Synthesis and evaluation of hedgehog signaling inhibitor with novel core system

Article abstract of DOI:10.1016/j.bmc.2015.05.036

As we previously reported, N-methylpyrrolo[3,2-c]pyridine derivatives 1 (TAK-441) was discovered as a clinical candidate of hedgehog (Hh) signaling inhibitor by modification of the upper part. We next focused on modification of the lower part including core skeletons to discover new Hh signaling inhibitors with novel core rings. Efforts to find novel chemotypes by using X-ray single crystal structure analysis led to some potent Hh signaling inhibitors (2c, 2d, 2e, 2f) with novel core ring systems, which had benzamide moiety at the 5-position as a key component for potent activity. The suppression of Gli1 expression with these new Hh signaling inhibitors were weaker than that of compound 1 (TAK-441) because of low pharmacokinetic property. We recognized again TAK-441 is a good compound as clinical candidate with good structural and pharmacokinetic advantages.

Full text of DOI:10.1016/j.bmc.2015.05.036

Bioorganic & Medicinal Chemistry 23 (2015) 4777–4791
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of hedgehog signaling inhibitor with novel
core system
⇑
Tomohiro Ohashi , Yuta Tanaka, Zenyu Shiokawa, Hiroshi Banno, Toshio Tanaka, Sachio Shibata,
Yoshihiko Satoh, Hiroko Yamakawa, Yukiko Yamamoto, Harumi Hattori, 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:
As we previously reported, N-methylpyrrolo[3,2-c]pyridine derivatives 1 (TAK-441) was discovered as a
clinical candidate of hedgehog (Hh) signaling inhibitor by modification of the upper part. We next
focused on modification of the lower part including core skeletons to discover new Hh signaling inhibi-
tors with novel core rings. Efforts to find novel chemotypes by using X-ray single crystal structure anal-
ysis led to some potent Hh signaling inhibitors (2c, 2d, 2e, 2f) with novel core ring systems, which had
benzamide moiety at the 5-position as a key component for potent activity. The suppression of Gli1
expression with these new Hh signaling inhibitors were weaker than that of compound 1 (TAK-441)
because of low pharmacokinetic property. We recognized again TAK-441 is a good compound as clinical
candidate with good structural and pharmacokinetic advantages.
Received 1 April 2015
Revised 20 May 2015
Accepted 22 May 2015
Available online 3 June 2015
Keywords:
Hedgehog
Hh signaling inhibitor
X-ray single crystal structure
Benzamide
Ó 2015 Elsevier Ltd. All rights reserved.
Dihedral angle
TAK-441
1. Introduction
The pyrrole ring of 1 was essential for potent inhibitory activity
because of the interaction by internal hydrogen bond between
The hedgehog (Hh) signaling pathway plays a significant role in
the regulation of cell growth and differentiation during embryonic
development. Abnormal activation of the Hh signaling pathway
has been linked to several types of human cancers including
pancreatic cancer,1 prostate cancer,2 colon cancer,3 basal cell carci-
noma4 and medulloblastoma,5 and the development of small-
molecule inhibitors of this pathway represents a promising route
toward novel anticancer therapeutics.6
2-amide NH and oxygen of 3-(2,2,2-trifluoro)methoxy group.8
Thus, we fixed this upper part and focused on the lower part of
core ring, especially six-membered rings. Besides novel core ring
modification, we evaluated the effect of 4- or 5-substituents to
in vitro inhibitory activity. In this report, we describe
syntheses and SAR of novel hedgehog signaling inhibitors with
multi-substituted bicyclic core rings (Fig. 2).
The pyrrolo[3,2-c]pyridine-4-one derivative 1 (TAK-441) we
had reported as a clinical candidate was discovered by modifica-
tion of 2-, 3-, 6-substituents of a lead compound to improve activ-
ity and physicochemical property as hedgehog (Hh) signaling
inhibitors (Fig. 1).7,8 However, it was still unclear that exact role
of the core skeleton including substituents at the 4- and 5-posi-
tions. Thus, we evaluated the modification of central core to con-
firm these properties and discovered new potent Hh signaling
inhibitors with novel skeletons compared to that of 1.
⇑
Corresponding authors. Tel.: +81 466 32 1151; fax: +81 466 29 4458 (T.O.); tel.:
+81 466 32 1146; fax: +81 466 29 4448 (S.S.).
E-mail
addresses:
tomohiro.oohashi@takeda.com
(T.
Ohashi),
satoshi.sasaki@takeda.com (S. Sasaki).
Figure 1. Chemical structure of 1 (TAK-441).
http://dx.doi.org/10.1016/j.bmc.2015.05.036
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
Figure 2. Change to novel core ring in 1 (TAK-441).
The synthesis of pyrrolo[2,3-d]pyrimidine 3a is shown in
2. Chemistry
Scheme 1. Addition of propionitrile 7 with ammonia in acidic con-
dition provided amidine hydrochloride 8, followed by cyclization
with diethyl malonate to afford 9 in 67% yield. The preparation
of a key intermediate 6a was conducted by stepwise conversion;
introduction of formyl group, oxidation to carboxylic acid and
esterification. After addition of sarcosine ethyl ester hydrochloride,
subsequent Dieckmann condensation provided the bicyclic com-
pound 4a in 91% yield. The 3-hydroxyl group of 4a was treated
with (2,2,2-trifluoroethyl)trifluoromethanesulfonate to afford 10.
The stepwise saponification of chloropyridine and ethoxycarbonyl
moieties gave carboxylic acid 3a.
In our previous papers, we reported the synthesis of 1 using an
intermediate with requisite phenacyl moiety on six-membered
ring of the bicyclic core. The similar synthetic route using the inter-
mediate 11 was initially attempted (Scheme 2). The phenacylation
of 11 gave 12 in 57% yield.10 The following hydrolysis under basic
As we previously reported, the intramolecular hydrogen bond
formed between (2,2,2-trifluoro)ethoxy group at 3-position and
N-[1-(hydroxyacetyl)piperidinyl] amide at the 2-position on N-
methyl pyrrole ring was important for potent in vitro activity.7
Figure 3 illustrates our retrosynthetic analysis to access novel
bicyclic cores with these two moieties. To construct the unique
bicyclic core of 2, it was necessary to synthesize intermediate 4.
We considered the Dieckmann condensation would be promising
because the important substituents as mentioned before could be
introduced in the desired position. Therefore, o-halo arylcarboxylic
acid ester 6 was selected for a key intermediate which was con-
verted to di-ester 5 by addition of sarcosine ester unit.9 We
thought this strategy could be utilized for the synthesis of various
useful core rings. The syntheses for five types of carboxylic acid 3
are detailed in the next section.
Figure 3. Retrosynthetic analysis of novel core.
Scheme 1. Synthesis of pyrrolo[2,3-d]pyrimidine derivative 3a. Reagents and conditions : (a) (1) HCl, EtOH; (2) NH3, MeOH, 70%; (b) (COOEt)2, NaOMe, MeOH, 67%; (c) (1)
DMF, POCl3, 0 °C to reflux, 73%; (2) NaClO2, NH2SO3H, tert-BuOH/H2O; (3) (COCl)2, DMF, THF; (4) Et3N, EtOH, 53%; (d) MeNHCH2COOEtꢀHCl, Et3N, THF, 99%; (e) NaOEt, EtOH,
91%; (f) CF3CH2OTf, Cs2CO3, DMF, 91%; (g) AcONa, AcOH, reflux, 96%; (h) NaOH, EtOH, 60 °C, 79%.

T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
4779
Scheme 2. Synthesis of carboxylic acid 13 using similar route to TAK-441. Reagents and conditions: (a) phenacyl bromide, tert-BuONa, LiBr, DME, DMF, 60 °C, 57%.
conditions did not work well and gave only tricyclic product 14 or
15, which was supposed by LC–MS analysis. It was considered the
undesired product was obtained by cyclization of 6-ethylene moi-
ety with carbonyl at the 5-phenacyl group prior to saponification
(Scheme 3).11 We speculated this cyclization was triggered by
higher acidity of methylene in the ethyl group of compound 12
than that of 1. On the other hand, the hydrolysis of 12 under acidic
condition gave dealkylated compound 16 as a major product sup-
posed by LC–MS analysis. These results strongly suggested the
hydrolysis should be conducted before introduction of 5-phenacyl
moiety as described above.
Next, the pyrrolo[2,3-b]pyridine ring was constructed along the
synthetic route below (Scheme 4). The hydrogenation of 4-
chloropyridine-2-one derivative 177 by palladium-carbon was
achieved in 90% yield. Stepwise halogenation of 18 was conducted
to afford a key intermediate 6b. Addition of sarcosine ethyl ester
hydrochloride and cyclization along our strategy provided 4b.
Alkylation
of
the
3-hydroxy
group
with
(2,2,2-
Scheme 3. Presumed reaction mechanism of tricyclic compound.
Scheme 4. Synthesis of pyrrolo[2,3-b]pyridine derivatives 3b and 3c. Reagents and conditions: (a) Pd-C, H2, Et3N, EtOH, THF, 90%; (b) NBS, DMF, 86%; (c) POCl3, reflux, 76%;
(d) (1) MeNHCH2COOEtꢀHCl, Et3N, THF, reflux; (2) NaOEt, EtOH, 73%; (e) CF3CH2OTf, Cs2CO3, DMF, 96%; (f) (1) acetophenone, tBuONa, Pd(OAc)2, MePhos, toluene, 70 °C; (2)
NaOH, EtOH, 50 °C, 22%; (g) (1) benzophenoneimine, Pd2(dba)3, xantphos, Cs2CO3, toluene, 100 °C; (2) 2 M HCl, THF, 91%; (h) BzCl, Py, THF, 94%; (i) NaOH, EtOH, 95%.

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trifluoroethyl)triflate led to 20 in 96% yield. Coupling with ace-
tophenone in Buchwald condition gave 3b in 22% yield, but intro-
duction of amino group to benzophenoneimine gave 21 in 91%
yield. Acylation with benzoyl chloride followed by hydrolysis led
to carboxylic acid 3c.
The synthesis of indole derivative 3d is shown in Scheme 5. The
key intermediate 6d was synthesized from ethyl 4-bromo-
2-chlorobenzoate 23 as follows; introduction of ethyl group by
Stille coupling followed by hydrogenation, and nitration. The
construction of pyrrole ring proceeded directly in 41% yield. After
alkylation of 4d, subsequent hydrogenation of nitro group led to
aniline 25 in 97% yield. Acylation of 25 with benzoyl chloride
followed by saponification were conducted to afford the desired
carboxylic acid 3d.
bromo to ethyl moiety, successive introduction of hydroxyl and
bromo groups. The direct addition of sarcosine ethyl ester was dif-
ficult because of its low reactivity of bromo group against nucle-
ophilic substitution, thus we conducted stepwise introduction.
The amination of 6e with benzophenoneimine afforded 30 under
Buchwald condition. Following monomethylation was achieved
via Boc protection. The precursor for Dieckmann condensation 5e
was prepared by addition of ethyl bromoacetate. The cyclization
of 5e with sodium ethoxide afforded 4e in 77% yield. After the alky-
lation of 3-hydroxyl group, the hydrogenation was carried out to
remove benzyl group. Conversion to triflate with trifluo-
romethanesulfonic anhydride was successively conducted to give
31. The similar reaction in the synthesis of pyrrolo[2,3-b]pyridine
3c afforded carboxylic acid 3e with N-benzoyl group at the
5-position.
The synthetic route of pyrrolo[3,2-b]pyridine derivative 3e is
shown in Scheme 6. The key intermediate 6e was successfully pre-
pared from ethyl 3-bromopicolinate 27 in 3 steps; conversion of
The synthesis of pyrrolo[2,3-b]pyrazine derivative 3f is shown
in Scheme 7. Cyclization of 33 with diethyl ketomalonate gave
Scheme 5. Synthesis of indole derivative 3d. Reagents and conditions: (a) (1) (vinyl)SnBu3, Pd(PPh3)4, DMF, 100 °C, 100%; (2) Pd-C, Ba(OH)2, H2, AcOEt, 98%; (b) NaNO3,
H2SO4, 0 °C, 70%; (c) MeNHCH2COOEtꢀHCl, Et3N, EtOH, reflux, 41%; (d) (1) CF3CH2OTf, Cs2CO3, DMF, 100%; (2) Pd-C, H2, EtOH, THF, 97%; (e) BzCl, Py, THF, 86%; (f) NaOH, EtOH,
95%.
Scheme 6. Synthesis of pyrrolo[3,2-b]pyridine derivative 3e. Reagents and conditions: (a) (1) (vinyl)SnBu3, Pd(PPh3)4, DMF, 100 °C, 100%; (2) Pd-C, H2, EtOH, 100%; (b) (1)
mCPBA, MeCN, 83%; (2) TFAA, DMF, 96%; (c) (1) NBS, DMF, 79%; (2) BnBr, Ag2CO3, toluene, 40 °C, 99%; (d) (1) benzophenoneimine, Pd2(dba)3, xantphos, Cs2CO3, toluene; (2)
2 M HCl, THF, 98% (for 30), 92% (for 32); (e) (1) Boc2O, tert-BuOH, 90 °C; (2) MeI, Cs2CO3, DMF, 87%; (3) 4 M HCl, AcOEt, 0 °C, 87%; (4) BrCH2COOEt, iPr2NEt, DMF, 110 °C, 79%;
(f) NaOEt, EtOH, 77%; (g) (1) CF3CH2OTf, Cs2CO3, DMF, 97%; (2) Pd-C, H2, EtOH, THF, 97%; (3) Tf2O, pyridine, 60 °C, 92%; (h) (1) BzCl, pyridine, THF; (2) NaOH, EtOH, 50 °C, 88%.

T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
4781
Scheme 7. Synthesis of pyrrolo[2,3-b]pyrazine derivative 3f. Reagents and conditions: (a) (COOEt)2CO, iPr2NEt, EtOH, reflux, 25%; (b) (1) NBS, DMF; (2) BnBr, Ag2CO3, acetone,
91%; (c) (1) benzophenoneimine, Pd2(dba)3, xantphos, Cs2CO3, toluene, 100 °C; (2) 2 M HCl, THF, 59%; (3) BzCl, pyridine, THF, 0 °C, 93%; (d) (1) Pd/C, H2, EtOH, 77%; (2) Tf2O,
pyridine, 0 °C, 94%; (e) (1) MeNHCH2COOEtHCl, Et3N, EtOH; (2) NaOEt, EtOH, 80%; (f) (1) CF3CH2OTf, Cs2CO3, DMF, 0 °C, 74%; (2) NaOH, THF, EtOH, 99%.
Scheme 8. Amidation reaction in each core ring. Reagents and conditions: (a) amine 37, EDC, HOBt, Et3N, DMF, 46–82%; (b) BrCH2COPh, LiBr, K2CO3, DME/DMF, 60–80 °C,
14%.
pyrazine 34 in 25% yield. Bromination followed by benzylation of
34 provided 35. Conversion to amino group using Buchwald condi-
tion and following acylation with benzoyl chloride afforded 36. The
benzyloxy moiety was converted to triflate by hydrogenation and
triflation. Addition with sarcosine ethyl ester hydrochloride was
achieved at the triflate moiety, and cyclization of pyrrole moiety
was prepared in one-pot to give 4f. The obtained alcohol was alky-
lated with (2,2,2-trifluoro)ethyl triflate, following hydrolysis to
give carboxylic acid 3f.
Finally, the amidation reaction is listed in Scheme 8. The
compound 2a–f was obtained by condensation of corresponding
carboxylic acid 3a–f with amine 378 in 46–82% yield. For the com-
pound 2a, the alkylation with phenacyl bromide was carried out in
14% yield.
suppression assay of transcription factor Gli1 mRNA expression
was conducted by the treatment of mice bearing PAN-04 human
pancreatic xenograft tumors with the compounds after oral admin-
istration (0 h and 9 h). Quantification of mouse stromal Gli1 mRNA
as a pharmacodynamic (PD) marker extracted from the tumors was
performed 24 h after the first administration of drug (in vivo PD
assay).
We initially assessed the novel two types of core ring bearing
2-{N-[1-(hydroxyacetyl)piperidinyl]} amide, 3-[(2,2,2-trifluoro)
ethoxy], 5-phenacyl, and 6-ethyl groups at the same positions as
1 (TAK-441) (Table 1). Pyrrolo[2,3-d]pyrimidine 2g maintained
potent inhibitory activity compared to 1, but the pyrrolo[2,3-b]
pyridine 2b showed 10-fold drop. This result indicated it might
be important for potent inhibitory activity to bear carbonyl group
at the 4-position in the core ring. However, the compound 2b still
showed 10ꢁ8 order inhibition without carbonyl moiety. It was
suggested that the pyrrolo[2,3-b]pyridine ring could be promising
core as novel Hh signaling inhibitor by further modification.
We next focused on enhancement of inhibitory activity in
compound 2b.
3. Results and discussions
In vitro activities of compounds 2b–g were evaluated using a
luciferase reporter in NIH3T3 cells carrying a stably transfected
Gli-reporter construct (Gli-luc reporter cell line). An in vivo

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Table 1
Effect of core ring with 5-phenacyl group
a IC50 values are the mean of four measurements.
resulted in decrease of activity.8 Considering our previous knowl-
edge and necessity of regulation, the benzamide moiety would
be a proper substituent at 5-position.
The benzamide was provided the planarity to whole in the 5-
position, and regulated the conformation compare to phenacyl
group. This structural regulation newly introduced in the 5-posi-
tion as a surrogate of regulation by steric hindrance in compound
1 brought us expectation that benzamide side chain took the
appropriate conformation. Moreover, the rigid benzamide group
was advantaged in entropy compared to more flexible phenacyl
group because the flexibility loss in binding the target protein
was small.12 Thus, our next effort was shifted to investigation of
5-benzamide derivatives.
The compound 2c with benzamide moiety instead of phenacyl
group showed dramatic increase of Gli reporter inhibitory activity
compared to that of 2b as we expected. We next adapted this find-
ing to other novel core skeleton without 4-carbonyl group.
Consequently, as the indole 2d, pyrrolo[3,2-b]pyridine 2e and pyr-
rolo[2,3-b]pyrazine 2f also showed potent inhibitory activity.
These results demonstrated that 5-benzamide was quite effective
side chain for our novel core skeletons without 4-carbonyl group
(Table 2). For further consideration, the X-ray single crystal struc-
tures of 2d and 2e were obtained and its overlaid figure was illus-
trated in Figure 5.
They also indicated that 5-substituents of 2d and 2e took simi-
lar position to that of 1 despite the absence of 4-carbonyl moiety.
This conformation was considered to be necessary for potent Gli
reporter inhibitory activity as we expected. The dihedral angles
between central core ring and 5-substituent are also described in
Table 2. To notify the relation between inhibitory activity and X-
ray single structure, the potent activity requested almost vertical
dihedral angle, and its value in 1, 2d, and 2e were 84°, 90° and
91°, respectively. We considered that amide linker formed ade-
quate conformation with dihedral angle of about 90°, and fitted
well to whole molecule to the pocket of smo protein.
Figure 4. Overlay of 1 (black) and 2b (gray) in X-ray single crystal structure. The
figure was described based on X-ray structural analysis data using PyMOL Software.
(PyMOL version 1.4.1).
For this purpose, we obtained X-ray single crystal structure of 1
and 2b (Fig. 4). The upper parts of these two compounds were well
overlaid. On the other hand, the conformations of terminal ben-
zenes in the 5-phenacyl group were quite different. The 5-phenacyl
group of 1 might be influenced by the steric hindrance of both 6-
ethyl and 4-carbonyl groups, but lack of the 4-carbonyl group led
2b to take the different conformation from that of 1. Based on these
analyses of X-ray single crystal structures, the enhancement of
inhibitory activity in 2b could be achieved by adjusting the confor-
mation of terminal benzene ring similar to compound 1. The
decrease of steric hindrance by the lack of 4-carbonyl group
induced flexible conformation at the 5-position, thereby an alter-
native regulation was necessary for enhancement of inhibitory
activity as a surrogate of steric hindrance. In our previous report,
the lack of carbonyl group in the side chain or benzene ring
The in vivo PD profiles of 2c–f 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 signif-
icant stroma-derived Gli1 and cancer-derived Shh activity. We
measured the reduction of Gli1 mRNA expression levels as a phar-
macodynamic marker in this model. The suppression of Gli1 mRNA

T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
4783
Table 2
The inhibitory activity and dihedral angle of each novel core ring
a IC50 values are the mean of four measurements.
b A: least-squares surface of central core, B : least-squares surface of 5-phenacyl or benzamide group.
c Not detected.
Table 3
The results of in vivo PD study and corresponding PK data
a IC50 values are the mean of four measurements.
b Gli1 mRNA expression at 25 mg/kg BID, 24 h post-dose. The value indicates Gli1
mRNA expression levels compared to controls. Test compounds were dosed at
25 mg/kg, BID.
c Cassette dosing at 10 mg/kg, po in mice. AUC: area under the plasma concentra-
tion versus time curve from 0 to 8 h.
4. Conclusions
We explored new Hh signaling inhibitors with novel core rings
by the modification of 5-substituent including core ring, and found
pyrrolo[2,3-b]pyridine derivative 2b with 10ꢁ8 order activity in Gli
reporter assay. To enhance in vitro activity, we considered the
preferable conformation in X-ray single crystal structure, and dis-
covered 2c with benzamide moiety at the 5-position. This sub-
stituent worked well in the core ring without 4-carbonyl moiety,
the indole 2d, pyrrolo[3,2-b]pyridine 2e and pyrrolo[2,3-b]pyra-
zine 2f also showed potent inhibitory activity. In these compounds,
vertical dihedral angle between 5-substituent and core ring might
be important for potent inhibitory activity from X-ray crystal
structure. The suppression of Gli1 mRNA expression in these novel
compounds 2c–f was unfortunately inefficient compare to that of 1
in the result of pharmacokinetics. Based on the results of potent Gli
reporter inhibitory activity including structural characteristics and
good PK profiles, we confirmed that 1 (TAK-441) is the best candi-
date as our Hh signaling inhibitor.
Figure 5. Overlay of 1 (black), 2b (gray), 2d (pink) and 2e (green) in X-ray single
crystal structure. The figure was described based on X-ray structural analysis data
using PyMOL Software. (PyMOL version 1.4.1).
expression in novel compounds 2c–f was weaker than 1 despite of
their potent in vitro inhibitory activity. We considered it was
caused by their pharmacokinetics, which was well correlated to
suppression of Gli1 mRNA expression. Considering these results,
compound 1 (TAK-441) was well-balanced compound in the point
of good PK profile while potent inhibitory activity.

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pale yellow powder. To a mixture of obtained aldehyde (11 g,
5. Experimental section
52 mmol), amidosulfuric acid (7.0 g, 72 mmol), tert-butanol
(100 mL) and water (40 mL) was added dropwise a solution of
sodium chlorite (6.6 g, 72 mmol) in water (20 mL). After stirring
at room temperature for 30 min, the reaction mixture was diluted
with water, and extracted twice with AcOEt. The extracts were
combined, washed with water and brine, dried over MgSO4 and
concentrated in vacuo. The residue was dissolved in THF (60 mL),
oxalyl chloride (9.7 mL, 111 mmol) was added dropwise at 0 °C,
then DMF (1 drop) was added. After stirring at room temperature
for 3 h, the reaction mixture was concentrated in vacuo. EtOH
(60 mL) and Et3N (16 mL, 111 mmol) were added under ice-cool-
ing, and the mixture was stirred at room temperature for 3 h.
The reaction mixture was diluted with saturated NaHCO3 aq and
water, and extracted twice with AcOEt. The extracts were com-
bined, washed with brine, dried over MgSO4 and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy (hexane/AcOEt = 97/3 to 17/3) to give the title compound
(6.7 g, 53%) as pale yellow oil. 1H NMR (CDCl3) d 1.31–1.48 (6H,
m), 2.97 (2H, q, J = 7.6 Hz), 4.48 (2H, q, J = 7.2 Hz).
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 Bruker Avance 300
(300 MHz) or Bruker DPX300 (300 MHz) instruments. Chemical
shifts are reported as d values (ppm) down-field from internal
tetramethylsilane of the indicated organic solution. Peak multiplic-
ities are expressed as follows. Abbreviations are used as follows: 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 theo-
retical values. LC–MS spectra were obtained on a SHIMADZU cor-
poration LC–MS system (LCMS-2010A). Column chromatography
was carried out on silica gel columns (Kieselgel 60, 63–200 mesh,
Merck) or basic silica gel columns (ChromatorexÒ NH-DM1020,
100–200 mesh, Fuji Silysia Chemical Ltd.) or Purif-PackÒ (SI
60 lM or NH 60 lM, Fuji Silysia Chemical Ltd.). Reaction progress
was determined by thin layer chromatography (TLC) analysis on
silica gel 60F254plate (Merck) or NH TLC plates (Fuji Silysia
Chemical Ltd). The purities of compounds submitted for biological
evaluation were P95% as determined by LC/MS and elemental
analysis. Yields are not optimized.
5.4. Ethyl 4-chloro-6-[(2-ethoxy-2-oxoethyl)(methyl)amino]-2-
ethylpyrimidine-5-carboxylate (5a)
A
solution of 6a (425 mg, 1.7 mmol), ethyl sarcosinate
hydrochloride (262 mg, 1.7 mmol) and Et3N (0.57 mL, 4.1 mmol)
in THF (13 mL) was stirred at room temperature for 19.5 h. The
reaction mixture was diluted with saturated NaHCO3 aq, and
extracted twice with AcOEt. The extracts were combined, washed
with brine, dried over MgSO4 and concentrated in vacuo to give
the title compound (560 mg, 99%) as colorless oil. 1H NMR
(CDCl3) d 1.17–1.32 (6H, m), 1.41 (3H, t, J = 7.2 Hz), 2.72 (2H, q,
J = 7.6 Hz), 3.11 (3H, s), 4.21 (2H, q, J = 7.1 Hz), 4.28 (2H, s), 4.40
(2H, q, J = 7.2 Hz).
5.1. Propanimidamide hydrochloride (8)
To a solution of propionitrile (10 g, 142 mmol) in EtOH (8.4 mL)
was blown hydrogen chloride gas (total amount increase 7.9 g), and
the mixture was stirred at room temperature for 21.5 h. The mixture
was concentrated in vacuo, the residue was suspended in EtOH
(5.5 mL), and cooled to ꢁ10 °C. To the suspension was added 8 M
MeOH solution of ammonia (17.8 mL), and the mixture was stirred
at room temperature for 28 h. The insoluble material was filtered
off, and the filtrate was concentrated in vacuo to give the title com-
pound (10.8 g, 70%) as a white solid. 1H NMR (DMSO-d6) d 1.17 (3H,
t, J = 7.6 Hz), 2.40 (2H, q, J = 7.7 Hz), 8.77 (2H, br s), 9.07 (2H, br s).
5.5. Ethyl 4-chloro-2-ethyl-5-hydroxy-7-methyl-7H-pyrrolo[2,3-
d]pyrimidine-6-carboxylate (4a)
A solution of 5a (810 mg, 2.5 mmol) and 20% solution of NaOEt
in EtOH (836 mg, 2.5 mmol) in EtOH (25 mL) was stirred at room
temperature for 15 min. The reaction mixture was diluted with
water, neutralized with 1 M HCl aq, and stirred at 0 °C for
30 min. The precipitated solid was collected by filtration, washed
with water, and dried in vacuo to give the title compound
(637 mg, 91%) as a pale yellow powder. 1H NMR (DMSO-d6) d
1.21–1.43 (6H, m), 2.90 (2H, q, J = 7.6 Hz), 3.88 (3H, s), 4.37 (2H,
q, J = 7.0 Hz), 9.50 (1H, br s).
5.2. 2-Ethylpyrimidine-4,6-diol (9)
To a solution of 8 (11 g, 99 mmol) in MeOH (20 mL) was added
28% solution of NaOMe in MeOH (57 g, 296 mmol), and the mix-
ture was stirred at room temperature for 10 min. Diethyl malonate
(15 mL, 99 mmol) was added dropwise, and the mixture was fur-
ther stirred at room temperature for 16 h. The reaction mixture
was concentrated in vacuo, the residue was dissolved in water
and acidified with concd HCl. The precipitated solid was collected
by filtration, washed with water and Et2O to give the title com-
pound (9.3 g, 67%) as a white solid. 1H NMR (DMSO-d6) d 1.16
(3H, t, J = 7.6 Hz), 2.36–2.60 (2H, m), 5.03 (1H, s), 11.62 (2H, br s).
5.6. Ethyl 4-chloro-2-ethyl-7-methyl-5-(2,2,2-trifluoroethoxy)-
7H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (10)
To
a mixture of 4a (1.0 g, 3.7 mmol) and Cs2CO3 (1.3 g,
5.3. Ethyl 4,6-dichloro-2-ethylpyrimidine-5-carboxylate (6a)
4.0 mmol) in DMF (27 mL) was added dropwise 2,2,2-trifluoroethyl
trifluoromethanesulfonate (0.64 mL, 4.4 mmol) at 0 °C, and the
mixture was stirred at room temperature for 4 h. The mixture
was diluted with water, and the precipitated solid was collected
by filtration. The obtained solid was washed with water, and dried
in vacuo to give the title compound (1.2 g, 91%) as a pale yellow
powder. 1H NMR (CDCl3) d 1.34–1.49 (6H, m), 3.02 (2H, q,
J = 7.6 Hz), 4.06 (3H, s), 4.39–4.57 (4H, m).
DMF (5.5 mL) was added dropwise to POCl3 (60 mL, 641 mmol)
at 0 °C, the mixture was stirred at 0 °C for 1 h. 9 (10 g, 71 mmol)
was added, the mixture was stirred at room temperature for 1 h
and heated under reflux for 16 h. The mixture was concentrated
in vacuo, the residue was added to ice water by small portions.
The mixture was extracted three times with a mixed solvent of
Et2O/AcOEt. The extracts were combined, washed with brine, dried
over MgSO4 and concentrated in vacuo. The residue was suspended
in AcOEt, and the insoluble material was filtered off. The filtrate
was concentrated in vacuo, and the residue was purified by silica
gel column chromatography (hexane/AcOEt = 99/1 to 9/1) to give
4,6-dichloro-2-ethylpyrimidine-5-carbaldehyde (10.6 g, 73%) as a
5.7. Ethyl 2-ethyl-7-methyl-4-oxo-5-(2,2,2-trifluoroethoxy)-4,7-
dihydro-3H-pyrrolo[2,3-d]pyrimidine-6-carboxylate (11)
A
solution of 10 (1.2 g, 3.3 mmol) and AcONa (272 mg,
3.3 mmol) in AcOH (20 mL) was heated under reflux for 2.5 h.

T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
4785
After allowing to cool to room temperature, the reaction mixture
was poured into water, and the precipitated solid was collected
by filtration. The obtained solid was washed with water, and dried
in vacuo to give the title compound (1.10 g, 96%) as a pale yellow
solid. 1H NMR (DMSO-d6) d 1.16–1.37 (6H, m), 2.63 (2H, q,
J = 7.6 Hz), 3.83 (3H, s), 4.25 (2H, q, J = 7.1 Hz), 5.01 (2H, q,
J = 9.4 Hz), 12.02 (1H, br s).
compound (21.2 g, 86%) as a brown powder. 1H NMR (DMSO-d6)
d 1.14 (3H, t, J = 7.6 Hz), 1.26 (3H, t, J = 7.2 Hz), 2.64 (2H, q,
J = 7.6 Hz), 4.20 (2H, q, J = 7.2 Hz), 8.06 (1H, s), 12.42 (1H, br s).
5.12. Ethyl 5-bromo-2-chloro-6-ethylpyridine-3-carboxylate (6b)
A
mixture of 19 (28.0 g, 102 mmol) and POCl3 (28 mL,
309 mmol) was heated under reflux for 12 h. The mixture was con-
centrated in vacuo, ice water (100 mL) was added at 0 °C, and the
mixture was extracted with AcOEt (100 mL ꢂ 3). The extract was
washed successively with saturated NaHCO3 aq (50 mL) and brine,
dried over MgSO4 and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (hexane/AcOEt = 99/1 to
9/1) to give the title compound (22.8 g, 76%) as pale yellow oil. 1H
NMR (DMSO-d6) d 1.22 (3H, t, J = 7.5 Hz), 1.33 (3H, t, J = 7.2 Hz),
2.91 (2H, q, J = 7.5 Hz), 4.34 (2H, q, J = 7.2 Hz), 8.42 (1H, s).
5.8. 2-Ethyl-7-methyl-4-oxo-5-(2,2,2-trifluoroethoxy)-4,7-dihyd
ro-3H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (3a)
A mixture of 11 (4.0 g, 11.5 mmol) and 8 M NaOH aq (8 mL) in
EtOH was stirred at room temperature for 15 min and at 60 °C
for 4 h. After cooling at 0 °C, the mixture was neutralized with
6 M HCl aq. The solvent was evaporated, the precipitate was col-
lected by filtration washed with water, and dried in vacuo to give
the title compound (2.91 g, 79%) as a pale yellow powder. 1H NMR
(DMSO-d6) d 1.22 (3H, t, J = 7.6 Hz), 2.62 (2H, q, J = 7.4 Hz), 3.83
(3H, s), 5.01 (2H, q, J = 9.3 Hz), 12.07 (1H, s), 12.71 (1H, br s).
5.13. Ethyl 5-bromo-6-ethyl-3-hydroxy-1-methyl-1H-pyrrolo[2,
3-b]pyridine-2-carboxylate (4b)
5.9. Ethyl 2-ethyl-7-methyl-4-oxo-3-(2-oxo-2-phenylethyl)-5-
(2,2,2-trifluoroethoxy)-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidine-6-carboxylate (12)
A mixture of 6b (22.8 g, 78.0 mmol), ethyl sarcosinate
hydrochloride (18.0 g, 117 mmol), Et3N (54 mL, 387 mmol) and
EtOH (200 mL) was heated under reflux for 22 h. Then, ethyl sar-
cosinate hydrochloride (6.00 g, 39.1 mmol) and Et3N (22 mL,
158 mmol) were added, and the mixture was heated under reflux
for 17 h. To the reaction mixture was added water (250 mL), and
the mixture was extracted three times with AcOEt (300 mL). The
extract was washed successively with water (100 mL) and brine
(100 mL), and dried over MgSO4 and concentrated in vacuo. To
the residue were added EtOH (200 mL) and a 20% solution of
NaOEt in EtOH (32.0 g, 94.0 mmol), and the mixture was stirred
at room temperature for 1 h. The mixture was concentrated in
vacuo, diluted with water (250 mL), and the mixture was acidified
with 5 M HCl aq (20 mL). The precipitate was collected by filtra-
tion, and washed with water to give the title compound (18.8 g,
73%) as a pale orange solid. 1H NMR (DMSO-d6) d 1.27 (3H, t,
J = 7.6 Hz), 1.33 (3H, t, J = 7.1 Hz), 2.96 (2H, q, J = 7.6 Hz), 3.88
(3H, s), 4.33 (2H, q, J = 7.1 Hz), 8.36 (1H, s), 9.76 (1H, br s).
To
a suspension of 11 (124 mg, 0.357 mmol) in DME
(4 mL)/DMF (1 mL) was added portionwise potassium tert-butox-
ide (44 mg, 0.462 mmol) and the mixture was stirred at 0 °C for
15 min. LiBr (62 mg, 0.714 mmol) was added, and the mixture
was stirred at room temperature for 15 min. Phenacyl bromide
(142 mg, 0.714 mmol) was added, and the mixture was stirred at
60 °C for 16 h. The mixture was diluted with brine, and extracted
twice with AcOEt. The combined organic layer was dried over
MgSO4 and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane/AcOEt = 97/3 to 7/3) to
give the title compound (95 mg, 57%) as a yellow solid. 1H NMR
(DMSO-d6) d 1.17–1.36 (6H, m), 2.74 (2H, q, J = 7.2 Hz), 3.90 (3H,
s), 4.27 (2H, q, J = 7.2 Hz), 4.91 (2H, q, J = 9.1 Hz), 5.72 (2H, s),
7.56–7.68 (2H, m), 7.69–7.83 (1H, m), 8.03–8.24 (2H, m).
5.10. Ethyl 6-ethyl-2-oxo-1,2-dihydropyridine-3-carboxylate
(18)
5.14. Ethyl 5-bromo-6-ethyl-1-methyl-3-(2,2,2-trifluoroethoxy)
-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (20)
A mixture of 17 (32.1 g, 140 mmol), Et3N (39 mL, 280 mmol),
10% Pd-C (1.60 g) in EtOH (180 mL)/THF (180 mL) was stirred at
room temperature for 5 h under H2 atmosphere. The catalyst was
filtered off, washed with MeOH, and the filtrate was concentrated
in vacuo. To the residue was added AcOEt, the insoluble material
was filtered off, and washed with AcOEt. The filtrate was washed
with water containing 6 M HCl aq (1 mL), and the aqueous layer
was extracted with AcOEt. The combined organic layer was washed
with brine, dried over MgSO4 and concentrated in vacuo. The pre-
cipitate was collected by filtration, and washed with mixed solvent
of hexane/AcOEt to give the title compound (24.7 g, 90%) as a
brown powder. 1H NMR (CDCl3) d 1.32 (3H, t, J = 7.6 Hz), 1.38
(3H, t, J = 7.2 Hz), 2.74 (2H, q, J = 7.6 Hz), 4.37 (2H, q, J = 7.2 Hz),
6.29 (1H, d, J = 7.2 Hz), 8.18 (1H, d, J = 7.6 Hz), 12.50 (1H, br s).
In the same manner as in the preparation of 10, the title com-
pound (1.80 g, 96%) was obtained as a pale yellow powder from
4b (1.50 g, 4.58 mmol). 1H NMR (DMSO-d6) d 1.22–1.41 (6H, m),
3.00 (2H, q, J = 7.4 Hz), 3.96 (3H, s), 4.35 (2H, q, J = 7.1 Hz), 4.86
(2H, q, J = 9.1 Hz), 8.36 (1H, s).
5.15. Ethyl 5-amino-6-ethyl-1-methyl-3-(2,2,2-trifluoroethoxy)-
1H-pyrrolo[2,3-b]pyridine-2-carboxylate (21)
To a mixture of 20 (2.53 g, 6.18 mmol), benzophenoneimine
(1.5 mL, 8.94 mmol), Cs2CO3 (3.98 g, 12.2 mmol) and toluene
(30 mL) were added Pd2(dba)3 (389 mg, 0.425 mmol) and xantphos
(499 mg, 0.862 mmol), and the mixture was stirred at 100 °C for
22 h under Ar atmosphere. The mixture was filtered through
Celite pad, and washed with AcOEt. The filtrate was washed with
water (20 mL) and brine (10 mL), dried over MgSO4 and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography (hexane/AcOEt = 99/1 to 9/1) to give crude ethyl
5-[(diphenylmethylidene)amino]-6-ethyl-1-methyl-3-(2,2,2-triflu-
oroethoxy)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate. The obtained
crude compound was dissolved in THF (20 mL), then 2 M HCl aq
(5 mL) was added, and the mixture was stirred at room temperature
for 1 h. To the mixture was added aqueous NaHCO3 solution
(40 mL), and the mixture was extracted three times with mixed
5.11. Ethyl 5-bromo-6-ethyl-2-oxo-1,2-dihydropyridine-3-carbo
xylate (19)
To a solution of 18 (17.5 g, 89.7 mmol) in DMF (100 mL) was
added N-bromosuccinimide (16.0 g, 89.9 mmol) at 0 °C, and the
mixture was stirred at room temperature for 1 h. To the mixture
was added dropwise water (250 mL) at 0 °C, and the mixture was
stirred at room temperature for 30 min. The precipitate was
collected by filtration, and washed with water to give the title

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T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
solution of AcOEt/THF (50 mL). The extract was washed with brine
(20 mL), dried over MgSO4 and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hexane/
AcOEt = 99/1 to 2/1) to give the title compound (1.95 g, 91%) as a
yellow powder. 1H NMR (DMSO-d6) d 1.25 (3H, t, J = 7.4 Hz), 1.34
(3H, t, J = 7.1 Hz), 2.75 (2H, q, J = 7.4 Hz), 3.91 (3H, s), 4.32 (2H, q,
J = 7.1 Hz), 4.66 (2H, q, J = 9.1 Hz), 4.86 (2H, s), 7.16 (1H, s).
was stirred at 100 °C for 1 h. After cooling, the mixture was diluted
with water (150 mL), and extracted twice with AcOEt. The extracts
were combined, washed with brine, dried over MgSO4 and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane/AcOEt = 20/1) to give ethyl 2-chloro-4-
ethenylbenzoate (820 mg, 100%) as colorless oil. A mixture of ethyl
2-chloro-4-ethenylbenzoate (7.70 g, 36.55 mmol), 5% barium
hydroxide on palladium (1.5 g) in AcOEt (150 mL) was stirred at
room temperature for 8 h under H2 atmosphere. The catalyst was
filtered off, and the filtrate was concentrated in vacuo to give the
title compound (7.61 g, 98%) as pale yellow liquid. 1H NMR
(CDCl3) d 1.24 (3H, t, J = 7.5 Hz), 1.40 (3H, t, J = 7.2 Hz), 2.65 (2H,
q, J = 7.5 Hz), 4.38 (2H, q, J = 7.2 Hz), 7.13 (1H, dd, J = 8.1, 1.2 Hz),
7.28 (1H, d, J = 1.2 Hz), 7.76 (1H, d, J = 8.1 Hz).
5.16. Ethyl 6-ethyl-1-methyl-5-[(phenylcarbonyl)amino]-3-(2,2,
2-trifluoroethoxy)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate
(22)
To a mixture of 21 (135 mg, 0.391 mmol), pyridine (63.4
0.784 mmol) and THF (3 mL) was added benzoyl chloride (54.4
l
l
L,
L,
0.469 mmol) at 0 °C, and the mixture was stirred at room tempera-
ture for 1.5 h. To the reaction mixture was added water (5 mL), and
the mixture was extracted four times with AcOEt (5 mL). The
extracts were combined, dried over MgSO4 and concentrated in
vacuo. The precipitate was collected by filtration to give the title
compound (140 mg, 80%) as a white powder. The filtrate was con-
centrated in vacuo, and the residue was purified by basic silica gel
column chromatography (hexane/AcOEt = 49/1 to 2/1) to give the
title compound (24.9 mg, 14%) as a white powder. 1H NMR
(DMSO-d6) d 1.25 (3H, t, J = 7.4 Hz), 1.36 (3H, t, J = 7.1 Hz), 2.89
(2H, q, J = 7.4 Hz), 4.01 (3H, s), 4.37 (2H, q, J = 7.1 Hz), 4.82 (2H, q,
J = 9.1 Hz), 7.46–7.72 (3H, m), 7.90–8.13 (3H, m), 10.13 (1H, s).
5.20. Ethyl 2-chloro-4-ethyl-5-nitrobenzoate (6d)
To a solution of 24 (3.0 g, 14.1 mmol) in concd H2SO4 (10 mL)
was added dropwise a solution of NaNO3 (1.20 g, 14.1 mmol) in
concd H2SO4 (10 mL) at 0 °C, and the mixture was stirred at 0 °C
for 30 min. The mixture was poured into water (300 mL) and
extracted with AcOEt. The extract was washed with brine, dried
over MgSO4 and concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane/AcOEt = 20/1) to
give the title compound (2.56 g, 70%) as a pale yellow solid. 1H
NMR (DMSO-d6) d 1.22 (3H, t, J = 7.5 Hz), 1.34 (3H, t, J = 7.1 Hz),
2.88 (2H, q, J = 7.5 Hz), 4.36 (2H, q, J = 7.1 Hz), 7.82 (1H, s), 8.38
(1H, s).
5.17. 6-Ethyl-1-methyl-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluo
roethoxy)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (3b)
5.21. Ethyl 6-ethyl-3-hydroxy-1-methyl-5-nitro-1H-indole-2-
To a mixture of sodium tert-butoxide (264 mg, 2.75 mmol),
Pd(OAc)2 (25.9 mg, 0.115 mmol), 2-(dicyclohexylphosphino)-20-
methylbiphenyl (86.1 mg, 0.236 mmol) and toluene (6 mL) were
added a solution of 20 (450 mg, 1.10 mmol) and acetophenone
(0.256 mL, 2.20 mmol) in toluene (4 mL), and the mixture was stir-
red at 70 °C for 17 h. After allowing to cool to room temperature,
1 M NaOH aq (3 mL) and EtOH (5 mL) were added, and the mixture
was stirred at 50 °C for 2 h. To the reaction mixture was added
aqueous NH4Cl solution (30 mL), and the mixture was extracted
four times with AcOEt (20 mL). The extracts were combined,
washed with brine (10 mL), dried over anhydrous MgSO4 and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (hexane/AcOEt = 1/1 to AcOEt/MeOH = 19/1) and
preparative HPLC (0.1% TFA-containing MeCN/0.1% TFA-containing
water = 1/1 to 7/3) to give the title compound (104 mg, 22%) as a
pale yellow powder. 1H NMR (DMSO-d6) d 1.22 (3H, t, J = 7.5 Hz),
2.70 (2H, q, J = 7.5 Hz), 3.98 (3H, s), 4.63 (2H, s), 4.77 (2H, q,
J = 9.3 Hz), 7.51–7.63 (2H, m), 7.64–7.74 (1H, m), 7.82 (1H, s),
8.04–8.17 (2H, m), 13.35 (1H, br s).
carboxylate (4d)
A mixture of 6d (1.2 g, 4.66 mmol), ethyl sarcosinate hydrochlo-
ride (2.86 g, 18.6 mmol) and Et3N (3.77 g, 37.3 mmol) in EtOH
(20 mL) was heated under reflux for 20 h. After cooling, the mixture
was partitioned between HCl aq and AcOEt. The organic layer was
separated, washed with brine, dried over MgSO4 and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy (hexane/AcOEt = 3/1) to give the title compound (557 mg,
41%) as
a d 1.25 (3H, t,
yellow solid. 1H NMR (DMSO-d6)
J = 7.5 Hz), 1.33 (3H, t, J = 7.1 Hz), 2.99 (2H, q, J = 7.5 Hz), 3.90 (3H,
s), 4.33 (2H, q, J = 7.1 Hz), 7.53 (1H, s), 8.57 (1H, s), 9.92 (1H, br s).
5.22. Ethyl 5-amino-6-ethyl-1-methyl-3-(2,2,2-trifluoroethoxy)-
1H-indole-2-carboxylate (25)
To a mixture of 4d (150 mg, 0.51 mmol) and Cs2CO3 (200 mg,
0.62 mmol) in DMF (3 mL) was added 2,2,2-trifluoroethyl trifluo-
romethanesulfonate (131 mg, 0.56 mmol), and the mixture was
stirred at room temperature for 1 h. The mixture was diluted with
saturated NaHCO3 aq and extracted with AcOEt. The extract was
washed with brine, dried over MgSO4 and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane/AcOEt = 3/1) to give the ethyl 6-ethyl-1-methyl-5-nitro-
3-(2,2,2-trifluoroethoxy)-1H-indole-2-carboxylate (190 mg, 100%)
5.18. 6-Ethyl-1-methyl-5-[(phenylcarbonyl)amino]-3-(2,2,2-trifl
uoroethoxy)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (3c)
In the same manner as in the preparation of 3a, the title com-
pound (75.4 mg, 95%) was obtained as a white powder from 22
(84.5 mg, 0.188 mmol). 1H NMR (DMSO-d6)
d
1.25 (3H, t,
as a yellow solid. A mixture of obtained compound above
J = 7.6 Hz), 2.88 (2H, q, J = 7.6 Hz), 4.01 (3H, s), 4.78 (2H, q,
J = 9.1 Hz), 7.47–7.73 (3H, m), 7.96 (1H, s), 7.98–8.09 (2H, m),
10.12 (1H, s), 13.28 (1H, br s).
(180 mg, 0.48 mmol) and 10% Pd-C (40 mg) in EtOH (10 mL)/THF
(4 mL) was stirred at room temperature for 3 h under H2 atmo-
sphere. The catalyst was removed by filtration and the filtrate
was concentrated in vacuo to give the title compound (160 mg,
97%) as a pale brown solid. 1H NMR (DMSO-d6) d 1.21 (3H, t,
J = 7.4 Hz), 1.33 (3H, t, J = 7.1 Hz), 2.59 (2H, q, J = 7.4 Hz), 3.84
(3H, s), 4.31 (2H, q, J = 7.1 Hz), 4.61 (2H, q, J = 9.3 Hz), 4.67 (2H,
s), 6.77 (1H, s), 7.16 (1H, s).
5.19. Ethyl 2-chloro-4-ethylbenzoate (24)
A mixture of 23 (1.00 g, 3.79 mmol), tributyl(vinyl)tin (2.41 g,
7.59 mmol) and Pd(PPh3)4 (439 mg, 0.38 mmol) in DMF (15 mL)

T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
4787
5.23. Ethyl 6-ethyl-1-methyl-5-[(phenylcarbonyl)amino]-3-(2,2,
2-trifluoroethoxy)-1H-indole-2-carboxylate (26)
5.27. Ethyl 6-(benzyloxy)-3-bromo-5-ethylpyridine-2-carboxy-
late (6e)
In the same manner as in the preparation of 22, the title com-
pound (89 mg, 86%) was obtained as a beige powder from 25
(80 mg, 0.23 mmol). 1H NMR (DMSO-d6) d 1.20 (3H, t, J = 7.4 Hz),
1.36 (3H, t, J = 7.1 Hz), 2.74 (2H, q, J = 7.4 Hz), 3.96 (3H, s), 4.36
(2H, q, J = 7.1 Hz), 4.74 (2H, q, J = 9.0 Hz), 7.49–7.61 (5H, m), 8.01
(2H, d, J = 7.2 Hz), 9.97 (1H, s).
To a solution of 29 (1.10 g, 5.63 mmol) in DMF (11 mL) was added
N-bromosuccinimide (1.30 g, 7.33 mmol), and the mixture was stir-
red at room temperature for 1 h. The mixture was diluted with water
(50 mL), and the resulting precipitate was collected by filtration to
give ethyl 3-bromo-5-ethyl-6-oxo-1,6-dihydropyridine-2-carboxy-
late (1.21 g, 79%) as a white solid. To a solution of obtained bromide
(17.1 g, 62.3 mmol) in toluene (342 mL) were added Ag2CO3 (29.2 g,
106 mmol) and benzyl bromide (14.8 mL, 125 mmol), and the mix-
ture was stirred at 40 °C for 2 h. The insoluble material was filtered,
and the filtrate was concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane to
hexane/AcOEt = 47/3) to give the title compound (22.4 g, 99%) as
yellow oil. 1H NMR (DMSO-d6) d 1.14 (3H, t, J = 7.4 Hz), 1.34 (3H, t,
J = 7.0 Hz), 2.60 (2H, q, J = 7.4 Hz), 4.36 (2H, q, J = 7.0 Hz), 5.35 (2H,
s), 7.31–7.43 (3H, m), 7.45–7.53 (2H, m), 7.92 (1H, s).
5.24. 6-Ethyl-1-methyl-5-[(phenylcarbonyl)amino]-3-(2,2,2-trifl
uoroethoxy)-1H-indole-2-carboxylic acid (3d)
In the same manner as in the preparation of 3a, the title com-
pound (72 mg, 95%) was obtained as a white powder from 26
(80 mg, 0.18 mmol). 1H NMR (DMSO-d6) d 1.20 (3H, t, J = 7.5 Hz),
2.73 (2H, q, J = 7.5 Hz), 3.96 (3H, s), 4.72 (2H, q, J = 9.1 Hz), 7.46–
7.60 (5H, m), 8.01 (2H, d, J = 6.9 Hz), 9.96 (1H, s), 13.20–13.40
(1H, br).
5.28. Ethyl 3-amino-6-(benzyloxy)-5-ethylpyridine-2-carboxy-
late (30)
5.25. Ethyl 5-ethylpyridine-2-carboxylate (28)
To a solution of 27 (20.0 g, 86.9 mmol) in DMF (100 mL) were
added tributyl(vinyl)tin (28.1 mL, 95.6 mmol) and Pd(PPh3)4 (2.
01 g, 1.74 mmol), and the mixture was stirred at 100 °C for 2 h
under Ar atmosphere. The mixture was diluted with water
(200 mL), and extracted with AcOEt (400 mL). The extracts were
combined, washed with brine (200 mL), dried over Na2SO4 and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane/AcOEt = 49/1 to 4/1) to give ethyl
5-ethenylpyridine-2-carboxylate (16.5 g, quant.) as pale yellow
oil. To a solution of obtained compound above (7.71 g, 43.5 mmol)
in EtOH (77 mL) was added 10% Pd-C (1.54 g) and the mixture was
stirred at room temperature for 3 h under H2 atmosphere. The mix-
ture was filtered through membrane filter, and the filtrate was con-
centrated in vacuo to give the title compound (7.92 g, quant.) as
pale yellow oil. 1H NMR (DMSO-d6) d 1.22 (3H, t, J = 7.6 Hz), 1.33
(3H, t, J = 7.1 Hz), 2.71 (2H, q, J = 7.6 Hz), 4.33 (2H, q, J = 7.1 Hz),
7.83 (1H, dd, J = 8.0, 2.2 Hz), 7.98 (1H, d, J = 8.0 Hz), 8.58 (1H, d,
J = 2.2 Hz).
In the same manner as in the preparation of 21, the title com-
pound (17.8 g, 98%) was obtained as yellow oil from 6e (22.0 g,
60.4 mmol). 1H NMR (DMSO-d6) d 1.12 (3H, t, J = 7.5 Hz), 1.33 (3H,
t, J = 7.1 Hz), 2.45–2.58 (2H, m), 4.27 (2H, q, J = 7.1 Hz), 5.26 (2H,
s), 6.33 (2H, s), 7.08 (1H, s), 7.27–7.40 (3H, m), 7.48–7.57 (2H, m).
5.29. Ethyl 6-(benzyloxy)-3-[(2-ethoxy-2-oxoethyl)(methyl)ami
no]-5-ethylpyridine-2-carboxylate (5e)
To a solution of 30 (771 mg, 2.57 mmol) in tert-butanol (7.7 mL)
was added di-tert-butyl dicarbonate (1.77 mL, 7.71 mmol), and the
mixture was stirred at 90 °C for 18 h. The mixture was concentrated
in vacuo, and the residue was dissolved in DMF (10 mL). To the
solution were added Cs2CO3 (2.93 g, 9.00 mmol) and iodomethane
(480 lL, 7.71 mmol), and the mixture was stirred at room temper-
ature for 4 h. The mixture was diluted with water (50 mL), and
extracted with AcOEt (100 mL). The organic layer was washed with
brine (50 mL), dried over Na2SO4 and concentrated in vacuo. To this
residue was added 4 M HCl/AcOEt (5.4 mL), and the mixture was
stirred at 0 °C for 2 h. The mixture was diluted with saturated
NaHCO3 aq (50 mL) and extracted with AcOEt (100 mL). The organic
layer was washed with brine (50 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by column chro-
matography (hexane/AcOEt = 49/1 to 47/3) to give ethyl 6-(benzy-
5.26. Ethyl 5-ethyl-6-oxo-1,6-dihydropyridine-2-carboxylate (29)
To a solution of 28 (1.28 g, 7.14 mmol) in MeCN (13 mL) was
added 3-chloroperoxybenzoic acid (2.84 g, 10.7 mmol), and the
mixture was stirred at room temperature for 20 h. The mixture
was concentrated to half volume, and diluted with AcOEt
(50 mL). The solution was poured into saturated NaHCO3 solution
(100 mL), and extracted with AcOEt (200 mL). The organic layer
was washed with brine (50 mL), dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane/AcOEt = 9/1 to AcOEt) to give ethyl 5-
ethylpyridine-2-carboxylate 1-oxide (1.15 g, 83%) as yellow oil.
To a solution of obtained compound in DMF (12 mL) was added tri-
fluoroacetic anhydride (8.19 mL, 58.9 mmol), and the mixture was
stirred at room temperature for 12 h under Ar atmosphere. The
mixture was concentrated in vacuo, and diluted with water
(50 mL). The mixture was extracted with AcOEt (100 mL). The
organic layer was washed with brine (50 mL), dried over Na2SO4
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/AcOEt = 19/1 to 1/1) to give the
title compound (1.10 g, 96%) as yellow oil. 1H NMR (DMSO-d6) d
1.11 (3H, t, J = 7.5 Hz), 1.30 (3H, t, J = 7.1 Hz), 2.41–2.49 (2H, m),
4.29 (2H, q, J = 7.1 Hz), 6.99 (1H, d, J = 7.0 Hz), 7.41 (1H, d,
J = 7.0 Hz), 11.53 (1H, br s).
loxy)-5-ethyl-3-(methylamino)pyridine-2-carboxylate
(705 mg,
87%) as yellow oil. To a solution of the compound obtained above
in DMF (14 mL) were added diisopropylethylamine (2.35 mL,
13.5 mmol) and ethyl bromoacetate (1.49 mL, 13.5 mmol), and
the mixture was stirred at 110 °C for 24 h. The mixture was diluted
with water (50 mL), and extracted with AcOEt (100 mL). The
organic layer was washed with brine (50 mL), dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane to hexane/AcOEt = 4/1) to give the
title compound (711 mg, 79%) as yellow oil. 1H NMR (DMSO-d6) d
1.09–1.21 (6H, m), 1.30 (3H, t, J = 7.1 Hz), 2.57 (2H, q, J = 7.4 Hz),
2.81 (3H, s), 3.88 (2H, s), 4.07 (2H, q, J = 7.1 Hz), 4.28 (2H, q,
J = 7.1 Hz), 5.28 (2H, s), 7.26–7.42 (3H, m), 7.42–7.51 (3H, m).
5.30. Ethyl 5-(benzyloxy)-6-ethyl-3-hydroxy-1-methyl-1H-pyrr
olo[3,2-b]pyridine-2-carboxylate (4e)
To a solution of 5e (711 mg, 1.78 mmol) in EtOH (14 mL) was
added 20% solution of NaOEt in EtOH (1.42 mL), and the mixture

4788
T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
was stirred at room temperature for 1 h. The mixture was concen-
trated in vacuo, acidified with 1 M HCl aq, and extracted with
AcOEt (100 mL). The organic layer was washed with brine
(50 mL), dried over Na2SO4 and concentrated in vacuo. The precip-
itate was collected by filtration, and washed with IPE to give the
title compound (485 mg, 77%) as a pale orange powder. 1H NMR
(DMSO-d6) d 1.22 (3H, t, J = 7.4 Hz), 1.34 (3H, t, J = 7.1 Hz), 2.69
(2H, q, J = 7.4 Hz), 3.84 (3H, s), 4.35 (2H, q, J = 7.1 Hz), 5.44 (2H,
s), 7.27–7.44 (3H, m), 7.46–7.56 (2H, m), 7.78 (1H, s), 8.84 (1H, s).
compound (152 mg, 88%) as a white solid. 1H NMR (DMSO-d6) d
1.23 (3H, t, J = 7.5 Hz), 2.71 (2H, q, J = 7.5 Hz), 3.97 (3H, s), 5.10
(2H, q, J = 9.1 Hz), 7.49–7.65 (3H, m), 7.97–8.06 (3H, m), 10.56
(1H, s), 13.17 (1H, br s).
5.34. Ethyl 5-ethyl-3-hydroxypyrazine-2-carboxylate (34)
To a suspension of 33 (6.40 g, 39.7 mmol) in EtOH (60 mL) was
added diisopropylethylamine (14 mL, 80.4 mmol), and the mixture
was stirred at room temperature for 10 min. To this solution was
added dropwise diethyl ketomalonate (6 mL, 39.3 mmol) at 0 °C,
and the mixture was stirred at room temperature for 1.5 h and
with heating under reflux for 20 h. The reaction mixture was con-
centrated in vacuo, and the residue was dissolved in a small
amount of EtOH. The solution was passed through silica gel
(hexane/AcOEt = 1/1 to AcOEt). The eluate was concentrated in
vacuo, and the residue was purified by silica gel column chro-
matography (hexane/AcOEt = 2/1 to AcOEt) to give the title com-
pound (1.94 g, 25%) as a pale yellow powder. 1H NMR (DMSO-d6)
d 1.19 (3H, t, J = 7.6 Hz), 1.27 (3H, t, J = 7.2 Hz), 2.53–2.62 (2H,
m), 4.26 (2H, q, J = 7.2 Hz), 7.42 (1H, br s), 12.68 (1H, br s).
5.31. Ethyl 6-ethyl-1-methyl-3-(2,2,2-trifluoroethoxy)-5-{[(trifl
uoromethyl)sulfonyl]oxy}-1H-pyrrolo [3,2-b]pyridine-2-carbox
ylate (31)
To a solution of 4e (485 mg, 1.37 mmol) in DMF (4.9 mL) were
added Cs2CO3 (534 mg, 1.64 mmol) and 2,2,2-trifluoroethyl
trifluoromethanesulfonate (217 lL, 1.51 mmol), and the mixture
was stirred at room temperature for 1 h. The mixture was diluted
with water (20 mL), and the precipitate was collected by filtration,
washed with water and IPE to give ethyl 5-(benzyloxy)-6-ethyl-
1-methyl-3-(2,2,2-trifluoroethoxy)-1H-pyrrolo[3,2-b]pyridine-2-
carboxylate (580 mg, 97%) as a pale orange powder. To a solution
of obtained powder (428 mg, 0.981 mmol) in EtOH (0.87 mL)/THF
(0.43 mL) was added 10% Pd-C (128 mg), and the mixture was stir-
red at room temperature for 1 h under H2 atmosphere. The mixture
was filtered through membrane filter, and the filtrate was concen-
trated in vacuo. The resulting solid was collected by filtration to
give ethyl 6-ethyl-1-methyl-5-oxo-3-(2,2,2-trifluoroethoxy)-4,5-
dihydro-1H-pyrrolo[3,2-b]pyridine -2-carboxylate (330 mg, 97%)
5.35. Ethyl 3-(benzyloxy)-6-bromo-5-ethylpyrazine-2-carboxy
late (35)
To a solution of 34 (3.75 g, 19.1 mmol) in DMF (40 mL) was added
N-bromosuccinimide (3.43 g, 19.3 mmol) at 0 °C. The mixture was
diluted with water (80 mL), and the precipitate was collected by fil-
tration washed with water to give ethyl 6-bromo-5-ethyl-3-hydrox-
ypyrazine-2-carboxylate. The filtrate was extracted with
Et2O/AcOEt = 1/1 solution, the extracts were washed with brine,
dried over MgSO4 and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (hexane/AcOEt = 99/1 to
4/1) to give ethyl 6-bromo-5-ethyl-3-hydroxypyrazine-2-carboxy-
late. To a solution of the obtained compound above (750 mg,
2.73 mmol) in toluene (10 mL) were added Ag2CO3 (1.05 g,
as
a
white solid. To
a
solution of obtained solid (330 mg,
L,
0.953 mmol) in pyridine (9.9 mL) was added Tf2O (384
l
2.28 mmol) at 0 °C, and the mixture was stirred at 60 °C for 16 h
under N2 atmosphere. The mixture was concentrated in vacuo,
the residue was diluted with saturated NaHCO3 aq (50 mL), and
extracted with AcOEt (100 mL). The organic layer was washed with
brine (50 mL), dried over Na2SO4 and concentrated in vacuo. The
resulting solid was collected by filtration, and washed with hexane
to give the title compound (418 mg, 92%) as a pale yellow powder.
1H NMR (DMSO-d6) d 1.24–1.39 (6H, m), 2.79 (2H, q, J = 7.6 Hz),
3.99 (3H, s), 4.36 (2H, q, J = 7.2 Hz), 5.05 (2H, q, J = 8.9 Hz), 8.35
(1H, s).
3.81 mmol) and benzyl bromide (390 lL, 3.28 mmol) at 0 °C, the
mixture was stirred at room temperature for 3 h. The insoluble
material was removed by filtration through Celite pad, and the fil-
trate was concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/AcOEt = 99/1 to 4/1) to give
the title compound (908 mg, 91%) as colorless oil. 1H NMR (DMSO-
d6) d 1.25 (3H, t, J = 7.5 Hz), 1.29 (3H, t, J = 7.1 Hz), 2.89 (2H, q,
J = 7.5 Hz), 4.33 (2H, q, J = 7.1 Hz), 5.50 (2H, s), 7.28–7.44 (3H, m),
7.44–7.56 (2H, m).
5.32. Ethyl 5-amino-6-ethyl-1-methyl-3-(2,2,2-trifluoroethoxy)-
1H-pyrrolo[3,2-b]pyridine-2-carboxylate (32)
In the same manner as in the preparation of 21, the title com-
pound (141 g, 90%) was obtained as a white powder from 31
5.36. Ethyl 3-(benzyloxy)-5-ethyl-6-[(phenylcarbonyl)amino]-
pyrazine-2-carboxylate (36)
(218 mg, 0.456 mmol). 1H NMR (DMSO-d6)
d 1.20 (3H, t,
J = 7.5 Hz), 1.29 (3H, t, J = 7.1 Hz), 2.51–2.61 (2H, m), 3.83 (3H, s),
4.26 (2H, q, J = 7.1 Hz), 5.10 (2H, q, J = 9.1 Hz), 5.75 (2H, s), 7.55
(1H, s).
To a mixture of 35 (906 mg, 2.48 mmol), benzophenoneimine
(624 lL, 3.72 mmol), Cs2CO3 (1.62 g, 4.97 mmol) in toluene
(13 mL) were added xantphos (294 mg, 0.508 mmol) and
5.33. 6-Ethyl-1-methyl-5-[(phenylcarbonyl)amino]-3-(2,2,2-trifl
Pd2(dba)3 (228 mg, 0.249 mmol), and the mixture was stirred at
uoroethoxy)-1H-pyrrolo[3,2-b]pyridine-2-carboxylic acid (3e)
100 °C for 14 h. Benzophenoneimine (416 lL, 2.48 mmol),
Pd2(dba)3 (120 mg, 0.131 mmol) and xantphos (148 mg,
0.256 mmol) were further added, the mixture was stirred at
100 °C for 3.5 h. The mixture was filtered through Celite pad using
AcOEt, and the filtrate was washed with water and brine. The
organic layer was dried over MgSO4, and concentrated in vacuo.
The residue was dissolved in THF (10 mL) /EtOH (2 mL), and 2 M
HCl aq (3 mL) was added. After stirring for 3 h, the mixture was
quenched with aqueous NaHCO3 solution, and extracted with
AcOEt. The extracts were washed with brine, dried over MgSO4
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/AcOEt = 19/1 to 1/2) to give ethyl
6-amino-3-(benzyloxy)-5-ethylpyrazine-2-carboxylate (441 mg,
To a solution of 32 (141 mg, 0.408 mmol) in THF (1.4 mL) were
added pyridine (85.8 lL, 1.06 mmol) and benzoyl chloride (104 lL,
0.898 mmol), and the mixture was stirred at 50 °C for 2 h. The mix-
ture was diluted with water (30 mL) and extracted with AcOEt
(50 mL). The organic layer was washed with brine (30 mL), dried
over Na2SO4 and concentrated in vacuo. To a solution of the residue
in EtOH (3.7 mL) was added aqueous 8 M NaOH aq (0.366 mL), and
the mixture was stirred at 50 °C for 1 h. The mixture was concen-
trated in vacuo, the precipitate was diluted with water (20 mL) and
acidified with aqueous 1 M HCl aq. The resulting precipitate was
collected by filtration, and washed with water to give the title

T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
4789
59%) as a brown powder. To a solution of obtained product
(250 mg, 0.828 mmol) in THF (3 mL) were added pyridine
(0.5 mL), and the mixture was stirred at room temperature for
overnight. The mixture was acidified with 1 N HCl aq, and diluted
with water. The precipitate was collected by filtration using water
to give the title compound (99.6 mg, 99%) as a pale yellow powder.
1H NMR (DMSO-d6) d 1.29 (3H, t, J = 7.4 Hz), 2.89 (2H, q, J = 7.4 Hz),
4.01 (3H, s), 5.10 (2H, q, J = 8.9 Hz), 7.45–7.72 (3H, m), 7.96–8.10
(2H, m), 10.82 (1H, s), 13.47 (1H, br s).
(100
at 0 °C, and the mixture was stirred at 0 °C for 2.5 h. Benzoyl chlo-
ride (53 L, 0.457 mmol) was added, and the mixture was stirred at
0 °C for 1 h. Further benzoyl chloride (53 L, 0.457 mmol) and pyr-
idine (55 L, 0.680 mmol) were added, the mixture was stirred at
lL, 1.24 mmol) and benzoyl chloride (106 lL, 0.913 mmol)
l
l
l
0 °C for 1 h. The mixture was quenched with aqueous NaHCO3
solution, and the mixture was extracted with AcOEt. The organic
layer was dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(hexane/AcOEt = 19/1 to 1/1) to give the title compound (312 mg,
5.40. 2-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-7-methyl-4-
oxo-5-(2,2,2-trifluoroethoxy)-4,7-dihydro-3H-pyrrolo[2,3-d]pyr
imidine-6-carboxamide (2a)
93%) as an orange oil. 1H NMR (DMSO-d6)
d
1.22 (3H, t,
To a mixture of 3a (1.0 g, 3.1 mmol), 2-(4-aminopiperidin-1-yl)-
2-oxoethanol hydrochloride (732 mg, 3.7 mmol) and HOBt
(635 mg, 4.7 mmol) in DMF (15 mL) were added EDC (900 mg,
4.7 mmol) and Et3N (1.2 mL, 8.5 mmol) at 0 °C, and the mixture
was stirred at room temperature for 4.5 h. The reaction mixture
was diluted with saturated NaHCO3 aq, and the precipitated solid
was collected by filtration. The solid was washed with water, and
dried in vacuo to give the title compound (1.1 g, 79%) as a white
powder. 1H NMR (DMSO-d6) d 1.13–1.52 (5H, m), 1.76–1.98 (2H,
m), 2.63 (2H, q, J = 7.5 Hz), 2.75–2.95 (1H, m), 3.00–3.19 (1H, m),
3.61–3.75 (1H, m), 3.83 (3H, s), 3.93–4.17 (3H, m), 4.17–4.34
(1H, m), 4.53 (1H, t, J = 5.5 Hz), 5.20 (2H, q, J = 9.1 Hz), 7.43 (1H,
d, J = 7.7 Hz), 12.10 (1H, s).
J = 7.5 Hz), 1.28 (3H, t, J = 7.1 Hz), 2.73 (2H, q, J = 7.5 Hz), 4.33
(2H, q, J = 7.1 Hz), 5.55 (2H, s), 7.28–7.47 (3H, m), 7.47–7.71 (5H,
m), 7.95–8.09 (2H, m), 10.76 (1H, s).
5.37. Ethyl 5-ethyl-6-[(phenylcarbonyl)amino]-3-{[(trifluorome
thyl)sulfonyl]oxy}pyrazine-2-carboxylate (6f)
To a solution of 36 (310 mg, 0.765 mmol) in EtOH (5 mL) was
added 10% Pd-C (32.7 mg), and the mixture was stirred at room
temperature for 3 h under H2 atmosphere. The catalyst was
removed by filtration through Celite pad, and the filtrate was con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (hexane/AcOEt = 9/1 to 1/4) to give ethyl 5-
ethyl-3-hydroxy-6-[(phenylcarbonyl)amino]pyrazine-2-carboxy-
late (185 mg, 77%) as a pale yellow powder. To a solution of the
compound obtained above (183 mg, 0.580 mmol) in pyridine
The following compounds 2b–f were prepared in a same man-
ner similar to that described for 2a.
5.41. 6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-5-
(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-1H-
pyrrolo[2,3-b]pyridine-2-carboxamide (2b)
(2 mL) was added dropwise Tf2O (147
lL, 0.871 mmol) at 0 °C,
and the mixture was stirred at 0 °C for 1 h. Further Tf2O (30
lL,
0.178 mmol) was added, and the mixture was stirred at 0 °C for
1 h. The mixture was diluted with water (15 mL), and the mixture
was extracted three times with AcOEt (15 mL). The extracts were
combined, washed with water (20 mL) and brine (10 mL), dried
over MgSO4 and concentrated in vacuo. The residue was purified
by silica gel column chromatography (hexane/AcOEt = 49/1 to
1/1) to give the title compound (244 mg, 94%) as yellow oil. 1H
NMR (DMSO-d6) d 1.21 (3H, t, J = 7.3 Hz), 1.34 (3H, t, J = 7.1 Hz),
2.83 (2H, q, J = 7.3 Hz), 4.43 (2H, q, J = 7.1 Hz), 7.49–7.74 (3H, m),
7.99–8.10 (2H, m), 11.42 (1H, s).
Yield 47%, white crystals. Mp 150 °C (recrystallized from
hexane/AcOEt) 1H NMR (DMSO-d6) d 1.22 (3H, t, J = 7.4 Hz), 1.28–
1.58 (2H, m), 1.82–2.02 (2H, m), 2.70 (2H, q, J = 7.2 Hz), 2.78–
2.94 (1H, m), 3.02–3.21 (1H, m), 3.60–3.78 (1H, m), 3.93 (3H, s),
4.00–4.19 (3H, m), 4.20–4.36 (1H, m), 4.53 (1H, t, J = 5.4 Hz), 4.63
(2H, s), 4.91 (2H, q, J = 8.9 Hz), 7.52–7.64 (2H, m), 7.64–7.74 (1H,
m), 7.79 (1H, d, J = 7.7 Hz), 8.03 (1H, s), 8.11 (2H, d, J = 7.4 Hz).
Anal. Calcd for C28H31N4O5F3: C, 59.99; H, 5.57; N, 9.99. Found: C,
59.75; H, 5.51; N, 9.81. LC–MS: m/z = 561 (MH+).
5.38. Ethyl 3-ethyl-7-hydroxy-5-methyl-2-[(phenylcarbonyl)-
amino]-5H-pyrrolo[2,3-b]pyrazine-6-carboxylate (4f)
5.42. 6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-5-
[(phenylcarbonyl)amino]-3-(2,2,2-trifluoroethoxy)-1H-pyrrolo-
[2,3-b]pyridine-2-carboxamide (2c)
In the same manner as in the preparation of 4b, the title com-
pound (159 mg, 80%) was obtained as a pale yellow powder from
6f (242 mg, 0.541 mmol). 1H NMR (DMSO-d6) d 1.28 (3H, t,
J = 7.6 Hz), 1.35 (3H, t, J = 7.2 Hz), 2.88 (2H, q, J = 7.6 Hz), 3.95
(3H, s), 4.37 (2H, q, J = 7.2 Hz), 7.45–7.72 (3H, m), 7.94–8.12 (2H,
m), 9.91 (1H, br s), 10.72 (1H, s).
Yield 53%, white crystals. Mp 200 °C (recrystallized from
hexane/AcOEt). 1H NMR (DMSO-d6) d 1.24 (3H, t, J = 7.5 Hz),
1.32–1.55 (2H, m), 1.84–1.99 (2H, m), 2.79–2.92 (1H, m), 2.86
(2H, q, J = 7.5 Hz), 3.02–3.19 (1H, m), 3.63–3.78 (1H, m), 3.94
(3H, s), 4.00–4.18 (1H, m), 4.10 (2H, t, J = 5.7 Hz), 4.21–4.37 (1H,
m), 4.53 (1H, t, J = 5.5 Hz), 4.94 (2H, q, J = 8.8 Hz), 7.46–7.71 (3H,
m), 7.86 (1H, d, J = 7.6 Hz), 8.03 (2H, d, J = 6.8 Hz), 8.13 (1H, s),
10.11 (1H, s). Anal. Calcd for C27H30N5O5F3: C,57.75; H,5.38;
N,12.47. Found: C, 58.03; H,5.47; N,12.44. LC–MS: m/z = 562 (MH+).
5.39. 3-Ethyl-5-methyl-2-[(phenylcarbonyl)amino]-7-(2,2,2-tri-
fluoroethoxy)-5H-pyrrolo[2,3-b]pyrazine-6-carboxylic acid (3f)
To a solution of 4f (59.2 mg, 0.161 mmol) in DMF were added
Cs2CO3 (72.0 mg, 0.220 mmol) and 2,2,2-trifluoroethyl trifluo-
romethanesulfonate (28 lL, 0.166 mmol) at 0 °C, and the mixture
5.43. 6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-5-
[(phenylcarbonyl)amino]-3-(2,2,2-trifluoroethoxy)-1H-indole-2
-carboxamide (2d)
was stirred at 0 °C for 2 h. The mixture was diluted with water,
and the precipitate was collected by filtration. The collection was
dissolved in hexane/AcOEt, and purified by silica gel column chro-
matography (hexane/AcOEt = 49/1 to 2/1) to give ethyl 3-ethyl-
5-methyl-2-[(phenylcarbonyl)amino]-7-(2,2,2-trifluoroethoxy)-5H
-pyrrolo[2,3-b]pyrazine-6-carboxylate (53.2 mg, 74%) as a pale yel-
low powder. To a solution of obtained compound above (107 mg,
0.238 mmol) in THF (1 mL)/EtOH (2 mL) was added 1 M NaOH aq
Yield 82%, white crystals. Mp 227 °C (recrystallized from
hexane/AcOEt). 1H NMR (DMSO-d6) d 1.20 (3H, t, J = 7.5 Hz),
1.30–1.50 (2H, m), 1.91 (2H, d, J = 9.9 Hz), 2.72 (2H, q, J = 7.5 Hz),
2.82 (1H, t, J = 11.9 Hz), 3.10 (1H, t, J = 11.9 Hz), 3.71 (1H, d,
J = 12.9 Hz), 3.89 (3H, s), 4.04–4.12 (3H, m), 4.30 (1H, d,
J = 13.8 Hz), 4.54 (1H, t, J = 5.4 Hz), 4.85 (2H, q, J = 8.9 Hz), 7.44

4790
T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
(1H, s), 7.51–7.62 (4H, m), 7.84 (1H, d, J = 7.5 Hz), 8.01 (2H, d,
J = 6.9 Hz), 9.96 (1H, s). Anal. Calcd for C28H31N4O5F3: C; 59.99, H;
5.57, N; 9.99. Found: C; 59.79, H; 5.71, N; 9.73. LC–MS: m/z = 561
(MH+).
overnight in 25
37 °C. After incubation, 20
(2.5 g/mL in DMEM containing 2% FBS) and 5
diluted the compounds 10ꢂ solution (0.0003–10
were added to the culture to achieve the final concentrations of
5.8% FBS, 1- g/mL of Shh-N, and 0.03–1000 nM of the compounds
(n = 4 wells per concentration). The cells were then incubated for
an additional 48 h. To determine the window of the assay, the cells
were incubated in the media containing 0.1% DMSO with or with-
l
L of DMEM containing 10% FBS under 5% CO2 at
of recombinant mouse Shh-N
L of a serially
M in DMEM)
lL
l
l
l
5.44. 6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-5-
[(phenylcarbonyl)amino]-3-(2,2,2-trifluoroethoxy)-1H-pyrrolo-
[3,2-b]pyridine-2-carboxamide (2e)
l
Yield 46%, white crystals. Mp 165 °C (recrystallized from
hexane/AcOEt). 1H NMR (DMSO-d6) d 1.23 (3H, t, J = 7.5 Hz),
1.31–1.53 (2H, m), 1.84–1.96 (2H, m), 2.71 (2H, q, J = 7.5 Hz),
2.80–2.96 (1H, m), 3.04–3.20 (1H, m), 3.62–3.77 (1H, m), 3.94
(3H, s), 4.02–4.16 (3H, m), 4.19–4.30 (1H, m), 4.54 (1H, t,
J = 5.5 Hz), 5.23 (2H, q, J = 8.9 Hz), 7.47–7.65 (3H, m), 7.80 (1H, d,
J = 7.7 Hz), 7.95–8.05 (3H, m), 10.52 (1H, s). Anal. Calcd for
out 1 lg/mL Shh-N (0% or 100% inhibition control, respectively
(n = 10 wells). The luciferase activities of reporter cells were mea-
sured by Bright-Glo™ (Promega Corp [Madison, WI, USA]) using
the EnVisionÒ plate leader (PerkinElmer, Inc [Waltham, MA, USA]).
5.48. In vivo PD assay
C
27H30F3N5O5ꢀ0.30H2O: C, 57.20; H, 5.44; N, 12.35. Found: C,
In vivo PD assay was conducted by using the nude mice bearing
human primary pancreatic tumor (PAN-04). The tumor line was
established by Central Institute for Experimental Animals. The test
compounds were orally administered twice a day. After 24 h from
the first administration, the tumors were extirpated and treated
with RNAlater (Ambion). Total RNA samples were isolated using
RNeasy Mini kit (Qiagen), and the first strand cDNA samples were
prepared using the high capacity cDNA transcription kit (Applied
Biosystems). The primer sets ofqPCR for quantification of stromal
57.32; H, 5.58; N, 12.12. LC–MS: m/z = 562 (MH+).
5.45. 3-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-5-methyl-2-
[(phenylcarbonyl)amino]-7-(2,2,2-trifluoroethoxy)-5H-pyrrolo-
[2,3-b]pyrazine-6-carboxamide (2f)
Yield 61%, pale yellow crystals. Mp 142 °C (recrystallized from
hexane/AcOEt). 1H NMR (DMSO-d6) d 1.28 (3H, t, J = 7.5 Hz),
1.33–1.58 (2H, m), 1.84–1.99 (2H, m), 2.81–2.98 (3H, m), 3.06–
3.21 (1H, m), 3.62–3.76 (1H, m), 3.97 (3H, s), 4.01–4.18 (3H, m),
4.18–4.32 (1H, m), 4.54 (1H, t, J = 5.5 Hz), 5.20 (2H, q, J = 8.9 Hz),
7.49–7.69 (3H, m), 7.95 (1H, d, J = 7.6 Hz), 7.99–8.10 (2H, m),
10.79 (1H, s). Anal. Calcd for C26H29N6O5F3ꢀH2O: C, 54.64; H,
5.29; N, 14.70. Found: C, 55.02; H, 5.18; N, 14.81. LC–MS:
m/z = 563 (MH+).
Gli1
mRNA
were
as
follows
(Applied
Biosystems):
Mm00494645_m1 (mouse Gli1), 4352339E (mouse GAPDH).
5.49. Pharmacokinetic studies
Test compounds were administered at a dose of 10 mg/kg as a
cassette dosing to nonfasted mice. After the oral administration,
blood samples were collected. The blood samples were centrifuged
to obtain the plasma fraction. The plasma samples were depro-
teinized with MeCN containing an internal standard. After centrifu-
gation, the supernatant was diluted with a mixture of 10 mmol/L
HCOONH4 containing 0.2% formic acid and MeCN containing 0.2%
formic acid (9:1, v/v), and centrifuged again. The compound con-
centrations in the supernatant were measured by LC/MS/MS.
5.46. 2-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-7-methyl-4-
oxo-3-(2-oxo-2-phenylethyl)-5-(2,2,2-trifluoroethoxy)-4,7-dihy
dro-3H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (2g)
A
mixture of 2a (300 mg, 0.65 mmol), K2CO3 (135 mg,
0.98 mmol), DMF (1.0 mL)/DME (4.0 mL) was stirred at 0 °C for
30 min. LiBr (114 mg, 1.3 mmol) was added, and the mixture was
stirred at room temperature for 30 min. Phenacyl bromide
(260 mg, 1.3 mmol) was added, and the reaction mixture was stir-
red at 60 °C for 13.5 h followed by addition of DMF (1.0 mL). The
mixture was stirred at 60 °C for 1.5 h, at 70 °C for 2.5 h, and at
80 °C for 20 h. The mixture was diluted with water, and extracted
twice with AcOEt. The extracts were combined, washed with brine,
dried over MgSO4 and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography (hexane/AcOEt = 2/3 to
AcOEt) and then aminosilica gel column chromatography
(hexane/AcOEt = 2/3 to AcOEt), and recrystallized from hexane/
AcOEt to give the title compound (53 mg, 14%) as white crystals.
mp 154 °C. 1H NMR (DMSO-d6) d 1.16–1.55 (5H, m), 1.77–2.00
(2H, m), 2.66–2.93 (3H, m), 3.03–3.20 (1H, m), 3.60–3.78 (1H,
m), 3.83–3.95 (3H, m), 3.97–4.16 (3H, m), 4.16–4.35 (1H, m),
4.53 (1H, t, J = 5.5 Hz), 5.09 (2H, q, J = 9.1 Hz), 5.73 (2H, s), 7.50
(1H, d, J = 7.7 Hz), 7.56–7.68 (2H, m), 7.69–7.83 (1H, m), 8.12
(2H, d, J = 7.2 Hz). Anal. Calcd for C27H30F3N5O6: C; 56.15, H; 5.24,
N; 12.13. Found: C; 55.87, H; 5.28, N; 11.98. LC–MS: m/z = 578
(MH+).
5.50. X-ray structure analysis
Crystal data for 1: C28H31F3N4O6ꢀ0.1H2O, MW = 578.37; crystal
size, 0.41 ꢂ 0.20 ꢂ 0.14 mm; colorless, prism; monoclinic, space
group P21/c, a = 11.6375(2) Å, b = 18.6948(3) Å, c = 25.8181(5) Å,
a = c
= 90°, b = 100.8640(7)°, V = 5516.33(17) Å3, Z = 8, Dx = 1.393
g/cm3, T = 100 K,
wR2 = 0.181.
l
= 0.956 mmꢁ1
,
k = 1.54187 Å, R1 = 0.064,
Crystal data for 2b: C28H31F3N4O5ꢀ2H2O, MW = 596.60; crystal
size, 0.22 ꢂ 0.09 ꢂ 0.08 mm; colorless, block; triclinic, space
group P-1, a = 9.88809(18) Å, b = 11.9889(3) Å, c = 13.0792(3) Å,
a
= 112.612(8)°, b = 100.466(7)°,
Å3, Dx = 1.417 g/cm3,
Z = 2,
k = 1.54187 Å, R1 = 0.041, wR2 = 0.095.
c
= 91.845(7)°, V = 1398.57(11)
T = 100 K,
l
= 0.982 mmꢁ1
,
Crystal data for 2d: C28H31F3N4O5, MW = 560.57; crystal size,
0.17 ꢂ 0.13 ꢂ 0.04 mm; colorless, platelet; triclinic, space
group P-1, a = 9.29308(17) Å, b = 9.62000(17) Å, c = 16.0576(3) Å,
a
= 75.445(6)°, b = 81.749(6)°,
c
= 74.270(6)°, V = 1333.05(7) Å3,
= 0.938 mmꢁ1, k = 1.54187 Å,
Z = 2, Dx = 1.396 g/cm3, T = 100 K,
R1 = 0.043, wR2 = 0.111.
l
5.47. Gli-luciferase assay
Crystal data for 2e: C27H30F3N5O5, MW = 561.56; crystal size,
0.16 ꢂ 0.08 ꢂ 0.03 mm; colorless, platelet; triclinic, space
group P-1, a = 9.2592(3) Å, b = 9.6488(3) Å, c = 16.0297(4) Å,
NIH3T3/Gli-luc cells were maintained in DMEM containing 10%
FBS, 500-
lg/mL G418, and 0.1% gentamicin solution (Invitrogen
a
= 76.213(6)°, b = 82.549(6)°,
c
= 73.877(6)°, V = 1333.03(8) Å3,
= 0.951 mmꢁ1, k = 1.54187 Å,
Corp [Carlsbad, CA, USA]). The cells were plated onto collagen-
Z = 2, Dx = 1.399 g/cm3, T = 100 K,
R1 = 0.055, wR2 = 0.155.
l
coated 384-well plates at 7.5 ꢂ 103 cells/well and cultured

T. Ohashi et al. / Bioorg. Med. Chem. 23 (2015) 4777–4791
4791
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All measurements were made on a Rigaku R-AXIS RAPID-191R
diffractometer using graphite monochromated Cu-K
a radiation.
The structure was solved by direct methods with SHELXS-9713
and was refined using full-matrix least-squares on F2 with
SHELXL-97.13 All non-H atoms were refined with anisotropic dis-
placement parameters.
CCDC 865214 for compound 1, 1027998 for compound 2b,
1027990 for compound 2d and 1028036 for compound 2e contain
the supplementary crystallographic data for this paper. These data
can be obtained free of charge from The Cambridge
Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/
Community/Requestastructure/Pages/DataRequest.aspx?.
Acknowledgements
We thank Mr. Nagano, Mr. Iida and Mr. Nishitani for the X-ray
crystal structure analyses of 1, 2b, 2d and 2e.
7. 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. 2012, 20, 5496.
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