Procedure:

General procedure for synthesis of derivatives (1 to 9)

Synthesis of pyrazine-2-ester (Stage -I) :

Taken 0.0805 M of pyrazine-2-carboxylic acid(starting material) and 100ml of Methanol, Stirred for 10 min at RT under nitrogen atmosphere and cooled to <15°c then slowly added 0.161M of thionyl chloride drop wise about 30 min under the same conditions. Later refluxed at 60-65°c for 2.30 hrs and completion of reaction was conformed by TLC (M.P – 30% methanol-DCMand 2drops of NH₃). Reaction mass was distilled under vaccum <50°c ST-I compound was extracted with DCM and dried.⁴

Synthesis of pyrazinamide (Stage –II) :

Taken 100ml of Methanolic Ammonia, cooled to -5 to 0°c and Slowly added 0.0762 M of pyrazine-2-ester (st -1) compound. Kept for stirring at -5 to 0°c for 2hrs completion of reaction was confirmed by TLC (M.P – Ethyl acetate). Reaction mass was distilled under vacuum <50°c and co-distilled with methanol and dried.

Synthesis of Amino pyrazine (Stage -III) :

Taken 0.4 M of NaOH, 125 ml of water and 0.2030 M of pyrazinamide (st-II) and added 0.22 M of NaOCl drop wise. kept for stirring at 10°c about 45 min. Then raise the temperature to RT and kept for reflux at 75-80°c for 2 hrs. Completion of reaction was confirmed by TLC (M.P– 30% methanol-DCMand 2drops of NH₃). Reaction mass was distilled under vaccum <50°c and extract with ethyl acetate and dried.

Synthesis of Imidazo[1,2-a] pyrazine (Stage -IV)

Taken 0.4731 M of 2-amino pyrazine (st-III) and 0.7097 M of chloro- acetaldehyde.Then slowly add 0.7097 M of sodium bicarbonate at room temperature. Kept reflux for 26 hrs at 98-100⁰C. completion of reaction was confirmed by TLC (M.P– 0.75% methanol-DCM). Filter it , saturated with potassium carbonate and extract with DCM.

Synthesis of Tetra hydro Imidazo[1,2-a] pyrazine (Final compound stage- V).

Taken 0.0840 M of imidazo(1,2-a) pyrazine,100 ml of methanol and 1 gm of 10% palladium on carbon in to vessel present in autoclave.15 kgs/cm² of hydrogen gas was passed over reaction mixture for reduction of duoble bonds in pyrazine ring at 50⁰C for 3 hrs. Completion of reaction was confirmed by TLC (M.P– 10% methanol-DCM).Reaction mixture was filtered and distilled. It was UV inactive

Synthesis of derivatives (I-IX) :

Taken 0.016 M of tetra hydro imidazo(1,2-a) pyrazine. 0.032 M of tri ethyl amine (TEA) 20ml of DCM. Then slowly added 0.032 M of different substituents. Stirring was kept at room temperature for 2 hrs. completion of reaction was conformed by TLC (M.P – 10% methanol-DCMand 2drops of NH₃). Reaction mass was distilled under vaccum <50°c.Derivatives were purified by re crystallization and column Chromatography.

From the above experimental procedure the following compounds (1-9) has been synthesized.

· tert-butyl 5,6-dihydroimidazo[1,2-a]pyrazine-7(8H)-carboxylate

· 5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl(phenyl)methanone

· (2-chlorophenyl)(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)methanone

· 5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl(2-fluorophenyl)methanone

· (4-bromophenyl)(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)methanone

· 1-(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)propan-1-one

· 7-[(4-nitrophenyl)sulfonyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine

· 1-(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)ethanone

· 1-(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)-2,2-dimethylpropan-1-one

Identification and Physical Characterization:

Compound code

Mol. Formula

Mol. Wt

Solubility

State

Melting Point

% yield

R_f

value

Column purification

IP₁

C₁₁H₁₇N₃O₂

223.27

Chloroform

DCM

Semisolid

77%

0.85

50% Ethyl acetate-Hexane

IP₂

C₁₃H₁₃N₃O

227.26

Chloroform

DCM

Semisolid

83%

0.79

5% methanol-DCM

IP₃

C₁₃H₁₂N₃OCl

262.76

Chloroform

DCM

Semisolid

66%

0.81

Ethyl acetate

IP₄

C₁₃H₁₂N₃OF

245.25

Chloroform

DCM

Semisolid

50%

0.84

3% methanol-DCM

IP₅

C₁₃H₁₂N₃OBr

307.16

Chloroform

DCM

solid

145 -146.9^₀C

98%

0.82

Recrystallisation-Ethyl acetate-Hexane (1:2)

IP₆

C₉H₁₃N₃O

179.22

Chloroform

DCM

Semisolid

68%

0.76

2% methanol-DCM

IP₇

C₁₂H₁₂N₄O₄S

308.30

Chloroform

DCM

solid

188 -190.2^₀C

92%

0.80

Ethyl acetate

IP₈

C₈H₁₁N₃O

163.17

Chloroform

DCM

solid

124 – 132^₀C

59%

0.85

IP₉

C₁₁H₁₇N₃O

207.27

Chloroform

DCM

solid

112 – 116 ^₀C

60%

0.88

2% methanol- DCM

pyrazinamide (Pyz-st-I) : IR (cm^-1 , KBr) : 1581.34 (C=C) , 1724.37(C=O) , 1526.19 (C=N),3015.77 (C-H) (Ar¹H NMR (400 MHz ) : Ar (3H) – 8.7- 9.3 , Ester (3H) - 4.0Mass (m/z) : 138.9.1 HPLC :100%

pyrazinamide (Pyz-st-II) :

IR (cm^-1 , KBr) :1377.81(C=C) , 1711.68(C=O) , 1609.98(C=N),3141.88(N-H), 3160.21 (C-H)¹H NMR (400 MHz ) : Ar (3H) – 8.7- 9.1 , N-H (2H) - 7.8.-8.2, Mass (n+1) :124.1 HPLC : 99.32%

2-amino pyrazine (Pyz-st-III) :

IR (cm^-1 , KBr) : 1426.86(C=C) , 1655.54(C=N), 3337.96(N-H), 3154.54 (C-H ¹H NMR (400 MHz ): Ar (3H) – 7.6- 7.8 , N-H (2H) - 6. Mass (n+1: 96.1)HPLC : 99.48%

imidazo(1,2-a) pyrazine (Pyz-st-IV)

IR (cm^-1 , KBr) : 1487.27(C=C) , 1616.03(C=N), 3328.27(C-N), 3136.03 (C-H)¹H NMR (400 MHz ) : Ar (5H) - 7.2-9.1 Mass (n+1) : 120.1HPLC : 100%

tetra hydro imidazo(1,2-a) pyrazine (Pyz-st-V)

IR (cm^-1 , KBr) : 1500.1(C=C) , 1655.08(C=N) , 2954.64 (C-H) (Ar)1302.02(C-N) , 3276.85 (N-H) , 2816.38 (C-H) (Ali)¹H NMR (400 MHz) : Ar(2H)- 6.7,7.2 . -CH₂ – 3.2-4.0,¹³C NMR : Ar-3C- 117-142 , -CH₂-3C-42-44 Mass : 124.1 HPLC : 96.07%

Compound1: tert-butyl 5,6-dihydroimidazo[1,2-a]pyrazine-7(8H)-carboxylate

IR (cm^-1 , KBr) : (C=C) 1413.43,(C-H) 3111.88, (C-H) 2977.15, (C=O) 1698.79, ( C-N) 1367.25, ( C=N ) 1499.39,¹H NMR (9H,s,-Aliphatic) 1.46, (6 H –CH₂) 3.82-4.67, (2H,Ar-H) 6.83-7.01,¹³C , (Ester C) 154, (Ar- 3C) 117-140, (CH₂ -3C) 40-44, (Ali-4C) 21-28, (Solvent-C) 76-77. Mass (n+1) : 224.3, HPLC: 90.52 %

Compound 2 :5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl(phenyl)methanone

IR (cm^-1 , KBr) (C=C) 1634.67, (C-H ) 3110.57,(C-H ) 2982.09,(C=O) 1730.93, (C-N) 1371.85, (C=N) 1423.21, ¹H NMR: (6 H -CH₂-)4.09-4.82, (7H,m,Ar-H) 6.87-7.45, ¹³C Ketone (C) 170, (Ar- 9C) 118-140, (CH₂-3C) 43, (Solvent-C) 76-77, Mass (n+1) : 228.1, HPLC : 91.03%

Compound 3 : (2-chlorophenyl)(5,6-dihydroimidazo [1,2-a]pyrazin-7(8H)-yl)methanon

IR (cm^-1 , KBr) (C=C) 1435.85, ( C-H ) 3009.02, (C-H)2936.21, (C=O) 1646.37, (C-N) 1286.48, (C=N) , 1593.78, C-Cl 752.04, ¹H NMR ( 6 H -CH₂₎- 3.6-4.5, (6H-Ar-H), 6.89-7.44, ¹³C NMR Ketone (C) 167, (Ar- 9C) 117-139, -(CH₂--3C) 39-45, (Solvent-C) 76-77, Mass (n) : 262.1, HPLC : 88.45 %

Compound 4: 5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl(2-fluorophenyl)methanone

IR (cm^-1 , KBr) (C=C) 1427.07, (C-H) 3010.28, (C-H) 2474.92, (C=O) 1643.04, (C-N) 1220.58, (C=N) 1453.43, (C-F) 754.23, ¹H NMR (6 H -CH₂-)3.74-4.66, (6H,m,Ar-H) 6.8-7.4, ¹³C NMR Ketone (C) 165, (Ar- 9C) 115-140, (-CH₂-3C) 39-45, (Solvent-C) 76-77, Mass (n+1) : 246.3, HPLC : 95.06%

Compound 5:(4-bromophenyl)(5,6-dihydroimidazo [1,2-a]pyrazin-7(8H)-yl)methanone

IR (cm^-1 , KBr) (C=C) 1422.36, (C-H) 3113.33, (C-H) 2836.86, (C=O) 1629.83,(C-N) 1360.74, (C=N) 1590.95, (C-Br) 586.10,¹H NMR (6 H -CH₂) 4.09-4.80, (6H,m,Ar-H) 6.8-7.6,¹³C NMR Ketone (C) 169, (Ar- 9C) 118-139, (CH₂-3C) 39-46, (Solvent-C) 76-77, Mass (n+1) : 306.1, HPLC : 97.63%

DISCUSSION OF RESULTS:

The present research involves the synthesis of series of 9 novel compounds of Tetra hydro imidazo[1,2-a] pyrazine. Here the Treatment of pyrazine-2-carboxylic acid with thionyl chloride in methanol yielded methyl pyrazine-2-carboxylate. Which on treatment with alcoholic ammonia gave pyrazinamide, further it was undergone Hoffman’s degradation and yielded 2-amino pyrazine , later it was treated with chloro acetaldehyde , sodium bicarbonate under reflux for 26 hrs, gave imidazo[1,2-a] pyrazine and hydrogen gas was passed on it in the presence of palladium on carbon yielded Tetra hydro imidazo[1,2-a] pyrazine , Which on further treatment with different substituents, 9 novel derivatives were obtained. Purification was done by recrystallization, column chromatography. Characterization of all derivatives were done by FT-IR , ¹H and ¹³C NMR , MASS and % purity was done by HPLC.

Intermediate compounds showed IR absorption at the region of C=O streching at 1724-1711cm^-1, C=N streching at 1699-1581cm^-1, C=C streching at 1426-1317cm^-1, N-H streching at 3276.85cm^-1. In ¹H NMR spectra delta values of intermediates compoundes were found in the range of 7-9 for aromatic protons, 13.7 for acid proton. Molecular weight of the compounds was confirmed by MASS spectroscopic studies.

Novel synthesized derivatives showed IR absorption at the region of C=O streching at 1629-1698cm^-1,C=N streching at 1499-1437cm^-1, C=C streching at 1634-1413cm^-1, C-N streching at 1367-1355cm^-1, C-H(Ar) streching at 3120-3000cm^-1, C-H(Ali) streching at 2990-2900cm^-1In ¹H NMR spectra delta values of compounds were found in the range of 6-8 for aromatic protons, 3-5 for -CH₂ - protons , 1-2 for aliphatic protons.¹³C NMR spectra values of compounds were found in the range of 128-140 for aromatic carbons , 40-45 for –CH₂ - carbons, 154 for ester , 165-176 for ketones , 21-27 for aliphatic carbons. Molecular weight of the compounds were confirmed by MASS spectroscopic studies.

Out of nine novel derivatives selected six pure (95-100%) compounds were selected for molecular docking , Q SAR studies and Biological evolution.¹²

Selected derivatives were docked by AUTO DOCK 4.2 version for theoritical prediction of Anti-oxidant activity using “Thioredoxin peroxidase B” as a target site, Ascorbic acid as a standard. Inhibitory activity of the most potent derivatives were explained by hydrogen bonding interactions, dipole- dipole interactions. Provide binding energy to the protein-ligand complex,the significance of a particular hydrogen bond to a particular protein-ligand complex was dependent on the geometry and distance of the bond , partial charges on the donor (or) acceptors. From the predicted IC₅₀ values of standard and synthesized derivatives, the order of anti-oxidant activity was found to be IP₇>IP₉> IP₈> IP₅>AA> IP₆> IP₁

So the compound IP₇was found to have highest anti-oxidant activity than standard and synthesized derivatives, significant inhibitory activity on “Thioredoxin peroxidase B” due to the more no.of hydrogen bonding interactions, dipole- dipole interactions and electron withdrawing groups. QSAR parametres like partition coefficient was studied, all showed partition coefficient below 2, From this result it was observed that, synthesised derivatives were hydrophilic in nature. They had polar surface area below 140 A⁰and passed ADME parameters.

The newly synthesized derivatives were evaluated for anti-oxidant activity by DPPH free radical scavenging assay method. Ascorbic acid as a standard. The effect of the standard and synthesized compounds were tested with different concentrations(25,50,75,100µg/ml) against free radicals produced by DPPH. The standard and selected derivatives were able to inhibit free radical production. The highest percentage inhibition of standard and synthesized derivatives (IP₇, IP_9,IP_8,IP_5,IP₆IP₁)were found to be 94.17% , 79.6% , 72.8%, 68.9%, 66%, 62.1%, 58.2% respectively.

The most effective one was IP₇(7-[(4-nitrophenyl)sulfonyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine) , due to the presence of nitro group at para position. The order of anti-oxidant activity of synthesized selected compounds against free radicals as follows

IP₇>IP₉> IP₈> IP₅> IP₆> IP₁

Thoeritical results of docking and experimental results were supporting for anti-oxidant activity. In both cases IP₇showed effective IC₅₀ values towards Anti-oxidant activity.

The newly synthesized selected compounds were screened for anti-diabetic activity by amylase inhibition assay method. Here acarbose (20, 40, 60, 80, 100 µg/ml)) taken as a standard and synthesized derivatives (50, 100, 150, 200, 250, 300 µg/ml) were tested against amylase enzyme activity. selected derivatives were able to inhibit amylase enzyme activity, The highest percentage inhibition of standard and synthesized derivatives(IP₅, IP_7,IP_9,IP_1,IP_6,IP₈)were found to be 72.06%, 61.04%, 59.62%, 56.92%, 53.18%, 51.92%, 48.26%. The most effective one was IP₅(4-bromophenyl) (5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)methanone) due to the presence of bromine, which is a e^-withdrawing group. The order of anti-diabetic activity of synthesized 6 selected compounds against alpha-amylase as follows

IP₅>IP₇> IP₉> IP₁> IP₆> IP₈

CONCLUSION AND FURTHER SCOPE OF RESEARCH:

New series of tetra hydro imidazo(1,2-a) pyrazine derivatives were synthesized by conventional methods. All the synthesized compounds were characterized by physical (TLC and M.P) and spectral data (IR , NMR, MASS). % of purity by HPLC. Computational methods like Docking and Q SAR studies were performed for selected derivatives IP₁ , IP₅ ,IP₆ , IP_{7 ,}IP₈ , IP₉ and explained molecular descriptors. Selected derivatives were screened for in-vitro Anti-oxidant and in-vitro anti-diabetic activity. Theoretically and practically the most effective anti-oxidant activity was shown by IP₇(7-[(4-nitrophenyl)sulfonyl]-5,6,7,8-tetrahydroimidazo[1,2-a]pyrazine) , due to the presence of nitro group. The better anti-diabetic activity was shown by IP₅(4-bromophenyl)(5,6-dihydroimidazo[1,2-a]pyrazin-7(8H)-yl)methanone) due to the presence of bromine which is a electronwithdrawing group.

The various pharmacophore groups present on tetra hydro imidazo(1,2-a) pyrazine moiety may results more potent activities than existing ones. Further research need to be carried out on IN VIVO studies of anti-oxidant and anti-diabetic activities and to know the relationship between biological activity and pharmacophore groups. There is further scope to study other Q SAR parameters like electronic and stearic parameters.

ACKNOWLEDGEMENTS:

I much thankful to our beloved Secretary Sri C. Gangireddy Garu - who provides such wonderful plat form to do this work , Principal Annamacharya College of Pharmacy Dr. C. Gopinath for his encouragement.

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Received on 17.04.2016 Modified on 04.05.2016

Asian J. Research Chem 9(6): June 2016; Page 271-276

DOI: 10.5958/0974-4150.2016.00044.4