Optimization of 2,4-diamino-5-fluoropyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-drug interactions and P-gp liability

Article abstract of DOI:10.1016/j.bmc.2015.04.050

Abstract Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and has therapeutic potential for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. Here, a series of 2,4-diamino-5-fluoropyrimidine derivatives were prepared and evaluated for their inhibition of PKCθ. Of these compounds, 14f was found to exhibit potent PKCθ inhibitory activity and significantly weak CYP3A4 time-dependent inhibition (TDI) and P-glycoprotein (P-gp) liability.

Full text of DOI:10.1016/j.bmc.2015.04.050

Accepted Manuscript
Optimization of 2,4-diamino-5-fluoropyrimidine derivatives as protein kinase
C theta inhibitors with mitigated time-dependent drug-drug interactions and P-
gp liability
Shigeki Kunikawa, Akira Tanaka, Koichiro Mukoyoshi, Shinya Nagashima,
Hiroaki Tominaga, Noboru Chida, Mamoru Tasaki, Fumiyuki Shirai
PII:
S0968-0896(15)00358-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.050
BMC 12262
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 March 2015
17 April 2015
18 April 2015
Please cite this article as: Kunikawa, S., Tanaka, A., Mukoyoshi, K., Nagashima, S., Tominaga, H., Chida, N.,
Tasaki, M., Shirai, F., Optimization of 2,4-diamino-5-fluoropyrimidine derivatives as protein kinase C theta
inhibitors with mitigated time-dependent drug-drug interactions and P-gp liability, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.050
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Optimization of 2,4-diamino-5-fluoropyrimidine derivatives as protein kinase C theta
inhibitors with mitigated time-dependent drug-drug interactions and P-gp liability
*
Shigeki Kunikawa, Akira Tanaka, Koichiro Mukoyoshi, Shinya Nagashima, Hiroaki Tominaga,
Noboru Chida, Mamoru Tasaki and Fumiyuki Shirai
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585,
Japan
*
Address correspondence regarding this manuscript to:
Shigeki Kunikawa
Chemistry Research Labs
Drug Discovery Research
Astellas Pharma Inc.
2
1 Miyukigaoka, Tsukuba,
Ibaraki 305-8585, Japan
Tel: +81-(0)29-829-6197
Fax: +81-(0)29-854-1519
E-mail: shigeki.kunikawa@astellas.com
1

Key words: protein kinase C theta (PKCζ), CYP3A4 time-dependent inhibition (TDI),
P-glycoprotein (P-gp)
2

Abstract
Protein kinase C theta (PKCζ) plays a critical role in T cell signaling and has therapeutic potential
for T cell-mediated diseases such as transplant rejection and rheumatoid arthritis. Here, a series of
2
,4-diamino-5-fluoropyrimidine derivatives were prepared and evaluated for their inhibition of
PKCζ. Of these compounds, 14f was found to exhibit potent PKCζ inhibitory activity and
significantly weak CYP3A4 time-dependent inhibition (TDI) and P-glycoprotein (P-gp) liability.
3

1
. Introduction
Immunological diseases such as transplant rejection and rheumatoid arthritis are characterized by
1
inappropriate T cell activation that results in a pathogenic response. In the field of transplantation,
immunosuppressive agents are indispensable for the prevention of graft rejection. The calcineurin
inhibitors (CNIs) such as FK506 and cyclosporine A, which reduce disease severity by preventing
IL-2 production in T cells, are used as immunosuppressive agents. Treatment with CNIs after
transplantation remarkably improves graft and patient survival. However, the clinical use of CNIs is
reported to induce mechanism-based adverse effects such as nephrotoxicity and hypertension.
Currently, combination therapy using CNIs and other immunosuppressive agents with an
alternative mechanism is widely used to reduce adverse effects of CNIs by decreasing dose of CNIs.
Regarding agents used in combination with CNIs, no concern of reversible and time-dependent
inhibition (TDI) against CYP3A4 is necessary, as CNIs are mainly metabolized by CYP3A4 and
the increased plasma concentration of CNIs due to CYP3A4 inhibition by a concomitant agent
induces the occurrence of adverse effects. Mycophenolate mofetil (MMF), which exerts
noncompetitive inhibition on the enzyme inosine monophosphate dehydrogenase (IMPDH) but
does not inhibit CYP3A4, is often used in combination with CNIs to reduce the adverse effects.
2
However, MMF also has side effects such as gastrointestinal tract disturbance. Therefore, a new
immunosuppressive drug with a distinct mechanism of action is urgently required.
The protein kinase C (PKC) family is one of the serine/threonine kinase families and has been
classified into three subfamilies: classical (α, β, and γ), novel (δ, ε, ꢀ, and ζ), and atypical (ꢁ and
4

3
λ). Of these, PKCζ has recently been shown to play an important role in T cell signaling leading to
4
,5
the production of IL-2, which is of key importance in the immune response. For example, the
3
phenotype of PKCζ knockout mice is specific to T cell inactivation. These results suggest the
potential utility of a PKCζ inhibitor as a new immunosuppressive drug.
6
Although a large number of PKCζ inhibitors have been reported, none have been launched as
an immunosuppressive drug. Only Novartis Pharmaceuticals have reported development of
7
Sotrastaurin (1) (Phase II, transplantation, Figure 1). Clinical trials revealed that Sotrastaurin was
effective in transplantation, but that it also increased area under the blood concentration-time curve
8
(
AUC) of FK506 as a combination drug by 2.0-fold, which may induce adverse effects. This
9
unfavorable outcome might be due to Sotrastaurin being a CYP3A4 inhibitor. In this paper, to
identify a novel PKCζ inhibitor with weak CYP3A4 inhibition, we focused on compound 2
1
0
described in the patent application from Rigel as our starting compound (Figure 1), due to its
good PKCζ inhibitory activity and synthetic accessibility. However, our investigation also revealed
that compound 2 had CYP3A4 TDI properties that may cause adverse effects of combination drugs.
In addition, compound 2 had a liability of being a substrate of P-glycoprotein (P-gp), which is an
intestinal export pump and limits the oral bioavailability of substrate drugs. We therefore attempted
to identify novel PKCζ inhibitors with weak CYP3A4 TDI and P-gp liability via modification of
compound 2. Here, we report the synthesis and biological evaluation of
2
,4-diamino-5-fluoropyrimidine derivatives as PKCζ inhibitors.
5

2
. Chemistry
As shown in Scheme 1, target compounds 5a and 5b were prepared from
,4-dichloro-5-fluoropyrimidine (3). Treatment of compound 3 with various amines in methanol at
2
room temperature in the presence of N,N-diisopropylethylamine (DIPEA) selectively gave 4a and
b. Ipso substitution of these compounds with 3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)aniline
4
under microwave irradiation with a catalytic amount of HCl gave target products 5a and 5b.
Compounds 9c and its enantiomer 10c were prepared via the alternative synthetic route
depicted in Scheme 2. Treatment of compound 3 with sodium thiomethoxide selectively gave
2
3
-chloro-5-fluoro-4-(methylsulfanyl)pyrimidine
(6), which
was
by
then reacted with
oxidation with
-methoxy-5-(5-methyl-1H-tetrazol-1-yl)aniline,
followed
m-chloroperoxybenzoic acid (m-CPBA) to give a sulfoxide 8. Displacement of the sulfoxide moiety
by (3R) and (3S)-3-(aminomethyl)piperidine-1-carboxylic acid tert-butyl ester gave
tert-butoxycarbonyl (Boc)-protected compounds 9a and 10a. Deprotection of the Boc group using
trifluoroacetic acid (TFA) furnished compounds 9b and 10b, followed by reductive alkylation with
formalin and sodium triacetoxyborohydride to give target compounds 9c and 10c, respectively.
The syntheses of various N-alkyl piperidine derivatives 11a–11d are described in Scheme 3.
Alkylation of compound 10b with various alkyl bromides or triflates in the presence of DIPEA
gave 11a–11d.
Conversion of the tetrazole moiety on the phenyl ring to other heteroaryl rings is shown in
Scheme 4. Treatment of compound 3 with (3S)-3-(aminomethyl)piperidine-1-carboxylic acid
6

tert-butyl ester and deprotection of Boc group following reductive alkylation with formalin and
sodium triacetoxyborohydride gave the key intermediate 13 which was then reacted with various
heteroaryl-substituted anilines to give 14a–14f under acidic conditions.
The synthesis of aliphatic amine 17 and heteroaryl-substituted anilines 20 are described in
Scheme 5 and 6, respectively. Ketone 15 was reacted with p-toluenesulfonylmethyl isocyanide
(
TosMIC) to give cyano compound 16, which was then reduced with lithium alminium hydride
LAH) to give target product 17. 3-Fluoronitrobenzene (18) was reacted with 4-chloro-1H-pyrazole,
(
11
and nitro group was reduced with SnCl ∙2H O to give compound 20. Intermediates otherwise
2
2
noted were commercially available.
7

3
. Results and discussion
We evaluated the inhibitory activity of the compounds against human PKCζ in vitro by
measuring fluorescence intensity after incubation with full-length human PKCζ and ATP. We also
assessed the inhibitory activity of compounds against CYP3A4, the metabolic activity of human
liver microsomes (HLMs) for midazolam was measured at 0 and 30 min after preincubation with
test compounds. In the present study, the residual activity of CYP3A4 is shown as the percentage of
metabolic activity remaining after 30 min preincubation, based on that at 0 min (i.e. without
preincubation). In addition, we evaluated P-glycoprotein (P-gp) liability in LLC-PK1-MDR1 cells.
Efflux ratio (ER) is the ratio of basolateral-to-apical permeability over apical-to-basolateral
permeability of a compound. Net efflux ratio (NER) is the ratio of ER of MDR1 to wild type.
Compound 2 showed potent PKCζ inhibitory activity, but the residual activity of CYP3A4
was only 43% (Table 1). This result shows that compound 2 exhibits TDI for CYP3A4. Further, the
NER of 2 was 40 in an LLC-PK1-MDR1 permeability assay, implying that compound 2 might be a
1
2
P-gp substrate that induces low in vivo exposure.
To minimize TDI, we focused on the distance between the piperidine nitrogen atom and the
pyrimidine core, as CYP3A4 substrates are reported to have a hydrogen bond acceptor atom 5.5-7.8
13
Å from the site of metabolism. We therefore modified the distance between the piperidine
nitrogen atom and pyrimidine core (hydrogen bond acceptor). Although insertion of a methylene
linker between piperidine moiety and pyrimidine maintained PKCζ inhibitory activity, TDI of 5a
remained unchanged. We next investigated an alternative strategy for reducing lipophilicity, as
8

14
correlations between lipophilicity of compounds and CYP3A4 substrates have been reported.
Removal of four methyl groups of 5a subsequently proved effective. Compound 5b showed weak
TDI and potent PKCζ inhibitory activity. Changing the position of the piperidine nitrogen atom to
the 3-position also maintained good activity and weak TDI (9c and 10c). Isomer preference was
noted for these two compounds. The S-isomer 10c had 10-fold more active than that of the
R-isomer 9c. Our investigation demonstrated that structural modifications reducing the lipophilicity
of compounds successfully ameliorated TDI. In addition, removal of four methyl groups also
improved P-gp liability by reducing the pKa of compound (P-gp NER of 10c was 9.3). This
improvement was consistent with previous reports that indicated good correlation between basicity
1
5
of compounds and P-gp NER. We therefore reduced the pKa of the nitrogen in the piperidine to
further improve P-gp NER (Table 2). Although introduction of a methoxyethyl group into the
piperidine moiety (11b) reduced the pKa, improvement of P-gp NER was not observed. In contrast,
2
,2-difluoromethyl piperidine (11c) and trifluoromethyl peperidine (11d) showed reduced pKa
1
6
values 6.29 and 4.81 respectively, and both improved the P-gp NER. While this investigation
demonstrated the importance of lowering pKa to decrease P-gp NER value, basicity was also
deemed important for PKCζ inhibitory activity.
We next focused on the high topological polar surface area (TPSA) of compound 10c with an
2
16,17
associated cost to P-gp NER (TPSA of 10c: 105.9 Å ).
As an opening strategy to reduce the
TPSA, we removed the methoxy substituent on the phenyl ring, but P-gp NER of desmethoxy
derivative 14a was not improved. We then replaced the N atom of tetrazole with a C or O atom to
9

further reduce TPSA, resulting in triazole 14b, oxazole 14c and pyrazole 14d. Triazole 14b did not
substantially change the P-gp NER. In contrast, while oxazole 14c and pyrazole 14d decreased
PKCζ inhibitory activity, they provided a sought after improvement in P-gp NER to 3.5 and 4.5.
Our investigation demonstrated that structural modifications reducing the TPSA of compounds
successfully improved the P-gp NER of compounds.
Of these compounds, we selected 14d with good P-gp NER for further optimization to improve
PKCζ inhibitory activity. Introduction of substituents to the pyrazole ring and their effect on PKCζ
inhibitory activity are shown in Table 4. Introduction of a methyl group to 14d slightly decreased
PKCζ inhibitory activity (14e), while the chlorine derivative 14f exhibited 9-fold more potent
activity than 14d, maintaining good P-gp NER and CYP3A4 TDI. Docking studies of other
diaminopyrimidine compounds with PKCζ have suggested that the diaminopyrimidine binds to the
hinge region at the ATP binding site via 2-point binding of the 2-amino substituent and the nitrogen
of the pyrimidine at the 1 position with Leu461, i.e. first between the NH of the 2-amino substituent
and carbonyl „O‟ of Leu461 (NH∙∙∙CO), second between the N of pyrimidine and NH of Leu461
1
8,19
(N∙∙∙NH).
Electron withdrawal by the chlorine atom of 14f is considered to decrease the
electron density of the NH of 2-amino substituent, which might strengthen the hydrogen bond with
2
0
Leu461 and improve in vitro activity.
As a result of the above optimization, we identified novel PKC inhibitors such as compound 14f
with mitigated CYP3A4 TDI and P-gp liability. These results indicated that 14f will be a promising
immunosuppressive combination agent with CNIs such as FK506, because 14f has little concern
1
0

about TDI compared to compound 2. Further optimization and biological evaluation of this
compound is being conducted at present and studies of diaminopyrimidine analogues will be
disclosed in due course.
4
. Conclusions
We investigated the SAR of PKCζ inhibitory activity, CYP3A4 TDI and P-gp liability to obtain
a promising PKCζ inhibitor. We first reduced the lipophilicity of 2 to obtain 10c with mitigated
CYP3A4 TDI. We then investigated the optimization of P-gp liability by lowering pKa or reducing
TPSA. Although we demonstrated the importance of lowering pKa to improve P-gp liability,
basicity was also required for PKCζ inhibition (11c and 11d). In contrast, the alternative strategy of
reducing TPSA gave compound 14f with improved P-gp liability and maintained PKCζ inhibitory
activity. As a result of our optimization, we discovered the novel PKCζ inhibitor 14f, which
presented little concern of CYP3A4 TDI or P-gp liability.
5
5
. Experimental section
.1. Chemistry
1
H NMR spectra were recorded on a Varian VNS-400, JEOL JNM-LA400, or JEOL
JNM-AL400 and chemical shifts were expressed in δ (ppm) values with tetramethylsilane as an
internal reference (s=singlet, d=doublet, t=triplet, m=multiplet, dd=double doublet, tt=triple triplet,
q=quartet, ddd=double double doublet, and br=broad peak). Mass spectra (MS) were recorded on a
11

Waters UPLC/SQD[413W]-LC/MS system. Elemental analyses were performed using a Yanaco
MT-6 (C, H, N), Elementar Vario EL III (C, H, X), and Dionex ICS-3000 (S, halogene) and were
within ± 0.4% of theoretical values. Electrospray ionization positive high-resolution mass spectrum
(
HRMS) was obtained using a Waters LCT Premier. Unless otherwise noted, all reagents and
solvents obtained from commercial suppliers were used without further purification. The following
abbreviations are used: m-CPBA, m-chloroperoxybenzoic acid; DIPEA,
N,N-diisopropylethylamine; DME, 1,2-dimethoxy ethane; DMF, N,N-dimethylformamide; DMI,
,3-dimethyl-2-imidazolidinone; IPA, isopropyl alcohol (2-propanol); LAH, lithium alminium
1
hydride; NMP, N-methylpyrrolidone; TFA, trifluoroacetic acid; and THF, tetrahydrofuran. TosMIC,
p-tolylsulfonylmethyl isocyanide.
5
.1.1. 2-Chloro-5-fluoro-N-[(1,2,2,6,6-pentamethylpiperidin-4-yl)methyl]pyrimidin-4-amine
4a)
To a solution of 2,4-dichloro-5-fluoropyrimidine 3 (400 mg, 2.4 mmol) in MeOH (6 mL),
-(1,2,2,6,6-pentamethylpiperidin-4-yl)methanamine 16 (480 mg, 2.6 mmol) and DIPEA (820 μL,
.8 mmol) were added. The reaction mixture was stirred at room temperature for 16 h, concentrated
(
1
4
in vacuo and triturated with (n-hexane-EtOAc) (50:50) to give 4a (500 mg, 66%) as a colorless
1
solid. H NMR (DMSO-d ) δ 1.28 (6H, s), 1.47 (6H, s), 1.58–1.92 (5H, m), 2.64 (3H, s), 3.22–3.29
6
+
(
2H, m), 8.09 (1H, d, J = 3.4 Hz), 8.13–8.36 (1H, m); MS (ESI) m/z 315, 317 [M+H] .
1
2

5
.1.2. 2-Chloro-5-fluoro-N-[(1-methylpiperidin-4-yl)methyl]pyrimidin-4-amine (4b)
To a solution of 3 (500 mg, 3.0 mmol) in MeOH (5 mL), (1-methyl-4-piperidinyl)methanamine
(380 mg, 3.0 mmol) was added. The reaction mixture was stirred at room temperature for 16 h and
then diluted with CHCl and basified with saturated aqueous NaHCO . The organic layer was
3
3
washed with brine, dried over Na SO4 and concentrated in vacuo. The residue was
2
chromatographed on silica gel with elution using (CHCl -MeOH) (97:3) to give 4b (295 mg, 38%)
3
1
as a colorless solid. H NMR (DMSO-d ) δ 1.09–1.23 (2H, m), 1.48–1.66 (3H, m), 1.72–1.83 (2H,
6
m), 2.12 (3H, s), 2.67–2.77 (2H, m), 3.17–3.25 (2H, m), 8.04 (1H, d, J = 3.6 Hz), 8.18 (1H, br s);
+
MS (ESI) m/z 259, 261 [M+H] .
5
5
.1.3.
-Fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-N -[(1,2,2,6,6-pentamethylpipe
2
4
ridin-4-yl)methyl]pyrimidine-2,4-diamine (5a)
To solution of 4a (200 mg,
-methoxy-5-(5-methyl-1H-tetrazol-1-yl)aniline (110 mg, 0.54 mmol) and 4 M HCl-EtOAc (310
a
0.64
mmol)
in
IPA
(2
mL),
3
μL, 1.3 mmol) were added in a 10 mL microwave vial. The reaction mixture was heated with
stirring at 140 °C in a Biotage auto-sampling microwave reactor for 1 h. The reaction mixture was
diluted with CHCl and washed with H O. The organic layer was dried over Na SO and
3
2
2
4
concentrated in vacuo. The residue was chromatographed on amino silica gel with elution using
1
(
CHCl -MeOH) (90:10) to give 5a (100 mg, 33%) as a colorless solid. H NMR (DMSO-d ) δ
3
6
1
3

0
2
.81–1.12 (2H, m), 0.86 (6H, s), 1.03 (6H, s), 1.40–1.52 (2H, m), 1.92–2.08 (1H, m), 2.14 (3H, s),
.58 (3H, s), 3.14–3.23 (2H, m), 3.80 (3H, s), 6.77 (1H, t, J = 2.0 Hz), 7.56–7.74 (3H, m), 7.90 (1H,
+
d, J = 3.8 Hz), 9.43 (1H, s); MS (ESI) m/z 484 [M+H] ; HRMS (ESI) calcd for C H FN O
2
4
34
9
+
[
M+H] : 484.2949, Found; 484.2946.
5
5
.1.4.
-Fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-N -[(1-methylpiperidin-4-yl)me
2
4
thyl]pyrimidine-2,4-diamine dihydrochloride (5b)
To solution of 4b (120 mg,
a
0.48
mmol)
in
IPA
(3
mL),
3
0
-methoxy-5-(5-methyl-1H-tetrazol-1-yl)aniline (98 mg, 0.48 mmol) and 4 M HCl-EtOAc (240 μL,
.96 mmol) were added in a 10 mL microwave vial. The reaction mixture was heated with stirring
at 140 °C in a Biotage auto-sampling microwave reactor for 1 h. The reaction mixture was washed
with IPA to give an ivory solid. The solid was washed with EtOAc and MeOH to give 5b (86 mg,
1
3
1
3
6%) as a colorless solid. H NMR (DMSO-d ) δ 1.41–1.93 (2H, m), 1.74–1.77 (2H, m),
6
.84–1.94 (1H, m), 2.60 (3H, s), 2.67–2.71 (3H, m), 2.78–2.87 (2H, m), 3.27–3.30 (2H, m),
.34–3.37 (2H, m), 3.38 (3H, s), 6.96 (1H, br), 7.50–7.51 (1H, m), 7.58–7.59 (1H, m), 8.12–8.13
+
(
1H, m), 8.66 (1H, br), 10.31 (1H, br); MS (ESI) m/z 428 [M+H] ; HRMS (ESI) calcd for
+
C H FN O [M+H] :428.2322, Found: 428.2326.
20
26
9
5
.1.5. 2-Chloro-5-fluoro-4-(methylsulfanyl)pyrimidine (6)
1
4

To a solution of 3 (3.0 g, 18.0 mmol) in THF (30 mL), sodium thiomethoxide (1.35 g, 19.3
mmol) was added under -30 °C in dry ice-acetone bath and stirred at -30 °C for 2 h. The reaction
mixture was warmed to room temperature and poured into H O and extracted with EtOAc. The
2
organic layer was then washed with H O, brine, dried over Na SO and concentrated in vacuo to
2
2
4
1
give 6 (24 g, 96%) as a colorless solid. H NMR (CDCl ) δ 2.62 (3H, s), 8.09 (1H, d, J = 1.4 Hz);
3
+
MS (ESI) m/z 179, 181 [M+H] .
5
5
.1.6.
-Fluoro-N-[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-4-(methylsulfanyl)pyrimidin-2-
amine (7)
To
a
solution
of
6
(550
mg,
3.1
mmol)
in
IPA
(5
mL),
3
3
1
-methoxy-5-(5-methyl-1H-tetrazol-1-yl)aniline (710 mg, 3.5 mmol) and 4 M HCl-EtOAc (850 μL,
.4 mmol) were added in a 25 mL microwave vial. The reaction mixture was heated with stirring at
30 °C in a Biotage auto-sampling microwave reactor for 1 h. The reaction mixture was poured
1
into H O and the precipitate was washed with H O to give 7 (890 mg, 83%) as a colorless solid. H
2
2
NMR (DMSO-d ) δ 2.58 (3H, s), 2.59 (3H, s), 3.82 (3H, s), 6.88 (1H, t, J = 2.0 Hz), 7.56 (1H, t, J =
6
2
.0 Hz), 7.70 (1H, t, J = 1.8 Hz), 8.31 (1H, d, J = 2.0 Hz), 10.02 (1H, s); MS (ESI) m/z 348
+
[
M+H] .
1
5

5
5
.1.7.
-Fluoro-N-[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-4-(methylsulfinyl)pyrimidin-2-a
mine (8)
To a solution of 7 (800 mg, 2.3 mmol) in CH Cl (8 mL), m-CPBA (650 mg, 2.9 mmol) was
2
2
added at 0 °C and stirred for 3h at 0 °C. The reaction mixture was poured into saturated aqueous
NaHCO and extracted with CHCl . The organic layer was washed with brine, dried over Na SO
3
3
2
4
and concentrated in vacuo The residue was chromatographed on silica gel with elution using
1
EtOAc to give 8 (450 mg, 54%) as a colorless solid. H NMR (DMSO-d ) δ 2.62 (3H, s), 2.94 (3H,
6
s), 3.84 (3H, s), 6.91 (1H, t, J = 1.9 Hz), 7.71–7.75 (2H, m), 8.77 (1H, d, J = 1.5 Hz), 10.52 (1H, s);
+
MS (ESI) m/z 364 [M+H] .
5
.1.8.
3R)-3-{[(5-fluoro-2-{[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]amino}pyrimidin-4-yl)
amino]methyl}piperidine-1-carboxylate (9a)
To solution of (300
3R)-3-(aminomethyl)piperidine-1-carboxylic acid tert-butyl ester (210 mg, 0.99 mmol) and
tert-Butyl
(
a
8
mg,
0.83
mmol)
in
NMP
(3
mL),
(
DIPEA (180 μL, 1.1 mmol) were added in a 10 mL microwave vial. The reaction mixture was
heated with stirring at 110 °C in a Biotage auto-sampling microwave reactor for 0.5 h. The reaction
mixture was poured into saturated aqueous NH Cl and collected by vacuum filtration to give 9a
4
1
(
420 mg, 99%) as a colorless solid. H NMR (DMSO-d ) δ 1.18–1.83 (5H, m), 1.32 (9H, s), 2.58
6
1
6

(3H, s), 2.63–2.84 (2H, m), 3.17–3.29 (2H, m), 3.62–3.93 (2H, m), 3.80 (3H, s), 6.78 (1H, t, J = 2.1
Hz), 7.54 (1H, t, J = 1.9 Hz), 7.59–7.64 (1H, m), 7.71–7.74 (1H, m), 7.92 (1H, d, J = 3.8 Hz), 9.44
+
(
1H, s); MS (ESI) m/z 514 [M+H] .
5
5
.1.9.
-Fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-N -[(3S)-piperidin-3-ylmethyl]
2
4
pyrimidine-2,4-diamine (9b)
To a solution of 9a (400 mg, 0.78 mmol) in CH Cl (4 mL), TFA (2.0 mL, 26 mmol) was added
2
2
and stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo. The
residue was added to saturated aqueous K CO and extracted with CHCl . The organic layer was
2
3
3
dried over Na SO and concentrated in vacuo The residue was chromatographed on amino silica gel
2
4
1
with elution using (CHCl -MeOH) (96:4) to give 9b (260 mg, 81%) as a colorless solid. H NMR
3
(
(
(
DMSO-d ) δ 0.80–1.71 (6H, m), 2.05–2.40 (2H, m), 2.57 (3H, s), 2.71–2.87 (2H, m), 3.14–3.24
6
2H, m), 3.80 (3H, s), 6.77 (1H, t, J = 2.0 Hz), 7.52–7.75 (3H, m), 7.89 (1H, d, J = 3.8 Hz), 9.41
+
1H, s); MS (ESI) m/z 414 [M+H] .
5
5
.1.10.
-Fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-N -{[(3R)-1-methylpiperidin-3-
2
4
yl]methyl}pyrimidine-2,4-diamine (9c)
To a solution of 9b (50 mg, 0.12 mmol) in CH Cl (1 mL), 36% aqueous formaldehyde solution
2
2
1
7

(11 μL, 0.15 mmol) and sodium triacetoxyborohydride (33 mg, 0.16 mmol) were added and the
mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with CHCl and
3
basified with saturated aqueous NaHCO . The organic layer was washed with brine, dried over
3
Na SO and concentrated in vacuo. The residue was chromatographed on amino silica gel with
2
4
1
elution using EtOAc to give 9c (32 mg, 62%) as a colorless solid. H NMR (DMSO-d ) δ 0.74–1.93
6
(7H, m), 2.09 (3H, s), 2.48–2.64 (2H, m), 2.57 (3H, s), 3.18–3.33 (2H, m), 3.79 (3H, s), 6.77 (1H, t,
+
J = 2.0 Hz), 7.52–7.75 (3H, m), 7.89 (1H, d, J = 3.8 Hz), 9.42 (1H, s); MS (ESI) m/z 428 [M+H] ;
2
1
+
HRMS (ESI) calcd for C H FN O [M+H] : 428.2322, Found: 428.2331. [α] (c = 0.1, MeOH)
20
26
9
D
-5.5 °.
5
.1.11.
tert-Butyl
(3S)-3-{[(5-fluoro-2-{[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]amino}pyrimidin-4-yl)a
mino]methyl}piperidine-1-carboxylate (10a)
Compound 10a was prepared from compound 8 in 85% yield as a colorless solid, using a
1
similar approach to that described for 9a. H NMR (DMSO-d ) δ 1.16–1.85 (5H, m), 1.32 (9H, s),
6
2
.31–2.78 (2H, m), 2.58 (3H, s), 3.15–3.29 (2H, m), 3.60–3.94 (2H, m), 3.80 (3H, s), 6.78 (1H, t, J
=
2.1 Hz), 7.54 (1H, t, J = 2.1 Hz), 7.59–7.67 (1H, m), 7.71–7.76 (1H, m), 7.92 (1H, d, J = 3.7 Hz),
+
9
.44 (1H, s); MS (ESI) m/z 514 [M+H] .
5
.1.12.
1
8

2
4
5
-Fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-N -[(3R)-piperidin-3-ylmethyl]
pyrimidine-2,4-diamine (10b)
Compound 10b was prepared from compound 10a in 99% yield as a colorless solid, using a
1
similar approach to that described for 9b. H NMR (DMSO-d ) δ 0.80–1.77 (5H, m), 2.05–2.53
6
(3H, m), 2.58 (3H, s), 2.72–2.88 (2H, m), 3.14–3.24 (2H, m), 3.80 (3H, s), 6.78 (1H, t, J = 2.0 Hz),
+
7
.52–7.76 (3H, m), 7.89 (1H, d, J = 3.8 Hz), 9.42 (1H, s); MS (ESI) m/z 414 [M+H] .
5
5
.1.13.
2
4
-Fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-N -{[(3S)-1-methylpiperidin-3-
yl]methyl}pyrimidine-2,4-diamine (10c)
Compound 10c was prepared from compound 10b in 61% yield as a colorless solid, using a
1
similar approach to that described for 9c. H NMR (DMSO-d ) δ 0.73–1.93 (7H, m), 2.09 (3H, s),
6
2
7
.44–2.70 (2H, m), 2.57 (3H, s), 3.12–3.36 (2H, m), 3.80 (3H, s), 6.78 (1H, t, J = 2.0 Hz),
+
.52–7.76 (3H, m), 7.89 (1H, d, J = 3.8 Hz), 9.42 (1H, s); MS (ESI) m/z 428 [M+H] ; HRMS (ESI)
+
calcd for C H FN O [M+H] : 428.2322, Found: 428.2332; Anal. Calcd for C H FN O: C,
2
0
26
9
20 26
9
21
5
6.19; H, 6.13; N, 29.49; F, 4.44. Found: C, 56.28; H, 6.15; N, 29.45; F, 4.47. [α] D (c = 0.1,
MeOH) 6.0 °.
5
.1.14.
4
2
N -{[(3S)-1-Ethylpiperidin-3-yl]methyl}-5-fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)
1
9

phenyl]pyrimidine-2,4-diamine (11a)
To a solution of 10b (52 mg, 0.13 mmol) in NMP (500 μL), ethylbromide (12 μL, 0.16 mmol)
and DIPEA (28 μL, 0.16 mmol) were added and the reaction mixture was stirred at 80 °C for 1h.
To the reaction mixture CHCl was added and chromatographed on amino silica gel with elution
3
1
using (n-hexane-EtOAc) (1:6) to give 11a (38 mg, 68%) as a colorless solid. H NMR (DMSO-d )
6
δ 0.77–2.36 (9H, m), 0.94 (3H, t, J = 8.0 Hz), 2.40–2.78 (2H, m), 2.57 (3H, s), 3.17–3.37 (2H, m),
3
.80 (3H, s), 6.78 (1H, t, J = 2.0 Hz), 7.52–7.76 (3H, m), 7.89 (1H, d, J = 3.8 Hz), 9.42 (1H, s); MS
+
+
(
ESI) m/z 442 [M+H] ; HRMS (ESI) calcd for C H FN O [M+H] : 442.2479, Found: 442.2479.
21 28 9
5
5
.1.15.
-Fluoro-N -{[(3S)-1-(2-methoxyethyl)piperidin-3-yl]methyl}-N -[3-methoxy-5-(5-methyl-1H-t
4
2
etrazol-1-yl)phenyl]pyrimidine-2,4-diamine (11b)
Compound 11b was prepared from compound 10b in 64% yield as a colorless solid, using a
1
similar approach to that described for 11a. H NMR (DMSO-d ) δ 0.79–1.93 (7H, m), 2.36–2.42
6
(2H, m), 2.57 (3H, s), 2.65–2.76 (2H, m), 3.18 (3H, s), 3.19–3.39 (4H, m), 3.80 (3H, s), 6.78 (1H, t,
+
J = 2.0 Hz), 7.52–7.76 (3H, m), 7.89 (1H, d, J = 3.8 Hz), 9.42 (1H, s); MS (ESI) m/z 472 [M+H] ;
+
HRMS (ESI) calcd for C H FN O [M+H] : 472.2585, Found: 472.2575.
2
2
30
9
2
5
2
.1.16.
-[(3S)-3-{[(5-Fluoro-2-{[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]amino}pyrimidin-4-
2
0

yl)amino]methyl}piperidin-1-yl]ethanol (11c)
To a solution of 10b (59 mg, 0.14 mmol) in THF (1.2 ml), 2,2-difluoroethyl
trifluoromethanesulfonate (37 mg, 0.17 mmol) and DIPEA (49 μL, 0.29 mmol) were added. The
reaction mixture was refluxed for 14 h. After cooling to room temperature, the reaction mixture
was diluted with CHCl and basified with saturated aqueous NaHCO . The organic layer was
3
3
washed with brine, dried over Na SO4 and concentrated in vacuo. The residue was
2
chromatographed on silica gel with elution using (CHCl -MeOH) (97:3) to give 11c (35mg, 51%)
3
1
as a colorless solid. H NMR (DMSO-d ) δ 0.80–0.88 (1H, m), 1.31–1.42 (1H, m), 1.53–1.59 (2H,
6
m), 1.82–1.86 (2H, m), 2.05–2.11 (1H, m), 2.57 (3H, s), 2.61–2.70 (2H, m), 2.73–2.80 (2H, m),
3
.16–3.31 (2H, m), 3.80 (3H, s), 6.07 (1H, tt, J = 55.9, 4.3 Hz), 6.78 (1H, t, J = 2.1 Hz), 7.54–7.59
+
(
2H, m), 7.73 (1H, t, J = 1.8 Hz), 7.90 (1H, d, J = 3.7 Hz), 9.42 (1H, s). MS (ESI) m/z 478 [M+H] ;
+
HRMS (ESI) calcd for C H F N O [M+H] : 478,2291, Found: 478.2288.
2
1
26
3
9
5
5
.1.17.
-Fluoro-N -[3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl]-N -{[(3S)-1-(2,2,2-trifluoroethy
2
4
l)piperidin-3-yl]methyl}pyrimidine-2,4-diamine (11d)
Compound 11d was prepared from compound 10b in 64% yield as a colorless solid, using a
1
similar approach to that described for 11c. H NMR (DMSO-d ) δ 0.81–0.91 (1H, m), 1.33–1.43
6
(1H, m), 1.53–1.59 (2H, m), 1.84–1.91 (1H, m), 1.98–2.03 (1H, m), 2.21–2.27 (1H, m), 2.57 (3H,
s), 2.77–2.80 (1H, m), 2.83–2.86 (1H, m), 3.10 (2H, q, J = 10.3 Hz), 3.21–3.25 (2H, m), 3.79 (3H,
2
1

s), 6.78 (1H, t, J = 2.1 Hz), 7.55–7.60 (2H, m), 7.72 (1H, t, J = 1.8 Hz), 7.90 (1H, d, J = 3.8 Hz),
+
+
9
.43 (1H, s). MS (ESI) m/z 496 [M+H] ; HRMS (ESI) calcd for C H F N O [M+H] : 496.2196,
21 25 4 9 2
Found: 496.2206.
5
.1.18. 2-Chloro-5-fluoro-N-[(3R)-piperidin-3-ylmethyl]pyrimidin-4-amine (12)
To solution of (3.4 g, 20 mmol) in DMF
a
3
(34
mL),
(3S)-3-(aminomethyl)piperidine-1-carboxylic acid tert-butyl ester (5.2 g, 24 mmol) and DIPEA
(4.5 mL, 26 mmol) were added. The reaction mixture was stirred at room temperature for 14 h. The
reaction mixture was poured into saturated aqueous NH Cl and extracted with EtOAc. The organic
4
layer was washed with H O and brine and then dried over Na SO and concentrated in vacuo to
2
2
4
give a brown solid. To a solution of the residue in CH Cl (60 mL), TFA (13 mL, 170 mmol) was
2
2
added and stirred at room temperature for 4h. The reaction mixture was concentrated in vacuo. The
residue was added to saturated aqueous K CO and extracted with (CHCl -MeOH) (80/20). The
2
3
3
organic layer was dried over Na SO and concentrated in vacuo to give 12 (4.2 g, 87%) as a pale
2
4
1
solid. H NMR (DMSO-d ): 0.99–1.12 (1H, m), 1.24–1.39 (1H, m), 1.52–1.61 (1H, m), 1.66–1.79
6
(2H, m), 2.21 (1H, dd, J = 11.8, 9.4 Hz), 2.38–2.48 (1H, m), 2.77–2.85 (1H, m), 2.85–2.93 (1H, m),
+
3
.15–3.24 (3H, m), 8.04 (1H, d, J = 3.2 Hz), 8.15-8.24 (1H, m); MS (ESI) m/z 245, 247 [M+H] .
5
.1.19. 2-Chloro-5-fluoro-N-{[(3S)-1-methylpiperidin-3-yl]methyl}pyrimidin-4-amine (13)
To a solution of 12 (2.1 g, 8.6 mmol) in CH Cl (21 mL) , 36% aqueous formaldehyde solution
2
2
2
2

(1.0 g, 13 mmol) and sodium triacetoxyborohydride (2.7 g, 13 mmol) were added and then stirred
at room temperature for 1 h. The reaction mixture was diluted with CHCl and basified with
3
saturated aqueous NaHCO . The organic layer was washed with brine, dried over Na SO and
3
2
4
concentrated in vacuo. The residue was chromatographed on amino silica gel with elution using
1
CHCl to give 13 (1.3 g, 59%) as a colorless solid. H NMR (DMSO-d ) δ 0.82–0.97 (1H, m),
3
6
1
3
.34–1.49 (1H, m), 1.54–1.70 (3H, m), 1.78–1.94 (2H, m), 2.12 (3H, s), 2.53–2.69 (2H, m),
+
.12–3.29 (2H, m), 8.04 (1H, d, J = 3.5 Hz), 8.14–8.24 (1H, m); MS (ESI) m/z 259, 261 [M+H] .
5
5
.1.20.
4
2
-Fluoro-N -{[(3S)-1-methylpiperidin-3-yl]methyl}-N -[3-(5-methyl-1H-tetrazol-1-yl)phenyl]p
yrimidine-2,4-diamine (14a)
To a solution of 13 (355 mg, 1.4 mmol) in IPA (14 mL), 3-(5-methyl-1H-tetrazol-1-yl)aniline
(313 mg, 1.3 mmol) and 4 M HCl-EtOAc (1.0 mL, 4.1 mmol) were added and stirred at 120 °C for
9
h. The reaction mixture was diluted with CHCl and washed with saturated aqueous NaHCO .
3
3
The organic layer was washed with brine, dried over Na SO and concentrated in vacuo. The
2
4
residue was chromatographed on amino silica gel with elution using (CHCl -MeOH) (90:10) to
3
1
give 14a (320 mg, 59%) as a colorless solid. H NMR (DMSO-d ) δ 0.74–0.87 (1H, m), 1.28–1.41
6
(1H, m), 1.54–1.64 (3H, m), 1.74–1.92 (2H, m), 2.10 (3H, s), 2.52–2.70 (2H, m), 2.57 (3H, s),
3
.15–3.28 (2H, m), 7.10–7.18 (1H, m), 7.47 (1H, t, J = 8.1 Hz), 7.55–7.63 (1H, m), 7.77–7.83 (1H,
+
m), 7.90 (1H, d, J = 3.8 Hz), 8.24 (1H, t, J = 2.1 Hz), 9.49 (1H, s); MS (ESI) m/z 398 [M+H] ;
2
3

+
HRMS (ESI) calcd for C H FN [M+H] : 398.2217, Found: 398.2208.
1
9
24
9
5
5
.1.21.
-Fluoro-N -{[(3S)-1-methylpiperidin-3-yl]methyl}-N -[3-(1H-1,2,3-triazol-1-yl)phenyl]pyrimi
4
2
dine-2,4-diamine (14b)
Compound 14b was prepared from compound 13 in 74% yield as a colorless solid, using a
1
similar approach to that described for 14a. H NMR (DMSO-d ) δ 0.81–0.95 (1H, m), 1.27–1.42
6
(1H, m), 1.50–1.64 (3H, m), 1.71–1.82 (1H, m), 1.83–1.94 (1H, m), 2.07 (3H, s), 2.47–2.66 (2H,
m), 3.23–3.37 (2H, m), 7.27–7.33 (1H, m), 7.41 (1H, t, J = 8.1 Hz), 7.52–7.61 (1H, m), 7.65–7.72
(1H, m), 7.90 (1H, d, J = 3.8 Hz), 7.95 (1H, d, J = 3.8 Hz), 8.57 (1H, d, J = 2.1 Hz), 8.71 (1H, d, J
+
+
=
1.2 Hz), 9.4 (1H, s); MS (ESI) m/z 383 [M+H] ; HRMS (ESI) calcd for C H FN [M+H] :
19 23 8
3
83.2108, Found: 383.2106.
5
5
2
.1.22.
4
2
-Fluoro-N -{[(3S)-1-methylpiperidin-3-yl]methyl}-N -[3-(1,3-oxazol-5-yl)phenyl]pyrimidine-
,4-diamine (14c)
Compound 14c was prepared from compound 13 in 72% yield as a colorless solid, using a
1
similar approach to that described for 14a. H-NMR (DMSO-d ) δ 0.80–0.95 (1H, m), 1.30–1.46
6
(1H, m), 1.52–1.71 (3H, m), 1.74–1.84 (1H, m), 1.84–1.98 (1H, m), 2.08 (3H, s), 2.42–2.70 (2H,
m), 3.23–3.39 (2H, m), 7.21–7.27 (1H, m), 7.31 (1H, t, J = 7.9 Hz), 7.50–7.56 (1H, m), 7.57 (1H, s),
2
4

7
.63–7.69 (1H, m), 7.88 (1H, d, J = 3.8 Hz), 8.20–8.24 (1H, m), 8.42 (1H, s), 9.19 (1H, s); MS
+
+
(
ESI) m/z 383 [M+H] ; HRMS (ESI) calcd for C H FN O [M+H] : 383.1996, Found: 383.1994.
20 23 6
5
5
.1.23.
-Fluoro-N -{[(3S)-1-methylpiperidin-3-yl]methyl}-N -[3-(1H-pyrazol-1-yl)phenyl]pyrimidine
4
2
-2,4-diamine (14d)
Compound 14d was prepared from compound 13 in 86% yield as a colorless solid, using a
1
similar approach to that described for 14a. H NMR (DMSO-d ) δ 0.82–0.97 (1H, m), 1.31–1.45
6
(
1H, m), 1.53–1.68 (3H, m), 1.74–1.97 (2H, m), 2.08 (3H, s), 2.50–2.71 (2H, m), 3.21–3.41 (2H,
m), 6.52 (1H, dd, J = 2.4, 1.6 Hz), 7.24–7.34 (2H, m), 7.48–7.59 (2H, m), 7.71 (1H, d, J = 1.6 Hz),
.88 (1H, d, J = 4.0 Hz), 8.34 (1H, d, J = 2.4 Hz), 8.44 (1H, dd, J = 1.6, 1.6 Hz), 9.24 (1H, s); MS
7
+
+
(
ESI) m/z 382 [M+H] ; HRMS (ESI) calcd for C H FN [M+H] : 382.2155, Found: 382.2148.
20 24 7
5
5
.1.24.
-Fluoro-N -{[(3S)-1-methylpiperidin-3-yl]methyl}-N -[3-(4-methyl-1H-pyrazol-1-yl)phenyl]p
4
2
yrimidine-2,4-diamine (14e)
Compound 14e was prepared from compound 13 in 93% yield as a white solid, using a similar
1
approach to that described for 14a. H NMR (DMSO-d ) δ 0.83–0.97 (1H, m), 1.32–1.45 (1H, m),
6
1
.53–1.69 (3H, m), 1.74–1.84 (1H, m), 1.84–1.96 (1H, m), 2.08 (3H, s), 2.10 (3H, s), 2.52–2.62
(1H, m), 2.62–2.70 (1H, m), 3.26–3.38 (2H, m), 7.17–7.23 (1H, m), 7.27 (1H, dd, J = 8.0, 8.0 Hz),
2
5

7
.46–7.56 (3H, m), 7.88 (1H, d, J = 3.6 Hz), 8.10 (1H, s), 8.38 (1H, dd, J = 2.0, 2.0 Hz), 9.21 (1H,
+
+
s); MS (ESI) m/z 396 [M+H] ; HRMS (ESI) calcd for C H FN [M+H] : 396.2312, Found:
2
1
26
7
3
96.2308.
5
.1.25.
2
4
N -[3-(4-Chloro-1H-pyrazol-1-yl)phenyl]-5-fluoro-N -{[(3S)-1-methylpiperidin-3-yl]methyl}p
yrimidine-2,4-diamine (14f)
Compound 14j was prepared from compound 13 in 92% yield as a white solid, using a similar
1
approach to that described for 14a. H NMR (DMSO-d ) δ 0.80–0.95 (1H, m), 1.30–1.44 (1H, m),
6
1
7
.52–1.68 (3H, m), 1.73–1.96 (2H, m), 2.08 (3H, s), 2.49–2.70 (2H, m), 3.24–3.39 (2H, m),
.21–7.27 (1H, m), 7.32 (1H, dd, J = 8.0, 8.0 Hz), 7.49–7.58 (2H, m), 7.83 (1H, d, J = 0.4 Hz), 7.89
(1H, d, J = 3.6 Hz), 8.45 (1H, dd, J = 2.2, 2.2 Hz), 8.65 (1H, d, J = 0.4 Hz), 9.28 (1H, s); MS (ESI)
+
+
m/z 416, 418 [M+H] ; HRMS (ESI) calcd for C H ClFN [M+H] : 416.1766, Found: 416.1770;
2
0
23
7
Anal. Calcd for C H ClFN : C, 57.76; H, 5.57; N, 23.57; Cl, 8.52; F, 4.57. Found: C, 57.79; H,
2
0
23
7
2
1
5
.46; N, 23.64; Cl, 8.49; F, 4.59. [α] D (c = 0.1, MeOH) -4.5 °.
5
.1.26. 1,2,2,6,6-Pentamethylpiperidine-4-carbonitrile (16)
Under argon atmosphere, to a solution of 15 (3.0 g, 18 mmol) and TosMIC (3.8 g, 19 mmol) in
DME (63 mL) and tert-BuOH (21 mL), tert-BuOK (4.0 g, 35 mmol) was added at 0 °C. The
reaction mixture was allowed to room temperature and stirred for 2 h. The reaction mixture was
2
6

poured into H O and extracted with EtOAc. The organic layer was washed with brine and dried
2
with anhydrous Na SO and concentrated under reduced pressure. The residue was triturated with
2
4
1
(
(
(
n-hexane-EtOAc) (50:50) to give 16 (1.8 g, 56%) as a colorless solid. H NMR (CDCl ) δ 1.00
3
6H, s), 1.16 (6H, s), 1.63–1.74 (2H, m), 1.78–1.88 (2H, m), 2.22 (3H, s), 2.73–2.84 (1H, m); MS
+
ESI) m/z 181 [M+H] .
5
.1.27. 1-(1,2,2,6,6-Pentamethylpiperidin-4-yl)methanamine (17)
Under argon atmosphere, to a suspension of LAH (210 mg, 5.5 mmol) in THF (20 mL), 16 (1.0
g, 5.5 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 3 h then cautiously
quenched with saturated aqueous Rochelle's salt and stirred at room temperature for 15 min. The
precipitate was filtrated and concentrated under reduced pressure. The residue was
chromatographed on amino silica gel with elution using (CHCl -MeOH) (98:2) to give 17 (1.0 g,
3
1
9
1
8%) as a colorless oil. H NMR (CDCl ) δ 0.97–1.17 (4H, m), 1.01 (6H, s), 1.14 (6H, s),
3
.48–1.58 (2H, m), 1.61–1.75 (1H, m), 2.25 (3H, s), 2.53 (2H, d, J = 6.4 Hz); MS (ESI) m/z 185
+
[
M+H] .
5
.1.28. 4-Chloro-1-(3-nitrophenyl)-1H-pyrazole (19)
To a solution of 20 (400 mg, 2.8 mmol) in DMI (2.4 mL), 4-chloro-1H-pyrazole (440 mg, 4.3
mmol) and K CO (0.78 g, 5.7 mmol) were added and stirred at 130 °C for 16 h. The mixture was
2
3
partitioned with EtOAc and H O. The organic layer was washed with brine, dried over anhydrous
2
2
7

Na SO and concentrated under reduced pressure. The residue was chromatographed on amino
2
4
silica gel with elution using (n-hexane-EtOAc) (90:10) to give 21 (583 mg, 92%) as a pale yellow
1
solid. H NMR (DMSO-d ) δ 7.82 (1H, dd, J = 8.0, 8.0 Hz), 8.00 (1H, d, J = 0.4 Hz), 8.16–8.21
6
(1H, m), 8.27–8.32 (1H, m), 8.64 (1H, dd, J = 2.4, 2.4 Hz), 9.04 (1H, d, J = 0.4 Hz); MS (ESI) m/z
+
2
24, 226 [M+H] .
5
.1.29. 3-(4-Chloro-1H-pyrazol-1-yl)aniline (20)
To a solution of 19 (580 mg, 2.6 mmol) in EtOH (14 mL), SnCl ∙2H O (2.3 g, 10 mmol) was added
2
2
and refluxed for 6 h. The mixture was partitioned with EtOAc and saturated aqueous NaHCO and
3
filtered with celite. The residue was extracted, washed with brine, dried over anhydrous Na SO
2
4
and concentrated under reduced pressure. The residue was chromatographed on amino silica gel
1
with elution using (n-hexane-EtOAc) (45:55) to give 22 (467 mg, 94%) as a pale yellow solid. H
NMR (DMSO-d ) δ 5.38 (2H, s), 6.49–6.54 (1H, m), 6.84–6.88 (1H, m), 7.01 (1H, dd, J = 2.0, 2.0
6
Hz), 7.10 (1H, dd, J = 8.0, 8.0 Hz), 7.79 (1H, d, J = 0.8 Hz), 8.59 (1H, d, J = 0.8 Hz); MS (ESI) m/z
+
1
5
5
94, 196 [M+H] .
.2. Biology
.2.1. PKCζ inhibitory activity (enzyme assay)
The reaction mixture contained STK Substrate 1-biotin, full-length human PKCζ and ATP. Our
2
8

compound was dissolved in dimethyl sulfoxide and added to the reaction mixture. The reaction
mixture was incubated at room temperature for 60 min, followed by incubation with Sa-XL665 and
STK Antibody Cryptate for 60 min. The enzyme reaction rate was measured in fluorescence
intensity at 620 nm (Cryptate) and 665 nm (XL665). The activity is expressed as “PKCζ IC (nM)”
5
0
in Tables.
5
.2.2. Assessment of inhibition potency of CYP3A4 using human liver microsomes
The test compounds (5 μM) were added to human liver microsomes (HLMs) (0.1 mg/mL) in
potassium sodium phosphate buffer (100 mM, pH 7.4) containing NADPH (1 mM) and
ethylenediaminetetraacetic acid (EDTA, 0.1 mM). For reversible inhibition study, midazolam was
added to the reaction mixture without pre-incubation. For time-dependent inhibition study,
midazolam was added to the mixture after pre-incubation for 30 min at 37 °C. The reaction was
stopped by addition of acetonitrile after incubation for 20 min at 37 °C. The metabolite of
midazolam (1'-hydroxymidazolam) was analyzed by LC/MS/MS and the metabolic activity was
determined. The residual metabolic activity of HLMs for midazolam in the time-dependent
inhibition study was expressed as the percentage of that in the reversible inhibition study.
5
.2.3. Transcellular transport study in LLC-PK1-MDR1 cells
Wild type or MDR1-expressing LLC-PK1 cells (LLC-PK1-WT or LLC-PK1-MDR1,
respectively) cultured for 5 days on a BD Falcon 96-Multiwell 1.0 m Insert System were
2
9