Synthesis of tryptoline-3-carboxylic Acid derivatives a novel antidiabetic agent.

The compounds, 2-(methylsulfonyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylic acid (DM3), 2-(phenylsulfonyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylic acid (DM(4)), and 2-(p-toluenesulfonyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylic acid (DM(5)) were synthesized by coupling of 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylic acid (DM(2)) with methanesulfonyl chloride, benzenesulfonyl chloride, and toluenesulfonyl chloride, which in turn, was synthesized by dissolving dilute aqueous ammonia with 2-(N-hydroxy methyl amino)-indol-3-yl-propanoic acid (DM(1)) which is the reaction product of l-tryptophan and formalin. All the intermediates and title compounds were characterized by physical, chemical, analytical, and spectral data. All the title compounds have been screened for in vivo antidiabetic activity in streptozotocin-induced diabetic rats, and serum glucose was estimated spectrophotometrically at 505 nm by glucose oxidase/peroxidase method. Compound DM(5) showed potent antidiabetic activity.

INTRODUCTION

Diabetes mellitus is a major health concern, especially in the urban world.[1] Over 90% of the diabetes mellitus patients are type-2 patients.[2] Type-2 diabetes mellitus is characterized by insulin resistance and cardiovascular dysmetabolic syndrome.[3] The conventional therapy of type-2 diabetes mellitus has not been satisfactory as it is not successful in treating associated cardiovascular risk factors, which is the main cause of morbidity.[4] The current trend is therefore, to make therapy better by choosing appropriate combination of available drugs. A parallel search for newer drugs is also being made.

Thiazolidine-2,4-diones are the class of oral hypoglycemic agents which increase insulin sensitivity at target tissues such as liver and skeletal muscles. In addition, it also improves the markers of cardiovascular risk factors by decreasing the free fatty acids and altering the lipoprotein metabolism.[5] Thiazolidine-2,4-diones act on peroxisome proliferators activating receptor-γ (PPAR-γ) receptors which regulate the gene expression mainly in the adipose tissues.[6-8] Rosiglitazone and pioglitazone molecules from the class of thioglitazone available in the market are showing severe adverse effects.[910]

A survey of literature revealed that replacement of the thiazolidine-2,4-dione ring by various acidic groups such as α-heteroatom-substituted carboxylic acids and α-carbon-substituted carboxylic acids can results in qualitative and quantitative changes in the activity,[11-13] which prompted us to undertake the synthesis of various new derivatives with the aim of having improved activity and less toxicity.

MATERIALS AND METHODS

The melting points were recorded in open sulphuric acid or oil bath using thermometer and were uncorrected. IR spectra were recorded using Hitachi 270-30 infrared and Bruker Vector 22 spectrophotometers using KBr pellet techniques. 1H-NMR spectrum was recorded using DMSO-d 6 on Bruker Avance DPX-200 at 300 MHz, and their chemical shifts are recorded in δ (parts per million) units with respect to tetramethyl silane (TMS) as an internal standard. Atmospheric pressure ionization (API) mass spectra were recorded on Bruker Ion Trap Esquire 3000 spectrometer with the ionization potential 3000 V. Progress of the reactions was monitored using TLC, performed on aluminum plates precoated with silica gel-G, using chloroform–methanol (92:8) as the solvent systems, and the spots were visualized by exposure to iodine vapors. The physical constants of the title compounds are reported in Table 1

Table 1

Physical constants of intermediates and title compounds

Compound code	Molecular formula	Molecular weight	Yield, %	Melting point (ºC)	Solvent system chloroform:methanol	Rf values
DM1	C12H12N2O3	234.32	75	230–231	92:8	0.50
DM2	C12H12N2O2	216.23	80	312–313	92:8	0.72
DM3	C12H14N2O4S	294.32	70	240–242	92:8	0.62
DM4	C18H16N2O4S	356.39	78	262–263	92:8	0.69
DM5	C19H18N2O4S	370.42	74	274–275	92:8	0.67

Procedure for the preparation of 2-(N-hydroxymethyl amino)-indol-3-yl-propanoic acid (DM1)

In a 250-mL conical flask, 5 g (0.0245 M) of l-tryptophan was dissolved in 120 mL of water. To this, 20 mL formalin was added and the mixture was incubated at 38°C for 6 h. The colorless, crystalline, but rather granular solid that had then separated was collected, washed with cold water, and dried in vacuum. The crude product was recrystallized from hot water.

IR (KBr) (cm 3600–3400 (m) broad O–H stretching, 3300 (m) N–H stretching, 3099 (m) aromatic C–H stretching (asymmetric), 3056 (m) aromatic C–H stretching (symmetric), 2989 (m) aliphatic C–H stretching (asymmetric), 2852 (m) aliphatic C–H stretching (symmetric), 1680 (s) carbonyl stretching 1620, 1552, 1447 (m) skeleton in plane vibrations C=C, 1408–1425 (s) C=C and C=N ring stretching, 750 (m) N–H wagging, 741 (s) out of plane C=C bending.

7.36–7.45 (m, 4H, ArH), 7.10 (s, 1H, α proton of indole ring), 3.70–3.80 (s, 1H, aromatic N-H, D2O exchangeable), 2.80 {d, 2H, –CH2 (Jvic = 7.2)}, 3.88 {t, 1H, –CH (Jvic = 7.2)}, 11.00 (s, 1H, COOH), 2.10 (s, 1H, NH, D2O exchangeable), 4.60 (s, 2H, –CH2), 4.10 (s, 1H, OH).

Mass spectra: Molecular ion peak M at m/e 234 (23.8%), [M–(OH)]+and [M–(H2O)]+shows peak at m/e 217 (32.4%), 216 (100%). The other important ions are at m/e 190 (10.5%), 158 (8.4%), 157 (11.8%), 156 (15.3), 144 (10.8%), 117 (18.6%), and 91 (8.5%).

Procedure for the preparation of 1,2,3,4-tetrahydro- 9 In a 250-mL conical flask, 150 mL of dilute aqueous ammonia was placed and 5 g (0.0214 M) of α-hydroxymethylamino-α-3-indolylpropionic acid (DM1) was added and dissolved. Then, this solution was refluxed for 3 h and concentrated to a small volume. The white-to-cream colored crystals obtained were given charcoal treatment and filtered by suction, washed with cold water and dried at 80°C. The product was obtained in colorless leaflets and was purified by recrystallization from hot water.

IR (KBr) (cm 3650–3300 (m) broad O–H stretching, 3350 (m) N–H stretching, 3100 (m) aromatic C–H stretching (asymmetric), 3056 (m) aromatic C–H stretching (symmetric), 2990 (m) aliphatic C–H stretching (asymmetric), 2850 (m) aliphatic C–H stretching (symmetric), 1690 (s) carbonyl stretching 1600, 1582, 1450 (m) skeleton in plane vibrations C=C, 1410–1430(s) C=C and C=N ring stretching, 760 (m) N–H wagging, 752 (s) out of plane C=C bending.

7.10–7.56 (m, 4H, ArH), 3.70–3.80 (s, 1H, aromatic N–H, D2O exchangeable), 2.80 {d, 2H, –CH2(Jvic = 7.2)}, 3.88 {t, 1H, CH (J = 7.2)}, 11.00 (s, 1H, COOH), 2.10 (s, 1H, NH of piperidine ring, D2O exchangeable), 4.10 (s, 2H, CH2).

Mass spectra: Molecular ion peak M at m/e 216 (100%), [M–(OH)] +and [M–(C2H2)] +shows peak at m/e 199 (18.4%), 190 (27.2%). The other important ions are at m/e 177 (19.7%), 144 (14.3%), 118 (5.8%), 117 (20.5%), 91 (6.2%), 91 (5.8%).

Procedure for the preparation of 2-(methylsulfonyl)- 1,2,3,4-tetrahydro-9H-pyrido [3,4-b]indole-3- carboxylic acid (DM3)

Treated 3 g (0.01389 M) of the amine (DM2) with 50 mL of 10% sodium hydroxide solution and 2 mL (3.5 g, 0.0303 M) of methanesulfonyl chloride was added in small proportions. The reaction mixture was stirred vigorously for 30 min and warmed it slightly with continued shaking. When the odor of methanesulfonyl chloride was dissipated, the mixture was cooled in an ice bath. As oil separated, crystallization was induced by rubbing the inner wall of the beaker with the glass rod in an ice bath. Any solid produced should be separated by filtration; and the crude product was recrystallized from dilute ethanol to give pure DM3 as light brown crystals.

IR (KBr)(cm 3600–3400 (Broad O–H stretching), 3300 (N–H stretching), 3099 (aromatic C–H stretching asymmetric), 3056 (aromatic C–H stretching symmetric), 2989 (aliphatic C–H stretching asymmetric), 2852 (aliphatic C–H stretching symmetric), 1680 (carbonyl stretching), 1620, 1552, 1447 (skeleton in plane vibrations C=C), 748 (N–H wagging), 741 (s) out of plane C=C bending, 1330 (s) SO2 stretching asymmetric, 1120 (s) SO2 stretching symmetric, 560 (s) SO2 rocking, 530 (s) SO2 scissoring.

7.10–7.40 (m, 4H, ArH), 3.70–3.80 (s, 1H, aromatic N–H, D2O exchangeable), 2.80 {d, 2H, –CH2 Jvic = 7.2)}, 3.88 {t, 1H, –CH Jvic = 7.2)}, 11.00 (s, 1H, COOH), 2.90 (s, 3H, –SO2CH3), 3.80–3.85 (m, 2H, –CH2).

Mass spectra: Molecular ion peak M at m/e 294 (43.9%), [M–(OH)] +shows peak at m/e 277 (26.2%). The other important ions are at m/e 249 (10.8%), 216 (100%), 144 (12.2%), 118 (6.4%), 117 (20.5%), 107 (6.2%), 91 (16.5%), 79 (8.4%).

Procedure for the preparation of 2-(phenylsulfonyl)- 1,2,3,4-tetrahydro-9H-pyrido [3,4-b] indole-3- carboxylic acid (DM4)

To a 100-mL round bottom flask was added 3 g (0.01389 M) of amine (DM2), 20 mL of dry pyridine, and 2 mL (2.75 g, 0.01559 M) of benzene sulfonylchloride. The reaction mixture was refluxed for 2 h on heating mantle and poured into crushed ice to give a light-brownish solid. As oil separated, crystallization was induced by rubbing the inner wall of the beaker with the glass rod in an ice bath. Any solid produced should be separated by filtration, and the crude product was recrystallized from dilute ethanol to give pure DM4 as light brown crystals.

IR (KBr) (cm 3600–3400 (m) (broad O–H stretching), 3300 (m) (N–H stretching), 3099 (m) (aromatic C–H stretching asymmetric), 3056 (m) (aromatic C–H stretching symmetric), 2989 (m) (aliphatic C–H stretching asymmetric), 2852 (m) (aliphatic C–H stretching symmetric), 1680 (s) (carbonyl stretching), 1620, 1552, 1447 (m) (skeleton in plane vibrations –C=C), 1408–1425 (s) (C=C and C=N ring stretching), 750 (N–H wagging), 741 (s) out of plane C=C bending, 1338 (s) SO2 stretching asymmetric, 1118 (s) SO2 stretching symmetric, 565 (s) SO2 rocking, 532 (s) SO2 scissoring.

H 7.05–7.10 (m, 4H, ArH), 3.70–3.80 (s, 1H, aromatic N–H, D2O exchangeable), 2.80 {d, 2H, –CH2 (Jvic = 7.2)}, 3.86 {t, 1H, –CH (Jvic = 7.2)}, 11.00 (s, 1H, COOH), 7.95 (d, 2H, o-protons of ArH with respect to SO2 group), 7.30–7.50 (m, 3H, ArH with respect to SO2), 4.55–4.60 (m, 2H, –CH2).

Mass spectra: Molecular ion peak M at m/e 356 (43.9%), [M–(OH)] +at m/e 339 (18.8%). The other important ions are at m/e 330 (21.2%), 311 (13.5%), 216 (100%), 169 (16.44%), 144 (20.54%), 141 (6.5%), 118 (22.4%), 117 (23.7%), 91 (8.5%), 77 (9.4%), 51 (6.2%)

Procedure for the preparation of 2-(p-toluenesulfonyl)- 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3- carboxylic acid (DM5)

Treated 3g (0.01389 M) of the amine (DM2) with 50 mL of 10% sodium hydroxide solution and 3 g (0.01574 M) of p-toluenesulfonyl chloride was added in small proportions. The reaction mixture was stirred vigorously for 30 min and warmed it slightly and shaking was continued. When the odor of p-toluenesulfonyl chloride was dissipated, the mixture was cooled in an ice bath. As oil started separating, crystallization was induced by rubbing the inner wall of the beaker with the glass rod in an ice bath. Any solid produced should be separated by filtration, and the crude product was recrystallized from dilute ethanol to give pure DM5 as light brown crystals.

IR (KBr) cm 3600–3400 (m) (broad O–H stretching), 3300 (m) (N–H stretching), 3099 (m) (aromatic C–H stretching asymmetric), 3056 (m) (aromatic C–H stretching symmetric), 2989 (m) (aliphatic C–H stretching asymmetric), 2852 (m) (aliphatic C–H stretching symmetric), 1680 (s) (carbonyl stretching), 1620, 1552, 1447 (m) (skeleton in plane vibrations C=C), 1408–1425 (s) (C=C and C=N ring stretching), 748 (m) (N–H wagging), 740 (s) out of plane C=C bending, 1328 (s) SO2 stretching asymmetric, 1122 (s) SO2 stretching symmetric, 560 (s) SO2 rocking, 532 (s) SO2 scissoring.

7.05–7.10 (m, 4H, ArH), 3.70–3.80 (s, 1H, aromatic N–H, D2O exchangeable), 2.80 {d, 2H, –CH2 (Jvic = 7.2)}, 3.88 {t, 1H, –CH (Jvic = 7.2)}, 11.00 (s, 1H, COOH), 7.80 (d, 2H, o-protons of ArH with respect to SO2 group), 7.30–7.45 (m, 2H, m-protons of ArH with respect to SO2 group), 2.45 (s, 3H, –CH3), 4.55–4.62 (m, 2H, –CH2).

Mass spectra : Molecular ion peak M at m/e 370 (44.2%) and [M–(OH)] + shows peak at m/e 353 (11.9%). The other important ions are at m/e 330 (21.4%), 325 (13.9%), 216 (100%), 183 (6.44%), 155 (20.5%), 144 (14.2%), 118 (9.2%), 117 (22.88%), 91 (8.5%), 91 (9.4%).

Evaluation of antidiabetic activity

The synthesized compounds were screened for in vivo antidiabetic activity in streptozotocin-induced diabetic rats. After administration of standard drug (pioglitazone) and synthesized compounds (DM1–5), serum glucose was estimated spectrophotometrically at 505 nm by glucose oxidase/peroxidase method using a commercially available kit (Span Diagnostic Ltd, Surat, India).

Concentration of glucose (mg/dL) = Optical density of test/Optical density of std. × 100.

The results are summarized in Table 2.

Table 2

Antidiabetic activity of synthesized compounds against streptozotocin-induced diabetes in rats

Groups	Blood glucose concentration (mg/dL)
DM1	176
DM2	179
DM3	154
DM4	167
DM5	145
Control	118
Streptozotocin treated	187
Pioglitazone treated	135

RESULTS AND DISCUSSION

The structures of synthesized compounds were confirmed by thin layer chromatography (TLC), m.p, IR, 1H-NMR, and mass spectrometry (MS) spectral analysis. The compounds (DM3–DM5) were synthesized by the treatment of l-tryptophan with formaldehyde which resulted in the formation of an intermediate α-hydroxymethylamino-α-3-indolylpropionic acid (DM1) which after refluxing with ammonia solution for 3 h resulted in cyclized product DM2 (Scheme 1). The compound DM2 on further treatment with methanesulfonyl chloride, benzenesulfonyl chloride, and toluenesulfonyl chloride yields different 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylic acid derivatives (Scheme 2). The yield was found to be in range of 70–80%. The title compounds were confirmed by IR spectral data showing characteristic bands at 3650-3300 cm -1corresponding to OH stretching, sharp band at 1690 cm-1 indicated the presence of C=O group. Compounds (DM3–DM5) were confirmed by stretching at 1330 and 1120 cm -1due to the presence of the SO2 group. Compounds DM1–DM5 was confirmed by 1H-NMR spectral analysis. The NMR proton singlet peak at δ 11 ppm and 3.70–3.80 revealed the presence of carboxylic acid and aromatic N–H groups. Further appearance of molecular ion peak M at m/e 294 (43.9%) 356 (43.9%), and 370 (44.2%) confirmed the structures of compounds (DM3–DM4). The compounds DM1 and DM2 do not significantly decrease blood glucose level in streptozotocin-induced diabetes. Compounds DM3 and DM4 exhibited less activity, whereas compound DM5 i.e., was found to possess better antidiabetic activity.

Synthesis of DM2

Synthesis of DM3, DM4 and DM5

CONCLUSIONS

The yield of all 1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylic acid derivatives were found to be in the range 70-80%. The purity of compounds was ascertained by meting point and TLC. The assigned structures were further established by IR, 1H-NMR, and mass spectral studies. The antidiabetic activity of the synthesized compounds was screened using streptozotocin-induced diabetes in rats. Pioglitazone was used as a standard drug. Compounds DM3 [2-(methylsulfonyl)-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole-3-carboxylic acid] and DM4 [2-(phenylsulfonyl)-1,2,3,4-tetrahydro-9H-pyrido [3,4-b]indole-3-carboxylic acid] showed moderate activity, whereas compound DM5 [2-(p-toluenesulfonyl)-1,2,3,4-tetrahydro-9H-pyrido [3,4-b]indole-3-carboxylic acid] exhibited the highest antidiabetic activity.

From this study, it may be concluded that the 1,2,3,4-tetrahydro-9H-pyrido [3,4-b]indole-3-carboxylic acid compounds can be potentially be developed into useful antidiabetic agents that can prompt future researchers to synthesize a series of 1,2,3,4-tetrahydro-9H-pyrido [3,4-b]indole-3-carboxylic acid derivatives containing a wide variety of substituents with the aim of obtaining novel compounds with enhanced activity.