Spiro-(indoline-isoxazolidines) compounds having inhibitory effects on cytokineinduced
cell adhesion molecule ICAM-1 expression onto human endothelial cells
Field of the invention:
The present invention relates to compounds having inhibitory effects on cytokine-induced
cell adhesion molecule ICAM-1 expression onto human endothelial cells. The present
invention in particular relates to compounds, belonging to spiro-(indoline-isoxazolidines)
class, which have inhibitory effects on cytokine-induced cell surface expression of
ICAM-1 and are useful as anti-inflammatory agents for various inflammatory and like
conditions.
Background of the invention
Inflammation is an integral part of immune surveillance mechanism. It is a complex
response to local injury and involves various immune cells and numerous inflammatory
mediators. Localized leukocyte accumulation is the cellular hallmark of inflammation.
Endothelial cells are key players in the recruitment of leukocytes to the site of
inflammation. Inflammatory cytokines like IL-1P, TNF-a and bacterial
lipopolysaccharide (LPS) "activate" the endothelium to become adhesive for peripheral
blood leukocytes. These adhesive interactions are mediated by glycoproteins expressed
on the surface of all cells, known as Cell Adhesion Molecules (CAMs). Some of these
CAMs are expressed constitutively, while others are expressed only in response to local
concentrations of cytokines, such as IL- 1 j3 and TNF-a, produced during an inflammatory
response. Thus, vascular endothelium serves as an important "gate-keeper", regulating the
movement of leukocytes to the tissues.
Once the leukocytes, primarily neutrophils have migrated across the endothelial barrier
into the site of injury, additional chemoattractants stimulate neutrophils to release
peroxides and proteases, which function to destroy the offending organism. Leukocyte
recruitment thus, is critical for host defense. Hence the primary function of CAMs is to
4
allow tissue specific accumulation of leukocytes. CAMs also serve to increase the
strength of the functional interactions between various cells of the immune system. Based
on the structural homology and functional similarities, the adhesion molecules have been
classified into three catagories: i) Selectins ii) Integrins and iii) Immunoglobulin
Superfamily.
In addition to being involved in host defense, excessive leukocyte endothelial interactions
can generate pathologic inflammation in a variety of conditions. The cell surface
expression of many adhesion molecules is upregulated following activation during a
microbial invasion or an inflammatory response. It may be responsible for both cell
activation and migration during a protective immune response on one hand, and
exacerbating the pathology of the disease in other cases. In most animal models of
inflammation, E-selectin is expressed early and correlates with neutrophil influx, whereas
ICAM-1 and VCAM-1 expression increases with a more prolonged time course and
correlates with leukocyte infiltration. A promising therapeutic approach to diminish
aberrant leukocyte adhesion is, therefore, to inhibit cytokine-induced expression of
CAMs
Interruption of the leukocyte-endothelial cell cascade has become a major focus of
developing anti-inflammatory molecules. Several approaches are being developed to deal
with the growing therapeutic needs. The blockade of CAM function by use of specific
blocking monoclonal antibodies is one strategy. For example, anti-LFA-1 monoclonal
antibodies have been used to prevent graft failure in patients receiving HLA-mismatched
bone marrow transplants. Clinical trials are currently under way using anti-ICAM-1
antibodies to inhibit kidney allograft rejection. It is obvious that the use of monoclonal
antibodies in the treatment of human diseases will be limited by the generation of '
antimurine antibodies by the recipient. However, the use of monoclonal antibodies may
be useful for problems that would benefit from short-term therapies, such as acute
transplant rejection, prevention of ischemia reperfusion injury, or in well-defined
episodes of sepsis or tissue injury. Humanization of murine antibodies may provide some
advantage by limiting the production of antimurine antibodies.
Because of problems such as immunogenicity, the potential for causing immune complex
disease, and requirements for intravenous administration, a number of alternative ways to
inhibit cell adhesion molecules, are being explored. Soluble forms of adhesion molecules
represents one such approach. Neutrophil influx has been inhibited in vitro with soluble
P-selectin and in murine and rat models using soluble selectin-IgG chimeras.
Unfortunately, the bioactivity of other soluble recombinant adhesion receptors, Eselectin,
for example, seems to be quite low. The difficulty in bulk preparation of these
receptors and the requirement for intravenous delivery further limit their potential utility.
The synthesis of peptide mimetics of active binding regions of CAMS is also being
explored. Short peptides based on sequences of ICAM-1, P-selectin, and PECAM-1 have
been shown to block certain in-vitro adhesion events and could potentially be useful in
animal models.
Of more practical use would be small molecular weight compounds with the potential for
oral administration. Such compounds could include spiro-(indoline-isoxazolidines).
Oxindoles fimctionalised at C-3 as spirocarbon- and heterocyclics, spirolactones and
spirocyclic ethers are elegant targets in organic synthesis due to their significant
biological activities. Spiro-(indoline-isoxazolidines) have been reported earlier to have
antitubercular and anti-invasive activities.
None of the aforementioned prior art has been found to suggest the use of these
compounds or like compounds as anti-inflammatory agents.
Object and Summary of the Invention
The object of the present invention is to provide compounds having inhibitory effects on
cytokine-induced cell adhesion molecule ICAM- 1 expression onto human endothelial
cells.
d
Another object of the present invention is to provide low molecular weight compounds
having inhibitory effects on cytokine-induced cell adhesion molecule ICAM-1 expression
onto human endothelial cells.
In order to achieve the aforesaid objectives, present invention provides novel compounds
which inhibit cell adhesion molecule ICAM-1 expression on human endothelial cells. The
present invention identifies a number of novel spiro-(indoline-isoxazolidines)
compounds, belonging to different chemical classes, which have excellent ICAM-1
expression inhibiting effects and can be useful as anti-inflammatory agents. If one
considers the ICs0 values for ICAM-1 inhibition on HUVECs of lead molecules or drugs
that are in clinical use, it seems that the ICso values for our novel compounds are
comparatively lower. Therefore, these results indicate that our novel compounds are
potentially effective and, therefore could be useful for further pharmaceutical studies.
The present invention provides compounds of Formula I including pharmaceutically
acceptable optical and geometrical isomers and salts, having inhibitory effects on
cytokine-induced cell adhesion molecule ICAM-I expression onto human endothelial
cells;
Formula I
wherein:
X may independently be 0, S
Y may independently be ORIS, SR15, NR15R16
R1-RI4c an independently be H, (CH~),halogen, alkyl, (CHz),ql, (CH2),0H, (CH2),SH,
(CH2)nOalkyl, (CHz)nSalkyl, (CHz)noql, (CH2)nSqL (CH2)nNH2, (CHz)nNHalkyl,
(CH2)nN(alkyl)z, (CHz)nNHacyl, (CH2)nCN, (CH2)nN02, (CH2)nCOOH, (CH2)nCSOH,
5
X X
I I I I
(CH2)nCON(alkyl)2R,I ~-C-Z-(CHZ),- and Rq5-(CHz)nZ-CZ
may independently be 0, S, NR15
A can be rb: and B can be 0 or
A can be 0 and B can be 64
Rl5-RI7c an independently be H, alkyl, (CH2)nql
including pharmaceutically acceptable optical and geometrical isomers and salts.
The invention mher provides a process for preparing compound of formula 1,
comprising microwave mediated 1,3-dipolar cycloaddition of ethyl (5-fluoro-2-0x0- 1,2-
dihydro-3(H)-indol-3-ylidene) acetate (compound of formula 4) and substituted a,Ndiphenylnitrones
(compound of formula 5-8) at 850W, 1.2Cft.
Detailed Description of the Invention
Chemical synthesis, detailed description of spiro-(indoline-isoxazolidines) with general
structures:
i Y-C
12
rt R17 R11
Formula I
wherein:
X may independently be 0, S
Y may independently be ORl5, SR15, NRl5RI6
Rl-Rl4 can independently be H, (CHz),halogen, alkyl, (CH2),aryl, (CH2),0H, (CH2),SH,
(CHz),Oalkyl, (CH2),Salkyl, (CHz),Oaryl, (CHz),Saryl, (CH2),NH2, (CHz),NHalkyl,
(CHz)nN(alkyl)z, (CHz)nNHacyl, (CH2)nCN, (CH2),N02, (CH2),COOH, (CH2),CSOH,
(CH2),COSH, (CH2),COOalkyl, (CH2),COOaryl, (CH2),CSOalkyl, (CH2),COSaryl,
(CH2),COSalkyl, (CH2),CSOaryl, (CH2),CONH2, (CH2),CONHalkyl,
X X
I I I I
(CH2),CON(alkyl)2, RI~-C-Z-(CH~)~ and R15-(CH2)7Z-CZ
may independently be 0, S, NR15
A can be & and B can be 0 or
A can be 0 and B can be r;,
3
R15-Rc1an7 i ndependently be H,a lkyl, (CHz),aryl
including pharmaceutically acceptable optical and geometrical isomers and salts.
Figure 1: Spiro-(indoline-isoxazolidines)
The list of novel compounds belonging to spiro-(indoline-isoxazolidines) with antiinflammatory
activity is provided in Table 1.
Table 1
* The maximum tolerable dose is the concentration of the compound at which
the cell viability is > 95 %
The compounds of formula 1 are selected form the group consisting of:
3'-(2,3-Dimethoxyphenyl)-5'-ethoxycarbonyl-2'-phenylspiro-[5-floroindoline3-, 4'-
isoxazolidine]-2-one (9);
5'-Ethoxycarbonyl-3'-(4-N, N-dimethylaminophenp1)-2'-phenylspiro- [5-floroindoline-3,4'-
isoxazolidine]-2-one (10);
3'-(4-Ethoxyphenyl)-5'-ethoxycarbonyl-2'-phenylspiro-[5-floroindoline-3,4'-
isoxazolidine]-2-one (1 1);
5'-Ethoxycarbonyl-2'-phenyl-3'-(3,4,5-trimethoxyphenyl)-spiro-[5-floroindoline-3,4'-
isoxazolidine]-2-one (12); and
ICS0
(pM)
3 2
27
11
29
27
% ICAM-1
Inhibition
at MTD
(PM)
98
96
97
97
99
Maximum
tolerable
dose (MTD)
(PM)*
102
110
105
96
102
Molecular
weight
492
475
476
522
492
Compound
No.
9.
10.
11.
12.
13.
Compound
code
7k
7q
7r
7v
8 k
According to another aspect of the invention, the spiro-(indoline-isoxazolidines) 9-13
having the general structure shown in Figure 1 were synthesized by the microwave
mediated 1,3- dipolar cycloaddition reaction between ethyl (5-fluoro-2-0x0- l,2-dihydro-
3(H)-indol-3-ylidene) acetate (4) and various substituted a,N-diphenylnitrones 5-8
(Scheme-1). Cycloaddition of nitrone 5 and compound 4 yielded both regioisomers 9 &
13, where as the cycloaddition of nitrones 6-8 and the compound 4 resulted in the
formation of single regioisomers 10-12, respectively. Nitrones 5-8 were prepared
according to the procedures reported in the literature, viz. by the condensation of
appropriately substituted aromatic aldehydes with phenylhydroxylamine in ethanol at
room temperature.
F 0 - ($ Conc. H2S04, '- do
Conc.HC1, Water, 80 OC H
NH2 reflux NHCOCHNOH
Microwave
Compound R1 R2 R3 Rq
OMe OMe H H
H H NMe2 H
H H OEt H
H OMe OMe OMe
OMe OMe H H
Compound 4 was synthesized in three steps, starting with the synthesis of N-(4-
fluoropheny1)-2-hydroxyiminoacetamide (2) by refluxing equimolar amounts of
commercially available 4-floroaniline (I), chloral hydrate and hydroxyl mine in water
along with few drops of concentrated hydrochloric acid in 90 % yield; compound 2 was
then cyclized by dissolving it in concentrated H2S04 and heating the reaction mixture at
80 OC to get 5-fluoro-indoline-2, dione (3) in 72 % yield (Scheme-1). Compound 4 was
then synthesized in 72 % yield by the condensation of commercially available
ethoxycarbonylmethylenetriphenyl phosphorane with the 5-fluoro-indoline-2,3 dione (3)
in acetic acid (glacial) at 80 OC (Scheme-1).
Biological evaluation
The present invention showed that the compounds are not toxic to endothelial cells at
their determined maximum tolerant doses, however, the maximum tolerant dose for each
compound is different as assessed by MTT assay.
The compounds of the present invention were screened for their cytokine-induced ICAM-
1 expression inhibitory activity on human endothelial cells using the modified Cell-
ELISA. The ICso (the concentration of compound that shows 50 % inhibition) values for
the compounds (Table 1) with respect to their inhibition of ICAM-1 expression on
endothelial cells were calculated.
4
If one considers the ICso values for ICAM-1 inhibition on HUVECs of lead molecules or
drugs that are in clinical use (Table 2), it seems that the ICsa values for our compounds
are comparatively lower. Therefore, these results indicate that our novel compounds are
potentially effective and, therefore, could be useful for further experimental studies.
Table 2 : Commonly used Anti-inflammatory Drugs.
Drug name Class of Drug I Inhibition/cell I 1C. I Reference
N-acetyl cysteine
PDTC
Diclofenac
ANTIOXIDANT
ANTIOXIDANT
Aspirin
I Rolipram I PDE4 INHIBITOR ( ICAM-IJHUVEC 1 >>I0 pM I Br J Pharmacol. 1998 I
NSAID
Sodium salicylate
Dexamethasone
Methylprednisolone
I ( May; 124(1):229-3 7.
*These examples are from literatures and not possible for them to mention.
type
ICAM-l/ECV304
ICAM- l/HUVEC
NSAID
The present invention will hereinafter be described in more detail by the following
ICAM-l/HUVEC
NSAID
GLUCOCORTICOID
GLUCOCORTICOID
examples. However, the invention is not limited to these examples.
10 mM
100 pM
I Biol. 1994;14;1665-1673
750 pM I Life Sci.1996,58,2377-
ICAM- 1 IHUVEC
EXAMPLE 1
J. Physiol Pharmacol.
2006,57, Supp 4,325-334
Arterioscler. Thromb. Vasc.
ICAM-l/HUVEC
ICAM- 1lHUVEC
ICAM- 1 /HUVEC
General Method for the preparation of spiro-(indoline-3,4'/3,S1-isoxazolidines) 9-13
5 mM
A mixture of ethyl (5-fluoro-2-oxo-l,2-dihydro-3(H)-indol-3-ylidenea)c etate (4, 1.84
2387.
Circulation. 1995;9 1 : 19 14-
1 mM
100 pM
High
dose
mmol) and variously substituted a,N-diphenylnitrones 5-8 (1.84 rnmol) was irradiated in
1917
J Immunol. 1996 May
15;156(10):3961-9
Cell Immunol. 1996 Jan
10; 167(1):79-85.
Journal of the neurological
sciences 1998, vol. 157,2,
117-121
a microwave (850 W 1.2 Cft). The progress of the reaction was monitored by TLC after
irradiating the reaction mixture for 4-5 min (one minute at a time with 10 sec interval),
the mixture was chromatographed over silica gel using ethyl acetate-petroleum ether as
eluent to afford the spiro compounds 9-13 as light yellow solids, which were
i
recrystallized from ethyl acetate- petroleum ether to afford the white to light yellow
crystals of the regioisomeric spiro derivatives in 5-58 % yields.
EXAMPLE 2
9
Obtained as a white solid (412 mg, 45 %), melting point: 120-121 OC (recrystallization
from petroleum ether-ethyl acetate), Rf: 0.35 (3:2 petroleum ether-ethyl acetate).
IR (film) v,,: 3297 (NH), 1749 (COOC2H5), 1619 (CONH), 1463, 1377, 1270
and 1073 cm-' .
'H NMR (300 MHz, CDC13): 6 0.85 (3H, t, J = 7.0 Hz, COOCH2CH3), 3.72 &
3.75 (6H, 2s, 2xOCH3), 3.87-3.93 (2H, m, COOCH2CH3), 5.12 (lH, s, C-3'H), 5.56
(lH, s, C-5'H), 6.60-6.62 (lH, m, C-4"H), 6.71-6.74 (2H, m, C-3"H and C-5"H),
6.87-6.90 (lH, m, C-7H), 6.95-7.02 (4H, m, C-2"H, C-5"'H, C-6"H and C-6"'H),
7.29-7.32 (2H, m, C-4"'H and C-6H), 7.40-7.42 (lH, m, C-4H), 8.25 (lH, s, NH).
13c NMR (75.5 MHz, CDC13): 6 13.46 (COOCH2CH3), 55.54 & 59.62 (2xOCH3),
61.3 1 (COOCH2CH3), 66.43 (C-4'), 74.45 (C-3'), 83.66 (C-57, 109.60 (C-6), 1 12.37
(C-4"'), 113.72 (C-2" and C-6"), 114.34 (d, J = 25.3 Hz, C-7), 115.1 1 (d, J = 23.4
Hz, C-4), 120.24 (C-1"' and C-6"'), 122.08 (C-4"), 123.95 (C-5"'), 129.08 (C-3" and
C-5"), 130.54 (C-8), 137.10 (C-9), 15 1.32 (C- 1 "), 15 1.82 (C-3"'), 154.24 (C-2"'),
154.42 (C-5), 166.20 (CONH), 175.20 (COOC2H5).
HRMS: m/z Calcd. for C27H25m20[6M I' : 492.1697. Found: 492.1703.
.I
EXAMPLE 3
5'-Ethoxycarbonyl-3'-(4-N,N-dimethylaminophenyl)-2'-phenylspiro-[5-
floroindoline-3,4'-isoxazo1idine)-2-one (10)
C H ~
10
Obtained as a light brown solid (490 mg, 54 %), melting point: 126-127 OC
(recrystallization from petroleum ether-ethyl acetate), Rf: 0.32 (3:2 petroleum ether-ethyl
acetate).
IR ( f:i,,lvm) 3306 (NH), 1740 (COOC~HS)1, 613 (CONH), 1487,1377, 1232 and
1077 cm-'.
'H NMR (300 MHz, DMSO-ds): 6 0.80 (3H, t, J = 7.1 Hz, COOCH2CH3), 2.83
(6H, s, N(CH3)2), 3.80-3.89 (2H, m, COOCH2CH3), 5.08 (1 H, s, C-3'H), 5.12 (1 H,
s, C-5'H), 6.45-6.48 (2H, m, C-3"'H and C-5"'H), 6.64-6.68 (lH, m, C-4"H), 6.84-
6.86 (lH, m, C-7H), 7.01-7.03 (SH, m, C-2"H, C-3"H , C-5"H, C-6H and C-6"H),
7.19-7.27 (3H, m, C-2"'H, C-4H and C-6"'H), 10.66 (lH, s, NH).
"C NMR (75.5 MHz, DMSO): 6 13.60 (COOCH2CH3), 39.80 (-N(CH3h), 61.01
(COOCH2CH3), 66.64 (C-4'), 78.49 (C-3'), 81.62 (C-57, 110.60 (d, J = 8.3 HZ, C-
6), 1 12.04 (C-3"' and C-5"'), 1 14.13 (d, J = 25.6 HZ, C-7), 1 15.68 (d, J = 23.7 Hz,
C-4), 116.69 ((2-2" and C-69, 121.34 (C-1"' and C-49, 123.62 (C-8), 127.65 (C-3"
and C-57, 129.28 (C-2"' and C-6"'), 138.76 (C-9), 150.20 (C-4"'), 151.37 (C-1"),
156.10 (C-5), 165.82 (CONH), 173.67 (COOC2HS).
HRMS: m/z Calcd. for C27H27FN304[ M+H]+: 476.1980. Found: 476.197 1.
EXAMPLE 4
3'-(4-Ethoxyphenyl)-5'-ethoxycarbonyl-2'-phenylspiro-[5-floroindoline-3,4'-
isoxazolidine]-2-one (1 1)
11
Obtained as a light brown solid (455 mg, 50 %), melting point: 124-125 OC
(recrystallization from petroleum ether-ethyl acetate), Rf: 0.42 (3:2 petroleum ether-ethyl
acetate).
IR (film) v,,: 3300 (NH), 1724 (COOC2H5), 1620 (CONH), 146 1,1377,1232 and
1079 cm-' .
'H NMR (300 MHz, DMSO-ds): 6 0.81 (3H, t, J = 6.9 Hz, COOCH2CH3), 1.3
(3H, t, J = 6.5 Hz, OCH2CH3), 3.86-3.91 (4H, m, COOCH2CH3 and OCH2CH3),
5.12 (lH, s, C-3'H), 5.16 (lH, s, C-5'H), 6.65-6.67 (3H, m, C-3"H, C-4"H and C-
5"H), 6.75-6.78 (lH, m, C-7H), 7.00-7.03 (3H, m, C-2"'H, C-4H, C-6"'H), 7.1 1-
7.14 (3H, m, C-2"H, C-6H and C-6"H), 7.23-7.27 (2H, m, C-3"'H and C-5"'H),
10.25 (1 H, s, NH).
13c NMR (75.5 MHz, CDC13): 6 13.98 (COOCH2CH3), 15.07 (OCH2CH3), 61.83
(COOCH2CH3), 63.68 (OCH2CH3), 67.37 (C-47, 77.57 (C-37, 82.08 (C-57, 110.35
(d, J = 7.9 HZ, C-6), 114.74 (C-2" and C-6"), 115.38 (d, J = 25.4 HZ, C-7), 115.85
(d, J = 23.6 Hz, C-4), 116.56 (C-3" and C-5"), 123.61 (C-1"' and C-4"), 126.81 (C-
8), 128.16 (C-2"' and C-6"'), 129.26 (C-3"' and C-59, 136.74 (C-9), 151.33 (C-1"),
158.55 (d, J = 237.1 HZ, C-5), 160.12 (C-4"'), 166.17 (CONH), 174.62 (COOC2H5).
HRMS: d z Calcd. for C27H26FN20[5M +H]+: 477.1826. Found: 477.1838.
EXAMPLE 5
5'-Ethoxycarbonyl-2'-pheny1-3'-(3,4,5-trimethoxyphenyl)-spiro-[5-floroindoline-
3,4'-isoxazolidine]-2-one (12)
OCH~
12
Obtained as a white solid (527 mg, 58 %), melting point: 125-126 OC (recrystallization
from petroleum ether-ethyl acetate), Rf: 0.32 (3:2 petroleum ether-ethyl acetate).
IR (film) v,,: 3275 (NH), 1734 (COOC2H5), 1590 (CONH), 1460, 1377 and 1235
cm-1 .
'H NMR (300 MHz, DMSO-ds): 6 0.82 (3H, brs, COOCH2CH3), 3.65 (9H, s,
OCH3), 3.83-3.93 (2H, m, COOCH2CH3), 5.12 (lH, s, C-3'H), 5.16 (1 H, s, C-5'H),
6.49 (2H, S, C-3"H and C-5"H), 6.69 (lH, S, C-4"H), 6.82 (1 H, S, C-7H), 7.04-7.14
(4H, m, C-2"H, C-2"'H, C-6"H and C-6"'H), 7.29 (2H, brs, C-4H and C-6H), 10.72
(1 H, s, NH).
13c NMR (75.5 MHz, DMSO): 6 13.56 (COOCH2CH3), 56.06 and 60.22
(3xOCH3), 61.06 (COOCH2CH3), 66.96 (C-4'), 78.77 (C-3'), 81.83 (C-57, 103.82
(C-2"' and C-6"'), 1 10.67 (d, J = 7.4 HZ, C-6), 1 14.06 (d, J = 25.1 HZ, C-7), 1 15.49
(C-4), 115.84 (C-2" and C-6"), 123.37 (C-4"), 126.94 (C-1"'), 129.42 (C-3" and C-
5'7, 13 1.05 (C-8), 137.26 (C-4"'), 138.79 (C-9), 15 1.39 (C-I"), 152.94 (C-3"' and C-
5"'), 157.55 (d, J = 237.2 HZ, C-5), 165.44 (CONH), 173.79 (COOC2H5).
HRMS: m/' Calcd. for C2*H27FN2o7N[aM +N~]:+ 5 45.1695. Found: 545.1699.
EXAMPLE 6
3'-(2 J-Dimethoxyphenyl)-4'-ethoxycarbonyl-2'-phenyIspiro-[5-floroindoline-3,5'-
isoxazolidine]3-one (13)
13
Obtained as a white solid (46 mg, 5 %), melting point: 126-127 OC (recrystallization
from petroleum ether- ethyl acetate), Rf: 0.39 (3:2 petroleum ether-ethyl acetate).
IR (film) v,,: 3270 (NH), 1723 (COOC2H5), 1587 (CONH), 1463,1377,1268 and
1192 cm-'.
'H NMR (300 MHz, CDC13): 8 0.75 (3H, t, J= 7.1 Hz, COOCH2CH3), 3.71-3.79
(2H, m, COOCH2CH3), 3.81 & 3.85 (6H, 2s, 2 x -OCH3,) 4.29 (lH, d, J= 7.7 Hz,
C-4'H), 5.84 (lH, d, J = 7.7 Hz, C-3'H), 6.80-6.86 (3H, m, C-3"H, C-4"H and C-
5"H), 6.90-6.92 (2H, m, C-6"'H and C-7H), 6.95-6.98 (2H, m, C-2"H and C-6"H),
7.05-7.07 (lH, m, C-4"'H), 7.10-7.17 (2H, m, C-5"'H and C-6H), 7.4 1-7.44 (1H, m,
C-4H), 10.20 (lH, brs, NH).
13c NMR (75.5 MHz, CDC13): 8 13.27 (COOCH2CH3), 55.56 (C-4'), 60.88 &
60.94 (2x -OCH3), 63.85 (COOCH2CH3), 64.03 (C-3'),83.00 (C-57, 110.79 (d, J =
7.7 Hz, C-6), 11 1.82 (C-47, 114.07 (d, J = 25.9 Hz, C-7), 116.46 (C-2" and C-69,
116.73 (C-4), 120.46 (C-1"' and C-6"'), 122.62 (C-4"), 124.36 (C-57, 128.34 (C-3"
and C-57, 131.87 (C-8), 138.65 (C-9), 148.70 (C-1"), 152.30 (C-3"'), 156.55 (C-
2"'), 159.85 (C-5), 167.60 (CONH), 174.80 (COOC2H5).
HRMS: d z Calcd. for C27H25FN206N[aM +N~]:+ 5 15.1594. Found: 5 15.1642.
EXAMPLE 7
Cell Culture: Primary endothelial cells were isolated from human umbilical cord using
mild trypsinization (28). The cells were grown in M199 medium (Sigma, USA)
supplemented with 15 % heat inactivated fetal calf serum (Biological Industries, Israel), 2
mM L-glutamine (Sigma, USA), 100 unitslml penicillin (Sigma, USA), 100 pglml
streptomycin (Sigma, USA), 0.25 pglml amphotericin B (Sigma, USA), endothelial cell
-7
growth factor (50 pglml) ( Sigma, USA). At confluence, the cells were subcultured using
0.05 % trypsin-0.01 M EDTA solution and were used between passages three to four.
EXAMPLE 8
Cell Viability Assay: The cytotoxicity of these compounds was analyzed by colorimetric
MTT (methylthiazolydiphenyl-tetrazoliurn bromide, Sigma, USA) assay as described
(28). Briefly, endothelial cells were treated with DMSO alone (0.25 % as vehicle) or with
different concentrations of compounds for 24h. The medium was removed and 100 p1
MTT solution (2.5 mglml in serum free medium) was added to each well. The MTT
solution was removed after 4h, cells were washed with PBS and 100 pl 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. From this assay, the percentage
viability (% viability) of the cells at various concentrations of each compound was
determined by normalization to cells incubated in vehicle (0.25% DMSO in cell culture
medium) and which were considered 100% viable. The highest concentration at which
the viability of the cells was >95% was denoted as the maximum tolerable concentration
for that compound.
% Viability = (A570 of extract or compound(s) treated cells1 AS7o~f vehicle treated cells)
x 100
EXAMPLE 9
Cell-ELISA for measurement of ICAM-1: 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
17
Y
period, followed by treatment with LPS (lpg/ml)(BD, USA) for 16 h for ICAM-1
expression. The cells were fixed with 1.0 % glutaraldehyde (Sigma, USA). Non-specific
binding of antibody was blocked by using skimmed milk (3.0 % in PBS). Cells were
incubated overnight at 4 OC with anti-ICAM-1 mAb(BD, USA), diluted in blocking
buffer, the cells were further washed with PBS and incubated with peroxidase-conjugated
goat anti-mouse secondary antibody (Sigma, USA). After washings, cells were exposed
to the peroxidase substrate (0-phenylenediamine dihydrochloride 40 mg1100 ml in citrate
phosphate buffer, pH 4.5). Reaction was stopped by the addition of 2 N sulfuric acid and
absorbance at 490 nrn was measured using microplate reader (Spectramax 190, Molecular
Devices, USA). The percentage inhibition (% Inhibition) was calculated using the
following mathematical formula:
(1 - CIB) x 100
where, B = The change in the relative level of induction in controls (no compound treatment)
calculated by subtracting the A490v alues for induced wells from that of uninduced wells.
C = The change in the relative level of induction in presence of compound (at various
concentrations)-treated wells was calculated by subtracting the values for induced
wells from that of uninduced wells.
The percentage inhibitions of each compound at its various log concentrations were
plotted to generate a sigmoidal curve. The concentration at which a compound showed
50% inhibiton was taken as the ICso value of the compound.

We claim:
1. Spiro-(Indoline-isoxazolidines) compounds of formula I including
pharmaceutically acceptable optical and geometrical isomers and salts, having inhibitory
effects on cytokine-induced cell adhesion molecule ICAM-1 expression onto human
endothelial cells;
Y-C
h@TiTl@ Ra / lN 2 X
7 Rl0 2'. 1 4 " 12
3"
rt Rt7 R11
Formula I
wherein:
X may independently be 0 , S
Y may independently be ORl5, SR15, NRl5RI6
RI-RI~c an independently be H, (CH2),halogen, alkyl, (CH2),aryl, (CH2),0H,
(CH2InSH, (CHz),Oalkyl, (CH2),Salkyl, (C&),Oaryl, (CHz),Saryl, (CH2),NH2,
(CHz),NHalkyl, (CH2),N(alkyl)2, (CHz),NHacyl, (CH2),CN, (CH2)nN02, (CH2)nCOOH,
(CH2),CSOH, (CH2),COSH, (CH2),C00alkyl, (CH2),C00aryl, (CH2),CSOalkyl,
(CH2),COSaryl, (CH2),COSalkyl, (CH2),CSOaryl, (CH2),CONH2, (CH2)nCONHalkyl,
Z may independently be 0, S, NR15
A can be and B can be 0 or
R4
A can be 0 and B can be ria
R15-R17ca n independently be H, alkyl, (CHz),aryl
including pharmaceutically acceptable optical and geometrical isomers and salts.
2. The compounds as claimed in claim 1, wherein the compounds of formula 1 are
selected form the group consisting of:
3'-(2,3-Dimethoxyphenyl)-5'-ethoxycarbonyl-2-phenylspiro-[5-floroindolin3e-,4 '-
isoxazolidine]-2-one (9);
5'-Ethoxycarbonyl-3'-(4-N,N-dimethylaminopheny1)-2'-phenylspiro[-5 -floroindoline-
3,4'-isoxazolidine]-2-one (1 0);
3 ' - ( 4 - E t h o x y p h e n y l ) - 5 ' - e t h o x y c a r b o n y l - 2 ~ i n d o l i n e - 3 , 4 ' -
isoxazolidine]-2-one (1 1);
5'-Ethoxycarbonyl-2'-phenyl-3'-(3,4,5-trimethoxyphenyl)-spiro-[5-floroindoline-3,4'-
isoxazolidine]-2-one (1 2); and
3'-(2,3-Dimethoxyphenyl)-4'-ethoxycarbonyl-2'-phenylspiro-[5-floroindoline-3,5'-
isoxazolidine]-2-one (1 3).
3. A process for preparing compound of formula I, comprising microwave mediated 1,3-
dipolar cycloaddition of ethyl (5-fluoro-2-oxo-l,2-dihydro-3(H)-indol-3-ylidene)
acetate (compound of formula 4) and substituted a,N-diphenylnitrones (compound of
formula 5-8) at 850W, 1.2Cft.
4. The process as claimed in claim 3, wherein 1,3-dipolar cycloaddition results in the
formation of regioisomers.
5. The process as claimed in claim 3, wherein ethyl (5-fluoro-2-0x0- l,2-dihydro-3(H)-
indol-3-ylidene) acetate (compound of formula 4) is prepared in three steps
comprising:
- refluxing equimolar amounts of 4-floroaniline, chloral hydrate and hydroxyl
arnine in acidic medium to obtain N-(4-fluoropheny1)-2-hydroxyiminoacetamide;
- cyclizing N-(4-fluoropheny1)-2-hydroxyiminoacetamide by heating the reaction
mixture at 80 "C in presence of an acid to obtain 5-fluoro-indoline-2,3 dione;
- condensing ethoxycarbonylmethylenetriphenyl phosphorane with the 5-fluoroindoline-
2,3 dione in acetic acid at 80 O C to obtain ethyl (5-fluoro-2-0x0-1,2-
dihydro-3(H)-indol-3-ylidene) acetate.
6. The process as claimed in claim 3, wherein substituted a,N-diphenylnitrones
(compound of formula 5-8) are prepared according to the conventional procedures.
7. The compounds of Formula I having inhibitory effects on cytokine-induced cell
adhesion molecule ICAM-1 expression onto human endothelial cells antiinflammtory
activity, as and when used for treating one or more diseases caused by cell adhesion
andlor cell infiltration selected from the group consisting of allergy, asthma,
inflammation, rheumatism, COPD and arteriosclerosis.