Innovative Disulfide Esters of Dithiocarbamic Acid as Women-Controlled Contraceptive Microbicides: A Bioisosterism Approach

DOI: 10.1002/cmdc.201500291

Full Papers

Innovative Disulfide Esters of Dithiocarbamic Acid as

Women-Controlled Contraceptive Microbicides: A

Bioisosterism Approach

[a]

[b]

[a]

Dhanaraju Mandalapu, Nand Lal, Lokesh Kumar, Bhavana Kushwaha, Sonal Gupta,

[

a]

[a, e]

[b]

[c]

[d]

Lalit Kumar, Veenu Bala,

Santosh K. Yadav, Pratiksha Singh, Nidhi Singh,

[b]

[f]

[c]

Jagdamba P. Maikhuri, Satya N. Sankhwar, Praveen K. Shukla, Imran Siddiqi,

Gopal Gupta, and Vishnu L. Sharma*

[b]

[a, e]

In an ongoing effort to discover an effective, topical, dual-func-

tion, non-surfactant contraceptive vaginal microbicide, a novel

series of 2,2’-disulfanediylbis(3-(substituted-1-yl)propane-2,1-

diyl) disubstituted-1-carbodithioates were designed by using

a bioisosterism approach. Thirty-three compounds were syn-

thesized, and interestingly, most demonstrated multiple activi-

ties: they were found to be spermicidal at a minimal effective

concentration of 1–0.001%, trichomonacidal against drug-sus-

ceptible and resistant Trichomonas strains at minimal inhibitory

concentration (MIC) ranges of 10.81–377.64 and 10.81–

These compounds were also found to be non-cytotoxic to

human cervical (HeLa) epithelial cells and vaginal microflora

(Lactobacilli) in vitro. The most promising compound, 2,2’-disul-

fanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)dipyrrolidine-1-

carbodithioate (5), exhibited spermicidal activity 15-fold higher

than that of the marketed spermicide Nonoxynol-9 (N-9) and

also demonstrated microbicidal potency. To identify common

structural features required for spermicidal activity, a 3D-QSAR

analysis was carried out, as well as in vivo efficacy studies and

fluorescent labeling studies to determine the biological targets

of compound 5.

7

54.14 mm, respectively, and fungicidal at MIC 7.93–86.50 mm.

Introduction

The world’s population explosion is a current matter of grow-

ing concern. According to a United Nations report, the global

population has increased from 5.7 to 7.2 billion since 1994 and

[

1]

could reach up to 9.6 billion by 2050. Most of this growth

has been, and will continue to be, in the less developed coun-

[

2]

[3]

+

tries. The global epidemics of unwanted pregnancies,

[

a] D. Mandalapu, Dr. N. Lal, S. Gupta, Dr. L. Kumar, Dr. V. Bala,

Dr. V. L. Sharma

[

4]

unsafe abortions, and sexually transmitted diseases (STDs)

Medicinal and Process Chemistry Division

CSIR-Central Drug Research Institute (CSIR-CDRI)

Jankipuram Extension, Sitapur Road, Lucknow 226031 (India)

E-mail: vl_sharma@cdri.res.in

[5]

[6,7]

such as opportunistic Candida and Trichomonas infections

have left women with inadequate options for safety. It is well

known that heterosexual contacts are the major cause of STD

[

8]

vlscdri@gmail.com

transmission and unintended pregnancies among women.

[

b] L. Kumar, B. Kushwaha, Dr. S. K. Yadav, Dr. J. P. Maikhuri, Dr. G. Gupta

Endocrinology Division

Male condoms can provide protection, but are beyond the

[9]

control of the woman. Other methods of pregnancy protec-

CSIR-Central Drug Research Institute (CSIR-CDRI)

Jankipuram Extension, Sitapur Road, Lucknow 226031 (India)

tion other than condoms may not prevent STDs, and microbi-

[

10]

cides without spermicidal action have limited use. A contra-

ceptive agent that is capable of immobilizing 100% of human

spermatozoa immediately upon contact under physiological

conditions in vitro is likely to provide pregnancy protection in

vivo. Hence, women-controlled vaginal contraceptive microbi-

[

c] P. Singh, Dr. P. K. Shukla

Microbiology Division

CSIR-Central Drug Research Institute (CSIR-CDRI)

Jankipuram Extension, Sitapur Road, Lucknow 226031 (India)

[

d] N. Singh, Dr. I. Siddiqi

Molecular and Structural Biology Division

CSIR-Central Drug Research Institute (CSIR-CDRI)

Jankipuram Extension, Sitapur Road, Lucknow 226031 (India)

[

11]

cides may offer a suitable alternative to condoms for dual

protection by targeting sperm and STDs such as Trichomonas

and Candida. These microbicides could prove to be an ideal

strategy to avoid heterosexual transmission of STDs and fertile

sperm.

[

e] Dr. V. Bala, Dr. V. L. Sharma

Academy of Scientific and Innovative Research

New Delhi 110001 (India)

[

f] Dr. S. N. Sankhwar

Department of Urology

King George Medical University, Lucknow 226003 (India)

Even though the human vagina is resistant to HIV infections

due to its low pH levels, five million new cases are added

every year to the 40 million people living with HIV worldwide,

+

[

] These authors contributed equally to this work.

[

12]

half of which are women. This is also largely due to STDs

like trichomoniasis that increase susceptibility to HIV. To date,

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201500291.

ChemMedChem 2015, 10, 1739 – 1753

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

all commercially available micro-

bicidal spermicides, like Nonoxy-

nol-9 (N-9; an over-the-counter

spermicide), have surfactant

action that disrupts the integrity

of

cervicovaginal

epithelial

[

12]

tissue and causes localized in-

flammatory responses, which re-

sults in increased rates of STD/

[

13]

HIV

transmission.

Health

Recent

Organization

World

(

WHO) reports and clinical trials

have cautioned against the use

of N-9-based spermicides by

women vulnerable to STD/HIV

infection. Due to its surfactant

action, N-9 not only fails to pre-

vent STD/HIV transmission but

may even enhance transmission

[

14]

if used frequently.

We have discovered thiols as Figure 1. Graphical depiction of the development of DTCs leading to the current series.

a common target on sperm and

some STD pathogens that may

be vulnerable to designed chemical attack. Free sulfhydryl

groups play an important role in the endurance of most anae-

signed, Figure 1). Following this strategy, the newly synthe-

sized compounds (disulfide esters of dialkylaminocarbodithioic

acid) not only exhibited microbicidal activities but also showed

improved spermicidal activity. Design, synthesis, in vitro and in

vivo biological activities, fluorescence labeling of biological tar-

gets, and safety evaluation against vaginal epithelium and Lac-

tobacilli are reported in this paper.

[15]

robic cells like spermatozoa,

Trichomonas vaginalis,

and

[

16]

Candida albicans. Because of the lower physiological pH, the

normal human vagina inhibits growth of several pathogens,

and the probability of existence of free functional sulfhydryls is

[

15]

also negligible. However, it has been found that during the

deposition of alkaline semen, the vaginal pH is disturbed,

making it vulnerable to pathogenic infection. Hence, synthesis

of multitargeting, non-surfactant new chemical entities (NCEs)

to simultaneously attack sperm, Trichomonas, and Candida at

seminal pH could provide novel dual-action agents. Keeping in

mind the drawbacks of surfactant spermicides, attempts were Results and Discussion

made to develop non-surfactant, vaginal contraceptive micro-

Chemistry

bicides using various pharmacophores embedded in diverse

chemical structures, which resulted in some very promising

Designed compounds 4–36 were synthesized according to

[

15,17–26]

[18]

compounds.

However, the most promising spermicide

Scheme 1 in a three-step procedure. Oxiranes 2a–2e were syn-

thesized by reacting epichlorohydrin and secondary amines

1a–1e. These oxiranes were further reacted with an excess of

carbon disulfide and aqueous sodium hydroxide in ethanol at

0–58C to afford 1,3-dithiolane-2-thiones 3a–3e in very good

was only mildly microbicidal against Trichomonas and Candida

[

27]

strains (Figure 1).

Although agents separately targeting sperm, Trichomonas, or

Candida are available, no lead has been reported to date

which can target the three simultaneously. In this study, we at-

tempted to unite these three unique capabilities through ra-

tional drug design and bioisosterism. During the ongoing ef-

forts to develop a multitargeted vaginal contraceptive microbi-

cide, we planned to synthesize a compound having both di-

thiocarbamate (DTC) and a disulfide bond within the same

molecule. The microbicidal properties of DTCs are well docu-

[

29]

yields. Finally, 3a–3e gave 2,2’-disulfanediylbis (3-(substitut-

ed-1-yl) propane-2,1-diyl) disubstituted-1-carbodithioates 4–36

through a ring-opening reaction with various secondary

amines under catalyst-free conditions in ethyl acetate

[

30]

(Scheme 1).

Dual-action agents are presently need to prevent vaginal in-

[

31]

fections as well as pregnancy. Therefore, the newly synthe-

sized compounds (4–36, Table 1) were evaluated for spermici-

dal, anti-Trichomonas, and antifungal activity in vitro, safety

toward the cervicovaginal epithelium (HeLa), and compatibility

with vaginal microflora Lactobacillus jensenii (ATCC25258)

(Tables 1 and 2) as these are envisioned for topical use in the

female genital tract.

[

17–27]

mented,

and disulfide bonds play a crucial role in rapid

[15,22]

immobilization of spermatozoa.

Keeping in mind the con-

[28]

cept of bioisosterism, as thiocarbamate and DTC are bioisos-

teres, we therefore decided to transform a thiocarbamate

group of the disulfide ester of dialkylaminocarbothioic acid

into DTC without altering the rest of scaffold (prototype de-

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Furthermore, replacement of

1

2

the NR R group by azepane

21–27, MEC: 0.001–1%) or mor-

(

pholine (28–34, MEC: 0.005–1%)

also reduced the overall activity

and showed different sperm im-

mobilization effect with different

3

4

substitution patterns at NR R .

1

2

When NR R was 4-methylpipera-

zine (35 or 36, MEC: 0.01–1%),

the structure–activity relation-

ship (SAR) study suggested that

this molecular framework pos-

sessed mild sperm immobiliza-

tion properties. A closer glimpse

Scheme 1. Reagents and conditions: a) epichlorohydrin, triethylamine, neat, 0–58C, 2 h; b) excess carbon disulfide,

aq. NaOH, EtOH, 0–58C, 20–22 h; c) various secondary amines, EtOAc, 72–80 h, RT.

Biological assays

at the SAR for spermicidal activity indicated that pyrrolidine or

1

2

azepane as the NR R group and pyrrolidine, piperidine, 4-

Spermicidal activity

3

4

methyl piperidine, or morpholine as the NR R group produce

desirable scaffolds in these synthesized compounds.

All compounds (4–36, Table 1) were found to be spermicidal at

concentration ranges from 1–0.001% (MEC) within 30 seconds.

Four compounds (4, 5, 9, and 22) demonstrated activity at an

MEC value of 0.001% (m/v), which is fifteen-fold higher than

the activity of the clinically used spermicide N-9, while com-

pounds 7, 10, 11, 15, and 19 showed the same activity at an

MEC value of 0.002% (7.5-fold higher than for N-9). Another

five compounds (6, 18, 20, 23, and 33) immobilized 100% of

human spermatozoa at an MEC value of 0.005%, which is

threefold higher than for N-9, whereas eight compounds (8,

Cytotoxicity toward cervical cells and compatibility with

vaginal flora

All of the test compounds exhibited a high safety profile rela-

tive to N-9 toward both human cervical (HeLa) cells and com-

patibility with the vaginal flora Lactobacillus jensenii, with IC₅₀

values ranging from 130.06 to >1650.16 mm and 402.03 to

>2212.99 mm (Table 1), respectively. N-9 exhibited IC₅₀values

of 53.83 and 52.70 mm toward both HeLa cells and Lactobacil-

lus, respectively. These test compounds have no effect on the

viability of HeLa cells or the growth of Lactobacilli over a 24 h

incubation period.

16, 25–28, 31, and 35) were as active as N-9, at an MEC value

of 0.0125%. The remaining six compounds (13, 17, 21, 29, 30,

and 36) exhibited spermicidal action at MEC values of 0.10–1%

(

Table 1).

The spermicidal action of compounds 4–36 revealed that

the amine substituents in 2,2’-disulfanediylbis (3-(substituted-

-yl) propane-2,1-diyl) disubstituted-1-carbodithioates were

Anti-Trichomonas activity

1

2

detrimental for biological activity. A combination of NR R as

The anti-Trichomonas data for compounds 4–36 (Table 2) illus-

trated good to moderate activity against a metronidazole

(MTZ)-susceptible strain, and most of the compounds showed

remarkably better activity than MTZ against an MTZ-resistant

strain of Trichomonas vaginalis. The compounds were found to

be trichomonacidal against an MTZ-susceptible strain, with

MIC values ranging from 10.81–377.64 mm and at MIC values of

10.81–754.14 mm against an MTZ-resistant strain.

Compound 5 demonstrated trichomonacidal activity at an

MIC value of 10.81 mm against both MTZ-susceptible and -re-

sistant strains of Trichmonas, while MTZ demonstrated this

effect at 11.70 mm and 292.0 mm, respectively. The MIC value of

compound 5 was similar to that of MTZ in an MTZ-susceptible

strain and 27-fold better than MTZ in an MTZ-resistant strain. It

is apparent from the trichomonicidal activity of the synthesized

3

4

pyrrolidine (4–12) and NR R as pyrrolidine (5), piperidine (4),

or 4-methylpiperidine (9) gave the most active compounds

(

MEC: 0.001%), but the activity was diminished by either

changing the position of the methyl substituent in piperidine

10, MEC: 0.002%) or introducing another heteroatom into the

(

piperidine moiety (7 and 11, MEC: 0.002%; 8, MEC: 0.0125%).

Further, ring expansion and addition of a benzyl substituent to

the piperidine ring also resulted in decreased activity (6, MEC:

1

2

0

.005%; 12, MEC: 0.05%). On the other hand, when NR R was

piperidine (13–20), the overall activity decreased relative to

the pyrrolidine analogues.

The sperm immobilization efficacy of these compounds was

3

4

gradually enhanced by replacement of the NR R group of 13

MEC: 0.1%) with morpholine (14, MEC: 0.05%), azepane (16,

MEC: 0.01%), 4-methylpiperidine or 4-butylpiperazine (18 or

(

1

2

compounds against an MTZ-susceptible strain, when the NR R

amine residue was pyrrolidine (4–12), that the ring size and

2

1

0, MEC: 0.005%), or pyrrolidine or 3-methylpiperidine (15 or

9, MEC: 0.002%) and was retained with 4-methylpiperazine

3

4

presence of a heteroatom on the NR R amine affected the

anti-Trichomonas activity to a large extent. Substitution of the

piperidine of 4 (MIC: 41.25 mm) with pyrrolidine (5, MIC:

10.81 mm), azepane (6, MIC: 19.71 mm), morpholine (8, MIC:

25.47 mm), or 4-methylpiperidine (9, MIC: 24.50 mm) enhanced

(

17, MEC: 0.1%). These results exhibited that the ring size, the

3

4

NR R substitution pattern on this ring, and introduction of

a heteroatom in the ring play an important role in the spermi-

cidal action of these compounds.

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[a]

Table 1. Spermicidal potential (MEC), cytotoxicity (IC50), and selectivity indexes (SI) of synthesized compounds 4–36.

1

2

3 4

Compd

NR R

MEC [%]

IC50 [mm]

SI (IC50/MEC)

[

b]

[c]

(

mm)

HeLa

L. jensenii

HeLa

L. jensenii

0

.001

4

5

6

7

8

9

387.80

1138.63

23.54

69.13

(

16.47)

.001

17.27)

.005

78.74)

.002

31.39)

.012

196.39)

.001

15.74)

0

237.00

555.20

548.40

>1577.30

1163.50

13.72

7.05

31.75

>20

0

1572.32

1204.91

1247.63

50.08

6.13

37.06

7.02

0

1380.30

(

0

1140.30

79.26

72.44

(

0.002

1

0

1577.28

1058.33

544.16

50.08

38.16

17.28

31.49)

0.002

11

>1388.88

>50

27.73)

0

.050

1

2

3

4

5

6

7

8

947.83

>1577.28

505.64

402.03

>1577.28

>1567

1.49

>1

0.63

>1

(

635.32)

0.100

(

1574.8)

0.050

0.64

>2

(

782.47)

0.002

>1650.16

1510.57

1048.19

1277.94

1226.07

1182.77

>50

8.34

37.22

6.53

(

32.94)

0.012

180.99)

0.100

832.83

0.69

0.55

(

1503.7)

0.005

>1510.57

16.94

20.03

(

75.41)

0.002

1

2

9

0

1510.57

1203.20

>2212.99

50.08

18.02

>73.37

30.16)

0.005

1336.89

20.02

66.75)

1

.00

2

1

2

3

4

5

130.06

528.52

507.55

587.98

0

0.03

39.22

>19.06

>2

(

15082)

0.001

35.25

19.06

1.82

5.44

(

14.99)

.005

78.74)

.050

723.58)

.012

0

1501.00

1321.00

943.64

>1501

(

0

>1449.2

861.27

(

0

4.97

173.16)

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Table 1. (Continued)

1

2

3 4

Compd

NR R

MEC [%]

IC50 [mm]

SI (IC50/MEC)

[

b]

[c]

(

mm)

HeLa

L. jensenii

HeLa

L. jensenii

0

.012

2

6

7

1449.27

939.13

8.34

5.40

(

173.66)

0.012

2

1449.27

1131.88

8.34

6.51

(

173.66)

0

.012

2

3

8

9

0

1

548.58

616.39

333.83

1221.00

1079.93

593.44

699.10

1924.3

2.92

0.37

0.02

6.54

5.75

0.36

(

187.79)

0.100

(

1636.6)

1.00

0.04

(

14947)

0.012

10.31

(

186.62)

0.050

3

2

3

1501.00

>1501.5

2

>2

749.62)

0.005

20.02

>20

(

74.96)

0

.050

3

4

5

6

1501.00

>1506.02

>1572.32

>1501.5

1463.85

1047.31

2

>2

(

749.62)

0.012

>8.34

>1

8.11

0.66

180.45)

0.100

(

1569.8)

0

.015

[d]

N-9

53.830

52.70

0.22

0.21

(

243.50)

[

9

a] All experiments were carried out in triplicate. [b] Minimum effective concentration; vehicle (control) had >85% motility. [c] ATCC25258. [d] Nonoxynol-

(positive control).

1

2

the activity. On the other hand, replacement of the piperidine

of 4 with 4-methylpiperazine (7, MIC: 196.23 mm), 3-methylpi-

peridine (10, MIC: 98.42 mm), 4-butylpiperazine (11, MIC:

NR R amine was a pyrrolidine residue (4–12), the trichomona-

cidal effect decreased with increasing ring size from a five-

membered ring (5, MIC: 10.81 mm) to six- or seven-membered

3

4

8

6.80 mm), or 4-benzylpiperidine (12, MIC: 79.51 mm) decreased

rings (4 or 6, MIC: 411.86 or 41.25 mm, respectively) at NR R .

Upon ring substitution of piperidine 4 with a 3-methyl (10,

MIC: 98.42 mm), 4-benzyl (12, MIC: 159.03 mm), or 4-methyl (9,

MIC: 196.85 mm) group, activity increased relative to the un-

substituted piperidine 4. Further, these results showed that

any replacement of the NR R amine from pyrrolidine (4–12) to

piperidine (13–20, MIC: 167.11–411.86 mm), azepane (21–27,

MIC: 180.89–754.14 mm), morpholine (28–34, MIC: 46.78–

391.23 mm), or 4-methylpiperazine (35 or 36, MIC: 94.12–

392.46 mm) resulted in decreased trichomonacidal activity. The

comparison of anti-Trichomonas activity of these compounds

the anti-Trichomonas activity. The overall trichomonacidal

action decreased when the pyrrolidine moiety of 4–12 was re-

placed with piperidine (13–20, MIC: 78.86–377.64 mm), azepane

(

21–27; MIC: 90.58–197.16 mm), morpholine (28-34, MIC:

1

2

4

1

6.78–195.92 mm) or 4-methylpiperazine (35 or 36, MIC: 94.12–

96.54 mm), similar to the observations for spermicidal activity

(

Table 1).

Seventeen compounds (5–13, 20–21, 26, 29, 30, and 33–35)

were found to be 1.2- to 27-fold more active than MTZ against

an MTZ-resistant strain of Trichomonas vaginalis. When the

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[a]

Table 2. Anti-Trichomonas and antifungal activities of synthesized compounds 4–36.

[

b]

[b]

Compd

Trichomonacidal MICÆSEM [mm]

Antifungal MIC [mm]

S. schenckii

[

c]

[d]

MTZ-susceptible

MTZ-resistant

C. albicans

C. parapsilosis

T. mentagrophytes

C. neoformans

4

5

6

7

8

9

41.25Æ4.65

10.81Æ0.79

19.71Æ1.45

196.23Æ0.01

25.47Æ7.65

24.50Æ3.63

98.42Æ14.67

86.80Æ12.95

79.51Æ0.02

78.86Æ5.83

195.92Æ0.08

206.27Æ46.14

377.64Æ0.16

376.50Æ0.16

377.64Æ0.16

94.41Æ14.09

83.55Æ12.47

188.82Æ0.08

93.84Æ14.00

197.16Æ0.09

181.16Æ0.07

180.63Æ26.84

90.58Æ0.03

195.92Æ0.08

102.45Æ0.04

46.78Æ0.02

194.70Æ0.08

93.70Æ13.96

187.40Æ27.93

94.12Æ14.05

196.54Æ0.09

11.70Æ3.30

–

411.86Æ68.64

10.81Æ4.47

41.25

10.21

39.43

78.49

40.91

78.74

34.67

7.93

41.25

10.21

78.86

78.49

10.22

78.74

17.33

7.93

>82.50

86.50

78.86

78.49

40.91

78.74

34.67

31.72

>78.86

78.24

82.37

75.41

37.59

>75.41

>66.75

>75.52

18.76

>78.98

72.35

36.07

72.35

>72.35

>78.36

20.49

37.42

>77.76

74.96

>74.96

18.74

75.18

39.49

–

>82.50

43.25

78.86

78.49

20.45

39.37

17.33

31.72

78.86

78.24

82.37

75.41

18.79

>75.41

>66.75

>75.52

37.53

78.98

72.35

18.03

72.35

78.36

40.98

74.85

77.76

74.96

18.74

75.18

39.49

–

>82.50

43.25

78.86

39.24

10.22

39.37

17.33

7.93

>78.86

78.24

10.29

37.70

18.79

75.41

>75.41

66.75

75.52

37.53

78.98

72.35

18.03

36.17

72.35

>78.36

20.49

18.71

>77.76

74.96

9.37

41.25Æ2.37

196.23Æ58.50

102.29Æ30.49

196.85Æ58.68

98.42Æ29.34

86.8Æ25.82

1

0

1

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

159.03Æ47.29

236.22Æ46.95

391.84Æ58.41

411.86Æ92.09

377.64Æ56.29

376.5Æ56.12

377.64Æ56.29

377.64Æ56.37

167.11Æ49.72

188.82Æ56.16

754.14Æ1.34

394.32Æ0.18

724.63Æ108.17

721.5Æ107.55

180.89Æ53.93

362.31Æ54.01

391.23Æ58.32

102.45Æ30.47

46.78Æ0.02

78.86

78.24

41.18

75.41

37.59

75.41

66.75

75.52

37.53

78.98

72.35

36.07

72.35

>78.36

20.49

18.71

>77.76

74.96

>74.96

18.74

>75.18

>78.49

–

39.43

78.24

82.37

37.70

37.59

75.41

>66.75

75.52

75.07

78.98

72.35

36.07

72.35

78.36

40.98

37.42

77.76

74.96

18.74

>75.18

78.49

–

388.8Æ57.95

374.81Æ0.01

187.4Æ0.01

187.4Æ55.87

94.12Æ28.57

392.46Æ58.50

>75.18

>78.49

–

[

e]

MTZ

FLZ

292Æ32.57

[

f]

–

1.63

3.26

6.53

3.26

[

a] All experiments were carried out in triplicate. [b] Minimum inhibitory concentration. [c] ATCC50143. [d] Candida parapsilosis (ATCC22019). [e] Metronida-

zole. [f] Fluconazole.

against clinical and resistant strains revealed that compounds

Antifungal activity

4

–36 were equipotent or lost activity by only two- to tenfold

relative to the MTZ-sensitive strain, whereas MTZ had fivefold

lower activity against the MTZ-resistant strain.

The antifungal activity for the synthesized compounds (4–36,

Table 2) revealed that all compounds were found to be fungici-

dal against at least two of the five different fungal strains

tested. Pyrrolidine-derived compounds (4–12) displayed activi-

ties within MIC range of 7.93–86.50 mm. The compounds with

The nitro group of MTZ, the only available drug for Tricho-

monas treatment, is activated through anaerobic reduction by

the hydrogenosomes of Trichomonas vaginalis, resulting in the

[32]

1 2

generation of nitro radical ion intermediates. This activation

piperidine as the NR R group (13–20) exhibited antifungal ac-

tivity with MIC values of 10.29-82.37 mm, while replacement of

[32]

step is nonoperational in resistant pathogens. As the nitro

group was absent in these test compounds, this indicated that

a mechanism different from that of MTZ was operational in

these compounds to make them nearly equally effective

against both MTZ-susceptible and -resistant Trichomonas

strains responsible for vaginal trichomoniasis. The most active

compound, 5, exhibited nearly similar activity to MTZ in all

clinical Trichomonas strains and was 27-fold more potent than

MTZ against resistant Trichomonas strains.

1

2

the NR R group with azepane (21–27) and morpholine (28–

34) showed fungicidal effects at 18.03–78.98 mm and 9.37-

78.36 mm, respectively. The two remaining compounds in

1

2

which NR R was 4-methylpiperazine (35, 36) demonstrated an-

tifungal activity at MIC: 39.49–78.49 mm. By comparison, the

standard drug, fluconazole, inhibited the growth of all five

tested strains at MIC: 1.63–6.53 mm. The antifungal results ex-

hibited that all compounds except 35 were active against the

two Candida strains tested, with MIC values of 7.93–82.37 mm.

Thirty-four compounds were found to be active against one or

ChemMedChem 2015, 10, 1739 – 1753

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

more of three opportunistic fungal strains—Cryptococcus neo-

formans, Sporothrix schenckii, and Trichophyton mentagro-

phytes—at MIC: 7.93–86.50 mm.

27.73 mm), 9 (0.001%, 15.74 mm), 10 (0.002%, 31.49 mm), and 4

3

4

(0.001%, 16.47 mm) having bulkier substitutions at the NR R

position embedded in the green contour region. The small

1

2

Many vulvovaginitis cases have been attributed to Candida

infection, and more importantly, this infection it is present

asymptomatically in healthy women. It adheres to the epithe-

lial tissue and causes itching, which may facilitate the transmis-

sion of STDs and HIV. This SAR study revealed that a pyrrolidine

green contour near the NR R position suggests the orientation

of a favored bulky substituent that may contribute an increase

of activity, as exemplified by 35 (0.012%, 180.45 mm), with

a bulkier group inclined toward this green contour, and 36

(0.10%, 1569.85 mm), with a bulkier group oriented in the op-

posite direction.

1

2

3 4

residue at NR R and a 4-benzyl piperidine at NR R (12) was

most desirable in the 2,2’-disulfanediylbis (3-(substituted-1-yl)

propane-2,1-diyl) disubstituted-1-carbodithioate scaffold for

antifungal activity against multiple strains.

The other small blue and red contours indicate the preferred

orientation of electronegative and electropositive groups. For

example, an electronegative group is oriented toward the red

contour for 33 (0.005%, 74.96 mm), 35 (0.012%, 180.45 mm), 31

(0.012%, 186.62 mm), 28 (0.012%, 187.79 mm), 34 (0.05%,

749.62 mm), and 32 (0.05%, 749.62 mm) while it is oppositely

oriented for 29 (0.10%, 1636.6 mm; see structures in Support-

ing Information).

The azole class (fluconazole) is commonly used to treat can-

didiasis, with these drugs inhibiting the biosynthesis of ergo-

[

33]

sterol. As there is no azole moiety in this molecular frame-

work, the activity may be due to the presence of a dithiocarba-

mate group and/or a disulfide linkage as the key pharmaco-

phore.

The 3D-QSAR study performed on the dataset using CoMFA

led to the identification of regions of importance for steric and

electrostatic interactions. The derived model explained the ob-

served variance in the activity and provided guidelines for the

structural requirements for compounds possessing enhanced

spermicidal activity.

3

D-QSAR analysis

Herein, comparative molecular field analysis (CoMFA) was em-

ployed to study the SAR of novel compounds (4–36) with

regard to spermicidal activities. The results showed that 3D-

QSAR models could be used to explain structural requirements

for activity. The alignment of active spermicidal compound 22

[34]

In vivo spermicidal activity of compound 5

(

0.001%, 14.99 mm) was embedded in the CoMFA contour

On the basis of the SAR study, it may be concluded that the

most promising compound for multiple activities (spermicidal,

anti-Trichomonas, anti-Candida) was 5. It appeared to be non-

toxic against cervicovaginal cells (HeLa) and the microflora Lac-

tobacillus jensenii and, therefore, is evidently much safer for

vaginal use. There was apparently a dose-dependent decrease

in pregnancy rate, as evidenced by rabbits receiving intravagi-

nal instillation of compound 5; however, the effect was more

consistent with the average litter size. About 50–80% of ani-

mals who received vaginal doses of compound 5 (10–30 mg)

did not conceive at all, while this efficacy was only 33.3% in

the case of N-9 (20 mg). The average litter size of animals re-

ceiving compound 5 before mating was decreased by 71, 46,

and 94% in 10, 20, and 30 mg groups, respectively. On the

other hand, the marketed spermicide N-9 at a 20 mg dose

caused a 41% reduction in the average litter size of rabbits

maps to demonstrate its affinity for the steric and electrostatic

regions of inhibitors (Figure 2). From contour analysis, it is

clear that the contours have greater distribution surrounding

3

4

the substitutions at the NR R positions of the central core

1

2

than the NR R position (Figure 2). Contour analysis near the

3

4

NR R regions showed the presence of a medium-sized electro-

static red contour, indicating that the introduction of more

electronegative groups in this region may improve the activity.

For example, compounds 22 (0.001%, 14.99 mm), 31 (0.012%,

1

86.62 mm), and 8 (0.012%, 196.39 mm), with electronegative

substitutions near the red contour, have higher activity than

1 (1.00%, 15082 mm) and 30 (1.00%, 14947 mm), which

2

showed a drastic decrease in activity. The large green contour

suggests favorability for the substitution having steric bulki-

ness. This is exemplified by 7 (0.002%, 31.39 mm), 11 (0.002%,

(

Table 3). It was found that, at a dosage of 30 mg, compound 5

demonstrated a maximum spermicidal effect in vivo.

Table 3. In vivo vaginal contraceptive efficacy of compound 5 in Belgian

rabbits.

Treatment Dose [mg] No. Preg- Efficacy [%] Avg. No.

Decrease in

Litter

Size [%]

nant/

Pups per

Animal

Mated

[a]

Vehicle

5

0

10

20

30

20

5/5

2/4

1/5

4/6

0

7.00

2.00

3.75

0.40

4.16

0

71

46

94

41

Figure 2. CoMFA steric and electrostatic contours displayed with potent

compound 22. The green areas in the figure indicate regions where steric

bulk is well accommodated with an increase in activity, whereas yellow

areas indicate regions where steric bulk is unfavorable. The red contours in-

dicate regions where substitution with more electronegative substituents in-

crease the activity, whereas the blue contour show the opposite.

50.0

80.0

33.3

N-9

[a] K-Y Jelly (Johnson & Johnson) was used as a vehicle.

ChemMedChem 2015, 10, 1739 – 1753

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

Inhibition of free sulfhydryl groups on sperm and

Trichomonas by compound 5

model, and a 3D-QSAR study explained the necessary key

structural features for spermicidal activity. This study also sug-

gested that DTC, along with the disulfide linkage, may be

a key factor in these compounds for spermicidal, as well as mi-

crobicidal activity. Fluorescence labeling studies further con-

firmed the inhibition of sulfhydryls on sperm and Trichomonas

by these compounds. The embodiment of microbicidal activity

into the 2,2’-disulfanediylbis (3-(substituted-1-yl) propane-2,1-

diyl) disubstituted-1-carbodithioates may have considerable

implications for their impending use as prophylactic, topical,

women-controlled, vaginal contraceptive microbicides.

The aptness of new compounds to target sulfhydryl groups on

sperm and Trichomonas at seminal pH may considerably im-

prove their multiple activities and safety. Due to the lack of

glutathione (the intracellular redox buffer), Trichomonas vagi-

nalis largely depends on cysteine to overcome redox stress.

This makes it very susceptible to sulfhydryl-manipulating

[

35]

agents. The compounds were designed to target free sulf-

hydryl groups, and the inhibition of sperm and Trichomonas

sulfhydryl groups was qualitatively localized by fluorescence

detection after labeling the free sulfhydryl groups with a fluori-

metric thiol detector. Motile human sperm/Trichomonas vagi-

nalis (control) and compound 5-treated human sperm/Tricho-

monas were digitally imaged for qualitative assessment. It is

clear from Figure 3 that there was significant inhibition of free

thiol fluorescence in compound 5-treated sperm/Trichomonas.

Fluorescence intensities were higher in controls, due to the

greater number of free sulfhydryl groups than in compound 5-

treated samples. The diminished fluorescence in the latter sug-

gested the interaction of compound 5 with free sulfhydryl

groups present in sperm and Trichomonas, which could be the

mechanism of spermicidal and trichomonicidal action.

Experimental Section

General procedures

In general, all reagents and solvents were of commercial quality

and were used without further purification. Chromatography was

carried on silica gel (60–120 and 100–200 mesh). All reactions were

monitored by thin-layer chromatography (TLC) using F₂₅₄silica gel

plates with fluorescence (Aldrich). Melting points were determined

in open capillary tubes on an electrically heated block and are un-

À1

corrected. IR spectra (n_m_a_xin cm ) of the compounds were record-

1

ed on a PerkinElmer FT-IR RX1 PC spectrophotometer. H NMR

spectra were recorded on Bruker Supercon Magnet Avance DPX-

2

1

00/DRX-300 spectrometers (operating at 400 and

00 MHz, respectively, for H and C) in deuterated sol-

1

13

vents with TMS as an internal reference (chemical shifts

d in ppm, J in Hz). Electrospray ionization mass spectra

(

ESI-MS) were recorded on a Thermo Lcq Advantage

Max-IT. High-resolution mass spectra (HRMS) were re-

corded on a 6520 Agilent Q Tof LC–MS/MS (accurate

mass). Elemental analyses were performed on a Carlo

Erba EA-1108 microanalyzer/Vario EL-III C, H, N analyzer.

All compounds were analyzed for C, H, N, and the results

obtained were within Æ0.4% of calculated values. All

final compounds were found to have >95% purity.

Figure 3. Fluorescent labeling of A) human sperm and B) Trichomonas sulfhydryls: left

panels: labeling with fluorimetric thiol detector specifically binds free sulfhydryls; right

panels: phase contrast images.

Synthesis

1-(Oxiran-2-ylmethyl)pyrrolidine compounds 2a–2e were

synthesized according to a reported procedure.

[

21]

4

-(Pyrrolidin-1-ylmethyl)-1,3-dithiolane-2-thione (3a):

Conclusions

Sodium hydroxide (0.274 g, 11.81 mmol) in water (1 mL)

was added to an ice-cooled solution of 1-(oxiran-2-ylmethyl)pyrroli-

dine (2a) (3 g, 23.62 mmol) in EtOH (20 mL). Carbon disulfide

In summary, dual-action spermicides are presently needed, as

these can be used to control the population as well as the

spread of STDs. Attempts have been made to incorporate mul-

tiple activities within the same molecular framework by inhibit-

ing common targets in sperm and Trichomonas. This quest for

a novel non-surfactant spermicidal microbicide yielded a novel

series of compounds that were designed and synthesized on

the basis of previous analogy and bioisosterism. Most com-

pounds were found to be dually active, with high safety pro-

files against cervicovaginal epithelium (HeLa) cells and micro-

flora. The results indicated that compound 5 was the most

promising candidate for multiple activities, illustrating excep-

tional spermicidal (MEC: 0.001%), anti-Trichomonas (MIC:

(

2.47 mL, 70.86 mmol) in EtOH (5 mL) was added dropwise to the

above reaction mixture while stirring at 0–58C, and the resulting

mixture was stirred at 0–58C for 20–22 h. The EtOH was removed

under vacuum, and the residue was partitioned between EtOAc

(40 mL) and excess water (330 mL). The organic layer was sepa-

rated and dried over anhydrous sodium sulfate. The sodium sulfate

was removed by filtration, and the solvent was concentrated in

vacuo. The residue was purified by column chromatography over

silica gel (60–120 mesh) using chloroform/hexane (2:8) as an

eluent to yield title compound 3a as a pale-yellow oil (yield: 91%,

1

4

.70 g): H NMR (400 MHz, CDCl ): d=4.55–4.48 (m, 1H), 3.94 (dd,

3

J=3.6 and 5.8 Hz, 2H), 2.97 (dd, J=7.8 and 12.3 Hz, 1H), 2.81 (dd,

J=7.6 and 12.3 Hz, 1H), 2.59–2.54 (m, 4H), 1.78–1.75 ppm (m, 4H);

1

3

1

0.81 mm), and anti-Candida (MIC: 10.21 mm) activities. It also

C NMR (100 MHz, CDCl₃): d=228.5, 59.7, 58.0, 54.1, 45.8,

À1

+

exhibited remarkable in vivo spermicidal activity in a rabbit

23.7 ppm; IR (neat): n˜ =1042 cm ; ESI-MS (m/z): 220 [M+H] ;

ChemMedChem 2015, 10, 1739 – 1753

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1746

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

+

HRMS-ESI m/z [M+H] calcd for C H NS : 220.0288, found:

sodium sulfate was removed by filtration, and the filtrate was con-

centrated in vacuo. The residue was purified by column chroma-

tography with CH OH/CHCl (1:9) as eluent to yield title compound

8

13

3

2

20.0281.

3

4

-(Piperidin-1-ylmethyl)-1,3-dithiolane-2-thione (3b): This com-

1

4

as a light-yellow oil (yield: 78%, 0.34 g): H NMR (400 MHz,

pound was synthesized by the method described for compound

a, employing 1-(oxiran-2-ylmethyl)piperidine (2b; 5 g,

5.46 mmol), sodium hydroxide (0.709 g, 17.73 mmol), and carbon

disulfide (6.41 mL, 106.38 mmol) to yield title compound 3b as

a light-yellow oil (yield: 87%, 7.18 g): H NMR (400 MHz, CDCl ): d=

.59–4.52 (m, 1H), 3.93 (d, J=6.2, 2H), 2.79 (dd, J=8.0 and

2.8 Hz, 1H), 2.63 (dd, J=7.4 and 12.8 Hz, 1H), 2.44–2.43 (m, 4H),

.58–1.53 (m, 4H), 1.45–1.25 ppm (m, 2H); C NMR (100 MHz,

CDCl ): d=4.25–3.80 (m, 9H), 3.70–3.58 (m, 2H), 3.33–3.25 (m, 3H),

3

2

.88–2.71 (m, 4H), 2.60–2.55 (m, 8H), 1.75–1.68 ppm (m, 20H);

13

C NMR (100 MHz, CDCl ): d=195.1 (C=S), 59.6, 54.3, 52.9, 51.4,

3

5

0.1, 40.4, 25.9, 24.3, 23.6 ppm; IR (neat): n˜ =2979, 1478, 1204,

1

3

À1

+

1130 cm ; ESI-MS (m/z): 607 [M+H] ; HRMS-ESI m/z [M+H]

4

1

calcd for C H N S : 607.2125, found: 607.2122; Anal. calcd for

26

46

4 6

C H N S : C 51.44, H 7.64, N 9.23, found: C 51.57, H 7.80, N 9.04.

1

3

26 46

4 6

CDCl ): d=228.7 (C=S), 60.6, 58.5, 54.8, 45.7, 26.0, 24.2 ppm; IR

3

(Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl) di-

pyrrolidine-1-carbodithioate (5): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3a (0.37 g, 1.68 mmol) and pyrroli-

À1

+

(

[

neat): n˜ =1045 cm ; ESI-MS (m/z): 234 [M+H] ; HRMS-ESI m/z

+

M+H] calcd for C H NS : 234.0445, found: 234.0429.

9

15

3

4

-(Azepan-1-ylmethyl)-1,3-dithiolane-2-thione (3c): This com-

dine (0.15 mL, 1.84 mmol) to afford compound 5 as a colorless oil

pound was synthesized by the method described for compound

a, employing 1-(oxiran-2-ylmethyl)azepane (2c) (4 g, 25.80 mmol),

sodium hydroxide (0.516 g, 12.90 mmol), and carbon disulfide

1

(

yield: 65%, 0.631 g): H NMR (400 MHz, CDCl ): d=3.93–3.89 (m,

3

5

2

H), 3.70–3.58 (m, 5H), 3.33–3.30 (m, 2H), 2.88–2.75 (m, 4H), 2.60–

.56 (m, 8H), 2.10–2.00 (m, 6H), 1.98–1.95 (m, 4H), 1.76–1.75 ppm

(

4.66 mL, 77.40 mmol) to yield title compound 3c as a yellow oil

13

1

(m, 8H); C NMR (100 MHz, CDCl

): d=192.3 (C=S), 59.5, 58.0, 54.1,

3

yield: 86%, 5.48 g): H NMR (400 MHz, CDCl ): d=4.53–4.48 (m,

3

4

5.7, 39.7, 24.3, 23.6 ppm; IR (neat): n˜ =2927, 1473, 1227,

1

5

H), 3.93 (dd, J=3.0 and 6.5 Hz, 2H), 2.97 (dd, J=7.8 and 13.0 Hz,

H), 2.83 (dd, J=7.6 and 13.0 Hz, 1H), 2.70 (t, J=5.2 Hz, 4H), 1.64–

À1

+

1

115 cm ; ESI-MS (m/z): 579 [M+H] ; HRMS-ESI m/z [M+H]

calcd for C24 : 579.1812, found: 579.1811; Anal. calcd for

C H N S : C 49.78, H 7.31, N 9.68, found: C 49.62, H 7.48, N 9.59.

24 42 4 6

1

3

H N S

42 4 6

.56 ppm (m, 8H); C NMR (100 MHz, CDCl ): d=228.8 (C=S), 59.9,

3

À1

9.7, 55.5, 45.3, 28.5, 27.0 ppm; IR (neat): n˜ =1046 cm ; ESI-MS (m/

+

z): 248 [M+H] ; HRMS-ESI m/z [M+H] calcd for C H NS :

10

19

3

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl) dia-

2

48.0601, found: 248.0599.

zepane-1-carbodithioate (6): This compound was synthesized by

following the procedure described for the preparation of com-

pound 4, employing compound 3a (0.18 g, 0.82 mmol) and aze-

4

-(Morpholinomethyl)-1,3-dithiolane-2-thione (3d): This com-

pound was synthesized by the method described for compound

a, employing 4-(oxiran-2-ylmethyl)morpholine (2d) (2.96 g,

0.69 mmol), sodium hydroxide (0.413 g, 10.34 mmol), and carbon

pane (0.11 mL, 0.90 mmol) to afford compound 6 as a light-brown

3

2

1

oil (yield: 68%, 0.35 g): H NMR (400 MHz, CDCl ): d=4.20–4.17 (m,

3

4

H), 3.93–3.88 (m, 6H), 3.69–3.59 (m, 2H), 3.37–3.31 (m, 2H), 2.90–

disulfide (3.74 mL, 62.07 mmol) to yield title compound 3d as

1

2.74 (m, 4H), 2.64–2.59 (m, 8H), 1.88–1.77 (m, 16H), 1.60–1.58 ppm

yellow solid (yield: 79%, 4.86 g): mp: 61–638C; H NMR (400 MHz,

13

(

m, 8H); C NMR (100 MHz, CDCl ): d=195.9 (C=S), 59.6, 55.6, 54.3,

3

CDCl ): d=4.57–4.53 (m, 1H), 3.95 (dd, J=1.3 and 5.8 Hz, 2H), 3.70

3

5

2.8, 40.3, 27.4, 26.6, 23.6 ppm; IR (neat): n˜ =2940, 1465, 1223,

(

t, J=4.6, 4H), 2.86 (dd, J=7.9 and 12.7 Hz, 1H), 2.71 (dd, J=7.6

À1

+

1

3

1145 cm ; ESI-MS (m/z): 635 [M+H ]; HRMS-ESI m/z [M+H]

calcd for C H N S : 635.2438, found: 635.2429; Anal. calcd for

and 12.7 Hz, 1H), 2.53 ppm (t, J=4.5 Hz, 4H); C NMR (100 MHz,

28

50

4 6

CDCl ): d=227.9 (C=S), 66.8, 60.2, 57.6, 53.7, 45.5 ppm; IR (KBr): n˜ =

3

À1

+

C H N S : C 52.95, H 7.94, N 8.82, found: C 52.86, H 7.84, N 8.70.

28 50 4 6

1

048 cm ; ESI-MS (m/z): 236 [M+H] ; HRMS-ESI m/z [M+H]

calcd for C H NOS : 236.0238, found: 236.0232.

8

13

3

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)

bis(4-methylpiperazine-1-carbodithioate) (7): This compound was

synthesized following the procedure described for the preparation

of compound 4, employing compound 3a (0.2 g, 0.91 mmol) and

4

-((4-Methylpiperazin-1-yl)methyl)-1,3-dithiolane-2-thione (3e):

This compound was synthesized by the method described for

compound 3a, employing 1-methyl-4-(oxiran-2-ylmethyl)piperazine

4

-methylpiperazine (0.13 mL, 1.18 mmol) to afford compound 7 as

(

2e) (4.80 g, 30.76 mmol), sodium hydroxide (0.615 g, 15.38 mmol),

1

a light-brown oil (yield: 72%, 0.41 g): H NMR (400 MHz, CDCl ): d=

4

6

and carbon disulfide (5.56 mL, 92.28 mmol). The crude product was

purified using methanol/chloroform (2:8) as eluent to yield title

compound 3e as a light-yellow solid (yield: 82%, 6.25 g): mp: 56–

5

(

1

(

4

3

.33 (bs, 4H), 3.98–3.86 (m, 6H), 3.70–3.60 (m, 2H), 3.35–3.20 (m,

H), 2.84–2.72 (m, 3H), 2.62–2.50 (m, 15H), 2.32 (s, 6H), 1.77 ppm

13

1

(

bs, 6H); C NMR (100 MHz, CDCl ): d=196.5 (C=S), 59.5, 54.3,

3

88C; H NMR (400 MHz, CDCl ): d=4.57–4.50 (m, 1H), 3.93–3.91

3

5

1

1.5, 50.0, 49.9, 45.5, 40.4, 23.6 ppm; IR (neat): n˜ =2935, 1461,

m, 2H), 2.85 (dd, J=7.9 and 12.7 Hz, 1H), 2.69 (dd, J=7.6 and

À1

+

13

290, 1146 cm ; ESI-MS (m/z): 637 [M+H] ; HRMS-ESI m/z [M+

2.7 Hz, 1H), 2.55–2.42 (m, 8H), 2.27 ppm (s, 3H); C NMR

+

H] calcd for C H N S : 637.2343, found: 637.2360; Anal. calcd for

C H N S : C 49.02, H 7.59, N 13.19, found: C 49.17, H 7.64, N

1

100 MHz, CDCl ): d=228.1 (C=S), 59.8, 57.9, 54.9, 53.2, 45.9,

26 48

6 6

3

À1

+

5.5 ppm; IR (KBr): n˜ =1041 cm ; ESI-MS (m/z): 249 [M+H] ;

26 48

6 6

+

3.05.

HRMS-ESI m/z [M+H] calcd for C H N S : 249.0554, found:

9

16

2 3

2

49.0546.

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl) di-

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl) dipi-

morpholine-4-carbodithioate (8): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3a (0.2 g, 0.91 mmol) and mor-

pholine (0.10 mL, 1.18 mmol) to afford compound 8 as a yellow oil

(yield: 77%, 0.42 g): H NMR (400 MHz, CDCl ): d=4.52–3.87 (m,

10H), 3.75–3.55 (m, 10H), 3.34–3.31 (m, 2H), 2.89–2.75 (m, 4H),

2.62–2.57 (m, 8H), 1.76 ppm (bs, 8H); C NMR (100 MHz, CDCl₃):

d=197.1 (C=S), 66.2, 59.6, 54.3, 50.8, 49.9, 40.2, 23.6 ppm; IR

(neat): n˜ =2953, 1430, 1230, 1128, 1019 cm ; ESI-MS (m/z): 611

peridine-1-carbodithioate (4): Synthesis of compound 4 was car-

[30]

ried out through a reported procedure with slight modifications.

Piperidine (0.086 mL, 0.87 mmol) was added to a solution of pyrro-

lidine trithiocarbonate (3a) (0.16 g, 0.73 mmol) in EtOAc (20 mL),

and contents were stirred at room temperature until completion of

the reaction (72–80 h, monitored by TLC). After completion of the

reaction, EtOAc (15 mL) was added, and the organic layer washed

with excess water (20 mL3) and dried over sodium sulfate. The

1

3

13

À1

ChemMedChem 2015, 10, 1739 – 1753

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1747

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

+

[

M+H] ; HRMS-ESI m/z [M+H] calcd for C H N O S : 611.1710,

(Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl) dipi-

peridine-1-carbodithioate (13): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3b (0.2 g, 0.85 mmol) and piperi-

2

4

42

4

2 6

found: 611.1698; Anal. calcd for C H N O S : C 47.18, H 6.93, N

.17, found: C 47.26, H 6.95, N 9.26.

2

4

42

4

2 6

9

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)

dine (0.11 mL, 1.11 mmol) to afford compound 13 as a light-brown

bis(4-methylpiperidine-1-carbodithioate) (9): This compound was

synthesized following the procedure described for the preparation

of compound 4, employing compound 3a (0.2 g, 0.91 mmol) and

1

oil (yield: 79%, 0.42 g): H NMR (400 MHz, CDCl ): d=4.27–3.91 (m,

3

8

2

1

H), 3.69–3.55 (m, 2H), 3.38–3.34 (m, 2H), 2.66–2.54 (m, 4H), 2.43–

.42 (m, 8H), 2.21–2.05 (m, 2H), 1.71 (bs, 12H), 1.57 (bs, 8H), 1.43–

4

-methylpiperidine (0.14 mL, 1.18 mmol) to afford compound 9 as

a colorless oil (yield: 68%, 0.39 g): H NMR (400 MHz, CDCl ): d=

.49 (bs, 2H), 4.65–4.49 (m, 2H), 3.97–3.63 (m, 4H), 3.36–3.13 (m,

H), 2.83–2.77 (m, 4H), 2.59 (bs, 8H), 1.77 (bs, 13H), 1.29–1.26 (m,

H), 0.99–0.97 ppm (m, 6H); C NMR (100 MHz, CDCl ): d=195.3

C=S), 59.6, 54.3, 52.2, 50.5, 50.1, 40.4, 33.8, 30.9, 23.6, 21.2 ppm; IR

neat): n˜ =2953, 1432, 1220, 1141 cm ; ESI-MS (m/z): 635 [M+H] ;

HRMS-ESI m/z [M+H] calcd for C H N S : 635.2438, found:

35.2442; Anal. calcd for C H N S : C 52.95, H 7.94, N 8.82, found:

13

.41 ppm (m, 4H); C NMR (100 MHz, CDCl ): d=195.4 (C=S), 62.5,

3

1

3

54.9, 52.9, 51.4, 48.9, 40.5, 25.9, 24.4, 24.3 ppm; IR (neat): n˜ =2921,

5

6

5

(

À1

+

1

[

459, 1219, 1124 cm ; ESI-MS (m/z): 635 [M+H] ; HRMS-ESI m/z

+

M+H] calcd for C H N S : 635.2438, found: 635.2437; Anal.

28 50 4 6

1

3

calcd for C H N S : C 52.95, H 7.94, N 8.82, found: C 52.82, H 8.10,

28 50 4 6

N 8.73.

À1

+

(Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl)

di-

2

8

50

4 6

6

morpholine-4-carbodithioat (14): This compound was synthesized

by following the procedure described for the preparation of com-

pound 4, employing compound 3b (0.2 g, 0.85 mmol) and mor-

2

8

50

4 6

C 52.80, H 7.99, N 8.95.

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)

pholine (0.10 mL, 1.11 mmol) to afford compound 14 as a light-

bis(3-methylpiperidine-1-carbodithioate) (10): This compound

was synthesized following the procedure described for the prepa-

ration of compound 4, employing compound 3a (0.2 g,

1

yellow oil (yield: 82%, 0.44 g): H NMR (400 MHz, CDCl ): d=4.49–

3

1

.86 (m, 8H), 3.71–3.53 (m, 10H), 3.28–3.06 (m, 2H), 2.63–2.39 (m,

4H), 1.52 (bs, 8H), 1.38 ppm (bs, 4H); C NMR (100 MHz, CDCl₃):

13

0

.91 mmol) and 3-methylpiperidine (0.11 mL, 1.00 mmol) to afford

compound 10 as a brown (yield: 59%, 0.34 g): H NMR (400 MHz,

CDCl ): d=5.36 (bs, 2H), 4.58–4.51 (m, 2H), 3.94–3.90 (m, 2H),

.67–3.58 (m, 2H), 3.36–3.33 (m, 2H), 3.20–3.14 (m, 2H), 2.91–2.77

m, 6H), 2.64–2.59 (m, 8H), 1.90–1.86 (m, 2H), 1.77 (bs, 12H), 1.64–

.60 (m, 2H), 1.27–1.22 (m, 2H), 0.98 ppm (bs, 6H); C NMR

d=197.1 (C=S), 66.2, 62.3, 54.8, 50.9, 48.8, 40.3, 25.8, 24.3 ppm; IR

1

À1

(

neat): n˜ =2934, 2858, 1225, 1115, 1070 cm ; ESI-MS (m/z): 639

+

3

[M+H] ; HRMS-ESI m/z [M+H] calcd for C H N O S : 639.2023,

26 46 4 2 6

3

(

1

found: 639.1976; Anal. calcd for C H N O S : C 48.87, H 7.26, N

26

46

4

2 6

8

.77, found: C 48.85, H 7.40, N 8.68.

13

(

5

2

100 MHz, CDCl ): d=195.2 (C=S), 59.6, 58.8, 57.4, 54.3, 52.5, 50.9,

(Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl)

di-

3

0.1, 40.4, 32.9, 31.7, 31.2, 25.3, 24.6, 23.6, 18.8 ppm; IR (neat): n˜ =

pyrrolidine-1-carbodithioate (15): This compound was synthe-

sized following the procedure described for the preparation of

compound 4, employing compound 3b (0.2 g, 0.85 mmol) and pyr-

À1

+

975, 1467, 1282, 1124 cm ; ESI-MS (m/z): 635 [M+H] ; HRMS-ESI

+

m/z [M+H] calcd for C H N S : 635.2438, found: 635.2417; Anal.

2

8

50

4 6

calcd for C H N S : C 52.95, H 7.94, N 8.82, found: C 52.90, H 7.98,

N 8.79.

rolidine (0.09 mL, 1.11 mmol) to afford compound 15 as a colorless

2

8

50

4 6

1

oil (yield: 71%, 0.36 g): H NMR (400 MHz, CDCl ): d=3.94–3.91 (m,

3

4

2

1

6

1

H), 3.71–3.54 (m, 6H), 3.36–3.33 (m, 2H), 2.66–2.54 (m, 5H), 2.45–

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)

.42 (m, 9H), 2.10–2.00 (m, 4H), 1.98–1.96 (m, 4H), 1.57 (bs, 8H),

bis(4-butylpiperazine-1-carbodithioate) (11): This compound was

synthesized following the procedure described for the preparation

of compound 4, employing compound 3a (0.2 g, 0.91 mmol) and

13

.43–1.41 ppm (m, 4H); C NMR (100 MHz, CDCl ): d=192.5 (C=S),

3

2.4, 54.8, 50.6, 48.9, 39.8, 26.0, 25.9, 24.2 ppm; IR (neat): n˜ =2933,

À1

+

431, 1226, 1161 cm ; ESI-MS (m/z): 607 [M+H] ; HRMS-ESI m/z

4

-butylpiperazine (0.16 g, 1.18 mmol) to afford compound 11 as

+

[M+H] calcd for C H N S : 607.2125, found: 607.2100; Anal.

26 46 4 6

1

a brown oil (yield: 63%, 0.41 g): H NMR (300 MHz, CDCl ): d=

4

3

calcd for C H N S : C 51.44, H 7.64, N 9.23, found: C 51.49, H 7.60,

26 46 4 6

N 9.28.

.51–3.87 (m, 6H), 3.63–3.57 (m, 4H), 3.33–3.31 (m, 2H), 3.12–2.83

(

m, 4H), 2.63–2.35 (m, 20H), 1.98 (bs, 4H), 1.77 (bs, 6H), 1.47 (bs,

4

2

H), 1.33–1.24 (m, 4H), 0.93–0.88 ppm (m, 6H); IR (neat): n˜ =2954,

932, 1463, 1225, 1150 cm ; ESI-MS (m/z): 721 [M+H] ; Anal.

(Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl) dia-

zepane-1-carbodithioate (16): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3b (0.2 g, 0.85 mmol) and azepane

À1

+

calcd for C H N S : C 53.29, H 8.38, N 11.65, found: C 53.34, H

8

3

2

60

6 6

.30, N 11.79.

(

3

2

1

6

0.13 mL, 1.11 mmol) to afford compound 16 as a light-yellow oil

(

Æ)-2,2’-Disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)

1

yield: 63%, 0.35 g): H NMR (400 MHz, CDCl ): d=4.22–4.12 (m,

3

bis(4-benzylpiperidine-1-carbodithioate) (12): This compound

was synthesized following the procedure described for the prepa-

ration of compound 4, employing compound 3a (0.2 g,

H), 3.93–3.87 (m, 5H), 3.62–3.54 (m, 2H), 3.37–3.33 (m, 2H) 2.74–

.54 (m, 7H), 2.43–2.41 (m, 7H), 1.86 (bs, 7H), 1.58–1.57 (m, 17H),

13

.42–1.41 ppm (m, 4H); C NMR (100 MHz, CDCl ): d=196.0 (C=S),

3

0

.91 mmol) and 4-benzylpiperidine (0.2 g, 1.18 mmol) to afford

2.5, 55.6, 54.9, 52.8, 48.9, 40.4, 27.4, 26.5, 25.9, 24.4 ppm; IR (neat):

1

compound 12 as a colorless oil (yield: 52%, 0.373 g): H NMR

400 MHz, CDCl ): d=7.32–7.28 (m, 4H), 7.24–7.20 (m, 2H), 7.16–

À1

+

n˜ =2929, 1445, 1237, 1160 cm ; ESI-MS (m/z): 663 [M+H] ; HRMS-

ESI m/z [M+H] calcd for C H N S : 663.2751, found: 663.2722;

(

+

3

30

54

4 6

7

3

1

.14 (m, 4H), 5.55 (bs, 2H), 4.69 (bs, 2H), 3.93–3.90 (m, 2H), 3.67–

.65 (m, 2H), 3.36–3.33 (m, 2H), 3.11–3.01 (m, 3H), 2.83–2.77 (m,

H), 2.59–2.57 (m, 10H), 2.26 (bs, 4H), 1.93–1.87 (m, 2H), 1.79 (bs,

Anal. calcd for C H N S : C 54.33, H 8.21, N 8.45, found: C 54.43,

H 8.29, N 8.40.

30

54

4 6

1

3

2H), 1.37–1.34 ppm (m, 4H); C NMR (100 MHz, CDCl ): d=195.4

(Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl) bis(4-

methylpiperazine-1-carbodithioate) (17): This compound was

synthesized following the procedure described for the preparation

of compound 4, employing compound 3b (0.2 g, 0.85 mmol) and

3

(

3

C=S), 139.7, 129.0, 128.3, 126.1, 59.7, 54.3, 52.1, 50.2, 42.5, 40.5,

8.1, 32.0, 23.6 ppm; IR (neat): n˜ =2926, 2854, 1560, 1448, 1226,

À1

+

1135 cm ; ESI-MS (m/z): 787 [M+H] ; HRMS-ESI m/z [M+H]

calcd for C H N S : 787.3064, found: 787.3064; Anal. calcd for

4-methylpiperazine (0.12 mL, 1.11 mmol) to afford compound 17

4

0

58

4 6

1

C H N S : C 61.02, H 7.43, N 7.12, found: C 59.98, H 7.57; N 7.16.

as a light-brown oil (yield: 75%, 0.42 g): H NMR (400 MHz, CDCl ):

40

58

4

6

3

ChemMedChem 2015, 10, 1739 – 1753

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

d=4.34–3.90 (m, 10H), 3.69–3.57 (m, 2H), 3.36–3.32 (m, 2H), 2.68–

663.2753; Anal. calcd for C H N S : C 54.33, H 8.21, N 8.45, found:

30

54

4 6

2

.42 (m, 20H), 2.34 (s, 6H), 1.57 (m, 8H), 1.43–1.42 ppm (m, 4H);

C 54.42, H 8.35, N 8.25.

13

C NMR (100 MHz, CDCl ): d=196.7 (C=S), 62.4, 54.8, 54.3, 50.9,

3

(Æ)-2,2’-Disulfanediylbis(3-(azepan-1-yl)propane-2,1-diyl) dimor-

4

9.6, 45.5, 40.5, 25.9, 24.3 ppm; IR (neat): n˜ =2930, 1460, 1231,

À1

+

pholine-4-carbodithioate (22): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3c (0.15 g, 0.60 mmol) and mor-

1150 cm ; ESI-MS (m/z): 665 [M+H] ; Anal. calcd for C H N S : C

2

8

52

6 6

5

0.56, H 7.88, N 12.63, found: C 50.45, H 7.98, N 12.56.

pholine (0.07 mL, 0.79 mmol) to afford compound 22 as a colorless

(

Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl) bis(4-

1

oil (yield: 68%, 0.27 g): H NMR (400 MHz, CDCl ): d=4.30–3.92 (m,

3

methylpiperidine-1-carbodithioate) (18): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3b (0.2 g, 0.85 mmol) and 4-

methylpiperidine (0.13 mL, 1.11 mmol) to afford compound 18 as

a light-yellow oil (yield: 68%, 0.38 g): H NMR (400 MHz, CDCl ): d=

5

2

8

4

8

2

H), 3.77–3.75 (m, 6H), 3.69–3.68 (m, 2H), 3.28–3.07 (m, 4H), 2.80–

.69 (m, 14H), 1.62–1.58 ppm (m, 16H); C NMR (100 MHz, CDCl₃):

13

d=197.2 (C=S), 66.2, 61.4, 55.5, 50.9, 45.2, 40.2, 28.4, 27.1 ppm; IR

À1

1

(neat): n˜ =2927, 1591, 1461, 1220, 1155, 1030 cm ; ESI-MS (m/z):

3

+

6

7

67 [M+H] ; HRMS-ESI m/z [M+H] calcd for C H N O S :

28 50 4 2 6

.49 (bs, 2H), 4.65–4.52 (m, 2H), 3.94–3.93 (m, 2H), 3.60–3.58 (m,

H), 3.35–3.34 (m, 2H), 3.13 (bs, 4H), 2.63–2.57 (m, 4H), 2.44 (bs,

H), 1.77–1.74 (m, 5H), 1.57 (bs, 9H), 1.43 (bs, 4H), 1.29–1.26 (m,

67.2336, found: 667.2322; Anal. calcd for C H N O S : C 50.41, H

.55, N 8.40, found: C 50.56, H 7.71, N 8.32.

28

50

4

2 6

1

3

H), 0.98–0.97 ppm (m, 6H); C NMR (100 MHz, CDCl ): d=195.4

3

(Æ)-2,2’-Disulfanediylbis(3-(azepan-1-yl)propane-2,1-diyl) dipyr-

rolidine-1-carbodithioate (23): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3c (0.15 g, 0.60 mmol) and pyrroli-

(

C=S), 62.5, 54.8, 52.1, 50.5, 48.8, 40.5, 33.9, 30.9, 25.8, 24.3,

À1

2

6

8

1.3 ppm; IR (neat): n˜ =2929, 1442, 1233, 1155 cm ; ESI-MS (m/z):

+

63 [M+H] ; HRMS-ESI m/z [M+H] calcd for C H N S :

30

54

4 6

63.2751, found: 663.2749; Anal. calcd for C H N S : C 54.33, H

3

0

54

4

6

dine (0.06 mL, 0.79 mmol) to afford compound 23 as a colorless oil

.21, N 8.45, found: C 54.38, H 8.29, N 8.40.

1

(

yield: 79%, 0.30 g): H NMR (400 MHz, CDCl ): d=3.92–3.89 (m,

3

6

2

H), 3.68–3.59 (m, 4H), 3.51–3.22 (m, 2H), 2.95–2.68 (m, 14H),

.10–2.00 (m, 4H), 1.98–1.95 (m, 4H), 1.62–1.57 ppm (m, 16H);

(

Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl) bis(3-

methylpiperidine-1-carbodithioate) (19): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3b (0.2 g, 0.85 mmol) and 3-

methylpiperidine (0.13 mL, 1.11 mmol) to afford compound 19 as

a colorless oil (yield: 79%, 0.44 g): H NMR (400 MHz, CDCl ): d=

13

C NMR (100 MHz, CDCl ): d=192.4 (C=S), 61.4, 55.8, 55.0, 50.6,

3

4

1

5.2, 39.7, 28.4, 27.1, 26.0, 24.3 ppm; IR (neat): n˜ =2927, 1432,

À1

+

225, 1166 cm ; ESI-MS (m/z): 633 [M+H] ; HRMS-ESI m/z [M+

+

H] calcd for C H N S : 635.2438, found: 635.2442; Anal. calcd for

C H N S : C 52.95, H 7.94, N 8.82, found: C 52.84, H 8.06, N 8.76.

28 50 4 6

28

50

4 6

1

3

5

3

.36 (bs, 2H), 4.58–4.52 (m, 2H), 3.96–3.94 (m, 1H), 3.62 (bs, 1H),

.38–3.34 (m, 2H), 3.21–3.13 (m, 2H), 2.88–2.58 (m, 8H), 2.45–2.44

(

Æ)-2,2’-Disulfanediylbis(3-(azepan-1-yl)propane-2,1-diyl) diaze-

pane-1-carbodithioate (24): This compound was synthesized fol-

lowing the procedure described for the preparation of compound

4

(

m, 8H), 1.90–1.87 (m, 2H), 1.79–1.76 (m, 4H), 1.59–1.58 (m, 11 H),

1

3

1

.44–1.43 (m, 5H), 0.98–0.97 ppm (m, 6H); C NMR (100 MHz,

, employing compound 3c (0.15 g, 0.60 mmol) and azepane

CDCl ): d=195.4 (C=S), 62.5, 60.6, 54.8, 50.2, 48.8, 40.5, 32.9, 31.6,

3

(

0.09 mL, 0.79 mmol) to afford compound 24 as a light-yellow oil

2

5.9, 24.3, 22.1, 18.8 ppm; IR (neat): n˜ =2937, 1428, 1230,

1

À1

+

(yield: 70%, 0.29 g): H NMR (400 MHz, CDCl ): d=4.25–4.12 (m,

3

(

1

IR (neat): n˜ =2926, 1451, 1217, 1138 cm ; ESI-MS (m/z): 691 [M+

H] ; HRMS-ESI m/z [M+H] calcd for C H N S :691.3064, found:

6

1121 cm ; ESI-MS (m/z): 663 [M+H] ; HRMS-ESI m/z [M+H]

H); 3.95–3.90 (m, 5H), 3.65–3.27 (m, 4H), 2.97–2.70 (m, 14H), 1.87

calcd for C H N S : 663.2751, found: 663.2744; Anal. calcd for

C H N S : C 54.33, H 8.21, N 8.45, found: C 54.28, H 8.39, N 8.54.

3

0

54

4 6

13

bs, 6H), 1.64–1.58 ppm (m, 26H); C NMR (100 MHz, CDCl ): d=

3

30

54

4 6

96.0 (C=S), 61.4, 55.8, 50.1, 45.2, 40.2, 28.4, 27.1, 26.7, 26.3 ppm;

À1

(

Æ)-2,2’-Disulfanediylbis(3-(piperidin-1-yl)propane-2,1-diyl) bis(4-

+

butylpiperazine-1-carbodithioate) (20): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3b (0.2 g, 0.85 mmol) and 4-

32 58

4 6

91.3045; Anal. calcd for C H N S : C 55.60, H 8.46, N 8.11, found:

32 58 4 6

C 55.75, H 8.16, N 8.19.

butylpiperazine (0.15 g, 1.11 mmol) to afford compound 20 as

(

Æ)-2,2’-Disulfanediylbis(3-(azepan-1-yl)propane-2,1-diyl) bis(4-

1

a brown oil (yield: 51%, 0.32 g): H NMR (300 MHz, CDCl ): d=

3

methylpiperazine-1-carbodithioate) (25): This compound was

synthesized following the procedure described for the preparation

of compound 4, employing compound 3c (0.15 g, 0.60 mmol) and

4

5

.31–3.57 (m, 8H), 3.35–2.93 (m, 5H), 2.85–2.39 (m, 20H), 1.97 (bs,

H), 1.57–1.22 (m, 20H), 0.91–0.86 ppm (m, 6H); C NMR (50 MHz,

1

3

CDCl ): d=196.3 (C=S), 61.8, 57.8, 57.6, 54.5, 52.2, 50.8, 48.2, 40.4,

3

4

-methylpiperazine (0.08 mL, 0.79 mmol) to afford compound 25

2

1

9.6, 28.3, 28.0, 25.3, 23.9, 22.1, 20.5, 13.9 ppm; IR (neat): n˜ =2932,

460, 1223, 1154 cm ; ESI-MS (m/z): 749 [M+H] ; Anal. calcd for

1

as a brown semisolid (yield: 70%, 0.29 g): H NMR (400 MHz,

CDCl ): d=4.49–3.91 (m, 9H), 3.68–3.62 (m, 2H), 3.35–3.25 (m, 2H),

2

À1

+

3

C H N S : C 54.50, H 8.61, N 11.22, found: C 54.62, H 8.58, N

34

64

6 6

.82–2.69 (m, 13H), 2.51–2.48 (m, 6H), 2.33 (s, 6H), 2.09–2.05 (m,

11.25.

13

H), 1.63–1.58 ppm (m, 16H); C NMR (100 MHz, CDCl ): d=196.6

3

(

C=S), 61.4, 55.7, 55.5, 54.4, 50.0, 45.6, 45.1, 40.3, 28.4, 27.0 ppm; IR

(

Æ)-2,2’-Disulfanediylbis(3-(azepan-1-yl)propane-2,1-diyl)

dipi-

À1

+

neat): n˜ =2926, 1459, 1233, 1141 cm ; ESI-MS (m/z): 693 [M+H] ;

peridine-1-carbodithioate (21): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3c (0.15 g, 0.60 mmol) and piperi-

dine (0.08 mL, 0.79 mmol) to afford compound 21 as a light-yellow

oil (yield: 78%, 0.31 g): H NMR (400 MHz, CDCl ): d=4.43 (bs, 4H),

+

HRMS-ESI m/z [M+H] calcd for C H N S :693.2969, found:

6

found: C 51.88, H 8.20, N 12.19.

30

56

6 6

93.2974; Anal. calcd for C H N S : C 51.98, H 8.14, N 12.12,

30 56 6 6

1

(Æ)-2,2’-Disulfanediylbis(3-(azepan-1-yl)propane-2,1-diyl) bis(4-

methylpiperidine-1-carbodithioate) (26): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3c (0.15 g, 0.60 mmol) and 4-

methylpiperidine (0.09 mL, 0.79 mmol) to afford compound 26 as

a light-yellow oil (yield: 75%, 0.31 g): H NMR (400 MHz, CDCl ): d=

3

4

1

.25 (bs, 5H), 3.91–3.26 (m, 5H), 2.88–2.78 (m, 4H), 2.68 (s, 8H),

1

3

.68–1.56 ppm (m, 28H); C NMR (100 MHz, CDCl ): d=195.3 (C=

3

S), 61.5, 55.8, 52.8, 51.3, 50.1, 40.3, 28.4, 27.1, 25.9, 24.3 ppm; IR

À1

+

(

neat): n˜ =2971, 1437, 1216, 1188 cm ; ESI-MS (m/z): 663 [M+H] ;

+

1

HRMS-ESI m/z [M+H] calcd for C H N S : 663.2751, found:

3

0

54

4

6

3

ChemMedChem 2015, 10, 1739 – 1753

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1749

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

À1

+

5

.50–4.09 (m, 4H), 3.95–3.93 (m, 3H), 3.30–3.13 (m, 5H), 3.01–2.82

1222, 1114, 1024 cm ; ESI-MS m/z: 669 [M+H] ; HRMS-ESI m/z

+

(

m, 4H), 2.73–2.70 (m, 10H), 1.64 (bs, 4H), 1.60–1.59 (m, 18H),

[M+H] calcd for C H N O S : 669.2241, found: 669.2242; Anal.

26

48

6

2 6

1

3

1

.33–1.27 (m, 4H), 0.99–0.98 ppm (m, 6H); C NMR (100 MHz,

calcd for C H N O S : C 46.67, H 7.23, N 12.56, found: C 46.53, H

26 48 6 2 6

CDCl ): d=195.4 (C=S), 59.7, 55.5, 52.1, 50.1, 45.2, 40.4, 33.9, 30.9,

7.44, N 12.65.

3

À1

2

8.5, 27.0, 21.3 ppm; IR (neat): n˜ =2986, 1432, 1234, 1131 cm ; ESI-

+

(Æ)-2,2’-Disulfanediylbis(3-morpholinopropane-2,1-diyl) dimor-

pholine-4-carbodithioate (31): This compound was synthesized

following the procedure described for the preparation of com-

pound 4, employing compound 3d (0.3 g, 1.27 mmol) and mor-

MS (m/z): 691 [M+H] ; HRMS-ESI m/z [M+H]

calcd for

C H N S : 691.3064, found: 691.3094; Anal. calcd for C H N S : C

32

58

4

6

32 58

4 6

5

5.60, H 8.46, N 8.11, found: C 55.68, H 8.38, N 8.29.

pholine (0.14 mL, 1.65 mmol) to afford compound 31 as a colorless

(

Æ)-2,2’-Disulfanediylbis(3-(azepan-1-yl)propane-2,1-diyl) bis(3-

1

oil (yield: 76%, 0.62 g): H NMR (400 MHz, CDCl ): d=4.26–3.87 (m,

methylpiperidine-1-carbodithioate) (27): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3c (0.15 g, 0.60 mmol) and 3-

methylpiperidine (0.09 mL, 0.79 mmol) to afford compound 27 as

a brown oil (yield: 73%, 0.30 g): H NMR (400 MHz, CDCl ): d=

3

1

2

6

1

0H), 3.74–3.58 (m, 18H), 3.35–3.29 (m, 2H), 2.71–2.59 (m, 4H),

13

.50–2.48 ppm (m, 8H); C NMR (100 MHz, CDCl ): d=196.8 (C=S),

3

6.8, 66.2, 62.0, 53.9, 50.9, 48.5, 39.9 ppm; IR (neat): n˜ =2957, 1453,

À1

+

1

419, 1231, 1118, 1031 cm ; ESI-MS (m/z): 643 [M+H] ; HRMS-ESI

3

+

m/z [M+H] calcd for C H N O S : 643.1609, found: 643.1583;

5

.34–4.10 (m, 4H), 3.95–3.92 (m, 2H), 3.71–3.63 (m, 2H), 3.45–3.14

24 42

4

4 6

Anal. calcd for C H N O S : C 44.83, H 6.58, N 8.71, found: C

(

m, 4H), 2.82–2.70 (m, 14H), 1.89–1.86 (m, 2H), 1.79–1.75 (m, 4H),

24 42

4

4 6

4

4.89, H 6.63, N 8.62.

1

.63–1.58 (m, 18H), 1.31–1.20 (m, 2H), 0.97–0.96 ppm (m, 6H);

13

C NMR (100 MHz, CDCl ): d=195.4 (C=S), 61.5, 55.8, 52.5, 50.8,

3

(

Æ)-2,2’-Disulfanediylbis(3-morpholinopropane-2,1-diyl)

bis(2-

5

0.2, 45.2, 40.3, 32.9, 31.6, 31.2, 28.4, 27.1, 25.4, 24.6, 18.8 ppm; IR

methylpiperidine-1-carbodithioate) (32): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3d (0.3 g, 1.27 mmol) and 2-

À1

+

(

neat): n˜ =2926, 1425, 1230, 1131 cm ; ESI-MS (m/z): 691 [M+H] ;

+

HRMS-ESI m/z [M+H] calcd for C H N S : 691.3064, found:

6

C 55.67, H 8.32, N 8.18.

3

2

58

4 6

91.3054; Anal. calcd for C H N S : C 55.60, H 8.46, N 8.11, found:

32 58 4 6

methylpiperidine (0.19 mL, 1.65 mmol) to afford compound 32 as

a colorless oil (yield: 48%, 0.40 g): H NMR (300 MHz, CDCl ): d=

1

3

.95–3.93 (m, 2H), 3.69–3.56 (bs, 10H), 3.33–3.14 (m, 4H), 2.88–2.02

(

Æ)-2,2’-Disulfanediylbis(3-morpholinopropane-2,1-diyl) dipiperi-

(

m, 16H), 1.75–1.42 (m, 10H), 1.28 ppm (m, 8H); IR (neat): n˜ =2948,

dine-1-carbodithioate (28): This compound was synthesized fol-

lowing the procedure described for the preparation of compound

4

À1

+

1

457, 1223, 1116, 1060 cm ; ESIMS (m/z): 667 [M+H] ); Anal.

calcd for C H N O S : C 50.41, H 7.56, N 8.42, found: C 50.37, H

28

50

4

2 6

, employing compound 3d (0.3 g, 1.27 mmol) and piperidine

7

.60, N 8.48.

(

0.16 mL, 1.65 mmol) to afford compound 28 as a colorless oil

yield: 68%, 0.55 g): H NMR (400 MHz, CDCl ): d=4.27–4.26 (m,

1

3

(

Æ)-2,2’-Disulfanediylbis(3-morpholinopropane-2,1-diyl)

bis(3-

4

2

H), 3.95–3.90 (m, 6H), 3.71–3.53 (m, 10H), 3.36–3.33 (m, 2H),

.73–2.59 (m, 4H), 2.52–2.50 (m, 8H), 1.88–1.70 ppm (m, 12H);

methylpiperidine-1-carbodithioate) (33): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3d (0.3 g, 1.27 mmol) and 3-

1

3

C NMR (100 MHz, CDCl ): d=194.8 (C=S), 66.9, 62.2, 53.9, 53.0,

3

5

1

1.4, 45.4, 40.0, 25.5, 24.2 ppm; IR (neat): n˜ =2927, 1456, 1220,

methylpiperidine (0.19 mL, 1.65 mmol) to afford compound 33 as

a light-yellow oil (yield: 62%, 0.52 g): H NMR (400 MHz, CDCl ): d=

5

À1

+

155, 1035 cm ; ESI-MS (m/z): 639 [M+H] ; HRMS-ESI m/z [M+

1

3

+

H] calcd for C H N O S : 639.2023, found: 639.2031; Anal. calcd

for C H N O S : C 48.87; H 7.26, N 8.77, found: C 48.75, H 7.42, N

8

2

6

46

4

2 6

.33–4.51 (m, 4H), 3.96–3.94 (m, 4H), 3.72–3.70 (m, 12H), 2.74–2.65

2

6

46

4

2 6

(

m, 4H), 2.55–2.52 (m, 12H), 1.90–1.60 (m, 8H), 0.98–0.92 ppm (m,

.54.

13

6

5

1

H); C NMR (100 MHz, CDCl ): d=194.9 (C=S), 66.8, 62.1, 60.2,

3

7.5, 53.9, 53.7, 52.6, 50.9, 45.4, 40.1, 32.8, 31.6, 25.4, 24.6,

(

Æ)-2,2’-Disulfanediylbis(3-morpholinopropane-2,1-diyl) dipyrro-

À1

8.8 ppm; IR (neat): n˜ =2957, 1452, 1372, 1229, 1117, 1043 cm

;

lidine-1-carbodithioate (29): This compound was synthesized fol-

+

ESI-MS (m/z): 667 [M+H] ; HRMS-ESI m/z [M+H] calcd for

C H N O S : 667.2336, found: 667.2328; Anal. calcd for

C H N O S : C 50.41, H 7.55, N 8.40, found: C 50.49, H 7.40, N

8.49.

lowing the procedure described for the preparation of compound

4

28

50

4

2 6

, employing compound 3d (0.3 g, 1.27 mmol) and pyrrolidine

28

50

4

2 6

(

0.13 mL, 1.65 mmol) to afford compound 29 as a colorless oil

yield: 78%, 0.60 g): H NMR (400 MHz, CDCl ): d=3.95–3.89 (m,

1

3

6

2

1

6

1

H), 3.71–3.64 (m, 12H), 3.59–3.54 (m, 2H), 3.37–3.30 (m, 2H),

.72–2.59 (m, 4H), 2.53–2.50 (m, 8H), 2.12–2.00 (m, 4H), 1.98–

(

Æ)-2,2’-Disulfanediylbis(3-morpholinopropane-2,1-diyl)

bis(4-

methylpiperidine-1-carbodithioate) (34): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3d (0.3 g, 1.27 mmol) and 4-

methylpiperidine (0.19 mL, 1.65 mmol) to afford compound 34 as

a yellow oil (yield: 76%, 0.64 g): H NMR (400 MHz, CDCl ): d=

1

3

.95 ppm (m, 4H); C NMR (100 MHz, CDCl ): d=192.0 (C=S), 66.9,

3

2.1, 53.8, 50.7, 48.7, 39.4, 26.0 ppm; IR (neat): n˜ =2920, 1434,

À1

+

217, 1132, 1029 cm ; ESI-MS (m/z): 611 [M+H] ); HRMS-ESI m/z

+

1

[M+H] calcd for C H N O S : 611.1710, found: 611.1707; Anal.

24 42 4 2 6

3

calcd for C H N O S : C 47.18, H 6.93, N 9.17, found: C 47.32, H

2

4

42

4

2

6

5.48–4.51 (m, 4H), 3.96–3.95 (m, 4H), 3.72–3.70 (m, 12H), 2.89–2.84

7

.13, N 9.20.

(

m, 4H), 2.74–2.52 (m, 12H), 1.79–1.76 (m, 6H), 1.33–1.27 (m, 2H),

13

1

6

3

1

.00–0.91 ppm (m, 6H); C NMR (100 MHz, CDCl ): d=194.9 (C=S),

3

(

Æ)-2,2’-Disulfanediylbis(3-morpholinopropane-2,1-diyl)

bis(4-

6.8, 62.2, 60.2, 57.5, 53.9, 53.6, 52.2, 50.5, 45.5, 40.1, 34.0, 33.5,

methylpiperazine-1-carbodithioate) (30): This compound was

synthesized by following the procedure described for the prepara-

tion of compound 4, employing compound 3d (0.3 g, 1.27 mmol)

0.9, 21.2 ppm; IR (neat): n˜ =2925, 1446, 1369, 1234, 1118,

À1

+

045 cm ; ESI-MS (m/z): 667 [M+H] ; HRMS-ESI m/z [M+H]

calcd for C H N O S : 667.2336, found: 667.2327; Anal. calcd for

C H N O S : C 50.41, H 7.55, N 8.40, found: C 50.57, H 7.63, N

8

28

50

4

2 6

and 4-methylpiperazine (0.18 mL, 1.65 mmol) to afford compound

3

d=4.33–3.90 (m, 10H), 3.71–3.70 (m, 8H), 3.59–3.57 (m, 2H), 3.36–

1

28 50

4

2 6

0 as a brown solid (yield: 60%, 0.51 g): H NMR (400 MHz, CDCl ):

3

.49.

3

.31 (m, 2H), 2.66–2.60 (m, 5H), 2.51 (bs, 15H), 2.33 ppm (s, 6H);

(Æ)-2,2’-Disulfanediylbis(3-(4-methylpiperazin-1-yl)propane-2,1-

diyl) dipiperidine-1-carbodithioate (35): This compound was syn-

thesized following the procedure described for the preparation of

1

3

C NMR (100 MHz, CDCl ): d=196.2 (C=S), 66.8, 62.0, 54.3, 53.8,

3

5

1.0, 49.7, 48.4, 45.5, 40.0 ppm; IR (neat): n˜ =2921, 1418, 1288,

ChemMedChem 2015, 10, 1739 – 1753

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

compound 4, employing compound 3e (0.3 g, 1.21 mmol) and pi-

the National Committee for Clinical Laboratory Standards using

RPMI 1640 media buffered with MOPS (3-[N-morpholino]propane-

sulfonic acid). Starting inoculums of test culture were 1–5

10 CFUmL . Microtiter plates were incubated at 358C. MIC values

were recorded after 48 h of incubation. All experiments were re-

peated three times (Table 2).

peridine (0.13 mL, 1.57 mmol) to afford compound 35 as a brown

1

oil (yield: 69%, 0.55 g): H NMR (400 MHz, CDCl ): d=4.26–4.11 (m,

3

À1

4

2

H), 3.96–3.89 (m, 6H), 3.60–3.54 (m, 2H), 3.35–3.28 (m, 2H), 2.70–

.45 (m, 20H), 2.27 (s, 6H), 1.70 ppm (bs, 12H); C NMR (100 MHz,

1

3

CDCl ): d=195.0 (C=S), 61.6, 55.0, 53.3, 48.8, 45.9, 40.2, 26.0,

3

À1

2

(

4.3 ppm; IR (neat): n˜ =2931, 2855, 1455, 1218, 1137 cm ; ESIMS

m/z): 665 [M+H] ; HRMS-ESI m/z [M+H] calcd for C H N S :

+

Cytotoxicity of compounds toward human cervical cells: The cytotox-

ic effect of test compounds were evaluated in an in vitro model of

cervicovaginal epithelium (HeLa) cells, using the MTT [3-(4, 5-dime-

28 52 6 6

6

7

65.2656, found: 665.2648; Anal. calcd for C H N S : C 50.56, H

28 52 6 6

.88, N 12.63, found: C 50.68, H 8.10, N 12.50.

[

37,38]

thylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay.

seeded in 96-well plates were incubated in culture medium

DMEM with 10% fetal calf serum) for 24 h at 378C in a 5% CO₂–

5% air atmosphere. After 24 h, the culture medium was replaced

Cells

(

Æ)-2,2’-Disulfanediylbis(3-(4-methylpiperazin-1-yl)propane-2,1-

diyl) dipyrrolidine-1 carbodithioate (36): This compound was syn-

thesized following the procedure described for the preparation of

compound 4, employing compound 3d (0.3 g, 1.21 mmol) and pyr-

(

9

with fresh medium containing dilutions of test compounds in ex-

perimental wells and 0.05% DMSO in culture medium in control

rolidine (0.15 mL, 1.57 mmol) to afford compound 36 as a light-

1

brown oil (yield: 71%, 0.54 g): H NMR (400 MHz, CDCl ): d=3.92–

3

wells. After incubation for another 24 h, MTT solution (5 mL of

À1

3

2

.89 (m, 5H), 3.68–3.51 (m, 7H), 3.33–3.29 (m, 2H), 2.71–2.47 (m,

0H), 2.28 (s, 6H), 2.10–2.05 (m, 4H), 1.99–1.96 ppm (m, 4H);

5

mgmL ) in phosphate-buffered saline (PBS; pH 7.4) was added

to each well. The formazan crystals formed inside the viable cells

were solubilized in DMSO, and the optical density at 540 nm

13

C NMR (100 MHz, CDCl ): d=192.2 (C=S), 61.5, 54.9, 53.2, 48.9,

3

À1

4

5.8, 39.5, 24.2 ppm; IR (neat): n˜ =2948, 1433, 1217, 1145 cm ; ESI-

(

OD₅₄₀) was recorded in a microplate reader (Microquant; BioTek).

+

MS (m/z): 637 [M+H] ; HRMS-ESI m/z [M+H]

C H N S : 637.2343, found: 637.2350; Anal. calcd for C H N S : C

calcd for

All experiments were repeated three times (Table 1)

26

48

6

26 48

6 6

Compatibility of compounds with commensal vaginal flora: Spores

of the vaginal flora Lactobacillus jensenii (ATCC25258, strain 62G)

were grown in 6% Rogosa SL broth medium (Hi Media, India) con-

taining 0.132% acetic acid at 378C. The effect of test compounds

4

9.02, H 7.59, N 13.19, found: C 49.15, H 7.76, N 13.32.

Biological methods

[15]

on L. jensenii was determined as detailed by us earlier. Briefly, Ro-

Spermicidal assay: Freshly ejaculated human semen was obtained

by masturbation from healthy and fertile volunteers, collected di-

rectly in a sterile plastic tube, and transported immediately to the

laboratory. Prior informed consent was obtained from the donors

for this study. The samples were allowed to liquefy at 378C for

gosa SL broth medium was prepared in Milli-Q water, boiled for 2–

À1

3

min, and distributed in 48-well plates (500 mLwell ). Serial dilu-

tions of test compounds were added to experimental wells, and

vehicle was added to control wells in triplicate. Approximately

1

000 CFU of L. jensenii were added to each well. The plates were

4

5 min. Semen characteristics and analysis were performed accord-

ⁱⁿ^c^u^b^a^t^e^d^a^t³⁷⁸^Cⁱⁿ^a^h^u^mⁱ^dⁱ^fⁱ^e^d^a^t^m^o^s^p^h^e^r^e^c^oⁿ^t^aⁱⁿⁱⁿ^g⁵^%^C^O2

[15]

ing to the normal criteria, as per WHO guidelines. Sperm count

and motility analyses were performed manually as well as in a com-

puter-automated semen analyzer (CASA, Hamilton Thorne) system

using a small drop of liquefied semen placed on a Makler counting

chamber (Sefy Medica, Hafia, Israel) pre-warmed to 378C. Semen

for 24 h. At the end of the experiment, the cultures were mixed

thoroughly, 100 mL from each well was transferred to the corre-

sponding well of a 96-well plate, and the number of Lactobacilli

were estimated by measuring the turbidity (OD₆₁₀) in a microplate

reader. All experiments were repeated three times (Table 1).

6

À1

samples with sperm counts of >6510 mL , >85% motility, and

normal sperm morphology were used to determine the minimum

effective spermicidal concentration (MEC) of test compounds in

3D-QSAR analysis: All calculations implicated in developing this

[39]

QSAR model were performed using SYBYL 2.1.

The molecules

[15]

vitro. Nonoxynol-9 (N-9) was purchased from Spectrum Chemical

Manufacturing (New Brunswick, NJ, USA) and used as a reference

standard (Table 1). This study was approved by the Institutional

Ethics Committee of CSIR-CDRI.

used in the study were drawn and energy-minimized using the

À1

Powell method with termination criterion of 0.05 Kcalmol for

1000 iterations, using the Tripos force field and Gasteiger–Hückel

partial charges. The energy-minimized conformation of the most

active compound, 22, was used as a template for alignment, and

all other molecules were overlaid using the database alignment

module. Activity values (MEC in mm) were transformed into

Àlog(MEC), and the whole dataset was divided into two randomly

generated sub-datasets composed of 27 compounds in the train-

ing set and six compounds in the test set. CoMFA calculations

were done using the Tripos force field with a distance-dependent

dielectric constant at all interactions in a regularly spaced (2 )

Anti-Trichomonas assay: The MTZ-susceptible T. vaginalis strain

used in this study was a clinical isolate obtained from the laborato-

[36]

ry of Dr. Divya Singh, and the MTZ-resistant strain of T. vaginalis

CDC085 [ATCC50143]) was procured from American Type Culture

Collection. In vitro drug susceptibility of Trichomonas vaginalis was

(

[15]

determined as detailed earlier. Briefly, parasites were incubated

at 378C in 48-well culture plates in the presence of test com-

pounds or MTZ, serially diluted from a stock solution in DMSO

using the culture medium. DMSO in culture media (0.05%, the

highest concentration of DMSO in test wells) was used as the vehi-

cle in control wells. Cell viability was checked after 48 h using the

trypan blue exclusion assay. The minimum concentration of the

test agent at which all cells were found dead was considered to be

the MIC. MTZ (the most widely used drug against Trichomonas vag-

inalis) was procured from Sigma–Aldrich and used as reference

standard. All experiments were repeated three times (Table 2).

3

grid taking a sp carbon atom as a steric probe and a +1 charge

À1

as an electrostatic probe. The cutoff was set to 30 kcalmol . The

[40]

partial least squares (PLS) method was used to linearly correlate

the CoMFA fields to biological activity values. The CoMFA descrip-

tor was used as an independent variable, and pIC₅₀values as de-

pendent variables in the PLS regression analysis and conducted

with the standard implementation in the SYBYL 2.1 package. Cross-

validation was performed using the leave-one-out (LOO) method

in which one compound was removed from the dataset and its ac-

tivity was predicted using the model derived from the rest of the

molecules in the dataset. Non-cross-validation was performed to

[21]

Antifungal assay:

The MIC values of compounds were deter-

mined by a broth micro-dilution technique as per the guidelines of

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

2

calculate the conventional r value using the same number of com-

phate-buffered saline, PLS: partial least squares, TLC: thin-layer

chromatography.

ponents. It was used in analysis of the CoMFA results and for

model prediction (see Supporting Information)

[

34]

Contraceptive activity (in vivo): Young adult female Belgian rab-

bits were given 200 IU of human chorionic gonadotropin (hCG) in-

tramuscularly, and 6 h after injection, the test compound (dissolved

in saline) was instilled in the vagina by inserting a 15 cm long cath-

eter attached to a plastic syringe. A vaginal dose of 0, 10, 20, and

Acknowledgements

We acknowledge Ms. Tara Rawat (Senior Technical Officer) for

technical assistance and the Sophisticated Analytical Instrument

Facility (SAIF) for spectral data. We are grateful to the Council of

Scientific and Industrial Research (CSIR) (D.M., N.S.), the Universi-

ty Grants Commission (UGC) (N.L., L.K., L.K.), and the Indian

Council of Medical Research (ICMR) (V.B., P.S., B.K.) for research

fellowships. This study was supported by the Department of

Health Research (DHR), ICMR, New Delhi, Government of India

3

0 mg of test compound was instilled in 2.0 mL saline. Ten minutes

after instillation, these rabbits were mated once with a fertile buck.

Mating was re-confirmed by presence of sperm in a vaginal smear.

The mated rabbits were then kept in separate cages and allowed

to complete gestation for 30–34 days. The pregnancies and litter

size were recorded at completion of gestation (Table 3). This study

was duly approved by the Institutional Animal Ethics Committee

(

GAP 00155) and the CSIR-Network Project PROGRAM, CSIR, New

(

IAEC) of CSIR-CDRI.

Delhi, India (BSC 0101). The authors declare no competing finan-

cial interests. CSIR-CDRI communication number: 9038.

[41]

Fluorescent labeling of sperm sulfhydryl groups: Free sulfhydryls

on human sperm (after treatment with vehicle or the most promis-

ing compound, 5) were examined and imaged using a fluorescence

microscope after labeling with a fluorimetric thiol detector using

a thiol detection assay kit (Cayman). The semen sample (0.5 mL)

was treated with 2.5 mL of compound 5 at MEC, as well as with an

equal volume of saline (control) in parallel, and the specimens

were then incubated for 15 min at room temperature. After incu-

bation, the sperm were pelleted at 700 g for 10 min and washed

Keywords: disulfide esters · dithiocarbamates · drug design ·

immobilization · spermicides · sulfhydryl groups

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Received: July 6, 2015

Published online on September 4, 2015

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Article Doi

Innovative Disulfide Esters of Dithiocarbamic Acid as Women-Controlled Contraceptive Microbicides: A Bioisosterism Approach

DOI: 10.1002/cmdc.201500291

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