Combretoxazolones: Synthesis, cytotoxicity and antitumor activity

Article abstract of DOI:10.1016/S0960-894X(01)00622-9

Two series of combretoxazolones including 3,4-diaryloxazolones (6) and 4,5-diaryloxazolones (7) were synthesized and evaluated for cytotoxicity and antitumor activity. Both series showed strong cytotoxicities against a variety of tumor cell lines. Compound 6g exhibited a significant antitumor activity in BDF1 mice bearing B16 murine melanoma cells with inhibition rates of 67 and 61% at 100 and 30 mg/kg/day, respectively.

Full text of DOI:10.1016/S0960-894X(01)00622-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 3073–3076
Combretoxazolones: Synthesis, Cytotoxicity and Antitumor
Activity
Nguyen-Hai Nam,a Yong Kim,a Young-Jae You,a Dong-Ho Hong,a
Hwan-Mook Kimb and Byung-Zun Ahna,*
aCollege of Pharmacy, Chungnam National University, Taejon 305-764, Republic of Korea
bKorea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Republic of Korea
Received 23July 2001; accepted 17 September 2001
Abstract—Two series of combretoxazolones including 3,4-diaryloxazolones (6) and 4,5-diaryloxazolones (7) were synthesized and
evaluated for cytotoxicity and antitumor activity. Both series showed strong cytotoxicities against a variety of tumor cell lines.
Compound 6g exhibited a significant antitumor activity in BDF1 mice bearing B16 murine melanoma cells with inhibition rates of
67 and 61% at 100 and 30 mg/kg/day, respectively. # 2001 Elsevier Science Ltd. All rights reserved.
Combretastatin A-4 (CA-4, 1), isolated from Com-
bretum caffrum, is one of the most potent antimitotic
agents and binds to tubulin on the colchicine binding
site.1 It shows strong cytotoxicity against a variety of
human cancer cells, including multi-drug resistant cell
lines.2 However, the low water solubility of CA-4 limits
its efficacy in vivo therefore, a water-soluble sodium
phosphate prodrug of CA-4 (2) was prepared and eval-
uated for clinical applications.3 Recently, the first com-
bretastatin A-4 analogues (3, 4) showing potent
antitumor activity in vivo were also described.4 From a
series of SAR studies, it was established that the cis-
orientation of two phenyl rings is essential to strong
cytotoxicity. However, cis-combretastatin analogues are
prone to isomerize to trans-forms during storage and
administration. The trans-forms of these compounds
show dramatic reduction in both antitubulin activity
and cytotoxicity. This prompted the syntheses of a
number of cis-restricted five-membered heterocyclic
analogues of CA-45 (5) (Fig. 1). Perusal of these analo-
gues revealed that there was an apparent lack of oxazo-
lone-type compounds (6, 3,4-diaryloxazolones; 7, 4,5-
diaryloxazolones) that can be readily synthesized. In
this paper we describe the synthesis and evaluation of
cytotoxicity of this compound class, hereafter given a
trivial name of combretoxazolone.
The combretoxazolones 6 were synthesized as shown
in Scheme 1. Acetophenones 86 were brominated to
give a-bromoacetophenones 9 which were converted to
a-hydroxyacetophenones 10 with betaine in moderate
yields. Reacting of 10 with respective arylisocyanates
and subsequent cyclization by refluxing in acetic acid
(AcOH) provided 6 in 60–70% yields. Under the cycli-
zation conditions, a 4-methoxybenzyl (PMB) group
used to protect a phenol in 8e was removed to afford 6e
directly. Reduction of a nitro group in 6f with zinc gave
6g in a good yield. The combretoxazolones 7 were con-
structed as shown in Scheme 2. Coupling of 11 with
various aryl benzaldehyde Ar2CHO7 by a reported
procedure8 gave a-hydroxyketones 12. Reacting of
a-hydroxyketones 12 with PMB-isocyanate and sub-
sequent cyclization provided the intermediates 13. The
N-PMB group was removed by refluxing in tri-
fluoroacetic acid (TFA) for 3h to give the expected
products 7. Under these conditions, a benzyl group used
to protect a phenol in 13e was removed to afford 7e
directly. Compound 7g was obtained from 7f as descri-
bed for 6g. All newly synthesized compounds were fully
characterized by spectral methods such as IR, 1H NMR,
13C NMR and HRMS.9
The synthesized combretoxazolones were evaluated10
against a small panel of tumor cell lines including mur-
ine melanoma (B16), human colon tumor (HCT116),
human breast tumor (MCF-7), human lung carcinomas
(A549) and prostate tumor (PC-3). The results are
summarized in Table 1.
*Corresponding author. Fax: +82-42-821-6566; e-mail: ahnbj@cnu.
ac.kr
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00622-9

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N.-H. Nam et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3073–3076
Figure 1.
Scheme 1. (a) Br2, CHCl3; (b) betaine, EtOH, 60 ꢀC; (c) (i) Ar2NCO, toluene, 80 ꢀC, 3h; (ii) AcOH, rfx, 8 h; (d) Zn, CH 3COOH; (e) 2 N HCl in
dioxane, 3h.
Scheme 2. (a) (i) TMS-CN, ZnI2, THF; (ii) LiHMDS. Ar2CHO, THF, À78 ꢀC; (b) (i) PMB-NCO, toluene, 80 ꢀC, 3h; (ii) AcOH, rfx, 8 h; (c) TFA,
rfx, 3h; (d) Zn, CH 3COOH.

N.-H. Nam et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3073–3076
Table 1. Cytotoxicity of the synthesized combretoxazolones against tumor cell linesa
3075
b
Compd
Cytotoxicity (IC50
,
nM)
A549
Compd
HCT116
Cytotoxicity (IC50, nM)
A549 PC-3
B16
HCT116
MCF-7
PC-3B16
MCF-7
c
6a
6b
6c
6d
6e
6g
CA-4d
1879
—
—
10.5
39.7
3.7
2.9
1.8
—
19.9
64.5
5.9
3.1
4.3
—
7.5
70.6
3.1
2.3
2.5
2.7
7a
7b
7c
7d
7e
7g
CA-4
1390
14.5
89.3101.1
7.4
5.4
—
21.7
—
—
16.9
63 112.1
—
20.1
7.9
11.0
57.4
4.9
1.7
3.2
28.4
78.5 .1
16.7
45.0
5.8
0.9
1.1
1.0
11.2
6.1
3.7
0.9
10.8
7.9
3.8
2.1
7.1
6.4
2.1
2.7
5.7
4.9
2.7
2.4
1.0
0.9
2.7
2.1
aCell lines: B16, murine melanoma; HCT116, human colon tumors; MCF-7, human breast tumors; A549, lung carcinomas and PC-3, prostate
tumors.
bThe concentration produces 50% reduction in cell growth.
cReduction in cell growth by 50% was not reached at the highest concentration assayed (10 mM).
dCA-4, combretastatin A-4, was synthesized as described previously.14
Previously, the Purdue group has studied structure–
activity relationships of CA-4 extensively11,12 and found
that a 3,4,5-trimethoxy group on the A ring was
essential for strong cytotoxicity. They also showed that
the 3-hydroxy group on the B-ring is not necessary for
potent activity.11 Therefore, for ease of preparation, we
chose 3,4,5-trimethoxyphenyl and 4-methoxyphenyl as
Ar1 and Ar2 and synthesized two isomers 6a and 6b. An
assay determined that 6b was active against a variety of
cancer cell lines with IC50 values in a range of 7.5–11
nM while the regio-isomer 6a was found relatively
inactive up to the highest concentration tested (10 mM).
From these results, we fixed Ar2 as 3,4,5-trimethoxy-
phenyl and examined several variations of Ar1 including
3,4-methylendioxyphenyl, 3-hydroxy-4-methoxyphenyl,
2-naphthyl and 3-amino-4-methoxyphenyl. Among the
synthesized combretoxazolone 6b–6g, it was not sur-
prising that compound 6e with Ar1 being 3-hydroxy-4-
methoxyphenyl, identical with B ring of CA-4, and
compound 6g with Ar1 being 3-amino-4-methoxy-
phenyl, identical with B ring of 3 and 4, were the most
potent ones. These two compounds showed comparable
cytotoxicities with CA-4 in all cell lines assayed with
IC50 values as low as 0.9 nM. Compound 6c bearing a
3,4-methylenedioxyphenyl group as Ar1 was the least
potent. Compound 6d with Ar1 being 2-naphthyl
retained much of the activity compared to 6e and 6g.
This result confirmed a surrogative role of a naphthyl
group for B ring of CA-4 reported previously.13
with the results reported previously.5 Moreover the
compounds 6 could adopt more flexible conformations
compared to 7. This conformational flexibility may
allow the correct positioning of the molecules for a
facile binding at the active site residues of receptors,
e.g., tubulins. Details of molecular modeling studies and
tubulin-binding activity of the synthesized compounds
will be reported elsewhere.
Although the synthesized combretoxazolones showed
very potent cytotoxicity in vitro, they proved to be of
limited solubility in aqueous system. Among the syn-
thesized compounds, only 6g and 7g were endowed with
reasonable water solubility. In vivo evaluation15 of a
representative compound 6g in the form of hydrochlo-
ride salt revealed that, when administered to BDF1 mice
bearing B16 murine melanoma cells, 6g inhibited the
growth of tumor mass by 67 and 61% at 100 and 30 mg/
kg/day, respectively (Table 2). At a lower dose (10 mg/
kg/day), 6g showed only a marginal activity with the
inhibition rate of 34%.
In summary, we have presented here the synthesis and
evaluation of cytotoxicity of two series of combretox-
azolones, including 3,4-diaryloxazolones (6) and 4,5-
diaryloxazolones (7). These combretoxazolones showed
potent cytotoxicity against a variety of tumor cell lines.
Structurally, compounds 7 are clearly cis-restricted and
compounds in series 6 can also be viewed as such in
term of position between the two aryl rings, and there-
fore, these analogues should be stable in term of iso-
merization. One of the synthesized combretoxazolones,
compound 6g, displayed a significant in vivo antitumor
activity.
In the series of compounds 7, initially two regio-isomers
7a and 7b were synthesized and evaluated for the cyto-
toxicity. Interestingly, despite the structural similarity
between the two isomers, only 7b showed strong cyto-
toxicity while 7a was found to be relatively inactive up
to the highest concentration tested (10 mM). The order
of cytotoxicity in this series was found to be similar with
6a–6g series; thus, compounds bearing a 3-hydroxy-4-
methoxy (7e) or a 3-amino-4-methoxy (7g) substituted
pattern being most potent while compound 7c, posses-
sing a 3,4-dioxymethylene group on the Ar2 ring was
found to be least cytotoxic. In overall, the compounds
in series 7 were less potent than those in series 6, sug-
gesting that a carbonyl group at position A of the five-
membered ring (Structure 5, Fig. 1) is more favorable
for the strong cytotoxicity. This finding is in consistent
Table 2. Antitumor activity of compound 6g
Compd
Dose (mg/kg/day)
IRa (%)
6g
100
3
67
0
10
355
34
ADRb
aThe inhibition rate; see ref 15.
bAdriamycin, used as a positive control.

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N.-H. Nam et al. / Bioorg. Med. Chem. Lett. 11 (2001) 3073–3076
References and Notes
J=8.8 Hz), 6.79 (1H, dd, J=8.8, 2.3Hz), 6.57 (2H, s), 3.86
(3H, s), 3.72 (3H, s), 3.70 (6H, s). HRMS: [M]+ 373.1173
(C19H19NO7) calcd 373.1161.
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rate=(1ÀTWt/TWc)Â100 (%). TWt, the mean tumor weight
of the treated group; TWc, the mean tumor weight of the
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