Selective biocatalytic aminolysis of (±)-epichlorohydrin: Synthesis and ICAM-1 inhibitory activity of (S)-(+)-3-arylamino-1-chloropropan-2-ols

Article abstract of DOI:10.1016/j.bmc.2011.02.029

Regio- and enantioselective synthesis of (S)-(+)-3-arylamino-1- chloropropan-2-ols has been achieved by the epoxide ring opening of (±)-epichlorohydrin with different aromatic amines in the presence of Candida rugosa lipase. Activities of seven model (S)-

Full text of DOI:10.1016/j.bmc.2011.02.029

Bioorganic & Medicinal Chemistry 19 (2011) 2263–2268
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective biocatalytic aminolysis of ( )-epichlorohydrin: Synthesis and ICAM-1
inhibitory activity of (S)-(+)-3-arylamino-1-chloropropan-2-ols
Pankaj Gupta a, Sumati Bhatia a, Ashish Dhawan a, Sakshi Balwani b, Shatrughan Sharma a, Raju Brahma a,c
,
Rajpal Singh c, Balaram Ghosh b, Virinder S. Parmar a, Ashok K. Prasad a,
⇑
a Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
b Molecular Immunogenetics Laboratory, Institute of Genomics & Integrative Biology, Mall Road, Delhi 110 007, India
c CFEES, Brig. SK Mazumdar Road, Timarpur 110 054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Regio- and enantioselective synthesis of (S)-(+)-3-arylamino-1-chloropropan-2-ols has been achieved by
the epoxide ring opening of ( )-epichlorohydrin with different aromatic amines in the presence of Can-
dida rugosa lipase. Activities of seven model (S)-(+)-3-arylamino-1-chloropropan-2-ols, out of 10 com-
Received 21 December 2010
Revised 15 February 2011
Accepted 16 February 2011
Available online 24 February 2011
pounds synthesized, have been evaluated for the inhibition of tumor necrosis factor-
a TNF-a) induced
expression of intercellular adhesion molecule-1 (ICAM-1), which is one of the factors responsible for
the modulation of inflammation in biological systems; (S)-(+)-1-chloro-3-(20-chlorophenylamino)-pro-
Keywords:
b-Amino alcohol
(S)-(+)-3-Arylamino-1-chloropropan-2-ols
Candida rugosa lipase
Regioselective
pan-2-ol has been found to exhibit highest activity, that is, 86% inhibition of TNF-
of ICAM-1 at a concentration of 40 g/ml.
a induced expression
l
Ó 2011 Elsevier Ltd. All rights reserved.
Enantioselective
TNF-a
ICAM-1
Anti-inflammatory
1. Introduction
maintaining the body fluidity. A promising therapeutic approach
for various inflammatory responses is to inhibit the cytokine in-
duced expression of cell adhesion molecules.4 Although, various
natural products, synthetic drugs, antibodies and peptides have
been demonstrated to inhibit the expression of these molecules,
they have their own limitations for usage because of side effects.5
b-Amino alcohols are versatile intermediates in the synthesis of
a vast range of biologically active natural and synthetic products,6
unnatural amino acids,7 and chiral auxiliaries for asymmetric syn-
thesis.8 b-Amino alcohols, like 3-arylamino-1-chloropropan-2-ols
have been found as useful intermediates for various drugs. Some
of the b-amino alcohols are important antihypertensive drugs too.
However compounds of this class have never been tested for inhibi-
tion of cell adhesion molecules implicated in inflammation.9,10
The nucleophilic opening of epoxide ring in epichlorohydrin
with amines constitutes a well recognized route for the synthesis
of b-amino alcohols.11 The classical approach,12 involving heating
of epoxides with amines, works less well with poorly nucleophilic
amines. Moreover, the lack of appreciable regioselectivity and the
need for an excess of amines in the classical methods of b-amino
alcohol synthesis have led to the necessity for activation of the
epoxides so as to increase their susceptibility to nucleophilic attack
by amines.13–15 However, many of these methods often involve the
use of expensive and stoichiometric amounts of reagents, where
Inflammation is caused by soluble antigen, live organisms and
chemical or mechanical stress upon tissue, which serves to destroy
and/or dilute the injurious materials, and remove the injured tis-
sues. It is characterized pathologically by an increased supply of
blood to the affected area, increased capillary permeability caused
by retraction of the endothelial cells and infiltration of phagocytic,
monocytic and polymorphonuclear cells into the site of tissue in-
sult. The accumulation and subsequent activation of leukocytes is
one of the central events in the pathogenesis of all forms of inflam-
mation. The migration of the leukocytes to the site of inflammation
is regulated in part by the expression of cell adhesion molecules,
such as ICAM-1 and E-selectin.1 These cell-adhesion molecules
are induced on endothelial cells by various pro-inflammatory cyto-
kines like IL-1a, and TNF-a
and also by bacterial LPS.2 The in-
creased expression of cell adhesion molecules on the endothelial
cells alters the adhesive property of the vasculature leading to
indiscriminate infiltration of the leukocytes across the blood ves-
sels and hence causes inflammation.3 A critically regulated expres-
sion of these adhesion molecules is therefore highly essential for
⇑
Corresponding author. Tel.: +91 11 27662486.
E-mail address: ashokenzyme@yahoo.com (A.K. Prasad).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.029

2264
P. Gupta et al. / Bioorg. Med. Chem. 19 (2011) 2263–2268
poor regioselectivity especially with metal amides derived from
primary amines, extended reaction times and undesirable side
reactions such as rearrangement of the oxiranes to allyl alcohols
under basic conditions16 or polymerization in strongly acidic con-
ditions often result in low yields of the desired products.
catalyzed epoxide ring opening of ( )-epichlorohydrin with appre-
ciable rate and almost exclusive selectivity leading to the forma-
tion of only one product on TLC.
In a typical reaction, equimolar mixture of racemic epichlorohy-
drin 1 and the aromatic amine 2a–2j was incubated with Candida
rugosa lipase in diisopropyl ether at 40–42 °C in an incubator sha-
ker and the reaction was monitored by analytical TLC. After 50%
consumption of the starting amine in 8–12 h, reaction became
too slow to complete even in one week of continued incubation.
Thus, the reaction was stopped by filtering off the enzyme after
about 45–50% conversion of the starting amine to a single product
in 8–12 h and solvent removed under reduced pressure to obtain
the crude product, which was passed through a silica gel column
to get the pure products 3a–3j in 72–90% yields (based on the
amount of the particular enantiomer in the racemic epichlorohy-
drin) on elution with 10–15% ethyl acetate in petroleum ether
(Scheme 1). The unreacted starting epichlorohydrin was not recov-
ered from the column.
The products of the CRL-catalyzed reaction of ( )-epichlorohy-
drin 1 with aromatic amines 2a–2j were unambiguously identified
as 3-arylamino-1-chloropropan-2-ols 3a–3j on the basis of their
spectral (IR, 1H NMR, 13C NMR and HRMS) analysis and comparison
of the spectral data with that of the known compounds 3a–3d and
3f–3j with those reported in the literature.20–27 It is worth men-
tioning here that the compounds reported in the literature are
racemic mixtures. The secondary nature of hydroxyl group in ami-
noalcohols 3a–3j was further confirmed by acetylation of one of
the compounds, 1-chloro-3-(20-methoxyphenylamino)-propan-2-
ol (3c) and comparison of the chemical shift value of the C-2H in
the 1H NMR spectrum of alcohol 3c with the chemical shift value
of the C-2H in the 1H NMR spectrum of the resultant acetate 4
(Scheme 2). In the 1H NMR spectrum of 3c, the C-2H resonated
at d 3.88, however the same proton in the corresponding acetate
4 resonated at d 5.13. The chemical shift values of other protons
in the 1H NMR spectra of the alcohol 3c and the acetate 4 remained
almost the same. The shift of d 1.25 ppm in the chemical shift
value of the C-2H of acetate with respect to the alcohol indicates
that the hydroxyl group in 3c is at the C-2 and thus secondary in
nature. This was further confirmed by the observation of a similar
Chirality is an important element of biological systems because
most of the biomolecules are chiral and therefore they recognize
the symmetry present in the foreign molecules. It is therefore
important to develop enantioselective synthetic methodologies
for the synthesis of drugs, drug like molecules or their precursors
and intermediates. In view of the biological importance of b-amino
alcohols and that the chirality plays an important role in biological
systems, development of simple, efficient and environmentally be-
nign processes for the synthesis of enantiomerically pure b-amino
alcohols is highly required. In continuation of our work on lipase
catalysis in organic synthesis,17–19 we report herein efficient syn-
thesis of (S)-(+)-3-arylamino-1-chloropropan-2-ols by highly re-
gio- and enantioselective ring opening of ( )-epichlorohydrin in
the presence of Candida rugosa lipase (CRL) in diisopropyl ether. Li-
pase-catalyzed reactions offer an attractive route for industrial
exploitation due to their ability to work in both aqueous & organic
solvents, low cost and high stability. Most of the chiral aminoalco-
hols synthesized have been examined for their ICAM-1 expression
inhibitory activity.
2. Results and discussion
2.1. Chemistry
Four different lipases, that is, Novozyme 435 (Candida antarctica
lipase immobilized on polyacrylate), Amano PS (Pseudomonas spe-
cies lipase), Porcine pancreatic lipase (PPL) and C. rugosa lipase
(CRL) were screened for regio- and enantioselective epoxide ring
opening of ( )-epichlorohydrin with different aromatic amines
2a–2j in different organic solvents. It was observed that Novozyme
435, Amano PS and PPL do not catalyze the reaction or if there is
any reaction, it was non-selective leading to the formation of two
products due to epoxide ring opening in epichlorohydrin from
either of the two sides. C. rugosa lipase (CRL) in diisopropyl ether
OH
OH
2
R-NH2
2a-2j
H
N
H
N
H
N
R-NH2
O
Cl
Cl
+
R
Cl
Cl
R
R
CRL
DIPE, 40-42 o
C
1
3
DIPE, 40-42 o
C
OH
( )-5a-5c
( )-3a-3c
( )-1
(+)-3a-3j
Compd.
2, 3 & 5
a
b
c
d
e
OMe
OMe
R
MeO
OMe
CH3
f
g
h
i
j
Cl
R
Cl
Cl
Br
Scheme 1.

P. Gupta et al. / Bioorg. Med. Chem. 19 (2011) 2263–2268
2265
O
1
OMe
OH
2
OMe
5'
O
2
CH3
Cl
H
N
H
N
DCM, Ac2O, DMAP
Cl
3'
1'
1
3
3
(+)-3c
4
Scheme 2.
downfield shift in the chemical shift value of the C-2 of the
acetate 4 with respect to that of the alcohol 3c in their 13C NMR
spectra. The formation of the mono O-acetylated compound 4 over
N-acetylated compound or the diacetate during acetylation of
compound 3c may be because of the secondary nature of the amino
function, which is also attached to the 2-methoxyphenyl ring that
exert inductive and resonance effects together with steric effects,
thereby reduces the nucleophilicity of –NH group present in the
compound.
5a–5c obtained by epoxide ring opening of ( )-epichlorohydrin 1
with amines 2a–2c in diisopropyl ether in the absence of lipase
CRL did not show any optical activity. Further, there was no reac-
tion between ( )-epichlorohydrin and 4-nitroaniline, 1,2-diamino-
benzene or aliphatic amines when incubated with the enzyme
under identical conditions. This may be due to the weaker nucleo-
philicity of these amines.
2.2. Antiinflammatory activity
This indicated that CRL-catalyzed epoxide ring opening of ( )-
epichlorohydrin with different aromatic amines is completely reg-
ioselective due to the attack of the amine nucleophile only on the
less hindered C-atom of the epoxide resulting in the formation of
3-arylamino-1-chloropropan-2-ols 3a–3j (Scheme 1). An attempt
on epoxide ring opening of ( )-epichlorohydrin with aniline (2a),
4-methylaniline (2b) and 2-methoxyaniline (2c) in diisopropyl
ether at 40–42 °C in the absence of lipase led to the formation of
two products, that is, 3-arylamino-1-chloropropan-2-ols 3a–3c in
45–68% yields and 2-arylamino-3-chloropropanols 5a–5c in 17 to
30% yields in 35–40 h due to the attack of the nucleophile at both
the two C-ends of the epoxide (Scheme 1). The major product
formed in the chemical reaction was due to the attack of the nucle-
ophile on less hindered C-atom of the epoxide as expected, but the
reaction was very slow and non-selective.
Ten 3-(arylamino)-1-chloro-propan-2-ols 3a–3j have been syn-
thesized, out of which seven b-aminoalcohols, that is, 3a, 3b, 3e, 3f,
3g, 3h and 3i were evaluated for their anti-inflammatory activity
using primary human umbilical vein endothelial cells. The inhibi-
tory activities of the aminoalcohols 3a, 3b and 3e–3i was evaluated
on the TNF-a tumor necrosis factor-a induced expression of cell
adhesion molecules, viz. ICAM-1 (intracellular adhesion mole-
cule-1) and E-selectin that play key roles in controlling various
inflammatory diseases. The results of anti-inflammatory activities
of b-amino alcohols are summarized in Table 2.
1-Chloro-3-(phenylamino)propan-2-ol (3a) shows an IC50 va-
lue of 39.8
mum tolerable dose of 30
l
g/ml with 38% of ICAM-1 inhibition at the maxi-
g/ml. Methyl group substitution at
l
the para position in the parent structure 1-chloro-3-(phenylami-
Further, evaluation of optical rotation values of 3-arylamino-1-
chloropropan-2-ols 3a–3j revealed that all the ten compounds
obtained by CRL-catalyzed epoxide ring opening of the ( )-epichlo-
rohydrin with different aromatic amines 2a–2j are optically active
and dextrorotatory. Thus the lipase-catalyzed epoxide ring opening
of ( )-epichlorohydrin is enantioselective also, together with regio-
selective (Table 1). The enantiomeric excess (ee) values of (+)-3-
arylamino-1-chloropropan-2-ol 3a–3j obtained by CRL-catalyzed
epoxide ring opening of ( )-epichlorohydrin (1) were determined
by calculations using specific rotation values of optically pure
(S)-(+)-b-amino alcohols 3a–3j prepared by the epoxide ring open-
ing of optically pure (S)-(+)-epichlorohydrin with the amines 2a–2j
and found to be in the range of 55–92% (Table 1). 3-Arylamino-1-
chloropropan-2-ols 3a–3c and 2-arylamino-3-chloropropanols
no)propan-2-ol (3a), that is, compound 3b shows an IC50 value
of 28.84
lg/ml with 81.5% ICAM-1 inhibition at the maximum
tolerant dose of 40
l
g/ml indicating that methyl substitution at
p-position results in the increase in inhibitory activity of the par-
ent compound. 1-Chloro-3-(4-bromophenylamino)propan-2-ol
(3f) shows an IC50 value of 31.62
tion at the maximum tolerant dose of 40
l
g/ml with 76% ICAM-1 inhibi-
g/ml indicating that
l
this substitution too results in increased inhibitory activity of
the parent compound 1-chloro-3-(phenylamino)propan-2-ol
(3a). 1-Chloro-3-(20,50-dimethoxyphenylamino)propan-2-ol (3e)
shows no significant inhibition of ICAM-1 activity at the maxi-
mum tolerant dose of 30 lg/ml indicating that the disubstitution
results in significant loss of inhibitory activity of the parent
compound.
Table 1
Regio- and enantioselective lipase-catalyzed epoxide ring opening of ( )-epichlorohydrin (1) with aromatic amines 2a–2j in diisopropyl ether (DIPE) in the presence of Candida
rugosa lipase at 40–42 °C
a 2D3
ꢀ
of product
½ ꢀ of product obtained
a 2D3
Amine 2a–2j
Product: (S)-(+)-3a–3j
Reaction
time (h)
Yielda (%)
ee (%)
½
from (S)-(+)-epichlorohydrin
2a
2b
2c
2d
2e
2f
2g
2h
2i
(S)-(+)-3a
(S)-(+)-3b
(S)-(+)-3c
(S)-(+)-3d
(S)-(+)-3e
(S)-(+)-3f
(S)-(+)-3g
(S)-(+)-3h
(S)-(+)-3i
(S)-(+)-3j
12
8
72
80
88
86
84
86
90
70
76
88
+5.6
+7.5
+23.0
+15.0
+11.7
+5.0
+22.3
+5.5
+6.2
75
73
92
89
75
90
55
80
82
76
+16.7
+13.7
+10.5
+3.7
+20.0
+3.0
+5.0
+11.2
+20.0
11
11
12
12
12
12
12
8
+13.7
+25.6
2j
a
Yields were calculated by assuming corresponding single enantiomer as 100% in the starting ( )-epichlorohydrin (1).

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P. Gupta et al. / Bioorg. Med. Chem. 19 (2011) 2263–2268
Table 2
TNF-
a induced ICAM-1 inhibitory activity of 3-arylamino-1-chloropropan-2-ols 3a, 3b and 3e–3i
Compound
Maximal tolerable
dose (MTD, lg/ml)
% Inhibition
at MTD
IC50 (l
g/ml)a
(S)-(+)-1-Chloro-3-phenylaminopropan-2-ol (3a)
30
40
30
40
40
30
30
38
81
40
76
86
24
60
39.8
28.8
—
31.6
33.1
—
(S)-(+)-1-Chloro-3-(40-methylphenylamino)-prop-an-2-ol (3b)
(S)-(+)-1-Chloro-3-(20,50-dimethoxyphenylamino)-propan-2-ol (3e)
(S)-(+)-1-Chloro-3-(40-bromophenylamino)-prop-an-2-ol (3f)
(S)-(+)-1-Chloro-3-(20-chlorophenylamino)-propan-2-ol (3g)
(S)-(+)-1-Chloro-3-(30-chlorophenylamino)-propan-2-ol (3h)
(S)-(+)-1-Chloro-3-(40-chlorophenylamino)-propan-2-ol (3i)
28.8
a
‘—’ Denotes no inhibition.
1-Chloro-3-(20-chlorophenylamino)propan-2-ol (3g) shows an
tively; C. rugosa lipase (CRL) and Porcine pancreatic lipase (PPL)
were purchased from Sigma Chemical Co. (USA). These lipases
were used after storing in vacuo over P2O5 for 24 h.
IC50 of 33.1
lg/ml with 86% ICAM-1 inhibition at the maximum tol-
erant dose of 40
lg/ml indicating that the ortho chloro substituents
increases inhibitory activity of the parent compound substantially.
1-Chloro-3-(30-chlorophenylamino)propan-2-ol (3h) shows no sig-
nificant inhibition of ICAM-1 activity at the maximum tolerant
4.1.1. General procedure for CRL-catalyzed epoxide ring
opening of ( )-epichlorohydrin (1)
dose of 30
pan-2-ol (3i) shows an IC50 of 28.8
tion at the maximum tolerant dose of 30
l
g/ml, while 1-chloro-3-(40-chlorophenylamino)pro-
To a solution of the aromatic amine (2a–2j, 10.0 mmol) and ( )-
epichlorohydrin (1, 10.0 mmol) in diisopropyl ether (50 ml), C. rug-
osa lipase (half of the amount of amine w/w) was added and the
resulting suspension was stirred at 40–42 °C for 8–12 h. The pro-
gress of the reaction was monitored periodically by TLC and at
45–50% completion, the reaction was quenched by filtering off
the enzyme. The solvent was removed under reduced pressure,
and the residue thus obtained was purified by column chromatog-
raphy on silica gel using petroleum ether–ethyl acetate (93:7 to
82:18) to obtain the pure products, (S)-(+)-3-arylamino-1-chloro-
propan-2-ols 3a–3j in 70–90% yields. Compounds 3a–3j were
unambiguously identified on the basis of their spectral (IR, 1H
NMR, 13C NMR and HRMS/EIMS) analysis and comparison of the
physical and the spectral data of the known compounds 3a–3d
and 3f–3j with those reported in the literature.20–27
l
g/ml with 60% ICAM-1 inhibi-
lg/ml indicating that the
chloro substituents at the ortho or the para position enhances the
inhibitory activity of the parent compound (Table 2).
3. Conclusion
It has been successfully demonstrated that lipase from C. rugosa
can be used for regio- and enantioselective synthesis of (S)-(+)-3-
arylamino-1-chloropropan-2-ols directly from ( )-epichlorohy-
drin, which is otherwise not possible by classical chemical
methods. This method may find general utility towards the synthe-
sis of enantiomerically enriched/pure b-aminoalcohols from race-
mic epichlorohydrin. It has also been discovered for the first time
that (S)-(+)-3-arylamino-1-chloropropan-2-ols inhibit the TNF-
a
4.1.1.1. (S )-(+)-1-Chloro-3-(20,50-dimethoxyphenylamino)-pro-
induced ICAM-1 expression. One of the compounds, (S)-(+)-1-
pan-2-ol (3e).
It was obtained as a light brown oil (500 mg)
chloro-3-(20-chlorophenylamino)-propan-2-ol exhibited 86% inhi-
in 84% yield. Rf: 0.50 (ethyl acetate/petroleum ether, 1:4); ½a D23
ꢀ
bition of TNF-a induced expression of ICAM-1 at 40 lM concentra-
tion which can be used for further development of anti-
inflammatory agents.
+3.7 (0.2, CHCl3); IR (nujol): 3413, 2940, 2834, 1614, 1522, 1462,
1219, 1169, 1048 cmꢁ1
;
1H NMR (300 MHz, CDCl3: d 3.22 and
3.35 (2H, 2dd, 1H each, J = 6.9 & 13.4 Hz and 4.6 & 13.4 Hz, C-
3H & C-3Hb), 3.64 (2H, m, C-1H), 3.74 and 3.79 (6H, 2s, 3H each,
a
4. Materials and methods
4.1. Chemistry
2ꢂ OCH3), 4.07 (1H, m, C-2H), 6.18 (1H, dd, J = 2.7 & 8.5 Hz,
C-40H), 6.26 (1H, d, J = 2.7 Hz, C-60H) and 6.66 (1H, d, J = 8.6 Hz,
C-30H); 13C NMR (75.5 MHz, CDCl3): d 46.70 (C-1), 47.52 (C-3),
55.46 and 55.93 (2ꢂ OCH3), 69.75 (C-2), 98.46 (C-60), 99.59 (C-
40), 110.15 (C-30), 138.65 (C-10), 141.67 (C-20), 154.66 (C-50); ESIMS
m/z calculated for C11H16NO3Cl [M+1]+ 246.71, observed [M+1]+
246.72.
Reactions were monitored by TLC on precoated Merck silica gel
60F254 aluminium plates. Flash column chromatography was car-
ried out using silica gel (100–200 mesh) for purification of the
compounds. The products on TLC were detected under UV light.
The 1H NMR and 13C NMR spectra were recorded on Bruker Avance
spectrometer at 300 MHz and 75.5 MHz, respectively. Chemical
shift values were reported as d ppm relative to TMS used as inter-
nal standard. Coupling constant (J values) are given in Hz. The IR
spectra were recorded on a Perkin–Elmer model 2000 FT–IR
instrument. The optical rotation values were measured on Rudolph
Research Analytical IV polarimeter. The FAB-HRMS spectra were
recorded on a JEOL JMS-AX505W high-resolution mass spectrome-
ter in positive mode using the matrix HEDS (bishydroxyethyldisul-
phide) doped with sodium acetate. The EIMS spectra were
recorded on a micromass KC 455 TOF mass spectrometer in posi-
tive ion mode. The organic solvents THF, dioxane and diisopropyl
ether were used after distillation over sodium pieces. Novozyme-
435 and Amano PS were obtained as gifts from Novozymes A/S
(Copenhagen, Denmark) and Rasayan Inc. (San Diego, USA), respec-
4.1.2. Synthesis of 2-acetoxy-1-chloro-3-(20-methoxyphenyl-
amino)-propane (4)
To a solution of (S)-(+)-1-chloro-3-(20-methoxyphenylamino)-
propan-2-ol (3c, 2.0 mmol) and catalytic amount of dimethylami-
nopyridine (DMAP) in dichloromethane (10 ml), acetic anhydride
(2.2 mmol) was added and the reaction mixture was stirred at
room temperature for 3 h until TLC examination indicated comple-
tion of the reaction. The reaction mixture was washed with a sat-
urated solution of sodium bicarbonate (3 ꢂ 5 ml) and brine
solution (3 ꢂ 5 ml), dried over sodium sulfate and evaporated un-
der reduced pressure to afford the crude product which was puri-
fied by column chromatography over silica gel using petroleum
ether and ethyl acetate as eluent to afford 2-acetoxy-1-chloro-3-
(20-methoxyphenylamino)-propane (4) as a brownish oil in 92%
yield (470 mg); Rf, 0.60 in 20% ethyl acetate in petroleum ether.

P. Gupta et al. / Bioorg. Med. Chem. 19 (2011) 2263–2268
2267
IR (nujol): 3419 (NH), 1742 (–O–C@O), 1602, 1515, 1224, 1043,
740 cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.02 (3H, s, OCOCH3),
6.87 (1H, t, J = 6.9 Hz, C-60H), 6.87 (1H, t, J = 5.7 Hz, C-50H), 6.98
(2H, m, C-30H and C-40H); 13C NMR (75.5 MHz, CDCl3) d 48.63
and 55.33 (C-1, C-3 and OCH3), 68.58 (C-2), 112.40 (C-60), 120.59
(C-30), 123.65 (C-40), 124.09 (C-50), 138.52 (C-10), 154.09 (C-20);
ESIMS m/z calculated for C10H14NO2Cl [M+1]+ 216.68 observed
[M+1]+ 216.64.
;
3.39 and 3.66 (2H, d, J = 5.7 Hz, C-3H), 3.66 (2H, m, C-1H), 3.75
(3H, s, OCH3), 4.38 (1H, m, NH), 5.13 (1H, m, C-2H), 6.65 and
6.77 (4H, m, C-30H, C-40H, C-50H and C-60H); 13C NMR (75.5 MHz,
CDCl3): d 20.82 (OCOCH3), 43.57 and 44.04 (C-1 and C-3), 55.38
(OCH3), 71.48 (C-2), 109.64 (C-60), 110.04 (C-30), 117.19 (C-40),
121.28 (C-50), 137.21 (C-10), 146.80 (C-20), 170.24 (OCOCH3); ESIMS
m/z calculated for C12H15NO3Cl [M+1]+ 257.72, observed [M+1]+
257.70.
4.2. Antiinflammatory activity
Anti-ICAM-1 and E-selectin were purchased from Pharmingen,
USA. M-199 media, L-glutamine, endothelial cell growth factor,
4.1.3. General procedure for non-enzymatic epoxide ring
opening of ( )-epichlorohydrin (1)
trypsin, Pucks saline, HEPES, o-phenylenediamine and anti-mouse
IgG-HRP were purchased from Sigma Chemical Co., USA. Fetal calf
serum was purchased from Biological Industries, Israel.
The aromatic amine (2a–2c, 10.0 mmol) was dissolved in diiso-
propyl ether (50 ml) and ( )-epichlorohydrin (1, 10.0 mmol) was
added into it. The resulting solution was stirred at 45 °C and pro-
gress of the reaction was monitored periodically by TLC. The TLC
examination of the reaction mixture revealed the formation of
two products and reaction remained incomplete even after 40 h
of stirring. The solvent was removed under reduced pressure, and
the residue thus obtained was purified by column chromatography
on silica gel using petroleum ether/ethyl acetate (93:7 to 82:18) to
obtain the pure products, 3-arylamino-1-chloropropan-2-ols 3a–
3c in 45–68% yields and 2-arylamino-3-chloropropanols 5a–5c in
17–30% yields. Compounds 3a–3c and 5a–5c were unambiguously
identified on the basis of their spectral (IR, 1H NMR, 13C NMR and
HRMS/EIMS) analysis and comparison of their physical and spec-
tral data with those reported in the literature. Further, the physical
characteristics and spectral data of 3a–3c were found identical
with the corresponding data of (S)-(+)-3-arylamino-1-chloropro-
pan-2-ols 3a–3c synthesized by the CRL-catalyzed epoxide ring
opening ( )-epichlorohydrin, except that their optical rotations
were zero.
4.2.1. Cells and cell culture
Primary endothelial cells were isolated from human umbilical
cord using mild trypsinization.28 The cells were grown in M 199
medium supplemented with 15% heat inactivated fetal calf serum,
2 mM
cin, 0.25
(50 g/ml) and heparin (5 U/ml). At confluence, the cells were sub-
L
-glutamine, 100 units/ml penicillin, 100
lg/ml streptomy-
lg/ml amphotericin B, endothelial cell growth factor
l
cultured using 0.05% trypsin–0.01 M EDTA solution and were used
between passages three to four. The viability of cells was deter-
mined by trypan blue exclusion test. The purity of endothelial cells
was determined by E-selectin expression.
4.2.2. Modified ELISA for measurement of ICAM-1 and E-selectin
Cell-ELISA was used for measuring the expression of ICAM-1
and E-selectin on the surface of the endothelial cells.29,30 Endothe-
lial cells were then incubated with or without the test compound
at desired concentrations for the required period, followed by
treatment with TNF-
a (10 ng/ml) or LPS (1 lg/ml) for 16 h for
ICAM-1 expression and 4 h for E-selectin expression. The cells were
fixed with 1.0% glutaraldehyde using non-fat dry milk (3.0% in PBS
blocked non-specific binding of the antibody). Following incuba-
tion overnight at 4 °C with ICAM-1 and E-selectin mAb, diluted
in blocking buffer, the cells were washed with PBS and incubated
with peroxidase-conjugated goat anti-mouse secondary Ab. The
cells were again washed with PBS and exposed to the peroxidase
substrate (o-phenylenediamine dihydrochloride 40 mg/100 ml in
citrate phosphate buffer, pH 4.5). The colour development reaction
was stopped by the addition of 2 N sulphuric acid and absorbance
at 490 nm was measured using an automated microplate reader
(Spectramax 190, Molecular Devices, USA).
4.1.3.1. 3-Chloro-2-phenylaminopropanol (5a).
It was ob-
tained as a brownish oil (330 mg) in 18% yield. Rf: 0.38 in 20% ethyl
acetate in petroleum ether; ½a D23
ꢀ
0.0 (c 0.2, CHCl3); IR (nujol): 3338
(NH), 3062, 1599, 1504, 1228, 852 and 751 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) d 3.32 (2H, dd, J = 7.8 and 15.6 Hz, C-3H), 3.60
(2H, m, C-1H), 3.93 (1H, br s, C-2H), 5.32 (1H, d, J = 4.8 Hz, OH),
5.46 (1H, d, J = 4.8 Hz, NH), 6.60 (1H, m, C-40H), 6.75 (2H, dd,
J = 1.8 and 8.7 Hz, C-20H and C-60H) and 7.15 (2H, m, C-30H and
C-50H); 13C NMR (75.5 MHz, CDCl3) d 48.12 and 55.08 (C-1 and C-
3), 67.64 (C-2), 112.07 (C-20 and C-60), 115.69 (C-40), 129.08 (C-30
and C-50) and 147.61 (C-10); ESIMS m/z calculated for C9H12NOCl
[M+1]+ 186.66, observed [M+1]+ 186.64.
Acknowledgements
4.1.3.2.
3-Chloro-2-(40-methylphenylamino)propanol
(5b).
It was obtained as a brownish oil (590 mg) in 30% yield. Rf: 0.36
We thank the University of Delhi, Department of Science and
Industrial Research (DSIR, New Delhi) and Defense Research and
Development Agency (DRDO, New Delhi) for financial support to
this work. Sumati Bhatia and Sakshi Balwani thank University
Grants Commission (UGC, New Delhi) and Council of Scientific
and Industrial Research (CSIR, New Delhi), respectively for the
award of senior research fellowships.
in 20% ethyl acetate in petroleum ether; ½a D23
ꢀ
0.0 (c 0.2, CHCl3);
IR (nujol): 3583 (NH), 3342 (OH), 1616, 1519, 1231, 805, 752 cmꢁ1
;
1H NMR (300 MHz, CDCl3) d 2.25 (3H, s, CH3), 3.37 (2H, dd,
J = 8.1 and 14.5 Hz, C-3H), 3.62 (2H, m, C-1H), 4.10 (1H, br s,
C-2H), 6.76 (2H, d, J = 8.1 Hz, C-20H and C-60H), 7.05 (2H, d,
J = 7.8 Hz, C-30H and C-50H); 13C NMR (75.5 MHz, CDCl3) d 27.93
(CH3), 54.46 and 54.81 (C-1 and C-3), 65.99 (C-2), 121.20 (C-40),
122.98 (C-20 and C-60), 137.68 (C-30 and C-50), 154.01 (C-10); ESIMS
m/z calculated for C10H14NOCl [M+1]+ 200.68, observed [M+1]+
200.58.
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