hampered by this increasing spread of antimalarial drug resistance. Therefore, strong interest has been directed at the search for new antimalarial agents.
During Intra-erythrocytic stages malaria parasites degrade vast amount of hemoglobin inside food vacuole and release a large quantities of redox active free heme (Goldberg et al. Semin Cell Biol. 1993;4:355-361, Akompon et al. Antimicrob Agents Chemother. 2000;44:88-96), which if allowed to accumulate may reach to 300-500 mM (Wright et al. J Med Chem. 2001;44:873-885).This Free heme (ferriprotoporphyrin IX) is very lethal to parasite and can produce reactive oxygen species (Kumar et al. Toxicol Lett. 2005; 157:175-188),which guided parasite towards death pathway (Vincent et al. Semin Hematol. 1989;26:105-113, Schmitt et al. Arch Biochem Biophys. 1993;307:96-103). Incorporation of this free heme to p hematin (Hemozoin) is one of the novel rescue mechanisms of this talented apicomplexan parasite (Rosenthal et al. Malaria: parasite biology, pathogenesis and protection. Washington, DC: ASM Press; 1998: 145-158).
Quinoline antimalarials such as chloroquine also target this biochemical route by making a complex with free heme (Cohen et al. Nature. 1964;202:805-806) and studies have shown that resistance to drug is not due to changes in the overall catalytic activity of heme polymerization (Martiney et al. Mol Med. 1996;2:236-246). A vast majority of antimalarial drugs such as quinoline, azoles, isonitriles, xanthones, methylene blue and their derivatives exhibit antimalarial activity by enhancing free heme toxicity through the inhibition of hemozoin formation (Kumar et al. Life Sci. 2007;80:813-828). Since Heme polymerization to hemozoin is a physical process not a genetic process which never approaches parasites to be modified genetically to access resistance. Hence targeting the heme polymerization activity of malaria parasite is one of the potential goals for novel antimalarials.
Trisubstituted methanes (TRSMs) with or without sulfur spacers have been reported to exhibit various biological activities as anti-breast cancer (Srivastava et al. Bioorg. Med. Chem. 2006,14: 1497-1505), antitubercular (Panda et al. Bioorg. Med. Chem. 2004;72:5269-5276; Panda et al. Indian Journal of Chemistry, 2009, Sec B, 48, 1121-1127, Parai, et al. Bioorganic & Medicinal Chemistry Letters, 2008, 18, 289-292, Das, S.K. et al. Bioorganic Medicinal Chemistry Letters; 2007, 17, 5586-5589, Shagufta et al. Journal of Molecular Modelling; 2007, 13, 99-109, Panda et al. European Journal of Medicinal Chemistry, 2007, 42, 410-419, Panda et al. Bioorganic Medicinal Chemistry Letters, 2005, 15, 5222-5225, Panda et al. Arkivoc, 2005, 2, 29-45), antiimplantation (Srivastava et al. Bioorg. Med. Chem. 2004; 12: 1011-1021), antiproliferative (Al-Qawasmeh et al. Bioorg. Med. Chem. Lett. 2004, 14, 347), etc. Aryl aryl methyl thio arenes (AAMTAs) belong to the class of TRSMs with sulfur spacer. Among TRSMs with sulfur spacers,
the antimalarial activity of several arylacridinyl sulfones has been reported. Initial antimalarial
activity of [(Aryl)-arylsulfanyl-methyl]-pyridines against chloroquine resistant P. yoelii in vivo
have been reported as well (Kumar et al. Antimicrobial Agents and Chemotherapy 2008; 52:
705-715). These preliminary findings encouraged us to synthesize and evaluate a new series of
AAMTAs with sulfur spacer for antimalarial efficacy. Here in this work, we have screened a large
number of Aryl aryl methyl thio arenes based on their heme binding affinity and subsequently
tested for antimalarial activity in vitro and in vivo using multidrug resistant strain (MDR strain P.
yoelii). These compounds offer antimalarial activity by promoting the development of oxidative
stress through the inhibition of heme polymerization and generting reactive oxygen species.
Following is the description of aryl aryl methyl thio arenes (AAMTAs) having antimalarial activity.
Objects of the invention:
Main objective of the present invention is to provide a process for the preparation of Aryl aryl
methyl thio arenes (AAMTAs).
Another objective of the present invention is to provide compounds with sulfur spacer having
significant pharmaceutical activity.
Further objective of the invention is to prepare a series of compounds of general formula I
having significant antimalarial activity.
Summary of the Invention:
Accordingly the present invention provides aryl aryl methyl thio arenes (AAMTAs) of general formula I
(Formula Removed)
wherein Aryl1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl; Aryl2 is selected from a group consisting of substituted phenyl, naphthalene, phenanthrene, pyridine, benzoxazolyl, benzthiazolyl; Aryl3 is selected from a group consisting of substituted phenyl groups such as methoxy phenyl, p-thiomethoxy phenyl, phenyl; R is H. In an embodiment of the present invention wherein the general formula I comprising:
2-((4-methoxyphenyl)(phenylthio)methyl)pyridine 4:
2-((4-methoxyphenyl)(o-tolylthio)methyl)pyridine 5: 3-[(4-Fluoro-phenylsulfanyl)-(4-methoxy-phenyl)-methyl]-pyridine 6: 2-[(4-Methoxy-phenyl)-pyridin-3-yl-methylsulfanyl]-benzooxazole 7: 4-((4-methoxyphenyl)(phenylthio)methyl)quinoline 8: 3-[(2-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 9: 3-[(3-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 10: 3-[(Naphthalen-2-ylsulfanyl)-phenyl-methyl]-pyridine 11: 3-[(4-Methylsulfanyl-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 12: 2-[(2-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 13: 2-[(3-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 14: 2-[(4-Methylsulfanyl-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 15: 2-((4-methoxyphenyl)(pyridin-3-yl)methylthio)pyridine 16: 2-Benzhydrylsulfanyl-pyridine 17: The representative compounds of general formula (I) comprising the following structures:
(Structure Removed)
In further embodiment of the present invention wherein the compounds of general formula I are useful in the treatment of chloroquine-sensitive ( P. falciparum) and MDR resistant (P.yoelli) strain of Plasmodium.
In an embodiment of the present invention wherein the compounds inhibited hemozoin
formation in a concentration dependent manner with an IC50 value of the most active AAMTAs
(compound 7, 9, 10, 12, 13, 16 and 17) were in the range of 5 ± 0.24 to 13±1.9 uM using
parasite lysate from P. yoelii (MDR strain).
In an embodiment of the present invention wherein the compounds showed the affinity towards
heme as evident from KD values for the binding of most active AAMTAs (compound 7, 10, 12,
and 16) ranging from 4.26 ±0.4 to 6.25 ±0.8.
In an embodiment of the present invention wherein the compounds showed that development of
oxidative stress is the antimalarial mode of action of AAMTAs.
In an embodiment of the present invention wherein the compounds inhibited the growth and
development of malaria parasite (P.falciparum) effectively as evident from the inhibition of
hypoxanthine uptake with an IC50 value of the most active AAMTAs (compound 7, 9,10,12,13,
16 and 17) were in the range of 1± 0.003 to 3.4±0.29 uM.
In an embodiment of the present invention wherein the compounds exhibit the apparent KD
(binding constant of AAMTAs with heme were studied by optical and optical differential
spectroscopy) values of compounds ranged between 4.26 ± 0.4 to 14.45±1.4 uM.
In an embodiment of the present invention wherein the compounds exhibit the apparent CSpKa
values of the compounds ranged between 3.411 ± 1.276 to 4.96 ± 0.8 mol/litre.
In an embodiment of the present invention wherein a process for the preparation of general
formula I wherein the process steps comprising:
i) reacting a compound having general formula 1a-e
(Formula Removed)
wherein R is selected from a group consisting of hydrogen, methoxy, thiomethoxy with general formula 2a-d,
(Formula Removed)
wherein Ar1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl under Grignard reaction conditions in the presence of organic solvent at 25 °C for a period ranging between 40 to 52 minutes to produce a compound of general formula 3a-k,
(Formula Removed)
Wherein R is selected from a group consisting of hydrogen, methoxy, thiomethoxy and Ar1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl, ii) reacting the compound of general formula 3a-k as obtained in step (i) with substituted aryl and heteroaryl thiol derivatives of formula
(Formula Removed)
wherein Ar2 is selected from a group consisting of Phenyl, 2-methyl-phenyl, 4-flouro-phenyl, 2-benzoxazolyl, naphthalenyl, 2-Pyridyl, 2-benzthiazolyl under Friedel-Crafts reaction conditions in the presence of Lewis acids in a solvent selected from group of benzene, toluene and xylene at a temperature in the range of 20°C to 40 °C for a period ranging between 1-2 hrs to obtain a compound of formula 4-17,
(Formula Removed)
Wherein R is selected from a group consisting of hydrogen, methoxy, thiomethoxy. Ar1 is
selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl; Ar2 is selected from
Phenyl, 2-methyl-phenyl, 4-flouro-phenyl, 2-benzoxazolyl, naphthalenyl, 2-Pyridyl, 2-
benzothioxazolyl.
In an embodiment of the present invention wherein a process employs the Lewis acids used in
Fridel-Crafts reaction may be selected from a group consisting of aluminium chloride and cone.
sulphuric acid.
In an embodiment of the present invention provides the active compounds are not showing in
vitro cytotoxicity on nucleated proliferating leukemia cell line U 937 with a selectivity index (S,)
value in range of 142-253.
In an embodiment of the present invention wherein the active compounds are capable of killing
malaria parasite.
In an embodiment of the present invention wherein the most active compound 12 and 16 shows
statistically significant protection of mice against experimental infection with MDR strain
(P.yoelli) of malaria parasite.
Brief Description of the Drawings:
Figure 1. Scheme 1. Synthesis of Aryl aryl methyl thio arenes (AAMTAs) (4-17). Synthesis of
Aryl aryl methyl thio arenes (AAMTAs) has been accomplished from a series of carbinols on
which S-alkylation of different aryl or heteroaryl thiols has been performed. Carbinols were
synthesized by Grignard reaction of arylmagnesium bromide with a series of arylcarbaldehydes.
Figure 2. Interaction of AAMTAs with heme. Optical and differential optical soret spectroscopy
for AAMTAs - hemin interaction at different concentrations of AAMTAs (1-20 µM).Compound
names are indicated in bracket. Inset shows the plot of 1/A360 nm vs 1/[ AAMTAs] to calculate
KD.
Figure 3. (A) AAMTAs favours the accumulation of free heme inside the parasite. Heme content
was measures as described under Experimental procedure. AAMTAs induce the generation of
intra-parasitic H2O2 and •OH. The levels of H2O2
(B) and •OH (C) in P. falciparum were measured at different concentrations of AAMTAs as
indicated.
Figure 4. AAMTAs develop oxidative stress in P. falciparum.
(A) Lipid peroxide formation
(B) Protein carbonyl formation and
(C) Decrease in total GSH content (relative to control) in P. falciparum were measured at different concentrations AAMTAs as indicated.
(D) Effect of hydroxyl radicals scavengers on AAMTAs -induced growth inhibition of P.
falciparum. P. falciparum growth was measured by following [3H] hypoxanthine uptake in
presence or absence of hydroxyl radical scavengers (PBN and mannitol) during AAMTAs (20
uM) treatment as described under 'EXPERIMENTAL PROCEDURE'. P. falciparum culture (4%
parasitemia) was treated with AAMTAs along with PBN (50 mM) or mannitol (10 mM) for 48
hours.
Figure 5. In vivo antimalarial activity of AAMTAs at different concentrations.
(A) BALB/c mice(rodent malarial model) was infected by MDR (chloroquine, mefloquine, and halofantrine) strain Plasmodium yoelii and subsequently treated intrapertonialy of selective AAMTAs at dose levels 5 mg/kg body weight,
(B) 10 mg/kg body weight and (C) 25 mg/kg body weight. Artimal (a/|3 arteether) was used as positive control at a dose of 50 mg/kg of body weight.
Detailed description of the Invention:
The present invention provides Aryl aryl methyl thio arenes substituted with various arenes and heteroarenes and a process for the preparation of the said compounds of general formula I useful in antimalarial activity wherein wherein Aryl1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl; Aryl2 is selected from a group consisting of substituted phenyl, naphthalene, phenanthrene, pyridine, benzoxazolyl, benzthiazolyl; Aryl3 is selected from a group consisting of substituted phenyl groups such as methoxy phenyl, p-thiomethoxy phenyl, phenyl; R is selected from a group consisting of H, OH, methyl.
The compounds of general formula I are new chemical entities and they have not been prepared earlier.
The compounds synthesized were tested for antimalarial activities. A number of these compounds showed inhibition of the malaria parasite. The compounds of general formula I were synthesized from various synthetic intermediates. They are described below.
(Formula Removed)
The starting materials will be compounds of general formula II wherein R, Aryl1 and Aryl3 have the meanings as stated above.
The most preferred compounds, represented by formula II are given below: (4-methoxyphenyl)(quinolin-4-yl)methanol:3c (2-methoxyphenyl)(pyridin-3-yl)methanol: 3d (2-methoxyphenyl)(pyridin-2-yl)methanol: 3e (3-methoxyphenyl)(pyridin-3-yl)methanol: 3f (3-methoxyphenyl)(pyridin-2-yl)methanol: 3g (4-(methylthio)phenyl)(pyridin-3-yl)methanol: 3h (4-(methylthio)phenyl)(pyridin-2-yl)methanol: 3i phenyl(pyridin-3-yl)methanol: 3j Diphenylmethanol: 3k
Following examples are given by way of illustration and should not construe to limit the scope of the present invention. Experimental Procedures: General procedure for preparation of carbinols (3a-k).
(Formula Removed)
Preparation of (3d):
To a suspension of Mg (1.5g, 61.70 mmol) in dry THF (40 mL) was added dropwise a solution of 2-bromoanisole (6.52 mL, 52.08 mmol) in dry THF (40 mL). After stirring the mixture for 30 min. a solution of pyridine 3-carboxaldehyde (45 mmol) in dry THF (30 mL) was added dropwise and the resulting solution was allowed to stir for an additional 30 min. After quenching by adding a saturated solution of NH4CI (20 mL), the reaction mixture was extracted with ethyl acetate (100
mL), washed with water (100 mL), brine (2x50mL) and then dried over Na2SO4. The organic layer was removed under reduced pressure. The crude product was purified by silica gel column chromatography.
Table 1.
(Table Removed)
(2-methoxyphenyl)(pyridin-3-yl)methanol (3d):
(Formula Removed)
Rf: 0.45 (ethyl acetate). Isolated as yellow solid (yield 80%, mp 104-105 °C) by elution with 41% ethyl acetate in hexane on silica gel. IR (KBr): cm-1 3185, 3008, 2366, 1593, 1488, 1465, 1432, 1243, 1029, 765.1H NMR (CDCI3, 300 MHz): δ 8.50 (d, J = 1.4 Hz, 1H), 8.32 (dd, J = 4.6, 0.9 Hz, 1H), 7.70-7.67 (m, 1H), 7.39-7.36 (m, 1H), 7.27-7.22 (m, 1H), 7.19-7.14 (m, 1H), 6.95 (m, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.06 (s, 1H), 3.73 (s, 3H). MS (ESI): m/z 216 [M+1]+. Anal. Cacld. for C13H13NO2: C, 72.54; H, 6.09; N, 6.51. Found: C, 72.69; H, 6.00; N, 6.62. (3-methoxyphenyl)(pyridin-3-yl)methanol (3f):
3f was synthesised following the procedure as described for 3d.
Rf: 0.50 (ethyl acetate). Isolated as yellowish white solid (yield 77%, mp 105-106 °C) by elution (Formula Removed)
with 43% ethyl acetate in hexane on silica gel. IR (KBr): cm-1 3402, 2373, 1595, 1429, 1262, 1221, 1039, 770. 1H NMR (CDCI3, 300 MHz): δ 8.50 (d, J = 1.77 Hz, 1H), 8.37 (dd, J = 4.75, 1.39 Hz, 1H), 7.69-7.67 (m, 1H), 7.27-7.19 (m, 2H), 6.92-6.90 (m, 2H), 6.83-6.79 (m, 1H), 5.79 (s, 1H), 3.77 (s, 3H). 13C NMR (CDCI3, 50 MHz): δ 159.72, 147.88, 147.70, 145.00, 139.94, 134.47, 129.55, 123.40, 118.79, 113.05, 111.99, 73.42, 55.10. MS (ESI): m/z 216 [M+1]+. Anal. Cacld. for C13H13NO2: C, 72.54; H, 6.09; N, 6.51. Found: C, 72.38; H, 6.18; N, 6.41.
phenyl(pyridin-3-yl)methanol (3j):
3j was synthesised following the procedure as described for 3d.
Rf: 0.45 (ethyl acetate). Isolated as white solid (yield 79%, mp 101-103 °C) by elution with 41% (Formula Removed)
ethyl acetate in hexane on silica gel. IR (KBr): cm-1 3440, 3025, 2365, 1630, 1430, 1217, 1027, 921, 770, 669, 501. 1H NMR (CDCI3, 300 MHz): £8.38 (d, J = 1.6 Hz, 1H), 8.23 (dd, J = 4.8, 1.3 Hz, 1H), 7.67-7.65 (m, 1H), 7.31-7.22 (m, 5H), 7.17-7.13 (m, 1H), 5.75 (s, 1H).13C NMR (CDCI3, 50 MHz): δ147.95, 147.77, 143.30, 139.94, 134.47, 128.57, 127.71, 126.49, 123.44, 73.62. MS (ESI): m/z 186 [M+1]+. Anal. Cacld. for C12H11NO: C, 77.81; H, 5.99; N, 7.56. Found: C, 72.92; H, 6.10; N, 7.45. diphenylmethanol (3k):
3k was synthesised following the procedure as described for 3a.
Rf: 0.45 (15% ethyl acetate in hexane). Isolated as yellowish white solid (yield 75%, mp 94-95 (Formula Removed)
°C) by elution with 5% ethyl acetate in hexane on silica gel. IR (neat): 3297.0, 2925.1, 2860.0, 2364.5, 1712.0, 1657.1, 1594.7, 1489.9, 1450.7, 1399.8, 1182.5, 1020.2, 759.6, 699.4. 1H NMR (CDCI3, 300 MHz): δ 7.28-7.12 (m, 10H), 5.68 (s, 1H), 2.65 (s, 1H). 13C NMR (CDCI3, 50 MHz): 8 143.71, 128.33, 127.37, 126.48, 76.00. MS (ESI): m/z 184 [M]+, 207 [M+Na]+. Anal. Cacld. for C13H12O: C, 84.75; H, 6.57; O, 8.68. Found: C, 84.70; H, 6.51.
General Procedure for Preparation of Aryl aryl methyl thio arenes (4-17). Preparation of compound 4:
Method a: To a solution of carbinol 3a (2.50 mmol) and thiophenol (3.75 mmol) in dry benzene (25 mL), a catalytic amount of cone. H2SO4 was added and the mixture was refluxed for half an hour. After adding water, the reaction mixture was extracted with ethyl acetate (25 mL), washed by brine (25 mL), and dried over Na2SO4. The combined organic layer was removed under
reduced pressure. The crude product was purified by silica gel column chromatography to furnish compound 4.
Method b: To a solution of carbinol 3a (2.50 mmol) and thiophenol (3.75 mmol) in dry benzene, anhydrous AICI3 (2.52 mmol) was added and the mixture was stirred at room temperature for half an hour. After adding ice-cooled water, the reaction mixture was extracted with ethyl acetate (25 mL), washed by brine (25 mL), and dried over Na2SO4. The combined organic layer was removed under reduced pressure. The crude product was purified by silica gel column chromatography to furnish compound 4.
Table 2.
(Table Removed)
Field of the Invention
The invention relates to aryl aryl methyl thio arenes (AAMTAs) as Antimalarial Agents. The invention particularly relates to a process for the preparation of substituted aryl and heteroaryl methanes with sulfur as spacer and their use as potential antimalarial agents. Novel substituted aryl aryl methyl thio arenes of formula I have been prepared.
The present invention provides aryl aryl methyl thio arenes substituted with various arenes and heteroarenes and a process for the preparation of the said compounds of general formula I useful in antimalarial activity wherein wherein Aryl1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl; Aryl2 is selected from a group consisting of substituted phenyl, naphthalene, phenanthrene, pyridine, benzoxazolyl, benzthiazolyl; Aryl3 is selected from a group consisting of substituted phenyl groups such as methoxy phenyl, p-thiomethoxy phenyl, phenyl; R is H.
Background of the Invention
Malaria is one of the most important infectious diseases and remains a major health problem in developing countries. According to the World Health Organization (WHO), it infects more than 300 million people per year and causes more than one million deaths annually, mostly among young children in the age group of less than five years (Sachs and Malaney. Nature. 2002;415:680-685). Malaria is re-emerging as the biggest infectious killer and is currently a first priority tropical disease of the WHO (World Health. Organ Tech Rep Ser. 2000; 892:1-74). The management of malaria relies solely on chemotherapeutics and chemoprophylaxis due to limitations associated with vaccine development and vector control (Kumar S et al. Life Sci. 2007;80:813-828). Rapid emergence of drug resistance in malaria parasite towards well established antimalarial drugs causes structural changes at molecular level of these drugs targets (Alam et al. Expert Review of Clinical Pharmacology. 2009;2:469-489, Musset et al. Microbes Infect. 2006;8:2599-2604, White et al. J Clin Invest. 2004;113:1084-1092, Plowe et al. J Infect Dis. 1997;176:1590-1596). Global efforts en route for malaria eradication are greatly
(Table Removed)
2-[(4-Methoxy-phenyl)-phenylsulfanyl-methyl]-pyridine 4:
Rf: 0.43 (50% ethyl acetate in hexane). Isolated as colourless semi-solid (yield 78%) by elution (Formula Removed)
with 12% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.51 (dd, 1H, J1 = 0.8 Hz, J2 = 4.7 Hz), 7.52-7.34 (m, 4H), 7.28-7.23 (m, 2H), 7.13-7.09 (m, 4H), 6.81-6.76 (m, 2H), 5.62 (s, 1H), 3.67 (s, 3H). MS (ESI): m/z 308 [M+1]+, 198 [M-C6H5S]+. Anal. Calcd for C19H17NOS: C, 74.23; H, 5.57; N, 4.56. Found: C, 74.15; H, 5.71; N, 4.46. 2-
[(4-Methoxy-phenyl)-o-tolylsulfanyl-methyl]-pyridine 5: Compound 5 was synthesised following the procedure as described for compound 4. Rf: 0.45 (50% ethyl acetate in hexane). Isolated as colourless semi-solid (yield 68%) by elution with 12% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.51-8.49 (m, 1H), 7.80-7.68 (m, 2H), 7.47 (d, 2H, J = 8.78 Hz), 7.34-7.19 (m, 5H), 6.80 (d, 2H, J = 8.8 Hz), 5.48 (s, 1H), 3.76 (s, 3H), 2.30 (s, 3H). MS (Formula Removed)
(ESI): m/z 322 [M+1]+, 198 [M- C7H7S]+. Anal. Calcd for C20H19NOS: C, 74.73; H, 5.96; N, 4.36. Found: C, 74.66; H, 6.14; N, 4.39.
3-[(4-Fluoro-phenylsulfanyl)-(4-methoxy-phenyl)-methyl]-pyridine 6: Compound 6 was synthesised following the procedure as described for compound 4. Rf: 0.48 (50% ethyl acetate in hexane). Isolated as pale yellow solid (yield 63%, mp 87-89 °C) by elution with 15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.57 (s, 2H), 8.41 (s, 1H), 7.68 (d, 1H, J = 7.8), 7.25-7.13 (m, 5H), 6.86- 6.77 (m, 3H), 5.66 (s, 1H), 3.74 (s, 3H). MS (ESI): m/z 326 (M++1). Anal. Calcd for C19H16FNOS: C, 70.13; H, 4.96, N, 4.30. Found: C, 70.23; H, 5.19; N, 4.30.
(Formula Removed)
2-[(4-Methoxy-phenyl)-pyridin-3-yl-methylsulfanyl]-benzooxazole 7: Compound 7 was synthesised following the procedure as described for compound 4. Compound 7 suppressed the mean parasitemia (day 8) by 50%, 68% and 79% at dose levels of 5 mg/kg, 10 mg/kg and 25 mg/kg, respectively.
Rf: 0.38 (50% ethyl acetate in hexane). Isolated as pale yellow solid (yield 61%, mp 83-85 °C) by (Formula Removed)
elution with 15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3) : δ 8.76 (d, 1H, J= 1.7), 8.48 (s, 1H), 7.83 (d, 2H, J= 7.9), 7.69- 7.65 (m, 1H), 7.40-7.21 (m, 5H), 6.88-6.83 (m, 2H), 6.34 (s, 1H), 3.75 (s, 3H). MS (ESI): m/z 349 (M++1). Anal. Calcd for C20H16N2O2S: C, 68.94; H, 4.63; N, 8.04. Found: C, 69.02; H, 4.42; N, 8.19.
[(4-Methoxy-phenyl)-phenylsulfanyl-methyl]-quinoline 8: Compound 8 was synthesised following the procedure as described for compound 4.
Rf: 0.44 (50% ethyl acetate in hexane). Isolated as pale yellow solid (yield 64%, mp 89-92 °C) by (Formula Removed)
elution with 15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.90 (d, 1H, J = 4.54), 8.16-8.03 (m, 2H), 7.80 (d, 1H, J = 4.52), 7.67 (m, 1H), 7.55 (m, 1H), 7.32-7.12 (m, 7H), 6.81 (d, 2H, J = 8.6), 6.24 (s, 1H),
3.74 (s, 3H). 13C NMR (50 MHz, CDCI3): δ 159.5, 150.6, 148.8, 146.5, 136.2, 131.4, 130.6,
130.2, 130.1, 129.6, 129.4, 127.9, 127.3, 127.2, 126.8, 123.8, 121.3, 114.6, 114.4, 55.6, 52.8.
MS (ESI): m/z 358 (M++1). Anal. Calcd for C25H43N3O4S: C, 62.34; H, 9.00; N, 8.72. Found: C,
62.52; H, 9.21; N, 8.46
3-[(2-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 9:
Compound 9 was synthesised following the procedure as described for compound 4.
Rf: 0.48 (50% ethyl acetate in hexane). Isolated as pale yellow solid (yield 64%, mp 102-104 °C) (Formula Removed)
by elution with 15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3):δ 8.65 (d, 1H, J= 1.94), 8.42 (m, 1H), 7.75-7.61 (m, 6H), 7.43-7.38 (m, 3H), 7.25-7.19 (m, 2H), 6.87 (m, 2H), 6.13 (s, 1H), 3.79 (s, 3H). 13C NMR (50
MHz, CDCI3): δ 156.7, 150.4, 148.5, 137.4, 136.2, 134.0, 133.5, 132.4, 129.4, 129.3, 128.7,
128.5, 128.0, 127.7, 126.8, 126.3, 123.6, 121.3, 111.2, 56.0, 48.0. MS (ESI): m/z 358 (M++1),
199.3 (M+-C10H7S). Anal. Calcd for C23H19NOS: C, 77.28; H, 5.36; N, 3.92. Found: C, 77.43; H,
5.48; N, 4.05.
3-[(3-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 10:
Compound 10 was synthesised following the procedure as described for compound 4.
Compound 10 suppressed the mean parasitemia (day 8) by 23%, 41% and 50% at dose levels
of 5 mg/kg, 10 mg/kg and 25 mg/kg, respectively.
Rf: 0.48 (50% ethyl acetate in hexane). Isolated as pale yellow solid (yield 65%, mp 100-102 °C) (Formula Removed)
by elution with 15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.63 (d, 1H, J = 2.2), 8.43 (d, 1H, J = 3.4), 7.74-7.61 (m, 5H), 7.43-7.36 (m, 5H), 7.32-7.24 (m, 1H), 6.86-6.82 (m, 2H), 5.62 (s, 1H), 3.76 (s,
3H). 13C NMR (50 MHz, CDCI3): 5 159.4, 150.2, 148.9, 137.4, 136.1, 133.9, 132.9, 132.6, 132.2,
130.3, 129.8, 128.8, 128.0, 127.7, 126.9, 126.5, 123.8, 114.6, 55.6, 54.8. MS (ESI): m/z 358
(M++1), 198 (M+-C10H7S). Anal. Calcd for C23H19NOS: C, 77.28; H, 5.36; N, 3.92. Found: C,
77.19; H, 5.51; N, 4.06.
3-[(Naphthalen-2-ylsulfanyl)-phenyl-methyl]-pyridine 11:
Compound 11 was synthesised following the procedure as described for compound 4.
Rf: 0.42 (45% ethyl acetate in hexane). Isolated as pale yellow solid (yield 68%) by elution with (Formula Removed)
20% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.48-8.43 (m, 2H), 7.42-7.12 (m, 12H), 7.12-7.08 (m, 2H), 5.55 (s, 1H). 13C NMR (50 MHz, CDCI3): δ 151.2, 148.1, 143.0, 137.0, 129.6, 128.9, 127.1, 123.6, 54.8. MS (ESI): m/z 328.2 (M++1). Anal. Calcd for C22H17NS: C, 80.70; H, 5.23; N, 4.28. Found: C, 80.88; H, 5.35; N, 4.39.
3-[(4-Methylsulfanyl-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 12: Compound 12 was synthesised following the procedure as described for compound 4. Compound 12 suppressed the mean parasitemia (day 8) by 65%, 75% and 85% at dose levels of 5 mg/kg, 10 mg/kg and 25 mg/kg, respectively.
Rf; 0.48 (50% ethyl acetate in hexane). Isolated as pale yellow solid (yield 64%) by elution with (Formula Removed)
15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.62 (d, 1H, J= 1.86 Hz), 8.44 (d, 1H), 7.74-7.65 (m, 4H), 7.45-7.32 (m, 5H), 7.26-7.17 (m, 4H), 5.61 (s, 1H), 2.45 (s, 3H). 13C NMR (50 MHz, CDCI3): 5 150.2, 149.0, 138.5, 136.1, 133.9, 132.6, 130.5, 129.1, 128.9, 128.0, 127.7, 127.1, 126.9, 126.6, 123.8, 55.1, 16.0. MS (ESI): m/z 274 (M++1), 215.4 (M+-C10H7S). Anal. Calcd for C23H19NS2: C, 73.95; H, 5.13; N, 3.75. Found: C, 74.04; H, 5.34; N, 3.93.
2-[(2-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 13: Compound 13 was synthesised following the procedure as described for compound 4. Rf: 0.38 (60% ethyl acetate in hexane). Isolated as pale yellow solid (yield 60%) by elution with 15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.51-8.49 (m, 1H), 7.56-7.51 (m, 1H), 7.32-7.19 (m, (Formula Removed)
4H), 6.94-6.85 (m, 2H), 6.20 (s, 1H), 3.82 (s, 3H). 13C NMR (50 MHz, CDCI3): δ 161.7, 157.0, 148.1, 137.0, 132.1, 129.1, 128.2, 122.5, 121.6, 121.3, 111.1, 69.6, 55.9, . MS(ESI): m/z (M++1), 230 (M+-C10H7S). Anal. Calcd for C23H19NOS: C, 77.28; H, 5.36; N, 3.92. Found: C, 77.48; H, 5.17; N, 4.07.
2-((3-methoxyphenyl)(naphthalen-2-ylthio)methyl)pyridine 14:
Compound 14 was synthesized following the procedure as described for compound 4.
Rf: 0.48 (50% ethyl acetate in hexane). Isolated as colourless oil (yield 81% ) by elution with (Formula Removed)
15% ethyl acetate in hexane from silica gel. IR (Neat): cm"1 2361, 1631, 1220, 1036, 771, 682. 1H NMR (300 MHz, CDCI3): δ 8.56 (d, J = 4.2 Hz,
1H), 7.73-7.50 (m, 6H), 7.42-7.35 (m, 3H), 7.24-7.07 (m, 4H), 6.77-6.74 (m, 1H), 5.75 (s, 1H), 3.73 (s, 3H). 13C NMR (75 MHz, CDCI3): δ 160.16, 159.71, 149.36, 141.46, 136.79, 133.56, 132.95, 132.02, 129.57, 129.32, 128.34, 128.21, 127.56, 127.28, 126.31, 125.87, 122.61, 122.17, 120.80, 114.04, 113.14, 58.95, 55.17. MS (ESI): m/z 358 (M++1). Anal. Calcd for C23H19NOS: C, 77.28; H, 5.36; N, 3.92. Found: C, 77.11; H, 5.23; N, 4.05.
2-[(4-Methylsulfanyl-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 15:
Compound 15 was synthesised following the procedure as described for compound 4.
Rf. 0.48 (50% ethyl acetate in hexane). Isolated as pale yellow solid (yield 64%, mp 102-104 °C) (Formula Removed)
by elution with 15% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.57-8.55 (m, 1H), 7.70-7.48 (m, 4H), 7.42-7.38 (m, 6H), 7.17-7.10 (m, 4H), 5.73 (s, 1H), 2.40 (s, 3H). 13C NMR (50 MHz, CDCI3): δ
160.5, 149.8, 138.2, 137.2, 134.0, 133.2, 132.5, 129.9, 129.3, 128.8, 128.7, 128.0, 127.7, 127.0,
126.7, 126.3, 123.0, 122.6, 59.0, 14.5. MS (ESI): m/z 374.5 (M++1), 215.3 (M+-C10H7S). Anal.
Calcd for C25H43N3O4S: C, 62.34; H, 9.00; N, 8.72. Found: C, 62.52; H, 9.21; N, 8.46.
2-((4-methoxyphenyl)(pyridin-3-yl)methylthio)pyridine 16:
Compound 16 was synthesised following the procedure as described for compound 4.
Compound 16 suppressed the mean parasitemia (day 8) by 55%, 72% and 80% at dose levels
of 5 mg/kg, 10 mg/kg and 25 mg/kg, respectively.
Rf: 0.52 (ethyl acetate in hexane). Isolated as colourless solid (60%, mp 139-140 °C) by elution (Formula Removed)
with 45% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3):δ 8.67 (s, 1H), 8.50 (d, 1H, J = 4.2), 8.36 (d, 1H, J = 4.4), 7.86 (d, 2H, J = 7.8), 7.08-7.06 (m, 1H), 6.88 (d, 2H, J = 8.52), 6.16 (s, 1H), 2.30 (s, 3H). 13C NMR (50 MHz, CDCI3): 6 160.39, 153.85, 149.87, 139.03, 138.29, 137.10, 134.61, 132.12,
130.01, 129.68, 128.76, 126.80, 125.09, 124.14, 123.54, 114.28, 96.54, 77.76, 55.56, 30.11,
21.55. MS (ESI): m/z 308 [M]+. Anal. Calcd for C18H16N2OS: C, 70.10; H, 5.23; N, 9.08. Found:
C, 70.21; H, 5.34; N, 9.27.
2-Benzhydrylsulfanyl-pyridine 17:
Compound 17 was synthesised following the procedure as described for compound 4.
Compound 17 suppressed the mean parasitemia (day 8) by 25%, 36% and 41% at dose levels
of 5 mg/kg, 10 mg/kg and 25 mg/kg, respectively
Rf: 0.52 (ethyl acetate in hexane). Isolated as colourless solid (60%, mp 139-140°C) by elution (Formula Removed)
with 45% ethyl acetate in hexane from silica gel. 1H NMR (200 MHz, CDCI3): δ 8.31-8.28 (m, 1H), 7.46-7.00 (m, 12H), 6.78-6.77 (m, 1H), 6.33 (s, 1H). 13C NMR
(50 MHz, CDCI3): δ 158.8, 149.9, 144.7, 136.5, 128.6, 127.6, 122.7, 120.3, 53.6. MS (ESI): m/z TIB [M]+. Anal. Calcd for C18H15NS: C, 77.94; H, 5.45; N, 5.05. Found: C, 78.02; H, 5.29; N, 5.19.
ANTIMALARIAL ACTIVITY: Material
Hemin, RPMI-1640, saponin, SDS, 2, chloroquine, glutathione (GSH), dichlorofluorescein diacetate, thiobarbituric acid (TBA), trichloroacetic acid (TCA), dichlorofluorescein diacetate, fetal calf serum (FCS); DMSO; 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) , Penicillin, streptomycin and tetraethoxypropane were purchased from Sigma (St. Louis, MO, USA). Albumax II was procured from Life Tecnologies, USA, Giemsa Stain was purchased from Qualigens Fine Chemicals, India. 3H hypoxanthine was purchased from Amersham Biosciences, USA. All other chemicals were of analytical grade purity.
P. falciparum culture
P. falciparum (clone NF54) was grown as described earlier (Trager W.Jensen J B. Science. 1976;193:673-675) Parasite culture was maintained at a hematocrit level of 5% in complete RPMI medium (CRPMI; RPMI 1640 medium, supplemented with 25 mM HEPES, 50 µg ml-1 gentamycin, 370 µM hypoxanthine and 0.5% (w/v) AlbuMaxll) using tissue-culture flasks (25 cm2 and 75 cm2) with loose screw caps. Old medium was changed with fresh medium once in 24 h and the culture was routinely monitored through Giemsa-staining of thin smears.
Preparation of parasite lysate
Parasite was isolated as described previously (Choubey et al. Antimicrob Agents Chemother. 2007;51:696-706). In brief, erythrocytes with ~10% parasitemia (P. falciparum) or 50% parasitemia (P.yoelii) were centrifuged at 800 x g for 5 mins, washed twice and resuspended in cold phosphate buffered saline (137 mM NaCI, 2.7 mM KCI, 5.3 mM Na2HPO4 and 1.8 mM KH2PO4). An equal volume of 0.5% saponin in PBS (final concentration 0.25%) was added to the erythrocyte suspension and kept on ice for 15-20 mins. Lysate was centrifuged at 1300 x g for 5 mins to get parasite pellet, and pellet was washed with PBS thrice and either used immediately or kept at -80°C. The isolated parasite was lysed in PBS by mild sonication (30 sec pulse, bath type sonicator) at 4°C and the whole lysate was then stored at -20°C for future use. Protein content of the parasite lysate was estimated by lowry method (Lowry et al. J Biol Chem. 1951;193:265-275).
Assay of hemozoin (ß-hematin) formation
In vitro hemozoin (ß-hematin) formation was assayed as described earlier (Pandey et al. J Pharm Biomed Anal. 1999;20:203-207, Trivedi et al. Biochim Biophys Acta. 2005; 1723:221-228, Sullivan et al. Science. 1996;271:219-222). In brief, the assay mixture contained final volume of 1 ml (100 mM sodium acetate buffer pH 5.2), 100 µM hemin, parasite lysate (20 µl), and different concentrations of AAMTAs compounds. The reaction was initiated by the addition of hemin and further incubated for 12 h at 37° C. The reaction was terminated by centrifugation at 15,000x g for 10 min at room temperature. The pellet was washed thrice with 100 mM Tris buffer pH 7.8 containing 2.5% SDS and finally with 100 mM bicarbonate buffer pH 9.2. The insoluble pellet (hemozoin) was solubilized in 50 µl of 2 N NaOH and diluted further to 1 ml with 2.5% SDS. The absorbance of the solution was recorded at 400 nm and an extinction coefficient of 91mM-1cm-1 was used to quantitate the heme converted to hemozoin (Pandey et al. J Pharm Biomed Anal. 1999;20:203-207).
To see the effect of AAMTAs on hemozoin formation in P. falciparum, the amount of hemozoin formed in presence or absence of AAMTAs was measured as described earlier (Coban et al. Infect Immun. 2002;70:3939-3943). In brief, D-sorbitol (5%) synchronized P. falciparum culture (5% parasitemia was treated with different concentrations of AAMTAs compounds and incubated further for 48 hours. The P. falciparum culture was then harvested and parasite was isolated from infected RBC by saponin (0.5%, 10 minutes) treatment. Parasite was washed three times with PBS and parasite lysate was prepared after mild sonication. Parasite lysate was washed three times with 2% SDS and the resulting pellet was suspended in a solution of 10 mM Tris-HCI (pH 8.0), 0.5% SDS, and 1 mM CaCI2 containing 2µg/ml of proteinase K and was then incubated at 37°C overnight. The pellet was then washed three times in 2% SDS and incubated in 6 M urea for 3 h at room temperature on a shaker. After incubation, it was centrifuged (4000 x g for 10 minutes) at room temperature and again washed three times with 2% SDS. Finally, the hemozoin pellet was dissolved in 20 mM NaOH containing 2% SDS and the OD of solution was measured at 400 nm to quantitate hemozoin.
Heme interaction studies
Heme interaction of synthesized AAMTAs compounds were analysed by optical and differential optical spectroscopy as described earlier (Kumar et al. Antimicrob Agents Chemother. 2008;52:705-715). In brief, reaction assembled in a total volume of 1 ml containing heme (1 µM) in 100 mM acetate buffer, pH 5.2 in a Perkin Elmer Lamda 15 UV/VIS spectrophotometer at 25 ± 1 °C with quartz cells of 1 cm light- path. Different concentrations of AAMTAs compounds (1-20 µM) were added successively. Soret spectrum without AAMTAs compounds and after addition of AAMTAs compounds was recorded immediately. Interaction of AAMTAs compounds
with native heme was also measured by optical difference spectroscopy as described earlier (Trivedi et al. J Biol Chem. 2005;280:41129-41136). For measurement of difference spectra of heme- AAMTAs compounds versus the heme, both the reference and sample cuvettes were filled with 1 ml of heme solution (1 µM) to provide the baseline trace. This was followed by addition of different concentration of AAMTAs compounds to the sample cuvette with concomitant addition of the same volume of DMSO to the reference cuvette. The contents were mixed well before the spectrum was recorded. The equilibrium dissociation constant (KD) for complex formation was calculated from the following expression as described by Schejter et. al (Schejter et al. Arch Biochem Biophys. 1976;174:36-44).
(Formula Removed)
is the dissociation constant of the heme - AAMTAs compound complex, S is the concentration of AAMTAs , AA is the observed absorption changes at a particular wavelength and Aa is the absorption changes at saturating concentration of the ligand (AAMTAs compounds).
In vitro antimalarial activity
Inhibition of P. falciparum growth was studied by following (3H) hypoxanthine uptake as described earlier (Desjardins et al. Antimicrob Agents Chemother. 1979;16:710-718). P. falciparum (NF-54 strain) was cultured in vitro as described earlier (Trager and Jensen. Science. 1976;193:673-675). D-sorbitol synchronized parasites culture was used to achieve uniform ring stages as described previously (Lambros et al. J Parasitol. 1979;65:418-420) To check the antimalarial activity of AAMTAs compounds, the ring synchronized P. falciparum (parasitemia 0.5% - 1%) was cultured in multiwells (200ul/well) plate in presence or absence of different concentrations of AAMTAs. Chloroquine was used as a positive control. After 48 hours (3H) hypoxanthine ( 0.7µCi/well) was added in each well and further incubated for 48 hours to monitor parasite viability by measuring incorporation of (3H ) hypoxanthine in parasite nucleic acids. After that culture was harvested and washed thrice in phosphate buffered saline (PBS). Parasite pellets were dissolved in 100 ul 3N NaOH by keeping at 37°C for 6 hours. After incubation, it was dissolved/ added in (10 ml/vial) scintillation fluid (PPO, 4 g; POPOP, 200mg; naphthalene, 60 g; ethylene glycol, 20ml; methanol 100 ml in one litre of 1, 4 dioxane). After 12 hours of incubation, (3H) hypoxanthine uptake was measured using p- scintillation counter.
Free heme quantitation in P. falciparum
The heme content in control and AAMTAs compounds-treated P. falciprum was measured as described earlier (Motterlini et al. Am J Physiol. 1995;269:H648-655, Kumar et al. Antimicrob Agents Chemother. 2008;52:705-715). In brief, P. falciparum culture was incubated in the
presence or absence of various concentrations of AAMTAs compounds for 48 h. The culture was then centrifuged to pellet the cells, and the cell pellet was washed in PBS. Concentrated formic acid (1 ml) was then added to solubilize pellets and then heme concentration of the formic acid solution was determined in a Shimadzu UV/VIS1700 spectrophotometer at 398 nm (extinction coefficient = 1.56 x 105 M-1 cm-1). The heme content was expressed as nmol/mg of cell protein.
Measurement of reactive oxygen species
P. falciparum (4% parasitemia) was cultured in the presence or absence of different concentrations of AAMTAs compounds for a period of 48 hours as described (Kumar et al. Antimicrob Agents Chemother. 2008;52:705-715). The culture was then further incubated for a period of 30 min in complete RPMI medium containing 10 µM 2', 7'-dichlorofluorescein diacetate. The culture was then washed thrice with phosphate-buffered saline, and the parasite was isolated from control and treated groups as described. Isolated parasites were lysed by mild sonication (5-sec pulse, bath type sonicator) at 4°C. Intra-paraitic H2O2 leve Iwas measured in control or AAMTAs compounds treated parasite by measuring the fluorescent dichlorofluorescein (DCF) oxidation product of non-fluorescent probe dichlorofluorescein diacetate by H2O2 (Munzel et al. Arterioscler Thromb Vase Biol. 2002;22:1761-1768). Fluorescent intensities were recorded from the lysate in a Hitachi F-7000 fluorescence spectrophotometer in a 5-mm path length quartz cell in a total volume of 1 ml at wavelength 502 and 523 nm for excitation and emission, respectively. H2O2 was measured as relative fluorescence and expressed as fluorescence intensity /mg of parasite lysate. 'OH radicals generated as a consequence of oxidative stress in the P. falciparum after AAMTAs compounds treatment at different concentrations was measured as described earlier( Babbs et al. Methods Enzymol. 1990;186:137-147, Biswas et al. J Biol Chem. 2003;278:10993-11001)using dimethyl sulfoxide (DMSO) as •OH scavenger. In brief, P. falciparum culture (200 ul) (2-4% parasitemia, ring + early trophozoites stage) was grown in multi-well plate in presence or absence of different concentrations of AAMTAs compounds containing 20 ul of 25% DMSO for 48 hours. DMSO (20 pi) was added in each time along with the specific concentrations of AAMTAs compounds when the medium was changed (once in 24 hours). Parasite alone (without DMSO and AAMTAs compounds) was used as negative control. After 48 hours, the culture was centrifuged at 800 xg for 5 min washed and resuspended in cold PBS. The parasite was isolated as described above and the isolated parasite was lysed in triple distilled water and processed for the extraction of methanesulfinic acid formed by the reaction of •OH with DMSO. Methanesulfinic acid formed was allowed to react it with Fast Blue BB salt and the intensity of
the resulting yellow chromophore was measured at 425 nm using benzene-sulfinic acid as
standard.
Assessment of oxidative stress in P. falciparum
To analyze the oxidative stress induced by AAMTAs, we have checked the formation of lipid peroxidation, protein carbonyl formation as well as total GSH content .P. falciparum culture (4% parasitemia) was incubated with different concentration of AAMTAs compounds for 48 hours. After incubation, parasite was isolated and mixed with PBS (500 ul) to prepare parasite lysate as described above and the lipid peroxidation product from these lysates was measured as described earlier (Buege et al. Methods Enzymol. 1978;52:302-310, Guha et al. Faseb J. 2006;20:1224-1226, Biswas et al. J Biol Chem. 2003;278:10993-11001). In brief, parasite lysate (500 ul) was treated with 1ml TCA-TBA mixture in 1N HCI and incubated 15 minutes at 100° C. After incubation, it was cooled and centrifuged (4000 rpm for 10 minutes). Then supernatant was collected and OD was measured at 535nm using Shimadzu UV-VIS spectrophotometer. Formation of lipid peroxide in parasite membrane was expressed as nmol/ mg protein. Tetraethoxypropane was used as a standard. Formation of protein carbonyl was measured as described previously (Levine et al. Methods Enzymol. 1990; 186,464-478).In brief Parasite lysate (500 ul) was treated with 10% trichloroacetic acid for protein precipitation and allowed to react with 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine for 1 h. After precipitation with 10% trichloroacetic acid, the protein was washed three times with a mixture of ethanol: ethylacetate (1:1), dissolved in 0.6 ml of a solution containing 6 M guanidine-HCI in 20 mM potassium phosphate adjusted to pH 2.3 with trifluoroacetic acid. The solution was centrifuged, and the supernatant was used for measurement of carbonyl content by measuring the OD at 362 nm.
Measurement of reduced glutathione (GSH)
P. falciparum (4% parasitemia) was cultured in the presence or absence of different concentrations of AAMTAs. GSH content of control and AAMTAs treated parasite culture was measured by using a fluorometric method using the commercially available glutathione assay kit (BioVision, 980 Linda Vista Avenue Mountain View, California, USA). To perform the experiments, essentially same protocol were followed as described in kit with slight modification. In the assay, kit provided probe o-phthalaldehyde (OPA) which reacts with GSH but not with GSSG, generating fluorescence to specifically quantify GSH. GSH was used as a standard. After 48 h of treatment, the culture was washed twice with PBS, and subsequently parasite was isolated. Isolated parasite was sonicated in 200 µl of 20 mM ice-cold using sonicator (by using a 9 s on/10 s off cycle for a period of 60 s) and centrifuged at 10,000 x g for 20 min to get clear
lysate. After that lysate (200 ul) was mixed with an equal volume of 10% trichloroacetic acid, and protein precipitate was removed by centrifugation. After that 10 ul of OPA was mixed with supernatant and incubated at room temperature for 40 min according to the manufacturer's instruction. Samples were then read on a fluorescence plate reader equipped with Ex/Em = 340/420 nm (HITACHI F7000, Nishi-Shimbashi 1-chome, Minato-ku, Tokyo, Japan). Each experiment was carried out in triplicate and the mean was used in calculations.
In vivo antimalarial activity
The in vivo antimalarial efficacy of AAMTAs was evaluated as described earlier (Kumar et al. Antimicrob Agents Chemother.2008;52:705-715). In brief, rodent malarial model BALB/c mice was infected by MDR (chloroquine, mefloquine, and halofantrine) strain Plasmodium yoelii and subsequently treated intrapertonialy at three dose levels (5 mg/kg, 10 mg/kg, 25 mg/kg body weight) of AAMTAs compounds (7, 10, 12 13 16 and 17). Briefly, for each dose level a group of six mice (25 ±5 g) were inoculated intraperitoneally (i.p.) with 1 x105 parasitized RBCs on day 0 and AAMTAs compounds derivatives was administered on days 2, 3 , 4 and 5 of post-infection via the i.p. route. The treatment was continued at each dose level from day 2 to 5 via the intraperitoneal route. The aqueous suspension (emulsion) of drugs was prepared in ground-nut oil so as to obtain the required drug dose per animal in 0.2 ml volume. The efficacy of active AAMTAs were assessed by continues monitoring the effects of the drugs on % parasitemia and survival. Levels of parasitemia from individual mice were evident in Giemsa-stained thin blood smears every day. The mean value resulted for each group of mice was used to calculate the percentage of suppression in parasitemia with respect to the vehicle control group. Arteemal (a-P-Arteether) treated mice were used as a positive control at a dose level of 50 mg/kg/day body weight.
In vitro cytotoxicity
Cytotoxicity of AAMTAs compounds was evaluated by monitoring (MTT) reduction assay using U 937 leaukemia cell line.The cytotoxic effect of different concentrations of AAMTAs on U 937 leaukemia cell line was evaluated as described earlier (Reed aaand Muench. Am J Hyg. 1938;27:493-497). In brief, U937 cells were obtained from culture medium by centrifugation at 500g for 5 minutes. The cell pellets were resuspended and loaded in a haemocytometer for cell count before plating of the cells. U 937 cells were seeded in 96-well flat-bottomed tissue culture microplate at a concentration of 105 cells per well treated with different concentrations of AAMTAs(1µM- 10 mM), DMSO (negative control) and sodium azide (1 mM) and incubated for 24 hrs at 37°C in a humidified atmosphere containing 5% CO2 respectively. At the end of incubation, 10ul of WST-1 reagent was added to each well and incubated for 3 h. After
incubation, the plates were thoroughly shaken on ELISA shaker for 30 seconds. The colour absorbance of each well was recorded at 450nm using an ELISA reader (Beckman Coulter, CA, USA) with a reference serving as blank. The IC50 value of each fraction was calculated for U937 cells. All experiment were performed in triplicate and presented as mean + standard error of mean of three different experiments.
Results
AAMTAs interact with heme and inhibit hemozoin (p-hematin) formation Malaria parasite possesses efficient mechanism of digestion of hemoglobin (Hb) and subsequently the detoxification of resultant heme (ferriprotoporphyrin IX (FP) to protect itself from heme-induced oxidative stress. The major heme detoxification system involved mainly hemozoin formation (Goldberg et al Proc Natl Acad Sci USA. 1990;87:2931-2935, Ziegler et al. Curr Med Chem. 2001;8:171-189) inside the food vacuole of parasite. In general, compound which interacts with heme inhibits hemozoin formation and inhibition of hemozoin formation is the most validated rationale to develop antimalarial (Kumar et al. Toxicol Lett. 2005; 157:175-188). In order to find new antimalarial compound, different AAMTAs compound were synthesized to evaluate their heme interacting property and effect on hemozoin formation. Interaction of AAMTAs with heme was followed at different concentrations. Addition of AAMTAs perturbed the heme spectrum (Fig 1), which is indicative of an interaction between the AAMTAs and the heme moieties. This is followed by titration with increasing amounts of AAMTAs into the heme solution produced spectra with well-defined isosbestic point in the Soret range with reduction in the heme Soret molar absorptivity, and a shift of the Soret band to longer wavelengths. Interaction of AAMTAs heme were determined at pH 5.2, approximating to the pH of the parasite food vacuole to mimic the cellular mileu inside parasite. Since heme have optical absorption at 362 nm , represented a broad peak , indicating that dimers of the u-oxo type or (3-hematin type predominate under our in vitro conditions (Brown et al. Biochem J. 1976; 153:279-285, Lemberg et al. New York: Interscience Publishers; 1949). The binding of AAMTAs to heme were also studied by optical difference spectroscopy to measure and calculate binding affinity of these compounds (Fig 1). The apparent KD values for the binding of AAMTAs compounds to heme, was calculated from the plot of 1/A362 against 1/ [AAMTAs] were shown in the insets (Fig 1). Out of various AAMTAs, compound 7, 10, 12, and 16 showed the highest affinity towards heme (Table 1)
Antimalrial drugs such as chloroquine and amodiaquine (type-1 blood schizontocides) act by forming complexes with heme (the hydroxo or aqua complex of ferriprotoporphyrin IX (Fe (III) PPIX). Such interaction with heme leads to the inhibition of hemozoin formation and
subsequently causes parasite death (antimalarial). Therefore, we checked whether interaction of AAMTAs with heme can cause any inhibition of hemozoin formation and offer antimalrial activity. Results clearly indicated that AAMTAs inhibited hemozoin formation in a concentration dependent manner with an IC 50 value in the uM range (Table 1). Compound 7, 10, 12, and 16 showed much higher activity in inhibiting hemozoin formation (IC50 ~ 5 and >5µM). However, some of AAMTAs showed less activity when compared with the others. Association with heme of any test compound is not fully sufficient to inhibit hemozoin formation, there is much concerned about the lipophilicity and pKa of the test compound (Egan et al. J Med Chem. 2000;43:283-291).
Inhibition of P. falciparum growth by AAMTAs
Inhibition hemozoin formation is a most validated antimalarial drug target; hence we were interested to test the in vitro antimalarial activity of AAMTAs. Interestingly, AAMTAs inhibited the growth and development of malaria parasite effectively as evident from the inhibition of 3Hhypoxantine uptake. But out of various AAMTAs, compound 7, 9, 10, 12, 13, 16 and 17 showed very potent antimalarial activity (IC50= > 2µM ) (Table 1).Since the dose used in in vitro experiment should not be correlated with the effective dose under in vivo conditions. Pharmacokinetic evaluations and analyses during pre clinical studies showing that under in vivo conditions effectiveness of any drugs based on absorption, distribution, metabolism, and excretion. That influence the drug levels and kinetics of drug exposure to the target region and hence influence the performance and pharmacological activity of the compound as a drug. Existing literature suggested that AAMTAs offers antiproliferation and anticancer properties along with promising antimalarial activity and shown to inhibit hemozoin formation (Nair et al. Tetrahedron. 2006; 62: 6731-6747, Shagufta et al, Bioorg. Med. Chem. 2006, 14, 1497-1505, Srivastava et al. Bioorg. Med. Chem.2004; 12: 1011-1021, Kumar et al. Antimicrob Agents Chemother. 2008; 52: 705-715). However compound showing antiproliferative activity in tumor cells follows some different mode of action. Therefore, we cannot exclude that antimalarial action of these molecules followed directly due to hemozoin formation or it may be targeted multiple sites of malaria parasite like tumor cells.
Table 3.
(Table Removed)
CSLogP and CSpKa of the compound AAMTAs compounds was calculated using property prediction software CSPredict developed by ChemSilico LLC which is a registered trademark of ChemSilico LLC, Tewksbury, MA 01876, USA (www.chemsilico.com) ( Veldkamp et al. J Phys Chem. 1976; 80: 2794, Votanoet al. Molecular Diversity. 2004; 8: 379, Joseph et a\.Chemistry & Biodiversity. 2004; 1: 1829)
AAMTAs promotes oxidative stress in P. falciparum
The inhibition of hemozoin formation as a result of heme interaction causes death of the parasite due to the accumulation of toxic free heme and subsequent development of the oxidative stress (Kannan et al. Biochem J. 2005;385:409-418, Portela et al. Bioorg Med Chem. 2004;12:3313-3321). It is well known that free heme is very toxic possess detergent-like properties interfering with membrane integrity and has the ability to undergo redox reactions causing the generation of reactive oxygen species (ROS) (Kumar et al. Toxicol Lett. 2005;157:175-188). In view of above finding, we were interested to check whether AAMTAs can develop oxidative stress in P. faciparum by favouring the accumulation of free heme. The selected AAMTAs (7, 9, 10, 12, 13, 16 and 17), which showed better anti-plasmodial activity were tested for this purpose. Results clearly indicated that the inhibition of hemozoin formation by AAMTAs allowed the accumulation of heme in the parasite (Fig 2A). Out of various AAMTAs tested, compound 7, 10, 12 and 16 were shown to be higly active. Intra-parasitic H2O2 was measured in control and AAMTAs treated parasite by measuring the fluorescent
dichlorofluorescein (DCF) oxidation product formed from non-fluorescent probe dichlorofluorescein diacetate after interation with ROS. Interestingly, AAMTAs treatment causes significant induction of the generation of intra-parasitic H2O2(Fig 2B)
The capabilities of iron of heme to accept and donate electrons contribute towards its potential toxicity. A possible route for degradation of the heam is by reacting with H2O2 under conditions designed to resemble those found in the food vacuole, i.e., at pH 5.2. However, the estimation of concentrations of heme and H2O2 in the food vacuole is difficult. The peroxidative degradation of haem is thought to involve a reaction with H2O2 to form a ferryl intermediate (Loria et al. Biochem. J. 1999; 339: 363-370). Transport of electrons within the Fe(IV) intermediate of heme causes widening of the porphyrin ring and release of iron, which can interact with H2O2 and formed hydroxyl radical (•OH) (Traylor et al. J. Am. Chem. Soc. 1995:117: 3468-3474, Brown et al. Biochem. J. 1978; 174;901 -907).
In view of above, we check the formation of •OH inside parasite after treating parasite in culture with different concentrations of AAMTAs. AAMTAs lead to the generation of highly reactive *OH in a concentration dependent manner (Fig 2C). The inhibition of heme detoxification to hemozoin by AAMTAs resulted in the accumulation of vast amount of reactive oxygen species (ROS) in P. falciparum. An excess of ROS will cause oxidative damage to critical biomolecules such as lipids and proteins leading to the formation of the lipid peroxidation product and protein carbonylation respectively. It was observed that incubation of parasite culture with AAMTAs leads to the formation of the lipid peroxidation product and protein carbonylation. Among AAMTAs compound 7, 9, 10, 12, 13, 16 and 17 caused significant formation of lipid peroxide and protein carbonyl (Fig 3A and 3B). GSH plays a pivotal role in the antioxidant defense through the maintenance of the redox state of protein-SH moieties, reduces the noxious hydrogen and lipid peroxides and the extrusion of heme (Muller et al. Mol Microbiol. 2004; 53: 1291-305). GSH uptake from erythrocyte cytosol through the process of pinocytosis and inside the food vacuole (parasite) binds with free heme and reduces its toxicity (Becker et al. Int J Parasitol. 2004; 34: 163-189) Apart from its role in heme detoxification, GSH acts as a cofactor for a variety of vital proteins including glutathione-dependent peroxidases, glutathione S-transferases (GSTs), glutaredoxins and glyoxalases of the parasite (http://plasmodb.org) (Sies et al. Free Radic Biol Med. 1999; 27: 916-921). It is also directly involved in antioxidant reactions - for instance, the termination of radical-based chain reactions where single electrons are transferred from thiyl radicals or disulphide radicals (Frey et al. Curr Opin Chem Biol. 1997; 1: 347-356), thus depletion of GSH would result in a less efficient detoxification of free FP IX and inactivation of many enzymes, development of oxidative stress and consequently the death
of parasite. To examine whether AAMTAs via stimulating the accumulation of intra-parasitic H2O2 reduced the cellular GSH level, selected AAMTAs were incubated with P. falciparum in culture and GSH level was measured. AAMTAs significantly decreased the GSH level concentration dependently (Fig. 3C).
In order to assess whether the oxidative stress induced by AAMTAs is actually responsible for the inhibition of parasite growth and development or any other factor is also involved. Therefore, we have checked the restoration of parasite growth and development after antioxidant therapy (effect of different antioxidant or •OH scavengers; such as mannitol and spin traps like PBN on AAMTAs induced P. falciparum death). Results clealy indicated that •OH scavengers significantly protected P. falciparum from AAMTAs induced growth inhibition (Fig. 3D). Thus from the above data it is evident that the development of oxidative stress is the antimalarial mode of action of AAMTAs.
In vivo antimalarial activity against multidrug resistant malaria parasite P. yoelii (MDR) In vitro antimalarial activity of AAMTAs encouraged us to evaluate the effect of efficient AAMTAs against multidrug resistant rodent malaria parasite Plasmodium yoelii (MDR) under in vivo conditions. Since here in this study we have found that AAMTAs showed potent antimalarial activity against chloroquine (CQ) sensitive strain of P. falciparum, but the whole world is concerned towards the identification of new antimalarial drugs against multi drug resistant parasite. Moreover, there is ample evidence in the literature that compounds, which are active in vitro even at lower concentration, did not show activity when tested in vivo. To overcome all these issues, we have used MDR strain of Plasmodium (P. yoelii) to test the antimalarial activity of AAMTAs under in vivo condition using mice model (balb/C). We have selected few in vitro screened AAMTAs, which showed highest activity. These issues comprises several aspects involves rapid metabolic degradation, selective uptake as well as ability of the drug to accumulate to pharmacologically relevant concentrations at the site of drug action. But, in vitro assay of antimalarial activity is beneficial to screen large number of drugs to select effective one. AAMTAs showed good antimalarial activity in vivo and out of various AAMTAs, compound most active one during in vivo study were compound 7, 12 and 16, which suppressed the mean parasitemia (day 8) by 50%, 68% and 79% (Compound 7) 65%, 75% and 85% (compound 12) and 55%, 72% and 80%(compound 16) at dose levels of 5 mg/kg, 10 mg/kg and 25 mg/kg, respectively (Table 2). While compound 10, 13 and 17 are comparatively less effective and suppressed the (day 8 ) mean parasitemia by 23%, 41% and 50% (compound 10), 33%, 53% and 58% (compound 13), 25%, 36% and 41% (compound 17) at dose levels of 5 mg/kg, 10 mg/kg and 25 mg/kg, respectively. Thus, results indicated that compound 7, 12 and
16 were highly effective but compound 10, 13 and 17 were found to be comparatively less effective. However, compound 10 showed best activity during different experiments like heme binding, hemozoin formation and in vitro parasite growth but not to be effective during in vivo testing. Table4.
(Table Removed)
Percentage suppression was calculatedas [(C-T)/C] x 100, where C is the parasitemia in the control group and T is the parasitemia in the treated group.
In vitro cytotoxicity assay
To study the toxicity of AAMTAs on nucleated rapidly proliferating cells, we used leukemia cell line U 937. Cells were incubated with 1mM-10 mM concentration of AAMTAs, followed by MTT reduction assay. It was found that AAMTAs had no significant toxicity on rapidly proliferating leukemia cells (Table 3) as evident from selectivity index, which were greater than 142. Table 5.
(Table Removed)
Advantages of the Inventions:
1. AAMTAs show antimalarial activity in vivo in animal model
2. Drug resistance of malaria parasite is a major public health problem which hinders the
control of malaria. AAMTAs show prominent antimalarial activity against multi drug
resistance strain of Plamodium yoelli under in vivo condition. Results of in vivo screening of
active AAMTAs compounds shows that these compounds suppress mean % parasitemia (in
AAMTAs treated group) more than 50% even at a dose of 25 mg/kg body weight in
experimental Balb/c mice model.
3. Since AAMTAs show interaction with heme and inhibits hemozoin formation, (which is
basically a physical process rather than genetic one) development of resistance against this
compound will not be easy and quick.
4. AAMTAs show interaction with heme effectively even at very low concentration and causes
free heme accumulation which ultimate leads to parasite death (as monitored by 3H
hypoxanthine incorporation assay using P. falciparum culture) via generation of oxidative
stress.
5. Selective active AAMTAs compounds do not show in vitro cytotoxicity on nucleated proliferating leukemia cell line U 937 and the selectivity index (Si) value was in a range of 142- 253, when analyzed by MTT reduction assay.

We claim:
1) Aryl aryl methyl thio arenes (AAMTAs) of general formula I
(Formula Removed)
wherein Aryl1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl; Aryl2 is selected from a group consisting of substituted phenyl, naphthalene, phenanthrene, pyridine, benzoxazolyl, benzthiazolyl; Aryl3 is selected from a group consisting of substituted phenyl groups such as methoxy phenyl, p-thiomethoxy phenyl, phenyl; R is H.
2) The compound as claimed in claim 1 wherein, the representative compounds of general
formula I comprising:
• 2-((4-methoxyphenyl)(phenylthio)methyl)pyridine 4:
• 2-((4-methoxyphenyl)(o-tolylthio)methyl)pyridine 5:
• 3-[(4-Fluoro-phenylsulfanyl)-(4-methoxy-phenyl)-methyl]-pyridine 6:
• 2-[(4-Methoxy-phenyl)-pyridin-3-yl-methylsulfanyl]-benzoxazole 7:
• 4-((4-methoxyphenyl)(phenylthio)methyl)quinoline 8:
• 3-[(2-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 9:
• 3-[(3-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine10
• 3-[(Naphthalen-2-ylsulfanyl)-phenyl-methyl]-pyridine 11:
• 3-[(4-Methylsulfanyl-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 12:
• 2-[(2-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 13:
• 2-[(3-Methoxy-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 14:
• 2-[(4-Methylsulfanyl-phenyl)-(naphthalen-2-ylsulfanyl)-methyl]-pyridine 15:
• 2-((4-methoxyphenyl)(pyridin-3-yl) methylthio) pyridine 16 :
• 2-Benzhydrylsulfanyl-pyridine 17:
3) The compound as claimed in claim 1 wherein the representative compounds of general
formula (I) comprising the following structures:
(Structure Removed)
4) The compound as claimed in claim 1 wherein, the compounds of general formula I are useful for the treatment of malaria.
5) The compound as claimed in claim 1 inhibited hemozoin formation in a concentration dependent manner with an IC50 value of the most active AAMTAs (Compound 7, 9, 10, 12, 13, 16 and 17) were in the range of 5 ± 0.24 to 13±1.9 uM using parasite lysate from P. yoelii (MDR strain).
6) The compound as claimed in claim 1 showed the affinity towards heme as evident from KD
values for the binding of most active AAMTAs (compound 7, 10, 12, and 16) ranging from 4.26
±0.4 to 6.25 ±0.8.
7) The compound as claimed in claim 1 inhibited the growth and development of malaria parasite effectively as evident from the inhibition of hypoxanthine uptake with an IC50 value of the active AAMTAs (Compound 7, 9, 10, 12, 13, 16 and 17) were in the range of 1 ± 0.003 to 3.4 ± 0.29 µM.
8) The compound as claimed in claim 1, having apparent KD (binding constant of AAMTAs with heme were studied by optical difference spectroscopy) values ranging between 4.26 ± 0.4 to 14.45 ± 1.4 µM.
9) The compound as claimed in claim 1, having apparent CSpKa ranging between 3.411 ±1.276
to 4.96±0.8 mol/litre.
10) A process for the preparation of general formula I wherein the process steps comprising:
i) reacting a compound having general formula 1a-e
(Formula Removed)
wherein R is selected from a group consisting of hydrogen, methoxy, thiomethoxy with general formula 2a-d,
(Formula Removed)
wherein Ar1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl under Grignard reaction conditions in the presence of organic solvent at 25 °C for a period ranging between 40 to 52 minutes to produce a compound of general formula 3a-k,
(Formula Removed)
Wherein R is selected from a group consisting of hydrogen, methoxy, thiomethoxy and Ar1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl. ii) reacting the compound of general formula 3a-k as obtained in step (i) with substituted aryl and heteroaryl thiol derivatives of formula
(Formula Removed)
wherein Ar2 is selected from a group consisting of Phenyl, 2-methyl-phenyl, 4-flouro-phenyl, 2-benzoxazolyl, naphthalenyl, 2-Pyridyl, 2-benzthiazolyl under Friedel-Crafts reaction conditions in the presence of Lewis acids in a solvent selected from group of benzene, toluene and xylene at a temperature in the range of 20°C to 40 °C for a period ranging between 1-2 hrs to obtain a compound of formula 4-17,
(Formula Removed)
Wherein R is selected from a group consisting of hydrogen, methoxy, thiomethoxy; Ar1 is selected from a group consisting of 2-pyridyl, 3-pyridyl, 4-quinolinyl, phenyl; Ar2 is selected from Phenyl, 2-methyl-phenyl, 4-flouro-phenyl, 2-benzoxazolyl, naphthalenyl, 2-Pyridyl, 2-benzothioxazolyl.
11) The process as claimed in claim 10 wherein, the Lewis acids used in Fridel-Crafts reaction
is selected from a group consisting of aluminium chloride and cone, sulphuric acid.
12) The process as claimed in claim 10 wherein, the solvent for Grignard reaction is dry THF.
13) The compound as claimed in claim 1 wherein, the active compounds do not show in vitro
cytotoxicity on nucleated proliferating leukemia cell line U 937 and the selectivity index (SI) is
more than 142.
14) The compounds as claimed in claim 1 wherein the most active compound 12 and 16 show
statistically significant protection of mice against experimental infection with malaria parasite.
15) Aryl aryl methyl thio arenes (AAMTAs) and a process for preparation thereof substantially as herein described with reference to the examples and drawings accompanying the specification.