DESC:Related Application
This application takes priority from the Indian Provisional Application 201741028011 filed on 7th August 2017, which is incorporated herein in its entirety.

Field of Invention
The present invention is related to providing derivatives of eugenol that have antileishmanial activity against both promastigotes and amastigotes ofL. donovani.

Background
Leishmaniasis, Chagas disease, and sleeping sickness affect 20 millionpeople worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Leishmania spp., Trypanosoma cruzi, and Trypanosoma brucei spp.(Nature 2016, 537:229). These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. (Science 2005, 309:404). Visceral leishmaniasis (VL) is one of the most devastating infectious diseases caused by obligate intracellular parasites, Leishmania donovani with a worldwide incidence of 0.3 million new cases per year. It mainly affects the proper functioning of host immune system and increases the risk of concomitant infections. The problems with contemporary medicines like pentavalent antimonials, amphotericin-B (AmpB), pentamidine and miltefosine are their low therapeutic index (TI), severe side effects, acute cytotoxicity, high cost and emergence of their resistance strains (See Clin.Microbiol. Rev. 19, 2006:111). In the absence of effective vaccination against VL, we are compelled to rely only on the available drugs to cure this disease. Although new entities have been reported in the recent literatures which act upon the type IB DNA topoisomerase, and pteridine reductase 1 of Leishmania parasite (See Eur. J. Med. Chem. 2016,123: 639).Several other compounds are also reported which effectively arrest the parasite cell cycle and kill the parasite by concomitant nitrite generation in host cells (See Eur. J. Med. Chem. 2016, 110:237; Eur. J. Med. Chem. 2016, 124:468).
There are two popular drug developmental approaches for VL treatment, (i) direct killing of the parasite and (ii) killing of the parasite by boosting the immune response of the host cell. It is already found that the second approach is very much effective compared to the first one in the sense of toxicity measure. For the clearance of the parasite inside the macrophage (host cell) specific cytokine responses need to be impaired like IL-10, TGF-ß, and at the same time other cytokine response like IL-12, IFN-? need to be enhanced. Similarly, in vivo model, leishmanial parasite enters into the host system and modulates the host immune system with strong Th2 cytokine response that causes severe immune suppression[9]. Therefore, it is quite challenging to develop such an immunomodulatory molecule for the treatment of VL.
In an attempt to develop a set of anti-visceral leishmanial immunomodulatory compounds inspired by natural products, applicants have selected eugenol as the parent scaffold. Eugenol, a phenolic phytochemical, is found as a major component of many essential oils extracted from various medicinal plants. It is known to have various medicinal properties, but the most intriguing one is its antileishmanial activity (See Parasitol. Int. 2006, 55: 99). Eugenol-rich essential oil from different plant sources has been documented to have high toxicity towards promastigotes and amastigotes of L. amazonensis andL. donovani. Simple chemical modifications of the eugenol scaffold by means of acetylation or benzoylation have also been found to improve the efficacy by 2 or 5 folds, respectively compared to eugenol (on L. infantum and L. chagasi promastigote) along with the reduction of their cytotoxicity level (See Med. Chem. 2014, 22; 6250).

Summary of the Invention
The present invention provides several novel cyclic and acyclic derivatives of eugenol by means of suitable chemical transformation. The invention is defined by Formula I:

wherein,
X is independently selected from the group consisting of -CH2-, -C=O, -O-CH2-CO-, -CH2CO- and -NH-C=O;
R1 is alkyl saturated or unsaturated, linear or branched,cycloalkyl, alkenyl, alkynyl, aryl, hetero aryl, unsubstituted or optionally substituted with F, Cl, Br, I, CN, OH, NH2, OMe;and wherein the said composition has antiparasitic activity.
Further, X is a linker selected from a group consisting of
wherein n is 0 to 6.
The invention also provides R1 selected from the group consisting of

wherein n is 0 to 6 and Y is F, Cl, Br, I, CN, OH, NH2, NO2 or OMe.
and the novel compounds listed as 3 to 59 were screened for their antileishmanial activity against promastigote and amastigote forms of L. donovani. Selected compounds were further studied to elucidate the immunomodulatory activity and mechanistic aspects of parasite killing in L. donovani infected peritoneal macrophages as in vitro model. The antileishmanial activity of the compounds in
L. donovani infected BALB/c mice was studied by estimating the hepatic and splenic parasite burden and other immunomodulatory effector responses.
The compounds of the invention show immunomodulation resulting in therapeutic activity against leishmanial and other related infections.

Brief Description of Figures
Figure 1. Hepatic and splenic parasite burden in L. donovani-infected BALB/c mice treated with compound 35.Parasite burden was determined in liver (A) and spleen (B) by the stamp smear method, subsequently expressed as Leishman-Donovan Unit (LDU). Results are expressed as mean ± S.E.M. from four mice per group. ***p<0.001, significant differences compared with infected mice.
Figure 2. Parasite burden in (A) Spleen and (B) Liver after oral administration of compound 35 for 7 and 14 days in 14 days post infected mice. *p<0.05; **p<0.01, significant differences compared with respective infected mice.
Figure 3. Comparative data of single dose of compound 35 given orally and by intravenous route in mice.

Detailed Description of the invention
The present invention provides novel cyclic or acyclic derivative of eugenol which have the utility as antimicrobial agents, particularly antileishmanial agents, more particularly having antileishmanial activity against parasitic infections such as the ones caused by Leishmania donovani, Leishmania infantum, Trypanosoma cruzi and Trypanosoma brucei
The novel composition of cyclic or acyclic derivative of eugenol of the present invention is provided herein as formula I:

wherein,
X is independently selected from the group consisting of -CH2-, -C=O, -O-CH2-CO-, -CH2CO- and -NH-C=O;
R1 is alkyl saturated or unsaturated, linear or branched,cycloalkyl, alkenyl, alkynyl, aryl, hetero aryl, unsubstituted or optionally substituted with F, Cl, Br, I, CN, OH, NH2, OMe;and wherein the said composition has antiparasitic activity.
Further, X is a linker selected from a group consisting of
wherein n is 0 to 6.
The invention also provides R1 selected from the group consisting of

wherein n is 0 to 6 and Y is F, Cl, Br, I, CN, OH, NH2, NO2 or OMe.
Provided below in Table A are structures of Compounds 3 to 59 all of which are claimed as compositions of the present invention. Compounds 1 and 2 are reference molecules used in all the studies carried out with regard to the novel compounds 3 to 59.
Table A
Compound Number Structure IUPAC Name
1
4-allyl-2-methoxyphenol (eugenol)
2
4-allyl-1,2-dimethoxybenzene (methyl eugenol)
3
4-allyl-1-(allyloxy)-2-methoxybenzene
4
4-allyl-2-methoxy-1-((3-methylbut-2-en-1-yl)oxy)benzene
5
4-allyl-1-ethoxy-2-methoxybenzene
6
4-allyl-2-methoxy-1-propoxybenzene
7
4-allyl-1-butoxy-2-methoxybenzene
8
4-allyl-2-methoxy-1-(pentyloxy)benzene
9
4-allyl-1-(hexyloxy)-2-methoxybenzene
10
4-allyl-1-(heptyloxy)-2-methoxybenzene
11
4-allyl-2-methoxy-1-(octyloxy)benzene
12
4-allyl-2-methoxy-1-(nonyloxy)benzene
13
4-allyl-1-(decyloxy)-2-methoxybenzene
14
4-allyl-1-(dodecyloxy)-2-methoxybenzene
15
4-allyl-1-(hexadecyloxy)-2-methoxybenzene
16
(Z)-4-allyl-2-methoxy-1-(octadec-9-en-1-yloxy)benzene
17
4-allyl-2-methoxyphenyl acetate
18
4-allyl-2-methoxyphenyl 4-fluorobenzoate
19
4-allyl-2-methoxyphenyl 2-chlorobenzoate
20
4-allyl-2-methoxyphenyl 4-nitrobenzoate
21
4-allyl-2-methoxyphenyl 3-nitrobenzoate
22
4-allyl-2-methoxyphenyl 1H-indole-3-carboxylate
23
4-allyl-2-methoxyphenyl 2-phenylacetate
24
4-allyl-2-methoxyphenyl 2-phenoxyacetate
25
4-allyl-2-methoxyphenyl propionate
26
4-allyl-2-methoxyphenyl butyrate
27
4-allyl-2-methoxyphenyl pentanoate
28
4-allyl-2-methoxyphenyl hexanoate
29
4-allyl-2-methoxyphenyl heptanoate
30
4-allyl-2-methoxyphenyl octanoate
31
4-allyl-2-methoxyphenyl nonanoate
32
4-allyl-2-methoxyphenyl decanoate
33
4-allyl-2-methoxyphenyl dodecanoate
34
4-allyl-2-methoxyphenyl dodecanoate
35
4-allyl-2-methoxyphenyl oleate
36
4-allyl-2-methoxyphenyl prolinate
37
4-allyl-2-methoxyphenyl piperidine-4-carboxylate
38
4-allyl-2-methoxyphenyl tryptophanate
39
4-allyl-2-methoxyphenyl decylcarbamate
40
4-allyl-2-methoxyphenyl tetradecylcarbamate
41
4-allyl-2-methoxyphenyl (Z)-hexadec-7-en-1-ylcarbamate
42
4-allyl-2-methoxyphenyl (Z)-hexadec-9-enoate
43
2-methoxy-4-propylphenyl palmitate
44
2-methoxy-4-propylphenyl (Z)-hexadec-9-enoate
45
4-allyl-2-methoxyphenyl non-8-enoate
46
4-allyl-2-methoxyphenyl (Z)-hexadec-9-en-1-ylcarbamate
47
4-allyl-2-methoxyphenyl (E)-hexadec-9-enoate
48
2-methoxyphenyl (Z)-hexadec-9-enoate
49
4-allyl-2-methoxyphenyl 2-(4-cyclohexylphenoxy)acetate
50
4-allyl-2-methoxyphenyl 2-(4-ethylphenoxy)acetate
51
4-allyl-2-methoxyphenyl 2-(4-nitrophenoxy)acetate
52
4-allyl-2-methoxyphenyl (E)-octadec-9-enoate
53
2-methoxy-4-propylphenyl oleate
54
4-allyl-2-methoxyphenyl 2-(4-methoxyphenoxy)acetate
55
4-allyl-2-methoxyphenyl (R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate
56
4-allyl-2-methoxyphenyl 2-(4-morpholinophenoxy)acetate
57
4-allyl-2-methoxyphenyl 4-(octyloxy)benzoate
58
4-allyl-2-methoxyphenyl 4-(decyloxy)benzoate
59
4-allyl-2-methoxyphenyl 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetate

The compounds of present invention represented as numbers 3 to 59 have antileishmanial activity and thus have the utility to serve as antileishmanial compounds for treatment of L. donovani parasitic infection.
The present invention provides three sets of eugenol derivatives using phenolic OH of eugenol (compound 1). For the first set of molecules (compounds 3 to 16), applicants have connected different aliphatic chains with the parent molecule via an ether linkage. The ether linkage was prepared under Mitsunobu reaction condition for 3, 4 and 7. However, for the other molecules of this set there obtained a low yield, under the same reaction condition and it was because of the moisture present with the corresponding alcohols. Therefore, to overcome this problem and to reduce the reaction time, remaining molecules were prepared by using alkyl/alkenyl halides in presence of NaH in quantitative yield (Scheme 1). Similarly, the second set of molecules (compounds 17–35) were prepared either by the reaction with anhydrides or the corresponding carboxylic acids. For esterification, two different coupling reagents DCC and EDCI.HCl were used. Selection of coupling agent was made based on the yield and the ease of purification. For the aliphatic carboxylic acids EDCI.HCl gave the best yield.
In an effort to introduce polar amino group, third set of molecules was prepared where N-Boc-L-proline, N-Boc-isonipecotic acid and Na-Boc-L-tryptophan were coupled with the eugenol moiety using DCC. However, under the usual condition at 5% TFA in DCM decomposition of the starting material was observed, thus, we used dry HCl gas in MeOH for the removal of Boc to yield 36, 37 and 38 respectively (Scheme 1).

Scheme 1: Reagents and conditions: (i) R1-OH, DIAD/PPh3, THF, 7-8 h, rt, or alkyl/alkenyl halide, NaH, DMF, 3-4 h, rt; (ii) (R2CO)2O*, Pyridine/CHCl3,6 h, rt, or R2CO2H, DCC/ DMAP, DMF, 8-10 h, rt, or R2CO2H, EDCI.HCl/ DMAP, DMF, 3 h, rt; (iii) N/Na-Boc-R3-CO2H, DCC/ DMAP, DMF, 6-8 h, rt and (iv) HCl gas (dry), MeOH, 3 h, rt.

The present invention provides several novel cyclic and acyclic derivatives of eugenol by means of suitable chemical transformation. These derivatives were screened for their antileishmanial activity against promastigote and amastigote forms of L. donovani. Selected compounds were further studied to elucidate the immunomodulatory activity and mechanistic aspects of parasite killing in L. donovani infected peritoneal macrophages as in vitro model.Theantileishmanial activity of the compounds in L. donovani infected BALB/c mice was studied by estimating the hepatic and splenic parasite burden and other immunomodulatory effector responses.

Uses
The compounds of the invention are useful for the treatment of infections in subjects, mammals in particular, including humans. In one embodiment, the compounds may be used for the treatment of infections of soft tissues, blood, skin, mouth, lungs, respiratory tract, urinary tract and reproductive tract.
In another embodiment, the compounds of the invention are useful for the treatment of parasitic infections, particularly leishmanial and other related infections and more particularly human infections caused by Leishmaniadonovani, Leishmania infantum,Trypanosoma cruzi and Trypanosoma brucei infections.

Route of Administration
The compounds of the present invention are delivered to the subjects by forms suitable for each administration route. For example, the compounds are administered as tablets, capsules, injection, drops, inhaler, ointment, foams suppository. In a preferred embodiment, the route of administration is oral, parenteral or topical. Topical or transdermal administration include powders, sprays, ointments, pastes creams, lotions, gels, solutions, patches and inhalants.

Dosage Forms
The composition of the present invention is presented in unit dosage form generally in an amount that produces a therapeutic effect in the subject.
The compounds of the present invention are administered at a daily dose that is the lowest dose effective to produce a therapeutic effect. Generally, the dosage will effect from about 0.0001 to about 100mg per kg body weight per day. Preferably, the dosage will range from about 0.001 to 75mg per kg body weight per day and more preferably, the dosage will range from about 0.1 to about 50mg per kg body weight per day. Each unit dose may be, for example, 5, 10, 25, 50, 100, 125, 150, 200 or 250 mg of the compound of the invention. As per the requirement of the subject, the effective daily dose of the compound is administered as two, three, four or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.

Formulation
The antiparasitic compositions of the present invention may be administered by any method known in the art. Some examples of suitable modes of administration include oral, intravenous, intramuscular topical or any other parenteral mode of administration.
In certain embodiments, the present invention is directed to a method of formulating compounds of the present invention in a pharmaceutically acceptable carrier or excipient and may be administered in a wide variety of different dosage forms e.g. tablets, capsules, sprays, creams, lotions, ointments, aqueous suspensions syrups, and the like. Such carriers may include one or more of solid diluents or fillers, sterile aqueous media, and various nontoxic organic solvents, etc.
For oral administration, tablets may contain various excipients such as one or more of microcrystalline cellulose, sodium citrate, calcium carbonate and the like, along with various dispersants such as starch and certain complex silicates, together with granulation binders like polyvinylpyrrolidone, sucrose and the like. Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
In another embodiment, the compounds of the present invention may be administered topically that include transdermal, buccal, or sublingual application. For topical applications, therapeutic compounds may be suitably admixed in a pharmacologically inert topical carrier such as a gel, an ointment, a lotion, and/or a cream. Such topical carriers may include water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, and/or mineral oils.
The timing of the administration of the pharmaceutical composition may also be regulated. For example the compounds may be administered intermittently or by modifying the release parameters.

Definitions
Promastigote is the motile, elongated, extracellular form in the life cycle of some protozoans (family Trypanosomatidae, and especially genus Leishmania) that is characterized by a single anterior flagellum and no undulating membrane
Amastigote is the nonmotile, parasitic form in the life cycle of some protozoans (family Trypanosomatidae, and especially genus Leishmania) that usually develops in the cells of vertebrate hosts and occurs as a minute, ovoid or spherical body with a prominent, rod-shaped kinetoplast and a rudimentary, internal flagellum arising from a basal body
Chain length as used in this application means the length of the alky chain, whose length is defined by the number of carbon atoms present in the alkyl chain.

The present invention is further illustrated and explained with the help of Examples. These are merely for better understanding and, however, not to be construed as limiting the scope of the compounds of present invention.

Examples
Example 1. Synthesis of the eugenol derivatives
Formation of the ether linkage: General method
Method A1: Eugenol was dissolved in dry THF and stirred at room temperature under nitrogen atmosphere. To the stirred solution triphenylphospine (TPP) and corresponding alcohol were added and the stirring was continued for 15 minutes at room temperature. Thereafter, diisopropylazodicarboxylate (DIAD) was added drop wise to the reaction mixture, and stirred at room temperature for another 8-12 hours. After completion of reaction (checked by TLC) volatiles were evaporated and extracted with ethylacetate. The combined organic layer was then dried over Na2SO4 and concentrated under reduced pressure. The compound thus obtained was purified by silica-gel column chromatography using ethyl acetate and hexanes as eluent.
Method B1: To a stirred solution of eugenol in DMF at 0°C, NaH was added and stirred for 10 min. Thereafter, measured amounts of alkyl/alkenyl halides were added and the reaction mixture was stirred for another 2 h at room temperature. After completion of the reaction it was quenched with water and diluted with EtOAc. The organic portion was then washed with water and brine solution. The organic portion was the dried over Na2SO4 and concentrated under reduced pressure. The compound thus obtained was purified by silica-gel column chromatography using ethyl acetate and hexanes as eluent.

Formation of the ester linkage: General method
Method A2: Eugenol was dissolved in chloroform and cooled to 0-5°C, to that pyridine was added followed by the addition of respective acid anhydride. The reaction mixture was then stirred at room temperature for 6 hours. After completion of reaction (checked by TLC) it was diluted with chloroform and washed with 5% aqueous HCl, water and brine. Combined organic layer was then dried over Na2SO4 and concentrated under reduced pressure. The compound thus obtained was purified by silica-gel column chromatography using ethyl acetate and hexanes as eluent.
Method B2: Eugenol was dissolved in DMF. To that 4-(dimethylamino) pyridine (DMAP) and respective acid were added and stirred at room temperature. After 15 minutes N, N-dicyclohexylcarbodiimide (DCC) was added dropwise and stirred at room temperature for 10 hours. After completion of reaction (checked by TLC), reaction mixture was diluted with water and extracted with Ethyl acetate (3x30 ml). Combined organic layer was then washed with saturated NaHCO3, water and then with brine solution. Combined organic layer was then dried over Na2SO4 and concentrated under reduced pressure. The compound thus obtained was purified by silica-gel column chromatography using ethyl acetate and hexanes.
Method C2: Carboxylic acid, EDCI.HCl and DMAP were suspended in 3 ml of THF and stirred for 20 min at room temperature. Then eugenol was added to the reaction mixture and allowed to stir for 4h at room temperature. After the completion of reaction (checked by TLC), all the volatile compounds were evaporated in vacuo. The crude was partitioned between ethyl acetate (3 × 10 ml) and 10% sodium bicarbonate. The combined organic layer was washed with water followed by brine and dried using Na2SO4. Then, the compound was purified by silica gel chromatography.

Example 2. Activity of Eugenol derivatives
Assays for establishing therapeutic potential of Compounds 1-59
a) Promastigote killing assay
Antipromastigote activity was determined by MTT assayreadily available for person skilled in the art from published literature. Promastigote viability was determined as a relative percentage to untreated control setsas per known protocols.
b) Amastigote killing assay
Mouse (BALB/c) peritoneal macrophages were cultured in 8 well chamber glass slide (Genetix), at a cell density of 1×105 cells per 200 µL of RPMI1640 supplemented with 10% FBS. Peritoneal macrophages were allowed to attach overnight and were infected with promastigotes at a macrophage: promastigote ratio of 1:10 for 6 h at 37°C with 5% CO2. Non-internalized promastigotes were removed by washing twice with RPMI1640 media. The infected macrophages were treated with increasing concentrations of eugenol derivatives (0 to 250 µg/ml) for another 42 h. Untreated and treated infected macrophages were washed, ?xed in methanol and stained with Giemsa stain. Parasite load was calculated as number of amastigotes/100 macrophages under light microscope.
c) Cytotoxicity assay
Cytotoxicity was determined by MTT assay in RAW264.7 murine macrophage cells and BALB/c derived peritoneal macrophages (1×105) cultured in 96 well plate with increasing concentrations of novel compounds of the invention (0 to 250 µg/ml). Absorbance of solubilized MTT formazan product was colorimetrically measured at 570 nm using a microplate reader.
d) Nitrite generation assay
Nitrite generation was estimated by using Griess reagent (sigma)as per standard protocols (J.Antimicrob.Chemother. 2012, 67:2892) known in the art which a person skilled in the art can readily identify.
e)In vivo anti-visceral leishmanial response
Male BALB/c mice (6-8 weeks, weight matched) were remained uninfected or infected with 2×107L. donovanipromastigotes intravenously. Uninfected control (n=4) and infected control mice (n=4) were injected intravenously via the tail-vein with phosphate-buffered saline. Another two infected groups (n=4 per group) were treated with compound 35 [10 mg/kg of body weight (BW) and 25 mg/kg of BW]. All treatments were started two weeks post infection with five days interval between each treatment. After completion of four weeks treatment, all mice were sacrificed and hepatic and splenic parasite burden were determined from tissue imprints after Giemsa staining. Results were expressed as Leishman Donovan Units (LDU) as described earlier.
Results
Structure activity relationship
The leishmanicidal activity of the synthesized molecules weas tested against both promastigote and amastigote forms of L. donovani. The nitric oxide production which is primarily responsible for the killing of parasite within the host macrophage was determined for individual compounds and is given as EC50 value.

Activity against promastigote
The activity (IC50) of the first set of molecules (3 to 16) against promastigote form of L. donovani was found to be improved with the elongation of chain length compared to the reference molecules 1 (441.66±13.28 ?M) and 2 (475.45±19.08 ?M) (Table 1). The IC50 value for compound 3 was 237.58±13.51, which was almost two folds better than the reference molecules. For further elongation of the chain length up to C18 we observed a sharp decrease of IC50 values. However, a limiting condition was observed for comp. 5 (C2) and 8 (C5) to 16 (C18). The best activity was found for compound 13 (49.92±7.06 ?M), with further elongation of the chain length the IC50 values were found to increase. This observation indicated that the C10 chain length was the most favoured selection in the first set (compounds 3-16). Similarly, for the second set of molecules (17 to 35), with aromatic substitution the IC50 values were found to be in the range of 82.38±6.19 ?M to 28.86±1.14 ?M and the best activity was observed for comp. 24 (28.86±1.14 ?M). For the aliphatic substitution, better activity was observed for compounds 32, 33, 34 and 35 with C9, C11, C15 and C17 chain length respectively (Table1). The IC50 values for the third set of molecules(compounds 36 to 38)were found in the range of 97.35±3.90 – 52.97±1.57 ?M (Table 1).
Compound 35 showed two and twenty times higher IC50 value compared to the known available drugs miltefosine and AmpB respectively (Table 1).

Table1: Antipromastigote and Antiamastigote activities against L. donovaniand nitric oxide generation of eugenol and its derivatives (Compound 1 to 38 and miltefosine & AmpB).
Promastigote Amastigote NO generation
(µM)
Comp. No. 1IC50
(?M) 2EC50
(?M) 3Cytotoxicity
EC50 (?M)
Milte 10.38±0.32 6.40±0.42 40.66±2.18 26.01±2.85
AmpB 1.36±0.16 1.18±0.13 9.18±0.85 n.d.
1 441.66±13.28 173.26±7.37 475.76±13.52 7.45±0.32
2 475.45±19.08 203.28±9.03 485.05±18.46 10.82±0.78
3 237.58±13.51 120.28±5.34 353.36±15.76 13.86±0.68
4 242.08±5.47 157.20±7.36 371.94±4.69 8.37±0.85
5 71.67±5.83 162.13±12.69 90.50±9.31 10.43±1.03
6 133.07±9.02 155.71±9.26 110.72±6.40 10.64±0.59
7 308.93±20.65 147.15±4.90 345.06±10.71 14.23±0.92
8 90.81±5.55 83.38±6.14 163.87±9.17 12.95±1.56
9 80.28±6.28 36.40±4.15 272.26±18.16 20.81±1.70
10 61.32±8.65 41.08±5.37 197.15±20.66 22.43±1.82
11 62.30±6.98 48.69±6.26 392.66±30.86 26.01±2.21
12 81.94±8.81 51.78±6.13 326.29±15.18 25.19±1.01
13 49.92±7.06 28.97±3.48 272.37±23.15 26.17±1.45
14 52.51±4.87 25.98±2.74 292.43±23.19 28.12±1.55
15 58.05±1.62 17.06±1.57 250.93±6.51 29.1±1.26
16 60.24±3.16 25.51±2.94 393.66±23.00 28.94±1.95
17 165.92±10.57 99.11±5.38 352.21±6.64 12.44±1.84
18 61.26±1.71 78.41±9.78 400.00±5.13 14.56±1.01
19 64.48±2.97 57.94±3.14 275.01±4.26 14.58±1.14
20 82.38±6.19 26.68±1.69 244.26±8.75 27.28±2.69
21 76.86±4.15 26.24±1.34 217.64±3.54 24.58±2.62
22 60.45±4.75 25.31±1.07 496.11±8.20 26.24±1.97
23 33.36±2.05 9.28±0.46 385.39±9.63 36.45±1.78
24 28.86±1.14 7.17±0.50 390.90±4.35 39.02±2.49
25 145.82±8.81 111.77±6.90 429.20±14.30 12.19±0.84
26 64.45±5.42 72.09±5.98 190.70±10.33 23.08±1.34
27 106.31±8.78 65.56±3.54 352.85±10.75 24.39±1.08
28 162.00±4.92 101.20±9.30 476.85±33.73 18.29±1.22
29 103.27±8.03 59.56±13.88 491.14±42.23 25.84±2.62
30 68.32±8.57 53.27±5.65 565.28±21.45 24.23±1.34
31 93.62±5.52 52.92±3.22 343.36±17.38 27.14±2.25
32 67.45±6.94 33.10±2.79 404.26±19.50 27.31±2.28
33 56.16±4.24 19.60±1.36 497.88±26.12 34.28±2.45
34 43.27±1.44 8.54±0.67 356.84±5.39 39.42±1.85
35 20.13±0.91 4.25±0.26 346.20±6.02 38.83±2.11
36 97.35±3.90 72.90±3.18 325.54±5.28 21.87±1.37
37 89.01±5.92 66.32±2.87 306.74±5.41 21.55±1.22
38 52.97±1.57 21.55±1.23 471.12±10.10 28.78±1.28
1 = 50% Inhibitory concentration (IC50); 2 = 50% Effective concentration (EC50);
3 = EC50 against mouse peritoneal macrophages

Activity against Amastigotes
The activity against amastigotes was determined to estimate their immunomodulatory activity. For the first set of molecules (3 to 16), EC50 values were found to be comparable with compounds 1 and 2 (references), except for 9 to 15.
For the second set of molecules with aromatic ring, the better activity (EC50) was obtained for compound 21 (26.24±1.34 ?M) and 22 (25.31±1.07 ?M) having the nitro group, although the nitric oxide generation was found to be comparable with 19 and 20. Nevertheless, for comp. 23 and 24 the EC50 values were reduced to 9.28±0.46 ?M and 7.17±0.50 ?M respectively. This enhanced activity explicitly showed the importance of the spacer length for the betterment of their activity. Now for the aliphatic substituents (17 and 25 to 35), the EC50 values were found to improve with the increase of hydrophobicity (carbon chain length) with two exceptions (25 and 28). The best activity was found for comp.35 (4.25±0.26 ?M) in this set of molecules. The nitric oxide generation bycomp.35 (38.83±2.11 ?M) was found to be comparable with miltefosine (26.01±2.85 ?M).
The third set of molecules 36, 37 and 38 showed moderate activity against amastigotes (Table 1).
All the synthesized molecules except 5, 6,18 and 26 showed better inhibitory activity against amastigote compared to their corresponding IC50 value against promastigote. This clearly suggested that the synthesized molecules preferentially clear the parasitic burden by immunomodulation rather than the direct killing of the L. donovani.
Comparison of antileishmanial activity between three set of molecules showed that the activity is mostly dependent of the hydrophobicity of the molecules except for 23 and 24. For the similar aliphatic substitutions (where R1 = R2), we observed an improvement in their antileishmanial (IC50 and EC50) activity for set I and set II molecules with few exceptions.

Compound 35 reduced the hepatic and splenic parasite burden in L. donovani infected BALB/c mice
The in vivo efficacy of 35 was tested in BALB/c mouse model to elucidate the antileishmanial and the immunomodulatory potential of5 in restricting L. donovani induced pathogenesis. AsL. donovani induced visceral leishmaniasis manifests as the enlargement of spleen and liver due to the accumulation of parasite. Hence, applicants selectively studied the hepatic and splenic parasite burden in infected BALB/c mouse treated with 35. The results indicate that comp.35was able to cause 81% and 92% of hepatic parasite clearance (Fig 2A) and 72% and 87% of splenic parasite clearance (Fig 2B) in the infected BALB/c mouse treated with 10 mg/kg body weight (B.W.) and 25 mg/kg B.W., respectively, after 28 days of treatment following two weeks of post infection. Hence, compound 35 not only increased the parasite killing in vitro, but was also equally effective in vivo experimental model of visceral leishmaniasis.

In addition, compound 35 was able to cause 81% and 92% of hepatic parasite clearance and 72% and 87% of splenic parasite clearance in the infected BALB/c mouse treated with 10 mg/kg body weight (B.W.) and 25 mg/kg B.W., respectively, after 28 days of treatment following two weeks of post infection.

Example 3. Amastigote killing
Percentage amastigote killing at 12.5µM concentration of the compounds 42 to 59 were tested by standard protocols and the results are provided below in Table 2.
Table 2.
Compound % of Amastigote killing at 12.5 µM Compound % of Amastigote killing at 12.5 µM
42 75 51 76
43 39 52 70
44 74 53 55
45 79 54 86
46 84 55 66
47 49 56 62
48 77 57 81
49 55 58 74
50 82 59 61

Example 4.
Efficacy of Compound 35 (4-allyl-2-methoxyphenyl oleate)
Three orally administered doses (3, 10 and 30 mg/kg b.wt) of Compound 35were tested in a pilot study with BALB/c mice model of VL. Miltefosine (20 mg/kg b.wt) served as standard oral drug against VL.
Two weeks (14 days post infection with 2X107 promastigotes) infected mice received oral treatment of compound 35 or miltefosine in treated groups (either with test compound or miltefosine) at indicated doses for 5 consecutive days for 7 days treatment experiment. Other sub-groups of 14 days treatment experiment received same doses and additional 5 doses from treatment day 8 to 12. BALB/c mice of uninfected and infected control groups received vehicle of E. oleate orally in the same volume at the same time of treatment.
After completion of the treatment all the mice were sacrificed and hepatic and splenic parasite burden were determined from tissue imprints after Giemsa staining. Results were expressed as Leishman Donovan Units (LDU) as described earlier. Results were expressed as mean ± S.D. from three mice per sub-group. The results show that compound 35 treated groups significantly cleared the parasites as against infected control and was comparable in leishmanicidal activity with that of the standard drug miltefosine thus establishing the oral efficacy of the novel compounds of the invention.

Example 5.
Pharmacokinetics: Oral and Intravenous PK Studies:
BALB/c mice aged 8-9 weeks of age were administered with compound 35 in a recommended vehicle (5 % DMA + 5% Tween 80 + 90% sterile water for injection) by oral or intravenous route with a dose of 10 mg/kg body weight at dose volume of 10 mL/kg body weight.
Blood specimens were collected at appropriate time and plasma were separated and stored at
-80ºC until analysis. Results of PK studies are provided in Tables 3.
Table 3. Single Dose Oral and Intravenous Pharmacokinetics Study of compound 35 in Male BALB/c Mice
PK Parameters Mean Plasma PK Parameters of Compound 35
Oral Intravenous
Dose(mg/kg b.w.) 10 5
Cmax(ng/mL) 4959.718 3290.644
C0(ng/mL) - 3485.818
Tmax(h) 0.500 0.080
AUClast(h*ng/mL) 17188.618 4839.977
AUCinf(h*ng/mL) 19264.313 7081.068
AUCextrap(%) 10.775 31.649
Vss(L/kg) - 1.226
CL(mL/min/kg) - 11.768
T1/2(h) 1.908 1.195
MRTlast(h) 1.908 0.815
F% (Oral bioavailability) >100
Table 3 provides comparative data on single dose oral and intravenous pharmacokinetics of compound 35. Figure 3 represents the oral and intravenous pharmacokinetic profiles of compound 35.
The pharmacokinetic data suggests that compound 35 is orally available and is suitable for development as an oral antileishmanial agent.
,CLAIMS:Claims
We Claim
1. A compound of formula I

wherein,
X is independently selected from the group consisting of -CH2-, -C=O, -O-CH2-CO-, -CH2CO- and -NH-C=O;
R1 is alkyl saturated or unsaturated, linear or branched,cycloalkyl, alkenyl, alkynyl, aryl, hetero aryl, unsubstituted or optionally substituted with F, Cl, Br, I, CN, OH, NH2, OMe;and wherein the said composition has antiparasitic activity.
2. The compound according to claim 1, wherein X is a linker selected from a group consisting of
wherein n is 0 to 6.
3. The compound according to claim 1, wherein R1 isselected from the group consisting of

wherein n is 0 to 6 and Y is F, Cl, Br, I, CN, OH, NH2, NO2or OMe.
4. A composition of cyclic or acyclic derivative of eugenol wherein the compound is
4-allyl-1-(allyloxy)-2-methoxybenzene; 4-allyl-2-methoxy-1-((3-methylbut-2-en-1-yl)oxy)benzene; 4-allyl-1-ethoxy-2-methoxybenzene; 4-allyl-2-methoxy-1-propoxybenzene; 4-allyl-1-butoxy-2-methoxybenzene; 4-allyl-2-methoxy-1-(pentyloxy)benzene; 4-allyl-1-(hexyloxy)-2-methoxybenzene; 4-allyl-1-(heptyloxy)-2-methoxybenzene; 4-allyl-2-methoxy-1-(octyloxy)benzene; 4-allyl-2-methoxy-1-(nonyloxy)benzene; 4-allyl-1-(decyloxy)-2-methoxybenzene; 4-allyl-1-(dodecyloxy)-2-methoxybenzene; 4-allyl-1-(hexadecyloxy)-2-methoxybenzene; (Z)-4-allyl-2-methoxy-1-(octadec-9-en-1-yloxy)benzene; 4-allyl-2-methoxyphenyl acetate; 4-allyl-2-methoxyphenyl 4-fluorobenzoate; 4-allyl-2-methoxyphenyl 2-chlorobenzoate; 4-allyl-2-methoxyphenyl 4-nitrobenzoate; 4-allyl-2-methoxyphenyl 3-nitrobenzoate; 4-allyl-2-methoxyphenyl 1H-indole-3-carboxylate; 4-allyl-2-methoxyphenyl 2-phenylacetate; 4-allyl-2-methoxyphenyl 2-phenoxyacetate; 4-allyl-2-methoxyphenyl propionate; 4-allyl-2-methoxyphenyl butyrate; 4-allyl-2-methoxyphenyl pentanoate; 4-allyl-2-methoxyphenyl hexanoate; 4-allyl-2-methoxyphenyl heptanoate; 4-allyl-2-methoxyphenyl octanoate; 4-allyl-2-methoxyphenyl nonanoate; 4-allyl-2-methoxyphenyl decanoate; 4-allyl-2-methoxyphenyl dodecanoate; 4-allyl-2-methoxyphenyl dodecanoate; 4-allyl-2-methoxyphenyl oleate; 4-allyl-2-methoxyphenyl prolinate; 4-allyl-2-methoxyphenyl piperidine-4-carboxylate; 4-allyl-2-methoxyphenyl tryptophanate; 4-allyl-2-methoxyphenyl decylcarbamate; 4-allyl-2-methoxyphenyl tetradecylcarbamate; 4-allyl-2-methoxyphenyl (Z)-hexadec-7-en-1-ylcarbamate; 4-allyl-2-methoxyphenyl (Z)-hexadec-9-enoate; 2-methoxy-4-propylphenyl palmitate; 2-methoxy-4-propylphenyl (Z)-hexadec-9-enoate; 4-allyl-2-methoxyphenyl non-8-enoate; 4-allyl-2-methoxyphenyl (Z)-hexadec-9-en-1-ylcarbamate; 4-allyl-2-methoxyphenyl (E)-hexadec-9-enoate; 2-methoxyphenyl (Z)-hexadec-9-enoate; 4-allyl-2-methoxyphenyl 2-(4-cyclohexylphenoxy)acetate; 4-allyl-2-methoxyphenyl 2-(4-ethylphenoxy)acetate; 4-allyl-2-methoxyphenyl 2-(4-nitrophenoxy)acetate; 4-allyl-2-methoxyphenyl (E)-octadec-9-enoate; 2-methoxy-4-propylphenyl oleate; 4-allyl-2-methoxyphenyl 2-(4-methoxyphenoxy)acetate; 4-allyl-2-methoxyphenyl (R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate; 4-allyl-2-methoxyphenyl 2-(4-morpholinophenoxy)acetate; 4-allyl-2-methoxyphenyl 4-(octyloxy)benzoate; 4-allyl-2-methoxyphenyl 4-(decyloxy)benzoate or 4-allyl-2-methoxyphenyl 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)acetate.

5. A pharmaceutical composition comprising a cyclic or acyclic derivative of eugenol as claimed in claims 1 or 4 wherein the said composition is formulated with at least one pharmaceutical excipient.

6. The compounds as in claims 1 or 4 for use in the treatment of patients suffering from infections caused by Leishmania infantum, Trypanosoma cruzi or Trypanosoma brucei.