Synthesis and biological activities of NB-506 analogues modified at the glucose group

Article abstract of DOI:10.1016/S0960-894X(00)00004-4

A new indolocarbazole compound, NB-506 (1), modified at the glucose group yielded a β-D-glucopyranoside, J-107,088 (2), which showed potent anticancer activity. A β-D-ribofuranoside, J-109,534 (3), was found to be 6 times more potent than J-107,088 at inhibiting topoisomerase I. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00004-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 419±422
Synthesis and Biological Activities of NB-506 Analogues Modi®ed
at the Glucose Group
Mitsuru Ohkubo,* Teruyuki Nishimura, Hiroshi Kawamoto, Masato Nakano,
Teruki Honma, Tomoko Yoshinari, Hiroharu Arakawa, Hiroyuki Suda,
Hajime Morishima and Susumu Nishimura
Banyu Tsukuba Research Institute in collaboration with Merck Research Laboratories, Okubo 3, Tsukuba, Ibaraki, 300-2611, Japan
Received 14 October 1999; accepted 16 December 1999
AbstractÐA new indolocarbazole compound, NB-506 (1), modi®ed at the glucose group yielded a b-d-glucopyranoside, J-107,088
(2), which showed potent anticancer activity. A b-d-ribofuranoside, J-109,534 (3), was found to be 6 times more potent than J-
107,088 at inhibiting topoisomerase I. # 2000 Elsevier Science Ltd. All rights reserved.
It is well known that DNA-topoisomerase I is an
attractive target for cancer chemotherapy.1,2 NB-506
(1),3±5 a new indolocarbazole anticancer agent devel-
oped via modi®cation of a natural compound, BE-
13793C (4),6 was found to be a potent topoisomerase I
inhibitor. Previous studies showed that synthetic NB-
506 analogues di€ered in the 6-N-amino formyl group,7
and that the presence of two hydroxyl groups at the
benzene ring8 yielded the most potent anticancer drug,
J-107,088 (2), against human stomach cancer cells,
MKN-45, implanted in mice. This compound had an
extremely broad safety margin.9,10 The potent anti-
cancer activity and wide safety margin of J-107,088 may
be due to its high concentration in the target cancer
cells. In order to enhance intracellular penetration, an
extensive study was conducted of the modi®cation of J-
107,088 at the glucose group. In this paper, we report
the synthesis and biological activities of a series of sugar
analogues of J-107,088, and discuss the in vivo anti-
cancer e€ects of several potent analogues (Fig. 1).
compound 613 as shown in Scheme 1. Most of the key
intermediate glycosides (7) were prepared by method A,
in which a glycosylation reaction with a 1-chloro-O-
benzyl-d-sugar14,15 was carried out using potassium
hydroxide (KOH) as a base in the presence of sodium
sulfate. In method B, the Mitsunobu reaction was very
useful when a halosugar such as d-erythrose or d-man-
nopyranose was unstable under the basic conditions
used in method A.
As shown in Table 1, the stereoselectivity of the glyco-
sylation reaction in method A was almost entirely b-
selective, but in the case of d-allopyranose, poor selec-
tivity (Run 9, a/b=2/3) was found because the b-chlo-
ride was easily inverted to a-chloride by steric hindrance
due to the 1,3-dipolar alignment in the six-membered
ring system. Interestingly, in the case of 2-deoxy-d-glu-
copyranose (Run 15, 16), stereoselectivity was com-
pletely controlled by the glycosylation method. The
reason for this is not clear, but it may be that the b-
conformer of the 1-chloro-2-deoxy-3,4,6-tri-O-benzyl-d-
glucopyranose is more stable than the a-conformer in
method A. The benzyl groups of the 6-N-methyl com-
pounds (7) were removed by hydrogenolysis with palla-
dium hydroxide followed by treatment with 2.0 M
aqueous potassium hydroxide to yield anhydride com-
pound 8. In the case of d-erythrose compound, 2,3-O-
isopropylidene-d-erythrose was used as a sugar source,
and deprotection of isopropylidene of compound 7
occurred at a hydrogenolysis step. Final compounds 2, 3
and 10±29 were obtained by a coupling reaction of 8
with hydrazine 9 in dimethylformamide (DMF) at
Chemistry
A series of sugar analogues of J-107, 088 (2, 3, 9±29)
was synthesized by applying two types of previously
reported11,12 glycosylation reactions starting from
indolocarbazole compound 513 or bisindolylmaleimide
*Corresponding author.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00004-4

420
M. Ohkubo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 419±422
Figure 1.
Scheme 1.
80 ꢀC. The ®nal structures of the analogues were deter-
mined by both 1H NMR and MS.16
Table 1. Results of the glycosylation reaction
Sugara
Method
Yield (%)
a/bb
1
2
3
4
5
6
7
8
Galactopyranose
Glucopyranose
Xylofuranose
Xylopyranose
Allopyranose
A
A
A
A
A
A
B
A
A
A
A
A
A
B
99
89
60
84
52
99
22
99
96
81
62
36
44
35
72
50
1:99
1:49
1:3.3
b onlyc
2:3
b only
1:2
b only
1:6
1:7
2:7
2:5
b only
2:5
a only
b only
Results and Discussion
As shown in Table 2, the b-d-ribofuranose derivative, J-
109,534 (3) was the most potent inhibitor of topo-
isomerase I in both the enzyme assay (Topo-I cleavage
EC50) and cellular assay (K+/SDS EC200). However, in
these sugar analogues, correlation between the enzyme
and cellular topoisomerase I inhibition assay was not
observed, probably due to the di€erence in penetration
into cells. Interestingly, the cytotoxicities of the com-
pounds did not correlate with the cellular assay (K+/
SDS EC200) results, probably because of the presence of
other mechanisms of cytotoxicity. Most of the com-
pounds showed complete selectivity for topoisomerase
II and protein kinase C (PKC); however, the 2-deoxy-d-
ribofuranose derivative 17 showed moderate inhibitory
activity against PKC, perhaps due to structural similar-
ity to adenosine triphosphate (ATP). To determine the
6-Deoxyglucopyranose
Mannopyranose
Arabinofuranose
Ribofuranose
9
10
11
12
13
14
15
16
Allofuranose
Glucofuranose
2-Deoxyribofuranose
Maltose
Erythrose
2-Deoxyglucopyranose
2-Deoxyglucopyranose
A
B
aAll sugars were d-enantiomers and protected with benzyl ether except
erythrose.
bDetermined by HPLC or 1H NMR.
1
cThe isomer was not detectable by H NMR measurement.

M. Ohkubo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 419±422
Table 2. In vitro activities of various compounds
421
R
Topo-Ia Cleavage
EC50 (nM)
Topo-IIa Cleavage
K+/SDSb (P388/S)
EC200 (nM)
PKCc
IC50 (mM)
CTXd P388/S
IC50 (nM)
CTXe MKN-45
IC50 (nM)
EC50 (mM)
10
11
12
13
3
14
15
16
17
18
b-Glucofuranose
b-Allofuranose
20
42
32
100
8
30
17
17
25
40
>50
>50
>50
>50
>50
>50
>50
>50
>50
NTf
1700
2000
430
2600
32
450
60
400
170
NT
>200
>200
120
>200
20
21
>200
>200
3
14
17
5.3
8.5
1.8
19
0.96
12
4.4
2.4
29
70
93
43
b-Arabinofuranose
a-Arabinofuranose
b-Ribofuranose
a-Ribofuranose
b-Xylofuranose
a-Xylofuranose
b-2-Deoxyribofuranose
b-Erythrose
50
130
6.0
68
45
37
12
2
b-Glucopyranose
a-Glucopyranose
b-Allopyranose
51
300
26
26
230
80
140
240
130
140
100
>50
>50
>50
NT
>50
>50
NT
>50
>50
>50
>50
100
450
160
200
23
17
72
20
50
>200
130
150
60
1.5
19
13
18
36
15
7.1
5.6
16
4.8
8.5
33
20
63
23
9.3
42
19
20
21
22
23
24
a-Allopyranose
1600
>10000
930
>10000
3000
1200
6500
300
b-Mannopyranose
a-Mannopyranose
b-Galactopyranose
25 b-2-Deoxyglucopyranose
26 a-2-Deoxyglucopyranose
27 b-6-Deoxyglucopyranose
33
38
50
3.0
3.8
28
b-Xylopyranose
>200
29
b-Maltose
>3000
>50
>10000
>200
3.5
73
aTopoisomerase-mediated DNA cleavage assay was carried out using supercoiled pBR322 plasmid DNA.4
bE€ects on the formation of protein-DNA complex in P388 cells were investigated by the K+/SDS method.4
cHistone II-As was used as a substrate for protein kinase C.4
dCytotoxicity (CTX) against murine leukemia cells (P388) was measured by the colorimetric tetrazolium±formazan method.4
eCTX against human stomach cancer cells (MKN-45) was measured by the colorimetric tetrazolium±formazan method and the sulforhodamine B
dye-staining method.4
fNT: not tested.
e€ect of stereochemistry at the anomeric carbon, a-iso-
mers were also synthesized and tested. As shown in
Table 2, a-isomers were less potent than the corre-
sponding b-isomers. Only one disaccharide analogue
was synthesized, but compound 29 did not inhibit either
topoisomerase I and II or PKC. In general, the furanose
analogues showed greater inhibitory activities against
topoisomerase I than did the pyranose analogues at the
enzyme level, possibly indicating that the sugar moiety
has some interaction with the 2-deoxy-d-ribofuranose
parts of DNA. Some analogues that showed potent
inhibition of topoisomerase I were tested for anticancer
e€ects in mice and compared with J-107,088 (2).
As shown in Table 3, b-d-ribofuranose derivative 3 and
b-d-xylofuranose derivative 16, though more potent
than J-107,088 (2) at inhibiting topoisomerase I, were
less potent with respect to anticancer e€ects against
MKN-45 xenografts in mice. Moreover, their safety
margins were not as wide as that of J-107,088 (2). The
greater anticancer e€ects of the b-isomers relative to
those of the corresponding a-isomers were re¯ected in the
potencies of topoisomerase I inhibition. These studies
revealed that a b-d-glucopyranose compound, J-107,088
(2), showed anticancer activity superior to that of other
glycosides, probably due to good distribution to the
target cancer cells, and had a very wide safety margin.
Table 3. Anticancer e€ects
Noa
R
Topo-I cleavage
EC50 (nM)
K+/SDS
(P388/S) EC200 (nM)
CTX MKN-45
IC50 (nM)
MKN-45
GID75b mg/m2
LD10c mg/m2
Safety margin
LD10 / GID75
d
3
b-Ribofuranose
a-Ribofuranose
b-Xylofuranose
b-Allofuranose
b-Glucopyranose
a-Glucopyranose
b-Allopyranose
8
30
17
42
51
300
26
40
450
60
2000
100
450
160
50
130
6.0
70
4.8
8.5
33
290
540
300
260
45
570
1000
420
2.0
1.9
1.4
1.4
14
15
11
2
19
20
370
>1600
170
990
>36.0
1.5
2.3
110
440
aCompounds were injected intravenously ®ve times/week for 2 weeks, and treatment was initiated when tumors grew to 0.2 cm3 or larger.
bGID75: approximate 75% growth inhibition dose re¯ects the anticancer e€ect on MKN-45 human stomach cancer cells implanted subcutaneously
into a side ¯ank of nude mice.
cLD10: approximate 10% lethal dose at the treatment schedule.
dSafety margin: the ratio LD10/GID75
.

422
M. Ohkubo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 419±422
8. Ohkubo, M.; Nishimura, T.; Honma, T.; Nishimura, I.; Ito,
Acknowledgements
S.; Yoshinari, T.; Arakawa, H.; Suda, H.; Morishima, H.;
Nishimura, S. Bioorg. Med. Chem. Lett. 1999, 9, 3307.
9. Yoshinari, T.; Ohkubo, M.; Fukasawa, K.; Egashira, S.;
Hara, Y.; Matsumoto, M.; Nakai, K.; Arakawa, H.; Mor-
ishima, H.; Nishimura, S. Cancer Res. 1999, 59, 4271.
10. Arakawa, H.; Morita, M.; Kodera, T.; Okura, A.;
Ohkubo, M.; Morishima, H.; Nishimura, S. Jpn. J. Cancer
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We are grateful to Atsushi Hirano, Ikuko Anma, Kat-
sumi Yamamoto, Kensuke Kobayashi, Katsuhisa Kojiri
and Masashi Morita of our institute for their colla-
boration and stimulating discussion. This paper is dedi-
cated to the memory of Dr. Akira Okura, who died
during the course of our research. We are also grateful
to Ms. Donna LeBlanc, Merck & Co., for her critical
reading of this manuscript.
11. Ohkubo, M.; Kawamoto, H.; Ohno, T.; Nakano, M.;
Morishima, H. Tetrahedron 1997, 53, 585.
12. Ohkubo, M.; Nishimura, T.; Jona, H.; Honma, T.; Ito, S.;
Morishima, H. Tetrahedron 1997, 53, 5937.
13. Compounds 5 and 6 were prepared from 6-benzyloxy-
indole and 2,3-dibromo-N-methylmaleimide, see ref.: Ohkubo,
M.; Nishimura, T.; Jona, H.; Honma, T.; Morishima, H. Tet-
rahedron 1996, 52, 8099.
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16. Physical data for a representative compound, J-109,534 [6-
N-(1-hydroxymetyl-2-hydroxy)ethylamino-12,13-dihydro-2,10-
dihydroxy-13-(b-d-ribofuranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-
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1
;
1H NMR (300 MHz, DMSO-d6), d 3.27
(1H, m), 3.49 (4H, m), 3.92±4.12 (4H, m), 4.34 (1H, m), 4.50
(2H, m), 5.22 (2H, m), 5.53 (2H, d, J=2.7 Hz), 6.13 (1H, d,
J=7.5 Hz),6.38 (1H, m), 6.81 (1H, d, J=1.8, 8.4 Hz), 6.87
(1H, d, J=1.8, 8.4 Hz), 7.03 (1H, d, J=1.8 Hz), 7.15 (1H, d,
J=1.8 Hz), 8.81 (1H, d, J=8.4 Hz), 8.93 (1H, d, J=8.4 Hz),
9.75 (1H, brs), 9.87 (1H, brs), 11.22 (1H, brs); HRMS (FAB)
calcd for C28H26N4O10 578.1649, found 578.1655.