Synthesis and antitumor activity of new retinobenzoic acids

Article abstract of DOI:10.1002/cbdv.201000116

New retinobenzoic acid derivatives have been synthesized starting from 1,2,3,4-tetrahydro-1,1,2,4,4,6-hexamethylnaphthalene and 1,1,2,3,3- pentamethylindane. Four of the synthetic compounds displayed potent cytotoxic activities in vitro against human breast cancer and leukemic cell lines. Thus, these molecules can be further evaluated for the treatment of human leukemia and breast cancer. Copyright

Full text of DOI:10.1002/cbdv.201000116

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
841
Synthesis and Antitumor Activity of New Retinobenzoic Acids
by Reena P. Khandare, Kedar R. Vaze, and Sujata V. Bhat*
Laboratory for Advanced Research in Natural and Synthetic Chemistry, V. G. Vaze College,
Mumbai University, Mithagar road, Mulund (East), Mumbai 400081, India
(phone: þ912221631421 ext 113; fax: þ912221634262; e-mail: sujata8b@gmail.com)
New retinobenzoic acid derivatives have been synthesized starting from 1,2,3,4-tetrahydro-
1,1,2,4,4,6-hexamethylnaphthalene and 1,1,2,3,3-pentamethylindane. Four of the synthetic compounds
displayed potent cytotoxic activities in vitro against human breast cancer and leukemic cell lines. Thus,
these molecules can be further evaluated for the treatment of human leukemia and breast cancer.
Introduction. – Retinoic acid (1 (all-E)) and its analogs (retinoids) modulate
various biological functions such as cell differentiation, proliferation, and embryonic
development in vertebrates. The most important activities of retinoids are certainly the
effects on the differentiation and proliferation of many types of cells and include the
treatment of the neoplastic disorders [1–4]. The (all-E)-retinoic acid (1) and
Isotretinoinꢀ (2; Fig.) have revolutionized the treatment of acute promyelocytic
leukemia (APL) by causing terminal differentiation of the malignant cells [5]. Further,
the inhibitory effect of retinoids on IL-6 production suggests their possible usefulness in
various IL-6-associated diseases including psoriasis and rheumatoid arthritis. Some
synthetic retinoids have been successfully used in the treatment of certain dermato-
logical conditions such as acne, psoriasis, eczema, and photo aging of skin [6][7].
The molecular basis for the activity of retinoic acid has been the subject of
considerable research. A major breakthrough was the discovery of the nuclear retinoic
acid receptors (RARs) and retinoid X receptors (RXRs), which have (all-E)- and
(9Z)-retinoic acids as ligand molecules, respectively [8]. Retinoids bind to these
proteins, then the ligand/protein complex binds to DNA, and the transcription of the
retinoid responsive genes is activated (or depressed). Three forms of the RARs and
RXRs have been termed as RARa, RARb, and RARg, and RXRa, RXRb, and RXRg
which belong to the steroid/thyroid nuclear subfamily and differ in their tissue
distribution. It is generally accepted that biological activities of retinoids are mediated
by RAR-RXR heterodimers, whose subunits follow a defined hierarchy of ligand
responsiveness. Various compounds with selective affinities for these receptors have
been reported.
Despite several beneficial properties, the use of available retinoids is associated
with a number of significant side-effects [9]. For example, systemic use of clinically
available retinoids such as isotretinoin causes an elevation of triglyceride levels in ca.
1/3 of the patients treated. Therefore, synthesis of potent new compounds with
retinoidal activities should facilitate the application of retinoids to medicinal and
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Figure. Tamibaroteneꢀ analogs
chemotherapeutic areas. Tamibaroteneꢀ (AM 80; 3) [10–14] and Tazaroteneꢀ (4) [15]
are synthetic retinobenzoic acid derivatives with considerable activity against acute
promyelocytic leukemia (APL). AM 80 has recently been approved for relapsed or
refractory APL treatment in Japan. It is chemically more stable and several times more
potent as an inducer of differentiation in promyelocytic leukemia cells. Furthermore, in
clinical trials of AM 80, adverse side-effects were milder than those of (all-E)-retinoic
acid (1). AM 80 is being investigated for the treatment of multiple myeloma and
Crohnꢃs disease in clinical trials [13]. The combination of AM 80 and glucocorticoids
was found to have synergistic growth inhibitory effect in human myeloma cells [16].
The present study involves the synthesis and evaluation of the antitumor activity of
AM 80 analogs, with the following structural modifications: i) additional Me group in
ring A; ii) incorporation of 1,1,2,3,3-pentamethylindane moiety in place of 1,2,3,4-
tetrahydro-1,1,4,4-tetramethylnaphthalene; iii) incorporation of ethyl nicotinate in

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
843
place of benzoic acid; iv) replacement of ꢀNHꢀCOꢀ linkage between two aromatic
residues with the ꢀCH2ꢀNHꢀ group. We have achieved the synthesis of new
retinobenzoic acid derivatives 5–10 starting from easily available 1,2,3,4-tetrahydro-
1,1,2,4,4,6-hexamethylnaphthalene and 1,1,2,3,3-pentamethylindane. The antitumor
activity has been studied against six important human breast cancer, leukemia, and T-
lymphoblast cell lines.
Results and Discussion. – The retinobenzoic acids 5–10 investigated in the present
study are shown in the Figure. The starting materials, namely 1,2,3,4-tetrahydro-
1,1,2,4,4,6-hexamethylnaphthalene (11), and 1,1,2,3,3-pentamethylindane (19), were
subjected to a series of reactions as described in Schemes 1–3 to obtain the target
retinobenzoic acids 5–10.
The synthesis of 4-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethylnaphthalen-2-ylcarba-
moyl)benzoic acid (5) was achieved through a parallel reaction sequence as reported
for the synthesis of 3 and its analogs [3][10]. Thus, the nitration of 1,2,3,4-tetrahydro-
1,1,2,4,4,6-hexamethylnaphthalene (11) yielded the mononitro derivative 12, which was
hydrogenated to the amine 13. The latter was condensed with terephthalic acid
monochloride monoester (14) leading to the ethyl ester 15, which, on base hydrolysis
with aqueous NaOH, gave the required acid 5 (Scheme 1).
Scheme 1. Synthesis of 4-(5,6,7,8-tetrahydro-3,5,5,6,8,8-hexamethylnaphthalen-2-ylcarbamoyl)benzoic
Acid (5)
a) HNO3, H2SO4, 60–708. b) H2/Pd, EtOH, 50 psi, 40–508. c) Pyridine. d) 10% NaOH, EtOH, 60–708.
The benzylic bromination of 11 with N-bromosuccinimide (NBS) in CCl4 gave the
compound 16 [17], which was condensed with p-aminobenzoic acid (PABA) to afford
the acid 6 (Scheme 2). The compound 16 was treated with hexamethylenetetramine
(¼1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane; HMT) to yield the aldehyde 17 [18], which
was further oxidized with KMnO4 to give the acid 18. The acid was converted to
aminocarbonyl derivative 7 by the treatment with SOCl2, followed by the condensation
with PABA in the presence of pyridine.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Scheme 2. Synthesis of 4-[(5,6,7,8-Tetrahydro-5,5,6,8,8-pentamethylnaphthalen-3-yl)methylamino]ben-
zoic Acid (6) and 4-[(5,6,7,8-Tetrahydro-5,5,6,8,8-pentamethylnaphthalen-3-yl)carbonylamino]benzoic
Acid (7)
a) N-Bromosuccinimide (NBS), CCl4, reflux. b) p-Aminobenzoic acid (PABA), acetone, anh. K2CO3,
reflux. c) Hexamethylenetetramine 1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane; HMT, CHCl3, reflux, 50%
gl. AcOH and HCl. d) KMnO4, H2O, 70–808. e) SOCl2, 45–508 f) PABA, pyridine.
1,1,2,3,3-Pentamethylindane (19) was nitrated to give the mononitro derivative 20,
which, on hydrogenation, gave amine 21 (Scheme 3). The latter was condensed with
terephthalic acid monochloride monoester (14) to yield the amide, which was
hydrolyzed with aqueous NaOH to 4-[(1,1,2,3,3-pentamethylindan-5-yl)carbamoyl]-
benzoic acid (8). Similarly, the treatment of 21 with phthalic anhydride gave the 2-
[(1,1,2,3,3-pentamethylindan-5-yl)carbamoyl]benzoic acid (9). Finally, ethyl 2-chloro-
6-[(1,1,2,3,3-pentamethylindan-5-yl)amino]nicotinate (10) was obtained by the con-
densation of 21 with ethyl 2,6-dichloronicotinate (22) in the presence of NaH [19].
Biology. The retinobenzoic acid analogues 5–10 were evaluated in vitro against
various tumor cell lines using SRB assay. The cell lines were grown in RPMI 1640
medium containing 10% fetal bovine serum and 2 mm l-glutamine. The doseꢀresponse
parameters were calculated for each test sample. Growth inhibition of 50%, IC50, which
is the drug concentration resulting in a 50% reduction in the net protein increase (as
measured by SRB staining) in control cells during the incubation, was calculated for
each sample in triplicate, and the mean values are given in Tables 1 and 2.
StructureꢀActivity Studies. Compound 7, with an additional Me group at C(6) as
compared to AM 80 and isomeric amide linkage, showed appreciable activity against
human erythroleukemia K562, T-lymphoblast-Jurkat, and human breast cancer Zr-75-1

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
845
Scheme 3. Synthesis of 4- or 2-(2,3-Dihydro-1,1,2,3,3-pentamethyl-1H-inden-5-ylcarbamoyl)benzoic
Acids (8 and 9, resp.) and Ethyl 2-Chloro-6-{[2,3-dihydro-1,1,2,3,3-pentamethyl-1H-inden-5-yl]amino}-
nicotinate (10)
a) HNO3, H2SO4, 60–708. b) H2, Pd/C, 50 psi, EtOH, 40–50. c) i) 14, pyridine; ii) NaOH, EtOH. d)
NaH, phthalic anhydride, THF, reflux. e) NaH, THF, reflux.
and MC-F7 cell lines. Compound 5, containing two additional Me groups at C(3) and
C(6), compared to AM 80, displayed better activity against breast cancer MC-F7 cell
line. Compound 8, the 1,1,2,3,3-pentamethylindane analog, showed potent activity
against breast cancer MC-F7 cell line, and finally, compound 10 showed appreciable
inhibitory activity against human leukemia HL-60 and U937 cell lines.
Table 1. Antitumor Activity of Compounds 5–10 against Leukemia Cell Lines
Compoundc) % Inhibition as compared to controla)b)
Erythroleukemia K 562
Leukemia U 937
Leukemia HL-60
d
10
20
40
80
IC50
)
10 20 40 80 IC50
10
20 40 80 IC50
5
6
7
8
9
10
24
4
41
17
66
39
22
35
37
52
58
48
84
60
57
51
48
57
72
58
78
74
69
75
52
68
30.8
49.6
15.2
30.2
36
38.5
56.5
–
27 36 48 53
21 46 52 54
31 44 52 61
28 24 41 71
55
33.5
11
2
20 34 44 >80
18 45 52
64.8
38
40
18
22
19
75
34.5 11
30 16
0.0 14 29 39 >80
28 43 49 >80
36 48 60
47.6
13 27 38 44 >80
35 65 68 67
23 53 71 77
70 63 72 77
15.5 16
19.4 35
80
49 64 83
45 60 80
71 68 66
22.5
26.5
–
AM-80
Doxe)
–
a) All values are averages of triplicate readings. b) In vitro antitumor activity was evaluated using SRB
c
assay. ) Concentration mg/ml. d) Concentration of drug required in mg/ml to reduce cell growth to 50%
of that obtained with control cells. e) Doxorubicin.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Table 2. Antitumor Activity of Compounds 5–10 against Lymphoma and Breast Cancer Cell Lines
Compoundc) % Inhibition as compared to controla)b)
T-Lymphoblast-Jurkat
10 20 40 80 IC50
Breast cancer Zr-75-1
Breast cancer MC F 7
d
)
10
20
40 80 IC50 10 20 40 80 IC50
5
6
7
8
8.2 17
9.3 25
26 51
65 85
69 63
41 70
79
32.5 0.0
5.9 11
47 42 >80 36 41 70 73 26.2
8
50
31
21 67
64 69
48 62
65 15 23 37 75 54.5
20 37 58 72 67 16.4
47 41 60 69 77 17.4
73 20 35 36 76 54.5
29 15 22 41 86 48
59 13 26 63 66 33.5
24
13
49
24
22
52
15
15
9
10
AM-80
Doxe)
02
7.4 15 15 NA
0.4
3.3 16 56
26
26
76
19
29
32 33 NA
63 82 <10
73 76 <10
17
17
72
78 41
36 64
80 84
70.3 62
64
73
–
4
68 77 80
–
a) All values are averages of triplicate readings. b) In vitro antitumor activity was evaluated using SRB
c
assay. ) Concentration mg/ml. d) Concentration of drug required in mg/ml to reduce cell growth to 50%
of that obtained with control cells. e) Doxorubicin.
In summary, we have disclosed appreciable antitumor activity of new retinobenzoic
acids against human leukemia and breast cancer cell lines. Thus, these molecules can be
further evaluated for the treatment of human leukemia and breast cancer.
Experimental Part
Chemistry. General. 1,2,3,4-Tetrahydro-1,1,3,4,4,6-hexamethylnaphthalene (11) and 1,1,2,3,3-penta-
methylindane (19) were obtained from S. H. Kelkar & Company, Mumbai. Products were obtained as
colorless to pale yellow needles from AcOEt/petroleum ether (unless mentioned otherwise). Column
chromatography (CC): silica gel (SiO2; 100–200 mesh) was activated by heating at 1208 for 4 h. M.p.:
Thermonik cap. instrument; are uncorrected. IR Spectra: Perkin-Elmer Spectrum One spectrometer. 1H-
and 13C-NMR spectra: Varian spectrometers at 400 and 75 MHz, resp., in CDCl3 with TMS as internal
standard. Elemental analyses: Carlo Erba Strumentazione 1106 analyzer.
Synthesis of 4-[(5,6,7,8-Tetrahydro-3,5,5,6,8,8-hexamethylnaphthalen-2-yl)carbamoyl]benzoic Acid
(5). To a mixture of cold conc. HNO3 and H2SO4 (1:1, 9 ml), 11 (10.0 g, 0.04 mol) was added dropwise,
and the resulting mixture was stirred at 08 for 10–15 min. The mixture was allowed to warm to r.t. and
was then heated at 60–708 for 30 min. The dark soln. was poured into ice and extracted with AcOEt. The
org layer was successively washed with H2O, NaHCO3, and brine, and dried (Na2SO4). Removal of the
solvent in vacuo and recrystallization of the residue from toluene gave 1,2,3,4-tetrahydro-1,1,2,4,4,7-
hexamethyl-6-nitronaphthalene (12; 6.20 g, 50%; m.p. 110–1208). Compound 12 (5.0 g) was subjected to
hydrogenation under H2 (pressure of 50 psi) at 408 in the presence of Pd/C (0.5 g) in EtOH (300 ml).
After 10 h, the mixture was filtered, and the solvent was evaporated in vacuo to give 5,6,7,8-tetrahydro-
3,5,5,6,8,8-hexamethylnaphthalen-2-amine (13; 3.2 g, 56%; m.p. 148–1508). A mixture of 13 (0.5 g) and
ethyl 4-(chlorocarbonyl)benzoate (14; 0.65 g) in pyridine (2 ml) was stirred at 60–808 for 6 h. The solvent
was removed, the residue was extracted with AcOEt, and the extract was washed with H2O, 1m HCl,
brine, and was dried (Na2SO4). The evaporation of the solvent gave 15 (0.38 g, 80%; m.p. 196–2008). The
mixture of 15 (0.5 g) and 4n aq. NaOH (4.0 ml) in EtOH (10 ml) was heated to reflux for 6 h. The solvent
was removed, the residue was diluted with AcOEt, and the soln. was washed with H2O, dil. HCl, and
brine, and dried (Na2SO4). After evaporation of the solvent, the residue was recrystallized from hexane
and AcOEt to give 5 (0.37 g, 70%). M.p. 128–1308. IR (KBr): 3275, 2965, 1696, 1649. 1H-NMR: 9.23 (s,
1 H); 8.13, 8.04 (AA’BB’, JAB ¼ 8.0, 4 H); 7.57 (s, 1 H); 7.40 (s, 1 H); 2.27 (s, 3 H); 1.89–1.66 (m, 2 H);
1.39 (d, J¼11.6, 1 H); 1.32 (s, 3 H); 1.29 (s, 3 H); 1.26 (s, 3 H); 1.25 (s, 3 H); 0.99 (d, J¼6.8, 3 H).
13C-NMR: 168.5; 167.4; 152.4; 146.4; 143.8; 132.9; 130.6; 129.2; 127.6; 121.3; 120.2; 117.5; 43.7; 37.5; 34.6;

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
847
34.4; 32.4; 32.1; 28.7; 25.0; 17.7; 16.8. Anal. calc. for C24H29NO3 (379.49): C 75.95, H 7.71, N 3.69; found: C
75.75, H 7.51, N 3.39.
Synthesis of 4-{[(5,6,7,8-Tetrahydro-5,5,7,8,8-pentamethylnaphthalen-2-yl)methyl]amino}benzoic
Acid (6). To a soln. of 11 (20.0 g, 0.09 mol) in CCl4 (200 ml), N-bromosuccinimide (NBS; 20.0 g,
0.1 mol) was added in small portions, and benzoyl peroxide (0.2 g) was added to initiate the reaction. The
mixture was refluxed for 8 h on a water bath. The resulting soln. was filtered in vacuo, and the filtrate on
evaporation gave 7-(bromomethyl)-1,2,3,4-tetrahydro-1,1,2,4,4-pentamethylnaphthalene (16; 36.7 g, 80%)
[17]. To the mixture of 16 (5.0 g, 0.01 mol) and p-aminobenzoic acid (PABA; 3.0 g, 0.02 mol) in acetone
(100 ml) anh. K2CO3 (6.0 g, 0.04 mol) was added. The resulting mixture was refluxed for 14 h. The solvent
was evaporated in vacuo, and the residue was acidified with 1n HCl, extracted with AcOEt, and the org.
layer was washed with H2O and dried (anh. Na2SO4). After evaporation of the solvent in vacuo, the crude
product was purified by CC. The product was recrystallized from hexane and AcOEt to give 6 (3.7 g,
80%). M.p. 174–1768. IR (KBr): 3417, 3369, 2964, 1725, 1667, 1602. 1H-NMR: 7.95, 6.65 (AA’BB’, JAB
¼
8.8, 4 H); 7.31 (dd, J¼6.0, 1.0, 1 H); 7.29 (d, J¼1, 1 H); 7.12 (d, J¼6.0, 1 H); 4.32 (s, 2 H); 1.80–1.90 (m,
2 H); 1.63 (t, J¼7.2, 1 H); 1.30 (s, 3 H); 1.29 (s, 3 H); 1.25 (s, 3 H); 1.05 (s, 3 H); 0.98 (d, J¼6.8, 3 H).
13C-NMR: 172.5; 152.6; 146.6; 144.3; 135.1; 132.4; 127.0; 126.4; 124.9; 117.5; 113.8; 111.6; 47.8; 43.6; 37.8;
34.5; 34.2; 32.4; 32.0; 29.7; 28.6; 25.0; 16.9. Anal. calc. for C23H29NO2 (351.48): C 78.61, H 8.31, N 3.98;
found: C 78.24, H 7.95, N 3.75.
Synthesis of 4-{[(5,6,7,8-Tetrahydro-5,5,7,8,8-pentamethylnaphthalen-2-yl)carbonyl]amino}benzoic
Acid (7). To a soln. of hexamethylenetetramine (¼1,3,5,7-tetraazatricyclo[3.3.1.13,7]decane, HMT;
12.9 g, 0.08 mol) in CHCl3 (100.0 ml) 16 (20.0 g, 0.04 mol) [17] was added. The resulting mixture was
refluxed for 2 h, and filtered and acidified with 50% AcOH (10.0 ml) and conc. HCl (5.0 ml). The
resulting soln. was again refluxed for 2 h. Two layers were separated, the aq. layer was extracted with
CHCl3, the org. layer was neutralized with Na2CO3, washed with H2O, dried (Na2SO4), and filtered. The
solvent was evaporated from the filtrate in vacuo to give 5,6,7,8-tetrahydro-5,5,7,8,8-pentamethylnaph-
thalene-2-carbaldehyde (17; 10.0 g, 68%) [18]. The soln. of 17 (5.0 g, 0.01 mol) in acetone (20 ml) was
stirred with an aq. soln. of KMnO4 (13.4 g, 0.02 mol) in H2O (50.0 ml) for 3 h at r.t. to give 5,6,7,8-
tetrahydro-5,5,7,8,8-pentamethylnaphthalene-2-carboxylic acid (18; 2.2 g, 40%). Then, 18 (2.0 g, 0.01 mol)
was converted to the acid chloride by using SOCl2 (5.0 ml, excess). The excess SOCl2 was recovered by
distillation. A mixture of acid chloride of 18 (1.5 g, 0.01 mol), PABA (0.75 g, 0.015 mol), and pyridine
(3.0 ml) was stirred at r.t. for 8 h. Pyridine was recovered in vacuo, and the residue was dissolved in
AcOEt. The org. layer was washed with H2O and 1n HCl and finally with brine, and was dried (Na2SO4).
After evaporation of the solvent in vacuo, the crude product was purified by CC to give 7 (1.2 g, 76%).
M.p. 234–2368 (hexane and AcOEt). IR (KBr): 3379, 3295, 2960, 1709, 1650, 1595. 1H-NMR: 8.07, 7.82
(AA’BB’, JAB ¼ 8.4, 4 H); 7.96 (d, J¼2.4, 1 H); 7.64 (dd, J¼8.4, 2.4, 1 H); 7.40 (d, J¼8.4, 1 H); 1.80–1.90
(m, 2 H); 1.60 (t, J¼7, 1 H); 1.39 (s, 3 H); 1.28 (s, 3 H); 1.25 (s, 3 H); 1.11 (s, 3 H); 1.0 (d, J¼6.8, 3 H).
13C-NMR: 167.5; 166.0; 146.4; 143.8; 130.8; 128.5; 126.6; 124.0; 119.7; 119.3; 43.1; 40.5; 39.9; 37.8; 34.5;
32.2; 28.4; 24.9; 16.6; 14.1. Anal. calc. for C23H27NO3 (365.46): C 75.58, H 7.44, N 3.83; found: C 75.88, H
7.74, N 3.54.
Synthesis of 4-[(2,3-Dihydro-1,1,2,3,3-pentamethyl-1H-inden-5-yl)carbamoyl]benzoic Acid (8). To a
mixture of cold conc. HNO3 and H2SO4 (1:1; 60 ml) 1,1,2,3,3-pentamethyl-2,3-dihydro-1H-indene (19;
50.0 g, 0.24 mol) was added dropwise, and the resulting mixture was stirred at 08 for 15 min. The mixture
was allowed to warm to r.t. and was heated at 60–708 for 30 min. The dark soln. was poured into ice-H2O
and extracted with AcOEt. The org. layer was successively washed with H2O, NaHCO3, and brine, and
dried (anh. Na2SO4). After removal of the solvent in vacuo, the residue was recrystallized from hexane to
give 1,1,2,3,3-pentamethyl-5-nitroindane (20; 7.2 g, 60%, m.p. 88–908). Then, 20 (5.0 g, 0.02 mol) was
subjected to hydrogenation under pressure of 50 psi at 408 in the presence of Pd/C (0.5 g) in EtOH
(100 ml). After 10 h, the mixture was filtered, and the solvent was evaporated to give 1,1,2,3,3-
pentamethylindan-5-amine (21; 2.7 g, 59%; m.p. 179–1808). The condensation of 21 with terephthalic acid
ethyl ester monochloride (14) and saponification were carried out as described above to give 8. IR (KBr):
3373, 3290, 2960, 1691, 1650, 1595. 1H-NMR: 8.14, 8.0 (AA’BB’, JAB ¼8.4, 4 H); 7.51 (d, J¼8.0, 1 H); 7.35
(s, 1 H); 7.13 (d, J¼8.0, 1 H); 1.88 (q, J¼7.2, 1 H); 1.29 (s, 3 H); 1.27 (s, 3 H); 1.09 (s, 3 H); 1.06 (s, 3 H);
0.99 (d, J¼7.2, 3 H). 13C-NMR: 168.7; 167.6; 152.4; 148.3; 136.3; 131.2; 130.5; 128.9; 127.5; 127.1; 123.1;

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
119.4; 115.1; 54.5; 44.9; 44.5; 29.7; 29.1; 29.0; 25.8; 8.5. Anal. calc. for C22H25NO3 (351.43): C 75.18, H 7.16,
N 3.98; found: C 75.35, H 7.33, N 3.68.
Synthesis of 2-[(2,3-Dihydro-1,1,2,3,3-pentamethyl-1H-inden-5-yl)carbamoyl]benzoic Acid (9). To a
suspension of NaH (0.9 g) in THF (25 ml), a soln. of 21 (1.2 g, 0.02 mol) in THF (50.0 ml) was added, and
the mixture was stirred for 15 min, then phthalic anhydride (3.6 g, 0.04 mol) was added. The resulting
mixture was refluxed for 6 h. THF was recovered, and the residue was diluted with AcOEt, the org. layer
was washed with 1n HCl and brine, and dried (Na2SO4). The evaporation of the solvent in vacuo and
purification of the residue by CC gave 9 (1.2 g, 70%). M.p. 158–1608 (hexane AcOEt, 1:1). IR (KBr):
3252, 2955, 1699, 1660, 1598. 1H-NMR: 8.06 (d, J¼6.8, 1 H); 7.80 (br. s, 1 H); 7.55–7.64 (m, 2 H); 7.50–
7.58 (m, 1 H); 7.38–7.42 (m, 2 H); 7.13 (d, J¼8.4, 1 H); 1.88 (q, J¼6.8, 1 H); 1.26 (s, 6 H); 1.07 (s, 3 H);
1.06 (s, 3 H); 0.99 (d, J¼6.8, 3 H). 13C-NMR: 167.6; 152.4; 151.4; 134.3; 131.0; 131.9; 131.2; 130.0; 125.0;
123.6; 123.3; 121.1; 54.3; 44.8; 44.7; 29.0; 28.9; 25.9; 25.8; 8.5. Anal. calc. for C22H25NO3 (351.43): C 75.18,
H 7.16, N 3.98; found: C 75.45, H 7.41, N 3.59.
Synthesis of Ethyl 2-Chloro-6-[(1,1,2,3,3-pentamethyl-2,3-dihydro-1H-inden-5-yl)amino]pyridine-3-
carboxylate (10). To a mixture of 21 (0.8 g, 0.02 mol) and ethyl 2,6-dichloropyridine-3-carboxylate (22;
0.20 g, 0.02 mol) in THF (20.0 ml) was added NaH (0.10 g). The resulting mixture was refluxed on a
water bath for 6 h, THF was recovered, the residue was diluted with AcOEt, and the org. layer was
washed with 1n HCl and brine, and dried (Na2SO4). Evaporation of the solvent in vacuo gave the crude
product, which was purified by CC to give 10 (0.68 g, 50%). M.p. 118–1208 (MeOH/AcOEt 1:3). IR
(KBr): 2979, 1694, 1585. 1H-NMR: 8.40 (d, J¼8.4, 1 H); 7.2 (s, 1 H); 7.12 (d, J¼8.0, 2 H); 7.0 (d, J¼8.4,
1 H); 4.68 (q, J¼6.8, 2 H); 1.86 (q, J¼6.8, 1 H); 1.54 (t, J¼6.8, 3 H); 1.50 (s, 3 H); 1.25 (s, 3 H); 1.24 (s,
3 H); 1.05 (s, 3 H); 0.98 (d, J¼6.8, 3 H). 13C-NMR: 164.5; 160.7; 153.4; 145.0; 131.0; 121.3; 118.1; 111.0;
65.0; 54.4; 44.4; 44.3; 29.7; 29.1; 28.9; 25.8; 14.3; 8.5. Anal. calc. for C22H27ClN2O2 (398.92): C 68.29, H
7.03, N 7.24; found: C 67.95, H 7.41, N 7.49.
Biology. Antitumor Activity. In vitro Anti-Tumor Activity Assay (SRB Assay) [20]. The cell lines
were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2 mm l-Gln (source of cell
lines: NCI, USA). The cells were inoculated into 96-well microtiter plates in 90 ml of medium and
incubated at 378 (5% CO2, 95% air, and 100% relative humidity) for 24 h prior to addition of compounds.
After 24 h, one 96-well plate containing 5ꢁ103 cells/well was fixed in situ with Cl3CCOOH (TCA) to
represent a measurement of the cell population at the time of drug addition (time-zero (Tz)).
Experimental drugs were initially solubilized in DMSO at 100 mg/ml and diluted to 1 mg/ml with H2O
and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate (1 mg/ml)
was thawed and diluted to 100, 200, 400, and 800 mg/ml with complete medium containing test sample.
Aliquots of 10 ml of these different compound dilutions were added to the appropriate microtiter wells
already containing 90 ml of medium, resulting in the required final drug concentration, i.e., 10, 20, 40, and
80 mg/ml, resp.
Endpoint Measurement. After compound addition, plates were incubated at standard conditions for
48 h, and the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle
addition of 50 ml of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 48.
The supernatant was discarded, and the plates were washed five times with tap H2O and air-dried.
Sulforhodamine B (SRB) soln. (50 ml) at 0.4% (w/v) in 1% AcOH was added to each of the wells, and the
plates were incubated for 20 min at r.t. After staining, unbound dye was recovered, and the residual dye
was removed by washing five times with 1% AcOH. The plates were dried in air. Bound stain was
subsequently eluted with 10 mm trizma base, and the absorbance was read on a plate reader at a
wavelength of 540 nm with 690-nm reference wavelength. Percent growth was calculated on a plate-by-
plate basis for test wells relative to control wells. Percent growth was expressed as the ratio of average
absorbance of test wells to the average absorbance of the control wells ꢁ100.
Using the six absorbance measurements (Tz, control growth (C), and test growth in the presence of
drug at the four concentration levels (Ti)), the percent growth was calculated at each of the drug
concentration levels. The dose–response parameters were calculated for each test sample. Growth
inhibition of 50% (IC50) was calculated from [(TiꢀTz)/(CꢀTz)]ꢁ100¼50, which is the drug
concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining)
in control cells during the incubation.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
849
We are grateful to Kelkar Education Trust, Mumbai, for encouragement and support. We are also
thankful to the Department of Chemistry and Sophisticated Analytical Instrumentation Facility, Indian
Institute of Technology Bombay, for NMR data, and the Advanced Centre for Treatment, Research and
Education in Cancer, Mumbai, for antitumor activity evaluations.
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Receveived April 10, 2010