CN114349738A

CN114349738A - Small molecule conjugate for targeted degradation of CDK2 and application thereof

Info

Publication number: CN114349738A
Application number: CN202011095654.9A
Authority: CN
Inventors: 饶燏; 王立国; 吴越; 应美丹; 何俏军
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-10-14
Filing date: 2020-10-14
Publication date: 2022-04-15
Anticipated expiration: 2040-10-14
Also published as: CN114349738B

Abstract

The invention provides a small molecule conjugate for degrading CDK2, and a preparation method and application thereof. The structural formula of the inhibitor is shown in a formula I, and the inhibitor has a mode of action different from that of the existing CDK2 inhibitor molecules at the protein level. In the formula I, X represents a ligand of CDK2 protein, which is also called a mother nucleus molecule, Z represents a ligand of E3 ligase, and Y represents a connecting part of X and Z, which is also called Linker. The compounds of formula I may be formed by a click reaction, amide condensation reaction or substitution reaction between Pomalidomide or Lenalidomide derivatives and derivatives of moiety X. The compounds achieve selective degradation of CDK2 on a variety of cells and exhibit a high degree of CDK2 selective degradation activity on a variety of tumor cell lines. In biological and therapeutic terms, it is expected to be a lead compound for the treatment of under-differentiated AML diseases and related diseases by inducing AML cell differentiation. X-Y-Z is formula I.

Description

Small molecule conjugate for targeted degradation of CDK2 and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a targeted degradation CDK2 small molecule conjugate and application thereof.

Background

Acute Myeloid Leukemia (AML) is a malignant disease of myeloid hematopoietic stem cells. The disease is mainly characterized by abnormal hyperplasia of primary and juvenile myeloid cells in bone marrow and peripheral blood, and accounts for 30% of the leukemia of children. Currently, the standard chemotherapeutic regimen for clinical treatment of AML is the anthracycline in combination with cytarabine. The treatment scheme has a certain desirable effect, but the symptoms such as infection, bleeding, hypodynamia and the like are very easy to appear in the chemotherapy process, the 5-year recurrence rate is up to 70 percent, the treatment aiming at AML is seriously influenced, and the life quality of patients is reduced.

In recent years, some drug treatment regimens, typified by all-trans retinoic acid, which attempt to treat Acute Promyelocytic Leukemia (APL) by inducing differentiation, have been developed and have achieved great success in addressing the characteristics of AML differentiation disorders. However, this treatment regimen still has major limitations, mainly manifested by unclear mechanisms and few drugs that can induce APL differentiation. In 2018, He group carries out mechanism research on APL, and provides a new possible scheme for the treatment of inducing APL differentiation. He Group found that Cyclin dependent kinase 2(CDK2) was significantly down-regulated during the induced differentiation of APL, and that APL differentiation was observed during the induction of CDK2 down by RNAi technology. At the same time, the knock-out of CDK2 did not cause embryonic lethality, suggesting that this treatment is safer.

CDKs are a family of cyclin dependent kinases within eukaryotic cells, comprising a set of kinase systems paired with cell cycle progression. The CDK family contains more than 20 subtypes, and is commonly involved in regulating the cell differentiation and division processes, wherein CDK1-6 directly plays a role in cell cycle regulation. CDK family proteins act synergistically with cyclin and are important factors in cell cycle regulation, e.g., CDK4 forms a complex with cyclin D1, regulating the G1 cycle of cells; for another example, the CDK2 complex with cyclin E controls the transition from G1 to S phase, while the CDK2 complex with cyclin A regulates the passage of cells through S phase. Currently, most of the approved clinical trials of the FDA and marketed CDK inhibitors are directed against various tumors, for example, CDK4 and the 6 inhibitor Palbociclib are approved for treating breast cancer, and Dinaciclib of CDK1, 2, 5 and 9 is in the third clinical stage, and the indications are multiple tumors such as CLL, AML, ALL, pancreatic cancer, breast cancer and melanoma. However, because the domains of CDK family kinases that bind ATP are highly similar, these CDK inhibitors are not highly selective, and most small molecules are often capable of simultaneously inhibiting multiple CDK kinase subtypes, as well as other non-CDK family kinases. Therefore, such off-target effects also have large side effects while bringing about strong cell killing or inhibition. The inhibition performance of the existing CDK2 inhibitor on cells is not reproduced by using shRNA technology, and the problem of targeting of the existing small molecules is also shown.

However, due to the highly similar ATP-binding domains of CDK family kinases, selective inhibitors against CDK2 are currently lacking. Most small molecules are often capable of inhibiting multiple CDK kinase subtypes, or even other non-CDK family kinases, simultaneously. Such off-target effects also have major side effects while causing potent cell killing or inhibition. In 2001, the introduction of PROTAC technology has made it possible for researchers to develop such CDK 2-selective degradants. The PROTAC technology is a technology for realizing target protein ubiquitination degradation by utilizing molecules containing bifunctional groups. The chemical structure of the protein comprises three sections, namely POI ligand, Linker and E3 ubiquitination ligase ligand, so that the small molecule can be drawn close to the E3 ubiquitination ligase to induce the non-natural ubiquitination degradation of target Protein (POI). In recent years, Nathanael S Gray Group achieved selective degradation of CDK6 and CDK9 by using different Binders, however, no case report on CDK2 degradation exists, and how to achieve selective degradation of CDK2 through reasonable design so as to achieve the aim of inducing APL differentiation under lower toxicity becomes the research target.

Disclosure of Invention

It is an object of the present invention to provide a small molecule conjugate that degrades CDK 2. The inhibitor molecule has a structure different from that of the existing inhibitor molecule, has a mode of action different from that of the CDK2 inhibitor molecule at the protein level, and shows properties and advantages which are obviously different from the original mother nucleus molecule (the mother nucleus molecule X in the figure below, which is also referred to as the item) at the molecular and cell level.

The structural formula of the small molecule conjugate degrading CDK2 provided by the invention is shown in formula I:

X-Y-Z

formula I

In the formula I, X represents a ligand of CDK2 protein, which is also called a mother nucleus molecule, Z represents a ligand of E3 ligase, and Y represents a connecting part of X and Z, which is also called Linker.

Specifically, X is selected from JNJ-7706621 and derivatives thereof, and the structural group of X is shown as formula II.

R in formula II includes, but is not limited to, the following groups:

r in the formula II_xAnd R_yCan be independently CH or N.

SO in formula II₂NH-may be in the 2,3,4 position

Further, the specific structure of X may be any one of the following groups:

in particular, said Z can be chosen from the group consisting of 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 (thalidoside 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 ] methanone group (RG-7112 group), etc.;

more specifically, Z includes, but is not limited to, any of the structures shown below:

in the formula III-1, R₁Is O and R₂Either NH, or R₁＝H₂And R is₂Either NH, or R₁＝H₂And R is₂＝CH₂Or R is₁＝H₂And R is₂＝C；

In the formula III-2, R₁Is O and R₂Either NH, or R₁＝H₂And R is₂Either NH, or R₁＝H₂And R is₂＝CH₂Or R is₁＝H₂And R is₂＝C；

In the formula III-3, R₁＝H₂And R is₂Either NH, or R₁＝H₂And R is₂＝CH₂Or R is₁＝H₂And R is₂＝C；

In the formula III-4, R₁Is O and R₂Either NH, or R₁＝H₂And R is₂Either NH, or R₁＝H₂And R is₂＝CH₂Or R is₁＝H₂And R is₂＝C；

In the formula III-5, R ═ O or H₂(ii) a In formula III-6, R is OH or H; in formula III-7, R is OH or H;

specifically, Y is a unit with the length of 1-30 atoms and any type.

More specifically, the Y includes, but is not limited to, the following structures, or any structural group consisting of the following structures.

Such as: unit consisting of ethylene glycol structure:

or units of fatty chains:

or units composed of unsaturated chains:

or units of aromatic compounds:

or heteroatom-containing units such as sulfur, nitrogen, phosphines, and the like, including but not limited to the following fragment units:

r in the heteroatom-containing unit may independently be: r in the heteroatom-containing unit is independently: alkyl, alkoxy or hydrogen;

preferably, the alkyl is specifically methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl; the alkoxy is-OEt, -OMe.

Specifically, the structural formula of the small molecule conjugate is shown as any one of formula IV-formula X:

in the formula IV, m is an integer between 0 and 6 (preferably an integer between 1 and 6), n is an integer between 0 and 6, and q is an integer between 1 and 15; a is CH₂NH, O or CO, G is CH₂Or CO, B is CO or CH₂E is CO or CH₂R is as defined for formula II, R_LIs H, methyl or ethyl.

In the formula V, m is an integer of 1-6, n is an integer of 0-6, R is defined as formula II, G is CH₂Or CO.

In formula VI, m is an integer of 1 to 6, n is an integer of 0 to 6, R is as defined in formula II, G is CH₂Or CO.

In formula VI I, m is an integer of 1 to 15, n is an integer of 0 to 6, and R is defined asIn the same formula II, G is CH₂Or CO.

In formula VIII, m is an integer of 1-15, n is an integer of 0-6, R is defined as formula II, G is CH₂Or CO.

In the formula IX, n is an integer between 0 and 6, R is as defined in the formula II, G is CH₂Or CO.

In the formula X, n is an integer between 0 and 6, R is defined as formula II, G is CH₂Or CO.

More specifically, the small molecule conjugate is a compound shown in the following formula 1-formula 138:

another objective of the invention is to provide a preparation method of the small molecule conjugate shown in the formula I.

The compound shown in the formula I can be synthesized by a click reaction, an amide condensation reaction, a substitution reaction or a cross-coupling reaction between a ligand (such as Pomalidomide derivatives, VHL ligand derivatives and MDM2 ligand suborganisms) of E3 ligase and a derivative of an X part.

Specifically, the small molecule conjugate described in formula IV (wherein G is CH)₂N is 1, q is 1, A is NH and B is CO) can be formed by click chemistry connection between Pomalidomide end derivative 1 (shown in a formula B) and JNJ-7706621 end derivative 1 (shown in a formula a);

r in formula a is as defined in formula II, R in formula b_LAnd m is as defined in formula IV.

Among them, the preparation methods of Pomalidomide terminal derivatives 1 can be referred to in Chemistry & Biology 22, 755-.

The small molecule conjugate (wherein G is CH) described by the formula V₂And n is 1) can be formed by connecting a VHL ligand derivative (shown in a formula c) and a click chemistry between JNJ-7706621 terminal derivative 1; m in the formula c is defined as the formula V.

Wherein, the preparation method of the VHL ligand derivative can be referred to the ACS Med.chem.Lett.2019,10(5), 767-.

The small molecule conjugate (wherein G is CH) described by the formula VI₂And n is 1) can be formed by connecting an MDM2 ligand derivative (shown in a formula d) with click chemistry between JNJ-7706621 terminal derivative 1; m in formula d is as defined in formula VI.

Among them, the preparation methods of the MDM2 ligand derivative (shown in formula d) can be referred to in the documents ACS Med.chem.Lett.2013,4(5),466-9 and ACS Med.chem.Lett.2019,10(5), 767-.

The small molecule conjugate shown in the formula VI I (wherein G is CH)₂N is 1) and a small molecule conjugate described by formula VIII (wherein, G is CH₂N is 1) can be formed by click chemistry connection between Lenalidomide derivative 1 (shown in formula e) and JNJ-7706621 terminal derivative 1 to obtain formula VI I; then further reducing alkyne in the formula VI I to obtain a formula VIII; m in the formula e is defined as formula VI I.

Among them, the preparation method of Lenalidomide derivative 1 is described as the preparation method of intermediate 17 below.

The small molecule conjugate shown in the formula IX (wherein G is CH)₂N is 3) and the small molecule conjugate described by the formula X (wherein, G is CH₂And n is 3) can be connected by Sonogashira cross-coupling reaction between Lenalidomide ligand derivative 2 (shown in formula g) and JNJ-7706621 terminal derivative 2 (shown in formula h) to obtain formula IX; then further reducing alkyne in the formula IX to obtain a formula X; r, G, n in formula h is as defined in formula IX or formula X.

The preparation method of the Lenalidomide ligand derivative 2 can be referred to in J.Med.chem.2018,61 (2)462-481.

The JNJ-7706621

terminal derivatives

1 and 2 also belong to the protection scope of the invention.

The synthesis routes of the JNJ-7706621

terminal derivatives

1 and 2 are shown in FIG. 2, and the specific processes are as follows:

1) reacting p-nitrobenzenesulfonyl chloride (formula 1b) with 2- (prop-2-yn-1-yloxy) ethane-1-amine (formula 3a) or 3,6,9, 12-tetraaptadienec-14-yn-1-amine (formula 3b) to obtain 4-nitro-N- (2- (prop-2-yn-1-yloxy) ethyl) benzanesulfonamide (formula 4a) or 4-nitro-N- (3,6,9, 12-tetraaptadienec-14-yn-1-yl) benzanesulfonamide (formula 4 b);

2) then reducing the nitro in the 4-nitro-N- (2- (prop-2-yn-1-yloxy) ethyl) benzasulfenamide (formula 4a) or the 4-nitro-N- (3,6,9, 12-tetraacetradec-14-yn-1-yl) benzasulfenamide (formula 4b) by using zinc powder and hydrochloric acid; then adding thiophosgene to react to prepare an intermediate 4-isothiocyanato-N- (2- (prop-2-yn-1-yloxy) ethyl) benzacetamide (formula 5a) or 4-isothiocyanato-N- (3,6,9, 12-tetraoxaprenec-14-yn-1-yl) benzacetamide (formula 5b) containing isothiocyanate;

3) reacting formula 5a or formula 5b with 3, 5-dimethyl-1-pyrazole carboxamidine ammonium nitrate to attack the isothiocyanate function to give intermediate 6a (formula 6a) or intermediate 6b (formula 6 b);

5) adding hydrazine hydrate into the formula 6a or the formula 6b for cyclization to obtain an intermediate 7a or 7b (the formula 7a or the formula 7 b);

6) reacting the formula 7a or the formula 7b with a compound containing acyl chloride respectively, and condensing different fragments through acyl chloride to obtain a JNJ-7706621 terminal derivative 1 or 2 (a formula 8a or a formula 8b), wherein the derivative contains alkyne sticky ends; wherein the structural formula of the acyl chloride-containing compound is shown as

The structure of R is the same as formula II.

The acyl chloride-containing compound is specifically as follows: 2, 6-difluorobenzoyl chloride and 3-methylthiophene-2-formyl chloride, and has the chemical structures as follows:

it is a further object of the present invention to provide uses of the above small molecule conjugates.

The application of the small molecule conjugate provided by the invention comprises at least one aspect of the following 1) to 5):

1) the use of CDK2 in the preparation of a selective degrader;

2) the application in preparing cancer cell proliferation inhibitor;

3) the application in preparing cancer cell differentiation inducer;

4) the application in preparing AML cell differentiation inducer;

5) the application in the preparation of the medicament for preventing and/or treating leukemia.

The CDK2 selective degraders selectively degrade CDK2 in cells including, but not limited to: acute myelogenous leukemia cells (e.g., U937, Kasumi-1, MV-4-11, OCI-AML2, OCI-AML3, NB4), T-lymphocytic leukemia cells (e.g., Jurkat, MOLT-4), chronic myelogenous leukemia cells, acute lymphocytic leukemia cells, B-lymphocytic leukemia cells (e.g., Ramos, Z138, HBL-1, Mino, Pfeiffer, DOHH2), chronic lymphocytic leukemia cells, chronic myelogenous leukemia cells (e.g., K562), myeloma cells (e.g., MM.1s), breast cancer cells (e.g., MDA-MB-231), and glioma cells (e.g., U251).

The cancer cells include leukemia cells, myeloma cells, breast cancer cells, and glioma cells. Such leukemia cells include, but are not limited to, the following: acute myelocytic leukemia cells (e.g., U937, Kasumi-1, MV-4-11, OCI-AML2, OCI-AML3, NB4), T-lymphocytic leukemia cells (e.g., Jurkat, MOLT-4), chronic myelocytic leukemia cells, acute lymphocytic leukemia cells, B-lymphocytic leukemia cells (e.g., Ramos, Z138, HBL-1, Mino, Pfeiffer, DOHH2), chronic lymphocytic leukemia cells, chronic myelocytic leukemia cells (e.g., K562)

The leukemia includes Acute Myelogenous Leukemia (AML), T lymphocyte leukemia, chronic myelogenous leukemia, acute lymphocyte leukemia, B lymphocyte leukemia, and chronic lymphocytic leukemia.

The Acute Myeloid Leukemia (AML) includes M1 acute myelocytic leukemia immature type, M2 acute myelocytic leukemia partially mature type, M3 Acute Promyelocytic Leukemia (APL), M4 acute myelomonocytic leukemia, M5 acute monocytic leukemia, M6 acute erythrocytic leukemia, M7 acute megakaryocytic leukemia, M0 acute differentiative myelocytic leukemia.

The AML cells include NB4, HL60, U937, Kasumi-1, MV-4-11, OCI-AML2, OCI-AML3 and the like.

Any product prepared by taking the small molecule conjugate shown in the formula I as an active ingredient also belongs to the protection scope of the invention; the product comprises: 1) CDK2 selective degraders; 2) an inhibitor of cancer cell proliferation; 3) a cancer cell differentiation inducer; 4) an AML cell differentiation inducer; 5) a medicament for preventing and/or treating leukemia.

The drug can be introduced into body such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue by injection, spray, nasal drop, eye drop, penetration, absorption, physical or chemical mediated method; or mixed or coated with other materials and introduced into body.

If necessary, one or more pharmaceutically acceptable carriers can be added into the medicine. The carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field.

The above medicine can be made into tablet, powder, granule, capsule, oral liquid, unguent, cream, injection, etc.; the medicaments in various dosage forms can be prepared according to the conventional method in the pharmaceutical field.

The invention also provides a pharmaceutical composition.

The small molecule conjugate (CDK2 degradation agent) provided by the invention not only can be independently used for differentiation induction treatment of AML and other diseases, but also can be used for combined medication, and can be more effectively used for treatment of diseases.

The invention provides a pharmaceutical composition, which comprises a small molecule conjugate (CDK2 degradation agent) shown as a formula I and at least one of the following drugs: BET inhibitors, CDK4&6 inhibitors, PI3K inhibitors, and all-trans retinoic acid (ATRA).

The BET inhibitors include ABBV-075, JQ1, dBET-1, ARV-825. The CDK4&6 inhibitor comprises YX-2-107, Palbociclib. The PI3K inhibitor includes Copanlisib.

The pharmaceutical composition may specifically be a small molecule conjugate of formula I (CDK2 degrader) and all inhibitors as above in a molar ratio of 1: 1-10: 1 in the ratio of the total weight of the composition.

There is also a lack of selective inhibitors or degradants against CDK 2. The invention obtains the selective CDK2 degradation agent by the PROTAC technology. Wherein the half Degradation concentration (DC 50) for the selective Degradation of CDK2 is about 8 nM. The degrading agent has good selectivity for other approximate target CDK1 and CDK5, no degrading activity or weak degrading activity, and good selectivity for molecular degradation of target protein. Compared with the existing CDK2 inhibitor, the inhibitor has good selectivity and low toxicity. Strong degradability to CDK2 was exhibited on a variety of cell lines. Exhibits strong differentiation-inducing activity on AML cell lines, and also exerts inhibitory activity on the growth of transplanted tumors in vivo in vivo experiments.

Drawings

FIG. 1 is a basic technical scheme of PROTACs.

FIG. 2 is a schematic diagram of the construction of the small molecule conjugate of the present invention by click reaction and amide condensation reaction and a synthetic route for synthesizing the alkyne end of the JNJ-7706621 derivative.

FIG. 3 is a graph of the degradation of CDK2 after 24 hours of treatment of JNJ, formula 1, formula 2, formula 3, formula 4, and formula 5 on a Ramos cell line, JNJ being an abbreviation for JNJ-7706621.

FIG. 4 is a graph of CDK2 degradation following 24 hours of treatment of

formulas

4 and 6 on a Ramos cell line.

FIG. 5 is the degradation of CDK2 after 24 hours of treatment of J2, formula 7, formula 8, formula 9 on a Ramos cell line, J2 being a terminal derivative of JNJ-7706621.

FIG. 6 shows the degradation of CDK1, CDK2, CDK4, CDK5, CDK6, CDK9-long form and CDK9-short form after treatment of J2, formula 7, formula 8, formula 9 on a Ramos cell line for 24 hours, J2 being the terminal derivative J2 of JNJ-7706621.

FIG. 7 shows the abundance of CDK2 in the cell lines of

formulas

11, 12, 13, 14, 15, 16, 17, 19, 20, 98, 8 and 26 at concentrations of 1nM and 5nM, in the Ramos cell line used.

FIG. 8 is a graph of the degradation of CDK2, CDK4, CDK6, CDK7, CDK9, with n being an abbreviation in nM (nanomolar) for formula 14 and formula 8, treated on Ramos cell lines for 6 hours.

FIG. 9 is the expression level of CDK2 in cells of formula 8 after 48 hours of treatment on the cell lines shown in the figure.

FIG. 10 is a schematic of the selectivity and low toxicity of formula 8; a-c. the cell line used was Ramos, a. formula 8 was used at a concentration of 250nM, b. the duration of the treatment was 6 hours, c.pre-treat means pretreatment was carried out 2 hours in advance, the treatment was done in the following order from left to right: no treatment is carried out; no treatment is carried out; 2 mu M J2 treatment; 2 μ M Pomalidomide treatment; 500nM Bortezomib treatment; 500nM Carfilzomib pretreatment, after which 250nM of formula 8, named in Pomalidomide: pomalidomide (from graduate medicine), Bortezomib chinese name: bortezomib (purchased from a source leafy organism); the viability of the four cells under the chinese name Carfilzomib (purchased from mcolin reagent), d, formula 8, Poma is an abbreviation for Pomalidomide, and is not described below, J2+ Poma is J2 and Pomalidomide is 1:1 molar concentration. Cells were treated for 3 days.

Fig. 11 shows the survival of Ramos cells treated with each drug molecule, and the cells were treated for three days.

FIG. 12 is a graph showing cell survival for combination experiments and FA-CI curves, a. cells were treated for 3 days on MV-4-11 with Copalisib (100nM) + formula 8 at a fixed Copanslisib concentration of 100nM in combination with formula 8; b. palbociclib (120nM) + formula 8 means that fixed Palbociclib is used at 120nM, in combination with formula 8, at MV-4-11, for 3 days of treatment; and (c) using open source software to calculate the FA-CI curve: CompuSynver.1.0. d, treatment for 3 days on Kasumi-1, Palbociclib (200nM) + formula 8 means that Palbociclib is used at a fixed concentration of 200nM, in combination with formula 8; e, HL60, the effect of the combination of the formula 8 and the ATRA on the induction of cell differentiation is shown schematically, the cell treatment time is 3 days, the use concentration is 8125 nM, and the ATRA concentration is 2.5 nM.

FIG. 13 shows the differentiation of NB4 cells treated according to formula 8.

FIG. 14 shows the results of the mouse transplantable tumor test and toxicity test under the treatment of formula 8; a-f, nude mouse NB4 cell axillary transplantation tumor experiment and related data, the injection frequency is 1 time per two days, the administration mode is tail vein injection, g, toxicity experiment of normal mouse model, the administration dosage of J2 and formula 8 is 400mg/kg, single administration and intraperitoneal injection.

Detailed Description

The present invention is described below with reference to specific embodiments, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

The Pomalidomide terminal derivatives used in the following examples were prepared according to the methods disclosed in the publications Chemistry & Biology 22, 755-.

J2, used in the examples below, is a terminal derivative of JNJ-7706621, having the structure:

the synthesis of this compound is described in J.Med.chem.2005,48, 4208-4211.

The synthetic route of the JNJ-7706621 terminal derivative 1 is shown in figure 2, and the specific process is as follows: 1) firstly, reacting p-nitrobenzenesulfonyl chloride with 2- (prop-2-yn-1-yloxy) ethane-1-amine to obtain 4-nitro-N- (2- (prop-2-yn-1-yloxy) ethyl) benzanesulfonamide; 2) then, reducing the nitro of the 4-nitro-N- (2- (prop-2-yn-1-yloxy) ethyl) benzasulfenamide by using zinc powder and hydrochloric acid to obtain 4-amino-N- (2- (prop-2-yn-1-yloxy) ethyl) benzasulfenamide; 3) then adding thiophosgene to prepare 4-isothiocyanato-N- (2- (prop-2-yn-1-yloxy) ethyl) benzanesulfonamide containing isothiocyanate intermediate; 4) further, adding 3, 5-dimethyl-1-pyrazole formamidine ammonium nitrate to attack an isothiocyanate functional group to obtain an intermediate; 5) adding hydrazine hydrate for cyclization to obtain an intermediate; 6) on the basis of this intermediate, the different fragments are condensed by acid chlorides. JNJ-7706621-terminated derivative 1 was obtained, which contained an alkyne cohesive terminus.

The preparation process comprises the following steps:

(1) preparation of intermediate 1a

A100 ml round bottom flask was charged with 610mg of 2-amino-1-ethanol and dissolved in 30ml of dichloromethane solvent. 61mg of 4-dimethylaminopyridine were added. With stirring in an ice bath, 2.6g of Boc anhydride was added slowly. After stirring at room temperature for 24 hours, TLC detection of the complete reaction of the starting materials, washing twice with a saturated brine bath, separation of the organic phase and spin drying. 1.47g of compound 1a was isolated by silica gel column in 91% yield.

LC-MS(ESI)m/z(MH)⁺called.for C₇H₁₅NO₃ ⁺＝162.11；found 162.23.

¹H NMR(400MHz,CDCl₃)δ5.13(s,1H),3.73–3.61(m,2H),3.24(dd,J＝10.0,5.0Hz,2H),3.12(s,1H),1.41(s,9H).

¹³C NMR(101MHz,CDCl₃)δ160.70,79.67,62.40,40.70,28.46.

(2) Preparation of intermediate 2a

A25 ml round bottom flask was charged with 161mg of intermediate 1a dissolved in 10ml of dry tetrahydrofuran solution. While stirring in an ice bath, 134mg of potassium tert-butoxide was slowly added, the temperature was slowly returned to room temperature, and stirring was continued for half an hour. After 125mg of bromopropyne were slowly added dropwise, the reaction was allowed to stir further at 40 ℃. The reaction progress is checked by TLC, and the solvent is removed by rotary evaporation after the reaction substrate is completely consumed. Ethyl acetate was added and the mixture was washed three times with saturated brine, and the organic phase was separated. The organic phase was removed by rotary drying. Purification by column separation on silica gel gave 120mg of Compound 2a in 61% yield.

LC-MS(ESI)m/z(M+H)⁺called.for C₁₀H₁₇NO₃ ⁺＝200.12；found 200.23.

¹H NMR(400MHz,CDCl₃)δ4.15(d,J＝2.2Hz,2H),3.58(t,J＝5.1Hz,2H),3.33(d,J＝5.0Hz,2H),2.44(s,1H),2.03(s,1H),1.43(s,9H).

¹³C NMR(101MHz,CDCl₃)δ79.57,74.78,69.23,60.51,58.40,40.45,28.52,14.32.

(3) Preparation of intermediate 3a

A25 mL round bottom flask was charged with 200mg of intermediate 2a, and 10mL of a trifluoroacetic acid-dichloromethane mixed solution (1:2, v/v) was added. After stirring for an additional 15 minutes, the progress of the reaction was checked by TLC until no starting material remained. The reaction solution was rotary evaporated to dryness. The sample bottle was connected to an oil pump and the sample was further dried to give intermediate 3 a. Intermediate 3a was used directly in the next step without further purification.

(4) Preparation of intermediate 4a

Intermediate 3a was added to a 25ml round bottom flask, intermediate 3a was prepared from intermediate 2a and the amount of intermediate 2a used to prepare intermediate 3a in the previous step was 200 mg. Intermediate 3a was dissolved in 10ml of DCM solution. The reaction was stirred in an ice bath, 277mg of p-nitrobenzenesulfonyl chloride was added, and 202mg of triethylamine solution was slowly added. The reaction was slowly brought to room temperature, stirred for 2 hours and checked by TLC to completion. The compound was purified by silica gel chromatography to give 210mg of intermediate 4 a.

LC-MS(ESI)m/z(M-H)^-called.for C₁₀H₁₀N₂O₅S^-＝283.05；found 283.26.

¹H NMR(400MHz,CDCl₃)δ8.36(d,J＝8.9Hz,2H),8.06(d,J＝8.9Hz,2H),5.19(s,1H),4.08(d,J＝2.4Hz,2H),3.68–3.37(m,2H),3.24(d,J＝4.6Hz,2H),2.43(s,1H).

¹³C NMR(100MHz,CDCl₃)δ150.17,146.01,128.45,124.53,78.92,75.38,68.04,58.49,43.13.

(5) Preparation of intermediate 5a

284mg of intermediate 4a were dissolved in 10ml of an ethanol-1M hydrochloric acid (1: 1v/v) mixed solution. While stirring in an ice bath, 262mg of zinc powder was slowly added, the reaction was checked by TLC until completion, and the unreacted zinc powder was removed by filtration. 172mg of thiophosgene was added and stirred overnight. Rotary drying to remove ethanol, adding ethyl acetate, separating, extracting, separating ethyl acetate phase, and drying with anhydrous sodium sulfate. The ethyl acetate solution was removed by rotary drying. Purification by silica gel chromatography gave 124mg of intermediate 5a in 42% yield.

LC-MS(ESI)m/z(M+H)⁺called.for C₁₁H₁₀N₂O₃S₂ ⁺＝297.03；found 297.23.

¹³C NMR(101MHz,CDCl₃)δ138.41,135.90,128.81,126.41,79.00,75.31,68.10,58.50,43.02.

(6) Preparation of intermediate 6a

296mg of intermediate 5a was added to a 25ml round bottom flask, and 10ml of anhydrous DMF was added. Under stirring in an ice bath, 201mg of 3, 5-dimethyl-1-pyrazole carboxamidine ammonium nitrate and 56mg of potassium hydroxide are added. Heating and stirring at 50 ℃ for 30 minutes, detecting by TLC that the raw material is completely consumed, and removing DMF by rotary evaporation. Ethyl acetate and saturated brine were added, and the mixture was extracted and washed. Separating and extracting an ethyl acetate phase, adding anhydrous sodium sulfate for drying, and carrying out rotary drying to obtain an intermediate 6a crude product. Purification by silica gel chromatography gave 209mg of intermediate 6a in 47% yield.

LC-MS(ESI)m/z(M+H)⁺called.for C₁₈H₂₂N₆O₃S₂ ⁺＝435.12；found 435.18.

¹H NMR(400MHz,CDCl₃)δ10.72(s,1H),8.33(s,1H),7.95(s,1H),7.82(d,J＝8.2Hz,2H),7.68(s,1H),5.96(s,1H),4.87(t,J＝5.6Hz,1H),4.09(s,2H),3.57(t,J＝4.8Hz,2H),3.18(d,J＝5.0Hz,2H),2.44(s,1H),2.24(s,3H),2.04(s,1H),1.70(s,1H),1.26(s,2H).

¹³C NMR(100MHz,CDCl₃)δ128.06,111.98,79.07,75.30,68.17,58.51,43.03,13.88.

(7) Preparation of intermediate 7a

434mg of intermediate 6a were charged into a 25ml round bottom flask, and 10ml of anhydrous tetrahydrofuran was added. Add 250mg of hydrazine hydrate. Heated and stirred at 65 ℃ for 3 hours. The THF was removed by rotary evaporation, dried under reduced pressure and washed with ethyl acetate petroleum ether (1:2, v/v) and filtered to give crude intermediate 7a as 130mg, 39% yield. Intermediate 7a was used directly in the next step without further purification.

(8) Preparation of intermediate 8a-1

337mg of the intermediate 7a was dissolved in 10ml of DMF solution, and 264mg of 2, 6-difluorobenzoyl chloride was slowly added dropwise under stirring in an ice bath, followed by 202mg of triethylamine solution. The reaction was slowly brought to room temperature and stirring was continued for 2 hours. Ethyl acetate and saturated brine were added for extraction, and the mixture was washed twice with saturated brine again. Separating and extracting the ethyl acetate phase, and adding anhydrous sodium sulfate for drying. After ethyl acetate was removed by rotary evaporation, compound 8a was purified by silica gel chromatography to give 308mg of pure product with a yield of 65%.

LC-MS(ESI)m/z(M+H)⁺called.for C₂₀H₁₈F₂N₆O₄S⁺＝477.11；found 477.07.

¹H NMR(400MHz,DMSO)δ9.91(s,1H),8.03(s,2H),7.72(dd,J＝11.7,5.0Hz,1H),7.54(d,J＝8.9Hz,2H),7.46(dd,J＝11.3,7.5Hz,3H),7.35(t,J＝8.3Hz,2H),4.06(d,J＝2.3Hz,2H),3.39(dd,J＝11.6,5.7Hz,2H),2.82(q,J＝5.9Hz,2H),1.99(s,1H).

(9) Preparation of intermediate 8a-2

337mg of the intermediate 7a is dissolved in 10ml of DMF solution, and after 240mg of 3-methylthiophene-2-carbonyl chloride is slowly added dropwise with stirring in an ice bath, 202mg of triethylamine solution is slowly added dropwise. The reaction was slowly brought to room temperature and stirring was continued for 2 hours. Ethyl acetate and saturated brine were added for extraction, and the mixture was washed twice with saturated brine again. Separating and extracting the ethyl acetate phase, and adding anhydrous sodium sulfate for drying. After ethyl acetate was removed by rotary evaporation, compound 8a was purified by silica gel chromatography to give 223mg of pure product with a yield of 49%.

LC-MS(ESI)m/z(M+H)⁺called.for C₁₉H₂₀N₆O₄S₂ ⁺＝461.01；found 461.11.

¹H NMR(400MHz,DMSO)δ9.94(s,1H),8.05(d,J＝5.0Hz,1H),7.87(s,2H),7.82(d,J＝8.8Hz,2H),7.73(d,J＝8.8Hz,2H),7.49(s,1H),7.17(d,J＝5.0Hz,1H),4.08(d,J＝2.3Hz,2H),3.45–3.38(m,2H),3.33(m,2H),2.89(d,J＝6.0Hz,3H),2.63(s,1H).

(10) Preparation of intermediates 9-1, 9-2, 9-3, 9-4, 9-5, 9-6, 9-7, 9-8, 9-9, 9-10, 9-11, 9-12, 9-13, 9-14

The bishydroxy compound (1.0eq) was stirred with 1.1eq of t-BuOK in THF for 15min, and 1eq of bromopropyne was added dropwise to give compound 9.

The dihydroxy compounds used for intermediates 9-1, 9-2, 9-3, 9-4, 9-5, 9-6, 9-7, 9-8, 9-9, 9-10, 9-11, 9-12, 9-13, 9-14 are respectively: diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, ethylene glycol, propane-1,3-diol, butane-1,4-diol, pentane-1,5-diol, hexane-1,6-diol, heptane-1,7-diol, octane-1,8-diol, nonane-1,9-diol, and cane-1, 10-diol.

(11) Preparation of intermediate 10

8g of sulfanilamide was dissolved in 30ml of DCM: THF 1: 1(v/v) mixed solvent, 30g of Boc anhydride was added slowly with stirring in ice bath and stirred overnight. Removing the solvent by rotary evaporation, adding water for washing and filtering, and discarding the filtrate. The intermediate 10 was obtained by silica gel column chromatography (petroleum ether/ethyl acetate 1:3, v/v) in 20% yield.

(12) Preparation of intermediate 11

3.7g of intermediate 10 and 2.8g of intermediate 9-4 were dissolved in 15ml of anhydrous DCM, and 3g of triphenylphosphine was added and dissolved with stirring. The reaction was placed in an ice bath with stirring and 2.58g of diethyl azodicarboxylate was added slowly. The reaction was then left to stir at room temperature for an additional 6 hours and after completion of the reaction was checked by TLC, the solvent was removed by rotary evaporation. The mixture was thoroughly washed with saturated brine and ethyl acetate, and the aqueous phase was discarded. Intermediate 11 was then isolated by silica gel chromatography in 58% yield.

(13) Preparation of intermediate 12

5g of intermediate 11 are dissolved in 10ml of DCM and 5ml of trifluoroacetic acid are added under ice bath. After stirring for 15min, TLC monitored no starting material remaining. DCM and trifluoroacetic acid were removed by rotary evaporation. The sample was further dried with an oil pump. Intermediate 12 will continue to be used for the next step without additional processing.

(14) Preparation of intermediate 13

Synthetic procedures reference compounds 8a-1 and 8 a-2.

(15) Preparation of intermediate 14

The synthesis of intermediate 14 is described in the literature: J.Med.chem.2018,61(2),462-481.

(16) Preparation of intermediate 15

Intermediate 14(627mg,2mmol), 2eq intermediate 9(400mg), 0.1eq Pd (PPh)₃)₂Cl₂(140mg)0.2eq CuI (76mg) were mixed well in DMF under argon and TEA was added and stirred at 80 ℃ for 3 h. Dissolving EA, washing twice with saturated salt solution, separating an organic phase, concentrating, and separating and purifying by a silica gel column to obtain 500mg of the intermediate 15 with the yield of 56-82%. The corresponding relation of the product is as follows: the intermediate 9 used, starting material number 9-a, gives the target product 15-b, where b ═ a-5 (a.gtoreq.6).

(17) Preparation of intermediate 16

The intermediate 15-n (500mg,1.12mmol), 3eq MsCl (400ul), 3eq TEA (750ul) were mixed well in DMF and reacted at room temperature for 3 h. Dissolving EA, washing twice with saturated saline solution, separating an organic phase, and concentrating to obtain a crude product intermediate 16-n, wherein n is 1, 2,3,4, 5, 6, 7 and 8.

(18) Preparation of intermediate 17

Intermediate 16-n (crude), 3eq NaN₃Dissolving in DMF, reacting at 70 ℃ for 3h, dissolving EA, washing twice with saturated saline solution, separating an organic phase, concentrating, separating and purifying by a silica gel column to obtain an intermediate 17-n, wherein the yield of the two steps is 49-88%, and the value of n is 1, 2,3,4, 5, 6, 7 and 8.

(19) Preparation of intermediates 18-1 to 18-5

The synthesis of intermediates 18-1 to 18-5 can be found in the following references: organic Letters 22,10,3838-3841(2019). Wherein, the intermediate 18-X, X and n have the corresponding relation: x is n +1, n takes the

values

0, 1, 2,3, 4.

(20) Preparation of intermediate 19

Preparation of intermediate 19 can be found in the literature: organic & Biomolecular Chemistry,13(31), 8500-8504; 2015.

(21) preparation of intermediate 20

Preparation of intermediate 20 is referenced: organic Letters 22,10,3838-3841(2019).

(22) Preparation of intermediate 21

368mg of intermediate 19 and 276mg of intermediate 20 were dissolved in 10ml of DMSO solution, and 200ul of triethylamine was added. Heated and stirred at 70 ℃ for 4 hours. Separating and purifying with silica gel chromatographic column to obtain 260mg of intermediate 21.

(23) Preparation of intermediate 22

And respectively stirring the intermediate 9-6, 9-1, 9-2, 9-3, 9-4, 9-5 and 1.1eq of t-BuOK in THF for 15min, and dropwise adding 1eq of bromopropyne to obtain the compound 22-1, 22-2, 22-3, 22-4, 22-5 and 22-6 in turn.

(24) Preparation of intermediate 23

Reference is made to the synthetic route for intermediate 15, intermediate 16, intermediate 17.

(25) Preparation of intermediate 24-1, 24-2, 24-3, 24-4

Tert-butyl acrylate (7.3ml,50mmol), 4eq ethylene glycol/diethylene glycol/triethylene glycol/ethylene glycol, 0.1eq benzyltrimethylammonium hydroxide were mixed well in dry THF and reacted for 24h at room temperature. Dissolving EA, washing twice with saturated saline solution, separating an organic phase, concentrating and purifying with a silica gel column to obtain an intermediate 24-n, wherein n is 1, 2,3 and 4.

(26) Preparation of intermediate 25-n

Reference intermediate 16 preparation method.

(27) Preparation method of intermediate 26-n

Reference intermediate 17 preparation method.

(28) Preparation of intermediate 27-n

1mmol of the corresponding 26-n was dissolved in 5ml of THF, 1ml of trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 30 minutes. After TLC detection of the raw material reaction is finished, the solvent and trifluoroacetic acid are removed by rotary evaporation to obtain an intermediate 27-n. The compound was used in the next reaction without purification.

(29) Intermediate 28 preparation method.

Intermediate 28 was synthesized as described in Chemical Communications (Cambridge, United Kingdom),55(12), 1821-; 2019.

(30) a preparation method of an intermediate 29-n.

Reacting the intermediate 28(43mg,0.1mmol), 1.5eq intermediate 27-n, 1.2eq HATU (46mg) and 2eq DIPEA (33ul) in DMF for 3h, dissolving EA, washing twice with saturated saline solution, separating an organic phase, concentrating, and purifying with a silica gel column to obtain the intermediate 29-n, wherein n is 0, 1, 2,3, 4.

(31) Process for the preparation of intermediate 30

Intermediate 30 synthesis methods are referenced to PCT int.appl.,2016146985,22Sep 2016.

(32) Process for the preparation of intermediate 31

Reference intermediate 16 preparation method.

(33) Process for the preparation of intermediate 32-n

Reference intermediate 17 preparation method. The value of n is 1, 2,3,4 and 5.

(33) Process for the preparation of intermediate 32

Reference intermediate 16 preparation method. The value of n is 1, 2,3,4 and 5.

(34) Process for the preparation of intermediate 33-n

Refluxing intermediate 30(47mg,0.1mmol), 1.5eq intermediate 32-n, 2eq DIPEA (35ul) in THF for 3h, dissolving EA, washing twice with saturated saline, separating the organic phase, concentrating and purifying with silica gel column to obtain intermediate 33-n, wherein n is 1, 2,3,4, 5.

(35) Process for the preparation of intermediate 34

Intermediate 34 synthesis methods refer to Nature Communications,8(1), 1-14; 2017

(36) Process for the preparation of intermediate 35-n

Refluxing intermediate 34(47mg,0.1mmol), 1.05eq intermediate 32-n, 2eq DIPEA (35ul) in THF for 3h, dissolving EA, washing twice with saturated saline, separating the organic phase, concentrating and purifying with silica gel column to obtain intermediate 35-n, wherein n is 1, 2,3,4, 5.

(37) Process for the preparation of intermediate 36

Synthesis of intermediate 36 can be referred to: journal of Medicinal chemistry, 61(2), 492; 2018

(38) Preparation method of intermediate 37-n

Reference is made to the intermediate 15-n preparation.

(39) Process for the preparation of intermediate 38-n

Reference is made to the intermediate 16-n preparation.

(40) Preparation method of intermediate 39-n

Reference is made to the intermediate 17-n preparation.

(41) Process for the preparation of intermediate 40

Reference Protein & Cell, 10(8), 606-; 2019 by the following synthetic route

(42) Process for the preparation of intermediate 41

Reference org.Lett.2019,21,10, 3838-

(43) Preparation method of intermediate 42-n

1mmol of 18-n was dissolved in 10ml of methanol solution, 1eq of HCl was added and 0.1mmol of Pd/C was added. And (3) filling hydrogen, stirring vigorously overnight, after TLC detection is completed, performing suction filtration to remove Pd/C, and performing spin drying to obtain an intermediate 42-n.

(44) Process for the preparation of intermediate 43

The intermediate 43 is obtained by repeating the preparation method of the intermediate 13 using methyl p-aminobenzoate as a raw material instead of the intermediate 12. 1mmol of intermediate 43 was dissolved in 20ml of THF, 5ml of water was added, 5mmol of KOH were added and stirring was carried out overnight at room temperature. The reaction was acidified and extracted and purified by silica gel chromatography to give intermediate 44. The yield was 87%.

Example 1: preparation of Compounds represented by formula 1 to formula 49

40mg of intermediate 18-150 mg, intermediate 8a-1, 6mg of CuSO were added to a 5ml round bottom flask₄40mg of sodium ascorbate, 5ml of DMF. After stirring overnight at room temperature, the mixture was washed with water and extracted with ethyl acetate. With dichloromethane: methanol 20: passing through silica gel column at 1(v/v) gave the compound of formula 1 in 75% yield.

LC-MS(ESI)m/z(M+H)⁺called.for C₃₅H₃₂F₂N₁₂O₈S⁺＝819.22；found 819.19.

¹H NMR(400MHz,DMSO)δ11.11(s,1H),9.92(s,1H),8.11(s,1H),8.03(s,1H),7.71(s,1H),7.54(d,J＝8.9Hz,3H),7.48(d,J＝8.7Hz,2H),7.43(s,1H),7.34(t,J＝8.7Hz,1H),7.04(d,J＝7.3Hz,1H),6.78(d,J＝8.1Hz,1H),5.04(s,1H),4.66–4.54(m,2H),4.42(s,2H),3.81(s,2H),3.51(s,2H),2.89–2.61(m,4H),2.05–1.92(m,2H).

Referring to the preparation method of formula 1, intermediate 18-2 and intermediate 8a-1 are used.

LC-MS(ESI)m/z(M+H)⁺called.for C₃₈H₃₈F₂N₁₂O₉S⁺＝863.24；found 862.98.

¹H NMR(400MHz,DMSO)δ11.11(s,1H),9.91(s,1H),8.02(s,1H),7.98(s,1H),7.69(d,J＝8.4Hz,1H),7.58–7.50(m,3H),7.47(d,J＝9.0Hz,2H),7.41(t,J＝6.0Hz,1H),7.34(t,J＝8.3Hz,2H),7.09(d,J＝8.6Hz,1H),7.06–6.99(m,1H),6.58(s,1H),5.08–4.99(m,1H),4.51(t,J＝5.2Hz,2H),4.40(s,2H),3.82(t,J＝5.2Hz,2H),3.63–3.53(m,3H),3.43(dd,J＝10.4,4.9Hz,3H),2.95–2.71(m,4H),2.61(s,2H),2.05–1.91(m,4H).

Referring to the preparation method of formula 1, intermediate 18-3 and intermediate 8a-1 are used.

LC-MS:calculated for C₃₉H₄₁F₂N₁₂O₁₀S[M+H]⁺:907.27,found 907.07.

¹H-NMR(400MHz,DMSO-d6,ppm):11.09(s,1H),9.88(s,1H),8.00(s,2H),7.97(s,1H),7.74-7.68(m,1H),7.58-7.51(m,3H),7.43-7.40(m,3H),7.35(t,J＝8.28,2H),7.11(d,J＝8.64,1H),7.04(d,J＝7.00,1H),6.58(t,J＝5.52,1H),5.05(dd,J＝5.44,J＝12.76,1H),4.48(t,J＝5.08,2H),4.39(s,2H),3.80(t,J＝5.08,2H),3.51-3.36(m,10H),2.90-2.55(m,5H),2.03(m,1H).

Referring to the preparation method of formula 1, intermediate 18-4 and intermediate 8a-1 are used.

LC-MS:calculated for C₄₁H₄₅F₂N₁₂O₁₁S[M+H]+:951.29,found 951.05.

¹H-NMR(400MHz,DMSO-d6,ppm):11.10(s,1H),9.88(s,1H),7.98(m,3H),7.74-7.68(m,1H),7.58-7.52(m,3H),7.47-7.41(m,3H),7.34(t,J＝8.28,2H),7.12(d,J＝8.56,1H),7.03(d,J＝7.00,1H),6.58(t,J＝5.28,1H),5.06(dd,J＝5.52,J＝12.84,1H),4.48(t,J＝5.04,2H),4.41(s,2H),3.78(t,J＝5.12,2H),3.58-3.37(m,14H),2.81-2.56(m,5H),2.02(m,1H).

Referring to the preparation method of formula 1, intermediate 18-5 and intermediate 8a-1 are used.

LC-MS:calculated for C₄₃H₄₈F₂N₁₂O₁₂S[M+H]+:995.32,found 995.95.

Referring to the preparation method of formula 1, intermediate 21 and intermediate 8a-1 are used.

LC-MS:calculated for C₄₄H₅₀F₂N₁₂O₈S[M+H]+:945.36,found 945.49.

Referring to the preparation method of formula 1, intermediate 18-2 and intermediate 8a-2 are used.

LC-MS:calculated for C₃₆H₃₉N₁₂O₉S₂[M+H]⁺:847.23,found 846.93.

¹H-NMR(400MHz,DMSO-d6):11.10(s,1H),9.93(s,1H),8.04(d,J＝5.04,1H),7.98(s,1H),7.86(s,2H),7.83(d,J＝8.92,2H),7.74(d,J＝8.96,2H),7.56(dd,J＝7.16,J＝8.48,1H),7.46(t,J＝6.00,1H),7.16(d,J＝5.08,1H),7.08(d,J＝8.56,1H),7.03(d,J＝6.96,1H),6.57(t,J＝5.72,1H),5.07(dd,J＝5.32,J＝12.80,1H),4.52(t,J＝5.12,2H),4.42(s,2H),3.83(t,J＝5.20,2H),3.60(t,J＝5.52,2H),3.43-3.35(m,6H),2.89-2.85(m,3H),2.62(s,3H),2.04(m,1H).

¹³C-NMR(100MHz,DMSO-d6):δ172.80,170.08,168.90,167.27,160.21,157.05,156.84,150.56,146.34,144.05,143.63,136.17,135.69,132.05,131.42,131.08,127.89,124.28,123.91,117.40,116.09,110.69,110.69,109.21,68.91,68.67,68.27,64.92,63.31,49.28,48.56,42.23,41.52,30.99,22.15,17.76.

Referring to the preparation method of formula 1, intermediate 18-3 and intermediate 8a-2 are used.

LC-MS:calculated for C₃₈H₄₃N₁₂O₁₀S₂[M+H]⁺:890.26,found 890.91.

¹H-NMR(400MHz,DMSO-d6):11.10(s,1H),9.94(s,1H),8.04(d,J＝5.00,1H),7.99(s,1H),7.87(s,2H),7.83(d,J＝8.76,2H),7.74(d,J＝8.80,2H),7.58(t,J＝7.96,1H),7.49(t,J＝5.96,1H),7.16(d,J＝5.00,1H),7.11(d,J＝8.60,1H),7.03(d,J＝7.04,1H),6.58(t,J＝5.48,1H),5.07(dd,J＝5.24,J＝12.76,1H),4.48(t,J＝5.00,2H),4.42(s,2H),3.80(t,J＝5.04,2H),3.57-3.34(m,12H),2.89-2.84(m,3H),2.62(s,3H),2.02(m,1H).

¹³C-NMR(100MHz,DMSO-d6):δ172.84,170.13,168.98,167.30,160.22,157.06,156.86,150.86,150.60,146.37,144.07,143.60,136.24,135.74,132.09,131.46,131.06,127.93,124.06,123.92,117.41,116.11,110.70,109.24.

Referring to the preparation method of formula 1, intermediate 18-4 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₀H₄₇N₁₂O₁₁S₂[M+H]⁺:934.29,found 935.09.

¹H-NMR(400MHz,DMSO-d6):11.10(s,1H),9.95(s,1H),8.05(d,J＝5.00,1H),8.00(s,1H),7.87(s,2H),7.83(d,J＝8.84,2H),7.74(d,J＝8.88,2H),7.57(t,J＝7.92,1H),7.50(t,J＝6.00,1H),7.16(d,J＝5.04,1H),7.11(d,J＝8.56,1H),7.03(d,J＝7.00,1H),6.59(t,J＝5.60,1H),5.07(dd,J＝5.48,J＝12.72,1H),4.48(t,J＝5.08,2H),4.44(s,2H),3.77(t,J＝5.12,2H),3.59(t,J＝5.48,2H),3.52-3.34(m,14H),2.90-2.85(m,3H),2.62(s,3H),2.02(m,1H).

¹³C-NMR(100MHz,DMSO-d6):172.84,170.11,168.93,167.29,160.21,157.05,156.85,150.58,146.37,144.06,143.59,136.20,135.73,132.08,131.44,131.06,127.91,124.27,123.91,117.40,116.09,110.67,109.22,69.74,69.66,69.52,68.84,68.66,68.32,63.39,49.29,48.54,42.23,41.66,30.98,22.13,17.78.

Referring to the preparation method of formula 1, intermediate 18-5 and intermediate 8a-2 are used.

Referring to the preparation method of formula 1, intermediate 16-1 and intermediate 8a-2 are used.

LC-MS:calculated for C₃₇H₃₈N₁₁O₈S₂[M+H]⁺:828.23,found 827.89.

¹H-NMR(400MHz,DMSO-d6,ppm):11.00(s,1H),9.93(s,1H),8.04(m,2H),7.86(s,2H),7.82(d,J＝8.72,2H),7.77-7.68(m,4H),7.56(t,J＝7.60,1H),7.48(t,J＝5.76,1H),7.16(d,J＝5.00,1H),5.17(dd,J＝4.80,J＝13.16,1H),4.58(t,J＝4.84,2H),4.48-4.29(m,4H),3.96(t,J＝4.92,2H),3.43-2.86(m,9H),2.62(s,3H),2.02(m,1H).

9mg of the product of formula 11 are dissolved in 5ml of methanol. 2mg Pd/C was added. The reaction solution was fully replaced with hydrogen, and hydrogen was continuously introduced. After 12 hours of reaction, mass spectrometry monitoring no raw materials remained, then the reaction was stopped. Separating and purifying with silica gel column chromatography to obtain 8mg of the product shown in formula 12.

LC-MS:calculated for C₃₇H₄₂N₁₁O₈S₂[M+H]⁺:832.26,found 831.92.

¹H-NMR(400MHz,DMSO-d6):10.99(s,1H),9.93(s,1H),8.04(d,J＝5.00,1H),8.01(s,1H),7.95(s,1H),7.87(s,2H),7.82(d,J＝8.88,2H),7.73(d,J＝8.92,2H),7.56(d,J＝7.04,1H),7.46-7.36(m,2H),7.16(d,J＝5.00,1H),5.14(dd,J＝5.00,J＝13.20,1H),4.51-4.36(m,4H),3.75(m,2H),3.32(m,6H),2.89-2.50(m,10H),2.00(m,1H),1.80(m,2H).

Referring to the preparation method of formula 1, intermediate 16-2 and intermediate 8a-2 are used.

LC-MS:calculated for C₃₈H₄₀N₁₁O₈S₂[M+H]⁺:842.24,found 841.92.

¹H-NMR(400MHz,DMSO-d6):11.00(s,1H),9.94(s,1H),8.04(m,2H),7.87(s,2H),7.82(d,J＝8.72,2H),7.77-7.69(m,4H),7.57(t,J＝7.56,1H),7.47(t,J＝5.76,1H),7.16(d,J＝4.92,1H),5.16(dd,J＝5.08,J＝13.24,1H),4.50-4.32(m,6H),3.53(t,J＝6.00,2H),3.40-3.35(m,4H),2.90-2.62(m,8H),2.12-1.98(m,3H).

9mg of the product of formula 13 were dissolved in 5ml of methanol solution. 2mg Pd/C was added. The reaction solution was fully replaced with hydrogen, and hydrogen was continuously introduced. After 12 hours of reaction, mass spectrometry monitoring no raw materials remained, then the reaction was stopped. Separating and purifying with silica gel column chromatography to obtain 8mg of the product shown in formula 14.

LC-MS:calculated for C₃₈H₄₄N₁₁O₈S₂[M+H]⁺:846.27,found 845.95.

¹H-NMR(400MHz,DMSO-d6):10.99(s,1H),9.94(s,1H),8.05(m,2H),7.95(s,1H),7.87(s,2H),7.83(d,J＝8.88,2H),7.74(d,J＝8.92,2H),7.57(t,J＝4.64,1H),7.48-7.44(m,2H),7.16(d,J＝4.96,1H),5.15(dd,J＝5.28,J＝13.24,1H),4.47-4.28(m,4H),3.42(t,J＝6.04,2H),3.39-3.30(m,6H),2.89-2.62(m,10H),2.03-1.99(m,3H),1.84(m,2H).

Referring to the preparation method of formula 1, intermediate 16-3 and intermediate 8a-2 are used.

LC-MS(ESI)m/z(M+H)⁺called for C₃₉H₄₁N₁₁O₈S₂ ⁺＝856.26；found 856.33.

¹H NMR(400MHz,DMSO)δ11.00(s,1H),9.95(s,1H),8.04(t,J＝2.5Hz,2H),7.88(s,2H),7.82(d,J＝8.9Hz,2H),7.78–7.68(m,3H),7.55(s,1H),7.47(s,1H),7.16(d,J＝5.0Hz,1H),5.14(dd,J＝13.4,5.0Hz,1H),4.48–4.38(m,4H),4.38–4.28(m,2H),3.53(t,J＝6.3Hz,3H),3.41(t,J＝5.9Hz,3H),2.89(dd,J＝7.5,4.3Hz,3H),2.74(s,1H),2.61(d,J＝9.9Hz,4H),2.06–1.91(m,3H),1.86(s,2H),1.54–1.39(m,3H),0.85(s,2H).

9mg of the product of formula 15 were dissolved in 5ml of methanol solution. 2mg Pd/C was added. The reaction solution was fully replaced with hydrogen, and hydrogen was continuously introduced. After 12 hours of reaction, mass spectrometry monitoring no raw materials remained, then the reaction was stopped. Separating and purifying with silica gel column chromatography to obtain 8mg of the product shown in formula 16.

LC-MS(ESI)m/z(M+H)⁺called.for C₃₉H₄₅N₁₁O₈S₂ ⁺＝860.29；found 860.23.

¹H NMR(400MHz,DMSO)δ11.01(s,1H),9.94(s,1H),8.05(t,J＝2.5Hz,1H),7.87(s,2H),7.81(d,J＝8.9Hz,2H),7.73(d,J＝8.92,2H),7.55(d,J＝7.04,1H),7.44(m,2H),7.16(d,J＝5.0Hz,1H),5.14(dd,J＝5.00,J＝13.20,1H),4.45(m,4H),4.39–4.26(s,2H),3.53(t,J＝6.8Hz,3H),3.41(t,J＝5.9Hz,3H),2.87(s,3H),2.61(d,J＝9.9Hz,4H),2.06–1.91(m,3H),1.86(s,2H),1.54–1.39(m,3H),0.85(s,2H).

Referring to the preparation method of formula 1, intermediate 16-4 and intermediate 8a-2 are used. LC-MS (ESI) M/z (M + H) + called. for C₄₀H₄₄N₁₁O₈S₂ ⁺＝870.27；found 870.39

9mg of the product of the formula 17 are dissolved in 5ml of methanol solution. 2mg Pd/C was added. The reaction solution was fully replaced with hydrogen, and hydrogen was continuously introduced. After 12 hours of reaction, mass spectrometry monitoring no raw materials remained, then the reaction was stopped. Separating and purifying with silica gel column chromatography to obtain 8mg of the product shown in formula 18.

LC-MS(ESI)m/z(M+H)+called.for C₄₀H₄₇N₁₁O₈S₂ ⁺＝874.31；found 874.29.

¹H NMR(400MHz,DMSO)δ11.01(s,1H),9.95(s,1H),8.06–7.99(m,1H),7.88(s,2H),7.82(d,J＝8.9Hz,2H),7.78–7.73(m,2H),7.73–7.68(m,2H),7.58–7.53(m,1H),7.48(s,1H),5.15(dd,J＝13.3,5.1Hz,1H),4.47–4.33(m,4H),4.33(s,2H),3.49(t,J＝6.3Hz,3H),3.44–3.37(t,J＝5.9Hz,4H),2.91(m,2H),2.62(s,5H),2.01-1.93(m,3H),1.80(s,4H),1.59–1.48(m,5H),1.18(m,2H),0.84(m,3H).

Referring to the preparation method of formula 1, intermediate 16-5 and intermediate 8a-2 are used.

LC-MS(ESI)m/z(M+H)⁺called.for C₄₁H₄₅N₁₁O₈S₂ ⁺＝884.29；found 884.17.

¹H NMR(400MHz,DMSO)δ11.02(s,1H),9.95(s,1H),8.08–8.00(m,2H),7.88(s,2H),7.82(d,J＝8.8Hz,2H),7.78–7.73(m,2H),7.71(d,J＝8.1Hz,3H),7.55(t,J＝7.6Hz,1H),7.47(t,J＝5.9Hz,1H),7.16(d,J＝5.0Hz,1H),5.15(dd,J＝13.3,5.1Hz,1H),4.41(d,J＝16.5Hz,4H),4.29(dd,J＝11.9,4.7Hz,3H),3.49(d,J＝6.4Hz,3H),3.40(t,J＝5.9Hz,3H),3.16(d,J＝5.1Hz,2H),2.88(d,J＝5.1Hz,3H),2.74(d,J＝6.7Hz,1H),2.62(s,4H),1.99(dd,J＝15.0,7.5Hz,3H),1.81–1.69(m,2H),1.56–1.41(m,3H),1.32(d,J＝6.7Hz,4H),0.85(t,J＝6.8Hz,2H).

9mg of the product of formula 19 are dissolved in 5ml of methanol. 2mg Pd/C was added. The reaction solution was fully replaced with hydrogen, and hydrogen was continuously introduced. After 12 hours of reaction, mass spectrometry monitoring no raw materials remained, then the reaction was stopped. Separating and purifying with silica gel chromatographic column to obtain 8mg of the product shown in formula 20.

LC-MS(ESI)m/z(M+H)⁺called.for C₄₁H₄₉N₁₁O₈S₂ ⁺＝888.32；found 888.06.

¹H NMR(400MHz,DMSO)δ11.00(s,1H),9.95(s,1H),8.11(s,1H),8.06–8.00(m,2H),7.95(s,1H),7.88(s,2H),7.81(d,J＝8.9Hz,2H),7.73(d,J＝8.9Hz,2H),7.55(s,1H),7.45(dd,J＝8.3,6.5Hz,2H),7.22(s,1H),7.16(d,J＝5.0Hz,1H),5.13(dd,J＝13.5,5.2Hz,1H),4.43(s,2H),4.32–4.25(m,3H),4.03(d,J＝7.1Hz,4H),3.43–3.37(m,6H),3.35–3.26(m,5H),2.99(s,1H),2.62(s,5H),1.44(s,4H),1.27(s,5H),1.17(t,J＝7.1Hz,3H),0.85(s,2H).

Referring to the preparation method of formula 1, intermediate 41 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₀H₄₇N₁₂O₁₀S₂[M+H]⁺:919.29,found 919.23.

¹H-NMR(400MHz,DMSO-d6):9.94(s,1H),8.04(d,J＝5.00,1H),7.99(s,1H),7.87(s,2H),7.83(d,J＝8.84,2H),7.74(d,J＝8.88,2H),7.58(t,J＝8.20,1H),7.49(t,J＝5.96,1H),7.16(d,J＝5.04,1H),7.11(d,J＝8.60,1H),7.04(d,J＝7.04,1H),6.59(t,J＝5.64,1H),5.14(dd,J＝5.32,J＝12.92,1H),4.49(t,J＝5.12,2H),4.42(s,2H),3.79(t,J＝5.20,2H),3.66-3.39(m,12H),2.94-2.86(m,3H),2.62(s,3H),2.02(m,1H),1.01(t,J＝6.92,3H).

¹³C-NMR(100MHz,DMSO-d6,ppm):δ171.39,169.36,167.27,150.59,136.25,135.73,132.07,131.45,131.05,127.92,124.24,123.92,117.43,116.10,110.72,109.20,69.58,69.54,68.80,68.75,68.32,64.94,63.37,49.33,49.11,42.23,41.63,40.14,39.93,39.73,39.52,39.31,39.10,38.89,34.67,31.18,21.44,17.79,15.19,12.92.

1eq intermediate 13, 2eq intermediate 14, 0.1eq Pd (PPh)₃)₂Cl₂0.2eq of CuI and DMF are mixed uniformly, the gas in the reaction system is changed into argon by a vacuum pump, the reaction is repeated for 3 times, then proper amount of TEA is added into the reaction system by an injector, the reaction is carried out for 2 times in the same way, and the reaction is carried out for 3 hours under the condition of 80 ℃ oil bath. The reaction was stopped, the solvent removed by rotary evaporation and purified through a 200-mesh 300-mesh silica gel column in DCM and MeOH (40:1, v/v) to give the final product.

LC-MS:calculated for C₃₈H₄₃N₈O₁₀S₂[M+H]⁺:835.25,found 834.96.

¹H-NMR(400MHz,CDCl₃,ppm):13.11(s,1H),8.39(s,1H),7.84(d,J＝7.56,1H),7.80(d,J＝8.56,2H),7.67(d,J＝4.96,1H),7.62(m,3H),7.47(t,J＝7.64,1H),7.08(s,2H),6.97(d,J＝5.00,1H),6.11(s,1H),5.16(dd,J＝4.00,J＝10.84,1H),4.63-4.47(m,4H),3.80(d,J＝2.28,2H),3.69-3.11(m,12H),2.83-2.56(m,8H),2.15(m,1H).

Into a 5ml round-bottom flask was added 9mg of the compound represented by formula 22 and 2mg of Pd/C. 3ml EtOH was added: DCM: DMF 1: 1:1 mixing the solution. The gas in the bottle was replaced sufficiently with hydrogen and continuously charged with hydrogen. After no starting material molecular weight was detected by LC-MS, the solvent was removed by rotary evaporation. With dichloromethane: methanol 20: passing through a silica gel column 1 gave the compound of formula 23 in 87% yield.

LC-MS:calculated for C₃₈H₄₇N₈O₁₀S₂[M+H]⁺:839.28,found 839.04.

¹H-NMR(400MHz,CDCl₃):13.37(s,1H),8.39(s,1H),7.83(d,J＝8.36,2H),7.74(d,J＝7.12,1H),7.68(d,J＝4.48,1H),7.61(d,J＝8.20,2H),7.43(m,2H),7.07(s,2H),6.98(d,J＝4.72,1H),6.63(s,1H),5.19(dd,J＝4.36,J＝13.32,1H),4.60(dd,J＝16.92,J＝51.36,2H),3.59-3.33(m,16H),3.10-2.55(m,10H),2.11-1.99(m,3H).

LC-MS:calculated for C₃₈H₄₅N₁₂O₉S₂[M+H]⁺:877.29,found 877.96.

Referring to the preparation method of formula 1, intermediate 23 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₁H₄₆N₁₁O₁₀S₂[M+H]⁺:917.00,found 917.56.

10mg of the product of formula 26 are dissolved in 5ml of methanol. 2mg Pd/C was added. The reaction solution was fully replaced with hydrogen, and hydrogen was continuously introduced. After 12 hours of reaction, mass spectrometry monitoring no raw materials remained, then the reaction was stopped. Separating and purifying with silica gel column chromatography to obtain 8mg of the product shown in formula 27.

LC-MS:calculated for C₄₁H₅₀N₁₁O₁₀S₂[M+H]⁺:920.31,found 920.77.

Referring to the preparation method of formula 1, intermediate 29-0 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₃H₅₂N₁₃O₈S₃[M+H]+:974.31,found 974.56.

Referring to the preparation method of formula 1, intermediate 29-1 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₆H₅₈N₁₃O₉S₃[M+H]+:1032.36,found 1034.03.

Referring to the preparation method of formula 1, intermediate 29-2 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₈H₆₂N₁₃O₁₀S₃[M+H]+:1076.38,found 1078.44.

Referring to the preparation method of formula 1, intermediate 29-3 and intermediate 8a-2 are used.

LC-MS:calculated for C₅₀H₆₆N₁₃O₁₁S₃[M+H]+:1120.41,found 1121.02.

Referring to the preparation method of formula 1, intermediate 29-4 and intermediate 8a-2 are used.

LC-MS:calculated for C₅₂H₇₀N₁₃O₁₂S₃[M+H]+:1164.44,found 1165.39.

Referring to the preparation method of formula 1, intermediate 33-1 and intermediate 8a-2 are used. LC-MS (calculated for C)₄₅H₅₆N₁₃O₈S₃[M+H]+:1002.35,found 1002.99.

Referring to the preparation method of formula 1, intermediate 33-2 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₇H₆₀N₁₃O₉S₃[M+H]+:1046.37,found 1047.11.

Referring to the preparation method of formula 1, intermediate 33-3 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₉H₆₄N₁₃O₁₀S₃[M+H]+:1090.40,found 1091.01.

Referring to the preparation method of formula 1, intermediate 33-4 and intermediate 8a-2 are used.

LC-MS:calculated for C₅₁H₆₈N₁₃O₁₁S₃[M+H]+:1134.42,found 1135.58.

Referring to the preparation method of formula 1, intermediate 35-1 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₅H₅₆N₁₃O₉S₃[M+H]+:1018.34,found 1019.32.

Referring to the preparation method of formula 1, intermediate 35-2 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₇H₆₀N₁₃O₁₀S₃[M+H]+:1062.37,found 1063.42.

Referring to the preparation method of formula 1, intermediate 35-3 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₉H₆₄N₁₃O₁₁S₃[M+H]+:1106.39,found 1107.12.

Referring to the preparation method of formula 1, intermediate 35-4 and intermediate 8a-2 are used.

LC-MS:calculated for C₅₁H₆₈N₁₃O₁₂S₃[M+H]+:1150.42,found 1151.03.

Referring to the preparation method of formula 1, intermediate 39-1 and intermediate 8a-2 are used.

LC-MS:calculated for C₃₇H₃₈N₁₁O₈S₂[M+H]+:828.23,found 828.44.

Referring to the preparation method of the formula 14,

LC-MS:calculated for C₃₇H₄₂N₁₁O₈S₂[M+H]+:832.26,found 832.96.

referring to the preparation method of formula 1, intermediate 39-2 and intermediate 8a-2 are used.

LC-MS:calculated for C₃₈H₄₀N₁₁O₈S₂[M+H]+:842.24,found 842.53.

Refer to formula 14.

LC-MS:calculated for C₃₈H₄₄N₁₁O₈S₂[M+H]+:842.24,found 842.96.

Referring to the preparation method of formula 1, intermediate 39-3 and intermediate 8a-2 are used.

LC-MS:calculated for C₃₉H₄₂N₁₁O₈S₂[M+H]+:856.26,found 856.76.

Refer to formula 14.

LC-MS:calculated for C₃₉H₄₆N₁₁O₈S₂[M+H]+:860.29,found 860.64.

Referring to the preparation method of formula 1, intermediate 39-4 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₀H₄₄N₁₁O₈S₂[M+H]+:870.27,found 870.93.

Refer to formula 14.

LC-MS:calculated for C₄₀H₄₈N₁₁O₈S₂[M+H]+:874.31,found 874.32.

Referring to the preparation method of formula 1, intermediate 39-5 and intermediate 8a-2 are used.

LC-MS:calculated for C₄₁H₄₆N₁₁O₈S₂[M+H]+:884.29,found 884.16.

Refer to formula 14.

LC-MS:calculated for C₄₁H₅₀N₁₁O₈S₂[M+H]+:878.32,found 878.15.

1mmol of intermediate 44-n and 1mmol of intermediate 12 are dissolved in 15ml of THF solution, 1.05mmol of HATU and 2mmol of TEA are added and stirred at room temperature overnight to obtain the product shown in formula 50-54.

Formula 50: LC-MS (calculated for C)₃₀H₂₇N₉O₆S[M+H]⁺:642.18,found 642.66.

Formula 51: LC-MS (calculated for C)₃₂H₃₁N₉O₇S[M+H]⁺:686.21,found 686.96.

Formula 52: LC-MS (calculated for C)₃₄H₃₅N₉O₈S[M+H]⁺:730.23,found 730.87.

Formula 53: LC-MS (calculated for C)₃₆H₃₉N₉O₉S[M+H]⁺:774.26,found 774.89.

Formula 54: LC-MS (calculated for C)₃₈H₄₃N₉O₁₀S[M+H]⁺:818.29,found 818.61.

The intermediate 42-n is used as a raw material to replace the intermediate 12, and the preparation method of the intermediate 13 is repeated to obtain the product shown in 55-57.

Formula 55: LC-MS (calculated for C)₂₇H₃₀N₈O₇S[M+H]⁺:611.20,found 611.53.

Formula 56: LC-MS (calculated for C)₂₉H₃₄N₈O₈S[M+H]⁺:655.22,found 655.83.

Formula 57: LC-MS (calculated for C)₃₁H₃₈N₈O₉S[M+H]⁺:699.25,found 699.47.

Example 2: biological activity test of small molecule conjugates shown in formula 1 to formula 57

First, the CDK2 degrading effect and other related protein degrading effects are verified through western blot

Mino, HBL1, Ramos, NB4, U937, MV-4-11, Kasumi-1, MM.1s, K562, Z138 and Jurkat cells were cultured at 37 ℃ in 5% CO₂The culture was carried out in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (CellMax) and 1% penicillin/streptomycin (macgene). OCI-AML2 cells were cultured in MEM medium (Hyclone) containing 15% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ with 5% CO₂Cultured in an incubator. OCI-AML3 cells were cultured in fetal bovine serum-MEM medium (Hyclone) containing 15% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ with 5% CO₂Cultured in an incubator. MDA-MB-231, Beas2b and 293T cells were placed in DMEM medium (Hyclone) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ with 5% CO₂Culturing in an incubator. HL60 cells were cultured in IMDM medium (Hyclone) containing 20% fetal bovine serum and 1% penicillin/streptomycin at 37 deg.C with 5% CO₂Culturing in an incubator. All cells as above can be purchased from commercial sources.

Cell treatment:

(1) suspension of cells:

the first day: spread 5X 10⁵Cells were divided into 6-well plates and 1ml of the corresponding medium was added. Adding the drug for incubation.

And on the third day: cells were harvested after 48 hours.

(2) Adherent cells

The first day: spread 5X 10⁵Cells were divided into 6-well plates and 1ml of the corresponding medium was added.

The next day: adding the drug for incubation.

The fourth day: cells were harvested after 48 hours.

Extracting cell whole protein:

collecting cells: the treated cells were scraped off from the medium, centrifuged at 1,000g for 5 minutes, collected, washed once with PBS, and discarded.

Cell lysis: 200ul of 2 × Loading buffer was added, and after blowing uniformly, the mixture was boiled in a metal bath at 100 ℃ for 20 minutes. Cooling and centrifuging for later use. The samples will be used directly in a western-blot.

The 2 × Loading Buffer has the following components: SDS at a final concentration of 0.5%, 3% beta-mercaptoethanol, 15% glycerol, and an appropriate amount of bromophenol blue.

Western Blot detection:

protein bands of the target protein and β -Actin (Tubulin or GAPDH) were visualized by Western blotting (Western-Blot). Beta-actin (tubulin) is an internal reference protein. In Table 1 below, CST is an abbreviation of Cell signaling technology, Inc., and Beyotime is the English name of Biyuntian, Inc.

TABLE 1

The degradation activity of the small molecule conjugate of the invention on CDK2 is shown in FIGS. 3-10, wherein JNJ is the abbreviation of JNJ7706621 molecule, and Poma is the abbreviation of Pomalidomide.

The structural formulas of JNJ7706621 and J2 are shown below:

as can be seen from the test results in fig. 3-10, most of the conjugates were able to degrade CDK2 at a concentration where the compound of formula 8 was most degraded.

We also demonstrated the universality of formula 8 on a variety of cell lines (see table 2), with down-regulation of CDK2 levels being observed on a variety of tumor cells. Formula 8, in this embodiment includes but is not limited to acute myelocytic leukemia cells, chronic myelocytic leukemia cells, acute lymphocytic leukemia cells, B-lymphoid leukemia cells, chronic lymphocytic leukemia cells, breast cancer cells, and glioma cells. In more detail, the DC50 values (concentration of 50% protein degradation) for each cell line of formula 8 are shown in table 2 below, using a quantitative method of grey scale analysis of Western blot images using ImageJ tool and nonlinear fitting of the degradation curve using GraphPad Prism7 tool:

TABLE 2

The molecular list, alias names and activities of the examples are shown in table 3 below (+ representing the strength of the degradation capacity, representing no degradation observed, n.d. representing no such experiment).

The CDK protein degradation activity in Ramos cells is determined by the following method: the quantitative method is characterized in that an ImageJ tool is adopted to carry out gray level analysis on the Western blot image, the relative DMSO ratio is set as A under the concentration of 100nM, and 80% < A < 100%: +; 60% < A ≤ 80%: + +; 40% < A ≦ 60%: + + + +; a is less than or equal to 40%: ++++.

TABLE 3

+ represents a degradation effect on the protein, the more + the more degradation is evident. -represents that the protein is not degraded.

Second, cell inhibition test

MTT test reagent:

reagent: RPIM 1640 medium; DMEM medium; 100 × non-essential amino acids (NEAA); 100 times streptomycin mixed liquor; 50mM beta mercaptoethanol; fetal bovine serum (FBS, previously inactivated).

Medium a (500 ml): RPIM 1640medium (450ml) +100 XNEAA (5ml) +100 Xstreptomycin mixed liquor (5ml) + fetal bovine serum (50ml) +50mM beta mercaptoethanol (0.5 ml).

B medium (500 ml): DMEM medium (450ml) +100 XNEAA (5ml) +100 Xstreptomycin mixture (5ml) + fetal bovine serum (50ml) +50mM beta mercaptoethanol (0.5 ml).

CCK-8 Kit (Cell Counting Kit-8)

MTT experimental procedure:

1) cells were collected in log phase and cell suspension concentration was adjusted to 6.6X 10 using A medium⁴/ml。

2) The small molecule solution is prepared by diluting the small molecule with a 2-fold gradient of the A culture medium to 50nM to 0.15 nM.

3) 45 μ L of cell suspension was added to a 96-well plate (marginal wells filled with sterile PBS, 3000 cells/well). Negative controls (45. mu.L of cell suspension and 45. mu.L of A medium) were set for each plate, and 3 wells were set for each group.

4) Standing at 37 deg.C for 5% CO₂After 1 hour of incubation, 45 μ L of the corresponding small molecule solution was added to each well of the 96-well plate. Then at 37 ℃ with 5% CO₂Incubate for 72-96 hours. Mu.l of CCK-8 solution was added to each well and incubation was continued for 4 h. The absorbance of each well was measured by direct enzyme-linked immunosorbent assay OD490 nm.

The results of the compounds of the invention for CDK2 protein degradation and inhibition of cell proliferation as determined by the methods described above are shown in figures 3-11 and in table 4. J2+ Poma is the composition of the two according to the molar ratio of 1: 1.

TABLE 4

	J2	J2+Poma	Formula	7	Formula 8	Formula 9	Poma
								IC50(nM)	115	101.9	1108	2305	2374	175.4

As can be seen from fig. 4, 8 and 10b, the molecule of formula 8 is highly selective, with the molecular degradation activity concentrated in CDK2 and CDK 9.

As can be seen in FIG. 7, the molecules of the present patent induce a decrease in the level of CDK2 in cells to varying degrees.

As can be seen from fig. 9, the molecule of formula 8 has a wide range of applications and can induce a decrease in CDK2 levels in a variety of cell lines.

As can be seen from fig. 10, formula 8 has a fast onset of action, is degraded by the ubiquitin-proteasome pathway, has low toxicity to normal cells, and can inhibit tumor cell proliferation.

From fig. 11, table 4 can be derived, and the molecules described in this patent are effective in inhibiting Ramos cell proliferation.

As can be seen in FIG. 12, the combination regimen of this patent is effective in inhibiting tumor cell proliferation and inducing tumor cells to differentiate.

As can be seen from fig. 13, the small molecules described in this patent were effective in inducing AML cells to differentiate.

As can be seen from fig. 14, the small molecule of formula 8 disclosed in this patent can effectively inhibit tumor proliferation in animals, and has the function of inducing CDK2 to be down-regulated, and has very low toxicity in vivo, and has certain clinical application value.

In particular, the small molecule conjugate (CDK2 degradation agent) provided by the invention can be used for the differentiation induction treatment of AML and other diseases independently, and can also be used for the combined medication, so that the small molecule conjugate can be used for the treatment of the diseases more effectively. More specifically, the CDK2 degradation agent is respectively combined with a BET inhibitor, a CDK4&6 inhibitor, a PI3K inhibitor and all-trans retinoic acid (ATRA), and has good combined effect. This patent includes, but is not limited to, the combination formulations recited in the above examples, and also includes any formulation or use containing the CDK2 degradant disclosed herein.

This example uses engineered cells 293T and Beas2b to mimic the toxicity to normal cells in humans. The toxicity of formula 8 against normal cells is very low. (see FIG. 10)

Example 3: schematic diagram of AML cell differentiation induced by molecule of formula 8

The implementation method comprises the following steps:

cell culture was as described in examples 1 and 2;

to determine expression of CD11b, after harvesting the cells, they were washed with PBS and then coupled to CD11-pe (bd biosciences) in a 1% solution of Bovine Serum Album (BSA) at 0 degrees celsius for 60 minutes. After incubation, the expression level of CD11b was detected by flow sorter

For NBT staining experiments:

NBT (1mg/ml) and TPA (1ug/ml) were dissolved in PBS, and cells were added and incubated at 37 ℃ for 30 minutes. After incubation, the cells were centrifuged and dispersed in methanol and averaged into 24-well plates. Then, the cell structure was observed by a microscope.

For cell morphology detection:

cells were incubated and prepared by cytospin. Then fixed with methanol and dried. The sections were stained with Wright-Giemsa solution, and then the cell structure was observed with a microscope.

The results are shown in FIGS. 12 and 13.

As can be seen in fig. 12e, the regimen described in this patent in combination with ATRA was effective in inducing differentiation of the AML cell line NB 4. Realizes obvious superposition and complementation with the existing clinical medicine.

As can be seen from fig. 13, the small molecules described in this patent were able to efficiently induce differentiation of AML cells NB4 when used alone.

Claims

1. A small molecule conjugate of formula I:

X-Y-Z

formula I

In formula I, X represents a ligand of CDK2 protein, Z represents a ligand of E3 ligase, and Y represents a linking moiety of X and Z.

2. The small molecule conjugate of claim 1, characterized in that: the X is selected from a group shown in formula II:

r in formula II includes, but is not limited to, the following groups:

r in the formula II_xAnd R_yIndependently is CH or N;

SO in formula II₂NH-is in the 2-, 3-or 4-position;

preferably, the specific structure of X is any one of the following groups:

3. the small molecule conjugate of claim 1 or 2, characterized in that:

z 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, thalidomide 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 ] methanone group;

specifically, Z includes, but is not limited to, the following structures:

In the formula III-5, R ═ O or H₂；

In the formula III-6, R is OH or H;

in the formula III-7, R is OH or H.

4. The small molecule conjugate of any one of claims 1-3, wherein: y is a unit with 1-30 atomic lengths and any type;

specifically, Y includes, but is not limited to, the following structures, or any structural group consisting of the following structures:

unit consisting of ethylene glycol structure:

or units of fatty chains:

or units composed of unsaturated chains:

or units of aromatic compounds:

or a heteroatom-containing group, the heteroatom being sulfur, nitrogen or phosphine, including but not limited to the following fragment units:

r in the heteroatom-containing unit is independently: alkyl, alkoxy or hydrogen;

5. The small molecule conjugate of any one of claims 1-4, wherein:

the structural formula of the small molecule conjugate is shown in any one of formula IV-formula X:

in the formula IV, m is an integer between 0 and 6 (preferably an integer between 1 and 6), n is an integer between 0 and 6, and q is an integer between 1 and 15; a is CH₂NH, O or CO, G is CH₂Or CO, B is CO or CH₂E is CO or CH₂R is as defined for formula II, R_LIs H, methyl or ethyl;

in the formula V, m is an integer between 1 and 6, n is an integer between 0 and 6, R is defined as the formula II, G is CH₂Or CO;

in formula VI, m is an integer of 1 to 6, n is an integer of 0 to 6, R is as defined in formula II, G is CH₂Or CO;

in the formula VI I, m is an integer between 1 and 15, n is an integer between 0 and 6, R is defined as the formula II, G is CH₂Or CO;

in the formula VI II, m is an integer between 1 and 15, n is an integer between 0 and 6, R is defined as the formula II, G is CH₂Or CO;

in the formula IX, n is an integer between 0 and 6, R is defined as the formula II, and G is CH₂Or CO;

in the formula X, n is an integer between 0 and 6, R is defined as the formula II, G is CH₂Or CO.

6. The small molecule conjugate of claim 5, which is a compound of formula 1-formula 138:

7. a method for preparing a small molecule conjugate of formula I according to claim 1, comprising the steps of: and (3) carrying out click reaction, amide condensation reaction, substitution reaction or cross coupling reaction on the ligand of the E3 ligase and the ligand of the X part CDK2 protein to obtain the small molecule conjugate shown in the formula I.

8. A method of preparing a small molecule conjugate of formula IV as described in claim 5, comprising the steps of: carrying out click reaction between Pomalidomide end derivative 1 shown in a formula b and JNJ-7706621 end derivative 1 shown in a formula a to obtain a small molecule conjugate shown in a formula IV;

wherein, in the small molecule conjugate shown in the formula IV, G is CH₂N is 1, q is 1, A is NH and B is CO;

r in formula a is as defined in formula II, R in formula b_LM is as defined in formula IV;

or, a method for preparing a small molecule conjugate of formula V as described in claim 5, comprising the steps of: carrying out click reaction between a VHL ligand derivative shown in a formula c and a JNJ-7706621 terminal derivative 1 shown in a formula a to obtain a small molecule conjugate shown in a formula V;

wherein, in the small molecule conjugate described in the formula V, G is CH₂N is 1;

m in the formula c is defined as the formula V;

or, a method for preparing a small molecule conjugate of formula vi as described in claim 5, comprising the steps of: carrying out click reaction between an MDM2 ligand derivative shown in a formula d and a JNJ-7706621 terminal derivative 1 shown in a formula a to obtain a small molecule conjugate shown in a formula VI;

wherein, in the small molecule conjugate described in the formula VI, G is CH₂N is 1;

m in the formula d is defined as formula VI;

or, a process for the preparation of a small molecule conjugate of formula vi as claimed in claim 5, comprising the steps of: click reaction is carried out between Lenalidomide derivative 1 shown in a formula e and JNJ-7706621 terminal derivative 1 shown in a formula a, so as to obtain the micromolecule conjugate shown in a formula VI I;

wherein, in the small molecule conjugate described in the formula V II, G is CH₂N is 1;

m in the formula e is defined as formula VI I;

or, a method of preparing a small molecule conjugate of formula VIII as described in claim 5, comprising the steps of: reducing alkyne in the small molecule conjugate shown in the formula VI I to obtain a small molecule conjugate shown in a formula VIII;

or, a method of preparing a small molecule conjugate of formula IX as described in claim 5, comprising the steps of: carrying out Sonogashira cross-coupling reaction between Lenalidomide ligand derivative 2 shown in formula g and JNJ-7706621 terminal derivative 2 shown in formula h to obtain a small molecule conjugate shown in formula IX;

r, G, n in the formula h is as defined in the formula IX;

or, a process for the preparation of a small molecule conjugate of formula X as claimed in claim 5, comprising the steps of: reducing alkyne in the small molecule conjugate shown in the formula IX to obtain a small molecule conjugate shown in the formula X;

9. a compound of formula a:

r in the formula a is as defined in the formula II.

10. A compound of formula h:

r, G, n in formula h is as defined for formula IX.

11. A process for the preparation of a compound of formula a as defined in claim 9 or a compound of formula h as defined in claim 10, comprising the steps of:

1) reacting p-nitrobenzenesulfonyl chloride shown in a formula 1b with a compound shown in a formula 3a or a formula 3b to obtain a compound shown in a formula 4a or a formula 4 b;

2) then reducing nitro in the compound shown in the formula 4a or 4b by using zinc powder and hydrochloric acid; then adding thiophosgene for reaction to prepare an intermediate containing isothiocyanate, namely a compound shown as a formula 5a or a formula 5 b;

3) reacting the intermediate 6a shown in the formula 6a or the intermediate 6b shown in the formula 6b with 3, 5-dimethyl-1-pyrazole formamidine ammonium nitrate to attack an isothiocyanate functional group;

5) adding hydrazine hydrate into the formula 6a or the formula 6b for cyclization to obtain an intermediate 7a of the formula 7a or an intermediate 7b of the formula 7 b;

6) reacting the compound of formula 7a or 7b with a compound containing acid chloride, respectively, and condensing different fragments through acid chloride to obtain the compound of formula a or the compound of formula hA compound; wherein the structural formula of the acyl chloride-containing compound is shown as

The structure of R is the same as formula II.

12. Use of a small molecule conjugate according to any one of claims 1 to 6 in at least one of the following 1) to 5):

1) the use of CDK2 in the preparation of a selective degrader;

2) the application in preparing cancer cell proliferation inhibitor;

3) the application in preparing cancer cell differentiation inducer;

4) the application in preparing AML cell differentiation inducer;

13. Use according to claim 12, characterized in that: the CDK2 selective degrader can selectively degrade CDK2 in at least one of the following cells: acute myelocytic leukemia cells, T-lymphocytic leukemia cells, chronic myelocytic leukemia cells, acute lymphocytic leukemia cells, B-lymphoid leukemia cells, chronic lymphocytic leukemia cells, chronic myelocytic leukemia cells, myeloma cells, breast cancer cells, and glioma cells;

the cancer cells comprise leukemia cells, myeloma cells, breast cancer cells and glioma cells; such leukemia cells include, but are not limited to, the following: acute myelocytic leukemia cells, T lymphocytic leukemia cells, chronic myelocytic leukemia cells, acute lymphocytic leukemia cells, B lymphocytic leukemia cells, chronic myelocytic leukemia cells;

the leukemia comprises acute myelogenous leukemia, T lymphocyte leukemia, chronic granulocytic leukemia, acute lymphocyte leukemia, B lymphocyte leukemia and chronic lymphocytic leukemia;

the acute myelogenous leukemia comprises M1 acute myelogenous leukemia immature type, M2 acute myelogenous leukemia partial mature type, M3 acute promyelocytic leukemia, M4 acute myelomonocytic leukemia, M5 acute monocytic leukemia, M6 acute erythrocytic leukemia, M7 acute megakaryocytic leukemia and M0 acute differentiation type myelocytic leukemia.

The AML cells include NB4, HL60, U937, Kasumi-1, MV-4-11, OCI-AML2 and OCI-AML 3.

14. A product prepared by using the small molecule conjugate of any one of claims 1-6 as an active ingredient; the product is selected from at least one of the following: 1) CDK2 selective degraders; 2) an inhibitor of cancer cell proliferation; 3) a cancer cell differentiation inducer; 4) an AML cell differentiation inducer; 5) a medicament for preventing and/or treating leukemia.

15. A pharmaceutical composition comprising a small molecule conjugate of any one of claims 1-6 and at least one of the following drugs: BET inhibitors, CDK4&6 inhibitors, PI3K inhibitors, and all-trans retinoic acid.

16. The pharmaceutical composition of claim 15, wherein: in the pharmaceutical composition, the small molecule conjugate of any one of claims 1-6 and the drug are present in a molar ratio of 1: 1-10: 1;

the BET inhibitors include ABBV-075, JQ1, dBET-1, ARV-825; the CDK4&6 inhibitor comprises YX-2-107, Palbociclib; the PI3K inhibitor includes Copanlisib.