CC-1065 analogues bearing different DNA-binding subunits: Synthesis, antitumor activity, and preliminary toxicity study

Article abstract of DOI:10.1021/jm0203433

CC-1065 analogues bearing different DNA-binding subunits were synthesized. A terminal C5-NO₂ and -F moiety at the DNA-binding subunit increased the drug's potency and antitumor efficacy. A C5-OCH₃ reduced the potency and antitumor efficacy. Compound (±)-7, bearing a trans double bond, had increased antitumor efficacy. A preliminary toxicity study indicated that terminal C5-OCH₃ and -acetamido moieties at the DNA-binding subunit caused delayed death in mice.

Full text of DOI:10.1021/jm0203433

634
J . Med. Chem. 2003, 46, 634-637
Brief Articles
CC-1065 An a logu es Bea r in g Differ en t DNA-Bin d in g Su bu n its: Syn th esis,
An titu m or Activity, a n d P r elim in a r y Toxicity Stu d y
Yuqiang Wang,*,† Lianfa Li,‡ Wenqing Ye,† Zhiming Tian,§ Wei J iang,† Hong Wang,† Susan C. Wright,† and
J ames W. Larrick†
Panorama Research, Inc., 2462 Wyandotte Street, Mountain View, California 94043, Analytical Center, Shandong Academy of
Sciences, J inan 250014, China, and Department of Medicinal Chemistry, Institute of Medicinal Biotechnology, Peking Union
Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China
Received August 12, 2002
CC-1065 analogues bearing different DNA-binding subunits were synthesized. A terminal C5-
NO2 and -F moiety at the DNA-binding subunit increased the drug’s potency and antitumor
efficacy. A C5-OCH3 reduced the potency and antitumor efficacy. Compound (()-7, bearing a
trans double bond, had increased antitumor efficacy. A preliminary toxicity study indicated
that terminal C5-OCH3 and -acetamido moieties at the DNA-binding subunit caused delayed
death in mice.
In tr od u ction
CC-1065 (Figure 1) is one of the most potent anti-
tumor agents discovered,1 and has potent antitumor
activity in vitro and in vivo.1-4 CC-1065 binds to double-
stranded B-DNA within the minor groove and alkylates
the N3 position of the 3′-adenine.5-7 CC-1065 also
inhibits gene transcription by interfering with binding
F igu r e 1. Structure of CC-1065.
of the TATA box binding protein to its target DNA.8 CC-
1065 cannot be used in humans because it caused
delayed death in experimental animals.9
CC-1065 comprises three indoles linked by amide
bonds. The left-hand cyclopropylindole subunit alkylates
DNA, and the middle and right-hand subunits enhance
the DNA-binding affinity and selectivity. Substituents
at the DNA-binding subunits have profound effects on
the compound’s antitumor potency and efficacy. A C5-
acyl group was found to increase the agent’s potency
by more than 1000-fold in vitro.10 A trans double bond
linking the left-hand DNA-reactive and the right-hand
DNA-binding subunits of CPI greatly increases the
agent’s potency in vitro.11-14 Duocarmycin analogues
bearing a trans double bond have increased antitumor
efficacy and reduced myelosuppression.15,16 We have
previously reported that CC-1065 analogues bearing a
C5-acetamido group had enhanced antitumor activity
and reduced hematological effects.17 Herein, we report
the synthesis, antitumor activity, and preliminary toxic-
ity study of new CC-1065 analogues bearing different
terminal moieties at the DNA-binding subunits (Figure
2).
* To whom correspondence should be addressed. Phone: (650) 694-
F igu r e 2. Structures of new CC-1065 analogues.
4996, extension 213. Fax: (650) 694-7717. E-mail: yuqiangwang@
worldnet.att.net.
† Panorama Research, Inc.
Ch em istr y
‡ Shandong Academy of Sciences.
Compound 1017 was treated with concentrated hy-
§ Peking Union Medical College and Chinese Academy of Medical
Sciences.
drochloric acid followed by sodium nitrite and tetrafluo-
10.1021/jm0203433 CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/11/2003

Brief Articles
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 635
Ta ble 1. Cytotoxicity against L1210 Leukemia Cellsa
Sch em e 1. Synthesis of Compound 12a
compd
IC50 (nM)
compd
IC50 (nM)
(+)-1
(()-1
(+)-2
(()-2
(()-3
(()-4
1.2 ( 0.73
2.5 ( 1.3
(()-5
0.29 ( 0.16
0.85 ( 0.28
1.0 ( 0.71
44 ( 5.7
(()-6
0.65 ( 0.35
1.2 ( 0.28
5.7 ( 1.6
(()-7
(()-8
doxorubicin
179 ( 94
a
(a) HNO2, HBF4; (b) NaOH, then HCl.
0.44 ( 0.13
Sch em e 2. Synthesis of Compounds 21-24a
a
Drugs were incubated with cells for 48 h.
Ta ble 2. Antitumor Activity in Mice Bearing L1210 Leukemia
dose
(µg /kg)
% weight
changea
30-day
survivors
compd
% ILS
(+)-1
25
42
70
25
42
70
45
67
100
45
67
100
45
67
100
30
+7
+5
-6
+12
-7
-18
+2
+3
-15
+2
-2
-5
+2
-2
-7
+5
+5
0
173
133
80
120
67
toxic
93
93
1
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
(+)-2
(()-3
(()-4
(()-5
(()-6
(()-7
(()-8
53
120
147
173
137
160
158
67
107
147
157
231
147
31
a
(a) HBTU; (b) NaOH, then HCl.
Sch em e 3. Synthesis of Compound 30a
45
67
298
498
620
250
500
1000
125 mg
0
0
-13
+7
+5
-6
-5
31
77
173
CPb
a
b
Group body weight change between days 0 and 6. CP )
Cyclophosphamide.
of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI),
affording 1-8.
a
Resu lts a n d Discu ssion
(a) EDCI; (b) MnO2; (c) Ph3PdCHCO2CH3; (d) NaOH, then
HCl.
Cytotoxicity. The compounds were tested in vitro
against L1210 leukemia cells (Table 1). As expected, the
chiral compounds (+)-1 and (+)-2, with the same con-
figuration as that of the natural CC-1065, are twice as
potent as their corresponding racemic counterparts
(()-1 and (()-2. It appears that an electron-withdrawing
moiety at the C5 position leads to an increased cyto-
toxicity. For example, compounds 4 (C5-NO2), 5 (C5-
F), and 6 (C5-F) are more potent than 1 (C5-amido),
2 (C5-amido), and 3 (C5-OCH3). The pyrrole derivative
(()-8 is much less potent than the indole and benzo-
furan derivatives 1-7. This is in agreement with what
has been reported before17 and is probably due to the
reduced binding between DNA and the compounds.
An titu m or Scr een in g in Mice. Antitumor activity
was tested against L1210 leukemia in mice (Table 2).
The chiral (+)-1 not only is more potent than its racemic
counterpart but also has improved antitumor efficacy.
For example, at an optimal dose (the dose at which the
drug produces the best antitumor efficacy and the body
weight loss is e15%), (+)-1 (25 µg/kg) has an ILS of
173% and the racemic (()-1 (42 µg/kg) has only 67%.17
The nitro analogue (()-4 and the fluorine agents (()-5
and (()-6 are not only more potent than the methoxy
roboric acid to afford an aryldiazonium tetrafluoro-
borate. Thermal decomposition of the latter produced
the required ester 11 (Scheme 1), which was hydrolyzed
to afford acid 12. The commercially available 13-15 and
12 were coupled to amine 17, respectively, in the
presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HBTU) to afford esters
18-21 (Scheme 2). Because of poor water solubility of
the esters, a mixture of solvents had to be used for the
subsequent saponification, and the yields varied from
40% to 80%. Acrylic acid 30 was synthesized using a
strategy we previously developed (Scheme 3).13,17 Alde-
hyde 28 was refluxed with methyl (triphenylphospho-
ranylidene)acetate in toluene for 4 days to afford ester
29 (83% yield), which was hydrolyzed to afford acid 30.
Acids 1, 2, and 8 were synthesized as we previously
reported.17 The targeted compounds 1-8 were synthe-
sized using a method we previously developed.17 CBI,
synthesized according to a published procedure,18 was
treated with anhydrous hydrogen chloride in ethyl
acetate to afford the seco-CBI, which was then coupled
to the corresponding acids, respectively, in the presence

636 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Brief Articles
toxicity,19,20 indicating that the C5 position of indole/
benzofuran is particularly sensitive to substitution in
terms of delayed toxicity. KW-2189, a duocarmycin
derivative, has a C5 methoxy group but does not cause
delayed toxicity.21 It is important to point out that KW-
2189 has only one DNA-binding subunit whereas CC-
1065, 1-3, and 8 have two. The delayed toxicity has no
relationship to the drug’s potency and therapeutic
efficacy. For example, compounds 4-6 are more potent
and more efficacious than 3 and the racemic 117 and
2;17 however, 4-6 did not have delayed toxicity. Further
structure-toxicity studies are needed to reveal the
structural features responsible for the delayed toxicity.
Ta ble 3. Time of Death in Mice Treated with Compounds
1-8a
dose
(µg/kg)
days until first
mouse died
no. of mice
surviving on day 180
compd
(+)-1
(+)-2
(()-3
(()-4
(()-5
(()-6
(()-7
(()-8
100
100
100
100
100
100
100
1000
2000
49
8
35
no death
no death
no death
no death
60
2/8
4/8
5/8
8/8
8/8
7/7
8/8
1/6
0/6
29
a
Drugs were administered on day 1, ip.
Con clu sion s
The chiral compound (+)-1 showed significant anti-
tumor activity against L1210. However, it caused de-
layed toxicity in mice. A C5-OCH3 resulted in reduced
potency and antitumor efficacy as well as delayed
toxicity. Compounds 4-6 had increased potency and
antitumor efficacy in vivo without delayed toxicity. A
trans double bond linking the DNA-reactive and -bind-
ing subunits led to an increased antitumor efficacy. A
preliminary toxicity study suggests that the terminal
C5 position of indole/benzofuran and the C4 position of
pyrrole substituents of the DNA-binding subunit of CC-
1065 analogues are very sensitive in terms of antitumor
efficacy and delayed toxicity.
Ack n ow led gm en t. This work was supported in part
by a grant from the National Institutes of Health (Grant
CA82949 to Y.W.).
F igu r e 3. Structural features responsible for delayed toxicity.
analogue (()-3 in vitro but also much more efficacious
than (()-3 against L1210 leukemia in mice. Compounds
with two indoles such as (+)-1 and (()-5 have better
antitimor efficacy than their counterparts with one
indole and one benzofuran such as (+)-2 and (()-6.
Compound (()-7 is approximately 3 times less toxic than
(()-5 in vitro but is 7 times less potent than the latter
in mice. Despite the low potency, (()-7 has the highest
therapeutic efficacy among 1-8. The pyrrole derivative
(()-8 has the lowest therapeutic efficacy.
P r elim in a r y Toxicity Stu d y. CC-1065 and some of
its analogues have delayed toxicity in mice; i.e., mice
die long after the normal observation period of 15 days
for the acute toxicity study. To investigate delayed
toxicity, 1-8 were given to non-tumor-bearing BDF1
mice ip on day 0 (Table 3). These animals were observed
for up to 180 days. Compounds (+)-1, (+)-2, and (()-8,
bearing a NHCOCH3, and (()-3, bearing a C5-OCH3,
caused delayed death, a characteristic of the phenomena
observed for CC-1065.9 The animals grew normally
initially and began to lose weight 1 or 2 weeks before
they died. Compounds (()-4, (()-5, and (()-6, bearing
a C5-substituted NO2 and F, and (()-7, bearing a trans
double bond in the middle of the molecule, did not cause
delayed toxicity.
Su p p or tin g In for m a tion Ava ila ble: Chemical synthesis,
spectral and analytical data for all new compounds, and
experimental details of antitumor activity evaluations in vitro
and in mice. This material is available free of charge via the
Internet at http://pubs.acs.org.
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