QSAR for phospholipase A2 inhibitions by 1-acyloxy-3-N-n- octylcarbamyl-benzenes

Article abstract of DOI:10.1016/j.bmcl.2005.02.092

1-Acyloxy-3-N-n-octylcarbamyl-benzenes are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A₂ with the K_i values from 9.6 to 119 μM. The pK_i values are correlated to both Taft substituent constant σ* and Hansch hydrophobicity constant π. The pre-steady state inhibition studies indicate that the pK_S values for the first inhibition step are linearly correlated to σ* alone with the ρ* of -0.09 for this correlation. Thus, the first inhibition step may involve the insertion of the inhibitor to hepta-coordinated Ca²⁺ ion of the enzyme to form the octa-coordinated Ca²⁺ ion of the enzyme. The log(k₂/k _-2) values for the second inhibition step are linearly correlated to π alone, and the ψ value for this correlation is 0.13. Therefore, the second step inhibition step may involve the van der Waals' interaction between the acyl group of the inhibitor and Tyr 69 of the enzyme.

Full text of DOI:10.1016/j.bmcl.2005.02.092

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2405–2408
QSAR for phospholipase A2 inhibitions by
1-acyloxy-3-N-n-octylcarbamyl-benzenes
Gialih Lin* and Gia-Yun Yu
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
Received 22 November 2004; accepted 21 February 2005
Abstract—1-Acyloxy-3-N-n-octylcarbamyl-benzenes are potent reversible competitive inhibitors of Naja mocambique mocambique
phospholipase A2 with the Ki values from 9.6 to 119 lM. The pKi values are correlated to both Taft substituent constant r* and
Hansch hydrophobicity constant p. The pre-steady state inhibition studies indicate that the pKS values for the first inhibition step
are linearly correlated to r* alone with the q* of ꢀ0.09 for this correlation. Thus, the first inhibition step may involve the insertion of
the inhibitor to hepta-coordinated Ca2+ ion of the enzyme to form the octa-coordinated Ca2+ ion of the enzyme. The log(k2/k
values for the second inhibition step are linearly correlated to p alone, and the w value for this correlation is 0.13. Therefore, the
)
ꢀ2
second step inhibition step may involve the van der WaalsÕ interaction between the acyl group of the inhibitor and Tyr 69 of the
enzyme.
Ó 2005 Elsevier Ltd. All rights reserved.
Phospholipase A2 (PLA2, EC 3.1.1.4) catalyzes hydroly-
sis of the sn-2 ester bonds of phospholipids, liberating
lysophospholipid, and free fatty acid products. Apart
from being the products of phospholipid digestion, fatty
acids and lysophopholipids may be transformed into
inflammatory lipid mediators, thereby implicating
PLA2s in the pathogenesis of many inflammatory dis-
ease states. In fact, PLA2s are found in a variety of
extracellular locations and are involved in a range of
physicological processes including phospholipid diges-
tion, signal transduction, and host defense.1 PLA2 has
served as an useful prototype for elucidating the mecha-
nism for an enzyme that functions at a lipid-aqueous
interface.2
The X-ray crystal structure of a complex between a tran-
sition state analogue, 1-O-octyl-2-heptylphosphonyl-sn-
glycero-3-phosphoethanolamine and Naja naja atra
PLA2 has shown that the active site Ca2+ binds to both
the sn-2 phosphonate oxygen and the sn-3 phosphodies-
ter oxygen of the inhibitor and that the dihedral angle
between the C(sn-2)-O and C(sn-3)-O bonds is about
180° (anti conformation).3 However, the X-ray structure
of the phosphonate inhibitor-porcine PLA2 complex has
revealed the same active site Ca2+ binding to those
oxygens but the dihedral angle is about 60° (gauche con-
formation).4 Thus, the glycerol sn-2-sn-3 backbond con-
formation plays an important role in the PLA2 catalysis.
We have found that 1,3-benzene-di-N-n-octylcarbamate
O
O
O
N
H
O
R
Inhibitors:
1
2
3
4
5
6
7
8
9
R = CH3, n-C4H9, n-C7H15, n-C15H31, Ph, CH2Ph, CPh3, t-Bu, CCl3
Figure 1. Structures of inhibitors 1–9.
Keywords: Phospholipase A2; QSAR; Enzyme mechanism; Carbamate inhibitor.
*
Corresponding author. Tel.: +886 930 383816; fax: +886 4 2286 2547; e-mail: gilin@dragon.nchu.edu.tw
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.092

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G. Lin, G.-Y. Yu / Bioorg. Med. Chem. Lett. 15 (2005) 2405–2408
Table 1. Inhibition constants for the PLA2 inhibitions by 1-acyloxy-3-N-n-octylcarbamyl-benzenes (1–9) and Taft substituent and Hansch
hydrophobicity constants
Inh.
R
r*
p
Ki (lM)
KS (lM)a
k2 (sꢀ1 a
)
k
(sꢀ1 b
)
ꢀ2
1
2
3
4
5
6
7
8
9
CH3
0
0.5
98
75
27
8
6
4
6.7 0.7
6.67 0.09
5.7 0.7
0.27 0.03
0.22 0.02
0.16 0.01
0.16 0.02
0.16 0.02
0.16 0.01
0.18 0.02
0.18 0.02
0.46 0.04
3.9 0.7
2.5 0.3
0.7 0.1
0.27 0.04
2.4 0.4
1.8 0.3
1.0 0.1
1.9 0.3
4.5 0.6
n-C4H9
n-C7H15
n-C15H31
Ph
ꢀ0.13
ꢀ0.13
ꢀ0.13
0.6
2
3.5
7.5
9.6 0.9
6.55 0.09
6.45 0.08
6.5 0.7
2.13
2.26
5.4c
1.98
1.67
95
73
38
55
9
8
3
6
CH2Ph
CPh3
0.215
0.89
7.1 0.1
5.13 0.07
12.1 0.3
C(CH3)3
CCl3
ꢀ0.3
3.15d
120 10
a The KS, k2 values were determined from nonlinear least squares curve fitting of the pre-steady state inhibition rate constant (kobs) on inhibitor
concentration [I] according to the following equation: kobs = kꢀ2 + k2 [I]/(KS + [I]).25–31 The kobs value was obtained from the initial burst phase of
the pH-stat titration.
b Calculated from 1/Ki = (1/KS)(k2/kꢀ2).25–31
c This value is calculated from 2.26 + 2(1.57), where 2.26 is the p value for benzyl and 1.57 is the p value difference between phenyl (2.13) and methyl
(0.5).
d Calculated from 3r*(CH2Cl).
Table 2. Correlation results for the PLA2 inhibitions by 1-acyloxy-3-
N-n-octylcarbamyl-benzenes (1–9)a
5.30
5.25
Parameters
pKi
pKS
log(k2/k )
ꢀ2
5.20
5.15
5.10
5.05
5.00
4.95
4.90
q*
w
ꢀ0.08 0.03
0.15 0.02
3.88 0.06
0.94095
ꢀ0.09 0.01
—
—
pKS=5.21+0.01-(0.09+0.01)σ*
R=0.94078
0.13 0.02
ꢀ1.29 0.07
0.92606
hb
R
5.21 0.01
0.94078
a Correlations of pKi, pKS, and log(k2/kꢀ2) against Eq. 1.
b Calculated pKi, pKS, and log(k2/kꢀ2) values for r = p = 0.
is a potent inhibitor of PLA2 due to the constrained
conformation between two C (benzene)-O bonds.5
In order to study quantitative structure–activity
relationship (QSAR) for PLA2, 1-acyloxy-3-N-n-octyl-
carbamyl-benzenes (1–9) (Fig. 1) are synthesized for this
purpose.
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
σ*
Figure 2. Plot of the pKS values against r*.
Inhibitors 1–6, 8, and 9 were synthesized from conden-
sation of resorcinol (1,3-benzene-diol) with 1 equiv of
n-octyl isocyanate in the presence of 1.2 equiv of trieth-
ylamine in THF at 25 °C for 1 day to yield 1-hydroxy-
3-N-n-octylcarbamyl-benzene (35–45% yield) then
followed by the condensation of the latter with 1.2 equiv
of the corresponding acyl chloride in the presence of
1 equiv of triethylamine in THF at 25 °C for 1 day to
yield inhibitors 1–6, 8, and 9 (70–90% yield). 1-N-n-Oct-
yl-carbamyl-3-triphenyl-acetoxy-benzene (7) was syn-
thesized from condensation of resorcinol with 1 equiv
of triphenylacetic acid in the presence of 1 equiv of
1,3-dicyclohexylcarbodiimide (DCC), 1 equiv of trieth-
ylamine, and a catalytic amount of 4-(dimethylamino)
pyridine (DMAP) in CH2Cl2 at 25 °C for 4 h to yield
1-hydroxy-3-triphenyl-acetoxy-benzene (20–25% yield)
then followed by the condensation of the latter with
1 equiv of n-octyl isocyanate in presence of 1 equiv tri-
ethylamine in THF at 25 °C for 2 h to yield inhibitor 7
-0.2
-0.4
-0.6
-0.8
log(k2/k-2)=-1.29+0.07+(0.13+0.02)π
R=0.92606
-1.0
-1.2
-1.4
0
1
2
3
4
5
6
7
8
π
Figure 3. Plot of log(k2/kꢀ2) values against p.
1
(50–60% yield). All products were characterized by H
and 13C NMR spectra, mass spectra, and elemental
analyses.
mocambique PLA2 (Sigma)-catalyzed hydrolysis of 1,2-
dimyristoyl-sn-glycero-3-phosphocholine (Sigma, 0.1
mM) in the presence of inhibitors 1–9 were followed
by pH-stat titration (Radiometer PHM 290) under a
The Ki values were obtained from the Lineweaver–Burk
plots. Steady-state initial rates of Naja mocambique

G. Lin, G.-Y. Yu / Bioorg. Med. Chem. Lett. 15 (2005) 2405–2408
2407
stream of nitrogen at pH 8.0 and 25 °C in a 5 mL reac-
In Eq. 1, the parameters h, q*, r*, ES, d, w, and p are
logko for the standard reaction, reaction constant to
substituent inductive effect, Taft substituent constant,
Taft steric constant, reaction intensity factor to substitu-
ent steric effect, reaction intensity factor to substituent
hydrophobicity, and Hansch hydrophobicity constant,10
respectively. Many successful QSAR studies for
cholesterol esterase,11–18 acetylcholinesterase,19–22 and
PSL11,23 inhibitions by aryl carbamates have been
reported. In this paper, we further report QSARs
for PLA2 inhibitions by carbamate inhibitors 1–9
(Fig. 1).
tion mixture containing NaCl (10 mM).5
Quantitative structure–activity relationships (QSARs)
represent an attempt to correlate structural properties
of compounds with biological activities and chemical
reactivities.6–9 These chemical descriptors, which include
parameters to account for hydrophobicity, electronic,
inductive, or polar properties, and steric effects, are
determined empirically or by calculations. Many drug
activities and chemical reactivities are linearly correlated
to three substituent parameters such as the Taft–Ingold–
Hanscdh–Ja¨rv equation (Eq. 1).9
All carbamate inhibitors 1–9 are reversible, competitive
inhibitors of Naja mocambique mocambique PLA2 with
log k ¼ h þ qÃrà þ dES þ wp
ð1Þ
D49
O
e
-
Y28
G32
e
a
e
O
e
Ca2+
e
G30
a
H
H48
inhibitors 1-9
H2O-12
H
O
6
5
D99
O
H
H
O
N
N
-O
KS
O
H
H
ρ* = -0.09
Y69
H48
N
D49
O
e
-
Y28
e
G32
e
a
e
O
Ca2+
G30
a
k2
H
e
e
H2O-12
H
O
+
6
O
5
O
NH2
D99
H
k-2
H
R
O
N
O
Ψ=0.13
-O
O
O
H
H
O
Y69
O
G32
D49
Y28
-
a
O
Ca2+
G30
a
e
H
H48
H2O-12
H
O
+
O
NH2
6
5
O
O
D99
H
H
R
O
N
O
N
O
-O
O
H
H
Y69
Figure 4. The proposed PLA2 inhibition mechanism by inhibitors 1–9. The first step (KS) is an insertion of the carbamyl carbonyl oxygen of the
inhibitor to the hepta-coordinated Ca2+ ion of the enzyme and then the formation of the octa-coordinated Ca2+ ion of the enzyme-inhibitor complex.
The second step (k2/kꢀ2) may involve the van der WaalsÕ interaction between the acyl R moiety and Tyr69 of the enzyme and therefore this
interaction stabilizes the enzyme-inhibitor complex.

2408
G. Lin, G.-Y. Yu / Bioorg. Med. Chem. Lett. 15 (2005) 2405–2408
2. Berg, O. G.; Gelb, M. H.; Tsai, M.-D.; Jain, M. K. Chem.
Rev. 2001, 101, 2613.
3. White, S. P.; Scott, D. L.; Otwinowski, Z.; Gelb, M. H.;
Sigler, P. B. Science 1990, 250, 1560.
the Ki values from 9.6 to 119 lM (Table 1). Moreover,
the pKi values are correlated to both r* and p values
against Eq. 2 (Table 2).
4. Sekar, K.; Kumar, A.; Liu, X.; Tsai, M.-D.; Gelb, M. H.;
Sundaralingam, M. Acta Crystallogr. Sect. D: Biol.
Crystallogr. 1998, 54, 334.
5. Lin, G.; Lin, Y.-F.; Hwang, M.-T.; Lin, Y.-Z. Bioorg.
Med. Chem. Lett. 2004, 14, 751.
pKi ¼ h þ qÃrà þ wp
ð2Þ
The pre-steady inhibitions indicate that each reversible
Ki step is further divided into two steps:25–31 the KS
(= kꢀ1/k1) step and the k2/k
step (1/Ki = (1/KS)k2/
ꢀ2) (Table 1). Moreover, the pKS values are linearly
6. March, J. Advanced Organic Chemistry, 4th ed.; John
Wiley & Sons: New York, 1992.
7. Isaacs, N. Physical Organic Chemistry, 2nd ed.; Longman:
UK, 1995.
8. Lowry, T. H.; Richardson, K. S. Mechanism and
Theory in Organic Chemistry, 3rd ed.; Harper & Row:
New York, 1992.
ꢀ2
k
correlated to the r* values alone with the q* value of
ꢀ0.09 (Fig. 2 and Table 2), and the log(k2/kꢀ2) values
are linearly correlated to the p values alone with the w
value of 0.13 (Fig. 3 and Table 2).
9. Ja¨rv, J.; Kesvatera, T.; Aaviksaar, A. Eur. J. Biochem.
1976, 67, 315.
10. Leo, A.; Hansch, C.; Elkins, D. Chem. Rev. 1971, 71,
525.
According to these QSAR results, a two-step inhibition
mechanism is proposed (Fig. 4). Thus, an insertion of
the carbamyl carbonyl oxygen of the inhibitor to the hepta-
coordinated Ca2+ ion of PLA22,24 (Fig. 4) may occur
in the KS step due to a small negative value of q*
(= ꢀ0.09) for the pKS–r* correlation (Fig. 2 and Table
2). This value also suggests that reaction centers are far
away from the varied substituents (R moieties in Fig.
1) of the inhibitors and that the products (octa-coordi-
nated Ca2+ ion of PLA2-inhibitor complexes)2,24 (Fig.
4) are more positively charged than the inhibitors them-
selves. The second inhibition step (k2/kꢀ2) may involve
the van der WaalsÕ interaction between the acyl R moiety
(Fig. 1) of the inhibitor and the Tyr69 residue of PLA22
due to a small value of w (= 0.13) for the log(k2/kꢀ2)–p
correlation (Fig. 3 and Table 2). Thus, the more the
inhibitor is hydrophobic at the acyl R moiety the more
the inhibitor interacts with the Tyr69 residue of PLA2.
Since the log(k2/kꢀ2)–r* correlation is poor (data not
shown), the electronic characters of the enzyme-inhibitor
11. Lin, G.; Shieh, C.-T.; Ho, H.-C.; Chouhwang, J.-Y.; Lin,
W.-Y.; Lu, C.-P. Biochemistry 1999, 38, 9971.
12. Lin, G. J. Phys. Org. Chem. 2000, 13, 313.
13. Lin, G.; Lai, C.-Y. Tetrahedron Lett. 1995, 36, 6117.
14. Lin, G.; Lai, C.-Y. Tetrahedron Lett. 1996, 37, 193.
15. Feaster, S. R.; Lee, K.; Baker, N.; Hui, D. Y.; Quinn,
D. M. Biochemistry 1996, 35, 16723.
16. Lin, G.; Shieh, C.-T.; Tsai, Y.-C.; Hwang, C.-I.; Lu, C.-P.;
Chen, G.-H. Biochim. Biophys. Acta 1999, 35905, 161.
17. Lin, G.; Lai, C.-Y.; Liao, W.-C.; Kuo, B.-H.; Lu, C.-P. J.
Chin. Chem. Soc. 2000, 47, 489.
18. Lin, G.; Liu, Y.-C.; Wu, Y.-G.; Lee, Y.-R. J. Phys. Org.
Chem. 2004, 17, 707.
19. Lin, G.; Liao, W.-C.; Chiou, S.-Y. Bioorg. Med. Chem.
2000, 8, 2601.
20. Lin, G.; Lai, C.-Y.; Liao, W.-C.; Liao, P.-S.; Chan, C.-H.
J. Chin. Chem. Soc. 2003, 50, 1259.
21. Lin, G. J. Chin. Chem. Soc. 2004, 51, 423.
22. Lin, G.; Liu, Y.-C.; Lin, Y.-F.; Wu, Y.-G. J. Enzyme
Inhib. Med. Chem. 2004, 19, 395.
complexes do not alter at all in the k2/k step.
ꢀ2
23. Lin, G.; Chouhwang, J.-Y. J. Biochem. Mol. Biol. Biophys.
2001, 5, 301.
24. Yu, B.-Z.; Rogers, J.; Nicol, G. R.; Theopold, K. H.;
Seshadri, K.; Visjweshwara, S.; Jain, M. K. Biochemistry
1998, 37, 12576.
25. Fersht, A. Enzyme Structure and Mechanism, 2nd ed.;
Freeman: New York, 1984.
26. Ikda, K.; Kunugi, S.; Ise, N. Arch. Biochem. Biophys.
1982, 217, 37.
Carbamates 3 and 4 are potential candidates for nonste-
roidal anti-inflammatory drugs (NSAIDs) due to the
fact that both carbamates are potent inhibitors of the
snake venom PLA2. Further investigations for the inhi-
bitions of pancreatic and bee venom PLA2s by carba-
mates 1–9 will be communicated in due course.
27. Nakatani, H.; Morita, T.; Hiromi, K. Arch. Biochem.
Biophys. 1978, 525, 423.
Acknowledgements
28. Hart, G. J.; OÕBrien, R. D. Pestic. Biochem. Physiol. 1974,
4, 239.
29. Nakatani, U.; Uehara, Y.; Hiromi, K. J. Biochem. 1975,
78, 611.
We thank the National Science Council of Taiwan for
financial support.
30. Nakatani, U.; Hanai, K.; Uehara, Y.; Hiromi, K. J.
Biochem. 1975, 78, 905.
References and notes
31. Nakatani, H.; Hujiwake, H.; Hiromi, K. J. Biochem. 1977,
81, 1269.
1. Murakami, M.; Kudo, I. Adv. Immunol. 2001, 77, 163.