Synthesis and preliminary biological evaluations of CC-1065 analogues: Effects of different linkers and terminal amides on biological activity

Article abstract of DOI:10.1021/jm990514c

CC-1065 analogues possessing a biologically active CBI functional group and amide-substituted indole and benzofuran were synthesized. The IC₅₀ values of compounds 26, bearing two indoles, and 25, bearing only one indole, are 0.4 and 3 nM, respectively, against U937 leukemia cells in vitro. The IC₅₀ values of compounds 28, bearing a butyramino group, and 27, bearing an acetamino group, are 0.008 and 0.4 nM, respectively, against U937 leukemia cells in vitro. Compound 29, bearing a double-bond linker, is about 4-fold more potent than 25, bearing no double-bond linker. Compound 26 is highly potent against all cell lines tested in the NCI in vitro screening with IC₅₀ values in the 0.1-5 nM range for most cell lines. Compounds 26 and 30 are highly active against L1210 leukemia in mice. Compound 26 is also active against B16BL6 melanoma in mice. Most importantly, 26 and 30 are not myelosuppressive at therapeutically effective doses. The mechanism of tumor cell death is through induction of apoptosis, and is accompanied by DNA fragmentation.

Full text of DOI:10.1021/jm990514c

J . Med. Chem. 2000, 43, 1541-1549
1541
Syn th esis a n d P r elim in a r y Biologica l Eva lu a tion s of CC-1065 An a logu es:
Effects of Differ en t Lin k er s a n d Ter m in a l Am id es on Biologica l Activity
Yuqiang Wang,* Huiling Yuan, Wenqing Ye, Susan C. Wright, Hong Wang, and J ames W. Larrick
Panorama Research, Inc., 2462 Wyandotte Street, Mountain View, California 94043
Received October 12, 1999
CC-1065 analogues possessing a biologically active CBI functional group and amide-substituted
indole and benzofuran were synthesized. The IC50 values of compounds 26, bearing two indoles,
and 25, bearing only one indole, are 0.4 and 3 nM, respectively, against U937 leukemia cells
in vitro. The IC50 values of compounds 28, bearing a butyramino group, and 27, bearing an
acetamino group, are 0.008 and 0.4 nM, respectively, against U937 leukemia cells in vitro.
Compound 29, bearing a double-bond linker, is about 4-fold more potent than 25, bearing no
double-bond linker. Compound 26 is highly potent against all cell lines tested in the NCI in
vitro screening with IC50 values in the 0.1-5 nM range for most cell lines. Compounds 26 and
30 are highly active against L1210 leukemia in mice. Compound 26 is also active against
B16BL6 melanoma in mice. Most importantly, 26 and 30 are not myelosuppressive at
therapeutically effective doses. The mechanism of tumor cell death is through induction of
apoptosis, and is accompanied by DNA fragmentation.
Ch a r t 1. Structures of CC-1065, CBI, and CPI
In tr od u ction
CC-1065 (Chart 1) was first isolated from Streptomy-
ces zelensis by scientists at the Upjohn Company1 and
was found to have potent antitumor and antimicrobial
activity both in vitro and in experimental animals.1-4
The duocarmycins discovered later were found to pos-
sess chemical structures and biological activities similar
to those of CC-1065.5-9 CC-1065 binds to double-
stranded B-DNA within the minor groove with the
sequence preference of 5′-d(A/GNTTA)-3′ and 5′-
d(AAAAA)-3′ and alkylates the N3 position of the 3′-
adenine with its left-hand CPI segment.10-12 CC-1065
also inhibits gene transcription by interfering with
binding of the TATA box binding protein to its target
DNA.13 Despite its high potency and broad spectrum of
antitumor activity, CC-1065 cannot be used in humans
because it causes delayed death in experimental ani-
mals.14
biologically active CBI group was linked to substituted
indole and benzofuran by different linkers and terminal
amides (Chart 2).
Resu lts
To pursue compounds retaining the potent antitumor
activity but devoid of the toxic side effects of the parent
compound, many CC-1065 analogues have been syn-
thesized and tested.15-41 Among them are the CC-1065
analogues adozelesin (U-73975), bizelesin (U-77779),
and carzelesin (U-080244), as well as the duocarmycin
analogue KW-2189. These compounds are currently in
clinical trials.42 CC-1065 analogues, in which the right-
hand indole was replaced by C4-substituted pyrrole
linked to the CPI unit by different linkers, were
synthesized by Wang et al.24,25,28,31,36 A trans double-
bond linking the CPI and the pyrrole substantially
increased the cytotoxicity of the agent. A C4-terminal
amide group also greatly increased cytotoxicity. Fur-
thermore, a trans double-bond and a C4-amide group
act synergistically to increase the potency in vitro.25,31
Herein, we report the synthesis and preliminary biologi-
cal evaluations of new CC-1065 analogues in which a
Ch em ica l Syn th esis. Synthesis of acids 11 and 12
is illustrated in Scheme 1. Ethyl 5-nitroindole-2-car-
boxylate, 1, and methyl 5-nitrobenzofuran-2-carboxy-
late, 2, were reductively transformed to their corre-
sponding amines 3 and 4, respectively, by hydrogenolysis
over Pd/C. Amines 3 and 4 were converted to their
corresponding acetamino esters 5 and 6 by treatment
with acetic anhydride in ethyl acetate with very high
yields. Esters 5 and 6 were then hydrolyzed, respec-
tively, using dilute sodium hydroxide solution (3 N),
followed by neutralization using dilute hydrochloric acid
(20%) to afford their corresponding acids 7 and 8. Acids
7 and 8 were coupled to amine 3 in the presence of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) to
afford esters 9 and 10, respectively. Esters 9 and 10
were then converted to their corresponding acids 11 and
12 by treatment with sodium hydroxide solution, fol-
lowed by neutralization using dilute hydrochloric acid.
Acid 17 was synthesized employing a procedure
similar to that for the synthesis of acid 12 (Scheme 2).
* To whom correspondence should be addressed. Tel: (650) 694-
4996. Fax: (650) 694-7717. E-mail: Yuqiangwang@worldnet.att.net.
10.1021/jm990514c CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/17/2000

1542 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Ch a r t 2. Structures of New CC-1065 Analogues
Wang et al.
Sch em e 2. Synthesis of Compound 17
Sch em e 3. Synthesis of Compound 23
Sch em e 1. Synthesis of Compounds 11-12
hydride in the presence of sulfuric acid at 0 °C for 30
min cleanly reduced the ester into alcohol 18 with high
yield (90%). The latter was hydrogenated over Pd/C to
afford amine 19. Treatment of 19 with acetyl chloride
followed by basic hydrolysis transformed the 5-amino
to a 5-acetamino group. The 2-hydroxymethyl group of
20 was oxidized using manganese dioxide in ethanol to
its corresponding aldehyde 21. The aldehyde was then
refluxed with methyl (triphenylphosphoranylidene)-
acetate in toluene for 3 days to afford ester 22. Basic
hydrolysis of the latter afforded 5-acetamino-2-indole-
acrylic acid, 23.
5-Nitrobenzofuran-2-carboxylic acid, 13, was treated
with amine 3 in the presence of EDCI to afford 14. The
latter was hydrogenated to give amine 15, which was
then reacted with butyric anhydride to afford but-
yramino ester 16. The latter was hydrolyzed using
sodium hydroxide solution to give 17, after neutraliza-
tion using dilute hydrochloric acid solution.
A strategy previously developed for the synthesis of
2-pyrroleacrylic acid was adapted to synthesize 2-in-
doleacrylic acid, 23 (Scheme 3).28,31 Treatment of ethyl
5-nitroindole-2-carboxylate, 1, with lithium aluminum
The CBI-bearing compounds 25-29 were synthesized
as shown in Scheme 4. Treatment of acids 7, 11, 12,

Synthesis and Evaluations of CC-1065 Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1543
Ta ble 2. Cytotoxicity of 26 against NCI Tumor Panelsa
Sch em e 4. Synthesis of Compounds 25-29
IC50
(nM)
IC50
(nM)
panel/cell line
leukemia:
panel/cell line
melanoma:
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
non-small-cell lung cancer:
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
colon cancer:
COLO 205
HCC-2998
HCT-116
HCT-15
3.14
1.40
3.76
0.375
8.98
0.562
LOX IMVT
M14
0.577
1.48
2.11
2.21
1.11
2.46
0.419
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.49 ovarian cancer:
2.39
1.25
2.11
1.36
IGROV1
OVCAR-3
OVCAR-8
SK-OV-3
1.01
3.51
1.60
4.33
Ta ble 1. Cytotoxicity against U937 Leukemia Cells
compd
IC50 (nM)a
compd
IC50 (nM)a
25
26
27
28
3.0
0.4
0.4
0.008
29
30
Adr
0.9
0.2
100
2.13 renal cancer:
1.48
0.238
786-0
A498
1.66
3.95
1.28
4.27
3.01
3.13
4.05
ACHN
CAKI-1
SN12C
TK-10
UO-31
2.99
4.03
0.495
a
IC50 values are defined as the minimal drug concentration
necessary to inhibit incorporation of [3H]thymidine by 50% and
are the averages of three experiments.
>10
HT29
1.34 prostate cancer:
17, and 23, respectively, with amine 2422 in the presence
of EDCI afforded targets 25-29. Compound 27 was
treated with triethylamine, acetonitrile, and water to
give 30 (85% yield).
KM12
SW-620
CNS cancer:
SF-268
SF-295
1.78
2.36
PC-3
DU-145
breast cancer:
MCF7
NCI/ADR-RES
2.03
0.982
0.212
2.64
0.476
8.56
Biological Stu dies. 1. Cytotoxicity. The new agents
were tested in vitro against U937 leukemia cells, and
the results are summarized in Table 1. Compound 26,
bearing two indoles with an IC50 value of 0.4 nM, is
7-fold more potent than 25, bearing only one indole with
an IC50 value of 3 nM. Compound 28, bearing a
butyramino group with an IC50 value of 0.008 nM, is
50-fold more potent than 27, bearing an acetamino
group with an IC50 value of 0.4 nM. Compound 26,
bearing two indoles, is as potent as 27, bearing one
indole and one benzofuran. Compound 29, bearing a
double-bond linker, is about 4-fold more potent than 25,
bearing no double-bond linker. Compound 30, with an
IC50 value of 0.2 nM, is 2-fold more potent than its
corresponding uncyclized precursor 27.
SF-539
SNB-19
SNB-75
U251
1.24
2.59
0.513
0.719
MDA-MB-231/ATCC 5.97
HS 578T
MDA-MB-435
MDA-N
1.63
1.86
2.22
3.29
1.68
BT-549
T-47D
a
The assay was performed by NCI using the SRB method (48-h
incubation).43
Cytotoxicity of 26 was also tested by the National
Cancer Institute (NCI), and the results are shown in
Table 2. As expected, 26 is highly potent against all cell
lines tested. For all cell lines IC50 values were less than
10 nM for a 48-h drug exposure, with most in the 0.1-5
nM range.
2. DNA F r a gm en ta tion a n d Ap op tosis. In general,
cell death occurs through either necrosis or apoptosis.
It is now recognized that most anticancer drugs, includ-
ing the CC-1065 and duocarmycin classes of agents,
induce apoptosis.44-46 To examine the mechanism of
cytotoxic action of these new compounds, 27 was chosen
for further studies using U937 cells. At a 10 nM
concentration, 27 caused DNA fragmentation in about
12%, 20%, 40%, and 80% of U937 cells for incubation
times of 2, 3, 4, and 6 h, respectively (Figure 1). Cells
exhibited the morphological changes typical of apoptosis.
The kinetics of appearance of morphologically apoptotic
cells correlated well with DNA fragmentation. More
than 80% of cells were in apoptotic state after 6 h of
incubation. Cell death was not obvious until 5 h of
incubation, and all cells were dead after 20 h of
incubation. These results agree with those found for
duocarmycin analogues.46
F igu r e 1. DNA fragmentation, apoptosis, and cell death.
cells (inoculated with 105 cells/mouse) in mice, and the
results are shown in Table 3. At a dose of 70 µg/kg, 26
and 30 produced an increase in life span (ILS) of 107%
and 93%, respectively. Cyclophosphamide (CP) produced
an ILS of 173% at a dose of 125 mg/kg and 100% cures
at a dose of 188 mg/kg.
The antitumor activity of 26, 28, and 30 was also
tested in mice inoculated with a low burden of L1210
leukemia cells (100 cells/mouse), and the results are
shown in Table 4. At a dose of 30 µg/kg, 26, 28, and 30
produced 40%, 20%, and 60% of long-term survivors (60
days), respectively. In contrast, at a dose of 30 mg/kg,
cyclophosphamide produced 50% of long-term survivors,
and taxol did not produce any long-term survivors.
Compound 26 was also tested in mice bearing B16
melanoma, and the results are shown in Table 5. In this
experiment, 26 is not as effective as adriamycin (Adr).
3. An titu m or Scr een in g in Mice. The antitumor
activity of 26 and 30 was tested against L1210 leukemia

1544 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Wang et al.
Ta ble 3. Antitumor Activity in Mice Bearing L1210 Leukemiaa
Ta ble 6. Hematological Effects in Micea
dose WBC platelet
compd dose (/kg) % weight changeb % ILS 30-day survivors
RBC
MCHC
drug
(/kg) (103/µL) (106/µL) (106/µL) (g/dL)
26
30
CP
25 µg
42 µg
+23
+16
+9
+23
+18
-15
-5
-16
-22
67
107
107
53
80
93
0
0
1
0
1
0
0
6
3
saline
12.20
12.85
1.13
1.16
9.56
8.50
33.70
34.45
70 µg
30% DMSO/
0.5% glucose
26
30
CP
taxol
5-FU
busulfan
25 µg
42 µg
50 µg
50 µg
30 mg
30 mg
30 mg
30 mg
13.10
13.95
11.22
13.40
12.40
7.00
1.10
1.01
0.77
1.04
1.24
0.71
8.72
7.81
7.67
8.33
8.82
8.45
36.70
35.50
37.25
36.50
35.40
35.65
70 µg
125 mg
188 mg
250 mg
173
a
BDF1 male mice (4-6 week old, 6/group) were used. Each
mouse was inoculated with 105 cells (0.1 mL) ip on day 0. Drugs
were administered on days 1, 5, and 9 ip. Antitumor activity was
determined by comparing the median survival time of the treated
groups (T) with that of a control group (C) and are expressed as a
percentage of ILS. The median number of days the vehicle-treated
a
Female CDF1 mice (10 mice/group) were used. Drugs were
administered on day 1 ip. On day 27, blood samples were taken
and hematological measurements were performed. Data are
presented as the median value calculated from 10 mice.
b
mice died was 7.5. Group body weight change between days 0
of compounds include hydrophobic interaction and van
der Waals contacts between the drug and DNA. The fact
that compounds 26, with two indoles, and 27, with one
indole and one benzofuran, are more potent than 25,
with only one indole, further supports this hypothesis.
That compound 28, with a more hydrophobic but-
yramino group, is approximately 50-fold more potent
than 27, with a less hydrophobic acetamino group, is
also in agreement with this hypothesis. Whereas in the
case of the CPI-pyrrole agents a trans double-bond
linker between the CPI and the right-hand pyrrole
drastically enhanced their cytotoxicity (IC50: 1.0 × 10-6
nM against KB cells in vitro),28,31 by contrast, 29 (IC50:
0.9 nM), also bearing a trans double-bond linker, is only
slightly more potent than 25 (IC50: 3 nM), bearing no
such linker. The reason for this dramatic effect of a
trans double-bond linker on CPI and CBI compounds
is not well-understood.
and the day when the animal weight was the lowest (days 9 for
CP and 12 for 26 and 30).
Ta ble 4. Antitumor Activity in Mice Bearing L1210 Leukemiaa
compd
dose (/kg)
60-day survivors (%)
26
28
30
CP
taxol
30 µg
30 µg
30 µg
30 mg
30 mg
40
20
60
50
0
a
One hundred L1210 leukemia cells were injected ip to female
CDF1 mice (10 mice/group) on day 0. Drugs were administered on
days 1, 5, and 9 ip. The median number of days the untreated
group of mice (given the vehicle only) died was 23.
Ta ble 5. Antitumor Activity in Mice Bearing B16BL6
Melanomaa
dose
(/kg)
weight change
(g/mouse)b
90-day
survivors
compd
% ILS
26
25 µg
50 µg
100 µg
5 mg
1.1 ( 0.6
0.3 ( 0.6
0.9 ( 0.9
0.5 ( 0.6
1.0 ( 1.3
50
59
0
0
1
0
2
Similar to other CC-1065 and duocarmycin classes of
agents,46 the new agents induced DNA fragmentation
and apoptosis. The link between DNA alkylation, DNA
fragmentation, and apoptosis caused by them is not
clear. We showed previously that apoptosis induced by
27 was inhibited by a serine protease inhibitor DK120.47
DK120 inhibits a novel serine protease termed AP24
(apoptotic protease of 24 kDa).48 AP24 apparently
functions downstream of a family of cysteine proteinases
called caspases in apoptosis induced by various chemo-
therapeutic agents.48-49 Activation of AP24 then trans-
mits signals to the nucleus to induce DNA fragmenta-
tion, and thus inhibition of this protease prevents
apoptotic cell death. The CC-1065 class of compounds
binds to DNA and alkylates N3 of adenine. However, it
is not clear whether 27 acts directly or indirectly via
DNA alkylation to activate AP24, which then triggers
apoptosis.
As seen from Tables 3 and 4, compounds 26 and 30
showed significant activity in mice bearing L1210
leukemia cells. Compound 26 was also active in B16
melanoma-bearing mice. Most importantly, in compari-
son to cyclophosphamide and busulfan, they had little
suppressive effects on WBC and platelet counts at a dose
of 50 µg/kg. However, CC-1065, at a dose of 25 µg/kg,
severely suppressed WBC.14 These results suggest that
a terminal acetamino group may preserve antitumor
activity and reduce myelosuppression, the main side
effects associated with this class of compounds. The four
CC-1065 and duocarmycin analogues, adozelesin, bize-
lesin, carzelesin, and KW-2189, currently under clinical
91c
44d
159
Adr
10 mg
a
BDF1 female mice (4-6 week old, 8/group) were used. Each
mouse was inoculated with 106 cells (0.1 mL) ip on day 0. Drugs
were administered on days 1, 5, and 9 ip. Antitumor activity was
determined by comparing the median survival time of the treated
groups (T) with that of a control group (C) and are expressed as a
percentage of ILS. The median number of days the untreated mice
b
(given the vehicle only) died was 16. Mean body weight change
between days 1 and 10. c One mouse died on day 12. One mouse
d
died on day 14.
However, 26 may be useful in the treatment of adria-
mycin-resistant cells because 26 is not cross-resistant
with adriamycin in B16 melanoma cells (data not
shown).
4. Hem a tologica l Mea su r em en ts. At a dose of 50
µg/kg, 27 days after the drugs were given to non-tumor-
bearing CDF1 mice, 26 and 30 had no effect on white
blood cell (WBC) and platelet counts compared with
controls (Table 6). However, the conventional alkylating
agents cyclophosphamide and busulfan significantly
suppressed both WBC and platelets. As expected, taxol
and 5-FU had little effect on WBC and platelets. All of
the compounds tested had little effect on red blood cell
counts (RBC) and the mean corpuscular hemoglobin
concentration (MCHC).
Discu ssion
Consistent with previous observations, the major
factors determining the potency of the CC-1065 class

Synthesis and Evaluations of CC-1065 Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1545
at room temperature. Ethyl acetate (40 mL) was added, and
the mixture was washed with saturated sodium carbonate
solution (10 mL) followed by water (20 mL × 2). The solution
was dried using sodium sulfate, and solvent was removed in
development, have remarkable antitumor activity in
mice. However, suppression of human bone marrow
undermines their clinical effectiveness. Further experi-
ments to confirm these preliminary results are in
progress, and we will report the results in due course.
1
vacuo. A gray powder 9 was obtained (129 mg, 69% yield). H
NMR (DMSO-d6, ppm): 11.82, (s, 1H, NH), 11.57 (s, 1H, NH),
10.09 (s, 1H, NH), 9.77 (s, 1H, NH), 8.13-7.15 (m, 8H, Ar-
H), 4.38-4.32 (q, 2H, J ) 7.0, 13.7 Hz, CH2CH3), 2.04 (s, 3H,
CH3CO), 1.37-1.33 (t, 3H, J ) 7.0 Hz, CH2CH3). MS (ion
spray, m/z): 404. Without further purification, a sodium
hydroxide solution (3 mL) was added to a solution of 9 (103
mg, 0.25 mmol) in DMF (3 mL) and methanol (15 mL). The
reaction mixture was stirred overnight at room temperature.
Methanol was removed, and water (5 mL) was added. The
solution was acidified to pH 2 using 20% hydrochloric acid.
The precipitate was filtered and washed with water. Com-
pound 11 was produced as a gray powder (45 mg, 48% yield),
mp >300 °C. 1H NMR (DMSO-d6, ppm): 12.80 (br, 1H, COOH),
11.69, (s, 1H, NH), 11.57 (s, 1H, NH), 10.06 (s, 1H, NH), 9.77
(s, 1H, NH), 8.12-7.09 (m, 8H, Ar-H), 2.05 (s, 3H, CH3CO).
Anal. (C20H16N4O4‚1.2H2O) C, H, N.
5-[(5-Aceta m in o-1H-ben zofu r a n -2-ylca r bon yl)a m in o]-
1H-in d ole-2-ca r boxylic Acid (12). EDCI (523 mg) was added
to a solution of 3 (186 mg, 0.91 mmol) and 8 (200 mg, 0.91
mmol) in DMF (3 mL) and THF (3 mL). The reaction mixture
was stirred overnight at room temperature. Ethyl acetate (40
mL) was added, and the mixture was washed with saturated
sodium carbonate solution (10 mL) followed by water (20 mL
× 2), diluted hydrochloric acid (10 mL), and water (20 mL).
The solution was dried using sodium sulfate, and solvent was
removed in vacuo. Ether (20 mL) was added, and a gray
powder 10 was obtained (270 mg, 73% yield): 1H NMR
(DMSO-d6, ppm): 11.84, (s, 1H, NH), 10.38 (s, 1H, NH), 10.03
(s, 1H, NH), 8.15-7.15 (m, 8H, Ar-H), 4.38-4.32 (q, 2H, J )
6.9, 13.7 Hz, CH2CH3), 2.08 (s, 3H, CH3CO), 1.37-1.33 (t, 3H,
J ) 6.8 Hz, CH2CH3). MS (ion spray, M + 2H): 406. Without
further purification, a 3 N sodium hydroxide solution (3 mL)
was added to a solution of 10 (140 mg, 0.35 mmol) in DMF (2
mL), acetone (10 mL), methanol (15 mL) and water (5 mL).
The reaction mixture was stirred overnight at room temper-
ature. Acetone and methanol were removed, and water (20 mL)
was added. The solution was acidified to pH 2 using 20%
hydrochloric acid, and the precipitate was filtered and washed
with water. Compound 12 was obtained as a gray powder (54
mg, 41% yield), mp >300 °C. 1H NMR (DMSO-d6, ppm): 12.80
(br, 1H, COOH), 11.72, (s, 1H, NH), 10.36 (s, 1H, NH), 10.03
(s, 1H, NH), 8.15-7.10 (m, 8H, Ar-H), 2.08 (s, 3H, CH3CO).
Anal. (C20H14N3O5‚1.1H2O) C, H, N.
Eth yl 5-[(5-Nitr o-1H-ben zofu r a n -2-ylca r bon yl)a m in o]-
1H-in d ole-2-ca r boxyla te (14). EDCI (612 mg) was added to
a solution of 3 (218 mg, 1.07 mmol) and 13 (221 mg, 1.07 mmol)
in DMF (8 mL) and THF (10 mL). The reaction mixture was
stirred overnight at room temperature. Solvent was removed,
and ethyl acetate (100 mL) was added. The reaction mixture
was washed with water (100 mL × 3). The organic solution
was dried using sodium sulfate, and solvent was removed in
vacuo. Ether was added. The product was filtered and washed
with ether to afford a yellow solid (220 mg, 52% yield). An
analytical sample was recrystallized in ethyl acetate. 1H NMR
(DMSO-d6, ppm): 11.87, (s, 1H, NH), 10.62 (s, 1H, NH), 8.84-
7.17 (m, 8H, Ar-H), 4.38-4.32 (q, 2H, J ) 7.2, 14.0 Hz, CH2-
CH3), 1.37-1.33 (t, 3H, J ) 7.2, 14.0 Hz, CH2CH3). Anal.
(C20H15N3O6) C, H, N.
5-[(5-Bu tyr am in o-1H-ben zofu r an -2-ylcar bon yl)am in o]-
1H-in d ole-2-ca r boxylic Acid (17). Compound 14 (200 mg,
0.51 mmol) was first dissolved in DMF (5 mL) and ethyl
acetate (20 mL) was then added. Pd/C (10%, 20 mg) was added
to the solution, and the reaction mixture was hydrogenated
at 1 atm for 1 h. Amine 15 was filtered over Celite, and the
filter cake was washed with methanol. Solvent was removed
in vacuo. Without further purification, the product was dis-
solved in DMF (2 mL). Butyric anhydride (1 mL) and pyridine
(1 mL) were added subsequently. The reaction mixture was
stirred at room temperature for 1 h. Ethyl acetate (50 mL)
Exp er im en ta l Section
Ch em istr y. 1H NMR spectra were recorded at ambient
temperature on an NT-360 spectrometer. Elemental analysis
was performed by Atlantic Microlab, Inc. at Norcross, GA.
High-resolution mass spectra (FABHRMS) were recorded on
a modified MS50 mass spectrometer equipped with a VG 11-
250J data system. Analytical thin-layer chromatography was
performed on silica-coated plastic plates (silica gel 60 F-254,
Merck) and visualized under UV light. Preparative separations
were performed by flash chromatography on silica gel (Merck,
70-230 mesh).
Eth yl 5-Aceta m in oin d ole-2-ca r boxyla te (5). To ethyl
5-nitroindole-2-carboxylate, 1 (500 mg, 2.14 mmol), in ethyl
acetate (100 mL) was added 5% Pd/C (100 mg) and the reaction
mixture was hydrogenated for 1 h at a pressure of 60 lb/in.2
at room temperature. The reaction mixture was filtered, and
the solvent was removed in vacuo to afford 3 as a yellow solid.
Without further purification, 3 was dissolved in ethyl acetate
(10 mL) and treated with acetic anhydride (2 mL). The reaction
mixture was stirred at room temperature for 1 h. Methanol (2
mL) was added, and the reaction mixture was stirred for an
additional 30 min. Solvent was removed and a gray powder
was obtained (540 mg, 100% yield). An analytical sample was
recrystallized in ethyl acetate, mp 202-203 °C. 1H NMR
(DMSO-d6, ppm): 11.74 (s, 1H, NH), 9.78 (s, 1H, NH), 7.99 (s,
1H, Ar-H), 7.38-7.30 (m, 2H, Ar-H), 7.09-7.08 (m, 1H, Ar-
H), 4.36-4.30 (q, 2H, J ) 7.0, 13.7 Hz, CH2CH3), 2.03 (s, 3H,
CH3CO), 1.36-1.31 (t, 3H, J ) 7.0 Hz, CH2CH3). Anal.
(C13H14N2O3) C, H, N.
Meth yl 5-Acetam in oben zofu r an -2-car boxylate (6). Meth-
yl 5-acetaminobenzofuran-2-carboxylate, 6, was made from
methyl 5-nitrobenzofuran-2-carboxylate, 2, using a procedure
similar to that used for synthesis of 5. The yield was 100%.
An analytical sample was recrystallized in ethyl acetate, mp
158-159 °C. 1H NMR (DMSO-d6, ppm): 10.03 (s, 1H, NH),
8.19-8.18 (d, 1H, J ) 1.6 Hz, Ar-H), 7.76-7.75 (d, 1H, J )
1.1 Hz, Ar-H), 7.65-7.63 (d, 1H, J ) 9.0 Hz, Ar-H), 7.55-
7.52 (dd, 2H, J ) 2.3, 8.9 Hz, Ar-H), 3.89 (s, 1H, OCH3), 2.07
(s, 3H, CH3CO). Anal. (C12H11NO4) C, H, N.
5-Aceta m in oin d ole-2-ca r boxylic Acid (7). A sodium
hydroxide solution (3 N, 2 mL) was added to a solution of 5
(250 mg, 1.02 mmol) in methanol (7 mL) and the reaction
mixture was stirred overnight at room temperature. Methanol
was removed, and water (5 mL) was added. The solution was
acidified to pH 2 using 20% hydrochloric acid. The precipitate
was filtered and washed with water. Compound 7 was obtained
as a gray powder (158 mg, 71% yield), mp 260 °C dec. 1H NMR
(DMSO-d6, ppm): 11.62 (s, 1H, NH), 9.77 (s, 1H, NH), 7.98-
7.97 (d, 1H, J ) 1.4 Hz, Ar-H), 7.36-7.28 (m, 2H, Ar-H),
7.02 (d, 1H, J ) 1.5 Hz, Ar-H), 2.03 (s, 3H, CH3CO). Anal.
(C11H10N2O3‚0.6H2O) C, H, N.
5-Aceta m in oben zofu r a n -2-ca r boxylic Acid (8). A so-
dium hydroxide solution (3 N, 2 mL) was added to a solution
of 6 (302 mg, 1.3 mmol) in methanol (20 mL) and the reaction
mixture was stirred for 48 h at room temperature. Solvent was
evaporated, and water (20 mL) was added. The solution was
acidified to pH 2 using 20% hydrochloric acid. The precipitate
was filtered and washed with water. Compound 8 was obtained
1
as a gray powder (231 mg, 81% yield), mp >300 °C. H NMR
(DMSO-d6, ppm): 13.30 (s, 1H, COOH), 10.01 (s, 1H, NH),
8.16-8.15 (d, 1H, J ) 1.9 Hz, Ar-H), 7.65-7.60 (m, 2H, Ar-
H), 7.53-7.50 (dd, 2H, J ) 1.6, 8.4 Hz, Ar-H), 2.07 (s, 3H,
CH3CO). Anal. (C11H9NO4‚0.3H2O) C, H, N.
5-[(5-Aceta m in o-1H-in d ol-2-ylca r bon yl)a m in o]-1H-in -
d ole-2-ca r boxylic Acid (11). EDCI (268 mg) was added to a
solution of 3 (94 mg, 0.46 mmol) and 7 (101 mg, 0.46 mmol) in
DMF (3 mL), and the reaction mixture was stirred overnight

1546 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Wang et al.
was added, and the solution was washed subsequently with
20 mL of water, sodium hydrogen carbonate solution and brine.
The solution was dried using sodium sulfate, and solvent was
removed in vacuo. To the flask was added ether, and the solid
was filtered and washed with ether to produce 150 mg of white
powder 16 (68% yield from 14). Without further purification,
ester 16 (100 mg, 0.23 mmol) was dissolved in DMF (3 mL),
and a 3 N sodium hydroxide solution (2 mL) was then added.
The reaction mixture was stirred overnight at room temper-
ature. Solvent was removed, and water was added to give a
suspension, which was then filtered. Hydrochloric acid (10%)
was added to the filtrate and the precipitate was filtered. The
resulting solid was washed with water and dried to afford an
off-white solid (12 mg, 13%). 1H NMR (DMSO-d6, ppm): 11.72
(br, 1H, NH), 10.37, (s, 1H, NH), 10.01 (s, 1H, NH), 8.18-7.10
(m, 8H, Ar-H), 2.34-2.30 (t, 2H, J ) 7.0, 14.4 Hz, CH2CH2-
CH3), 1.68-1.62 (m, 2H, CH2CH2CH3), 0.96-0.92 (t, 3H, J )
7.4, 14.7 Hz, CH2CH2CH3). Anal. (C22H18N3O5) C, H, N.
removed in vacuo to produce a gray solid (97 mg, 100% yield).
An analytical sample was recrystallized in ethyl acetate, mp
200 °C dec. 1H NMR (DMSO-d6, ppm): 11.80 (s, 1H, NH), 9.84
(s, 1H, NH), 9.81 (s, 1H, CHO), 8.09 (s, 1H, Ar-H), 7.38-7.32
(m, 3H, Ar-H), 2.04 (s, 3H, CH3). Anal. (C11H10N2O2‚0.4H2O)
C, H, N.
5-Aceta m in o-2-in d olea cr ylic Acid (23). Compound 21
(140 mg, 0.7 mmol) was added to a solution of methyl
(triphenylphosphoranylidene)acetate (257 mg, 0.77 mmol) in
toluene (30 mL), and the reaction mixture was heated to reflux
for 3 days. Solvent was removed after the mixture was cooled
to room temperature. Ethyl acetate (10 mL) was added to the
residue, and the resulting precipitate was filtered, MS (ion
spray, M + H) 259. Without further purification, the product
was dissolved in DMF (3 mL) and methanol (5 mL). Sodium
hydroxide solution (3 N, 2 mL) was added, and the reaction
mixture was stirred overnight. Methanol was removed, and
water (10 mL) was added. The solution was acidified to pH 2
using 20% hydrochloric acid. The resulting precipitate was
filtered and washed with water. The product (44 mg, 26%
yield) was obtained as a yellow solid, mp 238-239 °C. 1H NMR
(DMSO-d6, ppm): 12.25 (brs, 1H, COOH), 11.42 (s, 1H, NH),
9.74 (s, 1H, NH), 7.88 (s, 1H, Ar-H), 7.55-7.50 (d, 1H, J )
16.0 Hz, CHdCH), 7.28 (s, 2H, Ar-H), 6.80 (d, 1H, J ) 2.0
Hz, Ar-H), 6.44-6.40 (d, 1H, J ) 15.8 Hz, CHdCH), 2.03 (s,
3H, CH3). Anal. (C13H12N2O3‚0.3H2O) C, H, N.
2-Hyd r oxym eth yl-5-n itr oin d ole (18). Concentrated sul-
furic acid (1.27 mL) was added dropwise to lithium aluminum
hydride (1.89 g) in THF (100 mL) at 0 °C under nitrogen. The
reaction mixture was stirred for 20 min at 0 °C after the
addition was complete. A solution of 1 (2 g, 8.5 mmol) in THF
(80 mL) was then added slowly. The reaction mixture was
stirred for an additional 30 min at 0 °C. Ice (10 g) was added
carefully, and the mixture was filtered. The filter cake was
washed with ethyl acetate (200 mL). The mixture was then
washed with water (50 mL). The organic phase was dried using
sodium sulfate and filtered. Solvent was removed in vacuo to
produce a gray solid (1.48 g, 90% yield). An analytical sample
Compounds 25-29 were synthesized using a procedure
similar to that described below for the synthesis of 25.
3-[(5-Aceta m in o-1H-in d ol-2-yl)ca r bon yl]-1-(ch lor om e-
th yl)-5-h yd r oxy-1,2-d ih yd r o-3H-ben z[e]in d ole (25). An-
hydrous hydrochloride in ethyl acetate (3 N, 4 mL) was added
to CBI (20 mg, 0.1 mmol), which was synthesized according a
reported procedure.22 The reaction mixture was stirred for 30
min at room temperature in the dark. Solvent was removed
to produce 24. The latter was dissolved in DMF (1 mL).
Compound 7 (23 mg, 0.11 mmol) was added, followed by the
addition of EDCI (58 mg). The reaction mixture was stirred
overnight at room temperature. The product was purified by
thin-layer chromatography eluting with ethyl acetate. A gray
powder 25 was obtained (9.5 mg, 22% yield). 1H NMR (DMSO-
d6, ppm): 11.57 (s, 1H, NH), 10.52 (s, 1H, OH), 9.89 (s, 1H,
NH), 8.25-7.23 (m, 9H, Ar-H), 4.90-4.85 (dd, 1H, J ) 9.6,
11.0 Hz, NHH), 4.75-4.71 (dd, 1H, J ) 1.8, 10.6 Hz, NHH),
4.35-4.28 (m, 1H, ClCH2CHCH2), 4.14-4.10 (dd, 1H, J ) 3.4,
11.1 Hz, CHHCl), 3.97-3.92 (dd, 1H, J ) 8.0, 11.1 Hz, CHHCl),
2.12 (s, 3H, CH3). FABHRMS: calcd for (C24H21ClN3O3)
434.1271, found 434.1256.
1
was recrystallized in ethyl acetate, mp 156-157 °C. H NMR
(DMSO-d6, ppm): 11.77 (brs, 1H, NH), 8.49-8.48 (d, 1H, J )
3.5 Hz, Ar-H), 7.97-7.93 (dd, 1H, J ) 2.4, 9.0 Hz, Ar-H),
7.49-7.46 (d, 1H, J ) 9.2 Hz, Ar-H), 6.57 (s, 1H, Ar-H),
5.42-5.39 (t, 1H, J ) 5.6 Hz, OH), 4.66-4.64 (d, 1H, J ) 5.5
Hz, CH2OH). Anal. (C9H8N2O3) C, H, N.
5-Am in o-2-h yd r oxym eth ylin d ole (19). To a solution of
18 (862 mg, 4.49 mmol) in methanol (50 mL) was added 5%
Pd/C (50 mg). The reaction mixture was hydrogenated for 1 h
at a pressure of 50 lb/in.2. The reaction mixture was filtered
through Celite, which was washed with methanol. Solvent was
removed in vacuo, and 707 mg (97% yield) of gray powder was
obtained, mp 159-160 °C. 1H NMR (DMSO-d6, ppm): 10.44
(s, 1H, NH), 7.01-6.99 (d, 1H, J ) 8.5 Hz, Ar-H), 6.60 (d,
1H, J ) 2.0 Hz, Ar-H), 6.43-6.40 (dd, 1H, J ) 2.4, 8.5 Hz,
Ar-H), 5.97 (brs, 1H, Ar-H), 5.07 (brs, 1H, OH), 4.50 (brs,
2H, CH2OH), 4.30 (brs, 2H, NH2). Anal. (C9H10N2O) C, H, N.
3-[[5-[(5-Aceta m in o-1H-in d ol-2-ylca r bon yl)a m in o]-1H-
5-Aceta m in o-2-h yd r oxym eth ylin d ole (20). To a solution
of 19 (200 mg, 1.25 mmol), (dimethylamino)pyridine (20 mg)
and triethylamine (0.94 mL) in THF (10 mL) cooled to 0 °C
under nitrogen, was added a solution of acetyl chloride (0.30
mL, 4.16 mmol) in THF (5 mL) dropwise. The reaction mixture
was allowed to warm to room temperature and stirred for 3
h. THF was removed in vacuo, and ethyl acetate (40 mL) was
added. The solution was washed with water (20 mL × 2). The
organic phase was dried using sodium sulfate, and solvent was
removed in vacuo. The residue was dissolved in methanol (5
mL), and 3 N sodium hydroxide solution (1 mL) was added.
The reaction was allowed to proceed overnight. Methanol was
removed, and water (10 mL) was added. The product was
extracted with ethyl acetate (30 mL × 3), and the solvent was
removed in vacuo. The solution was dried using sodium sulfate.
The product was crystallized in ethyl acetate to afford 128 mg
(50% yield) of gray powder, mp 161 °C. 1H NMR (DMSO-d6,
ppm): 10.84 (s, 1H, NH), 9.62 (s, 1 H, NH), 7.75 (d, 1H, J )
2.1 Hz, Ar-H), 7.22-7.10 (m, 2H, Ar-H), 6.20 (s, 1H, Ar-H),
6.43-6.40 (dd, 1H, J ) 2.4, 8.5 Hz, Ar-H), 5.18-5.15 (t, 1H,
J ) 5.5 Hz, OH), 4.57-4.56 (d, 2H, J ) 5.4 Hz, CH2OH), 2.01
(s, 3H, CH3). Anal. (C11H12N2O2) C, H, N.
in d ol-2-yl]ca r bon yl]-1-(ch lor om eth yl)-5-h yd r oxy-1,2-d i-
1
h yd r o-3H-ben z[e]in d ole (26). Gray powder (80% yield). H
NMR (DMSO-d6, ppm): 11.63 (s, 1H, NH), 11.61 (s, 1H, NH),
10.51 (s, 1H, OH), 10.12 (s, 1H, NH), 9.84 (s, 1H, NH), 8.38-
7.29 (m, 13H, Ar-H), 4.92-4.86 (dd, 1H, J ) 9.6, 11.0 Hz,
NHH), 4.77-4.74 (dd, 1H, J ) 1.8, 10.6 Hz, NHH), 4.36-4.28
(m, 1H, ClCH2CHCH2), 4.15-4.11 (dd, 1H, J ) 3.0, 10.8 Hz,
CHHCl), 3.98-3.93 (dd, 1H, J ) 7.9, 11.0 Hz, CHHCl), 2.04
(s, 3H, CH3). FABHRMS: calcd for (C33H27ClN5O4) 592.1752,
found 592.1733.
3-[[5-[(5-Aceta m in o-1H-ben zofu r a n -2-ylca r bon yl)a m i-
n o]-1H-in d ol-2-yl]ca r bon yl]-1-(ch lor om eth yl)-5-h yd r oxy-
1,2-dih ydr o-3H-ben z[e]in dole (27). Gray powder (53% yield).
1H NMR (DMSO-d6, ppm): 11.66 (s, 1H, NH), 10.51 (s, 1H,
OH), 10.46 (s, 1H, NH), 10.10 (s, 1H, NH), 8.42-7.30 (m, 13H,
Ar-H), 4.92-4.88 (t, 1H, J ) 10.2 Hz, NHH), 4.77-4.74 (dd,
1H, J ) 1.8, 10.9 Hz, NHH), 4.36-4.28 (m, 1H, ClCH2CHCH2),
4.15-4.11 (dd, 1H, J ) 2.7, 10.7 Hz, CHHCl), 3.98-3.93 (dd,
1H, J ) 7.7, 11.0 Hz, CHHCl). FABHRMS: calcd for (C33H25
ClN4O5) 592.1508, found 592.1506.
-
3-[[5-[(5-Bu tyr am in o-1H-ben zofu r an -2-ylcar bon yl)am i-
n o]-1H-in d ol-2-yl]ca r bon yl]-1-(ch lor om eth yl)-5-h yd r oxy-
1,2-dih ydr o-3H-ben z[e]in dole (28). Gray powder (42% yield).
1H NMR (DMSO-d6, ppm): 11.75 (s, 1H, NH), 10.45 (s, 1H,
OH), 10.43 (s, 1H, NH), 10.00 (s, 1H, NH), 8.21-7.23 (m, 13H,
Ar-H), 4.85-4.80 (t, 1H, J ) 10.2 Hz, NHH), 4.60-4.56 (m,
5-Aceta m in o-2-in d oleca r boxa ld eh yd e (21). To a solution
of 20 (100 mg, 0.5 mmol) in ethanol (10 mL) was added
manganese dioxide (250 mg), and the reaction mixture was
stirred for 3 h at room temperature. The reaction mixture was
filtered, and the solid was washed with ethanol. Solvent was

Synthesis and Evaluations of CC-1065 Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1547
1H, NHH), 4.26-4.20 (m, 1H, ClCH2CHCH2), 4.06-4.00 (m,
1H, CHHCl), 3.92-3.88 (m, 1 H, CHHCl). FABHRMS: calcd
for (C35H30ClN4O5) 621.1905, found 621.1898.
is defined as the inability to exclude trypan blue. In most
experiments, the cultures were coded and counted blindly. At
least 100 cells were counted for each sample and data are
reported as the percentage of apoptotic cells.
3-[(5-Acetam in o-1H-in dol-2-yl)acr ylyl]-1-(ch lor om eth yl)-
5-h yd r oxy-1,2-d ih yd r o-3H-ben z[e]in d ole (29). Gray pow-
der (13% yield). 1H NMR (DMSO-d6, ppm): 11.69 (s, 1H, NH),
10.52 (s, 1H, OH), 9.82 (s, 1H, NH), 8.28-6.90 (m, 11H, Ar-
H), 4.55-4.54 (d, 1H, J ) 4.7 Hz, NHH), 4.27-4.23 (m, 2H,
NHH, ClCH2CHCH2), 4.12-4.08 (dd, 1H, J ) 3.1, 11.0 Hz,
CHHCl), 3.91-3.85 (dd, 1H, J ) 8.8, 11.1 Hz, CHHCl), 2.12
(s, 3H, CH3). FABHRMS: calcd for (C26H23ClN3O3) 460.1428,
found 460.1417.
An titu m or Scr een in g in Mice. L1210 leukemia cells (105-
cells/mouse, 0.1 mL) were injected ip to male BDF1 mice (6
mice/group) on day 0. Drugs dissolved in DMF and diluted with
a vehicle containing 30% DMSO in 0.5% glucose at different
doses were administered on days 1, 5, and 9 ip. Antitumor
activity was determined by comparing the median survival
time of the treated groups (T) with that of a control group (C)
and was expressed as a percentage of ILS [increase of life span,
where % ILS ) (T/C - 1) × 100]. These calculations considered
dying animals only. Long-term (30 days) survivors were noted
separately. Cyclophosphamide was used as a positive control.
The median number of days the untreated group of mice (given
the vehicle only) died was 7.5.
For the experiment with low number of tumor inoculation,
100 L1210 leukemia cells were injected ip to female CDF1 mice
(10 mice/group) on day 0. Drugs dissolved in DMSO and
diluted with a vehicle containing 30% DMSO in 0.5% glucose
at different doses were administered on days 1, 5, and 9 ip.
Long-term (60 days) survivors were expressed as a percentage
of the total number of mice in that group. Cyclophosphamide
and taxol were used as positive controls. The median number
of days the untreated group of mice (given the vehicle only)
died was 23.
2-[[5-[(5-Aceta m in o-1H-ben zofu r a n -2-ylca r bon yl)a m i-
n o]-1H-in d ol-2-yl]ca r bon yl]-1,2,9,9a -tetr a h yd r ocyclop r o-
p a [c]ben z[e]in d ol-4-on e (30). To a solution of 27 (10 mg) in
DMF (0.5 mL) were added triethylamine (0.5 mL), water (0.5
mL) and acetonitrile (0.5 mL) sequentially. The reaction
mixture was stirred at room temperature for 30 min. Solvent
was removed in vacuo and the product was washed with water.
Ether was added. The solid was filtered and washed with ether
1
to afford 30 (85% yield). H NMR (DMSO-d6, ppm): 11.77 (s,
1 H, NH), 11.45 (s, 1 H, NH),10.28 (s, 1 H, NH), 8.21-6.96
(m, 13 H, Ar-H), 4.66-4.61 (dd, 1 H, J ) 4.6, 10.0 Hz, NHH),
4.52-4.49 (d, 1 H, J ) 10.3 Hz, NHH), 3.05-2.95 (m, 1 H),
2.08 (s, 3 H), 1.77-1.73 (dd, 1H, J ) 4.4, 7.9 Hz), 1.72-1.70
(t, 1H, J ) 4.8 Hz). FABHRMS: calcd for (C33H24N4O5)
556.1741, found 556.1741.
Cell Lin es. The human monocytic leukemia, U937, was
obtained from ATCC and cultured in RPMI-1640 plus 10% FCS
in the absence of antibiotics. The cells were routinely tested
for mycoplasma contamination and always found to be nega-
tive.
Dr u gs. For in vitro studies, drugs were dissolved in DMF
to provide a stock solution of l mg/mL and were stored at -20
°C. For each experiment, drug solutions were freshly prepared
from the stock solution by addition of sterile H2O to afford
concentrations suitable for the experiment.
Cytotoxicity. Cytotoxic effects of the drugs were measured
by inhibition of DNA synthesis. U937 cells in RPMI-1640 plus
10% FCS medium were seeded at 5 × 104 cells/well in a 96-
well plate. Drugs (10 µL) at increasing concentrations were
added to each well and the total volume was adjusted to 0.1
mL/well using the same medium. The plate was incubated for
24 h at 37 °C followed by addition of 10 µL of [3H]thymidine
(20 µCi/mL). The plate was incubated for another 24 h. The
cells were harvested and radioactivity was counted using the
Packard Matrix 96 beta counter. The results are expressed as
the percentage growth inhibition (IC50) calculated as follows:
% growth inhibition (IC50) ) [(total cpm - experimental cpm)/
total cpm] × 100.
B16BL6 melanoma cells were grown in Dulbecco’s modified
Eagle’s complete minimal essential medium supplemented
with 10% heat-inactivated FBS, sodium pyruvate, nonessential
amino acids, 2-fold vitamin soluton, L-glutamine, 100 µg/mL
penicillin and 100 µg/mL streptomycin. Cultures incubated as
monolayers and harvested with EDTA/trypsin. Cells were
washed three times with medium and were adjusted to 107
cells/mL. BDF1 female mice (4-6 week old, 8/group) were used.
Each mouse was inoculated with 106 cells (0.1 mL) ip on day
0. Drugs were dissolved in dimethylacetamide (DMA) and
diluted with a vehicle containing DMA, ceremorphor and water
(1:5:44, v/v). Mice were injected with 0.1 mL of drug prepara-
tion ip on days 1, 5, and 9. Antitumor activity was determined
by comparing the median survival time of the treated groups
(T) with that of a control group (C) and was expressed as a
percentage of ILS [increase of life span, where % ILS ) (T/C
- 1) × 100]. These calculations considered dying animals only.
Adriamycin was used as a positive control. The median
number of days the untreated mice (given the vehicle only)
died was 16.
Hem a t ologica l Mea su r em en t s. Female CDF1 mice (10
mice/group) were used. Drugs were dissolved in DMSO,diluted
with a vehicle containing 30% DMSO in 0.5% glucose, and
administered on day 1 ip. On day 27, blood samples were taken
and hematological measurements were performed.
DNA F r a gm en ta tion . U937 cells (5 × 106 cells) in RPMI
1640 plus 10% FCS were labeled with 20 µCi of [3H]thymidine
for 20 h at 37 °C. Cells were washed three times and
resuspended at 1 × 106 cells/mL in the same medium contain-
ing 2.5% FCS. The cells (25-µL aliquot containing 2.5 × 103
cells/well) were then placed into a 96-well plate. Wells for total
counts received an additional 25 µL of medium, whereas
experimental wells received 10 µL of drug solution and 15 µL
of medium. The plates were incubated for the indicated length
of time at 37 °C. After incubation, 100 µL of 10 mM Tris, 10
mM of EDTA and 0.3% Triton X-100 was added to each well.
Intact DNA was collected on glass fiber filter paper using a
Packard harvester and samples were counted in a scintillation
counter. The percentage DNA release was calculated as
follows: % DNA release ) [(total cpm - experimental cpm)/
total cpm] × 100.
Ack n ow led gm en t. We thank Dr. Gary G. Meadows,
Department of Pharmaceutical Sciences, Washington
State University, Pullman, WA, for determining the
antitumor activity of compound 26 in mice bearing the
B16BL6 melanoma and Dr. Dieter Hermann, Boe-
hringer-Mannheim, Germany, for determining the an-
titumor activity of compounds 26, 28, and 30 in mice
inoculated with 100 L1210 leukemia cells and for
measuring the hematological effects. We also thank
J olande Murray for help with the manuscript. This work
was supported in part by a grant from the National
Institutes of Health (CA79357-01 to Y.W.).
Ap op tosis Assa y. U937 cells were cultured under the same
conditions as for the DNA fragmentation assay, except that
the cells were not labeled with [3H]thymidine. The plates were
incubated at 37 °C, and at different time points aliquots were
removed and examined microscopically in the presence of
trypan blue. Morphologically apoptotic cells are defined as
those exhibiting at least two or more prominent membrane
protuberances. This change occurs prior to cell death, which
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1548 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
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