Synthesis, characterization and derivatization of some novel types of mono- and bis-imidazolidineiminothiones and imidazolidineiminodithiones with antitumor, antiviral, antibacterial and antifungal activities - part I

European Journal of Medicinal Chemistry 44 (2009) 4315–4334

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European Journal of Medicinal Chemistry

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Original article

Synthesis, characterization and derivatization of some novel types

of mono- and bis-imidazolidineiminothiones and imidazolidineiminodithiones

with antitumor, antiviral, antibacterial and antifungal activities – part I

*

Ahmed M. Sh. El-Sharief , Ziad Moussa

Department of Chemistry, Faculty of Science, Taibah University, P.O. Box 30002, Almadinah Almunawarrah, Saudi Arabia

a r t i c l e i n f o

a b s t r a c t

Article history:

Halogenated and alkylated N-arylcyanothioformanilides were reacted with the nucleophilic reagents

triethylamine, hydrazine and diphenyldiazomethane to produce N-arylcyanothioformanilide ammonium

salts, a thiosemicarbazide and a 2-(arylamino)-3,3-diphenylacrylonitrile, respectively. They also under-

went several types of electrophilic reactions with aryl-, arylbisisocyanates and arylisothiocyanates to

yield mono- and bis-imidazolidineiminothiones and imidazolidineiminodithiones. Treatment of imida-

zolidineiminothiones with hydrogen sulﬁde, substituted ortho-phenylenediamines and thiocarbohy-

drazide afforded the corresponding thiohydantoin, quinoxaline and imidazotriazine derivatives. Several

of the synthesized products were subjected to in vitro biological evaluation against antitumor, antiviral,

antimicrobial and antifungal strains. Most tested compounds showed signiﬁcant activities.

Received 20 January 2009

Received in revised form

18 June 2009

Accepted 16 July 2009

Available online 23 July 2009

Keywords:

N-Arylcyanothioformanilides

Imidazolidineiminothiones

Bis-imidazolidineiminothiones

Imidazolidineiminodithiones

Antitumor

Antiviral

Antimicrobial

Antifungal

1. Introduction

[12,28]. In addition, bis-cyanothioformanilides derived from 1,4-

bis-isothiocyanatobenzene and 2,4-bis-isothiocyanatotoluene

Cyanothioformanilides are versatile reagents and have been

extensively utilized for the synthesis of various fused heterocycles

which display interesting medicinal and pharmacological proper-

ties. A variety of ‘‘one-pot’’ ring closure reactions of cyanothio-

formanilides [1–3] give rise to a plethora of heterocyclic cores

which include pyrroles [4,5], pyrrolothiazoles [6], imidazoles [7–9],

benzimidazoles [10], oxazoles [11,12], benzoxazoles [10,12], thia-

zoles [13,14], and quinazolinonethione [10,12].

Our keen interest in the chemistry of cyanothioformanilide I

[15,16] and derivatives thereof containing aliphatic, aromatic

[17,18], and various heterocyclic ring systems [6], as well as the

related cyanothiosulfonamide congeners [19] led us to synthesize

many bioactive heterocyclic compounds with a wide variety of

pharmacological effects [20–27]. In these transformations, various

derivatives of cyanothioformanilide I reacted with a range of

electrophiles, nucleophiles, and ortho-substituted aniline reagents

reacted with different reagents to produce novel bis-heterocycles

[5,29]. In the present study, the reactions of a series of previously

reported substituted cyanothioformanilides Ia–g [30] (Fig. 1) were

investigated with various electrophiles and nucleophiles, leading to

several new mono- and bis-imidazolidineiminothiones and imi-

dazolidineiminodithiones. Structures of the prepared compounds

were elucidated by the use of 1D/2D ¹H NMR, ¹³C NMR, IR and GC

mass spectral techniques. Finally, some selected compounds were

evaluated for cytotoxic activity against various tumor cell lines

(brain tumor cell line U251, human hepatocellular carcinoma cell

line HEPG2, human breast adenocarcinoma cell line MCF-7, human

epithelial carcinoma cell line HELA and colon carcinoma cell line

HCT116), antiviral (HAV, HSV1 and CoxB4), antibacterial (Bacillus

subtilis, Staphylococcus aureus and Pseudomonas aeruginosa) and

antifungal strains (Candida albicans and Aspergillus niger).

2. Chemistry

Prior to delving into the synthesis and biological evaluation of

heterocycles (vide post), we decided to investigate some of the

cyanothioformanilides shown in Fig. 1 by various 1D and 2D NMR

* Corresponding authors. Tel./fax: þ966 4 8611190.

E-mail addresses: alshraef_2008@hotmail.com (A.M.Sh. El-Sharief), zmousa@

taibahu.edu.sa (Z. Moussa).

doi:10.1016/j.ejmech.2009.07.019

4316

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

a; R = p-Me

b; R = p-EtO

c; R = p-Cl

d; R = p-Br

e; R = p-I

The chemical behavior of cyanothioformanilides Ia–g toward

a variety of basic and nucleophilic reagents such as triethylamine,

hydrazine hydrate and diphenyldiazomethane was studied. Thus,

room temperature reactions of equimolar amounts of p-bromo-

cyanothioformanilide Id or its iodo- and 3,4-dichloro-derivatives

with triethylamine in diethyl ether as a solvent immediately gave

the corresponding triethylammonium salts IIa–c which crystallized

S

R

N

H

CN

f; R = 3,4-Cl

g; R = p-MeO

I

Fig. 1. Structure of cyanothioformanilide I.

out of solution upon standing (Fig. 3). Interestingly, the ¹H and ¹³

C

techniques. Although these were reported in the literature by us and

others, they were not fully characterized and no NMR spectral data

has yet been reported. Analysis of cyanothioformanilides Ia–g by

NMR (¹H and ¹³C) indicated the presence of tautomeric mixtures of

thione and thiol forms, in which the former predominates. For

instance, the ¹H NMR spectrum of If (tautomeric mixture; major:-

spectra of these salts indicated that each comprised of a mixture of

two tautomeric products whose ratio slightly differed from that of

the starting cyanothioformanilide. For instance, whereas p-bro-

mocyanothioformanilide IIa consisted of a 75:25 mixture of

tautomers, its iodo analogue IIb exhibited a much higher tauto-

meric mixture of 91:9. The dichloro derivative IIc displayed an

intermediate tautomeric ratio of 80:20. The ¹H NMR spectrum of IIa

and the iodo analogue IIb each exhibited a set of two aromatic

doublets for each tautomer (4H), a set of two aliphatic quartets (6H,

J ¼ 7.3 Hz, 3XCH₂), and a set of two aliphatic triplets (9H, J ¼ 7.3 Hz,

3XCH₃). As expected, the ¹H NMR spectrum of IIc showed ﬁve sets

of signals for each tautomer. The proton of the triethylammonium

salt appeared as a broad signal at 6.97–6.65 ppm. The ¹³C spectrum

of the thione tautomer displayed ten signals which was assigned by

DEPT 90 and DEPT 135 to six aromatic carbons, two quaternary

thione and nitrile carbons, and two aliphatic methylene and methyl

carbons.

minor; 84:16) revealed at

d

¼ 11.01 ppm an exchangeable proton

(broad s, 1H, NH) which disappeared upon deuteration with D₂O.

The aromatic ring displayed a set of three signals for the major

tautomer as follows: Ar–H(2) as a doublet (1H, J ¼ 2.5 Hz), Ar–H(5)

as a doublet (J ¼ 8.8 Hz), and Ar–H(6) as doublet of doublets (J ¼ 8.8,

2.5 Hz) (Fig. 2). Protons for the minor tautomer exhibited well

separated chemical shifts and also appeared as a set of three signals

with similar multiplicity patterns and J-values. The ¹³C spectrum of

cyanothioformanilide If showed eight signals for the major form

which were assigned to the structure with the aid of DEPT 90/135 as

well as COSYand HSQC 2D techniques. Also, it showed a farthermost

downﬁeld signal for the thione group (C]S) at

signals corresponding to the CH groups, another three signals

stemming from the quaternary aromatic carbons, and an upﬁeld

d 164.2 ppm, three

Having 3,4-dichlorocyanothioformanilide (If) in hand, we

decided to explore its reactivity at room temperature with hydra-

zine hydrate using ethanol as a solvent. As one might anticipate, the

reaction led, via the intermediacy of A, to the formation of the

thiosemicarbazide derivative III, following the elimination of one

molecule of HCN (Scheme 1). The thiosemicarbazide product (III)

was analyzed by ¹H and ¹³C NMR techniques, where the former

showed the presence of three sets of aromatic proton signals and

two sets of exchangeable signals integrating for four protons,

whereas the latter revealed the presence of seven signals which

were assigned to the C]S group, three quaternary carbons and

three methine aromatic protons. As further evidence to support the

signal resulting from the nitrile group (

spectrum exhibited a molecular ion peak at m/z 203 (100%). In

addition, IR measurements displayed absorptions at:

(cm^ꢀ1) NH

(3250),

(cm^ꢀ1) SH (2600–2550), (cm^ꢀ1) CN (2119) and (cm^ꢀ1

d 114.1 ppm). The mass

n

)

CS–N (1450,1170). Interestingly, the extended conjugation of the

thiol form does not seem to impart enough thermodynamic stability

to drive the equilibrium forward, rendering it the major tautomer.

However, it is noted that cyanothioformanilides Ia, Ib and Ig

comprise a much higher ratio of the thiol form (thione:thiol; 58:42;

56:44; and 57:43 respectively) compared to compounds Ic–f (thio-

ne:thiol; 70:30; 68:32; 63:36; and 84:16 respectively). This is

understandable as the electron-donating substituents in the former

series place a charge in the para position of the aromatic ring which

can only be delocalized in the thiol form to give a stabilized anion

assigned structure, the infrared spectrum showed a broad band at

n

(cm^ꢀ1) 3400–3150 arising from overlapping absorptions of the NH

and NH₂groups.

Unexpectedly, room temperature reaction of an excess of ethe-

real diphenyldiazomethane with p-iodocyanothioformanilide (Ie)

produced the acrylonitrile derivative IV (Scheme 2). Interestingly,

a related reaction involving treatment of thiobenzophenone with

excess diazomethane at 20 ^ꢁC gave 4,4,5,5-tetraphenyl-1,3-dithio-

lane exclusively [39]. The reaction mode consisted of two 1,3-dipolar

cycloadditions separated by a 1,3-dipolar cycloreversion. It was

reported that reverse addition produced 22% yield of 1,1-dipheny-

lethene as a mixture with the corresponding thiirane precursor. A

plausible mechanistic rationale for the formation of product IV

involves a twofold extrusion reaction in which initial formation of

an intermediate 1,3,4-thiadiazoline via a 1,3-dipolar cycloaddition of

positioned

a to the nitrile group. In any case, it appears that the

superior basicity and electronegativity of the thioamide nitrogen

atom renders the thione form more favorable. Accordingly, such

reagents are ambident nucleophiles and may react via the sulfur or

nitrogen atom leading to different heterocyclic cores in ring closure

reactions. Indeed, Ketcham et al. observed that N-substituted

cyanothioformanilides react with alkyl or aryl isothiocyanates to

give 5-imino-l,3-disubstituted-2,4-imidazolidinedithiones or 5-

(phenylimino)-4-imino-2-thiazolidinethiones depending upon the

reaction conditions and the reactants [13].

d (8.16 ppm, J = 2.5 Hz)

d (7.67 ppm, J = 2.5 Hz)

H(2)

H(2) H

Cl

N

CN

SH

Cl

N

CN

S

H(6)

dd (7.70 ppm, J = 8.8, 2.5 Hz)

H(6)

H(5) dd (7.43 ppm, J = 8.8, 2.5 Hz)

d (7.66 ppm, = 8.8 Hz)

H(5)

d (7.62 ppm, J = 8.8 Hz)

J

If

(major)

(minor)

Fig. 2. ¹H NMR splitting pattern and coupling of the aromatic protons of cyanothioformanilide If.

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4317

_

+

Et₃NH

S

R

+

Et₃NH

N

CN

N

CN

_

(minor)

(major)

II

a; R = p-Br (ratio; major:minor 75:25)

b; R = p-I (ratio; major:minor 91:9)

c; R = 3,4-Cl (ratio; major:minor 80:20)

Fig. 3. Reaction products of cyanothioformanilides Ia–c with triethylamine in ether at room temperature.

diphenyldiazomethane to the thione function, is ensued by the

extrusion of molecular nitrogen and formation of a transient thio-

carbonyl ylide. Subsequent rapid electrocyclization of the ylide gives

rise to a thiirane. Related thiirane intermediates have been previ-

ously isolated by our group in the reaction of diphenyldiazomethane

with 5-bromo-2-p-tolylisoindoline-1,3-dithione and 2-phenyl-

isoindoline-1,3-dithione [40]. However, the resulting aromatic spi-

ro[isoindoline-1,2⁰-thiirane]-3-thiones were stable to sulfur

extrusion. In the current case, subsequent extrusion of sulfur affords

the corresponding tetra-substituted acrylonitrile derivative IV.

Clearly, this mode of cyclization leading to a thiirane is more

competitive as the alternative one would lead to a highly hindered

dithiolane product. The preceding transformation is particularly

noteworthy as both intermediates proved unstable to isolation and

directly extruded nitrogen and sulfur under ambient conditions.

This is in sharp contrast to twofold extrusion reactions where

a substrate must be induced thermally or photochemically to

extrude stable species, particularly when the chalcogen atom is

sulfur or selenium. Indeed as reported by Huisgen et al., a t_1/2w 16 h

at 20 ^ꢁC was reported for the sulfur extrusion from a thiirane ring

[39]. As such, this result marks the ﬁrst example of the conversion of

a cyanothioformanilide into an alkene at room temperature and

warrants a separate investigative study into its scope and potential

synthetic utility. It is possible that the driving force toward extrusion

is provided by the exocyclic nitrogen atom of the thiadiazoline and

thiirane intermediates which facilitates the extrusion process by

initial formation of an iminium ion intermediate in each step.

The ¹H NMR spectrum of IV showed an AB system typical of

a para-substituted aromatic system integrating for four protons.

Further, the aniline exchangeable proton appeared as a broad

singlet which disappeared upon treatment with D₂O. Finally, three

sets of aromatic signals integrating for ten protons were observed

for the two phenyl groups. Further analysis of the product by ¹³C

and DEPT experiments indicated the presence of ﬁfteen signals

(6 quaternary aromatic carbons, 8 CH⁰s, and CN). The mass spec-

trum of IV showed a molecular ion peak at m/z 422 (100%, base

peak), suggesting a high stability of the product against fragmen-

tation. The IR spectrum clearly showed the nitrile and NH

stretchings, and more informatively, did not display any C]S

stretch.

At the other end of their reactivity spectrum, the cyanothio-

formanilides were reacted with several electrophilic reagents such

as acetaldehyde. Thus, according to the general protocol of Ketcham

and Schaumann [11], reaction of 3,4-dichlorocyanothioformanilide

(If) with acetaldehyde in the presence of triethylamine produced

the corresponding oxazolidineiminothione V (Scheme 3). The ¹H

NMR spectrum of V exhibited six sets of signals, where the imine

proton displayed the usual broad exchangeable signal, and the

aromatic protons showed their anticipated pattern of two doublets

and one doublet of doublets. More informative were those signals

stemming for the methine and methyl groups which showed up as

a quartet at d 6.09 ppm and as a doublet at d 1.59 ppm, respectively.

Further analysis of the product by ¹³C and DEPT experiments

indicated the presence of ten signals (3 quaternary aromatic

carbons, 3 CH⁰s, O–CH, and CH₃). The IR spectrum clearly showed an

NH stretching at

1490, 1170, respectively.

n n )

(cm^ꢀ1) 3270 and a CS–N stretchings at (cm^ꢀ1

Imidazoles and their fused derivatives are key components of

great many bioactive compounds (such as histidine, purine, biotin

and hydantoin) of both natural and synthetic origin [31]. Hydantoin

derivatives are used in therapy as anticonvulsants and chemo-

therapeutic agents [32]. Antimicrobial activity was reported for

derivatives possessing aromatic substituents at the imidazole

nitrogen. Such active compounds include N-acyl and 5-arylidene

derivatives of hydantoin and 4-thiohydantoin [33–35]. Moreover,

previous reports demonstrated that synthetic imidazoles act either

as inhibitors of alpha-adrenoreceptor mediated events in platelets

[36] or inducers of platelets activation [37].

In continuation with our previous work in this area [22–27], we

decided to prepare and evaluate the biological activity of poly-

halogenated imidazolidineiminothiones containing trichlorophenyl,

chloro-triﬂuromethylphenyl and p-chloro, p-bromo or p-iodo phenyl

groups. As a synthetic entry point to access these derivatives,

heterocyclic ring closing reactions of various aromatic cyanothio-

formanilides (Ia–g) containing 3,4-dichloro-, p-chloro, p-bromo,

p-iodo, p-methyl, p-methoxy, and p-ethoxyphenyl groups were

carried out with aromatic phenyl-, p-chlorophenyl-, p-chloro-3-

I

N

S

Ph₂C=N₂

Ether, r.t.

NC

Cl

S

N

H

CN

S

N

NH₂NH₂

EtOH, r.t.

NH₂

H

Ie

N

H

N

H

N

H

CN

If

III

I

Ph

Ar NH

C

via

C

N

I

S

_

S

N₂

_

N

H

CN

S

NH-NH

+

2

N

IV

H

CN

A

Scheme 1. Reaction of cyanothioformanilide If with hydrazine hydrate in ethanol at

Scheme 2. Reaction of cyanothioformanilide Ie with diphenyldiazomethane in ether at

room temperature.

4318

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

O

Me

Cl

R

O

S

NCO

N

O

Et₃N

ether

Me

H

N

H

CN

Cl

S

Z

S

NH

Et₃N, ether

VII

VIII

V

If

+

a: R = OEt; Z = NH

b: R = Br; Z = NH

O

Scheme 3. Reaction of cyanothioformanilide If with acetaldehyde in ether at room

temperature.

dil. HCl

NH-C-CN

c: R = I;

Z = NH

boiling

EtOH

IX

R

triﬂuromethyl- and 2,4,5-trichlorophenylisocyanates in ether with

triethylamine as a base to furnish the corresponding 5-imino-4-thi-

oxo-2-imidazolidinones (VI) (Scheme 4). These products were puri-

ﬁed by crystallization and obtained in 65–70% yields. Compounds

VIa,h [8], VIb [21] and VIc [5] were previously reported by us but

were not evaluated in any biological assays.

a: R = OEt; Z = O

b: R = Br; Z = O

I

c: R = I;

Z = O

Scheme 5. Preparation of imidazolidineiminothiones containing 9H ﬂuoren moiety.

a broad exchangeable singlet for the imine proton, three aromatic

doublets and one broad signal, as well as two multiplets inte-

grating for eleven protons. More informative were those signals

stemming from the allylic methylene group which appeared as

a singlet and the ethoxy group which exhibited its usual multi-

plicity patter of a quartet and a triplet. The ¹³C NMR, displayed the

expected twenty two signals for C]S, C]N, C]O, C–O, six

quaternary aromatic carbons and nine aromatic CH protons, as

well as two methylene and one methyl carbons. Infrared

measurements of VIIIa showed the presence of an NH

(3251 cm^ꢀ1), C]O (1767 cm^ꢀ1) and C]N (1669 cm^ꢀ1) stretches at

their expected frequencies. The ¹³C spectrum corroborated the

proton and IR data, clearly displaying the expected 22 signals

(C]S, C–O, C]O, C]N, 6C, 9CH, CH₂O, CH₂, and CH₃). Further

structural evidence emerged from the hydrolysis of VIII with

dilute HCl in boiling ethanol which afforded diones IXa–c.

The ¹H NMR spectrum of VIe exhibited ﬁve sets of signals, where

the imine proton displayed the usual broad exchangeable signal,

and the aromatic protons for the p-iodophenyl and 2,4,5-tri-

chlorophenyl groups showed their anticipated pattern of two

doublets and two singlets, respectively. The ¹³C NMR, however,

displayed only twelve of the expected thirteen signals. Further

analysis by DEPT experiments revealed the presence of four CH

groups (6 carbons), one of C]S, C]O, C]N, and C–I, and four

quaternary aromatic carbons. When an inverse gated coupling

experiment was carried out on VIe, the aromatic carbons integrated

for ﬁve, suggesting that the missing aromatic quaternary carbon is

overlapping with one of the other four aromatic carbons. Infrared

measurements clearly showed the presence of NH, C]O, and CS–N

stretchings at their expected frequencies.

The ¹H NMR spectrum of VIf exhibited six sets of signals; broad

singlet for the imine proton, two doublets and one doublet of

doublets for the aromatic protons of 3,4-dichlorophenyl group, and

a multiplet for the p-iodophenyl group. The ¹³C NMR, displayed the

expected thirteen signals for C]S, C]N, C]O, ﬁve quaternary

aromatic carbons and seven aromatic CH protons. The mass spec-

trum exhibited a molecular ion peak at m/z 385 (70%) and a base

peak for a 3,4-dichloroisothiocyanate fragment at m/z 203. The

isotopic distribution of the molecular ion peak clearly corroborates

a molecular structure containing three atoms of chlorine.

In addition to the above compounds, we prepared other new

types of imidazolidineiminothiones (VIII) containing 9H ﬂuoren

moiety (Scheme 5). Thus, reactions of p-ethoxy-, p-bromo-, and p-

iodocyanothioformanilides Ib, Id and Ie with 9H ﬂuoren-2-yliso-

cyanate (VII) in the presence of triethylamine afforded imidazo-

lidineiminothiones VIIIa–c. These were all crystalline solids and

were therefore puriﬁed by recrystallization from ethanol. The ¹H

NMR spectrum of compound VIIIa exhibited ten sets of signals;

The ¹H NMR spectrum of IXc exhibited nine signals integrating

for thirteen protons as expected. This data, however, could not stand

on its own in providing unequivocal proof of structure for the parent

heterocycles VIIIa–c. More convincing evidence stemmed from the

¹³C NMR and DEPT spectra which showed the presence of 20 signals

representing 22 carbon atoms; C]S, 2XC]O, 6C, 9CH, C–I, and CH₃.

More signiﬁcantly, a chemical shift at 180 ppm was indicative of

a C]S moiety which is the only group that could possible possess

such a distinctive downﬁeld shift. The infrared spectrum proved as

useful in tracing the disappearance of the NH stretch in the parent

compound and the appearance of a C]O stretch. It is noted that all

the mass spectra of VIII and IX share a common base peak at m/z

207 stemming from a 2-isocyanato-9H-ﬂuorene fragment.

For the purpose preparing analogues for further structural

evidence of compounds VIIIa–c and their dione analogues, 4-thi-

oxoimidazolidine-2,5-dione (IXa) reacted successfully with excess

O

NCO

R₁

R₂

S

O

BnNH₂/heat

N

R₂

IXa

NH-C-CN

-H₂S

55%

N

R₁

O

Et₃N

ether, r.t.

S

NH

I

X

VI

80-85% yield

R₁

R₂

CF₃

a: p-Me

b: p-OEt

c: p-I

p-Cl

O

Br

O

Br

H

Cl

p-Cl

N

BnNH₂/heat

50%

N

d: p-Br

e: p-I

2,4,5-trichloro

p-Cl

N

O

S

Z

f: 3,4-dichloro

g: p-Br

h: p-OMe

p-Cl,3-(CF₃)

p-Cl

VIg Z = NH

dil. HCl

XII

XI Z = O

Scheme 4. Preparation of imidazolidineiminothiones.

Scheme 6. Preparation of imidazolidinebenzyliminodiones heterocycles.

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4319

benzylamine at the thione moiety to produce the corresponding

benzylimine derivative as a single geometric isomer, following the

elimination of H₂S (Scheme 6). Surprisingly, the ¹H and ¹³C data

were not consistent with the presence of a ﬂuoren moiety. For

instance, the ¹H NMR spectrum indicated the presence of 7 signals,

corresponding to 23 protons. The benzylimine methylene appears

equivalents of cyanothioformanilides Ib with one equivalent of

bis(3,5-diethyl-4-isocyanatophenyl)methane resulted in the

formation of the desired bis-imidazolidineiminothione XIV (Fig. 5).

Similarly, reaction of If and with 4,4⁰-oxybis(isocyanatobenzene)

afforded the novel bis-imidazolidineiminothione XV as a single

regioisomer. The ¹H NMR spectrum of XIV indicated the presence of

9 signals, corresponding to 46 protons. The benzyl methylene

as one singlet, at

d 5.35 ppm indicating one geometric isomer. The

¹³C NMR and DEPT spectra showed the presence of 27 signals

representing 31 carbon atoms; C]N, 2XC]O, 7C, 12 types of

protonated carbons corresponding to 8 CH types, 3 CH₂groups, and

a CH₃. Extensive 1D/2D NMR analysis led us to suggest structure X

as the product. Further structural evidence stemmed from the mass

spectrum which corroborates the spectral data and the proposed

structure, giving a molecular ion peak with m/z 413. In our further

attempts to prepare benzylimine derivatives, compound VIg was

subjected to iminohydrolysis to afford XI, which was subsequently

treated with excess benzylamine in boiling ethanol. Similarly, XII

was isolated as the major product of the reaction and was fully

characterized by spectroscopic and mass spectral techniques.

Proton NMR indicated the presence of two benzylic methylene

appears as one singlet, at d 4.04 ppm in the proton NMR and at

41.7 ppm in the ¹³C carbon, suggesting one regioisomeric product.

The ¹³C NMR and DEPT spectra showed the presence of 15 signals

representing 33 carbon atoms; 2XC]N, 2XC]O, 7C, 8 types of

protonated carbons corresponding to 3 CH types, 3 CH₂and 2 CH₃

groups. Mass spectral data strongly supports the proposed struc-

ture, giving

a molecular ion peak with m/z 774. Similarly,

compound XV was fully characterized and gave satisfactory NMR

and mass spectral data.

We also had a long standing interest in preparing derivatives of

thiohydantoins which have been shown to exhibit a spectrum of

biological and pharmacological activities. Earlier, we reported the

preparation of 4-thiohydantoin derivative XVIa [5] via reduction of

the imine function of the parent iminothione with hydrogen sulﬁde

[7]. We now report the preparation of its 3,4-dichloro derivative

XVIb following the same experimental protocol that had been used

earlier (Fig. 6). Its mass spectrum exhibited a molecular ion base

peak at m/z 370 and the IR clearly showed the absence of the NH

absorbance band present in the parent compound. The product

proved air sensitive as slow and progressive air oxidation to the

corresponding disulﬁde XVII was observed during preparation and

storage. Presumably, tautomerization of the thiohydantoin to the

enethiol, followed by air oxidation leads to the dihydantoin disul-

ﬁde as suggested by the mechanism shown in Scheme 8. Indeed,

when pure thione XVIb was left standing at room temperature, it

underwent partial conversion to give a 2.1:1 mixture of the enethiol

and disulﬁde as determined by the aid of ¹H NMR, ¹³C, DEPT 90/135

as well as COSY and HSQC 2D techniques. As such, we were able to

assign all the observed chemical shifts in the proton and carbon

NMR of the mixture to the enethiol hydantoin tautomer and its

dihydantoin disulﬁde oxidative product.

The imidazolidineiminothiones VIa, VIb and VIf underwent

cyclization with substituted ortho-phenylenediamines to give

sulfur-free products, following the elimination of H₂S and NH₃

molecules (Fig. 7). As anticipated, the products of VIa and VIf

(XVIIIa and XVIIIc, respectively) each consisted of two regioisom-

ers which were formed in almost equal amounts according to ¹H

NMR integration of the methyl and methoxy signals of the

respective mixtures. These were inseparable by column chroma-

tography and were therefore characterized as mixtures. The

molecular ion peak in the mass spectra of XVIIIa–c was the base

peak as well, pointing to the stability of such compounds.

singlets (

respective carbons appearing at

d

5.35 and 4.93 ppm) which were correlated to their

d

53.1 and 43.0. ¹⁹F and ¹³C NMR

data conﬁrmed the absence of the triﬂuoromethyl moiety which

often has a characteristic ﬂuorine chemical shift of ꢀ63 ppm and

a quartet multiplicity in the carbon that usually appears at

122.2 ppm (J ¼ 273.5 Hz).

Formation of X and XII can be rationalized according the

mechanism shown in Scheme 7 for XII. Reaction of the thione

moiety of XI with benzylamine leads to the formation of an imi-

dazolidinebenzyliminodione intermediate, which upon further

reaction with a second molecule of benzylamine affords XII.

Presumably, ring opening is facilitated by the longer reaction time

and excess benzylamine used for the reaction. Interestingly, the

increased steric bulkiness provided by the 9H-ﬂuoren moiety

compared to the p-chloro-3-triﬂuoromethylphenyl group does not

seem to maintain the intactness of the group under the same

reaction conditions.

Imidazolineiminothiones containing a naphthyl group were also

synthesized as single isomers and in good yields via reaction of

cyanothioformanilides Ic, Id and Ig with 1-isocyanatonaphthalene

to produce XIIIa and XIIIb, respectively (Fig. 4). The mass spectrum

of XIIIa exhibited a base peak at m/z 365 which clearly corre-

sponded to the molecular ion peak. In addition, proton and carbon

NMR data were consistent with the proposed structure.

Since a considerable number of bis-heterocyclic compounds

(e.g. bis-quinazoline derivatives) have been reported to exhibit

superior antibacterial and antifungal activities [38] than their

mono-heterocyclic counterparts, we opted to prepare some bis-

imidazolidineiminothione derivatives. Thus, reaction of two

CF₃

F₃C

Cl

O

Br

Cl

O

Br

N

BnNH₂/heat

-H₂S

N

XI

:NH₂Bn

H

N

O

N

CF₃

Cl

+

XII

H₂N

Scheme 7. Mechanism of formation of imine XII.

4320

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

O

R

O

Cl

R

N

S

NH

S

]

XVI

2

XIII

a: R = p-Cl

a: R = Cl

XVII

b: R = 3,4-Cl

b: R = Br

c: R = OMe

Fig. 6. Novel thiohydantoin derivative and its dihydantoin disulﬁde side product.

Fig. 4. Structures of naphthyl-containing imidazolineiminothiones.

We were also interested in preparing imidazotriazines of type

XIX due to an anticipated potential biological activity. Previously

we had anticipated that reactions of the iminothiones VI with

thiosemicarbazide would afford the desired triazine products XIX

(R]NH₂) through elimination of two moles of H₂S as shown in

Scheme 9 [22]. To our dismay, several attempts to prepare XIX

failed and we observed the evolution of only one mole of H₂S. Close

examination of the reaction mixture revealed that the non-cyclized

intermediate product XX had formed almost quantitatively and

apparently resisted cyclization. Attempted ring closure of XX by

reﬂuxing in ethanol also failed.

As an alternate route to the preparation of imidazotriazines,

imidazolidineiminothione VIc [5] and its p-chloro- and p-bromoi-

minothione derivatives [8] were reacted with benzophenehydrazone

and triethylamine in reﬂuxing ethanol. Unfortunately, cyclization did

not proceed under the reaction conditions and we were only able to

isolate the corresponding 4-((diphenylmethylene)hydrazono)-5-

iminoimidazolidin-2-one XXI products (Scheme 10).

R₁

O

R₂

N

+

N

R₃

XVIII

a: R₁= Me;

b: R₁= EtO;

R₂= Cl; R₃= CH₃

R₂= H; R₃= H

c: R₁= 3,4-Cl; R₂= Cl; R₃= H

Fig. 7. Cyclization reactions of imidazolidineiminothiones with ortho-phenylenediamines.

Because of stability issues, the former readily extrudes molecular

nitrogen at room temperature affording XXIV.

Previously, we reported preparation attempts toward imidazo-

triazine of type XXIII from the reactions of imidazolidineimino-

thiones VI with phenyl or diphenyldiazomethane [16] (Fig. 8). This

was based on the premise that addition of the diazomethane and

subsequent loss of sulfur would form the desired triazine. In the

present investigation, reactions of VIa,c,f and their p-ethoxy [21]

analogue with diphenyldiazomethane did not produce the

Finally after extensive experimentations, the desired imidazo-

triazine core could be constructed via the reactions of imidazolidi-

neiminothiones VI and related compounds with thiocarbohydrazide,

eliminating two molecules of hydrogen sulﬁde to produce triazines

XXV as shown in Scheme 11. While triazines XXVa–c are all novel,

XXVd has been reported earlier by our group [8], although it was

never evaluated in any biological assay. In fact, preliminary positive

results obtained by the evaluation of XXVd against certain types of

cancer cell lines (vide post) provided the impetus to search for

synthetic routes (vide supra) to construct these types of systems and

eventually led to the preparation of its triazine analogues XXVa–c.

As further addition to our pool of new heterocycles, we decided

to prepare a series of imidazolidineiminodithiones (Fig. 9). These

types of compounds are known to exhibit enhanced bioactivity

over their imidazolidineiminothione counterparts. Thus, several

dithiones were synthesized via the respective reactions of p-

ethoxy (Ib), p-bromo (Id) and p-iodocyanthioformanilide (Ie) with

phenyl, p-bromo and p-iodophenylisothiocyanate to furnish the

desired 5-imino-2,4-dithioxoimidazolidines (XXVIa–c). Whereas

XXVIa,c were produced and isolated as mixtures of 5-imino-2,4-

imidazolidinedithione and 4,5-diiminothiazolidine, XXVIb was

obtained as a single dithione isomer following recrystallization.

Thus, we elected to utilize the latter for biological testing and we

were also selective in the products we subjected to testing.

imidazotriazines.

Instead,

1-(4-chlorophenyl)-3-(3,4-dichlor-

ophenyl or 4-(substituted)-4-(diphenylmethylene)-5-iminoimida-

zolidin-2-ones (XXIV) was isolated and their structures were

conﬁrmed by spectroscopic and mass spectral techniques.

Presumably, a twofold extrusion reaction involving a [2 þ 3] dipolar

cycloaddition of diphenyldiazomethane to the thione function,

followed by the extrusion of molecular nitrogen and sulfur has

occurred, affording the tetra-substituted alkene XXIV. Alternatively

and more justifying to our approach, an intermediate triazine

structurally similar to XXIII is a plausible precursor to XXIV.

O

N

NH

HN

S

XIV

O

Cl

O

N

Cl

N

S

HS

S

Cl

XVIb

NH

HN

S

O₂oxidation

XV

XVII

Fig. 5. Structures of bis-imidazolidineiminothione derivatives.

Scheme 8. Air oxidation of a thiohydantoin derivative.

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4321

3. Results and discussion

3.2. Antiviral properties

3.1. Antitumor properties

Initially, the maximum non-toxic concentration (MNTC) of

compounds VIe–g, VIII-c, IXa,b and XXVIb on Vero cells was

determined using ﬁve 10-fold serial dilutions of each compound

starting from 10^ꢀ1until 10^ꢀ5dilution of the initial sample

concentration of 5 mg/mL (DMSO). Cells were monitored for any

physical signs of toxicity for three days. The MNTC data is shown in

Table 4 and was selected for further anti-infectivity studies against

hepatitis A virus (HAV), herpes simplex virus 1 (HSV1) and

Coxsackie B4 (COxB4) viral strain. The results are summarized in

Table 4. Imidazolidineiminothiones VIIIc containing a 9H ﬂuorene

moiety was the most effective against all three viral strains,

reducing virus plaque count of each one from 50 to 70%. Interest-

ingly, its bromo derivative VIIIa showed no effect, while its ethoxy

derivative VIIIa was active against HSV1. Notably, the hydrolysis

product of the latter thioxoimidazolidinedione IXa proved highly

active against all tested strains, while the hydrolysis derivative of

the former IXb affected HSV1 only. Imidazolidineiminothiones VIe

and VIg also showed activity against HAV and HSV1, respectively,

while the related analogue VIf was inactive. Finally, imidazolidi-

neiminodithione XXVIb was effective against HSV1.

The following set of compounds XIV, XVIa, XVIIIa,c, XXVd

and XXVIb were initially subjected to screening tests against

Ehrlich Ascites Carcinoma cells (EAC). They were then evaluated

for cytotoxic activity against various tumor cell lines (brain

tumor cell line U251, human hepatocellular carcinoma cell line

HEPG2, human breast adenocarcinoma cell line MCF-7, human

epithelial carcinoma cell line HELA and colon carcinoma cell line

HCT116).

3.1.1. Screening of compounds XIV, XVIa, XVIIIa,c, XXVd

and XXVIb against EAC cells

Compounds XIV, XVIa, XVIIIa,c, XXVd and XXVIb were screened

against EAC cells at concentrations of 100, 50 and 25

shown in Table 1. All compounds produced over 90% inhibition of

cell viability at the 100 g/mL concentration. Compounds XVIa and

mg/mL as

m

XXVd were highly effective at all concentration range and only

marginal drop in % inhibition of cell viability was observed with

decreasing concentration. On the other hand, % inhibition of cell

viability was more sensitive to concentration changes with the

remaining compounds as more pronounced drop was observed at

lower dose.

3.3. Antimicrobial properties

3.1.2. Cytotoxic activity of compounds XIV, XVIa, XVIIIa,c, XXVd

and XXVIb against various tumor cell lines

Two sets of heterocycles were selected and compounds within

each series were tested under the same conditions for antimicrobial

activity against Gram negative and Gram positive bacteria as

follows: series 1: VId–g, VIIIa–c and IXa–b; series 2: If, IIc, V, VIf,

XIIIa, XIV, XV, XVIb and XXVIb,c.

The cytotoxicity of compounds XIV, XVIa, XVIIIa,c, XXVd and

XXVIb was evaluated against brain tumor cell line U251, human

hepatocellular carcinoma cell line HEPG2, human breast adeno-

carcinoma cell line MCF-7, human epithelial carcinoma cell line

HELA and colon carcinoma cell line HCT116. The median inhibition

concentration (IC50) values are shown in Table 2 and the results are

summarized in Table 3 and represented graphically in Fig. 10.

The IC50 data shown in Table 2 and the dose response curves of

Fig. 10 clearly underscore the superiority of bis-imidazolidineimi-

nothione XIV against U251, MCF7, HELA and HCT116 and point to

the effectiveness of triazine XXVd against HEPG2 cell lines. Notably,

5-imino-2,4-imidazolidinedithione XXVIb was markedly active

against U251, MCF7 and HCT116 cell line. Generally, all cell lines

were responsive to dose increase as indicated by the ensuing

reduction in surviving fraction, although dose response was vari-

able as seen in Table 3.

3.3.1. Antimicrobial activities of compounds VId–g, VIIIa–c and

IXa–b against Gram negative and Gram positive bacteria

The following ﬁrst series of selected compounds VId–g, VIIIa–c

and IXa–b were tested in vitro for antibacterial activity against the

following bacterial strains: Gram-negative bacteria, Escherichia coli

NCTC 10416, Salmonella typhi NCIMB 9331, and Gram positive

bacteria, B. subtilis NCTC 1040, S. aureus NCTC 7447. The results are

summarized in Table 5 and represented graphically in Fig. 11. The

compounds were tested for antimicrobial activity by the agar disk

diffusion technique using a 1 cm microplate well diameter and

a 100 mL of each concentration. Chloramphenicol was used as

a standard antibacterial agent. All compounds were dissolved in

O

Ar

Ar'

N

R

N

EtOH

X

-2H₂S

XIX

O

H

boil. EtOH

O

N

NH₂

Ar

X

Ar'

+

-H₂S

H₂N

N

Ar

S

Ar'

S

N

S

NH

EtOH

-H₂S

N

NH

VI

NH

NH₂

XX

Scheme 9. Attempted preparation of imidazotriazines XIX.

4322

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

O

Ar

Ar'

EtOH/TEA

-H₂S

N

Ph

Ar'

Ar

+

H₂N

N

Ph

NH

N

NH

VIc

S

N

Ar

Ar'

Ph

a: p-C₆H₄I p-C₆H₄Cl

b: p-C₆H₄Cl p-C₆H₄Cl

c: p-C₆H₄Br p-C₆H₄Cl

Ph

XXI

O

N

O

Ar'

Ar

Ar'

Ar

N

+ B

X

+ B

-BH⁺

NH

Ph

NH

Ph

N

-BH⁺

N

Ph

XXII

Scheme 10. Attempted preparation of imidazotriazines XXII

N,N-dimethylformamide (5 mg/mL) except compounds VIg and

IXb which were concentrated at 4 and 1 mg/mL respectively.

The data displayed in Table 5 and Fig. 11 show that all hetero-

cycles tested in the antimicrobial assays possess signiﬁcant anti-

bacterial activities against the growth of E. coli and S. typhi on solid

media, where compounds VIf and IXa were found to possess the

highest antibacterial activities in the series against Gram-negative

bacteria. Substituting the para bromine substituent in VId with an

iodine atom as in VIe had negligible effect on activity. Further

reﬁnement of substituents on both aryl groups as in VIf,g produced

slight improvement in activity against E. coli. While compounds

VIe, VIIIa and IXa were equally as effective against B. subtilis, VIIIa

showed the highest antibacterial activity against both strains of

Gram-positive bacteria in the series. Interestingly, the hydrolysis

product (IXa) of compound VIIIa showed a relatively enhanced

activity against the Gram negative bacteria E. coli and reduced

activity against the Gram positive bacteria S. aureus. It is noted that

exchanging the ethoxy group of IXa with a bromine atom as in IXb

resulted in a signiﬁcant drop in activity against both classes of

bacteria.

responsive and increased signiﬁcantly with concentration increases

of compound V. It is noted that compounds XVIb and XXVIb show

distinct antimicrobial activities and rank third in activity against

P. aeruginosa and B. subtilis, respectively.

3.4. Antifungal properties

The same two sets of heterocycles that were tested for antimi-

crobial activity were selected (except IIc) and compounds within

each series were tested under the same conditions for antifungal

activity using C. albicans IMRU 3669 and A. niger IMI 31276 as

unicellular and ﬁlamentous fungi. The two sets are as follows:

series 1: VId–g, VIIIa–c and IXa,b; series 2: If, V, VIf, XIIIa, XIV, XV,

XVIb and XXVIb,c.

3.4.1. Antifungal activities of compounds VId–g, VIIIa–c

and IXa–b against C. albicans and A. niger

The following ﬁrst series of selected compounds VId–g, VIIIa–c

and IXa,b were tested in vitro for antifungal activity against the

following fungal strains: C. albicans IMRU 3669 and A. niger IMI

31276. The results are summarized in Table 7 and represented

graphically in Fig. 13. The compounds were tested for antifungal

activity by the agar disk diffusion technique using a 1 cm microplate

3.3.2. Antimicrobial activities of compounds If, IIc, V, VIf, XIIIa,

XIV, XV, XVIb and XXVIb,c against Gram negative and Gram

positive bacteria

well diameter and a 100 mL of each concentration. Grisoﬂuvin was

The following second series of selected compounds If, IIc, V, VIf,

XIIIa, XIV, XV, XVIb and XXVIb,c were tested in vitro for antibac-

terial activity against the following bacterial strains: Gram-negative

bacteria, Pseudomonas aeruginosa NCIB 9016, and Gram-positive

bacteria, B. subtilis NCIB 3610. The results are summarized in Table 6

and represented graphically in Fig. 12. The compounds were tested

for antimicrobial activity by the agar disk diffusion technique under

the same conditions of the ﬁrst series of compounds with the

exception that antimicrobial activities were measured using three

different sample concentrations. All compounds were dissolved in

N, N-dimethylformamide at 1, 2.5 and 5 mg/mL concentrations.

The data displayed in Table 6 and Fig. 12 show that all of the

compounds surveyed in the antimicrobial assays possess signiﬁcant

antibacterial activities against the growth of P. aeruginosa and

B. subtilis on solid media. Cyanothioformanilide If was remarkably

active at all concentrations and possessed the highest antibacterial

activity in the series against both, Gram negative and Gram positive

bacteria except at the 5 mg/mL concentration for P. aeruginosa.

Interestingly, oxazolidineiminothione V which is derived from

cyanothioformanilide If exhibited the highest activity against

P. aeruginosa at the 5 mg/mL concentration and ranked second in its

activity against B. subtilis. Further, antimicrobial activity was most

used as a standard antifungal agent. All compounds were dissolved

in N,N-dimethylformamide (5 mg/mL) except compounds VIg and

IXb which were concentrated at 4 and 1 mg/mL respectively.

All of the tested compounds were found to possess antifungal

activity against the two microorganisms. In particular, imidazoli-

dineiminothiones VIe and VIf exhibited the highest antifungal

activities and were almost equally as effective against C. albicans

and A. niger. It is noted that compound VIIIa was also highly

effective against A. niger but was not as potent against C. albicans.

R

O

Cl

N

Ar

Ar'

N

R

R₁

R₂

a: p-Me

b: p-I

NH

N

c: 3,4-Cl

d: p-OEt

XXIII

XXIV

Fig. 8. Reaction products of

a selection of imidazolidineiminothiones VI with

diphenyldiazomethane.

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4323

R₁

O

R₁

R₂

O

H

N

H

N

R₂

N

+

H₂N

NH₂

N

-H₂S

S

N

NH

S

NH

HS

R₂

HN NH₂

R₁

O

R₂

R₁

N

-H₂S

a: p-I

H

b: 3,4-Cl

c: p-MeO

d: p-Cl

H

N

XXV

Cl

NHNH₂

Scheme 11. Preparation of a series of imidazotriazines.

3.4.2. Antifungal activities of compounds If, V, VIf, XIIIa, XIV, XV,

XVIb and XXVIb,c against C. albicans and A. niger

Committee for Clinical and Laboratory Standards (NCCLS), against

a series of 4 bacterial strains and 2 fungal species. To carry out the

antimicrobial and antifungal evaluation, concentrations of

The following second series of selected compounds If, V, VIf,

XIIIa, XIV, XV, XVIb and XXVIb,c were tested in vitro for antifungal

activity against the same fungal organisms used earlier (C. albicans

and A. niger Ferm-BAM C-21). The results are summarized in Table 8

and represented graphically in Fig. 14. The compounds were tested

for antifungal activity by the agar disk diffusion technique under

the same conditions of the ﬁrst series of compounds with the

exception that antimicrobial activities were measured using three

different sample concentrations. All compounds were dissolved in

N,N-dimethylformamide at 1, 2.5 and 5 mg/mL concentrations.

All tested samples were found to possess antifungal activity

against both microorganisms. As observed earlier in antimicrobial

testing, cyanothioformanilide If was also remarkably active at all

concentrations and possessed the highest antifungal activity in the

series against both C. albicans and A. niger. Also as before, the

derivative of cyanothioformanilide If, oxazolidineiminothione V

showed high antifungal activity at all concentrations and ranked

second in activity and was relatively more potent against C. albicans.

Both If and V produced dramatic enhancement in antifungal activity

with concentration increase, whereas the remaining species

showed only marginal improvement with the exception of XVIb.

Although the latter was not tested against A. niger, it showed distinct

antimicrobial activity against C. albicans and ranked third in activity.

In addition to the preliminary evaluation of the potential bio-

logical activities of a large selection of the preceding heterocycles

by the agar disk diffusion technique, we tested the antimicrobial

and antifungal activity of a narrow selection of potential leads by

evaluating their minimum inhibitory concentrations (MIC). Thus,

the antibacterial and antifungal properties of compounds XXVIb,c,

XVIb and If were evaluated by the macrotube dilution procedure

(Table 9) following the guidelines of the Clinical and Laboratory

Standard Institute (CLSI) [41,42] (Table 9), formerly National

compounds up to 667 mg/mL were incorporated to growth media

according to published procedures [39,42]. As shown in Table 9, all

compounds tested showed strong inhibitory effect, low enough to

render them as targets for further studies. Interestingly, both 5-

imino-2,4-dithioxoimidazolidine XXVIb and it diiodo analogue

XXVIc showed similar MIC values, pointing to a negligible effect

achieved by exchanging the bromine by another halogen atom.

They both showed strong MIC values for the Gram negative bacteria

S. typho (MIC 1.95

mg/mL) and the fungi C. albicans (MIC 1.95

mg/mL)

and A. niger (MIC 0.49

mg/mL). Slightly higher MIC values were

observed for the rest of the bacterial strains, but low enough to

warrant further investigation. The thiohydantoin derivative XVIb

showed high activity against the Gram negative bacteria S. typho

(MIC 0.49 mg/mL) and the fungus C. albicans (MIC 0.97 mg/mL) and

slightly higher MIC values for B. subtilis and K. pneumonia. On the

other hand, moderate MIC values were observed for the remaining

strains. Finally, the cyanothioformanilide If showed the most

consistency in yielding very strong MIC values (ranging from as low

as 0.24–3.9 mg/mL) for the ﬁrst ﬁve strains shown in Table 9 and

a moderate value for A. niger (15.6 mg/mL).

4. Conclusion

In summary, the synthesis and characterization of a new series

of mono- and bis-imidazolidineiminothiones and imidazolidinei-

minodithiones have been described. Several heterocycles were also

derivatized for structural elucidation and biological evaluation

purposes. Most compounds displayed antitumor, antiviral, anti-

bacterial and antifungal activities and could therefore serve as lead

chemical entities for further modiﬁcation to render them clinically

useful drug agents.

Table 1

S

R₂

R₁

Inhibition of EAC cell viability.

N

Sample no.

% Inhibition of cell viability

g/mL

m

S

NH

100

50

80

100

60

70

100

70

25

XXVI

XIV

XVIa

XVIIIa

XVIIIc

XXVd

XXVIb

90

100

90

95

100

90

60

90

35

30

95

50

R₁

R₂

a: p-OEt

b: p-Br

c: p-I

p-I

p-Br

p-I

Fig. 9. A series of novel imidazolidineiminodithiones.

4324

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

Table 2

5.1.1. For the preparation of the known cyanothioformanilides Ia–g,

see Ref. [30]

IC50 values of compounds XIV, XVIa, XVIIIa,c, XXVd and XXVIb obtained against

cell lines U251, HEPG2, MCF7, HELA and HCT116.

5.1.1.1. 4-Tolylcarbamothioyl cyanide (Ia). Tautomeric ratio 58:42;

¹H NMR (CDCl₃, 400 MHz)

d 9.61 (br s, 1 H, NH, major and minor),

Sample no.

IC50 (mg/mL)

7.66 (d, J ¼ 8.5 Hz, 2H, Ar–H, major), 7.28–7.26 (m, 4H, Ar–H, minor),

Cell line

U251

7.24 (d, J ¼ 8.5 Hz, 2H, Ar–H, major), 2.38 (s, 3H, minor), 2.37 (s, 3H,

HEPG2

MCF7

HELA

HCT116

major); ¹³C NMR (CDCl₃, 100 MHz)

d 165.6 (C]S, major), 161.4

XIV

XVIa

XVIIIa

XVIIIc

XXVd

XXVIb

1.01

ND^a

NA^b

NA

ND

1.28

5.44

NA

4.43

7.11

1.54

NA

2.15

9.36

NA

2.42

NA

8.32

7.38

NA

4.36

ND

NA

(C]N, minor), 139.3 (Me–C, minor), 138.5 (Me–C, major), 134.4 (C–

N, major), 134.3 (C–N, minor), 130.5 (o-CH, minor), 129.8 (o-CH,

major), 122.8 (m-CH, minor), 122.4 (m-CH, major), 113.6 (CN,

major), 112.0 (CN, minor), 21.2 (CH₃, major), 21.1 (CH₃, minor).

3.09

8.93

4.56

a

ND: Not determined.

NA: Could not determine from data as surviving fraction was over 0.50 at the

5.1.1.2. (4-Ethoxyphenyl)carbamothioyl cyanide (Ib). Tautomeric

b

ratio 56:44; ¹H NMR (CDCl₃, 400 MHz)

d 9.78 (br s, 1 H, NH, major),

maximum sample dose of 10 mg/mL.

9.63 (br s, 1 H, NH, minor), 7.72 (d, J ¼ 9.0 Hz, 2H, Ar–H, major),

7.31–7.25 (m, 2H, Ar–H, minor), 6.97–6.89 (m, 2H, Ar–H, major and

minor), 4.05 (q, J ¼ 7.0 Hz, 2H, major and minor), 1.43 (t, J ¼ 7.0 Hz,

3H, minor),1.42 (t, J ¼ 7.0 Hz, 3H, major); ¹³C NMR (CDCl₃,100 MHz)

5. Experimental

d

165.7 (C]S, major), 160.6 (C]N, minor), 159.3 (O–C, minor),158.3

5.1. Chemistry

(O–C, major), 129.9 (C–N, major), 129.7 (C–N, minor), 124.8 (m-CH,

minor), 124.0 (m-CH, major), 115.5 (o-CH, minor), 114.9 (o-CH,

major), 113.6 (CN, major), 112.0 (CN, minor), 64.6 (OCH₂, minor),

63.9 (OCH₂, major), 14.7 (CH₃, major), 14.6 (CH₃, minor).

IR spectra were recorded (KBr) on a Perkin Elmer 1650 spec-

trophotometer. ¹H NMR and ¹³C NMR spectra were recorded on

Avance II Bruker FT NMR spectrometer 400 (400 MHz) using CDCl₃

or DMSO-d₆as solvents and TMS as an internal standard. Chemical

5.1.1.3. (4-Chlorophenyl)carbamothioyl cyanide (Ic). Tautomeric

ratio 70:30; ¹H NMR (CDCl₃, 400 MHz)

d 9.81 (br s, 1 H, NH,

shifts are expressed as d ppm units. Mass spectra were recorded on

Shimadzu GC-MS QP 100 EX (70 eV) at the Micro Analytical Center

at Cairo University. Found: C, H, N & S for all compounds were

within ꢂ0.3 from the theoretical value. Melting points were

obtained on a Fisher–Johns melting points apparatus and are

uncorrected. The physical data of the synthesized compounds are

shown in Table 10.

major), 9.54 (br s, 1 H, NH, minor), 7.77 (d, J ¼ 9.0 Hz, 2H, Ar–H,

major), 7.45 (d, J ¼ 9.0 Hz, 2H, Ar–H, minor), 7.41 (d, J ¼ 9.0 Hz,

2H, Ar–H, minor), 7.34 (d, J ¼ 9.0 Hz, 2H, Ar–H, major); ¹³C NMR

(CDCl₃, 100 MHz)

d 165.6 (C]S, major), 161.6 (C]N, minor),

135.4 (C–Cl, major), 135.3 (C–N, major), 134.8 (C–N, major), 133.5

(C–Cl, minor), 130.2 (o-CH, minor), 129.5 (o-CH, major), 124.2

(m-CH, minor), 123.6 (m-CH, major), 113.4 (CN, major), 111.8 (CN,

minor).

5.1.1.4. (4-Bromophenyl)carbamothioyl cyanide (Id). Tautomeric

ratio 68:32; ¹H NMR (CDCl₃, 400 MHz)

d 9.57 (br s, 1 H, NH, major

Table 3

Evaluation of compounds XIV, XVIa, XVIIIa,c, XXVd and XXVIb against brain tumor

cell line U251, human hepatocellular carcinoma cell line HEPG2, human breast

adenocarcinoma cell line MCF-7, human epithelial carcinoma cell line HELA and

colon carcinoma cell line HCT116.

and minor), 7.70 (d, J ¼ 9.0 Hz, 2H, Ar–H, major), 7.61 (d, J ¼ 9.0 Hz,

2H, Ar–H, minor), 7.57 (d, J ¼ 9.0 Hz, 2H, Ar–H, major), 7.29 (d,

J ¼ 9.0 Hz, 2H, Ar–H, minor); ¹³C NMR (CDCl₃, 100 MHz)

d 165.5

(C]S, major), 161.8 (C]N, minor), 135.8 (C–Br, major), 135.7 (C–Br,

minor), 133.2 (o-CH, minor), 132.5 (o-CH, major), 124.3 (m-CH,

minor), 123.8 (m-CH, major), 122.6 (C–N, minor), 121.4 (C–N,

major), 113.4 (CN, major), 111.8 (CN, minor).

Cell line

Sample conc.

g/mL)

Surviving fraction

(

m

XIV

XVIa

XVIIIa

XVIIIc

XXVd

XXVIb

U251

0

1

2.5

5.0

10.0

0

1.00

0.51

0.31

0.28

0.26

1.00

0.76

0.67

0.65

0.39

1.00

0.77

0.48

0.44

1.00

0.67

0.49

0.44

0.39

1.00

0.78

0.64

0.46

0.40

ND^a

ND

1.00

0.98

0.91

0.86

0.76

1.00

0.94

0.56

0.49

0.41

1.00

0.81

0.75

0.71

0.67

1.00

0.97

0.92

0.64

0.42

1.00

0.95

0.84

0.56

1.00

0.98

0.94

0.80

0.72

1.00

0.87

0.54

0.52

0.51

1.00

0.87

0.73

0.70

0.67

1.00

0.94

0.85

0.66

0.40

1.00

0.92

0.80

0.67

0.62

ND

1.00

0.53

0.46

0.33

0.31

1.00

0.92

0.85

0.84

0.71

1.00

0.83

0.54

0.36

0.28

1.00

0.67

0.65

0.64

0.46

1.00

0.88

0.64

0.44

0.42

5.1.1.5. (4-Iodophenyl)carbamothioyl cyanide (Ie). Tautomeric ratio

64:36; IR (KBr) 3250 (NH), 3080 (CH arom.), 2225 (CN) cm^ꢀ^{1 1}H

;

ND

NMR (CDCl₃, 400 MHz)

d 9.44 (br s, 1H, NH, both isomers), 8.11 (d,

HEPG2

MCF7

1.00

0.61

0.65

0.59

0.52

1.00

0.71

0.59

0.58

0.49

ND

1.00

0.56

0.46

0.50

0.49

1.00

1.01

0.66

0.67

1.00

0.97

0.92

0.64

0.42

1.00

0.95

0.84

0.56

1

J ¼ 8.8 Hz, 2H, Ar–H, minor), 7.78 (d, J ¼ 8.8 Hz, 2H, Ar–H, major),

2.5

5.0

10.0

0

7.56 (d, J ¼ 8.8 Hz, 2H, Ar–H, major), 7.16 (d, J ¼ 8.8 Hz, 2H, Ar–H,

minor); ¹³C NMR (CDCl3, 100 MHz)

d 160.4 (C]S), 152.1 (C]N),

152.0, 139.1 (o-CH, minor), 138.5 (o-CH, major), 124.3 (m-CH,

minor), 123.9 (m-CH, major), 125.7 (CH), 119.7 (CN), 93.8 (C–I), 92.7

(C–I); MS (m/z, %) 261 (33, M þ –HCN), 219 (30), 134 (14).

1

2.5

5.0

10.0

0

5.1.1.6. (3,4-Dichlorophenyl)carbamothioyl cyanide (If). Tautomeric

HELA

ratio 84:16; IR (KBr) 3275 (NH), 3090 (CH arom.), 2240 (CN) cm^ꢀ1

;

1

2.5

5.0

10.0

0

¹H NMR (CD₃CN, 400 MHz)

d

11.01 (br s, 1H, NH), 8.16 (d, J ¼ 2.5 Hz,

1H, Ar–H, major), 7.70 (dd, J ¼ 8.8, 2.5 Hz, 1H, Ar–H, major), 7.67 (d,

J ¼ 2.5 Hz, 1H, Ar–H, minor), 7.66 (d, J ¼ 8.8 Hz, 1H, Ar–H, minor),

7.62 (d, J ¼ 8.8 Hz, 1H, Ar–H, major), 7.43 (dd, J ¼ 8.8, 2.5 Hz, 1H, Ar–

HCT116

1

H, minor); ¹³C NMR (CD₃CN, 100 MHz)

d 164.2 (C]S, major), 138.0

2.5

5.0

10.0

(C, minor), 137.7 (C–N), 133.0 (C), 132.7 (C, minor), 132.3 (CH,

minor), 131.9 (CH, major), 131.5 (C), 126.1 (CH, minor), 125.1 (CH,

major), 124.2 (CH, minor), 123.5 (CH), 114.1 (CN, major), 114.1

ND

a

ND: Not determined.

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4325

1.2

1.1

1

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

1

2.5

5

10

Concentration (µg/mL)

XIV-U251

XIV-HEPG2

XVIIIa-U251

XVIIIc-HEPG2

XXVd-HELA

XIV-MCF7

XIV-HELA

XIV-HCT116

XVIIIa-HELA

XVIIIc-HCT116

XXVIb-HEPG2

XVIa-HEPG2

XVIIIa-HCT116

XXVd-HEPG2

XXVIb-MCF7

XVIa-MCF7

XVIIIc-U251

XXVd-MCF7

XXVIb-HELA

XVIIIa-HEPG2

XVIIIc-MCF7

XXVd-HCT116

XVIIIa-MCF7

XVIIIc-HELA

XXVIb-U251

XXVIb-HCT116

Fig. 10. Cytotoxicity of XIV, XVIa, XVIIIa,c, XXVd and XXVIb against brain tumor cell line U251, human hepatocellular carcinoma cell line HEPG2, human breast adenocarcinoma

cell line MCF-7, human epithelial carcinoma cell line HELA and colon carcinoma cell line HCT116.

(CN, minor). MS (m/z, %) 203 (100, M þ –HCN), 204 (19), 205 (73),

5.1.2.1. Triethylammonium (4-bromophenyl)carbamothioyl cyanide

(IIa). Tautomeric ratio 75:25; ¹H NMR (CDCl₃, 400 MHz)

7.47 (d,

206 (12), 207 (28), 161 (16).

d

J ¼ 8.8 Hz, 2H, Ar–H, major and minor), 7.19 (d, J ¼ 8.8 Hz, 2H, Ar–H,

major), 6.95 (d, J ¼ 8.8 Hz, 2H, Ar–H, minor), 6.37–5.89 (br s, 1H,

NH), 3.07 (q, J ¼ 7.3 Hz, 6H), 1.31 (t, J ¼ 7.3 Hz, 9H).

5.1.1.7. (4-Methoxyphenyl)carbamothioyl cyanide (Ig). Tautomeric

ratio 57:43; ¹H NMR (CDCl₃, 400 MHz)

d 9.62 (br s, 1 H, NH, major),

9.51 (br s, 1 H, NH, minor), 7.72 (d, J ¼ 9.0 Hz, 2H, Ar–H, major), 7.31

(d, J ¼ 9.0 Hz, 2H, Ar–H, minor), 9.67 (d, J ¼ 9.0 Hz, 2H, Ar–H,

minor), 6.95 (d, J ¼ 9.0 Hz, 2H, Ar–H, major), 3.85 (s, 3H, minor),

5.1.2.2. Triethylammonium (4-iodophenyl)carbamothioyl cyanide

(IIb). Tautomeric ratio 91:9; ¹H NMR (CD₃CN, 400 MHz)

d 7.67 (d,

3.84 (s, 3H, major); ¹³C NMR (CDCl₃, 100 MHz)

d

165.9 (C]S,

J ¼ 8.5 Hz, 2H, Ar–H, minor), 7.60 (d, J ¼ 8.8 Hz, 2H, Ar–H, major), 7.02

(d, J ¼ 8.8 Hz, 2H, Ar–H, major), 6.79 (d, J ¼ 8.8 Hz, 2H, Ar–H, minor),

3.83–2.76 (br s, 1H, NH), 3.09 (q, J ¼ 7.3 Hz, 6H), 1.22 (t, J ¼ 7.3 Hz,

minor), 161.0 (C]N), 159.9 (O–C, minor), 159.0 (O–C, major), 129.9

(C–N, major), 129.8 (C–N, minor), 124.9 (m-CH, minor), 124.2 (m-

CH, major), 115.0 (o-CH, minor), 114.4 (o-CH, major), 113.7 (CN,

major), 112.0 (CN, minor), 55.7 (OCH₃, minor), 55.6 (OCH₃, major).

9H); ¹³C NMR (CD₃CN, 100 MHz)

d 160.4 (C]S), 152.1, 152.0, 138.9

(CH, minor), 138.1 (CH), 125.7 (CH), 119.7 (CN), 86.4 (C–I), 47.8 (CH₂),

9.3 (CH₃).

5.1.2. Preparation of compounds IIa–c; the triethylammonium salts

were prepared according to the following general procedure

Equimolar amounts (5.0 mmol) of the corresponding cyano-

thioformanilide and triethylamine in dry diethyl ether (20 mL)

were stirred at room temperature for 25 min. The resulting salt was

ﬁltered off, washed with a minimum amount of ether, air-dried and

recrystallized to give IIa–c (Table 10).

5.1.2.3. Triethylammonium (3,4-dichlorophenyl)carbamothioyl

cyanide (IIc). Tautomeric ratio 80:20; ¹H NMR (CDCl₃, 400 MHz)

d

7.39 (d, J ¼ 2.3 Hz, 1 H, Ar–H), 7.38 (d, J ¼ 8.6 Hz, 1H, Ar–H), 7.07

(dd, J ¼ 8.6, 2.3 Hz, 1H, Ar–H), 7.03 (d, J ¼ 2.5 Hz, 1H, Ar–H, minor),

6.97–6.65 (br s, NH), 6.81 (dd, J ¼ 8.6, 2.5 Hz, 1H, Ar–H, minor),

3.83–2.26 (br s, 1H, NH), 3.15 (q, J ¼ 7.4 Hz, 2H), 1.36 (t, J ¼ 7.4 Hz,

3H); ¹³C NMR (CDCl₃, 100 MHz)

d 159.8 (C]S, major), 157.0 (C]S,

minor), 149.0 (C, major), 149.8 (C, minor), 132.7 (C, minor), 132.2 (C,

Table 4

Anti-infectivity effect of compounds VIe–g, VIII–c, IXa,b and XXVIb against HAV,

HSV1 and COxB4 viral strains.

Table 5

Selected dilution^a

Anti-infectivity effect

Viral strain

Antimicrobial effect of compounds VId–g, VIIIa–c and IXa–b against Gram negative

and Gram positive bacteria.

Sample no.

Compound no.

Mean values of inhibition zone diameter (mm)

HAV

HSV1

CoxB4

E. coli

S. typhi

B. subtilis

S. aureus

VIe

VIf

VIg

VIIIa

VIIIb

VIIIc

IXa

IXb

XXVIb

10^ꢀ4

10^ꢀ3

10^ꢀ4

10^ꢀ3

10^ꢀ2

10^ꢀ4

40%

ꢀve

50%

ꢀve

ꢀve^b

ꢀve

50%

ꢀve

70%

50%

40%

50%

ꢀve

50%

60%

ꢀve

VId

VIe

VIf

16.0

16.5

19.0

18.0

17.0

14.0

15.0

19.0

11.0

18.0

17.0

16.5

18.0

16.0

17.0

17.5

12.0

16.0

17.0

16.0

17.0

13.0

13.5

17.0

14.0

16.5

18.0

16.0

17.5

19.0

ND^a

13.5

15.5

13.0

VIg

VIIIa

VIIIb

VIIIc

IXa

IXb

a

Sample dilution at which no cytotoxicity on Vero cells was detected for 3 days.

No anti-infectivity effect was observed.

b

a

ND: Not determined.

4326

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

(m, 3H), 7.23–7.19 (m, 2H), 6.72 (d, J ¼ 8.8 Hz, 2H), 5.43 (br s, 1H,

NH); ¹³C NMR (CDCl3, 100 MHz)

143.7 (C), 141.5 (C), 138.2 (CH),

137.6 (C), 137.4 (C), 130.1 (CH), 129.3 (2CH), 129.2 (CH), 129.0

(CH), 128.4 (CH), 118.4 (CH), 115.8 (C), 109.7 (CN), 84.1 (C–I); MS

(m/z, %) 422 (100, M^þ), 295 (11), 77 (17)

20

18

16

14

12

10

8

d

5.1.2.6. 3-(3,4-Dichlorophenyl)-5-imino-2-methyloxazolidine-4-

thione (V). To a solution of If (5 mmol) in ether (25 mL), acetalde-

hyde (5 mmol) was added, followed by three drops of triethylamine.

The reaction mixture was stirred for 25 min. The obtained product

was ﬁltered off, washed with a minimum amount of ether, air-dried

and recrystallized to give V (Table 10): IR (KBr) 3270 (NH), 3090 (CH

6

4

2

arom.), 1490 & 1170 (CSN) cm^ꢀ1; ¹H NMR (CDCl3, 400 MHz)

d 8.83

0

VIf

VId

VIe

VIg VIIIa VIIIb VIIIc IXa

Compound number

IXb

(br s, 1 H, NH), 7.68 (d, J ¼ 2.5 Hz, 1 H, Ar–H), 7.64 (d, J ¼ 8.8 Hz, 1 H,

Ar–H), 7.38 (dd, J ¼ 8.8, 2.5 Hz, 1 H, Ar–H), 6.09 (q, J ¼ 5.6 Hz, 1 H,

CH),1.59 (d, J ¼ 5.6 Hz, 3 H, CH3); ¹³C NMR (CDCl3,100 MHz)

d 176.8

E. coli

B. subtilis

S. aureus

S. typhi

(C]S), 164.7 (C]N), 134.7 (C), 134.2 (C), 133.8 (C), 131.7 (CH), 127.1

(CH), 124.4 (CH), 91.2 (OCH), 20.3 (CH₃); MS (m/z, %) 274 (<1, M^þ),

127 (100).

Fig. 11. Antibacterial activities of compounds VId–g, VIIIa–c and IXa–b against Gram

negative and Gram positive bacteria.

5.1.3. Preparation of compounds VIa–g, VIIIa–c and XIIIa–c; the

preceding compounds were prepared according to the following

general procedure

A solution of the corresponding cyanothioformanilide (5 mmol)

in ether (15 mL) was treated with a solution of the respective

isocyanate (0.005 mol) in ether (10 mL), followed by three drops of

triethylamine. The reaction mixture was magnetically stirred for

25 min. The obtained product was ﬁltered off, washed with

a minimum amount of ether, air-dried and recrystallized to give

VIa–g, VIIIa–c and XIIIa–c (Table 10).

major), 130.8 (CH, minor), 130.3 (CH, major),128.2 (C, minor), 127.3

(C, major), 123.7 (CH, major), 123.3 (CH, minor), 121.9 (CH, major),

121.0 (CH, minor), 118.6 (CN, major), 113.4 (CN, minor), 46.3 (CH₂),

8.89 (CH₃).

5.1.2.4. N-(3,4-Dichlorophenyl)hydrazinecarbothioamide (III). A

solution of If (5 mmol) in ethanol (10 mL) was treated with excess

hydrazine hydrate (15 mmol) and the reaction mixture was stirred

at room temperature for 15 min, then diluted with cold water. The

obtained product was ﬁltered off, washed with cold water, air-dried

and recrystallized to give III (Table 10): IR (KBr) 3400–3150 (NH &

5.1.3.1. chlorophenyl)imidazolidin-2-one (VId). IR (KBr) 3250 (NH),

NH₂), 1490 & 1150 (CSN) cm^ꢀ^{1 1}H NMR (CD₃CN, 400 MHz)

; d 9.15

1765 (C]O), 1669 (C]N), 1500 & 1210 (CSN) cm^ꢀ^{1 1}H NMR

;

(br s, 1 H, NH), 7.17 (d, J ¼ 8.6 Hz, 1H, Ar–H), 6.77 (d, J ¼ 2.8 Hz, 1H,

(CDCl₃, 400 MHz)

d 9.53 (br s, 1H, NH), 7.72 (s, 1H), 7.69 (d,

Ar–H), 6.56 (dd, J ¼ 8.6, 2.8 Hz, 1 H, Ar–H), 4.41 (br s, 2H, NH₂); ¹³

C

J ¼ 8.8 Hz, 2H), 7.62 (s, 1H), 7.36 (d, J ¼ 8.8 Hz, 2H); ¹³C NMR (CDCl₃,

NMR (CD₃CN, 100 MHz)

d 189.6 (C]S), 149.3 (C), 132.7 (C), 128.6

100 MHz)

d 180.5 (C, C]S), 152.9 (C, C]O), 152.3 (C, C]N), 135.5

(C), 131.6 (CH), 116.3 (CH), 115.4 (CH).

(C), 132.8 (C–H), 132.3(C), 132.2 (C), 131.9 (CH), 131.8 (CH), 131.3 (C),

128.7 (C), 128.6 (CH); MS (m/z, %) 461 (7, M^þ), 229 (100), 428 (59),

213 (62), 75 (34).

5.1.2.5. 2-(4-Iodophenylamino)-3,3-diphenylacrylonitrile (IV). To

well stirred ethereal solution of diphenyldiazomethane

a

(excess), a solution of Ie (5 mmol) in dry ether (20 mL) was

added dropwise and the mixture was stirred for 25 min. The

obtained product was ﬁltered off, washed with a minimum

amount of ether, air-dried and recrystallized to give IV (Table

5.1.3.2. 4-Imino-1-(4-iodophenyl)-5-thioxo-3-(2,4,5-trichloro-

phenyl)imidazolidin-2-one (VIe). ¹H NMR (CDCl₃, 400 MHz)

d 9.53

(br s, 1H, NH), 7.89 (d, J ¼ 8.8 Hz, 2H), 7.71 (s, 1H), 7.62 (s, 1H), 7.23

(d, J ¼ 8.8 Hz, 2H); ¹³C NMR (CDCl₃, 100 MHz)

d 180.5 (C, C]S),

152.9 (C, C]O), 152.3 (C, C]N), 138.8 (C–H), 135.5 (C), 132.3(C),

10): IR (KBr) 3285 (NH), 2225 (CN) cm^ꢀ^{1 1}H NMR (CDCl₃,

;

400 MHz)

d

7.59 (d, J ¼ 8.8 Hz, 2H), 7.42–7.39 (m, 5H), 7.37–7.34

132.1 (C), 132.0 (C), 131.9 (CH), 131.8 (CH), 128.7 (CH), 95.7 (C–I).

5.1.3.3. 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-5-imino-4-thio-

Table 6

Antimicrobial effect of compounds If, IIc, V, VIf, XIIIa, XIV, XV, XVIb and XXVIb,c

against Gram negative and Gram positive bacteria.

xoimidazolidin-2-one (VIf). IR (KBr) 3257 (NH), 1761 (C]O), 1665

(C]N), 1495 & 1200 (CSN) cm^ꢀ1; ¹H NMR (CD₃CN, 400 MHz)

d 9.62

(br s, 1 H, NH), 7.76 (d, J ¼ 8.4 Hz, 1 H, Ar–H), 7.69 (d, J ¼ 2.4 Hz, 1 H,

Mean values of inhibition zone diameter (mm)

Ar–H), 7.58–7.56 (m, 4H), 7.48 (dd, J ¼ 8.4, 2.4 Hz, 1 H, Ar–H); ¹³C

Sample conc. Gram negative

Test organism

Gram positive

NMR (DMSO, 100 MHz)

d 182.5 (C]S), 153.7 (C]O), 153.2 (C]O),

133.1 (C), 132.7 (C), 132.5 (C), 131.6 (C), 131.5 (CH), 131.3 (C), 129.9

(CH), 129.1 (2XCH), 128.8 (2XCH), 128.5 (CH); Note: the aromatic

signals which are stemming from the p-substituted chlorobenzene

and having chemical shifts ranging from 7.58 to 7.52 appear as two

P. aerogenosa

B. subtilis

1 mg/mL 2.5 mg/mL 5 mg/mL 1 mg/mL 2.5 mg/mL 5 mg/mL

If

IIc

V

VIf

XIIIa

XIV

XV

XVIb

XXVIb

XXVIc

35

13

15

–

15

29

18

15

37

16

29

16

13

18

32

19

17

38

19

41

18

15

20

37

21

20

40

15

20

13

17

13

15

19

21

19

41

16

27

13

18

14

19

20

25

21

43

17

33

14

19

15

20

32

24

doublet with DMSO as a solvent

d 9.87 (br s, 1 H, NH), 7.92

(d, J ¼ 8.8 Hz, 1 H, Ar–H), 7.89 (d, J ¼ 2.3 Hz, 1 H, Ar–H),7.65

(d, J ¼ 8.8 Hz, 2 H, Ar–H), 7.59 (d, J ¼ 8.0 Hz, 2 H, Ar–H); Note, the

aromatic dd overlaps with one of the p-substituted chlorobenzene

signals. MS (m/z, %) 385 (70, M^þ), 203 (100), 153 (91), 126 (14).

5.1.3.4. 1-(4-Bromophenyl)-3-(4-chloro-3-(triﬂuoromethyl)phenyl)-

4-imino-5-thioxoimidazolidin-2-one (VIg). IR (KBr) 3263 (NH), 1770

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4327

50

P. aerogenosa

45

40

35

30

25

20

15

10

5

(1 mg/mL)

P. aerogenosa

(2.5 mg/mL)

P. aerogenosa

(5 mg/mL)

B. subtilis

(1 mg/mL)

B. subtilis

(2.5 mg/mL)

B. subtilis

(5 mg/mL)

0

Compound number

Fig. 12. Antibacterial activities of compounds If, IIc, V, VIf, XIIIa, XIV, XV, XVIb and XXVIb,c against Gram-negative and Gram-positive bacteria.

(C]O), 1674 (C]N), 1512 & 1238 (CSN) cm^ꢀ1

400 MHz)

;

¹H NMR (CDCl₃,

H), 7.35 (td, J ¼ 7.3, 1.3 Hz, 1H, Ar–H), 3.98 (s, 2 H, CH₂); ¹³C NMR

d

9.70 (br s, 1H, NH), 8.02 (d, J ¼ 2.4 Hz, 1H), 7.79 (dd,

(CDCl₃, 100 MHz) d 181.2 (C, C]S), 154.3 (C, C]O), 153.6 (C, C]N),

J ¼ 8.8, 2.4 Hz, 1H), 7.70 (d, J ¼ 8.8 Hz, 2H, Ar–H),7.67 (d, J ¼ 8.8 Hz,

144.3 (C), 143.6 (C), 142.4 (C), 140.6 (C), 132.8 (CH), 131.7 (C), 130.0

(C), 128.7 (CH), 127.4 (CH), 127.0 (CH), 125.3 (CH), 125.1 (CH), 123.7

(C, C–Br), 123.3 (CH), 120.5 (CH), 120.3 (CH), 37.0 (CH₂); MS (m/z, %)

447 (55, M^þ), 213 (13), 207 (100), 165 (59).

1H), 7.37 (d, J ¼ 8.8 Hz, 2H, Ar–H); ¹³C NMR (CDCl₃, 100 MHz)

d

180.4 (C, C]S), 153.2 (C, C]O), 153.0 (C, C]N), 132.9 (CH), 132.4

(CH), 132.3 (C), 131.3 (C–Cl), 130.7 (C–N), 130.3 (CH), 129.5 (q,

J ¼ 32.3 Hz, C), 128.6 (CH), 125.5 (q, J ¼ 5.3 Hz, CH), 124.0 (C–Br),

122.2 (q, J ¼ 273.5 Hz, CF₃). ¹⁹F NMR (CDCl₃, 376 MHz)

(m/z, %) 461 (48, M^þ), 213 (100).

d

–63.0; MS

5.1.3.7. 1-(9H-Fluoren-2-yl)-5-imino-3-(4-iodophenyl)-4-thio-

xoimidazolidin-2-one (VIIIc). IR (KBr) 3247 (NH), 1761 (C]O), 1670

(C]N), 1491 & 1195 (CSN) cm^ꢀ1; ¹H NMR (CDCl₃, 400 MHz)

d 9.61

5.1.3.5. 1-(4-Ethoxyphenyl)-3-(9H-ﬂuoren-2-yl)-4-imino-5-thio-

(br s, 1H, NH), 7.93–7.87 (m, 3H), 7.81 (d, J ¼ 7.7 Hz, 1H), 7.71 (d,

J ¼ 1.3 Hz,1H), 7.58–7.53 (m, 2H, Ar–H), 7.40 (t, J ¼ 7.4 Hz,1H, Ar–H),

7.35 (td, J ¼ 7.3, 1.3 Hz, 1H, Ar–H), 7.29–7.24 (m, 2H, Ar–H), 3.98 (s, 2

xoimidazolidin-2-one (VIIIa). IR (KBr) 3251 (NH), 1767 (C]O), 1669

(C]N), 1490 & 1200 (CSN) cm^ꢀ1; ¹H NMR (CDCl₃, 400 MHz)

d 9.51

(br s, 1H, NH), 7.93 (d, J ¼ 8.2 Hz, 1H), 7.83 (d, J ¼ 7.5 Hz, 1H), 7.74 (br

s, 1H, Ar–H), 7.76–7.55 (m, 2H, Ar–H), 7.44–7.32 (m, 4H, Ar–H), 7.05

(d, J ¼ 8.8 Hz, 2H, Ar–H), 4.10 (q, J ¼ 7.0 Hz, 2H), 3.99 (s, 2 H, CH₂),

H, CH₂); ¹³C NMR (CDCl₃,100 MHz)

d 181.1 (C, C]S),154.3 (C, C]O),

153.6 (C, C]N), 144.3 (C), 143.6 (C), 142.4 (C), 140.6 (C), 138.7 (CH),

132.4 (C), 130.0 (C), 128.8 (CH), 127.4 (CH), 127.0 (CH), 125.3 (CH),

125.1 (CH), 123.3 (CH), 120.5 (CH), 120.3 (CH), 95.5 (C, C–I), 37.0

(CH₂); MS (m/z, %) 495 (99, M^þ), 261 (22), 207 (100), 165 (49).

1.44 (t, J ¼ 7.0 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz)

d 182.4 (C, C]S),

159.7 (C, C–O), 154.8 (C, C]O), 154.4 (C, C]N), 144.3 (C), 143.7 (C),

142.2 (C), 140.7 (C), 130.6 (C), 128.5 (CH), 127.5 (CH), 127.1 (CH),

125.7 (CH), 125.4 (C),125.3 (CH), 123.7 (CH), 120.5 (CH), 120.4 (CH),

115.2 (CH), 64.0 (CH₂), 37.1 (CH₂), 14.8 (CH₃); MS (m/z, %) 413 (96,

M^þ), 207 (100), 179 (34).

5.1.4. Preparation of compounds IXa–c; the preceding compounds

were prepared according to the following general procedure

The corresponding diones VIIIa–c (5 mmol) were dissolved in

boiling ethanol (20 mL) and treated with dil. HCl (1:1 molar ratio).

The obtained product was ﬁltered off, washed with cold water, air-

dried and recrystallized to give IXa–c (Table 10).

5.1.3.6. 1-(4-Bromophenyl)-3-(9H-ﬂuoren-2-yl)-4-imino-5-thio-

xoimidazolidin-2-one (VIIIb). IR (KBr) 3255 (NH), 1770 (C]O), 1670

(C]N), 1500 & 1210 (CSN) cm^ꢀ1; ¹H NMR (CDCl₃, 400 MHz)

d 9.61

(br s, 1H, NH), 7.92 (d, J ¼ 8.4 Hz, 1H), 7.82 (d, J ¼ 7.2 Hz, 1H), 7.74–

25

7.67 (m, 3H, Ar–H), 7.59–7.53 (m, 2H, Ar–H), 7.44–7.38 (m, 3H, Ar–

C. albicans

20

A. niger

Table 7

Antifungal effect of compounds VId–g, VIIIa–c and IXa–b against C. albicans and

A. niger.

15

10

5

Compound no.

Mean values of inhibition zone diameter

(mm)

C. albicans

A. niger

VId

VIe

VIf

14.0

17.0

16.0

15.0

13.0

14.5

13.0

ND^a

20.0

21.0

16.0

20.0

ND

13.0

VIg

0

VIIIa

VIIIb

VIIIc

IXa

VId VIe

VIf VIg VIIIa VIIIb VIIIc IXa IXb

Compound number

IXb

Fig. 13. Antifungal activities of compounds VId–g, VIIIa–c and IXa,b against C. albicans

and A. niger.

a

ND: Not determined.

4328

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

Table 8

1H), 7.50 (dd, J ¼ 8.3, 2.0 Hz, 1H), 7.42 (t, J ¼ 7.4 Hz, 1H, Ar–H), 7.37

(td, J ¼ 7.5, 1.3 Hz, 1H, Ar–H), 7.27–7.23 (m, 2H, Ar–H), 4.00 (s, 2 H,

Antifungal effect of compounds If, V, VIf, XIIIa, XIV, XV, XVIb and XXVIb,c against

C. albicans and A. niger.

CH₂); ¹³C NMR (CDCl₃, 100 MHz)

d 181.9 (C, C]S), 153.0 (C, C]O),

Mean values of inhibition zone diameter (mm)

Sample conc. Test organism

152.8 (C, C]O), 144.4 (C), 143.6 (C), 142.8 (C), 140.4 (C), 138.9 (CH),

131.7 (C), 128.9 (CH), 128.3 (C), 127.6 (CH), 127.1 (CH), 125.2 (CH),

124.6 (CH), 122.6 (CH), 120.5 (CH), 120.4 (CH), 95.7 (C, C–I), 37.0

(CH₂).

C. albicans

A. niger

1 mg/mL 2.5 mg/mL 5 mg/mL 1 mg/mL 2.5 mg/mL 5 mg/mL

If

V

VIf

XIIIa

XIV

XV

XVIb

XXVIb

XXVIc

37

21

ND

12

15

25

18

19

40

33

13

12

14

17

29

20

44

39

14

15

18

32

23

21

29

45

11

15

ND

18

56

20

17

12

13

ND

20

17

5.1.4.4. 3-Benzyl-5-(benzylimino)-1-(4-ethoxyphenyl)imidazolidine-

2,4-dione (X). A solution of IXa (5 mmol) in ethanol (25 mL) was

treated with excess benzylamine (15 mmol). The reaction mixture

was heated under reﬂux for 1 h, cooled down and poured into cold

dil. HCl (1:1 molar ratio). The obtained product was ﬁltered off,

washed with cold water, air-dried and recrystallized to give X

ND^a

ND

15

(Table 10): ¹H NMR (CDCl₃, 400 MHz)

d

7.49 (dd, J ¼ 8.1, 2.0 Hz, 2H),

12

15

7.42–7.26 (m, 12H, Ar–H), 5.35 (s, 2 H, CH₂), 4.92 (s, 2 H, CH₂), 4.05

a

ND: Not determined.

(q, J ¼ 7.0 Hz, 2H), 1.43 (t, J ¼ 7.0 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz)

d

158.9 (C, C]N), 155.0 (C, C]N), 153.2 (C, C]O), 140.6 (C), 139.9

(C), 136.0 (C), 122.7 (C), 129.2 (CH), 128.6 (CH), 128.0 (CH),124.4 (C),

127.6 (CH), 127.4 (CH), 126.9 (CH), 115.0 (CH), 63.8 (CH₂), 53.0 (CH₂),

42.9 (CH₂), 14.7 (CH₃); MS (m/z, %) 413 (33, M^þ), 322 (42), 148 (79),

91 (100).

5.1.4.1. 1-(4-Ethoxyphenyl)-3-(9H-ﬂuoren-2-yl)-5-thioxoimida-

zolidine-2,4-dione (IXa). IR (KBr) 1770 (2XC]O), 1509 & 1205 (CSN)

cm^ꢀ1

;

¹H NMR (CDCl₃, 400 MHz)

d

8.05 (d, J ¼ 8.1 Hz, 1H), 7.96

(d, J ¼ 7.3 Hz, 1H), 7.82 (d, J ¼ 1.3 Hz, 1H, Ar–H), 7.72 (d, J ¼ 7.3 Hz,

2H, Ar–H), 7.65 (dd, J ¼ 8.1, 2.0 Hz, 2H, Ar–H), 7.59–7.47 (m, 4H, Ar–

H), 7.21–7.16 (m, 2H, Ar–H), 4.23 (q, J ¼ 7.0 Hz, 2H), 4.12 (s, 2 H, CH₂),

5.1.4.5. 1-(4-Bromophenyl)-3-(4-chloro-3-(triﬂuoromethyl)phenyl)-

5-thioxoimidazolidine-2,4-dione (XI). Imidazolidineiminothione

VIg (5 mmol) was dissolved in boiling ethanol (20 mL) and

treated with dil. HCl (1:1 molar ratio). The obtained product was

ﬁltered off, washed with cold water, air-dried and recrystallized

1.60 (t, J ¼ 7.0 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz)

d 182.7 (C, C]S),

159.8 (C, C–O), 153.6 (C, C]O), 153.1 (C, C]O), 144.3 (C), 143.6 (C),

142.7 (C), 140.4 (C), 128.5 (C), 128.4 (CH), 127.5 (CH), 127.0 (CH),

125.2 (CH), 124.6 (CH),124.4 (C), 122.7 (CH), 120.5 (CH), 120.4 (CH),

115.3 (CH), 63.9 (CH₂), 37.0 (CH₂), 14.8 (CH₃); MS (m/z, %) 414 (84,

M^þ), 207 (100), 179 (69), 165 (29), 151 (95).

to give XI (Table 10): ¹H NMR (CDCl₃, 400 MHz)

d 7.94 (d,

J ¼ 2.1 Hz, 1H), 7.73–7.69 (m, 3H), 7.55–7.48 (m, 1H), 7.34 (d,

J ¼ 8.8 Hz, 2H, Ar–H); ¹³C NMR (CDCl₃, 100 MHz)

d 180.8 (C]S),

152.3 (C]O), 151.7 (C]O), 133.0 (CH), 132.7 (CH), 130.6 (C),

129.9 (C), 129.6 (CH), 129.4 (q, J ¼ 32.0 Hz, C), 129.0 (C), 128.7

(CH), 124.8 (q, J ¼ 5.2 Hz, CH), 124.3 (C–Br), 122.0 (q, J ¼ 272.7 Hz,

CF₃).

5.1.4.2. 1-(4-Bromophenyl)-3-(9H-ﬂuoren-2-yl)-4-imino-5-thio-

xoimidazolidin-2-one (IXb). IR (KBr) 1770 (2XC]O) cm^ꢀ1; ¹H NMR

(CDCl₃, 400 MHz)

d

7.92 (d, J ¼ 8.3 Hz, 1H), 7.82 (d, J ¼ 7.2 Hz, 1H),

7.73–7.68 (m, 2H, Ar–H), 7.68–7.65 (m, 1H, Ar–H), 7.58 (d, J ¼ 7.6 Hz,

1H, Ar–H), 7.50 (dd, J ¼ 8.4, 2.0 Hz, 1H, Ar–H), 7.44–7.34 (m, 4H),

3.98 (s, 2 H, CH₂); ¹³C NMR (CDCl₃, 100 MHz)

d 182.0 (C, C]S), 153.1

5.1.4.6. 3-Benzyl-5-(benzylimino)-1-(4-bromophenyl)imidazolidin-

2,4-dione (XII). The benzylimine XII (Table 10) was prepared

following the same procedure used to prepare its analogue ben-

(C, C]O), 152.8 (C, C]O), 144.4 (C), 143.6 (C), 142.8 (C), 140.4 (C),

132.9 (CH), 131.0 (C), 128.8 (CH), 128.3 (C), 127.6 (CH), 127.1 (CH),

125.2 (CH), 124.6 (CH), 124.0 (C), 122.6 (CH), 120.5 (CH), 120.4 (CH),

37.0 (CH₂); MS (m/z, %) 448 (37, M^þ), 213 (11), 207 (100), 165 (52).

zylimine X: ¹H NMR (CDCl₃, 400 MHz)

d

7.61 (d, J ¼ 8.84 Hz, 2H),

7.51–7.42 (m, 2H), 7.41–7.28 (m,10H), 5.35 (s, 2H, NCH₂), 4.93 (s, 2H,

NCH₂); ¹³C NMR (CDCl₃, 100 MHz)

154.3 (C), 152.5 (C), 140.2 (C),

d

139.7 (C), 135.7 (C), 132.4 (CH), 129.5 (C), 129.3 (CH), 128.7 (CH),

128.5 (CH), 128.1 (CH), 127.6 (CH), 127.0 (CH), 127.3 (CH), 127.0 (CH),

122.4 (C), 53.1 (CH₂), 43.0 (CH₂).

5.1.4.3. 3-(9H-ﬂuoren-2-yl)-1-(4-iodophenyl)-5-thioxoimida-

zolidine-2,4-dione (IXc). ¹H NMR (CDCl₃, 400 MHz)

d 7.94–7.89 (m,

3H), 7.83 (d, J ¼ 7.3 Hz,1H), 7.67 (d, J ¼ 1.3 Hz,1H), 7.58 (d, J ¼ 7.4 Hz,

60

50

40

30

20

10

0

C. albicans

(1 mg/mL)

C. albicans

(2.5 mg/mL)

C. albicans

(5 mg/mL)

A. niger

(1 mg/mL)

A. niger

(2.5 mg/mL)

A. niger

(5 mg/mL)

If

V

VIf

XIIIa XIV

XV

XVIb XXVIb XXVIc

Compound number

Fig. 14. Antifungal activities of compounds If, V, VIf, XIIIa, XIV, XV, XVIb and XXVIb,c against C. albicans and A. niger.

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4329

Table 9

(CH), 63.8 (OCH₂), 41.7 (CH₂), 24.6 (CH₂), 14.7 (CH₃), 14.4 (CH₃); MS

(m/z, %) 774 (51, M^þ), 151 (100), 362 (19), 361 (26).

Minimum inhibitory concentrations of compounds XXVIb, XXVIc, XVIb and If

against various strains of Gram positive and Gram negative bacteria and fungi.

5.1.6.2. 3,3⁰-(4,4⁰-Oxybis(4,1-phenylene))bis(1-(3,4-dichlorophenyl)-

Minimum inhibitory concentration MIC (

Compound no. Test organism

mg/mL)

4-imino-5-thioxoimidazolidin-2-one) (XV). ¹H NMR (DMSO,

400 MHz)

d

9.80 (br s, 1H, NH), 7.92 (d, J ¼ 8.5 Hz, 2H), 7.91 (d,

Gram positive

bacteria

Gram negative

bacteria

Fungus

J ¼ 2.3 Hz, 2H), 7.62 (dd, J ¼ 8.5, 2.3 Hz, 2H), 7.61 (d, J ¼ 8.8 Hz, 4H),

7.28 (d, J ¼ 8.8 Hz, 4H); ¹³C NMR (DMSO, 100 MHz)

d 182.6 (C]S),

B. subtilis S. aureus K. pneumonia S. typhi C. albicans A. niger

155.9 (C),154.0 (C),153.3 (C), 133.1 (C),132.4 (C),131.5 (C),131.4 (CH),

129.9 (CH), 129.2 (CH), 128.4 (CH), 127.9 (C), 119.2 (CH).

XXVIb

XXVIc

XVIb

If

3.9

1.95

0.97

15.6

3.9

7.8

3.9

1.95

0.49

0.24

1.95

0.97

0.49

31.3

15.6

5.1.6.3. 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-4-thioxoimida-

zolidin-2-one (XVIb). To a solution of VIf (5 mmol) in benzene

(25 mL), triethylamine (3 drops) was added, then a stream of H₂S

gas was bubbled in for 15 min. The obtained product was ﬁltered

off, washed with a minimum amount of dry benzene and recrys-

tallized to give XVIb (Table 10). The benzene mother liquor upon

concentration yielded a product which crystallized to give XVII

(Table 10): IR (KBr) 1750 (C]O), 1490 & 1150 (CSN) cm^ꢀ1; ¹H NMR

5.1.5. Preparation of XIIIa–c: see the general procedure described

earlier for the preparation of compounds VIa–g, VIIIa–c

and XIIIa–c

5.1.5.1. 1-(4-Chlorophenyl)-4-imino-3-(naphthalen-1-yl)-5-thio-

xoimidazolidin-2-one (XIIIa). IR (KBr) 3261 (NH), 1760 (C]O),

1670 (C]N), 1510 & 1190 (CSN) cm^ꢀ^{1 1}H NMR (CDCl₃, 400 MHz)

;

(CDCl₃, 400 MHz)

H), 7.39–7.34 (m, 2 H, Ar–H), 4.83 (s, 2 H, CH₂); ¹³C NMR (CDCl₃,

100 MHz) 194.9 (C]S), 152.9 (C]O), 136.2 (C), 135.6 (C), 133.6 (C),

d 7.82–7.79 (m, 1 H, Ar–H), 7.55–7.48 (m, 4 H, Ar–

d

9.52 (br s, 1H, NH), 8.01 (dd, J ¼ 6.8, 2.4 Hz, 1H), 7.98–7.93

(m, 1H), 7.73–7.66 (m, 1H), 7.63–7.48 (m, 8H); ¹³C NMR (CDCl₃,

100 MHz) 181.3 (C]S), 154.5 (C]O), 153.7 (C]N), 135.6 (C),

d

131.4 (C), 131.1 (C), 131.1 (CH), 129.7 (CH), 128.9 (CH), 128.6 (C), 119.8

(CH),117.2 (CH), 59.4 (NCH₂); MS (m/z, %) 370 (62, M^þ),187 (24),153

(16).

134.6 (C), 131.1 (C), 130.7 (CH), 129.8 (CH), 129.7 (C), 128.9 (CH),

128.5 (CH), 128.1 (C), 127.5 (CH), 127.0 (CH), 126.9 (CH), 125.6 (CH),

122.1 (CH); MS (m/z, %) 365 (100, M^þ), 212 (85), 169 (63), 153 (11).

5.1.6.4. 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-4-mercapto-1H-

imidazol-2(3H)-one and 4,4⁰-disulfanediylbis(1-(4-chlorophenyl)-3-

(3,4-dichlorophenyl)-1H-imidazol-2(3H))-one (XVII). (mix. of thiol

5.1.5.2. 1-(4-Bromophenyl)-4-imino-3-(naphthalen-1-yl)-5-thio-

xoimidazolidin-2-one (XIIIb). IR (KBr) 3261 (NH), 1760 (C]O),

1670 (C]N), 1510 & 1190 (CSN) cm^ꢀ^{1 1}H NMR (CDCl₃, 400 MHz)

;

and disulﬁde: 68:32). IR (KBr) 1740 (C]O) cm^ꢀ^{1 1}H NMR (CDCl₃,

;

d

9.51 (br s, 1H, NH), 8.03–7.91 (m, 2H), 7.72–7.64 (m, 1H), 7.67 (d,

400 MHz)

d

7.64 (d, J ¼ 8.5 Hz, 1H, major), 7.61 (m, 1H, minor), 7.57–

J ¼ 8.8 Hz, 2H), 7.62–7.52 (m, 4H), 7.43 (d, J ¼ 8.8 Hz, 2H); ¹³C NMR

7.52 (m, 2H, both isomers), 7.44–7.35 (m, 3H, both), 7.33–7.29 (m,1H),

(CDCl₃, 100 MHz)

d 181.1 (C]S), 154.5 (C]O), 153.6 (C]N), 134.6

6.89 (d, J ¼ 2.5 Hz, 1H, major), 6.80 (d, J ¼ 2.5 Hz, 1H, minor), 6.75–

(C), 132.7 (CH), 131.6 (C), 130.6 (CH), 129.6 (C), 128.8 (CH), 128.6

(CH), 128.0 (C), 127.4 (CH), 126.9 (CH), 126.8 (CH), 125.5 (CH), 123.7

(C), 122.0 (CH); MS (m/z, %) 409 (84, M^þ), 212 (100), 168 (80), 153

(11).

6.64 (m, 2H, both); ¹³C NMR (CDCl₃, 100 MHz)

d 150.2 (C]O, minor),

150.1 (C]O, major), 135.3 (C),135.2 (C),134.8 (C),133.8 (C),133.6 (C),

133.1 (C, major),133.0 (C, minor),132.6 (C),131.8 (C),131.7 (2XC),131.2

(CH, major), 131.1 (CH, minor), 130.8 (CH, major), 130.7 (CH, minor),

130.2 (CH, major), 129.9 (CH, major), 129.8 (CH, minor), 129.6 (CH),

129.5 (CH),128.0 (CH),127.5 (CH, minor),127.4 (CH, major),126.5 (CH,

minor),126.3 (CH, minor),124.0 (CH, minor),123.9 (CH, major),123.4

(C, minor), 123.2 (C, major), 117.0 (C), 116.8 (C), 116.0 (C); MS (m/z, %)

740 (6, M^þ), 706 (17), 187 (5), 171 (19), 153 (6), 64 (100).

5.1.5.3. 1-(4-Methoxy)-4-imino-3-(naphthalen-1-yl)-5-thio-

xoimidazolidin-2-one (XIIIc). ¹H NMR (DMSO, 400 MHz)

d 9.58 (s,

1H, NH), 8.20–8.15 (m, 1H), 8.12 (d, J ¼ 8.4 Hz, 1H), 8.10–8.05 (m,

1H), 7.78 (dd, J ¼ 7.2, 1.2 Hz, 1H), 7.72–7.67 (m, 1H), 7.65–7.60 (m,

2H), 7.58 (d, J ¼ 9.2 Hz, 2H), 7.15 (d, J ¼ 9.2 Hz, 2H), 3.84 (s, 3H,

OCH₃); ¹³C NMR (DMSO, 100 MHz)

d 183.5 (C]S), 159.6 (C), 154.9

5.1.7. Preparation of compounds XVIIIa–c; the preceding

compounds were prepared according to the following

general procedure

(C), 154.3 (C), 133.8 (C), 130.1 (C), 129.6 (CH), 129.3 (C), 129.1 (CH),

128.1 (CH), 127.4 (CH), 126.9 (CH), 126.6 (CH), 126.0 (C), 125.7 (CH),

123.6 (CH), 114.3 (CH), 55.4 (OCH₃).

A mixture of the corresponding imidazolidineiminothione VI

(5 mmol) and the requisite o-phenylenediamine or its derivatives

(5 mmol) in ethanol (25 mL) was heated under reﬂux until the

evolution of ammonia and H₂S gases were ceased as detected by

their odor (approx. 7 h). The obtained product was ﬁltered off,

washed with a minimum amount of ethanol and crystallized to give

XVIIIa–c (Table 10).

5.1.6. Preparation of bis-imidazolidineiminothiones XIV and XV

To a solution of Ib or If (10 mmol, 2.0 equiv.) in ether (20 mL),

a solution of the corresponding bisisocyanate (5 mmol, 1.0 equiv.)

in ether (10 mL) was added, followed by 3 drops of triethylamine.

The reaction mixture was stirred for 25 min. The obtained product

was ﬁltered off, washed with a minimum amount of ether, air-

dried and re-crystallized to give XIV and XV (Table 10),

respectively.

5.1.7.1. 3-(4-Chlorophenyl)-6-methyl-1-p-tolyl-1H-imidazo[4,5-b]-

quinoxalin-2(3H)-one (XVIIIa). (two regioisomers: 1:1). IR (KBr)

1730 (C]O), 1630 (C]N) cm^ꢀ1; ¹H NMR (CDCl₃, 400 MHz)

d 7.89–

5.1.6.1. 3,3⁰-(4,4⁰-Methylenebis(2,6-diethyl-4,1-phenylene))bis(1-(4-

7.83 (m, 3H), 7.76 (s, 1H), 7.71–7.65 (m, 2H), 7.56 (d, J ¼ 8.8 Hz, 2H),

ethoxyphenyl)-4-imino-5-thioxoimidazolidin-2-one) (XIV). IR (KBr)

3228 (NH), 1779 (C]O), 1662 (C]N), 1531 & 1185 (CSN) cm^ꢀ^{1 1}H

;

7.46–7.38 (m, 3H), 2.54 (s, 3H, major), 2.53 (s, 3H, minor), 2.46 (s,

3H); ¹³C NMR (CDCl₃, 100 MHz)

d 152.1 (C]O), 139.2 (C), 138.9 (C),

NMR (CDCl₃, 400 MHz)

d

9.42 (br s, 2H, NH), 7.39 (d, J ¼ 8.8 Hz, 4H),

138.8 (C), 138.7 (C), 138.5 (C), 138.3 (C), 138.0 (C), 137.9 (C), 137.3 (C),

137.1 (C), 133.7 (C), 130.1 (C), 130.0 (CH), 129.7 (CH), 129.6 (CH),

129.5 (CH), 21.5 (CH), 21.3 (CH₃); MS (m/z, %) 400 (100, M^þ), 289

(11), 246 (27), 91 (19).

7.10 (s, 4H), 7.03 (d, J ¼ 8.8 Hz, 4H), 4.08 (q, J ¼ 7.0 Hz, 4H), 4.04 (s,

2H), 2.53 (q, J ¼ 7.5 Hz, 8H), 1.45 (t, J ¼ 7.0 Hz, 6H), 1.20 (t, J ¼ 7.5 Hz,

12H); ¹³C NMR (CDCl₃, 100 MHz)

d 181.9 (C]S), 159.6 (C), 154.4 (C),

154.2 (C), 142.4 (C), 128.3 (CH), 127.7 (CH), 126.8 (C),125.1 (C), 115.1

4330

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

Table 10

Physical data of the synthesized compounds.

Compd. no.

Cryst. solvent M.P [^ꢁC]

Yield %

Mol. formula M.Wt.

Elemental analysis

Calcd./Found [%]

C

H

N

S

IIa

^aC/H

80

73

75

50

30

40

55

65

63

67

70

65

60

70

45

50

55

50

53

51

69

72

75

65

60

55

50

63

60

65

75

77

73

55

51

59

C₁₄H₂₀BrN₃S

(341)

C₁₄H₂₀IN₃S

(389)

C₁₄H₁₉Cl₂N₃S

(331)

C₇H₇Cl₂N₃S

(235)

C₂₁H₁₅IN₂

(422)

49.12

49.00

43.19

43.10

50.60

50.50

35.61

35.50

59.73

59.60

43.65

43.50

38.86

38.70

35.29

35.20

46.76

46.70

41.54

41.50

69.71

69.60

58.94

58.80

53.34

53.20

69.55

69.40

58.76

58.60

53.24

53.10

72.64

72.60

41.51

41.40

63.69

63.60

62.38

62.20

55.62

55.50

66.48

66.40

66.67

66.50

50.43

50.30

48.47

48.40

48.65

48.60

69.00

68.80

72.25

72.10

57.27

57.10

55.72

55.60

65.75

65.70

60.54

60.50

63.76

63.70

58.43

58.40

64.99

64.90

5.89

5.80

5.18

5.20

5.76

5.60

2.99

2.90

3.58

3.50

2.93

2.90

1.52

1.40

1.38

1.20

2.29

2.20

1.74

1.60

4.63

4.50

3.15

3.10

2.85

2.80

4.35

4.30

2.89

2.80

2.64

2.60

5.57

5.50

1.51

1.40

3.82

3.70

3.28

3.20

2.93

2.80

4.15

4.10

5.94

5.90

2.26

2.20

2.44

2.30

2.16

2.10

4.25

4.10

4.71

4.60

2.50

2.40

3.15

3.10

3.72

3.60

3.42

3.30

3.60

3.50

3.30

3.20

3.48

3.40

12.28

12.20

10.79

10.65

12.65

12.60

17.80

17.65

6.63

9.37

9.20

8.24

8.20

9.65

9.50

13.58

13.50

–

IIb

C/H

100

C/H

120

^bE/W (1:1)

220

C/H

190

C/H

87

IIc

III

IV

6.55

–

V

C₁₀H₈Cl₂N₂OS

10.18

10.10

9.06

8.95

8.23

11.65

11.60

6.92

6.90

6.28

6.20

8.32

8.20

6.93

6.80

7.75

7.70

7.15

7.10

6.47

6.40

7.73

7.60

7.12

7.00

6.46

6.30

–

(274)

C₁₅H₇Cl₃BrN₃OS

(461.5)

C₁₅H₇Cl₃IN₃OS

(509.5)

C₁₅H₈Cl₃N₃OS

(383.5)

C₁₆H₈F₃ClBrN₃OS

(461.5)

C₂₄H₁₉N₃O₂S

(413)

C₂₂H₁₄BrN₃OS

(447)

VId

E^c

189

E

149

E

176

E

142

E

196

E

186

E

VIe

8.20

VIf

10.92

10.80

9.08

VIg

8.95

VIIIa

VIIIb

VIIIc

IXa

10.16

10.00

9.37

9.20

8.48

8.40

6.76

6.70

6.23

6.20

5.64

5.60

10.17

10.00

6.05

5.90

8.92

C₂₂H₁₄IN₃OS

(495)

202

E/W (1:1)

195

E/W (1:1)

205

E/W (1:1)

240

e/H^d

120

e/H

125

e/H

175

C/H

205

C/H

190

C/H

210

C/H

200

C/H

230

E

239

E

195

E

253

E

220

Dioxane

317

E

C₂₄H₁₈N₂O₃S

(414)

C₂₂H₁₃BrN₂O₂S

(448)

C₂₂H₁₃IN₂O₂S

(496)

C₂₅H₂₃N₃O₃

(413)

IXb

IXc

X

–

XI

C₁₆H₇F₃ClBrN₂O₂S

6.92

6.82

–

(462.5)

C₂₃H₈BrN₃O₂

(447)

C₁₉H₁₂ClN₃OS

(365.5)

C₁₉H₁₂BrN₃OS

(409)

XII

8.80

–

XIIIa

XIIIb

XIIIc

XIV

11.49

11.40

10.27

10.20

11.63

11.50

10.85

10.80

11.76

11.70

7.54

8.76

8.70

7.82

7.70

8.86

8.80

8.27

8.20

8.98

8.80

8.63

8.50

8.65

8.50

–

C₂₀H₁₅N₃O₂S

(361)

C₄₃H₄₆N₆O₄S₂

(774)

C₃₀H₁₆Cl₄N₆O₃S₂

(714)

C₁₅H₉Cl₃N₂OS

(371.5)

XV

XVIb

XVII

XVIIIa

XVIIIb

XVIIIc

XXIa

XXIb

XXIc

XXIVa

XXIVb

XXIVc

7.50

7.57

7.50

C₃₀H₁₆Cl₆N₄O₂S₂

(740)

C₂₃H₁₇ClN₄O

(400)

C₂₃H₁₈N₄O₂

(382)

14.00

13.90

14.66

14.50

12.73

12.60

11.61

11.50

13.70

13.60

12.61

12.50

7.97

C₂₁H₁₁Cl₃N₄O

(440)

C₂₈H₁₉ClIN₅O

(603)

C₂₈H₁₉Cl₂N₅O

(511)

C₂₈H₁₉ClBrN₅O

(555)

200

E

190

E

200

E

170

E

–

C₂₈H₁₉ClBrN₃O

(527)

C₂₈H₁₉ClIN₃O

(575)

C₂₈H₁₈Cl₃N₃O

(517)

7.70

7.30

7.20

8.10

157

E

191

8.00

–

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4331

Table 10 (continued )

Compd. no.

Cryst. solvent M.P [^ꢁC]

Yield %

Mol. formula M.Wt.

Elemental analysis

Calcd./Found [%]

C

H

N

S

XXIVd

XXVa

XXVb

XXVc

E

185

E

205

E

185

E

190

C/H

130

C/H

220

C/H

170

50

45

52

50

55

60

57

C₃₀H₂₄ClN₃O₂

(497)

73.02

72.90

43.24

43.10

49.61

69.70

55.59

55.60

43.68

43.60

39.74

39.60

32.78

32.80

4.87

4.80

2.70

2.60

2.84

2.80

3.82

2.70

3.00

2.90

1.99

1.90

1.64

1.50

8.59

–

8.50

22.07

21.90

25.32

25.20

26.70

26.60

8.99

8.90

9.27

9.20

7.65

C₁₆H₁₂IN₇O

(444)

C₁₆H₁₁Cl₂N₇O

(387)

C₁₇H₁₄ClN₇O

(367)

C₁₇H₁₄IN₄OS₂

(467)

XXVIa

XXVIb

XXVIc

13.71

13.60

14.13

14.10

11.66

11.50

C₁₅H₉Br₂N₃S₂

(453)

C₁₅H₉I₂N₃S₂

(549)

7.50

a

C/H: Chloroform/n-hexane.

E/W: Ethanol/water.

E: Ethanol.

b

c

d

e/H: Diethylether/n-hexane.

5.1.7.2. 1-(4-Ethoxyphenyl)-3-phenyl-1H-imidazo[4,5-b]quinoxalin-

2(3H)-one (XVIIIb). IR (KBr) 1735 (C]O), 1635 (C]N) cm^ꢀ^{1 1}H

NMR (CDCl₃, 400 MHz)

3271 (NH), 1760 (C]O), 1627 (C]N) cm^ꢀ1; MS (m/z, %) 557 (13,

M

;

^þþ 2), 402 (14), 361 (14), 165 (100), 77 (44).

d

7.96 (dd, J ¼ 6.2, 3.5 Hz, 2H), 7.88–7.84 (m,

2H), 7.70 (d, J ¼ 8.9 Hz, 2H), 7.63–7.56 (m, 4H), 7.50–7.45 (m, 1H),

7.10 (d, J ¼ 8.9 Hz, 2H), 4.12 (q, J ¼ 7.0 Hz, 2H), 1.47 (t, J ¼ 7.0 Hz, 3H);

5.1.9. Preparation of compounds XXIVa–d; the preceding

compounds were prepared according to the following

general procedure

¹³C NMR (CDCl₃, 100 MHz)

d 158.8 (C]O), 152.5 (C), 139.5 (C), 139.2

(2XC), 139.1 (C), 132.3 (C), 129.3 (CH), 128.2 (CH), 128.0 (2XCH),

127.5 (CH), 127.4 (CH), 125.8 (CH), 124.5 (C), 115.2 (CH), 63.8 (CH₂),

14.8 (CH₃); MS (m/z, %) 382 (100, M^þ), 354 (66), 353 (39).

To a solution of the respective imidazolidineiminothione VI

(5 mmol) in dry ether (20 mL), a solution of diphenyldiazo-

methane (15 mmol; excess) in dry ether (20 mL) was added. The

reaction mixture was stirred at room temperature for 30 min. The

obtained product was ﬁltered off, washed with a minimum

amount of dry ether, air-dried and crystallized to give XXIVa–

d (Table 10).

5.1.7.3. 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-1H-imidazo

[4,5-b]quinoxalin-2(3H)-one (XVIIIc). ¹H NMR (CDCl₃, 400 MHz)

d

8.11 (d, J ¼ 2.4 Hz, 1H), 8.03–7.95 (m, 2H), 7.86–7.81 (m, 1H), 7.83

(d, J ¼ 8.8 Hz, 2H), 7.70–7.63 (m, 3H), 7.59 (d, J ¼ 8.8 Hz, 2H); MS

(m/z, %) 440 (97, M^þ), 352 (29), 329 (13), 187 (10), 111 (10).

5.1.9.1. 1-(4-Chlorophenyl)-4-(diphenylmethylene)-5-imino-3-p-tol-

ylimidazolidin-2-one (XXIVa). IR (KBr) 3229 (NH), 1757 (C]O),

5.1.8. Preparation of compounds XXIa–c; the preceding compounds

were prepared according to the following general procedure

A solution of the corresponding imidazolidineiminothione VI

(5 mmol) in boiling ethanol was treated with benzophenone

hydrazone (5 mmol), followed by triethylamine (3 drops). The

reaction mixture was heated under reﬂux for 3 h. The obtained

product was ﬁltered off, washed with a minimum amount of

ethanol and crystallized to give XXIa–c (Table 10).

1657 (C]N), 1600 (C]C) cm^ꢀ1; ¹H NMR (CDCl₃, 400 MHz)

d

7.57–

7.29 (m, 11H, NH & Ar–H), 6.97–6.85 (m, 4H, Ar–H), 6.84–6.78 (m,

4H, Ar–H), 2.16 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz)

156.2 (C), 153.9

d

(C), 138.9 (C), 138.1 (C), 136.5 (C), 133.4 (C), 132.2 (C), 131.4 (C), 130.1

(CH),129.7 (CH), 129.6 (CH),129.1 (CH),129.0 (CH), 128.7 (CH),127.7

(C), 127.5 (CH), 127.3 (CH),126.3 (CH), 125.1 (C), 20.9 (CH₃); MS (m/z,

%) 463 (35, M^þ), 462 (100), 165 (23), 77 (44).

5.1.9.2. 1-(4-Chlorophenyl)-4-(diphenylmethylene)-5-imino-3-

5.1.8.1. (E)-1-(4-chlorophenyl)-4-((diphenylmethylene)hydrazono)-

(4-iodophenyl)imidazolidin-2-one (XXIVb). IR (KBr) 3229 (NH),

5-imino-3-(4-iodophenyl)imidazolidin-2-one (XXIa). IR (KBr) 3271

1757 (C]O), 1657 (C]N), 1600 (C]C) cm^ꢀ^{1 1}H NMR (CDCl₃,

;

(NH), 1760 (C]O), 1627 (C]N) cm^ꢀ1

;

¹H NMR (CDCl₃, 400 MHz)

400 MHz)

Ar–H), 7.08–7.02 (m, 2H, Ar–H), 6.99–693 (m, 2H, Ar–H), 6.85–

6.79 (m, 4H, Ar–H); ¹³C NMR (CDCl₃, 100 MHz)

155.8 (C), 153.5

d 7.50–7.41 (m, 7H, 1 NH & 6Ar–H), 7.40–7.33 (m, 4H,

d

10.5 (br s,1H, NH), 7.77–7.68 (m, 2H, Ar–H), 7.56 (d, J ¼ 8.8 Hz, 2H),

7.54–7.35 (m, 10H), 7.29–7.24 (m, 2H), 7.04 (d, J ¼ 8.8 Hz, 2H); ¹³C

d

NMR (CDCl₃, 100 MHz)

d

169.9 (C), 151.9 (C), 149.1 (C), 140.0 (C),

(C), 138.4 (C), 137.9 (C), 137.5 (C), 134.6 (C), 133.6 (C), 131.2 (C),

137.1 (CH), 130.0 (CH), 129.8 (CH), 129.6 (CH), 129.3 (CH), 129.1

(CH), 128.7 (CH), 128.1 (CH), 128.0 (CH), 127.7 (CH), 124.4 (C), 91.4

(C–I); MS (m/z, %) 575 (35, M^þ), 574 (100, M^þ–H), 447 (13), 165

(41).

136.7 (C), 134.9 (C), 134.0 (C), 131.5 (C), 137.6 (CH), 131.3 (CH), 130.2

(C), 130.0 (C), 129.6 (CH), 129.4 (CH), 129.3 (2XCH), 128.5 (CH), 128.2

(CH), 127.9 (2XCH), 92.8 (C-I); MS (m/z, %) 575 (48, M^þ–N₂), 574

(100), 526 (34), 357 (27), 77 (56).

5.1.8.2. (E)-1,3-bis(4-chlorophenyl)-4-((diphenylmethylene)

hydrazono)-5-iminoimidazolidin-2-one (XXIb). IR (KBr) 3271 (NH),

1760 (C]O), 1627 (C]N) cm^ꢀ1; MS (m/z, %) 511 (100, M^þ), 434 (80),

317 (15), 193 (27), 168 (18).

5.1.9.3. 1-(4-Chlorophenyl)-3-(3,4-dichlorophenyl)-4-(diphenyl-

methylene)-5 iminoimidazolidin-2-one (XXIVc). IR (KBr) 3229 (NH),

1757 (C]O), 1657 (C]N), 1600 (C]C) cm^ꢀ^{1 1}H NMR (CDCl₃,

;

400 MHz)

d 7.50 (br s, 1 H, NH),7.48–7.39 (m, 7H, Ar–H), 7.38–7.33

(m, 2H, Ar–H), 7.10 (d, J ¼ 8.8 Hz, 1H), 7.07 (d, J ¼ 2.3 Hz, 1H), 7.04–

5.1.8.3. (E)-1-(4-bromophenyl)-3-(4-chlorophenyl)-5-((diphenylme-

6.96 (m, 4H, Ar–H), 6.87–6.80 (m, 2H, Ar–H); ¹³C NMR (CDCl₃,

thylene)hydrazono)-4-iminoimidazolidin-2-one (XXIc). IR (KBr)

100 MHz) d 155. 5 (C), 153.4 (C), 138.2 (C), 138.0 (C), 134.0 (C), 133.8

4332

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

(C), 131.9 (C), 131.1 (C), 130.6 (C), 130.0 (CH), 129.9 (CH), 129.6 (CH),

129.5 (CH), 129.3 (CH), 129.1 (CH), 128.7 (CH), 128.6 (C), 128.4 (CH),

128.2 (CH), 127.8 (CH), 125.9 (CH), 124.3 (C); MS (m/z, %) 517

(32, M^þ), 516 (100, M^þ–H), 165 (30).

115.3 (CH, major), 115.1 (CH, minor), 95.7 (C, major), 95.3 (C,

minor), 63.8 (CH₂, major), 63.7 (CH₂, minor), 14.7 (CH₃);

5.1.11.2. 1,3-Bis(4-bromophenyl)-5-iminoimidazolidine-2,4-dithione

(XXVIb). IR (KBr) 3250 (NH₂), 1495 & 1150 (CS–N) cm^ꢀ^{1 1}H NMR

;

5.1.9.4. 1-(4-Chlorophenyl)-4-(diphenylmethylene)-3-(4-ethoxyphenyl)-

(CDCl₃, 400 MHz)

d

9.53 (br s, 1H, NH), 7.70 (d, J ¼ 8.8 Hz, 2H, Ar–

5-iminoimidazolidin-2-one (XXIVd). IR (KBr) 3220 (NH), 1750 (C]O),

H), 7.69 (d, J ¼ 8.8 Hz, 2H, Ar–H), 7.37 (d, J ¼ 8.8 Hz, 2H, Ar–H), 7.26

1655 (C]N), 1605 (C]C) cm^ꢀ1; ¹H NMR (CDCl₃, 400 MHz)

d

7.51–7.11

(d, J ¼ 8.8 Hz, 2H, Ar–H); ¹³C NMR (CDCl₃, 100 MHz)

d 179.6 (2XC,

(m, 10H, NH & Ar–H), 7.00–6.87 (m, 5H, Ar–H), 6.84–6.77 (m, 2H, Ar–

C]S), 156.0 (C, C]N), 134.0 (2XC–N), 132.9 (CH), 132.8 (CH), 130.0

(CH), 129.9 (CH), 124.4 (C–Br), 123.4 (C–Br); MS (m/z, %) 455 (14,

M^þ), 453 (17), 374 (12), 240 (16).

H), 6.57–6.49 (m, 2H, Ar–H), 3.88 (q, J ¼ 7.0 Hz, 2H), 1.32 (t, J ¼ 7.0 Hz,

3H);; ¹³C NMR (CDCl₃, 100 MHz)

d 157.3 (C), 156.1 (C), 154.0 (C), 138.9

(C), 138.0 (C), 133.3 (C), 131.5 (C), 127.5 (C), 127.4 (C), 130.0 (CH), 129.7

(CH), 129.6 (CH), 129.0 (CH), 128.9 (CH), 128.6 (CH), 127.7 (CH), 127.6

(CH), 127.3 (CH), 125.3 (C), 114.1 (CH), 63.6 (CH₂), 14.6 (CH₃); MS (m/z,

%) 493 (72, M^þ), 492 (100, M^þ–H), 165 (26).

5.1.11.3. 5-Imino-1,3-bis(4-iodophenyl)imidazolidine-2,4-dithione

&

4-imino-3-(4-iodophenyl)-5-(4-iodophenylimino)thiazolidine-

2-thione (XXVIc). Ratio prior to crystallization was

dithione:dimiminothiazole 35:65; ratio after re-crystallization

dithione:dimiminothiazole 68:32; IR (KBr) 3250 (NH₂), 1495 &

5.1.10. Preparation of compounds XXVa–c; the preceding

compounds were prepared according to the following

general procedure

A mixture of the requisite imidazolidineiminothione VI (5 mmol)

and thiocarbohydrazide (5 mmol) in ethanol (25 mL) was heated

under reﬂux for 7 h. Liberation of H₂S could be easily deleted

through reaction time. The obtained product was ﬁltered off,

washed with a minimum amount of ethanol, air-dried and crystal-

lized to give XXVa–c (Table 10).

1150 (CS-N) cm^ꢀ1; ¹H NMR (CDCl₃, 400 MHz)

d 9.57 (br s, 1H, NH,

minor), 9.52 (br s,1H, NH, major), 7.93–7.88 (app ddd consisting of

three overlapping doublets, 6H, minor 2H and major 4H), 7.80 (d,

J ¼ 8.8 Hz, 2H, Ar–H, minor), 7.24 (d, J ¼ 8.8 Hz, 2H, Ar–H, major),

7.13 (d, J ¼ 8.8 Hz, 2H, Ar–H, major), 7.10 (d, J ¼ 8.8 Hz, 2H, Ar–H,

minor), 6.93 (d, J ¼ 8.8 Hz, 2H, Ar–H, minor); ¹³C NMR (CDCl₃,

100 MHz)

d 191.0 (C]S, minor), 179.6 (C]S major), 179.5 (C]S

major), 160.7 (C]N, minor), 156.0 (C]N, major), 150.5 (C), 147.4

(C), 139.1 (CH), 138.9 (CH), 138.8 (CH), 138.8 (CH), 135.1 (C), 134.7

(C), 133.5 (C), 130.3 (CH), 130.1 (CH), 130.0 (CH), 122.7 (CH), 96.2

(C–I), 96.0 (C–I), 95.5 (C–I), 92.7 (C–I); MS (m/z, %) 549 (19, M^þ),

288 (5), 261 (57).

5.1.10.1. 3-Hydrazinyl-7-(4-iodophenyl)-5-phenyl-5H-imidazo

[4,5–e][1,2,4]triazin-6(7H)-one (XXVa). IR (KBr) 3431 (NH₂), 3297

(NH), 1760 (C]O) cm^ꢀ1; MS (m/z, %) 445 (1<, M^þ), 245 (71), 230

(16), 203 (15), 119 (44), 103 (27), 90 (100), 77 (91).

5.2. Biological evaluation

5.1.10.2. 5,7-Bis(4-chlorophenyl)-3-hydrazinyl-5H-imidazo

[4,5–e][1,2,4]triazin-6(7H)-one (XXVb). IR (KBr) 3431 (NH₂), 3297

(NH),1760 (C]O) cm^ꢀ1; MS (m/z, %) 388 (13, M^þþ 1), 387 (1<, M^þ),

353 (18), 268 (12), 200 (6), 187 (100), 119 (58).

5.2.1. Screening studies of compounds XIV, XVIa, XVIIIa,c, XXVd

and XXVIb against EAC cells

Anti tumor activity (in vitro) studies were done at Cairo Univer-

sity, National Cancer Institute, Cancer Biology Department, Phar-

macology Unit.

5.1.10.3. 5-(4-Chlorophenyl)-3-hydrazinyl-7-(4-methoxyphenyl)-5H-

imidazo[4,5–e][1,2,4]triazin-6(7H)-one (XXVc). IR (KBr) 3430 (NH₂),

3290 (NH), 1765 (C]O) cm^ꢀ1; MS (m/z, %) 384 (19, M^þþ 1), 383

(1<, M^þ), 353 (20), 235 (5), 200 (6), 153 (48), 149 (100).

5.2.1.1. Reagents.

1. RPMI 1640 medium (Sigma)

2. Ehrlich AscitesCarcinomacells (EAC) suspension(2.5 ꢃ10⁵/mL)

5.1.11. Preparation of compounds XXVIa–c; the preceding

compounds were prepared according to the following

general procedure

An equimolar mixture of the requisite cyanothioformamide I

and isothiocyanate (5 mmol) and triethylamine (3 drops) in dry

ether (25 mL) was stirred at room temperature for 30 min. The

reaction mixture was triturated several times with light petroleum

ether until solidiﬁcation. The resulting solid was collected and

recrystallized to give XXVIa–c (Table 10).

3. Trypan blue dye: A stock solution was prepared by dissolving

1 g of the dye in (100 mL) distilled water. The working solution

was then prepared by diluting (1 mL) of the stock solution with

(9 mL) of distilled water. The stain was used then for staining

the dead EAC cells.

4. Compounds tested: XIV, XVIa, XVIIIa,c, XXVd and XXVIb.

5.2.1.2. Procedure.

5.1.11.1. 3-(4-Ethoxyphenyl)-5-imino-1-(4-iodophenyl)imidazolidine

1. EAC cells were obtained by needle aspiration of ascetic ﬂuid

from the preinoculated mice under aseptic conditions.

2. The cells were tested for viability and contamination by

staining certain cell volume of this ﬂuid by an equal volume of

the working solution of trypan blue dye.

-2,4-dithione

nyl)thiazolidine-2-thione (XXVIa). Ratio dithione:dimiminothia-

zole 34:66; ¹H NMR (CDCl₃, 400 MHz)

9.50 (br s, 1 H, N–H,

& 5-(4-ethoxyphenylimino)-4-imino-3-(4-iodophe-

d

major), 9.48 (br s,1 H, N–H, minor), 7.93–7.84 (m, 3H, 2H major and

1H minor overlapping), 7.27–7.19 (m, 4H, 2H major and 2H minor

overlapping), 7.09 (d, J ¼ 8.7 Hz, 2H, major), 7.02 (d, J ¼ 9.0 Hz, 1H,

minor), 6.97 (d, J ¼ 8.8 Hz, 2H, major), 4.07 (q, J ¼ 7.0, 2 H, CH₂),

3. The ascetic ﬂuid was diluted to 1:10 with saline to contain

2.5 ꢃ10⁶cells on a hemocytometer.

4. In a set of sterile test tubes 0.1 mL of tumor cells suspension,

0.8 mL RPMI 1640 media and 0.1 mL of each tested compound

1.44 (t, J ¼ 7.0 Hz, 3H); ¹³C NMR (CDCl₃, 100 MHz)

d 192.1 (C, C]S,

major), 180.2 (C]S, minor), 180.0 (C]S, minor), 161.2 (C, major),

159.8 (C, major), 158.9 (C, major), 156.1 (C, minor), 145.1 (C, major),

139.7 (C, major), 138.9 (CH, major), 138.6 (CH, minor), 135.2 (C,

major), 133.6 (C, minor), 130.3 (CH, major), 130.0 (CH, minor), 129.2

(CH, minor), 127.3 (C, minor), 127.2 (C, minor), 123.9 (CH, major),

(corresponding to 25, 50 and 100 mg) were mixed. The test

tubes were incubated at 37 ^ꢁC for 2 h. Trypan blue exclusion

test was carried out to calculate the percentage of non viable

cells. Compounds producing more than 70% non-viable cells

are considered active [43].

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

4333

5.2.5. Antimicrobial and antifungal studies of compounds VId–g,

VIIIa–c and IXa–b

Antimicrobial and antifungal studies were carried out by the

diffusion agar technique [46] using a 1 cm microplate well diameter

No: of non-viable cells

% of non-viable cells ¼

ꢃ 100

No: of cells

5.2.2. Cytotoxic activity studies of XIV, XVIa, XVIIIa,c, XXVd

and XXVIb against various cell lines

and a 100 mL of a sample concentration of 5 mg/mL, unless other-

wise indicated. The tests were done by Dr. Samy El-Henawy, Tarik

M.A. Al-Raheem and Deya A. Fotoh at the Fermentation Biotech-

nology and Applied Microbiology (Ferm-BAM) Center, Al-Azhar

University, Cairo, Egypt. The tested organisms were Gram-negative

bacteria (E. coli, NCTC-10416 & S. typhi, NCIMB-9331), Gram-posi-

tive bacteria (S. aureus, NCTC-7447 & B. subtilis, NCTC-1040) and

fungi (C. albicans, IMRU-3669 & A. niger, IMI-31276). The bacteria

and fungi were maintained on nutrient agar and Czapek‘s Dox agar

medium, respectively. All chemical compounds were dissolved in

N,N-dimethylformamide (DMF) (5 mg/mL), except if noted other-

wise. DMF showed no inhibition activity.

Compounds XIV, XVIa, XVIIIa,c, XXVd and XXVIb were tested at

concentrations between 1 and 10 mg/mL using SRB assay for cyto-

toxic activity against the following tumor cell lines:

1. Brain tumor cell line (U251)

2. Liver carcinoma cell line (HEPG2)

3. Breast carcinoma cell line (MCF7)

4. Cervix carcinoma cell line (HELA)

5. Colon carcinoma cell line (HCT116)

5.2.3. Measurement of potential cytotoxicity by SRB assay

Potential cytotoxicity of compounds XIV, XVIa, XVIIIa,c, XXVd

and XXVIb was tested using the method of Skehan et al. [44], as

follows:

5.2.6. Antimicrobial and antifungal studies of compounds If, V, VIf,

XIIIa, XIV, XV, XVIb and XXVIb,c

The above compounds were tested in vitro for antimicrobial

activity against B. subtilis, MCIB-3610 and P. aeruginosa, NCIB9016

and for antifungal activity against C. albicans and A. niger Ferm-BAM

ꢄ Cells were plated in 96 multiwell plate (10⁴cells/well) for 24 h

before treatment with the compound(s) to allow attachment to

the wall of the plate.

C-21 using a 1 cm microplate well diameter and a 100 mL of each of

the following three concentrations: 1, 2.5 and 5 mg/mL. The

compounds were tested for antifungal activity by the agar disk

diffusion technique under the same conditions of the ﬁrst series of

compounds with the exception that antimicrobial activities were

measured using three different sample concentrations. All

compounds were dissolved in DMF.

ꢄ Different concentrations of the compounds (0, 1, 2.5, 5 and

10 mg/mL) were added to the cell monolayer triplicate wells

were prepared for each individual dose.

ꢄ Monolayer cells were incubated with the compound(s) for 48 h

at 37 ^ꢁC in atmosphere of 5% CO₂.

ꢄ After 48 h, cells were ﬁxed, washed and stained with Sulfo-

Rhodamine-B stain.

5.2.7. Antimicrobial and antifungal susceptibility testing

of compounds XXVIb,c, XVIb, and If

ꢄ Excess stain was washed with acetic acid and attached stain

was recovered with Tris EDTA buffer.

The Minimum Inhibitory Concentration (MIC) of the four tested

compounds was determined by the macrotube dilution technique

following the guidelines of the National Committee for Clinical

Laboratory Standards for bacteria [41] and for ﬁlamentous fungi

[42]. The four Compounds were tested for their Minimum Inhibi-

tory Concentration (MIC) against the Gram positive bacteria

B. subtilis, NCTC-1040 & S. aureus, NCTC-7447, the Gram negative

bacteria Klebsiella pneumonia, NCIMB-9111 & S. typhi, NCIMB-9331

and against ﬁlamentous fungi (A. niger, Ferm-BAM C-21) and

unicellular fungi (C. albicans, IMRU-3669). The results are

summarized in Table 9. The bacteria and fungi were maintained on

nutrient broth medium and Czapek‘s Dox medium, respectively.

ꢄ Color intensity was measured in an ELISA reader.

ꢄ The relation between surviving fraction and drug concentra-

tion is plotted to get the survival curve of each tumor cell line

after the speciﬁed compound.

5.2.4. Virucidal activity studies

Compounds VIe–g, VIII-c, IXa,b and XXVIb were tested against

HAV, HSV1 and COxB4 viral strains and viral activity was assayed by

the plaque formation method.

5.2.4.1. Determination of the anti-infectivity effect of compounds

VIe–g, VIII–c, IXa,b and XXVIb against HAV, HSV1 and COxB4 viral

strains.

5.2.7.1. Procedure

1. 0.1 mL of (10³) virus suspension were mixed with 0.1 mL of

sample at the selected dilution shown in Table 4 (initial

sample concentration 5 mg/mL DMSO). The mixtures were

incubated at room temperature for 1 h in sterile screw-cap-

ped vials.

2. Non-treated virus infected control set was done by mixing

0.1 mL of (10³) of virus suspension in MEM, with 0.1 mL of MEM

medium (virus control).

3. 12- well plates seeded with VERO cells were washed with wash

solution (total washes 2) then added 1 mL wash solution and

incubated at room temperature for 5–10 min.

4. 0.2 mL of either test or a control vial was inoculated. One well/

each plate was left neither inoculated with virus nor treated

with any compound as cell control.

5. Plates were incubated at 37 ^ꢁC to allow virus adsorption.

1) Prepare 15 sterile capped test tubes for each test

microorganism.

2) All of the following steps are carried out using aseptic technique.

3) Add 2.0 mL of dimethylformamide and 2 mg of the tested

compound to the ﬁrst tube and mix the contents well.

4) Add 1.0 mL of sterile broth medium to all other tubes.

5) Transfer 1.0 mL from the ﬁrst tube to the second tube.

6) Using a separate pipette, mix the contents of this tube and

transfer 1.0 mL to the third tube.

7) Continue dilutions in this manner to tube number 14, being

certain to change pipettes between tubes to prevent carry-

over of tested compound on the external surface of the

pipette.

8) Remove 1.0 mL from tube 14 and discard it. The ﬁfteenth tube,

which serves as a control, receives no tested compound.

9) Suspend to an appropriate turbidity several colonies of the

culture to be tested in 5.0 mL of broth medium to give a slightly

turbid suspension.

6. Cell monolayers were washed twice with PBS, then overlaid

with 2ꢃ MEM/agarose mixture. Viral infectivity was assayed by

the plaque formation method [45].

4334

A.M.Sh. El-Sharief, Z. Moussa / European Journal of Medicinal Chemistry 44 (2009) 4315–4334

[22] A.M.Sh. El-Sharief, Y.A. Ammar, Y.A. Mohamed, M.M. Aly, M.S.A. El-Gaby,

A.S. Aly, Phosphorus, Sulfur Silicon Relat. Elem 173 (2001) 39–58.

[23] A.M.Sh. El-Sharief, A.M. Hussein, M.S.A. El-Gaby, A.A. Atalla, A.A. Ahmed,

Phosphorus, Sulfur Silicon Relat. Elem 170 (2001) 47–63.

[24] A.M.Sh. El-Sharief, Y.A. Ammar, M.S.A. El-Gaby, M.A. Zahran, A.A. Khames,

Aﬁnidad 60 (2003) 47–54.

[25] A.M.Sh. El-Sharief, Y.A. Ammar, M.A. Zahran, H.Kh. Sabet, Phosphorus, Sulfur

Silicon Relat. Elem 179 (2004) 267–275.

[26] A.M.Sh. El-Sharief, Y.A. Ammar, M.S.A. El-Gaby, Aﬁnidad 61 (2004) 240–255.

[27] A.M.Sh. El-Sharief, M.S.A. El-Gaby, A.A. Atalla, B.-B.A.A.M. El-Adasy, Aﬁnidad

60 (2003) 475–481.

[28] A.M.Sh. El-Sharief, A.A. Atalla, A.M. Hussein, M.S.A. El-Gaby, A.A. Hassan,

Phosphorus, Sulfur Silicon Relat. Elem 160 (2000) 141–158.

[29] A.M.Sh. El-Sharief, Y.A. Ammar, M.A. Zahran, H.Kh. Sabet, J. Chem. Res., Synop.

3 (2003) 162–167.

[30] For a general procedure for the preparation of cyanothioformanilides see:

E.P. Papadopoulos J. Org. Chem. 44 (1979) 3858–3861 For the preparation of

Ia, see the following references;

10) Dilute this suspension by aseptically pipetting 0.2 mL of the

suspension into 40 mL.

11) Add 1.0 mL of the diluted culture suspension to each of the

tubes. The ﬁnal concentration of tested compound is now one-

half of the original concentration in each tube.

12) Incubate all tubes at 37 ^ꢁC overnight for bacteria and 30 ^ꢁC and

72 h for fungi.

13) Examine the tubes for visible signs of microbial and fungal

growth. The highest dilution without growth is the minimal

inhibitory concentration (MIC).

Acknowledgements

This work was supported by the Deanship of Scientiﬁc Research

of Taibah University (project number: 245/429). Ziad Moussa

(principle investigator) and Ahmed M. Sh. El-Sharief greatly

acknowledge this generous ﬁnancial support. We extend our

gratitude to Prof. M.M. Al-Nozha (Taibah University president), Dr.

A. Alharbe (Dean of Science).

(a) Ref. [1] (b) L. Hyunil, K. Kyongtae, Bull. Korean Chem. Soc. 13 (1992)

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Ib: A.D. Grabenko, P.S. Pel’kis Zh. Obshch. Khim. 30 (1960) 1222–1226;

Ic: commercially available; Also see: B. Kumelj, M. Tisler Vestn. Slov. Kem.

Drus. 5 (1958) 69–73;

For both Ic and Id; Also see Ref. [5] For Id; Ic–e: A.D. Grabenko, P.S. Pel’kis Inst.

Org. Chem., Kiev, Zh. Obshch. Khim. 31 (1961) 2739–2743;

If: Y. Kobayashi-Matsunaga, T. Ishiia, T. Hamaguchia, H. Osadab, M. Sato Lett.

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Article Doi

Synthesis, characterization and derivatization of some novel types of mono- and bis-imidazolidineiminothiones and imidazolidineiminodithiones with antitumor, antiviral, antibacterial and antifungal activities - part I

DOI: 10.1016/j.ejmech.2009.07.019

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