Thiourea derivatives incorporating a hippuric acid moiety: Synthesis and evaluation of antibacterial and antifungal activities

Article abstract of DOI:10.1016/j.ejmech.2013.04.002

New series of thiourea derivatives incorporating a hippuric acid moiety have been synthesized through the reaction of 4-hippuric acid isothiocyanate with various nitrogen nucleophiles such as aliphatic amines, aromatic amines, sulfa drugs, aminopyrazoles, phenylhydrazine and hydrazides. The synthesized compounds were tested against bacterial and fungal strains. Most of compounds, such as 2-(4-(3-(3-bromophenyl)thioureido)benzamido)acetic acid and 2-(4-(3-(4-(N-pyrimidin-2-ylsulfamoyl)phenyl)thioureido)benzamido)acetic acid, showed significant antibacterial and antifungal activities. These compounds comprise a new class of promising broad-spectrum antibacterial and antifungal agents.

Full text of DOI:10.1016/j.ejmech.2013.04.002

Accepted Manuscript
Thiourea derivatives incorporating a hippuric acid moiety: Synthesis and evaluation of
antibacterial and antifungal activities
Samir Y. Abbas, Marwa A.M. Sh. El-Sharief, Wahid M. Basyouni, Issa M.I. Fakhr,
Eman W. El-Gammal
PII:
S0223-5234(13)00234-1
10.1016/j.ejmech.2013.04.002
EJMECH 6112
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 November 2012
Revised Date: 1 April 2013
Accepted Date: 2 April 2013
Please cite this article as: S.Y. Abbas, M.A.M. Sh. El-Sharief, W.M. Basyouni, I.M.I. Fakhr, E.W.
El-Gammal, Thiourea derivatives incorporating a hippuric acid moiety: Synthesis and evaluation of
antibacterial and antifungal activities, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.04.002.
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ACCEPTED MANUSCRIPT
Thiourea derivatives incorporating a hippuric acid moiety: Synthesis and evaluation of
antibacterial and antifungal activities
Marwa A. M. Sh. El-Shariefb,*, Wahid M. Basyounia,
Issa M.I. Fakhrb and
a,
Samir Y. Abbas *,
Eman W. El-Gammalc
aOrganometallic and Organometalloid Chemistry Department, National Research Centre, Cairo, Egypt
bApplied Organic Chemistry Department, National Research Centre, Cairo, Egypt
cDepartment of the Chemistry of Natural and Microbial Products, National Research Centre, Cairo,
Egypt
Graphical abstract:
S
X = 3-Br-C6H4
X = C6H4SO2NH-2-pyrimidinyl
X = NHSO2C6H5
O
X
N
N
H
H
HN
COOH
MIC = 3.12 µg/mL
Antibacterial and antifungal activities
New series of thiourea derivatives were synthesized through reaction with 4-hippuric acid
isothiocyanate and evaluated for antibacterial and antifungal activity.
*Corresponding authors: samiryoussef98@yahoo.com (S.Y. Abbas); mrwa1976@hotmail.com
(M.A.M.Sh. El-Sharief)
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Thiourea derivatives incorporating a hippuric acid moiety: Synthesis and evaluation of
antibacterial and antifungal activities
Marwa A. M. Sh. El-Shariefb,*, Wahid M. Basyounia,
a,
Samir Y. Abbas *,
Issa M.I.
Eman W. El-Gammalc
Fakhrb and
aOrganometallic and Organometalloid Chemistry Department, National Research Centre,
Cairo, Egypt
bApplied Organic Chemistry Department, National Research Centre, Cairo, Egypt
cDepartment of the Chemistry of Natural and Microbial Products, National Research Centre,
Cairo, Egypt
*Corresponding
authors:
samiryoussef98@yahoo.com
(S.Y.
Abbas);
mrwa1976@hotmail.com (M.A.M.Sh. El-Sharief)
Abstract: New series of thiourea derivatives incorporating a hippuric acid moiety have been
synthesized through the reaction of 4-hippuric acid isothiocyanate with various nitrogen
nucleophiles such as aliphatic amines, aromatic amines, sulfa drugs, aminopyrazoles,
phenylhydrazine and hydrazides. The synthesized compounds were tested against bacterial
and
fungal
strains.
Most
of
compounds,
acid and
such
as
2-(4-(3-(3-
bromophenyl)thioureido)benzamido)acetic
2-(4-(3-(4-(N-pyrimidin-2-
ylsulfamoyl)phenyl)thioureido)benzamido)acetic acid, showed significant antibacterial and
antifungal activities. These compounds comprise a new class of promising broad-spectrum
antibacterial and antifungal agents.
Keywords: Thiourea; Hippuric acid; Antibacterial; Antifungal activity.
1. Introduction
Despite many significant progresses in antimicrobial therapy, infectious diseases
caused by bacteria and fungi remain a major worldwide health problem due to rapid
development of resistance to the existing antimicrobial drugs. In other words, the increasing
use and misuse of the existing antimicrobial drugs have resulted in the development of
resistant pathogens [1-4]. So, the medical community faces a serious problem against
infections caused by the pathogen microbes and needs an effective therapy and search for
novel antimicrobial agents. The emergence of multidrug resistant gram-positive and gram-
negative bacteria has caused life-threatening infectious diseases in many countries around the
world. In addition, systemic and dermal fungal infections have significantly increased,
specifically in individuals with suppressed immune systems such as cancer chemotherapy and
AIDS patients. Although there are different antifungal drugs used in the treatment of fungal
infections, some of them have undesirable side effects because of the biochemical similarity
to human cell. Moreover, some of them become less effective due to the development of
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resistance to these drugs. The search for new and effective antimicrobial agents, resistant to
the mechanisms of defense of these bacteria, is of paramount importance [5, 6].
Various 1,3-disubstitued thiourea derivatives are extremely versatile building blocks for the
synthesis of a variety of heterocyclic compounds and exhibit a wide spectrum of bioactivities.
Many thiourea derivatives showed significant antimicrobial activity [7]; 1,3-dialkyl or diaryl
thiourea derivatives exhibit significant antifungal activity against plant pathogens [8].
As a part of our extensive research program to rapidly assemble novel bioactive compounds
under mild conditions [9-13], we have employed 4-hippuric acid isothiocyanate to synthesize
various 1,3-disubstituted thiourea derivatives with structure modifications involving
incorporation of the hippuric acid moiety at position N1 with promising antimicrobial
properties.
2. Chemistry
The monosubstituted thiourea 2 was obtained through the reaction of 4-aminohippuric
acid 1 with ammonium thiocyanate in the presence of hydrochloric acid. The interest of 4-
aminohippuric acid 1 is based on the conversion possibility of the amino group in position 4
to isothiocyanate group. The isothiocyanate 3 was synthesized from the reaction of 4-
aminohippuric acid 1 with thiophosgene in the presence of dilute HCl at room temperature.
The scope of the reaction of isothiocyanate 3 with various nitrogen nucleophiles was studied
with the objective of obtaining biologically active compounds. When isothiocyanate 3 was
left to react with some selected aliphatic and aromatic amines, the corresponding thiourea
derivatives 4a-g were obtained in good yield. Supportive evidence the latter products was
obtained from their synthesis via another synthetic route. Thus, interaction of the amine 1
with isothiocyanate derivatives in dioxane gave the thiourea derivatives 4a-g (m.p. and mixed
m.p.) (Scheme 1).
Scheme 1
Moreover, our investigation was extended to include the behavior of isothiocyanate 3
towards different types of aromatic amines. Thus, interaction of 3 with 9-aminoacridine as
polynuclear aromatic amine gave the corresponding thiourea derivative 5. Interaction of 3
with 2-amino-N-(aryl)-benzamides gave the corresponding benzamide thiourea derivatives
6a,b. It’s well known that sulfonamides have enormous potential as pharmaceutical and
agricultural agents due to their biological activities; they are associated with antibacterial
properties. Some active sulfonamides as antibacterial agents are also known for their
immune-modifying effects. On the basis of these reports, we reported here the synthesis of
thiourea derivatives containing sulfonamide moiety to evaluate their antimicrobial activity.
Thus, interaction of 3 with some selected sulfa drugs gave the corresponding thiourea
derivatives 7a-f (Scheme 2). Symmetrical bis-hippuric acid 8a was obtained when one mole
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of compound 1 was treated with one mole of isothiocyanate 3 in dioxane under reflux. On the
other hand, symmetrical bis-compound 8b was obtained when two moles of compound 3
were reacted with one mole of 1,4-phenylenediamine in dioxane under reflux (Scheme 2).
Scheme 2
The pyrazole derivative 9 was obtained via the reaction of the isothiocyanate 3 with 4-
aminoantipyrene. Treatment of 3 with 5-amino-3-phenylamino-1H-pyrazole-4-carboxylic
acid amide as aminopyrazole afforded the corresponding pyrazole derivative 10a. Moreover,
treatment of
3
with 4-(4-chloro-phenylazo)-4H-pyrazole-3,5-diamine afforded the
corresponding pyrazole derivative 10b (Scheme 3). Treatment of 3 with phenylhydrazine in
dioxane furnished the novel disubstituted thiosemicarbazide derivative 11a. On the other
hand, interaction of 3 with hydrazide derivatives (namely, phenyl hyrazide, isonicotinic
hydrazide), the novel disubstituted thiosemicarbazide derivatives 11b,c were obtained in
good yield, respectively. Finally, the reaction of 3 with phenylsulphonylhydrazine afforded
the novel disubstituted thiosemicarbazide derivative 11d in high yield. (Scheme 3)
Scheme 3
3. Results and discussion
The search for newer antibacterial and antifungal agents continues due to the rapid
development the resistance among bacteria and fungi to the existing antimicrobial agents.
Even though novel broad spectrum antimicrobial agents were reported in recent years, the
emergence of resistance has become an obstacle. Thus, more effective classes of such agents
are desired. Thiourea derivatives may comprise a new class of antimicrobial agents. The aim
of the present investigation is to synthesize different series of thiourea derivatives which
bearing a 4-hippuric acid moiety at N1 and various substituent at N2. The authors measured
the anti (bacterial and fungal) activities of these derivatives. They studied the effect of each
substituent at N2 on these activities and make a comparative study between them to deduce a
structure activity relationship. The following selection of 23 compounds 4a-g, 5, 6a,b, 7a-f,
8a,b, 9, 10b and 11b-d were tested under the same conditions for antibacterial and antifungal
activity against Gram positive and Gram negative bacteria, and fungi.
3.1. Antibacterial activities
The synthesized compounds were tested in vitro for antibacterial activity against the
following bacterial strains: three Gram-positive bacteria, Staphylococcus aureus ATCC
29213, Bacillus subtilis ATCC 6633 and Bacillus megaterium ATCC 9885, and five Gram-
negative bacteria, Klebseilla peneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC
27953, Escherichia coli ATCC 25922, Sarcina lutea, Proteous vulgaris, and the results are
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summarized in Table 1. Antimicrobial tests were carried out by the agar well diffusion
method [14] using 100 ꢀL of tested compound solution prepared by dissolving 1 mg of the
chemical compound in 1 ml of dimethyl sulfoxide (DMSO). The inoculated plates were then
incubated for 24 h at 37 °C. The antibacterial agent ciprofloxacin (0.5 mg/ml) was used as a
standard. After incubation time, antimicrobial activity was evaluated by measuring the
inhibition zone diameters against the test organisms and compared with standard zone size
ranges that determine susceptibility, intermediate susceptibility, or resistance to the screened
compounds. Visual bacterial growth is observed only in areas in which the drug
concentrations are below those required for growth inhibition. The experiment was carried
out in triplicate and the average zone of inhibition was calculated.
Table 1
The mean values of the inhibition zone diameter obtained for these compounds suggest that
all of the thiourea derivatives evaluated possess significant antibacterial activity against most
of the test organisms used in these assays. Using the general structure provided in Figure 1,
certain aspects of the structure activity relationships for these compounds can be more clearly
highlighted.
Figure 1
As anticipated, a clear difference in antibacterial activity is noted between compounds within
and between each series, pointing to the reinforcing and opposing effects of X groups. The
results of the antimicrobial screening demonstrated the following assumptions about the
structural activity relationship (SAR). Structure 4, X were alkyl and aryl moieties (4a; n-
butyl, 4b; cyclohexyl, 4c; phenyl, 4d; 3-bromophenyl, 4e; 4-methylphenyl, 4f; 3-
fluorophenyl, 4g; 4-fluorophenyl): Regarding the effect of X group, it is evident that varying
such a unit may have a dramatic effect on antimicrobial activity, which may be augmented or
reduced depending on whether a matching or mismatching relationship exists with the X
group. The type of the substitutions on the benzene ring of aryl moiety is important. It was
noticed that the presence of electron withdrawing groups such as fluoride and bromide at the
benzene ring displayed strong effect on the antimicrobial activity. The presence of 3-
bromophenyl moiety (4d) resulted in the highest antimicrobial activity among all the
compounds investigated in this study. the presence of 3-bromophenyl moiety exhibited the
highest antibacterial activity against most of the organisms, 3-bromophenyl moiety showed
results greater than the reference drug reach 1.5 fold against most of the organisms. 4-
Fluorophenyl moiety exhibited activities near to the references drug. Also, 3-fluorophenyl
showed potent activities against Bacillus megaterium and good activity toward other
organisms. The presence of electron donating group such as methyl at the benzene ring or
leaving benzene ring without substitution had a detrimental effect on antibacterial activity.
Compounds 4c (phenyl moiety) showed moderate activity against four organisms and no
activates toward the other test organisms and 4e (4-methylphenyl moiety) showed strong
activities greater than reference drug against Sarcina lutea only and no activates toward the
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other test organisms. Also, comparing the zone inhibition diameter values obtained with 4d
(3-bromophenyl moiety) with those obtained with its 4c; phenyl, 4e; 4-methylphenyl, 4f; 3-
fluorophenyl, and 4g; 4-fluorophenyl against tested organisms indicates the superiority of 4d
and much improved activity. Whereas 4c, 4e and 4f produced values ranging from 0-19 mm,
0-25 mm and 18-25 mm, respectively, compound 4d afforded zone inhibition diameter values
ranging from 20-32 mm. This clear difference in antibacterial activity observed between the
preceding compounds is a direct reflection of the reinforcing effect induced by X group.
When X was alkyl moiety, such as n-butyl moiety in case of 4a, the activity was equipotent
to the reference drug while cyclohexyl moiety showed strong activities against Bacillus
megaterium and Sarcina lutea only and from weak to no activity towards the other test
organisms
Introduction of polynuclear aromatic ring such as acridine moiety in case of compound 5
showed strong activities against all of test organisms except against Sarcina lutea and
Saccharomyces cervesia.
Increasing the size of the substituent, as in structure 6, had a detrimental effect on
antibacterial activity. Structure 6, which has benzamide side chain ending with
trifluoromethylphenyl and ethoxyphenyl moieties showed good activity against two gram +ve
bacteria, moderate activity against one gram -ve bacteria only. The presence of ethoxyphenyl
moiety in case of 6b showed activity more than trifluoromethyl moiety in case of 6a.
Structure 7 has a sulfonamide moiety where X can be hydrogen; 7a, acetyl; 7b, C(NH)NH2;
7c, 2-thiazolyl; 7d, 2-pyridinyl, 7e, 2-pyrimidinyl; 7f. The type of the alkyl or heterocyclic
moieties on the sulfonamide side chain is important. The highest antibacterial activity of the
sulfonamide series was observed in case of compounds 7f. The presence of pyrimidinyl
moiety showed results greater than the reference drug reaching a 1.5 fold greater zone of
inhibition against most of the test organisms. The presence of thiazole nucleus resulted in the
lowest antimicrobial activity among all the tested compounds; 7d with thiazole moiety
showed good activity against two gram +ve bacteria and showed result greater than the
reference drug against one Gram -ve bacteria and no activates toward the other test
organisms. Replacement thiazole ring by pyridine ring improve the antibacterial activity; 7e
showed strong activities near to reference drug against most of the test strains. Further
introduction of polar functionality of the sulfonamide side chain, as in 7b with acetyl moiety
and 7c with C(NH)NH2 moiety, had a detrimental effect on antibacterial activity, 7b showed
strong activity against gram +ve bacteria only, 7c showed good activities against two bacteria
only. When X = was hydrogen in case of 7a had a good effect on antibacterial activity; 7a
showed strong activities near to reference drug against most of the test strains. A similar
comparison of relevant compounds from each series with the appropriate analogues clearly
demonstrates the influence of X group, although the optimum effect of a synergetic
combination against a particular strain may not necessarily extend to the strains.
Bis-compound 8a with two hippuric and one thiourea moieties showed activity equipotent to
the reference drug; but in case of bis-compound 8b with two hippuric and two thiourea
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moieties, the activity was increased and showed activity greater than the reference drug
against Bacillus subtilis, Escherichia coli.
Compounds 9, 10b, the X group have substituted pyrazole moiety; introduction of pyrazole
moiety resulted in increasing the antimicrobial activity. When a 4-chlorophenyldiazo moiety
and amino group were attached at position 3, 4 of the heterocyclic pyrazole system caused
better potency against most of the test organisms. Compound 10b showed activity greater
than reference drug against the most test organisms. The observed antimicrobial activity of
compounds 10b could be attributed to the synergistic effect of both the pyrazole and 4-
chlorophenyldiazo moieties.
Structure 11, X was NHCOPh in case 11b, X was pyridine amide in case 11c, X was
NHSO2Ph in case 11d. In general, treatment of 3 with hydrazide derivatives proved to be
detrimental to antibacterial activity. The effect on antibacterial activity was especially
apparent for the sulfonamide derivative (11d). Compound 11d with sulfonamide moiety
showed strong results greater than the reference drug against most of the test organisms.
Compound 11b showed good activity against most of the test organisms while 11c showed
strong activity against Bacillus megaterium and Sarcina lutea only.
The comparison between the antibacterial activities of our potent synthesized compounds and
ciprofloxacin as standard antibacterial reference drug against the used Gram positive and
Gram negative bacteria is represented graphically in Figure 2.
Figure 2
3.2. Antifungal activity
We chose Saccharomyces cerevisiae and Candida albicans NRRLY-477 in the antifungal
susceptibility tests. Antifungal agents are evaluated against clinical isolates of standard
strains of fungi by the agar well diffusion method [14] using 100 ꢀL of tested compound
solution prepared by dissolving 1 mg of the chemical compound in 1 ml of dimethyl
sulfoxide. The antifungal agent ketoconazole was used as a standard at concentration 0.5
mg/mL and was tested under the same conditions. The inoculated plates were then incubated.
After incubation time, antifungal activity was evaluated by measuring the inhibition zone
against the test organisms and compared with that of the standard. Antifungal activities were
expressed as inhibition zone diameter in millimeters (mm). The experiment was carried out in
triplicate and the average zone of inhibition was calculated. The results are tabulated in Table
1. In general, some compounds were very effective antifungal agents and in several cases
achieved similar or greater levels of activity as the standard antifungal agent ketoconazole
(23 and 22 mm). It is noted though that some fluctuation in antibacterial activity for
individual tested compound was observed across the two strains of tested fungi.
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As anticipated, a clear difference in antifungal activity is noted between compounds within
and between each series, pointing to the reinforcing and opposing effects of the X groups. For
instance, compound 4d (X = 3-bromophenyl) proved more effective against Saccharomyces
cerevisiae (28 mm) and Candida albicans (30 mm) than the standard antifungal agent (23 and
22 mm). In addition, 7f, 8b, 10b and 11d proved more effective than ketoconazole,
compound 7f with sulfonamide side chain ending pyrimidinyl moiety produced 30 mm and
25 mm, compound 8b gave 26 mm and 26 mm, compound 10b with pyrazole moiety gave 25
mm and 30 mm and compound 11d with sulfonamide moiety gave 24 mm and 32 mm
inhibition zone diameter values against the two fungal strains. Thus, these compounds
comprise a new class of potential broad-spectrum antifungal agents. The comparison between
the antifungal activities of our potent synthesized compounds and Ketoconazole as a
reference antifungal agent is illustrated graphically in Figure 3.
Figure 3
3.3. Minimum inhibitory concentrations against Gram positive bacteria, Gram negative
bacteria and fungi
Minimum inhibitory concentration (MIC) of the more active synthesized compounds 4d, 4f,
7f, 8a,b, 10b and 11d was then evaluated in vitro using the twofold serial dilution technique
[15]. The lowest concentration showing no growth was taken as the minimum inhibitory
concentration (MIC). The results of minimum inhibitory concentration were reported in Table
2.
Regarding the effect of X group (4d; 3-bromophenyl, 4f; 3-fluorophenyl, 7f, sulfonamide
side chain ending with 2-pyrimidinyl; 8a; hippuric acid moiety, 8b; phenyl thiourea side
chain ending with hippuric acid moiety, 10b; pyrazole moiety; 11d; NHSO2Ph) against
bacterial and fungal strains, results of antimicrobial activity in this study revealed that: The
presence of 3-bromophenyl moiety (4d) resulted in the highest antimicrobial activity among
all the compounds investigated in this study. Compound 4d showed better results when
compared with ciprofloxacin as revealed from its MIC values (3.12 - 12.5 ꢀg/mL).
Compound 4d showed twofold potency of ciprofloxacin in inhibiting the growth of B. subtilis
(MIC 3.12 ꢀg/mL), B. megaterium (MIC 3.12 ꢀg/mL), K. peneumoniae (MIC 6.25 ꢀg/mL),
E. coli (MIC 6.25 ꢀg/mL) and P. vulgaris (MIC 6.25 ꢀg/mL). Compound 4d was equipotent
to the ciprofloxacin in inhibiting the growth of S. aureus, P. aeruginosa and S. lutea (MIC
12.5 ꢀg/mL). Regarding the activity of 4d against fungal strains; 4d showed twofold potency
of ketoconazole in inhibiting the growth of C. albicans (MIC 3.12 ꢀg/mL), and it showed
50% less potent effect than ketoconazole against S. cerevisia (MIC 6.25 ꢀg/mL).
Also, compounds 7f, 10b and 11d showed better results when compared with ciprofloxacin
as revealed from their MIC values (3.12 - 12.5 ꢀg/mL). The presence of pyrimidinyl moiety
(7f) showed results greater than the reference drug reaching fourfold against P. aeruginosa
(MIC 3.12 ꢀg/mL), and twofold against S. aureus (MIC 3.12 ꢀg/mL) and E. coli (MIC 6.25
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ꢀg/mL). Compound 7f was equipotent to the ciprofloxacin in inhibiting the growth of K.
peneumoniae, S. lutea and P. vulgaris (MIC 12.5 ꢀg/mL), and displayed 50% less activity
compared to ciprofloxacin against the growth of the other organisms. Compound 10b with
pyrazole moiety showed twofold potency of ciprofloxacin in inhibiting the growth of B.
subtilis (MIC 3.12 ꢀg/mL), E. coli (MIC 6.25 ꢀg/mL) and P. vulgaris (MIC 6.25 ꢀg/mL), and
was equipotent to the ciprofloxacin toward other organisms. Compound 11d with
sulfonamide moiety showed twofold potency of ciprofloxacin in inhibiting the growth of B.
subtilis (MIC 3.12 ꢀg/mL), B. megaterium (MIC 3.12 ꢀg/mL) and E. coli (MIC 6.25 ꢀg/mL),
and was equipotent to the ciprofloxacin toward other organisms. Regarding the activity of
above compounds against fungal strains; compounds 7f, 10b and 11d showed good results
when compared with ketoconazole as revealed from their MIC values (3.12- 12.5 ꢀg/mL).
Compounds 7f was equipotent to the ketoconazole toward S. cerevisia (MIC 3.12 ꢀg/mL),
and it showed 50% less potent effect than ketoconazole against C. albicans (MIC 12.5
ꢀg/mL). Compounds 10b and 11d showed twofold potency of ketoconazole in inhibiting the
growth of C. albicans (MIC 3.12 ꢀg/mL), and 25% less potent effect than ketoconazole
against S. cerevisia (MIC 12.5 ꢀg/mL).
Compound 4f with 3-fluorophenyl moiety was equipotent to ciprofloxacin against the growth
of the different Gram-negative bacteria (MIC 12.5 ꢀg/mL). Compound 4f displayed 50% less
activity compared to ciprofloxacin against the growth of the different Gram-positive bacteria
(MIC 12.5 ꢀg/mL) except against S. aureus (MIC 50 ꢀg/mL). Regarding the activity of the
4f against fungal strains; it showed 50% less potent effect than ketoconazole against C.
albicans (MIC 12.5 ꢀg/mL), and moderate potency (MIC 25 ꢀg/mL) against S. cerevisia
when compared with the reference drug (MIC 3.12 ꢀg/mL).
Biscompounds 8a,b showed potency equipotent to the ciprofloxacin toward K. peneumoniae
and P. aeruginosa (MIC 12.5 ꢀg/mL), and moderate activity against the growth of the
different bacteria (MIC 12.5 - 50 ꢀg/mL). Regarding the activity of 8a,b against fungal
strains; biscompound 8a,b were 50% less potent effect than ketoconazole against C. albicans
(MIC 12.5 ꢀg/mL), and moderate potency (MIC 25 - 50 ꢀg/mL) against S. cerevisia when
compared with the reference drug (MIC 3.12 ꢀg/mL).
As shown in Table 2, most of the tested compounds revealed MIC 12.5 µg/ml against
Klebseilla peneumoniae, Pseudomonas aeroginosa and Sarcina lutea. The lowest MIC values
were obtained against the Candida albicans (MIC 3.12 - 12.5 ꢀg/mL) followed by Bacillus
megaterium, Pseudomonas aeroginosa, Klebseilla peneumoniae. Compounds 4d, 7f, 10b
and 11d were consistently most of the effective against all microbial strains, producing the
lowest MIC (3.12 µg/ml) against most of tested organisms. In certain cases, such values are
low enough to render such agents as potential candidates for further studies.
Table 2
4. Conclusion
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In summary, the aim of the present investigation is to synthesize different series of thiourea
derivatives which bearing a 4-hippuric acid moiety at N1 and various substituent at N2
through the reaction of 4-hippuric acid isothiocyanate with various nitrogen nucleophiles to
achieve the antimicrobial effect and reducing the toxicity associated with thiourea. This
coupling may lead to gives an opportunity in medicinal chemistry to improve the clinical and
therapeutic effectiveness of a drug that is suffering from some undesirable properties
hindering its clinical usefulness. The antimicrobial screening for the most of tested
compounds exhibited significant antimicrobial effect. The results of the antimicrobial
screening demonstrated the following assumptions about the structural activity relationship
(SAR). When aryl substituent at N2 containing electron withdrawing halogen such as fluoride
and bromide displayed strong effect on the antimicrobial activity, For example, the presence
of 3-bromophenyl moiety resulted in the highest antimicrobial activity among all the
compounds investigated in this study. the presence of 3-bromophenyl moiety exhibited the
highest antibacterial activity against most of the organisms, 3-bromophenyl moiety showed
results greater than the reference drug reach 1.5 fold against most of the organisms. While,
the presence of electron donating group such as methyl at the benzene ring or leaving
benzene ring without substitution had a detrimental effect on antibacterial activity. When X
was alkyl moiety the activity was equipotent to the reference drug. Introduction of
polynuclear aromatic ring showed strong activities against all of test organisms. Increase in
the size of X group had a detrimental effect on antibacterial activity. Coupling of thiourea
with sulfonamide showed strong activities near to reference drug against most of the test
strains. Biscompound with two hippuric and one thiourea moieties showed activity equipotent
to the reference drug. Introduction of pyrazole moiety resulted in increasing the antimicrobial
activity. In general, treatment of isothiocyanate derivative with hydrazide derivatives proved
to be detrimental to antibacterial activity. The effect on antimicrobial activity was especially
apparent for the sulfonamide derivative. Coupling with sulfonamide moiety showed strong
results greater than the reference drug against most of the test organisms.
5. Experimental Section
All melting points were determined on an electrothermal Gallenkamp apparatus and are
uncorrected. The IR spectra were measured on a Mattson 5000 FTIR Spectrometer in
potassium bromide discs. The NMR spectra were recorded in DMSO-d6 on a Bruker WP
spectrometer (500 MHz) and the chemical shifts δ downfield from TMS as an internal
standard. The mass spectra were recorded on Finnegan MAT 212 instrument, the ionizing
voltage was 70 ev, at Faculty of Science, Cairo University. Elemental analyses were carried
out by the Microanalytical unit of Faculty of Science, Cairo University.
5.1 Synthesis of 2-(4-thioureidobenzamido)acetic acid (2)
To a magnetically stirred solution of 4-aminohippuric acid 1 (0.01 mol) in 10 mL of 6 N HCl
was added ammonium thiocyanate (1.14 g, 0.015 mol). The mixture was heated at 80 °C
under reflux for 12 h until abundant precipitate appeared. The mixture was cooled to room
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temperature, the solid product obtained was collected by filtration, washed with water, and
dried under vacuum to produce the corresponding thiourea 2 as colorless crystals; yield 55%;
1
mp 135-136 °C; IR: ν/cm-1: 3412, 3270, 3160 (NH, NH2, OH), 1721, 1645 (C=O); HNMR:
δ/ppm: 3.90 (d, 2H, J = 6.22 Hz, CH2), 5.40 (br, 2H, NH2), 7.10 - 7.70 (m, 4H, Ar-H), 8.83
(t, 1H, J = 6.13 Hz, CONH), 10.84 (s, 1H, NH), 12.22 (br, 1H, OH); 13C NMR: 40.2
(NHCH2), 116.8, 117.8, 126.6 (2C), 130.2, 141.9, 166.5 (CONH), 171.9 (COOH), 176.6
(C=S); MS, m/z (%): 253 (M+, 12.8), 192 (10.4), 160 (23.4), 128 (33.1), 96 (19.86), 64
(100); Anal. calcd for C10H11N3O3S (253.05): C, 47.42; H, 4.38; N, 16.59. Found: C, 47.50;
H, 4.40; N, 16.70%.
5.2 Synthesis 2-(4-isothiocyanatobenzamido)acetic acid (3)
To an efficiently stirred solution of (4-amino-benzoylamino)-acetic acid 1 (0.12 mol) in 150
ml H2O and 50 ml conc. HCl, thiophosegene (0.12 mol) was added in one portion and the
stirring was continued until the red color of thiophosegene disappeared and white crystals of
isothiocyanate began to precipitate (0.5-1.0 h). The solid product obtained was collected by
filtration and crystallized from THF to give the corresponding isothiocyanato derivative 3 as
colorless crystals; yield 74%; mp 194-196 °C; IR: ν/cm-1: 3454 (OH), 3259 (NH), 1733, 1638
1
(C=O); H NMR: δ/ppm: 4.00 (d, 2H, J = 6.10 Hz, CH2), 7.58 (d, 2H, J = 8.45 Hz, Ar-H),
8.14 (d, 2H, J = 8.40 Hz, Ar-H), 9.04 (t, 1H, J = 6.15 Hz, CONH), 12.66 (br, 1H, OH); MS,
m/z (%): 236 (M+, 8.4), 192 (M-CO2; 24.4), 163 (M - NCH2COOH; 11.1), 162 (M -
HNCH2COOH; 100), 134 (C6H4NCS; 38.4); Anal. Calcd for C10H8N2O3S (236.03): C, 50.84;
H, 3.41; N, 11.86. Found: C, 51.00; H, 3.50; N, 11.70%.
5.3 Synthesis of disubstitued thiourea derivatives 4a-g
Method A: a solution of isothiocyanate 3 (0.01 mol) in dioxane was treated with the requisite
amine (0.01 mol) (namely n-butyl amine, cyclohexyl amine, aniline, 3-bromoaniline, 4-
methylaniline, 3-fluoroaniline, 4-fluoroaniline). The reaction mixture was heated under reflux
for 3 hours. The solution was concentrated under vacuum. The solid product so formed after
cooling was filtered off, washed with ethanol, dried well, and recrystallized from dioxane to
give 4a-g in good yield as shown below.
Method B: a solution of amine 1 (0.01 mol) and the requisite isothiocyanate (namely n-butyl
isothiocyanate, cyclohexyl isothiocyanate, phenyl isothiocyanate, 3-bromophenyl
isothiocyanate, 4-methylphenyl isothiocyanate, 3-fluorophenyl isothiocyanate, 4-
fluorophenyl isothiocyanate) (0.01 mol) in dioxane was heated under reflux for 3 hours. The
solution was concentrated under vacuum, left to cool. The solid product was filtered off,
washed with ethanol, dried well, and recrystallized (m.p. and mixed m.p.).
5.3.1 2-(4-(3-Butylthioureido)benzamido)acetic acid (4a) as colorless crystals; yield 74%; mp
169-171 °C; IR: ν/cm-1 = 3443, 3318, 3173 (NH, OH), 1709, 1641 (C=O); 1H NMR: δ/ppm:
0.93 (t, 3H, butyl-H at CH3), 1.30-1.60 (m, 4H, butyl-H at C2,3-H), 3.58 (m, 2H, butyl-H at
C1-H), 3.85 (d, 2H, J = 6.40 Hz, CH2), 7.54 (d, 2H, J = 8.40 Hz, Ar-H), 7.69 (d, 2H, J =
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8.40 Hz, Ar-H), 8.72 (t, 1H, J = 6.40 Hz, CONH), 9.35 (s, 1H, NH), 9.90 (s, 1H, NH), 12.30
(br, 1H, OH); 13C NMR: 14.18 (butyl CH3), 20.05 (butyl C3), 32.30 (butyl C2), 39.83
(NHCH2), 44.17 (butyl C1), 113.1 (2C), 117.0, 128.7, 129.3 (2C), 152.3 (CONH), 166.7
(COOH), 172.4 (C=S); Anal. calcd for C14H19N3O3S (309.38): C, 54.35; H, 6.19; N, 13.58.
Found: C, 54.20; H, 6.00; N, 13.40 %.
5.3.2 2-(4-(3-Cyclohexylthioureido)benzamido)acetic acid (4b) as colorless crystals; yield
76%; mp 180-181°C; IR: ν/cm-1 = 3478, 3235, 3192 (NH, OH), 1731, 1645 (C=O); 1H NMR:
δ/ppm: 1.10 - 1.95 (m, 10H, cycl-H), 3.93 (d, 2H, J = 5.49 Hz, CH2), 4.20 (m, 1H, cycl-C1-
H), 7.55 (d, 2H, J= 8.40 Hz, Ar-H), 7.77 (d, 2H, J= 8.40 Hz, Ar-H), 8.72 (t, 1H, J= 5.40 Hz,
CONH), 8.87 (s, 1H, NH), 9.90 (s, 1H, NH), 12.30 (br, 1H, OH); 13C NMR: 25.36 (2C),
25.57, 32.2 (2C), 41.23, 52.6, 113.1 (2C), 121.1, 129.3 (2C), 143.2, 166.5 (CONH), 172.0
(COOH), 179.4 (C=S); Anal. calcd for C16H21N3O3S (335.42): C, 57.29; H, 6.31; N, 12.53.
Found: C, 57.30; H, 6.20; N, 12.40 %.
5.3.3 2-(4-(3-Phenylthioureido)benzamido)acetic acid (4c): as colorless crystals; yield 76%;
mp 220-224 °C; IR: ν/cm-1 = 3425-3173 (NH, OH), 1708, 1645 (C=O); 1HNMR: δ/ppm: 3.87
(d, 2H, J = 6.40 Hz, CH2), 7.15 - 7.83 (m, 9H, Ar-H), 8.97 (t, 1H, J = 6.45 Hz, CONH), 9.77
(br, 1H, NH), 9.93 (br, 1H, NH), 12.12 (br, 1H, OH); Anal. calcd for C16H15N3O3S (329.08):
C, 58.34; H, 4.59; N, 12.76. Found: C, 56.37; H, 3.74; N, 18.28 %.
5.3.4 2-(4-(3-(3-Bromophenyl)thioureido)benzamido)acetic acid (4d): as colorless crystals;
1
yield 87%; mp > 300 °C; IR: ν/cm-1 = 3451, 3140 (NH, OH), 1711, 1645 (C=O); HNMR:
δ/ppm: 3.83 (d, 2H, J = 6.31 Hz, CH2), 6.85 - 7.88 (m, 8H, Ar-H), 8.77 (t, 1H, J = 6.39 Hz,
CONH), 9.85 (s, 1H, NH), 10.42 (s, 1H, NH), 12.22 (br, 1H, OH); Anal. calcd for
C16H14BrN3O3S (406.99): C, 47.07; H, 3.46; N, 10.29. Found: C, 47.00; H, 3.50; N, 10.20 %.
5.3.5 2-(4-(3-p-Tolylthioureido)benzamido)acetic acid (4e): as colorless crystals; yield 91%;
1
mp 205-207 °C; IR: ν/cm-1 = 3492, 3225, 3191 (NH, OH), 1703, 1645 (C=O); HNMR:
δ/ppm: 2.24 (s, 3H, CH3), 3.87 (d, 2H, J = 6.45 Hz, CH2), 7.10 (d, 2H, J = 8.40 Hz, Ar-H),
7.30 (d, 2H, J = 6.20 Hz, Ar-H), 7.58 (d, 2H, J = 8.40 Hz, Ar-H), 7.79 (d, 2H, J = 8.40 Hz,
Ar-H), 8.73 (t, 1H, J = 6.45 Hz, CONH), 9.85 (s, 1H, NH), 9.90 (s, 1H, NH), 12.32 (br, 1H,
OH); Anal. calcd for C17H17N3O3S (343.10): C, 59.46; H, 4.99; N, 12.24. Found: C, 59.30; H,
4.10; N, 12.20 %.
5.3.6 2-(4-(3-(3-Fluorophenyl)thioureido)benzamido)acetic acid (4f): as colorless crystals;
yield 87%; mp > 300 °C; IR: ν/cm-1 = 3440, 3212, 3173 (NH, OH), 1721, 1645 (C=O);
1HNMR: δ/ppm: 3.90 (d, 2H, J = 6.40 Hz, CH2), 6.95-7.91 (m, 8H, Ar-H), 8.87 (t, 1H, J =
6.40 Hz, CONH), 10.95 (s, 1H, NH), 11.18 (s, 1H, NH), 12.32 (br, 1H, OH); 13C NMR: 41.14
(NHCH2), 115.1, 116.3, 121.0, 122.4 (2C), 127.3, 128.5 (2C), 129.9, 137.3, 143.4, 160.7 (C-
F), 166.5 (CONH), 171.9 (COOH) 180.3(C=S); Anal. calcd for C16H14FN3O3S (347.07): C,
55.32; H, 4.06; N, 12.10. Found: C, 55.30; H, 4.10; N, 12.00 %.
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5.3.7 2-(4-(3-(4-Fluorophenyl)thioureido)benzamido)acetic acid (4g): as colorless crystals;
1
yield 80%; mp > 300 °C; IR: ν/cm-1 = 3343 - 3198 (NH, OH), 1724, 1640 (C=O); HNMR:
δ/ppm: 3.90 (d, 2H, J = 6.45 Hz, CH2), 6.91-7.89 (m, 8H, Ar-H), 8.85 (t, 1H, J = 6.45 Hz
CONH), 10.92 (s, 1H, NH), 11.42 (s, 1H, NH), 12.32 (br, 1H, OH); 13C NMR: 41.23
(NHCH2), 115.5, 115.7, 122.8 (2C), 126.8 (2C), 128.2 (2C), 129.7, 136.0, 142.8, 160.7 (C-
F), 166.5 (CONH), 171.9 (COOH) 180.3 (C=S); Anal. calcd for C16H14FN3O3S (347.07): C,
55.32; H, 4.06; N, 12.10. Found: C, 55.40; H, 4.00; N, 12.00 %.
5.4 Synthesis of disubstitued thiourea derivatives 5 and 6a,b
A solution of isothiocyanate 3 (0.01 mol) in 30 ml dioxane, 9-aminoacridine, 2-amino-N-(3-
trifluoromethylphenyl)-benzamide or 2-amino-N-(4-ethoxyphenyl)-benzamide (0.01 mol)
was added. The reaction mixture was heated under reflux for 3 hours. The solution was
concentrated under vacuum, left to cool. The solid product so formed was filtered off, washed
with ethanol, dried well, and recrystallized from ethanol/dioxane.
5.4.1 Synthesis of 2-(4-(3-acridin-9-ylthioureido)benzamido)acetic acid (5): as yellow
crystals, yield 85%; mp > 300 °C; IR: ν/cm-1 = 3415, 3218, 3187 (NH, OH), 1731, 1645
1
(C=O); HNMR: δ/ppm: 4.04 (d, 2H, J = 5.74 Hz, CH2), 6.98 - 8.94 (m, 13H, 12Ar-H +
CONH), 11.02 (s, 1H, NH), 11.51 (s, 1H, NH), 12.12 (br, 1H, OH); Anal. calcd for
C23H18N4O3S (430.11): C, 64.17; H, 4.21; N, 13.01. Found: C, 64.30; H, 4.50; N, 13.20 %.
5.4.2 2-(4-(3-(2-(3-(Trifluoromethyl)phenylcarbamoyl)phenyl)thioureido)benzamido)acetic
acid (6a): as colorless crystals; yield 87%; mp 265-270 °C; IR: ν/cm-1 = 3412- 3187 (NH,
OH), 1722, 1645 (C=O); 1H NMR: δ/ppm: 3.87 (d, 2H, J = 5.80 Hz, CH2), 6.40 - 8.88 (m,
13H, 12Ar-H + CONH), 9.92 (s, 1H, NH), 9.95 (s, 1H, NH), 10.62 (br, 1H, NH), 12.41 (br,
1H, OH); 13C NMR: 41.3 (NHCH2), 116.3 (2C),, 116.7, 124.9 (2C), 127.9, 128.5 (4C), 129.7
(4C), 134.2, 136.2 (2C), 140.1, 142.4, 160.2 (CONH), 166.6 (CONH), 171.9 (COOH), 176.2
(C=S); MS, m/z (%): 516 (M+, 84), 490 (89.0), 463 (94.0), 289 (88.1), 219 (99), 202 (99), 71
(51); Anal. calcd for C24H19F3N4O4S (516.11): C, 55.81; H, 3.71; N, 10.85. Found: C, 55.70;
H, 3.60; N, 10.60 %.
5.4.3 2-(4-(3-(2-(4-Ethoxyphenylcarbamoyl)phenyl)thioureido)benzamido)acetic acid (6b):
as colorless crystals; yield 86%; mp >300 °C; IR: ν/cm-1 = 3418, 3300, 3150 (NH,OH), 1715,
1
1642 (2C=O); H NMR: δ/ppm: 1.25 (t, 3H, J = 6.85 Hz, CH2CH3), 3.80 - 4.04 (m, 4H,
2CH2), 6.40 - 8.82 (m, 13H, 12Ar-H + CONH), 9.88 (s, 1H, NH), 9.98 (s, 1H, NH), 10.76
(br, 1H, NH), 12.52 (br, 1H, OH); Anal. calcd for C25H24N4O5S (492.15): C, 60.96; H, 4.91;
N, 11.37. Found: C, 61.10; H, 4.70; N, 11.60 %.
5.5 Synthesis of sulfonamido thiourea derivatives 7a-f
A mixture of 3 (0.01 mol), the appropriate sulfa drugs (namely sulfanilamide, sulfacetanilide,
sulfaguanidine, sulfathiazole, sulfapyridine and sulfadiazine) in dioxane (30 mL) was heated
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under reflux for 3 hours. The solution was concentrated under vacuum, left to cool. The
resulting solid was filtered off and recrystallized from diaxone.
5.5.1 2-(4-(3-(4-Sulfamoylphenyl)thioureido)benzamido)acetic (7a): as beige crystals; yield
79%; mp 205-207 °C; IR: ν/cm-1 = 3450, 3300, 3200 (NH, NH2, OH), 1710, 1642 (C=O);
1HNMR: δ/ppm: 3.95 (d, 2H, J = 5.94 Hz, CH2), 6.94 - 8.04 (m, 10H, 8Ar-H + SO2NH2),
8.82 (t, 1H, J = 6.11 Hz, CONH), 10.88 (s, 1H, NH), 11.32 (s, 1H, NH), 12.22 (br, 1H, OH);
13C NMR: 41.3 (NHCH2), 122.9 (2C), 123.3 (2C), 126.7 (2C), 128.2 (2C), 130.0, 139.8,
142.6, 143.0, 166.6 (CONH), 171.9 (COOH) 180.0(C=S); MS, m/z (%): 408 (M+, 7.0), 401
(11.0), 268 (12.1), 214 (34.7), 88 (100); Anal. calcd for C16H16N4O5S2 (408.06): C, 47.05; H,
3.95; N, 13.72. Found: C, 47.10; H, 4.10; N, 13.60 %.
5.5.2 2-(4-(3-(4-(N-Acetylsulfamoyl)phenyl)thioureido)benzamido)acetic acid (7b): as beige
crystals; yield 67%; mp >300 °C; IR: ν/cm-1 = 3423-3110 (NH, OH), 1714, 1641 (C=O);
1HNMR: δ/ppm: 1.87 (s, 3H, COCH3), 4.03 (d, 2H, J = 6.24 Hz, CH2), 7.50 - 8.74 (m, 9H,
8Ar-H + NH), 9.08, 10.32, 11.73, 12.43 (br, 4H, 3NH, OH); MS, m/z (%): 450 (M+, 62.0),
419 (94.0), 325 (92.1), 275 (100), 248 (95), 188 (94), 55 (98); Anal. calcd for C18H18N4O6S2
(450.07): C, 47.99; H, 4.03; N, 12.44. Found: C, 47.80; H, 4.10; N, 12.60 %.
5.5.3 2-(4-(3-(4-(N-Carbamimidoylsulfamoyl)phenyl)thioureido)benzamido)acetic acid (7c):
as beige crystals; yield 66%; mp >300 °C; IR: ν/cm-1 = 3400 - 3160 (NH, NH2, OH), 1763,
1645 (C=O); 1HNMR: δ/ppm: 3.87 (s, 1H, NH), 4.03 (m, 2H, CH2), 6.66 (br, 2H, NH2), 7.53
- 8.74 (m, 9H, 8Ar-H + CONH), 9.17 (s, 1H, NH), 10.33 (m, 2H, 2NH), 12.43 (br, 1H, OH);
MS, m/z (%): 450 (M+, 57.34), 419 (66.4), 399 (60.8), 306 (60.1), 261 (64.4), 194 (65.7), 80
(93.6), 64 (100); Anal. calcd for C17H18N6O5S2 (450.08): C, 45.32; H, 4.03; N, 18.66. Found:
C, 45.50; H, 4.20; N, 18.60 %.
5.5.4 2-(4-(3-(4-(N-Thiazol-2-ylsulfamoyl)phenyl)thioureido)benzamido)acetic acid (7d): as
beige crystals; yield 70%; mp > 300 °C; IR: ν/cm-1 = 3450, 3254, 3150 (NH, OH), 1733,
1645 (C=O); 1H NMR: δ/ppm: 3.88 (d, 2H, J = 5.89 Hz, CH2), 6.98 - 8.79 (m, 12H, 10Ar-H
+ 2NH), 10.79 (s, 1H, NH), 11.14 (s, 1H, NH), 12.08 (br, 1H, OH); Anal. calcd for
C19H17N5O5S3 (491.04): C, 46.42; H, 3.49; N, 14.25. Found: C, 46.60; H, 3.60; N, 14.50 %.
5.5.5 2-(4-(3-(4-(N-Pyridin-2-ylsulfamoyl)phenyl)thioureido)benzamido)acetic acid (7e): as
beige crystals; yield 74%; mp > 300 °C; IR: ν/cm-1 = 3386, 3250 (NH, OH), 1721, 1645
1
(C=O); H NMR: δ/ppm: 3.88 (d, 2H, J = 6.10 Hz, CH2), 7.03 - 8.86 (m, 14H, 12Ar-H +
2NH), 10.79 (s, 1H, NH), 11.14 (s, 1H, NH), 12.08 (br, 1H, OH); Anal. calcd for
C21H19N5O5S2 (485.08): C, 51.95; H, 3.94; N, 14.42. Found: C, 52.10; H, 3.70; N, 14.50 %.
5.5.6 2-(4-(3-(4-(N-Pyrimidin-2-ylsulfamoyl)phenyl)thioureido)benzamido)acetic acid (7f):
as beige crystals; yield 76%; mp 255-258 °C; IR: ν/cm-1 = 3325, 3200, 3160, (NH, OH), 1705
(C=O), 1691, 1645 (C=O); 1H NMR: δ/ppm: 3.88 (d, 2H, J = 5.79 Hz, CH2), 6.73 - 8.86 (m,
13H, 12Ar-H + 2NH), 11.64 (s, 2H, 2NH), 12.08 (br, 1H, OH); Anal. calcd for C20H18N6O5S2
(486.52): C, 49.37; H, 3.73; N, 17.27. Found: C, 49.50; H, 3.60; N, 17.10.
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5.6 Synthesis of bis-hippuric acid 8a,b
A mixture of isothiocyanate 3 (0.02 mol) and 4-aminohippuric acid 1 (0.02 mol) or 1,4-
phenylenediamine (0.01 mol) in dioxane (30 ml) was heated under reflux for 3 hours, then
allowed to cool. The solid product so formed was filtered off, washed with ethanol, dried
well, and recrystallized from dioxane to give the bis-compound 8a,b, respectively.
5.6.1 Synthesis of N,N’-bis(4-hippuric acid)thiourea (8a): colorless crystals; yield 75%; mp
260-261 °C; IR: ν/cm-1= 3243, 3198, 3161 (NH, OH), 1694, 1638 (C=O); 1HNMR: δ/ppm:
3.53 (m, 4H, 2CH2), 6.68 - 8.58 (m, 10H, 8Ar-H + 2NH), 11.27 (br, 2H, 2NH), 12.32 (br, 2H,
2OH); Anal. Calcd for C19H18N4O6S (430.09): C, 53.02; H, 4.22; N, 13.02. Found: C, 53.00;
H, 4.00; N, 13.00%.
5.6.2 Synthesis of 3,3'-bis(4-hippuric acid) 1,1'-(1,4-phenylene)dithiourea (8b): pale brown
crystals; yield 71%; mp 270-272 °C; IR: ν/cm-1 = 3450-3173 (NH, OH), 1712, 1641 (C=O);
1HNMR: δ/ppm: 3.53 (m, 4H, 2CH2), 6.68 - 8.58 (m, 14H, 12Ar-H + 2CONH), 11.27 (m,
4H, 4NH), 12.32 (br, 2H, 2OH); MS, m/z (%): 580 (M+, 50.8), 575 (100), 501 (87.7), 301
(85.1), 102 (99.1), 61 (95.1); Anal. Calcd for C26H24N6O6S2 (580.12): C, 53.78; H, 4.17; N,
14.47. Found: C, 53.70; H, 4.00; N, 14.40%.
5.7 Synthesis of pyrazole derivatives 9, 10a,b
A mixture of isothiocyanate 3 (0.01 mol) and aminopyrazole derivatives (namely 4-
aminoantipyrene, 5-amino-3-phenylamino-1H-pyrazole-4-carboxylic acid amide and 4-(4-
chloro-phenylazo)-4H-pyrazole-3,5-diamine ) (0.01mol) in dioxane (30 mL) was heated
under reflux for 3h. The reaction mixture was concentrated under vacuum, left to cool and
then cold ethanol was added. The solid product obtained was filtered off and crystallized
from ethanol.
5.7.1
2-(4-(3-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)thioureido)benzamido)acetic acid (9): as colorless crystals; yield 73%; mp 240-241°C;
IR: ν/cm-1: 3360, 3265, 3146 (NH, OH), 1697, 1665 (C=O); 1H NMR: δ/ppm: 2.05 (s, 3H,
CH3), 3.08 (s, 3H, CH3), 3.87 (d, 2H, J = 5.35 Hz, CH2), 7.29 - 7.84 (m, 9H, Ar-H), 8.74 (t,
1H, J = 5.40 Hz, CONH), 9.25 (br, 1H, NH), 9.85 (br, 1H, NH), 12.38 (br, 1H, OH); MS,
m/z (%): 439 (M+, 9.4), 162 (54.7), 120 (C6H4CONH2; 100), 56 (67.8); Anal. Calcd for
C21H21N5O4S (439.13): C, 57.39; H, 4.82; N, 15.94. Found: C, 57.40; H, 4.90; N, 16.00%.
5.7.2 2-(4-(3-(4-carbamoyl-3-(phenylamino)-1H-pyrazol-5-yl)thioureido)benzamido)acetic
acid(10a): as yellow crystals; yield 71%; mp 245-247 °C; IR: ν/cm-1: 3432 - 3110 (NH, NH2,
OH), 1709, 1640 (C=O); 1HNMR: δ/ppm: 3.88 (d, 2H, J = 6.10 Hz, CH2), 5.81 (br, 2H,
NH2), 6.69 - 7.45 (m, 9H, 7Ar-H + 2NH), 7.81 (d, 2H, J = 7.65 Hz, Ar-H), 8.77 (t, 1H, J =
6.10 Hz, CONH), 8.98 (s, 1H, NH), 11.27 (s, 1H, NH), 11.82 (br, 1H, OH); Anal. Calcd for
C20H19N7O4S (453.12): C, 52.97; H, 4.22; N, 21.62. Found: C, 52.90; H, 4.20; N, 21.50%.
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5.7.3
2-(4-(3-(3-amino-4-((4-chlorophenyl)diazenyl)-1H-pyrazol-5-
yl)thioureido)benzamido)acetic acid (10b): as yellow crystals; yield 76%; mp 201-203 °C;
IR: ν/cm-1: 3400-3160 (NH, NH2, OH), 1711, 1639 (C=O); 1H NMR: δ/ppm: 3.88 (d, 2H, J
= 6.10 Hz, CH2), 6.32 (br, 2H, NH2), 7.36 - 7.77 (m, 8H, Ar-H), 8.76 (t, 1H, J = 6.15 Hz,
CONH), 10.67 (br, 1H, NH), 11.26 (br, 1H, NH), 11.87 (br, 1H, NH), 11.92 (br, 1H, OH);
MS, m/z (%): 472 (M+, 5.7), 301 (5.8), 297 (4.1), 236 (6.6), 205 (6.7), 162 (18.1), 95 (23.1),
69 (100); Anal. calcd for C19H17ClN8O3S (472.08): C, 48.26; H, 3.62; N, 23.69. Found: C,
48.20; H, 3.60; N, 23.50%.
5.8 Synthesis of disubstituted thiosemicarbazide derivatives 11a-d
A mixture of isothiocyanate 3 (0.01 mol) and hydrazine derivatives (0.01 mol) (namely
phenylhydrazine, phenylhydrazide, isonicotinohydrazide and phenylsulfonylhydrazine) (0.01
mol) in ethanol (30 mL) was heated under reflux for one hour. The solid product was filtered
of, washed with ethanol, dried well, and recrystallized from ethanol/dioxane
5.8.1 2-(4-(2-Phenylhydrazinecarbothioamido)benzamido)acetic acid (11a): as colorless
o
crystals, yield (88%); mp 165-166 C; IR: ν/cm-1 = 3441 - 3161 (NH, OH), 1700, 1641
1
(C=O); ); HNMR: δ/ppm: 3.93 (d, 2H, J = 5.88 Hz, CH2), 6.65 - 8.75 (m, 11H, 9Ar-H +
2NH), 9.87 (br, 1H, NH), 10.72 (br, 1H, NH), 12.04 (br, 1H, OH); MS, m/z (%): 344 (M+,
14.7), 236 (11.4), 192 (27.4), 162 (100), 108 (92.5), 65 (85.6); Anal. Calcd for C16H16N4O3S
(344.09): C, 55.80; H, 4.68; N, 16.27. Found: C, 55.90; H, 4.50; N, 16.20%.
5.8.2 2-(4-(2-Benzoylhydrazinecarbothioamido)benzamido)acetic acid (11b): yellow crystals;
o
yield (78%); mp 216-218 C; IR: ν/cm-1 = 3440 - 3173 (NH, OH), 1698, 1638 (C=O);
1HNMR: δ/ppm: 3.87 (d, 2H, J = 4.95 Hz, CH2), 7.55 (d, 2H, J = 8.45 Hz, Ar-H), 7.80 (m,
5H, Ar-H), 8.75 (m, 3H, 2Ar-H and NH), 9.95 (br, 2H, NH), 10.88 (br, 1H, NH), 12.59 (br,
1H, OH); Anal. Calcd for C17H16N4O4S (372.09): C, 54.83; H, 4.33; N, 15.04. Found: C,
54.90; H, 4.50; N, 15.20%.
5.8.3 2-(4-(2-Isonicotinylhydrazinecarbothioamido)benzamido)acetic acid (11c): colorless
o
crystals, yield (73%); m.p. 233-235 C. IR: ν/cm-1 = 3462 - 3191 (NH, OH), 1703, 1664
1
(C=O); HNMR: δ/ppm: 3.87 (d, 2H, J = 4.95 Hz, CH2), 7.55- 8.75 (m, 10H, 8Ar-H and
2NH), 9.95 (s, H, NH), 10.88 (s, 1H, NH), 12.59 (br, 1H, OH); Anal. calcd for C16H15N5O4S
(373.08): C, 51.47; H, 4.05; N, 18.76. Found: C, 51.60; H, 4.20; N, 18.90%.
5.8.4 2-(4-(2-(Phenylsulfonyl)hydrazinecarbothioamido)benzamido)acetic acid (11d): as
colorless crystals; yield 71%; mp 161-163 °C; IR: ν/cm-1 = 3386-3150 (NH, OH), 1701, 1641
1
(C=O); ); HNMR: δ/ppm: 3.87 (d, 2H, J = 5.95 Hz, CH2), 6.94 - 8.89 (m, 11H, 9Ar-H and
2NH), 10.24 (s, 1H, 1NH), 11.34 (s, 1H, NH), 12.00 (br, 1H, OH); 13C NMR: 40.1 (NHCH2),
124.9 (2C), 127.7 (2C), 128.5 (2C), 129.7 (2C), 130.6, 134.0, 138.2, 142.2, 166.6, 171.9,
181.3; MS, m/z (%): 408 (M+, 23.8), 221 (40.4), 203 (39.3), 108 (100), 75 (37.1); Anal.
- 15 -

ACCEPTED MANUSCRIPT
Calcd for C16H16N4O5S2 (408.06): C, 47.05; H, 3.95; N, 13.72. Found: C, 47.20; H, 3.80; N,
13.90%.
5.9. Antimicrobial activity
Chemical compounds were individually tested against a panel of gram positive and gram
negative bacterial pathogens and fungi. Antimicrobial tests were carried out by the agar well
diffusion method using 100 ꢀL of suspension containing 1 x108 CFU/mL of pathological
tested bacteria and 1 x106 CFU/ml of yeast spread on nutrient agar (NA) and Sabouraud
dextrose agar (SDA) media, respectively. After the media had cooled and solidified, wells (10
mm in diameter) were made in the solidified agar and loaded with 100 ꢀL of tested
compound solution prepared by dissolving 1 mg of the chemical compound in one ml of
dimethyl sulfoxide (DMSO). The inoculated plates were then incubated for 24 h at 37 °C.
Negative controls were prepared using DMSO employed for dissolving the tested compound.
Ciprofloxacin (0.5 mg/ml) and Ketoconazole (0.5 mg/ml) were used as standard for
antibacterial and antifungal activity, respectively. After incubation time, antimicrobial
activity was evaluated by measuring the zone of inhibition against the test organisms and
compared with that of the standard. The observed zone of inhibition is presented in Table 1.
Antimicrobial activities were expressed as inhibition diameter zones in millimeters (mm).
The experiment was carried out in triplicate and the average zone of inhibition was
calculated.
5.10. Minimal inhibitory concentration (MIC) measurement
Screening was performed following the procedure outlined in the Manual of Clinical
Microbiology [15]. All the bacteria were incubated and activated at 30 °C for 24 h
inoculation into nutrient broth and the fungi were incubated in malt extract broth for 48 h.
The compounds were dissolved in DMSO and then diluted using cautiously adjusted Mueller-
Hinton broth. Two-fold serial concentrations of the compounds were employed to determine
the (MIC). The final concentrations of the solutions were 100, 50, 25, 12.5, 6.25, 3.12 and
1.56 ꢀg/ml. In each case triplicate tests were performed and the average was taken as the final
reading. The tubes were then inoculated with the test organisms, grown in their suitable broth
at 37 °C for 24 hours for tested microorganisms (1×108 CFU/ml for bacteria and 1 x106
CFU/ml of fungi), each 5 ml received 0.1 ml of the above inoculum and incubated at 37 °C
for 24 hours. The lowest concentration showing no growth was taken as the minimum
inhibitory concentration (MIC).
References
[1] D.K. Aneja1, P. Lohan, S. Arora, Ch. Sharma, K.R. Aneja, Om Prakash, Bioorg.
Med. Chem. Lett., (2011) 1-15
[2] I. Berber, C. Cokmus, E. Atalan, Microbiology 72 (2003) 42-47
[3] I. Bildirici, A. Sener and I. Tozlu, Med. Chem. Rev. 16 (2007) 418-426
[4] P.C. Appelbaum, Clin. Microbiol. Infect., 12 (2006) 16
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ACCEPTED MANUSCRIPT
[5] S.N. Shelke, G.R. Mhaske, V.D.B. Bonifácio, M.B. Gawande, Bioorg. & Med.
Chem. Letters, 22 (2012) 5727-5730
[6] V.J. Sattigeri, A. Soni, S. Singhal, S. Khan, M. Pandya, P. Bhateja, T. Mathur, A.
Rattan, J.M. Khanna, A. Mehta, ARKIVOC, ii (2005) 46-59
[7] M.S.A. El-Gaby, J.A. Micky, N.M. Taha, M.A.M.Sh. El-Sharief, J. Chin. Chem.
Soc., 49 (2002) 407-414
[8] K. Ramadas, G. Suresh, N. Janarthanan, S. Masilamani, Pestic Sci, 52 (1998) 145-
151
[9] Z. Moussa, M. A. M. Sh. El-Sharief, A.M.Sh. El-Sharief, Eur. J. Med. Chem. 46
(2011) 2280-2289.
[10] A.A. Farag, S.N. Abd-Alrahman, G.F. Ahmed, R.M. Ammar, Y.A. Ammar, S.Y.
Abbas, Arch. Pharm. Life Sci. 345 (2012) 703-712
[11] M.A.M.Sh. El-Sharief, Z. Moussa, A.M.Sh. El-Sharief, J. Fluorine Chem. 132
(2011) 596-611
[12] M.M. Aly, Y.A. Mohamed, Kh.A.M. El-Bayouki, W.M. Basyouni, S.Y. Abbas, Eur.
J. Med. Chem., 45 (2010) 3365-3373
[13] A.A. Farag, E.M. Khalifa, N.A. Sadik, S.Y. Abbas, A.G. Al-Sehemi, Y.A. Ammar
Med. Chem. Res. 22, (2013) 440-452
[14] A.C. Scott, Laboratory Control of Antimicrobial Therapy, in: , thirteenth ed.., in: J.G.
Collee, J.P. Duguid, A.G. Fraser, B.P. Marmion (Eds.), Mackie and Mac-Cartney
Practical Medical Microbiology, 2 Churchill, Livingstone, 1989, pp. 161-181.
[15] R.N. Jones, A.L. Barry, T.L. Gaven, J.A. Washington, in: E.H. Lennette, A. Balows,
W.J. Shadomy (Eds.), Manual of Clinical Microbiology, fourth ed. American Society
for Microbiology, Washington DC, 1984, pp. 972-977.
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•
•
•
Synthesis of N-(4-hippuric acid) isothiocyanate
Using the isothiocyanate for synthesizing variously substituted thioureas.
The antimicrobial activity assay was determined.

ACCEPTED MANUSCRIPT
Table 1: Antimicrobial activity of the synthesized compounds against the pathological organisms expressed as inhibition diameter zones in
millimeters (mm) based on well diffusion assay
Compd.
No.
4a
Gram +ve
S. aureus B. subtilis B. megaterium
Gram -ve
Fungi
S. cervesia C. albicans
K. peneumoniae P. aeroginosa E. coli
S. lutea P. vulgaris
22
0
0
31
0
20
20
20
0
20
14
15
0
13
9
17
30
0
18
0
12
16
0
18
18
16
15
12
26
18
31
15
32
12
0
16
23
13
32
19
27
15
18
14
0
20
20
20
14
0
27
17
24
18
27
10
23
31
24
-
20
12
0
30
0
23
20
23
0
20
22
0
22
0
12
22
0
21
22
15
0
22
11
19
27
9
20
22
15
0
0
18
0
0
0
21
32
22
24
14
28
15
0
9
24
0
21
0
0
18
9
0
28
0
17
13
0
15
18
17
0
15
0
20
30
18
26
17
25
13
0
10
0
0
30
18
22
9
11
0
9
0
0
0
4b
4c
4d
4e
4f
4g
5
6a
6b
7a
7b
7c
7d
7e
7f
8a
20
25
20
20
0
0
0
20
0
24
20
25
20
20
10
15
20
0
0
19
12
0
0
0
0
0
0
30
21
34
22
22
22
28
15
12
23
24
-
0
0
0
23
32
22
20
18
28
22
0
20
17
23
16
19
22
20
0
0
0
11
25
19
26
0
30
9
12
32
-
25
15
10
24
30
20
24
20
20
-
21
21
18
19
28
15
0
20
23
-
8b
9
10b
11b
11c
11d
CFL
KET
32
20
-
30
22
-
25
25
-
29
23
-
24
-
23
22

ACCEPTED MANUSCRIPT
Table 2: Minimum inhibitory concentration (µg/ml) of the more potent synthesized compounds against the pathological organisms
Gram +ve
S. aureus B. subtilis B. megaterium
Gram -ve
K. peneumoniae P. aeroginosa E. coli
Fungi
C. albicans
Compd.
No.
S. lutea
P. vulgaris
S. cervesia
6.25
50
3.12
12.5
12.5
50
3.12
12.5
12.5
12.5
12.5
6.25
3.12
6.25
---
6.25
12.5
12.5
12.5
12.5
12.5
12.5
12.5
---
12.5
12.5
3.12
12.5
12.5
12.5
12.5
12.5
---
6.25
12.5
6.25
12.5
25
12.5
12.5
12.5
25
6.25
12.5
12.5
25
6.25
25
3.12
12.5
12.5
12.5
12.5
3.12
3.12
---
4d
4f
3.12
50
3.12
50
7f
8a
50
12.5
3.12
3.12
6.25
---
50
25
25
8b
6.25
6.25
6.25
---
6.25
6.25
12.5
---
12.5
12.5
12.5
---
6.25
12.5
12.5
---
12.5
12.5
---
10b
11d
CFL
KET
3.12
6.25

ACCEPTED MANUSCRIPT
Scheme 1: Synthesis of mono- and disubstituted thiourea derivatives
Scheme 2: Synthesis of disubstitued thiourea and bisthiourea derivatives
Scheme 3: Synthesis of pyrazolyl thiourea and disubstituted thiosemicarbazide derivatives
Figure 1: General formula of the synthesized compounds
Figure 2: The comparison between the antibacterial activities of our potent synthesized
compounds and standard drug against the used Gram positive and Gram negative bacteria.
Figure 3: The comparison between the antifungal activities of our potent synthesized
compounds and standard drug.
Table 1: Antimicrobial activity of the synthesized compounds against the pathological
organisms expressed as inhibition diameter zones in millimeters (mm) based on well
diffusion assay
Table 2: Minimum inhibitory concentration (µg/ml) of the more potent synthesized
compounds against the pathological organisms
- 1 -

ACCEPTED MANUSCRIPT
Scheme 1: Synthesis of mono- and disubstituted thiourea derivatives
S
O
NH4SCN
N
H
NH2
HCl, 80 °C, 12 h
HN
O
COOH
NH2
2
HN
COOH
1
CSCl2
dil. HCl, r.t., 1h
NCS
S
XNCS, dioxane, reflux
XNH2, dioxane, reflux
O
X
N
N
H
O
H
HN
COOH
HN
4a X = C4H9
4b X = C6H11
4e X = 4-CH3-C6H4
4f X = 3-F-C6H4
4g
X = 4-F-C6H4
COOH
3
4c
X = C6H5
4d X= 3-Br-C6H4
Scheme 2: Synthesis of disubstitued thiourea and bisthiourea derivatives
S
O
N
N
H
N
H
HN
5
COOH
NH2
S
O
9-aminoacridine,
dioxane, reflux
N
N
CONHAr
H
H
HN
CONHAr
dioxane, reflux
COOH
O
6a Ar = 3-CF3-C6H4
6b Ar = 4-OC2H5-C6H4
NCS
HN
COOH
3
S
O
O
8a 1
Sulfa drugs
;
N
N
S NHX
O
8b
H
H
; 1,4-(NH2)2-C6H4
HN
dioxane, reflux
dioxane, reflux
COOH
7a X= H
7d X = 2-Thaizolyl
S
O
O
7b
7c
7e
7f
X= COCH3
X = C(=NH)NH2
X = 2-Pyridinyl
X = 2-Pyrimidinyl
X
N
N
H
H
HN
NH
COOH
8a X= 0
8b X= C6H4NHCSNH
HOOC
Scheme 3: Synthesis of pyrazolyl thiourea and disubstituted thiosemicarbazide derivatives
- 1 -

ACCEPTED MANUSCRIPT
S
O
CH3
CH3
4-aminoantipyrine
N
N
H
H
HN
N
dioxane, reflux
O
N
COOH
O
9
Ph
NCS
HN
H2N
HN
X
COOH
3
S
O
X
N
N
H
N
NHY
XNHNH2
dioxane, reflux
S
H
HN
HN
dioxane, reflux
N
NHY
COOH
10a X = CONH2
O
NHX
Y = Ph
10b X = N=NC6H4Cl-4 Y = H
N
N
H
H
HN
COOH
11a X= C6H5
11c X = isonicotinoyl
11b X = COC6H5 11d X = SO2C6H5
Figure 1
S
O
HN
X
N
N
H
H
COOH
- 2 -

ACCEPTED MANUSCRIPT
Figure 2: The comparison between the antibacterial activities of our potent synthesized
compounds and standard drug against the used Gram positive and Gram negative bacteria.
Figure 3: The comparison between the antifungal activities of our potent synthesized
compounds and standard drug.
- 3 -