Discovery of 6-ethyl-2,4-diaminopyrimidine-based small molecule renin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.04.052

Novel 2,4-diaminopyrimidine-based small molecule renin inhibitors are disclosed. Through high throughput screening, parallel synthesis, X-ray crystallography, and structure based drug design, we have developed the first non-chiral, non-peptidic, small mol

Full text of DOI:10.1016/j.bmcl.2007.04.052

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3575–3580
Discovery of 6-ethyl-2,4-diaminopyrimidine-based small
molecule renin inhibitors
Daniel D. Holsworth,a,* Mehran Jalaie,a Thomas Belliotti,a Cuiman Cai,a Wendy Collard,a
Suzie Ferreira,a Noel A. Powell,a Michael Stier,a Erli Zhang,a Pat McConnell,a
Igor Mochalkin,a Michael J. Ryan,a John Bryant,a Tingsheng Li,b Aparna Kasani,b
Rajendra Subedi,b Samarendra N. Maitib and Jeremy J. Edmundsa,
aPfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
bNaeja Pharmaceuticals, Inc., Edmonton, Alta., Canada T6E 5V2
Received 16 March 2007; revised 17 April 2007; accepted 18 April 2007
Available online 25 April 2007
Abstract—Novel 2,4-diaminopyrimidine-based small molecule renin inhibitors are disclosed. Through high throughput screening,
parallel synthesis, X-ray crystallography, and structure based drug design, we have developed the first non-chiral, non-peptidic,
small molecular template to possess moderate potency against renin. The designed compounds consist of a novel 6-ethyl-5-
(1,2,3,4-tetrahydroquinolin-7-yl)pyrimidine-2,4-diamine ring system that exhibit moderate potency (IC50: 91–650 nM) against renin
while remaining ‘Rule-of-five’ compliant.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Hypertension is a leading risk factor for cardiovascular
disease, such as congestive heart failure, stroke, myocar-
dial infarction, and is the leading cause of death in the
western world. It is estimated that there are approxi-
mately 1 billion people worldwide that suffer from high
blood pressure. Despite the current therapies available,
approximately 70% of those people with hypertension
do not reach their target blood pressure levels. In fact,
many patients must take three or more medicines to
control their blood pressure, yet some do not respond
fully to a combination of treatments. Therefore, oppor-
tunities remain for improved treatment options.1
cleavage of angiotensinogen by renin is the rate determin-
ing step in the RAS, it is of general consensus that inhibi-
tion of renin would be the optimal strategy for the control
of hypertension.2 Renin inhibitors not only should have
superior blood pressure reducing effects and thus, exhibit
optimal end organ protection, but also should diminish
‘dry cough’, a reflex demonstrated in some patients who
take ACE inhibitors.3a–c
Attempts to inhibit renin in the early 1980s centered on
high molecular weight transition state mimetics based
on the angiotensinogen backbone. These peptidomimet-
ic inhibitors suffered from poor PK properties such as
low oral bioavailability, short duration of action, and/
or cost of synthesis and all were eventually discontinued.
The rationale for the poor PK properties of the
peptidomimetic based inhibitors was their large size
(600–700 Da) and flexibility (>15 rotatable bonds). Fur-
thermore, cost of goods became an issue as all the com-
pounds contained multiple chiral centers, and required
lengthy and inefficient synthesis. These observations
led many to believe that renin was an ‘undruggable’
target since, at the time, only large flexible molecules
seemed to inhibit renin at nanomolar levels.4
The renin–angiotensin system (RAS) is well established as
an endocrine system involved in blood pressure (BP) and
fluid electrolyte balance. Renin, an aspartyl protease,
cleaves angiotensinogen to produce angiotensin I, which
is further converted by angiotensin converting enzyme
(ACE) to the potent vasoconstrictor, angiotensin II. Since
angiotensinogen is the only substrate known for renin and
Keywords: Renin inhibitor; Hypertension; 2,4-Diaminopyrimidine;
Structure based drug design; Renin–angiotensin system; RAS;
Conformational overlap; Scaffold hopping.
*
Corresponding author. Tel.: +1 734 622 1344; fax: +1 734 622
3909; e-mail: Daniel.Holsworth@pfizer.com
Present address: Abbott Labs, Worcester, MA 01605, USA.
In April 2006, Novartis Pharmaceuticals filed the first
NDA for an inhibitor of renin for the treatment of
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.052

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D. D. Holsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3575–3580
hypertension. Tekturna (Aliskerin) was approved on
March 6, 2007 and is the first of a new class of antihy-
pertensive drugs to enter the market in more than a
decade.3d Based on the positive results exhibited by
Tekturna, interest in renin as a viable drug target for
the control of hypertension has intensified.4 Herein, we
report our recent efforts in the discovery of novel small
molecule renin inhibitors for the treatment of
hypertension.
amino)phenyl) moiety occupies a portion of the large
S3 pocket and extends into the S4 pocket, toward
solvent.
Since the 2,4-diaminopyrimidine moiety can form such
an effective network of hydrogen bonds to the renin pro-
tein, it was regarded as the ‘recognition unit’ for binding
and was not further manipulated. In order to modify the
5-(4-(3,5-difluorobenzylamino)phenyl) structure to
potentially create more active analogs, the binding
modes of 2 and 4 to the renin protein were compared
(Fig. 2). The conformations of 2 (determined by dock-
ing)8 and 4 (determined by X-ray crystallography) were
overlapped. This overlap analysis revealed an opportu-
nity to further modify the 5-(4-(3,5-difluorobenzylami-
no)phenyl) section of the pyrimidine analog (Fig. 2).
We have described our efforts to develop non-peptidom-
imetic renin inhibitors based on 1, a trans,trans-4-arylpi-
peridine based scaffold (Scheme 1).5 These efforts
focused on replacing the piperidine C4 chiral center with
a nitrogen atom to achieve novel chemical matter, and
culminated in 2.6 Although 2 lowered blood pressure
in the double transgenic mouse when dosed orally,6a
the ketopiperazine series suffered from cytochrome
P450 3A4 inhibition and exhibited a poor PK profile.
In the ketopiperazine series, sub-nanomolar potency
had been achieved by extending a H-bond acceptor moi-
ety (methoxy ether or amide carbonyl) into the S3 sub-
pocket (S3sp).6f Conformational overlap analysis
revealed that replacing the 5-(4-(3,5-difluorobenzylami-
no)phenyl) section of 4 with either an N-3-methoxypro-
pyl-1,2,3,4-tetrahydro-quinoline ring or an N-ethyl
acetamide-1,2,3,4-tetrahydro-quinoline ring, the proper
vector orientation to place the side chains into the S3sp
Recently, we identified 3 (IC50 = 27,000 nM, MW =
387 Da) as a novel small molecule lead from a high
throughput screen (HTS) (Scheme 2). In the absence of
a crystal structure, parallel chemistry utilizing a reductive
amination reaction between aldehydes and 6-ethyl-5-(40-
amino-phenyl)-pyrimidine-2,4-diamine7
pounds) was conducted to further optimize the template.
(450
com-
This exercise led to the identification of 4 (IC50
4000 nM).
=
NH2
NH2
2
N
N
3
Compound 4 was 7-fold more potent than 3 and had a
molecular weight 32 Da less (MW = 355 Da) than the
original HTS lead compound. The crystal structure of
1
N
N
4
NH2
NH2
6
Parallel Chemistry
~450 analogs
5
˚
4 bound within the active site of renin (2.2 A, pdb code:
2IKO) revealed that the protein exists in the ‘flap-closed’
form (Fig. 1). Based on X-ray structure analysis, the 2,4-
diaminopyrimidine head group forms a hydrogen bond
network with various residues of renin. The 4-amino
group can interact with THR-77 and SER-76. The 2-
amino group can form bifurcated hydrogen bonds with
the side chain of ASP-215 and ASP-32. The pyrimidine
ring nitrogen at positions 1 and 3 can utilize interactions
with ASP-32 and a water solvent molecule, respectively.
The 6-ethyl substituent on the pyrimidine ring extends
into the S1 pocket, while the 5-(4-(3,5-difluorobenzyl-
HN
HN
Cl
F
F
Cl
3, IC50 = 27,000 nM
4, IC50 = 4,000 nM
Scheme 2. Optimization of 3 by parallel chemistry produced 4.
Numbering of the ring in 4 is provided for clarity.
O
NH
H
H
N
N
4
O
N
O
N
MeO
O
O
OH
MeO
O
O
O
O
OMe
OMe
1, IC50 = 0.067 nM
2, IC50 = 0.50 nM
CYP3A4 (BFC), IC50 = 82 nM
Scheme 1. Design of ketopiperazine framework based on a 4-arylpiperidine scaffold.

D. D. Holsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3575–3580
3577
could be achieved. Based on ease of synthesis, we chose
to first synthesize the N-3-methoxypropyl-1,2,3,4-
tetrahydro-quinoline analog. This modification would
result in a new template represented by compound 5
(Scheme 3).
The retrosynthetic analysis of 5 is outlined in Scheme 4.
It was envisioned that 5 could be constructed in a highly
convergent manner by employing an appropriately func-
tionalized boronate ester (7) and 5-bromo-6-ethyl-2,4-
diaminopyrimidine9 (6) via a Suzuki reaction (Scheme
4).
The synthesis of boronate ester 7 commenced with the
alkylation of 7-benzyloxy-1,2,3,4-tetrahydroquinoline 8
with 3-bromo-methoxypropane in 96% yield (Scheme
5). Next, de-benzylation of 9 was accomplished using
trifluoroacetic acid at reflux, to provide the phenol 10
in high yield. The resultant phenol was exposed to triflic
anhydride at ꢀ78 ꢁC for 10 min to afford triflate 11. Fi-
nally, palladium catalyzed borylation of 11 using
bis(pinacolatodiboron) provided 7.
Figure 1. X-ray co-crystal structure of 4 (yellow) bound within the
active site of the renin protein. ASP-32 and 215 of the renin protein are
shown forming a potential network of hydrogen bonds with the 2,4-
diaminopyrimidine moiety of 4. The protein surface has been colored
by atom type (red, O; blue, N; green, C).
Compound 5 was obtained by reacting 6 and 7 in the
presence of tetrakis(triphenylphosphine) palladium (0)
and sodium carbonate in a de-gassed solvent system
comprising of water/ethanol/toluene (1:1:1).
We were delighted to find that 5 (IC50 = 650 nM)
exhibited a 6-fold increase in potency against renin,
as compared to 4, while remaining ‘Rule-of-five’
compliant.10 X-ray crystallography (2.2 A, pdb code:
˚
2G21) revealed that 5 bound in the renin active site,
as anticipated, in the flap closed form, with the
3-methoxypropyl side-chain extending into the S3sp
and potentially making a hydrogen bond interaction
with the backbone of TYR-14 (Fig. 3).
Compound 5 exhibited low molecular weight (341 Da),
permeability
excellent
solubility
(59.3 lg/mL),
Figure 2. Conformation overlap of 4 (yellow) and 2 (green), bound in
the renin active site, as determined by X-ray crystallography (4) and
docking (2). ASP-32 and 215 of the renin protein are shown forming a
potential network of hydrogen bonds with the 2,4-diaminopyrimidine
moiety of 4 and ketopiperazine ring of 2. The protein has been
removed for clarity.
(26.5 · 10ꢀ6 cm/s), low clogP (3.34), and low Cyto-
chrome P450 3A4 inhibition (IC50 = 2.8 lM). Further-
more, compound
5
displayed moderate in-vivo
elimination half-life (1.11 h), systemic plasma clearance
(36.8 mL/min/kg), and volume of distribution
NH2
O
NH2
N
N
NH
H
N
N
N
NH2
Structure Based
Drug Design
NH2
N
O
N
O
N
+
HN
O
O
O
F
F
OMe
5
2
4
Scheme 3. Scaffold hopping of lead structure 4 to the tetrahydroquinoline framework 5 via structure based drug design and conformational overlap
analysis.

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D. D. Holsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3575–3580
NH2
N
O
O
NH2
N
N
B
N
NH2
N
NH2
N
O
Br
O
7
6
5
Scheme 4. Retrosynthetic approach to compound 5.
MeO
MeO
H
BnO
N
BnO
N
HO
N
c
b
a
8
9
10
MeO
NH2
MeO
MeO
H2N
N
O
N
B
N
N
TfO
N
O
d
e
5
11
7
Scheme 5. Reactions and conditions for synthesis of boronate ester 7. (a) 3-bromo-methoxy propane (2 equiv), sodium carbonate (2 equiv),
potassium iodide (0.15 equiv), acetonitrile, reflux, 74 h, 96%; (b) trifluoroacetic acid, reflux, 1 h, 99%; (c) triflic anhydride (1.5 equiv), triethylamine
(8 equiv), ꢀ78 ꢁC, 10 min, 74%; (d) bis(pinacolatodiboron) (1.1 equiv), potassium acetate (3 equiv), 1,10-bis(diphenylphosphino)ferrocine palladium
(II) chloride (0.2 equiv), dioxane, reflux, 18 h, 90%; (e) 5-bromo-6-ethyl pyrimidine 2,4-diamine (6)8 (1 equiv), Pd(PPh3)4 (0.10 equiv), Na2CO3
(3 equiv), toluene/ethanol/water (1:1:1), reflux 18 h, 32%.
(1.6 L/kg), but exhibited low oral bioavailability (<10%)
in Sprague–Dawley rats. Overall, 5 represented a novel,
non-peptidic small molecule renin inhibitor and served
as an excellent starting point for further investigation.
Next, the introduction of other side chains containing
hydrogen bond acceptors that could possibly penetrate
into the S3sp was evaluated (Table 1. Compounds 12–
15).
Compounds that contained ester side chains (12, 13, 14)
were installed to determine optimal hydrogen bond
acceptor location. The activity of the compounds in-
creased as the side-chain length increased. We chose to
limit the length of the side chain to five carbons (14,
IC50: 91 nM), in order to keep the molecular weight
low. Compound 14 was 7-fold more active than 5, with
only a 42 mass unit (dalton) increase. Since 12, 13, and
14 had metabolic liabilities (Human Liver Microsomal
Figure 3. X-ray co-crystal structures of 5 (green) and 4 (yellow), bound
(HLM) half-life of 12, 13, 14 = 5 min), 15 was designed.
in the active site of renin. The 3-methoxypropyl side chain of 5
The side chain of 15 contains a hydrogen bond donor
penetrates into the S3sp of renin. TYR-14 of the renin protein can form
and acceptor and was a key side chain in the ketopiper-
azine series (2).6f Compound 15 demonstrated an in-
a hydrogen bond with the oxygen of the side chain of 5. ASP-32 and
215 of the renin protein are shown forming a potential network of
crease in potency (3.5-fold) relative to 5 with a
molecular weight of 354 Da and was stable in HLM
up to 40 min.11 Compounds 5, 12–15 were also devoid
hydrogen bonds with the 2,4-diaminopyrimidine moiety of 5 and 4.
The protein surface has been colored by atom type (red, O; blue, N;
green, C).

D. D. Holsworth et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3575–3580
3579
Table 1. Brief SAR of compound 5
NH2
N
N
NH2
R
N
Entry no.
R
IC50 (nM)
650
MW
CYP3A4 (BFC) IC50 (nM)
2800
clogP
5
341
3.48
*
O
O
12
198
355
10,000
3.33
*
OMe
OMe
*
*
13
14
120
91
369
383
354
Not determined
12,000
3.65
3.80
2.36
O
O
OMe
O
H
N
15
178
8000
*
*
point of attachment.
2. (a) Stanton, A. JRAAS 2003, 4, 6; (b) Rosenberg, S. H.;
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of activity against the aspartic peptidases Cathepsin D,
Cathepsin E, and Pepsin (0–5% inhibition at 100 lM).
The identification of non-peptidic small molecule renin
inhibitors has historically been a major challenge.
Through high throughput screening, parallel synthesis,
X-ray crystallography, and structure based drug de-
sign we have developed the first non-chiral, non-pep-
tidic, small molecular template to possess moderate
potency against renin while remaining ‘Rule-of-five’
compliant.9 These novel compounds bind to renin in
the ‘flap closed’ conformation. The 6-ethyl-2,4-diami-
nopyrimidine moiety serves as the recognition unit
by interacting with the catalytic Aspartic acids 32
and 215 within the active site of renin. Further, the
compounds take advantage of the S3sp of renin for
added potency.6,12 Future efforts are to optimize po-
tency and pharmacokinetic/dynamic parameters with
this novel scaffold.
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A.; Sanwald-Ducray, P.; Scalone, M.; Schleimer, M.;
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Acknowledgments
The authors thank Diane Alessi, Jacqueline Day, Jim
Dyer, and Jeanne Hagadorn their efforts in supplying re-
nin protein.
References and notes
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