Discovery of 2,4-diamino-5-cyanopyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-drug interactions

Article abstract of DOI:10.1016/j.bmc.2019.01.019

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. PKCθ inhibitors have emerged as effective immunomodulative agents for

Full text of DOI:10.1016/j.bmc.2019.01.019

Accepted Manuscript
Discovery of 2,4-diamino-5-cyanopyrimidine derivatives as protein kinase C
theta inhibitors with mitigated time-dependent drug-drug interactions
Shigeki Kunikawa, Akira Tanaka, Yuji Takasuna, Mamoru Tasaki, Noboru
Chida
PII:
S0968-0896(18)32096-0
https://doi.org/10.1016/j.bmc.2019.01.019
BMC 14711
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 December 2018
14 January 2019
18 January 2019
Please cite this article as: Kunikawa, S., Tanaka, A., Takasuna, Y., Tasaki, M., Chida, N., Discovery of 2,4-
diamino-5-cyanopyrimidine derivatives as protein kinase C theta inhibitors with mitigated time-dependent drug-
drug interactions, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.01.019
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Discovery of 2,4-diamino-5-cyanopyrimidine derivatives as protein kinase C theta inhibitors
with mitigated time-dependent drug-drug interactions
Shigeki Kunikawa,* Akira Tanaka, Yuji Takasuna, Mamoru Tasaki and Noboru Chida
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585,
Japan
*Address correspondence regarding this manuscript to:
Shigeki Kunikawa
Advanced Biologics
Drug Discovery Research
Astellas Pharma Inc.
21 Miyukigaoka, Tsukuba,
Ibaraki 305-8585, Japan
Tel: +81-(0)29-829-307
Fax: +81-(0)29-854-1519
E-mail: shigeki.kunikawa@astellas.com
1

Key words: protein kinase C theta (PKCθ), CYP3A4 time-dependent inhibition (TDI), transplant
rejection.
2

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. PKCθ inhibitors
have emerged as effective immunomodulative agents for the prevention of transplant rejection. We
previously reported that the 2,4-diamino-5-cyanopyrimidine derivative 2 was a potent PKCθ
inhibitor; however, it exhibited CYP3A4 time-dependent inhibition (TDI). Here, we report the
structural modification of compound 2 into 34 focusing on mitigating CYP3A4 TDI. Compound 34
exhibited potent in vitro activity with mitigated CYP3A4 TDI and efficacy in vivo transplant
model.
3

1. Introduction
Organ transplant rejection is commonly treated with immunosuppressive drugs such as calcineurin
inhibitors.1 Calcineurin inhibitors (CNIs) such as tacrolimus and cyclosporin A inhibit the
phosphatase activity of calcineurin and the translocation of the nuclear factor of activated T cells
(NF-AT) into the nucleus. While treatment with CNIs after transplantation remarkably improves
graft and patient survival, the clinical use of CNIs reportedly induces mechanism-based adverse
effects such as nephrotoxicity and hypertension.2 Currently, CNI minimization protocols are widely
used to reduce the adverse effects of CNIs by combining other immunosuppressive agents with
different mechanisms of action. 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.
Protein kinase C (PKC) is a member of the family of serine/threonine kinases that consist of
classical (α, β, and γ), novel (δ, ε, η, and θ), and atypical (ζ and λ) isoforms.3 Of these isozymes,
PKCθ exhibits a selective expression pattern in T cells, platelets, and skeletal muscle. A recent
study showed that PKCθ plays an important role in T cell signaling, leading to the production of
interleukin-2 (IL-2), which plays a key role in the immune response.4 Moreover, PKCθ knockout
mice exhibit a T cell inactivation-specific phenotype. The unique role of PKCθ in the immune
system underlies its emergence as a potential target for the treatment of transplant rejection with
reduced risk of systemic side effects. A large number of PKCθ inhibitors have been reported to
4

date.5 For example, Novartis Pharmaceuticals developed Sotrastaurin (1) as an adjunctive drug in
kidney and liver transplant rejection (Fig. 1).6 Against this background, we previously reported the
2,4-diamino-5-cyanopyrimidine derivative 2 as an inhibitor of PKCθ (Fig. 1).9 However, upon
further investigation, we found that compound 2 had CYP3A4 TDI properties that may increase the
plasma concentration of CNIs such as tacrolimus to cause adverse effects as a combination therapy
in clinical treatment. In fact, clinical trials of Sotrastaurin (1) revealed that it was effective in
transplantation, but that it also increased area under the blood concentration-time curve (AUC) of
tacrolimus as a combination drug by 2.0-fold, which may induce adverse effects.7 This unfavorable
outcome might be due to Sotrastaurin being a CYP3A4 inhibitor.8 To our knowledge, none of the
reported PKCθ inhibitors have been launched.
Here, we attempted to identify a novel PKCθ inhibitor with mitigated CYP3A4 inhibition by
modifying our lead compound 2. We report the synthesis and biological evaluation of
2,4-diamino-5-cyanopyrimidine derivatives as PKCθ inhibitors.
Sotrastaurin (1)
2
Figure 1. Structure of PKC inhibitors.
5

2. Chemistry
As shown in Scheme 1, target compounds 4–6 and 9 were prepared from compound 3.9 Reductive
alkylation of compound 39 with various ketones and sodium triacetoxyborohydride (NaBH(OAc)3)
gave compounds 4–8. Subsequent chromatographic separation isolated the cis isomer 7 and trans
isomer 8. Deprotection of the trans isomer 8 using trifluoroacetic acid (TFA) furnished target
compound 9. Compounds 14 and 15 were prepared from 2,4-dichloro-5-cyanopyrimidine 10.
Treatment of 10 with 2-(trifluoromethoxy)benzylamine in the presence of DIPEA gave compound
11. Ipso substitution with amine 12 or 13 gave cis isomer 14 or trans isomer 15, respectively. The
synthesis of intermediate aliphatic amines 12, 13 are described in Scheme 2. Protection of
compound 169 with benzyl chloroformate (Cbz), followed by deprotection of the Boc group using
TFA gave 17. Reductive alkylation with 4-(tert-butyldimethylsilyloxy)cyclohexanone and
NaBH(OAc)3 resulted in the formation of a mixture of cis and trans isomers. Chromatographic
separation isolated the cis isomer 18 and trans isomer 19. The two isomers were identified on the
basis of the chemical shift of the proton at the -position with respect to the oxygen atom. This
proton resonates at higher ppm in the cis isomer (δ from 3.79 to 3.86 ppm) than the corresponding
proton in the trans isomer (δ from 3.50 to 3.61 ppm).9, 10 Deprotection of the TBDMS group using
tetrabutylammonium fluoride (TBAF), followed by hydrogenation with Pd on carbon gave cis
amine 12 and trans amine 13. Intermediates otherwise noted were commercially available.
Target compounds
–
were prepared from intermediate
(Scheme 3).9 Displacement of the
20
30 39
sulfoxide moiety by the corresponding amines, followed by deprotection of the Boc group gave
3
6

and – . Reductive alkylation with
21 29
trans-4-((tert-butyldimethylsilyl)oxy)cyclohexanecarbaldehyde11 and NaBH(OAc)3, followed by
deprotection of the tert-butyldimethylsilyl (TBDMS) group using HCl gave compounds – .
30 39
Compounds
and
were also prepared from . Displacement of the sulfoxide moiety by
44 20
43
[2-(methylsulfanyl)pyridin-3-yl]methanamine gave Boc protected compound , and subsequent
40
oxidation of m-chloroperoxy benzoic acid (m-CPBA) gave sulfoxide
or sulfone . Reductive
42
41
alkylation with trans-4-((tert-butyldimethylsilyl)oxy)cyclohexanecarbaldehyde and subsequent
deprotection gave or , respectively.
43 44
a
4
5
6
R =
7: R = Boc (cis)
8: R = Boc (trans)
9: R = H (trans)
b
3
c
d
14: (cis)
15: (trans)
10
11
Scheme 1. Reagents and conditions: (a) various ketones, NaBH(OAc)3, CH2Cl2 or THF, rt; (b) TFA,
CH2Cl2, rt; (c) 2-(trifluoromethoxy)benzylamine, DIPEA, DMF, -50 oC; (d) amine 12 or 13, DIPEA,
DMI, rt.
7

a, b
c
d, e
18: (cis)
16
17
12: (cis)
19: (trans)
13: (trans)
Scheme 2. Reagents and conditions: (a) CbzCl, NEt3, CH2Cl2, rt; (b) TFA, CH2Cl2, rt; (c)
4-(tert-butyldimethylsilyloxy)cyclohexanone, NaBH(OAc)3, CH2Cl2, rt; (d) TBAF, THF, 70 oC; (e)
H2 (1atm), 10% Pd/C (wet), MeOH, 35 oC.
a, b
c, d
20
3: X = Y = CH, R = OCF3
30: X = Y = CH, R = OCF3
31: X = Y = CH, R = CF3
21: X = Y = CH, R = CF3
22: X = Y = CH, R = Cl
32: X = Y = CH, R = Cl
23: X = Y = CH, R = SMe
24: X = N, Y = CH, R = SMe
25: X = N, Y = CH, R = OMe
26: X = N, Y = CH, R = SEt
27: X = N, Y = CH, R = SiPr
28: X = N, Y = CH, R = OiPr
29: X = Y = N, R = OMe
33: X = Y = CH, R = SMe
34: X = N, Y = CH, R = SMe
35: X = N, Y = CH, R = OMe
36: X = N, Y = CH, R = SEt
37: X = N, Y = CH, R = SiPr
38: X = N, Y = CH, R = OiPr
39: X = Y = N, R = OMe
e, b
or
f, b
c, d
a
20
43: R = SOMe
44: R = SO2Me
41:R = SOMe
42:R = SO2Me
40
Scheme 3. Reagents and conditions: (a) various amines, DIPEA, DMF, rt; (b) TFA, CH2Cl2, rt; (c)
trans-4-((tert-butyldimethylsilyl)oxy)cyclohexanecarbaldehyde, NaBH(OAc)3, CH2Cl2, DMF, rt;
(d) HCl, MeOH, rt; (e) m-CPBA (1.2 eq.), CH2Cl2, rt; (f) m-CPBA (3.8 eq.), CH2Cl2, rt.
8

.
3. Results and Discussion
We evaluated the in vitro inhibitory activity of the compounds against human PKCθ by measuring
the fluorescence intensity after incubation with the full-length human recombinant PKCθ and
adenosine triphosphate (ATP). An IL-2 production inhibitory activity assay was used to assess the
cellular potency of the compounds. We also assessed the inhibitory activity of the compounds
against CYP3A4 by measuring the metabolic activity of human liver microsomes for midazolam at
0 and 30 min after preincubation with the test compounds. The residual activity of CYP3A4 is
reported as the percentage of metabolic activity remaining after preincubation for 30 min compared
to that at 0 min (i.e. without preincubation).
Compound 2 showed potent PKCθ inhibitory activity but the residual activity of CYP3A4 was
only 36% (Table 1). This result suggests that compound 2 exhibits TDI for CYP3A4. To minimize
TDI, we focused on the secondary amine, which reportedly undergoes CYP3A4-mediated
N-hydroxylation to corresponding secondary hydroxylamines that inactivate CYP3A4 as metabolic
intermediate complex formation with the nitroso metabolite forms through the secondary
hydroxylamine and nitrone pathway.12 We previously reported that basicity and the location of the
amine at the 4-position of the pyrimidine derivative is essential for PKCθ inhibitor activity.9 We
therefore increased the steric hindrance around the adamantylamine moiety at the 4-position as an
alternative strategy for disrupting the formation of a metabolite-intermediate complex to reduce
TDI.13 Table 1 shows the effect of replacement of hydroxy ethyl group to other steric substituents.
9

While introduction of an isopropyl substituent did not improve TDI (4), more bulky substituents
such as cyclohexane slightly improved TDI (5) compared to 4. Conversion of the cyclohexane ring
to a tetrahydropyranyl (THP) ring (6) improved PKCθ inhibitory activity with maintaining TDI,
and trans cyclohexanol group improved both inhibitory activity and TDI (15). Isomer preference
was noted for these compounds. The trans isomer 15 had greater improvement in TDI than the cis
isomer 14. Replacing the hydroxy group with an amino group slightly decreased the TDI (9).
Insertion of a methylene linker between the cyclohexane moiety and pyrimidine proved slightly
effective for improving TDI (30).
The above described optimizations ameliorated TDI compared with the lead compound 2.
However, the improvement was not sufficient and the solubility of compound 30 was low (< 1 μM
in pH 6.8 buffer solution). They highlighted the need to improve the physicochemical properties.14
We therefore focused on reducing lipophilicity because the lipophilicity of compounds is reportedly
correlated with the solubility and CYP3A4 substrates.15, 16 Given that our previous docking study
suggested that the substituent of the phenyl ring at the 2-position was located at some distance from
the hinge binding region of PKCθ,9 modifications made here were expected to be acceptable. We
therefore modified the substituent of the phenyl ring of compound 30 to reduce lipophilicity (Table
2). Replacement of the OCF3 group with CF3 or Cl group resulted in 31 or 32 did not improve TDI.
On the other hand, we investigated SMe substituent (33) substantially improved TDI and water
solubility. Replacement of the phenyl ring with a pyridine ring resulted in compound 34, which had
improved CYP3A4 TDI and water solubility, as well as PKCθ inhibitory activity (IC50 = 0.44 nM)
10

and IL-2 inhibitory activity (IC50 = 10 nM). Replacement of the S atom with an O atom maintained
TDI and water solubility, but decreased PKCθ inhibitory activity and IL-2 inhibitory activity (35).
Oxidation of 34 produced 43 and 44, which exhibited decreased inhibitory activities. Increasing
lipophilicity such as in 36, 37 and 38 improved PKCθ inhibitory activity and IL-2 inhibitory
activity, but didn’t improve TDI compared with 34 and 35. Further, introduction of a nitrogen atom
to produce 39 resulted in a loss of PKCθ inhibitory activity.
As a result of the above optimizations, we selected 34, which had good in vitro potency and
solubility, for further evaluation. Pharmacokinetic parameters for compound 34 were measured in
rats (Table 3). Plasma concentration was measured after a single oral dose at 1 mg/kg. The dose
resulted in a maximum plasma concentration (Cmax) of 12.4 ng /mL, allowing bioavailability to be
calculated as a ratio of the area under the plasma concentration-time curve of 34 after intravenous
and oral administration; the bioavailability was 58.3%.
We also evaluated the inhibitory activity of 34 against other PKC isoforms and 27 serine/threonine
and tyrosine kinases,17 as some isozymes and kinases are known to cause side effects. For example,
PKCδ-deficient mice exhibit hyperproliferation of B cells and overproduction of inflammatory
cytokines.18 A near-linear relationship was also noted between the dose of pan PKC inhibitor
sotrastaurin and adverse effects such as gastrointestinal disorders and diarrhea.19 As a result of
evaluation, we found 34 was a potent and selective inhibitor of PKCθ.17 The expression profile and
selective inhibition exhibited by PKCθ may form a suitable balance between immunosuppression
and minimization of systemic side effects.
11

We subsequently investigated the effect of 34 in combination on cardiac graft survival in rats
(Table 4). In combination with tacrolimus (suboptimal dose: 0.02 mg/kg), 34 at 1 mg/kg b.i.d.
drastically reduced graft rejection. We also found the in vivo efficacy of 34 to prevent allograft
rejection in a monkey renal transplantation model.20
12

Table 1
Conversion of N-alkyl group on the adamantyl amine
Compound R
PKCθ
IC50a (nM) (residual activity, %)
CYP3A4 TDIb
2
4
5
-CH2CH2OH
-iPr
0.70
< 1.0
1.9
36
29
43
6
<1.0
0.38
0.66
0.10
0.42
41
64
43
59
70
15
14
9
30
a IC50 values were determined in triplicate in one experiment.
b Activities of HLMs for metabolism of midazolam were measured and residual activities are shown
as percentage of remained metabolic activity following preincubation for 30 min in presence of
test compounds (5 µM).
13

Table 2.
Conversion of the 2-trifluoromethoxy benzene ring
Compound R
X
Y
PKCθ IL-2 CYP3A4 TDIc Solubilityd ACDlogPe
a
b
IC50
IC50 (residual
(μM)
(nM) (nM) activity, %)
OCF3
CH CH 0.42
CH CH 0.57
CH CH 1.4
CH CH 0.94
11
17
22
16
10
43
70
63
77
92
98
94
<1
5.11
5.10
5.10
4.98
4.07
3.49
2.58
2.60
4.46
4.76
4.13
2.72
30
31
32
33
34
35
43
44
36
37
38
39
CF3
NTf
Cl
1.1
SMe
SMe
OMe
SOMe
45
N
N
N
CH 0.44
CH 1.9
CH 32
CH NTf
CH 0.46
CH 0.23
CH 0.59
>50
>50
>100
>100
16
170 NTf
140 NTf
<3.0 80
SO2Me N
SEt
N
N
N
N
SiPr
OiPr
OMe
3.0
66
<1
18
87
>100
>100
N
>100 NTf NTf
a IC50 values were determined in triplicate in one experiment.
b Inhibition of IL-2 production in Jurkat cells. IC50 values were determined in duplicate in one
experiment.
c Activities of HLMs for metabolism of midazolam were measured and residual activities are
shown as percentage of remained metabolic activity following preincubation for 30 min in presence
of test compounds (5 µM).
d Solubility of the test compound in a buffer solution of pH 6.8.
e ACDlogP values were calculated by ACD/Percepta. (version 14.0.0)
f Not tested.
14

Table 3
Pharmacokinetic parameters of compound 34 in ratsa
i.v. (1 mg/kg)
p.o. (1 mg/kg)
b
c
d
e
b
f
g
AUC24h
t1/2
Vss
(L/kg)
3.97 12.9
a Each value is an average of data from three animals.
CLtot
(mL/min/kg) (ng∙h/mL) (ng/mL) (h)
66.7 145 12.4 3.33
AUC24h
Cmax
tmax
Fh
(ng∙h/mL) (h)
248
(%)
58.3
b Area under the plasma concentration versus curve from time zero to 24 hours after dosing.
c Elimination half-life from plasma.
d Volume of distribution at steady state.
e Total body clearance.
f Maximum plasma concentration.
g Time to reach maximum plasma concentration.
h Absolute oral bioavailability.
Table 4
Effect of compounds 34 concomitant treatment with tacrolimus on graft survival of rat cardiac
transplantation modela
Combined
drug
Graft survival time
(days)
MSTb
(days)
6c
Compound
n
-
Vehicle
Vehicle
1 mg/kg
none
tacrolimusd 11
5, 5, 6, 6, 6, 7, 7, 7, 9, 11, 11
9, 11, 13, 17, 20, 23, 24, 24, 26 20e
7e
tacrolimusd
9
a Test compounds were orally administered.
b Median survival time.
c Standard data in our laboratory.
d 0.02 mg/kg, q.d., i.m.
e ref 19.
15

4. Conclusions
We investigated the structure activity relationship of novel compounds for PKCθ inhibitory activity
and CYP3A4 TDI to obtain a promising PKCθ inhibitor. We introduced steric hindrance and
reduced lipophilicity to mitigate CYP3A4 TDI. As a result of optimization, we discovered
compound 34 with an IC50 value in the nano molar range, excellent aqueous solubility and
mitigated CYP3A4 TDI. Compound 34 showed good selectivity for PKCθ over other PKC
isoforms and kinases. Administration of 34 in a rat model of cardiac transplantation and a monkey
model of renal transplantation demonstrated the potential of PKCθ as a new drug target for organ
transplantation. This potent and selective PKCθ inhibitor might exhibit a suitable balance between
immunosuppression and minimization of systemic side effects for improved treatment of transplant
rejection.
5. Experimental section
5.1. Chemistry
1H NMR spectra were recorded on a Bruker Avance 400, Bruker AV400M, Bruker Avance III HD,
Varian VNS-400, or Varian 400 MR; and chemical shifts were expressed in δ (ppm) values with
tetramethylsilane as an internal reference (s = singlet, d = doublet, t = triplet, m = multiplet, and br
= broad peak). Mass spectra (MS) were recorded on Agilent 1100, Thermo Electron LCQ
Advantage, or Waters UPLC/SQD. 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%
16

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; DMF,
N,N-dimethylformamide; DMI, 1,3-dimethyl-2-imidazolidinone; TBAF, tetrabutylammonium
fluoride; TFA, trifluoroacetic acid; and THF, tetrahydrofuran.
5.1.1.
4-[({(1s,3R,4s,5S,7s)-4-[(Propan-2-yl)amino]adamantan-1-yl}methyl)amino]-2-({[2-(trifluorom
ethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (4)
To a solution of 3 (50 mg, 0.11 mmol) in CH2Cl2 (2.5 mL), acetone (23 μL, 0.32 mmol) and
sodium triacetoxyborohydride (67 mg, 0.32 mmol) were added and then stirred at room temperature
for 5 h. The reaction mixture was basified with saturated aqueous NaHCO3 and extracted with
EtOAc. The organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo.
The residue was chromatographed on amino silica gel with elution using (CHCl3-MeOH) to give 4
(53 mg, 98%) as a colorless solid. 1H NMR (DMSO-d6) δ ppm 0.94 (6H, d, J = 6.2 Hz), 0.97–1.97
(14H, m), 2.46 and 2.65 (total 1H, each brs), 2.70–2.75 (1H, m), 2.94 and 3.15 (total 2H, each d, J =
6.3 Hz), 4.54 (2H, d, J = 6.0 Hz), 7.16–7.35 (5H, m), 7.90 and 8.14 (total 1H, each t, J = 6.3 Hz),
8.18 (1H, s); MS (ESI) m/z 515 [M+H]+; HRMS (ESI) calcd for C27H34F3N6O [M+H]+: 515.2741,
Found: 515.2740.
17

5.1.2.
4-[({(1s,3R,4s,5S,7s)-4-(Cyclohexylamino)adamantan-1-yl}methyl)amino]-2-({[2-(trifluoromet
hoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (5)
Compound 5 was prepared from compound 3 in 91% yield as a colorless solid, using similar
approach to that described for 4. 1H NMR (DMSO-d6) δ ppm 0.91–1.22 (14H, m), 1.45–1.99 (11H,
m), 2.32 (1H, m), 2.93 and 3.15 (total 2H, each d, J = 6.3Hz), 4.54 (2H, d, J = 6.0 Hz), 7.16–7.35
(5H, m), 7.89 and 8.14 (total 1H, each t, J = 6.3 Hz), 8.18 (1H, s); MS (ESI) m/z 555 [M+H]+;
HRMS (ESI) calcd for C30H38F3N6O [M+H]+: 555.3054, Found: 555.3047.
5.1.3.
4-[({(1s,3R,4s,5S,7s)-4-[(Oxan-4-yl)amino]adamantan-1-yl}methyl)amino]-2-({[2-(trifluoromet
hoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (6)
Compound 6 was prepared from compound 3 in 67% yield as a colorless solid, using similar
approach to that described for 4. 1H NMR (DMSO-d6) δ ppm 1.02–1.89 (18H, m), 2.54–2.74 (2H,
m), 2.94 and 3.17 (total 2H, each d, J = 6.3Hz), 3.27 (2H, t, J = 11.6 Hz), 3.81 (2H, d, J = 11.6 Hz),
4.54 (2H, d, J = 6.0 Hz), 7.16–7.34 (5H, m), 7.89 and 8.14 (total 1H, each t, J = 6.3Hz), 8.18 (1H,
s); MS (ESI) m/z 557 [M+H]+; HRMS (ESI) calcd for C29H36F3N6O2 [M+H]+: 557.2846, Found:
557.2839.
18

5.1.4.
4-[({(1s,3R,4s,5S,7s)-4-[(cis-4-tert-Butoxycarbonylaminocyclohexyl)amino]adamantan-1-yl}me
thyl)amino]-2-({[2-(trifluoromethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (7) and
4-[({(1s,3R,4s,5S,7s)-4-[(trans-4-tert-Butoxycarbonylaminocyclohexyl)amino]adamantan-1-yl}
methyl)amino]-2-({[2-(trifluoromethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (8)
To a solution of 3 (75 mg, 0.16 mmol) in CH2Cl2 (5 mL) and THF (2 mL),
4-(tert-butoxycarbonylamino)cyclohexanone (76 mg, 0.36 mmol) and sodium
triacetoxyborohydride (150 mg, 0.71 mmol) were added and then stirred at room temperature. The
reaction mixture was basified with saturated aqueous NaHCO3 and extracted with CHCl3. The
organic layer was washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue
was chromatographed on amino silica gel with elution using (n-hexane-EtOAc) (1:1) to give 7 (30
mg, 29%, less polar) and 8 (27 mg, 25%, high polar). 7 1H NMR (DMSO-d6) δ ppm 0.89–1.89
(24H, m), 1.37 (9H, m), 2.39–2.44 (1H, m), 2.90–2.97 (2H, m), 4.51–.4.58 (2H, m), 6.54–6.66 (1H,
m), 7.22–7.40 (5H, m), 8.10–8.16 (1H, m), 8.18 (1H, s); MS (ESI) m/z 670 [M+H]+; 8 1H NMR
(DMSO-d6) δ ppm 0.89–1.90 (24H, m), 1.37 (9H, m), 2.18–2.29 (1H, m), 2.88–2.98 (2H, m),
4.46–.4.59 (2H, m), 6.61–6.73 (1H, m), 7.22–7.42 (5H, m), 8.01–8.16 (1H, m), 8.18 (1H, s); MS
(ESI) m/z 670 [M+H]+;
5.1.5.
4-[({(1s,3R,4s,5S,7s)-4-[(trans-4-Aminocyclohexyl)amino]adamantan-1-yl}methyl)amino]-2-({[
19

2-(trifluoromethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (9)
To a solution of 8 (21 mg, 0.03 mmol) in CH2Cl2 (2 mL), TFA (0.12 mL, 1.6 mmol) was added
and stirred at room temperature. The reaction mixture was concentrated in vacuo. The residue was
chromatographed on amino silica gel with elution using (CHCl3-MeOH) to give 9 (13 mg, 72%). 1H
NMR (DMSO-d6) δ ppm 0.94–2.67 (27H, m), 2.93–3.15 (2H, m), 4.53–4.55 (2H, m), 7.15–7.35
(5H, m), 7.89–8.15 (1H, m), 8.18 (1H, s); MS (ESI) m/z 570 [M+H]+ ; HRMS (ESI) calcd for
C30H39F3N7O [M+H]+: 570.3163, Found: 570.3162.
5.1.6. 4-Chloro-2-({[2-(trifluoromethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (11)
To a solution of 2,4-dichloropyrimidine-5-carbonitrile 10 (5.1 g, 29 mmol) in DMF (50 mL),
2-(trifluoromethoxy)benzylamine (5.9 g, 31 mmol) in DMF (13 mL) and DIPEA (6.1 mL, 35
mmol) were added under -50 oC in dry ice-acetone bath and stirred at -50 oC. The reaction mixture
was diluted with EtOAc and washed with H2O, dried over MgSO4 and concentrated in vacuo. The
residue was chromatographed on silica gel with elution using CHCl3 to give 11 (4.0 g, 42%). 1H
NMR (DMSO-d6) δ ppm 4.58–4.69 (2H, m), 7.31–7.50 (4H, m), 8.75 and 8.77 (1H, each s),
9.23–9.33 (1H, br); MS (ESI) m/z 351, 353 [M+Na]+.
5.1.7.
4-[({(1s,3R,4s,5S,7s)-4-[(cis-4-Hydroxycyclohexyl)amino]adamantan-1-yl}methyl)amino]-2-({[
2-(trifluoromethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (14)
20

To a solution of 11 (43 mg, 0.13 mmol) in DMI (0.6 mL), 12 (30 mg, 0.11 mmol) and DIPEA (75
μL, 0.43 mmol) were added and stirred at room temperature over night. The reaction mixture was
diluted with CHCl3 and chromatographed on amino silica gel with elution using (CHCl3-MeOH) to
give 14 (42 mg, 68%) as a colorless solid. 1H NMR (DMSO-d6) δ ppm 0.71–2.70 (24H, m),
2.90–3.19 (2H, m), 3.56–3.64 (1H, m), 4.23–4.59 (3H, m), 7.14–7.41 (5H, m), 7.90 and 8.14 (total
1H, each t, J = 6.3 Hz), 8.18 (1H, s); MS (ESI) m/z 571 [M+H]+; HRMS (ESI) calcd for
C30H38F3N6O2 [M+H]+: 571.3003, Found: 571.3001.
5.1.8.
4-[({(1s,3R,4s,5S,7s)-4-[(trans-4-Hydroxycyclohexyl)amino]adamantan-1-yl}methyl)amino]-2-(
{[2-(trifluoromethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (15)
Compound 15 was prepared from compound 11 in 53% yield as a colorless solid, using similar
approach to that described for 14. 1H NMR (DMSO-d6) δ ppm 0.69–2.69 (24H, m), 2.88–3.17 (2H,
m), 3.22–3.41 (1H, m), 4.41–4.59 (3H, m), 7.13–7.43 (5H, m), 7.90 and 8.14 (total 1H, each t, J =
6.3 Hz), 8.18 (1H, s); MS (ESI) m/z 571 [M+H]+; HRMS (ESI) calcd for C30H38F3N6O2 [M+H]+:
571.3003, Found: 571.3001.
5.1.9. Benzyl {[(1s,3R,4s,5S,7s)-4-aminoadamantan-1-yl]methyl}carbamate (17)
To solution of 16 (500 mg, 1.8 mmol) and NEt3 (0.3 mL, 2.1 mmol) in CH2Cl2 (10 mL),
Carbobenzoxy Chloride (Cbz-Cl) 0.28 mL, 2.0 mmol) was added at 0oC and stirred at room
21

temperature for 4 h. The reaction mixture was diluted with EtOAc, washed with 0.1N aqueous HCl,
H2O, saturated aqueous NaHCO3 and brine. The separated organic layer was dried over MgSO4 and
concentrated in vacuo. The residue was chromatographed on amino silica gel with elution using
(n-hexane-EtOAc) (3:1) to give a white solid (739 mg). To a solution of the residue in CH2Cl2 (5.9
mL), TFA (5.9 mL, 76 mmol) was added at 0 oC and stirred at room temperature for 2 h. The
reaction mixture was concentrated, then diluted with CH2Cl2 and basified with aqueous K2CO3 and
extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4 and
concentrated in vacuo to give 17 (560 mg, 100%) as a colorless oil. 1H NMR (DMSO-d6) δ ppm
1.19–1.29 (2H, m), 1.33–1.51 (6H, m), 1.60–1.68 (2H, m), 1.74–1.82 (1H, m), 1.93–2.04 (2H, m),
2.28 (2H, br), 2.70 (2H, d, J = 6.4Hz), 2.77–2.83 (1H, m), 5.01 (2H, s), 7.18 (1H, t, J = 6.4Hz),
7.27–7.41 (5H, m); MS (ESI) m/z 315 [M+H]+.
5.1.10. Benzyl
({(1s,3R,4s,5S,7s)-4-[(cis-4-{[tert-butyl(dimethyl)silyl]oxy}cyclohexyl)amino]adamantan-1-yl}
methyl)carbamate (18) and Benzyl
({(1s,3R,4s,5S,7s)-4-[(trans-4-{[tert-butyl(dimethyl)silyl]oxy}cyclohexyl)amino]adamantan-1-yl
}methyl)carbamate (19)
To a solution of 17 (760 mg, 2.4 mmol) and 4-(tert-butyldimethylsilyloxy)cyclohexanone (1.1 g,
4.8 mmol) in CH2Cl2 (23 mL), sodium triacetoxyborohydride (1.0 g, 4.8 mmol) was added and then
stirred at room temperature for 4 h. The reaction mixture was extracted with EtOAc and saturated
22

aqueous NaHCO3. The organic layer was washed with H2O, brine, dried over MgSO4 and
concentrated in vacuo. The residue was chromatographed on amino silica gel with elution using
(n-hexane-EtOAc) (10:1 to 1:1) to give 18 (670 mg, 53%, less polar) as a colorless oil and 19 (440
mg, 34%, high polar) as a colorless oil. 18 1H NMR (DMSO-d6) δ ppm 0.02 (6H, s), 0.87 (9H, s),
1.08–1.29 (3H, m), 1.33–1.64 (14H, m), 1.70–1.82 (3H, m), 1.91–2.00 (2H, m), 2.42–2.54 (1H, m),
2.62–2.69 (1H, m), 2.70 (2H, d, J = 6.3 Hz), 3.79–3.86 (1H, m, -position of the oxygen atom),
5.00 (2H, s), 7.18 (1H, t, J = 6.3 Hz), 7.27–7.41 (5H, m); MS (ESI) m/z 527 [M+H]+. 19 1H NMR
(DMSO-d6) δ ppm 0.02 (6H, s), 0.85 (9H, s), 0.92–1.29 (7H, m), 1.32–1.48 (6H, m), 1.68–1.85 (7H,
m), 1.89–1.98 (2H, m), 2.31–2.44 (1H, m), 2.61–2.67 (1H, m), 2.70 (2H, d, J = 6.3 Hz), 3.50–3.61
(1H, m, -position of the oxygen atom)), 5.00 (2H, s), 7.18 (1H, t, J = 6.3 Hz), 7.27–7.41 (5H, m);
MS (ESI) m/z 527 [M+H]+.
5.1.11. cis-4-{[(1R,2s,3S,5s,7s)-5-(Aminomethyl)adamantan-2-yl]amino}cyclohexan-1-ol (12)
To a solution of 18 (640 mg, 1.2 mmol) in THF (13 mL), TBAF (1 M THF solution) (3.7 mL, 3.7
mmol) was added and stirred at 70 oC for 13 h. The reaction mixture was concentrated in vacuo. The
residue was extracted with CHCl3/H2O/brine. The organic layer was washed with brine 4 times,
dried over MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel with
elution using (CHCl3-MeOH-28% aqueous NH3) (20:1:0 to 8:1:0.1) to give a colorless solid (450
mg). To the obtained product (430 mg) in MeOH (13 ml), 10% Pd on carbon (85 mg, 50% wet) was
added and stirred at 35 oC under H2 for 2.5 h. The reaction mixture was diluted with MeOH and
23

filtered through a Celite® pad. The filtrate was concentrated in vacuo to give 12 (310 mg, 95% from
18) as a colorless solid. 1H NMR (DMSO-d6) δ ppm 1.08–3.28 (28H, m), 4.07–4.15 (1H, m),
4.46–4.72 (1H, m); MS (ESI) m/z 279 [M+H]+.
5.1.12. trans-4-{[(1R,2s,3S,5s,7s)-5-(Aminomethyl)adamantan-2-yl]amino}cyclohexan-1-ol (13)
Compound 13 was prepared from compound 19 in quantitative yield as a colorless solid, using
similar approach to that described for 12. 1H NMR (DMSO-d6) δ ppm 1.25–3.39 (28H, m),
3.75–3.83 (1H, m), 4.47 (1H, br s); MS (ESI) m/z 279 [M+H]+
5.1.13.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-{[2-(trifluoromethoxy)benzyl]
amino}pyrimidine-5-carbonitrile (3)
To a solution of 16 (2.1 g, 4.7 mmol) in DMI (20 mL), 2-(trifluoromethoxy)benzylamine (1.8 g,
9.4 mmol) was added at 0 oC and stirred at room temperature for 1 h. The reaction mixture was
poured into H2O, collected by vaccum filtration and chromatographed on silica gel with elution
using (n-hexane-EtOAc) (8:2 to 7:3) to give a colorless solid. To a suspension of obtained product
in CH2Cl2 (30 mL), TFA (14 mL) was added at 0 oC and stirred at room temperature for 1 h. The
reaction mixture was concentrated in vascuo. To the residue, saturated aqueous NaHCO3 was added
and the precipitate was washed with H2O and n-hexane to give 3 (1.7 g, 76%) as a colorless solid.
1H NMR (DMSO-d6) δ ppm 1.01–2.02 (15H, m), 2.63 and 2.79 (total 1H, each s), 2.94 and 3.14
24

(total 2H, each d, J = 6.4Hz), 4.54 (2H, d, J = 6.1Hz), 7.14–7.34 (5H, m), 8.90 and 8.14 (total 1H,
each t, J = 6.4Hz), 8.18 (1H, s); MS (ESI) m/z 473 [M+H]+; HRMS (ESI) calcd for C24H28F3N6O
[M+H]+: 473.2271, Found: 473.2276. Anal. Calcd for C24H27F3N6O: C, 61.01; H, 5.76; N, 17.79; F,
12.06. Found: C, 61.08; H, 5.84; N, 17.47; F, 11.84.
5.1.14.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-{[2-(trifluoromethyl)benzyl]a
mino}pyrimidine-5-carbonitrile (21)
Compound 21 was prepared from compound 20 in 80% yield as a colorless solid, using similar
approach to that described for 3. 1H NMR (DMSO-d6) δ ppm 0.95–2.09 (15H, m), 2.50–2.58 and
2.77–2.83 (1H, each m), 2.90 and 3.17 (2H, each d, J = 6.4Hz), 4.61–4.72 (2H, m), 7.14–7.76 (5H,
m), 7.92–8.01 and 8.16–8.27 (total 2H, each m); MS (ESI) m/z 457 [M+H]+; HRMS (ESI) calcd for
C24H28F3N6 [M+H]+: 457.2322, Found: 457.2321.
5.1.15.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-{[2-(chlorophenyl)methyl]ami
no}pyrimidine-5-carbonitrile (22)
Compound 22 was prepared from compound 16 in 69% yield as a colorless solid, using similar
approach to that described for 3. 1H NMR (DMSO-d6) δ ppm 1.07–2.03 (15H, m), 2.62 and 2.80
(total 1H, each br s), 2.94 and 3.16 (total 2H, each d, J = 6.3Hz), 4.51-4.54 (2H, m), 7.15–7.31 (4H,
25

m), 7.42 (1H, d, J = 7.4Hz), 7.92 and 8.19 (total 1H, each t, J = 6.3Hz), 8.18 (1H, s); MS (ESI) m/z
423 [M+H]+; HRMS (ESI) calcd for C23H28ClN6 [M+H]+: 423.2058, Found: 423.2059.
5.1.16.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-{[2-( methylsulfanyl)benzyl]a
mino}pyrimidine-5-carbonitrile (23)
Compound 23 was prepared from compound 20 in 79% yield as a colorless solid, using similar
approach to that described for 3. 1H NMR (DMSO-d6) δ ppm 1.07–2.03 (15H, m), 2.49 (3H, s),
2.63 and 2.81 (total 1H, each brs), 2.95 and 3.15 (total 2H, each d, J = 6.3Hz), 4.44 (2H, d, J =
5.8Hz), 7.06–7.27 (5H, m), 7.84 and 8.11 (total 1H, each t, J = 6.0Hz), 8.16 (1H, s); MS (ESI) m/z
435 [M+H]+; HRMS (ESI) calcd for C24H31N6S [M+H]+: 435.2325, Found: 435.2324.
5.1.17.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-({[2-(methylsulfanyl)pyridin-3
-yl]methyl}amino)pyrimidine-5-carbonitrile (24)
Compound 24 was prepared from compound 20 in 69% yield as a colorless solid, using similar
approach to that described for 3. 1H NMR (DMSO-d6) δ ppm 1.01–2.07 (15H, m), 2.54 and 2.55
(total 3H, each s), 2.58–2.82 (1H, m), 2.90–3.18 (2H, m), 4.29–4.41 (2H, m), 7.02–7.11 (1H, m),
7.14–7.41 (2H, m), 7.86–8.23 (2H, m), 8.29–8.37 (1H, m); MS (ESI) m/z 436 [M+H]+; HRMS
(ESI) calcd for C23H30N7S [M+H]+: 436.2278, Found: 436.2278.
26

5.1.18.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-({[2-(methoxy)pyridin-3-yl]me
thyl}amino)pyrimidine-5-carbonitrile (25)
Compound 25 was prepared from compound 20 in 80% yield as a colorless solid, using similar
approach to that described for 3. 1H NMR (DMSO-d6) δ ppm 1.02–2.05 (15H, m), 2.61–2.82 (1H,
m), 2.90–3.17 (2H, m), 3.89 and 3.92 (total 3H, each s), 4.34–4.41 (2H, m), 6.87–6.95 (1H, m),
7.10–7.31 (1H, m), 7.36–7.43 (1H, m), 7.78–8.09 (2H, m), 8.16 (1H, s); MS (ESI) m/z 420 [M+H]+;
HRMS (ESI) calcd for C23H30N7O [M+H]+: 420.2506, Found: 420.2507.
5.1.19.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-({[2-(ethylsulfanyl)pyridin-3-y
l]methyl}amino)pyrimidine-5-carbonitrile (26)
Compound 26 was prepared from compound 20 in 70% yield as a colorless solid, using similar
approach to that described for 3. 1H NMR (DMSO-d6) δ ppm 1.06–2.02 (15H, m), 1.32 (3H, t, J =
8.0 Hz), 2.61–2.79 (1H, m), 2.93–3.17 (2H, m), 3.22 (2H, q, J = 8.0 Hz), 4.31–4.34 (2H, m),
7.04–7.35 (3H, m), 7.87–8.17 (2H, m), 8.29–8.33 (1H, m); MS (ESI) m/z 450 [M+H]+; HRMS
(ESI) calcd for C24H32N7S [M+H]+: 450.2434, Found: 450.2434.
5.1.20.
27

4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-({[2-(isopropylsulfanyl)pyridi
n-3-yl]methyl}amino)pyrimidine-5-carbonitrile (27)
Compound 27 was prepared from compound 20 in 71% yield, using similar approach to that
described for 3. 1H NMR (DMSO-d6) δ ppm 1.07–2.02 (21H, m), 2.62–2.80 (1H, m), 2.94–3.16
(2H, m), 4.07–4.14 (1H, m), 4.29–4.33 (2H, m), 7.03–7.39 (3H, m), 7.82–8.11 (1H, m), 8.17 (1H, s),
8.30–8.33 (1H, m); MS (ESI) m/z 464 [M+H]+.
5.1.21.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-({[2-(isopropyloxy)pyridin-3-y
l]methyl}amino)pyrimidine-5-carbonitrile (28)
Compound 28 was prepared from compound 20 in 80% yield, using similar approach to that
described for 3. 1H NMR (DMSO-d6) δ ppm 1.06–2.03 (21H, m), 2.61–2.79 (1H, m), 2.94–3.15
(2H, m), 4.34–4.38 (2H, m), 5.26–5.34 (1H, m), 6.83–8.01 (5H, m), 8.17 (1H, s); MS (ESI) m/z 448
[M+H]+.
5.1.22.
4-({[(1s,3R,4s,5S,7s)-4-Aminoadamantan-1-yl]methyl}amino)-2-({[2-(methoxy)pyridazin-3-yl]
methyl}amino)pyrimidine-5-carbonitrile (29)
Compound 29 was prepared from compound 20 in 72% yield as a colorless solid, using similar
approach to that described for 3. 1H NMR (DMSO-d6) δ ppm 0.95–2.08 (13H, m), 2.58–3.19 (3H,
28

m), 3.22–3.40 (2H, m), 4.03–4.16 (3H, m), 4.32–4.48 (2H, m), 7.16–7.27 (1H, m), 7.28–8.23 (3H,
m), 8.71–8.84 (1H, m); MS (ESI) m/z 421 [M+H]+; HRMS (ESI) calcd for C22H29N8O [M+H]+:
421.2459, Found: 421.2460.
5.1.23.
4-({[(1s,3R,4s,5S,7s)-4-{[(trans-4-Hydroxycyclohexyl)methyl]amino}adamantan-1-yl]methyl}a
mino)-2-({[2-(trifluoromethoxy)phenyl]methyl}amino)pyrimidine-5-carbonitrile (30)
Under ice-cooling, to a solution of
(1S,4S)-4-((tert-butyldimethylsilyl)oxy)cyclohaxanecarbaldehyde (28 mg, 0.12 mmol) in CH2Cl2
(1.5 mL), 3 (50 mg, 0.11 mmol) and sodium triacetoxyborohydride (67 mg, 0.32 mmol) were added
and then stirred at room temperature for 2 h. To the reaction mixture,
(1S,4S)-4-((tert-butyldimethylsilyl)oxy)cyclohaxanecarbaldehyde (10 mg, 0.04 mmol) was added
and stirred at room temperature for 1 h. The reaction mixture was basified with saturated aqueous
NaHCO3 and extracted with CHCl3. The organic layer was washed with H2O, brine, dried over
MgSO4 and concentrated in vacuo. The residue was chromatographed on amino silica gel with
elution using (n-hexane- CHCl3) (2:8) to give a white solid (41 mg). To the obtained product (38
mg, 0.05 mmol) in MeOH (0.7 mL), 1N aqueous HCl (10 mL) was added under ice-cooling and
stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo and diluted
with CHCl3, basified with saturated aqueous NaHCO3. The mixture was extracted with 10%
MeOH/CHCl3, the organic layer was washed with H2O, brine, dried over MgSO4 and concentrated
29