Design, synthesis, and in vitro/vivo anticancer activity of 4-substituted 7-(3-fluoro-4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidines

Article abstract of DOI:10.1002/jccs.202100100

In this paper, we report the design and synthesis of 4-substituted 7-(3-fluoro-4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidines of scaffold 6 as anticancer agents. A total of 19 derivatives of scaffold 6 bearing a C-4 alkoxy, dialkylamino, alkyl, vinyl, or phenyl substituent were synthesized and evaluated. Among them, compound 6q having a C-4 ethyl group and a benzylic methyl group showed the most potent in vitro anticancer activity, inhibiting the proliferation of Hela, MDA-MB-231, and MDA-MB-426 cancer cells at submicromolar concentrations (GI₅₀: 0.11–0.58 μM). Compound 6q arrested the cell cycle of MDA-MB-231 at G₂/M phase, and showed in vivo activity on nude mice bearing MDA-MB-231 xenografts. Compound 6q has served as an anticancer lead for further optimization.

Full text of DOI:10.1002/jccs.202100100

Received: 10 March 2021
Revised: 18 April 2021
Accepted: 19 April 2021
DOI: 10.1002/jccs.202100100
A R T I C L E
Design, synthesis, and in vitro/vivo anticancer activity of
-substituted 7-(3-fluoro-4-methoxybenzyl)-7H-pyrrolo[2,3-d]
4
pyrimidines
1
2
2
1
Pei-Yi Tsai | Gong-Siang Hu | Po-Hsun Huang | Huei-Lin Jheng
|
2,3
2,3
1
1
Chi-Hsuan Lan
| You-Sin Chen
| Jia-Ming Chang | Shih-Hsien Chuang
|
2,3
Jiann-Jyh Huang
1
Development Center for Biotechnology,
National Biotechnology Research Park,
Taipei City, Taiwan
Abstract
In this paper, we report the design and synthesis of 4-substituted 7-(3-fluoro-
4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidines of scaffold 6 as anticancer
agents. A total of 19 derivatives of scaffold 6 bearing a C-4 alkoxy, dia-
lkylamino, alkyl, vinyl, or phenyl substituent were synthesized and evaluated.
Among them, compound 6q having a C-4 ethyl group and a benzylic methyl
group showed the most potent in vitro anticancer activity, inhibiting the prolif-
eration of Hela, MDA-MB-231, and MDA-MB-426 cancer cells at sub-
micromolar concentrations (GI : 0.11–0.58 μM). Compound 6q arrested the
2
Department of Applied Chemistry,
National Chiayi University, Chiayi City,
Taiwan
3
The Training and Research Institute of
Food and Agriculture, National Chiayi
University, Chiayi City, Taiwan
Correspondence
Jiann-Jyh Huang, Department of Applied
Chemistry, National Chiayi University,
No. 300, Syuefu Road, Chiayi City 60004,
Taiwan.
50
cell cycle of MDA-MB-231 at G /M phase, and showed in vivo activity on nude
2
mice bearing MDA-MB-231 xenografts. Compound 6q has served as an anti-
Email: lukehuang@mail.ncyu.edu.tw
cancer lead for further optimization.
Funding information
Ministry of Science and Technology of
R.O.C., Grant/Award Number:
K E Y W O R D S
anticancer, colchicine, combretastatin, ottelione A, pyrrolopyrimidine, tubulin
105-2113-M-415-001
1
| INTRODUCTION
Due to the promising anticancer activity, various
synthetic methods have been reported for the preparation
[
6–15]
[16]
Ottelione A (1, Figure 1), isolated from Ottelia alismoides
of 1
and are comprehensively reviewed.
Our
[1]
collected in the Nile Delta,
featuring a carbonyl-conjugated diene and a 3-hydroxyl-
-methoxybenzyl group. Compound 1 shows extremely
is a cis-hydrindanone
group has demonstrated a radical cyclization methodol-
[
13]
ogy for the enantioselective total synthesis for 1.
following study,
In the
[
17]
4
we established the structure–
potent anticancer activity, which can inhibit the prolif-
eration of NCI-60 cancer cells at picomolar to
activity relationship of 1 using the synthetic methodol-
ogy. Compound 2, the desvinyl and fluoro analog of 1,
was generated and found 6–38-fold more potent than
1. Inhibition of colchicine binding and tubulin
polymerization was observed for 2 in biochemical
[
1,2]
nanomolar concentrations.
binding site to inhibit tubulin polymerization,
mechanism of action of 1 is similar to that of colchicine
By binding to colchicine-
[
1,2]
the
[
3]
[4]
(
3) and combretastatin A-4 (4). Combretastatin A-4
assays, which suggested that 2 also bound to
phosphate (5), a prodrug of 4 classified as a vascular dis-
rupting agent, is currently investigated in phase-III
clinical trials for the treatment of cancers.
colchicine-binding site similar to 1 and 3–5. In addi-
[
5]
tion, our study demonstrated that the exocyclic double
[
17]
bond was essential for the potency of 1 and 2.
J Chin Chem Soc. 2021;1–10.
http://www.jccs.wiley-vch.de
© 2021 The Chemical Society Located in Taipei & Wiley-VCH GmbH
1

2
TSAI ET AL.
R1
MeO
MeO
H
H
NHCOMe
O
R2
MeO
OMe
O
OMe
1
2
1
2
. R = CH=CH , R = OH
2
3
1
2
1
. R = H, R = F (racemic)
R
4
N
MeO
MeO
F
'
3
N
N
4
'
OMe
R2
MeO
OR
OMe
1
6
. R = alkoxy, amino, alkyl
4
5
. R = H
. R = P(=O)(ONa)2
vinyl, phenyl
2
R = H, Me
FIGURE 1 Structures of ottelione A (1), ottelione A synthetic
derivative 2, colchicine (3), combretastatin A-4 (4), combretastatin
A-4 phosphate (5), and 4-substituted 7-(3-fluoro-4-methoxybenzyl)-
SCHEME 1 Synthesis of 6a–p bearing a C-4 alkoxy,
dialkylamino, alkyl, vinyl, or phenyl substituent. Reagents and
conditions: (i) NaBH , MeOH, 93%. (ii) SOCl , Et O, 95%.
iii) 4-chloro-7H-pyrrolo[2,3-d]pyrimidine, NaH, DMF, 93%.
4
2
2
7H-pyrrolo[2,3-d]pyrimidines 6 reported in this study
(
(
ꢀ
iv) For 6a–c: KOH, alcohol, 70 C; for 6d: KOH, cyclohexanol,
ꢀ
ꢀ
dioxane, 70 C; for 6e: NaH, BnOH, THF, 0 C; for 6f–h:
ꢀ
dialkylamine, EtOH, 70 C; for 6i–l and 6n–p: RMgBr, PdCl
2
(dppf),
An irreversible nucleophilic 1,6-addition would be
ꢀ
THF; for 6m: Bu
3
SnCH=CH
2
, Pd(PPh
3
)
2
Cl
2
, LiCl, DMF, 100 C
[
18,19]
responsible for their potency.
Although both
1
and 2 show potent in vitro activity, they might not be
active in vivo due to their inherent instability in serum.
However, the structural information of 2 suggested that
2 | RESULTS AND DISCUSSION
2.1 | Synthesis of 4-substituted
7
-(3-fluoro-4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidine
of scaffold 6 (a pyrrolopyrimidine, Figure 1) having a C-4
alkoxy, amino, alkyl, vinyl, and phenyl group could
mimic the structure of 2 to inhibit tubulin polymeriza-
7-(3-fluoro-4-methoxybenzyl)-7H-pyrrolo
[2,3-d]pyrimidines 6a–s
[
20,21]
[22]
tion. Several pyrrolopyrimidines
and indoles
Scheme 1 presents the synthesis of 6a–p bearing a C-4
alkoxy (O-series 6a–e), dialkylamino (N-series 6f–h), or
structurally similar to 2 have been reported to have anti-
cancer activity. For scaffold 6, a methyl group at the
benzylic position could create steric hindrance and
direct the benzyl group to the different positions.
Enhancement of anticancer activity might be observed.
Herein, we report the synthesis and biological
study for the pyrrolopyrimidines of scaffold 6. We first
screened various C-4 substituents, which could pro-
vide the anticancer activity of 6, and then explored the
effect of benzylic methyl on the potency of 6. Our
study eventually led to 6q having a C-4 ethyl group
and a benzylic methyl group that showed sub-
micromolar in vitro anticancer activity. The effect of
alkyl/vinyl/phenyl
(C-series
6i–p)
substituent.
3-Fluoro-4-methoxybenzaldehyde (7) served as the
starting material and was reduced by NaBH4.
The reaction gave benzyl alcohol 8 in 93% yield. The
hydroxyl group in 8 was transformed into a chloro group
using SOCl , providing benzyl chloride 9 in 95% yield. Alkyl-
2
ation of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine by 9 afforded
4-chloropyrrolopyrimidine 10 in 93% yield. To prepare 6a–e
(O-series), 10 was reacted with the corresponding alcohol to
replace its C-4 chloro group. The reaction of 10 with KOH in
ꢀ
MeOH, EtOH, or i-PrOH at 70 C gave 6a–c in 90–97%
yields. Heating 10 with cyclohexanol and KOH in dioxane
provided 6d in 60% yield. Compound 6e having a
4-benzyloxy substituent was obtained in 99% yield from the
reaction of 10 with BnOH and NaH in THF.
6
q on the cell cycle of cancer cells was studied to pre-
dict its possible mechanism of action. Finally, the
in vivo activity of 6q was investigated to evaluate
the potential of 6q as an anticancer lead for further
optimization.
To prepare 6f–h having a C-4 dialklylamino group (N-
series), 10 was heated with dimethylamine, piperidine, or

TSAI ET AL.
3
morpholine in EtOH. The reaction gave the corresponding
MDA-MB-468 (82–99%). Compound 6e with a C-4 OBn
substituent was the most potent analog among 6a–e to
inhibit Hela (viability: 63%). However, 6e was inactive for
MDA-MB-231 (105% viability) and MDA-MB-468 (92%
viability). Compounds 6a–e having a C-4 alkoxy group
were not further derived as they did not reduce the viabil-
ity of the three cells to below 50%.
6
f–h in 79–84% yields. The reaction of 10 with Grignard
[
23]
reagents in the presence of catalytic PdCl (dppf)
2
(Kumada coupling) gave the corresponding 6i–l and 6n–p
bearing an alkyl or a phenyl group in 26–88% yields.
For C-4 vinyl substituted 6m, 10 was coupled with
[24]
tributyl(vinyl)tin by Stille coupling
using Pd(PPh ) Cl /
3 2 2
LiCl/DMF catalytic system. The reaction gave 6m in 25%
yield.
We then evaluated the potency of C-4 dialkylamino
pyrrolopyrimidines 6f (dimethylamino), 6g (piperidinyl),
and 6h (morpholino) (Scheme 1 and Figure 2). Unfortu-
nately, they showed marginal effect on the cell viability
(90–100%) of the three cancer cells. Considering the inac-
tivity of C-4 alkoxy and dialkylamino derivatives 6a–h,
we synthesized and evaluated 6i–p that have a C-4 alkyl,
vinyl, or phenyl substituent. Compound 6i having a C-4
methyl substituent did not reduce the viability of the
three cancer cells to below 50%, though it was more
active for Hela (viability: 65%). On the other hand, 6j
with a C-4 ethyl group showed greatly improved potency,
reducing the viability of Hela, MDA-MB-231, and MDA-
MB-468 to 22, 38, and 13%, respectively. Increasing the
size of the C-4 substituent of 6j to isopropyl (6k),
cyclopropyl (6l), vinyl (6m), cyclohexyl (6o), and phenyl
Scheme 2 illustrates the synthesis of C-4 alkyl
substituted pyrrolopyrimidines 6q–s featuring a benzylic
methyl substituent. Aldehyde 7 (Scheme 1) also served
as the starting material, which was reacted with
methylmagnesium bromide to give secondary alcohol 11
in 96% yield. The hydroxyl group in 11 was converted to
a chloro group using SOCl , and the generated benzyl
2
chloride alkylated 4-chloro-7H-pyrrolo[2,3-d]pyrimidine
by NaH in DMF. The two consecutive reactions gave
4
-chloropyrrolopyrimidine 12 in 23% total yield. Coupling
of 12 with Grignard reagents gave the corresponding
racemic 6q–s having a C-4 ethyl, propyl, or butyl group
in 60–93% yields.
(6p) deactivated 6k–m, 6o, and 6p (viability: 67–99%),
2
.2 | Potency of 6a–p on the cell viability
except that 6n having a cyclopentyl was active for MDA-
MB-468 (viability: 23%). These results indicated that the
C-4 position of 6 only tolerates a narrow range of substit-
uents. Only ethyl group provided 6j with notable antican-
cer activity for Hela, MDA-MB-231, and MDA-MB-468
(GI : <1.0 μM).
of Hela, MDA-MB-231, and MDA-MB-468
We first studied the potency of 6a–p on the cell viability
on Hela (cervical), MDA-MB-231 (breast), and MDA-MB-
4
68 (breast) cancer cells at the concentration of 1.0 μM
50
after a 96-hr incubation. The results are summarized in
Figure 2. For 4-alkoxy derivatives 6a–d having a OMe,
OEt, O(i-Pr), and OC H substituent, the viability of the
As a result, the in vitro GI50 values of 6j were mea-
sured (Table 1). Compound 6j inhibited Hela, MDA-MB-
231, and MDA-MB-468 with GI50 of 0.81, 0.40, and
0.25 μM, respectively. As the benzyl group in 1 and 2 (Fig-
ure 1) should be more distant from the plane of fused
cyclohexenone–cyclopentane moiety, we synthesized and
evaluated 6q, the benzylic methylated analog of 6j. Com-
pound 6q showed around twofold increased potency
6
11
three cells was not reduced significantly (71–100%).
Although not potent, 6a–d were more active for Hela
(viability 71–97%) than MDA-MB-231 (94–100%) and
(
GI : 0.11–0.58 μM) compared to 6j (GI : 0.25–0.81 μM).
50
50
Nevertheless, increasing the size of the C-4 alkyl in 6q
did not improve the potency: 6r with a propyl group and
6
3
s with a butyl group have GI50 of 1.2–2.9 and 0.52–
.0 μM, respectively. Unfortunately, the more potent ana-
logs 6q–s from this study were less active than 3 (GI :
5
0
0.082–0.21 μM, Table 1).
2
2
.3 | Cell cycle study of 6q on MDA-MB-
31 using flow cytometry
SCHEME 2 Synthesis of 6q–s bearing a C-4 alkyl group and a
benzylic methyl group. Reagents and conditions: (i) MeMgBr, THF,
As 6q had the most potent in vitro anticancer activity for
the three cancer cells (GI : 0.11–0.58 μM, Table 1), its
9
6%. (ii) (a) SOCl
2
, Et
2
O; (b) 4-chloro-7H-pyrrolo[2,3-d]pyrimidine,
(dppf), THF
5
0
NaH, DMF, 23% (two steps). (iii) RMgBr, PdCl
2
effect on the cell cycle of MDA-MB-231 was explored

4
TSAI ET AL.
FIGURE 2 Cell viability of Hela (black), MDA-MB-231 (white), and MDA-MB-468 (gray) cancer cells treated with 6a–p at the
concentration of 1.0 μM for 96 hr
TABLE 1 In vitro anticancer activity of 6j, 6q–s, and 3
In vitro anticancer GI50 (μM)
1
2
Hela
0.81
0.58
1.2
MDA-MB-231
MDA-MB-468
Compd
R
R
6
6
6
6
3
j
Et
Et
Pr
Bu
—
H
0.40
0.20
2.9
0.25
0.11
1.7
q
r
s
Me
Me
Me
—
3.0
0.52
0.10
0.69
0.082
0.21
FIGURE 3 Cell cycle profile of
MDA-MB-231 cells treated with 0.3 μM
of 6q for 24 hr
using flow cytometry (Figure 3). Compared to control, 6q
to 39.3%. Similar populations of cells at the S phase were
observed in control (33.0%) and treatment (31.8%) groups.
arrested the cell cycle at the G /M phase at the concen-
2
[25,26]
tration of 0.3 μM after a 24-hr treatment. The population
The effect of 6q on cell cycle was similar to that of 3
[
27,28]
of cells at the G /M phase increased from 1.91 to 29.8%,
and 4.
The results suggested that 6q worked on the
2
while the population at the G phase decreased from 58.8
G /M phase to show its anticancer activity, possible due
2
1

TSAI ET AL.
5
4
4
| EXPERIMENTAL
.1 | General
Starting materials and reagents were used as purchased
without further purification. Column chromatography
was performed on Merck Reagents Silica Gel 60 (particle
size of 0.063–0.200 mm, 70–230 mesh ASTM) using
EtOAc and hexanes as the eluent. Proton (400 MHz) and
carbon-13 (100 MHz) NMR spectra were obtained on an
Agilent 400-MR NMR spectrometer using CDCl as the
3
solvent. EI-MS were recorded on an Agilent 5973 mass
spectrometer detector coupled with an Agilent 6890
Series GC System. High-resolution mass spectra were
obtained on an LTQ Orbitrap XL mass spectrometer
FIGURE 4 In vivo anticancer activity of 6q. The tumor growth
inhibition (1 À T/C) for 6q at day 14 was 27.6%
(
Thermo Fisher Scientific). Compounds 6q–s were found
not optically active by means of Jasco P-1010 polarimeter.
to inhibiting tubulin polymerization by binding to the
colchicine binding site.
4.2 | (3-Fluoro-4-methoxyphenyl)
methanol (8)
2.4 | In vivo anticancer activity of 6q
A
solution of 3-fluoro-4-methoxybenzaldehyde (7,
1.183 g, 7.675 mmol, 1.0 equiv) in MeOH (15 ml) was
To assess the potential of 6q as an anticancer lead for
further optimization, a pilot in vivo study for 6q was
conducted on nude mice bearing human MDA-MB-
slowly added with NaBH4 (0.4350 g, 11.50 mmol, 1.5
equiv) in an ice bath. The reaction mixture was stirred at
room temperature for 60 min. The solution was concen-
trated under reduced pressure and the residue was re-
dissolved in EtOAc. The organic layer was washed
2
31 xenografts. The result is shown in Figure 4. Com-
pound 6q showed moderate tumor growth inhibition
when administered PO once per day at a dose of
with brine, dried over anhydrous MgSO , filtered, and
4
2
0 mg/kg for 14 consecutive days. The tumor growth
concentrated under reduced pressure. The residue was
inhibition (1 À T/C) was 27.6% at day 14. No signifi-
cant weight loss was observed in mice administered
with 6q (compared to vehicle). Although the result
from in vivo study excluded 6q to enter development
phase, it suggested that the scaffold of 6q has the
potential to be optimized as an orally administered
drug. The ongoing study carried out in our group is to
separate the two enantiomers of 6q and derive the
unexplored C-3 position.
purified by column chromatography to give 8 (1.112 g,
7.121 mmol) in 93% yield: H NMR δ 7.08 (d, J = 12 Hz,
1
1 H), 7.03 (d, J = 8.5 Hz, 1 H), 6.92 (t, J = 8.5 Hz, 1 H),
13
4.57 (s, 2 H), 3.87 (s, 3 H), 2.11 (brs, 1 H); C NMR δ
152.3 (d, J = 244.7 Hz), 147.0 (d, J = 10.5 Hz), 133.9 (d,
J = 5.0 Hz), 122.7 (d, J = 4.0 Hz), 115.0 (d, J = 18.3 Hz),
+
113.3, 64.4, 56.3; EI-MS: m/z 156 (M , 100), 155 (44), 139
(50), 135 (16), 127 (70), 125 (51), 112 (43), 97 (28), 96 (17),
77 (21).
3
| CONCLUSIONS
4.3 | 4-(Chloromethyl)-2-fluoro-
1-methoxybenzene (9)
We designed and synthesized a series of 4-substituted
7-(3-fluoro-4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidines
A solution of 8 (3.013 g, 19.30 mmol, 1.0 equiv) in anhy-
6a–s as anticancer agents. Among them, 6q having a C-4
drous Et O (64 ml) was slowly added with SOCl (2.8 ml,
2
2
ethyl and a benzylic methyl was the most potent, which
inhibited the proliferation of Hela, MDA-MB-231, and
MDA-MB-468 cancers cells at submicromolar concentra-
tions. Compound 6q arrested the cell cycle of MDA-MB-231
38 mmol, 2.0 equiv) in an ice bath. The reaction mixture
was stirred at room temperature for 3.0 hr. The solution
was slowly poured into water and extracted with EtOAc.
The organic layer was washed with brine, dried over
at G /M phase and was active in vivo. It has served as a lead
MgSO , filtered, and concentrated under reduced pressure.
2
4
compound for further optimization.
The residue was purified by column chromatography to

6
TSAI ET AL.
1
+
give 9 (3.197 g, 18.31 mmol) in 95% yield: H NMR δ 7.13
d, J = 11.7 Hz, 1 H), 7.09 (d, J = 8.5 Hz, 1 H), 6.92 (t,
47.6; HRMS calcd for [C H ClFN O + H] : 292.0647,
14 11 3
found 292.0636.
(
13
J = 8.5 Hz, 1 H), 4.53 (s, 2 H), 3.89 (s, 3 H); C NMR δ
52.1 (d, J = 245.5 Hz), 147.5 (d, J = 10.5 Hz), 130.3 (d,
J = 7.0 Hz), 124.6 (d, J = 3.0 Hz), 116.5 (d, J = 18.8 Hz),
13.2 (d, J = 2.1 Hz), 56.2, 45.5; EI-MS: m/z 176 (34), 174
M , 91), 140 (47), 139 (100), 107 (22), 105 (22), 96 (64), 95
36), 77 (52), 75 (23).
1
4.6 | 4-Chloro-7-[1-(3-fluoro-
4-methoxyphenyl)ethyl]-7H-pyrrolo[2,3-d]
pyrimidine (12)
1
(
+
(
Compound 12 was prepared by the identical method
for 9 to 10 using 11 as the starting material. Yield: 23%;
1
4
.4 | 1-(3-Fluoro-4-methoxyphenyl)
H NMR δ 8.63 (s, 1 H), 7.24 (d, J = 3.6 Hz, 1 H), 7.01–
ethan-1-ol (11)
6.97 (m, 2 H), 6.91–6.87 (m, 1 H), 6.61 (d, J = 3.6 Hz, 1
H), 6.13 (q, J = 7.1 Hz, 1 H), 3.84 (s, 3 H), 1.87 (d,
J = 7.2 Hz, 3 H); C NMR δ 152.5 (d, J = 142.0 Hz),
150.8, 150.5, 150.4, 147.1 (d, J = 10.5 Hz), 133.4 (d,
J = 5.6 Hz), 126.3, 122.2 (d, J = 3.6 Hz), 117.4, 114.2 (d,
J = 19.0 Hz), 113.2 (d, J = 2.1 Hz), 99.7, 56.0, 52.2,
1
3
A solution of 7 (0.566 g, 3.67 mmol, 1.0 equiv) in THF
(
12 ml) was added with MeMgBr (11.0 ml, 1.0 M in
THF, 11.0 mmol, 3.0 equiv) in a period of 10 min at
0
ture for 1.0 hr. The solution was quenched with H O
ꢀ
C. The reaction mixture was stirred at room tempera-
+
20.2; HRMS calcd for [C H ClFN O + H] : 306.0804,
2
15 13
3
and extracted with EtOAc. The organic layer was dried
found 306.0791.
over MgSO , filtered, and concentrated under reduced
4
pressure. The residue was purified by column chroma-
tography to give 11 (0.603 g, 3.54 mmol) in 96% yield:
H NMR δ 7.09 (d, J = 12.3 Hz, 1 H), 7.04 (d,
J = 8.5 Hz, 1 H), 6.90 (t, J = 8.5 Hz, 1 H), 4.80
4.7 | Standard procedure for the
synthesis of 6a–e having a C-4 alkoxy
substituent
1
(
(
1
q, J = 6.4 Hz, 1 H), 3.86 (s, 3 H), 2.10 (brs, 1 H), 1.44
1
3
d, J = 6.4 Hz, 3 H); C NMR δ 152.3 (d, J = 244.0 Hz),
46.7 (d, J = 10.8 Hz), 138.9 (d, J = 5.3 Hz), 121.0 (d,
J = 3.5 Hz), 113.3, 113.2, 69.4, 56.3, 25.0; EI-MS: m/z
For 6a–c, 10 (ꢁ150 mg, 1.0 equiv) and NaOH (6.0 equiv)
were mixed in MeOH, EtOH, or i-PrOH (5.0 ml). For 6d,
10 (ꢁ150 mg, 1.0 equiv) was mixed with KOH (2.0 equiv)
and cyclohexanol (1.1 equiv) in dioxane. For 6e, 10
(ꢁ150 mg, 1.0 equiv) in THF (2.6 ml) was added with
+
1
70 (M , 78), 156 (23), 155 (100), 153 (30), 127 (93), 112
(
68), 109 (13), 95 (16), 83 (18), 77 (15).
ꢀ
NaH (3.0 equiv) and BnOH (1.2 equiv) at 0 C. For 6a–d,
ꢀ
the reaction mixture was heated at 70 C for 12 hr. For
4
4
.5 | 4-Chloro-7-(3-fluoro-
-methoxybenzyl)-7H-pyrrolo[2,3-d]
6e, the reaction mixture was stirred at room temperature
for 4.0 hr. The solution was quenched with H O and
2
pyrimidine (10)
extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO , filtered, and concentrated
4
A
solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine
under reduced pressure. The residue was purified by col-
umn chromatography to give the target 6a–e in 60–99%
yields.
(1.276 g, 8.150 mmol, 1.03 equiv) in DMF (16 ml) was
slowly added with NaH (0.351 g, 8.78 mmol, 1.1 equiv) in
an ice bath. The solution was then added with 9 (1.377 g,
7
.887 mmol, 1.0 equiv), and the resulting reaction mix-
ture was stirred at room temperature for 12 hr. The solu-
4.8 | 7-(3-Fluoro-4-methoxybenzyl)-
4-methoxy-7H-pyrrolo[2,3-d]
pyrimidine (6a)
tion was quenched with H O and extracted with EtOAc.
2
The organic layer was washed with brine, dried over
MgSO , filtered, and concentrated under reduced pres-
4
1
sure. The residue was purified by column chromatogra-
Yield: 90%; H NMR δ 8.50 (s, 1 H), 6.99 (d, J = 3.6 Hz, 1
1
phy to give 10 (2.147 g, 7.360 mmol) in 93% yield: H
H), 6.95–6.86 (m, 3 H), 6.54 (d, J = 3.6 Hz, 1 H), 5.34 (s, 2
13
NMR δ 8.66 (s, 1 H), 7.20 (d, J = 3.6 Hz, 1 H), 7.00–6.93
H), 4.12 (s, 3 H), 3.84 (s, 3 H); C NMR δ 162.9, 152.2 (d,
J = 245.7 Hz), 151.6, 150.9, 147.1 (d, J = 10.6 Hz), 129.9
(d, J = 6.0 Hz), 125.4, 123.2 (d, J = 3.0 Hz), 115.2 (d,
J = 18.7 Hz), 113.3 (d, J = 2.0 Hz), 105.3, 98.8, 56.1, 53.5,
(
m, 2 H), 6.90 (t, J = 8.2 Hz, 1 H), 6.62 (d, J = 3.6 Hz, 1
13
H), 5.37 (s, 2 H), 3.85 (s, 3 H); C NMR δ 152.3 (d,
J = 246.1 Hz), 152.2, 151.0, 150.9, 147.6 (d, J = 10.5 Hz),
+
1
1
29.0 (d, J = 6.0 Hz), 128.8, 123.6 (d, J = 4.0 Hz), 117.5,
15.6 (d, J = 18.9 Hz), 113.5 (d, J = 1.0 Hz), 100.1, 56.2,
47.2; HRMS calcd for [C H FN O + H] : 288.1143,
15
14
3 2
found 288.1141.

TSAI ET AL.
7
4
4
.9 | 4-Ethoxy-7-(3-fluoro-
-methoxybenzyl)-7H-pyrrolo[2,3-d]
J = 3.4 Hz), 115.1 (d, J = 18.6 Hz), 113.1, 105.2, 98.7,
+
67.5, 55.7, 47.0; HRMS calcd for [C H FN O + H] :
21
18
3 2
pyrimidine (6b)
364.1456, found 364.1453.
1
Yield: 93%; H NMR δ 8.50 (s, 1 H), 7.01 (d, J = 4.0 Hz, 1
H), 6.97–6.87 (m, 3 H), 6.58 (d, J = 4.0 Hz, 1 H), 5.37 (s, 2
4.13 | Standard procedure for the
H), 4.62 (q, J = 7.1 Hz, 2 H), 3.85 (s, 3 H), 1.50–4.60 (t,
synthesis of 6f–h having a C-4 dialkylamino
13
J = 7.1 Hz, 3 H); C NMR δ 162.7, 153.5, 151.7, 151.0,
47.2 (d, J = 10.6 Hz), 130.0 (d, J = 6.0 Hz), 125.3, 123.2
d, J = 4.0 Hz), 115.3 (d, J = 18.8 Hz), 113.3 (d,
substituent
1
(
A solution of 10 (ꢁ150 mg, 1.0 equiv) in EtOH (1.5 ml)
J = 2.0 Hz), 105.4, 99.0, 62.2, 56.1, 47.2, 14.5; HRMS calcd
was added with dimethylamine, piperidine, or mor-
+
ꢀ
for [C H FN O + H] : 302.1299, found 302.1299.
pholine (1.5 equiv). The solution was heated at 70 C for
16
16
3 2
8
.0 hr. The solution was quenched with H O and
2
extracted with EtOAc. The organic layer was washed
4
4
.10 | 7-(3-Fluoro-4-methoxybenzyl)-
-isopropoxy-7H-pyrrolo[2,3-d]
with brine, dried over MgSO , filtered, and concentrated
4
under reduced pressure. The residue was purified by col-
umn chromatography to give the target 6f–h in 79–84%
yields.
pyrimidine (6c)
1
Yield: 97%; H NMR δ 8.46 (s, 1 H), 6.97–6.86 (m, 4 H),
6
.54 (d, J = 3.6 Hz, 1 H), 5.58 (sept, J = 6.3 Hz, 1 H), 5.33
13
(s, 2 H), 3.85 (s, 3 H), 1.43 (d, J = 6.3 Hz, 6 H); C NMR
4.14 | 7-(3-Fluoro-4-methoxybenzyl)-N,N-
dimethyl-7H-pyrrolo[2,3-d]pyrimidin-
4-amine (6f)
δ 162.5, 152.3 (d, J = 245.4 Hz), 151.8, 151.1, 147.2 (d,
J = 10.6 Hz), 130.1 (d, J = 5.9 Hz), 125.2, 123.3 (d,
J = 3.6 Hz), 115.3 (d, J = 18.7 Hz), 113.4 (d, J = 2.0 Hz),
1
1
05.7, 99.1, 69.0, 56.1, 47.2, 22.1; HRMS calcd for
Yield: 79%; H NMR δ 8.40 (s, 1 H), 6.96–6.86 (m, 4 H),
+
[
C H FN O + H] : 316.1456, found 316.1455.
6.63 (d, J = 3.6 Hz, 1 H), 5.32 (s, 2 H), 3.85 (s, 3 H), 3.44
17
18
3 2
13
(
1
s, 6 H); C NMR δ 157.4, 152.2 (d, J = 245.2 Hz), 151.4,
50.6, 147.0 (d, J = 10.6 Hz), 130.4 (d, J = 5.8 Hz), 123.2
4
4
.11 | 4-(Cyclohexyloxy)-7-(3-fluoro-
-methoxybenzyl)-7H-pyrrolo[2,3-d]
(d, J = 3.8 Hz), 122.5, 115.2 (d, J = 18.7 Hz), 113.3 (d,
J = 1.9 Hz), 103.0, 102.2, 56.1, 46.9, 39.1; HRMS calcd for
+
pyrimidine (6d)
[C H FN O + H] : 301.1459, found 301.1460.
16
17
4
1
Yield: 60%; H NMR δ 8.46 (s, 1 H), 6.97–6.86 (m, 4 H),
6
.54 (d, J = 3.6 Hz, 1 Hz), 5.35–5.30 (m, 3 H), 3.84 (s,
H), 2.12–2.02 (m, 2 H), 1.58–1.79 (m, 2 H), 1.66–1.57
4.15 | 7-(3-Fluoro-4-methoxybenzyl)-
4-(piperidin-1-yl)-7H-pyrrolo[2,3-d]
pyrimidine (6g)
3
(
13
m, 2 H), 1.52–1.42 (m, 2 H), 1.34–1.28 (m, 2 H);
NMR δ 162.5, 152.3 (d, J = 245.5 Hz), 151.8, 151.1, 147.2
d, J = 10.6 Hz), 130.1 (d, J = 5.9 Hz), 125.1, 123.3 (d,
J = 3.6 Hz), 115.3 (d, J = 18.8 Hz), 113.4 (d, J = 2.0 Hz),
05.8, 99.2, 74.0, 56.2, 47.2, 31.8, 25.6, 23.9; HRMS calcd
C
1
(
Yield: 80%; H NMR δ 8.38 (s, 1 H), 6.97–6.86 (m, 4 H),
6.53 (d, J = 4.0 Hz, 1 H), 5.31 (s, 2 H), 3.96–3.90 (m, 4 H),
13
1
3.85 (s, 3 H), 1.79–1.67 (m, 6 H); C NMR δ 162.5, 152.2
(d, J = 245.5 Hz), 151.8, 151.1, 147.1 (d, J = 10.6 Hz),
+
for [C H FN O + H] : 356.1769, found 356.1766.
20
22
3 2
1
30.1 (d, J = 5.9 Hz), 125.1, 123.2 (d, J = 3.6 Hz), 115.3
d, J = 18.8 Hz), 113.3 (d, J = 10.7 Hz), 105.8, 99.1, 56.1,
47.2, 31.8, 25.5, 23.8; HRMS calcd for [C H FN O
(
4
4
.12 | 4-(Benzyloxy)-7-(3-fluoro-
-methoxybenzyl)-7H-pyrrolo[2,3-d]
19
21
4
+
+ H] : 341.1772, found 341.1772.
pyrimidine (6e)
1
Yield: 99%; H NMR δ 8.51 (s, 1 H), 7.52–7.51 (m, 2 H),
4.16 | 4-[7-(3-Fluoro-4-methoxybenzyl)-
7H-pyrrolo[2,3-d]pyrimidin-4-yl]
morpholine (6h)
7
.41–7.32 (m, 4 H), 7.00 (d, J = 3.6 Hz, 1 H), 6.96–6.86
m, 3 H), 6.58 (d, J = 3.6 Hz, 1 H), 5.60 (s, 2 H), 5.35 (s, 2
H), 3.85 (s, 2 H);
J = 245.5 Hz), 151.6, 150.7, 146.9 (d, J = 10.5 Hz), 136.5,
29.7 (d, J = 5.8 Hz), 128.2, 127.8, 127.7, 125.3, 123.1 (d,
(
13
C NMR δ 162.2, 152.0 (d,
1
Yield: 84%; H NMR δ 8.38 (s, 1 H), 6.95–6.86 (m, 4 H),
1
6.49 (d, J = 3.6 Hz, 1 H), 5.31 (s, 2 H), 3.97–3.94 (m, 4 H),

8
TSAI ET AL.
13
3
.86–3.84 (m, 7 H);
C NMR δ 157.0, 152.2 (d,
4.20 | 7-(3-Fluoro-4-methoxybenzyl)-
J = 245.5 Hz), 151.1, 151.0, 147.1 (d, J = 10.3 Hz), 130.2
d, J = 5.9 Hz), 123.5, 122.2 (d, J = 3.6 Hz), 115.2 (d,
4-isopropyl-7H-pyrrolo[2,3-d]
pyrimidine (6k)
(
J = 18.8 Hz), 113.3, 103.0, 101.1, 66.6, 56.1, 47.0, 45.8;
+
1
HRMS calcd for [C H FN O + H] : 343.1565, found
Yield: 72%; H NMR δ 8.77 (s, 1 H), 7.05 (d, J = 3.6 Hz, 1
18
19
4 2
3
43.1564.
H), 6.90–6.88 (m, 2 H), 6.80–6.76 (m, 1 H), 6.52 (d,
J = 3.6 Hz, 1 H), 5.27 (s, 2 H), 3.73 (s, 3 H), 3.35 (sept,
13
J = 6.8 Hz, 1 H), 1.33 (d, J = 6.8 Hz, 6 H); C NMR δ
167.8, 152.2 (d, J = 245.5 Hz), 151.5, 150.4, 147.2 (d,
J = 10.6 Hz), 129.8 (d, J = 5.9 Hz), 127.2, 123.5 (d,
J = 3.6 Hz), 116.1, 115.4 (d, J = 18.7 Hz), 113.4 (d,
J = 1.9 Hz), 99.6, 56.1, 46.9, 33.7, 21.4; HRMS calcd for
4
.17 | Standard procedure for the
synthesis of 6i–l and 6n–s having a C-4
alkyl or phenyl substituent
+
Compound 10 or 12 (ꢁ100 mg, 1.0 equiv) was mixed with
[C H FN O + H] : 300.1507, found 300.1499.
17
18
3
Pd(dppf)Cl (0.050 equiv) in THF (1.2 ml). The solution
2
was slowly added with the corresponding Grignard
ꢀ
reagent (6.0 equiv) at 0 C under N . The reaction mix-
4.21 | 4-Cyclopropyl-7-(3-fluoro-
4-methoxybenzyl)-7H-pyrrolo[2,3-d]
pyrimidine (6l)
2
ture was stirred at room temperature for 12 hr. The solu-
tion was quenched with H O and extracted with EtOAc.
2
The organic layer was washed with saturated aqueous
NH Cl, dried over MgSO , filtered, and concentrated
1
Yield: 79%; H NMR δ 8.71 (s, 1 H), 7.09 (d, J = 3.6 Hz, 2
4
4
under reduced pressure. The residue was purified by col-
H), 6.96–6.86 (m, 3 H), 6.65 (d, J = 3.6 Hz, 2 H), 5.35 (s, 2
umn chromatography to give the target 6i–l and 6n–s in
H), 3.85 (s, 3 H), 2.38–2.32 (m, 1 H), 1.37–1.34 (m, 2 H),
13
2
5–93% yields.
1.18–1.13 (m, 2 H);
J = 245.5 Hz), 151.6, 149.6, 147.2 (d, J = 10.6 Hz), 129.9
d, J = 5.8 Hz), 127.0, 123.3 (d, J = 3.5 Hz), 117.0, 115.3
C NMR δ 164.3, 152.2 (d,
(
4
4
.18 | 7-(3-Fluoro-4-methoxybenzyl)-
-methyl-7H-pyrrolo[2,3-d]pyrimidine (6i)
(d, J = 18.8 Hz), 113.3 (d, J = 2.1 Hz), 99.4, 56.1, 46.9,
+
14.4, 10.8; HRMS calcd for [C H FN O + H] :
17
16
3
298.1350, found 298.1339.
1
Yield: 94%; H NMR δ 8.82 (s, 1 H), 7.21 (d, J = 3.6 Hz, 1
H), 6.98–6.94 (m, 2 H), 6.91–6.87 (m, 1 H), 6.62 (d,
J = 3.6 Hz, 1 H), 5.37 (s, 2 H), 3.84 (s, 3 H), 2.82 (s, 3 H);
4.22 | 4-Cyclopentyl-7-(3-fluoro-
4-methoxybenzyl)-7H-pyrrolo[2,3-d]
pyrimidine (6n)
1
3
C NMR δ 159.0, 152.3 (d, J = 245.8 Hz), 150.8, 150.1,
47.3 (d, J = 10.6 Hz), 129.6 (d, J = 5.8 Hz), 127.3, 123.5
1
(d, J = 3.6 Hz), 117.7, 115.4 (d, J = 18.7 Hz), 113.4 (d,
1
J = 1.9 Hz), 100.0, 56.2, 47.1, 21.1; HRMS calcd for
Yield: 84%; H NMR δ 8.83 (s, 1 H), 7.09 (d, J = 3.6 Hz, 2
H), 6.96–6.93 (m, 2 H), 6.89–6.85 (m, 1 H), 6.58 (d,
J = 3.6 Hz, 2 H), 5.34 (s, 2 H), 3.83 (s, 3 H), 3.57–3.48 (m,
+
[
C H FN O + H] : 272.1194, found 272.1189.
15 14 3
1
H), 2.15–1.97 (m, 3 H), 1.94–1.85 (m, 2 H), 1.78–1.68
13
4
4
.19 | 4-Ethyl-7-(3-fluoro-
-methoxybenzyl)-7H-pyrrolo[2,3-d]
(m, 2 H); C NMR δ 166.7, 152.2 (d, J = 245.7 Hz), 151.6,
150.3, 147.2 (d, J = 10.6 Hz), 129.8 (d, J = 5.9 Hz), 127.0,
123.4 (d, J = 3.6 Hz), 116.9, 115.3 (d, J = 18.7 Hz), 113.4
pyrimidine (6j)
(
d, J = 1.7 Hz), 99.8, 56.2, 47.0, 44.8, 32.6, 26.2; HRMS
1
+
Yield: 67%; H NMR δ 8.97 (s, 1 H), 7.46 (d,
calcd for [C H FN O + H] : 326.1663, found 326.1652.
19 20 3
J = 4.0 Hz, 1 H), 7.05–7.01 (m, 2 H), 6.95–6.91 (m, 1
H), 6.87 (d, J = 3.6 Hz, 1 H), 5.45 (s, 2 H), 3.86 (s, 3
H), 3.39 (q, J = 7.6 Hz, 2 H), 1.57 (t, J = 7.6 Hz, 3 H);
4.23 | 4-Cyclohexyl-7-(3-fluoro-4-
methoxybenzyl)-7H-pyrrolo[2,3-d]
pyrimidine (6o)
1
3
C NMR δ 164.0, 152.2 (d, J = 245.6 Hz), 151.3, 150.2,
47.2 (d, J = 10.5 Hz), 129.6 (d, J = 5.8 Hz), 127.4,
23.4 (d, J = 3.5 Hz), 116.8, 115.4 (d, J = 18.8 Hz),
1
1
1
1
13.3 (d, J = 1.8 Hz), 99.6, 56.1, 47.0, 28.3, 21.8; HRMS
Yield: 26%; H NMR δ 8.82 (s, 1 H), 7.08 (d, J = 3.6 Hz, 2
+
calcd for [C H FN O + H] : 286.1350, found
H), 6.96–6.93 (m, 2 H), 6.85 (t, J = 8.5 Hz, 1 H), 6.58 (d,
J = 3.6 Hz, 2 H), 5.32 (s, 2 H), 3.81 (s, 3 H), 3.08–3.02 (m,
1
6
16
3
2
86.1335.

TSAI ET AL.
9
1
3
1
H), 1.94–1.73 (m, 8 H), 1.47–1.31 (m, 2 H); C NMR
4.27 | 4-Butyl-7-[1-(3-fluoro-
4-methoxyphenyl)ethyl]-7H-pyrrolo[2,3-d]
pyrimidine (6s)
δ 167.0, 151.5, 152.2 (d, J = 245.6 Hz), 150.5, 147.2 (d,
J = 10.6 Hz), 129.8 (d, J = 5.9 Hz), 127.0, 123.4
d, J = 3.6 Hz), 116.3, 115.4 (d, J = 18.7 Hz), 113.4 (d,
J = 2.0 Hz), 99.6, 56.1, 46.9, 44.0, 31.5, 26.4, 25.9;
HRMS calcd for [C H FN O + H] : 340.1820, found
(
1
Yield: 78%; H NMR δ 8.80 (s, 1 H), 7.16 (d, J = 3.6 Hz, 1
H), 7.02–6.09 (m, 2 H), 6.91–6.87 (m, 1 H), 6.57 (d,
J = 3.6 Hz, 1 H), 6.16 (q, J = 7.1 Hz, 1 H), 3.85 (s, 3 H),
+
2
0
22
3
3
40.1814.
3
2
.01 (t, J = 8.0 Hz, 2 H), 1.87–1.79 (m, 5 H), 1.48–1.38 (m,
13
H), 0.96 (t, J = 7.4 Hz, 3 H); C NMR δ 163.3, 152.2 (d,
4
4
.24 | 7-(3-Fluoro-4-methoxybenzyl)-
-phenyl-7H-pyrrolo[2,3-d]pyrimidine (6p)
J = 245.4 Hz), 151.2, 150.0, 147.0 (d, J = 10.7 Hz), 134.3
(d, J = 5.4 Hz), 124.6, 122.3 (d, J = 3.5 Hz), 117.5, 114.3
(
d, J = 18.9 Hz), 113.3, 99.5, 56.1, 51.5, 35.1, 30.9, 22.7,
1
+
Yield: 69%; H NMR δ 8.99 (s, 1 H), 8.11 (d, J = 6.8 Hz, 2
H), 7.57–7.49 (m, 3 H), 7.22 (d, J = 3.6 Hz, 1 H), 7.02–
20.5, 13.9; HRMS calcd for [C H FN O + H] :
19 22 3
328.1820, found 328.1805.
6
.99 (m, 2 H), 6.93–6.88 (m, 1 H), 6.83 (d, J = 3.6 Hz, 1
13
H), 5.42 (s, 2 H), 3.86 (s, 3 H); C NMR δ 157.5, 152.2 (d,
J = 245.7 Hz), 151.5, 151.5, 150.7, 147.2 (d, J = 10.6 Hz),
4.28 | 7-(3-Fluoro-4-methoxybenzyl)-
4-vinyl-7H-pyrrolo[2,3-d]pyrimidine (6m)
1
1
1
37.9, 129.9, 129.6 (d, J = 5.9 Hz), 128.7 (d, J = 7.9 Hz),
28.5, 123.4 (d, J = 3.6 Hz), 115.6, 115.4 (d, J = 18.8 Hz),
13.4 (d, J = 2.1 Hz), 100.9, 56.1, 47.0; HRMS calcd for
A solution of 10 (0.108 g, 0.370 mmol, 1.0 equiv),
+
[
C H FN O + H] : 334.1350, found 334.1339.
Pd(PPh ) Cl (14 mg, 0.020 mmol, 0.054 equiv), and LiCl
20
16
3
3 2
2
(
0.019 g, 0.45 mmol, 1.2 equiv) in DMF (1.2 ml)
was slowly added with tributyl(vinyl)tin (175 mg,
4
4
.25 | 4-Ethyl-7-[1-(3-fluoro-
-methoxyphenyl)ethyl]-7H-pyrrolo[2,3-d]
0.552 mmol, 1.5 equiv). The reaction mixture was heated
ꢀ
at 100 C for 19 hr under N . The solution were quenched
2
pyrimidine (6q)
with H O and extracted with EtOAc. The organic layer
2
was washed with brine, dried over MgSO , filtered, and
4
1
Yield: 60%; H NMR δ 8.88 (s, 1 H), 7.31 (d, J = 3.2 Hz, 1
concentrated under reduced pressure. The residue was
H), 7.03–7.01 (m, 2 H), 6.93–6.89 (m, 2 H), 6.71 (d,
J = 3.2 Hz, 1 H), 6.19 (q, J = 7.1 Hz, 1 H), 3.86 (s, 3 H),
purified by column chromatography to give 6m (0.026 g,
0.092 mmol) in 25% yield: H NMR δ 8.87 (s, 1 H), 7.17
1
3
.21 (q, J = 7.8 Hz, 2 H), 1.89 (d, J = 7.1 Hz, 3 H), 1.49 (t,
(d, J = 3.6 Hz, 1 H), 7.11 (dd, J = 17.6 Hz, 10.8 Hz, 1 H),
6.97–6.95 (m, 2 H), 6.91–6.87 (m, 1 H), 6.68 (d,
J = 3.6 Hz, 1 H), 6.64 (dd, J = 17.6 Hz, 1.2 Hz, 1 H), 5.79
(dd, J = 10.8 Hz, 1.2 Hz, 1 H), 5.38 (s, 2 H), 3.85 (s, 3 H);
C NMR δ 154.3, 152.3 (d, J = 245.9 Hz), 151.7, 151.5,
147.3 (d, J = 10.6 Hz), 133.5, 129.6 (d, J = 4.0 Hz), 128.4,
13
J = 7.8 Hz, 3 H);
J = 245.2 Hz), 151.1, 149.9, 146.9 (d, J = 10.5 Hz), 134.2
C NMR δ 164.0, 152.0 (d,
(
(
d, J = 5.4 Hz), 124.6, 122.1 (d, J = 3.4 Hz), 116.9, 114.2
d, J = 18.8 Hz), 113.1, 99.3, 56.0, 51.4, 28.3, 20.3, 12.7;
13
+
HRMS calcd for [C H FN O + H] : 300.1507, found
17
18
3
3
00.1499.
123.4 (d, J = 3.6 Hz), 123.2, 116.0, 115.4 (d, J = 18.7 Hz),
1
+
13.4, 99.5, 56.2, 47.0; HRMS calcd for [C H FN O
H] : 284.1194, found 284.1185.
16 14 3
+
4
.26 | 7-[1-(3-Fluoro-4-methoxyphenyl)
ethyl]-4-propyl-7H-pyrrlo[2,3-d]
pyrimidine (6r)
4.29 | Biological study
1
Yield: 93%; H NMR δ 8.80 (s, 1 H), 7.16 (d, J = 3.6 Hz, 1
The in vitro anticancer activity of compounds 6a–s
and 3 (Figure 1 and Table 1) for Hela, MDA-MB-231,
and MDA-MB-468 cancer cells was measured on
H), 7.02–6.98 (m, 2 H), 6.91–6.87 (m, 1 H), 6.57 (d,
J = 3.6 Hz, 1 H), 6.16 (q, J = 7.1 Hz, 1 H), 3.85 (s, 3 H),
2
.99 (t, J = 7.6 Hz, 2 H), 1.93–1.84 (m, 5 H), 1.01 (t,
CellTiter96 assay kit (Promega) after a 96-hr incuba-
13
[29]
J = 7.1 Hz, 3 H);
C NMR δ 163.0, 152.1 (d,
tion as we previously described.
The cell cycle
J = 245.2 Hz), 151.2, 149.9, 147.0 (d, J = 10.6 Hz), 134.2
analysis of 6q by flow cytometry (FACS) on MDA-MB-
231 cells was performed by our published method.
The in vivo anticancer activity of 6q was conducted on
[
29]
(d, J = 5.5 Hz), 124.6, 122.2 (d, J = 3.5 Hz), 117.6, 114.3
d, J = 18.9 Hz), 113.2 (d, J = 2.1 Hz), 99.5, 56.1, 51.4,
(
3
+
7.3, 22.1, 20.4, 14.1; HRMS calcd for [C H FN O
H] : 314.1663, found 314.1656.
nude mice bearing human MDA-MB-231 xenografts as
previously reported.
18
20
3
+
[30]

10
TSAI ET AL.
ACKNOWLEDGMENTS
[20] A. Gangjee, O. A. Namjoshi, S. N. Keller, C. D. Smith, Bioorg.
Med. Chem. 2011, 19, 4355.
We are grateful to the Ministry of Science and Technol-
ogy of R.O.C. (Taiwan, MOST 105-2113-M-415-001) for
financial support.
[
21] A. Gangjee, S. Kurup, C. D. Smith, Bioorg. Med. Chem. 2013,
1, 1180.
2
[
22] J.-P. Liou, Y.-L. Chang, F.-M. Kuo, C.-W. Chang, H.-Y. Tseng,
C.-C. Wang, Y.-N. Yang, J.-Y. Chang, S.-J. Lee, H.-P. Hsieh,
J. Med. Chem. 2004, 47, 4247.
ORCID
Jiann-Jyh Huang https://orcid.org/0000-0003-4336-
[23] T. Hayashi, M. Konish, Y. Kobori, M. Kumada, T. Higuchi, K.
7
913
Hirotsu, J. Am. Chem. Soc. 1984, 106, 158.
[
[
24] J. K. Stille, Angew. Chem. Int. Ed. Engl. 1986, 25, 508.
25] S.-K. Kim, S.-M. Cho, H. Kim, H. Seok, S.-O. Kim, T. K. Kwon,
J.-S. Chang, Exp. Mol. Med. 2013, 45, e19.
REFERENCES
[
[
1] S.-E. N. Ayyad, A. S. Judd, W. T. Shier, T. R. Hoye, J. Org.
Chem. 1998, 63, 8102.
2] C. Combeau, J. Provost, F. Lancelin, Y. Tournoux, F.
Prod'homme, F. Herman, F. Lavelle, J. Leboul, M. Vuilhorgne,
Mol. Pharmacol. 2000, 57, 553.
[
26] A. Ciupa, P. A. de Bank, M. F. Mahon, P. J. Wood, L.
Caggiano, Med. Chem. Commun. 2013, 4, 956.
[
[
27] M. Ma, L. Sun, H. Lou, M. Ji, Chem. Cent. J. 2013, 7, 179.
28] M.-Y. Song, Q.-R. He, Y.-L. Wang, H.-R. Wang, T.-C. Jiang, J.-
J. Tang, J.-M. Gao, Int. J. Mol. Sci. 2020, 21, 1817.
[
[
3] G. Nuki, Medicine 2006, 34, 417.
4] Y. Shan, J. Zhang, Z. Liu, M. Wang, Y. Dong, Curr. Med.
Chem. 2011, 18, 523.
[
29] C.-C. Chiang, Y.-H. Lin, S. F. Lin, C.-L. Lai, C. Liu, W.-Y. Wei,
S.-C. Yang, R.-W. Wang, L.-W. Teng, S.-H. Chuang, J.-M.
Chang, T.-T. Yuan, Y.-S. Lee, P. Chen, W.-K. Chi, J.-Y. Yang,
H.-J. Huang, C.-B. Liao, J.-J. Huang, J. Med. Chem. 2010, 53,
[
5] R. Smolarczyk, J. Czapla, M. Jarosz-Biej, K. Czerwinski, T.
Cicho, Eur. J. Pharmacol. 2021, 891, 173692.
5929.
[6] G. Mehta, K. Islam, Angew. Chem. Int. Ed. 2002, 41, 2396.
[7] G. Mehta, K. Islam, Tetrahedron Lett. 2003, 44, 6733.
[8] H. Araki, M. Inoue, T. Katoh, Org. Lett. 2003, 5, 3903.
[9] H. Araki, M. Inoue, T. Suzuki, T. Yamori, M. Kohno, K.
Watanabe, H. Abe, T. Katoh, Chem. A Eur. J. 2007, 13, 9866.
[
30] Y.-S. E. Lee, S.-H. Chuang, L. Y. L. Huang, C.-L. Lai, Y.-H.
Lin, J.-Y. Yang, C.-W. Liu, S.-c. Yang, H.-S. Lin, C.-c. Chang,
J.-Y. Lai, P.-S. Jian, K. Lam, J.-M. Chang, J. Y. N. Lau, J.-J.
Huang, J. Med. Chem. 2014, 57, 4098.
[
[
[
10] D. L. J. Clive, D. Liu, Angew. Chem. Int. Ed. 2007, 46, 3738.
11] D. L. J. Clive, D. Liu, J. Org. Chem. 2008, 73, 3078.
12] M. Y. Lee, K. H. Kim, S. Jiang, Y. H. Jung, J. H. Sim, G.-S.
Hwang, D. H. Ryu, Tetrahedron Lett. 2008, 49, 1965.
13] C.-H. Chen, Y.-K. Chen, C.-K. Sha, Org. Lett. 2010, 12, 1377.
14] D. R. Hookins, R. J. K. Taylor, Tetrahedron Lett. 2010, 51,
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.
[
[
6
619.
[
15] T. Suzuki, K. Ghozati, D.-Y. Zhou, T. Katoh, H. Sasai, Tetrahe-
dron 2010, 66, 7562.
How to cite this article: Tsai P-Y, Hu G-S,
Huang P-H, et al. Design, synthesis, and in vitro/
vivo anticancer activity of 4-substituted 7-(3-fluoro-
[
[
16] T. Katoh, Nat. Prod. Commun. 2013, 8, 973.
17] T.-Y. Chang, Y.-P. Tu, W.-Y. Wei, H. Y. Chen, C.-S. Chen, Y.-S. E.
Lee, J.-J. Huang, C.-K. Sha, ACS Med. Lett. 2012, 3, 1075.
18] A. G. Cs ꢀa kÿ, G. de la Herr ꢀa n, M. C. Murcia, Chem. Soc. Rev.
4-methoxybenzyl)-7H-pyrrolo[2,3-d]pyrimidines. J
[
[
Chin Chem Soc. 2021;1–10. https://doi.org/10.1002/
jccs.202100100
2
010, 39, 4080.
19] J. C. Powers, J. L. Asgian, Ö. D. Ekici, K. E. James, Chem. Rev.
002, 102, 4639.
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