Synthesis and Pseudomonas lipase inhibition study of stereoisomers of decahydro-2-naphthyl-N-n-butylcarbamate

Article abstract of DOI:10.2174/092986611797200913

(2S,4aR,8aS)-Cis,cis-, (2R,4aS,8aR)-cis,cis-, rac-cis,cis-, and rac-trans,cis-decahydro-2-naphthyl-N-n-butylcarbamates are synthesized from condensation of (2S,4aR,8aS)-cis,cis-, (2R,4aS,8aR)-cis,cis-, rac-cis,cis-, and rac-trans,cisdecahydro-2-naphthols, respectively, with n-butyl isocyanate in the presence of triethylamine in dichloromethane. Optically pure (2S,4aR,8aS)-(-)-and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthols are resolved by the porcine pancreatic lipase-catalyzed acetylation of decahydro-2-naphthols with vinyl acetate in t-butyl methyl ether. Absolute configurations of (2S,4aR,8aS)-(-)-and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthols are determined from the ¹⁹F NMR spectra of their Mosher's ester derivatives. (2S,4aR,8aR)-Trans,cis-and (2R,4aS,8aS)-trans,cis-decahydro-2- naphthols can't be resolved from the porcine pancreatic lipase-catalyzed acetylation of decahydro-2-naphthols with vinyl acetate in t-butyl methyl ether. For the inhibitory potency of Pseudomonas lipase, (2S,4aR,8aS)-cis,cis- decahydro-2-naphthyl-N-n-butylcarbamate is 3.5 times more potent than (2R,4aS,8aR)-cis,cis-decahydro-2-naphthyl-N-n-butylcarbamate; racemic cis,cis-decahydro-2-naphthyl-N-n-butylcarbamate is about the same with trans,cis-decahydro-2-naphthyl-N-n-butylcarbamate. These inhibitors also show similar effects on porcine pancreatic lipase.

Full text of DOI:10.2174/092986611797200913

1168
Protein & Peptide Letters, 2011, 18, 1168-1176
Synthesis and Pseudomonas Lipase Inhibition Study of Stereoisomers of
Decahydro-2-naphthyl-N-n-butylcarbamate
Ming-Cheng Lin1, Yu-Fang Shen2 and Gialih Lin2,*
1Department of Internal Medicine, Chung Shan Medical University Hospital, School of Medicine, Chung-Shan Medical
University, Taichung 402, Taiwan, 2Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
Abstract: (2S,4aR,8aS)-Cis,cis-, (2R,4aS,8aR)-cis,cis-, rac-cis,cis-, and rac-trans,cis-decahydro-2-naphthyl-N-n-butylcar-
bamates are synthesized from condensation of (2S,4aR,8aS)-cis,cis-, (2R,4aS,8aR)-cis,cis-, rac-cis,cis-, and rac-trans,cis-
decahydro-2-naphthols, respectively, with n-butyl isocyanate in the presence of triethylamine in dichloromethane. Opti-
cally pure (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthols are resolved by the porcine pancreatic li-
pase-catalyzed acetylation of decahydro-2-naphthols with vinyl acetate in t-butyl methyl ether. Absolute configurations of
(2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)- cis,cis-decahydro-2-naphthols are determined from the 19F NMR spectra of their
Mosher’s ester derivatives. (2S,4aR,8aR)-Trans,cis- and (2R,4aS,8aS)-trans,cis-decahydro-2-naphthols can’t be resolved
from the porcine pancreatic lipase-catalyzed acetylation of decahydro-2-naphthols with vinyl acetate in t-butyl methyl
ether. For the inhibitory potency of Pseudomonas lipase, (2S,4aR,8aS)-cis,cis-decahydro-2-naphthyl-N-n-butylcarbamate
is 3.5 times more potent than (2R,4aS,8aR)-cis,cis-decahydro-2-naphthyl-N-n-butylcarbamate; racemic cis,cis-decahydro-
2-naphthyl-N-n-butylcarbamate is about the same with trans,cis-decahydro-2-naphthyl-N-n-butylcarbamate. These inhibi-
tors also show similar effects on porcine pancreatic lipase.
Keywords: Lipase, stererospecificity, carbamates, inhibitors, resolutions.
INTRODUCTION
1-acyloxy-3-N-n-octyl- carbamyl-benzenes [21] have been
reported as potent inhibitors as Pseudomonas species lipase
(PSL). We have synthesized (R)-(+)-exo-, (S)-(-)-exo-, (R)-
Lipases are lipolytic enzymes, which primarily hydrolyze
ester bonds of triacylglycerols [1]. Lipases (EC 3.1.1.3) have
been widely used in organic synthesis especially in resolu-
tion of many chiral secondary alcohols as the enantiomeri-
cally pure starting materials in asymmetric synthesis [2,3].
(+)-endo-,
and
(S)-(-)-endo-2-norbornyl-N-n-butylcar-
bamates from optically pure (R)-(+)-exo-, (S)-(-)-exo-, (R)-
(+)-endo-, and (S)-(-)-endo-2-norborneols which are kineti-
cally resolved by porcine pancreatic lipase (PPL) in organic
solvent [22,23]. (R)-(+)-exo-, (S)-(-)-exo-, (R)-(+)-endo-, and
(S)-(-)-endo-2-Norbornyl N-n-butylcarbamates have shown
high enantioselectivity for the inhibition of acetylcho-
linesterase and butyrylcholinesterase. In this paper, we fur-
ther report that the stereoselectivity for lipase inhibitions by
stereoisomers of decahydro-2-naphthyl-N-n-butylcarbamate.
Many X-ray structures of lipases such as Pseudomonas
cepacia lipase (PCL) and Candida rugosa lipase (CRL) have
been reported [4-9]. Lipase usually contains a small ꢀ-helix
or loop, referred to as the lid, which covers the active site
pocket [4,6,7,10-14]. This conformation is termed the closed
conformation. When the lipase is absorbed to an interface,
the lid is displaced so that the active site becomes accessible
to substrate [10]. This conformation is termed the open con-
formation. The structures of the free and bound lipase are
believed to represent the start and end conformation in the
interfacial activation process [15].
Carbamate inhibitors have been characterized as the
pseudo substrate inhibitors of CEase (Scheme I) [17-30]. In
this paper, we also characterize all stereoisomers of decahy-
dro-2-naphthyl-N-n-butylcarbamate as the pseudo substrate
inhibitors of PSL and PPL.
Recently, there has been increased interest in lipase in-
hibitors due to the use of Orlistat (Xenical®) [16]. Orlistat,
whose original mechanism of action consists of the selective
inhibition of gastrointestinal lipases, has been commercial-
ized for the treatment of obesity. Orlistat is a chiral drug.
Therefore, the stereoselctivity for lipase inhibition plays an
important role in the design of novel drugs for management
of obesity.
Optically pure (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)-
cis,cis-decahydro-2- naphthols have been resolved from mi-
crobial hydrolysis of (±)-cis,cis-decahydro-2- naphthyl ace-
tate by Bacillus subtilis [31]. For resolution of enantiomers
of secondary alcohols, lipases (EC 3.1.1.3) have been widely
chosen to use because lipases can be applied in organic sol-
vent that is vey convenient to organic chemists [2, 3]. In this
paper, we apply the lipase-catalyzed stereospecifically acety-
lation of one of enantiomers of (2S,4aR,8aS)-(-)- and
(2R,4aS,8aR)-(+)-cis,cis-decahydro- 2-naphthols form a mix-
ture of stereoisomers of decahydro-2-naphthols with vinyl
acetate in t-butyl methyl ether.
Aryl carbamates [17,18], 1,2-ethylene-di-N-alkylcarba-
mates [19], 1,2-cyclopentane-di-N-alkylcarbamates [20], and
*Address correspondence to this author at the Department of Chemistry,
National Chung-Hsing University, Taichung 402, Taiwan;
Tel: +886-4-2284-0411; Ext 705; Fax: +886-4-2286-2547;
E-mail: gilin@dragon.nchu.edu.tw
0929-8665/11 $58.00+.00
© 2011 Bentham Science Publishers

Lipase Inhibition by Stereoisomers of decahydro-2-naphthyl-N-n-butylcarbamate
Protein & Peptide Letters, 2011, Vol. 18, No. 11 1169
Km
k2S
k3S
E + S
E-A
E + P''
ES
Acyl
P'
Enzyme-Substrate
Michaelis Complex
Enzyme
Intermediate
Ki
k3
k2
EI
E + Q
E-I'
E + I
P
Carbamyl Enzyme
Intermediate
Enzyme-Inhibitor
Michaelis Complex
aE: enzyme; E-A: acyl enzyme; EI: enzyme-inhibitor Michaelis complex; E-I’: carbamyl enzyme intermediate; ES: enzyme-substrate Michaelis complex; I: pseudo substrate inhibi-
tor; k2: carbamyl constant; k3: decarbamylation constant; k2S: formation rate constant of E-A; k3S: deacylation constant of E-A; Ki: inhibition constant; Km: Machaelis-Menten con-
stant; P: product, decahydro-2-naphthol; P’: product, p-nitrophenol; P’’: product, butyrate; Q: product, butylcarbamic acid; S: substrate, PNPB.
Scheme I. Kinetic scheme for pseudo-substrate inhibitions of PSL by decahydro-2-naphthyl- N-n-butylcarbamates in the presence of sub-
strate.a
8
H
H
H
H
8
2
N
N
2
O
O
8a
8a
4a
4a
O
O
4
4
H
H
(2R,4aS,8aR)-cis,cis-decahydro-2-naphthyl-
N-n-butylcarbamate
(2S,4aR,8aS)-cis,cis-decahydro-2-
naphthyl-N-n-butylcarbamate
H
H
H
H
H
N
N
O
O
+
O
O
H
racemic-trans,cis-decahydro-2-naphthyl-N-n-butylcarbamate
Figure 1. Structures of (2S,4aR,8aS)-cis,cis-, (2R,4aS,8aR)-cis,cis-, and racemic trans,cis- decahydro-2-naphthyl- N-n-butylcarbamate.
other chemicals were of the highest purity available com-
mercially.
MATERIALS AND METHODS
Materials
Instrumental Methods
Pseudomonas species lipase (PSL), porcine pancreatic
lipase (PPL), p-nitrophenyl butyrate (PNPB), and triton-X
1H, 13C, 19F NMR spectra were recorded in CDCl3 at 400,
100 (TX) were obtained from Sigma. Decahydro-2-naphthol,
n-butyl isocyanate, triethylamine, CDCl3, tetramethylsilane,
t-butyl methyl ether, butanol, vinyl acetate, butyryl chloride,
pyridine, and (S)-(+)-ꢀ-methoxy-ꢀ- trifluoromethyl- pheny-
lacetyl chloride were purchased from Aldrich (USA). Silica
gel and TLC plate were obtained from Merck (Germany).
Hexane, CH2Cl2, ethyl acetate, and tetrahydrofuran were
obtained from TEDIA (USA). Sodium dihydrogen phosphate
(NaH2PO4:2H2O), disodium hydrogen phosphate (Na2HPO4:
12H2O), hydrogen chloride (HCl), sodium hydroxide
(NaOH), potassium hydroxide (KOH), calcium chloride
(CaCl2), and sodium chloride (NaCl) were purchased from
UCW (Taiwan). Ethanol (95%) was obtained from Taiwan
Tobacco & Liquid Corporation (Taiwan). All other chemi-
cals were of the highest purity available commercially. All
100, and 377 MHz, respectively, with an internal reference
tetramethylsilane (TMS) at 25 C on a Varian Gemini 400
spectrometer. Mass spectra were recorded at 71 eV in a mass
spectrometer (Joel JMS-SX/SX 102A). Elemental analyses
were preformed on a Heraeus instrument. Optical rotation
was recorded on a polarimeter (Perkin-Elmer 241).
o
SYNTHESIS
Kinetic resolution of (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-
(+)-cis,cis- decahydro-2-naphthols (Fig. 2).
To a t-butyl methyl ether (50 mL) solution of decahydro-
2-naphthol (32.5 m mol) and vinyl acetate (10 mL), 30 g of
porcine pancreatic lipase were added. The reaction mixture
o
was shaken at 37 C at 200 rpm for 72 h. This reaction

1170 Protein & Peptide Letters, 2011, Vol. 18, No. 11
Lin et al.
H
H
H
H
OH
OH
+
rac-trans,cis-decahydro-2-naphthol
O
8
H
H
8
H
2
2
O
OH
OH
8a
8a
PPL
4a
4a
37oC
72 h
4
4
H
rac-decahydro-2-naphthol
(+)-(2R,4aS,8aR)-cis,cis-decahydro-2-naphthol
78% ee
H
8
2
O
8a
4a
O
4
H
(-)-(2S,4aR,8aS)-cis,cis-decahydro-2-naphthyl acetate
0.1 M KOH
EtOH
H
OH
H
(-)-(2S)-cis,cis-decahydro-2-naphthol
80% ee
Figure 2. Kinetic resolution of (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)-cis,cis- decahydro-2-naphthols from lipase catalyzed acetylation of
racemic (±)- cis,cis-decahydro-2-naphthol with vinyl acetate.
o
yielded (2S,4aR,8aS)-(-)-cis,cis-decahydro-2-naphthyl ace-
tate (33%) and recovered unreactive (2R,4aS,8aR)-(+)-
cis,cis-decahydro-2-naphthol (35%) and racemic (±)-
trans,cis-decahydro-2-naphthol (30%) (Fig. 2). The optical
purity of (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthol ([ꢀ]
ence of pyridine (5 mM) at 25 C for 24 h. The fluorine
chemical shifts at -73.982 and -74.237 ppm with the integra-
tion ratio of 89/11 were assigned to be the fluorine atoms of
(2R,4aS,8aR)- and (2S,4aR,8aS)- cis,cis-decahydro-2-naph-
thyl-(S)- ꢀ-methoxy-ꢀ-trifluoromethylphenyl acetates, re-
spectively (Fig. 3) [33, 34]. Therefore, the enantiomeric ex-
cess of (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naph-thol from
the kinetic resolution by lipase catalysis (Fig. 2) was calcu-
lated to be 78 % from integration of these two peaks (Fig. 3
and Table 1).
25 = +32.3 o) from this resolution was calculated to be 80%
D
25
o
([ꢀ]
= +40.4 from literature [31]. (2S,4aR,8aS)- (-)-
D
cis,cis-Decahydro-2-naphthol was obtained from the basic
hydrolysis (0.1 M KOH) of (2S)-(-)-cis,cis-decahydro-2-
naphthyl acetate in ethanol in 99% yield. The optical purity
of (2S,4aR,8aS)-(-)-cis,cis-decahydro-2-naphthol ([ꢀ] 25 = -
D
(2S,4aR,8aS)-(-)-cis,cis-Decahydro-2-naphthol (5 mM)
was condensed with the Mosher’s chiral derivatizing agent
(S)-(+)-ꢀ-methoxy-ꢀ-trifluoro-methylphenylacetyl chloride
[32] (5 mM) in CDCl3 in the presence of pyridine (5 mM) at
32.7o) from this resolution was calculated to be 81% ([ꢀ] D
25
= -40.4 o from literature [31].
The enantiomeric excess (e.e.) values of (2R,4aS,8aR)-
(+)- and (2S,4aR,8aS)-(-)- cis,cis-decahydro-2-naphthols
from the resolutions were calculated to be 78 and 80%, re-
spectively, from the 19F NMR spectra of their Mosher’s es-
ters (Fig. 3 and Table 1).
o
25 C for 24 h. The fluorine chemical shifts at -74.024 and -
74.279 ppm with the integration ratio of 10/90 were assigned
to be the fluorine atoms of (2R,4aS,8aR)- and (2S,4aR,8aS)-
cis,cis-decahydro-2-naphthyl-(S)-ꢀ-methoxy-ꢀ-trifluorome-
thylphenyl acetates, respectively. Therefore, the enanti-
omeric excess of (2S,4aR,8aS)-(-)-cis,cis- decahydro-2-naph-
thol from the kinetic resolution by lipase catalysis was calcu-
lated to be 80 % from integration of these two peaks (Fig. 3
and Table 1).
In a NMR tube, condensation reaction of (2R,4aS,8aR)-
(+)-cis,cis- decahydro-2-naphthol (5 mM) with the Mosher’s
chiral derivatizing agent (S)-(+)-ꢀ-methoxy-ꢀ-trifluorome-
thylphenylacetyl chloride [32] (5 mM) in CDCl3 in the pres-

Lipase Inhibition by Stereoisomers of decahydro-2-naphthyl-N-n-butylcarbamate
Protein & Peptide Letters, 2011, Vol. 18, No. 11 1171
Table 1. Enantiomeric Excess (%) and Optical Purity (%) for the Kinetic Resolution of Enantiomers of Cis,cis-decahydro-2-
Naphthanol (Fig. 1) by Lipase in Organic Solvent
Compound
Enantiomeric Excess (%)a
Optical Purity (%)b
(2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthol
(2S,4aR,8aS)-(-)-cis,cis-decahydro-2-naphthol
78
80
80
81
a Enantiomeric excess (%) was calculated from ratio of integration of fluorine chemical shifts of their Mosher’s ester derivatives of 19F NMR spectra.
25
25
bOptical purity (%) was calculated as 100 x [ꢄ] D observed / [ꢄ] D
.
literature
8
H
2
O
8
H
8a
O
2
NaH
THF
CF3
OH
4a
+
CF3
8a
OCH3
O
4a
Cl
4
OCH3
H
(2R,4aS,8aR)-cis,cis-decahydro-2-
naphthyl-(S)-a-methoxy-a-trifluoro
methylphenylacetate
4
(+)-(S)-a-methoxy-a-trifluoro
methylphenylacetyl chloride
H
(+)-(2R,4aS,8aR)-cis,cis-
decahydro-2-naphthol
H
8
H
8
2
OH
2
O
8a
8a
CF3
O
NaH
THF
4a
4a
+
CF3
OCH3
O
4
4
Cl
H
H
OCH3
(2S,4aR,8aS)-cis,cis-decahydro-2-naphthyl-
(S)-a-methoxy-a-trifluoro
methylphenylacetate
(+)-(S)-a-methoxy-a-trifluoro
methylphenylacetyl chloride
(-)-(2S,4aR,8aS)-cis,cis-
decahydro-2-naphthol
Figure 3. Determination of enantiomeric excess and absolute configuration of (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-
naphthols by 19NMR spectra of their Mosher’s ester derivatives. 19F NMR spectra after the reaction of with S-(+)-ꢄ-methoxy-ꢄ-trifluoro-
methylphenylacetyl chloride in the presence of pyridine in CDCl3. The peaks at -73.982 and -74.237 ppm were assigned to be the fluorine
chemical shifts of (2R,4aS,8aR)- and (2S,4aR,8aS)- cis,cis- (S)-ꢄ-methoxy-ꢄ-trifluoromethyl- phenylacetates, respectively.
(2R,4aS,8aR)-(+)- or (2S,4aR,8aS)-(-)-, or racemic (±)-
cis,cis-decahydro-2-naphthol H NMR (CDCl3) ꢀ0.80-1.80
butycarbamates (Fig. 1) were synthesized from condensation
of the corresponding alcohol with n-butyl isocyanate in the
presence of trethylamine in CH2Cl2 for 48h at 25 C (70-80
1
o
(m, 16H, 1,3-8,4a,8a-decahydro-2-naphthyl Hs), 3.03-3.09
(m, 1H, decahydro-2-naphthyl-C(2)H). 13C NMR (CDCl3)ꢀ
22.5 (C-4), 26.2, 26.4 (C-6 and C-7), 30.6, 30.9 (C-5 and C-
8), 32.0 (C-8a), 32.8 (C-3), 34.3, 34.7 (C-1 and C-4a), 74.0
(C-2). HRMS, exact mass: 154.1354; elemental analysis:
calculated for C10H18O: C, 77.87; H, 11.76, found C, 77.78;
H, 11.93.
yield).
Racemic
trans,cis-decahydro-2-naphthyl-N-n-butycar-
1
bamate H NMR (200 MHz, CDCl3) ꢀ 0.92 (t, J = 7.0 Hz,
3H, carbamate ꢁ-CH3), 1.20-2.00 (m, 20H, carbamate ꢂ- and
ꢃ-CH2 and decahydro-2-naphthyl Hs), 3.17 (dt, J = 6.2 and
6.8 Hz, 2H, carbamate ꢄ-CH2), 3.85-3.90 (m, 1H, decahy-
dro-2-naphthyl C(2)-H), 4.53 (br. s, 1H, carbamate NH). 13C
NMR (50.3 MHz, CDCl3) ꢀ 13.7 (carbamate ꢁ-CH3), 19.9
(carbamate ꢂ-CH2), 26.5, 22.6, 28.2, 30.3, 32.1, 33.7 (de-
cahydro-2-naphthyl C-3 to C-8), 33.7 (carbamate ꢃ-CH2),
37.2 (decahydro-2-naphthyl C-9), 37.6 (decahydro-2-naph-
thyl C-1), 40.6 (decahydro-2-naphthyl C-10), 42.7 (carbama-
te ꢄ-CH2), 70.4 (decahydro-2-naphthyl C-2), 156.4 (carba-
mate C=O). HRMS, exact mass: 239.1889; elemental analy-
sis: calculated for C14H25NO2: C, 70.25; H, 10.53, found C,
70.19; H, 10.61.
(2S,4aR,8aS)-cis,cis-Decahydro-2-naphthylacetate
1H
NMR (CDCl3) ꢀ0.80-1.80 (m, 16H, 1,3-8,4a,8a-decahydro-
2-naphthyl Hs), 2.00 (s, 3H, acetyl methyl), 3.87-3.92 (m,
1H, decahydro-2-naphthyl-C(2)H). 13C NMR (CDCl3)ꢀ 17.7
(acetyl methyl), 22.6 (C-4), 26.3, 26.4 (C-6 and C-7), 30.5,
30.8 (C-5 and C-8), 32.0 (C-8a), 32.7 (C-3), 34.2, 34.7 (C-1
and C-4a), 70.1 (C-2), 170.9 (acetyl C=O). HRMS, exact
mass: 196.1460; elemental analysis: calculated for C12H20O2:
C, 73.43; H, 10.27, found C, 73.31; H, 10.34.
Synthesis of racemic trans,cis-, (2R,4aS,8aR)-cis,cis-,
and (2S,4aR,8aS)- -cis,cis-decahydro-2-naphthyl-N-n-buty-
carbamates.
(2R,4aS,8aR)-, (2S,4aR,8aS)-, or racemic cis,cis-decahyd-
ro-2-naphthyl- N-n-butycarbamate 1H NMR (200 MHz,
CDCl3) ꢀ 0.92 (t, J = 7 Hz, 3H, carbamate ꢁ-CH3), 1.20-2.00
(m, 20H, carbamate ꢂ- and ꢃ-CH2 and decahydro-2-naphthyl
Racemic trans,cis-, (2R,4aS,8aR)-cis,cis-, (2S,4aR,8aS)-
cis,cis-, and racemic cis,cis-decahydro-2-naphthyl-N-n-

1172 Protein & Peptide Letters, 2011, Vol. 18, No. 11
Lin et al.
Hs), 3.15 (dt, J = 6.2 and 6.8 Hz, 2H, carbamate ꢀ-CH2),
4.53-4.59 (m, 1H, decahydro-2-naphthyl C(2)-H), 4.84 (br. s,
1H, carbamate NH). 13C NMR (50.3 MHz, CDCl3) ꢁ 13.6
(carbamate ꢂ-CH3), 19.7 (carbamate ꢃ-CH2), 26.4, 31.3,
33.0, 33.6, 34.4, 35.2 (decahydro-2-naphthyl C-3 to C-8),
32.0 (carbamate ꢄ-CH2), 38.5 (decahydro-2-naphthyl C-1),
40.6 (decahydro-2-naphthyl C-9), 40.9 (carbamate ꢀ-CH2),
42.2 (decahydro-2-naphthyl C-10), 73.2 (decahydro-2-
naphthyl C-2), 156.3 (carbamate C=O). HRMS, exact mass:
239.1890; elemental analysis: calculated for C14H25NO2: C,
70.25; H, 10.53, found C, 70.20; H, 10.59.
Cytotoxicity Test In Vitro
For this experiment in 96-well white plate, 10000 MDCK
cells were seeded per well and incubated for 24 hours at 37
oC. After 24 hours, MDCK cells were incubated with me-
dium containing 0.04 % (weight) inhibitors or drugs. The
number of metabolically viable cells was determined by the
Celltiter-GloTM luminescent cell viability assay (Promega)
after 48 hours. MDCK cells were grown in Dulbecco's Modi-
fied Eagle Medium (DMEM) containing 10% fetal bovine
serum (FBS). Cells were maintained at 37 °C and 5% CO2.
Data Reduction
RESULTS AND DISCUSSION
Origin (version 6.0) was used for linear and nonlinear
least-squares curve fittings.
Optically pure (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)-
cis,cis- decahydro-2-naphthols are resolved from the porcine
pancreatic lipase (PPL)-catalyzed acetylation of decahydro-
2-naphthols with vinyl acetate in t-butyl methyl ether (Fig.
2). The enantiomeric excess (e.e.) values of (2S,4aR,8aS)-(-)-
and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthols from
this resolution are calculated to be 78 and 80%, respectively,
from the 19F NMR spectra of their Mosher’s ester derivatives
(Fig. 3 and Table 1) [32-34]. Superimposition of (2S,4aR,
8aS)-(-)- and (2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naph-
thols into the active site of acetyl enzyme suggests that the
decahydro-2-naphthyl ring of (2S,4aR,8aS)-(-)- cis,cis-
decahydro- 2-naphthol is fit well into the leaving group bind-
ing site of the enzyme and the hydroxyl group of
(2S,4aR,8aS)-(-)- cis,cis-decahydro-2-naphthol is at the right
position to attack the carbonyl carbon of acetyl enzyme (Fig.
4). On the other hand, the decahydro-2-naphthyl ring of
(2R,4aS,8aR)-(+)-cis,cis-decahydro- 2-naphthol is not fit
well into the leaving group binding site of the enzyme, ex-
tending to the entrance (mouth) of the active site. Thus, the
hydroxyl group of (2R,4aS,8aR)-(+)-cis,cis- decahydro-2-
naphthol is away from the right position to attack the acetyl
enzyme.
Lipase Inhibition
Lipase inhibition reactions were determined as described
by Hosie et asl. [17-30]. PSL-or PPL-catalyzed hydrolysis of
PNPB in the presence of a carbamate inhibitor was followed
continuously at 410 nm on the UV-visible spectrometer. The
o
temperature was maintained at 25.0 C by a refrigerated cir-
culating water bath. All reactions were preformed in sodium
phosphate buffer (1 mL, 0.1 M, pH 7.0) containing NaCl
(0.1 M), CH3CN (2% by volume), detergent triton-X 100
(TX) (0.5% by weight), substrate PNPB (0.1 mM), and vary-
ing concentration of the inhibitors. Requisite volumes of
stock solution of substrate PNPB and the inhibitor in aceto-
nitrile were injected into reaction buffer via a pipet. PSL or
PPL was dissolved in sodium phosphate buffer (0.1 M, pH
7.0). Carbamate inhibitors racemic trans,cis-, (2R,4aS,8aR)-
cis,cis-, (2S,4aR,8aS)- -cis,cis-, and racemic cis,cis-decahyd-
ro-2-naphthyl-N-n-butycarbamates were characterized as the
pseudo or alternate substrate inhibitors of PSL and PPL
(Scheme I and Equation 1) [17-30]. The carbamylation stage
was rapid compared to subsequent decarbamylation (k2>>k3),
thus the two steps are easily resolved kinetically. The appar-
ent inhibition constant (1+[S]/Km) Ki and carbamylation con-
stant (k2) were obtained from the nonlinear least-squares
curve fitting of the kapp vs. [I] plot against Eq. (1) (Fig. 5).
The inhibition constant Ki was then calculated form the ap-
parent inhibition constant when both [S] and Km values for
the PSL-catalyzed hydrolysis of PNPB were known (Table
2). The Km value for the PSL catalyzed hydrolysis of PNPB
was 100±20 μM obtained from Michaelis-Menten equation.
The bimolecular rate constant, ki = k2/Ki, was related to over-
all inhibitory potency.
Optically pure (2S,4aR,8aR)- and (2R,4aS,8aS)-(+)-
trans,cis- decahydro-2-naphthols can not be resolved from
the porcine pancreatic lipase-catalyzed acetylation of deca-
hydro-2-naphthols with vinyl acetate in t-butyl methyl ether.
Therefore, the PSL inhibition by trans,cis-decahydro-2-
naphthyl-N-n- butylcarbamate (Fig. 1) is studied in its race-
mate form.
(2S,4aR,8aS)-Cis,cis-, (2R,4aS,8aR)-cis,cis-, racemic
cis,cis-, and racemic trans,cis-decahydro-2-naphthyl-N-n-
butylcarbamates (Fig. 1) are all characterized as pseudo sub-
strate inhibitors of PSL (Scheme I, Equation 1, Fig. 5, and
Table 2) [17-30]. Among all carbamate inhibitors in this
study, (2R,4aS,8aR)-cis,cis- decahydro-2-naphthyl-N-n-
butyl-carbamate is the most potent inhibitor of PSL (Table
2). Superimposition of (2R,4aS,8aR)-cis,cis-, (2S,4aR,8aR)-
cis,cis-, (2S,4aR,8aR)- trans,cis-, and (2R,4aS,8aS)-trans,cis-
decahydro-2-naphthyl-N-n-butylcarbamates and triacylglyc-
erol into the active site of PSL is modeled (Fig. 6). The sn-1
or sn-3 acyl chain of triacylglycerol and the carbamate moie-
ties of the inhibitors are fit into the acyl chain binding site
(ACS) of the enzyme [5]. The sn-1 or sn-3 acyl carbonyl
oxygen atom of the triacylglycerol and the carbamate
kapp = k2 [I] / (Ki (1 + [S] / Km)o + [I] )
(1)
Duplicate sets of data were collected for each inhibitor
concentration.
Molecular Modeling
Molecular structures shown in Figs. 4 and 6 were de-
picted from the molecular structures after MM-2 energy
minimization (minimum root mean square gradient was set
to be 0.01) by CS Chem 3D (version 6.0).

Lipase Inhibition by Stereoisomers of decahydro-2-naphthyl-N-n-butylcarbamate
Protein & Peptide Letters, 2011, Vol. 18, No. 11 1173
oxyanion hole
entrance (mouth)
O
H
O
H
(2R,4aS,8aR)-(+)-cis,cis-decahydro-2-naphthol
(2S,4aR,8aS)-(-)-cis,cis-decahydro-2-naphthol
O
acyl binding site
O
Ser
catalytic
triad
leaving group binding site
Figure 4. Superimposition of (2S,4aR,8aS)-(-)- and (2R,4aS,8aR)-(+)- cis,cis-decahydro-2-naphthols into the active site of acetyl PPL. The
decahydro-2-naphthyl ring of (2S,4aR,8aS)-(-)-cis,cis-decahydro-2-naphthol fits well into the leaving group binding site of lipase and the
hydroxyl group of (2S,4aR,8aS)-(-)- cis,cis-decahydro-2-naphthol is at the right position to attack the acetyl enzyme. On the other hand,
(2R,4aS,8aR)-(+)-cis,cis- decahydro-2-naphthol does not fit well into the leaving group binding site of lipase, extending to the entrance
(mouth) of the active site. Thus, the hydroxyl group of (2R,4aS,8aR)-(+)-cis,cis- decahydro-2-naphthol is away from the position to attack the
acetyl enzyme.
Table 2. The k2, Ki and ki Vlauesa of the PSL Inhibitions by Stereoisomers of Decahydro-2-naphthyl-N-n-butylcarbamates
Inhibitorsb
Ki (μM)
k2 (10-3s-1)
ki (103 M-1s-1)
Enantioselectivity
(2R,4aS,8aR)-cis,cis-
(2S,4aR,8aS)-cis,cis-
rac-(±)-cis,cis-
1.3±0.2
4.2±0.3
2.5±0.2
4.0±0.4
0.43±0.02
0.40±0.02
0.42±0.02
0.60±0.03
3.3±0.5
0.95±0.07
1.7±0.1
3.5
1.0
1.8
-
rac-(±)-trans,cis
1.5±0.2
a Obtained from the nonlinear least-squares curve fittings of kapp vs. [I] plot against Eq. (1).
b
(2R,4aS,8aR)- cis,cis-, (2S,4aR,8aS)-cis,cis-, and rac-(±)-cis,cis- stand for (2R,4aS,8aR)-, (2S,4aR,8aS)-, and racemic cis,cis-decahydro-2-naphthyl-N-n- butylcarbamates. Rac-(±)-
trans,cis stand for (2R,4aS,8aS)- and (2S,4aR,8aR)-trans,cis-decahydro-2-naphthyl-N-n- butylcarbamates.
0.0005
0.0005
0.0004
0.0004
0.0003
0.0003
0.0002
0.0002
0.0001
0.0000
0.0001
0.0000
0.0
0.1
0.2
0.3
0.4
0.5
0.0
0.1
0.2
0.3
0.4
0.5
[I] (mM)
[I] (mM)
A
B
Figure 5. Nonlinear least-squares curve fittings of kapp vs. inhibitor concentration ([I]) plot against Eq. (1) for the pseudo-substrate inhibition
of PSL by (A) (2R,4aS,8aR)- cis,cis-decahydro-2-naphthyl-N-n-butylcaarbamate and (B) (2S,4aR,8aS)- cis,cis-decahydro-2-naphthyl-N-n-
butylcaarbamate. The parameters of the fit were (A) k2 = 0.00043 ± 0.00002 s-1 , Ki = 1.3 ±0.2 μM, and R= 0.98064 and (B) k2 = 0.00040±
0.0003 s-1 , Ki =4.2 ± 0.3 μM, and R= 0.97847.
carbonyl oxygen atom are fit into the oxyanion hole of the
enzyme [5-7]. The sn-1 or sn-3 acyl carbonyl carbon atom of
the triacylglycerol and the carbamate carbonyl carbon atom
are fit into the correct position for Ser87 of the catalytic triad
of the enzyme to attack [5-7].
For stereospecificity of the PSL inhibition, (2R,4aS,8aR)-
cis,cis-decahydro-2- naphthyl-N-n-butylcarbamate is 3.5
times more potent than (2S,4aR,8aS)-cis,cis- decahydro-2-
naphthyl-N-n-butylcarbamate (Table 2). Superimposition of
both enantiomers of cis,cis-decahydro-2-naphthyl-N-n-

1174 Protein & Peptide Letters, 2011, Vol. 18, No. 11
Lin et al.
water lipid interface
entrance (mouth)
oxyanion
hole
T251
A247
least potent inhibitor
acyl chain binding site
Q88
L17
third acyl chain
binding site
(2S,4aR,8aS)-cis, cis
O
O
O
O
TG
O
N
N
O
N
OO
O
H
O
H
H
H
(2S,4aR,8aR)-trans ,cis-inhibitor
(2R,4aS,8aS)-trans, cis-inhibitor
O
OH
D264
H286
S87
(2R,4aS,8aR)-cis,cis
most potent inhibitor
catalytic triad
T18
Y23
L287
L293
second acyl chain
binding site
Figure 6. Superimposition of (2S,4aR,8aS)-cis,cis-, (2R,4aS,8aR)-cis,cis- , (2S,4aR,8aR)-trans,cis-, (2R,4aS,8aS)-trans,cis-decahydro-2-
naphthyl- N-n-butylcarbamates and triacylglycerol into the active site of PSL [5]. The decahydro-2-naphthyl ring of (2S,4aR,8aS)-cis,cis-
isomer unfavorably interacts with the oxyanion hole of the enzyme, but that of (2R,4aS,8aR)-cis,cis-isomer does not. Furthermore, the deca-
hydro-2-naphthyl ring of (2R,4aS,8aR)-cis,cis-isomer may bind well into the second acyl chain of the enzyme.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
control
orlistat atazanavir exelon 2S-cis,cis 2R-cis,cis rac-cis,cisrac-trans,cis
--
Figure 7. MCDK cell viability activity [relative light unit (RLU)] for Olistat, Atazanavir, Exelon, and (2S,4aR,8aS)-cis,cis-, (2R,4aS,8aR)-
cis,cis-, racemic-cis,cis-, and racemic-trans,cis-decahydro-2-naphthyl- N-n-butylcarbamates determined by the Celltiter-GloTM luminescent
cell viability assay (Promega).
butylcarbamate into the active site of PSL indicates that the
decahydro-2-naphthyl rings of (2S,4aR,8aS)-cis,cis-isomer is
strongly repulsive with the oxyanion hole of PSL [5] sug-
gesting that this unfavorable interaction makes (2S,4aR,8aS)-
cis,cis-isomer become the least potent inhibitor (Fig. 6). On
the other hand, the decahydro-2-naphthyl rings of (2R,4aS,
8aR)-cis,cis-isomer does not have this unfavorable repul-
sions and may interact well with the second acyl chain bind-
ing site (SACS) of the enzyme [5].
The inhibitory potency of rac-trans,cis-isomer is between
those of (2S,4aR,8aS)-cis,cis- and (2R,4aS,8aR)-cis,cis-
isomers (Table 2). Superimposition of (2S,4aR,8aR)-trans,
cis- and (2R,4aS,8aS)-trans,cis-isomers into the active site of

Lipase Inhibition by Stereoisomers of decahydro-2-naphthyl-N-n-butylcarbamate
Protein & Peptide Letters, 2011, Vol. 18, No. 11 1175
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[13]
These inhibitors also show similar effects on porcine
pancreatic lipase and bovine bile salt activated lipase (or
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In summary, optically pure (2S,4aR,8aS)-(-)- and (2R,
4aS,8aR)-(+)-cis,cis- decahydro-2-naphthols are resolved
from the lipase-catalyzed acetylation reaction. For PSL inhi-
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(2S,4aR,8aS)-cis,cis-decahydro-2-naphthyl-N-n-
butylcarbamate is 3.5 times less potent than (2R,4aS,8aR)-
cis,cis-decahydro-2-naphthyl-N-n- butylcarbamate due to the
unfavorable repulsion between the former inhibitor and the
enzyme.
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Received: March 22, 2011
Revised: May 2, 2011
Accepted: May 2, 2011