Synthesis and biological activity evaluation of N-protected isatin derivatives as inhibitors of ICAM-1 expression on human endothelial cells

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Synthesis and biological activity evaluation of N-protected isatin derivativesas inhibitors of ICAM-1 expression on human endothelial cells†

Shashwat Malhotra,a Sakshi Balwani,b Ashish Dhawan,a Brajendra K. Singh,ac Sarvesh Kumar,bd

Rajesh Thimmulappa,d Shyam Biswal,d Carl E. Olsen,e Erik Van der Eycken,c Ashok K. Prasad,a

Balaram Ghosh*b and Virinder S. Parmar*a

Received 15th December 2010, Accepted 6th May 2011

DOI: 10.1039/c0md00262c

Novel N-protected derivatives of substituted isatins have been synthesized and evaluated for their

potency in inhibiting TNF-a-induced ICAM-1 activity on human endothelial cells as a marker for anti-

inflammatory activity. Compound 3pwas found to be most potent in inhibiting the ICAM-1 expression

in a concentration- and time-dependent manner. The structure–activity relationship of these

compounds in inhibiting ICAM-1 expression activity is elucidated in the present study.

Introduction

Leukocytes are the key players in the pathogenesis of multiple

inflammatory disorders including asthma, COPD, atheroscle-

rosis and autoimmune diseases.1–3 Leukocytes in the inflamed

tissue secrete pro-inflammatory mediators, reactive oxygen

species and proteases that cause tissue damage, inflammation

and disease pathogenesis.4–6 Pharmaceutical companies are

designing drugs to limit leukocyte mediated inflammatory

response. However, current anti-inflammatory drugs show

limited efficacy and exhibit severe side effects.7 Therefore, more

specific and potent anti-inflammatory drugs are needed urgently.

Migration of leukocytes from circulating blood to the site of

infection or injury is a key event in the inflammatory response

that occurs through multiple step processes, which involve

sequential capture on, rolling along and firm adhesion to the

microvascular endothelium, followed by transmigration through

the vessel wall and further migration in extravascular tissue.

aBioorganic Laboratory, Department of Chemistry, University of Delhi,Delhi, 110 007, India. E-mail: [email protected].; Fax: +91-11-27667206; Tel: +91-11-27667206bMolecular Immunogenetics Laboratory, CSIR-Institute of Genomics andIntegrative Biology, University of Delhi Campus (North), Mall Road,Delhi, 110 007, India. E-mail: [email protected].; Fax: +91-11-27667471; Tel: +91-11-27662580cLaboratory for Organic & Microwave-Assisted Chemistry (LOMAC),Katholieke Universiteit Leuven, Celestijnenlaan200F, B-3001 Leuven,BelgiumdDepartment of Environmental Health Sciences, Bloomberg School ofPublic Health, Johns Hopkins University, Baltimore, Maryland, 21205,USAeDepartment of Basic Sciences and Environment, University ofCopenhagen, DK-1871 Frederiksberg C, Denmark

† Electronic supplementary information (ESI) available: 1H-NMR &13C-NMR spectra of compounds 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3j, 3l, 3m,3n, 3o and 3p and NOE-1H NMR and NOESY-1H NMR ofcompounds 2a, 2b, 2c and 2d. See DOI: 10.1039/c0md00262c

This journal is ª The Royal Society of Chemistry 2011

Rolling and extravasations of leukocytes are largely mediated by

the surface expression of cell adhesion molecules (CAM) that

includes E-selectin, ICAM-1 and VCAM-1 on endothelial cells.8

Pharmacological inhibition of CAM on endothelial cells is

a promising strategy for therapeutic intervention of inflamma-

tory disorders.9

Oxindoles, particularly isatin (1H-indole-2,3-dione) are endog-

enous compounds ubiquitously present in blood, central nervous

system, body fluids and other human tissues that show a wide

range of biological activities including antibacterial, antifungal,

anticonvulsant, antiviral, and antiproliferative activity.10 A variety

of N-protected isatin derivatives have been reported in the litera-

ture exhibiting a broad range of biological activities.11–18 N-alkyl

and N-acyl isatin derivatives with bromo and chloro substituents

and oxime derivatives have exhibited a broad-spectrum of bio-

logical activities including anti-inflammatory activity.19,20

Recognizing the beneficial pharmacological activities of 2,3-

oxindole derivatives and their Schiff andMannich bases, we have

synthesized novel N-protected derivatives of isatin having

different halogen substituents at the C-5 position in the isatin

ring and evaluated their anti-inflammatory activities using our

cell based assay that measures inhibition of TNF-a induced

ICAM-1 expression on human endothelial cells. The present

study reports anti-inflammatory activities and structure–activity

relationship of the novel isatin compounds.

Results and discussion

Different N-protected derivatives 3a–p were prepared in 70–80%

yields by treating compounds 2a–d with different acylating/

alkylating agents in tert-butanol using potassium tert-butoxide

as a base (Scheme 1). Compounds 2a–dwere obtained exclusively

as E-isomers by treating the commercially available different

substituted isatins 1a–d with ethoxycarbonylmethylene-

triphenyl-phosphorane in glacial acetic acid for 4 h at 80 �C in

Med. Chem. Commun., 2011, 2, 743–751 | 743

http://dx.doi.org/10.1039/c0md00262c






http://pubs.rsc.org/en/journals/journal/MD

http://pubs.rsc.org/en/journals/journal/MD?issueid=MD002008

Scheme 1 Synthetic route to N-protected isatin derivatives.

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65–72% yields (Scheme 1).21–23 The stereochemistry of the

compounds 2a–d was established from their 2D-NOESY and

1D-NOE NMR studies. As shown in Fig. 1, compounds 2a–

d could be formed as either E- or Z-isomers. Careful study of the

interactions of 2D-NOESY and 1D-NOE NMR spectral exper-

iments on compounds 2a–d (Fig. 1) revealed that there is no

interaction between the alkene proton and the aromatic proton

at the C-4 position of the benzene ring, thereby affirming the

exclusive formation of the E-isomers of the compounds 2a–d.

This is also reported in the literature23—a detailed study of the

1D-NOE NMR spectra of compounds having similar structures

was carried out and these were found to have E-geometry around

the double bond.23 If the geometry around the double bond in

these four compounds had been Z, there might have been

interactions between the alkene proton and the aromatic ring

proton at the C-4 position (Fig. 1), and the NOE effect would

have been observed between these two protons in the

2D-NOESY and 1D-NOE NMR spectral experiments on

compounds 2a–d. Compound 2c is novel and its melting point

and spectral data have not been reported earlier.

All the synthesized compounds 3a–p are novel and were

characterized by spectroscopic techniques like NMR, IR, mass

spectrometry, etc. The known compound 3a was characterized

by comparing its spectral data and melting point with that

Fig. 1 NOE interactions in E- and Z-isomers of compounds 2a–d.

744 | Med. Chem. Commun., 2011, 2, 743–751

reported in the literature.24 The synthesized compounds 2a–2d

and 3a–3p were then screened for their anti-inflammatory

activities by measuring TNF-a induced ICAM-1 expression

inhibition and the structure–activity relationship was studied.

Compounds 3a–p potently inhibited ICAM-1 expression.

Compound 3p showed highest inhibition (�93%) of ICAM-1

expression at the maximal tolerable dose of 100 mM (Table 1)

with an IC50 value of 10 mM. It is important to point out that the

IC50 values of 3a–p, particularly of 3p, are found to be much

lower than the commonly used anti-inflammatory agents, e.g.,

aspirin (IC50 5 mM), N-acetyl cysteine (IC50 10 mM), diclofenac

(IC50 0.75 mM) etc. indicating that these compounds could be

useful as lead molecules.25–27

Structure–activity relationship

To examine the role of different modifications (Fig. 2), such as

the effect of: (i) N-protection in compounds 2a–d, (ii) halogen

substituents at the C-5 carbon of the isatin ring, (iii) the length of

the alkyl chain in the alcohol part of the N-protected isatin

derivatives and (iv) the methylene linker between the ester

carbonyl moiety and the nitrogen atom of the isatin ring on the

Fig. 2 Different modifications on isatin ring.



Fig. 3 Effect of N-protection of isatin derivatives on ICAM-1 expres-

sion inhibitory activity.

Fig. 4 Effect of halogen substitution on ICAM-1 expression inhibitory

activity of N-protected derivatives of isatin.

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ICAM-1 expression inhibition, we have prepared different

N-protected derivatives, i.e. 3a–p of the substituted isatins 1a–d.

The results showed a good structure–activity relationship that

would be useful in understanding the functional components

responsible for the inhibitory activity of these compounds.

(i) Effect of N-protection. On comparing compound 2a with

compounds 3a, 3e, 3i and 3m in which the nitrogen atom of the

isatin ring has been protected (Table 1), we have found in each

case a decrease in IC50 value (Fig. 3), suggesting that ICAM-1

expression inhibitory activity increases with the protection of the

nitrogen atom of the isatin ring. Similar observations were

revealed on comparing the compound 2bwith the compounds 3b,

3f, 3j and 3n, 2c with the compounds 3c, 3g, 3k and 3o; and the

compound 2d with the compounds 3d, 3h, 3l and 3p.

(ii) Effect of halogen substituents at the C-5 carbon of the

isatin ring. From the data presented in Table 1, we have observed

that by increasing the atomic size of halogen substituent at the

C-5 carbon atom in the N-protected derivatives of isatin, the

ICAM-1 expression inhibitory activity gradually increases from

fluorine atom to bromine. Thus the IC50 value decreases from

fluorine to bromine in the same series of compounds, i.e. 3b–d,

3f–h, 3j–l and 3n–p as shown in Fig. 4.

(iii) Effect of alkyl chain length in alcohol part of the N-pro-

tected isatin derivatives. The ICAM-1 expression inhibitory

activity increased with increase in the length of the alkyl chain in

the alcohol part of isatin derivatives 3a–p. Comparison of IC50

values of compounds 3a–d and 3e–h revealed an increase in activity

when the methyl group was replaced by an ethyl group (Table 1). A

similar trend was observed between compounds 3i–l and 3m–p

when the methyl group is replaced by an ethyl group (Table 1).

(iv) Effect of methylene linker. The ICAM-1 expression

inhibitory activity increased with the addition of a methylene linker

between ester carbonyl moiety and the nitrogen atom (N-1) of the

Table 1 ICAM-1 expression inhibitory activity of isatin derivatives 2a–d an

S. No Compound (Structure given in Scheme 1) % Vi

1. 2a 962. 2b 953. 2c 964. 2d 975. 3a 986. 3b 977. 3c 968. 3d 989. 3e 9510. 3f 9611. 3g 9712. 3h 9813. 3i 9714. 3j 9615. 3k 9716. 3l 9917. 3m 9818. 3n 9719. 3o 9620. 3p 97


isatin ring. On comparing the IC50 values of compounds 3f–h with

those of compounds 3n–p, we found a decrease in the IC50 value in

each case, as shown in Fig. 5 and Table 1.

Time kinetics of ICAM-1 inhibition by compound 3p

The time kinetics of ICAM-1 inhibition by the most active

compound of the series, 3p on endothelial cells was also

d 3a–p at 100 mM Maximum Tolerable Dose (MTD)

ability% ICAM-1expression inhibition

ICAM-1 expressioninhibition IC50 (mM)

40 � 3.5 >10031 � 2.5 >10056 � 2.0 100 � 2.258 � 2.5 100 � 2.557 � 1.9 78 � 3.361 � 1.5 70 � 4.477 � 4.7 40 � 4.588 � 3.0 25 � 1.566 � 3.1 50 � 3.872 � 2.9 53 � 2.783 � 1.9 30 � 2.791 � 1.4 15 � 1.658 � 2.1 70 � 2.252 � 1.4 75 � 3.267 � 2.4 40 � 2.681 � 3.6 25 � 3.274 � 4.2 55 � 2.263 � 2.3 40 � 3.374 � 2.4 25 � 4.493 � 1.9 10 � 1.3

Med. Chem. Commun., 2011, 2, 743–751 | 745


Fig. 5 Effect of methylene linker on ICAM-1 expression inhibitory

activity of N-protected isatin derivatives.

Fig. 7 Effect of functional groups on ICAM-1 expression inhibitory

activity of the N-protected isatin derivatives.

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investigated. For this, the cells were incubated with the

maximum tolerable dose (100 mM) of 3p for time points before,

simultaneously and after TNF-a induction followed by the Cell-

Fig. 6 A. Time kinetics of ICAM-1 inhibition by 3p. The endothelial

cells were treated with 3p at various time points followed by induction

with TNF-a for 16 h. The ICAM-1 expression levels were measured by

Cell-ELISA. B. Dose-dependent inhibition of ICAM-1 expression on

human endothelial cells by 3p. The cells were treated with 3p at various

concentrations followed by induction with TNF-a for 16 h. The ICAM-1

expression levels were measured by Cell-ELISA. The results are expressed

as mean � SEM.

746 | Med. Chem. Commun., 2011, 2, 743–751

ELISA for measurement of ICAM-1 expression. A time-depen-

dent ICAM-1 inhibition pattern was observed where the

maximum inhibition of ICAM-1 expression occurred when 3p

was added prior to induction with TNF-a (Fig. 6A).

Dose-dependent inhibition of ICAM-1 expression by compound

3p

The dose-dependent effect of the most active compound of the

series, 3p on ICAM-1 expression was also investigated. For this,

the cells were pre-treated with various concentrations of 3p for

2 h followed by TNF-a induction for 16 h. The ICAM-1 protein

levels were measured by Cell-ELISA. The percentage ICAM-1

inhibition at each concentration of 3p were calculated. These

percentages were plotted against the log concentrations of

compound 3p to get a dose-response curve. The results showed

that compound 3p inhibited the TNF-a-induced ICAM-1

expression on human endothelial cells in a dose-dependent

manner with an IC50 value of 10 mM (Fig. 6B).

In summary, the ICAM-1 expression inhibitory activity of the

N-protected derivatives of isatin (i) increases with protection of

the nitrogen atom (N-1) of the isatin ring, (ii) increases with

increase in the atomic size of the halogen atom at the C-5 carbon

atom of the isatin ring, (iii) increases with increase in chain length

of the alcohol moiety, and (iv) increases with the introduction of

a methylene linker between the ester carbonyl moiety and the

nitrogen atom of the isatin ring (Fig. 7).

Conclusions

We have synthesized fifteen novel bioactive N-protected deriva-

tives of isatin. We report for the first time, the anti-inflammatory

activity of these novel compounds. Compound 3p was found to

be the most potent in inhibiting the ICAM-1 expression in

a concentration- and time-dependent manner. The structure–

activity relationship for these compounds has been discussed

extensively in the present study.

Experimental

Analytical TLCs were performed on Merck silica gel 60 F254

plates. All flash chromatographic separations were performed on

100–200 mesh silica gel. IR spectra were recorded on a Perkin-

Elmer 2000 FT-IR spectrometer. The 1H NMR and 13C NMR

spectra (in CDCl3) were recorded on a Bruker AC-300 Avance



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spectrometer operating at 300 MHz and at 75.5 MHz, respec-

tively using TMS as internal standard. The NOE 1H NMR and

NOESY 1H NMR spectra were recorded on 400, 500 and

700MHz instrument. The chemical shift values are on d scale and

the coupling constants (J) are in Hz. The HRMS determinations

were made in FAB positive mode on a JEOL JMS-AX505W

high-resolution mass spectrometer using bis-hydrox-

yethyldisulfide (HEDS) doped with sodium acetate as matrix.

Microwave reactions were performed in a microwave oven of

850 W 1.2 Cft (33 L, Infodisplay, Sharp Carosel). Melting points

were recorded in a sulfuric acid bath and are uncorrected.

Materials

Materials were procured from commercial vendors and were

used without further purification unless otherwise noted. Petro-

leum ether and ethyl acetate were distilled over P2O5 and K2CO3,

respectively prior to use.

General method for the preparation of compounds 2a–d

A mixture of compound 1a–d (0.05 mol) and commercially

available ethoxycarbonylmethylene-triphenyl-phosphorane

(0.05 mol) in glacial acetic acid (60 mL) was heated for 4 h at

80 �C.18 Acetic acid was removed under vacuum and the residue

was washed onto a filter funnel with a small quantity of meth-

anol. Recrystallization from ethanol gave compounds 2a–d as

orange solids in 65–72% yields.21–23

(E)-Ethyl (5-chloro-2-oxo-1,2-dihydro-3(H)-indol-3-ylidene)

acetate (2c)

Obtained as an orange solid (17.2 gm, 71% yield), mp 160–162 �C(from petroleum ether–ethyl acetate), Rf: 0.45(4:1 petroleum

ether–ethyl acetate); nmax(KBr)/cm�1: 3166, 1710, 1648, 1613,

1453, 1209, 1027, 819; 1H NMR (300MHz, CDCl3): d 1.29(3H, t,

J ¼ 6.9 Hz, ¼ CHCOOCH2CH3), 4.25(2H, q, J ¼ 6.9 Hz, ]

CHCOOCH2CH3), 6.59(1H, s, ]CHCO–), 6.85(1H, d, J ¼ 8.4

Hz, C-7H), 7.39(1H, d, J ¼ 6.6 Hz, C-6H), 8.35(1H, s, C-4H),

8.53(1H, s, NH); 13C NMR (75.5 MHz, CDCl3): d 13.9(]

CHCOOCH2CH3), 61.2(]CHCOOCH2CH3), 111.7(C-7), 120.9

(C-5), 122.2(]CH–), 125.7(C-4), 127.5(C-3), 132.3(C-6), 137.4

(C-8), 143.7(C-9), 164.9(]CHCOOC2H5) and 167.4(C-2).

HRMS m/z Calcd for C12H10ClNO3Na [M + Na]+: 274.0241.

Found: 274.0238.

General method for the preparation of N-protected isatin

derivatives 3a–p

Compound 2a–d (0.001 mol) was dissolved in tert-butanol

(15 mL) and potassium tert-butoxide (0.001 mol) was added to it

in an ice bath. The reaction mixture was stirred for 15 min

followed by dropwise addition of the appropriate acylating/

alkylating agent (0.001 mol) for 15 min.

The reaction mixture was then heated at 60 �C for 5–6 h and

the progress of the reaction was monitored by TLC; the reaction

mixture was then chromatographed over silica gel using ethyl

acetate–petroleum ether (30 : 70) as eluent to afford the

compounds 3a–p as yellow to orange solids in 70–80% yields.


(E)-Methyl 3-(2-ethoxy-2-oxoethylidene)-2-oxoindoline-1-

carboxylate (3a)

Obtained as a bright yellow solid (210 mg, 75%), mp 109–111 �C(lit. mp24 110–112 �C).

(E)-Methyl 3-(2-ethoxy-2-oxoethylidene)-5-fluoro-2-

oxoindoline-1-carboxylate (3b)

Obtained as a dark yellow solid (270 mg, 72% yield), mp 96–

98 �C (from petroleum ether–ethyl acetate), Rf: 0.54(4 : 1

petroleum ether–ethyl acetate); nmax(KBr)/cm�1: 3418, 2926,

1765, 1733, 1643, 1604, 1471, 1343, 1199, 1154, 1025; 1H NMR

(300 MHz, CDCl3): d 1.39(3H, t, J ¼ 7.2 Hz, ]

CHCOOCH2CH3), 4.04(3H, s, –OCH3), 4.32(2H, q,

J ¼ 7.2 Hz, ]CHCOOCH2CH3), 6.98(1H, s,]CHCO–), 7.14–

7.21(1H, m, C-6H), 7.97–8.02(1H, m, C-4H), 8.48–8.52(1H, m,

C-7H); 13C NMR (75.5 MHz, CDCl3): d 14.1(]

CHCOOCH2CH3), 54.1(–OCH3), 61.6(]CHCOOCH2CH3),

115.4(d, J¼ 26.4 Hz, C-4), 116.2(d, J¼ 8.3 Hz, C-6), 119.4(d, J¼23.4 Hz, C-7), 121.3(d, J¼ 10.5 Hz, C-3), 125.1(]CH–), 135.5(d,

J ¼ 3.2 Hz, C-8), 137.4(C-9), 151.0(NCOOCH3), 159.7(d, J ¼243.8 Hz, C-5), 164.9 (C-2) and 165.1(]CHCOOC2H5). HRMS

m/z Calcd for C14H13FNO5 [M + H]+: 294.0772. Found:

294.0767.

(E)-Methyl 5-chloro-3-(2-ethoxy-2-oxoethylidene)-2-

oxoindoline-1-carboxylate (3c)

Obtained as a light yellow solid (480 mg, 78% yield), mp 124–



1765, 1734, 1713, 1640, 1600, 1460, 1352, 1197, 1026, 774; 1H

NMR (300 MHz, CDCl3): d 1.40(3H, t, J ¼ 7.2 Hz, ]

CHCOOCH2CH3), 4.04(3H, s, –OCH3), 4.34(2H, q,

J ¼ 7.2 Hz, ]CHCOOCH2CH3), 6.97(1H, s, ]CHCO–), 7.41–

7.45(1H, m, C-6H), 7.95(1H, d, J ¼ 9.0 Hz, C-4H), 8.74(1H, d, J

¼ 2.1 Hz, C-7H); 13C NMR (75.5 MHz, CDCl3): d 14.1(]

CHCOOCH2CH3), 54.2(–OCH3), 61.7(]CHCOOCH2CH3),

116.2(C-7), 121.4(C-5), 125.2(]CH–), 128.2(C-4), 130.6(C-3),

132.5(C-6), 135.0(C-8), 139.7(C-9), 150.8(NCOOCH3) and 164.9

(C-2 and ¼ CHCOOC2H5). HRMS m/z Calcd for C14H13ClNO5

[M + H]+: 310.0477. Found: 310.0472.

(E)-Methyl 5-bromo-3-(2-ethoxy-2-oxoethylidene)-2-

oxoindoline-1-carboxylate (3d)




1766, 1734, 1712, 1638, 1595, 1458, 1352, 1195, 1107, 1026, 774;1H NMR (300 MHz, CDCl3): d 1.40(3H, t, J ¼ 7.2 Hz, ]

CHCOOCH2CH3), 4.04(3H, s, –OCH3), 4.33–4.40(2H, m, ]

CHCOOCH2CH3), 6.97(1H, s, ]CHCO–), 7.58(1H, d, J ¼ 8.7

Hz, C-6H), 7.91(1H, d, J ¼ 8.7 Hz, C-7H), 8.89(1H, brs, C-4H);13C NMR (75.5 MHz, CDCl3): d 14.1(]CHCOOCH2CH3), 54.2

(–OCH3), 61.7(]CHCOOCH2CH3), 116.5(C-7), 118.1(C-5),

121.8(C-4), 125.2(]CH–), 131.1(C-6), 134.8(C-3), 135.4(C-8),

140.2(C-9), 150.8(NCOOCH3), 164.7(C-2) and 164.9(]

Med. Chem. Commun., 2011, 2, 743–751 | 747


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CHCOOC2H5). HRMS m/z Calcd for C14H13BrNO5 [M + H]+:

353.9972. Found: 353.9969.

(E)-Ethyl 3-(2-ethoxy-2-oxoethylidene)-2-oxoindoline-1-

carboxylate (3e)




1758, 1734, 1710, 1638, 1597, 1464, 1371, 1198, 1090, 1027,

788; 1H NMR (300 MHz, CDCl3): d 1.31 & 1.39 (6H, 2t, J ¼ 6.9

Hz,]CHCOOCH2CH3 & –COOCH2CH3), 4.27 & 4.42(4H, 2q,

J ¼ 7.2‘ Hz, ]CHCOOCH2CH3 & –COOCH2CH3), 6.86(1H,

s, ]CHCO–), 7.12–7.19(1H, m, C-5H), 7.38(1H, t, J ¼ 7.8 Hz,

C-6H), 7.90(1H, d, J¼ 8.1 Hz, C-4H), 8.62(1H, d, J¼ 7.8 Hz, C-

7H); 13C NMR (75.5 MHz, CDCl3): d 13.1 & 13.2(]

CHCOOCH2CH3 & –COOCH2CH3), 60.4 & 62.6(]

CHCOOCH2CH3 & –COOCH2CH3), 114.0(C-7), 119.1(C-6),

122.5(C-4), 123.8(]CH–), 127.3(C-5), 131.8(C-3), 135.0(C-8),

140.4(C-9), 149.4(NCOOC2H5), 164.2(C-2) and 164.5(]

CHCOOC2H5). HRMSm/z Calcd for C15H15NO5Na [M +Na]+:

312.0842. Found: 312.0832.

(E)-Ethyl 3-(2-ethoxy-2-oxoethylidene)-5-fluoro-2-oxoindoline-

1-carboxylate (3f)




1763, 1735, 1712, 1642, 1598, 1476, 1371, 1334, 1208, 1159, 1027,

828; 1H NMR (300 MHz, CDCl3): d 1.39 & 1.46 (6H, 2t, J ¼ 7.2

Hz, ]CHCOOCH2CH3 & –COOCH2CH3), 4.35 & 4.49 (4H,

2q, J ¼ 7.2 Hz, ]CHCOOCH2CH3 & –COOCH2CH3), 6.96

(1H, s, ]CHCO–), 7.13–7.20 (1H, m, C-6H), 7.94–7.99 (1H, m,

C-4H), 8.48–8.51 (1H, m, C-7H); 13C NMR (75.5 MHz, CDCl3):

d 14.1 & 14.2 (]CHCOOCH2CH3 & –COOCH2CH3), 61.6 &

63.7 (]CHCOOCH2CH3 & –COOCH2CH3), 115.4 (d, J ¼ 26.4

Hz, C-4), 116.1 (d, J¼ 7.55 Hz, C-6), 119.3 (d, J¼ 24.1 Hz, C-7),

121.3 (d, J ¼ 9.8 Hz, C-3), 124.8 (]CH–), 135.6 (d, J ¼ 3.0 Hz,

C-8), 137.5 (d, J ¼ 2.2 Hz, C-9), 150.4 (NCOOC2H5), 159.6 (d,

J ¼ 243.1 Hz, C-5), 164.9 (C-2) and 165.1(]CHCOOC2H5).

HRMS m/z Calcd for C15H15FNO5 [M + H]+: 308.0929. Found:

308.0925.

(E)-Ethyl 5-chloro-3-(2-ethoxy-2-oxoethylidene)-2-oxoindoline-

1-carboxylate (3g)




1765, 1731, 1703, 1642, 1594, 1458, 1371, 1323, 1206, 1171, 1107,

1029, 776; 1H NMR (300 MHz, CDCl3): d 1.39 & 1.46 (6H, 2t,

J ¼ 7.2 Hz, ]CHCOOCH2CH3 & –COOCH2CH3), 4.35 & 4.44

(4H, 2q, J ¼ 7.2 Hz, ]CHCOOCH2CH3 & –COOCH2CH3),

6.97 (1H, s,]CHCO–), 7.41–7.45 (1H, m, C-6H), 7.93–7.99 (1H,

m, C-4H), 8.74 (1H, d, J ¼ 2.1 Hz, C-7H); 13C NMR (75.5 MHz,

CDCl3): d 14.1 & 14.2 (]CHCOOCH2CH3 & –COOCH2CH3),

61.7 & 63.8 (]CHCOOCH2CH3 & –COOCH2CH3), 116.2(C-7),

121.4(C-5), 125.0(]CH–), 128.2(C-4), 130.5(C-3), 132.5(C-6),

135.1(C-8), 139.8(C-9), 150.3(NCOOC2H5) and 164.9(C-2

748 | Med. Chem. Commun., 2011, 2, 743–751

and]CHCOOC2H5). HRMSm/zCalcd for C15H15ClNO5 [M +

H]+: 324.0533. Found: 324.0522.

(E)-Ethyl 5-bromo-3-(2-ethoxy-2-oxoethylidene)-2-oxoindoline-

1-carboxylate (3h)




1767, 1733, 1705, 1633, 1593, 1459, 1369, 1324, 1204, 1105, 1024,

821; 1H NMR (300 MHz, CDCl3): d 1.4 & 1.46(6H, 2t, J ¼ 7.2

Hz,]CHCOOCH2CH3& –COOCH2CH3), 4.36 & 4.49 (4H, 2q,

J ¼ 7.2 Hz, ]CHCOOCH2CH3 & –COOCH2CH3), 6.96(1H,

s, ]CHCO–), 7.58(1H, dd, J ¼ 1.8, 2.1 Hz, C-6H), 7.88(1H, d,

J ¼ 8.7 Hz, C-4H), 8.88(1H, d, J ¼ 2.1 Hz, C-7H); 13C NMR

(75.5 MHz, CDCl3): d 14.1 & 14.2 (]CHCOOCH2CH3 &

–COOCH2CH3), 61.7 & 63.8 (]CHCOOCH2CH3 &

–COOCH2CH3), 116.5 (C-7), 118.0 (C-5), 121.7 (C-4), 125.0 (]

CH–), 131.0 (C-6), 134.9 (C-3), 135.4 (C-8), 140.3 (C-9), 150.3

(NCOOC2H5), 164.8 (C-2) and 164.9 (]CHCOOC2H5). HRMS

m/z Calcd for C15H15BrNO5 [M + H]+: 368.0128. Found:

368.0119.

(E)-Methyl 2-[3-(2-ethoxy-2-oxoethylidene)-2-oxoindoline-1-yl]

acetate (3i)




1740, 1713, 1652, 1611, 1473, 1345, 1227, 1199, 1016, 755; 1H

NMR (300 MHz, CDCl3): d 1.37 (3H, t, J ¼ 7.2 Hz, ]

CHCOOCH2CH3), 3.75 (3H, s, –CH2COOCH3), 4.33 (2H, q,

J ¼ 7.2 Hz, ]CHCOOCH2CH3), 4.50 (2H, s, –CH2COOCH3),

6.69 (1H, d, J ¼ 7.8 Hz, C-7H), 6.94 (1H, s, ]CHCO–), 7.09

(1H, t, J ¼ 7.8 Hz, C-5H), 7.35 (1H, t, J ¼ 7.8 Hz, C-6H), 8.60

(1H, d, J ¼ 7.8 Hz, C-4H); 13C NMR (75.5 MHz, CDCl3): d 14.1

(]CHCOOCH2CH3), 41.2 (–CH2COOCH3), 52.6

(–CH2COOCH3), 61.2 (]CHCOOCH2CH3), 108.1 (C-7), 119.8

(C-6), 123.0 (C-4), 123.2 (]CH–), 128.9 (C-5), 132.4 (C-3), 137.1

(C-8), 144.5 (C-9), 165.4 (–CH2COOCH3), 167.53(C-2) and 167.8

(]CHCOOC2H5). HRMS m/z Calcd for C15H15NO5Na [M +

Na]+: 312.0842. Found: 312.0838.

(E)-Methyl 2-[3-(2-ethoxy-2-oxoethylidene)-5-flouro-2-

oxoindoline-1-yl] acetate (3j)




1766, 1749, 1709, 1652, 1617, 1491, 1351, 1204, 821; 1H NMR

(300 MHz, CDCl3): d 1.38 (3H, t, J ¼ 7.2 Hz, ]



6.60–6.64 (1H, m, C-7H), 6.97 (1H, s,]CHCO–), 7.04–7.10 (1H,

m, C-6H), 8.40–8.44 (1H, m, C-4H); 13C NMR (75.5 MHz,

CDCl3): d 14.1 (]CHCOOCH2CH3), 41.3 (–CH2COOCH3),

52.7 (–CH2COOCH3), 61.4 (]CHCOOCH2CH3), 108.5 (d, J ¼7.5 Hz, C-4), 116.6 (d, J ¼ 27.1 Hz, C-6), 118.7 (d, J ¼ 24.1 Hz,

C-7), 120.8 (d, J ¼ 9.8 Hz, C-3), 124.4 (]CH–), 136.8 (d, J ¼ 3.0

Hz, C-8), 140.6 (C-9), 159.1 (d, J ¼ 240.8 Hz, C-5), 165.2



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(–CH2COOCH3), 167.2 (C-2) and 167.6 (]CHCOOC2H5).

HRMS m/z Calcd for C15H14FNO5Na [M + Na]+: 330.0748.

Found: 330.0741.

(E)-Methyl 2-[5-chloro-3-(2-ethoxy-2-oxoethylidene)-2-

oxoindoline-1-yl] acetate (3k)




1755, 1713, 1651, 1607, 1477, 1347, 1200, 1025, 826; 1H NMR

(300 MHz, CDCl3): d 1.38 (3H, t, J ¼ 7.2 Hz, ]

CHCOOCH2CH3), 3.76 (3H, s, –CH2COOCH3), 4.35 (2H, q, J

¼ 7.2 Hz, ]CHCOOCH2CH3), 4.49 (2H, s, –CH2COOCH3),

6.63 (1H, d, J ¼ 8.4 Hz, C-7H), 6.97 (1H, s, ]CHCO–), 7.31–

7.35 (1H, m, C-6H), 8.63 (1H, d, J ¼ 2.1 Hz, C-4H); 13C NMR

(75.5 MHz, CDCl3): d 14.1 (]CHCOOCH2CH3), 41.2

(–CH2COOCH3), 52.7 (–CH2COOCH3), 61.5 (]

CHCOOCH2CH3), 109.0 (C-7), 121.0 (C-5), 124.5 (]CH–),

128.6 (C-3), 129.0 (C-4), 132.0 (C-6), 136.2 (C-8), 142.9 (C-9),

165.1 (–CH2COOCH3), 167.0 (C-2) and 167.5 (]

CHCOOC2H5). HRMS m/z Calcd for C15H14ClNO5Na [M +

Na]+: 346.0453. Found: 346.0451.

(E)-Methyl 2-[5-bromo-3-(2-ethoxy-2-oxoethylidene)-2-

oxoindoline-1-yl] acetate (3l)




1754, 1712, 1650, 1603, 1438, 1345, 1199, 1025, 823; 1H NMR

(300 MHz, CDCl3): d 1.39 (3H, t, J ¼ 7.2 Hz, ]



6.58 (1H, d, J ¼ 8.4 Hz, C-7H), 6.97 (1H, s, ]CHCO–), 7.47–

7.50 (1H, m, C-6H), 8.78 (1H, s, C-4H); 13C NMR (75.5 MHz,

CDCl3): d 14.1 (]CHCOOCH2CH3), 41.2 (–CH2COOCH3),

52.7 (–CH2COOCH3), 61.5 (]CHCOOCH2CH3), 109.5 (C-7),

115.9 (C-5), 121.4 (C-4), 124.6 (]CH–), 131.8 (C-6), 134.9 (C-3),

136.1 (C-8), 143.4 (C-9), 165.1 (–CH2COOCH3), 166.9 (C-2) and

167.5 (]CHCOOC2H5). HRMS m/z Calcd for C15H15BrNO5

[M + H]+: 368.0128. Found: 368.0118.

(E)-Ethyl 2-[3-(2-ethoxy-2-oxoethylidene)-2-oxoindoline-1-yl]

acetate (3m)




1735, 1711, 1649, 1606, 1474, 1344, 1222, 1025, 755; 1H NMR

(300 MHz, CDCl3): d 1.26 & 1.37 (6H, 2t, J ¼ 7.2 Hz, ]

CHCOOCH2CH3 & –CH2COOCH2CH3), 4.21 & 4.33 (4H, 2q,

J ¼ 7.2 Hz, ]CHCOOCH2CH3 & –CH2COOCH2CH3), 4.48

(2H, s, –CH2COOCH2CH3), 6.69 (1H, d, J¼ 7.8 Hz, C-7H), 6.94

(1H, s, ]CHCO–), 7.08 (1H, t, J ¼ 7.5 Hz, C-5H), 7.35 (1H, t,

J ¼ 7.5 Hz, C-6), 8.59 (1H, d, J ¼ 7.8 Hz, C-4H); 13C NMR (75.5

MHz, CDCl3): d 14.0 & 14.1 (]CHCOOCH2CH3 &

–CH2COOCH2CH3), 41.4 (–CH2COOCH2CH3), 61.2 & 61.8 (]

CHCOOCH2CH3 & –CH2COOCH2CH3), 108.1 (C-7), 119.8 (C-

6), 123.0 (C-4), 123.1 (]CH–), 128.9 (C-5), 132.3 (C-3), 137.2 (C-


8), 144.6 (C-9), 165.5 (–CH2COOC2H5), 167.2 (C-2) and 167.5

(]CHCOOC2H5). HRMS m/z Calcd for C16H17NO5Na [M +

Na]+: 326.0999. Found: 326.0985.

(E)-Ethyl 2-[3-(2-ethoxy-2-oxoethylidene)-5-flouro-2-

oxoindoline-1-yl] acetate (3n)


131 �C (from petroleum ether–ethyl acetate), Rf: 0.58 (4 : 1


1741, 1709, 1654, 1617, 1492, 1372, 1351, 1217, 1025, 777; 1H

NMR (300 MHz, CDCl3): d 1.26 & 1.38 (6H, 2t, J ¼ 7.2 Hz, ]



(2H, s, –CH2COOCH2CH3), 6.60–6.64 (1H, m, C-7H), 6.97 (1H,

s, ]CHCO–), 7.04–7.10 (1H, m, C-6H), 8.40–8.43 (1H, m, C-

4H); 13C NMR (75.5 MHz, CDCl3): d 14.0 & 14.1 (]

CHCOOCH2CH3 & –CH2COOCH2CH3), 41.4

(–CH2COOCH2CH3), 61.4 & 61.9 (]CHCOOCH2CH3 &

–CH2COOCH2CH3), 108.6 (d, J ¼ 8.3 Hz, C-4), 116.6 (d, J ¼36.9 Hz, C-6), 118.7 (d, J ¼ 24.9 Hz, C-7), 120.7 (d, J ¼ 7.1 Hz,

C-3), 124.3 (]CH–), 136.8 (d, J ¼ 2.2 Hz, C-8), 140.7 (C-9),

159.0 (d, J¼ 240.0 Hz, C-5), 165.2 (–CH2COOC2H5), 167.1 (C-2)

and 167.3 (]CHCOOC2H5). HRMS m/z Calcd for

C16H16FNO5Na [M + Na]+: 344.0905. Found: 344.0897.

(E)-Ethyl 2-[5-chloro-3-(2-ethoxy-2-oxoethylidene)-2-

oxoindoline-1-yl] acetate (3o)


165‘ �C (from petroleum ether–ethyl acetate), Rf: 0.58 (4 : 1


1741, 1709, 1652, 1610, 1443, 1369, 1202, 1020, 817; 1H NMR

(300 MHz, CDCl3): d 1.27 & 1.39 (6H, 2t, J ¼ 6.9, 7.2 Hz, ]



(2H, s, –CH2COOCH2CH3), 6.64 (1H, d, J¼ 8.4 Hz, C-7H), 6.98

(1H, s,]CHCO–), 7.32–7.36 (1H, m, C-6H), 8.65 (1H, d, J¼ 1.8

Hz, C-4H); 13C NMR (75.5 MHz, CDCl3): d 14.0 & 14.1 (]

CHCOOCH2CH3 & –CH2COOCH2CH3), 41.4

(–CH2COOCH2CH3), 61.5 & 61.9 (]CHCOOCH2CH3 &

–CH2COOCH2CH3), 109.1 (C-7), 121.0 (C-5), 124.5 (]CH–),

128.6 (C-3), 129.0 (C-4), 132.0 (C-6), 136.3 (C-8), 143.0 (C-9),

165.1 (–CH2COOC2H5), 167.0 (C-2) and 167.1 (]

CHCOOC2H5). HRMS m/z Calcd for C16H17ClNO5 [M + H]+:

338.0790. Found: 338.0782.

(E)-Ethyl 2-[5-bromo-3-(2-ethoxy-2-oxoethylidene)-2-

oxoindoline-1-yl] acetate (3p)


154 �C (from petroleum ether–ethyl acetate), Rf: 0.59 (4 : 1


1741, 1709, 1607, 1437, 1371, 1202, 1123, 1025, 816; 1H NMR

(300 MHz, CDCl3): d 1.26 & 1.38 (6H, 2t, J ¼ 7.2 Hz, ]


J ¼ 6.9, 7.5 Hz, ]CHCOOCH2CH3 & –CH2COOCH2CH3),

4.47 (2H, s, –CH2COOCH2CH3), 6.58 (1H, d, J¼ 8.1 Hz, C-7H),

6.96 (1H, s, ]CHCO–), 7.46–7.49 (1H, m, C-6H), 8.77 (1H, d,

J ¼ 1.5 Hz, C-4H); 13C NMR (75.5 MHz, CDCl3): d 14.0 & 14.1

Med. Chem. Commun., 2011, 2, 743–751 | 749


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(]CHCOOCH2CH3 & –CH2COOCH2CH3), 41.4

(–CH2COOCH2CH3), 61.5 & 61.9 (]CHCOOCH2CH3

& –CH2COOCH2CH3), 109.6 (C-7), 115.8 (C-5), 121.4 (C-4),

124.5 (]CH–), 131.7 (C-6), 134.8 (C-3), 136.1 (C-8), 143.5 (C-9),

165.1 (–CH2COOC2H5) and 166.9 (C-2 and ]CHCOOC2H5).

HRMSm/z Calcd for C16H17BrNO5 [M +H]+: 382.0285. Found:

382.0286.

Cells and cell culture

Primary endothelial cells were isolated from human umbilical

cord using mild trypsinization.28 The cells were grown in M199

medium supplemented with 15% heat inactivated fetal calf

serum, 2 mM L-glutamine, 100 units ml�1 penicillin, 100 mg ml�1

streptomycin, 0.25 mg ml�1 amphotericin B, endothelial cell

growth factor (50 mg ml�1). At confluence, the cells were sub-

cultured using 0.05% trypsin-0.01 M EDTA solution and were

used between passages three to four.

Cell viability assay

The cytotoxicity of these compounds was analyzed by colori-

metric MTT (methylthiazolydiphenyl-tetrazolium bromide)

assay as described.28 Briefly, endothelial cells were treated with

DMSO alone (0.25% as vehicle) or with different concentrations

of compounds for 24 h. The medium was removed and 100 ml

MTT (2.5 mgml�1 in serum free medium) was added to each well.

The MTT was removed after 4 h, cells were washed out with PBS

and 100 ml DMSO was added to each well to dissolve water

insoluble MTT-formazan crystals. Absorbance was recorded at

570 nm in an ELISA reader (Bio-Rad, Model 680, USA). All

experiments were performed at least 3 times in triplicate wells.

Cell based-ELISA for measurement of ICAM-1 activity

Cell-ELISA was used for measuring the expression of ICAM-1

on surface of endothelial cells.28 Endothelial cells were incubated

with or without the test compounds at desired concentrations for

the required period, followed by treatment with TNF-a (10 ng

ml�1) for 16 h for ICAM-1 expression. The cells were fixed with

1.0% glutaraldehyde. Non-specific binding of antibody was

blocked by using skimmed milk (3.0% in PBS). Cells were incu-

bated overnight at 4 �C with anti-ICAM-1 mAb, diluted in

blocking buffer, the cells were further washed with PBS and

incubated with peroxidase-conjugated goat anti-mouse

secondary Abs. After washings, cells were exposed to the

peroxidase substrate (o-phenylenediamine dihydrochloride

40 mg/100 ml in citrate phosphate buffer, pH 4.5). Reaction was

stopped by the addition of 2 N sulfuric acid and absorbance at

490 nm was measured using a microplate reader (Spectramax

190, Molecular Devices, USA). The potencies of the compounds

were compared on the basis of their IC50 values calculated from

the dose-response curves.28–30

Time kinetics of ICAM-1 inhibition by 3p

The confluent monolayer of human endothelial cells were treated

with or without 100 mMof 3p at time points ranging from 4 h, 2 h

and 1 h before TNF-a stimulation (pre-treatment), simulta-

neously along with TNF-a stimulation (co-treatment) and 4 h,

750 | Med. Chem. Commun., 2011, 2, 743–751

2 h and 1 h after the TNF-a stimulation (post-treatment). The

cells were incubated in the presence of TNF-a for 16 h before

ICAM-1 expression was measured by Cell-ELISA. The data are

representative of three independent experiments.

Acknowledgements

Authors acknowledge the help of St. Stephen’s Hospital, Delhi

for providing umbilical cords. This work was partly funded by

the Council of Scientific and Industrial Research, India grant

NWP0033 (to B.G.), the NIH grants HL081205, HL095420

(SCCOR), NIEHS-P50ES01590 & GM079239 (to S.B.) and the

University of Delhi grant under the Strengthening R&D

Doctoral Research Programme and the Department of Scientific

& Industrial Research (DSIR), Ministry of Science & Tech-

nology, India (to V.S.P. and A.K.P.).

Notes and references

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10 J. F.M. Silva, S. J. Garden andA. C. Pinto, J. Braz. Chem. Soc., 2001,12, 273.

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13 S. E. Webber, J. Tikhe, S. T. Worland, S. A. Fuhrman,T. F. Hendrickson, D. A. Matthews, R. A. Love, A. K. Patick,J. W. Meador, R. A. Ferre, E. L. Brown, D. M. DeLisle,C. E. Ford and S. L. Binford, J. Med. Chem., 1996, 39, 5072.

14 G. Filomeni, G. Cerchiaro, F. Da Costa, M. Ana, A. De Martino,J. Z. Pedersen, G. Rotilio and M. R. Ciriolo, J. Biol. Chem., 2007,282, 12010.

15 V. A. Muthukumar, S. George and V. Vaidhyalingam, Biol. Pharm.Bull., 2008, 31, 1461.

16 G. Filomeni, S. Piccirillo, I. Graziani, S. Cardaci, F. Da Costa,M. Ana, G. Rotilio and M. R. Ciriolo, Carcinogenesis, 2009, 30,1115.

17 G. Kiran, G. Rajyalakshmi, J. V. Rao and M. Sarangapani,Pharmacologyonline, 2009, 1, 303.

18 F. G. Salituro, G. W. Bemis, S. Wilke, J. Green, J. Cao, H. Gao andE. M. Harrington, PCT Int. Appl. WO Pat. 0064872.

19 M. Verma, S. N. Pandeya, K. N. Singh and J. P. Stables,Acta Pharm.,2004, 54, 49.

20 S. K. Sridhar, S. N. Pandeya, J. P. Stables and A. Ramesh, Eur. J.Pharm. Sci., 2002, 16, 129.

21 H. A. Bradman, J. Heterocycl. Chem., 1973, 10, 383.22 P. L. Julian, H. C. Printy, R. Ketcham and R. Doone, J. Am. Chem.

Soc., 1953, 75, 5305.23 R. Shimazawa, M. Kuriyama and R. Shirai, Bioorg. Med. Chem.

Lett., 2008, 18, 3350.24 B. M. Trost, N. Cramer and S. M. Silverman, J. Am. Chem. Soc.,

2007, 129, 12396.25 C. Weber, W. Erl, A. Pietsch and P. C Weber, Circulation, 1995, 91,

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26 D. M. Radomska-Le�sniewska, A. M. Sadowska, F. J. Van Overveld,U. Demkow, J. Zieli�nski and W. A. De Backer, Physiol. Pharmacol.,2006, 57, 325.

27 A. Sakai, Life Sci., 1996, 58, 2377.28 S. Kumar, P. Arya, C.Mukherjee, B. K. Singh, N. Singh, V. S. Parmar,

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29 S. Kumar, B. K. Singh, A. K. Pandey, A. Kumar, S. K. Sharma,H. G. Raj, A. K. Prasad, E. Van der Eycken, V. S. Parmar andB. Ghosh, Bioorg. Med. Chem., 2007, 15, 2952.

30 L. Pasquinucci, O. Prezzavento, A. Marrazzo, E. Amata,S. Ronsisvalle, Z. Georgoussi, D. D. Fourla, G. M. Scoto, C. Parenti,G. Aric�o and G. Ronsisvalle, Bioorg. Med. Chem., 2010, 18, 4975.

Med. Chem. Commun., 2011, 2, 743–751 | 751


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