3-Cyano-8-methyl-2-oxo-1,4-disubstituted-1,2,5,6,7,8-hexahydroquinolines: Synthesis and biological evaluation as antimicrobial and cytotoxic agents

Article abstract of DOI:10.3109/14756366.2011.637201

The synthesis, in vitro antimicrobial and cytotoxic activities of some novel hexahydroquinolines supported with various pharmacophores are described. The results revealed that 18 compounds displayed pronounced activity against Staphylococcus aureus and Escherichia coli bacteria beside a moderate antifungal activity. Compound 25 is the most active candidate with equipotency to ampicillin against S. aureus, E. coli and Pseudomonas aeruginosa, together with an obvious antifungal activity. Additionally, 12 compounds showed remarkable cytotoxic efficiency against human colon carcinoma HT29, hepatocellular carcinoma Hep-G2 and Caucasian breast adenocarcinoma MCF7 cell lines. Among these, the analogs 22 and 25 proved to be the most active cytotoxic members. Collectively, the results would suggest that compounds 22 and 25 could be considered as possible dual antimicrobial-anticancer agents.

Full text of DOI:10.3109/14756366.2011.637201

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(1): 123–130
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.637201
research arTIcLe
3-Cyano-8-methyl-2-oxo-1,4-disubstituted-1,2,5,6,7,8-
hexahydroquinolines: synthesis and biological evaluation
as antimicrobial and Cytotoxic agents
Hassan M. Faidallah1, Sherif A. F. Rostom2,3, Abdullah M. Asiri1, Khalid A. Khan1,
Mohammed F. Radwan2, and Hani Z. Asfour4
1Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia,
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia,
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt, and
4Department of Parasitology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
abstract
The synthesis, in vitro antimicrobial and cytotoxic activities of some novel hexahydroquinolines supported with
various pharmacophores are described. The results revealed that 18 compounds displayed pronounced activity
against Staphylococcus aureus and Escherichia coli bacteria beside a moderate antifungal activity. Compound 25 is the
most active candidate with equipotency to ampicillin against S. aureus, E. coli and Pseudomonas aeruginosa, together
with an obvious antifungal activity. Additionally, 12 compounds showed remarkable cytotoxic efficiency against
human colon carcinoma HT29, hepatocellular carcinoma Hep-G2 and Caucasian breast adenocarcinoma MCF7 cell
lines. Among these, the analogs 22 and 25 proved to be the most active cytotoxic members. Collectively, the results
would suggest that compounds 22 and 25 could be considered as possible dual antimicrobial-anticancer agents.
Keywords: Synthesis, hexahydroquinolines, antibacterial, antifungal, cytotoxic
Introduction
subjected cancer chemotherapy are mostly susceptible to
Over the past two decades, much attention has been
focused on addressing the problem of multi-drug resistant
(MDR) bacteria and fungi resulting from the widespread
use and misuse of classical antimicrobial agents1. In addi-
tion, fungal infection became a prominent complication
and a major cause of mortality in immuno-compromised
individuals such as those suffering from tuberculosis,
cancer or AIDS and in organ transplant cases2. erefore,
there is a real perceived need for the discovery of new com-
pounds endowed with antimicrobial activity, possibly act-
ing through non-traditional mechanisms of action. On the
other hand, the increasing number of neoplastic diseases
together with the accompanied high mortality rates3 has
stimulated an unprecedented level of research directed
towards the search for new structure leads that might be of
use in designing novel anticancer drugs. Patients that are
microbial infections due to subsequent lack of immunity.
Co-administration of multiple drugs for treating cancer
disease accompanied with microbial infections might
inflect some added health problems especially in patients
with impaired liver and/or kidney functions. erefore,
the concept of monotherapy by a single drug which would
possess dual utility might be advantageous from both
therapeutic and cost-effective stand points.
In recent years, pyridines and some derived fused-ring
systems such as quinolines have attracted much atten-
tion as versatile chemotherapeutic agents owing to their
reported potential antimicrobial activity4–6, in addition to
various antineoplastic7, antiproliferative8, and cytotoxic9
potentials. Furthermore, particular interest has been
given to cyanopyridine derivatives as anticancer and
antiviral10 agents.
Address for Correspondence: Dr. Sherif A.F. Rostom, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz
University, P.O. Box 80260, Jeddah 21589, Saudi Arabia. Tel: +966 507654566. E-mail: sherifrostom@yahoo.com
(Received 04 October 2011; revised 28 October 2011; accepted 29 October 2011)
123

124 H. M. Faidallah et al.
During our ongoing studies aimed at the discovery of
new structure leads endowed with diverse chemothera-
peutic activities, much concern has been given to the
antimicrobial and antitumour potentials of some pyri-
dine derivatives11–14, among which those comprising the
3-cyano-4,6-disubstituted-2(1H)-pyridinone scaffold12
exhibited promising broad spectrum antitumour activity.
e obtained results prompted further structure modifi-
cation of the disubstituted-2(1H)-pyridinone scaffold by
increasing compounds’ lipophilicity via the synthesis of
new 1,2,5,6,7,8-hexahydroquinoline analogs. e target
compounds were designed to encounter various phar-
macophores and functionalities at positin-1 that are
believed to be responsible for the biological significance
of some relevant antimicrobial and/or anticancer agents
such as the formyl, acetyl, nitroso, benzenesulfonyl,
thiocarbamoyl and alkyl groups. Moreover, it was con-
sidered worthwhile to utilize the N-acetyl derivatives as
precursors for the synthesis of the tricyclic fused-ring
system tetrahydro[1,2,4]triazolo[4,3-a]quinoline as an
interesting structural variation, hoping to improve the
anticipated chemotherapeutic activities.
3-Cyano-8-methyl-1-nitroso-2-oxo-4-substituted-1,2,5,6,7,8-
hexahydroquinolines (7,8)
To an ice-cooled stirred solution of the appropriate
starting material 4 or 5 (10 mmol) in acetic acid (15 mL),
was added dropwise a solution of sodium nitrite (1.05 g,
15 mmol) in water (5 mL) over a period of 2 h. Stirring
was maintained for further 2 h, and then the reaction
mixture was left aside at room temperature for an over-
night. e formed solid product was filtered, washed
with water, dried and recrystallized from ethanol as
needles. IR (cm−1): 2223-2217 (CN), 1675-1667 (C=O
pyridone). 13C NMR (δ-ppm) 7: 15.3 (CH3), 27.6, 27.7,
30.3, 34.2 (cyclohexyl C), 117.6 (CN), 95.8, 119.2, 133.0,
163.0 (CO), 170.2 (pyridone C), 125.9, 127.8, 128.6, 134.9
(Ar C).
3-Cyano-1-formyl-8-methyl-2-oxo-4-substituted-1,2,5,6,7,8-
hexahydroquinolines (9–11)
A solution of the appropriate starting compound 4–6 (3
mmol) in formic acid (5 mL) was heated under reflux for
3 h. e reaction mixture was poured on crushed ice (10 g)
and the separated solid product was filtered, washed with
water, dried and recrystallized from ethanol. IR (cm−1):
2220-2210 (CN), 1678-1670 (C=O pyridone), 1663-1657
(C=O aldehyde). 13C NMR (δ-ppm) 9: 15.1 (CH3), 27.5,
27.7, 34.3, 34.7 (cyclohexyl C), 117.2 (CN), 95.8, 120.0,
133.3, 160.9 (CO) 169.2 (pyridone C), 156.8 (formyl CO),
126.0, 127.5, 128.3, 134.7 (Ar C).
experimental
Chemistry
Melting points were determined on a Gallenkamp melt-
ing point apparatus and are uncorrected. e infrared
(IR) spectra were recorded on Shimadzu FT-IR 8400S IR
spectrophotometer using the KBr pellet technique. 1H
and 13C NMR spectra were recorded on a Bruker DPX-
400 FT NMR spectrometer using tetramethylsilane as the
internal standard and a mixture of CDCl3 and dimethyl
sulfoxide-d6 (DMSO-d6) as a solvent (Chemical shifts in
δ, ppm). Splitting patterns were designated as follows: s:
singlet; d: doublet; m: multiplet; q: quartet. Elemental
analyses were performed on a 2400 Perkin Elmer Series
2 analyzer and the found values were within 0.4% of the
theoretical values. Follow-up of the reactions and check-
ing the homogeneity of the compounds were made by
thin layer chromatography on silica gel-protected alu-
minum sheets (Type 60 F254, Merck) and the spots were
detected by exposure to UV-lamp at λ 254.
1-Acetyl-3-cyano-8-methyl-2-oxo-4-substituted-1,2,5,6,7,8-
hexahydroquinolines (12–14)
To a solution of the appropriate 4–6 (10 mmol) in acetic
anhydride (10 mL), was added anhydrous sodium acetate
(1.2 g, 15 mmol). e reaction mixture was heated under
reflux for 4 h, allowed to cool, and then poured on crushed
ice with vigorous stirring. e formed solid product was
filtered, thoroughly washed with water, dried and recrys-
tallized from aqueous ethanol. IR (cm−1): 2226-2215 (CN),
1725-1718 (C=O acetyl), 1678-1672 (C=O pyridone). 13C
NMR (δ-ppm) 12: 15.2 (CH3), 15.7 (CH3), 27.6, 27.8, 31.9,
34.2 (cyclohexyl C), 117.8(CN), 98.2, 120.0, 133.4, 160.9
(CO), 169.8 (pyridone C), 126.8, 127.9, 128.5, 134.8 (Ar C),
165.3 (CO).
3-Cyano-8-methyl-2-oxo-4-substituted-1,2,5,6,7,8-
hexahydroquinolines (4–6)
4-Cyano-1,9-dimethyl-5-Substituted-6,7,8,9-tetrahydro
[1,2,4]triazolo[4,3-a]quinolines (15–17)
A one-pot mixture of the appropriate aldehyde 1–3 (10
mmol), 2-methylcyclohexanone 1 (1.12 g, 10 mmol), ethyl
cyanoacetate (1.1 g, 10 mmol) and ammonium acetate
(6.2 g, 80 mmol) in absolute ethanol (50 mL), was refluxed
for 6 h. e reaction mixture was allowed to cool, and the
formed precipitate was filtered, washed with water, dried
and recrystallized. IR (cm−1): 3400-3250 (NH), 2225- 2210
(CN), 1682-1675 (C=O). 13C NMR (δ-ppm) 4: 15.1 (CH3),
27.7, 27.9, 34.4, 34.9 (cyclohexyl C), 117.2 (CN), 95.7,
119.5, 132.7, 165.9 (CO), 169.4 (pyridine C), 126.2, 127.7,
128.4, 134.9 (Ar C).
A mixture of the appropriate 1-acetylhexahydroquino-
line 12–14 (10 mmol) and hydrazine hydrate 99% (0.9g,
15 mmol) in ethanol (15 mL) was heated under reflux for
6–8 h. e reaction mixture was allowed to attain room
temperature, poured on crushed ice and the precipitated
solid product was filtered, washed with water, dried and
recrystallized from DMF/water. IR (cm−1): 2220-2215
(CN). 13C NMR (δ-ppm) 15: 12.3 (CH3), 22.3 (CH3), 24.7,
32.4, 29.1, 41.5 (cyclohexyl C), 118.0 (CN), 106.8, 135.8,
147.6, 154.3, 166.9 (pyridone C), 126.9, 128.9, 129.0, 138.2,
160.9 (Ar C).
Journal of Enzyme Inhibition and Medicinal Chemistry

1,4-Disubstituted hexahydroquinolines: Synthesis and biological evaluation 125
3-Cyano-8-methyl-2-oxo-1-(substituted sulfonyl)-4-
Minimal inhibitory concentration (MIC) measurement
substituted-1,2,5,6,7,8-hexahydro-quinolines (18–20)
A mixture of the appropriate start 4–6 (10 mmol) and
benzenesulfonyl chloride (10 mmol, 1.77 g) in pyridine
(10 mL), was heated under reflux for 4–6 h. After cooling
to room temperature, the reaction mixture was poured
on crushed ice and the separated solid product was fil-
tered, washed with water, dried and recrystallized from
ethanol. IR (cm−1): 2230-2221 (CN), 1670-1665 (C=O
pyridone), 1388-1370 and 1197-1175 (SO2). 13C NMR
(δ-ppm) 18: 15.1 (CH3), 27.6, 27.9, 31.3, 34.4 (cyclohexyl
C), 95.9, 119.8, 133.0, 163.2 (CO), 169.5 (pyridone C),
117.6 (CN), 125.6, 126.2, 127.7, 128.4, 128, 8, 131.7, 134.9,
139.3 (Ar C).
e MIC of the most active compounds were measured
using the twofold serial broth dilution method described
by Scott15. e MIC values in µg/mL (µM) of the com-
pounds are listed in Table 1.
In vitro MTT cytotoxicity assay
All of the newly synthesized compounds were investi-
gated for their in vitro cytotoxic effect via the standard
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide) method16,17 against a panel of three
human tumour cell lines namely; Caucasian breast ade-
nocarcinoma MCF7, hepatocellular carcinoma Hep-G2
and colon carcinoma HT29. e results are presented in
Table 2 as LC50 (μg/mL, μM) which is the lethal concen-
tration of the compound which causes death of 50% of
the cells in 24 h.
3-Cyano-8-methyl-2-oxo-1-(substituted-thiocarbamoyl)-4-
substituted-1,2,5,6,7,8-hexahydroquinolines (21–26)
A mixture of the appropriate starting compound 4–6 (10
mmol) and the appropriate isothiocyanate (11 mmol)
in pyridine (10 mL) was heated under reflux for 6–8 h.
After being cooled to room temperature, the reaction
mixture was poured on ice cold water and the separated
solid product was filtered, washed with water, dried and
recrystallized from ethanol as needles. IR (cm−1): 2230-
2215 (CN), 1678-1665 (C=O pyridone), 1232-1216 (C=S).
13C NMR (δ-ppm) 22: 15.7 (CH3), 27.9, 28.3, 32.4, 35.0
(cyclohexyl C), 117.5 (CN), 98.6, 120.2, 132.9, 162.7 (CO),
168 (pyridone C), 124.5, 125.3, 126.2, 127.7, 128.4, 128.8,
134.9, 139.4 (Ar C), 175.4 (CS).
results and discussion
Chemistry
e synthetic strategies adopted for the preparation of
the intermediate and target compounds are described
in Schemes 1 and 2. In Scheme 1, the key intermedi-
ates 3-cyano-8-methyl-2-oxo-4-substituted-1,2,5,6,7,8-
hexahydroquinolines 4–6 were synthesized via one-pot
multicomponent reaction (MCR) of the appropriate
aromatic aldehyde 1–3 and 2-methylcyclohexanone,
an excess of ammonium acetate and ethyl cyanoacetate
in boiling ethanol. Such type of reactions has received
considerable interest since it is easier to perform, gives
higher yields and less time consuming, when com-
pared with the traditional two-stepped procedure that
involved the formation of the 2-arylidene-6-methyl-
cyclohexanones (chalcones) via Claisen-Schmidt con-
densation followed by cyclocondensation with ethyl
cyanoacetate and ammonium acetate. Reacting the
starting compounds 4 and 5 with sodium nitrite in the
presence of cold acetic acid afforded the correspond-
ing the N-nitroso derivatives 7 and 8. Whereas, heating
compounds 4–6 with formic acid resulted in the forma-
tion of the N-formyl derivatives 9–11. In a similar fash-
ion, warming the starting compounds 4–6 with acetic
anhydride in the presence of anhydrous sodium acetate
furnished the N-acetyl derivatives 12–14. In their turn,
when the latter compounds were reacted with hydra-
zine hydrate, the targeted 5,6,7,8-tetrahydro[1,2,4]
triazolo[3,4-a]quinolines 15–17 were successfully
obtained. In Scheme 2, the same hexahydroquinolines
4–6 were utilized as key intermediates for the synthesis
of the target compounds 18–32. In this respect, react-
ing compounds 4–6 with benzenesulfonyl chloride in a
pyridine medium resulted in the introduction of a ben-
zenesulfonyl moiety at position-1 to yield compounds
18–20. Moreover, condensation of 4–6 with the appro-
priate isothiocyanate in alkaline medium afforded the
corresponding N-arylthiocarbamoyl analogs 21–26. On
the other hand, when compounds 4 were alkylated with
3-Cyano-1,4-disubstituted-8-methyl-2-oxo-1,2,5,6,7,8-
hexahydroquinolines (27–32)
To a solution of the appropriate hexahydroquinoline 4–6
(10 mmol) in pyridine (10 mL), was added the appropri-
ate alkyl halide (10 mmol) and the mixture was heated
under reflux for 3–4 h. e reaction mixture was allowed
to attain room temperature, poured on ice cold water and
the separated solid product was filtered, washed with
water, dried and recrystallized from ethanol. IR (cm−1):
2222-2216 (CN), 1675-1668 (C=O pyridone). 13C NMR
(δ-ppm) 27: 15.5 (CH3), 30.2 (N-CH3), 27.9, 28.0, 32.4, 34.7
(cyclohexyl C), 117.8 (CN), 95.7, 119.5, 132.9, 161.4 (CO),
169.6 (pyridone C), 126.5, 127.6, 129.2, 134.9 (Ar C).
In vitro antibacterial and antifungal activities
Inhibition-zone (IZ) measurements
All the newly synthesized compounds were evaluated
by the agar cup diffusion technique15 using a 1 mg/mL
solution in DMSO. e test organisms utilized were
Staphylococcus aureus (ATCC 6538), Bacillus subtilis
(ATCC 6633) and Micrococcus luteus (ATCC 21881) as
examples of Gram-positive bacteria and Escherichia coli
(ATCC 25922), Pseudomonas aeruginosa (ATCC 27853)
and Klebsiella pneumonia (clinical isolate) as examples
of Gram-negative bacteria. ey were also evaluated for
their in vitro antifungal potential against Candida albi-
cans (ATCC 10231) and Aspergillus niger (recultured)
fungal strains.
© 2013 Informa UK, Ltd.

126 H. M. Faidallah et al.
Table 1. Minimal inhibitory concentrations [MIC, µg/mL (μM)] of the tested compounds.
Compound no.
4
S. aureus
100
B. subtilis
100
M. luteus
E. coli
100
P. aeruginosa
K. pneumonia
C. albicans
a
–
–
–
–
(378.3)
25
(378.3)
50
(378.3)
25
5
–
–
–
–
–
–
–
–
50
(167.3)
100
–
–
–
–
–
–
(83.6)
50
(167.3)
100
(83.6)
25
6
(169.8)
100
(339.7)
200
(84.9)
100
(339.7)
200
10
(306)
25
(612)
100
(306)
25
(612)
50
13
200
(586.8)
–
100
(293.4)
–
(73.3)
100
(293.4)
100
(73.3)
50
(146.7)
50
14
(297.3)
200
(297.3)
200
(148.6)
100
(148.6)
–
16
–
–
–
–
–
–
(593.8)
200
(593.8)
–
(296.9)
200
17
–
(601.7)
50
(601.7)
100
18
100
(247.2)
25
100
(247.2)
25
(123.6)
12.5
(28.5)
25
(247.2)
6.25
(14.2)
25
19
100
(228)
200
100
(228)
–
25
(57)
100
(57)
100
(57)
50
20
(57.5)
12.5
(28.8)
25
(230)
50
(460)
100
(57.5)
12.5
(28.8)
25
(115)
25
(230)
25
22
–
–
(115.2)
100
(230.4)
100
(57.6)
100
(57.6)
50
23
(60)
(240)
25
(240)
25
(60)
(240)
12.5
(26.7)
50
(120)
12.5
(26.7)
12.5
(27.6)
–
25
6.25
(13.3)
12.5
(27.6)
50
6.25
(13.3)
12.5
(27.6)
100
50
(106.8)
100
(53.4)
50
(53.4)
–
26
(110.6)
100
(110.6)
100
(221.2)
–
28
–
(141)
12.5
(32)
(282)
50
(282)
6.25
(16)
(282)
25
30
50
(128)
50
100
(256)
200
50
(128)
100
(260)
–
(128)
100
(64)
25
32
25
12.5
(32.5)
6.25
(18)
(65)
(260)
12.5
(36)
–
(130)
12.5
(36)
(65)
12.5
(36)
–
(520)
12.5
(36)
Ampicillin
Clotrimazole
6.25
(18)
–
–
6.25
(18)
aMIC > 200 (600) µg/mL (μM).
the appropriate alkyl halide in the presence of sodium
hydroxide, the targeted N-alkyl hexahydroquinolines
27–32 were formed, but in low yields. Nevertheless, bet-
ter yields were obtained when the reaction was carried
out in pyridine as a basic solvent. At this stage, it was
thought of interest to study the effect of applying differ-
ent alkylating conditions on such type of compounds. In
this respect, compounds 4–6 were treated with the same
alkyl halides in the presence of ethanolic silver nitrate18
or sodium ethoxide19 as basic catalysts in an attempt
to obtain the O-alkyl derivatives 33–35. Unfortunately,
such procedures failed to produce the targeted com-
pounds, where the starting compounds were always
separated unreacted.
In vitro antibacterial and antifungal activities
As revealed from MIC data recorded in Table 1, at both
µg/mL and µM concentration levels, 18 out of the 29
newly synthesized compounds displayed variable inhibi-
tory effects on the growth of the tested Gram-positive and
Gram-negative microorganisms with special pronounced
activity against S. aureus and E. coli bacterial strains. In
addition, some members exhibited moderate antifungal
activity against C. albicans, whereas, all the tested com-
pounds lacked antifungal potential against Asp. Niger.
In terms of µg/mL concentration, among the tested
Gram-positive bacterial strains, two organisms namely;
S. aureus and B. subtilis showed relative high sensitivity
towardstheactivecompounds.Inthisview,compound25
Journal of Enzyme Inhibition and Medicinal Chemistry

1,4-Disubstituted hexahydroquinolines: Synthesis and biological evaluation 127
Table 2. Cytotoxic effects LC50; µg/mL (μM)a of the active
compounds on some human tumour cell lines using the
MTT assay.
A close examination of the structures of the active
compounds revealed that, their antimicrobial activity
is strongly bound to the nature of the substituent of the
aryl ring at C4, together with the substituent linked to
position-1 of the ring structure. In general, it could be
clearly recognized that potential antibacterial activity
was connected with the chlorinated derivatives (R = Cl),
whereas moderate activities were displayed by the meth-
oxylated analogs (R = OCH3). In this context, the key
hexahydroquinoline precursors 4–6 showed moderate
antimicrobial potential, with particular effectiveness
against S. aureus and E. coli. Nitrosation of position-1
of these compounds resulted in complete abolishment
of activity. Introduction of a formyl group furnished one
active compound (10; R = Cl), which was noticeably less
active than the parent compound 5. Whereas, acetylation
(12–14) led to a slight improvement in the antimicrobial
spectrum, especially 13 (R= Cl), which showed broad
antibacterial in addition to some antifungal activities.
However, cyclization of the latter N-acetyl compounds
into the tricyclic[1,2,4]triazolo[3,4-a]quinolines 15–17
resulted in a dramatic reduction in the antimicrobial
spectrum and potential. On the other hand, introduc-
ing a benzenesulfonyl moiety at position-1 gave rise to
three active compounds 18–20, among which the chloro
derivative 19 showed equipotent activity to ampicillin
against E. coli and 50% of its activity against S. aureus, B.
subtilis and P.aeruginosa, together with an appreciable
antifungal activity against C. albicans. Moreover, incor-
porating a substituted thiocarbamoyl counterpart as in
compounds 21–26, resulted in an obvious enhancement
in both the antimicrobial potential and spectrum. It could
be clearly recognized that the activity of these analogs is
connected with the aryl substituent (22, 23, 25 and 26).
Within this series, compound 25 (R = Cl; R1 = 4-Cl-C6H4)
was the most active member as it displayed a fourfold
increase in the antimicrobial activity against S. aureus,
E. coli and P. aeruginosa (MIC 6.25, 6.25 and 12.5 µg/
mL, respectively), together with a remarkable antifungal
activity (12.5 µg/mL), when compared with the parent
5. Finally, alkylation of the key intermediates 4–6 with
alkyl or arylalkyl groups furnished a series of compounds
27–32 with improved antimicrobial profiles, among
which the analogs 28, 30 and 32 (R2 = benzyl) showed a
variable degree of antimicrobial activity, whereas those
with the aliphatic substituent (R2 = CH3), were totally
inactive. When compared with the parent compounds 5
and 6, the active compounds 30 (R = Cl; R2 = benzyl) and
32 (R = OCH3; R2 = benzyl) revealed an obvious enhance-
ment in the activity against S. aureus, E. coli and P. aerugi-
nosa, together with a moderate antifungal activity.
Compound
5
HT29b
65.8
(220.2)
47.1
(160)
76.3
(237)
41.2
(122)
35.8
(108)
29.6
(68)
Hep-G2c
MCF 7d
46.3
(155)
34.1
(116)
–
57.2
(191.5)
26.3
6
(90)
e
11
–
14
39.4
(117)
21.3
(64)
18.5
(43)
20.6
(47)
48.7
(131)
11.6
(25)
31.8
(70)
–
–
17
41.6
(125)
25.2
(58)
12.1
(28)
64.9
(175)
9.7
20
22
8.4
(19)
24
26.3
(70)
25
14.9
(32)
(21)
–
26
34.9
(77)
29
41.5
(133)
54.6
(177)
21.1
(40)
–
–
31
–
Doxorubicinf
1.69
(3)
2.14
(4)
aLC50, lethal concentration of the compound which causes death
of 50% of cells in 24h µg/mL (µM).
bHT29 (Human colon carcinoma cell line).
cHep-G2 (Human hepatocellular carcinoma cell line).
dMCF7 (Human breast cancer cell line).
eTotally inactive against this cell line.
fPositive control cytotoxic agent.
was equipotent to ampicillin (MIC 6.25 µg/mL) against S.
aureus, whereas the analogs 19, 22, 26 and 30 (MIC 12.5
µg/mL) were 50% less active than ampicillin. With regard
to the activity against B. subtilis, compounds 19 and 25
(MIC 25 µg/mL) showed half the activity of ampicillin.
Concerning the antibacterial activity against the three
tested Gram-negative strains, three analogs namely;
19, 25 and 30 were able to produce a distinctive growth
inhibitory profile against E. coli (MIC 6.25 µg/mL) which
was equipotent to ampicillin, whereas, compounds 22,
26 and 32 (MIC 12.5 µg/mL), were 50% less active than
ampicillin against the same organism. Meanwhile, the
tested P. aeruginosa strain showed moderate sensitiv-
ity towards most of the active compounds, particularly
compound 25 (MIC 12.5 µg/mL) which was equipotent
to ampicillin. On the other hand, only nine compounds
13, 19, 20, 22, 23, 25, 26, 30 and 32 were able to display
a noticeable growth inhibitory potential against C. albi-
cans, among which compounds 25 and 26 (MIC 12.5 µg/
mL) were the most active members when compared with
clotrimazole (MIC 6.25 µg/mL).
In vitro MTT cytotoxicity assay
At both µg/mL and µM concentration levels, the
obtained data in Table 2 revealed that 12 compounds
namely; 3, 6, 11, 14, 17, 20, 22, 24, 25, 26, 29 and 31 were
able to affect cell viability of the tested tumour cell lines
particularly the human colon carcinoma HT29 cell line,
© 2013 Informa UK, Ltd.

128 H. M. Faidallah et al.
Scheme 1. Synthesis of compounds 4–17.
Scheme 2. Synthesis of compounds 18–32.
Journal of Enzyme Inhibition and Medicinal Chemistry

1,4-Disubstituted hexahydroquinolines: Synthesis and biological evaluation 129
whereas the rest of the compounds were totally inactive.
conclusion
In addition, the three tested human tumour cell lines
exhibited variable degree of sensitivity profiles towards
the active compounds. In terms of µg/mL concentration,
the human colon carcinoma HT29 cell line showed pro-
nounced sensitivity against compounds 22 and 25 (LC50
8.4 and 14.9 µg/mL, respectively) even higher than that
of doxorubicin (LC50 21.1 µg/mL). Moreover, a remark-
able cytotoxic potential was displayed by compounds 20
and 24 against the same cell line (LC50 29.6 and 26.3 µg/
mL respectively) comparable to that of doxorubicin. e
rest of the active compounds showed moderate to weak
activity profiles against the same cell line with LC50 range
of 34.9–76.3 µg/mL. On the other hand, the growth of the
human hepatocellular carcinoma Hep-G2 cell line was
found to be moderately inhibited by nine of the active
compounds with LC50 values range of 11.6–48.7 µg/
mL, among which compounds 20 and 25 (LC50 values
18.5 and 11.6 µg/mL, respectively) revealed the high-
est cytotoxic activity as compared to doxorubicin (LC50
1.69 μg/mL). Regarding the human breast cancer MCF7,
it was proved to be the least sensitive among the tested
cell lines, as its growth was inhibited by only seven of
the tested compounds. However, a remarkable growth
inhibitory potential was shown by compounds 22 and
25 as evidenced from their LC50 values (12.1 and 9.7 µg/
mL, respectively) when compared to doxorubicin (LC50
2.14 µg/mL).
A close examination of the structure of the active
compounds showed that the 4-chloro and 4-methoxy
groups at the C-4 aryl ring are the most favourable sub-
stituents. Although the key precursors 5 and 6 possessed
moderate cytotoxic profiles, yet formylation (11) or
nitrosation (7, 8) of these compounds led to a dramatic
reduction or even total loss of activity, respectively.
However, acetylation (14) and subsequent cyclization
into a tricyclic ring system (17) resulted in an obvious
improvement in the cytotoxic profile, especially against
the HT29 and Hep-G2 cell lines. Moreover, sulfonylation
as in (20), led to a noticeable enhancement in both the
cytotoxic spectrum and potential. On the other hand,
great improvement of the cytotoxic potential was linked
to the introduction of a thiocarbamoyl substituent at
position-1. It could be clearly recognized that the chlo-
rinated derivatives (22, 24, 25 and 26) showed distinc-
tive cytotoxic activities, whereas 21 and 23 were totally
inactive. Among these, the analogs 22 and 25 proved to
be the most active members in this study with a broad
spectrum of activity against the tested cell lines. Finally,
alkylation with a methyl or benzyl groups did not offer
any advantage to the cytotoxic profile of such type of
compounds. On the contrary, introduction of a methyl
group (29 and 31) resulted in a marginal enhancement
in the activity against HT29 cell line, whereas the effect
against the Hep-G2 and MCF7 cell lines was totally lost.
Meanwhile, alkylation with a benzyl group led to total
abolishment of activity.
Twenty nine novel 3-cyano-8-methyl-2-oxo-1,4-dis-
ubstituted-1,2,5,6,7,8-hexahydroquinolines supported
with various pharmacophores that are known to con-
tribute to potential chemotherapeutic effects were suc-
cessfully synthesized and evaluated for their biological
activity as antimicrobial agents against eight different
microorganisms and/or cytotoxic agents against three
different human tumour cell lines at both µg/mL and
µM concentration levels. e results revealed that eigh-
teen compounds displayed special pronounced activity
against S. aureus and E. coli bacterial strains, in addi-
tion to moderate antifungal activity against C. albicans.
Compounds 19, 22, 25, 30 and 32 could be considered
as the most active broad spectrum antimicrobial mem-
bers identified in this study. Among these, compound
25 proved to be the most active candidate as it showed
equipotency to ampicillin against S. aureus, E. coli and
P. aeruginosa, in addition to an obvious antifungal activ-
ity comparable with clotrimazole. e best antifungal
activity was demonstrated by compounds 25 and 26
which possessed almost half the activity of clotrimazole
against C. albicans. On the other hand, 12 compounds
were able show remarkable cytotoxic efficiency against
human colon carcinoma HT29, hepatocellular carci-
noma Hep-G2 and Caucasian breast adenocarcinoma
MCF7 cell lines. e results revealed that compounds
6, 17, 20, 22, 24 and 25 showed considerable broad
spectrum cytotoxic activity against the three tested cell
lines, among which the analogs 22 and 25 proved to
be the most active members in this study with special
effectiveness against the human colon carcinoma HT29
and human breast cancer MCF7 cell lines even higher
than that of doxorubicin, the reference standard cyto-
toxic agent utilized in this test. Finally, the obtained
antimicrobial and anticancer data makes such type of
compounds a fruitful matrix for further development
of more potent and selective cytotoxic and/or antimi-
crobial agents. At both µg/mL and µM concentration
levels, compounds 22 and 25 could be considered as
possible dual antimicrobial-anticancer candidates
that deserve further investigation and derivatization in
order to explore the scope and limitation of its biologi-
cal activities.
acknowledgement
e authors are deeply thankful to the authorities of the
Bioassay-Cell Culture laboratory, in vitro bioassays on
human tumour cell lines for drug discovery unit, National
Research Centre (NRC), Cairo, Egypt, for their efforts in
performing the MTT cytotoxicity assay.
Declaration of interest
e authors report no conflicts of interest.
© 2013 Informa UK, Ltd.

130 H. M. Faidallah et al.
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