ANGIOTENSIN I-CONVERTING ENZYME (ACE) INHIBITORS

BACKGROUND OF THE INVENTION

Angiotensin l-converting enzyme (ACE; EC 3.4.15.1) is a zinc-dipeptidyl carboxypeptidase that plays a key role in blood pressure regulation by converting the inactive decapeptide angiotensin I to the potent vasopressor octapeptide angiotensin Il by cleavage of the carboxy-terminal dipeptide. ¹ It also inactivates the vasodepressor nonapeptide bradykinin. ² ' ³ Thus, the inhibition of ACE has become a primary strategy in the treatment of hypertension, myocardical infarction, heart failure, and diabetic nephropathy. ⁴ There are a number of commecially available ACE inhibitors, including captopril, ⁵ ' ⁶ lisinopril, and enalapril, which have establised themselves in the therapy of hypertension and congestive heart failure. ⁷ ' ⁸ However, undesirable side effects such as persistant cough, loss of taste, and angioedema have been associated with current- generation ACE inhibitors. ⁹ ' ¹⁰ Therefore, the development of novel effective drugs for the treatment of hypertension and cardiovascular diseases still remains an important goal. The recent availability of the crystal structure of the testis ACE-lisinopril complex has opened the possibility of structure-guided design of second-generation, domain- selective ACE inhibitors. ¹¹

CONFIRMATION COPY

SUMMARY OF THE INVENTION

According to a first embodiment of the invention, there is provided a process for synthesising an amino acid derivative of a tripeptidomimetic, such as a tryptophan derivative of lisinopril, the process including the steps of:

peptide coupling a dipeptide, typically a carboxyl dipeptide, with a methyl ester having an amino moeity, typically a L-tryptophan methyl ester, to produce a dipeptide methyl ester ; and hydrolysing the dipeptide methyl ester to produce the tripeptidomimetic.

The dipeptide is preferably coupled to the P ₂ ' residue of the dipeptide methyl ester.

The carboxyl dipeptide may be that of lisinopril, enalaprilat, perindoprilat, cilazaprilat etc..

The dipeptide may be produced by reductively aminating a butyrate such as 2-oxo-aryl butyrate, typically ethyl 2-oxo-4-phenyl butyrate and a fe/t-butoxycarbonyl-L-amino acid such as N-ε-(ferf-butoxycarbonyl)-L-lysine.

The dipeptide methyl ester is preferably an amino methyl ester such as tryptophan or phenylalanine methyl ester.

According to a second embodiment of the invention, there is provided a compound produced by the process described above.

According to a third embodiment of the invention, there is provided a tripetidomemetic, preferably a derivative of lisinopril, incorporating a amino acid moeity , preferably tryptophan, at the P ₂ ' position.

The tryptophan derivative of lisinopril may be N-[N ⁶ -tert-butoxycarbonyl-L-lysyl-N ² - (R ₁ S)-(I -ethoxycarbonyl-3-phenylpropyl)]-L-tryptophan methyl ester or N ² -(R,S)-(1- Carbonyl-3-phenylpropyl)]-L-lysyl-L-tryptophan hydrochloride.

According to a fourth embodiment of the invention, there is provided a pharmaceutical composition including a compound described above. The compound may be for the treatment of hypertension and/or cardiovascular disease.

DETAILED DESCRIPTION OF THE INVENTION

The synthesis of lisinopril incorporating a tryptophan moiety at the P ₂ ' position is described herein.

ACE is a complex two-domain enzyme, comprising an N and a C domain, each containing an active site with similar but distinct substrate specificities and chloride- activation requirements. The N- and C-domain sites of ACE hydrolyze angiotensin 1 (Ang I) and bradykinin (BK) at comparable rates in vitro, but in vivo it appears that the C-domain is primarily responsible for regulating blood pressure. This might suggest that a C-selective inhibitor would have a profile comparable to current mixed inhibitors, but this is not necessarily the case.

First, while Ang I is hydrolyzed predominantly by the C domain in vivo, BK is hydrolyzed by both domains and therefore selective inhibition of the C-domain site will allow some level of BK degradation to continue, catalyzed by the N-domain. This could be sufficient to prevent the excessive BK accumulation that has been observed during attacks of angioedema. Second, BK potentiation by B ₂ receptor resensitization is maximal when both the N- and C-domains are inhibited, suggesting that a pure C- selective inhibitor will have a lower propensity for excessive BK stimulation. Third, the multiple Ang and non-Ang peptides known to be vaso-active are not hydrolyzed equally by the two domains, making it likely that the ratio of vasopressor to vasodilator peptides will differ between C-selective and mixed inhibitors. Thus, a highly selective C-domain inhibitor has the potential for effective blood pressure control with reduced vasodilator- related side effects.

In contrast to a C-selective inhibitor, an N-selective inhibitor may open up novel therapeutic areas. As discussed, the N-domain appears to play a minor role in blood pressure control in vivo. At least three physiologically important peptides are hydrolyzed preferentially or exclusively by the N domain: luteinizing hormone-releasing

hormone (LH-RH), Ang ₍ i _-7) , and AcSDKP (AZ-acetyl-seryl-aspartyl-lysyl-proline). The contribution of ACE to the metabolism of LH-RH and Ang^ in vivo is unclear, but there is increasing evidence that ACE is the principal metabolizing enzyme for AcSDKP, a natural hemoregulatory hormone. AcSDKP has antiproliferative and anti- fibrotic activities and may have utility in protecting hematopoietic stem cells against chemotherapy-induced injury and in limiting cardiac fibrosis. Administration of ACE inhibitors results in a 4-6-fold elevation of AcSDKP plasma levels. This may be the basis for the observed association between ACE inhibitors and anemia, and the effective treatment of altitude polycythemia by the ACE inhibitor enalaprilat.

Lisinopril, which is a commercially available ACE inhibitor, is active with a nanomolar inhibition constant and has been shown to be relatively safe and effective for the treatment of patients with acute myocardial infarction, ³² hypertension, and heart-related diseases. ^33"35

Lisinopril is chemically described as (S)-1-[λ/ ² -(1-carboxy-3-phenylpropyl)-L-lysyl]-L- proline dihydrate. Its empirical formula is C ₂₁ H ₃₁ N ₃ O ₅ ^» 2H ₂ O and its structural formula is:

Dive and co-workers ³⁶ have recently reported that a phosphinic ACE-inhibitor, RXP A380, is -3000-fold C-domain selective and these same researchers have further shown that one key feature of the C-domain selectivity is the tryptophan moiety at the P ₂ ' position.

Starting material 3 was prepared by the reductive amination of ethyl 2-oxo-4-phenyl butyrate 1 and N-ε-(terf-butoxycarbonyl)-L-lysine 2 using an ethanolic solution of NaBH ₃ CN. ³⁷ The ratio of the two diastereoisomers was observed as 60:40 from the ¹ H NMR spectrum. Peptide coupling of compound 3 with a L-tryptophan methyl ester 4 (prepared earlier by methylation of tryptophan in the presence of thionyl chloride and methanol according to the method described by Hvidt et a/. ³⁸ ) was effected using EDCHCI in the presence of HOBt and diisopropyl ethyl amine as a base to afford the

diastereomeric pseudopeptide 5 in 74% yield. The characterisation of this diastereomeric mixture was achieved from the EI-MS and spectroscopic data. The El- MS data indicated a molecular ion peak at 637 correponding to M ⁺ +H. The ¹ H NMR spectrum showed two singlets at δ 3.71 and 3.73 ppm corresponding to the two methoxy groups of the diastereomeric mixture 5. Acid hydrolysis of the resulting diastereomeric mixture 5 produced the hydrochloride salt of the diastereomeric mixture 6 in a quantitative yield. Evidence of the compound 6 was found from the disappearance of the Boc, methyl and ethyl signals on the ¹ H NMR spectrum. Purification and separation of the diastereomeric mixture 6 was done by HPLC.

Initial attempts to remove the Boc group with concommitent hydrolysis of the ethyl and methyl esters under acid conditions afforded a mixture of four products separated by HPLC. These four products P ₁ , P ₂ , P3 and P ₄ were generated as a result of incomplete hydrolysis of compound 5, since acid hydrolysis of ester at room temperature is relatively slow. The EI-MS data revealed molecular ion peaks at 522, 522, 536 and 536 for P ₁ , P ₂ , P ₃ and P ₄ , respectively. However, the diastereomeric mixture 5 was stirred in 4λ/ HCI at room temperature for 24 h, after which the solvent was evaporated and the mixture was then stirred with a solution of 0.5/V LiOH for a further 5 h to afford the desired product 6. The characterisation of this diastereomeric mixture 6 was achieved from the EI-MS and spectroscopic data that gave a molecular ion peak at 495 correponding to M ⁺ +H. Purification and separation of the diastereomeric mixture 6 by HPLC gave the required two diastereoisomers in a 60:40 ratio.

ACE inhibiton assays were carried out on the incomplete hydrolysis products P ₁ , P ₂ , P ₃ and P ₄ .

EC ₅₀ = 16 t/M EC ₅₀ = 419 ι/M

M ⁺ 536 M ⁺ 536 EC ₅₀ = 25 t/M EC ₅₀ = 79 ι/M

Incomplete hydrolysis products

Scheme

Reagents and conditions:

(i) ketone 1 (4.0 eq), amino acid 2 (1.0 eq), NaBH ₃ CN (2.0 eq), 50 % EtOH/H ₂ O, rt, 12 h; (ii) L-tryptophan methyl ester 4, EDCHCI, HOBt, JPr ₂ NEt (1.0 equiv), dry DMF, rt, 72 h; (iii)(a) 4λ/ HCI, EtOAc, rt, 24 h; (b) ) 0.5λ/ LiOH, THF-MeOH, rt, 5h.

It will be apparent to a person skilled in the art that it will also be possible to synthesize other compounds than those described above by the method of the invention. Lisinopril is a carboxyalkyl dipeptide, essentially a Phe-Lys-Pro analog. Compound 6 is a tryptophan derivative of lisinopril - i.e. the Pro in the P2 ¹ position has been substituted by a Trp. It should be possible to prepare numerous variations on this theme, with different Trp-like groups in the P2 ¹ position. Also, the Zn-binding carboxyl could be changed to a keto group or any one of the possibilities mentioned above.

Similarly, the functionalities in the PT and P1 position could be changed. In particular, the crystal structure indicates that the ST pocket can accomodate larger side chains, and the applicant is in the process of studying a lisinopril analog with an arginine (or arginine-like) residue replacing the lysine in the P1 ¹ position.

The invention will now be described in more detail with reference to the following non- limiting examples.

Examples

General procedure

All reactions were carried out under a nitrogen atmosphere, unless otherwise specified. Reactions were monitored by TLC using Merck 60 F _2S4 precoated silica gel plates. Detection was effected by observation under a UV lamp (wave-length of 254 nm) and developed with I ₂ . Column chromatography was carried out on silica gel and the eluent mixture used is specified in each experiment. Anhydrous solvents like dichloromethane were distilled from phosphorous pentoxide and stored over molecular sieves type 4 A. All other anhydrous solvents were obtained from Aldrich or Sigma or Merck Chemical Co. All melting points were determined using a Kofler hot plate apparatus and are uncorrected. Specific rotations ([α] _D ) were measured at 2O ⁰ C, unless otherwise specified, using a Perkin-Elmer 141 polarimeter and are recorded in units of 10 ^~1 ° cm ² g ^"1 . NMR spectra were obtained on a Varian Mercury 300 MHz or Varian Unity 400 MHz spectrometer. Chemical shifts are reported in ppm relative to the residual signal of the solvent used. The coupling constants, where specified, are given in hertz (Hz). Mass spectra were obtained using electron impact ionization.

Example 1: N-[N ⁶ -tert-butoxycarbonyl-L-lysyl-N ² -(R.S)-(1-ethoxycarbonyl-3- phenylpropyDl-L-tryptophan methyl ester 5

A solution of N-[N ⁶ -tert-butoxycarbonyl-N ² -(R,S)-(1-ethoxycarbonyl-3-phenylpropyl)]- L- lysine 3 (200 mg, 0.459 mmol, 1.0 eq.) and L-tryptophan methyl ester 4 (100 mg, 0.459 nnmol, 1.0 eq.) in dry DMF (10 ml_) was cooled at O ⁰ C. 1-Hydroxybenzotriazole hydrate (HOBt) (62 mg, 0.459 mmol, 1.0 eq.), A/-ethyl-λ/'-(dimethylaminopropyl)-carbodiimide hydrochloride (EDCHCI) (88 mg, 0.459 mmol, 1.0 eq.) and JPr ₂ NEt (0.079 ml_, 0.459 mmol, 1.0 eq.) were added and the resulting mixture stirred at O ⁰ C for 2 h. The cooling bath was removed and the reaction mixture then stirred for a further 72 h at room temperature. The reaction mixture was diluted with H ₂ O (30 ml_) and the extracted with EtOAc (3 x 30 mL). The combined organic extract were washed sequentially with saturated aqueous NaHCO ₃ (30 mL), brine (30 mL), and dried over anhydrous MgSO ₄ . Evaporation of the solvent gave a crude residue, which was subjected to column chromatography on silica gel elution with 66% EtOAc-hexane afforded the desired diastereomeric mixture N-[N ^B -tert-butoxycarhonyl'L-lysyl-N ² -(R,S)-(1-ethoxycarbon- yl~3-phenylpropyl)]-L-tryptophan methyl ester 5 as a pale yellow oil (0.215 g, 74%); R _f 0.27 (66% EtOAc-hexane); IR (liquid film) v 1718 (CO), 3300 (NH) cπV ¹ ; ¹ H NMR (300 MHz, CDCI ₃ ), duplication of the signals are due to the diastereomeric mixture, δ _H 1.23 (3H, t, J = 7.1 Hz, -OCH ₂ CH ₃ ), 1.25 (3H, t, J = 7.2 Hz, -OCH ₂ CH ₃ ), 1.47 (9H, s, (CH ₃ ) ₃ ), 1.48 (9H, s, (CH ₃ ) ₃ ), 1.52-1.87 (12H, m, 6 X -CH ₂ ), 1.91-2.21 (4H, m, 2 X -CH ₂ ), 2.49 (2H, m, -CH ₂ Ph), 2.58 (2H, t, J = 7.2 Hz, -CH ₂ Ph), 2.91-3.11 (4H, m), 3.12-3.45 (2H, m), 3.71 (3H, s, OCH ₃ ), 3.73 (3H, s, OCH ₃ ), 4.12 (2H, q, J = 7.2 Hz, -OCH ₂ CH ₃ ), 4.13 (2H, q, J = 7.0, -OCH ₂ CH ₃ ), 4.14 (2H, m, 2 x -CH-), 4.58 (2H, br s, 2 x -NH), 4.89 (1 H, m, -CH-), 4.95 (1H, m, -CH-), 6.97-7.48 (26H, m, Ar-H), 7.55 (1 H, t, J = 8.4 Hz ₁ ), 7.56 (1 H, t, J = 8.4 Hz ₁ ), 8.69 (1H, br s, -NH-pyrole) and 8.87 (1 H, br s, -NH-pyrole); ¹³ C NMR (75 MHz, CDCI ₃ ) δ _c 14.2, 21.0, 22.2, 22.5, 27.6, 27.7, 28.5, 30.0, 31.7, 32.0, 32.7, 33.6, 35.5, 40.3, 51.8, 52.3, 58.8, 60.2, 60.4, 61.0, 62.0, 111.3, 111.4, 118.5, 119.4, 119.5, 122.0, 122.1 , 122.9, 126.0, 126.1 , 127.6, 128.3, 128.4, 128.5, 136.2, 136.3, 141.0, 141.6, 156.2, 156.3, 162.53, 171.1, 172.4, 173.6 and 174.96; EI-MS m/z 637 (M ⁺ +H).

Example 2: N ² -(R,S)-(1-Carbonyl-3-phenylpropyl)]-L-lvsyl-L-tn/ptophan hydrochloride Incomplete hydrolysis products Pi, P ₂ , P3 and P ₄

To a solution of the diastereomeric mixture N-[N ⁶ -tert-butoxycarbonyl-L-lysyl-N ² - (R,S)-(1-ethoxycarbon-yl-3-phenylpropyl)]-L-tryptophan methyl ester 5 (100mg, 0.157 mmol) in EtOAc (5.0 mL) was added 4/V HCI (10 mL) and the resulting mixture was stirred at room temperature for 2 h. TLC showed the disappearance of the starting material. Evaporation of the solvent after neutralization with 2λ/ NaOH gave incomplete hydrolysis products Pi, P ₂ , P3 and P ₄ as a cream white solid material in quantitative yield; IR (liquid film) v 1720 (CO), 2980 (OH) and 3115 (NH) cm ^"1 . The El- MS data of indicated a molecular ion peaks at 522, 522, 536 and 536 for P ₁ , P ₂ , P ₃ and P ₄ respectively.

Example 3: N ^z -(R,S)-(1-Carbonyl-3-phenylpropyl)l-L-lvsyl-L-tryptophan hydrochloride 6

To a solution of the diastereomeric mixture N-[N ^β -tert-butoxycarbonyl-L-lysyl~N ² - (R,S)-(1-ethoxycarbon-yl-3-phenylpropyl)]~L-tryptophan methyl ester 5 (50 mg, 0.0785 mmol) in EtOAc (5.0 mL) was added AN HCI (10 mL) and the resulting mixture was stirred at room temperature for 24 h. Evaporation of the solvent and the residue was dissolved in 0.5N LiOH (5.0 mL) and stirred at room temperature for 5 h. TLC showed the disappearance of the starting material. Evaporation of the solvent after neutralization with 1λ/ HCI gave N ² -(R,S)-(1-Carbonyl'3-phenylpropyl)]-L-lysyl-L- tryptophan hydrochloride 6 as a cream white solid material in quantitative yield; IR

(liquid film) v 1720 (CO), 2980 (OH) and 3115 (NH) cm ^"1 ; IR (KBr) v 1715 (CO) and 3250 (broad, COOH/NH) crrf ¹ ; ¹ H NMR (400MHz, D ₂ O) δ _H 1.09-1.36 (2H, m, - CH ₂ CH ₂ CH ₂ NH ₂ ), 1.56-1.80 (4H, m, -CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ ), 2.27 (2H, q, J = 7.3 Hz ₁ - CH ₂ CH ₂ Ph), 2.34-2.49 (4H, m, -CH ₂ Ph and -CH ₂ NH ₂ ), 2.83 (1H, dd, J = 6.2 and 13.0 Hz, -CH _α Trp), 3.05 (1H, dd, J = 6.3 and 13.2 Hz, -CH/Trp), 3.31 (2H, m, 2 x -CH-), 4.63 (1 H, m, -CH-) and 7.01-7.70 (1OH, m, Ar-H); ¹³ C NMR (100MHz, D ₂ O) δ _c 20.9, 27.3, 30.7, 31.0, 31.4, 33.3, 39.9, 48.6, 55.0, 58.8, 59.5, 62.7, 110.0, 111.5, 118.3, 118.9, 121.5, 123.6, 125.7, 128.2, 128.3 (2C), 126.9, 135.9, 142.1 , 142.4, 175.6, 178.5 and 181.0 (duplication of peaks due the two diastereoisomers); EI-MS m/z calcd for C ₂₉ H ₃₄ N ₄ O ₅ (M ⁺ ) 494.2429, found 495 (M ⁺ +H). HPLC purification and separation give the required two diastereoisomers in a 60:40 ratio.

Example 4: HPLC purification and separation

RP-HPLC purification and separation of the incomplete hydrolysis products was performed using a Jupiter 5LJ C18 300A, size 250 x 4.60 mm column with gradient elution of t = 0 min (63% A, 37% B) and t = 30 min (50% A, 50% B); solvent A = 0.1 % TFA in H ₂ O and solvent B = 0.1% TFA and 75% CH ₃ CN in H ₂ O; a flow rate of 1.0 mL/min and a 215 and 280 nm UV wavelength detection.

t _R = 12.77 min for P ₁ , t _R = 14.5 min for P ₂ , t _R = 17.1 min for P ₃ and t _R = 21.1 min for P ₄ .

RP-HPLC purification and separation of the diastereomeric mixture 4 was performed using a Jupiter 5U C18 300A, size 250 x 4.60 mm column with gradient elution of t = 0 min (50% A, 50% B) and t = 50 min (40% A, 60% B); solvent A = 0.1% TFA in H ₂ O and solvent B = 0.1 % TFA and 75% CH ₃ CN in H ₂ O; a flow rate of 1.0 mL/min and a 215 and 280 nm UV wavelength detection.

t _R = 32.1 min for P ₁ and t _R = 41.0 min for P ₂ .

Example 5: ACE inhibitory activity

The ACE inhibitory activity (as tabulated in Table 1 ) was measured by the fluorometric determination of pthaldialdehyde-derivatised histidylleucine, a product of the enzyme reaction according to the method of Almquist et a/., ¹² with some modifications. For

tACEdelta36NJ and N-domain inhibition using Z-Phe-His-Leu as substrate:- 10 μL of 0.05 mg/mL enzyme + 120 μL inhibitor at ambient temperature for 3 hours. Inhibitor concentrations ranged from 2.0 μM to 500 μM . A 3.0 μL aliquot of this was assayed for enzyme activity using 30 μL of 1.0 mM Hip-His-Leu. This was incubated (in triplicate) for 30 mins at 37 ⁰ C and stopped with 180 μL solution of 0.28λ/ NaOH. To this alkalized mixture 12 μL of o-pthaldialdehyde (150 mM) was added and the mixture incubated for another 10 mins at room temperature. Reactions were stopped by adding 26 μL solution of 3λ/ HCI. Fluorescence was measured at Ex = 360 nm; Em = 486 nm, 5 x 5 slit width along with a His-Leu standard calibration curve fluorometer: Varian Gary Eclipse plate reader.

All assays included enzyme incubated with buffer in the absence of inhibitor (i.e. 0 μM inhibitor concentration). Lisinopril was also used as a positive control. The 50% inhibition (IC ₅₀ ) of ACE activity was calculated as the concentrations of samples that inhibited 50% of ACE activity under these conditions.

Table 1 : ACE inhibition of the Phe-Lys-Trp derivatives prepared above

Compounds 6 and 6a are two diastereoisomers of a lisinopril derivative incorporating a Trp at the P2' position. Their ACE inhibitory activities are shown in Table 1. Although they displayed Ki values in the low micromolar range, the introduction of a tryptophan moiety at the P2' position resulted in a marked increase in C domain-selectivity (>19- >72 fold) as compared with lisinopril (2.6-fold). Neither compound inhibited the N domain up to a concentration of 500 μM (Table 1). Compound 6a, with an S- configuration at the stereogenic centre bearing the zinc-binding carboxylate group, was more C-selective than its R-diastereoisomer (Compound 6).

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