Synthesis of some thiochromone derivatives and activity against Plasmodium falciparum in-vitro

Article abstract of DOI:10.1211/146080899128734541

A series of thiochromone derivatives were synthesized from 3 -cyano-2-methylthio-1-thiocromone with hydrazine and guanidine derivatives, and tested for antimalarial activity. Pyrimethamine was used as a reference drug. 3-Amino-[1]-benzothiopyran-[2,3-c]-p

Full text of DOI:10.1211/146080899128734541

Pharm. Pharmacol. Commun. 1999, 5: 107±110
Received October 27, 1998
Accepted November 11, 1998
# 1999 Pharm. Pharmacol. Commun.
Synthesis of some Thiochromone Derivatives and Activity
Against Plasmodium falciparum In-vitro
Â
J. CHARRIS, J. DOMINGUEZ, G. LOBO AND F. RIGGIONE*
Â
Â
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Laboratorio de Sõntesis Organica, Facultad de Farmacia and *Laboratorio de Investigacion en Malaria,
Facultad de Medicina, Universidad Central de Venezuela, Caracas 1051, Venezuela
Abstract
A series of thiochromone derivatives were synthesized from 3-cyano-2-methylthio-1-
thiocromone with hydrazine and guanidine derivatives, and tested for antimalarial activity.
Pyrimethamine was used as a reference drug.
3-Amino-[1]-benzothiopyran-[2,3-c]-pyrazol-4-one showed signi®cant antimalarial
activity against Plasmodium falciparum in-vitro. The remaining compounds had only
modest activity.
Material and Methods
Malaria is a mosquito-borne protozoal disease and
is endemic in many tropical and subtropical
regions. Plasmodium falciparum and Plasmodium
vivax are the most common causes of human
malaria. P. vivax causes the classic recur-
rent=relapsing febrile illness, widely recognized as
typical of malaria. Although it causes severe mor-
bidity, it is rarely fatal. P. falciparum presents a
much more variable picture. Symptoms may be
severe or deceptively mild but the disease is fatal in
approximately 1% of cases. Death may follow an
acute infection (endotoxic shock or cerebral
malaria) or may be the result of severe anaemia
following chronic infection. In 1997 the World
Health Organization estimated that world-wide up
to 300 million cases and 3 million deaths are
caused by malaria each year. The emergence and
spread of parasite resistance to present antimalarial
drugs urges for novel compounds to be discovered
and developed. Recently we have reported a series
of new quinolone, pyridopyrimidone and phe-
nothiazine derivatives with antimalarial activity
Chemistry
Melting points were recorded using a Thomas
Hoover Capillary Melting Point Apparatus and are
uncorrected. NMR was recorded using a Jeol JNM
EX 270 FT NMR spectrometer at 67Á8 MHz (13C)
and 270Á05 MHz (1H) and reported in ppm (d)
down®eld from tetramethylsilane as internal stan-
dard. Infrared spectra were determined as KBr
pellets on a Shimadzu model 470 spectro-
photometer. Mass spectra were recorded on a
Hewlett Packard 5995 mass spectrometer. The
purity of all compounds was determined by thin-
layer chromatography using several solvents of
different polarity. All solvents were distilled and
dried in the usual manner. Reactions were generally
performed under a nitrogen atmosphere.
O-Chlorobenzoylacetonitrile (1)
This was prepared according to a method reported
by Rudorf (1977).
Â
(Domõnguez et al 1996a, b, 1997). We have now
synthesized some thiochromone derivatives and
tested for in-vitro antimalarial activity against a
chloroquine-resistant strain of Plasmodium falci-
parum from Venezuela.
3-Cyan-2-methylthio-1-thiocromone (2)
A mixture of 1 (27Á8 mmol) and KOH (55Á6 mmol)
in dioxane (30 mL) was stirred at room temperature
for 30 min, and then treated with carbondisulphide
(27Á8 mmol). After 1 h, methyl iodide (27Á8 mmol)
in dioxane (1 mL) was added to the reaction mix-
ture and stirred at room temperature for a further
2 h. After the reaction mixture was re¯uxed for 1 h,
solvent was removed under vacuum. The resulting
Â
Â
Correspondence: J. Charris, Laboratorio de Sõntesis Orga-
nica, Facultad de Farmacia, Universidad Central de Venezuela,
Caracas 1051, Venezuela.
E-Mail: charrisj@camelot.rect.ucv.ve

108
J. CHARRIS ET AL
residue was added to water to give a solid which
was ®ltered and recrystallized from ethanol to yield
2 (83%). Mp 229±230ꢀC (Rudorf 1977: 233±
2,4-Diamino-[1]-benzothiopyrano-[2,3-d]-pyrimi-
din-5-one (5). Yield 81%. Mp 330±332ꢀC (Rudorf
1
& Augustin 1981: 334±336ꢀC). IR (KBr, cm ):
1
234ꢀC). IR (KBr, cm ): 2205 (CN); 1645 (CO);
1616 (CO), 1670, 3440, 3385, 3170 (NH); 1H NMR
(d6-DMSO): 7Á13 (br, 2H, NH2), 7Á39 (m, 1H, C±
H9), 7Á53 (dd, 1H, J:8Á16 Hz, C±H7), 7Á58 (m, 1H,
C±H8), 7Á82 (d, 1H, J: 4Á72 Hz, NH), 8Á31 (d, 1H, J:
7Á91 Hz, C±H6), 9Á45 (d, 1H, J: 4Á82 Hz, NH); 13C
NMR: 179Á72 (CO), 164Á91, 162Á31, 134Á67, 133Á15
(C8), 130Á53, 129Á25 (C6), 127Á52 (C9), 126Á65 (C7),
1H NMR (d6-DMSO): 2Á84 (s, 3H, SCH3), 7Á61±
7Á76 (m, 3H, Ar), 8Á45 (d, 1H, J: 8Á16 Hz, C±H5);
13C NMR: 14Á61 (SCH3), 178Á1 (CO), 125Á91 (C8),
128Á83 (C5), 129Á33 (C6), 132Á8 (C7); MS m=e 233
‡
(M 96%).
‡
101Á14; MS m=e 244 (M ).
General procedure for the synthesis of 3-amino-
[1]-benzothiopyrano-[2,3-c]-pyrazol-4-one deriva-
tives 3 and 4
A mixture of 2 (5Á4 mmol) was treated separately
with hydrazine hydrate or phenyl hydrazine
(6 mmol) in re¯uxing pyridine (8 mL) for 4 h. The
solvent was removed under vacuum. The resulting
residues were added to water and the solid obtained
was collected and crystallized from ethanol.
2,4-Diamino-2-cyanamino-[1]-benzothiopyrano-
[2,3-d]-pyrimidin-5-one (6). Yield 52%. Mp >
1
360ꢀC. IR (KBr, cm ): 1190 (CN), 1642 (CO),
3437, 3253, 3215 (NH); 1H NMR (d6-DMSO): 7Á19
(br, 1H, NHCN), 7Á56 (dd, 1H, J: 7Á67 Hz, C±H9),
7Á74 (m, 2H, C±H7), 8Á17 (d, 1H, NH, J: 5Á01 Hz),
8Á34 (dd, 1H, J: 7Á67 Hz, C±H8), 8Á71 (d, 1H, J:
7Á91 Hz, C±H6), 9Á58 (d, 1H, NH, J: 5Á16 Hz); 13C
NMR: 179Á83 (CO), 166Á55, 163Á89, 134Á5, 133Á5
(C8), 130Á76, 129Á31 (C6), 128Á04 (C9), 127Á1 (C7),
3-Amino-[1]-benzothiopyrano-[2,3-c]-pyrazol-
4(1H)-one (3). Yield 91%. Mp 257±259ꢀC (Rudorf
‡
102Á76; MS m=e 269 (M ). C12H7N5OS. 1=2 H2O;
C (51Á93), H (2Á67), N (25Á47).
1
& Augustin 1981: 260±261ꢀC). IR (KBr, cm ):
1632 (CO), 3416, 3282, 3113 (NH); H NMR (d6-
1
DMSO): 6Á62 (br, 2H, NH2), 7Á47±7Á51 (m, 3H,
Ar), 8Á4 (d, 1H, J:7Á91 Hz, C±H5), 12Á18 (br, 1H,
NH); 13C NMR: 177Á35 (CO), 151Á40, 145Á04,
135Á80, 131Á42 (C7), 130Á07, 127Á85 (C5), 126Á75
Biological evaluation
Field isolates. Isolates of Plasmodium falciparum
were collected between June and December 1996 in
Â
Bolõvar and Amazonas States, Venezuela, from six
patients with acute falciparum malaria who pre-
‡
(C6), 125Á46 (C8), 99Á56; MS m=e 217 (M ).
Â
sented voluntarily at the Direccion General Sec-
torial de Malariologõa y Saneamiento Ambiental.
Â
3-Amino-2-phenyl-[1]-benzothiopyrano-[2,3-c]-
pyrazol-4(2H)-one (4). Yield 75%. Mp 217±219ꢀC
(Rudorf & Augustin 1981: 220±222ꢀC). IR (KBr,
Parasitaemias (parasites=1000 erythrocytes) ranged
from 0Á1 to 1Á7%. Venous blood samples were
collected in Vacutainer tubes (Becton Dickinson,
Rutherford, NJ) before treatment. Blood samples
were transported at room temperature and were
used within 12 h of collection. Giemsa-stained thin
blood ®lms were examined to con®rm infection
with P. falciparum and to determine parasite den-
sity. The erythrocytes were washed three times in
RPMI 1640 medium (Gibco, Grand Island, NY) by
centrifugation. The washed erythrocytes were
resuspended (haematocrit 1Á5%) in RPMI 1640
medium supplemented with 10% human serum and
buffered with 25 mM HEPES and 0Á25% NaHCO3.
1
1
cm ): 1635 (CO), 3393, 3325, 3086 (NH); H
NMR: (CDCl3) 5Á99 (s, 2H, NH2), 7Á39±7Á60 (m,
8H, Ar), 8Á47 (d, 1H, J: 7Á67 Hz, C±H5). 13C NMR:
178Á86 (CO), 148Á77, 137Á31, 136Á59, 132Á09 (C7),
0
0
0
0
130Á22 (C3 ,5 ), 123Á93 (C2 ,6 ), 128Á80, 128Á58 (C5),
127Á33 (C6), 126Á07 (C8), 101Á30; MS m=e
‡
293(M ).
General procedure for the synthesis of 4-amino-
[1]-benzothiopyrano-[2,3-d]-pyrimidin-5-one deri-
vatives 5 and 6
Compound 2 (2Á3 mmol), was treated separately
with guanidine hydrochloride (2Á8 mmol) or cyano-
In-vitro assay of ®eld isolates. Drug effects on P.
falciparum proliferation were measured as inhibi-
tion of H-hypoxanthine (New England Nuclear,
Boston, MA) incorporation into nucleic acid, using
the method described by Desjardin et al (1979). All
compounds were dissolved in 10% dimethylsulph-
guanidine
(3 mmol),
potassium
carbonate
3
(4Á6 mmol) and DMF (10 mL) under re¯ux for 5 h.
The mixture was cooled and poured into water and
the solid obtained was collected and crystallized
from ethanol.

ANTIMALARIAL THIOCHROMONE DERIVATIVES
109
oxide (DMSO), subsequently diluted with RPMI
6
1640 medium and tested at 1 6 10 M in two
parallel columns of a ¯at-bottomed 96-well micro-
titre plate (Falcon plastics, Oxnard, CA). The ®nal
concentration of isotope in the medium was
1
2 mCi mL . Two hundred microlitres of the culture
were added to each well of the microculture plates
containing the drugs and the isotope. Plates were
incubated at 37ꢀC in candle jars for 24 and 48 h.
Just before reinvasion erythrocytes were frozen at
20ꢀC. Cells were lysed in an automatic cell har-
vester (LKB, Wallac oy, Finland). DNA was
retained in a glass ®lter, pore size 1 mm (LKB
Wallac oy, Finland), and the radioactivity incorpo-
rated in the DNA was measured in a beta counter
(Beta Plate, LKB, Wallac oy, Finland). Normal
non-infected erythrocytes were used as an addi-
tional control to measure radioactivity incorpora-
tion independent of parasite. The ®nal
concentration of DMSO never exceeded 1%
(v=v). DMSO alone had no effect on the prolifera-
tion of parasites. Control wells containing infected
erythrocytes without drugs and DMSO at the same
concentrations used by dissolving drugs were
included. The 50% inhibitory concentration
(IC50) was de®ned as the drug concentration cor-
responding to 50% inhibition of schizont develop-
ment in the control well.
Figure 1. Structures and synthetic pathways of 3-amino-
[1H]-benzothiopyrano[2,3-a]-pyrazol-4-one and 2,4-diamino-
[1]-benzothiopyrano[2,3-d]-pyrimidin-5-one analogues. i.
KOH, dioxane, CS2, CH3I; ii. RNHNH2, pyridine; iii.
R0CH3N2, DMF, K2CO3.
Table 1. Inhibition of biological activity by thiochromone
derivatives.
Compound
IC50 (mM)
Pyrimethamine
26Á4
25Á2
3
4
5
6
>100
>100
>100
IC50 was extrapolated from curves of percentage activity vs
concentration for each compound.
(J ˆ 4Á86 Hz). This provides clear evidence of the
presence of hydrogen bonding between one of the
NH2 protons and the carbonyl group attached to the
carbon at position 5. We have previously found the
Results and Discussion
3-Cyano-2-methylthio-1-thiochromone 2 (Figure
1), was prepared according with the procedure used
by Rudorf (1977), with some modi®cations. Reac-
tion of 2 with hydrazine hydrate or phenyl hydra-
zine in pyridine, gave the expected 3-amino-[1]-
benzothiopyrane-[2,3-c]-pyrazol-4-one derivatives
Â
same phenomenon in some quinolones (Domõnguez
et al 1998). This might be related to antimalarial
activity.
1
3 and 4 in good yields. The H NMR spectrum of
compounds 3 and 4 showed a singlet at 6Á62±
5Á99 ppm due to the NH2 protons. Two doublets in
the region of 7Á81 and 8Á47 (J ˆ 7Á91 and 7Á67 Hz)
were assigned to proton 5, and compound 3 showed
a broad singlet at 12Á18 ppm due to the NH proton.
Similarly, 4-amino-[1]-benzothiopyrano-[2,3-d]-
pyrimidin-5-one derivatives 5 and 6 were obtained
by treating 2 with guanidine hydrochloride or
dicyandiamide, and potassium carbonate in DMF.
The 1H NMR spectra of compounds 5 and 6
showed a singlet in the region 7Á13 and 7Á14 ppm
due to NH2 and NH proton, respectively; a doublet
in the region of 8Á31 and 8Á71 ppm (J ˆ 7Á91 Hz)
Biological results
Isolates of P. falciparum from South Venezuela
were characterized with respect to their suscept-
ibility to chloroquine. All were resistant to 100 mM
chloroquine (unpublished result). Table 1 shows the
effect of thiocromone derivatives on the ery-
throcytic cycle of six different P. falciparum iso-
lates. Compound 3 (IC50 25Á2 mM) had higher
activity, while compounds 4±6 showed no sig-
ni®cant antimalarial activity compared with the
parent compound, pyrimethamine. This is con-
sistent with our previous observations using the
quinolone analogues, indicating that the presence
of hydrogen bonding between one of the NH2
protons and the carbonyl group attached to the
carbon at C-5 reduces the antimalarial activity of 2,
4-diamino- [1] -benzothiopyrano- [2,3-d]-pyrimidin-
1
was assigned to proton 6. The H NMR for com-
pounds 5 and 6 showed two doublets around 9Á45,
7Á82 and 9Á58, 8Á17 ppm, respectively, which were
assigned to the amino group linked to C-4

110
J. CHARRIS ET AL
Â
Domõnguez, J., Basante, W., Charris, J., Riggione, F. (1996a)
Synthesis and activity of some quinolone derivatives against
Plasmodium falciparum in vitro. Farmaco 51: 407±412
5-one derivatives. Clearly compound 3 merits fur-
ther investigation.
Â
Â
Domõnguez, J., Charris, J., Iarruso, L., Lopez, S., Lobo, G.,
Riggione, F. (1996b) Synthesis of pyridopyrimidone deri-
vatives and their activity against P. Falciparum in vitro.
Farmaco 51: 781±784
Acknowledgements
We thank the Instituto de Investigaciones Farm-
Â
Â
Domõnguez, J., Lopez, S., Charris, J., Iarruso, L., Lobo, G.,
Semenov, A., Olson, P. J., Rosenthal, P. J. (1997) Synthesis
and antimalarial effects of phenothiazine inhibitors of a
Plasmodium falciparum cysteine protease. J. Med. Chem.
40: 2726±2732
Â
aceuticas, Facultad de Farmacia, Universidad
Central de Venezuela, project No IIF=01-94,
CONICIT projects No. RP-IV-130032-4, LAB.
97000665 and the technical assistance of Rafael
Paredes.
Â
Â
Domõnguez, J., Charris, J., Basante, W., Mendez, B. (1998)
13C NMR spectral characterization of some antimalarial
tricyclic quinolone derivatives. Magn. Reson. Chem. 36:
454±456
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