Design and synthesis of γ-butyrolactone derivatives as potential spermicidal agents

Article abstract of DOI:10.1016/j.bmcl.2014.06.045

A series of γ-butyrolactone derivatives has been designed and synthesized from commercially available 2-acetyl butyrolactone (3-acetyldihydrofuran-2(3H)-one, 1) by aminoalkylating its active methylene followed by condensation with different aldehydes. Compounds having amino group were further converted to their respective tartrate salts and were evaluated for spermicidal activity against human sperm in vitro. Compounds showing appreciable spermicidal activity at ≤0.5% [3c, 4d (0.5%); 2c, 3d (0.1%); 2d, 4c (0.05%)] were tested for safety studies against human cervical (HeLa) cell line. These compounds were found safer than, Nonoxynol-9. One of the two most active compounds was also found to be the safest (IC₅₀ = 961 μg/ml; 4c), while the second compound exhibited lower safety against HeLa (IC₅₀ = 269 μg/ml; 2d). The compound 4c significantly reduced the number of free thiols on human sperm. All the compounds were inactive against Trichomonas vaginalis.

Full text of DOI:10.1016/j.bmcl.2014.06.045

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3903–3906
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of
spermicidal agents q
c-butyrolactone derivatives as potential
Rishi Ranjan Pandey a, Akansha Srivastava a, Shakti Deep Pachauri a, Kiran Khandelwal a, Arshi Naqvi a,
Richa Malasoni a, Bhavana Kushwaha b, Lokesh Kumar b, Jagdamba Prasad Maikhuri b, Garima Pandey c,
Sarvesh Paliwal c, Gopal Gupta b, Anil Kumar Dwivedi a,
⇑
a Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
b Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
c Department of Pharmacy, Banasthali Vidyapith, Banasthali, Rajasthan, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of c-butyrolactone derivatives has been designed and synthesized from commercially available
Received 20 March 2014
Revised 30 May 2014
Accepted 17 June 2014
Available online 26 June 2014
2-acetyl butyrolactone (3-acetyldihydrofuran-2(3H)-one, 1) by aminoalkylating its active methylene fol-
lowed by condensation with different aldehydes. Compounds having amino group were further con-
verted to their respective tartrate salts and were evaluated for spermicidal activity against human
sperm in vitro. Compounds showing appreciable spermicidal activity at 60.5% [3c, 4d (0.5%); 2c, 3d
(0.1%); 2d, 4c (0.05%)] were tested for safety studies against human cervical (HeLa) cell line. These com-
pounds were found safer than, Nonoxynol-9. One of the two most active compounds was also found to be
Keywords:
Spermicides
Butyrolactone derivatives
Thiols
the safest (IC50 = 961
(IC50 = 269 g/ml; 2d). The compound 4c significantly reduced the number of free thiols on human
sperm. All the compounds were inactive against Trichomonas vaginalis.
lg/ml; 4c), while the second compound exhibited lower safety against HeLa
l
HeLa cells
Ó 2014 Elsevier Ltd. All rights reserved.
The world population is expanding at an alarming rate of ꢀ46
people per min causing an annual increase of ꢀ77 million. With
17% of the world’s population, India had become the second most
populous country in the world.1,2 With the global epidemic of
unintended pregnancies3 and pandemic of sexually transmitted
infections (STIs), the development of inexpensive, safe and effec-
tive contraceptive method is needed to face this threat. During
the last decade various physical and chemical methods of contra-
ception have been developed. The oral contraceptives have proven
their worth, but suffer the drawback of causing side effects. On the
other hand, topical use of vaginal spermicides lacks the systemic
side effects associated with the former, and may prove to be a con-
venient contraceptive choice.
The marketed spermicidal preparations usually comprise of
membrane surfactant, Nonoxynol-9 (N-9) as one of the major
ingredients. Due to its membrane disruptive properties it damages
the cervico vaginal epithelium and causes irritation to the vagina
and rectum and thereby increases the risk of HIV and STD infec-
tions.4,5 Unlike the detergent based microbicides that targets cell
membranes, a non-detergent spermicide lacking cytotoxicity to a
wide range of cells in the vaginal environment could be an ideal
replacement for N-9.
Our institute has developed a vaginal contraceptive cream
[CONSAP] containing spermicidal saponins isolated from the fruit
pericarp of Sapindus mukorossi6 with surfactant action and is safer
than N-9. Concurrently, we continued our efforts to design
non-detergent molecules acting specifically against sperm, thus
minimising the side effects associated with N-9 spermicides.7–16
It has been reported that various acrylonitriles,17 N-ethylmalei-
mide,18 5,5-dithiobis-(2-nitrobenzoic acid)19 etc. interact with
sulfhydryl groups present on sperm cells. This inter-conversion
to disulfide plays an important role in sperm function (mainly
maturation, motility and viability).20–22 Alternatively, several
derivatives of quinotoxin and cinchotoxin have been reported as
potent spermicidal agents.1,23,24
c
-Butyrolactone ring is an important structural component in
organic compounds as it is present in about 10% of all natural prod-
ucts. These lactones have an impressive biological profile including
strong antibiotic (e.g., Xanthatin, Fig. 1), antifungal (e.g., Encelin,
Fig. 1), antihelmitic, antitumor, antiviral, antiinflammatory and
cytostatic properties. They are bioactive due to the ability of their
a
-methylene group to bind with the nucleophilic sites of biomole-
q
CDRI communication No. 8724.
Corresponding author. Tel.: +91 522 2623405; fax: +91 522 2623938.
E-mail address: anilcdri@gmail.com (A.K. Dwivedi).
cules via conjugate addition.25
⇑
http://dx.doi.org/10.1016/j.bmcl.2014.06.045
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3904
R. R. Pandey et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3903–3906
Scheme 1. R@H (1), pyrrolidionoethyl (1a), N,N-diethylaminoethyl (1b), morpho-
linoethyl (1c), N,N-dimethylaminopropyl (1d). Reagents and conditions: (i) K2CO3,
aminoalkyl chloride HCl, acetone, reflux, 12 h. (ii) -tartaric acid, methanol, 2 h.
D
Figure 1. Structure of c-butyrolactone containing potent bioactive agents.
Moreover, sesquiterpene lactones such as 1-oacetylbritannilac-
tone (Fig. 1) and methylenolactocin (Fig. 1) exhibit impressive
cytotoxic activities because of their ability to interact with nucleic
acids. The O@CAC@CH2 moiety (the exocyclic, conjugated double
bond) acts as an alkylating agent via Michael-type reaction with
biological cellular nucleophiles or cysteine residues of functional
proteins to form covalent bonds.26
Scheme 2. Reagents and conditions: (i) vanillin, piperidine, dry benzene, reflux on
Dean Stark apparatus, 8 h. (ii) -tartaric acid, methanol, 2 h.
D
In the present study, we have designed and synthesized a series
of molecules having butyrolactone ring incorporated with
a,b-
unsaturated ketonic system. The former is potentially cytotoxic
and the later can interact with cellular thiols (Fig. 2). Compounds
having such properties have been reported earlier as potent sper-
micide.20–22 In addition, the synthesized compounds bear a tertiary
amino alkyl side chain, which can be converted to their respective
salts by reacting with a suitable acid to increase their water
solubility.
The synthetic scheme of all the compounds are represented in
Schemes 1–4. 2-Acetylbutyrolactone (3-acetyldihydrofuran-
2(3H)-one, 1) has acidic hydrogen flanked between two carbonyl
groups. This site was first amino alkylated by refluxing 1 with dif-
ferent amino alkyl chlorides and K2CO3 in acetone for 8 h to yield a
series of compounds 1a–1d with approximate yield of 70%
(Scheme 1). Acetyl group of compounds (1, 1a–1d) were further
condensed with different aldehydes (4-hydroxy-3-methoxybenzal-
dehyde, 4-(dimethylamino) benzaldehyde, 4-hydroxybenzalde-
hyde) by refluxing in dry benzene in presence of piperidine to
Scheme 3. Reagents and conditions: (i) 4-(N,N-Dimethylamino) benzaldehyde,
piperidine, dry benzene, reflux on Dean Stark apparatus, 8 h. (ii)
methanol, 2 h.
D-Tartaric acid,
yield exclusively (E)-
a,b-unsaturated ketones (2, 2a–2d, 3, 3a–3d,
4, 4a–4d).27
Compounds containing amino group were further converted to
their water soluble tartarate salts (1aT–1dT, 2aT–2dT, 3T, 3aT–
3dT, and 4aT–4dT) (Schemes 1–4). Compounds synthesised above
are summarised in Table 1. All the compounds were characterized
by spectroscopic method using mass, IR and NMR spectroscopy.
ESI–MS spectra of the above compounds have shown base peak
Scheme 4. Reagents and conditions: (i) 4-hydroxy benzaldehyde, piperidine, dry
benzene, reflux on Dean Stark apparatus, 8 h. (ii) -Tartaric acid, methanol, 2 h.
D
corresponding to m/z equals to (M++1) of the respective com-
pounds. In FT-IR spectra, peak around 3000 cmÀ1 is due to C@C
stretching, around 1750 is due to C@O stretching of lactone,
around 1700 cmÀ1 is due to conjugated C@O stretching, peaks
around 1580 cmÀ1 and 1530 cmÀ1 corresponds to aromatic ring.
In 1H NMR, two doublets around d 7.75 and 7.05 (or 6.50 in case
of compounds containing 4-hydroxy-3-methoxyphenyl moiety)
having coupling constant around 16 Hz indicated the presence of
trans alkenic protons thus indicating the formation of (E)-stereo-
isomer. Peaks due to aromatic protons were found between d
7.50 and 6.50. a-Methylene protons of butyrolactone ring are dia-
stereotropic, therefore, they appeared as two set of multiplets
between d 4.50 and 4.25. b-methylene protons which appeared
as two set of multiplets between d 2.30 and 2.10. Peaks due to
the substituents at position 3 of butyrolactone ring and that of
Figure 2. General structure of designed compounds.

R. R. Pandey et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3903–3906
3905
Table 1
Description of compounds
S. No.
1
Compound No.
R
R1
R2
MF, mass (M+1)
C14H14O5, 263.1
Tartrate salt
2
–H
–OH
–OCH3
—
2
2a
–OH
–OCH3
C20H25NO5, 360.2
2aT
3
4
2b
2c
–OH
–OH
–OCH3
–OCH3
C20H27NO5, 362.2
C20H25NO6, 376.2
2bT
2cT
5
6
2d
3
–OH
–OCH3
–H
C19H25NO5, 348.2
C15H17NO3, 260.2
2dT
3T
–H
–N(CH3)2
7
3a
–N(CH3)2
–H
C21H28N2O3,357.2
3aT
8
9
3b
3c
–N(CH3)2
–N(CH3)2
–H
–H
C21H30N2O3,359.2
C21H28N2O4, 373.2
3bT
3cT
10
11
3d
4
–N(CH3)2
–OH
–H
–H
C20H28N2O3, 345.2
C13H12O4, 233.1
3dT
—
–H
12
4a
–OH
–H
C19H23NO4,330.2
4aT
13
14
4b
4c
–OH
–OH
–H
–H
C19H25NO4, 332.2
C19H23NO5, 346.2
4bT
4cT
15
4d
–OH
–H
C18H23NO4, 318.2
4dT
aromatic ring were found in their respective regions in accordance
with their structure. Data of 1H NMR was fully supported by 13C
NMR spectroscopy.
lactone) were inactive. 3-Acetyl-3-(2-morpholinoethyl) dihydrofu-
ran-2(3H)-one (1c) and 3-acetyl-3-(3-(dimethylamino)propyl)
dihydrofuran-2(3H)-one (1d), when condensed with different alde-
hydes yielded potent spermicides (2c, 2d, 3c, 3d, 4c and 4d). The
Compounds were tested for in vitro spermicidal activity against
human spermatozoa. The spermicidal activity was determined by a
modified Sander–Cramer assay.28 Fresh human semen samples
were obtained from healthy and fertile volunteers and allowed to
liquefy at 37 °C for 30 min. Compounds showed 100% spermicidal
activity at a concentration range of 1.0–0.05% w/v. Compounds
active at 60.1% concentration are reported in Table 2. Other com-
pounds did not show spermicidal activity at 0.1% concentration
[data not shown].
result indicated that introduction of an
enhance the spermicidal activity.
a,b-unsaturation can
(E)-3-(3-(4-Hydroxy-3-methoxyphenyl) acryloyl) dihydrofuran-
2(3H)-one (2), (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)dihy-
drofuran-2(3H)-one (3) and (E)-3-(3-(4-hydroxyphenyl)acryloyl)
dihydrofuran-2(3H)-one (4) without aminoalkyl side chains were
found inactive against human spermatozoa. These findings indi-
cated that amino alkyl groups at position 3 along with a,b-unsatura-
The structure activity relationship (SAR) indicated that com-
pounds showing appreciable spermicidal activity (MEC 60.5%)
tion is an essential structural requisite for spermicidal activity of the
synthesized compounds.
Compounds (2c, 2d, 3c, 3d, 4c and 4d) were subjected to safety
studies against HeLa cell lines by MTT assay.8 These compounds
were found safer than N-9 (Table 2). Compounds (2d and 4c) were
less toxic than N-9 and 4c was found to be highly safe (IC50 = 961 -
contains an
a
,b-unsaturation along with 2-morpholinoethyl or
3-(dimethylamino) propyl as side chains at position 3 (2c, 2d, 3c,
3d, 4c and 4d. Compounds 1a–1d (aminoalkylated 2-acetylbutyro-
lg/ml). Thus warranting its further investigation as a potential
Table 2
vaginal contraceptive. Since interaction with free thiols could be
one of the mechanisms of spermicidal activity. Compounds (2d
and 4c) were tested for inhibition of thiols on human sperm. In a
qualitative assessment, compound 4c significantly inhibited free
thiols on human sperm, which indicated its mechanism involving
sulfhydryl inhibition (Fig. 3). Inhibition of free thiols was negligible
in case of compound 2d.
Thus a series of compounds (2, 2a–2d, 3, 3a–3d, 4, 4a–4d) were
synthesised and tested for spermicidal activity against human
sperm. Four compounds (2c, 3c, 3d, and 4d) were active at
Spermicidal activity and cytotoxicity of active compounds
S.
No.
Compounds
(tartarate)
Spermicidal
activity (%)
Cyto-toxicity HeLa
(IC50, lg/ml)
1
2
3
4
5
6
7
2c, (2cT)
2d, (2dT)
3c, (3cT)
3d, (3dT)
4c, (4cT)
4d, (4dT)
N-9
0.1
0.05
0.5
0.1
0.05
0.5
848
269
1334
1201
961
661
33.2
0.02

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R. R. Pandey et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3903–3906
Acknowledgments
Authors are thankful to staff members of SAIF Division of CDRI
for the spectral data, University Grant Commission, Council of Sci-
entific and Industrial Research, Ministry of Health and Family Wel-
fare, Government of India, New Delhi, for providing financial
assistance.
Supplementary data
Supplementary data (synthetic procedure of the reported com-
pounds and associated biological procedures) associated with this
article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmcl.2014.06.045.
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Figure 3. Human sperm exhibiting fluorescence due to the presence of free thiols
after treatment with vehicle (control, A); compound 2d (B) and compound 4c (C).
MEC = 0.5% and two compounds (2d and 4c) at MEC = 0.05%. Preli-
minary SAR analysis of the compounds indicated that substitution
of 2-morpholinoethyl and 3-(dimethylamino)propyl at position 3
increases spermicidal activity. These compounds were further
tested for safety studies against HeLa cell lines and were found
safer than N-9 (Table 2). Compounds (2d and 4c) came out to be
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and potent spermicidal agent.