Cytotoxic 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienes mounted on alicyclic scaffolds

Article abstract of DOI:10.1016/j.ejmech.2005.12.014

The 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienyl pharmacophore was incorporated into four series of compounds 1-4. Compounds 1a-g comprised a cluster of 3-arylidene-1-(4-nitrophenylmethylene)-2-oxo-3,4-dihydro-1H-naphthalenes while the analogues 2a-g co

Full text of DOI:10.1016/j.ejmech.2005.12.014

European Journal of Medicinal Chemistry 41 (2006) 577–585
http://france.elsevier.com/direct/ejmech
Original article
Cytotoxic 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienes
mounted on alicyclic scaffolds
Umashankar Das a, H. Inci Gul a,b, Jane Alcorn a, Anuraag Shrivastav c, Theresa George c,
Rajendra K. Sharma c, Kurt H. Nienaber d, Erik De Clercq e, Jan Balzarini e, Masami Kawase f,
Noriyuki Kan f, Toru Tanaka f, Satoru Tani f, Karl A. Werbovetz g, Adam J. Yakovich g,
Elias K. Manavathu h, James P. Stables i, Jonathan R. Dimmock a,â
a College of Pharmacy and Nutrition, University of Saskatchewan, 110 Science Crescent, Saskatoon, Saskatchewan S7N 5C9, Canada
b Ataturk University, Erzurum, Turkey
c Department of Pathology, College of Medicine and Cancer Research Unit,
Saskatoon Cancer Centre, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada
d Department of Biochemistry, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan S7N 5E5, Canada
e Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
f Faculty of Pharmaceutical Sciences, Josai University, Keyakida, Sakado, Saitama 350-0295, Japan
g College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, Ohio 43210-1291, USA
h Department of Medicine, Wayne State University, 550 E. Canfield, Detroit, Michigan 48201-1098, USA
i National Institute of Neurological Disorders and Stroke, 6001 Executive Boulevard, Rockville, Maryland 20852, USA
Received 2 August 2005; received in revised form 12 October 2005; accepted 12 December 2005
Available online 03 April 2006
Abstract
The 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienyl pharmacophore was incorporated into four series of compounds 1–4. Compounds 1a–g
comprised a cluster of 3-arylidene-1-(4-nitrophenylmethylene)-2-oxo-3,4-dihydro-1H-naphthalenes while the analogues 2a–g consisted of a
group of 6-arylidene-2-(4-nitrophenylmethylene)cyclohexanones. Three other compounds prepared in this study were 1-(4-nitrophenylmethy-
lene)-3-(3,4,5-trimethoxyphenylmethylene)-2-oxo-2,3-dihydro-1H-indene 3a as well as two 5-arylidene-2-(4-nitrophenylmethylene)cyclopenta-
nones 4a, b. The compounds were evaluated against human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 cells. In general,
the compounds in series 1 displayed marked cytotoxicity having IC50 values in the 1–5 μM range while the related cyclohexyl analogues in series
2 were slightly less potent (IC50 figures were mainly 5–10 μM). The relative locations of two aryl rings present in all four series were considered
to contribute significantly to bioactivity and may have accounted for the virtual absence of cytotoxic properties in series 3 and 4. Most of the
compounds were administered intraperitoneally to mice using doses up to and including 300 mg/kg. No mortalities were noted. The inhibiting
effect of most of the compounds towards Helicobacter pylori is noteworthy. The modes of action of representative compounds include the
induction of apoptosis while some compounds weakly inhibited tubulin polymerisation and human N-myristoyltransferase.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Conjugated unsaturated ketones; Molecular modelling; Cytotoxicity; Structure–activity relationships; Apoptosis; Rodent toxicity
1. Introduction
ent series of compounds have been designed which utilized the
1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore (Fig. 1) [1–3].
In particular, 1-(4-nitrophenylmethylene)-3-(3,4,5-trimethoxy-
phenylmethylene)-2-oxo-3,4-dihydro-1H-naphthalene 1a de-
monstrated marked cytotoxic potencies having IC50 values of
1–2 μM towards human Molt 4C8 and CEM T-lymphocytes
as well as murine L1210 cells (Table 1) and 0.59 μM when
evaluated against a panel of 56 human tumour cell lines [3].
The principal interest in our laboratory is focussed on studies
aimed at producing novel cytotoxic and anticancer agents. Var-
ious structural motifs have been investigated and recently differ-
â Corresponding author.
E-mail address: dimmock@skyway.usask.ca (J.R. Dimmock).
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.12.014

578
U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
emerge. These considerations led to the decision to prepare the
compounds in series 1–4.
An initial study was directed to determining if the com-
pounds in series 1–4 displayed greater toxicity to neoplasms
than normal cells and whether reversal of multidrug resistance
(MDR) in cancer cells would be demonstrated. The use of
HSC-2, HSC-3, HSG and HL-60 human tumour cells as well
as the non-malignant human HGF, HPC and HPLF cell lines
revealed that a number of the compounds were selectively
toxic to the neoplasms while MDR reversal was noted in both
human colon cancer Colo 320 and murine lymphoma L-5178
cells in several cases [7]. These encouraging results confirmed
that further experimentation on these compounds should be
conducted which is described herein. The development in-
cluded evaluating the compounds in series 1–4 against addi-
tional cancer cell lines which have been used previously in
evaluating candidate cytotoxics, examining the topography of
various molecules, probing as to the mode(s) of action of re-
presentative compounds and undertaking toxicity assessments
in mice.
Fig. 1. The 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore.
In order to develop further compounds based on 1a, the
molecular modification of this dienone was planned in two
general ways. First, conjugated unsaturated ketones are thiol
alkylators [4,5]. By varying the electronic properties of the
substituents in rings A and B (Fig. 1), sequential interactions
with cellular constituents is likely. Since successive chemical
insults may be more detrimental to malignant cells than the
corresponding normal cells [6], selective toxicity to neoplasms
may occur. Thus the nitro group was retained in ring A while
in general electron-donating substituents were placed in ring B.
Second, incorporation of the 1,5-diaryl-3-oxo-1,4-pentadienyl
pharmacophore onto various alicycles may cause the relative
positions of aryl rings A and B to vary. A correlation between
the topography of the molecule and cytotoxicity may therefore
2. Chemistry
The synthetic chemical routes employed which led to the
preparation of the compounds in series 1–4 are presented in
Scheme 1. Various strategies were used to prepare the inter-
mediates 5–8 which were then condensed with different aryl
aldehydes producing the compounds 1–4. The manner in
which the monoarylideneketones 5–8 were synthesized is as
follows. Condensation of 2-oxo-3,4-dihydro-1H-naphthalene
with 4-nitrobenzaldehyde led to the formation of the enone 5.
Reaction of cyclohexanone with 4-nitrobenzaldehyde under ba-
sic conditions led initially to the isolation of the corresponding
aldol. Subsequently the reaction was repeated whereby the in-
termediate aldol formed was dehydrated with acid giving rise
to 2-(4-nitrophenylmethylene)cyclohexanone 6. Reaction of 2-
oxo-2,3-dihydro-1H-indene with 4-nitrobenzaldehyde in the
presence of sodium hydroxide or hydrochloric acid led to the
formation of polymers and 1,3-bis(4-nitrophenylmethylene)-2-
oxo-2,3-dihydro-1H-indene one, respectively. An alternative
procedure was employed whereby an enamine from 2-oxo-
2,3-dihydro-1H-indene was prepared which reacted in good
yield with 4-nitrobenzaldehyde to give the desired product 7.
A similar route was undertaken in the conversion of cyclopen-
tanone into 2-(4-nitrophenylmethylene)cyclopentanone 8. The
compounds in series 1–4 were shown from 1H NMR spectro-
scopy to be stereoisomerically pure and exhibited the E,E con-
figuration. This observation is in accord with recent X-ray
crystallographic determinations of various 1,3-diarylidene-2-
oxo-3,4-dihydro-1H-naphthalenes [3] and 2,6-bis(arylidene)cy-
clohexanones [8–10] which revealed that the olefinic double
bonds adopted the E,E configuration. Molecular models of
1a, 2a, 3a and 4a were built in order to find the relative posi-
tions of different atoms and groups. These determinations are
illustrated in Fig. 2 while the data are presented in Table 2.
Table 1
Evaluation of various compounds in series 1–4 against human Molt 4/C8 and
CEM T-lymphocytes, murine L1210 cells and Helicobacter pylori
Compound
IC50(μM)a
Molt 4/C8
1.56 0.01
12.3 4.1
0.968
0.186
0.992
CEM
L1210
H. pylori
1ab
1b
1c
1.54 0.08
25.5 2.5
0.953
1.94 0.12
49.5 0.8
3.20 0.48
6.17 0.12
23.2 0.08
8.07 0.14
0.377
1d
1.64 0.61
2.52 0.01
6.63 0.15
0.672
1e
1.13 0.64
6.16 1.11
1.72 0.03
6.42 1.07
300 54
8.48 0.25
9.49 1.71
> 500
9.41 0.18
9.12 0.28
442 82
> 500
1.62 0.11
7.37 0.14
1.70 0.01
4.61 3.89
4.82 2.43
8.67 0.48
4.18 2.16
6.97 1.80
8.34 0.12
17.4 0.09
–
46.6 0.10
>100
14.2 0.11
5.80 0.13
>100
11.3 0.14
5.91 0.14
–
19.0 0.09
>100
–
–
–
1f
1gb
2a
2b
2c
2d
2e
2f
2g
3a
250
6
240
8
8.14 0.01
7.61 0.01
> 500
11.2 4.1
9.78 0.87
> 500
7.27 0.93
8.18 0.20
320 43
> 500
9.95 0.35
9.41 0.97
216 13
> 500
4a
4b
> 500
> 500
> 500
5b
34.0 5.9
14.9 0.5
3.24 0.79
–
25.5 14.8
14.5 0.4
2.47 0.30
–
39.4 0.0
54.5 1.7
2.13 0.03
–
6c
Melphalan
Metronidazole
309
a
The IC50 values are the concentrations required to inhibit the growth of
50% of the neoplastic cells (with standard deviation figures) and H. pylori
(standard errors are presented).
b
Data taken from Ref. [3] and reproduced with the permission of the copy-
right owner.
c
Data taken from Ref. [38] and reproduced with the permission of the copy-
right owner (www.tandf.co.uk).

U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
579
Scheme 1. The reagents used in the syntheses of the compounds in series 1–4 were as follows, namely i = piperidine/acetic acid/toluene; ii = dry HCl/chloroform/
ether; iii = NaOH/H2O; iv = HCl/ethanol; v = morpholine/PTSA/benzene. The aryl substituents were as follows: a: R1 = R2 = R3 = OCH3; b: R1 = R3 = H, R2 =
OCH3; c : R1 = R2 = OCH3, R3 = H; d: R1 = OCH3, R2 = OH, R3 = H; e : R1 = R2 = OCH2O, R3 = H; f : R1 = R3 = H; R2 = OH; g: R1 = R3 = H, R2 = Cl.
3. Bioevaluations
lon, central nervous system, ovarian, renal, prostate and breast
cancers and the results are summarized in Table 3. Most of the
compounds in series 1–4 were screened against Helicobacter
pylori and the IC50 values are given in Table 1. HepG2 cells
treated with 1 μM of 1e underwent apoptosis. The IC50 values
of 1a–f, 2a–g and 4a, b towards urease were greater than
200 μg/ml. The IC50 values of 1c, 2a and 4a when examined
for their efficacy in inhibiting tubulin polymerisation were in
The enones 1a–g, 2a–g, 3a, 4a, b, 5 and 6 were evaluated
against human Molt 4/C8 and CEM T-lymphocytes as well as
murine L1210 cells. These data are presented in Table 1. The
unsaturated ketones 1b, e, f were examined towards approxi-
mately 54 human tumour cell lines from nine different neopla-
sias, namely leukemia, melanoma and non-small cell lung, co-

580
U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
Table 2
Interatomic distances d1–d3, bond angle ψ1 and torsion angles θ1 and θ2 determined by molecular modelling in 1a, 2a, 3a and 4a
Compound
d1 (Å)
10.07
10.15
9.799
9.685
d2 (Å)
5.643
5.600
5.749
5.780
d3 (Å)
5.625
5.593
5.742
5.772
ψ1 (°)
126.8
130.1
117.0
113.9
θ1(°)
58.3
67.9
-89.7
-56.6
θ2(°)
80.3
-66.2
-90.0
-54.9
1a
2a
3a
4a
Over the time span of 0.25–4 hours, neither deaths nor neuro-
toxicity were observed in any of these experiments with rats.
4. Results and discussion
The compounds in series 1–4 were evaluated against human
Molt 4/C8 and CEM T-lymphocytes as well as murine L1210
cells. The results are summarized in Table 1.
The relative potencies between the lead molecule 1a and
related compounds in series 1 revealed a consistent pattern
using the Molt 4/C8 and CEM assays. These determinations
showed that in comparison to 1a, 1c was more potent, 1d, e
were equipotent and 1b, f, g had higher IC50 values than 1a.
This observation indicated that oxygen-containing substituents
at positions 3 and 4 in ring B were optimal in terms of potency
while a single substituent in the para location of this ring in
1b, f, g lowered cytotoxic potencies significantly. Somewhat
different structure–activity relationships were noted in the mur-
ine L1210 assay. For example, while 1g and 1a were equipo-
tent, other members of series 1 had higher IC50 values than 1a.
The IC50 values of 1a–g in these three cytotoxicity screens
were compared with those for melphalan. Greater potencies
than the reference drug were displayed by 1a, c–e, g, 1a, c,
e, g and 1a in the Molt 4/C8, CEM and L1210 screens, respec-
tively, while 1d, g and melphalan were equipotent in the CEM
and L1210 assays. Thus, in these three screens, over one half
of the compounds possessed greater or equal potencies com-
pared to the established alkylating agent melphalan revealing
the potential of this cluster of compounds for further develop-
ment.
Fig. 2. A. Designations of different parts of the 1,5-diaryl-3-oxo-1,4-
pentadienyl group in 2a. B. The interatomic distances d1-d3 and bond angle
ψ1 in 1a, 2a, 3a and 4a.
Table 3
Evaluation of 1b, e, f and reference drugs against human tumour cell lines
Compound
All cell lines
Leukemic cells
IC50
Colon cancer cells
a
GI50
SR
SIb
IC50
(μM)
8.32
3.39
1.98
49.9
5.82
SIb
(μM)
19.1
3.80
2.82
24.5
29.5
(μM)
7.76
1.35
1.51
3.98
52.9
1b
1e
1f
> 36.3
> 191
21.9
178
> 4365
2.46
2.82
1.87
6.16
0.56
2.30
1.12
1.42
0.49
5.07
Melphalan
5-Fluorouracil
a
The letters SR refer to the selectivity ratio viz the ratio of the IC50 values
of the least and most sensitive cell lines.
b
The letters SI indicate the selectivity index viz. the ratio of the GI50 figures
divided by the average IC50 values for leukemic and colon cancer cell lines.
A question to be resolved was whether greater cytotoxicity
was obtained using the 3,4-dihydro-1H-naphthalenyl or cyclo-
hexyl frameworks in series 1 and 2, respectively. A compari-
son was therefore made between the IC50 values of those com-
pounds bearing identical substituents in rings A and B in each
of the screens, e.g. the data for 1a and 2a were compared in the
Molt 4/C8 screen, then in the CEM test and so forth. With the
exception of 1f and 2f, which had statistically indistinguishable
IC50 values in the CEM screen, all the compounds in series 1
had greater cytotoxicity; the largest difference being between
the biodata generated in all three screens for 1e and 2e.
In order to seek explanations for the variation in cytotoxi-
city between series 1–4, molecular models of a representative
compound in each series were built, namely 1a, 2a, 3a and 4a.
Overlap of atoms 1–6 (Fig. 2A) were made between pairs of
compounds so that each unsaturated ketone was compared with
the other analogs in turn. The root mean square (RMS) values
generated were invariably less than 0.2 Å. Thus, this group
excess of 50 μM (1c, 2a) or 25 μM (4a). The enzyme human
N-myristoyltransferase (hNMT) catalyses the covalent attach-
ment of the myristoyl group to the N-terminal glycine residue
of a variety of proteins [11]. A representative compound 1a
was evaluated against hNMT and at the maximum concentra-
tion utilized, namely 1 mM, the percentage inhibition of this
enzyme was 23.2. Compounds 1b, e, f were screened against
five species of Aspergillus fumigatus and four Candida spe-
cies; however the minimal inhibitory concentration (MIC) va-
lues were in excess of 32 μg/ml. Doses of 30, 100 and 300 mg/
kg of 1a, b, e, f, 2a–g, 3a and 4a, b were administered intra-
peritoneally to mice and the animals were examined after 0.5
and 4 hours. No mortalities were noted, while minimal neuro-
toxicity was noted with 2a and 3a. Doses of 30 or 50 mg/kg of
1e, f and 2g were administered orally to rats while a further
study with 1e used a dose range of 25–250 mg/kg. A 30 mg/
kg dose of 1e and 2g was injected intraperitoneally into rats.

U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
581
occupied similar spatial arrangements in all four compounds.
Hence, the differences in cytotoxicity between the compounds
in series 1–4 are unlikely to be due to variations in the shapes
of the dienone substructure. However, the overlap of atoms 1–
6 revealed that rings A and B occupied significantly different
locations. In order to compare the locations of rings A and B in
1a, 2a, 3a and 4a, measurements were made between the cen-
tres of the aryl rings (I and III in Fig. 2A) and secondly, in
relation to the ketonic oxygen atom (II in Fig. 2A), taken as
the reference point. The distances d1–d3 and angle ψ1, as indi-
cated in Fig. 2B, enabled the relative positions of the aryl rings
in 1a, 2a, 3a and 4a to be found. These data are presented in
Table 2. In addition, the literature records a number of correla-
tions between the torsion angles between aryl rings and the
adjacent unsaturated groups with bioactivity [12]. Hence the
θ1 and θ2 angles as indicated in Fig. 2A were measured and
are presented in Table 2.
The cytotoxicity data in Table 1 revealed that 1a and 2a
have significant cytotoxic potencies (IC50 figures in the 1–7
μM range) while for 3a and 4a, the IC50 values are in excess
of 200 μM. The results in Table 2 indicate that the d1–d3 and
ψ1 figures are similar for 1a and 2a while little variation was
noted between 3a and 4a. Thus, the preferable locations of
rings A and B in series 1 and 2 likely contributed to the greater
toxicity of these compounds compared to the analogues con-
taining a five-member alicyclic ring. The θ1 values of 1a, 2a,
3a and 4a were 58°, 68°, 90° and 123° when considered in a
clockwise rotation of the deviation from coplanarity between
ring A and the adjacent olefinic linkage. Since the relative po-
tencies of these four compounds in the Molt 4/C8, CEM and
L1210 assays were 1a > 2a > 3a > 4a, an inverse relationship
between the size of the θ1 value and cytotoxic potency was
suggested. On the other hand, no correlations between the
IC50 figures in these cytotoxicity screens with the θ2 values
were discerned.
If cytotoxicity was caused by thiol alkylation, the possibility
of chemosensitivity occurring in the tumour cells was ad-
dressed by considering the cytotoxicity data in Table 1 for
the compounds 1a–g, 2a–g, 5 and 6. By chemosensitivity is
meant that initial depletion of thiols in the tumours sensitise
these cells to a further chemical insult which could be caused
by 1a–g and 2a–g but not 5 and 6. In the absence of chemo-
sensitivity, the potencies of the bis thiol alkylators 1a–g should
be twice that of the monoalkylator 5; similarly 2a–g would be
predicted to have double the potencies of 6. However, if sensi-
tisation of the cells occurred, then the second attack on cells
which had been made vulnerable by the initial thiol depletion
[6] will be demonstrated by the IC50 values of 1a–g and 2a–g
being greater than twice those of 5 and 6, respectively. The
data in Table 1 revealed that 1a–g were more than twice as
potent as 5 in 19 of the 21 comparisons made (91%). On the
other hand, 2a–g possessed more than twice the potency of 6 in
only 33% of the cases. Thus, while synergism was observed
62% of the time thus favouring the design of bifunctional al-
kylators, it is clearly dependent on the general structure of the
series of compounds under examination.
Three compounds in series 1, namely 1b, e, f, were evalu-
ated against 54 2 human tumour cell lines from nine different
neoplastic diseases. The concentration range employed was
10–4–10–8 M and on rare occasions the growth of the cells at
the maximum concentration of 10–4 M was not inhibited by
50%. Hence, the term GI50 was used and not IC50. The data
are presented in Table 3. The GI50 values demonstrated that
1b, e, f were more cytotoxic than melphalan and, in particular,
1e and 1f possessed 6.5 and 8.7 times the potency of the re-
ference drug. A positive feature of a candidate cytotoxin is ex-
hibition of varying potencies towards different cell lines. This
property may reveal that the compound in question possesses a
selective toxicity for one or more groups of cancers accompa-
nied by a much lower toxicity to the corresponding normal
cells. The enones 1b, e, f displayed varying potencies to the
cell lines and 1e possessed a greater selectivity ratio than mel-
phalan. An examination of the mean graphs [13] of 1b, e, f
revealed that, in general, a preferential toxicity for leukemic
and colon cancer cells was observed. The data in Table 3 in-
dicated that towards leukemic cells, 1e, f were more potent
than melphalan, which is effective towards chronic lymphatic
leukemia [14]. The average selectivity index (SI) value for 1b,
e, f was 2.4, which is significant although less than the value
for melphalan. 5-Fluorouracil is used in treating colon cancers
[15]. The enones 1e, f were more potent than 5-fluorouracil,
especially 1f, which possessed three times the potency of this
drug. The average SI value of 1b, e, f is 1.6, which is less than
for 5-fluorouracil. The conclusions that may be drawn from the
assay using a panel of human tumour cell lines is that 1e, f are
very useful lead molecules due to their potencies towards ma-
lignant cells in general and leukemic and colon cancers in par-
ticular.
A major challenge in cancer research is the successful treat-
ment of stomach cancer. This condition arises from various
causes, one of which is chronic infections with Helicobacter
pylori [16]. A useful drug used in treating these infections is
the nitroheterocycle metronidazole and thus the evaluation of
some of the nitroaromatics prepared in this study as candidate
drugs for treating H. pylori was considered a worthwhile inves-
tigation. A wide range of concentrations was employed viz.
approximately 2–5000 μM. In most cases the growth of the
microorganism was inhibited at lower concentrations up to
70–200 μM while higher concentrations led to stimulation of
the growth of H. pylori. The IC50 values of 1a–f, 2a–g and 4a,
b generated in this screen are presented in Table 1. The follow-
ing observations were made pertaining to structure–activity re-
lationships. First, in general, the compounds in series 1 had
lower IC50 values than the analogues in series 2. Thus 1a–c,
e were more potent than 2a–c, e and the average IC50 figures
for 1a–f and 2a–f were 11.6 and > 46.3 μM, respectively. Sec-
ond, the order of potency in series
1
was
1a > 1d > 1c > 1e > 1f > 1b revealing that the structures of
the four most potent compounds (IC50 values < 10 μM) each
possess substituents in both the 3 and 4 positions in ring B.
Third, the relationship between the nature of the aryl substitu-
ents in ring B of series 2 and antibacterial potencies was dif-
ferent from series 1 insofar as the relative potencies in series 2

582
U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
were 2d, g > 2f > 2c > 2a > 2b, e. Fourth, 4a, which contains a
cyclopentane ring, possessed more than twice the potency of
the cyclohexyl analogue 2a. Finally, most of the compounds
were far more potent than the established drug metronidazole.
An important conclusion to be drawn from these experiments
is that a number of the compounds prepared in this study are
novel prototypic anti-H. pylori agents.
With a view to identifying the way in which these com-
pounds exert their cytotoxic activity, experiments were in-
itiated to determine whether these enones would (i) induce
apoptosis, (ii) inhibit the sulphydryl enzyme urease, (iii) pre-
vent tubulin formation and (iv) exert any effect on hNMT.
Several years ago, compound 9, which contains two olefinic
groups in different electronic environments, as well as melpha-
lan, were shown to induce apoptosis in human Jurkat T leuke-
mia cells [17]. In fact a number of cytotoxic compounds are
apoptotic [18]. The question posed was whether a representa-
tive compound prepared in this study would also induce apop-
tosis in a malignant cell line. Compound 1e was chosen since
the data in Tables 1 and 3 reveal not only its excellent cyto-
toxicity but also its superior SR value compared to melphalan
(Table 3). A concentration of 10 μM of 1e was lethal to human
HepG2 liver cancer cells while the death of some of the cells
was noted at lower concentrations of 1 and 0.1 μM. A TUNEL
assay was conducted using 1 μM of 1e which provided unequi-
vocal evidence that 1e causes apoptosis. The concentrations at
which apoptosis is noted is similar to the quantities of com-
pound required to generate the data for 1e in Tables 1 and 3.
biocidal in general or whether some specificity for malignant
cells could be discerned. Two approaches were adopted to as-
sess this possibility, namely the examination of representative
compounds towards certain pathogenic fungi as well as toxicity
evaluations in mice and rats.
Second, ureases are sulphydryl enzymes which have a cy-
steine residue in the active site [19]. In particular, H. pylori
produces a urease which catalyses the hydrolysis of urea to
ammonia which makes possible the inhabitation of this micro-
organism in the stomach [20]. A number of α,β-unsaturated
ketones have demonstrated urease-inhibiting properties [21]
but the IC50 values of 1a–f, 2a–g, 4a, b towards this enzyme
were in excess of 200 μg/ml. Third, representative compounds
in series 1, 2 and 4 were examined for inhibition of tubulin
polymerisation. A concentration of 50 μM of 1c and 2a caused
16% and 40% inhibition, respectively, while solubility pro-
blems were encountered with 4a at this concentration. Using
25 μM of 4a, 16% inhibition of tubulin polymerisation was
observed. Thus, the compounds interact at one of the tubulin
binding sites [22]. Since the average IC50 values of 1c, 2a and
4a against the Molt 4/C8, CEM and L1210 cells is 1.71, 6.00
and > 500 μM, respectively, no correlation between inhibition
of tubulin polymerisation and cytotoxic potencies was ob-
served. Fourth, one of the differences between certain tumours,
such as some colon cancers and the surrounding normal tis-
sues, is the higher activity and expression of hNMT in the ma-
lignant cells [23]. The active site of a fungal NMT is believed
to contain a mercapto group [24] and hence the thiol alkylators
prepared in this study may exert their bioactivity, at least par-
tially, by this mechanism. Accordingly, the effect of the lead
molecule 1a on hNMT was undertaken. At the highest concen-
tration of 1a utilized, namely 1 mM, the enzyme was inhibited
by 23.2%. The IC50 figures of 1a in Table 1 are < 2 μM and
the average IC50 values of this compound towards human co-
lon and leukemic lines are < 0.4 μM [3]; hence the inhibition
of hNMT exerts, at the most, only a minor role in causing the
cytotoxicity of 1a.
Compounds 1b, e, f were examined for antifungal properties
using different strains of A. fumigatus and four Candida spe-
cies. In each case the MIC values of these compounds were in
excess of 32 μg/ml (78–91 μM). In view of the data presented
in Tables 1 and 3, 1b, e, f were clearly far more toxic to ma-
lignant cells than to the fungi. No acute mortalities were noted
when doses up to and including 300 mg/kg of 1a, b, e, f, 2a–g,
3a, 4a, b were administered intraperitoneally to mice. Studies
with rats involving intraperitoneal injection of 1e, 2g and oral
administration of 1e, f, 2g did not lead to acute mortalities or
neurotoxicity. These data suggested that, in contrast to many
cytotoxic and anticancer agents such as melphalan (the LD50
in mice is 21.7 mg/kg [25]), the enones were well tolerated in
rodents.
5. Conclusion
When the 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienyl
pharmacophore is aligned in a certain fashion as in series 1
and 2, the compounds exert potent cytotoxicity. These mole-
cules are structurally divergent from the anticancer drugs used
currently and may differ from them in their mechanisms of
action. Both the absence of rapid onset of toxicity in rodents
and the inhibition of the growth of H. pylori further enhance
the potential of thse compounds for development as therapeutic
agents. Further studies should be undertaken in order to find
the precise mechanisms whereby apoptosis is induced as well
as probing for other ways whereby the compounds exert their
cytotoxic effects.
The final experimentations were undertaken with the objec-
tive of discerning whether the compounds in this study were

U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
583
6. Experimental protocols
passage of hydrogen chloride gas. The m.p.s and yields for 2a–
g were as follows: 2a: 164–165, 61%; 2b: 197–199, 75%; 2c:
162–164, 80%; 2d: 191–192, 76%; 2e: 218–220, 65%; 2f:
199–200, 66%; 2g: 145–146, 82%. The 1H NMR spectrum
of a representative compound 2e is as follows: δ (CDCl3):
1.84 (qu, 2H, CH2), 2.91 (t, J = 5.13 Hz, J = 5.26 Hz, 2H,
CH2), 2.96 (t, J = 5.34 Hz, J = 5.22 Hz, 2H, CH2), 6.04 (s, 2H,
OCH2O), 6.88 (d, J = 8.03 Hz, 1H, C5′H), 7.03 (m, 2H, C2′H,
C6′H), 7.59 (d, J = 8.69 Hz, 2H, C2H, C6H), 7.76 (s, 1H, ole-
finic H ring B), 7.82 (s, 1H, olefinic H ring A), 8.27 (d,
J = 8.74 Hz, 2H, C3H, C5H).
6.1. Chemistry
The melting points, which are quoted in °C, were deter-
mined using a Gallenkamp melting point apparatus and are un-
corrected. Elemental analyses (C, H, N) were undertaken on
1b–f, 2a–g, 3a and 4a, b by Mr. K. Thoms, Department of
Chemistry, University of Saskatchewan and were within 0.4%
of the calculated values except for 2e (calcd. for C: 69.41%.
Found: C: 68.96%). 1H NMR spectra were determined on all
compounds using a Brucker AM 500 FT NMR machine
(500 MHz). The positions of the hydrogen atoms in the aryl
rings are designated as unprimed (ring A), single primed (ring
B) and, when present, double primed (ring C). The letters br, s,
d, t, qu, m refer to broad, singlet, doublet, triplet, quintet and
multiplet, respectively. The compounds were recrystallized
from chloroform/methanol except for chloroform which was
used to purify 2b and 4b while 3a was crystallised from
chloroform/acetone.
6.1.3. Synthesis of 3a
The intermediate 1-(4-nitrophenylmethylene)-2-oxo-2,3-di-
hydro-1H-indene 7 was prepared as follows. A solution of 2-
oxo-2,3-dihydro-1H-indene (0.02 mol), morpholine (0.022
mol), p-toluenesulfonic acid (20 mg) and benzene (100 ml)
was heated under reflux in a Dean-Stark apparatus for approxi-
mately 24 h until the stoichiometric amount of water was col-
lected. 4-Nitrobenzaldehyde (0.02 mol) was added to the reac-
tion mixture and heating under reflux was continued for 22 h.
On cooling, hydrochloric acid (37% w/v, 30 ml) was added
and the solution was stirred at room temperature for 8 h. Ben-
zene was removed in vacuo and the residue was extracted with
chloroform. The organic extract was washed with hydrochloric
acid (2 N, 3 × 50 ml), water (3 × 50 ml) and dried over anhy-
drous sodium sulphate. Removal of the chloroform led to a
solid which was recrystallized from chloroform–methanol to
give 7, m.p. 164–167 in 80% yield. The 1H NMR spectrum
was as follows: δ (CDCl3): 3.64 (s, 2H, CH2), 7.18 (t,
J = 7.62 Hz, J = 7.58 Hz, 1H, C5″H), 7.34 (s, 1H, C2′H),
7.37 (t, J = 7.49 Hz, J = 7.39 Hz, 1H, C2″H), 7.43 (d,
J = 7.53 Hz, 1H, C4″H), 7.49 (s, 1H, olefinic H), 7.53 (d,
J = 7.92 Hz, 1H, C7″H), 7.77 (d, J = 8.57, 2H, C2H, C6H),
8.35 (d, J = 8.63 Hz, 2H, C3H, C5H).
Compound 7 reacted with 3,4,5-trimethoxybenzaldehyde
using the same procedure as employed for the synthesis of
1a–e, g to give 3a, m.p. 220–222, in 80% yield. The 1H
NMR spectrum was as follows: δ (CDCl3): 4.02 (s, 6H, 2
xOCH3), 4.04 (s, 3H, OCH3), 7.16 (t, J = 7.61 Hz, J = 7.54
Hz, 1H, C5″H), 7.34 (s, 1H, C2′H), 7.40 (t, J = 7.59 Hz,
J = 7.51 Hz, 1H, C6″H), 7.46 (d, J = 7.85 Hz, C4″H), 7.61 (s,
1H, C6′H), 7.69 (d, J = 7.83 Hz, 1H, C7″H), 7.74 (s, 2H, ole-
finic H), 7.78 (d, J = 8.53 Hz, 2H, C2H, C6H), 8.33 (d, J = 8.65
Hz, 2H, C3H, C4H).
6.1.1. Synthesis of series 1
The preparation of 1a, g has been described previously [3];
however 1a was resynthesised for this project. The intermedi-
ate required in the preparation of 1a–g, namely 1-(4-nitrophe-
nylmethylene)-2-oxo-3,4-dihydro-1H-naphthalene
5, was
synthesized by a literature procedure [3] in 72% yield, m.p.
155 (lit. [3] m.p. 154–155). The general method followed in
preparing 1a–g was as follows. A stream of hydrogen chloride
gas was passed into a solution of 5 (0.01 mol) and the appro-
priate aryl aldehyde (0.01 mol) in chloroform (10 ml) and
diethyl ether (25 ml) for 10–15 min. For the synthesis of 1f,
2 ml of methanol was added to the reaction mixture prior to the
addition of hydrogen chloride gas. After stirring the reaction
mixture at room temperature for 3–4 h, the precipitate was col-
lected and recrystallized. The m.p.s and yields were as follows:
1a: (185 (lit. [3] 185–186) 72%; 1b : 212, 82%; 1c: 176–177,
68%; 1d: 207–208, 71%; 1e: 204, 76%; 1f: 251, 64%. The 1H
NMR spectrum of a representative compound 1e is as follows:
δ (CDCl3): 4.15 (s, 2H, CH2), 6.08 (s, 2H, OCH2O), 6.93 (d,
J = 7.99 Hz, 1H, C5′H), 7.01 (s, 1H, C2’H), 7.05 (m, 2H, C6′
H, C6″H), 7.20 (d, J = 7.76 Hz, 1H, C5″H), 7.28 (m, 2H, C7″H,
C8″H), 7.57 (s, 1H, olefinic H ring B), 7.62 (d, J = 8.70 Hz,
2H, C2H, C6H), 7.80 (s, 1H, olefinic H ring A), 8.15 (d,
J = 8.69, 2H, C3H, C5H).
6.1.4. Synthesis of 4a, b
6.1.2. Synthesis of series 2
The procedure employed in forming 7 was utilized in
synthesizing 2-(4-nitrophenylmethylene)cyclopentanone 8. Re-
crystallization of the crude product from chloroform-methanol
afforded 8, m.p. 138 (lit. [27] m.p. 145–146) in 60% yield.
Compound 8 was condensed with the appropriate aryl alde-
hyde using the procedure for preparing 1a–e, g to give 4a, m.p.
185–186 and 4b, m.p. 238–240 in yields of 81% and 88%,
respectively. The 1H NMR spectrum of a representative com-
pound 4a was as follows: δ (CDCl3): 3.18 (s, 4H, 2 xCH2),
The intermediate required in the synthesis of 2a–g, namely
2-(4-nitrophenylmethylene) cyclohexanone 6, was prepared by
a previously reported methodology [26] to give 6, m.p. 119 (lit.
[26] m.p. 118–120) in 72% yield with respect to 4-nitrobenzal-
dehyde.
The condensation of 6 with various aryl aldehydes was ac-
complished using the same methodology as the reaction of 5
with different aromatic aldehydes vide supra. In the case of 2f,
methanol (2 ml) was added to the reaction mixture prior to the

584
U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
3.94 (s, 9H, 3 xOCH3), 6.90 (s, 2H, olefinic H), 7.60 (d,
J = 9.15 Hz, 2H, C2′H, C6′H), 7.74 (d, J = 8.72 Hz, 2H,
C2H, C6H), 8.31 (d, J = 8.73 Hz, 2H, C3H, C5H).
6.2.4. Apoptosis experimentation
The HepG2 cells line was obtained from the ATCC. These
cells were grown in DMEM supplemented with 10% fetal calf
serum and a 1% antibiotic-antimycotic solution (GIBCO, Bur-
lington, Canada) which contained penicillin G sodium, strepto-
mycin sulphate and amphotericin B in an environment of hu-
midified air containing 5% carbon dioxide at 37 °C. For the
apoptosis assay, the HepG2 cells were cultured in chambered
slides in the presence of 1 μM of 1e. Control experiments were
undertaken with the HepG2 cells in the presence and absence
of dimethylsulphoxide. After 48 h, the cells were fixed and
permeabilized. The apoptotic cells were detected by the TU-
NEL assay. The apoptosis assay was carried out using the
Apoptosis Detection System, Fluorescein (Promega, Madison)
and following the protocol of the manufacturer. Apoptotic cells
having fluorescently labelled DNA were visualized under a
fluorescent microscope. The percentage of cells which are
apoptotic was 51.6 5.14.
6.1.5. Molecular modelling
Models of 1a, 2a, 3a and 4a were built using the Macro-
Model 8.0 programme [28] followed by a Monte Carlo search
for the conformations with the lowest energies using an Amber
force field of 1000 initial conformations. Overlaps were made
of atoms 1–6 as indicated in Fig. 2A between the following
pairs of compounds (RMS value in parenthesis) viz. 1a/2a
(0.1687), 1a/3a (0.1501), 1a/4a (0.1228), 2a/3a (0.1613), 2a/
4a (0.1746) and 3a/4a (0.0538).
6.2. Bioevaluations
6.2.1. Cytotoxic screens
The evaluations using human Molt 4/C8 and CEM T-lym-
phocytes as well as murine L1210 cells were undertaken by a
previously reported method [29].
6.2.5. Tubulin assembly assay
The effect of the compounds on the assembly of porcine
brain tubulin was assessed by measuring changes in turbidity
as described previously [34]. The IC50 for a reference com-
pound podophyllotoxin is 1.9 0.7 μM.
Evaluation against the panel of human cell lines was under-
taken using a literature procedure [30]. The concentrations of
compounds used were 10–4 to 10–8 M for 1b, 1e and 1f,
10–3.6–10–7.6 M for melphalan and 10–2.6–10–6.6 M for 5-fluor-
ouracil. The number of cell lines whereby 50% inhibition of
the growth of the cells at the highest concentrations was not
achieved/total number of cell lines were as follows, namely
1b (1/54), 1e (1/56), 1f (0/52), melphalan (0/55) and 5-fluor-
ouracil (3/57). The data for the leukemic and colon cancer cells
are IC50 values.
6.2.6. Assay of 1a versus hNMT
The evaluation of 1a towards hNMT was undertaken by a
previously reported procedure [35]. The percentage inhibition
of hNMT when 1 mM of 1a was used was 23.2 5.23.
6.2.7. Antifungal evaluations
Compounds 1b, e, f were examined against five strains of
Aspergillus fumigatus, namely H27023 (ATCC 280997),
W73355 (ATCC 280996), F55064 (ATCC 280995), M44251
and F69827. Evaluation was also undertaken using Candida
albicans (ATCC 90028), Candida glabrata (ATCC 66032),
Candida parapsilosis (ATCC 22019) and Candida tropicalis
(ATCC 13803). The broth microdilution assay was employed
[36]. The reference drug was voriconazole which had MIC
values of 0.25 μg/ml using the A. fumigatus isolates and
C. glabrata and 0.062 μg/ml towards C. albicans, C. para-
psilosis and C. tropicalis.
6.2.2. Anti-Helicobacter pylori assay
This determination was undertaken by a literature procedure
[31]. In brief, H. pylori was inoculated into brain heart infusion
broth containing 10% fetal bovine serum (Biofluid, Inc., Rock-
ville, USA) and 0.1% glucose and cultured at 37 °C. After
2 days, the bacterial colonies were collected and diluted with
medium to give 107 CFU/ml to which a solution of the com-
pound in dimethylsulphoxide was added. The mixture was in-
cubated at 37 °C for 5 days. Eight concentrations were used in
the range of 0.001–2.222 mg/ml. The experiment was per-
formed in triplicate for each compound except in the case of
metronidazole which was evaluated twice. The IC50 values
6.2.8. Rodent toxicity screens
Various compounds were evaluated for toxicity in mice and
rats using previously reported protocols [37]. The enones 1a,
b, e, f, 2a–g, 3a, 4a, b were injected intraperitoneally into mice
using doses of 30, 100 and 300 mg/kg with the view of deter-
mining if lethality and/or neurotoxicity could be demonstrated.
The animals were examined after 0.5 and 4 h. In addition, mice
receiving the 100 mg/kg dose of 1b, e, 2g were observed after
2 and 6 h (1e, 2g) and 0.25 and 1 h (1f). No deaths were noted,
while marginal neurotoxicity was displayed by 2a in 2/8 ani-
mals and 3a in 1/4 mice using doses of 100 and 300 mg/kg,
respectively.
(
S.E.M.) were obtained using nonlinear regression analysis
[32].
6.2.3. Urease assay
The assay was undertaken by a previously reported metho-
dology [33]. In brief, the test compound and Jack bean urease
(Sigma-Aldrich, Milwaukee, USA) were preincubated for
3 hours at room temperature after which time a solution of urea
and phenol red in phosphate buffer was added. The reaction
time was measured by a microplate reader (570 nm).

U. Das et al. / European Journal of Medicinal Chemistry 41 (2006) 577–585
585
[9] J.W. Quail, A. Doroudi, H.N. Pati, U. Das, J.R. Dimmock, Acta Crystal-
logr. E61 (2005) 1774–1776.
[10] J.W. Quail, U. Das, J.R. Dimmock, Acta Crystallogr. E61 (2005) 1150–
1152.
[11] P. Selvakumar, M.K. Pasha, L. Ashakumary, J.R. Dimmock, R.K. Shar-
ma, Int. J. Mol. Med. 10 (2002) 493–500.
[12] S.N. Pandeya, J.R. Dimmock, in: An Introduction to Drug Design, New
Age International, (P), Publishers, New Delhi, 1997, pp. 72–74.
[13] M.R. Grever, S.A. Schepartz, B.A. Chabner, Semin. Oncol. 19 (1992)
622–638.
[14] E.M. Greenspan, H.W. Bruckner, in: E.M. Greenspan (Ed.), Clinical
Cancer Chemotherapy, Raven Press, New York, 1975, p. 37.
[15] W.A. Remers, in: J.N. Delgado, W.A. Remers (Eds.), Wilson and Gis-
vold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry,
10th ed, Lippincott-Raven, Philadelphia, 1998, p. 359.
[16] J.L. Chase, D.K. Patel, V. Trinh, R.D. Lozano, in: E.T. Herfindal, D.R.
Gourley (Eds.), Textbook of Therapeutics Drug and Disease Manage-
ment, 7th ed, Lippincott, Williams and Wilkins, Philadelphia, 2000, p.
1827.
The enones 1e, f, 2g were administered orally to four rats
and observed after 0.25, 0.5, 1, 2 and 4 h using doses of 30 (1e,
2g) or 50 (1f) mg/kg. In addition, 1e was given to rats per os
using doses of 25, 60 and 120 mg/kg (four animals per dose)
and 250 mg/kg (eight animals were employed). Using a dose of
30 mg/kg, 1e and 2g were each injected intraperitoneally to
four rats and observed after 0.25, 0.5, 1, 2 and 4 h. In all of
these experiments, neither deaths nor neurotoxicity were ob-
served.
These bioevaluations were undertaken by the National Insti-
tutes of Neurological Disorders and Stroke, USA, under the
Anticonvulsant Screening Program. The animals were housed,
fed and handled following the procedures documented in the
National Research Council publication “Guide for the Care and
Use of Laboratory Animals”. The mice and rats were eutha-
nised using the policies of the Institute of Laboratory Re-
sources.
[17] S.C. Vashishtha, A.J. Nazarali, J.R. Dimmock, Cell. Mol. Neurobiol. 18
(1998) 437–445.
[18] M. Tsurusawa, K. Saeki, T. Fujimoto, Int. J. Hematol. 66 (1997) 79–88.
[19] G. Mamiya, K. Takishima, M. Masakuni, T. Kayumi, K. Ogawa, T. Se-
kita, Proc. Jpn. Acad. 61B (1985) 395–398.
Acknowledgements
[20] L.T. Mobley, M.D. Island, R.P. Hausinger, Microbiol. Rev. 59 (1995)
451–480.
[21] T. Tanaka, M. Kawase, S. Tani, Life Sci. 73 (2003) 2985–2990.
[22] M. Medarde, A.B.S. Maya, C. Pérez-Melero, J. Enz. Inhib. Med. Chem.
19 (2004) 521–540.
The authors thank the following organizations for financial
contributions to this project, namely the Canadian Institutes of
Health Research (awards of operating grants to J.R.D. and R.K.
S. and a postdoctoral fellowship to AS), NATO (a visiting
scholar grant to H.I.G. distributed by the Scientific and Tech-
nical Research Council of Turkey) and the Flemish Fonds voor
Geneeskundig Wetenschappelijk Onderzoek (E.D.C., J.B.).
Thanks are also extended to the U.S. National Cancer Institute
who generated the data in Table 3, the National Institute of
Neurological Disorders and Stroke who undertook the rodent
toxicity studies (J.P.S.), and Ms. B. McCullough who typed the
manuscript.
[23] B.A. Magnuson, R.V.S. Raju, T.N. Moyana, R.K. Sharma, J. Natl. Can-
cer Inst. 87 (1995) 1630–1635.
[24] S.M. Peseckis, M.D. Resh, J. Biol. Chem. 269 (1994) 30888–30892.
[25] F.R. Quinn, G.W.A. Milne, Fundam. Appl. Toxicol. 6 (1986) 270–277.
[26] H. Vieweg, G. Wagner, Pharmazie 34 (1979) 785–788.
[27] T. Sugasawa, T. Toyoda, K. Sasakura, Synth. Commun. 9 (1979) 583–
602.
[28] F. Mohamadi, N.G.J. Richards, W.C. Gude, M.L. Liskamp, C. Canfield,
G. Change, T. Hendrickson, W.C. Still, Comput. Chem. 11 (1990) 440–
467.
[29] J. Balzarini, E. De Clercq, M.P. Mertes, D. Shugar, P.F. Torrence, Bio-
chem. Pharmacol. 31 (1982) 3673–3682.
References
[30] M.R. Boyd, K.D. Paull, Drug Dev. Res. 34 (1995) 91–109.
[31] N. Motohashi, H. Wakabayashi, T. Kurihara, H. Fukushima, T. Yamada,
M. Kawase, Y. Sohara, T. Tani, Y. Shirataki, H. Sakagami, K. Satoh, N.
Nakashima, A. Molnár, G. Spengler, N. Gyémánt, K. Ugocsai, J. Molnár,
Phytother. Res. 18 (2004) 212–223.
[32] GraphPad Prism 4.0, GraphPad Softward, San Diego, CA, USA, 2003.
[33] D.D. van Slyke, R.M. Archibald, J. Biol. Chem. 154 (1944) 623–642.
[34] K.A. Werbovetz, D.L. Sackett, D. Delfin, G. Bhattacharya, M. Salem, T.
Obrzut, D. Rattendi, C. Bacchi, Mol. Pharmacol. 64 (2003) 1325–1333.
[35] J.R. Dimmock, A. Jha, P. Kumar, G.A. Zello, J.W. Quail, E.O. Oloo, J.
Oucharek, M.K. Pasha, D. Seitz, R.K. Sharma, T.M. Allen, C.L. Santos,
E.K. Manavathu, E. De Clercq, J. Balzarini, J.P. Stables, Eur. J. Med.
Chem. 37 (2002) 35–44.
[1] J.R. Dimmock, M.P. Padmanilayam, G.A. Zello, K.H. Nienaber, T.M.
Allen, C.L. Santos, E. De Clercq, J. Balzarini, E.K. Manavathu, J.P.
Stables, Eur. J. Med. Chem. 38 (2003) 169–177.
[2] J.R. Dimmock, M.P. Padmanilayam, R.N. Puthucode, A.J. Nazarali, N.L.
Motaganahalli, G.A. Zello, J.W. Quail, E.O. Oloo, H.-B. Kraatz, J.S.
Prisciak, T.M. Allen, C.L. Santos, J. Balzarini, E. De Clercq, E.K. Man-
avathu, J. Med. Chem. 44 (2001) 586–593.
[3] J.R. Dimmock, M.P. Padmanilayam, G.A. Zello, J.W. Quail, E.O. Oloo,
J.S. Prisciak, H.-B. Kraatz, A. Cherkasov, J.S. Lee, T.M. Allen, C.L.
Santos, E.K. Manavathu, E. De Clercq, J. Balzarini, J.P. Stables, Eur. J.
Med. Chem. 37 (2002) 813–824.
[4] A. Baluja, A.M. Municio, S. Vega, Chem. Ind. (1964) 2053–2054.
[5] B. Mutus, J.D. Wagner, C.J. Talpas, J.R. Dimmock, O.A. Phillips, R.S.
Reid, Anal. Biochem. 177 (1989) 237–243.
[36] National Committee for Clinical Laboratory Standards, Publications
M38-P and M27-A, NCCLS, Wayne, PA, USA, 1997.
[37] J.P. Stables, H.J. Kupferberg, In Molecular and Cellular Targets for Anti-
epileptic Drugs, in: G. Vanzini, P. Tanganelli, M. Avoli (Eds.), John Lib-
bey and Company Ltd, London, 1997, pp. 191–198.
[38] J.R. Dimmock, M. Chamankah, U. Das, G.A. Zello, J.W. Quail, J. Yang,
K.H. Nienaber, R.K. Sharma, P. Selvakumar, J. Balzarini, E. De Clercq,
J.P. Stables, J. Enz. Inhib. Med. Chem. 19 (2004) 1–10.
[6] J.R. Dimmock, K.K. Sidhu, M. Chen, R.S. Reid, T. Allen, G.Y. Kao,
G.A. Truitt, Eur. J. Med. Chem. 28 (1993) 313–322.
[7] J.R. Dimmock, U. Das, H.I. Gul, M. Kawase, H. Sakagami, Z. Baráth, I.
Ocsovsky, J. Molnár, Bioorg. Med. Chem. Lett. 15 (2005) 1633–1636.
[8] J.W. Quail, A. Doroudi, H.N. Pati, U. Das, J.R. Dimmock, Acta Crystal-
logr. E61 (2005) 1795–1797.