Identification and characterization of 3-substituted pyrazolyl esters as alternate substrates for cathepsin B: The confounding effects of DTT and cysteine in biological assays

Article abstract of DOI:10.1016/j.bmcl.2007.06.091

Substituted pyrazole esters were identified as hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the enzyme cathepsin B. Members of this class, along with functional group analogs, were synthesized in an effort to define the structural requirements for activity. Analog characterization was hampered by the need to include a reducing agent such as dithiothreitol (DTT) or cysteine in the assay, highlighting the caution required in interpreting biological data gathered in the presence of such nucleophiles. Despite the confounding effects of DTT and cysteine, our studies demonstrate that the pyrazole 1 acts as alternate substrate for cathepsin B, rather than as an inhibitor.

Full text of DOI:10.1016/j.bmcl.2007.06.091

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4761–4766
Identification and characterization of 3-substituted pyrazolyl
esters as alternate substrates for cathepsin B: The
confounding effects of DTT and cysteine in biological assays
Michael C. Myers,a,b Andrew D. Napper,a,c Nuzhat Motlekar,a,c
Parag P. Shah,a,c Chun-Hao Chiu,a,c Mary Pat Beavers,a,c Scott L. Diamond,a,c
a,b,
Donna M. Huryna,b, and Amos B. Smith, III
*
*
aPenn Center for Molecular Discovery, University of Pennsylvania, 1024 Vagelos Research Laboratories,
Philadelphia, PA 19104-6383, USA
bDepartment of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, USA
cInstitute for Medicine and Engineering, University of Pennsylvania, 1024 Vagelos Research Laboratories, Philadelphia,
PA 19104-6383, USA
Received 29 March 2007; revised 20 June 2007; accepted 21 June 2007
Available online 5 July 2007
Abstract—Substituted pyrazole esters were identified as hits in a high throughput screen (HTS) of the NIH Molecular Libraries
Small Molecule Repository (MLSMR) to identify inhibitors of the enzyme cathepsin B. Members of this class, along with functional
group analogs, were synthesized in an effort to define the structural requirements for activity. Analog characterization was hampered
by the need to include a reducing agent such as dithiothreitol (DTT) or cysteine in the assay, highlighting the caution required in
interpreting biological data gathered in the presence of such nucleophiles. Despite the confounding effects of DTT and cysteine, our
studies demonstrate that the pyrazole 1 acts as alternate substrate for cathepsin B, rather than as an inhibitor.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Cathepsins play an important role in cellular protein
degradation through the lysosomal pathway,1 and as
such, represent a significant class of drug targets, having
been implicated in a variety of degenerative and invasive
processes including: asthma, Alzheimer’s disease,2 can-
cer,3,4 diabetes, inflammatory diseases,5 liver disorders,
multiple sclerosis, muscular dystrophy, and pancreati-
tis.6 The large number of disease states associated with
the biological effects of the cathepsin protease family,
and specifically cathepsin B, demands an understanding
of their biological function. The availability of small
molecule probes of the cathepsins thus holds the prom-
ise both for characterization of this ubiquitous enzyme
class and for the discovery of new cysteine protease
inhibitors of considerable biomedical value.3,7,8
Recently, the Penn Center for Molecular Discovery
(PCMD)9 completed
high throughput screen
a
(HTS) to identify small molecule probes (i.e., inhibi-
tors) for the papain-like cysteine protease family,
including cathepsins B, L, and S. While a number
of both potent and selective inhibitors have been de-
scribed previously,2–8 this project presented an oppor-
tunity to annotate the NIH Molecular Libraries
Small Molecule Repository (MLSMR) through depo-
sition of data in PubChem.10 As such, this work rep-
resents one of the first efforts to create
a
comprehensive, publicly available profile of small-
molecule inhibitors of the cysteine protease class.
Screening 63,332 members of the MLSMR against
human liver cathepsin B resulted in a number of
hits.11 Further confirmatory assays included IC50
determination and elimination of false positives
resulting from non-specific redox chemistry.12 Based
on these results, a family of substituted pyrazole
esters was identified that displayed promising activity
as inhibitors of cathepsin B (Table 1).13
Keywords: Cathepsin B; DTT; Cysteine; Alternate substrate; HTS;
MLSMR; Pyrazole esters.
*
Corresponding authors. E-mail: huryn@sas.upenn.edu
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.091

4762
M. C. Myers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4761–4766
Table 1. Pyrazole HTS hits in the cathepsin B assay
occurred upon treatment with an aqueous solution of
NaOH, followed by acidification (10% HCl) to precipi-
tate the pyrazolones.18 The pyrazolones were used in
the subsequent acylation step without purification. For
example, treatment of 12–15 with 2-thiophenecarbonyl
chloride or 2-furancarbonyl chloride in chloroform re-
sulted in chemoselective19 acylation at the C3-oxygen
versus the C5-amino group to furnish pyrazoles 1–6 in
moderate to good yields after silica gel chromatography.
X
O
O
N
H2N
1- 6
N
S
O
O
R
a
PubChem SID
X
R
IC50 (lM)
The availability of newly synthesized pyrazoles 1–6 al-
lowed confirmation of the structures and observed bio-
logical activity of the initial hits. Pleasingly, both the
structures and re-assay IC50 values of synthetic pyraz-
oles 1–6 were in agreement with the results obtained
from the original screening experiments (Table 1).
4249135 (1)
4247730 (2)
4245669 (3)
844213 (4)
849441 (5)
845259 (6)
S
H
F
0.25 0.03
0.44 0.11
0.69 0.10
1.99 0.17
1.75 0.06
1.26 0.04
S
S
OMe
Me
Me
F
S
O
O
a IC50 values are reported as means standard deviation (number of
determinations = 3).
R
O
O
1. NaOH (aq)
0.5 h
H
ArSO2Cl
NH2
N
N
N
S
EtOH, 24 h
30-40%
2. HCl, filter
63-71%
H
H
The pyrazole series (1–6) displayed suitable physico-
chemical properties (MW 347–379 and XlogP = 2.7–
3.3) with structurally related analogs revealing a range
of activities in our HTS analysis. Based on these attri-
butes, members of this series were viewed as potential
small molecule lead compounds for the inhibition of
cathepsin B, and as such were selected for further study
to understand and improve their biological profile.
O
O
CN
CN
7
8
9
R = H 10 R = OMe
R = F 11 R = Me
O
X
NH
O
O
H2N
N
S
X
O
O
O
Cl
N
H2N
N
1- 6
Et3N, CHCl3
2 h
O
R
S
O
12 R = H 14 R = OMe
13 R = F 15 R = Me
R
Structurally related pyrazoles that were inactive in the
HTS assay were compared to compounds 1–6 in a pre-
liminary effort to understand the requirements for activ-
ity. Based on this analysis, we pinpointed the ester group
as a functional moiety important for activity; we rea-
soned that the ester could act as an electrophilic site that
would react with the active site cysteine. Alternatively,
the compound could behave as an alternate substrate
of the enzyme resulting in transesterification or hydroly-
sis of the ester functionality. To differentiate these two
scenarios, a series of analogs were constructed and sub-
jected to biochemical and analytical studies to define the
mechanism of action. Herein we report the results of this
effort.
compound X
yield (%)
R
yield (%)
compound X
R
1
2
3
S
S
S
4
5
6
S
O
O
H
F
53
68
Me
Me
F
64
77
48
OMe 85
Scheme 1. Synthetic procedure to prepare pyrazole esters 1–6.
The ester carbonyl carbon in 1, activated by the adjacent
pyrazole, is a likely site for nucleophilic attack by the ac-
tive site cysteine-25 sulfur in cathepsin B. In order to as-
sess the electrophilicity of this carbon atom, equilibrium
geometries and electrostatic potential surfaces were cal-
culated for 1 using PC Spartan software.20 The known
cysteine protease inhibitor E6421 was also analyzed for
comparison to pyrazole ester 1 (Fig. 1).
To confirm both the activity and identity of the original
hits, we resynthesized compounds 1–6.14,15 This exercise
permitted both the development of an effective method
to construct the pyrazole scaffold and the preparation
of analogs. The synthesis of 1,3-disubstituted-5-amino-
pyrazoles (1–6) began with commercially available cya-
noacetohydrazide (7) and the appropriate aryl-
substituted sulfonyl chlorides, according to the proce-
dure of Elgemeie et al.16,17 Reaction of 7 with the aryl-
substituted sulfonyl chlorides in ethanol furnished the
sulfonamides 8–11 which conveniently precipitated from
solution (Scheme 1).
O
S
O
O
CO2H
O
Me
NH
N
H2N
N
S
O
O
Me
(CH2)4 NH
HN
O
N
H
NH2
1
E64
Figure 1. Compounds for which electrostatic potential surfaces were
calculated: pyrazole ester 1 and E64.
As expected, the major by-products, aryl ethyl sulfo-
nates, remained soluble and were easily removed from
8–11 via filtration. Thus, on a 50 mmol scale, gram
quantities of the required sulfonamide hydrazides could
be readily prepared in high purity. Efficient cyclizations
of sulfonamide hydrazides (8–11) to pyrazolones (12–15)
As expected, the most electrophilic center for pyrazole
ester 1 was the carbonyl carbon of the ester, with an
electrostatic potential value of 128.0 kJ/mol. The car-

M. C. Myers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4761–4766
4763
bons of the epoxide ring in E64 had values of 104.3 kJ/
mol and 104.7 kJ/mol for their electrostatic potential
surface energies. The greater positive value of 128.0 kJ/
mol suggests that the carbonyl carbon of 1 is more prone
to nucleophilic attack than the epoxide carbons in the
irreversible cathepsin B inhibitor E64. This difference
in electrophilicity was confirmed experimentally (vide in-
fra). Presumably, the stability of the pyrazolone by-
product of 1 provides the driving force for attack of this
atom center.
Figure 3. Kinetic characterization of inhibition of cathepsin B by 1.
Cathepsin B was assayed with combinations of 20–350 lM Z-Arg-Arg-
AMC substrate and 0.17–1.3 lM inhibitor (1). Initial rates of product
release were determined by measuring increase in fluorescence over
15 min. Data were fit by non-linear regression using IDBS XLfit. (a)
Rate of enzyme-catalyzed substrate hydrolysis as a function of its
concentration. Substrate concentration was varied from 20 to 350 lM
in the presence of 0 lM (Æ), 0.17 lM (h), 0.25 lM (n), 0.39 lM (j),
0.58 lM (m), 0.87 lM (s), and 1.3 lM (Ç) compound 1. (b) Replot of
Km values calculated by non-linear fit of curves in (a). Linear
regression gave a Ki value of 0.9 lM with a standard error of 0.25 lM.
We reasoned that, modulating the electronic parameters
of the electrophilic ester functionality in this series of
compounds would quickly validate or eliminate the
importance of this structural feature. To test this
hypothesis, pyrazole 14 was employed as a common
intermediate to construct ether 16, sulfonate 17, and
isonicotinoyl ester 18 (Fig. 2).
R
S
O
O
R
16
17
O
O
that 1 acted as a substrate of cathepsin B, however, re-
mained. That is, under the conditions employed for
the kinetic analyses, a competitive inhibitor is indistin-
guishable from an alternate substrate.23 Thus, experi-
ments were carried out to evaluate the reactivity of 1
under the assay conditions.
N
S
NH
a, b, or c
S
H2N
H2N
N
S
N
S
O
O
O
O
O
14
OMe
OMe
N
18
As a control, a biochemical time course experiment was
designed. Pyrazole 1 was combined with the assay buffer
(phosphate, pH 6.8) over a series of time points (0, 2, 17,
and 24 h). Samples at each time point were then incu-
Figure 2. Reagents and conditions: (a) 2-bromomethyl-thiophene,
K2CO3, DMF, 0–23 ꢁC, 3 h, 50%; (b) thiophene-2-sulfonyl chloride,
Et3N, CHCl3, 23 ꢁC, 2 h, 76%; (c) isonicotinoyl chloride, Et3N, CHCl3,
23 ꢁC, 2 h, 43%.
bated with cathepsin
B (0.1 nM) in the buffer
Ether 16 was formed via an alkylation of 14 with 2-bro-
momethyl-thiophene22 in moderate yield. Sulfonate 17
and isonicotinoyl ester 18 were constructed under
chemoselective acylation conditions similar to those em-
ployed to prepare pyrazoles 1–6.19 In support of the
requirement of the ester functionality for biological
activity, ether 16 and sulfonate 17 were found to be inac-
tive. Surprisingly, however, related ester analog pyridine
18 was devoid of activity, despite its steric and electronic
similarity to the thiophene moiety found in 1. This unex-
pected result led us to question the stability of the pyra-
zole hits (e.g., hydrolysis or transesterification) under
the assay conditions. To probe these issues, we under-
took further biological characterization of 1, as well as
a detailed analysis of assay by-products. Kinetic analysis
of the inhibition of cathepsin B by 1 revealed a compet-
itive inhibition pattern, in which 1 increased the Km of
the substrate but had no effect on the Vmax. The Ki of
1 was determined to be 0.9 lM ( ) 0.25 lM (Fig. 3).23
(100 mM) containing dithiothreitol (DTT) (2 mM) un-
der the standard assay conditions, and IC50 data were
measured.13 Results from these experiments indicated
a significant loss of inhibitory activity which correlated
with the time of pre-incubation (Table 2).
Table 2. Time dependent pre-incubation of 1 with and without DTT:
an analysis of 1 in the cathepsin B assay
a
Pre-incubation
time (h)
IC50 (lM)
Assay buffer
with DTT
Assay buffer
without DTT
Non-buffered
without DTT
0
2
1.4
9.0
0.8
1.4
0.7
3.2
0.8
1.7
1.8
5.0
17
24
45.4
>50
a IC50 values are calculated from a dose response curve of 16 2-fold
dilutions.
Reversibility of inhibition was next demonstrated by
incubating cathepsin B with 1 at a concentration that
gave complete inhibition followed by determination of
enzyme activity after 100-fold dilution. As expected for
reversible inhibition, the enzyme regained full activity
upon dilution.24
The IC50 values of pyrazole 1 increased from 1.42 lM at
a pre-incubation time of 0 h to >50 lM at an incubation
time of 24 h. Two additional experiments were con-
ducted to pinpoint the cause of the time dependent loss
of activity: (1) pyrazole 1 was added to an assay buffer
that did not contain DTT (Column 2, Table 2); and
(2) pyrazole 1 was added to a non-buffered solution also
without DTT (Column 3, Table 2). Both experiments re-
vealed that the IC50 values changed slightly from the 0
These experiments ruled out the possibility that 1 acts
similar to E64, an irreversible inhibitor. The possibility

4764
M. C. Myers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4761–4766
to 24 h incubation time. Thus, the presence of DTT was
deemed largely responsible for the significant loss in
activity.
The molecular ion corresponding to 19 (M+1 = 265)
was observed in the LC–MS analysis of the assay buffer
with DTT. As further verification, adduct 19 was syn-
thesized independently using DTT and 2-thiophenecar-
bonyl chloride, and used as a standard in the LC–MS
analysis. Bioassay of 19 with cathepsin B revealed no
activity. Taken together, these data reveal the basis for
the diminished IC50 values following extended pre-incu-
bation times.
With these results in hand, we set out to define more pre-
cisely the role of DTT in the inactivation of 1. Analytical
experiments were designed to mimic the biochemical
pre-incubation experiment, wherein the aqueous solu-
tions of 1 with and without DTT would be analyzed
via LC–MS for hydrolysis products (e.g., pyrazolone
12). The LC–MS analysis corroborated the previous
study and also provided an additional detail, the loss
of 1 was the result of its conversion to pyrazolone 12
(Table 3).
The inherent redox chemistry associated with DTT is
well known, both in connection with the identification
of false positives in cysteine protease assays and with en-
zymes such as phosphatases.12,26–28 To avoid the redox
issues of DTT, cysteine is often used in place of DTT
due to its lower oxidation potential. Less well appreci-
ated, however, is the propensity of DTT to act as a
nucleophile, thereby confounding bioassay results.29,30
Unfortunately, in our case, replacement of DTT with
cysteine does not alleviate the problem. Indeed, the same
fast conversion of 1–12 was observed, via LC–MS, in the
presence of cysteine. Thus, the series of pyrazole esters is
susceptible to both the hydrolysis and transesterification
pathways. We were, however, intrigued by the apparent
lack of any activity of 18, despite the similar size and
electronic attributes of the ester moiety. To explore this
observation, we analyzed the rate of hydrolysis and
transesterification of 18. Surprisingly, incubation of 18
in assay buffer, with and without DTT, and a non-buf-
fered aqueous solution, revealed rapid conversion (ca.
1 h) to pyrazolone 14, regardless of the presence of
DTT (Table 4).25
Table 3. Time dependent pre-incubation of 1 with and without DTT:
an analysis of 1 by LC–MSa
S
O
O
O
NH
conditions
H2N
N
S
N
+
1
H2N
O
O
N
S
O
O
1
12
Incubation
time (h)
% conversion of 1–12
Assay buffer
with DTT
Assay buffer
without DTT
Non-buffered
without DTT
1
2
72
80
87
88
5
5
5
6
16
24
13
17
11
9
a LC–MS analysis conducted on a 6.5 min run time (100 lM sample
concentration).
Table 4. Time dependent pre-incubation of 18 with and without DTT:
an analysis of 18 by LC–MSa
N
For example, when 1 was incubated in the DTT contain-
ing assay buffer, conversion to 12 occurred quickly with
28% of 1 remaining after 1 h, and only 12% after 24 h.25
Hydrolysis of 1 to 12 also occurred, albeit slowly, in
both buffer without DTT and non-buffered water. How-
ever, on the time-scale of the HTS assay, the contribu-
tion of this degradation pathway to the activity was
negligible. Additional experiments indicated that when
E64 was exposed to DTT (under the same conditions
as listed in Table 3.) it was found to be stable with no
E64 decomposition and no DTT-E64 adduct formed,
as monitored by LC–MS.
O
NH
O
O
H2N
conditions
N
S
N
+
18
H2N
O
O
N
S
O
O
18
14
OMe
OMe
Incubation
time (h)
% conversion of 18–14
Assay buffer
with DTT
Assay buffer
without DTT
Non-buffered
without DTT
1
2
>99
>99
>99
>99
41
80
15
24
95
99
16
24
>99
>99
Anticipating that 12 could arise via nucleophilic dis-
placement of the thiophene ester with DTT, we
examined the LC–MS data for the anticipated transeste-
rified DTT-thiophene ester product 19 (Scheme 2).
a LC–MS analysis conducted on a 6.5 min run time (100 lM sample
concentration).
The ease of hydrolysis and esterification of 18 is likely a
reflection of the fact that the pyridine ring is partially
protonated under the conditions.31 Thus, within the
timescale of the cathepsin B assay (1 h incubation with
the enzyme), a negligible amount 18 would be present.
OH
O
OH
thiophene-2-carbonyl
chloride, Et3N, CHCl3
HS
HS
S
S
SH
OH
OH
DTT =
DL-1,4-dithiothreitol
HPLC purification
19
Taken together, the results support the hypothesis that 1
acts as an alternate cathepsin B substrate rather than an
Scheme 2. Synthesis of DTT-thiophene adduct 19.

M. C. Myers et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4761–4766
4765
4. Frlan, R.; Gobec, S. Curr. Med. Chem. 2006, 13, 2309.
inhibitor. Presumably, the active site cysteine acts much
like DTT to transesterify 1, and thereby forms a tran-
sient thiophenoyl-enzyme intermediate.7,32,33 To test this
scenario, a stoichiometric reaction was devised to incu-
bate pyrazole ester 1 (1 equiv) with and without cathep-
sin B (2 equiv) (Table 5).
5. Greenspan, P. D.; Clark, K. L.; Tommasi, R. A.; Cowen,
S. D.; McQuire, L. W.; Farley, D. L.; van Duzer, J. H.;
Goldberg, R. L.; Zhou, H.; Du, Z.; Fitt, J. J.; Coppa, D.
E.; Fang, Z.; Macchia, W.; Zhu, L.; Capparelli, M. P.;
Goldstein, R.; Wigg, A. M.; Doughty, J. R.; Bohacek, R.
S.; Knap, A. K. J. Med. Chem. 2001, 44, 4524.
6. Schirmeister, T.; Kaeppler, U. Mini-Rev. Med. Chem.
2003, 3, 361.
Table 5. Standard assay conditions with and without cathepsin B: a
stoichiometric reaction analyzed by LC–MSa
7. Otto, H.-H.; Schirmeister, T. Chem. Rev. 1997, 97, 133.
8. Hernandez, A. A.; Roush, W. R. Curr. Opin. Chem. Biol.
2002, 6, 459.
S
O
9. Penn Center for Molecular Discovery: http://www.seas.
upenn.edu/~pcmd/ Molecular Library Screening Center
Network: http://nihroadmap.nih.gov/molecularlibraries/.
10. PubChem: http://pubchem.ncbi.nlm.nih.gov/.
11. PubChem web address for PCMD cathepsin B hits: http://
www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pcassay&
term=488,453,523.
O
standard assay conditions
O
NH
H2N
with and without cathepsin B
N
S
N
+
1
H2N
O
O
N
S
O
O
15 min then LC-MS analysis
1
12
´
12. Tjernberg, A.; Hallen, D.; Schultz, J.; James, S.; Benke-
Assay conditions
with cathepsin B
Assay conditions
without cathepsin B
stock, K.; Bystro¨m, S.; Weigelt, J. Bioorg. Med. Chem.
Lett. 2004, 14, 891.
0% remaining 1
50% remaining 1
13. Compounds were serially diluted in DMSO and trans-
ferred into the assay microplate using a 100-nL pintool to
give 16 dilutions ranging from 50 lM to 1.5 nM. Triplicate
plates were set up in this manner to give three indepen-
dently calculated IC50 values for each compound. Cathep-
sin B (Calbiochem 219362) was activated by incubating
with assay buffer for 15 min. Assay buffer consisted of
100 mM sodium-potassium phosphate, pH 6.8 (86 mM
potassium phosphate, monobasic; 7 mm sodium phos-
phate, monobasic; 7 mM sodium phosphate, tribasic),
1 mM EDTA, and 2 mM DTT. Upon activation, cathep-
sin B was incubated with Z-Arg-Arg-AMC substrate
(15 lM) and test compound in 10 lL of assay buffer for
1 h at room temperature. Fluorescence of AMC released
by enzyme-catalyzed hydrolysis of Z-Arg-Arg-AMC was
read on a Perkin-Elmer Envision microplate reader
(excitation 355 nm, emission 460 nm).
a LC–MS analysis conducted on a 6.5 minute run time (20 lM
cathepsin B; 10 lM 1).
Under these stoichiometric reaction conditions, in the
presence of cathepsin B, pyrazole 1 was fully converted
to 12 after only 15 min. However, under identical condi-
tions, in the absence of cathepsin B, 50% of pyrazole 1
remained.34 We conclude that pyrazole esters such as 1
are competitive substrates for the enzyme cathepsin B.
In summary, we have demonstrated that pyrazole 1 acts
as an alternate substrate for the cysteine protease,
cathepsin B. Synthesis and evaluation of related analogs
revealed the potential reactivity of the ester functionality
with the nucleophilic enzyme active site cysteine to form
a transient thiphenoyl-enzyme intermediate. Initially,
the similar reactivity of DTT and cysteine in the bioas-
say confounded the HTS and subsequent assay results
due to the nucleophilic properties of the thiol sulfur.
Thus, it is important for the biological and chemical
communities to consider the potential of DTT and cys-
teine to act as nucleophiles in assay systems where sub-
strates contain electrophilic functionality.
14. Characterization data for 1. Mp = 167 ꢁC; IR (thin film,
CH2Cl2) 3479, 3328, 1733, 1621, 1189, 610 cmÀ1 1H
;
NMR (500 MHz, DMSO-d6) d 8.11 (m, 1H), 7.98 (m, 1H),
7.94 (m, 2H), 7.79 (m, 1H), 7.69 (m, 2H), 7.29 (m, 1H),
6.57, (s, 3H), 5.36 (s, 1H); 13C NMR (125 MHz, DMSO-
d6) d 158.4, 158.0, 152.4, 136.5, 136.2, 135.9, 134.9, 130.4,
129.8, 128.9, 127.2, 80.5; high resolution mass spectrum
(ES+) m/z 350.0265 [(M+H)+; calcd for C14H11N3O4S2H+:
350.0269].
15. A single crystal X-ray structure of pyrazole ester 2 was
also obtained to verify its structure.
16. Elgemeie, G. H.; Metwally, N. H. J. Chem. Res. 1999, 6,
384.
Acknowledgments
17. Elgemeie, G. H.; Elghandour, A. H.; Elzanate, A. M.;
Masoud, W. A. Phosphorus, Sulfur Silicon 2000, 163, 91.
18. For the cyclization of hydrazide-substituted ureas using
this protocol, see: Drummond, J. T.; Johnson, G.
J. Heterocycl. Chem. 1988, 25, 1123.
Financial support for this work was provided by the
NIH (5U54HG003915-02). We thank Professor Barry
S. Cooperman for helpful discussions. We also thank
Dr. Patrick J. Carroll for X-ray structural determination
of pyrazole ester 2.
19. Vostrova, L. M.; Grenad’orova, M. V.; Klad’ko, L. G.
Ukr. Khim. Zh. 2004, 9–10, 115.
20. PC Spartan is available from Wavefunction, Inc., 18401
Von Karman Avenue, Suite 370 Irvine, CA 92612.
21. Kim, M. J.; Yamamoto, D.; Matsumoto, K.; Inoue, M.;
Ishida, T.; Mizuno, H.; Sumiya, S.; Kitamura, K. Bioche.
J. 1992, 287, 797.
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