Nonclassical antifolates, part 4. 5-(2-Aminothiazol-4-yl)-4-phenyl-4H-1,2, 4-triazole-3-thiols as a new class of DHFR inhibitors: Synthesis, biological evaluation and molecular modeling study

Article abstract of DOI:10.1016/j.ejmech.2013.05.039

A new series of compounds possessing 5-(2-aminothiazol-4-yl)-4-phenyl-4H-1, 2,4-triazole-3-thiol skeleton was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, antitumor and schistosomicidal activities. Four active compounds were allocated, the antibacterial 22 (comparable to gentamicin and ciprofloxacin), the schistosomicidal 29 (comparable to praziquantel), the DHFR inhibitor 34 (IC₅₀ 0.03 μM, 2.7 fold more active than MTX), and the antitumor 36 (comparable to doxorubicin). Molecular modeling studies concluded that recognition with key amino acid Leu4 and Val1 is essential for DHFR binding. Flexible alignment and surface mapping revealed that the obtained model could be useful for the development of new class of DHFR inhibitors.

Full text of DOI:10.1016/j.ejmech.2013.05.039

Accepted Manuscript
Nonclassical antifolates, Part 4. 5-(2-Aminothiazol-4-yl)-4-phenyl-4H-1,2,4-triazole-3-
thiols as a new class of DHFR inhibitors: Synthesis, biological evaluation and
molecular modeling study
Ghada S. Hassan, Shahenda M. El-Messery, Fatmah A.M. Al-Omary, Sarah T. Al-
Rashood, Marwa I. Shabayek, Yasmin S. Abulfadl, El-Sayed E. Habib, Salwa M. El-
Hallouty, Walid Fayad, Khaled M. Mohamed, Bassem S. El-Menshawi, Hussein I. El-
Subbagh
PII:
S0223-5234(13)00357-7
10.1016/j.ejmech.2013.05.039
EJMECH 6217
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 February 2013
Revised Date: 19 May 2013
Accepted Date: 25 May 2013
Please cite this article as: G.S. Hassan, S.M. El-Messery, F.A.M. Al-Omary, S.T. Al-Rashood, M.I.
Shabayek, Y.S. Abulfadl, E.-S.E. Habib, S.M. El-Hallouty, W. Fayad, K.M. Mohamed, B.S. El-Menshawi,
H.I. El-Subbagh, Nonclassical antifolates, Part 4. 5-(2-Aminothiazol-4-yl)-4-phenyl-4H-1,2,4-triazole-3-
thiols as a new class of DHFR inhibitors: Synthesis, biological evaluation and molecular modeling study,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.039.
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1
Nonclassical antifolates, Part 4. 5-(2-Aminothiazol-
Leave this area blank for abstract info.
4-yl)-4-phenyl-4H-1,2,4-triazole-3-thiols as a new
class of DHFR inhibitors: Synthesis, biological
evaluation and molecular modeling study.
Ghada S. Hassan,1,2 Shahenda M. El-Messery,3 Fatmah A.M. Al-Omary,1 Sarah T. Al-Rashood,1 Marwa I.
Shabayek,4 Yasmin S. Abulfadl,4 El-Sayed E. Habib,5 Salwa M. El-Hallouty,6 Walid Fayad,6 Khaled M.
Mohamed,6 Bassem S. El-Menshawi,7 Hussein I. El-Subbagh,8*
N
N
HS
HS
N
N
N
N
O
N
N
CH3
CH3
NH2
N
S
S
O
O
O
29 (Schistosomicidal)
22 (Antifungal, antibacterial)
N
N
HS
CH3O
HS
N
OCH3
N
O
N
N
S
S
N
N
N
H
N
H
N
H
OCH3
N
H
S
S
CH3O
O
34 (DHFR inhibitor, IC50 0.03 µM)
36 (Antitumor)
Compounds 22, 29, 34 and 36 showed remarkable activities. Recognition with key amino acids Leu4
and Val1is essential for the new DHFR inhibitor 34 (IC50 0.03 µM)

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Nonclassical antifolates, Part 4. 5-(2-Aminothiazol-4-yl)-4-phenyl-4H-1,2,4-
triazole-3-thiols as a new class of DHFR inhibitors: Synthesis, biological
evaluation and molecular modeling study. #
Ghada S. Hassan,1,2 Shahenda M. El-Messery,3 Fatmah A.M. Al-Omary,1 Sarah T. Al-Rashood,1
Marwa I. Shabayek,4 Yasmin S. Abulfadl,4 El-Sayed E. Habib,5 Salwa M. El-Hallouty,6 Walid
Fayad,6 Khaled M. Mohamed,6 Bassem S. El-Menshawi,7 Hussein I. El-Subbagh,8*
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P.O.Box 2457, Riyadh 11451, Saudi Arabia.
2Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, P.O.Box
35516, Mansoura, Egypt.
3Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University,
P.O.Box 35516, Mansoura, Egypt.
4Department of Pharmacology (Biochemistry Section), Faculty of Pharmaceutical Sciences &
Pharmaceutical Industries, Future University, 12311 Cairo, Egypt.
5Department of Pharmaceutics and pharmaceutical technology (Microbiology), College of
Pharmacy, Taibah University, Almadinah Almunawwarah, 344, Saudi Arabia.
6Drug Bioassay-Cell Culture Laboratory, Department of Pharmacognosy, Pharmaceutical and
Drug Industries Division, National Research Center, Dokki, Giza, 12622, Egypt.
7Department of Pharmacognosy, Faculty of Pharmaceutical Sciences & Pharmaceutical
Industries, Future University, 12311 Cairo, Egypt.
8Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences &
Pharmaceutical Industries, Future University, 12311 Cairo, Egypt.
Correspondence: Hussein I. El-Subbagh, Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical
Sciences & Pharmaceutical Industries, Future University, 12311 Cairo, Egypt.
E-mail: subbagh@yahoo.com.
Tel &Fax: +20-2-26186111.
Abstract
A new series of compounds possessing 5-(2-aminothiazol-4-yl)-4-phenyl-4H-1,2,4-triazole-3-
thiol skeleton was designed, synthesized, and evaluated for their in vitro DHFR inhibition,
antimicrobial, antitumor and schistosomicidal activities. Four active compounds were allocated,
the antibacterial 22 (comparable to gentamicin and ciprofloxacin), the schistosomicidal 29
(comparable to praziquantel), the DHFR inhibitor 34 (IC50 0.03 µM, 2.7 fold more active than
MTX), and the antitumor 36 (comparable to doxorubicin). Molecular modeling studies
concluded that recognition with key amino acid Leu4 and Val1 is essential for DHFR binding.
Flexible alignment and surface mapping revealed that the obtained model could be useful for the
development of new class of DHFR inhibitors.
Key words: Synthesis, Substituted thiazoles, DHFR inhibitors, Molecular modeling study.
________________
#For parts 1-3 see references 24-26.

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1.
Introduction
Dihydrofolate reductase (DHFR) is one of the key enzymes in the process of DNA replication, it
catalyzes the transformation of 7,8-dihydrofolate into tetrahydrofolate [1]. The latter serves as a
necessary cofactor in the important one-carbon transfer reactions involved in the pyrimidine,
purine, and amino acid biosynthetic pathways. The lower levels of tetrahydrofolate result in
decreased conversion of glycine to serine, reduced methionine synthesis, and lower thymidylate
levels, with a subsequent arrest of DNA replication [2, 3]. DHFR has a proven record as a drug
target in the treatment of bacterial, parasitic infections and cancer chemotherapy [4, 5].
Numerous studies showed that the structure of the classical DHFR inhibitor methotrexate (MTX)
can be modified considerably while significant inhibitory potency still retained [6, 7]. Parasitic
diseases are one of the causes of mortality and morbidity worldwide. The WHO estimates that
one billion people are affected by parasitic infections, such as malaria, leishmaniasis,
trypanosomiasis and schistosomiasis. The annual mortality associated with parasitic infections, is
estimated 500,000 deaths [8]. The majority of the drugs used to treat these diseases is old and has
several limitations, including high cost, poor efficacy, and toxicity [9]. Moreover, the
development of drug resistance makes the search for new molecules able to act as selective and
effective antiparasitic chemotherapeutic agents a very important task for medicinal chemists.
Thiazole heterocycle represents an important synthon which is responsible for numerous
biological activities such as the antimicrobial agent Sulfatiazol [10], and the antineoplastic
agents Tiazofurin (A), Netropsin (B), Thia-netropsin (C), Chart 1 [11-15]. On the other hand,
1,2,4-Triazole represents an important pharmacophore that plays a vital role as medicinal active
agents exhibiting variety of biological potencies such as antimicrobial [16, 17], antifungal [18],
antitubercular [19] and anticancer agents [20, 21]. Recently, hybrids of 4-phenyl-thiazole-1,3,5-
triazines (D, Chart 1) are approved as effective parasitic and microbial dihydrofolate reductase
(DHFR) inhibitors that selectively inhibit biochemical processes which are vital for parasite
growth [22, 23].
In view of these facts, and in continuation to our previous efforts [24-33], the present study
reports an efficient and reproducible synthesis of a new series of 2,4-substituted-thiazoles
bearing 1,2,4-triazole-3-thione, acetylamino, and thioureido moieties known to contribute to
biological potency [27-29], with anticipated DHFR inhibitory activity. The aim of this study is to

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identify novel synthetic lead compound(s) tackling the folate pathway. Structure modification of
MTX has been performed in this study by designing new compounds of different structure class
and reduced molecular weight to define the structure requirements and features that enhance
selectivity and specificity for the tight binding to DHFR. The synthesized derivatives were tested
for their in vitro DHFR inhibition, and in vitro antimicrobial activity against a panel of standard
strains of Gram-positive, Gram-negative bacteria and pathogenic fungi, in addition to the in-vitro
antitumor activity using cell-based disease oriented approach [34-37] against Human
hepatocellular carcinoma HepG2, caucasian breast adenocarcinoma MCF7, colon carcinoma
HCT116, and lung carcinoma A549 cell lines. Furthermore, the synthesized compounds were
screened for their antiparasitic activity using schistosoma mansoni as a model.
2.
Results and Discussion
2.1
Chemistry
The synthesis of the target compounds is depicted in scheme 1. 2-Acetamido-thiazole-4-
carboxyhydrazide (1) was prepared adopting reported procedures [38-41] and treated with a
variety of phenylisothiocyanate derivatives (2-8) to give the N-{4-[2-(4-substituted-phenyl-
carbamothioyl)hydrazinecarbonyl]thiazol-2-yl}acetamide derivatives (9-15) which were then
cyclized and deacetylated using NaOH solution to produce the 5-(2-aminothiazol-4-yl)-4-
substituted-phenyl-4H-1,2,4-triazole-3-thiols (16-22). The latter compounds were either
acetylated by the aid of acetic anhydride to give compounds (23-29), or treated with the
phenylisothiocyante derivatives (2-8) to produce the 1-[4-(5-mercapto-4-substituted-phenyl-4H-
1,2,4-triazol-3-yl)thiazol-2-yl]-3-(4-substituted-phenyl)thiourea derivatives (30-36); (Scheme 1,
1
Table 1). The H NMR spectra of the synthesized intermediates 9-15 proved the presence of the
phenylcarbamothioyl-hydrazinecarbonyl moiety in their structures by the appearance of aromatic
protons signals appeared in the region 7.21–7.93 ppm which confirmed the structure of the
molecule, while the signal for NH groups appeared as set of singlet at down field which could be
1
attributed to the intra-molecular hydrogen-bonding. H NMR spectra for compounds 23-29
proved the inclusion of diacetyl group into their structures by the appearance of two singlet peaks
integrated for a couple of acetyl functions along with the absence of singlet peak of NH2. The
structures of compounds 30-36 were inferred by the presence of singlet peaks for two NH groups

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at the expected range. The structures of the new compounds were verified by the aid of elemental
analyses, mass spectrometry, 1H, and 13C-NMR spectra.
2.2
Dihydrofolate Reductase Inhibition
The synthesized compounds 16-19, 21-27, 29, 31-36 were evaluated as inhibitors of bovine
liver DHFR using reported procedure [42-44]. Results were reported as IC50 values (Table 2).
All compounds showed weak to moderate DHFR inhibition with IC50 values range of 5.0-40.0
µM, except compound 34 (IC50, 0.03 µM) which proved to be 2.7 fold more active than the
positive control MTX (IC50, 0.08 µM).
2.3
Antimicrobial Screening
The synthesized compounds 16-36 were tested for their in vitro antimicrobial activity against a
panel of standard strains of the Gram-positive bacteria (Staphylococcus aureus and Bacillus
subtilis), the Gram-negative bacteria (Escherichia coli and Pseudomonas aeuroginosa), and the
yeast-like pathogenic fungus Candida albicans. The primary screen was carried out using the
agar disc-diffusion method using Müller-Hinton agar medium [45]. Results of the preliminary
antimicrobial testing of the synthesized compounds, the antibacterial and the antifungal drugs are
shown in Table 2. The results revealed that the majority of the synthesized compounds showed
varying degrees of inhibition against the tested microorganisms. Gram-positive bacteria B.
subtilis and Gram negative E. coli are considered the most sensitive among the tested
microorganisms. Compounds 18, 22, 24, 25, 29, 32, and 36 were moderate to strong active
against the Gram-positive bacteria S. aureus and B. subtilis. The inhibitory activity against the
tested Gram-negative bacteria P. aeuroginosa was rather lower than the other tested
microorganisms. The minimal inhibitory concentration (MIC) for the most active compounds 18,
22, 24, 25, 29, 32, and 36 against the same microorganism used in the primary screening was
carried out using the micro-dilution susceptibility method in Müller-Hinton Broth as shown in
Table 2 [46]. However, it could be concluded that the Gram-positive bacteria B. subtilis and to a
certain extent S. aureus and the Gram-negative bacteria E. coli are sensitive to majority of the
synthesized compounds. Compound 22, 32, and 36 showed remarkable antibacterial potency
comparable to the known drugs gentamicin and ciprofloxacin antibiotics. None of the active
compounds exert their activity through DHFR inhibition.

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2.4
In vitro Antitumor Screening
The synthesized compounds 16-36 were subjected to in-vitro antitumor screening against human
cancer cell lines using cell-based disease oriented approach [34-37]. Test compounds were used
to evaluate their antitumor potency on four human tumor cell lines namely: hepatocellular
carcinoma HepG2, caucasian breast adenocarcinoma MCF7, colon carcinoma HCT116, and lung
carcinoma A549. Cell viability was assessed by the mitochondrial dependent reduction of yellow
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] to purple Formosan
[47,48]. A probit analysis was carried for LC50 determination using SPSS 11 program. The
antitumor drug doxorubicin was used as positive control. In regard to the antitumor selectivity
among the used tumor cell lines, compounds 23 and 26 proved to be selective toward the lung
carcinoma A549 cell line with LC50 values of 33.2 and 35.4 µM, respectively; while compound
24 proved to be selective towards hepatocellular carcinoma HePG2 cell line with LC50 value of
29.1 µM. Compounds 28 and 36 exhibited antitumor potency comparable to the standard drug
doxorubicin. Compound 28 showed LC50 values of 25.0, 18.9 and 23.8 µM against lung
carcinoma A549, hepatocellular carcinoma HepG2, and caucasian breast adenocarcinoma MCF7
cell lines, respectively; while compound 36 exhibited LC50 values of 23.5 and 17.4 µM against
colon carcinoma HCT116 and caucasian breast adenocarcinoma MCF7 cell lines, respectively.
2.5
Antischistosomal Screening
The synthesized compounds 16-36 were subjected to in vitro antischistosomal screening using
reported procedures [49, 50]. The bio-screening results revealed that compounds 16, 17, 20, 21,
22, 26, and 29 possessed reproducible and confirmed in vitro antischistosomal activity with
percentage mortality range of 5-100% at concentration of 50 ꢀg/ml. Compound 27 proved to
possess marginal activity manifested as worm deformation causing intensive contraction,
bending and swelling of the worms, however, it did not cause mortality, compound 22 showed
moderate activity of 55.6 % worm mortality; while 29 exihibted schistosomicidal potency
comparable to praziquantel (the known antischistosomal drug) with 100% worm mortality.

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3.
Structure-Activity Correlation
Three different series of compounds were synthesized, namely: 5-(2-aminothiazol-4-yl)-4-
substituted-phenyl-4H-1,2,4-triazole-3-thiols (16-22); N-acetyl-N-[4-(5-mercapto-4-substituted-
phenyl-4H-1,2,4-triazol-3-yl)thiazol-2-yl]acetamides (23-29); and 1-[4-(5-mercapto-4-substit-
uted-phenyl-4H-1,2,4-triazol-3-yl)thiazol-2-yl]-3-(4-substituted-phenyl)thiourea derivatives (30-
36). The structure core skeleton of all of the synthesized compounds is 5-(2-aminothiazol-4-yl)-4-
phenyl-4H-1,2,4-triazole-3-thiol moiety, where it seemed to be that the type of substituent at the
2-amino group of the thiazole nucleus or at the 4-phenyl ring attached to the 1,2,4-triazole
function manipulate the activity.
5-(2-Aminothiazol-4-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol (16) was tested and proved to be
devoid of DHFR inhibition potency. Substitution at the 4-phenyl ring of the 1,2,4-triazole
function of 16 either with electron withdrawing, (4-Cl, 17) or electron donating functions, (2-
CH3O-, 18; 4-CH3O-, 19; 4-CH3-, 21) brought the compounds into the activity side with IC50
range of 18-24 µM. Diacetylation of the 2-amino-thiazole function of 16 produced the moderate
active compounds 24, 26, and 27 with IC50 range of 5-10 µM. The 2-amino function of 16 was
used to synthesize the 3-phenyl-thioureido compounds with the production of the most active
DHFR inhibitor in the present study, 1-(2,4-dimethoxyphenyl)-3-(4-(4-(2,4-dimethoxyphenyl)-5-
mercapto-4H-1,2,4-triazol-3-yl)thiazol-2-yl)thiourea (34, IC50 value of 0.003 µM) which proved
to be 2.7 fold more active than MTX. Compound 16 did not exert any antimicrobial activity. The
introduction of electron withdrawing chlorine function to the 4-phenyl ring of the 1,2,4-triazole
function produced 17 with antifungal activity. Replacing the chlorine atom of 17 with electron
donating function as methoxy or methyl abolished the antimicrobial potency, but upon the
introduction of phenoxy moiety to postion 4- of the phenyl ring of 16 produced 22 with a
remarkable broad spectrum antimicrobial activity (comparable to gentamicin and ciprofloxacin).
The 2-amino function of 16 was used to synthesize the 2-methoxy-phenyl-thioureido analogue 32
and the 4-phenoxy-phenyl-thioureido analogue 36 with a remarkable broad spectrum antibacterial
activity. It looks that the antitumor potency is confined to the N-acetyl-acetamide series (23-29).
The type of substituent on the 4-phenyl ring of the 1,2,4-triazole function of 23-29 manipulate the
magnitude of activity. The tested compounds exhibited certain pattern of selectivity as shown in
the un-substituted 4-phenyl analogue 23 proved toward lung carcinoma A549 cell line; the 4-(4-

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chlorophenyl)- derivative 24 toward hepatocellular carcinoma HepG2; and the 4-(4-
methoxyphenyl)- analogue 26 toward lung carcinoma A549. The introduction of 4-(4-
phenoxyphenyl)- function produced 29 with abolished antitumor activity. Replacing the 2-
diacetylamino function of 29 by 4-phenoxyphenyl-thioureido moiety produced 36 with broad
spectrum antitumor activity comparable to doxorubicin. The antibacterial 22 showed moderate
antischistosomal potency of 55.6 % worm mortality. Acetylation of the 2-amino function of 22
produced 29 with schistosomicidal activity comparable to praziquantel (100% worm mortality).
4.
Molecular Modeling Study
The DHFR inhibitory activity of the new synthesized compounds 16-19, 21-27, 29, 31-36 was
experimentally determined showing that compound 34 (IC50, 0.03 µM), in particular, is the most
active derivative with 2.7 fold more active than the positive control MTX (IC50, 0.08 µM).
Molecular modeling study was essentially needed to understand and interpret the unusual
DHFR inhibitory pattern of this new class of compounds. It was interesting to start a
comparative modeling study of the most active DHFR inhibitor 34 and the least active
compound 31 against MTX. The tertiary complex of human dihydrofolate reductase (hDHFR),
NADPH and MTX were used as references for modeling and docking. The obtained results
were quite interesting.
The binding mode of MTX to hDHFR is a complex interaction where the enzyme undergoes
conformational changes resulting in tight binding; in addition to the ionic bonding of N1 and the
2-NH2 group of MTX to Glu30, Figure 1a, [51-55]. The 3D binding mode and residues involved
in the recognition of the most active compound 34 (IC50, 0.03 µM) docked and minimized in the
hDHFR binding pocket are shown in Figure 1b. The amino acid Leu4, which is not one of the
key residues in the recognition of the parent ligand MTX, plays an essential role in the binding
recognition of 34, beside the involved hydrogen bonding network. Moreover, interaction to Val1
residue occurred in the hDHFR binding pocket seemed to be crucial for binding which explains
the difference in the magnitude of activity of 31 and 34. Compound 34 interacts with Val1
residue through the 1,2,4-triazole moiety, while compound 31 interacts surprisingly through the
thiazole function with complete absence of binding to Leu4, and hence the loss of its DHFR
inhibition potency (IC50, 40.0 µM), Figure 1c. In addition, each of compound 34 or 31 has its

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own unique pattern of binding profile, where the most active compound 34 is located deeply
inside the pocket (Figure 2a), while the least active 31 is mostly exposed out of the binding
pocket to the solvent surface (Figure 2b). Ligand-based active site alignment is a widely adopted
technique for the structural analysis of protein–ligand complexes. A good alignment occurs if the
strain energy of each molecule is small; molecules having a similar shape and their aromatic
parts overlap [56]. The objective of computing a multiple ligands alignment is to maximize the
similarity among those ligands, while keeping their sound conformations. So to probe similarity
between the 3D structures of the most active compound 34 and MTX, flexible alignment was
employed. The initial approach was to employ MOE/MMFF94 flexible alignment to
automatically generate superposition of the compounds under investigation with minimal user
bias [57]. 200 conformers of each compound were generated and minimized with a distance-
dependant dielectric model. A low energy set of 100 was selected for further analysis. The top
scoring alignment with the least strain energy is shown in Figure 3a; where a good alignment
between compound 34 (IC50, 0.03 µM) and MTX explains its activity. On the contrary, Figure 3b
obviously indicated a different alignment profiles for compound 31 (IC50, 40.0 µM) and MTX.
These findings are in consistent with the obtained DHFR inhibition experimental data. In a
further attempt to reveal the reasons behind the diminished DHFR inhibition of 31, in
comparison to the remarkable activity of 34, hydrophobic surface mapping study was conducted.
Compound 34, showed more hydrophobic regions which could be attributed to the presence of
the two methoxy groups which are responsible for the interaction with amino acid residues inside
the enzyme active pocket, Figure 4a. On the other hand, the hydrophobic regions of the least
active compound 31 are far less due to the presence of chlorine atoms, hence the required
lipophilicity for the effective binding to DHFR is diminished, Figure 4b.
The performed molecular modeling studies and the proposed binding mode analysis for 34
revealed common features which highlight the importance of hydrophobicity, aromaticity and
hydrogen bonding in such a crucial interaction with the active site. Recognition of 34 by Leu4
residue and the interaction of its 1,2,4-triazole moiety by Val1 residue of the DHFR active site
are of primary importance for activity.

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5.
Conclusion
Compounds 16-22, 23-29, and 30-36 represent three different series of compounds designed and
synthesized, with a structure core skeleton of 5-(2-aminothiazol-4-yl)-4-phenyl-4H-1,2,4-
triazole-3-thiol moiety. It seemed to be that the type of substituent at the 2-amino group of the
thiazole nucleus or at the 4-phenyl ring attached to the 1,2,4-triazole function manipulate the
type and magnitude of activity. Molecular modeling study performed and concluded that
recognition with key amino acid Leu4 and Val1 is essential for binding and the DHFR inhibition
activity. Flexible alignment with minimal user bias showed a good alignment between compound
34 (IC50, 0.03 µM) and MTX explaining its activity. Four active compounds were allocated in
the present study, the broad spectrum antibacterial 22 (comparable to gentamicin and
ciprofloxacin), the schistosomicidal 29 (comparable to praziquantel), the DHFR inhibitor 34
(IC50, 0.03 µM; 2.7 fold more active than MTX), and the antitumor 36 (comparable to
doxorubicin); Figure 5. 5-(2-Aminothiazol-4-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol skeleton as a
new class of DHFR inhibitors, could be used as a useful template for future development.
6.
Experimental Part
Melting points (°C) were determined on Mettler FP80 melting point apparatus and are
uncorrected. Microanalyses were performed on a Perkin-Elmer 240 elemental analyzer at the
Central Research Laboratory, College of Pharmacy, King Saud University. All of the new
compounds were analyzed for C, H and N and agreed with the proposed structures within ± 0.4%
1
of the theoretical values. H, 13C-NMR spectra were recorded on a on Bruker 500 MHz FT
spectrometer (the Central Research Laboratory, College of Pharmacy, King Saud University);
chemical shifts are expressed in δ ppm with reference to TMS. Mass spectral (MS) data were
obtained on a Perkin Elmer, Clarus 600 GC/MS and Joel JMS-AX 500 mass spectrometers. Thin
layer chromatography was performed on precoated (0.25 mm) silica gel GF254 plates (E. Merck,
Germany), compounds were detected with 254 nm UV lamp. Silica gel (60−230 mesh) was
employed for routine column chromatography separations. DHFR inhibition activity experiments
were performed at Pharmacology Department, Facult of Pharmacy; Future University in Egypt.
Bovine liver DHFR enzyme and methotrexate (MTX) were used in the assay (Sigma Chemical
Co, USA). Elisa reader SN208125 Bio Tek MQX200 and Software program GEN5 at wave

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length 340 nm were used to measure the changes in absorbance. The in vitro antimicrobial
testing was performed at Department of Microbiology, Faculty of Pharmacy, Mansoura
University, Egypt. The agar disc-diffusion method and a panel of standard strains (S. aureus IFO
3060, B. subtilis IFO 3007, M. luteus IFO 3232, E. coli IFO 3301, and P. aeuroginosa IFO 3448)
were employed. In vitro antitumor and schistosomicidal activities were conducted at Drug
Bioassay-Cell Culture Laboratory, Pharmacognosy Department, Pharmaceutical and Drug
Industries Division, National Research Center, Giza, Egypt. Schistosoma mansoni adult worms
were obtained from the Schistosomes Biological Supply Center at Theodor Bilharz Research
Institute, Cairo, Egypt. Concerning the molecular modeling study, all experiments were
conducted with Hyperchem 8.0.5 package from Hypercube running on a PC computer. The
docking of test compounds into DHFR pocket was performed with MOE software. Enzyme
structure, starting coordinate of hDHFR enzyme in tertiary complex with reduced-nicotinamide
adenine dinucleotide phosphate (NADPH) and MTX, code ID 3EIG, were obtained from the
Protein Data Bank of Brookhaven National Laboratory [58].
6.1.
Chemistry
6.1.1. N-{4-[2-(4-Substituted-phenylcarbamothioyl)hydrazinecarbonyl]thiazol-2-yl}acetamide
derivatives (9-15)
A solution of the acid hydrazide 1 (0.5 g, 2.5 mmol), the appropriate phenylisothiocynate (2-8,
3.5 mmol) in ethanol was heated under reflux for 3 hrs, and then cooled. The separated solid was
filtered, washed with aqueous ethanol and recrystallized from ethanol to yield the required
products 9-15 (Table 1). 9: 1H-NMR (DMSO-d6) δ 2.18 (s, 3H, CH3), 7.15 (s, 1H, thiazole-H),
7.33 (d, 2H, J = 7.5 Hz, Ar-H), 7.48 (d, 2H, J = 7 Hz, Ar-H), 7.94 (s, 1H, Ar-H), 9.81 (brs, 2H, 2
1
+
NH), 10.10 (s, 1H, NH), 12.34 (s, 1H, NH). MS m/z (%): 335 (2.5, M ). 10: H-NMR (DMSO-
d6) δ 2.16 (s, 3H, CH3), 7.39 (s, 1H, thiazole-H), 7.51 (d, 2H, J = 8 Hz, Ar-H), 7.93 (d, 2H, J =
7.5 Hz, Ar-H), 9.12 (brs, 1H, NH), 9.84 (s, 1H, NH), 10.12 (s, 1H, NH), 10.33 (s, 1H, NH). MS
1
+
m/z (%): 370 (1.1, M ). 11: H-NMR (DMSO-d6) δ 2.18 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 6.91
(t, 2H, J = 13.5 Hz, Ar-H), 7.05 (s, 1H, thiazole-H), 7.14 (d, 2H, J = 7 Hz, Ar-H), 7.96 (s, 2H,
1
+
2NH), 10.27 (s, 1H, NH), 12.38 (s, 1H, NH). MS m/z (%): 365 (0.16, M ). 12: H-NMR (DMSO-
d6) δ 2.19 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.89 (d, 2H, J = 6 Hz, Ar-H), 7.30 (d, 2H, J = 7
Hz, Ar-H), 7.94 (s, 1H, thiazole-H), 9.70 (s, 2H, 2NH), 10.10 (s, 1H, NH), 12.34 (s, 1H, NH).

ACCEPTED MANUSCRIPT
11
13C-NMR δ 22.5, 55.2, 113.3, 118.5, 127.2, 131.9, 132.6, 142.8, 149.3, 156.6, 157.7, 161.5,
1
+
169.1, 180.9. MS m/z (%): 365 (1.0, M ). 13: H-NMR (DMSO-d6) δ 2.19 (s, 3H, CH3), 3.77 (s,
6H, OCH3), 6.51 (d, 2H, J = 8 Hz, Ar-H), 6.60 (s, 1H, Ar-H), 7.62 (brs, 1H, NH), 7.94 (s, 1H,
thiazole-H), 9.24 (s, 1H, NH), 10.15 (s, 1H, NH), 12.35 (s, 1H, NH). 13C-NMR δ 22.5, 55.3,
55.7, 98.8, 104.0, 112.9, 113.2, 118.5, 122.3, 127.4, 132, 133.4, 148.7, 158, 169.1. MS m/z (%):
1
+
395 (4.8, M ). 14: H-NMR (DMSO-d6) δ 2.18 (s, 3H, CH3), 2.29 (s, 3H, CH3), 7.13 (d, 2H, J =
6.5 Hz, Ar-H), 7.33 (d, 2H, J = 6.9 Hz, Ar-H), 7.93 (s, 1H, thiazole-H), 9.71 (s, 2H, 2NH),
13
10.06 (s, 1H, NH), 12.32 (s, 1H, NH). C-NMR δ 20.5, 22.4, 98.4, 110.2, 111.2, 112.8, 112.9,
1
+
113.5, 117.4, 118.5, 128.5, 133.9, 136.6, 169.1. MS m/z (%): 349 (5.2, M ). 15: H-NMR
(DMSO-d6) δ 2.18 (s, 3H, CH3), 7.10-7.14 (m, 5H, Ar-H), 7.41 (d, 4H, J = 25.5 Hz, Ar-H), 7.93
(s, 1H, thiazole-H), 9.73 (brs, 2H, 2NH), 10.10 (s, 1H, NH), 12.33 (s, 1H, NH). 13C-NMR δ 22.5,
100.4, 101.5, 104.1, 111.0, 112.4, 113.8, 118.3, 118.6, 123.3, 127.4, 130.1, 131.0, 134.7, 156.9,
+
157.7, 169.1. MS m/z (%): 427 (3.3, M ).
6.1.2. 5-(2-Aminothiazol-4-yl)-4-substituted-phenyl-4H-1,2,4-triazole-3-thiols (16-22)
The thiosemicarbazides 9-15 (10 mmol) were refluxed in NaOH solution (2N, 30 ml) for 3 hrs.
The resultant solution was cooled, neutralized to pH 6 using dilute hydrochloric acid to give the
crude form of the target compounds. This crude compounds were filtered, washed with water
1
and crystallized from aqueous ethanol (Table 1). 16: H-NMR (DMSO-d6) δ 6.27 (s, 1H,
thiazole-H), 7.13 (s, 2H, NH2), 7.34 (d, 2H, J = 3.5 Hz, Ar-H), 7.53 (s, 3H, Ar-H), 14.00 (s, 1H,
SH).13C-NMR δ 109.7, 128.5, 129.2, 129.4, 134.7, 136.2, 146.6, 168.2, 168.3. MS m/z (%): 275
1
+
(8.7, M ). 17: H-NMR (DMSO-d6) δ 6.02 (s, 1H, thiazole-H), 6.51 (s, 1H, SH), 7.11 (s, 2H,
NH2), 7.38 (d, 2H, J = 8.5 Hz, Ar-H), 7.59 (d, 2H, J = 8.5 Hz, Ar-H). 13C-NMR δ 110.0, 129.2,
1
+
130.4, 130.5, 131.9, 133.7, 136.1, 146.5, 151.8, 164.1, 168.3. MS m/z (%): 309 (2.2, M ). 18: H-
NMR (DMSO-d6) δ 3.67 (s, 3H, OCH3), 6.15 (s, 1H, thiazole-H), 7.11-7.13 (m, 3H, NH2 and Ar-
H), 7.22 (d, 1H, J = 8 Hz, Ar-H), 7.32 (d, 1H, J = 7 Hz, Ar-H), 7.52 (t, 1H, J = 14.5 Hz, Ar-
13
H),13.91 (s, 1H, SH). C-NMR δ 55.8, 108.3, 112.9, 120.6, 120.8, 123.3, 130.6, 131.3, 136.5,
1
+
146.9, 154.8, 168.1. MS m/z (%): 305 (7.1, M ). 19: H-NMR (DMSO-d6) δ 3.68 (s, 3H, OCH3),
4.55 (s, 1H, SH), 6.77 (d, 2H, J = 7.5 Hz, Ar-H), 6.95-7.00 (m, 3H, NH2 and thiazole-H), 7.13
13
(d, 1H, J = 8 Hz, Ar-H), 7.26 (d, 1H, J = 8.5 Hz, Ar-H). C-NMR δ 55.4, 106.6, 114.2, 114.5,
1
+
124.8, 128.1, 143.1, 149.3, 150.3, 150.6, 159.7, 170.5. MS m/z (%): 305 (5.0, M ). 20: H-NMR
(DMSO-d6) δ 3.67 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.14 (s, 1H, thiazole-H), 6.69 (d, 1H, J =

ACCEPTED MANUSCRIPT
12
8.5 Hz, Ar-H), 6.75 (s, 1H, Ar-H), 7.13 (s, 2H, NH2), 7.21 (d, 1H, J = 8.5 Hz, Ar-H), 13.85 (s,
1H, SH). 13C-NMR δ 55.5, 56.0, 99.7, 105.5, 108.3, 116.1, 130.7, 136.6, 147.1, 155.8, 161.4,
1
+
168.0, 168.8. MS m/z (%): 335 (9.2, M ). 21: H-NMR (DMSO-d6) δ 2.39 (s, 3H, CH3), 6.24 (s,
1H, thiazole-H), 7.14 (s, 2H, NH2), 7.22 (d, 2H, J = 8 Hz, Ar-H), 7.33 (d, 2H, J = 8 Hz, Ar-H),
13.96 (s, 1H, SH). 13C-NMR δ 20.8, 100.5, 104.8, 109.7, 128.2, 129.8, 132.1, 136.2, 139, 146.7,
1
+
168.2, 168.3. MS m/z (%): 289 (11.4, M ). 22: H-NMR (DMSO-d6) δ 6.38 (s, 1H, thiazole-H),
7.09 (d, 4H, J = 9 Hz, Ar-H), 7.15 (s, 2H, NH2), 7.23 (t, 1H, J = 6.5 Hz, Ar-H), 7.34 (d, 2H, J =
8.5 Hz, Ar-H), 7.46 (t, 2H, J = 7.5 Hz, Ar-H), 13.99 (s, 1H, SH). 13C-NMR δ 109.4, 118.3,
119.5, 124.2, 128.5, 130.2, 138.7, 144.5, 155.4, 156.0, 156.5, 157.3, 158.4, 168.1, 168.9. MS m/z
+
(%): 367 (2.8, M ).
6.1.3. N-Acetyl-N-[4-(5-mercapto-4-substituted-phenyl-4H-1,2,4-triazol-3-yl)thiazol-2-yl]-
acetamide derivatives (23-29)
A solution of compounds 16-22 (2.5 mmol) in acetic anhydride (5.0 ml) were heated for 2 hours,
after which the solution was poured in ice-water solution. The products formed were filtered,
washed with excessive amount of water, and finally crystallized from ethanol to yield the target
1
compounds 23-29 (Table 1). 23: H-NMR (DMSO-d6) δ 2.13 (s, 3H, CH3CO), 2.71 (s, 3H,
CH3CO), 6.63 (s, 1H, thiazole-H), 7.39 (d, 2H, Ar-H), 7.60 (t, 3H, Ar-H), 12.50 (s, 1H, SH). 13C-
NMR δ 22.4, 25.4, 60.3, 65.0, 117.8, 128.8, 130.2, 134.2, 145.4, 158.5, 167.8, 169.1, 171.4. MS
1
+
m/z (%): 359 (9.4, M ). 24: H-NMR (DMSO-d6) δ 2.14 (s, 3H, CH3CO), 2.70 (s, 3H, CH3CO),
6.88 (s, 1H, thiazole-H), 7.50 (d, 2H, J = 8.5 Hz, Ar-H), 7.67 (d, 2H, J = 8 Hz, Ar-H), 12.45 (s,
1H, SH). 13C-NMR δ 22.4, 24.7, 62.9, 63.4, 118.4, 129.9, 130.8, 133.1, 134.0, 134.8, 141.0,
1
+
145.3, 158.6, 169.3, 172.0. MS m/z (%): 394 (12.5, M ). 25: H-NMR (DMSO-d6) δ 2.14 (s,
3H, CH3CO), 2.70 (s, 3H, CH3CO), 3.63 (s, 3H, OCH3), 6.66 (s, 1H, thiazole-H), 7.18 (t, 1H, J =
14 Hz, Ar-H), 7.28 (d, 1H, J = 8.5 Hz, Ar-H), 7.44 (d, 1H, J = 7 Hz, Ar-H), 7.61 (t, 1H, J = 15
Hz, Ar-H), 12.51 (s, 1H, SH). 13C-NMR δ 22.4, 24.7, 56.0, 62.3, 113.3, 116.8, 122.3, 130.3,
1
+
132.1, 134.2, 145.7, 154.6, 158.5, 167.8., 169.1, 171.9. MS m/z (%): 389 (7.2, M ). 26: H-NMR
(DMSO-d6) δ 2.14 (s, 3H, CH3CO), 2.70 (s, 3H, CH3CO), 3.85 (s, 3H, OCH3), 6.63 (s, 1H,
thiazole-H), 7.13 (d, 2H, J = 8.5 Hz, Ar-H), 7.35 (d, 2H, J = 8 Hz, Ar-H), 12.53 (s, 1H, SH).
13C-NMR δ 21.1, 22.4, 24.7, 55.5, 61.8, 115.0, 117.9, 126.6, 130.1, 134.2, 145.7, 158.5, 160.1,
1
+
167.8, 169.1, 169.6. MS m/z (%): 389 (15.1, M ). 27: H-NMR (DMSO-d6) δ 2.14 (s, 3H,
CH3CO), 2.73 (s, 3H, CH3CO), 3.60 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 6.69 (s, 1H, thiazole-

ACCEPTED MANUSCRIPT
13
H), 6.73 (d, 1H, J = 9.5 Hz, Ar-H), 6.81 (s, 1H, Ar-H), 7.33 (d, 1H, J = 9 Hz, Ar-H), 12.55 (s,
1H, SH). 13C-NMR δ 22.4, 24.7, 55.6, 56.1, 99.8, 105.6, 106.1, 114.4, 115.1, 130.9, 134.3,
1
+
145.9, 158.5, 162.0, 167.8, 169.0, 172.0. MS m/z (%): 419 (3.6, M ). 28: H-NMR (DMSO-d6) δ
2.14 (s, 3H, CH3CO), 2.26 (s, 3H, CH3), 2.71 (s, 3H, CH3CO), 6.64 (s, 1H, thiazole-H), 7.31 (d,
2H, J = 7 Hz, Ar-H), 7.40 (d, 2H, J = 7 Hz, Ar-H), 12.52 (s, 1H, SH). 13C-NMR δ 20.9, 22.4,
24.7, 55.9, 58.3, 117.9, 128.5, 130.3, 131.6, 134.2, 139.2, 145.5, 158.5, 167.9, 169.1, 169.4. MS
1
+
m/z (%): 373 (4.1, M ). 29: H-NMR (DMSO-d6) δ 2.15 (s, 3H, CH3CO), 2.70 (s, 3H, CH3CO),
6.76 (s, 1H, thiazole-H), 7.17 (d, 4H, J = 6.5 Hz, Ar-H), 7.23 (t, 1H, J = 14 Hz, Ar-H), 7.43 (d,
2H, J = 8 Hz, Ar-H), 7.47 (t, 2H, J = 14 Hz, Ar-H), 12.53 (s, 1H, SH). 13C-NMR δ 22.4, 24.7,
55.3, 56.9, 118.2, 118.9, 119.5, 124.4, 128.8, 130.3, 130.6, 134.1, 145.6, 155.6, 158.1, 158.5,
+
167.8, 169.1, 169.4. MS m/z (%): 451 (8.6, M ).
6.1.4. 1-[4-(5-Mercapto-4-substituted-phenyl-4H-1,2,4-triazol-3-yl)thiazol-2-yl]-3-(4-
substituted-phenyl)thiourea derivatives (30-36)
The 1,2,4-triazole derivatives (16-22, 2.5 mmol) were heated with phenylisothiocyanate
derivatives (2-8, 3.5 mmol) in ethanol under reflux for 4 hrs. The separated solid obtained upon
cooling was filtered, washed with water and crystallized from ethanol to yield the pure
compounds 30-36 (Table 1). 30: 1H-NMR (DMSO-d6) δ 3.37 (brs, 1H, NH), 3.44 (brs, 1H, NH),
6.26 (s, 1H, thiazole-H), 7.13 (s, 2H, Ar-H), 7.36 (s, 2H, Ar-H), 7.53-7.56 (m, 6H, Ar-H), 14.00
(s, 1H, SH). 13C-NMR δ 99.9, 104.8, 109.7, 111.0, 114.2, 116.9, 118.7, 119.0, 128.5, 129.2,
1
+
129.4, 134.7, 136.2, 146.6, 168.2, 168.8. MS m/z (%): 410 (4.4, M ). 31: H-NMR (DMSO-d6) δ
3.71 (brs, 2H, 2NH), 6.02 (s, 1H, thiazole-H), 7.30 (d, 2H, J = 8 Hz, Ar-H), 7.39 (d, 2H, J = 8
13
Hz, Ar-H), 7.49 (d, 2H, J = 7 Hz, Ar-H), 7.62 (d, 2H, J = 8 Hz, Ar-H), 12.85 (s, 1H, SH). C-
NMR δ 102.3, 110.9, 112.5, 115.6, 117.1, 117.9, 119.3, 128.5, 129.4, 129.5, 130.5, 131.1, 131.9,
1
+
133.7, 147.9, 151.7, 164.1, 168.5. MS m/z (%): 479 (0.9, M ). 32: H-NMR (DMSO-d6) δ 3.79
(s, 6H, OCH3), 6.92 (t, 2H, J = 14 Hz, Ar-H), 7.05 (d, 2H, J = 6.5 Hz, Ar-H), 7.14 (t, 2H, J = 14
Hz, Ar-H), 7.22 (brs, 2H, 2NH), 7.39 (s, 1H, thiazole-H), 8.13 (d, 2H, J = 6.5 Hz, Ar-H), 10.79
(s, 1H, SH). 13C-NMR δ 55.5, 55.8, 100.0, 101.5. 106.0, 107.9, 111.3, 113.2, 114.2, 115.8, 119.8,
1
+
124.1, 125.4, 127.6, 138.1, 139.0, 143.3, 150.9, 164.8, 168.4. MS m/z (%): 470 (2.8, M ). 33: H-
NMR (DMSO-d6) δ 3.82 (s, 6H, OCH3), 6.09 (brs, 2H, 2NH), 7.03 (d, 4H, J = 8.5 Hz, Ar-H),
13
7.10 (s, 1H, thiazole-H), 7.20 (d, 4H, J = 8.5 Hz, Ar-H), 13.89 (brs, 1H, SH). C-NMR δ 55.3,
55.8, 101.3, 104.8, 107.7, 108.5, 111.4, 114.2, 115.2, 117.5, 119.0, 129.6, 130.5, 135.4, 137.3,

ACCEPTED MANUSCRIPT
14
1
+
139.0, 146.9, 159.2, 164.3, 138.1. MS m/z (%): 470 (5.4, M ). 34: H-NMR (DMSO-d6) δ 3.75
(s, 6H, OCH3), 3.84 (s, 6H, OCH3), 6.14 (s, 1H, thiazole-H), 6.64 (d, 2H, J = 8.5 Hz, Ar-H), 6.68
(s, 2H, Ar-H), 7.10 (d, 2H, J = 8.5 Hz, Ar-H), 12.63 (s, 2H, 2NH), 13.90 (s, 1H, SH). 13C-NMR
δ 55.2, 55.5, 55.8, 56.0, 99.5, 99.7, 105.3, 105.5, 108.4, 114.0, 116.0, 130.7, 131.2, 146.8, 152.5,
1
+
155.8, 156.2, 161.3, 161.5, 164.9, 168.2, 168.7. MS m/z (%): 530 (2.4, M ). 35: H-NMR
(DMSO-d6) δ 2.38 (s, 6H, CH3), 6.24 (s, 1H, thiazole-H), 4.15 (s, 2H, 2NH), 7.22 (d, 4H, J = 14
Hz, Ar-H), 7.32 (d, 4H, J = 14 Hz, Ar-H), 13.98 (s, 1H, SH). 13C-NMR δ 20.8, 21.3, 55.5, 101.5.
107.9, 109.7, 124.1, 125.4, 127.6, 128.2, 129.8, 132.1, 136.2, 138.1, 139.0, 139.3, 143.3, 146.7,
1
+
168.2, 168.4. MS m/z (%): 438 (12.8, M ). 36: H-NMR (DMSO-d6) δ 6.37 (s, 1H, thiazole-H),
6.87-6.94 (m, 4H, Ar-H), 7.04 (t, 3H, J = 8.5 Hz, Ar-H), 7.22 (d, 4H, J = 8 Hz, Ar-H), 7.44 (d,
4H, J = 6.5 Hz, Ar-H),7.65 (t, 3H, J = 6.5 Hz, Ar-H), 12.81 (s, 2H, 2NH), 14.00 (s, 1H, SH). 13C-
NMR δ 109.9, 117.5, 118.6, 119.2, 119.5, 119.7, 122.7, 124.1, 124.2, 127.4, 130.2, 130.3, 136.1,
136.1, 146.8, 148.6, 150.3, 152.1, 155.7, 155.8, 156.0, 157.2, 157.5, 157.7, 164.2, 168.3. MS m/z
+
(%): 594 (2.5, M ).
6.2.
Dihydrofolate Reductase (DHFR) Inhibition Assay
The assay mixture contained 0.5 mol/l Tris buffer (A), pH 7.5 and 0.05 mol/l Tris buffer (B), pH
7.5. Stock solutions of FH2 (25 mg in 1.5 ml of 2-mercaptoethanol and 6.0 ml of buffer A in 0.25
ml aliquots), NADPH (50 mg in 10 ml of buffer (A) in 0.4 ml aliquots), and DHFR (2.1 U in 10
ml of buffer (B) in 0.5 ml aliquots) stored at −70 °C. 130 ꢀl FH2 reaction solutions, 0.25 ml
aliquot of FH2 stock solution in 8.0 ml of buffer B, yielding a final working concentration of
0.104 g/l, were added to each well of the 96-well flat-bottom microplate reader. 20 ꢀl MTX
calibrators or unknown samples and blank of different concentrations 10-11 to 10-5 (adjusted by
DMSO) were added into duplicate wells. The microplate was shaken in a shaking incubator for 1
min, after which 50 ꢀl NADPH/DHFR reaction solution, 0.4-ml aliquot of NADPH stock
solution and 0.5 ml aliquot of DHFR stock solution in 6.0 ml of buffer B, yielding a final
working concentration of 0.29 g NADPH/l and 15u DHFR/l were added to each well, and the
microplate was again shaken in a shaking incubator for 1 min. The absorbance of each well was
read in the microplate reader at room temperature at wavelengths of 340 nm [42-44], using the
kinetic mode with a reading interval of 1 min for duration of 10 min. The changes in absorbance
were downloaded directly into an ELISA reader SN208125 Bio Tek MQX200 computer and

ACCEPTED MANUSCRIPT
15
analyzed with software Gen5 wave length 340 nm. Results were reported as % inhibition of
enzymatic activity calculated using the following formula:


ꢁ
A/mintest



% Inhibition = 1 −
×100


ꢁ
A/minDMSO

The linear decrease of absorbance (∆A/min) between 0 and 10 min for each MTX or sample was
calculated and presented by % of inhibition then plotted against their concentrations (log scale)
to obtain a calibration curve [44]. The 50% inhibitory concentration (IC50) of each compound
was obtained.
6.3.
Determination of in vitro Antimicrobial Activity
The primary screen was carried out using the agar disc-diffusion method [45] using Müller-
Hinton agar medium. Sterile filter paper discs (8 mm diameter) were moistened with the
compound solution in dimethylsulphoxide of specific concentration 200 µg/disc, the antibacterial
antibiotic gentamicin, ciprofloxacin, (200 µg/disc) and the antifungal drug clotrimazole (200 µg
/disc) were carefully placed on the agar cultures plates that had been previously inoculated
separately with the microorganisms. The plates were incubated at 37 oC, and the diameter of the
growth inhibition zones was measured after 24 hours in case of bacteria and at 25 oC for 48 hours
in case of C. albicans. The minimal inhibitory concentrations (MIC) for the compounds against
the same microorganisms used in the primary screening were carried out using the microdilution
susceptibility method in Müller-Hinton Broth [46]. The compounds, gentamicin, and
ciprofloxacin were dissolved in dimethylsulphoxide at concentration of 64 µg /mL. The twofold
dilutions of the solution were prepared (64, 32, …, 0.5, 0.25 and 0.125 µg/ml). The
microorganism suspensions at 106 CFU/ml (colony forming unit/ml) concentrations were
inoculated to the corresponding wells. The plates were incubated at 37 oC for 24 hours. The MIC
values were determined as the lowest concentration that completely inhibited visible growth of
the microorganism as detected by unaided eye.
6.4.
Antitumor Screening
The in vitro Antitumor screening was performed adopting previously reported procedures
[37,47,48]. Cells were suspended in RPMI 1640 medium for HepG2, MCF7 and HCT116 and
DMEM for A549, 1% antibiotic-antimycotic mixture (10,000 u/ml potassium penicillin, 10,000

ACCEPTED MANUSCRIPT
16
µg/ml streptomycin sulfate and 25 µg/ml amphotericin B) and 1% L-glutamine at 37°C, under
5% CO2 and 95% humidity. Cells were seeded at concentration of 10 x 103 cells/well in fresh
complete growth medium in 96-well microtiter plates for 24 hrs. Media was aspirated, fresh
medium (without serum) was added and cells were incubated with different concentrations of
sample to give a final concentration of (100-50-25-12.5-6.25-3.125- 0.78 and 1.56 ꢀM). 0.5%
DMSO was used as negative control and 100 µg/ml of doxorubicin was used as positive control.
MTT assay was used for assessment of cytotoxicity [37,47,48]. After 48 h of incubation, medium
was aspirated, 40 ꢀl MTT salt (2.5 ꢀg/ml) were added to each well and incubated for further 4 h.
To stop the reaction and dissolving the formed crystals, 200 ꢀl of 10% sodium dodecyl sulphate
(SDS) in deionized water were added to each well and incubated overnight at 37°C. The
absorbance was then measured at 595 nm and a reference wavelength of 620 nm. A statistical
significance was tested between samples and negative control (cells with vehicle) using
independent t-test by SPSS 11 program. The % cytotoxicity was calculated according to the
formula:
[1-(OD compound / OD negative control)] x 100
A probit analysis was carried for LC50 determination using SPSS 11 program.
6.5
Antischistosomal Screening
The bioassay was performed as previously described [49,50], where the experiment was
performed in 24-well sterile culture plates. Six worms (3 males and 3 females) were introduced
into each well in triplicates. The compounds were dissolved in DMSO and were tested at 50
µg/ml. The respective amount of DMSO (0.5%) was used as negative control. The culture plates
were incubated at 37°C for 24 h. Worms were examined for the toxic effect under a dissecting
microscope. Worms that did not show any movement through at least a minute especially
associated with coiling and becoming much darker in colour, were considered dead and counted.
The activity of each of the test compounds was measured by calculating the % of dead worms
per well. Praziquantel was used as the positive control at 0.1 µg/ml. Five different experiments at
five different dates, each in triplicate, were done to calculate different validation parameters.
6.6
Docking and Molecular Modeling Study

ACCEPTED MANUSCRIPT
17
The three-dimensional structures of the 2,4-substituted thiazole derivatives, in their neutral
forms, were constructed using the MOE of Chemical Computing Group Inc software. Lowest
energy conformer of each of the new analogue ‘global-minima’ was docked into the hDHFR
enzyme-binding domain. All the hydrogens were added and enzyme structure was subjected to a
refinement protocol in which the constraints on the enzyme were gradually removed and
minimized until the rms gradient was 0.01 kcal/mol Ao. The energy minimization was carried out
using the molecular mechanics force field ‘AMBER.’ The energy-minimized structure was used
for molecular dynamics studies. For each of the thiazole analogues, energy minimizations (EM)
were performed using 1000 steps of steepest descent, followed by conjugate gradient
minimization to a RMS energy gradient of 0.01 Kcal/mol Å. The active site of the enzyme was
defined using a radius of 10.0 Ao around MTX. Energy of binding was calculated as the
difference between the energy of the complex and individual energies of the enzyme and ligand
[59-62].
6.7
Flexible Alignment
The investigated compounds were subjected to flexible alignment experiment using ‘Molecular
Operating Environment’ software (MOE of Chemical Computing Group Inc., on a Core 2 duo
2.3 GHz workstation). The molecules were built using the Builder module of MOE. Their
geometry was optimized by using the MMFF94 forcefield followed by a flexible alignment using
systematic conformational search. . Lowest energy aligned conformation(s) were identified [59-
62].
Acknowledgment
This research project was supported by a grant from the “Research Center of the Center for
Female Scientific and Medical Colleges”, Deanship of Scientific Research, King Saud
University.
7.
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Captions
Table 1: Physicochemical properties of the newly synthesized compounds 9-36
Table 2: DHFR inhibition (IC50, µM), and the antimicrobial activity results of compounds 16-
19, 21-29, 31-36
Table 3: Median lethal concentration (LC50, µM) of the most active antitumor compounds 23,
24, 26, 28 and 36
Table 4: Antischistosomal testing results of compounds 16, 17, 20-22, 26, 27 and 29 at
concentration of 50 µg/ml
Chart 1: Structures of Tiazofurin (A), Netropsin (B), Thia-netropsin (C) and 4-Phenyl-thiazole-
1,3,5-triazines (D).
Figure 1: (a) 2D binding mode and residues involved in the recognition for MTX; (b) 3D
binding mode and residues involved in the recognition for the most active compound
34 (IC50 0.03 µM); (c) 2D binding mode and residues involved in the recognition and
the least active compound 31 (IC50 40.0 µM) docked and minimized in the DHFR
binding pocket.
Figure 2: The aligned conformation of (a) the most active compound 34 (red, IC50 0.03 µM)
occupying the DHFR binding pocket; (b) the most inactive compound 31 (cyan, IC50
40.0 µM) exposing out of the DHFR binding pocket surface map.
Figure 3: Flexible alignment of (a) the most active compound 34 (red); (b) the least active
compound 31 (cyan); and MTX (gray).
Figure 4: Hydrophobic surface map for (a) the most active compound 34 (red); (b) the least
active compound 31 (cyan) in pocket side. Pink: hydrogen bond; blue: mild polar;
green: hydrophobic area.
Figure 5: Structures of the broad spectrum antibacterial 22 (comparable to gentamicin and
ciprofloxacin), the schistosomicidal 29 (comparable to praziquantel), the DHFR
inhibitor 34 (IC50 0.03 µM, 2.7 fold more active than MTX), and the antitumor 36
(comparable to doxorubicin).
Scheme 1: Synthesis of the target compounds 16-22, 23-29, and 30-36.

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24
Table 1: Physicochemical properties of the newly synthesized compounds 9-36
H
H
N
N
N
N
S
HS
HS
HS
R
N
N
N
N
O
R
N
N
N
N
R
R
R
S
O
O
N
N
N
H
N
N
H
CH3
CH3
CH3
N
H
N
N
NH2
S
S
S
S
H
O
9-15
30-36
16-22
23-29
Compound
R
Solvent
Yield %
Mp 0C
Molecular Formulae
H
4-Cl
2-CH3O
4-CH3O
2,4-(CH3O)2
4-CH3
EtOH
EtOH
EtOH
EtOH/H2O
EtOH/H2O
EtOH/H2O
EtOH
EtOH/H2O
EtOH/H2O
EtOH
65
70
73
69
54
55
90
48
82
46
33
52
67
69
66
51
52
49
59
63
75
50
54
63
66
67
58
49
123-5
138-40
124-6
141-3
150-2
144-6
169-72
149-52
151-3
142-5
163-7
165-8
121-3
135-7
98-100
121-4
115-7
139-42
129-32
158-60
144-7
152-5
143-5
161-3
145-7
149-51
163-5
158-60
C13H13N5O2S2
C13H12ClN5O2S2
C14H15N5O3S2
C14H15N5O3S2
C15H17N5O4S2
C14H15N5O2S2
C19H17N5O3S2
C11H9N5S2
C11H8ClN5S2
C12H11N5OS2
C12H11N5OS2
C13H13N5O2S2
C12H11N5S2
C17H13N5OS2
C15H13N5O2S2
C15H12ClN5O2S2
C16H15N5O3S2
C16H15N5O3S2
C17H17N5O4S2
C16H15N5O2S2
C21H17N5O3S2
C18H14N6S3
C18H12Cl2N6S3
C20H18N6O2S3
C20H18N6O2S3
C22H22N6O4S3
C20H18N6S3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
4-C6H5O
H
4-Cl
2-CH3O
4-CH3O
2,4-(CH3O)2
4-CH3
EtOH
EtOH/H2O
EtOH/H2O
EtOH
EtOH/H2O
EtOH
EtOH/H2O
EtOH
EtOH
EtOH/H2O
EtOH/H2O
EtOH
EtOH
EtOH
EtOH/H2O
EtOH
EtOH/H2O
EtOH
4-C6H5O
H
4-Cl
2-CH3O
4-CH3O
2,4-(CH3O)2
4-CH3
4-C6H5O
H
4-Cl
2-CH3O
4-CH3O
2,4-(CH3O)2
4-CH3
24
25
26
27
28
29
30
31
32
33
34
35
36
4-C6H5O
C30H22N6O2S3

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25
Table 2: DHFR inhibition (IC50, µM), and the antimicrobial activity results of compounds 16-
19, 21-29, 31-36
Compound
DHFR inhibition S. aureus
B. subtilis
E. coli
P. aeuroginosa C. albicans
-
-
-
-
-
-
16
18.0
24.0
21.0
24.0
-
35.0
10.0
-
5.0
8.0
-
15.0
40.0
-
-
12
-
-
28 (0.5)
-
16 (4.0)
-
12
-
-
18 (4.0)
-
16
-
12
16 (4.0)
12
-
32 (0.5)
-
21 (4.0)
20 (4.0)
14
-
-
20 (2.0)
14
18
-
-
-
-
-
-
12
-
-
-
14
-
-
-
-
-
-
-
-
-
-
-
-
-
17
18
19
21
22
23
24
25
26
27
28
29
31
32
33
34
15 (8.0)
-
-
20 (0.5)
-
-
16 (0.8)
-
-
-
-
-
16 (1.0)
-
-
-
-
-
-
-
-
-
-
-
-
-
18 (0.8)
-
-
-
30.0
0.03
10.0
35.0
-
-
-
-
-
35
36
16 (4.0)
26.5 (2.0)
32 (0.5)
Nd
18 (2.0)
25 (2.0)
35 (0.5)
Nd
14
20.8 (0.5) 19 (1.0)
38 (0.25) 36 (1.0)
Nd
-
Nd
Nd
21
-
Gentamicin
Ciprofloxacin
Clotrimazole
Methotrexate
-
-
Nd
-
0.08
-
-
Inhibition Zone (mm): (-) Not active (8 mm), Weak activity (8-12 mm), Moderate activity (12-15 mm), Strong activity (> 15
mm). Solvent: DMSO (8 mm). MICs showed in parentheses. Nd, not determined.

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Table 3: Median lethal concentration (LC50, µM) of the most active antitumor
compounds 23, 24, 26, 28 and 36
Compound.
A549
HCT116
HepG2
MCF7
33.2
-
35.4
25.0
-
-
-
-
-
-
-
-
23
24
26
28
29.1
-
18.9
-
-
23.8
17.4
26.1
23.5
37.7
36
28.3
21.4
Doxorubicin

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Table 4: Antischistosomal testing results of compounds 16, 17, 20-
22, 26, 27 and 29 at concentration of 50 µg/ml.
Resultsa
Compound
% Mortality
3/18
1/18
16.6
5.0
16
17
2/18
2/19
10/18
11.1
10.5
55.6
6.0
20
21
22
26
1/16
0/18 (deformation)b
0.0
27
18/18
0/18
0/18
100
0.0
0.0
29
DMSO
Untreated
Praziquantel
18/18
100
a Number of dead worms/Total number of worms.
b Worm morphological deformities (intensive contraction, bending and swelling).

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O
NH2
HN
CH3
N
H
NH2
CH3
N
O
N
N
OH
S
NH
HN
N
O
H2N
O
H
NH
OH OH
O
B
A
NH2
O
H
H2N
N
N
N
O
N
N
H
N
N
S
N
Cl
N
S
S
H
H2N
O
R
D
C
Chart 1: Structures of Tiazofurin (A), Netropsin (B), Thia-netropsin (C)
and 4-Phenyl-thiazole-1,3,5-triazines (D).