Discovery of the investigational drug TAK-441, a pyrrolo[3,2-c]pyridine derivative, as a highly potent and orally active hedgehog signaling inhibitor: Modification of the core skeleton for improved solubility

Article abstract of DOI:10.1016/j.bmc.2012.07.034

We recently reported the discovery of the novel pyrrolo[3,2-c]quinoline-4- one derivative 1 as a potent inhibitor of Hedgehog (Hh) pathway signaling. However, the PK evaluation of 1 at high dosage (100 mg/kg) revealed the C _max value 3.63 μg/mL

Full text of DOI:10.1016/j.bmc.2012.07.034

Bioorganic & Medicinal Chemistry 20 (2012) 5507–5517
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of the investigational drug TAK-441, a pyrrolo[3,2-c]pyridine
derivative, as a highly potent and orally active hedgehog signaling
inhibitor: Modification of the core skeleton for improved solubility
Tomohiro Ohashi, Yuya Oguro, Toshio Tanaka, Zenyu Shiokawa, Yuta Tanaka, Sachio Shibata,
Yoshihiko Sato, Hiroko Yamakawa, Harumi Hattori, Yukiko Yamamoto, Shigeru Kondo, Maki Miyamoto,
⇑
Mitsuhiro Nishihara, Yoshimasa Ishimura, 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:
We recently reported the discovery of the novel pyrrolo[3,2-c]quinoline-4-one derivative 1 as a potent
inhibitor of Hedgehog (Hh) pathway signaling. However, the PK evaluation of 1 at high dosage
(100 mg/kg) revealed the Cmax value 3.63 lg/mL, likely due to poor solubility of this compound. Efforts
to improve solubility by reducing the aromatic ring count of the core system led to N-methylpyrrol-
o[3,2-c]pyridine derivative 11. Further optimization of the 3-alkoxy group led to compound 11d with
acceptable solubility and potent Hh inhibitory activity. Compound 11d suppressed transcription factor
Gli1 mRNA expression in tumor-associated stromal tissue and inhibited tumor growth (treatment/control
ratio, 3%) in a mouse medulloblastoma allograft model owing to the improved PK profile based on
increased solubility. Compound 11d (TAK-441) is currently in clinical trials for the treatment of advanced
solid tumors.
Received 25 May 2012
Revised 17 July 2012
Accepted 18 July 2012
Available online 27 July 2012
Keywords:
Hedgehog
Hh signaling inhibitor
Pyrrolo[3,2-c]pyridine-4-one
TAK-441
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ties. Our prior investigations demonstrated that substituents at the
2- and 5-positions were fixed because they played important roles
We reported pyrrolo[3,2-c]quinoline-4-one derivative
1
in potency and metabolic stability as mentioned above. Herein, we
describe the design and synthesis of a novel series of pyrrolo[3,2-
c]pyridine derivatives leading to the identification of our clinical
candidate, 11d (TAK-441).
(Table 1) as a potent Hh signaling inhibitor.1 The phenacyl group
at the 5-position is important for its potent in vitro activity, and
the N-[1-(hydroxyacetyl)piperidinyl] amide side chain at the 2-po-
sition provided both high metabolic stability and reduction of
human ether-a-go-go related gene (hERG) inhibition, which is
important in cardiac repolarization, based on its low basicity.
Compound 1 exhibited potent suppression of tumor growth in
in vivo experiments in a mouse medulloblastoma allograft model.
However, the pharmacokinetic (PK) profile of 1 at high dose in
mice (100 mg/kg) showed poor oral absorption that was not pro-
2. Chemistry
We considered that the removal of the benzene ring from com-
pound 1 would improve solubility by reducing planarity. Retrosyn-
thetic analysis (Fig. 2) demonstrated that ketoesters bearing R1 and
R2 substituents would allow investigation of replacements for the
phenyl ring that was being removed.
The general synthesis of the compounds in this study is outlined
below. Pyridone 4, except for commercially available 4c, was pre-
pared from 2. Ketoester 2 was converted to 3 by amination. Cycli-
zation of 3 with diethyl malonate afforded 4 in 60–92% yield.
Monochlorination of 4 was achieved using phosphoryl chloride in
17–60% yields. Alkylation of 5 with phenacyl bromide and potas-
sium carbonate proceeded in low yield (7–23%) because of the pro-
portional to dose (AUC0–24h 32.3
that this poor oral absorption could be attributed to low solubility
(8.4 g/mL at pH 6.8), we sought to improve the solubility while
lgh/mL; Table 1). Considering
l
maintaining Hh pathway inhibitory activity as shown in Figure 1.
In general, compounds with a tricyclic core show poor solubility
and often low bioavailability.2 Thus, we removed the benzene ring
on pyrrolo[2,3-c]quinoline-4-one in order to decrease planarity. In
addition, we speculated that the residual substituent at the 3-posi-
tion was available for modification of the physicochemical proper-
duction of O-phenacylated compound as
a major product.
Substitution and cyclization of 6 was conducted with sarcosine
ethyl ester hydrochloride under basic conditions3 to obtain pyrrol-
o[3,2-c]pyridine 7. The hydroxyl group at the 3-position of 7 was
⇑
Corresponding author. Tel.: +81 466 32 1146; fax: +81 466 29 4448.
E-mail address: satoshi.sasaki@takeda.com (S. Sasaki).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.034

5508
T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
Table 1
Activity profile and solubility of compound 1
a
Gli-luc reporter IC50 (nM)
In vivo PD Gli1 mRNAb (% of ctrl)
Solubilityc
8.4
(lg/mL)
Mouse PK 10 mg/kgd
Mouse PK 100 mg/kge
Cmax
AUC (
l
gh/mL)
Cmax
AUC (lgh/mL)
4.6
5
2.65
12.1
3.63
32.3
a
b
c
IC50 values are the mean of four measurements.
The value is the Gli1 mRNA expression level at 25 mg/kg BID of compound 1 compared to controls.
Measured at pH 6.8 (Japanese Pharmacopoeia second fluid).
d
e
Cassette dosing, AUC0–8h
.
BALB/c-nu/nu mice, AUC0–24h
.
the 3-ethyl derivative 8h. Finally, saponification of 8 afforded the
corresponding carboxylic acid 9 in 23–99% yield (Scheme 1).
Amidation of carboxylic acid 9 is shown in Scheme 2. Com-
pounds 11e and 11j were synthesized from corresponding carbox-
ylic acids 9e and 9j by stepwise acylation. Compounds 11a–d, 11h,
and 11i were obtained by condensation of corresponding carbox-
ylic acid 9 with amine 13 directly. The requisite 13 was prepared
from 4-Boc-aminopiperidine (12) in three steps: acylation with
acetoxyacetyl chloride, followed by removal of the acetyl and
tert-butoxycarbonyl groups in 80% yield.
3. Results and discussion
In vitro activities of compounds 11a–j were evaluated using
luciferase reporter activities in NIH3T3 cells carrying a stably-
transfected Gli-reporter construct, designated the Gli-luc reporter
cell line. First, modification of the benzene ring to cyclohexane in
the core structure was examined. In the Gli-luc reporter assay,
cyclohexene 11i showed an approximately 20-fold decreased
activity compared with that of 1 (Table 2). This result implied that
a substituent with specific size and shape would be required for
maintaining the potent Gli-luc reporter activity. Downsizing the
cyclohexene ring to a monomethyl group (R1) was effective in
maintaining activity (11j vs 11i). In addition, solubility of 11j
Figure 1. Compound design strategy for improving solubility and maintaining
potency.
treated with various alkylating reagents to afford corresponding
alkoxy derivatives 8a–e, 8i, and 8j. Ethyl derivative 8h was pre-
pared by treatment of 7a with trifluoromethanesulfonic anhydride
and afforded triflate 8f. Following Stille coupling of the obtained
triflate 8f with vinyltributyltin, ethenyl derivative 8g was treated
with palladium on carbon under hydrogen atmosphere to give
Figure 2. Synthetic plan.

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
5509
Scheme 1. Reagents and conditions: (a) NH4OAc, MeOH, rt; (b) diethyl malonate, NaOEt, EtOH, xylene, 120 °C, then 150 °C; (c) POCl3 for 4a,b or POCl3, BnEt3NBr, MeCN, 40 °C,
then reflux for 4c; (d) PhCOCH2Br, K2CO3, DMF, rt for 5a,c or PhCOCH2Br, NaH, DMA, rt for 5b; (e) MeNHCH2COOEtꢀHCl, Et3N, EtOH, reflux; (f) R0X, DBU, DMF, rt for 8b
(R0 = CH2CH2F), 8e, j (R0 = Me); (g) R02SO4, K2CO3, acetone, reflux for 8a (R0 = Et), 8i (R0 = Me); (h) R0OTf, Cs2CO3, DMF, rt for 8c (R0 = CH2CHF2), 8d (R0 = CH2CF3); (i) Tf2O,
pyridine, 60 °C for 8f; (j) vinyltributyltin, Pd(PPh3)4, DMF, 100 °C; (k) Pd/C, H2, THF/MeOH, rt; (l) NaOH, EtOH, H2O, 60 °C.
Scheme 2. Reagents and conditions: (a) (1) 4-amino-1-Boc-piperidine, EDC, HOBt, DMF, rt, (2) 4 M HCl in AcOEt, AcOEt, rt; (b) (1) ClCOCH2OAc, Et3N, THF, rt, (2) NaOH, EtOH,
H2O, rt for 11e, 11j; (c) 13, EDC, HOBt, DMF, rt for 11a–d, 11h, 11i; (d) (1) ClCOCH2OAc, Et3N, THF, 0 °C; (2) 8 M NaOH, EtOH, H2O, rt; (3) 4 M HCl in AcOEt, AcOEt, rt.
was improved compared with that of 1, as expected. Introduction
of an ethyl group at the 6-position resulted in further enhancement
of Hh inhibitory activity without loss of solubility (11e vs 11j).
Among the compounds listed in Table 2, we selected for further
investigation the 6-ethyl derivative 11e which had potent activity
in the Gli-luc reporter assay and good solubility.

5510
T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
Table 2
Table 3
Modification of the core ring
Optimization of pyrroro[3,2-c]pyridine at the 3-position
Compds
R1
–(CH2)4–
R2
Gli-luc reporter IC50 (nM)
Solubilityb
(lg/mL)
a
Compds R4
Gli-luc reporter IC50
(nM)
Solubilityb
g/mL)
AUCc
mL)
(lgh/
a
(
l
11i
11j
11e
1
96
14
5.7
4.6
80
77
63
8.4
Me
Et
H
H
11e
11h
11a
11b
11c
11d
OMe
Et
OEt
OCH2CH2F 4.9
OCH2CHF2 4.4
OCH2CF3
5.7
95
6.9
63
31.647
58
69
84
81
20.678
5.149
5.404
a
IC50 values are the mean of four measurements.
Measured at pH 6.8 (Japanese Pharmacopoeia second fluid).
b
4.4
28.346
a
b
c
IC50 values are the mean of four measurements.
Measured at pH 6.8 (Japanese Pharmacopoeia second fluid).
Cassette dosing at 10 mg/kg, po in mice. AUC: area under the plasma concen-
Compound 11e was examined in an in vivo PD study at 25 mg/kg,
BID, but efficacy was relatively poor. We surmised that the solubil-
ity of compound 11e was insufficient to show good efficacy at the
higher dose. To further improve solubility, substitutions were
made at the 3-position (Table 3). Exchange of the methoxy group
in 11e to an ethyl group significantly decreased activity (11h vs
11e) suggesting that the oxygen atom in the 3-methoxy group in
11e plays an important role in activity. In general, fluorination of
alkyl substituents is known to impact the physicochemical profile
by changing lipophilic and electronic factors.4 Therefore, we inves-
tigated the effect of fluorination on solubility and potency using
the 3-ethoxy derivative 11a as a standard compound with good
activity and solubility.
tration versus time curve from 0 to 8 h.
The PK profiles of 11d in other species are shown in Table 5.
Compound 11d showed favorable oral bioavailability (F), which
was 32% in rats and 90% in dogs. Moreover, low drug clearance
(CL) and high AUC were observed in dogs. These results suggested
that compound 11d is able to achieve sufficient exposure following
oral administration in rats and dogs to enable appropriate toxico-
logical studies.
Both 2-fluoroethoxy (11b) and 2,2-difluoroethoxy (11c) groups
effectively increased solubility, but exposure (AUC) of these com-
pounds in mice following oral dosing was decreased compared
with that of 11a. On the other hand, trifluoroethoxy derivative
11d exhibited favorable exposure compared to those of 11a and
11e. Furthermore, the oral absorption of 11d was better than that
of 1, suggesting that the improvement of solubility contributed to
the improved PK profile at the higher dose (Table 4). Thus, we se-
lected 11d as a compound for further investigation.
As mentioned above, the oxygen atom in the alkoxy group at the
3-position was important for inhibitory activity. An X-ray structural
analysis of 11d suggested an explanation for this finding (Fig. 3).5
An intramolecular hydrogen bond interaction was observed be-
tween the oxygen atom in the 2,2,2-trifluoroethoxy group and the
amide hydrogen atom at the C2 side chain. This conformation
would not be favored in the C3-ethyl derivative. We speculated that
the oxygen atom would be necessary for hydrogen bond formation
with the amide, thereby stabilizing the position of the side chain.
We evaluated the in vivo efficacy of 11d using a Ptc1+/ꢁ p53ꢁ/
ꢁ medulloblastoma allograft model in mice in which the Hh path-
way was activated. Plasma and tumor concentrations of 11d after a
single oral dose are shown in Figure 4 indicating good tumor per-
meability and dose linearity. Repeated oral administration of com-
pound 11d (QD, 14 days) resulted in moderate antitumor activity
with a treated/control (T/C) value of 46% at a dose of 1 mg/kg
(Fig. 5); complete growth inhibition was observed at 25 mg/kg
(T/C = 1%) without excessive accumulation. These data suggest that
improved solubility resulted in a dose-dependent PK profile for
compound 11d.
4. Conclusions
With the aim of improving solubility, chemical modification of
the previously developed compound 1 was examined. Decreasing
the planarity by reducing the number of aromatic rings led to the
identification of the novel pyrrolo[3,2-c]pyridine-4-one derivative
11j which had good solubility. Further optimization of the 3-alkoxy
group resulted in discovery of 11d with good physicochemical pro-
files and potent activity. Compound 11d exhibited strong antitu-
mor activity in an in vivo study in Ptc1+/ꢁ p53ꢁ/ꢁ mice bearing
medulloblastoma allografts; improved solubility of compound
11d enabled dose-dependent plasma and tumor concentrations
to be achieved in this model. On the basis of the overall pharma-
ceutical profile, compound 11d was identified as a potential candi-
date for clinical development, and a clinical trial of 11d (TAK-441)
is currently ongoing.
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
(300 MHz) or Bruker DPX300 (300 MHz) instrument. Chemical
shifts are reported as d values (ppm) down-field from internal tet-
ramethylsilane of the indicated organic solution. Peak multiplici-
ties are expressed using the following abbreviations: 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,

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
5511
Table 4
The mixture was concentrated in vacuo. The residue was diluted
with water (500 mL) and extracted with AcOEt. The extract was
washed with brine, dried over MgSO4, concentrated in vacuo, and
dried to give the title compound (68.5 g, 92%) as a pale yellow
oil. 1H NMR (DMSO-d6) d 1.06 (3H, t, J = 7.6 Hz), 2.09 (2H, q,
J = 7.6 Hz), 3.49 (3H, s), 4.34 (1H, s), 6.94 (1H, s), 7.72 (1H, br s).
Pharmacokinetic data of 1 and 11d
Compd
Mouse PK 10 mg/kga
Mouse PK 100 mg/kgb
Cmax
(lg/mL)
AUC (
l
gh/mL)
Cmax
3.63
21.5
(
lg/mL)
AUC (lgh/mL)
1
11d
2.65
5.62
12.1
28.3
32.3
206
a
Cassette dosing, AUC0–8h
BALB/c-nu/nu mice, AUC0–24h
.
5.2. Ethyl 2-aminocyclohex-1-ene-1-carboxylate (3b)
b
.
In the same manner as in the preparation of 3a, the title com-
pound (32.3 g, 60%) was obtained as a white solid from 2b
(53.9 g, 0.317 mol). 1H NMR (DMSO-d6) d 1.16 (3H, t, J = 7.1 Hz),
1.45–1.59 (4H, m), 2.09–2.21 (4H, m), 4.00 (2H, q, J = 7.1 Hz),
7.09 (2H, br s).
5.3. Ethyl 6-ethyl-4-hydroxy-2-oxo-1,2-dihydropyridine-3-
carboxylate (4a)
A mixture of 3a (100 g, 774 mmol), diethyl malonate (130 mL,
856 mmol), 20% solution of NaOEt in EtOH (290 g, 852 mmol),
EtOH (400 mL), xylene (800 mL) was stirred at 120 °C for 2 h, at
150 °C for 17 h with a Dean–Stark trap. The precipitate was col-
lected by filtration and washed with hexane. The filtered material
was dissolved in water (800 mL) and filtered. The filtrate was acid-
ified with 5 M HCl aq at 0 °C. The precipitate was collected by fil-
tration, successively washed with water and hexane/IPE to give
the title compound (75.4 g, 46%) as a pale yellow powder. 1H
NMR (DMSO-d6) d 1.12 (3H, t, J = 7.6 Hz), 1.26 (3H, t, J = 7.1 Hz),
2.42 (2H, q, J = 7.6 Hz), 4.25 (2H, q, J = 7.1 Hz), 5.79 (1H, s), 11.37
(1H, br s), 12.56 (1H, s).
Figure 3. X-ray crystal structure of compound 11d. The figure was described based
on X-ray structural analysis data using MOE Software. Molecular Operating
Environment (MOE version 2010.10); Chemical Computing Group, Inc.: Montreal,
Quebec, Canada; http://www.chemcomp.com.
5.4. Ethyl 4-hydroxy-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-
carboxylate (4b)
In the same manner as in the preparation of 4a, the title com-
pound (24.4 g, 58%) was obtained as a yellow solid from 3b
(30.0 g, 0.177 mol). 1H NMR (DMSO-d6) d 1.28 (3H, t, J = 7.1 Hz),
1.57–1.73 (4H, m), 2.24–2.35 (2H, m), 2.41–2.48 (2H, m), 4.30
(2H, q, J = 7.1 Hz), 11.19 (1H, s), 13.49 (1H, s).
5.5. Ethyl 4-chloro-6-ethyl-2-oxo-1,2-dihydropyridine-3-
carboxylate (5a)
A mixture of 4a (15.0 g, 71.0 mmol) and POCl3 (19.9 mL,
213 mmol) was stirred at 80 °C for 30 min. The mixture was con-
centrated in vacuo and ice water was added to the residue at
0 °C. The resulting solid was collected by filtration and washed
with water and AcOEt to give the title compound (9.80 g, 60%) as
a white solid. 1H NMR (DMSO-d6) d 1.15 (3H, t, J = 7.5 Hz), 1.26
(3H, t, J = 7.1 Hz), 2.44–2.55 (2H, m), 4.25 (2H, q, J = 7.1 Hz), 6.26
(1H, s), 12.28 (1H, s).
Figure 4. Concentrations of compound 11d in the plasma and tumor after oral
administration at doses of 1 and 25 mg/kg in medulloblastoma allografted mice.
Mean standard deviation, SD (n = 3).
multiplet. Coupling constants (J values) are given in hertz (Hz). Ele-
ment analyses were carried out by Takeda Analytical Laboratories
and the results were within 0.4% of theoretical values. LC–MS spec-
tra were obtained on a Shimadzu Corporation LC–MS system
(LCMS-2010A). Column chromatography was carried out on silica
gel columns (Kieselgel 60, 63–200 mesh, Merck, Darmstadt, Ger-
many) or basic silica gel columns (ChromatorexÒ NH-DM1020,
100–200 mesh, Fuji Silysia Chemical Ltd, Kasugai, Japan) or Purif-
5.6. Ethyl 4-chloro-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-
carboxylate (5b)
A
mixture of 4b (26.0 g, 110 mmol) and POCl3 (51.3 mL,
552 mmol) was stirred at 130 °C for 1.5 h. After cooling, the reac-
tion mixture was concentrated in vacuo and ice was added to the
residue. The mixture was neutralized with saturated NaHCO3 aq
and extracted with AcOEt. The extract was washed with saturated
NaHCO3 aq water and brine, dried over MgSO4, and concentrated in
vacuo. The residual solid was collected by filtration and washed
with hexane/AcOEt to give the title compound (4.65 g, 17%) as a
white solid. 1H NMR (DMSO-d6) d 1.25 (3H, t, J = 7.1 Hz), 1.61–
1.74 (4H, m), 2.37–2.45 (2H, m), 2.51–2.57 (2H, m), 4.24 (2H, q,
J = 7.1 Hz), 12.09 (1H, s).
PackÒ columns (SI 60
lM or NH 60 lM, Fuji Silysia). Reaction pro-
gress was determined by thin layer chromatography (TLC) analysis
on silica gel 60F254 plate (Merck) or NH TLC plates (Fuji Silysia).
5.1. Methyl (2Z)-3-aminopent-2-enoate (3a)
A mixture of 2a (75.0 g, 576 mmol), NH4OAc (222 g, 2.88 mmol)
and MeOH (750 mL) was stirred at room temperature for 3 days.

5512
T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
Figure 5. Efficacy of compound 11d in medulloblastoma in a Ptc1+/ꢁ p53ꢁ/ꢁ mouse allograft model (left) and body weight after 14 days, QD (right). Oral administration of
11d in mice once daily (QD) for 14 days at 1 mg/kg (N) or 25 mg/kg (d) compared with vehicle-treated animals (s). Data are shown as mean SD (n = 5). Antitumor effects
were expressed as the ratio of treatment versus control (T/C, %) which was calculated by comparison of the mean change in tumor volume over the treatment period for the
control and treated groups. ⁄P 60.025 by a 1-tailed Shirley-Williams test compared to vehicle control.
Table 5
Pharmacokinetic profilea in rats and dogs
Vss (mL/kg)
CL (mL/h/kg)
AUC0–24h,iv (ng h/mL)
AUC0–24h,po (ng h/mL)
F (%)
Ratb
681.6 81.6
2181.3 82.8
397.9 10.1
161.3 35.6
2532.3 69.1
5101.5 685.5
8031.8 1218.6
45405.6 5812.0
31.7
90.3 8.8
Dogc
a
Dose: iv, 1 mg/kg; po, 10 mg/kg.
b
Each value except for bioavailabilty (F%) represents the mean SD of three non-fasted animals. The F value is calculated from mean values of AUC0–24h after oral and
intravenous administration.
c
Each value represents the mean SD of four fed animals. The F value is calculated from the individual value of AUC0–24h after oral and intravenous administration for the
same animal.
5.7. Ethyl 4-chloro-6-methyl-2-oxo-1,2-dihydropyridine-3-
carboxylate (5c)
5.61 (2H, s), 6.44 (1H, s), 7.54–7.66 (2H, m), 7.70–7.79 (1H, m),
8.05–8.13 (2H, m).
5.9. Ethyl 4-chloro-2-oxo-1-(2-oxo-2-phenylethyl)-1,2,5,6,7,8-
hexahydroquinoline-3-carboxylate (6b)
A
mixture of 4c (3.00 g, 15.2 mmol), POCl3 (7.75 mL,
83.3 mmol), and benzyltriethylammonium chloride (13.8 g,
60.8 mmol) in MeCN (60 mL) was stirred at 40 °C for 30 min and
at reflux for 30 min. After cooling, the reaction mixture was con-
centrated in vacuo, water was added to the residue, and the mix-
ture was extracted with AcOEt. The extract was washed with
brine, dried over MgSO4, and concentrated in vacuo. The residual
solid was collected and washed with hexane/AcOEt to give the title
compound (1.45 g, 44%) as a white powder. 1H NMR (DMSO-d6) d
1.26 (3H, t, J = 7.0 Hz), 2.20 (3H, s), 4.25 (2H, q, J = 7.0 Hz), 6.26
(1H, s), 12.29 (1H, s).
Compound 5b (3.00 g, 11.7 mmol) was added to a suspension of
NaH (60% in oil, 516 mg, 12.9 mmol) in DMA (30 mL) and the mix-
ture was stirred at room temperature for 30 min. Phenacyl bro-
mide (2.57 g, 12.9 mmol) was added, and the mixture
was stirred at room temperature for 15 h. The mixture was diluted
with water, extracted with AcOEt, and washed with water and
brine. The organic layer was dried over MgSO4, and
concentrated in vacuo. The residue was purified by basic silica
gel column chromatography (hexane/AcOEt = 9/1 to AcOEt) to give
the title compound (710 mg, 16%) as a white powder. 1H NMR
(DMSO-d6) d 1.25 (3H, t, J = 7.1 Hz), 1.60–1.80 (4H, m), 2.50–2.65
(4H, m), 4.26 (2H, q, J = 7.1 Hz), 5.66 (2H, s), 7.61 (2H, t,
J = 7.4 Hz), 7.74 (1H, t, J = 7.4 Hz), 8.09 (2H, d, J = 7.4 Hz).
5.8. Ethyl 4-chloro-6-ethyl-2-oxo-1-(2-oxo-2-phenylethyl)-1,2-
dihydropyridine-3-carboxylate (6a)
A mixture of 5a (9.50 g, 41.4 mmol), K2CO3 (13.7 g, 99.2 mmol),
phenacyl bromide (9.88 g, 49.6 mmol) and DMF (100 mL) was stir-
red at room temperature for 15 h. The reaction mixture was diluted
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 chromatography (hexane/
AcOEt = 9/1 to 3/7) to give the title compound (1.00 g, 7%) as a
white powder. 1H NMR (DMSO-d6) d 1.14 (3H, t, J = 7.4 Hz), 1.24
(3H, t, J = 7.1 Hz), 2.62 (2H, q, J = 7.4 Hz), 4.25 (2H, q, J = 7.1 Hz),
5.10. Ethyl 4-chloro-6-methyl-2-oxo-1-(2-oxo-2-phenylethyl)-
1,2-dihydropyridine-3-carboxylate (6c)
Inthe same mannerasinthe preparation of6a, the titlecompound
(3.18 g, 23%) was obtained as a white powder from 5c (9.00 g,
41.7 mmol). 1H NMR (DMSO-d6) d 1.25 (3H, t, J = 7.0 Hz), 2.30 (3H,
s), 4.25 (2H, q, J = 7.0 Hz), 5.63 (2H, s), 6.54 (1H, d, J = 0.6 Hz), 7.61
(2H, t, J = 7.5 Hz), 7.72–7.78 (1H, m), 8.07–8.10 (2H, m).

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
5513
5.11. Ethyl 6-ethyl-3-hydroxy-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo-[3,2-c]pyridine-2-
carboxylate (7a)
J = 7.3 Hz), 3.85 (3H, s), 4.26 (2H, q, J = 7.1 Hz), 4.32–4.36 (1H, m),
4.42–4.47 (1H, m), 4.53–4.59 (1H, m), 4.69–4.75 (1H, m), 5.58
(2H, s), 6.51 (1H, s), 7.61 (2H, t, J = 7.6 Hz), 7.70–7.77 (1H, m),
8.08–8.14 (2H, m).
A mixture of 6a (7.00 g, 19.3 mmol), sarcosine ethyl ester
hydrochloride (5.94 g, 38.7 mmol), Et3N (29.7 mL, 193 mmol),
and EtOH (100 mL) was stirred at reflux for 2 days. After cooling,
the mixture was diluted with water and acidified with 5 M HCl
aq. The resulting solid was collected and washed with water and
hexane/AcOEt to give the title compound (5.70 g, 74%) as a white
solid. 1H NMR (DMSO-d6) d 1.18 (3H, t, J = 7.4 Hz), 1.31 (3H, t,
J = 7.1 Hz), 2.56 (2H, q, J = 7.4 Hz), 3.80 (3H, s), 4.30 (2H, q,
J = 7.1 Hz), 5.55 (2H, s), 6.43 (1H, s), 7.60 (2H, t, J = 7.6 Hz), 7.68–
7.79 (1H, m), 8.00–8.21 (2H, m), 8.90 (1H, s).
5.16. Ethyl 3-(2,2-difluoroethoxy)-6-ethyl-1-methyl-4-oxo-5-(2-
oxo-2-phenylethyl)-4,5-dihydro-1H-pyrrolo-[3,2-c]pyridine-2-
carboxylate (8c)
2,2-Difluoroethyl
trifluoromethanesulfonate
(336 mg,
1.57 mmol) was added to a mixture of 7a (500 mg, 1.31 mmol)
and Cs2CO3 (554 mg, 1.70 mmol) in DMF (5 mL) and the mixture
was stirred at room temperature for 2 h. The mixture was diluted
with water (15 mL) and the precipitate was collected by filtration.
The collected material was washed with water, EtOH and Et2O, and
dried in vacuo to give the title compound (0.45 g, 77%) as a white
solid. 1H NMR (DMSO-d6) d 1.19 (3H, t, J = 7.4 Hz), 1.30 (3H, t,
J = 7.2 Hz), 2.59 (2H, q, J = 7.4 Hz), 3.86 (3H, s), 4.26 (2H, q,
J = 7.2 Hz), 4.42 (2H, td, J = 14.9, 3.9 Hz), 5.61 (2H, s), 6.27 (1H, tt,
J = 54.9, 3.9 Hz), 6.54 (1H, s), 7.61 (2H, t, J = 7.5 Hz), 7.74 (1H, t,
J = 7.5 Hz), 8.10–8.13 (2H, m).
5.12. Ethyl 3-hydroxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-
4,5,6,7,8,9-hexahydro-1H-pyrrolo[3,2-c]quinoline-2-
carboxylate (7b)
A mixture of the compound 6b (270 mg, 0.724 mmol), sarcosine
ethyl ester hydrochloride (222 mg, 1.45 mmol), Et3N (1.00 mL,
7.24 mmol), and EtOH (5 mL) was stirred at reflux for 2 days. After
cooling, water (10 mL) was added to the mixture and the resulting
solid was collected. This was used for the next reaction without
further purification.
5.17. Ethyl 6-ethyl-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-
(2,2,2-trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-
2-carboxylate (8d)
5.13. Ethyl 3-hydroxy-1,6-dimethyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxylate (7c)
In the same manner as in the preparation of 8c, the title com-
pound (7.81 g, 86%) was obtained as a white solid from 7a
(7.47 g, 19.5 mmol). 1H NMR (DMSO-d6) d 1.19 (3H, t, J = 7.4 Hz),
1.29 (3H, t, J = 7.1 Hz), 2.59 (2H, q, J = 7.4 Hz), 3.86 (3H, s), 4.26
(2H, q, J = 7.1 Hz), 4.83 (2H, q, J = 9.3 Hz), 5.61 (2H, s), 6.56 (1H,
s), 7.61 (2H, t, J = 7.6 Hz), 7.69–7.79 (1H, m), 8.06–8.18 (2H, m).
In the same manner as in the preparation of 7a, the title com-
pound (1.50 g, 68%) was obtained as a white powder from 6c
(2.00 g, 5.99 mmol). 1H NMR (DMSO-d6) d 1.31 (3H, t, J = 7.1 Hz),
2.26 (3H, s), 3.77 (3H, s), 4.30 (2H, q, J = 7.1 Hz), 5.57 (2H, s), 6.52
(1H, s), 7.54–7.67 (2H, m), 7.69–7.79 (1H, m), 8.03–8.18 (2H, m),
8.90 (1 H, s).
5.18. Ethyl 6-ethyl-3-methoxy-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxylate (8e)
5.14. Ethyl 3-ethoxy-6-ethyl-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxylate (8a)
In the same manner as in the preparation of 8b, the title com-
pound (156 mg, 23%) was obtained as a white powder from 7a
(650 mg, 1.70 mmol). 1H NMR (DMSO-d6)
d 1.19 (3H, t,
J = 7.3 Hz), 1.30 (3H, t, J = 7.1 Hz), 2.58 (2H, q, J = 7.4 Hz), 3.84
(3H, s), 3.85 (3H, s), 4.27 (2H, q, J = 7.0 Hz), 5.58 (2H, s), 6.50 (1H,
s), 7.61 (2H, t, J = 7.6 Hz), 7.67–7.82 (1H, m), 8.03–8.19 (2H, m).
K2CO3 (10.6 g, 76.8 mmol) and Et2SO4 (4.03 mL, 30.1 mmol) were
added to a solution of 7a (9.80 g, 25.6 mmol) in acetone (196 mL)
and the mixture was refluxed for 1 h. Et2SO4 (4.03 mL, 30.1 mmol)
and acetone (70 mL) were added, and the mixture was refluxed for
17 h. The mixture was diluted with water (400 mL) and the resulting
precipitate was collected by filtration and washed with water and
hexane/AcOEt to give the title compound (8.58 g, 82%) as a white so-
lid. 1H NMR (DMSO-d6) d 1.13–1.27 (6H, m), 1.31 (3H, t, J = 7.1 Hz),
2.57 (2H, q, J = 7.3 Hz), 3.84 (3H, s), 4.15 (2H, q, J = 7.1 Hz), 4.26
(2H, q, J = 7.1 Hz), 5.57 (2H, s), 6.49 (1H, s), 7.56–7.66 (2H, m),
7.69–7.77 (1H, m), 8.07–8.16 (2H, m).
5.19. Ethyl 6-ethyl-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-
{[(trifluoromethyl) sulfonyl]oxy}-4,5-dihydro-1H-pyrrolo[3,2-
c]pyridine-2-carboxylate (8f)
A
mixture of 7a (300 mg, 0.784 mmol), Tf2O (158 lL,
0.941 mmol), and pyridine (6 mL) was stirred at 60 °C for 3 h under
N2 atmosphere. The mixture was diluted with water (50 mL) and
extracted with AcOEt (100 mL). The extract was washed with brine
(50 mL), dried over MgSO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hexane/
AcOEt = 1/1 to AcOEt) to give the title compound (239 mg, 59%)
as a pale yellow powder. 1H NMR (DMSO-d6) d 1.20 (3H, t,
J = 7.3 Hz), 1.33 (3H, t, J = 7.2 Hz), 2.60 (2H, q, J = 7.3 Hz), 3.95
(3H, s), 4.34 (2H, q, J = 7.2 Hz), 5.64 (2H, s), 6.64 (1H, s), 7.57–
7.68 (2H, m), 7.70–7.81 (1H, m), 8.07–8.19 (2H, m).
5.15. Ethyl 6-ethyl-3-(2-fluoroethoxy)-1-methyl-4-oxo-5-(2-
oxo-2-phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxylate (8b)
2-Fluroroethyl iodide (512 mg, 2.94 mmol) was added to a mix-
ture of 7a (750 mg, 1.96 mmol) and DBU (0.440 mL, 2.94 mmol) in
DMF (10 mL) and the mixture was stirred at room temperature for
15 h. The mixture was diluted with water and extracted with
AcOEt. The organic layer was washed with water and brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified
by basic silica gel column chromatography (AcOEt) to give the title
compound (649 mg, 77%) as a white solid. 1H NMR (DMSO-d6) d
1.19 (3H, t, J = 7.3 Hz), 1.30 (3H, t, J = 7.1 Hz), 2.58 (2H, q,
5.20. Ethyl 3-ethenyl-6-ethyl-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo-[3,2-c]pyridine-2-
carboxylate (8g)
A
mixture of 8f (218 mg, 0.424 mmol), vinyltributyltin
(328
lL, 1.28 mmol), Pd(PPh3)4 (148 mg, 0.128 mmol) and DMF

5514
T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
(5.9 mL) was stirred at 100 °C for 3 h under Ar atmosphere. The
mixture was diluted with water (50 mL) and extracted with
AcOEt (100 mL). The extract was washed with brine (50 mL),
dried over MgSO4, and concentrated in vacuo. The insoluble
material was removed by filtration and the filtrate was concen-
trated in vacuo. The precipitate was collected by filtration and
washed with IPE to give the title compound (120 mg, 72%) as a
grey solid. 1H NMR (DMSO-d6) d 1.20 (3H, t, J = 7.3 Hz), 1.33
(3H, t, J = 7.1 Hz), 2.61 (2H, q, J = 7.3 Hz), 3.85 (3H, s), 4.33 (2H,
q, J = 7.1 Hz), 5.29–5.43 (1H, m), 5.59 (2H, s), 6.41–6.59 (2H,
m), 7.23 (1H, dd, J = 17.7, 11.8 Hz), 7.55–7.67 (2H, m), 7.68–
7.81 (1H, m), 8.01–8.20 (2H, m).
5.25. 6-Ethyl-3-(2-fluoroethoxy)-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo-[3,2-c]pyridine-2-
carboxylic acid (9b)
Yield 56%, white powder. 1H NMR (DMSO-d6) d 1.19 (3H, t,
J = 7.4 Hz), 2.58 (2H, q, J = 7.4 Hz), 3.85 (3H, s), 4.32–4.36 (1H, m),
4.42–4.47 (1H, m), 4.54–4.59 (1H, m), 4.70–4.74 (1H, m), 5.58
(2H, s), 6.50 (1H, s), 7.61 (2H, t, J = 7.6 Hz), 7.70–7.77 (1H, m),
8.09–8.15 (2H, m), 12.48 (1H, br s).
5.26. 3-(2,2-Difluoroethoxy)-6-ethyl-1-methyl-4-oxo-5-(2-oxo-
2-phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxylic acid (9c)
5.21. Ethyl 3,6-diethyl-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]-pyridine-2-
carboxylate (8h)
Yield 83%, white powder. 1H NMR (DMSO-d6) d 1.19 (3H, t,
J = 7.2 Hz), 2.58 (2H, q, J = 7.3 Hz), 3.85 (3H, s), 4.40 (2H, td,
J = 14.6, 1.2 Hz), 5.60 (2H, s), 6.26 (1H, tt, J = 55.0, 3.9 Hz), 6.77 (1H,
s), 7.58–7.76 (3H, m), 8.12 (2H, d, J = 7.5 Hz), 12.50–12.70 (1H, m).
A mixture of 8g (1.33 g, 3.40 mmol), 10% Pd-C (270 mg) and THF
(50 mL) /MeOH (25 mL) was stirred at room temperature for 7 h
under H2 atmosphere. The catalyst was removed by filtration and
the filtrate was concentrated in vacuo. The precipitate was col-
lected by filtration to give the title compound (1.35 g, 100%) as a
white solid. 1H NMR (DMSO-d6) d 1.09 (3H, t, J = 7.2 Hz), 1.19
(3H, t, J = 7.5 Hz), 1.33 (3H, t, J = 7.2 Hz), 2.58 (2H, q, J = 7.2 Hz),
3.07–3.19 (2H, m), 3.87 (3H, s), 4.30 (2H, q, J = 7.2 Hz), 5.59 (2H,
s), 6.48 (1H, s), 7.58–7.64 (2H, m), 7.70–7.77 (1H, m), 8.08–8.14
(2H, m).
5.27. 6-Ethyl-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-
trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxylic acid (9d)
Yield 93%, white powder. 1H NMR (DMSO-d6) d 1.19 (3H, t,
J = 7.4 Hz), 2.58 (2H, q, J = 7.4 Hz), 3.86 (3H, s), 4.82 (2H, q,
J = 9.2 Hz), 5.61 (2H, s), 6.54 (1H, s), 7.61 (2H, t, J = 7.6 Hz), 7.70–
7.77 (1H, m), 8.07–8.16 (2H, m), 12.74 (1H, br s).
5.28. 6-Ethyl-3-methoxy-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo-[3,2-c]-pyridine-2-
carboxylic acid (9e)
5.22. Ethyl 3-methoxy-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5,6,7,8,9-hexahydro-1H-pyrrolo[3,2-c]quinoline-
2-carboxylate (8i)
Yield 79%, white powder. 1H NMR (DMSO-d6) d 1.19 (3H, t,
J = 7.3 Hz), 2.57 (2H, q, J = 7.3 Hz), 3.84 (3H, s), 3.85 (3H, s), 5.58
(2H, s), 6.48 (1H, s), 7.61 (2H, t, J = 7.6 Hz), 7.67–7.86 (1H, m),
7.97–8.31 (2H, m), 12.51 (1H, br s).
In the same manner as in the preparation of 8a, the title com-
pound (34.0 mg, 11% in two steps) was obtained as a white powder
from 7b. 1H NMR (DMSO-d6) d 1.31 (3H, t, J = 7.1 Hz), 1.60–1.77
(4H, m), 2.45–2.55 (2H, m), 2.94–3.02 (2H, m), 3.81 (3H, s), 4.03
(3H, s), 4.27 (2H, q, J = 7.1 Hz), 5.62 (2H, s), 7.61 (2H, t,
J = 7.5 Hz), 7.73 (1H, t, J = 7.5 Hz), 8.10 (2H, d, J = 7.5 Hz).
5.29. 3,6-Diethyl-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-
dihydro-1H-pyrrolo[3,2-c]-pyridine-2-carboxylic acid (9h)
5.23. Ethyl 3-methoxy-1,6-dimethyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo-[3,2-c]pyridine-2-
carboxylate (8j)
Yield 99%, white powder. 1H NMR (DMSO-d6) d 1.07 (3H, t,
J = 7.3 Hz), 1.19 (3H, t, J = 7.4 Hz), 2.57 (2H, q, J = 7.4 Hz), 3.11 (2H,
q, J = 7.3 Hz), 3.87 (3H, s), 5.58 (2H, s), 6.46 (1H, s), 7.56–7.66 (2H,
m), 7.68–7.78 (1H, m), 8.05–8.17 (2H, m), 12.80 (1H, br s).
In the same manner as in the preparation of 8b, the title com-
pound (390 mg, 26%) was obtained as a white powder from 7c
(1.45 g, 3.94 mmol). 1H NMR (DMSO-d6) d 1.30 (3H, t, J = 7.1 Hz),
2.28 (3H, s), 3.81 (3H, s), 3.86 (3H, s), 4.27 (2H, q, J = 7.1 Hz), 5.60
(2H, s), 6.59 (1H, s), 7.54–7.66 (2H, m), 7.69–7.79 (1H, m), 8.05–
8.16 (2H, m).
5.30. 3-Methoxy-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-
4,5,6,7,8,9-hexahydro-1H-pyrrolo-[3,2-c]-quinoline-2-
carboxylic acid (9i)
Yield 23%, white powder. 1H NMR (DMSO-d6) d 1.67 (4H, m),
2.44–2.52 (2H, m), 2.94–3.02 (2H, m), 3.81 (3H, s), 4.05 (3H, s),
5.62 (2H, s), 7.56–7.65 (2H, m), 7.70–7.78 (1H, m), 8.10 (2H, d,
J = 7.6 Hz), 12.56 (1H, br s).
5.24. 3-Ethoxy-6-ethyl-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]-pyridine-2-
carboxylic acid (9a)
5.31. 3-Methoxy-1,6-dimethyl-4-oxo-5-(2-oxo-2-phenylethyl)-
4,5-dihydro-1H-pyrrolo[3,2-c]-pyridine-2-carboxylic acid (9j)
8 M NaOH aq (42.9 mL) was added to
a solution of 8a
(8.58 g, 20.9 mmol) in EtOH (257 mL) and the mixture was stir-
red at 60 °C for 30 min. The mixture was diluted with water
and acidified with 5 M HCl aq The resulting precipitate was
collected by filtration, and washed with water and Et2O to give
the title compound (7.12 g, 89%) as a beige powder. 1H NMR
(DMSO-d6) d 1.12–1.27 (6H, m), 2.57 (2H, q, J = 7.4 Hz), 3.84
(3H, s), 4.16 (2H, q, J = 7.1 Hz), 5.58 (2H, s), 6.48 (1H, s),
7.55–7.66 (2H, m), 7.68–7.78 (1H, m), 8.05–8.19 (2H, m),
12.41 (1H, br s).
Yield 89%, white powder. 1H NMR (DMSO-d6) d 2.28 (3H, s), 3.81
(3H, s), 3.86 (3H, s), 5.60 (2H, s), 6.58 (1H, s), 7.61 (2H, t, J = 7.4 Hz),
7.74 (1H, t, J = 7.3 Hz), 8.11 (2H, d, J = 7.7 Hz), 12.50 (1H, br s).
5.32. 3-Methoxy-1,6-dimethyl-4-oxo-5-(2-oxo-2-phenylethyl)-
N-piperidin-4-yl-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxamide hydrochloride (10a)
A
mixture of 9j (290 mg, 0.818 mmol), EDC (236 mg,
The following compounds 9b–j were prepared in a same man-
ner similar to that described for 9a.
1.23 mmol), HOBt (166 mg, 1.23 mmol), and DMF (5 mL) was

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
5515
stirred at room temperature for 15 h, after which 4-amino-1-Boc-
piperidine (212 mg, 1.06 mmol) was added and the mixture was
stirred at room temperature for 15 h. The mixture was diluted with
water and extracted with AcOEt. The organic layer was washed
with water and brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by basic silica gel column
chromatography (AcOEt) to give tert-butyl 4-({[3-methoxy-1,6-
dimethyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-pyrrolo
[3,2-c]pyridin-2-yl]carbonyl}amino)piperidine-1-carboxylate. 4 M
HCl in AcOEt (4 mL) was added to a solution of the compound ob-
tained above in AcOEt (4 mL) and the mixture was stirred at room
temperature for 2 h. The precipitated solid was collected by filtra-
tion, and washed with AcOEt to give the title compound (240 mg,
62%) as a white powder. 1H NMR (DMSO-d6) d 1.61–1.84 (2H, m),
1.95–2.11 (2H, m), 2.28 (3H, s), 2.92–3.12 (2H, m), 3.20–3.32
(2H, m), 3.83 (3H, s), 3.94–4.15 (4H, m), 5.62 (2H, s), 6.59 (1H, s),
7.61 (2H, t, J = 7.6 Hz), 7.68–7.81 (2H, m), 8.04–8.19 (2H, m), 8.64
(1H, br s.), 8.87 (1H, br s).
m). Anal. Calcd for C28H33FN4O6: C, 62.21; H, 6.15; N, 10.36. Found:
C, 62.03; H, 6.24; N, 10.18. LC–MS: m/z = 541 (MH+).
5.36. 3-(2,2-Difluoroethoxy)-6-ethyl-N-[1-
(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-
carboxamide (11c)
Yield 65%, white crystals; mp 188 °C (recrystallized from ace-
tone/H2O). 1H NMR (DMSO-d6) d 1.19 (3H, t, J = 7.4 Hz), 1.20–
1.50 (2H, m), 1.90 (2H, d, J = 9.9 Hz), 2.59 (2H, q, J = 7.4 Hz), 2.83
(1H, t, J = 12.2 Hz), 3.09 (1H, t, J = 11.7 Hz), 3.68 (1H, d,
J = 12.9 Hz), 3.90 (3H, s), 3.95–4.20 (3H, m), 4.26 (1H, d,
J = 11.7 Hz), 4.51 (1H, t, J = 5.4 Hz), 4.67 (2H, td, J = 16.1, 3.0 Hz),
5.23 (1H, s), 5.61 (2H, s), 6.33 (1H, tt, J = 54.2, 3.0 Hz), 7.49 (1H,
d, J = 7.8 Hz), 7.61 (2H, t, J = 7.5 Hz), 7.73 (1H, t, J = 7.5 Hz), 8.10–
8.12 (2H, m). Anal. Calcd for C28H32F2N4O6: C, 60.21; H, 5.77; N,
10.03. Found: C, 60.23; H, 5.78; N, 10.01. LC–MS: m/z = 559 (MH+).
5.33. 6-Ethyl-3-methoxy-1-methyl-4-oxo-5-(2-oxo-2-
phenylethyl)-N-piperidin-4-yl-4,5 -dihydro-1H-pyrrolo[3,2-
c]pyridine-2-carboxamide hydrochloride (10b)
5.37. 6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-
oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-
dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (11d)
In the same manner as in the preparation of 10a, the title com-
pound (97.2 mg, 74%) was obtained as a white powder from 9e
Yield 76%, white crystals; mp 169 °C (recrystallized from EtOH/
H2O). 1H NMR (DMSO-d6) d 1.19 (3H, t, J = 7.3 Hz), 1.24–1.51 (2H,
m), 1.79–1.98 (2H, m), 2.59 (2H, q, J = 7.3 Hz), 2.75–2.94 (1H, m),
2.98–3.20 (1H, m), 3.57–3.77 (1H, m), 3.86 (3H, s), 3.94–4.17
(3H, m), 4.17–4.38 (1H, m), 4.53 (1H, t, J = 5.3 Hz), 5.05 (2H, q,
J = 9.3 Hz), 5.63 (2H, s), 6.54 (1H, s), 7.51 (1H, d, J = 7.7 Hz), 7.55–
7.67 (2H, m), 7.68–7.82 (1H, m), 8.01–8.21 (2H, m). Anal. Calcd
for C28H31F3N4O6: C, 58.33; H, 5.42; N, 9.72. Found: C, 58.32; H,
5.55; N, 9.63. LC–MS: m/z = 577 (MH+).
(100 mg, 0.271 mmol). 1H NMR (DMSO-d6)
d 1.19 (3H, t,
J = 7.3 Hz), 1.66–1.85 (2H, m), 1.97–2.09 (2H, m), 2.58 (2H, q,
J = 7.4 Hz), 2.95–3.10 (2H, m), 3.19–3.30 (2H, m), 3.87 (3H, s),
3.96–4.14 (4H, m), 5.60 (2H, s), 6.49 (1H, s), 7.55–7.66 (2H, m),
7.69–7.79 (2H, m), 8.12 (2H, d, J = 7.7 Hz), 8.92 (2H, br s).
5.34. 3-Ethoxy-6-ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-
methyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-
pyrrolo[3,2-c]pyridine-2-carboxamide (11a)
5.38. 3,6-Diethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-
4-oxo-5-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-
c]pyridine-2-carboxamide (11h)
EDC (55.6 mg, 0.290 mmol) at 0 °C was added to a mixture of
the compound 10a (74.0 mg, 0.194 mmol), 13 (49.1 mg,
0.252 mmol), HOBt (39.2 mg, 0.290 mmol), and Et3N (34.9 mL,
0.252 mmol) in DMF (3 mL) and the mixture was stirred at room
temperature for 15 h. The reaction mixture was diluted with water
and extracted twice with AcOEt. The combined extract was washed
with water and saturated brine, dried over MgSO4, and concen-
trated in vacuo. The residue was purified by basic silica gel column
chromatography (AcOEt) and the obtained solid was recrystallized
from AcOEt to give the title compound (61.2 mg, 60%) as white
crystals; mp 204 °C. 1H NMR (DMSO-d6) d 1.13–1.29 (6H, m),
1.32–1.56 (2H, m), 1.83–1.96 (2H, m), 2.57 (2H, q, J = 7.4 Hz),
2.80–2.94 (1H, m), 3.04–3.19 (1H, m), 3.59–3.72 (1H, m), 3.90
(3H, s), 3.95–4.14 (3H, m), 4.17–4.27 (1H, m), 4.34 (2H, q,
J = 7.2 Hz), 4.51 (1H, t, J = 5.4 Hz), 5.59 (2H, s), 6.49 (1H, s), 7.56–
7.65 (2H, m), 7.66–7.77 (2H, m), 8.07–8.14 (2H, m). Anal. Calcd
for C28H34N4O6: C 64.35; H 6.56; N 10.72. Found: C, 64.20; H,
6.52; N, 10.61. LC–MS: m/z = 523 (MH+).
Yield 47%, white crystals; mp 141 °C (recrystallized from hex-
ane/AcOEt). 1H NMR (DMSO-d6) d 1.06 (3H, t, J = 7.2 Hz), 1.18
(3H, t, J = 7.3 Hz), 1.30–1.54 (2H, m), 1.81–1.93 (2H, m), 2.54–
2.63 (2H, m), 2.78–2.92 (3H, m), 3.01–3.17 (1H, m), 3.59–3.73
(4H, m), 3.99–4.16 (3H, m), 4.20–4.31 (1H, m), 4.51 (1H, t,
J = 5.5 Hz), 5.58 (2H, s), 6.42 (1H, s), 7.56–7.65 (2H, m), 7.69–7.77
(1H, m), 8.07–8.15 (2H, m), 8.21 (1H, d, J = 7.4 Hz). Anal. Calcd
for C28H34N4O5ꢀ0.5AcOEt: C, 65.44; H, 6.96; N, 10.17. Found: C,
65.46; H, 7.01; N, 10.27. LC–MS: m/z = 507 (MH+).
5.39. N-[1-(Hydroxyacetyl)piperidin-4-yl]-3-methoxy-1-methyl-
4-oxo-5-(2-oxo-2-phenylethyl)-4,5,6,7,8,9-hexahydro-1H-
pyrrolo[3,2-c]quinoline-2-carboxamide (11i)
Yield 76%, colorless oil. 1H NMR (DMSO-d6) d 1.22–1.25 (2H, m),
1.35–1.56 (2H, m), 1.60–1.74 (4H, m), 1.80–1.92 (2H, m), 2.83–3.22
(4H, m), 3.59–3.70 (1H, m), 3.90 (3H, s), 3.98–4.12 (7H, m), 4.51
(1H, t, J = 5.4 Hz), 5.63 (2H, s), 7.56–7.65 (2H, m), 7.69–7.82 (2H,
m), 8.10 (2H, d, J = 8.3 Hz). LC–MS: m/z = 535 (MH+).
The following compounds 11b–d, h, i were prepared in a same
manner similar to that described for 11a.
5.35. 6-Ethyl-3-(2-fluoroethoxy)-N-[1-(hydroxyacetyl)piperidin-
4-yl]-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-
pyrrolo[3,2-c]pyridine-2-carboxamide (11b)
5.40. N-[1-(Hydroxyacetyl)piperidin-4-yl]-3-methoxy-1,6-
dimethyl-4-oxo-5-(2-oxo-2-phenylethyl)-4,5-dihydro-1H-
pyrrolo[3,2-c]pyridine-2-carboxamide (11j)
Yield 80%, white crystals; mp 208 °C (recrystallized from AcOEt/
THF). 1H NMR (DMSO-d6) d 1.19 (3H, t, J = 7.4 Hz), 1.26–1.48 (2H,
m), 1.87 (2H, s), 2.59 (2H, q, J = 7.4 Hz), 2.76–2.91 (1H, m), 3.01–
3.17 (1H, m), 3.57–3.73 (1H, m), 3.92 (3H, s), 3.97–4.13 (3H, m),
4.18–4.32 (1H, m), 4.45–4.81 (5H, m), 5.59 (2H, s), 6.49–6.56
(1H, m), 7.55–7.66 (3H, m), 7.69–7.78 (1H, m), 8.06–8.16 (2H,
Acetoxyacetyl chloride (61.4
to a mixture of 10a (225 mg, 0.476 mmol) and Et3N (198
1.43 mmol) in THF (5 mL) and the mixture was stirred at room
temperature for 15 h. The mixture was diluted with AcOEt, washed
with water and brine, and dried over MgSO4. After removal of
l
L, 0.571 mmol) at 0 °C was added
lL,

5516
T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
MgSO4 by filtration, the filtrate was concentrated in vacuo. The res-
idue was purified by basic silica gel column chromatography
(AcOEt) to give 2-[4-({[3-methoxy-1,6-dimethyl-4-oxo-5-(2-oxo-
2-phenylethyl)-4,5-dihydro-1H-pyrrolo[3,2-c]-pyridin-2-yl]car
bonyl}amino)piperidin-1-yl]-2-oxoethyl acetate. 8 M NaOH aq
(0.5 mL), THF (2.5 mL) and EtOH (5 mL) were added to a mixture
of the compound obtained above and stirred at room temperature
for 2 h. The mixture was neutralized with 1 M HCl aq and diluted
with water, and extracted with AcOEt/THF. The organic layer was
washed with water and brine and dried over MgSO4. After removal
of MgSO4 by filtration, the filtrate was concentrated in vacuo. The
residue was purified by basic silica gel column chromatography
(AcOEt) to give the title compound (156 mg, 66%) as a white solid;
mp 237 °C (recrystallized from hexane/AcOEt/THF). 1H NMR
(DMSO-d6) d 1.32–1.61 (2H, m), 1.79–1.93 (2H, m), 2.28 (3H, s),
2.82–2.98 (1H, m), 3.04–3.20 (1H, m), 3.57–3.71 (1H, m), 3.86
(3H, s), 3.96–4.25 (7H, m), 4.50 (1H, d, J = 5.3 Hz), 5.61 (2H, s),
6.58 (1H, s), 7.55–7.67 (3H, m), 7.69–7.78 (1H, m), 8.07–8.16
(2H, m). Anal. Calcd for C26H30N4O6: C, 63.15; H, 6.11; N, 11.33.
Found: C, 63.03; H, 6.15; N, 11.07. LC–MS: m/z = 495 (MH+).
each well to achieve the final concentrations of 5.8% FBS, 1 lg/
mL Shh-N and 0.03–1000 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 med-
ia containing 0.1% DMSO with or without 1 lg/mL Shh-N (0% or
100% inhibition control, respectively (n = 10 wells). The luciferase
activities of reporter cells were measured by Bright-Glo™ (Prome-
ga Corp., Madison, WI) using the EnVisionÒ plate reader (PerkinEl-
mer, Inc., Waltham, MA).
5.44. In vivo PD assay
The in vivo PD assay was conducted using nude mice bearing
human primary pancreatic tumors (PAN-04). The tumor line was
established by the Central Institute for Experimental Animals
(Kanagawa, Japan). Compound 11d was administered orally twice
daily. Twenty four hours after the first dose, the tumors were ex-
cised and treated with RNAlater (Ambion, Foster, CA). Total RNA
samples were isolated using RNeasy Mini kit (Qiagen, Valencia,
CA), and first strand cDNA samples were prepared using the high
capacity cDNA transcription kit (Applied Biosystems, Carlsbad,
CA). The primer sets ofqPCR for quantification of stromal Gli1
mRNA (Applied Biosystems, Carlsbad, CA) were as follows:
Mm00494645_m1 (mouse Gli1), 4352339E (mouse GAPDH).
5.41. 6-Ethyl-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]pyridine-2-carboxamide (11e)
In the same manner as in the preparation of 11j, the title com-
pound (61.2 mg, 65%) was obtained as a white powder from 10b
(90.0 mg, 0.185 mmol); mp 146 °C (recrystallized from AcOEt). 1H
NMR (DMSO-d6) d 1.13–1.23 (3H, m), 1.31–1.59 (2H, m), 1.80–
1.94 (2H, m), 2.58 (2H, q, J = 7.4 Hz), 2.83–2.99 (1H, m), 3.04–
3.21 (1H, m), 3.59–3.70 (1H, m), 3.89 (3H, s), 3.97–4.26 (7H, m),
4.49 (1H, t, J = 5.4 Hz), 5.59 (2H, s), 6.49 (1H, s), 7.56–7.67 (3H,
m), 7.70–7.78 (1H, m), 8.06–8.15 (2H, m). Anal. Calcd for
5.45. In vivo anti-tumor test
Anti-tumor effects of compounds were evaluated using a mouse
medulloblastoma allogeneic transplantation model6 in which the
medulloblastoma spontaneously occurred in the cerebellum of 7–
9-week-old Patch 1 (+/ꢁ) and p53 (ꢁ/ꢁ) double mutant mice.
Patch 1 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 tumors were subcutane-
ously transplanted into nude mice from Charles river laboratories
(Yokohama, Japan; CAnN.Cg-Foxn1nu/CrlCrlj), and allograft tu-
mors, following several serial passages in vivo, were used for com-
pound testing. To examine the anti-tumor activity of compound
11d, animals bearing tumors with an average size of 150–
250 mm3 were treated with compound 11d (0.5% methylcellulose
suspension) twice daily for 2 weeks. The 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 V = (a ꢂ b2)/2. The tumor growth rate (T/C %)
was calculated as the mean values for [Treated (VendꢁVstart)/Con-
trol (VendꢁVstart)] ꢂ 100.
C
27H32N4O6: C, 63.77; H, 6.34; N, 11.02. Found: C, 63.58; H, 6.31;
N, 10.87. LC–MS: m/z = 509 (MH+).
5.42. 2-(4-Aminopiperidin-1-yl)-2-oxoethanol hydrochloride
(13)
Acetoxyacetyl chloride (3.10 mL, 28.8 mmol) was added drop-
wise at 0 °C to a mixture of 12 (4.80 g, 24.0 mmol) and Et3N
(9.96 mL, 71.9 mmol) in THF (50 mL) and the mixture was stirred
for 2 h. The mixture was diluted with AcOEt, washed with water
and brine, dried over MgSO4, and concentrated in vacuo. The resid-
ual solid was collected by filtration and washed with hexane/AcOEt
solution. A mixture of the solid obtained, 8 M NaOH aq (5 mL) and
EtOH (35 mL) was stirred at room temperature for 2 h. The mixture
was acidified with 6 M HCl aq and concentrated in vacuo. The res-
idue was dissolved in AcOEt (30 mL) and 4 M HCl in AcOEt (30 mL)
was added. After stirring at room temperature for 6 h, the resulting
solid was collected by filtration and washed with AcOEt to give the
title compound (3.73 g, 80%) as a white solid. 1H NMR (DMSO-d6) d
1.31–1.51 (2H, m), 1.93 (2H, d, J = 12.9 Hz), 2.69 (1H, t, J = 12.2 Hz),
3.00 (1H, t, J = 12.3 Hz), 3.10–3.30 (1H, m), 3.73 (1H, d, J = 13.8 Hz),
4.08 (2H, q, J = 13.9 Hz), 4.33 (1H, d, J = 12.9 Hz), 8.38 (3H, br s).
5.46. Solubility determination
Small volumes of the compounds in DMSO were added to the
aqueous buffer (pH 6.8). After incubation, precipitates were sepa-
rated from by filtration through a filter plate. The filtrates were
analyzed for the amount of compound in solution by HPLC
analysis.
5.43. Gli-luc reporter assay
5.47. Pharmacokinetic studies
NIH3T3/Gli-luc cells were maintained in DMEM containing 10%
FBS, 500-lg/mL G418, and 0.1% gentamicin solution (Invitrogen
Test compounds were administered at a dose of 10 mg/kg as
cassette dosing to non-fasted mice. After oral administration, blood
samples were collected and centrifuged to obtain the plasma frac-
tion that was deproteinized with MeCN containing an internal
standard. After centrifugation, the resulting supernatant was di-
luted with a mixture of 0.01 mol/L HCOONH4 solution and MeCN
(9:1, v/v) and centrifuged again. The concentrations of compound
in the supernatant were measured by LC/MS/MS.
Corp., Carlsbad, CA). The cells were seeded onto collagen-coated
384-well plates at 7.5 103cells/well and cultured overnight in
25
After incubation, 20
in DMEM containing 2% FBS) and 5
pounds in 10ꢂ solution (0.0003–10
l
L /well of DMEM containing 10% FBS under 5% CO2 at 37 °C.
L of recombinant mouse Shh-N (2.5 g/mL
L of a serially diluted the com-
M in DMEM) were added to
l
l
l
l

T. Ohashi et al. / Bioorg. Med. Chem. 20 (2012) 5507–5517
5517
5.48. Pharmacokinetic profile of rats and dogs
References and notes
1. Ohashi, T.; Oguro, Y.; Tanaka, T.; Shiokawa, Z.; Shibata, S.; Sato, Y.; Yamakawa,
H.; Hattori, H.; Yamamoto, Y.; Kondo, S.; Miyamoto, M.; Tojo, H.; Baba, A.;
Sasaki, S. Bioorg. Med. Chem. in press. http://dx.doi.org/10.1016/j.bmc.2012.
07.039.
2. (a) Ishikawa, M.; Ajito, K. In Soyaku Shien Kenkyu no Tenbo; Torisawa, Y., Ed.;
CMC Publishing: Tokyo, 2008; pp 3–13; (b) Fujita, Y.; Yonehara, M.; Tetsuhashi,
M.; Noguchi-Yachide, T.; Hashimoto, Y.; Ishikawa, M. Bioorg. Med. Chem. Lett.
2010, 18, 1194.
3. Dropinski, D. F.; Akiyama, T.; Einstein, M.; Habulihaz, B.; Doebber, T.; Berger, J.
P.; Meinke, P. T.; Shi, G. Q. Bioorg. Med. Chem. Lett. 2005, 15, 5035.
4. (a) Hans-Joachim, B.; David, B.; Stefanie, B.; Manfred, K.; Bernd, K.; Klaus, M.;
Ulrike, O.; Martin, S. ChemBioChem 2004, 5, 637; (b) William, K. H. J. Med. Chem.
2008, 51(15), 4359.
5. X-ray crystallographic data for compound 11d has been deposited with the
Cambridge Crystallographic Data Center as CCDC 865214. The crystallographic
data can be obtained, free of charge, on application to CCDC; 12 Union Road,
Cambridge CB2 1EZ, UK.
Test compounds were administered to fed rats and dogs. Plasma
samples were collected after oral (10 mg/kg) and intravenous
(1 mg/kg) administration and were deproteinized with methanol
containing an internal standard. After centrifugation, the superna-
tant was diluted with a mixture of 0.01 mol/L ammonium formate
solution and [MeCN/formic acid (100:0.2, v/v)] (7:3, v/v) and cen-
trifuged again. The compound concentrations in the supernatant
were measured by LC–MS/MS.
Acknowledgements
We thank Mr. Nagano, Mr. Iida and Mr. Iwata for the X-ray crys-
tal structure analysis of TAK-441.
6. Sasai, K.; Romer, J. T.; Lee, Y.; Finkelstein, D.; Fuller, C.; McKinnon, P. J.; Curran, T.
Cancer Res. 2006, 66, 4215.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.034.