Synthesis and cytotoxicity of a novel 1-alkylaminomethyl-2,4-diaryl- butadiene-1,3 fragment integrated within cyclohex(pent)enes

Article abstract of DOI:10.1016/j.farmac.2004.01.010

A group of 1-alkylaminomethyl-2-aryl-3-arylidenecyclohex(pent)enes 3a-n with a 1-alkylaminomethyl-2,4-diaryl-1,3-butadiene fragment and a group of their congeners 3-alkylaminomethyl-1,2-diarylcyclohexene 7a-f have been synthesised for the first time. The conjugated system in 1-alkylaminomethyl-2-aryl-3- arylidenecyclohex(pent)enes 3a-n was unambiguously confirmed by X-ray crystallography. Cytotoxicity tests revealed that 3a-n possess inconsistent cytotoxicity against cancer cells, not their congeners 7a-f.

Full text of DOI:10.1016/j.farmac.2004.01.010

IL FARMACO 59 (2004) 373–379
www.elsevier.com/locate/farmac
Synthesis and cytotoxicity of a novel
1-alkylaminomethyl-2,4-diaryl-butadiene-1,3 fragment integrated
within cyclohex(pent)enes
Kejun Zhao a,b,Yongfeng Wang a,*, Linxiang Zhao b, Zhizhong Ji b, Dan Li a,
Michael J. Tisdale a, Carl H. Schwalbe a
a School of Pharmacy, Aston University, Birmingham, B4 7ET, UK
b Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang City, China
Received 20 October 2003; accepted 23 January 2004
Abstract
A group of 1-alkylaminomethyl-2-aryl-3-arylidenecyclohex(pent)enes 3a-n with a 1-alkylaminomethyl-2,4-diaryl-1,3-butadiene fragment
and a group of their congeners 3-alkylaminomethyl-1,2-diarylcyclohexene 7a-f have been synthesised for the first time. The conjugated
system in 1-alkylaminomethyl-2-aryl-3-arylidenecyclohex(pent)enes 3a-n was unambiguously confirmed by X-ray crystallography. Cytotox-
icity tests revealed that 3a-n possess inconsistent cytotoxicity against cancer cells, not their congeners 7a-f. All rights reserved.
Keywords: Cyclohex(pent)enes; Cyclohex(pent)anone; a,b-unsaturated ketone; X-ray structure and Cytotoxicity
1. Introduction
whether or not a ketone is essential to the cytotoxicity of the
cytotoxic centre and whether or not it could be replaced by
another fragment, which possesses a better selectivity to
cancer cells and a lower toxicity. In this communication, for
the first time, we report the synthesis of 1-alkylaminomethyl-
2-aryl-3-arylidenecyclohex(pent)enes 3a-n and their conge-
ners of 3-alkylaminomethyl-1,2-diarylcyclohexenes 7a-f,
the crystal structure of the conjugated system in
1-alkylaminomethyl-2-aryl-3-arylidenecyclohex(pent)enes
3a-n and inconsistent cytotoxicity among 3a-n against mu-
rine cancer cells.
It is well known that fragments of a,b-unsaturated ketone
in cyclopentenones [1,2], cyclohexenones [3], a-alkyl(aryl)
idenecyclohex(pent)anone a’-alkylaminomethyl [4] (hidden
a,b and a’,b’-unsaturated ketone form) and a-methylene
lactone [5,6,7] are responsible for the cytotoxicity in a num-
ber of natural and synthetic compounds. Though this has
been discovered over three decades and the mechanisms of
their actions have been recorded [8], none of these com-
pounds have been introduced into the clinic. The reasons for
these are because at most of the time the toxicities are too
severe to be of any therapeutic value.
2. Results and discussion
In conjunction with the previous studies of anticancer
activities of cyclopentenones and a-alkylaminomethyl-
cyclohexanone a’-alkyl(aryl)idene [4,9], we designed and
synthesised a group of 1-alkylaminomethyl-2-aryl-3-aryli-
denecyclohex(pent)enes 3a-n with a 1-alkylaminomethyl-
2,4-diaryl-1,3-butadiene fragment in which the known cyto-
toxic fragment of a,b-unsaturated ketones has been
abolished. The purpose of the study is to explore further
2.1. Chemistry
The starting materials Mannich bases 1a-i were prepared
by use of the procedure reported previously [4,9].A Grignard
reagent was considered as a convenient and economical ap-
proach for the introduction of an aryl substitutent on the
carbonyl carbon in 1a-i. However, preparation of the Grig-
nard reagents 2a-d was hampered by inertness of aromatic
rings with a methoxy group. In contrast to phenyl bromide,
* Corresponding author.
E-mail address: y.f.wang@aston.ac.uk (Y.F. Wang).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.01.010

374
K. Zhao et al. / IL FARMACO 59 (2004) 373–379
Scheme 1
Synthesis of the products 3a-n
R
4
R
5
R
4
R
O
2
R
2
R
3
R
5
B
+
.
B HCl
(CH )n
2
R
3
R
1
(CH )n
2
MgCl
R
1
3a-n
1a. R1=R2=H, n=1, B=-N(CH3)2
2a. R3=R4=R5=H
1b. R1=OCH3; R2=H, n=1, B=-N(CH3)2
1c. R1=Cl; R2=H, n=1, B=-N(CH3)2
1d. R1=R2=H, n=2, B=-N(CH3)2
2b. R3=R5=H, R4= OCH3
2c. R3=R5=OCH3, R4= H
2d. R3=R4=H, R5= OCH3
1e. R1=OCH3; R2=H, n=2, B=-N(CH3)2
1f. R1=H; R2= OCH3, n=2, B=-N(CH3)2
1g. R1=H; R2= OCH3, n=2, B=pyrrolidine-1
1h. R1=R2=H, n=2, B=piperidine-1
1i. R1=Cl; R2=H, n=2, B=piperidine-1
Compound
n
1
1
1
1
1
1
1
2
2
2
2
2
2
2
R1
R2
H
R3
H
R4
H
R5
H
B
Yield (%)
m.p (oC)
158–160
200–202
186-188
178-180
197-200
181–182
207-210
211-213
188-190
184-188
210-212
216-218
157-158
188-189
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
H
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
-N(CH3)2
28
16
21
30
11
29
15
58
OCH3
OCH3
H
H
H
H
H
H
H
OCH3
H
H
H
OCH3
H
OCH3
Cl
H
OCH3
H
H
OCH3
H
H
H
Cl
H
OCH3
H
H
OCH3
H
H
H
H
H
H
H
OCH3
H
H
47
OCH3
H
H
H
H
32
OCH3
OCH3
H
H
H
OCH3
OCH3
H
20
H
H
H
pyrrolidine-1 10
piperidine-1 60
piperidine-1 60
H
H
H
Cl
H
H
H
OCH3
which reacts pretty fast in dry ether at 35°C, high temperature
and a prolonged reaction time were required for a methox-
yphenyl bromide. We mixed initiating reagents, 1,2-
dibromoethane and iodine, with magnesium and 1/4 portion
of a methoxyphenyl bromide in dry toluene with dry THF
and heated to reflux at 70°C. Once the reaction was initiated,
the remained methoxyphenyl bromide toluene and THF solu-
tion was added gradually and kept for refluxing. In most
cases the reflux had to last for 5 to 10 hrs. In cases of the
starting materials 1a-c with cyclopentanone (n=1), 3a-g were
afforded in a one-pot manner (Scheme 1). However the
starting materials 1d-i with cyclohexanone (n=2), to obtain
3h-n required two steps, because the dehydration did not
happen readily. Particularly when one of R1 or R2 is a meth-
oxy group, a stronger acidic condition, hydrochloric acid in
ether, was required for the dehydration; for the rest hydro-
chloric acid in ethanol were employed. The low yields of
3a-n were attributed to sensitivity of the Mannich bases to
the basic reaction media.
In order to evaluate the importance of the conjugated
diene in 3a-n for the cytotoxicity, a group of 3-alkyl-
aminomethyl-1,2-diarylcyclohexenes 7a-f was designed and
synthesised. A number of methods have been reported for the
synthesis of a-phenyl cyclohexanones, including a conden-
sation of a diazoaryl with a cyclohexanone [10], oxidation of
1-arylcyclohexenes [11], substitution of a-chlorocyclo-
hexanones with arylmetallic agents [12], diazoaryls with
cyclohexen-1-ol acetates [13], and a reaction of aryl halides
with cyclohexanone under strongly basic conditions, such as
NaNH2 [14]. As aryl halides are cheap and readily available
chemicals, we performed the reaction of chlorobenzene with
cyclohexanone to give a moderate yield of a-phenyl-
cyclohexanone 5, followed by Mannich reactions with dim-
ethylamine, pyrrolidine and piperidine to give a-phenyl cy-
clopentanone a’-Mannich bases 6a-c. Compounds 7a-f were
obtained directly from the addition reactions of Grignard
reagents 2a-d with 6a-c followed by the elimination of water
in situ (Scheme 2). Spectroscopic data for compounds 6a-c
and 7a-f are in Table 2.
The 2,4-bisaryl conjugated butadiene fragment in 3a-n
was unambiguously confirmed by X-ray crystallography of
3b (Figure 1). The 2-aryl group is obviously out of the plane
of the butadiene, by 58.7(1)o. Coplanarity would be impos-
sible due to the interaction of the vinyl H15 with the 2-aryl
H14, but this contact is a generous 2.57Å. Although the
4-aryl group is conjugated with the butadiene, their planes
still intersect at 13.7(2)o, most of the twist occurring around
the C15-C16 bond. The summary of the crystal data and
refinement are given in Table 1 and spectroscopic data for
compounds 3a-n in Table 2.
Fig. 1. ORTEP diagram of 3b

K. Zhao et al. / IL FARMACO 59 (2004) 373–379
375
Table 1
ity, and interestingly the compound 3h without any substitu-
ent on aryls showed the highest cytotoxicity in the tests.
Crystal data and structure refinement for 3b
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
C22 H25 N1O1
In summary, this communication has described for the
first time the synthesis, crystal structure and cytotoxicity of
1-alkylaminomethyl-2-aryl-3-arylidenecyclohex(pent)enes
3a-n. The precise mechanism of their cytotoxic effect is not
known; however it is perceptible that the 1-alkylamino-
methyl-2,4-diaryl-butadiene-1,3 fragment is involved in the
cytotoxic action.
319.45
293(2)
0.71073
triclinic
P-1
Unit cell dimensions
a (Å)
8.3290(18)
b (Å)
10.1207(12)
12.0593(11)
96.750(9)
c (Å)
␣ (o)
b (o)
100.873(12)
111.989(12)
905.7(2)
3. Experimental
c (o)
V (Å3)
3.1. Chemistry
Z
2
DCaled (Mg/m3)
Absorption coefficient (mm-1)
F(000)
1.171
Melting points were determined on a Reichert-Jung Mi-
crothermal apparatus and are uncorrected. NMR spectra
were recorded on a Bruker AC250 spectrometer at 1H
(250.1 MHz) and 13C (62.9 MHz). Chemical shifts are down-
field of tetramethylsilane. Mass spectroscopic analysis was
carried out on a Hewlett Packard 5989B MS machine with an
HP 5998A API Electrospray LC/MS interface; the LC being
an HP1100 system with autosampler. High resolution mass
spectra (HRMS) were measured on a Finnigan MAT
900 XLT high resolution double focussing mass spectrom-
eter using an electrospray method. Infrared spectra were
recorded on a Mattson 3000 FTIR Spectrometer; solid
samples were prepared as KBr discs and liquids as thin films
between sodium chloride plates. Flash column chromatogra-
phy was performed using Sorbsil C60 silica gel. TLC was
carried out using aluminum backed Merck Silica Gel 60 F254
plates and visualised under UV (254 nm). Potassium per-
manganate was used where appropriate to develop TLC
plates. Elemental analyses (C, H, N) were performed on a
Leeman 440 analyzer. Dry THF and dry ether were prepared
by refluxing with sodium metal chips and distillation. Mag-
nesium powder was activated using 3%HCl, washed with
distilled water then acetone and dried in vacuum.
0.071
344
Crystal size(mm)
2h Range (o)
0.45 x 0.40 x 0.20
2.22-24.98
Limiting ind.
-9 = h = 9, -12 = k = 12, -14 = l = 14
6155/3186
full-matrix least squares on F2
3186/0/221
Reflection collected/unique
Refinement method
Data/restraints/parameters
Final R indices [I > 2r (I)]
Goodness-of-fit on F2
Largest difference peak and hole
R1 = 0.0542, wR2 = 0.0886
1.072
0.173 and -0.136
(e Å -3
)
2.2. Cytotoxicity
The cytotoxicity of the compounds was tested against
cancer cell lines MAC13 (sensitive to most cytotoxic agents)
and MAC16 (resistant to most cytotoxic agents) and the
results are summarised in Table 3. [15] The compounds 3a-n
demonstrated inconsistent cytotoxicities; some members of
them are very potent, such as 3h with IC50 less than 1µM
against both resistant and sensitive cell lines, while most of
them showed a moderate activity against sensitive cell line
MAC13 and a weak activity against resistant MAC16.
The cytotoxicity of 3a-n was surprising and encouraging
because in these compounds there is no longer an a,b-
unsaturated ketone, the known cytotoxic core. Instead there
is a 2,4-bisaryl conjugated butadiene, which has never been
demonstrated as a cytotoxic core. In line with the previously
proposed action mechanism for the a,b-unsaturated ketone
and the nature of the conjugated diene system, one possible
action model responding to the cytotoxicity of 3a-n through
an alkylating effect is suggested in Scheme 3. As a proto-
nated amino group is electron withdrawing and a good leav-
ing group in an aqueous environment, position 4 is likely to
first undergo a nucleophilic attack, such as with a thiol; as a
result, an intermediate 4 may be formed, which may be
viable for a subsequent addition [3]. Since the numbers of
compounds tested are limited, it is difficult to see a clear
correlation between substituents on both aryls and cytotoxic-
3.1.1. Synthetic protocol for 1-dimethylaminomethyl-2-
aryl-3-arylidenecyclopentenes 3a-g
a. For phenyl bromide Grignard reagent 2a: Magne-
sium powder (0.5g, 20.5mmol) and one granule of io-
dine were put in a flask fitted with a dropping funnel and
condenser, phenyl bromide (20mmol) in dry ether
(20ml) was added in the dropping funnel and 2~3ml of
this mixture was let through into the flask, the flask was
heated up slowly with stirring until the reaction was
initiated. The remaining aryl bromide solution was
added over a period of 30min while the reaction mixture
was kept refluxing. After refluxing for another 30min,
and the magnesium powder had nearly disappeared,
then the solution was cooled to -5°C, a 2(E)-arylidene-
5-dimethylaminomethylcyclopentanone hydrochloride

Table 2
Spectroscopic data of compound 6a-c, 3a-n and 7a-fa
Compound 1H-NMR(CDCl3) d(ppm)
13C-NMR(CDCl3) d(ppm)
IR (cm-1)
MS (m/z)b
3a
3b
3c
3d
3e
3f
2.20(1, s, 6H), 2.80(9, m, 2H), 3.00(2, s 2H), 3.05(8, s, 2H), 5.95(3, s, 1H), CH3: 45.5 (2CH3); CH2: 29.5, 33.7, 58.4; CH: 119.7, 125.6, 127.0, 128.0, 128.2, 1610, 1588, 1501, 290(M+1),
7.10-7.50(4, 5, m, 10H);
129.8 C: 135.6, 138.6, 144.8, 146.2, 151.0
1035, 762, 696
245(M - NMe2)
2.20(1, s, 6H), 2.76(9, m, 2H), 3.01(2, s 2H), 3.05(8, s, 2H), 3.80(11, s, 3H), 5.87(3, CH3: 45.5 (2CH3), 55.1CH2: 29.4, 33.6, 58.5;CH: 113.6, 119.0, 126.9, 128.1, 1602, 1512, 1239, 320(M+1),
s, 1H), 6.82, 7.10-7.50(4, 5, m, 9H); 129.1, 129.8 C: 131.4, 135.8, 144.7, 145.2, 148.9, 157.4 1035, 796, 700 275(M - NMe2)
2.15(1, s, 6H), 2.74(9, m, 2H), 3.01(2, s 2H), 3.05(8, s, 2H), 3.78(11, s, 3H), CH3: 45.5 (2CH3), 55.1; CH2: 29.4, 33.6, 58.5; CH: 113.5, 113.6, 118.9, 129.1, 1600, 1508, 1243, 350(M+1),
3.84(11, s, 3H), 5.89(3, s, 1H), 6.82, 7.10-7.30(4, 5, m, 9H); 130.9; C: 127.8, 131.5, 144.3, 145.0, 149.1, 157.5, 158.5. 1182, 1029, 830 305(M - NMe2)
2.20(1, s, 6H), 2.75(9, m, 2H), 3.00(2, s 2H), 3.05(8, s, 2H), 3.85(11, s, 3H), 5.95(3, CH3: 45.5 (2CH3), 55.1; CH2: 29.5, 33.6, 58.5; CH: 113.6, 119.4, 125.5, 128.0, 1602, 1512, 1241, 320(M+1),
s, 1H), 6.93, 7.10-7.30(4, 5, m, 9H); 128.1, 130.9; C: 127.7, 138.6, 135.8, 146.2, 151.2, 158.5 1033, 810, 701 275(M - NMe2)
2.10(1, s, 6H), 2.77(9, m, 2H), 2.86, 2.93(2, d, 2H), 3.03(8, s, 2H), 3.67(11, s, 3H), CH3: 45.6 (2CH3), 55.1, 55.6, 56.2; CH2: 29.5, 33.5, 58.8; CH: 112.3, 113.2, 113.6, 1602, 1506, 1285, 380(M+1),
3.85(11, s, 6H), 5.80(3, s, 1H), 6.62, 6.79-6.87, 7.22-7.25(4, 5, m, 7H); 117.0, 118.0, 129.1; C: 125.7, 131.6, 140.9, 145.9, 148.2, 151.6, 153.3, 157.3 1022 335(M - NMe2)
2.10(1, s, 6H), 2.77(9, m, 2H), 2.99(2, s 2H), 3.02(8, s, 2H), 5.87(3, s, 1H), CH3: 45.5 (2CH3); CH2: 29.5, 33.7, 58.4; CH: 118.4, 127.1, 128.2, 129.1, 129.7; C: 1611, 1486, 1091, 324(M+1),
7.10-7.50(4, 5, m, 10H);
128.1, 135.6, 138.6, 144.8, 146.2, 151.0
795, 707
279(M - NMe2)
3g
2.11(1, s, 6H), 2.78(9, m, 2H), 2.87, 2.93(2, d, 2H), 3.01(8, s, 2H), 3.68(11, s, 3H), CH3: 45.6 (2CH3), 55.6, 56.1; CH2: 29.4, 33.5, 58.8; CH: 112.2, 113.2, 117.0, 1608.1600.1506,
384(M+1),
3.77(11, s, 3H), 5.79(3, s, 1H), 6.60, 6.87, 7.20-7.25(4, 5, m, 7H);
117.5, 128.1 129.1; C: 125.2, 130.7, 137.3, 140.7, 147.9, 151.0, 151.5, 153.3
1419,1218, 1046, 339(M - NMe2)
1040,
3h
3i
1.55(m, 2H), 1.73(dd, 1H), 1.90(m, 1H), 2.03 (dd, 1H), 2.13(s, 6H), 2.30(m, 1H), CH3: 45.5, 45.8; CH2: 25.8, 26.2, 27.5, 62.7; CH: 121.7, 125.7, 126.1, 127.0, 127.5, 1614, 1596, 1014, 322(M+1+H2O)
2.66(t, 1H), 2.89(d, 1H), 6.95, 7.15, 7.73, 7.93 (m, 11H); 127.7, 129.2; C: 78.7, 138.5, 145.6, 146.4. 701
1.55(m, 2H), 1.70(dd, 1H), 1.83(m, 1H), 2.07 (dd, 1H), 2.13(s, m, 6H), 2.25(1, m, CH3: 45.5, 45.8, 55.1; CH2: 25.8, 26.2, 27.5, 62.7; CH: 112.8, 121.3, 125.7, 127.7, 1608, 1509, 1253, 352(M+1+H2O),
1H), 2.67(t, 1H), 2.85(d, 1H), 3.79(s, 3H), 6.86, 7.20, 7.60, 7.89 (m, 10H); 128.1, 129.1; C: 78.5, 138.6, 145.9, 157.7. 1190, 829 277
3j
1.54(m, 2H), 1.70(dd, 1H), 1.84(m, 1H), 2.02 (dd, 1H), 2.14(s, m, 6H), 2.20(m, CH3: 45.5, 45.8, 55.4; CH2: 25.8, 26.0, 27.6, 63.6; CH: 113.2, 121.0, 126.0, 127.0, 1604, 1507, 1243, 352(M+1+H2O),
1H), 2.62(t, 1H), 2.89(d, 1H), 3.75(s, 3H), 6.78, 6.88, 7.15, 7.70, (m, 10H); 127.5, 130.3, 131.0; C: 78.6, 141.4, 144.6, 155.5. 1031, 761, 707 277
3k
3l
1.67(m, 1H), 1.88(m, 2H), 2.15(m, 3H), 2.50 (d, 1H), 2.80(m, 6H), 3.20(m, 3H), CH3: 55.4, 55.7, 42.1, 45.6; CH2: 18.0, 24.0, 25.0, 28.8; CH: 104.4, 109.0, 120.7, 1612, 1239, 1025, 364(M+1)
3.80(s, 3H), 6.60, 6.71, 6.95, 7.20(m, 9H);
127.8; C: 60.1, 144.4, 157.7, 158.9.
CH3: 55.5, 55.8; CH2: 18.1, 23.1, 23.2, 24.0, 25.0, 53.4, 55.9, 57.0; CH: 104.5, 1612, 1239, 1025, 390(M+1)
108.4, 120.7, 127.7; C: 127.9, 157.8, 158.9. 720
764
1.5-3.0(m, 16H), 3.25(m, 3H), 3.90(m, 3H), 6.60, 6.74, 6.84, 6.91, 7.20(m, 9H);
3m
1.0-3.0(m, 18H), 6.94, 7.20, 7.70, 8.17(m, 11H);
CH2: 23.9, 25.8, 26.1, 27.0, 54.7, 61.9; CH: 121.8, 125.7, 126.1, 126.8, 127.5, 1601, 1505, 1465, 362(M+1+H2O)
127.7, 129.1; C: 78.4, 138.5, 145.6.
1440, 853, 763,
703
3n
6a
6b
6c
1.0-3.0(m, 15H), 3.47(d, 1H), 3.71(m, 1H), 3.80(s, 3H), 3.84(m, 1H), 6.77, 6.95, CH3: 54.0; CH2: 20.8, 21.0, 21.1, 25.9, 44.0, 52.0, 53.9, 60.4; CH: 110.2, 112.3, 1601, 1486, 1247, 426(M+1+H2O)
7.15, 7.25 (m, 9H);
119.7, 120.4, 128.0, 128.5, 129.0; C: 79.8, 130.5, 134.4, 141.9, 144.4, 158.2.
791
1.60(m, 1H), 1.95(m, 3H), 2.33(m, 1H), 2.51(m, 1H), 2.70(m, 6H), 3.43(m, 1H),
3.61 (m, 1H), 3.82(m, 1H), 7.11, 7.25(m, 5H).
1701, 1425, 1093, 232(M+1)
694
1.55(m, 1H), 1.74(m, 1H), 2.04(m, 6H), 2.31(m, 1H), 2.53(m, 1H), 2.80(s, 2H), CH3: 22.9, 24.9, 34.6, 36.1, 54.1; CH: 47.8, 57.2, 127.0, 128.1, 128.6; C: 137.7, 1706, 1129, 700
2.94(d, 1H), 3.52 (m, 3H), 3.80(m, 2H), 7.11, 7.32(m, 5H). 209.1.
258(M+1)
1.40(m, 1H), 1.57(m, 1H), 1.77(m, 3H), 2.04(m, 4H), 2.32( m, 2H), 2.51(m, 2H), CH3: 21.7, 22.4, 22.6, 25.1, 35.3, 36.2, 52.7, 55.5, 56.2; CH: 47.2, 57.3, 127.1, 1708, 750, 695
2.69(d, 2H), 3.19 (m, 1H), 3.40(m,1H), 3.64(m, 2H), 3.82(m,1H), 7.14 , 7.29(m, 128.2, 128.6; C: 137.5, 209.1.
5H).
272(M+1)
7c
1.74(2,3 m, 3H), 2.08(3, m, 1H), 2.40, 2.60(9, d, 6H), 2.33, 2.55, (8, m, 2H), CH3: 42.0, 46.0, 55.1; CH2: 25.1, 27.1, 27.3, 61.0; CH: 43.2, 53.5, 126.5, 127.9, 1610, 1508, 1259, 340(M+1+H2O)
2.59(4, m, 1H), 2.94 (1, m, 1H), 3.12(4, dd, 1H), 3.74(10, s, 3H) 6.75, 7.06 (7, m, 128.6; C: 77.0, 136.3, 140.0, 158.2.
9H);
1001, 699
7d
7e
7f
1.71, 2.14 (2, 8, 9 m, 10H), 2.25, 2.75 (9, m, 2H), 2.53(3, dd, 2H), 3.07(1, 4, m, 2H), CH3: 55.0; CH2: 22.9, 23.0, 25.1, 26.9, 27.3, 53.0, 56.3, 58.9; CH: 44.0, 53.5, 1596, 1241, 1149, 366(M+1+H2O)
3.57(10, s, 3H), 3.80(4, m, 1H), 6.77, 7.05 (7, m, 9H); 126.5, 127.8, 128.5, 129.0; C: 77.2, 140.0, 159.0. 700
1.00-3.00(2,3,4,8,9, m, 17H) 3.10(1, m, 1H), 3.20(1, m, 1H), 3.58(10, s, 3H), CH3: 55.3; CH2: 21.6, 21.7, 25.0, 27.3, 27.8, 51.2, 55.9, 56.0, 59.9;CH: 42.4, 53.4, 1608, 1595, 1242, 380(M+1+H2O)
3.85(4, m, 1H), 6.77, 7.03 (7, m, 9H); 126.5, 127.8, 128.6, 129.2; C: 77.5, 140.0, 159.5. 1149, 700
1.20-2.50(2,3,4,8,9, m, 17H), 2.75(4, m, 1H), 3.40(1, dd, 1H), 3.47(10, s, 3H), 55.4, 55.5; CH2: 23.6, 26.2, 26.9, 28.1, 28.6, 56.7, 61.6; CH: 42.5, 49.5, 111.3, 1606, 1498, 1220, 410(M+1+H2O)
3.83(10, s, 3H), 6.55, 6.71, 6.90 (7, m, 8H); 112.9, 114.1, 125.2, 126.8, 128.8; C: 79.5, 136.6, 143.5, 149.8, 153.2. 1047, 697
a Elemental analysis of the compounds are within 0.4% of the calculated values; b m/z excludes HCl.

K. Zhao et al. / IL FARMACO 59 (2004) 373–379
377
Scheme 2
Synthesis of products 7a-f
Cl
O
O
O
HCHO
NaNH2
B .HC l
+
HB
6a. B =N(CH3)2
5
6b. B =pyrrolidine-1
6c. B =piperidine-1
R3
R3
R4
R2
R1
R4
R2
+
R1
2a-d
B .HC l
HO
B
H
7a-f
Compound
R1
H
R2
R3
R4
H
H
H
H
H
B
Yield (%)
m.p (oC)
240 (d)
7a
7b
7c
7d
7e
7f
H
H
-N(CH3)2
34
19
14
21
24
20
H
H
OCH3
-N(CH3)2
216-220
152-154
216-220
204-208
214-220
H
OCH3
OCH3
OCH3
H
H
H
H
H
-N(CH3)2
H
pyrrolidine-1
piperidine-1
piperidine-1
H
OCH3
OCH3
Table 3
dried over magnesium sulfate. The clear solution was
treated with dry HCl ether solution dropwise and the
resultant oil was kept at room temperature for 1 day,
then it was dissolved in water (10ml), basified to pH12
with 10% sodium hydroxide and extracted with ether
(3 x 20ml). The ether solution was titrated with dry HCl
ether solution to give a white solid, which was recrys-
tallized to afford the titled products.
Cytotoxicity of 3a-b, 3d, 3f-h, 3k-l, and 7a-f
Compound
MAC16(72hr)
IC50(µM)*
43.88
MAC13(48hr)
IC50(µM)*
18.33
3a
3b
3d
3f
3g
3h
3k
3l
27.86
6.93
51.17
29.17
8.31
7.31
18.23
18.23
0.9
0.96
b. For methoxyphenyl bromide Grignard reagents 2b,
2c and 2d: Magnesium powder (0.5g, 20.5mmol), one
granule of iodine and 4 drops of 1,2-dibromoethane
were put in a flask. 10ml of a methoxyphenyl bromide
(20mmol) in a mixed solvent of dry THF (30ml) and dry
toluene (20ml) was added and the flask was heated up
with stirring until the reaction was initiated. The re-
maining methoxyphenyl bromide solution was added
over a period of 30min while the reaction mixture was
kept refluxing. When the magnesium powder had nearly
disappeared (It usually took 5-10 hrs.), the solution was
cooled to -5°C. A 2(E)-arylidene-5-dimethylamino-
methylcyclopentanone hydrochloride [9] (1.5g,
5.6mmol) was added by portions and the mixture was
stirred at room temperature for 6 hours. The following
operation procedure was same to a. For phenyl bro-
mide Grignard reagent 2a.
31.7
4.35
58.32
4.15
7a
7b
7c
7d
7e
7f
>100
>100
>100
>100
92.69
>100
67.18
60.27
30.69
46.37
37.34
33.36
* Values are mean of triplicate determinations.
Nu
Cl
Nu
4
+
H2N(CH3)
2Cl
H
N
2
1
3
CH3
CH3
3h
4
Scheme 3. A proposed action model for 3a-n (Nu is a nucleophile. 3a-n
were in a hydrogen chloride salt form dissolved in a phosphate buffer.)
3.1.2. Synthetic protocol for1-alkylaminomethyl-2-aryl-3-
arylidenecyclohexenes 3h-n
[9] (1.5g, 5.6mmol) was added by portions and the
mixture was stirred at room temperature for 6 hours.
Each 1-alkylaminomethyl-2-aryl-3-arylidenecyclohexa-
nol (3.3mmol) was dissolved in ethanol (15ml), then 37%
hydrochloric acid (1.5ml) was added. The reaction solution
was heated to 60°C and kept at this temperature for 1 hour
with constant stirring. After the solution was cooled to room
temperature, it was diluted with water (40ml) and washed
with ether (20ml). The aqueous solution was basified using
50% sodium hydroxide and extracted with ether (2 x 20ml).
The ether layer was washed with brine (2 x 20ml), and dried
over magnesium sulfate. On titration with dry HCl ether
The resulting mixture was poured into a mixture of
ammonium chloride (5g), water (20ml) and ice (30-
40g). The ether layer was separated, while the water
layer was extracted with ether (20ml). The combined
organic layer was washed with brine. The organic layer
was extracted by cold 5% hydrochloric acid (2 x 20ml),
and the combined acid solution was adjusted to pH14
using 10% sodium hydroxide solution and extracted
with ether (3 x 20ml), the ether layer washed with brine,

378
K. Zhao et al. / IL FARMACO 59 (2004) 373–379
solution a white solid was given which was recrystallized
from acetone to afford the titled products.
3.2.2. Cytotoxicity
MAC13 cells were seeded at 5x103cells/ml. MAC16 cells
were seeded at 2x104cells/ml. Both cell lines were seeded in
24 well plates and incubated for 24 hours before the test
compound addition. The test compounds were dissolved in
phosphate-buffered saline (PBS) to make a 1ml stock solu-
tion and added to wells in 10µl aliquots to give the concen-
trations of the tested compounds ranging from 0.1µM to
100µM. Each concentration had two replicates and a control
well. MAC13 cell counts were made 72 hr after drug addi-
tion. MAC16 cell counts were made 48hr and 72hr after drug
addition using a ZM electronic particle Coulter Counter.
Coulter Counter settings are upper threshold TU (15µm),
lower threshold TL (8µm), attenuation (A) 16 and current I
(200).
3.1.3. Synthetic protocol for ␣-arylcyclohexanone
␣’-Mannich bases hydrochloride 6a-c
2-Phenylcyclohexanone [12] (5g, 28.7mmol), paraform-
aldehyde (3g, 100mmol) and a base (dimethylamine hydro-
chloride, pyrrolidine or piperidine) (29mmol) was stirred in
ethanol (80ml). The pH value of the mixture was adjusted to
3 using 37% hydrochloric acid, and the reaction mixture was
refluxed for 12 hours. The resulting solution was diluted with
water (50ml), washed with ether (2 x 30ml), and the aqueous
layer was basified with 20% sodium hydroxide to pH10
followed by extraction with ether (3 x 30ml). The organic
layer was washed with water, and dried over magnesium
sulfate. On titration with dry HCl ether solution a white solid
was given which was recrystallized from ethanol to afford the
product, 2-phenyl-6-dimethylaminomethylcyclohexanone
HCl (6a), 2-phenyl-6-(pyrrolidin-1-yl)methylcyclohexa-
none HCl (6b) or 2-phenyl-6-(piperidin-1-yl)methylcyclo-
hexanone hydrochloride HCl (6c).
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3.2.1. Cell culture
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