Spiro compound, pharmaceutical composition containing same and use thereof

Abstract

Disclosed in the present invention are a spiro compound, a pharmaceutical composition containing same and the use thereof. The spiro compound has the structure as shown in the following formula (I). The compound having the spiro structure of the present invention has a strong inhibitory activity on WRN, can mediate the degradation of WRN, shows excellent selectivity with regard to the anti-proliferation activity of MSI cells and MSS cells, and can be used in the preparation of a drug for preventing or treating WRN-related diseases, in particular cancers with MSI-H or dMMR characteristics.

Description

Spirocyclic compound, pharmaceutical composition containing the same and use thereof Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a class of spirocyclic compounds targeting WRN, a pharmaceutical composition containing the same, and uses thereof. Background Art

[0002] The limitations of current cancer treatments can lead to adverse side effects that are not limited to tumor cells, such as indiscriminate killing of normal tissue cells. Therefore, therapeutic strategies that can specifically and accurately target tumors and have high clinical trial success rates are crucial for the development of anti-cancer drugs.

[0003] The synthetic lethality strategy that can selectively kill tumor cells has achieved varying degrees of success in the clinic as one of the means of treating cancer. Synthetic lethality refers to the fact that a single mutation of any one of the two genes in a cell can survive, but when both genes mutate at the same time, it will cause cell death (Genetics 1968, 59, 37-44; Am.Nat.1922, 56, 51-63). In 2019, a series of related reports revealed the existence of a synthetic lethal relationship between the RecQ-like family helicase protein WRN and MSI tumors (Nature 2019, 568, 551-556; Nature 2019, 568, 511-516; Elife 2019, 8, e43333), emphasizing that WRN is a potential target for tumor therapy.

[0004] Microsatellite sequences are short (1-6 base pairs) repetitive DNA sequences distributed along coding and non-coding regions, accounting for about 3% of the human genome (Genome Biol.2003, 4, R13). Due to the repetitiveness of its sequence, DNA polymerase slippage events are prone to occur in microsatellite regions, resulting in insertion or deletion mutations, which are often identified and repaired by DNA mismatch repair (DNA mismatch repair, MMR) (Nat.Rev.Genet 2004, 5, 435-445). MMR maintains the stability of the genome by eliminating the errors generated during DNA replication, long-chain DNA repair synthesis and recombination (Front Mol.Biosci.2020, 7, 122). When MMR is defective, insertion or deletion mutations frequently occurring in microsatellite regions cause microsatellite lengths to change, referred to as microsatellite instability (MSI) (Mol.Med Today 1997, 3, 61-68). MSI is present in at least 27 different tumor types, primarily in colon cancer (15%), gastric cancer (22%), endometrial cancer (20-30%), and ovarian cancer (12%) (Nature 2019, 568, 551-556).

[0005] WRN, the synthetic lethal target of MSI cells, is a member of the helicase family and is located in the cell nucleus. It is a multifunctional protein with helicase activity and exonuclease activity that is different from other members of the helicase family (J.Biol.Chem.2007, 282(13), 9941-9951). It plays a role in various cellular processes such as DNA replication, transcription, repair, and telomere maintenance to maintain genomic stability (Biogerontology 2009, 10, 235-252). WRN depletion prevents DNA damage and repair in MSI cancer cells (Nature 2020, 586, 292-298), leading to subsequent growth arrest and cell death accompanied by characteristics of apoptosis. Studies have shown that the dependence of MSI cells on WRN is only related to its helicase domain (Elife 2019, 8, e43333). Mechanistically, microsatellite sequences in MMR-deficient cells are highly unstable and undergo large-scale amplification, ultimately forming non-B-type DNA secondary structures. The presence of WRN can decompose these DNA structures (Nature 2020, 586, 292-298). When WRN is missing, the DNA secondary structures generated by microsatellite repeat sequences lead to replication fork stalling, resulting in extensive chromosome fragmentation and subsequent cell death. Therefore, the development of inhibitors and degraders targeting WRN for precise tumor cell killing strategies has great value.

[0006] Currently, there are only a handful of WRN inhibitors reported, and WRN degraders also need to be explored and developed. Inhibiting WRN or mediating WRN protein degradation is an attractive strategy for treating mismatch repair-deficient cancers. In short, there is an urgent clinical need to develop new WRN inhibitors or degraders. Summary of the Invention

[0007] In response to the problems existing in the prior art, the compound containing a spirocyclic structure represented by Formula I of the present invention or a pharmaceutically acceptable salt thereof has potent WRN inhibitory activity and can mediate WRN degradation, and can therefore be used to prepare drugs for preventing or treating WRN-related diseases.

[0008] Based on this, one of the objects of the present invention is to provide a compound containing a spirocyclic structure as shown in Formula I, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, or metabolite thereof.

[0009] A second object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned compound containing a spirocyclic structure, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, or metabolite thereof.

[0010] A third object of the present invention is to provide a class of regulators that inhibit WRN or mediate WRN degradation, which comprises the above-mentioned compound containing a spirocyclic structure, or its pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, metabolite, or the above-mentioned pharmaceutical composition.

[0011] A fourth object of the present invention is to provide the use of the above-mentioned compound containing a spirocyclic structure, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, metabolite thereof, or the above-mentioned pharmaceutical composition in the preparation of a drug for preventing and / or treating diseases related to WRN.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] In the first aspect, the present invention provides a compound containing a spirocyclic structure as shown in the following formula I, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, or metabolite thereof:

[0014] in:

[0015] Ring A is selected from: a benzene ring, a 5-6 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O, and S; in particular, Ring A is a benzene ring or a pyridine ring;

[0016] Ring B is selected from a 5-7 membered saturated or partially saturated heterocyclic ring containing 1-2 heteroatoms selected from N, O, and S; in particular, Ring B is

[0017] L is selected from: -C(O)-, -S(O)-, -S(O)2-,

[0018] X is CH or N;

[0019] n is an integer between 0 and 5, for example, 0, 1, 2, 3, 4 or 5;

[0020] Each R2 is the same or different and is independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-6 Alkyl, C 1- 6 alkoxy, saturated or partially unsaturated C 3-8 Alicyclic group, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2- 4-alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group containing 1-3 heteroatoms selected from N, O, S, C 6-10 Aryl, 5- to 6-membered heteroaryl, in particular, each R 2 Independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl; the C 1-6 Alkyl, C1-4 alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy, saturated or partially unsaturated C 3-8 Alicyclic group, C 3-6 Cycloalkyl, C 3-8 Cycloalkoxy, C 3-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 1-4 Alkyl, -SC 3-8 Cycloalkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 4-6 membered or 5-6 membered saturated or partially unsaturated heterocyclic group, C 6-10 Aryl, phenyl, 5-6 membered heteroaryl are optionally substituted by 1-4 independently selected R 2a replaced; or

[0021] Two adjacent substituted R 2 Together with the A ring atoms to which they are attached, they form a 5-6 membered saturated or partially unsaturated alicyclic ring, a 5-6 membered saturated or partially unsaturated heterocyclic ring, a 5-6 membered heteroaromatic ring or a benzene ring; in particular, two adjacent substituted R 2 Together with the A ring atoms to which they are attached, they form the following structure: Two adjacent R 2 The formed ring is optionally substituted by 1 to 4 R 2a replace;

[0022] Among them, each R 2a Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1- 6 alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl; the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; in particular, each R 2a Independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3- 6 cycloalkyl, C 1-4 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl; the C 1- 4 alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 1-4 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; more particularly, each R 2aIndependently selected from halogen (e.g., F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3;

[0023] More specifically, each R 2 Independently selected from: halogen (eg, Cl), methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, cyclopropyl, cyclohexyl, pentafluorosulfenyl (-SF5);

[0024] R 5 and R 6 Independently selected from: H, halogen, C 1-4 Alkyl; or R 5 With R 6 Together with the C atom to which they are attached, they form a cyclopropyl group; or R 5 With R 6 Together they form =O; in particular, R 5 and R 6 Independently selected from: H, halogen, methyl, especially H;

[0025] R 7 Selected from: C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 Alkoxy, -SC 1-4 Alkyl, -NH2, -NH(C 1-4 Alkyl), -N(C 1-4 Alkyl)(C 1-4 alkyl); the aforementioned group is optionally replaced by 1 to 3 independently selected R 7a Replace, each R 7a are the same or different and are independently selected from halogen or -OH; in particular, R 7 is methyl or ethyl, more particularly ethyl;

[0026] m is an integer between 0 and 3, for example, 0, 1, 2 or 3;

[0027] Each R 3 Same or different, independently selected from: halogen, -OH, -CN, -NR 3a R 3b 、C 1-6 Alkyl, C 1-6 Alkoxy, saturated or partially unsaturated C 3-8 Alicyclic group, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4-alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group containing 1-3 heteroatoms selected from N, O, S, C 6-10 Aryl, 5-6 membered heteroaryl; in particular, each R 3 Independently selected from: halogen, -OH, -CN, -NR 3a R 3b 、C 1-4 Alkyl, C 1- 4 alkoxy, saturated or partially unsaturated C 3-6 Alicyclic group, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2- 4-alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl; the C 1-6 Alkyl, C 1-4 Alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy, saturated or partially unsaturated C 3-8 Alicyclic, saturated or partially unsaturated C 3-6 Alicyclic group, C 3-8 Cycloalkoxy, C 3-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 1-4 Alkyl, -SC 3-8 Cycloalkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl, optionally substituted by 1-4 independently selected R 3c replace;

[0028] Among them, R 3a and R 3b Independently selected from: H, C 1-4 Alkyl; or, R 3a and R 3b Together with the nitrogen atom to which they are attached, they form a 4-6 membered heterocyclic ring containing 1-2 heteroatoms selected from N, O, and S;

[0029] Each R 3c Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8Cycloalkyl, C 2-4 Alkenyl, C 2- 4 alkynyl; in particular, each R 3c Independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl; the C 1-6 Alkyl, C 1-4 Alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy, C 3-8 Cycloalkyl, C 3-6 Cycloalkyl, C 3-8 Cycloalkoxy, C 3-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 1-4 Alkyl, -SC 3-8 Cycloalkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; more particularly, each R 3c Independently selected from: halogen (e.g., F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3;

[0030] More specifically, R 3 is selected from methyl, methoxy, and cyclopropyl;

[0031] p is an integer from 0 to 5, for example, 0, 1, 2, 3, 4 or 5;

[0032] Each R4 is the same or different and is independently selected from: halogen, -OH, -CN, C 1-4 Alkyl, C 3-5 Cycloalkyl; or, two R4 attached to the same atom together with the atom to which they are attached form C 3-5 or a 4- to 6-membered spiro ring containing 1 to 3 heteroatoms selected from N, O, and S; two adjacent substituted R 4 Together with the atoms to which they are attached, they form a 3- to 6-membered alicyclic ring; two non-adjacent substituted R 4 Together with the atoms to which they are attached, they form a bridged ring; in particular, R 4 is methyl; or two adjacent substituted R4 Together with the atoms to which they are attached, they form a 4-6 membered alicyclic ring;

[0033] W is selected from: C, CH, N;

[0034] J, G, K, V, Q, Y are independently selected from: none (i.e., absent), C, CH, -(CH2) i -, N, NH, O, S; E, M are independently selected from: none (i.e., absent), -(CH2) i -, NH, O, S;

[0035] i is an integer from 1 to 3, for example, i is 1, 2, or 3;

[0036] Each independently represents a single bond or a double bond;

[0037] The ring where W, E, G, and J are located and the ring where K, V, Q, Y, and M are located are independently a ring having 4 to 8 ring atoms, for example, a ring having 4, 5, 6, or 7 ring atoms;

[0038] q is an integer from 0 to 5, for example, 0, 1, 2, 3, 4 or 5;

[0039] Each R 8 Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, C 1-4 Alkyl; or, two R attached to the same atom 8 Together with the atoms to which they are attached, they form a cyclopropyl spiro ring. Two adjacently substituted R8 together with the atoms to which they are attached form a cyclopropyl group. Two non-adjacently substituted R8 as described above form a cyclopropyl group. 8 Together with the atoms to which they are attached, they form a bridged ring; in particular, each R 8 Independently selected from: halogen, -OH, -NH2, =O, -CN, methyl;

[0040] y is an integer from 0 to 6, for example, 0, 1, 2, 3, 4, 5 or 6;

[0041] Each R 9 Same or different: each R 9 Independently selected from: halogen, -OH, =O, -CN, -N(R 9a )(R 9b ),-C(O)R 9c 、-S(O)R 9c 、-S(O)2R 9c 、C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, C6-10 Aryl or 5- to 6-membered heteroaryl; in particular, each R 9 Independently selected from: halogen, -OH, =O, -CN, -N(R 9a )(R 9b ),-C(O)R 9c 、-S(O)R 9c 、-S(O)2R 9c 、C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, phenyl or 5- to 6-membered heteroaryl;

[0042] The C 1-6 Alkyl, C 1-4 Alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy, C 3-8 Cycloalkyl, C 3-6 Cycloalkyl, C 3-8 Cycloalkoxy, C 3-6 Cycloalkoxy, C 6-10 Aryl, phenyl or 5-6 membered heteroaryl is optionally replaced by 1-4 independently selected R 9d Replace; or

[0043] Two R's attached to the same atom 9 Together with the atoms to which they are attached, they form C 3-5 spirocyclic ring or a 4-6 membered spirocyclic ring containing 1-3 heteroatoms selected from N, O, and S; two adjacent substituted R9 together with the atoms to which they are attached form a 5-6 membered saturated or partially unsaturated alicyclic ring, a 5-6 membered saturated or partially unsaturated heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S, a 5-6 membered heteroaromatic ring or a benzene ring containing 1-3 heteroatoms selected from N, O, and S; two non-adjacent R9 as described above together with the atoms to which they are attached form a bridged ring; the two R9 as described above 9 The rings involved in the formation are optionally replaced by 1 to 3 R 9d replace;

[0044] Each R 9a The same or different, independently selected from: H, C 1-6 Alkyl, C 3-8 Cycloalkyl; in particular, each R 9a Independently selected from: H, C 1-4 Alkyl, C 3-6 Cycloalkyl; the C 1-6 Alkyl, C 1-4 Alkyl, C 3-8 Cycloalkyl, C 3- 6-cycloalkyl is optionally substituted with one or more halogens;

[0045] R 9b Selected from: H, C substituted or unsubstituted by one or more halogens 1-6 Alkyl (especially C 1-4 alkyl), -C(O)R 9b-1 、-S(O)2R 9b-1 ; Each R 9b-1 Independently selected from: C 1-6 Alkyl, C 3-8 Cycloalkyl, C 1-6 Alkoxy, in particular selected from C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 Alkoxy; each R 9c The same or different, independently selected from: H, halogen, -OH, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl; in particular, each R 9c Independently selected from: H, halogen, -OH, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl; the C 1-6 Alkyl, C 1-4 Alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy, C 3-8 Cycloalkyl C 3-6 Cycloalkyl is optionally substituted with one or more halogens;

[0046] Each R 9d Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2- 4 alkynyl; in particular, each R 9d Independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C2-4 Alkynyl; the C 1-6 Alkyl, C 1-4 Alkyl, C 1-6 Alkoxy, C 1-4 Alkoxy, C 3-8 Cycloalkyl, C 3-6 Cycloalkyl, C 3-8 Cycloalkoxy, C 3-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 1-4 Alkyl, -SC 3-8 Cycloalkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl groups are optionally substituted with one or more halogens.

[0047] In some embodiments, ring A is a benzene ring or a pyridine ring;

[0048] n is an integer between 0 and 3;

[0049] Each R 2 Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1- 4 alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3- 6 cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl, optionally substituted by 1-4 independently selected R 2a replaced; or

[0050] Two adjacent substituted R 2 Together with the A ring atoms to which they are attached, they form the following structure: Two adjacent R 2 The formed ring is optionally substituted by 1 to 4 R 2a replace;

[0051] Among them, each R 2a Independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 1-4 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 1-4 Cycloalkoxy, -SC 1-4 Alkyl, -5-C 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; preferably, each R 2a Independently selected from halogen (eg, F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3.

[0052] In some embodiments, each R 2 Independently selected from: halogen (eg, Cl), methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, cyclopropyl, cyclohexyl, pentafluorosulfenyl (-SF5); or

[0053] Two adjacent substituted R 2 Together with the A ring atoms to which they are attached, they form

[0054] In some embodiments, Ring B is

[0055] p is an integer from 0 to 4;

[0056] R4 is methyl; or two adjacent substituted R 4 Together with the atoms to which they are attached, they form a 4-6 membered alicyclic ring.

[0057] In some embodiments, X is CH or N;

[0058] m is 0 or 1;

[0059] R 3 Selected from: halogen, -OH, -CN, -NR 3a R 3b 、C 1-4 Alkyl, C1-4 Alkoxy, saturated or partially unsaturated C 3- 6 alicyclic group, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl; the C 1-4 Alkyl, C 1-4 Alkoxy, saturated or partially unsaturated C 3-6 Alicyclic group, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl, optionally substituted by 1-4 independently selected R 3c replace;

[0060] Among them, R 3a and R 3b Independently selected from: H, C 1-4 Alkyl; or, R 3a and R 3b Together with the nitrogen atom to which they are attached, they form a 4-6 membered heterocyclic ring containing 1-2 heteroatoms selected from N, O, and S;

[0061] Each R 3c Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2- 4 alkynyl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3- 6 cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; preferably, each R 3cIndependently selected from: halogen (eg, F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3.

[0062] In some embodiments, R 3 Selected from methyl, methoxy, cyclopropyl.

[0063] In some embodiments, L is -C(O)-.

[0064] In some embodiments, R 5 and R 6 Independently selected from: H, halogen, methyl, preferably H.

[0065] In some embodiments, R 7 It is a methyl group or an ethyl group, preferably an ethyl group.

[0066] In some embodiments, the ring where W, E, G, and J are located and the ring where K, V, Q, Y, and M are located are both rings having 4 to 6 ring atoms.

[0067] In some embodiments, the spirocyclic structure where W, E, G, J, K, V, Q, Y, and M are located is selected from the following structures:

[0068] wherein each Z is independently selected from: O, NH, S, preferably O or NH;

[0069] Each i is independently 1, 2 or 3; R 8 、R 9 The definitions of , q, and y are as described above.

[0070] In some embodiments, q is an integer from 0 to 5;

[0071] Each R 8 Independently selected from: halogen, -OH, -NH2, =O, -CN, methyl.

[0072] In some embodiments, y is an integer from 0 to 6;

[0073] Each R 9 Independently selected from: halogen, -OH, =O, -CN, -N(R 9a )(R 9b ),-C(O)R 9c 、-S(O)R 9c 、-S(O)2R 9c 、C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6Cycloalkoxy, phenyl or 5- to 6-membered heteroaryl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, phenyl or 5-6 membered heteroaryl is optionally replaced by 1-4 independently selected R 9d Replace; or

[0074] Two R's attached to the same atom 9 Together with the atoms to which they are attached, they form C 3-5 or a 4- to 6-membered spiro ring containing 1 to 3 heteroatoms selected from N, O, and S; two adjacent substituted R 9 Together with the atoms to which they are attached, they form a 5-6 membered saturated or partially unsaturated alicyclic ring, a 5-6 membered saturated or partially unsaturated heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S, a 5-6 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O, and S, or a benzene ring; two non-adjacent R 9 Together with the atoms to which they are attached, they form a bridged ring; as described above, two R 9 The rings involved in the formation are optionally replaced by 1 to 3 R 9c

[0075] replace;

[0076] Each R 9a The same or different, independently selected from: H, C 1-4 Alkyl, C 3-6 Cycloalkyl; the C 1-4 Alkyl, C 3- 6 cycloalkyl is optionally substituted with one or more halogens; preferably, each R 9a Independently selected from: H, methyl, trifluoromethyl;

[0077] R 9b Selected from: H, C substituted or unsubstituted by one or more halogens 1-4 Alkyl, -C(O)R 9b-1 、-S(O)2R 9b- 1 ; Each R 9b-1 Independently selected from: C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 alkoxy;

[0078] Each R 9c The same or different, independently selected from: H, halogen, -OH, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl; the C 1-4 Alkyl, C1-4 Alkoxy, C 3-6 Cycloalkyl is optionally substituted with one or more halogens; preferably, each R 9c Independently selected from: H, halogen, -OH, methyl, trifluoromethyl;

[0079] Each R 9d Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2- 4 alkynyl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkyne is optionally substituted with one or more halogens; preferably, each R 9c Independently selected from: halogen (eg, F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3.

[0080] In some embodiments, each R 9 Independently selected from: halogen, -OH, -NH2, =O, -CN, methyl; or two adjacent substituted R 9 Together with the atoms they are attached to form a benzene ring.

[0081] In some embodiments, the spirocyclic structure where W, E, G, J, K, V, Q, Y, and M are located is selected from the following structures:

[0082] In some embodiments, the compound containing a spiro ring structure represented by Formula I has a structure selected from the following:

[0083] in,

[0084] W, E, G, J, K, V, Q, Y, M, n, R 2 ,m,R 3 ,q,R 8 ,y,R 9 、 The definitions of are as mentioned above.

[0085] In some embodiments, the compound containing a spiro ring structure represented by Formula I is selected from any one of the following compounds:

[0086] The present invention includes pharmaceutically acceptable salt forms of the compounds of the present invention. The pharmaceutically acceptable salts of the compounds may be acid addition salts of at least one of the following acids: galactaric acid, D-glucuronic acid, glycerophosphoric acid, hippuric acid, isethionic acid, lactobionic acid, maleic acid, 1,5-naphthalenedisulfonic acid, naphthalene-2-sulfonic acid, pivalic acid, terephthalic acid, thiocyanic acid, bile acid, n-dodecylsulfuric acid, benzenesulfonic acid, citric acid, D-glucose, glycolic acid, lactic acid, malic acid, malonic acid, mandelic acid, phosphoric acid, propionic acid, hydrochloric acid, sulfuric acid, tartaric acid, succinic acid, formic acid, Hydroiodic acid, hydrobromic acid, methanesulfonic acid, nicotinic acid, nitric acid, orotic acid, oxalic acid, picric acid, L-pyroglutamic acid, saccharinic acid, salicylic acid, gentisic acid, p-toluenesulfonic acid, valeric acid, palmitic acid, sebacic acid, stearic acid, lauric acid, acetic acid, adipic acid, carbonic acid, benzenesulfonic acid, ethanedisulfonic acid, ethylsuccinic acid, fumaric acid, 3-hydroxynaphthalene-2-carboxylic acid, 1-hydroxynaphthalene-2-carboxylic acid, oleic acid, undecylenic acid, ascorbic acid, camphoric acid, camphorsulfonic acid, dichloroacetic acid, ethanesulfonic acid. On the other hand, the pharmaceutically acceptable salt of the compound can also be a salt formed by a compound of the present invention and a metal (including sodium, potassium, calcium, etc.) ion or a pharmaceutically acceptable amine (including ethylenediamine, tromethamine, etc.), ammonium ion, or choline.

[0087] The present invention also includes solvates of the compounds of the present invention.

[0088] The present invention includes the various deuterated forms of the compounds of the present invention. Each available hydrogen atom attached to a carbon atom can independently be replaced with a deuterium atom.

[0089] The compounds of the present invention or their pharmaceutically acceptable salts can be prepared by referring to the methods described in the examples or by modified methods.

[0090] In a second aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of a compound containing a spirocyclic structure as shown in Formula I according to the first aspect, or one or more of its pharmaceutically acceptable salts, stereoisomers, deuterated substances, solvates, prodrugs, and metabolites, and an optional pharmaceutically acceptable carrier.

[0091] The carrier can vary depending on the dosage form, administration mode, etc. Examples of carriers include excipients, binders, disintegrants, lubricants, flavoring agents, fragrances, colorants, or sweeteners. The pharmaceutical composition can be in the form of capsules, powders, tablets, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories, or patches, etc., which are conventional pharmaceutical formulations.

[0092] In a third aspect, the present invention provides a WRN regulator comprising a compound containing a spirocyclic structure as shown in Formula I described in the first aspect, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, metabolite thereof, and one or more of the pharmaceutical compositions described in the second aspect.

[0093] Specifically, the WRN regulator is a WRN inhibitor or degrader.

[0094] In a fourth aspect, the present invention provides the use of one or more of the compounds containing a spirocyclic structure represented by Formula I described in the first aspect or their pharmaceutically acceptable salts, stereoisomers, deuterated substances, solvates, prodrugs, metabolites, and the pharmaceutical compositions described in the second aspect as WRN inhibitors or degraders.

[0095] In a fifth aspect, the present invention provides a compound containing a spirocyclic structure as shown in Formula I described in the first aspect, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, metabolite thereof, and one or more of the pharmaceutical compositions described in the second aspect for use in the preparation of WRN inhibitors or degraders.

[0096] In a sixth aspect, the present invention provides a compound containing a spirocyclic structure as shown in Formula I described in the first aspect, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, metabolite thereof, and one or more of the pharmaceutical compositions described in the second aspect for use in the preparation of a medicament for preventing or treating WRN-related diseases.

[0097] In a seventh aspect, the present invention provides a method for treating WRN-related diseases, comprising administering to a patient an effective dose of a compound containing a spirocyclic structure as shown in Formula I described in the first aspect, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, metabolite thereof, and one or more of the pharmaceutical compositions described in the second aspect.

[0098] The compounds of the present invention can be used alone or in combination with other therapeutic agents, such as targeted drugs, immunotherapy drugs, or chemotherapy drugs.

[0099] As modulators that inhibit WRN or mediate WRN degradation, the compounds of the present invention can be used as monotherapy or in combination with other therapeutic agents to treat diseases associated with WRN.

[0100] The WRN-related diseases include diseases that can be treated by inhibiting WRN or mediating WRN degradation, especially cancers characterized by high microsatellite instability (MSI-H) or DNA mismatch repair deficiency (dMMR). The cancers include but are not limited to colorectal cancer, gastric cancer, prostate cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer and ovarian cancer.

[0101] In an eighth aspect, the present invention provides a compound containing a spirocyclic structure as shown in Formula I described in the first aspect, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, metabolite thereof, and the use of the pharmaceutical composition described in the second aspect in the preparation of an immune adjuvant drug.

[0102] Group Definition

[0103] The term "C 1-6 "Alkyl" refers to a straight or branched alkyl group containing 1 to 6, preferably 1 to 4, or 1 to 3 carbon atoms. For example, it includes but is not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, hexyl, etc.

[0104] The term "C 1-6 "Alkoxy" refers to a linear, branched or cyclic alkoxy group containing 1 to 6, preferably 1 to 4, or 1 to 3 carbon atoms. For example, it includes but is not limited to methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy, cyclopentyloxy, hexyloxy, cyclohexyloxy, etc.

[0105] The term "saturated or partially unsaturated C 3-8 "Alicyclic" means a saturated or partially unsaturated alicyclic ring containing 3-8, preferably 3-7, 3-6, 3-5 ring carbon atoms, including C 3-8 Cycloalkyl and non-aromatic alicyclic groups containing unsaturated bonds, the hydrogen atoms on the ring carbon atoms of which may be optionally replaced by C 1-6 Alkyl substitution.

[0106] The term "C 3-8 "Cycloalkyl" means a cyclic alkyl group containing 3 to 8 carbon atoms, wherein the hydrogen atoms on the ring carbon atoms may be optionally replaced by C 1-6 Alkyl substituted. C 3-8 Cycloalkyl groups include, but are not limited to, cyclopropyl, methylcyclopropyl, ethylcyclopropyl, dimethylcyclopropyl, cyclobutyl, methylcyclobutyl, ethylcyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.

[0107] The term "C 3-8 "Cycloalkoxy" means a C 3-8 Cycloalkane, preferably C 3-7 Cycloalkoxy, C 3-6 Cycloalkoxy, C 3-4 Cycloalkoxy includes, but is not limited to, cyclopropyloxy, methylcyclopropyloxy, ethylcyclopropyloxy, dimethylcyclopropyloxy, cyclobutyloxy, methylcyclobutyloxy, ethylcyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

[0108] The term "-SC 1-6 "Alkyl" means a C group as defined herein attached through a sulfur 1-6 Alkyl, preferably -SC 1-4 Alkyl, -SC 1-3 Alkyl groups include -S-methyl, -S-ethyl, -S-propyl, -S-isopropyl, -S-n-butyl, -S-isobutyl, -S-tert-butyl and the like.

[0109] The term "-SC 3-8 "Cycloalkyl" means a C 3-8 Cycloalkyl, preferably -SC 3-7 Cycloalkyl, -SC 3-6 Cycloalkyl, -SC 3-5 Cycloalkyl groups include, but are not limited to, -S-cyclopropyl, -S-methylcyclopropyl, -S-cyclobutyl, -S-cyclopropyl, -S-cyclobutyl, and the like.

[0110] The term "C 2-4 "Alkenyl" refers to a linear or branched monovalent hydrocarbon group containing 2 to 4 carbon atoms and at least one double bond, including but not limited to ethenyl, propenyl, isopropenyl, butenyl, and isobutenyl.

[0111] The term "C 2-4 "Alkynyl" refers to a linear or branched monovalent hydrocarbon group containing 2 to 4 carbon atoms and at least one triple bond, including but not limited to ethynyl, propynyl, isopropynyl, butynyl, and isobutynyl.

[0112] The term "saturated or partially unsaturated heterocycle" refers to a partially or fully hydrogenated non-aromatic ring that can exist as a monocycle, a bicycle (including fused rings), or a spirocycle. Unless otherwise specified, the heterocycle is typically a 4- to 6-membered, preferably a 4- to 5-membered, monocyclic or bicyclic ring system containing 1 to 3, for example 1 or 2, heteroatoms independently selected from S, O, and N, such as -O-, -N=, -NR-, or -S-, wherein R is hydrogen, a C1-4 alkyl group, or a nitrogen protecting group. When the term "saturated or partially unsaturated heterocyclyl" is used, it is intended to include "heterocycloalkyl" and "partially saturated heterocyclyl".

[0113] The term "C 6-10 The term "aryl" refers to an aromatic group having 6 to 10 ring carbon atoms and composed of one or more rings fused together, including a monocyclic aromatic group (e.g., phenyl) or a fused ring system (e.g., naphthalene, anthracenyl).

[0114] The term "5- to 6-membered heteroaryl" refers to a 5- to 6-membered aromatic ring system containing 1-3 ring heteroatoms selected from N, O or S, including but not limited to: pyrrolyl, furanyl, thienyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, isothiazolyl, oxazolyl, pyridyl, pyranyl, pyrazinyl, pyridazinyl, pyrimidinyl, oxazinyl, oxadiazinyl, etc.

[0115] The term "halogen" is selected from fluorine, chlorine, bromine, and iodine, preferably fluorine and chlorine.

[0116] The term "substituted" refers to the replacement of one or more hydrogen atoms on a group with one or more (eg, 2, 3, 4, 5) substituents.

[0117] Compared with the prior art, the present invention has the following advantages:

[0118] (1) Currently, there are few reported WRN-targeting regulators, especially those with potent regulatory effects. The spirocyclic compounds represented by Formula I of the present invention are a novel class of WRN regulators with potent WRN inhibitory activity and significant WRN degradation-mediating activity. They exhibit excellent selectivity for anti-proliferative activity against MSI cells HCT116 and MSS cells DLD-1. The compounds of the present invention can be used as WRN regulators for the preparation of drugs for preventing or treating WRN-related diseases.

[0119] (2) The synthetic route of the compound of formula I of the present invention is ingeniously designed, the raw materials are cheap and readily available, the synthetic process is safe and environmentally friendly, and it is easy to produce on a large scale. DETAILED DESCRIPTION

[0120] The present invention will be described in detail below by way of examples. In the present invention, the following examples are provided to better illustrate the present invention and are not intended to limit the scope of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0121] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are conventional raw materials and reagents in the art and are purchased commercially.

[0122] Example 1

[0123] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(2-oxa-6-azaspiro[3.3]hept-6-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-1)

[0124] (1) Synthesis of intermediate D-1

[0125] 3,5-diamino-1,2,4-triazole (5 g, 50.5 mmol) was dissolved in 48% aqueous hydrobromic acid solution (40 mL), and an aqueous solution (10 mL) of sodium nitrite (NaNO2) (2.52 g, 12 mmol) was added dropwise at 0°C. After stirring at room temperature for 1 hour, the reaction was heated to 100°C and the reaction was continued overnight. After the reaction was completed, sodium hydroxide (NaOH) was added to the reaction solution at 0°C to adjust the pH to neutral. The mixture was extracted with a mixed solvent of ethyl acetate (EA) and tetrahydrofuran (THF) (80 mL x 3). The organic phases were combined, washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was slurried with 1,4-dioxane and filtered to obtain intermediate D-1 (white solid, 3.5 g): 1 H NMR (600MHz, DMSO-d6) δ12.26 (s, 1H), 6.30 (s, 2H).

[0126] (2) Synthesis of intermediate D-2

[0127] Methyl 3-oxopentanoate (1.3 g, 10 mmol) was dissolved in dichloromethane (DCM) (20 mL), and sulfonyl chloride (SO2Cl2) (1.52 g, 13 mmol) was added dropwise. The reaction was allowed to react overnight at room temperature. After completion of the reaction, saturated aqueous sodium bicarbonate solution was added to quench the reaction. The mixture was extracted with dichloromethane (30 mL x 3), and the organic phases were combined, washed with saturated brine (30 mL x 1), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Intermediate D-2 (colorless oil, 1.59 g) was obtained.

[0128] (3) Synthesis of intermediate D-3

[0129] Intermediate D-2 (1.49 g, 9.08 mmol) was dissolved in acetonitrile (ACN) (10 mL), and 1-Boc-piperazine (1.69 g, 9.08 mmol) and triethylamine (TEA) (2.7 g, 27 mmol) were added. The mixture was stirred at 60°C for 8 hours. After completion of the reaction, the solvent was evaporated under reduced pressure, and 50 mL of water was added to the residue. The mixture was extracted with dichloromethane (50 mL x 3). The organic phase was washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain intermediate D-3 (yellow solid, 1.43 g).

[0130] (4) Synthesis of Compound D-4

[0131] 2-Methyl-4-trifluoromethylaniline (724 mg, 4.14 mmol) was dissolved in DCM (10 mL), and triethylamine (TEA) (836 mg, 8.27 mmol) was added. Chloroacetyl chloride (514 mg, 4.55 mmol) was added dropwise at 0°C. After the reaction, 15 mL of water was added for dilution. The mixture was extracted with dichloromethane (15 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 5:1) to obtain intermediate D-4 (white solid, 933 mg): 1 H NMR (600MHz, DMSO-d6) δ9.82 (s, 1H), 7.74 (d, J=8.4Hz, 1H), 7.62 (s, 1H), 7.56 (d, J=8.4Hz, 1H), 4.37 (s, 2H), 2.31 (s, 3H).

[0132] (5) Synthesis of Compound D-5

[0133] Intermediate D-4 (73 mg, 0.29 mmol) was dissolved in acetone (3 mL), and potassium iodide (KI) (62 mg, 0.374 mmol) was added. The mixture was reacted at 60°C for 1 hour. After the reaction, the mixture was filtered and the solvent was evaporated under reduced pressure to obtain the crude intermediate D-5, which was used directly in the next reaction without purification.

[0134] (6) Synthesis of Compound D-6

[0135] Intermediate D-1 (2.96 g, 18 mmol) was dissolved in ethanol (EtOH) (30 mL), and intermediate D-3 (6.27 g, 20 mmol) and polyphosphoric acid (PPA) (7.3 g, 21.6 mmol) were added. The mixture was reacted at 90 ° C overnight. After the reaction was completed, the system was cooled to room temperature, N, N-diisopropylethylamine (DIPEA) (7 g, 54 mmol) and di-tert-butyl dicarbonate (Boc anhydride) (5.9 g, 27 mmol) were added, and the mixture was stirred at room temperature for 6 hours. After the reaction was completed, ethanol was evaporated under reduced pressure, the residue was diluted with water (50 mL), extracted with ethyl acetate (50 mL x 3), and the organic phase was washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Methanol was added to the residue and slurried, stirred at room temperature for 2 hours, and filtered to obtain intermediate D-6 (white solid, 1.75 g): 1 H NMR (600MHz, DMSO-d6) δ13.26 (s, 1H), 3.91 (s, 2H), 3.29 (m, 2H), 2.87 (m, 2H), 2.76 (q , J=7.5Hz, 2H), 2.62 (d, J=9.3Hz, 2H), 1.42 (s, 9H), 1.18 (t, J=7.6Hz, 3H). ESI-MS: m / z 449.2[M+Na] + .

[0136] (7) Synthesis of Compound D-7

[0137] Intermediate D-6 (500 mg, 1.17 mmol) was dissolved in dioxane (4 mL), and intermediate D-5 (462 mg, 1.35 mmol) and N,N-diisopropylethylamine (DIPEA) (453 mg, 3.51 mmol) were added. The mixture was allowed to react at 80°C for 3 hours. After the reaction, the system was cooled to room temperature, and DIPEA (7 g, 54 mmol) and Boc anhydride (5.9 g, 27 mmol) were added. The mixture was stirred at room temperature for 6 hours. After the reaction, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (30 mL x 3). The organic phase was washed with saturated brine (10 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol = 20:1) to obtain compound D-7 (white solid, 580 mg): 1H NMR (600MHz, CDCl3) δ8.81 (s, 1H), 8.00 (d, J=8.7Hz, 1H), 7.45 (m, 2H), 5.18 (s, 2H), 4.13 (q, J=7.1Hz, 2H), 3. 60 (t, J=10.6Hz, 2H), 3.15 (s, 2H), 2.97 (s, 2H), 2.65 (m, 2H), 2.37 (s, 3H), 1.49 (s, 9H), 1.27 (t, J=7.1Hz, 3H).

[0138] (8) Synthesis of Compound D-8

[0139] Intermediate D-7 (100 mg, 0.16 mmol) was dissolved in dimethyl sulfoxide (DMSO) (2 mL), and 2-oxa-6-azaspiro[3,3]heptane (80 mg, 0.78 mmol) and potassium acetate (92 mg, 0.94 mmol) were added. The mixture was allowed to react overnight at 120°C. After completion of the reaction, the reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Methanol was added to the residue, stirred at room temperature for 2 hours, and filtered to obtain compound D-8 (a white solid, 88 mg).

[0140] (9) Synthesis of Compound D-9

[0141] Intermediate D-8 (80 mg, 0.12 mmol) was dissolved in DCM (2 mL), and trifluoroacetic acid (0.5 mL) was added. The mixture was reacted at room temperature for 10 minutes. After completion of the reaction, the DCM was evaporated under reduced pressure, and 10 mL of water and 1 mL of TEEA were added. The mixture was extracted with ethyl acetate (15 mL x 2). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain the crude intermediate D-9, which was used directly in the next reaction without further purification.

[0142] (10) Synthesis of Compound I-1

[0143] 3-Hydroxy-2-pyridinecarboxylic acid (33 mg, 0.24 mmol) was dissolved in dichloromethane (3 mL), and 1-chloro-N,N,2-trimethylpropenamine (35 mg, 0.26 mmol) was added dropwise. The mixture was stirred at room temperature for 1.5 hours. The entire residue from the post-treatment of the previous reaction containing intermediate D-9 was dissolved in dichloromethane (DCM) (1 mL) and DIPEA (77 mg, 0.6 mmol). This solution was added dropwise to the reaction system and allowed to react at room temperature for 1 hour. After the reaction, the reaction solution was diluted with water (10 mL) and extracted with DCM (10 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol = 20:1) to obtain compound I-1 (white solid, 30 mg): 1 H NMR (600MHz, DMSO-d6) δ10.38 (s, 1H), 9.97 (s, 1H), 8.06 (dd, J=3.2, 2.6Hz, 1H), 7.73 (d, J=8.5Hz , 1H), 7.63 (s, 1H), 7.54 (d, J=8.1Hz, 1H), 7.30-7.27 (m, 2H), 5.14 (s, 2H), 4.70 (s, 4H), 4.53 (d, J =12.2Hz, 1H), 4.13(s, 4H), 3.46(q, 2H), 3.40-3.37(m, 1H), 3.24-3.16(m, 1H), 2.99-2.87(m, 3H) , 2.75 (d, J=10.5Hz, 1H), 2.57 (d, J=11.0Hz, 1H), 2.36 (s, 3H), 1.15 (t, J=7.5Hz, 3H). ESI-MS: m / z 682.3[M+H] + .

[0144] Example 2

[0145] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-8-azaspiro[4.5]dec-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-2)

[0146] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-8-aza-spiro[4,5]decane hydrochloride to prepare compound I-2: 1H NMR (600MHz, DMSO-d6) δ10.37 (s, 1H), 9.98 (s, 1H), 8.06 (dd, J=3.4, 2.5Hz, 1H), 7.69 (d, J=8.5Hz, 1H), 7.63 (s, 1H), 7.5 3 (d, J=8.4Hz, 1H), 7.31-7.25 (m, 2H), 5.13 (s, 2H), 4.53 (d, J=12.3Hz, 1H), 3.74 (t, J=6.7Hz, 2H), 3.62-3.55 (m, 2H), 3. 51-3.43 (m, 2H), 3.40-3.35 (m, 3H), 3.20 (t, J=10.9Hz, 1H), 3.00-2.87 (m, 3H), 2.75 (d, J=10.9Hz, 1H), 2.57 (d, J=10.4H z, 1H), 2.34 (s, 3H), 1.91-1.82 (m, 2H), 1.67 (t, J = 7.4Hz, 2H), 1.55 (t, J = 5.6Hz, 4H), 1.16 (t, J = 7.5Hz, 3H). ESI-MS: m / z 724.4[M+H] + .

[0147] Example 3

[0148] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(5-oxa-2-azaspiro[3.4]octan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-3)

[0149] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 5-oxa-2-azaspiro[3.4]octane oxalate to prepare compound I-3: 1H NMR (600MHz, DMSO-d6) δ10.38 (s, 1H), 9.96 (s, 1H), 8.09-8.02 (m, 1H), 7.72 (d, J=8.6Hz, 1H), 7.63 (s, 1H), 7.53 (d, J=8 .6Hz, 1H), 7.28 (d, J=2.8Hz, 2H), 5.14 (s, 2H), 4.53 (d, J=12.0Hz, 1H), 3.99 (d, J=8.7Hz, 2H), 3.93 (d, J=8.6Hz, 2H), 3.7 5 (t, J=6.8Hz, 2H), 3.52-3.43 (m, 2H), 3.41-3.35 (m, 1H), 3.20 (t, J=11.5Hz, 1H), 3.00-2.85 (m, 3H), 2.75 (d, J=10.7Hz , 1H), 2.58 (d, J=10.8Hz, 1H), 2.35 (s, 3H), 2.08 (t, J=7.3Hz, 2H), 1.91-1.78 (m, 2H), 1.16 (t, J=7.4Hz, 3H). ESI-MS: m / z 696.4[M+H] + .

[0150] Example 4

[0151] 2-(5-ethyl-6-(4-(3-hydroxyoctanoyl)piperazin-1-yl)-7-oxo-2-(1,4-dioxaspiro[4.5]dec-7-en-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-4)

[0152] Synthesis of compound D-10

[0153] Intermediate D-7 (60 mg, 0.093 mmol) was dissolved in 4M HCl / EA (1.5 mL) and reacted at room temperature for 1 hour. After the reaction, the solvent was evaporated under reduced pressure to obtain the crude intermediate D-10, which was used directly in the next reaction without purification.

[0154] Synthesis of compound D-11

[0155] 3-Hydroxy-2-pyridinecarboxylic acid (26 mg, 0.186 mmol) was dissolved in dichloromethane (3 mL), and 1-chloro-N,N,2-trimethylpropenamine (27 mg, 0.2 mmol) was added dropwise. The mixture was stirred at room temperature for 1.5 hours. The entire residue from the post-treatment of the previous reaction containing Intermediate D-10 was dissolved in dichloromethane (DCM) (1 mL) and DIPEA (60 mg, 0.46 mmol). This solution was added dropwise to the reaction system and allowed to react at room temperature for 1 hour. After completion of the reaction, the reaction solution was diluted with water (10 mL) and extracted with DCM (10 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol = 20:1) to obtain Intermediate D-11 (white solid, 60 mg).

[0156] Synthesis of compound I-4

[0157] Intermediate D-11 (60 mg, 0.09 mmol), 1,4-dioxa-spiro[4,5]dec-7-ene-8-boronic acid pinacol ester (31 mg, 0.117 mmol), potassium phosphate (57 mg, 0.27 mmol), XPhos Pd G3 (3.8 mg, 0.0045 mmol), dioxane (3 mL), and H2O (1 mL) were mixed and stirred at 80°C under an argon atmosphere for 9 hours. After completion of the reaction, water (10 mL) was added to dilute the reaction solution, which was then extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol = 30:1) to obtain compound I-4 (white solid, 3 mg): 1 H NMR (400MHz, CDCl3) δ11.82 (s, 1H), 8.70 (s, 1H), 8.19-8.02 (m, 2H), 7.46 (d, J=9.0Hz, 1H), 7.41 (s, 1H), 7.36-7.31(m, 1H), 7.30-7.27(m, 1H), 6.90-6.84(m, 1H), 5.71-5.50(m, 1H), 5.04(s, 2H), 4.79( s, 1H), 4.02 (s, 4H), 3.86-3.73 (m, 2H), 3.55-3.38 (m, 1H), 3.30-3.18 (m, 2H), 3.12-2.99 (m, 1H), 2. 89-2.67 (m, 4H), 2.51 (s, 2H), 2.19 (s, 3H), 1.89 (t, J=6.5Hz, 2H), 1.34 (t, J=7.5Hz, 3H). ESI-MS: m / z 723.3[M+H] +.

[0158] Example 5

[0159] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-5)

[0160] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride to prepare compound I-5: 1 H NMR (400MHz, DMSO-d6) δ10.40 (s, 1H), 9.99 (s, 1H), 8.09-8.04 (m, 1H), 7.70 (d, J=8.4Hz, 1H), 7.64 (s, 1H), 7.5 4 (d, J=8.4Hz, 1H), 7.30 (d, J=2.6Hz, 2H), 5.13 (s, 2H), 4.54 (d, J=12.2Hz, 1H), 3.78-3.66 (m, 2H), 3.63-3.56 ( m, 2H), 3.53-3.42 (m, 3H), 3.27-3.10 (m, 3H), 3.03-2.89 (m, 3H), 2.76 (d, J=9.7Hz, 1H), 2.64-2.54 (m, 1H), 2.3 5 (s, 3H), 1.84 (d, J = 13.4Hz, 2H), 1.58 (d, J = 4.3Hz, 2H), 1.50-1.34 (m, 6H), 1.17 (t, J = 7.5Hz, 3H). ESI-MS: m / z 738.5[M+H] + .

[0161] Example 6

[0162] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(2-oxa-8-azaspiro[4.5]dec-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-6)

[0163] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 2-oxazole-8-azaspiro[4.5]decane hydrochloride to prepare compound I-6: 1H NMR (400MHz, DMSO-d6) δ10.38 (s, 1H), 9.98 (s, 1H), 8.09-8.02 (m, 1H), 7.69 (d, J=8.3Hz, 1H), 7.62 (s, 1 H), 7.53 (d, J=8.2Hz, 1H), 7.28 (d, J=2.8Hz, 2H), 5.13 (s, 2H), 4.53 (d, J=12.0Hz, 1H), 3.75 (t, J=7.1Hz , 2H), 3.57-3.37(m, 9H), 3.25-3.13(m, 1H), 2.94(d, J=12.7Hz, 3H), 2.75(d, J=11.3Hz, 1H), 2.57(d, J= 10.6Hz, 1H), 2.34 (s, 3H), 1.73 (t, J=7.1Hz, 2H), 1.56-1.48 (m, 4H), 1.16 (t, J=7.4Hz, 3H). ESI-MS: m / z 724.5[M+H] + .

[0164] Example 7

[0165] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxo-8-azaspiro[4.5]dec-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-7)

[0166] Synthesis of compound D-12

[0167] tert-Butyl 1-oxo-8-azaspiro[4.5]decane-8-carboxylate (100 mg, 0.39 mmol) was dissolved in 4M HCl / dioxane (2 mL) and stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure to obtain intermediate D-12 (white solid, 73 mg).

[0168] Synthesis of compound I-7

[0169] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with D-12 to obtain compound I-7: 1HNMR (400 MHz, DMSO-d6) δ10.49 (s, 1H), 9.99 (s, 1H), 8.08 (dd, J = 3.6, 2.3 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.63 (s, 1H), 7.56-7.50 (m, 1H), 7.34-7.30 (m, 2H), 5.13 (s, 2H), 4.54 (d, J = 12.1 Hz, 1H), 3.93-3.81 (m, 2H), 3.53-3.50 ( m, 2H), 3.27-3.20 (m, 1H), 3.18-3.09 (m, 2H), 3.04-2.89 (m, 3H), 2.77 (d, J = 10.6Hz, 1H), 2.59 (d, J = 10.8Hz, 1H), 2.53-2.52 (m, 1H), 2.35 (s , 3H), 2.26 (t, J=7.5Hz, 2H), 1.97-1.92 (m, 2H), 1.91-1.83 (m, 2H), 1.56-1.48 (m, 2H), 1.42-1.34 (m, 2H), 1.17 (t, J=7.4Hz, 3H). ESI-MS: m / z 736.5[M+H] + .

[0170] Example 8

[0171] 2-(5-ethyl-6-(4-(3-hydroxyoctanoyl)piperazin-1-yl)-7-oxo-2-(1-oxaspiro[4.5]dec-7-en-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-8)

[0172] Synthesis of compound D-13

[0173] 1-Oxaspiro[4.5]-8-decanone (100 mg, 0.65 mmol) was dissolved in tetrahydrofuran (1 mL) and added dropwise to a 1M solution of LiHMDS (770 μL, 0.77 mmol) in tetrahydrofuran at -50°C. After 15 minutes of reaction, N-phenylbis(trifluoromethanesulfonyl)imide (244 mg, 0.68 mmol) in tetrahydrofuran (1 mL) was added dropwise to the reaction system. The reaction was allowed to proceed at -50°C for 1 hour, then the mixture was allowed to warm to room temperature and continued for another hour. After completion of the reaction, the reaction mixture was quenched with saturated ammonium chloride (5 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (10 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 30:1) to yield compound D-13 (colorless liquid, 119 mg): 1 H NMR (600MHz, CDCl3) δ5.69 (td, J=4.0, 2.0Hz, 1H), 3.96-3.82 (m, 2H), 2.64-2.50 (m, 1H), 2.44-2.23 (m, 3H), 2.05-1.87 (m, 3H), 1.83-1.71 (m, 3H).

[0174] Synthesis of compound D-14

[0175] Intermediate D-13 (115 mg, 0.4 mmol), bis(pinacolato) borate (122 mg, 0.48 mmol), DPPF palladium dichloride (29 mg, 0.04 mmol), potassium acetate (117 mg, 1.2 mmol), and dioxane (3 mL) were mixed and reacted at 100°C under an argon atmosphere for 12 hours. After completion of the reaction, the reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 20:1) to obtain compound D-14 (colorless liquid, 72 mg): 1 H NMR (600MHz, CDCl3) δ6.51-6.44(m, 1H), 3.93-3.79(m, 2H), 2.41-2.30(m, 1H), 2. 26-2.11(m, 3H), 1.97-1.88(m, 2H), 1.75-1.65(m, 3H), 1.55(m, 1H), 1.25(s, 12H).

[0176] Synthesis of compound D-15

[0177] Intermediate D-7 (64 mg, 0.1 mmol), intermediate D-14 (32 mg, 0.12 mmol), potassium phosphate (64 mg, 0.3 mmol), Xphos Pd G3 (4 mg, 0.005 mmol), dioxane (2.4 mL), and H2O (0.8 mL) were mixed and reacted at 80°C under an argon atmosphere for 4 hours. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol = 20:1) to obtain compound D-15 (white solid, 60 mg).

[0178] Synthesis of compound I-8

[0179] Referring to the method of Example 1, D-8 was replaced with D-15 to prepare compound I-8: 1 H NMR (600MHz, DMSO-d6) 610.40 (s, 1H), 10.00 (s, 1H), 8.09-8.03 (m, 1H), 7.72 (d, J=8.4Hz, 1H), 7.63 (s, 1H), 7.53 (d, J= 8.2Hz, 1H), 7.31-7.26(m, 2H), 6.77-6.71(m, 1H), 5.23(s, 2H), 4.54(d, J=12.1Hz, 1H), 3.79-3.69(m, 2H), 3.52-3.43(m , 2H), 3.42-3.39(m, 2H), 3.26-3.19(m, 1H), 3.05-2.91(m, 3H), 2.80(s, 1H), 2.65-2.56(m, 2H), 2.35(s, 3H), 2.34-2.23 (m, 2H), 1.94-1.86 (m, 2H), 1.79-1.73 (m, 1H), 1.73-1.69 (m, 1H), 1.68-1.62 (m, 2H), 1.19 (t, J=7.5Hz, 3H). ESI-MS: m / z 721.5[M+H] + .

[0180] Example 9

[0181] 2-(5-ethyl-6-(4-(3-hydroxypyridinyl)piperazin-1-yl)-7-oxo-2-(spiro[isochromatic hexahydrocyclohexane-1,4′-piperidin]-1′-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-9)

[0182] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with spiro[isochromatic hexahydroane-1,4′-piperidine] hydrochloride to prepare compound I-9: 1 H NMR (600MHz, DMSO-d6) δ10.39 (s, 1H), 10.00 (s, 1H), 8.08-8.05 (m, 1H), 7.71 (d, J = 8.4Hz, 1H), 7.62 (d, J = 2.3Hz, 1H ), 7.55-7.50 (m, 1H), 7.32-7.26 (m, 2H), 7.16-7.10 (m, 4H), 5.16 (s, 2H), 4.55 (d, J=12.2Hz, 1H), 4.02-3.97 (m, 2H) , 3.89 (t, J=5.5Hz, 2H), 3.54-3.44 (m, 2H), 3.41 (d, J=12.5Hz, 1H), 3.25-3.18 (m, 3H), 3.02-2.91 (m, 3H), 2.78 (t, 3 H), 2.63-2.57(m, 1H), 2.34(s, 3H), 1.98-1.90(m, 2H), 1.84(d, J=13.3Hz, 2H), 1.19(t, J=7.5Hz, 3H).HRMS(ESI)for C 40 H 43 F3N9O5[M+H] + : calcd, 786.3334; found, 786.3337.

[0183] Example 10

[0184] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(3-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-10)

[0185] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 3-oxa-9-azaspiro[5.5]undecane hydrochloride to prepare compound I-10: 1H NMR (400MHz, DMSO-d6) δ10.38 (s, 1H), 9.99 (s, 1H), 8.11-8.01 (m, 1H), 7.70 (d, J=8.3Hz, 1 H), 7.63 (s, 1H), 7.54 (d, J = 7.8Hz, 1H), 7.36-7.21 (m, 2H), 5.13 (s, 2H), 4.54 (d, J = 12.6Hz , 1H), 3.63-3.36 (m, 10H), 3.25-3.15 (m, 1H), 3.05-2.87 (m, 3H), 2.76 (d, J=10.5Hz, 1H), 2 .58(d, J=9.7Hz, 1H), 2.35(s, 3H), 1.55-1.40(m, 8H), 1.17(t, J=7.4Hz, 3H).HRMS(ESI)for C 36 H 43 F3N9O5[M+H] + :calcd, 738.3334; found, 738.3336.

[0186] Example 11

[0187] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(2-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-11)

[0188] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 2-oxa-9-azaspiro[5.5]undecane hydrochloride to prepare compound I-11: 1 H NMR (400MHz, DMSO-d6) δ10.38 (s, 1H), 9.99 (s, 1H), 8.10-8.03 (m, 1H), 7.70 (d, J=8.4Hz, 1 H), 7.63 (s, 1H), 7.54 (d, J = 8.5Hz, 1H), 7.33-7.26 (m, 2H), 5.13 (s, 2H), 4.54 (d, J = 12.0Hz, 1H), 3.62-3.35(m, 10H), 3.27-3.15(m, 1H), 3.03-2.84(m, 3H), 2.76(d, J=10.6Hz, 1H), 2. 58(d, J=10.9Hz, 1H), 2.35(s, 3H), 1.58-1.36(m, 8H), 1.17(t, J=7.4Hz, 3H).HRMS(ESI)for C 36H 43 F3N9O5[M+H] + :calcd, 738.3334; found, 738.3334.

[0189] Example 12

[0190] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(3-azaspiro[5.5]undec-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-12)

[0191] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 3-azaspiro-[5.5]-undecane hydrochloride to prepare compound I-12: 1 H NMR (400MHz, DMSO-d6) δ10.38 (s, 1H), 9.98 (s, 1H), 8.09-8.03 (m, 1H), 7.74-7.67 (m, 1H) , 7.63 (s, 1H), 7.54 (d, J = 8.9Hz, 1H), 7.34-7.25 (m, 2H), 5.13 (s, 2H), 4.54 (d, J = 12.3Hz, 1 H), 3.57-3.35 (m, 7H), 3.24-3.14 (m, 1H), 3.04-2.89 (m, 3H), 2.76 (d, J=10.7Hz, 1H), 2.58 (d, J=10.5Hz, 1H), 2.35 (s, 3H), 1.62-1.22 (m, 14H), 1.17 (t, J=7.4Hz, 3H).HRMS (ESI) for C 37 H 45 F3N9O4[M+H] + :calcd, 736.3541; found, 736.3540.

[0192] Example 13

[0193] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(6-oxa-2-azaspiro[4.5]decan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-13)

[0194] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 6-oxa-2-azaspiro[4.5]decane hydrochloride to prepare compound I-13: 1 H NMR (600MHz, DMSO-d6) δ10.39 (s, 1H), 9.99 (s, 1H), 8.08-8.04 (m, 1H), 7.72 (d, J=8.5Hz, 1H), 7.65-7.62 (m, 1H), 7 .55-7.51(m, 1H), 7.32-7.27(m, 2H), 5.14(s, 2H), 4.54(d, J=12.4Hz, 1H), 3.67-3.58(m, 2H), 3.54-3.43(m, 4H), 3. 43-3.37 (m, 2H), 3.25-3.17 (m, 2H), 3.00-2.89 (m, 3H), 2.76 (d, J = 11.2Hz, 1H), 2.58 (d, J = 11.0Hz, 1H), 2.36 (s, 3H) , 2.17-2.11(m, 1H), 1.87-1.79(m, 1H), 1.67-1.50(m, 4H), 1.48-1.44(m, 2H), 1.17(t, J=7.5Hz, 3H).HRMS(ESI)for C 35 H 41 F3N9O5[M+H] + : calcd, 724.31 77; found, 724.3173.

[0195] Example 14

[0196] 2-(5-ethyl-6-(4-(3-hydroxy-6-methylpicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-14)

[0197] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 3-hydroxy-2-pyridinecarboxylic acid was replaced with 3-hydroxy-6-methylpyridine-2-carboxylic acid to prepare compound I-14: 1H NMR (600MHz, DMSO-d6) δ10.08 (s, 1H), 9.98 (s, 1H), 7.74-7.66 (m, 1H), 7.63 (s, 1H), 7.53 (d, J=8.4Hz, 1H), 7.21-7 .16 (m, 1H), 7.12 (d, J = 8.4Hz, 1H), 5.13 (s, 2H), 4.52 (d, J = 11.9Hz, 1H), 3.75-3.65 (m, 2H), 3.62-3.55 (m, 2H), 3.5 2-3.40 (m, 3H), 3.24-3.14 (m, 3H), 2.99-2.86 (m, 3H), 2.75 (d, J = 10.5Hz, 1H), 2.58 (d, J = 10.0Hz, 1H), 2.37 (s, 3H) , 2.34 (s, 3H), 1.83 (d, J = 13.2Hz, 2H), 1.61-1.54 (m, 2H), 1.47-1.36 (m, 6H), 1.16 (t, J = 7.5Hz, 3H). HRMS (ESI) for C 37 H 45 F3N9O5[M+H] + : calcd, 752.3490; found, 752.3491.

[0198] Example 15

[0199] 2-(5-ethyl-6-(4-(5-hydroxy-6-methoxypyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-15)

[0200] Synthesis of compound D-16

[0201] Referring to the synthesis method of intermediate D-9, intermediate D-16 was prepared by replacing 2-oxa-6-azaspiro[3,3]heptane with 1-oxa-9-azaspiro[5.5]undecane hydrochloride.

[0202] Synthesis of compound D-17

[0203] Intermediate D-16 (40 mg, 0.065 mmol), 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid (12.4 mg, 0.07 mmol), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH) (22 mg, 0.078 mmol), N-methylimidazole (NMI) (19 mg, 0.228 mmol), and acetonitrile (2 mL) were mixed and stirred at room temperature for 1 hour. After completion of the reaction, the reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol = 15:1) to obtain compound D-17 (white solid, 48 mg).

[0204] Synthesis of compound I-15

[0205] Intermediate D-17 (45 mg, 0.052 mmol) was dissolved in TFA (1 mL) and stirred at 50°C for 10 hours. After the reaction, TFA was evaporated under reduced pressure, and the residue was diluted with saturated sodium bicarbonate (10 mL). The reaction mixture was extracted with EA (20 mL x 3). The organic phase was washed with saturated brine (20 mL x 1), dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol = 20:1) to obtain compound I-15 (white solid, 21 mg): 1 H NMR (600MHz, DMSO-d6) 610.24 (s, 1H), 9.98 (s, 1H), 8.32-8.29 (m, 1H), 7.69 (d, J=8.5Hz, 1H), 7.63 (s, 1H), 7 .56-7.51(m, 1H), 5.13(s, 2H), 4.49(d, J=12.5Hz, 1H), 3.99(s, 3H), 3.75-3.68(m, 2H), 3.62-3.52(m, 2H), 3 .49-3.36 (m, 3H), 3.25-3.12 (m, 3H), 2.99-2.87 (m, 3H), 2.76 (d, J = 10.5Hz, 1H), 2.58 (d, J = 10.8Hz, 1H), 2.3 4(s, 3H), 1.83(d, J=13.5Hz, 2H), 1.62-1.54(m, 2H), 1.48-1.34(m, 6H), 1.16(t, J=7.5Hz, 3H).HRMS(ESI)for C 36 H 44 F3N 10 O6[M+H] + : calcd, 769.3392; found, 769.3395.

[0206] Example 16

[0207] 2-(5-ethyl-6-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-16)

[0208] Referring to the method of Example 15, 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid was replaced with 5-(benzyloxy)-6-methylpyrimidine-4-carboxylic acid to prepare compound I-16: 1 H NMR (600MHz, DMSO-d6) δ10.23 (s, 1H), 9.98 (s, 1H), 8.57 (s, 1H), 7.69 (d, J=8.6Hz, 1H), 7.63 (s, 1H), 7.53 (d, J=8 .3Hz, 1H), 5.13 (s, 2H), 4.51 (d, J=12.5Hz, 1H), 3.71 (d, J=12.8Hz, 2H), 3.62-3.54 (m, 2H), 3.54-3.41 (m, 3H), 3. 20 (dt, J=21.8, 11.7Hz, 3H), 3.01-2.88 (m, 3H), 2.77 (d, J=10.8Hz, 1H), 2.59 (d, J=11.4Hz, 1H), 2.44 (s, 3H), 2.3 4 (s, 3H), 1.83 (d, J = 13.3Hz, 2H), 1.58 (d, J = 4.2Hz, 2H), 1.48-1.36 (m, 6H), 1.16 (t, J = 7.5Hz, 3H). HRMS (ESI) for C 36 H 44 F3N 10 O5[M+H] + : calcd, 753.3443; found, 753.3441.

[0209] Example 17

[0210] 2-(6-(4-(6-cyclopropyl-3-hydroxypicolinyl)piperazin-1-yl)-5-ethyl-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-17)

[0211] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 3-hydroxy-2-pyridinecarboxylic acid was replaced with 6-cyclopropyl-3-hydroxypyridinecarboxylic acid to prepare compound I-17: 1 H NMR (600MHz, DMSO-d6) δ10.09 (s, 1H), 9.98 (s, 1H), 7.69 (d, J = 8.4Hz, 1H), 7.63 (s, 1H), 7.53 (d, J = 8.5Hz, 1H), 7.22-7.1 4(m, 2H), 5.13(s, 2H), 4.51(d, J=11.9Hz, 1H), 3.75-3.67(m, 2H), 3.63-3.55(m, 2H), 3.51-3.44(m, 3H), 3.21-3.13(m, 3 H), 2.99-2.87 (m, 3H), 2.74 (d, J=10.2Hz, 1H), 2.58 (d, J=10.7Hz, 1H), 2.34 (s, 3H), 2.04-1.98 (m, 1H), 1.83 (d, J=13.4H z, 2H), 1.63-1.53 ​​(m, 2H), 1.47-1.36 (m, 6H), 1.16 (t, J=7.5Hz, 3H), 0.90-0.84 (m, 2H), 0.82-0.73 (m, 2H).HRMS (ESI) for C 39 H 47 F3N9O5[M+H] + :calcd, 778.3647; found, 778.3646.

[0212] Example 18

[0213] 2-(5-ethyl-6-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-2-(1-oxo-8-azaspiro[4.5]decan-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-18)

[0214] Referring to the method of Example 15, 2-oxa-6-azaspiro[3,3]heptane was replaced with D-12, and 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid was replaced with 5-(benzyloxy)-6-methylpyrimidine-4-carboxylic acid to prepare compound I-18: 1H NMR (600MHz, DMSO-d6) δ10.22 (s, 1H), 9.98 (s, 1H), 8.57 (s, 1H), 7.68 (d, J=8.4Hz, 1H), 7.62 (s, 1H), 7.53 (d, J=8.2H z, 1H), 5.13 (s, 2H), 4.51 (d, J=11.5Hz, 1H), 3.91-3.83 (m, 2H), 3.55-3.47 (m, 3H), 3.26-3.20 (m, 1H), 3.17-3.10 (m, 2 H), 3.02-2.89 (m, 3H), 2.78 (d, J = 10.4Hz, 1H), 2.62-2.58 (m, 1H), 2.44 (s, 3H), 2.33 (s, 3H), 2.26 (t, J = 7.6Hz, 2H), 1 .96-1.91(m, 2H), 1.89-1.82(m, 2H), 1.55-1.47(m, 2H), 1.37(d, J=13.4Hz, 2H), 1.17(t, J=7.5Hz, 3H).HRMS(ESI)for C 36 H 42 F3N 10 O5[M+H] + :calcd, 751.3286; found, 751.3286.

[0215] Example 19

[0216] N-(5-chloro-2-methyl-4-(trifluoromethyl)phenyl)-2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Compound I-19)

[0217] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 5-chloro-2-methyl-4-trifluoromethylaniline to prepare compound I-19: 1H NMR (400MHz, DMSO-d6) δ10.38 (s, 1H), 10.08 (s, 1H), 8.06 (dd, J=3.4, 2.4Hz, 1H), 7.94-7.89 (m, 1H), 7.76 (s, 1H), 7.30-7.26 (m, 2H), 5.15 (s, 2H), 4.53 (d, J=12.1Hz, 1H), 3.70 (d, J=12.9Hz, 2H), 3.62-3.54 (m, 2H), 3.54-3.42 (m, 2H), 3.39 (d, J=12.7Hz, 1H), 3.23-3.12 (m, 3H), 3.02-2.87 (m, 3H), 2.76 (d, J=10.9Hz, 1H), 2.58 (d, J=10.8Hz, 1H) , 2.34 (s, 3H), 1.83 (d, J = 13.4Hz, 2H), 1.62-1.53 ​​(m, 2H), 1.49-1.33 (m, 6H), 1.15 (t, J = 7.5Hz, 3H). HRMS (ESI) for C 36 H 42 ClF3N9O5[M+H] + :calcd, 772.2944; found, 772.2941.

[0218] Example 20

[0219] N-(4-cyclopropylphenyl)-2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Compound I-20)

[0220] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 4-cyclopropylaniline to prepare compound I-20: 1H NMR (400MHz, DMSO-d6) δ10.38 (s, 1H), 10.33 (s, 1H), 8.06 (dd, J=3.3, 2.5Hz, 1H), 7.42 (d, J=8.6Hz, 2H), 7.31-7.27 (m, 2H), 7.02 (d, J=8.6H z, 2H), 5.01 (s, 2H), 4.53 (d, J = 11.8Hz, 1H), 3.70-3.64 (m, 2H), 3.59-3.54 (m, 2H), 3.47 (dd, J = 26.4, 12.2Hz, 2H), 3.39 (d, J = 12.3Hz, 1H), 3 .19 (t, J=11.1Hz, 1H), 3.17-3.10 (m, 2H), 2.96-2.83 (m, 3H), 2.75 (d, J=10.5Hz, 1H), 2.62-2.53 (m, 1H), 1.88-1.84 (m, 1H), 1.84-1.77 (m, 2 H), 1.56 (d, J = 4.5Hz, 2H), 1.47-1.40 (m, 3H), 1.40-1.35 (m, 4H), 1.13 (t, J = 7.5Hz, 3H), 0.92-0.88 (m, 2H), 0.62-0.58 (m, 2H). HRMS (ESI) for C 37 H 46 N9O5[M+H] + :calcd, 696.3616; found, 696.3617.

[0221] Example 21

[0222] N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Compound I-21)

[0223] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 2-chloro-4-trifluoromethylaniline to prepare compound I-21: 1H NMR (400MHz, DMSO-d6) δ10.46 (s, 1H), 10.33 (s, 1H), 8.08 (dd, J=3.8, 1.9Hz, 1H), 8.03 (d, J=8.6Hz, 1H), 7.97 (d, J=1.4Hz, 1H), 7. 75-7.69 (m, 1H), 7.34-7.28 (m, 2H), 5.20 (s, 2H), 4.53 (d, J=11.8Hz, 1H), 3.70 (dd, J=9.2, 3.7Hz, 3H), 3.57 (t, 2H), 3.47 (dd, J=25 .3, 12.2Hz, 2H), 3.40 (d, J = 12.5Hz, 1H), 3.24-3.11 (m, 3H), 2.98-2.87 (m, 3H), 2.76 (d, J = 10.4Hz, 1H), 2.58 (d, J = 10.5Hz, 1H), 1. 83 (d, J = 13.3Hz, 2H), 1.57 (d, J = 4.5Hz, 2H), 1.47-1.42 (m, 3H), 1.39 (dd, J = 12.5, 6.5Hz, 3H), 1.16 (t, J = 7.5Hz, 3H). HRMS (ESI) for C 35 H 40 ClF3N9O5[M+H] + :calcd,758.2788; found.758.2789.

[0224] Example 22

[0225] 2-(5-ethyl-6-(4-(3-hydroxypyridinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(4-(trifluoromethoxy)phenyl)acetamide (Compound I-22)

[0226] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 4-(trifluoromethoxy)aniline to prepare compound I-22: 1H NMR (400MHz, DMSO-d6) δ10.65 (t, 1H), 10.38 (s, 1H), 8.06 (dd, J=3.4, 2.4Hz, 1H), 7.71-7.62 (m, 2H), 7.34 (d, J=8.7Hz, 2H ), 7.31-7.26 (m, 2H), 5.05 (s, 2H), 4.53 (d, J=11.6Hz, 1H), 3.67 (dd, J=9.0, 3.8Hz, 2H), 3.56 (t, 2H), 3.52-3.42 (m, 2H), 3. 39 (d, J=12.1Hz, 1H), 3.20 (t, 1H), 3.17-3.11 (m, 2H), 2.98-2.83 (m, 3H), 2.76 (d, J=10.3Hz, 1H), 2.58 (d, J=10.3Hz, 1H), 1.81 (d, J=13.3Hz, 2H), 1.56 (d, J=4.4Hz, 2H), 1.48-1.40 (m, 3H), 1.39-1.34 (m, 3H), 1.14 (t, J=7.5Hz, 3H).HRMS (ESI) for C 35 H 41 F3N9O6[M+H] + :calcd, 740.3126; found, 740.3126.

[0227] Example 23

[0228] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(4-(trifluoromethyl)phenyl)acetamide (Compound I-23)

[0229] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 4-(trifluoromethyl)aniline to prepare compound I-23: 1H NMR (400MHz, DMSO-d6) δ10.82 (t, 1H), 10.43 (s, 1H), 8.07 (dd, J=3.6, 2.2Hz, 1H), 7.77 (d, J=8.6Hz, 2H), 7.70 (d, J=8.7Hz, 2H) , 7.30 (d, J=3.9Hz, 2H), 5.08 (s, 2H), 4.53 (d, J=11.9Hz, 1H), 3.70-3.63 (m, 3H), 3.47 (d, J=14.7Hz, 2H), 3.40 (d, J=12.5Hz, 1H ), 3.21 (t, J=11.9Hz, 1H), 3.16-3.10 (m, 2H), 2.97-2.85 (m, 3H), 2.76 (d, J=10.4Hz, 1H), 2.58 (d, J=10.7Hz, 1H), 1.80 (d, J=13 .2Hz, 2H), 1.56 (d, J=4.5Hz, 2H), 1.48-1.39 (m, 3H), 1.37 (d, J=5.7Hz, 3H), 1.24 (s, 1H), 1.14 (t, J=7.5Hz, 3H).HRMS (ESI) for C 35 H 41 F3N9O5[M+H] + : calcd, 724.3177; found, 724.3176.

[0230] Example 24

[0231] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(3-(trifluoromethyl)phenyl)acetamide (Compound I-24)

[0232] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 3-(trifluoromethyl)aniline to prepare compound I-24: 1H NMR (600MHz, DMSO-d6) δ10.81 (s, 1H), 10.38 (s, 1H), 8.06 (dd, J=3.5, 2.4Hz, 2H), 7.73 (d, J=8.2Hz, 1H), 7.63-7.56 (m, 1H), 7.4 5 (d, J=7.8Hz, 1H), 7.33-7.25 (m, 2H), 5.07 (s, 2H), 4.53 (d, J=12.1Hz, 1H), 3.67 (dd, J=9.0, 3.8Hz, 2H), 3.56 (t, 2H), 3.52-3.4 2 (m, 2H), 3.40 (d, J = 12.9Hz, 1H), 3.20 (t, 1H), 3.17-3.10 (m, 2H), 2.98-2.86 (m, 3H), 2.76 (d, J = 10.7Hz, 1H), 2.59 (d, J = 10.5Hz , 1H), 1.80 (d, J=13.3Hz, 2H), 1.56 (d, J=4.9Hz, 2H), 1.48-1.40 (m, 3H), 1.39-1.34 (m, 3H), 1.14 (t, J=7.5Hz, 3H).HRMS (ESI) for C 35 H 41 F3N9O5[M+H] + : calcd, 724.3177; found, 724.3177.

[0233] Example 25

[0234] 2-(6-(4-(6-cyclopropyl-5-hydroxypyrimidine-4-carbonyl)piperazin-1-yl)-5-ethyl-7-oxo-2-(1-oxo-8-azaspiro[4.5]decan-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-25)

[0235] Referring to the method of Example 15, 2-oxa-6-azaspiro[3,3]heptane was replaced with D-12, and 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid was replaced with 5-(benzyloxy)-6-cyclopropylpyrimidine-4-carboxylic acid to prepare compound I-25: 1H NMR (600MHz, DMSO-d6) δ10.32 (s, 1H), 9.98 (s, 1H), 8.52 (s, 1H), 7.68 (d, J = 8.4Hz, 1H), 7.62 (s, 1H), 7.53 (d, J = 8.2Hz , 1H), 5.13 (s, 2H), 4.51 (d, J=11.3Hz, 1H), 3.94-3.83 (m, 2H), 3.56-3.50 (m, 3H), 3.26-3.21 (m, 1H), 3.16-3.09 (m, 2H) ), 3.01-2.90 (m, 3H), 2.78 (d, J=1O.4Hz, 1H), 2.65-2.58 (m, 1H), 2.33 (s, 3H), 2.26 (t, J=7.6Hz, 2H), 1.95-1.91 (m, 2H ), 1.89-1.83(m, 2H), 1.56-1.47(m, 2H), 1.41-1.33(m, 2H), 1.17(t, J=7.5Hz, 3H), 1.11-1.01(m, 4H).HRMS(ESI)forC 38 H 44 F3N 10 O5[M+H] + :calcd, 777.3443; found, 777.3446.

[0236] Example 26

[0237] 2-(5-ethyl-6-(4-(5-hydroxy-6-methoxypyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-2-(1-oxo-8-azaspiro[4.5]decan-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-26)

[0238] Referring to the method of Example 15, 2-oxa-6-azaspiro[3,3]heptane was replaced with D-12, and 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid was replaced with 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid to prepare compound I-26: 1H NMR (600MHz, DMSO-d6) δ10.24 (s, 1H), 9.98 (s, 1H), 8.30 (s, 1H), 7.68 (d, J=8.5Hz, 1H), 7.62 (s, 1H), 7.53 (d, J=8.4H z, 1H), 5.13 (s, 2H), 4.49 (d, J=11.9Hz, 1H), 3.99 (s, 3H), 3.92-3.82 (m, 2H), 3.51-3.36 (m, 3H), 3.24-3.17 (m, 1H), 3. 16-3.10 (m, 2H), 3.00-2.87 (m, 3H), 2.77 (d, J=10.6Hz, 1H), 2.59 (d, J=11.0Hz, 1H), 2.33 (s, 3H), 2.26 (t, J=7.6Hz, 2H ), 1.97-1.91(m, 2H), 1.88-1.82(m, 2H), 1.54-1.47(m, 2H), 1.40-1.33(m, 2H), 1.16(t, J=7.5Hz, 3H).HRMS(ESI)forC 36 H 42 F3N 10 O6[M+H] + : calcd, 767.3235; found, 767.3239.

[0239] Example 27

[0240] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxo-2-oxa-8-azaspiro[4.5]dec-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-27)

[0241] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 2-oxa-8-azaspiro[4.5]decan-1-one to prepare compound I-27: 1H NMR (600MHz, DMSO-d6) δ10.38 (s, 1H), 9.98 (s, 1H), 8.08-8.03 (m, 1H), 7.69 (d, J=8.6Hz, 1H), 7.64-7.61 (m, 1H) ), 7.55-7.51(m, 1H), 7.29-7.28(m, 2H), 5.14(s, 2H), 4.53(d, J=12.4Hz, 1H), 4.31(t, J=7.0Hz, 2H), 3.96-3.90 (m, 2H), 3.51-3.36 (m, 3H), 3.24-3.13 (m, 3H), 2.99-2.90 (m, 3H), 2.76 (d, J=11.2Hz, 1H), 2.62-2.56 (m, 1H), 2 .34(s, 3H), 2.25(t, J=7.0Hz, 2H), 1.72-1.65(m, 2H), 1.64-1.58(m, 2H), F1.17(t, J=7.5Hz, 3H).HRMS(ESI)for C 35 H 38 F3N9O6[M+H] + :calcd, 737.2970; found, 737.2969.

[0242] Example 28

[0243] 2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(4-methoxyphenyl)acetamide (Compound I-28)

[0244] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 4-methoxyaniline to prepare compound I-28: 1H NMR (600MHz, DMSO-d6) δ10.47 (s, 1H), 10.28 (s, 1H), 8.11-8.03 (m, 1H), 7.46 (d, J=9.1Hz, 2H), 7.35-7.27 (m, 2H), 6.89 (d, J= 9.1Hz, 2H), 5.01 (s, 2H), 3.72 (s, 3H), 3.70-3.65 (m, 2H), 3.57 (t, 2H), 3.47 (dd, J=25.7, 12.2Hz, 2H), 3.40 (d, J=12.4Hz, 1H), 3.20 (t, J=11.2Hz, 1H), 3.14 (t, J=17.4, 6.7Hz, 2H), 2.97-2.83 (m, 3H), 2.76 (d, J=10.7Hz, 1H), 2.58 (d, J=10.5Hz, 1H), 1.82 ( d, J=13.3Hz, 2H), 1.60-1.54(m, 2H), 1.47-1.41(m, 3H), 1.39-1.35(m, 3H), 1.24(s, 1H), 1.14(t, J=7.5Hz, 3H).HRMS(ESI)for C 35 H 44 N9O6[M+H] + :calcd, 686.3409; found, 686.341.

[0245] Example 29

[0246] N-(Benzo[d][1,3]dioxol-5-yl)-2-(5-ethyl-6-(4-(3-hydroxypyridinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Compound I-29)

[0247] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with benzo[d][1,3]dioxol-5-amine to prepare compound I-29: 1H NMR (600MHz, DMSO-d6) δ10.50 (d, 1H), 10.35 (s, 1H), 8.11-8.05 (m, 1H), 7.36-7.29 (m, 2H), 7.23 (s, 1H), 6.95-6.90 (m, 1H), 6.8 7 (d, J=8.4Hz, 1H), 5.98 (s, 2H), 5.00 (s, 2H), 3.71-3.63 (m, 2H), 3.57 (t, 2H), 3.47 (dd, J=25.7, 12.2Hz, 2H), 3.40 (d, J=12.2Hz , 1H), 3.20 (t, J = 11.2Hz, 1H), 3.17-3.10 (m, 2H), 2.98-2.83 (m, 3H), 2.76 (d, J = 10.7Hz, 1H), 2.58 (d, J = 10.7Hz, 1H), 1.82 (d, J = 13.3Hz, 2H), 1.61-1.53(m, 2H), 1.48-1.41(m, 3H), 1.40-1.34(m, 3H), 1.28-1.20(m, 1H), 1.13(t, J=7.5Hz, 3H).HRMS(ESI)for C 35 H 42 N9O7[M+H] + :calcd, 700.3202; found, 700.3204.

[0248] Example 30

[0249] N-(2-chloro-4-(pentafluoro-λ 6 -thio)phenyl)-2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Compound I-30)

[0250] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 2-chloro-4-(pentafluorosulfanyl)aniline to prepare compound I-30: 1H NMR (600MHz, DMSO-d6) δ10.46 (s, 1H), 10.38 (s, 1H), 8.15 (d, J=2.6Hz, 1H), 8.11-8.04 (m, 2H), 7.91 (dd, J=9.2, 2.6H z, 1H), 7.35-7.26 (m, 2H), 5.21 (s, 2H), 4.53 (d, J=11.8Hz, 1H), 3.71-3.69 (m, 2H), 3.60-3.54 (m, 2H), 3.51-3.42 (m, 2H), 3.39 (d, J=12.4Hz, 1H), 3.25-3.19 (m, 1H), 3.18-3.11 (m, 2H), 3.01-2.85 (m, 3H), 2.76 (d, J=10.5Hz, 1H), 2.58 ( d, J=10.6Hz, 1H), 1.82 (d, J=13.3Hz, 2H), 1.61-1.53 ​​(m, 2H), 1.48-1.34 (m, 6H), 1.15 (t, J=7.5Hz, 3H).HRMS (ESI) for C 34 H 40 ClF5N9O5S[M+H] + :calcd, 81 6.2476; found, 816.2474.

[0251] Example 31

[0252] N-(4-cyclohexylphenyl)-2-(5-ethyl-6-(4-(3-hydroxypicolinyl)piperazin-1-yl)-7-oxo-2-(1-oxa-9-azaspiro[5.5]undec-9-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Compound I-31)

[0253] Referring to the method of Example 1, 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-9-azaspiro[5.5]undecane hydrochloride, and 2-methyl-4-trifluoromethylaniline was replaced with 4-cyclohexylaniline to prepare compound I-31: 1H NMR (600MHz, DMSO-d6) δ10.39 (s, 1H), 10.34 (s, 1H), 8.06 (dd, J=3.4, 2.4Hz, 1H), 7.45 (d, J=8.5Hz, 2H), 7.31-7.26 (m, 2H), 7.16 (d, J=8.5Hz , 2H), 5.06-4.98 (m, 2H), 4.53 (d, J=11.8Hz, 1H), 3.67 (d, J=12.9Hz, 2H), 3.58-3.54 (m, 2H), 3.47 (dd, J=27.4, 13.0Hz, 2H), 3.20 (t, J=11.2H z, 1H), 3.13 (dd, J=17.6, 6.8Hz, 2H), 2.96-2.83 (m, 3H), 2.75 (d, J=10.7Hz, 1H), 2.58 (d, J=10.9Hz, 1H), 2.46-2.40 (m, 1H), 1.81 (t, J=11.6H z, 2H), 1.78-1.72 (m, 4H), 1.69 (d, J=12.0Hz, 1H), 1.57 (s, 2H), 1.46-1.31 (m, 10H), 1.26-1.18 (m, 2H), 1.13 (t, J=7.5Hz, 3H).HRMS (ESI) for C 40 H 52 N9O5[M+H] + :calcd, 738.4086; found, 738.4085.

[0254] Example 32

[0255] 2-(5-ethyl-6-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-2-(2-oxo-1-oxa-8-azaspiro)[4.5]dec-8-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-32)

[0256] Referring to the method of Example 15, 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid was replaced with 5-(benzyloxy)-6-methylpyrimidine-4-carboxylic acid, and 2-oxa-6-azaspiro[3,3]heptane was replaced with 1-oxa-8-azaspiro[4.5]decan-2-one hydrochloride to prepare compound I-32: 1H NMR (600MHz, DMSO-d6) δ10.23 (s, 1H), 10.00 (s, 1H), 8.57 (s, 1H), 7.69 (d, J=8.5Hz, 1H), 7.63 (s, 1H), 7.54 ( d, J=8.5Hz, 1H), 5.14 (s, 2H), 4.51 (d, J=12.4Hz, 1H), 4.06-3.99 (m, 1H), 3.72-3.67 (m, 3H), 3.53-3.42 (m, 3H) ), 3.40-3.35(m, 2H), 3.24-3.21(m, 1H), 2.99-2.93(m, 3H), 2.77(d, J=11.2Hz, 1H), 2.64-2.58(m, 3H), 2.44( s, 3H), 2.34 (s, 3H), 2.03 (t, J=8.2Hz, 2H), 1.99 (s, 1H), 1.84-1.78 (m, 2H), 1.78-1.71 (m, 2H).HRMS (ESI) for C 35 H 40 F3N 10 O6[M+H] + :calcd, 753.3079; found, 753.3075.

[0257] Example 33

[0258] 5-Ethyl-6-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-2-(1-oxaspiro[5.5]undecen-8-en-9yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl(2-methyl-4-(trifluoromethyl)phenyl)carbamate (Compound I-33)

[0259] Referring to the methods of Examples 8 and 15, 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid was replaced with 5-(hydroxy)-6-methylpyrimidine-4-carboxylic acid, and 1-oxaspiro[4.5]dec-7-ene was replaced with 1-oxa-8-azaspiro[4.5]dec-2-one hydrochloride to prepare compound I-33: 1H NMR (400MHz, DMSO-d6) δ10.24 (s, 1H), 10.01 (s, 1H), 8.58 (s, 1H), 7.72 (d, J=8.4Hz, 1H), 7.63 (s, 1H) , 7.56-7.49 (m, 1H), 6.69 (s, 1H), 5.24 (s, 2H), 4.52 (d, J = 12.4Hz, 1H), 3.60-3.42 (m, 5H), 3.31-3.16 ( m, 1H), 3.05-2.91 (m, 3H), 2.81 (d, J=11.3Hz, 1H), 2.70-2.59 (m, 1H), 2.44 (s, 5H), 2.36 (s, 3H), 2.29 ( s, 2H), 2.10-2.02 (m, 1H), 1.76-1.51 (m, 3H), 1.49-1.39 (m, 4H), 1.19 (t, J=7.4Hz, 3H).HRMS (ESI) for C 37 H 43 F3N9O5[M+H] + :calcd, 750.3334; found, 750.3334.

[0260] Example 34

[0261] 2-(5-ethyl-6-(4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl)-7-oxo-2-(1-oxo-1,3-dihydrospiro[indene]-2,4′-piperidin]-1′-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)-N-(2-methyl-4-(trifluoromethyl)phenyl)acetamide (Compound I-34)

[0262] Referring to the methods of Examples 8 and 15, 5-(benzyloxy)-6-methoxypyrimidine-4-carboxylic acid was replaced with 5-(hydroxy)-6-methylpyrimidine-4-carboxylic acid, and 1-oxaspiro[4.5]dec-7-ene was replaced with spiro[indene-2,4'-piperidinyl]-1(3H)-one hydrochloride to prepare compound I-34: 1H NMR (400MHz, DMSO-d6) δ9.99 (s, 1H), 8.58 (s, 1H), 7.76-7.65 (m, 3H), 7.63-7.59 (m, 2H), 7.54-7.51 (m, 1H), 7.48-7.38 (m, 2H), 5.16 (d, J=7.4Hz, 2H), 4.53-4.49 (m, 1H), 4.13-4.07 (m, 2H), 3.54-3.46 (m, 2H), 3.28-3.12(m, 5H), 3.01-2.94(m, 3H), 2.79(d, J=11.1Hz, 1H), 2.65-2.59(m, 1H), 2.44(s, 3H), 2.33(s, 3H), 1.78-1.71(m, 2H), 1.40(d, J=13.0Hz, 2H), 1.29-1.23(m, 1H), 1.18(t, J=7.5Hz, 3H).HRMS(ESI)for C 40 H 42 F3N 10 O5[M+H] + :calcd,799.3286; found.799.3286.

[0263] Test Example 1: Determination of the half inhibition rate IC of the compound on WRN helicase activity 50

[0264] Experimental principle: WRN is an ATP-dependent helicase that consumes ATP and generates ADP during the unwinding process. TM ADP-Glo ​​in Kinase Assay Kit TM The reagent terminates the reaction and consumes any remaining ATP. The Kinase Detection Reagent then converts the product, ADP, into ATP, which is then detected via the luciferase / luciferin reaction. The resulting light signal correlates with the amount of ADP produced in the reaction, reflecting WRN helicase activity and, therefore, determining the compound's inhibitory effect on WRN helicase.

[0265] Experimental reagents and materials: Tris-HCl (Sinopharm Reagent), NaCl (Sinopharm Reagent), MgCl2 (Sinopharm Reagent), dithiothreitol (DTT) (Sinopharm Reagent), Tween-20 (Sinopharm Reagent), isopropyl-β-D-thiogalactopyranoside (IPTG) (MCE), PMSF (Meilun Biotechnology), HEPES (Sangong Biotechnology), ATP (Biode Pharmaceuticals), glycerol (Sinopharm Reagent), reduced glutathione (GSH) (Sangong Biotechnology), GSTrap (Cytiva), Superdex 200increase10 / 300GL (Cytiva), calfthymus DNA (MCE), ADP-Glo TM Kinase Assay kit (Promega), 384-well black microplate (Corning), OptiPlateTM-384-well white plate (Perkin Elmer).

[0266] Experimental methods:

[0267] (1) Protein purification: GST-WRN 500-946 The recombinant protein (Uniprot ID: Q14191, human) was expressed in Escherichia coli BL21(DE3) strain. Cells were cultured at 37°C and induced overnight with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 18°C. Cells were harvested by centrifugation at 3000 rpm for 30 min and lysed by sonication in lysis buffer (50 mM HEPES, 600 mM NaCl, 5% glycerol, 1 mM TCEP, pH 7.4) supplemented with 1 mM PMSF. After centrifugation, the supernatant was loaded onto a GSTrap column and eluted with elution buffer (50 mM HEPES, 600 mM NaCl, 5% glycerol, 10 mM GSH, 1 mM TCEP, pH 7.4). The flow-through was collected, concentrated, and further purified using Superdex 200 increase 10 / 300 GL (buffer: 50 mM HEPES, 600 mM NaCl, pH 7.4). The purified protein was concentrated, and after the protein concentration was determined, the protein was aliquoted and frozen in a -80°C refrigerator for storage.

[0268] (2) Prepare reaction buffer: 25 mM Tris-HCl (pH 8.0), 5 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol (DTT), 0.01% Tween-20, 2.5 μg / mL calf thymus DNA.

[0269] (3) Detection: Add 28 μL of GST-WRN with a final concentration of 200 nM to a 384-well black plate.500-946 , add 2 μL of serially diluted compounds and incubate at room temperature for 15 minutes, then add 5 μL of double-stranded DNA (OLIGO-A: TTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTACCCGATGTGTTCGTTC (SEQ ID NO: 1); OLIGO-B: GAACGAACACATCGGGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (SEQ ID NO: 2)) with a final concentration of 300 nM and 5 μL of ATP with a final concentration of 3 mM, shake the reaction for 3 hours, and mix the reaction system. Dilute the reaction system 5000 times with reaction buffer, take 10 μL of the diluted reaction system and add it to the OptiPlate TM - Add 10 μL ADP-Glo ​​to a 384-well white plate TM Reagent, centrifuge at 1000 rpm for 1 min, incubate at room temperature for 40 min, add 20 μL Kinase Detection Reagent (protect from light), centrifuge at 1000 rpm for 1 min, incubate in the dark for 40 min, and use a multifunctional microplate reader to perform chemiluminescence detection.

[0270] (4) Data processing: The WRN helicase activity was calculated based on the chemiluminescence readings, and the half-maximal inhibition rate (IC) of the compound on the WRN helicase activity was determined by curve fitting with a variable slope (four parameters) nonlinear regression in Graphpad Prism (8.0.1). 50 ).

[0271] Experimental results: The results of the inhibitory activity of the compounds on WRN helicase are shown in Table 1.

[0272] Test Example 2: Evaluation of the selective proliferation inhibitory activity of compounds on HCT116 and DLD-1 cells

[0273] Principle: Microsatellite instability-high (MSI-H) cancer cells are specifically dependent on WRN. WRN inactivation selectively kills MSI-H cancer cells, while having no significant effect on microsatellite stable (MSS) cancer cell lines. HCT-116 is a microsatellite instability-high cell line, while DLD-1 is a microsatellite stable cell line. By measuring cell viability, the selectivity of compounds in inhibiting proliferation against MSI-H and MSS cells can be evaluated. CellTiter-Glo (CTG) is a rapid cell viability assay based on ATP detection. ATP is present in living cells, and its level directly reflects cell number and cell status. The CTG detection reagent contains recombinant luciferase and luciferin. The detection reagent automatically lyses cells, releasing ATP from living cells and preventing ATP degradation. ATP in living cells participates in the luciferase reaction, and the resulting fluorescence intensity is proportional to the ATP content. ATP content is positively correlated with viable cell count, thus measuring cell viability.

[0274] Experimental reagents and materials: fetal bovine serum (Gibco), RPMI 1640 medium (Shanghai Yuanpei Biotechnology), McCoy's 5A medium (Shanghai Yuanpei Biotechnology), penicillin-streptomycin double antibody (Gibco), 0.25% Trypsin-EDTA (Meilun Biotechnology), CellTiter-Meiluncell luminescent cell viability detection kit (Meilun Biotechnology), HCT116 cells (Punosai), DLD-1 cells (Punosai), OptiPlateTM-96-well white bottom plate (Perkin Elmer).

[0275] Experimental methods:

[0276] (1) Cell plating: Trypsinize well-grown HCT 116 / DLD-1 cells for 3-5 minutes, then terminate the digestion with complete medium (McCoy's 5A / RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin-streptomycin). Centrifuge, resuspend, and count. Mix an appropriate number of cells with complete medium, and add 50 μL to a 96-well plate, seeding 2000 cells per well. Incubate in a 37°C, 5% CO2 incubator overnight.

[0277] (2) Cell administration: The test compound was prepared into a 20 mM stock solution, diluted to 20 μM with the corresponding cell culture medium, and then diluted 3-fold with a culture medium containing 0.2% DMSO to 6.67, 2.22, 0.74, 0.25, 0.082, 0.027, 0.0091, 0.0030, and 0 μM solutions. 50 μL was added to the corresponding cell plate, and the plate was cultured in an incubator for 72 hours before detection.

[0278] (3) Detection: Add 20 μL of CellTiter-Meiluncell luminescent cell viability detection reagent to each well of the cell culture plate, place on a shaker and mix for 10 min. After mixing, transfer 65 μL to an OptiPlateTM-96-well white bottom plate, read the luminescence value on a microplate reader and save it.

[0279] (4) Data processing: The inhibition rate of the compound on cell proliferation at a fixed concentration was calculated according to the following formula: inhibition rate of the compound at a certain concentration = (reading value of the DMSO well - reading value of the compound well at a certain concentration) / reading value of the DMSO well × 100%; the inhibition rate at each concentration of the compound was calculated, and the analysis and graphing were performed using GraphPad Prism (8.0.1) software to obtain the half inhibition rate (GI) of the compound in inhibiting cell proliferation. 50 ).

[0280] Experimental results: The selective proliferation inhibitory activity of the compounds on HCT116 and DLD-1 cells is shown in Table 1.

[0281] Table 1. Results of biological activity evaluation of compounds

[0282] Test Example 3: Compound-mediated WRN protein degradation in HCT116 cells

[0283] Experimental principle: Western Blot is used to detect the content of WRN protein in HCT116 cells after compound treatment, and the degradation effect of the compound is indirectly inferred based on the content of WRN protein.

[0284] Experimental reagents and materials: fetal bovine serum (Gibco), McCoy's 5A medium (Shanghai Yuanpei Biotechnology), penicillin-streptomycin dual antibody (Mei Lun Biotechnology), 0.25% Trypsin-EDTA (Mei Lun Biotechnology), HCT116 cells (Punosai), PBS buffer (Mei Lun Biotechnology), SDS lysis buffer (Biyuntian), phosphatase inhibitor cocktail (Selleck), protease inhibitor cocktail (Selleck), BCA quantitative reagent (Thermo Fisher), 5× SDS-PAGE protein loading buffer (Biyuntian), SDS-PAGE gel preparation kit (Mei Lun Biotechnology), prestained protein marker (Thermo Fisher), nitrocellulose membrane (Merck), skim milk (Yazyme), 20× TBS buffer (Mei Lun Biotechnology), Tween-20 (Sinopharm Reagent), primary antibody diluent (Biyuntian), secondary antibody diluent (Biyuntian), WRN monoclonal antibody (Cell Signaling Technology), β-tubulin antibody (Cell Signaling Technology), Anti-Mouse IgG (H+L) antibody (Promega), Anti-Rabbit IgG (H+L) antibody (Promega), and Fector ultrasensitive ECL luminescent solution (Meilun Biotechnology).

[0285] Experimental methods:

[0286] (1) Cell plating: HCT 116 cells in good growth condition were trypsinized for 3 minutes, and then digested with complete culture medium (McCoy's 5A medium + 10% fetal bovine serum + 1% penicillin-streptomycin). The cells were centrifuged and resuspended, and the cell number was counted. The cells were evenly plated at a density of 500,000 cells / well in a 12-well plate. The cell culture plate was shaken evenly and then placed in a 37°C, 5% CO2 incubator for overnight culture.

[0287] (2) Cell administration: Prepare the test compound into a 20 mM stock solution and serially dilute the compound with DMSO to 10, 1, and 0.1 mM solutions. Add appropriate amounts of the corresponding concentrations to the experimental wells to achieve final compound concentrations of 10, 1, and 0.1 μM. Then, add an equal volume of DMSO to the control wells. Incubate the cells in an incubator for 48 h before collecting the samples.

[0288] (3) Detection: Remove the cell culture plate, discard the supernatant, add PBS to wash the cells twice, then add 100 μL SDS lysis buffer to each well and aspirate the cell lysate into an EP tube. After boiling the sample in a 100℃ metal bath for 15 minutes, take 5 μL of sample for BCA protein quantification. After quantification, the total amount of protein loaded is kept consistent. All samples are loaded onto the prepared SDS-PAGE gel for electrophoresis separation (130V, 80 minutes), and then the protein blot is transferred to a nitrocellulose (NC) membrane using a wet transfer instrument. Next, block the NC membrane with 5% skim milk prepared in 1×TBST for 1 hour at room temperature, and then rinse the NC membrane three times with 1×TBST, each for 10 minutes. According to the position of the protein marker, the WRN protein and β-tubulin protein bands of corresponding molecular weight are cut, and the target bands are incubated with the corresponding primary antibody dilution (dilution ratio is 1:1000) at 4℃ overnight. The primary antibody is recovered the next day, and the NC membrane is rinsed three times with 1×TBST, each for 10 minutes. The strips were incubated with secondary antibody dilution (1:5000) at room temperature for 1 hour, after which the secondary antibody was recovered. The NC membrane was then rinsed three times with 1× TBST for 10 minutes each. Finally, the target strips were placed in the prepared Fecton ultrasensitive ECL luminescent solution and incubated for 10 seconds. The strips were then developed using a developer, and the images were saved and exported.

[0289] (4) Data processing: All developed images were labeled with compounds and concentrations, and the images were organized. Grayscale quantification was performed using Image J, and the degradation rate of the compound on WRN protein at a fixed concentration was calculated according to the following formula: Degradation rate of the compound at a certain concentration = (grayscale value of the DMSO well - grayscale value of the compound well at a certain concentration) / grayscale value of the DMSO well × 100%. The degradation rate of the compound at each concentration was then calculated and analyzed and plotted using the nonlinear regression curve fitting method with a variable slope (four parameters) in GraphPad Prism (8.0.1) software to obtain the half-degradation rate (DC) of the compound degrading WRN protein. 50 ).

[0290] Experimental results: The results of compound-mediated WRN protein degradation activity in HCT 116 cells are shown in Table 2.

[0291] Table 2. Compounds mediate WRN degradation activity

[0292] The experimental results (Tables 1 and 2) show that the spirocyclic compounds represented by Formula I of the present invention can significantly inhibit the helicase activity of WRN and also exhibit potent anti-cell proliferation activity against HCT 116 cells with MSI characteristics. In addition, some compounds can significantly mediate the degradation of WRN protein in HCT 116 cells. This shows that the spirocyclic compounds represented by Formula I of the present invention are potent WRN regulators and can inhibit the proliferation of cells with MSI characteristics by inhibiting WRN or mediating WRN degradation.

Claims

1. A spirocyclic compound of formula I, or a pharmaceutically acceptable salt, stereoisomer, deuterated form, solvate, prodrug, or metabolite thereof, in: Ring A is selected from: a benzene ring, a 5- to 6-membered heteroaromatic ring containing 1 to 3 heteroatoms selected from N, O, and S; Ring B is selected from a 5-7 membered saturated or partially saturated heterocyclic ring containing 1-2 heteroatoms selected from N, O, and S; L is selected from: -C(O)-, -S(O)-, -S(O)2-, X is CH or N; n is an integer between 0 and 5; Each R 2 Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-6 Alkyl, C 1- 6 alkoxy, saturated or partially unsaturated C 3-8 Alicyclic group, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2- 4-alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group containing 1-3 heteroatoms selected from N, O, S, C 6-10 Aryl, 5-6 membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkoxy, 4-6 membered saturated or partially unsaturated C 3-8 Alicyclic group, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered saturated or partially unsaturated heterocyclic group, C 6-10 Aryl, 5-6 membered heteroaryl are optionally substituted by 1-4 independently selected R 2a replaced; or Two adjacent substituted R 2 Together with the A ring atoms to which they are connected, they form a 5- to 6-membered saturated or partially unsaturated alicyclic ring, a 5- to 6-membered saturated or partially unsaturated heterocyclic ring, a 5- to 6-membered heteroaromatic ring or a benzene ring; the two adjacent R 2 The formed ring is optionally substituted by 1 to 4 R 2a replace; Among them, each R 2a Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1- 6 alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl; the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 1-6 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; R 5 and R 6 Independently selected from: H, halogen, C 1-4 Alkyl; or R 5 With R 6 Together with the C atom to which they are attached, they form a cyclopropyl group; or R 5 With R 6 Together they form =O; R 7 Selected from: C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 Alkoxy, -SC 1-4 Alkyl, -NH2, -NH(C 1-4 Alkyl), -N(C 1-4 Alkyl)(C 1-4 alkyl); the aforementioned group is optionally replaced by 1 to 3 independently selected R 7a Replace, each R 7a the same or different, independently selected from halogen or -OH; m is an integer between 0 and 3; Each R 3 Same or different, independently selected from: halogen, -OH, -CN, -NR 3a R 3b 、C 1-6 Alkyl, C 1-6 Alkoxy, saturated or partially unsaturated C 3-8 Alicyclic group, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2- 4-alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group containing 1-3 heteroatoms selected from N, O, S, C 6-10 Aryl, 5-6 membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkoxy, saturated or partially unsaturated C 3-8 Alicyclic group, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group, C 6-10 Aryl, 5-6 membered heteroaryl are optionally substituted by 1-4 independently selected R 3c replace; Among them, R 3a and R 3b Independently selected from: H, C 1-4 Alkyl; or, R 3a and R 3b Together with the nitrogen atom to which they are attached, they form a 4-6 membered heterocyclic ring containing 1-2 heteroatoms selected from N, O, and S; Each R 3c Same or different, independently selected from: halogen (such as F), -OH, -NH2, =O, -CN, -SF5, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2- 4-alkenyl, C 2-4 Alkynyl; the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, -SC 1- 6-alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; p is an integer from 0 to 5; Each R 4 Same or different, independently selected from: halogen, -OH, -CN, C 1-4 Alkyl, C 3-5 Cycloalkyl; or, two R attached to the same atom 4 Together with the atoms to which they are attached, they form C 3-5 or a 4- to 6-membered spiro ring containing 1 to 3 heteroatoms selected from N, O, and S; two adjacent substituted R 4 Together with the atoms to which they are attached, they form a 3- to 6-membered alicyclic ring; two non-adjacent substituted R 4 Together with the atoms to which they are attached, they form a bridged ring; W is selected from C, CH, N; J, G, K, V, Q, and Y are independently selected from the group consisting of: none, C, CH, -(CH2) i -, N, NH, O, S; E and M are independently selected from: none, -(CH2) i -, NH, O, S; i is an integer from 1 to 3; Each independently represents a single bond or a double bond; The ring where W, E, G, and J are located and the ring where K, V, Q, Y, and M are located are independently a ring having 4 to 8 ring atoms; q is an integer from 0 to 5; Each R 8 Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, C 1-4 Alkyl; or, two R attached to the same atom 8 Together with the atoms to which they are attached, they form a cyclopropyl spiro ring. Two adjacently substituted R8 together with the atoms to which they are attached form a cyclopropyl group. Two non-adjacently substituted R8 as described above form a cyclopropyl group. 8 Together with the atoms to which they are attached, they form a bridged ring; y is an integer from 0 to 6; Each R 9 Same or different: each R 9 Independently selected from: halogen, -OH, =O, -CN, -N(R 9a )(R 9b ),-C(O)R 9c 、-S(O)R 9c 、-S(O)2R 9c 、C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, C 6-10 Aryl or 5- to 6-membered heteroaryl; the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, C 6-10 Aryl or 5-6 membered heteroaryl is optionally substituted by 1-4 independently selected R 9d Replace; or Two R's attached to the same atom 9 Together with the atoms to which they are attached, they form C 3-5 or a 4- to 6-membered spiro ring containing 1 to 3 heteroatoms selected from N, O, and S; two adjacent substituted R 9 Together with the atoms to which they are attached, they form a 5-6 membered saturated or partially unsaturated alicyclic ring, a 5-6 membered saturated or partially unsaturated heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S, a 5-6 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O, and S, or a benzene ring; two non-adjacent R 9 Together with the atoms to which they are attached, they form a bridged ring; as described above, two R 9 The rings involved in the formation are optionally replaced by 1 to 3 R 9d replace; Each R 9a The same or different, independently selected from: H, C 1-6 Alkyl, C 3-8 Cycloalkyl; the C 1-6 Alkyl, C 3- 8-cycloalkyl is optionally substituted with one or more halogens; R 9b Selected from: H, C substituted or unsubstituted by one or more halogens 1-6 Alkyl, -C(O)R 9b-1 、-S(O)2R 9b- 1 ; Each R 9b-1 Independently selected from: C 1-6 Alkyl, C 3-8 Cycloalkyl, C 1-6 alkoxy; Each R 9c The same or different, independently selected from: H, halogen, -OH, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl; the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl is optionally substituted with one or more halogens; Each R 9d Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2- 4 alkynyl; the C 1-6 Alkyl, C 1-6 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkoxy, -SC 1-6 Alkyl, -SC 3-8 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl groups are optionally substituted with one or more halogens.

2. The spirocyclic compound of formula I according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, or metabolite thereof, characterized in that: Ring A is a benzene ring or a pyridine ring; n is an integer between 0 and 3; Each R 2 Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1- 4 alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3- 6 cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 5-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl, optionally substituted by 1-4 independently selected R 2a replaced; or Two adjacent substituted R 2 Together with the A ring atoms to which they are attached, they form the following structure: Two adjacent R 2 The formed ring is optionally substituted by 1 to 4 R 2a replace; Among them, each R 2a Independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 1-4 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 1-4 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; preferably, each R 2a Independently selected from: halogen (e.g., F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3; Preferably, each R 2 Independently selected from: halogen (eg, Cl), methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, cyclopropyl, cyclohexyl, pentafluorosulfenyl (-SF5); or Two adjacent substituted R 2 Together with the A ring atoms to which they are attached, they form 3. The compound containing a spiro ring structure represented by formula I according to claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, or metabolite thereof, characterized in that: Ring B is p is an integer from 0 to 4; R 4 is methyl; or two adjacent substituted R 4 Together with the atoms to which they are attached, they form a 4-6 membered alicyclic ring; and / or X is CH or N; m is 0 or 1; R 3 Selected from: halogen, -OH, -CN, -NR 3a R 3b 、C 1-4 Alkyl, C 1-4 Alkoxy, saturated or partially unsaturated C 3- 6 alicyclic group, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl; the C 1-4 Alkyl, C 1-4 Alkoxy, saturated or partially unsaturated C 3-6 Alicyclic group, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, 4-6 membered saturated or partially unsaturated heterocyclic group, phenyl, 5-6 membered heteroaryl, optionally substituted by 1-4 independently selected R 3c replace; Among them, R 3a and R 3b Independently selected from: H, C 1-4 Alkyl; or, R 3a and R 3b Together with the nitrogen atom to which they are attached, they form a 4-6 membered heterocyclic ring containing 1-2 heteroatoms selected from N, O, and S; Each R 3c Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2- 4 alkynyl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3- 9 cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkynyl is optionally substituted with one or more halogens; preferably, each R 3c Independently selected from: halogen (e.g., F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3; Preferably, R 3 Selected from: methyl, methoxy, cyclopropyl.

4. The compound containing a spiro ring structure represented by formula I according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, or metabolite thereof, characterized in that: L is -C(O)-; and / or R 5 and R 6 Independently selected from: H, halogen, methyl, preferably H; and / or R 7 is methyl, ethyl, preferably ethyl; and / or The ring where W, E, G, and J are located and the ring where K, V, Q, Y, and M are located are all rings having 4 to 6 ring atoms; Preferably, the spirocyclic structure where W, E, G, J, K, V, Q, Y, and M are located is selected from the following structures: wherein each Z is independently selected from: O, NH, S, preferably O or NH; Each i is independently 1, 2 or 3; R 8 、R 9 , q, y are defined as in any one of claims 1-3; In particular, q is an integer from 0 to 5; Each R 8 Independently selected from: halogen, -OH, -NH2, =O, -CN, methyl; and / or y is an integer from 0 to 6; Each R 9 Independently selected from: halogen, -OH, =O, -CN, -N(R 9a )(R 9b ),-C(O)R 9c 、-S(O)R 9c 、-S(O)2R 9c 、C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, phenyl or 5- to 6-membered heteroaryl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, phenyl or 5-6 membered heteroaryl is optionally replaced by 1-4 independently selected R 9d Replace; or Two R's attached to the same atom 9 Together with the atoms to which they are attached, they form C 3-5 or a 4- to 6-membered spiro ring containing 1 to 3 heteroatoms selected from N, O, and S; two adjacent substituted R 9 Together with the atoms to which they are attached, they form a 5-6 membered saturated or partially unsaturated alicyclic ring, a 5-6 membered saturated or partially unsaturated heterocyclic ring containing 1-3 heteroatoms selected from N, O, and S, a 5-6 membered heteroaromatic ring containing 1-3 heteroatoms selected from N, O, and S, or a benzene ring; two non-adjacent R 9 Together with the atoms to which they are attached, they form a bridged ring; as described above, two R 9 The rings involved in the formation are optionally substituted by 1 to 3 R 9c replace; Each R 9a The same or different, independently selected from: H, C 1-4 Alkyl, C 3-6 Cycloalkyl; the C 1-4 Alkyl, C 3- 6 cycloalkyl is optionally substituted with one or more halogens; preferably, each R 9a Independently selected from: H, methyl, trifluoromethyl; R 9b Selected from: H, C substituted or unsubstituted by one or more halogens 1-4 Alkyl, -C(O)R 9b-1 、-S(O)2R 9b- 1 ; Each R 9b-1 Independently selected from: C 1-4 Alkyl, C 3-6 Cycloalkyl, C 1-4 alkoxy; Each R 9c The same or different, independently selected from: H, halogen, -OH, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl is optionally substituted with one or more halogens; preferably, each R 9c Independently selected from: H, halogen, -OH, methyl, trifluoromethyl; Each R 9d Same or different, independently selected from: halogen, -OH, -NH2, =O, -CN, -SF5, C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2- 4 alkynyl; the C 1-4 Alkyl, C 1-4 Alkoxy, C 3-6 Cycloalkyl, C 3-6 Cycloalkoxy, -SC 1-4 Alkyl, -SC 3-6 Cycloalkyl, C 2-4 Alkenyl, C 2-4 Alkyne is optionally substituted with one or more halogens; preferably, each R 9c Independently selected from: halogen (e.g., F), -OH, -NH2, =O, -CN, -SF5, methyl, ethyl, difluoromethyl, trifluoromethyl, methoxy, difluoromethoxy, trifluoromethoxy, -SCF3; Preferably, each R 9 Independently selected from: halogen, -OH, -NH2, =O, -CN, methyl; or two adjacent substituted R 9 Together with the atoms to which they are attached, they form a benzene ring; Further preferably, the spirocyclic structure where W, E, G, J, K, V, Q, Y, and M are located is selected from the following structures:

5. The compound containing a spiro ring structure represented by formula I according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, deuterated substance, solvate, prodrug, or metabolite thereof, characterized in that: The compound containing a spiro ring structure shown in Formula I has a structure selected from the following: in, W、E、G、J、K、V、Q、Y、M、n、R 2 、m、R 3 、q、R 8 、y、R 9 、 The definitions are as described in any one of claims 1 to 4; In particular, the compound containing a spiro ring structure represented by Formula I is selected from any one of the following compounds:

6. A pharmaceutical composition comprising a therapeutically effective dose of a compound containing a spirocyclic structure as represented by Formula I according to any one of claims 1 to 5, or one or more of its pharmaceutically acceptable salts, stereoisomers, deuterated forms, solvates, prodrugs, and metabolites, and optionally a pharmaceutically acceptable carrier.

7. A WRN modulator, comprising one or more of the spirocyclic compound of formula I according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, stereoisomer, deuterated form, solvate, prodrug, metabolite thereof, and the pharmaceutical composition according to claim 6; Preferably, the WRN regulator is a WRN inhibitor or degrader.

8. Use of one or more of the spirocyclic compound of formula I according to any one of claims 1 to 5, or pharmaceutically acceptable salts, stereoisomers, deuterated forms, solvates, prodrugs, metabolites thereof, and the pharmaceutical composition according to claim 6 in the preparation of a WRN inhibitor or degrader.

9. Use of one or more of the spirocyclic compound of formula I according to any one of claims 1 to 5, or pharmaceutically acceptable salts, stereoisomers, deuterated forms, solvates, prodrugs, metabolites thereof, and the pharmaceutical composition according to claim 6 in the preparation of a medicament for preventing or treating diseases associated with WRN; Preferably, the WRN-related diseases include diseases that can be treated by inhibiting WRN or mediating the degradation of WRN, especially cancers characterized by high microsatellite instability or DNA mismatch repair function deficiency; in, Such cancers include, but are not limited to, colorectal cancer, gastric cancer, prostate cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, and ovarian cancer.

10. Use of the spirocyclic compound of formula I according to any one of claims 1 to 5, or one or more of its pharmaceutically acceptable salts, stereoisomers, deuterated forms, solvates, prodrugs, metabolites, and the pharmaceutical composition according to claim 6 in the preparation of an immune adjuvant.

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