Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel trichomonicidal fungicides: Design, synthesis, and biology

Accepted Manuscript

Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel

trichomonicidal fungicides: Design, synthesis, and biology

Dhanaraju Mandalapu, Bhavana Kushwaha, Sonal Gupta, Shagun Krishna, Nidhi

Srivastava, Mahendra Shukla, Pratiksha Singh, Bhavana S. Chauhan, Ravi Goyani,

Jagdamba P. Maikhuri, Koneni V. Sashidhara, Brijesh Kumar, Renu Tripathi, Praveen

K. Shukla, Mohammad I. Siddiqi, Jawahar lal, Gopal Gupta, Vishnu L. Sharma

PII:

S0223-5234(17)30963-7

10.1016/j.ejmech.2017.11.060

EJMECH 9935

DOI:

Reference:

To appear in: European Journal of Medicinal Chemistry

Received Date: 1 September 2017

Revised Date: 7 November 2017

Accepted Date: 22 November 2017

Please cite this article as: D. Mandalapu, B. Kushwaha, S. Gupta, S. Krishna, N. Srivastava, M. Shukla,

P. Singh, B.S. Chauhan, R. Goyani, J.P. Maikhuri, K.V. Sashidhara, B. Kumar, R. Tripathi, P.K. Shukla,

M.I. Siddiqi, J. lal, G. Gupta, V.L. Sharma, Substituted carbamothioic amine-1-carbothioic thioanhydrides

as novel trichomonicidal fungicides: Design, synthesis, and biology, European Journal of Medicinal

Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.060.

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ACCEPTED MANUSCRIPT

Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel

trichomonicidal fungicides: Design, synthesis, and Biology^#

Dhanaraju Mandalapu,^aBhavana Kushwaha,^bSonal Gupta,^a,iShagun Krishna,^cNidhi Srivastava,^aMahendra

Shukla,^dPratiksha Singh,^eBhavana S. Chauhan,^gRavi Goyani,^hJagdamba P. Maikhuri,^bKoneni V. Sashidhara,^a

Brijesh Kumar,^fRenu Tripathi,^gPraveen K. Shukla,^eMohammad I. Siddiqi,^cJawahar lal,^dGopal Gupta,^bVishnu L.

Sharma^a,i,*

b

c

^aMedicinal & Process Chemistry Division, Endocrinology Division, Molecular and Structural Biology Division,

e

f

^dPharmacokinetic & Metabolism Division, Microbiology Division, Sophisticated Analytical Instrument Facility,

^gParasitology Division, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow-226031 (India),

^hDepartment of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Raebareli

229 010, India, ⁱAcademy of Scientific and Innovative Research (AcSIR), New Delhi-110001 (India).

Abstract:

Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like

candidiasis are major global health burden in female reproductive health. In this context a novel

non-nitroimidazole class of substituted carbamothioic amine-1-carbothioic thioanhydride series

was designed, synthesized, evaluated for trichomonacidal and fungicidal activities, and was

found to be more active than the standard drug Metronidazole (MTZ). Compounds were

trichomonicidal in the MIC ranges of 4.77–294.1 µM and 32.46–735.20 µM against MTZ-

susceptible and -resistant strains, respectively. Further, compounds inhibited the growth of at

least two out of ten fungal strains tested at MIC of 7.50-240.38 µM. The most active compound

(20) of this series was 3.8 and 9.5 fold more active than the MTZ against the two Trichomonas

strains tested. Compound 20 also significantly inhibited the sulfhydryl groups present over

Trichomonas vaginalis and was found to be more active than the MTZ in vivo. Further, a

docking analysis carried out with cysteine proteases supported their thiol inhibiting ability and

preliminary pharmacokinetic study has shown good distribution and systemic clearance.

Keywords: Carbamothioic, Thioanhydrides, Trichomoniasis, Candidiasis, Thiol inhibition.

*Corresponding author: Medicinal and Process Chemistry Division, CSIR-Central Drug Research

Institute, Sector 10, Jankipuram ext., Lucknow, Uttar Pradesh 226031, India.

Tel.: 91-522-2772450; Ext. 4671; Fax: 91-522-2771941 E-mail address: vl_sharma@cdri.res.in.

^#CDRI Communication No. 162/2017/VLS

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1. Introduction:

Sexually transmitted infections (STIs) have a major impact on reproductive health of millions of

people worldwide.[1, 2] Among the 30 infections identified to be sexually transmitted, incidence

and prevalence rate of trichomoniasis is higher than the remaining.[3] Trichomoniasis is a very

common, curable, non-viral sexually transmitted urogenital infection in humans caused by the

anaerobic, microaerophilic parasite Trichomonas vaginalis (TV).[4] Most of the cases in United

States and India occur among women of reproductive age[5] and a recent World Health

Organization (WHO) report estimates that more than 276 million people are afflicted with

trichomoniasis every year.[6] Women and men both can be affected but in men the symptoms are

mild and mostly go unreported, which results in its horizontal spread through heterosexual

contacts to sexual partners.[2] In men, the observed complications include urethral discharge,

dysuria, prostatitis, epididymitis, infertility and benign prostatic hyperplasia.[7-9] In women it is

often called “nuisance infection” and may cause endometritis, pyosalpinx, vaginitis, adnexitis,

preterm delivery, infertility, bacterial vaginosis and threat of cervical cancer.[10-13] However,

TV infections increase the risk of HIV and candidiasis transmissions through colonization of the

human urogenital tract epithelium.[14] In sexually active women opportunistic vulvovaginal

candidiasis, caused by Candida albicans has a high prevalence rate which often co-exists along

with TV.[15] The US-FDA approved drug Metronidazole (MTZ), a 5-nitroimidazole, has limited

efficacy against drug-resistant Trichomoniasis infections. For decades, the use of MTZ has been

notable for its effectiveness but the increasing drug-resistance and cross-resistance to MTZ and

other nitroimidazoles (NIs) has been limiting their application in recent times.[16, 17] In 21^st

century the incidence of drug resistance cases augmented by 17 fold which are being treated

typically with increased and probably toxic doses of MTZ.[18, 19] Prolonged treatment or high

doses of MTZ may cause side effects such as headache, dry mouth, metallic taste, glossitis and

urticaria.[20] Since women are at the receiving end with long term consequences of STIs, lack of

effective clinical candidates necessitates the researchers to discover highly effective molecules,

preferably non-nitroimidazoles to treat TV and associated infections.[21] Reactive oxygen

species (ROS-) sensitive anaerobes TV and Candida albicans largely depend on cysteine to

overcome redox stress [22] making these infections very susceptible to thiol-inhibiting agents

which signifies thiol as vulnerable target for antitrichomonal and antifungal agents.

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Our previous research work has established that thiol modifying molecules (dithiocarbamates; a-

d) are effective against TV and fungal infections. [23-29] Dithiocarbamate (DTC) incorporated

MTZ hybrids (c) resulted with increased trichomonacidal and spermicidal activities than

MTZ.[30] The possibility of drug resistance to nitroimidazole class of compounds necessitates

the discovery of non-nitroimidazole compounds. Recently, disulfiram like compounds with two

DTCs linked together directly at sulfur atom (a) also yielded potent topical dual function

microbicides.[31] However like Disulfiram, (which is a drug with two DTC groups and is in

clinical use for more than 50 years [32]), these compounds underwent rapid cleavage of S-S

bond and failed to show up in pharmacokinetic evaluation after vaginal administration. Since

pharmacokinetic evaluation is a mandatory step in modern drug development, it was envisaged

that modification of S-S bond to -S- bond which might lead to the discovery of a new class of

compounds with effective anti-Trichomonas and antifungal activities, and with suitable stability

for pharmacokinetic evaluation.

In this present study we evaluated the new compounds against MTZ-susceptible and -resistant

TV infections and some fungi that infect the female vaginal tract. A novel series of molecules

with improved efficacy over the previous drug designs is being reported along with their in vivo

biological activities, fluorescence labelling of biological targets and safety evaluation against

human epithelium cells (HeLa).

(Figure 1)

2. Results and Discussion:

2.1 Chemistry

The general procedure for the synthesis of substituted carbamothioic amine-1-carbothioic

thioanhydrides (3-21, 25-29 and 33-38) has been depicted in scheme 1. Initially the intermediates

sodium substituted amine-1-carbodithioates (2a-2s, 24a-24e and 31a-31f) were synthesized by

the earlier reported methods.[28, 33] In general, various secondary amines (1a-1s) on reaction

with carbon disulfide and aq. sodium hydroxide yield corresponding carbodithioates (2a-2s).

(Scheme 1)

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Further sodium 4-(tert-butoxycarbonyl)piperazine-1-carbodithioate on reaction with different

alkyl halides gave tert-butyl 4-(substitutedthiocarbonothioyl)piperazine-1-carboxylate (22a-22e).

Furthermore Boc removal followed by reaction with carbon disulfide and aq. NaOH yielded

sodium 4-(substituted thiocarbonothioyl)piperazine-1-carbodithioates (24a-24e). Piperazine on

reaction with different substituted isothiocyanates/isocyanates in ethanol yielded different

urea/thiourea derivatives (31a-31f) which on reaction with carbon disulfide and aq. NaOH gave

sodium 4-(substituted carbomothioyl)piperazine-1-carbodithioates (32a-32f). Further

intermediate compounds 2a-2s, 24a-24e and 31a-31f were on reaction with

o

dimethylcarbamothioic chloride in acetonitrile at 0 C for 1-2 hr yielded the final desired

products (3-21, 25-29 and 33-38) in good yields.

2.2 Biological Evaluation

2.2.1 Anti-Trichomonas activity of synthesized compounds

All the synthesized compounds (3-21, 25-29 and 33-38) were evaluated for their anti-

Trichomonas activity against MTZ-susceptible, -resistant strains and safety against cervico

epithelium cells (HeLa). All the compounds were found to be active at MIC range of 4.77–

294.11 µM and 32.46–735.20 µM against MTZ-susceptible and -resistant strains, respectively,

where MTZ showed activity at MIC 18.24 and 365.40 µM. Among these four compounds (12,

20, 21 and 34) were 1 to 3.8 fold more active against -susceptible strain while twenty-six

compounds (3-5, 7-15, 17-21, 26, 28, 29, and 33-38) were 1.3 to 11.2 fold more active against -

resistant strain than the standard MTZ. The most active compound (20) was 3.8 and 9.5 fold

more active than the MTZ against two strains tested. It was also interesting to note that while the

standard drug MTZ was twenty folds less active in resistant strain than in susceptible, compound

20 was only 8-fold less active. This could be attributed to the entirely new drug design.

Structure activity relationship (SAR) study: The scaffold studied was substituted carbamothioic

amine-1-carbothioic thioanhydrides (3-21, 25-29 and 33-38), which contained four prototypes by

varying substituents at -NR¹R². Firstly, a single nitrogen amines with cyclic (3-7) and acyclic (8-

10) framework were synthesized and then a two nitrogen system i.e., substituted piperazines (11-

21) were incorporated at -NR¹R². Thereafter a dithiocarbamate (25-29) and a thiourea (33-38)

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group were integrated at N⁴-position of piperazine core. The anti-Trichomonas activity data

revealed that amine substituents -NR¹R²played vital role for the biological activity.

(Table 1) (Figure 2)

Cyclic (3-7) and acyclic (8-10) amines at -NR¹R²showed good to moderate activity (MIC 25.20-

112.61 µM) against -susceptible strain while against -resistant strain all the compounds except

compound 6 showed 1.5 to 3.8 fold better activity than MTZ irrespective of cyclic or acyclic

amines. The replacement of single nitrogen amines by a two nitrogen containing piperazines the

compounds exhibited better activity profile. Of the eleven compounds (11-21) four compounds

were comparable (12, 13 and 21) or more active (20) than MTZ at MIC 4.77-20.42 µM. The

activity data suggested that a 2-pyrimidyl (20) substituent at N⁴-position was more desirable over

alkyl (11, 13)/aryl (15-19)/carboxy (12, 14)/furoyl (21) substitutents. Against –resistant strain

except compound 16 remaining ten compounds were 1.3 to 9.5 times more active than MTZ.

Here again 2-pyrimidyl (20) substituent at N⁴-position was essential for resistant anti-

Tichomonas activity. The order of activity with substituents -NR¹R²was 2-pyrimidyl (20)>Boc

(12)>phenyl (15)> n-butyl (13)>methyl (11)>3-CF₃phenyl (17)>2,3-dichlorophenyl (19)>furoyl

(21)>ethoxycarbonyl (14)>3-cholorophenyl (18)>2-methoxyphenyl (16). In the scaffold

carbamothioic amine-1-carbothioic thioanhydride attempts were made to hybridize active

pharmacophores, dithiocarbamate (25-29) and thiourea (33-37) moieties at N⁴-position of

piperazine. Against MTZ-susceptible strain the incorporation of thiourea moiety was more

beneficial over dithiocarbamate as most active compound i.e., with phenyl thiourea (38, MIC

8.10 µM) at N⁴-position was 2.2 fold more active and rest of the compounds were less active

than standard MTZ. Again the same pattern of activity was observed with resistant strain as

compound 34 was 11.4 fold more active than MTZ. The order of activity among dithiocarbamate

containing compounds was benzyl (29)>butyronitrile (28)>ethyl (26)>butyl (27)>methyl (25)

while in thiourea derivatives it was phenyl (34)>cyclohexyl (37)>butyl (33)>phenethyl

(36)>benzyl (35). Furthermore a modification of thiourea moiety with urea moiety (38) resulted

in loss of activity in both the strains tested suggests it least favorable for desired activity.

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2.2.2 Antifungal activity of synthesized compounds

All the synthesized compounds (3-21, 25-29 and 33-38) were also evaluated for antifungal

activity against ten fungal strains including three opportunistic fungal strains and seven Candida

strains. The activity data showed that twelve compounds (3-7, 9-13, 20 and 21) were active

against all the ten fungal strains while remaining compounds inhibited the growth of at least two

fungal strains tested in the range of 7.50-240.38 µM. The amine substituents at -NR¹R²played a

significant role for antifungal activity as like in case of anti-Trichomonas activity. Substitutions

at -NR¹R²with cyclic/acyclic secondary amines (3-11) were found to be active at MIC 11.90-

240.38 µM where various substituted piperazine compounds (12-21) showed activity in a range

of MIC 8.66-163.39 µM. Further a DTC incorporated piperazine compounds (25-29) have shown

activity at MIC 7.50-147.05 µM while thiourea/urea hybridized compounds (33-38) exhibited

fungicidal activity at MIC 125.31-136.98 µM except compound 36 which was inactive in all the

ten strains tested. These fungicidal results suggested that in the prototype where DTC was

incorporated in piperazine core at -NR¹R²was more favorable over other three prototypes. It was

interesting to note that except one compound (36), all the remaining twenty nine compounds

were found to be active against at least two opportunistic fungal starins; cryptococcus

neoformans, sporothrix schenckii, trichophyton mentagrophytes at MIC 15.86-240.38 µM. One

compound 7 against Candida glabrata and four compounds (18, 26, 27 and 29) against Candida

parapsilosis had activity comparable to standard Flucanazole. Many volvovaginal infections

were associated with Candida infections which coheres to vaginal epithelium cells enhances the

transmission of STIs. Among compounds evaluated, twelve (3-7, 9-13, 20 and 21) have shown

activity against all the candida strains tested. Rest of the compounds except compounds 28 and

34-36 were active against at least one out of seven different candida strains evaluated.

(Table 2)

Since the compounds exhibited good activity against the protozoan STD-pathogen (T. vaginalis).

We also tested some of the promising structures (12, 20, 21, 34) against P. falciparum to observe

if the effects are universal to all kinds of protozoa or specific only to Trichomonas.

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These compounds were less active than standard antimalarial chloroquine (Supplementary Data

Table 1).

2.2.3 Thiol Inhibition by Active Compound 20

:

The lack of glutathione makes T. vaginalis to depend on cysteine to overcome redox stress,

making very susceptible to sulfhydryl manipulating agents.[22] The DTC compounds were

known to be sulfhydryl binding agents. With the presence of DTC in the most active compound

20 it was evaluated for its sulfhydryl binding ability. The inhibition of sulfhydryl groups over

Trichomonas vaginalis were localized qualitatively by fluorescence detection after labeling them

with mBBr dye. Motile Trichomonas vaginalis (control) and compound 20 treated Trichomonas

were digitally imaged for qualitative assessment. From figure 2, a significant inhibition of free

thiol fluorescence was observed with compound 20 treatment. In control group due to the

presence of higher number of free sulfhydryl groups the fluorescence intensities was higher than

in compound 20 treated groups. The decreased fluorescence indicates the interaction of

compound 20 with the sulfhydryls present over Trichomonas, which might be its

trichomonacidal mechanism.

(Figure 3)

2.2.4 Docking study

Further a docking study was also carried out with most active compound (20) to ascertain their

cysteine biosynthesis pathway. Cysteine is vital for all living life forms for protein synthesis, as a

precursor for glutathione and biomolecules also as a source for synthesis of iron-sulfur clusters.

This series of compounds possibly exhibit anti-Trichomonas activity by inhibiting the cysteine

biosynthesis pathway. To acquire knowledge about the binding mode of the most promising

compound 20 found in this study, we have performed the docking study with the homology

model of Cysteine Synthase (CS). The validation of the resulting model was done with the

Structural Analysis and Verification Server (SAVS) [42]. In the selected model the majority of

the residues (97%) occupy the most favourable region of Ramachandran plot and 2% and 1%

residue lie in additionally allowed region and generously allowed region [31]. This model was

used for docking studies. The Pyridoxal-5'-phosphate (PLP) binding site was taken for the

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docking of the most active compound. It was observed that compound 20 docked well in this

cavity with binding energy of -8.40 kcal/mol. The binding mode of docked complex is shown in

Figure 4. It was found that compound 20 is involved in forming a hydrogen bond with

catalytically important residue Lys43, which is supposed to interact covalently with PLP. The

docked complex was further stabilized by interactions with residues Gly178, Thr182, Thr179 and

Ser180 present in the active site.

(Figure 4)

2.2.5 In vivo anti-Trichomonas activity of compound 20:

Due to its better in vitro activity and safety against HeLa cells, compound 20 was further

screened for in vivo anti-Trichomonas activity. The in vivo efficacy of compound was evaluated

using the mouse abscess assay. Subcutaneous injection of live trichomonads resulted in a small

pustule of ~50 mm²area on day-7 of injection (day-1 of treatment) in experimental and control

animals that grew to ~96.55 mm²in area in controls but was reduced to ~1-2.5 mm²after 5-days

of treatment with compound and MTZ (Figure 4). Thereafter the growth of abscess was

exponential in controls and it was >100 mm²in area after 7 days. However, it was reduced to

~0.8–2.5 mm²in compound-20 treated animals. On the day of autopsy (i.e. the day following

seven days of treatment), the abscess area measured ~96.5 mm²in control animals and 2.35 mm²

in 50 mg of MTZ, 1.57 and 0.79 mm²in 50 mg and 100 mg of compound-20 treated animals,

respectively.

(Figure 5)

2.2.6 Pharmacokinetics study

A preliminary pharmacokinetic study of compound 20 involved monitoring drug substance in

blood plasma. The animals tolerated the treatment well as no peculiarities in the animals’

behaviour were observed. Following 50 mg/kg oral dose of compound 20, peak serum

concentration C_max= 47.1±2.8 ng/mL was achieved at 2 hr and could be monitored in serum up to

6 hr post dose. The concentration-time profile was analyzed using non-compartmental approach

and the calculated pharmacokinetic parameters are shown in Table 3. The pharmacokinetic

models were compared according to maximal correlation between observed and predicted

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concentration, minimal sum of squared residuals, Akaike’s Information Criterion (AIC) and

Schwarz Bayesian Criterion (SBC) [34, 35]. The volume of distribution (12.6±0.6 L/kg) was

larger than the total blood volume of rat (0.054 L/kg; [36]) and systemic clearance (4.9±0.2

L/h/kg) was higher than the total hepatic blood flow in rats (2.9 L/h/kg; [36]) indicating

extravascular distribution along with the extra hepatic elimination of the compound.

(Table 3)

3. Conclusion

Sexually transmitted diseases like trichomoniasis along with opportunistic fungal infections like

candidiasis are major global concern in female reproductive health now days. The increased drug

resistance to available 5-nitroimidazole drugs i.e., Metronidazole, Tinidazole necessitates the

researchers for new drug development. Developing non-nitro imidazole molecules to overcome

the drug resistance would be a useful strategy. In the present study we developed a novel series

of non-nitroimidazole molecules and evaluated their trichomonicidal and fungicidal efficacies.

All the compounds were found to be trichomonicidal at MIC range of 4.77–294.11 µM and

32.46–735.20 µM against MTZ-susceptible and -resistant strains, respectively. MTZ showed

activity at MIC of 18.24 and 365.40 µM respectively against the strains. Four compounds (12,

20, 21 and 34) were up to 3.8 fold more active against -susceptible strain and twenty-six

compounds (3-5, 7-15, 17-21, 26, 28, 29 and 33-38) were up to 11.2 fold more active against

resistant strain than the standard MTZ. The most active compound (20) was 3.8 and 9.5 fold

more active than MTZ against susceptible and resistant strains, respectively. The absence of nitro

group in this novel series of compounds could be the reason for the activity of compounds

against resistant trichomoniasis. Twenty-nine compounds were found to be active against at least

two opportunistic fungal strains at MIC 15.86-240.38 µM. Many vulvovaginal infections are

associated with Candida infections, which cohere to vaginal epithelium cells and enhance the

transmission of STIs. Among the compounds evaluated twelve (3-7, 9-13, 20 and 21) have

shown activity against all the ten candida strains tested at MIC 7.50-200.00 µM. Compound 20

significantly inhibited the free sulfhydryl groups presented over Trichomonas in vitro, and

demonstrated significant elimination of infection in in vivo assay at doses 50 mg/kg and 100

mg/kg compound. Most active compounds also have shown moderate antimalarial activity. A

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preliminary pharmacokinetic study has shown good distribution, systemic clearance of

compound 20 in SD-rats. The present study has identified promising structures that could

conquer the drug-resistance in Trichomonas and their further lead optimization may lead to the

identification of more effective non-nitro imidazole class of anti-microbial compounds.

Experimental Section

Chemistry

In general, all reagents and solvents were of commercial quality and were used without

further purification. 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 uncorrected. IR spectra (ν_max

in cm^-1) of the compounds were recorded on Perkin Elmer FT-IR RX1 PC

1

spectrophotometer. H NMR spectra were recorded on Bruker Supercon Magnet Avance

1

DPX-200/DRX-300 spectrometers (operating at 400 and 100 MHz, respectively, for H

and ¹³C) in deuterated solvents with TMS as internal reference (chemical shifts

in Hz.). Electrospray ionization mass spectra (ESI-MS) were recorded on Ion Trap LCQ

δ in ppm,

J

Advantage Max-IT (Thermo Electron Corporation). High-resolution mass spectra

(HRMS) were recorded on a 6520 Agilent Q Tof LC MS/MS (accurate mass). Elemental

analyses were performed on a Carlo Erba EA-1108 micro analyzer/Vario EL-III C, H, N

analyzer. All compounds were analyzed of C, H, N and the results obtained were within ±

0.4% of calculated values. All final compounds were found to have >95% purity.

Intermediate compounds 2a-2s, 24a-24e and 31a-31f Compounds were synthesized

according to a previously reported procedure.[28, 33]

General procedure for the synthesis of dimethylcarbamothioic pyrrolidine-1-

carbothioic thioanhydride (3):

To a solution of sodium pyrrolidine-1-carbodithioate (2a; 2 equiv.) in 10 mL acetonitrile

o

at 0-5 C was added dimethylcarbamothioic chloride (1 equiv.) and stirred the contents

for 1-2 hr. After completion of reaction (as monitored by TLC) acetonitrile was

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evaporated and excess distilled water (3 x 30 mL), 20 mL of EtOAc were added to the

reaction mixture. Organic layer was separated, dried (over Na₂SO₄) and concentrated

under reduced pressure to get the title compound

81 °C; IR (KBr)

(cm^-1): 3019, 1218; ¹H NMR (400 MHz, CDCl₃):

2H), 3.73 (t,

MHz, CDCl₃):

3 in 79% yields as yellow solid. mp: 79-

ν

δ

3.89 (t,

J

= 7.0 Hz,

13

J

= 6.8 Hz, 2H), 3.54 (s, 3H), 3.47 (s, 3H), 2.12–2.00 (m, 4H); C (100

δ

186.9, 183.2, 55.0, 52.8, 45.0, 44.3, 26.3, 24.7; ESI-MS: m/z 235

(M+H⁺); HRMS (ESI): m/z calculated for C₈H₁₄N₂S₃+ H⁺(M+H⁺): 235.0392. Found:

235.0389. Elemental analysis (%) for C₈H₁₄N₂S₃: Calcd.: C, 40.99; H, 6.02; N, 11.95;

Found, C, 40.63; H, 6.38; N, 11.79.

The compounds 4-21, 25-29 and 33-38 were synthesized by using a procedure similar to

described for compound 3.

Dimethylcarbamothioic piperidine-1-carbothioic thioanhydride (4)

The title compound (4) was synthesized from sodium piperidine-1-carbodithioate (2b; 2

equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 71% yield as yellow solid. mp:

1

90-92 °C; IR (KBr)

ν

(cm^-1): 3019, 1214; H NMR (400 MHz, CDCl₃):

δ

8.02 (s, 1H),

4.26 (bs, 2H), 3.89 (bs, 2H), 3.53 (s, 3H), 3.44 (s, 3H), 1.74–1.61 (m, 6H); ¹³C (100 MHz,

CDCl₃): 187.6, 185.7, 54.0, 52.2, 44.9, 44.0, 26.3, 25.2, 23.8; ESI-MS: m/z 249

δ

(M+H⁺); HRMS (ESI): m/z calculated for C₉H₁₆N₂S₃+ H⁺(M+H⁺): 249.0548. Found:

249.0549. Elemental analysis (%) for C₉H₁₆N₂S₃: Calcd.: C, 43.51; H, 6.49; N, 11.28;

Found, C, 43.15; H, 6.76; N, 11.39.

Dimethylcarbamothioic 3-methylpiperidine-1-carbothioic thioanhydride (5)

The title compound (5) was synthesized from sodium 3-methylpiperidine-1-

carbodithioate (2c; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 82% yield

1

as yellow solid. mp: 95-97 °C; IR (KBr)

CDCl₃):

2.83 (m, 1H), 1.91–1.83 (m, 3H), 1.72–1.65 (m, 2H), 0.98 (bs, 3H); C (100 MHz,

ν

(cm^-1): 3021, 1216; H NMR (400 MHz,

δ

5.24 (bs, 1H), 4.42–4.30 (m, 1H), 3.54–3.44 (m, 6H), 3.27–3.19 (m, 1H), 3.01–

13

CDCl₃):

δ

187.3, 185.8, 59.8, 58.0, 53.5, 51.8, 44.9, 44.0, 32.4, 32.0, 31.1, 25.7, 24.4,

11

ACCEPTED MANUSCRIPT

18.7; ESI-MS: m/z 263 (M+H⁺); HRMS (ESI): m/z calculated for C₁₀H₁₈N₂S₃+ H⁺

(M+H⁺): 263.0705. Found: 263.0724. Elemental analysis (%) for C₁₀H₁₈N₂S₃: Calcd.: C,

45.76; H, 6.91; N, 10.67; Found, C, 45.99; H, 7.21; N, 10.80.

Dimethylcarbamothioic morpholine-4-carbothioic thioanhydride (6)

The title compound (6) was synthesized from sodium morpholine-4-carbodithioate (2d; 2

equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 78% yield as yellow solid. mp:

1

128-130 °C; IR (KBr)

ν

(cm^-1): 3019, 1215, 1104; H NMR (400 MHz, CDCl₃):

δ

4.31–

13

3.99 (m, 4H), 3.83 (bs, 4H), 3.52 (s, 3H), 3.42 (s, 3H); C (100 MHz, CDCl₃):

δ

187.5,

186.5, 66.1, 52.9, 50.9, 44.9, 43.6; ESI-MS: m/z 251 (M+H⁺); HRMS (ESI): m/z

calculated for C₈H₁₄N₂OS₃+ H⁺(M+H⁺): 251.0341. Found: 251.0334. Elemental analysis

(%) for C₈H₁₄N₂OS₃: Calcd.: C, 38.37; H, 5.64; N, 11.19; Found, C, 38.01; H, 5.98; N,

11.32.

Azepane-1-carbothioic dimethylcarbamothioic thioanhydride (7)

The title compound (

equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 85% yield as yellow solid. mp:

75-77 °C; IR (KBr) 4.14 (t, = 6.0

(cm^-1): 3010, 1216; ¹H NMR (400 MHz, CDCl₃):

= 6.0 Hz, 2H), 3.54 (s, 3H), 3.47 (s, 3H), 1.91–1.84 (m, 4H), 1.64–1.61

187.2, 187.0, 55.0, 54.8, 45.0, 44.5, 28.0, 26.9, 26.4,

7) was synthesized from sodium azepane-1-carbodithioate (2e; 2

ν

δ

J

Hz, 2H), 3.87 (t,

J

13

(m, 4H); C (100 MHz, CDCl₃):

δ

25.8; ESI-MS: m/z 263 (M+H⁺); HRMS (ESI): m/z calculated for C₁₀H₁₈N₂S₃+ H⁺

(M+H⁺): 263.0705. Found: 263.0702. Elemental analysis (%) for C₁₀H₁₈N₂S₃: Calcd.: C,

45.76; H, 6.91; N, 10.67; Found, C, 45.45; H, 7.22; N, 10.49.

Diethyl-1-carbothioic dimethylcarbamothioic thioanhydride (8)

The title compound (8) was synthesized from sodium diethylcarbamodithioate (2f; 2

equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp:

1

84-86 °C; IR (KBr)

ν

(cm^-1): 3020, 1211; H NMR (400 MHz, CDCl₃):

δ

4.02–3.97 (m,

13

2H), 3.76–3.68 (m, 2H), 3.53 (s, 3H), 3.45 (s, 3H), 1.35–1.29 (m, 6H); C (100 MHz,

CDCl₃):

δ

187.1, 186.5, 49.0, 48.9, 45.0, 44.4, 13.2, 11.1; ESI-MS: m/z 237 (M+H⁺);

12

ACCEPTED MANUSCRIPT

Elemental analysis (%) for C₈H₁₆N₂S₃₂: Calcd.: C, 40.64; H, 6.82; N, 11.85; Found, C,

40.50; H, 6.99; N, 11.61.

Dimethyl-1-carbothioic thioanhydride (9)

The title compound (9) was synthesized from sodium dimethylcarbamodithioate (2g; 2

equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 68% yield as yellow solid. mp:

1

89-91 °C; IR (KBr)

ν

(cm^-1): 3019, 1218; H NMR (400 MHz, CDCl₃):

δ

3.53 (s, 6H),

13

3.44 (s, 6H); C (100 MHz, CDCl₃):

δ

187.3, 44.9, 44.0; ESI-MS: m/z 209 (M+H⁺);

HRMS (ESI): m/z calculated for C₆H₁₂N₂S₃+ H⁺(M+H⁺): 209.0241. Found: 209.0282.

Elemental analysis (%) for C₆H₁₂N₂S₃: Calcd.: C, 34.59; H, 5.80; N, 13.44; Found, C,

34.37; H, 5.99; N, 13.56.

Dimethylcarbamothioic ethyl(methyl)-4-carbothioic thioanhydride (10)

The title compound (10) was synthesized from sodium ethyl(methyl)carbamodithioate

(

2h; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 84% yield as yellow

solid. mp: 85-87 °C; IR (KBr) 4.12–

(cm^-1): 3010, 1210; ¹H NMR (400 MHz, CDCl₃):

4.03 (m, 1H), 3.82–3.77 (m, 1H), 3.55–3.37 (m, 9H), 1.31 (t,

MHz, CDCl₃): 187.3, 187.1, 51.8, 51.5, 44.9, 44.0, 42.4, 41.1, 12.9, 10.7; ESI-MS: m/z

ν

δ

13

J

= 7.1 Hz, 3H); C (100

δ

223 (M+H⁺); Elemental analysis (%) for C₇H₁₄N₂S₃: Calcd.: C, 37.80; H, 6.35; N, 12.60;

Found, C, 37.54; H, 6.70; N, 12.84.

Dimethylcarbamothioic 4-methylpiperazine-1-carbothioic thioanhydride (11)

The title compound (11) was synthesized from sodium 4-methylpiperazine-1-

carbodithioate (2i; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 88% yield

as yellow solid. mp: 70-72 °C; IR (KBr)

CDCl₃):

(100 MHz, CDCl₃):

ν

(cm^-1): 3011, 1218, 1167; ¹H NMR (400 MHz,

13

δ

4.34–3.98 (m, 4H), 3.53 (s, 3H), 3.44 (s, 3H), 2.58 (s, 4H), 2.36 (s, 3H); C

δ

187.4, 186.7, 54.4, 53.9, 52.2, 50.5, 45.3, 44.9, 43.8; ESI-MS: m/z

264 (M+H⁺); HRMS (ESI): m/z calculated for C₉H₁₇N₃S₃+ H⁺(M+H⁺): 264.0657. Found:

264.0656. Elemental analysis (%) for C₉H₁₇N₃S₃: Calcd.: C, 41.03; H, 6.50; N, 15.95;

Found, C, 41.25; H, 6.76; N, 15.59.

13

ACCEPTED MANUSCRIPT

4-(tert-Butoxycarbonyl)piperazine-1-carbothioic

thioanhydride (12)

The title compound

butoxycarbonyl)piperazine-1-carbodithioate (2j; 2 equiv.) and dimethylcarbamothioic

dimethylcarbamothioic

(12

)

was

synthesized

from

sodium

4-(tert

-

chloride (1 equiv.) in 85% yield as yellow solid. mp: 201-203 °C; IR (KBr)

ν

(cm^-1):

1

3010, 1730, 1216, 1020; H NMR (400 MHz, CDCl₃):

δ

4.29–3.97 (m, 4H), 3.64–3.61

187.5, 186.7,

13

(m, 4H), 3.53 (s, 3H), 3.42 (s, 3H), 1.49 (s, 9H); C (100 MHz, CDCl₃):

δ

154.3, 80.6, 52.1, 50.6, 44.9, 43.6, 42.7, 28.3; ESI-MS: m/z 350 (M+H⁺); HRMS (ESI):

m/z calculated for C₁₃H₂₃N₃O₂S₃+ H⁺(M+H⁺): 350.1025. Found: 350.1029. Elemental

analysis (%) for C₁₃H₂₃N₃O₂S₃: Calcd.: C, 44.67; H, 6.63; N, 12.02; Found, C, 44.39; H,

6.89; N, 12.15.

4-Butylpiperazine-1-carbothioic dimethylcarbamothioic thioanhydride (13)

The title compound (13) was synthesized from sodium 4-butylpiperazine-1-carbodithioate

(

2k; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 77% yield as yellow

1

solid. mp: 102-104 °C; IR (KBr)

ν

(cm^-1): 3015, 1215, 1165; H NMR (400 MHz,

CDCl₃):

δ

4.32–3.96 (m, 4H), 3.53 (s, 3H), 3.43 (s, 3H), 2.59 (bs, 4H), 2.42–2.38 (m,

13

2H), 1.53–1.45 (m, 2H), 1.40–1.33 (m, 2H), 0.94 (t,

J = 7.3 Hz, 3H); C (100 MHz,

CDCl₃):

δ

187.4, 186.4, 57.6, 52.7, 52.1, 50.6, 44.9, 43.8, 28.9, 20.5, 13.9; ESI-MS: m/z

306 (M+H⁺); Elemental analysis (%) for C₁₂H₂₃N₃S₃: Calcd.: C, 47.17; H, 7.59; N, 13.75;

Found, C, 47.00; H, 7.86; N, 13.57.

4-(Ethoxycarbonyl) piperazine-1-carbothioic dimethylcarbamothioic thioanhydride

(14)

The title compound (14) was synthesized from sodium 4-(ethoxycarbonyl)piperazine-1-

carbodithioate (2l; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 80% yield

1

as yellow solid. mp: 112-114 °C; IR (KBr)

ν

(cm^-1): 3011, 1736, 1216; H NMR (400

MHz, CDCl₃):

δ

4.25–3.99 (m, 6H), 3.67–3.64 (m, 4H), 3.50 (s, 3H), 3.40 (s, 3H), 1.27 (t,

13

J

= 7.1 Hz, 3H); C (100 MHz, CDCl₃):

δ

187.5, 186.8, 155.5, 61.8, 51.9, 50.6, 44.9,

14

ACCEPTED MANUSCRIPT

43.6, 42.9, 14.6; ESI-MS: m/z 322 (M+H⁺); Elemental analysis (%) for C₁₁H₁₉N₃O₂S₃:

Calcd.: C, 41.10; H, 5.96; N, 13.07; Found, C, 41.00; H, 6.06; N, 13.10.

Dimethylcarbamothioic 4-phenylpiperazine-1-carbothioic thioanhydride (15)

The title compound (15) was synthesized from sodium 4-phenylpiperazine-1-

carbodithioate (2m; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 79% yield

as yellow solid. mp: 88-90 °C; IR (KBr)

ν

(cm^-1): 3410, 1427, 1215, 1150; ¹H NMR (400

MHz, CDCl₃):

δ

7.33–7.29 (m, 2H), 6.95–6.92 (m, 3H), 4.46–4.11 (m, 4H), 3.54 (s, 3H),

13

3.45 (s, 3H), 3.39–3.37 (m, 4H); C (100 MHz, CDCl₃):

δ 187.5, 186.5, 150.1, 129.3,

120.6, 116.3, 50.6, 49.1, 48.4, 44.9, 43.8; ESI-MS: m/z 326 (M+H⁺); HRMS (ESI): m/z

calculated for C₁₄H₁₉N₃S₃+ H⁺(M+H⁺): 326.0814. Found: 326.0820. Elemental analysis

(%) for C₁₄H₁₉N₃S₃: Calcd.: C, 51.66; H, 5.88; N, 12.91; Found, C, 51.59; H, 6.11; N,

12.83.

4-(2-Methoxyphenyl)piperazine-1-carbothioic dimethylcarbamothioic thioanhydride

(16)

The title compound (16) was synthesized from sodium 4-(2-methoxyphenyl)piperazine-1-

carbodithioate (2n; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 81% yield

1

as yellow solid. mp: 170-172 °C; IR (KBr)

ν

(cm^-1): 2925, 1468, 1225, 1026; H NMR

(400 MHz, CDCl₃):

δ

7.09–7.05 (m, 1H), 6.98–6.91 (m, 3H), 4.51 (bs, 2H), 4.17–4.11 (m,

13

2H), 3.90 (s, 3H), 3.55–3.46 (m, 6H), 3.23 (s, 4H); C (100 MHz, CDCl₃):

δ 187.5,

186.5, 152.2, 139.9, 123.8, 121.1, 118.5, 111.4, 55.4, 52.8, 50.9, 50.4, 49.8, 44.9, 43.8;

ESI-MS: m/z 356 (M+H⁺); HRMS (ESI): m/z calculated for C₁₅H₂₁N₃OS₃+ H⁺(M+H⁺):

356.0920. Found: 356.0919. Elemental analysis (%) for C₁₅H₂₁N₃OS₃: Calcd.: C, 50.67;

H, 5.95; N, 11.82; Found, C, 50.50; H, 6.15; N, 11.78.

4-(2-(Trifluoromethyl)phenyl)piperazine-1-carbothioic

thioanhydride (17)

dimethylcarbamothioic

The

title

compound

(

17

)

was

synthesized

2o

from

2

sodium

equiv.)

4-(2-

and

(trifluoromethyl)phenyl)piperazine-1-carbodithioate

(

;

dimethylcarbamothioic chloride (1 equiv.) in 75% yield as yellow solid. mp: 95-97 °C; IR

15

ACCEPTED MANUSCRIPT

1

(KBr)

ν

(cm^-1): 3020, 1429, 1215, 1028; H NMR (400 MHz, CDCl₃):

δ

7.40 (t,

J

= 7.9

13

Hz, 1H), 7.16–7.06 (m, 3H), 4.48–4.16 (m, 4H), 3.54 (s, 3H), 3.46–3.45 (m, 7H); C

(100 MHz, CDCl₃):

δ 187.5, 186.6, 150.2, 132.0, 131.7, 131.4, 131.0, 129.8, 125.5,

122.8, 118.8, 116.5, 116.5, 112.1, 112.1, 51.9, 50.4, 48.1, 44.9, 43.7; ESI-MS: m/z 394

(M+H⁺); HRMS (ESI): m/z calculated for C₁₅H₁₈F₃N₃S₃+ H⁺(M+H⁺): 394.0688. Found:

394.0692. Elemental analysis (%) for C₁₅H₁₈F₃N₃S₃: Calcd.: C, 45.78; H, 4.61; N, 10.68;

Found, C, 45.47; H, 4.90; N, 10.50.

4-(3-Chlorophenyl)piperazine-1-carbothioic dimethylcarbamothioic thioanhydride

(18)

The title compound (18) was synthesized from sodium 4-(2-chlorophenyl)piperazine-1-

carbodithioate (2p; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 73% yield

1

as yellow solid. mp: 90-92 °C; IR (KBr)

ν

(cm^-1): 3021, 1215, 1152, 758; H NMR (400

MHz, CDCl₃):

δ

7.23–7.19 (m, 1H), 6.90–6.87 (m, 2H), 6.82–6.77 (m, 1H), 4.48–4.43

13

(m, 2H), 4.18–4.11 (m, 2H), 3.54 (s, 3H), 3.44 (m, 3H), 3.41–3.38 (m, 4H); C (100

MHz, CDCl₃):

δ 187.5, 186.6, 151.0, 135.1, 130.3, 120.1, 115.9, 113.9, 51.1, 48.6, 48.5,

44.9, 43.7; ESI-MS: m/z 360 (M+H⁺); HRMS (ESI): m/z calculated for C₁₄H₁₈ClN₃S₃+

H⁺(M+H⁺): 360.0424. Found: 360.0424. Elemental analysis (%) for C₁₄H₁₈ClN₃S₃₂:

Calcd.: C, 46.71; H, 5.04; N, 11.67; Found, C, 46.90; H, 5.37; N, 11.50.

4-(2,3-Dichlorophenyl)piperazine-1-carbothioic

thioanhydride (19)

dimethylcarbamothioic

The title compound (19) was synthesized from sodium 4-(2,3-dichlorophenyl)piperazine-

1-carbodithioate (2q; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 80%

1

yield as yellow solid. mp: 235-237 °C; IR (KBr)

NMR (400 MHz, CDCl₃): 7.25–7.17 (m, 2H), 6.96 (d/d,

4.51 (m, 2H), 4.18–4.11 (m, 2H), 3.55 (s, 3H), 3.45 (m, 3H), 3.22–3.20 (m, 4H); ¹³C (100

MHz, CDCl₃): 187.5, 186.8, 149.9, 134.2, 127.7, 127.6, 125.5, 118.8, 52.8, 51.0, 50.4,

ν

(cm^-1): 3019, 1423, 1215, 757; H

δ

J = 1.7 & 7.8 Hz, 1H), 4.52–

δ

44.9, 43.7; ESI-MS: m/z 394 (M+H⁺); HRMS (ESI): m/z calculated for C₁₄H₁₇Cl₂N₃S₃+

H⁺(M+H⁺): 394.0034. Found: 394.0035. Elemental analysis (%) for C₁₄H₁₇Cl₂N₃S₃:

Calcd.: C, 42.63; H, 4.34; N, 10.65; Found, C, 42.79; H, 4.60; N, 10.39.

16

ACCEPTED MANUSCRIPT

Dimethylcarbamothioic 4-(pyrimidin-2-yl)piperazine-1-carbothioic thioanhydride

(20)

The title compound (20) was synthesized from sodium 4-(2,3-dichlorophenyl)piperazine-

1-carbodithioate (2r; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 84%

1

yield as yellow solid. mp: 180-182 °C; IR (KBr)

NMR (400 MHz, CDCl₃): 8.36 (d, = 4.7 Hz, 2H), 6.59 (t,

(m, 8H), 3.54 (s, 3H), 3.45 (m, 3H); ¹³C (100 MHz, CDCl₃):

ν

(cm^-1): 3019, 1500, 1427, 1215; H

δ

J

= 4.7 Hz, 1H), 4.39–4.03

δ

187.4, 186.6, 161.1, 157.8,

110.7, 52.0, 50.7, 44.9, 43.7, 42.5; ESI-MS: m/z 328 (M+H⁺); HRMS (ESI): m/z

calculated for C₁₂H₁₇N₅S₃+ H⁺(M+H⁺): 328.0719. Found: 328.0718. Elemental analysis

(%) for C₁₂H₁₇N₅S₃: Calcd.: C, 44.01; H, 5.23; N, 21.38; Found, C, 44.30; H, 5.55; N,

21.09.

4-(furan-2-carbonyl)piperazine-1-carbothioic dimethylcarbamothioic thioanhydride

(21)

The title compound (21) was synthesized from sodium 4-(furan-2-yl)piperazine-1-

carbodithioate (2s; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 77% yield

as yellow solid. mp: 84-86 °C; IR (KBr)

ν

(cm^-1): 3017, 1422, 1216, 1090; ¹H NMR (400

MHz, CDCl₃):

δ

7.52 (d,

J

= 0.7 Hz, 1H), 7.13–7.11 (m, 1H), 6.54–6.52 (m, 1H), 4.36–

13

4.03 (m, 8H), 3.53 (s, 3H), 3.43 (m, 3H); C (100 MHz, CDCl₃):

δ 187.6, 186.8, 159.0,

147.4, 144.2, 117.5, 111.6, 51.8, 50.7, 45.6, 44.9, 43.5; ESI-MS: m/z 418 (M+H⁺);

Elemental analysis (%) for C₁₃H₁₇N₃O₂S₃: Calcd.: C, 45.46; H, 4.99; N, 12.23; Found, C,

45.29; H, 5.33; N, 12.00.

Dimethylcarbamothioic

thioanhydride (25)

4-(methylthiocarbonothioyl)piperazine-1-carbothioic

The

title

compound

(

25

)

was

synthesized

24a

from

2

sodium

equiv.)

4-

(methylthiocarbonothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 77% yield as yellow solid. mp: 235-237 °C;

IR (KBr)

ν

(cm^-1): 3022, 1215; ¹H NMR (400 MHz, CDCl₃):

δ

4.35–4.11 (m, 8H), 3.53–

17

ACCEPTED MANUSCRIPT

13

3.42 (m, 6H), 2.70 (s, 3H); C (100 MHz, CDCl₃):

δ 199.0, 187.6, 186.5, 50.4, 48.5,

45.0, 43.5, 20.0; ESI-MS: m/z 340 (M+H⁺); Elemental analysis (%) for C₁₀H₁₇N₃S₅:

Calcd.: C, 35.37; H, 5.05; N, 12.37; Found, C, 35.09; H, 5.31; N, 12.12.

4-(Ethylthiocarbonothioyl)piperazine-1-carbothioic

thioanhydride (26)

dimethylcarbamothioic

The

title

compound

(

26

)

was

synthesized

24b

from

2

sodium

equiv.)

4-

(ethylthiocarbonothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp: 98-100 °C;

IR (KBr)

3.53–3.42 (m, 6H), 3.33 (q,

MHz, CDCl₃):

ν

(cm^-1): 3015, 1218, 1150; ¹H NMR (400 MHz, CDCl₃):

= 7.4 & 14.8 Hz, 2H), 1.38 (t, J = 7.4 Hz, 3H); C (100

δ

4.43–4.18 (m, 8H),

13

J

δ

198.1, 187.6, 186.5, 50.3, 48.0, 45.0, 43.5, 31.5, 13.6; ESI-MS: m/z 354

(M+H⁺); Elemental analysis (%) for C₁₁H₁₉N₃S₅: Calcd.: C, 37.36; H, 5.42; N, 11.88;

Found, C, 37.45; H, 5.75; N, 11.70.

4-(Butylthiocarbonothioyl)piperazine-1-carbothioic

thioanhydride (27)

dimethylcarbamothioic

The

title

compound

(

27

)

was

synthesized

24c

from

2

sodium

equiv.)

4-

(butylthiocarbonothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 76% yield as yellow solid. mp: 85-87 °C; IR

1

(KBr)

ν

(cm^-1): 3022, 1220, 1129; H NMR (400 MHz, CDCl₃):

δ

4.34–4.18 (m, 8H),

3.53–3.42 (m, 6H), 3.33 (t,

J

= 7.4 Hz, 2H), 1.75–1.67 (m, 2H), 1.51–1.42 (m, 2H), 0.96

13

(t,

J = 7.3 Hz, 3H); C (100 MHz, CDCl₃): δ 198.4, 187.6, 50.4, 45.0, 43.5, 37.0, 30.5,

22.1, 13.6; ESI-MS: m/z 382 (M+H⁺); HRMS (ESI): m/z calculated for C₁₃H₂₃N₃S₅+ H⁺

(M+H⁺): 382.0568. Found: 382.0567. Elemental analysis (%) for C₁₃H₂₃N₃S₅: Calcd.: C,

40.91; H, 6.07; N, 11.01; Found, C, 40.70; H, 6.30; N, 11.13.

4-((3-cyanopropylthio)carbonothioyl)piperazine-1-carbothioic

dimethylcarbamothioic thioanhydride (28)

The

title

compound

(

28

)

was

synthesized

from

24d

sodium

4-((3-

cyanopropylthio)carbonothioyl)piperazine-1-carbodithioate

(

;

2

equiv.) and

18

ACCEPTED MANUSCRIPT

dimethylcarbamothioic chloride (1 equiv.) in 69% yield as yellow solid. mp: 91-93 °C; IR

1

(KBr)

3.54–3.42 (m, 8H), 2.51 (t,

MHz, CDCl₃):

ν

(cm^-1): 3020, 2231, 1216; H NMR (400 MHz, CDCl₃):

δ

4.37–4.12 (m, 8H),

13

J

= 7.2 Hz, 2H), 2.12 (q, J = 7.1 & 14.2 Hz, 2H); C (100

δ

196.6, 187.6, 187.3, 51.2, 50.1, 45.0, 43.5, 35.0, 28.8, 16.3; ESI-MS: m/z

393 (M+H⁺); Elemental analysis (%) for C₁₃H₂₀N₄S₅: Calcd.: C, 39.77; H, 5.13; N, 14.27;

Found, C, 39.51; H, 5.41; N, 14.12.

4-(Benzylthiocarbonothioyl)piperazine-1-carbothioic

thioanhydride (29)

dimethylcarbamothioic

The

title

compound

(

29

)

was

synthesized

24e

from

2

sodium

equiv.)

4-

(benzylthiocarbonothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 80% yield as yellow solid. mp: 83-85 °C; IR

1

(KBr)

ν

(cm^-1): 3410, 1639, 1442, 1216; H NMR (400 MHz, CDCl₃):

δ

7.40 (d,

J

= 7.5

Hz, 2H), 7.36–7.29 (m, 3H), 4.60 (s, 2H), 4.35–4.13 (m, 8H), 3.54–3.53 (m, 3H), 3.45–

13

3.42 (m, 3H); C (100 MHz, CDCl₃):

δ 197.4, 187.6, 186.5, 135.5, 129.3, 128.6, 127.7,

50.1, 45.0, 43.5, 42.1; ESI-MS: m/z 416 (M+H⁺); HRMS (ESI): m/z calculated for

C₁₆H₂₁N₃S₅+ H⁺(M+H⁺): 416.0412. Found: 416.0412. Elemental analysis (%) for

C₁₆H₂₁N₃S₅: Calcd.: C, 46.23; H, 5.09; N, 10.11; Found, C, 46.01; H, 5.28; N, 10.25.

4-(Butylcarbamothioyl)piperazine-1-carbothioic

thioanhydride (33)

dimethylcarbamothioic

The title compound (33) was synthesized from sodium 4-(butylcarbamothioyl)piperazine-

1-carbodithioate (32a; 2 equiv.) and dimethylcarbamothioic chloride (1 equiv.) in 76%

1

yield as yellow solid. mp: 108-110 °C; IR (KBr)

ν

(cm^-1): 3018, 1216, 1192; H NMR

(400 MHz, CDCl₃):

δ

5.48 (s, 1H), 4.35–4.08 (m, 8H), 3.71–3.66 (m, 2H), 3.53 (s, 3H),

13

3.43 (s, 3H), 1.68–1.60 (m, 2H), 1.46–1.36 (m, 2H), 0.97 (t,

J = 7.3 Hz, 3H); C (100

MHz, CDCl₃):

δ

187.4, 186.5, 181.9, 50.6, 50.0, 46.1, 45.0, 43.6, 31.3, 20.1, 13.8; ESI-

MS: m/z 365 (M+H⁺); HRMS (ESI): m/z calculated for C₁₃H₂₄N₄S₄+ H⁺(M+H⁺):

365.0957. Found: 365.0948. Elemental analysis (%) for C₁₃H₂₄N₄S₄: Calcd.: C, 42.82; H,

6.63; N, 15.37; Found, C, 42.59; H, 6.88; N, 15.09.

19

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Dimethylcarbamothioic

thioanhydride (34)

4-(phenylcarbamothioyl)piperazine-1-carbothioic

The

title

compound

(

34

)

was

synthesized

32b

from

2

sodium

equiv.)

4-

(phenylcarbamothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 83% yield as yellow solid. mp: 82-84 °C; IR

(KBr)

(cm^-1): 3410, 1490, 1216, 1194, 1127; ¹H NMR (400 MHz, CDCl₃+DMSO-d₆):

8.83 (s, 1H), 7.14–7.13 (m, 4H), 6.99 (bs, 1H), 4.15–3.92 (m, 8H), 3.32–3.23 (m, 6H);

¹³C (100 MHz, CDCl₃+DMSO-d₆):

192.1, 191.1, 187.1, 145.3, 133.1, 130.7, 130.1,

ν

δ

55.8, 55.2, 51.9, 49.7, 48.4; ESI-MS: m/z 385 (M+H⁺); HRMS (ESI): m/z calculated for

C₁₅H₂₀N₄S₄+ H⁺(M+H⁺): 385.0644. Found: 385.0635. Elemental analysis (%) for

C₁₅H₂₀N₄S₄: Calcd.: C, 46.84; H, 5.24; N, 14.57; Found, C, 46.50; H, 5.49; N, 14.40.

4-(Benzylcarbamothioyl)piperazine-1-carbothioic

thioanhydride (35)

dimethylcarbamothioic

The

title

compound

(

35

)

was

synthesized

32c

from

2

sodium

equiv.)

4-

(benzylcarbamothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 83% yield as yellow solid. mp: 218-220 °C;

1

IR (KBr)

ν

(cm^-1): 3410, 1639, 1402, 1216, 1185; H NMR (400 MHz, CDCl₃):

δ

7.35–

7.27 (m, 4H), 6.20–6.07 (m, 1H), 4.87–4.86 (m, 2H), 4.27–4.08 (m, 8H), 3.50–3.39 (m,

6H); ¹³C (100 MHz, CDCl₃):

187.4, 186.5, 182.0, 137.6, 128.8, 128.0, 50.2, 46.3, 45.0,

δ

43.6, 30.9; ESI-MS: m/z 399 (M+H⁺); HRMS (ESI): m/z calculated for C₁₆H₂₂N₄S₄+ H⁺

(M+H⁺): 399.0800. Found: 399.0791. Elemental analysis (%) for C₁₆H₂₂N₄S₄: Calcd.: C,

48.21; H, 5.56; N, 14.05; Found, C, 48.02; H, 5.85; N, 14.29.

Dimethylcarbamothioic

thioanhydride (36)

4-(phenethylcarbamothioyl)piperazine-1-carbothioic

The

title

compound

(

36

)

was

synthesized

32d

from

2

sodium

equiv.)

4-

(phenethylcarbamothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp: 85-87 °C; IR

(KBr) 7.36–7.31

(cm^-1): 3410, 1639, 1402, 1216, 1185; ¹H NMR (400 MHz, CDCl₃):

ν

δ

(m, 2H), 7.26–7.22 (m, 3H), 5.54–5.53 (m, 1H), 4.38–3.93 (m, 10H), 3.52–3.41 (m, 6H),

20

ACCEPTED MANUSCRIPT

13

3.00–2.96 (m, 2H); C (100 MHz, CDCl₃):

δ 187.4, 186.5, 181.8, 138.7, 128.7, 126.7,

50.6, 50.0, 47.0, 45.9, 45.0, 43.7, 35.1; ESI-MS: m/z 413 (M+H⁺); HRMS (ESI): m/z

calculated for C₁₇H₂₄N₄S₄+ H⁺(M+H⁺): 413.0957. Found: 413.0946. Elemental analysis

(%) for C₁₇H₂₄N₄S₄: Calcd.: C, 49.48; H, 5.86; N, 13.58; Found, C, 49.32; H, 6.11; N,

13.31.

4-(Cyclohexylcarbamothioyl)piperazine-1-carbothioic

thioanhydride (37)

dimethylcarbamothioic

The

title

compound

(

37

)

was

synthesized

32e

from

2

sodium

equiv.)

4-

(cyclohexylcarbamothioyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 70% yield as yellow solid. mp: 238-240 °C;

IR (KBr) 5.32–5.30 (m, 1H),

(cm^-1): 3019, 1216, 1190; ¹H NMR (400 MHz, CDCl₃):

ν

δ

4.36–4.06 (m, 8H), 3.68–3.60 (m, 1H), 3.53–3.42 (m, 6H), 2.14–2.09 (m, 2H), 1.76–1.65

13

(m, 3H), 1.48–1.38 (m, 2H), 1.15–1.24 (m, 3H); C (100 MHz, CDCl₃):

δ 187.5, 186.5,

180.7, 54.6, 50.6, 49.9, 45.8, 45.0, 43.6, 33.0, 25.5, 24.9; ESI-MS: m/z 391 (M+H⁺);

HRMS (ESI): m/z calculated for C₁₅H₂₆N₄S₄+ H⁺(M+H⁺): 391.1113. Found: 391.1123.

Elemental analysis (%) for C₁₅H₂₆N₄S₄: Calcd.: C, 46.12; H, 6.71; N, 14.34; Found, C,

46.20; H, 6.99; N, 14.06.

4-(Cyclohexylcarbamoyl)piperazine-1-carbothioic

thioanhydride (38)

dimethylcarbamothioic

The

title

compound

(

38

)

was

synthesized

32f

from

2

sodium

equiv.)

4-

(cyclohexylcarbamoyl)piperazine-1-carbodithioate

(

;

and

dimethylcarbamothioic chloride (1 equiv.) in 73% yield as yellow solid. mp: 109-111 °C;

1

IR (KBr)

ν

(cm^-1): 3020, 1215, 1150; H NMR (400 MHz, CDCl₃):

δ

4.30 (s, 3H), 4.14–

4.02 (m, 2H), 3.67–3.60 (m, 4H), 3.54–3.53 (m, 3H), 3.45–3.43 (m, 3H), 1.98–1.96 (m,

2H), 1.74–1.71 (m, 2H), 1.64 (s, 1H), 1.43–1.35 (m, 2H), 1.30 (s, 1H), 1.27–1.08 (m,

13

3H); C (100 MHz, CDCl₃):

δ 187.5, 186.7, 156.5, 51.6, 50.4, 49.6, 44.9, 43.7, 42.6,

33.9, 25.6, 25.0; ESI-MS: m/z 418 (M+H⁺); HRMS (ESI): m/z calculated for

C₁₅H₂₆N₄OS₃+ H⁺(M+H⁺): 375.1341. Found: 375.1348. Elemental analysis (%) for

C₁₅H₂₆N₄OS₃: Calcd.: C, 48.10; H, 7.00; N, 14.96; Found, C, 48.22; H, 7.31; N, 14.64.

21

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Biological Materials and Methods:

Anti-Trichomonas assay

Metronidazole-susceptible T. vaginalis strain was a clinical isolate obtained from the

laboratory of Divya Singh [37] and metronidazole-resistant strain of T. vaginalis

(CDC085 [ATCC 50143]) was procured from American Type Culture Collection. In vitro

drug susceptibility of Trichomonas vaginalis was assayed as detailed earlier.[16] Briefly,

parasites were incubated at 37°C in the presence of test compounds or MTZ, serially

diluted from a stock-solution in DMSO using the culture medium, in 48-well culture

plates. 0.05% DMSO in culture media (the highest concentration of DMSO in test wells)

was used as vehicle in control wells. Cell viability was checked after 48 h by trypan blue

exclusion assay. The minimum concentration of the test agent at which all cells were

found dead was considered as its MIC. MTZ (the most widely used drug against

Trichomonas vaginalis) was procured from Sigma-Aldrich, and used as reference

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

Antifungal assay

The MIC of compounds were determined by broth micro-dilution technique as per the

guidelines of National Committee for Clinical Laboratory Standards using RPMI 1640

media buffered with MOPS [3-(N-Morpholino)propanesulfonic acid]. Starting inoculums

of test culture was 1–5 x 10³CFU/mL. Micro titer plates were incubated at 35 °C. MICs

were recorded after 48h of incubation. All experiments were repeated three times.[22]

(Table 2).

Cytotoxicity of compounds toward human cervical (HeLa) cells

The cytotoxic effect of test compounds were evaluated in an in vitro model of

cervicovaginal epithelium (HeLa) cells, using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,

5-diphenyl tetrazolium bromide] assay.[24] Cells seeded in 96 well plates were incubated

in culture medium (DMEM with 10% fetal calf serum) for 24 h at 37°C in a 5% CO2–

95% air atmosphere. After 24 h, the culture medium was replaced with fresh medium

containing serial dilutions of test compounds starting with 1000 µg/ml in experimental

22

ACCEPTED MANUSCRIPT

wells and 0.05% DMSO in culture medium in control wells. After incubation for another

24 h, 5 µL of 5 mg/mL MTT solution in 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 (OD540) was recorded in a microplate reader (Microquant;

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

Qualitative estimation of inhibition of free sulfhydryl groups on Trichomonas

vaginalis

The effect of test compound on Trichomonas free sulfhydryl groups was examined and

imaged by a method published earlier with slight modification [38] using a fluorescence

microscope, after labelling with the fluorometric thiol detector using a thiol –detection

assay kit (Cayman). Trichomonas vaginalis was treated with the vehicle or the test

compound at MIC and incubated for 24 h at 37°C. After incubation, trichomonads were

pelleted at 700 xg for 10 min at 4°C and washed 2-3 times with PBS. Thereafter 50 µL

fluorometric thiol detector (pre-diluted 100x with dilution buffer), was added and

incubated in the dark. A drop of this sample was taken on a microscope slide, covered

with a coverslip and imaged on a Nikon Eclipse 80i microscope equipped with

epifluoresence illumination, using the UV-1A filter. Exposure times were the same for all

samples.

Subcutaneous abscess assay for measuring anti-Trichomonal efficacy of test compounds in

the mouse model

The subcutaneous abscess assay of Krieger et. al. [39] was used. Briefly, the parasites (T.

vaginalis) were cultured under partial anaerobic condition in TYM medium and on attaining

concentration of approximately 2x10⁶cells/mL (in ~48 hrs) trichomonads were harvested from

the culture by centrifugation at 250 xg for 10 min and then re-suspended in sterile saline. Six

week old mice were inoculated subcutaneously with T. vaginalis (50 µL of 2 x 10⁶organisms per

mL) into the left hind flank. Control animals were injected with sterile saline only. Five groups

were used for each experiment (n=3). The abscess / lesion formation was determined by

palpation 7 days after injection, and measured daily thereafter. Fine needle biopsy specimens

23

ACCEPTED MANUSCRIPT

were taken from the lesion and examined microscopically to ensure infection. Infected animals

were then treated with compounds (orally) with a dose of 50 mg/kg and 100 mg/kg for 7 days

and abscess size measured longitudinally and the area calculated as πr². MTZ was used as

positive control. The assay was approved by the Institutional Animal Ethics Committee.²⁸

Docking study

The sequence of Trichomonas vaginalis cysteine synthase (TvCS) was retrieved from

Uniprot (A2GMG5). Since crystal structure of TvCS is not available, therefore a

homology model was constructed using crystal structure of cysteine synthase from

Escherichia coli (PDB ID-2BHS) [40] as template with the help of MODELLER package.

[41] All docking studies were carried using AUTODOCK4.2 Package.51 For molecular

visualization and structure manipulation Chimera was used. [42]

In vivo pharmacokinetic assay

The pharmacokinetic studies of compound 20 was carried out in young and healthy male

Sprague Dawley rats weighing 250±25 g obtained from laboratory animal division, CSIR-CDRI,

Lucknow. The animals were housed in plastic cages in standard laboratory conditions with a

regular 12 hr day-night cycle. Standard pelleted laboratory chow (Goldmohar Laboratory Animal

Feed, Lipton India Ltd, Chandigarh, India) and water were allowed ad libitum. The rats were

acclimatized to this environment for at least five days before conducting the experiment. The

oral dose pharmacokinetic study was conducted in overnight fasted (12-16 hr) rats (n = 4 per

time point). All experiments, euthanasia and disposal of carcasses were carried out as per the

guidelines of Local Ethics Committee for animal experimentation. Suspension formulations

containing 12.5 mg/mL of compound was prepared separately by triturating the compound, gum

acacia (1% w/v) and water (drop wise addition) in mortar and pestle. A single 50 mg/kg oral

dose was given to conscious rats using rat feeding needle. Blood samples were withdrawn at

various predefined times up to 24 hr post dose. Serum samples were harvested and stored at -80

°C until analysis. A Shimadzu UFLC pump (LC-20AD) with online degasser (DGU-20A3), an

auto-sampler (SIL-HTc) with a temperature-controlled peltier-tray and a triple quadrupole API

4000 mass spectrometer (Applied Biosystems, Toronto, Canada) was used for analysis.

Chromatographic separation was made on a Discovery HS C-18 column (5 µm, 50x4.6 mm id)

24

ACCEPTED MANUSCRIPT

preceded with a guard column (5 µm, 20 x 4.0 mm, id) packed with the same material with

mobile phase [acetonitrile: aqueous ammonium acetate buffer (0.01M; pH, 4.5) (80:20, %v/v)]

pumped at a flow rate of 0.6 mL/min under isocratic condition. The mobile phase was degassed

by ultra sonication for 15 min before use. LC-MS/MS system was equilibrated for

approximately 20 min before commencement of analysis. Total analysis time was 3 min per

sample. The mass spectral analysis was performed in positive ionization mode at 5500 V using

multiple reaction monitoring technique to monitor the transitions m/z 328.3→ m/z 206.9 for

compound 20 and m/z 180.1 →m/z 138.1 for phenacetin (internal standard). Data acquisition and

quantitation were performed using analyst software (version 1.4.2; AB Sciex, Toronto, Canada).

The method utilizes 50 µL of serum. For sample clean up protein precipitation was used. The

method showed linearity over the range of 1- 200 ng/mL with recovery of >50% and acceptable

accuracy and precision [43] [FDA, Guidance for Industry: Bioanalytical Method Validation].

Supporting Information:

¹H NMR, ¹³C NMR spectra of the compounds, HRMS spectra and supplementary were available

free of charge via internet at ……………..

ACKNOWLEDGEMENTS

We acknowledge Mrs. Tara Rawat (Senior Technical Officer) for technical assistance and

SAIF Division for providing spectral data. We are grateful to CSIR (D.M., M.S.), and

ICMR (B.K., S.G., S.K., N.S.) for research fellowships. This study was supported by

DHR, ICMR, New Delhi, Government of India (GAP 00155) and CSIR-Network project

PROGRAM, CSIR, New Delhi, INDIA (BSC 0101).

Abbreviations:

STDs, sexually transmitted disease; TV, Trichomonas vaginalis; DTC, dithiocarbamate; SAR,

structure activity relationship; MTZ, Metronidazole; NI, nitroimidazoles; HeLa, epithelium cells;

3D-QSAR, 3D-quantitative structure–activity relationship; SAR, structure–activity relationship;

MIC, minimum inhibitory concentration; IC₅₀, half maximal inhibitory concentration; CoMFA,

comparative molecular field analysis; PLS, partial least squares; LDH, lactate dehydrogenase;

DMEM, dulbecco’s modified eagle’s medium; TLC, thin layer chromatography.

25

ACCEPTED MANUSCRIPT

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Article Doi

Substituted carbamothioic amine-1-carbothioic thioanhydrides as novel trichomonicidal fungicides: Design, synthesis, and biology

DOI: 10.1016/j.ejmech.2017.11.060

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Full text of DOI:10.1016/j.ejmech.2017.11.060

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