Rapid synthesis of 4-benzylidene and 4-[bis-(4-methoxyphenyl)-methylene-2-substituted phenyl-benzopyrans as potential selective estrogen receptor modulators (SERMs) using McMurry coupling reaction

Article abstract of DOI:10.1016/j.bmcl.2006.08.126

7-Methoxy-4-(4-methoxybenzylidene)-2-substituted phenyl-benzopyrans I and 4-[bis-(4-methoxyphenyl)-methylene-2-substituted phenyl-benzopyrans II carrying different alkylamino residues, designed as estrogen receptor (ER) binding ligands, were successfully synthesized through the McMurry coupling reaction of substituted benzaldehyde/substituted benzophenones and 2-hydroxyphenyl-7-methoxy-chroman-4-one in presence of lithium aluminum hydride and titanium (IV) chloride (LAH-TiCl₄). Self-coupling of carbonyl reactants led to the formation of several side products. The prototypes were evaluated for their relative binding affinity (RBA), as well as their estrogenic and antiestrogenic activities. High order of estrogenic activity (>50% gain) observed with compounds 3, 7a, 7b, 7c, 8, and 10a and also their partial estrogen antagonistic activity (≥15%) at the uterine level points toward successful designing of the compounds. Compounds 4, 7a, 7b, 7c, and 10a also possessed significant anticancer activity against human adenocarcinoma cell line (MCF-7 cell line) that may be related to their estrogen-dependent action.

Full text of DOI:10.1016/j.bmcl.2006.08.126

Bioorganic & Medicinal Chemistry Letters 16 (2006) 6006–6012
Rapid synthesis of 4-benzylidene and 4-[bis-(4-methoxyphenyl)-
methylene-2-substituted phenyl-benzopyrans as potential
selective estrogen receptor modulators (SERMs) using
McMurry coupling reaction
Atul Gupta,a,* Anila Dwivedy,b Govind Keshri,b Ramesh Sharma,c
Anil Kumar Balapure,c Man Mohan Singhb and Suprabhat Raya,*
aMedicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226001, India
bEndocrinology Division, Central Drug Research Institute, Lucknow 226001, India
cTissue Culture and Animal Laboratory, Central Drug Research Institute, Lucknow 226001, India
Received 29 June 2006; revised 14 August 2006; accepted 30 August 2006
Available online 18 September 2006
Abstract—7-Methoxy-4-(4-methoxybenzylidene)-2-substituted phenyl-benzopyrans I and 4-[bis-(4-methoxyphenyl)-methylene-2-
substituted phenyl-benzopyrans II carrying different alkylamino residues, designed as estrogen receptor (ER) binding ligands, were
successfully synthesized through the McMurry coupling reaction of substituted benzaldehyde/substituted benzophenones and 2-
hydroxyphenyl-7-methoxy-chroman-4-one in presence of lithium aluminum hydride and titanium (IV) chloride (LAH–TiCl4).
Self-coupling of carbonyl reactants led to the formation of several side products. The prototypes were evaluated for their relative
binding affinity (RBA), as well as their estrogenic and antiestrogenic activities. High order of estrogenic activity (>50% gain)
observed with compounds 3, 7a, 7b, 7c, 8, and 10a and also their partial estrogen antagonistic activity (P15%) at the uterine level
points toward successful designing of the compounds. Compounds 4, 7a, 7b, 7c, and 10a also possessed significant anticancer activ-
ity against human adenocarcinoma cell line (MCF-7 cell line) that may be related to their estrogen-dependent action.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The importance of selective estrogen receptor modula-
tors (SERMs)1–4 as contraceptive and in the treatment
of various estrogen dependent diseases such as breast
cancer, osteoporosis, Alzheimer’s disease (AD), and
coronary heart disease has led to the development
of structurally diverse estrogen receptor binding
molecules.
the prevention and treatment of osteoporosis. Orme-
loxifene,8 which is a benzopyran-based SERM devel-
oped by our group, is the first non-steroidal, post
coital, oral contraceptive.
Ormiloxifene is a racemate mixture and is marketed un-
der the trade name Saheli. Use of ormeloxifene and its
different analogues as antiosteoporotic and antibreast
cancer agents is under investigation.8
Four main types of SERMs that have been investigated
are triphenylethylenes (including tamoxifen,5 toremi-
fene, droloxifene, and idoxifene), benzothiaphenes
(e.g., raloxifene,6 and arzoxifene), naphthalenes (nafox-
idine), and benzopyrans (ormeloxifene7 and related
derivatives) (Fig. 1). Tamoxifen5 is being used frequently
as a drug for the prevention and treatment of breast can-
cer. Raloxifene6, another SERM, is commonly used in
However, the SERMs that have been introduced in the
market are not totally free from several undesirable
side effects such as increased incidence of endometrial
cancer and hot flashes.9,10 Therefore, further work in
this area is needed to develop SERM for a specific
disorder.
It was found that introduction of an aryl moiety at C-2
of a benzopyran nucleus leads to potent estrogen antag-
onists. Two such important compounds, CDRI-85/28711
and EM-652,12 are shown below in Figure 2.
Keywords: Antiestrogens; Estrogen antagonists; Antifertility; RBA.
*
Corresponding authors. Tel.: +91 522 2629558; e-mail addresses:
atisky2001@yahoo.co.in; suprabhatray@yahoo.co.in
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.08.126

A. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6006–6012
6007
N
O
O
O
N
N
O
OH
OH
O
S
S
HO
HO
Tamoxifen
Raloxifene
Arzoxifene
N
N
O
O
N
O
HO
H3CO
O
H3CO
Nafoxidine
Lasofoxifene
Ormeloxifene
Figure 1. Non-steroidal SERM molecules.
OH
HO
O
O
N
O
N
O
CDRI 85/287
EM-652
Figure 2.
Keeping in view all the above-mentioned compounds
and their biological activities, it was thought worthwhile
to design and synthesize compounds belonging to proto-
types I and II (Fig. 3) which are reported herein.
preparation.14 Lithium aluminum hydride (LAH), a
far stronger reducing agent as compared to zinc has
been successfully used for such reactions. We therefore
used LAH–TiCl4 in place of Zn–TiCl4 in anhydrous
THF. Thus, reductive coupling of compound 2 with 4-
methoxybenzaldehyde was carried out with LAH–TiCl4
in dry THF under inert reaction condition giving
compound 4 in satisfactory yield. In this reaction,
dimeric products of 4-methoxybenzaldehyde (5) and
The synthesis of prototype I was initially attempted
through Grignard reaction on 2-(4-hydroxyphenyl)-7-
methoxy chroman-4-one 2, prepared in 35% yield from
2-hydroxy-4-methoxy-acetophenone 1 and substituted
benzyl bromide. This reaction led to a mixture of prod-
ucts containing 1-(2-hydroxy-4-methoxyphenyl)-3-(4-
hydroxyphenyl)-propenone as a result of chromanone
ring opening and possibly due to the formation of prod-
ucts with endo- and exocyclic double bonds, which
could not be separated. Chromans and chromanones
are known to undergo ring opening under Mg/THF.13
The desired compounds were finally synthesized through
the McMurry coupling reaction. When condensation of
4-benzyloxy benzaldehyde with 2-(4-hydroxyphenyl)-7-
methoxy-chroman-4-one 2 was carried out in presence
of Zn–TiCl4 in dry THF at reflux temperature, the de-
sired compound 4-[7-methoxy-4-(4-benzyloxybenzylid-
ene)-chroman-2-yl]-phenol 3 was obtained in very poor
yield along with dimeric product of reactants. It is
reported that in McMurry reaction the efficacy of reac-
tion depends on the reactivity of low valent titanium
reagent which ultimately depends on its method of
2-(4-hydroxyphenyl)-7-methoxy-chroman-4-one
2 (6)
were formed as byproducts. Dimer of 4-methoxybenzal-
dehyde was isolated as white solid which was a mixture
of cis- and trans isomers, whereas dimer of 2-(4-
hydroxyphenyl)-7-methoxy-chroman-4-one 2 (6) ap-
peared to be unstable and was detected only through
mass spectroscopy of partly purified sample. Condensa-
tion of compound 4 with 1-(2-chloroalkyl) amine hydro-
chloride in acetone in the presence of anhydrous K2CO3
under reflux gave the desired product 7 (prototype I)
(Scheme 1).20
The synthesis of prototype II was carried out through
the McMurry coupling reaction using 4,40-dimethoxy
benzophenone and 2-(4-hydroxyphenyl)-7-methoxy-
chroman-4-one 2 (Scheme 2).20 In this case, besides the
desired compound 8, dimers of chromanone 2 (6) and
4,40-dimethoxybenzophenone (9) were formed as

6008
A. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6006–6012
OCH3
H3CO
OCH3
H3CO
O
H3CO
O
R
R
O
O
Prototype I
Prototype II
Figure 3.
OCH2Ph
O
O
a
b
H3CO
OH
H3CO
O
2
H3CO
HO
O
1
OH
3
OH
c
OCH3
OCH3
O
O
OCH3
+
+
H3CO
H3CO
O
H3CO
OH
OH
4
5
6
d
OCH3
H3CO
O
OCH2CH2R
7
Scheme 1. Reagents: (a) 4-hydroxybenzaldehyde, dry piperidine, dry benzene, reflux; (b) 4-benzyloxybenzaldehyde, Zn–TiCl4, dry THF, reflux; (c) 4-
methoxybenzaldehyde, TiCl4–LAH, dry THF, reflux; (d) 2-chloroethyl alkylamine hydrochloride, K2CO3, dry acetone, reflux. For structure 7, R =
(a) NC4H8, (b) NC5H10, (c) N(CH3)2.
byproducts. Dimer 9 was isolated as crystalline solid,
whereas dimer 6 seemed to be unstable and was detected
through mass spectroscopy. Condensation of compound
8 with 1-(2-chloroalkyl) amine hydrochloride in acetone
in the presence of anhydrous K2CO3 under reflux gave
the desired product 10 (prototype II).
sessed significant anticancer activity similar to that of
Tamoxifen against MCF-7 cancer cell line.
In conclusion, this study presents an efficient method for
the preparation of 4-[7-methoxy-4-(4-methoxy-benzyli-
dene)-chroman-2-yl]-phenol and 4-[bis-(4-methoxyphe-
nyl)-methylene]-7-methoxy-chroman-2-yl]-phenol substi-
tuted with different alkyl amine chains, using the
McMurry coupling reaction. Significant estrogenic and
anti-estrogenic activities exhibited by these compounds
point to their ability to bind to estrogen receptor as such
or as possible hydroxy metabolite. Their low RBA val-
ues are likely due to the absence of free phenolic groups
Estrogen receptor binding affinity, estrogenic, antiestro-
genic, and antiproliferative activity data of compounds
are shown in Table 1. Compounds 3, 7a, 7b, 7c, 8, and
10a administered orally showed high estrogenic activity
(>50% gain) and also possessed estrogen antagonistic
activity (P15%). Compounds 4, 7a, 7b, 7c, and 10a pos-

A. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6006–6012
6009
H3CO
OCH3
H3CO
OCH3
+
a
2
6
H3CO
O
H3CO
OCH3
9
8
OH
b
H3CO
H3CO
OCH3
O
R
O
10
Scheme 2. Reagents: (a) 4,40-dimethoxybenzophenone, TiCl4–LAH, dry THF, reflux; (b) 2-chloroethyl alkylamine hydrochloride, K2CO3, dry
acetone, reflux. For structure 10, R = (a) NC5H10, (b) N(CH3)2.
Table 1. Biological activity data
Compound
Dose (oral)
(mg/kg/day)
Estrogen antagonistic
activity16
Estrogen agonistic activity16
RBA15 % of E2
Antiproliferative
activity17 IC50 (lM)
Uterine weighta
(mg)
Inhibitionb
(%)
Uterine weighta (mg)
Gainc (%)
Vehicle
EE
10
18.20 0.50
18.20 0.50
102.60 2.20
46.32 2.92
15.00 0.57
66.35 0.44
38.30 3.66g
14.00 1.52
85.00 0.57
17.33 2.40f
26.00 2.30g
16.03 0.99
108.43 4.14
39.57 4.75g
24.63 0.90g
46.30 1.61
19.60 0.44
106.96 7.05
52.50 4.60f
—
0.02
3
102.60 2.20
52.30 5.30
15.00 0.57
66.35 0.44
46.33 2.16e
14.00 1.52
85.00 0.57
88.33 10.47
70.33 6.88d
16.03 0.99
108.43 4.14
92.30 13.17d
104.4 1.00d
85.40 2.88
19.60 0.44
106.96 7.05
81.70 4.00e
—
464
155
TAM
Vehicle
EE
49
29
2g
5.09
10
0.02
10
10
342
153
3
0.02
Inactive
Vehicle
EE
0.02
10
10
507
24
4
—
17
0.013
0.097
8.77
Inactive
8
86
Vehicle
EE
7a
10
0.02
10
10
576
147
54
15
4
0.095
0.095
0.006
7.39
4.39
2.00
7b
7c
10
10
21
189
Vehicle
EE
10a
0.02
10
10
445
168
ND
26
ND
<0.001
<0.001
6.68
Inactive
10b
ND, not determined; EE = 17a-ethynylestradiol; E2 = 17b-estradiol.
a Values represent means SEM of a minimum of six observations in each group.
b Percent of 17a-ethynylestradiol per se treated group.
c Percent of vehicle control group.
d P < 0.05.
e P < 0.01 versus corresponding EE per se treated group; compound 4 exhibited an additive effect on ethnylestradiol induced uterine weight gain, but
lacked any estrogen antagonistic activity.
f P < 0.05.
g P < 0.01, versus corresponding vehicle control group, all other relevant comparisons were statistically not significant, g = Ref. 18,
TAM = tamoxifene.

6010
A. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6006–6012
16. Ghosh, R.; Kamboj, V. P.; Singh, M. M. Contraception
2001, 64, 261.
at the estrogen receptor binding subsite. Free phenolic
groups present in compounds 4 and 8 are possibly at
the anti-estrogen binding subsite. A preliminary study
of their anti-proliferative activity, showing promising
activity, suggests a more detailed investigation of these
prototype molecules as anticancer agents.
17. (a) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.;
McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Acad. Sci. 1990, 82, 1107;
(b) Srivastava, S.; Sharma, R.; Balapure, A. K. Ind. J.
Pharm. 2004, 36, 238.
18. Sudo, K.; Munsmaj, F. J.; Katzenellenbogen, B. S.
Endocrinology 1983, 112, 425.
19. Selected Physical data:
Acknowledgments
Compound 3: Yield: 20%, mp138–140 ꢁC. FABMS: 450;
IR (cmꢀ1): 3383, 2945, 1606, 1509, 1238, 836; 1H NMR
(d): 2.84 (m, 1H, CH2), 3.15 (dd, 1H, CH2), 3.79 (s, 3H,
OCH3), 4.90 (dd, 1H, CH), 5.07(s, 1H, CH), 6.48 (d,
J = 2.40 Hz, 1H, ArH), 6.57 (dd, 1H, ArH), 6.84 (d,
J = 8.40 Hz, 2H, ArH), 6.94 (d, J = 8.70 Hz, 2H, ArH),
7.32 (m, 8H, ArH), 7.89 (d, J = 8.70 Hz, 1H, ArH). Anal.
Calcd for C30H26O4: C, 80.00; H, 5.78. Found: C, 80.25;
H, 6.02.
Compound 4: Yield: 30%, mp 162–164 ꢁC. FABMS: 374;
IR (cmꢀ1): 3398, 2943, 1601, 1352, 1242, 1165, 1024, 836;
1H NMR (d): 2.86 (t, 1H, CH), 3.15 (dd, 1H, CH), 3.83
(s, 6H, 2· OCH3), 4.89 (dd, 1H, CH), 6.49 (s, 1H, CH),
6.57 (d, J = 8.81 Hz, 1H, ArH), 6.85 (m, 5H, ArH), 7.29
(m, 4H, ArH), 7.60 (d, J = 8.60 Hz, 1H, ArH); 13C NMR
(d): 32, 54, 76, 100, 107, 112, 114, 118.4, 124, 125.8,
126.6, 127.2129.4, 130.1, 155, 156.4, 157, 159.2. Anal.
Calcd for C24H22O4: C, 77.00; H, 5.88. Found: C, 76.81;
H, 5.93.
Compound 7a: Yield: 85%, mp of oxalate salt 183–
185 ꢁC. FABMS: 472(M+1); IR (cmꢀ1): 2950, 2832,
1603, 1510, 1244, 1030; 1H NMR (d): 1.39 (br s, 4H,
2CH2), 2.49 (br s, 4H, 2CH2), 2.75 (t, 2H, CH2), 2.83
(dd, 1H, CH), 3.09 (dd, 1H, CH), 3.72 (s, 3H, OCH3),
3.74 (s, 3H, OCH3), 4.08 (t, 2H, CH2), 4.84 (dd, 1H,
CH), 6.42 (s, 1H, CH), 6.51 (d, J = 8.21 Hz, 1H, ArH),
6.84 (m, 5H, ArH), 7.15 (d, J = 8.06 Hz, 2H, ArH), 7.27
(d, J = 8.04 Hz, 2H, ArH), 7.53 (d, J = 8.64 Hz, 1H,
ArH). Anal. Calcd for C30H33NO4: C, 76.43; H, 7.01; N,
2.97. Found: C, 76.51; H, 7.33; N, 3.21.
Atul Gupta thanks CSIR (India) for senior research fel-
lowship. The authors thank Miss. Mohini Chhabra for
efficient technical assistance and the Ministry of Health
and Family Welfare, Government of India, for financial
support.
References and notes
1. (a) A. Gruber, C. J.; Tschugguel, W.; Schneeberger, C.;
Huber, J. C. N. Eng. J. Med. 2002, 346, 304; (b) Grese, T.
A.; Dodge, J. A. Annu. Rep. Med. Chem. 1996, 19, 181.
2. Riggs, B. L.; Hartmann, L. C. N. Eng. J. Med. 2003, 348,
618.
3. Pearce, S. T.; Jordan, V. C. Crit. Rev. Oncol./Heamatol.
2004, 50, 3.
4. (a) Jordan, V. C. J. Med. Chem. 2003, 46, 883; (b) Ray, S.;
Dwivedy, I. Adv. Drug Res. 1997, 29, 171; (c) Jordan, V. C.
J. Med. Chem. 2003, 46, 1081.
5. (a) Andersson, M.; Storm, H. H.; Mouridsen, H. J. Acta
Oncol. 1992, 31, 259; (b) Anzai, Y.; Holinka, C. F.;
Kuramoto, H.; Gurpide, E. Cancer Res. 1989, 49, 2362.5;
(c) Sacco, A.J.; Gendre, I. Tamoxifene, Beyond the
antiestrogens. In Jhon A Kellen, Ed.; 1996, pp. 59–92.
6. (a) Black, L. J.; Goode, R. L. Life Sci. 1980, 26, 1453; (b)
Black, L. J.; Sato, M.; Rowley, E. R., et al. J. Clin. Invest.
1994, 93, 63; (c) Ettinger, B.; Black, D. M.; Mitlak, B. H.,
et al. J. Am. Med. Assoc. 1999, 282, 637.
7. (a) Ray, S.; Tandon, A.; Dwivedy, I.; Wilson, S. R.;
O’Neil, J. P.; Katzenellenbogen, J. A. J. Med. Chem. 1994,
37, 696; (b) Kamboj, V. P.; Ray, S.; Dhawan, B. N. Drugs
Today 1992, 28, 227.
8. (a) Singh, M. M. Med. Res. Rev. 2001, 21, 302; (b) Misra,
N. C.; Nigam, P. K.; Gupta, R.; Agarwal, A. K.; Komboj,
V. P. Int. J. Caner 1989, 43, 781.
9. Fisher, B.; Costantino, J. P.; Wickerham, D. L.; Red-
mond, C. K.; Kavanah, M.; Cronin, W. M.; Vogel, V.;
Robidoux, A.; Dimitrov, N.; Atkins, J.; Daly, M.;
Wieand, S.; Tan-Chiu, E.; Ford, L.; Wolmark, N. J. Natl.
Cancer Inst. 1998, 90, 1371.
10. Walsh, B. W.; Kuller, L. H.; Wild, R. A.; Paul, S.; Farmer,
M.; Lawrence, J. B.; Shah, A. S.; Anderson, P. W. J. Am.
Med. Assoc. 1998, 279, 1445.
Compound 7b: Yield: 82%, mp 93–95 ꢁC. FABMS: 486
(M+1).
Compound 7c: Yield: 80%, mp of oxalate salt 138–
140 ꢁC. FABMS: 535.
Compound 8: Yield: 35%, mp 182–184 ꢁC. FABMS: 480;
IR (cmꢀ1): 3430, 2969, 1643, 1601, 1317, 1255, 1162, 849,
1
767; H NMR (d): 2.80 (br s, 2H, CH2), 3.78 (s, 9H, 3·
OCH3), 5.16 (dd, 1H, CH), 5.29 (s, 1H, OH), 6.14 (d,
1H, J = 3.60 Hz, ArH), 6.43 (s, 1H, ArH), 7.01 (m, 13H,
ArH). Anal. Calcd for C31H28O5: C, 77.50; H, 5.83.
Found: C, 77.82; H, 5.72.
Compound 10a: Yield: 84%, mp 167–169 ꢁC. FABMS: 591;
IR(cmꢀ1): 2933, 1603, 1506, 1383, 1248, 1166, 1028, 832:
1H NMR (d): 1.43 (br s, 6H, 3CH2), 2.51 (br s, 4H, 2CH2),
2.68 (t, 2H, CH2), 2.81 (dd, 1H, CH), 3.12 (dd, 1H, CH),
2.80 (br s, 2H, CH2), 3.78 (s, 9H, 3· OCH3), 5.16 (dd, 1H,
CH), 6.16 (d, J = 3.60 Hz, 1H, ArH), 6.44 (s, 1H, ArH),
7.14 (m, 13H, ArH). Anal. Calcd for C38H41NO5: C, 77.16;
H, 6.94; N, 2.37. Found: C, 77.23; H, 7.02; N, 2.53.
Compound 10b: Yield: 75%, mp 170–172 ꢁC. FABMS: 566
(M+1).
11. Saeed, A.; Sharma, A. P.; Durani, N.; Jain, R.;
Durani, S.; Kapil, R. S. J. Med .Chem. 1990, 33,
3210, 3216, 3211.
12. Gauthier, S.; Caron, B.; Cloutier, J.; Dory, Y. K.; Favre,
A.; Larouche, D.; Mailhot, J.; Ouellet, C.; Schwerdtfeger,
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Belanger, A.; Labrie, C.; Labrie, F. J. Med. Chem. 1997,
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20. Experimental:
40, 2117.
General procedures for the synthesis of 1-(2-{4-[7-methoxy-4-
(4-methoxybenzylidene)-chroman-2-yl]-phenoxy}-ethyl)-alkyl-
amines (7):
Synthesis of 4-[7-methoxy-4-(4-methoxybenzylidene)-
chroman-2-yl]-phenol (4):
Under an inert atmosphere of N2, a stirred mixture of
THF (60 ml) and TiCl4 (5 ml, 8.65 g, 0.05 mol) at 0 ꢁC was
13. Katritzy and Rees, Comprehensive Heterocyclic Chemis-
try, Vol. 3, Chapter 2.22, British Cataloguing in Publica-
tion Data, pp. 573–883.
14. McMurry, J. E. Chem. Rev. 1989, 89, 1513.
15. Dhar, J. D.; Dwivedi, A.; Srivastava, A.; Setty, B. S.
Contraception 1994, 49, 609.

A. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6006–6012
6011
treated with LAH (1 g, 0.03 mol). The resulting black
slurry was allowed to reflux for an hour, then the reaction
mixture was gradually cooled to 0 ꢁC. To this mixture was
added a mixture of 2-hydroxy-7-methoxy-chroman-4-one
2 (0.50 g, 0.002 mol) and 4-methoxybenzaldehyde (0.95 g,
0.007 mol) in dry THF. The reaction mixture was refluxed
at 70 ꢁC for 3 h. On completion of reaction, the mixture
was poured onto 10% aqueous solution of potassium
carbonate. The whole content was then filtered through
silica gel under suction. Filtrate was extracted with ethyl
acetate, the organic layer was dried over sodium sulfate
and concentrated to oily residue. The crude material was
chromatographed over silica gel using ethyl acetate–
hexane (7:93) as eluent to yield (30%) pure compound 4.19
Synthesis of 1-(2-{4-[7-Methoxy-4-(4-methoxy-benzyli-
dene)-chroman-2-yl]-phenoxy}-ethyl)-pyrrolidine (7a, R =
NC4H8):
To a solution of 4-[7-methoxy-4-(4-methoxybenzylidene)-
chroman-2-yl]-phenol (4) (0.374 g, 0.001 mol) in dry
acetone, anhydrous potassium carbonate (1.0 g) and 2-
chloroethylpyrrolidine hydrochloride (0.26 g, 0.002 mol)
were added. The solution was heated under reflux for
6 h. The reaction mixture was filtered and evaporated to
dryness. The residue was taken into ethyl acetate,
washed with water, dried over anhydrous sodium
sulfate, and concentrated. The crude material was then
chromatographed over a column of silica gel eluting
with methanol–chloroform (3:97) to afford compound 7a
in 85 % yield. Pure compound 7a was then treated with
oxalic acid to obtained the oxalate salt of compound
7a.19
for 15 min. DCC was precipitated by centrifugation
(800g · 10 min) and the supernatants counted for radio-
activity in 10 ml of a dioxane-based scintillation fluid.
RBA of the text compound was computed from a graph
plotted between percent bound radioactivity versus log
concentration of the test substance. At 50% inhibition,
log of the competitor concentration relative to that of
estradiol gave the affinity of the test compound to
estrogen receptor relative to estradiol. This when mul-
tiplied with 100 gave the percentage value designated as
RBA.
Estrogen agonistic activity16
Twenty-one-day-old immature female Sprague–Dawley
rats were bilaterally ovariectomized under light ether
anesthesia and after post-operative rest for 7 days were
randomized into different treatment groups. Each rat
received the compound of the invention once daily for
three consecutive days on days 28–30 of age by oral
route. A separate group of animals received only the
vehicle for similar duration served as control. At
autopsy 24 h after the last treatment on day 31 of
age, vaginal smear of each rat was taken and uterus was
carefully excised, gently blotted, and weighed. Premature
opening of vagina, cornification of vaginal epithelium,
and increase in uterine fresh weight were taken as
parameters for evaluation of estrogen agonistic activity
in comparison to rats of vehicle control group. The
objective was to evaluate estrogen agonistic effect of the
compounds on the uterus and vagina.
Estrogen antagonistic activity16
Twenty-one-day-old immature female Sprague–Dawley
rats were bilaterally ovariectomized under light ether
anesthesia and after post-operative rest for 7 days were
randomized into different treatment groups. Each rat
received the compound of the invention and
0.02 mg kgꢀ1 dose of 17a-ethynylestradiol in 10% etha-
nol-distilled water once daily for 3 consecutive days on
days 28–30 of age by oral route. A separate group of
General procedure for the synthesis of 1-[2-(4-{4-[bis-(4-
methoxyphenyl)-methylene]-7-methoxy-chroman-2-yl}-
phenoxy)-ethyl]-alkylamine (10):
Synthesis
of
4-[bis-(4-methoxyphenyl)-methylene]-7-
methoxy-chroman-2-yl]-phenol (8):
The title product, compound 8,19 was prepared using the
same procedure as for the preparation of derivative 4
using 4,40-dimethoxybenzophenone (0.726 g, 0.003 mol)
instead of 4-methoxybenzaldehyde. Compound 8 was
obtained in 35% yield.
animals
receiving
only
17a-ethynylestradiol
(0.02 mg kgꢀ1) in 10% ethanol-distilled water for similar
duration were used for comparison. At autopsy on day
31 of age, vaginal smear of each rat was taken and
uterus was carefully excised, gently blotted, weighed,
and fixed for histology. Inhibition in ethynylestradiol
induced cornification of vaginal epithelium and increase
in uterine fresh weight were taken as parameters for
evaluation of estrogen antagonistic effect of the com-
pounds.
Synthesis of 1-[2-(4-{4-[bis-(4-methoxyphenyl)-methy-
lene]-7-methoxy-chroman-2-yl}-phenoxy)-ethyl]-piper-
idine (10a, R = NC5H10):
The title product10a19 was prepared using the same
procedure as for the preparation of derivative 7a using
derivative 8 (0.48 g, 0.001 mol) and 2-chloroethylpiperi-
dine hydrochloride (0.04 g, 0.002 mol) instead of 2-
chloroethylpyrrolidine hydrochloride. Compound 10a
was obtained in 84%.
Antiproliferative activity assay of compounds17
The test produce for the evaluation of antiprolifer-
ative cytotoxic activity in vitro was accomplished
following method proposed by Skehan et al.17a
Briefly, the confluent flask of MCF-7 cells was
trypsinized using 0.05% Trypsin–EDTA solution in
Estrogen receptor binding affinity15
The relative binding affinity (RBA) of the compounds
for the estrogen receptor was determined15 by compe-
tition assay, employing radiolabeled estradiol (3H-E2) as
the reference compound. The test ligands and (3H-E2)
were incubated (4 ꢁC) with cytosol estrogen receptors
obtained from immature 20 to 21-day-old rat uteri.
Aliquots of the uterine cytosol (200 ll concentrated 1
uterus per ml) prepared in TEA buffer (10 mM Tris,
1.5 mM EDTA, and 0.02% sodium azide, pH 7.4) were
incubated in triplicate with a fixed concentration of
radiolabeled estradiol with or without various concen-
trations of the competitor substance dissolved in 60 ll of
the TEA buffer containing DMF as co-solvent (final
concentration of DMF in the incubation medium never
exceeded 5%) for 18 h at 4 ꢁC. At the end of this period,
dextran-coated charcoal (DCC) (5% Norit 0.5% dextran)
suspension in 100 ll of TEA buffer was added into each
tube, which was briefly vortexed and allowed to stand
PBS, 104 cells/well plated in
a 96-welled flat-bot-
tomed plate in 200 ll Minimum Essential Medium,
pH 7.4, and allowed to attach for 24 h at 37 ꢁC in
a humidified CO2 incubator essentially according to
Srivastava et al.17b The test compounds were dis-
solved in appropriate solution (ethanol/DMSO), add-
ed at specified concentration, and further incubated
for 48 h as before. The cells were fixed with chilled
10% TCA and incubated for 1 h at 4 ꢁC. The
supernatant was then discarded and the cells washed
five times with deionized water and air-dried. One
Hundred microliters of 0.4% (w/v) sulforhodamine B
(SRB) in 1% acetic acid was added to each well and
incubated at room temperature for 30 min. Unbound
SRB was removed by five washes with 1% acetic

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A. Gupta et al. / Bioorg. Med. Chem. Lett. 16 (2006) 6006–6012
acid and the plate was air-dried. Two hundred OD read at 560 nM in plate reader. IC50 values
microliters of unbuffered Tris base, pH 10.5, was
added to extract the bound stain for 5 min and the
were determined with respect to Tamoxifen as
positive control.
a