WO2016059647A2 - Triaminotriazine picolinonitrile derivatives as potent reverse transcriptase inhibitor of hiv-1 - Google Patents

Triaminotriazine picolinonitrile derivatives as potent reverse transcriptase inhibitor of hiv-1 Download PDF

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WO2016059647A2
WO2016059647A2 PCT/IN2015/000386 IN2015000386W WO2016059647A2 WO 2016059647 A2 WO2016059647 A2 WO 2016059647A2 IN 2015000386 W IN2015000386 W IN 2015000386W WO 2016059647 A2 WO2016059647 A2 WO 2016059647A2
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triazin
picolinonitrile
ylamino
formula
phenylamino
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PCT/IN2015/000386
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WO2016059647A3 (en
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Dhananjay Vithalrao MANE
Sandip Namdev GAVADE
Smita Shrikant KULKARNI
Ramesh Shivram PARANJAPE
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SHRI CHHATRAPTI SHIVAJI COLLEGE Maharashtra, India)
National Aids Research Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links

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  • the present invention relates to triamino triazine picolinonitriles of formula- I useful for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of human immunodeficiency virus (HIV) infections.
  • the invention further relates to process for preparation of compounds of formula-I and pharmaceutical composition thereof.
  • HIV-1 human immunodeficiency virus type 1
  • Retroviridae family The human immunodeficiency virus type 1 (HIV-1) is a retrovirus belonging to the Retroviridae family that was first identified in 1983 and was shown to be the cause of acquired immune deficiency syndrome (AIDS).
  • AIDS acquired immune deficiency syndrome
  • HAART Highly active antiretroviral therapy incorporating at least three antiretroviral agents has been the standard therapy for HIV/AIDS for more than 10 years.
  • HAART includes protease inhibitors (Pis), nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), entry inhibitors and integrase inhibitors.
  • NNRTIs for the treatment of HIV/AIDS include (a)Nevirapine, (b)Delavirdinemesilate, (c)Efavirenz, (d)Etravirine, (e)Rilpivirine, (f)Amdoxovir, (g)BILR-355 (BILR-355-BS), (h)UK-453061 , (i) UC-781 and (j)Thiofoscarnet (ANX-
  • NNRTIs are however associated with the rapid emergence of resistance mutations that surround the NNRTI binding pocket, thus rendering the molecules ineffective. Also, extensive cross-resistance is well documented among the available NNRTIs. Combination therapy has also led to the development of adherence problems, reduced antiretroviral activity and drug toxicity.
  • diaryltriazine analogues DATAs
  • diarylpyrimidine analogues DAPYs
  • DATAs diaryltriazine analogues
  • DAPYs diarylpyrimidine analogues
  • WO 99/50250 and WO 00/27825 disclose substituted amino pyrimidine derivatives hich are used in the systemic treatment and prophylaxis of HIV infection. They function by blocking the multiplication process of HIV particularly they block the reverse transcriptase enzyme that plays a vital role in the viral multiplication process.
  • WO99/50256 discloses 1,3,5 trisubstituted triazine derivatives having HIV replication inhibiting properties. wherein A may be selected from N; R 3 is H; , R 4 is -CN, L is -X-R 5 , R 5 is (un) - substituted phenyl; Ri and R 2 represent amino, or R 1 and R 2 taken together may form pyrrolidinyl, morpholidinyl.
  • WO'256 however specifically relates to triazine benzonitrile compounds.
  • WO01/85700 also relates to substituted amino triazine derivatives having HIV replication inhibiting properties of the formula iven below.
  • R 2 is cyano,
  • Q is selected from halo, amino Ci-Ce alkyl, Ci-C 6 alkyloxy, hetero which is pyrazolidinyl, L is -X-R 3 , R 3 is (un) substituted phenyl.
  • the compounds disclosed in WO'256 and WO'700 inhibit replication of HIV virus in the human T-4 cells via an interaction with HIV reverse transcriptase enzyme.
  • EP0834507 discloses substituted diamino triazine derivatives having HIV replication inhibiting properties.
  • the present invention focusses in designing triazine derivatives by introducing a pyridine moiety in the side chain. Introducing a pyridine ring increases the polarity and subsequently the bioavailability of the compounds thus enhancing the treatment of HIV with convenience.
  • the invention is based on the unexpected finding that the compounds described herein below are antagonists of the chemokine CXCR4 receptor which are useful to mediate the medical condition that is modulated by CXCR4 receptor signalling, and in particular for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
  • the other object is to provide novel triamino triazine compounds with a polar pyridine moiety in the side chain with reduced toxicity and increased bioavailability.
  • Yet another object of the invention is to provide a process for synthesis of novel triamino triazine compounds with a polar pyridine moiety in the side chain under mild conditions.
  • the present invention provides triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable addition salts or enantiomers or mixtures of enantiomers or stereoisomers or racemates or solvates or hydrates useful for treating or reducing the severity of hyper proliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections with reduced toxicity, comprising ;
  • Y and Y' represent NH, ' ⁇ ' independently is selected from hydrogen, halo, NH 2 , NMe 2 , OMe, NH-Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR';
  • Ri, R 2 and R 3 independently are selected from hydrogen, , alkyl, cycloalkyl, aryl, heteroaryl, acyl, halo, OH, OR', NH 2 , NHR', SR', COOH, COOR', wherein R' independently is selected from straight or branched alkyl, cyclic alkyl or aralkyl, aryl or heteroaryl groups.
  • the present invention provides a process for preparation of compounds of formula-I comprising catalytic coupling of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin- 2-ylamino) picolinonitrile (formula -5)with a suitable reagent in presence of a base, ligand as promoter and solvent at a temperature in the range of 50-80°C.
  • the suitable reagent is optimised by selecting the appropriate catalyst and promoter ligand to improve the yield and selectivity.
  • the suitable reagent is selected from aq. ammonia, or an amino derivative of general formula NHR'"; wherein R'" is selected from (un) substituted or substituted alkyl, cycloalkyl, aryl, heteroaryl; preferably the reagent is aq. ammonia.
  • the catalyst is selected from copper catalyst such as Cu(I) chloride, Cu(I) iodide, CuS0 4 .5H 2 0, [CuBr(PPh 3 ) 2 ] and the like.
  • the ligand as promoter is selected from 1,10- phenanthroline, picolinic acid, 4-hydroxy-proline and ⁇ , ⁇ -dimethylglycine.
  • the solvent is selected from polar aprotic solvent. wherein 'Z' independently is selected from hydrogen, halogen, NH 2 , NMe 2 , OMe, NH- Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR';
  • R independently is selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halogen, OH, OR, NH 2 , NHR', SR', COOH, COOR'; wherein R' is selected from straight or branched alkyl, cyclic alkyl, aralkyl, aryl or heteroaryl groups.
  • the compound of formula -5 was prepared from cyanuric chloride by a process described in WO99/50256. Accordingly, the process for preparation of triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable addition salt under mild conditions comprises; 1. Adding cyanuric chloride (1) to 5-amino-picolinonitrile (2) in presence of a solvent at a temperature 0-5 °C with constant stirring to obtain 5 -(4, 6-dichloro- l,3,5-triazin-2-yl amino) picolinonitrile (3);
  • the present invention provides pharmaceutical composition comprising triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable addition salt along with pharmaceutically acceptable excipients for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
  • the pharmaceutically acceptable addition salts as mentioned hereinabove and herein after are meant to comprise the therapeutically active non-toxic acid addition salt forms which the compounds of formula-I are able to form.
  • the compounds of formula-I which have basic properties can be converted to their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid.
  • Appropriate acids comprise, for example, inorganic acids such as hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like acids.
  • the present invention provides triamino triazine derivatives by introducing a pyridine moiety in the side chain to achieve the desired properties and activity.
  • the invention further provides a process for the synthesis of triamino triazine derivatives with a pyridine ring in the side chain.
  • the process of the present invention further uses promoter ligand that facilitates to complete the reaction at an ambient temperature in the range of 50-80°C.
  • the present invention relates to triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable acid addition salts or enantiomers or mixtures of enantiomers or stereoisomers or racemates or solvates or hydrates useful for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections with reduced toxicity, comprising;
  • 'Z' independently is selected from hydrogen, halo, NH 2 , NMe 2 , OMe, NH-Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR';
  • Ri, R 2 and R 3 independently are selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halo, OH, OR', NH 2 , NHR', SR', COOH, COOR', wherein R' independently is selected from straight or branched alkyl, cyclic alkyl or aralkyl, aryl or heteroaryl groups.
  • compounds of formula-I or its pharmaceutically acceptable acid addition salts comprises;
  • the triamino triazine picolinonitriles of the present invention comprises;
  • the present invention discloses a process for preparation of compounds of formula-I comprising catalytic coupling of 5-(4-chloro-6-(phenylamino)- l,3,5-triazin-2-ylamino) picolinonitrile (formula -5) with a suitable reagent in presence of a base, ligand as promoter and solvent at a temperature in the range of 50-80°C.
  • the suitable reagent is optimised by selecting the appropriate catalyst and promoter ligand to improve the yield and selectivity.
  • the suitable reagent is selected from aq. ammonia, or an amino derivative of general formula NHR'"; wherein R'" is selected from (un) substituted or substituted alkyl, cycloalkyl, aryl, heteroaryl; preferably the reagent is aq. ammonia.
  • the catalyst is selected from copper catalyst such as Cu(I) chloride, Cu(I) iodide, CuS0 4 .5H 2 0, [CuBr(PPh 3 ) 2 ] and the like; preferably the catalyst is Cu(I) iodide.
  • the ligand as promoter is selected from 1 ,10-phenanthroline, picolinic acid, 4-hydroxy-proline and ⁇ , ⁇ -dimethylglycine, most preferably the ligand is 4-hydroxy-proline.
  • the solvent is selected from polar aprotic solvent such as DMSO, acetonitrile, DMF, DCM and the like; preferably DMSO.
  • 'Z' independently is selected from hydrogen, halogen, NH 2 , NMe 2 , OMe, NH- Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR';
  • R independently is selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halogen, OH, OR, NH 2 , NHR', SR', COOH, COOR'; wherein R' is selected from straight or branched alkyl, cyclic alkyl, aralkyl, aryl or heteroaryl groups.
  • the compounds of formula 7(a-e) or its pharmaceutically acceptable acid salts are obtained by catalytic coupling of 5-(4-chloro- 6-(phenylamino)-l ,3,5-triazin-2-ylamino) picolinonitrile of formula 5( a-e) with aq. ammonia in presence of K 2 C0 3 , 4-hydroxy proline and DMSO as solvent at a temperature of 55°C.
  • the compound of formula -5 was prepared from cyanuric chloride by a process described in WO99/50256. Accordingly, the process for preparation of triamino triazine picolinonitriles of formula- 1 or its pharmaceutically acceptable acid addition salt under mild conditions comprises the steps of;
  • step (i) cyanuric chloride was added slowly to 5-amino-picolinonitrile (2) in dry solvent with constant stirring for about an hour at a temperature in the range of 0- 5°C followed by stirring at room temperature for about 4.5-5.5 hrs.
  • the solvent was removed under reduced pressure and the resultant residue was dissolved in an organic solvent.
  • the organic phase was washed with water several times followed by evaporation of the solvent and dried to obtain 5-(4, 6-dichloro-l,3,5-triazin-2-yl amino) picolinonitrile (3).
  • Step (ii) includes reacting compound (3) with substituted phenyl amine compound of formula 4 in presence of a base and a solvent at room temperature. On completion of the reaction, reaction mixture was diluted in an organic solvent followed by several washings, drying to obtain 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile (formula -5) The compound of formula 5 optionally were further converted to its salt by a process known in the art.
  • R is as defined above.
  • Step (iii) comprises heating a mixture of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2- ylamino) picolinonitrile (formula -5), suitable reagent, catalyst selected from Cul, ligand selected from 4-hydroxy-proline in presence of a base such as potassium carbonate and DMSO as solvent to a temperature in the range 50-60°C until completion of the reaction.
  • a base such as potassium carbonate and DMSO as solvent
  • the reaction mixture was further diluted in an organic solvent followed by several washings, drying to obtain compounds of formula I.
  • the compounds of formula I were further converted to their hydrochloride salts by a process known in the art.
  • the solvents for step (i) and step (ii) are selected from dry dioxane, dry acetone, dry DMF and dry THF; preferably the solvent is dry acetone.
  • the solvent for step (iii) is selected from polar aprotic solvent such as DMSO, acetonitrile, DMF, DCM and the like; preferably DMSO.
  • the base for step (ii) and step (iii) is selected from t-BuOK, TEA, DIPEA, K 2 C0 3 , K 3 P0 4 ; preferably the base is K 2 C0 3 .
  • the compound (5a-e) is treated with aq. ammonia in presence of Cul, 4- hydroxy proline in DMSO at a temperature of 55°C for about 16 hours to obtain compounds (7a-e) and further converted to their hydrochloride salt (8a-e).
  • the compound (5a-e) is treated with piperidine, morpholine, 2- methoxyethanamine and pyrolidine under optimum conditions to obtain compounds (9a- e), (lla-e), 13(a-e), 15(a-e) and further converted to their hydrochloride salt (10 a-e), (12a-e), (14a-e) and (16a-e) which are depicted in Scheme 3 below:
  • R 15a) 4-F; 15b) 4-OMe; 15c) 4-CH 3 ; 15d) 3-OMe; 15e) 2,4 >6 -CH 3 .
  • R 12a) 4-F; 12b) 4-OMe; 12c) 4-CH 3 ; 12d) 3-OMe; 12e) 2,4,6-CH 3 .
  • 14(a-e) Z pyrolidine
  • R 14a) 4-F; 14b) 4-OMe; 14c) 4 ⁇ CH 3 ; 14d) 3-OMe; 14e) 2,4, 6 -CH 3 .
  • 16(a-e) Z 2-methoxyethanamine
  • R 16a) 4-F; 16b) 4-OMe; 16c) 4-CH 3 ; 16d) 3-OMe; 16e) 2,4,6-CH 3 .
  • the present invention relates to pharmaceutical composition
  • pharmaceutical composition comprising triamino triazine picolinonitriles of formula I or its pharmaceutically acceptable addition salt along with pharmaceutically acceptable excipients for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
  • the compound triamino triazine picolino nitriles of formula SI (5-(4- chloro-6-(mesityl amino)- 1, 3, 5-triazin-2-yl amino) picolino nitrile hydrochloride and S2 (5-(4-amino-6-(mesityl amino)-l,3,5-triazin-2-ylamino) picolino nitrile hydrochloride were observed to inhibit cell associated CXCR4 tropic HlV-lms , HIV-1UGO70, CCR5 tropic HIV- 1 Adas, HIV-1VB59 in TZM-bl assay and HIV- I VBS I in PBMC assay to maximum level as depicted in Table 1 and Table 2 below.
  • the present invention discloses a method for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections comprising administering an effective amount of triamino triazine picolinonitriles of formula-I or its pharmaceutically acceptable acid addition salt along with pharmaceutically acceptable excipients to a subject in need thereof.
  • the pharmaceutical composition of the present invention may be administered orally, topically or systemically.
  • the present invention discloses use of triamino triazine picolinonitriles of formula-I or its pharmaceutically acceptable acid addition salt along with pharmaceutically acceptable excipients for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
  • Cyanuric chloride 1 (2.19 g, 0.012 mole) was added slowly to 5-aminopicolinonitrile 2 (1.19 g, 0.01 mole) in dry acetone (30 ml) with constant stirring for 1 h at 0-5 °C; then left to stir at room temperature for 5 hr. The solvent was removed under reduced pressure and the resultant residue was dissolved in ethyl acetate (100 ml). The organic phase was washed with water (3 x 30 ml), dried over anhydrous Na 2 S0 4 ; organic layer was evaporated to dryness under vacuum.
  • Example 2 General experimental procedure for the synthesis of 5-(4-chloro-6- (phenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile [5(a-e)J:
  • Example 12 General experimental procedure for the synthesis of 5-(4-(2- methoxyethylamino)-6-(phenylamino)-l,3 ? 5-triazin-2 ylamino)picolinonitrile [15(a- e)]:
  • Example 13 General experimental procedure for the synthesis 5-(4-(2- methoxyethylamino)-6-(phenylaminO)-l,3,5-triazin-2-ylamino)picolinonitrile hydrochloride [16(a-e)]:
  • Cytotoxicity assay Both compounds SI and S2 were first tested for cytotoxicity using a series of concentrations and the toxicity was determined using MTT assay. CC 50 (concentration showing 50% cytotoxicity) value was determined.
  • Anti-HIV 1 activity The anti-HIVl activity was assessed using sub-toxic concentrations (concentrations showing > 50% cell viability) of AZT (control), and the compound SI and S2. The activity was tested against CXCR4 (HIV- im) and CCR5 (HIV- 1 Adas) tropic lab adapted strains. The compounds SI and S2 showing inhibition against lab adapted strains were further confirmed against CXCR4 (HIV-1 uG07o) and CCR5 (HIV-1VB 5 9) tropic primary isolates.
  • Cytotoxicity assay The compounds SI and S2 were first tested for cytotoxicity using a series of concentrations in PBMCs. The viability was determined by MTT assay and the CC 50 values were calculated.
  • Anti-HIV assay using PBMC The sub toxic concentrations of the compounds SI and S2 were added to PBMCs previously infected with pre titrated virus (HIV- 1VB5I). After 5 days, the inhibition of virus growth was monitored by inhibition of p24 antigen levels in culture supernatant. IC 50 values were calculated. Each experiment was carried out in duplicate on three separate occasions. Results are tabulated in Table 2.
  • Table 1 Anti-HIV activity against CCR4 tropic HIV-I HIB, HIV-1UGO70 and CCR5 tropic HIV- l Ada 5 , HIV-1VB 5 9 strains in TZM-bl assay (Primary screening)
  • CC50 (Concentration showing 50% viability): Determined by fitting logarithmic trend line to the data.
  • CC50 (Concentration showing 50% viability): Determined by fitting logarithmic trend line to the data.
  • the present invention relating to novel triamino triazine picolinonitriles of formula-I or its pharmaceutically acceptable addition salts or enantiomers thereof or mixtures of enantiomers or stereoisomers or racemates or solvates or hydrates are useful in treating, or reducing the severity of hyper proliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
  • the compounds of formula -I as antagonists of chemokine CXCR4 receptor exhibit enhanced activity and reduced toxicity and are more effective than the known antiviral AZT (Atazanavir) as shown hereinabove.

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Abstract

The present invention discloses triamino triazine picolinonitriles of formula- I useful for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of human immunodeficiency virus (HIV) infections. The invention further discloses process for preparation of compounds of formula-I and pharmaceutical composition thereof.

Description

"Triaminotriazine Picolinonitrile Derivatives As Potent Reverse Transcriptase
Inhibitor of HIV-1"
Technical field of the invention
The present invention relates to triamino triazine picolinonitriles of formula- I useful for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of human immunodeficiency virus (HIV) infections. The invention further relates to process for preparation of compounds of formula-I and pharmaceutical composition thereof.
Background and prior art
The human immunodeficiency virus type 1 (HIV-1) is a retrovirus belonging to the Retroviridae family that was first identified in 1983 and was shown to be the cause of acquired immune deficiency syndrome (AIDS).
Highly active antiretroviral therapy (HAART) incorporating at least three antiretroviral agents has been the standard therapy for HIV/AIDS for more than 10 years. HAART includes protease inhibitors (Pis), nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), entry inhibitors and integrase inhibitors.
FDA approved NNRTIs for the treatment of HIV/AIDS include (a)Nevirapine, (b)Delavirdinemesilate, (c)Efavirenz, (d)Etravirine, (e)Rilpivirine, (f)Amdoxovir, (g)BILR-355 (BILR-355-BS), (h)UK-453061 , (i) UC-781 and (j)Thiofoscarnet (ANX-
Figure imgf000003_0001
The highly specific NNRTIs are however associated with the rapid emergence of resistance mutations that surround the NNRTI binding pocket, thus rendering the molecules ineffective. Also, extensive cross-resistance is well documented among the available NNRTIs. Combination therapy has also led to the development of adherence problems, reduced antiretroviral activity and drug toxicity.
In continuation to develop new potent, selective and non-toxic anti HIV agents, scientists worldwide found a new class of compounds belonging to s-tirazines, which are known for their anti-inflammatory, antispasmodic, diuretic and for modifying adreno-cortico hormone secretion, with promising antiviral and anti HIV activity.
Among the triazine compounds that belong to NNRTI category, diaryltriazine analogues (DATAs) and diarylpyrimidine analogues (DAPYs) are known for their activity against wild-type and various mutant strains of HIV- 1.
WO 99/50250 and WO 00/27825 disclose substituted amino pyrimidine derivatives hich are used in the systemic treatment and prophylaxis of HIV infection. They function by blocking the multiplication process of HIV particularly they block the reverse transcriptase enzyme that plays a vital role in the viral multiplication process.
WO99/50256 discloses 1,3,5 trisubstituted triazine derivatives having HIV replication inhibiting properties.
Figure imgf000004_0001
wherein A may be selected from N; R3 is H; , R4 is -CN, L is -X-R5 , R5 is (un) - substituted phenyl; Ri and R2 represent amino, or R1 and R2 taken together may form pyrrolidinyl, morpholidinyl. WO'256 however specifically relates to triazine benzonitrile compounds.
WO01/85700 also relates to substituted amino triazine derivatives having HIV replication inhibiting properties of the formula iven below.
Figure imgf000004_0002
wherein, -a'=a2-a3=a4-represents a bivalent radical of formula -N=CH-CH=CH-; R2 is cyano, Q is selected from halo, amino Ci-Ce alkyl, Ci-C6 alkyloxy, hetero which is pyrazolidinyl, L is -X-R3 , R3 is (un) substituted phenyl.
The compounds disclosed in WO'256 and WO'700 inhibit replication of HIV virus in the human T-4 cells via an interaction with HIV reverse transcriptase enzyme.
EP0834507 discloses substituted diamino triazine derivatives having HIV replication inhibiting properties.
Figure imgf000004_0003
wherein the variables are as described in the patent.
With a need to improve antiretroviral activity against both wild-type and drug-resistant HIV and to overcome the drawback of the currently used compounds which are toxic and have poor bioavailability, and further with the promising biological activity of triazine scaffold, the present invention focusses in designing triazine derivatives by introducing a pyridine moiety in the side chain. Introducing a pyridine ring increases the polarity and subsequently the bioavailability of the compounds thus enhancing the treatment of HIV with convenience.
Further, the invention is based on the unexpected finding that the compounds described herein below are antagonists of the chemokine CXCR4 receptor which are useful to mediate the medical condition that is modulated by CXCR4 receptor signalling, and in particular for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
Summary of the invention
It is therefore the primary object of the present invention to provide novel triamino triazine compounds by introducing a polar pyridine moiety in the side chain for the treatment or prevention of HIV infections and for treating or reducing the severity of hyperproliferative diseases.
The other object is to provide novel triamino triazine compounds with a polar pyridine moiety in the side chain with reduced toxicity and increased bioavailability.
Yet another object of the invention is to provide a process for synthesis of novel triamino triazine compounds with a polar pyridine moiety in the side chain under mild conditions.
In an aspect, the present invention provides triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable addition salts or enantiomers or mixtures of enantiomers or stereoisomers or racemates or solvates or hydrates useful for treating or reducing the severity of hyper proliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections with reduced toxicity, comprising ;
Figure imgf000005_0001
(I)
wherein, Y and Y' represent NH, 'Ζ' independently is selected from hydrogen, halo, NH2, NMe2, OMe, NH-Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR';
Ri, R2 and R3 independently are selected from hydrogen, , alkyl, cycloalkyl, aryl, heteroaryl, acyl, halo, OH, OR', NH2, NHR', SR', COOH, COOR', wherein R' independently is selected from straight or branched alkyl, cyclic alkyl or aralkyl, aryl or heteroaryl groups.
In another aspect, the present invention provides a process for preparation of compounds of formula-I comprising catalytic coupling of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin- 2-ylamino) picolinonitrile (formula -5)with a suitable reagent in presence of a base, ligand as promoter and solvent at a temperature in the range of 50-80°C. The suitable reagent is optimised by selecting the appropriate catalyst and promoter ligand to improve the yield and selectivity.
Accordingly, the suitable reagent is selected from aq. ammonia, or an amino derivative of general formula NHR'"; wherein R'" is selected from (un) substituted or substituted alkyl, cycloalkyl, aryl, heteroaryl; preferably the reagent is aq. ammonia. The catalyst is selected from copper catalyst such as Cu(I) chloride, Cu(I) iodide, CuS04.5H20, [CuBr(PPh3)2] and the like. The ligand as promoter is selected from 1,10- phenanthroline, picolinic acid, 4-hydroxy-proline and Ν,Ν-dimethylglycine. The solvent is selected from polar aprotic solvent.
Figure imgf000006_0001
wherein 'Z' independently is selected from hydrogen, halogen, NH2, NMe2, OMe, NH- Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR'; and
R independently is selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halogen, OH, OR, NH2, NHR', SR', COOH, COOR'; wherein R' is selected from straight or branched alkyl, cyclic alkyl, aralkyl, aryl or heteroaryl groups.
In another aspect, the compound of formula -5 was prepared from cyanuric chloride by a process described in WO99/50256. Accordingly, the process for preparation of triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable addition salt under mild conditions comprises; 1. Adding cyanuric chloride (1) to 5-amino-picolinonitrile (2) in presence of a solvent at a temperature 0-5 °C with constant stirring to obtain 5 -(4, 6-dichloro- l,3,5-triazin-2-yl amino) picolinonitrile (3);
2. Reacting 5-(4,6-dichloro-l,3,5-triazin-2-ylamino) picolinonitrile (3) with substituted phenyl amine of formula -4 in presence of a base and solvent at room temperature to obtain 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile of formula -5 and optionally converting to its salt;
Figure imgf000007_0001
wherein R is as defined above; and
3. catalytic coupling of compound of formula -5 with a suitable reagent in presence of a base, ligand and solvent at ambient temperature to obtain compounds of formula- 1.
In yet another aspect, the present invention provides pharmaceutical composition comprising triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable addition salt along with pharmaceutically acceptable excipients for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
Detailed description of the invention
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.
As used herein , the pharmaceutically acceptable addition salts as mentioned hereinabove and herein after are meant to comprise the therapeutically active non-toxic acid addition salt forms which the compounds of formula-I are able to form. The compounds of formula-I which have basic properties can be converted to their pharmaceutically acceptable acid addition salts by treating said base form with an appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like acids. With the need for new antiretroviral agents that can enhance bioavailability, reduce toxicity and improve antiretroviral activity against both wild-type and drug-resistant HIV, the present invention provides triamino triazine derivatives by introducing a pyridine moiety in the side chain to achieve the desired properties and activity.
The invention further provides a process for the synthesis of triamino triazine derivatives with a pyridine ring in the side chain. The process of the present invention further uses promoter ligand that facilitates to complete the reaction at an ambient temperature in the range of 50-80°C.
In an embodiment, the present invention relates to triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable acid addition salts or enantiomers or mixtures of enantiomers or stereoisomers or racemates or solvates or hydrates useful for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections with reduced toxicity, comprising;
Figure imgf000008_0001
(I)
wherein, Y and Y' represent NH,
'Z' independently is selected from hydrogen, halo, NH2, NMe2, OMe, NH-Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR';
Ri, R2 and R3 independently are selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halo, OH, OR', NH2, NHR', SR', COOH, COOR', wherein R' independently is selected from straight or branched alkyl, cyclic alkyl or aralkyl, aryl or heteroaryl groups.
In another embodiment, compounds of formula-I or its pharmaceutically acceptable acid addition salts comprises;
i. 5-(4-chloro-6-(p-tolylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile ;
ii. 5-(4-chloro-6-(3-methoxyphenylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile; iii. 5-(4-amino-6-(p-tolylamino)-l,3,5-triazin-2-ylamino)picolinonitrile;
iv. 5-(4-amino-6-(3-methoxyphenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile; v. 5-(4-amino-6-(mesitylamino)-l , 3, 5-triazin-2-ylamino) picolinonitrile; vi. 5-(4-moipholino-6-(p-tolylamino)-l,3,5-triazin-2-ylamino)picolinonitrile;
vii. 5-(4-(4-fluoro henylamino)-6-nlO holino- 1 ,3,5-triazin-2-ylamino)picolinonitrile; viii. 5-(4-(3-methoxyphenylamino)-6-(piperidin- 1 -yl)- 1 ,3,5-triazin-2ylamino)
picolinonitrile;
ix. 5-(4-(3-methoxyphenylamino)-6-(pyrrolidin-l-yl)-l,3,5-triazin-2ylamino)
picolinonitrile;
x. 5-(4-(2-methoxyethylamino)-6-(p-tolylamino)-l,3,5-triazin-2ylamino)
picolinonitrile;
xi. 5-(4-(phenylamino)-6-(pyrrolidin-l -yl)-l ,3,5-triazin-2ylamino) picolinonitrile; xii. 5-(4-(2-methoxyethylamino)-6-(phenylamino)-l,3,5-triazin-2ylamino)picolino nitrile;
xiii. 5-(4-(phenylamino)-6-(piperidin-l-yl)-l,3,5-triazin-2-ylamino) picolinonitrile; xiv. 5-(4-(phenylamino)-6-(morpholine- 1 -yl)- 1 ,3,5-triazin-2-ylamino)
picolinonitrile;
xv. 5-(4-amino-6-(phenylamino)-l,3,5-triazin-2- ylamino) picolinonitrile;
xvi. 5-(4-chloro-6-(mesityl amino)-l,3,5-triazin-2-ylamino) picolinonitrile.
In a preferred embodiment, the triamino triazine picolinonitriles of the present invention comprises;
i. 5-(4-chloro-6-(mesityl amino)-l,3,5-triazin-2-ylamino) picolinonitrile; ii. 5-(4-amino-6-(mesitylamino)-l , 3, 5-triazin-2-ylamino) picolinonitrile
In an embodiment, the present invention discloses a process for preparation of compounds of formula-I comprising catalytic coupling of 5-(4-chloro-6-(phenylamino)- l,3,5-triazin-2-ylamino) picolinonitrile (formula -5) with a suitable reagent in presence of a base, ligand as promoter and solvent at a temperature in the range of 50-80°C. The suitable reagent is optimised by selecting the appropriate catalyst and promoter ligand to improve the yield and selectivity.
Accordingly, the suitable reagent is selected from aq. ammonia, or an amino derivative of general formula NHR'"; wherein R'" is selected from (un) substituted or substituted alkyl, cycloalkyl, aryl, heteroaryl; preferably the reagent is aq. ammonia. The catalyst is selected from copper catalyst such as Cu(I) chloride, Cu(I) iodide, CuS04.5H20, [CuBr(PPh3)2] and the like; preferably the catalyst is Cu(I) iodide. The ligand as promoter is selected from 1 ,10-phenanthroline, picolinic acid, 4-hydroxy-proline and Ν,Ν-dimethylglycine, most preferably the ligand is 4-hydroxy-proline. The solvent is selected from polar aprotic solvent such as DMSO, acetonitrile, DMF, DCM and the like; preferably DMSO.
Figure imgf000010_0001
wherein 'Z' independently is selected from hydrogen, halogen, NH2, NMe2, OMe, NH- Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR'; and
R independently is selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halogen, OH, OR, NH2, NHR', SR', COOH, COOR'; wherein R' is selected from straight or branched alkyl, cyclic alkyl, aralkyl, aryl or heteroaryl groups.
In an embodiment of the present invention, the compounds of formula 7(a-e) or its pharmaceutically acceptable acid salts are obtained by catalytic coupling of 5-(4-chloro- 6-(phenylamino)-l ,3,5-triazin-2-ylamino) picolinonitrile of formula 5( a-e) with aq. ammonia in presence of K2C03, 4-hydroxy proline and DMSO as solvent at a temperature of 55°C.
Figure imgf000010_0002
-i .'H ?<a-e> R 7 a.) 7J» -l-O!vfc: 7c> 4-CH i.
7:1) M) c- 7 i■J,4,i. :H,.
In another aspect, the compound of formula -5 was prepared from cyanuric chloride by a process described in WO99/50256. Accordingly, the process for preparation of triamino triazine picolinonitriles of formula- 1 or its pharmaceutically acceptable acid addition salt under mild conditions comprises the steps of;
i. Adding cyanuric chloride (1) to 5-amino-picolinonitrile (2) in presence of a solvent at a temperature 0-5°C with constant stirring to obtain 5-(4, 6-dichloro- l ,3,5-triazin-2-yl amino) picolinonitrile (3);
ii. Reacting 5-(4,6-dichloro-l,3,5-triazin-2-ylamino) picolinonitrile (3) with substituted phenyl amine of formula 4 in presence of a base and solvent at room temperature to obtain 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile (formula -5) optionally converting to its acid salt; and
iii. catalytic coupling of compound of formula -5 with a suitable reagent in presence of a base, ligand and solvent at a temperature in the range of 50-60°C to obtain compounds of formula I.
According to step (i), cyanuric chloride was added slowly to 5-amino-picolinonitrile (2) in dry solvent with constant stirring for about an hour at a temperature in the range of 0- 5°C followed by stirring at room temperature for about 4.5-5.5 hrs. The solvent was removed under reduced pressure and the resultant residue was dissolved in an organic solvent. The organic phase was washed with water several times followed by evaporation of the solvent and dried to obtain 5-(4, 6-dichloro-l,3,5-triazin-2-yl amino) picolinonitrile (3).
Figure imgf000011_0001
Step (ii) includes reacting compound (3) with substituted phenyl amine compound of formula 4 in presence of a base and a solvent at room temperature. On completion of the reaction, reaction mixture was diluted in an organic solvent followed by several washings, drying to obtain 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile (formula -5) The compound of formula 5 optionally were further converted to its salt by a process known in the art.
Figure imgf000011_0002
Wherein R is as defined above.
Step (iii) comprises heating a mixture of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2- ylamino) picolinonitrile (formula -5), suitable reagent, catalyst selected from Cul, ligand selected from 4-hydroxy-proline in presence of a base such as potassium carbonate and DMSO as solvent to a temperature in the range 50-60°C until completion of the reaction. The reaction mixture was further diluted in an organic solvent followed by several washings, drying to obtain compounds of formula I. The compounds of formula I were further converted to their hydrochloride salts by a process known in the art.
Figure imgf000012_0001
Wherein Z and R are as defined above.
The solvents for step (i) and step (ii) are selected from dry dioxane, dry acetone, dry DMF and dry THF; preferably the solvent is dry acetone. The solvent for step (iii) is selected from polar aprotic solvent such as DMSO, acetonitrile, DMF, DCM and the like; preferably DMSO. The base for step (ii) and step (iii) is selected from t-BuOK, TEA, DIPEA, K2C03, K3P04; preferably the base is K2C03.
In a preferred embodiment, the preparation of various triamino triazine compounds from compound (5a-e) by process step (iii) using the various reagents is described herein below:
In one aspect, the compound (5a-e) is treated with aq. ammonia in presence of Cul, 4- hydroxy proline in DMSO at a temperature of 55°C for about 16 hours to obtain compounds (7a-e) and further converted to their hydrochloride salt (8a-e).
The process step (iii) for preparation of compounds of formula (I) is depicted in Scheme 2 and Scheme 3 below:
Scheme 2:
-CH3;
Figure imgf000012_0002
7(a-e) R = 7a) 4-F; 7b) 4-OMe; 7c) 4-CH3; 8(a-e) R = 8a) 4-F; 8b) 4-OMe; 8c) 4-CH3;
7d) 3-OMe; 7e) 2,4,6-CH3. 8d) 3-OMe; 8e) 2,4,6-CH3.
In the second aspect, the compound (5a-e) is treated with piperidine, morpholine, 2- methoxyethanamine and pyrolidine under optimum conditions to obtain compounds (9a- e), (lla-e), 13(a-e), 15(a-e) and further converted to their hydrochloride salt (10 a-e), (12a-e), (14a-e) and (16a-e) which are depicted in Scheme 3 below:
Scheme 3:
RT 1
Figure imgf000013_0001
9(a-e) Z = mo holine
S(a-e) R = 5a) 4-F; 5b) 4-OMe; 5c) 4-CH3; R = 9a) 4.F 9b) 4_OM 9c) 4_CH 9d) 3_OM 9g) 2 4 6.CH
5d) 3-0Me; Se) 2,4,6-CH3. 1 l(a-e) Z = pipendine
R = l la),4-F; l ib) 4-OMe; 11c) 4-CH3; l ld) 3-OMe; lie) 2,4,6-CH3. 13 (a-e) Z = pyrolidine
R = 13a) 4-F; 13b) 4-OMe; 13c) 4-CH3; 13d) 3-OMe; 13e) 2,4,6-CH3. 15 (a-e) Z = 2-methoxyethanamine
R = 15a) 4-F; 15b) 4-OMe; 15c) 4-CH3; 15d) 3-OMe; 15e) 2,4>6-CH3.
HCI in Dioxane
Figure imgf000013_0002
10(a-e) Z = morpholine
R = 10a) 4-F; 10b) 4-OMe; 10c) 4-CH3; lOd) 3-OMe; lOe) 2,4,6-CH3. 12(a-e) Z = piperidine *
R = 12a) 4-F; 12b) 4-OMe; 12c) 4-CH3; 12d) 3-OMe; 12e) 2,4,6-CH3. 14(a-e) Z = pyrolidine
R = 14a) 4-F; 14b) 4-OMe; 14c) 4÷CH3; 14d) 3-OMe; 14e) 2,4,6-CH3. 16(a-e) Z = 2-methoxyethanamine
R = 16a) 4-F; 16b) 4-OMe; 16c) 4-CH3; 16d) 3-OMe; 16e) 2,4,6-CH3.
In yet another embodiment, the present invention relates to pharmaceutical composition comprising triamino triazine picolinonitriles of formula I or its pharmaceutically acceptable addition salt along with pharmaceutically acceptable excipients for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
In an embodiment, the compound triamino triazine picolino nitriles of formula SI (5-(4- chloro-6-(mesityl amino)- 1, 3, 5-triazin-2-yl amino) picolino nitrile hydrochloride and S2 (5-(4-amino-6-(mesityl amino)-l,3,5-triazin-2-ylamino) picolino nitrile hydrochloride were observed to inhibit cell associated CXCR4 tropic HlV-lms , HIV-1UGO70, CCR5 tropic HIV- 1 Adas, HIV-1VB59 in TZM-bl assay and HIV- I VBS I in PBMC assay to maximum level as depicted in Table 1 and Table 2 below. The data reveals that the compounds of the present invention exhibit enhanced activity against the strains CXCR4 tropic HIV-l niB , HIV-1UGO70, CCR5 tropic HIV-1 Adas, HIV-1VB59 and also are more effective than the known antiviral AZT (Atazanavir). In yet another embodiment, the present invention discloses a method for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections comprising administering an effective amount of triamino triazine picolinonitriles of formula-I or its pharmaceutically acceptable acid addition salt along with pharmaceutically acceptable excipients to a subject in need thereof. The pharmaceutical composition of the present invention may be administered orally, topically or systemically.
In another embodiment, the present invention discloses use of triamino triazine picolinonitriles of formula-I or its pharmaceutically acceptable acid addition salt along with pharmaceutically acceptable excipients for treating or reducing the severity of hyperproliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections.
The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.
Examples:
(A) Example 1: General experimental procedure for the synthesis of 5-(4, 6- dichloro-l,3,5-triazin-2-ylamino)picolinonitrile (3):
Cyanuric chloride 1 (2.19 g, 0.012 mole) was added slowly to 5-aminopicolinonitrile 2 (1.19 g, 0.01 mole) in dry acetone (30 ml) with constant stirring for 1 h at 0-5 °C; then left to stir at room temperature for 5 hr. The solvent was removed under reduced pressure and the resultant residue was dissolved in ethyl acetate (100 ml). The organic phase was washed with water (3 x 30 ml), dried over anhydrous Na2S04; organic layer was evaporated to dryness under vacuum. The compound obtained was purified with flash column chromatography using hexane to hexane/ethyl acetate 8/2 as gradient elution to obtain the final compounds (3). Example 2: General experimental procedure for the synthesis of 5-(4-chloro-6- (phenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile [5(a-e)J:
The mixture of compound 3 (0.01 mole), various substituted aniline 4 (a-e) (0.01 mole) and K2C03 (0.012 mole) in dry acetone (30 ml) was left to stir at room temperature for 6 h. When the reaction was completed, reaction mixture was diluted with ethyl acetate (250 ml), the organic phase was washed with water (3 χ 75 ml), dried over anhydrous Na2S04; organic layer was evaporated to dryness under vacuum. The compound obtained was purified with flash column chromatography using hexane to hexane/ethyl acetate 8/2 as gradient elution to obtain the final compounds 5(a-e).
Example 3: General experimental procedure for the synthesis of 5-(4-chIoro-6- (phenylamino)-l ,3>5-triazin-2-ylamino)picolinonitrile h drochloride [6(a-e)] :
The mixture of compound 5a (1.0 gm) and HCl in dioxane (10 ml, 90 % HCl) was stirred at room temperature for 5 hr to afford compound 6a. Same procedure was followed to synthesize compounds (6b-6e).
Example 4: General experimental procedure for the synthesis of 5-(4-amino-6- (phenylamino)-l,3»5-triazin-2-ylamino)picolinonitrile [7(a-e)]:
The mixture of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile (1.0 mmol), aq. ammonia (28 %, 2 ml), Cul (0.2 mmol), 4-Hydroxy Proline (0.2 mmol), K2C03 (2.0 mmol) in 5 ml of DMSO was heated at 55°C for 16 h. When the reaction was completed, reaction mixture was diluted with ethyl acetate (150 ml), the organic phase was washed with water (3 χ 50 ml), dried over anhydrous Na2S04; organic layer was evaporated to dryness under vacuum. The compound obtained was purified with flash column chromatography using hexane to hexane/ethyl acetate 2/8 as gradient elution to obtain the final compounds 7(a-e).
Example 5: General experimental procedure for the synthesis of 5-(4-amino-6-
(phenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile hydrochloride [8(a-e)]:
■i
The mixture of compound 7a (1.0 gm) and HCl in dioxane (10 ml, 90 % HCl) was stirred at room temperature for 5 hr to afford compound 8a. Same procedure was followed to synthesize compounds (8b-8e). Example 6: General experimental procedure for the synthesis of 5-(4-morpholino-6- (phenylamino)-l ,3,5-triazin-2-ylamino)picolinonitrile [9(a-e)] :
The mixture of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile (1.0 mmol), morpholine (1.5 mmol) and K2C03 (1.5 mmol) in dry DMF (10 ml) was left to stir at room temperature for 4 h. When the reaction was completed, reaction mixture was diluted with ethyl acetate (150 ml), the organic phase was washed with water (3 χ 50 ml), dried over anhydrous Na2S04; organic layer was evaporated to dryness under vacuum. The compound obtained was purified with flash column chromatography using hexane to hexane/ethyl acetate 5/5 as gradient elution to obtain the final compounds (9a-9e).
Example 7: General experimental procedure for the synthesis 5-(4-morpholino-6- (phenylamino)-l,355-triazin-2-ylamino)picolinonitrile hydrochloride [10(a-e)]:
The mixture of compound 9a (1.0 gm) and HCl in dioxane (10 ml, 90 % HCl) was stirred at room temperature for 5 hr to afford compound 10a. Same procedure was followed to synthesize compounds (lOa-lOe).
Example 8: General experimental procedure for the synthesis of 5-(4-(phenylamino)- 6-(piperidin-l-yI)-l,3?5-triazin-2-ylamino)picolinonitrile [ll(a-e)]:
The mixture of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile (1.0 mmol), piperidine (1.5 mmol) and K2C03 (1.5 mmol) in dry DMF (10 ml) was left to stir at room temperature for 5 h. When the reaction was completed, reaction mixture was diluted with ethyl acetate (150 ml), the organic phase was washed with water (3 χ 50 ml), dried over anhydrous Na2S04; organic layer was evaporated to dryness under vacuum. The compound obtained was purified with flash column chromatography using hexane to hexane/ethyl acetate 5/5 as gradient elution to obtain the final compounds (lla-lle).
Example 9: General experimental procedure for the synthesis 5-(4-(phenylamino)-6- (piperidin-l-yl)-l,3,5-triazin-2-ylamino)picolinonitrile hydrochloride [12(a-e)] :
The mixture of compound 11a (1.0 gm) and HCl in dioxane (10 ml, 90 % HCl) was stirred at room temperature for 5 hr to afford compound 12a. Same procedure was followed to synthesize compounds (12a-12e). Example 10: General experimental procedure for the synthesis of 5-(4- (phenylamino)-6-(pyrrolidin-l-yl)-l,3,5-triazin-2-yIamino)picolinonitrile [13(a-e)]:
The mixture of 5-(4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino)picolinonitrile (1.0 mmol), pyrrolidine (1.5 mmol) and K2C03 (1.5 mmol) in dry DMF (10 ml) was left to stir at room temperature for 8 h. When the reaction was completed, reaction mixture was diluted with ethyl acetate (150 ml), the organic phase was washed with water (3 x 50 ml), dried over anhydrous Na2S04; organic layer was evaporated to dryness under vacuum. The compound obtained was purified with flash column chromatography using hexane to hexane/ethyl acetate 2/8 as gradient elution to obtain the final compounds (13a-13e).
Example 11: General experimental procedure for the synthesis of 5-(4- (phenylamino)-6-(pyrrolidin-l-yl)-l,3,5-triazin-2-ylamino) picolinonitrile hydrochloride [14(a-e)]:
The mixture of compound 13a (1.0 gm) and HC1 in dioxane (10 ml, 90 % HC1) was stirred at room temperature for 5 hr to afford compound 14a. Same procedure was followed to synthesize compounds (14b-14e).
Example 12: General experimental procedure for the synthesis of 5-(4-(2- methoxyethylamino)-6-(phenylamino)-l,3?5-triazin-2 ylamino)picolinonitrile [15(a- e)]:
The mixture of 5-(4-chloro-6-( henylamino)-l,3,5-triazin-2-ylamino)picolinonitrile (1.0 mmol), 2-methoxyethanamine (1.5 mmol) and K2C03 (1.5 mmol) in dry DMF (10 ml) was left to stir at room temperature for 6 h. When the reaction was completed, reaction mixture was diluted with ethyl acetate (150 ml), the organic phase was washed with water (3 x 50 ml), dried over anhydrous Na2S04; organic layer was evaporated to dryness under vacuum. The compound obtained was purified with flash column chromatography using ethyl acetate as gradient elution to obtain the final compounds (15a-15e). Example 13: General experimental procedure for the synthesis 5-(4-(2- methoxyethylamino)-6-(phenylaminO)-l,3,5-triazin-2-ylamino)picolinonitrile hydrochloride [16(a-e)]:
The mixture of compound 15a (1.0 gm) and HCl in dioxane (10 ml, 90 % HCl) was stirred at room temperature for 5 hr to afford compound 16a. Same procedure was followed to synthesize compounds (16b-16e).
(B) Spectral analysis: 5-(4-chloro-6-(p-tolylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile (5c)
Figure imgf000018_0001
lK NMR (300 MHz, DMSO-i¾) S: 10.66 (br. s., lH), 10.39 (br. s., 1H), 8.90 (br. s., mi l 8.28 (br. s., 1H), 7.94 (m, 1H), 7.45 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 2.28 (st 3H); 13C NMR (300 MHz, DMSO-i¾) S: 168.69, 164.42, 143.15, 139.29, 135.66, 133.75^ 129.60, 129.53, 126.96, 126.54, 125.82, 121.66, 118.25, 20.91. IR (KBr, cm "'): 3321, 2233, 1521, 1257, 987, 798; LC-ESMS m/z 338.13 [M+H]+.
5-(4-chloro-6-(3-methoxyph
Figure imgf000018_0002
5d
!H NMR (300 MHz, DMSO-i tf) δ: 10.83 (br. s., 1H), 10.42 (br. s., 1H), 8.94 (br. s., 1H), 8.45 (br. s., 1H), 8.30 (br. s., 1H), 7.95 (br. s., 1H), 7.13 (m, 2H), 6.70 (d, J = 7.2 Hz, 1H), 3.-73 (s, 3H); LC-ESMS m/z 354.18 [M+H]+.
5-(4-chloro-6-(p-tolylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile hydrochloride (6c)
Figure imgf000018_0003
6c Ή NMR (300 MHz, DMSO-i¾) < :10.63 (br. s., IH), 10.36 (br. s., IH), 8.92 (br. s., IH), 8.36 (br. s., IH), 8.11 (m, IH), 7.45 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 2.28(s, 3H); 13C NMR (300 MHz, OMSO-d6) δ: 168.69, 164.42, 143.15, 139.29, 135.66, 133.75, 129.60, 129.53, 126.96, 126.54, 125.82, 121.66, 118.25, 20.91. IR (KBr, cm "'): 3321 , 2233, 1521, 1257, 987, 798; LC-ESMS m/z 338.13 [M+H]+.
5-(4-chIoro-6-(3-methoxyphenylamino)-l,3,5-triazin-2-yIamino)picolinonitrile hydrochloride (6d)
Figure imgf000019_0001
6d
¾ NMR (300 MHz, DMSO-i¾) δ 10.76 (br. s., IH), 10.44 (br. s., IH), 8.95 (br. s., IH), 8.54 (br. s., IH), 8.39 (br. s., IH), 7.95 (br. s., IH), 7.13 (m, 2H), 6.70 (d, J = 7.2 Hz, IH), 3.73 (s, 3H); LC-ESMS m/z 354.34 [M+H]+.
5-(4-amino-6-(p-tolylamino)-l,355-triazin-2-ylamino)picoIinonitrile (7c):
Figure imgf000019_0002
7c
'H NMR (300 MHz, DMSO-^): δ 9.77 (s, IH), 9.17 (s, IH), 9.11 (s, IH), 8.52 (d, J- 8.3 Hz, IH), 7.87 (d, J= 8.7 Hz, IH), 7.60 (d, J = 7.9 Hz, 2H), 7.16 (d, J- 7.9 Hz, 2H), 6.83 (br. s., 2H), 2.28 (s, 3H). ,3C NMR (75 MHz, DMSO-<¾: δ 167.21, 164.78, 164.65, 142.52, 141.22, 137.69, 131.40, 129.51 , 129.24, 125.49, 123.87, 120.81, 118.68, 20.83.IR (KBr, cm -1): 3186, 2229, 1505, 1245, 991, 810.LC-ESMS m/z 319.13 [M+H]+HPLC Purity: 98 %.
5-(4-amino-6-(3-methoxyphenyIamino)-1 j5-triazin-2-ylamino)picolinonitrile (7d):
Figure imgf000019_0003
7d Ή NMR (300 MHz, DMSO-<fc): δ 9.80 (s, 1H), 9.25 (s, 1H), 9.13 (d, J = 2.3 Hz, 1H), 8.53 (dd, J = 8.7, 2.3 Hz, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.42 (br. s., 1H), 7.34 (d, J= 7.9 Hz, 1H), 7.17 (t, J= 8.1 Hz, 1H), 6.90 (br. s., 2H), 6.55 (dd, J = 7.9, 1.9 Hz, 1H), 3.73 (s, 3H). 13C NMR (75 MHz, DMSO-<¾: δ 166.74, 164.32, 164.19, 159.41, 142.15, 141.04, 140.72, 129.33, 129.06, 125.13, 123.50, 118.21, 112.45, 107.33, 106.05, 54.89. IR (KBr, cm 3377, 2231, 1492, 1205, 1035, 806. LC-ESMS m/z 333.33 [M-H]+ HPLC Purity: 98.9 %.
■amino-6-(p-toIylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile hydrochloride
Figure imgf000020_0001
8c
!H NMR (300 MHz, DMSO-c¾) δ: 9.79 (s, 1H), 9.29 (s, 1H), 9.23 (s, 1H), 8.68 (d, J = 8.3 Hz, 1H), 7.85 (d, J = 8.7 Hz, 1H), 7.62 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 2H), 2.28 (s, 3H); 13C NMR (300 MHz, DMSO-i¾) δ: 167.21 , 164.78, 164.65, 142.52, 141.22, 137.69, 131.40, 129.51, 129.24, 125.49, 123.87, 120.81, 118.68, 20.83; LC-ESMS m/z 319.13 [M+H]+.
5-(4-amino-6-(3-methoxyphenylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile hydrochloride (8d)
Figure imgf000020_0002
8d
¾ NMR (300 MHz, DMSO-i¾) δ: 9.78 (s, 1H), 9.33 (s, 1H), 9.21 (d, j = 2.3 Hz, 1H), 8.61 (dd, J = 8.7, 2.3 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.44 (br. s., 1H), 7.34 (d, J = 7.9 Hz, 1H), 7.17 (t, J = 8.1 Hz, 1H), 6.55 (dd, J = 7.9,1.9 Hz, 1H), 3.73 (s, 3H); 13C NMR (300 MHz, DMSO-<¾) δ: 170.30, 166.74, 164.32, 164.19, 159.41, 142.15, 141.04, 140.72, 129.06, 125.13, 123.50, 118.21, 112.45, 107.33, 106.05, 54.89; LC-ESMS m/z 335.33 [M+H]+. 5-(4-chloro-6-(mesityl amino)- 1 ,3 ,5-triazin-2-y lamino) picolinonitrile hydrochloride:-
Figure imgf000021_0001
'H NMR (300 MHz, DMSO): δ = 9.76 (br. s., IH), 9.53 (br. s., IH), 9.42 (br. s., IH),
9.21 (m IH), 8.70 (br. s., IH), 6.95 (m, 2H), 2.11(m, 9H).
HRMS (ESI): m/z [M] + calcd for Ci8Hi6ClN7: 365.1212; found: 366.2025.
5-(4-amino-6-(mesitylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile hydrochloride (8e)
Figure imgf000021_0002
8e
'H NMR (300 MHz, DMSO-<fc) δ: 9.70 (br. s., IH), 9.25 (br. s., IH), 8.63 (br. s., IH), 8.49 (s, IH), 7.77 (m, IH), 6.99 (d, J = 9.8Hz, 2H), 2.1 l(m, 9H); l3C NMR (300 MHz, DMSO-*) δ: 165.31, 163.74, 160.48, 159.50, 141.96, 138.76, 135.74, 135.63, 133.34, 128.74, 128.33, 126.58, 125.54, 118.36, 118.20, 20.98, 18.43; LC-ESMS m/z 347.35 [M+H]+.
5-(4-morpholino-6-(p-tolylamino)-l,355-triazin-2-ylamino)picolinonitrile (9c):
Figure imgf000021_0003
9c
1 H NMR (300 MHz, DMSO-*): δ 9.89 (s, IH), 9.35 (s, IH), 9.07 (br. s., IH), 8.43 (d, J = 7.6 Hz, IH), 7.90 (d, J = 8.3 Hz, IH), 7.55 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 8.3 Hz, 2H), 3.75 (m, 4H), 3.65 (m, 4H), 2.25 (s, 3H). 13C NMR (75 MHz, DMSO-*): δ 165.01, 164.32, 164.19, 142.60, 140.96, 137.41, 131.67, 129.59, 129.32, 125.71, 124.12, 120.86, 118.58, 66.34, 43.90, 20.82.IR (KBr, cm 3386, 3313, 2222, 1554, 1234, 1018, 894, 803.LC-ESMS m z 389.13 [M+H]+HPLC Purity: 98.5 %.
5-(4-(4-fluorophenylamino)-6-morpholino-l, 3, 5-triazin-2-ylamino) picolinonitrile hydrochloride (10a)
Figure imgf000022_0001
10a
Ή NMR (300 MHz, DMSO-i¾) δ: 9.96 (s, IH), 9.49 (s, IH), 9.19 (br. s., IH), 8.55 (d, J = 7.2Hz, IH), 8.13 (d, J = 8.3 Hz, IH), 7.68 (dd, J - 8.3, 5.1 Hz, 2H), 7.15 (m, 2H), 3.73 (m, 4H), 3.67 (m, 4H);LC-ESMS m/z 393.13 [M+H]+.
5-(4-morpholino-6-(p-tolyIamino)-l,3, 5-triazin-2-yIamino) picolinonitrile hydrochloride (10c)
Figure imgf000022_0002
10c
Ή NMR (300 MHz, DMSO-fik) 3: 9.86 (s, IH), 9.32 (s, IH), 9.19 (br. s., IH), 8.56 (d, J = 7.6Hz, IH), 7.99 (d, J = 8.3 Hz, IH), 7.65 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 8.3 Hz, 2H), 3.75 (m, 4H), 3.65 (m, 4H), 2.25 (s, 3H); 13C NMR (300 MHz, DMSO-^) δ: 165.01, 164.32, 142.60, 140.96, 137.41, 131.67, 129.59, 129.32, 125.71, 124.12, 120.86, 118.58, 112.22, 66.34, 43.90, 20.82; LC-ESMS m/z 389.13 [M+H]+. . 5-(4-(3-methoxyphenylamino)-6-(piperidin-l-yl)-l,3,5-triazin-2ylamino) picolinonitrile (lid):
Figure imgf000023_0001
lid
Ή NMR (300 MHz, DMSO-i¾: δ 9.84 (s, 1H), 9.36 (s, 1H), 9.10 (d, J = 2.3 Hz, 1H), 8.43 (m, 1H), 7.89 (d, J = 8.7 Hz, 1H), 7.46 (br. s., 1H), 7.21 (m, 2H), 6.56 (dd, J = 9.1, 2.6 Hz, 1H), 3.76 (m, 4H), 3.73 (s, 3H), 1.65 (m, 2H), 1.52 (m, 4H). 13C NMR (75 MHz, DMSO- e): δ 164.15, 164.07, 163.91 , 159.38, 142.13, 141.03, 140.62, 129.13, 129.10, 125.15, 123.58, 118.15, 112.22, 107.76, 105.53, 54.80, 43.88, 25.32, 24.20.IR (KBr, cm " !): 3369, 2937, 2221, 1537, 1238, 1039, 958, 806, 777.LC-ESMS m/z 403.53 [M+H]+HPLC Purity: 100 %.
5-(4-(3-methoxyphenylamino)-6-(piperidin-l-yl)-l, 3, 5-triazin-2ylamino) picolinonitrile hydrochloride (12d)
Figure imgf000023_0002
12d
Ή NMR (300 MHz, DMSO-i&) δ: 9.87 (s, 1H), 9.39 (s, 1H), 9.23 (d, J = 2.3 Hz, 1H), 8.56 (m, 1H), 7.98 (d, J = 8.7 Hz, 1H), 7.48 (br. s., 1H), 7.21 (m, 2H), 6.56 (dd, J = 9.1, 2.6 Hz,^ 1H), 3.76 (m, 4H), 3.73 (s, 3H), 1.65 (m, 2H), 1.52 (m, 4H);13C NMR (300 MHz, DMSO-ί/ί) δ: 164.07, 163.91, 159.38, 142.13, 141.03, 140.62, 129.13, 129.10, 125.15, 123.58, 120.54, 118.15, 112.22, 107.76, 105.53, 54.80, 43.88, 25.32, 24.20; LC-ESMS m/z 403.53 [M+H]+. 5-(4-(3-methoxyphenylamino)-6-(pyrrolidin-l-yl)-l,3,5-triazin-2yl amino) picolinonitrile (13d):
Figure imgf000024_0001
13d
!H NMR (300 MHz, DMSO-i¾): δ 9.82 (s, IH), 9.31 (s, IH), 9.14 (d, J = 2.3 Hz, IH), 8.51 (dd, J = 8.7, 2.3 Hz, IH), 7.87 (d, J = 8.7 Hz, IH), 7.59 (br. s., IH), 7.30 (d, J = 7.9 Hz, IH), 7.16 (t, J = 8.1 Hz, IH), 6.54 (dd, J = 8.1, 1.7 Hz, IH), 3.73 (s, 3H), 3.53(m, 4H), 1.91 (m, 4H). 13C NMR (75 MHz, DMSO-<¾): δ 163.93, 163.36, 159.85, 142.56, 141.72, 141.20, 129.49, 129.00, 125.51, 123.89, 118.64, 112.47, 112.34, 107.87, 105.85, 54.85, 46.06, 24.75. IR (KBr, cm -'): 3375, 2975, 2223, 1545, 1207, 1041, 914, 808. LC- ESMS m/z 387.07 [M-H]+ HPLC Purity: 98.9 %.
5-(4-(3-methoxyphenylamino)-6-(pyrroIidin-l-yl)-l, 3, 5-triazin-2picolinonitrile hydrochloride (14d)
Figure imgf000024_0002
14d
'H NMR (300 MHz, DMSO-ik) δ: 9.82 (s, IH), 9.31 (s, IH), 9.24 (d, J = 2.3 Hz, IH), 8.61 (dd, J = 8.7, 2.3 Hz, IH), 7.92 (d, J = 8.7 Hz, IH), 7.59 (br. s., IH), 7.30 (d, J = 7.9 Hz, IH), 7.16 (t, J = 8.1 Hz, IH), 6.54 (dd, J = 8.1, 1.7 Hz, IH), 3.73 (s, 3H), 3.53 (m, 4H), 1.91 (m, 4H); 13C NMR (300 MHz, DMSO-i¾) δ: 163.50, 162.94, 159.41, 142.13, 141.28, 140.77, 129.08, 129.00, 125.10, 123.48, 120.76, 118.20, 112.02, 107.45, 105.45, 54.85, 46.06, 24.75; LC-ESMS m/z 389.07 [M+H]+. 5-(4-(2-methoxyethylamino)-6-(p-tolyIamino)-l,3,5-triaz ^
(15c):
Figure imgf000025_0001
15c
Ή NMR (300 MHz, DMSO-^): δ 9.85 (s, 1H), 9.35 (s, 1H), 9.15 (br. s., 1H), 8.53 (m, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.35 (br. s., 1H), 7.11 (d, J = 8.3 Hz, 2H), 3.55 (s, 4H), 3.25 (s, 3H), 2.25 (s, 3H). 13C NMR (75 MHz, DMSO-ifc): δ 166.13, 164.45, 164.21, 142.55, 141.23, 137.69, 131.40, 129.51, 129.24, 125.52, 123.99, 120.67, 118.68, 70.94, 58.34, 20.82.IR (KBr, cm 3340, 2924, 2229, 1512, 1107, 1011, 894, 806.LC-ESMS m/z 377.25 [M+H]+HPLC Purity: 100 %.
5-(4-(2-methoxyethylamino)-6-(p-tolylamino)-l, 3, 5-triazin-2-yIamino)
picolinonitrile hydrochloride (16c)
Figure imgf000025_0002
16c
Ή NMR (300 MHz, DMSO-i¾ S: 9.87 (s, 1H), 9.37 (s, 1H), 9.23 (br. s., 1H), 8.56 (m, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.35 (br. s., 1H), 7.11 (d, J = 8.3 Hz, 2H), 3,55 (s, 4H), 3.25(s, 3H), 2.25(s, 3H); 13C NMR (300 MHz, DMSO-<&) S: 166.13, 164.45, 142.55, 141.23, 137.69, 131.40, 129.51, 129.24, 125.52, 123.99, 121.33, 120.67, 118.68, 70.94, 58.34, 20.82; LC-ESMS m/z 377.25 [M+H]+.
5-(4-(phenylamino)-6-(pyr ino) picolinonitrile :-
Figure imgf000025_0003
Ή NMR (300 MHz, DMSO): δ = 9.65 (s, 1H), 9.23 (s, lH), 8.62 (d, J = 2.3 Hz, 1H), 7.95 (d, J = 8.7 Hz, 1H), 7.74 (m, 2H), 7.56 (dd, J = 8.7, 2.3 Hz, 1H), 7.30 (m, 2H), 7.16(m, 1H), 3.44 (m, 4H), 1.91(m, 4H). HRMS (ESI): m/z [M] " calcd for C19H18N8: 358.4560; found: 357.4723.
(C ) Anti-HIV assay using TZM-bl cells: (Primary screening)
i) Solubility and preparation of working stock: The compounds SI (5-(4-chloro- 6-(mesityl amino)- l,3,5-triazin-2-yl amino) picolinonitrile hydrochloride and S2 (5-(4-amino-6-(mesityl amino)- 1 ,3,5-triazin-2-ylamino) picolinonitrile hydrochloride were dissolved in the solvent DMSO. The working stocks of required concentration were prepared in growth medium, filtered and used for the respective assays.
ii) Cytotoxicity assay: Both compounds SI and S2 were first tested for cytotoxicity using a series of concentrations and the toxicity was determined using MTT assay. CC50 (concentration showing 50% cytotoxicity) value was determined.
iii) Anti-HIV 1 activity: The anti-HIVl activity was assessed using sub-toxic concentrations (concentrations showing > 50% cell viability) of AZT (control), and the compound SI and S2. The activity was tested against CXCR4 (HIV- im) and CCR5 (HIV- 1 Adas) tropic lab adapted strains. The compounds SI and S2 showing inhibition against lab adapted strains were further confirmed against CXCR4 (HIV-1 uG07o) and CCR5 (HIV-1VB59) tropic primary isolates.
Cell Associated assay: The sub toxic concentrations were added to TZM-bl cells previously infected with pre titrated virus. After 48 hours, the RLU were measured and percent inhibition and IC50 (Concentration showing 50% inhibition) values were calculated. Results are tabulated in Table 1.
The 2nd batch of compounds SI and S2 was requested in a coded manner and tested in the TZM-bl assay. Both compounds SI and S2 showed reduction of HIV growth upon retesting.
II. Anti-HIV assay using PBMCs; (Secondary screening)
i) Cytotoxicity assay: The compounds SI and S2 were first tested for cytotoxicity using a series of concentrations in PBMCs. The viability was determined by MTT assay and the CC50 values were calculated. ii) Anti-HIV assay using PBMC: The sub toxic concentrations of the compounds SI and S2 were added to PBMCs previously infected with pre titrated virus (HIV- 1VB5I). After 5 days, the inhibition of virus growth was monitored by inhibition of p24 antigen levels in culture supernatant. IC50 values were calculated. Each experiment was carried out in duplicate on three separate occasions. Results are tabulated in Table 2.
III. Results:
Two compounds SI (5-(4-chloro-6-(mesityl amino)- l,3,5-triazin-2-yl amino) picolinonitrile hydrochloride and S2 (5-(4-amino-6-(mesityl amino)-l,3,5-triazin-2- ylamino) picolinonitrile hydrochloride were found to inhibit cell associated CXCR4 tropic HIV-luiB, HIV-1UGO70, CCR5 tropic HIV- da5, HIV-1VB59 in TZM-bl assay and HIV-1VB5I in PBMC assay to maximum level. Both compounds SI and S2 exhibited enhanced activities against the strains CXCR4 tropic HIV- I JIIB , HIV-1UGO70, CCR5 tropic HIV-1 Adas, HIV-1VB59 and also are more effective than the known antiviral AZT (Atazanavir).
Table 1: Anti-HIV activity against CCR4 tropic HIV-I HIB, HIV-1UGO70 and CCR5 tropic HIV- l Ada5, HIV-1VB59 strains in TZM-bl assay (Primary screening)
Figure imgf000027_0001
(Control) (SD) (±0.0005) (±0.001) (±0.001) (±0.005)
TI 198182 46029 117689 34352
(SD) (±34326) (±2498) (±474908) (±7474)
CC50 (Concentration showing 50% viability): Determined by fitting logarithmic trend line to the data.
IC50 (Concentration showing 50% inhibition): Average of two independent
experiments. Determined by using Luc software
TI: Therapeutic Index (CC50 / IC5o). ( ) Standard deviation
Table 2: Anti-HIVl activity against CCR5 tropic HIV-IVB SI in PBMC assay (Secondary screening)
Figure imgf000028_0001
CC50 (Concentration showing 50% viability): Determined by fitting logarithmic trend line to the data.
ICso (Concentration showing 50% inhibition) Determined by fitting logarithmic trend line to the p24 data
TI: Therapeutic Index (CC50 / IC50)
Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.
Industrial Advantage:
The present invention relating to novel triamino triazine picolinonitriles of formula-I or its pharmaceutically acceptable addition salts or enantiomers thereof or mixtures of enantiomers or stereoisomers or racemates or solvates or hydrates are useful in treating, or reducing the severity of hyper proliferative diseases by inhibiting metastasis, or in the treatment or prevention of HIV infections. Further, the compounds of formula -I as antagonists of chemokine CXCR4 receptor exhibit enhanced activity and reduced toxicity and are more effective than the known antiviral AZT (Atazanavir) as shown hereinabove.

Claims

We claim;
1. Triamino triazine picolinonitriles of formula- I or its pharmaceutically acceptable addition salts or enantiomers or mixtures of enantiomers or stereoisomers or racemates or solvates or hydrates comprising ;
Figure imgf000030_0001
(I)
wherein, Y and Y' represent NH,
'Z' independently is selected from hydrogen, halogen, NH2, NMe2, OMe, NH-Ar, morpholine, piperidine, pyrolidine, NHR, COOH or COOR;
Ri, R2 and R3 independently are selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halogen, OH, OR, NH2, NHR, SR, COOH, COOR; 'R' independently is selected from straight or branched alkyl, cyclic alkyl or aralkyl, aryl or heteroaryl groups.
2. The triamino triazine picolinonitriles according to claim 1 , comprising;
i. 5-(4-chloro-6-(p-tolylamino)-l , 3, 5-triazin-2-ylamino) picolinonitrile ; ii. 5-(4-chloro-6-(3-methoxyphenylamino)- 1 ,3,5-triazin-2-ylamino)picolino nitrile;
iii. 5-(4-amino-6-(p-tolylamino)-l,3,5-triazin-2-ylamino)picolinonitrile;
iv. 5-(4-amino-6-(3-methoxyphenylamino)-l,3,5-triazin-2-ylamino)picolino nitrile;
v. 5-(4-amino-6-(mesitylamino)-l, 3, 5-triazin-2-ylamino) picolinonitrile; vi. 5-(4-morpholino-6-(p-tolylamino)-l,3,5-triazin-2-ylamino)picolinonitrile; vii. 5-(4-(4-fluorophenylamino)-6-morpholino-l,3,5-triazin-2-ylamino)
picolino nitrile;
viii. 5-(4-(3-methoxyphenylamino)-6-(piperidin- 1 -yl)- 1 ,3,5-triazin-2yl
amino) picolinonitrile;
ix. 5-(4-(3-methoxyphenylamino)-6-(pyrrolidin- 1 -yl)- 1 ,3 ,5-triazin-2ylamino) picolinonitrile; x. 5-(4-(2-methoxyethylamino)-6-(p-tolylamino)-l,3,5-triazin-2ylamino) picolinonitrile;
xi. 5-(4-(phenylamino)-6-(pyrrolidin-l-yl)-l,3,5-triazin-2ylamino)
picolinonitrile;
xii. 5-(4-(2-methoxyethylamino)-6-(phenylamino)- 1 ,3,5-triazin-
2ylamino)picolino nitrile;
xiii. 5-(4-(phenylamino)-6-(piperidin- 1 -yl)- 1 ,3,5-triazin-2-ylamino)
picolinonitrile;
xiv. 5-(4-(phenylamino)-6-(morpholine- 1 -yl)- 1 ,3,5-triazin-2 -ylamino)
picolinonitrile;
xv. 5-(4-amino-6-(phenylamino)-l,3,5-triazin-2- ylamino) picolinonitrile; xvi. 5-(4-chloro-6-(mesityl amino)-l,3,5-triazin-2-ylamino) picolinonitrile.
3. The triamino triazine picolinonitriles according to claim 2, comprising; i. 5-(4-chloro-6-(mesityl amino)-l ,3,5-triazin-2-ylamino) picolinonitrile; ii. 5-(4-amino-6-(mesitylamino)-l, 3, 5-triazin-2 -ylamino) picolinonitrile
4. A process for preparation of compounds of formula-I or its pharmaceutically acceptable addition salts according to claim 1, comprising catalytic coupling of 5- (4-chloro-6-(phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile of formula-5 with a suitable reagent in presence of a base, ligand as promoter and solvent at an ambient temperature in the range of 50-80°C;
Figure imgf000031_0001
wherein 'Z' independently is selected from hydrogen, halogen, NH2, NMe2, OMe, NH-Ar, morpholine, piperidine, pyrolidine, NHR', COOH or COOR'; and
R independently is selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, acyl, halogen, OH, OR, NH2, NHR', SR', COOH, COOR'; wherein R' is selected from straight or branched alkyl, cyclic alkyl, aralkyl, aryl or heteroaryl groups.
5. The process according to claim 4, comprising catalytic coupling of 5-(4-chloro-6- (phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile of formula-5(a-e) with aqueous ammonia in presence of a base, ligand as promoter and solvent at an ambient temperature in the range of 50-80°C to obtain compounds of formula 7(a- e) and optionally converting to acid salts thereof;
Figure imgf000032_0001
5(;t-M R = Sill 4- : 5ti) -OMe; ¾ I
7(l 5-0>fK ¾) 2.4,i -CH
6. The process according to claim 4, comprising catalytic coupling of 5-(4-chloro-6- (phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile of formula-5(a-e) with piperidine in presence of a base, ligand as promoter and solvent at an ambient temperature in the range of 50-80°C to obtain compounds of formula 10(a-e) and optionally converting to acid salts thereof.
(*"¾ f*' ~»" »S¾ tec; wtvwjt χ χ ^"
i - I Hi> «;: l lb) J-OMfe UcM- CItj: i ttli 'J-OMe: t ki 2A6-CH,.
7. The process according to claim 3, comprising catalytic coupling of 5-(4-chloro-6- (phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile of formula-5(a-e) with morpholine in presence of a base, ligand as promoter and solvent at an ambient temperature in the range of 50-80°C to obtain compounds of formula l l(a-e) and optionally converting to acid salts thereof.
Figure imgf000032_0002
li Vt -i- Me. ¾M-Ol ,; %ij »-<)Μκ; ¾·! 3,4,ή-Π ;.
8. The process according to claim 3, comprising catalytic coupling of 5-(4-chloro-6- (phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile of formula-5(a-e) with pyrolidine in presence of a base, ligand as promoter and solvent at an ambient temperature in the range of 50-80°C to obtain compounds of formula 13(a optionally converting to acid salts thereof.
Figure imgf000033_0001
y!iCiite y,(..ei: <w¾ 1 l.JcJ - tHji JSJ> .H> e; l ¾) 2A(-i'Hi.
9. The process according to claim 3, comprising catalytic coupling of 5-(4-chloro-6- (phenylamino)-l,3,5-triazin-2-ylamino) picolinonitrile of formula-5(a-e) with 2- methoxyethanamine in presence of a base, ligand as promoter and solvent at an ambient temperature in the range of 50-80°C to obtain compounds of formula 15(a-e) and optionally converting to acid salts thereof.
,.
Figure imgf000033_0002
«::«5 R »R ».*: ?I<! * ifei; ] l-i:H!; J-mcihtsvci!uninmitiir
1 ' ' R i&M-F: Ι 5Ιϊ) 4-ΟΜ¾; 1 Ϊ) 4 ¾: i¾l) 34") e: I Sc) JA6- H,.
10. The process according to any of the preceding claims, wherein, the catalyst is selected from copper catalyst such as Cu(I) chloride, Cu(I) iodide, CuS04.5H20, [CuBr(PPh3)2] and the like.
11. The process according to any of the preceding claims, wherein, the ligand is selected from 1,10-phenanthroline, picolinic acid, 4-hydroxy-proline or N,N- dimethylglycine.
12. The process according to any of the preceding claims, wherein, the solvent is selected from polar aprotic solvent such as DMSO, acetonitrile, DMF, DCM and the like.
13. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or its pharmaceutically acceptable addition salts along with pharmaceutically acceptable excipients.
14. The triamino triazine picolinonitrile of formula- I or its pharmaceutically acceptable addition salts according to claim 1 for use in treating or reducing the severity of hyper proliferative diseases by inhibiting metastasis, or for treating or preventing HIV infections with reduced toxicity.
15. A method for treating or reducing the severity of hyper proliferative diseases by inhibiting metastasis, or for treating or preventing HIV infections with reduced toxicity comprising administering therapeutically effective amount of a compound of formula (I) or its pharmaceutically acceptable addition salts to a subject in need thereof.
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