2,7-Disubstituted amidofluorenone derivatives as inhibitors of human telomerase

Article abstract of DOI:10.1021/jm990084q

Telomerase is a major new target for the rational design of novel anticancer agents. We have previously identified anthraquinone-based molecules capable of inhibiting telomerase by stabilizing G-quadruplex structures formed by the folding of telomeric DNA

Full text of DOI:10.1021/jm990084q

J . Med. Chem. 1999, 42, 2679-2684
2679
2,7-Disu bstitu ted Am id oflu or en on e Der iva tives a s In h ibitor s of Hu m a n
Telom er a se
Philip J . Perry,†,§ Martin A. Read,† Rhian T. Davies,† Sharon M. Gowan,‡ Anthony P. Reszka,† Alexis A. Wood,†
Lloyd R. Kelland,‡ and Stephen Neidle*,†
Cancer Research Campaign Biomolecular Structure Unit and Centre for Cancer Therapeutics, The Institute of Cancer
Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K.
Received February 19, 1999
Telomerase is a major new target for the rational design of novel anticancer agents. We have
previously identified anthraquinone-based molecules capable of inhibiting telomerase by
stabilizing G-quadruplex structures formed by the folding of telomeric DNA. In the present
study we describe the synthesis and biological evaluation of a series of analogous fluorenone-
based compounds with the specific aims of, first, determining if the anthraquinone chromophore
is a prerequisite for activity and, second, whether the conventional cytotoxicity inherent to
anthraquinone-based molecules may be reduced by rational design. This fluorenone series of
compounds exhibits a broad range of telomerase inhibitory activity, with the most potent
inhibitors displaying levels of activity (8-12 µM) comparable with other classes of G-quadruplex-
interactive agents. Comparisons with analogous anthraquinone-based compounds reveal a
general reduction in the level of cellular cytotoxicity. Molecular modeling techniques have been
used to compare the interaction of fluorenone- and analogous anthraquinone-based inhibitors
with a human G-quadruplex structure and to rationalize their observed biological activities.
In tr od u ction
The telomerase enzyme is a reverse transcriptase
which elongates the 3′ ends of telomeric DNA.1,2 It is
not activated in normal somatic cells, which progres-
sively lose telomeric repeats during successive rounds
of cell division leading to the nonreplicating state of
senescence and ultimately to cell crisis. In contrast,
some 80-90% of tumor cells have activated levels of
telomerase resulting in stabilization of telomere length
and the ability for sustained cellular proliferation.3
Telomerase is thus an essential factor in cellular im-
mortalization and consequent tumorigenesis.4 There is
currently much interest in the inhibition of telomerase
F igu r e 1. Structures of 2,7-disubstituted anthraquinones 1
and 2,7-disubstituted fluorenones 2 examined in this study.
as a novel anticancer strategy.5 Recent studies have
demonstrated inhibition of telomerase activity by anti-
sense oligonucleotides6 or small-molecule inhibitors,7
which in turn can result in the onset of senescence and
antitumor activity. In order for telomerase inhibitors
to be effective as anticancer agents, they may well
require long-term administration in order for effective
attrition of telomere length to occur after ca. 20 rounds
of cell division have taken place. Thus, such agents must
have low toxicity toward normal human cells.8
telomerase activity.11-14 We have systematically inves-
tigated the nature of the terminal amino substituents,
the alkyl side-chain linker length,10a and the effect of
regioisomerism10b on telomerase activity for these an-
thraquinone-based inhibitors. Generally, simple alkyl-
amino, piperidine, and piperazine derivatives attached
via propionamido substituents gave optimal activity
(telIC50 values in the 1-10 µM range) with regioisomer
variation showing little effect. Conventional cytotoxici-
ties against a panel of human ovarian tumor cell lines
were at levels broadly comparable to telomerase activity.
We now report studies on the nature of the chromophore
itself to determine, first, if the anthraquinone chro-
mophore is essential for telomerase inhibitory activity
and, second, if a component of conventional cytotoxicity
(i.e., redox cycling) inherent to quinone-based molecules
could be moderated by rational design. This paper
describes a series of analogous 2,7-disubstituted fluo-
renone derivatives (2) (Figure 1) in which the central
quinone ring has been replaced by a five-membered ring
with just one carbonyl substituent.
Several classes of planar aromatic compounds have
been shown to act as telomerase inhibitors.9 We have
recently shown10 that inhibition of telomerase can be
achieved with appropriately substituted diamidoan-
thraquinone derivatives (1) (Figure 1). This inhibition
is believed to occur as a result of the stabilization of
telomeric DNA by these compounds as folded guanine-
quadruplex structures, which are known to inhibit
* Correspondence to: Prof. Stephen Neidle. Tel/fax: +44 181 643
1675. E-mail: steve@iris5.icr.ac.uk.
† CRC Biomolecular Structure Unit.
‡ CRC Centre for Cancer Therapeutics.
§ Present address: School of Pharmacy and Pharmaceutical Sci-
ences, De Montfort University, The Gateway, Leicester LE1 9BH, U.K.
10.1021/jm990084q CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/17/1999

2680 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Sch em e 1. Synthesis of 2,7-Diamidofluorenone Compoundsa
Perry et al.
a
Reagents: (i) Na2S‚9H2O/NaOH/EtOH/reflux/6 h; (ii) ClCH2CH2COCl/reflux/4 h; (iii) R2NH/NaI/EtOH/reflux; (iv) excess MeI/CH2Cl2/
24 h.
Ta ble 1. Chemical and Physical Properties of the 2,7-Disubstituted Fluorenone Derivatives 6a -21a
compd
substituent (NR2)
reaction time (h)
yield (%)
mp (°C)a
formula
C23H28N4O3
C27H36N4O3‚0.3H2O
anal.b
6a
7a
8a
NMe2
NEt2
8
4
18
3
88
84
90
86
84
83
73
77
60
43
84
78
52
55
67
54
245 (191-192)
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
151 (136-137)
4-morpholinyl
1-piperidinyl
249 (185-186)
C
C
C
C
C
C
C
C
C
C
C
C
C
C
27H32N4O5
9a
245-246 (216)
29H36N4O3‚0.5H2O
31H40N4O3‚1.0H2O
31H40N4O3‚0.5H2O
33H44N4O3‚0.5H2O
31H40N4O5‚0.3H2O
33H44N4O5‚0.5H2O
29H36N4O5‚1.0H2O
29H38N6O3‚0.4H2O
31H42N6O3‚0.5H2O
39H42N6O3‚0.25H2O
41H46N6O3‚0.25H2O
37H40N8O3
10a
11a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
1-(2-methyl)piperidinyl
1-(4-methyl)piperidinyl
1-(2-ethyl)piperidinyl
1-(2-hydroxymethyl)piperidinyl
1-(2-hydroxyethyl)piperidinyl
1-(4-hydroxy)piperidinyl
1-(4-methyl)piperazinyl
1-(4-ethyl)piperazinyl
1-(4-phenyl)piperazinyl
1-(4-benzyl)piperazinyl
1-[4-(2-pyridyl)]piperazinyl
1-[4-(2-pyrimidyl)]piperazinyl
7
196-198 (103-105)
179-180 (207-209)
180-182 (61-63)
171-173 (66-68)
163-164 (62-64)
230-232 (184-185)
255-256 (218-220)
247-248 (>300)
264-265 (260 dec)
207 (240 dec)
22
20
24
48
72
24
20
24
24
72
72
258-259 (204-206)
281-283 (102 dec)
35H38N10O3‚0.25H2O
a
b
Mp of acid addition salts given in parentheses. Indicated elemental analyses were within (0.4% of theoretical values.
We have previously reported10b on the use of molec-
ular modeling to study diamidoanthraquinone interac-
tions with the folded structure formed by the four-repeat
sequence d[AG3(T2AG3)3] of the human telomere.15
Ligands were docked into the intercalation site at the
5′-AG step, in accord with NMR data based upon other
ligands binding to this sequence.9b,c The present study
employs an automated docking procedure to locate low-
energy positions for fluorenones in this G-quadruplex
structure, which we have compared with the corre-
sponding anthraquinone-quadruplex complex.
ing 2,7-disubstituted anthraquinone derivatives where
available. The fluorenone derivatives examined show a
broad range of telomerase inhibitory activity with the
most potent inhibitor having a telIC50 value of 8 µM.
Some DNA polymerase selectivity is also suggested,
with no inhibition of Taq DNA polymerase being
observed at equivalent concentrations. The acid addition
salts (6a -12a ) were consistently more active and
generally less cytotoxic than the corresponding quater-
nary dimethiodide derivatives (6b-12b). Compound 8a ,
containing a morpholine substituent, was found to be
totally inactive at concentrations up to 50 µM and
follows a trend previously observed for anthraquinone-
based molecules.10 In addition, compounds bearing
extended substituents containing an aryl moiety (18a -
21a ) were also found to be inactive. Direct comparisons
for compounds 6-9 with analogous 2,7-disubstituted
anthraquinone derivatives reveal slightly reduced levels
of telomerase inhibition with a similar reduction in
cytotoxicity also being evident.
Ch em istr y
Synthesis of the bis(aminopropionamido)fluorenones
6a -21a was accomplished as described in Scheme 1.
Briefly, Zinin reduction of 2,7-dinitrofluorenone (3)
followed by acylation afforded the intermediate chloro-
propionamide 5 in good yield. Subsequent aminolysis
by reflux treatment with the appropriate secondary
amine gave the fluorenones 6a -21a as indicated in
Table 1. In addition, quaternary dimethiodide salts of
the bis(amino) derivatives 6b-15b were prepared by
treatment with iodomethane.
The molecular modeling studies found a single low-
energy position (with a total potential energy of -2210
kcal mol-1) for the 2,7-disubstituted diamidoanthraqui-
none bound to the G-quadruplex complex (Figure 2d).
In contrast, two low-energy positional orientations were
possible for the analogous fluorenone derivative 9a ,
related by approximately 180° of rotation around the
molecular long axis. The first of these orientations
Resu lts
Telomerase and cytotoxic activity for the 2,7-disub-
stituted fluorenones 6a -21a and 6b-15b are given in
Table 2 and are compared with data for the correspond-

Amidofluorenones as Inhibitors of Human Telomerase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2681
Ta ble 2. Telomerase, Cytotoxicity, and Taq Polymerase Inhibition Data for Fluorenones 6-21
IC50 (µM)c
Taq inhibitiond
20 µM
compda
telIC50 (µM)b
A2780
CH1
SKOV-3
10 µM
50 µM
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
17a
18a
19a
20a
21a
16.2 (4.7)
18.5 (14.5)
15.5 (4.3)
25.4 (>20)
.50 (.50)
27.3 (16.5)
9 (3.1)
21 (7.8)
12
12 (2.1)
2.25 (0.44)
14 (2.15)
3.7 (0.33)
>25 (5.3)
2.55 (0.52)
11 (0.48)
4.3 (0.18)
10.5
4.85
2.2
2.5
2.2
2.9
13
3.75
8.5 (4.4)
1.95 (1.3)
8 (4.6)
8.2 (4.15)
1.95 (4)
9.65 (7.2)
2.4 (2)
>25 (>100)
2.45 (2.95)
11 (3.7)
3.9 (2.2)
12
3.95
7.6
3.75
3.15
2.6
15
4.3
20
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
+
-
+
-
-
-
++
-
+
-
-
-
-
-
-
2.45 (1.13)
>25 (7.5)
2.15 (1.1)
7.6 (3.15)
5.2 (1.05)
1.8
>20
2.6
>20
>25
>25
2.15
2.35
14
>20
14
>20
8
33
16.5
>20
18.5
>20
44
17
4.2
14.5
>25
5.2
3.15
>25
1.95
>25
>25
>25
>25
>25
>25
5.2
>25
>25
2.4
4.7
>25
>25
>25
>25
.50
>25
>25
.50
>25
>25
.50
>25
>25
.50
>25
>25
a
Where available, data for the corresponding 2,7-disubstituted anthraquinone derivatives are given in parentheses as published
elsewhere.10b Concentration required to inhibit telomerase activity by 50% relative to controls. c Concentration required to inhibit cell
growth by 50% relative to controls. Key: (+++) total, (++) significant, (+) slight, or (-) no inhibition.
b
d
(Figure 2a,b) is closely related to that of the correspond-
ing anthraquinone (Figure 2d) and has a potential
energy of -2168 kcal mol-1. The second orientation
given their structural similarities. However, the change
from a six-membered to a five-membered central ring
has a significant effect on the shape of the chromophore
rendering it crescent-shaped. This, in turn, affects the
angular disposition of the aminopropionamido substit-
uents compared to the 2,7-disubstituted anthraqui-
nones, so that a fit of the disubstituted fluorenones into
the putative binding site can only be achieved with a
degree of distortion of the site and a resultant energy
cost. This change also reduces the electron deficiency
of the chromophore, so weakening the strength of
π-stacking interactions and thus contributing to the
reduced affinity of the fluorenone analogue for the
G-quadruplex structure. The correlation of lowered
activity with the computed reduced potential energy
(and, by implication, reduced affinity) for the telomere
binding site is further evidence that telomerase inhibi-
tion by bis(amido)-substituted tricyclic aromatic mol-
ecules proceeds via interaction with and stabilization
of folded telomeric DNA. Furthermore, the ability of the
molecular modeling to explain the reduced telomerase
activity of the fluorenones is yet more evidence in favor
of the structural model outlined here.
(Figure 2c) has a potential energy of -2163 kcal mol-1
.
The higher energies of the two fluorenone-G-quadru-
plex complexes, by 42-47 kcal mol-1, are not due to
differences in stacking or other nonbonded energies.
Indeed, the intermolecular energy of the fluorenone
complex in the first orientation (Figure 2b) is slightly
more favorable than that of the anthraquinone complex,
due to improved chromophore-base overlap. The ob-
served differences in energies have their origin in the
necessity for the fluorenones to induce a measure of
distortion in nucleotide backbone conformation at the
binding site in order to fit adequately. Thus these
differences in potential energy are almost entirely due
to increased torsion angle strain energies in the two
fluorenone complexes. This also correlates with an
observed 3-fold reduction in telomerase activity for the
fluorenone compared to the analogous anthraquinone
derivative.
Discu ssion
This study has shown that other tricyclic systems in
addition to anthraquinone-based molecules can inhibit
telomerase activity. Furthermore, we have demon-
strated that conventional cytotoxicity may be moderated
through rational design without the loss of telomerase
activity. The most potent compounds described here
(telIC50 values of 8-12 µM) show levels of telomerase
inhibitory activity that compare favorably with other
classes of G-quadruplex-interactive agents.9,10 Where
direct comparisons with analogous 2,7-disubstituted
anthraquinone derivatives are possible (compounds
6-9), the slightly reduced levels of activity shown by
the fluorenone derivatives is at first sight surprising
The results presented here on the fluorenone series
of telomerase inhibitors also highlight the need to
discover new inhibitors with superior potency against
telomerase coupled with lower levels of acute cytotox-
icity, to achieve high therapeutic indices. The exploita-
tion of rationally derived structure-activity relation-
ships, such as have been found in this study, is likely
to be the key to future success.
Exp er im en ta l Section
Syn th etic Ch em istr y. Melting points (mp) were recorded
on a Leica Galen III hot-stage melting point apparatus and
are uncorrected. NMR spectra were recorded at 250 MHz on

2682 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Perry et al.
F igu r e 2. Comparison of binding orientations for the analogous 2,7-disubstituted fluorenone 9a and anthraquinone derivatives:
(a) view of the fluorenone ligand interacting at the 5′-AG step region of the human telomeric G-quadruplex structure; (b) orientation
of the fluorenone ligand viewed perpendicular to the plane of the adjacent G-quartet; (c) second orientation of the fluorenone
ligand, rotated by approximately 180° around the molecular long axis, and similarly viewed perpendicular to the plane of the
adjacent G-quartet; (d) comparative view of the analogous 2,7-disubstituted anthraquinone.
a Bruker AC250 spectrometer in either Me2SO-d6 or CDCl3
solution at 303 ( 1 K using Me4Si as internal standard. J
values are given in hertz (Hz). EI (70 eV), FAB, and high-
resolution accurate mass spectra were determined by The
School of Pharmacy (University of London, U.K.). Elemental
analyses were carried out by Medac Ltd. (Brunel Science
Center, Egham, Surrey, U.K.); results for elements indicated
by symbols were within (0.4% of theoretical values. TLC was
carried out on silica gel (Merck 60F-254) using CHCl3-MeOH
(0-20% MeOH) as eluent, with visualization at 254 and 366
nm. Organic solutions were dried over sodium sulfate. Acid
addition salts were prepared by standard literature methods.
The 2,7-disubstituted anthraquinone derivative described in
this study was prepared as described previously.10b
2,7-Dia m in o-9-flu or en on e (4). To a stirred suspension of
2,7-dinitro-9-fluorenone (3) (9.45 g, 35 mmol) in ethanol (375
mL) was added a solution of sodium sulfide nonahydrate (37.85
g, 157.5 mmol) and sodium hydroxide (15 g, 375 mmol) in
water (650 mL). The mixture was heated at reflux for 5 h and
left to stand overnight. The mixture was cooled to 0-5 °C and
the resulting precipitate collected by filtration. The crude
product was washed successively with water (2 × 100 mL),
aqueous NaOH (2 × 100 mL, 5% w/v), water (3 × 100 mL),
cold EtOH (2 × 50 mL), ether (50 mL), and hexane (50 mL)
and dried in vacuo. Recrystallization from acetone-ethanol
(1:1 v/v) afforded the fluorenone 4 (6.30 g, 86%) as purple
needles: mp 278-279 °C; NMR δ (DMSO) 5.32 (4H, br s, NH2)
6.57 (2H, dd, J ) 7.9, 2.1, H-3,6), 6.69 (2H, d, J ) 2.1, H-1,8),
7.09 (2H, d, J ) 7.9, H-4,5); MS (rel intensity) m/z 210 (100);
calcd ([M + 1]+) 210.0793, found 210.0780. Anal. (C13H10N2O)
C, H, N.
2,7-Bis(3-ch lor op r op ion a m id o)-9-flu or en on e (5). A
stirred suspension of 2,7-diamino-9-fluorenone (4) (6.3 g, 30
mmol) and 3-chloropropanoyl chloride (50 mL, 0.525 mol) in
xylene (200 mL) was heated at reflux for 1.5 h and cooled to
0-5 °C. The crude product was collected by filtration, washed
with toluene (2 × 100 mL), acetone (100 mL), EtOH (100 mL),
and ether (100 mL), and dried in vacuo. Recrystallization from
DMF-EtOH (2:1 v/v) afforded chloroamide 5 (9.39 g, 80%) as
salmon-pink crystals: mp 328 °C dec; NMR δ (DMSO) 2.85
(4H, t, J ) 6.1, COCH2), 3.90 (4H, t, J ) 6.1, CH2Cl), 7.62
(2H, t, J ) 8.1, H-4,5), 7.70 (2H, dd, J ) 8.1, 1.9, H-3,6), 7.92
(2H, d, J ) 1.9, H-1,8), 10.31 (2H, s, NH); MS (rel intensity)
m/z 391 (100), 373 (46), 355 (72), 301 (76), 289 (84), 231 (96),
210 (91); calcd ([M + 1]+) 391.0616, found 391.0610. Anal.
(C19H16N2O3Cl2‚0.1H2O) C, H, N.
2,7-Bis[3-(d im e t h yla m in o)p r op ion a m id o]-9-flu or e -
n on e (6a ). Gen er a l Am in olysis P r oced u r e. Dimethylamine

Amidofluorenones as Inhibitors of Human Telomerase
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2683
(5.4 mL of 33% w/w solution in EtOH, 30 mmol) was added
during 15 min to a stirred, refluxing suspension of chloroamide
5 (0.936 g, 2.4 mmol) and NaI (0.25 g) in EtOH (50 mL). After
refluxing for the indicated time period (see Table 1), the
mixture was cooled to 0-5 °C and KOH pellets (5 g) were
added. The mixture was stirred for 1 h, and the resulting
precipitate was collected by filtration, washed with ether (50
mL), and dried in vacuo. The crude product was dissolved in
CHCl3 (100 mL), washed with water (2 × 50 mL), and dried.
Evaporation and recrystallization from EtOH gave the amide
6a (0.86 g, 88%) as an orange solid: mp 245 °C; NMR δ (CDCl3)
2.39 (12H, s, CH3), 2.52 (4H, m, CH2N), 2.64 (4H, m, COCH2),
7.38 (2H, d, J ) 8.1, H-4,5), 7.47 (2H, d, J ) 1.9, H-1,8), 7.96
(2H, dd, J ) 8.1, 1.9, H-3,6), 11.21 (2H, s, NH); MS (rel
intensity) m/z 409 (100); calcd ([M + 1]+) 409.2240, found
409.2211. Anal. (C23H28N4O3) C, H, N. Maleate salt: mp 191-
192 °C.
2,7-Bis[3-(d im e t h yla m in o)p r op ion a m id o]-9-flu or e -
n on e N,N′-Dim eth iod id e (6b). Gen er a l P r oced u r e. To a
solution of 6a (0.408 g, 1 mmol) in dichloromethane (50 mL)
was added iodomethane (3.3 mL, 50 mmol), and the solution
stirred at room temperature for 24 h. The resulting precipitate
was collected by filtration, washed with dry ether, and dried
in vacuo at 25 °C to give dimethiodide 6b (0.67 g, 97%) as an
orange solid: mp 234-235 °C; NMR δ (DMSO) 2.92 (4H, t, J
) 7.5, COCH2), 3.10 (18H, s, N+CH3), 3.67 (4H, t, J ) 7.5,
CH2N+), 7.65 (4H, br m, ArH), 7.91 (2H, br s, ArH), 10.39 (2H,
br s, NH). Anal. (C25H34N4O3I2‚2H2O) C, H, N, I.
allowed to attach overnight. Agents (acid addition and qua-
ternary dimethiodide salts) were dissolved at 500 µM in water
and immediately added to wells in quadruplicate at final
concentrations of 0.05, 0.25, 1, 5, and 25 µM. Following an
incubation period of 96 h, remaining cells were fixed with ice-
cold 10% w/v trichloroacetic acid (30 min) and stained with
0.4% SRB in 1% v/v acetic acid (15 min). Mean absorbance at
540 nm for each drug concentration was expressed as a
percentage of the control untreated well absorbance, and IC50
values (concentration required to inhibit cell growth by 50%)
were determined for each agent.
Molecu la r Mod elin g Stu d ies. The Macromodel 5.0 pro-
gram17 using the AMBER* force field with continuum solvation
treatment (GB/SA model of water solvation, van der Waals
cutoff 8 Å, electrostatic cutoff 20 Å, dielectric constant 1) was
employed for initial model building and calculations. Coordi-
nates from the solution NMR structure of the human telomeric
repeat d[AG3(T2AG3)3] G-quadruplex15 were used to give an
initial low-energy starting model. Molecular mechanics energy
minimization (1000 steps steepest descent with line searching
and 3000 steps Polak Ribiere conjugate gradient with deriva-
tive convergence of 0.05 kJ /Å mol) followed by dynamics (1.5-
fs time step, 40 ps at 300 K equilibrium, 100 ps at 300 K
production with time averaging of 100 sampled structures) and
subsequent mechanics (minimization of time-averaged dynam-
ics structure) was used. An intercalation site was introduced
between the diagonal T2A loop and the G-quartet segment of
the structure (the 5′-AG step) by breaking the phosphate
backbones and separating the structure while monitoring the
distance between the segments. The sugar-phosphate chains
were reconnected, and molecular mechanics energy minimiza-
tion (1000 steps steepest descent with line searching followed
by 1000 steps conjugate gradient) was used to relieve any
resulting steric distortion while retaining the G-quartet and
loop motifs with positional restraints.
Models for the fluorenone and anthraquinone molecules
with 2,7-bis(3-piperidinopropionamido) substituents were cre-
ated, minimized, and docked into the intercalation site using
the DOCKING module within the INSIGHTII package.18 This
enables molecular orientation to be explored while monitoring
electrostatic and van der Waals ligand and DNA interactions.
Both ligand and intercalation site geometries were allowed to
vary during the search. Possible starting orientations were
chosen by means of a Monte Carlo algorithm. Individual bases
were constrained at this initial docking stage, although the
backbone around the intercalation site was unconstrained. The
best chromophore positions were subjected to 100 steps of
unconstrained molecular mechanics minimization. The final
ranking order of structures was based on the resulting energy
values.
Ta q P olym er a se Assa y. Compounds were tested as their
acid addition (6a -15a as hydrochlorides, 16a -21a as male-
ates) and quaternary dimethiodide (6b-15b) salts at 10, 20,
and 50 µM final concentrations in a PCR 50-µL master mix
containing 10 ng of pCI-neo mammalian expression vector
(Promega, Southampton, U.K.) and forward (GGAGTTC-
CGCGTTACATAAC) and reverse (GTCTGCTCGAAGCAT-
TAACC) primers (200 nmol) as described previously.10a The
product of approximately 1 kb was visualized on a 2% w/w
agarose gel following amplification (30 cycles of 94 °C for 1
min, 55 °C for 1 min, and 72 °C for 2.5 min).
Mod ified Telom er ic Rep ea t Am p lifica tion P r otocol
(TRAP ) Assa y. The ability of agents to inhibit telomerase in
a cell-free assay was assessed with a modified TRAP assay
using extracts from exponentially growing A2780 human
ovarian carcinoma cells as described previously.10a The TRAP
assay was performed in two steps: (a) telomerase-mediated
extension of the forward primer (TS: 5′-AATCCGTCGAGCA-
GAGTT; Oswel Ltd., Southampton, U.K.) contained in a 40-
µL reaction mix comprising TRAP buffer (20 mM Tris-HCl (pH
8.3), 68 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 0.05% v/v
Tween 20), 0.05 µg of bovine serum albumin, 50 µM of each
deoxynucleotide triphosphate, 0.1 µg of TS primer, and 3 µCi
of [R-32P]dCTP (Amersham plc, U.K.). Protein (0.04 µg) was
then incubated with the reaction mix ( agent (acid addition
and quaternary dimethiodide salts) at final concentrations of
up to 50 µM for 20 min at 25 °C. A lysis buffer (no protein)
control, heat-inactivated protein control, and 50% protein (0.02
µg) control were included in each assay. (b) While heating at
80 °C in a PCR block of a thermal cycler (Hybaid, U.K.) for 5
min to inactivate telomerase activity, 0.1 µg of reverse CX
primer (3′-AATCCCATTCCCATTCCCATTCCC-5′) and 2 units
of Taq DNA polymerase (“red hot”, Advanced Biotechnologies)
were added. A three-step PCR was then performed: 94 °C for
30 s, 50 °C for 30 s, and 72 °C for 1 min for 31 cycles.
Telomerase-extended PCR products in the presence or absence
of compounds were then determined either by electrophoretic
separation using 8% w/w acrylamide denaturing gels and
analysis by phosphorimaging or autoradiography or by har-
vesting on Whatman filters (25-mm glass microfiber) and
analysis by liquid scintillation counting.
Ack n ow led gm en t. This work was supported by the
Cancer Research Campaign and Institute of Cancer
Research.
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