Diaza- and triazachrysenes: potent topoisomerase-targeting agents with exceptional antitumor activity against the human tumor xenograft, MDA-MB-435.

Article abstract of DOI:10.1016/S0960-894X(02)00737-0

Several 5,12-diazachrysen-6-ones and 5,6,11-triazachrysen-12-ones were synthesized with varied substituents at the 5- or 11-position, respectively. Each compound was evaluated for its potential to stabilize the cleavable complex formed with TOP1 and DNA.

Full text of DOI:10.1016/S0960-894X(02)00737-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3333–3336
Diaza- and Triazachrysenes: Potent Topoisomerase-Targeting
Agents with Exceptional Antitumor Activity against the
Human Tumor Xenograft,MDA-MB-435
Alexander L. Ruchelman,a Sudhir K. Singh,a Xiaohua Wu,a,b Abhijit Ray,a
Jin-Ming Yang,b,c Tsai-Kun Li,c Angela Liu,c Leroy F. Liub,c
and Edmond J. LaVoiea,b,
*
aDepartment of Pharmaceutical Chemistry, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020, USA
bThe Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA
cDepartment of Pharmacology, The University of Medicine and Dentistry of New Jersey,
Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
Received 26 June 2002; accepted 9 August 2002
Abstract—Several 5,12-diazachrysen-6-ones and 5,6,11-triazachrysen-12-ones were synthesized with varied substituents at the 5- or
11-position, respectively. Each compound was evaluated for its potential to stabilize the cleavable complex formed with TOP1 and
DNA. Two analogues with very potent TOP1-targeting activity, 3a and 4a, exhibited cytotoxic activity with IC50 values at or below
2 nM against RPMI8402. Compound 3a was active in vivo by either ip or po administration in the human tumor xenograft athymic
nude mice model.
# 2002 Elsevier Science Ltd. All rights reserved.
DNA topology is regulated by DNA topoisomerases
that catalyze the breaking and rejoining of DNA
strands.1À3 Topoisomerases are critical to both replica-
tion and transcription. There are two major types of
topoisomerases, type I [e.g., topoisomerase I (TOP1)]
and type II [e.g., topoisomerase II (TOP2)], based upon
differences in their initial mechanisms wherein a single-
or double-stranded DNA break is implicated.1À3
Topoisomerase-targeting agents that can stabilize the
cleavable complex formed between the enzyme and
DNA have proved to be effective in the treatment of
cancer. This drug-induced stabilization of the enzyme–
DNA cleavable complex effectively converts these
nuclear enzymes into cellular poisons.
and its structurally related analogues have resulted in
the clinical development of two TOP1-targeting drugs,
topotecan (Hycamptin1) and irinotecan (CPT-11/
Camptosar1). Both of these drugs have incorporated
within their structure the camptothecin-ring system,
which includes the presence of a g-lactone. Hydrolysis
of this lactone moiety results in an inactive derivative
that possesses high affinity for human serum
albumin.5À7 The metabolic instability of this lactone and
the observation that both topotecan and irinotecan are
substrates for efflux transporters associated with resis-
tance have prompted further studies on the development
of novel TOP1-targeting agents.8À10 Recently, bi- and
terbenzimidazoles,11,12 benz[a]anthracenes,13 certain
benzo[c]phenanthridine and protoberberine alkaloids
and their synthetic analogues,14,15 indolocarbazoles,16
the fungal metabolites bulgarein,17 and saintopin,18 sev-
eral indenoisoquinolines19 and benzophenazines20 have
been identified as TOP1-targeting agents.
Camptothecin was the first agent identified as a TOP1-
targeting agent.4 The extensive studies on camptothecin
*Corresponding author at: Ernest Mario School of Pharmacy, Rutgers
University, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA.
Tel.: +1-732-445-2674; fax: +1-732-445-6312; e-mail: elavoie@rci.
rutgers.edu
Studies in our laboratory originating with proto-
berberine analogues and more recently with non-
charged heterocyclic analogues have revealed that
appropriately substituted benzo[i]phenanthridines and
Website: http://www.rci.rutgers.edu/ꢀlayla/Faculty/LaVoie.htm
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00737-0

3334
A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3333–3336
dibenzo[c,h]cinnolines can exhibit significant TOP1-
targeting activity and cytotoxicity. Structure–activity
studies have revealed that the presence of a methylene-
dioxy group on the benzo-ring fused adjacent to the
nitrogen heteroatom is associated with enhanced activ-
ity. In addition, the presence of two methoxyl groups on
the aromatic ring distal from the methylenedioxy group
favors both TOP1-targeting activity and cytotoxicity.
These structural characteristics associated with
enhanced activity are embodied in the benzo[i]phenan-
thridine, 1, and the dibenzo[c,h]cinnoline, 2, illustrated
in Figure 1.
Several 5,12-diazachrysen-6-ones, 3a–c, and 5,6,11-triaza-
chrysen-12-ones, 4a–d, were selected for synthesis. It was
envisioned that a similar approach to that used by Har-
ayama et al. for benzo[c]phenanthridine alkaloids could be
used for the synthesis of either series of compounds.21 The
synthetic approach that was employed is outlined in
Scheme 1. The starting material for compounds 3a–c and
4a–d was either 4-chloro-6,7-methylenedioxyquinoline, 5,
or 4-chloro-6,7-methylenedioxycinnoline, 6. Intermediates
5 and 6 were prepared from 4-hydroxy-6,7-methylene-
dioxyquinoline22 and
4-hydroxy-6,7-methylenediox-
ycinnoline23 as previously described.22,24,25 For the
formation of the 4-aminoquinoline derivatives, 7a–c, reac-
tions were performed in the presence of phenol as detailed
in the literature.26,27 The preferred method for the forma-
tion of 4-aminocinnolines, 8a–d, was to use the appro-
priate primary amine, RNH2, as solvent.28,29
Further studies on aza-derivatives of 2 demonstrated
that the presence of an additional nitrogen heteroatom
at the 11- or 12-position did not negatively affect rela-
tive potency with regard to either TOP1 targeting
activity or cytotoxicity. Because of the poor solubility of
both 1 and 2, difficulties were encountered in developing
suitable formulations for assessment of their relative
antitumor activity in vivo. Efforts were directed toward
the preparation of analogues that would allow for
incorporation of functionality to enhance solubility.
One suitable modification would be to develop 5,12-
diazachrysen-6-ones and 5,6,11-triazachrysen-12-ones
that possessed varied substituents at either the 5- or 11-
position, respectively. The synthesis and the pharma-
cological evaluation of these agents are discussed in the
present study.
The synthesis of 2-iodo-4,5-dimethoxybenzoic acid was
performed as described in the literature.30 Conversion
of this acid to the acid chloride using oxalyl chloride,
followed by immediate reaction with the appropriate 4-
(alkylamino)-6,7-methylenedioxyquinoline or 4-(alkyla-
mino)-6,7-methylenedioxycinnoline provided variable
yields of the amides, 9a–c and 10a–d. The Heck reaction
was employed for the cyclization of the o-iodobenza-
mides to provide the 5,12-diazachrysen-6-ones, 3a–c, as
well as the 5,6,11-triazachrysen-12-ones, 4a–d.31
Compounds 3a–c and 4a–d were evaluated for the abil-
ity to induce DNA cleavage in the presence of TOP1
and TOP2. The results of these analyses are listed in
Table 1. The influence of structure differences on TOP1-
targeting activity is evident from these data. Enhanced
intrinsic TOP1-targeting activity is observed with 4a
relative to 3a. This trend is consistent among each pair
of similarly substituted diazachysen-6-one and tria-
zachysen-12-one. Thus, the relative potency of 4a >3a,
4b >3b, and 4c >3c. These data are consistent with
data obtained with benzo[i]phenanthridines and diben-
Figure 1. Benzo[i]phenanthridine and dibenzo[c,h]cinnoline deriva-
tives with TOP1-targeting activity.
Scheme 1. (i) 5: Reflux in C6H5OH for 2.5 h, then add RNH2 at 100 ꢁC; 6: RNH2, Cu powder, reflux; (ii) (COCl)2, anhyd CH2Cl2 and TEA, reflux;
(iii) Pd(OAc)2, P(o-tolyl)3, Ag2CO3, in DMF, reflux.

A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3333–3336
3335
Table 1. TOP1-targeting activity and cytotoxicity of 5,12-diazachry-
sen-6-ones, 3a–c, and 5,6,11-triazachrysen-12-ones, 4a–d
Compd
TOP1-mediated DNA cleavagea Cytotoxicity IC50 (mM)
RPMI8402b CPT-K5c
3a
3b
3c
4a
4b
4c
4d
0.5
>1000
200
0.3
1000
30
1.0
25
1.0
>1000
0.002
0.57
0.15
0.001
0.26
0.010
0.004
0.57
0.012
0.22
0.90
3.4
>10
0.60
3.6
>10
10
>10
>10
0.28
CPT-11
Topotecan
VM-26
aTopoisomerase I cleavage values are reported as REC, relative effec-
tive concentration, that is concentrations relative to topotecan, whose
value is arbitrarily assumed as 1, that are able to produce the same
cleavage on the plasmid DNA in the presence of human topoisome-
rase I. TOP2-mediated cleavage was >100 relative to VM26 for all
compounds listed.
bRPMI8402 is a human lymphoblast tumor cell line.
cCPT-K5 is a camptothecin-resistant variant of RPMI8402 that pos-
sesses a functional, but mutant TOP1.
Figure 2. Stimulation of enzyme-mediated DNA cleavage by 4a, 3a,
3b and topotecan (TPT) using human TOP1. The first lane is DNA
control without enzyme. The second lane is the control with enzyme
alone. The rest of the lanes contain human TOP1 and serially (10-fold
each) diluted compound from 0.01 to 10 mM.
zo[c,h]cinnolines. In these studies, dibenzo[c,h]cinnolines
proved to be significantly more potent in inducing DNA
cleavage in the presence of TOP1 than similarly sub-
stituted benzo[i]phenanthridines. The relative potency
of the 5,12-diazachrysen-6-ones with respect to inducing
DNA fragmentation in the presence of TOP1 is 3a >3c
> >3b. Each of the varied 5-substituents in 3a–c had a
similar effect on activity among the triazachrysen-12-
ones. The relative TOP1-targeting activity of the 5,6,11-
triazachrysen-12-ones is 4a >4d >4c >>4b.
cin-resistant variant, CPT-K5 are consistent with the
relative intrinsic potency of these compounds as TOP1-
targeting agents. The basis for the resistance of CPT-K5
cells to camptothecin is related to a mutant, but func-
tional form of TOP1. In contrast to results observed
with some benzo[i]phenanthridine derivatives, all of the
compounds in the present study exhibited significant
cross-resistance in the CPT-K5 cell line.
While the n-butyl analogue 4d is slightly less active than
4a, the retention of potent TOP1-targeting activity by
this derivative suggests that the 2-(N,N-dimethylami-
noethyl) group of compound 4a is not a critical require-
ment for potent activity among these 5,6,11-
triazachrysen-12-ones. The effect of the b-methyl sub-
stituent in the case of both 3b and 4b is dramatic. Clearly,
the presence of this substituent had a decisively negative
impact on the ability of these compounds to effectively
stabilize cleavable complex formation in the presence of
enzyme and DNA. None of these compounds exhibited
induced DNA cleavage in the presence of TOP2 (Fig. 2).
Compounds 3a,b and 4a,b were found to have excellent
solubility in chloroform and to have water solubility
>2.0 mg/mL as their citrate salts. Despite the fact that
substituents at the 11-position do disrupt the planarity of
these diazachrysene and triazachrysene derivatives, com-
pounds 3c and 4c,d had limited solubility and were diffi-
cult to formulate for in vivo studies. Thus, compounds 3a
and 4a were selected for the initial in vivo assessment of
the efficacy of these novel TOP1- targeting agents. NCR-
nude-nude mice obtained from Taconic Farms (Albany,
NY, USA) were inoculated with 1.2–1.5 Â 106 MDA-MB-
435 cells. Mice were administered 2.0 mg/kg of 3a po or ip.
Mice treated with 4a received 1.0 mg/kg ip and po per
injection. Mice treated with 3a or 4a received doses ip or
po as tolerated, followed by periods of 1–3 days of no
treatment. The total average dose administered per mouse
is provided in Table 2. Irinotecan (CPT-11) was adminis-
tered ip for 5 consecutive days per week at a dose of
25mg/kg. Treatment of all groups was continued for 31
days. The results of this assay are summarized in Table 2.
The presence of an alkyl substituent at N5 of 5,12-dia-
zachrysen-6-ones or N11 of 5,6,11-triazachrysen-12-ones
was shown in modeling studies to result in a twist within
these pentacyclic compounds. The lack of planarity
associated with 3a–c and 4a–d could, in part, be respon-
sible for an absence of activity observed with TOP2. For
example for 3a–c, models, geometry optimized at the
AM1 level using Spartan (Wavefunction, Inc.) suggest
that there exists an approximately 14–22ꢁ torsion angle
involving C4,C4a,C4b,N5.32 Previous studies on cor-
alyne and benzo[i]phenanthridine derivatives have indi-
cated that non-planar analogues generally are not as
effective in stabilizing the cleavable complex with TOP2.
These data demonstrate that select 5,12-diazachrysen-6-
ones and 5,6,11-triazachrysen-12-ones are potent TOP1-
targeting agents with exceptional cytotoxic activity.
Based upon the studies performed with 3a, 5,12-diaza-
chrysen-6-one derivatives have the potential to exhibit
exceptional potency in vivo as antitumor agents by
either parenteral or oral administration.
The cytotoxicity data obtained using the human lym-
phoblastoma cell line, RPMI8402, and its camptothe-

3336
A. L. Ruchelman et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3333–3336
Table 2. Antitumor activity observed in athymic nude mice with the human tumor xenograft MDA-MB-435
Compd
Route
Average tumor volume (mm3)
Average total dose (mg/mouse)
Day 7
Day 13
Day 21
Day 31
3a
3a
poa
ipa
159
123
148
84
99
65
62
40
0.78
0.73
4a
4a
poa
ipa
154
167
183
211
207
252
200
403
0.27
0.22
CPT-11
Vehiclec
ipb
ipb
118
199
116
234
36
11
13.88
356
472
aSeven mice per group.
bSix mice per group.
cVehicle controls consisted of 0.01% citrate administered 5 days per week.
gen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2002, 45,
242.
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6.17 (s, 2H), 7.46 (s, 1H), 7.66 (s, 1H), 7.77 (s, 1H), 7.87 (s,
1H), 9.35 (s, 1H); (3c) 1.80–2.21 (m, 4H), 3.63–3.82 (m, 2H),
4.07 (s, 3H), 4.14 (s, 3H), 4.52–4.84 (m, 3H), 6.18 (s, 2H), 7.48
(s, 1H), 7.70 (s, 1H), 7.90 (s, 1H), 8.04 (s, 1H), 9.39 (s, 1H);
(4a) 2.42(s, 6H), 3.04 (t, 2H, J=7.2 Hz), 4.08 (s, 3H), 4.17 (s,
3H), 4.64 (t, 2H, J=7.2 Hz), 6.25 (s, 2H), 7.81 (s, 1H), 7.84 (s,
1H), 8.07 (s, 1H), 8.65 (s, 1H); (4b) 1.96–1.98 (m, 9H), 2.79
(dd, 1H, J=12.2, 7.2 Hz), 3.26 (dd, 1H, J=12.0, 7.8 Hz), 4.08
(s, 3H), 4.18 (s, 3H), 4.80–4.88 (m, 1H), 6.22 (s, 2H), 7.66 (s,
1H), 7.80 (s, 1H), 7.82 (s, 1H), 8.58 (s, 1H); (4c) 1.74–2.33 (m,
4H), 3.74–4.0 (m, 2H), 4.09 (s, 3H), 4.18 (s, 3H), 4.56–4.70 (m,
3H), 6.25 (s, 2H), 7.80 (s, 1H), 7.84 (s, 1H), 8.32 (s, 1H), 8.63
(s, 1H); (4d) 1.07 (t, J=7.4 Hz, 3H), 1.56 (m, 2H), 2.14 (m,
2H), 4.09 (s, 3H), 4.17 (s, 3H), 4.49 (m, 2H), 6.26 (s, 2H), 7.62
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