Novel phenylazo derivatives of condensed and uncondensed thiophene. Synthesis, characterization, and antimicrobial studies

Article abstract of DOI:10.1515/znb-2011-0606

In the search for new therapeutic agents against microbial infections, two novel series of monocyclic and tricyclic 5-(phenylazo)thiophene systems were synthesized based on 3-amino-2-thioxopyrimidinone and 2-cyanoacetamidothiophene derivatives 4 and 6. Functionalization of the pyrimidine ring in precursor 4 resulted in the formation of the target tricyclic condensed thiophenes 7, 12, and 13a, b, by the application of a variety of addition, substitution, and condensation reactions. On the other hand, derivatization of the versatile cyanoacetylated compound 6 led to a second series of monocyclic N-substituted aminothiophenes 15, 17, 19, and 20, through convenient methods. The new thiophene-based derivatives were tested for their antimicrobial activity with reference to relevant standard drugs. They displayed different levels of antibacterial activity, with compound 7 showing essentially the highest antipseudomonal activity. As for antifungal action, the compounds under investigation, unfortunately, had no inhibitory effects against test fungi isolates except for 7 and 20, that revealed slight inhibition.

Full text of DOI:10.1515/znb-2011-0606

Novel Phenylazo Derivatives of Condensed and Uncondensed Thiophene.
Synthesis, Characterization, and Antimicrobial Studies
Hatem M. Gabera, Mark C. Bagleyb, Sherif M. Sherifc, and Mohsen A. Sayedd
a National Organization for Drug Control and Research (NODCAR), P. O. Box 29, Cairo, Egypt
b School of Chemistry, Main Building, Cardiff University, Park Place, Cardiff, CF10 3AT, U. K.
c Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
d Department of Botany, Faculty of Science, Cairo University, Giza 12613, Egypt
Reprint requests to Dr. H. M. Gaber. Fax: +2 02 35728843. E-mail: hatem.gaber@yahoo.com
Z. Naturforsch. 2011, 66b, 585 – 596; received March 5, 2011
In the search for new therapeutic agents against microbial infections, two novel series of mono-
cyclic and tricyclic 5-(phenylazo)thiophene systems were synthesized based on 3-amino-2-thioxo-
pyrimidinone and 2-cyanoacetamidothiophene derivatives 4 and 6. Functionalization of the pyrim-
idine ring in precursor 4 resulted in the formation of the target tricyclic condensed thiophenes 7,
12, and 13a, b, by the application of a variety of addition, substitution, and condensation reactions.
On the other hand, derivatization of the versatile cyanoacetylated compound 6 led to a second se-
ries of monocyclic N-substituted aminothiophenes 15, 17, 19, and 20, through convenient methods.
The new thiophene-based derivatives were tested for their antimicrobial activity with reference to
relevant standard drugs. They displayed different levels of antibacterial activity, with compound 7
showing essentially the highest antipseudomonal activity. As for antifungal action, the compounds
under investigation, unfortunately, had no inhibitory effects against test fungi isolates except for 7
and 20, that revealed slight inhibition.
Key words: Thienopyrimidinones, Hydrazinolysis, Cyclocondensation, Cyclodesulfurization,
Antibacterial Activity
Introduction
the development of safe and effective antifungals [6].
In order to overcome these emerging resistance prob-
lems, there is an urgent need to discover novel antimi-
crobial agents in structural classes distinct from exist-
ing antimicrobial drugs.
In an effort to identify such compounds, we con-
tinuously review various structures which may be of
Almost all of the major classes of antibiotics have
encountered resistance in clinical applications [1, 2].
Antibiotic resistance among Gram-positive bacteria
(Staphylococci, Enterococci and Streptococci) is be-
coming increasingly serious [2, 3]. In particular, the
emergence of multidrug-resistant Gram-positive bac- use in the design of novel antimicrobial agents. In the
teria, such as methicillin-resistant Staphylococcus au- course of this screening, some thiophene derivatives
have attracted much of our attention because of their
synthetic and biological importance. A number of them
reus (MRSA) has made treatment of infectious dis-
eases difficult, and over the last decades, has emerged
as a major worldwide health problem [4]. There has have been recently reported to display significant bac-
tericidal and fungicidal properties [7 – 11]. The thio-
phene ring system is notably a structural component of
also been significant increase in the frequency of
systematic fungal infection in humans. Patients af-
fected with AIDS and cancer, and transplant patients the commercial imidazole antifungal agent sertacon-
are immunosuppressed and very susceptible to life- azole [12], and the antimicrobial profile of condensed
thiophenes, particularly their thieno[2,3-d]pyrimidin-
4-one analogs, is also well categorized in the litera-
threatening systematic fungal infections like Candidi-
asis, Cryptococcosis and Aspergillosis [5]. It is known
that the biochemical similarity of the human cell and ture [13 – 15].
fungi provides a significant obstacle to the discovery of
Based on these observations and as part of our ongo-
therapeutic agents with selective activity, but the emer- ing studies in the development of new chemotherapeu-
gence of resistance is the main problem encountered in tic agents [16 – 18], we embarked upon the synthesis of
c
0932–0776 / 11 / 0600–0585 $ 06.00 ꢀ 2011 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
Scheme 1. Reagents and conditions:
(a) CS2, K2CO3, DMF, r. t. then
reflux, 100 ◦C; (b) Me2SO4, r. t.;
(c) N2H4·H2O, r. t.; (d) CSCl2, K2CO3,
CH2Cl2, r. t.; (e) (i) N2H4·H2O,
CH2Cl2, r. t.; (ii) KOH, EtOH, reflux
then HCl, r. t.; (f) NCCH2CO2Et,
NaOEt, EtOH, reflux. r. t. = room
temperature.
a series of novel condensed tricyclic compounds con- similar transformation of a thiophene o-aminoester
taining the thieno[2,3-d]pyrimidin-4-one skeleton by moiety to a pyrimidinone-type o-aminothione frag-
annelation and a second series of novel monocyclic N- ment [20]. It is worth mentioning that compound 4
substituted-aminothiophenes with the objective to de- could also be obtained by an alternative synthetic
velop new leads and to improve the efficacy of existing route involving initial treatment of the 2-aminothio-
motifs.
phene derivative 1 with thiophosgene and subsequent
cyclization of the isolated isothiocyanate product 5
with hydrazine hydrate. Compound 4 prepared by the
latter route was found to be identical to that obtained
by the former method. Similar transformations of het-
erocyclic amines to the corresponding isothiocyanate
analogs have been reported previously [21 – 23]. On
the other hand, cyanoacetylation of compound 1
with ethyl cyanoacetate in ethanolic sodium ethoxide
solution led to the respective 2-cyanoacetamide
derivative 6 with an active methylene group for further
derivatization (Scheme 1). The IR spectrum of the
latter product was informative in establishing the
proposed structure. The spectrum showed absorption
bands belonging to stretching vibrations of a cyano
function (ν = 2260 cm−1) and carbonyl groups (ν =
1694 and 1667 cm−1). The structure assigned to
the reaction product was additionally supported by
its 1H NMR spectrum. A diagnostically important
signal in the spectrum was a singlet at δH = 3.85 ppm
attributable to the methylene protons, whereas the
NH proton (exchangeable with D2O) resonated in
the downfield region of the spectrum, typical for a
secondary amide NH. Other resonances were also
observed as expected and are recorded in the Exper-
imental Section. In addition, the mass spectrum of 6
showed a molecular ion peak at m/z = 418, which is
indicative of an increase by 67 from the parent 1, pro-
viding strong evidence for cyanoacetylation of 1 to 6.
Results and Discussion
Synthesis
Treating ethyl 2-amino-4-phenyl-5-(phenylazo)-
thiophene-3-carboxylate (1) [19] with carbon disulfide
in the presence of anhydrous potassium carbonate
and subsequent methylation of the in situ generated
monopotassium salt 2 with dimethyl sulfate produced
the 2-mercapto(methylthio)methyleneaminothiophene
derivative 3 which was reacted without isolation.
Hydrazinolysis at r. t., followed by acidification caused
an intramolecular cyclization to afford the final amino-
thioxo derivative 4 (Scheme 1). All spectroscopic data
fitted perfectly with the proposed structure 4. In its
IR spectrum, the ring carbonyl stretching frequency
was observed at ν = 1685 cm−1. Also, its H NMR
1
spectrum contained no signals for the protons of an
ethyl ester group, but did contain characteristic D2O-
exchangeable signals for the amino and thioamidic
protons at δH = 6.02 and 12.45 ppm, respectively,
besides aromatic protons. Furthermore, the 13C NMR
spectrum of 4 displayed distinct shifts of fourteen
carbon atoms. Among these, two signals at δC = 158.6
and 172.8 ppm were characteristic for the C=O and
C=S carbons, respectively, thus lending additional
support to the structural assignments. This observation
was also supported by a recent literature report on a
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
587
Scheme 2. Reagents and condi-
tions: (a) CS2, C5H5N, reflux;
(b) MeI, NaOEt, EtOH, reflux;
(c) N2H4·H2O, EtOH, reflux;
(d) HC(OEt)3, glacial AcOH,
reflux; (e) (MeO)2CHNMe2,
glacial AcOH, r. t.; (f) NaNO2,
AcOH, 0 – 5 ◦C; (g) excess
NaNO2, AcOH, 0 – 5 ◦C. r. t. =
room temperature.
The α-aminothione derivative 4 was submitted to the N-amino group on the thione carbon atom, fol-
a series of transformations that provided further ev- lowed by cyclodesulfurization in a fashion similar to
idence in support of compound identity (Scheme 2). established α-aminothione derivatizations [34]. The
Thus, heterocyclization of 4 with carbon disulfide was constitution of compound 4 was also well supported
carried out to afford a tricyclic product with an an- by its conversion into the corresponding 2-methylthio
nelated thiadiazole ring for which the thione structure 7 analog 8 on alkylation with methyl iodide. On reaction
was assigned based on analytical and spectral data. The with hydrazine hydrate, compound 8 gave the target 2-
IR spectrum of the latter product lacked an absorp- hydrazino derivative 9 (Scheme 2).
tion band due to the thiol function, which is consis-
Cyclocondensation of hydrazino compound 9
tently found at around ν = 2600 – 2550 cm−1 [24, 25], with one-carbon building blocks such as triethyl
but contained stretching frequencies at ν = 3198
and 1682 cm−1 attributable to the NH and C=O groups,
respectively. In the 1H NMR spectrum of the ob-
orthoformate and dimethylformamide dimethylacetal
(DMFDMA) resulted in the formation of a tricyclic
product. Theoretically, three isomeric reaction prod-
tained product, the signal for the NH proton resonated ucts (triazole/tetrazine) 10, 11 and 12 might be
as a D2O-exchangeable singlet at a typical downfield
expected. However, the reaction product was identified
as tricyclic tetrazine derivative 12 on the basis of the
shift (δH = 13.54 ppm), whereas the signal associ-
ated with the SH proton was not detectable in the ex- 1H NMR spectrum of the isolated product, which
pected higher field region of the spectrum (δH = 1.6 – gave unequivocal proof in favor of structure 12. One
4.0 ppm), confirming the assigned thione structure.
Similar assignments have been reported on closely re-
diagnostic signal is due to the methine proton; the
obtained product displayed a methine doublet at
lated heterocyclic thioamides [24 – 30]. Moreover, the δH = 6.94 ppm. Two other diagnostically important
13C NMR spectrum, recorded for compound 7, re-
vealed a signal at δC = 183.9 ppm, which corresponds
signals in the spectrum appeared as a singlet at δH
=
9.85 ppm assignable to the N-1 proton and a doublet
to the typical chemical shift of a thione carbon atom, at δH = 9.62 ppm attributable to the N-4 proton which
in line with prior observations of spectra of closely re- was coupled with the C-3 proton. In the presence
lated compounds [26, 27, 31 – 33]. Compound 7 is as-
sumed to be formed via initial nucleophilic attack by
of deuterium oxide, the signals for the NH groups
disappeared whereas the signal associated with 3-H
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
Scheme 3. Reagents and
conditions: (a) HSCH2CO2H,
AcOH, reflux; (b) S8, Ph-
NCS, EtOH, Et3N, reflux;
(c) ArN2Cl, EtOH, AcONa,
0 – 5 ◦C; (d) PhNCS, EtOH,
Et3N, reflux; (e) o-HOPhCHO,
EtOH, piperidine, reflux;
(f) EtOH, HCl, reflux.
became a singlet, providing unambiguous evidence N-4-amino group at the typical shift (δH = 5.68 ppm)
in support of tetrazine formation. On the contrary, the characteristic for an N-NH2 resonance, whereas the
alternative triazole structures 10 and 11, if isolated, corresponding protodeamino analog 13b showed no
would be expected to display the methine signal as N-NH2 resonance, which is generally found at around
a singlet together with only one D2O-exchangeable δH ∼ 5.75 ppm by analogy with related N-3-amino-
singlet signal corresponding to the NH2 protons. quinazolin-4-one analogs [35], thus indicating the dis-
Accordingly, it can be concluded that the studied appearance of that N-4-amino group in the spectrum
reaction occurrs with the regioselective formation of of 13b. The remaining resonances were also observed
the tetrazine derivative 12 as the sole product.
at the expected frequencies (cf. Experimental Section).
Furthermore, compound 13b displayed a molecular ion
peak which clearly confirmed its molecular formula.
This observation is also supported by prior literature
reports on the well established nitrous acid deamina-
tion of the exocyclic amino group in closely related
cyclic N-aminoamides [36 – 39].
The hydrazino compound 9 proved to be a use-
ful precursor for the synthesis of other tricyclic thio-
phenes. By treatment of 9 with in situ generated nitrous
acid in a molar ratio of 1 : 1, the 4-amino derivative
of a tricyclic system 13a was formed, whilst by treat-
ment with an excess of nitrous acid, deamination of
the amino group at position 4 occurred with the forma-
This protocol was also suitable for the synthesis
tion of the corresponding 4-protodeamino analog 13b. of an N-substituted aminothiophene system through
1H NMR spectroscopy could firmly discriminate be- a series of transformations of the 2-cyanoacetamide
tween the structure of these products. The amino derivative 6 (Scheme 3). Thus for example, reaction
derivative 13a displayed a diagnostic signal for the of compound 6 with thioglycolic acid afforded the
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
589
product of addition/cyclodehydration for which two of 16 can then occur readily with the formation of the
tautomeric forms, 15 and 15ꢀ, might be present. In isolable 4-aminothiazole derivative 17. This assump-
the IR spectrum of the isolated product, the absence tion is consistent with similar observations [46 – 49a].
of a band associated with the CN function and the
presence of an additional band corresponding to the
C=O group, which was not present in the original ni-
trile 6, verified unequivocally ring closure through cy-
clodehydration. The 1H NMR spectrum of the reaction
product showed one proton signal at δH = 5.56 ppm.
This signal can only be interpreted in terms of a me-
thine proton of the 2-substituted methylidine form 15,
thus excluding the presence of the tautomeric structure
of the 2-functionally substituted-methyl-2-thiazolin-4-
one form 15ꢀ. Moreover, the 13C NMR spectrum gave
added proof for the assigned structure (cf. Experimen-
tal Section). It is worthwhile to report here that the
hydrogen-bonded tautomer is the most stable form in
which compound 15 predominantly exists as indicated
by spectroscopic studies. In the IR spectrum of com-
pound 15, an amidic functional group was clearly ob-
served with a low carbonyl absorption frequency at ν =
1656 cm−1. This large shift to lower wavenumbers can
be explained by intramolecular NH hydrogen bond-
ing and conjugation with the C=C double bond [40 –
Moreover, the activated methylene group in the 2-
cyanoacetamide derivative 6 readily took part in the
diazo coupling reaction of compound 6 with diazo-
tized aryl amines, affording the corresponding arylhy-
drazonoacetamide derivatives 18a, b. On the basis of
1
the IR and H NMR spectral studies, the latter prod-
ucts were recommended to exist mainly, in each case,
in the hydrazone configuration with intramolecular hy-
drogen bonding. The IR spectra indicated the presence
of intramolecularly hydrogen-bondedcarbonyl groups,
their bands appearing at the typical low frequencies,
whereas their 1H NMR spectra confirmed the presence
of highly deshielded NH protons (exchangeable with
D2O), showing clear evidence for internal hydrogen
bonding. Undoubtedly, the six-membered hydrogen-
bonded ring structure in 18a, b enhances their relative
stability. Similar results were found by other investiga-
tors in their studies on arylhydrazones[40 – 42, 50]. On
treatment with phenyl isothiocyanate, the hydrazono
compound 18a afforded the respective 5-imino-3-thi-
oxo-1,2,4-triazine derivative 19 via a nucleophilic ad-
dition/ring closure sequence (Scheme 3).
1
43]. In a similar fashion, the H NMR spectrum ex-
hibited two downfield NH resonances at δH = 11.85
and 12.50 ppm corresponding to the thiazolidine NH
and the CONH protons, respectively. The deshielding
of these protons strongly supports the existence of in-
tramolecular hydrogen bonding, which stabilizes this
form [40 – 43]. These findings affirm that the isolated
product exists predominantly in the configuration 15,
which permits an intramolecular hydrogen bond. As il-
lustrated in Scheme 3, initially the sulfur nucleophile
adds to the electron deficient cyanocarbon, forming
an acyclic imino adduct 14 as a first step. Subsequent
elimination of water leads eventually to the final cyclo-
condensation product 15. This hypothesis is consistent
with similar observations [44, 45].
By reaction of nitrile 6 with o-salicylaldehyde in
ethanol in the presence of a catalytic amount of piperi-
dine, we obtained the desired coumarin derivative 20
via Knoevenagel reaction, in which the initially formed
iminocoumarin 20ꢀ was subsequently hydrolyzed with
hydrochloric acid to the final coumarin 20 via imino-
lactone ring opening followed by recyclization with
loss of ammonia, in accordance with the findings of re-
lated reports [48, 49b, 51, 52]. Elucidation of structure
for the latter products was established on the basis of
elemental and spectroscopic analyses in each case (cf.
Experimental Section).
Antimicrobial evaluation
The reactivity of the cyanomethylene moiety in
the cyanoacetylated compound 6 was demonstrated
The biological evaluation of antibacterial and an-
by its facile heterocyclization with elemental sulfur tifungal effects are summarized in Tables 1 and 2.
and phenyl isothiocyanate to give another new thi- As shown by these results, the new thiophene-based
azole derivative 17. The pathway of the studied reac- derivatives under investigation displayed different lev-
tion may involve an initial Gewald reaction of nitrile 6 els of in vitro antimicrobial activity. In general, the
with elemental sulfur, followed by nucleophilic attack chemical structure, comprising the nature of the het-
by the formed thiol adduct on the electrophilic thione erocyclic system as well as the substituted function
carbon atom of the isothiocyanate moiety to generate present in the heterocyclic ring, has a pronounced
an acyclic 2-(phenylcarbamothioylthio)acetamide in- effect on the antimicrobial activity. In particular, it
termediate 16. Subsequent intramolecular cyclization was found that the attachment of a coumarin-3-carb-
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
Table 3. MIC (µg mL−1) of compound 7 and streptomycin
against P. aeruginosa.
Table 1. Antibacterial activity of test compounds and refer-
ence drug.
IZDa (mm)
Sample
Compound 7
Streptomycin
MIC
23
17
Sample
G –negative
G +positive
E. coli P. aeruginosa
B. subtilis S. aureus
Controlb
0
19
16
3
0
20
18
2
0
22
12
0
0
21
14
0
Standardc
rimidinone scaffold can improve antipseudomonal ac-
tivity as observed for compound 7, which can serve as
a lead compound in this field.
7
12
13a
13b
15
17
19
20
3
4
0
0
2
4
2
5
0
0
0
0
The overall results of the present study can be con-
sidered promising in the perspective of new antibac-
terial drug discovery, considering the clinical impor-
tance of Pseudomonas aeruginosa, which was found to
be sensitive to most of the test compounds, especially
the 2-thioxothiadiazole derivative 7 (Table 1). Such a
pathogen has emerged as one of the most problematic
Gram-negative pathogens, with alarmingly high antibi-
otic resistance rates [53]. Even with the most effective
antibiotics against this pathogen, namely carbapenems
(imipenem and meropenem), the resistance rates were
detected as 15 – 20.4 % amongst 152 P. aeruginosa
strains [53].
3
2
4
3
0
0
0
0
9
11
3
5
a IZD: inhibition zone diameter; b DMSO has no antibacterial activ-
ity; c standard for bacteria: streptomycin.
Table 2. Antifungal activity of test compounds and reference
drug.
IZDa (mm)
Sample
Mould
Yeast
A. niger A. flavus
C. albicans C. parapsilosis
Controlb
0
13
4
0
11
4
0
12
5
0
10
4
Standardc
7
12
13a
13b
15
17
19
20
0
0
0
0
Conclusion
0
0
0
0
0
0
0
0
In summary, we have described in the present work
a thorough study on the design, synthesis and an-
timicrobial activity of two series of novel phenyl-
azo derivatives of condensed and N-substituted thio-
phenes. The thiophene-based derivatives were ob-
tained through a convenient route, based on versatile
amino-thioxo and 2-cyanoacetamide building blocks.
The biological potential of the newly obtained com-
pounds was further investigated by screening their an-
timicrobial activity against four pathogenic bacteria
and four pathogenic fungi. The biological study of
the compounds under investigation indicated that the
highest antipseudomonal activity was obtained with
compound 7 carrying a 2-thioxothiadiazole moiety an-
nelated to the thienopyrimidinone skeleton. Its MIC
value (23 µg mL−1) towards P. aeruginosa is very sig-
nificant. Compound 20 showed an appreciable broad
0
0
0
0
0
0
0
0
0
3
0
2
0
2
0
3
a IZD: inhibition zone diameter; b DMSO has no antifungal activity;
c
standard for fungi: amphoterisin B.
oxamide pharmacophore to the 5-(phenylazo)thio-
phene fragment favored antibacterial activity espe-
cially against P. aeruginosa, as observed for com-
pound 20, whereas the annelation of a 2-thioxothiadi-
azole moiety to the thienopyrimidinone core produced
an inhibitory effect against P. aeruginosa almost simi-
lar to the reference drug streptomycin as observed for
compound 7 (Table 1). As for antifungal action, there
was no essential in vitro activity of the test compounds.
Only compounds 7 and 20 showed poor activity.
Based on the biological evaluation, compound 7 spectrum of action against both Gram-negative and
was selected for further assessment. The mini- Gram-positive bacteria. In light of the results presented
mum inhibitory concentration (MIC) of the 2-thi- in this work and taking into account that this prelimi-
oxothiadiazole derivative 7 against P. aeruginosa nary study does not produce conclusive evidence re-
was 23 µg mL−1, which is very close to that of strep- garding structure-antimicrobial relationships, we have
tomycin as a reference antibacterial drug as depicted focused our attention on the most promising com-
in Table 3. From the structure-activity relationship pound 7 as an interesting starting point for the devel-
(SAR), we can conclude that the heteroannelation of opment of a new class of antibacterial agents. Further
a 2-thioxothiadiazole pharmacophore to the thienopy- structural modifications might lead to the discovery
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
591
of more potent antibacterial agents, and this work is then added to a solution of potassium hydroxide (0.002 mol)
in absolute ethanol (10 mL), and the mixture was refluxed
under stirring for 3 h. The reaction mixture was cooled,
diluted with iced water, neutralized with concentrated hy-
drochloric acid and stirred at r. t. for 1 h. The separated
solid product was filtered off, washed repeatedly with wa-
ter and recrystallized from dioxane to give a solid prod-
uct, in 43 % yield, identical in every respect (melting point,
mixed melting point, and IR data) to that obtained above
from method A.
in progress. We believe that research in this direction
should be encouraged in order to broaden the applica-
bility of these new heterocyclic frameworks to serve as
leads for designing novel chemotherapeutic agents.
Experimental Section
Melting points are uncorrected. IR spectra were recorded
(KBr) on a Pye Unicam SP-1000 spectrophotometer. NMR
spectra were obtained on a Varian Gemini 300 MHz spec-
trometer using tetramethylsilane (TMS) as an internal ref-
erence. Chemical shifts are expressed in δ/ppm. Splitting
patterns were designated as follows: s: singlet; d: doublet;
t: triplet; q: quartet; m: multiplet. EI mass spectra were
recorded on a Shimadzu GC MS-QP 1000 EI mass spectrom-
eter at 70 eV. Compound 1 was prepared by a published pro-
cedure [19].
Ethyl 2-isothiocyanato-4-phenyl-5-(phenylazo)thiophene-3-
carboxylate (5)
A suspension of aminoester 1 (0.002 mol) in dichloro-
methane (5 mL) was added to a stirred suspension of
potassium carbonate (0.003 mol) in water (10 mL) and
dichloromethane (20 mL) at r. t. To the stirred mixture, thio-
phosgene (0.0025 mol) was added slowly in an ice bath. The
temperature of the reaction mixture was allowed to reach
r. t. Stirring was continued for 6 h. Inorganic salts were re-
moved by filtration, the solvent was removed under reduced
pressure, and the residue was washed with petroleum ether
and purified by recrystallization from dioxane to give yel-
low needles of the 2-isothiocyanate derivative 5 (0.50 g;
64 %). M. p. 94 ◦C. – IR (KBr): ν = 3056 (arom. CH),
3-Amino-5-phenyl-6-(phenylazo)-2-thioxo-2,3-dihydro-
thieno[2,3-d]pyrimidin-4(1H)-one (4)
Method A: A mixture of aminoester 1 (0.005 mol), car-
bon disulfide (5 mL, 0.08 mol) and anhydrous potassium
carbonate (10 g) was dissolved in 35 mL of dimethylform-
amide and stirred for 12 h at r. t. The reaction mixture was
further heated on a water bath for 12 h, cooled and poured
over iced water. The reaction mixture was treated dropwise
with dimethyl sulfate (7.5 mL, 0.08 mol) under stirring, then
allowed to stand at r. t. for 12 h and extracted with chloro-
form. The solvent was evaporated under reduced pressure,
and the crude product 3 was obtained. The latter product
was then mixed with hydrazine hydrate (5 mL, 0.1 mol), and
the mixture was stirred for 3 h at r. t. The reaction mixture
was cooled, poured over iced water and acidified with hy-
drochloric acid. The precipitate was collected by filtration,
washed with water, dried and recrystallized from dioxane to
give the title compound 4 as a brown solid (0.85 g; 45 %).
M. p. 206 – 208 ◦C. – IR (KBr): ν = 3301 – 3200 (NH, NH2),
3064 (arom. CH), 1685 (C=O) cm−1. – 1H NMR (300 MHz,
[D6]DMSO): δ = 6.02 (s, 2H, NH2, D2O-exchangeable),
7.16 – 7.50 (m, 10H, 2PhH), 12.45 (s, br, 1H, NH, D2O-ex-
changeable). – 13C NMR (75 MHz, [D6]DMSO): δ = 117.9,
127.0, 127.4, 127.9, 128.3, 128.7, 129.5, 132.3, 134.6, 142.2,
152.8, 153.1, 158.6 (C=O), 172.8 (C=S). – C18H13N5OS2
(379.459): calcd. C 56.97, H 3.45, N 18.46, S 16.90; found
C 56.78, H 3.31, N 18.25, S 16.65.
1
2075 (N=C=S), 1712 (C=O) cm−1. – H NMR (300 MHz,
[D6]DMSO): δ = 0.98 (t, 3H, J = 7.2 Hz, ester Me), 4.20 (q,
2H, J = 7.2 Hz, ester CH2), 7.12 – 7.47 (m, 10H, 2PhH). –
MS (EI, 70 eV): m/z(%) = 393 (17) [M]+. – C20H15N3O2S2
(393.482): calcd. C 61.05, H 3.84, N 10.68, S 16.30; found
C 60.82, H 3.72, N 10.47, S 16.13.
Ethyl 2-(2-cyanoacetamido)-4-phenyl-5-(phenylazo)thio-
phene-3-carboxylate (6)
The 2-aminothiophene 1 (0.005 mol) and ethyl cyanoac-
etate (0.005 mol) were added to a solution of sodium ethox-
ide (from 0.12 g, 0.005 mol, of sodium metal and 15 mL of
absolute ethanol). The reaction mixture was heated at reflux
for 6 h with stirring, and the solvent was evaporated under re-
duced pressure. The residue was dissolved in water and neu-
tralized with concentrated hydrochloric acid. The separated
solid was collected by filtration, dried and recrystallized from
ethanol to obtain the cyanoacetylated compound 6 as color-
less crystals (0.65 g; 31 %). M. p. 160 – 161 ◦C. – IR (KBr):
ν = 3298 (NH), 3061 (arom. CH), 2260 (CN), 1694, 1667
(2C=O) cm−1. – 1H NMR (300 MHz, [D6]DMSO): δ = 1.12
Method B: A solution of the isothiocyanate derivative 5 (t, 3H, J = 7.2 Hz, ester Me), 3.85 (s, 2H, CH2CN), 4.26
(0.002 mol) in dichloromethane (30 mL) was added under (q, 2H, J = 7.2 Hz, ester CH2), 7.22 – 7.54 (m, 10H, 2PhH),
stirring to a solution of hydrazine hydrate (0.003 mol) in 12.65 (s, br, 1H, NHCO, D2O-exchangeable). – 13C NMR
dichloromethane (10 mL). The reaction mixture was stirred (75 MHz, [D6]DMSO): δ = 13.8 (ester Me), 24.5 (CH2),
at r. t. for 2 h, whereupon a precipitate of the corresponding 59.6 (ester CH2), 109.0 (C-3), 116.9 (CN), 127.0, 127.5,
hydrazinothioxo derivative was separated from the original 127.8, 128.1, 128.6, 129.2, 131.7, 134.3, 142.4, 149.2, 152.6,
solution and collected by filtration. The isolated solid was 162.1 (amide C=O), 166.3 (ester C=O). – MS (EI, 70 eV):
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
m/z(%) = 418 (21) [M]+. – C22H18N4O3S (418.468): calcd. dimethylformamide to give canary-yellow crystals of the hy-
C 63.14, H 4.34, N 13.39, S 7.66; found C 62.90, H 4.25, drazino compound 9 (0.55 g; 49 %). M. p. 192 – 193 ◦C. – IR
N 13.21, S 7.53.
(KBr): ν = 3342 – 3155 (NH, NH2), 3071 (arom. CH), 1674
(C=O) cm−1. – 1H NMR (300 MHz, [D6]DMSO): δ = 4.06
(s, 2H, NH2, D2O-exchangeable), 5.84 (s, 2H, N-NH2, D2O-
exchangeable), 7.13 – 7.49 (m, 10H, 2PhH), 9.87 (s, 1H, NH,
D2O-exchangeable). – MS (EI, 70 eV): m/z(%) = 377 (26)
[M]+. – C18H15N7OS (377.423): calcd. C 57.28, H 4.01,
N 25.98, S 8.50; found C 57.06, H 3.85, N 25.79, S 8.37.
7-Phenyl-6-(phenylazo)-2-thioxo-1H-[1,3,4]thiadiazolo-
[3,2-a]thieno[2,3-d]pyrimidin-8(2H)-one (7)
A mixture of the N-amino compound 4 (0.0025 mol)
and carbon disulfide (1 mL, 0.017 mol) in dry pyridine
(10 mL) was heated under reflux until the evolution of hy-
drogen sulfide ceased (7 h). The reaction mixture was al-
lowed to cool, poured onto iced water and acidified with
concentrated hydrochloric acid. The precipitated product
was filtered off, washed with water and dried. Recrystal-
lization from a mixture of dimethylformamide and wa-
ter gave the corresponding thione derivativ◦e 7 as reddish-
brown crystals (0.63 g; 60 %). M. p. > 300 C. – IR (KBr):
ν = 3198 (NH), 3069 (arom. CH), 1682 (C=O) cm−1. –
1H NMR (300 MHz, [D6]DMSO): δ = 7.14 – 7.51 (m, 10H,
2PhH), 13.54 (s, 1H, NH, D2O-exchangeable). – 13C NMR
(75 MHz, [D6]DMSO): δ = 117.3 (C-7a), 127.1, 127.5,
127.9, 128.3, 128.6, 129.3, 132.5, 134.5, 142.2, 152.4,
156.7, 158.0, 159.4 (C=O), 183.9 (C=S). – C19H11N5OS3
(421.519): calcd. C 54.14, H 2.63, N 16.61, S 22.82; found
C 54.01, H 2.44, N 16.38, S 22.62.
7-Phenyl-8-(phenylazo)-1,4-dihydro-6H-thieno[2ꢀ ,3ꢀ:4,5]-
pyrimido[1,2-b][1,2,4,5]tetrazin-6-one (12)
Method A: A mixture of the hydrazino compound 9
(0.0025 mol) and triethyl orthoformate (0.5 mL, 0.003 mol)
in glacial acetic acid (12 mL) was heated under reflux for 1 h.
After cooling to r. t., the formed precipitate was filtered off,
washed with diethyl ether and dried. Recrystallization from
an ethanol and dioxane mixture gave the condensed tetrazine
derivative 12 as a yellow solid (0.57 g; 59 %). M. p. 240 –
242 ◦C. – IR (KBr): ν = 3326 (NH), 3064 (arom. CH),
1678 (C=O) cm−1. – 1H NMR (300 MHz, [D6]DMSO):
δ = 6.94 (d, 1H, J = 2.5 Hz, tetrazine 3-H), 7.20 – 7.53 (m,
10H, 2PhH), 9.62 (d, 1H,J = 2.5 Hz, tetrazine 4-H, D2O-
exchangeable), 9.85 (s, 1H, tetrazine 1-H, D2O-exchange-
able). – C19H13N7OS (387.418): calcd. C 58.90, H 3.38,
N 25.31, S 8.28; found C 58.71, H 3.21, N 25.07, S 8.13.
3-Amino-2-(methylthio)-5-phenyl-6-(phenylazo)thieno[2,3-
d]pyrimidin-4(3H)-one (8)
Method B:
A mixture of the hydrazino com-
pound 9 (0.0025 mol) and dimethylformamide dimethylac-
etal (0.003 mol) in glacial acetic acid (5 mL) was stirred
at r. t. for 5 h. After cooling to r. t., the suspended solid
product was collected by filtration, washed with ethanol and
dried. Recrystallization from a mixture of ethanol and diox-
ane gave the same product (48 % yield) as obtained above
from method A.
To a solution of ethanolic sodium ethoxide [prepared by
dissolving sodium metal (0.005 mol) in absolute ethanol
(20 mL)], compound 4 (0.005 mol) was added, and the so-
lution was then heated at reflux for 10 min. Methyl iodide
(0.0075 mol) was added, and refluxing was continued for ad-
ditional 2 h. The reaction mixture was then cooled, poured
onto cold water, neutralized with concentrated hydrochlo-
ric acid, whereby the product that separated out was fil-
tered off, dried and recrystallized from aqueous dioxane to
give the methylthio derivative 8 as a colorless solid (0.69 g;
35 %). M. p. 151 – 153 ◦C. – IR (KBr): ν = 3337, 3215
(NH2), 3071 (arom. CH), 1680 (C=O) cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 2.49 (s, 3H, SMe), 5.59 (s, 2H,
N-NH2, D2O-exchangeable), 7.20 – 7.52 (m, 10H, 2PhH). –
C19H15N5OS2 (393.485): calcd. C 58.00, H 3.84, N 17.80,
S 16.30; found C 57.75, H 3.71, N 17.58, S 16.12.
4-Amino-6-phenyl-7-(phenylazo)tetrazolo[1,5-a]thieno[3,2-
e]pyrimidin-5(4H)-one (13a)
The hydrazino compound 9 (0.005 mol) was dissolved in
acetic acid (30 mL), a small amount of insoluble material was
filtered off, then the filtrate was cooled in an ice bath at 0 –
5 ◦C. The solution was stirred at this temperature and treated
gradually with a cold saturated solution of sodium nitrite
(0.005 mol of sodium nitrite in 3.5 mL of water) over a period
of 15 min. Stirring was continued for further 30 min, then the
reaction mixture was left to stand at r. t. for 3 h. The resulting
solid was filtered off, washed with water and dried. Recrys-
tallization from dimethylformamide gave the N-amino com-
3-Amino-2-hydrazinyl-5-phenyl-6-(phenylazo)thieno[2,3-
d]pyrimidin-4(3H)-one (9)
Compound 8 (0.003 mol) was mixed with hydrazine hy- pound 13a as light-brown crystals (1.09 g; 56 %). M. p. 224 –
drate (1.5 mL, 0.03 mol) in absolute ethanol (15 mL). The 225 ◦C. – IR (KBr): ν = 3321, 3210 (NH2), 3060 (arom. CH),
reaction mixture was heated at reflux for 5 h and then 1680 (C=O) cm−1. – 1H NMR (300 MHz, [D6]DMSO): δ =
held overnight at r. t. After cooling and dilution with wa- 5.68 (s, 2H, NH2, D2O-exchangeable), 7.15 – 7.49 (m, 10H,
ter, the obtained precipitate was filtered off, dried and pu- 2PhH). – C18H12N8OS (388.406): calcd. C 55.66, H 3.11,
rified by recrystallization from a mixture of ethanol and N 28.85, S 8.26; found C 55.41, H 2.97, N 28.62, S 8.14.
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
593
6-Phenyl-7-(phenylazo)tetrazolo[1,5-a]thieno[3,2-e]pyr-
imidin-5(4H)-one (13b)
(0.0025 mol) in absolute ethanol (10 mL) was stirred at r. t.
for 30 min. Then, phenyl isothiocyanate (0.0025 mol) was
added gradually, and stirring was continued for 20 min. Next,
the reaction mixture was heated at reflux with constant stir-
ring for 3 h. After cooling to r. t., the reaction mixture was
poured over iced water and stirred for several hours until
crystallization was complete. The material which separated
out was isolated by filtration, washed with ether and dried.
It was recrystallized from benzene to give the correspond-
ing dihydrothiazole derivative 17 as yellow crystals (0.50 g;
34 %). M. p. 229 – 232 ◦C. – IR (KBr): ν = 3418 – 3296
(NH, NH2), 3058 (arom. CH), 1661, 1652 (2C=O) cm−1. –
1H NMR (300 MHz, [D6]DMSO): δ = 1.24 (t, 3H, J =
7.2 Hz, ester Me), 4.32 (q, 2H, J = 7.2 Hz, ester CH2),
7.08 – 7.55 (m, 15H, 3PhH), 7.61 (s, br, 2H, NH2, D2O-ex-
changeable), 12.30 (s, br, 1H, NH, D2O-exchangeable). –
MS (EI, 70 eV): m/z(%) = 585 (23) [M]+. – C29H23N5O3S3
(585.720): calcd. C 59.47, H 3.96, N 11.96, S 16.42; found
C 59.23, H 3.81, N 11.77, S 16.25.
The hydrazino compound 9 (0.003 mol) was dissolved
in acetic acid (20 mL), a small amount of insoluble mate-
rial was filter◦ed off, then the filtrate was cooled in an ice
bath at 0 – 5 C. The solution was stirred at this tempera-
ture and treated gradually with a cold saturated solution of
sodium nitrite [1 g of sodium nitrite (0.015 mol) in water
(10 mL)] over a period of 15 min. Stirring was continued for
further 30 min, then the reaction mixture was left to stand
at r. t. for 3 h. The resulting solid was filtered off, washed
with water and dried. Recrystallization from dimethylform-
amide gave the N-deamino compound 13b as a dark-brown
◦
solid (0.57 g; 51 %). M. p. > 300 C. – IR (KBr): ν = 3200
(NH), 3060 (arom. CH), 1669 (C=O) cm−1. – 1H NMR
(300 MHz, [D6]DMSO): δ = 7.10 – 7.46 (m, 10H, 2PhH),
12.24 (s, 1H, NH, D2O-exchangeable). – MS (EI, 70 eV):
m/z(%) = 373 (14) [M]+. – C18H11N7OS (373.391): calcd.
C 57.90, H 2.97, N 26.26, S 8.59; found C 57.67, H 2.81,
N 26.09, S 8.45.
General procedure for the synthesis of ethyl 2-[2-cyano-2-
(2-arylhydrazono)acetamido]-4-phenyl-5-(phenylazo)thio-
phene-3-carboxylates 18a,b
Ethyl 2-[2-(4-oxothiazolidin-2-ylidene)acetamido]-4-
phenyl-5-(phenylazo)thiophene-3-carboxylate (15)
A solution of either aniline or the corresponding p-
chloro analog (0.0025 mol) in concentrated hydrochloric
acid (5 mL) was cooled to 0 – 5 ◦C with stirring. A sec-
ond well cooled saturated aqueous solution of sodium ni-
trite (0.0026 mol) was added portionwise to the first solution
Equimolecular amounts (0.002 mol) of the cyanomethy-
lene compound 6 and mercaptoacetic acid were heated at
reflux in 10 mL of acetic acid for 5 h. The solution was
then poured onto iced water and stirred for several hours
until crystallization was complete. The material which sep-
arated out was isolated by filtration, washed with an aqueous
sodium bicarbonate solution to remove the unreacted thio-
glycolic acid and dried. It was recrystallized from ethanol
to give the thiazolidinone derivative 15 as a yellow solid
◦
at 5 – 10 C, and the reaction mixture was stirred for 1 h at
the same temperature. Excess nitrous acid was decomposed
by the addition of urea, and the solution was cooled to 0 –
◦
5 C. The resulting clear diazonium salt solution was then
◦
added dropwise over 20 min with consta◦nt stirring and with
frequent addition of ice to a cold (0 – 5 C) stirred solution
of the coupling component cyanomethylene 6 (0.0023 mol)
dissolved in 20 mL of ethanol containing sodium acet◦ate
(0.013 mol) while maintaining the temperature at 0 – 5 C.
After addition of the diazonium salt, the mixture was stirred
for an additional 3 h at 5 – 10 ◦C. The precipitated product, in
each case separated upon dilution with cold water (30 mL),
was filtered off, washed with hot water and with cold wa-
ter, and dried. Recrystallization from the appropriate solvents
gave the hydrazones 18a (0.73 g; 61 %) and 18b (0.67 g;
52 %), respectively.
(0.65 g; 66 %). M. p. 215 – 217 C. – IR (KBr): ν = 3251,
3175 (2NH), 3066 (arom. CH), 1705 (ring C=O), 1664, 1656
(2C=O) cm−1. – 1H NMR (300 MHz, [D6]DMSO): δ = 1.20
(t, 3H, J = 7.2 Hz, ester Me), 3.99 (s, 2H, thiazolidine CH2),
4.33 (q, 2H, J = 7.2 Hz, ester CH2), 5.56 (s, 1H, methylenic
CH), 7.12 – 7.51 (m, 10H, 2PhH), 11.85 (s, br, 1H, thiazo-
lidine NH, D2O-exchangeable), 12.50 (s, br, 1H, CONH,
D2O-exchangeable). – 13C NMR (75 MHz, [D6]DMSO): δ =
14.0 (ester Me), 32.4 (thiazolidine CH2), 59.5 (ester CH2),
89.0 (methylenic CH), 109.2 (thiophene C-3), 127.1, 127.4,
127.8, 128.2, 128.7, 129.3, 132.0, 134.3, 141.6, 142.8, 149.7,
152.5, 165.3 (amide C=O), 166.5 (ester C=O), 174.6 (thi-
azolidine C=O). – C24H20N4O4S2 (492.570): calcd. C 58.52,
H 4.09, N 11.37, S 13.02; found C 58.32, H 3.98, N 11.19,
S 12.80.
Ethyl 2-[2-cyano-2-(2-phenylhydrazono)acetamido]-4-
phenyl-5-(phenylazo)thiophene-3-carboxylate (18a)
◦
Yellow powder (EtOH). M. p. 198 – 199 C. – IR (KBr):
Ethyl 2-(4-amino-3-phenyl-2-thioxo-2,3-dihydrothiazole-5-
carboxamido)-4-phenyl-5-(phenylazo)thiophene-3-carbox-
ylate (17)
ν = 3408 – 3281 (2NH), 3051 (arom. CH), 2218 (CN), 1662,
1655 (2C=O) cm−1. – 1H NMR (300 MHz, [D6]DMSO): δ =
1.32 (t, 3H, J = 7.2 Hz, ester Me), 4.28 (q, 2H, J = 7.2 Hz,
A mixture of the nitrile 6 (0.0025 mol), finely pow- ester CH2), 7.02 – 7.53 (m, 15H, 3PhH), 12.41 (s, br, 1H, NH,
dered elemental sulfur (0.0025 mol) and triethylamine D2O-exchangeable), 12.90 (s, br, 1H, NH, D2O-exchange-
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
able). – C28H22N6O3S (522.578): calcd. C 64.35, H 4.24, 2H, J = 7.2 Hz, ester CH2), 7.18 – 7.80 (m, 14H, 2PhH, ArH),
N 16.08, S 6.14; found C 64.17, H 4.11, N 15.86, S 5.98.
8.27 (s, 1H, coumarin 4-H), 12.46 (s, br, 1H, NH, D2O-ex-
changeable). – MS (EI, 70 eV): m/z(%) = 523 (28) [M]+. –
C29H21N3O5S (523.559): calcd. C 66.53, H 4.04, N 8.03,
S 6.12; found C 66.28, H 3.87, N 7.88, S 6.01.
Ethyl 2-{2-[2-(4-chlorophenylhydrazono)]-2-cyanoacet-
amido}-4-phenyl-5-(phenylazo)thiophene-3-carboxylate
(18b)
Antimicrobial activity
Orange crystals (DMF/H2O). M. p. 237 – 238 ◦C. – IR
(KBr): ν = 3400 – 3266 (2NH), 3059 (arom. CH), 2219
(CN), 1663, 1658 (2C=O) cm−1. – 1H NMR (300 MHz,
[D6]DMSO): δ = 1.27 (t, 3H, J = 7.2 Hz, ester Me), 4.36
(q, 2H, J = 7.2 Hz, ester CH2), 7.11 – 7.54 (m, 14H, 2PhH,
ArH), 12.58 (s, br, 1H, NH, D2O-exchangeable), 13.02 (s, br,
1H, NH, D2O-exchangeable). – C28H21ClN6O3S (557.023):
calcd. C 60.37, H 3.80, Cl 6.36, N 15.09, S 5.76; found
C 60.15, H 3.61, Cl 6.19, N 14.89, S 5.62.
The antimicrobial activity was investigated on eight newly
obtained phenylazo derivatives of condensed and uncon-
densed thiophenes. The antimicrobial profile was evalu-
ated by measuring the inhibitory effects of such compounds
against two Gram-negative (Escherichia coli ATTC 11775
and Pseudomonas aeruginosa ATTC 10145) and two Gram-
positive (Bacillus subtilis ATTC 6051 and Staphylococcus
aureus ATTC 12600) bacteria, two moulds (Aspergillus niger
and A. flavus) and two yeasts (Candida albicans ATTC
26555 and C. parapsilosis ATCC 22019) using the disk diffu-
sion technique. Streptomycin and amphoterisin B were used
as reference drugs. Solutions of the tested compounds and
reference drugs were prepared by dissolving 0.5 g of the
compound in DMSO (10 mL). The bacterial strains were
cultured on nutrient agar, Candida spp. were maintained on
Sabouraud dextrose agar, while Aspergillus spp. were main-
tained on Czapeck-Dox medium.
Ethyl 2-(5-imino-2,4-diphenyl-3-thioxo-2,3,4,5-tetrahydro-
1,2,4-triazine-6-carboxamido)-4-phenyl-5-(phenylazo)thio-
phene-3-carboxylate (19)
To equimolar amounts (0.003 mol) of compound 18a and
phenyl isothiocyanate in 15 mL of ethanol, triethylamine
(two drops) was added. The reaction mixture was heated at
reflux with stirring for 3 h. Upon cooling, the solvent was
removed under reduced pressure, and the residue was trit-
urated with diethyl ether and purified by recrystallization
from dimethylformamide to obtain the imino compound 19
as light-brown crystals (0.66 g; 34 %). M. p. 255 – 256 ◦C. –
IR (KBr): ν = 3384, 3275 (2NH), 3062 (arom. CH), 1660 –
1657 (2C=O) cm−1. – 1H NMR (300 MHz, [D6]DMSO):
δ = 1.35 (t, 3H, J = 7.2 Hz, ester Me), 4.42 (q, 2H, J =
7.2 Hz, ester CH2), 6.91 – 7.48 (m, 20H, 4PhH), 10.40 (s, 1H,
NH, D2O-exchangeable), 12.34 (s, br, 1H, CONH, D2O-ex-
changeable). – MS (EI, 70 eV): m/z(%) = 657 (15) [M]+. –
C35H27N7O3S2 (657.764): calcd. C 63.91, H 4.14, N 14.91,
S 9.75; found C 63.72, H 4.02, N 14.67, S 9.60.
Disk diffusion assay for bacteria: Disk diffusion test-
ing was performed by standard NCCLS methods [54] using
Mueller-Hinton plates supplemented with sheep blood and
inoculated with 0.5 McFarland standard. Disks with 10 µg of
the test compounds were applied. After overnight incubation
at 35 ◦C, zone diameters were measured.
Disk diffusion assay for fungi: The in vitro antifun-
gal activity of newly obtained phenylazo derivatives was
tested against two Aspergillus spp. (Aspergillus flavus and
Aspergillus niger). The test organisms were identified to
the species level by standard methods [55] and maintained
at −70 ◦C on Sabouraud dextrose agar slant until tested.
This assay employed an investigational method that used
the reference RPMI 1640 agar supplemented with 2 % glu-
cose as the test medium [56]. 10 µg mL−1 solutions of the
test compounds were prepared by using blank paper disks
(6.3 mm in diameter). The disks were allowed to dry at r. t.
prior to their use in disk diffusion assays. The inoculum den-
sity to be used in the test was adjusted spectrophotometri-
cally to 106 CFUmL−1. The previously prepared disks were
then placed on the inoculated plates, and the plates were in-
cubated at 35 ◦C. The results were examined after 5 d of in-
cubation, and the % of inhibition zone was compared to that
of the control.
Ethyl 2-(2-oxo-2H-chromene-3-carboxamido)-4-phenyl-5-
(phenylazo)thiophene-3-carboxylate (20)
To a mixture of equivalent amounts (0.0025 mol) of ni-
trile 6 and o-salicylaldehyde in ethanol (40 mL), piperidine
(3 drops) was added. The reaction mixture was refluxed with
stirring for 30 min. The solvent was evaporated to give a
residue, which was treated with ethanol (20 mL) and concen-
trated hydrochloric acid (0.75 mL). This mixture was heated
at reflux for 30 min. After cooling, the formed precipitate
was collected by filtration, washed with water, dried and re-
crystallized from dimethylformamide to obtain bright-yellow
Determination of minimum inhibitory concentration
crystals of compound 20 (0.92 g; 70 %). M. p. 246 – 249 ◦C. – (MIC): The MIC50 was defined as the lowest drug con-
IR (KBr): ν = 3307 (NH), 3058 (arom. CH), 1715 (lactone centration that inhibits growth by 50 %; serial dilutions (as
C=O), 1692, 1670 (2C=O) cm−1. – 1H NMR (300 MHz, µg mL−1) of compound 7 were prepared and tested against
[D6]DMSO): δ = 1.25 (t, 3H, J = 7.2 Hz, ester Me), 4.21 (q, P. aeruginosa.
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H. M. Gaber et al. · Phenylazo Derivatives of Condensed and Uncondensed Thiophene
595
Acknowledgements
ship. Financial support from Cardiff University, the Engi-
neering and Physical Sciences Research Council (EPSRC)
and the Biotechnology and Biological Sciences Research
Council (BBSRC), in collaboration with the University of
Wales College of Medicine, U. K., is gratefully acknow-
ledged.
The first author (H. M. Gaber) would like to express
his sincere gratitude to the School of Chemistry, Cardiff
University, U. K., for a Post-Doctoral Research Fellow-
ship grant and for all facilities provided to this fellow-
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