Amide conjugates of ketoprofen and indole as inhibitors of Gli1-mediated transcription in the Hedgehog pathway

Article abstract of DOI:10.1016/j.bmc.2010.05.001

We have previously reported small-molecule inhibitors of Gli1-mediated transcription, an essential down-stream element of the Hh pathway. We created new derivatives of the previous compounds aiming to improve the druggable properties. The new compounds, a

Full text of DOI:10.1016/j.bmc.2010.05.001

Bioorganic & Medicinal Chemistry 18 (2010) 4801–4811
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Amide conjugates of ketoprofen and indole as inhibitors of Gli1-mediated
transcription in the Hedgehog pathway
*
Neeraj Mahindroo, Michele C. Connelly, Chandanamali Punchihewa, Lei Yang, Bing Yan, Naoaki Fujii
Departments of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously reported small-molecule inhibitors of Gli1-mediated transcription, an essential
down-stream element of the Hh pathway. We created new derivatives of the previous compounds aiming
to improve the druggable properties. The new compounds, amide conjugates of ketoprofen and indole,
showed inhibitory activity and membrane permeability, while also improving the microsome stability.
Among them, 33 and 42 inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously trans-
Received 22 March 2010
Revised 30 April 2010
Accepted 1 May 2010
Available online 7 May 2010
fected with Gli1 with 2.6
express Gli1, and were selective to Gli1 over Gli2.
lM and 1.6 lM of IC50, respectively, and in Rh30 cells that endogenously over-
Keywords:
Hedgehog
Gli
Ó 2010 Elsevier Ltd. All rights reserved.
Anticancer
Metabolic stability
1. Introduction
pediatric cancers. To overcome this problem, previously we have
designed ketoprofen amide derivatives including 5 and 6 that inhi-
bit transcription by Gli1,10 an oncogenic down-stream transcrip-
tion factor of the Hh pathway.11,12 These compounds later were
proven to be unstable in liver microsomes and this instability
might be due to their phenol moiety (observation presented in this
paper). There have been several efforts to improve metabolic sta-
bility and bioavailability of lead compounds possessing a phenol
moiety, by replacing it with an indole13 or other NH-containing
Inappropriate activation of the Hedgehog (Hh) signaling can re-
sult in cancer.1 Inhibitors of smoothened (Smo), a transmembrane
protein that promotes this pathway, have shown anticancer activ-
ity and in a recent clinical trial have proven to be effective.2 How-
ever, this Smo inhibitor, GDC0449 (1), rapidly acquired resistance
by mutation of Smo.3 Another Smo inhibitor, HhAntag (2) was
shown to eliminate medulloblastoma from a mouse model in
which Ptch1 (a suppressor of the Hh signaling upstream of the
Smo) is inactivated,4 but not to be effective in another mouse mod-
el in which SuFu (a suppressor of the Hh signaling downstream of
the Smo) is inactivated.5 Furthermore, HhAntag caused permanent
defects in bone development in young mice.6 This is believed to be
a mechanism-based toxicity because Smo inhibitors mechanisti-
cally also inhibit Indian Hh-Gli2 signaling, an essential pathway
for bone development,7 which could be affected also by non-selec-
tive Gli inhibitors such as GANT618 (3) and Physalin9 (4). This
observation represented a very serious concern in their use for
Cl
Cl
O
N
N
N
H
O
N
1
2
NMe2
NMe2
S
3
Me
Cl
O
O
N
MeO
N
O
N
O
H
HN
HO
O
O
NMe2
OMe
O
O
O
O
Abbreviations: Hh, hedgehog; Gli, glioma-associated oncogene homolog; Smo,
smoothened; Ptch1, patched 1; SuFu, suppressor of fused; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; Gli-Luc, Gli-responsible firefly luciferase; HATU,
2-(1H-7-azabenzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluoro-
phosphate; HBTU, 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate; DIPEA, N,N-diisopropylethylamine; RT-PCR, reverse tran-
scription polymerase chain reaction; PAMPA, parallel artificial membrane perme-
ability assay.
4
O
R
N
O
N
OH
H
5
6
R = H; R = n-propyl
Figure 1. Panel A: GDC0449 (1) and HhAntag (2) are inhibitors of Smo, and GANT61
(3) and Physalin (4) are inhibitors of Gli-mediated transcription. Panel B: inhibitors
of Gli1-mediated transcription (5 and 6) and lead optimization in this study.
* Corresponding author. Tel.: +1 901 595 5854; fax: +1 901 595 5715.
E-mail address: naoaki.fujii@stjude.org (N. Fujii).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.001

4802
N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
heterocyclic rings.14 These studies motivated us to adopt a similar
approach to improve druggability of our lead compounds. Here we
report new compounds in which an indole is substituted in place of
the phenol moiety of 5 and 6 (Fig. 1B).
Table 1
Compounds that were designed by replacing the phenol moiety of 5 with different
indoles
O
H
N
R
2. Chemistry
O
Compounds were prepared as outlined below (Scheme 1). Keto-
profen 7 was coupled with several indole primary amines to afford
amides 20–24. An amine 8 was coupled with several indole carbox-
ylic acids to afford reverse amides 25–31. Also, the indole amines
were alkylated with several alkyl halides to afford secondary
amines 9–19, which were coupled with 7 to afford N-alkyl amides
32–39 and 42–44. Preparation of compounds 40 and 41 were de-
scribed in Section 5.
R
.
5
OH
.
20
N
H
H
3. Results and discussion
N
21
.
Compounds were tested in a reporter assay using C3H10T1/2
mouse embryo fibroblasts exogenously transfected with vectors
encoding human full-length Gli1 and Gli-responsive luciferase
(Gli-Luc) reporter. Endogenous Gli1 expression in C3H10T1/2 cells
is undetectable and Gli-Luc activity in these cells is negligible with-
out the exogenous Gli1, thus inhibitors of Gli-Luc activity in this as-
say are believed to be inhibitors of the Gli1-mediated transcription
but not of upstream mediators of the Hh pathway, for example,
Smo. Consistently, HhAntag,15 a Smo inhibitor with nanomolar le-
vel of IC50, is almost inactive in this assay at a concentration up to
H
N
22
.
.
H
N
23
H
N
24
40
Gli-mediated transcription,8 showed activity with an IC50 of
ꢀ20 M (Supplementary Fig. S1).
lM; while GANT61 (3), a known non-selective inhibitor of
l
Compound 20, an indole variant of 5 having two methylenes be-
tween the amide group and the phenol, showed much weaker
activity than 5. Compounds 21 and 22 with only one or no methy-
lene spacer showed higher activity than 20. We investigated the
preferable position of the indole substitution and found that the
6-position is best among the tested analogs (compound 23)
(Table 1 and Fig. 2).
Next, we investigated the effect of reversal of the amide group.
Compound 25 has a linker length the same as 5 and showed only
weak activity, as observed in compound 20. In contrast, removal
of the methylene spacer between the amide and 5- or 6-position
of the indole in compounds 26 and 27 afforded equipotent activity
to those of 23 and 24. Moving the substitution to the 2- or 3-posi-
tion on the indole also gave equipotent inhibitors 28 and 29,
though moving to the 4- or 7-position in compound 30 and 31 de-
creased the potency (Table 2 and Fig. 3).
Figure 2. Replacement of the phenol in
equipotent inhibition of Gli1-mediated transcription. Percent inhibition of Gli-
responsive firefly luciferase reporter activity in Gli1-transfected C3H10T1/2 cells
5 with an indole moiety afforded
O
O
NHR1
R1NH2
a
CO2H
24 h after addition of 10
lM (open bar) or 20
l
M (closed bar) of the test compound.
Renilla luciferase
O
Each Gli-Luc firefly luciferase signal is normalized with
transfection control and the normalized Gli-Luc activity with DMSO treatment
serves as 0% inhibition. Error bars represent standard error of triplicate data.
a
7
20-24
O
O
H
N
R1
NH2
R1CO2H
a
O
Next, we explored the effect of an alkyl group substitution on
the amide moiety. We had previously observed that substitution
of the amide nitrogen of 5 with an n-propyl group (compound 6)
improved potency.10 To attain optimal substitution on its amide
nitrogen, we had chosen 23 but not 26, because of better stability
over liver microsomes (Fig. 9B). Little improvement was shown by
a methyl group (compound 32) while substitution of an n-propyl
group improved the potency (compound 33). A similar effect was
observed with several other alkyl groups that are larger than
n-propyl, but no further potency improvement was observed
25-31
8
O
R2
N
7
b
R1
R1NH2
R1NHR2
a
O
9-19
32-39, 42-44
Scheme 1. Reagents and conditions: (a) HBTU (for 20–31) or HATU (for 32–39 and
42–44), N,N-diisopropylethylamine, DMF, room temperature; (b) R2Br, DMF,
microwave, 5 min, 150 °C. Compound 8 was previously prepared from ketoprofen
7.10

N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
4803
Table 2
Overall, clear SAR trend is observed. The effect of linker length is
similar among two scaffolds; both amides 20, 21 and reverse
amide 25 having a longer linker showed much lower activity than
the corresponding shorter variants (22 and 26, respectively). Incor-
porating an n-propyl group on the amide nitrogen increased po-
tency in both of the phenol derivatives (5 vs 6) and the indole
derivatives (23 vs 33). Effect of the n-propyl is observed as a clear
SAR; moving it to the adjacent methylene (40) or indole nitrogen
(41) reduced the activity. These observations may suggest presence
of a specific target that those compounds could bind to suppress
the Gli1-mediated transcription.
Compounds designed by reversing the amide group substitution of compounds
O
H
N
R
O
R
.
25
26
27
28
N
H
H
N
Selected indole compounds were assayed for inhibition of
Gli2-mediated transcription by using a vector encoding Gli2. In this
assay, IC50 of 33 and 42 were estimated as 29 lM and 21 lM, respec-
.
.
tively (Fig. 6); showing approximately 10-fold Gli1-selectivity. To
further ensure that these lead compounds are not non-specific
inhibitors of transcription, lead compounds were tested also in an
unrelated transcription reporter, AP-1, in the C3H10T1/2 cells under
the same conditions as for the Gli1/Gli-Luc assay. They showed no
inhibition (Supplementary Fig. S3). Histone deacetylase (HDAC)
strongly promotes transcriptional activation by both of Gli1 and
Gli2.16 However, compounds 5, 6, 33, and 42 do not inhibit HDAC
N
H
N
H
.
up to 40 lM (data not shown), suggesting that the Gli1-inhibition
by these compounds is not due to HDAC inhibition.
29
N
H
To exclude the possibility that these compounds are inhibitors
of the specific promoter for the exogenous Gli1 transgene expres-
sion, and also to corroborate that lead compounds selected by
assaying against artificially overexpressed Gli1 can inhibit
endogenously overexpressed Gli1 in cancers, they were evaluated
by inhibition of the Gli-Luc activity in Rh30, a rhabdomyosarcoma
cell line that highly overexpresses Gli1 gene.17 HhAntag showed al-
most no Gli-Luc inhibition in these cells at a concentration as high
30
31
.
.
NH
HN
as 40
ꢀ40
l
M, meanwhile GANT61 showed activity with an IC50 of
l
M (Supplementary Fig. S2). Compounds 23, 33, and 42
showed inhibition with similar or slightly weaker potency to those
in the Gli1-transfected C3H10T1/2 cells (Fig. 7A). The inhibitory ef-
fect in the Rh30 cells of 33 was observed also in regulating the
messenger transcripts of some Gli1-target genes,18 which were
quantified by RT-PCR (Fig. 7B). This chemical scaffold could sup-
press growth of Rh30 but is generally nontoxic to a human normal
fibroblast cell line BJ (Fig. 8).
Finally, stability of the lead compounds in mouse liver micro-
somes was evaluated. The phenol-based compounds (5 and 6)
were completely consumed within less than 1 h whereas ketopro-
fen (7) was very stable; suggesting that the phenol moiety might
be responsible for the instability (Fig. 9A). In contrast, the indole-
based compounds, especially 6-indole derivatives (23 and 33),
were much more stable than 5 and 6 (Fig. 9B). Cell membrane per-
meability of the lead compounds was evaluated in a PAMPA assay.
The phenol-based amide 5 and indole-based amides 23 and 33 are
permeated to a comparable extent, suggesting that the higher po-
tency of 33 than 23 is not simply due to higher permeability.
Meanwhile, the indole-based reverse amide 26 was less permeable
(Table 4). This trend was also confirmed in a Caco-2 cell assay, in
which all of the tested compounds had much lower efflux ratios,
that is, lower excretion than digoxin (Table 5).
Figure 3. The effect of position of the indole substitution of reverse amides. Data
are measured and presented as in Figure 1.
(compounds 34–36). Meanwhile, increasing hydrophilicity of the
alkyl group decreased the potency (compounds 37–39). The posi-
tional effect of the n-propyl and indole was also investigated. Intro-
duction of an n-propyl group on the benzyl carbon of the
ketoprofen moiety (compound 40) or indole-1-position (com-
pound 41) did not increase potency. Compounds 42 and 43, in
which the indole is substituted on the 5- and 4-position respec-
tively, are equipotent to 33. Compound 44, a variant of 42 in which
the indole substitution is homologated with one methylene,
showed weaker potency than 42, as observed in 20 and 25. (Table
3 and Fig. 4) In separate dose–response titrations, IC50 of 33 and 42
4. Conclusion
To date, several compounds targeting Gli-transcription were
found in a high-throughput screen,8,20 but their druggable proper-
ties such as metabolic stability, cell permeability and non-specific
toxicity were not addressed. Compounds from natural product (such
as Physalin9 4) have another limitation; they cannot be easily
reproduced or synthesized to yield the additional large amounts of
were estimated as 2.6 lM and 1.6 lM, respectively (Fig. 5).

4804
N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
Table 3
Table 3 (continued)
Compounds designed by incorporating an alkyl on the amide nitrogen or other
positions
R
O
43
44
R
NH
N
.
O
H
R
N
H
N
N
32
.
N
O
.
O
H
N
33
34
N
.
.
O
H
N
N
O
35
36
H
N
N
N
N
.
O
O
O
H
N
.
.
N
37
H
N
O
N
38
39
H
N
N
N
.
O
N
H
N
.
.
.
.
O
O
O
O
Figure 4. (Two panels) Alkyl substitution on the amide nitrogen increases the
potency. Data are measured and presented as in Figure 1.
H
N
H
40
41
N
materials needed for validating in the other assays, animal testing,
and clinical trials. In contrast, compounds such as 33 are very easily
prepared in large scale from ketoprofen, a clinically used drug.
C3H10T1/2 cells express endogenous Gli2 and are differentiated into
osteoblasts by Indian Hh; and this differentiation is inhibited by
cyclopamine.21 In our hands, inhibitory effect of compounds 23,
33, and 42 in inducing Hh signal-mediated alkaline phosphatase,
an osteoblastic differentiation marker, was much smaller than that
of HhAntag in C3H10T1/2 cells (Supplementary Fig. S4). This sug-
gests that selective inhibitors of the Gli1-mediated transcription
could less affect mouse bone development than HhAntag did.6 Stud-
iesareongoingto createadditionalleadswith improvedpotencyand
H
N
N
N
42
N
H

N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
4805
Figure 5. Dose–response of Gli-Luc signal in Gli1-transfected C3H10T1/2 cells treated with 33 (panel A) or 42 (panel B). Data was plotted and curve fitted by Prism 4.03
(GraphPad Software). IC50 was estimated as 2.6 M (1.77–3.75 M (0.82–3.05 M for 95%
M for 95% confidence intervals) for 33 with curve fitting: R2 = 0.93 and 1.6
confidence intervals) for 42 with curve fitting: R2 = 0.90. Error bars represent standard error of triplicate data.
l
l
l
l
Figure 6. Compounds show smaller inhibitory activity for Gli2-mediated transcription than for Gli1-mediated transcription. IC50 was estimated as 29
42 by Prism 4.03 (GraphPad Software). Error bars represent standard error of triplicate data.
lM for 33 and 21 lM
Figure 7. Panel A: lead compounds inhibit transcription by endogenous Gli in Rh30 cells. Percent inhibition of Gli-reporter activity in Rh30 cells (without Gli1 transfection)
24 h after addition of 10 M (open bar) or 20 M (closed bar) of the test compound. Each firefly luciferase signal is normalized with Renilla luciferase transfection control, and
l
l
then normalized by firefly luciferase activity with DMSO treatment serving as 0% inhibition. Error bars represent standard error of triplicate data. Panel B: compound 33
inhibits expression of Gli1-target genes18 in Rh30 cells. Expression levels of each indicated Gli1-mediated transcription products in Rh30 cells were measured by real-time
RT-PCR 24 h after addition of 33. Error bars represent standard error of duplicate data. Average
treatment to calculate DDCt. Ct, cycle time.
D
Ct (Ct(target gene) ꢁ Ct(GAPDH)) were subtracted with that of DMSO

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N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
Figure 8. Effect of lead compounds in growth of Rh30 (panel A) and normal human fibroblast cell line BJ-TERT19 (panel B). Cells were incubated with indicated compound for
72 h, and the viability was measured by Alamar blue assay. Data are normalized with signals of DMSO treatment (= 100%) and no cells (= 0%). Error bars represent standard
error of triplicate data.
Figure 9. Stability of compounds (1
lM) in CD-1 female mouse liver microsomes at 37 °C. Panel A: phenol compounds and parent scaffold ketoprofen (7). Panel B: indole
compounds. Half-lives are estimated as 2.8 h (23), 0.6 h (26), 1.8 h (33), and 0.8 h (42).

N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
4807
Table 4
d 7.82 (s, 1H), 7.39 (d, J = 9.0, 1H), 6.93 (dd, J = 3.2, 2.3, 1H), 6.57–
6.46 (m, 2H), 6.45–6.33 (m, 1H), 3.59 (s, 1H), 2.94 (d, J = 6.8, 2H),
1.92 (dt, J = 13.4, 6.7, 1H), 1.00 (d, J = 6.7, 6H).
PAMPA permeability of selected compounds at pH 7.4. Ranitidine and carbamazepine
served as controls
Compound
Average P (nm/s)
Average permeability (%)
5.2.4. N-(Cyclohexylmethyl)-1H-indol-6-amine (12)
5
23
26
33
602
778
521
714
55
92
75
47
80
2
Compound 12 was synthesized from 1H-indol-6-amine and
bromomethylcyclohexane in 48% yield. 1H NMR (400 MHz, CDCl3)
d 7.85 (s, 1H), 7.38 (d, J = 8.4, 1H), 6.95 (dd, J = 3.1, 2.3, 1H), 6.59–
6.46 (m, 2H), 6.40 (ddd, J = 3.0, 2.0, 0.8, 1H), 3.63 (s, 1H), 2.97 (d,
J = 6.7, 2H), 1.90–1.79 (m, 2H), 1.79–1.55 (m, 4H), 1.33–1.10 (m,
3H), 1.00 (qd, J = 12.1, 3.1, 2H).
Ranitidine
Carbamazepine
199
33
Table 5
5.2.5. N-Phenethyl-1H-indol-6-amine (13)
Caco-2 cell permeability of selected compounds at pH 7.4. Digoxin served as a control
Compound 13 was synthesized from 1H-indol-6-amine and
2-bromoethylbenzene in 55% yield. 1H NMR (400 MHz, CDCl3) d
7.85 (s, 1H), 7.40 (d, J = 8.4, 1H), 7.36–7.28 (m, 2H), 7.23 (ddd,
J = 4.3, 3.4, 2.4, 3H), 6.96 (dd, J = 3.2, 2.3, 1H), 6.56 (d, J = 1.9, 1H),
6.50 (dd, J = 8.4, 2.1, 1H), 6.40 (ddd, J = 3.1, 2.0, 0.9, 1H), 3.51 (s,
1H), 3.41 (t, J = 7.0, 2H), 2.94 (t, J = 7.0, 2H).
Compound Average Papp A?B Average Papp B?A Efflux ratio (B?A)/
(nm/s)
(nm/s)
(A?B)
5
23
26
Digoxin
130
122
58
58
39
27
92
0.45
0.31
0.46
1.15
80
5.2.6. N-(2-(Piperidin-1-yl)ethyl)-1H-indol-6-amine (14)
Compound 14 was synthesized from 1H-indol-6-amine and N-
(2-chloroethyl)piperidine hydrochloride in 50% yield. 1H NMR
(400 MHz, CDCl3) d 8.28 (s, 1H), 7.39 (d, J = 8.7, 1H), 6.95 (dd,
J = 3.0, 2.4, 1H), 6.60–6.48 (m, 2H), 6.44–6.29 (m, 1H), 4.46 (s,
1H), 4.12 (q, J = 7.1, 1H), 3.17 (t, J = 6.1, 2H), 2.60 (t, J = 6.1, 2H),
2.44 (s, 4H), 1.68–1.53 (m, 4H), 1.48–1.38 (m, 2H).
Gli1/Gli2-selectivity, which will be tested in animal models of pedi-
atric cancers for antitumor efficacy and bone growth safety.
5. Experimental section
5.1. Materials
5.2.7. N-(2-Morpholinoethyl)-1H-indol-6-amine (15)
Compound 15 was synthesized from 1H-indol-6-amine and N-
(2-chloroethyl)morpholine hydrochloride in 50% yield. 1H NMR
(400 MHz, CDCl3) d 8.00 (s, 1H), 7.41 (d, J = 8.2, 1H), 6.97 (dd,
J = 3.2, 2.3, 1H), 6.56 (ddd, J = 5.1, 3.3, 1.7, 2H), 6.41 (ddd, J = 3.0,
2.0, 0.8, 1H), 3.81–3.65 (m, 4H), 3.24–3.10 (m, 2H), 2.72–2.58 (m,
2H), 2.53–2.37 (m, 4H).
HhAntag was prepared by the method reported in the litera-
ture.15 GANT61 was purchased from Enzo Life Sciences (Plymouth
Meeting, PA). Other chemicals and solvents were purchased from
Sigma–Aldrich (St. Louis, MO) and used as received.
5.2. General procedure for preparation of amines 9–19
5.2.8. N1-(1H-Indol-6-yl)-N3,N3-dimethylpropane-1,3-diamine
(16)
Compound 16 was synthesized from 1H-indol-6-amine and
3-chloropropyldimethylamine hydrochloride in 55% yield. 1H
NMR (400 MHz, CDCl3) d 8.03 (s, 1H), 7.39 (d, J = 8.4, 1H), 6.96
(dd, J = 3.1, 2.4, 1H), 6.59–6.45 (m, 2H), 6.44–6.33 (m, 1H), 3.19
(t, J = 6.8, 2H), 2.41 (t, J = 6.9, 2H), 2.25 (s, 6H), 1.81 (p, J = 6.8, 2H).
The mixture of appropriate 1H-indolamine (1.0 equiv) and halo-
alkane (0.5 equiv) in DMF (0.5 mL) was exposed to microwave irra-
diations for 5 min at 150 °C. When the haloalkane is used as
hydrochloride (14–16), triethylamine (2.5 equiv) was included in
the mixture prior to the irradiation. The reaction mixture was
cooled to room temperature, quenched with water (10 mL) and ex-
tracted with ethyl acetate (2 ꢂ 10 mL). The organic layer was suc-
cessively washed with water (2 ꢂ 10 mL) and brine (1 ꢂ 10 mL)
and dried over anhydrous sodium sulfate. The solvent was re-
moved in vacuo and the residue was chromatographed over silica
gel (Biotage SP4, 12+S Column, eluting with dichloromethane:
methanol gradient 0–10%) to give N-alkyl-1H-indolamine in 50–
55% yield.
5.2.9. N-Propyl-1H-indol-5-amine (17)
Compound 17 was synthesized from 1H-indol-5-amine and
1-bromopropane in 54% yield.
5.2.10. N-Propyl-1H-indol-4-amine (18)
Compound 18 was synthesized from 1H-indol-4-amine and
1-bromopropane in 56% yield.
5.2.1. N-Methyl-1H-indol-6-amine (9)
Compound 9 was synthesized from 1H-indol-6-amine and iodo-
methane in 52% yield. 1H NMR (400 MHz, CDCl3) d 7.87 (s, 1H),
7.44–7.36 (m, 1H), 6.97 (dd, J = 3.2, 2.3, 1H), 6.57–6.48 (m, 2H),
6.44–6.38 (m, 1H), 2.86 (s, 3H).
5.2.11. N-((1H-Indol-5-yl)methyl)propan-1-amine (19)
Compound 19 was synthesized from (1H-indol-5-yl)methan-
amine and 1-bromopropane in 52% yield.
5.2.2. N-Propyl-1H-indol-6-amine (10)
5.3. General procedure for preparation of compounds 20–39
and 42–44
Compound 10 was synthesized from 1H-indol-6-amine and
1-bromopropane in 55% yield. 1H NMR (400 MHz, CDCl3) d 7.85
(s, 1H), 7.39 (d, J = 8.4, 1H), 7.04–6.86 (m, 1H), 6.64–6.46 (m, 2H),
6.40 (dd, J = 3.6, 1.6, 1H), 3.56 (s, 1H), 3.10 (t, J = 7.1, 2H), 1.82–
1.54 (m, 2H), 1.02 (t, J = 7.4, 3H).
A mixture of (S)-ketoprofen (1 equiv), HBTU or HATU (2.5 equiv),
and DIPEA(3 equiv)in DMF (0.3 mL) was allowedto standfor 30 min
at rt. Appropriate amine (9–19, 2.0 equiv) was then added to the
mixture and stirred for 2 h at rt. Water was added to the reaction
mixture and extracted with ethyl acetate followed by successive
washings with water and brine. The organic layer was dried over
anhydrous sodium sulfate. The solvent was removed in vacuo to give
5.2.3. N-Isobutyl-1H-indol-6-amine (11)
Compound 11 was synthesized from 1H-indol-6-amine and
1-iodo-2-methylpropane in 54% yield. 1H NMR (400 MHz, CDCl3)

4808
N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
the residue, which was purified by a chromatography over silica gel
(Biotage SP4, 12+S column, eluting with hexanes/ethyl acetate gra-
dient from 10% to 80%) to give the desired compound in 60–80%
yield. The purity of all the compounds was determined by HPLC on
a Waters Alliance HT LC–MS system (Waters 2795 Separation Mod-
ule linked to a Waters 2996 Photodiode Array Detector) using a
7.58–7.49 (m, 3H), 7.46–7.39 (m, 3H), 7.35–7.31 (m, 1H), 7.24–
7.22 (m, 1H), 6.59–6.54 (m, 1H), 6.15 (t, J = 5.7, 1H), 3.83 (dt,
J = 13.1, 6.4, 1H), 3.51 (ddd, J = 18.8, 9.0, 4.8, 1H), 3.20 (dd,
J = 14.7, 6.9, 1H), 1.37 (d, J = 7.0, 3H). HRMS (ESI (M+H)+ m/z) calcd
for C25H23N2O2 383.1760, found 383.1761.
Waters XBridge C18, 3.5
l
m (4.6 ꢂ 50 mm) column by running a
5.3.8. N-(2-(3-Benzoylphenyl)propyl)-1H-indole-6-carboxamide
(27)
0–95% gradient for water (+0.05% TFA)/MeOH. All the compounds
showed P95% HPLC purity (see Supplementary data). Optical activ-
ities of the products were not determined.
Yield 80%. 1H NMR (400 MHz, CDCl3) d 9.21 (s, 1H), 7.90 (s, 1H),
7.80–7.75 (m, 2H), 7.72 (dd, J = 4.6, 2.9, 1H), 7.61 (dt, J = 7.5, 1.5,
1H), 7.59–7.52 (m, 2H), 7.47 (dt, J = 7.6, 1.4, 1H), 7.41 (ddd,
J = 7.6, 3.4, 2.1, 3H), 7.33–7.28 (m, 2H), 6.53 (ddd, J = 3.0, 2.0, 0.9,
1H), 6.24 (t, J = 5.8, 1H), 3.85–3.74 (m, 1H), 3.46 (ddd, J = 13.6,
8.5, 5.4, 1H), 3.17 (dd, J = 14.8, 6.8, 1H), 1.35 (d, J = 7.0, 3H). HRMS
(ESI (M+H)+ m/z) calcd for C25H23N2O2 383.1760, found 383.1751.
5.3.1. N-(2-(1H-Indol-5-yl)ethyl)-2-(3-benzoylphenyl)propana-
mide (20)
Compound 20 was synthesized by coupling 7 with 2-(1H-in-
dole-5-yl)ethanamine in 66% yield. 1H NMR (400 MHz, CDCl3) d
8.22 (s, 1H), 7.75–7.69 (m, 2H), 7.67–7.62 (m, 2H), 7.61–7.55 (m,
1H), 7.51–7.42 (m, 4H), 7.40–7.34 (m, 1H), 7.23 (d, J = 8.3, 1H),
7.18–7.13 (m, 1H), 6.84 (dd, J = 8.3, 1.6, 1H), 6.43 (ddd, J = 3.0,
2.0, 0.9, 1H), 5.39 (s, 1H), 3.63–3.37 (m, 3H), 2.82 (m, 2H), 1.50
(d, J = 7.2, 3H). HRMS (ESI (M+H)+ m/z) calcd for C26H25N2O2
397.1916, found 397.1928.
5.3.9. N-(2-(3-Benzoylphenyl)propyl)-1H-indole-2-carboxamide
(28)
Yield 75%. 1H NMR (400 MHz, CDCl3) d 9.53 (s, 1H), 7.80–7.76
(m, 2H), 7.72 (t, J = 1.7, 1H), 7.66 (dt, J = 7.3, 1.6, 1H), 7.61–7.57
(m, 1H), 7.55 (dt, J = 2.6, 1.6, 1H), 7.49 (dt, J = 7.6, 1.5, 1H), 7.47–
7.43 (m, 2H), 7.43–7.39 (m, 2H), 7.27 (ddd, J = 8.3, 7.0, 1.1, 1H),
7.12 (ddd, J = 8.0, 7.0, 1.0, 1H), 6.69 (dd, J = 2.1, 0.8, 1H), 6.21 (t,
J = 5.9, 1H), 3.84 (dt, J = 13.3, 6.5, 1H), 3.59–3.47 (m, J = 13.7, 8.4,
5.5, 1H), 3.25–3.13 (m, J = 14.9, 6.8, 1H), 1.39 (d, J = 7.0, 3H). HRMS
(ESI (M+H)+ m/z) calcd for C25H23N2O2 383.1760, found 383.1764.
5.3.2. N-((1H-Indol-5-yl)methyl)-2-(3-benzoylphenyl)propana-
mide (21)
Yield 73%. 1H NMR (400 MHz, CDCl3) d 8.34 (s, 1H), 7.79–7.72
(m, 3H), 7.68–7.63 (m, 1H), 7.61–7.53 (m, 2H), 7.48–7.39 (m,
4H), 7.32–7.23 (m, 1H), 7.22–7.15 (m, 1H), 7.00 (dd, J = 8.4, 1.6,
1H), 6.47 (ddd, J = 3.0, 2.0, 0.9, 1H), 5.72 (s, 1H), 4.48 (qd, J = 14.3,
5.5, 2H), 3.61 (q, J = 7.1, 1H), 1.57 (d, J = 7.1, 3H). HRMS (ESI
(M+H)+ m/z) calcd for C25H23N2O2 383.1760, found 383.1756.
5.3.10. N-(2-(3-Benzoylphenyl)propyl)-1H-indole-3-carboxam-
ide (29)
Yield 41%. 1H NMR (400 MHz, CDCl3) d 9.60 (s, 1H), 7.95 (s, 1H),
7.76 (dd, J = 8.2, 1.1, 2H), 7.72 (s, 1H), 7.68–7.64 (m, 1H), 7.59 (dd,
J = 10.5, 4.4, 1H), 7.53 (t, J = 5.0, 2H), 7.48–7.42 (m, 3H), 7.41–7.34
(m, 1H), 7.33–7.28 (m, 2H), 5.89 (t, J = 5.8, 1H), 3.97–3.85 (m, 1H),
3.57–3.39 (m, 1H), 3.22 (dd, J = 15.4, 6.6, 1H), 1.41 (d, J = 7.0, 3H).
HRMS (ESI (M+H)+ m/z) calcd for C25H23N2O2 383.1760, found
383.1760.
5.3.3. 2-(3-Benzoylphenyl)-N-(1H-indol-5-yl)propanamide (22)
Yield 69%. 1H NMR (400 MHz, CDCl3) d 8.41 (s, 1H), 7.84 (t, J = 1.6,
1H), 7.81–7.72 (m, 3H), 7.70–7.62 (m, 2H), 7.60–7.53 (m, 1H), 7.49–
7.40(m, 4H), 7.19 (d, J = 8.7, 1H), 7.14–7.06(m, 2H), 6.42 (ddd, J = 2.9,
2.0, 0.8, 1H), 3.76 (q, J = 7.1, 1H), 1.60 (d, J = 7.1, 3H). HRMS (ESI
(M+H)+ m/z) calcd for C24H21N2O2 369.1603, found 369.1598.
5.3.4. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)propanamide (23)
Yield 62%. 1H NMR (400 MHz, CDCl3) d 8.61 (s, 1H), 8.03 (s, 1H),
7.86 (s, 1H), 7.81–7.73 (m, 2H), 7.70–7.62 (m, 2H), 7.61–7.51 (m,
2H), 7.44 (m, 4H), 7.09 (dd, J = 3.1, 2.5, 1H), 6.77 (dt, J = 8.4, 1.7,
1H), 6.44 (ddd, J = 3.0, 2.0, 0.9, 1H), 3.79 (q, J = 7.1, 1H), 1.61 (d,
J = 7.1, 3H). HRMS (ESI (M+H)+ m/z) calcd for C24H21N2O2
369.1603, found 369.1613.
5.3.11. N-(2-(3-Benzoylphenyl)propyl)-1H-indole-4-carboxam-
ide (30)
Yield 79%. 1H NMR (400 MHz, CDCl3) d 8.81 (s, 1H), 7.80–7.70
(m, 3H), 7.66 (d, J = 7.6, 1H), 7.55 (dd, J = 14.7, 7.5, 2H), 7.47–7.37
(m, 5H), 7.19 (t, J = 2.8, 1H), 7.14 (t, J = 7.8, 1H), 6.58 (s, 1H), 6.21
(t, J = 5.4, 1H), 3.89 (dt, J = 13.1, 6.4, 1H), 3.56 (ddd, J = 13.8, 8.8,
5.3, 1H), 3.24 (dd, J = 15.2, 6.8, 1H), 1.39 (d, J = 7.0, 3H). HRMS
(ESI (M+H)+ m/z) calcd for C25H23N2O2 383.1760, found 383.1762.
5.3.5. 2-(3-Benzoylphenyl)-N-(1H-indol-2-yl)propanamide (24)
Yield 62%. 1H NMR (400 MHz, CDCl3) d 10.34 (s, 1H), 8.22 (d,
J = 4.6, 1H), 7.83 (s, 1H), 7.78 (dd, J = 5.1, 3.3, 2H), 7.72–7.55 (m,
3H), 7.53–7.36 (m, 4H), 7.32–7.20 (m, 1H), 7.15–7.01 (m, 2H),
5.78 (d, J = 1.6, 1H), 3.81 (q, J = 7.1, 1H), 1.61 (d, J = 7.1, 3H). HRMS
(ESI (M+H)+ m/z) calcd for C24H21N2O2 369.1603, found 369.1606.
5.3.12. N-(2-(3-Benzoylphenyl)propyl)-1H-indole-7-carboxam-
ide (31)
Yield 71%. 1H NMR (400 MHz, CDCl3) d 10.26 (s, 1H), 7.80–7.70
(m, 4H), 7.66 (dt, J = 7.4, 1.5, 1H), 7.58–7.52 (m, 1H), 7.50 (dt,
J = 7.6, 1.4, 1H), 7.45 (d, J = 7.5, 1H), 7.43–7.36 (m, 2H), 7.33–7.28
(m, 1H), 7.18 (d, J = 7.5, 1H), 7.03 (t, J = 7.7, 1H), 6.55 (dd, J = 3.2,
2.3, 1H), 6.38 (t, J = 5.5, 1H), 3.83 (dt, J = 13.1, 6.5, 1H), 3.54 (ddd,
J = 13.6, 8.4, 5.4, 1H), 3.26–3.13 (m, 1H), 1.38 (d, J = 7.0, 3H). HRMS
(ESI (M+H)+ m/z) calcd for C25H23N2O2 383.1760, found 383.1760.
5.3.6. N-(2-(3-Benzoylphenyl)propyl)-2-(1H-indol-5-yl)acetam-
ide (25)
Yield 63%. 1H NMR (400 MHz, CDCl3) d 8.27 (s, 1H), 7.71 (dd,
J = 5.2, 3.2, 2H), 7.64–7.57 (m, 1H), 7.54 (m, 1H), 7.50–7.43 (m, 3H),
7.34 (s, 1H), 7.25 (d, J = 7.5, 1H), 7.20 (m, 3H), 6.87 (dd, J = 8.3, 1.6,
1H), 6.46 (m, 1H), 5.30 (s, 1H), 3.60 (s, 2H), 3.52 (m, 1H), 3.24–3.12
(m, 1H), 2.91 (dd, J = 15.0, 6.8, 1H), 1.20 (d, J = 7.0, 3H). HRMS (ESI
(M+H)+ m/z) calcd for C26H25N2O2 397.1916, found 397.1924.
5.3.13. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)-N-methylpropan-
amide (32)
Yield 60%. 1H NMR (400 MHz, CDCl3) d 8.69 (s, 1H), 7.71 (d,
J = 7.4, 2H), 7.66–7.49 (m, 4H), 7.45 (t, J = 7.8, 2H), 7.36 (dd,
J = 9.1, 6.2, 2H), 7.33–7.28 (m, 1H), 7.10–6.91 (m, 1H), 6.83 (s,
1H), 6.64–6.54 (m, 1H), 3.79 (q, J = 6.9, 1H), 3.30 (s, 3H), 1.41 (d,
J = 6.9, 3H). HRMS (ESI (M+H)+ m/z) calcd for C25H23N2O2
383.1760, found 383.1760.
5.3.7. N-(2-(3-Benzoylphenyl)propyl)-1H-indole-5-carboxamide
(26)
Yield 60%. 1H NMR (400 MHz, CDCl3) d 8.72 (s, 1H), 7.97 (d,
J = 0.7, 1H), 7.80–7.74 (m, 2H), 7.72 (s, 1H), 7.68–7.63 (m, 1H),

N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
4809
5.3.14. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)-N-propylpropan-
amide (33)
4H), 7.35 (t, J = 7.6, 2H), 7.31–7.16 (m, 2H), 6.97 (d, J = 19.3, 1H),
6.57 (s, 1H), 3.85–3.53 (m, 3H), 1.60–1.45 (m, 2H), 1.40 (d, J = 6.9,
3H), 0.86 (t, J = 7.4, 3H). HRMS (ESI (M+H)+ m/z) calcd for
C27H27N2O2 411.2073, found 411.2065.
Yield 73%. 1H NMR (400 MHz, CDCl3) d 8.73 (s, 1H), 7.87–7.64
(m, 3H), 7.65–7.48 (m, 4H), 7.47–7.40 (m, 2H), 7.39–7.27 (m,
3H), 7.04–6.68 (m, 1H), 6.58 (s, 1H), 3.87–3.67 (m, 2H), 3.66–
3.51 (m, 1H), 1.58–1.46 (m, 2H), 1.39 (d, J = 6.9, 3H), 0.84 (t,
J = 7.4, 3H). HRMS (ESI (M+H)+ m/z) calcd for C27H27N2O2
411.2073, found 411.2075.
5.3.22. 2-(3-Benzoylphenyl)-N-(1H-indol-4-yl)-N-propylpropan-
amide (43)
Yield 62%. 1H NMR (400 MHz, CDCl3) d 7.88 (s, 1H), 7.83–7.68
(m, 5H), 7.61–7.55 (m, 1H), 7.52 (t, J = 7.7, 1H), 7.49–7.42 (m,
2H), 7.29 (s, 1H), 7.19–7.09 (m, 2H), 7.01 (d, J = 3.2, 1H), 6.06 (d,
J = 2.9, 1H), 4.04 (t, J = 7.1, 2H), 3.90 (q, J = 7.1, 1H), 1.88–1.77 (m,
2H), 1.69 (d, J = 7.1, 3H), 0.89 (t, J = 7.4, 3H).
5.3.15. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)-N-isobutylpro-
panamide (34)
Yield 60%. 1H NMR (400 MHz, CDCl3) d 8.65 (s, 1H), 7.81–7.64
(m, 3H), 7.64–7.50 (m, 3H), 7.50–7.40 (m, 3H), 7.36 (t, J = 7.7,
1H), 7.33–7.28 (m, 1H), 6.96 (s, 1H), 6.76 (s, 1H), 6.62–6.55 (m,
1H), 3.87–3.33 (m, 3H), 1.78 (dt, J = 13.7, 6.8, 1H), 1.39 (d, J = 6.9,
3H), 0.85 (dd, J = 23.3, 5.9, 6H). HRMS (ESI (M+H)+ m/z) calcd for
C28H29N2O2 425.2229, found 425.2226.
5.3.23. N-((1H-Indol-5-yl)methyl)-2-(3-benzoylphenyl)-N-pro-
pylpropanamide (44)
Yield 72%. 1H NMR (400 MHz, CDCl3) (a mixture of rotamers)
d 8.17 (d, J = 25.6, 1H), 7.78 (t, J = 4.2, 1H), 7.72 (dd, J = 5.1, 3.3,
1H), 7.68–7.61 (m, 2H), 7.61–7.53 (m, 2H), 7.49–7.39 (m, 3H),
7.37–7.26 (m, 2H), 7.24–7.16 (m, 1H), 6.96 (ddd, J = 65.9, 8.4,
1.5, 1H), 6.48 (dt, J = 9.4, 2.1, 1H), 4.90 (d, J = 14.5, 0.5H), 4.70
(d, J = 16.8, 0.5H), 4.53 (d, J = 14.5, 0.5H), 4.42 (d, J = 16.7,
0.5H), 4.01 (dq, J = 17.3, 6.9, 1H), 3.73–3.59 (m, 0.5H), 3.20 (dd,
J = 9.7, 5.7, 0.5H), 3.15–2.96 (m, 1H), 1.56–1.44 (m, 5H), 0.82
(dt, J = 10.2, 7.4, 3H). HRMS (ESI (M+H)+ m/z) calcd for
C28H29N2O2 425.2229, found 425.2229.
5.3.16. 2-(3-Benzoylphenyl)-N-(cyclohexylmethyl)-N-(1H-indol-
6-yl)propanamide (35)
Yield 60%. 1H NMR (400 MHz, CDCl3) d 8.61 (s, 1H), 7.80–7.62
(m, 3H), 7.58 (t, J = 7.4, 2H), 7.50–7.40 (m, 3H), 7.36 (t, J = 7.7,
1H), 7.32–7.26 (m, 3H), 6.95 (s, 1H), 6.75 (s, 1H), 6.62–6.56 (m,
1H), 3.85–3.42 (m, 3H), 1.50 (ddd, J = 18.0, 12.2, 9.8, 4H), 1.39 (d,
J = 6.9, 3H), 1.19–0.81 (m, 6H). HRMS (ESI (M+H)+ m/z) calcd for
C31H33N2O2 465.2542, found 465.2545.
5.3.24. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)-2-methylpen-
tanamide (40)
5.3.17. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)-N-phenethylpro-
panamide (36)
Yield 65%. 1H NMR (400 MHz, CDCl3) d 8.58 (s, 1H), 7.71 (d,
J = 7.5, 2H), 7.54 (ddd, J = 16.9, 14.5, 8.1, 4H), 7.44 (t, J = 7.7, 2H),
7.40–7.26 (m, 3H), 7.24–6.99 (m, 5H), 6.89 (s, 1H), 6.59 (d,
J = 2.2, 1H), 4.06–3.55 (m, 3H), 2.88 (t, J = 7.8, 2H), 1.40 (d, J = 6.9,
3H). HRMS (ESI (M+H)+ m/z) calcd for C32H29N2O2 473.2229, found
473.2238.
Thionyl chloride (0.043 mL, 0.590 mmol) was added to a solu-
tion of compound 7 (0.030 g, 0.118 mmol) in methanol (2 mL) at
0 °C and allowed to warm to room temperature. The reaction mix-
ture was refluxed for 2 h and then the solvent was removed in va-
cuo, dissolved in ethyl acetate and washed with water and brine to
give the 2-(3-benzoylphenyl)-2-methylacetate (0.032 g, 100%
yield). To a solution of the 2-(3-benzoylphenyl)-2-methylacetate
(0.032 g, 0.119 mmol) in DMF, NaH (60% in paraffin oil) (0.011 g,
0.298 mmol) was added and the mixture was stirred for 30 min
at room temperature followed by addition of n-propyl iodide
(0.023 mL, 0.238 mmol). The mixture was stirred for 2 h at room
temperature and then water (10 mL) was added. The mixture
was extracted with ethyl acetate (2 ꢂ 10 mL) and the organic layer
was successively washed with water (2 ꢂ 10 mL) and brine
(1 ꢂ 10 mL). The organic layer was dried over anhydrous sodium
sulfate and the solvent was removed in vacuo to give methyl
2-(3-benzoylphenyl)-2-methylpentanoate (0.033 g, 89% yield).
Methyl 2-(3-benzoylphenyl)-2-methylpentanoate (0.033 g, 0.106
mmol) was stirred with 1 M aqueous lithium hydroxide (0.425
mL, 0.425 mmol) in MeOH (10 mL) at room temperature for 2 h.
The solvent was removed and the residue was dissolved in ethyl
acetate and washed with 1 M hydrochloric acid followed by water
and brine. The organic layer was dried over anhydrous sodium sul-
fate and reduced in vacuo to give 2-(3-benzoylphenyl)-2-methyl-
pentanoic acid (0.030 g, 0.101 mmol, 95% yield). 1H NMR
(400 MHz, CDCl3) d 7.85 (t, J = 1.7, 1H), 7.79 (dd, J = 5.1, 3.3, 2H),
7.70–7.65 (m, 1H), 7.63–7.55 (m, 2H), 7.50–7.42 (m, 3H), 2.10–
1.88 (m, 2H), 1.61 (s, 3H), 1.34–1.16 (m, 2H), 0.92 (t, J = 7.3, 3H).
This acid was coupled with 1H-indol-6-amine following the gen-
eral procedure to give 40 in 72% yield. 1H NMR (400 MHz, CDCl3)
d 8.52 (s, 1H), 8.08 (s, 1H), 7.89 (t, J = 1.7, 1H), 7.82–7.75 (m, 2H),
7.75–7.69 (m, 1H), 7.66 (ddd, J = 7.9, 1.9, 1.1, 1H), 7.56 (ddd,
J = 8.7, 2.5, 1.2, 1H), 7.53–7.45 (m, 2H), 7.42 (t, J = 7.8, 2H), 7.14
(dd, J = 3.1, 2.5, 1H), 6.97 (s, 1H), 6.63 (dd, J = 8.4, 1.9, 1H), 6.46
(ddd, J = 3.0, 2.0, 0.9, 1H), 2.20–2.00 (m, 2H), 1.69 (s, 3H),
1.44–1.29 (m, 1H), 1.29–1.15 (m, 1H), 0.95 (t, J = 7.3, 3H). HRMS
(ESI (M+H)+ m/z) calcd for C27H27N2O2 411.2073, found 411.2065.
5.3.18. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)-N-(2-(piperidin-
1-yl)ethyl)propanamide (37)
Yield 63%. 1H NMR (400 MHz, CDCl3 + CD3OD) d 7.71 (d, J = 7.8,
2H), 7.59 (t, J = 7.4, 3H), 7.50–7.41 (m, 3H), 7.38–7.28 (m, 2H), 7.21
(d, J = 7.8, 1H), 6.95 (br s, 1H), 6.52 (d, J = 2.9, 1H), 6.38 (br s, 1H),
3.90–3.75 (m, 1H), 3.16–2.91 (m, 4H), 1.97–1.67 (m, 6H), 1.57 (s,
1H), 1.41 (d, J = 6.9, 2H), 1.23–1.14 (m, 1H). HRMS (ESI (M+H)+
m/z) calcd for C31H34N3O2 480.2651, found 480.2661.
5.3.19. 2-(3-Benzoylphenyl)-N-(1H-indol-6-yl)-N-(2-morpholino-
ethyl)propanamide (38)
Yield 58%. 1H NMR (400 MHz, CDCl3) d 8.68 (br s, 1H), 7.71 (d,
J = 7.3, 2H), 7.66–7.48 (m, 4H), 7.44 (dd, J = 10.6, 4.7, 2H), 7.40–
7.27 (m, 3H), 7.00 (br s, 1H), 6.85 (br s, 1H), 6.58 (ddd, J = 2.9,
1.9, 0.8, 1H), 4.09–3.95 (m, 1H), 3.81–3.65 (m, 2H), 3.59 (m, 4H),
2.43 (dd, J = 14.9, 8.8, 6H), 1.40 (d, J = 6.9, 3H). HRMS (ESI (M+H)+
m/z) calcd for C25H23N2O2 383.1760, found 383.1760.
5.3.20. 2-(3-Benzoylphenyl)-N-(3-(dimethylamino)propyl)-N-
(1H-indol-6-yl)propanamide (39)
Yield 67%. 1H NMR (400 MHz, CDCl3) d 7.73 (d, J = 8.2, 2H), 7.68–
7.50 (m, 4H), 7.48 (t, J = 7.7, 2H), 7.42–7.29 (m, 3H), 7.18 (br s, 1H),
6.92 (br s, 1H), 6.55 (d, J = 2.5, 1H), 3.97–3.76 (m, 1H), 3.14 (s, 1H),
2.16–1.88 (m, 5H), 1.44 (d, J = 6.7, 1H). HRMS (ESI (M+H)+ m/z)
calcd for C30H32N3O3 482.2444, found 482.2439.
5.3.21. 2-(3-Benzoylphenyl)-N-(1H-indol-5-yl)-N-propylpropan-
amide (42)
Yield 81%. 1H NMR (400 MHz, CDCl3) d 8.61 (s, 1H), 7.76–7.66
(m, 2H), 7.62 (d, J = 7.5, 1H), 7.56 (t, J = 7.4, 1H), 7.51–7.38 (m,

4810
N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
5.3.25. 2-(3-Benzoylphenyl)-N-(1-propyl-1H-indol-6-yl)propan-
amide (41)
with 100 lL RPMI-1640 (10% FBS) containing the compound or
the DMSO control. After cells were exposed to compound for
The mixture of 23 (0.015 g, 0.041 mmol) and potassium hydrox-
ide (2.5 mg, 0.045 mmol) in DMF (0.2 mL) was stirred for 10 min at
room temperature followed by addition of 1-bromopropane
24 h at 37 °C in 5% CO2, the media was aspirated, and the cells were
lysed with 20
l
L/well of Passive Lysis Buffer (1ꢂ) by placing the
plate on a shaking platform for 15 min at 600 rpm. The luciferase
activity was determined as described above in the method using
C3H10T1/2 cells.
(3.7 lL, 0.041 mmol). The mixture was stirred for another 2 h.
The reaction was quenched by addition of water (5 mL) and ex-
tracted with ethyl acetate (2 ꢂ 5 mL). The organic layer was
washed with water (2 ꢂ 5 mL) followed by brine (5 mL) and dried
over anhydrous sodium sulfate. The solvent was removed in vacuo
and the residue purified by flash chromatography (Biotage SP4,
25+M silica gel column, eluting with hexane ethyl acetate gradient
10–100%) to give 41 in 54% yield. 1H NMR (400 MHz, CDCl3) d 8.05
(s, 1H), 7.85 (s, 1H), 7.83–7.78 (m, 2H), 7.74–7.65 (m, 2H), 7.59 (t,
J = 7.4, 1H), 7.48 (dt, J = 7.4, 6.3, 4H), 7.16 (s, 1H), 7.06 (d, J = 3.1,
1H), 6.69 (dd, J = 8.4, 1.9, 1H), 6.41 (d, J = 2.4, 1H), 4.05 (t, J = 7.1,
2H), 3.81 (q, J = 7.0, 1H), 1.85 (dd, J = 14.4, 7.2, 2H), 1.67 (d,
J = 7.1, 3H), 0.91 (t, J = 7.4, 3H). HRMS (ESI (M+H)+ m/z) calcd for
C27H27N2O2 411.2073, found 411.2072.
5.6. Real-time RT-PCR
Rh30 cells were grown in six-well plates and treated with com-
pound or DMSO control. Plates were incubated for 24 h and cells
were harvested by trypsinization. Total RNA extraction was carried
out using Qiagen RNeasy kit (QIAGEN, Valencia, CA). Each RNA
sample (250 ng) was used for cDNA synthesis using the QuantiTect
Reverse Transcription kit (QIAGEN, Valencia, CA), following DNase
removal according to the manufacturer’s protocol. The real time
PCR reaction was carried out in 10 lL reactions using the Qunati-
Fast SYBR Green PCR kit (QIAGEN, Valencia, CA). one microliter of
each 10 times-diluted cDNA sample was used along with each pri-
mer at 200 nM concentration. The relative quantity of Gli1-target
mRNA level in each sample was determined using the comparative
Ct method where the amount of target, normalized to GAPDH
(endogenous control) and relative to a calibrator, is calculated
5.4. Gli-mediated transcription reporter assay in C3H10T1/2
cells transfected with Gli1 or Gli2
C3H10/T1/2 cells were plated at 2.5 ꢂ 105 cells/dish in two
DD
60 mm culture dishes in 5 mL of basal medium Eagle (BME) (Invit-
using the formula 2ꢁ Ct. The DDCt was calculated by subtracting
rogen Corporation, Carlsbad, CA) containing 2 mM
L
-glutamine and
the DCt (DMSO control) from the DCt (target gene) where DCt is
10% fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT). The
cells were maintained in 5% CO2 at 37 °C in a humidified incubator.
After 24 h, the cells were transfected at approximately 75% conflu-
ence by replacing the media with 5 mL BME (10% FBS) containing
the mixture of plasmids encoding cDNAs of GliLuc-BS (250 ng/
mL), hGli1 (250 ng/mL), and pRL-TK (12.5 ng/mL) (Promega, Mad-
ison, WI) and the transfection reagent Fugene6 (1:3 w/v of DNA)
(F. Hoffmann-La Roche, Basel, Switzerland) and incubated over-
night. A simultaneous control was transfected with empty vector,
pcDNA3, GliLuc, pRL-TK, and Fugene6 in similar ratios as used for
hGli1. Eighteen hours after transfection, the cells were trypsinized
and reconstituted in 10 mL BME (10% FBS) and plated in a white
the value of Ct (target gene or DMSO control) ꢁ Ct (GAPDH). Primer
sequences are:Ptch1: forward 50-TCCCAAGCAAATGTACGAGCA, re-
verse 50-TCCCAAGCAAATGTACGAGCABcl2: forward 50-TGTGTGGA-
GAGCGTCAACC, reverse 50-GCCGTACAGTTCCACAAAGGCyclin-D1:
forward 50-CTCTGTGCCACAGATGTGAA, reverse 50-TCCTGTCCTACT
ACCGCCTCAGAPDH: forward 50-GGAGTCAACGGATTTGGTC, reverse
50-CCACCCATGGCAAATTCCA
5.7. Cell growth suppression assay
The cells were plated in the appropriate medium in a 384-well
plate at 1.0 ꢂ 103 (BJ) or 1.6 ꢂ 103 (other cells) cells/well 24 h be-
fore the addition of the compounds. The 10 mM stock solution of
each compound in DMSO was diluted in EMEM media to prepare
96-well cell culture plate at 100
lL/well. Six hours after plating,
the media in the wells was replaced with 100
lL BME (10% FBS)
containing the compound or the DMSO control. After exposing
the cells to compound for 24 h at 37 °C in 5% CO2, the media was
carefully aspirated to remove the compound, and the cells were
160
licate to get a final concentration of 40
lowed to incubate at 37 °C for 68 h. Alamar Blue (4
l
M solution (4ꢂ), and 10
l
L of that solution was added in trip-
M/well. The cells were al-
L) (Biotium,
l
l
lysed with 20
l
L/well of Passive Lysis Buffer (1ꢂ) by placing the
Inc., Hayward, CA) was added to each well, and the plates were
incubated for 4 h at 37 °C. The fluorescence was measured with
excitation wavelength at 510 nm and emission wavelength at
590 nm using an EnVision Multilabel Plate Reader (Perkin–Elmer,
Waltham, MA, USA). The cell viability was determined by compar-
ing the fluorescence of experimental wells with that of the DMSO
control wells.
plate on a shaking platform for 15 min at 600 rpm. The luciferase
activity was determined using Dual-Luciferase Reporter Assay Sys-
tem (Promega) according to manufacturer’s instructions. The activ-
ity was determined by dividing the luminescence of the firefly
luciferase by that of Renilla luciferase. The assay for mGli2 was car-
ried out following the protocol for the hGli1-transactivation assay
except replacing hGli1 with mGli2.
5.8. Microsomal stability assay
5.5. Gli-mediated transcription reporter assay in Rh30 cells
The NADPH regenerating agent solutions A (catalog#: 451220)
and B (catalog#: 451200) and mouse liver microsomes (CD-1,
female, catalog#: 452702) were obtained from BD Gentest (Woburn,
MA). The microsomal stability assay was carried out as described.22
The 2 mM solutions in 20% DMSO/80% acetonitrile were prepared
from DMSO stock solutions (10 mM) of test compounds. Mouse liver
microsomal solution was prepared by adding 1.27 mL of concen-
trated mouse liver microsomes (20 mg/mL protein concentration)
to 38.6 mL of pre-warmed (37 °C) 0.1 M potassium phosphate buffer
Rh30 cells were plated at 3 ꢂ 105 cells/dish in two 60 mm cul-
ture dishes in 5 mL RPMI-1640 (ATCC, Manassas, VA) containing
10% FBS (Hyclone Laboratories) and were maintained in 5% CO2
at 37 °C in a humidified incubator. After 24 h, the cells were trans-
fected at approximately 70% confluence by replacing the media
with 5 mL RPMI-1640 (10% FBS) containing the DNA mixture of
GliLuc-BS (500 ng/mL) and pRL-CMV (2.5 ng/mL) (Promega), and
Fugene6 (1:3 w/v of DNA) (F. Hoffmann-La Roche, Basel, Switzer-
land) and incubated overnight. Six hours after transfection, the
cells were trypsinized and reconstituted in 10 mL RPMI-1640
(10% FBS) and plated in a white 96-well cell culture plate at
(pH 7.4) containing 102
(protein) microsomal solution. Each test compound (30.4
2 mM solution) was added directly to 2.4 mL of rat liver microsomal
solution and 180 L was transferred to 12 wells of a 96-well plate
l
L of 0.5 M EDTA to make a 0.6329 mg/mL
lL of
100
lL/well. Eighteen hours after plating, the media was replaced
l

N. Mahindroo et al. / Bioorg. Med. Chem. 18 (2010) 4801–4811
4811
(0, 0.5, 1, and 2 h time points each in triplicate). The NADPH regener-
ating agent was prepared by mixing 2.5 mL of NADPH regenerating
agent Solutions A, 0.5 mL of solution B and 7 mL of 0.1 M potassium
B?A) apparent permeability coefficients (Pappa, cm/s) of each com-
pound were calculated using the equation, Pappa = dQ/dt ꢂ 1/AC0.
The flux of a drug across the monolayer is dQ/dt (
lmol/s). The initial
phosphate buffer (pH 7.4). To each well of the 96-well plate, 45
l
L of
drug concentration on the apical side is C0 (
lmol/L). The surface area
the NADPH regenerating agent was added to initiate the reaction,
and the plate was incubated at 37 °C for each time point (0, 0.5, 1,
and 2 h each in triplicate wells). The reaction was quenched by add-
of the monolayer is A (cm2).
Acknowledgments
ing 450
nal concn) as internal control to each well. All of the plates were
centrifuged at 3000 rpm for 15 min and the supernatants (200 L)
were transferred to another 96-well plates for analysis on UPLC–
MS (Waters Acquity UPLC linked to Waters Acquity Photodiode Ar-
ray Detector and Waters Acquity Single Quadrupole Mass Detector)
lL of cold acetonitrile containing ketoconazole (500 nM fi-
We thank Mr. Marcelo Actis and Dr. Anand Mayasundari for
excellent technical support, Dr. Shunsuke Ishii (RIKEN, Tsukuba, Ja-
pan) and Dr. Hiroshi Sasaki (RIKEN, Kobe, Japan) for the gift of Gli1,
Gli2 and Gli-Luc plasmids. This work was supported by the Amer-
ican Lebanese Syrian Associated Charities (ALSAC) and NCI Cancer
Center Support Grant 5 P30 CA021765-31.
l
on Acquity UPLC BEH C18 1.7
lM (2.1 ꢂ 50 mm) column by running
90–5% gradient for water (+0.1% formic acid) and acetonitrile (+0.1%
formicacid). Theareaunderthesingleionrecording(SIR)channelfor
the test compounddivided by the area under theSIR for internalcon-
trol at 0 time concentration was considered as 100% to calculate
remaining concentration at each time point. The terminal phase rate
constant (ke) was estimated by linear regression of logarithmic
transformed concentration versus the data, where ke = slope x
(ꢁln10). The half life t1/2 was calculated as ln2/ ke.
Supplementary data
Supplementary data (author contributions, method for the alka-
line phosphatase assay, data of control experiments (Figs. S1–S4)
and HPLC traces for compounds 20–44) associated with this article
can be found, in the online version, at doi:10.1016/
j.bmc.2010.05.001.
5.9. Parallel artificial membrane permeability assay (PAMPA)
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homogeneous artificial lipid membrane using the normal Double-
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