WO2013138951A1 - 喹唑啉衍生物及其作为细胞凋亡抑制剂的用途 - Google Patents

喹唑啉衍生物及其作为细胞凋亡抑制剂的用途 Download PDF

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WO2013138951A1
WO2013138951A1 PCT/CN2012/000363 CN2012000363W WO2013138951A1 WO 2013138951 A1 WO2013138951 A1 WO 2013138951A1 CN 2012000363 W CN2012000363 W CN 2012000363W WO 2013138951 A1 WO2013138951 A1 WO 2013138951A1
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group
compound
alkoxy
alkyl
terazosin
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PCT/CN2012/000363
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English (en)
French (fr)
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刘磊
许晓椿
李笑宇
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无锡新融合药业有限公司
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Priority to EP12871851.7A priority Critical patent/EP2924037A1/en
Priority to SG11201501212QA priority patent/SG11201501212QA/en
Priority to PCT/CN2012/000363 priority patent/WO2013138951A1/zh
Publication of WO2013138951A1 publication Critical patent/WO2013138951A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to a novel pharmaceutical use of a quinazoline derivative and a quinazoline derivative such as terazosin, in particular, a step and a use of a quinazoline derivative such as terazone as a drug for apoptosis inhibitor, Also relates to the use of quinazoline derivatives, such as terazosin, in the treatment and/or prevention of septicemia and its complications, and the use of quinazoline derivatives such as terazosin to activate new targets of action .
  • Background technique
  • Apoptosis is a fundamental biological phenomenon of cells that plays a necessary role in the removal of unwanted or abnormal cells by multicellular organisms. It plays an important role in the evolution of organisms, the stability of the internal environment, and the development of multiple systems. Apoptosis is not only a special type of cell death, but also has important biological significance and complex molecular biological mechanisms. Apoptosis is a process in which multiple genes are strictly controlled. These genes are very conserved among species, such as the Bcl-2 family, the caspase family, oncogenes such as C-myc, and the tumor suppressor gene P53. With the development of molecular biology techniques, there are many processes for apoptosis.
  • the disorder of the apoptotic process may be directly or indirectly related to the occurrence of many diseases. It is understood that brain death due to cerebral thrombosis is also associated with apoptosis. In addition, it is believed that activation of phosphoglycerate kinase 1 (pgkl) contributes to the treatment or prevention of diseases such as cell apoptosis.
  • pgkl phosphoglycerate kinase 1
  • Terazosin is usually used clinically as its hydrochloride salt, and the specifications of the marketed tablets or capsules are 1 mg, 2 mg and 5 mg.
  • Terazosin hydrochloride can be used to treat benign prostatic hyperplasia and can also be used to treat hypertension. It can be used alone or in combination with other antihypertensive drugs such as diuretics or al-adrenergic blockers.
  • the dose range of terazosin for adults is usually 1 ⁇ 10mg.
  • Terazosin is used to treat benign prostatic hyperplasia (BPH), and the reduction of benign prostatic hyperplasia symptoms and improvement in urine flow rate are associated with smooth muscle relaxation caused by al-adrenergic receptor blockade in the bladder neck and prostate.
  • terazosin can reduce blockage of the bladder outlet without affecting bladder contraction.
  • terazosone lowers blood pressure by reducing total peripheral vascular resistance.
  • the vasodilation and blood pressure lowering effects of terazosin appear to be mainly caused by blockade of al-adrenergic receptors.
  • potent inhibitors of apoptosis the development of novel anti-apoptotic compounds for use in, for example, the treatment and/or prevention of sepsis and its complications, and stroke septicemia and its Complications are compelling research goals. Summary of the invention
  • the present inventors have surprisingly found that a class of quinazoline derivatives such as the clinical use of the drug terazosin for benign prostatic hyperplasia and hypertension alone or in combination with antibiotics can effectively treat and/or prevent septicemia and its complication.
  • the present invention has been completed based on this finding.
  • the first aspect of the invention relates to the combination of formula I,
  • Ria and R lb each independently selected from 11, NH 2, OH, C 1-6 alkyl -, C l-6 alkoxy, -C ⁇ alkyl -, C 2-6 alkenyl -, C 2-6 Alkynyl-, C L -6 methoxy-, C 1 -6 alkyl acyl-, aryl acyl-, C 6-1() aryl-, C 5-6 cycloalkyl-, or R la and Rib, together with the nitrogen atom to which they are attached, form a 5- or 6-membered ring wherein the alkyl group is optionally substituted with from 1 to 3 substituents selected from the group consisting of: hydroxy, halo;
  • R 2 and R 3 are each independently selected from the group consisting of 11, halo, C 1-6 alkyl-, halo C 1-6 alkyl-, C 2 . 6 alkenyl-, C 2 - 6 alkynyl-, CN, N0 2, NH 2, OH, C 1-6 alkoxy -, C 1-6 alkoxy, -C ,.
  • R 4 and R 5 are each independently selected from H, halogen, CN, N0 2 , NH 2 , OH, C L-6 alkyl-, C 1-6 alkoxy-C alkoxy-, C 1-6 Alkanoyloxy-, halogenated C 1-6 fluorenyl-, C 2-6 alkenyl-, C 2-6 alkynyl-, C 1-6 alkoxy-, d. 6 alkanoylamino-, aromatic Acylamino-, saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic group, saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic oxy-, alkyl acyl group.
  • a second aspect of the invention relates to the use of a compound of formula I, such as terazosin, for the preparation of a medicament as an inhibitor of apoptosis, the use of a compound of formula I, for example, terazosin, for the preparation of a medicament as a pgkl activator, or Use of a compound of I, such as terazosin, for the manufacture of a medicament for the treatment and/or prevention of sepsis and its complications.
  • a compound of formula I such as terazosin
  • a third aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically and/or prophylactically effective amount of a compound of formula I, such as terazosin or a pharmaceutically acceptable salt or solvate thereof, and optionally pharmaceutically acceptable Plant.
  • a fourth aspect of the invention provides a kit product comprising a therapeutically and/or prophylactically effective amount of a compound of formula I
  • a compound of formula I for example, terazosin or a pharmaceutically acceptable salt or solvate thereof, and at least one antimicrobial agent.
  • a fifth aspect of the invention relates to a method of inhibiting apoptosis in a subject or biological sample in need thereof, a method of activating pgk1 in a subject or biological sample in need thereof, or treating in a subject in need thereof And/or methods of preventing septicemia and its complications.
  • a sixth aspect of the invention relates to a compound of formula I, such as terazosone, for use as an inhibitor of apoptosis, as a pgkl activator, or as a therapeutic and/or prophylactic septicemia and its complications.
  • Figure 2 is a list of compounds selected for drug use. These compounds are selected based on Cmap. Grouped according to the positive or negative correlation of their apoptotic microarray chip technology.
  • Figure 3 depicts the screening of drugs using the apoptotic Drosophila model.
  • the survival rate of each drug in apoptotic fruit flies is expressed as mean + SE.
  • Control no medication given
  • white bars Different drugs are given as white or black bars.
  • Test ⁇ 5.
  • Figure 4 depicts that terazosin (4 g/ml) significantly prevented cell apoptosis due to LPS and IFNy toxicity.
  • Apoptotic cells were stained by Annexin V.
  • the statistical results obtained by the experiment show that terazosin can inhibit cell apoptosis.
  • the number of cells per group was counted as 200-250 cells.
  • Figure 5a depicts the survival rate curve for the effect of terazosin (0.4 mg/kg) on the LPS model of septicemia. Survival curve analysis was performed by the & ⁇ 131 ⁇ 161 ⁇ 1" test using the 1 ⁇ ( ⁇ 1 ⁇ 11
  • Figure 5b depicts the agarose gel DNA breaks after LPS treatment are shown. Each band represents genomic DNA from one thymus of the mouse. The treatment of each mouse is labeled on top of each band.
  • Figure 6 depicts the survival of mice after treatment with terazosin (0.04 mg/kg) after 12 hours of LPS injection (13.5 mg/kg). The analysis was carried out by 1 ⁇ ( ⁇ 1 ⁇ 111 4 algorithm by 1 ⁇ 18 ⁇ ] ⁇ 61 ⁇ 1 "test. The number of mice used and the p value are shown in the figure, where T represents terazosin.
  • Figure 7 depicts the survival curve of the E. coli model of septicemia. After 1 hour of E. coli injection, terazosin (0.4 mg/kg) was injected. The analysis was performed by [( ⁇ ! ⁇ ! ⁇ algorithm by ! ⁇ !-! ⁇ ! ⁇ ! ⁇ ). The number of mice used and the p value are shown in the figure.
  • Figure 8 depicts the effect of terazosin on the growth of E. coli. After 24 hours of terazosin, the effect of it with ampicillin on bacterial growth was compared by bacteriostatic. The results in the figure indicate that the untreated control and different doses of terazosin (0.2 g T, 2 g T, 200 ⁇ 8 ⁇ , 2 mg ⁇ ) did not inhibit E. coli growth, while ampicillin (2 mg Amp) ) shows inhibition of E. coli growth.
  • Figure 9 depicts the survival curve of the effect of terazosin on the CLP model of septicemia.
  • Terazosin was injected twice by subcutaneous injection at 0.08 mg/kg at 1.5 and 24 hours after CLP.
  • the use of the antibiotic Co-Am alone has increased mortality in the CLP model.
  • the combination of terazosin and the antibiotic Co-Am has a protective effect on the CLP model of septicemia.
  • Analysis was performed by Kaplan-Merier test using the Log Rank algorithm. The number of mice used and the p value are shown in In the picture.
  • T represents terazosin
  • Co-Am represents a mixture of amoxicillin and potassium clavulanate in a weight ratio of 4:1.
  • Figure 10 depicts a Western blot of the active form of apoptosis protease 3 in LPS and IFNy treated Raw 264.7 cells. The amount of protein loaded in the three differently treated groups was the same.
  • Figure 11 depicts the chemical modification of terazosin and the chemical flow of the Affi-gel linkage.
  • Figure 12 depicts the use of terazosin agarose beads to obtain a protein band that is apparently only present in the experimental group (neutral bands marked with arrows between 43 kd and 55 kd). It was identified as Pgkl by protein profiling.
  • Figure b depicts the passage of Pkgl on the terazosin agarose beads column with in vitro expressed Pgkl, indicating that terazosin and Pgkl are directly bindable.
  • Figure 13 depicts the effect of different concentrations of terazosin on Pgkl activity.
  • Figure 14 depicts that Raw 264.7 cells expressing Pgk1 antagonize apoptosis.
  • the apoptotic cell membrane phospholipidylserine turns from the inside of the lipid membrane to the outside.
  • Annexin V binds to phospholipids, thereby labeling apoptotic cells.
  • Figure 15 depicts the protective effect of the mouse in the CLP model after infection with Pgkl lentivirus. Each mouse was injected with 1 x 10 7 active units of virus and the experiment was performed one week later.
  • Figure 16 depicts the effect of terazosin on reducing blood glucose in mice.
  • Figure 17 depicts the effect of trachyzine against cerebral thrombosis.
  • a first aspect of the invention relates to a compound of formula I,
  • Ria and Rib are each independently selected from H, NH 2, OH, C 1-6 alkyl -, C 1-6 alkoxy, -C l-6 alkyl -, C 2 - 6 alkenyl -, C 2 _ 6 alkynyl-, d-6 alkoxy-, C 1-6 alkyl acyl-, aryl acyl-, C 6 - , ⁇ -aryl-, C 5-6 cycloalkyl, or! ⁇ and! ⁇ together with the nitrogen atom to which they are attached form a 5- or 6-membered ring wherein the alkyl group is optionally substituted with from 1 to 3 substituents selected from the group consisting of: hydroxy, halo;
  • R 2 and R 3 are each independently selected from H, halogen, C 1-6 alkyl-, halo C 1-6 alkyl-, C 2 - 6 alkenyl-, C 2 -6 alkynyl-, CN, N0 2, NH 2, OH, Ci -6 alkoxy -., Cj 6 kang -C ⁇ alkoxy group -, C, -6 alkanoyloxy -, C l -6 alkanoylamino -, aryl An acylamino-, a saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic group, a C 1-6 alkyl acyl-, or R 2 and R 3 together with the ring atom to which they are attached form a 5- or 6- a carbon ring or a heterocyclic ring;
  • R 4 and R s are each independently selected from H, halogen, CN, N0 2 , NH 2 , OH, C 1-6 alkyl-, c 1-6 alkoxy-c 1 -6 alkoxy-, c 1-6 alkanoyloxy-, halo c 1-6 alkyl-, c 2-6 alkenyl-, c 2-6 alkyne a base-, alkoxy-, alkanoylamino-, aroylamino-, a saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic group, a saturated or unsaturated 5- or 6-membered carbocyclic ring or Heterocyclyloxy-, alkyl acyl.
  • R la and R lb are each independently selected from H, NH 2 , OH,
  • said R and R lb are each independently selected from H, -NH 2 , -OH.
  • CH 3 C(0)-, -(CH 2 ) 2 -0-(CH 2 ) 2 -OH -CH 2 -CH CH 2 , -CH 2 -C ⁇ CH, -(CH 2 ) S CH 3 . -(CH 2 ) 4 -CF 3 , cyclohexyl, -CH 2 -(CH 2 ) 3 - CH 2 -, -(CH 2 ) 2 0(CH 2 ) 2 -OH, -Ph. CH 3 , -C(0)-CF 3 , -C(0)-Ph.
  • each and independently is selected from the group consisting of H, halogen, C 1-6 alkyl-, haloalkyl-, C 1-6 alkoxy-, C 1-6 alkoxy-C 1-6 alkoxy-, C 1-6 alkanoyloxy-, C 1-6 alkanoylamino-, aroylamino-, a saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic group, Or R 2 and R 3 together with the ring atom to which they are attached form a 5- or 6-membered carbocyclic or heterocyclic ring.
  • said R 2 and R 3 are each independently selected from the group consisting of H, CH 3 0-, -CH 2 -0-CH 2 -, -0(CH 2 ) 2 OC 2 H s , -OC ( 0) CH 3 , -F,
  • R 4 and R 5 are each independently selected from H, halogen, C 1-6 alkyl-, pitoxy-C 6 alkoxy-, .6 alkanoyloxy- , haloalkyl-, alkoxy-, alkanoylamino-, aroylamino-, saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic, saturated or unsaturated 5- or 6-membered carbon Ring or heterocyclic oxy.
  • the R 4 and R s are each independently selected
  • the alkyl, alkenyl and alkynyl groups are linear or branched.
  • the c 1-6 group is selected from the group consisting of c, _ 5 alkyl, c 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl Base, tert-butyl.
  • the C 2 -6 alkenyl group is selected from the group consisting of C 2 -s alkenyl, C 2 -4 alkenyl, such as ethenyl, propenyl, allyl.
  • the C 2-6 alkynyl group is selected from the group consisting of c 2-5 alkynyl, c 2-4 alkynyl.
  • the compound according to the first aspect of the invention wherein the c 5-6 cycloalkyl group is selected from the group consisting of a cyclopentyl group and a cyclohexyl group.
  • the compound according to the first aspect of the invention, wherein the saccharin is selected from the group consisting of fluorine, chlorine, bromine and iodine, preferably fluorine and Chlorine.
  • the compound according to the first aspect of the present invention which is a compound selected from the following numbers Co.l to Co.33 (the structures of which are as described in the preparation section of the compound):
  • a second aspect of the invention relates to the use of a compound of formula I, such as traviline, for the manufacture of a medicament as an inhibitor of apoptosis.
  • a second aspect of the invention also relates to the use of a compound of formula I, such as terazosin, for the manufacture of a medicament as a pgkl activator.
  • a second aspect of the invention also relates to the use of a compound of formula I, such as terazosone, for the manufacture of a medicament for the treatment and/or prevention of septicemia and its complications.
  • a compound of formula I such as terazosone
  • a second aspect of the invention also relates to the use of a compound of formula I, such as terazosin, for the manufacture of a medicament for the treatment and/or prophylaxis of hyperglycemia, cerebral thrombosis and complications thereof.
  • a compound of formula I such as terazosin
  • apoptosis inhibitor is for the treatment and/or prevention of clinical diseases and for laboratory diagnosis and/or detection.
  • the pgk1 activator is for the treatment and/or prevention of clinical diseases as well as laboratory diagnosis and/or detection.
  • the septicemia is a septicemia caused by infection with bacteria and/or other microorganisms.
  • the complication of the septicemia is selected from the group consisting of: renal failure, respiratory failure, coagulopathy, organ damage (including but not limited to toxic cardiomyopathy, encephalopathy, liver disease, and toxic bowel Paralysis, etc.), purulent meningitis, pneumonia, lung abscess, cellulitis, osteomyelitis, pyelonephritis.
  • the daily dose of the compound of the formula I, for example, terazosin, for human or animal is 0.001 to 5 mg/kg, preferably 0.002 to 4 mg/kg, It is preferably 0.003 to 3 mg/kg, preferably 0.005 to 2.5 mg/kg, preferably 0.0075 to 2 mg/kg, preferably 0.01 to 2 mg kg, preferably 0.01 to 1 mg/kg.
  • terazosin is a pharmaceutically acceptable salt of terazosin or a solvate thereof.
  • terazosin is the hydrochloride salt of terazosin.
  • terazosin is a hydrate of terazosin hydrochloride.
  • the terrazine is the dihydrate of terazosin hydrochloride.
  • the medicament further comprises at least one antimicrobial drug.
  • the medicament further comprises at least one antimicrobial drug selected from the group consisting of: penicillin antibiotics, cephalosporin antibiotics, P-lactamase inhibitors, amino groups Glycoside antibiotics, tetracycline antibiotics, amide alcohol antibiotics, macrolide antibiotics, sulfonamides, trimethoprims, quinolones.
  • the medicament further comprises at least one antimicrobial drug selected from the group consisting of: amoxicillin, penicillin, penicillin V, oxacillin, cloxacillin, chlorofluorocarbon Xilin, ampicillin, piperacillin, azlocillin, potassium clavulanate, sulbactam, sultamicillin, tazobactam, aztreonam, meropenem.
  • the medicament further comprises at least one antimicrobial agent selected from the group consisting of: amoxicillin, clavulanic acid clock.
  • the drug further comprises amoxicillin and potassium clavulanate.
  • a third aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically and/or prophylactically effective amount of a compound of formula I, such as terazosin or a pharmaceutically acceptable salt or solvate thereof, and optionally pharmaceutically acceptable Carrier.
  • a pharmaceutical composition according to the third aspect of the present invention which further comprises at least one antimicrobial drug.
  • the pharmaceutical composition according to the third aspect of the present invention which further comprises at least one antimicrobial drug selected from the group consisting of: a blue-collagen antibiotic, a cephalosporin antibiotic, a ⁇ -lactamase inhibitor, and a gas Glycoside antibiotics, tetracycline antibiotics, amide alcohol antibiotics, macrolide antibiotics, sulfonamides, trimethoprims, quinolones.
  • the pharmaceutical composition according to the third aspect of the present invention which further comprises at least one antimicrobial drug selected from the group consisting of: amoxicillin, penicillin, quercetin V, oxacillin, cloxacillin, chlorofluorocarbon Xilin, ampicillin, lacillin, azlocillin, potassium clavulanate, sulbactam, sultamicillin, tazobactam, aztreonam, meropenem.
  • the pharmaceutical composition further comprises at least one antimicrobial agent selected from the group consisting of: amoxicillin, clavulanic acid clock.
  • the pharmaceutical composition further comprises amoxicillin and clavulanic acid clock.
  • a pharmaceutical composition according to the third aspect of the invention which is for use in an apoptosis inhibitor.
  • the apoptosis inhibitor is for use in the treatment and/or prevention of clinical diseases as well as laboratory diagnosis and/or detection.
  • composition according to the third aspect of the present invention which is used as a p g k1 activator.
  • the pgk1 activator is used in the treatment and/or prevention of clinical diseases as well as in laboratory diagnosis and/or detection.
  • a pharmaceutical composition according to the third aspect of the invention for use in the treatment and/or prevention of septicemia and its complications.
  • the septicemia is a septicemia caused by a bacterial and/or other microbial infection.
  • the complication of the septicemia is selected from the group consisting of: renal failure, respiratory failure, coagulopathy, organ damage (including but not limited to toxic cardiomyopathy, encephalopathy, liver disease, and toxic intestinal paralysis) , purulent meningitis, pneumonia, lung abscess, cellulitis, osteomyelitis, pyelonephritis.
  • a pharmaceutical composition according to the third aspect of the present invention for use in the treatment and/or prevention of hyperglycemia, cerebral thrombosis and complications thereof.
  • a pharmaceutical composition according to the third aspect of the present invention wherein the compound of the formula I, for example, terazosin, is administered to a human or an animal (eg, a mammal) at a daily dose of 0.001 to 5 mg/kg, preferably 0.002 to 4 Mg/kg, preferably 0.003 ⁇ 3 mg/kg, preferably 0.005 ⁇ 2.5 mg/kg, preferably 0.0075 ⁇ 2 mg/kg, preferably 0.01 ⁇ 2 mg/kg, preferably 0.01 ⁇ 1.
  • the fourth aspect of the present invention provides a kit A product, which comprises a therapeutically and/or prophylactically effective amount of a compound of formula I, such as terazosin or a pharmaceutically acceptable salt or solvate thereof, and at least one antimicrobial agent.
  • kits product according to the fourth aspect of the present invention, wherein the compound of the formula I, such as tertimarazine or a pharmaceutically acceptable salt or solvate thereof, and the at least one antimicrobial agent are in the same composition or In the separated composition.
  • the compound of the formula I such as tertimarazine or a pharmaceutically acceptable salt or solvate thereof
  • the at least one antimicrobial agent are in the same composition or In the separated composition.
  • kits according to the fourth aspect of the present invention wherein the compound of the formula I, such as terrazine or a pharmaceutically acceptable salt or solvate thereof, and the at least one antimicrobial agent are in separate compositions.
  • a kit product comprising a first composition and a second composition separated from each other, the first composition comprising a therapeutically and/or prophylactically effective amount of a compound of formula I, such as terazosin or A pharmaceutically acceptable salt or solvate thereof and optionally a pharmaceutically acceptable carrier, the second composition comprising a therapeutically and/or prophylactically effective amount of an antimicrobial agent and optionally a pharmaceutically acceptable carrier.
  • a compound of formula I such as terazosin or A pharmaceutically acceptable salt or solvate thereof
  • the second composition comprising a therapeutically and/or prophylactically effective amount of an antimicrobial agent and optionally a pharmaceutically acceptable carrier.
  • kits product according to a fourth aspect of the present invention, wherein the antimicrobial drug is selected from the group consisting of penicillin antibiotics, cephalosporin antibiotics,
  • kits product wherein the antimicrobial drug is selected from the group consisting of: amoxicillin, chlorhexidine, penicillin V, oxacillin, cloxacillin, flucloxacillin, ampicillin, and valacillin, Azlocillin, potassium clavulanate, sulbactam, sultamicillin, tazobactam, aztreonam, meropenem.
  • the antimicrobial drug is selected from the group consisting of: amoxicillin, potassium clavulanate.
  • the antimicrobial drug comprises amoxicillin and potassium clavulanate.
  • kits product which is for use in an apoptosis inhibitor.
  • the apoptosis inhibitor is for use in the treatment and/or prevention of clinical diseases as well as laboratory diagnosis and/or detection.
  • a kit product according to the fourth aspect of the invention is for use as a pgk1 activator.
  • the pgk1 activator is used in the treatment and/or prevention of clinical diseases as well as in laboratory diagnosis and/or detection.
  • kits product for use in the treatment and/or prevention of septicemia and its complications.
  • the septicemia is a septicemia caused by a bacterial and/or other microbial infection.
  • the complication of the septicemia is selected from the group consisting of: renal failure, respiratory failure, coagulopathy, organ damage (including but not limited to toxic cardiomyopathy, encephalopathy, liver disease, and toxic intestinal paralysis) , purulent meningitis, pneumonia, lung abscess, cellulitis, osteomyelitis, pyelonephritis.
  • kits product according to the fourth aspect of the present invention for use in the treatment and/or prevention of hyperglycemia, 'cerebral thrombosis and complications thereof.
  • a kit according to the fourth aspect of the present invention wherein the compound of the formula I, for example, terazosin, is administered to a human or an animal (e.g., a mammal) at a daily dose of 0.001 to 5 mg/kg, preferably 0.002 to 4 mg/ Kg, preferably 0.003 to 3 mg/kg, preferably 0.005 2.5 mg/kg, preferably 0.0075 to 2 mg/kg, preferably 0.01 to 2 mg/kg, preferably 0.01 to 1 mg/kg.
  • a fifth aspect of the invention relates to a method of inhibiting apoptosis in a subject or biological sample in need thereof, the method comprising administering to said subject or biological sample an effective amount of a compound of formula I, such as terazosin.
  • a fifth aspect of the invention also relates to a method of activating pgk1 in a subject or biological sample in need thereof, the method comprising administering to the subject or biological sample an effective amount of a compound of formula I, such as terazosin.
  • a fifth aspect of the invention also relates to a method of treating and/or preventing septicemia and its complications in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula I, such as terazos Oxazine.
  • a fifth aspect of the invention also relates to a method of treating and/or preventing hyperglycemia, cerebral thrombosis and complications thereof in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula I, for example Terazosin.
  • septicemia is septicemia caused by infection with bacteria and/or other microorganisms.
  • the complication of the septicemia is selected from the group consisting of: renal failure, respiratory failure, coagulopathy, organ damage (including but not limited to toxic cardiomyopathy, encephalopathy, liver disease, and toxic bowel Paralysis, etc.), purulent meningitis, pneumonia, lung abscess, cellulitis, osteomyelitis, pyelonephritis.
  • the daily dose of the compound of the formula I, such as travidin, for human or animal is 0.001 to 5 mg/kg, preferably 0.002 to 4 mg/kg, Preferably, 0. 003 to 3 mg/kg, preferably 0.005 to 2.5 mg/kg, preferably 0.0075 2 mg/kg, preferably 0.01 to 2 mg/kg, preferably 0.01 to 1 mg/kêt according to the method of the fifth aspect of the invention, Wherein the compound of formula I is terazosin.
  • terazosin is a pharmaceutically acceptable salt of terazosin or a solvate thereof.
  • terazosin is the hydrochloride salt of terazosin.
  • the terazosin is a hydrate of terazosin hydrochloride.
  • the oxazosin is a dihydrate of terazosin hydrochloride.
  • a method according to the fifth aspect of the invention further comprising administering to said subject or biological sample an effective amount of at least one antimicrobial agent.
  • the method according to the fifth aspect of the invention further comprising administering to said subject or biological sample an effective amount of at least one antimicrobial agent selected from the group consisting of: a penicillin antibiotic, a cephalosporin antibiotic, --lactamase inhibitors, aminoglycoside antibiotics, tetracycline antibiotics, amide alcohol antibiotics, macrolide antibiotics, chloramines, trimethoprims, quinolones.
  • at least one antimicrobial agent selected from the group consisting of: a penicillin antibiotic, a cephalosporin antibiotic, --lactamase inhibitors, aminoglycoside antibiotics, tetracycline antibiotics, amide alcohol antibiotics, macrolide antibiotics, chloramines, trimethoprims, quinolones.
  • the method according to the fifth aspect of the present invention further comprising administering to said subject or biological sample an effective amount of at least one antimicrobial drug selected from the group consisting of: amoxicillin, penicillin, penicillin V, benzene Oxacillin, cloxacillin, flucloxacillin, ampicillin, indapcillin, azlocillin, potassium clavulanate, sulbactam, sultamicillin, tazobactam, aztreonam, meropenem.
  • the antimicrobial agent The substance is selected from the group consisting of: amoxicillin, potassium clavulanate.
  • the antimicrobial is amoxicillin and potassium clavulanate.
  • a sixth aspect of the invention relates to a compound of formula I, such as terazosin, for use as an inhibitor of apoptosis.
  • a sixth aspect of the invention also relates to a compound of formula I, such as terazosin, for use as a pgk1 activator.
  • the sixth aspect of the invention also relates to a compound of formula I for use as a therapeutic and/or prophylactic septicemia and a complication thereof, such as terazos. Qin.
  • the sixth aspect of the invention also relates to a compound of formula I, such as terazos, for use in the treatment and/or prevention of hyperglycemia, cerebral thrombosis and complications thereof.
  • a compound of formula I such as terazos
  • a compound of formula I according to the sixth aspect of the invention wherein the complication of the septicemia is selected from the group consisting of: renal failure, respiratory failure, coagulopathy, organ damage (including but not limited to toxic cardiomyopathy, encephalopathy, liver disease, and Toxic intestinal paralysis, etc., purulent meningitis, pneumonia, lung abscess, cellulitis, osteomyelitis, pyelonephritis.
  • a compound of formula I according to the sixth aspect of the invention wherein the daily dose of the compound of formula I for use in a human or animal (e.g., a mammal) is 0.001 to 5 mg/kg, preferably 0. 002 to 4 mg/kg, preferably 0.003 ⁇ 3 mg/kg, preferably 0.005-2.5 mg/kg, preferably 0.0075-2 mg/kg, preferably 0.01-2 mg/kg, preferably 0.01-1 mg/kg.
  • a compound of formula I according to the sixth aspect of the invention which is terazosin.
  • a compound of formula I according to the sixth aspect of the invention which is a pharmaceutically acceptable salt of trachymazine or a solvate thereof.
  • a compound of formula I according to the sixth aspect of the invention which is the hydrochloride salt of terazosin.
  • a compound of formula I according to the sixth aspect of the invention which is a hydrate of terazosin hydrochloride, such as the dihydrate of terazosin hydrochloride.
  • a compound of formula I according to the sixth aspect of the invention which is further combined with at least one antimicrobial agent.
  • a compound of formula I according to the sixth aspect of the invention which is further combined with at least one antimicrobial agent selected from the group consisting of: penicillin antibiotic cephalosporin antibiotics, P-lactamase inhibitors, aminoglycosides Antibiotics, tetracycline antibiotics, amide alcohol antibiotics, macrolide antibiotics, guanamines, trimethoprims, quinolones.
  • at least one antimicrobial agent selected from the group consisting of: penicillin antibiotic cephalosporin antibiotics, P-lactamase inhibitors, aminoglycosides Antibiotics, tetracycline antibiotics, amide alcohol antibiotics, macrolide antibiotics, guanamines, trimethoprims, quinolones.
  • a compound of formula I according to the sixth aspect of the invention which is further combined with at least one antimicrobial agent selected from the group consisting of: amoxicillin, penicillin, penicillin V, oxacillin, cloxacillin, flucloxacillin , ampicillin, piperacillin, azlocillin, clavulanic acid, sulbactam, sulbactam, tazobactam, aztreonam, meropenem.
  • the antimicrobial drug is selected from the group consisting of: amoxicillin, potassium clavulanate.
  • the antimicrobial drug is amoxicillin and potassium clavulanate.
  • the amount of the compound of formula I can be referred to the existing clinical dose.
  • terazosin when used for human treatment and/or prevention of septicemia and its complications, can be used in a dose of 0.01 to 100 times the dose of the drug currently used for other diseases such as hypertension. It is preferably 0.02 to 80 times, preferably 0.05 to 20 times, preferably 0.1 to 10 times, preferably 0.1 to 5 times, preferably 0.2 to 5 times, preferably 0.2 to 2 times.
  • the amount of the antimicrobial agent used can be referred to an existing clinical dose.
  • the dosage when used for human treatment and/or prevention of septicemia and its complications, the dosage may be 0.01 to 100 times, preferably 0.02, of the dose of the antibacterial drug currently used for other diseases (for example, anti-infection). ⁇ 80 times, preferably 0.05 to 20 times, preferably 0.1 10 times, preferably 0.2 to 5 times.
  • the amount of amoxicillin and/or potassium clavulanate used can be referred to an existing clinical dose.
  • the dose may be the dose of the clinically useful amoxicillin and/or potassium clavulanate for other diseases (eg, anti-infection). 0.01 to 100 times, preferably 0.02 80 times, preferably 0.05 to 20 times, preferably 0.1 to 10 times, preferably 0.2 to 5 times.
  • the compound of Formula I does not include a compound of the number Co.33 - any of the aspects of any aspect of the invention or any of the aspects of the invention is equally applicable to the other In one aspect or any of the other aspects of the invention, as long as they do not contradict each other, and of course, when applied to each other, the corresponding features may be suitably modified as necessary.
  • septicemia also known as "sepsis”
  • serpsis has the meaning commonly known to those skilled in the art, and generally refers to a condition in which bacteria and/or other microorganisms enter the blood circulation, and A systemic serious infection caused by growth and reproduction of toxins.
  • Gram-positive cocci sepsis is prone to migratory lesions; Gram-negative bacilli are susceptible to septic shock. When sepsis is accompanied by multiple abscesses, it is called sepsis or sepsis.
  • pgkl activator refers to an activator of phosphoglycerate kinase 1, abbreviated as pgkl, which activates pgk1 and thus is understood by those skilled in the art to be useful for correlation. Treatment, prevention, alleviation and/or alleviation of a disease or condition.
  • the term “effective amount” refers to a dose that can achieve treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • pharmaceutical composition which is used interchangeably with “composition,” is meant to be useful in the treatment, prevention, amelioration, and/or alleviation of the diseases, disorders, and conditions of the invention in a subject. , the substance of the symptoms.
  • the term "subject” or “patient” may refer to an animal that receives the compositions and extracts of the invention to treat, prevent, ameliorate and/or alleviate the diseases, disorders, symptoms of the invention, particularly It is a mammal, such as a person, a dog, a monkey, a cow, a horse, and the like.
  • disease or condition refers to a physical condition of the subject that is associated with the disease or condition of the present invention.
  • % generally refers to a weight/weight percentage for a solid material and a weight/volume percentage for a liquid when the total material is liquid.
  • the percentage characterizing the liquid shield is generally a percentage of volume/volume.
  • terazosin when terazosin is mentioned, it includes not only the compound represented by the above structure, but also a pharmaceutically acceptable salt (for example, hydrochloride) of the above structural compound, and the above structural compound and a salt thereof. Solvates such as hydrates.
  • the terazosin refers to terazosin hydrochloride dihydrate.
  • a number of studies have been carried out using trathionazine as a typical example of the compound of the formula I to demonstrate the surprising effect of the present invention; in the following tests, in particular biological tests, the test reagents used, unless otherwise indicated, terazosin Refers to terazosin hydrochloride dihydrate.
  • the present invention attempts to screen for apoptosis inhibitors from clinical drugs.
  • the present invention analyzes gene expression by microarray chip technology using a cell death model of Drosophila.
  • the present invention determines a candidate apoptosis blocker by bioinformatics analysis using a connectivity map (Cmap).
  • Cmap connectivity map
  • the present invention screens a drug candidate for inhibiting apoptosis of Diptera insect Drosophila (Z)mw)/ ⁇ //fl) cells.
  • Z diptera insect Drosophila
  • terazosin which is a clinical drug for relieving hypertension.
  • terazosin inhibits apoptosis in macrophages mediated by bacterial endotoxin (lipopolysaccharide, LPS, 2 g/ml) and interferon Y (IFN Y, 50 U/ml).
  • LPS lipopolysaccharide
  • IFN Y interferon Y
  • terazosin can greatly reduce mortality in mice in three experimental models of septicemia.
  • the combination of terrazine and antibiotics resulted in better protection than antibiotics alone.
  • the results of the present invention indicate that terazosin is a novel inhibitor of apoptosis. It can be used in combination with antibiotic treatment.
  • Drosophila is an important animal model system for screening drugs. Furthermore, the caspase-mediated apoptotic pathway is sufficiently conserved between Diptera insects and humans. For example, ⁇ op ⁇ yr) plays a major role in the apoptosis of ⁇ (White et al., 1994). Although ⁇ / ⁇ expression from heat-induced promoters causes widespread distribution Abnormal apoptosis and organ death (White et al., 1996). The present invention investigated whether the present invention is capable of screening for apoptosis blockers using a Drosophila apoptosis model.
  • HS-GaW a systemically expressed and heat-activated promoter
  • UAS upstream activating sequence
  • HS>rpr 45-Refl/;er Drosophila
  • the female offspring developed normally from beginning to end.
  • heat shock was given at 37 °C. 2-3 hours, approximately 50-70% of the offspring die after 14-24 hours ( Figure 1).
  • the present invention selected 25 of the top compounds ( Figure 2) and further determined their lethality to HS > ⁇ The effect of the rate.
  • the present inventors have found that only terazosin can significantly increase the survival of HS>y;r Drosophila (Fig. 3).
  • the present invention induces apoptosis by LPS and IFNy in macrophage RAW264.7 cells, which are induced in cultured cells.
  • the present invention observes cell death by staining with Annexin V (Fig. 4).
  • the present inventors have found that cells treated with LPS (2 pg/ml) and IFNy (50 U/ml) cause cell membrane damage, eversion, and are stained by Annexin V. This is a classic pattern of apoptosis.
  • the use of terazosin (4 pg/ml) reduced apoptosis by 50% (Fig. 4).
  • the present inventors have surprisingly found that terazosone can effectively inhibit apoptosis and can be further used to treat and/or prevent septicemia and its complications.
  • the present inventors investigated mice that received LPS treatment.
  • the present inventors found that injection of terazosin (0.4 mg/kg i.p.) at 1.5 hours after LPS injection (13.5 mg/kg, i.p.) significantly increased mouse survival (Fig. 5a).
  • the present invention detects the DNA fragmentation of apoptosis markers.
  • the thymus is sensitive to apoptosis during septicemia (Ayala et al., 1998).
  • the present invention compares genomic DNA from the thymus of the mouse treated with LPS to the genomic DNA of the mouse thymus treated with LPS plus terazosin.
  • the results showed that a significant DNA ladder occurred in LPS-treated mice, but was greatly reduced in mice treated with terazosin (Fig. 5b)terrorism
  • terazosin can be used in septicemia
  • the present invention injects terazosin 12 hours after the use of LPS. The results show that terazosin still has a satisfactory effect (Fig. 6).
  • the present invention further tests terazosin in a cecal ligation and puncture (CLP) model, which is considered to be the gold standard model for experimental septicemia ( Parker and Watkins, 2001; Wichterman et al., 1980).
  • CLP cecal ligation and puncture
  • the present invention detects 0.4 mg/kg of terazosin (the same concentration used in the LPS model).
  • mice injected with terazosin died faster than the control group (data not shown).
  • the present invention concludes that this may be due to severe cardiac dysfunction and hypotension caused by CLP. Therefore, the ability of drugs to lower blood pressure may mask their anti-apoptotic effects.
  • the present invention reduced the concentration of terazosin to 0.08 mg/kg.
  • the inventors measured the effect of this concentration on the blood pressure of mice, and found that the blood pressure of the mice was completely normal. This result is consistent with its use in rats without lowering blood pressure (Kyncl et al., 1985).
  • terazosin is significantly promoted when injected 1.5 times and 24 hours after surgery.
  • the survival of mice in the CLP model was advanced (Fig. 9).
  • the present invention tested terazosin in combination with antibiotics.
  • the antibiotics amoxicillin and potassium clavulanate (Co-Am, amoxicillin and clavulanate potassium were selected to have a weight ratio of 4:1).
  • references to specific examples or examples elsewhere in this invention also refer to such a 4:1 ratio.
  • the weight ratio of the cilar and the clavulanic acid clock can be from 1:1 to 10:1, especially from 1:1 to 7:1, for example about 1:1, about 4:1, about 7:1) with terazosin. combination.
  • the present inventors have found that the combination of terazosin and Co-Am shows a stronger protective effect than Co-Am alone (Fig. 9). In addition, this combination therapy still demonstrates the therapeutic effect of administration 6 hours after CLP.
  • terazosin In order to test whether the protective effect of terazosin is related to its function as an ⁇ -adrenergic receptor inhibitor, the present invention examined Brazosin (0.4 mg/kg, ip), which is another ⁇ - xian Adenin receptor blockers (Cavero et al., 1977). The present inventors have found that brazosin has no effect on sepsis caused by LPS. These results indicate that terazosin significantly increases the survival rate of inhibition of LPS-mediated septicemia, and its function is likely to be through inhibition of apoptosis rather than targeting ⁇ -adrenergic receptors.
  • the present invention explores its potential role in the inhibition of apoptosis proteases, since apoptosis in Drosophila is achieved mainly by intrinsic activation of apoptosis proteases. (McCall and Sachr, 1997). However, Western blot analysis of the present invention showed that the active form of the daughter cell apoptosis protein 3 (the homologous gene of Dpc-1 and drICE in Drosophila) was reduced by terazosin (Fig. 10). The present invention concludes that terazosin modulates the upstream target of apoptotic protease 3.
  • the present invention binds terazosin to agarose beads, and Figure 11 shows its chemical synthesis pathway.
  • the protein was then taken in the Raw 264.7 cell extract and it was found that one more band was compared to the blank control and the drug competition control group (Fig. 12a). According to the corpus, this band is Pgkl.
  • the inventors expressed and purified the mouse Pgk1 protein in the bacteria, and found that terazosin can directly bind to the Pgk1 protein in vitro (Fig. 12b).
  • the results of the present inventors showed that the RAW264.7 cell line established by expressing Pgkl lentivirus significantly reduced the cell death after LPS and IFNy treatment; whereas the apoptosis of cells overexpressing EGFP did not change (Fig. 14). Further, the inventors expressed Pgkl or EGFP as a control in mice by subcutaneous injection of lentivirus. After one week of CLP surgery, it was found that mice injected with Pgk1 virus significantly improved survival compared to a group of mice infected with green fluorescent protein (Fig. 15). The present invention is not limited by any particular theory, and the inventors believe that Pgkl is a new target for terazosin.
  • Step 1 Ammonia gas was introduced into a solution of the compound la (50 mmol) in 200 mL of tetrahydrofuran, and the reaction was carried out at 25 ° C for 36 hours. A large amount of white solid precipitated in the system, and the white solid obtained by filtration was washed with tetrahydrofuran to give the final product lf. The yield was 63%.
  • Step 2 15 mL of acetic anhydride was added to the compound lf (10 mmol), and the mixture was refluxed for 2 hr. A large amount of white solid was precipitated in the system to be cooled, and the obtained white solid was washed with tetrahydrofuran to give a final product yield: 63%.
  • Step 3 In the case of an argon atmosphere, lh (2 mmol) was added to a solution in which lg (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 4.5 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product li as compound Co.l. The yield was 60%.
  • Step 1 Compound lb (20 mmol) was added to a solution of compound la (20 mmol) in 100 mL of methanol, and the reaction was carried out at 25 ° C for 4 hours. After the sheet chromatography indicated that the conversion of la was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in an environment of -20 ° C to stand for crystallization. The obtained white solid was recrystallized from petroleum acid / ethyl acetate to give the final product lc. The yield was: 41%.
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of lc (2 mmol) of 1-pentanol. After refluxing for 4.5 hours, the solution was placed at 0-5 ° C. The crystal was allowed to stand in the middle. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from acetonitrile / methanol to give the final product le as compound Co. The yield is 62%.
  • Step 1 To a solution of compound la (20 mmol) in 100 mL of methanol was added hydrazine hydrate (20 mmol), and the reaction was carried out at 25 ° C for 4 hours. After the thin plate chromatography indicated that the conversion of la was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in an environment of -20 ° C to stand for crystallization. The obtained white solid was recrystallized from petroleum ether / ethyl acetate to give the final product 3a. The yield was: 61%.
  • Step 2 In a hydrogen atmosphere, a solution of 3a (2 mmol) of 1-pentanol was added to the solution of ld (2 mmol) a for 4.5 hours, and then placed at 0-5 ° C. The crystal was allowed to stand in the middle. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 3b as Compound Co. The yield was: 31%.
  • Step 1 Hydrochloric acid hydrate (20 mmol) was added to a solution of the compound la (20 mmol) in 100 mL of methanol, and the reaction was carried out at 25 ° C for 3 hours. Thin plate chromatography indicated that after la conversion was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in a -20 ° C environment to stand for crystallization. The resulting white solid was recrystallized from petroleum acid / ethyl acetate to give the final product 4a. The yield was: 88%.
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 4a (2 mmol) of 1-pentanol. After refluxing for 4.5 hours, the solution was placed at 0-5 ° C. The crystal was allowed to stand in the middle. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / methanol to give the final product 4b as Compound Co. The yield is 75%.
  • Step 1 To a solution of compound la (20 mmol) in 100 mL of methanol was added EtOAc (22 mmol). After the thin plate chromatography indicated that the conversion of la was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in an environment of -20 ° C to stand for crystallization. The obtained white solid was recrystallized from petroleum acid / ethyl acetate to give the final product 5a. The yield was: 31%.
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 4a (2 mmol) of 1-pentanol. After the reaction system was refluxed for 4.5 hours, it was placed at 0-5. Crystallization is allowed to stand in the C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 5b as Compound Co. The yield is 75%.
  • Step 1 Allylamine (28 mmol) was added to a solution of the compound la (20 mmol) in 100 mL of methanol, and the reaction was carried out at 25 ° C for 10 hours. Thin plate chromatography indicated that after la conversion was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in a -20 ° C environment to stand for crystallization. The resulting white solid was recrystallized from petroleum ether / ethyl acetate to give the final 6a. The yield is: 44%
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 4a (2 mmol) of 1-pentanol. After the reaction system was refluxed for 4.5 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / dec. The yield is 75%.
  • Step 1 To a solution of the compound la (20 mmol) in 100 mL of methanol was added Compound 7a (20 mmol), and the reaction was carried out at 25 ° C for 3 hours. Thin plate chromatography indicated that after la conversion was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in a -20 ° C environment to stand for crystallization. The resulting white solid was reconstituted with petroleum ethyl acetate
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 7b (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 4.5 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with lOinL acetone, and then recrystallized from diethyl ether / methanol to give the final product 7c as compound Co. The yield is 62%.
  • Step 1 To a solution of the compound la (20 mmol) in 100 mL of methanol was added Compound 8a (20 mmol), and the reaction was carried out at 25 ° C for 1.5 hours. Thin plate chromatography indicated that after la conversion was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in a -20 ° C environment to stand for crystallization. The resulting white solid was reconstituted with petroleum acid / ethyl acetate
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 8b (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 4.5 hours, it was placed at 0-5. Crystallization is allowed to stand in the C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / methanol to give the final product 8c as compound Co. Production The rate is: 42%.
  • Step 1 To a solution of compound la (20 mmol) in 100 mL of decyl alcohol was added compound 9a (20 mmol). It was carried out for 2.5 hours under C. After the thin plate chromatography indicated that the conversion of la was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in an environment of -20 ° C to stand for crystallization. The obtained white solid was recrystallized from petroleum acid / ethyl acetate to give the final product 9b. The yield was: 21%.
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 9b (2 mmol) of 1-pentanol. After refluxing for 4.5 hours, the system was allowed to stand for crystallization at 0-5 ° C. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 9c as compound Co.9. The yield is 43%.
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 9b (2 mmol) of 1-pentanol. After the reaction system was refluxed for 4.5 hours, it was placed at 0-5. Crystallization is allowed to stand in the C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from acetic acid / methanol to give the final product 10c as compound Co.lO. The yield was: 43%.
  • Step 1 Compound lb (20 mmol) was added to a solution of compound la (20 mmol) in 100 mL of methanol, and the reaction was carried out at 25 ° C for 5 hours. After the thin plate chromatography indicated that the conversion of la was completed, 100 mL of diethyl ether was added to the system, mixed, and placed in an environment of -20 ° C to stand for crystallization. The obtained white solid was recrystallized from petroleum acid / ethyl acetate to give the final product, ⁇ / RTI> yield: 41%.
  • Step 2 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of llb (2 mmol) of 1-pentanol. After the reaction system was refluxed for 4.5 hours, it was left to stand in an environment of 0 to 5 ° C, and the obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from ethyl acetate / decyl alcohol to obtain the final product 11c. Ll. The yield was 62%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 12a (2 mmol) of 1-pentanol. After the reaction system was refluxed for 4.5 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from acetic acid / methanol to give the final product 12b as Compound Co.l. The yield was 62%.
  • Step 1 20 mL of 13a was added to compound lf (10 mmol), and the mixture was refluxed for 2 hr. After cooling to room temperature, a large amount of white solid was precipitated from the system, and the obtained white solid was washed with tetrahydrofuran to give the final product 13b. The yield was 82%.
  • Step 2 In the case of an argon atmosphere, ld was added to a solution of 13b (2nunol) of 1-pentanol (2mmol reaction system was refluxed for 5.5 hours, and then it was left to stand in a 0-5 C environment for crystallization. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 13c as the compound Co yield: .
  • Step 1 22 mL of 14a was added to the compound lf (10 mmol), and the reaction was refluxed for 2 hr. After cooling to room temperature, a large amount of white solid was precipitated from the system, and the obtained white solid was filtered and washed with tetrahydrofuran to give the final product 14b. Yield
  • Step 2 In the case of an argon atmosphere, a solution of 14b (2 mmol) of 1-pentanol was added to the solution of ld (2 mmol) o for 4.5 hours, and then placed in a 0-5 ° C environment. Crystallize. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 14c as compound Co.l. The yield was: 48%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 15b (2 minol) of 1-pentanol. After the reaction system was refluxed for 4.5 hours, it was placed at 0-5. Crystallization is allowed to stand in the C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / decyl alcohol to give the final product 15c as Compound Co. The yield was: 55%.
  • Step 1 In the case of an argon atmosphere, ld (2mraol) was added to a solution in which 16a (2 mmol) of 1-pentanol was dissolved. After refluxing for 4.5 hours, the reaction system was placed in an environment of 0-5 ° C. Crystallize. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / decyl alcohol to give the final product 16a as compound Co.l. The yield was: 45%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 17a (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 4 hours, it was allowed to stand in a 0-5 C environment. Crystallization. The obtained white crystals were washed twice with 10 mL of propyl ketone, and then recrystallized from diethyl ether / methanol to give the final product 17a as Compound Co. The yield was: 55%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 18 mmol (2 ramol) of 1-pentanol, and the reaction system was refluxed for 3 hours, and then placed in a 0-5 ° C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / decyl alcohol to give the final product 18b as compound Co.l8. Yield: 75%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 19a (2 mmol) of 1-pentanol. After the reaction system was refluxed for 3 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of acetone and then recrystallized from diethyl ether / dec. The yield was: 54%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 20a (2 mmol) of 1-pentanol was dissolved, and the reaction system was refluxed for 3 hours, and then placed in a 0-5 ° C environment. The crystals were allowed to stand still. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the product 20b as compound Co. 20. Yield: 54%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 21a (2 mmol) of 1-pentanol was dissolved, and the reaction system was refluxed for 4 hours, and then placed in a 0-5 ° C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / dec.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 22a (2 imnol) of 1-pentanol. After the reaction system was refluxed for 5 hours, it was placed at 0-5. Crystallization is allowed to stand in the C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / methanol to give the compound 22b as compound. The yield was: 61%.
  • Step 1 In the case of an argon atmosphere, was added to a solution of the ld 23a (2mmol) of 1-pentanol solution (2mmol) 0 After reaction was refluxed for 5 hours, placed in a static environment 0-5 ° C Crystallize. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 23b as Compound Co. The yield was: 48%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 25a (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 4 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of acetone and then recrystallized from acetic acid / methanol to give the final product 25b as Compound Co. The yield was: 24%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 25a (2 mmol) of 1-pentanol was dissolved. After refluxing for 4.5 hours, the reaction system was placed in a 0-5 ° C environment. Crystallize. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 25b as Compound Co. The yield was: 45%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 26a (2 mmol) of 1-pentanol. After the reaction system was refluxed for 4 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the final product 26a as compound Co.l. The yield was: 55%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 27a (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 3 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of acetone and then recrystallized from diethyl ether/methanol to give the compound 27b as Compound Co. The yield was: 53%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 28a (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 3 hours, it was placed at 0-5. Crystallization is allowed to stand in the C environment. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether/methanol to give the product 28b as compound Co. The yield was: 75%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 29a (2 mmol) of 1-pentanol. After the reaction system was refluxed for 3 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of hexanes and then recrystallized from diethyl ether/methanol to give the product 29b as compound compound.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 30a (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 5 hours, it was allowed to stand in an environment of 0 to 5 ° C to stand for crystallization. The obtained white crystals were washed twice with 10 mL of propyl ketone, and then recrystallized from diethyl ether/methanol to give the final product 30b as Compound Co. The yield was: 65%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution in which 31a (2 mmol) of 1-pentanol was dissolved. After the reaction system was refluxed for 4 hours, it was placed in an environment of 0-5 ° C. Crystallize. The obtained white crystals were washed twice with 10 mL of acetone, and then recrystallized from diethyl ether / methanol to give the compound 31b. The yield was: 24%.
  • Step 1 In the case of an argon atmosphere, ld (2 mmol) was added to a solution of 32 a (2 imnol) of 1-pentanol. After refluxing for 4 hours, the reaction system was placed in an environment of 0-5 ° C. Crystallize. The obtained white crystals were washed twice with 10 mL of propyl ketone, and then recrystallized from diethyl ether/methanol to give the product 32b as compound Co.32. The yield is: 45% plausible
  • UAS-rpr and HS-GaM fruit flies were purchased from the Bloomington Storage Center in the United States.
  • the F1 progeny (HS>rpr) of these two germline hybrids were used to screen for drugs. These progeny flies were raised at 18 °C.
  • Various drugs were dissolved in a 5% dextrose solution at the concentration used in the cell culture in cmap.
  • the drug solution (150 ⁇ M) was placed on the three layers of the filter paper in the small tube. Then, 20 1-3 day old adult H5>r/7 fruit flies were placed in the bottle for 1 day. To induce apoptosis, these flies were heat shocked for 2 hours at 37 °C. After 12 hours of heat shock, Calculate the survival rate.
  • RNA samples 10 bottles of 1-3 day old fruit flies (gene background matching control) were prepared from hs>rpi" and progeny of hs-Gal4 hybridized with w67c?. Each bottle contained 35 female flies. Flies were frozen to liquid nitrogen before heat shock (0 hours) and heat shock 1, 2, 3, 4, 5, 6, 7, 8 and 12 hours originate Total preparation by RNeasy mini kit (Quiagen, Inc) RNA. The TURBO DNase kit (Ambion, Inc) was used for further processing to remove chromosomal DNA from the sample. The quality of the RNA sample detection and the microarray chip technology processing steps were performed in accordance with the Affymetrix DNA microarray technology standard protocol.
  • Drosophila 2.0 DNA microarray technology from Affymetrix (Affymetrix, Inc) was used. These arrays are processed simultaneously to reduce the effects between different batches. Data analysis was performed using Arrayassist 5.0 (Affymetrix, Inc) software. The "RMA" algorithm is used to analyze the data. Since only one experimental data point is collected at each time point, three adjacent time points are analyzed together, and the intermediate value of each time point is determined as the data of the group. For example, time 0, 1, and 2 are called “time 1" in the result.
  • the present invention designs microarray chip technology data in this process to increase the instantaneous resolution of gene expression changes. The present invention concludes that if the gene shows a change in transcription, it can be reflected in adjacent time points. For functional gene enrichment, the present invention uses a database tool (http: ⁇ david.abcc.ncifcrf.gov/).
  • the present invention converts up-regulated and down-regulated genes from Drosophila microarray chip technology into mammalian homologous genes (www.affymetrix.com) via a database. Then, a signal file is generated based on the scheme provided by cmap (Lamb et al., 2006). Thereafter, positively and negatively correlated compounds are obtained. These drugs were purchased from Sigma-Aldrich.
  • RAW 264.7 cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS (Gibco), 0.1 g/L streptomycin, and 0.06 g/L penicillin (Amresco). To induce apoptosis, 90% confluent RAW 264.7 cells were cultured for 24 hours in DMEM (10% FBS) containing 2 g/ml lipopolysaccharide (LPS; Escherichia coli OUl: B4, Sigma).
  • DMEM 10% FBS
  • LPS lipopolysaccharide
  • Cells were first fixed with Carnoy fixative (75% ethanol, 25% acetic acid) and then stained with Hoechst 33342 (10 g/ml, Sigma) for 10 min. After washing three times with ice-cold PBS, the cells were observed by a confocal microscope (TCS SP5, Leica).
  • mice Male BALB/C (20 ⁇ 3 g) mice purchased from Vital River (Beijing Weitong Lihua Company, China) were raised at the Peking University Animal Center. All trials were approved by the Peking University Institutional Animal Care and Use Committee.
  • LPS LPS (13.5 mg/k g , ip)-time for septicemia.
  • E. coli model of disease each mouse was injected (ip) with 1 x 10 s CFU of bacteria.
  • terazosin 0.4 mg/kg or 0.04 mg/kg
  • vehicle buffer was injected (ip) 1.5 hours after LPS administration. The mouse lethality rate was recorded every 12 hours for 7 days.
  • mice were anesthetized with hydrated chlorine (300 mg/kg, ip). The midline of the abdomen was cut 1.5-2.0 cm, the cecum was exposed, and after returning to the cecal artery, the cecum was puncture once with a standard needle No. 22, and ligation was performed 5 mm from the top of the cecum. The abdomen is then closed by continuous suturing. Finally, the animals were resuscitated by injection of pre-warmed saline (37 C; 5 ml/100 g, sc). For antibiotic treatment, Co-Am (Lunan Pharmaceutical Group, Shandong, China) (30 mg/kg) was administered by gavage. The survival rate of the mice was observed every 12 hours for 7 days.
  • the apoptotic protein Su 3 was detected by an antibody recognizing activated Apoptosis protease 3 (#9664, Cell Signaling), and the antibody to actin was purchased from Wuhan Boster Company.
  • terazosin The potential antibiotic activity of terazosin was analyzed using an Oxford Cup. Different masses of terazosin were added to the Oxford Cup and placed in the LB culture subfamily covered with Escherichia coli 0111:B4. After 24 hours, the saliva, sputum and ampicillin were grown on E. coli. Impact.
  • the present invention washes off the hybrid protein bound to Affi-gd by attaching terazosin to Affi-gel and then mixing with the protein extract of RAW 264.7 cells.
  • the bound protein was eluted with a loading buffer, and the gel was heated after heating (15% protein gel). Specific protein bands were excised and characterization was performed. ⁇
  • the mouse Pgkl recombinant protein (His-Pgkl) was expressed in E. coli, and then His-Pgkl was purified using a nickel column. The purified protein was diluted to 0.15 units per ml, and a gradient concentration of terazosin was added to measure the activity of Pgkl.
  • Kaplan-Meier test was used under the Log Rank algorithm.
  • a one-sided ANOVA test was used.
  • the present invention uses a student-t test.
  • terazosin new properties have been discovered, in particular, they are useful as inhibitors of apoptosis, as well as for the treatment and/or prevention of septicemia and its complications.
  • the activity of the compound of formula I is expressed as a multiple of activated Pgkl, wherein 0.02 ⁇ 1 terazosin activates Pgkl activity 2.9-fold; compounds Co.l to Co.32 are tested at 0.02 g/ml, activating multiples of Pgkl activity See above, for example, the multiples of Co.l and Co.2 activation of Pgkl activity are 2.9 and 2.3, respectively.
  • RAW 264.7 cells were cultured in DMEM medium (Gibco), and 5% fetal bovine serum and double antibody (0.1 mg/ml of streptavidin and 0.06 mg/ml of penicillin) were added.
  • Apoptosis was induced by the addition of 2 g/ml lipopolysaccharide and (LPS; Escherichia coli OHl: B4, Sigma-Aldrich) 50 U/ml gamma interferon (Peprotech). Add a certain concentration of compound at the same time.
  • Western blot was used to assess apoptosis, and the antibody was Caspase 3 active form antibody (#9661, Cell Signaling). >3 ⁇ 4 ⁇ -Actin was used as the internal reference (Boster). The extent of apoptosis was assessed by the value of Caspase 3/p-Actin.
  • mice were injected with terazosin (0.4 mg/kg ip) once, and the blood glucose of the mice was measured 18 hours later. It was found that the blood glucose was reduced by nearly 30% (/-test, P ⁇ 0.01); See significant differences. The result is shown in Figure 16.
  • other exemplary compounds of the invention such as Co.l, Co.3>Co.7, Co.9, Co.l3, Co.l6, Co.l9 Co.24, Co.27, Co .32 shows a reduction in blood glucose by 20% to 50%.
  • terazosin can significantly reduce the area of brain dead tissue.
  • other exemplary compounds of the invention such as Co.l, Co. 4 , Co.7, Co.8, Co.13-14, Co.l6, Co.l9, Co.24-27, Co.29, Co.31-32 all showed significant reduction in dead tissue area, ⁇ 0.05.
  • mice (about 20 g) were intraperitoneally injected with lipopolysaccharide (13.5 mg/kg). After half an hour, a certain concentration of the drug was injected. The survival of the mice was observed every 12 hours. A total of 7 days were observed. There was no significant difference.
  • CLP cecal puncture and ligation

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Abstract

本发明涉及式I化合物例如特拉唑嗪的新用途和新靶点。具体地，本发明涉及式I化合物在制备作为细胞凋亡抑制剂的药物中的用途，或者在制备治疗和/或预防败血病及其并发症的药物中的用途。本发明的发现为抑制细胞凋亡以及治疗和/或预防败血病及其并发症提供了新的方法和方向。

Description

喹唑啉衍生物及其作为细胞凋亡抑制剂的用途 技术领域
本发明涉及喹唑啉衍生物以及喹唑啉衍生物例如特拉唑嗪的新制药用途，特别；步 及喹唑啉衍生物例如特拉峻嗪作为细胞凋亡抑制剂的药物中的用途，还涉及喹唑淋衍 生物例如特拉唑嗪在治疗和 /或预防败血病及其并发症的药物中的用途， 以及喹唑啉 衍生物例如特拉唑嗪激活新的作用靶点的用途。 背景技术
败血病 (Sepsis)是全球特护病房中死亡的首要原因。 败血病据认为是一种复杂的 炎症的异常调节(80!^ 6( 31.，1996)。 然而， 细胞因子或抗炎特异性药剂的许多临床试 验未能显著改善败血病患者的存活， 重新考虑治疗败血病的策略是必要的。 尽管效果 是有限的， 但是抗生素治疗是治疗败血病的重要途径。 基础研究表明， 抑制细胞冽亡 可有效阻断试验性败血病。在败血病患者中，免疫细胞的凋亡可进一步削弱免疫应答。 此外， 阻断细胞凋亡已被证明是一种救治败血病的重要策略 (Braun et al., 1999; Hotchkiss et al., 2000; Chung et al., 2001 ; Weaver et al., 2004; Wesche-Soldato et al., 2005)。
细胞凋亡是细胞的一种基本生物学现象，在多细胞生物去除不需要的或异常的细 胞中起着必要的作用。 它在生物体的进化、 内环境的稳定以及多个系统的发育中起着 重要的作用。 细胞凋亡不仅是一种特殊的细胞死亡类型， 而且具有重要的生物学意义 及复杂的分子生物学机制。 凋亡是多基因严格控制的过程。 这些基因在种属之间非常 保守， 如 Bcl-2家族、 caspase家族、 癌基因如 C-myc、 抑癌基因 P53等， 随着分子 生物学技术的发展对多种细胞凋亡的过程有了相当的认识 ,但是迄今为止凋亡过程确 切机制尚不完全清楚。 而凋亡过程的紊乱可能与许多疾病的发生有直接或间接的关 系。 人们理解由于脑血栓造成的脑死亡亦与细胞凋亡有关。 此外， 据信磷酸甘油酸酯 激醉 1 (phosphoglycerate kinase 1 ,简称为 pgkl)的激活有助于一些疾病例如与细胞凋 亡相关的治疗或预防。
特拉唑嗪 (Terazosin)通常以其盐酸盐用于临床， 已上市片剂或胶囊剂的规格有 lmg、 2mg和 5mg。 盐酸特拉唑嗪可用于治疗良性前列腺增生症， 也可用于治疗高血 压， 可单独使用或与其它抗高血压药物如利尿剂或 al-肾上腺素能阻滞剂合用。 针对 现有的临床适应症， 特拉唑嗪用于成人的曰剂量常用范围为 l~10mg。 特拉唑嗪用于 治疗良性前列腺增生 (BPH), 用药后良性前列腺增生症状减轻和尿流速改善与膀胱颈 和前列腺中的 al-肾上腺素能受体阻断所引起的平滑肌松弛有关。 因为在膀胱体中有 相对少的 al-肾上腺素能受体，因此特拉唑嗪能够减轻膀胱出口的阻塞而不影响膀胱 的收缩。 此外， 特拉唑螓通过减少总外周血管阻力从而使血压降低。 特拉唑嗪的血管 舒张、 血压降低作用似乎主要是由 al-肾上腺素能受体阻断所引起的。 迄今为止， 临床上仍然缺少有效的细胞凋亡抑制剂， 开发新颖的抗 -细胞凋亡的 化合物以用于例如治疗和 /或预防败血病及其并发症 , 以及脑卒中败血病及其并发症 是引人注目的研究目标。 发明内容
本发明的目的是提供治疗和 /或预防败血病及其并发症的新方法和新靶点。 本发 明人令人惊奇地发现，一类喹唑琳衍生物例如临床用于良性前列腺增生症和高血压的 药物特拉唑嗪单独或与抗生素组合可以有效治疗和 /或预防败血病及其并发症。 本发 明基于此发现而得以完成。
发明概述
本发明第一方面涉及式 I化合 ，
Figure imgf000003_0001
或其药学可接受的盐、 溶剂合物、 酯、 前药， 其中
Ria和 Rl b 各自独立地选自 11、 NH2、 OH、 C1-6烷基 -、 Cl-6烷氧基 -C^烷基 -、 C2-6烯基 -、 C2-6炔基-、 CL -6垸氧基-、 C1 -6烷基酰基-、 芳基酰基-、 C6-1()芳基-、 C5-6 环烷基-,或者 Rla和 Rib与它们连接的氮原子一起形成 5-或 6-元环， 其中所述烷基任 选被 1-3个选自下列的取代基取代： 羟基、 卤素；
R2和 R3 各自独立地选自 11、 卤素、 C1-6烷基-、 卤代 C1-6烷基-、 C2.6烯基 -、 C2-6炔基-、 CN、 N02、 NH2 , OH、 C1-6烷氧基-、 C1-6烷氧基 -C,.6烷氧基-、 C,-6 烷酰基氧基-、 Cl-6烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环 基、 C 烷基酰基-,或者 R2和 R3与它们连接的环原子一起形成 5-或 6-元碳环或杂环；
R4和 R5 各自独立地选自 H、 卤素、 CN、 N02、 NH2、 OH、 CL-6烷基 -、 C1-6烷氧基 -C 烷氧基-、 C1-6烷酰基氧基-、 卤代 Cl-6浣基 -、 C2-6烯基 -、 C2-6炔基 -、 C1-6烷氧基-、 d.6烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环 基、 饱和或不饱和的 5-或 6-元碳环或杂环基氧基-、 烷基酰基。
本发明第二方面涉及式 I化合物例如特拉唑嗪在制备作为细胞凋亡抑制剂的药物 中的用途， 式 I化合物例如特拉唑嗪在制备作为 pgkl激活剂的药物中的用途， 或者 式 I化合物例如特拉唑嗪在制备治疗和 /或预防败血病及其并发症的药物中的用途。
本发明第三方面提供一种药物组合物，其中包含治疗和 /或预防有效量的式 I化合 物例如特拉唑嗪或其药学可接受的盐或溶剂合物， 以及任选的药学可接受的栽体。
本发明第四方面提供一种药盒产品，其中包括治疗和 /或预防有效量的式 I化合物 例如特拉唑嗪或其药学可接受的盐或溶剂合物， 以及至少一种抗微生物药物。
本发明第五方面涉及在有需要的受试者或生物样品中抑制细胞凋亡的方法，在有 需要的受试者或生物样品中激活 pgkl的方法， 或者在有需要的受试者中治疗和 /或预 防败血病及其并发症的方法。
本发明第六方面涉及用作细胞凋亡抑制剂、 用作 pgkl激活剂、 或者用作治疗和 / 或预防败血病及其并发症的式 I化合物例如特拉唑溱。 附图说明
图 1 描迷了诱导 ffS-Ga^表达 1-3小时之后凋亡 HS>r/H"果蝇的存活率。 各点表 示平均值 + SE。 试验 n = 6。
图 2是药物 选中使用的化合物列表。 这些化合物是基于 Cmap选择的。 根据它们 的细胞凋亡的微阵列芯片技术的正相关性或负相关性来分组的。
图 3 描绘了用凋亡果蝇模型筛选药物。 凋亡果蝇对每种药物的存活率以均值 + SE表示。 对照 (未给予药物)是以白色棒条显示的。 给予不同的药物分别以白色或黑色 棒条表示。 试验 π=5。
图 4 描绘了特拉唑喙 (4 g/ml)可显著阻止由于 LPS和 IFNy毒性所致的细胞凋 亡。 被 Annexin V染色的为凋亡细胞。 试验得到的统计结果显示特拉唑嗪可抑制细胞 凋亡。 每组细胞数目计数为 200-250个细胞。
图 5 中， 图 5a描述了特拉唑嗪 (0.4 mg/kg)对败血病的 LPS模型效果的存活率曲 线。 用 1^(^ 1^11|4算法通过 & 1311-1\161^1"检验进行存活率曲线分析。 使用的小鼠数和 p 值显示于该图中。 图 5b描述了琼脂糖凝胶显示 LPS治疗之后的 DNA断裂。 每条带表示 来自小鼠的一个胸腺的基因组 DNA。 各小鼠的处理方法标记于每条带的顶部。
图 6 描述了小鼠在 LPS注射 (13.5 mg/kg)12小时之后使用特拉唑嗪 (0.04 mg/kg) 后的存活率。 用 1^(^ 1^1114算法通过1^ 18^]^61^1"检验进行分析。 使用的小鼠数和 p值 显示于该图中， 图中 T表示特拉唑嗪。
图 7 描述了败血病的大肠杆菌模型的存活率曲线。 大肠杆菌注射 1.5 小时之后 注入特拉唑嗪 (0.4 mg/kg)。用 [(^！^！^算法通过！^叩 ！！-！^^！^！^检验进行分析。使用的 小鼠数和 p值显示于该图中。
图 8 描述了特拉唑嗪对大肠杆菌生长的影响。 使用特拉唑嗪 24小时之后， 通过 抑菌囷比较它与氨苄青霉素对细菌生长的作用。 图中结果表明， 未加药物的对照 (control)和不同剂量的特拉唑嗪 (0.2 g T、 2 g T、 200μ8 Τ、 2mg Τ)均不能抑制大肠杆 菌生长， 而氨苄青霉素 (2mg Amp)显示抑制大肠杆菌生长。
图 9描述了特拉唑嗪对败血病的 CLP模型作用的存活率曲线。 在 CLP之后于 1.5和 24小时以 0.08 mg/kg通过皮下注射两次特拉唑嗪。 单独使用抗生素 Co-Am对 CLP模型 有增加死亡现象。而特拉唑嗪与抗生素 Co-Am的组合对败血病的 CLP模型有保护作用。 用 Log Rank算法通过 Kaplan-Merier检验进行分析。 使用的小鼠的数量和 p值显示于该 图中。 图中 T表示特拉唑嗪， Co-Am表示阿莫西林和克拉维酸钾的重量比为 4: 1的混 合物。
图 10描述了在 LPS和 IFNy处理的 Raw 264.7细胞中细胞凋亡蛋白酶 3的活性形式的 蛋白质印迹。 三个不同处理的组中的蛋白上样量相同。
图 11描绘了特拉唑嗪化学修饰及产生 Affi-gel连接的化学流程。
图 12中，图 a描绘了用特拉唑嗪琼脂糖小珠钓蛋白得到了一条明显只在实验组中出 现的蛋白条带 (在 43kd至 55kd之间用箭头标记的奈带）。 经过蛋白质谱鉴定为 Pgkl。 图 b 描绘了用体外表达的 Pgkl通过特拉唑嗪琼脂糖小珠蛋白柱， 仍然获得了 Pkgl奈带， 说 明特拉唑嗪与 Pgkl是可直接结合。
图 13描绘了不同浓度的特拉唑嗪对 Pgkl活性的影响。
图 14描绘了表达 Pgkl的 Raw 264.7细胞可拮抗细胞凋亡。 发生凋亡的细胞膜磷脂 酰丝氨酸由脂膜内側翻向外侧。 Annexin V与磷脂结合， 从而标记凋亡的细胞。
图 15描绘了小鼠感染 Pgkl慢病毒后， 在 CLP模型中表现出保护作用。 每只小鼠注 射 lxlO7个活性单位的病毒， 一周后进行实验。
图 16描绘了特拉唑嗪降低小鼠血糖的作用
图 17描绘了特拉峻嗪抗脑血栓的作用 具体实施方式
本发明第一方面涉及式 I化合物，
Figure imgf000005_0001
或其药学可接受的盐、 溶剂合物、 酯、 前药， 其中 .
Ria和 Rib 各自独立地选自 H、 NH2、 OH、 C1-6烷基-、 C1-6烷氧基 -Cl-6烷基 -、 C2-6烯基-、 C2_6炔基 -、 d-6烷氧基-、 C1-6烷基酰基-、 芳基酰基-、 C6— , ο芳基-、 C5-6 环烷基， 或者！^^和！^^与它们连接的氮原子一起形成 5-或 6-元环， 其中所述烷基任 选被 1-3个选自下列的取代基取代： 羟基、 卤素；
R2和 R3 各自独立地选自 H、 卤素、 C1-6烷基-、 卤代 C1-6烷基 -、 C2-6烯基 -、 C2-6炔基-、 CN、 N02、 NH2、 OH、 Ci-6烷氧基-、 Cj.6烷氧基 -C^炕氧基-、 C,-6 烷酰基氧基-、 Cl -6烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环 基、 C1-6烷基酰基-，或者 R2和 R3与它们连接的环原子一起形成 5-或 6-元碳环或杂环；
R4和 Rs 各自独立地选自 H、 卤素、 CN、 N02、 NH2、 OH、 C1-6烷基 -、 c1-6烷氧基 -c1 -6烷氧基-、 c1-6烷酰基氧基-、 卤代 c1-6烷基 -、 c2-6烯基 -、 c2-6炔基-、 烷氧基-、 烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环 基、 饱和或不饱和的 5-或 6-元碳环或杂环基氧基-、 烷基酰基。
根据本发明第一方面的化合物， 其中 Rla和 Rl b各自独立地选自 H、 NH2、 OH、
Cl_6 基 -、 Cl_4坑氧基 -C】_4坑基靈、 C2-4婦基-、 C2-4快基-、 C 4燒 基-、 Cl_4燒基醜 基-、 苯基酰基-、 苯基-、 Cs_6环烷基， 或者 Rla和 Rlb与它们连接的氮原子一起形成 5-或 6-元环， 其中所述垸基任选被 1-3个选自下列的取代基取代： 羟基、 卤素。 在一 个实施方案中， 所述 R 和 Rlb各自独立地选自 H、 - NH2、 -OH . CH3C(0)-、 -(CH2)2-0-(CH2)2-OH、 -CH2-CH=CH2、 -CH2-C≡CH、 -(CH2)SCH3. -(CH2)4-CF3、 环 己基、 -CH2-(CH2)3-CH2-、 -(CH2)20(CH2)2-OH、 -Ph. CH3、 -C(0)-CF3、 -C(0)-Ph。
根据本发明第一方面的化合物， 其中 和 各自独立地选自 H、 卤素、 C1-6烷 基-、 卤代 烷基-、 C1-6烷氧基-、 C1-6烷氧基 -C1-6烷氧基-、 C1-6烷酰基氧基-、 C1-6 烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环基， 或者 R2和 R3 与它们连接的环原子一起形成 5-或 6-元碳环或杂环。 在一个实施方案中， 所述 R2和 R3各自独立地选自 H、 CH30-、 -CH2-0-CH2-、 -0(CH2)2OC2Hs、 -OC(0)CH3、 -F、
-CF3、
Figure imgf000006_0001
、 并 1，2-吡啶环、 -NHCOCH3、 （CH2)2CH3、 -NHCOPh, 、· 、。
根据本发明第一方面的化合物， 其中 R4和 R5各自独立地选自 H、 鹵素、 C1-6 烷基-、 坑氧基 -C 6烷氧基-、 .6烷酰基氧基-、 卤代 烷基-、 烷氧基 -、 烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环基、 饱和或不 饱和的 5-或 6-元碳环或杂环基氧基。在一个实施方案中， 所述 R4和 Rs各自独立地选
自 H、 -0(CH2)2OC2Hi；、 -OC(0)CH3、 -OCH3、
Figure imgf000006_0002
-CF3 -F、 -NHCOCH;
、Q
(CH2)3CH3、 -NHCOPh, i。
才艮据本发明第一方面的化合物， 其中所述烷基、 烯基和炔基是直链或者支链的。 在一个实施方案中， 所述 c1-6 基选自 c,_5烷基、 c1-4烷基， 例如甲基、 乙基、 正丙 基、 异丙基、 正丁基、 仲丁基、 叔丁基。 在一个实施方案中， 所述 C2-6烯基选自 C2-s 烯基、 C2-4烯基， 例如乙烯基、 丙烯基、 烯丙基。 在一个实施方案中， 所述 C2-6炔基 选自 c2-5炔基、 c2-4炔基。
根据本发明第一方面的化合物， 其中所迷 c5-6环烷基选自环戊基和环己基。 根据本发明第一方面的化合物， 其中所述芳基选自苯基、 萘基， 优选苯基。 根据本发明第一方面的化合物， 其中所述 素选自氟、 氯、 溴和碘， 优选氟和 氯。
根据本发明第一方面的化合物， 其为选自下列编号为 Co.l至 Co.33的化合物 (它 们的结构如化合物制备例部分所述)：
Figure imgf000007_0002
Figure imgf000007_0001
Figure imgf000008_0001
注： 活性 *表示各化合物在 0.02 g/ml浓度下测试， 激活 Pgkl活性的倍数。 麥发明第二方面涉及式 I化合物例如特拉嗤嗪在制备作为细胞凋亡抑制剂的药物 中的用途。
本发明第二方面还涉及式 I化合物例如特拉唑嗪在制备作为 pgkl激活剂的药物 中的用途。
本发明第二方面还涉及式 I化合物例如特拉唑嚷在制备治疗和 /或预防败血病及 其并发症的药物中的用途。
本发明第二方面还涉及式 I化合物例如特拉唑嗪在制备治疗和 /或预防高血糖、脑 血栓及其并发症的药物中的用途。
根据本发明第二方面的用途， 其中所述细胞凋亡抑制剂用于临床疾病的治疗和 / 或预防以及实验室诊断和 /或检测。
根据本发明第二方面的用途， 其中所述 pgkl激活剂用于临床疾病的治疗和 /或预 防以及实验室诊断和 /或检测。
根据本发明第二方面的用途， 其中所述败血病是细菌和 /或其它微生物感染引起 的败血病。
根据本发明第二方面的用途， 其中所述败血病的并发症选自： 肾功能衰竭、 呼吸 衰竭、 凝血障碍、 器官损害（包括但不限于中毒性心肌病变、 脑病、 肝病及中毒性肠 麻痹等)、 化脓性脑膜炎、 肺炎、 肺脓肿、 蜂窝組织炎、 骨髓炎、 肾盂腎炎。
根据本发明第二方面的用途，其中所述式 I化合物例如特拉唑嗪用于人或动物 (例 如哺乳动物）的每日剂量是 0.001~5 mg/kg，优选 0.002~4 mg/kg,优选 0.003~3 mg/kg, 优选 0.005~2.5 mg/kg,优选 0.0075~2 mg/kg,优选 0.01~2 mg kg,优选 0.01~1 mg/kg。
根据本发明第二方面的用途， 其中所述式 I化合物是特拉唑嗪。
根据本发明第二方面的用途，其中特拉唑嗪是特拉唑嗪的药学可接受的盐或者其 溶剂合物。
根据本发明第二方面的用途， 其中所述特拉唑嗪是特拉唑嗪的盐酸盐。 根据本发明第二方面的用途， 其中所述特拉唑嗪是特拉唑嗪盐酸盐的水合物。 在 一个实施方案中， 特拉 嗪是特拉唑嗪盐酸盐的二水合物。
根据本发明第二方面的用途， 其中所述药物中还包括至少一种抗微生物药物。 根据本发明第二方面的用途， 其中所述药物中还包括至少一种抗微生物药物， 所 迷抗微生物药物选自： 青霉素类抗生素、 头孢菌素类抗生素、 P-内酰胺酶抑制剂、 氨 基糖苷类抗生素、 四环素类抗生素、 酰胺醇类类抗生素、 大环内酯类类抗生素、 磺胺 类、 甲氧苄啶类、 喹诺酮类。
根据本发明第二方面的用途， 其中所述药物中还包括至少一种抗微生物药物， 所 述抗微生物药物选自： 阿莫西林、 青霉素、 青霉素 V、 苯唑西林、 氯唑西林、 氟氯西 林、 氨苄西林、 哌拉西林、 阿洛西林、 克拉维酸钾、 舒巴坦、 舒他西林、 三唑巴坦、 氨曲南、 美罗培南。 在一个实施方案中， 所述药物中还包括至少一种抗微生物药物， 所述抗微生物药物选自： 阿莫西林、 克拉维酸钟。 在一个实施方案中， 所述药物中还 包括阿莫西林和克拉维酸钾。
本发明第三方面提供一种药物组合物，其中包含治疗和 /或预防有效量的式 I化合 物例如特拉唑嗪或其药学可接受的盐或溶剂合物 , 以及任选的药学可接受的载体。
根据本发明第三方面的药物组合物， 其中还包括至少一种抗微生物药物。
根据本发明第三方面的药物组合物， 其中还包括至少一种抗微生物药物， 所述抗 微生物药物选自： 青審素类抗生素、 头孢菌素类抗生素、 β-内酰胺酶抑制剂、 氣基糖 苷类抗生素、 四环素类抗生素、 酰胺醇类类抗生素、 大环内酯类类抗生素、 磺胺类、 甲氧苄啶类、 喹诺酮类。
根据本发明第三方面的药物组合物， 其中还包括至少一种抗微生物药物， 所述抗 微生物药物选自： 阿莫西林、 青霉素、 青霹素 V、 苯唑西林、 氯唑西林、 氟氯西林、 氨苄西林、 拉西林、 阿洛西林、 克拉维酸钾、 舒巴坦、 舒他西林、 三唑巴坦、 氨曲 南、美罗培南。在一个实施方案中，所述药物组合物中还包括至少一种抗微生物药物， 所述抗微生物药物选自： 阿莫西林、 克拉维酸钟。 在一个实施方案中， 所述药物组合 物中还包括阿莫西林和克拉维酸钟。
根据本发明第三方面的药物组合物， 其是用于细胞凋亡抑制剂。 在一个实施方案 中， 其中所述细胞凋亡抑制剂用于临床疾病的治疗和 /或预防以及实验室诊断和 /或检 测。
才艮据本发明第三方面的药物组合物，其是用作 pgkl激活剂。在一个实施方案中， 其中所述 pgkl激活剂用于临床疾病的治疗和 /或预防以及实验室诊断和 /或检测。
根据本发明第三方面的药物组合物， 其是用于治疗和 /或预防败血病及其并发症。 在一个实施方案中， 其中所述败血病是细菌和 /或其它微生物感染引起的败血病。 在 一个实施方案中，其中所述败血病的并发症选自： 肾功能衰竭、呼吸衰竭、凝血障碍、 器官损害（包括但不限于中毒性心肌病变、 脑病、 肝病及中毒性肠麻痹等)、 化脓性脑 膜炎、 肺炎、 肺脓肿、 蜂窝组织炎、 骨髓炎、 肾盂肾炎。 根据本发明第三方面的药物组合物， 其是用于治疗和 /或预防高血糖、 脑血栓及 其并发症。
根据本发明第三方面的药物组合物， 其中所述式 I化合物例如特拉唑嗪用于人或 动物（例: ^哺乳动物)的每日剂量是 0.001~5 mg/kg, 优选 0.002~4 mg/kg, 优选 0.003~3 mg/kg,优选 0.005~2.5 mg/kg,优选 0.0075~2 mg/kg,优选 0.01~2 mg/kg,优选 0.01~1 本发明第四方面提供一种药盒产品，其中包括治疗和 /或预防有效量的式 I化合物 例如特拉唑嗪或其药学可接受的盐或溶剂合物， 以及至少一种抗微生物药物。
根据本发明第四方面的药盒产品， 其中所迷式 I化合物例如特拉峻嗪或其药学可 接受的盐或溶剂合物和所述至少一种抗微生物药物是在同一组合物中或者在分离的 组合物中。
根据本发明第四方面的药盒产品， 其中所迷式 I化合物例如特拉峻嗪或其药学可 接受的盐或溶剂合物和所述至少一种抗微生物药物是在分离的组合物中。
根据本发明第四方面的药盒产品， 其中包括相互分离的第一组合物和第二组合 物，所述第一组合物包含治疗和 /或预防有效量的式 I化合物例如特拉唑嗪或其药学可 接受的盐或溶剂合物和任选的药学可接受的载体， 所述第二组合物包含治疗和 /或预 防有效量的抗微生物药物和任选的药学可接受的栽体。
根据本发明第四方面的药盒产品，其中所述抗微生物药物选自：青霉素类抗生素、 头孢菌素类抗生素、 |ί-内酰胺酶抑制剂、 氨基糖苷类抗生素、 四环素类抗生素、 酰胺 醇类类抗生素、 大环内酯类类抗生素、 磅胺类、 甲氧苄啶类、 喹诺酮类。
根据本发明第四方面的药盒产品， 其中所述抗微生物药物选自： 阿莫西林、 青審 素、 青霉素 V、 苯唑西林、 氯唑西林、 氟氯西林、 氨苄西林、 哝拉西林、 阿洛西林、 克拉维酸钾、舒巴坦、舒他西林、 三唑巴坦、氨曲南、 美罗培南。 在一个实施方案中， 所述抗微生物药物选自： 阿莫西林、 克拉维酸钾。 在一个实施方案中， 所述抗微生物 药物包括阿莫西林和克拉维酸钾。
根据本发明第四方面的药盒产品，其是用于细胞凋亡抑制剂。在一个实施方案中， 其中所述细胞凋亡抑制剂用于临床疾病的治疗和 /或预防以及实验室诊断和 /或检测。
根据本发明第四方面的药盒产品， 其是用作 pgkl激活剂。 在一个实施方案中， 其中所述 pgkl激活剂用于临床疾病的治疗和 /或预防以及实验室诊断和 /或检测。
根据本发明第四方面的药盒产品， 其是用于治疗和 /或预防败血病及其并发症。 在一个实施方案中， 其中所述败血病是细菌和 /或其它微生物感染引起的败血病。 在 一个实施方案中， 其中所迷败血病的并发症选自： 腎功能衰竭、呼吸衰竭、凝血障碍、 器官损害（包括但不限于中毒性心肌病变、 脑病、 肝病及中毒性肠麻痹等)、 化脓性脑 膜炎、 肺炎、 肺脓肿、 蜂窝组织炎、 骨髓炎、 肾盂肾炎。
根据本发明第四方面的药盒产品， 其是用于治疗和 /或预防高血糖、 '脑血栓及其 并发症。 根据本发明第四方面的药盒产品，其中所述式 I化合物例如特拉唑嗪用于人或动 物 (例如哺乳动物)的每日剂量是 0.001~5 mg/kg, 优选 0.002~4 mg/kg, 优选 0.003~3 mg/kg,优选 0.005 2.5 mg/kg,优选 0.0075~2 mg/kg,优选 0.01~2 mg/kg,优选 0.01~1 mg/kg。
本发明第五方面涉及在有需要的受试者或生物样品中抑制细胞凋亡的方法，该方 法包括给所述受试者或生物样品施用有效量的式 I化合物例如特拉唑嗪。
本发明第五方面还涉及在有需要的受试者或生物样品中激活 pgkl的方法， 该方 法包括给所述受试者或生物样品施用有效量的式 I化合物例如特拉唑嗪。
本发明第五方面还涉及在有需要的受试者中治疗和/或预防败血病及其并发症的 方法， 该方法包括给所述受试者施用有效量的式 I化合物例如特拉唑嗪.
本发明第五方面还涉及在有需要的受试者中治疗和 /或预防高血糖、 脑血栓及其 并发症症的方法， 该方法包括给所述受试者施用有效量的式 I化合物例如特拉唑嗪。
根据本发明第五方面的方法， 其中所述败血病是细菌和 /或其它微生物感染引起 的败血病。
根据本发明第五方面的方法， 其中所述败血病的并发症选自： 肾功能衰竭、 呼吸 衰竭、 凝血障碍、 器官损害（包括但不限于中毒性心肌病变、 脑病、 肝病及中毒性肠 麻痹等)、 化脓性脑膜炎、 肺炎、 肺脓肿、 蜂窝组织炎、 骨髓炎、 腎盂肾炎。
根据本发明第五方面的方法，其中所述式 I化合物例如特拉嗤嗪用于人或动物 (例 如哺乳动物）的每日剂量是 0.001~5 mg/kg,优选 0.002〜4 mg/kg,优选 0·003~3 mg/kg, 优选 0.005~2.5 mg/kg,优选 0.0075 2 mg/kg,优选 0.01~2 mg/kg,优选 0.01~1 mg/k „ 根据本发明第五方面的方法， 其中所述式 I化合物是特拉唑嗪。
根据本发明第五方面的方法，其中特拉唑嗪是特拉唑嗪的药学可接受的盐或者其 溶剂合物。
根据本发明第五方面的方法， 其中所述特拉唑嗪是特拉唑嗪的盐酸盐。
根据本发明第五方面的方法， 其中所述特拉唑嗪是特拉唑嗪盐酸盐的水合物。 在 一个实施方案中， 特掉唑嗪是特拉唑嗪盐酸盐的二水合物。
根据本发明第五方面的方法，其中还包括给所述受试者或生物样品施用有效量的 至少一种抗微生物药物。
根据本发明第五方面的方法，其中还包括给所述受试者或生物样品施用有效量的 至少一种抗微生物药物， 所述抗微生物药物选自： 青霉素类抗生素、 头孢菌素类抗生 素、 β-内酰胺酶抑制剂、 氨基糖苷类抗生素、 四环素类抗生素、 酰胺醇类类抗生素、 大环内酯类类抗生素、 磅胺类、 甲氧苄啶类、 喹诺酮类。
根据本发明第五方面的方法，其中还包括给所述受试者或生物样品施用有效量的 至少一种抗微生物药物， 所迷抗微生物药物选自： 阿莫西林、 青霉素、 青霉素 V、 苯 唑西林、 氯唑西林、 氟氯西林、 氨苄西林、 哝拉西林、 阿洛西林、 克拉维酸钾、 舒巴 坦、 舒他西林、 三唑巴坦、 氨曲南、 美罗培南。 在一个实施方案中， 所述抗微生物药 物选自： 阿莫西林、 克拉维酸钾。 在一个实施方案中， 所述抗微生物是阿莫西林和克 拉维酸钾。
本发明第六方面涉及用作细胞凋亡抑制剂的式 I化合物例如特拉唑嗪。
本发明第六方面还涉及用作 pgkl激活剂的式 I化合物例如特拉唑嗪。
本发明第六方面还涉及用作治疗和 /或预防败血病及其并发症的式 I化合物例如 特拉唑。秦。
本发明第六方面还涉及用作治疗和 /或预防高血糖、脑血栓及其并发症的式 I化合 物例如特拉唑 。
·据本发明第六方面的式 I化合物， 其中所述细胞凋亡抑制剂用于临床疾病的治 疗和 /或预防以及实验室诊断和 /或检测。
根据本发明第六方面的式 I化合物， 其中所述 pgkl激活剂用于临床疾病的治疗 和 /或预防以及实验室诊断和 /或检测。
根据本发明第六方面的式 I化合物，其中所述败血病是细菌和 /或其它微生物感染 引起的败血病。
根据本发明第六方面的式 I化合物，其中所述败血病的并发症选自：肾功能衰竭、 呼吸衰竭、 凝血障碍、 器官损害（包括但不限于中毒性心肌病变、 脑病、 肝病及中毒 性肠麻痹等)、 化脓性脑膜炎、 肺炎、 肺脓肿、 蜂窝组织炎、 骨髓炎、 肾盂腎炎。
根据本发明第六方面的式 I化合物 ,其中所述式 I化合物用于人或动物 (例如哺乳 动物）的每日剂量是 0.001~5 mg/kg, 优选 0·002~4 mg/kg, 优选 0.003~3 mg/kg, 优选 0.005-2.5 mg/kg, 优选 0.0075~2 mg/kg, 优选 0.01~2 mg/kg， 优选 0.01~1 mg/kg。
根据本发明第六方面的式 I化合物， 其是特拉唑嗪。
根据本发明第六方面的式 I化合物， 其是特拉峻嗪的药学可接受的盐或者其溶剂 合物。
根据本发明第六方面的式 I化合物， 其是特拉唑嗪的盐酸盐。
根据本发明第六方面的式 I化合物， 其是特拉唑嗪盐酸盐的水合物， 例如特拉唑 嗪盐酸盐的二水合物。
根据本发明第六方面的式 I化合物， 其还与至少一种抗微生物药物組合。
根据本发明第六方面的式 I化合物， 其还与至少一种抗微生物药物組合， 所述抗 微生物药物选自： 青霉素类抗生素 头孢菌素类抗生素、 P-内酰胺酶抑制剂、 氨基糖 苷类抗生素、 四环素类抗生素、 酰胺醇类类抗生素、 大环内酯类类抗生素、 磧胺类、 甲氧苄啶类、 喹诺酮类。
根据本发明第六方面的式 I化合物， 其还与至少一种抗微生物药物组合， 所述抗 微生物药物选自： 阿莫西林、 青霉素、 青霉素 V、 苯唑西林、 氯唑西林、 氟氯西林、 氨苄西林、 哌拉西林、 阿洛西林、 克拉维酸卸、 舒巴坦、 舒他西林、 三唑巴坦、 氨曲 南、 美罗培南。 在一个实施方案中， 所述抗微生物药物选自： 阿莫西林、克拉维酸钾。 在一个实施方案中， 所述抗微生物药物是阿莫西林和克拉维酸钾。 根据本发明任一方面， 其中所述的式 I化合物例如特拉唑嗪的使用量可以参考现 有临床用药剂量。 例如在用于人治疗和 /或预防敗血病及其并发症时， 特拉唑嗪的使 用剂量可以是目前临床上该药物用于其它疾病 (例如高血压）的剂量的 0.01~100倍，优 选 0.02~80倍， 优选 0.05~20倍， 优选 0.1~10倍， 优选 0.1~5倍， 优选 0.2~5倍， 优 选 0.2~2倍。
根据本发明任一方面，其中所述的抗微生物药物的使用量可以参考现有临床用药 剂量。 例如在用于人治疗和 /或预防败血病及其并发症时， 其使用剂量可以是目前临 床上该抗微生物药物用于其它疾病 (例如抗感染)的剂量的 0.01~100倍， 优选 0.02~80 倍， 优选 0.05~20倍， 优选 0.1 10倍， 优选 0.2~5倍。
根据本发明任一方面， 其中所述的阿莫西林和 /或克拉维酸钾的使用量可以参考 现有临床用药剂量。 例如在用于人治疗和 /或预防败血病及其并发症时， 其使用剂量 可以是目前临床上该阿莫西林和 /或克拉维酸钾用于其它疾病 (例如抗感染)的剂量的 0.01~100倍， 优选 0.02 80倍， 优选 0.05~20倍， 优选 0.1~10倍， 优选 0.2~5倍。
在本发明任一方面的一些实施方案中， 所述式 I化合物不包括编号 Co.33的化合 物- 本发明任一方面或该任一方面的任一实施方案所具有的特征同样适用于其它任 一方面或该其它任一方面的任一实施方案， 只要它们不会相互矛盾， 当然在相互之间 适用时， 必要的话可对相应特征作适当修饰。
下面对本发明的各个方面和特点作进一步的描述。
本发明所引述的所有文献， 它们的全部内容通过引用并入本文， 并且如果这些文 献所表达的含义与本发明不一致时， 以本发明的表述为准。 此外, 本发明使用的各种 术语和短语具有本领域技术人员公知的一般含义， 即便如此， 本发明仍然希望在此对 这些术语和短语作更详尽的说明和解释， 提及的术语和短语如有与公知含义不一致 的， 以本发明所表述的含义为准。
如本文所述的， 术语"败血病，，亦称为"败血症，，， 其具有本领域技术人员通常已知 的含义， 并且通常是指一种由于细菌和 /或其它微生物进入血循环， 并在其中生长繁 殖、产生毒素而引起的全身性严重感染。 临床表现为发热、严重毒血症状、皮疹瘀点、 肝脾肿大和白细胞数增高等。 革兰阳性球菌败血症易发生迁徙病灶； 革兰阴性杆菌败 血症易合并感染性休克。 当败血症伴有多发性脓肿时称为脓毒败血症或称为脓毒症。
如本文所述的， 术语" pgkl激活剂 "是指鱗酸甘油酸酯激酶 1 (phosphoglycerate kinase 1， 简称为 pgkl)的激活剂 , 其可激活 pgkl并且因此本领域技术人员理解其可 用于相关的疾病或病症的治疗、 预防、 减轻和 /或緩解。
本文中使用的术语"约"， 其通常是指包括本领域允许的误差范围， 例如 ±10% , 例如 ±5% , 例如 ±2%„
如本文所述的， 术语"有效量，，是指可在受试者中实现治疗、 预防、 减轻和 /或緩 解本发明所述疾病或病症的剂量。 如本文所述的， 术语"药物组合物"， 其可与 "组合物"互换使用， 是指可用于在受 试者中实现治疗、 预防、 减轻和 /或緩解本发明所述疾病、 病症、 症状的物质。
如本文所述的， 术语"受试者 "或"患者"，可以指接受本发明组合物和提取物以治 疗、 预防、 减轻和 /或緩解本发明所述疾病、 病症、 症状的动物， 特别是哺乳动物， 例如人、 狗、 猴、 牛、 马等。
如本文所述的， 术语"疾病或症状"是指所述受试者的一种身体状态， 该身体状态 与本发明所述疾病或症状有关。
如本文所述的， "％", 如未特别指明， 对于总物料是固体时一般是指重量 /重量 的百分比， 对于总物料是液体时一般是指重量 /体积的百分比。 当然， 对于总物料是 液体并且溶质是液体时， 表征该液态溶盾的百分比一般是指体积 /体积的百分比。
特拉唑嗪 (Terazosin，（4-(4-氨基 -6,7-二甲氧基喹唑啉 -2-基)旅 #-1-基) (四氢呋喃 -2- 基)甲酮， C19H2SN504) , 其具有以下化学结构式：
Figure imgf000014_0001
在本发明中， 当提及特拉唑嗪时， 其不但包括以上结构所示的化合物， 还包括上 述结构化合物的药学可接受的盐 (例如盐酸盐）， 以及上述结构化合物及其盐的溶剂合 物例如水合物。在本发明的一个优选实施方案中， 所述特拉唑嗪是指盐酸特拉唑嗪二 水合物。 本发明下文使用作为式 I化合物典型示例的特拉峻嗪进行了大量研究以表明 本发明出人意料的效果； 在下文试验特别是生物学试验中， 如未另外指明， 所用的试 药特拉唑嗪是指盐酸特拉唑嗪二水合物。
本发明尝试从临床药物中筛选细胞凋亡抑制剂。 首先， 本发明使用果蝇的细胞凋 亡模型通过微阵列芯片技术分析基因表达。 然后， 本发明使用联系图（connectivity map, Cmap)通过生物信息学分析法断定候选的细胞凋亡阻断剂。 接着， 本发明筛选 了抑制双翅目昆虫果蝇 (Z)mw)/^//fl)细胞凋亡的候选药物。 结果， 本发明确定了特拉 唑嗪， 其为缓解高血压的临床用药。 此外， 本发明发现特拉唑嗪可抑制巨噬细胞中由 细菌内毒素 (脂多糖， LPS , 2 g/ml)和干扰素 Y (IFN Y， 50 U/ml)介导的细胞凋亡。 此 外， 本发明发现， 在败血病的三个试验模型中， 特拉唑嗪可大大减小小鼠的死亡率。 有趣的是，与单独使用抗生素相比，使特拉峻嗪与抗生素组合会产生更好的保护作用。 本发明的结果表明， 特拉唑嗪是一种新的细胞凋亡抑制剂。 其可用于与抗生素治疗组 合。
果蝇是筛选药物的重要的动物模型系统。 此外， 细胞凋亡蛋白酶 (caspase)-介导的 凋亡途径在双翅目昆虫与人之间是充分保守的。 例如^op^ yr)在^ ϋ的细胞凋亡中 发挥主要作用（White et al.， 1994)。 虽然， 来自热诱导启动子的 φ/·表达会引起广泛分布 的异常细胞凋亡和器官死亡 (White et al.， 1996). 本发明研究了本发明方案是否能够使 用果蝇凋亡模型来筛选细胞凋亡阻断剂。 HS-GaW (—种全身表达并且热激活的启动 子）， 可以促使含有 UAS (上游激活序列）的 5，重复的任何转基因的表达。 当在 18°C下使 该 IS-Gu"杲蝇与 45-Refl/;er果蝇杂交 (简称为 HS>rpr)时，雌性后代自始至终是正常发 育。然而，在 37°C下给予热休克 2-3 小时，大约 50-70%的后代在 14-24 小时之后死亡（图 1)。 本发明选择了 25种排名靠前的化合物 (图 2), 并进一步测定它们对 HS> ^的致死率 的影响。 本发明发现仅有特拉唑嗪可以显著地提高 HS> y;r果蝇的存活（图 3)。
为了测试特拉唑嗪是否亦在培养的哺乳动物细胞中抑制细胞凋亡， 本发明在巨噬 细胞 RAW264.7细胞中通过 LPS和 IFNy诱导细胞凋亡，该 LPS和 IFNy是在培养细胞中诱 发细胞凋亡的公知药剂。 本发明通过用 Annexin V 染色来观察细胞^亡 (图 4)。 本发明 人发现， LPS (2 pg/ml)和 IFNy(50 U/ml) 处理的细胞导致细胞膜损伤、外翻， 从而被 Annexin V染色。 这是一种经典的细胞凋亡模式。 而应用特拉唑嗪 (4 pg/ml)可降低 50% 的细胞凋亡（图 4)。 重要的是， 本发明令人意外地发现特拉唑嚎可以有效抑制细胞凋亡 并且可以进一步用于治疗和 /或预防败血病及其并发症。
为了进一步在败血病的哺乳动物模型中研究特拉唑嗪的效果， 本发明研究了接受 LPS治疗的小鼠。本发明发现，在注射 LPS(13.5 mg/kg，i.p.)之后的 1.5 小时注射特拉唑 嗪 (0.4 mg/kg i.p)会显著增加小鼠存活（图 5a)。 为了确认特拉唑嗪对细胞凋亡的效果， 本发明检测了细胞凋亡的标记物 DNA断裂。 作为产生免疫细胞的器官， 败血病期间胸 腺对细胞凋亡是敏感的（Ayala et al.， 1998)。 因此， 本发明比较了来自用 LPS治疗的小 鼠胸腺的基因组 DNA与用 LPS加特拉唑嗪治疗的小鼠胸腺的基因组 DNA。 结果显示， 在 LPS处理的小鼠中发生了明显 DNA梯型， 但在用特拉唑嗪治疗的小鼠中大大减小（图 5b)„ 为了测试特拉唑嗪是否可以在败血病的后期发挥作用， 本发明在使用 LPS之后 12 小时注射特拉唑嗪。 结果显示， 特拉唑嗪仍具有满意的效果 (图 6)。
接着， 在注射大肠杆菌的败血病模型中测试了特拉峻嗪。 结果显示， 特拉嗤嗪可 保护小鼠由大肠杆菌诱导的死亡 (图 7)。 为了测定潜在的抗菌效果， 本发明人检测了在 特拉唑嗪存在下的大肠杆菌生长情况。 结果显示， 相对于氨苄青霉素， 特拉唑嗪不能 抑制大肠杆菌生长 (图 8)。
由于 LPS和大肠杆菌模型仅能模拟革兰阴性菌感染， 本发明进一步在盲肠结扎和 穿刺 (CLP)模型中测试了特拉唑嗪， 该模型据认为是试验性敗血病的金标准模型 (Parker and Watkins, 2001; Wichterman et al., 1980)。首先，本发明检测 0.4 mg/kg的特 拉唑嗪 (用于 LPS模型相同的浓度)。 然而， 在最初 5天， 注射特拉唑嗪的小鼠比对照组 死的更快 (数据未显示)。本发明推断这可能是由于由 CLP引起的严重的心脏功能障碍和 低血压导致的。 因此， 药物降低血压的功能可能掩盖了其抗凋亡的作用。 为了解决此 问题，本发明将特拉唑嗪浓度降低到 0.08 mg/kg,本发明人测量了小鼠注射此浓度对血 压的影响，发现小鼠血压完全正常。这一结果与其用在大鼠中不会降低血压一致 (Kyncl et al., 1985) 有趣的是， 当在手术之后 1.5 小时和 24 小时注射 2次时， 特拉唑嗪显著促 进了 CLP模型中小鼠的存活 (图 9)。 其次， 本发明测试了特拉唑嗪与抗生素组合， 在本 发明中， 选择了抗生素阿莫西林和克拉维酸钾 (Co-Am, 阿莫西林和克拉维酸钾的重量 比为 4:1， 在本发明其它地方以具体示例或实例的方式提及时， 亦是指这种 4:1的配比。 本领域技术人员理解， 当泛指阿莫西林克拉维酸钾的组合时， 阿莫西林和克拉维酸钟 的重量比可以为 1:1至 10:1 , 特别是 1:1至 7:1， 例如约 1:1、 约 4:1、 约 7:1)与特拉唑嗪组 合。 本发明发现， 特拉唑嗪与 Co-Am组合比之于单独的 Co-Am显示出更强的保护作用 (图 9)。 此外， 该组合治疗方法仍然证明了 CLP之后 6小时给药的治疗效果。
为了测试特拉唑嗪的保护效果是否与其作为 αΐ-肾上腺素受体抑制剂的功能有关， 本发明检查了布拉唑嗪 (0.4 mg/kg, i.p.),其为另一种 αΐ-贤上腺素受体阻滞剂 (Cavero et al.，1977)。 本发明发现布拉唑嗪对 LPS导致的败血症无任何疗效。这些结果表明， 特拉 唑嗪可显著增加抑制 LPS-介导的败血病的存活率， 并且其功能是有可能通过细胞凋亡 的抑制作用而不是靶向 αΐ-肾上腺素受体。
为了研究特拉唑嗪阻断细胞凋亡的生物学机理， 本发明探寻了其对抑制细胞凋亡 蛋白酶的潜在作用， 由于果蝇中的细胞凋亡实现主要是通过内在的细胞凋亡蛋白酶活 化 (McCall and Steller, 1997)。 然而， 本发明的蛋白印迹分析显示， 执行子细胞凋亡蛋 白酶 3的活性形式 (果蝇中 Dpc-1和 drICE的同源基因）因特拉唑嗪而减少（图 10)。本发明 推断， 特拉唑嗪可调节细胞凋亡蛋白酶 3的上游靶标。 为了鉴别基因， 本发明将特拉唑 嗪连到琼脂糖小珠上， 图 11显示了其化学合成途径。 然后在 Raw 264.7 细胞提取液中 钓蛋白， 结果发现相对于空白对照和药物竟争对照組， 多出一条带（图 12a)。 经质语鉴 定， 这条带为 Pgkl。 为了确定特拉 嗪与 Pgkl的结合是否为直接关系， 本发明人在细 菌中表达并纯化了小鼠的 Pgkl蛋白， 结果发现特拉唑嗪可体外直接结合 Pgkl蛋白（图 12b)。 因为在酵母中过表达 Pgkl可抑制细胞凋亡 (Mazzoni et al., 2009), 本发明人猜测 特拉唑嗪可能激活了 Pgkl的酶活性。 为了证明这个猜测， 本发明人检测了 Pgkl体外酶 活性。 结果发现， 0.05μΜ的特拉唑嗪可激活 Pgkl的酶活性近 3倍， 而增加特拉唑嗪浓 度到 0.4μΜ会降低对酶活性的激活（图 13)。如果 Pgkl酶活性是抑制细胞凋亡的原因， 过 表达 Pkgl就应该抑制细胞凋亡。 本发明人的实验结果表明， 用表达 Pgkl慢病毒建立的 RAW264.7细胞系，显著降低了 LPS和 IFNy处理后 ^细胞 ¾亡； 而过表达 EGFP的细胞 凋亡发生没有改变 (图 14)。更进一步，本发明人用皮下注射慢病毒在小鼠中表达了 Pgkl 或 EGFP做对照。一周后做 CLP手术，结果发现，相对于感染绿色荧光蛋白的一组小鼠， 注射 Pgkl病毒的小鼠明显的提高了存活率（图 15)。 本发明不受任何特定理论的束縛, 本发明人认为 Pgkl是特拉唑嗪新的靶点。
本发明的研究表明， 特拉唑嗪可以容易地转变到临床应用中， 而无需修改现有的 抗生素治疗程序。 下面对本发明的生物学试验进行详细说明。 实施例
A、 化合物制备例部分 以下制备例示例性地制备了本发明的部分式 I化合物， 各化合物分别以 Co.l至 Co.32表示， 而 Co.33表示特拉唑嗪。 以下制备例的反应流程中， "reflux"表示回流， "l-pentanol"表示 1-戊醇。
制备例 1: Co.l的制备
Figure imgf000017_0001
步骤 1: 向溶有化合物 la(50 mmol)的 200 mL四氢呋喃溶液中通入氨气， 反应在 25°C下进行 36小时。 体系中有大量白色固体析出， 过滤所得白色固体用四氢呋喃洗 涤后得最终产品 lf。 产率为： 63%。
Figure imgf000017_0002
步骤 2: 向化合物 lf(10 mmol)加入 15 mL醋酸酐， 反应回流 2小时。 冷却到室 体系中有大量白色固体析出， 过滤所得白色固体用四氢呋喃洗涤后得最终产品 产率为： 63%。
Figure imgf000017_0003
步骤.3:在氩气氛的情况下，向溶有 lg(2mmol)的 1-戊醇的溶液中加入 lh(2mmol)。 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙酮洗涤所得 白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 li即为化合物 Co.l。产率为：60%。
1H NMR (300 MHz, CDCI3): 1.84 (m, 2H， thf-H), 2.03 (m, 2H, thf-H), d 2.61 (s, 3H, CH3), 3.47-4.07 (m， 11H, thf-H， pip-H和 CH2CH20), 3.93 (s, 3H, OCH3), 3.97 (s， 3H, OCH3), 4.82 (m, 1H， thf-2H), 7.16 (s， 1H， Ar-5H), 7.72 (s, 1H, Ar-8H); HR-MS (ESI-正性)： 430.20928 (M+H) (计算值： 430.20904); 元素分析: (C, 58.74; H, 6.34; , 16.30; 0, 18.63); 计算值： (C, 58.73; H, 6.34; N, 16.31; 0, 18.63). 制备例
Figure imgf000017_0004
步驟 1: 向溶有化合物 la(20 mmol)的 100 mL 曱醇溶液中加入化合物 lb(20 mmol),反应在 25°C下进行 4小时。薄板层析指示 la转化完全后，向体系中加入 100 mL 乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油酸 /乙酸乙酯重结 晶后得最终产品 lc。 产率为： 41%。
Figure imgf000018_0001
步骤 2: 在氩气氛的情况下， 向溶有 lc(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol). 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙酮 洗涤所得白色晶体两次， 再用乙髄 /甲醇重结晶后得最终产品 le即为化合物 Co.2。 产 率为： 62%。
1H NMR (300 MHz, DMSO-d6): 1.84 (m, 2H， thf-H), 2.03 (m, 2H, thf-H), 3.47-4.07 (m, 14H, thf-H， pip-H和 CH2CH20>， 3.84 (s， 3H, OCH3)， 3.87 (s, 3H, OCH3) 4.73 (m, 1H， thf-2H), 7.16 (s, 1H， Ar-5H), 7.72 (s， 1H， Ar-8H); 13 C NMR (DMSO-d6): d 25.2, 27.9, 40.9, 44.1, 44.6, 55.8， 56.2, 60.1， 68.1, 68.2, 72.1, 74.9， 102.4, 104.4, 106.9, 146.2, 154.4, 154.2, 158.6， 169.6; HR-MS (ESI-正性)： 476.25120 (M+H) (计算值: 476.25091); 元素分析： C, 58.08; H, 7.00; N, 14.73; 0， 20.19; 计算值： （C， 58.09; H， 6.99; N， 14.73; 0， 20.19)。 制备例 3: Co.3的制备
Figure imgf000018_0002
步骤 1: 向溶有化合物 la(20 mmol)的 100 mL甲醇溶液中加入水合肼 (20 mmol), 反应在 25°C下进行 4小时。 薄板层析指示 la转化完全后， 向体系中加入 100 mL乙 醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油醚 /乙酸乙酯重结晶后得 终产品 3a。 产率为： 61%。
Figure imgf000018_0003
步骤 2: 在氫气氛的情况下， 向溶有 3a(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol)a 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 lOmL丙酮 洗涤所得白色晶体两次， 再用乙醚 /甲醇重结晶后得最终产品 3b即为化合物 Co.3。产 率为： 31%。 IH NMR (300 MHz, DMSO-d6): d 1.87 (m, 2H， thf-H), 2.03 (m, 2H, thf-H), 3.43-4.07 (m, 6H, thf-H， pip-H), 3.86 (s， 3H, OCH3), 3.88 (s, 3H, OCH3)， 4.63 (m， 1H， thf-2H), 7.16 (s, IH, Ar-5H)， 7.82 (s, 1H， Ar-8H); HR-MS (ESI-正性)： 03.20938 (M+H) (计算值： 403.20946)。 制备例 4: Co.4的制备
Figure imgf000019_0001
步骤 1: 向溶有化合物 la(20 mmol)的 100 mL 甲醇溶液中加入水合羟氨 (20 mmol),反应在 25°C下进行 3小时。薄板层析指示 la转化完全后，向体系中加入 100 mL乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油酸 /乙酸乙酯重结 晶后得最终产品 4a。 产率为： 88%。
丫
Τ 丫 HN r、
ΗΝ、 、OH 1-pentanol, reflux
Figure imgf000019_0002
步骤 2: 在氩气氛的情况下， 向溶有 4a(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol). 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙酮 洗涤所得白色晶体两次，再用乙醚 /曱醇重结晶后得最终产品 4b即为化合物 Co.4。产 率为： 75%。
IH NMR (300 MHz, DMSO-d6): 1.81 (m, 2H, thf-H), 2.03 (m, 2H， thf-H), 3.47-4.07 (m, 6H, thf-H， pip-H), 3.87 (s, 3H, OCH3)， 3.94 (s, 3H, OCH3)， 4.43 (m, IH, thf-2H), 7.26 (s, 1H， Ar-5H), 7.74 (s， 1H， Ar-8H); HR-MS (ESI-正性)： 404.19339 (M+H) (计算值： 404.19341)。 制备例 5: Co.5的制备
Figure imgf000019_0003
步骤 1: 向溶有化合物 la(20 mmol)的 100 mL 甲醇溶液中加入烯丙基胺 (22 mmol),反应在 25°C下进行 8小时。薄板层析指示 la转化完全后，向体系中加入 100 mL乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油酸 /乙酸乙酯重结 晶后得最终产品 5a。 产率为： 31%。
Figure imgf000020_0001
步骤 2: 在氩气氛的情况下， 向溶有 4a(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol). 反应体系回流 4.5小时后， 将其置于 0-5。C环境中静置结晶。 用 10mL丙酮 洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 5b即为化合物 Co.5。产 率为： 75%。
IH NMR (300 MHz, DMSO-d6): 1.82 (m, 2H, thf-H), 2.03 (m, 2H, thf-H), 3.47-4.07 (m, 6H, thf-H， pip-H), 3.87 (s， 3H, OCH3)， 3.94 (s, 3H, OCH3)， 4.04 (d， 2H)， 4.43 (m, 1H， thf-2H), 5.19-5.23 (m, 2H)， 5.82-5.88 (m, 1H)， 7.26 (s， IH, Ar-5H), 7.74 (s, IH, Ar-8H); HR-MS (ESI-正性)： 428.22978 (M+H) (计算值： 428.22986)。 制备例 6: Co.6的制备
Figure imgf000020_0002
步驟 1: 向溶有化合物 la(20 mmol)的 100 mL 甲醇溶液中加入烯丙基胺 (28 mmol), 反应在 25°C下进行 10小时。 薄板层析指示 la转化完全后， 向体系中加入 100 mL乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油醚 /乙酸乙酯 重结晶后得最终 6a。 产率为： 44%
Figure imgf000020_0003
步骤 2: 在氩气氛的情况下， 向溶有 4a(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol)。 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙酮 洗涤所得白色晶体两次， 再用乙醚 /曱醇重结晶后得最终产品 6b即为化合物 Co.6。产 率为： 75%。
IH NMR (300 MHz, DMSO-d6): 1.82 (m, 2H， thf-H), 2.03 (m, 2H， thf-H), 3.47-4.07 (m, 6H, thf-H， pip-H), 3.80 (s, 2H), 3.87 (s， 3H， OCH3)， 3.94 (s, 3H, OCH3), 4.04 (d， 2H), 4.43 (m, 1H， thf-2H)， 7.26 (s, 1H， Ar-5H), 7.74 (s， 1H， Ar-8H); HR-MS (ESI-正性)： 426.21413 (M+H) (计算值： 426.21408)。 制备例 7: Co.7的制备
Figure imgf000021_0001
步骤 1: 向溶有化合物 la(20 mmol)的 100 mL 曱醇溶液中加入化合物 7a(20 mmol) ,反应在 25°C下进行 3小时。薄板层析指示 la转化完全后，向体系中加入 100 mL乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油 乙酸乙酯重结
Figure imgf000021_0002
步骤 2: 在氩气氛的情况下， 向溶有 7b(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol)。 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 lOinL丙酮 洗涤所得白色晶体两次， 再用乙醚 /曱醇重结晶后得最终产品 7c即为化合物 Co.7。 产 率为： 62%。
1H NMR (300 MHz, DMSO-d6): 0.86-1.62 (m, ΙΙΗ,) , 1.84 (m, 2Η, thf-H), 2.03 (m， 2H, thf-H), 3.46-4.07 (m, 8H， thf-H， pip-H), 3.86 (s, 3H， OCH3)， 3.83(s, 3H, OCH3)， 4.75 (m, 1H， thf-2H), 7.16 (s, 1H， Ar-5H)， 7.72 (s, 1H， Ar-8H); HR-MS (ESI-正性): 472.29238 (M+H) (计算值： 472.29229)。 制备例 8: Co.8的制备
Figure imgf000021_0003
步骤 1 : 向溶有化合物 la(20 mmol)的 100 mL 甲醇溶液中加入化合物 8a(20 mmol), 反应在 25°C下进行 1.5小时。 薄板层析指示 la转化完全后， 向体系中加入 100 mL乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油酸 /乙酸乙酯 重结
Figure imgf000021_0004
步骤 2: 在氩气氛的情况下， 向溶有 8b(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol)。 反应体系回流 4.5小时后， 将其置于 0-5。C环境中静置结晶。 用 lOmL丙酮 洗涤所得白色晶体两次， 再用乙醚 /曱醇重结晶后得最终产品 8c即为化合物 Co.8。 产 率为： 42%。
1H NMR (300 MHz, DMSO-d6): 1.22-1.72 (m, 11H，）， 1.84 (m, 2H， thf-H), 2.03 (m, 2H, thf-H), 3.47-4.05 (m, 8H， thf-H , pip-H), 3.82 (s， 3H， OCH3), 3.73 (s, 3H, OCHj), 4.43 (m, 1H， thf-2H), 7.54 (s, 1H， Ar-5H), 7.86 (s, 1H， Ar-8H); HR-MS (ESI-正性)： 512.24846 (M+H) (计算值： 512.24859)。 制备例 9: Co.9的制备
Figure imgf000022_0001
步骤 1: 向溶有化合物 la(20 mmol)的 100 mL 曱醇溶液中加入化合物 9a(20 mmol), 反应在 25。C下进行 2.5小时。 薄板层析指示 la转化完全后， 向体系中加入 100 mL乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油酸 /乙酸乙酯 重结晶后得最终产品 9b。 产率为： 21%。
Figure imgf000022_0002
步骤 2: 在氩气氛的情况下， 向溶有 9b(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol)。 反 体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙酮 洗涤所得白色晶体两次， 再用乙醚 /甲醇重结晶后得最终产品 9c即为化合物 Co.9。 产 率为： 43%.
1H NMR (300 MHz, DMSO-d6): 1.32-1.52 (m, 10H，)， 1.86 (m， 2H， thf-H), 2.03 (m 2H， thf-H), 3.47-4.07 (m, 9H, thf-H， pip-H), 3.82 (s， 3H, OCH3), 3.79 (s， 3H, OCH3), 4.46 (m, 1H， thf-2H), 7.33 (s, 1H， Ar-5H)， 7.78(s, 1H， Ar-8H); HR-MS (ESI-正性): 470.27673 (M+H) (计算值： 470.27658)。
Figure imgf000022_0003
步骤 1: 向溶有化合物 la(20 mmol)的 100 mL 甲醇溶液中加入化合物 10a(20 mmol),反应在 25°C下进行 5小时。薄板层析指示 la转化完全后，向体系中加入 100 mL乙醜、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油 it/乙酸乙酯重结 晶后得 终产品 9b。 产率为： 36%。
Figure imgf000023_0001
步骤 2: 在氩气氛的情况下， 向溶有 9b(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol). 反应体系回流 4.5小时后， 将其置于 0-5。C环境中静置结晶。 用 10mL丙酮 洗涤所得白色晶体两次， 再用乙酸 /甲醇重结晶后得最终产品 10c即为化合物 Co.lO。 产率为： 43%。
1H NMR (300 MHz, DMSO-d6): 1.33-1.47 (m， 6H，）， 1.86 (m, 2H， thf-H), 2.04 (m, 2H, thf-H), 3.57^1.09 (m, 12H, thf-H， pip-H), 3.82 (s, 3H， OCH3)， 3.77 (s, 3H, OCHj), 4.48 (m， 1H, thf-2H)， 7.37 (s， 1H， Ar-5H), 7.79(s, 1H， Ar-8H); HR-MS (ESI-正性): 456.26108 (M+H) (计算值： 456.26119)。 制备例 11: Co.ll的制备
Figure imgf000023_0002
步骤 1: 向溶有化合物 la(20 mmol)的 100 mL 甲醇溶液中加入化合物 llb(20 mmol),反应在 25°C下进行 5小时。薄板层析指示 la转化完全后，向体系中加入 100 mL乙醚、 混匀后放入 -20°C环境中静置结晶。 所得白色固体用石油酸 /乙酸乙酯重结 晶后得最终产品 llc„ 产率为： 41%。
Figure imgf000023_0003
步骤 2: 在氩气氛的情况下， 向溶有 llb(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol)。 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶， 用 lOmL丙酮 洗涤所得白色晶体两次， 再用乙 /曱醇重结晶后得最终产品 11c即为化合物 Co.ll。 产率为： 62%。
1H NMR (300 MHz, DMSO-d6): 1.79 (m， 2H, thf-H), 2.06 (m, 2H, thf-H), 3.27-4.08 (m, 17H, thf-H， pip-H和 CH2CH20)， 3.85 (s， 3H, OCH3)， 3.88 (s, 3H, OCH3) 4.73 (m, 1H， thf-2H)， 7.26 (s， 1H， Ar-5H), 7.72 (s， 1H, Ar-8H); HR-MS (ESI-正性): 490.26656 (M+H) (计算值： 490.26658)。 制备例 12: Co.12的制备
Figure imgf000024_0001
1-pentanol, reflux
步骤 1: 在氩气氛的情况下， 向溶有 12a(2 mmol)的 1-戊醇的溶液中加入 ld(2 mmol)。 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙酮 洗涤所得白色晶体两次， 再用乙酸 /甲醇重结晶后得最终产品 12b即为化合物 Co.l2。 产率为： 62%。
1H 画 R (300 MHz, D SO-d6): 1.79 (m, 2H, thf-H), 2.06 (m, 2H， thf-H), 3.47-4.08 (m, 11H, thf-H， pip-H), 3.85 (s， 3H, OCH3), 3.88 (s, 3H, OCH3), 4.73 (m, 1H， thf-2H), 6.86 -7.62 (m, 7H， Ar-H); HR-MS (ESI-正性)： 464.22978 (M+H) (计算值： 464.22996)。 制备例 13: Co.1 的制备
Figure imgf000024_0002
步骤 1: 向化合物 lf(10 mmol)加入 20 mL13a, 反应回流 2小时。 冷却到室温， 体系中有大量白色固体析出， 过滤所得白色固体用四氢呋喃洗涤后得最终产品 13b。 产率为： 82%。
Figure imgf000024_0003
步骤 2 : 在氩气氛的情况下， 向溶有 13b(2nunol)的 1-戊醇的溶液中加入 ld(2mmol 反应体系回流 5.5小时后， 将其置于 0-5。C环境中静置结晶。 用 lOmL 丙酮洗涤所得白色晶体两次， 再用乙醚 /甲醇重结晶后得 终产品 13c 即为化合物 Co 产率为: 。
IH NMR (300 MHz, CDC13): 1.80 (m, 2H, thf-H), 2.06 (m, 2H, thf-H), 3.47-4.07 (m, 13H, thf-H , pip-H )， 3.93 (s, 3H, OCH3)， 3.97 (s, 3H, OCH3), 4.82 (m, 1H， thf-2H), 7.16 (s, 1H， Ar-5H), 7.72 (s, IH, Ar-8H); HR-MS (ESI-正性)： 484.18078 (M+H) (计算 值： 484.18084)。 制备例 14: Co.14的制备
Figure imgf000025_0001
步骤 1: 向化合物 lf(10 mmol)加入 22 mL14a, 反应回流 2小时。 冷却到室温， 体系中有大量白色固体析出， 过滤所得白色固体用四氢呋喃洗涤后得最终产品 14b。 产率
Figure imgf000025_0002
步骤 2 : 在氩气氛的情况下， 向溶有 14b(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)o 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL 丙酮洗涤所得白色晶体两次， 再用乙醚 /甲醇重结晶后得最终产品 14c 即为化合物 Co.l4。 产率为： 48%。
1H NMR (300 MHz, CDC13): d 1.84 (m, 2H， thf-H), 2.03 (m, 2H， thf-H), 3.45-4.47 (m, 13H, thf-H， pip-H ), 3.93 (s， 3H, OCH3), 3.97 (s， 3H, OCH3), 4.82 (m， 1H, thf-2H), 6.89 -7.72 (m, 7H, Ar-H); HR-MS (ESI-正性)： 492.22469 (M+H) (计算值： 492.22478)。 制备例 15: Co.15的制备
Figure imgf000025_0003
-pentanol, reflux
步骤 1 : 在氩气氛的情况下， 向溶有 15b(2minol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 4.5小时后， 将其置于 0-5。C环境中静置结晶。 用 10mL 丙酮洗涤所得白色晶体两次， 再用乙醚 /曱醇重结晶后得最终产品 15c 即为化合物 Co.15。 产率为: 55%。
1H NMR (300 MHz, CDC13): 1.84 (m, 2H, thf-H), 2.03 (m, 2H, thf-H), 3.49-4.27 (m, 13H, thf-H， pip-H )， 4.82 (m， 1H， thf-2H), 6.07(s, 2H， CH2OCH2) 7.27 (s， 1H， Ar-5H)， 7.68 (s, 1H， Ar-8H); HR-MS (ESI-正性）： 372.16718 (M+H) (计算值: 372.16726).
Figure imgf000026_0001
步骤 1 : 在氩气氛的情况下， 向溶有 16a(2mmol)的 1-戊醇的溶液中加入 ld(2mraol). 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL 丙酮洗涤所得白色晶体两次， 再用乙醚 /曱醇重结晶后得最终产品 16a 即为化合物 Co.l6。 产率为： 45%。
1H NMR (300 MHz, CDC13): 1.15-1.26 (t, 6H， CH3-H) 1.87 (m, 2H， thf-H), 2.03 (m, 2H, thf-H), 3.45-4.17 (m, 25H， thf-H， pip-H )， 4.79 (m, 1H， thf-2H), 7.27 (s, 1H， Ar-5H), 7.68 (s, 1H， Ar-8H); HR-MS (ESI-正性）： 504.28215 (M+H) (计算值: 504.28221).
Figure imgf000026_0002
步骤 1 : 在氩气氛的情况下， 向溶有 17a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol). 反应体系回流 4小时后， 将其置于 0-5 C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 17a即为化合物 Co.17。 产率为： 55%。
1H NMR (300 MHz, CDC13): 1.83 (m, 2H, thf-H), 2.03 (m， 2H, thf-H), d 2.61 (s, , CH3), 3.47-4.07 (m, 11H, thf-H， pip-H和 CH2CH20), 3.94 (s, 3H, OCH3), 4.82 (m 1H， thf-2H)， 7.18 (s, 1H， Ar-5H), 7.72 (s, 1H, Ar-8H); HR-MS (ESI-正性)： 416.19344 (M+H) (计算值： 416.19339)。 制备例 18: Co.18的制备
Figure imgf000026_0003
步骤 1 : 在氩气氛的情况下， 向溶有 18a(2ramol)的 1-戊醇的溶液中加入 ld(2mmol)„ 反应体系回流 3小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /曱醇重结晶后得最终产品 18b即为化合物 Co.l8。 产率为： 75%。
IH NMR (300 MHz, CDC13): 1.80 (m, 2H, thf-H), 2.03 (m, 2H, thf-H), 3.57-4.07 (m, 11H, thf-H， pip-H), 3.94 (s, 3H, OCH3), 4.85 (m, 1H， thf-2H), 7.23 (s, 1H， Ar-5H), 7.80 (s， 1H， Ar-8H); HR-MS (ESI-正性)： 376.17853 (M+H) (计算值： 376.17849)。
Figure imgf000027_0001
步骤 1 : 在氩气氛的情况下， 向溶有 19a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 3小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /曱醇重结晶后得最终产品 19b即为化合物 Co.l9。 产率为： 54%。
IH NMR (300 MHz, CDC13): 1.15-1.26 (m, 6H), 1.89 (m, 2H， thf-H), 2.03 (m, 2H， thf-H), 3.07-4.07 (m, 15H， thf-H， pip-H), 4.85 (m, 1H， thf-2H), 7.63 (s, 1H， Ar-5H), 7.89 (s, 1H， Ar-8H); HR-MS (ESI-正性)： 479.23836 (M+H) (计算值： 479.23823)。
Figure imgf000027_0002
步骤 1 : 在氩气氛的情况下， 向溶有 20a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)„ 反应体系回流 3小时后， 将其置于 0-5°C!环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 20b即为化合物 Co.20。 产率为： 54%。
IH NMR (300 MHz, CDC13): 1.83 (m, 2H， thf-H), 2.03 (m, 2H, thf-H), 3.07-4.07 (m, 11H, thf-H , pip-H), 4.88 (m， IH, thf-2H), 7.58 (m, IH, Ar-H), 7.63 (s, IH, Ar-H), 7.89 (s， 1H， Ar-H), 8.38(d, 1H， Ar-H) 8.83(d, IH, Ar-H); HR-MS (ESI-正性)： 379.18833 (M+H) (计算值： 379.18825)。 制备例 21: Co.21的制备
Figure imgf000028_0001
步骤 1 : 在氩气氛的情况下， 向溶有 21a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)„ 反应体系回流 4小时后， 将其置于 0-5°C环境中静置结晶。 用 lOmL丙 酮洗涤所得白色晶体两次，再用乙醚 /曱醇重结晶后得最终产品 21b即为化合物 Co.21。 产率为： 54%。
IH NMR (300 MHz, CDC13): 1.86 (m, 2H, thf-H), 2.01 (m, 2H, thf-H), 2.62 (s, 3H, CH3)， 3.17 - 4.07 (m, 11 H, thf-H , pip-H), 3.97 (s, 3H, OCH3), 4.85 (m, IH, thf-2H), 7.89 (s, 1H， Ar-H), 8.83(d, 1H， Ar-H); HR-MS (ESI-正性)： 379.18833 (M+H) (计算值: 379.18825)。 制备例 22: Co.22的制备
Figure imgf000028_0002
步骤 1 : 在氩气氛的情况下， 向溶有 22a(2imnol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 5小时后， 将其置于 0-5。C环境中静置结晶。 用 lOmL丙 酮洗涤所得白色晶体两次，再用乙醚 /曱醇重结晶后得最终产品 22b即为化合物 Co.22。 产率为： 61%。
IH NMR (300 MHz, CDC13): 0.88-0.93(t, 3H), 1.62(m, 2H), 1.88(m， 2H, thf-H), 2.03 (m, 2H, thf-H), 2.64(t， 2H), 3.57-4.07 (m, 11H, thf-H， pip-H), 3.94 (s, 3H， OCH3), 4.85 (m, IH, thf-2H), 7.23 (s, IH, Ar-5H), 7.82 (s, 1H， Ar-8H); HR-MS (ESI-正性): 400.23464 (M+H) (计算值： 400.23486)。 制备例 23: C
Figure imgf000028_0003
步骤 1 : 在氩气氛的情况下， 向溶有 23a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)0 反应体系回流 5小时后， 将其置于 0-5°C环境中静置结晶。 用 lOmL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 23b即为化合物 Co.23。 产率为： 48%。 1H NMR (300 MHz, CDC13): 1.87 (m, 2H， thf-H), 2.01 (m, 2H， thf-H), 3.17-4.07 (m, 11H, thf-H， pip-H), 3.97 (s， 3H, OCH3), 4.87 (m, 1H， thf-2H), 6.86 -7.62 (m, 7H， Ar-H); HR-MS (ESI-正性)： 477.22497 (M+H) (计算值： 477.22503)。
Figure imgf000029_0001
步骤 1 : 在氩气氛的情况下， 向溶有 25a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 4小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙酸 /甲醇重结晶后得最终产品 25b即为化合物 Co.25。 产率为： 24%。
1H NMR (300 MHz, CDCI3): 1.86 (m, 2H, thf-H), 1.94-2.58 (m, 8H)， 3.17-4.07 (m, 12H, thf-H， pip-H), 3.97 (s, 3H, OCH3), 4.85 (m, 1H, thf-2H), 5.56 (m, 2H), 7.15 (s, 1H， Ar-5H), 7.71 (s， 1H, Ar-8H); HR-MS (ESI-正性）: 454.24499 (M+H) (计算值: 454.24543)。 制备例 25: Co.25
Figure imgf000029_0002
步骤 1 : 在氩气氛的情况下， 向溶有 25a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol). 反应体系回流 4.5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL 丙酮洗涤所得白色晶体两次， 再用乙醚 /甲醇重结晶后得最终产品 25b 即为化合物 Co.25。 产率为： 45%。
1H NMR (300 MHz, CDC13): 1.15-1.26 (t, 6H， CH3-H) 1.88 (m, 2H， thf-H), 2.03 (m, 2H, thf-H), 3.44-4.17 (m, 25H, thf-H， pip-H ) , 3.85 (s, 3H, OCH3), 3.87 (s， 3H, OCH3),4.87 (m, 1H， thf-2H)， 7.27 (s, 1H， Ar-5H), 7.68 (s, 1H， Ar-8H); HR-MS (ESI-正 性)： 564.30330 (M+H) (计算值： 564.30334)。 制备例 26: Co.26的制备
Figure imgf000030_0001
步骤 1 : 在氩气氛的情况下， 向溶有 26a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 4小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 26a即为化合物 Co.l7。 产率为： 55%。
1H NMR (300 MHz, CDCI3): 1.86 (m， 2H， thf-H), 2.03 (m, 2H， thf-H), d 2.61 (s, 3H， CH3)， 3.47-4.07 (m， 11H， thf-H， pip-H和 CH2CH20), 3.91 (s, 3H， OCH3)， 3.95 (s, 3H， OCH3), 3.99 (s, 3H， OCH3), 4.82 (m， 1H， thf-2H), 7.16 (s， 1H， Ar-5H)， 7.72 (s， 1H， Ar-8H); HR-MS (ESI-正性)： 476.21445 (M+H) (计算值： 476,21452)。
Figure imgf000030_0002
步骤 1 : 在氩气氛的情况下， 向溶有 27a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 3小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 27b即为化合物 Co.27。 产率为： 53%。
1H NMR (300 MHz, CDC13): 1.15-1.26 (m， 6H)， 1.88 (m， 2H, thf-H), 2.03 (m， 2H， thf-H), 3.07-4.07 (m， 15H， thf-H， pip-H)， 3.86 (s， 3H， OCH3)， 3.88 (s, 3H， OCH3)， 4.85 (m, 1H， thf-2H), 7.63 (s， 1H， Ar-5H), 7.89 (s， 1H， Ar-8H); HR-MS (ESI-正性）: 539.25942 (M+H) (计算值： 539.25936)。
Figure imgf000030_0003
步骤 1 : 在氩气氛的情况下， 向溶有 28a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 3小时后， 将其置于 0-5。C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 28b即为化合物 Co,28。 产率为： 75%。
1H NMR (300 MHz, CDC13): 1.78 (m, 2H, thf-H), 2.03 (m, 2H, thf-H), 3.57-4.07 (m, 11H, thf-H， pip-H), 3.86 (s， 3H， OCH3), 3.88 (s, 3H, OCH3), 3.94 (s， 3H， OCH3), 4.85 (m， 1H， thf-2H)， 7.23 (s, 1H， Ar-5H), 7.82 (s, 1H， Ar-8H); HR-MS (ESI-正性): 436.19955 (M+H) (计算值： 436.19962)。
Figure imgf000031_0001
步骤 1 : 在氩气氛的情况下， 向溶有 29a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 3小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 29b即为化合物 Co.29, 产率为： 44%。
1H NMR (300 MHz, CDC13): 1.84 (m, 2H, thf-H), 2.01 (m, 2H， thf-H), 2.62 (s, 3H, CH3)， 3.17—4.07 (m, 11H, thf-H， pip-H), 3.82 (s, 3H, OCH3), 3.85 (s， 3H, OCH3), 3.97 (s, 3H, OCH3), 4.85 (m, 1H， thf-2H), 7.89 (s, 1H， Ar-H), 8.83(d, 1H， Ar-H); HR-MS (ESI-正性)： 75.23050 (M+H) (计算值： 475.23051)。 制备例 30: Co.30的制备
Figure imgf000031_0002
1-pentanol, reflux e 2
步骤 1 : 在氩气氛的情况下， 向溶有 30a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol)。 反应体系回流 5小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 30b即为化合物 Co.30。 产率为： 65%。
1H NMR (300 MHz, CDC13): 0.88-0.93(t, 3H), 1.62(m, 2H)， 1.84 (m， 2H， thf-H), 2.03 (m, 2H， thf-H), 2.64(t, 2H)， 3.57-4.07 (m, 11H, thf-H， pip-H), 3.71 (s， 3H, OCH3), 3.76 (s, 3H, OCH3), 3.91 (s, 3H， OCH3), 4.85 (m, 1H， thf-2H), 7.23 (s, 1H, Ar-5H), 7.82 (s, 1H， Ar-8H); HR-MS (ESI-正性)： 400.23464 (M+H) (计算值: 474.27164)。 制备例 31: Co.31的制备
Figure imgf000032_0001
步骤 1 : 在氩气氛的情况下， 向溶有 31a(2mmol)的 1-戊醇的溶液中加入 ld(2mmol). 反应体系回流 4小时后， 将其置于 0-5°C环境中静置结晶。 用 lOmL丙 酮洗涤所得白色晶体两次，再用乙醚 /曱醇重结晶后得最终产品 31b即为化合物 Co.31。 产率为： 24%。
IH NMR (300 MHz, CDC13): 1.864 (m, 2H, thf-H), 1.93-2.58 (m, 8H), 3.17-4.07 (m, 12H, thf-H , pip-H), 3.80 (s， 3H, OCH3), 3.83 (s, 3H, OCH3), 3.94 (s, 3H， OCH3)， 4.85 (m， 1H， thf-2H), 5.56 (m, 2H), 7.15 (s， 1H， Ar-5H), 7.71 (s, 1H， Ar-8H); HR-MS (ESI-正性)： 536.25085 (M+H) (计算值： 536.25091)。 制备例 32: Co.32的制备
Figure imgf000032_0002
步骤 1 : 在氩气氛的情况下， 向溶有 32a(2imnol)的 1-戊醇的溶液中加入 ld(2mmol). 反应体系回流 4小时后， 将其置于 0-5°C环境中静置结晶。 用 10mL丙 酮洗涤所得白色晶体两次，再用乙醚 /甲醇重结晶后得最终产品 32b即为化合物 Co.32。 产率为： 45%„
IH NMR (300 MHz, CDCI3): 1.860 (m, 2H， thf-H), 1.95-2.58 (m, 8H)， 3.17-4.07 (m, 12H, thf-H , pip-H), 3.80 (s, 3H, OCH3), 3.86 (s, 3H， OCH3), 3.93 (s, 3H, OCH3), 4.85 (m, 1H， thf-2H), 5.56 (m, 2H)， 7.15 (s, 1H， Ar-5H)， 7.71 (s， IH, Ar-8H); HR-MS (ESI-正性)： 514.26655 (M+H) (计算值： 514.26656)。
B、 生物学试验例部分
1、 果蝇的饲养和化合物筛选
UAS-rpr和 HS-GaM果蝇购自美国 Bloomington贮存中心。 这两种种系杂交的 F1子代 (HS>rpr)用于筛选药物。使这些子代果蝇在 18°C下养育。将各种药物以 cmap 中的细胞培养物中使用的浓度溶解于 5%葡萄糖溶液中。将药物溶液 (150 μΐ)放置在小 管中的滤纸板的三层上。 然后， 将 20只 1-3日龄成年 H5>r/7 果蝇放置在该瓶中达 1 天。 为了诱导细胞凋亡， 使这些果蝇在 37°C下热休克 2小时。 热休克 12小时以后， 计算存活率。
2、 微阵列芯片技术处理和数据分析
从 hs>rpi"和与 w67c ?杂交的 hs-Gal4的子代制备 10瓶 1-3日龄的果蝇 (基因背 景与对照系匹配)。 每瓶中含有 35只雌性果蝇。 使该果蝇在热休克前 (时间为 0 小时） 以及热休克 1， 2, 3， 4， 5， 6， 7， 8和 12 小时冷冻至液氮中„ 通过 RNeasy mini试剂盒 (Quiagen, Inc)制备总 RNA。 用 TURBO DNase试剂盒 (Ambion, Inc)作进一步处理以 去除样品中的染色体 DNA。 RNA样品检测的质量和微阵列芯片技术处理步骤是按照 Affymetrix DNA微阵列芯片技术标准方案进行。 使用来自 Affymetrix 的果蝇 2.0 DNA微阵列芯片技术 (Affymetrix, Inc)。 同时处理这些阵列以减少不同批次间影响。 使用 Arrayassist 5.0 (Affymetrix, Inc)软件进行数据分析。 "RMA"的算法用于分析数 据。 由于各时间点仅收集一个实验数据点， 三个相邻时间点共同分析， 该各时间点的 中间值认定为该组的数据。例如时间 0、 1和 2在结果中称为 "时间 1"。本发明在此过 程中设计了微阵列芯片技术数据以增加基因表达变化瞬时分辨率。本发明推断如果基 因在转录中显示了变化， 其可以反映在相邻时间点中。 对于功能基因富集， 本发明使 用数据库工具 (http：〃 david.abcc.ncifcrf.gov/)。
3、 通过联系图发现候选化合物
本发明将来自果蝇微阵列芯片技术的上调和下调基因通过数据库转变成哺乳动 物同源基因（www.affymetrix.com)。然后，基于 cmap提供的方案生成信号文件 (Lamb et al.，2006)。 此后， 得到正相关和负相关的化合物。 这些药物购自 Sigma-Aldrich。
4、 细胞培养和凋亡诱导
使 RAW 264.7细胞在 DMEM培养基 (Gibco)中培养， 该培养基中补充有 10% FBS (Gibco)、 0.1g/L链霉素和 0.06g/L青霉素 (Amresco)。 为了诱导细胞凋亡， 将 90%融合的 RAW 264.7细胞在含有 2 g/ml 脂多糖 （LPS; Escherichia coli OUl :B4， Sigma)的 DMEM (10% FBS)中培养 24小时。
5、 Hoechst染色
首先用 Carnoy固定液 (75%乙醇， 25%乙酸)固定细胞， 然后用 Hoechst 33342 (10 g/ml， Sigma)染色 10min。用冰冷的 PBS洗涤 3次之后，通过共焦显微镜 (TCS SP5, Leica) 观察细胞。
6、 败血病的小鼠模型
购自 Vital River (北京维通利华公司， 中国）的雄性 BALB/C (20±3 g)小鼠饲养于北 京大学动物中心。 所有试验由北京大学动物养护和使用委员会 (Peking University Institutional Animal Care and Use Committee)批准， 对于败血病的 LPS模型， 将小鼠 注射 LPS (13.5 mg/kg, i.p.)—次， 对于败血病的大肠杆菌模型， 各小鼠注射 (i.p.) 1x10s CFU的细菌。 对于药物试验， 在给予 LPS之后 1.5 小时注射 (i.p.)特拉唑嗪 (0.4 mg/kg或 0.04 mg/kg)或溶媒緩冲液。 每隔 12小时记录小鼠致死率 7天。
对于败血病的盲肠结扎和穿刺 (CLP)模型， 用水合氯 (300 mg/kg, i.p.)麻醉小鼠。 腹中线切开 1.5-2.0 cm , 暴露盲肠， 分离回盲肠动脉之后， 用 22号标准针头对盲肠穿刺 一次， 并在离盲肠顶部 5 mm处进行结扎。 然后通过连续缝合法使腹部封闭。 最后， 通 过注射预温的生理盐水 (37。C; 5 ml/100 g， s.c.)使动物复苏。 对于抗生素治疗， 通过灌 胃法给予 Co-Am (鲁南制药集团公司， 山东， 中国) (30 mg/kg)。 每隔 12小时观察小鼠存 活率达 7天。
7、 蛋白质印迹
对于蛋白质印迹，通过识别激活的细胞凋亡蛋白酶 3 (#9664, Cell Signaling)的抗体 检测细胞凋亡蛋白蘇 3， actin的抗体购自武汉博士德公司。
8、 DNA 片段化分析
在 LPS注射 24小时之后， 从接受不同治疗的小鼠取出一个胸腺。 然后通过染色体 DNA纯化试剂盒 (Biofuture,北京，中国）提取染色体 DNA。通过 DNA琼脂糖胶评价 DNA 片段程度。
9、 细菌抑制活性的分析
利用牛津杯分析特拉唑嗪潜在的抗生素活性。 将不同质量的特拉唑嗪加入牛津杯 中，放入铺满大肠杆菌 (Escherichia coli 0111:B4)的 LB培养亚中， 24小时之后观察特拉 唾，溱与氨苄青審素对大肠杆菌生长的影响。
10、 特拉峻嗪靶标的分析
本发明通过将特拉唑嗪连接到 Affi-gel上， 然后与 RAW 264.7细胞的蛋白提取液混 合， 将结合在 Affi-gd上的杂蛋白洗掉。 用上样緩冲液将结合的蛋白洗脱， ％度加热后 跑胶 (15%浓度的蛋白胶)。 将特异的蛋白条带切下， 进行质语鉴定。 ―
11、 特拉唑嗪对 Pgkl活性的影响
在大肠杆菌中表达小鼠的 Pgkl重组蛋白（His-Pgkl)， 然后用镍柱纯化 His-Pgkl。 将纯化蛋白稀释到 0.15单位每毫升， 加入梯度浓度的特拉唑嗪， 检测 Pgkl的活性。
12、 特拉唑嗪与 Pgkl亲和力的研究
用不同处理的 Affi-gel与纯化的 His-Pgkl孵化， 4个小时后洗去非特异结合的蛋白， 用上样緩冲液将结合的蛋白洗脱， 96度加热后跑胶 (15%浓度的蛋白胶)。
13、 统计分析
对于存活率分析， 在 Log Rank算法下使用 Kaplan-Meier检验。 对于组比较， 使用 单侧 ANOVA检验。 为了比较两个数据组， 本发明使用 student-t检验。
根据本发明， 已经发现特拉唑嗪的新的性能， 特别是其可用作细胞凋亡抑制剂， 以及用于治疗和 /或预防败血病及其并发症。
14、 Pgkl活性检测
利用 Colorimetric GAPDH Assay Kit (ScienCell)的成分， 以及购买并配制的 GAPDH溶液， HEPES溶液， MgS04溶液。 然后在反应液 (30 mM Hepes, 3 mM ATP, 0.22 mM NADH, 10 mM MgS04 , 10 mM 3-PGA, 3-4 U/ml的 GAPDH, pH 7.5)中加入 一定浓度的式 I化合物， 在 25°C下用分光光度计 (340nm)检测吸收。 根据 1-10分钟 内吸收值的变化， 计算 Pgkl相对活性。 变化越快， 活性越大。 同时注意实验组要减 去同等浓度的化合物在 340nm的吸收。
式 I化合物的活性以激活 Pgkl的倍数表示，其中 0.02μ^ηι1特拉唑嗪可激活 Pgkl 活性 2.9倍； 化合物 Co.l至 Co.32在 0.02 g/ml浓度下测试， 激活 Pgkl活性的倍数 分别见上文， 例如 Co.l和 Co.2激活 Pgkl活性的倍数分别为 2.9和 2.3。
15、 细胞凋亡诱导及检测
将 RAW 264.7 细胞培养于 DMEM培养基中（Gibco), 加入 5%胎牛血清以及双 抗（0.1 mg/ml 的链審素和 0.06mg/ml 的青霉素）。 加入 2 g/ml 的脂多糖和 (LPS;Escherichia coli OHl:B4, Sigma-Aldrich)50 U/ml的伽马干扰素 (Peprotech)诱导 凋亡。同时加入一定浓度的化合物。用 western blot的方法来评估凋亡，抗体为 Caspase 3的活性形式抗体 (#9661， Cell Signaling). >¾以卩-Actin为内参 (Boster)。 以 Caspase 3/p-Actin的值来评估凋亡发生的程度。
16、 特拉唑嗪的降血糖作用
给小鼠一次注射特拉唑嗪 (0.4 mg/kg i.p.)， 18个小时后测量小鼠血糖， 发现血糖 降低了近 30%左右 (/-检验， P < 0.01); 而同时给予盐水时未见显著性差异。 结果见 图 16。在相同的试验方法中，本发明其它示例性化合物例如 Co.l、 Co.3> Co.7、 Co.9、 Co.l3、 Co.l6、 Co.l9 Co.24、 Co.27、 Co.32均显示可使血糖降低 20%至 50%。
17、 特拉唑嗪抗脑血栓的作用
制作小鼠脑血栓模型， 在小鼠因血栓死亡后， 对脑组织进行染色， 结果显示在脑 片中部呈现白色（其为脑死亡的典型现象）； 在一次注射特拉唑 (0.4 mg/kg i.p.)时可 明显降低死亡组织面积。 对结果进行统计， 以％死亡组织=白色区域面积 /总面积为考 察指标， 每种条件下小鼠数量 = 6。 经 检验， < 0.05„ 结果见图 17, 从图中结果可 见， 特拉唑嗪可以明显降低脑死亡组织的面积。 在相同的试验方法中， 本发明其它示 例性化合物例如 Co.l、 Co.4、 Co.7、 Co.8、 Co.13-14, Co.l6、 Co.l9、 Co.24-27, Co.29、 Co.31-32均显示可明显降低死亡组织面积， < 0.05。
18、 抗败血病效果
BALB/C小鼠 （20克左右)腹腔注射脂多糖 (13.5 mg/kg) , —个半小时后注射一定 浓度的药物， 每 12小时观察一次小鼠的存活， 共观察 7天， 统计存活有无显著性差 异。
另一模型为盲肠穿刺与结扎 (CLP)。小鼠麻醉后，在其腹部剪开 1.5-2厘米的小口， 将盲肠取出。 将粪便挤到盲肠底部， 在离盲肠顶端 5毫米处结扎， 而后用 22号针头 穿一小孔， 挤出一些粪便。 将盲肠放回腹腔， 用线缝好并封闭伤口。 一个半小时后注 射一定浓度的药物， 每 12小时观察一次小鼠的存活， 共观察 7天， 统计存活有无显 著性差异。
结果表明， 在以上两种模型的试验中， 化合物 Co.l、 Co.4、 Co.9> Co.l3、 Co.l8、 Co.21、 Co.28、 Co.29、 Co.33分别以每天一次注射 (0.4 mg/kg i.p.)连续给药 3天后， 在 7天观察期结束时， 显示这些化合物均可显著提高小鼠的存活率（P < 0.05 , 与不 给药物的对照组比较）。
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Claims

权刊 ^求
1. 式 I化合物，
Figure imgf000037_0001
或其药学可接受的盐、 溶剂合物、 酯、 前药， 其中
Ria和 Rib 各自独立地选自 H、 NH2、 OH、 C1-6烷基 -、 C1-6烷氧基 -C 烷基 -、 C2_6烯基-、 2_6炔基-、 C"烷氧基-、 C"烷基酰基-、 芳基酰基-、 C6-ie芳基 -、 C5_6 环烷基， 或者 Rla和 Rl b与它们连接的氮原子一起形成 5-或 6-元环， 其中所述烷基任 选被 1-3个选自下列的取代基取代： 羟基、 卤素；
和 各自独立地选自 H、 鹵素、 C1-6烷基-、 鹵代 C1-6烷基 -、 C2-6烯基 -、 C2-6炔基-、 CN、 N02、 NH2、 OH、 C1-6烷氧基-、 d— 6烷氧基 -C1-6烷氧基-、 C1-6 烷酰基氧基-、 烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环 基、 烷基貌基-，或者 R2和 R3与它们连接的环原子一起形成 5-或 6-元破环或杂环；
R4 7fn R5 各自独立地选自 H、 卤素、 CN、 N02、 NH2、 OH、 C,.6烷基-、 C1-6烷氧基 -Ci-6烷氧基-、 C1-6烷酰基氧基-、 卤代 C1-6烷基 -、 C2.6烯基-、 C2-6炔基-、 C1-6烷氧基-、 C1-6烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环 基、 饱和或不饱和的 5-或 6-元碳环或杂环基氧基-、 C1-6綻基酰基。
2. 根据权利要求 1的化合物， 其中 Rla和 Rlb各自独立地选自 H、 NH2、 OH、 C "烷基-、 d.4烷氧基 -C1-4烷基-、 C2-4烯基 -、 C2.4炔基-、 Ci-4烷氧基-、 C1-4烷基酰 基-、 苯基酰基-、 苯基-、 C5.6环烷基， 或者 Rla和 Rlb与它们连接的氮原子一起形成 5-或 6-元环， 其中所述垸基任选被 1-3个选自下列的取代基取代： 羟基、 卤素。
3. 根据权利要求 1的化合物, 其中 和 各自独立地选自 H、 卤素、 C1-6烷 基-、 卤代 C1-6烷基-、 C1-6烷氧基-、 C1-6烷氧基 -C1-6烷氧基-、 烷酰基氧基-、 c,-6 烷酰基氛基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环基， 或者 R2和 R3 与它们连接的环原子一起形成 5-或 6-元碳环或杂环。
4. 根据权利要求 1的化合物， 其中 和 各自独立地选自 H、 卤素、 d.6烷 基-、 C1-6烷氧基 -C1-6烷氧基-、 C1-6烷酰基氧基-、 卤代 C1-6烷基-、 C1-6烷氧基-、 C,-6 烷酰基氨基-、 芳酰基氨基-、 饱和或不饱和的 5-或 6-元碳环或杂环基、 饱和或不饱和 的 5-或 6-元碳环或杂环基氧基。
5.根据权利要求 1的式 I化合物，其为选自下列编号为 Co.1至 Co.33的化合物：
Figure imgf000038_0001
Figure imgf000039_0001
6. 权利要求 1-5任一项所述化合物在制备作为细胞凋亡抑制剂或 pgkl激活剂的 药物中的用途， 或者在制备治疗和 /或预防败血病及其并发症的药物中的用途， 或者 在制备治疗和 /或预 ^高血糖、 脑血栓及其并发症的药物中的用途。
7. 根据权利要求 6的用途，其中所述细胞凋亡抑制剂或 pgkl激活剂用于临床疾 病的治疗和 /或预防以及实验室诊断和 /或检测。
8. 根据权利要求 6的用途， 其中所述败血病是细菌和 /或其它微生物感染引起的 欧血病。
9. 根据权利要求 6的用途， 其中所述败血病的并发症选自： 肾功能衰竭、 呼吸 衰竭、 凝血障碍、 器官损害、 中毒性心肌病变、 脑病、 肝病及中毒性肠麻痹、 化脓性 脑膜炎、 肺炎、 肺脓肿、 蜂窝組织炎、 骨髓炎、 肾 肾炎。
10. 根据权利要求 6的用途， 其中所述式 I化合物是特拉唑嗪或其药学可接受的 盐或者其溶剂合物。
11. 根据权利要求 6的用途， 其中所述药物中还包括至少一种抗微生物药物。
12. '根据权利要求 11的用途， 其中所迷抗微生物药物选自： 阿莫西林、 青霉素、 青霉素 V、 苯唑西林、 氯唑西林、 氟氯西林、 氨苄西林、 哌拉西林、 阿洛西林、 克拉 维酸钾、 舒巴坦、 舒他西林、 三唑巴坦、 氨曲南、 美罗培南。
13. 一种药物组合物， 其中包含治疗和 /或预防有效量的权利要求 1-5任一项所述 化合物，、 以及任选的药学可接受的栽体。
14. .根据权利要求 13的药物组合物， 其中还包括至少一种抗微生物药物。
15. 根据权利要求 14的药物组合物， 其中所述抗微生物药物选自： 阿莫西林、 青霉素 青霉素 V、 苯唑西林、 氯唑西林、 氟氯西林、 氨苄西林、 哝拉西林、 阿洛西 林、 克拉维酸钾、 舒巴坦、 舒他西林、 三唑巴坦、 氨曲南、 美罗培南。
16. 根据权利要求 13的药物组合物， 其是用作细胞凋亡抑制剂、 用作 pgkl激活 剂、 或者用于治疗和 /或预防败血病及其并发症、 或者用于治疗和 /或预防高血糖、 脑 血栓及其'并发症。
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