Design and synthesis of 3-(azol-1-yl)phenylpropanes as microbicidal spermicides for prophylactic contraception

Article abstract of DOI:10.1016/j.bmcl.2010.11.042

We designed a series of 25 3-(azol-1-yl)phenylpropanes which yielded 10 compounds (3, 4, 7, 8, 13, 14, 19, 21, 23, 26) that irreversibly immobilized 100% human sperm at 1% (w/v) concentration in 60 s; 12 compounds (8, 9, 15, 16, 19-21, 23-25, 27, 28) that showed potent microbicidal activity at 12.5-50 μg/mL against Trichomonas vaginalis; and 17 compounds (3-11, 13, 15, 19, 21, 23, 26, 28, 30) that exhibited potent anticandida activity with minimum inhibitory concentration (MIC) of 12.5-50 μg/mL. Almost all the compounds exhibited high level of safety towards normal vaginal flora (Lactobacillus) and human cervical (HeLa) cells in comparison to the marketed spermicide nonoxynol-9 (N-9). All the biological activities were evaluated in vitro. Two compounds (4, 8) with good safety profile exhibited multiple (spermicidal, antitrichomonas and anticandida) activities, warranting further lead optimization for furnishing a prophylactic vaginal contraceptive.

Full text of DOI:10.1016/j.bmcl.2010.11.042

Bioorganic & Medicinal Chemistry Letters 21 (2011) 176–181
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 3-(azol-1-yl)phenylpropanes as microbicidal
spermicides for prophylactic contraception q
Lalit Kumar a, Amit Sarswat a, Nand Lal a, Ashish Jain b, Sumit Kumar c, S. T. V. S. Kiran Kumar a,
Jagdamba P. Maikhuri b, Atindra K. Pandey d, Praveen K. Shukla d, Gopal Gupta b, Vishnu L. Sharma a,
⇑
a Division of Medicinal and Process Chemistry, Central Drug Research Institute (CSIR), Lucknow 226 001, India
b Division of Endocrinology, Central Drug Research Institute (CSIR), Lucknow 226 001, India
c National Institute of Pharmaceutical Education and Research (NIPER), Raebareli 229 010, India
d Division of Fermentation Technology, Central Drug Research Institute (CSIR), Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed a series of 25 3-(azol-1-yl)phenylpropanes which yielded 10 compounds (3, 4, 7, 8, 13, 14,
19, 21, 23, 26) that irreversibly immobilized 100% human sperm at 1% (w/v) concentration in 60 s; 12
compounds (8, 9, 15, 16, 19–21, 23–25, 27, 28) that showed potent microbicidal activity at
Received 25 August 2010
Revised 12 October 2010
Accepted 5 November 2010
Available online 2 December 2010
12.5–50
lg/mL against Trichomonas vaginalis; and 17 compounds (3–11, 13, 15, 19, 21, 23, 26, 28, 30) that
exhibited potent anticandida activity with minimum inhibitory concentration (MIC) of 12.5–50
l
g/mL.
Almost all the compounds exhibited high level of safety towards normal vaginal flora (Lactobacillus)
and human cervical (HeLa) cells in comparison to the marketed spermicide nonoxynol-9 (N-9). All the
biological activities were evaluated in vitro. Two compounds (4, 8) with good safety profile exhibited
multiple (spermicidal, antitrichomonas and anticandida) activities, warranting further lead optimization
for furnishing a prophylactic vaginal contraceptive.
Keywords:
Spermicides
Antitrichomonas
Anticandida
Fluoxetine
Ó 2010 Elsevier Ltd. All rights reserved.
Nonoxynol-9
Good reproductive health is the basis for intimate relationship,
happy family and healthy children, which ensures that every child
is wanted, every birth is safe, and every person is free of sexually
transmitted infection (STI) and human immunodeficiency virus
(HIV).1 On the other hand, some 340 million new cases of curable
STIs occur every year [excluding HIV and other viral sexually trans-
mitted diseases (STDs)], amongst which trichomoniasis has the
highest incidence. The presence of one or more STI increases the
risk of becoming infected with HIV by 2–9 times. Furthermore, wo-
men’s health can also be affected adversely by reproductive tract
infections like candidiasis that are not sexually transmitted. There-
fore, developing user-friendly, self-administrable, anti-STI vaginal
spermicides has become an urgent global priority.2
The inter-conversion of sulfhydryl groups to disulfide plays an
important role in sperm maturation, its motility and viability.3–5
This free SH to S–S interchange protects sperm from oxidative dam-
ages.6 Thus a sulfhydryl binding agent should possess spermicidal
activity as exemplified by N-ethylmaleimide,7 acrylonitriles,8 acryl-
ophenones,9 quinolines,10 5,50-dithiobis(2-nitrobenzoic acid),11
dithiocarbamates12 and disulfide esters of carbothioic acid.13–16
In our efforts to design dually active spermicides, benzenepro-
panamines17 and selective serotonin reuptake inhibitor (SSRI)
antidepressants fluoxetine and paroxetine18 exhibited potent sper-
micidal activity, possibly by interacting with sulfhydryl groups
present over sperm cell membrane.19,20 Metronidazole21–23 is the
only FDA approved drug for trichomonasiasis. On the other hand
various azoles being used clinically as microbicidal agents.24–28
N
HN
CH3
O2N
N
CF3
O
OH
(ii)
(i)
R1
N
N
2
1
X
R2
O2N
CH3
N
3
CF3
X = CO, CHOH, CH(O)C6H4-p-CF3
O
NR1R2 = azoles
(iii)
(iv)
q
C.D.R.I. Communication no. 7988.
⇑
Corresponding author. Tel.: +91 522 2612411x4320; fax: +91 522 2623405.
E-mail address: vlscdri1@rediffmail.com (V.L. Sharma).
Figure 1. Chemical structures of phenylpropanamine (i), metronidazole (ii), hybrid
compound (iii) and general structures of synthesized compounds (iv).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.042

L. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 176–181
177
HO
Cl
a, b
i
j,k
Cl
O
Cl
O
OH
O
2
CF
1
3
c
30
29
1
1
R
1
R
R
h
d
N
2
N
N
2
R
2
R
R
OH
O
for compd.
19-23
10-16
f
26-28
3-9
e
1
R
1
R
N
N
g
2
R
2
R
for compd.
24, 25
O
Br
F C
3
17, 18
19-25
CH
N
3
CH
3
N
N
N
N
N
N
N
NR1R2 :
N
N
N
N
N
N
N
N
O N
2
NO
2
Scheme 1. Synthetic route of the compounds. Reagents and conditions: (a) SOCl2, C6H6, reflux, 4 h; (b) C6H6, AlCl3, 0–5 °C; (c) HNR1R2, toluene, Et3N, reflux, 5 h; (d) NaBH4,
MeOH, 0–5 °C, 2 h; (e) NaH, p-chlorobenzotrifluoride, DMAc/DMF, 110–115 °C, 7–10 h; (f) PBr3, diethylether, rt, 3 h; (g) K2CO3, dry acetone, 4-(trifluoromethyl)phenol, rt,
16 h; (h) PBr3, toluene, reflux, 1 h; (i) NaBH4, MeOH, 0–5 °C, 1 h; (j) PBr3, diethylether, rt, 3 h; (k) 4-(trifluoromethyl)phenol, NaH, DMF, 110–115 °C, 8 h.
Table 1
Spermicidal and antitrichomonas activity of the compounds (3–16) against human spermatozoa and Trichomonas vagainalis
R1
N
X
R2
3-16
Compound
X
NR1R2
Immobilization of human
spermatozoaa,b (%)
Antitrichomonas activity
(MIC in
lg/mL) at 48 h
N
N
3
4
5
100
100
60
>200
O
O
N
N
200
N
>200
N
O
O
N
N
N
N
N
6
80
200
CH3
7
8
100
100
>200
25
O
N
N
N
O
O
O2N
CH3
N
N
9
90
50
NO2
N
N
N
10
11
12
90
90
70
>200
>200
>200
OH
OH
N
N
N
N
OH
OH
N
N
N
13
100
>200
(continued on next page)

178
L. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 176–181
Table 1 (continued)
Compound
X
NR1R2
Immobilization of human
spermatozoaa,b (%)
Antitrichomonas activity
(MIC in
lg/mL) at 48 h
CH3
14
15
100
95
>200
N
OH
N
N
N
25
50
OH
OH
O2N
CH3
N
N
16
70
NO2
Metronidazole
Nonoxynol-9
—
—
—
—
—
100
2.0
37.4
a
Vehicle (control) has 100% motility at the time of testing.
At 1% concentration of test compound.
b
Table 2
Spermicidal and antitrichomonas activity of the compounds (20–29, 31) against human spermatozoa and Trichomonas vagainalis
R1
N
R2
O
F3C
19-25
R1
N
R2
26-28
O
CF3
30
Compound
NR1R2
Immobilization of human
spermatozoaa,b (%)
Antitrichomonas activity
(MIC in
lg/mL) at 48 h
N
N
19
20
100
50
12.5
N
N
25
50
N
21
22
100
70
N
N
N
N
N
N
>200
CH3
23
24
100
40
25
N
N
N
12.5
O2N
CH3
N
N
25
70
25
NO2
N
26
27
N
N
100
80
>200
25
N
O2N

L. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 176–181
179
Table 2 (continued)
Compound
NR1R2
Immobilization of human
spermatozoaa,b (%)
Antitrichomonas activity
(MIC in
lg/mL) at 48 h
CH3
N
N
28
90
25
NO2
30
—
—
—
90
—
100
>200
2.0
37.4
Metronidazole
Nonoxynol-9
a
Vehicle (control) has 100% motility at the time of testing.
At 1% concentration of test compound.
b
We hypothesized that hybrid molecules (Fig. 1, iii) incorporating
both phenylpropanamine (Fig. 1, i) and metronidazole (Fig. 1, ii)
structures may yield dually active spermicidal microbicides. Thus
the compounds synthesized (Fig. 1, iv) were evaluated in vitro for
their spermicidal, antitrichomonas and anticandida activities as
well as their safety against vaginal micro flora and cervical cells.
The structure activity relationship (SAR) is being discussed in this
communication.
standard drugs metronidazole and nonoxynol-9 inhibited the
growth of T. vaginalis at 2.0 and 37.5 g/mL, respectively. Among
1-oxo (3–9) and 1-hydroxyl (10–16) derivatives of phenylpro-
panes, significant antitrichomonas activity was exhibited by com-
pounds substituted with 4-nitroimidazolyl (8, 15) and 2-methyl-
5-nitroimidazolyl (9, 16) groups at position-3 (MIC 25–50 lg/
mL). Whereas amongst 1-[4-(trifluoromethyl)]phenoxy (19–25)
derivatives, six compounds exhibited significant activity (MIC
l
The compounds (3–16, 19–28, 30) were synthesized according
to Scheme 1. Compounds 19–25 were obtained by using the pub-
lished reaction procedure,17 however, nitro group containing com-
pounds 24 and 25 could not be obtained by this method.
Furthermore, unusual reactions were observed while preparing
compounds 24 and 25, resulting in formation of compounds 26–
28 and 30 due to dehydrobromination.
12.5–50 lg/mL) while compound 22 was inactive (MIC >200 lg/
mL). The compounds with 4-nitroimidazolyl (27) and 2-methyl-
5-nitroimidazolyl groups (28) at position-3 were more active
among 1,2-dehydrophenylpropanes (26–28). The significant activ-
ity in compounds containing nitroimidazole group confirmed the
role of nitro group in antitrichomonas activity of metronidazole.29
All the compounds were tested against five strains of candida.
Seventeen compounds (3–11, 13, 15, 19, 21, 23, 26, 28, 30) exhib-
The study included the incorporation of four phenylpropane
moieties viz., 1-oxo (3–9), 1-hydroxyl (10–16), 1-[4-(trifluoro-
methyl)]phenoxy (19–25) and 1,2-dehydro (26–28, 30) with five
diazoles and two triazoles. All the 25 compounds exhibited sper-
micidal activity (Tables 1 and 2) as these compounds immobilized
40–100% normal human spermatozoa. Ten compounds (3, 4, 7, 8,
13, 14, 19, 21, 23, 26) caused 100% immobilization while six com-
pounds (9–11, 15, 28, 30) caused 90–95% immobilization of human
sperm at 1% concentration (w/v). The spermicidal activity of 3, 10,
19 and 26 among the imidazolo derivatives ranged between 90%
and 100%, which suggested that imidazole group imparts the activ-
ity. Whereas in benzimidazolo derivatives (4, 11, 20), the sperm
immobilizing potential reduced progressively from oxo (100%)
and hydroxy (90%) to 1-[4-(trifluoromethyl)]phenoxy (50%). Con-
versely, in triazolo derivatives (5, 12, 21) the spermicidal activity
increased from 60% (oxo) to 70% (hydroxyl) and 100% 1-[4-(trifluo-
romethyl)]phenoxy. Among benzotriazolo compounds (6, 13, 22),
the hydroxyl derivative was 100% active while the other two
showed 70–80% activity. The three 2-methylimidazolyl com-
pounds (7, 14, 23) caused 100% immobilization of human sperms.
Among 4-nitroimidazolyl compounds, the oxo (8) and hydroxyl
(15) derivatives immobilized 95–100%, whereas 1-[4-(trifluoro-
methyl)]phenoxy (24) and 1,2-dehydro (27) exhibited reduced
spermicidal activity of 40% and 80%, respectively. The compounds
with 2-methyl-5-nitroimidazolyl group exhibited 90% (9, 28) and
70% (16, 25) spermicidal activity. On the other hand, 1,2-dehydro
compound (30) in which the 3-azol-1-yl group was replaced by
1-[4-(trifluoromethyl)]phenoxy moiety; the spermicidal activity
was 90%. Thus it could be inferred that in 3-azol-1-ylphenylpro-
panes an imidazole or 2-methylimidazole group at position-3 with
an oxo group at position-1 was more desirable for spermicidal
activity.
ited anticandida activity (Table 3) with MIC of 25–50 lg/mL
against one or more strains. Out of these, two compounds (19,
Table 3
Anticandida activity of compounds (3–16, 19–28, 30) against five different strains of
candida
Compound
Anticandida activity (MIC in lg/mL)
1
2
3
4
5
3
4
5
6
7
8
9
10
11
12
13
14
15
16
19
20
>50
25
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
12.5
>50
50
>50
50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
50
25
50
>50
50
50
50
50
50
>50
50
>50
50
>50
50
>50
>50
>50
50
>50
50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
50
>50
>50
>50
50
>50
50
>50
25
>50
>50
>50
50
21
22
23
>50
25
24
25
26
27
28
30
>50
>50
>50
>50
50
>50
0.5
>50
>50
>50
50
>50
>50
>50
0.5
>50
>50
>50
>50
>50
>50
0.25
>50
>50
>50
25
>50
>50
50
>50
>50
>50
>50
>50
>50
0.25
>50
Fluconazole
Nonoxynol-9
0.25
>50
All the 25 compounds were evaluated for antitrichomonas
activity, out of which 14 compounds (4, 6, 8, 9, 15, 16, 19–21,
23–25, 27, 28) were found to be active against Trichomonas vaginal-
50
1, Candida albicans; 2, Candida parapsilosis; 3, Candida albicans MTCC-183; 4, Patient
isolates CDRI; 5, NCIM-3557; MIC, minimum inhibitory concentration.
is with MIC ranging from 12.5 to 200 lg/mL (Tables 1 and 2). The

180
L. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 176–181
Figure 2. Cytotoxicity of compounds (3–16, 19–28, 30) towards human cervical (HeLa) cells.
Figure 3. Compatibility of compounds (3–16, 19–28, 30) with normal vaginal flora Lactobacillus.
23) inhibited the growth of all the five candida strains tested. The
activity was found to be much better than nonoxynol-9 (MIC
has resulted into the discovery a structural class that may lead to a
potent dually active microbicidal spermicide.
>50 lg/mL). This suggests that imidazole and 2-methylimidazole
was important for anticandida activity.
All the compounds were evaluated for their safety towards hu-
man cervical cell line (HeLa, Fig. 2) and vaginal flora (Lactobacillus,
Acknowledgements
Authors are thankful to Mrs. Tara Rawat for her technical assis-
tance. L.K. and N.L. are thankful to the University Grants Commis-
sion, A.S. is thankful to the Council of Scientific and Industrial
Research and A.J. is thankful to the Indian Council of Medical
Research, New Delhi, for the grant of research fellowships. Authors
acknowledge the SAIF Division for the spectral data.
Fig. 3) at 200 lg/mL concentration by cell-viability assay. Most of
the compounds were highly safe as compared to nonoxynol-9.
However, the toxicity of four compounds (19–21, 23) towards HeLa
cells and Lactobacillus indicates that incorporation of 1-[4-(trifluo-
romethyl)]phenoxy group at position-1 may not be desirable.
Of the 10 compounds (3, 4, 7, 8, 13, 14, 19, 21, 23, 26) that
immobilized 100% human sperms, nine of these have been found
to be dually active (except compound 14). Compounds 3, 13 and
26 exhibited anticandida activity as well and were safe towards
both HeLa cell and Lactobacillus as compared to nonoxynol-9. How-
ever, compound 7 showed safety only towards Lactobacillus. Five
compounds (4, 8, 19, 21, 23) exhibited spermicidal, anticandida
and antitrichomonas activities. However, amongst these com-
pounds 4 and 8 had high safety profile against HeLa cells and Lac-
tobacillus, compound 26 was safe towards HeLa cells only while the
remaining two (19, 21) exhibited toxicity towards both the HeLa
cells and Lactobacillus. The activities and safety profile of com-
pounds 4 and 8 suggested that a keto group at position-1 with
an imidazole or a nitroimidazole at position-3 in phenylpropane
framework were most suited for dual activity and safety. The study
Supplementary data
Supplementary data (the general procedures for the synthesis
and biological evaluation of compounds) associated with this arti-
cle can be found, in the online version, at doi:10.1016/j.bmcl.
2010.11.042.
References and notes
1. http://www.unfpa.org/swp/2009/en/pdf/EN_SOWP09.pdf; http://www.unfpa.
org/rh/ (Accessed June 2010).
2. http://data.unaids.org/pub/Outlook/2010/
20100713_outlook_report_web_en.pdf (Accessed August 2010).
3. Storey, B. T. Mol. Hum. Reprod. 1997, 3, 203.

L. Kumar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 176–181
181
4. Nivsarkar, M.; Cherian, B.; Patel, S. Biochem. Biophys. Res. Commun. 1998, 247,
716.
5. Seligman, J.; Newton, G. L.; Fahey, R. C.; Shalgi, R.; Kosower, N. S. J. Androl. 2005,
26, 629.
6. Maan, T.; Lutwak-Maan, C. Male Reproductive Function and Semen; Springer:
Berlin, 1981. Chapter X, p 288.
7. Solanki, S.; Potter, W. D.; Anderson, L. U.S. Patent U.S. 2005131238, 2005.
8. Cavins, J. F.; Friedmanthe, M. J. Biol. Chem. 1968, 243, 3357.
9. Kumaria, N.; Dwivedi, A. K.; Maikhuri, J. P.; Gupta, G.; Habib, S.; Dhar, J. D.;
Singh, S. Eur. J. Med. Chem. 2002, 37, 855.
17. Kiran Kumar, S. T. V. S.; Sharma, V. L.; Kumar, M.; Shukla, P. K.; Tiwari, P.; Singh,
D.; Jain, R. K.; Maikhuri, J. P.; Gupta, G.; Singh, M. M. Bioorg. Med. Chem. 2006,
14, 6593.
18. Kiran Kumar, S. T. V. S.; Sharma, V. L.; Tiwari, P.; Singh, D.; Maikhuri, J. P.;
Gupta, G.; Singh, M. M. Bioorg. Med. Chem. Lett. 2006, 16, 2509.
19. Calvin, H. I.; Yu, C. C.; Bedford, J. M. Exp. Cell Res. 1973, 81, 333.
20. de Lamerande, E.; Gagon, C. Free Radiac. Biol. Med. 2003, 35, 1271.
21. http://www.urologychannel.com/std/trichomoniasis.shtml (Accessed August
2010).
22. Upcroft, J. A.; Dunn, L. A.; Wright, J. M.; Benakli, K.; Upcroft, P.; Vanelle, P.
Antimicrob. Agents Chemother. 2006, 50, 344.
23. Kumar, L.; Sarswat, A.; Nandlal; Sharma, V. L.; Jain, A.; Kumar, R.; Verma, V.;
Maikhuri, J. P.; Kumar, A.; Shukla, P. K.; Gupta, G. Eur. J. Med. Chem. 2010, 45,
817.
10. Maikhuri, J. P.; Dwivedi, A. K.; Dhar, J. D.; Setty, B. S.; Gupta, G. Contraception
2003, 67, 403.
11. Hughes, L. M.; Griffith, R.; Aitken, R. J. Curr. Med. Chem. 2007, 14, 775.
12. Kiran Kumar, S. T. V. S.; Kumar, L.; Sharma, V. L.; Jain, A.; Jain, R. K.; Maikhuri, J.
P.; Kumar, M.; Shukla, P. K.; Gupta, G. Eur. J. Med. Chem. 2008, 43, 2247.
13. Jain, R. K.; Maikhuri, J. P.; Kiran Kumar, S. T. V. S.; Sharma, V. L.; Dwivedi, A. K.;
Mitra, K.; Bajpai, V. K.; Gupta, G. Hum. Reprod. 2007, 22, 708.
14. Dwivedi, A. K.; Sharma, V. L.; Kumaria, N.; Kiran Kumar, S. T. V. S.; Srivastava, P.
K.; Ansari, H.; Maikhuri, J. P.; Gupta, G.; Dhar, J. D.; Roy, R.; Joshi, B. S.; Shukla, P.
K.; Kumar, M.; Singh, S. Bioorg. Med. Chem. 2007, 15, 6642.
15. Jain, R. K.; Jain, A.; Maikhuri, J. P.; Sharma, V. L.; Dwivedi, A. K.; Kiran Kumar, S.
T. V. S.; Mitra, K.; Bajpai, V. K.; Gupta, G. Hum. Reprod. 2009, 24, 590.
16. Jain, R. K.; Jain, A.; Kumar, R.; Verma, V.; Maikhuri, J. P.; Sharma, V. L.; Mitra, K.;
Batra, S.; Gupta, G. Hum. Reprod. 2010, 25, 1156.
24. Meerpoel, L. et al J. Med. Chem. 2005, 48, 2184.
25. Sheng, C.; Zhang, W.; Ji, H.; Zhang, M.; Song, Y.; Xu, H.; Zhu, J.; Miao, Z.; Jiang,
Q.; Yao, J.; Zhou, Y.; Zhu, J.; Lu, J. J. Med. Chem. 2006, 49, 2512.
26. Silvestri, R.; Artico, M.; Regina, G. L.; Pasquali, A. D.; Martino, G. D.; D’Auria, F.
D.; Nencioni, L.; Palamara, A. T. J. Med. Chem. 2004, 47, 3924.
27. Santo, R. D.; Tafi, A.; Costi, R.; Botta, M.; Artico, M.; Forte, F. M.; Caporuscio, F.;
Angiolella, L.; Palamara, A. T. J. Med. Chem. 2005, 48, 5140.
28. Tafi, A.; Costi, R.; Botta, M.; Santo, R. D.; Corelli, F.; Massa, S.; Ciacci, A.; Manetti,
F.; Artico, M. J. Med. Chem. 2002, 45, 2720.
29. http://www.medic8.com/medicines/Metronidazole.html (Accessed August
2010).