Indole-5-phenylcarbamate derivatives as human non-pancreatic secretory phospholipase A2 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2005.07.006

The synthesis of the human non-pancreatic secretory phospholipase A2 inhibitor (IC₅₀ = 1.81 ± 0.59 μM) is reported.

Full text of DOI:10.1016/j.bmcl.2005.07.006

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4540–4542
Indole-5-phenylcarbamate derivatives as human
non-pancreatic secretory phospholipase A2 inhibitor
Ying Liu,* Xiao-feng Han, Chang-kang Huang, Xin Hao and Lu-Hua Lai*
State Key Laboratory for Structural Chemistry of Unstable and Stable Species, Institute of Physical Chemistry
and College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
Received 21 April 2005; revised 1 July 2005; accepted 4 July 2005
Available online 18 August 2005
Abstract—The synthesis of the human non-pancreatic secretory phospholipase A2 inhibitor (IC50 = 1.81 0.59 lM) is reported.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
pocket. Different types of fused heterotricyclic com-
pounds were chosen to be candidates. In the present
study, we prepared new 1-benzyl-3-(carbamoylmethyl)-
2-methyl-1H-indol-5-yl-phenylcarbamate derivatives by
multistep synthesis and tested their inhibitory activity.
The compound had showed good inhibition as expected.
A study of rheumatoid arthritis (RA) patients found that
serum secretory phospholipase A2 (PLA2) activity corre-
lates with disease activity.1 When purified PLA2 enzyme
from synovial fluid and snake venom were injected into
animal joints, an acute inflammatory response with swell-
ing of synovial cells, hyperplasia, and edema have been
aggravated.2,3 High levels of PLA2 are observed in a vari-
ety of severe inflammatory diseases and have been con-
sidered to be accountable for some inflammatory
reactions. Thus, the inhibitors of PLA2 would have great
potential for the treatment of inflammatory diseases.
2. Design
All molecular modeling and docking calculations were
performed on the Linux workstation by using SYBYL
6.917 molecule modeling software and Autodock3.05.8
The crystal structure of hnps PLA2 complexed with in-
dole inhibitor4 (PDB code:1db4) was used as the starting
complex structure. Compounds 1, 3, and 5 were built
using SKETCH option in SYBYL. The atomic charges
The first indole inhibitor of human non-pancreatic
secretory (hnps) PLA2 was reported by Eli Lilly and
Company.4 Then many indole-3-acetamides have been
synthesized and showed inhibition of PLA2.5 Two doses
of the indole inhibitors (LY315920Na/S-5920) have been
tested for the efficacy and safety, from clinical pharma-
cology studies, in critically ill patients with sepsis.6
were calculated with the Gasteiger–Huckel method.
¨
The molecules were geometry-optimized in standard
Tripos force field using the steepest descent and conju-
gate gradients methods.
Based on the crystal structure of hnps PLA2 complexed
with indole inhibitor4 (PDB code: 1db4), we confirmed
that ligation of the catalytic calcium, hydrogen bond,
and hydrophobic interaction are the necessary condi-
tions for sPLA2 inhibitorsꢀ potency, and the most
important condition is the ligand having the right shape
for interacting with the hnps PLA2 substrate binding
3. Synthesis
Indomethacin 1 was used as starting material. Using
mixed anhydride method, 1 reacted with ethyl chlorofor-
mate, followed by mixing with ammonia, NaOH, and
then acidification of the mixture, 5-methoxy-2-methyl-
1H-indole-3-acetamide 2 was obtained in high yields.
Compound 2 was alkylated on the indole nitrogen by
forming the sodium salt with sodium hydride and treated
with benzyl chloride, 5-methoxy-2-methyl-1-(phenylm-
ethyl)1H-indole-3-acetamide 3 was obtained. Boron
Keywords: Indole-5-phenylcarbamate; PLA2; Inhibitor.
*
Corresponding authors. Tel.: +86 106 27 56 833; fax: +86 10 62 75
1725 (Y.L.); e-mail addresses: liuying@pku.edu.cn; lhlai@pku.
edu.cn
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.006

Y. Liu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4540–4542
4541
Scheme 1. Synthetic approach. Reagents: (A) ClCO2C2H5, NH3ÆH2O,
NaOH; (B) NaH, PhCH2Cl, DMF; (C) BBr3, CH2Cl2; (D) 3-ClPhCNO,
NEt3, CH3CN.
tribromide demethylation of 3 gave the hydroxyl indole 4,
which was treated with substituted phenylisocyanates and
Et3N in CH3CN to give the title compounds 5.
Our general synthetic approach to the title compounds
is shown in Scheme 1.
Figure 1. The binding mode of indole inhibitor to hnps-PLA2 (drawn
by Ligplot11).
compound for potent and selective inhibitors of hnps
PLA2.5 First, 4-methoxy phenylhydrazine was convert-
ed by the classical Fischer-indole synthesis to give indole
acetic acid esters; second, the indole-3-acetic acid ester
was alkylated by forming the sodium salt and treating
with an arylmethyl halide to give 5-methoxy-2-methyl-
1-(phenylmethyl)1H-indole-3-acetic acid ester; third, by
heating with hydrazine hydrate in ethanol, 1-(arylmeth-
yl)indole-3-acetic acid hydrazide was got; finally, heat-
ing the hydrazides with Raney nickel in ethanol gave
the corresponding 1-(arylmethyl)indole-3-acetamides.
In this procedure, products from two out of four steps
were of lower yield, and needed chromatography for
purification. The overall yield is 5.88%. Our method
here only contains two-step reactions at lower or room
temperature with rare side reactions. The overall yield
is 78.3%, which is 13.3 times as much as the reported
method. In our experiment, compounds 4 and 512 were
obtained very easily with high yields.
4. Biological evaluation
The fluorescence-based assay9 method was used to eval-
uate bioactivity of the compounds. Human recombinant
hnps PLA2 was used as the target enzyme. The expres-
sion and purification of the PLA2 followed a method
similar to the one given in literature10 with the synthe-
sized gene inserted into pET 21a plasmid. The assay
was carried out in a 96-well plate utilizing a multiwell
fluorometer (SpectraMax GeminiXS, Molecular Devic-
es). All solutions were prepared with high purity
(18 MX conductivity) water. The buffer used in all the
experiments was 0.25 M Tris, pH 9.0, 2 mM CaCl2.
The substrate was 0.25 mg/mL NBD-C12-PG buffer
solution, which was sonicated for 1 min to get the micell
suspension. All the samples were dissolved in DMSO. A
200 lL reaction solution contained 2.6 nM PLA2,
0.0125 mg/mL substrate, 0.1 mg/mL BSA, and 10 lL
inhibitor solution. The reaction was monitored by exci-
tation at 485 nm and emission at 535 nm. Fluorescent
signals were monitored by kinetics mode program.
Reported IC50 values, determined by plotting concentra-
tion–velocity curves are the mean of at least three sepa-
rate experiments.
A preliminary bioassay study in vitro showed that
compound 5 displayed inhibition to synovial-fluid
PLA2 with the average IC50 of 1.81 0.59 lM, which
is better to the reference compoundꢀs average IC50
5. Discussion
The potential binding modes of compounds 1, 3, and 5
were obtained by molecular docking. When the ligands
formed hydrogen bonds and liganded to the active site
calcium in suitable distance, the pKi were calculated to
be 5.37, 6.13, and 7.09, respectively. It is clear that com-
pound 5 will be a better candidate. It formed hydrogen
bonds with Gly29, His47, and Asp48 with distances of
0.305, 0.290, and 0.257 nm, respectively. The calcium
ion was liganded by the oxygen atom of indole-3-car-
bamoylmethyl group at a distance of 0.244 nm (Fig. 1).
Figure 2. The concentration–velocity curve. (The curves were induced
by equation I = IC50(V0ÀV)/V. I is the concentration of the inhibitor.
V0 is the max-velocity of the enzyme without inhibitor. V is the max-
velocity of the enzyme with certain concentration of inhibitor. The
slope of the curve is the value of IC50.)
Compound 3 was prepared through four steps by Nich-
olas J. Bachꢀs group, and it has been shown to be a lead

4542
Y. Liu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4540–4542
4. Schevitz, R. W.; Bach, N. J.; Carlson, D. G.; Chirgadze,
N. Y.; Clawson, D. K.; Dillard, R. D.; Draheim, S. E.;
Hartley, L. W.; Jones, N. D.; Mihellch, E. D.; Olkowski, J.
L.; Snyder, D. W.; Sommers, C.; Wery, J. P. Nat. Struct.
Biol. 1995, 2, 458.
(4.26 0.72 lM) in the system we used. The concentra-
tion–velocity curve of compound 5 and reference are
shown in Figure 2.
5. Dillard, R. D.; Bach, N. J.; Draheim, S. E.; Berry, D. R.;
Carlson, D. G.; Chirgadze, N. Y.; Clawson, D. K.;
Hartley, L. W.; Johnson, L. M.; Jones, N. D.; McKinney,
E. R.; Mihelich, E. D.; Olkowski, J. L.; Schevitz, R. W.;
Smith, A. C.; Snyder, D. W.; Sommers, C. D.; Wery, J. P.
J. Med. Chem. 1996, 39, 5119.
6. Abraham, E.; Naum, C.; Bandi, V.; Gervich, D.; Lowry,
S. F.; Wunderink, R.; Schein, R. M.; Macias, W.;
Skerjanec, S.; Dmitrienko, A.; Farid, N.; Forgue, S. T.;
Jiang, F. Crit. Care Med. 2003, 31, 718.
6. Conclusion
We have developed a new type of indole compounds as
hnps PLA2 inhibitor. The compound was shown to be
active for human recombinant hnps PLA2, which is
comparable to the reported indole compounds from
Eli Lilly and Company. The reported compounds here
contain more cyclic component and can be easily
synthesized.
7. Sybyl, version 6.91; Tripos Associates: St. Louis, MO, 1998.
8. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.;
Hart, W. E.; Belew, R. K.; Olson, A. J. J. Comput. Chem.
1998, 19, 1639.
Acknowledgment
9. Blanchard, S. G.; Harris, C. O.; Parks, D. J. Anal.
Biochem. 1994, 222, 435.
10. Othman, R.; Baker, S.; Li, Y.; Worrall, A. F.; Wilton, D.
C. Biochim. Biophys. Acta 1996, 1303, 92.
This work was supported by the 863 High Technology
Program of China (863-103-13-04-02, 2001AA235061,
2003AA235010).
11. Wallace, A. C.; Laskowski, R. A.; Thomton, J. M. Protein
Eng. 1995, 8, 127.
References and notes
12. All compounds gave satisfactory structural data. The
physical data and spectrum data of compound 5 are as
follows: mp: 181–182 °C; yield: 74%; IR: 1655 (NH2), 1716
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1
(NH), 3458, 3410, 3327, 3196 (NH and NH2); H NMR
(400 MHz, DMSO): 2.34 (s, 3H, 2-CH3), 3.34 (s, 2H, 3-
CH2CONH2), 5.42 (s, 2H, 1-CH2Ph), 6.84–7.81 (m, 12H,
Ar and NH2), 10.51 (s, 1H, NH2); MS: m/z 482 (M+) calcd
for C25H21N2O3Cl2 482.4; Anal (calcd) %: C, 62.04
(62.25); H, 4.269 (4.388); N, 8.405 (8.711).
3. Bomalaski, J. S.; Lawton, P.; Browining, J. L. J. Immunol.
1991, 146, 3904.