CN116675731A - Small molecule conjugate of targeted cyclin-dependent kinase 12/13 and application thereof - Google Patents

Small molecule conjugate of targeted cyclin-dependent kinase 12/13 and application thereof Download PDF

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CN116675731A
CN116675731A CN202310479552.4A CN202310479552A CN116675731A CN 116675731 A CN116675731 A CN 116675731A CN 202310479552 A CN202310479552 A CN 202310479552A CN 116675731 A CN116675731 A CN 116675731A
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formula
small molecule
compound shown
molecule conjugate
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曹戟
朱成梁
杨波
何俏军
应美丹
邵雪晶
蒋莉
陈丹妮
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Zhejiang University ZJU
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Zhejiang University ZJU
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The invention discloses a small molecule conjugate for degrading CDK12/13, a preparation method and application thereof, and the structure is as follows: T-L-E, wherein T represents ligand of mother nucleus molecule CDK12/13 protein, L represents connecting part of T and E, also called Linker, E represents ligand of E3 ligase. The compound shown in the formula I can be synthesized through amide condensation reaction, substitution reaction or click reaction between the derivative of the T part and the E3 enzyme ligand and the derivative thereof. The compound shows high-efficiency CDK12/13 degradation activity on various cell lines, high-specificity tumor cell proliferation inhibition activity and high-efficiency treatment effect when other antitumor drugs are combined. In biological meaning and treatment, can be used for lead compounds for treating various cancers and other CDK12/13 abnormal diseases.

Description

Small molecule conjugate of targeted cyclin-dependent kinase 12/13 and application thereof
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a small molecule conjugate targeting cyclin dependent kinase 12/13 (CDK 12/13) and application thereof.
Background
Cyclin-dependent kinases (cyclin-dependent kinases, CDKs) are major mediators of various cell cycle processes and are divided into two major classes: one class is associated with the cell cycle, such as CDK1,2,4,6, which directly regulate cell cycle progression; the other class is related to gene transcription, such as CDK7,8,9, 11, 12, 13, which mainly regulates the gene transcription process. It has been found that CDKs are often dysfunctional in tumor cells and thus are promising therapeutic targets for these tumors.
Of these, CDK12 and CDK13 are transcriptional-related CDKs that form complexes with Cyclin K (Cyclin K) and are involved in regulating multiple stages of the transcription process of genes such as transcriptional elongation, mRNA splicing, transcription termination, etc., by mediating serine phosphorylation of the C-terminal domain (RNAPII CTD) of RNA polymerase II. Recent studies have found that CDK12/13, which is a specific transcription regulator of DNA damage homologous recombination repair genes, promotes the process of intron polyadenylation and plays an important role in maintaining the genomic stability of cells. Most of the kinase functions of CDK12/13 compensate for each other and inhibition or silencing of both at the same time results in accumulation of intracellular DNA damage and ultimately cell death.
The tumor cells need to carry out a large amount of DNA replication in the process of rapid amplification, so that DNA damage and instability of genome are increased, and CDK12/13 is more dependent on the function of CDK12/13 than normal cells, and obvious gene amplification exists in various tumor cells, so that CDK12/13 has been clearly taken as an effective treatment target for diseases such as breast cancer, lymphoma and the like. Drug development against CDK12/13 not only helps to delve into its biological regulatory mechanisms, but also can be a powerful strategy for tumor treatment. However, CDK12/13 inhibitors are currently in preclinical research. Because the homologous protein CDKs have highly similar kinase activity domains, all the developed small molecule inhibitors can indiscriminately inhibit a plurality of CDK activities, and meanwhile, because the small molecule inhibitors lack tissue and cell targeting, the small molecule inhibitors have great toxic and side effects on normal cells.
PROTAC is an advanced protein degradation technology, and can utilize a naturally occurring protein degradation system in a body to reduce protein level instead of inhibiting protein function and play a role in treating diseases. The principle is that the ubiquitination of the target protein is realized by approaching the distance between the target protein and E3 ubiquitin ligase, and the degradation of the target protein is specifically induced, so that the aim of treating diseases is fulfilled. Through continuous perfection and development for 20 years, PROTAC has become a powerful means of drug development. Due to subtle differences in intracellular environment between different cell lines, the pro tac mediated formation of the target protein-pro tac-E3 ligase ternary complex is complicated and the like, and pro tac function is often accompanied by selective degradation or proliferation inhibitory activity of the cell lines. Therefore, the rational design and development of novel CDK12/13 degradants to achieve tissue or cell selective effects, and the research of therapeutic and pharmacological mechanisms for the related diseases remains to be explored further.
Disclosure of Invention
The primary object of the invention is to provide a small molecule conjugate which can efficiently degrade CDK12/13 and selectively inhibit tumor cell proliferation. Unlike conventional inhibitors and degradants targeting CDK12/13, the small molecule conjugates provided herein efficiently degrade target proteins while having highly specific tumor cell line proliferation inhibitory activity, exhibiting properties and advantages that are significantly different from those of the prokaryotic molecule (the parent molecule T in the following formula, hereinafter also referred to as such) at both the molecular and cellular level.
The structural formula of the small molecule conjugate for degrading CDK12/13 provided by the invention is shown in a formula I or pharmaceutically acceptable salts, co-crystals, tautomers, stereoisomers, solvates, hydrates, polymorphs, isotopically enriched derivatives or prodrugs thereof:
T-L-E
i is a kind of
In the formula I, T represents a ligand of CDK12/13 protein, which is also called a parent nucleus molecule, E represents a ligand of E3 ligase, and L represents a connecting part or a linking chain of T and E, which is also called a Linker.
Specifically, T is selected from a CDK12/13 inhibitor and derivatives thereof, and the structural group is shown as a formula II.
Or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative or prodrug thereof, wherein:
R in formula II 1 One (n is not 0) or two (n is 0); each R 1 Each independently is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally when there are two R 1 When two R 1 Forms a substituted or unsubstituted heterocyclic group or a substituted or unsubstituted heteroaryl group together with the intermediate atom (N) thereof;
preferably, R 1 Is specifically composed of ethyl,Or a nitrogen protecting group;
R 1-a there may be a plurality or one (R when w is 0 1-a Not present), each R 1-a Each independently is halogen,An optionally substituted acyl group, an optionally substituted alkyl group;
w is 0, 1, 2, 3, 4, 5 or 6.
R in formula II 2 Is halogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, -CN, -OR D1 、-N(R D1a ) 2 or-SR D1 The method comprises the steps of carrying out a first treatment on the surface of the Wherein R is D1 Is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom;
Wherein each R is D1a Each independently is hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally, two R D1a Forms together with the intermediate atom (C) thereof a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring;
R 1 、R 2 in (a) and (b); the acyl is preferably a C1-C6 acyl; the alkyl is preferably a C1-C6 alkyl; the alkyl is preferably a C1-C6 alkenyl; the preferable C1-C6 alkynyl; the carbocyclyl is 3-8 membered carbocyclyl; the heterocyclic group is 3-8 membered heterocyclic group; the aryl is 5-10 membered aryl; the heteroaryl is a 5-10 membered heteroaryl;
the nitrogen protecting group comprises tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl and p-methoxybenzyl. The oxygen protecting group comprises dihydropyran, methoxymethyl, benzyl, methoxybenzyl, trimethylsilyl, and tert-butyldimethylsilyl. The sulfur protecting group comprises acetamidomethyl, phenylacetylaminomethyl and tert-butylthio.
The carbocyclyl may be a 3-8 membered alkyl ring or a 3-8 membered carbocyclyl with one or two double bonds and a triple bond.
The optional substitution includes the case where there is no substitution or there is substitution, where there is substitution, the substituent may be one or more, and each of the plurality of substituents is independently selected from one or more of hydroxyl, cyano, halogen, nitro, C1-C4 alkyl, C1-C4 alkoxy, 3-6 membered cycloalkyl, 3-8 membered heterocyclyl, aryl, heteroaryl, and the like.
Preferably, R 2 is-CN.
Rx and Ry in formula II are each independently CH or N;
n in formula II is 0, 1, 2, 3, 4 or 5; when n is 0, the ring A has an open-loop chain structure, that is, it becomes:
two R in the formula (II-0-2) 1 Independent of each other, may be the same or different.
Further, the specific structure of T may be any of the following groups:
specific E may be selected from 3-amino-N- (2, 6-dioxo-3-piperidyl) phthalimide group (Pomalidomide group), 3- (7-amino-3-oxo-1H-isoindol-2-yl) piperidine-2, 6-dione group (Lenalidomide group), thalidomide group (Thalidomide group), (2S, 4R) -1- ((S) -2- (3-aminopropionamido) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide group (VH 032 group), [ (4R, 5S) -4, 5-bis (4-chlorophenyl) -2- [4- (1, 1-dimethylethyl) -2-ethoxyphenyl ] -4, 5-dihydro-4, 5-dimethyl-1H-imidazol-1-yl ] [4- [3- (methylsulfonyl) propyl ] -1-piperazinyl ] pentanoyl group (RG-12-phenylimide group) and the like;
The said certain group refers to the structure of the group formed by removing the leaving H or other leaving groups in the compound corresponding to the group. For example, taking the 3-amino-N- (2, 6-dioxo-3-piperidyl) phthalimide group as an example, this refers to the structure of the compound 3-amino-N- (2, 6-dioxo-3-piperidyl) phthalimide with the leaving H or other leaving group being removed.
As an embodiment, E may be selected from one of the following groups:
in the formulae III-a to III-d, III-a-0 to III-d-0, R 1 Is NH or O or CH 2 ；
In the formula III-e, R 1 =h or OH, R 2 =h or CH 3 The method comprises the steps of carrying out a first treatment on the surface of the In the formula III-f described above,wherein R is 3 ＝-CN，-F，-COCH 3 ，R 2 =h or CH 3 The method comprises the steps of carrying out a first treatment on the surface of the In the formula III-g, R 1 =h, CN, F, cl, br, or formula: />In said formula III-h, -/-, is->Wherein R is 3 ＝-CN，-F，-COCH 3 ；R 2 Is H or CH 3 The method comprises the steps of carrying out a first treatment on the surface of the In the formula III-i described above,wherein R is 3 ＝-CN，-F，-COCH 3 ；R 2 Is H or CH 3 The method comprises the steps of carrying out a first treatment on the surface of the In the formula III-l, X=N or CH.
Specifically, L is a unit of 1-30 atoms in length and of any type;
more specifically, the L includes, but is not limited to, the following structure, or any structural group consisting of the following structures:
such as units of ethylene glycol structure:or a unit consisting of a fatty chain: Or units consisting of unsaturated chains: />Or a unit consisting of a non-aromatic heterocycle: />Or a unit consisting of an aromatic compound:-CH 2 -、-(C＝O)-、/> or a heteroatom-containing group that is sulfur, nitrogen or phosphine, including but not limited to the following fragment units: /> R in the heteroatom-containing units is independently: alkyl, alkoxy or hydrogen; preferably, the alkyl is specifically methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl; the alkoxy is-OEt, -OMe.
Preferably, the L is selected from the following structures:
preferably, the L is selected from the following structures:
in the above structural formula, each X, Y, Z, V, W is independently selected from N, CH. Further preferably, X, Y, Z is not simultaneously CH.
Preferably, m 1 0,1,2,3,4,5,6, m 2 0,1,2,3,4,5,6.
Specifically, the structural formula of the small molecule conjugate is shown in any one of the formulas IV-X:
in formula IV, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, A is CH 2 NH, O or CO, G being absent, CH 2 NH, O, CONH or NHCO, B is CO or CH 2 X, Y, Z are each independently CH or N, R 1 N is defined as formula II; as one embodiment, G is O;
in the formula V, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, A is CH 2 NH, O or CO, G being absent, CH 2 NH, O, CONH or NHCO, B is CO or CH 2 X, Y are each independently CH or N, R 1 N is defined as formula II; as one embodiment, G is absent when Y is N; when Y is CH, G is a heteroatom (NH, O, etc.);
in formula VI, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15A is CH 2 NH, O or CO, G being absent, CH 2 NH, O, CONH or NHCO, B is CO or CH 2 X, Y, Z are each independently CH or N, V, W are each independently CH or N, R 1 N is defined as formula II; as one embodiment, G is absent when W is N; when W is CH, G is a heteroatom (NH, O, etc.);
in formula VII, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; rx is H or CH 3 X, Y, Z are each independently CH or N, R 1 N is defined as formula II; as one embodiment, G is O;
in formula VIII, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO, rx is H or CH 3 X, Y is independently CH or N, R 1 N is defined as formula II; as one embodiment, G is absent when Y is N; when Y is CH, G is a heteroatom (NH, O, etc.);
In formula IX, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; rx is H or CH 3 X, Y, Z is independently CH or N, V, W is independently CH or N, R 1 N is defined as formula II; as one embodiment, G is absent when W is N; when W is CH, G is a heteroatom (NH, O, etc.);
in the formula X, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; a is CH 2 Or CO, X, Y, Z are each independently CH or N,
R 1 n is defined as formula II; as one embodiment, G is O;
in formula XI, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; a is CH 2 Or CO, X, Y are each independently CH or N; r is R 1 N is defined as formula II; as one embodiment, G is absent when Y is N; when Y is CH, G is a heteroatom (NH, O, etc.);
in formula XII, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; a is CH 2 Or CO, X, Y, Z are each independently CH or N, V, W are each independently CH or N, R 1 N is defined as formula II; as one embodiment, G is absent when W is N; when W is CH, G is a heteroatom (NH, O, or the like).
As a specific embodiment, the small molecule conjugate has a structure as shown in formula IX, wherein n=0; r is R 1 Is benzyl; x is CH; at least one of Y and Z is N, and when the other is not N, the other is CH; g is absent; m is m 1 Is 0; m is m 2 0,1,2, rx is methyl.
More specifically, the small molecule conjugates are compounds represented by the following formulas 1 to 138:
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it is another object of the present invention to provide a method for preparing a small molecule conjugate according to any one of the above-mentioned embodiments (preferably, any one of the structures of formula I, formula IV-formula XII, and formulas 1 to 86).
Taking the structure shown in the formula I as an example, the compound shown in the formula I (or any one of the structures shown in the formulas IV-XII and formulas 1-86) provided by the invention can be synthesized through amide condensation reaction, substitution reaction, click reaction or cross coupling reaction between the corresponding ligand of E3 ligase (such as Pomalidomide derivative, VHL ligand derivative and MDM2 ligand derivative) and the corresponding T part derivative.
Specifically, the preparation method of the small molecule conjugate shown in the formula IV (taking G as O, A and NH, X as CH and Y as N, Z and CH as an example) comprises the following steps: obtaining a small molecule conjugate shown in a formula IV through nucleophilic substitution reaction between a CDK12/13 ligand end derivative (shown in a formula a) and a Pomalidomide end derivative (shown in a formula b);
Wherein R in formula a 1 、m 1 、m 2 N is defined as formula IV, and B is defined as formula IV;
the small molecule conjugate of formula V (G is CH 2 A is NH and X is N, Y is N), comprising the steps of: carrying out nucleophilic substitution reaction between CDK12/13 ligand end derivative (shown in formula c) and Pomalidomide end derivative (shown in formula b) to obtain small molecule conjugate shown in formula V;
wherein R in formula c 1 、m 1 、m 2 N is defined as formula V;
the small molecule conjugate of formula VI (G is CH 2 A is NH, X is CH, Y is N, Z is CH, V is N, W is N), comprising the steps of: nucleophilic substitution reaction is carried out between CDK12/13 ligand end derivative (shown as formula d) and Pomalidomide end derivative (shown as formula b), so as to obtain the small molecule conjugate shown as formula VI;
wherein R in formula d 1 、m 1 、m 2 N is as defined for formula VI;
a method for preparing a small molecule conjugate of formula VII (G O, X CH and Y N, Z CH) comprising the steps of: performing condensation reaction between CDK12/13 ligand end derivative (shown as formula e) and VHL ligand derivative (shown as formula f) to obtain a small molecule conjugate shown as formula VII;
wherein R in formula e 1 、m 1 、m 2 N is defined as formula VII;
the small molecule conjugate of formula VIII (G is CH 2 X is N, Y N), comprising the following steps: performing condensation reaction between CDK12/13 ligand end derivative (shown as formula g) and VHL ligand derivative (shown as formula f) to obtain a small molecule conjugate shown as formula VIII;
wherein R in formula g 1 、m 1 、m 2 N is defined as formula VIII, and Rx in formula f is defined as formula VIII;
the preparation method of the small molecule conjugate (G is empty, X is CH, Y is N, Z CH, V is N, W N) shown in the formula IX comprises the following steps: performing condensation reaction between CDK12/13 ligand end derivative (shown as formula h) and VHL ligand derivative (shown as formula f) to obtain a small molecule conjugate shown as formula X;
wherein R in formula h 1 、m 1 、m 2 N is defined as formula IX, and Rx in formula f is defined as formula IX;
the preparation method of the small molecule conjugate (G is O, X CH, Y is N, Z CH and A is CO) shown in the formula X comprises the following steps: performing condensation reaction between CDK12/13 ligand end derivative (shown in formula e) and MDM2 ligand derivative (shown in formula j) to obtain a small molecule conjugate shown in formula X;
wherein, in the small molecule conjugate shown in the formula X, R in the formula e 1 、m 1 、m 2 N is defined as formula X;
the small molecule conjugate shown in formula XI (G is CH 2 N, Y for X N, A for CO), comprising the steps of: performing condensation reaction between CDK12/13 ligand end derivative (shown in formula g) and MDM2 ligand derivative (shown in formula j) to obtain a small molecule conjugate shown in formula XI;
Wherein R in formula g 1 、m 1 、m 2 N is defined as in formula XI.
The small molecule conjugate of formula XII (G is CH 2 X is CH, Y is N, Z is CH, V is N, W is N, A is CO), comprising the steps of: performing condensation reaction between CDK12/13 ligand end derivative (shown as formula h) and MDM2 ligand derivative (shown as formula j) to obtain a small molecule conjugate shown as formula XII;
wherein R in formula h 1 、m 1 、m 2 N is as defined in formula XII.
The CDK12/13 ligand-end derivatives (represented by formula a, formula c, formula d, formula e, formula g, formula h) are also within the scope of the present invention. The CDK12/13 ligand-end derivatives (represented by formula a, formula c, formula d, formula e, formula g, formula h) are synthesized as follows:
when n is 0 in formula II: the compounds corresponding to the formula a, the formula c, the formula d, the formula e, the formula g and the formula h are structures shown as the formula a0, the formula c0, the formula d0, the formula e0, the formula g0 and the formula h 0:
1) CDK12/13 ligand end derivative (formula a 0) is obtained by coupling the ligand end derivative shown in formula 1a with bisboronic acid pinacol ester to obtain a compound shown in formula 2 a; nucleophilic reaction of 5-bromo-2-fluoropyridine (formula 3 a) with a compound shown in formula 4a to obtain a compound shown in formula 5 a; removing protecting groups from the compound shown in the formula 2a and the compound shown in the formula 5a after coupling reaction to obtain a compound shown in the formula a 0;
The preparation method of the compound shown in the formula 1a comprises the following steps: coupling 1-bromo-4-iodobenzene (formula 1 aa) with trans- (4-aminocyclohexyl) carbamic acid tert-butyl ester (formula 2 aa) to obtain ((1 r,4 r) -4- ((4-bromophenyl) amino) cyclohexyl) carbamic acid tert-butyl ester (formula 3 aa), subjecting a compound shown in formula 3aa and benzyl isocyanate (formula 4 aa) to nucleophilic reaction to obtain and then deprotecting to obtain 1- ((1 r,4 r) -4-aminocyclohexyl) -3-benzyl-1- (4-bromophenyl) urea (formula 5 aa), and subjecting a compound shown in formula 5aa and 6-fluoronicotinonitrile (formula 6 aa) to nucleophilic reaction to obtain a compound shown in formula 1 a;
2) A CDK12/13 ligand end derivative (shown as a formula c 0) is prepared by coupling reaction of a formula 1a and tert-butyl 1-piperazine carboxylate (shown as a formula 1 c) and deprotection to obtain a formula 3c; carrying out nucleophilic substitution reaction on the compound shown in the formula 2c and the compound shown in the formula 3c, and removing a protecting group to obtain a compound shown in the formula c 0;
3) CDK12/13 ligand-end derivatives (formula d 0): carrying out nucleophilic substitution reaction on the compound shown in the formula 3a and the compound shown in the formula 1c to obtain a compound shown in the formula 1d, and carrying out coupling reaction on the compound shown in the formula 1d and the compound shown in the formula 2a and removing a protecting group to obtain a compound shown in the formula 2 d; carrying out nucleophilic substitution reaction on the compound shown in the formula 2c and the compound shown in the formula 2d, and removing a protecting group to obtain a compound shown in the formula d 0;
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4) CDK12/13 ligand-end derivatives (formula e 0): obtaining e2 through nucleophilic substitution reaction between the formula e1 and the formula 3 a; coupling reaction is carried out on the compound shown in the formula e2 and the compound shown in the formula 2a, and protective groups are removed to obtain a compound shown in the formula e 0;
5) CDK12/13 ligand-end derivatives (formula g 0): the compound shown in the formula 3c and the compound shown in the formula g1 are subjected to nucleophilic substitution reaction and the protecting group is removed to obtain the compound shown in the formula g 0.
6) CDK12/13 ligand-end derivatives (formula h 0): and carrying out nucleophilic substitution reaction on the compound shown in the formula 2d and the compound shown in the formula g1, and removing the protecting group to obtain the compound shown in the formula h 0.
When n is other than 0 in formula II (n is 1 for example): the compounds corresponding to the formula a, the formula c, the formula d, the formula e, the formula g and the formula h are structures shown as the formula ab1, the formula cb1, the formula db1, the formula eb1, the formula gb1 and the formula hb 1:
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1) Coupling the compound shown in the formula 1b-1 with the bisboronic acid pinacol ester to obtain the compound shown in the formula 2b-1
Wherein, taking R1 as benzyl as an example, the preparation method of the compound shown in the formula 1b-1 comprises the following steps: selectively reducing cyano groups in a compound shown in a formula 1bb to obtain a compound shown in a formula 2bb, carrying out reductive amination on the compound shown in the formula 2bb and the compound shown in a formula 3bb to obtain a compound shown in a formula 4bb, reducing the compound shown in the formula 4bb to obtain a compound shown in a formula 5bb, carrying out intramolecular urea formation on the compound shown in the formula 5bb to obtain a compound shown in a formula 6bb, carrying out nucleophilic substitution reaction on the compound shown in the formula 6bb and the compound shown in a formula 7bb, and then carrying out deprotection to obtain a compound shown in a formula 8bb, and carrying out nucleophilic reaction on the compound shown in the formula 8bb and the compound shown in a formula 6aa to obtain a compound shown in a formula 1 b-1;
2) The synthesis of the compound shown in the formula 3cb-1 is obtained by coupling reaction and deprotection of the compound shown in the formula 1b-1 and the compound shown in the formula 1 c; synthesizing a compound shown in a formula 2db-1, and obtaining the compound shown in the formula 2b-1 and the compound shown in a formula 1d through coupling reaction and removing a protecting group;
3) Synthesizing a compound shown in a formula ab1, and referring to a formula a0, performing coupling reaction on the compound shown in a formula 2b-1 and the compound shown in a formula 5a, and removing a protecting group;
synthesizing a compound shown in a formula cb1, and referring to a formula c0, carrying out nucleophilic substitution reaction on the compound shown in the formula 3cb-1 and the compound shown in a formula 2c, and removing a protecting group;
synthesizing a compound shown in a formula db1, and referring to a formula d0, carrying out nucleophilic substitution reaction on the compound shown in the formula 2db-1 and the compound shown in a formula 2c, and removing a protecting group to obtain the compound;
synthesizing a compound shown in a formula eb1, and referring to a formula e0, performing a coupling reaction on the compound shown in a formula 2b-1 and a compound shown in a formula e2, and removing a protecting group to obtain the compound;
synthesizing a compound shown in a formula gb1, referring to a formula g0, and carrying out nucleophilic substitution reaction on the compound shown in a formula 3cb-1 and the compound shown in the formula g1 to remove a protecting group;
the synthesis of the compound shown in the formula hb1 is obtained by nucleophilic substitution reaction of the compound shown in the formula 2db-1 and the compound shown in the formula g1 and removal of the protecting group by referring to the formula h 0.
It is a further object of the present invention to provide the use of the above small molecule conjugates.
Further, a small molecule conjugate according to any one of the above embodiments is at least one of the following 1) -3):
1) Use in the preparation of a CDK12/13 degradant;
2) Use in the preparation of an inhibitor of cancer cell proliferation;
3) Use in the manufacture of a medicament for the prevention and/or treatment of a CDK12/13 disorder.
The CDK12/13 degrading agent can selectively degrade CDK12/13 including, but not limited to, the following cells: colorectal cancer cells (e.g., HCT 116), breast cancer cells (e.g., MDA-MB-231) and brain glioma cells (e.g., U251), acute myelogenous leukemia cells (e.g., U937, kasumi-1, MV-4-11, NB 4), T lymphocytic leukemia cells (e.g., jurkat, MOLT-4), myeloma cells (e.g., MM.1 s).
Such cancer cells include, but are not limited to, myeloma cells, breast cancer cells, leukemia cells, and glioma cells of the following cells.
The CDK12/13 abnormal diseases include, but are not limited to, breast cancer, prostate cancer, gastric cancer, ewing sarcoma, leukemia.
The product comprises: 1) CDK12/13 protein degrading agents; 2) Tumor cell proliferation inhibitors; 3) A medicament for preventing and/or treating CDK12/13 abnormality.
The degradation agent drug, cancer cell proliferation inhibitor drug or CDK12/13 abnormal disease drug can be introduced into body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, nasal drop, eye drop, permeation, absorption, physical or chemical mediated method; or mixed or wrapped with other substances and introduced into the body. If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier includes diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. which are conventional in the pharmaceutical field.
The medicine can be prepared into various forms such as tablets, powder, granules, capsules, oral liquid, ointment, cream, injection and the like; the medicaments of the various formulations can be prepared according to the conventional method in the pharmaceutical field.
The invention also provides a pharmaceutical composition (combination drug) containing the small molecule conjugate according to any one of the above technical schemes.
The small molecule conjugate (CDK 12/13 degrading agent) provided by the invention can be independently used for treating tumors, can be used in combination, and is more efficient for treating diseases.
As an embodiment, when combined, the combination pharmaceutical composition (or combination drug) provided by the invention comprises a small molecule conjugate (CDK 12/13 degrading agent) shown in formula I and at least one of the following drugs: PARP inhibitors, BET inhibitors, CDK4&6 inhibitors, PI3K inhibitors, and all-trans retinoic acid (ATRA).
The PARP inhibitors include Olaparib, rucapparib, nirapparib, talazoparib. BET inhibitors include ABBV-075, JQ1, dBET-1, ARV-825. The CDK4&6 inhibitor comprises YX-2-107, palbociclib. The PI3K inhibitor comprises Copanlisib.
The combined drug composition can be specifically a composition which is formed by a small molecule conjugate (CDK 12/13 degrading agent) shown in a formula I and all the inhibitors according to a molar ratio of 1:1-10:1.
Currently there is a lack of inhibitors and degradants of CDK12/13 with cell type selectivity. The invention obtains the high-activity CDK12/13 degradation agent by utilizing the PROTAC technology, and has the capability of highly specifically inhibiting the proliferation of cancer cells. Wherein the half-degradation concentration (Degradation concentration%, DC for short) for CDK12 and CDK13 50 ) About 30nM and 21nM, half-inhibitory concentration (Inhibition concentration rate%, IC for short) against tumor cells KASUMI-1 and MV4-11 50 ) About 170nM and 90nM, respectively, but IC in normal cells L-02 and HK-2 50 All about 1000nM. The degradation agent provided by the invention has strong degradation capability on CDK12/13 on various cell lines, and highly selective proliferation inhibition activity on tumor cell lines, and also has high therapeutic effect on tumor cells under a combined administration scheme.
The compound shows high-efficiency CDK12/13 degradation activity on various cell lines, high-specificity tumor cell proliferation inhibition activity and high-efficiency treatment effect when other antitumor drugs are combined. In biological meaning and treatment, can be used for lead compounds for treating various cancers and other CDK12/13 abnormal diseases.
Drawings
FIG. 1 is a schematic representation of the PROTACs technology.
FIG. 2 shows degradation of CDK12 and CDK13 after Jurkat cells were treated with IN-2 (parent molecule), formula 46, formula 47, formula 48, formula 49, formula 60, formula 61, formula 59, formula 32, formula 33, formula 34, and formula 38 at a concentration of 3. Mu.M for 24 hours.
FIG. 3 shows degradation of CDK12 and CDK13 after Jurkat cells were treated for 24h IN the concentrations of 3. Mu.M IN-2, formula 57, formula 58, formula 59, formula 1, formula 2, formula 3, formula 4, formula 6, formula 7, and formula 8.
FIG. 4 shows degradation of CDK12 and CDK13 after MV4-11 cells were treated with formula 40, formula 39, formula 18, formula 13, formula 11, formula 12, formula 48, formula 45 and formula 1 at a concentration of 2. Mu.M for 24 hours.
FIG. 5 shows degradation of CDK12 and CDK13 after KASUMI-1 and MV4-11 cells of formula 18, formula 54, formula 53, formula 55, formula 21, formula 22, formula 17, formula 15, formula 71, formula 40, formula 38, formula 45, formula 23, formula 44, formula 37 and formula 30 were treated for 24 hours at a concentration of 0.125. Mu.M, respectively.
FIG. 6 shows the degradation of CDK12 and CDK13 at various concentrations of formula 18 and formula 45 on MDA-MB-231 cell line for 24 hours.
FIG. 7 shows the change in CDK12 and CDK13 protein abundance over time in MDA-MB-231 cells treated with formula 18 and formula 45 at a concentration of 0.3. Mu.M.
FIG. 8 shows the degradation of CDK12, CDK13, CDK9, CDK2 at various concentrations of formula 18 and formula 45 on MDA-MB-231 cell lines for 24 hours.
FIG. 9 shows that formulas 18 and 45 were treated on MDA-MB-231 cell line for 24 hours, and the proteasome inhibitor MG132 was added 12 hours prior to sample collection, and that the degradation of CDK12, CDK13 by formulas 18 and 45 was reversed, indicating that the small molecule conjugates degraded CDK12, CDK13 via the ubiquitin-proteasome pathway.
FIG. 10 shows the survival of tumor cells MDA-MB-231, MV4-11, KASUMI-1 and normal cells L-02, HK-2 after 72 hours under the action of THZ-531 (classical CDK12/13 inhibitor), IN-2, formula 18 IN a concentration gradient.
FIG. 11 shows the proliferation inhibiting effect of formula 18 in combination with Olaparib on MDA-MB-231 cells at various concentrations.
Fig. 12 shows the results of stability test of formula 18 and formula 45 under liver microsomes of four different animals, rat, mouse, dog and monkey.
Detailed Description
The present invention is illustrated by the following specific examples, but the present invention is not limited thereto, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The quantitative tests in the following examples were all performed in triplicate, and the results were averaged. IN-2 (parent molecule) used IN the following examples was structured as follows:
the synthetic route of the compound is as follows: the compound shown in the formula 1a is obtained by coupling reaction of 1-methyl-5- (4, 5-tetramethyl l-1,3, 2-dioxaborane-2-yl) pyridine-2 (1H) -ketone (formula N1).
Specific procedures for the synthesis of CDK12/13 ligand-end derivatives (represented by formula a 0) (exemplified by a0-1 (R) 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) is: coupling 1-bromo-4-iodobenzene (1 aa) with trans-tert-butyl (4-aminocyclohexyl) carbamate (2 aa) to obtain tert-butyl ((1 r,4 r) -4- ((4-bromophenyl) amino) cyclohexyl) carbamate (3 aa), nucleophilic reacting the formula ((1 r,4 r) -4- ((4-bromophenyl) amino) cyclohexyl) carbamate with benzyl isocyanate (4 aa) and removing the Boc protecting group with hydrochloric acid to obtain 1- ((1 r,4 r) -4-aminocyclohexyl) -3-phenyl-1- (4-bromophenyl) urea (5 aa), and nucleophilic reacting 1- ((1 r,4 r) -4-aminocyclohexyl) -3-benzyl-1- (4-bromophenyl) urea with 6-fluoronicotinonitrile (6 aa) to obtain 3-benzyl-1- (4-bromophenyl) -1- ((1 r,4 r) -4- ((5-cyanopyridin-2 yl) amino) urea (formula 1 a); coupling 3-benzyl-1- (4-bromophenyl) -1- ((1 r,4 r) -4- ((5-cyanopyridin-2-yl) amino) cyclohexyl) urea with pinacol ester to give 3-benzyl-1- ((1 r,4 r) -4- ((5-cyanopyridin-2-yl) amino) cyclohexyl) -1- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) urea (formula 2 a); nucleophilic reaction of 5-bromo-2-fluoropyridine (3 a) and a compound shown as 4a to obtain a compound shown as formula 5 a; coupling reaction is carried out on the compound shown in the formula 2a and the compound shown in the formula 5a, and finally protective groups are removed in an acidic environment to obtain a compound shown in the formula a 0-1;
In particular, when n is 0, the specific preparation process (taking a0-1 as an example (R 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) as follows:
(1) Preparation of intermediate 3 aa:
into a 250ml round bottom flask was charged 5g of 1-bromo-4-iodobenzene (1 aa), 3.2g of t-butyl trans- (4-aminocyclohexyl) carbamate (2 aa), 2.2g of sodium t-butoxide, 856mg of Xantphos (4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene) in 40ml of toluene solvent. After three nitrogen substitutions 542mg Pd was added 2 (dba) 3 After nitrogen was again replaced three times, heated to 70 ℃ and stirred for 2 hours, TLC detected complete reaction of the starting material, followed by filtration through celite, washing the filter cake with ethyl acetate, concentrating the filtrate under reduced pressure and spin-drying. Pure 2.8g of compound 3aa were isolated on a silica gel column with 67% yield.
LC-MS(ESI)m/z(M+H) + called for C 17 H 26 BrN 2 O 2 + ＝369.1；found 369.2。
1H NMR(400MHz,DMSO-d6)δ7.15(d,J＝8.8Hz,2H),6.79(d,J＝7.9Hz,1H),6.49(d,J＝8.9Hz,2H),5.62(d,J＝8.0Hz,1H),3.25–3.15(m,1H),3.13–2.99(m,1H),1.93(d,J＝11.5Hz,2H),1.78(d,J＝11.0Hz,2H),1.37(s,9H),1.30–1.19(m,2H),1.18–1.07(m,2H).
(2) Preparation of intermediate 5 aa:
in a 125ml round bottom flask was added 2.8g of intermediate 3aa dissolved in 30ml of anhydrous tetrahydrofuran and 2.4g of anhydrous triethylamine. After three times of nitrogen substitution, stirring for 5 minutes at room temperature, adding 4.0g of benzyl isocyanate (4 aa) dropwise into the reaction solution, heating to 60 ℃, stirring for 8 hours, filtering with diatomite after TLC detection of complete reaction of the raw materials, washing a filter cake with ethyl acetate, concentrating the filtrate under reduced pressure, and spin-drying. After separation and purification on a silica gel column, the reaction solvent was distilled under reduced pressure after completion of the reaction by TLC using a 2N hydrogen chloride-ethyl acetate solution to obtain 3.4g of compound 5aa in 90% yield.
LC-MS(ESI)m/z(M+H) + called for C 20 H 25 BrN 3 O + ＝402.1；found 402.1。
1H NMR(400MHz,DMSO-d6)δ7.61(d,J＝8.5Hz,2H),7.25(t,J＝7.4Hz,2H),7.15(t,J＝8.9Hz,3H),7.09(d,J＝8.5Hz,2H),6.67(d,J＝7.9Hz,1H),5.99(t,J＝5.9Hz,1H),4.19–4.09(m,3H),2.99–2.87(m,1H),1.71(d,J＝9.6Hz,4H),1.32(s,9H),1.25–1.17(m,2H),1.03–0.94(m,2H).
(3) Preparation of intermediate 1 a:
3.4g of intermediate 5aa and 1.8g of 6-fluoronicotinonitrile (6 aa) were added to a 100ml round bottom flask and dissolved in anhydrous N-methylpyrrolidone solution, the reaction system was cooled to 0 ℃, 5.3g of DIPEA (N, N-diisopropylethylamine) was added dropwise to the reaction solution, the temperature was then raised to 100 ℃, after stirring for 8 hours, after TLC detection of complete reaction of the starting materials, the reaction solution was poured into 100ml of water, extracted three times with 60ml of methylene chloride, the organic phase was concentrated under reduced pressure, the obtained solid was suspended in ethyl acetate and stirred at room temperature for 15 minutes, the suspension was suction-filtered through a suction filter funnel, the cake was washed with 50% ethyl acetate/petroleum ether, and the obtained cake solid was dried under vacuum to obtain 2.2g of product 1a, yield 64%.
LC-MS(ESI)m/z(M+H) + called for C 26 H 27 BrN 5 O 1 + ＝504.1；found 504.1。
1H NMR(400MHz,Chloroform-d)δ8.27(s,1H),7.56(d,J＝8.5Hz,2H),7.40(dd,J＝8.8,2.1Hz,1H),7.29(t,J＝7.2Hz,2H),7.23(d,J＝7.1Hz,1H),7.16(d,J＝7.1Hz,2H),7.06(d,J＝8.5Hz,2H),6.26(d,J＝8.8Hz,1H),5.19(d,J＝8.0Hz,1H),4.64–4.49(m,1H),4.36(d,J＝5.8Hz,2H),4.27(t,J＝5.8Hz,1H),3.53(s,1H),2.10(d,J＝11.8Hz,2H),1.95(d,J＝11.5Hz,2H),1.41(q,J＝13.4Hz,2H),1.25(q,J＝15.1,13.8Hz,2H).
(4) Preparation of intermediate 2 a:
into a 100ml round bottom flask were charged 1.0g of intermediate 1a,1.3g of pinacol biborate, 779mg of potassium acetate, dissolved in anhydrous dioxane solution, replaced three times with nitrogen and 325mg of Pd (dppf) was added 2 Cl 2 After nitrogen substitution three times again, heating to 80 ℃, stirring for 1.5 hours, detecting complete reaction of the raw materials by TLC, washing twice with saturated brine, separating an organic phase, concentrating the organic phase under reduced pressure, and rotary drying. Pure 1.0g of compound 2a was isolated on a silica gel column in 91% yield.
LC-MS(ESI)m/z(M+H) + called for C 32 H 39 BrN 5 O 3 + ＝552.3；found 552.3。
1H NMR(400MHz,DMSO-d6)δ8.28(d,J＝2.4Hz,1H),7.75(d,J＝8.1Hz,2H),7.60(dd,J＝8.9,2.3Hz,1H),7.48(d,J＝7.7Hz,1H),7.27(t,J＝7.5Hz,2H),7.23–7.12(m,4H),6.46(d,J＝8.9Hz,1H),5.83(t,J＝6.1Hz,1H),5.76(s,1H),4.34–4.21(m,1H),4.13(d,J＝6.0Hz,2H),3.45(m,J＝26.2Hz,1H),1.90(d,J＝12.1Hz,2H),1.83–1.75(d,2H),1.30(s,12H),1.17–1.07(m,J＝35.8Hz,4H).
(5) Preparation of intermediate 4a (in the case of 4a-1 (m 1 ＝1，m 2 ＝0))：
2.0g of 2- (2-aminoethoxy) ethanol and 2.8g of triethylamine are added into a 100ml round bottom flask, the reaction system is cooled to 0 ℃, boc anhydride is added dropwise into the reaction liquid, the reaction liquid is slowly warmed to room temperature and stirred for 3 hours, after TLC detects that the raw materials are completely reacted, the reaction liquid is washed twice by a saturated salt water bath, an organic phase is separated, the organic phase is concentrated under reduced pressure and dried under vacuum, and the obtained reaction liquid is directly subjected to the next reaction.
LC-MS(ESI)m/z(M+H) + called for C 9 H 20 NO 4 + ＝205.1；found205.2。
1H NMR(400MHz,Chloroform-d)δ5.29(s,1H),4.22(t,2H),3.71(t,2H),3.48(t,2H),3.21-3.26(m,2H),2.26(s,1H),1.41(s,9H).
Preparation of other intermediates (4 a-0, 4a-2, 4a-3, 4a-4, 4 a-5) reference 4a-1:
4a-0(m 1 ＝0，m 2 =0): the raw material is N-Boc-ethanolamine;
4a-2(m 1 ＝2，m 2 =0): starting from 2- [2- (2-aminoethoxy) ethoxy ]]Ethanol;
4a-3(m 1 ＝3，m 2 =0): the raw material is 2- (2- (2- (2-amino ethoxy) ethanol;
4a-4(m 1 ＝4，m 2 =0): the raw material is 14-amino-3, 6,9, 12-tetraoxatetradecan-1-ol;
4a-5(m 1 ＝5，m 2 =0): the raw material is 17-amino-3, 6,9,12, 15-pentaoxa heptadecanol.
(6) Preparation of intermediate 5a-1 (taking 5a-1 as an example (m 1 ＝1，m 2 ＝0))：
1g of 2- (2-Boc-aminoethoxy) ethanol (4 a-1), 1.1g of 2-fluoro-5-bromopyridine and 4.8g of cesium carbonate were added to a 100ml round bottom flask, the reaction system was warmed to 100℃and Boc anhydride was added dropwise to the reaction solution, followed by slowly warming to room temperature and stirring for 6 hours, after the completion of the TLC detection of the starting material reaction, the reaction solution was poured into 100ml of saturated saline, extracted three times with 60ml of ethyl acetate, and the organic phase was concentrated under reduced pressure and dried by rotary drying. Pure 1.42g of Compound 5a-1 was isolated on a silica gel column in 82% yield.
LC-MS(ESI)m/z(M+H) + called for C 14 H 22 BrN 2 O 4 + ＝361.1；found 361.1。
1 H NMR(400MHz,Chloroform-d)δ7.48(d,J＝2.7Hz,1H),7.38(dd,J＝9.7,2.8Hz,1H),6.50(d,J＝10.2Hz,1H),4.99(s,1H),4.28(t,2H),3.75(t,2H),3.50(t,2H),3.22-3.25(m,2H),1.40(s,9H).
Preparation of other intermediates (5 a-0, 5a-2, 5a-3, 5a-4, 5 a-5) reference 5a-1:
5a-0(m 1 ＝0，m 2 =0): the raw material is 4a-0 instead of 4a-1;
5a-2(m 1 ＝2，m 2 =0): the raw material is 4a-2 instead of 4a-1;
5a-3(m 1 ＝3，m 2 =0): the raw material is 4a-3 instead of 4a-1;
5a-4(m 1 ＝4，m 2 =0): the raw material is 4a-4 instead of 4a-1;
5a-5(m 1 ＝5，m 2 =0): the starting material was 4a-5 instead of 4a-1.
(7) Preparation of a Compound of formula a 0-1:
into a 25ml round bottom flask were charged 80mg of intermediate 2a,63mg of intermediate 5a-1 and 142mg of cesium carbonate, dissolved in toluene: ethanol: water = 4:4:1, and after three nitrogen substitutions, 16mg Pd (PPh) 3 ) 4 After nitrogen substitution three times again, heating to 100 ℃, stirring for 3 hours, TLC detects complete reaction of the starting material, extraction three times with 20ml dichloromethane, concentration of the organic phase under reduced pressure, rotary drying. Pure 46mg was separated on a silica gel column and treated with 2N hydrogen chloride-ethyl acetate solution to give compound a0-1 in 47% yield.
LC-MS(ESI)m/z(M+H) + called for C 35 H 40 N 7 O 3 + ＝606.3；found 606.4。
Preparation of other intermediates (a-0, a-2, a-3, a-4, a-5) reference a-1:
a0-0(m 1 ＝0，m 2 =0): 5a-0 is used for replacing 5a-1 as a raw material;
a0-2(m 1 ＝2，m 2 =0): the raw material is 5a-2 instead of 5a-1;
a0-3(m 1 ＝3，m 2 =0): 5a-3 is used for replacing 5a-1 as a raw material;
a0-4(m 1 ＝4，m 2 =0): 5a-4 is used for replacing 5a-1 as a raw material;
a0-5(m 1 ＝5，m 2 =0): the raw material is 5a-5 instead of 5a-1.
When n is not 0 (taking ab1-1 as an example (R 1 Benzyl, n=1, m 1 ＝1，m 2 =0)), the specific preparation process is as follows:
(1) Preparation of intermediate 2bb
Into a 100ml round bottom flask was added 2.0g of 2-nitro-5-bromoxynil (1 bb), after three nitrogen substitutions, 18ml of borane tetrahydrofuran complex (1M) was slowly added dropwise under ice bath, again three nitrogen substitutions, stirred overnight, after TLC detection of complete reaction of the starting material, extracted three times with 20ml of ethyl acetate, the filtrate was concentrated under reduced pressure and dried by spin. Pure 1.1g of compound 2bb was isolated on a silica gel column in 54% yield.
LC-MS(ESI)m/z(M+H) + called for C 7 H 8 BrN 2 O2+＝230.9；found 230.9。
1 H NMR(400MHz,Chloroform-d)δ7.84–7.77(m,1H),7.54(t,J＝1.8Hz,1H),7.49(dd,J＝8.7,2.2Hz,1H),3.02(s,2H),1.53(s,2H).
(2) Preparation of intermediate 4bb
460mg of benzaldehyde (3 bb) was added to a 100ml round bottom flask, dissolved in anhydrous tetrahydrofuran, 1.0g of intermediate 2bb was dissolved in anhydrous tetrahydrofuran solution, the round bottom flask was added dropwise under ice bath, after three nitrogen substitutions, stirring was performed at room temperature for 1h, 545mg of sodium cyanoborohydride was added, stirring was performed overnight, after TLC detection of completion of the reaction of the starting materials, 20ml of ice water was added to quench the reaction, extraction was performed three times with 20ml of ethyl acetate, the filtrate was concentrated under reduced pressure, and rotary-dried. Pure 1.2g of compound 4bb was isolated on a silica gel column in 92% yield.
LC-MS(ESI)m/z(M+H) + called for C 14 H 14 BrN 2 O 2 + ＝321.0；found 321.0。
1 H NMR(400MHz,Chloroform-d)δ7.55(d,J＝2.1Hz,1H),7.49(dd,J＝8.7,2.2Hz,1H),7.35–7.28(m,4H),7.25(d,J＝4.8Hz,1H),3.81(s,2H),3.09(t,J＝7.1Hz,2H),1.49(m,1H).
(3) Preparation of intermediate 5bb
1.2g of intermediate 4bb was added to a 100ml round bottom flask, dissolved in anhydrous ethyl acetate, replaced with 2.4g of stannous chloride dihydrate, and after three nitrogen substitutions, heated to 80 ℃ and stirred for 3h, after TLC detection of complete reaction of starting material, 20ml of ice water was added, extracted three times with 20ml of ethyl acetate, the filtrate concentrated under reduced pressure and spin-dried. Pure 840mg of compound 5bb was isolated on a silica gel column in 77% yield.
LC-MS(ESI)m/z(M+H) + called for C 14 H 16 BrN 2 + ＝291.0；found 291.1。
1 H NMR(400MHz,DMSO-d 6 )δ7.57–7.50(m,2H),7.47–7.42(m,3H),7.15–7.08(m,2H),6.63(d,J＝9.2Hz,1H),4.15(s,2H),3.04(dd,J＝9.8,6.1Hz,2H),1.32–1.16(m,1H).
(4) Preparation of intermediate 6bb
840mg of intermediate 5bb is added to a 100ml round bottom flask, dissolved in anhydrous tetrahydrofuran solution, 447mg of CDI (1, 1' -carbonyldiimidazole) is added, stirring is carried out for 3 hours at room temperature, heating is carried out to 60 ℃ and stirring is continued for 12 hours, after TLC detection of complete reaction of the raw materials, extraction is carried out three times with 20ml of ethyl acetate, the filtrate is concentrated under reduced pressure and dried by rotation. Pure 706mg of compound 6bb was isolated on a silica gel column in 76% yield.
LC-MS(ESI)m/z(M+H) + called for C 15 H 14 BrN 2 O + ＝317.0；found 317.0。
1 H NMR(400MHz,DMSO-d 6 )δ9.46(s,1H),7.40–7.20(m,7H),6.75(d,J＝8.2Hz,1H),4.52(s,2H),4.35(s,2H).
(5) Preparation of intermediate 8bb
700mg of intermediate 6bb is added into a 100ml round bottom flask, dissolved in anhydrous dioxane solution, 97mg of sodium hydride is added in batches under ice bath, after stirring for 0.5h at 0 ℃, 1.2g of cis-4- ((tert-butoxycarbonyl) amino) cyclohexyl 4-methylbenzenesulfonate (7 bb) is slowly added dropwise into the reaction system after dissolving in anhydrous dioxane, heating to 60 ℃ for continuous stirring for 8h, after TLC detects that the raw materials are completely reacted, 20ml of ice water is added for quenching reaction, extraction is carried out three times by 20ml of ethyl acetate, and the filtrate is concentrated under reduced pressure and dried in a rotating way. After separation and purification on a silica gel column, the reaction was completed by TLC detection using a 2N hydrogen chloride-ethyl acetate solution, and the reaction solvent was distilled under reduced pressure to obtain 572mg of Compound 8bb in 63% yield.
LC-MS(ESI)m/z(M+H) + called for C 21 H 25 BrN 3 O + ＝414.1；found 414.1。
(6) Preparation of intermediate 1b-1 referring to the preparation of formula 1a, starting materials were intermediate 8bb and 6-fluoronicotinonitrile.
LC-MS(ESI)m/z(M+H) + called for C 27 H 27 BrN 5 O + ＝516.1；found 516.1。
(7) Preparation of intermediate 2b-1 referring to the preparation of formula 2a, starting materials are intermediate 1b and pinacol borate.
LC-MS(ESI)m/z(M+H) + called for C 33 H 39 BrN 5 O 3 + ＝564.3；found 564.3。
(8) Preparation of the Compounds of formula ab1-1: referring to the preparation of formula a0-1, the raw materials are a compound shown in formula 2b and
a compound represented by formula 5a-1;
LC-MS(ESI)m/z(M+H) + called for C 36 H 40 BrN 7 O 3 + ＝618.3；found 618.3。
LC-MS(ESI)m/z(M+H) + called for C 35 H 40 N 7 O 3 + ＝606.3；found 606.4。
preparation of other intermediates (ab 1-0, ab1-2, ab1-3, ab1-4, ab 1-5) reference ab1-1:
ab1-0(m 1 ＝0，m 2 =0): 5a-0 is used for replacing 5a-1 as a raw material;
ab1-2(m 1 ＝2，m 2 =0): the raw material is 5a-2 instead of 5a-1;
ab1-3(m 1 ＝3，m 2 =0): 5a-3 is used for replacing 5a-1 as a raw material;
ab1-4(m 1 ＝4，m 2 =0): 5a-4 is used for replacing 5a-1 as a raw material;
ab1-5(m 1 ＝5，m 2 =0): the raw material is 5a-5 instead of 5a-1.
Specific procedures for the synthesis of CDK12/13 ligand-end derivatives (represented by formula c) (exemplified by c0-1 (R) 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) is:
3-benzyl-1- (4-bromophenyl) -1- ((1 r,4 r) -4- ((5-cyanopyridin-2-yl) amino) cyclohexyl) urea (represented by formula 1 a) and tert-butyl piperazine-1-carboxylate (represented by formula 1 c) were reacted to give 4- (4- { [ (benzylamino) carbonyl ] {4- [ (5-cyanopyridin-2-yl) amino ] cyclohexyl } amino) phenyl) piperazine-1-carboxylic acid 2-methylpropan-2-yl ester, and the Boc protecting group was removed to give 1- (benzylamino) -N- {4- [ (5-cyanopyridin-2-yl) amino ] cyclohexyl } -N- [4- (piperazin-1-yl) phenyl ] methanamide (represented by formula 3 c); substitution reaction of 2- (2-Boc-aminoethoxy) ethanol (formula 4 a-1) with p-methylphenyl sulfonyl chloride to give 2- (2- ((tert-butoxycarbonyl) amino) ethoxy) ethyl 4-methylbenzenesulfonate (formula 2 c-1); nucleophilic reaction of 1- (benzylamino) -N- {4- [ (5-cyanopyridin-2-yl) amino ] cyclohexyl } -N- [4- (piperazin-1-yl) phenyl ] methanamide and 2- (2- ((tert-butoxycarbonyl) amino) ethoxy) ethyl 4-methylbenzenesulfonate, and final removal of protecting groups in acidic environment to give compounds of formula c 0-1;
When n is 0 (taking c0-1 as an example (R 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) specifically prepared as follows:
(1) Preparation of intermediate 3 c:
200mg of intermediate 1a,110mg of tert-butyl piperazine-1-carboxylate, 150mg of sodium tert-butoxide, 23mg of Xantphos, dissolved in 5ml of toluene solvent, were placed in a 25ml round bottom flask. After three nitrogen substitutions 22mg Pd was added 2 (dba) 3 After nitrogen was replaced three times again, heated to 80 ℃ and stirred for 2 hours, TLC detected complete reaction of the starting materials, the filtrate was concentrated under reduced pressure and dried by rotary drying. 15mg of the purified product was subjected to a silica gel column separation and treatment with a 2N hydrogen chloride-ethyl acetate solution to obtain compound 1c in 48% yield.
LC-MS(ESI)m/z(M+H) + called for C 30 H 36 N 7 O + ＝510.3；found 510.3。
1 H NMR(400MHz,Chloroform-d)δ8.25(s,1H),7.30–7.13(m,6H),7.03(d,J＝8.8Hz,2H),6.89(d,J＝8.9Hz,2H),6.23(d,J＝8.8Hz,1H),5.21(d,J＝8.0Hz,1H),4.59–4.48(m,1H),4.41–4.32(m,3H),3.57(t,J＝5.2Hz,4H),3.48(m,J＝19.3Hz,1H),3.16(t,J＝5.1Hz,4H),2.05(d,J＝12.5Hz,2H),1.92(d,J＝12.1Hz,2H),1.44–1.28(m,4H).
(2) Preparation of intermediate 2c (in the case of 2c-1 (m 1 ＝1，m 2 ＝0))：
1.0g of 4a-1 and 743mg of triethylamine, 1g of 4-toluenesulfonyl chloride are added to a 100ml round bottom flask, dissolved in methylene chloride and stirred at room temperature for 3 hours, after TLC detection of complete reaction of the starting materials, washed twice with a saturated brine bath, the organic phase is separated, concentrated under reduced pressure, dried under vacuum and spun dry. After separation and purification by a silica gel column, 1.7g of compound 2c-1 was obtained in 98% yield.
LC-MS(ESI)m/z(M+H) + called for C 16 H 26 NO 6 S + ＝360.2；found 360.3。
1H NMR(400MHz,Chloroform-d)δ＝7.80(d,J＝8.7Hz,2H),7.36(d,J＝8.7Hz,2H),4.18–4.15(m,2H),3.63(t,J＝4.6Hz,2H),3.45(t,J＝5.0Hz,2H),3.24(q,J＝5.0Hz,2H),2.45(s,3H),1.45(s,9H).13C NMR(100MHz,Chloroform-d)δ＝156.24,144.98,133.09,129.91,128.01,79.36,70.40,69.18,68.41,40.29,28.46,21.68
Preparation of other intermediates (2 c-0, 2c-2, 2c-3, 2c-4, 2 c-5) reference 2c-1:
2c-0(m 1 ＝0，m 2 =0): the raw material is 4a-0 instead of 4a-1;
2c-2(m 1 ＝2，m 2 =0): the raw material is 4a-2 instead of 4a-1;
2c-3(m 1 ＝3，m 2 =0): the raw material is 4a-3 instead of 4a-1;
2c-4(m 1 ＝4，m 2 =0): the raw material is 4a-4 instead of 4a-1;
2c-5(m 1 ＝5，m 2 =0): the starting material was 4a-5 instead of 4a-1.
(3) Preparation of the Compound of formula c0-1:
100mg of intermediate 1c,141mg of intermediate 2c-1 and 30mg of triethylamine are added into a 25mg round bottom flask, the mixture is dissolved in an anhydrous acetonitrile solvent, the mixture is heated to 60 ℃ and stirred for 8 hours, after TLC detection of complete reaction of the raw materials, the mixture is washed twice with a saturated brine bath, an organic phase is separated, the organic phase is concentrated under reduced pressure, dried in vacuum and dried in a rotary manner. 86mg of the purified compound was treated with 2N of hydrogen chloride-ethyl acetate solution to give compound c0-1 in 63% yield.
LC-MS(ESI)m/z(M+H) + called for C 34 H 45 N 8 O 2 + ＝597.4；found 597.5。
Preparation of other intermediates (c 0-0, c0-2, c0-3, c0-4, c 0-5) reference c0-1:
c0-0(m 1 ＝0，m 2 =0): the raw material is 2c-0 instead of 4a-1;
c0-2(m 1 ＝2，m 2 =0): the raw material is 2c-2 instead of 4a-1;
c0-3(m 1 ＝3，m 2 =0): the raw material is 2c-3 instead of 4a-1;
c0-4(m 1 ＝4，m 2 =0): 2c-4 replaces 4a-1 as raw material;
c0-5(m 1 ＝5，m 2 =0): the raw material is 2c-5 instead of 4a-1.
When n is not 0 (taking cb1-1 as an example (R 1 Benzyl, n=1, m 1 ＝1，m 2 =0)), the specific preparation process is as follows:
(1) Preparation of the compound of formula c-1: preparation of intermediate 3cb referring to preparation of formula 3c, starting from a compound represented by formula 1b and piperazine-1-carboxylic acid tert-butyl ester (represented by formula 1 c);
LC-MS(ESI)m/z(M+H) + called for C 31 H 36 N 7 O + ＝522.3；found 522.3。
(2) Preparation of the Compound of formula cb 1-1: referring to the preparation of the formula c0-1, the raw materials are a compound shown in the formula 3cb and a compound shown in the formula 2 c-1;
LC-MS(ESI)m/z(M+H) + called for C 35 H 45 N 8 O 2 + ＝609.4；found 609.4。
Preparation of other intermediates (cb 1-0, cb1-2, cb1-3, cb1-4, cb 1-5) reference cb1-1:
cb1-0(m 1 ＝0，m 2 =0): 2c-0 is used as a raw material to replace 2c-1;
cb1-2(m 1 ＝2，m 2 =0): 2c-2 is used as a raw material to replace 2c-1;
cb1-3(m 1 ＝3，m 2 =0): 2c-3 is used as a raw material to replace 2c-1;
cb1-4(m 1 ＝4，m 2 =0): 2c-4 is used as a raw material to replace 2c-1;
cb1-5(m 1 ＝5，m 2 =0): the raw material is 2c-5 instead of 2c-1.
Specific procedures for the synthesis of CDK12/13 ligand-end derivatives (formula d) (exemplified by d0-1 (R) 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) is: performing affinity substitution reaction on piperazine-1-carboxylic acid tert-butyl ester (shown as a formula 1 c) and 5-bromo-2-fluoropyridine (shown as a formula 3 a) to obtain 4- (5-bromopyridine-2-yl) piperazine-1-carboxylic acid-2-methylpropan-2-yl ester (shown as a formula 1 d); coupling reaction is carried out on 4- (5-bromopyridine-2-yl) piperazine-1-formic acid-2-methylpropan-2-yl ester and an intermediate 2a, and protective groups are removed under an acidic environment to obtain 1- (benzylamino) -N- {4- [ (5-cyanopyridine-2-yl) amino]Cyclohexyl } -N- {4- [6- (piperazin-1-yl) pyridin-3-yl]Phenyl } methanamide (formula 2 d), 1- (benzylamino) -N- {4- [ (5-cyanopyridin-2-yl) amino group]Cyclohexyl } -N- {4- [6- (piperazin-1-yl) pyridin-3-yl]Phenyl } methanamide and 2- (2- ((tert-butoxycarbonyl) amino) ethoxy) ethyl 4-methylbenzenesulfonate (shown as a formula 2 c-1) undergo nucleophilic reaction to obtain a compound shown as a formula d 0-1.
When n is 0, the specific preparation process (taking d0-1 as an example (R 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) as follows:
(1) Preparation of intermediate 1 d:
200mg of piperazine-1-carboxylic acid tert-butyl ester, 227mg of 2-fluoro-5-bromopyridine and 416mg of DIPEA are added into a 50ml round bottom flask, dissolved in anhydrous dimethyl sulfoxide solution, heated to 120 ℃ and stirred for 3 hours, after TLC detects that the raw materials are completely reacted, the reaction is washed twice by a saturated salt water bath, an organic phase is separated, the organic phase is concentrated under reduced pressure, dried in vacuum and rotated and dried. 347mg of compound 1d was obtained after purification by silica gel column chromatography, and the yield was 94%.
LC-MS(ESI)m/z(M+H) + called for C 14 H 21 BrN 3 O 2 + ＝342.1；found 342.1。
1H NMR(400MHz,Chloroform-d)δ＝8.21(d,J＝2.4Hz,1H),7.56(dd,J＝9.2,2.8Hz,1H),6.56(d,J＝4.0Hz,1H),3.51-3.56(m,8H),1.64(s,9H).
(2) Preparation of intermediate 2 d:
into a 25ml round bottom flask were charged 80mg of intermediate 2a,60mg of intermediate 1d and 142mg of cesium carbonate, dissolved in toluene: ethanol: water = 4:4:1, and after three nitrogen substitutions, 16mg Pd (PPh) 3 ) 4 After nitrogen substitution three times again, heating to 100 ℃, stirring for 3 hours, TLC detects complete reaction of the starting material, extraction three times with 20ml dichloromethane, concentration of the organic phase under reduced pressure, rotary drying. Pure 77mg was isolated on a silica gel column and treated with 2N hydrogen chloride-ethyl acetate solution to give compound 2d in 78% yield.
LC-MS(ESI)m/z(M+H) + called for C 35 H 39 N 8 O + ＝586.3；found 586.3。
(3) Preparation of a Compound of formula d 0-1:
to a 25mg round bottom flask were added 60mg of intermediate 2d,44mg of intermediate 2c-1 and 31mg of triethylamine, dissolved in anhydrous acetonitrile solvent, heated to 60 ℃ and stirred for 8 hours, after TLC detection of complete reaction of the starting material, washed twice with a saturated brine bath, the organic phase was separated, concentrated under reduced pressure, dried under vacuum and spun dry. After 50mg of the purified compound d0-1 was treated with a 2N hydrogen chloride-ethyl acetate solution in a silica gel column, the yield was 63%.
LC-MS(ESI)m/z(M+H)+called for C 39 H 48 N 9 O 2 + ＝674.4；found 674.5。
Preparation of other intermediates (d 0-0, d0-2, d0-3, d0-4, d 0-5) reference d0-1:
d0-0(m 1 ＝0，m 2 =0): 2c-0 is used as a raw material to replace 2c-1;
d0-2(m 1 ＝2，m 2 =0): 2c-2 is used as a raw material to replace 2c-1;
d0-3(m 1 ＝3，m 2 =0): 2c-3 is used as a raw material to replace 2c-1;
d0-4(m 1 ＝4，m 2 =0): 2c-4 is used as a raw material to replace 2c-1;
d0-5(m 1 ＝5，m 2 =0): the raw material is 2c-5 instead of 2c-1.
When n is not 0 (taking db1-1 as an example (R 1 For benzyl, n=1, m1=1, m2=0)), the specific preparation process is as follows:
(1) Preparation of the compound of formula 2 db: preparation of intermediate 2db referring to preparation of formula 2d, starting material is a compound represented by formula 2b and piperazine-1-carboxylic acid tert-butyl ester (represented by formula 1 c);
LC-MS(ESI)m/z(M+H) + called for C 36 H 39 N 8 O + ＝599.3；found 599.3。
(2) Preparation of the compound of formula db1-1: referring to the preparation of the formula d0-1, the raw materials are a compound shown as the formula 2db and
a compound represented by the formula 2c-1;
LC-MS(ESI)m/z(M+H) + called for C 40 H 48 N 9 O 2 + ＝686.4；found 686.5。
preparation of other intermediates (db 1-0, db1-2, db1-3, db1-4, db 1-5) reference db1-1:
db1-0(m 1 ＝0，m 2 =0): 2c-0 is used as a raw material to replace 2c-1;
db1-2(m 1 ＝2，m 2 =0): 2c-2 is used as a raw material to replace 2c-1;
db1-3(m 1 ＝3，m 2 =0): 2c-3 is used as a raw material to replace 2c-1;
db1-4(m 1 ＝4，m 2 =0): 2c-4 is used as a raw material to replace 2c-1;
db1-5(m 1 ＝5，m 2 =0): the raw material is 2c-5 instead of 2c-1.
Specific procedures for the synthesis of CDK12/13 ligand-end derivatives (represented by formula e) (exemplified by e0-1 (R 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) is: the 2- (2-hydroxy ethoxy) acetic acid tert-butyl ester and 5-bromo-2-fluoropyridine undergo an affinity substitution reaction to obtain ({ 2- [ (5-bromopyridin-2-yl) oxy) ]Ethyl } oxy) acetic acid-2-methylpropan-2-yl ester (represented by formula 1 e-1); then ({ 2- [ (5-bromopyridin-2-yl) oxy]Ethyl } oxy) acetic acid-2-methylpropan-2-yl ester and the intermediate 2a undergo a coupling reaction, and finally, the protecting group is removed in an acidic environment, so that a compound shown as a compound e0-1 is obtained.
When n is 0, the specific preparation process (taking e0-1 as an example (R 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) as follows:
(1) Preparation of intermediate 1e (1 e-1 as an example (m 1 ＝1，m 2 ＝0))：
To a 100ml round bottom flask was added 860mg of the intermediate tert-butyl 2- (2-hydroxyethoxy) acetate, 1.1g of 2-fluoro-5-bromopyridine and 4.8g of cesium carbonate, dissolved in anhydrous dimethyl sulfoxide, the reaction system was heated to 100℃and stirred for 6 hours, after the completion of the reaction by TLC, the reaction solution was poured into 100ml of saturated brine, extracted three times with 60ml of ethyl acetate, and the organic phase was concentrated under reduced pressure and dried by rotation. Pure 1.41g of Compound 1e-1 was isolated on a silica gel column in 87% yield.
LC-MS(ESI)m/z(M+H) + called for C 13 H 19 BrNO 4 + ＝332.1；found 332.1。
Preparation of other intermediates (1 e-0, 1e-2, 1e-3, 1e-4, 1 e-5) reference 1e-1:
1e-0(m 1 ＝0，m 2 =0): the raw material is 2-glycollic acid tert-butyl ester instead of 2- (2-hydroxy ethoxy) acetic acid tert-butyl ester;
1e-2(m 1 ＝2，m 2 =0): the raw material is 2- (2- (2-hydroxyethoxy) ethoxy) tert-butyl acetate instead of 2- (2-hydroxyethoxy) acetic acid tert-butyl ester;
1e-3(m 1 ＝3，m 2 =0): the raw material is 2- (2- (2- (2-hydroxyethoxy) ethoxy) tert-butyl acetate instead of 2- (2-hydroxyethoxy) acetic acid tert-butyl ester;
1e-4(m 1 ＝4，m 2 =0): the raw material is 14-hydroxy-3, 6,9, 12-tetraoxatetradecane-1-carboxylic acid tert-butyl ester instead of 2- (2-hydroxyethoxy) acetic acid tert-butyl ester;
1e-5(m 1 ＝5，m 2 =0): the raw material is 17-hydroxy-3, 6,9,12, 15-pentaoxa-1-heptadecanoic acid tert-butyl ester instead of 2- (2-hydroxyethoxy) acetic acid tert-butyl ester 1.
(2) Preparation of the Compound of formula e0-1:
into a 25ml round bottom flask were charged 80mg of intermediate 2a,58mg of intermediate 1e-1 and 142mg of cesium carbonate, dissolved in toluene: ethanol: water = 4:4:1, and after three nitrogen substitutions, 16mg Pd (PPh) 3 ) 4 After nitrogen substitution three times again, heating to 100 ℃, stirring for 3 hours, TLC detects complete reaction of the starting material, extraction three times with 20ml dichloromethane, concentration of the organic phase under reduced pressure, rotary drying. Pure 61mg was isolated on a silica gel column and treated with 50% TFA in dichloromethane to give compound e0-1 in 62% yield.
LC-MS(ESI)m/z(M+H) + called for C 35 H 37 N 6 O 5 + ＝621.3；found 621.4。
Preparation of other intermediates (e 0-0, e0-2, e0-3, e0-4, e 0-5) reference e0-1:
e0-0(m 1 ＝0，m 2 =0): 1e-0 is used for replacing 1e-1 as a raw material;
e0-2(m 1 ＝2，m 2 =0): 1e-2 is used for replacing 1e-1 as a raw material;
e0-3(m 1 ＝3，m 2 =0): 1e-3 is used for replacing 1e-1 as a raw material;
e0-4(m 1 ＝4，m 2 =0): 1e-4 is used for replacing 1e-1 as a raw material;
e0-5(m 1 ＝5，m 2 =0): 1e-5 is used as raw material instead of 1e-1.
When n is not 0 (taking eb-1 as an example (R 1 Benzyl, n=1, m 1 ＝1，m 2 =0)), the specific preparation process is as follows:
preparation of the Compound of formula eb1-1: referring to the preparation of the formula e0-1, the raw materials are a compound shown in the formula 2b and a compound shown in the formula 1e-1;
LC-MS(ESI)m/z(M+H) + called for C 36 H 37 N 6 O 5 + ＝632.3；found 632.3。
preparation of other intermediates (eb 1-0, eb1-2, eb1-3, eb1-4, eb 1-5) reference eb1-1:
eb1-0(m 1 ＝0，m 2 =0): 1e-0 is used for replacing 1e-1 as a raw material;
eb1-2(m 1 ＝2，m 2 =0): 1e-2 is used for replacing 1e-1 as a raw material;
eb1-3(m 1 ＝3，m 2 =0): 1e-3 is used as raw material to replace1e-1；
eb1-4(m 1 ＝4，m 2 =0): 1e-4 is used for replacing 1e-1 as a raw material;
eb1-5(m 1 ＝5，m 2 =0): 1e-5 is used as raw material instead of 1e-1.
Specific procedures for the synthesis of CDK12/13 ligand-end derivatives (represented by formula g) (exemplified by g0-1 (R) 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) is: substitution reaction of tert-butyl 2- (2-hydroxyethoxy) acetate with 4-toluenesulfonyl chloride to obtain tert-butyl 2- (2- (p-toluenesulfonyloxy) ethoxy) acetate (formula 1 g-1); and carrying out nucleophilic reaction on tert-butyl 2- (2- (p-toluenesulfonyloxy) ethoxy) acetate and the intermediate 3c, and removing the protecting group in an acidic environment to obtain a compound shown as a compound g 0-1.
When n is 0, the specific preparation process (taking g0-1 as an example (R 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) as follows:
(1) Preparation of intermediate 1g (1 g-1 as example (m 1 ＝1，m 2 ＝0))：
In a 100ml round bottom flask were added 1.0g of tert-butyl 2- (2-hydroxyethoxy) acetate and 861mg of triethylamine, 1.1g of 4-toluenesulfonyl chloride, dissolved in dichloromethane, stirred at room temperature for 3 hours, after TLC detection of complete reaction of the starting material washed twice with a saturated brine bath, the organic phase was separated, concentrated under reduced pressure, dried under vacuum and spun dry. After separation and purification by a silica gel column, 1.8g of compound 1g-1 is obtained, and the yield is 96%.
LC-MS(ESI)m/z(M+H) + called for C 15 H 23 O 6 + ＝331.1；found 331.2。
1H NMR(400MHz,Chloroform-d)δ＝7.77-7.83(m,2H),7.44(d,J＝7.83Hz,2H),4.14-4.19(m,2H),3.93(s,2H),3.68-3.74(m,2H),2.46(s,3H),1.46(s,9H)。
Preparation of other intermediates (1 g-0, 1g-2, 1g-3, 1g-4, 1g-5 …) reference 1g-1:
1g-0(m 1 ＝0，m 2 =0): the raw material is 2-glycollic acid tert-butyl ester instead of 2- (2-hydroxy ethoxy) acetic acid tert-butyl ester;
1g-2(m 1 ＝2，m 2 =0): the raw material is 2- (2- (2-hydroxyethoxy) ethoxy) tert-butyl acetate instead of 2- (2-hydroxyethoxy) acetic acid tert-butyl ester;
1g-3(m 1 ＝3，m 2 =0): the raw material is 2- (2- (2- (2-hydroxyethoxy) ethoxy) tert-butyl acetate instead of 2- (2-hydroxyethoxy) acetic acid tert-butyl ester;
1g-4(m 1 ＝4，m 2 =0): the raw material is 14-hydroxy-3, 6,9, 12-tetraoxatetradecane-1-carboxylic acid tert-butyl ester instead of 2- (2-hydroxyethoxy) acetic acid tert-butyl ester;
1g-5(m 1 ＝5，m 2 =0): the raw material adopts 17-hydroxy-3, 6,9,12, 15-pentaoxa-1-heptadecanoic acid tert-butyl ester to replace 2- (2-hydroxyethoxy) acetic acid tert-butyl ester 1 …
(2) Preparation of a Compound of formula g0-1:
100mg of intermediate 3c,78mg of intermediate 1g-1 and 30mg of triethylamine are added into a 25mg round bottom flask, the mixture is dissolved in an anhydrous acetonitrile solvent, the mixture is heated to 60 ℃ and stirred for 8 hours, after TLC detection of complete reaction of the raw materials, the mixture is washed twice with a saturated brine bath, an organic phase is separated, the organic phase is concentrated under reduced pressure, dried in vacuum and dried in a rotary manner. 76mg of the purified product was subjected to silica gel column separation and treatment with 50% TFA in methylene chloride to obtain compound g0-1 in 57% yield.
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LC-MS(ESI)m/z(M+H) + called for C 34 H 41 N 7 O 4 + ＝612.3；found 612.3。
Preparation of other intermediates (g 0-0, g0-2, g0-3, g0-4, g 0-5) reference g0-1:
g0-0(m 1 ＝0，m 2 =0): 1g-0 is used for replacing 1g-1 of raw materials;
g0-2(m 1 ＝2，m 2 =0): raw materials1g-2 is used to replace 1g-1;
g0-3(m 1 ＝3，m 2 =0): 1g-3 is used for replacing 1g-1 of raw materials;
g0-4(m 1 ＝4，m 2 =0): 1g-4 is used for replacing 1g-1;
g0-5(m 1 ＝5，m 2 =0): 1g-5 is used as raw material instead of 1g-1.
When n is not 0 (taking gb1-1 as an example (R 1 Benzyl, n=1, m 1 ＝1，m 2 =0)), the specific preparation process is as follows:
(1) Preparation of the Compound of formula gb1-1: referring to the preparation of the formula g0-1, the raw materials are a compound shown in the formula 3cb and a compound shown in the formula 1g-1;
LC-MS(ESI)m/z(M+H) + called for C 35 H 42 N 7 O 4 + ＝624.3；found 624.3。
preparation of other intermediates (gb 1-0, gb1-2, gb1-3, gb1-4, gb 1-5) reference gb1-1:
gb1-0(m 1 ＝0，m 2 =0): 1g-0 is used for replacing 1g-1 of raw materials;
gb1-2(m 1 ＝2，m 2 =0): 1g-2 is used for replacing 1g-1 of raw materials;
gb1-3(m 1 ＝3，m 2 =0): 1g-3 is used for replacing 1g-1 of raw materials;
gb1-4(m 1 ＝4，m 2 =0): 1g-4 is used for replacing 1g-1;
gb1-5(m 1 ＝5，m 2 =0): 1g-5 is used as raw material instead of 1g-1.
Specific procedures for the synthesis of CDK12/13 ligand-end derivatives (formula h) (exemplified by h0-1 (R) 1 For benzyl, n=0, m=1)) is: substitution reaction of 2-methylpropan-2-yl 4- (5-bromopyridin-2-yl) piperazine-1-carboxylate (represented by formula 1 d) with tert-butyl 2- (2- (p-toluenesulfonyloxy) ethoxy) acetate (represented by formula 1 g-1) gives ({ 2- [4- (5-bromopyridin-2-yl) piperazin-1-yl)]Ethyl } oxy) acetic acid-2-methylpropan-2-yl ester (formula 1 h-1); then the ({ 2- [4- (5-bromopyridin-2-yl) piperazin-1-yl)]Ethyl } oxy) acetic acid-2-methylpropan-2-yl ester and the intermediate 2a undergo a coupling reaction, and finally the protective group is removed in an acidic environment, so as to obtain a compound shown as a compound h 0-1.
When n is 0, the specific preparation process (taking h0-1 as an example (R 1 Benzyl, n=0, m 1 ＝1，m 2 =0)) as follows:
(1) Preparation of intermediate 1h (taking 1h-1 as an example (n=0, m=1)):
200mg of 1d solution of 2N hydrogen chloride-ethyl acetate is added into a 50ml round bottom flask, after vacuum drying, 325mg of 1g-1 and 125mg of anhydrous triethylamine are added, the mixture is dissolved in anhydrous acetonitrile solution, the temperature is raised to 60 ℃ and stirred for 8 hours, after TLC detection of complete reaction of the raw materials, the mixture is washed twice with saturated salt water bath, the organic phase is separated, the organic phase is concentrated under reduced pressure, vacuum dried and rotary dried. 268mg of compound 1h-1 was isolated and purified by silica gel column with 81% yield.
LC-MS(ESI)m/z(M+H) + called for C 17 H 26 BrN 3 O 3 + ＝400.1；found 400.2。
Preparation of other intermediates (1 h-0, 1h-2, 1h-3, 1h-4, 1 h-5) reference 1h-1:
1h-0(m 1 ＝0，m 2 =0): 1g-0 is used for replacing 1g-1 of raw materials;
1h-2(m 1 ＝2，m 2 =0): 1g-2 is used for replacing 1g-1 of raw materials;
1h-3(m 1 ＝3，m 2 =0): 1g-3 is used for replacing 1g-1 of raw materials;
1h-4(m 1 ＝4，m 2 =0): 1g-4 is used for replacing 1g-1;
1h-5(m 1 ＝5，m 2 =0): 1g-5 is used as raw material instead of 1g-1.
(2) Preparation of the Compound of formula h0-1:
at 25ml round bottom80mg of intermediate 2a,70mg of intermediate 1h-1 and 142mg of cesium carbonate were added to the flask and dissolved in toluene: ethanol: water = 4:4:1, and after three nitrogen substitutions, 16mg Pd (PPh) 3 ) 4 After nitrogen substitution three times again, heating to 100 ℃, stirring for 3 hours, TLC detects complete reaction of the starting material, extraction three times with 20ml dichloromethane, concentration of the organic phase under reduced pressure, rotary drying. Pure 72mg was isolated on a silica gel column and treated with 50% TFA in dichloromethane to give compound h0-1 in 67% yield.
LC-MS(ESI)m/z(M+H) + called for C 39 H 44 N 8 O 4 + ＝689.4；found 689.5。
Preparation of other intermediates (h 0-0, h0-2, h0-3, h0-4, h 0-5) reference h0-1:
h0-0(m 1 ＝0，m 2 =0): 1h-0 is used for replacing 1h-1 of the raw materials;
h0-2(m 1 ＝2，m 2 =0): 1h-2 is used for replacing 1h-1 as a raw material;
h0-3(m 1 ＝3，m 2 =0): 1h-3 is used for replacing 1h-1 as a raw material;
h0-4(m 1 ＝4，m 2 =0): 1h-4 is used for replacing 1h-1 of raw materials;
h0-5(m 1 ＝5，m 2 =0): the raw material is replaced by 1h-5 for 1h-1.
When n is not 0 (taking hb1-1 as an example (R 1 Benzyl, n=1, m 1 ＝1，m 2 =0)), the specific preparation process is as follows:
(1) Preparation of the Compound of formula hb1-1: referring to the preparation of the formula h0-1, the raw materials are a compound shown in the formula 2b-1 and a compound shown in the formula 1h-1;
LC-MS(ESI)m/z(M+H) + called for C 40 H 45 N 8 O 4 + ＝701.4；found 701.4。
preparation of other intermediates (hb 1-0, hb1-2, hb1-3, hb1-4, hb 1-5) reference hb1-1:
hb1-0(m 1 ＝0，m 2 =0): 1h-0 is used for replacing 1h-1 of the raw materials;
hb1-2(m 1 ＝2，m 2 =0): 1h-2 is used for replacing 1h-1 as a raw material;
hb1-3(m 1 ＝3，m 2 =0): 1h-3 is used for replacing 1h-1 as a raw material;
hb1-4(m 1 ＝4，m 2 =0): 1h-4 is used for replacing 1h-1 of raw materials;
hb1-5(m 1 ＝5，m 2 =0): the raw material is replaced by 1h-5 for 1h-1.
Example 1: preparation of Compounds represented by formulas 1 to 86
Into a 10ml round bottom flask was charged 40mg of intermediate e0-1, 29mg of intermediate V, 37mg of HATU, 25mg DIPEA,3ml DMF. Stirring at room temperature for 20 min, and TLC detection of complete reaction of the raw materials, water washing and extraction with ethyl acetate. The organic phase was concentrated under reduced pressure and dried by rotary drying. With dichloromethane: methanol=20: 1 (v/v) was passed through a silica gel column to give the compound represented by formula 1 in a yield of 68%.
The preparation of (2S, 4 r) -1- ((S) -2-amino-3, 3-dimethylbutyryl) -4-hydroxy-N- ((S) -1- (4- (4-methylthiazol-5-yl) phenyl) ethyl) pyrrolidine-2-carboxamide (formula V) can be referred to as: proc Natl Acad Sci U S a.2016jun 28;113 (26):7124-9.
LC-MS(ESI)m/z(M+H) + called for C 58 H 67 N 10 O 7 S + ＝1047.5；found 1047.5。
1 H NMR(400MHz,Chloroform-d)δ8.63(s,1H),8.34(d,J＝1.8Hz,1H),8.26(dd,J＝2.0Hz,1H),7.75(dd,J＝8.6,2.6Hz,1H),7.53(d,J＝8.4Hz,2H),7.50(d,J＝7.7Hz,2H),7.42(dd,J＝8.9,2.3Hz,1H),7.39–7.34(m,5H),7.29–7.17(m,6H),6.92(dd,J＝8.6,0.7Hz,1H),6.27(dd,J＝8.8,0.8Hz,1H),5.16–5.04(m,1H),4.66–4.50(m,5H),4.38(d,J＝5.8Hz,2H),4.10(d,J＝2.5Hz,2H),3.93(t,J＝4.2Hz,2H),2.49(m,4H),2.10(d,J＝13.9Hz,2H),2.00(d,J＝8.4Hz,2H),1.89(s,3H),1.48(d,J＝6.9Hz,3H),1.45-1.41(m,2H),1.34–1.29(m,2H),1.25-1.24(m,5H),1.07(s,9H).13C NMR(100MHz,DMSO-d6)δ170.5,169.1,168.5,162.7,159.3,156.5,153.1,151.5,147.8,144.7,141.3,138.4,137.6,137.4,136.4,131.8,131.1,129.7,128.9,128.7,128.0,127.2,126.8,126.4,126.3,126.3,119.2,111.1,109.0,94.0,69.5,68.8,64.8,59.8,58.6,56.6,55.7,53.3,48.6,47.8,43.5,37.8,35.9,31.3,30.3,26.3,22.5,16.0.
Referring to the preparation method of formula 1, intermediate e0-2 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 10 O 8 S + ＝1091.5；found 1091.6。
Referring to the preparation method of formula 1, intermediate e0-3 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 62 H 75 N 10 O 9 S + ＝1135.5；found 1135.6。
Referring to the preparation method of formula 1, intermediate e0-4 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 64 H 79 N 10 O 10 S + ＝1179.6；found 1179.6。
Referring to the preparation method of formula 1, intermediate e0-5 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 66 H 83 N 10 O 11 S + ＝1223.6；found 1223.6。
Referring to the preparation method of formula 1, intermediate e0-0 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 56 H 63 N 10 O 6 S + ＝1003.5；found 1003.5。
1 H NMR(400MHz,Chloroform-d)δ8.65(s,1H),8.36(d,J＝2.5Hz,1H),8.26(d,J＝2.2Hz,1H),7.82(dd,J＝8.6,2.6Hz,1H),7.58–7.51(m,2H),7.45–7.33(m,6H),7.31–7.16(m,6H),7.09(dd,J＝8.8,7.0Hz,1H),6.94(d,J＝8.6Hz,1H),6.27(d,J＝8.8Hz,1H),5.14–5.05(m,2H),4.99(d,J＝15.6Hz,1H),4.82–4.64(m,3H),4.54(s,1H),4.39(m,2H),4.09(m,J＝16.7,9.3Hz,1H),3.65(dd,J＝11.3,3.8Hz,1H),3.51(m,J＝24.5Hz,1H),2.50(s,3H),2.10(d,J＝12.2Hz,2H),2.00(d,J＝8.9Hz,2H),1.86(m,J＝14.3,7.2Hz,4H),1.48(d,J＝6.9Hz,3H),1.39(m,J＝21.1Hz,4H),1.01(s,9H).
Referring to the preparation method of formula 1, intermediate g0-1 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 57 H 72 N 11 O 6 S + ＝1038.5；found 1038.6。
1 H NMR(400MHz,Chloroform-d)δ8.67(s,1H),8.26(d,J＝2.3Hz,1H),7.50(d,J＝5.7,3.3Hz,1H),7.46(dd,J＝8.8,2.3Hz,1H),7.35(m,J＝7.6,6.8,4.2Hz,6H),7.22–7.16(m,3H),7.03–7.00(m,2H),6.88(d,J＝8.7Hz,2H),6.27(d,J＝8.8Hz,1H),5.21(d,J＝8.1Hz,1H),5.08(t,J＝7.0Hz,1H),4.73(t,J＝7.8Hz,1H),4.57(d,J＝8.6Hz,1H),4.56–4.44(m,3H),4.36(d,J＝5.9Hz,2H),4.11–4.00(m,3H),3.68–3.59(m,4H),3.46(s,1H),3.25–3.21(m,4H),2.68(m,J＝6.7Hz,4H),2.51(s,3H),2.50–2.44(m,1H),2.09(d,J＝11.7Hz,2H),1.95(d,J＝12.0Hz,2H),1.47(d,J＝7.0Hz,3H),1.43–1.34(m,4H),1.07(s,9H).
Referring to the preparation method of formula 1, intermediate g0-2 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 59 H 76 N 11 O 7 S + ＝1082.6；found 1082.6。
1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.26(d,J＝2.2Hz,1H),7.45(d,J＝7.9Hz,1H),7.37(q,J＝8.1Hz,6H),7.25–7.14(m,4H),7.00(d,J＝8.6Hz,2H),6.86(d,J＝8.6Hz,2H),6.25(d,J＝8.9Hz,1H),5.18(d,J＝8.1Hz,1H),5.07(t,J＝7.0Hz,1H),4.73(t,J＝7.8Hz,1H),4.57(d,J＝8.6Hz,1H),4.54–4.42(m,3H),4.34(d,J＝5.9Hz,2H),4.11–4.00(m,3H),3.73–3.57(m,8H),3.47(s,1H),3.25–3.21(m,4H),2.68(m,J＝6.7Hz,4H),2.51(s,3H),2.50–2.44(m,1H),2.09–1.97(m,4H),1.96–1.89(d,2H),1.47(d,J＝7.0Hz,3H),1.43–1.34(m,4H),1.09(s,9H).
Referring to the preparation method of formula 1, intermediate g0-3 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 61 H 80 N 11 O 8 S + ＝1126.6；found 1126.6。
1 H NMR(400MHz,Chloroform-d)δ8.67(s,1H),8.27(d,J＝2.3Hz,1H),7.53(dd,J＝5.7,3.3Hz,1H),7.46(dd,J＝8.8,2.3Hz,1H),7.37(qd,J＝7.6,6.8,4.2Hz,6H),7.22–7.16(m,3H),7.03–7.00(m,2H),6.88(d,J＝8.7Hz,2H),6.27(d,J＝8.8Hz,1H),5.34(t,J＝3.4Hz,1H),5.07(m,J＝17.4,15.9,8.8Hz,2H),4.76(t,J＝8.0Hz,1H),4.57(d,J＝8.6Hz,1H),4.52–4.42(m,2H),4.34(d,J＝5.9Hz,2H),4.21(t,J＝6.0Hz,1H),4.11–4.02(m,3H),3.72–3.64(m,8H),3.28(m,2H),2.80(m,4H),2.52(s,3H),2.46(m,1H),2.30(t,J＝7.4Hz,2H),2.21(t,J＝7.7Hz,2H),2.08(d,J＝11.7Hz,2H),1.93(d,J＝12.0Hz,2H),1.66(m,J＝21.9,14.5,7.6,7.0Hz,4H),1.48(d,J＝6.9Hz,3H),1.44–1.37(m,4H),1.04(s,9H).
Referring to the preparation method of formula 1, intermediate g0-0 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 55 H 68 N 11 O 5 S + ＝994.5；found 994.5。
1 H NMR(400MHz,Chloroform-d)δ8.67(s,1H),8.32(d,J＝2.2Hz,1H),7.50(dd,J＝5.7,3.2Hz,1H),7.45(dd,J＝8.6,2.5Hz,1H),7.39(m,6H),7.20–7.14(m,3H),7.03–7.00(m,2H),6.92(d,J＝8.7Hz,2H),6.44(d,J＝8.0Hz,1H),6.27(d,J＝8.6Hz,1H),5.23(d,J＝7.8Hz,1H),5.11–5.05(m,1H),4.76(t,J＝7.9Hz,1H),4.69–4.56(m,1H),4.54(d,J＝8.5Hz,2H),4.48–4.42(m,1H),4.40(d,J＝5.7Hz,2H),4.20–4.08(m,2H),3.76–3.50(m,7H),3.08(d,J＝3.3Hz,2H),2.68(t,J＝5.6Hz,4H),2.52(s,3H),2.12(d,J＝9.0Hz,2H),1.99(d,J＝10.8Hz,2H),1.50(d,J＝7.1Hz,3H),1.38–1.30(m,4H),1.08(s,9H).
Referring to the preparation method of formula 1, intermediate e0-a is used instead of intermediate e0-1. Wherein, the preparation method of the intermediate e0-a refers to the formula e0-1, and the intermediate 1e-1 is replaced by the intermediate 1 e-a; the preparation of intermediate 1e-a is described with reference to formula 1e-1, replaced with tert-butyl 2- (4-hydroxypiperidin-1-yl) acetate.
LC-MS(ESI)m/z(M+H) + called for C 61 H 72 N 11 O 6 S + ＝1086.5；found 1086.6。
1 H NMR(400MHz,Chloroform-d)δ8.64(s,1H),8.32(d,J＝2.5Hz,1H),8.25(d,J＝2.2Hz,1H),7.76(dd,J＝8.6,2.6Hz,1H),7.54(d,J＝8.3Hz,2H),7.50(d,J＝8.2Hz,1H),7.41(dd,J＝8.9,2.3Hz,1H),7.39–7.33(m,4H),7.30–7.15(m,7H),6.80(d,J＝8.6Hz,1H),6.26(d,J＝8.8Hz,1H),5.19(s,1H),5.14(d,J＝8.1Hz,1H),5.06(p,J＝7.0Hz,1H),4.75(t,J＝8.0Hz,1H),4.61(tt,J＝11.6,3.6Hz,1H),4.48(d,J＝7.8Hz,2H),4.41–4.35(m,3H),4.12(d,J＝11.5Hz,1H),3.59(dd,J＝11.4,3.5Hz,1H),3.55(s,1H),3.23(s,1H),2.96(d,J＝16.4Hz,2H),2.63(m,J＝21.9Hz,2H),2.50(s,3H),2.46(m,J＝6.9Hz,1H),2.14(m,2H),2.09(d,J＝14.3Hz,2H),1.99(d,J＝11.6Hz,2H),1.47(d,J＝6.9Hz,3H),1.41(m,5H),1.37–1.27(m,4H),1.06(s,9H).
Referring to the preparation method of formula 1, intermediate e0-b is used instead of intermediate e0-1. Wherein, the preparation method of the intermediate e0-b refers to the formula e0-1, and the intermediate 1e-1 is replaced by the intermediate 1 e-b; preparation of intermediate 1e-b referring to formula 1e-1, tert-butyl 2- (2-hydroxyethoxy) acetate is replaced with tert-butyl 2- (4- (hydroxymethyl) piperidin-1-yl) acetate.
LC-MS(ESI)m/z(M+H) + called for C 62 H 74 N 11 O 6 S + ＝1100.6；found 1100.6。
1 H NMR(400MHz,Chloroform-d)δ8.65(s,1H),8.36(dd,J＝2.5,0.8Hz,1H),8.27(dd,J＝2.3,0.7Hz,1H),7.78(dd,J＝8.6,2.6Hz,1H),7.55(d,J＝8.5Hz,2H),7.46(dd,J＝8.9,2.2Hz,2H),7.42–7.34(m,5H),7.33–7.26(m,2H),7.24–7.16(m,4H),6.83(dd,J＝8.6,0.7Hz,1H),6.28(dd,J＝8.9,0.8Hz,1H),5.07(p,J＝7.0Hz,1H),4.93(d,J＝7.9Hz,1H),4.77(t,J＝8.0Hz,1H),4.67–4.57(m,1H),4.51(s,1H),4.45(d,J＝8.3Hz,1H),4.38(s,3H),4.22(d,J＝6.4Hz,3H),4.16–4.07(m,2H),3.59(dd,J＝11.4,3.5Hz,1H),3.09(d,J＝11.4Hz,1H),2.97(d,J＝10.8Hz,3H),2.52(s,4H),2.33(dd,J＝18.0,7.5Hz,2H),2.11(d,J＝11.5Hz,3H),2.00(d,J＝11.0Hz,1H),1.91(d,J＝8.9Hz,2H),1.74(m,7H),1.47(d,J＝7.0Hz,3H),1.43–1.36(m,4H),1.07(s,9H).
Referring to the preparation method of formula 1, the intermediate e0-c is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate e0-c refers to the formula e0-1, and the intermediate 1e-1 is replaced by the intermediate 1 e-c; the preparation of intermediate 1e-c refers to formula 1e-1, substituting tert-butyl 2- (2-hydroxyethoxy) acetate with tert-butyl 6-hydroxy-3-alkynoate.
LC-MS(ESI)m/z(M+H) + called for C 61 H 68 N 9 O 6 S + ＝1054.5；found 1054.5。
Referring to the preparation method of formula 1, the intermediate e0-d is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate e0-d refers to the formula e0-1, and the intermediate 1e-1 is replaced by the intermediate 1 e-d; the preparation of intermediate 1e-d refers to formula 1e-1, substituting tert-butyl (3-hydroxyazetidin-1-yl) acetate for tert-butyl 2- (2-hydroxyethoxy) acetate.
LC-MS(ESI)m/z(M+H) + called for C 59 H 68 N 11 O 6 S + ＝1058.5；found 1058.5。
Referring to the preparation method of formula 1, the intermediate e0-e is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate e0-e refers to the formula e0-1, and the intermediate 1e-1 is replaced by the intermediate 1 e-e; the preparation method of the intermediate 1e-e comprises the steps that the intermediate 1a and butyl-3-tert-butyl alkynoate undergo a gashira coupling reaction, and specific conditions can be referred to in the literature: chemistry Letters,2005,vol.34,issue 9,1294-1295.
LC-MS(ESI)m/z(M+H) + called for C 58 H 63 N 10 O 5 S + ＝1011.5；found 1011.6。
1 H NMR(400MHz,Chloroform-d)δ8.74–8.69(d,1H),8.65(s,1H),8.25(d,J＝2.2Hz,1H),7.80(dd,J＝8.1,2.4Hz,1H),7.62–7.56(d,2H),7.46(d,J＝8.1Hz,1H),7.44–7.39(m,2H),7.37(m,J＝5.8Hz,4H),7.30–7.16(m,6H),6.59(d,J＝8.8Hz,1H),6.27(d,J＝8.8Hz,1H),5.14–5.03(m,2H),4.73(t,J＝8.0Hz,1H),4.68(d,J＝8.9Hz,1H),4.61(m,1H),4.53(s,1H),4.37(s,3H),3.62(dd,J＝11.3,3.6Hz,1H),3.53(m,1H),2.88–2.69(m,2H),2.62–2.51(m,3H),2.50(s,3H)2.11(d,J＝6.1Hz,2H),2.04(m,5H),1.99(d,J＝13.2Hz,2H),1.47(d,J＝6.9Hz,3H),1.36(m,J＝39.3,2.9Hz,5H),1.05(s,9H).
Referring to the preparation method of formula 1, the intermediate e0-f is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate e0-f refers to the formula e0-1, and the intermediate 1e-1 is replaced by the intermediate 1 e-f; the preparation of intermediate 1e-f is described with reference to formula 1e-1, substituting tert-butyl 2- (2-hydroxyethoxy) acetate with tert-butyl 5-hydroxypentane-3-isocyanate.
LC-MS(ESI)m/z(M+H) + called for C 59 H 65 N 10 O 6 S + ＝1041.5；found 1041.6。
Referring to the preparation method of formula 1, intermediate h0-a is used instead of intermediate e0-1. Wherein, the preparation method of the intermediate h0-a refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-a; the preparation of intermediate 1h-a is described with reference to formula 1d, substituting tert-butyl piperidine-4-carboxylate for tert-butyl piperazine-1-carboxylate.
LC-MS(ESI)m/z(M+H) + called for C 60 H 70 N 11 O 5 S + ＝1056.5；found 1056.6。
1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.40(d,J＝2.6Hz,1H),8.24(d,J＝2.3Hz,1H),7.68(dd,J＝8.9,2.6Hz,1H),7.53(d,J＝8.4Hz,2H),7.42–7.34(m,5H),7.27(d,J＝5.5Hz,3H),7.24–7.16(m,4H),6.73(d,J＝9.0Hz,1H),6.35(d,J＝8.7Hz,1H),6.26(d,J＝8.8Hz,1H),5.38(d,J＝8.0Hz,1H),5.14–5.02(m,1H),4.71(t,J＝7.8Hz,1H),4.59(m,J＝8.7Hz,2H),4.53–4.49(m,1H),4.46(t,J＝5.9Hz,1H),4.37(m,J＝6.0Hz,4H),4.04(d,J＝11.3Hz,1H),3.66–3.58(dd,1H),3.54(s,1H),2.99–2.86(m,2H),2.51(s,3H),2.49–2.36(m,2H),2.06(m,J＝15.1,9.3Hz,3H),2.02–1.87(m,4H),1.79(m,J＝4.0Hz,2H),1.49(d,3H),1.41(m,J＝3.1Hz,2H),1.39–1.32(m,2H),1.25(m,2H),1.05(s,9H).
Referring to the preparation method of formula 1, intermediate h0-0 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
1H NMR(400MHz,Chloroform-d)δ8.64(s,1H),8.43(d,J＝2.5Hz,1H),8.25(dd,J＝2.3,0.7Hz,1H),7.93(d,J＝8.5Hz,1H),7.71(dd,J＝8.8,2.6Hz,1H),7.54(d,J＝8.4Hz,2H),7.49(d,J＝7.7Hz,1H),7.44–7.34(m,4H),7.31–7.14(m,7H),6.75(d,J＝8.8Hz,1H),6.27(dd,J＝8.9,0.8Hz,1H),5.23(d,J＝8.0Hz,1H),5.13–5.02(m,1H),4.76(t,J＝7.9Hz,1H),4.67–4.56(m,1H),4.51(d,J＝8.6Hz,2H),4.48–4.42(m,1H),4.38(d,J＝5.8Hz,2H),4.18–4.06(m,2H),3.73–3.59(m,7H),3.11(d,J＝3.5Hz,2H),2.70(t,J＝5.4Hz,4H),2.50(s,3H),2.10(d,J＝8.7Hz,2H),1.99(d,J＝11.2Hz,2H),1.47(d,J＝6.9Hz,3H),1.42–1.29(m,4H),1.08(s,9H).
Referring to the preparation method of formula 1, intermediate h0-b is used instead of intermediate e0-1. Wherein, the preparation method of the intermediate h0-b refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-b; preparation of intermediate 1h-b referring to formula 1d, tert-butyl piperazine-1-carboxylate was replaced with 4- (hexahydropyridin-4-yl) but-3-ynoic acid-2-methylpropan-2-yl ester.
LC-MS(ESI)m/z(M+H) + called for C 63 H 72 N 11 O 5 S + ＝1094.5；found 1094.6。
Referring to the preparation method of formula 1, intermediate g0-a is used instead of intermediate e0-1. Wherein the preparation method of the intermediate g0-a refers to a formula 1c, and 4- (hexahydropyridine-4-yl) but-3-alkynoic acid-2-methylpropan-2-yl ester is used for replacing piperazine-1-carboxylic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 58 H 69 N 10 O 5 S + ＝1017.5；found 1017.5。
Referring to the preparation method of formula 1, intermediate h0-c is used instead of intermediate e0-1. Wherein, the preparation method of the intermediate h0-c refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-c; the intermediate 1h-c was prepared by substituting tert-butyl piperazine-1-carboxylate with tert-butyl 1- (piperidine-4-carbonyl) piperidine-4-carboxylate according to formula 1 d.
LC-MS(ESI)m/z(M+H) + called for C 66 H 79 N 12 O 6 S + ＝1167.6；found 1167.7。
Referring to the preparation method of formula 1, intermediate g0-b is used instead of intermediate e0-1. Wherein the preparation method of the intermediate g0-b refers to the formula 1c, and the piperazine-1-carboxylic acid tert-butyl ester is replaced by the 1- (piperidine-4-carbonyl) piperidine-4-carboxylic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 61 H 76 N 11 O 6 S + ＝1090.6；found 1090.7。
Referring to the preparation method of formula 1, the intermediate h0-d is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate h0-d refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-d; preparation of intermediate 1h-d referring to formula 1d, tert-butyl piperazine-1-carboxylate was replaced with tert-butyl 4- (azetidin-3-yl) but-3-ynoate.
LC-MS(ESI)m/z(M+H) + called for C 61 H 68 N 11 O 5 S + ＝1066.5；found 1066.5。
Referring to the preparation method of formula 1, intermediate g0-c is used instead of intermediate e0-1. Wherein the preparation method of the intermediate g0-c refers to the formula 1c, and the tert-butyl piperazine-1-carboxylate is replaced by the tert-butyl 4- (azetidin-3-yl) but-3-ynoate.
LC-MS(ESI)m/z(M+H) + called for C 56 H 65 N 10 O 5 S + ＝989.5；found 989.5。
Referring to the preparation method of formula 1, intermediate g0-d is used instead of intermediate e0-1. The preparation method of the intermediate g0-d comprises the following steps: intermediate 1a and tert-butyl 2- (4-ethynyl-1-piperidine) acetate undergo a sonogashira coupling reaction, and specific conditions can be referred to in the literature: chemistry Letters,2005,vol.34,issue 9,1294-1295.
LC-MS(ESI)m/z(M+H) + called for C 58 H 69 N 10 O 5 S + ＝1017.5；found 1017.5。
Referring to the preparation method of formula 1, intermediate g0-e is used instead of intermediate e0-1. The preparation method of the intermediate g0-e comprises the following steps: intermediate 1a and tert-butyl 2- (4-ethynyl-1-piperidine) acetate undergo a sonogashira coupling reaction, and specific conditions can be referred to in the literature: chemistry Letters,2005,vol.34,issue 9,1294-1295.
LC-MS(ESI)m/z(M+H) + called for C 56 H 65 N 10 O 5 S + ＝989.5；found 989.5。
Referring to the preparation method of formula 1, intermediate g0-f is used instead of intermediate e0-1. Wherein the preparation method of the intermediate g0-f refers to the formula 1c, and the piperazine-1-carboxylic acid tert-butyl ester is replaced by the 2-amino spiro [3.3] heptane-6-carboxylic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 57 H 69 N 10 O 5 S + ＝1005.5；found 1005.6。
Referring to the preparation method of formula 1, the intermediate h0-e is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate h0-e refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-e; the preparation of intermediate 1h-e was carried out with reference to formula 1d, substituting tert-butyl piperazine-1-carboxylate with tert-butyl 2-aminospiro [3.3] heptane-6-carboxylate.
LC-MS(ESI)m/z(M+H) + called for C 62 H 72 N 11 O 5 S + ＝1082.5；found 1082.6。
Referring to the preparation method of formula 1, the intermediate h0-f is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate h0-f refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-f; the preparation of intermediate 1h-f refers to formula 1d, substituting tert-butyl 2- (azetidin-3-yl) acetate for tert-butyl piperazine-1-carboxylate.
LC-MS(ESI)m/z(M+H) + called for C 59 H 68 N 11 O 5 S + ＝1042.5；found 1042.5。
Referring to the preparation method of formula 1, intermediate h0-g is used instead of intermediate e0-1. Wherein, the preparation method of the intermediate h0-g refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-g; the preparation of intermediate 1h-g refers to formula 1d, substituting pyrrolidine-3-acetic acid tert-butyl ester for piperazine-1-carboxylic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 60 H 70 N 11 O 5 S + ＝1056.5；found 1056.6。
Referring to the preparation method of formula 1, the intermediate h0-h is used instead of the intermediate e0-1. Wherein, the preparation method of the intermediate h0-h refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-h; the preparation method of the intermediate 1H-H refers to a formula 1H-1, and the raw materials are 4- (4-bromo-1H-pyrazol-1-yl) piperidine and bromoacetic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 59 H 71 N 12 O 5 S + ＝1059.5；found 1059.6。
70mg of intermediate a0-1 and 35mg of intermediate P were added to a 10ml round bottom flask and dissolved in 4ml anhydrous DMSO. To the system was slowly added dropwise 95mg of DIPEA with stirring at room temperature, followed by heating to 100 ℃ and reaction for 8h. After the completion of the reaction, TLC was followed by washing with water and extraction with ethyl acetate. The organic phase was concentrated under reduced pressure and dried by rotary drying. With dichloromethane: methanol=20: 1 (v/v) was passed through a silica gel column to give the compound represented by formula 32 in 36% yield.
The preparation of 2- (2, 6-dioxopiperidin-3-yl) -4-fluoroisoindoline-1, 3-dione (formula P) can be referred to in the literature: organic Letters 22,10,3838-3841 (2019).
LC-MS(ESI)m/z(M+H) + called for C 48 H 48 N 9 O 7 S + ＝862.36；found 862.4。
Referring to the preparation method of formula 32, intermediate a0-2 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 50 H 52 N 9 O 8 + ＝906.39；found 906.4。
Referring to the preparation method of formula 32, intermediate a0-3 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 52 H 56 N 9 O 9 + ＝950.4；found 950.5。
Referring to the preparation of formula 32, intermediate a0-4 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 54 H 60 N 9 O 10 + ＝994.4；found 994.5。
Referring to the preparation of formula 32, intermediate a0-5 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 56 H 64 N 9 O 11 + ＝1038.5；found 1038.6。
Referring to the preparation method of formula 32, intermediate a0-0 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 46 H 44 N 9 O 6 + ＝818.3；found 818.4。
Referring to the preparation method of formula 32, intermediate c0-1 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 47 H 53 N 10 O 6 + ＝853.4；found 853.4。
Referring to the preparation method of formula 32, intermediate c0-2 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 49 H 57 N 10 O 7 + ＝897.4；found 897.5。
Referring to the preparation of formula 32, intermediate c0-3 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 51 H 61 N 10 O 8 + ＝941.5；found 941.6。
Referring to the preparation of formula 32, intermediate c0-4 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 53 H 65 N 10 O 9 + ＝985.5；found 985.6。
Referring to the preparation method of formula 32, intermediate c0-0 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 45 H 49 N 10 O 5 + ＝809.4；found 809.4。
Referring to the preparation of formula 32, intermediate a0-3-Et is used instead of intermediate a0-1. Wherein the preparation method of the intermediate a0-3-Et refers to the formulas a0-3, the intermediate 5aa-Et is used instead of the intermediate 5aa, wherein the preparation method of the intermediate 5aa-Et refers to the formula 5aa, and the ethyl isocyanate is used instead of the intermediate 4aa.
LC-MS(ESI)m/z(M+H) + called for C 47 H 54 N 9 O 9 + ＝888.4；found 888.4。
Referring to the preparation of formula 32, intermediate h0-0-Et is used instead of intermediate e0-1. Wherein the preparation method of the intermediate h0-0-Et refers to the formula h0-0, the intermediate 5aa-Et is used instead of the intermediate 5aa, wherein the preparation method of the intermediate 5aa-Et refers to the formula 5aa, and the ethyl isocyanate is used instead of the intermediate 4aa.
LC-MS(ESI)m/z(M+H) + called for C 55 H 69 N 12 O 5 S + ＝1009.5；found 1009.5。
Referring to the preparation method of formula 32, intermediate h0-0-N is used instead of intermediate e0-1. Wherein the preparation method of the intermediate h0-0-N refers to the formula h0-0, the intermediate 1d-N is used for replacing the intermediate 1d, wherein the preparation method of the intermediate 1d-N refers to the formula 1d, and 5-bromo-2-fluoropyrimidine is used for replacing 2-fluoro-5-bromopyridine.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.55(s,2H),8.27(d,J＝2.2Hz,1H),7.92(d,J＝8.4Hz,1H),7.54–7.35(m,8H),7.32–7.17(m,6H),6.27(d,J＝8.7Hz,1H),5.06(dt,J＝14.4,7.5Hz,2H),4.77(t,J＝7.8Hz,1H),4.62(s,1H),4.53(s,1H),4.49(d,J＝8.4Hz,1H),4.38(s,3H),4.17(d,J＝11.5Hz,1H),3.94(s,4H),3.61(dd,J＝11.4,3.7Hz,1H),3.11(d,J＝2.7Hz,2H),2.66(t,J＝5.1Hz,4H),2.58(ddd,J＝13.5,7.5,4.7Hz,1H),2.52(s,3H),2.10(d,J＝12.3Hz,2H),2.00(d,J＝11.7Hz,2H),1.78(s,2H),1.48(d,J＝6.9Hz,3H),1.45–1.41(m,2H),1.38–1.27(m,4H),1.09(s,9H).
Referring to the preparation method of formula 32, intermediate a0-1-N is used instead of intermediate a0-1. The preparation method of the intermediate a0-1-N refers to the formula a0-1, and the intermediate 5a-1-N is used for replacing the intermediate 5a-1, wherein the preparation method of the intermediate 5a-1-N is that 2c-1 and 5-bromo-2-hydroxypyridine undergo nucleophilic reaction to obtain the intermediate.
LC-MS(ESI)m/z(M+H)+called for C 48 H 48 N 9 O 7 + ＝862.4；found 862.4。
Referring to the preparation method of formula 32, intermediate a0-2-N is used instead of intermediate a0-1. The preparation method of the intermediate a0-2-N refers to the formula a0-1, and the intermediate 5a-2-N is used for replacing the intermediate 5a-1, wherein the preparation method of the intermediate 5a-2-N is that 2c-2 and 5-bromo-2-hydroxypyridine undergo nucleophilic reaction.
LC-MS(ESI)m/z(M+H) + called for C 50 H 52 N 9 O 8 + ＝906.3；found 906.4。
Referring to the preparation method of formula 32, intermediate a0-3-N is used instead of intermediate a0-1. The preparation method of the intermediate a0-3-N refers to the formula a0-1, and the intermediate 5a-3-N is used for replacing the intermediate 5a-1, wherein the preparation method of the intermediate 5a-3-N is that 2c-3 and 5-bromo-2-hydroxypyridine undergo nucleophilic reaction.
LC-MS(ESI)m/z(M+H) + called for C 52 H 56 N 9 O 9 + ＝950.4；found 950.5。
Referring to the preparation method of formula 32, intermediate a0-4-N is used instead of intermediate a0-1. The preparation method of the intermediate a0-4-N refers to the formula a0-1, and the intermediate 5a-4-N is used for replacing the intermediate 5a-1, wherein the preparation method of the intermediate 5a-4-N is that 2c-4 and 5-bromo-2-hydroxypyridine undergo nucleophilic reaction.
LC-MS(ESI)m/z(M+H) + called for C 54 H 60 N 9 O 10 + ＝994.4；found 994.5。
Referring to the preparation method of formula 32, intermediate a0-4-N is used instead of intermediate a0-1. The preparation method of the intermediate a0-4-N refers to the formula a0-1, and the intermediate 5a-4-N is used for replacing the intermediate 5a-1, wherein the preparation method of the intermediate 5a-4-N is that 2c-4 and 5-bromo-2-hydroxypyridine undergo nucleophilic reaction.
LC-MS(ESI)m/z(M+H) + called for C 56 H 64 N 9 O 11 + ＝1038.4；found 1038.5。
In a 10ml round bottom flask was added 18mg of intermediate V,6mg of CDI and dissolved in 2ml of anhydrous dichloromethane. Stirring for 1 hour at room temperature, then adding 18mg of intermediate g0-h, and reacting for 18h. After the completion of the reaction, TLC was followed by washing with water and extraction with ethyl acetate. The organic phase was concentrated under reduced pressure and dried by rotary drying. With dichloromethane: methanol=20: 1 (v/v) was passed through a silica gel column to give the compound represented by formula 51 in 47% yield.
Wherein the preparation method of the intermediate g0-h refers to the formula 1c, and 9-diazaspiro [5.5] undecane-3-carboxylic acid tert-butyl ester is used for replacing piperazine-1-carboxylic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 59 H 74 N 11 O 5 S + ＝1048.5；found 1048.6。
1 H NMR(400MHz,Chloroform-d)δ8.67(s,1H),7.63(d,J＝7.9Hz,1H),7.46(dd,J＝8.8,2.3Hz,1H),7.43–7.36(m,4H),7.29–7.15(m,4H),7.04–6.98(m,2H),6.92–6.86(m,2H),6.26(dd,J＝8.8,0.8Hz,1H),5.08(p,J＝7.1Hz,1H),4.99(d,J＝8.9Hz,1H),4.92(d,J＝8.0Hz,1H),4.80(t,J＝8.2Hz,1H),4.60–4.50(m,1H),4.46(s,1H),4.42–4.30(m,5H),3.68(s,1H),3.54–3.47(m,2H),3.43–3.30(m,4H),3.20(t,J＝5.7Hz,4H),2.59(ddd,J＝13.1,8.0,4.3Hz,1H),2.53(s,3H),2.09(m,J＝13.8,9.0Hz,3H),1.94(d,J＝12.0Hz,2H),1.53(t,J＝5.8Hz,4H),1.46(d,J＝7.0Hz,2H),1.41–1.28(m,4H),1.07(s,9H).
Referring to the preparation method of formula 51, intermediates h0-i are used instead of intermediates g0-h. Wherein, the preparation method of the intermediate h0-i refers to the formula h0-1, and the intermediate 1h-1 is replaced by the intermediate 1 h-i; preparation of intermediate 1h-g referring to formula 1d, tert-butyl piperazine-1-carboxylate was replaced with tert-butyl 3, 9-diazaspiro [5.5] undecane-3-carboxylate.
LC-MS(ESI)m/z(M+H) + called for C 64 H 77 N 12 O 5 S + ＝1125.6；found 1125.6。
Referring to the preparation method of formula 51, intermediates g0-j are used instead of intermediates g0-h. Wherein the preparation method of the intermediate g0-j refers to the formula 1c, and tert-butyl piperazine-1-carboxylic acid tert-butyl ester is replaced by tert-butyl piperazine-1-carboxylic acid ester.
LC-MS(ESI)m/z(M+H) + called for C 54 H 66 N 11 O 5 + ＝980.5；found 980.5。
1 H NMR(400MHz,Chloroform-d)δ8.66(s,1H),8.43(d,J＝2.5Hz,1H),8.26(dd,J＝2.3,0.7Hz,1H),7.72(dd,J＝8.8,2.5Hz,1H),7.60(d,J＝7.9Hz,1H),7.57–7.52(m,2H),7.43–7.36(m,5H),7.31–7.26(m,2H),7.24–7.17(m,5H),6.70(d,J＝8.8Hz,1H),6.26(dd,J＝8.9,0.8Hz,1H),5.10(dt,J＝14.5,7.3Hz,2H),4.78(t,J＝8.1Hz,1H),4.62(td,J＝11.8,6.0Hz,1H),4.49(s,1H),4.45–4.35(m,4H),4.25(d,J＝11.6Hz,1H),3.71–3.61(m,4H),3.60–3.50(m,4H),2.57(m,J＝8.3,4.9Hz,1H),2.52(s,3H),2.15–2.07(d,2H),2.03–1.96(d,2H),1.89(m,2H),1.47(d,J＝6.9Hz,3H),1.43–1.22(m,6H),1.08(s,9H).
Referring to the preparation method of formula 51, intermediates g0-k are used instead of intermediates g0-h. Wherein the preparation method of the intermediate g0-k refers to the formula 1c, and the piperazine-1-carboxylic acid tert-butyl ester is replaced by the 2, 7-diazaspiro [3.5] nonane-7-carboxylic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 57 H 70 N 11 O 5 S + ＝1020.5；found 1020.6。
Referring to the preparation method of formula 51, intermediates g0-l are used instead of intermediates g0-h. Wherein the preparation method of the intermediate g0-l refers to the formula 1c, and 2-tert-butoxycarbonyl-2, 7-diazaspiro [3.5] nonane is used for replacing piperazine-1-carboxylic acid tert-butyl ester.
LC-MS(ESI)m/z(M+H) + called for C 57 H 70 N 11 O 5 S + ＝1020.5；found 1020.6。
1 H NMR(400MHz,Chloroform-d)δ8.67(s,1H),8.27(d,J＝2.2Hz,1H),7.56(d,J＝7.9Hz,1H),7.45(dd,J＝8.9,2.3Hz,2H),7.38(dd,J＝8.7,6.3Hz,4H),7.27(s,1H),7.01(d,J＝8.5Hz,2H),6.89(d,J＝8.8Hz,2H),6.26(d,J＝8.8Hz,1H),5.08(p,J＝7.1Hz,1H),4.97(d,J＝7.9Hz,1H),4.77(t,J＝8.0Hz,1H),4.70(d,J＝9.3Hz,1H),4.58–4.50(m,1H),4.48(d,J＝4.8Hz,1H),4.37(dt,J＝14.1,4.0Hz,4H),4.23(d,J＝11.6Hz,1H),3.74–3.64(m,5H),3.57–3.46(m,2H),3.16(t,J＝5.4Hz,4H),2.53(s,3H),2.12–1.83(m,10H),1.47(d,J＝6.9Hz,3H),1.27(d,J＝12.3Hz,12H),1.05(s,9H).
Referring to the preparation method of formula 1, intermediate e0-1-N is used instead of intermediate a0-1. Wherein the preparation method of the intermediate e0-1-N refers to the formula e0-1, and the intermediate 1e-1-N is used for replacing the intermediate 1e-1, wherein the preparation method of the intermediate 1e-1-N is that 1g-1 and 5-bromo-2-hydroxypyridine undergo nucleophilic reaction.
LC-MS(ESI)m/z(M+H) + called for C 58 H 67 N 10 O 7 S + ＝1047.5；found 1047.5。
1 H NMR(400MHz,Chloroform-d)δ8.98(s,1H),8.43(d,J＝7.8Hz,1H),8.28(d,J＝2.4Hz,1H),8.18(d,J＝2.9Hz,1H),7.89(dd,J＝9.5,2.6Hz,1H),7.69(d,J＝8.5Hz,2H),7.59(dd,J＝8.9,2.5Hz,1H),7.53–7.46(m,1H),7.45–7.40(m,2H),7.38–7.31(m,3H),7.29–7.21(m,4H),7.20–7.16(m,3H),6.52(d,J＝9.5Hz,1H),6.46(d,J＝8.9Hz,1H),5.84(t,J＝6.2Hz,1H),5.14(d,J＝3.5Hz,1H),4.95–4.84(m,1H),4.49(d,J＝9.5Hz,1H),4.43(t,J＝8.2Hz,1H),4.36–4.19(m,4H),4.17(d,J＝5.9Hz,2H),4.05–3.94(m,2H),3.88–3.76(m,2H),3.56(s,2H),2.45(s,3H),2.10–2.00(m,1H),1.95–1.73(m,5H),1.35(d,J＝7.0Hz,3H),1.32–1.10(m,5H),0.84(s,9H)；13C NMR(100MHz,DMSO-d6)δ170.5,169.0,168.1,160.8,159.3,156.5,153.1,151.5,147.8,144.7,141.3,139.0,138.4,137.3,136.7,135.5,131.6,131.1,129.7,128.9,128.1,126.8,126.3,126.3,126.2,119.7,119.1,117.0,108.9,94.0,69.4,68.8,68.3,58.6,56.5,55.8,53.3,48.6,48.5,47.8,43.5,37.7,35.6,31.3,30.3,26.2,22.5,16.0.
Referring to the preparation method of formula 1, intermediate e0-2-N is used instead of intermediate a0-1. Wherein the preparation method of the intermediate e0-2-N refers to the formula e0-1, the intermediate 1e-2-N is used for replacing the intermediate 1e-2, wherein the preparation method of the intermediate 1e-2-N refers to the formula 1e-1-N, and the intermediate 1g-2 is used for replacing the intermediate 1g-1.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 10 O 8 S + ＝1091.5；found 1091.6。
Referring to the preparation method of formula 1, intermediate e0-3-N is used instead of intermediate a0-1. Wherein the preparation method of the intermediate e0-3-N refers to the formula e0-1, the intermediate 1e-3-N is used for replacing the intermediate 1e-1, wherein the preparation method of the intermediate 1e-3-N refers to the formula 1e-1-N, and the intermediate 1g-3 is used for replacing the intermediate 1g-1.
LC-MS(ESI)m/z(M+H) + called for C 62 H 75 N 10 O 9 S + ＝1135.5；found 1135.6。
Referring to the preparation method of formula 1, intermediate e0-4-N is used instead of intermediate a0-1. Wherein the preparation method of the intermediate e0-4-N refers to the formula e0-1, the intermediate 1e-4-N is used for replacing the intermediate 1e-1, wherein the preparation method of the intermediate 1e-4-N refers to the formula 1e-1-N, and the intermediate 1g-4 is used for replacing the intermediate 1g-1.
LC-MS(ESI)m/z(M+H) + called for C 64 H 79 N 10 O 10 S + ＝1179.6；found 1179.6。
Referring to the preparation method of formula 1, intermediate e0-5-N is used instead of intermediate a0-1. Wherein the preparation method of the intermediate e0-5-N refers to the formula e0-1, the intermediate 1e-5-N is used for replacing the intermediate 1e-1, wherein the preparation method of the intermediate 1e-5-N refers to the formula 1e-1-N, and the intermediate 1g-5 is used for replacing the intermediate 1g-1.
LC-MS(ESI)m/z(M+H) + called for C 66 H 83 N 10 O 11 S + ＝1223.6；found 1223.6。
Referring to the preparation method of formula 1, intermediate e0-0-N is used instead of intermediate a0-1. Wherein the preparation method of the intermediate e0-0-N refers to the formula e0-1, the intermediate 1e-0-N is used for replacing the intermediate 1e-1, wherein the preparation method of the intermediate 1e-0-N refers to the formula 1e-1-N, and the intermediate 1g-0 is used for replacing the intermediate 1g-1.
LC-MS(ESI)m/z(M+H) + called for C 56 H 63 N 10 O 6 S + ＝1003.5；found 1003.5。
Referring to the preparation method of formula 1, intermediate eb1-1 is used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 59 H 67 N 10 O 7 S + ＝1059.5；found 1059.6。
Referring to the preparation method of formula 1, intermediate eb2-1 is used instead of intermediate e0-1, wherein the preparation method of formula eb2-1 refers to formula eb1-1, intermediate 6bb-2 is used instead of intermediate 6bb, wherein the preparation of formula 6bb-2 refers to formula 6bb, and 2- (5-bromo-2-nitrophenyl) acetonitrile is used instead of 2-nitro-5-bromoxynil (formula 1 bb).
LC-MS(ESI)m/z(M+H) + called for C 60 H 69 N 10 O 7 S + ＝1073.5；found 1073.6。
Referring to the preparation method of formula 1, intermediate eb3-1 is used instead of intermediate e0-1, wherein the preparation method of formula eb3-1 refers to formula eb1-1, intermediate 6bb-3 is used instead of intermediate 6bb, wherein the preparation of formula 6bb-3 refers to formula 6bb, and 2- (5-bromo-2-nitrophenyl) propionitrile is used instead of 2-nitro-5-bromoxynil (formula 1 bb).
LC-MS(ESI)m/z(M+H) + called for C 61 H 71 N 10 O 7 S + ＝1087.5；found 1087.6。
Referring to the preparation of formula 32, intermediate ab1-2 was used instead of intermediate a0-1.
LC-MS(ESI)m/z(M+H) + called for C 51 H 52 N 9 O 8 + ＝918.4；found 918.4。
Referring to the preparation method of formula 32, intermediate ab2-2 is used instead of intermediate a0-1, wherein the preparation method of formula ab2-2 refers to formula ab1-2, intermediate 6bb-2 is used instead of intermediate 6bb, wherein the preparation of formula 6bb-2 refers to formula 6bb, and 2- (5-bromo-2-nitrophenyl) acetonitrile is used instead of 2-nitro-5-bromoxynil (formula 1 bb).
LC-MS(ESI)m/z(M+H) + called for C 52 H 54 N 9 O 8 + ＝932.4；found 932.4。
Referring to the preparation method of formula 32, intermediate ab3-2 is used instead of intermediate a0-1, wherein the preparation method of formula ab3-2 refers to formula ab1-2, intermediate 6bb-3 is used instead of intermediate 6bb, wherein the preparation of formula 6bb-3 refers to formula 6bb, and 2- (5-bromo-2-nitrophenyl) propionitrile is used instead of 2-nitro-5-bromoxynil (formula 1 bb).
LC-MS(ESI)m/z(M+H) + called for C 53 H 56 N 9 O 8 + ＝946.4；found 946.4。
Referring to the preparation method of formula 1, intermediate ab1-0 is used instead of intermediate e0-1.
LC-MS(ESI)m/z(M+H) + called for C 61 H 71 N 12 O 5 S + ＝1083.5；found 1083.6。
Referring to the preparation method of formula 1, intermediate hb2-0 is used instead of intermediate e0-1, wherein the preparation method of formula hb2-0 refers to formula hb1-0, intermediate 6bb-2 is used instead of intermediate 6bb, wherein the preparation of formula 6bb-2 refers to formula 6bb, and 2- (5-bromo-2-nitrophenyl) acetonitrile is used instead of 2-nitro-5-bromoxynil (formula 1 bb).
LC-MS(ESI)m/z(M+H) + called for C 62 H 73 N 12 O 5 S + ＝1097.6；found 1097.6。
Referring to the preparation method of formula 1, intermediate hb3-0 is used instead of intermediate e0-1, wherein the preparation method of formula hb3-0 refers to formula hb1-0, intermediate 6bb-3 is used instead of intermediate 6bb, wherein the preparation of formula 6bb-3 refers to formula 6bb, and 2- (5-bromo-2-nitrophenyl) propionitrile is used instead of 2-nitro-5-bromoxynil (formula 1 bb).
LC-MS(ESI)m/z(M+H) + called for C 63 H 75 N 12 O 5 S + ＝1111.6；found 1111.6。
640mg of the compound shown in formula 18 and 800mg of L-malic acid are dissolved in 5mL of absolute ethyl alcohol, the mixture is reacted overnight at normal temperature, white solid is separated out, 699mg of the compound shown in formula 71 is obtained through suction filtration, and the yield is 97%.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of formula 71, the raw materials are a compound shown in formula 18 and D-malic acid.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of the formula 71, the raw materials are a compound shown in the formula 18 and (+ -) -malic acid.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of formula 71, the raw materials are the compound shown in formula 18 and L-tartaric acid.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of formula 71, the raw materials are the compound shown in formula 18 and D-tartaric acid.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of formula 71, the raw materials are a compound shown in formula 18 and methanesulfonic acid.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of formula 71, the raw materials are a compound shown in formula 18 and p-toluenesulfonic acid.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of formula 71, the raw materials are a compound shown in formula 18 and citric acid.
LC-MS(ESI)m/z(M+H) + called for C 60 H 71 N 12 O 5 S + ＝1071.5；found 1071.6。
Referring to the preparation method of formula 71, the raw materials are a compound shown in formula 45 and L-malic acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Referring to the preparation method of formula 71, the raw materials are a compound shown in formula 45 and D-malic acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Referring to the preparation method of the formula 71, the raw materials are a compound shown in the formula 45 and (+ -) -malic acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Referring to the preparation method of formula 71, the raw materials are the compound shown in formula 45 and L-tartaric acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Referring to the preparation method of formula 71, the raw materials are the compound shown in formula 45 and D-tartaric acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Referring to the preparation method of formula 71, the raw materials are the compound shown in formula 45 and methanesulfonic acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Referring to the preparation method of formula 71, the raw materials are the compound shown in formula 45 and p-toluenesulfonic acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Referring to the preparation method of formula 71, the raw materials are the compound shown in formula 45 and citric acid.
LC-MS(ESI)m/z(M+H) + called for C 59 H 70 N 13 O 5 S + ＝1072.5；found 1072.6。
Example 2: biological Activity test of small molecule conjugates represented by formulas 1 to 86
1. Western blot verification of CDK12/13 degradation effect and other related protein degradation effects
And (3) determining the degradation condition of the compound on CDK12/13 proteins on leukemia cells KASUMI-1, MV4-11 and Jurkat by adopting a Western blot method, wherein two compounds with the best degradation effect verify the capability of degrading CDK12/13 proteins on breast cancer cells MDA-MB-231 by using a concentration gradient.
Experimental materials:
cell lines: KASUMI-1, MV4-11, jurkat or MDA-MB-231
Culture medium: DMEM/RIPM-1640+10% Hyclone serum
Compound dissolution method: the compound was dissolved in DMSO to make a 50mM stock solution and diluted in a proportion to give the corresponding concentration.
1. Cell lines (KASUMI-1 or MV4-11 or MDA-MB-231 or Jurkat or HCT 116) were cultured and administered in vitro
(1) Cell strain in vitro culture:
the selected cells (KASUMI-1 or MV4-11 or MDA-MB-231 or Jurkat or HCT 116) were treated with 5% CO 2 Culturing in a constant temperature incubator at 37 deg.C until the cell density reaches 70-90%, and usingAs required for subsequent experiments.
(2) Cell seed plates: the cells were digested and the cell suspension was added to a 1.5mL Eppendorf tube, centrifuged at 1,000rpm for 5min, resuspended in 2mL of culture medium and counted, the cell suspension was seeded in 24 well plates, 20 thousand cells per well, and left for 8-12h until the cells were all adherent.
(3) Compound action: diluting 50mM of the compound with cell culture medium to a final concentration of 0.01 μm-3. Mu.M, and concentrating at 37deg.C with 5% CO 2 The cells are incubated for 3h-48h in a cell incubator, and a DMSO group is used as a negative control.
2. Cell lysis
At the end of the time of action, the cells are collected and washed once with PBS; adding 4% SDS (sodium dodecyl sulfate) of corresponding volume according to the cell quantity to lyse the cells, and performing ultrasonic treatment until the cells are not sticky; centrifuging at room temperature for 30min at 12,000 rpm; the supernatant was transferred to a new EP tube for protein quantification.
3. Protein quantification
Taking 2mg/mL BSA standard substance for sesquidilution to obtain the concentration used by a standard curve, wherein the concentration is sequentially 2mg/mL,1mg/mL,0.5mg/mL,0.25mg/mL,0.125mg/mL and 0.0625mg/mL; according to 200 mu L of A solution and 4 mu L B solution in the BCA quantitative kit for each sample, taking corresponding volumes of A solution and B solution (the volume ratio is 50:1) and uniformly mixing; taking 10 mu L of BSA with different concentrations and samples in the ep tube obtained in the step 2, respectively adding the samples into a 96-well plate, then adding 200 mu L of uniformly mixed solution A and solution B, gently photographing the mixture, and then placing the mixture at 37 ℃ to react for 30min in a dark place; after the completion of the reaction, absorbance values were measured at 562nm, and the sample protein concentration was calculated using a standard curve.
4.Western Blot
(1) Protein sample preparation
Taking a sample with the total amount of 20 mu g of protein from the sample obtained in the step 2, placing the sample in a new EP tube, adding 10 mu L of 6 xLoding Buffer, and adding distilled water to make the total volume of liquid in the new EP tube be 30 mu L; heating and denaturing at 95 ℃ for 10min, cooling, centrifuging and mixing uniformly, performing Western Blot experiment, and freezing the rest sample at-80 ℃.
(2) Glue making
a. And (3) separating glue: selecting the concentration of the separating gel to be prepared according to the molecular weight of the target protein, and firstly preparing a gel clamp: clamping the thick glass and the thin glass and keeping the ground level; sequentially adding a separating gel reagent into the centrifuge tube, and uniformly mixing by vortex; after mixing, adding the separating glue between two pieces of glass, adding proper amount of isopropanol on the upper layer of the glue surface, and waiting for solidification.
b. Concentrated glue: discarding isopropanol on the upper layer of the separating gel, washing residual liquid with triple distilled water, preparing concentrated gel, vortex mixing, adding the concentrated gel between two pieces of glass, inserting gel-making comb, and waiting for solidification.
(3) Electrophoresis
a. Preparation: the prepared gel frame is arranged in an electrophoresis tank and put in an electrophoresis apparatus, diluted 1 Xtricine buffer solution is added in the middle electrophoresis tank, 1 Xrunning buffer solution is added in the outer tank, and the gel frame stands for a plurality of minutes.
b. Loading: and (3) vortex mixing the samples obtained in the step (1), sucking a certain amount of samples (5-10 mu L), loading the samples, starting an electrophoresis apparatus after loading, firstly adjusting a constant voltage mode, carrying out electrophoresis for 10min at a voltage of 70V, confirming that the current is normal, adjusting the voltage to 130V after the samples enter the separation gel, waiting for the samples to run to a proper position, and stopping electrophoresis.
(4) Transfer film and closure
a. Transferring: sequentially adding sponge, thick filter paper and thin filter paper into the film transferring clamp; taking glue: removing the concentrated gel blocks at the edge by using a gel stripping shovel, carefully taking down the separation gel, and placing the separation gel in the center of filter paper; sticking film: pasting a PVDF film which is activated by methanol in advance on the adhesive from one side, and expelling bubbles; then Bao Lvzhi, thick filter paper and sponge are sequentially added, the membrane transferring clamp is clamped, and the membrane transferring clamp is sequentially assembled. And adding a proper amount of film transfer liquid into the film transfer tank, adding a proper amount of water into the outer layer of the film transfer tank, and putting into an ice bag, so that the film transfer tank is positioned in ice water to prevent a great amount of heat released in the film transfer process from affecting the film transfer effect.
b. Closing: after the transfer, the transfer clamp is opened to remove the glue and PVDF membrane (if the transfer is successful, the protein Marker is completely transferred to the PVDF membrane), then the PVDF membrane is cut off according to the molecular weight of the target protein, and the membrane is placed in 5% skimmed milk and sealed at room temperature for 60min.
(5) Incubation of antibodies
a. Incubating primary antibodies: after blocking, the milk was discarded and the PVDF membrane was washed 3 times with TBST (15 min,5 min), and an appropriate amount of primary antibody (typically diluted as primary antibody: antibody diluent=1:1000) was added and incubated overnight at 4℃in a shaker.
b. Incubating a secondary antibody: the primary antibody was recovered and washed 3 times with TBST (15 min,5 min), and secondary antibody (typically formulated as secondary antibody: 5% skim milk = 1:5000) was added and incubated for 60min at room temperature on a horizontal shaker.
(6) Exposure to light
a. Washing the film: the secondary antibody was discarded and washed 3 times with TBST (15 min,5 min),
b. exposure: preparing ECL luminous liquid: and (3) solution A: solution B = 1:1. the residual TBST on the strip was first blotted with filter paper, and the strip was clamped to an exposure plate with forceps, and ECL color development solution was dropped onto the strip to cover the strip, and left standing for about 1 min. The exposure plate is placed in an AI800 exposure machine, and exposure is performed at a proper time.
The degradation rate calculating method comprises the following steps: quantitative analysis is carried out on the exposure result through Image J software, namely, gray value calculation is carried out on the exposure strip through the Image J software, then the gray value of the target protein is divided by the gray value of the reference protein, normalization processing is carried out, and the calculation formula is D experiment group= {1- [ (A) Experimental group, target protein /A Experimental group, internal reference protein )÷(A Negative control group, target protein /A Negative control group, internal reference protein )]100% (A is gray scale value, D is degradation rate).
The degradation activity of the small molecule conjugates of the invention on CDK12, CDK13 is shown in FIGS. 3-10; CDK12 and CDK13 are target proteins, beta-action and GAPDH are internal reference proteins, and the internal reference proteins are used for indicating the total protein amount of each group in the experiment so as to perform quantitative analysis.
As can be seen from the test results in FIGS. 3-9, most of the conjugates were able to degrade CDK12/13 at a concentration where the compound of formula 45 degraded most.
TABLE 1
Cell name Cell type CDK12 DC 50 CDK13 DC 50
MDA-MB-231 Breast cancer cells 29nM 18nM
HCT116 Colorectal cancer cells 34nM 24nM
KASUMI-1 Leukemia cells 28nM 17nM
MV4-11 Leukemia cells 30nM 21nM
The list of molecules, aliases and activities of the examples are shown in Table 2 below (+represents the strength of the degradability, -represents no degradation observed, N.D. represents that no such experiment was performed).
TABLE 2
/>
+ represents the degradation effect against the protein, with more+ degradation being more pronounced. -representing not degrading the protein. Specific:
++ and represents DC 50 <100nM
++ + + and represents 100nM<DC 50 <0.5μM
++ represents 0.5 mu M<DC 50 <1μM
++ represents 1. Mu.M<DC 50 <3μM
+ represents 3. Mu.M<DC 50 <10μM
-represents a non-degrading activity
2. Dye detection of proliferation inhibition effect of compound on tumor cells
Measuring proliferation inhibition of compounds on leukemia cell lines MV4-11 and KASUMI-1 by CCK8 staining; proliferation inhibition of the solid tumor cell line MDA-MB-231 by the compounds was determined using Sulforhodamine B (SRB) staining.
CCK8 staining-MV 4-11, KASUMI-1 cells were first seeded in 96-well plates at 5000 per well, followed by gradient concentration (0.03. Mu.M-10. Mu.M) of compound for 72h, while solvent DMSO was administered as a negative control. At the end of the time of action, 10ul of CCK8 reagent was added to each well, incubated at 37℃for 4 hours in the absence of light, and absorbance at 450nm was measured after mixing.
SRB staining: MDA-MB-231, L-02, HK-2 cells were first seeded separately in 96-well plates at a density of 3000 per well, and after 8-12h of cell attachment, a gradient concentration (62.5 nM-10. Mu.M) of the compound was administered for 72h, while solvent DMSO was administered as a negative control. At the end of the time of action, 70. Mu.L of PBS was added for one wash, and then 70. Mu.L of 10% trichloroacetic acid was added to fix the cells at 4℃for 1 hour; washing trichloroacetic acid clean by tap water, and then placing the trichloroacetic acid in an oven for drying; then 70 mu L of SRB staining solution is added for staining for 20min at room temperature; discarding the dyeing liquid, cleaning with 1% glacial acetic acid, and oven drying; mu.L of 10mM Tris-base solution was added thereto, and absorbance was measured at 540nm after shaking for 15 minutes.
Drug combination methodology: MD-MB-231 cells were seeded at a density of 2000 per well in 6 well plates, and after 24h cell attachment, PARP inhibitor olaparib at a specific concentration (0.5. Mu.M, 1. Mu.M) was administered separately to react with a compound of formula 18 at a specific concentration (0.5. Mu.M, 1. Mu.M) for 10 days, while solvent DMSO was administered as a negative control. At the end of the time of action, 100. Mu.L of PBS was added for one wash, and then 700. Mu.L of 10% trichloroacetic acid was added to fix the cells at 4℃for 1 hour; washing trichloroacetic acid clean by tap water, and then placing the trichloroacetic acid in an oven for drying; then 70 mu L of SRB staining solution is added for staining for 20min at room temperature; discarding the dyeing liquid, cleaning with 1% glacial acetic acid, and oven drying; the well plates were then photographed and the cell clones of each group were counted using Image J software.
The calculation formula of the proliferation inhibition of CCK8 staining and SRB staining cells is as follows: r experimental group= (1-OD experimental group/OD negative control group) ×100% (OD is absorbance value, R is cell proliferation inhibition rate).
The calculation formula of cell proliferation inhibition for drug combination is as follows: r experimental group= (1-K experimental group/K negative control group) ×100% (K is the number of cell clones, R is the cell proliferation inhibition rate).
The results are shown in FIGS. 10 and 11.
As can be seen from fig. 10, the small molecule conjugates provided herein selectively inhibit proliferation of tumor cells KASUMI-1, mv4-11, while THZ-531 (classical CDK12/13 inhibitor) and IN-2 (mother molecule) showed no differential proliferation inhibitory activity on normal cells and tumor cells.
As can be seen from fig. 11, the combined administration scheme of the small molecule conjugate and the PARP inhibitor can effectively synergistically enhance the proliferation inhibition activity on breast cancer cells MDA-MB-231, and achieve obvious superposition and complementation with the existing clinical medicines.
3. Compound liver microsome stability test
Experimental materials:
a compound: ketone color forest, formula 18, formula 45
Liver microsomes: human liver microsomes (purchased from BioreclamationIVT); dog, mouse, rat, monkey liver particles (purchased from Xenotech)
PBS was warmed up and brought to pH 7.4.
Compound solution with concentration of 500 μm was prepared: mu.L of a 10mM compound solution and 95. Mu.L of DMSO were added to the ep tube.
A compound solution containing 0.75mg/mL liver microsomes at a concentration of 1.5. Mu.M was prepared: to the ep tube were added 1.5. Mu.L of 500. Mu.M compound solution, 18.75. Mu.L of liver microparticle solution at a concentration of 20mg/mL and 479.75. Mu.L of PBS solution.
NADPH solution with concentration of 6mM was prepared: 5mg of NADPH was dissolved in 1mL of PBS solution.
mu.L of a compound solution containing 0.75mg/mL of liver microsomes and a concentration gradient was added to a 96-well plate, 15. Mu.L of a 6mM NADPH solution, 150. Mu.L of an IS-containing acetonitrile-methanol (1:1) solution was added, vortexed and centrifuged at 6,000rpm for 10min. Samples of each concentration were taken and 140. Mu.L of pure water was added to 80. Mu.L of supernatant for LC/MS detection (sample injection amount 10. Mu.M), and a standard curve was drawn according to the final concentration of the compound and the corresponding mass spectrum peak area.
mu.L of a compound solution containing 0.75mg/mL of liver microsomes at a concentration of 1.5. Mu.M was added to each 96-well plate for detection at different time points (0, 5, 15, 30, 45 minutes, two groups each).
For the 0 minute group, 150. Mu.L of acetonitrile in methanol (1:1) containing IS was added to the wells, and 15. Mu.L of 6mM NADPH solution was added thereto, and the mixture was vortexed and centrifuged at 6,000rpm for 10 minutes.
For the 5, 15, 30, 45 minute groups, 15. Mu.L of 6mM NADPH solution was added to the wells to initiate the reaction. After 5, 15, 30, 45 minutes, the reaction was terminated by adding 50. Mu.L of acetonitrile/methanol (1:1) solution containing IS to the corresponding group, respectively. Vortex mixing and centrifuge at 6,000rpm for 10min.
80 mu L of supernatant is taken from each well, a 96-well plate containing 140 mu L of pure water is added for LC/MS detection (sample injection amount is 10 mu M), and the corresponding mass spectrum peak area of the compound is compared with a standard curve to obtain the concentration value of the compound.
The calculation formula is as follows:
cancellation rate k= (Inc 1 -Inc 2 )/(t 2 -t 1 )(t 1 ，t 2 For the time point, c 1 ，c 2 Respectively the time is t 1 ，t 2 Concentration of drug at the time)
Half-life t 1/2 ＝0.693/K
The results are shown in FIG. 12.
As can be seen from fig. 12, the liver microsomes of the four species of human, rat, dog and monkey were incubated together in the formulas 18 and 45, and the half-life period was more than 45min, so that the liver microsomes were excellent in stability.

Claims (23)

1. A small molecule conjugate characterized by having a structure represented by formula i, or a pharmaceutically acceptable salt, co-crystal, tautomer, stereoisomer, solvate, hydrate, polymorph, isotopically enriched derivative or prodrug thereof:
T-L-E
I
In formula I, T represents a ligand of CDK12/13 protein, E represents a ligand of E3 ligase, and L represents a linking chain of T and E.
2. The small molecule conjugate of claim 1, wherein: t is selected from the group represented by formula II:
r in formula II 1 Selected from the group consisting of hydrogen, N-substituted amides, C1-C4 alkyl substituted acyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-to 8-membered carbocyclyl, 3-to 8-membered heterocyclyl, 5-to 10-membered aryl, 5-to 10-membered heteroaryl, or nitrogen protecting groups; or optionally when R 1 When two, two R1 together with the intermediate atom (N) thereof form a substituted or unsubstituted heterocyclyl or substituted or unsubstituted heteroaryl; the nitrogen protecting group comprises tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl and p-methoxybenzyl.
R in formula II 2 Selected from the group consisting of hydrogen, N-substituted amides, C1-C4 alkyl substituted acyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-to 8-membered carbocyclyl, 3-to 8-membered heterocyclyl, 5-to 10-membered aryl, 5-to 10-membered heteroaryl, -CN, -OR D1 、-N(R D1a ) 2 or-SR D1 The method comprises the steps of carrying out a first treatment on the surface of the Wherein R is D1 Is hydrogen, N-substituted amide, C1-C4 alkyl substituted acyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 3-to 8-membered carbocyclyl, 3-to 8-membered heterocyclyl, 5-to 10-membered aryl, 5-to 10-membered heteroaryl, an oxygen protecting group when attached to an oxygen atom, or a sulfur protecting group when attached to a sulfur atom; wherein the oxygen protecting group is selected from dihydropyran, methoxymethyl, benzyl, methoxybenzyl, trimethylsilyl, tert-butyldimethylsilyl, and the sulfur protecting group is selected from acetamidomethyl, phenylacetylaminomethyl, tert-butylthio.
Wherein two R D1a Each independently of the others is selected from the group consisting of hydrogen, N-substituted amides, C1-C4 alkyl substituted acyl groups, C1-C6 alkyl groups, C1-C6 alkenyl groups, C1-C6 alkynyl groups, 3-to 8-membered carbocyclyl groups, 3-to 8-membered heterocyclyl groups, 5-to 10-membered aryl groups, 5-to 10-membered heteroaryl groups or nitrogen protecting groups; or optionally, two R D1a Together with the intervening atoms, form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring;
rx and Ry in formula II are each independently selected from CH or N;
n in formula II is 0, 1, 2, 3, 4 or 5;
and->Indicating that the two N's attached are in trans configuration relative to the six-membered ring attached.
3. The small molecule conjugate of claim 2, wherein: r is R 1 Selected from ethyl groups、
Or a nitrogen protecting group; w is 0, 1, 2, 3, 4, 5 or 6; r is R 1-a When one or more, one or more R 1-a Each independently selected from the group consisting of halogen, N-substituted amides, C1-C4 alkyl substituted acyl, C1-C6 alkyl; r is R 2 Selected from halogen, cyano, trifluoromethyl; the nitrogen protecting group comprises tert-butoxycarbonyl, benzyloxycarbonyl, allyloxycarbonyl and p-methoxybenzyl.
4. The small molecule conjugate of claim 2, wherein: the T has any structure shown in the following formula:
5. The small molecule conjugate of claim 1 or 2, characterized in that:
said E is selected from any one of the following groups: 3-amino-N- (2, 6-dioxo-3-piperidyl) phthalimide group, 3- (7-amino-3-oxo-1H-isoindol-2-yl) piperidine-2, 6-dione group, (2S, 4 r) -1- ((S) -2- (3-aminopropionamido) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide group, [ (4 r, 5S) -4, 5-bis (4-chlorophenyl) -2- [4- (1, 1-dimethylethyl) -2-ethoxyphenyl ] -4, 5-dihydro-4, 5-dimethyl-1H-imidazol-1-yl ] [4- [3- (methylsulfonyl) propyl ] -1-piperazinyl ] methanone group, phenyl glutarimide.
6. The small molecule conjugate of claim 1 or 2, characterized in that:
the E is selected from one of the following structures:
in the formulae III-a to III-d, III-a-0 to III-d-0, R 1 Is NH or O or CH 2 ；
In the formula III-e, R 1 Is H or OH, R 2 Is H or CH 3 ；
In the formulae III-f, III-h and III-i, R 1 Is thatWherein R is 3 is-CN, -F, -COCH 3 ，R 2 Is H or CH 3 ；
In the formula III-g, R 1 Is H, CN, F, cl, br, or formula:
in the formula III-l, X=N or CH.
7. A small molecule conjugate according to any one of claims 1-3, characterized in that: the L is one of the following structural units, or,
Any structural group consisting of the following structural units:
-CH 2 -、-(C＝O)-、/>
r in the heteroatom-containing units are each independently selected from alkyl, alkoxy or hydrogen;
the alkyl is specifically methyl, ethyl, propyl, isopropyl, n-butyl and tert-butyl; the alkoxy is-OEt, -OMe.
8. The small molecule conjugate of claim 1 or 2, characterized in that: the L is selected from the following structures:
9. a small molecule conjugate according to claim 2 or 3, characterized in that:
the structural formula of the small molecule conjugate is shown in any one of the formulas IV-XII:
in formula IV, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, A is CH 2 NH, O or CO, G being absent, CH 2 NH, O, CONH or NHCO, B is CO or CH 2 X, Y, Z are each independently CH or N, R 1 N is defined as formula II;
in the formula V, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, A is CH 2 NH, O or CO, G being absent, CH 2 NH, O, CONH or NHCO, B is CO or CH 2 X, Y eachIndependently CH or N, R 1 N is defined as formula II;
in formula VI, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, A is CH 2 NH, O or CO, G being absent, CH 2 NH, O, CONH or NHCO, B is CO or CH 2 X, Y, Z are each independently CH or N, V, W are each independently CH or N, R 1 N is defined as formula II;
in formula VII, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; rx is H or CH 3 X, Y, Z are each independently CH or N, R 1 N is defined as formula II;
in formula VIII, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO, rx is H or CH 3 X, Y is independently CH or N, R 1 N is defined as formula II;
in formula IX, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; rx is H or CH 3 X, Y, Z is independently CH or N, V, W is independently CH or N, R 1 N is defined as formula II;
in the formula X, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; a is CH 2 Or CO, X, Y, Z are each independently CH or N, R 1 N is defined as formula II;
in formula XI, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; a is CH 2 Or CO, X, Y are each independently CH or N, R 1 N is defined as formula II;
in formula XII, m 1 Is an integer between 0 and 6, m 2 Is an integer of 0 to 15, G is absent, CH 2 NH, O, CONH or NHCO; a is CH 2 Or CO, X, Y, Z are each independently CH or N, V, W are each independently CH or N, R 1 N is as defined in formula II.
10. The small molecule conjugate of claim 9, wherein: the small molecule conjugate has a structure as shown in formula IX, wherein n=0; r is R 1 Is benzyl; x is CH; at least one of Y and Z is N, and when the other is not N, the other is CH; g is absent; m is m 1 Is 0; m is m 2 0,1,2, rx is methyl.
11. The small molecule conjugate of claim 1, wherein: the small molecule conjugate is a compound represented by the following formulas 1 to 70:
1 (1)
2, 2
3
4. The method is to
5. The method is to
6. The method is to
7. The method of the invention
8. The method is used for preparing the product
9. The invention is applicable to
10. The method of the invention
11. The method of the invention
12. Fig.
13 of the group
14, of the order of magnitude/>
15 of the formula
16, respectively
17 (17)
18, 18
19, of the order of magnitude
20 (20)/>
21, a combination of
22, of the type
23 of the group
24 of the formula
25 of the group
26, respectively/>
27. The method of the invention
28, respectively
29
30, 30
31, of the order of magnitude
32, of the shape of a triangle/>
33, 33
34, of the group
35, of the type
36, respectively
37, of the type
38, respectively
39 of the formula/>
40, of the order of magnitude
41 of the order of magnitude
42 of the die
43, respectively
44, respectively
45. The device is a kind of device
46, of the shape of a cone/>
47, of the material
48, respectively
49 of the formula
50, respectively
51
52, of the shape/>
53
54, respectively
55, of the shape of a cylinder
56
57, respectively
58/>
59, and method for manufacturing the same
60 of the formula
61
62, of the group
63, respectively
64 of the shape of a cylinder/>
65 of the type
66, respectively
67 for the treatment of cancer
68, respectively
69, 69
70, of the order of magnitude
12. The small molecule conjugate of any one of claims 1-11, wherein the pharmaceutically acceptable salt is a salt with an acid selected from the group consisting of: citric acid, fumaric acid, acetic acid, oxalic acid, tartaric acid, malic acid, maleic acid, lactic acid, camphorsulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, succinic acid, citric acid, hydrohalic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, glutamic acid, aspartic acid.
13. The small molecule conjugate of claim 12, wherein: the small molecule conjugate is a compound represented by the following formulas 71 to 86:
71, of the shape of a sphere
72, of the order of magnitude
73, of the group
74, respectively
75 of the group
76, respectively/>
77, of the group
78, a pair of arms
79 for preventing and treating cancer
80, respectively
81 of the type
82, respectively/>
83 of the following description
84 parts of a machine
85
86, of the order of magnitude
14. A method of preparing a small molecule conjugate according to any one of claims 1 to 12, comprising the steps of: the ligand precursor compound of E3 ligase and the ligand precursor compound of the chain-linked precursor compound and the T-part CDK12/13 protein are respectively subjected to amide condensation reaction, substitution reaction, click reaction or cross-coupling reaction to obtain the small molecule conjugate shown in the formula I.
15. The method of preparing a small molecule conjugate according to claim 14, wherein each of the following is:
a method of preparing a small molecule conjugate described by formula IV: obtaining a small molecule conjugate shown in a formula IV through nucleophilic substitution reaction between a CDK12/13 ligand end derivative shown in a formula a and a Pomalidomide end derivative shown in a formula b;
wherein, in the small molecule conjugate shown in the formula IV, G is O, A NH, X is CH, Y is N, Z CH; r in formula a 1 、m 1 、m 2 N is defined as formula IV, and B is defined as formula IV;
or, the preparation method of the small molecule conjugate shown in the formula V comprises the following steps: carrying out nucleophilic substitution reaction between CDK12/13 ligand end derivative shown in formula c and Pomalidomide end derivative shown in formula b to obtain small molecule conjugate shown in formula V;
wherein, in the small molecule conjugate of the formula V, G is CH 2 A is NH, X is N, Y and N; r in c 1 、m 1 、m 2 N is defined as formula V, and B is defined as formula V;
or, the preparation method of the small molecule conjugate shown in the formula VI comprises the following steps: carrying out nucleophilic substitution reaction between CDK12/13 ligand end derivative shown in formula d and Pomalidomide end derivative shown in formula b to obtain small molecule conjugate shown in formula VI;
Wherein, in the small molecule conjugate of the formula VI, G is CH 2 A is NH, X is CH, Y is N, Z is CH, V is N, W is N; r in d 1 、m 1 、m 2 N is defined as formula VI, and B is defined as formula VI in formula B;
/>
or, the preparation method of the small molecule conjugate shown in the formula VII comprises the following steps: performing condensation reaction between CDK12/13 ligand end derivatives shown in formula e and VHL ligand derivatives shown in formula f to obtain a small molecule conjugate shown in formula VII;
wherein, in the small molecule conjugate shown in the formula VII, G is O, X CH, Y is N, Z CH; r in e 1 、m 1 、m 2 N is defined as formula VII, wherein R x Definition is the same as formula VII;
or, the preparation method of the small molecule conjugate shown in the formula VIII comprises the following steps: performing condensation reaction between CDK12/13 ligand end derivatives shown in a formula g and VHL ligand derivatives shown in a formula f to obtain a small molecule conjugate shown in a formula VIII;
wherein, in the small molecule conjugate of formula VIII, G is CH 2 X is N, Y and N; r in g 1 、m 1 、m 2 N is defined as formula VIII, wherein R x The definition is the same as that of formula VIII;
or, the preparation method of the small molecule conjugate shown in the formula IX comprises the following steps: performing condensation reaction between CDK12/13 ligand end derivatives shown in a formula h and VHL ligand derivatives shown in a formula f to obtain a small molecule conjugate shown in a formula X;
Wherein, in the small molecule conjugate of formula IX, G is CH 2 X is CH, Y is N, Z CH, V is N, W N; r in h 1 、m 1 、m 2 N is defined as formula IX, formula f, wherein R x The definition is the same as formula IX;
or, the preparation method of the small molecule conjugate shown in the formula X comprises the following steps: performing condensation reaction between CDK12/13 ligand end derivatives shown in formula e and MDM2 ligand derivatives shown in formula j to obtain a small molecule conjugate shown in formula X;
wherein, in the small molecule conjugate shown in the formula X, G is O, X CH, Y is N, Z CH, and A is CO; r in e 1 、m 1 、m 2 N is defined as formula X;
or, the preparation method of the small molecule conjugate shown in the formula XI comprises the following steps: performing condensation reaction between CDK12/13 ligand end derivatives shown in a formula g and MDM2 ligand derivatives shown in a formula j to obtain a small molecule conjugate shown in a formula XI;
wherein, in the small molecule conjugate shown in the formula XI, G is CH 2 X is N, Y N, A is CO; r in g 1 、m 1 、m 2 N is defined as formula XI;
or, the preparation method of the small molecule conjugate shown in the formula XII comprises the following steps: performing condensation reaction between CDK12 ligand end derivatives shown in formula h and MDM2 ligand derivatives shown in formula j to obtain a small molecule conjugate shown in formula XII;
Wherein, in the small molecule conjugate of formula XII, G is CH 2 X is CH, Y is N, Z CH, V is N, W, and N, A is CO; r in h 1 、m 1 、m 2 N is as defined in formula XII.
16. An intermediate for preparing a compound shown in claim 2, which is characterized by having a structure shown in a formula a, a formula c, a formula d, a formula e, a formula g and a formula h:
in the above formulas: m is m 1 Is an integer between 0 and 6, m 2 R is an integer of 0 to 15 1 Selected from the group consisting of hydrogen, optionally substituted acyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, optionally substituted heteroaryl, or a nitrogen protecting group; or optionally when R 1 When there are two, two R 1 Together with the intervening atoms, form a substituted or unsubstituted heterocyclic ring or a substituted or unsubstituted heteroaryl ring; n is 0, 1, 2, 3, 4 or 5.
17. A method for producing the compound represented by formula a or the compound represented by formula c or the compound represented by formula d according to claim 15, comprising the steps of:
when n is 0 in formula II:
1) Coupling the compound shown in the formula 1a with the bisboronic acid pinacol ester to obtain the compound shown in the formula 2a
2) Nucleophilic reaction between the compound shown in the formula 3a and the compound shown in the formula 4a to obtain a compound shown in the formula 5 a; coupling reaction is carried out on the formula 1a and the formula 1c, and deprotection is carried out to obtain a formula 3c; carrying out nucleophilic substitution reaction on the compound shown in the formula 3a and the compound shown in the formula 1c to obtain a compound shown in the formula 1d, and carrying out coupling reaction on the compound shown in the formula 1d and the compound shown in the formula 2a and removing a protecting group to obtain 2d;
3) Coupling reaction is carried out on the compound shown in the formula 2a and the compound shown in the formula 5a, and a protecting group is removed, so that a compound shown in the formula a0 is obtained;
carrying out nucleophilic substitution reaction on the compound shown in the formula 2c and the compound shown in the formula 3c, and removing a protecting group to obtain a compound shown in the formula c 0;
carrying out nucleophilic substitution reaction on the compound shown in the formula 2c and the compound shown in the formula 2d, and removing a protecting group to obtain a compound shown in the formula d 0;
the compounds shown in the formula a0, the formula c0 and the formula d0 respectively correspond to the compounds shown in the formula a, the formula c and the formula d when n=0;
when n is other than 0 in formula II:
1) Coupling the compound shown in the formula 1b with the bisboronic acid pinacol ester to obtain the compound shown in the formula 2b
2) The synthesis of the compound shown in the formula 3cb is obtained by coupling reaction and deprotection of the compound shown in the formula 1b and the compound shown in the formula 1 c; synthesizing a compound shown in a formula 2db, and carrying out coupling reaction on the compound shown in the formula 2b and the compound shown in a formula 1d to remove a protecting group;
3) Synthesizing a compound shown in a formula ab, and removing a protecting group from the compound shown in a formula 2b and a compound shown in a formula 5a after coupling reaction;
synthesizing a compound shown in a formula cb, wherein the compound shown in the formula 3cb and the compound shown in the formula 2c are subjected to nucleophilic substitution reaction and a protecting group is removed;
synthesizing a compound shown in a formula db, wherein the compound shown in the formula 2db and the compound shown in a formula 2c are subjected to nucleophilic substitution reaction and a protecting group is removed;
the compounds shown in the formulas ab, cb and db respectively correspond to the compounds shown in the formulas a, c and d when n is not equal to 0;
/>
18. a method for producing the compound represented by formula e or the compound represented by formula g or the compound represented by formula h according to claim 15, comprising the steps of:
when n is 0 in formula II:
1) Obtaining e2 through nucleophilic substitution reaction between the formula e1 and the formula 3 a;
2) Coupling reaction is carried out on the compound shown in the formula e2 and the compound shown in the formula 2a, and protective groups are removed to obtain a compound shown in the formula e 0; carrying out nucleophilic substitution reaction on the compound shown in the formula 3c and the compound shown in the formula g1, and removing a protecting group to obtain a compound shown in the formula g 0; carrying out nucleophilic substitution reaction on the compound shown in the formula 2d and the compound shown in the formula g1, and removing a protecting group to obtain a compound shown in the formula h 0; the compounds shown in the formulas e0, g0 and h0 respectively correspond to the compounds shown in the formulas e, g and h when n=0;
/>
When n is other than 0 in formula II:
synthesizing a compound shown in a formula eb, and referring to a formula e, wherein the raw materials are a compound shown in a formula 2b and a compound shown in a formula e 2;
synthesizing a compound shown in a formula gb, and referring to a formula c, wherein the raw materials are a compound shown in a formula 3cb and a compound shown in a formula g 1;
synthesizing a compound shown in a formula hb, and referring to a formula d, wherein the raw materials are a compound shown in a formula 2db and a compound shown in a formula g 1; the compounds shown in the formulas eb, gb and hb respectively correspond to the compounds shown in the formulas e, g and h when n is not equal to 0;
/>
19. a composition comprising the small molecule conjugate of any one of claims 1-13 and one or more pharmaceutically acceptable carriers.
20. The composition of claim 19, wherein the carrier comprises one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants conventional in the pharmaceutical arts.
21. A formulation derived from the composition of claim 19 or 20, comprising a tablet, powder, granule, capsule, oral liquid, paste, cream, injection.
22. Use of a small molecule conjugate according to any one of claims 1-13 in at least one of the following 1) -3):
1) Use in the preparation of a CDK12/13 degradant;
2) Use in the preparation of an inhibitor of cancer cell proliferation;
3) Use in the manufacture of a medicament for the prevention and/or treatment of a CDK12/13 disorder.
23. The use according to claim 22 wherein the CDK12/13 degrading agent selectively degrades CDK12/13 in: colorectal cancer cells, breast cancer cells, and brain glioma cells, acute myelogenous leukemia cells, T-lymphocytic leukemia cells, myeloma cells;
the cancer cells include cell myeloma cells, breast cancer cells, leukemia cells and brain glioma cells;
the CDK12/13 abnormal diseases comprise breast cancer, prostatic cancer, gastric cancer, ewing sarcoma and leukemia.
CN202310479552.4A 2023-04-28 2023-04-28 Small molecule conjugate of targeted cyclin-dependent kinase 12/13 and application thereof Pending CN116675731A (en)

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