Structure-activity relationship and improved hydrolytic stability of pyrazole derivatives that are allosteric inhibitors of West Nile Virus NS2B-NS3 proteinase

Article abstract of DOI:10.1016/j.bmcl.2009.07.150

West Nile Virus (WNV) is a potentially deadly mosquito-borne flavivirus which has spread rapidly throughout the world. Currently there is no effective vaccine against flaviviral infections. We previously reported the identification of pyrazole ester derivatives as allosteric inhibitors of WNV NS2B-NS3 proteinase. These compounds degrade rapidly in pH 8 buffer with a half life of 1-2 h. We now report the design, synthesis and in vitro evaluation of pyrazole derivatives that are inhibitors of WNV NS2B-NS3 proteinase with greatly improved stability in the assay medium.

Full text of DOI:10.1016/j.bmcl.2009.07.150

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5773–5777
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship and improved hydrolytic stability of
pyrazole derivatives that are allosteric inhibitors of West Nile Virus NS2B-NS3
proteinase
Shyama Sidique a,b, Sergey A. Shiryaev b, Boris I. Ratnikov b, Ananda Herath a,b, Ying Su a,b
,
Alex Y. Strongin b, Nicholas D. P. Cosford a,b,
*
a Conrad Prebys Center for Chemical Genomics, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
b Burnham Institute for Medical Research, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
West Nile Virus (WNV) is a potentially deadly mosquito-borne flavivirus which has spread rapidly
throughout the world. Currently there is no effective vaccine against flaviviral infections. We previously
reported the identification of pyrazole ester derivatives as allosteric inhibitors of WNV NS2B-NS3 pro-
teinase. These compounds degrade rapidly in pH 8 buffer with a half life of 1–2 h. We now report the
design, synthesis and in vitro evaluation of pyrazole derivatives that are inhibitors of WNV NS2B-NS3
proteinase with greatly improved stability in the assay medium.
Received 26 March 2009
Revised 28 July 2009
Accepted 30 July 2009
Available online 3 August 2009
Keywords:
West Nile virus
Flavivirus
Ó 2009 Elsevier Ltd. All rights reserved.
Proteinase inhibitors
NS2B-NS3 proteinase
Antiviral
West Nile virus (WNV) is a mosquito-borne pathogen of the
genus, Flavivirus.1 This genus also contains many other human
pathogens, such as Dengue virus (Den), Japanese encephalitis virus
(JE), and yellow fever virus (YF). First isolated in 1937 from Ugan-
da, WNV has since been widely distributed around the world.2
Infections in humans are usually asymptomatic or cause a mild
flu-like illness for a few days called West Nile fever. However, re-
cent infections of WNV have been associated with much higher
fatality rates particularly among the senior population.3 Since the
identification of WNV in New York in 1999 the virus has distrib-
uted itself widely throughout North America, infecting over
28,000 people (see the Center for Disease Control and Prevention
site on the World Wide Web at www.cdc.gov/ncidod/dvbid/west
nile/index.htm). Currently there is no effective vaccine against fla-
viviral infections.2 WNV has a single-stranded RNA genome, which
encodes a single polyprotein. This consists of three structural (C,
prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3,
NS4A, NS4B, and NS5) proteins. Post translational processing of
the polyprotein precursor is required to produce functional viral
proteins.4 In particular, the WNV NS2B-NS3 proteinase holds
promise as a potential target for therapeutic intervention with
small molecule drugs.5–9 Thus the identification of potent small
molecule inhibitors of WNV would be a highly useful starting point
for drug discovery and development.
We previously reported the discovery of pyrazole derivatives as
allosteric inhibitors of WNV NS2B-NS3 proteinase identified by
high-throughput screening (HTS) of a small molecule library
through the NIH Molecular Libraries Initiative (MLI).10 The struc-
tures and data for compounds were deposited to the PubChem
database under AID 577 (http://pubchem.ncbi.nlm.nih.gov/assay/
assay.cgi?aid=577&loc=ea_ras) and AID 653 (http://pubchem.ncbi.
nlm.nih.gov/assay/assay.cgi?aid=653&loc=ea_ras). The hit com-
pounds exhibited potencies ranging from 0.105 to 1.353
lM
(Figure 1). However, these pyrazolyl benzoic acid ester derivatives
S
O
O
O
O
O
H2N
O
H2N
N
N
N
S
N
S
O
H3C
O
N
N
O
O
N
OMe
OMe
1
2
3
SID-852843
SID-4245669
SID-3717586
IC50 = 1.353 µM
IC50 = 0.105 µM
IC50 = 0.110 µM
* Corresponding author.
E-mail address: ncosford@burnham.org (N.D.P. Cosford).
Figure 1. WNV NS2B-NS3 proteinase inhibitor hits from screening.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.150

5774
S. Sidique et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5773–5777
Cl
Table 1 (continued)
O
S
OMe
O
H
O
N
Compound
R
IC50 (lM)
1)KOH, EtOH
2) AcOH, filter
O
NC
NH2
O
N
NC
S
N
H
H
O
EtOH, rt
7f
3.55
4
5
OMe
F
F
R
O
O
O
7g
7h
7i
4.28
4.90
5.00
O
R
Cl
NH
N
S
N
H2N
H2N
Et3N, CHCl3
N
O
O
S
O
O
F
OMe
OMe
6
7
F
Scheme 1. Synthetic route to prepare pyrazole esters.
(1–3) were rapidly hydrolyzed in an aqueous buffer (pH 8) to the
corresponding pyrazol-3-ol in approximately 1–2 h. Clearly these
compounds, while potent, were of limited use as tools to investi-
gate the biochemistry and enzymatic kinetics of WNV NS2B-NS3
proteinase in vitro because of their instability. We hypothesized
S
F
F
F
F
7j
10.69
11.92
26.18
45.49
>100
>100
>100
Table 1
Summary of in vitro data from first focused library.
R
F
F
O
O
7k
7l
O
O
F
F
R1
N
N
H2N
N
N
N
R2
O
S
O S
O
F
O
OMe
OMe
R1 = CH3 , R2 =H
7r R1= R2 = CH3
7
7q
F
Compound
R
IC50 (lM)
7m
7n
7o
7p
F3C
1
0.105
0.11
F
Cl
Cl
S
2
F
F
7a
7b
7c
7d
7e
0.31
0.97
1.22
1.83
1.96
OEt
F
F
NO2
F
S
that it might be possible to design and synthesize analogues of
the hits which retain potency as inhibitors of WNV NS2B-NS3 pro-
teinase and, in addition, exhibit improved hydrolytic stability in
the assay medium. Herein, we report the synthesis and struc-
ture–activity relationship (SAR) of pyrazole WNV NS2B-NS3 pro-
teinase inhibitors with substantially increased stability towards
hydrolysis in an aqueous medium.
The initial phase of studies to address this problem focused on
the development of modified benzoate ester analogues of the lead
structures. Our approach to the design of ester derivatives with the
potential for improved stability in buffer was to introduce substit-
uents in the ortho positions of the aryl ring in an attempt to
Cl
F
F
F
F
F

S. Sidique et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5773–5777
5775
Cl
NHNH2
+
We therefore designed and synthesized a focused library of pyra-
zole ester analogues. The synthetic chemistry used for the prepara-
tion of the pyrazole ester analogues of 1 and 2 is outlined in
Scheme 1.11
The reaction of commercially available cyanoacetohydrazide 4
with 4-methoxyphenylsulfonyl chloride in ethanol yielded the sul-
fonamide 5 which precipitated from solution. Cyclization of sulfon-
amide 5 to pyrazolone 6 was achieved in an ethanolic solution of
KOH, followed by neutralization with AcOH. Lastly, pyrazolone 6
was treated with an arylcarbonyl chloride to obtain the pyrazole
O
O
AcOH
reflux
anhyd. NH2NH2
OH
O
N
N
MeOH
0°C- rt
R
R
9
R
O
R1
8
O
N
Et3N
N
R1COCl
CHCl3
R = H or OMe
R
10
F
(a)
Scheme 2. Synthetic route to prepare pyrazole esters 10.
O
F
O
Table 2
O
pH = 8 buffer
DMSO
NH
Summary of in vitro data for pyrazole esters 10.
H2N
N
N
O
S
H2N
N
O
H3C
O S
O
R2
OMe
O
O
N
6
N
OMe
7e
R3
10
R4
7
Compound + Buffer
6
5
4
3
2
1
0
R1
Compound + Buffer + Enzyme
Compounds
R1
R2
R3
R4
IC50 (lM)
10a
10b
10c
10d
10e
OMe
H
OMe
OMe
H
H
F
F
F
F
H
F
F
F
F
H
H
H
CH3
CH3
4.03
8.04
8.71
9.26
9.43
prevent hydrolysis of the ester. Compounds 1 and 2 were used as
starting points because they were the most potent in that series.
0
100
200
300
400
Time/min
Table 3
(b)
Time-dependant degradation of pyrazole series 7 and 10.a
O
OH
N
O
N
N
pH = 8 buffer
DMSO
N
O
pH = 8 buffer
DMSO
NH
7
H2N
N
O
N
S
O
10a
8
MeO
MeO
OMe
6
5
Compound + Buffer
Compound + Buffer + Enzyme
4.5
pH = 8 buffer
DMSO
OH
4
3.5
3
10
N
2.5
2
MeO
8
t1/2 (min)b
1.5
1
Compounds
7a
90
90
7b
7d
7e
0.5
0
255
450
150
300
900
90
0
100
200
Time/min
300
400
500
7g
7n
10a
10c
Figure 2. Stability of compounds 7e and 10a in the presence and absence of WNV
NS2B-NS3 proteinase. (a) Stability studies on compound 7ea. (b) Stability studies on
compound 10aa. a10 mM Tris–HCl buffer, pH 8.0, containing 20% (v/v) glycerol and
a
The compounds were dissolved in <10% DMSO, pH
8
buffer (1 mM) and
Chlorpromazine (15 mM).
0.005% Brij 35. The substrate and enzyme concentrations were 25
respectively.
lM and 10 nM,
b
Degradation of compound w.r.t. standard.

5776
S. Sidique et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5773–5777
esters 7. The analogues in this library were designed to probe both
the steric and electronic requirements of the benzoic acid ester
moiety and the in vitro data for selected examples are shown in Ta-
ble 1. Although the introduction of ortho difluoro substituents on
the aryl ring was tolerated (e.g., 7a, 7b) with a small loss of po-
tency, more bulky substituents such as dichloro (7n) led to a com-
plete loss of activity. Similarly, the presence of electron donating
(7o) or withdrawing (7p) substituents in the ortho position gave
inactive analogues, although the ortho methyl derivative (7f) re-
tained its micromolar inhibitory activity. In general the addition
of substituents led to a reduction in potency for both the benzoate
and the thiophenecarboxylate (7c, 7i) analogues (Table 1). To test
the effect of N-alkylation of the pyrazole 5-amino group on inhib-
itory activity we synthesized the monomethylamino (7q) and
dimethylamino (7r) derivatives which were prepared by methyla-
tion of compound 1 using (CH3)2SO4. Unfortunately this led to loss
standard. The corresponding half lives are reported in Table 3. We
were gratified to observe a significant improvement in aqueous
stability for compounds 7e (t = 450 min) and 10a (t = 900 min)
½
½
compared with the initial hits10 (Table 3). To help address whether
compounds appearing to be inactive under the assay conditions
were in fact being rapidly hydrolyzed we also tested the stability
of the inactive 2,6-dichloro derivative 7n (IC50 >100 lM). Interest-
ingly, as shown in Table 3, this compound exhibited significantly
improved stability (t = 300 min) compared with the initial hit (1).
½
Based on the data for the stability experiments in a pH 8 buffer,
we selected 7e and 10a for additional stability studies in the
in vitro assay buffer with or without the enzyme WNV NS2B-NS3
proteinase (Fig. 2). The goal in this study was to mimic the assay
conditions used for the in vitro experiments and to gain insight
into any differences in stability for the two chemical series. As
shown in Figure 2, the results were quite different for each com-
pound. Thus, while 7e was relatively stable in the assay buffer, it
degraded rapidly in the presence of the enzyme (Fig. 2a). On the
other hand, the stability of 10a was approximately the same in
the presence or absence of the enzyme (Fig. 2b). Taken together,
the stability data suggest that the relative stability of the benzoate
ester derivatives may be related to electronic rather than steric ef-
fects of substituents. These results are in agreement with Charton’s
studies on the hydrolysis of ortho-substituted benzoate esters.14,15
Although at this juncture we had made significant progress in
enhancing the stability of the pyrazole ester derivatives, we next
investigated pyrazole analogues containing non-hydrolyzable ester
isosteres. In particular, we wanted to determine whether it was
possible to replace the ester moiety with alcohol, ketone, alkene
or amide functional groups in the pyrazole derivatives while
retaining potency as inhibitors of WNV NS2B-NS3 proteinase.
The pyrazole analogues containing ester isosteres were prepared
according to the procedures outlined in Schemes 3 and 4. Thus,
commercially available 3,5-dimethylisoxazole was reacted with
LDA followed by reaction with the corresponding substituted benz-
aldehyde to yield compound 11 (Scheme 3). The isoxazole deriva-
tive 11 was converted to pyrazole 1216 using Raney Ni and 12 was
then oxidized using the Dess–Martin reagent to produce the ke-
tone derivative 13. Acid catalyzed dehydration to afford the alkene
of inhibitory activity (IC50 >100 lM). It should be noted that the
products of ester hydrolysis 6 and 8 are inactive (IC50 >100 lM)
as inhibitors of WNV NS2B-NS3 proteinase.10
We also investigated the structural requirements important for
potency and stability of 3-substituted pyrazolyl esters related to
compound 3. Pyrazole derivative 8 was selected as the key synthon
for the preparation of ester analogues. The synthetic chemistry
used to prepare the second series of pyrazole ester derivatives is
outlined in Scheme 2. The reaction of commercially available chlo-
romethyl methoxybenzene with anhydrous hydrazine in MeOH
provided the benzyl hydrazine derivative 9, which reacted with
ethylacetoacetate in AcOH under reflux to obtain pyrazole 8 in
excellent yield. Lastly, the treatment of 8 with the appropriate aryl-
carbonyl chloride in the presence of Et3N in CHCl3 furnished the
corresponding pyrazole esters 10.12
The in vitro data for the pyrazole ester analogues based on com-
pound 3 are shown in Table 2. The potency of the second set of
analogues ranged from 4.03 to 9.43 l
M in the in vitro assay13, with
compound 10a being the most potent.
The stability of the most potent compounds in each series in pH
8 buffer was determined by analyzing the amount of compound
remaining with time using LC/MS detection. To ensure an accurate
quantitation of degradants chlorpromazine was used as an internal
1) LDA, THF
R1
R1
H2, Ni(Ra)
N
N
H2N
O
O
O
2)
O
MeOH
HO
HO
R2
R2
R
11
THF
R = 2,6 di F or H
R1
R1
Dess martin reagent
N
N
N
N
O
CH2Cl2
rt
HO
MeO
AcOH, rt
R2
R2
NHNH2
MeO
12
MeO
13
pTsOH
toluene
reflux
R1
N
N
R2
14
MeO
Scheme 3. Synthetic route to prepare ester isosteres.

S. Sidique et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5773–5777
5777
O
ated the in vitro activity of a series of pyrazole derivatives contain-
O
NH2
ing ester isosteres. Of these analogues, the alkene 14 (IC50
=
N
H
N
N
N
Cl
Pyridine, dioxane
N
13.8 M) and amide 15 (IC50 = 16.0 M) derivatives are highly sta-
l
l
ble inhibitors of WNV NS2B-NS3 proteinase. These compounds,
which interact with an allosteric site on the enzyme,10 are promis-
ing leads for additional optimization studies and may find utility in
in vitro studies to elucidate the biochemistry and enzyme kinetics
of WNV NS2B-NS3 proteinase.
3-methyl-1-phenyl-1H-pyrazol-5-amine
15
O
S
O
AcOH
EtOH
H2N
H3C
CN
NHNH2
MeO
N
H2N
N
O
S O
Acknowledgments
O
O
Cl
This work was supported by NIH grants U01 AI078048 and U54
HG005033.
N
N
H
OMe
16
N
Pyridine
dioxane
O
S O
References and notes
17
1. Campbell, G. L.; Marfin, A. A.; Lanciotti, R. S.; Gubler, D. J. Lancet Infect. Dis.
2002, 2, 519.
OMe
2. Van der Meulen, K. M.; Pensaert, M. B.; Nauwynck, H. J. Arch. Virol. 2005, 150,
637.
Scheme 4. Synthetic routes to prepare pyrazole amide analogues.
3. Hayes, C. G. Ann. N. Y. Acad. Sci. 2001, 951, 25.
4. Bera, A. K.; Kuhn, R. J.; Smith, J. L. J. Biol. Chem. 2007, 282, 12883.
5. Ekonomiuk, D.; Su, X.; Ozawa, K.; Bodenreider, C.; Lim, S. P.; Yin, Z.; Keller, T.
H.; Beer, D.; Patel, V.; Otting, G.; Caflisch, A.; Huang, D. PLoS Negl. Trop. Dis.
2009, 3, 356.
6. Tomlinson, S. M.; Watowich, S. J. Biochem 2008, 47, 11763.
7. Stoermer, M. J.; Chappell, K. J.; Liebscher, S.; Jensen, C. M.; Gan, C. H.; Gupta, P.
K.; Xu, W.; Young, P. R.; Fairlie, D. P. J. Med. Chem. 2008, 51, 5714.
8. Mueller, N. H.; Pattabiraman, N.; Ansarah-Sobrinho, C.; Viswanathan, P.;
Pierson, T. C.; Padmanabhan, R. Antimicrob. Agents Chemother 2008, 52, 3385.
9. Chappell, K. J.; Stoermer, M. J.; Fairlie, D. P.; Young, P. R. Curr. Med. Chem 2008,
15, 2771.
10. Johnston, P. A.; Phillips, J.; Shun, T. Y.; Shinde, S.; Lazo, J. S.; Huryn, D. M.;
Myers, M. C.; Ratnikov, B.; Smith, J. W.; Su, Y.; Dahl, R.; Cosford, N. D. P.;
Shiryaev, S. A.; Strongin, A. Y. Assay. Drug Dev. Technol. 2007, 5, 737.
11. Myers, M. C.; Napper, A. N.; Motlekar, N.; Shah, P. P.; Chiu, C.; Beavers, M. P.;
Diamond, S. L.; Huryn, D. M.; Smith, A. B., III Bioorg. Med. Chem. Lett. 2007, 17,
4761.
derivative 14 was accomplished using pTsOH in hot toluene
(Scheme 3).
Treatment of 3-methyl-1-phenyl-1H-pyrazol-5-amine with
benzoyl chloride in the presence of pyridine and dioxane furnished
the amide derivative 15. N-(1-(4-Methoxyphenylsulfonyl)-3-
methyl-1H-pyrazol-5-yl)benzamide (17) was prepared by the
condensation of 3-aminocrotononitrile with 4-methoxybenzene-
sulfonohydrazide to afford pyrazole 16 followed by reaction of
benzoyl chloride (Scheme 4). The in vitro data for some of the tar-
get compounds are shown in Table 4. All of the alcohol derivatives
were inactive up to the highest concentration tested (100 lM)
while the two ketone derivatives exhibited IC50 values in the high
micromolar range. Encouragingly, however, the alkene derivative
12. Synthesis of 10a: a solution of (4-methoxybenzyl)hydrazine (2.0 g, 0.013 mol)
and ethyl acetoacetate (1.8 mL, 0.014 mol) in glacial acetic acid (54.0 mL) was
stirred and heated at 100 °C over night. The solvent was evaporated and the
product purified using automated medium pressure silica gel chromatography
(ISCO) eluting with 20–80% EtOAc/CH2Cl2 to obtain 1-(4-methoxybenzyl)-3-
methyl-1H-pyrazol-5-ol (8) (1.28 g, 46%) as a white solid. 1H NMR (300 MHz,
DMSO-d6): d 7.12 (d, J = 8.70 Hz, 2H), 6.86 (d, J = 8.40 Hz, 2H), 5.15 (s, 1H), 4.86
(s, 2H), 3.71 (s, 3H), 2.00 (s, 3H). A mixture of 8 (53.4 mg, 0.24 mmol), Et3N
(0.2 mL, 1.43 mmol) and benzoyl chloride (0.03 mL, 0.29 mmol) were dissolved
in CHCl3 (2 mL) and allowed to react at room temperature for 5 min. The crude
reaction mixture was dissolved in water (10 mL) and extracted with CH2Cl2
(20 mL). The organic layer was separated and the solvent was removed in
vacuo. The crude residue was purified using preparative HPLC to obtain the
title compound (65.3 mg, 84%) as a white solid. 1H NMR (300 MHz, DMSO-d6):
d 8.09 (d, J = 7.80 Hz, 2H), 7.80–7.75 (m, 1H), 7.64–7.59 (m, 2H), 7.14 (d,
J = 8.40 Hz, 2H), 6.85 (d, J = 8.40 Hz, 2H), 6.07 (s, 1H), 5.14 (s, 2H), 3.69 (s, 3H),
2.16 (s, 3H) 13C NMR (75 MHz, DMSO-d6): d 161.6, 158.6, 146.1, 143.9, 134.6,
130.0, 129.1, 128.9, 128.8, 127.4, 113.9, 94.0, 55.0, 50.2, 14.1. LRMS (ESI):
323.00 (M+1)+.
14 was more potent with IC50 = 13.8
l
M, while the amide deriva-
tives 15 (IC50 = 16.0 M) and 17 (IC50 = 9.2
l
lM) showed activity
in a comparable range in the in vitro enzyme assay. Compounds
14, 15 and 17 were also tested for stability in pH 8 buffer (Table
5). Interestingly, while the alkene (14) and amide 15 were highly
stable, the amide 17 possessed a relatively short half-life of 1.25 h.
In conclusion, we have described the design and synthesis of 3-
substituted pyrazole ester derivatives which are active as allosteric
inhibitors of West Nile Virus NS2B-NS3 proteinase. Two com-
pounds, 7a and 10a, while less potent than the original hits (1.96
and 4.03 lM IC50, respectively) are significantly more stable in
pH 8 buffer. In addition, we have designed, synthesized and evalu-
Table 4
Summary of in vitro data for ester isosteres.
13. WNV NS2B-NS3 proteinase activity assay with Pyr-RTKR-AMC fluorogenic
substrate: the assay for WNV NS2B-NS3 protease activity was performed in
10 mM Tris–HCl buffer, pH 8.0, containing 20% (v/v) glycerol and 0.005% Brij
Compounds
R1
R2
IC50 (lM)
12a
12b
13a
13b
13c
14
H
F
H
F
F
F
H
F
H
F
H
F
>100
>100
>100
23.9
38.8
13.8
35. The substrates and enzyme concentrations were 25 lM and 10 nM,
respectively. The total assay volume was 0.1 mL. Initial reaction velocities
were monitored continuously at kex (excitation wavelength) of 360 nm and kem
(emission wavelength) of 465 nm on a Spectramax Gemini EM fluorescence
spectrophotometer (Molecular Devices). All assays were performed in
triplicate in wells of a 96-well plate. The Km and kcat values were derived
from a double-reciprocal plot of 1/V0 against 1/[S], using the Lineweaver–Burk
transformation: 1/V0 = Km/Vmax  1/[S] + 1/Vmax, where V0 is the initial velocity
of substrate hydrolysis, [S] is the substrate concentration, Vmax is the maximum
rate of hydrolysis, and Km is the Michaelis–Menten constant. The concentration
of the catalytically active proteinase was measured using the fluorescent assay
by titration against a standard aprotinin solution of known concentration. The
concentration of active NS2B-NS3 was close to 100% when compared with the
total protein in the sample. For the determination of the IC50 value of the
inhibitors, NS2B-NS3 proteinase was pre-incubated for 60 min at 18 °C with
increasing concentrations of the inhibitors. Following addition of the Pyr-
Table 5
Time-dependant degradation of pyrazoles.a
Compounds
t1/2 (h)b
14
15
17
13
>96
1.25
RTKR-AMC substrate (25 lM), the rate of substrate hydrolysis was monitored,
and IC50 values were determined using routine kinetics software.
14. Charton, M. J. Am. Chem. Soc. 1969, 91, 619.
15. Charton, M. J. Am. Chem. Soc. 1969, 91, 624.
a
The compounds were dissolved in <10% DMSO, pH
Chlorpromazine (15 mM).
8
buffer (1 mM) and
b
Degradation of compound w.r.t standard.
16. Sviridov, S. I.; Vasil’ev, A. A.; Shorshnev, S. V. Tetrahedron 2007, 63, 12195.