HK1212675B - Mixed inhibitors of aminopeptidase n and of neprilysine - Google Patents

Description

Mixed inhibitors of aminopeptidase N and enkephalinase

Technical Field

The present invention relates to mixed inhibitors of aminopeptidase N and enkephalinase (enzymes involved in enkephalin degradation).

Background

Enkephalin, Tyr-Gly-Phe-met (leu), is an endogenous ligand for the opioid receptors μ and is involved in the regulation of pain impulses in the central and peripheral nervous systems. However, despite their affinity for opioid receptors similar to morphine, these peptides induce only a very short analgesic response when administered intracerebroventricularly in rodents due to their very rapid inactivation in vivo (including humans) (Mosnaim et al (2008) neurochem. res., 33, 81-86). Two metallopeptidases cause this inactivation: aminopeptidase N (APN, EC3.4.11.2) and enkephalinase (NEP, EC3.4.24.11), which cleave Tyr of enkephalin, respectively¹–Gly²Bond and Gly³–Phe⁴Bonds, thus leading to inactivation of metabolites (Roques et al (1993) Pharmacol. Rev., 45, 87-146).

Mixed inhibitors of these two enzymes are known to completely protect endogenous enkephalins from enzymatic degradation, thus showing pharmacological activity, in particular analgesic and antidepressant activity of enkephalins (Noble et al (2007) expert. opin. ther. targets, 11, 145-149). Such inhibitors disclosed in the prior art include hydroxamates (FR2518088 and FR2605004), aminophosphinic acid compounds (FR2755135, FR2777780, FR0855015), amino acids with a thiol functional group (FR2651229, FR0510862, FR0604030, FR0853092), endogenous peptides (Wisner et al PNAS (2006), 103, 17979-. These various molecules have physicochemical (solubility) and pharmacodynamic (bioavailability) properties that confer them pharmacological efficacy, intravenously or orally, against different types of pain, particularly acute or chronic pain with excessive injury (Noble et al (2007) expert. opin. ther. targets, 11, 145-149) and neuropathic pain (Menendez et al (2008) Eur J Pharmacol, 596, 50-55; Thibault et al (2008) eur.j. Pharmacol., 600, 71-77).

However, none of the mixed inhibitors disclosed to date have expanded the low doses that can be used for infusion in clinically suitable vehicles after intravenous administration in cases of acute pain (post-surgery, cancer, trauma, dentistry, etc.), enabling a rapid, strong analgesic response with a sufficiently long duration of action.

It is an object of the present invention to provide compounds having the advantageous properties of morphine substances for the central nervous system, in particular analgesia, behavioral effects (reduction of the emotional elements of pain and antidepressant responses), without their major drawbacks for the central nervous system (habituation, physical and mental dependence, respiratory depression) and the peripheral nervous system (constipation). In addition, it would be beneficial for the compounds to have favorable peripheral effects (anti-inflammatory and anti-neurological) without the drawbacks as described above.

Disclosure of Invention

The present invention relates to compounds having the following general formula (1):

(1)R–NH–CH(R₁)–CH₂–S–S–C(R₂)(R₃)–COCH₂–CH(R₄)–COR₅

r, R therein₁、R₂、R₃、R₄And R₅As defined in claim 1.

The invention also relates to pharmaceutical compositions containing at least one compound of the invention.

The invention also relates to pharmaceutical compositions containing at least one compound according to the invention and at least one compound selected from morphine and derivatives thereof, inhibitors of endocannabinoids and endocannabinoid metabolites, GABA derivatives such as gabapentin or pregabalin, duloxetine or methadone.

Finally, the present invention relates to the compounds of the invention or pharmaceutical compositions containing them for use as analgesics, anxiolytics, antidepressants or anti-inflammatory agents.

Drawings

FIG. 1: analgesic response induced after intravenous injection of a compound according to the invention (10mg/kg) -mouse hot plate test.

Detailed Description

Definition of

Alkyl means a straight or branched C1, C2, C3, C4, C5 or C6 hydrocarbon chain, in particular methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

Examples of aromatic or saturated 5-or 6-membered heterocyclic rings containing at least one sulfur, oxygen or nitrogen atom include the following groups: thienyl, pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, thiadiazolyl, furanyl, pyranyl, isoxazolyl, morpholinyl, furazanyl, oxazolyl, oxazolidinyl, and oxazolinyl.

The term "halogen" is designated herein as chlorine, bromine, iodine or fluorine.

Detailed Description

The compounds of the invention are linked by a disulfide bond corresponding to an APN inhibitor and a NEP inhibitor, which are capable of inhibiting the activity of these two enzymes at nanomolar concentrations. This disulfide bond breaks in vivo and releases two inhibitors that are capable of interacting with their respective target sites (NEP or APN) (Fournie-Zaluski et al (1992) J. Med. chem., 35, 2473-2481).

NEP inhibitors disclosed to date typically have a peptide pattern and thus have one or more amide bonds. For example, peptide-mimetic derivatives containing at least one amide bond and additionally a disulfide bond pattern are disclosed in Roques et al (Nature Rev. drug Discov. (2012)11, 292-. In addition, they generally have a molecular weight (MW (abbreviated PM in french)) of 500Da or more. These characteristics are not very advantageous for crossing physiological barriers, such as the intestinal barrier, and as a result these products have a rather low oral bioavailability. The same is true for crossing the blood brain barrier.

The NEP inhibitors used according to the invention and which make it possible to overcome these drawbacks are structurally characterized in that: 1) a novel pattern of thiones capable of interacting with the zinc of NEP in monodentate or bidentate form, ii) a pattern of peptides (and therefore amide bonds) lacking, iii) a backbone containing minimal groups enabling nanomolar affinity for NEP, iv) low molecular weight.

The compounds of the present invention have the following general formula (1):

(1)R–NH–CH(R₁)–CH₂–S–S–C(R₂)(R₃)–COCH₂–CH(R₄)–COR₅

wherein R is:

a) r is:

-hydrogen;

-an alkoxyalkyl carbonyl group R 'c (o) OCH (R ") oc (o) -, wherein R' and R" are independently alkyl groups containing 1 to 6 carbon atoms;

b)R₁is a straight OR branched alkyl group of1 to 6 carbon atoms, unsubstituted OR substituted with a-OR '", -SOR'" OR-SR '"group, wherein R'" is an alkyl group of1 to 6 carbon atoms, unsubstituted OR substituted with one OR more halogen atoms;

c)R₂comprises the following steps:

-a linear or branched alkyl group of1 to 6 carbon atoms, unsubstituted or substituted with:

■–OR₆、–SR₆or-SOR₆Group, wherein R₆Is hydrogen, straight or branched chain alkyl of1 to 4 carbon atoms, phenyl or benzyl;

■–CO₂R₇group, wherein R₇Is hydrogen, a linear or branched alkyl group containing 2 to 4 carbon atoms, or a benzyl group;

■–NR₈R₉group, wherein R₈And R₉Independently hydrogen, straight or branched alkyl of1 to 4 carbon atoms, phenyl or benzyl, or wherein-NR₈R₉Together are a saturated 5-or 6-membered heterocyclic ring containing one or more heteroatoms selected from N or O, preferably morpholine or piperidine; or

■ carboxamide group-CONR₈R₉wherein-NR₈R₉As defined above;

■ phenyl which is unsubstituted OR substituted by one OR more halogen, alkoxy-OR selected from fluoro OR bromo₆Is substituted in which R₆Have the same definition as above, or are substituted by phenyl;

■ an aromatic 5-or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

■ saturated 5-or 6-membered rings or heterocycles containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

-saturated 5-or 6-membered rings or heterocycles containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur; or

-phenyl, unsubstituted OR substituted by one OR more halogens selected from fluorine OR bromine, OR by-OR₅Is substituted by radicals in which R₅Have the same definitions as above;

and R is₃Is hydrogen; or

R₂And R₃Identical and is a linear or branched alkyl group of1 to 6 carbon atoms; or

–C(R₂)(R₃) -aThe method comprises the following steps:

■ saturated 5-membered ring compounds fused or unfused with an aromatic ring (e.g., to form an indane ring);

■ saturated 6-membered ring compounds;

■ contains at the 4-position 1 heteroatom selected from oxygen, nitrogen and sulfur saturated 6-membered heterocyclic compounds, wherein when the heteroatom is nitrogen, the nitrogen is unsubstituted or substituted by alkyl, phenyl, benzyl or alkanoyl of1 to 6 carbon atoms;

d)R₄comprises the following steps:

-a linear or branched alkyl group of1 to 6 carbon atoms, unsubstituted or substituted with:

■–OR₆、–SR₆or-SOR₆Group, wherein R₆Is hydrogen, straight or branched chain alkyl of1 to 4 carbon atoms, phenyl or benzyl;

■–CO₂R₇group, wherein R₇Is hydrogen, straight or branched chain alkyl containing 2 to 4 carbon atoms, benzyl;

■–NR₈R₉group, wherein R₈And R₉Independently hydrogen, straight or branched alkyl of1 to 4 carbon atoms, phenyl or benzyl, or wherein-NR₈R₉Together are a saturated 5-or 6-membered heterocyclic ring containing one or more heteroatoms selected from N or O, preferably morpholine or piperidine;

■ carboxamide group-CONR₈R₉wherein-NR₈R₉As defined above;

■ phenyl, which is unsubstituted or substituted with:

-one or more halogens selected from fluorine or bromine;

-–OR₆group, R₆Have the same definitions as above;

-phenyl or thienyl;

■ an aromatic 5-or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur; or

■ contains 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur saturated 5-or 6-membered rings or heterocyclic compounds;

-phenyl, unsubstituted or substituted with:

■ one or more halogens, especially fluorine or bromine;

■–OR₆group, wherein R₆Have the same definitions as above;

■ a phenyl group;

■ an aromatic 5-or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

e)R₅comprises the following steps:

-a hydroxyl group;

-–NR₈R₉group, wherein R₈And R₉Independently hydrogen, straight or branched alkyl of1 to 4 carbon atoms, phenyl or benzyl, or wherein-NR₈R₉Together are a 5-or 6-membered heterocyclic ring containing one or more heteroatoms selected from N or O, preferably morpholine or piperidine;

-alkoxy-OR₁₀Wherein R is₁₀Comprises the following steps:

■ contain a straight or branched chain alkyl group of 2 to 6 carbon atoms;

■ benzyl group;

■–CHR₁₁–COOR₁₂、–CHR₁₁–O–C(＝O)R₁₂、–CHR_11–OR-C (═ O) -OR₁₂Group, wherein R₁₁And R₁₂Independently a straight or branched chain alkyl group of1 to 6 carbon atoms.

The compounds of the invention may be in the form of pharmaceutically acceptable addition salts, for example addition salts of the compounds of formula (I) with inorganic or organic acids when the amino function is free, or with inorganic or organic bases when the acid function is free.

R and R are usually carried out₅Protection of the N-terminal and/or C-terminal portion of the group to facilitate bioavailability for various routes of administration.

In a particular embodiment of the invention, the compounds have the general formula (1), wherein R₂Is a straight or branched alkyl group of1 to 6 carbon atoms or a straight or branched alkyl group of1 to 6 carbon atoms substituted with:

■ a phenyl group;

■ phenyl substituted with one or more halogens selected from fluorine or bromine;

■ an aromatic 5-or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

■ saturated 5-or 6-membered rings or heterocycles containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

and R is₃Is hydrogen; or

R₂And R₃Identical and is a straight-chain or branched alkyl radical of1 to 6 carbon atoms, or

–C(R₂)(R₃) -together are:

■ saturated 5-membered ring compounds;

■ saturated 5-membered ring compounds fused to aromatic rings;

■ saturated 6-membered ring compounds; or

■ saturated 6-membered heterocyclic compound containing 1 heteroatom selected from oxygen, nitrogen and sulfur at the 4-position;

and R, R₁、R₄、R₅As described above or as described below.

More particularly, R₂May represent an isobutyl group or a methyl group substituted by:

■ a phenyl group;

■ phenyl substituted at the 4-position with a halogen selected from fluorine or bromine;

■ phenyl substituted at the 4-position with phenyl;

and R is₃Is hydrogen; or

R₂And R₃Is identical and is methyl or ethyl, or

–C(R₂)(R₃) -together are:

■ saturated 5-or 6-membered ring groups; or

■ saturated 5-membered ring groups fused to aromatic rings.

In a particular embodiment of the invention, the compounds have the general formula (1), wherein R₄Is a straight or branched chain alkyl group of1 to 6 carbon atoms substituted with:

■ phenyl which is unsubstituted OR substituted by one OR more halogen, alkoxy-OR selected from fluoro OR bromo₆Is substituted in which R₆Have the same definition as above, or are substituted by phenyl;

■ an aromatic 5-or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

■ saturated 5-or 6-membered ring compounds;

■ saturated 5-or 6-membered heterocyclic compounds containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulfur;

and R, R₁、R₂、R₃、R₅As described above or below.

Advantageously, R₄Is a straight or branched chain alkyl group of1 to 6 carbon atoms substituted with:

■ a phenyl group; or

■ phenyl substituted with:

-one or more halogens selected from fluorine or bromine;

-a phenyl group or a thienyl group,

and R, R₁、R₂、R₃、R₅As described above or below.

More particularly, R₄May represent a carbon alkyl group substituted with:

■ a phenyl group;

■ phenyl substituted at the 4-position with a halogen selected from fluorine or bromine;

■ phenyl substituted at the 4-position with phenyl.

In a particular embodiment of the invention, the compounds have the general formula (1), wherein R₅Comprises the following steps:

-a hydroxyl group; or

-alkoxy-OR₁₀Wherein R is₁₀Comprises the following steps:

■ contain a straight or branched chain alkyl group of 2 to 6 carbon atoms;

■ benzyl group;

–CHR₁₁–COOR₁₂、–CHR₁₁–O–C(＝O)R₁₂、–CHR_11–OR-C (═ O) -OR₁₂Group, wherein R₁₁And R₁₂Independently a straight or branched alkyl group of1 to 6 carbon atoms;

and R, R₁、R₂、R₃、R₄As described above or below.

Is advantageous forEarth, R₅Is hydroxy and R, R₁、R₂、R₃、R₄As described above or below.

Preferred compounds have the following general formula (1) wherein:

a)R₁is selected from-CH₂CH₂SCH₃、–CH₂CH₂CH₂CH₃、–CH₂CH₂SOCH₃；

b)R₂Is a straight or branched alkyl group of1 to 6 carbon atoms or 1 to 6 carbons substituted with

Linear or branched alkyl of atoms:

■ a phenyl group;

■ phenyl substituted with one or more halogens selected from fluorine or bromine;

■ an aromatic 5-or 6-membered heterocyclic ring containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

■ saturated 5-or 6-membered rings or heterocycles containing 1 or 2 heteroatoms selected from oxygen, nitrogen and sulphur;

and R is₃Is hydrogen; or

R₂And R₃Identical and is a linear or branched alkyl group of1 to 6 carbon atoms; or

–C(R₂)(R₃) -together are:

■ saturated 5-membered ring compounds;

■ saturated 5-membered ring compounds fused to aromatic rings;

■ saturated 6-membered ring compounds; or

■ saturated 6-membered heterocyclic compound containing 1 heteroatom selected from oxygen, nitrogen and sulfur at the 4-position;

c)R₄is a quiltStraight or branched chain alkyl of1 to 6 carbon atoms substituted with:

■ a phenyl group; or

■ phenyl substituted with:

-one or more halogens selected from fluorine or bromine;

-phenyl or thienyl;

d)R₅is a hydroxyl group.

Preferred compounds have formula (1), wherein:

–R₂＝CH₂Ph；R₃＝H；R₄＝CH₂ph; or

–R₂＝iBu；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂Ph; or

–R₂＝CH₂Ph；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Ph–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₃；R₃＝CH₃；R₄＝CH₂(4-Br-Ph); or

–R₂＝C₂H₅；R₃＝C₂H₅；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₅H₈；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₆H₁₀；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₉H₈；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₆H₁₀；R₄＝CH₂(4-Ph-Ph); or

–C(R₂)(R₃)＝C₅H₈；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₉H₈；R₄＝CH₂(4–Ph–Ph)。

In a particular embodiment, the compounds of the invention have formula (1), wherein R is₁Is selected from-CH₂CH₂SCH₃、–CH₂CH₂CH₂CH₃、–CH₂CH₂SOCH₃。

In a preferred embodiment, the compounds of the present invention have formula (1), wherein R is₁Is selected from-CH₂CH₂SCH₃、–CH₂CH₂CH₂CH₃、–CH₂CH₂SOCH₃And, and

–R₂＝CH₂Ph；R₃＝H；R₄＝CH₂ph; or

–R₂＝iBu；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂Ph；

–R₂＝CH₂Ph；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Ph–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₃；R₃＝CH₃；R₄＝CH₂(4-Br-Ph); or

–R₂＝C₂H₅；R₃＝C₂H₅；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₅H₈；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₆H₁₀；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₉H₈；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₆H₁₀；R₄＝CH₂(4-Ph-Ph); or

–C(R₂)(R₃)＝C₅H₈；R₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃)＝C₉H₈；R₄＝CH₂(4–Ph–Ph)。

In other embodiments, the compounds of the present invention have formula (1), wherein:

–R₁＝CH₂CH₂SCH₃；R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂CH₂CH₃；R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂SOCH₃；R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂CH₂CH₃；–C(R₂R₃) - ═ cyclohexyl; r₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂SCH₃；–C(R₂)(R₃) - ═ cyclohexyl; r₄＝CH₂(4-Ph-Ph); or

–R₁＝CH₂CH₂CH₂CH₃；–C(R₂)(R₃) - ═ cyclohexyl; r₄＝CH₂(4–Ph–Ph)；

And R₅As described above.

In a particular embodiment of the invention, the compounds of the invention have formula (1) wherein R is hydrogen or R is a R 'C (O) OCH (R') OC (O) -group, wherein R 'is isopropyl and R' is methyl, and R₁、R₂、R₃、R₄And R₅As described above.

The compounds of the invention can be used as medicaments. More particularly, the compounds may be used for the preparation of a pharmaceutical composition comprising at least one compound as described above as active ingredient and at least one pharmaceutically acceptable excipient. The excipients are selected from typical excipients well known to those skilled in the art depending on the dosage form and mode of administration desired.

Since the compounds of the invention jointly inhibit the enzymatic activity responsible for the degradation of enkephalins, they increase the rate of extracellular endogenesis of enkephalins, thus demonstrating an effective analgesic and/or antidepressant action. The analgesic effect of the compounds is manifested in various acute or chronic pains such as neurogenic pain, neuropathic pain, neuroinflammatory pain or nociceptive pain, or general pain such as fibromyalgia. Examples of pain include mechanical pain (e.g., muscle pain, vascular ischemia), phantom limb pain, pain caused by herpes zoster, cancer pain associated with the cancer itself or the outcome of its treatment, pain associated with inflammatory diseases (e.g., arthritis, rheumatoid arthritis, osteoarthritis, gout), pain associated with type I diabetes, pain associated with migraine, facial neuralgia, headache, pain associated with peripheral nerve injury (e.g., post-operative), back neuralgia, dental pain, pain associated with burns, sunburn, mosquito bites or stings, pain associated with infections, metabolic diseases (diabetes, alcoholism), nerve compression (hernia, carpal tunnel, fibrosis … …), bone fractures, burns, hematomas, cuts and inflammation.

Finally, typically and advantageously, the compounds of the present invention do not have the major drawbacks of morphine substances (tolerance, physical dependence, respiratory depression, nausea, sedation, constipation … …).

Thus, the compounds of the present invention and pharmaceutical compositions comprising the same may be used for at least one use selected from the following: analgesic, anxiolytic, antidepressant or anti-inflammatory.

The invention also relates to the use of the compounds of formula (I) as defined above and of the pharmaceutical compositions comprising them for the preparation of analgesic, anxiolytic, antidepressant or anti-inflammatory drugs, more particularly for the preparation of drugs for the treatment of pain. The pain may be in particular chronic or acute pain as defined above.

The compounds of the present invention may be used alone or in combination with compounds known to have analgesic properties. This combination results in an enhanced pharmacological effect, and high doses of analgesic compounds are known to often have undesirable side effects.

Such enhancement of pharmacological effects (synergy) has been shown in the past by combining mixed inhibitors differing in chemical structure from the mixed inhibitor of the present invention with known analgesic compounds. Thus, for example, strong potentiation of the analgesic response is obtained in combination with morphine (MasNieto et al (2001) Neuropharmacol.41, 496-506, THC (Valverde et al (2001) Eur.J.Neurosci., 13, 1816-1824), gabapentin (Menendez et al (2007) Eur.J.Pharmacol., 596, 50-55) and analogs thereof such as pregabalin.) these combinations enable a 3 to 10 fold lower dosage of the combination partner (e.g., morphine and inhibitor) for equivalent pharmacological effects.

Thus, in one embodiment, the pharmaceutical composition comprises at least one compound of the invention in combination with at least one analgesic as active ingredient, and at least one pharmaceutically acceptable excipient. The analgesic may be selected from:

-morphine and derivatives thereof,

-endocannabinoids,. DELTA.⁹THC, synthetic cannabinoid receptor agonists or cannabinoid degradation inhibitors (FAAH), or

GABA analogs, such as gabapentin or pregabalin, or

Duloxetine, a serotonin and norepinephrine reuptake inhibitor.

In another embodiment, the pharmaceutical composition comprises at least one compound of the present invention in combination with methadone as an active ingredient, and at least one pharmaceutically acceptable excipient.

In another embodiment, the present invention relates to a composition comprising:

a) at least one compound of formula (1) as defined above, and

b) at least one analgesic agent, for example selected from morphine and derivatives thereof, endocannabinoids, delta⁹THC, synthetic cannabinoid receptor agonists or cannabinoid degradation inhibitors (FAAH), or GABA analogs such as gabapentin or pregabalin, or duloxetine,

as a combination product for simultaneous, separate or sequential use in the treatment of pain, in particular chronic or acute pain.

Previously, it has been shown that combination of a mixed inhibitor of a compound different in chemical structure from the compounds of the present invention with methadone enables synergistic enhancement of the action of the ingredients (Le Guen et al (2003) Pain, 104, 139-148). The combination reduces the addictive processes of opioids and cocaine.

The pharmaceutical composition according to the invention can be administered parenterally, for example intravenously or intradermally, or topically, orally or nasally.

Forms for parenteral administration include aqueous suspensions, isotonic saline solutions or sterile injectable solutions, which may contain pharmacologically compatible dispersing and/or wetting agents. Forms that can be administered orally include tablets, soft or hard capsules, powders, granules, oral solutions and suspensions. Forms that can be administered nasally include aerosols. Forms that can be administered topically include patches, gels, creams, ointments, lotions, sprays, eye washes.

The effective dosage of the compounds of the invention will vary according to a number of parameters, such as the chosen route of administration, the weight, age, sex, progress of the pathology to be treated and the sensitivity of the individual to be treated.

According to another aspect, the present invention also relates to a method of treatment of a pathology as described above, comprising administering to a patient in need thereof an effective dose of a compound according to the present invention or a pharmaceutically acceptable salt thereof or a composition according to the present invention, preferably parenterally, orally or nasally.

Mixed NEP-APN inhibitor 1 can be prepared in two steps. In a first step, Boc β -aminothiol 11 (Fournie-Zalusky M-C. et al (1992) J.Med.chem., 35, 2473-2481) is activated with methoxycarbonylsulphonyl chloride and then condensed with mercaptoalkanoic acid 10 in a second stage to give compound 12.

By reaction of the acid 12 with the corresponding alcohol R₅OH reaction or by reaction of acid 12 with chlorinated derivative R₅Cl in Et₃Refluxing in ethyl acetate in the presence of N afforded ester 13.

Deprotection of the N-terminal Boc group of 13 by the action of formic acid releases 1.

Alternatively, the N-terminal Boc group can be deprotected by the action of formic acid and then in SOCl₂Esterification with the corresponding alcohol in the presence of 12 at room temperature gives the ester 1.

Alternatively, the N-terminal Boc group can be deprotected by the action of formic acid and then on CH₃In CN 2NNaHCO₃Condensation with 1- ((2, 5-dioxopyrrolidin-1-yloxy) carbonyloxy) ethyl isobutyrate in the presence of water afforded the N-protected compound 1(R ═ iPrCOOCH (CH): from 12₃) OCO) (Cundy et al (2004) j.pharm.exp.thermal, 311,315–323)。

where R is obtained in 5 steps from amino acid 2 which defines the absolute configuration, preferably (R)₃H and R₅Compounds of formula 10 ═ OH.

Step 1:

amino acid 2 is converted to the brominated derivative 3 by a deamination-halogenation reaction, which generally results in a retention of the configuration (Claesong G. et al (1968) Acta chem. Scand.22, 3155-.

Brominated derivative 3 is converted to thioether 4 by a nucleophilic substitution reaction of a conformational flip-flop in basic medium by the action of 4-methoxy- α -toluenethiol (PMBSH).

Step 2: preparation of halomethylketones 6 from 4

The method comprises the following steps:compound 4 is converted to enone 5 from either a mixed anhydride of 4 (prepared by the action of isobutyl chloroformate and N-methylmorpholine) or from an acid chloride (prepared by the action of thionyl chloride on 4).

Ketene 5 is then converted to halomethyl ketone 6 by bubbling HCl gas or HBr gas in 1, 4-dioxane, diethyl ether or ethyl acetate.

The method 2 comprises the following steps:alternatively, chloromethyl ketone 6(X ═ Cl) can be obtained by the action of chloroiodomethane on methyl ester of 4 (prepared in the presence of DMAP and EDCI or by the action of acetyl chloride in methanol) in the presence of freshly prepared LDA (Chen et al (1997) tet.lett.38, 18, 3175-.

Step 3

The method comprises the following steps:treatment of halomethyl ketone 6 with NaI, undergoing halogen exchange, and then reacting with a sodium dialkylmalonate salt yields compound 7. R₁₃It may be methyl, ethyl or tert-butyl.

By bromination of derivative R₄Effect of Br on the aforementioned anions of malonate 7 (in situ deprotonation with NaH) introduction of substituent R₄. Compound 8 was obtained.

The method 2 comprises the following steps:if X ═ Br, the substituent R4 can also be introduced directly onto the halomethyl ketone 6. The latter is treated with NaI, undergoes halogen exchange, and then reacts with a substituted sodium dialkylmalonate to afford compound 8.

This reaction maintains the configuration of the carbon bearing the thiol group.

Step 4

By action of TFA (when R is present₁₃T-butyl) or by saponification (when R is₁₃Methyl or ethyl) hydrolysis of the ester of 8 followed by decarboxylation, e.g. at reflux in toluene, gives compound 9.

Step 5

Deprotection of the thiol group on compound 9 was carried out in 2 stages by the action of DTNP (2, 2' -dimercaptobis (5-nitropyridine)) in trifluoroacetic acid followed by reaction with TCEP (tris (2-carboxyethyl) phosphine) (harrisk.m. et al (2007) j.pept.sci. (2), 81-93) or heating at 50 ℃ directly in trifluoroacetic acid in the presence of anisole to give mercaptoalkanoic acid 10.

Wherein R is₂＝R₃Alkyl, R ═ H and R₅Compound 10 ═ OH can be obtained as follows: to obtain chloromethyl ketone 6, bromoacetic acid and 4-methoxybenzyl mercaptan are reacted, then dialkylated and converted to chloromethyl ketone 6 as described above.

Wherein R is₂And R₃The cyclic compound 10 can be obtained as follows: chloromethyl ketone 6 is prepared directly from the corresponding ester (e.g., methyl ester) by alkylation using 4-methoxybenzyl mercaptan disulfide (or from another activated form of the mercaptan) and conversion to chloromethyl ketone 6.

Alternatively, cyclic geminal chloromethyl ketones 6 can be prepared directly from the corresponding esters (e.g. methyl esters) by alkylation using activated 4-methoxybenzylmercaptan, and TMSN is used₂The solution is converted to bromomethyl ketone 6.

The rest of the synthesis was performed as described above.

When R is₄In the case of 4-bromobenzyl, compound 9 can be subjected to a Suzuki reaction to introduce an aromatic group on the benzyl group.

Compound 9 (when R is₃H) has 2 asymmetric centers and 4 stereoisomers. When R is₂＝R₃Compound 9 has only one asymmetric center and is thus a mixture of 2 stereoisomers when either alkyl or cyclic.

Deprotection of 9 affords compound 10.

If compound 9 is chiral, it can be isolated by selective precipitation using a chiral amine such as α -methylbenzylamine or demethyl ephedrine or by chiral column HPLC.

Examples

The invention is further illustrated below, but is not in any way restricted to the following examples.

1.Synthesis of NEP-APN mixed inhibitors

1.1Synthesis of NEP inhibitors

1.1.1Step 1: synthesis of (R) -2-bromocarboxylic acid

The (R) -configured amino acid (39.3mmol) was dissolved in 50mL of water. KBr (3.5eq, 31.8g) and then H were added dropwise at 0 ℃ respectively₂SO₄(7.73mL) while maintaining the temperature below 5 ℃. The mixture was cooled to-10 ℃ and NaNO dissolved in 17mL of water was added dropwise₂(1.3eq,3.59 g). The mixture was stirred at-5 ℃ for 2 hours.

After returning to room temperature, use CH₂Cl₂(2 × 50mL) the mixture was extracted with H₂O, washing the organic phase with saturated NaCl, Na₂SO₄Drying to obtain the expected (R) configuration product.

3a R₂＝CH₂Ph: a pale yellow oil; (yield: 50%); rf (CH)₂Cl₂/MeOH):0.62

NMR (abbreviated as RMN in French) (CDCl)₃,200MHz):3.15–3.40(2H,dd)；4.69(1H,m)；7.20–7.40(5H,m)

3b R₂＝CH₂CH(CH₃)₂: an oil; (yield: 82.5%); rf (CH)₂Cl₂/MeOH)：0.49

NMR(CDCl₃,200MHz):0.90–1.0(6H,m)；1.55(2H,m)；2.40(1H,m)；4.29(1H,d)

3c R₂＝CH₂(4-Br-Ph): a pale yellow oil; (yield: 50%); rf (CH)₂Cl₂/MeOH):0.62

NMR(CDCl₃,200MHz):3.15–3.40(2H,dd)；4.70(1H,m)；7.20–7.40(4H,m)

3d R₂＝CH₂(4-Ph-Ph): light yellowAn oil; (yield: 60%); rf (CH)₂Cl₂/MeOH):0.7

NMR(CDCl₃,200MHz):3.15–3.40(2H,dd)；4.70(1H,m)；7.20–7.60(9H,m)

Synthesis of (S) -2- (4-methoxybenzylthio) carboxylic acid

Under an inert atmosphere, 4-methoxybenzylthiol (4.2 mL; 30.06 mmol; 1eq) was dissolved in 70mL of anhydrous THF, and 1.1eq of 60% NaH (1.33 g; 33.07mmol) was added. The mixture was stirred at room temperature for 15 minutes, and then the brominated derivative 3(1eq,30.06mmol) dissolved in 30mL of THF was added dropwise to the bromine using a dropper. The mixture was stirred at room temperature overnight. The mixture was evaporated to dryness and then taken up in AcOEt. By H₂O, washing the organic phase with saturated NaCl, Na₂SO₄Dried and evaporated under reduced pressure to give the crude product. The latter was purified by silica gel chromatography using CHex/AcOEt 5/5 as the elution system to give compound 4 in the (S) configuration as an oil.

4a R₂＝CH₂Ph: an oil; (yield: 40%); rf (CH)₂Cl₂/MeOH/9/1):0.5

NMR(CDCl₃,200MHz):2.80–3.10(2H,m)；3.68(2H,s)；3.76(3H,s)；4.53(1H,t)；6.84(2H,d)；7.09–7.49(7H,m)

4b R₂＝CH₂CH(CH₃)₂: an oil; (yield: 26%); rf (CHex/AcOEt) 0.65

NMR(CDCl₃,200MHz):0.90–1.0(6H,m)；1.55(2H,m)；2.25(1H,m)；3.40(1H,d)；3.70(2H,s)；3.90(3H,s)；6.8–6.9(4H,m)

4c R₂＝CH₂(4-Br-Ph): an oil; (yield: 40%); rf (CH)₂Cl₂/MeOH/9/1):0.5

NMR(CDCl₃,200MHz):3.0–3.30(2H,m)；3.60(1H,q)；3.70(2H,s)；3.90(3H,s)；6.80–7.20(8H,m)

4d R₂＝CH₂(4-Ph-Ph): an oil; (yield: 50%); rf (CH)₂Cl₂/MeOH/9/1):0.6

NMR(CDCl₃,200MHz):3.0–3.30(2H,m)；3.60(1H,q)；3.70(2H,s)；3.90(3H,s)；6.80–7.2(13H,m)

1.1.2Step 2:

procedure 1:

the method comprises the following steps: synthesis of diazoketones from mixed anhydrides

To a solution of acid 4(18.5mmol) in 20mL of dry THF under an inert atmosphere at-20 deg.C were added N-methylmorpholine (2.15 mL; 1.05eq) and iBuOCOCOCl (2.52 mL; 1.05eq) sequentially. The mixture was stirred at-20 ℃ for 5-10 minutes, then the precipitate was filtered on celite and washed with 20mL THF.

Reacting CH at 0 ℃₂N₂Ether solution (2.5eq) (fromAnd KOH pre-made in carbitol) to the activated ester solution. The solution turned yellow. The mixture was stirred at room temperature for 2 hours.

The method 2 comprises the following steps: synthesis of diazoketones from acid chlorides

Dissolve acid 4(14.5mmol) in 23mL anhydrous CH₂Cl₂In (1). In roomWarm addition of SOCl₂(1.5 eq; 21.75mmol) and the mixture was refluxed for 2 hours under an inert atmosphere. The mixture was then evaporated to dryness to give a brown oil. The product was dissolved in anhydrous THF at a concentration of 5 mmol/mL.

The previously prepared CH is added at 0 deg.C₂N₂(2.5eq) the ethereal solution was transferred to the acid chloride solution. The solution turned yellow. The mixture was stirred at room temperature under an inert atmosphere for 2 hours.

Synthesis of chloromethyl ketones

The solution of compound 5 was placed in a three-necked flask maintained at 0 ℃ under an inert atmosphere. The mixture was saturated with HCl at 0 ℃ by bubbling.

After 30 minutes, the solvent and excess HCl were evaporated under reduced pressure. The product was taken up in AcOEt (150mL) and then with 10% NaHCO₃、H₂Washed with O and Na₂SO₄Drying to obtain the crude product. The latter was used as such in the next step without purification.

Procedure 2: from the synthesis of methyl esters

Acetyl chloride (3 eq; 2mL) was added dropwise to a solution of acid 4(9.1mmol) in 50mL of anhydrous MeOH at 0 deg.C under an inert gas. The mixture was stirred at room temperature overnight. The mixture was concentrated under reduced pressure and taken up in MTBE (methyl-tert-butyl ether) (200 mL). With 10% NaHCO₃(100mL)、H₂The organic phase was washed with O (100mL) and saturated NaCl (100 mL). Through Na₂SO₄The organic phase was dried and then concentrated under reduced pressure to give crude methyl ester. The latter was purified by silica gel flash chromatography.

Over 30 minutes, to a solution of methyl ester (4.42mmol) and chloroiodomethane (1.3 mL; 4eq) in 25mL THF was added dropwise a solution of LDA (5eq) in THF (55mL) (freshly prepared from 1.6M BuLi in hexane (15 mL)) and diisopropylamine (3.4 mL). During the addition, the internal temperature of the reaction was maintained below-70 ℃ and at-75 ℃ for 10 minutes. The temperature was maintained below-65 ℃ while adding an acetic acid solution (6mL in 44mL THF) to neutralize the medium. The mixture was then extracted with AcOEt. With 10% NaHCO₃The organic phase was washed with 10% citric acid, saturated NaCl and then concentrated under reduced pressure to give the crude product, which was used as such in the following step.

6a R₂＝CH₂Ph；R₃H: an orange oil; (yield: 93.0%); rf (CHex/AcOEt 6/4) 0.73

HPLC:Kromasil C18CH₃CN(0.1％TFA)/H₂O(0.1％TFA)60/40Rt:19.18min

NMR(CDCl₃,200MHz):2.85(1H,dd)；3.2(1H,dd)；3.55(1H,d)；3.6(2H,d)；3.7(3H,s)；4.1(2H,d)；6.7(2H,d)；7.2–7.4(7H,m)

6b R₂＝CH₂CH(CH₃)₂；R₃H: an orange oil; (yield: 94.0%); rf (CHex/AcOEt 5/5) 0.68

NMR(CDCl₃,200MHz):0.90–1.0(6H,m)；1.55(2H,m)；2.25(1H,m)；3.40(1H,d)；3.70(2H,s)；3.90(3H,s)；4.25(2H,d)；6.80(2H,d)；7.15(2H,d)

6c R₂＝CH₂(4–Br–Ph)；R₃H: an orange oil; (yield: 85.0%); rf (CHex/AcOEt 6/4) 0.80

NMR(CDCl₃,200MHz):2.85(1H,dd)；3.2(1H,dd)；3.55(1H,d)；3.6(2H,d)；3.7(3H,s)；4.1(2H,s)；6.7(2H,d)；7.2–7.4(6H,m)

6d R₂＝CH₂(4–Ph–Ph)；R₃H orange oil; (yield: 90.0%); rf (CHex/AcOEt 6/4) 0.73

NMR(CDCl₃,200MHz):2.85(1H,dd)；3.2(1H,dd)；3.55(1H,d)；3.6(2H,d)；3.7(3H,s)；4.1(2H,s)；6.7(2H,d)；7.2–7.5(11H,m)

Synthesis of geminal chloromethyl ketones

Bromoacetic acid (10g,72mmol) was dissolved in 50mL MeOH under an inert atmosphere. At 4 ℃ 11mL (1.1eq) of 4-methoxybenzylthiol were added and an alcoholic sodium hydroxide solution (6.4g NaOH (2.2eq) in 100mL MeOH) was added dropwise. The mixture was stirred at room temperature for 40 minutes. The solvent was evaporated under reduced pressure. The product was taken up in Et₂O (200mL) and 350mL 10% NaHCO₃In (1). The aqueous phase was acidified to pH 1 and then treated with 350mL Et₂And (4) extracting. By H₂The organic phase was washed with O (100mL), saturated NaCl (100mL) and Na₂SO₄Drying was carried out to obtain 15g of a crude white solid (yield: 98%), which was used as it was in the following step.

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 10-90% 15min, Rt 10.64min

NMR(CDCl₃,200MHz):3.0(2H,s)；3.75(3H,s)；3.75(2H,s)；6.80(2H,d)；7.20(2H,d)

Acetyl chloride (1.5 eq; 7.6 mL; 106mmol) is added dropwise to a solution of the aforementioned acid (70.8mmol) in 150mL of anhydrous MeOH at 4 ℃ under an inert atmosphere the mixture is stirred at room temperature overnight the mixture is concentrated under reduced pressure, taken up in MTBE (methyl-tert-butyl ether) (350mL) and the mixture is taken up in 0.5N HCl (2 × 100mL), 10% NaHCO₃(2×100mL)、H₂The organic phase was washed with O (100mL) and saturated NaCl (100 mL). Through Na₂SO₄The organic phase was dried and then concentrated under reduced pressure to give the methyl ester.

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) ladderDegree of 50-90% for 10min, Rt 5.24min

NMR(CDCl₃,200MHz):3.1(2H,s)；3.75(3H,s)；3.83(2H,s)；3.85(3H,s)；6.85(2H,d)；7.30(2H,d)。

To a solution of the aforementioned ester (1 g; 4.4 mmol; 1eq) dissolved in 5mL of anhydrous THF was added dropwise a solution of 1MLiHMDS in THF (4.4 mL; 1eq) under an inert atmosphere at-78 ℃. The mixture was stirred at-78 ℃ for 1 hour and then a solution of the derivative RX (1eq) was added at-78 ℃ under an inert atmosphere. The temperature was returned to room temperature, and the mixture was stirred for 3 hours. The mixture was again cooled to-78 ℃ and 1eq LiHMDS was added followed by 1.5eq RX. The temperature was returned to room temperature, and the mixture was stirred for 4 hours. The mixture was then partitioned between 200mL of 1N HCl and 300mL of AcOEt. Through Na₂SO₄The organic phase was dried and then concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography.

R₂＝CH₃，R₃＝CH₃: oil (yield: 44%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 50-90% 10min, Rt 7.39min

NMR(CDCl₃,200MHz):1.60(6H,s)；3.70(3H,s)；3.80(2H,s)；3.80(3H,s)；6.85(2H,d)；7.25(2H,d)

R₂＝C₂H₅，R₃＝C₂H₅: oil (yield: 55%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 50-90% 10min, Rt 9.72min

NMR(CDCl₃,200MHz):0.90(6H,t)；1.85(4H,m)；3.68(2H,s)；3.70(3H,s)；3.80(3H,s)；6.82(2H,d)；7.22(2H,d)

Chloromethyl ketones were synthesized as described in 4 b.

6e R₂＝CH₃，R₃＝CH₃: amber oil

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 50-90% 10min, Rt 7.91min

NMR(CDCl₃,200MHz):1.60(6H,s)；3.55(1H,d)；3.60(2H,s)；3.80(3H,s)；4.45(2H,s)；6.7(2H,d)；7.2(2H,d)

6f R₂＝C₂H₅，R₃＝C₂H₅: amber oil

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 50-90% 10min, Rt 10.04min

NMR(CDCl₃,200MHz):0.85(6H,t)；1.80(4H,q)；3.55(1H,d)；3.60(2H,s)；3.80(3H,s)；4.45(2H,s)；6.7(2H,d)；7.2(2H,d)

Synthesis of cyclic chloromethyl ketones

The method comprises the following steps:

A2.5M solution of BuLi in hexane (2.77 mmol; 1.1 mL; 1.2eq) was added to a solution of DIPA (diisopropylethylamine) (3 mmol; 1.3 eq; 420. mu.L) in THF (9mL) at-10 ℃ under an inert atmosphere. The mixture was stirred at 0 ℃ for 1 hour. This LDA solution, freshly prepared, was added dropwise to a solution of methyl pivalate in 5mL of THF at-55 ℃. The mixture was stirred at-55 ℃ for 1 hour under an inert atmosphere. HMPA (hexamethylphosphoramide) (3.46 mmol; 1.5 eq; 610. mu.L) was added and the mixture was stirred at the same temperature for 10 min. Then, a solution of 4-methoxybenzylthiol disulfide (3 mmol; 1.3 eq; 920mg) in 12mL of THF was added dropwise at-55 ℃. After returning to room temperature, the mixture was stirred overnight. The mixture was partitioned between 10mL of saturated NH₄Between Cl and 20mL AcOEt. With saturated NH₄The organic phase was washed with Cl (2 × 10mL), saturated NaCl (2 × 15mL)Over Na₂SO₄Dried and then concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography.

C(R₂R₃) Cyclopentyl: oil (yield: 40%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 30-90% 10min, Rt 9.68min

NMR(CDCl₃,200MHz):1.60–2.40(8H,m)；3.67(3H,s)；3.77(2H,s)；3.80(3H,s)；6.83(2H,d)；7.24(2H,d)

Chloromethyl ketones were synthesized as described in 4 b.

6g C(R₂)(R₃) Cyclopentyl: amber oil

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 60-90% 10min, Rt 6.47min

NMR(CDCl₃,200MHz):1.60–1.80(8H,m)；3.54(2H,s)；3.80(3H,s)；4.47(2H,s)；6.78(2H,d)；7.22(2H,d)

6h C(R₂)(R₃) Cyclohexyl: amber oil

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 8.11min

NMR(CDCl₃,200MHz):1.40–2.20(10H,m)；3.45(2H,s)；3.80(3H,s)；4.40(2H,s)；6.80(2H,d)；7.20(2H,d)

The method 2 comprises the following steps:

A2.5M solution of BuLi in hexane (16 mmol; 6.4 mL; 1.15eq) was added to DIPA (diisopropylethylamine) (16.7 mmol; 1.2 eq; 2.3. mu.l) at-10 ℃ under an inert atmosphere4mL) in THF (10 mL). The mixture was stirred at 0 ℃ for 1 hour. This freshly prepared LDA solution was neutralized HMPA (hexamethylphosphoramide) (1.0 eq; 2.5mL) by the addition of methyl phenylcyclopentanoate (THF) in 5mL of THF dropwise at-78 deg.C. The mixture was stirred at-78 ℃ for 1 hour under an inert atmosphere. Solid activated 4-methoxybenzylthiol (18 mmol; 1.3 eq; 6.37mg) was added at-78 ℃. The mixture was stirred at-78 ℃ for 1.5 hours. The mixture was partitioned between 200mL of 1N HCl and 200mL of AcOEt. The organic phase was diluted with 200mL AcOEt, washed with saturated NaCl (200mL), over Na₂SO₄Dried and then concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography.

C(R₂R₃) Phenyl cyclopentyl (indanyl): white solid (yield: 25%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 50-90% 10min, Rt 9.59min

NMR(CDCl₃,200MHz):2.90–3.70(6H,m)；3.55(3H,s)；3.65(3H,s)；6.60–7.10(8H,m)

The product of the previous step (1.13 g; 3.44mmol) was dissolved in 14mL of THF/MeOH mixture. 14mL of 2N NaOH was added and the mixture was stirred at room temperature for 3 hours. Using 40mL of H₂The mixture was diluted O, the mixture was concentrated under reduced pressure, the aqueous phase was acidified with 1N HCl, then extracted with MTBE (3 × 100mL), washed with saturated NaCl (100mL), and Na₂SO₄Dried and then concentrated under reduced pressure to obtain a white solid (yield: 98%).

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 50-90% 10min, Rt 6.50min

Under an inert atmosphere, at 0 ℃ in the presence of 20. mu.L DMF (0.2eq) in 10mL CH of the aforementioned acid (1.38 mmol; 434mg)₂Cl₂Oxalyl chloride (2 mmol; 1.5 eq; 177. mu.L) was added dropwise to the solution in (1). The mixture was allowed to return to room temperature and then stirred at room temperature for 30 minutes.

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 6.70 min.

After evaporation under reduced pressure, the acid chloride is absorbed in anhydrous CH under an inert atmosphere₃CN (5mL) and TMSCHN was added dropwise at 0 ℃₂(1M in Et₂O) (1.5 eq; 1 mL). The mixture was allowed to return to room temperature and then stirred for 1.5 hours. The mixture was then captured with 300. mu.L (1.65 mmol; 1.2eq) of 33% HBr in acetic acid. The mixture was stirred at room temperature for 15 minutes. The mixture was concentrated under reduced pressure and then taken up in MTBE (200 mL). With 10% NaHCO₃The organic phase was washed (100mL), saturated NaCl (50mL), and Na₂SO₄Drying and then concentration under reduced pressure gave compound 6h as a brown oil (yield: 83%).

6h C(R₂R₃) Phenyl cyclopentyl (indanyl): white solid

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 5.30min

NMR(CDCl₃,200MHz):2.90–3.70(6H,m)；3.65(3H,s)；4.15(2H,s)；6.60–7.10(8H,m)

1.1.3Step 3

Method 1

980mg of 60% NaH (1eq) were added to a dialkyl malonate (1eq) in 25mL (1mL/mmol) of DME (1, 2-dimethoxyethane) under an inert atmosphere. The mixture was stirred at room temperature for 1 hour.

A mixture of chloromethyl ketone 6(24.42mmol) and NaI (24.42mmol,3.66g,1eq) in 50mL DME was stirred at room temperature for 15 minutes before it was added to the freshly prepared solution of the sodium dialkylmalonate. The mixture was stirred at room temperature for 4 hours.

At the end of the reaction, the solvent was evaporated under reduced pressure. The product was taken up in dichloromethane. The organic phase is washed with water and Na₂SO₄And (5) drying. The product was purified by silica gel column chromatography with CHex/AcOEt9/1 as the eluent.

7a1 R₂＝CH₂Ph，R₃＝H，R₁₃＝CH₂CH₃: an orange oil; (yield: 60%); rf (CHex/AcOEt 9/1): 0.16

HPLC:Kromasil C18CH₃CN/H₂O(0.1％TFA)80/20Rt:6.57min

NMR(CDCl₃,200MHz):1.30(6H,t)；2.70–3.40(4H,m)；3.45(1H,t)；3.50(2H,d)；3.60(1H,t)；3.65(3H,s)；4.15(4H,q)；6.75(2H,d)；7.2–7.4(7H,m)

7a2 R₂＝CH₂Ph，R₃＝H，R₁₃tBu: an orange oil; (yield: 62%); rf (CHex/AcOEt 9/1): 0.36

HPLC:Kromasil C18CH₃CN/H₂O(0.1％TFA)85/15Rt:15.51min

NMR(CDCl₃,200MHz):1.40(18H,s)；2.70–3.40(4H,m)；3.45(1H,t)；3.50(2H,d)；3.60(1H,t)；3.65(3H,s)；6.75(2H,d)；7.2–7.4(7H,m)

7b R₂＝CH₂CH(CH₃)₂，R₃＝H，R₁₃tBu: an orange oil; (yield: 30%); rf (CHex/AcOEt 9/1): 0.49

HPLC:Kromasil C18CH₃CN/H₂O(0.1％TFA)90/10Rt:6.49min

NMR(CDCl₃,200MHz):0.90–1.0(6H,m),1.40(18H,s)；1.55(2H,m)；2.15(1H,m)；3.19(2H,m)；3.40(2H,m)；3.50(2H,d)；3.80(3H,s)；6.75(2H,d)；7.2(2H,d)

7c R₂＝CH₂(4–Br–Ph)，R₃＝H，R₁₃tBu: an orange oil; (yield: 62%); rf (CHex/AcOEt 9/1): 0.36

HPLC:Kromasil C18CH₃CN/H₂O(0.1％TFA)85/15Rt:15.51min

NMR(CDCl₃,200MHz):1.40(18H,s)；2.70–3.40(4H,m)；3.45(1H,t)；3.50(2H,d)；3.60(1H,t)；3.65(3H,s)；6.75(2H,d)；7.2–7.4(6H,m)

7d R₂＝CH₂(4–Ph–Ph)，R₃＝H，R₁₃tBu: an orange oil; (yield: 60%); rf (CHex/AcOEt 9/1): 0.36

HPLC:Kromasil C18CH₃CN/H₂O(0.1％TFA)85/15Rt:16.71min

NMR(CDCl₃,200MHz):1.40(18H,s)；2.70–3.40(4H,m)；3.45(1H,t)；3.50(2H,d)；3.60(1H,t)；3.65(3H,s)；6.75(2H,d)；7.2–7.5(11H,m)

7e R₂＝CH₃，R₃＝CH₃，R₁₃Et (yield: 40%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 4.87min

NMR(CDCl₃,200MHz):1.30(6H,t)；1.55(6H,s)；3.40(2H,d)；3.55(2H,s)；3.75(1H,t)；3.82(3H,s)；4.25(4H,q)；6.85(2H,d)；7.25(2H,d)

7f R₂＝C₂H₅，R₃＝C₂H₅，R₁₃Et (yield: 30%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 50-90% 10min then 90-50%, Rt 11.56min

NMR(CDCl₃,200MHz):0.80(6H,t)；1.20(6H,t)；1.75(4H,m)；3.30(2H,s)；3.38(2H,d)；3.65(1H,t)；3.72(3H,s)；4.15(4H,q)；6.72(2H,d)；7.15(2H,d)

7g C(R₂)(R₃) Is cyclopentyl, R₁₃: et (yield: 24%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 60-90% 10min, Rt 8.04min

NMR(CDCl₃,200MHz):1.29(6H,t)；1.60–2.30(8H,m)；3.39(2H,d)；3.52(2H,s)；3.74(1H,t)；3.79(3H,s)；4.22(4H,q)；6.83(2H,d)；7.20(2H,d)

7h C(R₂)(R₃) Cyclohexyl radical, R₁₃: et (yield: 33%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 7.76min

NMR(CDCl₃,200MHz):1.29(6H,t)；1.60–2.30(10H,m)；3.45(2H,d)；3.50(2H,s)；3.75(1H,t)；3.80(3H,s)；4.25(4H,q)；6.85(2H,d)；7.20(2H,d)

7i C(R₂)(R₃) Phenyl cyclopentyl (indanyl), R₁₃: et (yield: 80%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 6.57min

NMR(CDCl₃,200MHz):1.20(6H,t)；2.90–3.60(9H,m)；3.65(3H,s)；4.15(4H,q)；6.60–7.10(8H,m)

Alkylation of malonic esters

To a solution of product 7 in 15mL DME (dimethoxyethane) was added 1.5eq 60% NaH.

The mixture was stirred at room temperature for 1 hour, then bromine was addedChemical derivative R₄Br (3 eq). The mixture was stirred at room temperature overnight.

The solvent was evaporated under reduced pressure and the mixture was taken up in H₂O and AcOEt. By H₂O washing the organic phase over Na₂SO₄Dried and then concentrated under reduced pressure.

The product was purified by silica gel chromatography, using CHex/AcOEt9/1 as eluent, to give the desired product 8.

8a R₂＝CH₂Ph；R₃＝H；R₄＝CH₂(4–Br–Ph)；R₁₃Et: orange oil

8b R₂＝CH₂Ph；R₃＝H；R₄＝CH₂Ph；R₁₃tBu: orange oil

8c R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Ph–Ph)；R₁₃Et: orange oil

8d R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂Ph；R₁₃Et: orange oil

8e R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Br–Ph)；R₁₃Et: orange oil

8f R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂Ph；R₁₃tBu: orange oil

8g R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂(4–Br–Ph)；R₁₃tBu: orange oil

8h R₂＝CH₂(4–Ph–Ph)；R₃＝H；R₄＝CH₂(4–Br–Ph)；R₁₃tBu: orange oil

8i R₂＝CH₃；R₃＝CH₃；R₄＝CH₂(4–Br–Ph)；R₁₃Et: orange oil

8j R₂＝C₂H₅；R₃＝C₂H₅；R₄＝CH₂(4–Br–Ph)；R₁₃Et: orange oil

8k C(R₂)(R₃) Cyclopentyl; r₄＝CH₂(4–Br–Ph)；R₁₃Et: orange oil

8l C(R₂)(R₃) Cyclohexyl group; r₄＝CH₂(4–Br–Ph)；R₁₃Et: orange oil

8m C(R₂)(R₃) Phenyl cyclopentyl (indanyl); r₄＝CH₂(4–Br–Ph)；R₁₃Et: orange oil

1.1.4Step 4

When R is₁₃For methylethyl, compound 8(0.585mmol) was dissolved in 10mL EtOH and 2N NaOH (6eq) was added. The mixture was stirred at room temperature overnight. The ethanol was evaporated under reduced pressure. The product was taken up in water and Et₂And (4) extracting. The aqueous phase was acidified with 3N HCl and Et₂And (4) extracting. The organic phase is passed through Na₂SO₄Dried and then evaporated under reduced pressure to give a yellow oil.

When R is₁₃For t-butyl, dissolve product 8 in 10mL CH₂Cl₂To this was added 10mL of TFA. The mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure. The product was taken up in water and Et₂And (4) extracting. The organic phase is passed through Na₂SO₄Dried and then evaporated under reduced pressure to give a yellow oil.

The resulting product was then dissolved in 6mL of toluene and heated to 150 ℃ for 12 hours.

The solvent was evaporated under reduced pressure to give compound 9.

9a R₂＝CH₂Ph；R₃＝H；R₄＝CH₂(4-Br-Ph); orange oil

9b R₂＝CH₂Ph；R₃＝H；R₄＝CH₂Ph; orange oil

9c R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4-Ph-Ph); orange oil

9d R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂Ph; orange oil

9e R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4-Br-Ph); orange oil

9f R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂Ph; orange oil

9g R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); orange oil

9h R₂＝CH₂(4–Ph–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); orange oil

9i R₂＝CH₃；R₃＝CH₃；R₄＝CH₂(4-Br-Ph); orange oil

9j R₂＝C₂H₅；R₃＝C₂H₅；R₄＝CH₂(4-Br-Ph); orange oil

9k C(R₂)(R₃) Cyclopentyl; r₄＝CH₂(4-Br-Ph); orange oil

9l C(R₂)(R₃) Cyclohexyl group; r₄＝CH₂(4-Br-Ph); orange oil

9m C(R₂)(R₃) Phenyl cyclopentyl (indanyl); r₄＝CH₂(4-Br-Ph); orange oil

Suzuki reaction

Compound 9l (180 mg; 0.356mmol) was dissolved in 2mL of toluene under an inert atmosphere. Adding Pd (PPh)₃)₄(11 mg; 3% mol) and the mixture was stirred at room temperature for 5 minutes. Phenylboronic acid (46 mg; 0.374 mmol; 1.05eq) was added to 1mL of MeOH followed by 500. mu.L of 2M Na₂CO₃. The mixture was refluxed for 1.5 hours, and then concentrated under reduced pressure. The reaction mixture was taken up in 30mL Et₂O and 30mL of 1N HCl. The organic phase was washed with 20mL of 1N HCl, 10mL of saturated NaCl and Na₂SO₄Dried and concentrated under reduced pressure. The product was purified by silica gel chromatography, eluting with Hept/AcOEt 65/35 to give the desired product 9 n.

9n C(R₂)(R₃) Cyclohexyl group; r₄＝CH₂(4-Ph-Ph); 75mg of an orange oil (yield: 47%)

ESI(+)：[M+Na]⁺＝525.2

HPLC：Atlantis T3CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 8.60min

The following compounds were obtained according to the same scheme.

9o C(R₂)(R₃) Cyclopentyl; r₄＝CH₂(4-Ph-Ph); orange oil (yield: 25%)

ESI(–)：[M–H]^–＝487.2

HPLC：Atlantis T3CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 6.60min

9p C(R₂)(R₃) Phenyl cyclopentyl (indanyl); r₄＝CH₂(4-Ph-Ph); orange oil (yield: 55%)

ESI(–)：[M–H]^–＝535.2

HPLC：Atlantis T3CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 70-90% 10min, Rt 7.45min

1.1.5Step 5

The method comprises the following steps:compound 9 from step 7 (0.53mmol) was dissolved in 2.1mL BTFA (tris (trifluoroacetic acid) borane) (1M) (previously prepared from BBr)₃And TFA preparation) and stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure and the mixture was purified by semi-preparative HPLC.

10a R₂＝CH₂Ph；R₃＝H；R₄＝CH₂Ph

ESI(+)：[M+H]⁺＝329

HPLC：ACE C18CH₃CN/H₂Gradient O (0.1% TFA) over 60/9030 min; rt: 7.50min

10b R₂＝iBu；R₃＝H；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺372 and 374

HPLC：ACE C18CH₃CN/H₂Gradient of O (0.1TFA) over 60/9030 min; rt: 8.60min

10c R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺＝384

HPLC：ACE C18CH₃CN/H₂Gradient O (0.1% TFA) over 60/9030 min; rt: 10.52min

10d R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂Ph

ESI(+)：[M+H]⁺406 and 408

HPLC：ACE C18CH₃CN/H₂Gradient O (0.1% TFA) over 60/9030 min; rt: 9.01min

10e R₂＝CH₂Ph；R₃＝H；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺406 and 408

HPLC：Kromasil C18CH₃CN/H₂O(0.1％TFA)80/20；Rt：6.23min

10f R₂＝CH₂(4–Ph–Ph)；R₃＝H；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺482 and 484

HPLC：ACE C18CH₃CN/H₂Gradient O (0.1% TFA) over 60/9030 min; rt: 11.4min

The method 2 comprises the following steps:the product 9c (2.03mmol) was dissolved in 10mL TFA in the presence of 5eq anisole (1.1 mL). The mixture was heated at 50 ℃ for 2.5 hours. The solvent was evaporated under reduced pressure and the mixture purified by semi-preparative HPLCTo give compound 10 b.

10b R₂＝iBu；R₃＝H；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺372 and 374

HPLC：ACE C18CH₃CN/H₂Gradient O (0.1% TFA) over 60/9030 min; rt: 8.60min

The method 3 comprises the following steps:to a solution of compound 9c (0.447 mmol; 120mg) in 4.5mL TFA under an inert atmosphere was added thioanisole (2.8 eq; 0.936 mmol; 110. mu.L) followed by 2, 2' -dithiobis (5-nitropyridine) (3 eq; 1.0 mmol; 417mg) and the solution turned orange. The mixture was stirred at room temperature for 1 hour. The mixture was concentrated at 30 ℃ under reduced pressure. The disulfide formed was purified by semi-preparative HPLC to give 146mg of the product (yield: 62%).

Dissolving the disulfide in CH₃CN(720μL)/H₂O (180. mu.L). Tris (2-carboxyethyl) phosphine hydrochloride (1.2 eq; 96mg) was then added and the mixture was stirred at room temperature for 10 minutes. The solvent was evaporated under reduced pressure and the compound was purified by semi-preparative HPLC to give 10 b.

If chirality is present, the synthetic scheme preserves the chirality of the molecule.

10b R₂＝iBu；R₃＝H；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺372 and 374

HPLC：ACE C18CH₃CN/H₂O (0.1% TFA) 60/9030 min; rt: 8.60min

NMR(CDCl₃,200MHz):0.87(3H,d)；0.89(3H,d)；1.50–1.80(3H,m)；2.7–3.5(6H,m)；7.08(2H,d)；7.43(2H,d)

The following compounds were obtained according to the same scheme.

10g R₂＝CH₃；R₃＝CH₃；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+Na]⁺366 and 368

HPLC：Atlantis T3CH₃CN/H₂O (0.1% TFA) 50/9010 min; rt: 6.51min

NMR(DMSO d6,200MHz):1.45(3H,s)；1.47(3H,s)；2.7–3.2(5H,m)；7.18(2H,d)；7.50(2H,d)

10h R₂＝C₂H₅；R₃＝C₂H₅；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+Na]⁺394 and 396

HPLC：Atlantis T3CH₃CN/H₂O (0.1% TFA) 70/9010 min; rt: 3.9min

NMR(DMSO d6,200MHz):1.30(6H,t)；1.75(4H,q)；2.6–3.1(5H,m)；7.0(2H,d)；7.40(2H,d)

10i C(R₂)(R₃)＝C₅H₈；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺370 and 372

HPLC：Atlantis T3CH₃CN/H₂O (0.1% TFA) 60/9010 min; rt: 5.0min

NMR(CDCl₃,200MHz):1.60–2.25(8H,m)；2.75–3.20(5H,m)；7.10(2H,d)；7.44(2H,d)

10j C(R₂)(R₃)＝C₆H₁₀；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+Na]⁺382 and 384

HPLC：Atlantis T3CH₃CN/H₂O (0.1% TFA) 70/9010 min; rt: 8.97min

NMR(CDCl₃,200MHz):1.60–2.40(10H,m)；2.75–3.20(5H,m)；7.10(2H,d)；7.44(2H,d)

10k C(R₂)(R₃)＝C₉H₈；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+Na]⁺442 and 444

HPLC：Atlantis T3CH₃CN/H₂O (0.1% TFA) 70/9010 min; rt: 3.84min

NMR(CDCl₃,200MHz):2.70–3.50(5H,m)；3.60(2H,d)；3.70(2H,d)；7.10–7.6(8H,m)

10l C(R₂R₃)＝C₆H₁₀；R₄＝CH₂(4–Ph–Ph)

ESI(+)：[M+H]⁺＝383

HPLC：Atlantis T3CH₃CN/H₂O (0.1% TFA) 70/9010 min; rt: 5.35min

NMR(CDCl₃,200MHz):1.60–2.40(10H,m)；2.75–3.20(5H,m)；7.20–7.60(9H,m)

10m C(R₂R₃)＝C₅H₈；R₄＝CH₂(4–Br–Ph)

ESI(+)：[M+H]⁺368 and 370

HPLC：Atlantis T3CH₃CN/H₂O(0.1％TFA)60/40；Rt：9.79min

NMR(CDCl₃,200MHz):1.60–2.25(8H,m)；2.75–3.20(5H,m)；7.05–7.55(9H,m)

10n C(R₂)(R₃)＝C₉H₈；R₄＝CH₂(4–Ph–Ph)

ESI(+)：[M+H]⁺＝415

HPLC：Atlantis T3CH₃CN/H₂O (0.1% TFA) 70/9010 min; rt: 4.73min

NMR(CDCl₃,200MHz):2.70–3.50(5H,m)；3.60(2H,d)；3.70(2H,d)；7.10–7.8(13H,m)

1.2Synthesis of NEP-APN mixed inhibitors

1.2.1Synthesis of boc- β -aminothiol 11

Compounds were prepared according to the protocol described in j.med.chem., 35, 1992, 2473.

11a R₁：CH₂CH₂SCH₃(ii) a White solid

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂O (0.1% TFA) gradient 50-90% 10min, Rt 6.45min

NMR(CDCl₃,200MHz):1.45(9H,s)；1.85(2H,m)；2.12(3H,s)；2.52(2H,t)；2.75(2H,dd)；3.90(1H,t)；4.80(1H,NH)

11b R₁：CH₂CH₂CH₂CH₃(ii) a White solid

HPLC：ACE C18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)30％–70％，Rt＝13.53min

NMR(DMSO d6,200MHz):0.95(3H,t)；1.20–1.60(6H,m)；1.40(9H,s)；2.30(2H,m)；3.40(1H,t)；6.80(1H,NH)

11c R₁：CH₂CH₂OCH₃(ii) a White solid

HPLC：ACE C18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)50％–50％，Rt＝5.58min

NMR(DMSO d6,200MHz):1.40(9H,s)；1.60(2H,m)；2.75–3.10(2H,m)；3.20(3H,s)；3.30(2H,t)；3.60(1H,m)；6.80(1H,NH)

11d R₁：CH₂CH₂OCH₂CH₃(ii) a White solid

HPLC：ACE C18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)40％–60％，Rt＝13.33min

NMR(DMSO d6,200MHz):1.10(3H,t)；1.40(9H,s)；1.60(2H,m)；2.60(2H,m)；3.10–3.30(2H,m)；3.30(2H,q)；3.60(1H,m)；6.80(1H,NH)

11e R₁：CH₂OCH₂CH₃(ii) a White solid

HPLC：ACE C18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)40％–60％，Rt＝14.00min

NMR(DMSO d6,200MHz):1.10(3H,t)；1.40(9H,s)；3.20–3.40(4H,m)；3.30(2H,q)；3.60(1H,m)；6.70(1H,NH)

1.2.2Synthesis of asymmetric disulfide 12

Boc-aminothiol 11(9.86mmol,2.5g) was dissolved in 20mL degassed MeOH under an inert atmosphere at 0 deg.C. Et was added₃N (2 eq; 2.79mL) was added followed by methoxycarbonylsulfonyl chloride (2eq,1.78mL) in 20mL degassed CHCl₃The solution of (1). The mixture was stirred at 0 ℃ for 15 minutes, then 100mL of CHCl was added₃With 10% citric acid (2 × 100mL), H₂The organic phase was washed with O (100mL), saturated NaCl (100mL), and Na₂SO₄Dried and concentrated under reduced pressure.

The product was purified on silica gel.

R₁：CH₂CH₂SCH₃(ii) a White solid (yield: 40%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70-90% within 10min, Rt 3.66min

NMR(CDCl₃,200MHz):1.45(9H,s)；1.85(2H,m)；2.12(3H,s)；2.52(2H,t)；2.75(2H,dd)；3.90(1H,t)；3.90(3H,s)；5.0(1H,NH)

R₁：CH₂CH₂CH₂CH₃(ii) a White solid (yield: 41%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70-90% within 10min, Rt 5.57min

NMR(CDCl₃,200MHz):0.90(3H,t)；1.34(4H,m)；1.45(9H,s)；1.62(2H,m)；2.99(2H,d)；3.78(1H,m)；3.90(3H,s)；4.77(1H,NH)

Under an inert atmosphere, compound 10 was added to the previous compound (0.754mmol,1eq) in 8mL degassed CHCl₃In the solution of (1). The mixture was cooled to-10 ℃ and degassed Et was added₃N (0.754mmol, 105. mu.l, 1 eq). The mixture was stirred at-10 ℃ for 30 minutes, then 10mL CH was used₂Cl₂Dilute, wash the organic phase with 10% citric acid (5mL), saturated NaCl (2 × 10mL), and Na₂SO₄Drying to give the crude product, which was purified by semi-preparative HPLC to give compound 12.

12a–b R₁：CH₂CH₂SCH₃；R₂：iBu；R₃：H；R₄：CH₂(4-Br-Ph) (yield: 64%)

ESI(+)：[M+Na]⁺644 and 646 ═

12b–b R₁：CH₂CH₂CH₂CH₃；R₂：iBu；R₃：H；R₄：CH₂(4-Br-Ph) (yield: 87%)

ESI(+)：[M+Na]⁺626 and 628

12b–l R₁：CH₂CH₂CH₂CH₃；C(R₂R₃)：C₆H₁₀；R₄：CH₂(4-Ph-Ph) (yield: 85%)

ESI(+)：[M+Na]⁺＝637

12b–j R₁：CH₂CH₂CH₂CH₃；C(R₂R₃)：C₆H₁₀；R₄：CH₂(4-Br-Ph) (yield: 90%)

ESI(+)：[M+Na]⁺638 and 640 ═ b

12a–l R₁：CH₂CH₂SCH₃；C(R₂R₃)：C₆H₁₀；R₄：CH₂(Ph-Ph) (yield: 90%)

ESI(+)：[M+H]⁺＝632

1.2.3Preparation of Compound 1

Method 1

Part 1: synthesis of esters

Compound 13 a-b-1: 1- (ethoxycarbonyloxy) ethyl 2- (4-bromobenzyl) -5- (((S) -2- (tert-butoxycarbonylamino) -4- (methylthio) butyl) disulfide) -7-methyl-4-oxooctanoate

Compounds 12 a-b (640 mg; 1.03mmol) and Et₃N (730. mu.L, 5eq) was dissolved in 10mL AcOEt. The mixture was stirred at room temperature for 15 minutes. Ethyl-1-chloroethyl carboxylate (prepared according to Barcelo et al Synthesis, 1986, 627) (800. mu.L; 5eq) and NaI (800mg,5eq) were added. The mixture was refluxed for 3 hours. Using 10mL of H₂Dilute the mixture with 20mL AcOEt extract the aqueous phase with 3 × 30mL AcOE 10% citric acid(2×15mL)、10％NaHCO₃(2 × 15mL), the organic phase was washed with saturated NaCl and Na₂SO₄Dried and evaporated under reduced pressure to give the crude product. The mixture was purified by semi-preparative HPLC to give 80mg of a yellow oil.

13a–b–1 R₁：CH₂CH₂SCH₃；R₂：iBu；R₃：H；R₄：CH₂(4–Br–Ph)；R₅：CH(CH₃)OCOOC₂H₅(yield 10.5%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70-90% within 10min, Rt 12.85min

ESI(+)：[M+Na]⁺760 and 762 ═ e

Compound 13 b-b-1: 2- (4-bromobenzyl) -5- (((S) -2- (tert-Butoxycarbonylamino) hexyl) dithio) -7-methyl-4-oxooctanoic acid ethyl ester

Compound 12 b-b (395 mg; 0.653mmol) was dissolved in 4.3mL CH at 4 deg.C under an inert atmosphere₂Cl₂In (1). EDCI-HCl (138 mg; 0.718 mmol; 1.1eq) was added, followed by DMAP (88 mg; 0.718 mmol; 1.1eq) and EtOH (0.784 mmol; 1.2 eq). The mixture was stirred at room temperature for 4 hours. With 10mL of 10% citric acid and 20mL of CH₂Cl₂Dilute the mixture with 10% citric acid (2 × 10mL), 10% NaHCO₃(2 × 10mL), the organic phase was washed with saturated NaCl and Na₂SO₄Dried and evaporated under reduced pressure to give the crude product. The mixture was purified by semi-preparative HPLC to give a yellow oil.

13b–b–1 R₁：CH₂CH₂CH₂CH₃；R₂：iBu；R₃：H；R₄：CH₂(4–Br–Ph)；R₅：C₂H₅(yield 60%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 80-90% within 10min, Rt ═12.20–12.65min

Part 2: deprotection of amines

Compound 1 a-b-1: 1- (ethoxycarbonyloxy) ethyl 5- (((S) -2-amino-4- (methylthio) butyl) dithio) -2- (4-bromobenzyl) -7-methyl-4-oxooctanoate

Compound 13 a-b-1 (130 mg; 0.176mmol) was stirred in 2mL of HCOOH for 1 h. The mixture was evaporated under reduced pressure to give the crude product, which was purified by semi-preparative HPLC to give 13mg of compound 1 a-b-1.

1a–b–1R₁：CH₂CH₂SCH₃；R₂：iBu；R₃：H；R₄：CH₂(4–Br–Ph)；R₅：CH(CH₃)OCOOC₂H₅(yield 11%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 10-90% 15min, Rt 14.50min

ESI(+)：[M+H]⁺638 and 640 ═ b

Compound 1 b-b-1: 5- (((S) -2-Aminohexyl) dithio) -2- (4-bromobenzyl) -7-methyl-4-oxooctanoic acid ethyl ester

Compound 13 b-b-1 (141 mg; 0.223mmol) was stirred in 2mL of HCOOH for 1 h. The mixture was evaporated under reduced pressure to give the crude product, which was purified by semi-preparative HPLC to give 92mg of compound 1 b-b-1.

1b–b–1R₁：CH₂CH₂CH₂CH₃；R₂：iBu；R₃：H；R₄：CH₂(4–Br–Ph)；R₅：C₂H₅(yield 64%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) over 50-90% 10min, Rt 7.79min

ESI(+)：[M+H]⁺532 and 534

Compound 1 f-b-1: 5- ((2-amino-4- (methylsulfinyl) butyl) dithio) -2- (4-bromobenzyl) -7-methyl-4-oxooctanoic acid ethyl ester

1f–b–1R₁：CH₂CH₂SOCH₃；R₂：iBu；R₃：H；R₄：CH₂(4–Br–Ph)；R₅：C₂H₅(yield 47.9%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 10-90% within 15min, Rt 12.45min

ESI(+)：[M+H]⁺566 and 568

Method 2

Compound 12(0.323mmol) was stirred in 4mL HCOOH for 1 h. The mixture was evaporated under reduced pressure to give the crude product, which was purified by semi-preparative HPLC to give the acid.

R₁：CH₂CH₂SCH₃；R₂：iBu；R₃：H；R₄：CH₂(4-Br-Ph) (69% yield)

HPLC：ACEC18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)60/40％，Rt＝8.0min

ESI(+)：[M+H]⁺522 and 524

R₁：CH₂CH₂CH₂CH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4-Ph-Ph) (yield 60%)

HPLC：Luna T3，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)60/40％，Rt＝2.83min

ESI(+)：[M+H]⁺＝514

R₁：CH₂CH₂CH₂CH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4-Br-Ph) (69% yield)

HPLC：ACE C18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)60/40％，Rt＝8.0min

ESI(+)：[M+H]⁺516 and 518

R₁：CH₂CH₂SCH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4-Ph-Ph) (yield 70%)

HPLC：Luna C18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)50/50％，Rt＝5.43min

ESI(+)：[M+H]⁺＝532

Under an inert atmosphere, at 0 ℃ SOCl₂(30. mu.L, 6eq) was added to the acid (0.068mmol) in 400. mu.L of anhydrous EtOH (R)₅Et). The solution became clear. The mixture was stirred at room temperature overnight. The mixture was evaporated under reduced pressure to give the crude product, which was purified by semi-preparative HPLC to give 35mg of the expected product.

1a–b–2R₁：CH₂CH₂SCH₃；R₂：iBu；R₃：H；R₄：CH₂(4–Br–Ph)；R₅：C₂H₅(yield 82%)

HPLC：ACE C18，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 50/90% within 30min, Rt 11.98min

ESI(+)：[M+H]⁺550 and 552

1b–l–1R₁：CH₂CH₂CH₂CH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4–Ph–Ph)；R₅：C₂H₅(yield 80%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70/90% within 10min, Rt 4.0min

ESI(+)：[M+H]⁺＝542

1b–j–1R₁：CH₂CH₂CH₂CH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4–Br–Ph)；R₅：C₂H₅(yield 85%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70/90% within 10min, Rt 3.28min

ESI(+)：[M+H]⁺546 and 548 ═ c

1a–l–1R₁：CH₂CH₂SCH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4–Ph–Ph)；R₅：C₂H₅(yield 86%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 50/90% within 10min, Rt 8.42min

ESI(+)：[M+H]⁺＝560

1b–l–2R₁：CH₂CH₂CH₂CH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4–Ph–Ph)；R₅：CH₂Ph (yield 71%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70/90% within 10min, Rt 5.71min

ESI(+)：[M+H]⁺＝604

Method 3

Compounds 1 b-l-3: 2- (Biphenyl-4-ylmethyl) -4- (1- ((2- ((1- (isobutyryloxy) ethoxy) carbonylamino) (S) -hexyl) disulfide) cyclohexyl) -4-oxobutanoic acid

Compound 12 b-l (590 mg; 0.961mmol) is stirred in 10mL of HCOOH for 1 h. The mixture was evaporated under reduced pressure to give the crude product, which was purified by semi-preparative HPLC to give 2 diastereomers.

R₁：CH₂CH₂CH₂CH₃；C(R₂R₃)：C₆H₁₀；R₄：CH₂(4-Ph-Ph) (89% yield)

HPLC Dia 1：LunaC18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)55/35％，Rt＝3.90min

HPLC Dia 2：Luna C18，CH₃CN(0.1％TFA)/H₂O(0.1％TFA)55/35％，Rt＝4.10min

ESI(+)：[M+H]⁺＝522.2

The aforementioned compound (155mg,0.246mmol) was dissolved in 2mL of anhydrous CH₃In CN. 370 μ L of 2N NaHCO was added₃Then 1mL of H was added₂And O. The mixture was stirred at room temperature for 10 minutes and CH was added₃1- ((2, 5-dioxopyrrolidin-1-yloxy) carbonyloxy) ethylisobutyrate (90 mg; 0.329 mmol; 1eq) in CN (1mL) (Cundy et al (2004) J. pharm. exp. thermal, 311, 315-323). The mixture was stirred at 60 ℃ for 30 minutes. The solvent was evaporated under reduced pressure. The product was taken up in AcOEt, 1N HCl. Washing the organic phase over Na₂SO₄Dried and concentrated under reduced pressure to give the crude product, which was purified by semi-preparative HPLC to give the desired product.

1b–l–3dia 1R₁：CH₂CH₂CH₂CH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4-Ph-Ph) (yield 85%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70/90% within 15min, Rt 10.50min and 10.80min

ESI(+)：[M+H]⁺＝672.1

1b–l–3dia 2R₁：CH₂CH₂CH₂CH₃；C(R₂)(R₃)：C₆H₁₀；R₄：CH₂(4-Ph-Ph) (yield 85%)

HPLC：Atlantis T3，CH₃CN(0.1％TFA)/H₂Gradient of O (0.1% TFA) 70/90% within 15min, Rt 10.80min and 11.10min

ESI(+)：[M+H]⁺＝672.1

2.Measurement of inhibition intensity

The assay is performed in 96-well plates in the presence of a specific fluorogenic substrate. The emitted fluorescence was read in a Berthold Twinkle LS970B plate reader. Inhibition was then plotted against inhibitor concentration using GraphPad software, and Ki was then determined from the Cheng Prusoff equation: ki ═ IC₅₀/(1+(S/Km))。

The inhibition intensity for 2 target enzymes was determined as follows:

cleavage of the N-and/or C-terminal protecting groups and disulfide bonds in situ starting from prodrug 1

The aforementioned values were confirmed by the intermediate selective inhibitors in the prodrug synthesis, compound 10 (for enkephalin) and 11 for the elimination of amine functions (for APN).

Inhibition of enkephalinase (NEP) activity

Neprilysin purified from rabbit kidney was used at a final concentration of 200ng/mL in 50mM Tris buffer (pH7.4) (AubryM. et al 1987, biochem. cell. biol.65, 398-404). The substrate Dansyl-Gly- (NO)₂) Phe- β -Ala (Goudreau N, et al (1994) anal. biochem., 219, 87-95) (Km 37. mu.M) was dissolved in ethanol and used at a final concentration of 20. mu.M.In 50mM Tris buffer (pH7.4) at 37 ℃ with NEP-1 preincubated to increase the concentration of inhibitor (10)^–10To 10^–3M)15 minutes. Substrate was then added and incubation continued for 60 minutes. The reaction was quenched by placing the well plate in ice for 10 minutes. The emitted fluorescence was read in a fluorimeter, where λ ex ═ 355nm and λ em ═ 535 nm. The results are presented in the table below.

Inhibition of APN activity

Measurement of the inhibition of aminopeptidase N (APN) by Using L-Ala ↓, β -NA substrate (50. mu.M, Sigma Aldrich.) recombinant human enzyme (Rh) (50 ng/mL; R)&D System) determines the inhibition intensity. At 37 ℃ in 50mM Tris buffer (pH)7.4) Pre-incubation increasing concentrations of inhibitor (10)^–10To 10^–3M)30 minutes. Substrate was then added and incubation continued for 30 minutes at 37 ℃. The reaction was quenched by placing the well plate in ice for 10 minutes. The emitted fluorescence was read in a fluorimeter where λ ex ═ 340nm and λ em ═ 405 nm. The results are presented in the table below.

Compound (I)	R1	Ki APN(nM)
			a	CH2CH2SCH3	11±2
b	CH2CH2CH2CH3	13±2
			c	CH2CH2OCH3	35±2
d	CH2CH2OCH2CH3	47±8
			e	CH2OCH2CH3	55±10

3、Pharmacology: hot plate test

The bounce reflex of a mouse placed on a 52 ℃ heating plate was measured by the time required for the animal to jump to escape pain (bounce delay) (Eddy, N.B et al J.pharm.exp.Therap., 1953, 107, 385- "389).

After dissolving in ethanol/Tween 80/water (1/1/8) mixture, the compound of formula (1 a-b-1 Dia 3) or (1 b-l-1 Dia

1) Compounds were injected intravenously into male OF1 mice (23-26 g) (10 mg/kg).

Injection volume: 10mL/kg

Jump delay time was measured 10 minutes after intravenous injection.

Results are expressed as percent analgesia using the following equation:

pain-relieving%(measured delay time-control delay time)

(maximum delay time-control delay time)

Maximum delay time 240 seconds

Results are expressed as mean ± SEM. The results are presented in fig. 1.

Claims (19)

1. A compound of the general formula (1):

(1)R–NH–CH(R₁)–CH₂–S–S–C(R₂)(R₃)–COCH₂–CH(R₄)–COR₅

wherein R is:

a) r is:

-hydrogen;

-an alkoxyalkyl carbonyl group R 'c (o) OCH (R ") oc (o) -, wherein R' and R" are independently alkyl groups containing 1 to 6 carbon atoms;

b)R₁is a straight or branched alkyl group of1 to 6 carbon atoms, unsubstituted or substituted with a-SOR ' "or-SR '" group, wherein R ' "is an alkyl group of1 to 6 carbon atoms, unsubstituted or substituted with one or more halogen atoms;

c)R₂comprises the following steps:

-a linear or branched alkyl group of1 to 6 carbon atoms, unsubstituted or substituted with:

■ phenyl which is unsubstituted OR substituted by one OR more halogen, alkoxy-OR selected from fluoro OR bromo₆Is substituted in which R₆Have the same definition as above, or are substituted by phenyl;

and R is₃Is hydrogen; or

R₂And R₃Identical and is a linear or branched alkyl group of1 to 6 carbon atoms; or

–C(R₂)(R₃) -together are:

■ saturated 5-membered ring compounds fused or unfused with aromatic rings;

■ saturated 6-membered ring compounds;

d)R₄comprises the following steps:

-a linear or branched alkyl group of1 to 6 carbon atoms, unsubstituted or substituted with:

■ phenyl, which is unsubstituted or substituted with:

-one or more halogens selected from fluorine or bromine;

-phenyl or thienyl;

e)R₅comprises the following steps:

-a hydroxyl group;

-alkoxy-OR₁₀Wherein R is₁₀Comprises the following steps:

■ contain a straight or branched chain alkyl group of 2 to 6 carbon atoms.

2. The compound of claim 1, wherein R₂Is a straight or branched alkyl group of1 to 6 carbon atoms or a straight or branched alkyl group of1 to 6 carbon atoms substituted with:

■ a phenyl group;

■ phenyl substituted with one or more halogens selected from fluorine or bromine;

and R is₃Is hydrogen, or

R₂And R₃Identical and is a straight-chain or branched alkyl radical of1 to 6 carbon atoms, or

–C(R₂)(R₃) -together are:

■ saturated 5-membered ring compounds;

■ saturated 5-membered ring compounds fused to aromatic rings;

■ saturated 6-membered ring compounds.

3. The compound of claim 1, wherein R₅Is a hydroxyl group.

4. The compound of claim 1, wherein:

a)R₁is selected from-CH₂CH₂SCH₃、–CH₂CH₂SOCH₃、–CH₂CH₂CH₂CH₃；

b)R₂Is a straight or branched alkyl group of1 to 6 carbon atoms or a straight or branched alkyl group of1 to 6 carbon atoms substituted with:

■ a phenyl group;

■ phenyl substituted with one or more halogens selected from fluorine or bromine;

and R is₃Is hydrogen; or

R₂And R₃Identical and is a linear or branched alkyl group of1 to 6 carbon atoms; or

–C(R₂)(R₃) -together are:

■ saturated 5-membered ring compounds;

■ saturated 5-membered ring compounds fused to aromatic rings;

■ saturated 6-membered ring compounds; or

■ saturated 6-membered heterocyclic compound containing 1 heteroatom selected from oxygen, nitrogen and sulfur at the 4-position;

c)R₄is a group ofSubstituted straight or branched chain alkyl of1 to 6 carbon atoms:

■ a phenyl group; or

■ phenyl substituted with:

-one or more halogens selected from fluorine or bromine;

-phenyl or thienyl;

d)R₅is a hydroxyl group.

5. The compound of claim 1, wherein R₂Is an isobutyl group or a methyl group substituted with:

■ a phenyl group;

■ phenyl substituted at the 4-position with a halogen selected from fluorine or bromine;

■ phenyl substituted at the 4-position with phenyl;

and R is₃Is hydrogen; or

R₂And R₃Identical and is methyl or ethyl; or

–C(R₂)(R₃) -together are:

a saturated 5-or 6-membered ring group;

■ saturated 5-membered ring groups fused to aromatic rings.

6. The compound of claim 1, wherein R₄Is a carbon alkyl group substituted with the following groups:

■ a phenyl group;

■ phenyl substituted at the 4-position with a halogen selected from fluorine or bromine;

■ phenyl substituted at the 4-position with phenyl.

7. The compound of claim 1, wherein:

–R₂＝CH₂Ph；R₃＝H；R₄＝CH₂ph; or

–R₂＝iBu；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Br–Ph)；R₃＝H；R₄＝CH₂Ph；

–R₂＝CH₂Ph；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₂(4–Ph–Ph)；R₃＝H；R₄＝CH₂(4-Br-Ph); or

–R₂＝CH₃；R₃＝CH₃；R₄＝CH₂(4-Br-Ph); or

–R₂＝C₂H₅；R₃＝C₂H₅；R₄＝CH₂(4-Br-Ph); or

––C(R₂)(R₃) Cyclopentyl; r₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃) Cyclohexyl group; r₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃) Indanyl; r₄＝CH₂(4-Br-Ph); or

–C(R₂)(R₃) Cyclohexyl group; r₄＝CH₂(4-Ph-Ph); or

–C(R₂)(R₃) Indanyl; r₄＝CH₂(4–Ph–Ph)。

8. The compound of claim 1, wherein R₁Is selected from-CH₂CH₂SCH₃、CH₂CH₂SOCH₃、–CH₂CH₂CH₂CH₃。

9. The compound of claim 1, wherein:

–R₁＝CH₂CH₂SCH₃；R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂CH₂CH₃；R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂SOCH₃；R₂＝CH₂CH(CH₃)₂；R₃＝H；R₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂CH₂CH₃；C(R₂)(R₃) - ═ cyclohexyl; r₄＝CH₂(4–Br–Ph)；

–R₁＝CH₂CH₂SCH₃；C(R₂)(R₃) Cyclohexyl group; r₄＝CH₂(4–Ph–Ph)；

–R₁＝CH₂CH₂CH₂CH₃；C(R₂)(R₃) Cyclohexyl group; r₄＝CH₂(4–Ph–Ph)。

10. Use of a compound according to any one of claims 1 to 9 in the manufacture of a medicament.

11. The use of claim 10, wherein the medicament is an analgesic, an anxiolytic, an antidepressant, or an anti-inflammatory.

12. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 9 and at least one pharmaceutically acceptable excipient.

13. The pharmaceutical composition according to claim 12, comprising at least one compound selected from morphine and derivatives thereof, inhibitors of endocannabinoids and endocannabinoid metabolites, GABA derivatives, duloxetine or methadone.

14. The pharmaceutical composition of claim 13, wherein the GABA derivative is gabapentin or pregabalin.

15. The pharmaceutical composition of claim 12, for parenteral, topical, oral, or nasal administration.

16. Use of a pharmaceutical composition according to claim 12 for the preparation of an analgesic, an anxiolytic, an antidepressant or an anti-inflammatory agent.

17. The use according to claim 16, wherein the pharmaceutical composition further comprises at least one compound selected from morphine and derivatives thereof, inhibitors of endocannabinoids and endocannabinoid metabolites, GABA derivatives, duloxetine or methadone.

18. The use of claim 17, wherein the GABA derivative is gabapentin or pregabalin.

19. The use of claim 16, wherein the pharmaceutical composition is for parenteral, topical, oral, or nasal administration.