Synthesis and cytotoxic activities of a new benzo[c]phenanthridine alkaloid, 7-hydroxynitidine, and some 9-oxygenated benzo[c]phenanthridine derivatives.

Article abstract of DOI:10.1021/ol990775g

[formula: see text] A new benzo[c]phenanthridine alkaloid, 7-hydroxynitidine, was synthesized by a novel synthetic procedure. The cytotoxic activity of this compound against HeLa S3 cells was strong, but not greater than those of its mother compounds, nitidine and NK109. We also synthesized other 9-oxygenated benzo[c]phenanthridine alkaloids, 7-methoxynitidine, 9-demethylnitidine, nitidine, and fagaronine, and tested their cytotoxic activities. These results suggest that the 7-hydroxy group enhances antitumor activity and an 8- or 9-hydroxy group weakens this activity.

Full text of DOI:10.1021/ol990775g

ORGANIC
LETTERS
1999
Vol. 1, No. 7
985-988
Synthesis and Cytotoxic Activities of a
New Benzo[c]phenanthridine Alkaloid,
7-Hydroxynitidine, and Some
9-Oxygenated Benzo[c]phenanthridine
Derivatives
Takeshi Nakanishi* and Masanobu Suzuki
Pharmaceuticals Group, Nippon Kayaku Company, Ltd., 31-12, Shimo 3-chome,
Kita-ku, Tokyo 115-8588, Japan
kendama@kk.iij4u.or.jp
Received June 30, 1999
ABSTRACT
A new benzo[c]phenanthridine alkaloid, 7-hydroxynitidine, was synthesized by a novel synthetic procedure. The cytotoxic activity of this
compound against HeLa S3 cells was strong, but not greater than those of its mother compounds, nitidine and NK109. We also synthesized
other 9-oxygenated benzo[c]phenanthridine alkaloids, 7-methoxynitidine, 9-demethylnitidine, nitidine, and fagaronine, and tested their cytotoxic
activities. These results suggest that the 7-hydroxy group enhances antitumor activity and an 8- or 9-hydroxy group weakens this activity.
Quaternary benzo[c]phenanthridiniums are widely known
alkaloids with some antitumor activities.1 Among them,
nitidine (1a) and fagaronine (1b) have shown the most
promise as practical antitumor drugs and were the subject
of preclinical studies at the National Cancer Institute in the
1970s.2 In 1992, we reported that NK109 (1c) had antitumor
activity3 that was superior to those of 1a and 1b.4 We are
now conducting clinical evaluations of NK109 in Japan. The
superior activity of NK109 is due to its structure under
biological conditions; i.e., NK109 exists as a resonance
hybrid due to the loss of its acid moiety (Scheme 1). The
resonance hybrid retains molecular planarity and a cationic
property, both of which are essential for the expression of
antitumor activity. This structural property of NK109 is
derived from the hydroxy group at the 7-position. We were
interested in a compound with both NK109 and nitidine
substituents and therefore investigated a novel procedure for
synthesizing a new benzo[c]phenanthridine alkaloid, 7-hy-
droxynitidine (1d, Figure 1). Since our procedure can be
(1) Suffness, M.; Cordell, G. A. The Alkaloids; Academic: New York,
1985; Vol. 25, pp 178-189.
(2) (a) Cordell, G. A.; Farnsworth, N. R. Heterocycles 1976, 4, 393-
427. (b) Phillips, S. D.; Castle, R. N. J. Heterocycl. Chem. 1981, 18, 223-
232.
(3) Hanaoka, M.; Ekimoto, H.; Kobayashi, M. F.; Irie, Y.; Takahashi,
K.Chem. Abstr. 1992, 116, 718d.
(4) Nakanishi, T.; Suzuki, M.; Saimoto, A.; Kabasawa, T. J. Nat. Prod.
1999, 62, 864-867.
Figure 1. Structures of benzo[c]phenanthridine alkaloids.
10.1021/ol990775g CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/04/1999

Scheme 1. Equilibrium of 7-Hydroxybenzo[c]phenanthridine Alkaloids
applied to other 9-oxygenated derivatives, we also synthe-
5-position, rather than 6-position, to yield 3e. To change the
reaction site, we introduced an electron-withdrawing group
at the 2-position. First, by treatment with lithium chloride,7
2e was demethylated selectively to give 2-hydroxy-3,4-
dimethoxybenzaldehyde 2d. After phenolic hydroxide was
protected by methyl carbonate, it was treated with bromine.
However, no brominated compound was obtained and the
starting material was recovered. Bromination also did not
proceed when the temperature was increased to 80 °C. Thus,
bromination at the 6-position of 2,3,4-trialkoxy aldehyde was
very difficult. We could not obtain a precursor for the radical
cyclization and did not continue the synthesis by this route.
sized 7-methoxynitidine (1e), 9-demethylnitidine (1f), and
the natural benzo[c]phenanthridine alkaloids 1a and 1b. In
this paper, we report the synthetic investigation and the
cytotoxic activity of these compounds (Table 1).
Table 1. Substituent Patternsa
R1
R2
R3
R4
H
R5
a
b
c
d
e
f
g
h
i
H
H
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OMe
OMe
OH
OMe
OBzl
OMe
-CH2-
Me
OH
OH
OMe
H
OBzl
H
-CH2-
-CH2-
-CH2-
-CH2-
-CH2-
-CH2-
Scheme 3. Attempt To Synthesize the 6-Bromoaldehyde Unita
H
iPr
Me
a This pattern is applied to all compounds.
7,8,9-Trialkoxybenzo[c]phenanthridine alkaloids have never
been isolated from plants and have only been reported by
Ishii et al. as a synthetic compound, 7-methoxynitidine (1e).5
Since it is difficult to apply their method to our target
compound 1d, we decided to develop a procedure for
synthesizing 1d.
We previously established the synthetic procedure for
NK109 outlined in Scheme 2.6 We initially attempted to
obtain a corresponding 6-bromoaldehyde unit for the syn-
thesis of 1d (Scheme 3). When 2,3,4-trimethoxybenzaldehyde
(2e) was reacted with bromine, it was brominated at the
a Reagents and yield: (a) Br2, NaOAc, AcOH, 50 °C; (b) LiCl,
DMSO, 110 °C, 67%; (c) MeOCOCl, Et3N, toluene, rt, 98%; (d)
Br2, NaOAc, AcOH, 50-80 °C.
As noted above, 2e was easily brominated to 3e. We
decided to use 3e as a new aldehyde unit (Scheme 4).
Accordingly, to obtain the aldehyde unit for 1d, phenolic
benzaldehyde 2d was protected by a benzyl group. The
resulting trialkoxybenzaldehyde 2g was converted into the
aldehyde unit 3g (Scheme 5). Synthesis of the aldehyde units
for 1a, 1b, and 1f was much simpler. 5-Bromo-4-hydroxy-
Scheme 2. Synthetic Procedure of NK109 (1c)
(5) Ishii, H.; Ichikawa, Y.; Kawanabe, E.; Ishikawa, M.; Ishikawa, T.;
Kuretanim K.; Inomata, M.; Hoshi, A. Chem. Pharm. Bull. 1985, 33, 4139-
4151.
(6) Nakanishi, T.; Suzuki, M.; Mashiba, A.; Ishikawa, K.; Yokotsuka,
T. J. Org. Chem. 1998, 63, 4235-4239.
(7) Bernard, A. M.; Ghiani, M. R. G.; Piras, P. P.; Rivoldini, A. Synthesis
1989, 287-289.
(8) (a) Stermitz, F. R.; Gillespie, J. P.; Amoros, L. G.; Romero, R.;
Stermitz, T. A. J. Med. Chem. 1975, 18, 708-713. (b) Clark, J. H.; Holland,
H. L.; Miller, J. M. Tetrahedron Lett. 1976, 41, 3361-3364.
986
Org. Lett., Vol. 1, No. 7, 1999

Scheme 4. Synthetic Procedure of 9-Oxygenated Benzo[c]phenanthridine Alkaloids
3-methoxybenzaldehyde 3f is readily available from com-
mercial suppliers, and 3a and 3h were obtained by meth-
ylation and benzylation of 3f, respectively.
5-bromobenzylamine 5 as a precursor instead of 6-bro-
mobenzylamine (precursor of the radical cyclization). In this
study, 5 was lithiated at -78 °C. This intermediate was
allowed to stand at room temperature to give a cyclized
product. Subsequent oxidization with manganese dioxide
gave benzo[c]phenanthridine 6. The resulting reaction mix-
ture contained many unknown byproducts, and the yield of
6 was 17-47%. However, with 5i, the yield of cyclization
reached 74%. Since 6i was precipitated from the reaction
medium, undesirable reactions did not proceed and 6i was
obtained in good yield.
Scheme 5. Synthesis of 5-Bromoaldehyde Unitsa
We also attempted cyclization of 5 by a radical-mediated
reaction. Although 5c was treated with n-Oct3SnH and
azobis(2-methylbutyronitrile),6 no cyclized product was
obtained. The product was mainly a 5-debromo compound.
Thus, 5 was not available for radical cyclization; this
supported the notion that the reaction with LDA proceeded
via benzyne as an intermediate.
Benzo[c]phenanthridine 6 was methylated with methyl
2-nitrobenzenesulfonate. The resulting product was neutral-
ized with sodium hydroxide to remove acid residue. After
purification, it was acidified with dilute hydrochloric acid
to give 1a, 1e, and 1g-i. Finally, the benzyl or isopropyl
group was deprotected with concentrated hydrochloric acid
to give 1b, 1d, and 1f.
a Reagents and yield: (a) BzlCl, KI, K2CO3, MeOH, 80 °C, 98%;
(b) Br2, NaOAc, AcOH, 50 °C, 93%; (c) MeI, K2CO3, DMF, 50
°C, 100%; (d) BzlBr, K2CO3, DMF, 50 °C, 94%.
The aldehyde unit (3) and amine units (4), which were
derived from 2,3-dihydroxynaphthalene,8 were condensed in
toluene to Schiff base. The imine portion of the Schiff base
was reduced to amine with dimethylamineborane9 to give
the key intermediate 5 in good yield (Table 2).
A growth inhibition assay against HeLa S3 cells was
performed as described previously.4 Table 3 shows the
cytotoxic activities of some phenolic benzo[c]phenanthridine
Table 2. Yield of Each Process
Table 3. Cytotoxic Activities of Several
Benzo[c]phenanthridine Alkaloids against HeLa S3
starting materials
product (yield, %)
3
4
5
6
1
phenolic
alkaloid
(IC50
µg/mL)
,
methylated
alkaloid
(IC50,
3a
3a
3g
3e
3h
4a
4i
4a
4a
4a
5a (76)
5i (58)
5g (79)
5 (83)
6a (17)
6i (74)
6g (17)
6e (29)
6h (47)
1a (55)
µg/mL)
1i (65), 1b (92)
1g (82), 1d (99)
1e (77)
1d
1f
0.23
>10
1e
1a
0.82
0.05
1.24
1.24
fagaridinea
1c
1b
3.99
0.08
0.39
chelerythrineb
chelerythrineb
5 (70)
1h (66), 1f (92)
a Synthesized in our laboratory (R1 ) OMe, R2 ) OH, R3 ) H, R4 +
R5 ) -CH2-).11 b Perchased from Sigma Chemical Company (R1 ) R2 )
OMe, R3 ) H, R4 + R5 ) -CH2-).
We previously examined the cyclization of the precursor
of NK109 with LDA.6 This reaction proceeds via benzyne
as an intermediate. Accordingly, it is reasonable to use
Org. Lett., Vol. 1, No. 7, 1999
987

alkaloids compared with those of the corresponding meth-
ylated derivatives. The derivative with a hydroxy group at
the 9-position had no activity (1f), and that with 8-OH had
merely weaker activity (fagaridine). On the other hand, that
with 7-OH showed stronger activity (1c). Compound 1c has
been reported to possess a unique property in its structure
due to its 7-OH,4 and the cytotoxic activity of 1d is greater
than that of 1e. These facts suggest that the effect of the
7-hydroxide of 1d is similar to that of 1c. We measured UV
spectra of 1d under several pH conditions and observed some
isosbestic points. The results show that 1d is in acid-base
equilibrium, the same as 1c (Scheme 1).10 We determined
the pKa value of 1d (5.5) by an absorbance method. On the
other hand, no isosbestic point was observed in UV spectra
of phenolic alkaloid 1f. This suggests that 1f dose not exhibit
acid-base equilibrium like 1c and 1d. This could explain
why 1f had no cytotoxic activity.
In conclusion, we have established a new procedure for
synthesizing 7,8,9-trioxygenated-benzo[c]phenanthridiniums,
which have not yet been isolated from plants. This procedure
is very useful, since the aldehyde units are readily available;
they were easily obtained either by bromination of the
corresponding benzaldehyde or by alkylation of a 5-bromo
aldehyde obtained from commercial sources. The benzo[c]-
phenanthridine rings were constructed via benzyne as
intermediates which were produced by treatment of substi-
tuted benzylamine with LDA. The yield of this reaction was
about 20%, while the yield of 6i, an intermediate of
fagaronine, was excellent. Accordingly, our procedure may
also be a practical approach to fagaronine. Subsequently, we
tested the cytotoxicity of our synthetic benzo[c]phenanthri-
diniums 1d-f and confirmed the effect of the 7-hydroxide
on the antitumor activity of benzo[c]phenanthridiniums.
While 1d showed strong antitumor activity, it was not greater
than those of its mother compounds nitidine and NK109.
(9) Billman, J. H.; McDowell, J. W. J. Org. Chem. 1961, 26, 1437-
1440.
1
(10) NMR data of 1d: quaternary cation, H NMR (DMSO-d6) δ 3.87
(s, 3H), 4.23 (s, 3H), 4.86 (s, 3H), 6.34 (s, 2H), 7.75 (s, 1H), 7.96 (s, 1H),
8.25 (d, 1H, J ) 9.1 Hz), 8.27 (s, 1H), 8.83 (d, 1H, J ) 9.1 Hz), 9.85 (s,
1H), 11.65-11.80 (br s, 1H); 13C NMR (DMSO-d6) δ 51.7, 57.9, 61.5,
96.9, 103.2, 104.9, 106.1, 112.3, 119.9, 120.5, 124.3, 130.3, 133.1, 133.2,
133.3, 136.6, 148.9, 149.2, 149.4, 150.4, 163.2; resonance hybrid, 1H NMR
(DMSO-d6) δ 3.72 (s, 3H), 4.01 (s, 3H), 4.46 (s, 3H), 6.26 (s, 2H), 7.03 (s,
1H), 7.59 (s, 1H), 7.94 (d, 1H, J ) 9.0 Hz), 8.03 (s, 1H), 8.42 (d, 1H, J )
9.0 Hz), 8.99 (s, 1H); 13C NMR (DMSO-d6) δ 48.2, 56.0, 58.4, 88.3, 102.0,
103.4, 105.1, 117.2, 119.3, 120.1, 123.6, 127.1, 130.9, 131.7, 132.6, 137.7,
147.5, 147.7, 148.2, 161.0, 168.8.
Supporting Information Available: Synthetic procedures
for all compounds and 1H NMR and HPLC charts of
evaluated compounds 1a-f. This material is available free
of charge via the Internet at http://pubs.acs.org.
(11) Nakanishi, T.; Suzuki, M. J. Nat. Prod. 1998, 61, 1263-1267.
OL990775G
988
Org. Lett., Vol. 1, No. 7, 1999