Synthesis, in vitro and in silico evaluation of l-tyrosine containing PPARα/γ dual agonists

Article abstract of DOI:10.1016/j.bmc.2006.06.063

A novel series of l-tyrosine derivatives have been reported with potential PPARα/γ dual agonistic activity. In vitro cell based PPARα/γ transactivation studies have shown compound 4a and compound 4f to be the most potent PPARγ and PPARα activators, respec

Full text of DOI:10.1016/j.bmc.2006.06.063

Bioorganic & Medicinal Chemistry 15 (2007) 1547–1555
Synthesis, in vitro and in silico evaluation of L-tyrosine containing
PPARa/c dual agonists
Rakesh Kumar,a Uma Ramachandran,a Smriti Khanna,b Prasad V. Bharatam,b,*
Suryaprakash Raichurc and Ranjan Chakrabartic
aDepartment of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research (NIPER),
Sector 67, S. A. S. Nagar 160 062, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER),
Sector 67, S. A. S. Nagar 160 062, India
cDr. Reddy’s Laboratory-Discovery Biology, Hyderabad 500 050, India
Received 6 June 2006; revised 26 June 2006; accepted 28 June 2006
Available online 12 December 2006
Abstract—A novel series of L-tyrosine derivatives have been reported with potential PPARa/c dual agonistic activity. In vitro cell
based PPARa/c transactivation studies have shown compound 4a and compound 4f to be the most potent PPARc and PPARa acti-
vators, respectively. Molecular docking studies performed on these series of compounds have complemented the experimental results
and have led to interesting inferences.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
inedione insulin sensitizers, there are many groups
working on PPARa/c dual agonists as they are involved
in both insulin sensitization and improving lipidemic
profile of the diabetic patients.10–15 Many compounds
belonging to the category of dual activators have
advanced to clinical trials.16
Diabetes is the root cause of several chronic and pro-
gressive diseases that adversely affect a number of
organs including nervous and vascular systems.1 Type
2 diabetes mellitus (T2DM) is a complex metabolic dis-
order that affects 90% of total diabetic population.2 This
disease is characterized by insulin resistance in liver and
peripheral tissues accompanied by a defect in pancreatic
b-cells.3 The drugs that reverse insulin resistance fulfill
the major medical need in treating T2DM and hence
have the potential to reduce long-term complications
of T2DM.4 Insulin sensitizers bind to peroxisome
proliferator-activated receptor (PPARs) belonging to
the hormone receptors superfamily of ligand-activated
transcription factors that govern glucose and lipid
homeostasis.5 The PPARs have three subtypes, PPARa,
PPARc, and PPARd each of which is differentially
expressed in a tissue specific manner.6–8 Two pharma-
ceutically important PPARc agonists, pioglitazone and
rosiglitazone, are blockbuster drugs (Chart 1).9 Though
current research trend has shifted toward non-thiazolid-
Based on the crystal structure analysis of PPARa and
PPARc and the molecular modeling studies, a common
U-shaped pharmacophore has been derived for PPARc
agonists which is constituted by an acidic moiety and a
bulky hydrophobic fragment, on the two terminals of
the active compounds connected by a flat aromatic link-
er. A similar pharmacophoric pattern holds true for the
PPARa/c dual agonists except that there are differences
in the size of the acidic head group. The thiazolidinedi-
one (TZD) head group is anchored by four important
H-bonds in the active site—Ser289, His323, His449,
and Tyr473. However, His323 is replaced by Tyr314 in
PPARa. The relatively larger size of Tyr314 leads to ste-
ric problems in accommodating bulkier TZD ring. Steri-
cally favorable open chain acid derivatives have been
found to bind comfortably in the active sites of both
PPARa and PPARc and therefore show better dual
activity.17,24
Keywords: PPAR; Dual agonists; L-Tyrosine; Transactivation; Molec-
ular docking.
*
Recently, we reported carbazole derivatives with the
acyclic acidic head groups as PPARa/c dual agonists
Corresponding author. Tel.: +91 0 172 214682–87; fax: +91 0 172
214692; e-mail: pvbharatam@niper.ac.in
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.063

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R. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 1547–1555
O
O
NH
NH
S
S
N
N
O
N
O
O
O
O
Rosiglitazone
Pioglitazone
H
O
N
N
S
COOH
NH
S
O
N
O
HO
Cl
Troglitazone
WY 14643
COOH
COOH
O
O
HN
HN
N
N
O
O
O
O
GW 409544
Farglitazar
Chart 1. Compounds known to be PPARa, PPARc and PPARa/c dual agonists.
with antioxidant property.18 Antioxidant is an added
property of such compounds, as it scavenges free radi-
2. Chemistry
cals generated and thus offers additional benefits to the
diabetic patients.19 In this work, we have made an at-
tempt to improve the potency of the active PPARa/c
dual agonists identified from the previously published
work. It is an N-derivatized L-tyrosine analogue. Since
compounds based on tyrosine derivatives have reached
up to phase II clinical trials,20–23 so the compounds with
this moiety were explored further. Two types of N-der-
ivatised tyrosine derivatives have been synthesized
(Scheme 1). Different substituents have been tried in
the hydrophobic side chains of the molecules. This exer-
cise has lead to some potent PPARa/c dual agonists that
have been evaluated by in vitro assays. Molecular dock-
ing studies were performed to evaluate and analyze these
compounds for important interactions at the active sites
of PPARa and PPARc.
Two series of compounds were synthesized as
described in Scheme 1 in chiral non-racemic form
starting with L-tyrosine methyl ester hydrochloride
1, which was converted to its free ester form 2 by
treatment with sodium bicarbonate in methanol. 2
was refluxed with 2-benzoylacetone (series I) or
2-benzoylcyclohexanone (series II) in toluene in pres-
˚
ence of 4 A molecular sieves, to adsorb in situ gener-
ated water, to afford
3 and 5, respectively, in
excellent yields. The intermediates 3 and 5 were con-
densed with various haloalkoxyheteroaryl moieties
(HET substituents in Table 1) by refluxing in acetone
in presence of K2CO3 to furnish 4a–i and 6a–d,
respectively. Saponification with LiOH in THF/
MeOH afforded corresponding acids 4a–i and 6a–d
(Table 1) in enantiomerically pure fashion. The struc-
COOR
COOCH
3
HN
HET
HN
O
HO
d,e
O
O
b
COOCH
3
4a-i
Series I
a
3
1
NH
2
HO
c
2
COOCH
COOR
3
HN
HET
HN
O
HO
O
O
d,e
5
6a-d
Series II
˚
Scheme 1. Reagents and conditions: (1) L-tyrosine methyl ester HCl; (a) NaHCO3, MeOH, 1 h; (b) benzoylacetone, toluene, 4 A molecular sieves,
˚
reflux, 12 h; (c) benzoylcyclohexanone, toluene, 4 A molecular sieves, reflux, 12 h; (d) haloalkoxyheteroryl groups (HET), K2CO3, acetone, reflux,
14 h; (e) 1.5 equiv LiOH in H2O, THF/CH3OH (3:1).

R. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 1547–1555
1549
Table 1. List of synthesized compounds, their PPAR fold activation values, and their FlexX docking scores in 1K7L (PPARa) and 1FM9 (PPARc)
Sr. No.
Compound
HET
R
PPARa fold
activation
1K7L docking
score
PPARc fold
activation
1FM9 docking
score
O
1
2
3
4
5
6
4a
4b
4c
4d
4e
4f
H
4.6
1.2
1.4
1.9
2.0
5.1
À33.91
ND
48.2
1.9
À41.17
ND
HN
CH3
N
H
À35.77
ND
1.7
À39.05
ND
N
O
O
CH3
1.8
N
H
H
À29.69
À34.55
4.2
À37.90
À29.5
N
H
O
H
15.1
N
H
O
7
4g
H
1.1
À28.18
2.3
À35.03
N
CF
3
CF
3
O
8
4h
4i
H
2.8
0.3
1.4
1.2
0.5
1.0
À29.16
À28.86
ND
22.6
0.7
À34.49
À30.97
ND
Cl
O
F3C
9
H
O
10
11
12
13
6a
6b
6c
6d
CH3
4.7
HN
O
H
À29.24
ND
11.2
3.0
À34.05
ND
HN
CH3
N
H
ND
8.4
À37.25
N
PPAR fold activation at 50 lM and FlexX docking scores in kcal/mol, ND = not docked.
tures of all the synthesized compounds were con-
firmed by various spectral analysis techniques and
3. Biological evaluation
their enantiomeric purity was confirmed by chiral
HPLC.
In vitro screening of compounds was performed to eval-
uate their PPARa/c dual agonistic activity. The cells

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R. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 1547–1555
were transfected with an expression plasmid for PPAR
receptors and activation of luciferase gene was mea-
sured. Potencies of PPAR gene activation were evaluat-
ed in cell based transcription assays using GAL4-PPAR
chimeric receptors.25 Transactivation studies were done
for all the compounds at 50 lM concentration both
for PPARa and PPARc using WY14,643 and rosiglitaz-
one as references, respectively. The comparative poten-
cies were determined in terms of fold activation at
50 lM concentration.
The results of the transactivation studies are listed in Ta-
ble 1. In the first series of molecules, the lead compound
4a showed maximum agonistic activity for both the
receptors followed by compounds 4f and 4h. The lead
compound 4a exhibits more than 2-fold activity for
PPARc receptor (48.2) as compared to rosiglitazone
(22.5) and also shows very good activity for PPARa.
The compound 4f, with three carbon chain linker,
showed higher potency for PPARa receptor (5.1) as
compared to WY14643 (4.0) and moderate PPARc fold
activation. Compound 4h showed good PPARc fold
agonistic activity while showing moderate activity for
PPARa. The compounds 4a and 4f constitute the carba-
zole derivatives, whereas the compound 4h has 4-chloro-
benzophenone unit of fenofibrate in place of carbazole
as the hydrophobic side chain. Other substitutions were
also tried in the hydrophobic region of the compounds.
Replacing carbazole ring with trifluoromethyl-substitut-
ed ring systems (4g and 4i) did not improve the potency
of these compounds. In the second series, the compound
6b showed the best results.
4. Molecular docking
Molecular docking studies were performed on two
proteins, viz. PPARa and PPARc, employing the
FlexX docking procedure using Sybyl6.9 program in-
stalled on silicon graphics Octane2 workstation. FlexX
is a fast automated program based on incremental
construction procedure. In this method flexibility of
the ligands is considered by including several confor-
mations of ligands, while maintaining a rigid structure
for the biomolecule. The 3D coordinates of the active
sites were taken from the X-ray crystal structures of
PPARa and PPARc proteins reported as complexes
with their corresponding agonists, GW409544 and
farglitazar deposited in Brookhaven Protein Data
Bank with PDB codes 1K7L and 1FM9, respectively.
WY14643 and rosiglitazone, the reference compounds
used for PPARa and PPARc transactivation studies,
respectively, were docked into the active sites of the
two receptors to judge the discriminatory strength of
the docking procedures and the choice of the crystal
structures. The result of this initial docking exercise
was fruitful as WY14643 docked well in the active site
of PPARa but failed to dock in the active site of
PPARc. Similarly rosiglitazone was able to dock well
in the active site of PPARc, while it failed to do so in
PPARa. Encouraged by these results, all the synthe-
sized molecules were docked into the active sites of
the PPARa and PPARc. The active sites were as-
Molecular docking studies were carried out on the synthe-
sized compounds to get insight into their binding prefer-
ences at the active sites of the two receptors.
Rosiglitazone when docked in the active site of PPARc
receptor attained a score of À15.96 kcal/mol, however,
it could not fit well in the active site of PPARa receptor.
It showed the important H-bonding interactions with
the residues at the active site of PPARc. Similarly,
WY14643 docked well and achieved the score of
À19.92 kcal/mol in the active site of PPARa but could
not bind well in the active site of PPARc. These results
are on the expected lines as rosiglitazone is a PPARc ago-
nist, while WY14643 is a PPARa agonist. These mole-
cules in their docked conformations are shown in the
Figure 1a and b indicating the important interactions they
make at the active site. Similar FlexX based docking stud-
ies were carried out to evaluate the synthesized com-
pounds. The FlexX docking scores of these compounds
are listed in Table 1. From the observed results, the fol-
lowing general conclusions can be drawn: the acidic deriv-
atives docked well in both the receptors and maintained
all the essential H-bonding interactions required for good
anchoring at the active site. However, the ester analogues
of these compounds could not fit into active sites of the
two receptors, as they were devoid of the acidic factor
and could not interact through the H-bonds. All the active
compounds docked very well in the active sites of both the
receptors. Scores of some of these molecules correlated
well with their in vitro activity profiles. The most active
compound 4a fitted the best in the active site of PPARc
and attained the best score of À41.17 kcal/mol amongst
all the molecules. It also docked well in the active site of
PPARa and scored a FlexX score of À33.91 kcal/mol. It
showed all the prime interactions to anchor well in the ac-
tive sites of both the receptors (Fig. 1d). The other active
compounds in the series I, 4f and 4 h, also achieved very
good docking scores and could fit well into the active sites
of both the receptors. The compound 4f has a three-car-
bon linker between the hydrophobic region and the cen-
tral aromatic unit. The extended length introduces a
strong H-bond with Thr279 which is not observed with
˚
signed at a radius of 8 A around the reference ligand.
FlexX run was submitted and the docking scores were
obtained and analyzed.
5. Results and discussion
The results obtained from the transactivation studies
exhibited that series I (4a–i) showed better PPARa/c
dual agonistic activity as compared to series II (6a–d).
In both the series, the acid derivatives have been found
to be much more active as compared to their ester ana-
logues. It reinforces the fact that PPARc agonistic activ-
ity requires trivial amount of acidic character. Some
changes in the hydrophobic side chain were introduced
by substitution of the carbazole ring. The results indicat-
ed that the carbazoles substituted at the C4 position are
more active than those substituted at other positions.
The compounds obtained with 4-hydroxy carbazoles
as the starting material and substituted at this hydroxyl
group showed better activity than the compounds
substituted at the –NH of the carbazole moiety.

R. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 1547–1555
1551
Figure 1. (a) WY14643 with PPARa (1K7L). (b) Rosiglitazone with PPARc (1FM9). (c) Compound 4f with PPARa (1K7L). (d) Compound 4a with
PPARc (1FM9). The molecules in orange are the reference ligands found as complexes with the crystal structures (farglitazar in 1FM9 and
GW409544 in 1K7L).
the other molecules (Fig. 1c). The new H-bond provides it
a better score over the others. In case of the compound 4h,
it shows a good p-stacking interaction with the Phe287
that makes this particular hydrophobic unit more active
than the others. In the second series, compounds 6b and
6d docked well andattained good docking scores at the ac-
tive site of PPARa and PPARc. But no additional interac-
tions were observed in these molecules. The reason for the
better activity of 4-hydroxy-substituted carbazole deriva-
tives over compounds with N-substitution and 2-hydroxy
substituents can be attributed to the fact that the 4-hy-
droxy substituent orients its oxygen such that there is an
additional H-bonding interaction that is observed with
this oxygen atom and a water molecule lying in close vicin-
ity. Hence the strategic position of this group is important
for its activity. Thus, with these studies we could get some
insights into the nature of binding of the ligands and these
studies can be applied for designing better molecules hav-
ing dual PPARa/c agonistic activity.
melting point apparatus and are uncorrected. Parr sha-
ker used for hydrogenation is from Perfit-India. Infrared
(IR) spectra were recorded on a Nicolet Impact-410
FTIR spectrometer. Proton magnetic resonance
(NMR) spectra were recorded on a Bruker 300 MHz
spectrometer in CDCl3, CD3OD, D2O or DMSO-d6
solution. The chemical shifts are reported in d (ppm) rel-
ative to internal standard tetramethylsilane (TMS) and
coupling constants J are given in Hertz. Mass spectros-
copy was conducted using Shimadzu QP5000 mass spec-
trometer, LCQ Finnigan MAT, and Bruker Daltonics
MALDI Tandem TOF mass spectrometer. Elemental
analyses were obtained from Elementar VarioÒEL.
6.1.1. (S)-3-{4-[2-(9H-Carbazol-4-yloxy)-ethoxy]-phen-
yl}-2-(1-methyl-3-oxo-3-phenyl-propenylamino)-propionic
acid (4a). Step A. Synthesis of (S)-3-(4-hydroxy-phenyl)-
2-(methyl-3-oxo-3-phenyl-propenylamino)-propionic
acid methyl ester. To a 10 ml dry toluene suspension of
L-tyrosine methyl ester (2.0 g, 10.2 mmol), benzoylace-
tone (1.7 g, 10.2 mmol) was added, in presence of 4°A
molecular sieve. The suspension was refluxed for 12 h.
Toluene was removed and residue was extracted with
dichloromethane. The organic layer was separated and
dried over anhydrous sodium sulfate, decanted, concen-
trated, and chromatographed on silica gel. Elution with
a gradient mixture of ethyl acetate/hexane yielded the ti-
tle compounds (90–95%). IR (cmÀ1) 3177.8, 1744.1,
6. Experimental
6.1. General experimental techniques
Thin-layer chromatography analyses were performed on
precoated silica gel plates (GF254, Merck). Chromatog-
raphy was performed on flash silica gel (230–400 mesh).
Melting points were recorded on a Buchi capillary
1
1596.1, 1438.5, 1118.2, 745.6 H NMR (CDCl3) d 1.81

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R. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 1547–1555
(s, 3H), 2.99–3.16 (m, 4H), 3.72 (s, 3H), 5.65 (m, 1H),
6.74 (d, J = 8.34 Hz, 1H), 7.02 (d, J = 6.57 Hz, 1H),
7.38 (d, J = 6.78 Hz, 1H), 7.84 (d, J = 7.02 Hz, 1H),
11.63 (d, J = 9.00 Hz, 1H). MS (APCI): 339 (m/z M+1).
2H), 7.87 (m, 3H), 8.01 (s, 1H), 8.25 (d, J = 8.4 Hz,
1H) 13C NMR d (CDCl3) 13.24, 20.02, 28.60, 29.08,
60.09, 60.47, 68.79, 100.05, 100.24, 103.41, 103.64,
109.48, 114.26, 114.76, 118.21, 122.35, 124.04, 125.71,
126.41, 127.74, 128.09, 129.17, 130.00, 131.86, 132.37,
138.81, 141.08, 143.94, 154.55, 171.49 MS (MALDI):
535 M+. Anal. Calcd for C33H30N2O5; C, 74.14; H,
5.66; N, 5.24; Found: C, 73.93; H, 5.53; N, 5.04.
Step B. Synthesis of 4-(2-bromo-ethoxy)-9H-carbazole.
To a 10 ml acetone solution of 4-hydroxycarbazole
(2.0 g, 10.8 mmol) and 1, 2-dibromoethane (2.0 g,
12.0 mmol) was added K2CO3 (2.2 g, 12.0 mmol). The
suspension was refluxed overnight. The reaction mixture
was concentrated, and residue was extracted with ethyl
acetate. The organic layer was separated and dried over
anhydrous sodium sulfate, decanted, concentrated and
chromatographed on silica gel. Elution with a gradient
mixture of ethyl acetate/hexane yielded the title com-
pounds as white powder mp 115 °C (60–70%). 1H
NMR (CDCl3) d 3.84 (t, J = 6 Hz, 2H), 4.55 (t,
J = 6 Hz, 2H), 6.64 (d, J = 8.1 Hz, 1H), 7.07 (d,
J = 8.1 Hz, 1H), 7.25 (m, 2H), 7.36 (d, J = 9 Hz, 1H),
8.06 (s, 1H), 8.38 (d, J = 6.9 Hz, 1H) MS (EI): 289,
291 (m/z M+1, M+2).
6.1.2.
(S)-3-[4-(2-Carbazol-9-yl-ethoxy)-phenyl]-2-(1-
methyl-3-oxo-3-phenyl-propenylamino)-propionic acid
methyl ester (4b). (S)-3-(4-Hydroxy-phenyl)-2-(methyl-
3-oxo-3-phenyl-propenylamino)-propionic acid methyl
ester (1 equiv), carbazole-9-ethyl mesylate (1 equiv),
and potassium carbonate (2 equiv) in ethanol and tolu-
ene (1:1) were refluxed for 10 h. The reaction mixture
was concentrated and extracted with DCM. The organic
layer was washed with water, brine and dried over sodi-
um sulfate. Purification with column chromatography
afforded 4b. IR (cmÀ1) 2920, 1592, 1458, 1079, 1018,
1
750 H NMR (CDCl3) d 1.98 (s, 3H), 3.91 (m, 4H),
4.11 (m, 3H), 4.31 (m, 4H), 7.20 (m, 5H), 7.42 (m,
8H), 8.05 (d, J = 7.6 Hz, 4H) MALDI: 535 (m/z M+)
Anal. Calcd for (C34H32N2O4) C, 76.67; H, 6.06; N,
5.26; Found: C, 76.61; H, 5.93; N, 5.04.
Step C. To a 10 ml acetone solution of (S)-3-(4-Hy-
droxy-phenyl)-2-(methyl-3-oxo-3-phenyl-propenylami-
no)-propionic acid methyl ester (1.0 g, 2.9 mmol) and 4-
(2-bromo-ethoxy)-9H-carbazole (0.9 g, 3.19 mmol) was
added K2CO3 (0.6 g, 4.35 mmol). The suspension was
refluxed for 14 h, concentrated, and residue was extract-
ed with ethyl acetate. The organic layer was separated
and dried over anhydrous sodium sulfate, decanted,
concentrated, and chromatographed on silica gel. Elu-
tion with a gradient mixture of ethyl acetate/hexane
yielded the (S)-3-{4-[2-(9H-carbazol-4-yloxy)-ethoxy]-
phenyl}-2-(1-methyl-3-oxo-3-phenyl-propenylamino)-
propionic acid methyl ester. 1H NMR (CDCl3) d 1.94 (s,
3H), 3.16 (m, 1H), 3.20 (m, 1H), 3.54 (t, J = 6 Hz, 1H),
3.76 (s, 3H), 4.49 (t, J = 6 Hz, 2H), 4.57 (t, J = 6 Hz,
2H), 4.74 (m, 1H), 5.6 (s, 1H), 6.74 (d, J = 8.4 Hz,
2H), 6.93 (t, J = 8.4 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H),
7.20 (m, 5H), 7.33 (d, J = 9 Hz, 2H), 7.87 (m, 3H),
8.01(s, 1H), 8.25 (d, J = 8.4 Hz, 1H) MS (EI): 548 (m/z
M+) Anal. Calcd for C34H32N2O5 (548.23) C, 74.43;
H, 5.88; N, 5.11; Found: C, 74.27; H, 5.76; N, 5.08.
6.1.3.
(S)-3-[4-(2-Carbazol-9-yl-ethoxy)-phenyl]-2-(1-
methyl-3-oxo-3-phenyl-propenylamino)-propionic acid
(4c). (S)-3-[4-(Carbazol-9-yloxy)-phenyl]-2-(1-methyl-3-
oxo-3-phenyl-propenylamino) propionic acid methyl es-
ter was saponified with 1.5 equiv aqueous LiOH in
THF/CH3OH to give (S)-3-[4-(carbazol-9-yloxy)-phen-
yl]-2-(1-methyl-3-oxo-3-phenyl-propenylamino) propi-
1
onic acid H NMR (CDCl3) d 1.97 (s, 3H), 3.90 (m,
1H), 4.13 (m, 3H), 4.33 (m, 4H), 7.21 (m, 5H), 7.42
(m, 8H), 8.03 (d, J = 7.6 Hz, 4H) MALDI: 518 (m/z
M+) Anal. Calcd for (C33H30N2O4) C, 76.43; H, 5.83;
N, 5.40; Found: C, 76.35; H, 5.73; N, 5.02.
6.1.4. (S)-3-{4-[2-(9H-carbazol-2-yloxy)-ethoxy]-phenyl}-
2-(1-methyl-3-oxo-3-phenyl-propenylamino)-propionic
acid methyl ester (4d). Compound 4d was synthesized
according to procedure as mentioned in 6.1.2 from 2-
(2-bromo-ethoxy)-9H-carbazole (1 equiv) and (S)-3-(4-
hydroxy-phenyl)-2-(methyl-3-oxo-3-phenyl-propenyla-
Step D. To ethanol solution of compound (S)-3-{4-[2-
(9H-carbazol-4-yloxy)-ethoxy]-phenyl}-2-(1-methyl-3-
oxo-3-phenyl-propenylamino)-propionic acid methyl es-
ter (1.0 g, 1.8 mmol) was added 1.5 equiv aqueous LiOH
in THF/CH3OH and stirred at room temperature over-
night. The reaction mixture was concentrated, acidified
with acetic acid (pH 4). The acidic solution was extract-
ed with ethyl acetate. The organic layer was separated
and dried over anhydrous sodium sulfate, decanted,
and concentrated to yield the title compounds (S)-3-
{4-[2-(9H-carbazol-4-yloxy)-ethoxy]-phenyl}-2-(1-meth-
1
mino)-propionic acid methyl ester (1 equiv). H NMR
(CDCl3) d 1.98 (s, 3H), 3.04–3.05 (m, 2H), 3.57 (t,
J = 6 Hz, 1H), 3.72 (s, 3H), 4.35 (t, J = 6 Hz, 2H), 4.46
(t, J = 6 Hz, 2H), 5.61 (s, 1H), 6.75 (d, J = 8.4 Hz,
2H), 6.93 (t, J = 8.4 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H),
7.20 (m, 5H), 7.32 (d, J = 9 Hz, 2H), 7.89 (m, 4H),
8.12 (s, 1H). MS (MALDI): 549 (m/z M+) Anal. Calcd
for (C34H32N2O5) C, 74.43; H, 5.88; N, 5.11; Found:
C, 74.22; H, 5.68; N, 5.07.
yl-3-oxo-3-phenyl-propenylamino)-propionic
acid
6.1.5. (S)-3-{4-[2-(9H-Carbazol-2-yloxy)-ethoxy]-phen-
yl}-2-(1-methyl-3-oxo-3-phenyl-propenylamino)-propionic
acid (4e). Compound 4e was synthesized according to
procedure as mentioned in 6.1.3 from (S)-3-{4-[2-(9H-
carbazol-2-yloxy)-ethoxy]-phenyl}-2-(1-methyl-3-oxo-3-
phenyl-propenylamino)-propionic acid methyl ester. H
NMR (CDCl3) d 1.99 (s, 3H), 2.99 (m, 1H), 3.05 (m,
1H), 3.58 (t, J = 6 Hz, 1H), 4.34 (t, J = 6 Hz, 2H), 4.45
(75%). Chiral HPLC (chiral-AGP, 150 4 mm, 5 lm,
*
hexane/isopropanol (4:1), 1 ml/min), tR = 8.2, 98% ee.
1H NMR (CDCl3) d 1.94 (s, 3H), 3.16 (m, 1H), 3.25–
3.30 (m, 1H), 3.54 (t, J = 6 Hz, 1H), 3.60 (m, 1H), 4.49
(t, J = 6 Hz, 2H), 4.57 (t, J = 6 Hz, 2H), 5.6 (s, 1H),
6.74 (d, J = 8.4 Hz, 2H), 6.93 (t, J = 8.4 Hz, 1H), 7.09
(d, J = 8.4 Hz, 2H), 7.20 (m, 5H), 7.33 (d, J = 9 Hz,
1

R. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 1547–1555
1553
(t, J = 6 Hz, 2H), 5.59 (s, 1H), 6.74 (d, J = 8.4 Hz, 2H),
6.93 (t, J = 8.4 Hz, 1H), 7.09 (d, J = 8.4 Hz, 2H), 7.20
(m, 5H), 7.33 (d, J = 9 Hz, 2H), 7.87–7.91 (m, 4H),
8.13 (s, 1H). MS (MALDI): 535 (m/z M+) Anal. Calcd
for (C33H30N2O5) C, 74.14; H, 5.66; N, 5.24; Found:
C, 74.02; H, 5.32; N, 4.95.
Calcd for (C34H30ClNO6) C, 69.92; H, 5.18; N, 2.40;
Found: C, 69.67; H, 5.01; N, 2.13.
6.1.9. (S)-2-(1-Methyl-3-oxo-3-phenyl-propenylamino)-3-
{4-[2-(4-trifluoromethyl-phenyl)-ethoxy]-phenyl}-propion-
ic acid (4i). Compound 4i was synthesized according to
procedure as mentioned in 6.1.2 and 6.1.3 by condensing
(S)-3-(4-hydroxy-phenyl)-2-(methyl-3-oxo-3-phenyl-
propenylamino)-propionic acid methyl ester with
methanesulfonic acid 2-(4-trifluoromethyl-phenyl)-ethyl
ester. 1H NMR (CDCl3) d 1.94 (s, 3H), 2.93 (t,
J = 6 Hz, 2H), 3.04–3.16 (m, 2H), 4.14 (t, J = 6 Hz,
2H), 4.38 (m, 1H), 5.65 (s, 1H), 6.77 (d, J = 8.4 Hz,
2H), 6.90 (d, J = 8.4 Hz, 2H), 7.39 (m, 3H), 7.53 (d,
J = 9 Hz, 2H), 7.72 (d, J = 8.7 Hz, 2H), 7.85 (d,
J = 8.7 Hz, 2H). MS (MALDI): 497 (m/z M+) Anal.
Calcd for (C28H26F3NO4) C, 67.61; H, 5.27; N, 2.82;
Found: C, 67.21; H, 5.18; N, 2.74.
6.1.6. (S)-3-{4-[3-(9H-Carbazol-4-yloxy)-propoxy]-phen-
yl}-2-(1-methyl-3-oxo-3-phenyl-propenylamino)-propionic
acid (4f). Step A. Synthesis of 4-(3-chloro-propoxy)-9H-
carbazole: 4-hydroxycarbazole was refluxed with 3-bro-
mo-1-chloropropane in acetone in presence of K2CO3 to
afford 4-(3-chloro-propoxy)-9H-carbazole with 55%
yield. 1H NMR (CDCl3) d 2.43 (tt, J = 6.0 Hz, 2H),
3.89 (t, J = 6.0 Hz, 2H), 4.38 (t, J = 6.0 Hz, 2H), 6.67
(d, J = 8.1 Hz, 1H), 7.03 (d, J = 7.8 Hz, 1H), 7.23 (m,
2H), 7.37 (m, 2H), 8.02 (s, 1H), 8.23 (d, J = 7.5 Hz,
1H). MS (MALDI): 259 (m/z M+).
Step B. (S)-3-{4-[3-(9H-Carbazol-4-yloxy)-propoxy]-
phenyl}-2-(1-methyl-3-oxo-3-phenyl-propenylamino)-
propionic acid was synthesized according to procedure
as mentioned in 6.1.2 and 6.1.3 by condensing (S)-3-
(4-hydroxy-phenyl)-2-(methyl-3-oxo-3-phenyl-prope-
nylamino)-propionic acid methyl ester with 4-(3-chloro-
propoxy)-9H-carbazole. 1H NMR (CDCl3) d 1.94 (s,
3H), 2.43 (t, J = 6 Hz, 2H), 3.16 (m, 1H), 3.25–3.30
(m, 1H), 3.54 (t, J = 6 Hz, 1H), 4.49 (m, 4H), 5.6 (s,
1H), 6.73 (d, J = 8.4 Hz, 2H), 6.92 (t, J = 8.4 Hz, 1H),
7.09 (d, J = 8.4 Hz, 2H), 7.19–7.24 (m, 5H), 7.31 (d,
J = 9 Hz, 2H), 7.85 (m, 3H), 8.22 (d, J = 8.4 Hz, 2H).
MS (MALDI): 549 (m/z M+) Anal. Calcd for
(C34H32N2O5) C, 74.43; H, 5.88; N, 5.11; Found: C,
74.21; H, 5.63; N, 5.01.
6.1.10. (S)-2-(2-Benzoyl-cyclohex-1-enylamino)-3-{4-[2-
(9H-carbazol-4-yloxy)-ethoxy]-phenyl}-propionic
acid
methyl ester (6a). Step A. Synthesis of (S)-2-(2-benzo-
yl-cyclohex-1-enylamino)-3-(4-hydroxy-phenyl)-propi-
onic acid methyl ester: L-tyrosine ester was refluxed with
˚
2-benzoyl-cyclohexanone in toluene in presence of 4 A
molecular sieve for 12 h. Reaction mixture was concen-
trated under vacuum and extracted with methylene chlo-
ride, washed with brine, and then dried over sodium
sulfate. 1H NMR (CDCl3) d 1.32 (m, 4H), 2.18 (m,
4H), 3.04 (m, 2H), 3.72 (s, 3H), 4.42 (m, 1H), 6.73 (d,
J = 9.0 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 7.32–7.43
(m, 5H), 12.14 (d, J = 9.0 Hz, 1H). MS (MALDI): 379
(m/z M+).
Step B. (S)-2-(2-Benzoyl-cyclohex-1-enylamino)-3-{4-[2-
(9H-carbazol-4-yloxy)-ethoxy]-phenyl}-propionic acid
methyl ester was synthesized from (S)-2-(2-benzoyl-
cyclohex-1-enylamino)-3-(4-hydroxy-phenyl)-propionic
acid methyl ester and 4-(2-bromo-ethoxy)-9H-carbazole
according to procedure as mentioned in 6.1.1. 1H NMR
(CDCl3) d 1.40–1.45 (m, 2H), 1.55 (m, 2H), 1.75–1.79
(m, 2H), 2.34 (m, 2H), 2.91 (m, 1H), 3.00–3.05 (m,
1H), 3.72 (s, 3H), 4.30–4.45 (m, 5H), 6.56 (m, 2H),
6.81 (m, 1H), 6.95 (m, 2H), 7.15 (m, 2H), 7.32–7.39
(m, 5H), 7.41 (m, 2H), 7.86 (m, 1H), 8.25 (d,
J = 7.8 Hz, 2H), 12.22 (d, J = 8.7 Hz, 1H). 13C NMR
(CDCl3) d 21.93, 23.69, 27.74, 29.56, 32.65, 39.16,
52.51, 57.55, 67.56, 100.90 (2), 104.20 (2), 110.01 (2),
115.60 (2), 119.40, 122.76 (2), 124.88, 126.53 (2),
127.53 (2), 128.43 (2), 130.46 (2), 138.76, 142.54,
148.15, 155.57, 157.86, 163.17, 171.91, 195.34 MS (EI):
589 (m/z M+) Anal. Calcd for (C37H36N2O5) C, 75.49;
H, 6.16; N, 4.76; Found: C, 75.21; H, 5.98; N, 4.74.
6.1.7.
(S)-3-{4-[2-(2,8-Bis-trifluoromethyl-quinolin-4-
yloxy)-ethoxy]-phenyl}-2-(1-methyl-3-oxo-3-phenyl-
propenylamino)-propionic acid (4g). Compound 4g was
synthesized according to procedure as mentioned in
6.1.2 and 6.1.3 by condensing (S)-3-(4-hydroxy-phen-
yl)-2-(methyl-3-oxo-3-phenyl-propenylamino)-propionic
acid methyl ester with 4-(2-bromo-ethoxy)-2,8-bis-tri-
fluoromethyl-quinoline. 1H NMR (CDCl3) d 1.94 (s,
3H), 3.04–3.14 (m, 2H), 4.26 (m, 4H), 4.38 (m, 1H),
5.65 (s, 1H), 6.92 (d, J = 8.4 Hz, 2H), 7.21 (d,
J = 8.4 Hz, 2H), 7.44 (m, 3H), 7.64 (m, 3H), 8.13 (m,
2H), 8.47 (m, 1H). MS (MALDI): 632 (m/z M+) Anal.
Calcd for (C32H26F6N2O5) C, 60.76; H, 4.14; N, 4.43;
Found: C, 60.26; H, 4.08; N, 4.17.
6.1.8.
(S)-3-(4-{2-[4-(4-Chloro-benzoyl)-phenoxy]-eth-
oxy}-phenyl)-2-(1-methyl-3-oxo-3-phenyl-propenylami-
no)-propionic acid (4h). Compound 4h was synthesized
according to procedure as mentioned in 6.1.2 and
6.1.3 by condensing (S)-3-(4-hydroxy-phenyl)-2-(meth-
6.1.11. (S)-2-(2-Benzoyl-cyclohex-1-enylamino)-3-{4-[2-
(9H-carbazol-4-yloxy)-ethoxy]-phenyl}-propionic acid
(6b). Compound 6b was synthesized from (S)-2-(2-ben-
zoyl-cyclohex-1-enylamino)-3-{4-[2-(9H-carbazol-4-
yloxy)-ethoxy]-phenyl}-propionic acid methyl ester
according to procedure as mentioned in 6.1.3. 1H
NMR (CDCl3) d 1.41–1.46 (m, 2H), 1.67–1.77(m, 4H),
2.31 (m, 2H), 2.99 (m, 2H), 4.30–4.45 (m, 5H), 6.64
(m, 2H), 7.00 (m, 1H), 7.09 (m, 2H), 7.18 (m, 2H),
yl-3-oxo-3-phenyl-propenylamino)-propionic
acid
methyl ester with [4-(2-bromo-ethoxy)-phenyl]-(4-chlo-
ro-phenyl)-methanone. 1H NMR (CDCl3) d 1.94 (s,
3H), 3.04–3.14 (m, 2H), 4.26 (m, 4H), 4.38 (m, 1H),
5.65 (s, 1H), 6.89 (d, J = 8.4 Hz, 2H), 7.07 (d,
J = 9 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 7.5 (d,
J = 8.1 Hz, 2H), 7.58–7.77 (m, 7H), 7.93 (d,
J = 9 Hz, 2H). MS (MALDI): 584 (m/z M+) Anal.

1554
R. Kumar et al. / Bioorg. Med. Chem. 15 (2007) 1547–1555
7.41–7.48 (m, 5H), 7.67 (d, J = 8.7 Hz, 1H), 7.76 (d,
J = 7.8 Hz, 1H), 7.86 (m, 1H), 7.93 (d, J = 8.7 Hz,
1H), 8.06 (d, J = 7.8 Hz, 1H), 12.23 (d, J = 8.7 Hz,
1H). MS (MALDI): 575 (m/z M+) Anal. Calcd for
(C36H34N2O5) C, 75.24; H, 5.96; N, 4.87; Found: C,
75.11; H, 5.48; N, 4.72.
HEK 293T cells were grown in Dulbecco’s modified Ea-
gle’s medium supplemented with 10% fetal bovine serum
(DMEM-FBS) at 37 °C in 5% CO2. At 1 day prior to
transfection, cells were plated to 50–60% confluence in
DMEM containing 10% delipidated FBS (DMEM-
DFBS). Cells were transfected by Superfect as per the
manufacturer’s protocol. At 3 h after transfection, the
reagent was removed and cells were maintained in
DMEM-DFBS. At 42 h after transfection, the cells were
placed in phenol red-free DMEM-DFBS and treated for
18 h with the test compounds or vehicle alone. The cells
were lysed and assayed for luciferase activity. Luciferase
activity was determined by using Lucite kit (Packard,
CT, USA) in a Packard Top count and expressed as fold
activation relative to untreated cells.
6.1.12. (S)-2-(2-Benzoyl-cyclohex-1-enylamino)-3-[4-(2-
carbazol-9-yl-ethoxy)-phenyl]-propionic acid methyl ester
(6c). Compound 6c was synthesized from (S)-2-(2-ben-
zoyl-cyclohex-1-enylamino)-3-(4-hydroxy-phenyl)-pro-
pionic acid methyl ester and carbazole-9-ethyl mesylate
1
according to procedure mentioned in 6.1.2. H NMR
(CDCl3) d 1.39–1.46 (m, 2H), 1.53–1.61 (m, 2H), 2.16–
2.23 (m, 4H), 2.90 (m, 1H), 3.01–3.07 (m, 1H), 3.67 (s,
3H), 4.50 (m, 3H), 4.56 (t, J = 6 Hz, 2H), 6.69 (d,
9i = 7.8 Hz, 2H), 6.95 (d, J = 7.8 Hz, 2H), 7.23–7.26
(m, 2H), 7.32–7.35 (m, 5H), 7.37–7.44 (m, 4H), 8.06
(d, J = 7.8 Hz, 2H), 12.15 (d, J = 8.4 Hz, 1H). 13C
NMR d (CDCl3) 14.05, 20.92, 22.91, 27.16, 36.86,
38.94, 41.89, 52.41, 65.81, 101.30, 108.44 (2), 108.71,
115.56, 115.78 (2), 119.03, 119.51 (2), 120.36 (2),
125.60, 126.43 (2), 127.06 (2), 127.96 (2), 128.51,
130.29 (2), 139.99, 142.22, 155.84, 163.35, 171.81,
194.93 MS (EI): 573 (m/z M+) Anal. Calcd for
(C37H36N2O4) C, 77.60; H, 6.34; N, 4.89; Found: C,
77.21; H, 6.18; N, 4.69.
Acknowledgments
Rakesh Kumar thanks CSIR, New Delhi, for his senior
research fellowship. We thank CSIR and DST, New
Delhi, for funding this project. S.K. acknowledges the
Laboratoire de Dynamique Moleculaire (LDM), IBS,
CEA, Grenoble, France.
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1
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