H. Khatra et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
by 1 (Fig. S2). Further, cell viability tests were performed using
ity than 1 and the other derivatives as seen from the luciferase
MTT assay in Shh-LIGHT2 cells. No sign of cytotoxicity was
assay, where it inhibited Gli-mediated transcription with an
observed post 40 h treatment with doses upto 150
ditions used for Hh reporter assays (Fig. S3).
Thus, we decided to prepare the compounds di-Boc-Piz (6), d-N-
Cbz-Piz (7), di-Me-Piz (8), d-N-Me-Piz (9) focusing our modifica-
l
M under con-
IC50 = 10
expression (IC50 = 30
best among the tested derivatives (Fig. 4a). The derivatives were
nontoxic up to 150 M when their cytotoxicity was evaluated
l
M. Amide (10) also showed suppression of firefly
l
M). Hence, we found that amide (11) is the
l
tions at the
a
/b-nitrogen positions or compounds Piz-amide (10)
using MTT assay in Shh-LIGHT2 cells (Fig. S3). Western blot analy-
sis in the presence of the derivatives showed that amide (10) and
amide (11) reduced Gli1 protein levels with amide (11) depicting
better potency (Fig. 4b).
and Piz-amide (11) at the carboxylic group (Fig. 3). These com-
pounds were synthesized according to Scheme 1 and screened
using luciferase assay as mentioned previously. Compounds 6
and 7 were synthesized following our previously reported proce-
dures. Our initial attempt to synthesis di-Me-Piz (8) using MeI in
the presence of K2CO3 in dry DMF gave a mixture of N-methylated
esters which were impossible to separate by column chromatogra-
phy. Our next attempt was to selectively protect the 1-N with Cbz,
followed by methylation using HCHO/NaBH4 method at the 2-posi-
tion, however, selective Cbz protection of piperazic acid (1) was
not achieved. Instead, we isolated di-Cbz-protected piperazic acid.
Finally we started with benzyl ester of piperazic acid 13 which
was prepared from 6. After methylation by MeI/K2CO3, it gave di-
Me-Piz-OBz 14. Hydrogenolysis of 14 by 10% Pd/C gave the desired
dimethylated compound 8. For the preparation of 2-methylpiper-
azine-3-carboxylic acid, mono Cbz-protected benzyl ester 15 was
used as a starting material. Unlike free piperazic acid 1, benzyl ester
13 underwent mono Cbz protection at the 1-position selectively to
give compound 15. After treatment with MeI/K2CO3, 2-methylated
product 16 was obtained in the protected forms which after
hydrogenolysis gave the desired compound 9. None of the analogues
6, 7, 8 and 9 showed inhibitory activity in luciferase assay (Fig. S4).
We investigated the effect of amides 10 and 11 on hedgehog
signaling which were then synthesized from 6 by the activation
of carboxylic acid 6 using HBTU followed by the treatment with
amine (Scheme 1). Interestingly, amide (11) exhibited higher activ-
To understand the mechanism of action of 10 and 11 and exam-
ine the possibility of their interaction with Smo, luciferase assay
was performed in the presence of varying concentrations of the
compounds, where pathway stimulation was carried out using
500 nM SAG (a known agonist of the pathway which acts by bind-
ing to Smo).5,24 Both compounds significantly suppressed Gli tran-
scriptional activity even in the presence of SAG. This was
ascertained by western blot analysis of Gli1 expression in presence
and absence of compounds upon pathway activation by SAG. Both
compounds inhibited Gli1 with 11 showing comparatively better
inhibition (Fig. 5a and b). This indicated that the compounds act
either independent of Smo binding or bind to a site other than
the Smo heptahelical bundle (where SAG is known to bind)
although protein levels of Smo remained unaffected (Fig. S2). Fur-
ther, protein expression of other components of the pathway (SHH
and SuFu) was unaffected post 24 h treatment with amide (10) and
amide (11) when analyzed by western blotting (Fig. S2).
Next, their activity was evaluated in Ptch1ꢀ/ꢀ cells derived from
mouse embryos where Ptch1 is functionally absent25 and hence,
hedgehog signaling is constitutively active. Pathway activation in
this cell line is indicated by b-galactosidase activity as its expres-
sion is under control of the Ptch1 promoter and Ptch1 itself is a
transcriptional target of Hh signaling. No significant suppression
Figure 4. (a) Plot showing percentage inhibition of luciferase expression in presence of 10 and 11. (b) Downregulation of Gli1 protein post 24 h treatment with 10: 50
lM;
11: 25, 50 M; 1: 50 M. 10 M cyclopamine (Cyc) is used as a positive control. (c) Plot showing quantification of Gli1 bands obtained in (b) using ImageJ software. Control
l
l
l
set (C) denotes Shh-N stimulated cells treated with DMSO only. GAPDH is used as loading control. Data represents an average of three independent experiments performed in
triplicate, with the error bars denoting standard deviation (SD).