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B. Cherfaoui et al. / Bioorg. Med. Chem. 24 (2016) 2423–2432
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1
:R = N,N-dimethylsulfonyl amide
;
O
6a
:R = acetyl;
OH
O
6b
:R = isopropyl;
a
R
6c:R = cyclopentyl;
6d:R = cyclohexyl;
OH
N
Br
Br
O
N
2
3
6e
:R = benzyl;
d
1
6f:R = 4-hydroxyphenyl;
6g:R = 3-hydroxyphenyl;
6h:R = 2-hydroxyphenyl;
Br
(6a-m)
NH
NH
b, c
N
N
Boc
R
6i
: R = 4-methylphenyl;
4
6j: R = 4-chlorophenyl;
6k:R = phenyl;
5
6l: R = 4-methoxyphenyl;
6m
:R = 2-pyridyl;
Scheme 1. Reagents and conditions: (a) NaH, THF, epichlorohydrin, N2; (b) dimethylsulfamoyl chloride, EDTA, DCM, rt; (c) TFA/DCM, rt; (d) EtOH, reflux.
Table 2
Hsp90. This loss of inhibitory effect on Hsp90 ATPase activity was
accompanied with absence of anti-proliferative activity against
cancer cell lines. From results described above, it was supposed
that analogue 6f unveiled on existence of key hydrogen-bonding
interaction between the 4-hydroxyphenyl and protein target
Hsp90, which was critical for anti-proliferative activity.
Anti-proliferative activity of analogues 8a–c
O
n
N
N
Br
n= 1, 2, 3
OH
Basing the results on Hsp90 inhibitory effect of those analogues,
we continued our study and selected 6f for further modifications.
In the next series, we were keen to explore the significance of
the hydroxyl group on the carbon chain spacer between the
6-bromonaphthalene and the 1-(4-hydroxyphenyl)piperazine,
since that this hydroxyl group might induce unstable metabolism.
The length of this spacer was also investigated. Therefore, three
compounds lacking the hydroxyl group on the linker were
designed and synthesized as described in Scheme 2.
Compd no.
Antiproliferative activity on cancer cell lines (IC50, lM)
MCF-7
HCT-116
A549
MDA-MB-231
8a (n = 1)
8b (n = 2)
8c (n = 3)
56.52 0.8
>200
>250
16.83 0.7
88.22 1.3
112 4.2
20.56 0.7
>200
>250
5.26 0.9
173.7 2.3
49.86 1.2
Finally, alteration of 6-bromonaphthalenyl to 1,3-benzodioxolyl
(9l) was favorable over replacement with the indolyl (9m).
Results from structural alteration studies reported above, illus-
trated that the hydroxyl group on fourth position of the phenyl sig-
nificantly modulated both cytotoxicity and potency against Hsp90.
We concluded that compound 6f was the optimal analogue among
others, maintaining the best balance between Hsp90 inhibition and
cytotoxicity.
To further investigate whether the antiproliferative activity was
the functional consequence of Hsp90 inhibition, we determined
the expression of Hsp90 client proteins which are crucial for tumor
growth. MCF-7 cancer cells were exposed to a range of concentra-
tions of 6f for 24 h and various client proteins were explored by
Western blot.
As shown in Figure 2, compound 6f significantly down-regu-
lated the level of client proteins including Akt, Erk1/2 and HER-2
in a dose-dependent manner. As expected, the expression level of
Hsp90 was dose-dependently enhanced, which was considered as
the feedback of Hsp90 inhibition. Together, these results indicated
that 6f inhibited proliferation of cancer cells through Hsp90 inhibi-
tion, thus degrading its client proteins.
We next explored whether 6f could induce the apoptosis of
MCF-7 cells and evaluated its influence on cell skeleton by a mor-
phological observation. Treatment of MCF-7 cells with 6f resulted
in phenotypic changes including shrinkage, membrane blebbing
and distortion and large proportion of cells turned round in shape
and necrosis (Fig. 3D–F). In contrast, untreated cells (control) grew
The synthesis of analogues (8a–c) began with the preparation
of intermediate compounds (7a–c) through reaction of
6-bromonaphthelen-2-ol with the corresponding
a,ῳ-dibromoa-
clane derivative (alkyl bromides) in DMF and presence of K2CO3
at room temperature. These intermediates were coupled with
4-(piperazin-1-yl)phenol in DMF at reflux with presence of K2CO3
(detailed synthetic procedures and analytical data of all com-
pounds were described in Section 5). Unfortunately, we found that
removal of the hydroxyl group from the spacer was detrimental for
both cytotoxic and Hsp90 inhibition activities (Table 2).
In continuation of our work, we turned our attention to the
6-bromonaphthalene in compound 6f. Several analogues with dis-
place of 6-bromonaphthalenyl were prepared through the same
synthetic sequence and evaluated as Hsp90 inhibitors (Table 3).
As anticipated, most of analogues from this series showed similar
inhibition activities against Hsp90 ATP hydrolysis function, com-
pared to compound 6f. Removing the bromine in compound 9a
resulted in gain of potency against Hsp90 (IC50 0.4 1.2 lM).
Alteration of naphthalene ring to benzene ring was tolerated. The
bromine, chlorine or electron donating substituents on the
benzene ring, were all tolerated (9b, 9c and 9h) with more or less
increase in potency compared to each other. Moreover, substitu-
tion on the forth position was more favorable than that on other
positions (9d < 9c, 9f < 9e), and analogue 9j containing a 4-amino
group showed slight increase in potency comparing to other ana-
logues. Meanwhile, steric substitution (9i, 9k) was unfavorable.
OH
O
O
Br
N
Br
n
e
n
f
n
+
Br
N
Br
Br
Br
2
7a-c
α,ω−dibromoalkane
8a-c
n= 1, 2, 3
OH
derivatives
Scheme 2. Reagents and conditions: (e) DMF, K2CO3, rt, N2, 2 h; (f) 4-(piperazin-1-yl)phenol, DMF, K2CO3, reflux, 5 h.