Regioselective synthetic approaches towards 1,2,8,9-tetraazadispiro[4.1.4.2]trideca-2,9-dien-6-ones of potential antimicrobial properties

Article abstract of DOI:10.1016/j.ejmech.2009.01.008

Reaction of 2,5-bis(arylmethylidene)cyclopentanones 1a-d with nitrilimines (generated in situ via triethylamine dehydrohalogenation of the corresponding hydrazonoyl chlorides 2a,b) in 1:2 molar ratio proceeds in a high regioselective manner affording monocycloadducts 3 and dicycloadducts in the form of two isomers 4, 5. Single crystal X-ray diffraction studies of the isolated crystalline form of 3c support the established structure and indicate that the formed product is 7E, 4S, 5R. Antimicrobial activity screening of the synthesized compounds 3-5, utilizing a variety of Gram-positive (Staphylococcus aureus, Enterococcus fecalis and Streptococcus agalactiae), Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris) and yeast (Candida albicans), exhibited that all the prepared analogues acquire promising activities against both Gram-positive and Gram-negative bacteria especially compounds 3b, 4a (antimicrobial active agents against Gram-positive bacteria) and 3c (antimicrobial active agent against Gram-negative bacteria).

Full text of DOI:10.1016/j.ejmech.2009.01.008

European Journal of Medicinal Chemistry 44 (2009) 2447–2451
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Regioselective synthetic approaches towards 1,2,8,9-tetraazadispiro[4.1.4.2]
trideca-2,9-dien-6-ones of potential antimicrobial properties
,
b
c
Adel S. Girgis a , Flora F. Barsoum , Ahmed Samir
*
a Pesticide Chemistry Department, National Research Centre, El-Behoos Street, Dokki, 12622 Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Microbiology Department, Faculty of Veterinary Medicine, Cairo University, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Reaction of 2,5-bis(arylmethylidene)cyclopentanones 1a–d with nitrilimines (generated in situ via trie-
thylamine dehydrohalogenation of the corresponding hydrazonoyl chlorides 2a,b) in 1:2 molar ratio
proceeds in a high regioselective manner affording monocycloadducts 3 and dicycloadducts in the form
of two isomers 4, 5. Single crystal X-ray diffraction studies of the isolated crystalline form of 3c support
the established structure and indicate that the formed product is 7E, 4S, 5R. Antimicrobial activity
screening of the synthesized compounds 3–5, utilizing a variety of Gram-positive (Staphylococcus aureus,
Enterococcus fecalis and Streptococcus agalactiae), Gram-negative bacteria (Escherichia coli, Klebsiella
pneumoniae and Proteus vulgaris) and yeast (Candida albicans), exhibited that all the prepared analogues
acquire promising activities against both Gram-positive and Gram-negative bacteria especially
compounds 3b, 4a (antimicrobial active agents against Gram-positive bacteria) and 3c (antimicrobial
active agent against Gram-negative bacteria).
Received 20 August 2008
Received in revised form
15 December 2008
Accepted 9 January 2009
Available online 20 January 2009
Keywords:
1,2,8,9-Tetraazadispiro[4.1.4.2]trideca-2,
9-dien-6-ones
2,5-Bis(arylmethylidene)cyclopentanones
Nitrilimines
1,3-Dipolar cycloaddition reactions
Antimicrobial properties
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
In the present work, it is intended to investigate the reaction of
2,5-bis(arylmethylidene)cyclopentanones 1 with nitrilimines in an
Spiro-compounds have been known for a long time to be
present in phytochemicals either in alkaloids, lactones or terpe-
noids. Many spirocyclic alkaloids were found to be very potent
nicotinic receptor antagonists [1]. Spiroketals were reported to be
the sub-units of many natural biological interest substances such as
insect pheromones, antifeedants and polyether antibiotics [2].
Many spiro-oxindole derivatives were found to possess very wide
biological applications as antimicrobial, antitumor and inhibitors of
human NK-1 receptor [3–5]. Several azaspiro-compounds were
reported to be tachykinin antagonists and of particular use in the
treatment of depression, anxiety, pain, inflammation, migraine,
emesis or postherpetic neuralgia [6]. The most developed proce-
dure for construction of spiro-containing compounds depends
mainly on cycloaddition reactions, especially 1,3-dipolar cycload-
ditions to exocyclic double bonds [7–9]. Nitrilimines are considered
one of the most important dipole systems which used intensively
for preparation of diazaspiro-compounds where, great attention
was directed towards their reactions due to their high regio- as well
as stereoselective properties [10–12].
attempt not only to study the regioselectivity of these dipole
compounds towards neighbouring olefinic linkages but also to
prepare novel dispiro-containing pyrazoline nucleus. The antimi-
crobial properties of the obtained spiro-containing compounds will
be screened utilizing a variety of Gram-positive, Gram-negative
bacteria and yeast. The interest in synthesis of pyrazoline con-
taining compounds is attributed to their well known properties as
antidepressant [13], monoamine oxidase [14,15] and low density
lipoprotein oxidation [16] inhibitory activities in addition to their
antimycobacterial ‘‘Mycobacterium tuberculosis’’ [17–20] as well as
antifungal [21] properties.
2. Results and discussion
2.1. Chemistry
Reaction of 2,5-bis(arylmethylidene)cyclopentanones 1a–d with
nitrilimines (generated in situ via triethylamine dehydrohalogena-
tion of the corresponding hydrazonoyl chlorides 2a,b) in 1:2 molar
ratio in refluxing dry benzene afforded monocycloadducts 3 and
dicycloadducts 4, 5. The structures of the isolated products 3–5 were
* Corresponding author. Tel.: þ202 22352405; fax: þ202 33370931.
E-mail address: girgisas10@yahoo.com (A.S. Girgis).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.01.008

2448
A.S. Girgis et al. / European Journal of Medicinal Chemistry 44 (2009) 2447–2451
O
PhCCl=NNHR'
+
R
R
1a-d
2a,b
1a, R = Ph
2a, R' = Ph
1b, R = 4-H3CC6H4
1c, R = 4-FC6H4
1d, R= 4-ClC6H4
2b, R' = 4-H3CC6H4
C6H6, TEA
R'
R'
N
R
R'
O
R'
O
O
Ph
N
R
N
N
N
R
N
N
+
Ph
Ph
N
+
N
R
Ph
N
Ph
H
R'
R
R
5
3
4
Compd.
R
R'
3 (mp, °C)
4 (mp, °C)
5 (mp, °C)
a
b
c
d
e
f
Ph
Ph
Ph
53 % (199-201) 19 % (280-282)
---
---
---
---
4-H3CC6H4 21 % (234-236) 41 % (287-289)
4-H3CC6H4
4-FC6H4
Ph
Ph
74 % (227-229)
---
16 % (211-213) 29 % (274-276)
4-FC6H4
4-H3CC6H4
Ph
---
---
---
---
---
---
45 % (232-234)
56 % (270-272)
43 % (260-262)
4-ClC6H4
g
4-ClC6H4 4-H3CC6H4
Scheme 1.
established through different spectroscopic studies (IR,1H,13C NMR)
as well as elemental analyses data (Scheme 1).
(nitrilimine) approaches the dipolarophile (enone system, E-form
configuration) 1 from the less hindered face with retention of the
starting geometrically isomeric configuration for the other
unreacted double bond residue.
The IR spectra of monocycloadducts 3a–d reveal the presence of
a strong carbonyl stretching vibration band at
n
¼ 1712–1707 cmꢀ1
region excluding any cycloaddition reaction pathway that may take
place with this moiety. 1H NMR spectra of 3a–d exhibit the
heterocyclic H-4 as a sharp singlet signal at
d
¼ 4.88–4.92. The
appearance of this signal at the mentioned chemical shift value
region is consistent with many similar previously reported struc-
tures, supporting the assigned regio-isomeric form [22,23]. In
addition, the aliphatic two methylene group protons appear as
multiplet signals at
d
¼ 1.87–2.93. However, the olefinic ylidene
proton is hidden under the multiplet aromatic protons which
makes identification of the geometrical isomerism for the isolated
products so difficult based on this spectroscopic technique. Single
crystal X-ray diffraction studies of 3c (Fig. 1) indicated that the
isolated product is of 7E-configuration. Also, based on these data
the established regio-isomeric form structure is completely
confirmed and the isolated crystalline form product is inferred to
be 4S, 5R. 13C NMR spectrum (APT) of 3c reveals the presence of
a methine carbon (C-4) at
spiro-carbon (C-5) at
with many reported similar structures [22,23] confirming the
established regio-isomeric form. From all the above mentioned
data, it could be concluded that the attacking dipole compound
d
¼ 61.35, in addition to a quaternary
d
¼ 79.09. These observed data are in accord
Fig. 1. Single crystal X-ray diffraction of 3c.

A.S. Girgis et al. / European Journal of Medicinal Chemistry 44 (2009) 2447–2451
2449
The IR spectra of 4 exhibit the carbonyl band at
1749 cmꢀ1. Meanwhile, 1H NMR spectra of 4 show a singlet signal
integrated for two protons at
chemically and magnetically equivalent H-4 and H-11. These
chemical shift values are highly related to the observed ones of
monocycloadducts 3 confirming the established regio-isomeric
form structure. 13C NMR ‘‘APT’’ spectrum of 4b also adds a conclu-
sive evidence for the assumed structure. Where the pyrazoline
n
¼ 1751–
Gram-negative bacteria. However, all the tested compounds seem
completely inactive against the used yeast strain (C. albicans). It has
also generally been noticed that, compounds 3 and 4 are more
effective antibacterial agents than compounds 5. Additionally,
compound 3b seems to be the most effectively prepared Gram-
positive antibacterial agent. Otherwise, compound 3c seems to be
the most effectively prepared Gram-negative antibacterial agent.
This may be attributed to the role of tolyl function attachment to
either 1- or 4-position of pyrazoline ring system as the most
observed enhancing antibacterial properties moiety comparable
with the other adopted residues. However, this assumed role seems
not a general applicable rule governing the observed antibacterial
properties concerning tetraazadispiro-ring system, as exhibited in
compounds 4a and 4b, where the former one ‘‘possessing no tolyl
residue’’ is considered more Gram-positive active agent.
d
¼ 4.57–4.61 assignable to the
HC-4/11 are observed as one signal at
d
¼ 60.46 beside the spiro C-
5/7 which were exhibited at
d
¼ 80.51. From all the aforementioned
data, it could be concluded that the 1,3-dipolar cycloaddition
reaction of nitrilimines (dipoles) attack both the two olefinic
linkages of dipolarophiles 1 superficially (i.e. from the less hindered
face as described in the case of monocycloadduct formation 3)
affording eventually 4.
On the otherhand, structureof dicycloadducts 5e–g was deduced
from their IR spectra which reveal a strong carbonyl stretching
3. Experimental
vibration band at
n
¼ 1749–1741 cmꢀ1. In addition 1H NMR spectra of
5 exhibit the pyrazoline H-4/11 as a singlet signal at
d
¼ 3.52–3.55.
Melting points are uncorrected and recorded on a digital Elec-
trothermal 9100 melting point apparatus. IR spectra (KBr) were
recorded on a JASCO FT/IR 300E spectrophotometer. 1H NMR
spectra were recorded on Varian GEMINI 200 MHz and Varian
MERCURY 300 MHz spectrometers. 13C NMR spectra were recorded
on a Varian MERCURY 300 (75 MHz). The starting compounds 1a–
d [27–29] and 2a,b [30,31] were prepared according to the previ-
ously reported procedures.
The appearance of this signal at the mentioned chemical shift value
region is consistent with many similar reported structures, where
the dipole molecules attack the olefinic linkages of diene system in
a regioselective manner from two opposite faces [24,25]. 13C NMR
(APT) spectrum of 5g adds a good support for the assigned regio-
isomeric form structure which reveals the pyrazoline methine
carbons HC-4/11 as one signal at
5/7) at
previously described similar structures [24,25].
d
¼ 56.70 and the spiro-carbons (C-
d
¼ 80.12. These observed values are highly related to many
From all the above, it could be concluded that 1,3-dipolar
cycloaddition reaction of nitrilimines with 2,5-bis(ar-
ylmethylidene)cyclopentanones 1 under the described reaction
conditions afforded either monocycloadducts 3 or dicycloadducts 4,
5 in a high regioselective manner where many efforts have been
made for either isolation or identification of any different regio-
isomers from the reaction medium but were unsuccessful. The
isolation of two dicycloadduct isomeric forms could be attributed
to the way of nitrilimine (dipole) attack the diene system at either
the same face affording 4 or at different faces giving rise to 5.
3.1. Synthesis of 3a–d, 4a,b,d and 5e–g (general procedure)
A mixture of 1a–d (2.5 mmol) and the corresponding 2a,b
(5 mmol) in dry benzene (20 ml) containing triethylamine
(7.5 mmol) was boiled under reflux for the appropriate time. The
separated triethylammonium chloride was collected and the
reaction mixture was evaporated till dryness under reduced
pressure. The formed solid upon triturating the residue with
methanol (5 ml), was collected and crystallized from a suitable
solvent affording the corresponding 3 or 5. Upon concentrating
the solvent of crystallization to the least amount (z5 ml), 4 was
separated. In case of 5e,g the products were purified by silica gel
(F254) TLC.
2.2. Antimicrobial activity
Antimicrobial activity screening of the synthesized compounds
3–5 was determined by the agar dilution technique as recom-
mended by the Clinical and Laboratory Standard Institute (CLSI) [26]
utilizing a variety of Gram-positive bacteria (Staphylococcus aureus,
Enterococcus fecalis and Streptococcus agalactiae), Gram-negative
bacteria (Escherichia coli, Klebsiella pneumoniae and Proteus vulgaris)
and yeast (Candida albicans). From the obtained results (Table 1)
it has been noticed that, all the tested compounds exhibit
promising antimicrobial properties against both Gram-positive and
3.1.1. 7-(Phenylmethylidene)-1,3,4-triphenyl-1,2-
diazaspiro[4.4]non-2-en-6-one (3a)
Reaction time 40 h, orange crystals from n-butanol ‘‘through
reaction of 1a and 2a affording 3a which was crystallized, filtered
and dried, upon concentrating the filterate 4a was obtained as
almost colourless crystals’’, mp 199–201 ꢁC, yield 53%. IR: nmax
/
cmꢀ1 1711, 1615, 1595. 1H NMR (CDCl3):
d
1.89 (dd, J ¼ 8.4, 13.8 Hz,
1H, upfield H of CH2), 2.06–2.25 (m, 1H, downfield H of CH2), 2.34–
2.49 (m, 1H, upfield H of CH2), 2.79–2.93 (m, 1H, downfield H of
CH2), 4.92 (s, 1H, hetero. H-4), 6.90–7.71 (m, 21H, 20 arom.
H þ olefinic CH). Anal. Calcd. for C32H26N2O (454.55): C, 84.55; H,
5.77; N, 6.16. Found: C, 84.78; H, 5.96; N, 6.25.
Table 1
Minimum inhibitory concentrations (MIC,
different organisms.
mg/ml) of the tested compounds against
3.1.2. 1,3,4,8,10,11-Hexaphenyl-1,2,8,9-
tetraazadispiro[4.1.4.2]trideca-2,9-dien-6-one (4a)
Compounds
Organisms
3a 3b 3c 3d 4a 4b 4d 5e 5f 5g CIP* AMP**
Almost colourless crystals from n-butanol ‘‘through reaction of
1a and 2a affording 3a which was crystallized, filtered and dried,
upon concentrating the filterate 4a was obtained as almost col-
ourless crystals’’, mp 280–282 ꢁC, yield 19%. IR: nmax/cmꢀ1 1749,
Staphylococcus aureus
Enterococcus fecalis
Streptococcus agalactiae 2.5 1.3
Escherichia coli
Klebsiella pneumoniae 2.5 2.5 1.3 2.5 10
5
0.6 2.5 10
1.3 2.5
2.5
2.5 2.5 10 2.5 10 10 0.6 (—)
10 10 10 0.3 (—)
10 10 10 1.3 (—)
10 10 10 10 0.3 (—)
5
10 10 10 0.3 (—)
2.5 1.3
5
5
5
5
5
10 10 10 10 0.3 (—)
5
5
0.6 10 10 10
5
1593, 1560. 1H NMR (CDCl3):
d 1.50–1.60 (m, 2H, CH2), 1.88–1.99 (m,
5
5
5
Proteus vulgaris
Candida albicans
2.5
5
1.3
5
10
2H, CH2), 4.61 (s, 2H, H-4/11), 6.90–7.53 (m, 30H, arom. H). Anal.
Calcd. for C45H36N4O (648.77): C, 83.30; H, 5.59; N, 8.64. Found: C,
83.14; H, 5.51; N, 8.48.
(—) (—) (—) (—) (—) (—) (—) (—) (—) (—) (—)
4
CIP* ¼ Ciprofloxacin; AMP** ¼ Amphotericin B; (—) ¼ inactive.

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A.S. Girgis et al. / European Journal of Medicinal Chemistry 44 (2009) 2447–2451
3.1.3. 3,4-Diphenyl-1-(4-methylphenyl)-7-(phenylmethylidene)-
1,2-diazaspiro[4.4]non-2-en-6-one (3b)
Calcd. for C45H34F2N4O (684.75): C, 78.93; H, 5.00; N, 8.18. Found: C,
79.01; H, 5.11; N, 8.35.
Reaction time 45 h, orange crystals from ethanol ‘‘through
reaction of 1a and 2b affording 3b which was crystallized, filtered
and dried, upon concentrating the filterate 4b was obtained as pale
yellow crystals’’, mp 234–236 ꢁC, yield 21%. IR: nmax/cmꢀ1 1707,
3.1.8. 4,11-Bis(4-fluorophenyl)-1,8-bis(4-methylphenyl)-3,10-
diphenyl-1,2,8,9-tetraazadispiro[4.1.4.2]trideca-2,9-dien-6-one (5e)
Reaction time 50 h, pale yellow crystals ‘‘through reaction of 1c
and 2b affording 5e’’ purified by silica gel TLC using chloroform–light
petroleum (60–80 ꢁC) mixture as 2:3 v/v for elution, mp 232–234 ꢁC,
1610, 1595. 1H NMR (CDCl3):
d
1.87 (dd, J ¼ 8.8 13.2 Hz,1H, upfield H
of CH2), 2.10–2.23 (m, 1H, downfield H of CH2), 2.26 (s, 3H, CH3),
2.32–2.44 (m, 1H, upfield H of CH2), 2.75–2.87 (m, 1H, downfield H
of CH2), 4.88 (s, 1H, hetero. H-4), 7.02–7.67 (m, 20H, 19 arom.
H þ olefinic CH). Anal. Calcd. for C33H28N2O (468.57): C, 84.58; H,
6.02; N, 5.80. Found: C, 84.69; H, 6.14; N, 6.06.
yield 45%. IR: nmax/cmꢀ11749,1604,1556.1H NMR (CDCl3):
d 1.50–1.54
(m, 2H, CH2),1.81–1.83 (m, 2H, CH2), 2.46 (s, 6H, 2CH3), 3.55 (s, 2H, H-
4/11), 6.91–7.32 (m, 26H, arom. H). Anal. Calcd. for C47H38F2N4O
(712.80): C, 79.19; H, 5.37; N, 7.86. Found: C, 79.35; H, 5.44; N, 7.67.
3.1.4. 1,8-Bis(4-methylphenyl)-3,4,10,11-tetraphenyl-1,2,8,9-
tetraazadispiro[4.1.4.2]trideca-2,9-dien-6-one (4b)
3.1.9. 4,11-Bis(4-chlorophenyl)-1,3,8,10-tetraphenyl-1,2,8,9-
tetraazadispiro[4.1.4.2]trideca-2,9-dien-6-one (5f)
Pale yellow crystals from ethanol ‘‘through reaction of 1a and
2b affording 3b which was crystallized, filtered and dried, upon
concentrating the filterate 4b was obtained as pale yellow crys-
tals’’, mp 287–289 ꢁC, yield 41%. IR: nmax/cmꢀ1 1749, 1591, 1560. 1H
Reaction time 50 h, yellow crystals from n-butanol ‘‘through
reaction of 1d and 2a affording 5f’’, mp 270–272 ꢁC, yield 56%. IR:
nmax/cmꢀ1 1741, 1595, 1560. 1H NMR (CDCl3):
d 1.54–1.61 (m, 2H,
CH2), 1.83–1.88 (m, 2H, CH2), 3.53 (s, 2H, H-4/11), 6.84–7.55 (m,
28H, arom. H). Anal. Calcd. for C45H34Cl2N4O (717.66): C, 75.31; H,
4.78; N, 7.81. Found: C, 75.40; H, 4.91; N, 8.06.
NMR (CDCl3):
2.35 (s, 6H, 2CH3), 4.58 (s, 2H, H-4/11), 6.80–7.52 (m, 28H, arom.
H). 13C NMR ‘‘APT’’ (CDCl3):
21.03 (CH3), 25.80 (CH2), 60.46 (C-4/
d 1.40–1.53 (m, 2H, CH2), 1.81–1.90 (m, 2H, CH2),
d
11), 80.51 (spiro C-5/7), 124.55, 126.25, 128.15, 128.19, 128.33,
129.14, 129.33, 129.70 (arom. CH), 131.97, 134.24, 134.72, 141.00,
148.89 (quaternary arom. C), 209.95 (C]O). Anal. Calcd. for
C47H40N4O (676.82): C, 83.40; H, 5.96; N, 8.28. Found: C, 83.15; H,
5.82; N, 8.49.
3.1.10. 4,11-Bis(4-chlorophenyl)-1,8-bis(4-methylphenyl)-3,10-
diphenyl-1,2,8,9-tetraazadispiro[4.1.4.2]trideca-2,9-dien-6-one (5g)
Reaction time 48 h, pale yellow crystals ‘‘through reaction of 1d
and 2b affording 5g’’ purified by silica gel TLC using chloroform–
light petroleum (60–80 ꢁC) mixture as 2:3 v/v for elution, mp 260–
262 ꢁC, yield 43%. IR: nmax/cmꢀ1 1741, 1593, 1560. 1H NMR (CDCl3):
3.1.5. 1,3-Diphenyl-4-(4-methylphenyl)-7-[(4-
d
1.47–1.50 (m, 2H, CH2), 1.71–1.79 (m, 2H, CH2), 2.46 (s, 6H, 2CH3),
3.52 (s, 2H, H-4/11), 6.80–7.31 (m, 26H, arom. H). 13C NMR ‘‘APT’’
(CDCl3): 20.95 (CH3), 25.94 (CH2), 56.70 (HC-4/11), 80.12 (spiro C-
methylphenyl)methylidene]-1,2-diazaspiro[4.4]non-2-en-6-one (3c)
Reaction time 45 h, orange crystals from n-butanol ‘‘through
reaction of 1b and 2a affording 3c’’, mp 227–229 ꢁC, yield 74%. IR:
d
5/7), 125.99, 126.13, 128.14, 128.56, 129.10, 129.52, 130.23 (arom.
CH), 131.23, 133.13, 133.81, 135.68, 142.03, 151.39 (quaternary arom.
C), 208.83 (C]O). Anal. Calcd. for C47H38Cl2N4O (745.71): C, 75.70;
H, 5.14; N, 7.51. Found: C, 75.49; H, 5.04; N, 7.34.
nmax/cmꢀ1 1707, 1620, 1599. 1H NMR (CDCl3):
d
1.90 (dd, J ¼ 8.4,
13.4 Hz, 1H, upfield H of CH2), 2.10–2.29 (m, 1H, downfield H of
CH2), 2.34 (s, 3H, CH3), 2.41 (s, 3H, CH3), 2.38–2.50 (m, 1H, upfield H
of CH2), 2.80–2.93 (m, 1H, downfield H of CH2), 4.89 (s, 1H, hetero.
H-4), 6.85–7.69 (m, 19H, 18 arom. H þ olefinic CH). 13C NMR ‘‘APT’’
3.2. Single crystal X-ray crystallographic data of 3c
(CDCl3): d 21.14, 21.51 (CH3), 24.01, 25.26 (CH2), 61.35 (HC-4), 79.09
(spiro C-5), 116.77, 120.61, 126.52, 127.97, 128.05, 128.77, 129.45,
129.60, 131.11, 136.50 (arom. CH þ olefinic CH), 131.78, 132.03,
132.26, 132.34, 137.82, 140.64, 142.58, 147.12 (arom. quaternary C),
204.70 (C]O). Anal. Calcd. for C34H30N2O (482.60): C, 84.61; H,
6.27; N, 5.81. Found: C, 84.42; H, 6.16; N, 5.88.
Compound 3c was recrystallized as prismatic orange crystals
from n-butanol. The crystallographic data were collected at
T ¼ 298 K on a Kappa CCD Enraf Nonius FR 590 diffractometer using
a graphite monochromator with Mo-Ka radiation (
l
¼ 0.71073 Å).
The crystal structure was determined by SIR97 [32] and refined by
maXus [33] (Bruker Nonius, Delft and MacScience, Japan). Chemical
formula C34H30N2O, Mr ¼ 482.627, triclinic, crystallizes in space
group P-1, Cell lengths ‘‘a ¼ 10.4100(3), b ¼ 11.0315(4),
3.1.6. 1,3-Diphenyl-4-(4-fluorophenyl)-7-[(4-
fluorophenyl)methylidene]-1,2-diazaspiro[4.4]non-2-en-6-one (3d)
Reaction time 48 h, orange crystals from n-butanol ‘‘through
reaction of 1c and 2a affording 3d which was crystallized, filtered
and dried, upon concentrating the filterate 4d was obtained as
almost colourless crystals’’, mp 211–213 ꢁC, yield 16%. IR: nmax/cmꢀ1
c ¼ 12.4118(5) Å’’, Cell angles ‘‘
a
¼ 101.044(2),
b
¼ 104.167(2),
g
¼ 101.047(2)ꢁ’’, V ¼ 1313.11(8) Å3, Z ¼ 4, Dc ¼ 2.441 mg/m3,
q
values
2.910–27.485ꢁ,
absorption
coefficient
m
(Mo-
Ka) ¼ 0.15 mmꢀ1, F(000) ¼ 1024. The unique reflections measured
1712, 1622, 1595. 1H NMR (CDCl3):
d
1.87 (dd, J ¼ 8.2, 13.4 Hz, 1H,
9858 of which 2862 reflections with threshold expression I > 3s(I)
upfield H of CH2), 2.04–2.23 (m, 1H, downfield H of CH2), 2.35–2.51
(m, 1H, upfield H of CH2), 2.77–2.90 (m, 1H, downfield H of CH2),
4.90 (s, 1H, hetero. H-4), 6.88–7.65 (m, 19H, 18 arom. H þ olefinic
CH). Anal. Calcd. for C32H24F2N2O (490.53): C, 78.35; H, 4.93; N,
5.71. Found: C, 78.49; H, 5.09; N, 5.93.
were used in the structural analysis. Convergence for 334 variable
parameters by least-squares refinement on F2 with w ¼ 1/
[
s
2(Fo2) þ 0.10000Fo2]. The final agreement factors were R ¼ 0.048
and wR ¼ 0.083 with a goodness-of-fit of 1.391. Full crystallographic
details excluding structure factors; have been deposited at Cam-
bridge Crystallographic Data Centre (CCDC) as supplementary
publication number CCDC 713181.
3.1.7. 4,11-Bis(4-fluorophenyl)-1,3,8,10-tetraphenyl-1,2,8,9-
tetraazadispiro[4.1.4.2]trideca-2,9-dien-6-one (4d)
Almost colourless crystals from n-butanol ‘‘through reaction of
1c and 2a affording 3d which was crystallized, filtered and dried,
upon concentrating the filterate 4d was obtained as almost col-
ourless crystals’’, mp 274–276 ꢁC, yield 29%. IR: nmax/cmꢀ1 1751,
3.3. Antimicrobial activity screening
Antimicrobial activity screening of the synthesized compounds
3–5 was determined by the agar dilution technique as recom-
mended by the Clinical and Laboratory Standard Institute (CLSI)
[26]. The tested compounds were dissolved in dimethyl sulfoxide
1595, 1560. 1H NMR (CDCl3):
d
1.45–1.56 (m, 2H, CH2), 1.95–2.05 (m,
2H, CH2), 4.57 (s, 2H, H-4/11), 6.87–7.51 (m, 28H, arom. H). Anal.

A.S. Girgis et al. / European Journal of Medicinal Chemistry 44 (2009) 2447–2451
2451
(DMSO). An inoculum of about 1.5 ꢂ108 colony forming unit (CFU)
per spot was applied to the surfaces of Mueller–Hinton agar plates
containing graded concentrations of the respective compound;
plates were incubated at 37 ꢁC for 18 h. The spot with the lowest
concentration of compound showing no growth was defined as the
minimum inhibitory concentration (MIC). All organisms used in
this study were standard strains obtained from American Type
Culture Collection (ATCC). The organisms included representatives
of Gram-positive bacteria (S. aureus 25923, E. fecalis 29212 and S.
agalactiae 123860), Gram-negative bacteria (E. coli 25922, K.
pneumoniae 33495 and P. vulgaris 13315) and yeast (C. albicans
20260). The MIC of Ciprofloxacin and Amphotericin B was deter-
mined concurrently as reference for antibacterial and antifungal
activities, respectively (Table 1). Control DMSO was carried out
with each experiment.
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Acknowledgment
This work is sponsored by the U.S.-Egypt Science and Tech-
nology Joint Fund under Project No. MAN10-007-002.
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