Synthesis, spectral analysis, and in vitro microbiological evaluation of ethyl 7,9-diaryl-1, 4-diazaspiro[4.5]dec-9-ene-6-carboxylates as a new class of antibacterial and antifungal agents

Article abstract of DOI:10.1007/s10593-011-0801-5

A series of novel ethyl 7,9-diaryl-1,4-diazaspiro[4.5]dec-9-ene-6- carboxylates was synthesized by the reaction of ethyl 4,6-diaryl-2-oxocyclohex- 3-ene-1-carboxylates with ethylenediamine in the presence of p-toluenesulfonic acid without a solvent under focused microwave irradiation. The title compounds were screened for their antimicrobial activities against a spectrum of clinically isolated microorganisms.

Full text of DOI:10.1007/s10593-011-0801-5

Chemistry of Heterocyclic Compounds, Vol. 47, No. 5, August 2011 (Russian original Vol. 47, No. 5, May, 2011)
SYNTHESIS, SPECTRAL ANALYSIS, AND
IN VITRO MICROBIOLOGICAL EVALUATION OF
ETHYL 7,9-DIARYL-1,4-DIAZASPIRO[4.5]DEC-9-ENE-6-
CARBOXYLATES AS A NEW CLASS OF ANTIBACTERIAL
AND ANTIFUNGAL AGENTS
J. Thanusu1, V. Kanagarajan1, and M. Gopalakrishnan1*
A series of novel ethyl 7,9-diaryl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylates was synthesized by the
reaction of ethyl 4,6-diaryl-2-oxocyclohex-3-ene-1-carboxylates with ethylenediamine in the presence of
p-toluenesulfonic acid without a solvent under focused microwave irradiation. The title compounds were
screened for their antimicrobial activities against a spectrum of clinically isolated microorganisms.
Keywords: ethyl 7,9-diaryl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylates, ethylenediamine, p-toluene-
sulfonic acid, antibacterial activity, antifungal activity.
Heterocycles form by far the largest of the classical divisions of organic chemistry and are of immense
importance biologically and industrially. The majority of pharmaceuticals and biologically active agrochemicals
are heterocyclic, while countless additives and modifiers used in industrial applications, including cosmetics,
reprography, information storage, and plastics, are also heterocyclic in nature. The development of simple,
efficient, and environmentally benign chemical processes for widely used organic compounds from readily
available reagents is one of the major challenges for chemists in organic synthesis.
Imidazoline is a nitrogen-containing heterocycle derived from imidazole. The ring contains an imine
bond, and the carbon atoms at the positions 4 and 5 are singly rather than doubly bonded as in the case of
imidazole. The importance of the imidazoline unit rests on its presence in many biologically active compounds
[1, 2]. Imidazoline units are also used in organic synthesis as synthetic intermediates [3, 4], chiral auxiliaries [5],
chiral catalysts [6], and ligands for asymmetric catalysis [7]. Like imidazole, imidazoline-based compounds
have been used as N-heterocyclic carbene ligands [8] with various transition metals, for example, in the
commercially available second generation Grubbs' catalyst.
A good number of bioactive imidazoline derivatives bear a substituent (aryl or alkyl group) on the car-
bon atom between the nitrogen centers. Oxymetazoline, generally available as a nasal spray [9], is a selective
_______________
* To whom correspondence should be addressed, e-mail: profmgk@yahoo.co.in.
1 Synthetic Organic Chemistry Laboratory, Department of Chemistry, Annamalai University, Annamalai Nagar-
608 002, Tamil Nadu, India
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 5, pp. 696–705, May, 2011. Original
article received November 2, 2009, in revised form April 6, 2011.
0009-3122/11/4705-0575©2011 Springer Science+Business Media, Inc.
575

α1-adrenoceptor agonist and partial α2-adrenoceptor agonist topical decongestant, used in the form of
hydrochloride in products such as Afrin. Oxymetazoline causes down-regulation of α2-adrenoceptors, and
therefore becomes less effective after a few doses. Xylometazoline (also known as Xylomethazoline) is a drug
which is used as a nasal decongestant. It is a topical decongestant that is applied directly into the nose either as a
spray or as drops. Tetrahydrozoline, a derivative of imidazoline, is an α-adrenoceptor agonist [10], and its main
mechanism of action is the constriction of conjunctival blood vessels. This serves to relieve the redness of the
eye caused by minor ocular irritants. Naphazoline is a sympathomimetic agent [11] with marked α-adrenergic
activity. It is a vasoconstrictor with a rapid action in reducing swelling when applied to mucous membrane.
It acts on α-receptors in the arterioles of the conjunctiva to produce constriction, resulting in decreased con-
gestion. It is an active ingredient in Naphcon-A and Clearine eye drops. Clonidine is a medication used to treat
several medical conditions. It is a direct-acting 2-adrenergic agonist. An imidazole ring forms a part of some
antitumor drugs such as dacarbazine (5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide) and Azathioprine
(Imuran, 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)sulfanyl]-7H-purine) [12], as well as the tumor growth-
inhibiting iminoimidazolidine (Cyclocreatine) [13].
Me
Me
Cl
H
H
H
H
N
N
N
N
N
N
N
Me
Bu-t
Me
Bu-t
Cl
OH
Oxymetazoline
Xylometazoline
Clonidine
H2N
N
O
NH
N
Me
H
OH
N
N
N
HN
N
N
NH
Tetrahydrozoline
Me
N
Me
O
N
H
Naphazoline
Dacarbazine
Cyclocreatine
Me
O
HN
NH
N
N
Me
OEt
Me
Me
Me
Cl
Ru
Cl
Ph
Y
X
P(Cy)3
Second generation Grubbs’ catalyst
Imidazolidine derivative. Target molecule
A variety of methods has been reported for the synthesis of imidazolines, which include conversion of
esters using aluminum reagents [14], the reaction between N-ethoxycarbonylthioamides with 1,2-diamines [15],
and the reaction of aldehydes with 1,2-diamines followed by N-halosuccinimides [16]. Recently, several
methods have been developed where azalactones [17], 2-aryl-1,1-dibromoethanes [18], nitriles [19], and amino
amides [20] are used as starting materials for this synthesis. However, many of the synthesis protocols reported
so far suffer from disadvantages such as the need for anhydrous conditions [14], use of organic solvents [14–20],
severe reaction conditions [14], prolonged reaction time [16], use of metals, and expensive reagents [14].
Therefore the development of a cost-effective, safe, and environmentally friendly reagent system is desirable.
In recent years, there has been a great deal of interest in exploiting more than one proximal functional
group for designing novel structures capable of performing a variety of functions. The present study describes
576

the use of ethyl 4,6-diaryl-2-oxocyclohex-3-ene-1-carboxylates [21], intermediates with three versatile
functional groups, i.e., ketone, olefin, and ester, for the synthesis of imidazoline derivatives. In continuation of
our earlier work on the synthesis of structurally diverse biologically active hybrid heterocyclic ring systems and
as part of our ongoing research program [22–26], we plan to design imidazoline derivatives bearing a
cyclohexene substituent bound by a spiro-linkage to the carbon atom between the nitrogen centers to obtain a
new series of heterocycles, namely, ethyl 7,9-diaryl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylates since most of
the bioactive imidazoline derivatives bear a substituent (aryl or alkyl group) on this carbon center [11–13]. In
order to evaluate the effect of the structure on the biological activity of the target compounds, we chose different
substituents at the phenyl rings of the starting reactants.
Condensation of appropriate acetophenone derivatives with appropriate benzaldehydes in the presence
of sodium hydroxide yielded the respective 1,3-diarylprop-2-en-1-ones 1–10. On treatment with ethyl
acetoacetate in the presence of sodium ethoxide, α,β-unsaturated ketones 1–10 gave cyclohex-2-ene derivatives
11–20 by the Knoevenagel reaction. Synthesis of ethyl 7,9-diaryl-1,4-diazaspiro[4.5]-deca-9-ene-6-carboxylates
21–30 was carried out by the reaction of compounds 11–20 with ethylenediamine in the presence of
p-toluenesulfonic acid under solvent-free conditions and under focused microwave irradiation. The applications
of microwave technology to rapid synthesis of biologically significant heterocyclic molecules under solvent-free
conditions are very promising and numerous and have recently been recognized as a useful tool for a drug-
discovery program, especially in combinatorial chemistry [27–29]. The reactions were performed at 120°C
under 4 bar pressure within 5–10 min. After completion of the reaction, as indicated by the TLC, the reaction
mixture was poured into ice water and worked up using standard methods.
The synthetic route for the formation of compounds 21–30 and their physical and analytical data are
given in Scheme 1 and Table 1, respectively. The structures of all the synthesized compounds 21–30 are
1
determined with the help of elemental analysis, FT-IR, MS, one-dimensional H NMR, D2O-exchanged
1H NMR, 13C NMR, and two-dimensional HSQC spectra. In order to investigate the spectral assignments, ethyl
7,9-diphenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate 21 was chosen as a representative compound.
The FT-IR spectrum of ethyl 7,9-diphenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate 21 shows charac-
teristic absorption frequencies in the region of 3267–3528 cm–1 (NH stretching frequency). The absorption
bands at 1738 cm–1 and 1607 cm–1 are due to carbonyl stretching of the ester group and C=C stretching,
respectively. The presence of the band at 1445 cm–1 (C–N) adds more evidence for the formation of compound
21. The mass spectrum of compound 21 shows a protonated molecular ion peak at m/z 363. This is consistent
with its proposed empirical formula which is confirmed also by elemental analysis.
Scheme 1
O
O
O
O
OEt
O
Me
OEt
NaOEt, EtOH,
reflux, 1 h
Y
X
Y
X
11–20
1–10
NH2
O
HN
NH
H2N
OEt
Monomode MW,
0.4 MPa, 120oC, 5–10 min
Y
X
21–30
1–5, 11–15, 21–25 X = H, 1, 11, 21 Y = H, 2, 12, 22 Y = Cl, 3, 13, 23 Y = F,
4, 14, 24 Y = Me, 5, 15, 25 Y = OMe, 6, 16, 26 X = Cl, Y = H, 7, 17, 27 X = OMe, Y = H,
8, 18, 28 X = Cl, Y = Me, 9, 19, 29 X = OMe, Y = Cl, 10, 20, 30 X = Cl, Y = OMe
577

TABLE 1. Physical and Analytical Data for the Title Compounds 21–30
Found, %
Calculated, %
Com-
pound
m/z [M+1]+
Empirical formula
mp,
°C
Yield,
%
C
H
N
21
22
23
24
25
26
27
28
29
30
363
76.11
76.21
7.15
7.23
7.63
7.73
77
100
65
92
93
92
90
93
91
92
93
94
93
C23H26N2O2
397
C23H25ClN2O2
69.48
69.60
6.24
6.35
6.95
7.06
381
C23H25FN2O2
72.52
72.61
6.57
6.62
7.23
7.36
377
C24H28N2O2
76.49
76.56
7.43
7.50
7.32
7.44
117
112
68
393
C24H28N2O3
73.33
73.44
7.10
7.19
7.01
7.14
397
C23H25ClN2O2
69.52
69.60
6.30
6.35
6.97
7.06
393
C24H28N2O3
73.32
73.44
7.11
7.19
7.03
7.14
65
411
C24H27Cl2O2
70.04
70.15
6.53
6.62
6.78
6.82
73
427
C24H27ClN2O3
67.42
67.52
6.25
6.37
6.51
6.56
69
427
C24H27N2O3Cl
67.44
67.52
6.29
6.37
6.48
6.56
99
1
13
The assignments of signals in the H and C NMR spectrum have been done with the help of HSQC
one-bond 1H–13C correlations, which allowed us to confirm the formation of the imidazolidine ring. Among the
signals important for the structure determination are two NH protons of the imidazoline moiety (H-1, H-4) in the
form of a broad singlet at 5.98 ppm which disappears upon addition of D2O.
The methylene protons (H-2, H-3) of the imidazolidine moiety were observed as one multiplet at 4.02–
4.13 ppm. The 13C resonance of a quaternary carbon at 80.1 ppm is due to the spiro carbon (C-5).
The antibacterial activity of compounds 21–30 was tested in vitro against Bacillus subtilis, Vibrio cho-
lerae, Shigella flexneri, Salmonella typhi, and Escherichia coli. The potency of the synthesized compounds
expressed as the minimum inhibitory concentration (MIC) was compared with a broad-spectrum antibiotic,
Ciprofloxacin (Table 2). A close survey of the MIC values indicate that all the compounds exhibited a varied
range (6.25–200 μg/ml) of antibacterial activity against all the tested bacterial strains except 21 and 29 which
did not show activity against E. coli and S. flexneri, respectively, even at the maximum concentration of
200 μg/ml. Compound 22, having an electron-withdrawing p-chloro substituent at the phenyl ring attached to
C-7 of the cyclohexenone moiety, shows moderate activity against B. subtilis, V. cholerae, and S. typhi and good
activity against S. flexneri and E. coli. Compound 23 with an electron-withdrawing p-fluoro substituent at the
same position was more active than Ciprofloxacin against all the tested bacterial strains, thus showing a broad
spectrum of antibacterial activity.
The in vitro antifungal activity of compounds 21–30 was studied against the fungal strains Aspergillus
flavus, Aspergillus niger, Mucor, and Rhizopus. Fluconazole was used as the standard drug. The corresponding
MIC values are given in Table 3. All the compounds 21–30 exhibited a varied range (6.25–200 μg/ml) of
antifungal activity against all the tested fungal strains except compounds 21 and 25 which were not active
against A. niger and A. flavus, respectively. Compound 22 with a p-chloro substituent at the 7-phenyl ring shows
good activity against A. niger and Rhizopus and moderate activity against Mucor, being more active than
Fluconazole against those three species. Likewise, compound 29, which has an electron-withdrawing chloro
substituent at the same position and an electron-donating p-methoxy substituent at the phenyl ring attached to
C-9, performed better than Fluconazole against A. flavus, A. niger, and Rhizopus.
578

TABLE 2. In vitro Antibacterial Activity of Compounds 21–30 against
Clinically Isolated Bacterial Strains
MIC, µg/ml
Compound
B. subtilis
V. cholerae
S. flexneri
S. typhi
E. coli
21
200
25
25
25
25
12.5
12.5
50
100
25
–*
22
6.25
12.5
6.25
100
100
100
50
23
6.25
100
100
100
50
25
25
24
50
100
6.25
50
25
50
50
26
6.25
100
12.5
12.5
6.25
50
50
27
100
100
–*
12.5
100
6.25
100
50
28
100
12.5
100
25
29
30
50
25
100
25
Ciprofloxacin
25
_______
* No inhibition even at concentration 200 µg/ml.
TABLE 3. In vitro Antifungal Activity of Compounds 21–30 against
Clinically Isolated Fungal Strains
MIC, µg/ml
Compound
A. flavus
A. niger
Mucor
Rhizopus
21
200
100
6.25
50
–*
6.25
100
100
50
50
12.5
6.25
50
25
6.25
25
22
23
24
100
100
25
25
–*
50
26
25
100
12.5
100
12.5
6.25
50
12.5
50
27
100
50
100
50
28
6.25
25
29
30
25
6.25
25
50
50
Fluconazole
_____________
50
25
25
* No inhibition even at concentration 200 µg/ml.
Thus we have synthesized a series of novel biologically active ethyl 7,9-diaryl-1,4-diazaspiro[4.5]dec-
9-ene-6-carboxylates and established their structures by their spectral data. A survey of their in vitro
antibacterial and antifungal activity was performed, which may be considered as an important step for
construction of novel chemical entities with comparable pharmacological profiles to that of the standard drugs.
EXPERIMENTAL
All the reported melting points were measured in open capillaries and were uncorrected. IR spectra were
recorded in KBr pellets on a Thermo Nicolet Avatar 330 FT-IR spectrophotometer, and noteworthy absorption
579

1
values alone are listed. One-dimensional H and 13C NMR spectra were recorded at 400 and 100 MHz,
respectively, on a Bruker AMX 400 NMR spectrometer using DMSO-d6 as solvent and TMS as internal
standard. Two-dimensional HSQC spectra were recorded at 500 MHz on a Bruker DRX 500 NMR spectrometer
using DMSO-d6 as solvent. The electron spray ionization (ESI) positive mode mass spectra were recorded on a
Bruker Daltonics Esquire 300 LC-MS spectrometer. Satisfactory microanalysis was obtained on a Carlo Erba
1106 CHN analyzer. TLC was used to control the reactions and the purity of the products. A Gyan Easi Flash
column chromatography system was used for flash chromatography. A Biotage Initiator microwave synthesizer
was used for the irradiation.
1,3-Diarylprop-2-en-1-ones 1–10 [30] and 4,6-diaryl-2-oxocyclohex-3-enecarboxylates 11–20 [21] were
prepared by adopting published procedures.
Synthesis of Ethyl 7,9-Diaryl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylates 21–30 (General Method).
An appropriate cyclohexenone 11–20 (0.01 mol), ethylenediamine (0.01 mol), and p-toluenesulfonic acid
(10 mg) were placed in a 10 ml Pyrex glass vial which was sealed with a Teflon cap. The vial was inserted in a
Teflon outer jacket and then placed into the holder in the microwave cavity. The sample was irradiated under
focused monomode irradiation at 120ºC for 5–10 min at 0.4 MPa. After allowing the mixture to cool to room
temperature, the reaction vessel was opened, and the contents were poured into ice water. The organic material
was extracted with ethyl acetate. The organic layer was washed with 10% sodium hydrogen carbonate and brine,
and dried over anhydrous Na2SO4. After evaporation of the ethyl acetate under reduced pressure, a solid mass
was obtained which was subjected to flash column chromatography using toluene–ethyl acetate as eluent.
Ethyl 7,9-Diphenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (21). IR spectrum, , cm–1: 3448,
3388, 3175, 3054, 3022, 2972, 2931, 2837, 1738, 1607, 1445, 1027, 827, 758, 698. 1H NMR spectrum, , ppm
(J, Hz): 0.90 (3H, t, J = 7.1, CH3 ester); 2.97–3.02 (2H, m, H-8); 3.04–3.22 (1H, m, H-7); 3.51–3.67 (1H, m,
H-6); 3.89 (2H, q, J = 6.8, CH2 ester); 4.02–4.13 (4H, m, 2CH2 imidazolidine); 5.98 (2H, s, 2NH imidazolidine);
6.54 (1H, s, H-10); 7.10–7.52 (8H, m, H Ar); 7.65–7.79 (2H, d, J = 2.7, H Ar). 13C NMR spectrum, δ, ppm: 13.7
(CH3 ester); 35.2 (C-7); 43.8 (C-8); 49.3 (C-2, 3); 58.7 (CH2 ester); 59.9 (C-6); 80.1 (C-5); 122.8 (C-10); 126.4
(p-C Ar); 127.0 (o-C Ar); 127.5 (p-C Ar); 128.3 (o-C Ar); 128.8 (m-C Ar); 130.0 (m-C Ar); 130.4 (C-9); 137.3
(ipso-C Ar); 141.4 (ipso-C Ar); 169.2 (C=O).
Ethyl 7-(4-Chlorophenyl)-9-phenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (22). IR spectrum, ,
1
cm–1: 3535, 3437, 3382, 3295, 3065, 2978, 2924, 2847, 1735, 1601, 1445, 1018, 824, 758, 693. H NMR
spectrum, , ppm (J, Hz): 0.92 (3H, t, J = 7.2, CH3 ester); 2.97–3.07 (2H, m, H-8); 3.10–3.14 (1H, m, H-7);
3.57–3.76 (1H, m, H-6); 3.92 (2H, q, J = 6.6, CH2 ester); 4.02–4.15 (4H, m, 2CH2 imidazolidine); 6.12 (2H, s,
13
2NH imidazolidine); 6.55 (1H, s, H-10); 7.13–7.48 (7H, m, H Ar); 7.69–7.79 (2H, d, J = 2.5, H Ar). C NMR,
δ, ppm: 13.7 (CH3 ester); 34.9 (C-7); 43.1 (C-8); 48.4 (C-2, 3); 58.5 (CH2 ester); 60.0 (C-6); 80.2 (C-5); 121.9
(C-10); 122.8 (o-C Ar); 128.1 (p-C Ar); 128.6 (m-C Ar); 128.8 (m-C Ar); 129.1 (o-C Ar); 130.6 (C-9); 135.6
(ipso-C Ar); 136.4 (p-C Ar); 140.4 (ipso-C Ar); 169.0 (C=O).
Ethyl 7-(4-Fluorophenyl)-9-phenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (23). IR spectrum, ,
1
cm–1: 3519, 3388, 3262, 3186, 3058, 2977, 2930, 2863, 1739, 1604, 1443, 1023, 832, 758, 694. H NMR
spectrum, , ppm (J, Hz): 0.89 (3H, t, J = 7.0, CH3 ester); 2.74–2.94 (2H, m, H-8); 2.98–3.13 (1H, m, H-7);
3.56–3.88 (1H, m, H-6); 3.90 (2H, q, J = 6.8, CH2 ester); 3.95–4.13 (4H, m, 2CH2 imidazolidine); 6.02 (2H, s,
13
2NH imidazolidine); 6.57 (1H, s, H-10); 6.83–7.28 (7H, m, H Ar); 7.80–7.94 (2H, d, J = 2.6, H Ar). C NMR
spectrum, δ, ppm: 13.7 (CH3 ester); 35.2 (C-7); 42.9 (C-8); 48.4 (C-2,3); 59.8 (CH2 ester); 61.0 (C-6); 113.1
(m-C Ar); 122.3 (C-10); 125.3 (o-C Ar); 127.9 (p-C Ar); 128.6 (m-C Ar); 128.7 (o-C Ar); 130.5 (C-9); 136.1
(ipso-C Ar); 139.8 (ipso-C Ar); 158.2 (p-C Ar); 169.1 (C=O).
Ethyl 7-(4-Methylphenyl)-9-phenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (24). IR spectrum, ,
cm–1: 3454, 3399, 3267, 3054, 3038, 2981, 2921, 2863, 1739, 1604, 1023, 832, 758, 694. 1H NMR spectrum, ,
ppm (J, Hz): 0.92–0.96 (3H, t, J = 6.9, CH3 ester); 2.26 (3H, s, CH3 tolyl); 2.72–2.93 (2H, m, H-8); 2.97–3.12 (1H,
m, H-7); 3.56–3.76 (1H, m, H-6); 3.91 (2H, q, J = 6.6, CH2 ester); 4.00–4.09 (4H, m, 2CH2 imidazolidine); 5.94
(2H, s, 2NH imidazolidine); 6.53 (1H, s, H-10); 6.88–7.48 (7H, m, H Ar); 7.82–7.91 (2H, d, J = 2.3, H Ar).
580

13C NMR spectrum, δ, ppm: 13.7 (CH3 ester); 20.5 (CH3 tolyl); 35.3 (C-7); 43.3 (C-8); 48.9 (C-2,3); 59.8 (CH2
ester); 60.9 (C-6); 80.2 (C-5); 121.9 (C-10); 122.5 (o-C Ar); 128.2 (p-C Ar); 128.4 (o-C Ar); 128.7 (m-C Ar);
129.3 (m-C Ar); 131.1 (C-9); 138.3 (ipso-C Ar); 138.4 (p-C Ar); 139.6 (ipso-C Ar); 169.1 (C=O).
Ethyl 7-(4-Methoxyphenyl)-9-phenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (25). IR spectrum, ,
cm–1: 3524, 3424, 3267, 3054, 2989, 2929, 2830, 1739, 1445, 1607, 1033, 832, 760, 694. 1H NMR spectrum, ,
ppm (J, Hz): 0.93 (3H, t, J = 7.1, CH3 ester); 2.69–2.93 (2H, m, H-8); 2.97–3.12 (1H, m, H-7); 3.58–3.64 (1H,
m, H-6); 3.72 (3H, s, OCH3); 3.90 (2H, q, J = 6.9, CH2 ester); 3.96–4.36 (4H, m, 2CH2 imidazolidine); 5.94 (2H,
s, 2NH imidazolidine); 6.61 (1H, s, H-10); 6.85–7.51 (7H, m, H Ar); 7.63–7.78 (2H, d, J = 2.6, H Ar). 13C NMR
spectrum, δ, ppm: 13.7 (CH3 ester); 35.4 (C-7); 43.0 (C-8); 48.5 (C-2,3); 54.9 (OCH3); 59.8 (CH2 ester); 60.9
(C-6); 80.3 (C-5); 113.1 (m-C Ar); 121.9 (C-10); 122.9 (o-C Ar); 128.2 (p-C Ar); 128.7 (m-C Ar); 129.2 (o-C Ar);
130.2 (C-9); 135.7 (ipso-C Ar); 140.3 (ipso-C Ar); 160.6 (p-C Ar); 169.2 (C=O).
Ethyl 9-(4-Chlorophenyl)-7-phenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (26). IR spectrum, ,
1
cm–1: 3524, 3386, 3175, 3059, 3029, 2978, 2927, 2858, 1738, 1584, 1448, 1013, 824, 762, 699. H NMR
spectrum, , ppm (J, Hz): 0.91 (3H, t, J = 7.2, CH3 ester); 2.71–2.97 (2H, m, H-8); 3.00–3.15 (1H, m, H-7);
3.60–3.78 (1H, m, H-6); 3.91 (2H, q, J = 6.9, CH2 ester); 3.97–4.13 (4H, m, 2CH2 imidazolidine); 6.08 (2H, s,
13
2NH imidazolidine); 6.59 (1H, s, H-10); 6.88–7.39 (7H, m, H Ar); 7.64–7.79 (2H, d, J = 2.4, H Ar). C NMR
spectrum, δ, ppm: 14.2 (CH3 ester); 35.1 (C-7); 43.0 (C-8); 48.3 (C-2,3); 59.9 (CH2 ester); 61.0 (C-6); 80.2 (C-5);
122.8 (C-10); 122.9 (o-C Ar); 128.0 (p-C Ar); 128.7 (m-C Ar); 129.3 (m-C Ar); 130.2 (o-C Ar); 130.9 (C-9);
137.6 (ipso-C Ar); 140.8 (ipso-C Ar); 141.4 (p-C Ar); 168.9 (C=O).
Ethyl 9-(4-Methoxyphenyl)-7-phenyl-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (27). IR spectrum, ,
1
cm–1: 3450, 3444, 3065, 3033, 2924, 2852, 1736, 1605, 1447, 1037, 757, 695. H NMR spectrum, , ppm
(J, Hz): 0.91 (3H, t, J = 7.0, CH3 ester); 2.72–2.98 (2H, m, H-8); 3.01–3.16 (1H, m, H-7); 3.59–3.71 (1H, m,
H-6); 3.78 (3H, s, OCH3); 3.91 (2H, q, J = 6.8, ester CH2); 3.96–4.08 (4H, m, 2CH2 imidazolidine); 5.93 (2H, s,
2NH imidazolidine); 6.51 (1H, s, H-10); 6.83 (7H, m, H Ar); 7.70–7.81 (2H, d, J = 2.1, H Ar). 13C NMR
spectrum, δ, ppm: 13.7 (CH3 ester); 36.0 (C-7); 43.7 (C-8); 49.2 (C-2,3); 54.3 (OCH3); 59.9 (CH2 ester); 61.0
(C-6); 80.2 (C-5); 116.2 (m-C Ar); 122.3 (C-10); 123.1 (o-C Ar); 128.3 (p-C Ar); 128.6 (m-C Ar); 129.3
(o-C Ar); 131.1 (C-9); 135.6 (ipso-C Ar); 141.4 (ipso-C Ar); 159.2 (p-C Ar); 169.0 (C=O).
Ethyl 9-(4-Chlorophenyl)-7-(4-methylphenyl)-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (28). IR spec-
1
trum, , cm–1: 3459, 3386, 3273, 3169, 3049, 2983, 2923, 2856, 1739, 1612, 1447, 1013, 817, 744, 711. H
NMR spectrum, , ppm (J, Hz): 0.93 (3H, t, J = 7.1, CH3 ester); 2.19 (3H, s, CH3 tolyl); 2.75–2.98 (2H, m, H-8);
2.98–3.15 (1H, m, H-7); 3.57–3.77 (1H, m, H-6); 3.90 (2H, q, J = 6.9, CH2 ester); 4.00–4.10 (4H, m, 2CH2
imidazolidine); 6.00 (2H, s, 2NH imidazolidine); 6.55 (1H, s, H-10); 6.89–7.52 (7H, m, H Ar); 7.83–7.98 (2H,
13
d, J = 2.5, H Ar). C NMR spectrum, δ, ppm: 14.2 (CH3 ester); 20.5 (CH3 tolyl); 35.5 (C-7); 43.2 (C-8); 48.8
(C-2,3); 59.8 (CH2 ester); 61.0 (C-6); 80.2 (C-5); 122.2 (C-10); 122.8 (o-C Ar); 128.2 (p-C Ar); 128.4 (o-C Ar);
128.7 (m-C Ar); 129.4 (m-C Ar); 130.8 (C-9); 137.2 (p-C Ar); 138.4 (ipso-C Ar); 140.7 (ipso-C Ar); 169.0 (C=O).
Ethyl 7-(4-Chlorophenyl)-9-(4-methoxyphenyl)-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (29).
1
IR spectrum, , cm–1: 3442, 3393, 3284, 3065, 2962, 2923, 2850, 1738, 1598, 1457, 1140, 826, 718. H NMR
spectrum, , ppm (J, Hz): 0.93 (3H, t, J = 7.3, CH3 ester); 2.77–2.98 (2H, m, H-8); 2.77–2.98 (1H, m, H-7);
3.48–3.75 (1H, m, H-6); 3.79 (3H, s, OCH3); 3.90 (2H, q, J = 6.9, CH2 ester); 4.01–4.06 (4H, m, 2CH2
imidazolidine); 5.96 (2H, s, 2NH imidazolidine); 6.55 (1H, s, H-10); 6.96–7.42 (6H, m, H Ar); 7.70–7.82 (2H,
d, J = 2.3, H Ar). 13C NMR spectrum, δ, ppm: 13.7 (CH3 ester); 36.0 (C-7); 43.7 (C-8); 49.2 (C-2,3); 55.2 (OCH3);
59.8 (CH2 ester); 60.9 (C-6); 80.2 (C-5); 119.9 (C-10); 120.9 (m-C Ar); 122.8 (o-C Ar); 128.7 (m-C Ar); 128.9
(ipso-C Ar); 129.4 (o-C Ar); 131.3 (C-9); 135.6 (ipso-C Ar); 140.6 (p-C Ar); 161.2 (p-C Ar); 169.2 (C=O).
Ethyl 9-(4-Chlorophenyl)-7-(4-methoxyphenyl)-1,4-diazaspiro[4.5]dec-9-ene-6-carboxylate (30).
1
IR spectrum, , cm–1: 3453, 3393, 3262, 3065, 2951, 2927, 2830, 1738, 1608, 1456, 1032, 824, 749, 711. H
NMR spectrum, , ppm (J, Hz): 0.93 (3H, t, J = 7.2, CH3 ester); 2.87–3.09 (2H, m, H-8); 3.12–3.19 (1H, m,
H-7); 3.59–3.70 (1H, m, H-6); 3.79 (3H, s, OCH3); 3.89 (2H, q, J = 6.7, CH2 ester); 4.05–4.20 (4H, m, 2CH2
imidazolidine); 5.96 (2H, s, 2NH imidazolidine); 6.55 (1H, s, H-10); 7.24–7.62 (6H, m, H Ar); 7.78–7.91 (2H,
581

d, J = 2.5, H Ar). 13C NMR spectrum, δ, ppm: 14.3 (CH3 ester); 35.6 (C-7); 43.1 (C-8); 48.4 (C-2,3); 55.2 (OCH3);
59.9 (CH2 ester); 61.0 (C-6); 79.9 (C-5); 119.9 (C-10); 120.9 (m-C Ar); 122.7 (o-C Ar); 128.2 (ipso-C Ar); 128.7
(m-C Ar); 129.9 (o-C Ar); 131.0 (C-9); 131.7 (ipso-C Ar); 139.8 (p-C Ar); 161.2 (p-C Ar); 169.1 (C=O).
In vitro Antibacterial and Antifungal Activity. All the clinically isolated bacterial and fungal strains
were obtained from the Faculty of Medicine, Annamalai University, Annamalainagar-608 002, Tamil Nadu,
India. MIC values were measured by the twofold serial dilution method [31]. The respective test compounds 21–
30 were dissolved in DMSO to obtain 1 mg/ml stock solution. Seeded broth (broth containing microbial spores)
was prepared in nutrient broth (NB) from 24 h old bacterial cultures on nutrient agar (Hi-media, Mumbai) at
37±1°C, while fungal spores from 1 to 7 days old Sabouraud agar (Hi-media, Mumbai) slant cultures were
suspended in Sabouraud dextrose agar broth (SDB). The colony-forming units (cfu) of the seeded broth were
determined by the plating technique and adjusted in the range of 104–105 cfu/ml. The final inoculum size was
105 cfu/mL for the antibacterial assay and 1.1–1.5×102 cfu/ml for the antifungal assay. Testing was performed at
pH 7.4±0.2 for bacteria (NB) and at pH 5.6 for fungi (SDB). Exactly 0.4 ml of the solution of the test compound
was added to 1.6 ml of seeded broth to form the first dilution. One milliliter of this was diluted with a further 1
ml of seeded broth to give the second dilution and so on until six such dilutions were obtained. A set of assay
tubes containing only seeded broth was kept as control. The tubes were incubated in BOD incubators at 37±1°C
for bacteria and 28±1°C for fungi. MIC values were determined by visual observation after 24 h (for bacteria)
and 72–96 h (for fungi) of incubation. Ciprofloxacin was used as standard for bacterial studies, and Fluconazole
was used as standard for fungal studies.
The authors are thankful to the NMR Research Centre, Indian Institute of Science, Bangalore for re-
cording spectra. One of the authors (V. K.) is grateful to the Council of Scientific and Industrial Research
(CSIR), New Delhi, Republic of India for providing financial support in the form of a CSIR-Senior Research
Fellowship (SRF) in Organic Chemistry. Another author (J. T.) is highly thankful to Annamalai University
authorities for providing a University Studentship.
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