Synthesis and structure-activity relationship studies of cytotoxic vinorelbine amide analogues

Article abstract of DOI:10.1016/j.bmcl.2012.10.024

A series of 3-demethoxycarbonyl-3-acylamide methyl vinorelbine derivatives (compounds 7a-7z) were designed, synthesized, and evaluated for their inhibition activities against human non-small cell lung cancer cell line (A549). Most of the amide derivatives

Full text of DOI:10.1016/j.bmcl.2012.10.024

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7547–7550
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationship studies of cytotoxic
vinorelbine amide analogues
Lingjun Hu b, , Weibin Song a, , Yuhui Meng a, Dean Guo a, Xuan Liu a, , Lihong Hu a,b,
⇑
⇑
a Shanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
b College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-demethoxycarbonyl-3-acylamide methyl vinorelbine derivatives (compounds 7a–7z) were
designed, synthesized, and evaluated for their inhibition activities against human non-small cell lung
cancer cell line (A549). Most of the amide derivatives exhibited potent cytotoxicity, with the size of
the introduced substituents being the foremost factor in determining the resultant cytotoxic activity. Test
results in vivo against nude mice bearing A549 xenografts indicated that 7y showed comparable activities
compared to the parent NVB.
Received 4 September 2012
Revised 29 September 2012
Accepted 4 October 2012
Available online 17 October 2012
Keywords:
Vinorelbine
Ó 2012 Elsevier Ltd. All rights reserved.
C-22 amide analogues
Tubulin inhibitor
Structure–activity relationship (SAR)
Cytotoxicity
Vinblastine (1a) and vincristine (1b), isolated at the end of the
1950s from the periwinkle plant Catharanthus roseus,1 were a
group of antimitotic drugs inhibiting tubulin polymerization into
microtubules and inducing microtubule depolymerization, thereby
resulting in mitotic arrest of eukaryotic cells and eventually lead-
ing to apoptosis.2 Despite their similarities in structure and func-
tion, however, vinblastine and vincristine exhibited a significant
difference in clinical use and dose-limiting toxicities.3 Therefore,
since the early 1970s, numerous efforts in the fields of both chem-
istry and biology have been undertaken in an effort to obtain more
effective and less toxic Vinca alkaloid-type analogues exhibiting a
wider spectrum of antitumor efficacy.
Recently, our group have made further efforts to study the SAR
of vindoline C-22 site and synthesized a variety of C-22 substituted
carbamates, ether, ester and amide anhydrovinblastine analogues,
among which some of the amide anhydrovinblastine analogues
showed improved in vitro potency and in vivo efficacy compared
to positive control vinorelbine.8 In addition, the vinblastine-
binding studies showed that the vindoline moiety of C-22 site
was involved in the binding interaction with tubulin heterodi-
mers.9 These encouraging results indicate that synthesis of C-22
substituted amide vinorelbine analogues seems desirable to us to
find new drug-like candidates with the desired characteristics.
To this day, these efforts have lead to three approved drugs,
vindesine (1c),4 vinorelbine (1d),5 vinflunine (1e).6 Of note,
vinorelbine, which was a semi-synthetic drug produced by biomi-
metic coupling of vindoline7 with catharanthine and subsequent
ring contraction of velbenamine moiety, showed markedly
improved clinical efficacy and reduced toxicity, and is now widely
used in the treatment of non-small cell lung cancer, metastatic
breast cancer and ovarian cancer. These very important results
prompt us to elaborate new vinorelbine analogues with better
anticancer therapeutic indices (Fig. 1).
R4
N
n
R6
R5
H
N
N
H
Et
OR3
MeOOC
OH
1
MeO
N
4
3
H
R1 COR2
22
R1
R2
R3
R4 R5
R6
n
1
1
1
0
0
1
1a CH3 OCH3 COCH3 OH
1b CHO OCH3 COCH3 OH
H
H
H
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CF2CH3
CH2CH3
⇑
Corresponding authors. Tel./fax: +86 21 2023 1965 (L.H); tel.: +86 2023 2221
(X.L.).
1c CH3
NH2 COCH3 OH
1d CH3 OCH3 COCH3
1e CH3 OCH3 COCH3
1f CH3 OCH3 COCH3
=
=
=
E-mail addresses: xuanliu@mail.shcnc.ac.cn (X. Liu), simmhulh@mail.shcnc.ac.cn
(L. Hu).
These authors contributed equally to this work and should be considered co-first
authors.
Figure 1. Structures of known Vinca alkaloids.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.024

7548
L. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7547–7550
N
N
N
Four steps
a/b, c
Et
Et
Et
OH
OH
OH
O
N
H
OR'
Ref
O
N
OAc
O
N
OH
H
H
H
CO2CH3
NHCOR
NH2
5a-5z
2
4
Et
Et
N
N
H
N
N
3 Catharanthine
e
N
N
H
d
H
Et
Et
OR'
NHCOR
MeOOC
MeOOC
OH
OH
O
N
OR'
O
N
H
H
R' = Pro, Ac
R = Alkyl, aryl
NHCOR
6
7a-7z
Scheme 1. Reagents and conditions: (a) RCOCl, 33% CH3COONa aqueous solution, THF; (b) RCOOH, EDCI-HOBT, CH2Cl2, 0 °C to rt, 12 h; (c) Pyridine, Ac2O, rt, 8 h; (d) i—FeCl3,
aq NaHSO4–CF3CH2OH (pH 1.3), 25 °C, 8 h; ii—NaBH4, NH4OH, 0 °C, 30 min; (e) i—NBS, DEA, CH2Cl2, ꢀ70 °C, 1 h; ii—AgBF4, THF-H2O, 50 °C, 3 h.
Table 1 (continued)
Table 1
R0
R
A549a IC50 (nM)
6.3 0.1
Cytotoxic activity of vinorelbine analogues 7a–7z
Compound
Compound
R0
R
A549a IC50 (nM)
7t
Ac
NVB
7a
—
—
9.0 0.1
O
O
Ac
Ac
Ac
Ac
24.5 0.1
15.6 0.2
17.3 0.2
22.1 0.2
7b
7u
7v
7w
7x
Ac
Ac
Ac
Ac
6.5 0.1
O
Cl
7c
7d
25.6 0.3
20.7 0.4
41.1 0.5
7e
7f
Ac
Ac
Ac
11.4 0.1
22 0.2
37 0.4
CF3
NO2
7g
7h
7i
Ac
3.0 0.1
7y
7z
Ac
Ac
3.2 0.1
8.1 0.1
Pro
Ac
12.0 0.1
16.2 0.3
N
7j
a
7k
7l
Ac
Ac
Ac
5.0 0.1
Ditartrate of all compounds was used in bioassays.
15.3 0.1
17.2 0.1
Herein, we focus on the synthesis of C-22 amide vinorelbine ana-
logues and the evaluation of their potency in vitro and in vivo as
part of an extended SAR study.
7m
The preparation of the amide vinorelbine analogues was illus-
trated in Scheme 1. First, the initial substrate vindoline was con-
verted into the key intermediate amine 4 in good yield according
to a previously reported four-step procedure.8a Then, 4 was treated
with different acyl chloride in an aqueous tetrahydrofuran
solution, and/or different carboxylic acid in the presence of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
and 1-hydroxybenzotriazole (EDCI-HOBt) in dry dichloromethane
(DCM) to afford the corresponding N-acylation products, which
in turn, without purification, were further acetylated at 4-OH to
give the amide vindoline analogues 5a–5z. Later, each of these
amide vindoline analogues was coupled with catharanthine (3)
via a modified Potier/Kutney–Polonovski reaction to furnish the
amide anhydrovinblastine intimidates 6a–z,10 and which were
subsequently treated with the one-pot, two-step procedure of
bromination with NBS/CF3COOH at ꢀ70 °C followed by ring
contraction in the presence of AgBF4/H2O at 50 °C for 3 h, produced
the corresponding targeted vinorelbine analogues 7a–7z.11
F
7n
Ac
6.6 0.1
F
F
7o
7p
Ac
Ac
7.3 0.1
7.1 0.1
F
7q
Ac
7.9 0.1
F
O
7r
7s
Ac
Ac
6.1 0.1
6.1 0.1
O

L. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7547–7550
7549
Table 2
Anti-tumor efficacy of compounds 7h, 7k, 7r and 7ya against non-small cell lung cancer A549 xenografts in nude mice
Group
Dosage (mg/kg)
Mice (n)
Body weight (g)
Tumor volume (mm3) X SD
Relative TV (X SD)
Inhibition rate (%)
Initial/end
Initial/end
Initial
End
NS
7h
7k
7r
7y
NVB
—
9
9
9
9
9
6/6
6/6
6/6
6/6
6/5
6/6
26.03/27.61
25.10/26.53
25.00/24.60
25.02/25.32
25.13/25.45
25.92/26.22
262.75 15.42
258.04 22.92
259.30 5.63
260.15 5.89
260.46 18.87
262.76 24.79
1341.72 71.26
1057.20 27.33
750.34 96.01
766.08 22.15
797.27 38.56
743.91 48.82
5.17 0.21
4.24 0.34
2.92 0.12
2.91 0.21
3.00 0.31
2.89 0.17
17.99*
43.52*
43.71*
58.02*
55.90
a
The in vivo experiment was carried out in the nude mice bearing A549 xenografts and the compounds were intravenously given. Ditartrate of all compounds was used in
bioassays.
*
P < 0.01 versus control group.
The series of C-22 amide vinorelbine analogues were evaluated
for their antiproliferative activity in human non-small-cell lung
cancer (A549 cell) using the MTT cell viability assay,12 vinorelbine
1d was employed as standard for comparison purposes. The IC50
values for the inhibition of proliferation of the A549 cell line were
shown in Table 1.
Based on the in vitro activity, four C-22 amide vinorelbine ana-
logues (7h, 7k, 7r and 7y) were selected and tested further for their
antitumor efficacy in vivo. These three analogues were adminis-
tered by giving three doses intravenously (iv) once every seven
days (q7d ꢁ 3) at the doses of 9 mg/kg in nude mice bearing
A549 xenografts in comparison with NVB, and the results of the
in vivo study were summarized in Table 2.
Encouragingly, two compounds 7a and 7k, which were initially
synthesized for a preliminary prediction, showed desired potency
against A549 cell (IC50 = 24.5 and 6.0 nM, respectively) compared
to positive control NVB (IC50 = 9.0 nM). This result clearly indicated
that it was possible to improve the cytotoxicity by introducing dif-
ferent alkyl/aryl substituted amide groups. So, we first attempted
to study the alkyl-substituted effects on cytotoxicity, and the
results showed that the propionylamine compound 7b exhibited
an acceptable potency against A549 cell (IC50 = 15.6 nM), while
decreasing and/or increasing length of linear alkyl chains within
2, 4 and 5 carbons length showed decreased cytotoxic activity
(IC50 = 24.5, 17.3 and 22.1 nM, corresponding to 7a, 7c and 7d).
When the acetoxy at C-4 site of 7b was replaced with propionyloxy
to afford compound 7i (IC50 = 12.0 nM), an improved potency
against A549 cell was observed compared to 7b. Moreover, increas-
ing the steric hindrance of the substituent at the C-22 position led
to a gradual decrease in the cytotoxicity of the A549 cell line
(IC50 = 11.4, 22.0 and 37.0 nM, corresponding to 7e–7g). Particu-
larly, the cyclopropane carboxamide compound 7h was found to
be 3 times more potent against the A549 cell line (IC50 = 3.0 nM)
compared to the positive control NVB (IC50 = 9.0 nM), while all
the other alkyl-substituted analogues (7a–7g and 7i) displayed less
inhibitory potency than the positive control NVB.
As shown in Table 2, compounds 7y, 7k, and 7r showed compa-
rable or slightly lower antitumor activities in vivo (Inhibition
rate = 58.02, 43.52 and 43.71%, respectively) compared to the posi-
tive control NVB (Inhibition rate = 55.90%), while the compound 7h
displayed drastically reduced antitumor activity (Inhibition
rate = 17.99%). At the same time, compound 7y showed higher tox-
icity than that of NVB, for example, one mice died when treated
with 7y during the administration period. Generally, the amide
groups substituted at C-22 site have great influence on the antitu-
mor activity in vivo.
In conclusion, a series of C-22 amide vinorelbine analogues
(7a–7z) were designed, synthesized and evaluated for their anti-
proliferative activity in human non-small-cell lung cancer (A549
cell). The SAR information collected so far suggested that differ-
ent alkyl-substitution and aryl-substitution at the 22-position
had important influence on cytotoxic activity. Compounds 7h,
7k, 7r and 7y exhibited enhanced cytotoxic activities against
A549 cell line. The preliminary antitumor studies in vivo
indicated that 7y showed comparable activities compared to
the parent NVB.
Acknowledgments
Building on the result that the benzamide compound 7k
(IC50 = 6.0 nM) exhibited enhanced activity toward A549 cell com-
pared to NVB, we next turned our attention to study the substitu-
ent effects on the phenyl ring by introducing different substituents
at various positions and using benzyl and heteroaryl rings. The re-
sults showed that introduction of electron withdrawing [2/3/4-F,
2,6-(F)2] (IC50 = 6.6, 7.3, 7.1 and 7.9 nM, corresponding to 7n–7q)
or electron donating [2/3-OMe, 3,4-(OMe)2] (IC50 = 6.1, 6.1, 6.3
and 6.5 nM, corresponding to 7r–7u) groups on the phenyl group
showed retained potency against A549 cell compared to benzam-
ide compound 7k, while introduction of electron withdrawing (4-
Cl, 4-CF3, 4-NO2) (IC50 = 25.6, 20.7 and 40.1 nM, corresponding to
7v–7x) or electron donating (3/4-Me) (IC50 = 15.3 and 17.2 nM,
corresponding to 7l and 7k) groups on the phenyl ring showed sig-
nificantly decreased potency. In addition, no distinct effect on cyto-
toxic activity against the A549 cell was observed due to the
positions of F or OMe in the phenyl ring (7n–7u). The electron-rich
heteroaryl amide compounds 7y–7z (IC50 = 3.2 and 8.1 nM) dis-
played better potency against A549 than positive control NVB,
whereas the benzyl-substituted derivative 7j (IC50 = 16.2 nM) had
less potency. These results indicated that aryl substituents were
more suitable than benzyl substituent to maintain the potent cyto-
toxic activities.
This work was supported by the National Natural Science
Foundation of China (grants 30925040, 81102329, 81273430),
the Chinese National Science & Technology Major Project ‘Key
New Drug Creation and Manufacturing Program’ (grants
2012ZX09301001-001, 2011ZX09307-002-03), and the Science
Foundation of Shanghai (grant 12XD14057).
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.10.
024.
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