Structure-based design of a new series of N-(piperidin-3-yl)pyrimidine-5-carboxamides as renin inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.09.030

The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5–14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 (‘Leu-in’ to ‘Leu-out’) by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats.

Full text of DOI:10.1016/j.bmc.2016.09.030

Accepted Manuscript
Structure-based design of a new series of N-(piperidin-3-yl)pyrimidine-5-car-
boxamides as renin inhibitors
Yasuhiro Imaeda, Michiko Tawada, Shinkichi Suzuki, Masaki Tomimoto,
Mitsuyo Kondo, Naoki Tarui, Tsukasa Sanada, Ray Kanagawa, Gyorgy Snell,
Craig A. Behnke, Keiji Kubo, Takanobu Kuroita
PII:
S0968-0896(16)30717-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.030
BMC 13283
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 August 2016
12 September 2016
12 September 2016
Please cite this article as: Imaeda, Y., Tawada, M., Suzuki, S., Tomimoto, M., Kondo, M., Tarui, N., Sanada, T.,
Kanagawa, R., Snell, G., Behnke, C.A., Kubo, K., Kuroita, T., Structure-based design of a new series of N-
(piperidin-3-yl)pyrimidine-5-carboxamides as renin inhibitors, Bioorganic & Medicinal Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bmc.2016.09.030
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Structure-based design of a new series of N-
(piperidin-3-yl)pyrimidine-5-carboxamides as renin
inhibitors
Yasuhiro Imaedaa,*, Michiko Tawadaa, Shinkichi Suzukia,Masaki Tomimotoa, Mitsuyo Kondoa,
Naoki Taruia, Tsukasa Sanadaa,Ray Kanagawaa, Gyorgy Snellb, Craig A. Behnkeb,c, Keiji Kuboa,
Takanobu Kuroitaa
a Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-
Higashi, 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
b Takeda California, Inc., 10410, Science Center Drive, San Diego, California 92121, United
States
Corresponding Author
*Phone: +81-466-32-4112. Fax: +81-466-29-4471. E-mail: yasuhiro.imaeda@takeda.com.
Present Addresses
c 9363 Towne Centre Drive, San Diego, CA 92121, United States
Keywords: renin inhibitor; structure-based drug design (SBDD); crystal structure; N-(piperidin-
3-yl)pyrimidine-5-carboxamide
Abbreviations:
1

AcOH, acetic acid; Ang I, angiotensin I; Ang II, angiotensin II; AUC, area under the blood
concentration−time curve; C5min, concentration in plasma 5 min after administration; CLtotal, total
clearance; Cmax, maximum concentration in plasma; DIEA, N,N-diisopropylethylamine; Et3N,
triethylamine; EtOAc, ethyl acetate; EtOH, ethanol; F, rat bioavailability; HAC, heavy atom
count; HOBt, 1-hydroxybenzotriazole hydrate; hPRA, human plasma renin activity; i-PrOH,
isopropanol; LE, ligand efficiency; MeOH, methanol; MRT, mean residence time; Pd/C,
palladium on carbon; RAAS, renin-angiotensin-aldosterone system; Rt, retention time; Vd(ss),
volume of distribution at steady state
ethylcarbodiimide.
;
WSC, (1-[3-(dimethylamino)propyl]-3-
Abstract: The action of the aspartyl protease renin is the rate-limiting initial step of the renin-
angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood
pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine
derivatives 5−14 were designed in an attempt to enhance the renin inhibitory activity of
compound 3 identified by our previous fragment-based drug design approach. Introduction of a
basic amine essential for interaction with the two aspartic acids in the catalytic site and
optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114
(“Leu-in” to “Leu-out”) by a rational structure-based drug design approach led to the discovery
of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor
than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of
compound 14 has been achieved by the overall addition of only seven heavy atoms to compound
2

3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate
oral bioavailability in rats.
Graphical abstract
1. Introduction
The renin-angiotensin-aldosterone system (RAAS)1 is known to play a key role in the
regulation of blood pressure2 and homeostasis of body fluid volume, as well as in the
pathogenesis of many cardiovascular and renal diseases, such as congestive heart failure, chronic
kidney disease, and diabetic nephropathy. An aspartyl protease, renin cleaves its only known
substrate, angiotensinogen, to release the decapeptide angiotensin I (Ang I). The high substrate
selectivity of renin and its activity at the initial and rate-limiting step of the RAAS cascade
suggest that a direct renin inhibitor may offer an attractive alternative to angiotensin-converting
enzyme (ACE) inhibitors and Angiotensin II (Ang II) receptor blockers (ARBs).3−5 Renin
inhibitors should be possible to block this cascade completely even in situations of elevated
circulating or tissue active renin and prevent the formation of Ang I and Ang II. Therefore, renin
inhibitors may provide a therapeutic effect which is different from and superior to that of ACE
3

inhibitors and ARBs.6,7 A novel nonpeptidic renin inhibitor, aliskiren (1, CGP 60536)
discovered by Novartis, is the first new class of antihypertensive drugs to enter the market in
more than a decade (Figure 1).8−12 Based on the positive results reported for aliskiren, interest in
renin as a viable drug target for the control of hypertension has intensified. However, the oral
bioavailability of aliskiren is very low (bioavailability = 2.6% in human).13 Herein, we started to
discover novel and orally active nonpeptidic renin inhibitors with more potent antihypertensive
activity and improved organ protection compared to aliskiren.
Figure 1. Structure of the renin inhibitor 1 (aliskiren, CGP 60356)
Our search for more promising renin inhibitor lead scaffolds by use of a fragment-based drug
discovery (FBDD) approach14 resulted in the discovery of new pyridine amide compound 3 as a
weak human renin inhibitor (IC50 = 38 µM), derived from fragment-hit 215 We were interested in
the chemical structure of this scaffold, and thus initiated chemical modification of this
chemotype using a structure-based drug design (SBDD) approach. In this report, we describe
our further optimization to enhance renin inhibitory activity (IC50 < 1 nM).
Figure 2. Identification of compound 3 from fragment-hit 215.
4

To understand the binding mode of compound 3 and determine an approach to increase renin
inhibitory activity, X-ray crystallography study was carried out. The X-ray crystal structure
showed that compound 3 bound to the “closed” form of renin.16,17 The furfurylamino group
occupied the rather small S3sp cavity. In addition, the amino group at the pyridine 2-position
formed additional hydrogen bonding interactions with the Gly217 backbone carbonyl group.
The amide carbonyl oxygen atom of compound 3 is involved in a hydrogen bond with the
hydroxyl group of Thr79. The two carboxylic acids of two aspartic acids in the renin catalytic
site are close to the azepane ring of 3, however no interaction between the acids and the azepane
ring was observed. We hypothesized that introduction of a basic amino group onto the azepane
ring might be effective for interaction with the two aspartic acids and the enhancement of
inhibitory activity.
Figure 3. Crystal structure of 3 in complex with human renin (2.85 Å resolution): (a) cartoon
view (left), (b) binding mode (right)
Simultaneously, compound 4 as a renin inhibitor was identified by a manual structural
similarity search based on compound 2 from our compound library. 2-Phenylquinazoline 4
5

showed more potent renin inhibitory activity (IC50 = 11 µM) than thienopyrimidine 2 (IC50 43
µM). The difference in potency between compound 2 and 4 appeared to be derived from the
substituent on the pyrimidine ring.
Thus, we obtained the crystal structures of 2-
phenylquinazoline 4 bound to renin in order to clarify the difference in view of the binding
mode in complex of renin and inhibitors. Overlap of the crystal structures of 2 and 4 indicated
that the binding modes of these complexes are almost the same. The clear difference of them is
the position of the isobutyl group of Leu114. In complex with compound 2, the isobutyl group is
directed to the pyrimidine ring of 2 (“Leu-in”). On the other hand, the isobutyl group is pushed
to the opposite side of the 2-phenyl group of 4 (“Leu-out”). 2-Phenyl group on the pyrimidine
core could push Leu114 out to enhance the inhibitory potency (possibility of induced-fit).
Therefore, we assumed that introduction of bulky group on the 2-position of the pyridine ring of
compound 3 might enhance the renin inhibitory activity.
Figure 4. Overlap of two crystal structures of 2 (cyan) and 4 (orange) bound to human renin
On the basis of our structure-based hypotheses, we designed new pyrimidine derivatives
5−14 (Figure 5) in an attempt to further enhance renin inhibitory activity. The reason we chose
6

to use a pyrimidine core instead of a pyridine ring as scaffold is as follows: (1) a nitrogen atom
at the 1-position in the pyrimidine is likely expected to interact with the hydroxyl group of
Thr79, (2) the modification of the pyrimidine 2-position could be more easily achieved with
robust chemistry. As a first step toward our goal, the introduction of basic amines in place of the
azepane group brought additional important interactions with the two aspartic acids in the
catalytic site. Next, modifications of the pyrimidine 2-position and substituent (R3) on the amide
nitrogen atom were investigated to increase lipophilic interactions with lipophilic S3 and S1
sites. In this paper, we describe the optimization of pyrimidine derivatives 5−9 (Table 1) and
10−14 (Table 2) in order to enhance renin inhibitory activity by use of an SBDD approach.
Figure 5. Design of pyrimidine derivatives 5−14
2. Chemistry
The syntheses of the target structures 3 and 5−14 are depicted in Schemes 1−3. Compound 3
was synthesized as described in Scheme 1. 2-Chloronicotionic acid 15 was reacted with
furfurylamine to give the expected carboxylic acid 16. Amide coupling of carboxylic acid 16 and
hexamethyleneimine was performed using (1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
(WSC) and 1-hydroxybenzotriazole hydrate (HOBt).
Scheme 1. Synthesis of pyridine-3-carboxamide 3a
7

aConditions and reagents: (a) furfurylamine, 1-methylpyrrolidin-2-one, 36%; (b)
hexamethyleneimine, WSC, HOBt, DMF, 74%.
The pyrimidine core was constructed by the synthetic route shown in Scheme 2. Pyrimidine
ring formation from amidines 17a−c was accomplished by treatment with diethyl
(ethoxymethylidene)propanedioate in the presence of sodium ethoxide.18,19 Chlorination with
phosphoryl chloride followed by nucleophilic substitution with furfurylamine and hydrolysis
gave carboxylic acids 18a−c.
Scheme 2. Synthesis of pyrimidine-5-carboxylic acids 18a−ca
aConditions and reagents: (a) diethyl (ethoxymethylidene)propanedioate, sodium ethoxide,
EtOH; (b) POCl3; (c) furfurylamine, DIEA, i-PrOH; (d) NaOH, water, EtOH, THF, 7−57% for 4
steps.
The synthesis of various N-(piperidinyl)pyrimidine-5-carboxamides was conducted by two
main routes, methods A, B, and C, as shown in Scheme 3. Reaction of 4-oxopiperidine 19 or 3-
oxopiperidine 20 under reductive amination conditions with various alkylamines gave the
desired N-alkylated amines. Amide formation with these amines was performed using WSC and
8

HOBt, followed by deprotection of the N-Boc group, providing the desired pyrimidines 5−10
(Method A).
Compounds 11 and 12 were synthesized as shown in Method B. Reductive amination of 3-
oxopiperidine 20 with n-butylamine and isobutylamine gave the desired N-alkylated compounds
along with some undesired trialkyl amine as byproduct. This trialkylamine was removed by
silica gel chromatography after benzyloxycarbonyl (Cbz) group protection of the desired
compounds. Subsequent deprotection of Cbz group by catalytic hydrogenation provided
piperidines 21a and 21b, respectively. The condensation of isobutylamine 21b with carboxylic
acid 18c using WSC and HOBt did not proceed because of the steric hindrance, thus we used the
acyl chloride of 18c to improve the yield for this coupling. Amide formation with piperidine
amines 21a and 21b was performed by the treatment of carboxylic acid 18c with SOCl2 and
DMF, followed by deprotection of the N-Boc group, providing the desired amides 11 and 12,
respectively.
The synthesis of enantiomers 13 and 14 is depicted in Method C. 3-Aminopiperidines 22a
and 22b were treated under reductive alkylation conditions with isobutylaldehyde to give the
desired N-alkylated compounds. Condensation with the amines thus obtained was performed
using the acid chloride of carboxylic acid 18c followed by deprotection of the N-Boc group,
providing the desired amides 13 and 14, respectively
Scheme 3. Synthesis of N-(piperidinyl)pyrimidine-5-carboxamides 5−14a
9

aConditions and reagents: (a) R2NH2, NaBH(OAc)3, AcOH, MeOH; (b) 18a−c, WSC, HOBt,
Et3N, DMF; (c) 4 M HCl/EtOAc, 9−38% for 3 steps; (d) R2NH2, NaBH(OAc)3, AcOH, MeOH;
(e) benzyl chloroformate, NaOH, THF, water; (f) H2 (1 atm), 5% Pd/C, MeOH, 43−47% for 3
steps. (g) 1) 18c, SOCl2, DMF, toluene; (2) 21a and 21b, Et3N, THF; (h) 4 M HCl/EtOAc,
36−45% for 3 steps; (i) isobutylaldehyde, NaBH(OAc)3, AcOH, MeOH; (j) 1) 18c, SOCl2, DMF,
toluene; 2) Et3N, THF; (k) 4 M HCl/EtOAc, 22−35% for 3 steps.
3. Results and Discussion
10

The compounds listed in Figures 2 and 4 and Tables 1−3 have been evaluated for the
inhibition of recombinant human renin (rh-renin) by use of either a mobility shift assay or
ELISA (enzyme linked immunosolvent assay) method. Inhibitory activities of the compounds
are reported as IC50 values.
3-1. Introduction of a basic amine directed toward the catalytic aspartic acids.
In order to make an interaction with two aspartic acids of the renin catalytic site, we
investigated the introduction effect of basic amines instead of the azepane moiety. Replacement
of the azepane ring by a piperazine or homopiperazine ring might not be suitable to make
interactions between the basic amine and the aspartic acids since these groups are appeared to be
too far apart (approximately 5−7 Å). Therefore, we designed N-(piperidinyl)pyrimidine-5-
carboxamides 5−7, which are expected to make interactions.
The results of renin inhibition assay of N-(piperidinyl)pyrimidine-5-carboxamides 5−7 are
shown in Table 1. N-(Piperidin-4-yl)pyrimidine-5-carboxamide 5 had remarkably decreased
activity. We thought that the polar basic piperidine of 5 might decrease the affinity with the
target protein due to solvation penalty and charge repulsion. Meanwhile, N-(piperidin-3-
yl)pyrimidine-5-carboxamide 7 maintained the inhibitory activity despite the introduction of the
hydrophilic basic group (IC50 = 38 µM for 3 vs 11 µM for 7). Thus, we speculated that the basic
group on the piperidine ring of compound 7 might make an interaction with aspartic acids
because of the appropriate direction and position of the base toward the two aspartic acids.
Interestingly, N-methyl analog 6 showed less potent renin inhibitory activity compared to N-
ethyl analog 7. These results suggested that the alkyl group on the amide nitrogen atoms in
compounds 6 and 7 might make a lipophilic interaction with renin.
Table 1. Renin inhibitory potencies of N-(piperidinyl)pyrimidine-5-carboxamides 5−9
11

rh-renin
compd
R1
-
R2
IC50 (μM)a
-
>100
>100
5
6
7
8
9
Me
Me
iPr
t-Bu
Me (RS)
Et (RS)
Et (RS)
Et (RS)
11 (9.0-13)
0.47 (0.36-0.62)
0.073 (0.064-0.084)
aInhibitory activity against recombinant human renin. IC50 values shown are the means of
duplicate measurement by mobility shift assay. The 95% confidence intervals are shown in
parentheses.
The crystal structure of human renin soaked with compound 7 was determined to confirm the
binding mode (Figure 6). As we supposed, the structure revealed that the piperidine NH group
directly interacts with the catalytic two aspartic acids in the catalytic site of renin. The N-ethyl
group is located in the lipophilic S1 site. These results suggested that compound 7 appeared to
make the best balance between interaction gain and solvation penalty.
12

Figure 6. Crystal structure of 7 in complex with human renin (2.25 Å resolution): (a) cartoon
view (left), (b) binding mode (right)
3-2 Introduction of a bulky group toward S3 and S1 sites.
Utilization of the renin lipophilic S3 and S1 sites in the crystal structure of compound 7
bound to renin (Figure 6) provided important clues on how to enhance inhibitory activity.
Regarding the S3 site, the isobutyl group of Leu114 adopted the “Leu-in” form, like that of
compound 2 in Figure 4. We expected that introduction of a bulky group at the pyrimidine 2-
position of compound 7 might push the isobutyl group of Leu114 to adopt the “Leu-out” form in
an induced fit manner and enhance the inhibitory potency. Based on this idea, we focused on
modification of the pyrimidine 2-position. The assay results for 2-substituted pyrimidines are
shown in Table 1. Isopropyl derivative 8 showed 20-fold more potent renin inhibitory activity
than compound 7. As expected, the more bulky t-butyl derivative 9 exhibited 150-fold more
potent activity compared to compound 7. These results indicated that the introduction of a bulky
group at the pyrimidine 2-position could be effective at enhancing inhibitory activity.
13

Meanwhile, inspection of the lipophilic S1 site in the complex structure of 7 with renin
suggested additional opportunities to increase lipophilic interactions between the compound and
protein. An open space around the N-ethyl group of 7 appeared available to accommodate
additional modification, and thus we sought to elaborate the ethyl group. Our next set of
compounds incorporated a variety of bulky alkyl groups on the tertiary amide nitrogen. The
renin inhibitory potencies obtained for the compounds synthesized are listed in Table 2. Propyl
derivative 10, butyl derivative 11, and isobutyl derivative 12 exhibited more potent renin
inhibitory activities (IC50 values of 10-9 M order) than ethyl derivative 9. In particular, isobutyl
derivative 12 showed 26-fold more potent renin inhibitory activity than compound 9. In order to
determine the stereochemical preferences of the piperidine 3-position in 12, each individual
enantiomer 13 and 14 was synthesized and evaluated. As a result, the (3S)-enantiomer 14 was
determined to be the eutomer of 12, exhibiting the most potent renin inhibitory activity found in
this study (IC50 = 0.00058 µM). These results suggested that the isobutyl group of compound 14
might tightly occupy the lipophilic S1 site in renin. Thus, optimization of the S1/S3 binding
elements by addition of only five carbon atoms provided N-(piperidin-3-yl)pyrimidine-5-
carboxamide 14, which was found to be 20,000-fold more potent than parent compound 7.
Table 2. Renin inhibitory activity of N-(piperidin-3-yl)pyrimidine-5-carboxamides 10−14
rh-renin
IC50 (μM)a
compd
R3
14

n-Pr (RS)
n-Bu (RS)
i-Bu (RS)
i-Bu (R)
0.0045 (0.0033-0.0060)
0.0021 (0.0015-0.0029)
0.0012 (0.00068-0.0022)
0.0034 (0.0026-0.0045)
0.00058 (0.00039-0.00084)
10
11
12
13
14
i-Bu (S)
aInhibitory activity against recombinant human renin. IC50 values shown are the means of
duplicate measurement by enzyme-linked immunosorbent assay (ELISA). The 95% confidence
intervals are shown in parentheses.
In order to confirm its binding mode, the X-ray crystal structure of the potent renin inhibitor
14 in complex with renin was obtained (Figure 7). The stereochemistry of the central amide
bond of 14 in the binding site of renin is (Z)-form. This structure indicates that the large
contiguous hydrophobic S1/S3 specificity sites of the human enzyme are fully occupied with the
flap β-hairpin in a closed conformation.16,17 The isobutyl group of Leu114 adopts the “Leu-out”
form, like that of compound 4 in Figure 4. The amino group of the piperidine ring forms tight
hydrogen bonding interactions with the catalytic site (Asp32 and Asp215) of the enzyme. The
crystal structure also shows that the furfurylamino group occupies the S3sp cavity. In addition,
the amino group at the pyrimidine 4-position forms an additional hydrogen bonding interaction
with the Gly217 backbone carbonyl group. Moreover, the nitrogen atom at the 1-position in the
pyrimidine of 14 is involved in a hydrogen bond with the hydroxyl group of Thr79, as described
in the design of pyrimidine derivatives. The crystal structure suggested that compound 14 binds
to renin tightly through a combination of hydrophilic and hydrophobic interactions and our
hypothesis proved to be right to enhance the inhibitory activity.
15

Figure 7.
Crystal structure of 14 in complex with human renin (2.7 Å resolution): (a) cartoon view (left),
(b) binding mode (right)
To better understand quality of the new lead compounds, molecular weight (MW) and ligand
efficiency (LE) among compounds 3, 7, and 14 were compared (Table 3). MW of compound 14
(MW = 413.57) is obviously increased than those of compounds 3 and 7 (313.40 for 3, 343.43
for 7). The introduction of the basic moiety (3 => 7) appeared not to affect the LE values (0.19
for 3, 0.20 for 7). On the other hand, the LE value of compound 14 (0.31) was clearly higher
than those of 3 and 7. The result reflected that the hydrophobic interactions in the S1/S3 sites by
the addition of the five carbon atoms significantly and efficiently contributed on the
enhancement in renin inhibitory potency.
Table 3. Change in ligand efficiency of compounds 3, 7, and 14
16

Rh-renin
IC50 (μM)
38
compd
MW
HACa
23
LEb
0.19
0.20
0.31
313.40
343.43
413.57
3
7
11
25
0.00058
aHeavy atom count. bpIC50/HAC.
30
14
With its excellent in vitro enzyme potency, inhibitory activity of compound 14 against
endogenous renin in human plasma (human plasma renin activity, hPRA) was measured.
Measuring the hPRA of the compound proved to be an excellent means to take into account the
plasma protein binding and consequently the free fraction available in vivo for renin inhibition.
The hPRA IC50 value of compound 14 was 0.0046 µM, therefore the compound showed
promising inhibitory potency in hPRA assay. The IC50 value in hPRA assay was shifted upward
by about 10-fold over the value determined by ELISA assay. Compound 14 displayed
>100,000-fold selectivity for renin versus other aspartyl proteases, cathepsin D and pepsin (IC50
values > 100 µM). Compound 14 was also evaluated in a pharmacokinetic study in rats and the
pharmacokinetic profile of 14 is summarized in Table 4. Compound 14 showed moderate oral
bioavailability in rats. In vivo experiment using compound 14 was not performed, however the
in vivo result with further optimized compounds will be reported in a separate paper.
Table 4. Preliminary pharmacokinetic parameters for 14 in ratsa
iv
po
Cmax (ng/mL)
AUC0−24h,po (ng·h/mL)
MRTpo (h)
C5min (ng/mL)
Vd(ss) (mL/kg)
CLtotal (mL/h/kg)
29.0
4544
2766
9.3
53.3
3.35
13.8
F(%)
aCassette dosing. Rats were administered the drug intravenously at 0.1 mg/kg and orally at 1
mg/kg. Data are expressed as mean of three determinations. .
4. Conclusions
17

In order to enhance the renin inhibitory activity of compound 3, we were prompted to design
and synthesize new pyrimidine derivatives 5−14. We focused on introduction of a basic amine
essential for interaction with the two aspartic acids in the renin catalytic site and optimization of
the S1/S3 binding elements including an induced-fit structural change of Leu114 (from “Leu-in”
to “Leu-out”). The rational structure-based design approach led to the discovery of compound
14, a renin inhibitor 65,000-fold more potent than compound 3. Notably, this remarkable
enhancement in renin inhibitory potency was achieved through the addition of only seven heavy
atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl
proteases and moderate oral bioavailability in rats. Investigation of various substituents at the
piperidine 5-position of compound 14 will be reported separately.
5. Experimental Section
5-1. Chemistry
General procedures for chemistry. 1H NMR spectra were recorded on a Bruker DPX-300
(300 MHz) spectrometer, and are reported in parts per million (δ) relative to tetramethylsilane
(TMS: δ 0.0 ppm). Data are reported as follows: chemical shift, integration, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, ddd = doublet
of doublet of doublet, bs = broad singlet), and coupling constants (J, Hz). Unless otherwise
specified, all solvents and reagents were obtained from commercial suppliers and used without
further purification. Column chromatography was performed using Merck silica gel 60 (70–230
mesh). Thin-layer chromatography (TLC) was performed on Merck silica gel plates 60F254.
LC−MS analysis was performed on a Shiseido CAPCELL PACK C-18 UG120 S-3 column (1.5
mmφ x 35 mm) in a Waters Alliance 2795 or an Agilent 1100 LC system equipped with a Waters
2487 absorbance detector and a Micromass ZQ2000 mass spectrometer. All MS experiments
18

were performed using electrospray ionization (ESI) in positive ion mode. Analytes were eluted
using a linear gradient of water (0.05% trifluoroacetic acid (TFA))/acetonitrile (0.04% TFA) from
90:10 to 0:100 over 4 min at a flow rate of 0.5 mL/min. UV detection was at 220 nm.
Preparative HPLC was performed on a Shiseido CAPCELL PACK C-18 UG120 S-5 column (20
mmφ x 50 mm), eluting at 25 mL/min with a gradient of water (0.1% TFA)/acetonitrile (0.1%
TFA). UV detection was at 220 nm. Elemental analyses were performed by Takeda Analytical
Research Laboratories, Ltd. Melting points were determined on Yanagimoto micro melting-point
apparatus and are uncorrected. Purity measurements were carried out using a Shimadzu UFLC
system employing the following conditions: column; L-column2 ODS (3.0 mmIDx30 mmL, 2 m,
CERI, Japan); mobile phase; MeCN/H2O/TFA = 5:95:0.1 (0 min) → 90:10:0.1 (2.00 min) →
90:10:0.1 (3.3 min); flow rate; 1.2 mL/min; temperature; 40 ºC; detection; UV 220 mm. Each
compound was confirmed to be ≥90% pure by either LC-MS or elemental analysis. Yields are not
optimized.
2-[(Furan-2-ylmethyl)amino]-6-methylpyridine-3-carboxylic acid (16). A solution of 2-
chloro-6-methylnicotinic acid (1.72 g, 10.0 mmol) and 1-(2-furyl)methanamine (1.94 g, 20.0
mmol) in 1-methylpyrrolidin-2-one (25 mL) was heated at 150 ºC overnight. The solvent was
evaporated, and the residue was purified by silica gel chromatography (EtOAc/hexane = 4/1 to
1
EtOAc) to give 16 (838 mg, 36%). H NMR (CDCl3) : 2.45 (3H, s) 4.78 (2H, s) 6.26 (1H, d, J
= 3.2 Hz) 6.32 (1H, dd, J = 3.2 and 1.7 Hz) 6.46 (1H, d, J = 8.1 Hz) 7.33−7.40 (1H, m)
8.05−8.14 (2H, m).
Azepan-1-yl{2-[(furan-2-ylmethyl)amino]-6-methylpyridin-3-yl}methanone (3). To the
mixture of compound 16 (30 mg, 0.129 mmol) and hexamethyleneimine (19 mg, 0.192 mmol) in
DMF (2.0 mL), were added HOBt (21 mg, 0.155 mmol) and WSC-HCl (30 mg, 0.156 mmol)
and the mixture was stirred at room temperature overnight. To the reaction mixture EtOAc (7
19

mL) and 2% aqueous NaHCO3 solution (1.5 mL) were added and extraction was carried out. The
water phase was washed with EtOAc (7 mL). The combined organic phase was dried with
MgSO4 and evaporated. The residue was purified by silica gel chromatography (hexane to
1
EtOAc/hexane = 3/7) to give 3 (30 mg, 74%) as a clear oil. H NMR (CDCl3) δ: 1.55−1.86 (8H,
m), 2.41 (3H, s), 3.52 (4H, s), 4.65 (2H, d, J = 5.6 Hz), 5.81 (1H, t, J = 5.3 Hz), 6.23 (1H, d, J =
3.2 Hz), 6.27−6.34 (1H, m), 6.43 (1H, d, J = 7.6 Hz), 7.22 (1H, d, J = 7.3 Hz), 7.34 (1H, d, J =
1.7 Hz). LC−MS (ESI): m/z = 314 [M+H]+. Purity measurement by HPLC: 100%, Rt = 1.14
min.
2-tert-Butyl-4-[(furan-2-ylmethyl)amino]pyrimidine-5-carboxylic acid (18c). Compound
17c (2.90 g, 21 mmol) and diethyl (ethoxymethylidene)propanedioate (4.25 mL, 21 mmol) were
dissolved in EtOH (30 mL). A solution of sodium ethoxide (20% in EtOH; 14.3 g, 42 mmol) was
added to the solution dropwise at 0 ºC. After stirring at 80 ºC for 15 h, the reaction mixture was
concentrated in vacuo. The residue was acidified with 1 M HCl to pH 3. The precipitate was
collected by filtration and washed with small amount of water. The product obtained (2.90 g, 13
mmol) was dissolved in POCl3 (9.6 g). After stirring at 100ºC for 2 h, the reaction mixture was
concentrated in vacuo. The residue was cooled to 0 ºC, neutralized with a saturated aqueous
NaHCO3 solution to pH 7, and extracted with EtOAc. The extract was washed with brine, dried
over Na2SO4, and concentrated in vacuo. A solution of the product obtained, furfurylamine (1.20
mL, 12.9 mmol), and diisopropylethylamine (2.26 mL, 12.9 mmol) in 2-propanol (10 mL) was
stirred under reflux for 15 h. The reaction mixture was cooled to room temperature and
concentrated in vacuo. The residue was diluted with a saturated aqueous NaHCO3 solution and
extracted with EtOAc. The extract was washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatography (hexane/EtOAc = 9/1 to
5/1). The product obtained was dissolved in 2 M NaOH (15 mL, 30 mmol), EtOH (15 mL), and
20

THF (5 mL). After stirring at room temperature for 15 h, the mixture was acidified with 1 M HCl
to pH 3.0. The precipitate was collected by filtration and washed with water to give 18c as a
1
colorless powder (3.30 g, 57%). H NMR (CDCl3) δ: 1.32−1.40 (12H, m), 4.33 (2H, q, J = 7.1
Hz), 4.77 (2H, d, J = 5.7 Hz), 6.26 (1H, d, J = 3.2 Hz), 6.32 (1H, dd, J = 3.2 and 1.9 Hz), 7.36
(1H, dd, J = 1.8 and 0.8 Hz), 8.34 (1H, bs), 8.85 (1H, s). LC−MS (ESI): m/z = 287 [M+H]+.
The following compounds 18a and 18b were prepared in a manner similar to that described
for 18c.
4-[(Furan-2-ylmethyl)amino]-2-methylpyrimidine-5-carboxylic acid (18a). 1H NMR
(CDCl3) δ: 2.57 (3H, s), 4.78 (2H, s), 6.30 (2H, dd, J = 12.7 and 2.2 Hz), 7.37 (1H, d, J = 0.9
Hz), 8.47−8.72 (1H, m)
4-[(Furan-2-ylmethyl)amino]-2-(propan-2-yl)pyrimidine-5-carboxylic acid (18b). 1H
NMR (CDCl3) δ: 1.24 (6H, d, J = 5.1 Hz), 2.94−3.09 (1H, m), 4.78 (2H, d, J = 4.2 Hz), 6.33
(1H, d, J = 2.1 Hz), 6.41 (1H, s), 7.60 (1H, s), 8.70 (1H, s), 9.09 (1H, s).
4-[(Furan-2-ylmethyl)amino]-N,2-dimethyl-N-(piperidin-3-yl)pyrimidine-5-carboxamide
dihydrochloride (6). Compound 20 (398 mg, 2.0 mmol), AcOH (0.11 mL, 2.0 mmol), and
methylamine (2.0 M solution in THF; 1.0 mL, 2.0 mmol) were dissolved in MeOH (5 mL), and
the mixture was stirred at room temperature for 1 h. NaBH(OAc)3 (0.88 g, 4.0 mmol) was added
by portions, and the reaction mixture was stirred at room temperature for additional 15 h. After
the volatiles were removed by evaporation, the residue was dissolved in saturated aqueous
NaHCO3 and extracted twice with EtOAc. The combined organic layer was dried over Na2SO4,
and concentrated in vacuo. The product obtained was added to a solution of 18a (0.47 g, 2.0
mmol), HOBt (0.46 g, 3.0 mmol), WSC (0.58 g, 3.0 mmol), and Et3N (0.84 mL, 6.0 mmol) in
DMF (3 mL), and the resulting mixture was stirred at room temperature for 15 h. The reaction
mixture was partitioned between water and EtOAc. The organic layer was separated, washed
21

with water and brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/EtOAc = 2/1 to 0/1). The product obtained
was dissolved in 4 M HCl/EtOAc (3 mL), and the mixture was stirred at room temperature for 15
h. After concentration in vacuo, the residue was suspended in EtOAc, and the precipitate was
1
collected by filtration to give 6 (64 mg, 9%) as a beige amorphous powder. H NMR (DMSO-d6)
δ: 1.50−1.88 (3H, m), 2.58 (3H, s), 2.66−3.76 (8H, m), 4.75 (2H, bs), 6.35−6.44 (2H, m), 7.62
(1H, d, J = 0.8 Hz), 8.11 (1H, bs), 8.42 (1H, s), 9.06−9.80 (2H, m). LC−MS (ESI): m/z = 330
[M+H]+. Anal. Calcd for C17H25Cl2N5O2•3.5H2O•0.1EtOAc: C, 44.07; H, 6.97; N, 14.77.
Found: C, 44.20; H, 6.87; N, 14.62.
The following compounds 6−10 were prepared in a manner similar to that described for 6.
N-Ethyl-4-[(furan-2-ylmethyl)amino]-2-methyl-N-(piperidin-3-yl)pyrimidine-5-
1
carboxamide dihydrochloride (7). H NMR (DMSO-d6) δ: 0.80−1.20 (3H, m), 1.48−1.88 (2H,
m), 2.57 (3H, s), 2.73 (1H, bs), 2.87−3.70 (8H, m), 4.54−4.89 (2H, m), 6.29−6.47 (2H, m), 7.61
(1H, d, J = 0.9 Hz), 8.43 (1H, s), 9.17 (1H, bs), 9.36 (1H, bs). LC−MS (ESI): m/z = 344
[M+H]+. Anal. Calcd for C18H27Cl2N5O2•2H2O•0.2EtOAc: C, 48.04; H, 6.99; N, 14.90. Found:
C, 48.32; H, 6.95; N, 14.78.
N-ethyl-4-[(furan-2-ylmethyl)amino]-2-methyl-N-(piperidin-4-yl)pyrimidine-5-
1
carboxamide dihydrochloride (5). H NMR (DMSO-d6) δ: 0.94−1.24 (3H, m), 1.80 (2H, bs),
2.11 (2H, bs), 2.59 (3H, s), 2.85 (2H, bs), 3.46 (4H, bs), 4.71 (2H, d, J = 5.7 Hz), 6.43 (1H, d, J
= 1.7 Hz), 6.33−6.44 (1H, m), 7.60 (1H, d, J = 1.1 Hz), 8.30−8.45 (1H, m), 9.19 (3H, bs).
LC−MS (ESI): m/z = 344 [M+H]+. Anal. Calcd for C18H27Cl2N5O2•1.3H2O: C, 49.16; H, 6.78;
N, 15.93. Found: C, 49.31; H, 6.90; N, 15.63.
N-Ethyl-4-[(furan-2-ylmethyl)amino]-N-(piperidin-3-yl)-2-(propan-2-yl)pyrimidine-5-
1
carboxamide dihydrochloride (8). H NMR (DMSO-d6) δ: 0.92−1.22 (3H, m), 1.29 (6H, d, J
22

= 6.8 Hz), 1.67 (1H, bs), 1.76−1.96 (2H, m), 2.72 (1H, d, J = 1.9 Hz), 3.13 (3H, dt, J = 13.7and
6.7 Hz), 3.55 (3H, d, J = 13.9 Hz), 4.03 (1H, q, J = 7.2 Hz), 4.57−4.84 (2H, m), 6.35 (1H, d, J =
2.8 Hz), 6.42 (1H, dd, J = 3.2 and 1.9 Hz), 7.60 (1H, d, J = 0.9 Hz), 8.40 (1H, s), 9.18 (1H, bs),
9.29 (1H, bs), 9.70 (1H, bs). LC−MS (ESI): m/z = 372 [M+H]+. Anal. Calcd for
C20H31Cl2N5O2•2.3H2O: C, 49.44; H, 7.39; N, 14.42. Found: C, 49.58; H, 7.20; N, 14.17.
2-tert-Butyl-N-ethyl-4-[(furan-2-ylmethyl)amino]-N-(piperidin-3-yl)pyrimidine-5-
1
carboxamide dihydrochloride (9). H NMR (DMSO-d6) δ: 1.38 (9H, s), 1.91 (4H, bs), 2.73
(1H, bs), 3.12 (3H, bs), 3.36 (2H, bs), 3.54 (5H, bs), 4.54−4.83 (2H, m), 6.34 (1H, bs), 6.41 (1H,
dd, J = 3.2 and 1.9 Hz), 7.60 (1H, d, J = 0.9 Hz), 8.33 (1H, s), 9.22 (1H, bs). LC−MS (ESI):
m/z = 386 [M+H]+. Anal. Calcd for C21H33Cl2N5O2•2.5H2O: C, 50.10; H, 7.61; N, 13.91. Found:
C, 50.28; H, 7.47; N, 13.77. Purity measurement by HPLC: 98%, Rt = 2.55 min.
2-tert-Butyl-4-[(furan-2-ylmethyl)amino]-N-(piperidin-3-yl)-N-propylpyrimidine-5-
carboxamide dihydrochloride (10). 1H NMR (DMSO-d6) δ: 0.85 (3H, bs), 1.38 (9H, s),
1.50−1.96 (5H, m), 2.62−3.84 (8H, m), 4.00−4.34 (1H, m), 4.56−4.87 (2H, m), 6.33 (1H, bs),
6.41 (1H, dd, J = 3.2 and 1.9 Hz), 7.59 (1H, d, J = 0.9 Hz), 8.31 (1H, bs), 9.18 (1H, bs).
LC−MS (ESI): m/z = 400 [M+H]+. Anal. Calcd for C23H37Cl2N5O2•H2O: C, 54.76; H, 7.79; N,
13.88. Found: C, 54.76; H, 7.82; N, 13.75. Purity measurement by HPLC: 98%, Rt = 2.65 min.
tert-Butyl 3-[(2-methylpropyl)amino]piperidine-1-carboxylate (21b). Compound 20 (1.99
g, 10 mmol), AcOH (0.57 mL, 10 mmol), and isobutylamine (0.99 mL, 10 mmol) were dissolved
in MeOH (50 mL), and the mixture was stirred at room temperature for 1 h. Sodium
triacetoxyborohydride (4.23 g, 20 mmol) was added in portions, and the reaction mixture was
stirred at room temperature for additional 15 h. After the volatiles were removed by evaporation,
the residue was dissolved in saturated aqueous NaHCO3 solution and extracted twice with
EtOAc. The combined organic layer was dried over Na2SO4, and concentrated in vacuo. The
23

residue was dissolved in THF (20 mL) and 2 M NaOH (3 mL, 24 mmol), and then benzyl
chloroformate (2.86 mL, 20 mmol) was added dropwise. After stirring at room temperature for
15 h, the mixture was extracted with EtOAc. The extract was washed with water and brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column
chromatography (hexane/EtOAc = 1/9 to 1/4) to afford the Cbz-protected amine. A mixture of
the product obtained and 5% Pd/C (0.20 g) in EtOH (30 mL) was stirred at room temperature
under H2 atmosphere (1 kgf/cm2) for 15 h. The palladium catalyst was removed by filtration, and
1
the filtrate was concentrated in vacuo to give 21b as pale yellow syrup (1.21 g, 47%). H NMR
(CDCl3) δ: 0.90 (6H, d, J = 6.6 Hz), 1.22−1.40 (3H, m), 1.46 (9H, s), 1.55−1.78 (2H, m), 1.90
(1H, d, J = 13.6 Hz), 2.45 (3H, dd, J = 6.8 and 3.8 Hz), 2.87 (1H, bs), 3.54−4.24 (2H, m).
The following compound 21a was prepared in a manner similar to that described for 21b.
1
tert-Butyl 3-(butylamino)piperidine-1-carboxylate (21a). H NMR (CDCl3) δ: 0.91 (3H, t,
J = 7.2 Hz), 1.20−1.42 (4H, m), 1.42−1.52 (3H, m), 1.46 (9H, s), 1.67 (1H, td, J = 8.9 and 4.3
Hz), 1.78−2.04 (1H, m), 2.47−2.79 (3H, m), 2.87 (1H, bs), 3.79 (1H, d, J = 13.2 Hz), 4.04 (1H,
bs).
2-tert-Butyl-4-[(furan-2-ylmethyl)amino]-N-(2-methylpropyl)-N-(piperidin-3-
yl)pyrimidine-5-carboxamide dihydrochloride (12). To a suspension of compound 18c (138
mg, 0.5.mmol) in toluene (5 mL) was added thionyl chloride (0.11 mL, 1.5 mmol) and DMF (1
drop) at room temperature. After stirring at 100ºC for 1 h, the reaction mixture was concentrated
in vacuo. The residue was azeotroped with toluene and dissolved in THF (5 mL). Compound
21b (128 mg, 0.5 mmol) and Et3N (0.21 mL, 1.5 mmol) were added to the solution. After
stirring at room temperature for 2 h, the reaction mixture was concentrated in vacuo. The residue
was diluted with water and extracted with EtOAc. The extract was washed with brine, dried over
Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column
24

chromatography (hexane/EtOAc = 9/1 to 3/1). The product obtained was dissolved in 4 M
HCl/EtOAc (2 mL), and the mixture was stirred at room temperature for 15 h. After
concentration in vacuo, the residue was suspended in EtOAc, and the precipitate was collected by
filtration and washed with EtOAc to give 12 (85 mg, 36%) as a light-brown amorphous powder.
1H NMR (DMSO-d6) δ: 0.83 (5H, bs), 1.38 (9H, s), 1.57−2.12 (5H, m), 2.79 (1H, d, J = 7.3 Hz),
3.12 (4H, bs), 3.67 (3H, bs), 4.18 (1H, bs), 4.48−4.91 (2H, m), 6.33 (1H, bs), 6.41 (1H, dd, J =
3.1 and 1.8 Hz), 7.59 (1H, d, J = 0.9 Hz), 8.28 (1H, s), 9.23 (1H, bs). LC−MS (ESI): m/z = 414
[M+H]+. Anal. Calcd for C23H37Cl2N5O2•H2O: C, 54.76; H, 7.79; N, 13.88. Found: C, 54.76; H,
7.82; N, 13.75. Purity measurement by HPLC: 99%, Rt = 2.78 min.
The following compound 11 was prepared in a manner similar to that described for 12.
N-Butyl-2-tert-butyl-4-[(furan-2-ylmethyl)amino]-N-(piperidin-3-yl)pyrimidine-5-
1
carboxamide dihydrochloride (11). H NMR (DMSO-d6) δ: 0.92 (3H, bs), 1.38 (9H, s), 1.53
(1H, bs), 1.87 (4H, bs), 2.73 (1H, bs), 3.08 (3H, bs), 3.28 (3H, bs), 3.49 (3H, bs), 4.55−4.71
(1H, m), 4.71−4.83 (1H, m), 6.34 (1H, bs), 6.41 (1H, d, J = 1.9 Hz), 7.59 (1H, s), 8.33 (1H, bs),
9.21 (2H, bs). LC−MS (ESI): m/z = 414 [M+H]+. Anal. Calcd for C23H37Cl2N5O2•H2O: C,
54.76; H, 7.79; N, 13.88. Found: C, 55.15; H, 7.92; N, 13.86.
2-tert-Butyl-4-[(furan-2-ylmethyl)amino]-N-(2-methylpropyl)-N-[(3S)-piperidin-3-
yl]pyrimidine-5-carboxamide dihydrochloride (14). Compound 22a (200 mg, 1.0 mmol),
AcOH (0.07 mL, 1.2 mmol), and isobutylaldehyde (0.11 mL, 1.2 mmol) were dissolved in MeOH
(5 mL), and the mixture was stirred at room temperature for 1 h. Sodium triacetoxyborohydride
(530 mg, 2.5 mmol) was added in portions, and the reaction mixture was stirred at room
temperature for additional 15 h. After the volatiles were removed by evaporation, the residue was
dissolved in saturated aqueous NaHCO3 solution and extracted twice with EtOAc. The combined
organic layer was dried over Na2SO4, and concentrated in vacuo to give tert-butyl (3S)-3-[(2-
25

methylpropyl)amino]piperidine-1-carboxylate as crude. The obtained compound was dissolved
in THF (5 mL).
To a suspension of compound 18c (276 mg, 1.0.mmol) in toluene (5 mL) was added thionyl
chloride (0.18 mL, 2.5 mmol) and DMF (1 drop) at room temperature. After stirring at 80 ºC for
1 h, the reaction mixture was concentrated in vacuo. The residue was azeotroped with toluene
and dissolved in THF (5 mL).
The THF solution of tert-butyl (3S)-3-[(2-
methylpropyl)amino]piperidine-1-carboxylate described above and Et3N (0.42 mL, 3.0 mmol)
were added to the solution. After stirring at room temperature for 2 h, the reaction mixture was
concentrated in vacuo. The residue was diluted with water and extracted with EtOAc. The
extract was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/EtOAc = 9/1 to 3/1). The product obtained
was dissolved in 4 M HCl/EtOAc (2 mL), and the mixture was stirred at room temperature for 15
h. After concentration in vacuo, the residue was suspended in EtOAc, and the precipitate was
collected by filtration and washed with EtOAc to give 14 (105 mg, 22%) as a light-brown
amorphous powder. Measurement of the enantiomeric excess was performed on a liquid
chromatography system equipped with a CHIRALPACK AD (4.6 mm ID × 250 mm L, KF051,
Daicel, Japan), eluting with a flow rate of 1 mL/min hexane/ethanol/diethylamine = 950/50/1
1
(v/v/v) at 35 °C. Rt: 12.61 min (82% ee). H NMR (DMSO-d6) δ: 0.81 (6H, bs), 1.37 (9H, s),
1.63−2.05 (3H, m), 2.54−2.60 (1H, m), 3.57 (8H, bs), 4.16 (1H, bs), 4.54−4.74 (2H, m), 6.31
(1H, bs), 6.41 (1H, dd, J = 3.2 and 1.9 Hz), 7.58 (1H, s), 8.26 (1H, bs), 9.20 (1H, bs). LC−MS
(ESI): m/z = 414 [M+H]+. Anal. Calcd for C23H37Cl2N5O2•H2O: C, 54.76; H, 7.79; N, 13.88.
Found: C, 54.76; H, 8.07; N, 13.85. Purity measurement by HPLC: 98%, Rt = 2.75 min. [α]25
D
-13.5 (c 0.4390, MeOH).
The following compound 13 was prepared in a manner similar to that described for 14.
26

2-tert-Butyl-4-[(furan-2-ylmethyl)amino]-N-(2-methylpropyl)-N-[(3R)-piperidin-3-
yl]pyrimidine-5-carboxamide dihydrochloride (13). Measurement of the enantiomeric excess
was performed on a liquid chromatography system equipped with a CHIRALPACK AD (4.6 mm
ID × 250 mm L, KF051, Daicel, Japan), eluting with a flow rate of 1 mL/min
1
hexane/ethanol/diethylamine = 950/50/1 (v/v/v) at 35 °C. Rt: 11.13 min (91% ee). H NMR
(DMSO-d6) δ: 0.84 (6H, bs), 1.39 (9H, s), 1.69−2.10 (5H, m), 2.79 (1H, bs), 3.54 (6H, bs), 4.19
(1H, bs), 4.50−4.71 (1H, m), 4.71−4.84 (1H, m), 6.33 (1H, bs), 6.41 (1H, dd, J = 3.2 and 1.9
Hz), 7.59 (1H, s), 8.29 (1H, s), 9.28 (1H, bs). LC−MS (ESI): m/z = 414 [M+H]+. Anal. Calcd
for C23H37Cl2N5O2•H2O: C, 54.76; H, 7.79; N, 13.88. Found: C, 54.35; H, 7.78; N, 13.66.
Purity measurement by HPLC: 100%, Rt = 2.76 min. [α]25D +14.7 (c 0.4365, MeOH).
5-2. Assays of biological activities
Purification of recombinant human renin
Human preprorenin was expressed using the FreeStyle 293 Expression System (Invitrogen).
The recombinant prorenin was exported by FreeStyle 293 cells into the tissue culture media. The
cell culture supernatants were processed by filtration, concentration and dialysis in 20 mM Tris-
HCl buffer (pH 8.0). Prorenin was purified using Resource Q column (GE Healthcare) and
HiLoad 16/60 Superdex 200pg (GE Healthcare). Prorenin was activated to renin by trypsin
digestion. Renin was purified using TSKgel DEAE-5PW (Tosoh).
Renin activity assay using microchip-type capillary electrophoresis (Mobility shift assay).
In a 384−well plate (Nalgen−Nunc), 2 μL of test compound in 100% DMSO was incubated with
28 μL of enzyme in buffer (5 nM renin, 20 mM citric acid buffer (pH 6.0) and 0.004% Triton-
X100) at 37 ºC. After 15 min, 10 μL of substrate peptide (FITC-εAcp-Asp-Arg-Val-Tyr-Ile-His-
Pro-Phe-His-Leu-Val-Ile-His-Asn-Arg-NH2, 5 μM) was added to each well to start the reaction.
After incubation at 37 ºC for 30 min, the reaction was terminated by addition of 40 μL of stop
27

solution (1 μM CGP-29287,21 200 mM Tris-HCl (pH 8.0), 0.04% Triton-X 100, 0.4% Coating 3
reagent (Caliper Life Sciences)). After completion of the reaction, substrate and product peptide
were separated by electrophoresis and quantified by fluorescence detection (excitation wavelength
457 nm, measurement wavelength 530 nm) using a microchip-type capillary electrophoresis
apparatus 250HTS (Caliper Life Sciences). The rate of substrate conversion was calculated by
dividing the product peak height by the sum of the substrate and product peak height (P/(P+S)).
Inhibitory activity of each compound was calculated on the basis of 0% control wells with DMSO
and 100% control wells with 10 μM CGP-29287.
Human renin inhibition assay (enzyme-linked immunosorbent assay (ELISA)). The
inhibitory potency of the compounds against human renin was determined by the following
protocol. In 384−well plates (ABgene), 1 μL of test compound in 100% DMSO was incubated
with 14 μL of enzyme (at a final concentration of 40 pM human renin) in buffer (20 mM
Phosphate buffer, 1 mM EDTA, pH 7.4, with 0.004% Tween 20) at 37 ºC. After 10 min, 5 μL of
recombinant human angiotensinogen was added to a final concentration of 1.5 μM and incubated
at 37 ºC for 30 min. The enzymatic reaction was terminated by adding 20 μL of stop solution (1
μM CGP-29287 in diluent buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% BSA, 0.05%
Tween20)). The diluent buffer was used for diluting each reagent in the ELISA. Angiotensin I
generated during the incubation was measured by ELISA. Aliquots (10 μL) of the incubates or
angiotensin I peptide standards were transferred to 384−well immuno plates which were
previously coated with anti-angiotensin I antibody (Peninsula Laboratories) and incubated with 15
μL of 1.6 nM biotin-conjugated angiotensin I (AnaSpec) at room temperature for 1 h. After
washing the plates 5 times with wash buffer (0.05% Tween20 in PBS), 25 μL of 100 ng/ml
streptavidin-HRP (Pierce) was incubated at room temperature for 30 min. After washing, 25 μL
of substrate of HRP (Pierce) was added and chemiluminescence was detected using a microplate
28

reader. Compound 8: ELISA IC50 = 0.14 µM, Caliper IC50 = 0.47 µM. Compound 9: ELISA
IC50 = 0.032 µM, Caliper IC50 = 0.073 µM.
Human plasma renin activity (hPRA) assay
The inhibitory effect of each compound on the human plasma renin activity was tested using
the radioimmunoassay kit (SRL, Tokyo, Japan). IC50 values were calculated from concentration-
response curves with SAS software (SAS Institute Japan Ltd., Tokyo, Japan).
5-3. General procedure for X-ray crystallography of inhibitors with human renin
Crystallization of mature human renin (1-340) was carried out by the sitting drop vapor
diffusion method (Nanovolume CrystallizationTM methods22). Conditions for the reservoir
solution were 19.95%−40.95% PEG600, 100 mM citric acid buffer pH 4.5 – 6.0, or 24%−45%
PEG600, citric acid buffer pH 4.5 – 6.0, 50 mM NaH2PO4 aqueous solution. A mixture of the
reservoir solution and a solution of human renin (ca. 6 mg/mL in 25 mM Tris pH 7.9 and 150 mM
NaCl aqueous solution) was left at 20 ºC until crystals of apoprotein were generated. Crystals of
human renin in complex with inhibitors were prepared by the soaking method. The apoprotein
crystals were soaked into a soaking buffer, which was prepared by adding inhibitors to the
reservoir solution to 1 – 10 mM, for 30 min to 1 d. The crystals thus obtained were soaked into
the soaking buffer, to which was added ethylene glycol to 0 – 12%, and the mixture was frozen.
X-ray diffraction analysis was carried out by using beamline 5.0.3 at the Advanced Light Source
(ALS) and at beamline ID23B of the Advanced Photon Source (APS). The X-ray diffraction data
were processed with HKL2000.23 The structures were solved by molecular replacement using
MOLREP of CCP4 (Ver 4.0) CGP-29287. Iterative cycles of structure refinement and manual
building were done using the programs REFMAC and XFIT, respectively. Structure refinements
of the models, which were generated by Xfit24 based on the initial complex structures, were
carried out by using REFMAC.25
29