Synthesis and in-vitro anticancer evaluation of bis-tacrine congeners

Article abstract of DOI:10.1211/0022357011775046

In the search for potential new anticancer drugs, an efficient synthesis of bis-tetrahydro-aminoacridine (bis-tacrine) and its congeners was accomplished by bis-amination of 9-chloro-tetrahydroacridine and its congeners under heated conditions. The critical chlorides were efficiently prepared from o-aminoaromatic acids and cycloketones in-situ in the presence of phosphorus oxychloride. In-vitro cytotoxic evaluation of the compounds was carried out against a panel of 60 human cancer cell lines. Among them, butyl-linked bis-tacrine (5b) exhibited the strongest cytotoxic profile with G150 (concentration causing 50% growth inhibition) values of approximately 0.04-0.08 μm against breast, colon, melanoma and non-small lung cancer cells. Congeners bearing a longer alkyl chain were on average 30- to 100-fold less cytotoxic against these cancer cells. Shorter connecting alkyl chains of bis-tacrine or its congeners dramatically decreased the cytotoxic effects. Compound 5b has been selected for further biological evaluation of its anticancer profile.

Full text of DOI:10.1211/0022357011775046

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 83–88
#
2001 The Authors
Received July 18, 2000
Accepted September 27, 2000
ISSN 0022-3573
Synthesis and in-vitro anticancer evaluation of bis-
tacrine congeners
Ming-Kuan Hu
Abstract
In the search for potential new anticancer drugs, an efficient synthesis of bis-tetrahydro-
aminoacridine (bis-tacrine) and its congeners was accomplished by bis-amination of 9-chloro-
tetrahydroacridine and its congeners under heated conditions.
The critical chlorides were efficiently prepared from o-aminoaromatic acids and cyclo-
ketones in-situ in the presence of phosphorus oxychloride. In-vitro cytotoxic evaluation of the
compounds was carried out against a panel of 60 human cancer cell lines. Among them, butyl-
linked bis-tacrine (5b) exhibited the strongest cytotoxic profile with GI50 (concentration
causing 50% growth inhibition) values of approximately 0.04–0.08 µ against breast, colon,
M
melanoma and non-small lung cancer cells. Congeners bearing a longer alkyl chain were on
average 30- to 100-fold less cytotoxic against these cancer cells. Shorter connecting alkyl chains
of bis-tacrine or its congeners dramatically decreased the cytotoxic effects.
Compound 5b has been selected for further biological evaluation of its anticancer profile.
Introduction
School of Pharmacy, National
Defence Medical Center, PO Box
9
0048-508, Neihu, Taipei 114,
Various dimeric compounds have been shown to have broad spectrum activity
against a variety of human tumour cells (Wakelin 1986; Spicer et al 2000), and bis-
acridines and dimeric naphthalimides have been considered for clinical trials as
anticancer candidates (Goldin et al 1981; Brana et al 1997). A positive correlation
between DNA bis-intercalation and cytotoxicity was recently investigated (Bailley
et al 1996). However, some studies have shown that lipophilic dimeric acridines and
naphthalimides still showed significant undesirable toxicity (Segal-Bendirjian et al
1988; Diaz-Rubio et al 1994).
Taiwan, Republic of China
Ming-Kuan Hu
Correspondence: Email:
hmk!ndmctsgh.edu.tw
Acknowledgements and
funding: This work was
supported in part by the
Tetrahydroaminoacridine (1, tacrine; Figure 1) is currently one of the major
drugs approved for use in Alzheimer’s disease (McGeer & McGeer 1995). It has a
cyclohexyl-fused quinoline structure and is similar to the planar acridine moiety.
Taking advantage of the known ability of the acridine pharmacophore to interact
with DNA, a variety of bis-acridines with anticancer activity have been developed
(Chen et al 1978; Denny 1989). To my knowledge, there are no reports on the
cytotoxic evaluation of the analogous bis-tacrine derivatives. Therefore, a series of
bis-tacrine congeners, connected at the amino position of the fused-ring by alkyl
chains of varying length, were prepared as potential pharmacophores. Cytotoxic
evaluation of the bis-tacrines and their congeners was carried out using a sulf-
orhodamine B (SRB) protein assay against a panel of 60 human cancer cell lines
(National Cancer Institute drug-screening programme).
National Science Council of the
Republic of China (NSC 88-2314-
B-016-048). The author wishes to
thank the National Cancer
Institute (Bethesda, MD) for
testing the compounds in the
drug-screening programme.
Elemental analyses and high-
resolution mass spectra were
performed by the National
Science Council Regional
Instruments Center at National
Taiwan University and National
Tsing Hua University, and are
gratefully appreciated.
83

84
Ming-Kuan Hu
NH2
(3.11 mL, 30 mmol) were condensed to give the title
compound (3.92 g, 86%) as a brown solid: mp
1
1
46–149mC; H NMR (300 MHz, CDCl ) δ 9.02 (dd,
3
J l 1.7, 4.2 Hz, 1H, H-6), 8.49 (dd, J l 1.8, 8.4 Hz, 1H,
H-8), 7.46 (dd, J l 4.2, 8.3 Hz, 1H, H-7), 3.19 (s br, 2H,
CH ), 3.00 (t, J l 6.1 Hz, 2H, CH ), 2.03–1.90 (m, 4H,
N
Tacrine (1)
2
2
Figure 1 Structure of tacrine (1).
CH CH ); EIMS: m\z 220 [Mj2+, 33], 218 [M+, 100].
2
2
10-Chloro-2,3,4,5-tetrahydro-1H-cyclohepta[1,2-b]
quinoline (4c).
Materials and Methods
According to the above procedure, anthranilic acid
(2.05 g, 14.9 mmol) and cycloheptanone (1.27 mL,
Chemistry
1
4.9 mmol) were condensed to give the title compound
1
as a pale brown solid (1.55 g, 45%): mp 87–89mC; H
NMR (300 MHz, CDCl ) δ 8.56 (d, J l 8.4 Hz, 1H,
ArH), 8.20 (d, J l 8.2 Hz, 1H, ArH), 7.77 (t, J l 6.7 Hz,
1H, ArH), 7.69 (t, J l 7.6 Hz, 1H, ArH), 3.54 (t, J l
4.5 Hz, 2H, CH ), 3.24–3.16 (m, 2H, CH ), 1.90–1.70
All reagents were commercial materials and were used
directly unless otherwise stated. Dimethylformamide
3
/
was dehydrated over a 4-A molecular sieve. NMR
spectra were recorded on a Varian Gemini at 300 MHz
1
13
2
2
for H NMR and at 75 MHz for C NMR. Elemental
analyses were determined using a Perkin-Elmer 240 EA
analyser. Chromatography refers to flash chromato-
graphy on silica gel (silica gel 60, 230–400 mesh ASTM;
E. Merck). Melting points were recorded on a Thomas
Hoover capillary melting-point apparatus in open ca-
pillary tubes and are uncorrected.
(m, 6H, CH CH CH ); FABMS (NBA as matrix): m\z
2 2 2
+
[MjH] 232.0, HR-FABMS: exact mass calculated for
+
C H NCl [MjH] 232.0869; found 232.0888.
1
4
15
General procedure for the synthesis of alkyl-linked bis-
tacrinyl congeners.
A mixture of 9-chloro-1,2,3,4-tetrahydroacridine (4a)
or its congeners (4b, c; 1.0 equiv.), 1,n-diaminoalkane
9
-Chloro-1,2,3,4-tetrahydroacridine (4a).
(0.5 equiv.), phenol (2.0 equiv.), and NaI (0.025 equiv.)
To a mixture of anthranilic acid (2a; 7.4 g, 53.9 mmol)
and cyclohexanone (3a; 5.36 mL, 51.7 mmol), 30 mL
phosphoryl chloride was carefully added in an ice bath.
The resulting mixture was heated under reflux for 2 h,
then cooled at room temperature, and concentrated to
give a viscous slurry. The residue was diluted with
EtOAc, neutralized with aqueous K CO , and washed
was heated at 180mC in an oil bath for 1.5–5.0 h. The
reaction mixture was cooled to room temperature, then
diluted with EtOAc and made basic with 10% KOH
solution. The organic layer was washed with water, then
brine, dried over MgSO and concentrated in-vacuo to
4
remove solvent. The resulting residue was purified by
flash chromatography (CH Cl to CH Cl :CH OH l
2
3
2
2
2
2
3
with brine. The organic layer was dried over anhydrous
K CO and concentrated in-vacuo to give a pale brown
10:1 as eluents) to afford alkyl-linked bis-tacrines in
moderate yields.
2
3
solid, which was recrystallized with acetone to give the
title compound (11.4 g, 94%): mp 68–70mC (lit. mp Ethyl-linked bis-tacrine (5a).
1
6
6–68mC; Sargent & Samll (1946)); H NMR (300 MHz, According to the general procedure, chloride 4a
CDCl ) δ 8.13 (d, J l 7.5 Hz, 1H, ArH), 8.00 (d, J l (0.75 g, 3.50 mmol) and 1,2-diaminoethane (0.12 mL,
3
8
7
2
.3 Hz, 1H, ArH), 7.63 (dd, J l 9.2, 7.5 Hz, 1H, ArH), 1.75 mmol) were condensed under heat for 1.5 h to
.51 (dd, J l 9.2, 8.3 Hz, 1H, ArH), 3.10 (t, J l 6.3 Hz, afford, after flash chromatography (CH Cl to CH Cl \
2
2
2
2
H, CH ), 2.97 (t, J l 4.8 Hz, 2H, CH ), 1.91 (s br, 4H, CH OH l 10:1 as eluents), the title compound (0.40 g,
2
2
3
1
3
CH -CH ); C NMR (75 MHz, CDCl ) δ 160.1, 147.2, 54%) as an amber solid: mp 123–125mC; R 0.27
2
2
3
f
1
1
29.9, 129.5, 129.1, 127.1, 124.3, 34.7, 28.1, 23.2, 23.0; (CH Cl \CH OH\NH OH l 10:1:1);
H
NMR
2
2
3
4
+
+
EIMS: 217 (M , 100), 219 (Mj2 , 33); HR-EIMS: (300 MHz, CDCl ) δ 7.93 (d, J l 8.2 Hz, 2H, Ar-H),
exact mass calculated for C H NCl [M] 217.0659; 7.88 (d, J l 8.2 Hz, 2H, Ar-H), 7.55 (t, J l 7.1 Hz, 2H,
3
+
1
3
12
found 217.0648.
Ar-H), 7.33 (t, J l 7.3 Hz, 2H, Ar-H), 4.20 (s br, 2H,
NH), 3.71 (s br, 4H, CH CH ), 3.04 (t, J l 6.3 Hz, 4H,
2
2
2
9
[
-Chloro-1,2,3,4-tetrahydro-cyclohexa[1,2-b]pyrido
2,3-b]pyridine (4b).
2 CH ), 2.59 (t, J l 5.7 Hz, 4H, 2 CH ), 1.95–1.70
2
2
(m, 8H, 2 CH CH ); FABMS (NBA as matrix): m\z
2
2
+
According to the above reaction procedure, 2-amino- [MjH] 423.2; HR-FABMS: exact mass calculated for
+
nicotinic acid (4.14 g, 30 mmol) and cyclohexanone C H N [MjH] 423.2584; found 423.2577.
2
8
31
4

Anticancer bis-tacrine congeners
85
Butyl-linked bis-tacrine (5b).
According to the general procedure, chloride 4a (0.75 g, the title compound (0.14 g, 24%) as an amber solid: mp
graphy (CH Cl to CH Cl \CH OH l 10:1 as eluents),
2
2
2
2
3
3
1
.5 mmol)
and
1,4-diaminobutane
.75 mmol) were condensed under heat for 2.5 h to 10:1:1); H NMR (300 MHz, CDCl ) δ 8.79 (s br, 2H,
(0.18 mL, 131–133 mC; Rf 0.22 (CH Cl \CH OH\NH OH l
1
2
2
3
4
3
afford, after flash chromatography (CH Cl to CH Cl \ Ar-H), 8.41 (d, J l 8.4 Hz, 2H, Ar-H), 7.26–7.09
2
2
2
2
CH OH l 10:1 as eluents), the title compound (m, 2H, Ar-H), 3.80 (s br, 4H, 2 N-CH ), 2.99 (t, J l
3
2
(
0.40 g, 51%) as an amber glass foam: mp 156–158 mC, 4.5 Hz, 4H, 2 CH ), 2.53 (t, J l 5.4 Hz, 4H, 2 CH ),
2
2
1
Rf 0.34 (CH Cl \CH OH\NH OH l 10:1:1);
NMR (300 MHz, CDCl ) δ 7.93 (d, J l 4.7 Hz, 2H, matrix): m\z [MjH] 425.1; HR-FABMS: exact mass
Ar-H), 7.90 (d, J l 4.4 Hz, 2H, Ar-H), 7.56 (t, J l calculated for C H N [MjH] 425.2454; found
H
1.82–1.74 (m, 8H, 2 CH CH ); FABMS (NBA as
2 2
2
2
3
4
+
3
+
2
6
29
6
7
.4 Hz, 2H, Ar-H), 7.346 (t, J l 7.8 Hz, 2H, Ar-H), 3.51 423.2454.
s br, 4H, 2 N-CH ), 3.07 (s br, 4H, 2 CH ), 2.67 (s br,
(
2
2
4
H, 2 CH ), 1.90 (s br, 8H, 2 CH CH ), 1.75 (s br, 4H, N,Nh-Bis-(1,2,3,4-tetrahydro-cyclohexa[1,2-b]pyrido-
2
2
2
+
CH CH ); FABMS (NBA as matrix): m\z [MjH]
[2,3-b]pyrid-9-yl)-1,4-diaminobutane (6b).
2
2
451.2; HR-FABMS: exact mass calculated for C H N
3
According to the general procedure, chloride 4b (0.51 g,
2.34 mmol) and 1,4-diaminobutane (0.12 mL, 1.17
mmol) were condensed under heat for 1.5 h to afford,
after flash chromatography (CH Cl to CH Cl \CH OH
0
35
4
+
[MjH] 451.2864; found 451.2840.
Hexyl-linked bis-tacrine (5c).
2
2
2
2
3
Chloride 4a (0.75 g, 3.5 mmol) and 1,7-diaminoheptane l 10:1 as eluents), the title compound (70 mg, 13%) as
0.20 g, 1.75 mmol) were condensed under heat for 2 h an amber solid: mp 121–123 mC; R 0.29 (CH Cl
(
f
2
2
1
to afford the title compound (0.47 g, 56%) as an amber \CH OH\NH OH l 10:1:1); H NMR (300 MHz,
3
4
glass foam: R 0.38 (CH Cl \CH OH\NH OH l CDCl ) δ 8.84 (s br, 2H, Ar-H), 8.28 (d, J l 8.4 Hz, 2H,
f
2
2
3
4
3
1
1
0:1:1); H NMR (300 MHz, CDCl ) δ 7.94 (d, J l Ar-H), 7.27 (dd, J l 4.1, 8.4 Hz, 2H, Ar-H), 4.20 (s br,
3
4
.1 Hz, 2H, Ar-H), 7.91 (d, J l 4.1 Hz, 2H, Ar-H), 7.54 2H, 2 NH), 3.50 (t, J l 4.2 Hz, 4H, 2 N-CH ), 3.07 (s br,
2
(
t, J l 7.0 Hz, 2H, Ar-H), 7.32 (t, J l 7.0 Hz, 2H, 4H, 2 CH ), 2.58 (s br, 4H, 2 CH ), 1.85 (s br, 8H, 2
2
2
Ar-H), 3.47 (t, J l 7.1 Hz, 4H, 2 N-CH ), 3.06 (s br, 4H, CH CH ), 1.72–1.60 (m, 4H, CH CH ); FABMS (NBA
2
2
2
2
2
+
2
CH ), 2.68 (s br, 4H, 2 CH ), 1.90–1.86 (m, 8H, as matrix): m\z [MjH] 453.2; HR-FABMS: exact
2
2
+
2
CH CH ), 1.65 (s br, 4H, CH CH ), 1.41 (s br, 4H, mass calculated for C H N [MjH] 453.2767; found
2
2
2
2
28 33
6
+
CH CH ); FABMS (NBA as matrix): m\z [MjH]
453.2768.
2
2
479.2; HR-FABMS: exact mass calculated for C H N
3
2
39
4
+
[
MjH] 479.3173; found 479.3183.
N,Nh-Bis-(1,2,3,4-tetrahydro-cyclohexa[1,2-b]pyrido
2,3-b]pyrid-9-yl)-1,6-diaminohexane (6c).
Chloride 4b (0.43 g, 2.0 mmol) and 1,6-diaminohexane
Chloride 4a (0.75 g, 3.5 mmol) and 1,8-diaminooctane (0.12 g, 1.0 mmol) were condensed under heat for 2 h to
0.25 g, 1.75 mmol) were condensed under heat for 4 h furnish, after flash chromatography (CH Cl to CH Cl \
[
Octyl-linked bis-tacrine (5d).
(
2
2
2
2
to afford the title compound (0.45 g, 51%) as an amber CH OH l 10:1 as eluents), the title compound (0.12 g,
3
1
oil: R 0.48 (CH Cl \CH OH\NH OH l 10:1:1); H 25%) as an amber glass foam: R 0.28 (CH Cl
f
2
2
3
4
f
2
2
1
NMR (300 MHz, CDCl ) δ 7.96 (d, J l 8.7 Hz, 2H, \CH OH\NH OH l 10:1:1); H NMR (300 MHz,
3
3
4
Ar-H), 7.92 (d, J l 8.5 Hz, 2H, Ar-H), 7.55 (t, CDCl ) δ 8.86 (d, J l 2.7 Hz, 2H, Ar-H), 8.38 (d, J l
3
J l 7.3 Hz, 2H, Ar-H), 7.35 (t, J l 7.0 Hz, 2H, Ar-H), 8.2 Hz, 2H, Ar-H), 7.26–7.21 (m, 2H, Ar-H), 4.50 (s br,
3
CH ), 2.69 (s br, 4H, 2 CH ), 1.91 (s br, 8H, 2 CH CH ), 2 CH ), 2.70–2.60 (m, 4H, 2 CH ), 1.95–1.80 (m, 8H, 2
.49 (t, J l 7.4 Hz, 4H, 2 N-CH ), 3.07 (s br, 4H, 2 2H, 2 NH), 3.60–3.50 (m, 4H, 2 N-CH ), 3.07 (s br, 4H,
2
2
2
2
2
2
2
2
1
.75–1.55 (m, 4H, CH CH ), 1.50–1.20 (m, 8H, 2 CH CH ), 1.70–1.60 (m, 4H, 2 CH ), 1.45–1.35 (m, 4H,
2 2 2 2 2
+
+
CH CH ); FABMS (NBA as matrix): m\z [MjH]
2 CH ); FABMS (NBA as matrix): m\z [MjH]
481.3; HR-FABMS: exact mass calculated for C H N
30 37
2
2
2
5
07.3; HR-FABMS: exact mass calculated for C H N
43
3
4
4
6
+
+
[
MjH] 507.3487; found 507.3470.
[MjH] 481.3080; found 481.3057.
N,Nh-Bis-(1,2,3,4-tetrahydro-cyclohexa[1,2-b]pyrido
2,3-b]pyrid-9-yl)-1,2-diaminoethane (6a).
N,Nh-Bis-(1,2,3,4-tetrahydro-cyclohexa[1,2-b]pyrido
[2,3-b]pyrid-9-yl)-1,8-diaminooctane (6d).
[
According to the general procedure, chloride 4b (0.60 g, According to the general procedure, chloride 4b (0.57 g,
.75 mmol) and 1,2-diaminoethane (90 µL, 1.38 mmol) 2.60 mmol) and 1,8-diaminooctane (0.19 mL, 1.30
were condensed for 1.5 h to afford, after flash chromato- mmol) were condensed under heat for 2.5 h to afford,
2

86
Ming-Kuan Hu
Cl
N
CO H
2
O
i
(
)
(
)
m
X
X
NH2
m
2a, b X = CH, N
3a, b m = 1, 2
4a X = CH, m = 1
b X = N, m = 1
c X = CH, m = 2
Figure 2 Synthesis of 9-chlorotetrahydroacridine (4a) and its congeners 4b and 4c. Reagents: i. POCL , reflux 2 h.
3
after flash chromatography (CH Cl to CH Cl \CH OH 4H, 2 CH ), 2.90 (s br, 4H, 2 CH ), 1.95–1.65 (m, 16H,
2
2
2
2
3
2
2
l 10:1 as eluents), the title compound (0.34 g, 51%) as 8 CH ), 1.45–1.20 (m, 8H, 4 CH ); FABMS (NBA as
2
2
+
an amber solid: mp 115–117 mC; R 0.38 (CH Cl
matrix): m\z [MjH] 535.1; HR-FABMS: exact mass
f
2
2
1
+
\
CH OH\NH OH l 10:1:1); H NMR (300 MHz, calculated for C H N [MjH] 535.3801; found
3
4
36 47
4
CDCl ) δ 8.84 (d, J l 1.8 Hz, 2H, Ar-H), 8.37 (d, J l 535.3777.
3
8
.3 Hz, 2H, Ar-H), 7.19 (dd, J l 4.0, 8.3 Hz, 2H, Ar-H),
4
.46 (s br, 2H, 2 NH), 3.50 (s br, 4H, 2 N-CH ), 3.06
2
National Cancer Institute drug-screening
programme
(
s br, 4H, 2 CH ), 2.62 (s br, 4H, 2 CH ), 1.85 (s br, 8H,
2 2
2
8
CH CH ), 1.60 (s br, 4H, CH CH ), 1.45–1.20 (s br,
2 2 2 2
These synthetic compounds were selected and evaluated
in a drug-screening programme at the National Cancer
Institute (Bethesda, MD). Cytotoxicity tests were per-
formed on 60 human cancer cell lines derived from nine
different tissues and compounds were tested at a mini-
mum of five concentrations at 10-fold dilutions. A 48-h
continuous drug exposure protocol was used, and a
SRB protein assay (Skehan et al 1990) was used to
estimate cell viability or growth. The GI50 (concen-
tration causing 50% growth inhibition) was determined,
which corresponds to the IC50 value defined elsewhere
H, 2 CH CH ); FABMS (NBA as matrix): m\z
2
2
+
[MjH] 509.3; HR-FABMS: exact mass calculated
+
for C H N [MjH] 509.3393; found 509.3392.
3
2
41
6
N,Nh-Bis-(2,3,4,5-tetrahydro-1H-cyclohepta[1,2-b]
quinolin-10-yl)-1,6-diaminohexane (7a).
According to the general procedure, chloride 4c (0.25 g,
1.08 mmol) and 1,6-diaminohexane (63 mg, 0.54 mmol)
were condensed under heat for 2.5 h to give, after flash
chromatography (CH Cl to CH Cl \CH OH l 10:1 as
eluents), the title compound (75 mg, 26%) as an amber
1
glass foam: H NMR (300 MHz, CDCl ) δ 7.98 (d, J l
2
2
2
2
3
(Alley et al 1988).
3
7
.8 Hz, 2H, Ar-H), 7.88 (d, J l 7.9 Hz, 2H, Ar-H), 7.55
t, J l 6.9 Hz, 2H, Ar-H), 7.41 (t, J l 7.0 Hz, 2H,
Ar-H), 3.30 (t, J l 4.6 Hz, 4H, 2 N-CH ), 3.20–3.10
(
Results and Discussion
2
(
m, 4H, 2 CH ), 2.90–2.80 (m, 4H, 2 CH ), 1.95–1.60 (m,
2 2
The synthesis of 9-chlorotetrahydroacridine (4a) and its
congeners 4b and 4c was accomplished in 84–96% yields
by heating the o-aminoaromatic acids 2a and 2b and
1
6H, 2 (CH ) & CH CH ), 1.50–1.30 (m, 4H, CH CH );
2 3 2 2 2 2
+
FABMS (NBA as matrix): m\z [MjH] 507.2; HR-
FABMS: exact mass calculated for C H N [MjH]
+
3
4
43
4
cycloketones 3a and 3b in the presence of POCl as
3
5
07.3488; found 507.3483.
illustrated in Figure 2 (Hu & Lu 2000). Bis-amination of
the chloride 4a was conducted by heating a mixture of
4a and a variety of 1,n-diaminoalkanes (0.5 equiv.) in
the presence of phenol and catalytic amounts of sodium
N,Nh-Bis-(2,3,4,5-tetrahydro-1H-cyclohepta[1,2-b]
quinolin-10-yl)-1,8-diaminooctane (7b).
According to the general procedure, chloride 4c (0.29 g, iodide at 180mC under an argon atmosphere to furnish
.25 mmol) and 1,8-diaminooctane (90 mg, 0.63 mmol) the desired bis-tacrines 5a–d (Figure 3). The bis-tacrine
1
were condensed under heat for 5 h to furnish, after flash congeners 6a–d were then obtained by the same ap-
chromatography (CH Cl to CH Cl \CH OH l 10:1 as proach with moderate yields. However, the reaction of
eluents), the title compound (94 mg, 28%) as an amber 1,n-diaminoalkanes and heptyl-fused chloride 4c pro-
2
2
2
2
3
1
glass foam: H NMR (300 MHz, CDCl ) δ 8.05 (d, J l vided congeners 7a and 7b with relatively lower yields
3
8
.4 Hz, 2H, Ar-H), 7.94 (d, J l 8.5 Hz, 2H, Ar-H), 7.54 even with an extension of the reaction time. These bis-
(
t, J l 7.3 Hz, 2H, Ar-H), 7.41 (t, J l 8.8 Hz, 2H, tacrines and their congeners were characterized spectro-
1
Ar-H), 3.37 (t, J l 4.4 Hz, 4H, 2 N-CH ), 3.20 (s br, scopically ( H NMR, MS, and HR-FABMS).
2

Anticancer bis-tacrine congeners
87
Cl
X
N
X
N
i
NH
(CH2)n
HN
(
)
m
X
N
4a–c
(
)
(
)
m
m
5
6
7
a–d, X = CH, m = 1, n = 2, 4, 6, 8
a–d, X = N, m = 1, n = 2, 4, 6, 8
a–b, X = CH, m = 2, n = 6, 8
Figure 3 Synthesis of bis-tacrines 5a–d, by bis-amination of the chloride 4a. Reagents: i. 1,n-diaminoalkane, Phenol, NaI, 180 mC.
Table 1 Cytotoxicity of bis-tacrine congeners in the National Cancer Institute drug-screening
programme.
a
Cell line
GI50 (µm)
5
a
5b
5d
6a
6b
6c
6d
7a
7b
Non-small cell lung cancer
NCI-H226
NCI-H23
31.5
22.7
0.06
–
49.3
59.4
53.9
70.2
16.4
13.1
6.67
4.50
2.14
1.82
1.55
1.66
0.06 1.69
Colon cancer
COLO 205
HCT-116
10.9
11.2
0.04 1.22
1.23 0.37
0.05 1.23
16.7
14.9
18.9
20.6
5.24
2.72
0.92
0.45
4.36
0.92
0.90
4.54
0.75
0.72
4.13
HCT-15
1.69
" 100 " 100 2.75
CNS cancer
SF-268
17.3
b
0.33 1.22
16.8
25.0
59.0
26.1
9.99
7.89
10.5
0.38
1.72
1.46
1.86
1.50
SF-539
–
0.03
–
Melanoma
LOX IMVI
SK-MEL-5
Ovarian cancer
OVCAR-8
SK-OV-3
15.0
18.6
0.06 1.36
0.06 1.54
17.5
14.7
18.6
18.9
11.4
11.6
5.69
5.92
1.47
1.82
1.50
1.44
18.0
34.4
0.07 1.36
24.5
35.8
25.1
53.1
6.73
10.1
0.61
10.5
1.34
1.53
1.34
1.75
18.2
1.45
Renal cancer
CAKI-1
23.6
22.6
1.88
32.8
21.8
–
6.19
2.25
8.64
2.69
2.21
Breast cancer
MCF7
16.4
18.6
19.0
16.84
3.75 1.65
0.04 1.86
0.08 1.69
4.66 1.32
–
5.45
1.58
1.06
5.36
1.18
2.22
0.30
1.52
1.17
2.07
0.22
1.65
NCI\ADR RES
BT-549
85.1
17.0
" 100 19.0
18.7 1.66
c
mid
31.47 31.67 8.80
a
b
Values are the average of duplicate testing. GI50 l 50% growth inhibition; indicates a default value;
the average GI50 value for the drug over the whole cell line panel.
c
The synthetic bis-tacrine congeners were selected and 7a, and 7b exhibited moderate selectivity toward colon
evaluated against a panel of 60 human cancer cell lines and melanoma cells, in which colon 205, HCT-116,
using the standard SRB assay, and the results for fifteen LOX IMVI, and SK-MEL-5 were selected as a rep-
of those cell lines are presented in Table 1 as GI50 values resentative cell line sub panel. Selectivity for colon 205
(equivalent to IC50), together with the mid-value (the (GI50 (mid)\GI50 (colon 205)) varied from 1.1 to 116.5,
average GI50 value for the whole cell line). From the with an average of 18-fold, whereas an average of 4.0-
representative data shown, bis-tacrine congeners 5b, 5d, fold selectivity for HCT116 was obtained. In general,

88
Ming-Kuan Hu
the MCF7 and NCI\ADR RES lines could be used to
evaluate the extent to which bis-tacrine congeners are
affected by P-glycoprotein-mediated multidrug resist-
ance (Roninson 1992). The extent of resistance indicated
by the P-glycoprotein over-producing cell line
screening with panels of human tumor cell lines using a
microculture tetrazolium assay. Cancer Res. 48: 589–601
Brana, M. F., Castellano, J. M., Perron, D., Maher, C.,
Conlon, D., Bousquet, P. F., George, J., Qian, X. -D.,
Robinson, S. P. (1997) Chromophore-modified bis-naph-
thalimides: synthesis and antitumor activity of bis-dibenz-
(
measured as GI50 (NCI-ADR)\GI50 (MCF7)]
[deh]isoquinoline-1,3-diones. J. Med. Chem. 40: 449–454
changed from 0.01 (5b) to 8.4 (6c), with an average value
over all the tested compounds of 2.2-fold. These data
indicate a less significant degree of resistance for the
class. All of the tested compounds had weak cytotoxicity
against both the CAKI-1 renal tumour line and SKOV3
ovarian carcinoma, which were selected as represen-
tative refractory solid tumours. Interestingly, butyl-
Canellakis, E. S., Shaw, Y. H., Hanners, W. E. Schwartz, R.
A. (1976) Diacridines: bifunctional intercalators. I. Chem-
istry, physical chemistry and growth inhibitory pro-
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Chen, T. K., Fico, R., Canellakis, S. E. (1978) Diacridines,
bifunctional intercalators. Chemistry and antitumor ac-
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linked bis-tacrine (5b) exhibited broad and strong cyto- Denny, W. A. (1989) DNA-intercalating agents as antitumour
toxicity, with GI50 values at sub-micromolar levels
particularly against non-small cell lung and colon can-
cers and melanoma. Its aza congener, 6b, was almost
devoid of activity against these cell lines. These results
suggest that the decreased cytotoxic effects might result
from the electronic properties of pyridyl nitrogen. The
short connecting alkyl chain of bis-tacrine and some
congeners (e.g. 5a, 6a) induced a dramatic decrease in
cytotoxic effects. The cytotoxic profiles of dimers with a
longer alkyl chain (e.g. n l 8; congeners 5d, 6d, 7b) were
drugs: prospects for future design. Anticancer Drug Des. 4:
2
41–263
Diaz-Rubio, E., Martin, M., Lopez-Vega, J. M., Casado, A.,
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1
0: 25–26
Goldin, A., Vendetti, M. M., McDonald, J. S., Muggia, F.
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broadly identical, with most GI50 values in the region of Hu, M. K., Lu, C. F. (2000) A facile synthesis of bis-tacrine
–10 µ. The strong cytotoxicity of 5b suggests that this
isosteres. Tetrahedron Lett. 41: 1815–1818
compound might act as a bifunctional intercalator. McGeer, P. L., McGeer, E. G. (1995) Alzheimer’s disease:
1
This suggestion might explain the moderate potency of
congeners 5d, 6d and 7b, in which the longer connect-
ing chains correspond to the intercalating actions on
different binding grooves that are also required for
anticancer activity. Given the excellent in-vitro results,
compound 5b has been selected for further biological
evaluation of its anticancer profile.
arthritis of the brain? Drug News Perspect. 8: 80–83
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Segal-Bendirjian, E., Coulaud, D., Roques, B. P., Le Pecq,
J. -B. (1988) Selective loss of mitochondrial DNA after
treatment of cells with ditercalinium (NSC 335153),
an antitumor bis-intercalating agent. Cancer Res. 48:
4982–4992
It has been reported that the fully aromatic bis-
acridines are potent inhibitors of the growth of various
tumour cells in culture (Canellakis et al 1976). The bis-
tacrine moieties reported here might act as potential
bifunctional intercalators, as do the well known bis- Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon,
acridines. The prominent cytotoxicity of bis-tacrines
might reveal an alternative approach toward potent
anticancer drugs.
J., Vistica, D., Warren, J. R., Bokesch, H., Kenney, S.,
Boyd, M. R. (1990) New colorimetric cytotoxicity assay for
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1
112
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Bis(phenazine-1-carboxamides): structure-activity relation-
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