Title of Invention

A NOVEL COMBINATORIAL LIBRARY OF 3 AND 30-SUBSTITUTED LUP-20(29)- ENE DERIVATIVES USEFUL AS ANTI-MALARIAL AGENTS

Abstract The present invention relates to a novel combinatorial library of 3 and 30-substituted lup of -20(29)-ene derivatives useful as anti-malaria agents . Therefore, in order to invent new class of anti-malarial agents, the triterpenoid 3-D-hydroxy lup-20 (29)-ene has been chemically modified utilizing solid-phase organic synthesis in combinatorial chemistry approach. The present invention describes the synthesis of highly active novel series of 3 and 30-substituted lup-20(29)-ene and evaluation for their activity against resistant malarial parasite Plasmodium falciparum strain NF54. We have synthesized compound3-succinamate 30-(N-Cyclohexylamino)-lup-20(29)-ene, 3-glutatrimate 30-(N-benzylamino)-lup-20(29)-ene, 3-adipamate 30-(N,N-Dicyclohexylamino)-lup-20(29)-ene, 3-suberamate 30[N-(Methyl)N'-piperazinyl]-lup-20(29)-ene,3-succinamate 30-benzyloxy-lup-20(29)-ene,3-glutarimate 30[4-(bromo)benzyloxy]-lup-20(29)-ene and 3-adipamate 30-amino-lup-20(29)-ene as a lead structure for a novel class of anti-malarial agents. Compound 4 a key intermediate represents an important step in the development of these lead structures into an anti-malarial drug candidate.
Full Text The present invention relates to a novel combinatorial library of 3 and 30-substituted lup-20(29)-ene useful as anti-malarial agents.
Main utility of present invention relates to novel class of 3 and 30-substituted lup-20(29)-ene derivatives as anti-malarial agents active against malarial parasite in vitro maintained cell culture of Plasmodiumfalciparum strain NF54.
Reference may be made to the patent and primary scientific literature pertaining to research on anti-malarial agents that encompasses most widely used aryl amino alcohols, 4 and 8-aminoquinolines, peroxides, naphthoquinones and various miscellaneous compounds (D.M. Panisko and J.S. Keystone, Drugs, 1990, 39, 160-189). The drawbacks of these arylamino alcohols, 4, 8- amino alcohols, peroxides anti-malarial drugs are associated with adverse effect that vary from mild to strong. Reference may be made to several other classes of compounds were reported with weak anti-malarial activity, with different structural features may be useful as anti-malarial agents if these chemically modified to display stronger anti-malarial activity and less adverse effects. Reference may be made to the lupane class of triterpenoid 3β-hydroxy lup-20(29)-ene exhibited 45% inhibition at 25µg/mL against chloroquin resistant P. falcipaRUm, reference may be made to (Phytother. Res. 13, 115-119,1999).
Todate, no triterpenoid class of compound 3β-hydroxy Iup20(29)-ene have been suggested as anti-malarial drugs. Therefore, in order to invent new class of anti-malarial agents, the triterpenoid 3-p-hydroxy lup-20 (29)-ene has been chemically modified utilizing solid-phase organic synthesis in combinatorial chemistry approach and The present invention describes the synthesis of highly active novel series of 3 and 30-substituted lup-20(29)-ene and evaluation for their activity against resistant malarial
parasite Plasmodium falciparum strain NF54. We have synthesized compound 6a, 6b, 8a, 8b, l0a, 105 and 12a as a lead structure for a novel class of anti-malarial agents. Compound 4 represents an important step in the development of these lead structures into an anti-malarial drug candidate.
The main objective of the present invention is to provide novel combinatorial library of 3 and 30-substituted lup-20(29)-ene derivatives useful as antimalarial agents.
Another objective of the present invention is to provide a process for the preparation of novel derivatives of 3 and 30-substituted lup-20(29)-ene.
Yet another objective of the present invention is to provide compounds useful in
the treatment of malaria.In the drawings accompanying this specification figure 1 represents loading of
symmetrical anhydrides of the general formula 1, to Rink amide resin to get formula 2, condensation of 3 p-hydroxy lup-20(29)-ene to get formula 3. Figure 2 represents introduction of bromo group at C-30 position of 3β-hydroxy lup-20(29)-ene to get formual4. Figure 3 represents substitution of bromo group by primary amines (formula 5), cleavage of products from solid-phase to get formula 6a and 6b. Figure 4 represents substitution of bromo group by secondary amines (formula 7), cleavage of product from solid-phase to get formula 8a and 8b. Figure 5 represents substitution of bormo group by of primary alcohols (formula 9), cleavage of products to get formula lOa and l0b. Figure 6 represents reduction of formula 11 and cleavage of product from solid-phase to get formula 12 Accordingly, the present invention provides a novel combinatorial library of 3 and 30-substituted lup of -20(29)-ene derivatives useful as anti-malaria agents are selected from 3-succinamate 30-(N-Cyclohexylamino)-lup-20(29)-ene, 3-glutatrimate 30-(N-benzylamino)-lup-20(29)-ene, 3-adipamate 30-(N,N-Dicyclohexylamino)-lup-20(29)-ene, 3-suberamate 30[N-(Methyl)N'-piperazinyl]-lup-20(29)-ene,3-succinamate 30-benzyloxy-lup-20(29)-ene,3-glutarimate 30[4-(bromo)benzyloxy]-lup-20(29)-ene and 3-adipamate 30-amino-lup-20(29)-ene.
In an embodiment of the present invention the reaction vessel used is selected from the group consisting of syringe, automated robotic synthesizer, Merrifield's vessel, and three way sintered vessel.
In yet another embodiment the symmetrical anhydride used is selected from the group consisting of succinic anhydride, glutaric anhydride, suberic anhydride and adipic anhydride.
In yet another embodiment the coupling agent used for loading lupeol to solid phases is selected from the group consisting of TBTu, DCC, HOBt, DMAP and DIPEA.
In yet another embodiment the primary amine used is selected from the group consisting of benzyl amine, cyclohexyl amine, furfural amine, and N,N'diethyl ethyl amine. In yet another embodiment the secondary amine used is selected from the group consisting of N-methyl piperazine, morpholine, N-methyl amine, dicyclohexyl amine, and 4-benzylpiperidine.
In still another embodiment the aromatic and aliphatic alcohol used is selected may be selected from the group consisting of benzyl alcohol, ethyl alcohol, 2-methoxybenzyl alcohol, and Fmoc-3-amino propanol.
In still another embodiment the organic solvent used for coupling reaction is selected from the group consisting of DMF, THF, CHC13, and DCM.
thiophenol in presence of triethylamine as base for 12 hrs at room temperature to get 3-0
(resin-alkanoyl) 30-amino lup-20(29)-ene of formula 12.

Finally cleavage of the reaction products from the solid phase using TFA and/or DCM-TFA at the temperature in the range of 0-40°C for 0.5 to1 hr to give the combinatorial library of lupeol of the general formula 6a, 6b, 8a,8b, 10a,10b and 12a.
The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
Example-1

The library of synthetic compounds was tested against malarial parasite in vitro maintained cell culture of Plasmodium falciparum strain NF54. The in vitro microassay technique has been used for evaluation of the anti-malarial activity of the test compounds (WHO, 1990, Tech. Rep. Ser. No. 711). The assay was carried out against in vitro maintained cell culture of P. falciparum strain NF54 using medium RPM1-1640 as the growth medium. The assay was performed in 96 well microtitre plate. At the start of assay, the synchronized ring stage parasites were incubated in presence of different concentrations of test agent ranging between 2µg/ml-50 µg/ml for 4-44 hrs. The initial Parasitaemia in the test wells ranged between 1-2% at 3% hematocrit. The test is based on the principle that in the absence of any inhibitory agent, ring stage parasites mature into the schizont stage in 40-44hrs. Thin blood smears were prepared from each well of
the microtitre plate stained with Giemsa and examined microscopically. The test concentration showing complete inhibition of maturation to schizont was recorded as the minimum inhibitory concentration (MIC). The MIC of chloroquine used as reference drug in this assay is 40ng/ml.

(Table Removed)
The representative compounds of the formula 6a, 6b, 8a, 8b, 10a, lOb and 12a prepared by the process of the present invention are as follows:
The following example broadly illustrates the nature of this invention, the manner in which it is to be performed without limiting the nature and scope of the invention.
Example-2
3-succinamate 30-(N-Cyclohexylamino)-lup-20(29)-ene (Formula 6a)
The 50 mg Rink amide resin ( 0.025mM) swelled with solvents such as DCM,
DMF. Next, deprotection of Fmoc-NH to corresponding NH2 using 20% piperidine in
DMF was carried out twice for 5 min and 25 min. After successive washings with DMF, to the resin was added 300µl of DMF, 300µl of pyridine, and succinic anhydride (25mg, 0.25mM). The reaction mixture was allowed to shake at r.t for 6 hrs and then resin filtered. The resin was successively washed with 5 ml each of 1% AcOH (3 x 2min), DMF (3x2min) MeOH (3x2min) and again with DMF (3x2min) to get resin linked acids of general formula 2.
General formula 2 was coupled with lupeol (64mg, O.5mM) in THF (200}µl), DMF (200½1) in the presence of DMAP (6mg, O.015mM) in DMF (200µ1) and DIC (0.15mM, 25µl) at rt for 24 hrs. Finally the resin was successively washed 5mL each with
DMF (3 x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et2O (3x2min).
Then dried under vaccum to provide resin loaded with lupeol of general formula 3.
General formula 3 (0.025mM) was swelled with DCM: CC14 (300µ1, 1:1) and then treated with NBS (18mg, 0.lmM) in DCM: CC14 (300µl, 1:1) and reaction was allowed to be shaken on orbital shaker for 4 hrs at rt. Finally the resin was successively washed with DMF (3x2min), THF (3x2min), DCM (3x2min) and Et2O (3x2min). Then dried under vaccum to get 3-0 (resin-alkanoyl) 30-bromo lup-20(29)-ene of general formula 4.
Compound 4 (0.025 mM) was treated cyclohexyl amine (29µl, 0.25mM) in presence of DBU (19 µl, 0.125mM) using DMSO as solvent at rt for 24 hrs. The resin was wahsed successively 5mL each with DMF (3x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et2O (3x2min) and then dried under vaccum to get 3-0 (resin-alkanoyl) 30-cyclohexyl amine lup-20(29)-ene of general formula 6.
Finally general formula 6 was subjected to acidolytic cleavage using TFA:DCM (1.5:8.5 ) for (2 xl0 min) and cleavage was evaporated to dryness under vaccum. The residue so obtained was freeze dried by dissolving in tBuOH:water (4:1) to get 3-succinamate 30-(N-Cyclohexylamino)-lup-20(29)-ene, of formula 6a, MS(FAB): 628 (M+H)+. This was characterized using HPLC, MS(FAB) and NMR spectral data. The derivatives were obtained in 85-90% purity that was sufficient for in vitro evaluation.
Example-3 3-glutatrimate 30-(N-benzylamino)-lup-20(29)-ene (Formula 6b)
The 50mg Rink amide resin ( 0.025mM) swelled with solvents such as DCM, DMF. Next, deprotection of Fmoc-NH to corresponding NHi using 20% piperidine in DMF was carried out for 5 min and 25 min twice. After successive washings with DMF, to the resin was added DMF, pyridine300 µl of each, and glutaric anhydride (29mg, 0.25mM). The reaction mixture was allowed to shake at it for 6 hrs and then resin filtered. The resin was successively washed with 5 ml each of 1% AcOH (3x2 min), DMF(3x2min) MeOH (3x2 min) and again with DMF (3x2min) to get general formula 2.
General formula 2 was coupled with lupeol (64mg, 0.5mM) in THF (200(µl), DMF (200µl) in the presence of DMAP (6mg, 0.015mM) in DMF (200µl) and DIC (0.15mM, 25µl) at it for 24 hrs. Finally the resin was successively washed with DMF (3 x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et2O (3x2min). Then dried under vaccum to provide resin loaded with lupeol of general formula 3.
General formula 3 (0.025mM) was swelled with DCM: CC14 (300µl, 1:1) and then treated with NBS (18mg, O.lmM) in DCM: CC14 (300µl, 1:1) and reaction was allowed to be shaken on orbital shaker for 4 hrs at rt. Finally the resin was successively washed
with DMF (3x2min), THF (3x2min), DCM (3x2min) and Et2O (3x2min). Then dried under vaccum to get 3-0 (resin-alkanoyl) 30-bromo lup-20(29)-ene of general formula 4.
General formula 4- (0.025mM) was treated benzyl amine (27µl, 0.25mM) in presence of DBU (19 µ1, 0.125mM) using DMSO as solvent at it for 24 hrs. The resin was washed successively 5mL each with DMF (3 x 2min), THF (3 x 2min), MeOH (3x2min), DCM (3x2min) and Et20 (3x2min) and then dried under vaccum to get 3-O (resin-alkanoyl) 30-benzyl amine Lup-20(29)-ene of general formula 6.
Finally general formula 6 was subjected to acidolytic cleavage using TFA:DCM (1.5:8.5) for (2xlOmin) and cleavage was evaporated to dryness under vaccum. The residue so obtained was freeze dried by dissolving in tBuOH-water (4:1) to get 3-glutatrimate 30-(N-Benzylamino)-Lup-20(29)-ene of formula 6b, MS(FAB): 645 (M+H)+. This was characterized using HPLC, MS(FAB)and NMR spectral data. The derivatives were obtained in 85-90% purity that was sufficient for in vitro evaluation.
Example-4 3-adipamate 30-(N,N-DicyclohexyIamino)-lup-20(29)-ene (Formula 8a)
The 50 mg rink amide resin (0.025mM) swelled with solvents such as DCM, DMF. Next, deprotection of F moc-NH to corresponding NH2 using 20% piperidine in DMF was carried out for 5 min and 25 min twice. After successive washings with DMF, to the resin was added DMF, pyridine 300 µl of each and adipic anhydride (35mg, 0.25mM). The reaction mixture was allowed to shake at rt for 6 hrs and then resin filtered. The resin was successively washed with 5 ml each of 1% AcOH (3x2 min), DMF(3x2min) MeOH (3x2 min) and again with DMF (3 x 2min) to get general formula 2.
General formula 2 was coupled with lupeol (64mg, O.5mM) in THF (200µ1), DMF (200(al) in the presence of DMAP(6mg, O.015mM) in DMF (200µl) and DIC (0.15 mM, 25µ1) at rt for 24 hrs. Finally the resin was successively washed with DMF (3 x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et20 (3x2min). Then dried under vaccum to provide resin loaded with lupeol of general formula 3.
General formula 3 (0.025mM) was swelled with DCM: CC14 (300µl, 1:1) and then treated with NBS (18mg, 0.lmM) in DCM: CC14 (300µ1, 1:1) and reaction was allowed to be shaken on orbital shaker for 4 hrs at rt. Finally the resin was successively washed with DMF(3x2min), THF(3x2min), DCM(3x2min) and Et20(3x2min). Then dried under vaccum to get 3-O (resin-alkanoyl) 30-bromo lup-20(29)-ene of general formula 4.
General formula 4 (0.025 mM). was treated N,N'-dicyclohexylamine (50µl, 0.25mM) in presence of DBU (I9µl, 0.125mM) using DMSO as solvent at rt for 24 hrs. The resin was washed successively 5mL each with DMF (3x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et20 (3x2min) and then dried under vaccum to get 3-0 (resin-alkanoyl) 30- N,N'-dicyclohexylamino-Lup-20(29)-ene of general formula 8
Finally general formula 8 was subjected to acidolytic cleavage using TFA:DCM (1.5:8.5) for (2 x 10 min) and cleavage was evaporated to dryness under vaccum. The residue so obtained was freeze dried by dissolving in tBuOH-water (4:1) to get 3-adipamate 30-(N,N'-dicyclohexylamino)-Lup-20(29)-ene of formula 8a, MS(FAB): 733 (M+Hy1".. This was characterized using HPLC, MS(FAB)and NMR spectral data. The derivatives were obtained in 85-90% purity that was sufficient for in vitro evaluation.
Example-5 3-suberamate 30[N-(Methyl)N'-piperazinyl]-lup-20(29)-ene (Formula 8b)
The 50mg Rink amide resin (0.025mM) swelled with solvents such as DCM, DMF. Next, deprotection of Fmoc-NH to corresponding NH2 using 20% piperidine in DMF was carried out for 5 min and 25 min twice. After successive washings with DMF, to the resin was added DMF, pyridine 300µl of each, and suberic anhydride (39mg, 0.25mM). The reaction mixture was allowed to shake at rt for 6 hrs and then resin filtered. The resin was successively washed with 5 ml each of 1% AcOH (3x2 min), DMF(3x2min) MeOH (3x2 min) and again with DMF (3x2min) to get general formula 2.
General formula 2 was coupled with lupeol (64mg, O.5mM) in THF (200µl),
DMF (200µl) in the presence of DMAP(6mg, O.O15mM) in DMF (200µl) and DIC
1
(O.l5mM, 25µl) at rt for 24 hrs. Finally the resin was successively washed with DMF (3 x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et2O (3x2min). Then dried under vaccum to provide resin loaded with lupeol of general formula 3.
General formula 3 (0.025mM) was swelled with DCM: CC14 (300jal, 1:1) and then treated with NBS (18mg, 0.1 mM) in DCM: CC14 (300(4 1:1) and reaction was allowed to be shaken on orbital shaker for 4 hrs at rt. Finally the resin was successively washed with DMF(3x2min), THF(3x2min), DCM(3x2min) and Et20(3 x 2min). Then dried under vaccum to get 3-0 (resin-alkanoyl) 30-bromo lup-20(29)-ene of general formula 4.
Compound 4 (0.025mM) was treated N-Methyl N-piperazinyl amine (28µl, 0.25mM) in presence of DBU (19µ1, 0.125mM) using DMSO as solvent at rt for 24 hrs. The resin was washed successively with 5mL each DMF (3x2min), THF (3x2min),
MeOH (3 x 2min), DCM (3x2min) and Et2O (3x2min) and then dried under vaccum to get 3-0 (resin-alkanoyl) 30- N-Methyl N'-piperazinyl amine lupeol of general formula 8
Finally general formula 8 was subjected to acidolytic cleavage using TFA:DCM (1.5:8.5) for (2 x 10 min) and cleavage was evaporated to dryness under vaccum. The residue so obtained was freeze dried by dissolving in tBuOH-water (4:1) to get 3-suberamate 30-(N-Methyl N'-piperazinyl amine)lup-20(29)-ene of formula 8b, MS(FAB): 680 (M+H)+. This was characterized using HPLC, MS(FAB) and NMR spectral data. The derivatives were obtained in 85-90% purity that was sufficient for in vitro evaluation.
Example-6
3-succinamate 30-benzyloxy-lup-20(29)-ene (Formula 10a)
The 50mg Rink amide resin (0.025mM) swelled with solvents such as DCM, DMF. Next, deprotection of Fmoc-NH to corresponding NH2 using 20% piperidine in DMF was carried out for 5 min and 25 min twice. After successive washings with DMF, to the resin was added DMF, pyridine 300 µl of each and succinic anhydride (25 mg, 0.25 mM). The reaction mixture was allowed to shake at rt for 6 hrs and then resin filtered. The resin was successively washed with 5 ml each of 1% AcOH (3x2 min), DMF (3 x 2min) MeOH (3x2 min) and again with DMF (3 x 2min) to get general formula 2.
General formula 2 was coupled with lupeol (64mg, 0.5mM) in THF (200ul), DMF (200ul) in the presence of DMAP (6mg, O.OlSmM) in DMF (200^1) and DIG (0.15mM, 25(4.1) at rt for 24 hrs. Finally the resin was successively washed 5mL each with
piperidine in DMF was carried out for 5 min and 25 min twice. After successive washings with DMF, to the resin was added DMF, pyridine 300 µl of each and glutaric anhydride (29mg, 0.25mM). The reaction mixture was allowed to shake at rt for 6 hrs and then resin filtered. The resin was successively washed with 5 ml each of 1% AcOH (3 x2 min), DMF (3 x 2min) MeOH (3 x2 min) and again with DMF (3 x 2min) to get general formula 2.
General formula 2 was coupled with lupeol (64mg, O.5mM) in THF (200µ1), DMF (200µl) in the presence of DMAP(6mg, O.015mM) in DMF (200µl) and DIC (0.15mM, 25µl) at rt for 24 hrs. Finally the resin was successively washed with DMF (3 x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et2O (3x2min). Then dried under vaccum to provide resin loaded with lupeol of general formula 3.
General formula 3 (0.025mM) was swelled with DCM: CC14 (300µ1, 1:1) and then treated with NBS (18mg, 0.1 mM) in DCM: CC14 (300µl, 1:1) and reaction was allowed to be shaken on orbital shaker for 4 hrs at rt. Finally the resin was successively washed with 5 mL each of DMF(3x2min), THF(3x2min), DCM(3x2min) and Et2O(3x2min). Then dried under vaccum to get 3-0 (resin-alkanoyl) 30-bromo lup-20(29)-ene of general formula 4.
General formula 4 (0.025 mM) was treated 4-bromobenzyl alcohol (46mg, 0.25mM) in presence of DBU (19 µl, 0.125mM) using DMSO as solvent at it for 24 hrs. The resin was washed successively with 5mL each of DMF (3 x 2min), THF (3 x 2min), MeOH (3 x 2min), DCM (3x2min) and Et2O (3x2min) and then dried under vaccum to get 3-O (resin-alkanoyl) 30-(4-bromobenzyloxy) lupeol of general formula 10
DMF (3 x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et2O (3x2min). Then dried under vaccum to provide resin loaded with lupeol of general formula 3.
General formula 3 (0.025mM) was swelled with DCM: CCl4, (300µ1, 1:1) and then treated with NBS (18mg, O.lmM) in DCM: CC14 (300µl, 1:1) and reaction was allowed to be shaken on orbital shaker for 4 hrs at it. Finally the resin was successively washed with 5 mL each DMF(3x2min), THF(3x2min), DCM(3x2min) and Et2O(3x2min). Then dried under vaccum to get 3-O (resin-alkanoyl) 30-bromo lup-20(29)-ene of general formula 4.
General formula 4 (0.025mM) was treated with benzyl alcohol (26µ1, 0.25mM) in presence of DBU (19µl, 0.125mM) using DMSO as solvent at rt for 24hrs. The resin was washed successively 5mL each with DMF (3x2min), THF (3x2min), MeOH (3x2min), DCM (3x2min) and Et2O (3x2min) and then dried under vaccum to get 3-O (resin-alkanoyl) 30-benzyloxy lupeol of general formula 10
Finally general formula 10 was subjected to acidolytic cleavage using TFA:DCM (1.5:8.5) for (2 x 10 min) and cleavage was evaporated to dryness under vaccum. The residue so obtained was freeze dried by dissolving in tBuOH-water (4:1) to get 3-succinamate- 30-benzyloxy-lup-20(29)-ene of formula10a, MS (FAB): 632 (M+H)+. This was characterized using HPLC, MS(FAB) and NMR spectral data. The derivatives were obtained in 85-90% purity, which was sufficient for in vitro evaluation.
Example-7 3-glutarimate 30[4-(bromo)benzyloxy]-lup-20(29)-ene (Formula lOb)
The 50mg Rink amide resin (0.025mM) swelled with solvents such as DCM, DMF. Next, deprotection of Fmoc-NH to corresponding NH2 using 20%
Finally general formula 10 was subjected to acidolytic cleavage using TFA:DCM (1.5:8.5) for (2x10 min) and cleavage was evaporated to dryness under vaccum. The residue so obtained was freeze dried by dissolving in tBuOH-water (4:1) to get 3-glutarimate 30[4-(bromo)benzyloxy]-lup-20(29)-ene of formula lOb, MS(FAB): 725 (M+H)+. This was characterized using HPLC, MS(FAB) and NMR spectral data. The derivatives were obtained in 85-90% purity, which was sufficient for in vitro evaluation.
Example-8 3-adipamate 30-amino-lup-20(29)-ene (Formula 12a)
The 50 mg rink amide resin (0.025mM) swelled with DCM. Next, deprotection of Fmoc-NH to corresponding NH2 using 20% piperidine in DMF was carried out for 5 min
and 25 min twice. After successive washings with DMF, to the resin was added DMF,
pyridine 300 µl of each and adipic anhydride (35mg, 0.25mM). The reaction mixture was
allowed to shake at rt for 6 hrs and then resin filtered. The resin was successively washed with 5 ml each of 1% AcOH (3x2 min), DMF (3x2min) MeOH (3x2 min) and again with DMF (3x2min) to get general formula 2.
General formula 2 was coupled with lupeol (64mg, O.5mM) in THF (200(µ1), DMF (200µ1) in the presence of DMAP (6mg, 0.015mM) in DMF (200µl) and DIC (0.15mM, 25µl) at rt for 24 hrs. Finally the resin was successively washed with DMF(3 x2min), THF(3x2min), MeOH(3x2min), DCM (3x2min) and Et2O (3x2min). Then dried under vaccum to provide resin loaded with lupeol of general formula 3.
General formula 3 (0.025mM) was swelled with DCM: CC14 (300µl, 1:1) and then treated with NBS (18mg, 0.1 mM) in DCM: CC14 (300µl, 1:1) and reaction was allowed to be shaken on orbital shaker for 4 hrs at rt. Finally the resin was successively washed
with DMF(3x2min), THF(3x2min), DCM(3x2min) and Et2O(3x2min). Then dried under vaccum to get 3-O (resin-alkanoyl) 30-bromo lup-20(29)-ene of general formula 4.
Formula 4 (0.025 mM) was treated with, sodium azide (16mg, 0.25mM) and heated at 80°C using DMSO as solvent for 24hrs. The resin was successively washed 5 mL each with H2O (3x2min), DMF (3x2min), MeOH (3x2min), DCM (3x2min) and Et20 (3x 2min), and finally dried under vaccum to get 3-O (resin-alkanoyl) 30-azido lupeol of general formula 11. It was then subjected to reduction of azide to amine by treating with TEA (90µl), PhSH (51µl) and SnCl2 (24 mg) at rt for 2hr. The resin was successively washed with DMF (3x2min), Et2O (3x2min), DCM (3x2min), Et2O (3x2min) to get 3-O (resin-alkanoyl) 30-amino lupeol of formula 12. Finally general formula 12 was subjected to acidolytic cleavage using TFA:DCM (1.5:8.5) for (2x10 min) and cleavage was evaporated to dryness under vaccum. The residue so obtained was freeze dried by dissolving in tBuOH-water (4:1) to get 3-adipamate- 30-amino-lup-20(29)-ene of formula 12a, MS(FAB): 541 (M+H)+. This was characterized using HPLC, MS(FAB)and NMR spectral data. The derivatives were obtained in 85-90% purity, which was sufficient for in vitro evaluation.
In conclusion, this new class of anti-malarial compounds may be potential precursor for effective anti-malarial drug with new mode of action. 3□-hydroxy Iup20(29)-ene derivatives represents the first triterpenoid compounds to be suggested as anti-malarial agents. Todate no 3β-hydroxy lup20(29)-ene have been suggested as anti-malarial drugs. Further studies are underway in order to determine the precise and

potential derivative to become new anti-malarial drug. Current synthetic efforts are focused toward the synthesis of selected 3β-hydroxy lup20(29)-ene derivatives with improved anti-malarial activity.
The main advantages of the invention are:
1. First, and novel synthetic approach toward which involves solid-phase organic
synthesis and combinatorial chemistry.
2. 3β-hydroxy lup20(29)-ene derivatives as a new class of anti-malarial agents.
3. Key intermediate formula 4 has potential to generate several 3p-hydroxy
lup20(29)-ene derivatives as a anti-malarial agents.
4. The simple methods utilized to generate complex compounds.

















We claim
1. A novel combinatorial library of 3 and 30-substituted lup of -20(29)-ene derivatives
useful as anti-malaria agents are selected from 3-succinamate 30-(N-
Cyclohexylamino)-lup-20(29)-ene, 3-glutatrimate 30-(N-benzylamino)-lup-20(29)-
ene, 3-adipamate 30-(N,N-Dicyclohexylamino)-lup-20(29)-ene, 3-suberamate 30[N-
(Methyl)N'-piperazinyl]-lup-20(29)-ene,3-succinamate 30-benzyloxy-lup-20(29)-
ene,3-glutarimate 30[4-(bromo)benzyloxy]-lup-20(29)-ene and 3-adipamate 30-amino-lup-20(29)-ene.
2. A novel combinatorial library of 3 and 30-substituted lup of -20(29)-ene derivatives useful as anti-malaria agents substantially as herein described with reference to the examples and drawing accompanying the specification.

Documents:

153-DEL-2004-Abstract-(15-06-2010).pdf

153-DEL-2004-Abstract-(16-06-2010).pdf

153-del-2004-abstract.pdf

153-DEL-2004-Claims-(15-06-2010).pdf

153-DEL-2004-Claims-(16-06-2010).pdf

153-del-2004-claims.pdf

153-DEL-2004-Correspondence-Others-(15-06-2010).pdf

153-DEL-2004-Correspondence-Others-(16-06-2010).pdf

153-del-2004-correspondence-others.pdf

153-DEL-2004-Description (Complete)-(15-06-2010).pdf

153-DEL-2004-Description (Complete)-(16-06-2010).pdf

153-del-2004-description (complete).pdf

153-DEL-2004-Drawings-(15-06-2010).pdf

153-DEL-2004-Drawings-(16-06-2010).pdf

153-del-2004-drawings.pdf

153-del-2004-form-1.pdf

153-del-2004-form-18.pdf

153-DEL-2004-Form-2-(15-06-2010).pdf

153-DEL-2004-Form-2-(16-06-2010).pdf

153-del-2004-form-2.pdf

153-DEL-2004-Form-3-(15-06-2010).pdf

153-DEL-2004-Form-3-(16-06-2010).pdf

153-DEL-2004-Form-3.pdf

153-del-2004-form-5.pdf


Patent Number 243559
Indian Patent Application Number 153/DEL/2004
PG Journal Number 44/2010
Publication Date 29-Oct-2010
Grant Date 26-Oct-2010
Date of Filing 30-Jan-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 MISBAH ALAM FAROOQ BIABANI, THANGATHIRUPATHI SRINIVASAN, SUNIL KUMAR PURI, KANWAL RAJ, BIJOY KUNDU CENTRAL DRUG RESEARCH INSTITUTE, CHATTAR MANZIL PALACE, LUCKNOW-226 001, (U.P.), INDIA.
PCT International Classification Number A61K 31/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA