Design and synthesis of novel human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR) dual inhibitors bearing a pyrrolo[3,2-d]pyrimidine scaffold

Article abstract of DOI:10.1021/jm2008634

Dual inhibitors of human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) have been investigated for breast, lung, gastric, prostate, and other cancers; one, lapatinib, is currently approved for breast cancer. To develop novel HER2/EGFR dual kinase inhibitors, we designed and synthesized pyrrolo[3,2-d]pyrimidine derivatives capable of fitting into the receptors' ATP binding site. Among the prepared compounds, 34e showed potent HER2 and EGFR (HER1) inhibitory activities as well as tumor growth inhibitory activity. The X-ray cocrystal structures of 34e with both HER2 and EGFR demonstrated that 34e interacts with the expected residues in their respective ATP pockets. Furthermore, reflecting its good oral bioavailability, 34e exhibited potent in vivo efficacy in HER2-overexpressing tumor xenograft models. On the basis of these findings, we report 34e (TAK-285) as a promising candidate for clinical development as a novel HER2/EGFR dual kinase inhibitor.

Full text of DOI:10.1021/jm2008634

Article
pubs.acs.org/jmc
Design and Synthesis of Novel Human Epidermal Growth Factor
Receptor 2 (HER2)/Epidermal Growth Factor Receptor (EGFR) Dual
Inhibitors Bearing a Pyrrolo[3,2-d]pyrimidine Scaffold†
Tomoyasu Ishikawa,*,‡ Masaki Seto,‡ Hiroshi Banno,‡ Youichi Kawakita,‡ Mami Oorui,‡
Takahiko Taniguchi,‡ Yoshikazu Ohta,‡ Toshiya Tamura,‡ Akiko Nakayama,‡ Hiroshi Miki,‡
Hidenori Kamiguchi,‡ Toshimasa Tanaka,‡ Noriyuki Habuka,‡ Satoshi Sogabe,‡ Jason Yano,§
Kathleen Aertgeerts,§ and Keiji Kamiyama‡
‡Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1 Muraoka-Higashi 2-chome, Fujisawa,
Kanagawa 251-8555, Japan
§Structural Biology, Takeda San Diego, Inc., 10410 Science Center Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Dual inhibitors of human epidermal growth
factor receptor 2 (HER2) and epidermal growth factor
receptor (EGFR) have been investigated for breast, lung,
gastric, prostate, and other cancers; one, lapatinib, is currently
approved for breast cancer. To develop novel HER2/EGFR
dual kinase inhibitors, we designed and synthesized pyrrolo-
[3,2-d]pyrimidine derivatives capable of fitting into the
receptors’ ATP binding site. Among the prepared compounds,
34e showed potent HER2 and EGFR (HER1) inhibitory
activities as well as tumor growth inhibitory activity. The X-ray
cocrystal structures of 34e with both HER2 and EGFR demonstrated that 34e interacts with the expected residues in their
respective ATP pockets. Furthermore, reflecting its good oral bioavailability, 34e exhibited potent in vivo efficacy in HER2-
overexpressing tumor xenograft models. On the basis of these findings, we report 34e (TAK-285) as a promising candidate for
clinical development as a novel HER2/EGFR dual kinase inhibitor.
As for EGFR inhibitors, small molecules such as gefitinib5
INTRODUCTION
■
and erlotinib6 (Figure 1), as well as anti-EGFR antibodies
Protein kinases play important roles in signal transduction
cetuximab7 and panitumumab,8 have been approved for the
pathways that regulate numerous cellular functions, including
treatment of EGFR-overexpressing lung cancers and have been
proliferation, differentiation, migration, apoptosis, and angio-
shown to be effective in a subset of patients. HER2 is
genesis. Because signal transduction pathways are upregulated
overexpressed in a number of human cancers, including 20−
in many tumor cells, protein kinase inhibitors that target these
upregulated pathways are attractive candidates for cancer
therapy.1 The targeting of human epidermal growth factor
receptor (HER) or epidermal growth factor receptor (EGFR) by
tyrosine kinase inhibitors (TKIs) represents one such therapeutic
approach. The HER kinase family contains 4 members (EGFR
(HER1 or ErbB-1), HER2 (ErbB-2 or neu), HER3 (ErbB-3),
and HER4 (ErbB-4)) that are multidomain proteins consisting
of an extracellular ligand binding domain, a single trans-
membrane domain, and an intracellular tyrosine kinase
domain.2 Several ligands that bind to the extracellular portion
of EGFR, HER3, and HER4 have been identified. Upon ligand
binding, these receptors form homo- or heterodimers to
undergo autophosphorylation of each tyrosine residue within
the intracellular kinase domain.3 Although there is no known
ligand for HER2, this receptor also undergoes spontaneous
homo- or heterodimerization and activates downstream signaling.4
40% of solid tumors, e.g., breast, ovarian, lung, gastric, and oral
cancers, in which overexpression of this receptor correlates with
poor prognosis.9 In addition, because HER2 is only expressed
at low levels in normal human tissues, it is an attractive target
for tumor-specific therapies. On the basis of this, two
approaches have been developed for blocking the upregulated
HER2 signal pathway. One approach utilizes the anti-HER2
monoclonal antibody, trastuzumab,10 which blocks the
extracellular ligand-binding region of the receptor, thereby
interfering with its activation and modulating the resultant
intracellular signal cascade. The other approach for blocking the
upregulated HER2 signal pathway involves the use of orally
active small molecule TKIs several of which have been
developed. Many types of malignancies are characterized by
overexpression of HER2 and EGFR. Lapatinib,11 a dual TKI for
Received: July 1, 2011
Published: October 17, 2011
© 2011 American Chemical Society
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Figure 1. Structure of EGFR and/or HER2 small molecule inhibitors.
HER2 and EGFR, was approved in 2007 in combination with
capecitabine or letrozole in patients with metastatic breast
cancer that overexpress the HER2 receptor. Irreversible
inhibitors (Neratinib/HKI-27212 and Afatinib/BIBW-299213)
and reversible inhibitors (AEE-78814 and BMS-59962615) have
also been tested in clinical trials. In addition to breast cancer,
these HER2/EGFR dual kinase inhibitors are being investigated
in a number of solid tumors including lung, gastric, and prostate
cancers.9,11−15 In addition, CP-72471416 has been reported as a
HER2-specific TKI. All of these small molecule inhibitors
share a common structure characterized by a pyrimidine or
pyrimidine analogue at its core.
but this pyrimidine core had not been previously utilized for
drug development. Molecular modeling studies using HER2
homology models based on the EGFR cocrystal structure
For the purpose of developing a novel HER2/EGFR TKI, we
focused on the pyrrolo[3,2-d]pyrimidine scaffold, which
contains a pyrimidine core that interacts with Met801 and
Thr862 in the ATP-binding site of the HER2 hinge region
(Figure 2). Nucleic acid research using pyrrolo[3,2-d]-
pyrimidine derivatives has been conducted in our laboratories,17
Figure 2. Design of pyrrolo[3,2-d]pyrimidine scaffold as HER2 kinase
inhibitors.
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a
Scheme 1. Synthesis of Pyrrolo[3,2-d]pyrimidine Derivatives 6a−n
a
Reagents: (a) 2a−n, K2CO3, DMF, 80 °C, 2 h; (b) H2, Pt−C, AcOEt, rt, 2 h, or Fe, CaCl2, EtOH, 100 °C, 15 h, 56−99% for 2 steps; (c) 5, NMP,
100 °C, 3 h, 31−83%.
with erlotinib6 suggested that the pyrrolo[3,2-d]pyrimidine
scaffold would fit into the ATP-binding site of the HER2/
EGFR protein. In this model, introduction of a phenoxy
anilino group at the C-4 position could occupy a lipophilic
back pocket and enhance anti-HER2/EGFR kinase activity.
In addition, because the linkages at the N-5 or C-6 positions
are directed toward the solvent contact region, we
envisioned that chemical modification of the R2 or R3
groups at these positions could be utilized to further improve
the cellular activity and pharmacokinetic properties of this
chemotype.
(93% yield). The methylated adduct 7 was converted into the
desired products 8e−l by coupling with aniline 4e (92%), 4j
(75%), and 4l (77%), respectively. Similarly, alkylation of
Scheme 2. Synthesis of Pyrrolo[3,2-d]pyrimidine Derivatives
a
6o−q
In this paper, we describe the structure−activity relationships
(SAR) and in vivo efficacy of our pyrrolo[3,2-d]pyrimidine
derivatives. Crystallographic studies of 34e (TAK-285),18 our
candidate for clinical development, with HER2 and EGFR
revealed key molecular details related to compound binding
and specificity.
a
Reagents: (a) 4o−4q, NMP, 100 °C, 3 h, 66−80%.
CHEMISTRY
■
With the aim of examining substituent effects on the terminal
phenoxy rings, 4-substituted anilino-5H-pyrrolo[3,2-d]-
pyrimidine derivatives (6a−n) were synthesized by the method
shown in Scheme 1. Condensation of commercially available
2-chloro-1-fluoro-4-nitrobenzene (1) with phenols (2a−n) was
carried out in the presence of potassium carbonate in
dimethylformamide (DMF), followed by reduction of the nitro
group with platinum on carbon under hydrogen atmosphere to
afford substituted anilines (4a−e, 4i−n) in good yields (61−
99% in 2 steps). In the case of 4f−h containing a cyano group,
the nitro group was reduced using reduced-iron and calcium
chloride in ethanol (EtOH) (56−71% in 2 steps). The obtained
anilines 4a−n were reacted with 4-chloro-5H-pyrrolo[3,2-d]-
pyrimidine (5)19 in 1-methyl-2-pyrrolidone (NMP) at 100 °C
to afford 6a−n in moderate yields (31−83%). In Scheme 2, the
pyrrolo[3,2-d]pyrimidine derivatives 6o−q were prepared from
reported anilines 4o−q11c,12a,16c in a similar manner to that
shown in Scheme 1.
compound 5 with 2-iodoethyl benzoate or 2-(2-iodoethoxy)-
ethyl benzoate in the presence of cesium carbonate in DMF
gave the adducts 9 and 11 in 70% and 55% yield, respectively.
After reaction of 9 or 11 with aniline (4e, 4j, 4l), the terminal
benzoyl group was hydrolyzed by 1N aqueous sodium
hydroxide (NaOH) to afford the desired target compounds
(10e−l, 12e−l) in 46−72% yields. A similar reaction was
carried out for the synthesis of 12r, which is a pyridine analogue
of 12j.
On the basis of this SAR study, we focused on the 2-(2-
hydroxyethoxy)ethyl group of compound 12 at the N-5 position.
We selected a trifluoromethyl group j as an anilino substituent on
the terminal phenoxy ring, and carried out further optimization of
the 2-(2-hydroxyethoxy)ethyl group at the N-5 position.
The synthesis of the N-5-hydroxypentyl derivative 15, where
the ether linkage in 12j was replaced with a methylene bridge, is
shown in Scheme 4. Alkylation of compound 5 with 5-
chloropentyl acetate in the presence of cesium carbonate in
DMF gave 5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl
acetate (13) in 69% yield. The obtained compound 13 was
reacted with aniline 4j in 2-propanol followed by hydrolysis of
the acetate group with 1N aqueous NaOH to afford the desired
compound 15 in 79% yield in two steps.
Next, we selected 3 anilino groups (e: −Cl; j: −CF3; l:
−OCF3) and examined the introduction of methyl (8e−l),
2-hydroxyethyl (10e−l), and 2-(2-hydroxyethoxy)ethyl (12e−l)
moieties as representative alkyl substituents at the N-5 position.
The synthesis is shown in Scheme 3. The 4-chloro derivative 5
was reacted with iodomethane under basic conditions using
potassium carbonate in DMF to afford 5-methyl intermediate 7
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a
Scheme 3. Synthesis of Pyrrolo[3,2-d]pyrimidine Derivatives (8, 10, and 12)
a
Reagents: (a) iodomethane, K2CO3, DMF, rt, 3 h, 93%; (b) aniline 4e or 4j or 4l, NMP, 140 °C, 4 h, 75−92%; (c) 2-iodoethyl benzoate, Cs2CO3,
DMF, rt, 15 h, 70%; (d) (i) aniline 4e or 4j or 4l NMP, 140 °C, 2.5 h, (ii) 1N NaOH, MeOH, rt, 46−72%; (e) 2-(2-iodoethoxy)ethyl benzoate,
Cs2CO3, DMF, rt, 15 h, 55%; (f) (i) 2,3-dichloro-5-nitropyridine, NaH, THF, rt, 2.5 h, (ii) Fe, CaCl2, EtOH, 80 °C, 8 h, 39% from 2j.
a
Scheme 4. Synthesis of N-5 Hydroxypentyl Pyrrolo[3,2-d]pyrimidine derivative 15
a
Reagents: (a) 5-chloropentyl acetate, Cs2CO3, DMF, 40 °C, 4 days, 69%; (b) 4j, 2-PrOH, 80 °C, 14 h; (c) 1N NaOH, rt, 1 h, MeOH, 79% for 2 steps.
The pyrrolo[3,2-d]pyrimidine derivative 20 with substitution
of the nitrogen atom for N-5 ether oxygen in 12j was
synthesized as shown in Scheme 5. Reaction of compound 5
with 2-bromo-1,1-diethoxyethane gave alkylated product 16 in
72% yield. The obtained compound 16 was heated with phenol
in the presence of potassium carbonate in NMP to afford
phenoxy diethylacetal derivative (17) in excellent yield. After
deprotecting the diethylacetal moiety with trifluoroacetic acid,
reductive amination of the obtained ethanediol derivative (18)
with 2-hydroxyethylamine provided 2-{[2-(4-phenoxy-5H-
pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]amino}ethanol (19) in
good yield. Finally, compound 19 was coupled with aniline 4j
using pyridinium chloride as an acidic promoter in phenol to
afford the desired product 20 in 19% yield. The low yield was
due to formation of tricyclic compound 21, resulting from
intramolecular cyclization of molecule 19, as the major product.
The pyrrolo[3,2-d]pyrimidine derivatives 28−30, possessing
a sulfur atom-containing side chain, were synthesized as shown
in Scheme 6. The alkylbromide (25) was prepared from
2-iodoethyl benzoate derivative (22) and 2-mercaptoethanol
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a
Scheme 5. Synthesis of a N-5 Hydroxyethylaminoethyl Pyrrolo[3,2-d]pyrimidine Derivative 20
a
Reagents: (a) 2-bromo-1,1-diethoxyethane, Cs2CO3, DMF, 80 °C, 4.5 h, 72%; (b) PhOH, K2CO3, NMP, 140 °C, 6 h, 95%; (c) TFA, CH2Cl2, rt,
16 h, 91%; (d) 2-aminoethanol, NaB(OAc)3H, AcOH, DMF, rt, 16 h, 52%; (e) 4j, pyridinium chloride, phenol, 140 °C, 16 h, 19%.
a
Scheme 6. Synthesis of a N-5 Sulfur-Containing Pyrrolo[3,2-d]pyrimidine Derivatives (28, 29, and 30)
a
Reagents: (a) iso-Pr2EtN, 40 °C, 3 days, 77%; (b) PPh3, CBr4, rt, 3 days, 76%; (c) 5, Cs2CO3, DMF, rt, 15 h, 80%; (d) 4j, 2-PrOH, 80 °C, 15 h;
(e) 1N NaOH, MeOH, rt, 1 h, 59% for 2 steps; (f) mCPBA, CH2Cl2, −78 °C, 1 h, 85%; (g) tert-BuO2H, Ti(OisoPr)4, MeOH, H2O, rt, 2 days, 74%.
derivative (23), which were coupled in the presence of
ethyldiisopropylamine to give the thioether derivative (24) in
77% yield. Bromination of compound 24 with carbon
tetrabromide and triphenylphosphine provided the desired
alkylbromide (25) in 76% yield. The obtained alkylbromide
derivative (25) was introduced into the N-5 position of
compound 5 in the usual manner to afford adduct 26 in 80%
yield. Compound 26 was reacted with aniline derivative 4j in
2-propanol followed by hydrolysis of the benzoyl ester with 1N
aqueous NaOH to afford the thioether derivative 28 in 59% yield
in 2 steps. The sulfoxide (29) was obtained by oxidization of
compound 28 with 3-chloroperbenzoic acid (mCPBA) in 85%
yield. Further oxidization of sulfoxide (29) with 70% aqueous
tert-butyl hydroperoxide in the presence of titanium tetraisoprop-
oxide provided its sulfone derivative (30) in 74% yield.
compound 31a with aniline 4j was carried out in 2-propanol,
followed by deprotecting the terminal Boc group of the
obtained molecule 32a with 2N aqueous hydrochloric acid
(HCl) to afford key intermediate (33a) in 85% yield in 2 steps.
Amidation of the key intermediate 33a with a variety of carboxylic
acids in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide hydrochloride (water-soluble carbodiimide,
WSC) and 1-hydroxybenzotriazole monohydrate (HOBt)
provided the corresponding desired carboxamides (34a, 34c,
and 34e, Scheme 7). Carboxamide 34d was isolated a
methanesulfonic acid salt. The synthesis of carboxamide 34b
was achieved using 2-[(tert-butoxycarbonyl)(methyl)amino]-
ethyl methanesulfonate as an N-5 alkylating agent in a similar
manner, shown in Scheme 8.
Next, we examined the introduction of representative alkyl
groups at the C-6 position in 12j. The synthesis of the C-6
methyl derivative 40 is shown in Scheme 9. The Sonogashira
cross-coupling reaction of 4-iodo-6-phenoxypyrimidine-5-
amine (35) with trimethyl(prop-1-yn-1-yl)silane gave acetylene
Preparation of the N-5 carboxamide derivatives 34a−e has
been summarized in Schemes 7 and 8. Alkylation of compound
5 with tert-butyl (2-bromoethyl)carbamate in the usual manner
gave intermediate (31a) in 71% yield. Condensation of
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a
Scheme 7. Synthesis of a N-5 Carboxamido Pyrrolo[3,2-d]pyrimidine Derivatives (34a and 34c−34e)
a
Reagents: (a) tert-butyl (2-bromoethyl)carbamate, Cs2CO3, DMF, 40 °C, 4 days, 71%; (b) 4j, 2-PrOH, 80 °C, 12 h, 85%; (c) 2N HCl, THF, 60 °C,
20 h, quant; (d) WSC, HOBt, Et3N, carboxylic acid, DMF, 3 days, 74−86%; (e) MsOH, AcOEt, rt, 2 h, 25% from 33a.
a
Scheme 8. Synthesis of a N-5 Carboxamido Pyrrolo[3,2-d]pyrimidine Derivative 34b
a
Reagents: (a) 2-[(tert-butoxycarbonyl)(methyl)amino]ethyl methanesulfonate, Cs2CO3, DMF, 40 °C, 4 days, 33%; (b) 4j, 2-PrOH, 80 °C, 12 h,
76%; (c) 2N HCl, THF, 60 °C, 20 h, 91%; (d) WSC, HOBt, Et3N, hydroxyacetic acid, DMF, 3 days, 62%.
derivative 36 in 73% yield. Intramolecular cyclization of
compound 36 with sodium tert-butoxide afforded 6-methyl-4-
phenoxy-5H-pyrrolo[3,2-d]pyrimidine (37) in 67% yield. The
obtained molecule (37) was reacted with 2-{2-[(methylsulfonyl)-
oxy]ethoxy}ethyl benzoate in the usual manner to afford adduct
38 in 93% yield. Reaction of compound 38 with aniline 4j
followed by hydrolysis of the benzoyl ester with 1N aqueous
NaOH afforded the desired product 40 in good yield.
Scheme 10 shows the preparation of the 6-cyano derivative
49. The Sonogashira reaction of compound 35 with 3,3-
diethoxyprop-1-yne, followed by intramolecular cyclization
using sodium tert-butoxide, provided intermediate 42. After
deprotecting the diethylacetal group in compound 42 with 1N
aqueous HCl, the aldehyde group of the resulting product 43
was oxidized with sodium chlorite to provide carboxylic acid
(44) in good yield. Conversion of compound 44 into the acid
chloride using thionyl chloride was followed by reaction with
ammonia to afford carboxamide (45) in 92% yield. Treatment
of compound 45 with phosphorus oxychloride gave carbonitrile
(46) in 61% yield. The obtained product 46 led to the desired
6-cyano pyrrolo[3,2-d]pyrimidine derivative (49) in a manner
similar to that shown in Scheme 9.
BIOLOGICAL RESULTS AND DISCUSSION
■
To examine the potential of a pyrrolo[3,2-d]pyrimidine scaffold
for use as a HER2/EGFR kinase inhibitor, a variety of
substituted 4-anilino derivatives (6a−q) were evaluated. Table 1
shows their HER2/EGFR kinase and tumor cell growth (BT-474,
HER2-overexpressing human breast cancer cell line) inhibitory
activities as well as their in vitro human metabolic stability.
Almost all of the derivatives showed good HER2/EGFR
inhibitory activities with IC50 values less than 1 μM, especially
against HER2. In addition, the reported anilino derivatives 6o
and 6p showed HER2/EGFR dual inhibitory activity, and
derivative 6q showed HER2-selective activity similar to those of
quinazoline analogues.16 These results suggested that the
N-1/N-3 nitrogen atoms of the pyrrolo[3,2-d]pyrimidine scaffold
can interact with the Met801 and Thr862 residues in the
hinge region of the HER2 ATP-binding pocket and that the
4-anilino group fits into the lipophilic back pocket as
expected by our designs. All anilino substituents at the C-2 or
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a
Scheme 9. Synthesis of a C-6 Methyl Pyrrolo[3,2-d]pyrimidine Derivative 40
a
Reagents: (a) trimethyl(prop-1-yn-1-yl)silane, Pd(PPh3)2Cl2, CuI, KF, DMF, Et3N, 60 °C, 16 h, 73%; (b) tert-BuOK, THF, 0 °C, 40 min, 67%;
(c) 2-{2-[(methylsulfonyl)oxy]ethoxy}ethyl benzoate, K2CO3, DMF, 60 °C, 21 h, 93%; (d) 4j, pyridinium chloride, phenol, 140 °C, 17 h, 58%;
(e) 1N NaOH, MeOH, rt, 2 h, 90%.
a
Scheme 10. Synthesis of a C-6 Cyano Pyrrolo[3,2-d]pyrimidine Derivative 49
a
Reagents: (a) 3,3-diethoxyprop-1-yne, Pd(PPh3)2Cl2, CuI, MeCN, Et3N, rt, 16.5 h, 89%; (b) tert-BuOK, THF, rt, 1.5 h, 51%; (c) 1N HCl, THF, rt,
2 h, 90%; (d) NaClO2, NaH2PO4, DMSO, H2O, rt, 2 h, quant; (e) (i) SOCl2, 75 °C, 2 h, (ii) NH3, THF, 0 °C, 92%; (f) POCl3, 70 °C, 1.5 h, 61%;
(g) 2-{2-[(methylsulfonyl)oxy]ethoxy}ethyl benzoate, K2CO3, DMF, 60 °C, 7 h, 61%; (h) 4j, pyridinium chloride, phenol, 140 °C, 17 h, 74%;
(i) 1N NaOH, MeOH, rt, 1 h, 61%.
C-3 positions of the phenoxy ring gave rise to better than 130
nM HER2 inhibitory activity, although the cyano group at
the C-4 position in 6h resulted in reduced HER2/EGFR
inhibitory activity. Reflecting their potent inhibitory activities,
especially against HER2, a variety of compounds (6b, 6e, 6f, 6g,
6j, 6l, and 6m) showed good growth inhibitory (GI) activity
against BT-474 cells with GI50 values of 480−1100 nM,
comparable to those of the reported anilino derivatives 6o−q.
Among them, we selected the 3-chloro (6e), 3-trifluoromethyl
(6j), and 3-trifluoromethoxy (6l) anilino derivatives because
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Table 1. Biological Data for C-4-Anilino Substituted Pyrrolo[3,2-d]pyrimidine Derivatives
metabolic
a
ab
,
a
enzyme
IC50 (nM) (95% CI)
cell growth
stability
GI50 (nM) (95% CI)
BT-474
μL/min/mg
compd
R1
HER2
EGFR
human
6a
6b
6c
6d
6e
6f
H
16 (13−19)
9.3 (7.3−12)
20 (15−26)
11 (7.2−17)
8.3 (6.4−11)
8.0 (5.9−11)
8.2 (6.1−11)
720 (590−880)
120 (110−130)
30 (23−39)
55 (40−76)
41(31−56)
4.6 (4−5.3)
26 (19−36)
13 (9.8−17)
22 (16−30)
60 (49−73)
110 (93−130)
61 (41−90)
150 (94−250)
27 (18−42)
35 (23−51)
51 (43−60)
150 (84−280)
590 (460−740)
150 (110−220)
67 (40−110)
210 (110−390)
220 (130−370)
20 (13−31)
290 (220−400)
7.7 (5−12)
18 (12−27)
869 (730−1034)
737 (601−903)
1267 (1070−1501)
1413 (1181−1692)
459 (385−544)
956 (835−1094)
787 (548−1131)
4473 (3795−5279)
2328 (1918−2828)
875 (750−1020)
2543 (2198−2944)
1063 (905−1250)
762 (605−959)
122
87
56
66
20
97
42
2-F
3-F
2-Cl
3-Cl
2-CN
3-CN
4-CN
2-CF3
3-CF3
2-OCF3
3-OCF3
3-CH3
3-OCH3
6g
6h
6i
6j
7
22
6k
6l
15
6m
6n
6o
6p
6q
187
150
36
1216 (1067−1386)
650 (426−994)
1160 (890−1510)
2049 (1806−2324)
23
1900 (1300−2800)
b
70
a
These measurements were showed in the Experimental Section. IC50 values and 95% confidence intervals (CI) were calculated by nonlinear
regression analysis of the percentage inhibitions (n = 2).
these compounds showed good in vitro oxidative metabolic
stability against human hepatic microsomes.
Considering the in vivo oxidative metabolism in the ether
linkage of N-5 2-(2-hydroxyethoxy)ethyl moiety in 12j, we
focused on replacement of its ether oxygen with a carbon,
nitrogen, and sulfur atom. Furthermore, to investigate
substituent effects at the C-6 position of the pyrrolo[3,2-
d]pyrimidine scaffold, we introduced representative methyl
(40) and cyano groups (49) to 12j. These results are shown in
Table 3. Similar to the 2-(2-hydroxyethoxy)ethyl moiety in 12j,
replacement of its ether oxygen with carbon (15), nitrogen
(20), and sulfur (28−30) maintained good HER2/EGFR
kinase inhibitory activities. On the other hand, these derivatives
showed reduced GI activity compared with 12j, although polar
linkages such as sulfoxide (29) and sulfone (30) moieties improved
the human metabolic stability. We believed that log D at pH 7.4
would be an important factor for enhancing their GI activity. In
addition, introduction of the methyl (40) and cyano (49) group at
the C-6 position led to a decrease of the cellular GI activities.
With the aim of pursuing compatibility between cellular
activity and metabolic stability, we envisioned that replacement
of the ether with a carboxamide linkage could provide a good
profile with favorable polarity for our purpose (Table 4). These
prepared derivatives (34a−e) were found to show potent
HER2/EGFR inhibitory activities. Although the 2-hydroxyacet-
amide derivatives 34a,b (log D at pH 7.4: 3.45−3.51) showed
slightly reduced cellular activity, higher lipophilicity, compara-
ble to that of 12j, could be achieved by the addition of a methyl
or methylene group to the side chain to recover cellular GI
activity (34c−e, log D at pH 7.4: 3.54−4.18).
Next, with the aim of further enhancing their cellular activity,
we examined the introduction of N-5 substituents into the
pyrrolo[3,2-d]pyrimidine scaffold. As representative N-5 alkyl
substituents, methyl (8), 2-hydroxyethyl (10), and 2-(2-
hydroxyethoxy)ethyl (12) moieties were selected and their
corresponding derivatives with a 3-chloro (e), 3-trifluoromethyl
(j), or 3-trifluoromethoxy (l) anilino group were evaluated.
These results are summarized in Table 2. Introduction of
these substituents at the N-5 position maintained the potent
HER2/EGFR inhibitory activities. This result was consistent
with our design that N-5 chemical modifications would be
directed toward the solvent contact region of the HER2/EGFR
kinases. On the other hand, their cellular activity had a
tendency to be higher when the molecule had a longer side
chain. Among the prepared compounds, the 2-(2-
hydroxyethoxy)ethyl derivatives 12e−l showed significantly
more potent cell growth inhibitory activity than those of the
corresponding derivatives 8 and 10. In addition, compounds
12e−l showed acceptable metabolic stability and PK profiles in
mice. On the basis of its potent GI activity and PK profile in
mice, we selected the 3-trifluoromethyl derivative 12j as lead
compound for further optimization. Compared with 12j, the
pyridine analogue 12r reduced EGFR inhibitory activity together
with cellular GI activity. As cocrystal structure20 of 12r with
HER2 was successfully obtained, we focused on structural
discussions of EGFR.
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Table 2. Biological Data for N5- and C4-Substituted Pyrrolo[3,2-d]pyrimidine Derivatives
metabolic
a
ab
,
a
enzyme
IC50 (nM) (95% CI)
cell growth
stability
PK
c
GI50 (nM) (95% CI)
BT-474
μL/min/mg
AUC0−8 h μg·h/mL
compd
X
R1
Cl
R2
HER2
EGFR
mice
34
human
37
mice
8e
8j
8l
CH
CH
CH
CH3
3.3 (2.6−4.3)
11 (8.9−14)
17 (12−25)
9.2 (5.9−14)
27 (20−37)
50 (36−69)
184 (142−232)
692 (565−846)
1884 (1750−2028)
CF3
−13
−9
14
1
4.545
OCF3
10e
10j
10l
CH
CH
CH
Cl
2- hydroxy ethyl
4.1 (3.5−4.7)
12 (11−14)
29 (23−38)
9.5 (5.9−15)
34 (26−44)
110 (68−180)
97 (81−115)
212 (179−249)
582 (481−704)
28
9
23
35
16
1.882
6.381
CF3
OCF3
11.546
12e
CH
Cl
2-(2- hydroxy
ethoxy) ethyl
2.1 (1.8−2.5)
5.7 (4.1−8)
54 (46−63)
41
73
2.633
12j
12l
12r
CH
CH
N
CF3
5.1 (4.1−6.3)
8.4 (5.8−12)
11 (7.8−15)
15 (11−21)
49 (31−78)
420 (170−1000)
b
27 (21−33)
116 (100−132)
2170 (1978−2382)
39
11
64
63
53
96
3.324
3.342
3.996
OCF3
CF3
a
These measurements were showed in the Experimental Section. IC50 values and 95% confidence intervals (CI) were calculated by nonlinear
c
regression analysis of the percentage inhibitions (n = 2). The animals used in the study were female BALB/cAJcl mice (7-weeks old; CLEA Japan,
Inc.). A mixture of 5 test compounds was suspended in 0.5% (w/v) methylcellulose solution for oral administration at a dose of 10 mg each/10 mL/kg.
The concentrations of compounds in the plasma were determined by LC/MS/MS.
Table 3. Biological Data for C6-, N5-, and C4-Substituted Pyrrolo[3,2-d]pyrimidine Derivatives
metabolic
ab
,
a
a
enzyme
IC50 (nM) (95% CI)
cell growth
stability
PK
c
GI50 (nM) (95% CI)
μL/min/mg
AUC0−8h μg·h/mL
log D
compd
X
Y
HER2
EGFR
BT-474
mice
39
human
63
mice
pH 7.4
12j
15
20
28
29
30
40
49
O
H
5.1 (4.1−6.3)
15 (12−19)
7.7 (5.3−11)
9.4 (7.4−12)
12 (11−15)
12 (10−14)
12 (10−15)
19 (12−29)
15 (11−21)
33 (25−45)
22 (15−32)
44 (26−73)
31 (25−40)
40 (32−49)
38 (26−56)
230 (75−690)
27 (21−33)
3.324
0.856
2.476
0.284
2.272
2.080
1.607
4.425
3.86
4.90
3.50
4.77
2.97
3.41
4.28
3.62
CH2
NH
S
H
406 (337−487)
601 (561−643)
699 (604−807)
1378 (1284−1479)
1076 (991−1168)
69 (37−105)
16
59
34
45
88
17
17
30
34
H
H
163
SO
SO2
O
H
H
CH3
CN
77
28
O
1143 (1025−1274)
a
b
These measurements were showed in the Experimental Section. IC50 values and 95% confidence intervals (CI) were calculated by nonlinear
c
regression analysis of the percentage inhibitions (n = 2). The animals used in the study were female BALB/cAJcl mice (7-weeks old; CLEA Japan,
Inc.). A mixture of 5 test compounds was suspended in 0.5% (w/v) methylcellulose solution for oral administration at a dose of 10 mg each/10 mL/kg.
The concentrations of compounds in the plasma were determined by LC/MS/MS.
Compounds 34c−e, together with 12j, were selected for in vivo
efficacy studies in a HER2-overexpressing BT-474 tumor xenograft
mouse model (100 mg/kg, twice daily, orally for 14 days). Among
these compounds, 34e and 12j exhibited significant in vivo
efficacy (tumor/control ratio [T/C]): 29% and 48%, respectively,)
without body weight loss (data not shown). On the basis of its
potent in vivo efficacy and good in vitro profiles, 34e was selected
as a candidate for further investigation.
As shown in Figures 3 and 4, 34e was evaluated using 4−1ST
(HER2-overexpressing human gastric cancer tumor) xenograft
models in mice and rats. Similar to the BT-474 model, 34e
exhibited dose-dependent tumor growth inhibition (T/C: 44%
and 11% at 50 and 100 mg/kg, twice daily, respectively)
without significant body weight loss in mice (Figure 3).
Furthermore, 34e showed potent in vivo efficacy in rats in a
dose-dependent manner and inhibited the growth of 4−1ST
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Table 4. Biological Data for N5- and C4-Substituted Pyrrolo[3,2-d]pyrimidine Derivatives
a
b
These measurements were showed in the Experimental Section. IC50 values and 95% confidence intervals (CI) were calculated by nonlinear
c
regression analysis of the percentage inhibitions (n = 2). The animals used in the study were female BALB/cAJcl mice (7-weeks old; CLEA Japan,
Inc.). A mixture of 5 test compounds was suspended in 0.5% (w/v) methylcellulose solution for oral administration at a dose of 10 mg each/10 mL/kg.
d
The concentrations of compounds in the plasma were determined by LC/MS/MS. Compounds suspended in a 0.5% (w/v) methylcellulose solution
was administered orally twice daily (100 mg/kg) to the BT-474 xenograft mice for 14 days.
tumors with T/C of 38% and 14% at doses of 6.25 and 12.5
mg/kg and, particularly noteworthy, tumor regression with
T/C of −12% and −16% at doses of 25 and 50 mg/kg (Figure 4).
During the study, 34e caused no significant body weight loss in
rats (data not shown).
To clarify the potent efficacy of 34e, we investigated the
pharmacokinetic profile of 34e in mice and rats, which is shown
in Table 5. The oral bioavailability of 34e was 97.7% in rats and
72.2% in mice at a dose of 50 mg/kg. In addition, we found that
the concentration of 34e in the tumor was much higher than
that in the plasma of rats (Figure 5). Consequently, we
concluded that the potent efficacy of 34e was achieved by a
combination of good PK profiles and high concentration levels
of 34e in the tumor. These results suggested that 34e is a
promising candidate for further evaluation.
The inhibitory activity of 34e against various kinases is
summarized in Table 6. Besides HER2 and EGFR, 34e ex-
hibited HER4 inhibitory activity with an IC50 value of 260 nM.
With these results, we confirmed that 34e is a selective HER
family inhibitor.
Structural Analysis and Discussion for Compound 34e
and 12r. The crystal structures of a pyridine analogue 12r in
complex with HER2 and 34e in complex with EGFR were
described previously by Aertgeerts et al.20 Although the re-
solution is still low (3.21 Å), we also determined the cocrystal
structure of 34e with HER221 and a cartoon presentation is
shown in Figure 6A. This is the first time that crystal structures
have been reported for both HER2 and EGFR in complex with
the same inhibitor. From the two cocrystal structures of 34e
(Figure 6A,B), we confirmed that the N-1 nitrogen of the
pyrrolo[3,2-d]pyrimidine scaffold was hydrogen-bonded to the
main chain NH of the hinge region (Met801 in HER2 or
Met793 in EGFR), whereas the N-3 nitrogen formed a water-
mediated hydrogen bond network only with the side chain of
Thr854 in EGFR. In the case of 34e with HER2, this hydrogen
bond network by the N-3 nitrogen was not observed, probably
because of a moderate resolution level. The bulky 3-
trifluoromethyl phenyl group of 34e was not accommodated
in the active form of both kinases and likely displaced the
C-helix away from its position in the active conformation.
Compared with the crystal structure of the pyridine analogue
12r with HER2 (2.25 Å, Figure 6C), 34e showed similar
interactions to that of 12r, which formed a water-mediated
hydrogen bond network with the side chain of Thr862. In
Figure 3. Antitumor efficacy of 34e in 4−1ST xenograft models in
mice. Dose levels 50 and 100 mg/kg; P ≤ 0.025 vs control at day 14
(one-tailed Shirley−Williams test).
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Figure 5. Concentration of 34e in tumor (μg/g) and plasma (μg/mL)
in rats.
Table 6. Kinase Selectivity of 34e
enzyme
IC50 (nM)
enzyme
Lck
IC50 (nM)
EGFR (HER1)
HER2
23
17
2400
4700
CSK
HER4
260
FAK
>10000
>10000
5200
MEK1
1100
Lyn A
MEK5
5700
Lyn B
c-Met
4200
ASK 1
TAK 1
MEKK 1
IKK bata
JNK 1
ERK 1
P38 alpha
PKA
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
Aurora B
VEGFR 1
VEGFR 2
FGFR1
FGFR 3
PDGFR α
PDGFR β
IRK
1700
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
Figure 4. Antitumor efficacy of 34e in 4−1ST xenograft models in
rats. (A) Dose levels 6.25 mg/kg and 12.5 mg/kg; P ≤ 0.025 vs control
at day 14 (one-tailed Shirley−Williams test). (B) Dose levels 25 and
50 mg/kg; P ≤ 0.025 vs control at day 14 (one-tailed Shirley−Williams
test).
PKC thete
GSK3 beta
B-raf
TIE2
c-kit
IGF-1R
Src
TTK
addition, the 4-anilino groups of 34e and 12r occupied the
same hydrophobic pocket formed by the side chain of amino
acids near the DFG motif, and a remarkable difference was
observed at the conformation of 3-trifluoromethyl phenyl
group. The 3-trifluoromethyl group in 12r was flipped by
approximately 150° relative to its orientation in both the
HER2/34e and EGFR/34e structures. In the costructure of 12r
with HER2, the nitrogen in the pyridine ring interacted with
the main chain of Asp863 in the DFG motif. Furthermore,
hydroxy group of R2 side chain of 12r was also hydrogen
bonded with the side chain of Asp863. As a result, the
conformation of the DFG motif in the HER2/12r was slightly
different from those of the HER2/34e and EGFR/34e
structures, which was considered to lead to the flip of
PLK 1
3-trifluoromethyl group. This difference may contribute to
the difference between HER2 and EGFR inhibitory activity in
12r (Table 2). It was noted that HER2/34e and EGFR/34e
structures were inactive conformations with a DFG-in and C-
helix-out conformation similar to that of the EGFR/Lapatinib
structure (PDB 1XKK).
The N-5-substituents containing a carboxamide moiety of
34e is suitably positioned at the solvent interface for both
HER2 and EGFR with small conformational differences. Thus,
a dramatic difference between the HER2 and EGFR cocrystal
structures of 34e was not observed. From the structural
Table 5. Pharmacokinetic Parameters of 34e in Rats and Mice
IV (5 mg/kg)
PO (50 mg/kg)
a
b
c
d
e
b
f
C5 min (μg/mL)
AUC0−24h (μg·h/mL)
MRT (h)
Cmax (μg/mL)
Tmax (h)
AUC0−24 h (μg·h/mL)
BA (%)
rat
2.277
4.060
5.63
3.39
0.96
4.157
4.364
4.00
0.50
55.03
97.7
72.2
mouse
2.72
19.64
a
b
c
Plasma concentration at 5 min after dosing. Area under the plasma concentration−time curve for 0−24 h after dosing. Mean residual time of 34e
d
e
f
in the plasma. Maximum plasma concentration after oral dosing. Time to reach Cmax. Bioavailability.
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and EGFR inhibitory activities. On the basis of results
from N-5 side chain optimization studies, we selected 34e for
further evaluation. From the X-ray cocrystal structures of
34e with both HER2 and EGFR, we confirmed that the
pyrrolo[3,2-d]pyrimidine scaffold fits into the ATP pocket
and that the N-1 nitrogen interact with Met801 of HER2 or
Met793 of EGFR. In a further investigation, 34e exhibited
potent and selective kinase inhibitory profiles against the HER
family as well as good cellular GI activity. Reflecting its
desirable cellular activity, metabolic stability, and good PK
profiles, 34e showed potent in vivo antitumor efficacy in mice
and rats. These data combined with our preliminary studies
revealing that the HER2 inhibitory activity of 34e compares
favorably to that of lapatinib11 and is superior to that of
erlotinib6 support the position that compound 34e is a
promising candidate for clinical development as a dual
HER2/EGFR inhibitor. Compound 34e (TAK-285) is
currently in phase 1 clinical trials.
EXPERIMENTAL SECTION
■
Melting points were determined on a Yanagimoto micro melting point
apparatus or SRS OptiMelt melting point apparatus and are
uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra
were recorded on a Varian Gemini-200 (200 MHz) spectrometer or
Varian Mercury-300 (300 MHz) spectrometer. Chemical shifts are
given in parts per million (ppm) with tetramethylsilane as an internal
standard, and coupling constants (J values) are given in hertz (Hz).
Splitting patterns and apparent multiplicities are designated as s
(singlet), d (doublet), dd (double doublet), t (triplet), dt (double
triplet), q (quartet), m (multiplet), br s (broad singlet). Elemental
analyses were carried out by Takeda Analytical Research Laboratories,
Ltd., and the results obtained were within ±0.4% of the theoretical
values. MS spectra were collected with a Waters LC-MS system
(ZMD-1) and were used to confirm ≥95% purity of each compound.
The column used was an L-column 2 ODS (3.0 mm × 50 mm I.D.,
CERI, Japan) with a temperature of 40 °C and a flow rate of 1.2 mL/
min. Mobile phase A was 0.05% TFA in ultrapure water. Mobile phase
B was 0.05% TFA in acetonitrile, which was increased linearly from 5%
to 90% over 2 min, 90% over the next 1.5 min, after which the column
was equilibrated to 5% for 0.5 min.
Column chromatography was carried out on a silica gel column
(Kieselgel 60, 63−200 mesh, Merck or Chromatorex NH-DM1020,
100−200 mesh, Fuji Silysia Chemical, Ltd., Japan). Yields were not
optimized.
3-Chloro-4-phenoxyaniline (4a). To a mixture of 2-chloro-1-
fluoro-4-nitrobenzene (1, 1.75 g, 10.0 mmol) and potassium carbonate
(2.07 g, 10.0 mmol) in N,N-dimethylformamide (DMF) (20 mL) was
added phenol (2a, 941 mg, 10.0 mmol). After being stirred at 80 °C
for 2 h, water (40 mL) was added to the mixture and the mixture was
extracted with ethyl acetate (EtOAc). The organic layer was washed
with saturated brine (30 mL), dried over anhydrous magnesium sulfate
(MgSO4), and concentrated in vacuo to give 3a (2.32 g) as brown oil.
To a solution of 3a (2.32 g, 9.31 mmol) in EtOAc (50 mL) was added
5% platinum−carbon (Pt−C) (200 mg), and the mixture was stirred
under hydrogen atmosphere at room temperature for 2 h. After 5%
Pt−C was filtered off, the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography (eluent,
EtOAc:hexane = 1:9 to 5:5) to give 1.96 g (89%) of 4a as brown oil.
1H NMR (CDCl3) δ 3.65 (br s, 2H), 6.56 (dd, 1H, J = 8.6, 2.7 Hz),
6.79 (d, 1H, J = 2.7 Hz), 6.84−6.95 (m, 3H), 6.98−7.06 (m, 1H),
7.19−7.35 (m, 2H).
Figure 6. X-ray cocrystal structures of compound 34e (TAK-285) with
HER2 (A) and EGFR (B), together with that of compound 12r with
HER2 (C).20
similarities found for the binding mode of 34e to the HER2 and
EGFR kinases, we confirmed that 34e is an ATP-competitive
HER2/EGFR dual inhibitor.
The following compounds (4b−4e, 4i−4n) were prepared from
1 and the corresponding phenols (2b−2e, 2i−2n) by a method similar
to that described for 4a.
CONCLUSION
■
We designed pyrrolo[3,2-d]pyrimidine derivatives and modified
them chemically to develop novel HER2/EGFR TKIs. As
expected, the prepared derivatives showed good HER2
3-Chloro-4-(2-fluorophenoxy)aniline (4b). Yield 89%, brown
oil. 1H NMR (CDCl3) δ 3.65 (br s, 2H), 6.55 (dd, 1H, J = 8.6, 2.7 Hz),
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6.70−6.82 (m, 2H), 6.86 (d, 1H, J = 8.6 Hz), 6.96−7.04 (m, 2H), 7.10−
7.21 (m, 1H).
added 2,3-dichloro-5-nitropyridine (0.50 g, 2.61 mmol). The reaction
mixture was stirred at room temperature for 2.5 h. Water (100 mL)
was added to the reaction mixture, and then the mixture was extracted
with EtOAc (200 mL). The organic layer was washed with saturated
brine (50 mL), dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography (eluent, EtOAc:hex-
ane = 1:9 to 3:1) to give colorless oil. To a suspension of the oil and
reduced iron (0.65 g, 11.6 mmol) in a mixture of EtOH (19.5 mL) and
water (3.5 mL) was added calcium chloride (0.13 g, 1.17 mmol), and
the mixture was stirred at 80 °C for 8 h. The solid was removed by
filtration, and filtrate was poured into water (100 mL). The organic
layer was washed with saturated brine (100 mL), dried over MgSO4,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (eluent, EtOAc:hexane = 1:4 to 1:1) to give
0.29 g (39%) of 4r as brown oil. 1H NMR (CDCl3) δ 3.65 (br s, 2H),
7.20 (d, 1H, J = 2.9 Hz), 7.22−7.26 (m, 1H), 7.27−7.32 (m, 1H),
7.37−7.40 (m, 1H), 7.44−7.50 (m, 1H), 7.59 (d, 1H, J = 2.9 Hz).
N-(3-Chloro-4-phenoxyphenyl)-5H-pyrrolo[3,2-d]pyrimidin-
4-amine (6a). A solution of 4a (264 mg, 1.20 mmol) and 4-chloro-
5H-pyrrolo[3,2-d]pyrimidine18 (5, 154 mg, 1.00 mmol) in 1-methyl-2-
pyrrolidone (3.0 mL) was stirred with heating at 100 °C for 3 h. After
cooling to room temperature, the reaction mixture was diluted with
EtOAc (80 mL) and partitioned with saturated sodium hydrogen
carbonate (30 mL). The organic layer was washed with brine (30 mL),
dried over MgSO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (eluent, MeOH:EtOAc =
0:1 to 5:95) to give 105 mg (31%) of 6a as colorless crystals, mp
247−250 °C. 1H NMR (DMSO-d6) δ 6.51 (br s, 1H), 6.94 (d, 2H, J =
7.9 Hz), 7.10 (t, 1H, J = 7.3 Hz), 7.20 (d, 1H, J = 8.9 Hz), 7.31−7.45
(m, 2H), 7.63−7.73 (m, 2H), 8.32−8.43 (m, 2H), 9.47 (br s, 1H),
11.17 (br s, 1H). Anal. Calcd for C18H13ClN4O: C, 64.19; H, 3.89; N,
16.64. Found: C, 63.81; H, 3.91; N, 16.66.
The following compounds (6b−6q) were prepared from corre-
sponding 4b−4q and 5 by a method similar to that described for 6a.
N-[3-Chloro-4-(2-fluorophenoxy)phenyl]-5H-pyrrolo[3,2-d]-
pyrimidin-4-amine (6b). Yield 51%, colorless crystals, mp 282−
285 °C. 1H NMR (DMSO-d6) δ 6.50 (dd, 1H, J = 2.9, 2.0 Hz), 6.90−
7.03 (m, 1H), 7.09−7.25 (m, 3H), 7.33−7.49 (m, 1H), 7.55−7.73
(m, 2H), 8.39 (s, 2H), 9.41 (br s, 1H), 11.08 (br s, 1H). Anal. Calcd
for C18H12ClFN4O: C, 60.94; H, 3.41; N, 15.79. Found: C, 61.03; H,
3.43; N, 15.65.
N-[3-Chloro-4-(3-fluorophenoxy)phenyl]-5H-pyrrolo[3,2-d]-
pyrimidin-4-amine (6c). Yield 74%, colorless crystals, mp 239−
241 °C. 1H NMR (DMSO-d6) δ 6.46−6.54 (m, 1H), 6.70−6.85 (m, 2H),
6.89−7.02 (m, 1H), 7.29 (d, 1H, J = 8.9 Hz), 7.34−7.45 (m, 1H),
7.62−7.76 (m, 2H), 8.36−8.43 (m, 2H), 9.47 (s, 1H), 11.11 (br s,
1H). Anal. Calcd for C18H12ClFN4O: C, 60.94; H, 3.41; N, 15.79.
Found: C, 61.00; H, 3.41; N, 15.81.
3-Chloro-4-(3-fluorophenoxy)aniline (4c). Yield 76%, white
1
solid. H NMR (CDCl3) δ 3.69 (br s, 2H), 6.50−6.81 (m, 5H), 6.92
(d, 1H, J = 8.7 Hz), 7.14−7.28 (m, 1H).
3-Chloro-4-(2-chlorophenoxy)aniline (4d). Yield 97%, brown
oil. 1H NMR (CDCl3) δ 3.69 (br s, 2H), 6.56 (dd, 1H, J = 8.7, 2.8 Hz),
6.68 (dd, 1H, J = 8.0, 1.5 Hz), 6.79 (d, 1H, J = 2.8 Hz), 6.86 (d, 1H, J =
8.7 Hz), 6.94−7.04 (m, 1H), 7.07−7.17 (m, 1H), 7.42 (dd, 1H, J = 8.0,
1.5 Hz).
3-Chloro-4-(3-chlorophenoxy)aniline (4e). Yield 65%, white
solid. 1H NMR (CDCl3) δ 3.70 (br s, 2H), 6.58 (dd, 1H, J = 8.6, 2.7 Hz),
6.73−7.04 (m, 5H), 7.19 (t, 1H, J = 8.6 Hz).
3-Chloro-4-[2-(trifluoromethyl)phenoxy]aniline (4i). Yield
84%, yellow oil. 1H NMR (CDCl3) δ 3.70 (br s, 2H), 6.53−6.69
(m, 2H), 6.79 (d, 1H, J = 2.6 Hz), 6.92 (d, 1H, J = 8.7 Hz), 7.07 (t,
1H, J = 7.6 Hz), 7.34−7.42 (m, 1H), 7.64 (dd, 1H, J = 7.6, 0.9 Hz).
3-Chloro-4-[3-(trifluoromethyl)phenoxy]aniline (4j). Yield
1
72%, white solid. H NMR (CDCl3) δ 3.71 (br s, 2H), 6.59 (dd,
1H, J = 8.6, 2.7 Hz), 6.80 (d, 1H, J = 2.7 Hz), 6.92 (d, 1H, J = 8.6 Hz),
7.03 (dd, 1H, J = 8.0, 2.1 Hz), 7.10 (s, 1H), 7.23−7.32 (m, 1H), 7.38
(t, 1H, J = 8.0 Hz).
3-Chloro-4-[2-(trifluoromethoxy)phenoxy]aniline (4k). Yield
96%, brown oil. 1H NMR (CDCl3) δ 3.68 (br s, 2H), 6.57 (dd, 1H, J =
8.7, 2.6 Hz), 6.69 (dd, 1H, J = 8.2, 1.6 Hz), 6.79 (d, 1H, J = 2.6 Hz),
6.87 (d, 1H, J = 8.7 Hz), 6.97−7.08 (m, 1H), 7.11−7.20 (m, 1H),
7.28−7.35 (m, 1H).
3-Chloro-4-[3-(trifluoromethoxy)phenoxy]aniline (4l). Yield
1
61%, white solid. H NMR (CDCl3) δ 3.70 (br s, 2H), 6.59 (dd,
1H, J = 8.7, 2.8 Hz), 6.73 (s, 1H), 6.76−6.96 (m, 4H), 7.23−7.33 (m,
1H).
3-Chloro-4-(3-methylphenoxy)aniline (4m). Yield 92%, white
solid. 1H NMR (CDCl3) δ 2.31 (s, 3H), 3.65 (br s, 2H), 6.56 (dd, 1H,
J = 8.7, 2.6 Hz), 6.63−6.93 (m, 5H), 7.16 (t, 1H, J = 7.8 Hz).
3-Chloro-4-(3-methoxyphenoxy)aniline (4n). Yield 99%,
brown oil. 1H NMR (CDCl3) δ 3.66 (br s, 2H), 3.77 (s, 3H),
6.42−6.50 (m, 2H), 6.53−6.61 (m, 2H), 6.78 (d, 1H, J = 2.6 Hz), 6.91
(d, 1H, J = 8.7 Hz), 7.17 (t, 1H, J = 8.1 Hz).
2-(4-Amino-2-chlorophenoxy)benzonitrile (4f). To a mixture
of 1 (4.40 g, 184 mmol) and potassium carbonate (5.20 g, 221 mmol)
in DMF (60 mL) was added 2-hydroxybenzonitrile (2f, 3.00 g,
257 mmol). After being stirred at 100 °C for 2 h, water (300 mL) was
added to the mixture under ice-cooling and the mixture was stirred at
room temperature for 10 min. The resulting precipitate was collected
by filtration. The precipitate was washed with n-hexane and dried
under vacuum to give a yellow cake. To a suspension of the cake and
reduced iron (8.80 g) in EtOH (90 mL) was added a solution of
calcium chloride (1.50 g, 257 mmol) in water (10 mL), and the
reaction mixture was stirred at 100 °C for 15 h. The solid was removed
by filtration, and the resulting filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography (eluent,
EtOAc:hexane = 2:8 to 10:0) to give 3.50 g (65%) of 4f as brown oil.
1H NMR (CDCl3) δ 3.78 (br s, 2H), 6.58−6.65 (m, 2H), 6.76 (d, 1H,
J = 2.7 Hz), 6.97 (d, 1H, J = 8.4 Hz), 7.04−7.10 (m, 1H), 7.39−7.45
(m, 1H), 7.62−7.65 (m, 1H).
N-[3-Chloro-4-(2-chlorophenoxy)phenyl]-5H-pyrrolo[3,2-d]-
pyrimidin-4-amine (6d). Yield 33%, colorless crystals, mp 251−
1
254 °C. H NMR (DMSO-d6) δ 6.51 (dd, 1H, J = 2.8, 2.1 Hz), 6.88
(dd, 1H, J = 8.2, 1.4 Hz), 7.09−7.21 (m, 2H), 7.28−7.37 (m, 1H),
7.56−7.74 (m, 3H), 8.40 (s, 2H), 9.44 (br s, 1H), 11.09 (br s, 1H).
Anal. Calcd for C18H12Cl2N4O: C, 58.24; H, 3.26; N, 15.09. Found: C,
58.31; H, 3.32; N, 14.98.
N-[3-Chloro-4-(3-chlorophenoxy)phenyl]-5H-pyrrolo[3,2-d]-
pyrimidin-4-amine (6e). Yield 71%, colorless crystals, mp 229−
231 °C. 1H NMR (DMSO-d6) δ 6.51 (dd, 1H, J = 2.8, 2.2 Hz), 6.84−
6.95 (m, 1H), 6.99 (t, 1H, J = 2.2 Hz), 7.17 (dd, 1H, J = 8.0, 1.1 Hz),
7.30 (d, 1H, J = 8.9 Hz), 7.40 (t, 1H, J = 8.0 Hz), 7.65−7.75 (m, 2H),
8.32−8.42 (m, 2H), 9.47 (br s, 1H), 11.11 (br s, 1H). Anal. Calcd for
C18H12Cl2N4O: C, 58.24; H, 3.26; N, 15.09. Found: C, 58.25; H, 3.22;
N, 15.17.
The following compounds (4g,4h) were prepared from 1 and the
corresponding phenols (4g,4h) by a method similar to that described
for 4f.
3-(4-Amino-2-chlorophenoxy)benzonitrile (4g). Yield 71%,
yellow oil. 1H NMR (CDCl3) δ 3.75 (br s, 2H), 6.58−6.62 (m,
1H), 6.79 (d, 1H, J = 2.7 Hz), 6.92 (d, 1H, J = 8.4 Hz), 7.05−7.16 (m,
2H), 7.26−7.41 (m, 2H).
4-(4-Amino-2-chlorophenoxy)benzonitrile (4h). Yield 56%,
1
2-[2-Chloro-4-(5H-pyrrolo[3,2-d]pyrimidin-4-ylamino)-
beige solid. H NMR (CDCl3) δ 3.76 (br s, 2H), 6.60 (dd, 1H, J =
phenoxy]benzonitrile (6f). Yield 61%, yellow crystals, mp 264−
8.6, 2.7 Hz), 6.79 (d, 1H, J = 2.7 Hz), 6.84−7.00 (m, 3H), 7.51−7.62
(m, 2H).
1
266 °C. H NMR (DMSO-d6) δ 6.50 (s, 1H, s), 6.85 (d, 1H, J = 8.4
Hz), 7.22−7.60 (m, 2H), 7.65−7.74 (m, 3H), 7.88 (d, 1H, J = 7.8 Hz),
8.40 (s, 2H), 9.50 (br s, 1H), 11.09 (br s, 1H). Anal. Calcd for
C19H12ClN5O: C, 63.08; H, 3.34; N, 19.36. Found: C, 63.10; H, 3.25;
N, 19.15.
5-Chloro-6-[3-(trifluoromethyl)phenoxy]pyridin-3-amine
(4r). To a solution of 2j (0.42 g, 2.59 mmol) in THF (8.0 mL) was
added NaH (60% dispersion in mineral oil, 0.11 g, 2.75 mmol) under
ice-cooling. After being stirred for 1 h, to the reaction mixture was
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3-[2-Chloro-4-(5H-pyrrolo[3,2-d]pyrimidin-4-ylamino)-
phenoxy]benzonitrile (6g). Yield 71%, yellow crystals, mp 273−
N-{3-Methyl-4-[(6-methylpyridin-3-yl)oxy]phenyl}-5H-
pyrrolo[3,2-d]pyrimidin-4-amine (6q). Yield 66%, yellow crystals,
mp 111−113 °C. H NMR (CDCl3) δ: 2.16 (s, 3H), 2.51 (s, 3H),
1
1
275 °C. H NMR (DMSO-d6) δ 6.53 (s, 1H), 7.26 (m, 1H), 7.32 (d,
1H, J = 8.7 Hz), 7.45 (s, 1H), 7.58 (d, 2H, J = 5.7 Hz), 7.70−7.73 (m,
2H), 8.41 (s, 2H), 9.50 (br s, 1H), 11.10 (br s, 1H). Anal. Calcd for
C19H12ClN5O: C, 63.08; H, 3.34; N, 19.36. Found: C, 62.97; H, 3.30;
N, 19.48.
6.56 (d, 1H, J = 3.0 Hz), 6.80 (d, 1H, J = 9.0 Hz), 7.0−7.6 (m, 5H), 8
17 (m, 1H), 8.59 (s, 1H), 8.76 (br s, 1H), 11.08 (br s, 1H). Anal.
Calcd for C19H17N5O·0.2H2O: C, 68.13; H, 5.24; N, 20.91. Found: C,
68.18; H, 5.22; N, 20.82.
4-[2-Chloro-4-(5H-pyrrolo[3,2-d]pyrimidin-4-ylamino)-
phenoxy]benzonitrile (6h). Yield 48%, yellow crystals, mp 318 °C.
1H NMR (DMSO-d6) δ 6.52 (dd, 1H, J = 3.0, 1.9 Hz), 6.99−7.18 (m,
2H), 7.37 (d, 1H, J = 8.9 Hz), 7.68−7.79 (m, 2H), 7.79−7.90 (m,
2H), 8.41 (s, 2H), 9.55 (br s, 1H), 11.15 (br s, 1H). Anal. Calcd for
C19H12ClN5O·0.3H2O: C, 62.15; H, 3.46; N, 19.07. Found: C, 62.25;
H, 3.49; N, 18.84.
N-{3-Chloro-4-[2-(trifluoromethyl)phenoxy]phenyl}-5H-
pyrrolo[3,2-d]pyrimidin-4-amine (6i). Yield 69%, colorless crystals,
mp 273−276 °C. 1H NMR (DMSO-d6) 6.52 (dd, 1H, J = 3.0, 1.9 Hz),
6.83 (d, 1H, J = 8.5 Hz), 7.19−7.33 (m, 2H), 7.56−7.83 (m, 4H), 8.41
(s, 2H), 9.48 (br s, 1H), 11.10 (br s, 1H). Anal. Calcd for
C19H12ClF3N4O: C, 56.38; H, 2.99; N, 13.84. Found: C, 56.58; H,
2.97; N, 13.98.
4-Chloro-5-methyl-5H-pyrrolo[3,2-d]pyrimidine (7). To a
suspension of 5 (0.32 g, 2.09 mmol) and potassium carbonate (0.45 g,
3.27 mmol) in DMF (2.0 mL) was added iodomethane (0.44 g,
3.13 mmol), and the reaction mixture was stirred at room temperature
for 3 h. The reaction mixture was poured into water (25 mL), and
extracted with EtOAc (90 mL). The organic layer was washed with
brine (60 mL), dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography (eluent, EtOAc:hex-
1
ane = 1:4 to 9:1) to give 0.33 g (93%) of 7 as yellow solid. H NMR
(CDCl3) δ 4.16 (s, 3H), 6.70 (d, 1H, J = 3.9 Hz), 7.42 (d, 1H, J = 3.9
Hz), 8.69 (s, 1H).
N-[3-Chloro-4-(3-chlorophenoxy)phenyl]-5-methyl-5H-
pyrrolo[3,2-d]pyrimidin-4-amine Hydrochloride (8e). A solu-
tion of 4e (100 mg, 0.597 mmol) and 7 (227 mg, 0.893 mmol) in 1-
methyl-2-pyrrolidone (1.19 mL) was stirred at 140 °C for 4 h. After
cooling to room temperature, the reaction mixture was diluted with
EtOAc (30 mL). The resulting crystals were collected by filtration,
washed with EtOAc (10 mL), and dried under vacuum to give 231 mg
(92%) of 8e as colorless hydrochloride salt crystals, mp 168−170 °C.
1H NMR (DMSO-d6) δ 4.31 (d, 3H, J = 4.0 Hz), 6.65 (dd, 1H, J = 3.0,
1.0 Hz), 6.95 (dd, 1H, J = 9.0, 2.0 Hz), 7.03 (m, 1H), 7.22 (m, 1H),
7.34 (d, 1H, J = 9.0 Hz), 7.44 (t, 1H, J = 9.0 Hz), 7.69 (m, 1H), 7.97
(m, 2H), 8.72 (s, 1H), 9.94 (br s, 1H). Anal. Calcd for
C19H15Cl3N4O·0.5H2O: C, 52.98; H, 3.74; N, 13.01. Found: C,
53.00; H, 3.76; N, 13.26.
The following compounds (8j,8l) were prepared from correspond-
ing 4j,4l with 7 by a method similar to that described for 8e.
N-{3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-5-methyl-
5H-pyrrolo[3,2-d]pyrimidin-4-amine hydrochloride (8j). Yield
75%, colorless crystals, mp 175−177 °C. 1H NMR (DMSO-d6) δ
4.31 (s, 3H), 6.64 (dd, 1H, J = 3.0, 1.0 Hz), 7.26 (m, 2H),
7.37 (d, 1H, J = 9.0 Hz), 7.51 (d, 1H, J = 8.0 Hz), 7.67 (m, 2H), 7.96
(m, 2H), 8.71 (s, 1H), 9.89 (br s, 1H). Anal. Calcd for
C20H15Cl2F3N4O·0.5H2O: C, 51.74; H, 3.47; N, 12.07. Found: C,
52.20; H, 3.37; N, 12.25.
N-{3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-5H-
pyrrolo[3,2-d]pyrimidin-4-amine (6j). Yield 62%, colorless crys-
1
tals, mp 258−260 °C. H NMR (DMSO-d6) δ 6.42−6.56 (m, 1H),
7.15−7.27 (m, 2H), 7.33 (d, 1H, J = 8.9 Hz), 7.47 (d, 1H, J = 8.0 Hz),
7.61 (t, 1H, J = 8.0 Hz), 7.66−7.78 (m, 2H), 8.41 (s, 2H), 9.48 (br s,
1H), 11.11 (br s, 1H). Anal. Calcd for C19H12ClF3N4O: C, 56.38; H,
2.99; N, 13.84. Found: C, 56.34; H, 2.95; N, 13.91.
N-{3-Chloro-4-[2-(trifluoromethoxy)phenoxy]phenyl}-5H-
pyrrolo[3,2-d]pyrimidin-4-amine (6k). Yield 63%, colorless crys-
tals, mp 228−230 °C. 1H NMR (DMSO-d6) 6.51 (dd, 1H, J = 2.9, 2.0
Hz), 6.90 (dd, 1H, J = 8.3, 1.5 Hz), 7.15−7.26 (m, 2H), 7.32−7.43 (m,
1H), 7.49−7.57 (m, 1H), 7.63−7.77 (m, 2H), 8.35−8.48 (m, 2H),
9.46 (br s, 1H), 11.10 (br s, 1H). Anal. Calcd for C19H12ClF3N4O2: C,
54.23; H, 2.87; N, 13.32. Found: C, 54.43; H, 3.01; N, 13.13.
N-{3-Chloro-4-[3-(trifluoromethoxy)phenoxy]phenyl}-5H-
pyrrolo[3,2-d]pyrimidin-4-amine (6l). Yield 63%, colorless crys-
1
tals, mp 234−236 °C. H NMR (DMSO-d6) δ 6.52 (dd, 1H, J = 3.0,
1.9 Hz), 6.90−6.97 (m, 2H), 7.07−7.15 (m, 1H), 7.32 (d, 1H, J =
8.9 Hz), 7.49 (t, 1H, J = 8.9 Hz), 7.67−7.75 (m, 2H), 8.37−8.43 (m,
2H), 9.47 (br s, 1H), 11.10 (br s, 1H). Anal. Calcd for
C19H12ClF3N4O2: C, 54.23; H, 2.87; N, 13.32. Found: C, 54.23; H,
2.83; N, 13.33.
N-{3-Chloro-4-[3-(trifluoromethoxy)phenoxy]phenyl}-5-
methyl-5H-pyrrolo[3,2-d]pyrimidin-4-amine Hydrochloride
(8l). Yield 77%, colorless crystals, mp 169−171 °C. 1H NMR
(DMSO-d6) δ 4.30 (s, 3H), 6.63 (dd, 1H, J = 3.0, 2.0 Hz), 6.97 (m,
2H), 7.15 (d, 1H, J = 9.0 Hz), 7.35 (d, 1H, J = 9.0 Hz), 7.53 (t, 1H, J =
9.0 Hz), 7.68 (m, 1H), 7.96 (m, 2H), 8.70 (s, 1H), 9.87 (br s, 1H).
Anal. Calcd for C20H15Cl2F3N4O2: C, 50.97; H, 3.21; N, 11.89. Found:
C, 50.99; H, 3.13; N, 11.84.
N-[3-Chloro-4-(3-methylphenoxy)phenyl]-5H-pyrrolo[3,2-d]-
pyrimidin-4-amine (6m). Yield 70%, colorless crystals, mp 232−
1
234 °C. H NMR (DMSO-d6) δ 2.29 (s, 3H), 6.51 (dd, 1H, J = 2.9,
2.0 Hz), 6.67−6.81 (m, 2H), 6.92 (d, 1H, J = 7.5 Hz), 7.12−7.31 (m,
2H), 7.56−7.74 (m, 2H), 8.28−8.44 (m, 2H), 9.42 (br s, 1H), 11.10
(br s, 1H). Anal. Calcd for C19H15ClN4O: C, 65.05; H, 4.31; N, 15.97.
Found: C, 64.88; H, 4.36; N, 15.84.
N-[3-Chloro-4-(3-methoxyphenoxy)phenyl]-5H-pyrrolo[3,2-d]-
2-(4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl Benzoate
(9). To a suspension of 5 (0.31 g, 2.01 mmol) and cesium carbonate
(0.98 g, 5.08 mmol) in DMF (2.0 mL) was added 2-iodoethyl
benzoate (1.45 g, 5.25 mmol), and the reaction mixture was stirred at
room temperature for 15 h. The reaction mixture was poured into
saturated sodium hydrogen carbonate (100 mL) and extracted with
EtOAc (150 mL × 3). The organic layer was washed with brine
(100 mL) and dried over MgSO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography (eluent, EtOAc:hex-
pyrimidin-4-amine (6n). Yield 83%, colorless crystals, mp 204−
1
207 °C. H NMR (DMSO-d6) δ 3.74 (s, 3H), 6.38−6.56 (m, 3H),
6.69 (dd, 1H, J = 8.2, 2.0 Hz), 7.13−7.34 (m, 2H), 7.58−7.74 (m,
2H), 8.32−8.43 (m, 2H), 9.42 (br s, 1H), 11.10 (br s, 1H). Anal.
Calcd for C19H15ClN4O2: C, 62.21; H, 4.12; N, 15.27. Found: C,
62.11; H, 4.07; N, 15.33.
N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-5H-pyrrolo[3,2-
d]pyrimidin-4-amine (6o). Yield 80%, colorless crystals, mp 269−
1
270 °C. H NMR (CDCl3) δ: 5.15 (s, 2H), 6.52 (d, 1H, J = 3.0 Hz),
1
ane = 6:4 to 95:5) to give 0.42 g (70%) of 9 as white solid. H NMR
6.90−7.70 (m, 7H), 7.97 (d, 1H, J = 3.0 Hz), 8.44 (s, 1H), 8.84 (br s,
1H), 10.74 (br s, 1H). Anal. Calcd for C19H14ClFN4O: C, 61.88; H,
3.83; N, 15.19. Found: C, 61.83; H, 3.89; N, 15.08.
(CDCl3) δ4.71 (t, 2H, J = 5.3 Hz), 4.91 (t, 2H, J = 5.3 Hz), 6.76 (d,
1H, J = 3.2 Hz), 7.35−7.67 (m, 4H), 7.84−7.97 (m, 2H), 8.73 (s, 1H).
2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-5H-
pyrrolo[3,2-d]pyrimidin-5-yl)ethanol (10e). A solution of 9 (100 mg,
0.331 mmol) and 4e (126 mg, 0.496 mmol) in 1-methyl-2-pyrrolidone
(0.66 mL) was stirred at 140 °C for 2.5 h. After cooling to room
temperature, to the reaction mixture was added saturated sodium
hydrogen carbonate (80 mL), and the mixture was extracted with
EtOAc (80 mL). The organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was purified by silica gel column
N-[3-Chloro-4-(pyridin-2-ylmethoxy)phenyl]-5H-pyrrolo[3,2-
d]pyrimidin-4-amine (6p). Yield 78%, yellow crystals, mp 236−238
1
°C. H NMR (DMSO-d6) δ 5.27 (s, 2H), 6.48 (d, 1H, J = 2.4 Hz),
7.25 (d, 1H, J = 8.7 Hz), 7.37 (dd, 1H, J = 7.5, 5.1 Hz), 7.55−7.60 (m,
2H), 7.66 (s, 1H), 7.89 (t, 1H, J = 7.5 Hz), 8.20 (dd, 1H, J = 2.4, 1.5
Hz), 8.35 (d, 1H, J = 1.5 Hz), 8.60 (dd, 1H, J = 4.8, 0.6 Hz), 9.25 (br
s, 1H), 12.78 (br s, 1H). Anal. Calcd for C18H14ClN5O: C, 61.46; H,
4.01; N, 19.91. Found: C, 61.20; H, 4.04; N, 19.66.
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chromatography (eluent, EtOAc:hexane = 1:9 to 1:0) to give white
solid. To a solution of the solid in a mixture of MeOH (1.18 mL) and
THF (1.18 mL) was added 1 N NaOH (0.27 mL), and the mixture
was stirred at room temperature for 1.5 h. To the reaction mixture was
added 1 N HCl (0.27 mL), and the mixture was extracted with a
mixture of EtOAc (2.0 mL) and THF (1.8 mL). The organic layer was
washed with brine (30 mL), dried over MgSO4, and concentrated in
vacuo. The crystallized residue was recrystallized from EtOAc/
diisopropyl ether (2:1) to give 81 mg (72%) of 10e as colorless
crystals, mp 208−209 °C. 1H NMR (DMSO-d6) δ 3.87 (m, 2H), 4.53
(t, 2H, J = 4.5 Hz), 6.31 (br s, 1H,), 6.51 (d, 1H, J = 3.0 Hz), 6.88 (d,
1H, J = 9.0 Hz), 6.95 (s, 1H), 7.15 (d, 1H, J = 9.0 Hz), 7.28 (d, 1H, J =
9.0 Hz), 7.38 (t, 1H, J = 9.0 Hz), 7.60 (dd, 1H, J = 9.0, 2.0 Hz), 7.66
(d, 1H, J = 3.0 Hz), 7.97 (d, 1H, J = 2.0 Hz), 8.34 (s, 1H), 9.89 (br s,
1H). Anal. Calcd for C20H16Cl2N4O2: C, 57.84; H, 3.88; N, 13.49.
Found: C, 57.75; H, 3.66; N, 13.48.
C23H20ClF3N4O3: C, 56.05; H, 4.09; N, 11.37. Found: C, 56.05; H,
4.09; N, 11.28.
2-{2-[4-({3-Chloro-4-[3-(trifluoromethoxy)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}ethanol (12l). Yield
58%, colorless crystals, mp 148−150 °C. 1H NMR (CDCl3) δ 1.95 (br
s, 1H), 3.71−3.84 (m, 4H), 4.03 (t, 2H, J = 4.5 Hz), 4.57 (t, 2H, J =
4.5 Hz), 6.62 (d, 1H, J = 3.2 Hz), 6.80−6.95 (m, 3H), 7.08 (d, 1H, J =
8.8 Hz), 7.21 (d, 1H, J = 3.2 Hz), 7.30 (t, 1H, J = 8.2 Hz), 7.62 (dd,
1H, J = 8.8, 2.6 Hz), 7.90 (d, 1H, J = 2.6 Hz), 8.52 (s, 1H), 8.82 (br s,
1H). Anal. Calcd for C23H20ClF3N4O4: C, 54.29; H, 3.96; N, 11.01.
Found: C, 54.49; H, 3.93; N, 10.96.
2-{2-[4-({5-Chloro-6-[3-(trifluoromethyl)phenoxy]pyridin-3-
yl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}ethanol
(12r). Yield 58%, yellow crystals, mp 171−172 °C. 1H NMR (CDCl3)
δ 3.69−3.80 (m, 4H), 4.00−4.04 (m, 2H), 4.54−4.59 (m, 2H), 6.65
(d, 1H, J = 3.3 Hz), 7.23 (d, 1H, J = 3.3 Hz), 7.31−7.36 (m, 1H),
7.40−7.55 (m, 3H), 8.24 (d, 1H, J = 2.7 Hz), 8.47 (d, 1H, J = 2.7 Hz),
8.51 (s, 1H), 8.83 (s, 1H). Anal. Calcd for C22H19ClF3N5O3: C, 53.50;
H, 3.88; N, 14.18. Found: C, 53.47; H, 3.80; N, 14.08.
The following compounds (10j,10l) were prepared from 9,11 with
corresponding anilines (4e,4j,4l) by a method similar to that described
for 10e.
5-(4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)pentyl Acetate
(13). To a suspension of 5 (0.50 g, 3.26 mmol) and cesium carbonate
(1.59 g, 4.88 mmol) in DMF (5.0 mL) was added 5-chloropentyl
acetate (0.71 mL, 3.26 mmol) at 40 °C, and the reaction mixture was
stirred at 40 °C for 4 days. Water (150 mL) was added to the reaction
mixture, and the mixture was extracted with EtOAc (200 mL). The
organic layer was washed with brine (50 mL), dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica gel
chromatography (eluent, EtOAc:hexane = 1:3 to 3:2) to give 0.64 g
(69%) of 13 as white solid. 1H NMR (CDCl3) δ: 1.33−1.46 (m, 2H),
1.61−1.72 (m, 2H), 1.84−1.97 (m, 2H), 2.04 (s, 3H), 4.05 (t, 2H, J =
6.6 Hz), 4.48 (t, 2H, J = 7.5 Hz), 6.71 (d, 1H, J = 3.3 Hz), 7.46 (d, 1H,
J = 3.3 Hz), 8.69 (s, 1H).
5-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]pentan-1-ol (15). A
mixture of 13 (0.20 g, 0.71 mmol) and 4j (0.27 g, 0.94 mmol) in 2-
propanol (3.5 mL) was stirred at 80 °C for 14 h. After cooling to 0 °C,
to the reaction mixture was added 1 N NaOH (2.1 mL), and the
mixture was stirred at room temperature for 1 h. To the reaction
mixture was added 1 N HCl (2.1 mL) under ice-cooling, and the
mixture was extracted with EtOAc (150 mL). The organic layer was
washed with brine (50 mL), dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(eluent, MeOH:EtOAc = 0:1 to 1:19) to give 0.28 g (79%) of 15 as
colorless crystals, mp 146−148 °C. 1H NMR (CDCl3) δ: 1.35 (t, 1H,
J = 4.7 Hz), 1.50−1.69 (m, 4H), 1.92−2.05 (m, 2H), 3.63−3.71 (m,
2H), 4.32 (t, 2H, J = 7.4 Hz), 6.59 (d, 1H, J = 3.3 Hz), 6.70 (s, 1H),
7.08 (d, 1H, J = 8.7 Hz), 7.09−7.12 (m, 1H), 7.15−7.27 (m, 2H),
7.30−7.35 (m, 1H), 7.40−7.43 (m, 1H), 7.47 (dd, 1H, J = 8.7, 2.7
Hz), 7.82 (d, 1H, J = 2.7 Hz), 8.53 (s, 1H). Anal. Calcd for
C24H22ClF3N4O2: C, 58.72; H, 4.52; N, 11.41. Found: C, 58.73; H,
4.53; N, 11.40.
4-Chloro-5-(2,2-diethoxyethyl)-5H-pyrrolo[3,2-d]pyrimidine
(16). To a suspension of 5 (1.00 g, 6.51 mmol) and cesium carbonate
(6.37 g, 19.5 mmol) in DMF (13 mL) was added 2-bromo-1,1-
diethoxyethane (2.9 mL, 19.5 mmol) at room temperature, and the
reaction mixture was stirred at 80 °C for 4.5 h. After cooling to room
temperature, the reaction mixture was diluted with ethyl acetate
(100 mL) and washed with water (80 mL). The organic layer was
separated, dried over MgSO4 and concentrated in vacuo. The residue
was purified by silica gel chromatography (eluent, EtOAc:hexane = 1:1
to 1:0) to give 1.26 g (72%) of 16 as pale-yellow oil. 1H NMR
(CDCl3) δ 1.14 (t, 6H, J = 6.0 Hz), 3.40 (m, 2H), 3.72 (m, 2H), 4.08
(m, 1H), 4.56 (d, 2H, J = 5.0 Hz), 6.71 (d, 1H, J = 3.0 Hz), 7.55
(d, 1H, J = 3.0 Hz), 8.69 (s, 1H).
5-(2,2-Diethoxyethyl)-4-phenoxy-5H-pyrrolo[3,2-d]-
pyrimidine (17). A mixture of 16 (1.00 g, 3.71 mmol), phenol (0.42
g, 4.46 mmol), potassium carbonate (0.62 g, 4.46 mmol) in 1-methyl-
2-pyrrolidone (6.7 mL) was stirred at 140 °C for 6 h. After cooling to
room temperature, the reaction mixture was diluted with EtOAc (100
mL) and washed with water (80 mL). The organic layer was separated,
dried over MgSO4, and concentrated in vacuo. The residue was
2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethanol (10j). Yield
1
46%, colorless crystals, mp 193−194 °C. H NMR (CDCl3) δ 4.16
(t, 2H, J = 4.4 Hz), 4.38 (t, 2H, J = 4.4 Hz), 6.12 (d, 1H, J = 3.2 Hz),
6.97 (d, 1H, J = 3.2 Hz), 7.09 (d, 1H, J = 8.8 Hz), 7.10−7.17 (m, 1H),
7.21 (s, 1H), 7.32 (d, 1H, J = 7.7 Hz), 7.43 (t, 1H, J = 8.0 Hz), 7.52
(dd, 1H, J = 8.8, 2.6 Hz), 7.84 (d, 1H, J = 2.6 Hz), 8.24 (s, 1H), 9.59
(br s, 1H). Anal. Calcd for C21H16ClF3N4O2: C, 56.20; H, 3.59; N,
12.48. Found: C, 56.20; H, 3.54; N, 12.49.
2-[4-({3-Chloro-4-[3-(trifluoromethoxy)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethanol (10l). Yield
71%, colorless crystals, mp 168−170 °C. 1H NMR (CDCl3) δ
4.09−4.19 (m, 2H), 4.30−4.38 (m, 2H), 6.05 (d, 1H, J = 3.2 Hz),
6.79−6.97 (m, 5H), 7.10 (d, 1H, J = 8.9 Hz), 7.32 (t, 1H, J = 8.3 Hz),
7.51 (dd, 1H, J = 8.8, 2.5 Hz), 7.83 (d, 1H, J = 2.6 Hz), 8.18 (s, 1H),
9.67 (br s, 1H). Anal. Calcd for C21H16ClF3N4O3: C, 54.26; H, 3.47;
N, 12.05. Found: C, 54.56; H, 3.55; N, 12.06.
2-[2-(4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethoxy]ethyl
Benzoate (11). To a suspension of 5 (0.66 g, 4.31 mmol) and
cesium carbonate (3.13 g, 9.61 mmol) in DMF (5.0 mL) was added 2-
(2-iodoethoxy)ethyl benzoate (1.45 g, 4.53 mmol), and the reaction
mixture was stirred at room temperature for 15 h. The reaction
mixture was poured into saturated sodium hydrogen carbonate (100
mL) and extracted with EtOAc (450 mL). The organic layer was
washed with brine (100 mL) and dried over MgSO4, and concentrated
in vacuo. The residue was purified by silica gel chromatography
(eluent, EtOAc:hexane = 1:19 to 2:3) to give 0.82 g (55%) of 11 as
1
colorless oil. H NMR (CDCl3) δ3.71 (dt, 2H, J = 6.6, 3.0 Hz), 3.89
(t, 2H, J = 5.1 Hz), 4.41 (dt, 2H, J = 6.6, 3.0 Hz), 4.68 (t, 2H, J = 5.1
Hz), 6.56 (d, 1H, J = 3.3 Hz), 7.40−7.46 (m, 2H), 7.54−7.60 (m, 2H),
7.94−7.98 (m, 2H), 8.66 (s, 1H).
The following compounds (12e−12l) were prepared from 11 with
corresponding anilines (4e,4j,4l) by a method similar to that described
for 10e.
2-[2-(4-{[3-Chloro-4-(3-chlorophenoxy)phenyl]amino}-5H-
pyrrolo[3,2-d]pyrimidin-5-yl)ethoxy]ethanol (12e). Yield 68%,
1
colorless crystals, mp 135−137 °C. H NMR (CDCl3) δ 2.05 (br s,
1H), 3.71−3.84 (m, 4H), 4.03 (t, 2H, J = 4.5 Hz), 4.57 (t, 2H, J = 4.5
Hz), 6.61 (d, 1H, J = 3.0 Hz), 6.83−6.88 (m, 1H), 6.92 (t, 1H, J = 2.2
Hz), 7.01−7.06 (m, 1H), 7.06 (d, 1H, J = 8.9 Hz), 7.19−7.27 (m, 2H),
7.61 (dd, 1H, J = 8.9, 2.6 Hz), 7.89 (d, 1H, J = 2.6 Hz), 8.52 (s, 1H),
8.82 (br s, 1H). Anal. Calcd for C22H20Cl2N4O3: C, 57.53; H, 4.39; N,
12.20. Found: C, 57.28; H, 4.37; N, 12.16.
2-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}amino)-
5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}ethanol (12j). Yield
1
57%, colorless crystals, mp 130−132 °C. H NMR (CDCl3) δ 1.94
(br s, 1H), 3.71−3.85 (m, 4H), 4.03 (t, 2H, J = 4.4 Hz), 4.57 (t, 2H,
J = 4.4 Hz), 6.63 (d, 1H, J = 3.2 Hz), 7.07 (d, 1H, J = 8.9 Hz), 7.08−
7.14 (m, 1H), 7.19 (s, 1H), 7.22 (d, 1H, J = 3.2 Hz), 7.31 (d, 1H, J =
7.7 Hz), 7.42 (t, 1H, J = 8.0 Hz), 7.63 (dd, 1H, J = 8.9, 2.6 Hz), 7.91
(d, 1H, J = 3.0 Hz), 8.52 (s, 1H), 8.83 (br s, 1H). Anal. Calcd for
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purified by silica gel column chromatography (eluent, EtOAc:hexane =
1:9 to 6:4) to give 1.15 g (95%) of 17 as yellow oil. 1H NMR (CDCl3)
δ 1.13 (t, 6H, J = 7.0 Hz), 3.40 (m, 2H), 3.69 (m, 2H), 4.51 (d, 2H,
J = 6.0 Hz), 4.76 (t, 1H, J = 6.0 Hz), 6.65 (d, 1H, J = 3.0 Hz), 7.20−
7.50 (m, 6H), 8.45 (s, 1H).
2-(4-Phenoxy-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethane-1,1-
diol (18). A solution of 17 (1.10 g, 3.36 mmol) in a mixture of
dichloromethane (4.5 mL) and trifluoroacetic acid (4.5 mL) was
stirred at room temperature for 16 h. The reaction mixture was con-
centrated in vacuo, and the residue was dissolved in EtOAc (100 mL).
The solution was washed with saturated sodium hydrogen carbonate
(80 mL). The organic layer was dried over MgSO4 and concentrated
in vacuo to give 0.83 g (91%) of 18 as white solid. 1H NMR (DMSO-
d6) δ 4.35 (d, 2H, J = 6.0 Hz), 5.17 (t, 1H, J = 6.0 Hz), 6.14 (d, 2H, J =
6.0 Hz), 6.59 (d, 1H, J = 3.0 Hz), 7.20−7.60 (m, 5H), 7.75 (d, 1H, J =
3.0 Hz), 8.28 (s, 1H).
2-{[2-(4-Phenoxy-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]-
amino}ethanol (19). A solution of 18 (0.50 g, 1.84 mmol) and
2-hydroxyethylamine (167 mg, 2.73 mmol) in a mixture of DMF
(29 mL) and acetic acid (2.9 mL) was stirred at room temperature for
1.5 h. To the reaction mixture was added sodium triacetoxyborohy-
dride (579 mg, 2.73 mmol), and the mixture was stirred at room
temperature for 16 h. The reaction mixture was concentrated in vacuo,
and the residue was purified by silica gel column chromatography
(eluent, MeOH:EtOAc = 0:1 to 3:7) to give 286 mg (52%) of 19 as
gel. 1H NMR (CDCl3) δ 2.80 (t, 2H, J = 5.0 Hz), 3.19 (t, 2H, J = 6.0
Hz), 3.63 (t, 2H, J = 5.0 Hz), 4.59 (t, 2H, J = 6.0 Hz), 6.68 (d, 1H, J =
3.0 Hz), 7.20−7.35 (m, 3H), 7.40−7.51 (m, 3H), 8.46 (s, 1H).
2-({2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}amino)ethanol
(20). A mixture of 19 (154 mg, 0.516 mmol), 4j (223 mg, 0.775
mmol), pyridinium chloride (179 mg, 1.55 mmol), and phenol (396
mg, 4.21 mmol) was heated at 140 °C for 16 h. After cooling to room
temperature, the reaction mixture was diluted with dichloromethane
(40 mL), and washed with saturated sodium hydrogen carbonate (30
mL). The organic layer was separated, dried over MgSO4, and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (eluent, EtOAc:methanol = 1:0 to 7:3) to give 48 mg
(19%) of 20 as colorless crystals, mp 188−189 °C. 1H NMR (DMSO-
d6) δ 2.48 (m, 2H), 3.05 (m, 2H), 3.34 (m, 2H), 4.45 (m, 2H), 4.56
(t, 1H, J = 5.0 Hz), 6.49 (d, 1H, J = 3.0 Hz), 7.20 (m, 2H), 7.29 (d,
1H, J = 9.0 Hz), 7.45 (d, 1H, J = 8.0 Hz), 7.66 (m, 3H, m), 8.05 (d,
1H, J = 3.0 Hz), 8.32 (s, 1H). Anal. Calcd for C23H21ClF3N5O2: C,
56.16; H, 4.30; N, 14.24. Found: C, 56.35; H, 4.30; N, 14.03.
2-(5,6-Dihydro-4H-pyrrolo[3,2,1-de]pteridin-4-yl)ethanol
(21). 21 was obtained as a main product (29 mg, 28%) in this
reaction. 1H NMR (CDCl3) δ 3.78 (t, 2H, J = 4.0 Hz), 3.84 (t, 2H, J =
5.0 Hz), 3.94 (t, 2H, J = 4.0 Hz), 4.26 (t, 2H, J = 5.0 Hz), 6.49 (d, 1H,
J = 3.0 Hz), 7.14 (d, 1H, J = 3.0 Hz), 8.35 (s, 1H). LC-MS 205.09
(MH).
2-[(2-Hydroxyethyl)thio]ethyl benzoate (24). A solution of
2-iodoethyl benzoate (22, 6.00 g, 21.7 mmol), 2-sulfanylethanol (23,
1.5 mL, 22.0 mmol), and ethyldiisopropylamine (4.5 mL) in DMF
(60 mL) was stirred at 40 °C for 3 days. Water (300 mL) was added
to the reaction mixture, and the mixture was extracted with EtOAc
(300 mL). The organic layer was washed with brine (50 mL), dried
over MgSO4, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (eluent, EtOAc:hexane = 1:4 to 7:3)
to give 3.77 g (77%) of 24 as orange oil. 1H NMR (CDCl3) δ: 2.15 (t,
1H, J = 6.0 Hz), 2.83 (t, 2H, J = 5.9 Hz), 2.92 (t, 2H, J = 6.8 Hz), 3.79
(dt, 2H, J = 6.0, 6.0 Hz), 4.50 (t, 2H, J = 6.8 Hz), 7.43−7.48 (m, 2H),
7.55−7.61 (m, 1H), 8.03−8.08 (m, 2H).
2-[(2-Bromoethyl)thio]ethyl Benzoate (25). To a solution of
24 (1.00 g, 4.42 mmol) and carbon tetrabromide (2.78 g, 8.38 mmol)
in THF (30 mL) was added dropwise a solution of triphenylphosphine
(2.20 g, 8.38 mmol) in THF (10 mL) under ice-cooling. After stirring
at room temperature for 3 days, water (200 mL) was added, and then
the mixture was extracted with EtOAc (300 mL). The organic layer
was washed with brine (50 mL), dried over MgSO4, and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (eluent, EtOAc:hexane = 1:9 to 4:6) to give 0.98 g
(76%) of 25 as colorless oil. 1H NMR (CDCl3) δ: 2.95 (t, 2H, J = 6.8
Hz), 3.02−3.08 (m, 2H), 3.50−3.56 (m, 2H), 4.49 (t, 2H, J = 6.8 Hz),
7.43−7.48 (m, 2H), 7.55−7.61 (m, 1H), 8.03−8.06 (m, 2H).
2-{[2-(4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]thio}-
ethyl benzoate (26). Compound 26 was obtained as colorless oil
1
from 25 by a method similar to that described for 11. Yield 80%. H
NMR (CDCl3) δ: 2.81 (t, 2H, J = 6.8 Hz), 3.08 (t, 2H, J = 6.9 Hz),
4.45 (t, 2H, J = 6.8 Hz), 4.69 (t, 2H, J = 6.9 Hz), 6.73 (d, 1H, J = 3.3
Hz), 7.39−7.46 (m, 2H), 7.53−7.62 (m, 2H), 7.96−8.06 (m, 2H),
8.71 (s, 1H).
2-{[2-(4-{3-Chloro-4-[3-(trifluoromethyl)phenoxy]benzyl}-
5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]thio}ethanol (28). Com-
pound 28 was obtained as colorless crystals in 59% yield from 26 by a
1
method similar to that described for 15; mp 108−109 °C. H NMR
(CDCl3) δ: 1.92−2.00 (m, 1H), 2.52 (t, 2H, J = 5.6 Hz), 3.13 (t, 2H,
J = 6.5 Hz), 3.65−3.75 (m, 2H), 4.61 (t, 2H, J = 6.5 Hz), 6.67 (d, 1H,
J = 3.3 Hz), 7.08 (d, 1H, J = 8.7 Hz), 7.09−7.13 (m, 1H), 7.18−7.23
(m, 1H), 7.29 (d, 1H, J = 3.3 Hz), 7.32−7.35 (m, 1H), 7.41−7.46
(m, 1H), 7.51 (dd, 1H, J = 8.7, 2.7 Hz), 7.77 (d, 1H, J = 2.7 Hz), 7.80
(s, 1H), 8.55 (s, 1H). Anal. Calcd for C23H20ClF3N4O2S: C, 54.28; H,
3.96; N, 11.01. Found: C, 54.35; H, 3.94; N, 11.02.
2-{[2-(4-{3-Chloro-4-[3-(trifluoromethyl)phenoxy]benzyl}-
5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]sulfinyl}ethanol (29). To
a solution of 28 (0.10 g, 0.20 mmol) in dichloromethane (10 mL) was
added dropwise a 70% solution of 3-chloroperbenzoic acid (0.058 g,
0.34 mmol) in dichloromethane (5.0 mL) at −78 °C. The mixture was
stirred at −78 °C for 1 h, and aqueous sodium thiosulfate (100 mL)
was added. After stirring at room temperature for 30 min, the mixture
was extracted with EtOAc (200 mL). The organic layer was washed
with brine (50 mL) and dried over MgSO4. After concentration in
vacuo, the residue was purified by silica gel column chromatography
(eluent, MeOH:EtOAc = 0:1 to 1:4) to give 87 mg (85%) of 29 as
1
colorless crystals, mp 158−159 °C. H NMR (DMSO-d6) δ: 2.78−
3.01 (m, 2H), 3.27−3.40 (m, 1H), 3.42−3.58 (m, 1H), 3.71−3.79 (m,
2H), 4.80−4.90 (m, 2H), 5.02−5.09 (m, 1H), 6.58−6.63 (m, 1H),
7.16−7.25 (m, 2H), 7.27−7.31 (m, 1H), 7.44−7.50 (m, 1H), 7.59−
7.64 (m, 1H), 7.66−7.72 (m, 1H), 7.74−7.82 (m, 1H), 7.96−8.03 (m,
1H), 8.37 (s, 1H), 9.38 (s, 1H). Anal. Calcd for C23H20ClF3N4O3S: C,
52.62; H, 3.84; N, 10.67. Found: C, 52.62; H, 3.82; N, 10.67.
2-{[2-(4-{3-Chloro-4-[3-(trifluoromethyl)phenoxy]benzyl}-
5H-pyrrolo[3,2-d]pyrimidin-5-yl)ethyl]sulfonyl}ethanol (30). A
mixture of 29 (0.15 g, 0.29 mmol), titanium tetraisopropoxide (0.043 mL),
methanol (0.024 mL), and water (0.010 mL) in dichloromethane
(10 mL) was stirred at room temperature for 30 min. 70% aqueous
tert-butyl hydroperoxide (0.12 mL) was added to the reaction mixture,
and the mixture was stirred at room temperature for 2 days. Aqueous
sodium thiosulfate (100 mL) was added to the reaction mixture, and
the mixture was extracted with EtOAc (200 mL). The organic layer
was washed with brine (50 mL), dried over MgSO4, and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (eluent, MeOH:EtOAc = 0:1 to 1:4) to give 0.12 g
(74%) of 30 as colorless crystals, mp 196−197 °C. 1H NMR (DMSO-
d6) δ: 3.09−3.15 (m, 2H), 3.62−3.75 (m, 4H), 4.92−5.02 (m, 2H),
5.09−5.15 (m, 1H), 6.50−6.57 (m, 1H), 7.16−7.32 (m, 3H), 7.45−
7.48 (m, 1H), 7.58−7.74 (m, 3H), 7.91−7.97 (m, 1H), 8.37 (br s,
1H), 8.69−8.79 (m, 1H). Anal. Calcd for C23H20ClF3N4O4S: C, 51.07;
H, 3.73; N, 10.36. Found: C, 51.10; H, 3.63; N, 10.34.
tert-Butyl [2-(4-Chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-
ethyl]carbamate (31a). To a suspension of 5 (0.50 g, 3.26 mmol)
and cesium carbonate (1.59 g, 8.24 mmol) in DMF (10 mL) was
added tert-butyl (2-bromoethyl)carbamate (1.09 g, 4.89 mmol) at 40 °C,
and the mixture was stirred at 40 °C for 4 days. Water (100 mL) was
added to the reaction mixture, and the mixture was extracted with
EtOAc (200 mL). The organic layer was washed with brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified by silica
gel chromatography (eluent, EtOAc:hexane = 1:3 to 3:2) to give 0.69 g
(71%) of 31a as white solid. 1H NMR (CDCl3) δ: 1.31−1.46 (m, 9H),
3.55 (dt, 2H, J = 6.0 Hz), 4.51−4.68 (m, 3H), 6.74 (d, 1H, J = 3.2 Hz),
7.47 (d, 1H, J = 3.2 Hz), 8.71 (s, 1H).
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tert-Butyl{2-[4-({3-chloro-4-[3-(trifluoromethyl)phenoxy]-
phenyl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-
carbamate (32a). A mixture of 31a (0.71 g, 2.39 mmol) and 4j
(0.83 g, 2.79 mmol) in 2-propanol (7.1 mL) was stirred at 80 °C for
12 h. To the reaction mixture was added saturated sodium hydrogen
carbonate (50 mL) under ice-cooling, and the mixture was extracted
with EtOAc (200 mL). The organic layer was washed with brine and
dried over MgSO4. The solvent was concentrated in vacuo, and the
residue was purified by silica gel column chromatography (eluent,
EtOAc:hexane = 1:1 to 1:0) to give 1.12 g (85%) of 32a as white solid.
1H NMR (CDCl3) δ: 1.49 (s, 9H), 3.43−3.54 (m, 2H), 4.43−4.51 (m,
20.1 mmol), WSC (10.1 g, 52.7 mmol), HOBt (4.56 g 33.7 mmol),
and triethylamine (0.80 mL) in THF (17 mL) and DMF (17 mL) was
stirred at room temperature for 3 days. Water (300 mL) was added
to the reaction mixture, and the mixture was extracted with EtOAc
(400 mL). The organic layer was washed with water (100 mL) and
brine (100 mL) successively, dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatography
(eluent, MeOH:EtOAc = 0:1 to 1:9) to give white solid. To a solution
of the white solid in EtOAc (50 mL) was added methanesulfonic acid
(0.16 mL), and the mixture was stirred at room temperature for 2 h.
The reaction mixture was concentrated under reduced pressure, and
the residue was recrystallized from EtOAc to give 1.04 g (25%) of 34d
as colorless crystals, mp 154−155 °C. 1H NMR (DMSO-d6) δ: 2.22 (t,
2H, J = 6.3 Hz), 2.31 (s, 3H), 3.41−3.51 (m, 4H), 3.56 (t, 2H, J = 6.5
Hz), 6.67 (d, 1H, J = 3.0 Hz), 7.25−7.32 (m, 2H), 7.37 (d, 1H, J = 8.8
Hz), 7.50−7.56 (m, 1H), 7.62−7.74 (m, 2H), 7.98 (d, 1H, J = 2.8 Hz),
8.33 (t, 1H, J = 5.5 Hz), 8.75 (s, 1H), 10.11 (br s, 1H). Anal. Calcd for
C25H25ClF3N5O6S: C, 48.74; H, 4.09; N, 11.37. Found: C, 48.60; H,
4.08; N, 11.24.
2H), 5.10 (t, 1H, J = 5.6 Hz), 6.60 (d, 1H, J = 3.3 Hz), 7.07 (m, 1H),
7.09−7.14 (m, 1H), 7.16−7.22 (m, 2H), 7.25−7.30 (m, 1H), 7.37−
7.45 (m, 1H), 7.89 (dd, 1H, J = 8.7, 2.4 Hz), 8.02 (d, 1H, J = 2.4 Hz),
8.50 (s, 1H), 8.64 (br s, 1H).
5-(2-Aminoethyl)-N-{3-chloro-4-[3-(trifluoromethyl)-
phenoxy]phenyl}-5H-pyrrolo[3,2-d]pyrimidin-4-amine dihy-
drochloride (33a). A mixture of 32a (1.12 g, 2.04 mmol), 2 N HCl
(15 mL), and THF (30 mL) was stirred at 60 °C for 20 h. After the
mixture was concentrated under reduced pressure, ethanol
(50 mL) was added to the concentrate, and then the mixture was
further concentrated in vacuo. The residual crystals were collected by
filtration and washed with ethyl acetate to give 1.07 g (quant) of 33a as
tert-Butyl [2-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-
ethyl]methylcarbamate (31b). To a solution of 2-(methylamino)-
ethanol (1.00 g, 13.3 mmol) in THF (10 mL) was added di-tert-butyl
dicarbonate (3.6 mL) at room temperature. After stirring at room
temperature for 2 h, the mixture was concentrated under reduced
pressure. The residue was dissolved in THF (50 mL), and to the
solution was added dropwise triethylamine (3.7 mL) and meth-
anesulfonyl chloride (1.6 mL) at 0 °C successively, and the reaction
mixture was stirred at 0 °C for 1 h. Then, saturated sodium hydrogen
carbonate (10 mL) was added to the reaction mixture, and the mixture
was extracted with EtOAc (200 mL). The organic layer was washed
with brine (50 mL), dried over MgSO4, and concentrated in vacuo to
give colorless oil. To a suspension of 5 (1.34 g, 8.76 mmol) and
cesium carbonate (5.69 g, 29.5 mmol) in DMF (20 mL) was added the
obtained colorless oil, and the reaction mixture was stirred at 40 °C for
4 days. Water (100 mL) was added to the reaction mixture, and the
mixture was extracted with EtOAc (300 mL). The organic layer was
washed with brine (50 mL), dried over MgSO4, and concentrated in
vacuo. The residue was purified by silica gel chromatography (eluent,
EtOAc:hexane = 1:3 to 3:2) to give 0.90 g (33%) of 31b as pale-yellow
1
pale-yellow solid. H NMR (DMSO-d6) δ: 3.21−3.35 (m, 2H), 4.92−
5.02 (m, 2H), 6.71−6.76 (m, 1H), 7.24−7.32 (m, 2H), 7.37 (d, 1H, J =
9.0 Hz), 7.50−7.56 (m, 1H), 7.64−7.71 (m, 2H), 7.91−7.97 (m, 1H),
7.98−8.06 (m, 1H), 8.13−8.26 (m, 3H), 8.71 (br s, 1H), 9.90 (br s, 1H).
N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-2-hydroxyace-
tamide (34a). A mixture of 33a (0.11 g, 0.21 mmol), glycolic acid
(0.044 g, 0.58 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (WSC) (0.17 g, 0.89 mmol), 1-hydroxybenzotriazole
monohydrate (HOBt) (0.13 g 0.96 mmol), and triethylamine (0.40 mL)
in DMF (5.0 mL) was stirred at room temperature for 3 days. Water
(100 mL) was added to the reaction mixture, and the mixture was
extracted with EtOAc (200 mL). The organic layer was washed with
water (50 mL) and brine (50 mL), dried over MgSO4 and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (eluent, MeOH:EtOAc = 0:1 to 1:9) to give 0.11 g
(74%) of 34a as colorless crystals, mp 145−147 °C. 1H-NMR
(CDCl3) δ: 2.93−3.09 (m, 1H), 3.59−3.73 (m, 2H), 4.24 (s, 2H),
4.43−4.53 (m, 2H), 6.59 (d, 1H, J = 3.3 Hz), 7.07 (d, 1H, J = 8.7 Hz),
7.09−7.46 (m, 6H), 7.72 (dd, 1H, J = 8.7, 2.4 Hz), 8.06 (d, 1H, J = 2.4
Hz), 8.49 (s, 1H), 8.57 (1H, s). Anal. Calcd for C23H19ClF3N5O3: C,
54.61; H, 3.79; N, 13.84. Found: C, 54.77; H, 3.82; N, 13.89.
The following compounds (34c,34e) were prepared from 33a with
the corresponding carboxylic acid by a method similar to that
described for 34a.
1
oil. H NMR (CDCl3) δ: 1.12 (s, 4.5H), 1.43 (m, 4.5H), 2.55 (s,
1.5H), 2.81 (s, 1.5H), 3.58−3.60 (m, 2H), 4.54−4.69 (m, 2H), 6.73
(d, 1H, J = 3.0 Hz), 7.29−7.35 (m, 0.5H), 7.38−7.46 (m, 0.5H), 8.71
(s, 1H).
tert-Butyl{2-[4-({3-chloro-4-[3-(trifluoromethyl)phenoxy]-
phenyl}amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-
methylcarbamate (32b). Compound 32b was obtained as white
solid in 76% yield from 31b by a method similar to that described for
1
32a. H NMR (CDCl3) δ: 1.51 (s, 9H), 3.01 (s, 3H), 3.51−3.59 (m,
N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-2-hydroxy-2-
methylpropanamide (34c). Yield 86%, colorless crystals, mp 183−
185 °C. 1H NMR (CDCl3) δ: 1.49 (s, 6H), 2.12−2.27 (m, 1H), 3.56−
3.67 (m, 2H), 4.42−4.52 (m, 2H), 6.61 (d, 1H, J = 3.3 Hz), 7.06 (d,
1H, J = 9.0 Hz), 7.08−7.14 (m, 1H), 7.15−7.43 (m, 5H), 7.86 (dd,
1H, J = 9.0, 2.7 Hz), 8.10 (d, 1H, J = 2.7 Hz), 8.51 (s, 1H), 8.72 (s,
1H). Anal. Calcd for C25H23ClF3N5O3: C, 56.24; H, 4.34; N, 13.12.
Found: C, 56.33; H, 4.38; N, 13.10.
2H), 4.41−4.51 (m, 2H), 6.60 (d, 1H, J = 3.0 Hz), 7.06 (d, 1H, J =
8.7 Hz), 7.08−7.13 (m, 1H), 7.15−7.24 (m, 2H), 7.30 (d, 1H, J = 8.4
Hz), 7.38−7.44 (m, 1H), 7.85−7.93 (m, 1H), 7.99−8.04 (m, 1H),
8.50 (s, 1H), 8.82 (s, 1H).
N-{3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-5-[2-
(methylamino)ethyl]-5H-pyrrolo[3,2-d]pyrimidin-4-amine Di-
hydrochloride (33b). Compound 33b was obtained as white solid
in 91% yield from 32b by a method similar to that described for 33a.
1H NMR (DMSO-d6) δ: 2.54 (t, 3H, J = 5.3 Hz), 3.32−3.44 (m, 2H),
5.01−5.15 (m, 2H), 6.74 (d, 1H, J = 3.3 Hz), 7.22−7.27 (m, 2H), 7.36
(d, 1H, J = 8.7 Hz), 7.51 (d, 1H, J = 8.4 Hz), 7.60−7.69 (m, 2H),
7.91−7.96 (m, 1H), 8.01−8.07 (m, 1H), 8.72 (s, 1H), 9.00−9.18 (m,
2H), 10.06 (br s, 1H).
N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-2-hydroxy-N-
methylacetamide (34b). Compound 34b was obtained as colorless
crystals in 62% yield from 33b by a method similar to that described
for 34a; mp 176−178 °C. 1H NMR (CDCl3) δ: 3.02 (s, 3H), 3.29 (t,
1H, J = 4.5 Hz), 3.72−3.81 (m, 2H), 4.26 (d, 2H, J = 4.5 Hz), 4.45−
4.53 (m, 2H), 6.64 (d, 1H, J = 3.3 Hz), 7.08 (d, 1H, J = 8.7 Hz), 7.09−
7.15 (m, 1H), 7.23−7.28 (m, 1H), 7.32−7.37 (m, 1H), 7.30−7.35 (m,
1H), 7.39−7.46 (m, 1H), 7.74 (dd, 1H, J = 8.7, 2.7 Hz), 8.05 (d, 1H,
N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-3-hydroxy-3-
methylbutanamide (34e). Yield 77%, colorless crystals, mp 167−
1
169 °C. H NMR (CDCl3) δ: 1.33 (s, 6H), 2.49 (s, 2H), 2.65−2.77
(m, 1H), 3.57−3.68 (m, 2H), 4.44−4.53 (m, 2H), 6.61 (d, 1H, J = -
3.0 Hz), 6.93−7.01 (m, 1H), 7.07 (d, 1H, J = 9.0 Hz), 7.09−7.15 (m,
1H), 7.19 (d, 1H, J = 3.0 Hz), 7.23−7.35 (m, 2H), 7.40−7.45 (m,
1H), 7.77 (dd, 1H, J = 9.0, 2.7 Hz), 8.08 (d, 1H, J = 2.7 Hz), 8.52 (s,
1H), 8.66 (s, 1H). Anal. Calcd for C26H25ClF3N5O3: C, 56.99; H, 4.60;
N, 12.78. Found: C, 57.22; H, 4.61; N, 12.80.
N-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethyl}-3-hydroxypro-
panamide methanesulfonate (34d). A solution of 33a (3.50 g,
6.72 mmol), 3.6 M aqueous 3-hydroxypropanoic acid (5.6 mL,
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J = 2.7 Hz), 8.39 (s, 1H), 8.53 (s, 1H). Anal. Calcd for
C24H21ClF3N5O3: C, 55.44; H, 4.07; N, 13.47. Found: C, 55.54; H,
4.09; N, 13.36.
2-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}-
ethanol (40). To a suspension of 39 (220 mg, 0.36 mmol) in MeOH
(1.6 mL) was added 1 N NaOH (0.5 mL), and the reaction mixture
was stirred at room temperature for 2 h. The mixture was neutralized
with 1 N HCl (0.5 mL) and extracted with EtOAc (50 mL). The
organic layer was washed with brine (30 mL), dried over MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography (eluent, MeOH:EtOAc = 0:1 to 1:4) and crystallized
from diisopropylether to give 164 mg (90%) of 40 as colorless crystals,
mp 168−169 °C. 1H NMR (DMSO-d6) δ 2.46 (s, 3H), 3.49−3.51 (m,
4H), 3.84 (t, 2H, J = 4.0 Hz), 4.55 (br s, 2H), 4.76 (t, 2H, J = 4.0 Hz),
7.17−7.21 (m, 2H), 7.27 (d, 1H, J = 9.0 Hz), 7.44 (d, 1H, J = 8.0 Hz),
7.50−7.70 (m, 2H), 8.01 (d, 1H, J = 2.0 Hz), 8.29 (s, 1H), 8.98 (br s,
1H). Anal. Calcd for C24H22ClF3N4O3: C, 56.87; H, 4.37; N, 11.05.
Found: C, 56.89; H, 4.34; N, 11.05.
4-Iodo-6-phenoxypyrimidine-5-amine (35). To a solution of
4,6-diiodo-pyrimidine-5-amine (40.0 g, 115 mmol) in 1-methyl-2-
pyrrolidone (200 mL) were added phenol (11.9 g, 127 mmol) and
potassium carbonate (17.5 g, 127 mmol), and the reaction mixture was
stirred at 100 °C for 12 h. After cooling to room temperature, the
reaction mixture was diluted with EtOAc (600 mL), and washed with
water (700 mL) and brine (500 mL). The organic layer was dried over
MgSO4 and concentrated in vacuo. The residue was purified by silica
gel column chromatography (eluent, EtOAc:hexane = 1:4 to 1:1) to
1
give 33.0 g (91%) of 35. H NMR (CDCl3) δ 4.34 (br s, 2H), 7.14−
7.46 (m, 5H), 7.87 (s, 1H).
4-Phenoxy-6-prop-1-yn-1-ylpyrimidin-5-amine (36). A mix-
ture of 4-iodo-6-phenoxypyrimidine-5-amine (35, 5.00 g, 16.0 mmol),
1-(trimethylsilyl)-1-propyne (3.3 mL, 22.3 mmol), dichlorobis-
(triphenylphosphine)palladium(II) (558 mg, 0.795 mmol), triphenyl-
phosphine (421 mg, 0.161 mmol), copper iodide (303 mg, 0.159 mmol),
potassium fluoride (1.29 g, 22.2 mmol), and triethylamine (50 mL) in
DMF (100 mL) was stirred at 60 °C for 16 h. The reaction mixture was
diluted with diethyl ether (50 mL) and quenched with saturated sodium
hydrogen carbonate (100 mL). The mixture was extracted with diethyl
ether (100 mL). The organic layer was washed with brine (50 mL), dried
over MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography (eluent, EtOAc:hexane = 1:4 to 1:1) to give
4-(3,3-Diethoxyprop-1-yn-1-yl)-6-phenoxypyrimidin-5-
amine (41). A mixture of 35 (7.00 g, 22.4 mmol), 3,3-diethoxyprop-
1-yne (3.8 mL, 26.5 mmol), dichlorobis(triphenylphosphine)-
palladium(II) (783 mg, 1.12 mmol), copper iodide (255 mg, 1.34
mmol), and triethylamine (120 mL) in acetonitrile (160 mL) was
stirred at room temperature for 16.5 h. The mixture was concentrated
in vacuo, and the residue was purified by column chromatography
(eluent, EtOAc:hexane = 1:9 to 1:1) to give 6.20 g (89%) of 41 as
1
brown oil. H NMR (CDCl3) δ 1.29 (t, 6H, J = 7.2 Hz), 3.62−3.77
(m, 2H), 3.77−3.91 (m, 2H), 4.48 (br s, 2H), 5.56 (s, 1H), 7.14−7.21
(m, 2H), 7.27−7.33 (m, 1H), 7.39−7.50 (m, 2H), 8.11 (s, 1H).
6-(Diethoxymethyl)-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidine
(42). To a solution of 41 (6.20 g, 19.8 mmol) in THF (100 mL) was
added 1 N potassium tert-butoxide in THF (25 mL) at 0 °C, and the
reaction mixture was stirred at room temperature for 1.5 h. The
mixture was diluted with EtOAc (100 mL), quenched with saturated
aqueous ammonium chloride (100 mL), and extracted with EtOAc
(100 mL). The organic layer was washed with brine (50 mL), dried
over MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography (eluent, EtOAc:hexane = 1:9 to 1:1) to give
1
2.64 g (73%) of 36 as orange solid. H NMR (CDCl3) δ 2.20 (s, 3H),
4.37 (br s, 2H), 7.15−7.19 (m, 2H), 7.24−7.29 (m, 1H), 7.40−7.46 (m,
2H), 8.08 (s, 1H).
6-Methyl-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidine (37). To a
solution of 36 (2.56 g, 11.4 mmol) in THF (100 mL) was added
dropwise 1 N potassium tert-butoxide in THF solution (12 mL, 12.0 mmol)
at 0 °C, and the reaction mixture was stirred at 0 °C for 40 min. The
reaction mixture was diluted with EtOAc (100 mL) and quenched
with water (100 mL) at 0 °C. The mixture was extracted with EtOAc
(100 mL). The organic layer was washed with brine (50 mL), dried
over MgSO4, and concentrated in vacuo. The residue was purified by
column chromatography (eluent, EtOAc:hexane = 4:1 to 1:1) to give
1.71 g (67%) of 37 as pale-orange solid. 1H NMR (CDCl3) δ 2.54 (d,
3H, J = 0.9 Hz), 6.44 (q, 1H, J = 0.9 Hz), 7.21−7.30 (m, 3H), 7.42−
7.47 (m, 2H), 8.46 (m, 2H).
2-[2-(6-Methyl-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-
ethoxy]ethyl benzoate (38). A mixture of 37 (300 mg, 1.33
mmol), 2-{2-[(methylsulfonyl)oxy]ethoxy}ethyl benzoate (464 mg,
1.61 mmol), and potassium carbonate (431 mg, 3.19 mmol) in DMF
(7 mL) was stirred at 60 °C for 21 h. The reaction mixture was diluted
with EtOAc (10 mL), quenched with water (10 mL), and extracted
with EtOAc (10 mL). The organic layer was washed with brine
(10 mL), dried over MgSO4, and concentrated in vacuo. The residue
was purified by column chromatography (eluent: EtOAc:hexane = 1:4
to 4:1) to give 518 mg (93%) of 38 as yellow oil. 1H NMR (CDCl3) δ
2.50 (s, 3H), 3.62−3.74 (m, 2H), 3.92 (t, 2H, J = 5.3 Hz), 4.33−4.44
(m, 2H), 4.57 (t, 2H, J = 5.1 Hz), 6.36 (s, 1H), 7.15−7.34 (m, 3H),
7.34−7.51 (m, 4H), 7.51−7.65 (m, 1H), 7.87−8.00 (m, 2H), 8.40
(s, 1H).
1
3.15 g (51%) of 42 as orange solid. H NMR (CDCl3) δ 1.20−1.37
(m, 6H), 3.55−3.79 (m, 4H), 5.81 (d, 1H, J = 0.6 Hz), 6.69 (dd, 1H,
J = 2.2, 0.6 Hz), 7.18−7.36 (m, 3H), 7.39−7.52 (m, 2H), 8.50 (s, 1H),
8.92 (br s, 1H).
4-Phenoxy-5H-pyrrolo[3,2-d]pyrimidine-6-carbaldehyde
(43). To a solution of 42 (3.15 g, 10.1 mmol) in THF (40 mL) was
added 1 N HCl (40 mL), and the reaction mixture was stirred at room
temperature for 2 h. The mixture was neutralized with 1 N NaOH
(40 mL) and extracted with a mixture of EtOAc (30 mL)/THF (30 mL).
The organic layer was washed with brine (20 mL), dried over MgSO4,
and concentrated in vacuo. The residual solid was collected by
filtration and washed with diisopropylether to give 2.17 g (90%) of 43
1
as khaki powder. H NMR (CDCl3) δ 7.25−7.40 (m, 3H), 7.43−7.58
(m, 3H), 8.44 (s, 1H), 10.06 (s, 1H), 13.26 (br s, 1H).
4-Phenoxy-5H-pyrrolo[3,2-d]pyrimidine-6-carboxylic Acid
(44). To a mixture of 43 (2.17 g, 9.07 mmol) in DMSO (21 mL)
and sodium dihydrogen phosphate (5.45 g, 45.4 mmol) in water
(14 mL) was added dropwise a solution of sodium chlorite (2.06 g,
22.8 mmol) in water (14 mL), and the reaction mixture was stirred at
room temperature for 2 h. The mixture was quenched with saturated
sodium hydrogen carbonate (10 mL) gradually, and the pH was
adjusted to pH 2 by addition of 1 N HCl. The resulting precipitate was
collected by filtration and washed with diisopropylether to give 2.40 g
2-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}ethyl
benzoate (39). A mixture of 38 (270 mg, 0.65 mmol), 4j (279 mg,
0.97 mmol), pyridinium chloride (235 mg, 2.03 mmol), and phenol
(496 mg) was stirred at 140 °C for 17 h. After cooling to room
temperature, the mixture was diluted with dichloromethane (50 mL),
and the organic layer was washed with saturated sodium hydrogen car-
bonate (30 mL). The organic layer was dried over MgSO4 and concen-
trated in vacuo. The residue was purified by column chromatography
(eluent, EtOAc:hexane = 1:4 to 1:0) to give 228 mg (58%) of 39 as
1
(quant) of 44 as white powder. H NMR (CDCl3) δ 7.09 (s, 1H),
7.23−7.36 (m, 3H), 7.41−7.54 (m, 2H), 8.36 (s, 1H), 12.82 (br s,
1H).
4-Phenoxy-5H-pyrrolo[3,2-d]pyrimidine-6-carboxamide
(45). A mixture of 44 (465 mg, 1.82 mmol) and thionyl chloride
(7 mL) was stirred at 75 °C for 2 h. After cooling to room temperature,
the mixture was concentrated in vacuo and the residue was suspended in
THF (10 mL). The suspension was poured into 28% ammonia (20 mL)
at 0 °C, and the resulting precipitate was collected by filtration. The
filtrate was extracted with a mixture of EtOAc (10 mL)/THF (10 mL).
The organic layer was washed with brine (5 mL), dried over MgSO4,
and concentrated in vacuo. The residual solid was collected by filtration,
1
yellow oil. H NMR (CDCl3) δ 2.44 (s, 3H), 3.93−4.12 (m, 4H),
4.47−4.51 (m, 4H), 6.40 (s, 1H), 6.77 (d, 1H, J = 9.0 Hz), 7.01−7.03
(m, 1H), 7.15 (s, 1H), 7.20−7.50 (m, 5H), 7.75−7.86 (m, 3H), 8.44
(s, 1H), 8.72 (br s, 1H).
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and combined with the above precipitate to give 427 mg (92%) of 45 as
Accession Codes
1
†Protein Data Bank accession code: 3RCD.
pale-yellow powder. H NMR (DMSO-d6) δ 7.25 (s, 1H), 7.27−7.35
(m, 3H), 7.39−7.57 (m, 2H), 7.75 (s, 1H), 8.17 (s, 1H), 8.36 (s, 1H),
12.58 (br s, 1H).
AUTHOR INFORMATION
■
4-Phenoxy-5H-pyrrolo[3,2-d]pyrimidine-6-carbonitrile
(46). A mixture of 45 (427 mg, 1.68 mmol) in phosphoryl chloride
(5 mL) was stirred at 70 °C for 1.5 h. After cooling to room temperature,
the mixture was concentrated in vacuo. The residue was dissolved in
THF (10 mL), and to the solution were added water (5 mL) and 28%
ammonia (10 mL) at 0 °C. The mixture was extracted with a mixture
of EtOAc (10 mL)/THF (10 mL). The organic layer was washed with
brine (5 mL), dried over MgSO4, and concentrated in vacuo. The residue
was purified by column chromatography (eluent, EtOAc:hexane = 1:9 to
Corresponding Author
*Phone: +81-466-32-1155. Fax: +81-466-29-4449. E-Mail:
Ishikawa_Tomoyasu@takeda.co.jp.
ACKNOWLEDGMENTS
■
We are grateful to structural biology group members (Bi Ching
Sang, Hua Zou, Darbi Witmer, Gyorgy Snell, Ryan Bertsch, and
Jason Yano) and management at Takeda San Diego for
determining the first X-ray cocrystal structure of 34e with
HER2. We also acknowledge the editing assistance of Stephen
Mosley of KnowledgePoint360 Group Ltd. in the development
of this manuscript, which was funded by Millennium
Pharmaceuticals, Inc.
1
1:1) to give 240 mg (61%) of 46 as pale-yellow powder. H NMR
(DMSO-d6) δ 7.29−7.37 (m, 3H), 7.45−7.54 (m, 2H), 7.58 (s, 1H),
8.46 (s, 1H), 13.77 (br s, 1H).
2-[2-(6-Cyano-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidin-5-yl)-
ethoxy]ethyl benzoate (47). A mixture of 46 (240 mg, 1.02
mmol), 2-{2-[(methylsulfonyl)oxy]ethoxy}ethyl benzoate (354 mg,
1.23 mmol), and potassium carbonate (355 mg, 2.57 mmol) in DMF
(5 mL) was stirred at 60 °C for 7 h. To the mixture was added
additional 2-{2-[(methylsulfonyl)oxy]ethoxy}ethyl benzoate (133 mg,
0.462 mmol), and the reaction mixture was stirred at 60 °C for 16 h.
After cooling to room temperature, the mixture was diluted with
EtOAc (10 mL), quenched with aqueous ammonium chloride (20
mL), and extracted with EtOAc (20 mL). The organic layer was
washed with brine (5 mL), dried over MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography (eluent,
EtOAc:hexane = 1:4 to 4:1) to give 267 mg (61%) of 47 as colorless
oil. 1H NMR (CDCl3) δ 3.73−3.79 (m, 2H), 3.96 (t, 2H, J = 4.9 Hz),
4.37−4.43 (m, 2H), 4.83 (t, 2H, J = 4.9 Hz), 7.17 (s, 1H), 7.18−7.23
(m, 2H), 7.27−7.35 (m, 1H), 7.36−7.49 (m, 4H), 7.51−7.58 (m, 1H),
7.85−7.92 (m, 2H), 8.49 (s, 1H).
2-{2-[4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}-
amino)-6-cyano-5H-pyrrolo[3,2-d]pyrimidin-5-yl]ethoxy}ethyl
Benzoate (48). A mixture of 47 (262 mg, 0.610 mmol), 4j (264 mg,
0.919 mmol), pyridinium chloride (222 mg, 1.92 mmol), and phenol
(462 mg) was stirred at 140 °C for 17 h. After cooling to room
temperature, the mixture was diluted with dichloromethane (10 mL),
washed with brine (5 mL), dried over MgSO4, and concentrated in
vacuo. The residue was purified by column chromatography (eluent,
EtOAc:hexane = 1:4 to 1:1) to give 283 mg (74%) of 48 as yellow oil.
1H NMR (CDCl3) δ 3.96−4.06 (m, 2H), 4.16−4.22 (m, 2H), 4.45−
4.54 (m, 2H), 4.68−4.79 (m, 2H), 6.80 (d, 1H, J = 8.8 Hz), 7.01−7.09
(m, 1H), 7.14−7.20 (m, 1H), 7.24 (s, 1H), 7,27−7.53 (m, 6H), 7.68−
7.76 (m, 2H), 7.92 (d, 1H, J = 2.5 Hz), 8.53 (s, 1H), 8.95 (s, 1H).
4-({3-Chloro-4-[3-(trifluoromethyl)phenoxy]phenyl}amino)-
5-[2-(2-hydroxyethoxy)ethyl]-5H-pyrrolo[3,2-d]pyrimidine-6-
carbonitrile (49). To a solution of 48 (283 mg, 0.454 mmol) in
MeOH (3 mL) was added 1 N NaOH (0.6 mL), and the reaction
mixture was stirred at room temperature for 1 h. The mixture was
neutralized with 1 N HCl (0.6 mL) and extracted with EtOAc (5 mL).
The organic layer was washed with brine (20 mL), dried over MgSO4,
and concentrated in vacuo. The residue was purified by column
chromatography (eluent, EtOAc:hexane = 1:2 to 4:1) and crystallized
from diisopropylether to give 143 mg (61%) of 49 as colorless crystals,
mp 143−144 °C. 1H NMR (CDCl3) δ 3.74−3.88 (m, 4H), 4.08−4.16
(m, 2H), 4.70−4.80 (m, 2H), 7.05−7.15 (m, 2H), 7.16−7.21 (m, 1H),
7.25 (s, 1H), 7.30−7.36 (m, 1H), 7.43 (t, 1H, J = 7.8 Hz), 7.67 (dd,
1H, J = 8.8, 2.8 Hz), 7.96 (d, 1H, J = 2.8 Hz), 8.58 (s, 1H), 9.03 (br s,
1H). Anal. Calcd for C24H19ClF3N5O3: C, 55.66; H, 3.70; N, 13.52.
Found: C, 55.70; H, 3.54; N, 13.33.
ABBREVIATIONS USED
■
1H NMR, proton nuclear magnetic resonance; DMF, N,N-
dimethylformamide; EGFR, epidermal growth factor receptor;
EtOAc, ethyl acetate; EtOH, ethanol; GI, growth inhibitory;
HCl, hydrochloric acid; HER2, human epidermal growth factor
receptor 2; HOBt, 1-hydroxybenzotriazole monohydrate;
mCPBA, 3-chloroperbenzoic acid; MgSO4, magnesium sulfate;
NaOH, sodium hydroxide; NMP, 1-methyl-2-pyrrolidone;
ppm, parts per million; Pt−C, platinum−carbon; SAR,
structure−activity relationships; TKIs, tyrosine kinase inhib-
itors; WSC, water-soluble carbodiimide
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ASSOCIATED CONTENT
■
S
* Supporting Information
Methods used in molecular modeling, enzyme assays, cell line,
and animal models, pharmacokinetics, metabolic stability, and
structural analyses. This material is available free of charge via
the Internet at http://pubs.acs.org.
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This paper was published ASAP on November 4, 2011 with an
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