WO2018076537A1 - D-3-磷酸甘油酸脱氢酶别构抑制剂及其应用 - Google Patents

D-3-磷酸甘油酸脱氢酶别构抑制剂及其应用 Download PDF

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WO2018076537A1
WO2018076537A1 PCT/CN2016/113476 CN2016113476W WO2018076537A1 WO 2018076537 A1 WO2018076537 A1 WO 2018076537A1 CN 2016113476 W CN2016113476 W CN 2016113476W WO 2018076537 A1 WO2018076537 A1 WO 2018076537A1
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group
substituted
alkyl
halogen
pkumdl
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PCT/CN2016/113476
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French (fr)
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来鲁华
刘莹
王倩
刘培
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北京大学
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Priority claimed from CN201610941898.1A external-priority patent/CN106562951B/zh
Priority claimed from CN201610926351.4A external-priority patent/CN106562946A/zh
Application filed by 北京大学 filed Critical 北京大学
Priority to US16/344,799 priority Critical patent/US20200054593A1/en
Publication of WO2018076537A1 publication Critical patent/WO2018076537A1/zh
Priority to US16/405,569 priority patent/US10722489B2/en

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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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/38—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D307/52—Radicals substituted by nitrogen atoms not forming part of a nitro radical

Definitions

  • the present invention relates to a medicament for treating and preventing various diseases caused by a disorder of serine metabolism, and particularly to an N'-substituted benzoyl hydrazide compound as a D-3-phosphoglycerate dehydrogenase inhibitor, and the same
  • an N'-substituted benzoyl hydrazide compound as a D-3-phosphoglycerate dehydrogenase inhibitor
  • PHGDH D-3-phosphoglycerate dehydrogenase in humans catalyzes the first step of serine synthesis and is a key enzyme in the serine synthesis pathway.
  • PHGDH was confirmed to be overexpressed in 40% of human melanoma cells or 70% of triple-negative breast cancer cells in 2011.
  • the knockdown of PHGDH gene revealed that the growth of these cancer cells was greatly inhibited [1] Locasale, JW, et al. (2011). Nat. Genet. 43, 869-874. (2) Possemato, R., et al. (2011). Nature 476, 346-350. Therefore, the use of PHGDH as an anti-cancer target for drug design has broad prospects.
  • Allosteric regulation in proteins refers to the phenomenon that allosteric molecules bind to the inactive sites of the protein and cause changes in protein activity.
  • the advantage of allosteric drugs is that they are specific, regulate target protein activity without completely losing protein activity, and only exert effects in the presence of endogenous ligands.
  • the present invention finds potential allosteric sites suitable for small molecule binding (see FIG. 1), and performs virtual screening for predicted sites MDL-1, MDL-2 and the like.
  • a benzoyl hydrazide compound is provided as an allosteric inhibitor of PHGDH for the potential allosteric site MDL-1 of PHGDH, such compounds having the following structural formula:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 are the same or different and each independently represents hydrogen, halogen, nitro, hydroxy, amino or substituted amino, alkyl, alkoxy a benzyl, benzyloxy or halogen substituted alkyl group, or wherein two adjacent substituents (R 1 and R 2 , R 2 and R 3 , R 4 and R 5 , R 5 and R 6 and/or R 6 And R 7 ) can be looped.
  • the halogen includes F, Cl, Br, and I.
  • the substituted amino group is preferably a C1-C12 alkyl-substituted amino group, more preferably a C1-C6 alkyl-substituted amino group such as a methylamino group, an ethylamino group, a dimethylamino group or a diethylamino group.
  • the alkyl group is preferably a C1-C12 alkyl group, more preferably a C1-C6 alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group or the like.
  • the alkoxy group is preferably a C1-C8 alkoxy group, more preferably a C1-C4 alkoxy group such as a methoxy group, an ethoxy group, a propoxy group or the like.
  • the halogen-substituted alkyl group is preferably one or more halogen-substituted C1-C12 alkyl groups, more preferably one or more halogen-substituted C1-C6 alkyl groups such as a trifluoromethyl group and the like.
  • the compound of formula I can be prepared by the following method:
  • a furan compound is provided as an allosteric inhibitor of PHGDH for the potential allosteric site MDL-2 of PHGDH, such compounds having the following structural formula:
  • R 1 ', R 2 ', R 3 ' are the same or different and each independently represents hydrogen, halogen, nitro, hydroxy, amino, carboxy, alkyl, alkoxy, halogen-substituted alkyl, carboxylic acid ester a sulfonamide, amide or N-alkyl substituted amide group, or wherein two adjacent substituents (R 1 'and R 2 ' or R 2 ' and R 3 ') are cyclic; R 4 ' represents Alkyl, halogen substituted alkyl, amino, cycloalkyl, unsubstituted or substituted aryl; X is O, N or S.
  • the halogen includes F, Cl, Br, and I.
  • R 1 ', R 2 ' and R 3 ' is an alkyl group
  • it is preferably a C1 to C12 alkyl group, more preferably a C1 to C6 alkyl group, such as a methyl group, an ethyl group, a propyl group
  • it is an alkoxy group
  • it is preferably a C1-C8 alkoxy group, more preferably a C1-C4 alkoxy group, such as a methoxy group, an ethoxy group, a propoxy group or the like
  • it is one or more halogen-substituted C1-C12 alkyl groups, more preferably one or more halogen-substituted C1-C6 alkyl groups, often substituted by fluorine, such as trifluoromethyl.
  • R 1 ', R 2 ' and R 3 ' is the carboxylate group, it is preferably a C1 to C8 esteroxy group (-COOC n H 2n+1 , n is 1 to 7)
  • the integer is more preferably a C1-C4 esteroxy group such as a methoxy ester group, an ethoxylate group, a isopropyloxy ester group or the like.
  • R 1 ', R 2 ' and R 3 ' is the N-alkyl substituted amide group, it is preferably a C1 to C12 alkyl substituted amide group, more preferably a C1 to C6 alkyl group.
  • Substituted amide groups such as N-methylamido, N,N-dimethylamido and the like.
  • R 1 'and R 2 ' or R 2 ' and R 3 ' are ring-formed, the adjacent two substituents jointly represent 1,3-butadiene-1,4-subunit, 1,4-dibutyl Base.
  • R 4 ' is an alkyl group, it is preferably a C1 to C12 alkyl group, more preferably a C1 to C6 alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group or the like.
  • R 4 ' is a halogen-substituted alkyl group, it is preferably one or more halogen-substituted C1 to C12 alkyl groups, more preferably one or more halogen-substituted C1-C6 alkyl groups such as a trifluoromethyl group and the like.
  • R 4 ' is a cycloalkyl group, it is preferably a C5-C7 cycloalkyl group such as a cyclohexyl group.
  • R 4 ' is an unsubstituted or substituted aryl group
  • the aryl group is preferably a phenyl group
  • the substituted aryl group is preferably a 4-substituted phenyl group
  • the substituent on the phenyl group is preferably a C1 to C6 alkyl group, A halogen-substituted C1-C6 alkyl group, a nitro group, a C1-C4 alkoxy group or the like, for example, a 4-trifluoromethylphenyl group or a 4-nitrophenyl group.
  • the substituted furan aldehyde is reacted with a substituted semicarbazide (or a substituted thiosemicarbazide or a substituted amino oxime) to give an inhibitor of the formula II.
  • the chemical substances used in the synthetic route of the present invention are commercially available products or can be synthesized by the prior art.
  • the operation methods and operation steps, reaction conditions and intermediates, etc. are all well known to those skilled in the art.
  • the organic synthesis method is designed, implemented, and disclosed in the examples.
  • the present invention demonstrates by in vitro enzyme activity test, cell activity test and mouse xenograft model experiments that the compounds of Formula I and Formula II can specifically inhibit PHGDH activity. Inhibition of PHGDH by the isomorphism of the compound of formula I or formula II can reduce the overexpression of PHGDH in cancer cells, thereby delaying the growth of cancer cells.
  • the benzoyl hydrazide compound and the furan compound of the present invention can be prepared singly or in combination, or in combination with other PHGDH inhibitors or anticancer drugs, or a pharmaceutically acceptable salt thereof as an active ingredient, by adding a conventional pharmaceutical carrier.
  • the pharmaceutically acceptable salt of the benzoyl hydrazide compound and the furan compound of the present invention and a combination thereof means a pharmaceutically acceptable salt, for example, a salt formed with a mineral acid such as hydrochloric acid, sulfuric acid, phosphoric acid or nitric acid, or with a lemon. a salt formed from an organic acid such as acid, succinic acid, citric acid, acetic acid, tartaric acid or methanesulfonic acid.
  • compositions can be formulated into a variety of dosage forms depending on the purpose of the treatment, the route of administration, according to techniques well known in the art.
  • Figure 1 shows the allosteric site of PHGDH predicted by the protein surface property detection program CAVITY.
  • FIG. 2 is a molecular docking map of a compound of the present invention and PHGDH, wherein A: PKUMDL-WL-2101 docks with an MDL-1 site; BPKUMDL-WL-2201 docks with an MDL-2 site.
  • Figure 3 shows the effect of PKUMDL-WL-2101 (A) and PKUMDL-WL-2201 (B) on the mitosis cycle of breast cancer cells in Example 5.
  • Figure 4 shows the in vivo biological activity of PKUMDL-WL-2101 (A, C, E) and PKUMDL-WL-2201 (B, D, F) in a mouse xenograft model in Example 6.
  • the PHGDH (PDB code: 2G76) allosteric site was predicted using the protein surface exploration program CAVITY.
  • AdjustVolume and AdjustSurfaceArea are related to the hydrophobic area of the residue in the predicted site and the number of hydrogen bond acceptor donors.
  • MDL-1 is located near the active site and the prosthetic NAD + binding site, and the pocket volume is The predicted maximum pK D is 8.71.
  • MDL-1 shares 78-glycine, 79-valine, 80-aspartic acid, 81 asparagine, and 82-valine with the active site.
  • MDL-2 is located in the substrate binding domain and the pocket size is The predicted maximum pK D is 7.79.
  • the molecular mapping method is used to perform virtual screening of the SPECS database. Manually selected, purchased compounds were verified in in vitro enzyme activity assays.
  • the docking results of this compound with PHGDH are shown in Figure 2 (B).
  • Fig. 2(A) The interaction pattern between small molecules and PHDGH can be seen: two benzene rings occupy the hydrophobic cavity in the pocket, and the acyl group of the acyl group can be bonded to the phenyl phenyl alanine at position 261.
  • Hydrogen bond formation; 2-substituted hydroxyl group can be The glutamic acid at position 264 forms a hydrogen bond; the carbonyl oxygen in the hydrazide chain forms a hydrogen bond with the 57th lysine, and the hydrazide nitrogen forms a hydrogen bond with 264 glutamic acid; on the other benzene ring, the nitro group at the 3 position Hydrogen bonding can be formed with 134 arginine or 55 alanine; the ring also has an electrostatic interaction with the surrounding positively charged cavity. Accordingly, we optimized the substituent groups of two benzene rings and designed a series of PKUMDL-WL-2101 analogs.
  • the synthetic route is:
  • PKUMDLWL-2101 (E)-2,4-dihydroxy-N'-(2-hydroxy-5-nitrobenzylidene)benzoyl hydrazide;
  • PKUMDLWL-2102 (E)-N'-(4-fluorobenzylidene)benzoyl hydrazide;
  • PKUMDLWL-2103 (E)-N'-benzylidene-2,4-dihydroxybenzoyl hydrazide;
  • PKUMDLWL-2104 (E)-2,4-dihydroxy-N'-(naphthalene-1-methylene)benzoyl hydrazide;
  • PKUMDLWL-2105 (E)-2,4-dihydroxy-N'-(4-nitrobenzylidene)benzoyl hydrazide;
  • PKUMDLWL-2106 (E)-N'-(2-hydroxy-5-nitrobenzylidene)-4-nitrobenzoyl hydrazide;
  • PKUMDLWL-2107 (E)-N'-(2-hydroxy-5-nitrobenzylidene)-1-naphtholquinone;
  • PKUMDLWL-2109 (E)-2,4-dihydroxy-N'-(4-hydroxybenzylidene)benzoyl hydrazide
  • PKUMDLWL-2110 (E)-2-hydroxy-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide;
  • PKUMDLWL-2111 (E)-4-fluoro-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2112 (E)-2,4-dihydroxy-N'-(3-methoxybenzylidene)benzoyl hydrazide
  • PKUMDLWL-2113 (E)-N'-(4-ethoxy-3-nitrobenzylidene)-2,4-dihydroxybenzoyl hydrazide
  • PKUMDLWL-2114 (E)-2,4-dihydroxy-N'-(3-nitrobenzylidene)benzoyl hydrazide
  • PKUMDLWL-2115 (E)-3-hydroxy-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2116 (E)-2,4-dihydroxy-N'-(4-methoxy-3-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2117 (E)-2,4-dihydroxy-N'-(3-hydroxybenzylidene)benzoyl hydrazide
  • PKUMDLWL-2119 (E)-3-chloro-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2120 (E)-N'-(2-hydroxy-5-nitrobenzylidene)-3-nitrobenzoyl hydrazide
  • PKUMDLWL-2121 (E)-4-amino-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2122 (E)-N'-(2-hydroxy-5-nitrobenzyl)-2-methylbenzohydrazide
  • PKUMDLWL-2123 (E)-4-methoxy-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2124 (E)-4-(tert-butyl)-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2125 (E)-4-bromo-N'-(2-hydroxy-5-nitrobenzyl)benzoyl hydrazide
  • PKUMDLWL-2126 (E)-N'-(2-hydroxy-5-nitrobenzylidene)-3-methoxybenzoyl hydrazide
  • PKUMDLWL-2128 (E)-N'-(2-hydroxy-5-nitrobenzyl)-4-(trifluoromethyl)benzoyl hydrazide
  • PKUMDLWL-2129 (E)-N'-(4-chlorobenzylidene)-2,4-dihydroxybenzoyl hydrazide
  • PKUMDLWL-2130 (E)-N'-(4-chlorobenzylidene)-2,4-dihydroxybenzoyl hydrazide
  • PKUMDLWL-2131 E)-N'-(4-bromobenzylidene)-2,4-dihydroxybenzoyl hydrazide
  • PKUMDLWL-2132 (E)-2,4-dihydroxy-N'-(2-nitrobenzylidene)benzoyl hydrazide
  • Figure 2 (B) shows the interaction pattern of small molecules with PHGDH: the 2-phenylfuran aromatic ring occupies the hydrophobic cavity in the pocket, and the 4-position carboxyl oxygen in the benzene ring can be combined with the 11-position serine, the 35-position leucine or The asparagine at position 34 forms a hydrogen bond; the thiosemicarbazide group may interact with other negatively charged groups in PHGDH.
  • the synthetic route is:
  • PKUMDL-WL-2202 (E)-N-ethyl-2-((5-(4-(trifluoromethyl)phenyl)furan-2-yl)methylene)phosphonium carbamide;
  • PKUMDL-WL-2203 (E)-N-ethyl-2-((5-(4-methoxyphenyl)furan-2-yl)methylene)indenyl-1-thiocarboxamide;
  • PKUMDL-WL-2204 (E)-2-((5-(3-chlorophenyl)furan-2-yl)methyl)-N-ethylmercapto-1-thiocarboxamide;
  • PKUMDL-WL-2205 (E)-4-(5-((2-(phenylcarbamoyl) fluorenyl)methyl)furan-2-yl)benzoic acid;
  • PKUMDL-WL-2206 (E)-4-(5-((2-(methylcarbamoyl) fluorenyl)methyl)furan-2-yl)benzoic acid;
  • PKUMDL-WL-2207 (E)-N-ethyl-2-((5-phenylfuran-2-yl)methylene)phosphonium carbamide;
  • PKUMDL-WL-2208 (E)-2-((5-(4-(tert-butyl)phenyl)furan-2-yl)methyl)-N-ethylindenyl-1-thiocarboxamide ;
  • PKUMDL-WL-2209 (E)-2-chloro-5-(5-((2-(ethylcarbamoyl) fluorenyl)methyl)furan-2-yl)benzoic acid;
  • PKUMDL-WL-2210 methyl (E)-4-(5-((2-(ethylcarbamoyl)hydrazono)methyl)furan-2-yl)benzoic acid methyl ester;
  • PKUMDL-WL-2211 (E)-N-ethyl-2-((5-(p-tolyl)furan-2-yl)methylene)phosphonium carbamide;
  • PKUMDL-WL-2212 methyl (E)-4-(5-((2-((4-nitrophenyl))carbonyl)))methyl)furan-2-yl)benzoate;
  • PKUMDL-WL-2213 (E)-4-(5-((2-(cyclohexylcarbonyl)indolyl)methyl)furan-2-yl)benzoic acid;
  • PKUMDL-WL-2214 (E)-N-ethyl-2-((5-(naphthalen-1-yl)furan-2-yl)methylene)phosphonium carbamide;
  • PKUMDL-WL-2215 methyl (E)-4-(5-((2-(2-(4-(trifluoromethyl)phenyl)indol-1-carboxy)indolyl)methyl)furan Methyl-2-benzoate;
  • PKUMDL-WL-2216 (E)-N-ethyl-2-((5-(4-fluorophenyl)furan-2-yl)methylene)phosphonium carbamide;
  • PKUMDL-WL-2217 methyl (E)-2-amino-4-(5-((2-(indenyl))indolyl)methyl)furan-2-yl)benzoic acid methyl ester;
  • PKUMDL-WL-2218 (E)-2-((5-(4-bromophenyl)furan-2-yl)methyl)-N-ethylmercapto-1-thiocarboxamide;
  • PKUMDL-WL-2219 isopropyl (E)-4-(5-((2-(indolylcarbonyl)hydrazino)methyl)furan-2-yl)benzoic acid methyl ester;
  • PKUMDL-WL-2220 methyl (E)-4-(5-((2-(indolylcarbonyl)indolyl)methyl)furan-2-yl)benzoic acid methyl ester;
  • PKUMDL-WL-2221 (E)-2-((5-(4-chlorophenyl)furan-2-yl)methyl)-N-ethylmercapto-1-thiocarboxamide;
  • PKUMDL-WL-2222 (E)-4-(5-((2-(indolylcarbonyl)indolyl)methyl)furan-2-yl)benzoic acid;
  • PKUMDL-WL-2223 methyl (E)-4-(5-((2-(indolylcarbonyl)indolyl)methyl)furan-2-yl)-3-methylbenzoic acid methyl ester;
  • PKUMDL-WL-2224 methyl (E)-4-(5-((2-(ethylcarbamoyl)hydrazono)methyl)furan-2-yl)benzoic acid methyl ester;
  • PKUMDL-WL-2225 (E)-4-(5-((2-(indenyl))indolyl)methyl)furan-2-yl)benzenesulfonamide;
  • PKUMDL-WL-2226 (E)-4-(5-((2-(ethylcarbamoyl)indolyl)methyl)furan-2-yl)benzoic acid;
  • PKUMDL-WL-2227 ethyl (E)-4-(5-((2-(indenyl))indolyl)methyl)furan-2-yl)benzoic acid methyl ester;
  • PKUMDL-WL-2228 (E)-N-ethyl-2-((5-(4-nitrophenyl)furan-2-yl)methylene)phosphonium carbamide;
  • PKUMDL-WL-2229 (E)-N-ethyl-2-((5-(4-hydroxyphenyl)furan-2-yl)methylene)phosphonium carbamide;
  • PKUMDL-WL-2230 (E)-4-(5-((2-(indenyl))indolyl)methyl)furan-2-yl)-N-methylbenzamide;
  • PKUMDL-WL-2231 (E)-4-(5-((2-(4-(trifluoromethyl)phenyl)carbonyl)hydrazyl)methyl)furan-2-yl)benzoic acid;
  • the characterization data for the new compounds are listed in Table 2.
  • Example 4 Determination of in vitro enzymatic activity of PHGDH of compound by fluorescence kinetic method
  • PHGDH enzyme activity was achieved by detecting the fluorescence emission spectrum of NADH at 456 nm.
  • PHGDH final concentration 30 ng/ ⁇ L
  • HEPES buffer 25 mM, pH 7.1, 400 mM KCl
  • 5 ⁇ M PLP 5 ⁇ M PLP
  • 0.5 mM ⁇ KG 150 ⁇ M NADH
  • PSAT1 final concentration 30 ng/ ⁇ L
  • 10 ⁇ L of DMSO (control) or a small molecule-containing DMSO solution was added and shaken at 25 ° C for 5 minutes at 550 rpm.
  • the final concentration (v/v) of DMSO was maintained in the enzyme in vivo test system to be 5%.
  • an aqueous Pser solution (final concentration 0.5 mM) was added to initiate the reaction, and the change in NADH consumption at 456 nm over time was monitored by a UV-visible microplate reader. Protein activity was assessed using an initial rate of reaction within 30 s, at which time NADH consumption was linear over time.
  • the inhibition rate of enzyme activity was initially tested on 63 compounds, and the IC 50 values were obtained by intensive studies on compounds with an inhibition rate of enzyme activity greater than 50% at 50 ⁇ M, as shown in Table 3.
  • the biological activity of the compounds at the cellular level was investigated.
  • a series of cancer cells and normal mammary epithelial cells were selected and tested by MTT (3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide).
  • PHGDH-sensitive breast cancer cells MDA-MB-468 (5000 cells/well) and HCC70 (5000 cells/well)
  • PHGDH-insensitive breast cancer cells MCF-7 (3000) Cells/wells
  • MDA-MB-231 2000 cells/well
  • ZR-75-1 (4000 cells/well
  • colon cancer cells DLD-1 2000 cells/well
  • normal breast epithelial cells MCF-10A Transfer to 3000-well plates at 3000 cells/well and attach overnight.
  • various concentrations of the compound were added to a 96-well plate, and the cells were incubated for 72 hours to control the final concentration of DMSO (v/v) to be 0.2%.
  • DMSO without any compound was used as a control.
  • PKUMDL-WL-2101 and PKUMDL-WL-2201 exhibited micromolar inhibitory activity at the cellular level (see Table 4). Its PHGDH sensitive breast cancer cell line MDA-MB-468, and 50 exhibited EC HCC70 values were 7.70 and 10.8 ⁇ M, for PHGDH insensitive breast cancer cells MDA-MB-231, ZR- 75-1 and MCF- 7 exhibited EC 50 values of 27.7,83.4 and 139 ⁇ M, colon cancer cells showed the EC 50 value of 18.3 ⁇ M. At the same time, PKUMDL-WL-2101 showed weak cytotoxicity and exhibited an EC 50 value of 45.8 ⁇ M for MCF-10A cells.
  • PKUMDL-WL-2201 has a EC 50 value of 6.9 and 10.0 ⁇ M for PHGDH-sensitive breast cancer cells MDA-MB-468 and HCC70, respectively, and is not sensitive to PHGDH breast cancer cells MDA-MB-231, ZR-75- 1 MCF-7 and exhibited EC 50 values of> 200,125 and> 200 ⁇ M, colon cancer cells showed the EC 50 value of 167 ⁇ M.
  • PKUMDL-WL-2201 showed weak cytotoxicity and exhibited an EC 50 value of 64.7 ⁇ M for MCF-10A cells, respectively.
  • MDA-MB-468 cells (3 ⁇ 10 5 cells/well) in an exponential growth cycle were transferred to a 6-well plate, and the compounds were incubated at different concentrations for 24 hours overnight, trypsinized, centrifuged, 70%. Pre-cooled ethanol fixation, PBS rinse, centrifuge, resuspend (0.5% triton-x-100, 50 ⁇ g/ml PI and 50 ⁇ g/ml DNase-free RNase in PBS), protected from light for 30 minutes at 37°C, using flow analyzer The results of the analysis indicate that both PKUMDL-WL-2101 and PKUMDL-WL-2201 can arrest the cell cycle in the G 0 /G 1 phase (see Figure 3), which may be the main cause of cancer death in compounds.
  • the MDA-MB-468 (2 ⁇ 10 5) cells were injected into the fourth mammary fat pad NOD.CB17-Prkdc scid / J mice (6-8 weeks), the mean tumor volume grew to be 30mm 3, Mice were randomly divided into 8 groups of 5 mice each. Subsequently, administration was started, and the control group was administered with a solvent (10% DMSO, 20% EL and 70% PBS) in which the compound was administered at a dose of 20 mg/kg/day. The tumor volume was measured every two days, and the tumor volume was calculated by the following formula.
  • the MDA-MB-468 (2x 10 5) cells were injected into NOD.CB17-Prkdc fourth mammary fat pad of scid / J mice (6-8 weeks), the mean volume of the tumor to be grown to 150mm 3, the Mice were randomly divided into 5 groups of 5 mice each. Subsequently, administration was started.
  • mice in the first group of mice was a control group, and only the solvent that dissolved PKUMDL-WL-2201 and dissolved doxorubicin (10% DMSO, 20% EL and 70% PBS) was injected; the second group of mice remained For the control group, only the solvent in which doxorubicin was dissolved (10% DMSO, 20% EL and 70% PBS); the mice in the third group to the fifth group were experimental groups, and the administration methods were as follows. (2.5mg/kg/4day), PKUMDL-WL-2201 (20mg/kg/day) and PKUMDL-WL-2201 (20mg/kg/day) combined with doxorubicin (2.5mg/kg/4day) medicine. Thereafter, the tumor volume growth curve and the mouse survival curve were monitored every two days, and the tumor size was measured by a caliper. The tumor volume is still calculated from the above formula.
  • the mice in the experimental group began to die 13 days after the start of administration, so the experimental results of the combination administration were only recorded 11 days after the start of administration.
  • the combination of PKUMDL-WL-2201 and doxorubicin can significantly inhibit tumor growth in mice and inhibit tumor growth caused by doxorubicin alone. There are significant differences in the effect.
  • the maximum efficacy of the combination drug occurred on the sixth day after the start of administration, and the inhibitory effect on tumor growth was 41% compared with the control group (see E-F in Fig. 4).
  • the compounds of the present invention specifically inhibit PHGDH activity as a result of comprehensive enzyme activity assays, cell assays, and mouse xenograft model experiments.

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Abstract

本发明公布了D-3-磷酸甘油酸脱氢酶别构抑制剂及其应用，一类是针对酶的别构位点MDL-1的苯甲酰肼类化合物，另一类是针对酶的别构位点MDL-2的呋喃类化合物。体外酶活性测试、细胞活性测试及小鼠异种移植模型实验证实，这两类别构抑制剂可特异性抑制D-3-磷酸甘油酸脱氢酶的活性，并通过降低该酶在癌细胞中的过表达延缓癌细胞的生长。它们单独或联用使用，或与其他抗癌药物组合应用，可治疗、预防或抑制乳腺癌、结肠癌、黑色素瘤及非小细胞肺癌等肿瘤疾病。

Description

D-3-磷酸甘油酸脱氢酶别构抑制剂及其应用 技术领域
本发明涉及治疗和预防由于丝氨酸代谢紊乱导致的各种疾病的药物，特别涉及作为D-3-磷酸甘油酸脱氢酶抑制剂的N’-取代苯甲酰肼类化合物，以及该化合物及其组合用药在治疗乳腺癌、结肠癌、黑色素瘤、非小细胞肺癌及其它疾病中的应用。
背景技术
人体中的D-3-磷酸甘油酸脱氢酶(PHGDH)催化丝氨酸合成第一步，是丝氨酸合成通路中的关键酶。PHGDH于2011年被证实在人类40％的黑色素瘤细胞或70％的三阴性乳腺癌细胞等过表达，进行PHGDH基因的敲除实验发现这些癌细胞的生长被大幅度抑制【(1)Locasale,J.W.,et al.(2011).Nat.Genet.43,869-874.(2)Possemato,R.,et al.(2011).Nature 476,346-350.】。因此，以PHGDH作为抗癌靶标进行药物设计具有广阔前景。由于PHGDH活性口袋体积较小、辅基NAD+在生物体内浓度高达0.3mM，PHGDH完整晶体结构至今未解出，使得基于PHGDH活性口袋的药物设计进展缓慢。新的思路是开展PHGDH的别构调控，设计PHGDH的别构抑制剂。
蛋白质中的别构调控指别构分子结合在蛋白的非活性位点而导致蛋白活性发生改变的现象。别构药物的优势在于其具有专一性，调控靶标蛋白活性而不完全丧失蛋白活性，且只在内源配体存在的情况下发挥效应等。
有文献指出：PHGDH基因的敲除与抗癌药物顺铂、阿霉素等联用可在体内及体外显著提高抗癌药物的生物活性【(3)Jing,Z.,et al.(2015)Cancer Biol.Ther.16,541-548.(4)Zhang,X.,and Bai,W.(2016)Cancer Chemother.Pharmacol.78,655-659.】，这为开展PHGDH抑制剂与抗癌药物联合使用提供了参考。迄今为止，未见PHGDH抑制剂进入临床研究的报道，也未见其与抗癌药物联合使用的药物效果被报道。针对PHGDH别构位点开展药物设计并将别构抑制剂用于肿瘤预防与治疗，具有新颖性和创造性。
发明内容
本发明的目的是提供作为PHGDH的别构抑制剂的化合物，应用于治疗和预防某些乳腺癌、结肠癌、黑色素瘤及非小细胞肺癌等疾病。
本发明的目的还在于提供上述化合物与其它PHGDH抑制剂或抗癌药物组合用药在制备治疗和预防某些乳腺癌、结肠癌、黑色素瘤及非小细胞肺癌等疾病药物中的应用。
本发明通过对PHGDH蛋白表面性质分析，发现适合小分子结合的潜在别构位点(参见图1)，并针对所预测位点MDL-1、MDL-2等进行虚拟筛选。
在本发明的第一方面，针对PHGDH潜在的别构位点MDL-1提供了一种苯甲酰肼类化合物作为PHGDH的别构抑制剂，这类化合物具有如下结构通式：
Figure PCTCN2016113476-appb-000001
式I中，R1、R2、R3、R4、R5、R6、R7相同或不同，各自独立代表氢、卤素、硝基、羟基、氨基或取代氨基、烷基、烷氧基、苄氧基或卤素取代烷基，或者，其中相邻的两个取代基(R1与R2、R2与R3、R4与R5、R5与R6和/或R6与R7)可以成环。
所述卤素包括F、Cl、Br和I。
所述取代氨基优选为C1～C12烷基取代氨基，更优选为C1～C6烷基取代氨基，例如甲胺基、乙胺基、二甲胺基、二乙胺基等。
所述烷基优选为C1～C12烷基，更优选为C1～C6烷基，例如甲基、乙基、丙基、异丙基、丁基等。
所述烷氧基优选为C1～C8烷氧基，更优选为C1～C4烷氧基，例如甲氧基、乙氧基、丙氧基等。所述卤素取代烷基优选一个或多个卤素取代的C1～C12烷基，更优选为一个或多个卤素取代的C1～C6烷基，例如三氟甲基等。
式I中，R1与R2、R2与R3、R4与R5、R5与R6和/或R6与R7成环时，联合代表1,3-丁二烯-1,4-二基，1,4-二丁基，与所在的苯环联合形成萘、四氢化萘等。
式I化合物可以通过下述方法制备得到：
Figure PCTCN2016113476-appb-000002
用取代苯甲酰肼与取代苯甲醛脱水缩合，得到相应的式I化合物。式I化合物的具体例子可参见实施例2。
在本发明的第二方面，针对PHGDH潜在的别构位点MDL-2提供了一种呋喃类化合物作为PHGDH的别构抑制剂，这类化合物具有如下结构通式：
Figure PCTCN2016113476-appb-000003
式II中，R1'、R2'、R3'相同或不同，各自独立代表氢、卤素、硝基、羟基、氨基、羧基、烷基、烷氧基、卤素取代烷基、羧酸酯基、磺酰胺基、酰胺基或N-烷基取代酰胺基，或者其中相邻的两个取代基(R1'和R2'或者R2'和R3')成环；R4'代表烷基、卤素取代烷基、氨基、环烷基、未取代或取代芳基；X是O、N或S。
所述卤素包括F、Cl、Br和I。
当R1'、R2'和R3'中的一个或多个为烷基时，优选为C1～C12烷基，更优选为C1～C6烷基，例如甲基、乙基、丙基、异丙基等；为烷氧基时，优选为C1～C8烷氧基，更优选为C1～C4烷氧基，例如甲氧基、乙氧基、丙氧基等；为卤素取代烷基时，优选为一个或多个卤素取代的C1～C12烷基，更优选为一个或多个卤素取代的C1～C6烷基，常为氟取代，例如三氟甲基。
当R1'、R2'和R3'中的一个或多个为所述羧酸酯基时，优选为C1～C8酯氧基(-COOCnH2n+1，n为1～7的整数)，更优选为C1～C4酯氧基，例如甲氧酯基、乙氧酯基、异丙氧酯基等。
当R1'、R2'和R3'中的一个或多个为所述N-烷基取代酰胺基时，优选为C1～C12烷基取代的酰胺基，更优选为C1～C6烷基取代的酰胺基，例如N-甲基酰胺基、N,N-二甲基酰胺基等。
当R1'和R2'或者R2'和R3'成环时，相邻的两个取代基联合代表1,3-丁二烯-1,4-亚基、1,4-二丁基等。
当R4'为烷基时，优选为C1～C12烷基，更优选为C1～C6烷基，例如甲基、乙基、丙基、异丙基等。
当R4'为卤素取代烷基时，优选为一个或多个卤素取代的C1～C12烷基，更优选为一个或多个卤素取代的C1～C6烷基，例如三氟甲基等。
当R4'为环烷基时，优选为C5～C7环烷基，例如环己基。
当R4'为未取代或取代芳基时，所述芳基优选为苯基，所述取代芳基优选为4-取代的苯基，苯基上的取代基优选为C1～C6烷基、卤素取代的C1～C6烷基、硝基、C1～C4烷氧基等，例如4-三氟甲基苯基、4-硝基苯基。
上述式II化合物可以通过下述方法制备得到：
Figure PCTCN2016113476-appb-000004
将取代呋喃醛与取代氨基脲(或取代氨基硫脲、或取代氨基胍)反应，得到式II所示的抑制剂。
式II化合物的具体例子可参见实施例3。
本发明合成路线所用的化学物质为市售产品或者可通过现有技术合成得到，进行反应时，所采用的操作方法和操作步骤以及反应条件和中间体等，都是依据本领域技术人员熟知的有机合成方法设计、实施的，并公开于实施例中。
本发明通过体外酶活测试、细胞活性测试和小鼠异种移植模型实验证实，式I与式II所示的化合物可特异性抑制PHGDH活性。通过式I或式II化合物别构抑制PHGDH，可降低PHGDH在癌细胞中的过表达，从而延缓癌细胞的生长。
将本发明的苯甲酰肼类化合物和呋喃类化合物单独或联合使用，或者与其它PHGDH抑制剂或抗癌药物组合用药，或者将它们的药用盐作为有效成分，添加常规药物载体，可制备用于治疗或预防各种癌症的药物。
本发明的苯甲酰肼类化合物和呋喃类化合物及其组合用药的药用盐是指药学上可接受的盐，例如与盐酸、硫酸、磷酸、硝酸等无机酸形成的盐，或是与柠檬酸、琥珀酸、枸橼酸、醋酸、酒石酸、甲磺酸等有机酸形成的盐。
常规药物载体指无毒固态、半固态或液态填充剂、稀释剂、佐剂、包裹材料或其他制剂辅料。根据本领域的公知技术，可以根据治疗目的、给药途径的需要将药物组合物制成各种剂型。
附图说明
图1显示了蛋白表面性质探测程序CAVITY预测的PHGDH的别构位点。
图2是本发明化合物与PHGDH的分子对接图，其中A：PKUMDL-WL-2101与MDL-1位点对接；BPKUMDL-WL-2201与MDL-2位点对接。
图3显示了实施例5中PKUMDL-WL-2101(A)以及PKUMDL-WL-2201(B)对乳腺癌细胞有丝分裂周期的影响。
图4显示了实施例6中PKUMDL-WL-2101(A、C、E)以及PKUMDL-WL-2201(B、D、F)在小鼠异种移植模型中的体内生物活性。
具体实施方式
以下实施例用于说明本发明，表示实践本发明的方法，其对本发明的范围无任何限制。本领域技术人员可能找到对于他们而言显而易见的实现本发明的其他方法，均应认为那些方法被包括在本发明的范围之内。
实施例1、PHGDH别构抑制剂的发现
一、PHGDH别构位点的预测
PHGDH(PDB code:2G76)别构位点的预测，使用的是蛋白表面探索程序CAVITY。首先，该程序通过擦除球方法对蛋白表面进行探测，发现蛋白表面潜在的结合位点；随后，程序根据经验公式(CavityScore＝(Volume-AdjustVolume)/(SurfaceArea-AdjustSurfaceArea))对蛋白结合小分子的能力进行打分。AdjustVolume和AdjustSurfaceArea与所预测位点中残基疏水面积和氢键受体给体数量相关。通过对已知结合位点-配体结合对的最大pKD进行打分，并与已知实验pKD值进行拟合，得到较好的线性相关性。根据pKD的数值以及口袋体积的大小，最终选取合适的潜在的别构位点。对于PHGDH，我们首次获得了两个全新的潜在的别构位点：MDL-1以及MDL-2。如图1所示，MDL-1位于活性位点以及辅基NAD+结合位点的附近，口袋体积为
Figure PCTCN2016113476-appb-000005
所预测的最大pKD为8.71。MDL-1与活性位点共用78位甘氨酸、79位缬氨酸、80位天冬氨酸、81位天冬酰胺以及82位缬氨酸。MDL-2位于底物结合结构域，口袋体积大小为
Figure PCTCN2016113476-appb-000006
所预测的最大pKD为7.79。
二、PHGDH别构分子的虚拟筛选
针对预测的别构位点，采用分子对接的方法，对SPECS数据库进行虚拟筛选。经人工挑选，购买化合物在体外酶活性测试中验证。
从MDL-1位点得到的IC50值小于50μM的化合物，即化合物(E)-2,4-二羟基-N'-(2-羟基-5-硝基亚苄基)苯甲酰肼，命名为PKUMDL-WL-2101。该化合物与PHGDH的对接结果见图2(A)。
从MDL-2位点得到的有活性的化合物(E)-2-氯-4-(5-((2-(乙基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸，命名为PKUMDL-WL-2201。该化合物与PHGDH的对接结果见图2(B)。
实施例2、针对MDL-1位点别构分子的合成
一、PKUMDL-WL-2101类似物的设计
分析见图2(A)，可见小分子与PHDGH的相互作用模式：两个苯环占据口袋中的疏水空腔，连酰基的苯环上，4位取代的羟基可与261位苯丙氨酸形成氢键；2位取代的羟基可与 264位谷氨酸形成氢键；酰肼链中的羰基氧与57位赖氨酸形成氢键，酰肼氮可与264谷氨酸形成氢键；另一个苯环上，3位的硝基可与134位精氨酸或55位丙氨酸形成氢键；该环还与周围带正电的空腔有静电作用。据此，我们优化了两个苯环的取代基团，设计出一系列PKUMDL-WL-2101类似物。
二、PKUMDL-WL-2101及类似物的合成
以(E)-2,4-二羟基-N'-(2-羟基-5-硝基亚苄基)苯甲酰肼(PKUMDL-WL-2101)为例，描述苯甲酰肼类PHGDH抑制剂的合成。
合成路线为：
Figure PCTCN2016113476-appb-000007
实验步骤为：
(1)将2,4-二羟基苯甲酸甲酯(1.781g,11.5mmol)溶于50mL甲醇溶液中，向其中加入85％的水合肼(2.031g,34.5mmol)，回流反应，TLC检测反应至原料消失。冷却，减压蒸馏除掉溶剂，残留物在甲醇中重结晶得到目标产物2,4-二羟基苯甲酰肼1.546g(白色固体，产率80％)，1H-NMR(400MHz,DMSO):6.35(2H,m),7.77(1H,d,J＝9.20Hz),10.06(1H,s),10.69(1H,s),11.86(1H,s)。
(2)将(1)所得产物2,4-二羟基苯甲酰肼(1.681g,10.0mmol)与5-硝基水杨醛(1.670g,10.0mmol)在50mL甲醇溶液中搅拌，常温反应至TLC检测反应至原料消失。减压蒸馏溶剂，残留物用甲醇中重结晶得到目标产物(E)-2,4-二羟基-N'-(2-羟基-5-硝基亚苄基)苯甲酰肼2.378g(黄色固体，产率：75％，熔点：296-298℃)，1H-NMR(DMSO):6.33(1H,d,J＝1.80Hz),6.39(1H,dd,J＝1.98,8.92Hz),7.12(1H,d,J＝8.99Hz),7.81(1H,d,J＝8.75Hz),8.18(1H,dd,J＝2.64,9.20Hz),8.57(1H,d,J＝2.57Hz),8.73(1H,s),10.26(1H,s),12.02(1H,s),12.15(1H,s),12.32(1H,s)；13C NMR(101MHz,DMSO-d6)δ165.32,162.95,162.55,162.19,144.51,139.85,129.84,126.47,123.83,119.78,117.06,107.58,105.83,102.81。质谱：ESI-MS测量值(理论值[M+H+])318.1(318.1)。
采用上述方法制备31个苯甲酰肼类化合物，新化合物的表征数据列入表1。核磁共振波谱数据(1H-NMR)由美国Varian Mercury 400M测得。使用含有四甲基硅烷为内标的氘代二甲基亚砜作为溶剂，耦合常数以Hz作为单位，所用缩写示意为：s＝单峰，d＝双峰，t＝三重峰，q＝四重峰，m＝多重峰，br＝单宽峰。熔点由北京泰克仪器有限公司X-4数字显示 显微熔点测定仪测得。
表1.苯甲酰肼类新化合物表征
Figure PCTCN2016113476-appb-000008
各编号对应的化合物名称为：
1)PKUMDLWL-2101：(E)-2,4-二羟基-N'-(2-羟基-5-硝基亚苄基)苯甲酰肼；
2)PKUMDLWL-2102：(E)-N'-(4-氟亚苄基)苯甲酰肼；
3)PKUMDLWL-2103：(E)-N'-亚苄基-2,4-二羟基苯甲酰肼；
4)PKUMDLWL-2104：(E)-2,4-二羟基-N'-(萘-1-亚甲基)苯甲酰肼；
5)PKUMDLWL-2105：(E)-2,4-二羟基-N'-(4-硝基亚苄基)苯甲酰肼；
6)PKUMDLWL-2106：(E)-N'-(2-羟基-5-硝基亚苄基)-4-硝基苯甲酰肼；
7)PKUMDLWL-2107：(E)-N'-(2-羟基-5-硝基亚苄基)-1-萘酚酰肼；
8)PKUMDLWL-2108：(E)-N'-(2-羟基-5-硝基亚苄基)-4-甲基苯甲酰肼
9)PKUMDLWL-2109：(E)-2,4-二羟基-N'-(4-羟基亚苄基)苯甲酰肼
10)PKUMDLWL-2110：(E)-2-羟基-N'-(2-羟基-5-硝基苄基)苯甲酰肼；
11)PKUMDLWL-2111：(E)-4-氟-N'-(2-羟基-5-硝基苄基)苯甲酰肼
12)PKUMDLWL-2112：(E)-2,4-二羟基-N'-(3-甲氧基亚苄基)苯甲酰肼
13)PKUMDLWL-2113：(E)-N'-(4-乙氧基-3-硝基亚苄基)-2,4-二羟基苯甲酰肼
14)PKUMDLWL-2114：(E)-2,4-二羟基-N'-(3-硝基亚苄基)苯甲酰肼
15)PKUMDLWL-2115：(E)-3-羟基-N'-(2-羟基-5-硝基苄基)苯甲酰肼
16)PKUMDLWL-2116：(E)-2,4-二羟基-N'-(4-甲氧基-3-硝基苄基)苯甲酰肼
17)PKUMDLWL-2117：(E)-2,4-二羟基-N'-(3-羟基亚苄基)苯甲酰肼
18)PKUMDLWL-2118：(E)-4-羟基-N'-(2-羟基-5-硝基苄基)苯甲酰肼
19)PKUMDLWL-2119：(E)-3-氯-N'-(2-羟基-5-硝基苄基)苯甲酰肼
20)PKUMDLWL-2120：(E)-N'-(2-羟基-5-硝基亚苄基)-3-硝基苯甲酰肼
21)PKUMDLWL-2121：(E)-4-氨基-N'-(2-羟基-5-硝基苄基)苯甲酰肼
22)PKUMDLWL-2122：(E)-N'-(2-羟基-5-硝基苄基)-2-甲基苯甲酰肼
23)PKUMDLWL-2123：(E)-4-甲氧基-N'-(2-羟基-5-硝基苄基)苯甲酰肼
24)PKUMDLWL-2124：(E)-4-(叔丁基)-N'-(2-羟基-5-硝基苄基)苯甲酰肼
25)PKUMDLWL-2125：(E)-4-溴-N'-(2-羟基-5-硝基苄基)苯甲酰肼
26)PKUMDLWL-2126：(E)-N'-(2-羟基-5-硝基亚苄基)-3-甲氧基苯甲酰肼
27)PKUMDLWL-2127：(E)-4-氯-N'-(2-羟基-5-硝基苄基)苯甲酰肼
28)PKUMDLWL-2128：(E)-N'-(2-羟基-5-硝基苄基)-4-(三氟甲基)苯甲酰肼
29)PKUMDLWL-2129：(E)-N'-(4-氯亚苄基)-2,4-二羟基苯甲酰肼
30)PKUMDLWL-2130：(E)-N'-(4-氯亚苄基)-2,4-二羟基苯甲酰肼
31)PKUMDLWL-2131：E)-N'-(4-溴亚苄基)-2,4-二羟基苯甲酰肼
32)PKUMDLWL-2132：(E)-2,4-二羟基-N'-(2-硝基亚苄基)苯甲酰肼
实施例3、针对MDL-2位点别构分子的合成
一、PKUMDL-WL-2201类似物的设计
从图2(B)可见小分子与PHGDH的相互作用模式：2-苯基呋喃芳环占据口袋中的疏水空腔，苯环中4位羧基氧可与11位丝氨酸、35位亮氨酸或34位天冬酰胺形成氢键；氨基硫脲基团则可能与PHGDH中的其它负电基团存在相互作用。我们通过分子等排等策略设计一系列化合物。
二、PKUMDL-WL-2201及类似物的合成
以(E)-2-氯-4-(5-((2-(乙基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸(PKUMDLWL-2201)为例，描述呋喃类PHGDH抑制剂分子的合成方法。
合成路线为：
Figure PCTCN2016113476-appb-000009
实验步骤为：
(1)将4-羧基-3-氯苯硼酸(1.342g,6.70mmol)，5-溴-2-糠醛(1.406g,8.04mmol)，TBAB(2.160g,6.70mmol),Pd(OAc)2(0.015g,0.07mmol)和K2CO3(1.420g,13.4mmol)加入到250mL圆底烧瓶中，加入100mL水。在Ar下保护下室温搅拌，TLC检测至呋喃原料点消失。用50mL×3乙酸乙酯萃取，水相加入3N HCl酸化，产生大量沉淀。过滤，收集固体，烘干得到目标产物2-氯-4-(5-甲酰基呋喃-2-基)苯甲酸(1.055g，黄色固体，63％)。1H-NMR(400MHz,DMSO):7.55(1H,d,J＝3.76Hz),7.70(1H,d,J＝3.76Hz),7.92(2H,m),8.05(1H,d,J＝1.28Hz),9.67(1H,s).
(2)将步骤(1)所得产物2-氯-4-(5-甲酰基呋喃-2-基)苯甲酸(0.100g,0.4mmol)与4-乙基-3-硫代氨基脲(0.048g,0.40mmol)在20mL甲醇溶液中室温搅拌反应，TLC检测至原料消失。减压蒸馏出去溶剂，残留物在甲醇中重结晶得到目标产物(E)-2-氯-4-(5-((2-(乙基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸0.126g(黄色固体，熔点：271-273℃，产率：90％)。1H-NMR(DMSO):1.18(3H,t,J＝7.08Hz),3.62(2H,m,J＝6.81Hz),7.13(1H,d,J＝3.65Hz),7.39(1H,d,J＝3.60Hz),7.87(2H,q,J＝9.09Hz),7.98(1H,s),8.01(1H,s),8.39(1H,t,J＝5.85Hz),11.54(1H,s),13.40(1H,s)；13C NMR(101MHz,DMSO-d6)δ176.46,166.09,151.84,150.43,133.19,132.89,131.84,131.18,129.57,125.36,122.15,115.10,111.42,38.32,14.51。HRMS(ESI):352.0(352.0，理论值[(M+H)+])。
采用上述方法制备另外30个呋喃类化合物，所合成化合物名称如下：
PKUMDL-WL-2202：(E)-N-乙基-2-((5-(4-(三氟甲基)苯基)呋喃-2-基)亚甲基)肼硫代甲酰胺；
PKUMDL-WL-2203：(E)-N-乙基-2-((5-(4-甲氧基苯基)呋喃-2-基)亚甲基)肼基-1-硫代甲酰胺；
PKUMDL-WL-2204：(E)-2-((5-(3-氯苯基)呋喃-2-基)甲基)-N-乙基肼基-1-硫代甲酰胺；
PKUMDL-WL-2205：(E)-4-(5-((2-(苯基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸；
PKUMDL-WL-2206：(E)-4-(5-((2-(甲基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸；
PKUMDL-WL-2207：(E)-N-乙基-2-((5-苯基呋喃-2-基)亚甲基)肼硫代甲酰胺；
PKUMDL-WL-2208：(E)-2-((5-(4-(叔丁基)苯基)呋喃-2-基)甲基)-N-乙基肼基-1-硫代甲酰胺；
PKUMDL-WL-2209：(E)-2-氯-5-(5-((2-(乙基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸；
PKUMDL-WL-2210：甲基(E)-4-(5-((2-(乙基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2211：(E)-N-乙基-2-((5-(对甲苯基)呋喃-2-基)亚甲基)肼硫代甲酰胺；
PKUMDL-WL-2212：甲基(E)-4-(5-((2-((4-硝基苯基)碳酰)肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2213：(E)-4-(5-((2-(环己基碳酰)肼基)甲基)呋喃-2-基)苯甲酸；
PKUMDL-WL-2214：(E)-N-乙基-2-((5-(萘-1-基)呋喃-2-基)亚甲基)肼硫代甲酰胺；
PKUMDL-WL-2215：甲基(E)-4-(5-((2-(2-(4-(三氟甲基)苯基)肼-1-羰)亚肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2216：(E)-N-乙基-2-((5-(4-氟苯基)呋喃-2-基)亚甲基)肼硫代甲酰胺；
PKUMDL-WL-2217：甲基(E)-2-氨基-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2218：(E)-2-((5-(4-溴苯基)呋喃-2-基)甲基)-N-乙基肼基-1-硫代甲酰胺；
PKUMDL-WL-2219：异丙基(E)-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2220：甲基(E)-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2221：(E)-2-((5-(4-氯苯基)呋喃-2-基)甲基)-N-乙基肼基-1-硫代甲酰胺；
PKUMDL-WL-2222：(E)-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)苯甲酸；
PKUMDL-WL-2223：甲基(E)-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)-3-甲基苯甲酸甲酯；
PKUMDL-WL-2224：甲基(E)-4-(5-((2-(乙基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2225：(E)-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)苯磺酰胺；
PKUMDL-WL-2226：(E)-4-(5-((2-(乙基氨基甲酰基)亚肼基)甲基)呋喃-2-基)苯甲酸；
PKUMDL-WL-2227：乙基(E)-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)苯甲酸甲酯；
PKUMDL-WL-2228：(E)-N-乙基-2-((5-(4-硝基苯基)呋喃-2-基)亚甲基)肼硫代甲酰胺；
PKUMDL-WL-2229：(E)-N-乙基-2-((5-(4-羟基苯基)呋喃-2-基)亚甲基)肼硫代甲酰胺；
PKUMDL-WL-2230：(E)-4-(5-((2-(肼羰)亚肼基)甲基)呋喃-2-基)-N-甲基苯甲酰胺；
PKUMDL-WL-2231：(E)-4-(5-((2-((4-(三氟甲基)苯基)碳酰)肼基)甲基)呋喃-2-基)苯甲酸；新化合物的表征数据列入表2。
表2.呋喃类新化合物表征
Figure PCTCN2016113476-appb-000010
Figure PCTCN2016113476-appb-000011
Figure PCTCN2016113476-appb-000012
Figure PCTCN2016113476-appb-000013
实施例4、荧光动力学方法测定化合物的PHGDH体外酶活性
PHGDH酶活性的测定是通过检测NADH在456nm处的荧光发射谱而实现的。首先，PHGDH(终浓度30ng/μL)在96孔板中与HEPES缓冲液(25mM，pH 7.1，400mM KCl),5μM PLP,0.5mMαKG,150μM NADH以及PSAT1(终浓度30ng/μL)孵育10分钟。随后，加入10μL DMSO(对照组)或者含小分子的DMSO溶液，在25℃下以550rpm的转速震荡平衡5分钟。酶活体测试体系中保持DMSO的终浓度(v/v)为5％。最后，加入Pser水溶液(终浓度0.5mM)，启动反应，并用紫外可见酶标仪监测456nm下NADH的消耗量随时间的变化。使用30s以内反应初速率对蛋白活性进行评估，此时NADH消耗随时间增长呈线性。对63个化合物初步测试了酶活性抑制率％，并对50μM时酶活性抑制率大于50％的化合物做深入研究得到IC50数值，如表3所示。
表3.化合物的IC50数值
编号 IC50(μM) 编号 IC50(μM)
PKUMDL-WL-2101 34.8±3.6 PKUMDL-WL-2222 16.7±1.6
PKUMDL-WL-2128 36.1±4.2 PKUMDL-WL-2225 38.9±1.2
PKUMDL-WL-2201 35.7±8.6 PKUMDL-WL-2226 35.5±1.1
PKUMDL-WL-2212 29.8±9.4 PKUMDL-WL-2228 37.0±2.8
PKUMDL-WL-2220 8.9±1.8 PKUMDL-WL-2229 30.3±4.5
实施例5、化合物的癌细胞抑制活性
对化合物在细胞水平上的生物活性进行了研究。选用一系列的癌细胞以及正常乳腺上皮 细胞，采用MTT(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide)的实验方法。具体做法是：首先，将处于指数期生长的PHGDH敏感的乳腺癌细胞MDA-MB-468(5000细胞/孔)及HCC70(5000细胞/孔)，PHGDH不敏感的乳腺癌细胞MCF-7(3000细胞/孔)、MDA-MB-231(2000细胞/孔)、ZR-75-1(4000细胞/孔)，结肠癌细胞DLD-1(2000细胞/孔)及正常乳腺上皮细胞MCF-10A(3000细胞/孔)转移至96孔板中，过夜贴壁。然后，加入不同浓度的化合物至96孔板中，与细胞孵育72小时，控制DMSO终浓度(v/v)为0.2％。不含任何化合物的DMSO作为对照。随后，72小时后，向各个实验孔中加20μL 5mg/mL MTT，孵育至少4小时后，移去各个实验孔中的液体，加入200μL DMSO，37℃缓慢摇晃10分钟，使用酶标仪检测490nm处的可以光吸收。实验数据使用细胞存活率％来表示，细胞半数致死率EC50值由Hill方程拟合得到。
PKUMDL-WL-2101与PKUMDL-WL-2201在细胞水平上表现出微摩尔的抑制活性(见表4)。其对PHGDH敏感的乳腺癌细胞MDA-MB-468以及HCC70表现出的EC50值分别为7.70以及10.8μM，对PHGDH不敏感的乳腺癌细胞MDA-MB-231，ZR-75-1以及MCF-7表现出的EC50值分别为27.7，83.4以及139μM，对结肠癌细胞表现出的EC50值为18.3μM。同时，PKUMDL-WL-2101表现出微弱的细胞毒性，对MCF-10A细胞表现出的EC50值为45.8μM。PKUMDL-WL-2201对PHGDH敏感的乳腺癌细胞MDA-MB-468以及HCC70表现出的EC50值分别为6.9以及10.0μM，对PHGDH不敏感的乳腺癌细胞MDA-MB-231，ZR-75-1以及MCF-7表现出的EC50值分别为>200，125以及>200μM，对结肠癌细胞表现出的EC50值为167μM。同样，PKUMDL-WL-2201表现出微弱的细胞毒性，对MCF-10A细胞表现出的EC50值分别为64.7μM。
表4.PKUMDL-WL-2101以及PKUMDL-WL-2201的癌细胞致死活性
Figure PCTCN2016113476-appb-000014
通过将处于指数生长周期的MDA-MB-468细胞(3×105细胞/孔)转移至6孔板中，过夜贴壁加入不同浓度的化合物孵育24小时后，胰酶消化，离心，70％预冷的乙醇固定，PBS冲洗，离心，重悬(0.5％triton-x-100,50μg/ml PI以及50μg/ml DNase-free RNase的PBS)，37℃避光30分钟，使用流式分析仪分析结果表明，PKUMDL-WL-2101以及PKUMDL-WL-2201均可使细胞周期停滞在G0/G1期(见图3)，这可能是化合物造成癌细胞死亡的主要原因。
以上实验结果表明，PKUMDL-WL-2101和PKUMDL-WL-2201对PHGDH敏感的乳腺癌细胞表现出较好的细胞致死活性。
实施例6、化合物在小鼠异种移植模型中的生物活性效果
1.PKUMDL-WL-2101或PKUMDL-WL-2201单独给药实验方案及结果
首先，将MDA-MB-468(2×105)细胞注射至NOD.CB17-Prkdcscid/J小鼠(6-8周)的第四乳房脂肪垫中，待肿瘤平均体积长至30mm3，将小鼠随机分成8组，每组5只小鼠。随后，开始给药，对照组给药为溶解化合物的溶剂(10％DMSO,20％EL and 70％PBS)，给药剂量均为20mg/kg/day。每两天检测肿瘤体积大小，肿瘤体积通过如下公式计算得到。
短径2(mm)×长径(mm)×0.5
实验结果如图4所示，可以看出，相较于对照组，无论是PKUMDL-WL-2101给药组还是PKUMDL-WL-2201给药组，小鼠体内的肿瘤生长被显著抑制(见图4中A-B)。实验过程中小鼠均保持较好的生长状态(见图4中C-D)。实验结果具有统计显著性，P值小于0.05。
2.PKUMDL-WL-2201与阿霉素组合给药实验方案及结果
首先，将MDA-MB-468(2x 105)细胞注射至NOD.CB17-Prkdcscid/J小鼠(6-8周)的第四乳房脂肪垫中，待肿瘤平均体积长至150mm3，将小鼠随机分成5组，每组5只小鼠。随后，开始给药，第一组小鼠为对照组，只注射溶解PKUMDL-WL-2201以及溶解阿霉素的溶剂(10％DMSO,20％EL和70％PBS)；第二组小鼠仍为对照组，只注射溶解阿霉素的溶剂(10％DMSO,20％EL和70％PBS)；第三组至第五组小鼠为实验组，给药方式分别为，阿霉素给药(2.5mg/kg/4day)，PKUMDL-WL-2201(20mg/kg/day)给药以及PKUMDL-WL-2201(20mg/kg/day)与阿霉素(2.5mg/kg/4day)组合给药。之后，每两天监测肿瘤体积生长曲线以及小鼠生存曲线，肿瘤大小由卡尺测量。肿瘤体积仍由上述公式计算得到。
因抗癌药物阿霉素具有较强的细胞毒，实验组的小鼠在给药开始后的13天开始死亡，因此组合用药的实验结果只记录到给药开始后的11天。相较于PKUMDL-WL-2201或者阿霉素单独用药，PKUMDL-WL-2201与阿霉素的组合用药可以显著抑制小鼠体内的肿瘤生长，并与阿霉素单独用药时产生的肿瘤生长抑制效果存在显著性差异。组合用药的最大药效发生在给药开始后的第六天，相较于对照组，其对肿瘤生长的抑制效果达到41％(见图4中E-F)。
综合酶活性测试、细胞实验和小鼠异种移植模型实验结果，本发明的化合物可特异性抑制PHGDH活性。

Claims (16)

  1. 式I所示化合物及其药用盐在制备用于治疗、预防或抑制肿瘤的药物中的用途：
    Figure PCTCN2016113476-appb-100001
    其中，R1、R2、R3、R4、R5、R6、R7相同或不同，各自独立代表氢、卤素、硝基、羟基、
    氨基或取代氨基、烷基、烷氧基、苄氧基或卤素取代烷基，或者，其中的R1与R2、R2与R3、R4与R5、R5与R6、和/或R6与R7成环。
  2. 如权利要求1所述的用途，其特征在于，所述取代氨基为C1～C12烷基取代氨基。
  3. 如权利要求1所述的用途，其特征在于，所述烷基为C1～C12烷基；所述烷氧基为C1～C8烷氧基。
  4. 如权利要求1所述的用途，其特征在于，所述卤素取代烷基为一个或多个卤素取代的C1～C12烷基。
  5. 如权利要求1所述的用途，其特征在于，所述R1与R2、R2与R3、R4与R5、R5与R6、和/或R6与R7成环是指，相邻的两个取代基连接成1,3-丁二烯-1,4-二基或1,4-二丁基。
  6. 如权利要求1所述的用途，其特征在于，所述式I所示化合物是下列化合物PKUMDL-WL-2101至PKUMDL-WL-2132之一：
    Figure PCTCN2016113476-appb-100002
    Figure PCTCN2016113476-appb-100003
  7. 式II所示化合物及其药用盐在制备用于治疗、预防或抑制肿瘤的药物中的用途：
    Figure PCTCN2016113476-appb-100004
    式II中，R1'、R2'、R3'相同或不同，各自独立代表氢、卤素、硝基、羟基、氨基、羧基、烷基、烷氧基、卤素取代烷基、羧酸酯基、磺酰胺基、酰胺基或N-烷基取代酰胺基，或者其中相邻的两个取代基成环；R4'代表烷基、卤素取代烷基、氨基、环烷基、芳基或取代芳基；X是O、N或S。
  8. 如权利要求7所述的用途，其特征在于，当R1'、R2'和R3'中的一个或多个为烷基时，所述烷基为C1～C12烷基；当R1'、R2'和R3'中的一个或多个为烷氧基时，所述烷氧基为C1～C8烷氧基；当R1'、R2'和R3'中的一个或多个为卤素取代烷基时，所述卤素取代烷基为一个或多个卤素取代的C1～C12烷基；当R1'、R2'和R3'中的一个或多个为羧酸酯基时，所述羧酸酯基为C1～C8酯氧基；当R1'、R2'和R3'中的一个或多个为N-烷基取代酰胺基时，所述N-烷基取代酰胺基为C1～C12烷基取代的酰胺基。
  9. 如权利要求7所述的用途，其特征在于，当R1'和R2'或者R2'和R3'成环时，相邻的两个取代基联合代表1,3-丁二烯亚基或1,4-二丁基。
  10. 如权利要求7所述的用途，其特征在于，当R4'为烷基时，所述烷基为C1～C12烷基；当R4'为卤素取代烷基时，所述卤素取代烷基为一个或多个卤素取代的C1～C12烷基；当R4'为环烷基时，所述环烷基为C5～C7环烷基。
  11. 如权利要求7所述的用途，其特征在于，当R4'为芳基或取代芳基时，所述芳基为苯基；所述取代芳基为4-取代的苯基。
  12. 如权利要求11所述的用途，其特征在于，所述4-取代的苯基的4-位上的取代基为C1～C6烷基、卤素取代的C1～C6烷基、硝基或C1～C4烷氧基。
  13. 如权利要求1所述的用途，其特征在于，所述式II所示化合物是下列化合物PKUMDL-WL-2201至PKUMDL-WL-2231之一：
    Figure PCTCN2016113476-appb-100005
    Figure PCTCN2016113476-appb-100006
    Figure PCTCN2016113476-appb-100007
  14. 如权利要求1或7所述的用途，其特征在于，所述肿瘤为乳腺癌、结肠癌、黑色素瘤或非小细胞肺癌。
  15. 权利要求1中式I所示化合物或权利要求7中式II所示化合物或它们的药用盐在制备D-3-磷酸甘油酸脱氢酶的抑制剂中的应用。
  16. 权利要求1中式I所示化合物与权利要求7中式II所示化合物或它们的药用盐联合用于制备治疗、预防或抑制肿瘤的药物的用途。
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US20100035932A1 (en) * 2008-08-07 2010-02-11 Schepetkin Igor A Novel formyl peptide receptor like 1 agonists that induce macrophage tumor necrosis factor alpha and computational structure-activity relationship analysis of thereof
WO2011069039A1 (en) * 2009-12-04 2011-06-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Hydrazone and diacyl hydrazine compounds and methods of use
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WO2011069039A1 (en) * 2009-12-04 2011-06-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Hydrazone and diacyl hydrazine compounds and methods of use
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