Solvent-free synthesis of nitrilotriacetamide and diketopiperazines from nitrilotriacetic acid under microwave irradiation and their antimicrobial activity

Article abstract of DOI:10.1080/00397910701572795

Efficient synthesis of dioxopiperazines-diketopiperzines (DKPs)- and amide from nitrilotriacetic acid (NTA) using microwave irradiation and classical heating were described. All compounds were characterized using 1H NMR, 13C NMR and elemental analysis. An

Full text of DOI:10.1080/00397910701572795

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Solvent-Free Synthesis of Nitrilotriacetamide
and Diketopiperazines from Nitrilotriacetic
Acid under Microwave Irradiation and Their
Antimicrobial Activity
Gülşen Öztürk a , Bahattin Gümgüm a , Murat Kizil a & Sevil Emen a
a University of Dicle, Faculty of Science and Art, Department of Chemistry ,
Diyarbakır, Turkey
Published online: 19 Nov 2007.
To cite this article: Gülşen Öztürk , Bahattin Gümgüm , Murat Kizil & Sevil Emen (2007) Solvent-Free
Synthesis of Nitrilotriacetamide and Diketopiperazines from Nitrilotriacetic Acid under Microwave
Irradiation and Their Antimicrobial Activity, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 37:22, 3981-3988, DOI: 10.1080/00397910701572795
To link to this article: http://dx.doi.org/10.1080/00397910701572795
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Synthetic Communicationsw, 37: 3981–3988, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910701572795
Solvent-Free Synthesis of
Nitrilotriacetamide and Diketopiperazines
from Nitrilotriacetic Acid under Microwave
Irradiation and Their Antimicrobial
Activity
¨
Gu¨ls¸en Oztu¨rk, Bahattin Gu¨mgu¨m, Murat Kizil,
and Sevil Emen
Faculty of Science and Art, Department of Chemistry, University of
Dicle, Diyarbakır, Turkey
Abstract: Efficient synthesis of dioxopiperazines-diketopiperzines (DKPs)- and amide
from nitrilotriacetic acid (NTA) using microwave irradiation and classical heating were
described. All compounds were characterized using 1H NMR, 13C NMR and elemental
analysis. Antimicrobial effects of these compounds are also investigated. All tested
compounds showed moderate antimicrobial activity.
Keywords: amide, antimicrobial activity, diketopiperazine, microwave irradiation,
solvent-free synthesis
The formation of the amide bond by the direct combination of carboxylic acids
and amines is highly desired, as the methods employed may also be utilized in
a peptide and lactam synthesis. In general, the formation of carboxyamides
from amines and carboxylic acids implies the activation of the carboxyl
groups.[1,2] Nitrilotriacetamide is an ubiquitous tripodal ligand for metal
coordination, but there are very few reports of nitrilotriacetamide in the
literature.[3,4] An efficient method to prepare 2,6-diketopiperazine (DKP)
Received in the U.K. December 1, 2006
¨
Address correspondence to Gu¨ls¸en Oztu¨rk, Faculty of Science and Art, Department
of Chemistry, University of Dicle, 21280 Diyarbakır, Turkey. E-mail: gozturk@dicle.
edu.tr
3981

¨
G. Oztu¨rk et al.
3982
involves the tandem formation of the N1–C2 and N4–C5 bonds. The intramo-
lecular N-acylation reaction to form the N1–C2 bond is a common method of
2,6-DKP synthesis. The acyclic precursor generally contains a primary or
secondary amide, frequently derived from a nitrile, as the nucleophilic
nitrogen source and an acid as the acylating group. An early synthesis by
Cignarella illustrates the efficiency with which one can construct the 2,6-DKP
ring system by the cyclization of an amide onto an ester group. The acid
bromide was sequentially coupled to an amine and an a-amino ester to give
amide, which was simply heated to provide 2,6-DKP in good yield.[5]
Previously, D’Angeli and coworkers reported the treatment of a-bromo-
carboxamide with sodium hydride to produce the hexasubstituted 2,6-DKP.
Early reports focused on elevated dehydration of diacids in the presence of
various nitrogen sources, such as urea,[6] ammonia,[7] ammonium format,[8]
and a primary amine.[9] 1-Aryl-2,6-DKPs was prepared by Li et al.[10] and
evaluated for antitumor activity in a report by Abdel-Hamide directed
toward the synthesis of new antimicrobial agents.[11] DKPs remain
important in drug discovery because they contain constrained amino acids
imbedded within their structures, without the unwanted physical and
metabolic properties of peptides. The regioisomeric dioxopiperzines are
found in numerous natural and synthetic organic compounds, and this
substance possesses a multitude of important biological properties.[12–18]
Nonetheless, dioxopiperazines of this class exhibit synergistic antitumor
effects[19,20] in combination with clinically efficacious antineoplastic drug.
Research on the synthesis of ether, ester, etc., using microwave irradiation
is going on in our laboratory.[21–23] In the present study, to avoid the prelimi-
nary and often expensive synthesis, pyrolitic preparation of amides as method
for their synthesis in the absence of any catalyst and solvent was considered.
Therefore, we aimed to simplify the well-known procedure [Eq. (1)] of amide
synthesis from carboxylic acid and amine reaction by microwave application.
GENERAL PROCEDURE FOR SYNTHESIS OF AMIDE AND
DIKETOPIPERAZINES
All chemicals were reagent grade unless otherwise specified. Amines and nitri-
lotriacetic acid were purchased from Fluka Chemical Company. The START
labstation (Milestone labstation for microwave enhanced chemistry) was used.
Silica gel/TLC cards (F254) were used for thin-layer chromatography (TLC).
Melting points were determined with a Gallenkamp apparatus with open
capillaries. Infrared spectra were recorded on a Mattson 1000 FTIR model spec-
trophotometer. Elemental analyses were obtained with a Carlo-Erba 1108 model
apparatus. 1H (400-MHz) and 13C (100-MHz) NMR spectra were recorded on a
Bruker DPX-400 high-performance digital FT-NMR spectrometer.
Nitrilotriacetic acid (0.50 g, 2.62 mmol) and amine (1.25 g, 11.70 mmol)
were taken in the reaction vessel. The resultant mixture was heated at 1508C

Nitrilotriacetamide and Diketopiperazines
3983
with microwave irradiation and conventional heating. The mixture was then
cooled and extracted with chloroform. The extract was washed successively
with solution of 2 M HCl (50 mL) and a solution of 5% NaHCO3 (50 mL).
The organic layer was dried over MgSO4 and concentrated under reduced
pressure. The crude product was crystallized from a mixture of dichloro-
methane and hexane.
SYNTHESIZED COMPOUNDS
2,20,20-Nitrilotris (N-Benzyl Acetamide) (1a)
Mp 143–1448C (lit. mp ¼ 145),[24] 1H NMR (400 MHz, CDCl3): d 3.25
(s, 2H), 4.30 (d, 2H, J ¼ 5.90 Hz), 7.21–7.31 (m, 5H), 7.70 (broad, 1H);
13C NMR (100 Hz, CDCl3): d 43.38, 60.42, 127.52, 127.71, 128,71, 137.97,
170.17; IR (KBr): 3294 (NH), 1655 (C55O), 1537 (Ar-H), 1074 (C-N).
Anal. calcd. for C27N4H30O3: C, 70.74; N, 12.23; H, 6.55. Found: C, 72.25;
N, 11.93; H, 6.88.
N-Dibenzyl-2-(4-dibenzyl-3,5-dioxopiperazine-1-yl)acetamide (1b)
1
Mp 144–1458C; H NMR (400 MHz, CDCl3): d 3.20 (s, 2H), 3.50 (s, 4H),
4.50 (d, 2H, J ¼ 8.10 Hz), 5.02 (s, 2H), 7.03 (broad, 1H), 7.25–7.39
(m, 10H); 13C NMR (100 MHz, CDCl3): d 42.48, 43.26, 56.79, 59.35,
77.03, 77.35, 127.77, 129.00, 136.29, 137.76, 167.55, 168.69; IR (KBr):
3321 (NH), 1695 (C55O), 1663 (C55O), 1537 (Ph), 1074 (C-N). Anal.
calcd. for C20N3H21O3: C, 68.37; N, 11.96; H, 5.98. Found: C, 68.70; N,
11.27; H, 6.00.
N-Hexzyl-2-(4-hexzyl-3,5-dioxopiperazine-1-yl)acetamide (2)
1
Mp 177–1788C; H NMR (400 MHz, CDCl3): d 0.90 (t, 6H, J ¼ 0.94 Hz),
1.31 (m, 8H), 1.65 (m, 8H), 3.21 (m, 4H), 3.30 (s, 2H), 3.50 (s, 4H), 6.66
(broad, 1H); 13C NMR (100 MHz, CDCl3): d 13.97, 22.49, 26.56, 27.82,
29.55, 31.37, 38.25, 39.42, 56.87, 59.48, 167.66, 168.80; IR (KBr): 3281
(NH), 1694 (C55O), 1576 (C55O), 1537, 1054 (C-N).
N-Buthyl-2-(4-buthyl-3,5-dioxopiperazine-1-yl)acetamide (3)
1
Mp 179–1808C; H NMR (400 MHz, CDCl3): d 0.90 (t, 6H, J ¼ 16.60 Hz),
1.32 (m, 4H), 1.65 (m, 4H), 3.20 (m, 4H,), 3.30 (s, 2H), 3.51 (s, 4H), 6.66
(broad, 1H); 13C NMR (100 MHz, CDCl3): d 14.07, 19.97, 20.03, 29.85,
29.55, 31.52, 38.55, 38.65, 54.92, 57.50, 166.87, 168.72.

¨
G. Oztu¨rk et al.
3984
TEST MICROORGANISMS
The in vitro antimicrobial activities of amide (1a) and diketopiperazines
(1b–3) were tested against laboratory control strains belonging to the
American Type Culture Collection (Maryland, USA): Escherichia coli
(ATCC 25922) and Salmonella typhimurium (ATCC 14028), except for
Klebsiella oxyloca, Basillus cereus, Streptecocus pyogenes, and Candida
albicans, which were obtained from the Microbiology Department at the
University of Dicle (Diyarbakır, Turkey).
EVALUATION OF ANTIMICROBIAL ACTIVITY
Antimicrobial activity was assayed by the disc diffusion susceptibility test
according to the recommendation of the National Committee for Clinical
Laboratory Standards (NCCLS).[25] The disk diffusion test was performed
on Muller–Hinton agar plates. Plates were dried at 35 to 368C for about
30 min in an incubator before inoculation. Three to five freshly grown
colonies of bacterial strains were inoculated into 25 mL of Mullar–Hinton
broth medium in a shaking water bath for 4 to 6 h until a turbidity of 0.5
McFarland (1 ꢀ 108 CFU/mL) was reached. Final inocula were adjusted to
5 ꢀ 105 CFU/mL. Three to five colonies of C. albicans were inoculated
into 25 mL of Sabouraud dextrose broth in a shaking water bath for 8 to
10 h until a turbidity of 0.5 McFarland was reached. The final inocula were
adjusted to 5 ꢀ 105 CFU/mL using a spectrophotometer.[26] The inoculum
(50 mL) from the final inocula was applied to each agar plate and uniformly
spread with a sterilized cotton spreader over the surface. Absorption of
excess moisture was allowed to occur for 30 min before application of
sterile filter-paper discs. Sterile filter-paper disks (Oxoid, England, 6 mm in
diameter) were impregnated with 20 mL of the sample solutions in dimethyl-
sulphoxide (DMSO), 1.5 mg per 1 mL of DMSO (all solutions were filter-
sterilized using a 0.20 mm membrane filter) and placed on inoculated plates.
These plates were incubated at 378C for 24 h for bacteria and 48 h for
fungi. Standard disks of Amoxycillin/clavulanic acid (AMC, 30 mg/disc),
and imipenem (IMP, 10 mg/disc) were individually used as positive
controls, and the disks imbued with 20 mL of pure DMSO were a negative
control. The diameters of the inhibition zones were measured in millimeters
using an inhibition zone ruler.
RESULTS AND DISCUSSION
The reactions of carboxylic acid with amines were carried out without catalyst
and solvent. Reaction temperature was around 1508C to favor shifting of
equilibrium by water removal (Eq. 1). To compare the efficiency of

Nitrilotriacetamide and Diketopiperazines
3985
Scheme 1. Synthesis of amide and diketopiperazines from NTA and amines.
microwave heating with conventional heating, amide 2,6-DKP derivatives
(NTA) tricarboxylic acid and amines were synthesized by both methods
(Scheme 1). Compound 1a was obtained by excess of amine within 45 min
under microwave irradiation with excellent yield (94%) and conventional
heating at the same time with low yield (less than 5%), but 1b was obtained
within 15 min under microwave irradiation with an excess of nitrilotriacetic
acid (NTA) with good yield (80%) and conventional heating at the same
time with low yield (less than 5%) (Table1). Tang et al. have synthesized
1a using ethyl ester of nitrilotriacetic acid with benzylamine at 120–1258C
within 12 h with 50% yield.[27] DKP derivatives were synthesized under
microwave irradiation in a very short time without solvent or reagent at
one step. In contrast, 2,6-DKP derivative was reported from dicarboxylic
acid–amine with three steps using DCC (dicyclohexylcarbodiimide)
in THF in 2 h with 43% yield by Prakash et al. to prepare a neuroleplic
agent.[13] This clearly shows that our method is very efficient for the
synthesis of amides.
Diketopiperazines have been repeatedly shown to exhibit strong biological
activities.[28–30] Amide (1a) and diketopiperazines (1b–3) showed moderate
antibacterial activity and antifungal activity (Table 2). Diketopiperazines 2
indicated 14-mm inhibition zones against Escherichia coli (ATCC 25922)
and 13-mm zones against Candida albicans. Diketopiperazines 1b, 2, and 3
showed the same inhibition zones (12 mm) against Streptecocus pyogenes.
Compound 1a and 1b indicate the same inhibition zones (10, 8, 10, and
8 mm against Escherichia coli (ATCC 25922), Bacillus cereus, Klebsiella

¨
G. Oztu¨rk et al.
3986
Table 1. Optimization of the reaction condition of microwave-assisted (600 W) and
conventional amide and DKP formation [nitrilotriacetic acid (NTA)/amine]
Yielda (%)
Relative amount
(mmol) NTA/amine
Reaction
temp. (8C)
Reaction
time (min)
Compound
MW
D
1a
1a
1a
1a
1a
1a
1a
1b
1b
1b
1b
1b
2
2.6/11.7
2.6/13.4
2.6/7.8
2.6/7.8
2.6/9.3
2.6/7.8
2.6/18.2
4.2/7.3
2.6/7.3
2.6/2.6
2.6/3.6
2.6/5.4
2.6/12.6
2.6/8.7
2.6/7.8
2.6/10
150
150
150
180
180
180
180
150
150
150
180
150
150
150
200
150
200
15
45
60
20
60
60
60
15
15
15
60
20
30
15
60
20
60
76
94
þ
þ
NT
NT
NT
NT
NT
80
8
41
61
59
41
þ
50
34
—
—
16
—
—
—
12
—
13
NT
50
7
11
2
2
NT
7
3
3
2.6/7.8
NT
aIn isolated products.
2: no reaction.
þ: low yield (less than 5%).
NT: not tested.
Table 2. Antimicrobial activity of amide (1a,b) and diketopiperazines (1b,2,3) and
standard antibiotics
Zones of inhibition (mm)
Amide and diketopiper-
azines (20 mL/paper Standard antibiotics
disc)
(mg/paper disc)
Tested organisms
1a
1b
2
3
AMC 30 IPM 10
Escherichia coli, ATCC 25922
Salmonella typhimurium, ATCC 14028
Streptecocus pyogenesa
Bacillus cereusa
10
10
10
8
10 14
12 —
12 12 12
12
10 12 —
13
8
20
24
28
26
8
.30
16
.30
30
8
8
Klebsiella oxylocaa
10
8
18
NT
26
NT
Candida albicansa
8
8
aClinical isolates.
Notes. IPM: imipenem; AMC: amoxycillin/clavulanic acid; NT: not tested; —: not active.

Nitrilotriacetamide and Diketopiperazines
3987
oxyloca, and Candida albicans respectively. The antimicrobial activity of the
diketopiperazines 2 was found to be similar to amoxycillin against Bacillus
cereus. All organisms are susceptible to imipenem. The DMSO (20 mL)
negative control showed no inhibiting effect.
The synthesis of nitrilotriacetamide and diketopiperazines using
microwave irradiation was achieved for the first time by our group. Nitrilotria-
cetamide and diketopiperazines were synthesized in very short time and
without any reagent and solvent. All tested compounds were found to have
moderate antimicrobial activity.
ACKNOWLEDGMENT
¨
Financial support by University of Dicle (grant no: DUAPK 04-FF-51) is
gratefully acknowledged.
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