Synthesis and antifungal activity of some s-mercaptotriazolobenzothiazolyl amino acid derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.10.033

A series of s-triazolobenzothiazolylthioacetyl/propionyl amino acid derivatives were synthesized with the aim of evaluating their antifungal activity. Their chemical structures were confirmed by ¹H, ¹³C NMR, IR, mass spectrometry and elemental analyses. The synthesized derivatives were screened for their antifungal activity against Aspergillus flavus and Candida albicans. Five compounds (3, 5, 7c, 8 and 17) were found to possess high activity comparable to fluconazole at 100 μg/mL against C. albicans.

Full text of DOI:10.1016/j.ejmech.2012.10.033

European Journal of Medicinal Chemistry 60 (2013) 503e511
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antifungal activity of some s-mercaptotriazolobenzothiazolyl amino
acid derivatives
,
a
a
b
A. Aboelmagd a , Ibrahim A.I. Ali , Ezzeldin M.S. Salem , M. Abdel-Razik
*
a Department of Chemistry, Faculty of Science, Suez Canal University, Ismailia 41511, Egypt
b Department of Botany, Microbiology Division, Faculty of Science, Suez Canal University, Ismailia 41511, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of s-triazolobenzothiazolylthioacetyl/propionyl amino acid derivatives were synthesized with
the aim of evaluating their antifungal activity. Their chemical structures were confirmed by 1H, 13C NMR,
IR, mass spectrometry and elemental analyses. The synthesized derivatives were screened for their
antifungal activity against Aspergillus flavus and Candida albicans. Five compounds (3, 5, 7c, 8 and 17)
Received 4 July 2012
Received in revised form
14 October 2012
Accepted 18 October 2012
Available online 26 October 2012
were found to possess high activity comparable to fluconazole at 100 mg/mL against C. albicans.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Amino acids
Antifungal activity
Azide and carbodiimide coupling methods
s-Mercaptotriazolobenzothiazole
1. Introduction
Other amino acids with nonpolar hydrocarbon side chains (leucine,
valine, alanine and glycine) have been also coupled to the hetero-
cyclic ring system for comparison. The above mentioned selected
amino acids are actually constituents of some antifungal poly-
peptide antibiotics [6e11]. The synthesized triazolobenzothiazolyl
derivatives were screened for their antifungal activity against
Candida albicans and Aspergillus flavus (Tables 1 and 2).
Triazoles and benzothiazoles are well known for their high
antimicrobial activities [1,2]. Thus, fusion of the two antimicrobial
agents, i.e. triazole and benzothiazole moieties could afford new
interesting fused tricyclic drugs, analogs to the fungicide tricycla-
zole. Tricyclazole (5-methyl-1,2,4-triazolo[3,4-b]benzothiazole)
patented by Eli Lilly, is commercial antifungal product that acts by
inhibiting melanin synthesis. Tricyclazole is one of the few
commercial compounds which are used for control of some
phytopathogenic fungi and is also active on dematiaceous human
pathogenic fungi [3,4]. From our previous study it is obvious that 2-
benzothiazolylthioacetyl amino acid derivatives showed, in
general, good activity against mold and yeast fungi [5] (Fig. 1).
Accordingly, in the present study, it is planned to synthesize
a series of amino acid derivatives of s-mercaptotriazolo[3,4-b]
benzothiazole 3 to evaluate their antifungal action as shown in
Schemes 1 and 2. The amino acids are attached to the starting
2. Results and discussion
2.1. Chemistry
The starting compound triazolobenzothiazole derivative 3 was
prepared by the reaction of 2-hydrazinobenzothiazole (2) with
carbon disulfide in alcoholic potassium hydroxide by the methods
recorded in literature [12,13].
As has been previously mentioned, it was planned to attach the
amino acids to the fused tricyclic nucleus via flexible link, namely
acetyl or propionyl group. This anchor will permit free stereo-
chemical reactions between the bulky triazolobenzothiazole ring
and the amino acids. Such type of attachment is provided via
alkylation or Michael addition reactions.
Ethyl s-triazolo[3,4-b]benzothiazol-3-ylthioacetate (4) (Scheme
1) was prepared in good yield by alkylation of 3 with ethyl chlor-
oacetate in the presence of anhydrous potassium carbonate. s-Tri-
azolo[3,4-b]benzothiazol-3-ylthioacetic acid (6) was prepared by
alkylation of compound 3 with chloroacetic acid in the presence of
compound
3 via S- or N-acetyl/propionyl link. Therefore,
compound 3 was further derivatized to 3-thioacetic/propionic or N-
propionic acids 6, 9 and 16 respectively. The amino acids used in the
synthesis contain the physiologically active hydroxyl, sulfur and
phenolic groups (serine, threonine, methionine and tyrosine).
* Corresponding author. Tel.: þ20 1221834049.
E-mail address: ahmedaelmagd@gmail.com (A. Aboelmagd).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.033

504
A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
S
S
N
N
N
N
N
N
SH
CH3
Tricyclazole
[3]
(5-methyl-1,2,4-triazolo[3,4-b]benzothiazole) 3-mercapto-s-triazolo[3, 4-b]benzothiazole
Fig. 1. Structures of tricyclazole and compound 3.
sodium hydroxide or by alkaline hydrolysis of the corresponding
ester 4 with potassium hydroxide in aqueous ethanol. The struc-
tures of 4 and 6 were proved by elemental analyses, IR, 13C NMR and
1H NMR spectra (see the Experimental part).
In the present investigation methionine methyl ester derivative
7a was prepared by three different coupling procedures i.e. azide,
mixed anhydride and carbodiimide coupling methods for
comparison (Scheme 1). In the azide coupling method the azide,
obtained from the hydrazide 5 by treatment with nitrous acid, is
unstable which, in turn without isolation, is allowed to react with
methionine methyl ester to afford thioacetyl methionyl methyl
ester derivative 7a in moderate yield (59.7%). In the second method,
i.e. mixed anhydride method, the acid derivative 6 was treated with
ethyl chloroformate in toluene in the presence of triethyl amine,
followed by addition of a cold solution (0 ꢀC) of
L
-methionine
methyl ester hydrochloride and triethyl amine in toluene. The s-
triazolo[3,4-b]benzothiazole-3-thioacetyl -methionine methyl
L
ester (7a) was obtained in lower yield (43.7%) from this method
after purification by crystallization in the presence of charcoal. The
third one is the DCCI-procedure, by coupling the acid derivative 6
with L-methionine methyl ester hydrochloride in acetonitrile in the
presence of dicyclohexylcarbodiimide and triethyl amine. The iso-
lated product was impure and contained a mixture from the
desired compound 7a and a side-product i.e. N-acylurea derivative
S
S
S
N
i
SH
NHNH2
N
N
O
N
N
[1]
S
N
[2]
S
N
H
ii
O
[8]
S
S
N
N
N
N
vii
N
N
iii
N
N
N
SCH2CO2C2H5
SH
[6]
SCH2CO2H
[3]
[4]
S
iv
viii
N
N
N
xi
SCH2CON2H3
x
[5]
x
i
[7a]
v
[7a ] + [8]
S
N
N
N
SCH2CON3
7
n
R1
-CH2CH2SCH3
vi
-a (L-Met)
-b (L-Ser)
-c (L-Tyr)
-d (DL-Thr)
-e (Gly)
1
1
1
1
1
1
1
2
-CH2OH
-CH -Ph-4-OH
S
N
N
-CH2(OH)CH
3
-H
N
R
O
-f (L-Val)
-g (L-Leu)
-CH(CH3)CH3
-
CH2CH(CH3)CH3
S
OCH3
β
-h ( -Ala)
N
H
-H
n
O
[7a-h]
(i) N2H4.H2O, reflux in EtOH; (ii) a) CS2, KOH, EtOH, reflux, b) HCl; (iii) ethyl chloroacetate,K2CO3, reflux in dry acetone;
(iv) N2H4.H2O, reflux in EtOH; (v) NaNO2 / HCl, at - 5 oC; (vi) HCl.H2NCHR1CO2CH3, Et3N at 0 oC; (vii) a) chloroaceticacid,
NaOH, reflux in EtOH, b) HCl; (viii) a) KOH / EtOH 95%, b) HCl; (ix) L-Met-OMe, DCC; (x) L-Met-OMe, DCC / HOBt;
(xi) ClCO2Et, Et3N, L-Met-OMe; (xii) DCC, Et3N.
Scheme 1. Synthesis of some triazolo[3,4-b]benzothiazol-3-ylthioacetyl amino acid methyl esters.

A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
505
S
S
N
N
N
N
v
N
NHNH2
N
N3
O
S
O
S
[14]
iv
vi
S
S
N
N
N
N
vii
S
N
OCH3
N
OH
N
N
O
O
O
S
S
S
N
[16]
viii
[13]
S
OCH3
N
H
O
[15]
S
S
iii
N
N
S
N
N
N
N
N
H
N
N
N
N
OCH3
S
SH
[3]
O
S
O
[12]
S
O
[17]
i
N
S
N
S
S
N
N
ii
N
S
N
N
viii
N
N
H
N
S
N
OCH3
OH
S
[9]
N
H
O
O
O
O
O
[11]
[10]
(i) a) 3-chloropropionic acid,NaOH, reflux in EtOH, b) HCl; (ii) L-Met-OMe, DCC / HOBt; (iii) methyl acrylate,Et3N,
reflux in MeOH;(iv) N2H4.H2O, reflux in EtOH; (v) NaNO2 / HCl, at - 5oC; (vi) HCl.L-Met-OMe,Et3N at 0oC;
(vii) a) NaOH, aq. EtOH, b) conc. HCl;(viii) DCC / Et3N
Scheme 2. Synthesis of some triazolo[3,4-b]benzothiazole propionyl derivatives.
8. The two compounds 7a and 8 were separated by preparative TLC
on silica gel and their ratio was found to be 1:2. The structure of the
N-acylurea 8 was confirmed by elemental analysis, IR, 13C NMR and
1H NMR spectrum in addition to its synthesis from the reaction of
the acid 6 with DCCI.
Formation of N-acylurea by-product was suppressed when the
reaction was carried out in the presence of 1-hydroxybenzotriazole
(HOBt) to afford solely the desired methionine methyl ester 7a in
54.1% yield. It is clear that, the azide method is more convenient
than the other two activating methods with its simplicity, purity of
the product and the relatively higher yield (59.7%).
Accordingly the synthesis of the remaining s-triazolo[3,4-b]
benzothiazol-3-ylthioacetyl amino acid methyl esters 7beh was
carried out by the azide method. The chemical structures of the
methyl esters 7aeh were confirmed by 1H NMR, 13C NMR and
elemental analyses. The 1H NMR spectra of these compounds
exhibited the following common data: signals of the protons of
CONH groups of the peptide bonds appeared in the range 8.66e
7.99 ppm, multiplet signals between 8.20 and 7.46 ppm are of the
four aromatic protons, and singlet signal from 3.52 to 3.72 ppm for
the three protons of OCH3 of the ester groups. Also 13C NMR spectra
exhibited the characteristic common signals: 171.7 and 167.6 ppm
for the two carbonyl carbons, the aromatic carbon signals appeared
between 132.0 and 114.5 ppm, NCH a-carbons of the amino acids
are found around 52.4 ppm, and the signal at 52.0 ppm attributed
to (OCH3), in addition to other signals corresponding to protons and
carbons of the individual side chains of the amino acids (see the
Experimental part).
To study the effect of a longer link between the amino acids and
the heterocycle on antifungal activity, the alkylation was carried
with b-chloropropionic acid under basic condition to afford b-(s-
triazolo[3,4-b]benzothiazole-3-thio)propionic acid (9) (Scheme 2).
The structure of the acid obtained 9 was assigned by elemental
analysis, 1H NMR and mass spectrum (see the Experimental part).
Trials to obtain ester derivative 12 by Michael addition of tri-
azolobenzothiazole derivative 3 to methyl acrylate failed to give the
desired S-derivative, but instead, the N-derivative 13 i.e. methyl b-
(3-thio-s-triazolo[3,4-b]benzothiazol-2-yl)propionate was ob-
tained. It is evident that 3 reacted in the thione form. The structure
of the methyl ester 13 was established by elemental analysis, 1H
NMR, 13C NMR and mass spectrum. 1H NMR spectrum showed the
characteristic singlet of the three protons of the methyl ester group

506
A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
Table 1
The antifungal effects of s-triazolo[3,4-b]benzothiazole derivatives
Compd.
Aspergillus flavus
Candida albicans
100ppm
300ppm
500ppm
1000ppm
100ppm
300ppm
500ppm
1000ppm
1
3
4
5
6
7a
7c
7d
7e
7g
7h
8
9
10
11
13
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þ
e
þ
e
e
e
e
e
e
e
e
e
þ
e
e
e
e
e
e
e
þ
þþ
þ
þþ
þþþ
e
þ
þ
e
þ
e
e
þ
e
e
e
e
þ
e
e
e
e
e
e
e
þ
þ
þ
þþ
þþþ
þ
þþþ
þþþþ
þþ
þþ
e
þþ
e
e
e
þ
þþ
þ
þþþ
þþ
e
þ
þ
e
e
e
e
þ
e
þ
þ
þþþ
þ
þþþþ
þþ
e
þþ
þ
e
e
e
þ
þþ
e
e
e
þ
þþ
e
þ
e
e
þ
þþ
þ
þþþþ
þþ
þ
þ
þþ
þþ
þ
þþþþ
þþþ
þþ
þ
e
e
e
þ
þ
þþ
þ
þþþ
þþþ
þ
e
þ
þ
14
15
16
17
e
e
e
e
e
þ
e
þ
þþ
þ
þþ
þþþ
þþþ
þ
þþ
þþþ
þþþ
þþþ
þþþþ
þþþ
þþ
þþþ
þþ
þþ
FluconazoleÒ
Zone diameter of growth inhibition: (e) inactive, (þ) <10 mm, (þþ) 10e15 mm, (þþþ) >16 mm.
at 3.73 ppm. The four protons of the two CH2 groups of the propi-
onate moiety appeared as two triplets at 4.62 and 2.96 ppm.
Comparing these values with those of the S-derivative of propionic
acid 9 (the two triplets at 3.37 ppm and 2.75 ppm) indicates the
formation of the N-derivative 13 and not the expected S-derivative
12 (Fig. 2). The high down field shift of the N-derivative is due to the
deshielding effect of the thiocarbonyl group. These values are
consistent with a previously published data on similar compounds
[14]. These structures were confirmed also, by 13C NMR spectra
where the spectrum of compound 13 showed the carbon signals at
52.0 ppm for methoxy carbon OCH3 of the ester group, in addition
to signals at 44.6 and 32.4 ppm attributable to the methylene
carbons of the propionyl group indicative for the formation of the
N-derivative 13 whereas the signals of the corresponding
methylene groups of the S-derivative 9 appeared at 34.5 and
29.3 ppm (Fig. 2).
Also, from 1H NMR of compound 13, it is evident that the
aromatic proton on C5 which is closer to the thione group appeared
at higher down field signal (9.05 ppm) than the remaining aromatic
protons (7.62e7.40 ppm) due also to the deshielding effect of thione
group (C]S). On the other hand, in case of the S-derivative 9, the
signal of the same proton (position 5) appeared at the normal ex-
pected value (8.13 ppm), which added further support, for the
presence of 13 in the N-form.
The thiopropionyl
coupling the acid derivative 9 with
L
-methionine methyl ester 10 was prepared by
-methionine methyl ester by the
L
DCCI activating method, in the presence of 1-hydroxybenzotriazole
to prevent the occurrence of the side reaction. The expected side-
product N-acylurea derivative 11 or 17 was prepared from the
reaction of the corresponding acid 9 or 16 with dicyclohex-
ylcarbodiimide to add further support to the formation of the
Table 2
The MIC and the MFC of the s-triazolo[3,4-b]benzothiazole derivatives compared
with 1 and Fluconazole.
desired ester 10 on reaction of 9 with L-methionine methyl ester/
DCCIeHOBt. 1H NMR spectrum of compound 10 showed the char-
acteristic signals of methionine protons: multiplet signals at 4.41e
Compd.
A. flavus
C. albicans
MIC
MFC
MIC
MFC
4.31 ppm for the a-CH, singlet at 3.22 ppm for the three protons
values (
mg/mL) values (
m
g/mL) values (
m
g/mL) values (mg/mL)
of OCH3 group, two multiplets at 2.48e2.41 ppm and 2.02e1.85 ppm
corresponding to SCH2CH2 and SCH2CH2 respectively, singlet signal
at 2.00 ppm for the three protons of SCH3. The signals of the four
protons of the thiopropionyl appeared as multiplet at 3.4e3.3 ppm
corresponding to the two protons of SCH2CH2CO and triplet at 2.70e
2.50 ppm for the other two protons SCH2CH2CO. Moreover, the four
aromatic protons appeared as multiplet signals at 8.10e7.49 region.
1
3
4
5
500
300
e
e
100
100
500
100
300
1000
100
500
500
500
1000
100
300
500
300
300
1000
500
300
100
100
500
500
e
500
e
e
e
500
e
6
1000
e
500
e
7a
7c
7d
7e
7g
7h
8
e
1000
1000
1000
e
e
e
e
e
e
e
The azide coupling method was used to prepare the N-propionyl L-
e
e
methionine derivative 15 from the hydrazide 14 for comparison
with the isoster S-derivative 10.1H NMR spectrum of this compound
showed the characteristic signals of methionine protons in addition
to the signals of the four protons of the propionyl moiety as ex-
pected at higher values than the corresponding protons of the S-
isomer 10 (see the Experimental part).
e
e
e
300
500
1000
1000
1000
e
500
e
e
9
e
10
11
13
14
15
16
17
e
e
e
e
e
e
e
e
1000
500
300
e
e
e
e
2.2. Biological evaluation
e
500
300
FluconazoleÒ 100
300
The features of the synthesized triazolobenzothiazole deriva-
tives are similar to antifungal azoles. Azoles inhibit ergosterol
MIC: minimum inhibitory concentration MFC: minimum fungicidal concentration
(e): denotes inactive.

A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
507
7.62
8.08
1112 instrument, and IR spectra were recorded on a Perking
Elmer 1430 ratio recording infrared spectrophotometer with CDS
data station using KBr Wafer technique at the Microanalytical
Laboratory, Faculty of Science, Suez Canal University, Ismailia,
Egypt. NMR spectra were measured on Bruker (200 MHz/300 MHz)
and TMS was used as internal standard, and Mass spectra were
measured on a GC-MSQP 1000EX Schimadzu at microanalytical
laboratory, Cairo University, Giza, Egypt.
S
S
N
N
3.73
OCH3
2.96
4.62
N
N
N
N
12.40
OH
2.75
1H NMR
S
S
S
9.05
[13]
8.13
O
3.37
[9]
O
S
N
N
52.0
OCH3
32.4
N
N
N
N
13C NMR
3.2. Chemical method
29.3
44.6
OH
O
S
S
34.5
3.2.1. Synthesis of 2-hydrazinobenzothiazole (2)
[9]
O
[13]
To a solution of MBT (10.0 mmol) in ethanol (30 mL), hydrazine
hydrate (5 mL, 80.0 mmol) was added. The reaction mixture was
refluxed for 20 h, after cooling to room temperature the precipitate
was filtered off, washed with cold ethanol, and ether followed by
recrystallization from ethanol. Colorless crystals (1.23 g, 73.5%
yield), m.p. 198e200 ꢀC, lit mp 199e200 ꢀC [15].
Fig. 2. 1H NMR and 13C NMR shifts of compounds 9 and 13.
biosynthesis in the fungal cell membranes at the C-14 demethyla-
tion step. The basic nitrogen of the azole ring forms a tight bond
with the heme iron of the fungal cytochrome P450 14a-demethy-
lase preventing substrate and oxygen binding [1]. From the recor-
ded data (Tables 1 and 2), it is clear that the starting compound 3-
mercaptotriazolobenzothiazole 3 had similar antifungal activity
3.2.2. Synthesis of s-triazolo[3,4-b]benzothiazole-3-thiol (3)
To a suspension of hydrazine derivative 2 (10.0 mmol) in
ethanol (40 mL), a solution of potassium hydroxide (0.56 g,
10 mmol) in water (5 mL) was added. After addition of carbon
disulfide (3.8 mL, 50 mmol) the reaction mixture was refluxed
gently on water bath at 70 ꢀC for 7 h. Afterward, the solvent was
evaporated under reduced pressure to complete dryness. When the
cold aqueous solution of the obtained solid residue was acidified
with conc. HCl to pH w5, the formed precipitate was filtered,
washed with cold water several times, and crystallized from
ethanol to yield yellow crystals (1.90 g, 91.0% yield), m.p. 251e
253 ꢀC, lit mp 255 ꢀC [13].
(100
From structureeactivity relationship of the amino acids deriv-
atives 7a, 7ceh, the tyrosine methyl ester derivative 7c (100 g/mL)
mg/mL) against C. albicans as compared with fluconazole.
m
had equivalent activity to fluconazole. At the same time, activity of
7c was from 5 to 10 fold more efficient than the other studied
aliphatic amino acid ester derivatives. This result is in agreement
with published data on the activity of benzothiazolyl tyrosine
methyl ester (100
mg/mL) toward the same fungi [5]. The high
activity of the tyrosine derivatives probably could be attributed to
the high lipophilicity of the six-membered hydrocarbon ring in
their side-chains, facilitating their transport through the lipophilic
regions of the fungal membrane.
Moreover, the high antifungal activity of the tested N-acyl
dicyclohexylurea derivatives 8, 11 and 17 adds further support for
the importance of the presence of the hydrocarbon six-membered
ring in their structures. The acyl urea derivatives 8 and 17 had
3.2.3. Synthesis and characterization of ethyl s-triazolo[3,4-b]
benzothiazol-3-ylthioacetate (4)
To a solution of s-triazolo[3,4-b]benzothiazole-3-thiol (3) (1.0 g,
4.8 mmol) in dry acetone (20 mL), anhydrous potassium carbonate
(0.47 g, 4.8 mmol) and ethyl chloroacetate (0.59 g, 4.8 mmol) were
added. The reaction mixture was refluxed for 9 h, filtered while hot,
afterward the solvent was evaporated to dryness and the oily
residue was cooled in refrigerator overnight. The resulting solid
was crystallized from ethanol to afford colorless crystals (0.91 g,
similar activity (100
mg/mL) to fluconazole, whereas compound 11
was three times weaker (300
mg/mL). These results suggest that
such types of compounds are worthy of more detailed study on
different aromatic amino acid ester derivatives and other N-acyl
dialkyl urea derivatives to clarify the role of lipophilicity on bio-
logical activity.
It is noteworthy to mention that most of the intermediates; 2-N
and 3-S-carboxyalkyl derivatives 5, 6, 9, 13 and 16 exhibited higher
78.9% yield), m.p. 90e93 ꢀC. 1H NMR (300 MHz, CDCl3):
d 8.19 (1H,
d, J ¼ 8.1 Hz, ArH), 7.75 (1H, d, J ¼ 8.1 Hz, ArH), 7.55e7.40 (2H, m,
ArH), 4.17e4.14 (4H, m, OCH2, SCH2), 1.23e1.18 (3H, t, J ¼ 7.2 Hz,
CH3). 13C NMR (75 MHz, CDCl3):
d 167.9 (CO), 156.6, 142.4 (2SCN),
132.0, 129.6, 127.0, 126.1, 123.8, 114.5 (AreC), 62.1 (OCH2), 37.0
antifungal activities (100e300 mg/mL) when compared with the
(SCH2), 13.9 (CH3). IR (KBr,
n
/cmꢁ1) 1735 (C]O, ester). Anal. Calcd.
amino acid ester derivatives. This can be explained on the basis of
the polarity of these short S- and N-alkyl groups, which plays
important role in penetration of these compounds through
hydrophilic regions of the biological membranes.
for C12H11N3O2S2 (293.36): C, 49.13; H, 3.78; N, 14.32; found C,
49.50; H, 3.70; N, 14.63.
3.2.4. Synthesis and characterization s-triazolo[3,4-b]benzothiazol-
3-ylthioacetyl hydrazide (5)
3. Experimental
To a solution of 4 (2.93 g, 10.0 mmol) in ethanol (20 mL),
hydrazine hydrate (1.8 mL, 30.0 mmol) was added. The reaction
mixture was refluxed for 1 h, after cooling to room temperature the
precipitate was filtered off, washed with cold water, and ethanol
followed by recrystallization from aqueous ethanol. Colorless
crystals (2.48 g, 88.7% yield), m.p. 198e200 ꢀC. 1H NMR (300 MHz,
3.1. Materials and reagents
2-Mercaptobenzothiazole (MBT) was purchased from Fluka. The
boiling point range of the petroleum ether used was 40e60 ꢀC. Thin
layer chromatography (TLC) was carried out on silica gel 60 F254
plastic plates (E. Merck, layer thickness 0.2 mm) in the following
solvent systems, S1: chloroform/methanol (95:5); S2: chloroform/
methanol (90:10); S3: ethyl acetate/petroleum ether (2:1). The
spots on thin layer plates were detected by UV lamp. Melting points
were determined on a Buchi 510 melting-point apparatus and were
uncorrected. Elemental analyses were performed on a Flash EA-
DMSO):
J ¼ 8.4 Hz, ArH), 7.64e7.49 (2H, m, ArH), 4.23 (2H, s, NH2), 3.87 (2H,
s, SCH2). 13C NMR (75 MHz, DMSO):
166.3 (CO), 156.6, 142.8
(2SCN), 131.8, 129.9, 127.4, 126.8, 125.9, 114.8 (AreC), 36.2 (SCH2). IR
d
9.28 (1H, s, NH), 8.18 (1H, d, J ¼ 8.4 Hz, ArH), 8.09 (1H, d,
d
(KBr, n
/cmꢁ1) 3280 (NH2, NH), 1651 (C]O, amide). MS (m/z): 279
[M]þ. Anal. Calcd. for C10H9N5OS2 (279.34): C, 43.00; H, 3.25; N,
25.07; found C, 43.12; H, 3.24; N, 25.36.

508
A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
b- By mixed anhydride method:
3.2.5. Synthesis and characterization of s-triazolo[3,4-b]
benzothiazol-3-ylthioacetic acid (6)
To a cold solution of s-triazolo[3,4-b]benzothiazole-3-thioacetic
acid (6) (0.58 g, 2.2 mmol) and triethyl amine (0.31 mL, 2.2 mmol)
in toluene (30 mL) at ꢁ5 ꢀC, ethyl chloroformate (0.21 mL,
(a) By alkylation of 3 with chloroacetic acid:
2.2 mmol) was added. After 25 min a cooled solution (0 ꢀC) of
L-
To the solution of s-triazolo[3,4-b]benzothiazole-3-thiol (3)
(1.0 g, 4.8 mmol) in aqueous methanol (20 mL), sodium hydroxide
(0.38 g, 4.8 mmol) and chloroacetic acid (0.45 g, 4.8 mmol) were
added. The reaction mixture was refluxed for 8 h, cooled, filtered,
and acidified with conc. HCl. The formed precipitate was filtered off,
washed with cold water, and recrystallized from aqueous ethanol
(1.1 g, 85.0% yield), m.p. 260e263 ꢀC. 1H NMR (300 MHz, DMSO):
methionine methyl ester hydrochloride (0.43 g, 2.2 mmol) and
triethyl amine (0.31 mL, 2.2 mmol) in toluene (15 mL) was added to
the solution of the activated acid 6. After stirring at ꢁ5 ꢀC for 2 h,
the reaction mixture was kept aside at room temperature overnight
under stirring. The precipitated triethyl amine hydrochloride was
filtered off and the filtrate was washed with 5% NaHCO3 in 3% NaCl
and water, and then dried over anhydrous Na2SO4. The solvent was
evaporated and the remaining residue was crystallized from ethyl
acetateepetroleum ether to give the s-triazolo[3,4-b]benzothia-
d
13.20e12.51 (H, brs, CO2H), 8.13 (1H, d, J ¼ 8.1 Hz, ArH), 8.08 (1H,
d, J ¼ 8.1 Hz, ArH), 7.63e7.48 (2H, m, ArH), 4.03 (2H, s, SCH2). 13
C
NMR (75 MHz, DMSO):
d 169.1 (CO), 155.9, 142.4 (2SCN), 131.3,
129.3, 126.9, 126.3, 125.4, 114.2 (AreC), 35.8 (SCH2). IR (KBr, n )
/cmꢁ1
zole-3-thioacetyl -methionine methyl ester (7a), colorless crystals
L
(0.21 g, 43.7% yield), Rf ¼ 0.65 (S1), m.p. 98e100 ꢀC.
3500e2600 (OH), 1725 (C]O, acid). Anal. Calcd. for C10H7N3O2S2
(265.31): C, 45.27; H, 2.66; N, 15.84; found: C, 45.38; H, 2.91; N,
15.89.
c - By DCCI method:
(a) To a cold solution (ꢁ5 ꢀC) of
L-methionine methyl ester
(b) By alkaline hydrolysis of 4:
hydrochloride (0.86 g, 4.4 mmol) and triethyl amine (0.62 mL,
4.4 mmol) in acetonitrile (40 mL) s-triazolo[3,4-b]benzothiazole-3-
thioacetic acid (6) (1.16 g, 4.4 mmol) and dicyclohexylcarbodiimide
(0.92 g, 4.4 mmol) were added successively. The solution was stir-
red at 0 ꢀC for 2 h and at room temperature overnight. The
precipitated dicyclohexylurea (DCU) was filtered off and the filtrate
was evaporated under reduced pressure. The residue was extracted
with ethyl acetate, filtered off from the insoluble additional residual
DCU and the filtrate was washed successively with saturated NaCl
solution, 5% NaHCO3 solution, 1 M HCl and water. After dying over
anhydrous Na2SO4, the solvent was evaporated and the remaining
residue was crystallized from ethyl acetateepetroleum ether. TLC of
the product showed the presence of two compounds which were
separated by preparative TLC using solvent system (S1) to afford 7a
(0.44 g), Rf ¼ 0.65 (S1) identical with an authentic sample prepared
by the azide method and 1-(2-(s-triazolo[3,4-b]benzothiazol-3-
ylthio)acetyl)-1,3-dicyclohexylurea (8), Rf ¼ 0.84 (S1) colorless
The ester 4 (0.2 g, 0.7 mmol) was refluxed with potassium
hydroxide (0.04 g, 0.7 mmol) in aqueous ethanol (10 mL) for 5 h.
Afterward the reaction mixture was cooled, filtered, and acidified
with conc. HCl. The formed precipitate was filtered off, washed with
cold water, and recrystallized from aqueous ethanol (0.17 g, 86.6%
yield), m.p. 261e263 ꢀC.
3.2.6. Synthesis of s-triazolo[3,4-b]benzothiazol-3-ylthioacetyl L-
methionine methyl ester (7a)
a- By the azide method:
To a cold solution (ꢁ5 ꢀC) of hydrazide 5 (0.21 g, 0.8 mmol) in
acetic acid (6 mL), 5 N HCl (3 mL), and water (25 mL), was added
portion wise under stirring a cold solution (0 ꢀC) of sodium nitrite
(0.07 g, 1.0 mmol) in water (3 mL). After stirring at the same
temperature for 30 min, the azide was extracted with cold ethyl
acetate, and washed successively with cold water, 5% NaHCO3 and
water. After drying over anhydrous sodium sulfate, the azide was
used without further purification in the next step.
crystals (0.95 g), m.p. 182e184 ꢀC. 1H NMR (300 MHz, CDCl3):
d 8.02
(1H, d, J ¼ 7.8 Hz, ArH), 7.72 (1H, d, J ¼ 7.8 Hz, ArH), 7.54e7.46 (2H,
m, ArH), 3.93 (2H, s, SCH2), 3.90e3.65 (1H, m, NCH), 3.53e3.46 (1H,
m, NCH), 1.97e1.05 (20H, m, 10 CH2 cyclohexyl rings). 13C NMR
(75 MHz, CDCl3):
d 164.2, 156.4 (2CO), 153.3, 143.6 (2SCN), 131.9,
After stirring the reaction mixture of L-methionine methyl ester
129.4, 126.8, 126.5, 124.5, 114.6 (AreC), 55.5, 50.4 (2CH- cyclohexyl
hydrochlorides (0.17 g, 0.9 mmol), triethyl amine (0.2 mL) in ethyl
acetate (30 mL) at 0 ꢀC for 20 min, and the formed triethyl amine
hydrochloride was filtered off. The previously prepared cold dried
solution of the azide was added to the cold ethyl acetate solution of
rings), 35.2 (SCH2), 32.2, 30.5, 25.9, 25.3, 25.3, 24.7 (CH2-cyclohexyl
rings). IR (KBr, n
/cmꢁ1) 3280 (NH), 1690 (C]O, amide), 1615 (C]O,
urea). Anal. Calcd. for C23H29N5O2S2 (471.64): C, 58.57; H, 6.20; N,
14.85; found: C, 58.66; H, 6.21; N, 14.70.
the L-methionine methyl ester. Afterward the mixture was kept
(b) By DCCI/HOBt: to a cold solution (ꢁ5 ꢀC) of s-triazolo[3,4-b]
benzothiazole-3-thioacetic acid (6) (1.16 g, 4.4 mmol) in acetoni-
trile (40 mL) containing dicyclohexyl-carbodiimide (0.92 g,
12 h in the refrigerator and then at room temperature for additional
12 h. The reaction mixture was washed with 0.1 N HCl, water, 5%
NaHCO3 and water then dried over anhydrous sodium sulfate. The
solvent was evaporated in vacuum and the residue was crystallized
from ethyl acetateepetroleum ether to give the s-triazolo[3,4-b]
4.4 mmol), 1-hydroxybenzotriazole (0.6 g, 4.4 mmol), L-methionine
methyl ester hydrochloride (0.86 g, 4.4 mmol), and triethyl amine
(0.62 mL, 4.4 mmol) were added successively. The reaction mixture
was treated as mentioned above in (a) to afford 7a as a sole product
as colorless crystals (0.52 g, 54.1% yield), Rf ¼ 0.65 (S1), m.p. 99e
101 ꢀC.
benzothiazole-3-thioacetyl
colorless crystals (0.19 g, 59.7% yield), Rf ¼ 0.65 (S1), m.p. 99e
102 ꢀC. 1H NMR (300 MHz, CDCl3):
L-methionine methyl ester (7a) as
d
8.19 (1H, d, J ¼ 7.8 Hz, NH),
8.00 (1H, d, J ¼ 7.8 Hz, ArH), 7.73 (1H, d, J ¼ 7.8 Hz, ArH), 7.54e7.46
(2H, m, ArH), 4.71e4.63 (1H, m, NCH), 4.06 (2H, s, SCH2), 3.72 (3H, s,
OCH3), 2.53 (2H, t, J ¼ 7.4, SCH2), 2.21e2.02 (2H, m, CH2), 2.05 (3H, s,
3.2.7. General method for the synthesis of s-triazolo[3,4-b]
SCH3). 13C NMR (75 MHz, CDCl3):
d 171.7, 167.6 (2CO), 156.6, 143.8
benzothiazole-3-ylthioacetyl amino acid methyl esters (7beh)
To a cold solution (ꢁ5 ꢀC) of hydrazide 5 (0.8 mmol) in acetic
acid (6 mL), 5 N HCl (3 mL), and water (25 mL), was added portion
wise under stirring a cold solution (0 ꢀC) of sodium nitrite (0.07 g,
1.0 mmol) in water (3 mL). After stirring at the same temperature
for 30 min, the azide was extracted with cold ethyl acetate, and
(2SCN), 132.0, 129.4, 126.8, 126.5, 124.6, 114.5 (AreC), 52.4 (NCH),
52.0 (OCH3), 36.1 (SCH2), 31.3 (SCH2), 29.9 (CH2), 15.4 (SCH3). IR
(KBr, n
/cmꢁ1) 3390 (NH), 1730 (C]O, ester), 1660 (C]O, amide).
Anal. Calcd. for C16H18N4O3S3 (410.53): C, 46.81; H, 4.42; N, 13.65;
found C, 46.89; H, 4.60; N, 13.54.

A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
509
washed successively with cold water, 5% NaHCO3 and water. After
drying over anhydrous sodium sulfate, the azide was used without
further purification in the next step. The azide was mixed with the
amino acid methyl ester hydrochlorides (0.9 mmol) and triethyl
amine (0.2 mL, 0.9 mmol), afterward the reaction mixture was
treated as mentioned above in the preparation of 7a by the azide
method to afford the corresponding s-triazolo[3,4-b]benzothia-
zole-3-thioacetyl amino acid methyl esters (7beh).
CH), 1.00 (6H, d, J ¼ 6.2 Hz, 2CH3). 13C NMR (75 MHz, CDCl3):
d
171.7,
167.8 (2CO), 156.6 (SCN), 132.0, 129.4, 127.0, 126.8, 126.5, 124.6,
114.5 (AreC), 58.0 (OCH3), 52.0 (NCH), 36.2 (SCH2), 30.8 (CH), 19.0,
17.7 (2CH3). IR (KBr, n
/cmꢁ1) 3290 (NH), 1715 (C]O, ester), 1650
(C]O, amide). Anal. Calcd. for C16H18N4O3S2 (378.47): C, 50.78; H,
4.79; N, 14.80; found C, 50.78; H, 4.76; N, 14.92.
3.2.7.6. s-Triazolo[3,4-b]benzothiazol-3-ylthioacetyl
ester (7g). Colorless crystals (0.17 g, 55.6% yield), Rf ¼ 0.63 (S1), m.p.
121e124 ꢀC. 1H NMR (200 MHz, CDCl3):
7.99 (2H, m, NH, ArH),
L-leucine methyl
3.2.7.1. s-Triazolo[3,4-b]benzothiazol-3-ylthioacetyl
ester (7b). Colorless crystals (0.14 g, 49.5% yield), Rf ¼ 0.21 (S1), m.p.
155e157 ꢀC. 1H NMR (300 MHz, DMSO):
L
-serine methyl
d
7.68e7.40 (3H, m, ArH), 4.52 (1H, m, NCH), 4.10 (2H, s, SCH2), 3.65
d
8.58 (1H, d, J ¼ 7.8 Hz,
(3H, s, OCH3), 1.60 (3H, m, CH, CH2), 1.00 (6H, m, 2CH3). 13C NMR
NH), 8.20 (1H, d, J ¼ 8.4 Hz, ArH), 8.08 (1H, d, J ¼ 8.4 Hz, ArH), 7.62e
7.51 (2H, m, ArH), 5.05e5.00 (1H, m, NCH), 4.29e4.23 (1H, m,
CHOH), 3.99 (2H, s, SCH2), 3.67e3.48 (1H, m, CHOH), 3.58 (3H, s,
(75 MHz, CDCl3):
129.4,127.0,126.5,124.5,114.6 (AreC), 52.3 (OCH3), 51.4 (NCH), 40.9
(SCH2), 36.3 (CH2), 24.8 (CH), 22.7 (CH3), 21.7 (CH3). IR (KBr,
/cmꢁ1
d 172.7, 167.6 (2CO), 156.5, 143.9 (2SCN), 132.0,
n
)
OCH3). 13C NMR (75 MHz, DMSO):
d
171.0, 167.4 (2CO), 156.7, 142.7
(2SCN), 131.8, 130.0, 127.3, 126.8, 125.8, 114.9 (AreC), 61.5 (OCH2),
55.3 (NCH), 52.3 (OCH3), 37.9 (SCH2). IR (KBr,
/cmꢁ1) 3470 (OH,
3310 (NH), 1710 (C]O, ester), 1665 (C]O, amide). Anal. Calcd. for
C17H20N4O3S2 (392.5): C, 52.02; H, 5.14; N, 14.27; found C, 51.65; H,
4.97; N, 13.75.
n
NH), 1725 (C]O, ester), 1650 (C]O, amide). Anal. Calcd. for
C14H14N4O4S2 (366.42): C, 45.89; H, 3.85; N, 15.29; found C, 45.96;
H, 3.79; N, 15.41.
3.2.8. s-Triazolo[3,4-b]benzothiazol-3-ylthioacetyl b-alanine
methyl ester (7h)
Colorless crystals (0.18 g, 61.7% yield), Rf ¼ 0.56 (S1), m.p. 156e
158 ꢀC. 1H NMR (200 MHz, CDCl3):
8.04 (2H, m, NH, ArH), 7.71e
7.50 (3H, m, ArH), 4.13e4.02 (2H, m, SCH2), 3.68 (3H, s, OCH3), 2.16
(2H, m, NCH2), 1.40 (2H, m, CH2). IR (KBr,
/cmꢁ1) 3485 (NH), 1735
3.2.7.2. s-Triazolo[3,4-b]benzothiazol-3-ylthioacetyl-
methyl ester (7c). Colorless crystals (0.17 g, 47.2% yield), Rf ¼ 0.25
(S1), m.p. 208e210 ꢀC. 1H NMR (300 MHz, DMSO):
9.19 (1H, s, OH),
L
-tyrosine
d
d
n
8.63 (1H, d, J ¼ 7.8 Hz, NH), 8.16 (1H, d, J ¼ 8.1 Hz, ArH), 8.09 (1H, d,
J ¼ 8.1 Hz, ArH), 7.60e7.49 (2H, m, ArH), 6.92 (2H, d, J ¼ 8.4 Hz, ArH),
6.64 (2H, d, J ¼ 8.4 Hz, ArH), 4.40e4.31 (1H, m, NCH), 3.94 (2H, s,
SCH2), 3.52 (3H, s, OCH3), 2.85e2.70 (2H, m, CH2Ph). 13C NMR
(C]O, ester), 1650 (C]O, amide). Anal. Calcd. for C14H14N4O3S2
(350.42): C, 47.99; H, 4.03; N,15.99; found C, 47.75; H, 3.97; N,16.18.
3.2.9. Synthesis of b-(s-triazolo[3,4-b]benzothiazol-3-ylthio)
(75 MHz, DMSO):
130.4, 130.0, 127.4, 127.1, 126.8, 125.9, 115.5, 114.9 (AreC), 54.6
(NCH), 52.2 (OCH3), 37.8 (SCH2), 36.5 (CH2). IR (KBr,
/cmꢁ1) 3490
d
171.9, 167.2 (2CO), 156.4, 142.7 (2SCN), 131.8,
propionic acid (9)
To the solution of s-triazolo[3,4-b]benzothiazole-3-thiol (3)
(1.0 g, 4.8 mmol) in aqueous ethanol (20 mL), sodium hydroxide
n
(OH, NH), 1720 (C]O, ester), 1610 (C]O, amide). Anal. Calcd. for
C20H18N4O4S2 (442.51): C, 54.28; H, 4.10; N, 12.66; found C, 54.21;
H, 4.28; N, 12.41.
(0.38 g, 4.8 mmol) and b-chloropropionic acid (0.52 g, 4.8 mmol)
were added. The reaction mixture was refluxed for 12 h, cooled,
filtered, and acidified with conc. HCl. The formed precipitate was
filtered off, washed with cold water, and recrystallization from
aqueous ethanol to yield 9 as white amorphous powder (1.10 g,
79.2% yield), Rf ¼ 0.12 (S1), m.p. 209e211 ꢀC. 1H NMR (300 MHz,
3.2.7.3. s-Triazolo[3,4-b]benzothiazol-3-ylthioacetyl
methyl ester (7d). Colorless crystals (0.16 g, 53.4% yield), Rf ¼ 0.29
(S1), m.p. 171e174 ꢀC. 1H NMR (300 MHz, DMSO):
8.40 (1H, d,
DL-threonine
d
DMSO): d 12.40 (1H, brs, OH), 8.13e8.05 (2H, m, ArH), 7.62e7.51
J ¼ 8.4 Hz, NH), 8.20 (1H, d, J ¼ 7.5 Hz, ArH), 8.07 (1H, d, J ¼ 7.5 Hz,
ArH), 7.61e7.48 (2H, m, ArH), 5.03e4.97 (1H, m, NCH), 4.22e4.18
(1H, m, CH), 4.11e3.98 (2H, m, SCH2), 3.58 (3H, s, OCH3), 1.10 (3H,
(2H, m, ArH), 3.39e3.35 (2H, t, J ¼ 6.9 Hz, SCH2), 2.77e2.72 (2H,
t, J ¼ 6.6 Hz, CH2CO). 13C NMR (75 MHz, DMSO):
d 172.9 (CO), 156.6,
143.1 (2SCN), 131.9, 129.9, 127.4, 126.7, 125.9, 114.5 (AreC), 34.5
d, J ¼ 6.3 Hz, CH3). 13C NMR (75 MHz, DMSO):
d
171.1, 167.9 (2CO),
(NCH2), 29.3 (CH2). IR (KBr, n
/cmꢁ1) 3500e2600 (OH), 1735 (C]O,
156.7, 142.7 (2SCN), 131.8, 130.0, 127.3, 126.8, 125.8, 114.9 (AreC),
acid). MS (m/z): 279 [M]þ. Anal. Calcd. for C11H9N3O2S2 (279.34): C,
66.6 (OCH), 58.4 (NCH), 52.2 (OCH3), 37.8 (SCH2), 20.3 (CH3). IR
(KBr,
47.30; H, 3.25; N, 15.04; found C, 47.64; H, 3.30; N, 14.97.
n
/cmꢁ1) 3450 (OH, NH), 1730 (C]O, ester), 1645 (C]O,
amide). Anal. Calcd. for C15H16N4O4S2 (380.44): C, 47.36; H, 4.24; N,
14.73; found C, 47.61; H, 4.23; N, 14.78.
3.2.10. Synthesis of b-(s-triazolo[3,4-b]benzothiazol-3-ylthio)
propionyl -methionine methyl ester (10)
L
To a cold solution (ꢁ5 ꢀC) of
L-methionine methyl ester hydro-
chloride (0.86 g, 4.4 mmol) and triethyl amine (0.62 mL, 4.4 mmol)
3.2.7.4. s-Triazolo[3,4-b]benzothiazol-3-ylthioacetyl glycine methyl
ester (7e). Colorless crystals (0.18 g, 69.4% yield), Rf ¼ 0.51 (S1), m.p.
in acetonitrile (40 mL) b-(s-triazolo[3,4-b]benzothiazole-3-thio)
196e199 ꢀC. 1H NMR (200 MHz, DMSO):
d
8.66 (1H, m, NH), 8.18
propionic acid (9) (1.2 g, 4.4 mmol), 1-hydroxybenzotriazole (0.6 g,
4.4 mmol), and dicyclohexylcarbodiimide (0.92 g, 4.4 mmol) was
added successively. The solution was stirred at 0 ꢀC for 2 h and at
room temperature overnight. The precipitated dicyclohexylurea
(DCU) was filtered off and the filtrate was evaporated under
reduced pressure. The residue was extracted with ethyl acetate,
filtered off from the insoluble additional residual DCU and the
filtrate was washed successively with saturated NaCl solution, 5%
NaHCO3 solution, 1 M HCl and water. After dying over anhydrous
Na2SO4, the solvent was evaporated and the remaining residue was
crystallized from ethyl acetateepetroleum ether to yield 10 as
colorless crystals (0.86 g, 47.3% yield), Rf ¼ 0.58 (S1), m.p. 118e
(1H, d, J ¼ 7.0 Hz, ArH), 8.09 (1H, d, J ¼ 7.0 Hz, ArH), 7.60e7.52 (2H,
m, ArH), 4.00 (2H, s, NCH2), 3.81 (2H, m, SCH2), 3.58 (3H, s, OCH3).
13C NMR (75 MHz, DMSO):
d
170.3, 167.7 (2CO), 156.5, 142.8 (2SCN),
131.8, 130.0, 127.4, 126.8, 125.9, 114.9 (AreC), 52.1 (OCH3), 41.3
(NCH2), 37.6 (SCH2). IR (KBr,
/cmꢁ1) 3495 (NH), 1750 (C]O, ester),
n
1660 (C]O, amide). Anal. Calcd. for C13H12N4O3S2 (336.39): C,
46.42; H, 3.60; N, 16.66; found C, 46.12; H, 3.53; N, 16.82.
3.2.7.5. s-Triazolo[3,4-b]benzothiazol-3-ylthioacetyl
ester (7f). Colorless crystals (0.14 g, 49.1% yield), Rf ¼ 0.62 (S1), m.p.
141e143 ꢀC. 1H NMR (200 MHz, CDCl3):
8.13 (1H, m, NH), 8.01 (1H,
L-valine methyl
d
d, J ¼ 7.4 Hz, ArH), 7.73 (1H, d, J ¼ 7.4 Hz, ArH), 7.49 (2H, m, ArH),
121 ꢀC. 1H NMR (200 MHz, DMSO):
8.10e8.03 (2H, m, ArH), 7.59e7.49 (2H, m, ArH), 4.41e4.31 (1H, m,
d
8.40 (1H, d, J ¼ 7.4 Hz, NH),
4.46 (1H, m, NCH), 4.13 (2H, s, SCH2), 3.66 (3H, s, OCH3), 2.16 (1H, m,

510
A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
NCH), 3.62 (3H, s, OCH3), 3.44e3.33 (2H, m, SCH2), 2.70e2.50 (2H, t,
J ¼ 6.4 Hz, CH2CO), 2.48e2.41 (2H, m, SCH2), 2.02e1.85 (2H, m,
under stirring portion wise a cold solution (0 ꢀC) of sodium nitrite
(0.07 g, 1.0 mmol) in water (3 mL). After stirring at the same
temperature for 30 min, the azide was extracted with cold ethyl
acetate, and washed successively with cold water, 5% NaHCO3 and
water. After drying over anhydrous sodium sulfate, the azide was
used without further purification in the next step.
CH2), 2.00 (3H, s, SCH3). IR (KBr, n
/cmꢁ1) 3350 (NH), 1730 (C]O,
ester), 1655 (C]O, amide). Anal. Calcd. for C17H20N4O3S3 (424.56):
C, 48.09; H, 4.75; N, 13.20; found C, 48.51; H, 4.90; N, 12.96.
3.2.11. Synthesis of 1-(2-(s-triazolo[3,4-b]benzothiazol-3-ylthio)
After stirring the reaction mixture of L-methionine methyl ester
propionyl)-1,3-dicyclohexylurea (11)
hydrochloride (0.9 mmol), triethyl amine (0.2 mL) in ethyl acetate
(30 mL) at 0 ꢀC for 20 min, and the formed triethyl amine hydro-
chloride was filtered off. The previously prepared cold dried solu-
tion of the azide was added to the cold ethyl acetate solution of the
amino acid ester. Afterward the mixture was kept 12 h in the
refrigerator and then at room temperature for additional 12 h. The
reaction mixture was washed with 0.1 N HCl, water, 5% NaHCO3 and
water then dried over anhydrous sodium sulfate. The solvent was
evaporated in vacuum and the residue was crystallized from ethyl
acetateepetroleum ether to give 15 as colorless crystals (0.15 g,
44.7% yield), Rf ¼ 0.73 (S1), m.p. 165e167 ꢀC. 1H NMR (300 MHz,
A mixture of b-(s-triazolo[3,4-b]benzothiazole-3-thio)propionic
acid (9) (1.2 g, 4.4 mmol), dicyclohexylcarbodiimide (0.92 g,
4.4 mmol) and triethyl amine (0.62 mL, 4.4 mmol) in acetonitrile
(40 mL) was stirred at 0 ꢀC for 2 h and at room temperature over-
night. The solution evaporated under reduced pressure. The residue
was extracted with ethyl acetate, filtered off from the insoluble
additional residual DCU and the filtrate was washed successively
with saturated NaCl solution, 5% NaHCO3 solution, 1 M HCl and
water. After dying over anhydrous Na2SO4, the solvent was evap-
orated and the remaining residue was crystallized from ethanol to
yield 11 as colorless crystals (1.84 g, 88.2% yield), Rf ¼ 0.84 (S1), m.p.
CDCl3):
d
9.07 (1H, d, J ¼ 7.8 Hz, ArH), 7.64 (1H, d, J ¼ 7.8 Hz, ArH),
175e178 ꢀC. 1H NMR (300 MHz, CDCl3):
d
9.04 (1H, d, J ¼ 6.9 Hz,
7.51e7.27 (2H, m, ArH), 6.50 (1H, d, J ¼ 7.4 Hz, NH), 4.80e4.71 (1H,
m, NCH), 4.68e4.63 (2H, t, J ¼ 6.9 Hz, NCH2), 3.70 (3H, s, OCH3),
2.93e2.88 (2H, t, J ¼ 6.9 Hz, CH2CO), 2.49e2.46 (2H, m, SCH2),
2.02e1.92 (2H, m, CH2), 2.06 (3H, s, SCH3). 13C NMR (75 MHz,
ArH), 7.66 (1H, d, J ¼ 6.9 Hz, ArH), 7.54e7.42 (2H, m, ArH), 4.66e
4.64 (2H, m, SCH2), 3.92 (1H, m, NCH), 3.69 (1H, m, NCH), 3.10e
3.05 (2H, t, J ¼ 6.9 Hz, CH2CO), 1.99e1.14 (20H, m, 10 CH2 cyclo-
hexyl rings). IR (KBr,
n
/cmꢁ1) 3210 (NH), 1670 (C]O, amide), 1620
CDCl3):
(AreC), 52.4 (NCH), 51.6 (OCH3), 45.1 (NCH2), 34.5, 31.5, 29.9
(3CH2), 15.4 (SCH3). IR (KBr,
/cmꢁ1) 3210 (NH), 1710 (C]O, ester),
d 172.1, 169.2 (2CO), 131.7, 129.5, 126.8, 126.7, 123.4, 115.6
(C]O, urea). MS (m/z): 485 [M]þ. Anal. Calcd. for C24H31N5O2S2
(485.67): C, 59.35; H, 6.43; N, 14.42; found C, 59.60; H, 6.01; N,
14.55.
n
1670 (C]O, amide). Anal. Calcd. for C17H20N4O3S3 (424.56): C,
48.09; H, 4.75; N, 13.20; found C, 48.23; H, 4.74; N, 13.34.
3.2.12. Synthesis of methyl b-(3-thioxo-s-triazolo[3,4-b]
benzothiazol-2-yl)propionate (13)
3.2.15. Synthesis of b-(3-thioxo-s-triazolo[3,4-b]benzothiazol-2-yl)
To a solution of s-triazolo[3,4-b]benzothiazole-3-thiol (3) (1.0 g,
4.8 mmol) in methanol (20 mL), methyl acrylate (0.5 mL) and triethyl
amine (0.7 mL) were added. The reaction mixture was refluxed for
6 h, concentrated and cooled. The resulting precipitate was filtered
and crystallized from methanol to afford 13 as colorless crystals
(1.18 g, 83.6% yield) mp 109e112 ꢀC. 1H NMR (300 MHz, CDCl3):
propionic acid (16)
The ester 13 (0.5 g, 1.7 mmol) was refluxed with potassium
hydroxide (0.10 g, 1.7 mmol) in aqueous methanol (10 mL) for 8 h.
Afterward the reaction mixture was cooled, filtered, and acidified
with conc. HCl. The formed precipitate was filtered off, washed with
cold water, and recrystallized from aqueous ethanol. Colorless
crystals (0.31 g, 65.95% yield), Rf ¼ 0.77 (S1), m.p. 172e174 ꢀC. 1H
d
9.05 (1H, d, J ¼ 8.1 Hz, ArH), 7.62 (1H, d, J ¼ 8.1 Hz, ArH), 7.54e7.41
(2H, m, ArH), 4.65e4.60 (2H, t, J ¼ 6.8 Hz, NCH2), 3.73 (3H, s, OCH3),
NMR (300 MHz, DMSO): d 12.53 (1H, brs, OH), 8.89 (1H, d,
2.99e2.94 (2H, t, J ¼ 6.8 Hz, CH2). 13C NMR (75 MHz, CDCl3):
d
170.9
J ¼ 8.4 Hz, ArH), 8.07 (1H, d, J ¼ 8.4 Hz, ArH), 7.60e7.48 (2H, m,
(CO),161.2 (SCN),131.7,129.5,126.8,123.4,115.7 (AreC), 52.0 (OCH3),
ArH), 4.43e4.38 (2H, t, J ¼ 6.9 Hz, NCH2), 2.86e2.81 (2H, t,
44.6 (NCH2), 32.4 (CH2). IR (KBr,
n
/cmꢁ1) 1750 (C]O, ester). MS (m/
J ¼ 6.9 Hz, CH2CO). IR (KBr,
n
/cmꢁ1) 3500e2600 (OH), 1730 (C]O,
z): 293 [M]þ. Anal. Calcd. for C12H11N3O2S2 (293.36): C, 49.13; H,
acid). MS (m/z): 279 [M]þ. Anal. Calcd. for C11H9N3O2S2 (279.34): C,
3.78; N, 14.32; found C, 49.29; H, 3.60; N, 14.44.
47.30; H, 3.25; N, 15.04; found C, 47.59; H, 3.22; N, 15.19.
3.2.13. Synthesis of
propionyl hydrazide (14)
b
-(3-thioxo-s-triazolo[3,4-b]benzothiazol-2-yl)
3.2.16. Synthesis of 1-[
thione)propionyl]-1,3-dicyclohexylurea (17)
mixture of -(3-thioxo-s-triazolo[3,4-b]benzothiazol-2-yl)
b-(s-triazolo[3,4-b]benzothiazol-2-yl-3-
To a solution of 13 (1.0 g, 3.4 mmol) in methanol (20 mL),
hydrazine hydrate (0.9 mL, 15.0 mmol) was added. The reaction
mixture was refluxed for 6 h, after cooling to room temperature the
precipitate was filtered off, washed with cold water, and methanol
followed by recrystallization from aqueous ethanol. Colorless
crystals (0.81 g, 81.2% yield), m.p. 213e215 ꢀC. 1H NMR (300 MHz,
A
b
propionic acid (16) (1.2 g, 4.4 mmol), dicyclohexylcarbodiimide
(0.92 g, 4.4 mmol) and triethyl amine (0.62 mL, 4.4 mmol) in
acetonitrile (40 mL) was stirred at 0 ꢀC for 2 h and at room
temperature overnight. The solution evaporated under reduced
pressure. The residue was extracted with ethyl acetate, filtered off
from the insoluble additional residual DCU and the filtrate was
washed successively with saturated NaCl solution, 5% NaHCO3
solution, 1 M HCl and water. After dying over anhydrous Na2SO4,
the solvent was evaporated and the remaining residue was crys-
tallized from ethanol to yield 17 as colorless crystals (1.66 g, 79.81%
yield), Rf ¼ 0.83 (S1), m.p. 182e184 ꢀC. 1H NMR (300 MHz, CDCl3):
DMSO):
d
9.10 (1H, s, NH), 8.92 (1H, d, J ¼ 8.1 Hz, ArH), 8.08 (1H, d,
J ¼ 8.1 Hz, ArH), 7.62e7.49 (2H, m, ArH), 4.45e4.40 (2H, t, J ¼ 7.2 Hz,
NCH2), 4.19 (2H, s, NH2), 2.65e2.60 (2H, t, J ¼ 7.2 Hz, CH2). 13C NMR
(75 MHz, DMSO):
d 168.8 (CO), 160.3, 151.0 (2SCN), 131.7, 130.2,
127.3, 127.1, 125.3, 114.7 (AreC), 45.2 (NCH2), 32.2 (CH2). IR (KBr, n/
cmꢁ1) 3250 (NH2, NH), 1650 (C]O, amide). MS (m/z): 293 [M]þ.
Anal. Calcd. for C11H11N5OS2 (293.37): C, 45.03; H, 3.78; N, 23.87;
found C, 45.34; H, 3.77; N, 24.01.
d
9.07 (1H, d, J ¼ 8.7 Hz, ArH), 7.66 (1H, d, J ¼ 8.7 Hz, ArH), 7.54e7.42
(2H, m, ArH), 4.68e4.63 (2H, t, J ¼ 6.6 Hz, NCH2), 3.90 (1H, m, NCH),
3.68 (1H, m, NCH), 3.10e3.05 (2H, t, J ¼ 7.2 Hz, CH2CO), 1.96e1.19
3.2.14. Synthesis of
propionyl
b
-(3-thioxo-s-triazolo[3,4-b]benzothiazol-2-yl)
(20H, m, 10 CH2 cyclohexyl rings). IR (KBr, n
/cmꢁ1) 31,900 (NH),
L
-methionine methyl ester (15)
1665 (C]O, amide), 1610 (C]O, urea). MS (m/z): 485 [M]þ. Anal.
Calcd. for (485.67): C, 59.35; H, 6.43; N, 14.42; found C, 59.43; H,
6.28; N, 14.49.
To a cold solution (ꢁ5 ꢀC) of hydrazide 14 (0.23 g, 0.8 mmol) in
acetic acid (6 mL), 5 N HCl (3 mL), and water (25 mL), was added

A. Aboelmagd et al. / European Journal of Medicinal Chemistry 60 (2013) 503e511
511
3.3. Test of antifungal activity
amino acid ester derivatives via azide method. Coupling of s-tri-
azolo[3,4-b]benzothiazolyl-3-thioacetic acid with amino acid
esters by DCCI-method should be performed in the presence of
HOBt to suppress the formation of the by-product i.e. N-acylurea
derivatives. Michael addition of s-triazolo[3,4-b]benzothiazolyl-3-
thiol to methyl acrylate gave the N- instead of the S-derivative.
In general conjugation of the s-triazolo[3,4-b]benzothiazole
3.3.1. Fungal strains
Clinical isolates of mold fungi (A. flavus) and yeast (C. albicans)
were used to evaluate the antifungal activity of the synthesized
compounds.
3.3.2. Tested compounds
derivatives with amino acid esters (except
L-tyrosine methyl ester
The tested compounds were dissolved in dimethylsulphoxide to
derivative 7c) led to decrease in the antifungal action toward C.
albicans. Moreover, the length of the link between amino acids and
triazolobenzothiazole ring (compounds 7a, 12 and 14) did not alter
their antifungal activity. Based on the promising antifungal activity
of the two N-triazolobenzothiazolylthioacetyl/propionyl urea
derivatives (8 and 17), it is noteworthy to synthesize other new
derivatives of this series using different carbodiimides. This study
should be extended to include acidic, basic and aromatic amino acid
derivatives, such as, esters, hydrazides, hydrazones, and metal
complexes.
make a parent concentration of 103
trations were prepared by dilution.
mg/mL, and the other concen-
3.3.3. Determination of MIC
A standardized fungal spore suspension 104 CFU/mL was
prepared according to Grande and Artis [16]. The agar diffusion
method (well technique) was employed to determine the approx-
imate minimal inhibitory concentration (MIC) values. The concen-
tration considered inhibitory if minimal inhibition was found
around the well.
References
3.3.4. Determination of MFC
[1] K. Shalini, N. Kumar, S. Drabu, P.K. Sharma, Beilstein J. Org. Chem.
(2011) 668.
[2] N. Siddiqui, A. Rana, S.A. Khan, Indian J. Pharm. Sci. 69 (1) (2007) 10.
[3] M.H. Wheeler, A.A. Bell, Med. Mycol. 2 (1988) 338.
[4] D. Mares, C. Romangnoli, E. Andreotti, M. Manfrini, C.B. Vicentini, J. Agric. Food
Chem. 52 (2004) 2003.
[5] A. Aboelmagd, I.A.I. Ali, E.M.S. Salem, M. Abdel-Razik, Arkivoc. ix (2011) 337.
[6] C. Feignier, F. Besson, P. Hoet, M. Di Giambattista, C. Cocito, C. Sabatier,
G. Michel, Biotechnol. Tech. 7 (1993) 423.
[7] S.M. Eshita, N.M. Roberto, J. Antibiot. 48 (1995) 1240.
[8] T.J. Walsh, N. Giri, Eur. J. Clin. Microbiol. Infect. Dis. 16 (1997) 93.
[9] A.J. Delucca, T.J. Walsh, Rev. Iberoam. Micol. 17 (2000) 116.
[10] A.J. Carrillo-Munoz, G. Giusiano, P.A. Ezkurra, G. Quindos, Rev. Esp. Quimioter.
19 (2006) 130.
[11] Y. Yoon, C. Lee, Biotecnol. Bioprocess Eng. 14 (2009) 383.
[12] Y. Zhang, R. Zhong Qiao, C. Feng Dai, P. Fei Xu, Z.Y. Zhang, Chin. Chem. Lett. 13
(4) (2002) 287.
[13] J.D. Bower, F.D. Doyle, J. Chem. Soc. 3 (1957) 527.
[14] W. Fathalla, M. Cajan, P. Pazdera, Molecules 6 (2001) 557.
[15] I.A. Solov’eva, A.G. Guseva, J. Gen. Chem. U. S. S. R. 29 (1959) 2036.
[16] T.C. Grande, W.M. Artis, Antimicrob. Agents Chemother. 17 (1980) 725.
7
Sterilized test tubes containing standardized fungal spore
suspension were incorporated separately with the tested
compounds at different concentrations starting from those of the
MIC values and gradually multiplied with a control tube incorpo-
rated with the highest value of the solvent used as a control and
another incorporated with antifungal drug (fluconazole) for
comparison. Fungal colony forming unit (CFU) were counted and
their viability was checked at various times intervals from 0 to 24 h.
The minimal fungicidal concentration (MFC) was recorded as the
minimal concentration at which about 99% of the viable CFU was
destroyed.
4. Conclusion
This article afforded an easy and facile method for preparation of
s-triazolo[3,4-b]benzothiazolylthioacetyl/propionyl optically active