Synthesis of 5-spirocyclohexyl-2,4-dithiohydantoin derivatives: A potential anti-leishmaniasis agent

Article abstract of DOI:10.1007/s00706-008-0063-9

A series of spiro-dithiohydantoins were synthesized by heating a mixture of 5-spirocyclohexyl-2,4-dithiohydantoin potassium salt and 3-chloropropanoyl chloride. These compounds were synthesized and evaluated for their activity against five Leishmanial str

Full text of DOI:10.1007/s00706-008-0063-9

Monatsh Chem (2009) 140:243–249
DOI 10.1007/s00706-008-0063-9
ORIGINAL PAPER
Synthesis of 5-spirocyclohexyl-2,4-dithiohydantoin derivatives:
a potential anti-leishmaniasis agent
Abdel-Sattar S. Hamad Elgazwy Æ
Saad R. Atta-Allha Æ Sherif M. A. S. Keshk
Received: 10 July 2008 / Accepted: 28 August 2008 / Published online: 1 October 2008
Ó Springer-Verlag 2008
Abstract A series of spiro-dithiohydantoins were syn-
thesized by heating a mixture of 5-spirocyclohexyl-2,4-
dithiohydantoin potassium salt and 3-chloropropanoyl
chloride. These compounds were synthesized and evalu-
ated for their activity against five Leishmanial strains in the
promastigote stage in vitro. Seventy-two hours inoculation
of a variety of products gave an IC100 and average IC50
values of 1.25 and 0.376 mg/cm3 against all Leishmanial
strains tested.
(Dilantin) [13]. Small heterocyclic molecules are one of the
predominant types of building block in medicinal chemistry
[14, 15], and among the ‘‘drug-like’’ heterocycles, dith-
iohydantoins have been widely used for constructing
products with pharmaceutical applications [16]. Dith-
iohydantoins are sulfur analogs of hydantoins with both
carbonyl groups replaced by thiocarbonyl groups. Among
the known dithiohydantoins, 2-thiohydantoins are most
notably known due to their wide applications as hypolipi-
demic, anticarcinogenic, antimutagenic, antithyroidal, and
antiviral agents [17].
Keywords Dithiohydantoins ꢀ Spiro-dithiohydantoins ꢀ
1,3-Diazaspiro[4,5] decane-2,4-dithione ꢀ Leishmaniasis
We now report a simple method for the preparation of
5-spiro-cyclohexyl-2,4-dithiohydantoin derivatives that can
easily be scaled up in the laboratory. The reaction of
5-spirocyclohexyl-2,4-dithionhydantoin (1) with 1- or
2-mole equivalents of 3-chloropropanoyl chloride was
examined, and the synthesized compounds were tested
against five Leishmanial strains. In continuation [18, 19] of
our efforts toward the synthesis of heterocyclic compounds,
an efficient and practical preparation of 5-spirocyclohexy-
2,4-dithiohydantoin derivatives 1–9 is shown.
Introduction
In connection with our previous research work related to
aza-spiro compounds, such as 5-spirocyclohexyl-2,4-dith-
iohydantoin [1–7], spiro-thiopyrano[2,3-d]thiazolidines [8]
and isothiazoles [9], we aimed at the synthesis of compounds
with anti-leishmania activity based on the spiro-dithiohyd-
antoin scaffold. It seemed of interest to us to prepare new
5-spirocyclohexyl-2,4-dithiohydantoin derivatives bearing
some resemblance to the known drugs 5-ethyl-5-phen-
ylhydantoin (Nirvanol) [10–12] and 5,5-diphenylhydantoin
Results and discussion
Synthesis
A.-S. S. Hamad Elgazwy (&) ꢀ S. R. Atta-Allha
Department of Chemistry, Faculty of Science,
University of Ain Shams, Abbassia, 11566 Cairo, Egypt
e-mail: aselgazwy@yahoo.com; hamad@asunet.shams.edu.eg;
elgazwy@usa.com
The direct condensation between 5-spirocyclohexyl-2,4-
dithiohydantoin potassium salt 2 and 3-chloropropanoyl
chloride produces 5-spirocyclohexyl-2,4-dithiohydantoin
derivatives 3–9 in moderate to high yields (Scheme 1).
Spirocyclohexyl-2,4-dithiohydantoin (1) was synthesized
in high yield (96%) [1–5, 20]. The 5-spirocyclohexyl-2,4-
dithiohydantoin potassium salt (2) was readily obtained by
stirring 1 with ice-cooled KOH solution in ethanol [21, 22].
S. M. A. S. Keshk
Department of Basic Science,
Institute of Environmental Studies and Research,
University of Ain Shams, Abbassia, 11566 Cairo, Egypt
123

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A.-S. S. Hamad Elgazwy et al.
Scheme 1
7
8
O
S
6
i) KOH, EtOH
stirred 0oC
30 min
S
S
S
4
5
Cl
Cl
KI/Ethanol
refluxing 6h
O
N
N
O
N
N
HN NH
i) Ethanol
HN
N
K
1
K
3
refuxed/1h
S
2
ii) one equiv.
S
S
S
Cl
1
2
3
4
i) one equiv. 2
ethanol/KI
1N HCl
S
NH
N
S
S
HN
N
O
S
5
Then the reaction of 2 with 3-chloropropanoyl chloride was
investigated under different conditions.
(2) Similarly, when the reaction was performed with
2-mole equivalents of 3-chloropropanoyl chloride,
3-chloro-1-[1-(3-chloro-propionyl)-2,4-dithioxo-1,3-diaza-
spiro[4,5]dec-3-yl)-propan-1-one (6) was achieved in good
yield (75%). Compound 6 was reacted further with 2-mole
equivalents of 5-spirocyclohexyl-2,4-dithiohydantoin
potassium salt (2) in the presence of catalytic amounts of
KI to produce 1-{2,4-dithioxo-1-[3-(2-thioxo-1,3-diaza-spiro
[4, 5]dec-3-en-4-ylsulfanyl)-propionyl]-1,3-diaza-spiro[4,5]
dec-3yl)-3-(2-thioxo-1,3-diaza-spiro[4, 5]dec-3-en-4-ylsul-
fanyl)-propan-1-one (7) in 59% yield (Scheme 3). In
addition, we prepared 2,4-bis{3-[2-(4-methylphenyl)sulfo-
nyl]hydrazino-3-oxopropylthio}-1,3-diazaspiro[4,5] deca-
1,3-diene (9) (Scheme 4). For this purpose we developed an
efficient synthesis of 3-chloro-N0-[(4-methylphenyl)sulfo-
nyl] propanoylhydrazide 8 by the reaction of tosyl
hydrazide and 3-chlrorpropanoyl chloride in refluxing
benzene for 1 h [32].
(1) When the reaction was carried out with 1-mole
equivalent of 3-chloropropanoyl chloride in ethanol,
3-chloro-1-(2,4-dithioxo-1,3-diaza-spiro[4,
5]dec-3-yl)-
propan-1-one (3) was obtained in good yield (75%). Ring
closure of 3 by adding a catalytic amount of KI in refluxing
ethanol produced 30-thioxo-60,70-dihydro-50H-spiro[cyclo-
hexane-1,20-imidazo[2,1-b][1, 3]thiazin]-50-one 4 in excel-
lent yield (98%). When compound 3 was reacted with
1-mole equivalent of 5-spirocyclohexyl-2,4-dithiohydan-
toin potassium salt (2) in the presence of catalytic amounts
of KI, 1-(2,4-dithioxo-1,3-diaza-spiro[4, 5]dec-3yl)-3-
(2-thioxo-1,3-diaza-spiro[4, 5]dec-3-en-4-ylsulfanyl)-pro-
pan-1-one (5) was obtained in moderate yield (49%), as
depicted in Scheme 1.
The N-acylation and S-alkylation were confirmed by the
fact that boiling the dithiohydantoins with dilute hydro-
chloric acid produced the corresponding hydantoins
[10–15]. A mechanistic proposal depicting the formation of
4 including possible intermediates via ring closure of 3
undergoes competitive nucleophilic substitution reaction
by the preformed sulphur anion [A]. The condensation type
addition/substitution proceeds in a 1,4-fashion and results
in an intramolecular addition on precipitation during the
reaction at refluxing temperature under the action of a trace
amount of ethanol in the reaction mixture or during workup
on a silica gel column. A mechanistic proposal consistent
with literature data [23–31] is given in Scheme 2.
1
The chemical structure of 1 was established by H and
13C-NMR spectroscopies, employing the 1D and 2D
1
(1H/1H and H/13C correlation spectra) NMR techniques.
1
The H and 13C-NMR data obtained are presented in the
1
experimental part. The H-NMR spectra of 1, 2, 3, 4, 5, 6,
7, and 9 show resonance signals at 1.5–1.75 ppm (CH2
protons of cycloalkane residue, multiplet) and two broad
signals at 8.0–14.5 ppm characteristic of NH protons. The
NOEs observed for 1 exhibit an enhancement of the reso-
nance of the alkyl protons from the cycle, when resonances
at ca. d 11.0 ppm were irradiated. This result was used to
Scheme 2
i) Ethanol
refuxed/1h
ii) KI/ 1 equiv.
S
S
S
S
O
O
-KCl
N
O
N
N
N
N
N
O
N
N
K
K
K
S
S
S
Cl
Cl
K
S
2
[A]
4
3
Cl
Cl
123

Synthesis of 5-spirocyclohexyl-2,4-dithiohydantoin derivatives
245
Scheme 3
S
O
O
S
S
N
N
i) EtOH refuxed/ 1h
ii) two equiv.
i) KI/Ethanol
ii) two equiv of 2
O
O
S
HN
N
K
N
N
K
-KCl
1N HCl
O
S
S
HN
S
S
S
NH
S
Cl
Cl
N
2
N
Cl
Cl
6
7
Scheme 4
N
S
S
N
CH3
O
NHNH
H
HNHN
SH
EtOH/KI
refluxed/12 h
1 N HCl
O
O
N
N
+
O S O
O
S
O
S
Cl
N
K
K
N
O
H
O
HS
9
2
8
CH3
CH3
assign the signals at ca. 11.0 ppm to H-1 and those at ca.
13.0 ppm to H-3. The 13C-NMR chemical shifts assign-
ment of the spectra was facilitated by analyses of the
HMQC and HMBC spectra, which provide single and
their vertebrate hosts. Three major clinical manifestations
of leishmaniasis are recognized: visceral, cutaneous, and
muco-cutaneous leishmaniasis [33]. The disease usually
manifests itself as fever, weight loss, and hepato-spleno-
magaly with biochemical abnormalities of hyper-
globulinemia and pancytopenia [34]. It has received
increasing attention in developed countries because of the
growing number of cases seen in AIDS patients [33, 35]
and the occurrence of viescerotropic L. tropica disease
among Persian Gulf War participants [33–36].
1
multiple bond H/13C connectivities. On the basis of the
HMBC spectra, it was possible to assign the resonance
peaks of carbons C-2 and C-4. Resonance peaks of C-2 and
C-4 of the dithio analogues of spirodithiohydantoins 1, 3, 5,
7, and 9 appear at ca. d 180 and 210 ppm, respectively. In
the HMBC spectra of these compounds, cross peaks of C-2
with H-1 and H-3, and C-4 with H-3 were observed. The
13C-NMR spectra of 1, 3, 5, 7, and 9 show an up-field shift
of the resonance signals of C-4 as well as signals at 20.5
and 36.0 ppm characteristic of methylene protons of the
–CH2– group. The strong up-field shifts of the resonance
signals of carbons C-4 of cycloalkane 5-spiro-(2-dith-
iohydantoins) 1–7 confirm the presence of thiocarbonyl
group at C-4 position. The 1H and 13C-NMR data obtained
confirm the structure of the compounds 1, 2, 3, 4, 5, 6, 7,
and 9.
Pentavalent antimonial drugs have remained standard
treatment for visceral leishmaniasis since the 1940s [36].
These drugs not only have several adverse effects [36],
but drug resistance and treatment failures are becoming
increasingly common, especially in immune-compromised
patients who often fail to respond or relapse [35, 37, 38].
Amphotericin B and its new lipid formulations are used as
second line of treatment. However, there are limitations for
these drugs due to prolonged length of therapy and adverse
side reactions. Thus, there is still a need for the develop-
ment of new drugs [36, 39, 40]. In this article we report the
anti-Leishmanial effect of S- and N- heterocyclic com-
pounds in solution on Leishmanial promastigotes in vitro.
Each experiment was repeated at least three times.
Concentrations of dithiohydantoins ranging from 10 to
0.07 mg/cm3 were tested for anti-Leishmanial activity. As
shown in Table 1, concentration of 1.25 mg/cm3 was found
to be leishmanicidal for all the Leishmanial strains tested,
whereas 50% parasites of all strains were found to be dead
at an average value of 0.376 mg/cm3 after 72 h of
Biological studies
Evaluation of the Leishmanial activity
The different heterocyclic ring systems obtained in this
investigation are known to exhibit diverse biological
activities. This initiated our interest to evaluate the leish-
mania activity of some of these compounds. Leishmania
spp. are intracellular parasitic hemo-flagellates that infect
macrophages of the skin and viscera to produce disease in
123

246
A.-S. S. Hamad Elgazwy et al.
treatment. All five strains of Leishmania that were tested
were found to be sensitive to the spirocyclohexylhydantoins
1–7 and 9. No major differences in the degree of suscepti-
bility of parasites occurred during the study except for
L. mex mex, perhaps due to its very high multiplication rate.
A careful analysis of data suggests a susceptibility order of
L. major (Pak. Isolates) A [ L. major [ L. donovan-
i [ L. tropica [ L. mex mex. A concentration of 1.25 mg/
cm3 was leishmanicidal, whereas 0.25 mg/cm3 and below
was leishmaniastatic. Although these concentrations are not
low, it has to be taken into consideration that due to solu-
bility issues, the anti-leishmanial components may be
present in very low concentration [40]. It was interesting to
find that a methanol solution of the same dithiohydantoins
stock is 100% active at *200 lg/cm3 (unpublished data).
Dithiohydantoins 1–7 and 9 are rich in sulfur containing
active principles mainly in the form of cysteine derivatives,
as S-alkyl cysteine sulfoxides, which decompose into a
variety of thiosulfinates and polysulfides by the action of the
enzyme allinase on extraction. Decomposed products are
volatile and present in the oils of onion and garlic [41, 42].
They possess antidiabetic, antibiotic, hypocholesterolemic,
fibrinolytic, and various other biological actions. In addition
to free sulfoxides in onion, there are nonvolatile sulfur-
containing peptides and proteins, which possess various
activities and thus rendering it an important source of
therapeutic agents [41, 42]. Further investigations regarding
the principles of the anti-leishmanial activity of dith-
iohydantoins 1–7 and 9 merit consideration. Regarding
mammalian cell toxicity, the compounds 7, 8, and 9 were, in
general, less toxic than the other analogues.
Conclusions
In summary, we have described a novel synthetic method
for the synthesis of 5-spirocyclohexyl-2,4-dithiohydantoins
derivatives, which were tested for in vitro activity against
Leishmania parasites. The most active compounds to
emerge from this study were 5, 7, 8, and 9. They were
active against free and intracellular forms of T. cruzi with
SI higher than compounds 1–3. The results obtained offer
new possibilities for further improvements concerning the
anti-parasitic activity of other closely related derivatives of
these series of compounds. A more complete structure
activity relationship is currently being pursued in our
laboratory.
Experimental
Reactions were carried out without precautions to exclude
light, atmospheric oxygen, and moisture, unless otherwise
123

Synthesis of 5-spirocyclohexyl-2,4-dithiohydantoin derivatives
247
mp 202–205 °C dec.; IR (Nujol): ꢀm = (C=S) 1,115, (C=O)
1,741, (NH) 3,213 cm-1; 1H NMR (300 MHz, DMSO-d6):
d = 8.96 (s, 1H, NH), 3.75 (t, J = 6.5 Hz, 2H, –CH2), 2.76
(t, J = 6.5 Hz, 2H, –CH2), 1.54–1.75 (m, 10H, cyclohexyl)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 210.8 (C4),
181.2 (C2), 178.4 (C=O), 79.5 (C5), 38.7 (CH2–Cl), 37.57
(C6, C10), 36.3 (–CH2–), 26.5 (C8), 20.5 (C7, C9) ppm.
stated. Melting points were determined on a Reichert
apparatus. Elemental analyses were carried out with a
Carlo Erba 1106 microanalyzer at the University of Mur-
cia, Spain, and the microanalysis unit at Cairo University,
Egypt; results agreed favorably with calculated values. IR
spectra were recorded on a Perkin-Elmer FT-IR spec-
trometer with Nujol mulls between polyethylene sheets.
NMR spectra were recorded on a Bruker AC 200, Advance
300, or a Varian Unity 300 spectrometer at room temper-
ature unless otherwise stated. Chemical shifts were
referenced to TMS [1H and 13C (1H) and H3PO4 (31P)]. The
NMR probe temperature was calibrated using ethylene
30-Thioxo-60,70-dihydro-5’H-spiro[cyclohexane-1,
20-imidazo[2,1-b][1, 3]thiazin]-50-one (4, C11H14N2OS2)
Method (A). To a suspension of 1.16 g 3-(3-chloropropa-
noyl)-1,3-diazaspiro[4.5]decane-2,4-dithione 3 (0.004 mol)
in 10 cm3 ethanolic KOH (0.8 M) was added 0.002 g of KI
in 25 cm3 dry ethanol, and then this was heated under reflux
for 12 h. The reaction mixture was cooled and acidified by
the addition of two drops of 0.1 N hydrochloric acid 37% in
an ice bath. The formed KCl salts were removed by
filtration. The filtrate was diluted with Et2O, washed with
saturated aqueous solutions of Na2CO3, NH4Cl, and brine,
and dried over Na2SO4. After removal of the solvent, a solid
product was obtained, which was purified by chromatog-
raphy (silica gel, n-hexane/ether 1:3) to give 1.0 g (98%
yield) yellow powder. TLC Rf = 0.95 [n-hexane/ether 1:3],
1
glycol H NMR standard methods. Chromatographic sep-
arations were carried out by TLC on silica gel (70–230
mesh).
1,3-Diazaspiro[4, 5]decane-2,4-dithione (1)
A mixture of 9.8 g cyclohexanone (0.1 mol), 5.1 g sodium
cyanide (0.1 mol), 5.5 g ammonium chloride (0.1 mol),
and 7.6 g carbon disulfide (0.1 mol) in 100 cm3 ethanol
was heated in a water bath at 100 °C for 12 h. After
cooling to room temperature and evaporation of the
solvent, a solid product was formed and was purified by
recrystallization from ethyl alcohol [1, 2] to give 17.8 g
(89% yield) as needless yellow crystals, mp 270–272 °C
dec. (lit. [1–6] mp 267 °C, lit. [26] mp 269 °C).
ꢀ
mp 242–245 °C dec. IR (Nujol): m = (C=S) 1,115, (C=O)
1,741, (NH) 3,213 cm-1; 1H NMR (300 MHz, DMSO-d6):
d = 4.15 (t, J = 6.4 Hz, 2H, –CH2), 3.75 (t, J = 6.4 Hz,
2H, -CH2), 1.54–1.75 (m, 10H, cyclohexyl) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 210.2 (C=S), 177.2 (C=O), 163
(C=N), 79.1 (C5), 36.3 (C6, C10), 35.6 (–CH2), 24.9
(–CH2), 27.7 (C8), 20.0 (C7, C9) ppm.
1,3-Diazaspiro[4, 5]decane-2,4-dithione potassium salt
(2, C8H10N2S2K2)
A mixture of 1.104 g 5-spirocyclohexyl-2,4-dithiohydan-
toin (1) (0.04 mol) and 0.112 g of KOH (0.02 mol) in
25 cm3 ethanol was stirred at 0 °C for 30 min. The solid
residue was collected by filtration, washed with dry ethanol
and dried under reduced pressure to isolate 1.1 g (99%
Method (B). To a mixture of 2.765 g 5-spirocyclohexyl-
2,4dithiohydantoin potassium salt (2) (0.01 mol), 1 cm3 of
3-chloropropanoyl chloride (0.01 mol) was added 0.002 g
KI in 25 cm3 dry ethanol and then heated under reflux for
12 h. The obtained solid was collected by filtration, washed
with 50 cm3 boiling water followed by 25 cm3 boiling
ethanol, and recrystallized from DMF/ethanol to give
yellow crystals of 4 in similar yield.
yield) white powder, mp [ 300 °C dec. IR (Nujol):
1
m = (C=S) 1,117, (C=N and C=C) 1,536, 1,525 cm-1; H
ꢀ
NMR (300 MHz, DMSO-d6): d = 1.54–1.75 (m, 10H,
cyclohexyl) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 212.9 (C4), 180.9 (C2), 78.0 (C5), 36.6 (C6, C10),
26.3 (C8), 20.7 (C7, C9) ppm.
3-{3-[(2-Thioxo-1,3-diazaspiro[4.5]dec-3-en-4-
yl)thio]propanoyl}-1,3-diazaspiro[4.5]decane-2,
4-dithione (5, C19H26N4OS4)
3-(3-Chloropropanoyl)-1,3-diazaspiro[4.5]decane-2,
4-dithione (3, C11H15N2OS2Cl)
A mixture of 2.77 g 5-spirocyclohexyl-2,4dithiohydantoin
potassium salt (2) (0.01 mole), 2.9 g of 3-(3-chloro-
propanoyl)-1,3-diazaspiro[4.5]decane-2,4-dithione 3 (0.01
mole), and 0.002 g of KI was heated under reflux in
25 cm3 DMF for 12 h. The reaction mixture was cooled in
an ice bath and then acidified by the addition of 1 N
hydrochloric acid. The obtained solid was collected by
filtration, washed with 25 cm3 boiling water, and recrys-
tallized from DMF/ethanol to give 2.25 g (49% yield)
yellow powder, mp 258-260 °C dec. IR (Nujol): ꢀm = (C=S)
A mixture of 2.77 g 5-spirocyclohexyl-2,4dithiohydantoin
potassium salt (2) (0.01 mol) and 1 cm3 3-chloropropanoyl
chloride (0.01 mol) in 25 cm3 ethanol was stirred at 0 °C
for 30 min, then refluxed for 15 min and then stirred at RT
again overnight. The formed KCl salts were removed by
filtration. The filtrate was diluted with Et2O, washed with
saturated aqueous solutions of Na2CO3, NH4Cl, brine, and
dried over Na2SO4. After removal of the solvent a solid
product was obtained, which was purified by chromatog-
raphy (silica gel, n-hexane/ether 1:1) to give 2.2 g (75%
yield) yellow solid. TLC Rf = 0.145 [n-hexane/ether 1:1],
1
1,116, (C=N) 1,540, (C=O) 1,717, (NH) 3,219 cm-1; H
NMR (300 MHz, DMSO-d6): d = 10.16 (s, 1H, NH), 9.96
123

248
A.-S. S. Hamad Elgazwy et al.
(s, 1H, NH), 3.15 (t, J = 6.3 Hz, 2H, –CH2–S), 2.85 (t,
J = 6.3 Hz, 2H, –CH2–), 1.54–1.75 (m, 20H, cyclohexyl)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 210 (C=S),
183.2 (C=S), 187 (C=S), 177.2 (C=O), 163.7 (C=N), 80.7
(C5), 60.5 (C50), 37.1 (C6, C10), 33.4 (–CH2), 29.4 (C60,
C100), 23.9 (–CH2), 27.7 (C8), 27.1 (C80), 19.5 (C7, C9),
19.2 (C70, C90) ppm.
ropropanoyl chloride (4.77 cm3, 0.025 mol), and this was
heated under reflux for 1 h. After cooling to room
temperature, the formed crystals were collected by
filtration, washed with benzene/n-hexane, and recrystal-
lized from ethanol to produce 6.5 g (93% yield) white
ꢀ
crystals, mp 175–177 °C dec. IR (Nujol): m = (S=O)
1,159, (C=O and C=C) 1,678, 1,698, (NH–NH) 2,853,
1
2,953, 3,050, 3,300 cm-1; H NMR (300 MHz, DMSO-
1,3-Bis(3-chloropropanoyl)-1,3-diazaspiro[4,5]
decane-2,4-dithione (6, C11H15N2OS2Cl)
d6): d = 10.13–10.12 (d, J = 3.3 Hz, 1H, NH), 9.83–9.82
(d, J = 3.3 Hz, 1H, NH), 7.68 (d, J = 8.1 Hz, 2H, Ar–H),
7.34 (d, J = 8.3 Hz, 2H, Ar–H), 3.62 (t, J = 6.3 Hz, 2H,
–CH2), 2.46 (t, J = 6.3 Hz, 2H, –CH2), 2.36 (s, 3H,
–CH3) ppm; 13C NMR (75 MHz, DMSO-d6): d = 170.5
(C=O), 140.0, 143.1, 137.0, 130.1, 128.0, 39.9 (CH2–Cl),
37.5, 22.5 (CH3) ppm.
A mixture of 2.77 g 5-spirocyclohexyl-2,4dithiohydantoin
potassium salt (2) (0.01 mol) and 2 cm3 3-chloropropanoyl
chloride (0.02 mol) was heated under reflux in 25 cm3 dry
DMF for 12 h. The reaction mixture was cooled to room
temperature, and the obtained solid was collected by
filtration and then washed with 50 cm3 boiling water. The
solid was purified by chromatography (silica gel, n-hexane/
ether 1:1) to give 2.2 g (75% yields) as yellowish solid.
2,4-Bis{3-[2-(4-methylphenyl)sulfonyl]hydrazino-3-
oxopropylthio}-1,3-diazaspiro [4.5]deca-1,3-diene
(9, C28H36N6O6S4)
TLC Rf = 1.96 [n-hexane/ether 1:1], mp 218–220 °C dec.
1
IR (Nujol): m = (C=S) 1,118.5, (C=O) 1,746 cm-1; H
A mixture of 1.10 g 5-spirocyclohexyl-2,4-dithiohydan-
toin potassium salt (2) (0.004 mol), 1.10 g of 3-chloro-N0-
[(4-methylphenyl)sulfonyl]propanoylhydrazide (8) (0.004
mol), and 0.002 g KI in 25 cm3 DMF was heated under
reflux for 12 h. The reaction mixture was cooled in an ice
bath and then acidified by the addition of 1 N hydrochloric
acid. The obtained solid was collected by filtration, washed
with 25 cm3 boiling water, and recrystallized from DMF/
ethanol to give 1.5 g (55% yield) of 9 as a yellow powder,
ꢀ
NMR (300 MHz, DMSO-d6): d = 3.75 (t, J = 6.5 Hz, 4H,
–CH2), 2.76 (t, J = 6.5 Hz, 4H, –CH2), 1.54–1.75 (m, 10H,
cyclohexyl) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 210.8 (C4), 181.2 (C2), 178.4 (C=O)2, 79.5 (C5),
38.7 (CH2–Cl)2, 37.57 (C6, C10), 36.3 (–CH2–)2, 26.5
(C8), 20.5 (C7, C9) ppm.
1,3-Bis {3-[(2-thioxo-1,3-diazaspiro[4.5]dec-3-en-4-yl)
thio]propanoyl}-1,3-diazaspiro[4,5]decane-2,
4-dithione (7, C30H40N6O2S6)
ꢀ
mp [ 300 °C dec. IR (Nujol):m = (C=S) 1,116, 1,116,
(S=O) 1,159, (C=O) 1,678, (C=C) 1,698, (NH–NH) 3,050,
A mixture of 1.10 g 5-spirocyclohexyl-2,4-ithiohydantoin
potassium salt 2 (0.004 mol), 1.16 g of 6 (0.004 mol), and
0.002 g of KI was refluxed in 25 cm3 DMF for 12 h. The
reaction mixture was cooled in an ice bath and then
acidified by the addition of 1 N hydrochloric acid. The
obtained solid was collected by filtration, washed with
25 cm3 boiling water, and recrystallized from DMF/
ethanol to give 1.7 g (59% yield) yellow crystals, mp
3,178, 3,301 cm-1
;
1H NMR (300 MHz, DMSO-d6):
d = 10.11 (d, J = 3.3 Hz, 2H, NH), 9.81 (d, J = 3.3 Hz,
2H, NH), 7.85 (d, J = 8.1 Hz, 4H, Ar–Ho), 7.35
(d, J = 8.3 Hz, 4H, Ar–Hm), 3.64 (t, J = 6.3 Hz, 4H,
–CH2), 2.49 (t, J = 6.3 Hz, 4H, –CH2), 2.36 (s, 6H, two
–CH3), 1.54–1.75 (m, 10H, cyclohexyl) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 178.2 (C=O)2, 165.0 (C=N),
164 (C=N), 141.9 (Cipso)2, 136.9 (Cipso)2, 132.1 (CH-
ortho)2, 126.9 (CH-meta)2, 60.5 (C5), 38.9 (–CH2)2, 30.4
(C6, C10), 24.9 (–CH2)2, 28.7 (C8), 20.5 (C7, C9), 22.5
(CH3)2 ppm.
ꢀ
290–292 °C dec. IR (Nujol): m = (C=S) 1,116, 1,115.8,
(C=N) 1,541, (C=O) 1,654, 1,638, 1,718, 1,762, (NH)
3,218, 3,444, (CH2) 2,855, 2,926 cm-1
;
1H NMR
(300 MHz, DMSO-d6): d = 10.16 (s, 2H, NH), 3.15
(t, J = 6.3 Hz, 4H, –CH2–S), 2.85 (t, J = 6.3 Hz, 4H,
–CH2–), 1.21–1.75 (m, 30H, cyclohexyl) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 210 (C=S), 187 (C=S)2, 180
(C=S), 177.2 (C=O)2, 163.7 (C=N)2, 78.5 (C5), 60.5 (C50)2,
34.4 (C6, C10), 33.4 (–CH2)2, 29.4 (C60, C100)2, 23.9
(–CH2)2, 27.7 (C8), 27.1 (C80)2, 19.5 (C7, C9), 19.2
(C70, C90)2 ppm.
Biological testing
Parasite culture
All the promastigote cultures of both the reference and
local Egypt leishmanial strains were maintained in blood
agar-based bi-phasic Evan’s modified Tobie’s medium
supplemented with RPMI-1640 with 25 mM TES at 25 °C.
Leishmanial strains in promastigote stage that were used
include L. major (JISH118), L. major (MHOMyPKy88y-
DESTO), L. tropica (K27), L. infantum (LEM3437), L. mex
mex. (LV4), and L. donovani (H43).
3-Chloro-N0-[(4-methylphenyl)sulfonyl]
propanoylhydrazide (8, C10H13N2O3SCl)
To a solution of 4.65 g p-tosyl hydrazide (0.025 mol)
in 50 cm3 dry benzene was added 3.34 g of 3-chlo-
123

Synthesis of 5-spirocyclohexyl-2,4-dithiohydantoin derivatives
249
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Parasites in the promastigote stage were transferred from
Evan’s modified Tobie’s modified to RPMI-1640 supple-
mented with 5% fetal bovine serum, 1% sterile human
urine, and buffered with 25 mM TES, pH 7.2 (complete
medium). They were grown in bulk at 25 °C. They were
centrifuged at 2,500 rev./min for 10 min, and early log
phase promastigotes were collected. The parasites were
washed twice with RPMI (without FBS or urine) and
re-suspended in the complete medium to achieve a final
concentration of 106 parasites/cm3. In order to get the
100% and IC50 mortality concentration, serial dilutions of
heterocycles 1–9 were performed in 96-well micro-titer
plates. Subsequently, 103 promastigotes in 100 lcm3 of
culture medium were added to each well, and the plate was
incubated at 25 °C for 72 h. Negative controls (culture
without heterocycles) were on the same plate. At the end of
the incubation time, the plate was shaken mechanically
over a plate shaker, and parasites were counted with the
help of a hemocytometer. Dose-dependent viability curves
were obtained.
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Acknowledgments We thank the Institute of Biochemistry, Uni-
versity of Balochistan, Quetta, Pakistan, for facilities and Tropical
Medicine London for providing us with various Leishmanial strains.
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