Synthesis and biological evaluation of novel 4-substituted 1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazides as new class of potential antiprotozoal agents.

Abstract

A novel series of 4-substituted 1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide, 4a-4n, was synthesized in 9-21% yield by the condensation of 4-(10,15,20-triphenylporphyrin-5-yl)benzaldehyde (3) with various substituted thiosemicarbazides in presence of catalytic amount of AcOH. These compounds were assayed for in vitro antiamoebic activity, and the results showed that out of 14 compounds 9 were found with IC(50) values lower than metronidazole corresponding to 1.05- to 4.7-fold increase in activity. MTT Assay showed that all the compounds are nontoxic to human kidney epithelial cell line. 4-(m-Toluidinyl)-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide (4h) showed the highest antiamoebic activity with least cytotoxicity. Some of the compounds were screened for their antimalarial activities and ability to inhibit beta-haematin formation, but none of them showed an activity better than chloroquine and quinine. Only one compound out of six showed an activity comparable to standard drug.

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Synthesis and Biological Evaluation of Novel 4-Substituted 1-{[4-(10,15,20-
Triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazides as New
Class of Potential Antiprotozoal Agents
by Abdul R. Bhata), Fareeda Athara)b), Robyn L. Van Zylc), Chien-Teng Chenc), and Amir Azam*a)
a) Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi-110025, India
(fax: þ91-11-26980229; e-mail: amir_sumbul@yahoo.co.in)
b) Present address: Center for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia,
Jamia Nagar, New Delhi-110025, India
c) Pharmacology Division, Department of Pharmacy and Pharmacology, University of the
Witwatersrand, Johannesburg 2193, South Africa
A novel series of 4-substituted 1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiose-
micarbazide, 4a–4n, was synthesized in 9–21% yield by the condensation of 4-(10,15,20-triphenylpor-
phyrin-5-yl)benzaldehyde (3) with various substituted thiosemicarbazides in presence of catalytic
amount of AcOH. These compounds were assayed for in vitro antiamoebic activity, and the results
showed that out of 14 compounds 9 were found with IC50values lower than metronidazole corresponding
to 1.05- to 4.7-fold increase in activity. MTTAssay showed that all the compounds are nontoxic to human
kidney epithelial cell line. 4-(m-Toluidinyl)-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methyl-
idene}thiosemicarbazide (4h) showed the highest antiamoebic activity with least cytotoxicity. Some of
the compounds were screenedfortheir antimalarialactivities and abilityto inhibitb-haematinformation,
but none of them showed an activity better than chloroquine and quinine. Only one compound out of six
showed an activity comparable to standard drug.
Introduction. – The unmet need for the development of efficacious drugs against
amoebiasis is high, since its etiologic agent Entamoeba histolytica infects 300–500
million people annually causing up to 100,000 deaths worldwide [1–3]. The pathogenic
trophozoites of E. histolytica have the ability to cause amoebic dysentery, liver abscess
[4], and brain abscess [5–7]. The current therapeutic alternative for antiamoebic
chemotherapy includes nitroimidazoles, mainly metronidazole and tinidazole [8][9].
The problem associated with these infections is compounded by the occurrence of
resistance to these available antiprotozoal drugs and the adverse effects associated with
these drugs [10–12]. To ensure successful treatment, novel drugs are required to target
selective pathways to inhibit these resistant strains.
Porphyrins are of special interest in a variety of fields including biomedical imaging,
human immunodeficiency virus (HIV) and cancer research [13–16]. Due to their
unique electronic structure, they have been widely used as photosensitizers in the
treatment of neoplastic tissues [17–20]. These sensitizers? architecture required the
formation of unsymmetrically substituted tetra-mesophenyl porphyrins (TPPs) [21].
They have also the ability to photoinduce highly antibiotic-resistant Gram-positive and
Gram-negative bacteria [22]. These macrocycles prevent haemozoin production
(malaria pigment) by forming a heme–drug complex through p–p interactions
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 764
? 2008 Verlag Helvetica Chimica Acta AG, Z?rich

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[23][24], and thus, appear to be the ideal system to design an effective antimalarial
agent [25–28]. Thiosemicarbazides are rather small molecules and have great
importance due to their biological activities [29–33]. Our preceding studies on
thiosemicarbazides [34–36] showed that the compounds with cyclic and aromatic
substituents were better inhibitors of growth of E. histolytica. These studies have
encouraged us to synthesize a new series of compounds, viz. 4-substituted 1-{[4-
(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazides 4a–4n, which
possess unsymmetrical as well as core-extended properties of porphyrins. The
compounds were screened for in vitro antiamoebic activity against HM1:IMSS strain
of E. histolytica. Although porphyrins have been extensively screened for many
biological studies, they have not yet been explored for antiamoebic activity. The
promising results showed that 4a–4n might be used as potential antiamoebic agents.
We also screened few compounds for antimalarial activity, but, among six compounds,
only one compound was at par with chloroquine. Therefore, we did not screen more
compounds for antimalarial activity. They were also tested against human kidney
epithelial cell line to evaluate their toxicity. None of the compounds inhibited cell
growth at a concentration of 100 mm, and, therefore, were nontoxic to human cells.
Results and Discussion. – Synthesis. Two routes were proposed for the synthesis of
porphyrin thiosemicarbazides, as shown in the Scheme. Compound 1 was obtained by
using glycol to protect commercially available terephthalaldehyde. In Route 2, 1 was
condensed with thiosemicarbazide to yield A. The problem arose in the attempted
deprotection of compound A. It was neither soluble in organic solvents nor in H2O. A
number of reagents such as HCl, BF3·Et2O, CF3COOH (TFA), and cyclodextrin [37]
were used to deprotect these compounds in suspension, but all failed to do so. Thus,
only Route 1 was employed for the synthesis of desired compounds.
The unsymmetrical porphyrin was prepared by Lindsey?s method [38] with a
protected aldehyde group present in the para-position of one Ph ring. It was observed
that the quantity of acid affects the yield of the desired compound, which can alter the
degree of deprotection of the terephthalaldehyde. The synthesized porphyrin 2 was
purified by column chromatography and deprotected by treatment with TFA. The 4-
(10,15,20-triphenylporphyrin-5-yl)benzaldehyde (3) was condensed with various
thiosemicarbazides with AcOH as a catalyst to obtain the porphyrin thiosemicarba-
zides 4a–4n. The products were eluted with CHCl3/hexane 95 :5, and the structures
were elucidated by UV/VIS, IR, and1H-NMR spectroscopy, and mass spectrometry.
The formation of compound 3 from 2 was confirmed by their IR spectra. The
characteristic aldehyde bands were found in compound 3 at 1723 and 2894 cm?1,
indicating the deprotection of 5-[4-(1,3-dioxolan-2-yl)phenyl]-10,15,20-triphenylpor-
phyrin (2). The characteristic aldehyde bands were absent in the porphyrin
thiosemicarbazones 4a–4n. However, three sharp bands for n ˜ (N?H) at 3110–
3198 cm?1, n ˜ (C¼N) at 1608–1656 cm?1, and n ˜ (C¼S) at 1110–1152 cm?1were
observed in the IR spectra of compounds 4a–4n. The electronic spectra of 4a–4n
showed characteristic bands for C¼S at 278–260 nm due to n!p* transition and for
C¼N at 212–202, 230–218 nm due to n!s* and n!p* transitions, respectively. In
addition, the typical split Soret bands around 418–427 and 443–460, and two intense
(IV, III) and two less intense (II, I) visible Q bands were recorded in MeOH (Table 1).
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All the meso-substituted porphyrins gave etio spectra (eIV>eIII>eII>eI), and the
relative intensities of the QIand QIIbands show a standard type (eII/eI<1). The
extremely broadened split of the Soret band was due to the cofacial and edge-to-edge
interaction of self assembled aggregates [39]. The Soret bands were blue-shifted, which
could be attributed to face-to-face orientation of the porphyrin [40].
Spectroscopy was used for the characterization of the protected and unprotected
porphyrin aldehydes 2 and 3, respectively, and the meso-substituted porphyrins 4a–4n.
The appearance of a singlet at 9.46 ppm in the spectrum of 3, a characteristic aldehyde
signal, and its disappearance in the spectra of 4a–4n, indicates the deprotection of the
1H-NMR
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 766
Scheme
i) CH2Cl2, BF3·Et2O, DDQ, Et3N. ii) CH2Cl2, CF3COOH (TFA)/H2O, 16 h. iii) EtOH (abs.), AcOH,
reflux.

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acetal group of 2 in the former case and the formation of thiosemicarbazide in the
latter. The presence of one para-substituted Ph group reduces the symmetry of the
overall molecule, leading to the differentiation of the b-pyrrolic1H of the compound(s)
4a–4n, whose meso-positions were differently substituted with three identical meso-
phenyl groups. Thus, the b-pyrrolic H-atom signal splits into four doublets (with a J
value around 4.7 Hz) and two singlets. The appearance of a signal at 11.53–13.89 ppm
in the spectra of 4a–4n confirms the formation of a thiosemicarbazide, as the signal
corresponds to the C¼NH H-atom of the side chain of the porphyrin. The NH signal of
the porphyrin ring appears on the right side of the one of TMS between ?1.86 and ?
2.82 ppm.
Biological Activities. – Antiamoebic Activities. In vitro antiamoebic activity was
determined by using HM1:IMSS strain of E. histolytica cultured in TYIS-33 growth
medium, to which the compounds were added in a small amount of DMSO. The results
of the bioassays are collected in Table 2. The data are presented in terms of percent
growth inhibition relative to untreated controls, and plotted as probit values as a
function of drug concentration. The IC50values were interpolated in the corresponding
dose-response curves. The IC50values for compounds 4a (0.41 mm), 4b (0.70 mm), 4d
(1.07 mm), 4e (1.62 mm), 4g (0.49 mm), 4h (0.38 mm), 4i (0.42 mm), 4j (0.66 mm), 4m
(1.71 mm), and 4n (0.61 mm) were lower than metronidazole (1.8 mm), corresponding to
1.05- to 4.7-fold increase in activity. All these compounds were also more active than
the parent porphyrins 2 and 3. The results were statistically evaluated by analyses of
variance. The null hypothesis was tested using the t-test, and the significance of the
differences between the IC50value(s) of metronidazole vs. 4a–4n was evaluated. The
calculated t-values were higher than the values given in Table 2 at the 4% level. Hence,
the character under study was influenced by the treatment. The results showed that the
electron-donating groups attached to N(4) of the thiosemicarbazide moiety increase
the antiamoebic activity. However, compounds with cycloalkanes attached to N(4)
Table 1. UV/VIS Band Maxima and Extinction Coefficients for Porphyrins 4a–4n in MeOH (lmax[nm]
(e?10?3[cm?1mol?1l]))
Compound Soret 1Soret 2IVIII III
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
418 (112.9)
422 (81.2)
427 (91.5)
423 (81.9)
424 (65.5)
425 (70.2)
421 (85.5)
426 (99.9)
419 (118.5)
418 (120)
420 (95.5)
420 (92.8)
426 (95.1)
423 (80.3)
444 (51.9)
446 (61.4)
452 (47.8)
444 (52.2)
460 (47.2)
459 (48.9)
448 (79.1)
445 (63.4)
447 (62.1)
445 (70.5)
448 (80.1)
450 (46.1)
451 (49.2)
443 (50.9)
517 (29.8)
518 (30.5)
510 (36.9)
510 (35.3)
516 (18.9)
519 (26.4)
511 (36.1)
515 (39.4)
512 (20.5)
517 (29.5)
517 (18.5)
519 (26.9)
520 (27.1)
515 (40.1)
554 (25.7)
538 (15.5)
543 (20.8)
536 (29.3)
553 (21.4)
535 (19.1)
550 (22.8)
555 (27.3)
540 (17.9)
537 (16.3)
536 (13.3)
534 (24.8)
538 (18.2)
545 (18.7)
594 (4.1)
585 (7.9)
582 (6.1)
583 (5.8)
590 (2.3)
586 (4.3)
581 (6.8)
595 (4.5)
580 (6.5)
586 (3.2)
587 (5.1)
584 (4.2)
592 (6.7)
589 (5.7)
632 (6.1)
632 (6.8)
632 (3.3)
635 (2.8)
641 (4.2)
642 (2.1)
640 (1.7)
630 (2.3)
633 (5.9)
636 (2.3)
636 (1.8)
638 (5.4)
643 (2.5)
637 (2.6)
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CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)768
Table 2. In vitro Antiamoebic Activity against E. histolytica HM1:IMSS Strain and Cytotoxicity Profile
of Porphyrin Thiosemicarbazides
ProductR’/RAntiamoebic
activity IC50[mm]
Toxicity profile
IC50[mm]
Safety index
SIa)
2
>1.80?0.63
>100
>55.5
3
CHO
>1.80?0.69
1.41?0.41
>100
>55.5
4a
>100
>70.9
4b
0.70?0.59
>100
>143
4c
>1.8?0.71
>100
>55.5
4d
1.07?0.83
>100
>93.4
4e
1.62?0.53
>100
>61.7
4f
9.79?0.62
>100
>10.2
4g
0.49?0.21
>100
>204
4h
0.38?0.26
>100
>263
4i
0.42?0.32
>100
>238

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show better antiamoebic activity in comparison to those with a Me group attached to
N(4). The compound 4h with an electron-withdrawing group attached to N(4) showed
the best antiamoebic activity.
Antimalarial Activities. The porphyrin thiosemicarbazides displayed variable
activities against the Plasmodium protozoa (IC50 values ranging from 4.36 to
30.61 mm). These novel compounds were, however, not comparable with chloroquine
and quinine (Table 3). On assessment of a possible mechanism by which these
compounds inhibited parasite growth, it was initially proposed that these porphyrin
thiosemicarbazides would interfere with b-haematin formation. Of the six compounds,
only compounds 4h and 4j were able to show some degree of inhibition, with compound
4h being 94.4% as effective as chloroquine; whilst compound 4j was only 8.1% as active
as chloroquine. Thus it appears that overall the porphyrin thiosemicarbazide structure
could not hinder the planar p–p interactions between adjacent ferriprotoporphyrin IX
units. Compound 4h with a Me group on the cycloalkyl moiety attached to N(4) was as
effective as chloroquine in interfering with the ferriprotoporphyrin IX subunits from
forming the b-haematin crystalline structure. It is possible that the orientation of this
compound and that of the Me group allowed for cofacial p–p binding to occur between
the porphyrin structure and the ferriprotoporphyrin IX unit. Protoporphyrins IX,
which contain metal ions are able to inhibit the formation of b-haematin, with GaII,
SnIV, ZnII, and MgIIhaving comparable inhibitory properties as chloroquine; even
though they are unable to effectively inhibit parasite growth (at least a 1000 times less
active) [41]. The decrease in inhibition could be attributed to decreased accumulation
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)769
Table 2 (cont.)
ProductR’/RAntiamoebic
activity IC50[mm]
Toxicity profile
IC50[mm]
Safety index
SIa)
4j
0.66?0.29
>100
>151
4k
7.27?0.41
>100
>13.7
4l
>1.8?0.51
>100
>55.5
4m
1.71?0.34
>100
>58.4
4n
0.61?0.19
>100
>164
Metronidazole1.80?0.39
>100
>55.5
a) SI¼Toxicity IC50/antiamoebic activity IC50.

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of the large porphyrin molecule into the malaria parasite. In spite of the malaria
parasite incorporating new permeation pathways into the erythrocytic membranes, a
channel or uptake pathway for such large molecules is not generally accepted [42].
Nevertheless, the presence of a parasitophorous duct has been proposed to link the
extracellular environment with the parasitophorous vacuole which immediately
surrounds the parasite, and these compounds could then be endocytosed into the food
vacuole of the parasite to interact with haemozoin formation [43].
Toxicity Profile. To ensure that the porphyrin thiosemicarbazides were not toxic to
human cells, they were tested against a human kidney epithelial cell line. None of the
compounds inhibited cell growth at a concentration of 100 mm (Table 2). To investigate
the selectivity of the compounds, the ?safety index? (SI) was calculated and defined as
toxicity IC50/protozoal IC50, where toxicity IC50is the concentration of compound that
kills 50% of the human (kidney epithelial) cell line, and protozoal IC50 is the
concentration that kills 50% of either the malaria or amoeba protozoa. This allows an
estimate of which compounds might be efficacious or toxic against human cells and
potentially in vivo. The numerical results for each compound are given in Table 2.
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008) 770
Table 3. In vitro Antimalarial Activity against P. falciparum, Cytotoxicity Profile, and b-Haematin
Inhibition Activity of Porphyrin Thiosemicarbazides
ProductR Antimalarial
activity
IC50[mm]
Toxicity
profile
IC50[mm]
Safety
index
SI 1a)
b-Haematin
inhibitory activity
(relative to
chloroquine)
4b
8.02?1.13
>100
>12.5
<0.05
4d
5.36?0.10
>100
>18.7
<0.05
4f
30.61?3.20
>100
>3.27
<0.05
4h
5.66?0.50
>100
>17.70.94
4j
4.36?0.08
>100
>22.90.08
4k
23.15?6.19
>100
>4.32
<0.05
Chloroquine
Quinine
0.12?0.01
0.10?0.01
>240
>370
>833
>1000
1.00
0.51
a) SI 1¼Toxicity IC50/antimalarial IC50.

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These results show that the porphyrin thiosemicarbazide 4h has lowest cytotoxicity and
highest antiamoebic activity, and all compounds show more favorable safety profile
along with the most promising antiamoebic activity. Thus, the accumulation of the
porphyrin thiosemicarbazides will remain toxic to the parasite, whilst, in the human
host, there will be a decreased likelihood of toxicity.
Conclusions. – 4-Substituted 1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]meth-
ylidene}thiosemicarbazides 4a–4n were synthesized according to Lindsay?s method in
9–21% yields. All of the compounds were characterized by using spectroscopic
techniques. The cell-culture studies showed that these compounds possessed high in
vitro antiamoebic activities against E. histolytica. The IC50values for compounds 4a, 4b,
4d, 4e, 4g, 4h, 4i, 4k, 4m, and 4n were lower than that of metronidazole (1.8 mm)
corresponding to 1.05- to 4.7-fold increase in activity, thus proving them to be better
inhibitors of E. histolytica growth. In general, the compounds with electron-with-
drawing groups attached to N(4) showed better antiamoebic activity. In addition,
compound 4h is approximately five times more potent than metronidazole in inhibiting
E. histolytica growth with least cytotoxicity and warrants further investigation.
Experimental Part
General. CH2Cl2was pretreated with molecular sieves (4 ?). Moisture and photosensitive reactions
were conducted in oven-dried glassware under Ar and in a dark chamber. All chemicals were purchased
from Sigma-Aldrich (USA). Anal. TLC: precoated silica gel 60 F254plates. Flash column chromatog-
raphy (FC): silica gel 60 (200–400 mesh). Electronic spectra: in MeOH on a Shimadzu UV-1601PC UV/
VIS spectrophotometer; l in nm. IR Spectra (KBr): were recorded on a Perkin Elmer model 1620 FT-IR
spectrophotometer; in cm?1.1H-NMR Spectra: unless otherwise noted, in CDCl3at ambient temp.,
Bruker Spectrospin DPX-300 spectrophotometer with TMS as an internal standard; chemical shifts d in
ppm, J in Hz. ESI-MS: Micromass Quattro II triple quadrupole mass spectrometer; in m/z. Elemental
analyses: the Central Drug Research Institute (Lucknow, India); the results were within ?0.4% of the
theoretical values.
Synthesis of 5-[4-(1,3-Dioxolan-2-yl)phenyl]-10,15,20-triphenylporphyrin (2). In 500 ml of CH2Cl2,
dried over molecular sieves, a mixture of 4-(1,3-dioxolan-2-yl)benzaldehyde (1; 1 equiv.), benzaldehyde
(3 equiv.), and 1H-pyrrole (4 equiv.) was stirred at r.t. After 30 min, BF3·Et2O (cat. amount) was added,
and the mixture was stirred overnight before addition of DDQ (3 equiv.). After 2 h, the reaction was
quenched by the addition of Et3N. The resulting mixture was filtered and washed with H2O and CHCl3to
remove the H2O-soluble, green-colored compound. The org. layer was collected and dried (Na2SO4).
Removal of the solvent in vacuo gave a crude solid product. The solid was purified by CC (SiO2, CHCl3):
2 (23%). IR: 3395 (N?H), 2940, 2853 (C?H), 1583 (C¼N), 1564, 1540, 1433 (C¼C), 1076 (C?N).
1H-NMR (CDCl3): ?2.43 (s, 2 NH); 2.85 (s, 2 CH2O); 3.20 (s, OCHO); 7.39–7.76 (m, 19 arom. H); 7.89
(s, 2 H, pyrrole); 7.97 (d, J¼4.3, 1 H, pyrrole); 8.12 (d, J¼4.3, 1 H, pyrrole); 8.19 (s, 2 H, pyrrole); 8.26
(d, J¼4.3, 1 H, pyrrole); 8.38 (d, J¼4.3, 1 H, pyrrole). Anal. calc. for C47H34N4O2: C 82.02, H 4.25, N
8.14; found: C 82.02, H 4.23, N 8.15.
Synthesis of 4-(10,15,20-Triphenylporphyrin-5-yl)benzaldehyde (3). Compound 3 was prepared
according to a standard procedure [38]. Compound 2 in CH2Cl2was treated with TFA/H2O at r.t. for
16 h. The soln. was taken up in additional CH2Cl2. The org. layer was washed with 5% aq. NaHCO3,
followed by H2O, dried (Na2SO4), and chromatographed over silica gel (CHCl3/MeOH): 3 (52%). IR:
3443 (N?H), 2890 (CO?H), 1723 (C¼O), 1597 (C¼N), 1566, 1558, 1441 (C¼C), 1098 (C?N), 800
(1,4-disubstituted benzene ring).1H-NMR (CDCl3): ?2.76 (s, 2 NH); 7.51–7.84 (m, 19 arom. H); 7.95 (s,
2 H, pyrrole); 8.09 (d, J¼4.3, 1 H, pyrrole); 8.16 (d, J¼4.3, 1 H, pyrrole); 8.23 (s, 2 H, pyrrole); 8.29 (d,
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)771

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J¼4.3, 1 H, pyrrole); 8.36 (d, J¼4.3, 1 H, pyrrole); 9.46 (s, 1 H, CHO). Anal. calc. for C45H30N4O: C
83.90, H 4.69, N 8.70; found: C 83.89, H 4.66, N 8.67.
Synthesis of Thiosemicarbazides. Substituted thiosemicarbazides were prepared by a two-step
synthetic route as reported in [44].
General Procedure for the Synthesis of 4-Substituted 1-{[4-(10,15,20-triphenylporphyrin-5-yl)phe-
nyl]methylidene}thiosemicarbazides 4a–4n. Thiosemicarbazides (1 equiv.) were dissolvedin 10 ml of abs.
EtOH, followed by the addition of a mixture of 3 (1 equiv.) in EtOH. Then, AcOH was added in cat.
amount to increase the yield of the reaction [45]. The mixture was heated to reflux at 608 for 12 h. The
resultant mixture was evaporated in vacuo to give crude solid products, which were further purified by
CC (CH2Cl2/MeOH) to yield compounds 4a–4n.
4-Cyclopentyl-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide (4a).
Yield: 15%. UV/VIS: 209, 226, 261. IR: 3149 (NH), 1633 (C¼N), 1131 (C¼S).1H-NMR (CDCl3): ?
1.86 (s, 2 NH, pyrrole); 2.82–2.93 (m, 4 CH2); 3.95–4.05 (m, 1 H, NCH2); 6.7 (s, CH¼N); 6.85–7.57 (m,
19 arom. H); 7.97 (d, J¼4.7, 1 H, pyrrole); 8.09 (d, J¼4.7, 1 H, pyrrole); 8.15 (d, J¼4.7, 1 H, pyrrole);
8.25 (s, NH); 8.57 (s, 2 H, pyrrole); 8.63 (s, 2 H, pyrrole); 8.91 (d, J¼4.7, 1 H, pyrrole); 11.74 (s, NNH).
ESI-MS: 759.9 (Mþ; calc. 759.0). Anal. calc. for C49H41N7S: C 75.09, H 5.27, N 12.51; found: C 75.02, H
5.23, N 12.48.
4-Cyclohexyl-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide (4b).
Yield: 15%. UV/VIS: 211, 220, 270. IR: 3142 (NH), 1656 (C¼N), 1127 (C¼S).1H-NMR (DMSO):
?2.64 (s, 2 NH); 2.29–2.50 (m, 5 CH2); 4.03–4.12 (m, NCH); 6.87 (s, CH¼N); 7.13–7.93 (m, 19 arom.
H); 8.07(d, J¼4.5, 1 H, pyrrole); 8.26 (d, J¼4.4, 1 H, pyrrole); 8.31 (d, J¼4.5, 1 H, pyrrole); 8.43 (s, 2 H,
pyrrole); 8.67 (d, J¼4.5, 1 H, pyrrole); 8.91 (s, 2 H, pyrrole); 12.17 (s, NNH). ESI-MS: 797.53 (Mþ; calc.
797.34). Anal. calc. for C52H43N7S: C 78.12, H 5.43, N 12.27; found: C 78.14, H 5.41, N 12.23.
4-Cyclooctyl-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide (4c).
Yield: 16%. UV/VIS: 207, 225, 272. IR: 3135 (NH), 1643 (C¼N), 1119 (C¼S).1H-NMR (CDCl3): ?
2.58 (s, 2 NH, pyrrole); 2.09–2.71 (m, 6 CH2); 2.79–2.81 (m, CH); 6.58–7.12 (m, 19 arom. H); 7.34 (s,
CH¼N); 7.43 (d, J¼4.5, 1 H, pyrrole); 7.61 (d, J¼4.5, 1 H, pyrrole); 7.89 (s, NH); 7.95 (s, 2 H, pyrrole);
8.14 (d, J¼4.5, 1 H, pyrrole); 8.29 (s, 2 H, pyrrole); 8.36 (d, J¼4.5, 1 H, pyrrole); 11.53 (s, NNH). ESI-
MS: 801.6 (Mþ; calc. 801.0). Anal. calc. for C52H47N7S: C 75.26, H 5.74, N 11.87; found: C 75.21, H 5.76, N
11.82.
N’-{[4-(10,15,20-Triphenylporphyrin-5-yl)phenyl]methylidene}pyrrolidine-1-carbothiohydrazide
(4d). Yield: 13%. UV/VIS: 203, 228, 265. IR: 3110 (NH), 1636 (C¼N), 1148 (C¼S).1H-NMR (CDCl3):
?2.41 (s, 2 NH); 2.45–2.39 (m, 2 CH2); 3.41–3.36 (m, 2 CH2); 7.16 (s, CH¼N); 7.21–7.87 (m, 19 arom.
H); 7.93 (s, 2 H, pyrrole); 8.05 (d, J¼4.9, 1 H, pyrrole); 8.31 (s, 2 H, pyrrole); 8.41 (d, J¼4.9, 1 H,
pyrrole); 8.81 (d, J¼4.9, 1 H, pyrrole); 8.93 (d, J¼4.9, 1 H, pyrrole); 12.71 (s, NNH). ESI-MS: 769.62
(Mþ; calc. 769.31). Anal. calc. for C50H39N7S: C 77.86, H 5.10, N 12.71; found: C 77.84, H 5.12, N 12.69.
4-Methyl-N’-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}piperazine-1-carbothiohy-
drazide (4e). Yield: 19%. UV/VIS: 211, 221, 262. IR: 3154 (NH), 1632 (C¼N), 1136 (C¼S).1H-NMR
(CDCl3): ?1.94 (s, 2 NH, pyrrole); 1.23 (d, Me); 1.9–2.0 (m, CH); 2.21–2.68 (m, 4 CH2); 7.28 (s,
CH¼N); 7.57–7.91 (m, 19 arom. H); 7.97 (d, J¼4.6, 1 H, pyrrole); 8.09 (d, J¼4.6, 1 H, pyrrole); 8.15 (d,
J¼4.6, 1 H, pyrrole); 8.23 (s, 2 H, pyrrole); 8.33 (d, J¼4.6, 1 H, pyrrole); 8.39 (s, 2 H, pyrrole); 12.73 (s,
NNH). ESI-MS: 797.6 (Mþ; calc. 797.0.). Anal. calc. for C52H43N7S: C 78.29, H 5.43, N 12.29; found: C
78.25, H 5.47, N 12.31.
4-Cyclohexyl-4-methyl-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarba-
zide (4f). Yield: 18%. UV/VIS: 209, 230, 271. IR: 3157 (NH), 1633 (C¼N), 1110 (C¼S).1H-NMR
(DMSO): ?2.69(s, 2 NH); 1.23(d, Me); 2.39–2.56(m, 5 CH2); 4.20–4.32(m, N?CH); 6.91(s, CH¼N);
7.37–7.93 (m, 19 arom. H); 8.16 (d, J¼4.7, 1 H, pyrrole); 8.23 (d, J¼4.7, 1 H, pyrrole); 8.45 (d, J¼4.7,
1 H, pyrrole); 8.53 (s, 2 H, pyrrole); 8.62 (d, J¼4.7, 1 H, pyrrole); 8.89 (s, 2 H, pyrrole); 12.97 (s, NNH).
ESI-MS: 811.75 (Mþ; calc. 811.36). Anal. calc. for C53H45N7S: C 78.25, H 5.58, N 12.05; found: C 78.22, H
5.56, N 12.02.
4-(2-Methylphenyl)-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide
(4g). Yield: 9%. UV/VIS: 203, 222, 262. IR: 3198 (NH), 1656 (C¼N), 1121 (C¼S).1H-NMR (CDCl3):
?2.18 (s, 2 NH, pyrrole); 2.50 (s, Me); 6.95–7.34 (m, 23 arom. H); 7.12 (s, CH¼N); 7.65 (s, 2 H, pyrrole);
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)772

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7.95 (d, J¼4.5, 1 H, pyrrole); 8.15 (s, 1 H, NH); 8.17 (d, J¼4.9, 1 H, pyrrole); 8.39 (d, J¼4.9, 1 H,
pyrrole); 8.47 (d, J¼4.9, 1 H, pyrrole); 8.68 (s, 2 H, pyrrole); 11.93 (s, NNH). ESI-MS; 805.2 (Mþ; calc.
805.0). Anal. calc. for C53H39N7S: C 78.85, H 4.87, N 12.15; found: C 78.83, H 4.85, N 12.10.
4-(3-Methylphenyl)-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide
(4h). Yield: 20%. UV/VIS: 205, 223, 268. IR: 3139 (NH), 1621 (C¼N), 1130 (C¼S).1H-NMR (CDCl3):
?2.13 (s, 2 NH); 2.77 (s, Me); 6.4 (s, CH¼N); 6.68–7.73 (m, 23 arom. H); 7.84 (d, J¼4.7, 1 H, pyrrole);
8.05 (s, 1 H, NH); 8.13 (d, J¼4.7, 1 H, pyrrole); 8.25 (d, J¼4.7, 1 H, pyrrole); 8.46 (s, 2 H, pyrrole); 8.57
(s, 2 H, pyrrole); 8.79 (d, J¼4.7, 1 H, pyrrole); 11.68 (s, NNH). ESI-MS: 806.8 (Mþ; calc. 806.53). Anal.
calc. for C53H39N7S: C 78.85, H 4.87, N 12.15; found: C 78.82, H 4.90, N 12.11.
4-(4-Methylphenyl)-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide
(4i). Yield: 21%. UV/VIS: 212, 228, 275. IR: 3139 (NH), 1621 (C¼N), 1130 (C¼S).1H-NMR (CDCl3):
?2.82 (s, 2 NH, pyrrole); 2.05 (s, Me); 7.12–7.73 (m, 23 arom. H); 7.13 (s, CH¼N); 7.69 (d, J¼4.9, 1 H,
pyrrole); 7.78 (d, J¼4.9, 1 H, pyrrole); 8.05 (d, J¼4.9, 1 H, pyrrole); 8.31 (s, NH); 8.37 (s, 2 H, pyrrole);
8.46 (s, 2 H, pyrrole); 8.56 (d, J¼4.9, 1 H, pyrrole); 12.9 (s, NNH). ESI-MS: 805.6 (Mþ; calc. 805.0).
Anal. calc. for C53H39N7S: C 78.85, H 4.87, N 12.15; found: C 78.83, H, 4.91, N 12.17.
4-(2-Chlorobenzyl)-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide
(4j). Yield: 17 %. UV/VIS: 210, 229, 278. IR: 3121 (NH), 1608 (C¼N), 1152 (C¼S).
(DMSO): ?2.62 (s, 2 NH); 2.77 (s, Me); 6.76 (s, CH¼N); 6.88–7.43 (m, 23 arom. H); 7.91 (d, J¼4.7, 1 H,
pyrrole); 8.13 (d, J¼4.7, 1 H, pyrrole); 8.32 (d, J¼4.7, 1 H, pyrrole); 8.35 (s, 2 H, pyrrole); 8.43 (s, 2 H,
pyrrole); 8.52 (s, NH); 8.68 (d, J¼4.7, 1 H, pyrrole); 13.89 (s, NNH). ESI-MS: 839.63 (Mþ; calc. 839.80).
Anal. calc. for C53H38ClN7S: C 75.61, H 4.55, N 11.65; found: C 75.61, H 4.53, N 11.67.
4-Methyl-4-phenyl-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide
(4k). Yield: 23%. UV/VIS: 202, 224, 263. IR: 3157 (NH), 1612 (C¼N), 1150 (C¼S).1H-NMR (CDCl3):
?2.52 (s, 2 NH); 2.18 (s, Me); 7.05 (s, CH¼N); 7.13–7.75 (m, 24 arom. H); 7.85 (d, J¼4.5, 1 H, pyrrole);
8.12 (s, 2 H, pyrrole); 8.26 (d, J¼4.5, 1 H, pyrrole); 8.46 (d, J¼4.5, 1 H, pyrrole); 8.53 (s, 2 H, pyrrole);
8.86 (d, J¼4.5, 1 H, pyrrole); 12.04 (s, NNH). ESI-MS: 806.73 (Mþ; calc. 806.53). Anal. calc. for
C53H39N7S: C 78.85, H 4.87, N 12.15; found: C 78.83, H 4.84, N 12.11.
4-Benzyl-4-methyl-1-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}thiosemicarbazide
(4l). Yield: 21%. UV/VIS: 206, 218, 277. IR: 3131 (NH), 1627 (C¼N), 1137 (C¼S).1H-NMR (CDCl3):
?2.74 (s, 2 NH, pyrrole); 2.71 (s, Me); 4.9 (d, NCH2); 7.3 (s, CH¼N); 7.41–7.73 (m, 24 arom. H); 7.82 (s,
2 H, pyrrole); 7.95 (d, J¼4.7, 1 H, pyrrole); 8.07 (d, J¼4.7, 1 H, pyrrole); 8.35 (d, J¼4.7, 1 H, pyrrole);
8.39 (s, 2 H, pyrrole); 8.58 (d, J¼4.7, 1 H, pyrrole); 12.81 (s, NNH). ESI-MS: 795.5 (Mþ; calc. 795.0).
Anal. calc. for C52H41N7S: C 76.19, H 5.04, N 11.96; found: C 76.17, H 5.07, N 11.91.
4-Phenyl-N’-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}piperazine-1-carbothiohy-
drazide (4m). Yield: 14%. UV/VIS: 202, 226, 278. IR: 3179 (NH), 1651 (C¼N), 1141 (C¼S).1H-NMR
(CDCl3): ?2.68 (s, 2 NH, pyrrole); 2.49–2.65 (m, 2 CH2?N?CH2); 6.43 (s, CH¼N); 6.53–7.12 (m, 24
arom. H); 7.58 (d, J¼4.3, 1 H, pyrrole); 7.91 (d, J¼4.3, 1 H, pyrrole); 8.03 (d, J¼4.3, 1 H, pyrrole); 8.25
(s, 2 H, pyrrole); 8.39 (s, 2 H, pyrrole); 8.56 (d, J¼4.3, 1 H, pyrrole); 11.93 (s, NNH). ESI-MS: 836.8 (Mþ;
calc. 836.0). Anal. calc. for C54H44N8S: C 75.34, H 5.15, N 13.02; found: C 75.29, H 5.12, N 13.04.
1,2,3,4-Tetrahydro-N’-{[4-(10,15,20-triphenylporphyrin-5-yl)phenyl]methylidene}quinoline-1-carbo-
thiohydrazide (4n). Yield: 17%. UV/VIS: 212, 220, 260. IR: 3169 (NH), 1621 (C¼N), 1113 (C¼S).
1H-NMR (CDCl3): ?2.29 (s, 2 NH, pyrrole); 4.08 (m, 3 CH2); 6.4 (s, CH¼N); 7.73–6.68 (m, 26 arom.
H); 7.84 (d, J¼4.7, 1 H, pyrrole); 8.05(s, NH); 8.13(d, J¼4.7, 1 H, pyrrole); 8.25 (d, J¼4.7, 1 H, pyrrole);
8.46 (s, 2 H, pyrrole); 8.57 (s, 2 H, pyrrole); 8.79 (d, J¼4.7, 1 H, pyrrole); 11.68 (s, NNH). ESI-MS: 807.4
(Mþ; calc. 807.0). Anal. calc. for C53H41N7S: C 76.53, H 4.97, N 11.79; found: C 76.56, H 4.93, N 11.76.
Antiamoebic Activity. The compounds 4a–4n were screened in vitro for antiamoebic activity against
the HM1:IMSS strain of E. histolytica by the microdilution method [46]. E. histolytica trophozoites were
cultured in TYIS-33 growth medium as described previously, in a 96-well microtiter plate (Costar) [47].
The test compounds were dissolved in DMSO (40 ml), at which level no inhibition of the amoeba
occurred [48][49]. Culture medium was then added to obtain a concentration of 1 mg/ml, and twofold
serial dilutions were made. Each test included metronidazole as a standard amoebic drug, as well as
control wells (culture medium plus amoeba) and a blank (culture medium only). The number of amoeba
per milliliter was estimated with a haemocytometer, and trypan blue exclusion was used to confirm
1H-NMR
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viability. The cell suspension used was diluted to 105organisms/ml by adding fresh medium, and 170 ml of
this suspension was added to the test and control wells in the plate. An inoculum of 1.7?104organisms/
well was chosen, so that confluent, but not excessive, growth took place. The plates were sealed and
gassed for 10 min with N2, and then incubated at 378 for 72 h. After incubation, the growth of the amoeba
was checked with a low power microscope. Inverting the plate and shaking gently removed the culture
medium. The plates were immediately washed with 0.9% aq. NaCl soln. at 378. This procedure was
performed quickly, and the plate was not allowed to cool, to prevent the detachment of amoebae. After
the plate was dried at r.t., the amoebas were fixed with chilled MeOH, and the plates were kept on ice for
15 min, before being dried and stained with 0.5% aq. eosin for 15 min. The stained plate was washed
three times with distilled H2O, and allowed to air-dry. Then, 0.1n aq. NaOH soln. (200 ml) was added to
each well to dissolve the protein and to release the dye. The optical density of the resulting soln. in each
well was determined at 490 nm with a microplate reader. The inhibition (in %) of amoebal growth was
calculated from the optical densities of the control and test wells, and plotted against the logarithm of the
dose of the drug tested. Linear-regression analysis was used to determine the best-fitting straight line,
from which the IC50values were determined.
[3H]Hypoxanthine-Incorporation Assay. The chloroquine-resistant Gambian FCR-3 strain of the
malaria parasite Plasmodium falciparum was cultured in vitro as described in [50][51]. Briefly,
parasitized erythrocytes were suspended at a 5% haematocrit in supplemented RPMI-1640 and
maintainedat 378 in 5% CO2, 3% O2, and 92% N2. Cultures were synchronized with 5% d-sorbitol, when
the parasites were in the ring stage [52].
The antimalarial activity of the different compounds was determined by using the tritiated
hypoxanthine incorporation assay [53]. The parasite suspension, consisting predominately of the ring
stage,wasadjustedtoa 0.5%parasitaemiaand1%haematocrit, andexposedtovariousconcentrationsof
the compounds for 48 h. All assays were carried out using untreated parasites and uninfected red blood
cells as controls. Labelled [3H]hypoxanthine (Amersham) was added after 24 h, and the parasites?
[3H]DNA was harvested onto glass fibre filter mats, and the b-radioactivity was counted. The
concentration that inhibited 50% of parasite growth (IC50value) was determined from the log sigmoid
dose response curve generated by the Enzfitter?software. Chloroquine and quinine were used as the
reference antimalarial agents. The mean?S.D. values of IC50 values in Table 3 are from three
independent experiments.
Inhibition of b-Haematin Formation. A modification of the quantitative b-haematin inhibitory
activity (BHIA) assay [54] was used. The assay was performed under acidic conditions (pH 4.6–4.8),
similar to those in the parasitic vacuole. The method led to the determination of the concentration at
which 50% inhibition of b-haematin formation occurred, in units of molar ratio of the compound under
investigation with respect to haemin. A 0.767m haemin soln. in DMSO was distributed into 96-well flat-
bottom microplates (38.3 mmol/well). Solns. of test compounds, in varying molar ratios with respect to
haemin, were added to triplicate test wells; DMSO was used in the control wells. The final percentage of
DMSO per well was kept constant at 25%. b-Haematin formation was initiated by the addition of 0.5m
acetate buffer (pH 4.4), and the plates were then incubated at 378 for 24 h. Chloroquine and quinine
were used as positive controls. After centrifugation for 20 min to isolate the DMSO-insoluble b-
haematin, it was dissolved in 0.2m NaOH and diluted fourfold before being determined spectrophoto-
metrically at 405 nm. The haemin/compound molar ratio at which b-haematin formation was inhibited by
50% was calculated from log sigmoidal dose response curves generated by the Enzfitter?software.
Experiments were run in triplicat.
MTT Toxicity Assay. For the toxicity assay, transformed human kidney epithelium (Graham) cells
werecontinuouslymaintainedin cultureat 378in 5%CO2.TheMTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) cellular viability assay was used to determine the toxicity profile of the
compounds [55]. The trypsinized cell suspension was adjusted to 0.5 million cells/ml and plated out with
the various compounds. After 44 h of incubation, 2 mm MTTwas added to the plates and incubated for a
further 4 h. DMSO was then added to stop the reaction and dissolve the formazan crystals. The
absorbance was read at the test wavelength of 540 nm and reference wavelength of 690 nm, and the
percentage cellular viability calculated with appropriate controls taken into account. The mean?S.D.
values of IC50values in Table 2 are from three independent experiments.
CHEMISTRY & BIODIVERSITY – Vol. 5 (2008)774

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We are grateful to the Department of Science & Technology (Grant No. VII-PRDSF/44/2004-05/TT)
for funding the work. We thank Dr. Sudha Bhattacharya, School of Environmental Science, and Dr. Alok
Bhattacharya, School of Life Sciences, Jawaharlal Nehru University, New Delhi, for providing the
facilities for biological studies.
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Received June 27, 2007
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