M. Himaja et al.

IRJP 1 (1) 2010 436-440

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY

Available online http://www.irjponline.com
Research Article

SYNTHESIS AND BIOLOGICAL EVALUATION OF INDOLE-3-CARBOXYLIC

ACID DERIVATIVES OF AMINO ACIDS AND PEPTIDES
M. Himaja*1, Tesmine Jose1, M.V.Ramana2, Ranjitha Anand1 and Munirajasekhar. D1
1
Department of Pharmaceutical Chemistry, School of Advanced Sciences, VIT University,
Vellore 632 014, India
2
Department of Pharmacy, Aljabal Algharbi University, Zawia, Libya, SA

*Dr (Mrs).M.Himaja, Professor, Pharmaceutical Chemistry Division, School of Advanced Sciences

VIT University, Vellore-632014.Tamil Nadu, India. E-mail:[email protected]
Article Received on: 15/11/10 Revised on: 30/11/10 Approved for publication: 10/12/10

ABSTRACT
A novel series of Indole-3-carboxylic acid derivatives of amino acids and peptides were synthesized by
solution phase technique. The synthesized compounds were characterized by FTIR, 1H NMR and mass
spectral analysis and evaluated for their antibacterial and anthelmintic activities. The compounds
exhibited significant antibacterial and anthelmintic activities as compared to standard drugs Clotrimazole
and mebendazole, respectively.

KEYWORDS: Indole-3-carboxylic acid, amino acids/peptides, antibacterial activity.

INTRODUCTION
Indoles and their derivatives are found to be associated with various biological activities such as
anticancer1, antibacterial2, antifungal3, anthelmintic4 and antiinflammatory5,6 activities. In view of the
diverse biological activities associated with indoles, we wish to report the synthesis and antibacterial and
anthelimintic activities of amino acids and peptides incorporated with Indole-3-carboxylic acid. The
molecule with Indole-3-carboxylic acid derivatives of amino acids and peptides were synthesized by
using DCC/Et3N mediated solution phase technique of peptide synthesis. The acid group was protected
by esterification process. The Boc-amino acids were coupled with amino acid methyl ester hydrochlorides
by dicyclohexylcarbodiimide (DCC) as a coupling agent and triethylamine (Et3N) as a base to get
protected dipeptides. The with Indole-3-carboxylic acid was coupled with Boc-dipeptides using DCC to
get with Indole-3-carboxylic acid derivatives of amino acid and dipeptide followed by hydrolysis of Boc-
group with trifluoroacetic acid.

MATERIALS AND METHODS

All the reactions requiring anhydrous conditions were conducted in flame dried apparatus.The amino
acid used are L-amino acid, except D-alanine, purchased from Spectrochem Private Limited, Mumbai,
India. Solvents and reagents were purified by standard methods. Boc-amino acids, amino acid methyl
ester hydrochlorides and nitro-arginine were prepared by standard procedures7. N-methylated amino acids
were prepared using NaH/CH3I by Benoiton method8. Organic extracts were dried over anhydrous
sodium sulphate. Melting points were determined by an open capillary method and are uncorrected. The
completion of the reaction and purity of the compounds were checked by thin layer chromatography. IR
spectra were recorded on Nicolet impact 400 FT/IR spectrometer using KBr pressed pellet technique. 1H
NMR spectra were recorded on GEOL-JMS D–300 (MHz) NMR spectrometer. MASS spectra were
recorded on Shimandzu GC-MS (at 70 eV) Mass Spectrometer using xenon as the carrier gas.

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Preparation of the Dipeptides

Amino acid methyl ester hydrochloride (10mmol) was dissolved in chloroform (20ml). To this,
Triethylamine (1.3ml) was added at 00C and the reaction mixture was stirred for 15 minutes. Boc-amino
acid (10mmol) in CHCl3 (20ml) and DIPC (Diisopropylcarbodiimide) (10mmol) were added with stirring.
After 24 hours, the reaction mixture was filtered. The filtrate was washed with 5% NaHCO3 (20ml), 5%
HCl (20ml) and distilled H2O (20ml). The organic layer was dried over anhydrous Na2SO4, filtered and
evaporated in vacuum. The residue was purified by recrystallization from CHCl3 and petroleum ether.
Synthesis of Indole-3-carboxyl derivatives of Amino acids/peptides9
To the amino acid/peptides (10mmol) in CHCl3 (25ml), Indole-3-carboxylic acid (10mmol), Et3N
(2.69 ml, 20mmol) and DIPC was added at 00C and stirred for 24 hours. The reaction mixture was
washed with 10% NaHCO3 until the byproduct was removed completely and finally washed with 5%
HCl (5ml). The organic layer was dried over anhydrous Na2SO4. Chloroform and Et3N were distilled off
to get the crude product of the cyclised compound, which was then recrystallised from CHCl3. To the
above (1.2mmol) in CHCl3 (15ml), LiOH (0.274 g, 2.4mmol) was added, stirred for 2 hour at room
temperature and washed with 10% NaHCO3 solution. The organic layer was dried over anhydrous
Na2SO4. The physical data of the synthesized compound is mentioned in the tabular column-1.
Antimicrobial activity10: The antimicrobial activity was determined using disc diffusion method by measuring the
inhibition zone in mm. All the synthesized compounds were evaluated in vitro for their antibacterial and antifungal
activities. The compounds were tested at a concentration of 50 μg/mL against bacterial strains (Enterococcus
faecalis and Escherichia coli) diffusion method, respectively. Ampicillin and fluconazole were served as standard
drugs for comparison of the results. The culture media used were nutrient agar and sabourd's medium for bacteria
and fungus strains, respectively. The results are presented in Table-3.
Anthelmintic activity11: The synthesized compounds were evaluated for their anthelmintic activity against
Eudrilus eugenia by Garg's and Atal method11 using mebendazole as a standard drug. All the synthesized
compounds were found to be potent anthelmintics. The results are given in Table-2.
Spectral Data
Compound-1: Indole-3-carboxyl phenylalanine methyl ester: IR (KBr Pallets): 3361.53(N-H stretch),
3020.41 (aromatic C-H stretch), 2934.32, 2859.61(aliphatic C-H stretch), 1665.04(C=O stretch (amide
cm-1. 1H NMR (300MHz, CDCl3): δ7.186 - 7.819 (11 H, m, aromatic –H), δ6.491 (1H, d, -NH) δ4.407
(1H, m, αH), δ3.797 (3H, s, -OCH3), δ3.393 (2H, d, β H) FABMass: m/z = 323.1.
Compound-2: Indole-3-carboxyl tyrosine methyl ester: 3243.60 (N-H stretch), 2926.59, 2855.06
(aliphatic C-H stretch ),1734.06 (C=O stretch(ester)), 1668.90 (C=O stretch(amide) cm-1. 1H NMR
(300MHz, CDCl3): δ7.188 - 7.649 (10 H, m, aromatic –H), δ4.407 (1H, m, α H), δ3.797 (3H, s, -OCH3),
δ3.393 (2H, d, β H).FABMass: m/z = 337.20.
Compound-3 : Indole-3-carboxyl glycine methyl ester: IR (KBr Pallets): 3321.03 (N-H stretch),
2931.55, 2857.37 (aliphatic C-H stretch ), 1747.13 (C=O stretch(ester)), 1667.31 (C=O stretch(amide)
cm-1. 1H NMR (300MHz, CDCl3): δ7.280 - 8.228 (6H, m, aromatic –H, -NH-), δ4.086, 4.079 (2H, d, α
H), δ3.843 (3H, s, -OCH3) FABMass: m/z = 231.1.
Compound-4: Indole-3-carboxyl dipeptide methyl ester: IR (KBr Pallets): 3301.93 (N-H
stretch),3057.41 (aromatic C-H stretch), 2926.47,2855.41 ( aliphatic C-H stretch), 1651.53 (C=O stretch
(amide) cm-1. 1H NMR (300MHz, CDCl3): δ8.896 - 6.929 (15 H, m, aromatic –H, NH-), δ6.929 (2H, m,
-NH), δ4.128 (2H, m, α H), δ3.680 (3H, s, - OCH3), δ3.103 (4H, d, β H).FABMass: m/z 485.35.
Compound-5: Indole-3-carboxyl (N-Me) Phe methyl ester: IR (KBr Pallets): 3281.99 (N-H stretch),
2924.10, 2854.42 (aliphatic C-H stretch), 1371.69 (aliphatic C-H in plane bending), 1671.24 (C=O stretch
(amide) cm-1. 1H NMR (300MHz, CDCl3): δ7.187 - 8.228 (11 H, m, aromatic –H, NH-), δ4.128 (1H, m,
α H), δ3.680 (3H, s, -OCH3), δ3.103 (2H, d, β H) . FABMass: m/z = 358.2.

RESULTS AND DISCUSSION

Structural modification of Indole-3-carboxylic acid was carried out by coupling N-methylated amino
acids and dipeptides with the amino group of indole-3-carboxylic acid and the synthesized compounds
were characterized by FTIR, 1H NMR and Mass spectral analysis. The compounds were subjected to

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antimicrobial evaluation by Disk Diffusion method. All the compounds had shown potent antibacterial
activity against Enterococcus faecalis which can be comparable to the standard drug (Clotrimazole and
mebendazole). In fact all the bacterial strains used for the study were susceptible to the synthesized
compounds but Enterococcus faecalis was more sensitive than other strains to the synthesized compounds.
However the compound 4 and 5 having (N, dipep) phe-tyr unit as a substituent showed better activity
equally to the n-methylated compounds. Final conclusion was made, based on the antimicrobial activities
of the newly synthesized indole-3-carboxylic acid derivatives, the methylated and dipeptide compounds
had shown potent antibacterial and anthelimintic activities.

REFERENCES
1. John Porter , Simon Lumb, Richard J. Franklin, Jose M. Gascon-Simorte, Mark Calmiano, Kelly
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Discovery of 4-azaindoles as novel inhibitors of c-Met kinase, Bioorganic & Medicinal Chemistry
Letters 2009 ;19: 2780–2784.
2. Rakesh Kumar Tiwari,a Devender Singh,a Jaspal Singh,a Vibha Yadav, Ajay K. Pathak, Rajesh
Dabur,Anil K. Chhillar, Rambir Singh, G. L. Sharma, Ramesh Chandraa, and Akhilesh K.
Vermaa, Synthesis and antibacterial activity of substituted1,2,3,4-tetrahydropyrazino [1,2-a]
indoles, Bioorganic & Medicinal Chemistry Letters 2006; 16: 413–416.
3. Chung-Kyu Ryu, Jung Yoon Lee, Rae-Eun Park, Mi-Young Ma and Ji-Hee Nho,Synthesis and
antifungal activity of 1H-indole-4,7-diones, Bioorganic & Medicinal Chemistry Letters 2007 ;17:
127–131.
4. Yong Hai Nan, Jeong-Kyu Bang, Song Yub Shin, Design of novel indolicidin- derived
antimicrobial peptides with enhanced cell specificity and potent anti-inflammatory activity,
Peptides 2009; 30 :832–838.
5. Roman Mezencev, Melina Galizzi b, Peter Kutschy, Roberto Docampo, Trypanosoma cruzi:
Antiproliferative effect of indole phytoalexins on intracellular amastigotes in vitro, Experimental
Parasitology 2009; 122: 66–69.
6. Himaja M, Rajiv, Ramana MV, Synthesis of 6-nitrimidazolyl-1-acetyl amino acids and peptides as
potent anthelmintic agents, Ind J Hetero Chem 2002; 12: 121-124.
7. Webb RG, Haskell MW, Stammer CH. A novel method of protection for the carboxyl group, J.
Org. Chem 1994; 59: 5912.
8. Belagali SL, Mathew T, Himaja M, Kocienski P. A highly efficient method of N-methylation for
amino acid derivatives, Ind. J. Chem 1995; 34B:45-47.
9. Bodanszky M, Bodanszky A, Practice of Peptide synthesis (Springer- Verlag, New York), 1984:
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Table 1: Physical data of synthesized amino acid/dipeptide Indole-3-carboxylic acid

Mol. Physical
S.No Dipeptides Mol For. % yield
Wt state
Indole-3-carboxyl Yellow
1. C19H18N2O3 323.1 87.2
phenylalanine methyl ester semisolid
Indole-3-carboxyl tyrosine Orange
2. C19H18N2O4 337.2 82.4
methyl ester crystals
Indole-3-carboxyl glycine Brown
3. C12H12N2O4 231.1 78.9
methyl ester semisolid
Indole-3-carboxyl dipeptide Yellow
4. C28H26N3O5 485.3 84.6
methyl ester crystals
Pale
Indole-3-carboxyl (N-Me) Phe
5. C20H20N2O3 358.2 brown 81.2
methyl ester
crystals

Table-2 Results of Anthelimintic activity

S.No Synthesized compounds Conc. Paralyzing Death time

(mg/50ml) time

1. Indole-3-carboxyl phenylalanine methyl 100 01:10 02:00

ester

2. Indole-3-carboxyl tyrosine methyl ester 100 01:00 02:14

3. Indole-3-carboxyl glycine methyl ester 100 00:45 02:00

4. Indole-3-carboxyl dipeptide methyl ester 100 04:10 07:00

5. Indole-3-carboxyl (N-Me) Phe methyl ester 100 07:00 11:00

6. Mebendazole 100 01:00 02:10

7. Control - No effect No effect

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Table 3: Antibacterial Activity of Indole-3-carboxylic acid Derivatives

Sl. Compound no Diameter of zone of

No Inhibition (mm)
Enter. fae E. coli
1 Indole-3-carboxyl phenylalanine 10 8
methyl ester

2 Indole-3-carboxyl tyrosine 15 10
methyl ester

3 Indole-3-carboxyl glycine 12 8
methyl ester

4 Indole-3-carboxyl dipeptide 17 15
methyl ester

5 Indole-3-carboxyl (N-Me) Phe 18 18

methyl ester

6 Ampicillin 20 22

(–) indicates no inhibition zone (no activity)

H O O DCC, Et3N
O N O CHCl 3 ,24h H O
OH NH 2 O N O
O N
O H O
Boc-glycine glycine methyl ester Dipeptide
H
N DCC, Et3N
DCC, Et3N
CHCl 3 ,24h CHCl 3 ,24h
H 2N OHO
O OH O
O
Indole-3-carboxylic acid HN
HN OH OH
O H Phe-Tyr-OMe
N-Me-Phe
O N
DCC, Et3N
HO NH 2 CHCl ,24h
HN H Amino acid methyl ester 3
N O
NH
O O OH
O H
N HN O
O
N OH
O H O

Synthesized compounds - Indole-3-carboxyl-phe/tyr/gly; Indole-3-carboxyl-phe-tyr/N-Me-Phe

Scheme-I

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