Discovery of pyrrolo[3,2-c]quinoline-4-one derivatives as novel hedgehog signaling inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.07.039

The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differentiation during embryonic development. Since activation of the Hh signaling pathway is implicated in several types of human cancers, inhibitors of thi

Full text of DOI:10.1016/j.bmc.2012.07.039

Bioorganic & Medicinal Chemistry 20 (2012) 5496–5506
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of pyrrolo[3,2-c]quinoline-4-one derivatives as novel hedgehog
signaling inhibitors
⇑
Tomohiro Ohashi , Yuya Oguro, Toshio Tanaka, Zenyu Shiokawa, Sachio Shibata, Yoshihiko Sato,
Hiroko Yamakawa, Harumi Hattori, Yukiko Yamamoto, Shigeru Kondo, Maki Miyamoto, Hideaki Tojo,
Atsuo Baba, Satoshi Sasaki
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differ-
entiation during embryonic development. Since activation of the Hh signaling pathway is implicated in
several types of human cancers, inhibitors of this pathway could be promising anticancer agents. Using
high throughput screening, thieno[3,2-c]quinoline-4-one derivative 9a was identified as a compound of
interest with potent in vitro activity but poor metabolic stability. Our efforts focused on enhancement of
in vitro inhibitory activity and metabolic stability, including core ring conversion and side chain optimi-
zation. This led to the discovery of pyrrolo[3,2-c]quinoline-4-one derivative 12b, which has a structure
distinct from previously reported Hh signaling inhibitors. Compound 12b suppressed stromal Gli1 mRNA
expression in a murine model and demonstrated antitumor activity in a murine medulloblastoma allo-
graft model.
Received 25 May 2012
Revised 17 July 2012
Accepted 18 July 2012
Available online 31 July 2012
Keywords:
Hedgehog
Hh signaling inhibitor
Pyrrolo[3,2-c]quinoline-4-one
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
nism.6 This mechanism has been detected in a wide range of
solid tumors including colon,7 prostate,8 and pancreatic cancers.9
The hedgehog (Hh) signaling pathway plays an important role
in cell proliferation and control of survival signals1 in tumorigene-
sis as well as in embryogenesis. The mechanism of Hh signal acti-
vation has been elucidated. Three Hh family ligands, Sonic
hedgehog (Shh), Desert hedgehog (Dhh) and Indian hedgehog
(Ihh), bind to the twelve-transmembrane receptor protein Patched
(Ptch), which suppresses the activation of the seven-transmem-
brane signal component protein Smoothened (Smo).2 The activated
Smo upregulates Gli transcription activity, and subsequently, genes
under the control of Gli activate downstream signals that contrib-
ute to cellular proliferation and differentiation. This signaling path-
way is inactive in most adult tissues; however, aberrant activation
driven by Ptch-defective mutations or Hh-ligand overexpression
have been detected in certain types of cancers.3 In the mutation-
driven mechanism, tumor growth is enhanced by autonomous
activation of Gli caused by constitutive Smo activation due to loss
of Ptch function. This mechanism has been observed in medullo-
blastoma4 and basal cell carcinoma (BCC).5 In the Hh-ligand driven
mechanism, the overexpression of Hh ligand in cancer cells leads to
abnormal activation of Gli in neighboring stromal cells, and in-
duces the enhancement of tumor growth by a paracrine mecha-
Based on these results, it has been suggested that an Hh signaling
inhibitor may be useful as a therapeutic agent for these cancers.
To date, several small molecule Hh signaling inhibitors have
been identified (Fig. 1). Cyclopamine, a steroid jerveratum alkaloid
derived from corn lily, showed significant activity in Hh signaling
pathway inhibition10 and directly interacts with Smo protein,
thereby inhibiting tumor growth in several cancer cell types. The
synthetic small molecule Hh signaling inhibitor, vismodegib11
has been approved by FDA in advanced BCC. Other compounds in
development include a cyclopamine-derivative, saridegib (IPI-
926),12 a synthetic compound, NVP-LDE225,13 and small-molecule
inhibitor PF-04449913.14
Several compounds of interest, including thieno[3,2-c]quino-
line-4-one 9a, were identified as Hh signaling inhibitors by high
throughput screening (HTS) in a Gli-luc reporter assay. The chem-
ical structure of 9a is distinct from those of known Hh signaling
inhibitors currently in clinical trials (Figs. 1 and 2). Compound 9a
demonstrated potent inhibition of Hh activity with an IC50 of
5.1 nM in the Gli-luc reporter assay; however 9a was unstable in
mouse hepatic microsomes. In the metabolite analysis, the oxida-
tion of the amide moiety was observed in mouse hepatic micro-
some, thus we conducted chemical modifications of 9a focused
on amide moiety in an effort to enhance metabolic stability while
maintaining in vitro activity. In this report, we describe synthesis
and SAR of this new class of Hh signaling inhibitors.
⇑
Corresponding author. Tel.: +81 466 32 1151; fax: +81 466 29 4448.
E-mail address: tomohiro.oohashi@takeda.com (T. Ohashi).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.039

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
5497
Figure 1. Representative Hh signaling inhibitors.
3. Results and discussion
We initially assessed the in vitro effects of the tricyclic core on
activity (Table 1) using a luciferase reporter in NIH3T3 cells carry-
ing a stably transfected Gli-reporter construct (Gli-luc reporter cell
line).15 The furo analog 9b clearly had decreased Hh inhibitory
activity compared with 9a. On the other hand, a significant in-
crease in Hh inhibition was observed with N-methylpyrrolo analog
9c compared to 9b. We considered the N-methyl moiety of the 5-
membered ring to be essential for potent activity, and thus selected
N-methylpyrrolo[3,2-c]quinoline-4-one as a core ring system for
further investigation.
Although 9c showed potent in vitro activity as mentioned
above, the metabolic stability of this compound assessed by incu-
bation in mouse hepatic microsomes was still insufficient to sug-
gest that 9c would have in vivo efficacy in mice (Table 2). Thus,
our interest was shifted to improvement of the metabolic stability
by optimization of the substituents at the 2, 3, 5-positions of the
pyrrolo[3,2-c]quinoline-4-one.
Figure 2. Structure of compound 9a.
2. Chemistry
Thieno-, pyrrolo-, furo-[3,2-c]quinoline-4-one derivatives were
prepared as shown in Scheme 1 and Scheme 2. Treatment of
methyl anthranilate 1 with diethyl malonate under basic condi-
tions gave quinoline compound 2. Chlorination of 2 using phos-
phoryl chloride was achieved in 79% yield. Hydrolysis of 3 with
sodium acetate led to 4-chloroquinoline 4 selectively, which was
N-alkylated with phenacyl bromide or p-methoxybenzyl chloride
to afford 5a, b. The tricyclic core ring was constructed by treatment
of 5 with corresponding ester reagent under basic conditions.
Alkylation of hydroxyl group of 6 afforded alkoxy derivative 7,
and following hydrolysis gave derivative 8. Carboxylic acid 8 was
converted to various amides (9a–h) by condensation with corre-
sponding amines, 1-ethyl-3-(3-dimethylaminopropyl)carbodiim-
ide hydrochloride (EDC) and 1-hydroxybenzotriazole (HOBt) in
18–93% yield. Piperidine derivative 9i was afforded by removal of
the Boc group of 9h with HCl (Scheme 1).
N-Hydroxyethyl 12a and N-hydroxyacetyl 12b derivatives were
obtained from 9g by its alkylation or acylation in 33% and 51%
yield, respectively. 9i was alkylated with 2-bromoethanol to afford
10, and the removal of the p-methoxybenzyl group was carried out
with trifluoroacetic acid (TFA), anisole, and trifluoromethanesulfo-
nic acid (TfOH) at 80 °C, followed by acetylation to give 11. N-alkyl-
ation of 11 using the corresponding alkyl halides in the presence of
sodium tert-butoxide (tert-BuONa) followed by hydrolysis of the
acetyl group gave 12c, d in 15% and 2.4% yield, respectively
(Scheme 2).
Substitution of methoxy group at the 3-position reduced activ-
ity and was apparently unstable in mouse hepatic microsomes (9d
vs 9c). Therefore, we fixed the substituent at this position as meth-
oxy in the remaining studies. Several derivatives were prepared
with various cyclic amines at the terminal of the side chain at
the 2-position. Ethyl groups with piperidine (9e) or morpholine
(9f) derivatives resulted in decreased Gli-luc reporter activity
without improvement of metabolic stability. The 4-piperidinyl
derivative 9g showed dramatic improvement in metabolic stability
compared to 9c, indicating that the cyclic amine component at-
tached directly to the amide linker decreased lipophilicity and im-
proved metabolic stability. The side chain of 9g is shorter than that
of 9c and we speculated that introduction of the ethyl group on to
the nitrogen of the piperidine ring in 9g was responsible for
enhancing activity. The overlays of the stable conformation of 9g
and ethylated 9g (Et-9g) calculated by Molecular Operating Envi-
ronment (MOE) software were shown in Figure 3. It demonstrated
that the ethyl group of Et-9g could occupy the same space as the
pyrrolidine ring of 9c. The lipophilicity of Et-9g was predicted to
be higher than that of 9g (cLogP: Et-9g = 3.5 vs 9g = 2.5), suggest-
ing that Et-9g may have decreased microsomal stability as a result.
Thus we introduced a hydroxyethyl substituent (clogP value: 2.2)
onto the piperidine nitrogen of 9g instead of the ethyl group posi-
tion. As expected, 12a demonstrated potent Gli-luc reporter activ-
ity while retaining metabolic stability both in mouse and human

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T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
Scheme 1. Reagents and conditions: (a) diethyl malonate, NaOEt, EtOH, rt then 140 °C; (b) POCl3, 110 °C; (c) AcONa, AcOH, 120 °C; (d) PhCOCH2Br, NaH, DMF, rt for 5a, or
PMBCl, NaH, DMF, rt for 5b; (e) EtOOCCH2SH for 6a or EtOOCCH2OH for 6b, NaOEt, EtOH, rt; (f) (1) MeNHCH2COOEtÁHCl, Et3N, EtOH, 85 °C; (2) NaOEt, EtOH, 50 °C for 6c; (g)
Et3N, EtOH, 85 °C then NaOEt, EtOH, 60 °C for 6d; (h) MeI or EtI, DBU, DMF, rt; (i) NaOH, EtOH, H2O, rt; (j) R3NH2, EDC, HOBt, Et3N, DMF; (k) HCl, AcOEt, rt.
Scheme 2. Reagents and conditions: (a) 2-bromoethanol, K2CO3, DMF, 80 °C for 10, 100 °C for 12a; (b) (1) ClCOCH2OAc, Et3N, THF, 0 °C, (2) NaOH, EtOH, rt; (c) (1) TFA, anisole,
TfOH, 80 °C; (2) AcCl, Et3N, THF, 0 °C; (d) EtCOCH2Br or PhCH2CH2Br, tert-BuONa, LiBr, DME/DMF, 0 °C to rt. (e) NaOH, EtOH, H2O, rt.

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
5499
Table 1
not shown) which is involved in cardiac repolarisation. Since basi-
city of compounds is often correlated with hERG inhibition,16 the
acyl function was introduced to decrease basicity. Compound
12b, bearing a hydroxyacetylpiperidine moiety, achieved both po-
tent activity and good metabolic stability without hERG inhibition,
as expected.
Effect of tricyclic ring core in the Gli-luc reporter assay
Finally, the SAR around the 5-position was investigated. The 2-
butanoyl derivative 12c, which had an ethyl group in place of the
benzene ring in 12b, exhibited significantly decreased potency.
The phenethyl derivative 12d without the carbonyl group showed
30-fold drop in activity compared with 12a. These results sug-
gested that an aromatic substituent at the 5-position would be
necessary for tight binding to the Smo protein. Therefore, the
phenacyl group in 12b was selected as the best substituent at
the 5-position for further evaluation.
The in vivo pharmacodynamic and pharmacokinetic profiles of
12b are shown in Table 3. PAN-04 is a human pancreatic xenograft
tumor line derived from a clinical specimen established by the
Central Institute for Experimental Animals (Kanagawa, Japan). This
xenograft tumor expressed significant stroma-derived Gli1 and
cancer-derived Shh activity. We measured the reduction of Gli1
mRNA expression levels as a pharmacodynamic marker in this
model. Compound 12b showed potent activity in a Smo binding
assay (IC50: 41 nM), favorable in vitro metabolic stability in both
Compds
X
Gli-luc reporter IC50 (nM)a
9a
9b
9c
S
O
NMe
5.1
>1000
2.9
a
IC50 values represent the mean of four measurements.
microsomes. However, further evaluation of 12a showed strong
mouse and human microsomes, and
a
favorable mouse
inhibition of the human ether-a-go-go related gene (hERG; data
Table 2
Effect of substituents on pyrrolo[3,2-c]quinoline-4-one at 2, 3, and 5-position
Compds
R1
R2
R3
Gli-luc reporter IC50 (nM)a
In vitro clearanceb
Mouse
(
l
L/min/mg)
Human
9c
A
A
Me
Et
2.9
7.0
95
14
26
9d
155
9e
9f
A
A
A
Me
Me
Me
9.6
22
21
192
237
10
30
73
2
9g
12a
12b
A
A
Me
Me
6.6
4.6
9
6
11
<1
12c
12d
B
C
Me
Me
>100
150
<1
48
3
72
a
IC50 values are the mean of four measurements.
Hepatic microsomes.
b

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T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
Figure 3. Overlay of Et-9g (green), and 9c (purple) in stable conformation.
Table 3
Pharmacokinetic and pharmacodynamic characteristics of compound 12b
Compd
Smo binding (nM)
In vivo PD Gli mRNA (% of ctrl)a
In vitro clearanceb
(l
L/min/mg)
Mouse PKc
MRT (h)
2.99
Mouse
11
Human
<1
Cmax
(l
g/ml)
Tmax (h)
AUC (
12.1
lgh/mL)
C8h (h)
0.604
12b
41
5
2.65
1.00
a
Gli1 mRNA expression at 25 mg/kg BID, 24 h post-dose. The value indicates Gli1 mRNA expression levels compared to controls. Compound 12b was dosed at 25 mg/kg,
BID.
b
Hepatic microsomes.
Cassette oral dosing at 10 mg/kg.
c
Figure 4. Antitumor activity (left) and body weight change (right) upon treatment with 12b in nude mice carrying Ptch+/À p53À/À medulloblastoma allograft tumors.
Compound 12b was administrated orally at a dose of 6.25 mg/kg BID for 14 days (d); vehicle controls (s). Each point represents the mean SD of duplicate values. ⁄P <0.025
by a 1-tailed Dunnett’s test compared to controls.
pharmacokinetic profile. Consistent with the results of in vitro
activities and ADME profiles, 12b strongly suppressed Gli1 mRNA
expression after a single cycle of dosing at 25 mg/kg, twice daily
(BID).
4. Conclusions
In these studies, HTS to identify Hh signaling inhibitors resulted
in a novel lead compound 9a, which possesses a thieno[3,2-c]quin-
oline-4-one core structure. Our medicinal chemistry efforts, focus-
ing on maintaining in vitro activity while significantly improving
metabolic stability, led to pyrrolo[3,2-c]quinoline-4-one 12b with
a characteristic side chain at the 2-position. Compound 12b
showed suppression of Gli1 mRNA expression and virtually com-
plete growth suppression in the medulloblastoma allograft model.
This in vivo efficacy encouraged us to seek Hh signaling inhibitors
that might be considered as candidates for clinical research in pa-
tients with cancers that are characterized by aberrant Hh signaling
activity.
Compound 12b demonstrated marked suppression of Gli1
mRNA in the PAN-04 model. This supported in vivo efficacy studies
with compound 12b in a medulloblastoma allograft model gener-
ated from Ptch+/À p53À/À mice in which the Hh signaling path-
way was constitutively activated. Oral administration of
compound 12b for two weeks at 6.25 mg/kg BID resulted in virtu-
ally complete suppression of tumor growth (T/C ratio = 3%; Fig. 4,
left) without notable body weight loss (Fig. 4, right). It was sug-
gested that compound 12b might be effective in the treatment of
cancers that are activated by Hh signaling in mice.

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
5501
J = 7.2 Hz), 4.53 (2H, q, J = 7.2 Hz), 7.34 (1H, t, J = 7.8 Hz), 7.43
(1H, d, J = 8.1 Hz), 7.63 (1H, t, J = 7.8 Hz), 8.00 (1H, d, J = 8.1 Hz),
12.41 (1H, br s).
5. Experimental section
Melting points were determined on a Büchi melting point appa-
ratus and were not corrected. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded on
a Varian Gemini-300
5.4. Ethyl 4-chloro-2-oxo-1-(2-oxo-2-phenylethyl)-1,2-
dihydroquinoline-3-carboxylate (5a)
(300 MHz) or Bruker DPX300 (300 MHz) instrument. Chemical
shifts are reported as d values (ppm) downfield from internal tetra-
methylsilane of the indicated organic solution. Abbreviations for
peak multiplicities: s, singlet; d, doublet; t, triplet; q, quartet; dd,
doublet of doublet; ddd, doublet of doublet of doublets; dt, doublet
of triplet; br s, broad singlet; m, multiplet. Coupling constants (J
values) are given in hertz (Hz). Element analyses were carried
out by Takeda Analytical Laboratories, and the results were within
0.4% of theoretical values. LC–MS spectra were obtained on a Shi-
madzu Corporation LC–MS system (LCMS-2010A). Column chro-
matography was carried out on silica gel columns (Kieselgel 60,
63–200 mesh, Merck, Darmstadt, Germany) or basic silica gel col-
umns (ChromatorexÒ NH-DM1020, 100–200 mesh, Fuji Silysia
NaH (60% in oil, 1.70 g, 41.7 mmol) was added to a solution of
compound 4 (10.0 g, 39.7 mmol) in DMF (160 mL) under ice-cool-
ing, and the mixture was stirred for 15 min. Phenacyl bromide
(8.70 g, 43.7 mmol) was added under ice-cooling, and the resulting
mixture was stirred for 1 h. The reaction mixture was added to
water, and the mixture was extracted with AcOEt. The extract
was washed with brine, dried over MgSO4, and concentrated in va-
cuo. The residue was purified by silica gel column chromatography
(AcOEt/hexane = 1/2 to 1/1) to give the title compound (10.5 g,
71%) as a pale yellow solid. 1H NMR (DMSO-d6) d 1.42 (3H, t,
J = 7.2 Hz), 4.47 (2H, q, J = 7.2 Hz), 5.80 (2H, s), 7.02 (1H, d,
J = 8.1 Hz), 7.34 (1H, t, J = 7.8 Hz), 7.53–7.60 (3H, m), 7.65–7.70
(1H, m), 8.06–8.13 (3H, m).
Chemical Ltd., Kasugai, Japan) or Purif-PackÒ columns (SI 60
or NH 60 M, Fuji Silysia). Reactions were monitored by thin layer
lM
l
chromatography (TLC) analysis on silica gel 60F254 plates (Merck)
or NH TLC plates (Fuji Silysia). X-ray structural analyses were
determined using MOE version 2010.10; Chemical Computing
Group, Inc., Montreal, Quebec, Canada. ClogP values were calcu-
lated by Daylight Software ClogP, version 4.82, Daylight Chemical
Information Systems, Inc., Aliso Viejo, CA.
5.5. Ethyl 4-chloro-1-(4-methoxybenzyl)-2-oxo-1,2-
dihydroquinoline-3-carboxylate (5b)
Similar to the preparation of 5a, the title compound (10.8 g,
79%) was obtained as a white powder from 4 (10.0 g, 39.8 mmol).
1H NMR (DMSO-d6) d 1.34 (3H, t, J = 7.1 Hz), 3.70 (3H, s), 4.40
(2H, q, J = 7.1 Hz), 5.48 (2H, s), 6.89 (2H, d, J = 8.7 Hz), 7.19 (2H,
d, J = 8.7 Hz), 7.42 (1H, t, J = 7.8 Hz), 7.61 (1H, d, J = 8.7 Hz), 7.69–
7.75 (1H, m), 8.04 (1H, dd, J = 7.8, 1.2 Hz).
5.1. Ethyl 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate
(2)
A 20% solution of NaOEt in EtOH (274 g) was added to a solution
of methyl anthranilate (121 g, 0.800 mol) and diethyl malonate
(128 g, 0.800 mol) in EtOH (900 mL), and the mixture was stirred
at room temperature for 30 min. The mixture was heated at
140 °C under stirring for 14 h removing EtOH by a Dean-Stark trap.
After cooling, the residual solid was washed with Et2O and dis-
solved in water. After removal of insoluble solid by filtration, the
filtrate was acidified with 5 M HCl, and the precipitated solid
was collected by filtration. The solid was washed with water and
dried in vacuo to give the title compound (161 g, 86%) as a pale yel-
low powder. 1H NMR (DMSO-d6) d 1.31 (3H, t, J = 7.1 Hz), 4.34 (2H,
q, J = 7.1 Hz), 7.19–7.29 (2H, m), 7.63 (1H, dt, J = 1.2, 7.8 Hz), 7.94
(1H, d, J = 8.1 Hz), 11.51 (1H, br s), 13.40 (1H, br s).
5.6. Ethyl 3-hydroxy-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydrothieno[3,2-c]quinoline-2-carboxylate (6a)
A mixture of ethyl thioglycolate (6.10 g, 50.5 mmol), 20% solu-
tion of NaOEt in EtOH (17.2 g, 50.5 mmol), and EtOH (50 mL) was
stirred at room temperature for 5 min. Compound 5a (9.30 g,
25.3 mmol) was added and the mixture was stirred at room tem-
perature for 18 h. The mixture was acidified with 2 M HCl and
the resulting mixture was stirred for 30 min. The precipitated solid
was collected by filtration, washed with water and Et2O to give the
title compound (10.0 g, 96%) as a white powder. 1H NMR (DMSO-
d6) d 1.32 (3H, t, J = 7.0 Hz), 4.25–4.35 (2H, m), 6.01 (2H, s), 7.40
(1H, t, J = 7.2 Hz), 7.55–7.67 (4H, m), 7.77 (1H, t, J = 7.5 Hz), 8.00–
8.07 (1H, m), 8.07–8.19 (2H, m), 10.57 (1H, br s).
5.2. Ethyl 2,4-dichloroquinoline-3-carboxylate (3)
A mixture of 2 (75.0 g, 0.320 mol) and POCl3 (200 mL) was stir-
red at 110 °C for 6 h. After cooling, the reaction mixture was con-
centrated in vacuo. The residue was dissolved in a small amount
of AcOEt and the mixture was poured into ice water followed by
extraction with AcOEt. The extract was washed with 1 M NaOH,
water, and brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by basic silica gel column chromatogra-
phy (AcOEt/hexane = 1/5) to give the title compound (68.0 g,
5.7. Ethyl 3-hydroxy-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydrofuro[3,2-c]quinoline-2-carboxylate (6b)
Similar to the preparation of 6a, the title compound (1.10 g,
69%) was obtained as a white powder from 5a (1.50 g, 4.06 mmol).
1H NMR (DMSO-d6) d 1.34 (3H, t, J = 7.1 Hz), 4.35 (2H, q, J = 7.1 Hz),
5.96 (2H, s), 7.39 (1H, t, J = 7.7 Hz), 7.51 (1H, d, J = 8.4 Hz), 7.60–
7.67 (3H, m), 7.74–7.79 (1H, m), 8.05 (1H, dd, J = 7.7, 1.4 Hz),
8.15–8.18 (2H, m), 10.55 (1H, br s).
79%) as
a d 1.47 (3H, t,
white solid. 1H NMR (DMSO-d6)
J = 7.2 Hz), 4.54 (2H, q, J = 7.2 Hz), 7.71 (1H, t, J = 7.5 Hz), 7.85
(1H, t, J = 7.5 Hz), 8.06 (1H, d, J = 8.2 Hz), 8.24 (1H, d, J = 8.2 Hz).
5.8. Ethyl 3-hydroxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-
4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-2-carboxylate (6c)
5.3. Ethyl 4-chloro-2-oxo-1,2-dihydroquinoline-3-carboxylate
(4)
A mixture of 5a (52.0 g, 0.14 mol), sarcosine ethyl ester hydro-
chloride (25.6 g, 0.16 mol), Et3N (28.5 g, 0.28 mol), and EtOH
(500 mL) was stirred at 85 °C for 12 h. After cooling, the mixture
was diluted with water (2200 mL) and extracted with AcOEt. The
combined extracts were washed with brine, dried over MgSO4, and
concentrated in vacuo. The residue was crystallized with hexane/
AcOEt to give the ethyl 4-[(2-ethoxy-2-oxoethyl)(methyl)amino]-
A
mixture of 3 (68.0 g, 0.250 mol) and AcONa (21.7 g,
0.260 mol) in AcOH (200 mL) was stirred at 120 °C for 20 h. After
the reaction mixture was added to water, the precipitated solid
was collected and washed with water to give the title compound
(58.8 g, 94%) as a white solid. 1H NMR (CDCl3) d 1.46 (3H, t,

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T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
2-oxo-1-(2-oxo-2-phenylethyl)-1,2-dihydroquinoline-3-carboxyl-
ate (51.5 g) as pale yellow crystals. A mixture of the compound
(51.5 g) obtained above, 20% solution of NaOEt in EtOH (46.6 g,
0.14 mol), and EtOH (1000 mL) wasstirredat50 °C for 18 h. Themix-
ture was diluted with water (500 mL) then acidified with 1 M HCl
(140 mL), the resulting mixture was stirred for 30 min. The precipi-
tated solid was collected by filtration, washed with water and EtOH
successively, and dried in vacuo to give the title compound (37.5 g,
66%) as a white solid. 1H NMR (DMSO-d6) d 1.33 (3H, t, J = 7.2 Hz),
4.30–4.38 (5H, m), 5.93 (2H, s), 7.30–7.40 (2H, m), 7.48–7.53 (1H,
m), 7.61–7.66 (2H, m), 7.73–7.79 (1H, m), 8.15–8.18 (2H, m), 8.34–
8.37 (1H, m), 9.00 (1H, s). Anal. calcd for C23H20N2O5: C, 68.31; H,
4.98; N, 6.93. Found: C, 68.41; H, 5.08; N, 7.11.
m), 8.15–8.18 (2H, m), 8.38 (1H, dd, J = 8.3, 1.4 Hz). Anal. calcd
for C24H22N2O5: C, 68.89; H, 5.30; N, 6.69. Found: C, 68.52; H,
5.26; N, 6.61.
5.13. Ethyl 3-ethoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-
4,5-dihydro-1H-pyrrolo[3,2-c]-quinoline-2-carboxylate (7d)
Yield 100%, white powder. 1H NMR (DMSO-d6) d 1.27–1.37 (6H,
m), 4.16 (2H, q, J = 6.9 Hz), 4.29–4.36 (5H, m), 5.95 (2H, s), 7.27–
7.39 (2H, m), 7.50 (1H, t, J = 8.0 Hz), 7.63 (2H, t, J = 7.3 Hz), 7.76
(1H, t, J = 7.4 Hz), 8.17 (2H, d, J = 7.3 Hz), 8.38 (1H, d, J = 8.1 Hz).
5.14. Ethyl 3-methoxy-5-(4-methoxybenzyl)-1-methyl-4-oxo-
4,5-dihydro-1H-pyrrolo[3,2-c]-quinoline-2-carboxylate (7e)
5.9. Ethyl 3-hydroxy-5-(4-methoxybenzyl)-1-methyl-4-oxo-4,5-
dihydro-1H-pyrrolo[3,2-c]quinoline-2-carboxylate (6d)
Yield 61%, pale yellow solid. 1H NMR (DMSO-d6) d 1.35 (3H, t,
J = 7.1 Hz), 3.69 (3H, s), 3.94 (3H, s), 4.26 (3H, s), 4.34 (2H, q,
J = 7.1 Hz), 5.51 (2H, br s), 6.86 (2H, d, J = 8.7 Hz), 7.15 (2H, d,
J = 8.7 Hz), 7.25–7.31 (1H, m), 7.47 (2H, d, J = 3.6 Hz), 8.34 (1H, d,
J = 8.1 Hz).
A mixture of 5b (16.5 g, 44.47 mmol), sarcosine ethyl ester
hydrochloride (10.2 g, 66.5 mmol), Et3N (27.5 mL, 0.197 mol), and
EtOH (170 mL) was stirred at 85 °C for 18 h. A 20% solution of NaO-
Et in EtOH (75.3 g, 0.221 mol) was added, and the mixture was stir-
red at 60 °C for 1 h. The mixture was concentrated in vacuo, and
the residue was suspended with water (100 mL). The suspension
was acidified with 5 M HCl at 0 °C, and the mixture was stirred
at room temperature for 1.5 h. The precipitate was collected by fil-
tration, washed with water and EtOH to give the title compound
(14.6 g, 81%) as a brown solid. 1H NMR (DMSO-d6) d 1.33 (3H, t,
J = 7.0 Hz), 3.69 (3H, s), 4.28 (3H, s), 4.34 (2H, q, J = 7.0 Hz), 5.48
(2H, br s), 6.80–6.93 (2H, m), 7.16 (2H, d, J = 8.7 Hz), 7.23–7.35
(1H, m), 7.43–7.52 (2H, m), 8.33 (1H, d, J = 8.1 Hz), 9.10 (1H, s).
5.15. 3-Methoxy-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydrothieno[3,2-c]quinoline-2-carboxylic acid (8a)
A solution of 7a (5.70 g, 13.5 mmol) and 2 M NaOH (60 mL) in
THF (200 mL)/EtOH (100 mL) was stirred at room temperature
for 18 h. The reaction mixture was neutralized with 2 M HCl, and
the resulting solid was collected by filtration. The solid was
washed with water and Et2O to give the title compound (4.20 g,
79%) as a pale pink powder. 1H NMR (DMSO-d6) d 3.93 (3H, s),
5.98 (2H, s), 7.35 (1H, t, J = 7.8 Hz), 7.48 (1H, d, J = 8.7 Hz), 7.56–
7.67 (3H, m), 7.77 (1H, t, J = 7.2 Hz), 8.04 (1H, dd, J = 8.1, 1.2 Hz),
8.16–8.19 (2H, m), 13.43 (1H, s).
5.10. Ethyl 3-methoxy-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydrothieno[3,2-c]quinoline-2-carboxylate (7a)
The following compounds 8b–e were prepared in a manner
similar to that described for 8a.
DBU (4.30 g, 18.3 mmol) was added at room temperature to a stir-
red solution of compound 6a (7.00 g, 17.1 mmol) in DMF (150 mL).
Afterstirringfor10 min, MeI(4.00 g,28.0 mmol)wasadded.Themix-
ture was stirred at room temperature for 4 h, and concentrated in va-
cuo. The residue was diluted with water, and extracted with AcOEt/
THF. The organic layer was washed with brine, dried over MgSO4,
and concentrated in vacuo. The residual solid was collected and
washed with AcOEt/Et2O to give the title compound (5.70 g, 79%) as
a pale green powder. 1H NMR (DMSO-d6) d 1.34 (3H, t, J = 7.1 Hz),
3.95 (3H, s), 4.34 (2H, q, J = 7.1 Hz), 5.98 (2H, s), 7.37 (1H, t,
J = 7.5 Hz), 7.49 (1H, d, J = 8.7 Hz), 7.57–7.67 (3H, m), 7.76 (1H, t,
J = 7.5 Hz), 8.06 (1H, d, J = 7.8 Hz), 8.16–8.19 (2H, m).
5.16. 3-Methoxy-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydrofuro[3,2-c]quinoline-2-carboxylic acid (8b)
Yield 72%, white powder. 1H NMR (DMSO-d6) d 4.12 (3H, s), 5.99
(2H, s), 7.42 (1H, t, J = 7.6 Hz), 7.54 (1H, d, J = 7.8 Hz), 7.62–7.67
(3H, m), 7.77 (1H, t, J = 7.8 Hz), 8.07 (1H, dd, J = 7.6, 1.5 Hz),
8.16–8.18 (2H, m), 13.35 (1H, br s).
5.17. 3-Methoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydro-1H-pyrrolo[3,2-c]quinoline-2-carboxylic acid (8c)
Yield 91%, white powder. 1H NMR (DMSO-d6) d 3.88 (3H, s), 4.31
(3H, s), 5.95 (2H, s), 7.29–7.39 (2H, m), 7.46–7.52 (1H, m), 7.61–
7.66 (2H, m), 7.73–7.78 (1H, m), 8.16–8.18 (2H, m), 8.38 (1H, dd,
J = 8.3, 1.1 Hz), 12.92 (1H, s).
The following compounds 7b–e were prepared in a manner
similar to that described for 7a.
5.11. Ethyl 3-methoxy-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydrofuro[3,2-c]quinoline-2-carboxylate (7b)
5.18. 3-Ethoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydro-1H-pyrrolo[3,2-c]quinoline-2-carboxylic acid (8d)
Yield 23%, white powder. 1H NMR (DMSO-d6) d 1.35 (3H, t,
J = 7.1 Hz), 4.14 (3H, s), 4.37 (2H, q, J = 7.1 Hz), 5.99 (2H, s), 7.39–
7.45 (1H, m), 7.55 (1H, d, J = 8.7 Hz), 7.62–7.68 (3H, m), 7.74–
7.80 (1H, m), 8.09 (1H, dd, J = 7.8, 1.5 Hz), 8.16–8.18 (2H, m). Anal.
calcd for C23H19NO6: C, 68.14; H, 4.72; N, 3.46. Found: C, 67.91; H,
4.78; N, 3.42.
Yield 89%, white powder. 1H NMR (DMSO-d6) d 1.27 (3H, t,
J = 7.0 Hz), 4.16 (2H, q, J = 7.0 Hz), 4.31 (3H, s), 5.95 (2H, s), 7.27–
7.39 (2H, m), 7.49 (1H, t, J = 7.4 Hz), 7.63 (2H, t, J = 7.4 Hz), 7.76
(1H, t, J = 7.4 Hz), 8.15–8.18 (2H, m), 8.38 (1H, d, J = 7.2 Hz),
12.83 (1H, s). Anal. calcd for C23H20N2O5ꢀH2O: C, 65.39; H, 5.25;
N, 6.63. Found: C, 65.65; H, 4.95; N, 6.73.
5.12. Ethyl 3-methoxy-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-2-
carboxylate (7c)
5.19. 3-Methoxy-5-(4-methoxybenzyl)-1-methyl-4-oxo-4,5-
dihydro-1H-pyrrolo[3,2-c]quinoline-2-carboxylic acid (8e)
Yield 55%, white powder. 1H NMR (DMSO-d6) d 1.35 (3H, t,
J = 7.1 Hz), 3.89 (3H, s), 4.29–4.37 (5H, m), 5.96 (2H, s), 7.30–7.40
(2H, m), 7.48–7.53 (1H, m), 7.61–7.66 (2H, m), 7.73–7.78 (1H,
Yield 96%, pale yellow solid. 1H NMR (DMSO-d6) d 3.70 (3H, s),
3.94 (3H, s), 4.28 (3H, s), 5.51 (2H, br s), 6.86 (2H, d, J = 8.7 Hz), 7.15

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
5503
(2H, d, J = 8.7 Hz), 7.25–7.30 (1H, m), 7.46 (2H, d, J = 3.9 Hz), 8.34
(1H, d, J = 8.4 Hz), 12.90 (1H, br s).
5.24. 3-Methoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-N-(2-
piperidin-1-ylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-2-
carboxamide (9e)
5.20. 3-Methoxy-4-oxo-5-(2-oxo-2-phenylethyl)-N-(2-
pyrrolidin-1-ylethyl)-4,5-dihydrothieno[3,2-c]quinoline-2-
carboxamide (9a)
Yield 90%, white crystals; mp 230 °C (recrystallized from THF/
EtOH). 1H NMR (DMSO-d6) d 1.30–1.65 (6H, m), 2.30–2.60 (6H,
m), 3.40–3.45 (2H, m), 4.01 (3H, s), 4.38 (3H, s), 5.97 (2H, s),
7.29–7.39 (2H, m), 7.45–7.51 (1H, m), 7.63 (2H, t, J = 7.5 Hz),
7.73–7.78 (1H, m), 8.11–8.19 (3H, m), 8.38 (1H, dd, J = 8.4,
EDC (94 mg, 0.49 mmol) was added to a mixture of 8a (130 mg,
0.33 mmol), 1-(2-aminoethyl)-pyrrolidine (56 mg, 0.49 mmol), and
HOBt (67 mg, 0.49 mmol) in DMF (4 mL), and the mixture was stir-
red at room temperature for 18 h. The reaction mixture was diluted
with saturated NaHCO3 aq and extracted twice with AcOEt. The
combined organic extracts were washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by ba-
sic silica gel column chromatography (hexane/AcOEt = 1/1 to 0/1),
and crystallized from hexane/AcOEt to give the title compound
(100 mg, 62%) as white crystals; mp. 190 °C. 1H NMR (DMSO-d6)
d 1.72 (4H, br s), 2.49 (4H, m), 2.64 (2H, t, J = 6.3 Hz), 3.44 (2H, t,
J = 5.9 Hz), 4.01 (3H, s), 6.00 (2H, s), 7.35 (1H, t, J = 7.8 Hz), 7.49
(1H, d, J = 8.1 Hz), 7.55–7.67 (3H, m), 7.76 (1H, t, J = 7.5 Hz), 8.05
(1H, dd, J = 8.1, 1.5 Hz), 8.16–8.19 (3H, m). Anal. calcd for
1.2 Hz).
29H32N4O4Á0.2H2O: C, 69.08; H, 6.48; N, 11.11. Found: C, 69.06;
H, 6.45; N, 11.09.
LC–MS:
m/z = 501
(MH+).
Anal.
calcd
for
C
5.25. 3-Methoxy-1-methyl-N-(2-morpholin-4-ylethyl)-4-oxo-5-
(2-oxo-2-phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-
2-carboxamide (9f)
Yield 82%, white crystals; mp 228 °C (recrystallized from AcOEt/
THF). 1H NMR (DMSO-d6) d 2.40–2.55 (6H, m), 3.40–3.50 (2H, m),
3.61 (4H, t, J = 4.2 Hz), 4.01 (3H, s), 4.37 (3H, s), 5.97 (2H, s),
7.29–7.40 (2H, m), 7.48 (1H, t, J = 7.7 Hz), 7.64 (2H, t, J = 7.4 Hz),
7.76 (1H, t, J = 7.4 Hz), 8.12–8.19 (3H, m), 8.38 (1H, d, J = 8.4 Hz).
LC–MS: m/z = 503 (MH+). Anal. calcd for C28H30N4O5: C, 66.92; H,
6.02; N, 11.15. Found: C, 67.04; H, 5.93; N, 11.28.
C
27H27N3O4S: C, 66.24; H, 5.56; N, 8.58. Found: C, 66.08; H, 5.50;
N, 8.58.
The following compounds 9b-h were prepared in a manner sim-
ilar to that described for 9a.
5.26. 3-Methoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-N-
piperidin-4-yl-4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-2-
carboxamide (9g)
5.21. 3-Methoxy-4-oxo-5-(2-oxo-2-phenylethyl)-N-(2-
pyrrolidin-1-ylethyl)-4,5-dihydrofuro[3,2-c]quinoline-2-
carboxamide (9b)
Yield 85%, white crystals; mp 206 °C (recrystallized from AcOEt/
EtOH). 1H NMR (DMSO-d6) d 1.34–1.46 (2H, m), 1.78–1.84 (2H, m),
1.90–2.20 (1H, m), 2.50–2.60 (2H, m), 2.85–3.00 (2H, m), 3.80–3.95
(1H, m), 3.98 (3H, s), 4.32 (3H, s), 5.97 (2H, s), 7.29–7.39 (2H, m),
7.45–7.50 (1H, m), 7.63 (2H, t, J = 7.5 Hz), 7.76 (1H, t, J = 7.5 Hz),
7.86 (1H, d, J = 7.8 Hz), 8.16–8.18 (2H, m), 8.37 (1H, dd, J = 8.4,
Yield 66%, white crystals; mp 171 °C (recrystallized from
AcOEt). 1H NMR (DMSO-d6) d 1.69–1.73 (4H, m), 2.49–2.51 (4H,
m), 2.61 (2H, t, J = 6.6 Hz), 3.40–3.46 (2H, m), 4.15 (3H, s), 6.00
(2H, s), 7.43 (1H, t, J = 8.1 Hz), 7.54 (1H, d, J = 8.4 Hz), 7.60–7.67
(3H, m), 7.74–7.79 (1H, m), 8.06 (1H, t, J = 8.6 Hz), 8.15–8.19 (3H,
m). LC–MS: m/z = 474 (MH+). Anal. calcd for C27H27N3O5: C,
68.48; H, 5.75; N, 8.87. Found: C, 68.32; H, 5.68; N, 8.97.
1.2 Hz).
27H28N4O4Á1.0H2O: C, 66.11; H, 6.16; N, 11.42. Found: C, 65.82;
H, 6.05; N, 11.32.
LC–MS:
m/z = 473
(MH+).
Anal.
calcd
for
C
5.27. tert-Butyl 4-({[3-methoxy-5-(4-methoxybenzyl)-1-methyl-
4-oxo-4,5-dihydro-1H-pyrrolo[3,2-c]quinolin-2-
yl]carbonyl}amino)piperidine-1-carboxylate (9h)
5.22. 3-Methoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-N-(2-
pyrrolidin-1-ylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-
2-carboxamide (9c)
Yield 93%, white powder. 1H NMR (DMSO-d6) d 1.33–1.58 (11H,
m), 1.76–1.94 (2H, m), 2.83–3.09 (2H, m), 3.69 (3H, s), 3.79–3.95
(2H, m), 3.95–4.11 (4H, m), 4.27 (3H, s), 5.52 (2H, br s), 6.81–
6.91 (2H, m), 7.14 (2H, d, J = 8.7 Hz), 7.22–7.32 (1H, m), 7.38–
7.53 (2H, m), 7.97 (1H, d, J = 7.9 Hz), 8.33 (1H, d, J = 7.9 Hz). LC–
MS: m/z = 575 (MH+).
Yield 78%, white crystals; mp 204 °C (recrystallized from
AcOEt). 1H NMR (DMSO-d6) d 1.65–1.75 (4H, m), 2.48–2.55 (4H,
m), 2.62 (2H, t, J = 6.2 Hz), 3.39–3.45 (2H, m), 3.97 (3H, s), 4.37
(3H, s), 5.97 (2H, s), 7.29–7.39 (2H, m), 7.45–7.50 (1H, m), 7.61–
7.66 (2H, m), 7.73–7.78 (1H, m), 8.16–8.18 (3H, m), 8.39 (1H, dd,
J = 8.1, 1.2 Hz). LC–MS: m/z = 487 (MH+). Anal. calcd for
C
28H30N4O4: C, 69.12; H, 6.21; N, 11.51. Found: C, 69.16; H, 6.20;
5.28. 3-Methoxy-5-(4-methoxybenzyl)-1-methyl-4-oxo-N-
piperidin-4-yl-4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-2-
carboxamide hydrochloride (9i)
N, 11.59.
5.23. 3-Ethoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-N-(2-
pyrrolidin-1-ylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]quinoline-
2-carboxamide (9d)
4 M HCl in AcOEt solution (5 mL) was added to a solution of 9h
(500 mg, 0.87 mmol) in AcOEt (15 mL), and the mixture was stirred
at room temperature for 4 h. The precipitate was collected by fil-
tration, washed with diethyl ether, and dried in vacuo to give the
title compound (440 mg, 99%) as a pale pink powder. 1H NMR
(DMSO-d6) d 1.78 (2H, q, J = 10.3 Hz), 2.05 (2H, d, J = 10.8 Hz),
3.06 (2H, t, J = 10.7 Hz), 3.26–3.33 (2H, m), 3.63 (3H, s), 4.02 (3H,
s), 4.09–4.13 (1H, m), 4.24 (3H, s), 5.40–5.60 (2H, m), 6.86 (2H, d,
J = 8.7 Hz), 7.14 (2H, d, J = 8.7 Hz), 7.25–7.30 (1H, m), 7.42–7.49
(2H, m), 8.15 (1H, d, J = 7.5 Hz), 8.33 (1H, d, J = 7.8 Hz), 8.60–8.90
(2H, m). LC–MS: m/z = 475 (MH+-HCl).
Yield 18%, white crystals; mp 217 °C (recrystallized from AcOEt/
THF). 1H NMR (DMSO-d6) d 1.32 (3H, t, J = 7.0 Hz), 1.65–1.75 (4H,
m), 2.40–2.55 (4H, m), 2.62 (2H, t, J = 6.0 Hz), 3.05–3.47 (2H, m),
4.29 (2H, q, J = 7.0 Hz), 4.39 (3H, s), 5.96 (2H, s), 7.29–7.39 (2H,
m), 7.48 (1H, t, J = 7.8 Hz), 7.63 (2H, t, J = 7.7 Hz), 7.76 (1H, t,
J = 7.4 Hz), 8.12–8.19 (3H, m), 8.38 (1H, d, J = 8.4 Hz). LC–MS: m/
z = 501 (MH+). Anal. calcd for C29H32N4O4: C, 69.58; H, 6.44; N,
11.19. Found: C, 69.83; H, 6.26; N, 10.63.

5504
T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
5.29. N-[1-(2-Hydroxyethyl)piperidin-4-yl]-3-methoxy-5-(4-
methoxybenzyl)-1-methyl-4-oxo-4,5-dihydro-1H-pyrrolo[3,2-
c]quinoline-2-carboxamide (10)
7.63 (2H, t, J = 7.6 Hz), 7.76 (1H, t, J = 7.1 Hz), 7.87 (1H, d,
J = 7.5 Hz), 8.17 (2H, d, J = 7.6 Hz), 8.37 (1H, d, J = 8.4 Hz). LC–MS:
m/z = 517 (MH+). Anal. calcd for C29H32N4O5: C, 67.43; H, 6.24; N,
10.85. Found: C, 67.28; H, 6.20; N, 10.79.
A mixture of 9i (9.39 g, 18.4 mmol), 2-bromoethanol (3.25 mL,
45.6 mmol) and K2CO3 (10.2 g, 73.6 mmol) in DMF (250 mL) was
stirred at 80 °C for 16 h. After cooling, the reaction mixture was
concentrated in vacuo and the residue was partitioned between
saturated NaHCO3 aq and AcOEt. The aqueous layer was separated
and extracted with AcOEt/THF. The combined extracts were
washed with brine, dried over MgSO4, and concentrated in vacuo.
The residual precipitate was collected by filtration, and washed
with hexane/AcOEt to give the title compound (7.75 g, 81%) as
white powder. 1H NMR (DMSO-d6) d 1.43–1.71 (2H, m), 1.77–
1.94 (2H, m), 2.08–2.26 (2H, m), 2.40 (2H, t, J = 6.3 Hz), 2.70–2.86
(2H, m), 3.50 (2H, q, J = 6.2 Hz), 3.69 (3H, s), 3.73–3.94 (1H, m),
4.04 (3H, s), 4.29 (3H, s), 4.37 (1H, t, J = 5.4 Hz), 5.52 (2H, br s),
6.78–6.91 (2H, m), 7.14 (2H, d, J = 8.7 Hz), 7.22–7.33 (1H, m),
7.38–7.53 (2H, m), 7.90 (1H, d, J = 7.7 Hz), 8.33 (1H, d, J = 7.9 Hz).
LC–MS: m/z = 519 (MH+).
5.32. N-[1-(Hydroxyacetyl)piperidin-4-yl]-3-methoxy-1-methyl-
4-oxo-5-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-
c]quinoline-2-carboxamide (12b)
Acetoxyacetyl chloride (47.3
lL, 0.440 mmol) was added to a
mixture of 9g (200 mg, 0.423 mmol), Et3N (122
l
L, 0.880 mmol),
and THF (10 mL) at 0 °C and the mixture was stirred at room tem-
perature for 15 h. The reaction mixture was diluted with AcOEt,
washed with water and brine, dried over MgSO4, and concentrated
in vacuo. The residue was purified by basic silica gel column chro-
matography (AcOEt) followed by crystallization from AcOEt/THF to
give 2-[4-({[3-methoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-
4,5-dihydro-1H-pyrrolo[3,2-c]quinolin-2-yl]car-
bonyl}amino)piperidin-1-yl]-2-oxoethyl acetate (171 mg, 75%) as a
white powder. The compound obtained was dissolved in 8 M NaOH
aq (1 mL), THF (2 mL), and EtOH (7 mL). After stirring at room tem-
perature for 2 h, the reaction mixture was diluted with water, acid-
ified with 6 M HCl aq, and extracted with AcOEt. The extract was
washed with brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by basic silica gel column chromatogra-
phy (AcOEt) followed by crystallization from AcOEt/THF to give
the title compound (59.2 mg, 51%) as white crystals; mp 223 °C.
1H NMR (DMSO-d6) d 1.35–1.65 (2H, m), 1.80–2.00 (2H, m),
2.85–3.00 (1H, m), 3.05–3.25 (1H, m), 3.60–3.75 (1H, m), 3.97
(3H, s), 4.00–4.30 (4H, m), 4.31 (3H, s), 4.51 (1H, t, J = 5.3 Hz),
5.97 (2H, s), 7.29–7.39 (2H, m), 7.48 (1H, t, J = 7.6 Hz), 7.63 (2H,
t, J = 7.6 Hz), 7.76 (1H, t, J = 7.4 Hz), 7.95 (1H, d, J = 8.4 Hz), 8.17
(2H, d, J = 7.6 Hz), 8.37 (1H, d, J = 8.1 Hz). LC–MS: m/z = 531
(MH+). Anal. calcd for C29H30N4O6: C, 65.65; H, 5.70; N, 10.56.
Found: C, 65.60; H, 5.62; N, 10.46.
5.30. 2-(4-{[(3-Methoxy-1-methyl-4-oxo-4,5-dihydro-1H-
pyrrolo[3,2-c]quinolin-2-yl)carbonyl]-amino}piperidin-1-
yl)ethyl acetate (11)
A
mixture of 10 (498 mg, 0.959 mmol), anisole (0.4 mL,
3.68 mmol), TfOH (1 mL), and TFA (4 mL) was stirred at 80 °C for
1 h. After the mixture was concentrated in vacuo, the residue
was quenched with aqueous NaHCO3 solution and diluted with
AcOEt/THF = 1/1 solution (40 mL). The organic layer was separated
and the aqueous layer was extracted with AcOEt/THF = 1/1 solu-
tion. NaCl (15 g) was added to the aqueous layer and was extracted
with THF. The combined organic layer was dried over MgSO4, and
concentrated in vacuo. Et3N (0.4 mL, 2.87 mmol) and acetyl chlo-
ride (164
the residue in THF (10 mL). The mixture was stirred at 0 °C for
2 h, Et3N (0.4 mL, 2.87 mmol) and acetyl chloride (164 L,
lL, 2.31 mmol) were added at 0 °C to a suspension of
l
5.33. N-[1-(2-hydroxyethyl)piperidin-4-yl]-3-methoxy-1-
methyl-4-oxo-5-(2-oxobutyl)-4,5-dihydro-1H-pyrrolo[3,2-
c]quinoline-2-carboxamide (12c)
2.31 mmol) were further added. After stirring at 0 °C for 2 h, the
mixture was quenched with aqueous NaHCO3 solution (30 mL)
and extracted with AcOEt. The organic layer was washed with
brine, dried over MgSO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hexane/
AcOEt = 1/2 to AcOEt/MeOH = 4/1) to give the title compound
(221 mg, 52%) as a pale yellow powder. 1H NMR (DMSO-d6) d
1.46–1.66 (2H, m), 1.76–1.92 (2H, m), 2.02 (3H, s), 2.13–2.30
(2H, m), 2.55 (2H, t, J = 5.9 Hz), 2.69–2.86 (2H, m), 3.70–3.92 (1H,
m), 4.03 (3H, s), 4.11 (2H, t, J = 5.9 Hz), 4.29 (3H, s), 7.15–7.28
(1H, m), 7.35–7.52 (2H, m), 7.85 (1H, d, J = 7.7 Hz), 8.24 (1H, d,
J = 8.1 Hz), 11.33 (1H, s). LC–MS: m/z = 441 (MH+).
tert-BuONa (89.3 mg, 0.929 mmol) was added to a suspension of
11 (299 mg, 0.679 mmol) in DME (2.4 mL)/DMF (0.6 mL) and the
mixture was stirred at 0 °C for 30 min. LiBr (142 mg, 1.64 mmol)
was added, and the mixture was stirred at room temperature for
10 min. 1-Bromo-2-butanone (76.6 lL, 0.750 mmol) was added
and the mixture was stirred at room temperature for 65 h. After
addition of 1 M NaOH aq (1.5 mL) and MeOH (1 mL), the mixture
was stirred at room temperature for 30 min, and concentrated in va-
cuo. The residue was partitioned between AcOEt/THF = 3/1 solution
(20 mL) and aq NaHCO3 solution (20 mL). The organic layer was sep-
arated, and the aq layer was extracted with AcOEt/THF = 3/1 solu-
tion (20 mL Â 3). The organic layer was combined, washed with
brine (10 mL), dried over MgSO4, and concentrated in vacuo. The
residue was suspended with AcOEt (10 mL) and DMF (1 mL) at
60 °C, and insoluble material was removed by filtration. The filtrate
was concentrated in vacuo, and purified by basic silica gel column
chromatography (hexane/AcOEt = 1/2 to AcOEt/MeOH = 9/1). Crys-
tallization from IPE/AcOEt gave the title compound (48.7 mg, 15%)
as white crystals; mp 191 °C. 1H NMR (DMSO-d6) d 1.01 (3H, t,
J = 7.3 Hz), 1.45–1.67 (2H, m), 1.75–1.91 (2H, m), 2.08–2.25 (2H,
m), 2.39 (2H, t, J = 6.3 Hz), 2.70 (2H, q, J = 7.2 Hz), 2.70–2.85 (2H,
m), 3.42–3.57 (2H, m), 3.71–3.91 (1H, m), 3.98 (3H, s), 4.29 (3H, s),
4.38 (1H, t, J = 5.3 Hz), 5.29 (2H, s), 7.24–7.37 (2H, m), 7.43–7.56
(1H, m), 7.87 (1H, d, J = 7.9 Hz), 8.28–8.40 (1H, m). LC–MS: m/
z = 469 (MH+). Anal. calcd for C25H32N4O5: C, 64.09; H, 6.88; N,
11.96. Found: C, 63.73; H, 6.83; N, 11.81.
5.31. N-[1-(2-Hydroxyethyl)piperidin-4-yl]-3-methoxy-1-
methyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-
pyrrolo[3,2-c]quinoline-2-carboxamide (12a)
A
(42.5
mixture of 9g (200 mg, 0.399 mmol), 2-bromoethanol
lL, 0.599 mmol), K2CO3 (248 mg, 1.80 mmol), and DMF
(5 mL) was stirred at 100 °C for 15 h. The reaction mixture was di-
luted with water, and extracted with AcOEt. The extract was
washed with brine, dried over MgSO4, and concentrated in vacuo.
The residue was purified by basic silica gel column chromatogra-
phy (AcOEt) followed by crystallization from AcOEt/EtOH to give
the title compound (68.0 mg, 33%) as white crystals; mp 201 °C.
1H NMR (DMSO-d6) d 1.51–1.62 (2H, m), 1.75–1.90 (2H, m), 2.18
(2H, t, J = 10.5 Hz), 2.40 (2H, t, J = 5.9 Hz), 2.70–2.85 (2H, m),
3.47–3.52 (2H, m), 3.75–3.90 (1H, m), 3.98 (3H, s), 4.32–4.38
(4H, m), 5.97 (2H, s), 7.29–7.39 (2H, m), 7.47 (1H, t, J = 7.6 Hz),

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
5505
5.34. N-[1-(2-Hydroxyethyl)piperidin-4-yl]-3-methoxy-1-
5.37. In vivo pharmacodynamic assay
methyl-4-oxo-5-(2-phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-
c]quinoline-2-carboxamide (12d)
The in vivo pharmacodynamic assay was conducted in nude
mice bearing human primary pancreatic tumors (PAN-04). The tu-
mor line was established by Central Institute for Experimental Ani-
mals and test compounds were orally administered twice daily.
After 24 h from the first administration, tumors were excised and
treated with RNAlater (Ambion). Total RNA samples were isolated
using RNeasy Mini kit (Qiagen), and first strand cDNA samples
were prepared using a high capacity cDNA transcription kit (Ap-
plied Biosystems). The primer sets of qPCR for quantification of
stromal Gli1 mRNA (Applied Biosystems) were as follows:
Mm00494645_m1 (mouse Gli1), 4352339E (mouse GAPDH).
In a similar manner to the preparation of 12c, the title com-
pound (8.2 mg, 2.4%) was obtained as beige crystals from 11
(299 mg, 0.678 mmol); mp 163 °C (crystallized from IPE/AcOEt).
1H NMR (DMSO-d6) d 1.46–1.67 (2H, m), 1.76–1.91 (2H, m),
2.09–2.25 (2H, m), 2.40 (2H, t, J = 6.3 Hz), 2.67–2.84 (2H, m),
2.86–2.99 (2H, m), 3.44–3.55 (2H, m), 3.70–3.91 (1H, m), 3.98
(3H, s), 4.28 (3H, s), 4.37 (1H, t, J = 5.4 Hz), 4.45–4.58 (2H, m),
7.17–7.28 (1H, m), 7.28–7.40 (5H, m), 7.56–7.67 (1H, m), 7.69–
7.78 (1H, m), 7.86 (1H, d, J = 7.7 Hz), 8.36 (1H, dd, J = 8.4, 1.2 Hz).
LC–MS: m/z = 503 (MH+). Anal. calcd for C29H34N4O4Á0.5H2O: C,
68.08; H, 6.90; N, 10.95. Found: C, 67.77; H, 6.76; N, 10.72.
5.38. In vivo anti-tumor test
5.35. Gli-luc reporter assay
Anti-tumor effects of test compounds were evaluated using a
mouse medulloblastoma allogeneic transplantation model.17 The
medulloblastoma spontaneously occurred in the cerebellum of 7
to 9-week-old Ptch1 (+/À) and p53 (À/À) double-mutant mice.
Ptch1 gene mutant mice (Ptch1tm1Mps/J) were purchased from
The Jackson Laboratory, Bar Harbor, ME; and p53 gene mutant
mice (P53N4-M; B6.129-Trp53tm/BrdN4) were purchased from
Taconic, Hudson, NY. Medulloblastoma allograft tumors were
transplanted subcutaneously into nude mice (CAnN.Cg-Foxn1/nu/
CrlCrlj; Charles River Laboratories, Yokohama, Japan), and were
used for compound testing after several passages in vivo. When tu-
mors reached an average size of 150–250 mm3, animals were oral-
ly dosed with test compounds (0.5% methylcellulose suspension)
or vehicle alone twice daily for 2 weeks. Tumor size was measured
with an electronic vernier caliper, and tumor volume was calcu-
lated based on the longest (a) and shortest (b) tumor dimensions
using the formula volume (V) = (a  b2)/2. The tumor growth rate
(T/C%) was calculated as the mean values for [Treated (VendÀV-
start)/Control (VendÀVstart)] Â 100.
In vitro activities of these compounds were evaluated using a
luciferase reporter in NIH3T3 cells carrying a stably transfected
Gli-reporter construct (Gli-luc reporter cell line).16 IH3T3/Gli-luc
cells were maintained in DMEM containing 10% FBS, 500 lg/mL
G418, and 0.1% gentamicin solution (Invitrogen Corp., Carlsbad,
CA, USA). Cells were seeded onto collagen-coated 384-well plates
at 7.5 Â 103cells/well and cultured overnight in 25
lL of DMEM
containing 10% FBS under 5% CO2 at 37 °C. After incubation,
20
ing 2% FBS) and 5
0.0003–10 M in DMEM) were added to each well to achieve the
final concentrations of 5.8% FBS, 1 g/mL of Shh-N, and 0.03–
lL of recombinant mouse Shh-N (2.5
lg/mL in DMEM contain-
lL of serially diluted compounds (10Â solution;
l
l
1000 nM of the test compounds (n = 4 wells per concentration).
The cells were incubated for an additional 48 h. To determine the
assay window, cells were incubated in media containing 0.1%
DMSO with or without 1 lg/mL Shh-N to serve as 0% or 100% inhi-
bition controls, respectively (n = 10 wells). Luciferase activities of
reporter cells were measured by Bright-Glo™ (Promega Corp
[Madison, WI, USA]) using the EnVisionÒ plate reader (PerkinEl-
mer, Inc [Waltham, MA, USA]).
5.39. In vitro metabolism with hepatic microsomes
Metabolic stability was evaluated both in mouse and human
microsomes and used according to the manufacturer’s instructions.
Liver microsomes were purchased from Xenotech, LLC, Lenexa, KS,
USA. An incubation mixture consisted of microsomes in 50 mmol/L
5.36. Smo binding assay
293T cells were transfected with pCMV-HA/hSmo using Lipo-
fectamine reagent (Lipofectamine™ 2000, Invitrogen). In brief,
of KH2PO4/K2HPO4 phosphate buffer (pH 7.4) and 1 lmol/L of test
45
l
L of Lipofectamine 2000 and 22.5
l
g of pCMV-HA/hSmo were
compound. The concentration of microsomes was 0.2 mg protein/
mL. An NADPH-generating system containing 25 mmol/L MgCl2,
25 mmol/L glucose-6-phosphate, 2.5 mmol/L beta-NADP+, and
7.5 unit/mL glucose-6-phosphate dehydrogenase was added to
the incubation mixture with a 20% volume of the reaction mixture
to initiate the enzyme reaction. After the addition of the NADPH-
generating system, the mixture was incubated at 37 °C. The reac-
tion was terminated by the addition of MeCN equivalent to the vol-
ume of the reaction mixture. Test compounds in the reaction
mixtures were quantified by HPLC equipped with a UV detector
or LC–MS /MS. To determine metabolic stabilities, chromatograms
were analyzed for parent compound disappearance from the reac-
tion mixtures.
each incubated in 1.5 mL of Opti-MEM I media (Invitrogen) at room
temperature for 5 min and were mixed together. The mixture was
incubated for 20 min at room temperature after which it was
added to 6 Â 106 293T cells in 15 mL of culture medium in a
75 cm2 cell culture flask. After overnight incubation under 5%
CO2 at 37 °C, cells were detached using Versene (Invitrogen) and
resuspended in culture medium. The suspended cells were added
to a 96-well plate at 6 Â 104 cells/well. After removing the media
from each well, 25
and 25 L of BODIPY-cyclopamine (4 nM in DMEM containing 1%
FBS) were added to the culture which was incubated for 1 h
(n = 3 wells per concentration). Final concentrations were 0.03–
1000 nM for the compounds and 2 nM for BODIPY-cyclopamine.
lL of a 2Â stock solution of the compounds
l
To define the assay window, 25
amine solution (final concentration 1
in addition to 25 L of BODIPY cyclopamine to serve as 0% or 100%
inhibition controls (n = 5 wells), respectively. After incubation,
cells were detached using Versene and were resuspended in PBS
supplemented with 2% FBS. Cyclopamine binding competition with
the compounds was determined by measuring the intensity of cell
fluorescence using the Guava easyCyte System (Millipore Corp,
Billerica, MA, USA).
l
L of media or 25
l
L of 2Â cyclop-
5.40. Pharmacokinetic studies
lM) was added to the culture
l
Test compounds were administered at a dose of 10 mg/kg (in
0.5% methylcellulose) as cassette dosing to non-fasted mice. After
oral administration, blood samples were collected and centrifuged
to obtain the plasma fraction. Plasma samples were deproteinized
with MeCN containing an internal standard. After centrifugation,
the supernatant was diluted with
HCOONH4 solution and MeCN (9:1, v/v) and centrifuged again.
a mixture of 0.01 mol/L

5506
T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5496–5506
9. Thayer, S. P.; Magliano, M. P.; Heiser, P. W.; Nielsen, C. M.; Roberts, D. J.;
Lauwers, G. Y.; Qi, Y. P.; Gysin, S.; Castillo, C. F.; Yajnik, V.; Antoniu, B.;
McMahon, M.; Warshaw, A. L.; Hebrok, M. Nature 2003, 425, 851.
10. (a) Taipale, J.; Chen, J. K.; Cooper, M. K.; Wang, B.; Mann, R. K.; Milenkovic, L.;
Scott, M. P.; Beachy, P. A. Nature 2000, 406, 1005; (b) Karhadkar, S. S.; Bova, G.
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The compound concentrations in the supernatant were measured
by LC–MS/MS.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.039.
11. Vismodegiv (Erivedge™) Prescription Information, 2012, Genetech USA, Inc,
South San Francisco CA.
12. Tremblay, M. R.; Lescarbeau, A.; Grogan, M. J.; Tan, E.; Lin, G.; Austad, B. C.; Yu,
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