A practical chromatography-free synthesis of a 5,6-dihydroimidazolo[1,5-f]pteridine derivative as a polo-like kinase-1 inhibitor

Article abstract of DOI:10.1016/j.tet.2018.08.020

A practical chromatography-free synthesis of a potent polo-like kinase-1 inhibitor possessing a unique 5,6-dihydroimidazolo[1,5-f]pteridine structure has been developed. We showed that key cyanoimidazole ring formation could be conducted at benign temperature and obtained a chiral 5,6-dihydroimidazolo[1,5-f]pteridine derivative in good yield without epimerization. An aniline derivative containing a trans 1,4-cyclohexyl diamine structure was prepared by a synthesis that makes use of defined stereocenters of commercially available trans-cyclohexane-1,4-diamine via selective piperazine ring formation from a primary diamine. A coupling reaction of the 3-chloro-5,6-dihydroimidazolo[1,5-f]pteridine derivative and the aniline derivative in the endgame was closely investigated, and good yields were achieved both by palladium-catalyzed amination and acid-promoted coupling under benign reaction conditions. As a result of these investigations, the polo-like kinase-1 inhibitor was successfully obtained in a practical way without concern for generation/separation of stereoisomers.

Full text of DOI:10.1016/j.tet.2018.08.020

Accepted Manuscript
A practical chromatography-free synthesis of a 5,6-dihydroimidazolo[1,5-f]pteridine
derivative as a polo-like kinase-1 inhibitor
Kazuhisa Ishimoto, Keiichiro Nakaoka, Osamu Yabe, Atsuko Nishiguchi, Tomomi
Ikemoto
PII:
S0040-4020(18)30973-6
10.1016/j.tet.2018.08.020
TET 29740
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 21 June 2018
Revised Date: 12 August 2018
Accepted Date: 14 August 2018
Please cite this article as: Ishimoto K, Nakaoka K, Yabe O, Nishiguchi A, Ikemoto T, A practical
chromatography-free synthesis of a 5,6-dihydroimidazolo[1,5-f]pteridine derivative as a polo-like
kinase-1 inhibitor, Tetrahedron (2018), doi: 10.1016/j.tet.2018.08.020.
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A practical chromatography-free synthesis of
a 5,6-dihydroimidazolo[1,5-f]pteridine
derivative as a polo-like kinase-1 inhibitor
Leave this area blank for abstract info.
Kazuhisa Ishimoto, Keiichiro Nakaoka, Osamu Yabe, Atsuko Nishiguchi, Tomomi Ikemoto
Process Chemistry, Pharmaceutical Sciences, Takeda Pharmaceutical Company Limited, 17-85, Jusohonmachi
2-Chome, Yodogawa-ku, Osaka 532-8686, Japan

ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
A practical chromatography-free synthesis of a 5,6-dihydroimidazolo[1,5-f]pteridine
derivative as a polo-like kinase-1 inhibitor
Kazuhisa Ishimoto *, Keiichiro Nakaoka 1, Osamu Yabe 1, Atsuko Nishiguchi 1, Tomomi Ikemoto 1
Process Chemistry, Pharmaceutical Sciences, Takeda Pharmaceutical Company Limited, 17-85, Jusohonmachi 2-Chome, Yodogawa-ku, Osaka 532-8686, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A practical chromatography-free synthesis of a potent polo-like kinase-1 inhibitor possessing a
unique 5,6-dihydroimidazolo[1,5-f]pteridine structure has been developed. We showed that key
cyanoimidazole ring formation could be conducted at benign temperature and obtained a chiral
5,6-dihydroimidazolo[1,5-f]pteridine derivative in good yield without epimerization. An aniline
derivative containing a trans 1,4-cyclohexyl diamine structure was prepared by a synthesis that
makes use of defined stereocenters of commercially available trans-cyclohexane-1,4-diamine via
selective piperazine ring formation from a primary diamine. A coupling reaction of the 3-chloro-
5,6-dihydroimidazolo[1,5-f]pteridine derivative and the aniline derivative in the endgame was
closely investigated, and good yields were achieved both by palladium-catalyzed amination and
acid-promoted coupling under benign reaction conditions. As a result of these investigations, the
polo-like kinase-1 inhibitor was successfully obtained in a practical way without concern for
generation/separation of stereoisomers.
Available online
Keywords:
5,6-dihydroimidazolo[1,5-f]pteridine
polo-like kinase-1 inhibitor
chromatography-free synthesis
trans 1,4-cyclohexyldiamine
piperazine ring formation
amination
2009 Elsevier Ltd. All rights reserved
.
1. Introduction i
dihydroimidazolo[1,5-f]pteridine structure. Aniline 15 was
synthesized starting from commercially available ketone 9.
Amino ketone 10·HCl prepared by deprotection of 9 was
condensed with benzoic acid 11 to give ketone 12. Imine
formation of 12 with piperazine 13 followed by reduction using
NaBH4 provided trans 14 as the major product and cis isomer 14'
as the minor product, respectively. Hydrogenation of the mixture
of 14 and 14' provided aniline 15.
Polo-like kinases (PLKs) were identified as one of the kinase
families that play an essential role in various cell-cycle related
processes.1 Amongst five known PLKs (PLK1–5), PLK1 has
attracted much attention as an important target for a medicine,
and inhibitors of PLK1 have been developed and evaluated as
anticancer drugs in clinical studies.2 Compound 19 is a potent
PLK1 inhibitor that has strong enzyme and cellular activities
against PLK1, and also exhibits oral bioavailability.3
For the synthesis of 19 from 8, two routes have been reported
(Scheme 2). In one route (Route A), compound 8 was reacted
with aniline 15 in an acidic condition to afford 19. In the other
route (Route B), compound 19 was synthesized by sequential
reactions: compound 8 was first reacted with aniline 16 using a
palladium catalyst under a microwave condition, and then
condensed with known amine 184.
Compound 19 is characterized by its unique 5,6-
dihydroimidazolo[1,5-f]pteridine core structure, and medicinal
chemists have synthesized 19 as described in Scheme 1 and
Scheme 2.3 Unnatural amino acid 1 was converted to methyl ester
hydrochloric acid salt 2·HCl, and reductive amination of 2·HCl
with acetone followed by an SNAr reaction with 2,4-dichloro-5-
nitropyrimidine 4 gave 5 (Scheme 1). Reduction of the nitro
group of 5 led to cyclization to give 6. Activation of the amide
group of 6 by phosphorylation was followed by addition of an
anion derived from 7, and the subsequent acid-promoted
cyclization provided in one pot 8, which has the 5,6-
Three key issues need to be overcome for scale-up of the
medicinal chemistry syntheses. First, the cryogenic condition
(−78 °C) used for the synthesis of 8 from 6 is difficult to perform
in large-scale production. Second, construction of the trans
diamine structure on the cyclohexyl ring of 15/18 is a synthetic
challenge. In the patent filed by the medicinal chemists, 3a ratio of
14 to 14' synthesized from 12 and 13 by reductive amination is
not obviously mentioned. A mixture of 14 and 14' was
hydrogenated, and after simple workup, the obtained crude
aniline 15 was used for the final coupling with 8 without further
purification. Removal of the undesired cis isomer 19' seemingly
necessitated cumbersome workup procedures including column
chromatography separation (Route A) or HPLC separation
———
i * Corresponding author.
E-mail address: kazuhisa.ishimoto@takeda.com (K. Ishimoto).
1 Present address: SPERA PHARMA, Inc., 17-85, Jusohonmachi 2-
Chome, Yodogawa-ku, Osaka 532-0024, Japan.
E-mail address: tomomi.ikemoto@spera-pharma.co.jp (T. Ikemoto)

2
Tetrahedron
ACCEPTED MANUSCRIPT
Scheme 1. Medicinal chemistry synthesis of 8 and 15.
Reagents and Conditions: (a) SOCl2, MeOH; (b) acetone, NaBH(OAc)3, NaOAc, AcOH, THF; (c) 4, THF, rt; (d) 5% Pt/C, H2 (75 psi),
NH4VO3, P(OPh)3, 5 h, rt; (e) LHMDS, ClPO(OEt)2, THF, −78 °C; (f) 7, LHMDS, THF, −78 °C; (g) AcOH, THF; (h) 4 M HCl in dioxane, rt,
6 h; (i) 11, HATU, DIPEA, DMF, rt, 2.5 h; (j) 13, cat. methanesulfonic acid, toluene, reflux with a Dean-Stark reflux condenser, 5 h; (k)
NaBH4, EtOH, rt, overnight; (l) Pd/C, H2 (balloon), MeOH/AcOH (5:1), rt, 8 h.
Scheme 2. Medicinal chemistry synthesis of 19 from 8.
Reagents and Conditions: (a) conc. HCl, 2-propanol, 95 °C, 2 days; (b) Pd(OAc)2, Xantphos, Cs2CO3, N,N-dimethylacetamide/dioxane,
microwave, 160 °C, 15 min; (c) HATU, DIPEA, DMF, rt, 2 h.

(Route B).5 Finally, the syntheses of 19 from 8 required long
For preparation of 6, reduction of the nitro group of 5 using
inexpensive iron with acetic acid was examined to avoid the use
of an expensive Pt catalyst9. When the reduction was conducted
using acetic acid as the solvent, rapid and huge exotherm was
observed. During the course of investigation, addition of a
catalytic amount of AcOH was found to be sufficient for
completion of the reaction. The use of a catalytic amount of
AcOH decreased the reaction rate, which made the reaction
proceed without rapid exotherm. Also, it is noteworthy that
addition of a small amount of water was effective for improving
fluidity of the reaction mixture, presumably because water helped
acetic acid salt of 6 to dissolve into the reaction mixture. The
reaction was carried out in the presence of 4.0 equiv of iron, 0.3
equiv of acetic acid, and water (50 w/w% to 5) in toluene at
75 °C for 3 h, leading to the reduction of the nitro group and the
cyclization to provide 6 without notable exotherm. Upon
completion of the reaction, 6 M HCl was added to the reaction
mixture to dissolve iron. Following extraction with EtOAc, and
washing with water, brine, and saturated aqueous NaHCO3,
lactam 6 was crystallized from EtOH/H2O and isolated in 80%
yield.
ACCEPTED MANUSCRIPT
reaction time or the use of microwave irradiation, and suffered
from low yields. The acid-promoted coupling of 8 with 15 in 2-
propanol using concentrated HCl required 2 days for the reaction,
providing 19 in low yield (25%, Route A). The palladium-
catalyzed amination of 8 with aniline 16 was conducted using the
microwave system (160 °C, 15 min), and the subsequent
coupling with 18 gave 19, also in low yield (31%, Route B).
As a first step toward a future scale-up synthesis to supply 19
to preclinical/clinical studies, we initiated development of a
practical column chromatography-free/HPLC separation-free
synthesis of 19 that addresses these three key issues.
Retrosynthetic analysis of 19 is shown in Scheme 3. Since it is
known that a convergent synthesis is generally preferable from a
standpoint of cost and efficiency,6 we planned to conduct the
coupling reaction of 8 with 15 in the last step, and to obtain 19 in
a convergent manner. Compound 8 was expected to be
synthesized from 2 and 4 as in the medicinal chemistry synthesis,
though the synthesis was modified for scale-up, including revisit
of reaction conditions for the preparation of 8 from 6. For the
synthesis of 15, we adopted another synthetic route that makes
use of defined trans stereocenters of commercially available
trans diamine 22.7 Aniline 15 was envisioned to be synthesized
from 20 and 21 via piperazine ring formation. Aniline 20 was
anticipated to be synthesized from aniline 16 and 22, and 21 was
envisaged to be prepared by chlorination of diol 23 synthesized
by alkylation of diol 24 with 25.
10
Compound 73a,
aminoacetonitrile
was prepared by the reaction of
26·HCl with N,N-
hydrochloride
dimethylformamide dimethyl acetal in the presence of
triethylamine in THF, and isolated in 67% yield by distillation
(5.6 mmHg, 90 °C). During investigation into a workup
procedure, we found that treatment with a pad of NH silica gel
was effective for removing byproducts generated during the
reaction. After the reaction was completed, the solvent was
switched from THF to diisopropyl ether, and the mixture was
passed through a pad of NH silica gel (50 w/w% to 26·HCl),
which removed precipitated triethylamine hydrochloride salt and
brown tarry byproducts. Following evaporation of the solvent, 7
was obtained in 64% yield. It was possible to use the obtained 7
for the next reaction with 6 without further purification, which
allowed us to avoid time-consuming distillation.
2. Results and Discussion
Lactam 6 was prepared from commercially available 2·HCl
and 4 with a modified procedure (Scheme 4).8 The reductive
amination of 2·HCl with acetone using NaBH4 instead of
expensive NaBH(OAc)3 as the reducing agent proceeded
smoothly, and 3 was isolated as a hydrochloric acid salt in 81%
yield. The subsequent SNAr reaction of 4 with 3·HCl in toluene
using NaHCO3 as the base gave 5 in 73% yield.

4
Tetrahedron
ACCEPTED MANUSCRIPT
Scheme 3. Retrosynthetic analysis of 19.
Scheme 4. Synthesis of 6 and 7.
Scheme 5. Precedent work and synthesis of 8 from 6.
Next, the preparation of 8 from 6 was investigated. There
the anions to the iminochloride/iminophosphate was conducted at
–78 °C or at –30 °C, which largely restrict a manufacturing
facility for scale-up. To confirm whether this reaction could be
carried out under more benign conditions, we began by
examining the reaction conditions closely (Table 1).
have been some precedents for this interesting imidazole ring
formation reaction (Scheme 5). Rogers-Evans and co-workers
reported cyanoimidazole ring formation from an iminochloride.10
Addition of an anion of 28/7 generated by LHMDS to
iminochloride 27 gave intermediates 29 and 29', and the
subsequent acid-promoted isomerization of 29 to 29' and
cyclization provided 30.11 Fryer et al. reported that addition of an
anion of methyl ester analogue of 28 to an iminophosphate gave
a methoxycarbonyl imidazole.12 The medicinal chemists used
these chemistries in a very excellent manner and conducted the
three reactions (phosphorylation, addition of 7, and
isomerization/cyclization) in one pot. In these works, addition of
When we conducted the addition of the anion of 7 to 31 at
−70 °C, 8 was obtained in 56% yield (entry 1). To our surprise,
when the temperature for the addition of the anion of 7 was
increased to −35 °C and −5 °C, the yield increased to 72% and
77%, respectively (entries 2, 3). Next, we conducted the
phosphorylation of 6 at −5 °C. Under this condition, 8 was
obtained in good yield (71%, entry 4) comparable to the yield of
entry 3, where the phosphorylation was conducted at −70 °C.13

Under theses reaction conditions, phosphorylation of 6 did not
reach complete conversion, and 5–10% (HPLC area%) of 6
remained unreacted. On the basis of these results, we concluded
that both the phosphorylation and the addition of the anion of 7
could be performed at −5 °C. Fixing the temperatures for the
phosphorylation and the addition of the anion of 7 at −5 °C, we
ACCEPTED MANUSCRIPT
Table 1 Synthesis of 8 from 6.
phosphorylation
addition of 7
yielda
(%)
optical purity
(% ee)
entry
LHMDS
(equiv)
ClPO(OEt)2
(equiv)
temp
time
(h)
7
LHMDS
(equiv)
temp
(°C)
(equiv)
(°C)
1b
2 b
3 b
4
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
1.1
2
2
−70 → 0
−70 → 0
−70 → 0
−5
2.5
2.5
2.5
1
3
3
3
3
2
3
2
2
2
2
3
3
−70
−35
−5
−5
−5
−5
−5
−5
−5
−5
56
72
77
71
74
74
75
60
44
56
-
-
2
3
-
2
3
99.8
99.8
99.6
97.3
99.8
-
5
2
−5
1
3
6
1.5
1.5
1.5
1.5
1.2
−5
1
3
7
−5
1
3
8
−5
1
2.5
2
9
−5
1
10
−5
1
3
98.5
aIsolated yield. bCompound 7 isolated by distillation was used for the reaction.
then tried to decrease the amounts of reagents. Whereas decrease
of the amount of diethyl chlorophosphate from 2.0 equiv (entry
5) to 1.5 equiv did not affect the yield (75%, entry 7), decrease to
1.2 equiv caused large yield deterioration (56%, entry 10).
Decrease of the amount of 7 from 3.0 equiv (entry 4) to 2.0 equiv
was found to be acceptable (74%, entry 5). In contrast, decrease
of the amount of LHMDS used for the deprotonation of 7 had
crucial effects on the yields. When the amount of LHMDS for the
deprotonation of 7 was decreased from 3.0 equiv (entry 7) to 2.5
equiv and 2.0 equiv, the yield decreased to 60% and 44%,
respectively (entries 8, 9). Isolation of 8 was conducted by
crystallization, and typically 3–5% (HPLC assay yield) of 8 was
included in mother liquor.
optical purity decrease of 8. We also examined the possibility of
racemization of compound 6 by treating it with 1.1 equiv of
LHMDS in THF at –5 °C for 5 h. A small portion of the reaction
mixture was quenched with MeCN/20 mM aqueous KH2PO4
(30:70) and analyzed by chiral HPLC, which showed that the
optical purity of 6 (>99.7% ee) did not change during 5 h under
this reaction condition. Considering these results, the reaction
conditions of entry 5 were finally selected as the best reaction
conditions.
Aniline 15 was prepared by the synthesis described in Scheme
7. Mono N-Boc protected trans diamine 3314 was prepared from
commercially available trans-cyclohexane-1,4-diamine 22 by
treatment with Boc2O in MeCN. The obtained crude 33
containing di-Boc byproduct 34 (approximately 3:1) was used for
condensation with 16 without purification. The condensation of
16 with 33 was carried out using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCIꢀHCl)
and 1-hydroxybenzotriazole monohydrate (HOBtꢀH2O) in MeCN,
giving 35 exclusively without self-condensation of 16. Addition
of water to the reaction mixture led to crystallization, which
enabled isolation of 35 by filtration. The obtained 35 was
dissolved in MeOH, and the Boc group was deprotected with 6 M
HCl. After pH was adjusted to 7.8 with 8 M NaOH, the mixture
was treated with activated carbon. Following evaporation of
MeOH, filtration of the resulting aqueous slurry gave 20·HCl in
82% yield for 2 steps from 16. The di-Boc byproduct 34 was
completely removed during isolation of 20·HCl.
After screening the reaction conditions, we analyzed optical
purity of the obtained 8 and found that the optical purity of 8
prepared under the reaction conditions of entry 7 and entry 10
decreased to 97.3% ee and 98.5% ee, respectively. A plausible
mechanism for the optical purity deterioration is described in
Scheme 6. It is assumed that a strong base such as LHMDS could
bring about deprotonation at the γ position of the cyano group of
32 at −5 °C to give an anion, and reprotonation of the anion
would lead to the optical purity decrease. Under the reaction
conditions of entry 7 and entry 10, the total amounts of diethyl
chlorophosphate and 7 were less than the total amounts under the
conditions of entries 4, 5, and 6. This situation would result in an
increased amount of remaining active LHMDS in the reaction
mixture and would cause the deprotonation of 32, leading to the

6
Tetrahedron
ACCEPTED MANUSCRIPT
Scheme 6. Plausible mechanism for optical purity decrease of 8.
Scheme 7. Synthesis of 15.
Compound 21 was synthesized via alkylation of 24 with
(chloromethyl)cyclopropane followed by chlorination of the
hydroxyl groups. Diol 24 was reacted with
considering difference of nucleophilicity between an aromatic
amino group and an alkyl amino group.
For the synthesis of 15 from 20·HCl by the selective
piperazine ring formation, we screened several bases (NaHCO3,
K2CO3, NaOAc, N,N-diisopropylethylamine) and solvents (EtOH,
THF, acetone, MeCN) using 1.2 equiv of 21 at 50 °C for 3 h. The
reaction was rather slow, and in addition, generation of many
byproducts was observed, which resulted in low conversion of
the reaction. Among the screened solvents, EtOH was found to
be the best solvent; the conversion exceeded 50% only when
EtOH was used as the solvent. As for the bases, K2CO3 and N,N-
diisopropylethylamine gave good results. The best result was
achieved when K2CO3 and EtOH were used for the reaction with
NaI as an additive. The SN2 reaction of 20·HCl with 21 using 1.0
equiv of NaI and 3.0 equiv of K2CO3 in EtOH mainly occurred
not at the aromatic amino group, but at the alkyl amino group on
the cyclohexyl ring, and provided 15 as expected. During the
reaction, generation of several byproducts was observed, and
HPLC assay yield was found to be moderate (67%). Dialkylated
product 36 (the structure was suggested by LC-MS analysis) was
identified as the most notable byproduct for this reaction, and
even under the best reaction condition, approximately 17%
(HPLC area%) of 36 was observed. After workup, 15 was once
isolated as a hydrochloric acid salt, and then dissolved in water
and treated with activated carbon. The activated carbon was
(chloromethyl)cyclopropane in the presence of NaI and K2CO3 in
refluxing EtOH. After evaporation of EtOH, 23 was extracted
with EtOAc and washed with saturated brine, and the solvent was
switch to toluene. The obtained toluene solution of 23 was
treated with 2.0 equiv of SOCl2 at 50 °C for 3 h. On completion
of the reaction, the reaction mixture was neutralized with slow
addition of 8 M NaOH. Following phase separation, filtering off
insoluble matter with a pad of silica gel, and evaporation of the
solvent, the obtained 21 (66% for 2 steps from 24, determined by
1H NMR) was used for the piperazine ring formation without
further purification.
There have been many examples of piperazine ring formation
from a primary amine by a reaction with bis(2-chloroethyl)amine
derivatives.15 However, preparation of a piperazine ring from an
unsymmetrical primary diamine by reacting one of the primary
amino groups selectively with bis(2-chloroethyl)amine
derivatives without protection seems to be difficult. To the best
of our knowledge, there has been only one report (patent) that
includes this selective piperazine ring formation from an
unsymmetrical primary diamine, and the reported yield was quite
low (12%).16 We thought that protection of the aromatic amino
group of 20·HCl was not indispensable for the reaction with 21,

filtered off, and pH was adjusted to 9.0 with 25% aqueous NH3.
Filtration of the resulting slurry afforded pure 15 (36: 0.4 HPLC
area%) in 37% isolated yield with 17% of 15 in the mother liquor.
First, we tried to conduct the coupling reaction using a base;
however, the reaction did not proceed well in a basic condition.
When N,N-diisopropylethylamine was used for the reaction in
N,N-dimethylacetamide (DMAC) at 100 °C, the reaction did not
proceed at all. Additionally, when Cs2CO3 or t-BuOK was used
for the reaction in DMAC at 100 °C, decomposition of 8 was
mainly observed with a small amount of 19. Thus, we turned our
attention to the palladium-catalyzed amination18 (Table 2) and the
acid-promoted coupling reaction (Table 3).
ACCEPTED MANUSCRIPT
Having obtained 8 and 15 in hand, the last coupling reaction
of 8 with 15 was examined. Other than the patent filed by the
medicinal chemists3a, there has been only one report (patent) that
describes a coupling reaction of 3-halo-5,6-dihydroimidazo[1,5-
f]pteridine with an amine, and the reported yield was only
moderate (70 mg scale reaction, 52%).17 As described previously,
the acid-promoted coupling reaction required long reaction time
(>24 h) and the palladium-catalyzed amination needed high
reaction temperature (microwave, 160 °C), and in addition, the
yields were quite low; therefore, more benign reaction conditions
that afford the coupling product in good yield are highly
demanded for scale-up.
Screening of palladium catalysts and ligands using t-BuOH as
the solvent showed that the combination of Pd(OAc)2 and
Xantphos was good for the reaction (Table 2, entries 1–4). When
the reaction was conducted in t-BuOH using 10 mol% of
Pd(OAc)2 and 10 mol% of Xantphos in the presence of 2.0 equiv
of K2CO3, the reaction reached full conversion in 1 h. HPLC
assay yield was 86%, and after workup, 19 was isolated in 61%
Table 2 Synthesis of 19 by palladium-catalyzed amination.
catalyst
(mol%)
ligand
(mol%)
temp
(°C)
time
(h)
3
yielda
(%)
entry
solvent
1
2
Pd2dba3 (10)
Xphos (10)
Xantphos (10)
-
t-BuOH
t-BuOH
t-BuOH
t-BuOH
THF
reflux
reflux
reflux
reflux
reflux
reflux
85
82
Pd(OAc)2 (10)
Pd(dppf)Cl2 (10)
PdCl2[P(o-Tol)3]2 (10)
Pd(OAc)2 (10)
Pd(OAc)2 (10)
Pd(OAc)2 (10)
Pd(OAc)2 (1)
1
86 (61)b
3
4
35
4
-
4
<3
5
Xantphos (10)
Xantphos (10)
Xantphos (10)
Xantphos (1)
5
57
6c
7c
8c
s-BuOH
s-BuOH
s-BuOH
3
81
1
90 (78)b
N.D.d
90
3
aHPLC assay yield. bIsolated yield in parentheses. c1.1 equiv of 8 was used. dNot detected.
yield (entry 2). Several kinds of other solvents were screened for
the reaction. While the use of THF as the solvent decreased the
yield (57%, entry 5), s-BuOH was found to be a good solvent for
the reaction. HPLC assay yield reached 90%, and 19 was isolated
in 78% yield (entry 7). Although we tried to decrease the
amounts of Pd(OAc)2 and Xantphos to 1 mol%, the reaction
completely stalled (entry 8). This was probably because 8 and/or
15 contained a small amount of impurities that deactivated the
palladium catalyst.
concentrated HCl was very slow and reached as little as 6%
conversion after 8 h at reflux condition (Table 3, entry 1), it was
found that the use of MsOH and TsOHꢀH2O largely promoted the
conversion. When MsOH and TsOHꢀH2O were used for the
reaction, the conversion reached 44% and 58% after 8 h,
respectively (entries 2, 3). To achieve faster conversion, alcohols
that have a higher boiling point than 2-propanol were used for the
reaction. When the reaction was conducted in primary alcohol i-
BuOH (bp: 108 °C), a large amount of byproduct was formed by
addition of i-BuOH to 8 (byproduct 1), and a small amount of
hydrolyzed form of 8 (byproduct 2) was observed, which resulted
in moderate conversion (48%, entry 4). Although tertiary alcohol
t-amylalcohol (bp: 102 °C) did not give byproduct 1, the reaction
in t-amylalcohol was not so fast and conversion after 8 h was also
moderate (59%, entry 5). Fortunately, secondary alcohol s-
butanol (bp: 100 °C) was found to be a good solvent for this
reaction. The reaction in s-butanol with TsOHꢀH2O reached up to
In preliminary experiments of the acid prompted coupling
reaction of 8 with 15, several kinds of acids (concentrated HCl,
MsOH, TsOHꢀH2O, H2SO4, H3PO4, 6 M HCl, 48% aqueous HBr,
AcOH) and solvents (alcohols, DMF, DMAC, N-
methylpyrrolidone, toluene, dioxane, n-BuOAc) were screened.
Among the screened solvents, alcohols gave relatively good
conversion. Whereas the reaction in 2-propanol (bp: 82 °C) with

8
Tetrahedron
81% conversion after 8 h (entry 6). Furthermore, increase of the
conditions, taking into account that complete removal of 3.0
ACCEPTED MANUSCRIPT
amount of 8 to 2.0 equiv and 2.4 equiv led to higher conversions
(entries 7, 8). We also confirmed that the reaction did not
proceed in s-BuOH without acid (entry 9).
equiv of TsOH was found not to be easy.
Isolation of pure 19 required elaborated workup procedures.
On completion of the reaction, s-butanol was evaporated, and the
product was extracted with 1 M HCl and washed with EtOAc.
After pH of the aqueous extract was adjusted to approximately 9
with 8 M NaOH, the product was extracted with EtOAc and
washed with 5% aqueous NaHCO3. Following solvent switch to
2-propanol, 19 was crystallized from 2-propanol/n-heptane (1:3).
The obtained crude product was dissolved in MeOH and treated
with activated carbon (Darco® G-60) at 50 °C, which was very
effective for removing red color of the crude product. The
activated carbon was filtered off, and solvent was switched to 2-
propanol, and crystallization from 2-propanol gave pure 19 in
71% yield as an off-white solid (entry 12). The obtained 19 was
In the initial screening of acids, concentrated HCl was not
found to be effective for the reaction (entry 1, 10). During the
investigation, we happened to find that the reaction employing
concentrated HCl as the acid was largely accelerated by addition
of a small amount of water. When the reaction was carried out in
s-BuOH/H2O (95:5) using 3.0 equiv of concentrated HCl, the
reaction speed largely increased and became comparable to that
of the reaction using TsOHꢀH2O (entry 11, 12). We presume that
addition of a small amount of water helps hydrochloric acid salt
of 15 formed by addition of concentrated HCl to dissolve in the
solvent, which led to acceleration of the reaction. Finally, the
reaction conditions of entry 12 were selected as the best
Table 3 Synthesis of 19 by acid-promoted coupling reaction.
8
(equiv)
0.83
1.5
acid
temp
(°C)
time
(h)
8
conversion
yielda
entry
solvent
(equiv)
(%)
6
(%)
1b
2
c.HCl (3.0)
MsOH (3.0)
TsOH (3.0)
TsOH (3.0)
TsOH (3.0)
TsOH (3.0)
TsOH (3.0)
TsOH (3.0)
-
2-propanol (bp: 82 °C)
2-propanol
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
-
8
44
-
3
1.5
2-propanol
8
58
-
4
1.5
i-BuOH (bp: 108 °C)
t-amylalcohol (bp: 102 °C)
s-BuOH (bp: 100 °C)
s-BuOH
8
48
-
5
1.5
8
59
-
6
1.5
8
81
-
7
2.0
10
13
8
89
85 (56)c
8
2.4
s-BuOH
88
88 (60)c
9
1.5
s-BuOH
N.D.d
-
10
11
12
2.0
c.HCl (3.0)
c.HCl (3.0)
c.HCl (3.0)
s-BuOH
8
26
-
2.0
s-BuOH/H2O 95:5
s-BuOH/H2O 95:5
8
79
-
2.4
11
87
86 (71)c
aHPLC assay yield. bReagents were used based on the amount of 8 (15: 1.2 equiv, c.HCl: 3.0 equiv). cIsolated yield in parentheses. dNot detected.
analyzed by chiral SFC, which showed that the obtained 19 did
not contain three other potential stereoisomers ((S)-enantiomer,
(R)-cis isomer, (S)-cis isomer).
synthesis provided 15 in a simple way without concern for
generation of stereoisomers. The last coupling reaction of 3-
chloro-5,6-dihydroimidazolo[1,5-f]pteridine 8 with aniline 15
was scrutinized to find feasible reaction conditions and to
improve the low yields of the medicinal chemistry synthesis.
Consequently, much higher yields were achieved both by the
palladium-catalyzed amination and the acid-promoted coupling
under benign reaction conditions. Although the acid-promoted
coupling seems to be preferable for scale-up from the viewpoint
of cost at this point, future development of the palladium-
catalyzed amination might enable further decrease of the amount
of a palladium catalyst and render the palladium-catalyzed
amination a more cost-effective procedure. The obtained 19 did
not include stereoisomers, and column chromatography/HPLC
separation was not required. These findings will lead to a
successful future scale-up synthesis and development of a potent
anticancer drug candidate 19, and at the same time, will
3. Conclusions
A
practical chromatography-free synthesis of 19 was
successfully developed. We studied cyanoimidazole ring
formation closely and showed that the cryogenic condition was
not imperative for the reaction provided that appropriate care was
taken for the potential epimerization of 8. This result will allow
us to conduct this reaction on a large scale using a normal
manufacturing facility. An alternative synthesis of 15 utilizing
the advantage of the defined stereochemistry of commercially
available trans diamine 22 was also developed. The piperazine
ring was successfully constructed from primary diamine 20·HCl
without protection. Although the piperazine ring formation
requires some improvements to achieve higher yield, this

contribute to syntheses of related 5,6-dihydroimidazolo[1,5-
3 °C/min to 140 °C, 30 °C/min to 200 °C for 5 min. Retention
time: 7 (19.7 min).
ACCEPTED MANUSCRIPT
f]pteridine derivatives.
4. Experimental section
4.1 General
4.3 Methyl (2R)-2-(Propan-2-ylamino)butanoate Hydrochloride
(1:1) (3·HCl)
To a mixture of methyl (2R)-2-aminobutanoate hydrochloride
(1:1) (2·HCl) (14.7 g, 95.7 mmol) in THF (59 mL) were added
acetic acid (23.0 g, 383 mmol) and acetone (7.23 g, 124 mmol),
and the mixture was stirred at 20–30 °C for 1 h under N2
atmosphere. To the mixture was added sodium borohydride (3.62
g, 95.7 mmol) portionwise at 10 °C, maintaining the temperature
below 20 °C. After being stirred at 20–30 °C for 2 h, the mixture
was cooled to 0–10 °C, and H2O (44 mL) was slowly added. To
the mixture was added 8 M NaOH (54 mL), and pH was adjusted
to 9.0–9.5, maintaining the temperature below 30 °C. Toluene
(74 mL) was added, and the layers were separated. The organic
layer was filtered and insoluble matter was washed with toluene
(7 mL). To the combined filtrate were added seed crystals of
3·HCl (15.0 mg) at 0–10 °C. To the mixture was added 4 M HCl
in EtOAc (35.9 mL, 144 mmol) dropwise, and the mixture was
stirred at 0–10 °C for 1 h. The resulting solids were collected by
filtration and washed with EtOAc (29 mL) and dried in vacuo at
50 °C to give 3·HCl (15.1 g, 81%) as a white solid. Mp 155–
All materials were purchased from commercial suppliers and
used without any additional purification. NH silica gel
(CHROMATOREX) was purchased from Fuji Silysia Chemical
Ltd. Silica gel (Silica Gel 60, spherical) was purchased from
Nacalai Tesque, Inc. Celite® (No. 500) and DARCO® (G-60)
were purchased from Wako Pure Chemical Industries, Ltd.
Melting points were determined on a Stanford Research Systems
OptiMelt MPA 100, and are uncorrected unless otherwise noted.
1H and 13C NMR spectra were recorded on a BRUKER
AVANCE 600 spectrometer or a BRUKER AVANCE 500
spectrometer with tetramethylsilane as an internal standard.
Chemical shifts are shown in ppm. HPLC analysis of the
compounds and reaction monitoring was carried out on a
Shimadzu LC-2010CHT. High-resolution mass spectrometry
(HRMS) data was obtained on a Shimadzu Prominence UFLC
system with a Thermofisher LTQ Orbitrap Discovery. IR spectra
were recorded on a Thermo Electron FT-IR Nicolet 4700 (ATR).
Data of elemental analyses, data of melting points for 35 and
20·HCl (determined by differential scanning calorimetry: DSC),
data of HRMS for 7, 23 and 21, and IR spectra of 7, 35, 20·HCl,
23, 21 and 19 were obtained by Sumika Chemical Analysis
Service, Ltd.
1
157 °C; H NMR (600 MHz, DMSO-d6) δ 0.92 (t, J = 7.6 Hz,
3H), 1.28 (dd, J = 6.4, 4.2 Hz, 6H), 1.89 (dquin, J = 14.4, 7.5 Hz,
1H), 1.98–2.08 (m, 1H), 3.28 (br s, 1H), 3.78 (s, 3H), 4.04 (br s,
1H), 9.18 (br s, 1H), 9.87 (br s, 1H); 13C NMR (151 MHz,
DMSO-d6) δ 9.2, 18.1, 19.1, 22.6, 49.0, 52.8, 57.3, 169.3; IR
(ATR) 2969, 2650, 2504, 1736, 1561, 1470, 1436, 1389, 1314,
1264, 1219, 1165, 1117, 1011, 959, 924, 852, 798, 753, 653, 535,
455 cm−1; Anal. Calcd for C8H18ClNO2: C, 49.10; H, 9.27; Cl,
18.12; N, 7.16; O, 16.35. Found: C, 49.08; H, 9.03; N, 7.35.
4.2 HPLC, Chiral HPLC, chiral SFC, and GC conditions
HPLC conditions. (A) Inertsil ODS-3 column, 5 µm, 150 mm
× 4.6 mm i.d.; UV detector at 254 nm; isocratic elution with
MeCN/50 mM aqueous KH2PO4 (50:50) at 1.0 mL/min flow rate;
column temperature: 25 °C. Retention times: 6 (4.0 min), 31
(10.2 min).
4.4 Methyl (2R)-2-[(2-Chloro-5-nitropyrimidin-4-yl)(propan-2-
yl)amino]butanoate (5)
To a mixture of 2,4-dichloro-5-nitropyrimidine (4) (50.0 g,
258 mmol) and 3·HCl (55.5 g, 284 mmol) in toluene (500 mL)
was added NaHCO3 (54.1 g, 644 mmol), and the mixture was
stirred at 85–95 °C for 3 h under N2 atmosphere. After cooling to
room temperature, the mixture was filtered and insoluble matter
was washed with toluene (50 mL). To the combined filtrate was
added H2O (250 mL), and the layers were separated. To the
organic layer were added EtOAc (250 mL) and 1 M HCl (250
mL), and the layers were separated. The organic layer was
concentrated in vacuo until the weight of the mixture became
approximately 100 g. To this mixture was added 2-propanol (250
mL), and the mixture was concentrated in vacuo again until the
weight of the mixture became approximately 100 g. To the
resulting mixture were added 2-propanol (200 mL) and seed
crystals of 5 (50.0 mg), and the mixture was stirred at 20–30 °C
for 1 h. The mixture was cooled to 0–10 °C and stirred for 1 h,
and then filtrated. Wet solids were washed with 2-propanol/H2O
(1:1, 100 mL) and dried in vacuo at 50 °C to give 5 (59.7 g, 73%)
(B) YMC A-313 column, 5 µm, 250 mm × 6.0 mm i.d.; UV
detector at 254 nm; isocratic elution with MeCN/10 mM aqueous
AcONH4 (40:60) at 1.0 mL/min flow rate; column temperature:
25 °C. Retention times: 15 (8.3 min), 20·HCl (5.4 min), 36 (41
min).
Chiral HPLC conditions. (A) CHIRALCEL OJ-RH, 5 µm,
150 mm × 4.6 mm i.d.; UV detector at 254 nm; isocratic elution
with MeCN/20 mM aqueous KH2PO4 (40:60) at 1.0 mL/min flow
rate; column temperature: 25 °C. Retention times: 5 (11.8 min),
ent-5 (15.1 min).
(B) CHIRALCEL OJ-RH, 5 µm, 150 mm × 4.6 mm i.d.; UV
detector at 254 nm; isocratic elution with MeCN/20 mM aqueous
KH2PO4 (30:70) at 1.0 mL/min flow rate; column temperature:
25 °C. Retention times: 6 (8.2 min), ent-6 (5.7 min).
(C) CHIRALCEL OD-RH, 5 µm, 150 mm × 4.6 mm i.d.; UV
detector at 254 nm; isocratic elution with MeOH at 0.7 mL/min
flow rate; column temperature: 25 °C. Retention times: 8 (5.0
min), ent-8 (6.5 min).
1
as a yellow solid. Mp 89–91 °C; H NMR (600 MHz, CDCl3) δ
1.07 (t, J = 7.6 Hz, 3H), 1.31 (d, J = 6.4 Hz, 3H), 1.36 (d, J = 6.4
Hz, 3H), 1.86–2.04 (m, 1H), 2.37–2.56 (m, 1H), 3.55 (dt, J =
13.1, 6.5 Hz, 1H), 3.75 (s, 3H), 3.78 (t, J = 6.8 Hz, 1H), 8.63 (s,
1H); 13C NMR (151 MHz, CDCl3) δ 12.0, 19.5, 21.6, 23.2, 52.5,
53.7, 58.8, 131.0, 153.5, 156.5, 159.4, 170.8; IR (ATR) 1739,
1572, 1543, 1513, 1466, 1439, 1374, 1345, 1311, 1215, 1197,
1180, 1165, 1084, 993, 912, 866, 767, 559, 445 cm−1; HRMS
(ESI): [M+H]+ calcd for C12H18ClN4O4, 317.1011; found,
317.1008. Optical purity: 99.9% ee (chiral HPLC condition A).
Chiral SFC condition. CHIRALPAK AD-H, 5 µm, 250 mm ×
4.6 mm i.d.; UV detector at 220 nm; isocratic elution with
CO2/EtOH/diethylamine (700:300:3) at 3.0 mL/min flow rate;
pressure: 100 bar; column temperature: 35 °C. Retention times:
19 (20.3 min), ent-19 (13.3 min), cis isomer of 19 (9.3 min), cis
isomer of ent-19 (7.7 min).
GC condition. SUPELCO SPB®-5, Capillary GC Column, 30
m × 0.53 mm i.d., 5 µm film; FID detector; He carrier gas
(approximately 6 mL/min); oven heating 90 °C for 5 min,
4.5 (7R)-2-Chloro-7-ethyl-8-(propan-2-yl)-7,8-dihydropteridin-
6(5H)-one (6)

10
Tetrahedron
ACCEPTED MANUSCRIPT
To a mixture of 5 (20.0 g, 63.1 mmol) and reduced iron
chlorophosphate (6.76 g, 39.2 mmol) dropwise at −5 °C, and the
(14.1 g, 253 mmol) in toluene (100 mL) were added AcOH (1.14
g, 18.9 mmol) and H2O (10 mL) in this order, and the mixture
was stirred at 70–80 °C for 3 h under N2 atmosphere. To the
mixture was added 6 M HCl (100 mL) with vigorous stirring, and
the mixture was stirred at 70–80 °C for 1 h. After cooling to
room temperature, the mixture was filtered and insoluble matter
was washed with H2O (40 mL) and EtOAc (40 mL). To the
combined filtrate were added EtOAc (160 mL) and H2O (60 mL),
and the layers were separated. The aqueous layer was extracted
with EtOAc (2 × 200 mL), and the combined organic layer was
washed with 10% aqueous NaCl (2 × 100 mL) and saturated
aqueous NaHCO3 (200 mL). The organic layer was concentrated
in vacuo until the weight of the mixture became approximately
60 g. EtOH (100 mL) was added and the mixture was
concentrated in vacuo until the weight of the mixture became
approximately 60 g. EtOH (100 mL) was added and the mixture
was concentrated in vacuo again until the weight of the mixture
became approximately 60 g. To the resulting mixture was added
EtOH (20 mL), and H2O (90 mL) was added dropwise. The
mixture was cooled to 0–10 °C and stirred for 1 h, and then
filtrated. Wet solids were washed with EtOH/H2O (1:2, 40 mL)
and dried in vacuo at 50 °C to give 6 (12.7 g, 80%) as a white
solid. Mp 200–201 °C; 1H NMR (600 MHz, CDCl3) δ 0.94 (t, J =
7.6 Hz, 3H), 1.37 (d, J = 6.8 Hz, 3H), 1.41 (d, J = 6.8 Hz, 3H),
1.81 (dt, J = 14.5, 7.5 Hz, 1H), 1.99 (ddd, J = 14.5, 7.5, 3.0 Hz,
1H), 4.28 (dd, J = 7.4, 3.2 Hz, 1H), 4.59 (spt, J = 6.8 Hz, 1H),
7.71 (s, 1H), 9.80 (br s, 1H); 13C NMR (151 MHz, CDCl3) δ 8.6,
19.8, 20.9, 27.7, 49.2, 58.6, 117.8, 139.1, 151.9, 154.4, 165.8; IR
(ATR) 3229, 2964, 1692, 1655, 1604, 1476, 1410, 1365, 1269,
1237, 1194, 1155, 1127, 1088, 1002, 932, 773, 688, 558, 411,
401 cm−1; HRMS (ESI): [M+H]+ calcd for C11H16ClN4O,
255.1007; found, 255.1007. Optical purity: 99.4% ee (chiral
HPLC condition B).
mixture was stirred at −5 °C for 0.5 h. To the mixture was added
7 (4.96 g, 88.1 wt%, 39.3 mmol), and then LHMDS (1.1 M THF
solution, 53.5 mL, 58.9 mmol) was added dropwise at −5 °C, and
the mixture was stirred at −5 °C for 1 h. To the mixture was
added AcOH (30 mL) dropwise, maintaining the temperature
below 20 °C, and the mixture was stirred at 40–50 °C for 3 h.
After cooling to room temperature, H2O (50 mL), EtOAc (50
mL) and THF (50 mL) were added, and the layers were separated.
The organic layer was washed with 1 M NaOH (3 × 50 mL) and
H2O (50 mL), and concentrated in vacuo until the weight of the
mixture became approximately 30 g. EtOAc (50 mL) was added,
and the mixture was concentrated in vacuo until the weight of the
mixture became approximately 25 g. EtOAc (50 mL) was added
and the mixture was concentrated in vacuo again until the weight
of the mixture became approximately 25 g. To the mixture was
added diisopropyl ether (75 mL) dropwise at 40–50 °C, and the
mixture was stirred at 40–50 °C for 1 h. The mixture was
gradually cooled to 0–10 °C and stirred for 1 h, and then filtrated.
Wet solids were washed with diisopropyl ether (25 mL) and
dissolved in THF (150 mL). To the mixture was added activated
carbon (500 mg) at 40–50 °C, and the mixture was stirred at 40–
50 °C for 1 h. Activated carbon was filtered off and washed with
THF (10 mL). The combined filtrate was concentrated in vacuo
until the weight of the mixture became approximately 25 g.
EtOH (50 mL) was added and the mixture was concentrated in
vacuo again until the weight of the solution became
approximately 25 g. To the resulting mixture was added EtOH
(20 mL), and then H2O (50 mL) was added dropwise at 20–30 °C.
The mixture was stirred at 20–30 °C for 1 h and then filtrated.
Wet solids were washed with H2O (25 mL) and dried in vacuo at
50 °C to give 8 (4.40 g, 74%) as a brown solid. Mp 252–253 °C
(decomp); 1H NMR (600 MHz, CDCl3) δ 0.84 (t, J = 7.4 Hz, 3H),
1.44 (d, J = 6.8 Hz, 3H), 1.48 (d, J = 6.8 Hz, 3H), 1.84 (dquin, J
= 14.6, 7.5 Hz, 1H), 1.97 (ddd, J = 14.4, 7.6, 3.4 Hz, 1H), 4.68
(quin, J = 6.9 Hz, 1H), 5.11 (dd, J = 8.3, 3.4 Hz, 1H), 8.02 (s,
1H), 8.36 (s, 1H); 13C NMR (151 MHz, CDCl3) δ 8.6, 20.1, 20.8,
30.6, 50.6, 51.6, 109.9, 113.4, 114.3, 131.6, 133.1, 141.2, 152.8,
158.2; IR (ATR) 3106, 2970, 2938, 2223, 1605, 1536, 1492,
1419, 1402, 1377, 1362, 1341, 1331, 1236, 1142, 1114, 1081,
962, 755, 651, 586, 507, 463 cm−1; HRMS (ESI): [M+H]+ calcd
for C14H16ClN6, 303.1119; found, 303.1118. Optical purity:
99.8% ee (chiral HPLC condition C).
4.6 N'-(Cyanomethyl)-N,N-dimethylimidoformamide (7)
To a mixture of aminoacetonitrile hydrochloride (1:1)
(26·HCl) (10.0 g, 108 mmol) in THF (100 mL) were added N,N-
dimethylformamide dimethyl acetal (16.7 g, 141 mmol) and
triethylamine (16.4 g, 162 mmol), and the mixture was stirred at
45–55 °C for 5 h. After cooling to room temperature, the mixture
was concentrated in vacuo until the weight of the mixture became
approximately 40 g. Diisopropyl ether (50 mL) was added and
the mixture was concentrated in vacuo again until the weight of
the mixture became approximately 40 g. To the resulting mixture
was added diisopropyl ether (100 mL), and the mixture was
passed through a pad of NH silica gel (100–200 mesh, 5.00 g),
followed by washing with IPE (2 × 100 mL). The combined
filtrate was concentrated in vacuo to give 7 (8.75 g, 88.1 w/w%,
64% GC assay yield) as a pale yellow oil. The obtained 7 was
used for the next reaction without further purification. A small
portion of the product was purified by distillation (5.6 mmHg,
4.8 tert-Butyl (trans-4-Aminocyclohexyl)carbamate (33)14
To a mixture of trans-cyclohexane-1,4-diamine (22) (150 g,
1.31 mol) in MeCN (2.7 L) was added (Boc)2O (143 g, 657
mmol) in MeCN (300 mL) maintaining the temperature below
30 °C, and the mixture was stirred at 20–30 °C for 3 h. The
resulting slurry was concentrated in vacuo. To the residue was
added saturated aqueous NaHCO3 (3 L), and the mixture was
stirred at 20–30 °C for 1 h, and then filtrated. Wet solids were
washed with H2O (400 mL) and dried in vacuo at 50 °C to give
33 (33:34 = approximately 3:1, 129 g, 46%) as a white solid. The
obtained 33 was used for the next reaction without further
purification.
1
90 °C) to obtain a sample for analysis. H NMR (500 MHz,
CDCl3) δ 2.90 (br s, 6H), 4.18 (s, 2H), 7.44 (s, 1H); 13C NMR
(126 MHz, CDCl3) δ 34.6 (br s), 40.0 (br s), 41.5, 118.1, 157.5;
IR (ATR) 2917, 1639, 1489, 1432, 1415, 1377, 1326, 1260, 1233,
1114, 1066, 1042, 1017, 984, 970, 903, 865 cm−1; HRMS (ESI):
[M+H]+ calcd for C5H10N3, 112.0869; found, 112.0875.
4.9 4-Amino-N-(trans-4-aminocyclohexyl)-2-fluoro-5-
methoxybenzamide Hydrochloride (1:1) (20·HCl)
4.7 (6R)-3-Chloro-6-ethyl-5-(propan-2-yl)-5,6-
dihydroimidazo[1,5-f]pteridine-7-carbonitrile (8)
To a mixture of 4-amino-2-fluoro-5-methoxybenzoic acid 16
(65.8 g, 355 mmol), 33 (129 g, 604 mmol), N,N-
diisopropylethylamine (91.2 mL, 533 mmol) and 1-
hydroxybenzotriazole monohydrate (65.2 g, 426 mmol) in MeCN
To a mixture of 6 (5.00 g, 19.6 mmol) in THF (50 mL) was
added LHMDS (1.1 M THF solution, 19.6 mL, 21.6 mmol)
dropwise at −5 °C, and the mixture was stirred at −5 °C for 0.5 h
under N2 atmosphere. To the mixture was added diethyl
(1.32
L)
was
added
1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride (81.7 g, 426

mmol), and the mixture was stirred at 40 ˚C for 2 h. To the
layer were added Na2SO4 (500 g, 3.52 mol) and activated carbon
(30 g), and the mixture was stirred at room temperature for 30
min. The mixture was filtrated and insoluble matter was washed
with toluene, and the combined filtrate was concentrated in vacuo.
To the residue was added toluene (300 mL), and the mixture was
passed through a pad of silica gel, and insoluble matter was
washed with toluene (200 mL). The combined filtrate was
concentrated in vacuo to give 21 (343 g, 91.6 wt% determined by
1H NMR using diphenylmethane as an internal standard, 66% for
two steps from 24, pale brown oil). The obtained 21 was used for
the next reaction without further purification. A small portion of
the obtained 21 was purified by distillation (4 mmHg, 88 °C) to
ACCEPTED MANUSCRIPT
mixture was added H2O (1.32 L) dropwise, and the mixture was
stirred at 20–30 °C for 1 h, and then filtrated. Wet solids were
washed with H2O (526 mL) and dissolved in MeOH (1.32 L). To
the mixture was added 6 M HCl (526 mL), and the mixture was
stirred at 40 °C for 3 h. After cooling to 5 °C, 8 M NaOH (324
mL) was added dropwise and pH of the mixture was adjusted to
7.8, maintaining the temperature below 20 °C. To the mixture
was added activated carbon (6.60 g), and the mixture was stirred
at 20–30 °C for 0.5 h. Activated carbon was filtered off and
washed with MeOH (132 mL). The combined filtrate was
concentrated in vacuo to remove MeOH. The resulting slurry was
stirred at 20–30 °C for 0.5 h, and then filtrated. Wet solids were
washed with acetone (132 mL) and dried in vacuo at 50 °C to
give 20·HCl (92.0 g, 82% for 2 steps) as a brown solid. Mp
1
obtain a sample for analysis. H NMR (500 MHz, CDCl3) δ;
0.06–0.19 (m, 2H), 0.47–0.59 (m, 2H), 0.85 (ttt, J = 8.1, 6.5, 5.0
Hz, 1H), 2.49 (d, J = 6.6 Hz, 2H), 2.96 (t, J = 7.3 Hz, 4H), 3.54 (t,
J = 7.3 Hz, 4H); 13C NMR (126 MHz,CDCl3) δ 3.9 (2C), 9.2,
42.0 (2C), 56.5 (2C), 59.8; IR (ATR) 3079, 3001, 2960, 2817,
1462, 1447, 1299, 1251, 1102, 1051, 1019, 936, 828, 802, 765,
726, 661 cm−1; HRMS (ESI): [M+H]+ calcd for C8H16Cl2N,
196.0654; found, 196.0630. A small portion of the obtained 23
was purified by distillation (3 mmHg, 134 °C) to obtain a sample
1
309 °C (decomp.) (DSC); H NMR (500 MHz, DMSO-d6) δ
1.29–1.53 (m, 4H), 1.84–1.92 (m, 2H), 2.01 (br d, J = 10.4 Hz,
2H), 2.92 (ddd, J = 11.4, 7.5, 4.1 Hz, 1H), 3.60–3.72 (m, 1H),
3.77 (s, 3H), 5.61 (s, 2H), 6.40 (d, J = 13.2 Hz, 1H), 7.03 (d, J =
6.9 Hz, 1H), 7.32–7.54 (m, 1H), 8.30 (br s, 3H); 13C NMR (126
MHz, DMSO-d6) δ 29.6 (2C), 30.4 (2C), 47.6, 49.1, 56.3, 99.8
(2JCF = 29.0 Hz), 108.3 (2JCF = 13.9 Hz), 111.4 (3JCF = 5.0 Hz),
142.4, 142.9 (3JCF = 12.6 Hz), 156.0 (1JCF = 239.4 Hz), 163.3
(3JCF = 2.5 Hz); IR (ATR) 3428, 3310, 2926, 1622, 1607, 1541,
1510, 1319, 1256, 1217, 1169, 1022, 874, 853, 768, 729 cm−1;
HRMS (ESI): [M+H]+ calcd for C14H21FN3O2 (free form),
282.1612; found, 282.1610. A sample of tert-butyl {trans-4-[(4-
amino-2-fluoro-5-methoxybenzoyl)amino]cyclohexyl}carbamate
(35) for analysis was prepared by the reaction of 20·HCl with 1.0
equiv of Boc2O in EtOAc/5% aqueous NaHCO3. Data for 35: Mp
248 °C (DSC); 1H NMR (500 MHz, CDCl3) δ 1.22–1.38 (m, 4H),
1.44 (s, 9H), 2.01–2.08 (m, 2H), 2.08–2.16 (m, 2H), 3.45 (br s,
1H), 3.88 (s, 3H), 3.90–3.99 (m, 1H), 4.26 (s, 2H), 4.43 (br d, J =
6.3 Hz, 1H), 6.36 (d, J = 13.2 Hz, 1H), 6.50 (br dd, J = 15.0, 7.7
Hz, 1H), 7.44 (d, J = 6.9 Hz, 1H); 13C NMR (126 MHz, CDCl3) δ
28.4 (3C), 31.8 (2C), 32.1 (2C), 48.0, 49.0, 56.0, 79.2, 100.7
(2JCF = 30.2 Hz), 108.9 (2JCF = 11.3 Hz), 111.8 (3JCF = 3.8 Hz),
141.1 (3JCF = 12.6 Hz), 143.2, 155.3, 156.4 (1JCF = 239.4 Hz),
163.1 (3JCF = 2.5 Hz); IR (ATR) 3497, 3468, 3317, 2941, 1686,
1620, 1508, 1450, 1366, 1315, 1275, 1261, 1213, 1157, 1072,
1020, 766, 727, 652 cm−1; HRMS (ESI): [M+H]+ calcd for
C19H29FN3O4, 382.2137; found, 382.2131.
1
for analysis. Data for 23: H NMR (500 MHz, CDCl3) δ; 0.08–
0.16 (m, 2H), 0.45–0.60 (m, 2H), 0.89 (ttt, J = 8.1, 6.6, 5.0 Hz,
1H), 2.45 (d, J = 6.6 Hz, 2H), 2.73–2.79 (m, 4H), 3.57–3.71 (m,
4H); 13C NMR (126 MHz,CDCl3) δ 4.0 (2C), 8.8, 56.0 (2C), 59.6,
59.6 (2C); IR (ATR) 3347, 2939, 2876, 2820, 1033, 888, 873,
828 cm−1; HRMS (ESI): [M+H]+ calcd for C8H18NO2, 160.1332;
found, 160.1335.
4.11 4-Amino-N-{trans-4-[4-(cyclopropylmethyl)piperazin-1-
yl]cyclohexyl}-2-fluoro-5-methoxybenzamide (15)
To a mixture of 20·HCl (10.0 g, 31.5 mmol), K2CO3 (13.1 g,
94.8 mmol) and NaI (4.72 g, 31.5 mmol) in EtOH (50 mL) was
added 21 (7.40 g, 37.7 mmol), and the mixture was stirred at 20–
30 °C for 15 h. To the mixture were added EtOAc (50 mL), THF
(50 mL) and H2O (100 mL), and the layers were separated. To
the organic layer were added 10% aqueous NaCl (90 mL) and 12
M HCl (13 mL) maintaining the temperature below 30 °C, and
the layers were separated. The aqueous layer was concentrated in
vacuo to remove EtOH and THF. To the resulting aqueous layer
was added activated carbon (1.00 g), and the mixture was stirred
at 20–30 °C for 0.5 h. Activated carbon was filtered off, and
washed with H2O (20 mL). To the combined filtrate were added
EtOAc (50 mL) and THF (50 mL). To the mixture was added
25% aqueous NH3 (18 mL) dropwise maintaining the
temperature below 30 °C, and the layers were separated. The
organic layer was washed with saturated aqueous NaCl (50 mL),
dried over MgSO4, and concentrated in vacuo. To the residue was
added EtOH (60 mL), and 15% HCl in EtOH (40 mL) was added
dropwise at 20–30 °C. The resulting slurry was stirred at 20–
30 °C for 1 h, and 5–10 °C for 1 h, and then filtrated. Wet solids
were washed with EtOH (15 mL), and dissolved in H2O (65 mL).
To the mixture was added activated carbon (600 mg), and the
mixture was stirred at 20–30 °C for 0.5 h. Activated carbon was
filtered off, and washed with H2O (10 mL). To the combined
filtrate was added 25% aqueous NH3 (3.1 mL), and pH was
adjusted to 9.0. The resulting slurry was stirred at 20–30 °C for 1
h and then filtrated. Wet solids were washed with H2O (13 mL)
and dried in vacuo at 50 °C to give 15 (4.73 g, 37%) as a white
4.10 2-Chloro-N-(2-chloroethyl)-N-
(cyclopropylmethyl)ethanamine (21)
To a mixture of 2,2'-iminodiethanol 24 (400 g, 3.80 mol) in
EtOH (4 L) were added NaI (684 g, 4.57 mol),
(chloromethyl)cyclopropane (413 g, 4.57 mol), and K2CO3 (789
g, 5.71 mol), and the mixture was stirred under reflux for 5.5 h.
After cooling to room temperature, the resulting solids were
filtered off and washed with EtOAc. The combined filtrate was
concentrated in vacuo. To the residue were added EtOAc (8 L)
and saturated brine (1.6 L), and the layers were separated. The
aqueous layer was extracted with EtOAc (2 × 4 L), and to the
combined organic layer was added Na2SO4 (400 g, 2.82 mol),
and the mixture was stirred at room temperature for 10 min. The
mixture was filtrated and insoluble matter was washed with
EtOAc. The combined filtrate was concentrated in vacuo to give
2,2'-[(cyclopropylmethyl)imino]diethanol 23 (472 g, pale yellow
oil). The obtained 23 was used for the next reaction without
further purification. To a solution of 23 (300 g, 1.88 mol) in
toluene (3 L) was added thionyl chloride (448 g, 3.77 mol)
dropwise maintaining the temperature below 10 °C, and the
mixture was stirred at 50 °C for 3 h. After cooling to room
temperature, the mixture was neutralized by slow addition of 8 M
NaOH (1.2 L), and the layers were separated. To the organic
1
solid. Mp 184–185 °C; H NMR (600 MHz, CDCl3) δ 0.10 (q, J
= 4.9 Hz, 2H), 0.45–0.57 (m, 2H), 0.82–0.92 (m, 1H), 1.20–1.31
(m, 2H), 1.43 (qd, J = 12.5, 3.0 Hz, 2H), 1.97 (br d, J = 12.5 Hz,
2H), 2.16 (br d, J = 11.3 Hz, 2H), 2.25 (d, J = 6.8 Hz, 2H), 2.27–
2.32 (m, 1H), 2.64 (br s, 8H), 3.87 (s, 3H), 3.89–3.95 (m, 1H),
4.27 (s, 2H), 6.36 (d, J = 13.2 Hz, 1H), 6.50 (br dd, J = 14.9, 7.7
Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H); 13C NMR (151 MHz, CDCl3) δ

12
Tetrahedron
ACCEPTED MANUSCRIPT
3.9 (2C), 8.3, 27.3 (2C), 32.3 (2C), 48.7, 49.0 (2C), 53.7 (2C),
extracted with EtOAc (60 mL). The organic layer was washed
56.0, 62.7, 63.8, 100.6 (2JCF = 31.7 Hz), 108.9 (2JCF = 12.1 Hz),
with 5% aqueous NaHCO3 (3 × 60 mL) and concentrated in
vacuo. To the residue was added 2-propanol (30 mL), and the
mixture was stirred at 40–50 °C for 1 h. The mixture was
gradually cooled to 0–10 °C and stirred for 1 h, and then filtrated.
Wet solids were washed with 2-propanol/n-heptane (1:3, 9 mL)
and dissolved in MeOH (60 mL). To the mixture was added
DARCO® (G-60, 600 mg), and the mixture was stirred at 40–
50 °C for 1.5 h. DARCO® was filtered off, and the filtrate was
concentrated in vacuo. To the residue was added 2-propanol (15
mL), and the mixture was stirred at 40–50 °C for 1 h. The
mixture was gradually cooled to 0–10 °C and stirred for 1 h, and
then filtrated. Wet solids were washed with n-heptane (9 mL) and
dried in vacuo at 50 °C to give 19 (3.54 g, 71%) as a pale gray
solid.
111.7 (3JCF = 3.0 Hz), 141.0 (3JCF = 13.6 Hz), 143.1, 156.4 (1JCF
=
238.6 Hz), 163.0 (3JCF = 4.5 Hz); IR (ATR) 3481, 3346, 2937,
2809, 1738, 1619, 1512, 1451, 1309, 1236, 1214, 1160, 1005,
871, 836, 797, 766, 729, 584, 523 cm−1; HRMS (ESI): [M+H]+
calcd for C22H34FN4O2, 405.2660; found, 405.2659.
4.12 Synthesis of 4-{[(6R)-7-Cyano-6-ethyl-5-(propan-2-yl)-5,6-
dihydroimidazo[1,5-f]pteridin-3-yl]amino}-N-{trans-4-[4-
(cyclopropylmethyl)piperazin-1-yl]cyclohexyl}-2-fluoro-5-
methoxybenzamide (19) by coupling of 8 with 15 using a
palladium catalyst
To a mixture of 15 (1.00 g, 2.47 mmol), 8 (823 mg, 2.72
mmol) and potassium carbonate (683 mg, 4.94 mmol) in s-
butanol (10 mL) were added Xantphos (143 mg, 0.247 mmol)
and palladium(II) acetate (55.5 mg, 0.247 mmol), and the
mixture was stirred at 85 °C for 1 h. After cooling to room
temperature, H2O (15 mL) and 6 M HCl (3 mL) were added, and
the mixture was filtered through a pad of Celite® (No. 500, 2.00
g). To the filtrate was added EtOAc (10 mL), and the layers were
separated. To the aqueous layer was added 8 M NaOH (1.5 mL)
and pH was adjusted to 9.5. The resulting aqueous layer was
extracted with EtOAc/THF (3:2, 25 mL). The organic layer was
washed with 25% aqueous NH3 (10 mL) and saturated aqueous
NaCl (10 mL). The organic layer was filtrated to remove
insoluble matter, and the filtrate was concentrated in vacuo. To
the residue was added 2-propanol (10 mL), and the mixture was
concentrated in vacuo. To the residue was added 2-propanol (5
mL), and the mixture was stirred at 20–30 °C for 1 h, and then
filtrated. Wet solids were washed with 2-propanol (2 mL) and
H2O (2 mL), and dried in vacuo at 50 °C to give 19 (1.30 g, 78%)
Acknowledgments
We are grateful to Mr. Keiichi Satou for LC/MS analysis, and
Ms. Rie Kasai for chiral SFC analysis, and Dr. Makoto
Kanematsu for his advice on the manuscript.
Appendix A. Supplementary Data
Supplementary data related to this article can be found at
References and notes
1. For recent reviews, see: (a) Lens, S. M. A.; Voest, E. E.; Medema,
R. H. Nat. Rev. Cancer 2010, 10, 825–841. (b) Wurzenberger, C.;
Gerlich, D.W. Nat. Rev. Mol. Cell Biol. 2011, 12, 469–482. (c)
Bruinsma, W.; Raaijmakers, J. A.; Medema, R. H. Trends
Biochem. Sci. 2012, 37, 534–542. (d) Zitouni, S.; Nabais, C.; Jana,
S. C.; Guerrero, A.; Bettencourt-Dias, M. Nat. Rev. Mol. Cell Biol.
2014, 15, 433–452. (e) Archambault, V.; Lépine, G.; Kachaner, D.
Oncogene 2015, 34, 4799–4807.
2. For recent reviews, see: (a) Strebhardt, K. Nat. Rev. Drug Discov.
2010, 9, 643–660. (b) Christoph, D. C.; Schuler, M. Expert Rev.
Anticancer Ther. 2011, 11, 1115–1130. (c) Mclnnes, C.; Wyatt, M.
D. Drug Discov. Today 2011, 16, 619–625. (d) Craig, S. N.; Wyatt,
M. D.; Mclnnes, C. Expert Opin. Drug Discov. 2014, 9, 773–789.
(e) Palmisiano, N. D.; Kasner, M. T. Am. J. Hematol. 2015, 90,
1071–1076. (f) Berg, A.; Berg, T. Chembiochem 2016, 17, 650–
656. (f) Talati, C.; Griffiths, E. A.; Wetzler, M.; Wang, E. S. Crit.
Rev. Oncol. Hematol. 2016, 98, 200–210.
3. (a) Cao, S. X.; Ichikawa, T.; Kiryanov, A. A.; Mcbride, C.; Natala,
S. R.; Kaldor, S. W.; Stafford, J. A. PCT Int. Appl. WO
2010025073, 2010. (b) Kiryanov, A.; Natala, S.; Jones, B.;
McBride, C.; Feher, V.; Lam, B.; Liu, Y.; Honda, K.; Uchiyama,
N.; Kawamoto, T.; Hikichi, Y.; Zhang, L.; Hosfield, D.; Skene,
R.; Zou, H.; Stafford, J.; Cao, X.; Ichikawa, T. Bioorg. Med. Chem.
Lett. 2017, 27, 1311–1315.
4. (a) Hoffmann, M.; Grauert, M.; Brandl, T.; Breitfelder, S.;
Eickmeier, C.; Steegmaier, M.; Schnapp, G.; Baum, A.; Quant, J.
J.; Solca, F.; Colbatzky, F. PCT Int. Appl. WO 2004076454, 2004.
(b) Linz, G.; Kraemer, G. F.; Gutschera, L.; Asche, G. PCT Int.
Appl. WO 2006018220, 2006. (c) Schnaubelt, J.; Herter, R. PCT
Int. Appl. WO 2012049153, 2012.
1
as a pale yellow solid. Mp 240–242 °C; H NMR (600 MHz,
CDCl3) δ 0.07–0.14 (m, 2H), 0.47–0.55 (m, 2H), 0.81–0.92 (m,
4H), 1.21–1.35 (m, 2H), 1.44–1.50 (m, 5H), 1.54 (d, J = 6.8 Hz,
3H), 1.76–1.87 (m, 1H), 1.93 (dtd, J = 14.2, 7.3, 3.4 Hz, 1H),
1.99 (br d, J = 12.5 Hz, 2H), 2.15–2.22 (m, 2H), 2.26 (d, J = 6.8
Hz, 2H), 2.28–2.34 (m, 1H), 2.65 (br s, 8H), 3.90–3.96 (m, 1H),
3.97 (s, 3H), 4.79 (spt, J = 6.8 Hz, 1H), 5.08 (dd, J = 8.3, 3.4 Hz,
1H), 6.61 (br dd, J = 14.9, 7.7 Hz, 1H), 7.58 (d, J = 6.8 Hz, 1H),
7.86 (s, 1H), 7.96 (s, 1H), 8.30–8.37 (m, 1H), 8.43 (d, J = 15.1
Hz, 1H); 13C NMR (151 MHz, CDCl3) δ 3.9 (2C), 8.3, 8.8, 20.2,
21.4, 27.3 (2C), 30.6, 32.3 (2C), 48.8, 49.1 (2C), 49.1, 50.6, 53.7
(2C), 56.3, 62.7, 63.8, 104.9 (2JCF = 36.2 Hz), 109.2, 109.8, 111.1
(3JCF = 4.5 Hz), 112.2 (2JCF = 13.6 Hz), 114.0, 130.8, 133.0,
133.2 (3JCF = 13.6 Hz), 141.2, 143.7, 152.4, 155.6 (1JCF = 238.6
Hz), 157.2, 162.5 (3JCF = 4.5 Hz); IR (nujol) 3406, 2227, 1637,
1623, 1607, 1550, 1521, 1501, 1490, 1447, 1313, 1275, 1252,
1204, 1176, 1154, 1065, 1022, 1007, 964, 875, 780 cm−1; HRMS
(ESI): [M+H]+ calcd for C36H48FN10O2, 671.3940; found,
671.3943. Optical purity: >99.9% ee (chiral SFC condition).
4.13 Synthesis of 4-{[(6R)-7-Cyano-6-ethyl-5-(propan-2-yl)-5,6-
dihydroimidazo[1,5-f]pteridin-3-yl]amino}-N-{trans-4-[4-
(cyclopropylmethyl)piperazin-1-yl]cyclohexyl}-2-fluoro-5-
methoxybenzamide (19) by coupling of 8 with 15 in an acidic
condition
5. The ratio of 19 to 19' of a representative lot prepared by
palladium-catalyzed amination of 8 with 15 was found to be 93:7
before HPLC separation. Our initial target for the amount of 19'
was set as not more than 0.50%.
6. Anderson, N. G. Practical Process Research & Development – A
Guide for Organic Chemists, 2nd ed.; Academic Press: New York,
2012.
7. Leopoldo, M.; Lacivita, E.; Colabufo, N. A.; Berardi, F.; Perrone,
R. J. Pharm. Pharmacol. 2006, 58, 209–218.
8. For other syntheses of 6, see: (a) Duran, A.; Linz, G. PCT Int.
Appl. WO 2006058876, 2006. (b) Grauert, M.; Linz, G.; Schmid,
R.; Sieger, P. PCT Int. Appl. WO 2007090844, 2007. (c) Linz, G.;
Sieger, P.; Schmid, R.; Goepper, S. PCT Int. Appl. WO
2009019205, 2009.
To a mixture of 15 (3.00 g, 7.42 mmol) and 8 (5.39 g, 17.8
mmol) in s-BuOH/H2O (95:5, 30 mL) was added concentrated
hydrochloric acid (2.25 g, 22.3 mmol), and the mixture was
stirred at reflux for 11 h. After cooling to room temperature, the
mixture was concentrated in vacuo. To the residue were added
EtOAc (45 mL) and 1 M HCl (60 mL), and the layers were
separated. The organic layer was extracted with 1 M HCl (30
mL). To the combined aqueous layer was added 8 M NaOH (30
mL) and pH was adjusted to 9. The resulting aqueous layer was

9. Hoffmann, M.; Grauert, M.; Brandl, T.; Breitfelder, S.; Eickmeier,
C.; Steegmaier, M.; Schnapp, G.; Baum, A.; AQuCantC, J.EJ.;PSTolcEa,DF.; MANUSCRIPT
Colbatzky, F. U.S. Pat. Appl. US20040176380, 2004.
10. Rogers-Evans, M.; Spurr, P.; Hennig, M. Tetrahedron Lett. 2003,
44, 2425–2428.
11. Reference 10 reported that it was possible to isolate intermediate
29a after non-aqueous workup.
12. Fryer, R. I.; Kudzma, L. V.; Gu, Z.-Q.; Lin, K.-Y.; Rafalko, P. W.
J. Org. Chem. 1991, 56, 3715–3719.
13. We checked stability of 31 by extending the reaction time for
phosphorylation. Compound 6 was treated with 1.2 equiv of
LHMDS and 1.5 equiv of diethyl chlorophosphate in THF at
−5 °C for 3 h, and the reaction mixture was monitored by HPLC
every hour. HPLC area% of 31 at each hour was as follows: 80.1
area% after 1 h, 80.2 area% after 2 h, and 81.6 area % after 3 h.
These results indicate that 31 was stable up to 3 h at −5 °C.
14. (a) Liu, B.; Liu, G.; Xin, Z.; Serby, M. D.; Zhao, H.; Schaefer, V.
G.; Falls, H. D.; Kaszubska, W.; Collins, C. A.; Sham, H. L.
Bioorg. Med. Chem. Lett. 2004, 14, 5223–5226. (b) Wang, N.;
Xiang, J.; Ma, Z.; Quan, J.; Chen, J.; Yang, Z. J. Comb. Chem.
2008, 10, 825–834. (c) Caron, K.; Lachapelle, V.; Keillor, J. W.
Org. Biomol. Chem. 2011, 9, 185–197. (d) Bailey, S.; Barber, C.
G.; Glossop, P. A.; Middleton, D. S. PCT Int. Appl. WO
2005009966, 2005. (e) Lin, S.; Yang, Z. PCT Int. Appl. WO
2010022159, 2010.
15. For examples of piperazine ring formation, see: (a) Lazar, S.;
Soukri, M.; Leger, J. M.; Jarry, C.; Akssira, M.; Chirita, R.; Grig-
Alexa, I. C.; Finaru, A.; Guillaumet, G. Tetrahedron 2004, 60,
6461–6473. (b) Mutulis, F.; Yahorava, S.; Mutule, I.; Yahorau, A.;
Liepinsh, E.; Kopantshuk, S.; Veiksina, S.; Tars, K.; Belyakov, S.;
Mishnev, A.; Rinken, A.; Wikberg, J. E. S. J. Med. Chem. 2004,
47, 4613–4626. (c) Féau, C.; Klein, E.; Dosche, C.; Kerth, P.;
Lebeau, L. Org. Biomol. Chem. 2009, 7, 5259–5270. (d) Andrews,
M.; Brown, A.; Chiva, J.-Y.; Fradet, D.; Gordon, D.; Lansdell, M.;
MacKenny, M. Bioorg. Med. Chem. Lett. 2009, 19, 2329–2332.
(e) Zheng, Y.-Y.; Xie, P.; Xing, L.-X.; Wang, J.-Y.; Li, J.-Q. Org.
Process Res. Dev. 2012, 16, 1921–1926.
16. Bartkovitz, D. J.; Chu, X.-J.; Ding, Q.; Jiang, N.; Lovey, A. J.;
Moliterni, J. A.; Mullin, J. G. Jr.; Vu, B. T.; Wovkulich, P. M.
PCT Int. Appl. WO 2004069139, 2004
17. Charrier, J. D.; Kay, D.; Knegtel, R. PCT Int. Appl. WO
2008076392, 2008.
18. (a) Wolfe, J. P.; Wagaw, S.; Marcoux, J.-F.; Buchwald, S. L. Acc.
Chem. Res. 1998, 31, 805–818. (b) Hartwig, J. F. Acc. Chem. Res.
1998, 31, 852–860. (c) Hartwig, J. F. Angew. Chem. Int. Ed. 1998,
37, 2046–2067. (d) Yang, B. H.; Buchwald, S. L. J. Organomet.
Chem. 1999, 576, 125–146.