Synthesis of Pyrazolofuropyrazine via One-Pot S N Ar Reaction and Intramolecular Direct C-H Arylation

Article abstract of DOI:10.1055/s-0036-1591885

Fused-ring systems containing heterocycles are attractive templates for drug discovery. Biologically active 6-5-5+6 fused-ring systems that possess heterocycles are available, but these require a relatively large number of synthetic steps for preparation. Therefore, pyrazolofuropyrazine was designed as a 6-5-5+6 ring system template that incorporates ready accessibility for drug discovery. Pyrazolofuropyrazines were successfully constructed in only a few steps via one-pot S _N Ar reaction/intramolecular C-H direct arylation. As a drug candidate, pyrazolofuropyrazine has earned a favorable LogP, although significant biological activity has yet to be established; the ready accessibility of pyrazolofuropyrazine template, however, offers an opportunity for the rapid development of promising new drug candidates.

Full text of DOI:10.1055/s-0036-1591885

SYNTHESIS
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
© Georg Thieme Verlag Stuttgart · New York
2018, 50, A–F
paper
A
en
S. Fuse et al.
Paper
Synthesis
Synthesis of Pyrazolofuropyrazine via One-Pot SNAr Reaction and
Intramolecular Direct C–H Arylation
Shinichiro Fuse*
O
O
NH2
HN
N
N
Megumi Inaba
Shinichi Sato
+
HO
Manjusha Joshi
Hiroyuki Nakamura*
EtO
R
R
N
N
Cl
Laboratory for Chemistry and Life Science, Institute of Innova-
tive Research, Tokyo Institute of Technology, 4259 Nagatsuta-
cho, Midori-ku, Yokohama 226-8503, Japan
hiro@res.titech.ac.jp
Cl
N
N
N
N
Cl
N
N
N
O
R
pyrazolofuropyrazine
template for drug discovery
5 examples
up to 32% yield
(2 steps)
O
N
R
Received: 26.11.2017
Accepted after revision: 08.12.2017
Published online: 11.01.2018
The 6-5-5+6 ring system template for pyrazolofuropyra-
zines 1 is attractive for four reasons: 1) this framework has
never been reported; 2) the 1,4-pyrazine, furan, and pyra-
zole structures that constitute the framework of 1 are fre-
quently found in marketed drugs (47th, 25th, and 44th
among 351 ring systems in marketed drugs, respectively);7
3) pyrazine would suppress the hydrophobicity of com-
pounds;8 and 4) the structure of 1 offers the chance to de-
velop short and convergent synthetic approaches.
The coupling between readily available 5 and 6, and a
subsequent cyclization affords the desired framework in
only a few steps (Scheme 1). Reportedly, there are two ex-
amples for this type of reaction. Pal and co-workers have
reported a Lewis acid mediated C–C and C–O bond forma-
tion to synthesize tetracyclic compound 8.9 Liu and co-
workers have reported a Suzuki–Miyaura coupling between
9 and 5b, removal of the methyl group, and a subsequent
cyclization to synthesize tricyclic compound 10.10 The cou-
pling partners of 5 that have been reported thus far have
been limited to phenol or naphthol derivatives 7 and 9.
Herein, we wish to report a short-step synthetic ap-
proach to 1. The key reaction is a one-pot SNAr reaction/
intramolecular C–H direct arylation sequence. In addition, we
evaluated the cytotoxicity of these synthesized compounds.
The synthetic plan for 1 is shown in Scheme 2. We
planned to construct 5-hydroxypyrazole from condensation
between β-keto ester 11 and arylhydrazine 12 in accor-
dance with the Maes’ procedure.11
DOI: 10.1055/s-0036-1591885; Art ID: ss-2017-f0762-op
Abstract Fused-ring systems containing heterocycles are attractive
templates for drug discovery. Biologically active 6-5-5+6 fused-ring sys-
tems that possess heterocycles are available, but these require a rela-
tively large number of synthetic steps for preparation. Therefore, pyra-
zolofuropyrazine was designed as a 6-5-5+6 ring system template that
incorporates ready accessibility for drug discovery. Pyrazolofuropyra-
zines were successfully constructed in only a few steps via one-pot SNAr
reaction/intramolecular C–H direct arylation. As a drug candidate, pyra-
zolofuropyrazine has earned a favorable LogP, although significant bio-
logical activity has yet to be established; the ready accessibility of pyra-
zolofuropyrazine template, however, offers an opportunity for the rapid
development of promising new drug candidates.
Key words heterocycles, fused-ring systems, antitumor agents, cy-
clization, C–H activation
Fused-ring systems containing heterocycles are fre-
quently found in the framework of marketed drugs. For in-
stance, 6-5, 5-5, and 6-6-6 ring systems rank 6th, 27th, and
10th among a total of 1179 known frameworks. Also, 6-5
ring systems with a branched 6-membered ring rank 27th.1
We are interested in 6-5-5 ring systems with a branched 6-
membered ring that possesses these important frame-
works. Indenopyrazole 22 and indenone-fused pyrazole 33
have 6-5-5+6 ring systems that are known to exert potent
inhibitory activity against Chk1 and CDK, respectively (Fig-
ure 1). Our group has also reported the EGFR and VEFGR-2
inhibitor 44 (Figure 1) and the structurally similar 6-5-5+6
ring system compounds GN027075 and GN440286 (struc-
tures are not shown) as inhibitors against HIF-1 transcrip-
tional activity and tubulin polymerization. However, syn-
thetic difficulties encountered with the 6-5-5+6 ring sys-
tem compounds have slowed their speed of development as
viable drug candidates.
We originally anticipated that the SNAr reaction of alco-
hol 6 to 2,3-dichloropyrazine (5a) would afford 13, and a
subsequent intramolecular nucleophilic substitution of the
pyrazole to the chloropyrazine would afford the desired 1 in
a one-pot reaction.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

B
S. Fuse et al.
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Synthesis
5-Hydroxypyrazole 6a was prepared from acidic con-
densation between ethyl acetoacetate (11) and phenylhy-
drazine (12a) in accordance with a reported procedure
(Scheme 3).11
NaOAc, and DMA in accordance with the Maes’ procedure.14
The resultant mixture was heated under microwave irradia-
tion (160 °C, 1 h). The desired product 1a was obtained in
32% yield (one-pot, two-step yield). The structure of 1a was
unambiguously confirmed by X-ray crystallographic analy-
The key reaction for the synthesis of 1a was examined
as shown in Scheme 4. A mixture of dichloropyrazine 5a
and hydroxypyrazole 6a in DMF was heated at 100 °C for 10
minutes in the presence of K2CO3. Generation of an inter-
mediate 13a was indicated by TLC analysis. However, subse-
quent intramolecular nucleophilic substitution did not pro-
ceed even when heating time was extended either tradi-
tionally (100 °C, 5 h) or via microwave irradiation (100 to
120 °C, 1.5 h), which only produced an undesired reaction.
Therefore, our attention turned to the use of C–H direct
arylation (Scheme 5).13 After observing the generation of
13a by TLC analysis, we added a PdCl2(PPh3)2 catalyst,
one pot
or
N
N
N
N
N
N
X
X
N
N
one step
+
HO
O
R
R
5
6
1
Previously Reported Similar Reactions
Pal and co-workers
N
Cl
Cl
N
N
AlCl3
+
1,2-DCE
80 °C
HO
O
N
5a
7
8
5
5
6
5
6
6
6
rank 6
(119/5120)
rank 27
(22/5120)
rank 10
(81/5120)
Liu and co-workers
1) Pd(OAc)2, PPh3
K3PO4
MeCN, H2O
r.t. to 80 °C
N
N
Cl
Cl
N
6
5
(HO)2B
+
5
6
5
2) BBr3
3) CuTC, DMA
MeO
O
N
6
6
5b
9
10
rank 27
(22/5120)
Scheme 1 Short-step and convergent synthetic approach for con-
structing pyrazolofuropyrazines 1 and previously reported similar reac-
tions
This work
N
N
N
N
O
R
O
O
NH2
HN
N
N
pyrazolofuropyrazine 1
+
Previous reports
HO
EtO
R
R
N
NH
MeO
11
12
6
N
N
Cl
MeO
CN
Chk1 inhibitor 2
N
Cl
5a
N
NH
Cl
N
N
N
N
N
N
N
H
N
N
O
NH
OMe
N
O
O
R
R
O
O
CDK inhibitor 3
1
13
N
NH
Scheme 2 Synthetic plan for the synthesis of pyrazolofuropyrazines 1
O
O
H
N
O
NH
O
O
O
NH2
HN
N
N
AcOH
H2N
EGFR and VEGFR-2 inhibitor 4
+
100 °C
20 min
quant.
HO
Figure 1 Fused-ring frameworks and their ranking among 1179 frame-
works found in 5120 marketed drugs as well as structures of biologically
active compounds consisting of 6-5-5+6 fused ring system reported in
1996
EtO
11
12a
6a
Scheme 3 Preparation of 5-hydroxypyrazole 6a
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

C
S. Fuse et al.
Paper
Synthesis
sis (Figure 2),15 which indicated that all four of the aromatic
rings in 1a resided on a common plane.
Pyrazolofuropyrazines 1c–f were synthesized via our
originally developed procedure as shown in Scheme 6.
N-Arylhydroxypyrazoles 6c–f were prepared first. In the
syntheses of 6b–e, NaOAc was added in order to decrease
the acidity of the reaction mixture, because hydrazines
12b–e were commercially available in the form of HCl salts.
The product 6b was not obtained probably due to the steric
repulsion that originated from the ortho-methyl group of
12b. A subsequent key one-pot reaction was performed to
synthesize 1c–f. All the desired products 1c–f that have
electron-donating or -withdrawing groups in branched aryl
rings were obtained in moderate yields.
The effect of the synthesized compounds 1a, 1c, 1d, 1e,
and 1f on proliferative activity toward HeLa, A549, MDA-
MB-468, and SK-BR-3 cells was evaluated by MTT assay, and
no significant effects were observed (IC50 >20 μM). Other
biological evaluations of 1 are ongoing.
In summary, we have designed pyrazolofuropyrazines 1
as a novel 6-5-5+6 ring system template for drug discovery.
The designed compounds 1 were successfully constructed
in a few steps via our originally developed SNAr C–H direct
arylation sequence. Compounds 1 have a favorable LogP as
drug candidates. Although significant biological activity of
1 is yet to be revealed, the ready accessibility of template 1
offers opportunities for the development of promising drug
candidates.
The octanol/water partition coefficient of 1a was mea-
sured by an HPLC-based method. The LogP of 1a was 2.6
(cLogP16: 2.28). Compounds with LogPs between 1 and 3
are regarded as having a good balance of aqueous solubility
and cell permeability.17 Therefore, this newly designed tem-
plate 1 has favorable hydrophobicity as a drug candidate.
N
N
Cl
O
N
N
Cl
+
N
N
K2CO3
N
N
HO
DMF
100 °C, 10 min
Cl
5a
6a
13a
100 °C, 5 h
or
100 to 120 °C, MW, 1.5 h
N
N
N
N
O
1a
Scheme 4 The first attempt for a one-pot synthesis of 1a
N
N
Cl
O
N
N
Cl
+
N
N
NMR spectra were recorded on a Bruker Biospin Avance II 400 spec-
trometer (400 MHz for 1H, 100 MHz for 13C) or a Bruker Biospin
Avance III HD500 spectrometer (500 MHz for 1H, 125 MHz for 13C) in
the indicated solvent. Chemical shifts are reported in units of parts
per million (ppm) relative to the signal (0.00 ppm) for internal TMS
for solutions in CDCl3 (7.26 ppm for 1H, 77.2 ppm for 13C). Multiplici-
ties are given with standard abbreviations; coupling constants J in
hertz (Hz). Reactions were monitored by TLC on TLC plates (60-F254,
Merck) using UV light (254 or 365 nm) and ninhydrin AcOH solution
or p-anisaldehyde EtOH solution, and/or phosphomolybdic acid EtOH
solution. Flash column chromatography separations were performed
using silica gel (Fuji Silysia, Chromatorex PSQ 60B, 50-200 μm). Mi-
crowave-assisted synthesis were performed on microwave synthesiz-
er (Biotage, Initiator).
K2CO3
N
N
HO
DMA
100 °C, 10 min
Cl
5a
6a
13a
PdCl2(PPh3)2
NaOAc, DMA
160 °C, MW, 1 h
2 steps 32%
N
N
N
N
O
1a
Scheme 5 Construction of pyrazolofuropyrazine framework via one-
N-Arylpyrazolols 6; General Procedure
pot SNAr reaction and intramolecular C–H direct arylation
Method A: A solution of ethyl acetoacetate (11; 1.00 equiv) and aryl-
hydrazine 12 (1.00 equiv) in AcOH (1.10–1.50 mL/mmol) was stirred
under an argon atmosphere for 15 min to 5 h at r.t. to 100 °C. Then,
the reaction mixture was concentrated in vacuo. The residue was pu-
rified by column chromatography on silica gel to afford the desired N-
arylpyrazolol 6.
Method B: To a solution of arylhydrazine hydrochloride 12 (1.00
equiv) in EtOH (0.300–0.900 mL/mmol based on arylhydrazine hy-
drochloride 12) was added ethyl acetoacetate (11; 1.00 equiv) and
AcOH (0.300 mL/mmol based on hydrazine hydrochloride 12). The re-
sultant solution was stirred under an argon atmosphere for 3–17 h at
r.t. to 100 °C. Then, the reaction mixture was concentrated in vacuo.
The residue was purified by column chromatography on silica gel to
afford the desired N-arylpyrazolol 6.
Figure 2 ORTEP drawing of 1a
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

D
S. Fuse et al.
Paper
Synthesis
O
O
EtO
11
NH2
HN
NH2
NH2
HN
NH2
HN
NH2
HN
•HCl
•HCl
•HCl
•HCl
HN
OMe
F
CF3
12b
EtOH, NaOAc
then AcOH
r.t. to 100 °C, 18 h
(not obtained)
12d
12e
12c
EtOH, NaOAc
then AcOH
12f
AcOH
EtOH, NaOAc
then AcOH
r.t., 3 h
EtOH, NaOAc
then AcOH
r.t., 5 h
100 °C, 5 h
32%
80 °C, 16 h, 30%
58%
86%
N
N
N
N
N
N
N
N
N
N
HO
HO
HO
HO
HO
OMe
F
CF3
6b
6c
6d
6e
6f
i) 5a, K2CO3, DMA
100 °C, 10 min
ii) PdCl2(PPh3)2
NaOAc, DMA
160 °C, 1 h
i) 5a, K2CO3, DMA
100 °C, 10 min
ii) PdCl2(PPh3)2
NaOAc, DMA
160 °C, 1 h
i) 5a, K2CO3, DMA
100 °C, 10 min
ii) PdCl2(PPh3)2
NaOAc, DMA
160 °C, 1 h
i) 5a, K2CO3, DMA
100 °C, 10 min
ii) PdCl2(PPh3)2
NaOAc, DMA
160 °C, 1 h
2 steps
2 steps
2 steps
2 steps
one pot 21%
one pot 21%
one pot 10%
one pot 28%
N
N
N
N
N
N
O
O
N
N
F
N
N
N
1c
1e
N
N
N
O
O
N
N
OMe
CF3
1d
1f
Scheme 6 Synthesis of pyrazolofuropyrazines 1
3-Methyl-1-phenyl-1H-pyrazol-5-ol (6a)
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C11H12N2ONa: 211.0847;
found: 211.0839.
Prepared by following Method A; column chromatography: hexane/
EtOAc 5:2 to 100% EtOAc; yield: 403 mg (2.31 mmol, quant); orange
solid.
1-(4-Methoxyphenyl)-3-methyl-1H-pyrazol-5-ol (6d)
1H NMR (CDCl3, 400 MHz): δ = 7.86 (d, J = 7.6 Hz, 2 H), 7.39 (t, J = 7.2
Hz, 2 H), 7.18 (t, J = 7.6 Hz, 1 H), 3.43 (s, 2 H), 2.20 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 170.6, 156.3, 138.0, 128.8, 125.0,
Prepared by following Method B; column chromatography: hexane/
EtOAc 5:2 to 100% EtOAc; yield: 117 mg (0.571 mmol, 58%); orange
solid; mp 120–122 °C.
FT-IR (KBr): 2933, 2368, 1511, 1251, 1034, 832, 776 cm–1
.
118.9, 43.1, 17.0.
1H NMR (CDCl3, 400 MHz): δ = 7.72 (d, J = 9.2 Hz, 2 H), 6.91 (d, J = 9.2
1
The observed H NMR18 and 13C NMR19 spectra were consistent with
Hz, 2 H), 3.80 (s, 3 H), 3.40 (s, 2 H), 2.17 (s, 3 H).
previously reported data.
13C NMR (CDCl3, 125 MHz): δ = 170.3, 157.0, 156.2, 131.3, 120.9,
3-Methyl-1-(m-tolyl)-1H-pyrazol-5-ol (6c)
114.0, 55.5, 42.9, 17.0.
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C11H12N2O2Na: 227.0796;
found: 227.0791.
Prepared by following Method B; column chromatography: hex-
ane/EtOAc 5:2; yield: 110 mg (0.584 mmol, 30%); light brown solid;
mp 103–105 °C.
FT-IR (KBr): 2920, 2363, 1610, 1491, 1400, 1361, 779, 690 cm–1
.
1-(4-Fluorophenyl)-3-methyl-1H-pyrazol-5-ol (6e)
1H NMR (CDCl3, 500 MHz): δ = 7.66–7.64 (m, 2 H), 7.27 (t, J = 8.0 Hz, 1
H), 7.00 (d, J = 7.5 Hz, 1 H), 3.41 (s, 2 H), 2.38 (s, 3 H), 2.18 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 170.7, 156.4, 138.9, 138.0, 128.8,
Prepared by following Method B; column chromatography: hexane/
EtOAc 5:2 to 100% EtOAc; yield: 159 mg (0.824 mmol, 86%); orange
solid; mp 144–146 °C.
FT-IR (KBr): 2690, 2361, 1508, 1223, 833, 780 cm–1
.
126.1, 119.7, 116.3, 43.2, 21.7, 17.1.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

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Synthesis
1H NMR (CDCl3, 400 MHz): δ = 7.72 (d, J = 9.2 Hz, 2 H), 6.91 (d, J = 9.2
Hz, 2 H), 3.80 (s, 3 H), 3.40 (s, 2 H), 2.17 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 170.3, 157.0, 156.2, 131.3, 120.9,
114.0, 55.5, 42.9, 17.0.
1H NMR (CDCl3, 500 MHz): δ = 8.49 (d, J = 2.8 Hz, 1 H), 8.15 (d, J = 3.2
Hz, 1 H), 7.82–7.77 (m, 2 H), 7.41 (t, J = 8.0 Hz, 1 H), 7.14 (d, J = 7.6 Hz,
1 H), 2.70 (s, 3 H), 2.47 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 159.6, 156.2, 143.1, 140.8, 139.9,
137.1, 137.1, 135.2, 129.5, 127.4, 118.8, 115.4, 105.7, 21.6, 14.3.
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C15H12N4ONa: 287.0909;
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C10H9FN2ONa: 215.0597;
found: 215.0591.
found: 287.0903.
3-Methyl-1-[4-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol (6f)
1-(4-Methoxyphenyl)-3-methyl-1H-pyrazolo[4′,3′:4,5]furo[2,3-
b]pyrazine (1d)
Prepared by following Method A; column chromatography: hexane/
EtOAc 1:1; yield: 39 mg (0.16 mmol, 32%); orange solid.
Purification procedure B; column chromatography: hexane/EtOAc
5:3; preparative TLC: hexane/EtOAc 5:3; yield: 7.9 mg (0.028 mmol,
21% over 2 steps); yellow solid; mp 154–155 °C.
1H NMR (CDCl3, 400 MHz): δ = 8.05 (d, J = 8.4 Hz, 2 H), 7.64 (d, J = 8.8
Hz, 2 H), 3.47 (s, 2 H), 2.22 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 170.8, 156.9, 140.8, 126.5 (q, JC,F = 32.6
Hz), 126.1 (q, JC,F = 3.53 Hz), 124.1 (q, JC,F = 270 Hz), 118.1, 43.1, 17.1.
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C11H9F3N2ONa: 265.0565;
FT-IR (KBr): 2360, 1607, 1567, 1514, 1250, 1163, 1028, 823 cm–1
.
1H NMR (CDCl3, 400 MHz): δ = 8.48 (d, J = 2.8 Hz, 1 H), 8.13 (d, J = 3.2
Hz, 1 H), 7.87–7.85 (m, 2 H), 7.06–7.03 (m, 2 H), 3.87 (s, 3 H), 2.69 (s, 3
H).
13C NMR (CDCl3, 125 MHz): δ = 159.6, 158.2, 155.8, 142.8, 140.7,
137.3, 135.1, 130.6, 120.0, 114.8, 105.4, 55.6, 14.3.
found: 265.0559.
The observed 1H NMR spectrum was consistent with previously re-
ported data.20
(ESI-TOF): m/z [M + Na]+ calcd for C15H12N4O2Na: 303.0858; found:
303.0852.
Pyrazolofuropyrazines 1; General Procedure
A solution of N-arylpyrazolol 6 (1.00 equiv), 2,3-dichloropyrazine (5a;
1.00 equiv), and K2CO3 (1.20 equiv) in DMA (1.67 mL/mmol based on
N-arylpyrazolol 6) was stirred at 100 °C under argon atmosphere for
5–50 min. To the resultant solution was added Pd(PPh3)2Cl2 (10.0
mol%) and K2CO3 (1.50 equiv) at r.t. The mixture was stirred at 160 °C
under argon atmosphere for 20–90 min. The reaction mixture was
partitioned between H2O and EtOAc. The aqueous layer was separat-
ed, and extracted with EtOAc. The combined organic layers were
dried (anhyd Na2SO4), and concentrated in vacuo.
1-(4-Fluorophenyl)-3-methyl-1H-pyrazolo[4′,3′:4,5]furo[2,3-
b]pyrazine (1e)
Purification procedure C; column chromatography: hexane/EtOAc
5:2; preparative TLC: hexane/EtOAc 5:2; yield: 14 mg (0.051 mmol,
10% over 2 steps); colorless solid; mp 159–160 °C.
FT-IR (KBr): 2361, 1567, 1509, 1228, 1164, 1055, 828 cm–1
.
1H NMR (CDCl3, 500 MHz): δ = 8.49 (d, J = 2.4 Hz, 1 H), 8.15 (d, J = 2.4
Hz, 1 H), 7.95–7.92 (m, 2 H), 7.24–7.21 (m, 2 H), 2.68 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 160.9 (d, JC,F = 245 Hz), 159.5, 156.0,
143.3, 140.9, 137.1, 135.3, 133.4 (d, JC,F = 3.00 Hz), 120.0 (d, JC,F = 8.13
Hz), 116.5 (d, JC,F = 22.8 Hz), 105.8, 14.3.
Purification Procedure A: The residue was purified by column chroma-
tography on silica gel to afford pyrazolofuropyrazine 1.
Purification Procedure B: The residue was purified by column chroma-
tography on silica gel and preparative TLC to afford pyrazolofuropyra-
zine 1.
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C14H9FN4ONa: 291.0658;
found: 291.0653.
Purification Procedure C: The residue was purified by column chroma-
tography on silica gel, preparative TLC, and GPC to afford pyrazolofu-
ropyrazine 1.
3-Methyl-1-[4-(trifluoromethyl)phenyl]-1H-pyrazolo[4′,3′:4,5]fu-
ro[2,3-b]pyrazine (1f)
3-Methyl-1-phenyl-1H-pyrazolo[4′,3′:4,5]furo[2,3-b]pyrazine (1a)
Purification procedure B; column chromatography: hexane/EtOAc
5:2; preparative TLC: hexane/EtOAc 5:2; yield: 3.7 mg (0.012 mmol,
28% over 2 steps); yellow solid; mp 146–148 °C.
Purification procedure A; column chromatography: hexane/EtOAc
5:2; yield: 24 mg (0.095 mmol, 32% over 2 steps); colorless solid; mp
146–148 °C.
FT-IR (KBr): 2362, 1607, 1567, 1324, 1166, 1059, 752 cm–1
1H NMR (CDCl3, 400 MHz): δ = 8.50 (d, J = 2.8 Hz, 1 H), 8.15 (d, J = 2.8
Hz, 1 H), 7.98 (d, J = 8.0 Hz, 2 H), 7.54 (t, J = 7.6 Hz, 2 H), 7.33 (t, J = 7.6
Hz, 1 H), 2.71 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 159.6, 156.2, 143.2, 140.8, 137.2,
137.1, 135.3, 129.7, 126.6, 118.3, 105.8, 14.3.
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C14H10N4ONa: 273.0752;
found: 273.0749.
FT-IR (KBr): 2360, 1606, 1568, 1326, 1162, 1111, 1069, 839 cm–1
.
.
1H NMR (CDCl3, 400 MHz): δ = 8.53 (d, J = 2.8 Hz, 1 H), 8.19 (d, J = 2.8
Hz, 1 H), 8.13 (d, J = 8.6 Hz, 2 H), 7.80 (d, J = 8.4 Hz, 2 H), 2.71 (s, 3 H).
13C NMR (CDCl3, 125 MHz): δ = 159.5, 156.4, 144.2, 141.2, 139.7,
136.8, 135.7, 128.3 (q, JC,F = 32.9 Hz), 127.0 (q, JC,F = 3.68 Hz), 123.8 (q,
JC,F = 270 Hz), 118.0, 106.5, 14.3.
HRMS (ESI-TOF): m/z [M + Na]+ calcd for C15H9F3N4ONa: 341.0626;
found: 341.0621.
Cell Viability Assay (MTT Assay)
3-Methyl-1-(m-tolyl)-1H-pyrazolo[4′,3′:4,5]furo[2,3-b]pyrazine
Human cervical carcinoma HeLa cells, human lung carcinoma A549
cells, human breast carcinoma MDA-MB-468 cells, and human breast
carcinoma SK-BR3-3 cells were used for the cell viability assay. These
cells (5 × 103 cells per well of a 96-well plate) were incubated under
5% CO2 at 37 °C in RPMI-1640 media (Wako Pure Chemical) contain-
ing 10% fetal bovine serum (FBS, Thermo), 100 U/mL penicillin, 100
(1c)
Purification Procedure C; column chromatography: hexane/EtOAc
5:2; preparative TLC: hexane/EtOAc 5:2; yield: 10 mg (0.038 mmol,
21% over 2 steps); yellow solid; mp 144–146 °C.
FT-IR (KBr): 2361, 1611, 1563, 1327, 1163, 1017, 776 cm–1
.
© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F

F
S. Fuse et al.
Paper
Synthesis
μg/mL streptomycin (Thermo), and various concentrations of 1 (10
mM DMSO solution). After incubation, the medium was removed, a
solution of 3′-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) in PBS (5 mg/mL, 10 μL) were added to each well, and
the cells were further incubated at 37 °C for 2 h. After removal of the
medium, DMSO (100 μL) was added, and the plate was shaken to dis-
solve the formed formazan. The absorbance of formazan at 595 nm
was measured with a microplate reader. The drug concentration re-
quired to reduce cell viability by 50% (IC50) was determined from
semilogarithmic dose-response plots.
(6) Minegishi, H.; Futamura, Y.; Fukashiro, S.; Muroi, M.; Kawatani,
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Supporting Information
Supporting information for this article is available online at
(12) We speculated that another 6a reacted with 13a. This intermo-
lecular reaction seemed faster than the desired intramolecular
reaction.
https://doi.org/10.1055/s-0036-1591885.
S
u
p
p
ortiInfogrmoaitn
S
u
p
p
ortioInfgrmoaitn
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© Georg Thieme Verlag Stuttgart · New York — Synthesis 2018, 50, A–F