Design and Synthesis of 2-(1-Alkylaminoalkyl)pyrazolo[1,5-a]pyrimidines as New Respiratory Syncytial Virus Fusion Protein Inhibitors

Article abstract of DOI:10.1248/cpb.c19-00895

Respiratory syncytial virus (RSV) is one of the most common causes of lower respiratory tract infections and a significant pathogen for both adults and children. Although two drugs have been approved for the treatment of RSV infections, the low therapeutic index of these drugs have led pharmaceutical companies to develop safe and effective small-molecule anti-RSV drugs. The pyrazolo[1,5-a]pyrimidine series of compounds containing a piperidine ring at the 2-position of the pyrazolo[1,5-a]pyrimidine scaffold are known as candidate RSV fusion (F) protein inhibitor drugs, such as presatovir and P3. The piperidine ring has been revealed to facilitate the formation of an appropriate dihedral angle between the pyrazolo[1,5-a]pyrimidine scaffold and the plane of the amide bond for exertion of anti-RSV activity. A molecular-dynamic study on newly designed compounds with an acyclic chain instead of the piperidine ring proposed and demonstrated a new series of pyrazolo[1,5-a]pyrimidine derivatives, such as 9c with a 1-methyaminopropyl moiety, showing similar dihedral angle distributions to those in presatovir. Compound 9c exhibited potent anti-RSV activity with an EC₅₀ value of below 1nM, which was similar to that of presatovir. A subsequent optimization study on the benzene ring of 9c led to the potent RSV F protein inhibitor 14f with an EC₅₀ value of 0.15nM. The possibility of improving the biological properties of anti-RSV agents by modification at the 7-position of pyrazolo[1,5-a]pyrimidine is also discussed.

Full text of DOI:10.1248/cpb.c19-00895

Vol. 68, No. 4
Chem. Pharm. Bull. 68, 345–362 (2020)
345
Regular Article
Design and Synthesis of 2-(1-Alkylaminoalkyl)pyrazolo[1,5-a]pyrimidines
as New Respiratory Syncytial Virus Fusion Protein Inhibitors
,a
Toru Yamaguchi-Sasaki,* Yunoshin Tamura,b Yuya Ogata,a Takanori Kawaguchi,a
Jun Kurosaka,a Yutaka Sugaya,c Kanako Iwakiri,a Tsuyoshi Busujima,a Ryo Takahashi,a
Naoko Ueda-Yonemoto,c Eiji Tanigawa,c Tomoko Abe-Kumasaka,c Hiroyuki Sugiyama,c and
Kosuke Kanumaa
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama 331–
b
9530, Japan: Discovery Technologies Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku,
c
Saitama 331–9530, Japan: and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho,
Kita-ku, Saitama 331–9530, Japan.
Received October 10, 2019; accepted December 25, 2019
Respiratory syncytial virus (RSV) is one of the most common causes of lower respiratory tract infec-
tions and a significant pathogen for both adults and children. Although two drugs have been approved for
the treatment of RSV infections, the low therapeutic index of these drugs have led pharmaceutical companies
to develop safe and effective small-molecule anti-RSV drugs. The pyrazolo[1,5-a]pyrimidine series of com-
pounds containing a piperidine ring at the 2-position of the pyrazolo[1,5-a]pyrimidine scaffold are known as
candidate RSV fusion (F) protein inhibitor drugs, such as presatovir and P3. The piperidine ring has been
revealed to facilitate the formation of an appropriate dihedral angle between the pyrazolo[1,5-a]pyrimidine
scaffold and the plane of the amide bond for exertion of anti-RSV activity. A molecular-dynamic study on
newly designed compounds with an acyclic chain instead of the piperidine ring proposed and demonstrated a
new series of pyrazolo[1,5-a]pyrimidine derivatives, such as 9c with a 1-methyaminopropyl moiety, showing
similar dihedral angle distributions to those in presatovir. Compound 9c exhibited potent anti-RSV activity
with an EC50 value of below 1nM, which was similar to that of presatovir. A subsequent optimization study
on the benzene ring of 9c led to the potent RSV F protein inhibitor 14f with an EC50 value of 0.15nM. The
possibility of improving the biological properties of anti-RSV agents by modification at the 7-position of
pyrazolo[1,5-a]pyrimidine is also discussed.
Key words respiratory syncytial virus; fusion protein; pyrazolo[1,5-a]pyrimidine; dihedral angle
infection, and management primarily remains supportive,
Introduction
Respiratory syncytial virus (RSV) is one of the leading including oxygen supplementation, fluid administration, and
causes of lower respiratory tract infections, especially in respiratory management.20–23)
infants and young children.1) Primary infection with RSV is
Palivizumab (Synagis®) was initially expected as an effec-
always clinically apparent, and manifests with a variety of tive drug for the treatment of severe respiratory tract inflam-
symptoms, ranging from mild cold-like symptoms to lower mation caused by RSV; however, palivizumab was found to
respiratory tract illnesses, such as atopic asthma, severe not exert significant efficacy, and has been used as a preven-
bronchiolitis, and pneumonia.2–8) Infants under the age of one tive therapy in premature infants and children, who are at a
developing infection for the first time are likely to develop high risk for RSV infection, with underlying diseases of the
serious infection. RSV infection also causes lower respiratory heart and lungs.20,21,24–27) On the other hand, ribavirin is a
tract infection in adults and the elderly with chronic respira- synthetic guanosine nucleoside analogue and is an antiviral
tory and heart disease, and is a major reason for hospitaliza- agent with a broad range of antiviral and immunomodulatory
tion and cause of death.9–13) RSV infection is of particularly effects on DNA or RNA viruses.28–34) However, the usefulness
serious concern in immunocompromised patients, because in of ribavirin is limited by its high cost, by the demonstration
these patients, the infection is difficult to treat and the patients of its no benefit for decreasing the rate of hospitalization or
are at imminent risk of pneumonia and death.14,15) RSV is mortality, and by its relatively high toxicity for health care
distributed worldwide and repeated episodes of RSV infection workers.35–37)
can occur in humans. It has been suggested that more than a
RSV is an RNA virus that belongs to the genus Pneumo-
half of infants less than one year of age and most infants less virus of the family Paramyxoviridae.38) The viral genome
than two years of age are infected with RSV at least once.16,17) of RSV is approximately 12.5 kb in length, consisting of a
There were 33.1 million RSV-related episodes of acute lower negative-sense single-stranded RNA. Eleven proteins are
respiratory tract infection (RSV-ALRI) around the world in encoded in the RSV genome, including fusion (F) protein
2015, with 3.2 million hospitalizations and 59600 in-hospital interacting with nucleolin during viral fusion and attachment
deaths.18) The RSV infection-related mortality rate is next only (G) protein with a CX3C chemokine motif which interacts
to the rate of death from measles and is higher than that from with CX3CR1 in the host cells.39,40) The F protein forms a
influenza.19) There is currently no effective treatment for RSV trimer on the surface of the virus particle and plays a criti-
*To whom correspondence should be addressed. e-mail: too-yamaguchi@taisho.co.jp
© 2020 The Pharmaceutical Society of Japan

346
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
Fig. 1. Structures and Anti-RSV Activities of RSV F Protein Inhibitors
with the Pyrazolo[1,5-a]pyrimidine Scaffold
cal role in the fusion of its envelope membrane with the host
cell membrane, and entry of the virus genome into the host
cell. Palivizumab is a monoclonal antibody that binds to F
protein and inhibits infection by blocking virus binding to/
fusion with the host cells. Drugs that target several proteins
of RSV have been developed; inhibitors of RSV F protein are
also being developed as one class of anti-RSV agents. Inhibi-
tors of RSV F protein include several classes of compounds,
such as pyrazolo[1,5-a]pyrimidines,41–44) benzimidazoles,45–47)
and piperazinyl-quinolines.48) Among the RSV F protein in-
hibitors with the pyrazolo[1,5-a]pyrimidine scaffold, such as
presatovir (GS-5806, 1) developed by Gilead,41) and P3 (2)
reported by Janssen,44) 1 has advanced the furthest in clinical
trials (Fig. 1). The structure–activity relationships (SARs) of
1 and its derivatives have been well-studied and reported.41)
According to one report, the dihedral angle between the
pyrazolo[1,5-a]pyrimidine scaffold and the plane of the amide
bond on the piperidine ring is a key factor for the anti-RSV
activity. Through intensive SAR study of 1 and its derivatives
on the 2-position of the pyrazolo[1,5-a]pyrimidine scaffold, it
was concluded that the piperidine ring forces the maintenance
of an optimal dihedral angle for anti-RSV activity. There-
fore, we hypothesized that if an optimal dihedral angle of
the pyrazolo[1,5-a]pyrimidine scaffold with the plane of the
amide bond can be reproduced in newly designed compounds
by introducing an acyclic chain at the C-2 position of the
Fig. 2. Dihedral Angle Distributions of Compounds with Substitutions
between the C-2 Heteroaryl Ring and the Amide Plane Calculated by
REMD
pyrazolo[1,5-a]pyrimidine scaffold in place of the piperidine b), respectively. The most frequently observed dihedral angle
ring of 1, potent anti-RSV agents could be obtained. The in 1 was around 82°. Both 12a and 12b contain five carbon
development of safe and effective small-molecule anti-RSV atoms corresponding to the piperidine moiety of 1. The most
agents is required for patients, particularly for children and frequently encountered dihedral angle of 12a was around 82°,
infants. The ring-opening of the piperidine in 1 is a possible while that of 12b was bimodal at 84° and 89°. In addition,
idea to obtain compounds with short half-life, which would the angle in 9b and 9c was 87°. Both 9b and 9c contain four
allow us to manage individual risks of adverse effects.
or less carbons at the position corresponding to the piperi-
dine moiety of 1, and the α-position in their pyrazolo[1,5-a]-
pyrimidine scaffold was substituted by carbon. That is, these
Computational Chemistry
First, conformational analyses of 1 with a piperidine ring compounds (9b–c; 12a, b) were not much different from 1
and of the newly designed compounds (9a–d; 12a, b) with in terms of the most frequently noted dihedral angle or the
an acyclic chain at the C-2 position of the pyrazolo[1,5-a]- overall dihedral angle distribution. In contrast, 9a, without a
pyrimidine scaffold was performed by replica-exchange side chain at the α-position, was determined to have the most
molecular dynamics (REMD).49) REMD simulations of seven stable conformation with a dihedral angle of 89°. The dihedral
compounds, including 1, yielded relatively different dihedral angle of 9d with an isopropyl group at the α-position was
angle distributions between the pyrazolo[1,5-a]pyrimidine found to be 82°; however, the overall dihedral angle distribu-
scaffold and the amide plane (Fig. 2). The filled and lined tion of 9d was different from that of 1.
histograms in all the figures show the dihedral angle distribu-
These results indicate that the difference in the distributions
tions of 1 and the compounds with acyclic chains (9a–d; 12a, of the dihedral angles between the pyrazolo[1,5-a]pyrimidine

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
347
Chart 1. Synthesis of Pyrazolo[1,5-a]pyrimidin-2-yl Derivatives with Modified C-2 Substituents
scaffold and the amide plane mainly depended on the substitu- by the nucleophilic addition reactions of acetonitrile anions to
ent at the α-position of the pyrazolo[1,5-a]pyrimidine scaffold. 4a–f, followed by treating with hydrazine to obtain amino-
These findings also suggest that it may be possible to design pyrazoles 5a–f. Compounds 5a–f were treated with ethyl (E)-
new compounds with appropriate acyclic chains having simi- ethoxy-2-methylacrylate in the presence of sodium ethoxide
lar dihedral angle distributions as 1 with a piperidine ring, to obtain pyrazolopyrimidones 6a–f. Deprotection of the Boc
with anti-RSV activity similar or superior to that of 1.
group of 6a–f and chlorination with phosphorus oxychloride
The conformations of each compound (1; 9a–d; 12a, or triflation afforded pyrazolopyrimidines 7a–f. Compounds
b) were sampled using eight independent REMD simula- 7a–d were coupled with 5-chloro-2-(methylsulfonamido)-
tions. The dihedral angles of all compounds between the benzoic acid under standard amidation conditions. The desired
pyrazolo[1,5-a]pyrimidine scaffold and the amide plane were products 9a–d were obtained by nucleophilic addition of
calculated for each conformation sampled every 10 ps at (S)-tert-butyl pyrrolidin-3-ylcarbamate under basic conditions
310.0K.
and subsequent deprotection of the Boc group. Other desired
products 12a,b were synthesized from 7e–f. After the intro-
duction of (S)-tert-butyl pyrrolidin-3-ylcarbamate to 7e–f by
Chemistry
Compounds with an acyclic chain at the C-2 position (9a–d; nucleophilic aromatic substitution, monoalkylation with the
12a, b) were synthesized by the method shown in Chart 1. corresponding alkyl halides yielded 11a,b. Finally, 11a,b were
First, tert-butyloxycarbonyl (Boc)-protected amino acids 3a–f acylated with 5-chloro-2-(methylsulfonamido)benzoic acid,
with the appropriate substituents at the α-position were con- and the Boc group was deprotected to obtain 12a,b.
verted into methyl esters 4a–f. β-Ketonitriles were synthesized
The synthesis methods for the desired products (14a–s,

348
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
Chart 2. Synthesis of Pyrazolo[1,5-a]pyrimidin-2-yl Derivatives with Modified Aryl Groups
16–19) with modified aryl groups are shown in Chart 2. In- tection of the Boc group and dichlorination were simultane-
termediates 13a,b were obtained by nucleophilic addition of ously performed by treatment with phosphorus oxychloride
(S)-tert-butyl pyrrolidin-3-ylcarbamate or (S)-benzyl pyrro- to obtain 21. The intermediate 21 was monoaminated with
lidin-3-ylcarbamate to 7e under basic conditions, followed by various amines and then subjected to an amidation reaction
N-methylation via trifluoroacetylation and deprotection of the with 5-chloro-2-(methylsulfonamido)benzoic acid to obtain 23.
trifluoroacetyl group. The desired products 14a–s were ob- Finally, the desired products 24a–f were synthesized by intro-
tained from 13a by acylation with the corresponding carbox- ducing the (3S)-3-aminopyrrolidine moiety using microwaves
ylic acids and by deprotecting the Boc group after mesylation, under basic conditions, followed by deprotection under acidic
if necessary. Compounds 16–19 were synthesized from inter- conditions for compounds possessing a Boc group.
mediates 15a,b obtained by acylation of 13a,b with 2-amino-
5-chlorobenzoic acid. Acylation of 15a with acetic anhydride
and subsequent deprotection of the Boc group yielded 16.
Results and Discussion
The degrees of inhibition of the cytopathic effect (CPE)
Similarly, 15b was converted to carbamate, urea, and sulfo- induced by RSV infection were determined to evaluate the an-
nylurea, followed by deprotection of the carbobenzoxy (Cbz) tiviral activities of the synthesized compounds. Table 1 shows
group to obtain 17–19.
the anti-RSV activities of the test compounds (9a–d; 12a, b).
Subsequently, various substituent groups were introduced Compounds 9b, 9c, 12a, and 12b exhibited good anti-RSV
into the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold activities, with EC50 values of less than 4nM. As described
(Chart 3). A dihydropyrazolopyrimidine compound 20 was above, the distribution of the dihedral angles between the
synthesized by reaction of the 5-aminopyrazole derivative 5c pyrazolo[1,5-a]pyrimidine scaffold and the plane of the amide
with diethyl 2-methylmalonate under basic conditions. Depro- bond was similar to that in 1 (Fig. 2). In contrast, the dihedral

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
349
Chart 3. Synthesis of Pyrazolo[1,5-a]pyrimidin-2-yl Derivatives with Modified C-7 Substituents
angle distributions of 9a and 9d, which showed markedly de- Deletion of the methylsulfonamide group in the benzene ring
creased anti-RSV activities, were different from that of 1 (Fig. markedly attenuated the activity (14d). Compounds of vari-
2). These results indicate that the anti-RSV activities of 9a–d ous shapes and sizes were synthesized (14e–i) to investigate
and 12a,b without a piperidine ring were well correlated with the steric effect of the substituents at the 5-position in the
the dihedral angle distribution. Among these compounds, 9c benzene ring. The chlorine atom (9c) was replaceable with
with the 1-methyaminopropyl moiety showed the most potent other small substituents such as methyl (14e), ethyl (14f), and
anti-RSV activity, with an EC50 value in the sub-nanomolar methoxy (14g) at the 5-position, although longer or bulkier
range, which was similar to that of 1.41) These findings suggest substituents such as propyl (14h) and isopropyl (14i) reduced
that the piperidine ring of 1 plays a crucial role in control- the potency by two orders of magnitude. Among compounds
ling the dihedral angle between the pyrazolo[1,5-a]pyrimidine with a small substituent at the 5-position in the benzene ring,
scaffold and the plane of the amide bond, so as to allow ef- 14f exhibited the most potent anti-RSV activity, with an EC50
fective interaction of the compound with the RSV F protein. value of 0.15nM. The dihedral angle distribution of 14f was
Furthermore, these data suggest that potent anti-RSV activity calculated and found to be similar to that of presatovir (1)
could be achieved through controlling the dihedral angles of (Fig. S1 in Supplementary Materials). To estimate the pos-
compounds without a piperidine ring. The promising results sibility of replacement of the methylsulfonamide group 9c at
led us to further design new compounds with the acyclic the 2-position of the benzene ring with other groups, seven
chain, so as to obtain compounds with anti-RSV activity simi- compounds with no substituent, methyl, or chloro at the 5-po-
lar to or better than that of 9c.
sition were synthesized (14j–l, 16–19). It was found that meth-
Next, the SAR study was focused on substituents on the ylsulfone substitution (14j) drastically reduced the potency by
benzene ring, which could affect the dihedral angle of the four orders of magnitude as compared to that of 14c with a
pyrazolo[1,5-a]pyrimidine scaffold and the plane of the amide methylsulfonamide group in the same position. The antiviral
bond (Table 2). To investigate the presence and position of activities were also reduced by 12- and 28-fold, respectively,
a chlorine atom at the 5-position of the benzene ring in 9c, when the methylsulfonamide group (14e) was substituted with
two derivatives with a chlorine atom at different positions methoxy 14k and trifluoromethyl (14l) groups. Conversely,
14a,b were synthesized, as well as a compound without a replacement of methylsulfonamide (9c) with acetamide (16),
chlorine atom (14c). While the anti-RSV activities of 14a,b methoxycarboxamide (17), methylureido (18), or methylsul-
were more than 7-fold less active as compared to the anti-RSV fonylureido (19) was associated with reduction of the EC50
activity of 9c, 14c showed comparable activity to that of 9c. values (0.57–1.8nM). It appears that substituents having both

350
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
Table 1. RSV Inhibitory Activities of Compounds with
an Acyclic Chain at the C-2 Position of the Pyrazolo[1,5-
a]pyrimidine Scaffold
Table 2. RSV Inhibitory Activities of Compounds with Substitutions on
the Benzene Ring
Anti-RSV activity EC50
Compound
R
(nM)a)
9c
14a
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
16
2-NHSO2Me, 5-Cl
2-NHSO2Me, 3-Cl
2-NHSO2Me, 4-Cl
2-NHSO2Me
0.58
4.4
13
0.59
42
2-H
2-NHSO2Me, 5-Me
2-NHSO2Me, 5-Et
2-NHSO2Me, 5-OMe
2-NHSO2Me, 5-Pr
2-NHSO2Me, 5-iPr
2-SO2Me
0.33
0.15
0.43
39
51
1500
3.8
2-OMe, 5-Me
2-CF3, 5-Me
9.2
2-NHCOMe, 5-Cl
2-NHCO2Me, 5-Cl
2-NHCONHMe, 5-Cl
2-NHSO2NHMe, 5-Cl
0.84
0.57
1.0
17
18
19
1.8
a) EC50 values of the CPE inhibitory activities of all compounds were evaluated in
HEp-2 cells infected with RSV A2.
pyrimidine scaffold had little effect on the antiviral activity
in their patent.42–44) This result prompted us to investigate the
biological properties of 9c derivatives by introduction of vari-
ous substituents into the 7-position (Table 4). Compounds with
methylamino (24a) and dimethylamino (24b) groups at the
7-position of pyrazolo[1,5-a]pyrimidine showed a 7.8- and
21-fold reduction in the antiviral efficacy, respectively, as
a) EC50 values of the CPE inhibitory activities of all com-
pounds were evaluated in HEp-2 cells infected with RSV A2.
proton-donor and proton-acceptor capabilities at the 2-posi- compared to 9c, suggesting that a secondary amine structure
tion of the benzene ring are preferable for obtaining effective (24a) is preferable to a tertiary amine structure (24b). Com-
antiviral activity. The SAR of substituents on the benzoyl ring pounds having a cyclic moiety such as oxetane and azetidine
obtained in this study showed good consistency with that in via substitution of the amino group at the 7-position also
the previous report on 1 and its derivatives.41) However, little showed markedly reduced antiviral activities (data not shown).
attention has been paid to the replacement of the benzene ring In contrast, introduction of linear moieties such as ethylenedi-
by heterocycles. Therefore, compounds with a variety of five- amine and methyl ethylenediamine (24c,d) resulted in a mod-
and six-membered heterocycles (14m–s) were designed and erate decrease of the antiviral activity, i.e., a 4.2- to 4.7-fold
synthesized to investigate the effect on the anti-RSV activ- decrease in the activity as compared to 24a. Interestingly,
ity (Table 3). All heterocycles contained a methylsulfonamide compounds possessing sulfone (24e) and sulfonamide (24f)
group at the corresponding position of the benzene ring in moieties on the side chain at 7-position of the pyrazolo[1,5-a]-
14c, because the methyl sulfonamide group was essential for pyrimidine scaffold exhibited potent anti-RSV activities, with
obtaining potent antiviral activity. Compounds with a six- EC50 values of less than 1nM. These results suggest that sul-
membered heterocyclic ring, such as pyridine (14m,n), and a fone (24e) and sulfonamide (24f) moieties have some potential
five-membered heterocyclic ring, such as thiophene (14o,p), to interact with the RSV F-protein. Modification at the 7-posi-
thiazole (14q), and pyrazole (14r,s), were synthesized; howev- tion of pyrazolo[1,5-a]pyrimidine could be utilized for tuning
er, all of the compounds obtained showed a dramatic decrease the biological and physicochemical properties of the most
of the antiviral activity. In addition, nitrogen regioisomers in potent anti-RSV agent 14f obtained in this study.
the pyridine ring of 14m,n, and compounds with other hetero-
cycles, e.g., pyrazine and pyrimidine, showed remarkable loss
Conclusion
of antiviral activity (data not shown).
In summary, we designed and synthesized a new series
Previously, Gilead and Janssen reported that introduction of 2-(1-alkylaminoalkyl)pyrazolo[1,5-a]pyrimidine derivatives
of a substituent into the 7-position of the pyrazolo[1,5-a]- as RSV F protein inhibitors. The pyrazolo[1,5-a]pyrimidine

Vol. 68, No. 4 (2020)
Table 3. RSV Inhibitory Activity of Compounds with
Chem. Pharm. Bull.
351
Table 4. RSV Inhibitory Activity of Compounds with
Substitution at the 7-Position of the Pyrazolo[1,5-a]py-
rimidine Scaffold
Substitution of the Benzene Ring in 14c by Heterocycles
a) EC50 values of the CPE inhibitory activities of all com-
pounds were evaluated in HEp-2 cells infected with RSV A2.
be achieved through controlling the dihedral angle of com-
pounds with an acyclic chain. Subsequent optimization study
on the benzene ring located next to the amide bond led to
the potent anti-RSV agent 14f with an EC50 value of 0.15nM,
which showed a similar dihedral angle distribution compared
to that of 1. It is considered that introduction of a substituent
into the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold
has the potential to modify the anti-RSV activity. Compounds
24e and 24f with a sulfone and sulfonamide moiety on the
side chain at the 7-position of the pyrazolo[1,5-a]pyrimidine
a) EC50 values of the CPE inhibitory activities of all com-
pounds were evaluated in HEp-2 cells infected with RSV A2.
derivatives are well-known candidates as RSV F protein in- scaffold showed potent anti-RSV activities, with EC50 values
hibitor drugs, such as presatovir (1) and P3 (2), which have a of less than 1nM, suggesting the possibility that the biological
piperidine ring at the 2-position of pyrazolo[1,5-a]pyrimidine and physicochemical properties of the most potent anti-RSV
scaffold. The piperidine ring was considered to be an essential agent 14f obtained in this study could be improved further.
structure for anti-RSV activity, by providing an appropriate Currently, further optimization study is in progress.
dihedral angle between the pyrazolo[1,5-a]pyrimidine scaffold
and the plane of the amide bond. Conformational analysis of 1
Experimental
and newly designed compounds with an acyclic chain in place
Chemistry All solvents and reagents were purchased
of the piperidine ring by molecular dynamic study demon- from commercial suppliers and used without purification,
strated that several types of compounds with different dihe- or were prepared according to published procedures. The
dral angle distributions showed different levels of anti-RSV 1H-NMR and 13C-NMR spectra of compounds synthesized in
activity. Among compounds with an acyclic chain with ap- this study were recorded on a JNM-ECA600, JNM-ECA500
propriate dihedral angle distributions, 9c with 1-methylamino- (JEOL Ltd., Tokyo, Japan), or Avance III HD 400 (Bruker
propyl moiety showed potent anti-RSV activity, with an EC50 Corp., Billerica, MA, U.S.A.) and the chemical shifts were
value in the sub-nanomolar range, which was similar to that expressed in δ (:) values, with trimethylsilane as the inter-
of 1. These results suggest that potent anti-RSV activity could nal standard (s=singlet, d=doublet, t=triplet, q=quartet,

352
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
m=multiplet, and brs=broad singlet). Two sets of NMR 5.36 (1H, brs), 5.55 (1H, brs); MS (ESI/APCI dual) m/z: 241
signals were observed due to structural variations of the [M+H]+.
cis- and trans-amide rotamers. Mass spectra were recorded
on a Micromass Platform LC (Micromass Ltd., Manchester, as that described for 5b.
U.K.) or Shimadzu LCMS-2010EV (Shimadzu Corp., Kyoto, tert-Butyl [(5-Amino-1H-pyrazol-3-yl)methyl]methylcar-
Compounds 5a, c–f were obtained by the same procedure
1
Japan). High-resolution (HR) mass spectral data were acquired bamate (5a) Brown gum; H-NMR (600MHz, CDCl3,) δ:
using an LCMS-IT-TOF equipped with an electrospray ioniza- 1.41–1.57 (9H, m), 2.75–3.01 (3H, m), 4.26 (2H, brs), 5.59 (1H,
tion (ESI)/atmospheric pressure chemical ionization dual ion s); MS (ESI/APCI dual) m/z: 227 [M+H]+.
source (Shimadzu Corp.). Intermediates and final compounds
tert-Butyl [(1S)-1-(5-Amino-1H-pyrazol-3-yl)propyl]methy-
were purified by preparative HPLC using Agilent 1260 Infin- lcarbamate (5c) Orange amorphous; 1H-NMR (400MHz,
ity/Agilent 6130 (Agilent Technologies Inc., Santa Clara, CA, CDCl3) δ: 0.90–1.02 (4H, m), 1.48 (9H, s), 1.74–1.95 (2H, m),
U.S.A.) or a GX-281, UV/VIS-155, 331 PUMP, 332 PUMP, 2.63 (3H, s), 3.62 (2H, brs), 4.96–5.14 (1H, m), 5.54 (1H, s);
and SOFTA Model 300S ELSD (Gilson Inc., Middleton, WI, MS (ESI/APCI dual) m/z: 255 [M+H]+.
U.S.A.), under the following conditions: column, Sunfire prep
tert-Butyl [(1S)-1-(5-Amino-1H-pyrazol-3-yl)-2-methyl-
(5d) Colorless amorphous;
C18 OBD (5.0µm, 30×50mm) (Waters Corp., Milford, MA, propyl]methylcarbamate
U.S.A.), YMC-Actus Triart C18 (5.0µm, 30×50mm) (YMC 1H-NMR (400MHz, CDCl3) δ: 0.93 (3H, d, J=15.4Hz), 0.94
Co., Ltd., Kyoto, Japan), Xbridge Prep C18 OBD (5.0µm, (3H, d, J=15.4Hz), 1.46 (9H, brs), 2.22 (1H, brs), 2.67 (3H,
30×50mm) (Waters Corp.), or XSelect CSH C18 (5.0µm, s), 3.43–3.77 (2H, m), 4.52–4.63 (1H, m), 5.55 (1H, s); MS
30×50mm) (Waters Corp.); flow, 50mL/min; linear gradient, (ESI/APCI dual) m/z: 269 [M+H]+.
10–95% acetonitrile in water containing 0.1% formic acid for
7.5–11.5min; detection wavelength, 254nm.
tert-Butyl [(1S)-1-(5-Amino-1H-pyrazol-3-yl)propyl]car-
bamate (5e) Pale yellow amorphous; 1H-NMR (600MHz,
tert-Butyl [(1S)-1-(5-Amino-1H-pyrazol-3-yl)ethyl]methyl- CDCl3) δ: 0.95–1.06 (3H, m), 1.43–1.46 (9H, m), 1.63–1.77
carbamate (5b) To a solution of N-(tert-butoxycarbonyl)-N- (1H, m), 1.83–1.98 (1H, m), 4.35–4.58 (1H, m), 4.61–4.90 (1H,
methyl-L-alanine (6.0g, 29.3mmol) in toluene (18mL)–metha- m), 5.49 (1H, m); MS (ESI/APCI dual) m/z: 241 [M+H]+;
nol (12mL) was added 2.0M of trimethylsilyl diazomethane chiral HPLC, 99% ee (CHIRALPAK IC-3 5µm 4.6×250mm;
in diethyl ether (22.0mL, 43.9mmol) at 0°C, and the mixture flow, 1mL/min, 20% ethanol in hexane; detection wavelength,
was stirred for 1h at room temperature. Acetic acid was added 254nm), (S)-isomer tR =6.62min, (R)-isomer tR =5.13 min.
to the reaction solution until the solution became clear. The
tert-Butyl [(1S)-1-(5-Amino-1H-pyrazol-3-yl)ethyl]carba-
reaction mixture was basified with sat. sodium bicarbonate aq. mate (5f) Pale yellow amorphous; 1H-NMR (600MHz,
and extracted with chloroform. The organic layer was dried CDCl3) δ: 1.43–1.49 (12H, m), 4.71–4.81 (1H, m), 4.85 (1H,
over anhydrous magnesium sulfate and concentrated under brs), 5.49 (1H, s), 5.64 (1H, brs); MS (ESI/APCI dual) m/z:
reduced pressure to obtain methyl N-(tert-butoxycarbonyl)-N- 227 [M+H]+.
methyl-L-alaninate (6.5g, quant.) as a colorless oil, which was
used for the next reaction without further purification.
tert-Butyl Methyl[(1S)-1-(6-methyl-5-oxo-4,5-dihydropyra-
zolo[1,5-a]pyrimidin-2-yl)ethyl]carbamate (6b) To a so-
A 1.9M of sodium bis(trimethylsilyl)amide in tetrahydro- lution of 5b (1.7g, 6.87mmol) in N,N-dimethylformamide
furan (68mL, 88.6mmol) was added to a solution of aceto- (10mL) was added ethyl (E)-3-ethoxy-2-methylacrylate (1.6g,
nitrile (4.6mL, 88.6mmol) in tetrahydrofuran (75mL) at 10.3mmol) and cesium carbonate (6.7g, 20.6mmol). After
−78°C. After stirring at −50°C for 20min, methyl N-(tert- stirring at 120°C for 8h, the reaction mixture was poured into
butoxycarbonyl)-N-methyl-L-alaninate (6.0g, 29.5mmol) in water and extracted with chloroform. The organic layer was
tetrahydrofuran (45mL) at −78°C was added to the mixture. washed with brine, dried over anhydrous magnesium sulfate
After stirring at −50°C for 1h, acetic acid (5.2mL, 91.5mmol) and concentrated under reduced pressure. The residue was pu-
at −78°C was added to the mixture. The mixture was poured rified by silica gel column chromatography (0–10% methanol
into sat. ammonium chloride aq. and extracted with ethyl ace- in chloroform) to obtain 6b (1.4g, 4.41mmol, 64%) as a brown
1
tate. The organic layer was washed with brine, dried over an- amorphous. H-NMR (600MHz, CDCl3) δ: 1.49 (9H, s), 1.51
hydrous magnesium sulfate, and concentrated under reduced (3H, d, J=7.0Hz), 2.10 (3H, s), 2.67 (3H, brs), 5.28–5.62 (1H,
pressure to obtain tert-butyl (S)-(4-cyano-3-oxobutan-2-yl)- m), 5.78 (1H, s), 8.01 (1H, s), 10.85 (1H, brs); MS (ESI/APCI
methylcarbamate (7.2g, crude) as a brown oil, which was used dual) m/z: 307 [M+H]+.
for the next reaction without further purification.
To solution of hydrazine monohydrate (4.1mL, as that described for 6b.
84.8mmol), acetic acid (4.9mL, 84.8mmol), and ethanol
tert-Butyl Methyl[(6-methyl-5-oxo-4,5-dihydropyrazolo-
Compounds 6a, c–f were obtained by the same procedure
a
(20mL) was added tert-butyl (S)-(4-cyano-3-oxobutan-2-yl)- [1,5-a]pyrimidin-2-yl)methyl]carbamate (6a) Pale yellow
1
methylcarbamate (6.0g, 28.3mmol) in ethanol (20mL) at amorphous; H-NMR (600MHz, CDCl3) δ: 1.39–1.54 (9H, m),
0°C, and the mixture was stirred for 2d at room temperature. 2.09 (3H, s), 2.78–2.95 (3H, m), 4.31–4.51 (2H, m), 5.72–5.90
Then, the reaction mixture was concentrated under reduced (1H, m), 7.96 (1H, s), 10.77 (1H, brs); MS (ESI/APCI dual)
pressure and diluted with chloroform. The organic layer was m/z: 291 (M–H)-.
washed with sat. sodium bicarbonate aq. and brine, dried over
tert-Butyl Methyl[(1S)-1-(6-methyl-5-oxo-4,5-dihydropyra-
anhydrous magnesium sulfate and concentrated under reduced zolo[1,5-a]pyrimidin-2-yl)propyl]carbamate (6c) Colorless
1
pressure. The residue was purified by silica gel column chro- amorphous; H-NMR (400MHz, CDCl3) δ: 0.92–1.01 (3H, m),
matography (0–10% methanol in chloroform) to obtain 5b 1.49 (9H, s), 1.78–1.93 (1H, m), 1.99–2.13 (4H, m), 2.67 (3H, s),
1
(5.8g, 23.9mmol, 85%) as a brown gum. H-NMR (600MHz, 5.01–5.41 (1H, m), 5.79 (1H, brs), 8.00 (1H, s), 10.81 (1H, brs);
CDCl3) δ: 1.44 (3H, d, J=7.0Hz), 1.48 (9H, s), 2.65 (3H, s), MS (ESI/APCI dual) m/z: 321 [M+H]+.

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
353
tert-Butyl Methyl[(1S)-2-Methyl-1-(6-methyl-5-oxo-4,5-
Compound 8a was obtained by the same procedure as that
dihydropyrazolo[1,5-a]pyrimidin-2-yl)propyl]carbamate described for 8b.
(6d) Brown powder; 1H-NMR (400MHz, CDCl3) δ: 0.91
2-{[{5-Chloro-2-[(methanesulfonyl)amino]benzoyl}(meth-
(3H, d, J=6.5Hz), 0.97 (3H, d, J=6.5Hz), 1.47 (9H, brs), yl)amino]methyl}-6-methylpyrazolo[1,5-a]pyrimidin-5-yl
1
2.10 (3H, s), 2.31–2.45 (1H, m), 2.71 (3H, s), 4.64–5.03 (1H, Trifluoromethanesulfonate (8a) Pale yellow oil; H-NMR
m), 5.71–5.90 (1H, m), 8.01 (1H, s), 10.08–10.27 (1H, m); MS (600MHz, CDCl3) δ: 2.36–2.42 (3H, m), 2.83–3.14 (6H, m),
(ESI/APCI dual) m/z: 335 [M+H]+.
4.58 (0.6H, brs), 5.02 (1.4H, brs), 6.37–6.51 (1H, m), 7.15–7.78
tert-Butyl [(1S)-1-(6-Methyl-5-oxo-4,5-dihydropyrazolo- (3H, m), 8.72–8.87 (1H, m), 9.08 (0.7H, s), 10.09 (0.3H, s).
[1,5-a]pyrimidin-2-yl)propyl]carbamate (6e) Pale yellow 5-Chloro-N-[(1S)-1-(5-chloro-6-methylpyrazolo[1,5-a]-
1
powder; H-NMR (600MHz, CDCl3) δ: 0.88–0.97 (3H, m), pyrimidin-2-yl)propyl]-2-[(methanesulfonyl)amino]-N-
1.39–1.51 (9H, m), 1.78 (1H, dt, J=13.8, 7.1Hz), 1.90 (1H, dd, methylbenzamide (8c) To a solution of 6c (1.6g, 5.12mmol)
J=13.6, 7.0Hz), 2.07–2.14 (3H, m), 4.72 (1H, d, J=6.2Hz), in 1,4-dioxane (15mL) was added 4M hydrogen chloride in
5.06 (1H, d, J=7.0Hz), 5.82 (1H, s), 7.98 (1H, d, J=0.8Hz), 1,4-dioxane (15mL) and the mixture was stirred for 0.5h at
10.81 (1H, brs); MS (ESI/APCI dual) m/z: 307 [M+H]+.
room temperature. Then, the reaction mixture was concen-
tert-Butyl [(1S)-1-(6-Methyl-5-oxo-4,5-dihydropyrazolo- trated under reduced pressure to obtain (S)-6-methyl-2-[1-
[1,5-a]pyrimidin-2-yl)ethyl]carbamate (6f) Pale yellow (methylamino)propyl]pyrazolo[1,5-a]pyrimidin-5(4H)-one hy-
1
powder; H-NMR (600MHz, CDCl3) δ: 1.45 (9H, s), 1.50 (3H, drochloride (1.3g, 5.90mmol, quant.) as a colorless powder,
d, J=6.6Hz), 2.09 (3H, s), 4.88 (1H, brs), 5.06 (1H, brs), 5.81 which was used for the next reaction without further purifica-
(1H, s), 7.96 (1H, s), 10.23 (1H, brs); MS (ESI/APCI dual) m/z: tion.
293 [M+H]+.
The mixture of (S)-6-methyl-2-[1-(methylamino)propyl]-
2-{(1S)-1-[{5-Chloro-2-[(methanesulfonyl)amino]benzoyl}- pyrazolo[1,5-a]pyrimidin-5(4H)-one
hydrochloride (1.3g,
(methyl)amino]ethyl}-6-methylpyrazolo[1,5-a]pyrimidin- 5.90mmol) and phosphorus oxychloride (30mL) was stirred
5-yl Trifluoromethanesulfonate (8b) To a solution of 6b at 100°C for 3h. Then, the reaction mixture was concentrated
(717mg, 2.34mmol) and pyridine (0.95mL, 11.7mmol) in under reduced pressure to obtain 7c (1.4g, 5.91mmol, quant.)
chloroform (8.0mL) was added trifluoromethanesulfonic an- as black oil, which was used for the next reaction without fur-
hydride (0.79mL, 4.68mmol) at 0°C, and the mixture was ther purification.
stirred for 3.5h at room temperature. The reaction mixture
To a solution of 7c (1.4g, 5.91mmol) and 5-chloro-2-
was poured into sat. ammonium chloride aq. and extracted (methylsulfonamido)benzoic acid (1.8g, 7.08mmol) in N,N-
with chloroform. The organic layer was concentrated under dimethylformamide (15mL) was added 1-[bis(dimethylamino)-
reduced pressure. The residue was purified by silica gel col- methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
umn chromatography (5–100% ethyl acetate in hexane) to fluorophosphate (2.9g, 7.67mmol) and triethylamine (6.0mL,
obtain 2-{(1S)-1-[(tert-butoxycarbonyl)(methyl)amino]ethyl}-6- 59.0mmol). After stirring at room temperature for 2h, the
methylpyrazolo[1,5-a]pyrimidin-5-yl trifluoromethanesulfonate reaction mixture was added to water and extracted with ethyl
(0.87g, 1.98mmol, 84%) as a colorless powder.
acetate. The organic layer was washed with brine, dried over
To a solution of 2-{(1S)-1-[(tert-butoxycarbonyl)(methyl)- anhydrous magnesium sulfate, filtered, concentrated under re-
amino]ethyl}-6-methylpyrazolo[1,5-a]pyrimidin-5-yl trifluoro- duced pressure. The residue was purified by silica gel column
methanesulfonate (0.86g, 1.96mmol) in 1,4-dioxane (3.0mL) chromatography (20–65% ethyl acetate in hexane) to obtain 8c
1
was added 4M hydrogen chloride in 1,4-dioxane (10mL) and (0.88g, 1.88mmol, 32%) as a colorless amorphous. H-NMR
the mixture was stirred for 1h at room temperature. Then, the (400MHz, CDCl3) δ : 1.07–1.20 (3H, m), 1.86–2.35 (4H, m),
reaction mixture was concentrated under reduced pressure to 2.36–2.44 (3H, m), 2.60 (1H, s), 2.78 (2H, s), 2.96 (1H, s), 3.04
obtain 7b (1.1g, 2.60mmol, quant.) as a pale yellow oil. This (2H, s), 4.69–4.78 (0.5H, m), 6.03–6.13 (0.5H, m), 6.40–6.55
compound was used for the next reaction without further pu- (1H, m), 7.24–7.32 (2H, m), 7.33–7.43 (1H, m), 7.56–7.77 (1H,
rification.
m), 8.81 (0.5H, s), 8.92 (0.5H, s), 9.08 (0.5H, s), 10.52 (0.5H,
To a solution of 7b (0.81g, 1.96mmol) and 5-chloro-2- brs); MS (ESI/APCI dual) m/z: 470 [M+H]+.
(methylsulfonamido)benzoic acid (0.59g, 2.36mmol) in N,N-
Compound 8d was obtained by the same procedure as that
dimethylformamide (10mL) was added 1-[bis(dimethylamino)- described for 8c.
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
5-Chloro-N-[(1S)-1-(5-chloro-6-methylpyrazolo[1,5-a]-
hexafluorophosphate (1.1g, 2.95mmol) and triethylamine pyrimidin-2-yl)-2-methylpropyl]-2-[(methanesulfonyl)-
(1.7mL, 11.8mmol). After stirring at room temperature for amino]-N-methylbenzamide (8d) Colorless amorphous;
15h, the reaction mixture was added to water and extracted 1H-NMR (400MHz, CDCl3) δ: 0.95–1.34 (6H, m), 2.35–2.53
with ethyl acetate. The organic layer was concentrated under (4H, m), 2.55 (1H, s), 2.73 (2H, s), 2.94–3.03 (3H, m),
reduced pressure. The residue was purified by silica gel col- 4.32–4.44 (0.7H, m), 5.78–5.90 (0.3H, m), 6.52–6.63 (1H, m),
umn chromatography (10–100% ethyl acetate in hexane) to 7.17–7.50 (2H, m), 7.59–7.74 (1H, m), 8.36–8.49 (0.3H, m), 8.83
obtain 8b (0.71g, 1.24mmol, 63%) as a colorless amorphous. (0.7H, brs), 9.00 (0.3H, brs), 10.51–10.81 (0.7H, m); MS (ESI/
1H-NMR (600MHz, CDCl3) δ: 1.68 (1.4H, d, J=7.4Hz), 1.72 APCI dual) m/z: 484 [M+H]+.
(1.6H, d, J=7.4Hz), 2.39 (1.8H, s), 2.40 (1.2H, s), 2.66 (1.6H,
N-[(1S )-1-{5 -[(3S )-3 -Aminopyrrolidin-1-yl]- 6 -
s), 2.80 (1.4H, s), 2.96 (1.8H, s), 3.05 (1.2H, s), 5.00 (0.5H, methylpyrazolo[1,5-a]pyrimidin-2-yl}ethyl]-5-chloro-2-
q, J=7.4Hz), 6.31 (0.5H, q, J=7.4Hz), 6.45 (0.5H, s), 6.46 [(methanesulfonyl)amino]-N-methylbenzamide Hydrochlo-
(0.5H, s), 7.28–7.34 (1H, m), 7.35–7.42 (1H, m), 7.62 (0.5H, d, ride (9b) To a solution of 8b (0.70g, 1.23mmol) and
J=8.7Hz), 7.71 (0.5H, d, J=8.7Hz), 8.79 (1H, s), 8.93 (0.5H, (S)-tert-butyl pyrrolidin-3-ylcarbamate (0.46g, 2.46mmol) in
s), 9.10 (0.5H, s).
tetrahydrofuran (10mL) was added trimethylamine (0.87mL,

354
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
6.16mmol). After stirring at 80°C for 1h, the reaction mix- (1.5H, s), 2.72 (1.5H, brs), 2.97–3.06 (3H, m), 3.71–3.82 (2H,
ture was added to water and extracted with chloroform. The m), 3.82–3.90 (2H, m), 3.90–3.98 (1H, m), 4.54 (0.5H, dd,
organic layer was dried over ISOLUTE® Phase Separator and J=9.9, 5.0Hz), 5.73 (0.5H, dd, J=10.3, 5.4Hz), 6.13 (1H, s),
concentrated under reduced pressure. The residue was purified 7.36–7.57 (3H, m), 8.34 (3H, brs), 8.51 (0.5H, s), 9.21 (0.5H,
by silica gel column chromatography (5–100% ethyl acetate s); 13C-NMR (151MHz, DMSO-d6) δ: 10.27, 17.24, 21.98, 28.8,
in hexane) to obtain tert-butyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2- 30.83, 39.63, 40.15, 46.32, 48.31, 52.17, 89.59, 107.62, 126.15,
[(methanesulfonyl)amino]benzoyl}(methyl)amino]ethyl}-6- 126.63, 129.23, 129.81, 131.78, 133.55, 134.17, 146.53, 154.51,
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate 154.89, 167.01; HR-MS ESI/APCI dual m/z: 520.1873 [M+H]+
1
(0.73g, 1.21mmol, 98%) as a colorless amorphous. H-NMR (Calcd for C23H30ClN7O3S: 520.1892).
(600MHz, CDCl3) δ: 1.45 (9H, s), 1.60 (2H, d, J=7.0Hz),
1.64 (1H, d, J=7.0Hz), 1.86–1.97 (1H, m), 2.16–2.27 (1H, zolo[1,5-a]pyrimidin-2-yl}-2-methylpropyl]-5-chloro
m), 2.35–2.39 (3H, m), 2.63 (1H, s), 2.81 (2H, s), 2.95 (1H, s), 2-[(methanesulfonyl)amino]-N-methylbenzamide (9d) Col-
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
-
1
3.03 (2H, s), 3.50–3.58 (1H, m), 3.69–3.76 (1H, m), 3.77–3.85 orless powder; H-NMR (400MHz, DMSO-d6) δ: 0.73–1.10
(1H, m), 3.90–3.97 (1H, m), 4.24–4.34 (1H, m), 4.67 (1H, brs), (6H, m), 2.00–2.13 (1H, m), 2.19–2.31 (1H, m), 2.31–2.40 (3H,
4.88 (0.5H, q, J=7.0Hz), 5.97–6.04 (1H, m), 6.23 (0.5H, q, m), 2.60–2.81 (3H, m), 2.92–3.05 (3H, m), 3.69–3.99 (5H, m),
J=7.0Hz), 7.27–7.31 (1H, m), 7.33–7.40 (1H, m), 7.61 (0.6H, d, 4.09–4.21 (0.5H, m), 5.42–5.52 (0.5H, m), 6.13 (0.5H, s), 6.26
J=8.7Hz), 7.69 (0.4H, d, J=8.7Hz), 8.36 (1H, s), 8.64 (0.5H, (0.5H, brs), 7.21–7.61 (3H, m), 8.27–8.40 (3H, m), 8.50 (0.5H,
s), 9.40 (0.5H, s); MS (ESI/APCI dual) m/z: 606 [M+H]+.
s), 8.99 (0.5H, s); 13C-NMR (151MHz, DMSO-d6) δ: 17.53,
To a solution of tert-butyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2- 19.36, 20.55, 27.56, 29.21, 31.74, 40.75, 46.76, 48.72, 52.59,
[(methanesulfonyl)amino]benzoyl}(methyl)amino]ethyl}-6- 57.27, 90.95, 108.27, 126.52, 128.73, 129.58, 130.68, 132.17,
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate 134.60, 135.35, 146.34, 153.92, 155.28, 167.06; HR-MS ESI/
(0.69g, 1.14mmol) in 1,4-dioxane (6.9mL) was added 4M hy- APCI dual m/z: 534.2023 [M+H]+ (Calcd for C24H32ClN7O3S:
drogen chloride in 1,4-dioxane (6.9mL) and the mixture was 534.2049).
stirred for 5h at room temperature. Then, the reaction mixture
tert-Butyl [(3S)-1-{2-[(1S)-1-Aminopropyl]-6-methylpyrazolo-
was concentrated under reduced pressure to obtain 9b (0.62g, [1,5-a]pyrimidin-5-yl}pyrrolidin-3-yl]carbamate (10a) To
1
1.14mmol, 100%) as a colorless powder. H-NMR (600MHz, a solution of 6e (5.4g, 17.6mmol) in methanol (30mL) was
CDCl3) δ: 1.59 (1.8H, d, J=7.0Hz), 1.64 (1.2H, d, J=7.0Hz), added 4M hydrogen chloride in 1,4-dioxane (30mL) and
1.76–1.86 (1H, m), 2.11–2.22 (1H, m), 2.37 (1.2H, s), 2.38 the mixture was stirred for 2h at room temperature. Then,
(1.8H, s), 2.62 (1.2H, s), 2.81 (1.8H, s), 2.95 (1.2H, s), 3.03 the reaction mixture was concentrated under reduced pres-
(1.8H, s), 3.46 (1H, dd, J=10.9, 4.3Hz), 3.66–3.78 (2H, m), sure to obtain 2-[(1S)-1-aminopropyl]-6-methylpyrazolo[1,5-a]-
3.88 (2H, dd, J=10.3, 4.5Hz), 4.88 (0.6H, q, J=6.6Hz), 5.98 pyrimidin-5(4H)-one hydrochloride (4.7g) as a brown powder,
(0.6H, s), 5.99 (0.4H, s), 6.22 (0.4H, d, J=7.0 Hz), 7.22–7.32 which was used for the next reaction without further purifica-
(1H, m), 7.32–7.40 (1H, m), 7.61 (0.6H, d, J=8.7Hz), 7.68 tion.
(0.4H, d, J=8.7Hz), 8.34 (0.6H, s), 8.61 (0.4H, s); 13C-NMR
The mixture of 2-[(1S)-1-aminopropyl]-6-methylpyrazolo-
(151MHz, DMSO-d6) δ: 15.63, 17.66, 29.22, 31.35, 40.05, [1,5-a]pyrimidin-5(4H)-one hydrochloride (4.5g, 18.5mmol)
40.54, 46.73, 48.73, 52.61, 89.81, 107.96, 126.69, 126.77, 129.67, and phosphorus oxychloride (85g, 556mmol) was stirred
129.99, 132.40, 133.49, 134.61, 147.03, 155.29, 155.73, 166.67; at 100°C for 2h. Then, the reaction mixture was concen-
HR-MS ESI/APCI dual m/z: 506.1711 [M+H]+ (Calcd for trated under reduced pressure to obtain (1S)-1-(5-chloro-6-
C22H28ClN7O3S: 506.1736).
methylpyrazolo[1,5-a]pyrimidin-2-yl)propan-1-amine (7e) (5.0g,
Compounds 9a, c, d were obtained by the same procedure 19.2mmol) as a brown amorphous, which was used for the
as that described for 9b.
next reaction without further purification.
N-({5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyrazolo-
To a solution of 7e (4.8g, 18.4mmol) in methanol (100mL)
[1,5-a]pyrimidin-2-yl}methyl)-5-chloro-2-[(methanesul- was added (S)-tert-butyl pyrrolidin-3-ylcarbamate (17.1g,
fonyl)amino]-N-methylbenzamide Hydrochloride (9a) Col- 91.9mmol) and trimethylamine (20mL, 147mmol). After stir-
1
orless powder; H-NMR (600MHz, DMSO-d6) δ: 2.02–2.13 ring at 65°C for 4h, the reaction mixture was poured into
(1H, m), 2.19–2.30 (1H, m), 2.35 (3H, s), 2.82 (1.5H, s), 2.93 water and extracted with chloroform. The organic layer was
(1.5H, s), 3.00–3.06 (3H, m), 3.71–3.80 (2H, m), 3.82–3.90 washed with brine and dried over anhydrous magnesium
(2H, m), 3.90–3.99 (1H, m), 4.37 (1H, s), 4.75 (1H, s), 6.05 sulfate, filtered, and concentrated under reduced pressure.
(0.5H, s), 6.10 (0.5H, s), 7.42–7.56 (3H, m), 8.33–8.39 (3H, m), The residue was purified by silica gel column chromatog-
8.45 (0.5H, s), 8.50 (0.5H, s), 9.27 (0.5H, s), 9.65 (0.5H, s); raphy (50–100% ethyl acetate in hexane; 20% methanol in
13C-NMR (151MHz, DMSO-d6) δ: 18.05, 29.64, 37.12, 40.47, chloroform) to obtain 10a (4.5g, 12.0mmol, 65%) as a brown
1
45.00, 47.19, 49.12, 53.04, 90.16, 108.93, 126.41, 127.18, 127.54, amorphous. H-NMR (600MHz, CDCl3) δ: 0.88–0.99 (3H, m),
130.17, 133.06, 133.59, 135.07, 147.41, 152.54, 155.67, 167.05; 1.45 (9H, s), 1.72–1.97 (3H, m), 2.16–2.25 (1H, m), 2.33 (3H,
HR-MS ESI/APCI dual m/z: 492.1564 [M+H]+ (Calcd for s), 3.53 (1H, dd, J=10.7, 3.7Hz), 3.71 (1H, ddd, J=10.7, 7.8,
C21H26ClN7O3S: 492.1579).
5.8Hz), 3.80 (1H, dt, J=10.7, 7.4Hz), 3.88–3.97 (2H, m), 4.30
N-[(1S )-1-{5 -[(3S )-3 -Aminopyrrolidin-1-yl]- 6 - (1H, brs), 4.71 (1H, brs), 5.91–6.09 (1H, m), 8.02 (1H, s); MS
methylpyrazolo[1,5-a]pyrimidin-2-yl}propyl]-5-chloro-2- (ESI/APCI dual) m/z: 375 [M+H]+.
[(methanesulfonyl)amino]-N-methylbenzamide Hydrochlo-
ride (9c) Colorless powder; H-NMR (600MHz, DMSO-d6) described for 10a.
Compound 10b was obtained by the same procedure as that
1
δ: 0.91 (1.5H, brs), 1.03 (1.5H, t, J=7.4Hz), 1.82–2.11 (3H,
tert-Butyl [(3S)-1-{2-[(1S)-1-Aminoethyl]-6-methylpyrazolo-
m), 2.12–2.30 (2H, m), 2.35 (1.5H, s), 2.37 (1.5H, s), 2.58 [1,5-a]pyrimidin-5-yl}pyrrolidin-3-yl]carbamate (10b) Pale

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
355
1
yellow powder; H-NMR (600MHz, CDCl3) δ: 1.41–1.50 (12H, 14.8mmol) and trimethylamine (2.5mL, 18.2mmol). After
m), 1.89–1.95 (1H, m), 2.18–2.24 (1H, m), 2.33 (3H, m), 3.53 stirring overnight at room temperature, the reaction mixture
(1H, dd, J=11.4, 3.9Hz), 3.68–3.74 (1H, m), 3.77–3.83 (1H, was concentrated under reduced pressure to obtain tert-butyl
m), 3.88–3.95 (1H, m), 4.20 (1H, q, J=6.9Hz), 4.30 (1H, brs), [(3S)-1-{6-methyl-2-[(1S)-1-(2,2,2-trifluoroacetamido)propyl]-
4.69 (1H, brs), 6.02 (1H, s), 8.02 (1H, s); MS (ESI/APCI dual) pyrazolo[1,5-a]pyrimidin-5-yl}pyrrolidin-3-yl]carbamate (6.5g,
m/z: 361 [M+H]+.
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- reaction without further purification.
crude) as a pale yellow powder, which was used for the next
zolo[1,5-a]pyrimidin-2-yl}propyl]-5-chloro-N-ethyl-2-
To a solution of tert-butyl [(3S)-1-{6-methyl-2-[(1S)-1-(2,2,2-
[(methanesulfonyl)amino]benzamide Hydrochloride (12a) trifluoroacetamido)propyl]pyrazolo[1,5-a]pyrimidin-5-yl}
To a solution of 10a (50mg, 0.134mmol) in N,N-dimethyl- pyrrolidin-3-yl]carbamate (5.4g, 11.5mmol) in N,N-dimeth-
formamide (1.0mL) was added potassium carbonate (46mg, ylformamide (50mL) was added iodomethane (2.2mL,
0.334mmol) and iodoethane (0.011mL, 0.134mmol). After 34.4mmol) and cesium carbonate (15g, 45.9mmol). After
stirring at 80°C for 1h, 5-chloro-2-(methylsulfonamido)- stirring at 65°C for 2h, the reaction mixture was poured
benzoic acid (40mg, 0.160mmol), 1-[bis(dimethylamino)- into water and extracted with ethyl acetate. The organic
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa- layer was washed with brine and dried over anhydrous
fluorophosphate (66mg, 0.174mmol) and trimethylamine magnesium sulfate, filtered, and concentrated under re-
(0.093mL, 0.668mmol) were added to the reaction mix- duced pressure to obtain tert-butyl [(3S)-1-(6-methyl-2-
ture. After stirring at room temperature for 16h, the reac- {(1S)-1-[methyl(trifluoroacetyl)amino]propyl}pyrazolo[1,5-a]-
tion mixture was purified by reversed-phase preparative pyrimidin-5-yl)pyrrolidin-3-yl]carbamate (5.60g, crude) as a
HPLC to obtain tert-butyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2- pale yellow oil, which was used for the next reaction without
[(methanesulfonyl)amino]benzoyl}(ethyl)amino]propyl}-6- further purification.
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate
(13mg, 0.021mmol, 15%) as colorless powder.
To a solution of tert-butyl [(3S)-1-(6-methyl-2-{(1S)-1-
[methyl(trif luoroacetyl)amino]propyl}pyrazolo[1,5-a]-
To a solution of tert-butyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2- pyrimidin-5-yl)pyrrolidin-3-yl]carbamate (5.5g, 11.4mmol)
[(methanesulfonyl)amino]benzoyl}(ethyl)amino]propyl}-6- in tetrahydrofuran (40mL)–methanol (40mL) was added 1M
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate sodium hydroxide aq. (40mL). After stirring for 1h at room
(16mg, 0.025mmol) in 1,4-dioxane (1.0mL) was added 4M hy- temperature, the reaction mixture was poured into water and
drogen chloride in 1,4-dioxane (1.0mL) and the mixture was extracted with chloroform. The organic layer was washed
stirred for 1h at room temperature. Then, the reaction mix- with brine, dried over anhydrous magnesium sulfate, filtered,
ture was concentrated under reduced pressure to obtain 12a and concentrated under reduced pressure to obtain 13a (4.3g,
1
1
(15mg, 0.026mmol, 100%) as a colorless powder. H-NMR 11.1mmol, 98%) as a brown amorphous. H-NMR (600MHz,
(600MHz, DMSO-d6) δ: 0.80–0.98 (6H, m), 1.96–2.30 (4H, CDCl3) δ: 0.88 (3H, t, J=7.4Hz), 1.45 (9H, s), 1.68–1.98 (3H,
m), 2.32–2.40 (3H, m), 2.51–2.64 (3H, m), 2.98–3.05 (2H, m), 2.14–2.25 (1H, m), 2.28–2.42 (6H, m), 3.48–3.63 (2H,
m), 3.69–3.97 (5H, m), 4.47–4.55 (1H, m), 6.15–6.21 (1H, m), m), 3.66–3.85 (2H, m), 3.87–3.98 (1H, m), 4.30 (1H, brs),
7.45–7.56 (3H, m), 8.21 (3H, brs), 8.52 (0.5H, s), 9.20 (0.5H, s); 4.59–4.78 (1H, m), 5.91–6.07 (1H, m), 7.96–8.10 (1H, m); MS
13C-NMR (151MHz, DMSO-d6) δ: 10.85, 13.44, 17.86, 29.22, (ESI/APCI dual) m/z: 389 [M+H]+.
36.84, 40.05, 40.80, 46.70, 48.73, 52.57, 58.64, 90.56, 107.98,
Compound 13b was obtained by the same procedure as that
126.35, 127.32, 127.70, 129.49, 129.61, 133.63, 134.55, 146.97, described for 13a.
154.69, 155.59, 161.22; HR-MS ESI/APCI dual m/z: 534.2037
[M+H]+ (Calcd for C24H32ClN7O3S: 534.2049).
Benzyl [(3S)-1-{6-Methyl-2-[(1S)-1-(methylamino)propyl]-
pyrazolo[1,5-a]pyrimidin-5-yl}pyrrolidin-3-yl]carbamate
Compound 12b was obtained by the same procedure as that (13b) Colorless powder; 1H-NMR (400MHz, CDCl3) δ:
described for 12a.
0.82–0.94 (3H, m), 1.70–1.90 (2H, m), 1.90–2.01 (1H, m),
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyrazolo- 2.17–2.29 (1H, m), 2.32 (3H, s), 2.36 (3H, s), 3.53–3.62 (2H,
[1,5-a]pyrimidin-2-yl}ethyl]-5-chloro-2-[(methanesulfonyl)- m), 3.66–3.87 (2H, m), 3.88–4.00 (1H, m), 4.30–4.41 (1H, m),
amino]-N-propylbenzamide Hydrochloride (12b) Color- 4.86–4.97 (1H, m), 5.12 (2H, s), 6.03 (1H, s), 7.28–7.41 (5H,
1
less powder; H-NMR (600MHz, DMSO-d6) δ: 0.53 (1H, t, m), 8.04 (1H, s); MS (ESI/APCI dual) m/z: 423 [M+H]+.
J=7.2Hz), 0.77 (2H, t, J=7.4Hz), 1.21–1.54 (4H, m), 1.57
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
(2H, d, J=7.0Hz), 1.66 (1H, d, J=7.0Hz), 2.00–2.10 (1H, m), zolo[1,5-a]pyrimidin-2-yl}propyl]-3-chloro-2-[(methanesul-
2.20–2.30 (1H, m), 2.34–2.38 (3H, m), 2.85–2.98 (2H, m), 3.04 fonyl)amino]-N-methylbenzamide Hydrochloride (14a) To
(3H, s), 3.81–3.96 (5H, m), 4.74–4.82 (0.7H, m), 5.73 (0.3H, a solution of 13a (10mg, 0.026mmol) in N,N-dimethylfor-
brs), 6.13 (1H, s), 7.43–7.58 (3H, m), 8.18 (3H, brs), 8.52 (0.5H, mamide (1.0mL) was added 3-chloro-2-(methylsulfonamido)-
s), 9.34 (0.5H, s); 13C-NMR (151MHz, DMSO-d6) δ: 11.49, benzoic acid (6.4mg, 0.031mmol), 1-[bis(dimethylamino)-
17.04, 17.76, 21.44, 29.22, 40.68, 43.95, 46.72, 48.71, 52.59, methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa-
52.71, 89.79, 107.84, 125.19, 126.43, 127.48, 129.53, 129.61, fluorophosphate (13mg, 0.034mmol), and trimethylamine
132.18, 134.35, 146.95, 155.45, 156.56, 166.85; HR-MS ESI/ (0.018mL, 0.13mmol). After stirring at room temperature for
APCI dual m/z: 534.2011 [M+H]+ (Calcd for C24H32ClN7O3S: 3h, the reaction mixture was purified by reversed-phase pre-
534.2049).
parative HPLC to obtain tert-butyl [(S)-1-(2-{(S)-1-[3-chloro-
tert-Butyl [(3S)-1-{6-Methyl-2-[(1S)-1-(methylamino)propyl]- N-methyl-2-(methylsulfonamido)benzamido]propyl}-6-
pyrazolo[1,5-a]pyrimidin-5-yl}pyrrolidin-3-yl]carbamate methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate
(13a) To a solution of 10a (4.3g, 11.4mmol) in metha- (12mg, 0.019mmol, 72%) as a pale yellow powder.
nol (48mL) was added ethyl 2,2,2-trifluoroacetate (2.0g,
To a solution of tert-butyl [(S)-1-(2-{(S)-1-[3-chloro-

356
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
N-methyl-2-(methylsulfonamido)benzamido]propyl}-6- amino]-N-methyl-5-propylbenzamide Hydrochloride (14h)
1
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate Colorless powder; H-NMR (400MHz, DMSO-d6) δ: 0.81–1.06
(12mg, 0.019mmol) in 1,4-dioxane(1.0mL) was added 4M hy- (6H, m), 1.51–1.64 (2H, m), 1.83–2.31 (6H, m), 2.32–2.38 (3H,
drogen chloride in 1,4-dioxane (1.0mL) and the mixture was m), 2.57 (1.5H, s), 2.72 (1.5H, brs), 2.98 (3H, s), 3.45–3.96 (5H,
stirred for 1h at room temperature. Then, the reaction mixture m), 4.48–4.58 (0.5H, m), 5.73–5.81 (0.5H, m), 6.11 (1H, s), 7.14
was concentrated under reduced pressure to obtain 14a (10mg, (1H, s), 7.22–7.31 (1H, m), 7.33–7.41 (1H, m), 8.19–8.35 (3H,
1
0.019mmol, 100%) as a colorless powder. H-NMR (600MHz, m), 8.51 (0.5H, s), 8.95 (0.5H, s); 13C-NMR (151MHz, DMSO-
DMSO-d6) δ: 0.79–1.05 (3H, m), 1.84–2.30 (4H, m), 2.33–2.42 d6) δ: 10.72, 13.53, 17.63, 22.40, 23.92, 29.21, 31.22, 36.32,
(3H, m), 2.43–2.59 (3H, m), 2.98–3.17 (3H, m), 3.68–4.01 40.04, 40.34, 46.71, 48.70, 52.56, 89.94, 107.99, 125.54, 126.54,
(5H, m), 4.37 (0.5H, brs), 5.75 (0.5H, brs), 6.06–6.25 (1H, 127.33, 129.56, 130.80, 133.88, 140.55, 146.96, 154.66, 155.58,
m), 7.23–7.67 (3H, m), 8.17–8.27 (3H, m), 8.51 (0.5H, s), 9.43 169.15; HR-MS ESI/APCI dual m/z: 528.2731 [M+H]+ (Calcd
(0.5H, brs); 13C-NMR (151MHz, DMSO-d6) δ: 10.43, 17.48, for C26H37N7O3S: 528.2751).
22.81, 29.28, 31.23, 40.05, 42.69, 46.68, 48.77, 52.55, 90.10,
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
108.14, 125.89, 128.84, 129.39, 130.20, 130.25, 131.90, 134.53, zolo[1,5-a]pyrimidin-2-yl}propyl]-2-[(methanesulfonyl)-
146.89, 151.11, 154.89, 166.59; HR-MS ESI/APCI dual m/z: amino]-N-methyl-5-(propan-2-yl)benzamide Hydrochloride
1
520.1872 [M+H]+ (Calcd for C23H30ClN7O3S: 520.1892).
(14i) Pale pink amorphous; H-NMR (600MHz, DMSO-d6)
Compounds 14c–e, h–l, o, p, r, s were obtained by the δ: 0.87–0.96 (1.5H, m), 1.00–1.07 (1.5H, m), 1.13–1.24 (6H,
same procedure as that described for 14a. m), 1.84–2.30 (4H, m), 2.34–2.38 (3H, m), 2.56 (1.5H, s), 2.72
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- (1.5H, brs), 2.86–3.02 (4H, m), 3.70–4.07 (5H, m), 4.47–4.56
zolo[1,5-a]pyrimidin-2-yl}propyl]-2-[(methanesulfonyl)- (0.5H, m), 5.73–5.81 (0.5H, m), 6.12 (1H, s), 7.13–7.24 (1H, m),
amino]-N-methylbenzamide Hydrochloride (14c) Colorless 7.29–7.40 (2H, m), 8.36 (3H, brs), 8.51 (0.5H, s), 8.98 (0.5H,
powder; 1H-NMR (600MHz, DMSO-d6) δ: 0.85–1.06 (3H, s); 13C-NMR (151MHz, DMSO-d6) δ: 10.52, 17.65, 22.44,
m), 1.86–2.30 (4H, m), 2.33–2.40 (3H, m), 2.57 (1.5H, s), 2.73 23.66, 23.72, 29.23, 31.22, 32.71, 40.04, 40.42, 46.72, 48.70,
(1.5H, brs), 3.02 (3H, s), 3.76 (2H, brs), 3.82–3.89 (2H, m), 52.58, 89.98, 108.01, 124.63, 125.26, 125.72, 127.45, 130.82,
3.90–3.99 (1H, m), 4.55 (0.5H, brs), 5.77 (0.5H, dd, J=9.7, 132.51, 134.58, 146.94, 155.14, 155.52, 169.19; HR-MS ESI/
5.6Hz), 6.12 (1H, s), 7.26–7.39 (2H, m), 7.41–7.52 (2H, m), 8.39 APCI dual m/z: 528.2740 [M+H]+ (Calcd for C26H37N7O3S:
(3H, brs), 8.51 (0.5H, s), 9.08 (0.5H, s); 13C-NMR (151MHz, 528.2751).
DMSO-d6) δ: 10.71, 17.70, 22.43, 29.24, 31.31, 40.47, 46.81,
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
48.69, 52.63, 58.26, 89.95, 108.04, 125.11, 125.91, 127.07, zolo[1,5-a]pyrimidin-2-yl}propyl]-2-(methanesulfonyl)-N-
127.70, 129.81, 133.33, 134.73, 146.83, 155.15, 155.47, 169.04; methylbenzamide Hydrochloride (14j) Colorless powder;
HR-MS ESI/APCI dual m/z: 486.2257 [M+H]+ (Calcd for 1H-NMR (600MHz, DMSO-d6) δ: 0.98–1.11 (3H, m), 1.85–1.98
C23H31N7O3S: 486.2282).
(1H, m), 2.01–2.21 (2H, m), 2.21–2.29 (1H, m), 2.31–2.41 (3H,
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- m), 3.23–3.41 (6H, m), 3.69–4.08 (5H, m), 5.68–5.82 (1H,
zolo[1,5-a]pyrimidin-2-yl}propyl]-N-methylbenzamide Hy- m), 6.03–6.23 (1H, m), 7.38–7.64 (1H, m), 7.64–7.88 (3H, m),
drochloride (14d) Colorless powder; 1H-NMR (600MHz, 7.97–8.07 (1H, m), 8.27–8.41 (3H, m), 8.50–8.61 (1H, m);
DMSO-d6) δ: 0.75–0.85 (2H, m), 0.95–1.03 (1H, m), 1.85–2.13 13C-NMR (151MHz, DMSO-d6) δ: 10.76, 17.47, 22.24, 24.39,
(3H, m), 2.20–2.29 (1H, m), 2.36 (3H, s), 2.65 (1H, s), 2.77 29.21, 31.72, 45.01, 46.94, 48.72, 52.74, 90.57, 108.19, 127.55,
(2H, s), 3.72–3.80 (2H, m), 3.82–3.88 (2H, m), 3.91–3.97 (1H, 129.09, 129.80, 133.60, 134.22, 134.84, 136.83, 137.66, 154.84,
m), 4.67–4.73 (0.5H, m), 5.72–5.81 (0.5H, m), 6.04 (1H, s), 155.04, 169.11; HR-MS ESI/APCI dual m/z: 471.2179 [M+H]+
7.37–7.51 (5H, m), 8.43 (3H, brs), 8.51–8.59 (1H, m); 13C-NMR (Calcd for C23H30N6O3S: 471.2173).
(151MHz, DMSO-d6) δ: 10.51, 17.44, 23.49, 29.22, 40.05,
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
46.72, 48.73, 52.57, 58.40, 89.61, 108.32, 126.61, 128.38, zolo[1,5-a]pyrimidin-2-yl}propyl]-2-methoxy-N,5-dimeth-
129.14, 134.65, 136.92, 146.71, 155.11, 155.35, 171.21; HR-MS ylbenzamide Hydrochloride (14k) Colorless amorphous;
ESI/APCI dual m/z: 393.2393 [M +H]+ (Calcd for C22H28N6O: 1H-NMR (400MHz, DMSO-d6) δ: 0.72–0.89 (1.5H, m),
393.2397).
0.92–1.05 (1.5H, m), 1.77–1.95 (1H, m), 2.00–2.17 (2H, m),
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- 2.18–2.31 (4H, m), 2.31–2.40 (3H, m), 3.35–3.67 (3H, m),
zolo[1,5-a]pyrimidin-2-yl}propyl]-2-[(methanesulfonyl)- 3.70–4.05 (8H, m), 4.41–4.49 (0.5H, m), 5.74–5.84 (0.5H, m),
amino]-N,5-dimethylbenzamide Hydrochloride (14e) Col- 5.88–6.05 (1H, m), 6.84–7.08 (2H, m), 7.13–7.23 (1H, m),
1
orless powder; H-NMR (600MHz, DMSO-d6) δ: 0.86–0.95 8.43–8.59 (4H, m); 13C-NMR (151MHz, DMSO-d6) δ: 10.76,
(1.5H, m), 0.99–1.06 (1.5H, m), 1.84–2.29 (4H, m), 2.29–2.38 17.47, 19.86, 23.78, 29.21, 30.05, 46.80, 48.74, 52.64, 55.18,
(6H, m), 2.56 (1.5H, s), 2.72 (1.5H, brs), 2.96 (3H, s), 3.70–4.10 60.10, 89.78, 108.25, 111.31, 126.56, 129.58, 130.24, 130.30,
(5H, m), 4.51–4.58 (0.5H, m), 5.74–5.80 (0.5H, m), 6.11 (1H, 134.72, 134.84, 138.28, 152.55, 168.37, 171.74; HR-MS ESI/
s), 7.14 (1H, s), 7.22–7.29 (1H, m), 7.31–7.38 (1H, m), 8.35 APCI dual m/z: 437.2639 [M+H]+ (Calcd for C24H32N6O2:
(3H, brs), 8.51 (0.5H, s), 8.97 (0.5H, s); 13C-NMR (151MHz, 437.2660).
DMSO-d6) δ: 10.68, 17.63, 20.31, 22.38, 29.21, 31.20, 40.04,
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
40.28, 46.74, 48.68, 52.6, 89.92, 107.99, 125.64, 127.10, 130.22, zolo[1,5-a]pyrimidin-2-yl}propyl]-N,5-dimethyl-2-(trifluoro-
130.52, 132.59, 134.60, 135.69, 146.82, 155.14, 155.26, 169.05; methyl)benzamide Hydrochloride (14l) Colorless pow-
HR-MS ESI/APCI dual m/z: 500.2414 [M+H]+ (Calcd for der; 1H-NMR (400MHz, DMSO-d6) δ: 0.72–1.06 (3H, m),
C24H33N7O3S: 500.2438).
1.75–2.30 (4H, m), 2.31–2.46 (6H, m), 3.70–3.99 (5H, m),
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- 4.37–4.43 (0.5H, m), 5.73–5.81 (0.5H, m), 5.82–6.13 (1H,
zolo[1,5-a]pyrimidin-2-yl}propyl]-2-[(methanesulfonyl)- m), 7.18–7.50 (2H, m), 7.65–7.74 (1H, m), 8.33–8.47 (3H, m),

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
357
8.50–8.58 (1H, m); 13C-NMR (151MHz, DMSO-d6) δ: 10.81, fonyl)amino]-N-methylbenzamide Hydrochloride (14b) To
17.46, 20.70, 22.29, 29.22, 31.25, 46.89, 48.71, 52.71, 58.60, a solution of 13a (50mg, 0.13mmol) in N,N-dimethylfor-
89.91, 108.14, 121.93, 122.15, 123.02, 124.84, 126.41, 127.58, mamide (1.0mL) was added 4-chloroanthranilic acid (27mg,
128.46, 129.71, 129.91, 134.71, 134.79, 135.52, 142.39, 143.37, 0.15mmol),
146.45, 154.81, 155.17, 168.11; HR-MS ESI/APCI dual m/z: triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (64mg,
475.2402 [M+H]+ (Calcd for C24H29F3N6O: 475.2428).
0.17mmol) and trimethylamine (0.090mL, 0.64mmol).
1-[bis(dimethylamino)methylene]-1H-1,2,3-
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- After stirring at room temperature for 3h, the reac-
zolo[1,5-a]pyrimidin-2-yl}propyl]-3-[(methanesulfonyl)- tion mixture was purified by reversed-phase preparative
amino]-N-methylthiophene-2-carboxamide Hydrochloride HPLC to obtain tert-butyl ((S)-1-{2-[(S)-1-(2-amino-4-chloro-
(14o) Colorless powder; 1H-NMR (600MHz, DMSO-d6) N-methylbenzamido)propyl]-6-methylpyrazolo[1,5-a]-
δ: 0.88–0.97 (3H, m), 1.89–2.29 (4H, m), 2.35 (3H, s), 2.81 pyrimidin-5-yl}pyrrolidin-3-yl)carbamate (52mg, 0.10mmol,
(3H, brs), 3.06 (3H, brs), 3.69–3.96 (5H, m), 6.09 (1H, s), 7.16 75%) as a colorless powder.
(1H, s), 7.76 (1H, s), 8.23 (3H, s), 8.47 (1H, s), 10.11 (1H, s);
To a solution of tert-butyl ((S)-1-{2-[(S)-1-(2-amino-4-
13C-NMR (151MHz, DMSO-d6) δ: 10.63, 17.58, 22.95, 29.24, chloro-N-methylbenzamido)propyl]-6-methylpyrazolo[1,5-a]-
35.23, 40.29, 46.68, 48.75, 52.55, 58.16, 90.22, 110.98, 119.30, pyrimidin-5-yl}pyrrolidin-3-yl)carbamate (52mg, 0.10mmol)
122.85, 128.06, 128.18, 134.41, 146.89, 154.71, 155.43, 164.07; in chloroform (2.0mL) was added trimethylamine (0.14mL,
HR-MS ESI/APCI dual m/z: 492.1808 [M+H]+ (Calcd for 0.97mmol) and methanesulfonyl chloride (0.022mL,
C21H29N7O3S2: 492.1846).
0.29mmol) at 0°C. After stirring for 10min at 0°C, the re-
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- action mixture was concentrated under reduced pressure.
zolo[1,5-a]pyrimidin-2-yl}propyl]-2-[(methanesulfonyl)- Ethanol (2.0mL) and 2.94mol/L sodium ethoxide in ethanol
amino]-N-methylthiophene-3-carboxamide Hydrochloride (0.16mL, 0.48mmol) were added to the residue. After stir-
1
(14p) Colorless powder; H-NMR (600MHz, DMSO-d6) δ: ring at room temperature for 15min, the reaction mixture
0.78–1.02 (3H, m), 1.84–2.33 (4H, m), 2.35 (3H, s), 2.63–2.75 was poured into water and extracted with chloroform. The
(3H, m), 3.01 (3H, s), 3.68–4.17 (5H, m), 4.61–4.73 (0.5H, m), organic layer was dried over ISOLUTE® Phase Separator and
5.65–5.78 (0.5H, m), 6.05–6.20 (1H, m), 7.00 (1H, s), 7.39 concentrated under reduced pressure. The residue was puri-
(1H, s), 8.25 (3H, brs), 8.36–8.55 (1H, m), 9.87 (0.5H, brs), fied by reversed-phase preparative HPLC to obtain tert-butyl
10.62 (0.5H, brs); 13C-NMR (151MHz, DMSO-d6) δ: 10.54, [(S)-1-(2-{(S)-1-[4-chloro-N-methyl-2-(methylsulfonamido)-
17.57, 22.86, 29.21, 31.00, 40.04, 40.63, 46.72, 48.70, 52.58, benzamido]propyl}-6-methylpyrazolo[1,5-a]pyrimidin-5-yl)-
90.37, 108.01, 120.51, 125.14, 132.95, 134.10, 136.23, 146.72, pyrrolidin-3-yl]carbamate (40mg, 0.065mmol, 67%) as a
152.13, 155.28, 165.59; HR-MS ESI/APCI dual m/z: 492.1812 colorless powder.
[M+H]+ (Calcd for C21H29N7O3S2: 492.1846).
To a solution of tert-butyl [(S)-1-(2-{(S)-1-[4-chloro-N-methyl-2-
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- (methylsulfonamido)benzamido]propyl}-6-methylpyrazolo[1,5-a]-
zolo[1,5-a]pyrimidin-2-yl}propyl]-4-[(methanesulfonyl)- pyrimidin-5-yl)pyrrolidin-3-yl]carbamate (40mg, 0.065mmol)
amino]-N,1-dimethyl-1H-pyrazole-5-carboxamide Hydro- in 1,4-dioxane (1.0mL) was added 4M hydrogen chloride in
chloride (14r) Colorless powder; 1H-NMR (600MHz, 1,4-dioxane (1.0mL) and the mixture was stirred for 1h at
DMSO-d6) δ: 0.80–0.94 (1.5H, m), 0.95–1.05 (1.5H, m), room temperature. Then the reaction mixture was concentrat-
1.84–2.29 (4H, m), 2.33–2.37 (3H, m), 2.63–2.80 (3H, m), ed under reduced pressure to obtain 14b (36mg, 0.063mmol,
1
2.81–2.96 (3H, m), 3.70–3.80 (5H, m), 3.81–3.97 (3H, m), 98%) as a colorless powder. H-NMR (600MHz, DMSO-d6)
4.61–4.75 (0.5H, m), 5.67–5.75 (0.5H, m), 6.05–6.10 (0.5H, δ: 0.86–0.92 (1.5H, m), 1.02 (1.5H, t, J=7.2Hz), 1.86–2.30
m), 7.47 (1H, s), 8.21–8.30 (3.5H, m), 8.47–8.54 (0.5H, m), (4H, m), 2.33–2.38 (3H, m), 2.58 (1.5H, s), 2.73 (1.5H, brs),
9.07 (0.5h, brs); 13C-NMR (151MHz, DMSO-d6) δ: 10.70, 3.01–3.12 (3H, m), 3.68–4.15 (5H, m), 4.56 (0.5H, dd, J=9.9,
17.43, 24.04, 29.21, 30.09, 37.95, 40.02, 40.36, 46.69, 48.70, 4.5Hz), 5.73 (0.5H, dd, J=10.1, 5.6Hz), 6.11 (1H, s), 7.39 (2H,
52.54, 90.05, 108.17, 116.35, 119.53, 131.63, 134.50, 146.86, s), 7.49 (1H, d, J=11.6Hz), 8.23 (3H, brs), 8.51 (0.5H, s),
154.46, 155.36, 161.29; HR-MS ESI/APCI dual m/z: 490.2321 9.28 (0.5H, s); 13C-NMR (151MHz, DMSO-d6) δ: 10.70, 17.65,
[M+H]+ (Calcd for C21H31N9O3S: 490.2343).
22.40, 29.23, 31.34, 40.05, 40.77, 46.72, 48.70, 52.60, 89.98,
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- 108.03, 124.17, 125.66, 128.83, 129.32, 130.36, 133.88, 134.58,
zolo[1,5-a]pyrimidin-2-yl}propyl]-4-[(methanesulfonyl)- 146.98, 154.96, 155.54, 168.14; HR-MS ESI/APCI dual m/z:
amino]-N,1-dimethyl-1H-pyrazole-3-carboxamide Hydro- 520.1872 [M+H]+ (Calcd for C23H30ClN7O3S: 520.1892).
chloride (14s) Colorless powder; 1H-NMR (600MHz,
Compounds 14f, g, m, n, q were obtained by the same pro-
DMSO-d6) δ: 0.86–0.95 (3H, m), 1.82–2.28 (4H, m), 2.32 (3H, cedure as that described for 14b.
s), 2.71 (1.5H, s), 2.92 (1.5H, s), 2.96 (3H, s), 3.68–3.76 (2H,
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
m), 3.79–3.94 (6H, m), 5.73–5.78 (0.5H, m), 5.87–5.93 (0.5H, zolo[1,5-a]pyrimidin-2-yl}propyl]-5-ethyl-2-[(methanesulfonyl)-
m), 6.03 (0.5H, s), 6.07 (0.5H, s), 7.78–7.82 (1H, m), 8.31 (3H, amino]-N-methylbenzamide Hydrochloride (14f) Colorless
1
brs), 8.44 (0.5H, s), 8.49 (0.5H, s), 9.00 (0.5H, s), 9.15 (0.5H, powder; H-NMR (600MHz, DMSO-d6) δ: 0.86–0.95 (1.5H,
s); 13C-NMR (151MHz, DMSO-d6) δ: 10.45, 17.48, 22.43, m), 1.00–1.06 (1.5H, m), 1.14–1.22 (3H, m), 1.84–2.29 (4H,
29.20, 31.19, 38.93, 40.05, 46.83, 48.73, 52.67, 56.81, 90.12, m), 2.33–2.38 (3H, m), 2.57 (1.5H, s), 2.58–2.66 (2H, m), 2.72
108.28, 120.50, 125.79, 134.71, 138.31, 146.35, 155.13, 155.19, (1.5H, brs), 2.97 (3H, s), 3.71–3.99 (5H, m), 4.50–4.57 (0.5H,
163.51; HR-MS ESI/APCI dual m/z: 490.2316 [M+H]+ (Calcd m), 5.74–5.80 (0.5H, m), 6.12 (1H, s), 7.16 (1H, brs), 7.26–7.32
for C21H31N9O3S: 490.2343).
(1H, m), 7.34–7.40 (1H, m), 8.35 (3H, brs), 8.51 (0.5H, s), 8.98
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- (0.5H, s); 13C-NMR (151MHz, DMSO-d6) δ: 10.51, 15.41,
zolo[1,5-a]pyrimidin-2-yl}propyl]-4-chloro-2-[(methanesul- 17.64, 22.41, 27.37, 29.22, 31.23, 40.05, 40.35, 46.73, 48.69,

358
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
52.59, 89.95, 108.00, 125.69, 126.77, 129.02, 130.73, 132.58, 29.22, 31.05, 40.05, 46.72, 48.75, 52.57, 57.66, 90.44, 108.10,
134.57, 141.87, 146.93, 155.15, 155.53, 169.12; HR-MS ESI/ 132.16, 134.59, 138.59, 141.04, 146.71, 154.57, 155.31, 163.77;
APCI dual m/z: 514.2559 [M+H]+ (Calcd for C25H35N7O3S: HR-MS ESI/APCI dual m/z: 493.1780 [M+H]+ (Calcd for
514.2595); chiral HPLC, 99% ee (CHIRALCEL OZ-3 5µm C20H28N8O3S2: 493.1799).
4.6mm×150mm; flow, 1mL/min, 60% ethanol in hexane;
detection wavelength, 254nm), (S)-isomer tR =9.37min, (R)- (methyl)amino]propyl}-6-methylpyrazolo[1,5-a]pyrimidin-
isomer tR =7.31 min. 5-yl)pyrrolidin-3-yl]carbamate (15a) To solution of
tert-Butyl [(3S)-1-(2-{(1S)-1-[(2-Amino-5-chlorobenzoyl)-
a
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- 13a (50mg, 0.129mmol) and 2-amino-5-chloro-benzoic acid
zolo[1,5-a]pyrimidin-2-yl}propyl]-2-[(methanesulfonyl)- (24mg, 0.142mmol) in N,N-dimethylformamide (1.0mL)
amino]-5-methoxy-N-methylbenzamide Hydrochloride (14g
)
was added trimethylamine (0.090mL, 0.64mmol) and
Colorless powder; 1H-NMR (600MHz, DMSO-d6) δ: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
0.85–0.97 (1.5H, m), 0.99–1.06 (1.5H, m), 1.82–2.30 (4H, m), pyridinium 3-oxide hexafluorophosphate (59mg, 0.154mmol).
2.33–2.38 (3H, m), 2.56 (1.5H, s), 2.71 (1.5H, brs), 2.87–2.99 After stirring at room temperature for 1h, the reaction mix-
(3H, m), 3.72–3.81 (5H, m), 3.82–3.89 (2H, m), 3.90–3.98 ture was purified by reversed-phase preparative HPLC to
(1H, m), 4.47–4.58 (0.5H, m), 5.73–5.80 (0.5H, m), 6.09–6.16 obtain 15a (60mg, 0.117mmol, 91%) as a colorless amorphous.
(1H, m), 6.78–6.92 (1H, m), 6.97–7.06 (1H, m), 7.31–7.39 (1H, 1H-NMR (400MHz, CDCl3) δ: 0.87–1.19 (3H, m), 1.46 (9H, s),
m), 8.36 (3H, brs), 8.51 (0.5H, s), 8.96 (0.5H, s); 13C-NMR 1.85–2.30 (4H, m), 2.35 (3H, s), 2.83 (3H, brs), 3.49–3.60 (1H,
(151MHz, DMSO-d6) δ: 10.73, 17.62, 22.47, 29.22, 31.13, m), 3.66–3.86 (2H, m), 3.88–3.99 (1H, m), 4.23–4.48 (3H, m),
40.05, 40.21, 46.72, 48.69, 52.59, 55.55, 89.99, 108.04, 4.67 (1H, brs), 6.07 (1H, s), 6.58–6.68 (1H, m), 7.04–7.14 (1H,
111.66, 112.38, 115.00, 125.39, 128.76, 134.55, 135.23, 146.93, m), 7.22–7.30 (1H, m), 8.04 (1H, s); MS (ESI/APCI dual) m/z:
155.33, 157.56, 168.62; HR-MS ESI/APCI dual m/z: 516.2378 542 [M+H]+.
[M+H]+ (Calcd for C24H33N7O4S: 516.2387).
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- described for 15a.
zolo[1,5-a]pyrimidin-2-yl}propyl]-3-[(methanesulfonyl)- Benzyl [(3S)-1-(2-{(1S)-1-[(2-Amino-5-chlorobenzoyl)-
amino]-N-methylpyridine-4-carboxamide Hydrochloride (methyl)amino]propyl}-6-methylpyrazolo[1,5-a]pyrimidin-
Compound 15b was obtained by the same procedure as that
1
(14m) Colorless powder; H-NMR (600MHz, DMSO-d6) δ: 5-yl)pyrrolidin-3-yl]carbamate (15b) Colorless amorphous;
0.84–0.93 (1.5H, m), 0.97–1.05 (1.5H, m), 1.86–2.30 (4H, m), 1H-NMR (400MHz, CDCl3) δ: 0.85–1.15 (3H, m), 1.88–2.29
2.34–2.39 (3H, m), 2.58 (1.5H, s), 2.75 (1.5H, brs), 3.05–3.12 (4H, m), 2.32 (3H, s), 2.70–2.96 (3H, m), 3.52–3.63 (1H, m),
(3H, m), 3.69–3.99 (5H, m), 4.44–4.49 (0.5H, m), 5.70–5.77 3.67–3.87 (2H, m), 3.89–4.01 (1H, m), 4.28–4.43 (1H, m),
(0.5H, m), 6.12–6.17 (1H, m), 7.44–7.50 (1H, m), 8.27–8.33 4.89–5.01 (1H, m), 5.12 (2H, s), 6.06 (1H, s), 6.58–6.67 (1H,
(3H, m), 8.34–8.38 (0.5H, m), 8.51–8.53 (0.5H, m), 8.53–8.57 m), 7.04–7.22 (1.5H, m), 7.29–7.41 (5.5H, m), 8.04 (1H, s); MS
(1H, m), 8.66–8.72 (1H, m); 13C-NMR (151MHz, DMSO-d6) (ESI/APCI dual) m/z: 576 [M+H]+.
δ: 10.72, 17.61, 22.40, 29.21, 31.12, 34.11, 41.21, 46.84, 48.70,
2-Acetamido-N-[(1S)-1-{5-[(3S)-3-aminopyrrolidin-
52.62, 90.09, 108.15, 122.60, 123.47, 134.64, 142.44, 144.43, 1-yl]-6-methylpyrazolo[1,5-a]pyrimidin-2-yl}propyl]-
146.60, 146.84, 154.54, 155.18, 165.99; HR-MS ESI/APCI dual 5-chloro-N-methylbenzamide Hydrochloride (16) To a so-
m/z: 487.2217 [M+H]+ (Calcd for C22H30N8O3S: 487.2234).
lution of 15a (42mg, 0.081mmol) in chloroform (3.0mL) was
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- added acetic anhydride (0.022mL, 0.24mmol) and pyridine
zolo[1,5-a]pyrimidin-2-yl}propyl]-3-[(methanesulfonyl)- (0.033mL, 0.40mmol). After stirring at room temperature
amino]-N-methylpyridine-2-carboxamide
Hydrochloride for 4h, the reaction mixture was concentrated under reduced
1
(14n) Colorless powder; H-NMR (600MHz, DMSO-d6) δ: pressure and purified by reversed-phase preparative HPLC
0.88–0.94 (1.5H, m), 0.98–1.06 (1.5H, m), 1.87–2.30 (4H, m), to obtain tert-butyl [(3S)-1-(2-{(1S)-1-[(2-acetamido-5-chlo-
2.32–2.38 (3H, m), 2.59 (1.5H, s), 2.75 (1.5H, s), 3.06–3.11 (3H, robenzoyl)(methyl)amino]propyl}-6-methylpyrazolo[1,5-a]-
m), 3.65–3.97 (5H, m), 4.55–4.61 (0.5H, m), 5.72–5.80 (0.5H, pyrimidin-5-yl)pyrrolidin-3-yl]carbamate (26mg, 0.045mmol,
m), 6.10–6.16 (1H, m), 7.46–7.55 (1H, m), 7.87–7.93 (1H, m), 60%) as a colorless amorphous.
8.24 (3H, brs), 8.36 (0.5H, s), 8.44–8.49 (1H, m), 8.52 (0.5H,
s), 9.39 (0.5H, s), 10.18 (0.5H, s); 13C-NMR (151MHz, DMSO- 5-chlorobenzoyl)(methyl)amino]propyl}-6-methylpyra-
d6) δ: 10.62, 17.77, 22.40, 26.96, 29.19, 30.86, 40.89, 46.86, zolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate
(24mg,
To a solution of tert-butyl [(3S)-1-(2-{(1S)-1-[(2-acetamido-
48.74, 52.64, 90.09, 108.07, 124.69, 133.52, 134.70, 145.55, 0.045mmol) in chloroform (0.50mL) was added trifluoroacetic
145.88, 146.62, 147.95, 155.14, 155.36, 167.38; HR-MS ESI/ acid (0.50mL). After stirring at room temperature for 1h, the
APCI dual m/z: 487.2200 [M+H]+ (Calcd for C22H30N8O3S: reaction mixture was poured into sat. sodium bicarbonate aq.
487.2234).
and extracted with chloroform. The organic layer was washed
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- with brine, dried over anhydrous magnesium sulfate, filtered,
zolo[1,5-a]pyrimidin-2-yl}propyl]-4-[(methanesulfonyl)- and concentrated under reduced pressure to obtain 16 (25mg,
amino]-N-methyl-1,2-thiazole-5-carboxamide Hydrochloride 0.052mmol, quant.) as a colorless amorphous. 1H-NMR
(14q) Colorless powder; 1H-NMR (600MHz, DMSO-d6) (600MHz, CDCl3) δ: 0.79–0.99 (1.5H, m), 1.03–1.13 (1.5H,
δ: 0.84–0.91 (1.5H, m), 0.95–1.01 (1.5H, m), 1.88–2.31 (4H, m), 1.74–1.83 (1H, m), 1.89–2.08 (2H, m), 2.10–2.26 (4H, m),
m), 2.35 (3H, s), 2.73 (1.5H, s), 2.78 (1.5H, s), 3.07 (3H, s), 2.34–2.42 (3H, m), 2.73 (1.5H, s), 2.91 (1.5H, s), 3.40–3.47
3.68–3.95 (5H, m), 4.98–5.04 (0.5H, m), 5.70–5.76 (0.5H, m), (1H, m), 3.66–3.79 (2H, m), 3.85–3.94 (2H, m), 4.79–4.88
6.09–6.14 (1H, m), 8.21 (3H, brs), 8.44 (0.5H, s), 8.52 (0.5H, (0.5H, m), 5.90–5.97 (0.5H, m), 5.98–6.08 (1H, m), 7.23 (0.5H,
s), 8.81 (0.5H, s), 8.86 (0.5H, s), 9.62 (0.5H, s), 9.93 (0.5H, brs), 7.31–7.37 (1H, m), 8.01 (1H, brs), 8.06–8.21 (1H, m), 8.67
s); 13C-NMR (151MHz, DMSO-d6) δ: 10.63, 17.50, 22.45, (0.5H, brs); 13C-NMR (151MHz, DMSO-d6) δ: 10.73, 17.64,

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
359
22.23, 27.51, 31.03, 33.22, 47.49, 50.34, 52.61, 56.97, 89.77, perature for 1h, the reaction mixture was filtered through a
107.96, 126.35, 127.94, 129.02, 129.33, 133.10, 133.99, 134.15, membrane filter and concentrated under reduced pressure to
147.35, 154.63, 155.61, 167.37, 168.74; HR-MS ESI/APCI dual obtain 18 (30mg, 0.060mmol, 85%) as a colorless amorphous.
m/z: 484.2200 [M+H]+ (Calcd for C24H30ClN7O2: 484.2222).
Methyl
1H-NMR (400MHz, CDCl3) δ: 0.82–1.15 (3H, m), 1.75–1.88
(2-{[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6- (1H, m), 1.88–2.23 (3H, m), 2.32–2.41 (3H, m), 2.62–2.92 (6H,
methylpyrazolo[1,5-a]pyrimidin-2-yl}propyl](methyl)- m), 3.41–3.53 (1H, m), 3.67–3.79 (2H, m), 3.82–3.94 (2H, m),
carbamoyl}-4-chlorophenyl)carbamate (17) To a solution 4.69–4.85 (0.5H, m), 4.93–5.07 (0.5H, m), 5.51–5.64 (0.5H,
of 15b (60mg, 0.10mmol) in pyridine (1.0mL) was added m), 5.83–5.94 (0.5H, m), 5.95–6.10 (1H, m), 7.10–7.34 (2H, m),
methyl chloroformate (0.16mL, 2.1mmol). After stirring at 7.97–8.21 (2H, m); 13C-NMR (151MHz, DMSO-d6) δ: 10.79,
room temperature for 4h, the reaction mixture was concen- 17.66, 23.45, 26.02, 31.39, 33.24, 47.49, 50.36, 57.01, 58.84,
trated under reduced pressure. The residue was purified by 89.37, 107.94, 123.00, 124.34, 124.83, 125.91, 126.13, 129.00,
silica gel column chromatography (OH, 10–100% ethyl acetate 134.15, 147.25, 154.47, 155.43, 155.65, 167.99; HR-MS ESI/
in hexane) to obtain benzyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2- APCI dual m/z: 499.2306 [M+H]+ (Calcd for C24H31ClN8O2:
[(methoxycarbonyl)amino]benzoyl}(methyl)amino]propyl}-6- 499.2331).
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate
(26mg, 0.041mmol, 40%) as a colorless amorphous.
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra-
zolo[1,5-a]pyrimidin-2-yl}propyl]-5-chloro-N-methyl-2-
To a solution of benzyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2- [(methylsulfamoyl)amino]benzamide (19) To
a solution
[(methoxycarbonyl)amino]benzoyl}(methyl)amino]propyl}-6- of chlorosulfonyl isocyanate (0.020mL, 0.231mmol) in
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate chloroform (1.2mL) was added 2-chloroethanol (0.031mL,
(26mg, 0.041mmol) in methanol (2.1mL) was added 10% pal- 0.462mmol). After stirring at room temperature for 1h,
ladium on activated carbon (13mg). The reaction was flushed the reaction mixture was added trimethylamine (0.032mL,
with hydrogen and stirred under hydrogen atmosphere at room 0.231mmol) and 15b (67mg, 0.115mmol). After stirring at
temperature for 1h. The reaction mixture was filtered through room temperature for 6h, the reaction mixture was concen-
membrane filter and concentrated under reduced pressure to trated under reduced pressure. To a solution of the residue
obtain 17 (19mg, 0.037mmol, 91%) as a colorless amorphous. in chloroform (1.2mL) was added trimethylamine (0.048mL,
1H-NMR (400MHz, CDCl3) δ: 0.79–1.12 (3H, m), 1.83–2.28 0.346mmol) and methylamine (0.46mL, 0.923mmol). After
(4H, m), 2.28–2.38 (3H, m), 2.67–2.74 (1.5H, m), 2.77–2.86 stirring at 120°C under microwave irradiation for 30min,
(1.5H, m), 3.56–3.67 (1H, m), 3.70–3.80 (3H, m), 3.82–4.06 the reaction mixture was added to water and extracted
(4H, m), 4.67–4.82 (0.5H, m), 5.72–5.88 (0.5H, m), 5.91–6.07 with chloroform. The organic layer was concentrated under
(1H, m), 7.19–7.24 (0.5H, m), 7.27–7.38 (1H, m), 7.77–8.23 reduced pressure. The residue was purified by silica gel
(2H, m), 8.54–8.68 (0.5H, m); 13C-NMR (151MHz, DMSO- column chromatography (OH, 10–100% ethyl acetate in
d6) δ: 10.63, 17.52, 22.23, 29.60, 31.17, 40.05, 46.70, 48.88, hexane) to obtain benzyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2-
52.07, 52.89, 89.85, 107.94, 126.49, 126.89, 127.52, 128.20, [(methylsulfamoyl)amino]benzoyl}(methyl)amino]propyl}-6-
129.33, 133.51, 134.49, 147.19, 154.33, 154.91, 155.55, 167.57; methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate
HR-MS ESI/APCI dual m/z: 500.2145 [M+H]+ (Calcd for (23mg, 0.035mmol, 30%) as a colorless amorphous.
C24H30ClN7O3: 500.2171).
To a solution of benzyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2-
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methylpyra- [(methylsulfamoyl)amino]benzoyl}(methyl)amino]propyl}-6-
zolo[1,5-a]pyrimidin-2-yl}propyl]-5-chloro-N-methyl-2- methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate
[(methylcarbamoyl)amino]benzamide (18) To a solution of (22mg, 0.032mmol) in methanol (1.6mL) was added 10% pal-
15b (50mg, 0.087mmol) in chloroform (0.87mL) was added ladium on activated carbon (11mg). The reaction mixture was
pyridine (0.028mL, 0.347mmol) and 4-nitrophenyl chlorofor- flushed with hydrogen and stirred under hydrogen atmosphere
mate (19mg, 0.095mmol). After stirring at room temperature at room temperature for 1h. The reaction mixture was filtered
for 1h, the reaction mixture was concentrated under reduced through a membrane filter and concentrated under reduced
pressure.
pressure to obtain 19 (16mg, 0.030mmol, 92%) as a colorless
1
To a solution of the residue in chloroform (0.87mL) was amorphous. H-NMR (400MHz, CDCl3) δ: 1.00–1.16 (3H, m),
added methylamine (0.071mL, 0.694mmol). After stirring at 1.77–2.23 (4H, m), 2.34–2.40 (3H, m), 2.60–2.65 (1.5H, m),
room temperature for 2h, the reaction mixture was added to 2.71 (2H, s), 2.75–2.83 (2.5H, m), 3.39–3.54 (1H, m), 3.67–3.78
water and extracted with chloroform. The organic layer was (2H, m), 3.82–3.95 (2H, m), 4.60–4.70 (1H, m), 5.92–6.04 (1H,
concentrated under reduced pressure. The residue was purified m), 7.22–7.25 (0.5H, m), 7.30–7.39 (1H, m), 7.63–7.74 (1H, m),
by silica gel column chromatography (OH, 10–100% ethyl ace- 8.36 (0.5H, brs); 13C-NMR (151MHz, DMSO-d6) δ: 10.63,
tate in hexane) to obtain benzyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2- 17.87, 22.35, 28.17, 31.21, 31.97, 47.21, 49.86, 52.90, 55.67,
[(methylcarbamoyl)amino]benzoyl}(methyl)amino]propyl}-6- 89.61, 107.84, 122.29, 126.45, 127.28, 129.51, 130.19, 133.57,
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate 134.35, 147.29, 154.47, 155.69, 167.59; HR-MS ESI/APCI dual
(44mg, 0.070mmol, 81%) as a colorless amorphous.
To a solution of benzyl [(3S)-1-(2-{(1S)-1-[{5-chloro-2-
m/z: 535.1982 [M+H]+ (Calcd for C23H31ClN8O3S: 535.2001).
tert-Butyl [(1S)-1-(7-Hydroxy-6-methyl-5-oxo-4,5-
[(methylcarbamoyl)amino]benzoyl}(methyl)amino]propyl}-6- dihydropyrazolo[1,5-a]pyrimidin-2-yl)propyl]methylcar-
methylpyrazolo[1,5-a]pyrimidin-5-yl)pyrrolidin-3-yl]carbamate bamate (20) To a solution of 5c (0.55g 2.16mmol) in
(44mg, 0.070mmol) in methanol (3.5mL) was added 10% ethanol (10mL) was added diethyl 2-methylmalonate (0.55mL,
palladium on activated carbon (22mg). The reaction was 3.24mmol) and 2.94M sodium ethoxide in ethanol (3.7mL,
flushed with hydrogen and stirred under hydrogen atmosphere 10.81mmol), and the mixture was stirred at 90°C for 5h. The
at room temperature for 1h. After stirring at room tem- reaction mixture was concentrated under reduced pressure.

360
Chem. Pharm. Bull.
Vol. 68, No. 4 (2020)
The residue was acidified with 1N hydrogen chloride aq. and s), 6.07–6.16 (1H, m), 7.32–7.42 (2H, m), 7.52–7.58 (1H, m);
extracted with chloroform. The organic layer was dried over 13C-NMR (151MHz, DMSO-d6) δ: 10.81, 14.95, 22.85, 27.45,
ISOLUTE® Phase Separator and concentrated under reduced 31.63, 32.72, 40.49, 47.77, 49.96, 52.49, 55.29, 84.19, 90.42,
pressure. The residue was added to diethyl ether, precipitated, 123.82, 126.23, 127.44, 128.60, 129.12, 133.28, 146.87, 148.18,
and collected to obtain 20 (0.62g, 1.92mmol, 89%) as a pale 154.13, 159.73, 169.12; HR-MS ESI/APCI dual m/z: 549.2143
yellow powder. 1H-NMR (400MHz, CDCl3) δ: 0.90–1.01 [M+H]+ (Calcd for C24H33ClN8O3S: 549.2158).
(3H, m), 1.49 (9H, s), 1.69–1.75 (3H, m), 1.83–1.97 (1H, m),
Compounds 24b, e, f were obtained by the same procedure
2.10–2.22 (1H, m), 2.67 (3H, s), 3.58–3.69 (1H, m), 4.98–5.28 as that described for 24a.
(1H, m), 5.73 (1H, brs), 9.16 (1H, brs); MS (ESI/APCI dual)
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-7-(dimethyl-
amino)-6-methylpyrazolo[1,5-a]pyrimidin-2-yl}propyl]-
m/z: 337 [M+H]+.
(1S)-1-(5,7-Dichloro-6-methylpyrazolo[1,5-a]pyrimidin- 5-chloro-2-[(methanesulfonyl)amino]-N-methylbenzamide
2-yl)-N-methylpropan-1-amine Hydrochloride (21) The (24b) Pink powder; H-NMR (400MHz, CDCl3) δ: 0.96–1.04
1
mixture of 20 (4.6g, 13.6mmol) and phosphorus oxychloride (3H, m), 1.67–1.77 (1H, m), 1.93–2.06 (2H, m), 2.09–2.20 (4H,
(13mL, 136.1mmol) was stirred at 110°C for 3h, and the reac- m), 2.86–2.95 (6H, m), 3.11–3.19 (6H, m), 3.26–3.34 (1H, m),
tion mixture was concentrated under reduced pressure. After 3.57–3.70 (4H, m), 3.74–3.85 (0.3H, m), 4.58–4.69 (0.7H, m),
purifying by silica gel column chromatography (NH, 1–5% 6.05 (1H, s), 7.24–7.33 (1H, m), 7.36–7.41 (1H, m), 7.58–7.63
methanol in chloroform), 4M hydrogen chloride in AcOEt (1H, m); 13C-NMR (151MHz, DMSO-d6) δ: 10.83, 15.57,
was added to the residue and the mixture was concentrated 22.73, 30.73, 31.87, 40.54, 41.38, 47.59, 50.02, 52.59, 55.49,
under reduced pressure to obtain 21 (1.8g, 5.77mmol, 42%) 58.32, 90.22, 94.88, 124.36, 126.29, 127.12, 128.66, 129.02,
as a colorless powder. 1H-NMR (400MHz, DMSO-d6) δ: 133.45, 148.28, 149.48, 153.91, 159.41, 168.98; HR-MS ESI/
0.73–0.87 (3H, m), 1.94–2.20 (2H, m), 2.45 (3H, s), 2.48 (3H, APCI dual m/z: 563.2274 [M+H]+ (Calcd for C25H35ClN8O3S:
s), 4.34–4.46 (1H, m), 7.05 (1H, s), 9.38 (2H, brs); MS (ESI/ 563.2314).
APCI dual) m/z: 273 [M+H]+.
N-[(1S)-1-(5-[(3S)-3-Aminopyrrolidin-1-yl]-7-{[2-(methanesul-
N-[(1S)-1-{5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methyl-7- fonyl)ethyl]amino}-6-methylpyrazolo[1,5-a]pyrimidin-
(methylamino)pyrazolo[1,5-a]pyrimidin-2-yl}propyl]- 2-yl)propyl]-5-chloro-2-[(methanesulfonyl)amino]-N-meth-
1
5-chloro-2-[(methanesulfonyl)amino]-N-methylbenzamide ylbenzamide (24e) Colorless powder; H-NMR (400MHz,
(24a) To a solution of 21 (0.12g, 0.388mmol) in acetonitrile CDCl3) δ: 0.99 (3H, brs), 1.40–2.18 (4H, m), 2.24 (3H, brs),
(1.0mL) and water (1.0mL) was added 9.8M methylamine/ 2.81 (3H, brs), 2.97 (3H, brs), 3.01 (3H, brs), 3.21–4.09
methanol (0.20mL, 1.94mmol) and sodium bicarbonate (9H, m), 4.53 (1H, brs), 6.06 (2H, brs), 7.21–7.45 (2H, m),
(0.33g, 3.88mmol). After stirring at room temperature for 7.49–7.61(1H, m); 13C-NMR (151MHz, DMSO-d6) δ: 10.83,
22h, the reaction mixture was added to 20% potassium car- 14.57, 22.75, 30.67, 31.57, 38.61, 40.54, 40.96, 47.83, 49.92,
bonate aq. and extracted with chloroform. The organic layer 52.47, 54.08, 55.21, 86.26, 90.46, 124.04, 126.29, 127.22,
was dried over ISOLUTE® Phase Separator and concentrated 128.64, 129.00, 133.39, 146.29, 147.31, 154.39, 159.25, 169.08;
under reduced pressure to obtain 5-chloro-N,6-dimethyl-2- HR-MS ESI/APCI dual m/z: 641.2060 [M+H]+ (Calcd for
[(1S)-1-(methylamino)propyl]pyrazolo[1,5-a]pyrimidin-7-amine C26H37ClN8O5S2: 641.2090).
(0.10g, 0.372mmol, 96%) as a colorless powder.
To a solution of 5-chloro-N,6-dimethyl-2-[(1S)-1-(methyl- 7-{[2-(methylsulfamoyl)ethyl]amino}pyrazolo[1,5-a]pyrim-
amino)propyl]pyrazolo[1,5-a]pyrimidin-7-amine (97mg, idin-2-yl)propyl]-5-chloro-2-[(methanesulfonyl)amino]-N-
N-[(1S)-1-(5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methyl-
0.361mmol) and 5-chloro-2-(methylsulfonamido)benzoic acid methylbenzamide (24f) Colorless powder; 1H-NMR
(99mg, 0.397mmol) in N,N-dimethylformamide (1.0mL) (400MHz, CDCl3) δ: 0.98 (3H, t, J=7.0Hz), 1.70–1.82 (1H,
was added trimethylamine (0.25mL, 1.80mmol) and m), 1.94–2.06 (2H, m), 2.08–2.19 (4H, m), 2.21 (3H, brs),
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]- 2.82 (6H, brs), 2.97 (3H, s), 3.26–3.80 (7H, m), 3.98 (2H, q,
pyridinium 3-oxide hexafluorophosphate (0.17g, 0.433mmol). J=6.6Hz), 4.53 (1H, t, J=7.2Hz), 4.82 (1H, brs), 6.05 (1H,
After stirring at room temperature for 1h, the reaction mix- s), 6.21 (1H, brs), 7.32–7.44 (2H, m), 7.57 (1H, d, J=8.7Hz);
ture was purified by reversed-phase preparative HPLC to 13C-NMR (151MHz, DMSO-d6) δ: 10.83, 14.61, 22.75, 27.57,
obtain 5-chloro-N-{(1S)-1-[5-chloro-6-methyl-7-(methylamino)- 28.52, 30.67, 31.53, 40.54, 47.83, 49.84, 49.92, 52.51, 55.19,
pyrazolo[1,5-a]pyrimidin-2-yl]propyl}-2-[(methanesulfonyl)- 85.94, 90.48, 124.06, 126.29, 127.22, 128.64, 129.02, 133.39,
amino]-N-methylbenzamide (0.13g, 0.904mmol, 90%) as a 146.45, 147.29, 154.39, 159.31, 169.08; HR-MS ESI/APCI dual
colorless powder.
To a solution of 5-chloro-N-{(1S)-1-[5-chloro-6-methyl-7-
m/z: 656.2170 [M+H]+ (Calcd for C26H38ClN9O5S2: 656.2199).
N-[(1S)-1-{7-[(2-Aminoethyl)amino]-5-[(3S)-3-amino-
(methylamino)pyrazolo[1,5-a]pyrimidin-2-yl]propyl}-2- pyrrolidin-1-yl]-6-methylpyrazolo[1,5-a]pyrimidin-2-yl}-
[(methanesulfonyl)amino]-N-methylbenzamide (0.12mg, propyl]-5-chloro-2-[(methanesulfonyl)amino]-N-methyl-
0.252mmol) in 1-methyl-2-pyrrolidone (1.0mL) was added tri- benzamide (24c) To a solution of 21 (120mg, 0.38mmol)
methylamine (0.35mL, 2.52mmol) and (S)-pyrrolidin-3-amine in acetonitrile (1.0mL) and water (1.0mL) was added
(0.11mL, 1.26mmol). After stirring at 150°C under microwave 1-Boc-ethylenediamine (0.30mL, 1.91mmol) and sodium
irradiation for 30min, the reaction mixture was purified bicarbonate (0.32g, 3.81mmol). After stirring at room tem-
by reversed-phase preparative HPLC to obtain 24a (41mg, perature for 20h, the reaction mixture was poured into sat.
1
0.30mmol, 30%) as a pale yellow powder. H-NMR (400MHz, sodium bicarbonate aq. and extracted with chloroform. The
CDCl3) δ: 0.94–1.02 (3H, m), 1.74–2.19 (4H, m), 2.23 (3H, organic layer was dried over ISOLUTE® Phase Separator
s), 2.80 (3H, s), 2.97 (3H, s), 3.21–3.28 (3H, m), 3.29–3.38 and concentrated under reduced pressure. The residue was
(1H, m), 3.52–3.81 (4H, m), 4.49–4.60 (1H, m), 6.03 (1H, purified by reversed-phase preparative HPLC to obtain tert-

Vol. 68, No. 4 (2020)
Chem. Pharm. Bull.
361
butyl [2-({5-chloro-6-methyl-2-[(1S)-1-(methylamino)propyl]- 79.14, 90.54, 122.11, 125.99, 127.20, 128.50, 128.80, 132.00,
pyrazolo[1,5-a]pyrimidin-7-yl}amino)ethyl]carbamate (40mg, 146.75, 147.45, 154.23, 159.09, 170.04; HR-MS ESI/APCI dual
0.10mmol, 27%) as a colorless amorphous.
To solution of tert-butyl [2-({5-chloro-6-methyl-2-
m/z: 592.2546 [M+H]+ (Calcd for C26H38ClN9O3S: 592.2580).
a
[(1S)-1-(methylamino)propyl]pyrazolo[1,5-a]pyrimidin-7-yl}-
Computational Chemistry
amino)ethyl]carbamate (40mg, 0.10mmol) and 5-chloro-2-
The conformations of the compounds were sampled using
(methylsulfonamido)benzoic acid (30mg, 0.12mmol) in REMD simulation49) run on GROMACS 5.0.4.50) For each
N,N-dimethylformamide (1.0mL) was added trimethyl- compound, eight independent REMD simulations were per-
amine (0.070mL, 0.50mmol) and 1-[bis(dimethylamino)- formed under NVT conditions for 10ns each, to sample suf-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexa- ficient conformational space. Temperatures of the replicas
fluorophosphate (50mg, 0.13mmol). After stirring at room were set at 310.0, 366.5, 433.2, 512.1, 605.4, 715.6, 845.9, and
temperature for 2h, the reaction mixture was purified 1000K. GAFF forcefiled51) and GBSA model52) were applied
by reversed-phase preparative HPLC to obtain tert-butyl to the compounds and the solvent, respectively. The dihedral
{2-[(5-chloro-2-{(1S)-1-[{5-chloro-2-[(methanesulfonyl)amino]- angle between the pyrazolo[1,5-a]pyrimidine ring and the
benzoyl}(methyl)amino]propyl}-6-methylpyrazolo[1,5-a]- amide plane was calculated for each conformation sampled
pyrimidin-7-yl)amino]ethyl}carbamate (33mg, 0.052mmol, every 10 ps at 310.0K.
52%) as a colorless amorphous.
Cell and Virus HEp-2 cells were purchased from DS
To solution of tert-butyl {2-[(5-chloro-2-{(1S)-1- Pharma Biomedical Co., Ltd. (Osaka, Japan) and cultured
a
[{5-chloro-2-[(methanesulfonyl)amino]benzoyl}(methyl)- in minimum essential medium (MEM) supplemented with
amino]propyl}-6-methylpyrazolo[1,5-a]pyrimidin-7-yl)amino]- 10% fetal bovine serum (FBS), 50µg/mL gentamicin and
ethyl}carbamate (33mg, 0.052mmol) in 1-methyl-2-pyrrolidone 600µg/mL L-glutamine. RSV A2 (ATCC VR-1540) were pur-
(1.0mL) was added trimethylamine (0.12mL, 0.83mmol) and chased from American Type Culture Collection (Manassas,
(S)-pyrrolidin-3-amine (0.037mL, 0.42mmol). After stirring VA, U.S.A.).
at 150°C under microwave irradiation for 30min, the reaction
Antiviral Assay HEp-2 cells were cultured overnight in
mixture was purified by reversed-phase preparative HPLC to 96-well plates, each test compound diluted with MEM was
obtain tert-butyl {2-[(5-[(3S)-3-aminopyrrolidin-1-yl]-2-{(1S)-1- added, and the medium was supplemented with 2% FBS, 100
[{5-chloro-2-[(methanesulfonyl)amino]benzoyl}(methyl)- units/mL penicillin, 100µg/mL streptomycin and 300µg/mL
amino]propyl}-6-methylpyrazolo[1,5-a]pyrimidin-7-yl)amino]- L-glutamine. The cells were then infected with RSV A2. After
ethyl}carbamate (14mg, 0.021mmol, 40%) as a colorless incubation at 37°C in the presence of 5% CO2 for 4d, the
powder.
RSV-induced CPE was determined by adding XTT reagent.
To a solution of tert-butyl {2-[(5-[(3S)-3-aminopyrrolidin- The concentration of the test compound required to inhibit
1-yl]-2-{(1S)-1-[{5-chloro-2-[(methanesulfonyl)amino]- the CPE by 50% (EC50) was calculated by the least squares
benzoyl}(methyl)amino]propyl}-6-methylpyrazolo[1,5-a]- method. The EC50 values were determined in duplicate with at
pyrimidin-7-yl)amino]ethyl}carbamate (14mg, 0.021mmol) in least five concentrations (n=1–3).
chloroform (1.0mL) was added trifluoroacetic acid (1.0mL).
Acknowledgments The authors thank Dr. Taiji Asami
for carefully reading the manuscript and making useful sug-
gestions to improve it, Dr. Takashi Yoshizumi, Mr. Susumu
After stirring at room temperature for 1h, the reaction mix-
ture was poured into sat. sodium bicarbonate aq. and extracted
with chloroform. The organic layer was dried over ISOLUTE®
Phase Separator and concentrated under reduced pressure to Yamanobe and Dr. Tomohiro Sugimoto for their helpful dis-
obtain 24c (11mg, 0.019mmol, 91%) as a colorless powder. cussions, and Mr. Akira Higuchi for his assistance with the
1H-NMR (400MHz, CDCl3) δ: 0.94–1.03 (3H, m), 1.65–2.27 analytical and spectrum studies.
(7H, m), 2.82 (3H, s), 2.91–3.03 (5H, m), 3.22–3.31 (1H, m),
3.51–3.83 (7H, m), 4.52–4.63 (1H, m), 6.06 (1H, s), 6.08–6.17
Conflict of Interest The authors declare no conflict of
(1H, m), 7.32–7.43 (2H, m), 7.52–7.60 (1H, m); 13C-NMR interest.
(151MHz, DMSO-d6) δ: 10.73, 13.99, 22.37, 30.22, 32.90,
40.05, 41.14, 43.59, 47.95, 50.38, 51.41, 56.97, 88.43, 90.58,
Supplementary Materials The online version of this ar-
118.61, 120.78, 125.71, 126.07, 128.38, 131.26, 132.44, 146.51, ticle contains supplementary materials.
154.07, 158.99, 170.57; HR-MS ESI/APCI dual m/z: 578.2381
[M+H]+ (Calcd for C25H36ClN9O3S: 578.2423).
Compound 24d was obtained by the same procedure as that
described for 24c.
References
1) Lozano R., Naghavi M., Foreman K., et al., Lancet, 380, 2095–2128
(2012).
2) Sigurs N., Bjarnason R., Sigurbergsson F., Kjellman B., Am. J.
Respir. Crit. Care Med., 161, 1501–1507 (2000).
N-[(1S)-1-(5-[(3S)-3-Aminopyrrolidin-1-yl]-6-methyl-
7-{[2-(methylamino)ethyl]amino}pyrazolo[1,5-a]pyrimidin-
2-yl)propyl]-5-chloro-2-[(methanesulfonyl)amino]-N-meth-
3) Sigurs N., Aljassim F., Kjellman B., Robinson P. D., Sigurbergsson
F., Bjarnason R., Gustafsson P. M., Thorax, 65, 1045–1052 (2010).
4) Sigurs N., Gustafsson P. M., Bjarnason R., Lundberg F., Schmidt S.,
Sigurbergsson F., Kjellman B., Am. J. Respir. Crit. Care Med., 171,
137–141 (2005).
5) Stein R. T., Sherrill D., Morgan W. J., Holberg C. J., Halonen M.,
Taussig L. M., Wright A. L., Martinez F. D., Lancet, 354, 541–545
(1999).
1
ylbenzamide (24d) Colorless powder; H-NMR (400MHz,
CDCl3) δ: 0.94–1.02 (3H, m), 1.70–1.79 (1H, m), 1.94–2.24
(6H, m), 2.48 (3H, s), 2.83 (3H, s), 2.86–2.99 (5H, m),
3.22–3.31 (1H, m), 3.53–3.81 (7H, m), 4.52–4.65 (1H, m),
6.02–6.12 (2H, m), 7.32–7.43 (2H, m), 7.53–7.59 (1H, m);
13C-NMR (151MHz, DMSO-d6) δ: 10.75, 14.19, 22.61, 30.47,
32.01, 34.41, 40.05, 42.56, 47.85, 50.06, 50.93, 51.85, 55.87,
6) Bont L., Steijn M., van Aalderen W. M., Kimpen J. L., Pediatr. In-

362
Chem. Pharm. Bull.
249, 2666–2670 (1983).
Vol. 68, No. 4 (2020)
fect. Dis. J., 23, 414–417 (2004).
31) Streeter D. G., Witkowski J. T., Khare G. P., Sidwell R. W., Bauer
R. J., Robins R. K., Simon L. N., Proc. Natl. Acad. Sci. U.S.A., 70,
1174–1178 (1973).
32) Ohmit S. E., Moler F. W., Monto A. S., Khan A. S., J. Clin. Epide-
miol., 49, 963–967 (1996).
33) Graci J. D., Cameron C. E., Rev. Med. Virol., 16, 37–48 (2006).
34) Hultgren C., Milich D. R., Weiland O., Sällberg M., J. Gen. Virol.,
79, 2381–2391 (1998).
35) Smith D. W., Frankel L. R., Mathers L. H., Tang A. T., Ariagno R.
L., Prober C. G., N. Engl. J. Med., 325, 24–29 (1991).
36) Englund J. A., Piedra P. A., Ahn Y. M., Gilbert B. E., Hiatt P., J.
Pediatr., 125, 635–641 (1994).
7) Henderson J., Hilliard T. N., Sherriff A., Stalker D., Al Shammari
N., Thomas H. M., Pediatr. Allergy Immunol., 16, 386–392 (2005).
8) Nair H., Nokes D. J., Gessner B. D., et al., Lancet, 375, 1545–1555
(2010).
9) Fleming D. M., Cross K. W., Lancet, 342, 1507–1510 (1993).
10) Griffin M. R., Coffey C. S., Neuzil K. M., Mitchel E. F. Jr., Wright
P. F., Edwards K. M., Arch. Intern. Med., 162, 1229–1236 (2002).
11) Nicholson K. G., Epidemiol. Infect., 116, 51–63 (1996).
12) Neuzil K. M., Maynard C., Griffin M. R., Heagerty P., Clin. Infect.
Dis., 37, 201–207 (2003).
13) Thompson W. W., Shay D. K., Weintraub E., Brammer L., Cox N.,
Anderson L. J., Fukuda K., JAMA, 289, 179–186 (2003).
14) Resch B., Manzoni P., Lanari M., Paediatr. Respir. Rev., 10, 148–
153 (2009).
37) Meert K. L., Sarnaik A. P., Gelmini M. J., Lieh-Lai M. W., Crit.
Care Med., 22, 566–572 (1994).
38) Blount R. E. Jr., Morris J. A., Savage R. E., Proc. Soc. Exp. Biol.
Med., 92, 544–549 (1956).
39) Tayyari F., Marchant D., Moraes T. J., Duan W., Mastrangelo P.,
Hegele R. G., Nat. Med., 17, 1132–1135 (2011).
15) Hall C. B., Powell K. R., MacDonald N. E., Gala C. L., Menegus M.
E., Suffin S. C., Cohen H. J., N. Engl. J. Med., 315, 77–81 (1986).
16) Glezen W. P., Taber L. H., Frank A. L., Kasel J. A., Am. J. Dis.
Child., 140, 543–546 (1986).
40) Tripp R. A., Jones L. P., Haynes L. M., Zheng H., Murphy P. M.,
Anderson L. J., Nat. Immunol., 2, 732–738 (2001).
17) Committee on Infectious Diseases, Pediatrics, 124, 1694–1701
(2009).
41) Mackman R. L., Sangi M., Sperandio D., et al., J. Med. Chem., 58,
1630–1643 (2015).
18) Shi T., McAllister D. A., O’Brien K. L., et al., Lancet, 390, 946–
958 (2017).
42) Babaoglu K., Eisenberg E. J., Machkman R. L., Sangi M., Siegel D.,
Yang H., WO Patent WO/2011/163518, December 29 2011.
43) Tahri A., Vendeville S. M. H., Jonckers T. H. M., Raboisson P. J. M.,
Demin S. D., Hu L., Cooymans L. P., WO Patent WO/2016/091774,
June 16, 2016.
44) Tahri A., Vendeville S. M. H., Jonckers T. H. M., Raboisson P. J.
M., Demin S. D., Hu L., WO Patent WO/2016/091791, June 16, 2016.
45) Shi W., Jiang Z., He H., Xiao F., Lin F., Sun Y., Hou L., Shen L.,
Han L., Zeng M., Lai K., Gu Z., Chen X., Zhao T., Guo L., Yang C.,
Li J., Chen S., ACS Med. Chem. Lett., 9, 94–97 (2018).
46) Feng S., Hong D., Wang B., Zheng X., Miao K., Wang L., Yun H.,
Gao L., Zhao S., Shen H. C., ACS Med. Chem. Lett., 6, 359–362
(2015).
19) Weiss R. A., McMichael A. J., Nat. Med., 10 (Suppl.), S70–S76
(2004).
20) Smith D. K., Seales S., Budzik C., Am. Fam. Physician, 95, 94–99
(2017).
21) Kou M., Hwang V., Ramkellawan N., Emerg. Med. Clin. North Am.,
36, 275–286 (2018).
22) Offer I., Ashkenazi S., Livni G., Shalit I., Isr. Med. Assoc. J., 2,
108–110 (2000).
23) Livni G., Rachmel A., Marom D., Yaari A., Tirosh N., Ashkenazi
S., Pediatr. Infect. Dis. J., 29, 71–73 (2010).
24) Meissner H. C., N. Engl. J. Med., 374, 62–72 (2016).
25) Impact-RSV study group, Pediatrics, 102, 531–537 (1998).
26) Wu P., Escobar G. J., Gebretsadik T., Carroll K. N., Li S. X., Walsh
E. M., Mitchel E. F., Sloan C., Dupont W. D., Yu C., Horner J. R.,
Hartert T. V., Am. J. Epidemiol., 187, 1490–1500 (2018).
27) Obando-Pacheco P., Justicia-Grande A. J., Rivero-Calle I.,
Rodríguez-Tenreiro C., Sly P., Ramilo O., Mejías A., Baraldi E.,
Papadopoulos N. G., Nair H., Nunes M. C., Kragten-Tabatabaie
L., Heikkinen T., Greenough A., Stein R. T., Manzoni P., Bont L.,
Martinón-Torres F., J. Infect. Dis., 217, 1356–1364 (2018).
28) Revankar G. R., Robins R. K., Ann. N. Y. Acad. Sci., 255 (1 Chem-
istry, Bi), 166–176 (1975).
47) Feng S., Li C., Chen D., Zheng X., Yun H., Gao L., Shen H. C., Eur.
J. Med. Chem., 138, 1147–1157 (2017).
48) Zheng X., Wang L., Wang B., Miao K., Xiang K., Feng S., Gao L.,
Shen H. C., Yun H., ACS Med. Chem. Lett., 7, 558–562 (2016).
49) Sugita Y., Okamoto Y., Chem. Phys. Lett., 314, 141–151 (1999).
50) Abraham M. J., Murtola T., Schulz R., Páll S., Smith J. C., Hess B.,
Lindahl E., SoftwareX, 1–2, 19–25 (2015).
51) Wang J., Wolf R. M., Caldwell J. W., Kollman P. A., Case D. A., J.
Comput. Chem., 25, 1157–1174 (2004).
52) Onufriev A., Case D. A., Bashford D., J. Comput. Chem., 23, 1297–
1304 (2002).
29) Sidwell R. W., Huffman J. H., Khare G. P., Allen L. B., Witkowski
J. T., Robins R. K., Science, 177, 705–706 (1972).
30) Hall C. B., Walsh E. E., Hruska J. F., Betts R. F., Hall W. J., JAMA,