Optimization of naphthalimide-imidazoacridone with potent antitumor activity leading to clinical candidate (HKH40A, RTA 502)

Article abstract of DOI:10.1021/jm7009777

Unsymmetrical bifunctional antitumor agent WMC79 was further optimized to generate compound 7b that not only inhibited the growth of many tumor cell lines, but caused rapid apoptosis. Unlike the parent compound, 7b is toxic to both p53 positive and negati

Full text of DOI:10.1021/jm7009777

J. Med. Chem. 2007, 50, 5557-5560
5557
Optimization of Naphthalimide-imidazoacridone
Chart 1. Structures of 1 and New Derivatives (2)
with Potent Antitumor Activity Leading to
Clinical Candidate (HKH40A, RTA 502)
†
†
Humcha K. Hariprakasha, Teresa Kosakowska-Cholody,
§
†
‡
Colin Meyer, Wieslaw M. Cholody, Sherman F. Stinson,
Nadya I. Tarasova,* and Christopher. J. Michejda
,
†
†,|
Molecular Aspects of Drug Design Section, Structural Biophysics
Laboratory, Center for Cancer Research, NCI-Frederick, Frederick,
Maryland 21702, DeVelopmental Therapeutics Program,
NCI-Frederick, Frederick Maryland 21702, and Reata
Pharmaceuticals, 2801 Gateway DriVe, Suite 150, IrVing, Texas
ReceiVed August 6, 2007
Abstract: Unsymmetrical bifunctional antitumor agent WMC79 was
further optimized to generate compound 7b that not only inhibited the
growth of many tumor cell lines, but caused rapid apoptosis. Unlike
the parent compound, 7b is toxic to both p53 positive and negative
cancer cells. It has potent in vivo activity against xenografts of human
colon and pancreatic tumors in athymic mice.
Scheme 1. Synthesis of Derivatives of 1a
Many currently used anticancer drugs have multiple molecular
1
targets. Multitargeting may carry a significant advantage in
cancer therapy because tumor growth is caused by multiple
2
mutations. Frequently, activation of more than one signaling
pathway defines cancerous cell phenotype. DNA-binding com-
pounds present a class of multipotent antitumor agents. Com-
plexity and heterogeneity of DNA structure in the cells defines
significantly different effects of different DNA-binding com-
pounds on cellular events.
We have been working on a class of molecules that contain
two different DNA-binding moieties linked together with a
3-7
linker that can also facilitate binding to DNA.
Even slight
variations in the structure of these compounds tend to lead to
significant changes in their biological properties. Here we report
optimization of a previously reported compound 1 (WMC79;
5
,8
Chart 1) into a highly potent agent with remarkable and
selective antitumor activity. Cell toxicity of 1 is p53-dependent
and, thus, it has lower potency against p53-negative cells. The
compound is also poorly soluble. We focused our structure
optimization efforts on modifications in the positions 7 and 8
of the imidazoacridone ring as well as on substitutions in the
1
,8-naphthalimide moiety. The general synthetic routes for the
new compounds are presented in Scheme 1. The fluorinated
acridone 3a was obtained in an analogous manner from
9
4
-fluoroaniline, as described previously for 3b-d . Condensa-
tion of 2,6-dichloro-3-nitrobenzoic acid and 4-fluoroaniline led
to the acid 4, which upon acylation resulted in the fluorinated
acridone 3a. Heating the acridones 3 with excess of 1,4-bis(3-
8
aminopropyl)piperazine, as described earlier, in either dimeth-
a
ylformamide (DMF ) or dimethylsulfoxide (DMSO) gave the
nitroacridones 5, which were cyclized to the imidazoacridones
6
a-d. The unsubstituted compound 6e has been reported
8
a
recently. Imidazoacridones 6 were then condensed with equimo-
Reagents and conditions: (a) EtOH, reflux; (b) POCl3, CHCl3, reflux;
c)1,4-bis(3-aminopropyl)piperazine, DMF (for 5a) or DMSO (for 5b-d);
d) for 6a, Ra-Ni/H2, HCO2H; then HCl, reflux; for 6b-d, SnCl2, HCO2H,
(
(
*
To whom correspondence should be addressed. Tel.: 301-846-5225.
HCl, reflux; (e) naphthalic anhydride, DMF, 80 °C; (f) SnCl2, HCO2H,
HCl, reflux.
Fax: 301-846-6231. E-mail: tarasova@ncifcrf.gov.
†
Molecular Aspects of Drug Design Section, Structural Biophysics
Laboratory, Center for Cancer Research, NCI-Frederick.
‡
Developmental Therapeutics Program, NCI-Frederick.
Reata Pharmaceuticals.
Passed away on January 9, 2007.
lar amounts of appropriate naphthalic anhydrides, resulting in
compounds 7a-h. Compound 7a was further transformed to
the amino derivative 7i by reduction of the nitro group with
stannous chloride.
§
|
a
Abbreviations: DMF, N,N-dimethylformamide; DMSO, dimethyl sul-
foxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
1
0.1021/jm7009777 CCC: $37.00 © 2007 American Chemical Society
Published on Web 10/16/2007

5
558 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23
Letters
Table 1. In Vitro Activity of 1 and Derivatives 7 against Selected
Human Cancer Cell Lines
Table 2. In Vitro Activity of 1 and 7b against Selected Human Tumor
a
Cell Linesa
LC50 (nM)
cell line
cmpd 1
cmpd 7b
HCT116 (wild-type p53)
RKO (wild-type p53)
Colo205 (mutated p53)
HT29 (mutated p53)
ASPC-1 (p53 null)
40
40
500
550
350
30
25
80
80
70
90
45
10.05 (mutated p53)
300
PC3 (mutated p53)
>1000
tumor cell lines
a
Determined by a 5 day MTT assay.
HCT116
HT29
Hep3B
GI50
LC50
GI50 LC50 GI50 LC50
cmpd
R
X
Y
(nM) (nM) (nM) (nM) (nM) (nM)
7
7
7
7
7
7
7
7
7
1
a
b
c
d
e
f
g
h
i
8-F
NO2
H
1
1
3.5
3
15
3.5
40
30
30
30
2
1
600
80
4
2.5
400
50
8-OMe NO2
H
H
H
H
H
b
NTb
8-Me
7-Me
H
8-OMe
H
8-OMe
8-F
H
NO2
NO2
H
H
H
H
NH2
NO2
NT
NT
b
b
NT
1000
70
>1000 15
350
80
11
3
1000 22
1000 3.5
1000 18
1000
750
1000
330
1000
800
NO2 5.5
NO2
H
3
2.2
1
4
8
2
5
NTb
H
40
550
4
380
a
Measured by a 5 day MTT assay. The obtained data was used to
calculate GI50, TGI, and LC50 values presented. Values shown are means
SD of at least three independent experiments. The coefficients of variation
were between 10 and 20%. NT, not tested.
Figure 1. Time-dependent cytotoxicity of 1 and 7b, measured by MTT
assay in HCT116 colon cancer cells.
(
b
For biological testing, all new compounds (7) were trans-
formed into their tri(methanesulfonate) salts, that were suf-
ficiently soluble in water and in dilute buffers. All of the
compounds reported are very stable in the solid state, especially
in the methanesulfonate form, but suffer photochemical degra-
dation when dilute solutions are exposed to ambient light for
prolonged periods of time. The compounds are easily adsorbed
on glass and quartz surfaces, which affect the concentrations
of dilute solutions significantly. These solutions were not
affected by polyethylene or polycarbonate surfaces.
Figure 2. Effects of 1 and 7b on cell-cycle distribution in HCT116
cells exposed to compounds for 72 h.
All new derivatives had comparable growth inhibitory activi-
ties in p53-positive HCT116 colon tumor cell line (Table 1).
However, the activity in the ability to kill cancer cells differed
significantly. The nitro group in position 5 of the naphthalimide
ring was essential for the cell-killing activity of the compounds.
Eliminating it (as in 7f) resulted in about a 10-fold reduction in
LC50. Reduction of the nitro group into an amino group (7i)
caused a slight increase in LC50. Compounds with a nitro group
in position 6 rather than 5 (7g and 7h) had significantly lower
cell-killing activity. Position 8 of the imidazoacridone ring is
known to undergo metabolic hydroxylation that may have a
negative effect on the in vivo activity of the compounds. In an
attempt to improve the metabolic stability of the compounds,
we have evaluated derivatives with a fluorine atom in position
frequently requiring much more than 48 h, we evaluated the
cytotoxic effects in a 5 day assay. Table 2 presents results from
this experiment for selected cell lines that differ in the status of
p53. The growth inhibition effect (GI ) of both compounds was
very similar (1-5 nM) for all tested cell lines regardless of the
status of p53. In contrast, there was a significant difference
between the two compounds in their cell killing ability. For 1,
the LC50 is clearly about 10-fold higher for cell lines with
mutated or null p53, while the LC50 values for 7b were superior
in all tested cell lines.
Time course analysis of HCT116 cells exposed to 1 and 7b
performed at different concentrations indicated that 7b needed
much shorter incubation time to exhibit cytotoxic activity (Figure
1). Treatment of HCT116 cells with 7b at 50 nM caused visible
cell killing already after 48 h, whereas 1 required much longer
exposure to achieve similar effect. The cell-killing effects were
confirmed by cell cycle analysis (Figure 2). A 72 h treatment
of HCT116 cells with 50 nM of either drug resulted in the
50
8
(7a and 7i). The compounds were equal in activity to the
unsubstituted parent compound, 1; however, they turned out to
be much less soluble. Similarly, methyl derivatives (7c and 7d)
had significantly reduced solubility. On the contrary, methoxy
derivatives were more soluble than unsubstituted counterparts
and had much improved antitumor activity.
appearance of sub-G population corresponding to dead cells
1
that represented 15.5% and 48.0%, respectively, for 1 and 7b.
The unsubstituted naphthalimide ring gave rise to the least
active of the compounds (7e; Table 1). A comparison of
antitumor potency of 1 and 7b in the NCI 60 human tumor cell
line screen, which is based on a short 48 h continuous drug
exposure, showed about 30-fold higher cell killing activity of
At the same time, there was a significant accumulation of cells
in the G2/M phase (45.0% and 27.1% compared to 13% in
control cells), decrease in the G1 phase (from 65.9% in untreated
cells to 37.9% and 22.6%, respectively), and almost total
depletion of cells in the S-phase.
7
b (median LC50 ) 0.67 µM compared to 19.9 µM for 1).
Pilot pharmacokinetic studies conducted in CD2F1 mice after
i.v. injection of 1 or 7b showed that the total plasma clearance
Because killing of tumor cells by a drug can be a slow process

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 23 5559
Figure 4. Inhibition of colon tumor growth in mice. HCT-116 colon
cancer tumor fragments were implanted on the flank of nu/nu mice,
seven animals in each group. Treatment (iv administration, Q3Dx4)
Figure 3. Blood plasma concentrations of 7b following i.v. administra-
tion at a dose of 20 mg/kg body to ncr nu/nu mice and to a F344 rat.
Lines represent the respective lines of best fit determined by nonlinear
regression analysis.
3
was started when tumors reached 62 mm .
rate of 7b was 35% lower than that of 1 (238 vs 323 mL/min/
kg, respectively). Because of the more favorable pharmacoki-
netic and biological features of 7b compared to 1, more
comprehensive evaluations of 7b were performed. In both nu/
nu mice and F344 rats, disposition of 7b following i.v. injection
at a dose of 20 mg/kg was typified by a rapid initial “distribu-
tion” phase (6 and 2 min, respectively) and a long terminal
“
elimination” phase (4 and 8 h, respectively, Figure 3).
The terminal disposition phases accounted for a high propor-
tion (70% in mice, and 82% in rats) of the total areas under the
plasma concentration versus time curves. Initial (3 min) plasma
concentrations were 4.1 µM and 1.6 µM in mice and rats,
respectively. Plasma levels of 7b were 70 nM after 8 h in mice
and 90 nM after 4 h in rats. In both species, the apparent
volumes of distribution of 7b were very high relative to its
apparent volumes of the central (plasma) compartments (57 vs
Figure 5. Inhibition of pancreatic tumor growth in mice in comparison
and in combination with gemcitabine. PaCa-2 tumor fragments were
implanted on the flank of nu/nu mice (10 in each group). Treatment
(
intraperitoneal, Q3Dx4) started 12 days post-inoculation when tumors
3
reached 80 mm .
5
L/kg, respectively, in mice and 138 vs 6 L/kg, respectively,
in rats), suggesting that 7b is highly distributed into the tissues
in these species.
In both mice and rats, 7b was not detected in the plasma
Acknowledgment. We are very grateful to Dr. Larry Keefer
for the critical reading of the manuscript, to Dr. Dominic
Scudiero for the NCI 60 cell line assay data, and to Dr. Sergey
Tarasov for discussions. We thank the Structural Biophysics
Resource, SBL, NCI-Frederick, for the use of the mass
spectrometer. This research was supported in part by the
Intramural Research Program, the National Institutes of Health,
National Cancer Institute, Center for Cancer Research.
(l.o.d ) 50 nM) when it was given by the i.p. or p.o. routes,
strongly suggesting that 7b is rapidly metabolized and eliminated
by the liver after being absorbed into the hepatic portal
circulation. Also, 7b was not detected in plasma samples when
it was administered s.c. to nude mice, demonstrating that i.v.
injection offers the most promising route of administration for
in vivo efficacy studies with 7b. When given i.v. in a single
well-tolerated dose, plasma levels of 7b exceeded concentrations
required for in vitro efficacy for up to 8 h in mice and 4 h in
rats.
Supporting Information Available: Experimental details and
analytical data for the compounds described and NCI 60 cell lines
screening data for 7b. This material is available free of charge via
the Internet at http://pubs.acs.org.
Compound 7b dose-dependently inhibited growth of HCT116
xenografts in vivo, with a maximum TGI of 59% (Figure 4).
This compound significantly inhibited growth of PaCa-2
pancreatic xenografts, which has mutated p53, in vivo with
maximum growth inhibition of 53.5% at 15 mg/kg and
significantly outperformed gemcitabine, a standard agent for the
treatment of pancreatic tumors (TGI ) 85.3%). The combination
of 7b and gemcitabine performed better than both single agents
and induced regression over days 11 through 18 (Figure 5). No
toxicity was detected for 7b at the doses tested.
In conclusion, structure optimization of 1 revealed that
introduction of the methoxy group in position 8 of the
imidazoacridone ring improved in vitro and in vivo potency of
the parent compound. Based on its enhanced overall profile,
compound 7b (HKH40a, RTA502) was chosen for IND filing
and further clinical development.
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JM7009777