, 1H), 7.24–
7.28 (m, 1H), 7.34 (t, J = 7.6 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H). HRMS
(ESI) [M+H]+ calcd for C15H13N2OS, 269.0749; found 269.0748.
5.4.2.22. 1-(3-Nitrophenyl)thiourea (17). Compound 17 (742
mg, 52%, yellow solid) was prepared in a similar manner as de-
scribed for the synthesis of 11 and by substituting sulfanilamide
with 3-nitroaniline 24. 1H (DMSO-d6): 7.60 (t, J = 8.0 Hz, 1H),
7.83 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 8.62 (s, 1H), 10.11
(br, NH).
5.4.2.14. 3-(4-(4-Hydroxyphenyl)thiazol-2-ylamino)phenol
(4b). Compound 4b (305 mg, 96%, pale green solid) was prepared
from 14 and according to the manner described for 1b. 1H (DMSO-
d6): 6.37 (dd, J = 7.4, 1.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 2H), 7.01 (d,
J = 8.4 Hz, 1H), 7.04 (s, 1H), 7.09 (t, J = 8.0 Hz, 1H), 7.31 (t,
J = 2.2 Hz, 1H), 7.75 (d, J = 8.3 Hz, 2H), 10.09 (br, 1H). HRMS (ESI)
[M+H]+ calcd for C15H13N2O2S, 285.0698; found 285.0700.
5.4.2.23. N-(3-Nitrophenyl)-4-phenylthiazol-2-amine (7a). Com-
pound 7a (286 mg, 95%, orange solid) was prepared from 17 and
according to the method described for 1a. 1H (DMSO-d6): 7.20–
7.30 (m, 4H), 7.42 (dd, J = 0.8, 8.0 Hz, 1H), 7.60–7.67 (m, 2H), 7.76
(dd, J = 7.8, 8.0 Hz, 1H), 7.82 (t, J = 2.0 Hz, 1H), 7.89 (dd, J = 8.2,
1.0 Hz, 1H), 10.3 (br, 1H). HRMS (ESI) [M+H]+ calcd for C15H12N3O2S,
298.0650; found 298.0650.
5.4.2.15. 4-(2-(3-Hydroxyphenylamino)thiazol-4-yl)benzene-1,3-
diol (4c). Compound 4c (322 mg, 90%, brown solid) was prepared
from 14 and according to the manner described for 1c. 1H (DMSO-
d6): 6.30–6.32 (overlapped, m, 2H), 6.42 (dd, J = 8.0, 2.4 Hz, 1H),
6.92–6.90 (m, 1H), 7.01 (t, J = 2.0 Hz, 1H), 7.126 (s, 1H), 7.126 (t,
J = 8.0 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H), 10.25 (bs, 1H). HRMS (ESI)
[M+H]+ calcd for C15H13N2O3S, 301.0647; found 301.0656.
5.4.2.24. 4-(2-(3-Nitrophenylamino)thiazol-4-yl)phenol (7b). Com-
pound 7b (302 mg, 95%, yellow solid) was prepared from 17 and
according to the method described for 1b. 1H (DMSO-d6): 6.84 (d,
J = 8.4 Hz, 2H), 7.20 (s, 1H), 7.62 (t, J = 8.2 Hz, 1H), 7.79–7.82 (m,
3H), 7.95 (d, J = 8.0 Hz, 1H), 9.01 (t, J = 2.4 Hz, 1H), 9.62 (s, 1H),
10.78 (br, 1H). HRMS (ESI) [M+H]+ calcd for C15H12N3O3S, 314.0599;
found 314.0606.
5.4.2.16. 1-(2-Hydroxyphenyl)thiourea
(15). Compound
15
(720 mg, 47%, white solid) was prepared in a similar manner as de-
scribed for the synthesis of 11 and by substituting sulfanilamide
with 2-aminophenol 23. 1H (DMSO-d6): 6.76 (t, J = 7.2 Hz, 1H),
6.86 (d, J = 8.0 Hz, 1H), 6.93–6.99 (m, 2H), 7.76 (br, 1H), 8.92 (br,
1H), 9.75 (br, 1H).
5.4.2.25. N,4-Diphenylthiazol-2-amine
(8a). Compound
8a
(763 mg, 92%, yellow solid) was prepared from commercially avail-
able phenylthiourea 18 and according to the method described for
1a. 1H (DMSO-d6): 6.99 (t, J = 7.4 Hz, 1H), 7.31–7.39 (m, 4H), 7.45
(t, J = 7.6 Hz, 2H), 7.76 (d, J = 7.6 Hz, 2H), 7.93 (d, J = 7.2 Hz, 2H),
10.40 (br, 1H). HRMS (ESI) [M+H]+ calcd for C15H13N2S, 253.0799;
found 253.0802.
5.4.2.17. 2-(4-Phenylthiazol-2-ylamino)phenol (5a). Compound
5a (293 mg, 92%, white solid) was prepared from 15 and according

3922
S. Heng et al. / Bioorg. Med. Chem. 17 (2009) 3916–3922
5.4.2.26. N,4-Diphenylthiazol-2-amine
(8b). Compound
8b
College for providing computational support on their Linux re-
search cluster.
(820 mg, 93%, white solid) was prepared from 18 and according
to the method described for 1b. 1H (DMSO-d6): 6.81 (d, J = 8.4 Hz,
2H), 6.96 (t, J = 6.6 Hz, 1H), 7.06 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H),
7.35 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.6 Hz, 2H), 7.73 (d, J = 8.4 Hz,
2H), 10.22 (br, 1H). HRMS (ESI) [M+H]+ calcd for C15H13N2OS,
269.0749; found 269.0748.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.04.030.
References and notes
5.4.2.27. 4-(2-(Phenylamino)thiazol-4-yl)benzene-1,3-diol
(8c). Compound 8c (887 mg, 95%, light brown solid) was pre-
pared from 18, and according to the method described for 1c.
1H (DMSO-d6): 6.31 (d, J = 2.4 Hz, 1H), 6.33 (s, 1H), 7.02 (t,
J = 7.2 Hz, 1H), 7.14 (s, 1H), 7.37 (t, J = 7.8 Hz, 2H), 7.53 (d,
J = 7.2 Hz, 2H), 7.66 (d, J = 8.8 Hz, 1H), 10.43 (br, 1H). HRMS
(ESI) [M+H]+ calcd for C15H13N2O2S, 285.0698; found
285.0701.
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5.4.2.28. 2-(2-(Phenylamino)thiazol-4-yl)phenol
(8d). Com-
pound 8d (837 mg, 95%, white solid) was prepared from 18, and
according to the method described for 1b except using 2-bromo-
20-hydroxyacetophenone 29. 1H (DMSO-d6): 6.87–6.92 (over-
lapped, m, 2H), 7.01 (t, J = 7.4 Hz, 1H), 7.16 (t, J = 8.4 Hz, 1H), 7.37
(t, J = 7.4 Hz, 2H), 7.43 (s, 1H), 7.57 (d, J = 7.6 Hz, 2H), 7.91 (d,
J = 7.6 Hz, 1H), 10.40 (br, 1H). HRMS (ESI) [M+H]+ calcd for
C15H13N2OS, 269.0749; found 269.0751.
5.4.2.29. 4-(4-Nitrophenyl)-N-phenylthiazol-2-amine (8e). Com-
pound 8e (908 mg, 93%, orange solid) was prepared from 18, and
according to the method described for 1b except using 2-bromo-40-
nitroacetophenone 28. 1H (DMSO-d6): 7.00 (t, J = 7.4 Hz, 1H), 7.37
(t, J = 8.0 Hz, 2H), 7.741 (d, J = 7.2 Hz, 2H), 7.744 (s, 1H), 8.19 (d,
J = 9.2 Hz, 2H), 8.31 (d, J = 9.2 Hz, 2H), 10.42 (br s, 1H). HRMS (ESI)
[M+H]+ calcd for C15H12N3O2S, 298.0650; found 298.0659.
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the method described for 1a. 1H (DMSO-d6): 6.88 (d, J = 8.8 Hz,
2H), 7.01 (s, 1H), 7.56 (d, J = 8.8 Hz, 2H), 8.90 (br, 2H), 9.95 (br, 1H).
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5.4.2.31. 4-(2-Aminothiazol-5-yl)phenol (10b). Compound 10b
(480 mg, 95%, white solid) was prepared from 19 and according
to the method described for 1b. 1H (DMSO-d6): 7.25 (s, 1H),
7.44–7.52 (overlapped, m, 4H), 7.74 (d, J = 8.0 Hz, 2H), 8.80 (br,
2H). HRMS (ESI) [M+H]+ calcd for C9H9N2OS, 193.0436; found
193.0429.
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according to the method described for 1c. 1H (DMSO-d6): 6.34
(dd, J = 8.4, 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 6.94 (s, 1H), 7.36
(d, J = 8.8 Hz, 1H), 8.79 (br, 1H), 9.80 (br, 1H). HRMS (ESI) [M+H]+
calcd for C9H9N2O2S, 209.0385; found 209.0395.
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Acknowledgments
This work was supported by the Grant GM26237 from the Na-
tional Institutes of Health. We also thank the National Science
Foundation for financial support of the Boston College Mass Spec-
trometry Center (Grant DBI-0619576). Finally, we thank Boston
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