New renin inhibitors containing pseudodipeptidic units in P 3-P2 and P1-P1′ positions

Article abstract of DOI:10.1248/cpb.53.1305

A series of four non-peptidic renin inhibitors have been designed and synthesized. All of them contain in their molecule (3S,4S)-4-amino-5-cyclohexyl- 3-hydroxypentanoic acid (ACHPA), a hydrophobic portion at the C-terminus and a second dipeptide-like tra

Full text of DOI:10.1248/cpb.53.1305

October 2005
Notes
Chem. Pharm. Bull. 53(10) 1305—1309 (2005)
1305
New Renin Inhibitors Containing Pseudodipeptidic Units in P3–P2 and
P1–P1ꢀ Positions
Ryszard PARUSZEWSKI, ,a Pawel JAWORSKI,a Magdalena BODNAR,a
*
b
b
´
Jadwiga DUDKIEWICZ-WILCZYNSKA, and Iza ROMAN
a Department of Drug Chemistry, Medical University; 02–097 Warszawa, 1 Banacha Str, Poland: and b National Institute of
Public Health; 00–725 Warszawa, 30/34 Chelmska Str, Poland. Received October 21, 2004; accepted April 12, 2005
A series of four non-peptidic renin inhibitors have been designed and synthesized. All of them contain in
their molecule (3S,4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA), a hydrophobic portion at the C-
terminus and a second dipeptide-like transition state analog or unnatural dipeptidic fragment at the N-terminus.
Inhibitory activity of the compounds was measured in vitro by high performance liquid chromatography
(HPLC). Their IC50 (M/l) values were: ꢂ10ꢀ3 (12), 1.0ꢁ10ꢀ6 (19), 4.0ꢁ10ꢀ4 (23) and 1.0ꢁ10ꢀ6 (29), respectively.
All the compounds are stable against chymotrypsin.
Key words renin inhibitor; pseudodipeptidic unit; HPLC in vitro activity determination
Renin inhibitors may be useful drugs in the treatment of piperidine, which bind to a new enzyme active site confor-
hypertension and heart failure, especially because of their mation, are very potent renin inhibitors of a new type charac-
high specificity for angiotensinogen. In the past a large num- terized by a complete non-peptidic structure.7) This confor-
ber of peptidic renin inhibitors were designed and synthe- mation suggests that aspartic proteinase active sites have la-
sized. Unfortunately, a majority of them showed poor oral tent conformational flexibility.8) The most promising are two
absorption and rapid biliary uptake. The above limitations inhibitors with good pharmacodynamic and pharmacokinetic
may result from the substrate-like structure of the synthe- properties.9) However, there must be more possibilities for
sized inhibitors. Therefore, other structures of renin in- producing effective drugs free from side effects.
hibitors were also sought.
Our intention is search for new active inhibitors with sim-
Investigation of the crystal structure of human renin re- ple structure, good bioavailability, inexpensive and easy syn-
vealed that it is composed of two b-sheet domains and the thesis. We intend to reach this purpose by coupling non-pep-
cleft between them. This cleft extends over seven or eight tidic fragments with good affinity to some regions of the sub-
residues of the substrate (S4–S3ꢀ).1—3) Studies of the confor- strate. We previously obtained several in vitro active renin
mational structure of the cleft showed that the S3–S1 subsites peptidic inhibitors.10—12) Between them are compounds with
forming a hydrophobic pocket constitute a large hydrophobic pseudodipeptidic units at P1–P1ꢀ as well at P2ꢀ–P3ꢀ positions
cavity.4,5) Other subsites (S1ꢀ–S3ꢀ) are contained in the cleft, and activity in vitro in the limits of 10ꢁ5 M/l.12) Other previ-
but their pockets are not well defined.
ously obtained inhibitors with eAhx ethylamide11) or eAhx-
On the basis of these findings, inhibitors of a new type isoamylamide13) also at the P2ꢀ–P3ꢀ positions showed in-
were designed and synthesized. The peptide fragment P3–P1 hibitory activity at a concentration of 10ꢁ5 M/l. On the other
was replaced by a moderately hydrophobic moiety enclosing hand, substitution of only an isoamylamide group at this po-
an aliphatic or alicyclic fragment showing affinity to the S1 sition produces an inactive compound.14) Substitution of the
and aromatic fragment with affinity to the S3 subsites.3) It eAhx-amide fragment appeared equally effective as a pseu-
was recently determined that respective substituents of the dodipeptidic unit, but was obtained more easily and much
aromatic portion of the molecule are able strongly to enhance more cheaply. But the presence of a pseudodipeptidic unit,
the binding affinity to renin and the selectivity of the in- ideally with an alicyclic ring, is necessary at the P1–P1ꢀ posi-
hibitor. This moiety was directly linked to the C-terminus of tion.
the transition state analog containing a hydroxyl moiety. The
All things considered, we designed a series of inhibit
analog with a cyclohexyl substituent, used in numerous in- ors enclosing three different pseudodipeptidic units at the
hibitors, seemed especially worthy of note. This type of in- P3–P2 positions. We wanted to find the most effective one
hibitor did not contain residues at positions P2ꢀ and P3ꢀ, but at this position. The units are in the successive com-
rather an amide aliphatic chain. The inhibitors obtained pounds: (3S,4S)-4-amino-5-cyclohexyl-3-hydroxypentanoic
showed high in vitro affinity for renin and good bioavailabil- acid (ACHPA), (3S,4S)-4-amino-3-hydroxy-5-phenylpenta-
ity, and they were effective in lowering blood pressure in pri- noic acid (AHPPA), (3S,4S)-4-amino-3-hydroxy-6-methyl-
mates.
heptanoic acid (statine, Sta) and unnatural dipeptide Phe(4-
Aliskiren seems a very promising of new type com- OMe)-MeLeu. All of them contain at the P1–P1ꢀ positions
pound.6) It is a highly potent and selective non-peptidic renin ACHPA, whose hydroxyl group is presumed to be bound to
inhibitor in vitro and in vivo. Indeed, it contains only one two Asp residues. Chains of all four inhibitors are lengthened
peptide bond and is deprived of the P4–P1 fragment charac- at the C-terminus by linkage of e-aminohexanoic acid
teristic of the peptide inhibitors. Inhibiting of renin activity is (eAhx) isoamylamide (Iaa).
due to hydrogen binding of the hydroxyl groups of the transi-
Chemistry The inhibitors (12, 19, 23, 29) as well as
tion state analog to the catalytically active Asp 32 and Asp their intermediates were synthesized as shown in Schemata
215 residues of renin.3) Also, derivatives of 3,4-substituted 1—4 (Fig. 2). Their structures are shown in Fig. 1. General
∗ To whom correspondence should be addressed. e-mail: rpbw@farm.amwaw.edu.pl
© 2005 Pharmaceutical Society of Japan

1306
Vol. 53, No. 10
methods are given in the experimental section. Physicochem-
ical and analytical data of the newly synthesized compounds
(7, 11, 12, 15, 18, 19, 22, 23, 26, 28, 29) are presented in Ta-
bles 2 and 3. Properties of derivatives (5, 6, 9, 10, 13, 16, 17,
20, 21, 25, 27) obtained by removal of the substituent block-
ing functional groups of the parent compounds (N-tert-Boc
or ester) are not given. Data of the other synthesized com-
pounds are consistent with those described elsewhere (3, 8,10)
4,15) 14,11) 2416)).
Biochemical Assays. Determination of Inhibitor Stabil-
ity Stability was determined as described earlier12) against
chymotrypsin in a solution of ammonium carbonate (pH 6.9)
after incubation for 4 h at 37 °C. Analysis of incubates was
carried out by HPLC. The results of the experiments are
Table 1. Stability and Activity of the Synthesized Compounds
Stability t1/2 min
(chymotrypsin)
Compound
IC50 (M/l)
12
19
23
29
Stablea)
Stablea)
Stablea)
Stablea)
ꢂ10
1.0ꢃ10ꢁ6
ꢂ10ꢁ3
4.0ꢃ10ꢁ4
1.0ꢃ10ꢁ6
Boc-Phe-His-OMe
Boc-Val-Tyr-OMe
ꢂ10
Fig. 1. Structures of the Synthesized Renin Inhibitors
a) Stable means that no detectable degradation was found at 4 h.
Fig. 2. Schemata of the Synthesized Renin Inhibitors

October 2005
1307
determination is described in the previous paper.19) The activ-
ity is designed in terms of IC50, which is the molecular con-
centration of the tested inhibitor causing 50% inhibition of
the control renin activity. The results are given in Table 1.
given in Table 1.
Determination of Inhibition of Renin Activity Renin-
inhibiting activity of the compounds was determined in vitro
according to the method of Galen et al.17) with modification
consisting of a change from a spectrofluorimetric to chro-
matographic (HPLC)18) method of determination of the Leu-
Val-Tyr-Ser released from N-acetyl-Asp-Arg-Val-Tyr-Ile-His-
Pro-Phe-His-Leu-Leu-Val-Tyr-Ser effected by the action of
renin in the presence of the inhibitor tested. The course of
Results and Discussion
So far, activity of the synthesized inhibitors have been
tested in vitro, which it is not a expensive procedure. Our
modification18) of the method of Galen et al.17) allows for
Table 2. Physicochemical and Analytical Data of the Synthesized Compounds
Formula
Yield
(%)
mp
(°C)
[a]D20
(c, MeOH)
TLC, Rf H HPLC
Solv. syst. % purity
No
Compound
Log P
MW
7. Boc-Phe(4-OMe)-MeLeu-(SS)-ACHPA-OEt
11. Boc-Phe(4-OMe)-MeLeu-(SS)-ACHPA-eAhx-Iaa
12. HCl. Phe(4-OMe)-MeLeu-(SS))-ACHPA-eAhx-Iaa
15. Boc-(SS)-ACHPA-eAhx-Iaa
C36H58O8N3
660.84
C44H75O8N5
802.08
C39H68O6N5Cl
738.42
C27H51O5N3
497.70
C38H64O7N4
688.92
C33H57O5N4Cl
625.27
C38H70O7N4
625.27
C33H63O5N4Cl
631.31
74
71
85
73
79
78
70
67
54
69
72
45—46
About 58
61—64
ꢁ35.4 (1.0)
ꢁ25.7 (1.4)
ꢁ20.0 (1.2)
ꢁ35.0 (1.2)
ꢁ36.8 (1.3)
ꢁ26.0 (1.0)
ꢁ40.0 (1.6)
ꢁ23.6 (1.1)
ꢁ75.0 (1.2)
ꢁ38.0 (1.0)
ꢁ28.0 (1.2)
0.42 A
0.32 B
0.35 B
0.38 B
0.22 B
0.50 B
0.24 B
0.48 B
0.46 A
0.25 B
0.48 B
n.d.
n.d.
100
n.d.
n.d.
100
n.d.
100
n.d.
n.d.
100
—
—
5.55
—
Semi solid
60—62
18. Boc-(SS)-AHPPA-(SS)-ACHPA-eAhx-Iaa
19. HCl. (SS)-AHPPA-(SS)-ACHPA-eAhx-Iaa
22. Boc-(SS)-ACHPA-(SS)-ACHPA-eAhx-Iaa
23. HCl. (SS)-ACHPA-(SS)-ACHPA-eAhx-Iaa
26. Boc-(SS)-Sta-(SS)-ACHPA-OEt
—
86—90
4.39
—
55—58
89—94
4.33
—
C26H48O7N2
500.66
C35H66O7N4
654.91
C30H59O5N4Cl
591.25
Semi solid
53—56
28. Boc-(SS)-Sta-(SS)-ACHPA-eAhx-Iaa
29. HCl. (SS)-Sta-(SS)-ACHPA-eAhx-Iaa
—
71—74
3.65
(A)ꢄCHCl3 : MeOH 98 : 2, (B)ꢄCHCl3 : MeOH 80 : 20. The elemental analyses were within ꢅ0.4% of the theoretic value.
Table 3. 1H-NMR Spectra of the Synthesized Compounds
Compound
Chemical shifts d (ppm)
7
(CDCl3): 0.81—0.89 (m, 6H, 2ꢃCH3), 1.16 (t, Jꢄ9.4 Hz, 3H, –O–CH2–CH3), 1.24—1.68 (m, 12H, 6ꢃCH2), 1.40 (s, 9H, C4H9), 2.76
(s, 3H, N–CH3), 3.78 (d, Jꢄ5.7 Hz, 3H, –O–CH3), 4.16 (q, Jꢄ6.5 Hz, 2H, –O–CH2–CH3), 5.97 (d, Jꢄ9.3 Hz, 1H, NH), 6.85, 7.11 (dd,
Jꢄ9 Hz, 9 Hz, 4H, C6H4), 7.43 (d, Jꢄ9.3 Hz, 1H, NH).
11
(CDCl3): 0.80—0.92 (m, 12H, 4ꢃCH3), 1.16—1.80 (m, 21H, 10ꢃCH2, CH(CH3)2), 1.40 (s, 9H, C4H9), 2.14 (t, Jꢄ6.7 Hz, 2H, CH2),
2.84 (s, 3H, N–CH3), 3.23—3.28 (m, 4H, 2ꢃCH2), 3.78 (d, Jꢄ3.9 Hz, 3H, –O–CH3), 6.00 (d, Jꢄ9.3 Hz, 1H, NH), 6.50 (br s, 1H,
NH), 6.70 (br s, 1H, NH), 6.86, 7.13 (dd, Jꢄ9.4 Hz, 9.4 Hz, 4H, C6H4), 7.49 (d, Jꢄ9.3 Hz, 1H, NH).
(CD3OD): 0.90—1.00 (m, 12H, 4ꢃCH3), 1.20—1.90 (m, 21H, 10ꢃCH2, CH(CH3)2), 2.22 (t, Jꢄ7.1 Hz, 2H, CH2), 3.00 (s, 3H,
N–CH3), 3.18—3.22 (m, 4H, 2ꢃCH2), 3.79 (s, 3H, –O–CH3), 6.92, 7.25 (dd, Jꢄ9.5 Hz, 9.5 Hz, 4H, C6H4).
(CDCl3): 0.91 (d, Jꢄ5.8 Hz, 6H, 2ꢃCH3), 1.13—1.62 (m, 21H, 10ꢃCH2, CH(CH3)2), 1.44 (s, 9H, C4H9), 2.20 (t, Jꢄ7.0 Hz, 2H, CH2),
3.25—3.28 (m, 4H, 2ꢃCH2), 4.82 (d, Jꢄ8.5 Hz, 1H, NH), 5.67 (br s, 1H, NH), 6.52 (br s, 1H, NH).
(CDCl3): 0.91 (d, Jꢄ7.3 Hz, 6H, 2ꢃCH3), 1.16—1.58 (m, 21H, 10ꢃCH2, CH(CH3)2), 1.39 (s, 9H, C4H9), 2.15 (t, Jꢄ7.1 Hz, 2H, CH2),
3.22—3.26 (m, 4H, 2ꢃCH2), 5.02 (d, Jꢄ10.1 Hz, 1H, NH), 5.53 (br s, 1H, NH), 6.08 (d, Jꢄ10.0 Hz, 1H, NH), 6.46 (br s, 1H, NH),
7.23 (s, 5H, C6H5).
12
15
18
19
22
23
26
(CD3OD): 0.90 (d, Jꢄ6.0 Hz, 6H, 2ꢃCH3), 1.21—1.61 (m, 21H, 10ꢃCH2, CH(CH3)2), 2.16 (t, Jꢄ7.3 Hz, 2H, CH2), 3.16—3.20 (m,
4H, 2ꢃCH2), 7.26 (br s, 5H, C6H5).
(CDCl3): 0.91 (d, Jꢄ7.0 Hz, 6H, 2ꢃCH3), 1.11—1.58 (m, 33H, 16ꢃCH2, CH(CH3)2), 1.41 (s, 9H, C4H9), 2.15 (t, Jꢄ6.5 Hz, 2H, CH2),
3.21—3.29 (m, 4H, 2ꢃCH2), 4.81 (d, Jꢄ10.0 Hz, 1H, NH), 5.61 (br s, 1H, NH), 6.22 (d, Jꢄ8.5 Hz, 1H, NH), 6.71 (br s, 1H, NH).
(CD3OD): 0.90 (br s, 6H, 2ꢃCH3), 1.15—1.63 (m, 33H, 16ꢃCH2, CH(CH3)2), 2.17 (t, Jꢄ6.5 Hz, 2H, CH2), 3.16—3.30 (m, 4H,
2ꢃCH2).
(CDCl3): 0.92 (d, Jꢄ5.4 Hz, 6H, 2ꢃCH3), 1.26 (t, Jꢄ7.2 Hz, 3H, –O–CH2–CH3), 1.17—1.80 (m, 16H, 7ꢃCH2, 2ꢃCH(CH3)2), 1.44
(s, 9H, C4H9), 2.15 (t, Jꢄ6.9 Hz, 2H, CH2), 3.52—3.30 (m, 4H, 2ꢃCH2), 4.15 (q, Jꢄ6.8 Hz, 2H, –O–CH2–CH3), 4.85 (d, Jꢄ9.7 Hz,
1H, NH), 6.40 (d, Jꢄ9.7 Hz, 1H, NH).
28
29
(CDCl3): 0.91 (br s, 12H, 4ꢃCH3), 1.15—1.55 (m, 24H, 11ꢃCH2, 2ꢃCH(CH3)2), 1.43 (s, 9H, C4H9), 2.14 (t, Jꢄ6.7, 2H, CH2),
3.22—3.28 (m, 4H, 2ꢃCH2), 4.88 (d, Jꢄ8.6 Hz, 1H, NH), 5.73 (br s, 1H, NH), 6.36 (d, Jꢄ8.6 Hz, 1H, NH), 6.89 (br s, 1H, NH).
(CD3OD): 0.90 (d, Jꢄ5.8 Hz, 12H, 4ꢃCH3), 1.20—1.80 (m, 24H, 11ꢃCH2, 2ꢃCH(CH3)2), 2.22 (t, Jꢄ6.5 Hz, 2H, CH2), 3.23—3.26
(m, 4H, 2ꢃCH2).

1308
Vol. 53, No. 10
acetyl-tetradecapeptide was performed using the Shimadzu apparatus
equipped with two LC-10AT vp pumps, an automatic sampler SIL-10 AD
vp, detector UV-VIS SPD-10 A and controller/recorder SCL-10 A. The
peaks were recorded at 210 nm, and the following gradient systems—A:
0.01 C ammonium acetate, B: acetonitrile ꢆ1.5 ml acetic acid/1 l (0—
very precise determination. In vivo assay of the inhibitory ac-
tivity of the most promising compounds will be performed at
the end of the entire research project. Four obtained in-
hibitors are stable against chymotrypsin. Their molecular
weights are not too high (550—590 g/mol as free base, ex- 12 min. 10—60% B) at 25 °C were used.
Introduction of the N-tert-Boc Group This group was introduced by
cept for 12 which is about 700), and lipophilicity is moderate
(Log P values in the range of 3.65—5.55). The inhibitors
containing two pseudodipeptidic units have one peptide bond
between them and another one linking the unnatural eAhx.
An additional peptide bond couples the hydrophobic isoamy-
lamide (Iaa) substituent with the basic structure of the com-
pounds. These mentioned features seems to favor the activity
of the designed inhibitors. The rather high activity of com-
pound 29 (with eAhx-Iaa fragment at the C-terminus) sug-
gests that this linear hydrophobic portion of the inhibitor
prefers (SS)-ACHPA but not (SS)-Sta at the active site of
renin inhibitor. Therefore, eAhx-Iaa fragment appears to be
very important for the action. The potent activity of com-
pound 29 and rather poor activity of 19 and 23 shows that the
aliphatic chain of Sta at the N-terminal fragment of com-
pound 29 assures the desired effect, in contrast with the ali-
cyclic ACHPA (compound 23), and especially the aromatic
AHPPA (compound 19) rings. This probably results from a
higher affinity of the aliphatic portion to the S2 subpocket, in
spite of a strong preference of aromatic residues to the S3
subpocket. This hypothesis seems to be confirmed by the
presence of the 7(S)-isopropyl group in the structure of
Aliskiren, whose position suggests affinity to the S2 sub-
pocket.6) Also, the micromolar activity of compound 12 with
MeLeu in the P2 position supports our reasoning. It is a mat-
ter of debate whether the pseudodipeptide Sta could be sub-
stituted with a simpler (and cheaper) aliphatic fragment to
produce potent nonpeptidic inhibitors with improved
bioavailability. One conclusion from our investigation is that
it is possible to obtain with good yield a moderately active
renin inhibitor of a simple structure. The search for such
compound, but with higher potency, is under way.
the Schwyzer et al. method22) using Boc-azide.
Removal of the N-tert-Boc Group Boc-amino acid or Boc-peptide
(1 mmol) in a solution of 4 M HCl in dioxane (3—5 ml) was stirred at room
temperature for 30 min. The solution was concentrated in vacuo, then the
residue was re-evaporated twice with ethyl ether and dried in vacuo.
Esterification Reaction Boc-amino aids were esterified with CH3I as
described earlier.13) Boc-ACHPPA-OEt, Boc-AHPPA-OEt and Boc-Sta-OEt
were formed from mono-ethyl malonate used to prepare these compounds.16)
Alkaline Hydrolysis of Ester Group Hydrolysis was carried out as de-
scribed earlier.13)
Coupling Reaction with DCCI/HOBt The coupling was performed by
fragment condensation as shown in schemata 1—4, performed in a com-
monly used manner described earlier.11,12)
Acknowledgement This investigation was supported in part by the War-
saw Medical University (Grant FW-22/W/02-04).
References
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Experimental
The amino acids and reagents were purchased from Aldrich. Boc-
ACHPA-OEt, Boc-AHPPA-OEt and Boc-Sta-OEt were synthesized accord-
ing to the protocol of Maibaum et al.,16) Phe(4-OMe) using the method of
Behr’s et al.20) and Boc-MeLeu using that of Cheung et al.21) Renin from
porcine kidney, N-acetyl renin substrate tetradecapeptide and chymotrypsin
type I S from bovine pancreas were obtained from Sigma. Solvents were of
analytical purity. THF was distilled from Na/benzophenone under N2.
Dichloromethane and DMF were dried over 4 Å molecular sieves. The in-
hibitors were synthesized by the N,Nꢀ-dicyclohexylcarbodiimide/1-hydroxy-
benzotriazole (DCCI/HOBt) method of fragment condensation in solution.
All synthesized compounds were separated and purified by column chro-
matography (CC) on silica gel (Merck, grade 230 to 400 mesh). TLC was
carried out on 0.25 mm thickness silica gel plates (Merck, Kieselgel 60 F-
254). The solvents systems used in TLC and CC were CHCl3/MeOH in vari-
ous ratios. The spots were visualized with 0.3% ninhydrin in EtOH/AcOH
(97 : 3). Elemental analyses were performed on a Perkin-Elmer Micro-
10) Paruszewski R., Jaworski P., Tautt J., Dudkiewicz J., Boll. Chim.
Farm., 5, 301—308 (1994).
1
analyser. Melting points were determined in a Böetius apparatus. H-NMR
spectra were recorded on a Bruker DM 400 MHz spectrometer. Chemical 11) Paruszewski R., Jaworski P., Winiecka I., Tautt J., Dudkiewicz J.,
shifts were measured as d units (ppm) relative to tetramethylsilane. Optical
Pharmazie, 54, 102—106 (1999).
rotations were measured at the Na-D line with a Polamat (Carl Zeiss, Jena) 12) Paruszewski R., Jaworski P.,Winiecka I., Tautt J., Dudkiewicz J.,
polarimeter in a 5 cm polarimeter cell. HPLC analyses of the inhibitors were
performed on an apparatus equipped with a pump (Techma-Robot, Warsaw),
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