WO2020147624A1 - 环孢菌素类似物及其用途 - Google Patents

环孢菌素类似物及其用途 Download PDF

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WO2020147624A1
WO2020147624A1 PCT/CN2020/070852 CN2020070852W WO2020147624A1 WO 2020147624 A1 WO2020147624 A1 WO 2020147624A1 CN 2020070852 W CN2020070852 W CN 2020070852W WO 2020147624 A1 WO2020147624 A1 WO 2020147624A1
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Prior art keywords
compound
acid
organ
donor
kidney
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PCT/CN2020/070852
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English (en)
French (fr)
Inventor
麦靖邦
曾力
郁胜强
溥·迈克
费·汉斯
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Farsight Medical Technology Shanghai Co Ltd
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Farsight Medical Technology Shanghai Co Ltd
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Priority to KR1020217025827A priority Critical patent/KR102946658B1/ko
Priority to CN202080009966.5A priority patent/CN113329626A/zh
Priority to EP20741731.2A priority patent/EP3915366A4/en
Priority to SG11202107846XA priority patent/SG11202107846XA/en
Priority to US17/423,909 priority patent/US20220079139A1/en
Priority to JP2021541238A priority patent/JP7526189B2/ja
Priority to AU2020210046A priority patent/AU2020210046A1/en
Priority to CA3126569A priority patent/CA3126569A1/en
Publication of WO2020147624A1 publication Critical patent/WO2020147624A1/zh
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • C07K7/645—Cyclosporins; Related peptides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to cyclosporin analogs and their use as mitochondrial protective agents in organ donors.
  • the compounds of the present invention can be used for preservation (preservation) of organs or other body parts removed or excised from a subject before transplantation.
  • the present invention particularly but not exclusively relates to the use of cyclosporin analogues of Formula 1 as mitochondrial protective agents in organ donors. More specifically, the present invention relates to the use of compound 1 (Compound 1) as a mitochondrial protective agent in kidney donors.
  • Acute inflammation is recognized as a complex interaction between various cellular (neutrophils, macrophages) and extracellular (complement, histamine) factors that act in response to PAMP (the molecular pattern of pathogen activation) , Pathogen-activated molecular patterns) and DAMP (damage-activated molecular patterns) signals to eliminate the originating insult.
  • PAMP the molecular pattern of pathogen activation
  • DAMP damage-activated molecular patterns
  • mitochondrial membrane permeability transition pore (termed mitochondrial membrane permeability transition pore (MPTP)) is essential for the onset and maintenance of this necrotizing inflammation.
  • MPTP mitochondrial membrane permeability transition pore
  • the key regulator for the opening of this MPTP is cyclophilin D (CypD), and inhibitors of CypD have shown good activity in preventing tissue damage associated with necrotizing inflammation.
  • CypD cyclophilin D
  • the opening of MPTP and the subsequent initiation of necrotic cell death are triggered by increased intracellular calcium levels as a result of various factors, including oxidative stress, hypoxia, bile salt toxins, etc.
  • CypD gene ablation gene ablation
  • pharmacological inhibition pharmacological inhibition
  • Renal ischemia is caused by arterial occlusion, shock, and kidney transplantation, and renal ischemia can lead to renal cell death and renal failure.
  • Another source of tissue damage related to ischemia occurs during organ transplantation. After the donor organ is removed, the tissue inevitably suffers from oxygen deficiency as a result of the loss of blood flow, and damage to the ischemic tissue occurs after the blood flow restarts. Compounds that prevent tissue damage during resection and reperfusion will improve the viability of transplanted organs.
  • the preferred profile of the compound to be used as a tissue protective agent includes: effective inhibition of CypD; prevention of MPTP opening after ischemic stress; and formulation at a concentration high enough to protect the tissue for intravenous administration and It is used to add to the sufficient solubility of the preservation solution commonly used in organ transportation.
  • cyclophilin D knockout mice In studies using cyclophilin D knockout mice and pharmacological strategies using cyclophilin inhibitors, it has been clearly demonstrated that the mitochondrial membrane permeability transition pore-a non-specific channel in the inner mitochondrial membrane-is The basic event in cell death caused by multiple damages.
  • inhibiting cyclophilin D can prevent the opening of mPTP, which protects mitochondrial function and preserves cell viability.
  • the toxic damage to cells that can induce MPTP includes: ischemia, reactive oxygen species (ROS), bile salts, ⁇ -synuclein oligomers, and increased intracellular calcium levels.
  • ROS reactive oxygen species
  • Donated organs after removal from the donor can experience necrotizing inflammation, leading to tissue damage and impaired function when placed in the recipient.
  • the compounds described herein prevent degradation of the donated organ after removal while the organ is stored awaiting implantation in the recipient.
  • Cyclosporin A (Cyclosporin A) is a compound well known for its immunosuppressive properties, but other biological properties are also described. Cyclosporin A has the following chemical structure:
  • Biologically active derivatives of cyclosporin A have also been prepared.
  • EP 0 484 281 and EP 0 194 972 describe cyclosporin derivatives with various properties including immunosuppressive, antiparasitic and antiviral properties.
  • US 6,583,265 describes cyclosporin derivatives with a modification at position 3 of the cyclosporin macrocycle.
  • US 6,583,265 discloses compound 1:
  • Example 27 in the patent US 6,583,265, which includes hundreds of named compounds with modifications in different positions around the ring. However, there is no biological test data or specific use for this compound or related analogues. When the applicant tried to synthesize the compound using the route disclosed in US 6,583,265 for preparing compound 27, the method was ineffective. Many attempts have been made to repeat the method in US 6,583,265, but they have not been very successful. Without being bound by theory, it is believed that dimethylamino groups (which are basic) prefer to react with acid catalysts. This prevents the acid catalyst from activating the leaving group and suppresses the progress of the reaction. Therefore, there are some doubts as to whether Example 27 has been synthesized previously, and therefore whether the prior art actually fully discloses the preparation of Compound 1.
  • a compound as a mitochondrial protective agent can be administered to an organ donor to improve the preservation of the organ before implantation.
  • These compounds work through cyclophilin D inhibition.
  • Any known cyclophilin D inhibitor can be used as described herein.
  • Suitable inhibitors include: cyclosporine or cyclic depsipeptide analogues as disclosed in, for example, US 6,583,265, EP 0484281, EP 0194972, WO2010/076329 and WO2014/053834 Those reported in.
  • a compound for use as a mitochondrial protective agent in the preservation of body parts or organs removed from a donor before transplantation can be a limb, hand, foot, finger or toe.
  • the organ can be the kidney.
  • the donor may be a living donor.
  • n 2-5
  • R 1 and R 2 are independently selected from H or C 1 -C 4 alkyl, wherein R 1 and R 2 may be joined together to form a C 3 -C 5 heteroalkyl ring.
  • the compound is:
  • the compound of Formula 1 can be used to treat ischaemia-reperfusion injury related to re-attachment of excised body parts and organs.
  • the data provided in this application shows that Compound 1 is significantly better than the confirmed comparative example cyclosporin A and other closely related analogs, including those within the scope of Formula 1.
  • Compound 1 gave surprisingly good results, in fact showing an almost complete reversal in the expected damage to the affected organ.
  • Compound 1 showed significant efficacy as an inhibitor of cyclophilin D. As seen in Table 1, the compound 1 (entry 4) exhibits an EC 24nM for cyclophilin D 50. H substituted with a methyl group on the N atom (only 1 entry NHMe ve NMe2) reduce the effectiveness of about 100 times, with EC> 2000nM 50. Therefore, compound 1 is a surprisingly strong inhibitor of cyclophilin D and mitochondrial permeability transition (MPT).
  • MPT mitochondrial permeability transition
  • the dose of the compound is 0.1 to 10 mg/kg. In one embodiment of the compound, method or use as described above, the dose of the compound is 1 to 3 mg/kg. Within these dosage ranges, the compounds of the present invention are particularly effective.
  • the salt of the present invention can be produced by adding an acid to the compound of formula 1 or compound 1.
  • the acid addition salts produced include those formed with the following: acetic acid, 2,2-dichloroacetic acid, citric acid, lactic acid, mandelic acid, glycolic acid, adipic acid, alginic acid, aryl sulfonic acid (e.g.
  • Benzenesulfonic acid naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid and p-toluenesulfonic acid
  • ascorbic acid such as L-ascorbic acid
  • L-aspartic acid benzoic acid
  • 4-acetamido Benzoic acid butyric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, Lauryl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethyl sulfonic acid, formic acid, fumaric acid, mucic acid, gentisic acid, glucoheptonic acid, gluconic acid ( For example, D-gluconic acid),
  • acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid; organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, Malic acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, aryl sulfonic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid, and sulfuric acid
  • organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, Malic acid, benzoic acid, glycolic acid, gluconic acid, succinic acid, aryl sulfonic acid.
  • the compounds of the invention can be administered together with one or more other active substances.
  • a mitochondrial protective agent compound for use in the preservation of body parts or organs removed or excised from a subject before organ transplantation to a new individual or reattachment of the body part ,
  • the compound is a compound of formula 1:
  • n 2-5
  • R 1 and R 2 are independently selected from H or C 1 -C 4 alkyl, wherein R 1 and R 2 may be joined together to form a C 3 -C 5 heteroalkyl ring.
  • a method for preserving organs removed or resected from a subject before organ transplantation or reattachment which comprises exposing the organ to a mitochondrial protector compound of Formula 1:
  • n 2-5
  • R 1 and R 2 are independently selected from H or C 1 -C 4 alkyl, wherein R 1 and R 2 may be joined together to form a C 3 -C 5 heteroalkyl ring.
  • a mitochondrial protectant compound for preparing a medicament for preserving a body part or organ removed or excised from a subject before transplantation or reattachment, wherein ,
  • the compound is a compound of formula 1:
  • n 2-5
  • R 1 and R 2 are independently selected from H or C 1 -C 4 alkyl, wherein R 1 and R 2 may be joined together to form a C 3 -C 5 heteroalkyl ring.
  • a mitochondrial protective agent compound for preserving a body part or organ removed or excised from a subject before transplantation or reattachment, wherein the compound is a compound of formula 1:
  • n 2-5
  • R 1 and R 2 are independently selected from H or C 1 -C 4 alkyl, wherein R 1 and R 2 may be joined together to form a C 3 -C 5 heteroalkyl ring.
  • the compound can be used as a mitochondrial protective agent in an organ donor, wherein the compound can be administered to the organ donor to protect the organ before removing the organ from the donor.
  • the mitochondrial protectant compound is a cyclophilin inhibitor.
  • the compound can be used as a mitochondrial protective agent in an organ donor, wherein the compound is administered to the organ donor to protect the organ before removing the organ from the donor, wherein the compound Is a compound of formula 1:
  • n 2-5
  • R 1 and R 2 are independently selected from H or C 1 -C 4 alkyl, wherein R 1 and R 2 may be joined together to form a C 3 -C 5 heteroalkyl ring.
  • the compound of Formula 1 can be used as a mitochondrial protective agent in an organ donor, where the organ is the kidney.
  • the compound of Formula 1 may be used to preserve the kidney.
  • a method of preserving an organ in an organ donor comprising administering a mitochondrial protectant compound to the donor before removing the organ from the donor.
  • a method of preserving a kidney in a kidney donor comprising administering a mitochondrial protectant compound to the donor before removing the kidney from the donor.
  • a method of preserving a kidney in a kidney donor comprising administering a mitochondrial protector compound to the donor before removing the kidney from the donor, wherein the compound is of formula 1 compound.
  • the compound of Formula 1 is administered to a living donor prior to organ transplantation.
  • the compound of Formula 1 is administered to a living kidney donor before kidney transplantation.
  • a method of preserving a kidney in a kidney donor comprising administering a mitochondrial protector compound to the donor before removing the kidney from the donor, wherein the compound is a compound 1:
  • the compound can be used as a mitochondrial protective agent in an organ donor, wherein the compound is administered to the organ donor to protect the organ before the organ is removed from the donor, wherein the compound It is compound 1.
  • Compound 1 can be used to preserve the kidney.
  • a method of preserving a kidney in a kidney donor comprising administering a mitochondrial protector compound to the donor before removing the kidney from the donor, wherein the compound is a compound 1.
  • compound 1 is administered to a living kidney donor before kidney transplantation.
  • a mitochondrial protectant compound for preparing a medicine which is used to preserve the kidney.
  • the use of a mitochondrial protectant compound for the preparation of a medicament for the preservation of the kidney is provided, wherein the compound is a compound of Formula 1.
  • the use of a mitochondrial protectant compound for the preparation of a medicine for the preservation of the kidney is provided, wherein the compound is compound 1.
  • a method of preserving kidneys including treating a donor with Compound 1.
  • the organ or kidney donor is a living donor.
  • Ischemic injury occurs when the blood supply to the area of the tissue is cut off.
  • the incidence of ischemic injury is high: myocardial infarction, stroke, and other thrombotic events, and these events affect more than 1.3 million individuals each year in the United States alone.
  • ischemic damage can also occur during surgery where blood vessels are cross-clamped and in organs used for transplantation. The length of time that tissues can survive in hypoxic conditions varies, but eventually the ischemic tissue becomes necrotic tissue.
  • Reperfusion (reoxygenation) injury is tissue damage caused when the blood supply returns to the tissue after a period of ischemia or lack of oxygen (anoxia, hypoxia). Without being bound by theory, it is believed that the lack of oxygen and nutrients from the blood during ischemia creates a condition in which the restoration of circulation leads to inflammation and oxidative damage.
  • organ transplantation there is a period of time between the removal of the organ from the blood supply of the donor and the reconnection of the organ to the blood supply of the donor recipient. During this period, there is a potential ischemia-reperfusion injury. In some cases, organs may need to be transported long distances to the surgery site, increasing the possibility of organ damage.
  • the present invention is provided for administering the mitochondrial protective agent compound of the present invention to prevent such damage to body parts and organs. Especially in the period between the removal of the body part or organ from the blood supply of the donor and the reconnection of the blood supply to the donor recipient, or in the case of a severed body part to reattachment In time.
  • Those skilled in the art will know how to administer the compound of the present invention to an individual prior to removal of the body part or organ, or to a body part or organ removed from the individual, who is an organ donor or accident victim.
  • the compound of the present invention may be administered intravenously to the donor or accident victim before removing the body part or organ, or the compound of the present invention may be added to (or contained in) the fluid in which the organ is placed; and/or The compound of the present invention is added to (or contained in) a liquid that is recycled in an organ/body part or recycled through the organ/body part.
  • the compound of the invention is administered to the organ after the organ is removed from the individual and before transplantation or reattachment.
  • the compound of the invention is administered to the donor subject.
  • the compounds of the invention can be administered systemically. Injection is one way of administering systemic doses of the compounds of this invention.
  • the compounds of the present invention can also be administered to recipients after organ transplantation or to accident victims after reattachment.
  • the systemic dose of the compound of the present invention can be administered to the organ donor before organ removal. This allows the organ to receive a protective dose of the compound before being removed, thereby preserving the organ by protecting it from damage during the removal, and until the process of transplantation into the donor recipient and during the process. In cases where more than one organ is removed from the donor, the systemic dose ensures that each organ receives the dose of the compound.
  • the systemic dose is also more likely to provide a uniform dose of the compound to the organ tissue to be transplanted. In the case of a legal death of the donor, the dose may be greater than the dose normally administered to a living subject.
  • the compound can be administered shortly before the organ removal surgery or during the organ removal surgery.
  • the compound of the present invention can be administered 8, 7, 6, 5, 4, 3, 2, or 1 hour before surgery.
  • organ recipients or accident victims may receive a dose of the compound of the invention before receiving the organ or undergoing reattachment surgery, so that their blood supply contains a protective dose of the compound of the invention, thereby protecting The transplanted or reattached body part is protected from injury after surgery.
  • the body part can be severed and attached to the same individual, or can be given as a graft to a second individual.
  • the cut may be a complete cut or a partial cut.
  • partial severance can be to cut off the blood supply but the body part remains attached through, for example, skin, bone, or muscle tissue.
  • the compound can be administered to: (i) a severed body part; and/or (ii) a subject before reattaching the body part; and/or (iii) a subject during or after reattaching the body part.
  • the invention also has application in non-human subjects such as cats, dogs, horses and pigs.
  • the present invention also has application in transgenic animals (for example, transgenic pigs), where such animals have organs suitable for human transplantation.
  • the compound is compound 1:
  • compound 1 is an effective mitochondrial protective agent.
  • Table 1 confirm the unexpectedly high cyclophilin D inhibition and MPT of compound 1 (entry 4) relative to similar analogs known in the art (entries 1 to 3 and 5 to 7) .
  • a 100-fold improvement in MPT relative to the other three closely related compounds (entries 1, 5, and 7) was also observed, and a more than 25-fold improvement relative to the sub-best performing analog (entry 3) was observed.
  • Compound 1 also showed excellent cyclophilin D inhibition, with at least a 50-fold improvement over all other analogs tested.
  • the organ may be kidney, pancreas, liver, heart, lung, or intestine.
  • the body part may be limbs, hands, feet, fingers or toes.
  • the dose of the compound is 0.1 to 10 mg/kg; and alternatively 1 to 3 mg/kg.
  • the concentration of the compound can be increased or decreased as needed.
  • the compound is formulated in Cremophor (polyoxyethylene castor oil, Cremophor)/physiological saline/DMSO (dimethyl sulfoxide).
  • Cremophor polyoxyethylene castor oil, Cremophor
  • physiological saline/DMSO dimethyl sulfoxide
  • a method for preparing a compound of Formula 1 includes a product formation reaction, and the reaction includes copper triflate and an amino alcohol of Formula 2:
  • n 2-5
  • R 1 and R 2 are independently selected from H or C 1 -C 4 alkyl, wherein R 1 and R 2 may be joined together to form a C 3 -C 5 heteroalkyl ring.
  • the reaction includes a desiccant and/or is carried out under substantially anhydrous conditions.
  • the desiccant is a molecular sieve.
  • the molecular sieve is a 3A molecular sieve.
  • the amino alcohol is N,N-dimethylaminoethanol. This amino alcohol gives compound 1.
  • the amino alcohol is reacted with a cyclosporin precursor compound containing a labile group, wherein the labile group is released during the reaction.
  • the labile group is bonded to the precursor compound through an -S- bond.
  • the labile group is a thiopyridyl group or a mercaptobenzothiazole-2-ylthio group.
  • Figure 1 shows the inhibitory and/or protective effects of compound 1 and comparative compound CsA on acute kidney injury induced in rats by measuring serum creatinine concentration.
  • Figure 2 shows the inhibitory and/or protective effects of compound 1 and comparative compound CsA on acute kidney injury induced in rats by measuring blood urea nitrogen (BUN) concentration.
  • Figure 3 shows the inhibitory and/or protective effect of Compound 1 on LPS-induced acute kidney injury.
  • Figure 4 shows the effect of compound 1 on renal function.
  • the lower creatinine levels and blood urea nitrogen levels of the animals treated with compound 1 were consistent with the reduced level of kidney damage.
  • Figures 5 to 7 show the data saved in the isolated kidney after removal. 4 kidneys were used as controls, and there was no protective agent compound i.v. (intravenous injection) dose before renal perfusion; 5 kidneys received 5 mg/kg C4066 (compound 1) i.v. dose 1 hour before renal perfusion. The data shows the average scores of the studied kidneys at 0, 6, 24, and 48 hours after removal.
  • HE scoring standard according to the degree of inflammation from mild to severe, followed by a semi-quantitative score, negative for very small or no lesions is "-" 0; mild or small is "+" 1; moderate or moderate The big one is "+”2; the severe or big one is "++"3; the very severe or big one is "+++”4.
  • Figure 5 shows the inflammation score
  • Figure 6 shows the expansion of the renal capsule
  • Figure 7 shows the expansion of the renal ducts.
  • FIG. 3 LPS induced acute kidney injury: LPS induced acute kidney injury.
  • Control control; Compound: compound; Creatinine: creatinine.
  • Figure 5 Inflammation score: inflammation score.
  • FIG. 6 Dilatation of Renal Capsule: Dilatation of the kidney capsule.
  • FIG. 7 Renal tubular dilatation: renal duct dilatation.
  • the compound of Formula 1 can be prepared in a variety of ways.
  • the following route is just one example of the way that can be used to synthesize compound 1. Nevertheless, the route used to prepare compound 1 in US 6,583,265 is invalid. Many attempts have been made to repeat the method in US 6,583,265, but it has not been very successful. Without being bound by theory, it is believed that dimethylamino groups (which are basic) prefer to react with acid catalysts. This prevents the acid catalyst from activating the leaving group and suppresses the progress of the reaction.
  • the compound of Formula 1 can be conveniently prepared in a variety of ways.
  • the reaction of compound 2 (wherein R is a lower alkyl group) with a carbonyl compound and a reducing agent can undergo a reductive amination reaction process to give the desired compound.
  • the carbonyl compound is a lower alkyl aldehyde or ketone and the reducing agent is a metal borohydride.
  • the aldehyde is formaldehyde, acetaldehyde or propionaldehyde and the ketone is acetone or 2-butanone or the like.
  • the reducing agent is sodium triacetoxyborohydride or sodium cyanoborohydride.
  • Amine compound 2 can be conveniently prepared from a suitably protected ethanolamine compound such as compound 3, where R is hydrogen or a lower alkyl group, by treating the compound with known conditions for removing protective groups and producing a free amine compound.
  • Suitable protecting groups that can be removed in the presence of other functional groups in the molecule include: tert-butoxycarbonyl (BOC), 9-fluorenylmethoxycarbonyl (FMOC) and the like.
  • the protecting group is tert-butoxycarbonyl (BOC) and the conditions for removing the BOC group include treatment with an acid, such as trifluoroacetic acid.
  • Step 1 Preparation of [2'-(2-thiopyridyl)-Sar] 3 -cyclosporin A
  • Cyclosporin A (20g, 16.6mmol), anhydrous lithium chloride (21.1g, 499mmol) and anhydrous THF (500mL) were added to a dry 1L flask, then the flask was purged with argon and the mixture was cooled to -45°C.
  • diisopropylamine (13.5 g, 133 mmol) was dissolved in dry THF (120 mL) and cooled to -78°C.
  • n-butyllithium (53.2 mL of 2.5 M solution, 133 mmol)
  • the solution of lithium diisopropylamide was transferred to the solution of cyclosporin, and the resulting mixture was stirred at -45°C for 90 minutes.
  • a solution (20 mL) of 2-pyridyl disulfide (11 g, 49.9 mmol) in dry THF was added dropwise, and the resulting mixture was allowed to warm to room temperature overnight. The reaction was quenched by carefully adding saturated NaCl solution (200 mL), and the resulting organic layer was separated.
  • the aqueous layer was extracted with ethyl acetate (3 ⁇ 100 mL), and the combined organic fraction (fraction) was washed with 3N NaOH (2 ⁇ 100 mL), saturated NH 4 Cl (100 mL) and saturated NaCl (100 mL), and then washed with anhydrous Na 2 SO 4 is dried and evaporated.
  • the title compound was separated by silica gel chromatography as a solid, 7.18 g.
  • the celite was washed with ethyl acetate (3 ⁇ 25 mL) and the filtrate was added. The organic layer was separated; the aqueous layer was extracted with EtOAc (2 ⁇ 25 mL), and the combined organic fractions were dried over anhydrous Na 2 SO 4 and evaporated. The crude extract was purified on silica gel to provide the title compound, 86.4 mg.
  • analogs of compound 1 can be prepared by using different amino alcohol reagents.
  • the number of carbon atoms between alcohol and amine groups can be increased or decreased (examples of linking groups include: methylene, ethylene, propylene, butylene, pentalene) , And can include their branched forms, such as iso-propylene, sec-butylene, tert-butylene, 2-methylbutylene, 2 ,2-dimethylpropylene (2,2-dimethylpropylene).
  • N-amino substituent on the amino alcohol can also be changed to give other analogs of compound 1 (examples of N-amino substituents include: methyl, ethyl, propyl, isopropyl Group, n-butyl, sec-butyl, tert-butyl, butyl).
  • the cyclophilin inhibitory binding activity of the compounds disclosed herein was determined using a competitive ELISA adapted from the method described by Quesniaux et al. (Eur. J Immunol., 1987, 17:1359-1365). 8 and D-Lys - succinyl spacer activated ester of cyclosporin A (D-Lys 8 -Cs) bound by the first 8-bit D- lysyl residues coupled to bovine serum albumin (BSA ). BSA was dissolved in 0.1 M borate buffer, pH 9.0 (4 mg in 1.4 ml).
  • the microtiter plate was coated with D-Lys 8 -Cs-BSA conjugate (2 ⁇ g/ml in PBS at 4° C. for 24 hours). use Wash the titer plate with only PBS. To block non-specific binding, 2% BSA/PBS (pH 7.4) was added to the wells and allowed to incubate at 37°C for 2 hours. In a separate microtiter plate, prepare a five-fold dilution series of the compound to be tested in ethanol. For the determination with human recombinant cyclophilin, the initial concentration is 0.1 mg/mL.
  • cyclosporin A used as a reference compound
  • the reaction between the coated BSA-Cs conjugate, free cyclosporin A and cyclophilin was allowed to equilibrate overnight at 4°C.
  • Cyclophilin was detected with anti-cyclophilin rabbit serum diluted in PBS containing 1% BSA, and incubated overnight at 4°C. Wash the titer plate as described above.
  • the bound rabbit antibody was then detected by goat anti-rabbit IgG bound to alkaline phosphatase diluted in 1% BSA-PBS, and allowed to incubate at 37°C for 2 hours. Wash the titer plate as described above. After incubating with 4-nitrophenyl phosphate (1 g/l in diethanolamine buffer, pH 9.8) at 37°C for 1 to 2 hours, the enzymatic reaction was measured spectrophotometrically at 405 nm using a spectrophotometer. Results are expressed as EC 50, which is to achieve 50% inhibition concentration of the compounds of the present invention is required.
  • Compound 1 has an EC 50 value of less than 100 nM for cyclophilin A, cyclophilin B and cyclophilin D.
  • the Agilent 8453 spectrophotometer was used for the measurement.
  • the assay buffer composed of 35mM HEPES pH 7.8 and 50 ⁇ M DTT was cooled to 10°C (with stirring) in a precision glass cuvette, and inhibitors were added from the 100% DMSO stock solution.
  • Mitochondrial permeability transition is determined by measuring the swelling of mitochondria induced by Ca 2+ .
  • the program was adapted from the method described by Blattner et al., 2001, Analytical Biochem, 295:220.
  • Mitochondria are prepared from rat liver, which has been perfused with phosphate buffered saline (PBS) to remove blood, using standard methods that utilize gentle homogenization in a sucrose-based buffer and subsequent differential centrifugation to remove first Cell debris and then precipitate mitochondria.
  • Swelling was induced by 150 micromole Ca 2+ (added from a concentrated CaCl 2 solution) and monitored by measuring the scattering at 535-540 nm. Representative compounds were added 5 minutes before swelling was induced.
  • EC 50 is determined by comparing the swelling with and without the compounds disclosed herein. Compound 1 EC 50 of less than 0.2 ⁇ M inhibits mitochondrial swelling.
  • the formulations of these compounds were prepared by mixing compound 1 and cyclosporin A with Cremophor/Saline/DMSO.
  • the Sprague-Dawley rats were divided into 6 groups: group (i) was a sham operation group, and Cremophor/saline/DMSO was given with no active ingredients; group (ii) was a control Group, given Cremophor/Saline/DMSO, no active ingredients; group (iii) given compound 1 (3mg/kg); group (iv) given CsA (3mg/kg; group (v) given compound 1 (10mg/kg); group (vi) was given CsA (10mg/kg). Except for group (i), which is the "sham operation group", the rats were ligated through bilateral renal arteries for 30 minutes and then released Ligation induces renal ischemia-reperfusion-induced acute kidney injury (AKI).
  • AKI renal ischemia-reperfusion-induced acute kidney injury
  • the concentration of serum creatinine is an indicator of kidney injury.
  • the rats in the "sham operation group” had no induced AKI.
  • the "control group” represents rats with induced AKI but no treatment. Therefore, it can be seen that induced AKI leads to increased levels of serum creatinine from 25 ⁇ mol/ml (group i) to 195 ⁇ mol/ml group (group ii).
  • FIG 2 blood urea nitrogen (BUN) concentration is an indicator of kidney damage.
  • Figure 2 shows the same trend as that seen in Figure 1. That is, 3 mg/kg of CsA obtained a decrease in BUN level (compared with group iv), and compound 1 at 3 mg/kg showed a very significant decrease in BUN level (group iii compared with group ii) ) To obtain a BUN level close to that seen in the "sham operation group” (group i). Increasing the concentration of compound 1 and CsA from 3 mg/kg (group iii and v) to 10 mg/kg (group v and vi) proved to be less effective. This result supports the results seen in Figure 1.
  • LPS lipopolysaccharide
  • mice LPS-induced acute kidney injury (AKI) was induced by intraperitoneal injection of LPS (15 mg/kg). Twenty mice were randomly divided into two groups. Animals in the control group received the vehicle (cremophor/saline/DMSO) and the treatment group received compound 1 (3 mg/kg in Cremophor/saline/DMSO), each administered intraperitoneally. Animals were administered three times with vehicle or compound 1 (1h before LPS injection and 4h and 8h after LPS injection), and blood was taken from the animals 12h after LPS injection. The activity of the compound was determined by improved survival (Figure 3) and by evaluating markers of renal function ( Figure 4).
  • AKI LPS-induced acute kidney injury
  • Figure 4 shows the effect of compound 1 on renal function in this experiment.
  • the lower creatinine levels and lower blood urea nitrogen levels of the animals treated with compound 1 are consistent with the reduced levels of kidney damage.
  • the protective effect of compound 1 on organs undergoing transplantation conditions was exemplified by pig kidney.
  • a single dose of 5 mg/kg of Compound 1 was administered to pigs via intravenous delivery 1 h before nephrectomy.
  • the kidney was removed, perfused with standard hypertonic adenine citrate (HCA) preservation solution, and then stored in the HCA solution at low temperature (0°C-4°C).
  • HCA hypertonic adenine citrate
  • Histological evaluation was performed after hematoxylin and eosin (HE) staining using scoring criteria based on the degree of inflammation.
  • a semi-quantitative scoring system of "0 to 4" is adopted, in which very small or no lesions are assigned as “0”; mild or small as “1”; moderate as “2”; severe The distribution of "3”; the very severe distribution is "4".
  • the experiment was performed with a total of 9 pig kidneys, of which 4 kidneys were used as controls without the protectant compound, and 5 kidneys received an i.v. dose of 5 mg/kg of compound 1 1 hour before nephrectomy.
  • the data shows the average value of the studied kidney at each time after removal.
  • Figure 5 shows the results of the average inflammation score
  • Figure 6 shows the effect on the expansion of the kidney capsule
  • FIG. 7 shows the effect on renal tubule dilation.
  • Compound 1 is surprisingly effective in treating or preventing ischemia-reperfusion injury, especially at lower concentration levels.
  • Administration of the compound to the organ donor before removal of the organ (for subsequent implantation in the recipient) is also particularly effective.
  • Figures 5 to 7 show that by administering the compound to the donor before the organ is removed, the organ can be preserved ex vivo.

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Abstract

本发明提供环孢菌素类似物及其用途，具体涉及化合物及其用作用于保存捐赠器官的线粒体保护剂的用途，其中化合物是式1的化合物或其盐，其中，n为2-5，并且R1和R2独立地选自H或C1-C4烷基，其中R1和R2可以连接在一起以形成C3-C5杂烷基环。

Description

环孢菌素类似物及其用途 技术领域
本发明涉及环孢菌素类似物和它们在器官供体中作为线粒体保护剂的用途。本发明的化合物可以用于在移植前对从受试者上移除或切除的器官或其他身体部分的保存(防腐，preservation)。本发明特别但不排他地涉及式1(Formula 1)的环孢菌素类似物在器官供体中作为线粒体保护剂的用途。更具体地，本发明涉及化合物1(Compound 1)在肾脏供体中作为线粒体保护剂的用途。
背景技术
急性炎症被公认为参与各种细胞的(中性粒细胞，巨噬细胞)和细胞外(补体、组胺)因子之间的复杂相互作用，这些因子作用以响应于PAMP(病原体激活的分子模式，pathogen-activated molecular patterns)和DAMP(损伤激活的分子模式，damage-activated molecular patterns)信号以消除源头性损害(the originating insult)。已证实亲环素A(cyclophilin A)作为趋化因子以促进支持炎症应答的白细胞迁移，并且已经示出在急性炎症的动物模型中阻断亲环素A是有益的。最近，已经描述了一种伴随着细胞死亡和组织坏死的炎症的严重形式。现有大量证据支持在线粒体膜孔处的开放(术语称为线粒体膜通透性转换孔(MPTP))对于这种坏死性炎症的发作和维持至关重要。对该MPTP开放的关键调节子是亲环素D(CypD)，而CypD的抑制剂在预防与坏死性炎症相关的组织损伤方面已经表现出良好的活性。MPTP的开放，以及随后的坏死性细胞死亡的启动，是由作为多种因素的结果的升高的细胞内钙水平所引发的，多种因素包括氧化应激、缺氧、胆盐毒素等。值得注意的是，发现CypD的基因消融(genetic ablation)或药理抑制(pharmacological inhibition)对心肌组织的缺血-再灌注损伤所引起的组织降解是保护性的，这提示CypD抑制是更普遍的用于缺血-再灌注损伤的可行的药物靶点。
肾缺血是由动脉闭塞、休克和肾移植引起的，并且肾缺血可以导致肾细胞死亡和肾功能衰竭。与缺血相关的另一组织损伤源发生于器官移植的过程中。在移除供体器官后，作为血流损失的结果，组织不可避免地遭受氧气匮乏，而在血流重新开始后随之发生对缺血组织的损伤。能够在切除和再灌注过程中防止组织损伤的化合物会提高所移植的器官的生存能力。待用作组织保护剂的化合物的优选特性(preferred profile)包括：有效抑制 CypD；预防缺血性应激后的MPTP开放；和以足够高以保护组织的浓度被配制以用于静脉内施用和用于添加至在器官运输过程中通常使用的保存溶液的充分的溶解度。
在使用亲环素D敲除小鼠进行的研究以及使用亲环素抑制剂的药理学策略中，已经明确证明，线粒体膜通透性转换孔——线粒体内膜中的非特异性通道——是多种损害引起的细胞死亡中的基本事件。此外，抑制亲环素D可以防止mPTP的开放，其对线粒体功能起保护作用并保存细胞生存能力。可以诱导MPTP的对细胞的毒性损害包括：缺血、活性氧物质(ROS)、胆盐、α-突触核蛋白的寡聚体和升高的细胞内钙水平。从供体移除后的捐赠器官可以经历坏死性炎症，导致组织损伤和在置入受体时受损的功能。本文描述的化合物预防捐赠器官在移除后同时存储该器官等待植入受体期间的降解。
环孢菌素A(Cyclosporin A)是一种以其免疫抑制特性而众所周知的化合物，但也描述了其他生物学特性。环孢菌素A具有如下化学结构：
Figure PCTCN2020070852-appb-000001
也已制备了环孢菌素A的生物活性衍生物。例如，US 6,583,265、EP 0 484 281和EP 0 194 972描述了具有包括免疫抑制、抗寄生虫和抗病毒特性在内的多种特性的环孢菌素衍生物。US 6,583,265描述了在环孢菌素大环的第3位具有修饰的环孢菌素衍生物。特别是，US 6,583,265公开了化合物1：
Figure PCTCN2020070852-appb-000002
该化合物是专利US 6,583,265中的实例27，该专利包括数百种在环周围的不同位置具有修饰的已命名化合物。然而，没有对于该化合物或相关类似物的生物测试数据或具体用 途。当申请人试图采用在US 6,583,265中公开的用于制备化合物27的路线来合成所述化合物时，方法是无效的。已经进行了多次尝试以重复US 6,583,265中的方法，但并没有非常成功。在不受理论束缚的情况下，据信二甲基氨基基团(为碱性的)偏好与酸催化剂反应。由此防止酸催化剂对离去基团的脱离进行活化，抑制了反应的进行。因此，对于先前是否已合成实例27存在一些疑问，并且因此也怀疑现有技术是否实际上充分公开了化合物1的制备。
发明内容
申请人已经确定，可以将作为线粒体保护剂的化合物给予至器官供体以改善器官在植入前的保存。这些化合物通过亲环素D抑制发挥作用。可以如本文所描述使用任何已知的亲环素D抑制剂。合适的抑制剂包括：环孢菌素或环缩酚肽类似物(cyclic depsipeptide analogues)如在包括例如US 6,583,265、EP 0 484 281、EP 0 194 972、WO2010/076329和WO2014/053834在内的公开中所报道的那些。
申请人已经合成了先前在上述现有技术出版物中所提出的化合物，并且惊讶地发现这些化合物的小子集是特别好的亲环素D抑制剂。因此，下述化合物可以用于治疗得益于抑制亲环素D活性的病症，并且可以施用至器官供体以辅助离体器官保存。根据本发明的一个方面，提供了一种化合物，其用作对移植前从供体移除的身体部分或器官的保存中的线粒体保护剂。移除的身体部分可以是肢体、手、脚、手指或脚趾。器官可以是肾脏。供体可以是活体供体。
Figure PCTCN2020070852-appb-000003
或其盐，
其中n为2-5，并且
R 1和R 2独立地选自H或C 1-C 4烷基，其中R 1和R 2可以连接在一起以形成C 3-C 5杂烷基环(heteroalkyl ring)。
在一种优选的实施方式中，化合物是：
Figure PCTCN2020070852-appb-000004
式1的化合物可以用于治疗与已切除的身体部分和器官的重附着(re-attachment)相关的缺血-再灌注损伤(ischaemia-reperfusion injury)。本申请提供的数据(图1和图2中图示的)显示化合物1显著优于确认的对比例环孢菌素A和其他密切相关的类似物，包括在式1的范围内的那些。实际上，在进行的挑战性测试中，从器官除去血液持续三十分钟，化合物1给出了令人惊讶的良好结果，事实上示出在对受影响器官的预期损伤方面的几乎完全逆转。
化合物1表现出作为亲环素D的抑制剂的显著效力。如表1中可见的，化合物1(条目4)表现出针对亲环素D的24nM的EC 50。用H取代N原子上的一个甲基(条目1仅为NHMe ve NMe2)降低效力约100倍，具有>2000nM的EC 50。因此，化合物1是亲环素D和线粒体通透性转换(MPT)的令人惊讶的强抑制剂。
在如上所述的化合物、方法或用途的一种实施方式中，化合物的剂量为0.1至10mg/kg。在如上所述的化合物、方法或用途的一种实施方式中，化合物的剂量为1至3mg/kg。在这些剂量范围内，本发明的化合物是特别有效的。
可以通过向式1的化合物或化合物1加入酸来产生本发明的盐。所产生的酸加成盐包括用以下各项形成的那些：乙酸、2,2-二氯乙酸、柠檬酸、乳酸、扁桃酸、乙醇酸、己二酸、海藻酸、芳基磺酸(例如，苯磺酸、萘-2-磺酸、萘-1,5-二磺酸和对甲苯磺酸)、抗坏血酸(例如L-抗坏血酸)、L-天冬氨酸、苯甲酸、4-乙酰氨基苯甲酸、丁酸、(+)-樟脑酸、樟脑磺酸、(+)-(1S)-樟脑-10-磺酸、癸酸、己酸、辛酸、肉桂酸、柠檬酸、环拉酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙磺酸、2-羟乙基磺酸、甲酸、富马酸、粘酸、龙胆酸、葡庚糖酸、葡萄糖酸(例如，D-葡萄糖酸)、葡萄糖醛酸(例如，D-葡萄糖醛酸)、谷氨酸(例如，L-谷氨酸)]、α-酮戊二酸、乙醇酸、马尿酸、氢溴酸、盐酸、氢碘酸、羟乙基磺酸、乳酸(例如，(+)-L-乳酸和(±)-DL-乳酸)、乳糖 酸、马来酸、苹果酸(例如，(-)-L-苹果酸)、(±)-DL-扁桃酸、偏磷酸、甲磺酸、1-羟基-2-萘甲酸、烟碱酸、硝酸、油酸、乳清酸、草酸、棕榈酸、帕莫酸、磷酸、丙酸、L-焦谷氨酸、水杨酸、4-氨基水杨酸、癸二酸、硬脂酸、琥珀酸、硫酸、单宁酸、酒石酸(例如，(+)-L-酒石酸)、硫氰酸、十一烯酸和戊酸。特别地，酸加成盐包括衍生自以下各项的那些：无机酸如盐酸、氢溴酸、磷酸、偏磷酸、硝酸和硫酸；有机酸如酒石酸、乙酸、柠檬酸、苹果酸、乳酸、富马酸、苯甲酸、乙醇酸、葡萄糖酸、琥珀酸、芳基磺酸。
本发明的化合物可以与一种或多种其他活性物质一起施用。
根据本发明的一个方面，提供了一种线粒体保护剂化合物，用于在器官移植到新的个体或重附着身体部分之前，对从受试者移除或切除的身体部分或器官的保存中使用，其中化合物是式1的化合物：
Figure PCTCN2020070852-appb-000005
或其盐，
其中n为2-5，并且
R 1和R 2独立地选自H或C 1-C 4烷基，其中，R 1和R 2可以连接在一起以形成C 3-C 5杂烷基环。
根据本发明的一个方面，提供了一种用于在器官移植或重附着之前对从受试者移除或切除的器官进行保存的方法，包括将器官暴露于式1的线粒体保护剂化合物：
Figure PCTCN2020070852-appb-000006
或其盐，
其中n为2-5，并且
R 1和R 2独立地选自H或C 1-C 4烷基，其中，R 1和R 2可以连接在一起以形成C 3-C 5杂烷基环。
根据本发明的一个方面，提供了线粒体保护剂化合物用于制备药物的用途，所述药物用于在移植或重附着之前，对从受试者移除或切除的身体部分或器官进行保存，其中，化合物是式1的化合物：
Figure PCTCN2020070852-appb-000007
或其盐，
其中n为2-5，并且
R 1和R 2独立地选自H或C 1-C 4烷基，其中，R 1和R 2可以连接在一起以形成C 3-C 5杂烷基环。
根据本发明的一个方面，提供线粒体保护剂化合物用于在移植或重附着之前对从受试者移除或切除的身体部分或器官进行保存的用途，其中，化合物是式1的化合物：
Figure PCTCN2020070852-appb-000008
或其盐，
其中n为2-5，并且
R 1和R 2独立地选自H或C 1-C 4烷基，其中，R 1和R 2可以连接在一起形成C 3-C 5杂烷基环。
在一种实施方式中，化合物可以用作器官供体中的线粒体保护剂，其中，化合物可以在从所述供体移除所述器官之前被施用至器官供体以保护该器官。
在进一步的实施方式中，线粒体保护剂化合物是亲环素抑制剂。
在一种实施方式中，化合物可以用作器官供体中的线粒体保护剂，其中，化合物在从所述供体移除所述器官之前被施用至器官供体以保护所述器官，其中，化合物是式1的化合物：
Figure PCTCN2020070852-appb-000009
或其盐，
其中n为2-5，并且
R 1和R 2独立地选自H或C 1-C 4烷基，其中，R 1和R 2可以连接在一起形成C 3-C 5杂烷基环。
在一种实施方式中，式1的化合物可以用作器官供体中的线粒体保护剂，其中，器官是肾脏。
在一种实施方式中，式1的化合物可以被用于保存肾脏。
根据本发明的另一方面，提供一种保存器官供体中的器官的方法，包括在从所述供体移除所述器官之前将线粒体保护剂化合物施用至所述供体。
在一种实施方式中，提供一种保存肾脏供体中的肾脏的方法，包括在从所述供体移除所述肾脏之前将线粒体保护剂化合物施用至所述供体。
在一种实施方式中，提供一种保存肾脏供体中的肾脏的方法，包括在从所述供体移除所述肾脏之前将线粒体保护剂化合物施用至所述供体，其中，化合物是式1的化合物。
在一种实施方式中，在器官移植之前将式1的化合物施用至活体供体。
在一种优选的实施方式中，在肾脏移植之前将式1的化合物施用至活体肾脏供体。
在一种实施方式中，提供一种保存肾脏供体中的肾脏的方法，包括在从所述供体移除所述肾脏之前将线粒体保护剂化合物施用至所述供体，其中，化合物是化合物1：
Figure PCTCN2020070852-appb-000010
在一种实施方式中，化合物可用作器官供体中的线粒体保护剂，其中，化合物在从所述供体移除所述器官之前被施用至器官供体以保护所述器官，其中，化合物是化合物1。
在一种优选的实施方式中，化合物1可以用于保存肾脏。
在一种实施方式中，提供一种保存肾脏供体中的肾脏的方法，包括在从所述供体移除所述肾脏之前将线粒体保护剂化合物施用至所述供体，其中，化合物是化合物1。在一种优选的实施方式中，在肾脏移植之前将化合物1施用至活体肾脏供体。
根据本发明的一个方面，提供线粒体保护剂化合物用于制备药物的用途，所述药物用于保存肾脏。在一种实施方式中，提供线粒体保护剂化合物用于制备药物的用途，所述药物用于保存肾脏，其中，化合物是式1的化合物。在一种实施方式中，提供线粒体 保护剂化合物用于制备药物的用途，所述药物用于保存肾脏，其中，化合物是化合物1。
根据本发明的一个方面，提供一种保存肾脏的方法，包括用化合物1处理供体。在一种优选的实施方式中，器官或肾脏供体是活体供体。
当组织的区域的血液供应被切断时发生缺血性损伤。缺血性损伤的发生率很高：心肌梗死、中风和其他血栓性事件，并且仅在美国这些事件每年影响超过130万个体。此外，在血管被交叉夹住的手术期间以及在用于移植的器官中也会发生缺血性损伤。组织在缺氧条件中能够存活的时间长度不同，但最终缺血的组织变为坏死的组织。
再灌注(复氧)损伤是在缺血或缺少氧气(缺氧(anoxia)，低氧(hypoxia))一段时间后，当血液供应返回组织时所引起的组织损伤。在不受理论束缚的情况下，据信在缺血期间缺少来自血液的氧和营养物创造了一种条件，其中循环的恢复导致炎症和氧化损伤。
在器官移植中，从供体的血液供应移除器官到将器官重新连接至供体受体的血液供应之间存在一段时间。在此期间，存在潜在的缺血-再灌注损伤。在一些案例中，器官可能需要远距离运输至手术地点，增加了器官损伤的可能性。
在涉及断肢的事故中，从血液供应中切断身体部分直至将身体部分重新连接至血液供应之间存在一段时间。在此期间，存在潜在的缺血-再灌注损伤。在一些案例中，身体部分和患者可能需要长距离运输至手术地点，增加了重附着之前、期间和之后损伤的可能性。
提供本发明用于施用本发明的线粒体保护剂化合物以防止此类对身体部分和器官的损伤。特别是在从供体的血液供应移除身体部分或器官到重新连接至供体受体的血液供应之间的一段时间中，或者是在切断的身体部分的案例中到重附着之间的一段时间中。本领域技术人员会知晓在移除身体部分或器官之前将本发明的化合物施用至个体的方式，或者施用至从个体移除的身体部分或器官的方式，个体是器官供体或事故受害者。例如，可以在移除身体部分或器官之前将本发明的化合物静脉内施用至供体或事故受害者，或者可以将本发明的化合物添加至(或包含于)放置器官的液体中；和/或将本发明的化合物添加至(或包含于)在器官/身体部分中再循环或再循环通过器官/身体部分的液体中。
在如上所述的化合物、方法或用途的一种实施方式中，在从个体移除器官之后并在移植或重附着之前，将本发明的化合物施用至器官。可替代地，或另外地，在移除供体器官之前，将本发明的化合物施用至供体受试者。例如，本发明的化合物可以全身施用。 注射是施用本发明的化合物的全身剂量的一种方式。也可以将本发明的化合物施用至器官移植后的受体或者施用至重附着后的事故受害者。
可以在器官移除前将本发明的化合物的全身剂量施用至器官供体。这容许器官在被移除之前接受保护性剂量的化合物，从而在移除期间，以及直到移植进入供体受体的过程和在该过程期间，通过保护器官免于损伤来保存该器官。在从供体移除不止一个器官的案例中，该全身剂量确保每个器官接受化合物的剂量。全身剂量也更可能向待移植的器官组织提供均匀剂量的化合物。在供体合法死亡的情况下，剂量可以大于通常会给予至活体受试者的剂量。
可以在器官移除手术前不久或在器官移除手术期间施用化合物。例如，可以在手术前8、7、6、5、4、3、2或1小时施用本发明的化合物。
另外地，或可替代地，器官受体或事故受害者可以在接受器官或进行重附着手术之前接受本发明的化合物的剂量，使得他们的血液供应含有保护性剂量的本发明的化合物，从而保护移植的或重附着的身体部分免于手术后损伤。
身体部分可以切断自并重附着于相同个体，或者可以作为移植物给予至第二个体。身体部分从受试者切断的情况下，切断可以是完全切断或部分切断。例如，部分切断可以是切断血液供应但身体部分仍然通过例如皮肤、骨骼或肌肉组织等保持附着。可以将化合物施用至：(i)已切断的身体部分；和/或(ii)重附着身体部分之前的受试者；和/或(iii)身体部分的重附着期间或之后的受试者。
本发明还具有在非人类受试者例如猫、狗、马和猪中的应用。
本发明还具有在转基因动物(例如，转基因猪)中的应用，其中，这样的动物具有适用于人类移植的器官。
在如上所述的化合物、方法或用途的一种实施方式中，化合物是化合物1：
Figure PCTCN2020070852-appb-000011
在本申请中，已经示出化合物1是一种有效的线粒体保护剂。表1中所展示的结果 证实，相对于本领域已知的相似类似物(条目1至3和5至7)，化合物1(条目4)的出乎意料地高的亲环素D抑制和MPT。还观察到相对于其他三种密切相关的化合物(条目1、5和7)在MPT方面的100倍改善，以及观察到相对于次最佳表现的类似物(条目3)的超过25倍改善。化合物1也显示出优异的亲环素D抑制，具有相对于测试的所有其他类似物的至少50倍改善。
在如上所述的化合物、方法或用途的一种实施方式中，器官可以是肾脏、胰腺、肝脏、心脏、肺脏或肠。在切断的身体部分的情况下，身体部分可以是四肢、手、脚、手指或脚趾。
在如上所述的化合物、方法或用途的一种实施方式中，化合物的剂量是0.1至10mg/kg；并且可选地是1至3mg/kg。在其中器官或身体部分浸浴(bathed)在含有本发明的化合物的液体中或该液体被再循环的情况下，可以根据需要提高或降低化合物的浓度。
在如上所述的化合物、方法或用途的一个实施方式中，将化合物配制在克列莫佛(聚氧乙烯蓖麻油，Cremophor)/生理盐水/DMSO(二甲基亚砜)中。
根据本发明的一个方面，提供一种制备式1的化合物的方法，方法包括产物形成反应，反应包括三氟甲基磺酸铜(copper triflate)和式2的氨基醇：
Figure PCTCN2020070852-appb-000012
其中，n为2-5，并且
R 1和R 2独立地选自H或C 1-C 4烷基，其中，R 1和R 2可以连接在一起形成C 3-C 5杂烷基环。
令人惊奇地，已经发现，通过上述方法可以很容易地获得本发明的化合物。例如，本申请人进行了多次重复US 6,583,265中所公开的方法的失败尝试，并最终放弃了US 6,583,265中的不可行的方法。
在一种实施方式中，反应包括干燥剂和/或在基本无水的条件下进行。可选地，干燥剂是分子筛。可选地，分子筛是3A分子筛。在一种实施方式中，氨基醇是N,N-二甲基氨基乙醇。该氨基醇给出化合物1。在一种实施方式中，氨基醇与含有不稳定基团的环 孢菌素前体化合物反应，其中，不稳定基团在反应中脱离。可选地，不稳定基团通过-S-键与前体化合物键合。进一步可选地，不稳定基团是硫代吡啶基基团(thiopyridyl group)或巯基苯并噻唑-2-基硫代(mercaptobenzothiazole-2-ylthio)基团。
附图说明
图1示出通过测量血清肌酸酐浓度，化合物1和对比化合物CsA对大鼠中诱导的急性肾损伤的抑制和/或保护作用。
图2示出通过测量血尿素氮(BUN)浓度，化合物1和对比化合物CsA对大鼠中诱导的急性肾损伤的抑制和/或保护作用。
图3示出化合物1对LPS诱导的急性肾损伤的抑制和/或保护作用。
图4示出化合物1对肾功能的影响。用化合物1治疗的动物的较低的肌酸酐水平和血尿素氮水平与降低的对肾脏的损伤水平相一致。
图5至图7示出展示移除后离体肾脏保存的数据。4个肾脏用于对照，在肾脏灌注之前没有保护剂化合物i.v.(静脉注射)剂量；5个肾脏在肾脏灌注之前1小时接受5mg/kg C4066(化合物1)i.v.剂量。数据显示的是所研究的肾脏在移除后0、6、24和48小时的时间的平均评分。HE评分标准：根据炎症程度从轻度到重度，接着是半定量评分，对于非常小的或无病变的阴性为“-”0；轻度的或小的为“+”1；中度或中等大的为“+”2；严重的或大的为“++”3；非常严重或大的为“+++”4。图5示出炎症评分，图6示出肾囊的扩张，而图7示出肾管扩张。
附图中文字说明：
图1：Creatinine：肌酸酐。
图3：LPS induced acute kidney injury：LPS诱导的急性肾损伤。
图4：Control：对照；Compound：化合物；Creatinine：肌酸酐。
图5：Inflammation score：炎症评分。
图6：Dilatation of renal capsule：肾囊扩张。
图7：Renal tubular dilatation：肾管扩张。
具体实施方式
现在通过以下实施例来阐述本发明。
实验方法和实验结果
本领域的技术人员会认识到可以以多种方式制备式1的化合物。以下路线仅是可以用于合成化合物1的方式的一个示例。尽管如此，US 6,583,265中用于制备化合物1的路线是无效的。已进行多次尝试来重复US 6,583,265中的方法，然而不是非常成功。在不受理论束缚的情况下，据信二甲基氨基基团(为碱性的)偏好与酸催化剂反应。由此防止酸催化剂对离去基团的脱离进行活化，抑制了反应的进行。
可以通过多种途径方便地制备通式1的化合物。在一个实例(方案1)中，化合物2(其中R是低级烷基)与羰基化合物和还原剂的反应可以进行还原胺化反应过程以给出所需化合物。优选地，羰基化合物是低级烷基醛或酮并且还原剂是金属硼氢化物。更优选地，醛是甲醛、乙醛或丙醛并且酮是丙酮或2-丁酮等。优选地，还原剂是三乙酰氧基硼氢化钠或氰基硼氢化钠。
Figure PCTCN2020070852-appb-000013
胺化合物2可以方便地制备自适当保护的乙醇胺化合物如化合物3，其中，R是氢或低级烷基，通过用已知的用于除去保护基团的条件处理所述化合物并产生游离胺化合物。在分子中其他官能团存在的情况下可以被除去的合适的保护基团包括：叔丁氧基羰基(BOC)、9-芴基甲氧基羰基(FMOC)等。优选地，保护基团是叔丁氧基羰基(BOC)并且用于除去BOC基团的条件包括用酸处理，酸例如是三氟乙酸。
Figure PCTCN2020070852-appb-000014
第一步：制备[2'-(2-硫代吡啶基)-Sar] 3-环孢菌素A
Figure PCTCN2020070852-appb-000015
向干燥的1L烧瓶中加入环孢菌素A(20g，16.6mmol)、无水氯化锂(21.1g，499mmol)和无水THF(500mL)，然后用氩气吹扫烧瓶并将混合物冷却至-45℃。在另一个烧瓶中，将二异丙胺(13.5g，133mmol)溶解在无水THF(120mL)中并冷却至-78℃。向此烧瓶中加入正丁基锂(53.2mL的2.5M溶液，133mmol)，并将所得溶液在-78℃下搅拌20分钟。使用套管，将二异丙基氨基锂的溶液转移到环孢菌素的溶液中，并将所得到的混合物在-45℃下搅拌90分钟。逐滴加入2-吡啶基二硫化物(11g，49.9mmol)的无水THF溶液(20mL)，并允许所得到的混合物温暖至室温过夜。通过小心地加入饱和NaCl溶液(200mL)淬灭反应，并分离所得到的有机层。用乙酸乙酯(3×100mL)萃取水层，并用3N NaOH(2×100mL)、饱和NH 4Cl(100mL)和饱和NaCl(100mL)洗涤合并的有机级分(fraction)，然后用无水Na 2SO 4干燥并蒸发。通过硅胶色谱法分离标题化合物，为固体，7.18g。 1H NMR(400MHz,氯仿-d)d 8.45(ddd,J＝0.88,1.73,4.90Hz,1H),7.98(d,J＝9.66Hz,1H),7.65-7.73(m,1H),7.59(dt,J＝1.85,7.71Hz,1H),7.51(ddd,J＝0.76,1.68,6.44Hz,0H),7.45(d,J＝8.54Hz,1H),7.35(ddd,J＝1.73,6.97,8.77Hz,0H),7.25(s,0H),7.17(d,J＝7.96Hz,1H),7.09-7.15(m,2H),6.72(dt,J＝1.17,6.71Hz,0H),5.70(dd,J＝4.29,10.88Hz,1H),5.50(d,J＝6.39Hz,1H),5.32-5.38(m,1H),5.28(dd,J＝3.88,11.74Hz,1H),5.13(d,J＝10.88Hz,1H),4.97-5.11(m,2H),4.84(dq,J＝7.03,7.24Hz,1H),4.69(t,J＝9.15Hz,1H),4.54(quin,J＝7.31Hz,1H),4.13(q,J＝7.16Hz,0H),3.81(dt,J＝1.00,5.75Hz,1H),3.59-3.72(m,1H),3.50(s,2H),3.38(s,2H),3.26(s,2H),3.13(s,5H),2.70(d,J＝1.07Hz,5H),2.34-2.54(m,1H),1.92-2.23(m,4H),1.55-1.85(m,11H),1.19-1.54(m,11H),1.12(d,J＝6.54Hz,2H),0.78-1.07(m,30H),0.73(d,3H)。
第二步：制备[2’-(2-二甲基氨基乙氧基)-Sar] 3-环孢菌素A(化合物1)
Figure PCTCN2020070852-appb-000016
将三氟甲基磺酸铜(0.291g，0.8mmol)和3埃分子筛加入烧瓶中，加入无水THF(3mL)并用氩气吹扫烧瓶。在另一个烧瓶中，将[2’-(2-硫代吡啶基)-Sar] 3-环孢菌素A(1a)(0.293g，0.223mmol)、二甲基氨基乙醇(0.086g，0.96mmol)和3A分子筛在无水THF(2mL)中的混合物搅拌30分钟，然后加入至三氟甲基磺酸铜溶液中。允许反应在室温下搅拌过夜。加入NaHCO 3的饱和溶液(10mL)并且将混合物通过硅藻土(celite)过滤。将硅藻土用乙酸乙酯(3×25mL)洗涤并加入滤液。分离有机层；将水层用EtOAc(2×25mL)萃取，并将合并的有机级分经无水Na 2SO 4干燥和蒸发。在硅胶是纯化粗提物，提供标题化合物，86.4mg。 1H NMR(400MHz,氯仿-d)d 7.92(d,J＝9.61Hz,1H),7.75(d,J＝7.32Hz,1H),7.22(d,J＝8.15Hz),7.15(d,J＝7.86Hz,),6.01(s,1H),5.70(dd,J＝4.22,10.86Hz,1H),5.46(d,J＝6.10Hz,1H),5.35(q,J＝4.77Hz,1H),5.27(dd,J＝4.15,11.42Hz,1H),5.14(d,J＝10.83Hz,1H),5.05-5.11(m,1H),4.94-5.04(m,1H),4.77-4.90(m,1H),4.73(s),4.66(t,J＝8.83Hz,1H),4.46-4.57(m,1H),3.71-3.81(m,1H),3.58-3.67(m,J＝5.15,5.64,5.64,5.83Hz,1H),3.53-3.58(m,1H),3.51(s,2H),3.24(s,2H),3.20(s,2H),3.13(d,J＝2.10Hz,3H),2.71(d,J＝6.54Hz,3H),2.49-2.67(m,2H),2.33-2.46(m,1H),2.27(s,4H),1.88-2.20(m,4H),1.74(d,J＝0.29Hz,6H),1.57-1.68(m,5H),1.38-1.52(m,2H),1.35(d,J＝7.27Hz,3H),1.26(d,J＝2.88Hz,4H),0.77-1.12(m,30H),0.70(d,2H)。
例如，本领域技术人员会理解：可以通过使用不同的氨基醇试剂来制备化合物1的类似物。例如，醇和胺基团之间的碳原子数可以增加或减少(连接基团的实例包括：亚甲基、乙烯(ethylene)、丙烯(propylene)、丁烯(butylene)、戊搭烯(pentalene)，并且可以包括它们的支链形式，如异丙烯(iso-propylene)、仲丁烯(sec-butylene)、叔丁烯(tert-butylene)、2-甲基丁烯(2-methylbutylene)、2,2-二甲基丙烯(2,2-dimethylpropylene))。可替代地，或另外地，也可以改变氨基醇上的N-氨基取代基以给出化合物1的其他类似物(N-氨基取代基的实例包括：甲基、乙基、丙基、异丙基、正丁基、仲丁基、叔丁基、丁基)。
制备[2’-(2-N-Boc-氨基乙氧基)-Sar] 3-环孢菌素A
Figure PCTCN2020070852-appb-000017
将三氟甲基磺酸铜(4.95g，13.7mmol)和3A分子筛悬浮在无水THF(50mL)中并在氩气下搅拌30分钟。将[2’-(2-硫代吡啶基)-Sar] 3-环孢菌素A(1a)(5.0g，3.82mmol)和N-Boc-乙醇胺(2.64g，16.4mmol)的无水THF溶液(10mL)用3A分子筛干燥30分钟，然后加入三氟甲基磺酸铜悬浮液中。将得到的混合物在室温下搅拌过夜。加入饱和NaHCO 3(2×50mL)并将混合物通过硅藻土过滤。用EtOAc(4×100mL)洗涤硅藻土并分离有机层。用EtOAc(2×50mL)萃取水相，用饱和NaCl(50mL)洗涤合并的有机级分，用无水Na 2SO 4干燥并蒸发。在二氧化硅上纯化粗产物以产生标题化合物，4.18g。 1H NMR(400MHz,氯仿-d)d ppm 0.72(ddd,2H)0.91(m,31H)1.32(m,8H)1.48(dddd,J＝3.95,3.07,2.23,0.95Hz,2H)1.69(m,10H)2.10(m,4H)2.39(m,1H)2.70(m,4H)2.95(m,2H)3.12(d,J＝7.42Hz,4H)3.17(d,J＝9.37Hz,1H)3.20(s,2H)3.25(s,2H)3.29(m,J＝6.69,3.02,1.45,0.76,0.63Hz,1H)3.41(m,1H)3.51(s,2H)3.61(m,1H)3.75(dddd,J＝7.73,1.54,1.02,0.73Hz,1H)4.13(q,J＝7.11Hz,1H)4.50(m,1H)4.65(dd,J＝18.06,0.44Hz,1H)4.98(m,4H)5.30(m,2H)5.47(m,1H)5.70(m,1H)5.93(d,J＝0.34Hz)7.21(m,1H)7.71(m)8.03(m)。
制备[2’-(2-氨基乙氧基)-Sar] 3-环孢菌素A
Figure PCTCN2020070852-appb-000018
将[2’-(2-N-Boc-氨基乙氧基)-Sar] 3-环孢菌素A(3)(3.0g，2.2mmol)在无水CH 2Cl 2中 的溶液(30mL)冷却至0℃，并且逐滴加入三氟乙酸(6.54mL，10.03g，88mmol)，并将混合物搅拌30分钟。蒸发除去溶剂并且在二氧化硅上纯化粗产物以给出标题化合物，1.99g。
制备[2’-(2-二甲基氨基乙氧基)-Sar] 3-环孢菌素A(2)
Figure PCTCN2020070852-appb-000019
将[2’-(2-氨基乙氧基)-Sar] 3-环孢菌素A(0.273g，0.216mmol)溶解于CH 2Cl 2(5mL)，并加入甲醛(37％水溶液，0.048mL，0.69mmol)，然后加入NaB(OAc) 3H(0.138g，0.649mmol)，允许反应在约室温下搅拌18小时。将反应混合物过滤通过硅胶小垫，其用90:9:1的CH 2Cl 2:MeOH:浓NH 4OH(5×100mL)洗涤。蒸发溶剂，并且通过硅胶色谱法分离产物已提供标题化合物，0.214g。
亲环素抑制结合测定
使用改编自由Quesniaux等人(Eur.J Immunol.,1987,17:1359-1365)所描述的方法的竞争性ELISA测定本文所公开的化合物的亲环素抑制结合活性。与D-Lys 8-环孢菌素A(D-Lys 8-Cs)结合的经活化的琥珀酰基间隔子的酯通过第8位的D-赖氨酰残基连接至牛血清白蛋白(BSA)。将BSA溶解于0.1M硼酸盐缓冲液，pH 9.0中(4mg在1.4ml中)。在剧烈搅拌下，将溶解在二甲基甲酰胺(0.6ml)中的百倍摩尔过量的D-Lys 8-Cs逐滴加入BSA中。在温和搅拌的条件下，在室温下进行连接反应2至3小时，并将所得结合物(conjugate)对磷酸盐缓冲的盐水(PBS，pH7.4)进行充分透析。在用丙酮沉淀结合蛋白的等分试样后，没有共价结合的D-Lys 8-Cs保留在丙酮溶液中，然后计算环孢菌素共价结合的程度。
用D-Lys 8-Cs-BSA结合物涂覆微量滴定板(在4℃下，2μg/ml在PBS中，持续24小时)。用
Figure PCTCN2020070852-appb-000020
和仅PBS洗涤滴定板。为了阻断非特异性结合，向孔中加入2％BSA/PBS(pH7.4)并允许在37℃下孵育2小时。在单独的微量滴定板中，制备待测试化合物在乙醇中的五倍稀释系列。对于用人重组亲环素进行的测定，起始浓度为0.1mg/mL。将198μL的0.1μg/mL的亲环素溶液加入微量滴定板中，紧接着加入2μL已稀释的环孢菌素A(用作参比化合物)或本发明的化合物。允许涂覆的BSA-Cs结合物、游离环孢菌素A和亲环素之间的反应在4℃下平衡过夜。用稀释于含有1％BSA的PBS中的抗亲环素兔血清检测亲环素， 并在4℃下孵育过夜。如上所描述的那样洗涤滴定板。然后由稀释于1％BSA-PBS中的结合至碱性磷酸酶的山羊抗兔IgG检测结合的兔抗体，并允许在37℃下孵育2小时。如上所描述的那样洗涤滴定板。在37℃下用4-硝基苯基磷酸盐(1g/l在二乙醇胺缓冲液中，pH 9.8)孵育1至2小时后，使用分光光度计在405nm处分光光度法测量酶促反应。结果表示为EC 50，其是实现50％抑制所需的本发明的化合物的浓度。化合物1具有对亲环素A、亲环素B和亲环素D的小于100nM的EC 50值。
PPI酶(PPIase)抑制
基本上如Janowski等人以“未结合测定”{Jankowski et al.Anal.Biochem.(1997),252:299-307}所描述的那样，使用Agilent 8453分光光度计进行测定。在精密玻璃比色杯中将由35mM HEPES pH 7.8和50μM DTT组成的测定缓冲液冷却至10℃(伴随搅拌)，并且从100％DMSO原液加入抑制剂。获得空白光谱，然后加入纯化His标记的重组人亲环素酶(f/c 2nM)和四肽底物(已溶解于0.5M LiCl的三氟乙醇(Bachem，f/c 60μM)溶液中的Suc-Ala-Ala-Pro-Phe-对硝基苯胺)，并在5分钟内测量330nM处吸光度的变化。将吸光度数据进行一阶速率方程拟合以获得速率常数(由于混合最前10至15秒被排除)。从酶促速率减去背景速率计算催化速率。从速率常数对抑制剂浓度绘图获得抑制剂的K i。
线粒体通透性转换
线粒体通透性转换(MPT)是通过测量由Ca 2+诱导的线粒体的肿胀来确定的。程序改编自由Blattner et al.,2001,Analytical Biochem,295:220所描述的方法。从大鼠肝脏制备线粒体，大鼠肝脏已经用磷酸盐缓冲的盐水(PBS)灌注以除去血液，采用标准方法，其利用基于蔗糖的缓冲液中的温和匀化以及随后的差速离心以首先除去细胞碎片并然后沉淀线粒体。由150微摩尔Ca 2+(添加自浓缩的CaCl 2溶液)诱导肿胀，并通过测量535-540nm处的散射来进行监测。在诱导肿胀前5分钟加入代表性化合物。通过比较具有和不具有本文公开的化合物的肿胀来确定EC 50。化合物1以小于0.2μM的EC 50抑制线粒体肿胀。
Figure PCTCN2020070852-appb-000021
Figure PCTCN2020070852-appb-000022
表1–亲环素A抑制、亲环素D抑制和线粒体通透性转换(MPT)的测量结果
表1所示的结果表明，相对于相似的类似物(条目1至3和5至7)，化合物1(条目4)具有出乎意料地高的亲环素D抑制和MPT。相对于三种其他化合物(条目1、5和7)观察到在MPT方面的100倍改善，并且，相对于次最佳表现的类似物(条目3)观察到超过25倍改善。化合物1也显示出具有优异的亲环素D抑制，相对于测试的所有其他类似物具有至少50倍改善。
化合物1在器官损伤动物模型中的保护作用
由肾缺血-再灌注损伤诱导的急性肾损伤
通过将化合物1和环孢菌素A与克列莫佛/生理盐水/DMSO混合，制备了这些化合物的制剂。
将斯普拉-道来(Sprague-Dawley)大鼠分成6组：第(i)组为假手术组，给予克列莫佛/生理盐水/DMSO，无活性成分；第(ii)组为对照组，给予克列莫佛/生理盐水/DMSO，无活性成分；第(iii)组给予化合物1(3mg/kg)；第(iv)组给予CsA(3mg/kg；第(v)组给予化合物1(10mg/kg)；第(vi)组给予CsA(10mg/kg)。 除第(i)组外，即“假手术组”外，在大鼠中通过双侧肾动脉结扎30分钟然后释放结扎诱导肾缺血-再灌注诱导的急性肾损伤(AKI)。
对照组和治疗组中的动物给予腹膜内注射三次(结扎前1小时，结扎后4小时和8小时)。在结扎/释放过程后24小时从动物取血并分析血清肌酸酐和血尿素氮(BUN)浓度，作为肾损伤的测量。
如下示出这些实验的结果，并在图1和图2中图形显示。
Figure PCTCN2020070852-appb-000023
表2–第(i)组至第(vi)组的血清肌酸酐和BUN浓度的测量结果
结果讨论
在图1中，血清肌酸酐的浓度是肾损伤的指标。“假手术组”中的大鼠无诱导的AKI。“对照组”代表大鼠具有诱导的AKI，但没有治疗。因此，可以看出诱导的AKI导致血清肌酸酐从25μmol/ml(第i组)至195μmol/ml组(第ii组)的增加的水平。
与第ii组相比，用3mg/kg CsA治疗具有诱导的AKI的大鼠(第iv组)使得肌酸酐水平从195μmol/ml下降至115μmol/ml。因此理解的是CsA作用以预防缺血-再灌注损伤。
令人惊讶的是，当用3mg/kg的化合物1治疗具有诱导的AKI的大鼠时(第iii组)，给出了在肌酸酐水平方面的非常显著的降低，从195umol/ml下降至60umol/ml(与第ii组相比)，这接近于在“假手术组”(第i组)，即，没有诱导的AKI的大鼠中观察到的肌酸酐水平。当化合物1和CsA的剂量从3mg/ml(第iii组和第iv组)增加至10mg/ml(第v组和第vi组)时，似乎CsA和化合物1的益处被降低，但化合物1表现仍好于CsA。
在图2中，血尿素氮(BUN)浓度是肾损伤的指标。图2与图1中所见的趋势相同。即，3mg/kg的CsA获得BUN水平的下降(第iv组与第ii组相比)，而3mg/kg的化合物1显示在BUN水平方面非常显著的降低(第iii组与第ii组相比)，获得接近于在“假手术组”(第i组)中所见的BUN水平。将化合物1和CsA的浓度从3mg/kg(第iii组和第v组)增加至10mg/kg(第v组和第vi组)证明是较不有效的。该结果支持在图1中所见的结果。
由脂多糖(LPS)挑战诱导的急性肾损伤
在小鼠(C57)中，通过腹膜内注射LPS(15mg/kg)诱导了LPS诱导的急性肾损伤(AKI)。将20只小鼠随机分为两组。对照组中的动物接受媒介物(克列莫佛/生理盐水/DMSO)而治疗组接受化合物1(3mg/kg，在克列莫佛/生理盐水/DMSO中)，各自通过腹膜内给予。用媒介物或化合物1给予动物三次(LPS注射前1h以及LPS注射后4h和8h)，在LPS注射后12h从动物取血。通过提高的存活率(图3)和通过评价肾功能的标志物(图4)来确定化合物的活性。
Figure PCTCN2020070852-appb-000024
结果讨论
图3中，在动物存活率方面呈现了化合物1的保护作用。在3mpk的剂量水平下，化合物1施用获得该组中所有动物的存活，与此相比，在没有接受化合物1的对照组中，10只动物中仅4只存活。
图4示出在该实验中化合物1对肾功能的影响。用化合物1治疗的动物的较低的肌酸酐水平和血尿素氮水平更低与对肾脏的损伤的降低的水平相一致。
通过施用至器官供体在移植期间的器官保护
以猪肾脏例证了化合物1对经受移植条件的器官的保护作用。在所进行的实验中，在肾脏切除术前1h，经静脉递送，将5mg/kg的单剂量的化合物1施用至猪。切除肾脏，用标准高渗柠檬酸腺嘌呤(HCA)保存液灌注，然后在低温(0℃-4℃)下保存在HCA溶液中。在切除程序后的几个时间点，通过组织学评估和炎症标志物的测量来监测器官用于记录损伤：
○零点
○6h
○24h
○48h。
数据示于图5至图7中。
使用根据炎症程度的评分标准在苏木精和依红(HE)染色后进行组织学评估。采用“0至4”的半定量的评分系统，其中，非常小的或无病变的分配为“0”；轻度的或小的分配为“1”；中度的分配为“2”；严重的分配为“3”；非常严重的分配为“4”。
用总共9个猪肾脏进行实验，其中，将4个肾脏用作对照，没有保护剂化合物，而5个肾脏在肾脏切除之前1小时接受5mg/kg化合物1的i.v.剂量。数据显示所研究的肾脏在移除后的各时间的平均值。
图5示出平均炎症评分的结果；
图6示出对肾囊扩张的影响；并且
图7示出对肾小管扩张的影响。
结果讨论
综上所述，发现化合物1在治疗或预防缺血-再灌注损伤方面，特别是在较低浓度水平下是惊人有效的。在移除器官(用于随后植入受体)之前将该化合物施用至器官供体同样是特别有效的。图5至图7示出，通过在器官移除之前将化合物施用至供体，可以离体保存该器官。

Claims (10)

  1. 线粒体保护剂化合物用于保存器官的用途，其中，在从所述供体移除所述器官之前将所述化合物施用至器官供体以保护所述器官。
  2. 根据权利要求1所述的用途，其中，所述化合物是亲环素D抑制剂。
  3. 根据权利要求2所述的用途，其中，所述化合物是式1的化合物或其盐：
    Figure PCTCN2020070852-appb-100001
    其中n为2至5，并且
    R 1和R 2独立地选自H或C 1-C 4烷基，其中，R 1和R 2可以连接在一起以形成C 3-C 5杂烷基环。
  4. 根据权利要求3所述的化合物，其中，所述化合物是化合物1或其盐：
    Figure PCTCN2020070852-appb-100002
  5. 根据权利要求1至4中任一项所述的用途，其中，所述器官是肾脏。
  6. 一种保存来自肾脏供体的肾脏的方法，包括在从所述供体移除所述肾脏前将化合物施用至所述供体，其中，所述化合物是化合物1或其盐：
    Figure PCTCN2020070852-appb-100003
  7. 根据权利要求1至6中任一项所述的方法或用途，其中，所述供体是活体供体。
  8. 线粒体保护剂化合物在制备用于施用至活供体以保存肾脏的药物的用途，其中，所述化合物是化合物1或其盐：
    Figure PCTCN2020070852-appb-100004
  9. 根据权利要求1至7中任一项所述的方法或用途，其中，所述化合物的剂量为0.1至10mg/kg。
  10. 根据权利要求9所述的方法或用途，其中，所述化合物的剂量为1至3mg/kg。
PCT/CN2020/070852 2019-01-20 2020-01-08 环孢菌素类似物及其用途 Ceased WO2020147624A1 (zh)

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