New renin inhibitors with pseudodipeptidic units in P1-P 1′ and P2′-P3′ positions

Article abstract of DOI:10.1248/cpb.50.850

A series of four new potential renin inhibitors has been synthesized. The structure of the compounds was designed in such a way as to produce agents resistant to enzymatic degradation, metabolically stable, possibly potent and with improved oral absorptio

Full text of DOI:10.1248/cpb.50.850

850
Notes
Chem. Pharm. Bull. 50(6) 850—853 (2002)
Vol. 50, No. 6
New Renin Inhibitors with Pseudodipeptidic Units in P1–P1؅ and P2؅–P3؅
Positions
,a
a
a
b
⁄
*
Ryszard PARUSZEWSKI,
Jadwiga DUDKIEWICZ
Pawel JAWORSKI, Iwona WINIECKA, Jadwiga TAUTT, and
b
a Department of Drug Chemistry, Medical University; 02–097 Warszawa, Poland: and b Institute for Drug Research and
Control; 00–725 Warszawa, Poland. Received December 17, 2001; accepted March 25, 2002
A series of four new potential renin inhibitors has been synthesized. The structure of the compounds was
designed in such a way as to produce agents resistant to enzymatic degradation, metabolically stable, possibly po-
tent and with improved oral absorption. All positions of the 8—13 fragment of the human angiotensinogen were
occupied by unnatural units (two unnatural amino acids in positions P3 and P2 and two pseudodipeptides in posi-
tions P1–P1؅ and P2؅–P3؅). Both N- and C-terminal functions of the inhibitors were blocked with tert-Boc and ethyl
ester groups. Their hydrophobicity evaluated as a log P value, calculated by a computer method, was 6.57 and
6.08 respectively. All peptides were obtained by the carbodiimide method in solution and purified by chromatog-
raphy on the SiO2 column. Their resistance to enzymatic degradation was assayed by determination of stability
against chymotrypsin activity. The potency was measured in vitro by a spectrofluorimetric method (assay of Leu-
Val-Tyr-Ser released from the N-acetyltetradecapeptide substrate by renin in the presence of the inhibitor). All
inhibitors were stable to chymotrypsin. Their IC50 (M/l) values were: 9.6؋10؊4 (12), 1.6؋10؊5 (17), 1.0؋10؊5 (22)
and 1.0؋10؊5 (23) respectively.
Key words renin inhibitor; pseudodipeptide unit; spectrofluorimetric determination
So far a number of potent renin inhibitors have been ob- nounced lasting to 24 h and dose dependent reduction of
tained. Some of them were found to lower blood pressure mean arterial pressur. We designed a series of four com-
after intravenous administration. However, only a moderate pounds, looking for an effective inhibitor which ought to be
activity if any was observed when they were given orally. potent, metabolically stable and active after oral administra-
The likely reason was a poor absorption across the biological tion. Introduction of unnatural Phe(4-OMe) at P3 position
barriers, rapid metabolism before and after absorption as and MeLeu or MePhe at P2 position was to protect the P3–P2
well as fast excretion with bile. Probably also the molecule and P2–P1 bonds against enzymatic degradation. Substitution
size and peptidic character has limited the oral absorption. of His with MePhe or MeLeu could improve the oral
To improve the oral bioavailability many approaches have bioavailability, because basic His side chain causes rapid bil-
been attempted. Hashimoto et al.1) examined intestinal ab- iary elimination of inhibitor.6) Change in the P2 position is
sorption of 18 inhibitors after oral administration and inter- possible because the hydrogen atom of the P3–P2 peptide
dependence of absorption upon molecular stucture of the bonding is not involved in hydrogen bonding.7) Another aim
compounds. In the result of extensive research they found of our study was to check the result of simultaneous intro-
some relationship, especially connected with solubility of the duction of aromatic or aliphatic hydrophobic residue in the
inhibitors. Samanen et al.2) looking for possibility to improve position P2 (MePhe or MeLeu) and aromatic or aliphatic, but
the oral bioavailability of the inhibitors hypothesized, that in- with hydrophilic OH group, pseudodipeptide unit in the posi-
testinal permeability is increased by decreasing the desol- tion P1–P1, [(S,S)-AHPPA: (3S,4S)-4-amino-3-hydroxy-5-
vatation energy required to transfer a peptide from aqueous phenylpentanoic acid or (SS)-Sta: (3S,4S)-4-amino-3-hy-
environment to the lipid one. They give as a example two in- droxy-6-methylheptanoic acid)]. It is well known that intro-
hibitors differing one from another by N-methyl group on duction of a pseudodipeptidic fragment at the P1–P1Ј posi-
amino function of His. Intestinal permeability of N-methyl tions may produce compounds of improved activity, stable
analog was near twice higher. However, it is not always a against the hydrolytic action of renin. Previously some active
rule. The most important courses look to be using nonpep- inhibitors with P3...P2Ј sequence had been obtained in our
tidic structures. Maibaum et al.3) obtained two series of laboratory.8,9) Because it was found, that not only the P2Ј but
dipeptide transition state isosters containing a substituted P3 also the P3Ј, position was important for a high inhibitory po-
moiety directly linked to the P1 side chain and which lack the tency,10) in the present study a series of inhibitors of pro-
P4–P2 portion. These nonpeptidic inhibitors are highly potent longed chain was designed. Replacement of Leu-Leu at the
in vitro. Some of them show long time of duration and con- P1–P1Ј and Val-Tyr at the P2Ј–P3Ј positions with the transi-
siderable lower blood pressure after oral administration. It is tion-state mimetics: statine (Sta) or with AHPPA could pre-
necessary to cite here some nonpeptidic P2–P3 butanediamide sumably improve the metabolic stability of such inhibitors.
orally active rennin inhibitors with high bioavailability dis- Renin inhibitors with a free amino- and carboxyl function
covered by Simoneau et al.4) Also Rahuel et al.5) discovered show poor activity because of their polarity and limited pene-
new group of nonpeptidic, orally active inhibitors which are tration across biological membranes. Moreover, positively
binded to the S1/S3 hydrophobic pocket and in addition to charged compounds with a free amino group, well soluble in
subpocket extended from S3 toward the hydrophobic core of the gastric juice are precipitated in the small intestine at neu-
the enzyme. The inhibitors orally administered to conscious tral pH. On the other hand, negatively charged compounds
unrestrained and Naϩ depleted marmosets produced the pro- show a low intrinsic membrane permeability. Therefore the
To whom correspondence should be addressed. e-mail: rpbw@farm.amwaw.edu.pl
© 2002 Pharmaceutical Society of Japan

June 2002
851
potentially well-absorbed agents should be uncharged. It is Data of the other synthesized compounds are consistent with
known, that substitution at both the N and C terminals is es- those described elsewhere: 2,11) 3,12) 1, 8, 13, 15,13) 4.14,15)
sential for absorption from the small intestine.1) The N-buty-
Biochemical Assays Stability of the inhibitors was de-
loxycarbonyl (Boc) group and C-ethyl ester group to block termined against chymotrypsin in a solution of ammonium
N- and C- termini of the inhibitors were used. The designed carbonate (pH 6.9) after incubation for 4 h at 37 °C. Analysis
inhibitors are supposed to penetrate well across the biological of incubates was carried out by HPLC. Activity was assayed
barriers because of their unpolar character and moderate hy- in vitro by a spectrofluorimetric determination of Leu-Val-
drophobicity. Such blocked peptides are poorly soluble. Tyr-Ser released from N-acetyl-Asp-Arg-Val-Tyr-Ile-His-
However, it is possible to some extent to improve the solubil- Pro-Phe-His-Leu-Val-Tyr-Ser under action of rennin in the
ity of the new inhibitors obtained by increasing hydrophilic- presence of the inhibitor tested. Hydrophobicity of the in-
ity as a result of introduction of the Sta or AHPPA hydroxyl hibitors was calculated as log P value by a computer method.
group into the molecule. Hydrophobicity of Boc-[(SS)-Sta]-
[(SS)-AHPPA]-OEt and Boc-[(SS)-AHPPA]-[(SS)-Sta]-OEt Conclusion
is low (log Pϭ3.94 and 3.5). As a result the hydrophobicity
of the inhibitors designed is reduced (log Pϭ6.57 and 6.08).
Four synthesized renin inhibitors are stable against chy-
motrypsin as a result of replacing natural amino acids with
Chemistry The inhibitors (12, 17, 22, 23) as well as unnatural units. Inhibitory activity of all four compounds is
their intermediates were synthesized as shown in Charts 1— moderate and very similar. This moderate activity might re-
4. General methods are given in the experimental part. sult from poor solubility during the in vitro activity determi-
Physicochemical and analytical data of the new synthesized nation. It is necessary to mention, that sometimes there is no
compounds (9, 12, 17, 20, 22, 23) are presented in Tables 1 simple relation between in vitro and in vivo activity. Similar
and 2. Properties of derivatives (6, 7, 11, 18, 19, 21) obtained activity of the inhibitors irrespective of the structure of the
by removal of the substituents (N-tert-Boc or ester) blocking unit in P1–P1Ј position (Sta or AHPPA) as well C-terminus
functional groups of the parent compounds are not given. (Sta or AHPPA in P2Ј–P3Ј position) shows that aliphatic or
Chart 1
Chart 3
Chart 4
Chart 2
Table 1. Physicochemical and Analytical Data of the Synthesized Compounds
Yield
(%)
mp
[a]D20
(c, MeOH)
TLC, Rf
(solv. syst.)a)
HPLC
Compd.
Formula
MW
(°C)
(% of purity)
9
12
17
20
22
23
Boc-[(SS)-Sta]-[(SS)-AHPPA]-OEt
C26H42N2O7
494.62
833.00
798.99
494.62
833.00
798.99
70
34
25
62
30
32
Semisolid
80—83
Ϫ56.5 (1.7)
Ϫ50.4 (1.5)
Ϫ28.2 (1.6)
Ϫ55.5 (1.8)
Ϫ44.1 (1.7)
Ϫ18.2 (1.5)
0.25 (B)
0.33 (A)
0.35 (A)
0.23 (B)
0.30 (A)
0.38 (A)
nd
100
100
nd
Boc-[Phe(4-OMe)]-MePhe-[(SS)-Sta]-[(SS)-AHPPA]-OEt
Boc-[Phe(4-OMe)]-MeLeu-[(SS)-Sta]-[(SS)-AHPPA]-OEt
Boc-[(SS)-AHPPA]-[(SS)-Sta]-OEt
C46H64N4O10
C43H66N4O10
C26H42N2O7
C46H64N4O10
80—85
Semisolid
76—78
Boc-[Phe(4-OMe)]-MePhe-[(SS)-AHPPA]-[(SS)-Sta]-OEt
Boc-[Phe(4-OMe)]-MeLeu-[(SS)-AHPPA]-[(SS)-Sta]-OEt
98
C43H66N4O10
75—79
100
The elemental analyses were withinϮ0.4% of the theoretic value. a) Aϭhexane/EtOAc 50 : 50, Bϭhexane/EtOAc 60/40.

852
Vol. 50, No. 6
Table 2. 1H-NMR Spectra of the Synthesized Compounds
Compound
Chemical shifts d (ppm), CDCl3:
9
0.91 (6H, d, Jϭ6.3 Hz), 1.20—1.30 (6H, m), 1.45 (9H, s), 3.60 (1H, q, Jϭ6.2 Hz), 3.91—4.10 (2H, m), 4.12 (2H, q, Jϭ6.9 Hz), 4.84
(1H, d, Jϭ9.4 Hz), 6.54 (1H, d, Jϭ9.4 Hz), 7.24 (5H, s).
12
17
20
22
23
0.84—0.96 (6H, m), 1.19—1.40 (15H, m), 2.93, 2.97 (3H total, s, s), 3.81 (3H, s), 4.13 (2H, q, Jϭ7.1 Hz), 6.88, 7.18 (4H total, d, d,
Jϭ8.6, 8.6 Hz), 7.26 (10H, s).
0.83—0.97 (12H, m), 1.19—1.43 (15H, m, s at 1.39 ppm), 2.75, 2.87 (3H total, s, s), 3.78 (3H, s), 4.15 (2H, q, Jϭ7.1 Hz), 6.81, 7.13
(4H total, d, d, Jϭ8.6, 8.6 Hz), 7.26 (5H, s).
0.90 (6H, d, Jϭ6.1 Hz), 1.20—1.60 (15H, m, s at 1.39 ppm), 3.72 (1H, q, Jϭ7.0 Hz), 3.90—4.08 (2H, m), 4.15 (2H, q, Jϭ7.1 Hz), 5.04
(1H, d, Jϭ8.9 Hz), 6.00 (1H, d, Jϭ8.9 Hz), 7.25 (5H, s).
0.80—0.94 (6H m), 1.18—1.44 (15H, m), 2.86, 2.91 (3H total, s, s), 3.77 (3H, s), 4.12 (2H, q, Jϭ6.9 Hz), 6.81, 7.12 (4H total, d, d,
Jϭ8.2, 8.2 Hz), 7.26 (10H, s).
0.81—0.97 (12H, m), 1.20—1.42 (15H, m), 2.77, 2.83 (3H total, s, s), 3.77 (3H, s), 4.12 (2H, q, Jϭ7.0 Hz), 6.81, 7.12 (4H total, d, d,
Jϭ8.4, 8.4 Hz), 7.26 (5H, s).
Table 3. Biochemical Properties of the Synthsized Compounds, Some In-
termediates and the Reference Compounds
(Carl Zeiss, Jena) polarimeter in a 5 cm polarimeter cell. HPLC was per-
formed on a Techma-Robot Type 302 apparatus equipped with a UV detec-
tor LCD 2040 (Laboratorni Pristroje, Praha) and a computer registrator/
recorder CHROMA (POLLAB Warsaw). The peaks were recorded at
210 nm. Spectrofluorimetric determination of Leu-Val-Tyr-Ser released from
the substrate tetradecapeptide was performed on a Shimadzu apparatus with
Fluram solution. The fluorescence was detected at 395—495 nm.
Introduction of the N-tert-Boc-Group This group was introduced in a
commonly used manner.15)
Esterification Reaction Boc-amino acids were esterified with CH3I as
described earlier.18) Boc-Sta-OEt and Boc-AHPPA-OEt were formed from
mono-ethyl malonate used to prepare these pseudodipeptide units.11)
Coupling Reaction with DCCI/HOBt The amino acid or peptide ester
hydrochloride (1 mmol) was dissolved in dichloroethane (5 ml) and neutral-
ized at 0 °C with triethylamine (TEA). Boc-amino acid or Boc-peptide
(1 mmol) and HOBt (1.5 mmol) were added followed by a solution of DCCI
(1 mmol) in dichloromertane (5 ml). The reaction mixture was stirred at 0 °C
for 4 h and left at room temperature overnight. Dicyclohexylurea (DCU) was
filtered off and the filtrate was evaporated in vacuo. The residue was dis-
solved in EtOAc, left for 2 h and filtered again. The filtrate was washed suc-
cessively with 5% HCl solution, 5% NaHCO3 solution, saturated NaCl solu-
tion, dried over anhydrous MgSO4 and concentrated in vacuo. The raw com-
pound was purified by silica gel CC to yield the final product.
Stabilitya), t1/2 min.
(chymotrypsin)
Compound
IC50 (M/l) log Pb)
[(SS)-Sta]-[(SS)-AHPPA]-OEt
[(SS)-AHPPA]-[(SS)-Sta]-OEt
12
17
22
23
—
—
—
—
2.32
1.89
6.57
6.08
6.57
6.08
—
Stable
Stable
Stable
Stable
Ͻ10
9.6ϫ10Ϫ4
1.6ϫ10Ϫ5
1.0ϫ10Ϫ5
1.0ϫ10Ϫ5
—
Boc-Phe-His-OMe
Boc-Val-Tyr-OMe
Ͻ10
—
—
a) Stable means that no detectable degradation was found at 4 h. b) log Pϭvalue
calculated by computer method.
aromatic character of this fragment has no effect on the po-
tency of the inhibitor. Activity of the truncated structure de-
prived of P2Ј–P3Ј fragment was not determined. But compar-
ing activity of the inhibitors substituted and not substituted at
this fragment obtained by Hui et al.10) is possible conclude,
that pseudodipeptidic unit unsusceptible to enzymatic degra-
dation at C terminus of our compounds could increase their
inhibitory activity. In this study inspection in vitro of the ob-
tained compounds was designed. Examination in isolated tis-
sues and in animals is planned for the most promising in-
hibitors obtained to date and just lately synthesized.
Removal of the Boc-Group Boc-amino acid or peptide (1 mmol) in so-
lution of 4 M HCl in dioxane (3—5 ml) was stirred at room temperature for
30 min. The solution was concentrated in vacuo, the residue was re-evapo-
rated twice with ethyl ether and then dried in vacuo.19)
Alkaline Hydrolysis of Amino Acid or Peptide Ester Hydrolysis was
carried out as described earlier.18)
Inhibition of Renin Activity Inhibitory renin activity was determined
according to the method of Galen et al.20) The course of determination is de-
scribed in detail in a previous paper.18) The activity is designed in terms of
IC50, i.e. the molecular concentration of the tested inhibitor causing 50% in-
hibition of the control renin activity.
Experimental
The reagents were purchased from Aldrich. Phe(4-OMe) was obtained ac-
Stability Determination Chymotrypsin (1 mg) was dissolved in 0.5 M
cording to the method of Behr.14) Boc-MePhe and Boc-MeLeu were pre- NH4HCO3 solution (2.5 ml) adjusted with AcOH to pH 6.9. The inhibitor
pared using the method of Cheung et al.16) Sta and AHPPA were synthesized
(5 mm) was dissolved in MeOH (0.3—0.5 ml) and diluted with 0.5 M
according to the Maibaum protocol.11) Renin from porcine kidney, N-acetyl NH4HCO3 solution to a volume of 2.5 ml. Both solutions were mixed and in-
renin substrate tetradecapeptide and chymotrypsin type I S from bovine pan- cubated at 37 °C for 4 h. Aliquots were withdrawn after 2 and 4 h. The reac-
creas were obtained from Sigma. Solvents were of analytical purity. THF tion in each sample was stopped by addition of CH3CN (5 ml). Each solution
was distilled from Na/bezophenone under N2. Dichloromethane and DMF
was evaporated in vacuo, the residue was dissolved in 0.5 M pH 6.9
were dried over molecular sieves (4 Å). The peptides were synthesized by NH4HCO3 solution (2.5 ml). Analyses of incubates were carried out by
the N,NЈ-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCCI/HOBt) HPLC. Peak areas of inhibitors in incubates were compared with peak areas
method of the amino acid or fragment derivatives condensation in solu- of standard solutions containing 5 mm of inhibitor dissolved in MeOH
tion.17) All synthesized compounds were separated and purified by column
(0.3—0.5 ml) and diluted with 0.5 M pH 6.9 NH4HCO3 solution to a volume
chromatography (CC) on silica gel (Merck, grade 230 to 400 mesh). TLC of 2.5 ml. The values were determined in 3 separate assays. The differences
was carried out on a 0.25 mm thickness silica gel plates (Merck, Kieselgel between the peak areas in limits Ϯ5% were recognized as a determination
60 F-254). The solvent systems used in TLC and CC were: CHCl3/MeOH or error. Stability of Boc-Phe-His-OMe and Boc-Val-Tyr-OMe was determined
hexane/EtOAc in various ratios. The spots were visualized with 0.3% ninhy- in similar way. The differences were as follows: all reagents were used in
drin in EtOH/AcOH 97 : 3. Elemental analyses were performed on a Perkin- tenfold quantities, incubation was performed for 1 h, aliquots were with-
Elmer Microanalyser. Melting points were determined in a Böetius appara- drawn at intervals of 5 min., standard solutions contained the reference com-
tus. 1H-NMR spectra were recorded on a Varian, Unity 200 or 500 spectrom- pounds dissolved in MeOH (1 ml) and diluted as above. Results of the ex-
eter. Chemical shifts were measured as d units (ppm) relative to tetramethyl- periments are given in Table 3.
silane. Optical rotations were measured at the Na-D line with a Polamat
Log P Value Calculation Log P values were calculated by a computer

June 2002
853
method because of poor solubility of the compounds in water and in n-oc-
tanol. Structures of the compounds were built within the HyperChem 4.5
programme. The semiempirical method PM 3 was used for single point cal-
culation. Geometry optimization was performed by the Polak-Ribier
method. Log P as a measure of hydrophobicity of optimized structure was
calculated using the QSAR Properties programme, Chem Plus, extension for
HyperChem (Hypercube, Inc.). Calculation of log P in this programme was
carried out using atomic parameters derived by Ghose et al.21)
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