PIKfyve inhibitors

文档序号：704128 发布日期：2021-04-13 浏览：24次中文

阅读说明：本技术 PIKfyve抑制剂 (PIKfyve inhibitors ) 是由 R·J·布拉姆 A·B·平克顿 E·瑟古恩克于 2019-07-01 设计创作，主要内容包括：本申请尤其提供式(I)化合物或其药学上可接受的盐,其中Y、Ar、X～1、X～2、X～3、R～1、R～2、R～3、R～4、R～5和R～6如本文所描述。还提供制备这些化合物的方法和使用这些化合物治疗如癌症的疾病的方法。(The present application provides, inter alia, compounds of formula (I) or a pharmaceutically acceptable salt thereof, wherein Y, Ar, X 1 、X 2 、X 3 、R 1 、R 2 、R 3 、R 4 、R 5 And R 6 As described herein. Also provided are methods of making these compounds and methods of using these compounds to treat diseases such as cancer.)

1. A compound of the formula (I),

or a pharmaceutically acceptable salt thereof, wherein:

is shown asEach bond of (a) is a single bond or a double bond, with the proviso that

Is shown asThe bonds of (a) are not both double bonds;

X¹is selected from N and CR^A；

X²Is selected from N and CR^A；

X³Is selected from N and CR^A；

Each R^AIndependently selected from H, halo, C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Alkoxy and C_1-6A haloalkoxy group;

ar is selected from C_6-10Aryl and 5-10 membered heteroaryl, each of which is optionally selected from R through 1,2,3,4 or 5 independently⁷Substituted with the substituent(s);

each R⁷Independently selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, OR^a1、SR^a1、C(O)R^b1、C(O)NR^c1R^d1、C(O)OR^a1、OC(O)R^b1、OC(O)NR^c1R^d1、NR^c1R^d1、NR^c1C(O)R^b1、NR^c1C(O)OR^a1、NR^c1C(O)NR^c1R^d1、NR^c1S(O)₂R^b1、NR^c1S(O)₂NR^c1R^d1、S(O)₂R^b1And S (O)₂NR^c1R^d1(ii) a Wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Each alkynyl is optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、OR^a1、SR^a1、C(O)R^b1、C(O)NR^c1R^d1、C(O)OR^a1、OC(O)R^b1、OC(O)NR^c1R^d1、NR^c1R^d1、NR^c1C(O)R^b1、NR^c1C(O)OR^a1、NR^c1C(O)NR^c1R^d1、NR^c1S(O)₂R^b1、NR^c1S(O)₂NR^c1R^d1、S(O)₂R^b1And S (O)₂NR^c1R^d1；

R¹Selected from H and C_1-6Alkyl, wherein said C_1-6Alkyl is optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2、NR^c2R^d2、NR^c2C(O)R^b2、NR^c2C(O)OR^a2、NR^c2C(O)NR^c2R^d2、NR^c2S(O)₂R^b2、NR^c2S(O)₂NR^c2R^d2、S(O)₂R^b2And S (O)₂NR^c2R^d2；

R²Is C optionally substituted with 1,2 or 3 substituents independently selected from_1-6Alkyl groups: halogen radical, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2、NR^c2R^d2、NR^c2C(O)R^b2、NR^c2C(O)OR^a2、NR^c2C(O)NR^c2R^d2、NR^c2S(O)₂R^b2、NR^c2S(O)₂NR^c2R^d2、S(O)₂R^b2And S (O)₂NR^c2R^d2(ii) a Or

R¹And R²Together with the N to which they are attached form a 4-7 membered heterocycloalkyl group, optionally substituted with 1,2,3,4 or 5 independently selected R⁸Substituent group substitution;

each R⁸Independently selected from halo, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2、NR^c2R^d2、NR^c2C(O)R^b2、NR^c2C(O)OR^a2、NR^c2C(O)NR^c2R^d2、NR^c2S(O)₂R^b2、NR^c2S(O)₂NR^c2R^d2、S(O)₂R^b2And S (O)₂NR^c2R^d2；

R³Is selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

R⁴is selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

y is selected from N, C and CR^A；

When R is⁵When the bond to Y is a single bond, R⁵Is a 5-to 10-membered heteroaryl group,

optionally substituted with 1,2,3,4 or 5 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3(ii) a Wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Each alkynyl is optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3；

When R is⁵When the bond between Y and R is a double bond⁵Is CR^BR^C；

R^BIs selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

R^Cis selected from C_6-10Aryl and 5-10 membered heteroaryl, each of which is optionally substituted with 1,2,3,4 or 5 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3(ii) a Wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Each alkynyl is optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3(ii) a Or

R⁴And R⁵And R⁴The attached Y and N together form a 5-14 membered heteroaryl,optionally via 1,2,3,4 or 5 independently selected from R⁹Substituted with the substituent(s);

each R⁹Independently selected from halo, CN, NO₂、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3(ii) a Wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Each alkynyl is optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3；

R⁶Is selected from H, C_1-6Alkyl and C_1-6A haloalkyl group; or R⁶Is absent;

each R^a1、R^b1、R^a2、R^b2、R^a3And R^b3Independently selected from H, C_1-6Alkyl radical, C_1-4Haloalkyl, C_2-6Alkenyl and C_2-6Alkynyl, wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl is optionally selected from R through 1,2,3,4 or 5 independently^gSubstituted with the substituent(s);

each R^c1、R^d1、R^c2、R^d2、R^c3And R^d3Independently selected from H, C_1-6Alkyl radical, C_1-4Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C (O) R^b7、C(O)NR^c7R^d7、C(O)OR^a7、NR^c7R^d7、S(O)R^b7、S(O)NR^c7R^d7、S(O)₂R^b7And S (O)₂NR^c7R^d7(ii) a Wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl is optionally selected from R through 1,2,3,4 or 5 independently^gSubstituted with the substituent(s);

each R^a7、R^b7、R^c7And R^d7Independently selected from H, C_1-6Alkyl radical, C_1-4Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl and R^gWherein said C is_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl is optionally selected from R through 1,2,3,4 or 5 independently^gSubstituted with the substituent(s);

or any R^c1And R^d1Together with the N atom to which they are attached form optionally 1,2 or 3 independently selected from R^gA 4-, 5-, 6-or 7-membered heterocycloalkyl substituted with the substituent(s) of (a);

or any R^c2And R^d2Together with the N atom to which they are attached form optionally 1,2 or 3 independently selected from R^gA 4-, 5-, 6-or 7-membered heterocycloalkyl substituted with the substituent(s) of (a);

or any R^c3And R^d3Together with the N atom to which they are attached form optionally 1,2 or 3 independently selected from R^gA 4-, 5-, 6-or 7-membered heterocycloalkyl substituted with the substituent(s) of (a);

each R^gIndependently selected from OH, NO₂CN, halogen radical, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-4Haloalkyl, C_1-6Alkoxy radical, C_1-6Haloalkoxy, cyano-C_1-3Alkylene oxideGroup HO-C_1-3Alkylene, amino, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, thio, C_1-6Alkylthio radical, C_1-6Alkylsulfinyl radical, C_1-6Alkylsulfonyl, carbamoyl, C_1-6Alkylcarbamoyl, di (C)_1-6Alkyl) carbamoyl, carboxy, C_1-6Alkylcarbonyl group, C_1-6Alkoxycarbonyl, C_1-6Alkylcarbonylamino, C_1-6Alkanesulfonylamino, aminosulfonyl, C_1-6Alkylaminosulfonyl, di (C)_1-6Alkyl) aminosulfonyl, aminosulfonylamino, C_1-6Alkylamino sulfonylamino, di (C)_1-6Alkyl) aminosulfonylamino, aminocarbonylamino, C_1-6Alkylamino carbonylamino and di (C)_1-6Alkyl) aminocarbonylamino;

with the proviso that the compound of formula (I) is not:

2. the compound of claim 1, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

3. The compound of claim 2, wherein the compound of formula (I) is selected from:

or a pharmaceutically acceptable salt thereof.

4. The compound of claim 3, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

5. The compound of claim 3, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

7. The compound of claim 6, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

8. The compound of claim 7, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

9. The compound of claim 1, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

10. The compound of claim 9, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

11. The compound of claim 10, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

12. The compound of claim 10, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

13. The compound of claim 1, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

14. The compound of claim 13, wherein the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

15. The compound according to any one of claims 1-14, wherein R¹Is H and R²Is optionally via OR^a2Substituted C_1-6An alkyl group.

16. The compound according to any one of claims 1-14, wherein R¹And R²Each independently is optionally OR^a2Substituted C_1-6An alkyl group.

17. The compound of any one of claims 1-16, wherein R¹And R²Together form a 4-7 membered heterocycloalkyl group, optionally substituted with 1,2 or 3 independently selected R⁸And (4) substituent substitution.

18. The compound of claim 17, wherein R¹And R²Together with the N to which it is attached, form a ring selected from: morpholinyl, thiomorpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, azetidinyl and 3-oxa-8-azabicyclo [3.2.1]Octyl, each of which is optionally substituted with 1,2 or 3 independently selected R⁸And (4) substitution.

19. The compound of claim 18, wherein R¹And R²Together with the N to which it is attached, form a ring of the formula:

20. the compound according to any one of claims 1-19, wherein R⁸Is selected from halo, C_1-6Alkyl radical, C_1-6Haloalkyl, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2、NR^c2R^d2、NR^c2C(O)R^b2、NR^c2S(O)₂R^b2、S(O)₂R^b2And S (O)₂NR^c2R^d2。

21. The compound of any one of claims 1-20, wherein R⁸Is selected from C_1-6Alkyl and OR^a2。

22. The compound of any one of claims 1-21, wherein R^a2Is selected from H and C_1-6An alkyl group.

23. The compound of any one of claims 1-22, wherein R³And R⁴Each is H.

24. The compound of any one of claims 1-23, wherein R⁷Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a1、C(O)R^b1、C(O)NR^c1R^d1、C(O)OR^a1、NR^c1R^d1、NR^c1C(O)R^b1、NR^c1C(O)OR^a1、NR^c1S(O)₂R^b1、S(O)₂R^b1And S (O)₂NR^c1R^d1。

25. The compound of claim 24, wherein R⁷Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a1、NR^c1R^d1、NR^c1C(O)R^b1And NR^c1S(O)₂R^b1。

26. The compound of any one of claims 1-25, wherein R^a1、R^c1And R^d1Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group; and each R^b1Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group.

27. The compound of claim 25, wherein R⁷Is selected from methyl, trifluoromethyl, methoxy, fluorine, chlorine, bromine, CN, NO₂Amino group, dimethylamino group, NHC (O) CH₃And NHS (O)₂CH₃。

28. The compound according to any one of claims 1-27, wherein R^AIs selected from H, C_1-6Alkyl and C_1-6A haloalkyl group.

29. The compound of claim 28, wherein R^AIs H.

30. The compound of any one of claims 1-29, wherein R^BIs H.

31. The compound of any one of claims 1-30, wherein R^CIs a 5-10 membered heteroaryl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3。

32. The compound of claim 31, wherein the 5-10 membered heteroaryl is selected from indolyl, pyridinyl, pyrrolyl, and thienyl.

33. The compound of claim 32, wherein the 5-10 membered heteroaryl is indolyl.

34. The compound of any one of claims 1-30, wherein R^CIs C_6-10Aryl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3。

35. The compound of claim 34, wherein said C is_6-10Aryl is phenyl.

36. The compound of any one of claims 1-35, wherein R^a3、R^c3And R^d3Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group; and each R^b3Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group.

37. The compound of any one of claims 1-14, wherein:

R¹、R³and R⁴Each is H;

R²is optionally via OR³²Substituted C_1-6An alkyl group;

R^a2is selected from H and C_1-6An alkyl group;

R⁵and the bond between Y isA double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^C；

R⁷Is C_1-6An alkyl group;

R^Bis H; and is

R^CIs indolyl.

38. The compound of any one of claims 1-14, wherein:

R¹and R²Each independently selected from optionally via OR^a2Substituted C_1-6An alkyl group;

R³and R⁴Each is H;

R^a2is selected from H and C_1-6An alkyl group;

R⁵the bond between Y and Y is a double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^C；

R⁷Is C_1-6An alkyl group;

R^Bis H; and is

R^CIs indolyl.

39. The compound of any one of claims 1-14, wherein:

R¹and R²Together form a 4-7 membered heterocycloalkyl group, optionally substituted with 1,2 or 3 independently selected R⁸Substituent group substitution;

each R⁸Is selected from C_1-6Alkyl and OR^a2；

R^a2Is selected from H and C_1-6An alkyl group;

R³and R⁴Each is H;

R⁵the bond between Y and Y is a double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^C；

Each R⁷Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a1、C(O)R^b1、C(O)NR^c1R^d1、C(O)OR^a1、NR^c1R^d1、NR^c1C(O)R^b1、NR^c1C(O)OR^a1、NR^c1S(O)₂R^b1、S(O)₂R^b1And S (O)₂NR^c1R^d1；

R^a1、R^c1And R^d1Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group;

each R^b1Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group;

R^Ais selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

R^Bis a compound of formula (I) in the formula (H),

R^Cis a 5-10 membered heteroaryl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3；

R^a3、R^c3And R^d3Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group; and is

Each R^b3Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group.

40. The compound of any one of claims 1-14, wherein:

R¹and R²Together with the N to which it is attached, form a ring selected from: morpholinyl, thiomorpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, azetidinyl and3-oxa-8-azabicyclo [3.2.1]Octyl, each of which is optionally substituted with 1,2 or 3 independently selected R⁸Substitution;

R⁸is selected from C_1-6Alkyl and OR^a2；

R^a2Is selected from H and C_1-6An alkyl group;

R³and R⁴Each is H;

R⁷is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a1、NR^c1R^d1、NR^c1C(O)R^b1And NR^c1S(O)₂R^b1；

R^a1、R^c1And R^d1Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group;

each R^b1Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group;

R⁵the bond between Y and Y is a double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^C；

R^AIs H;

R^Bis H; and is

R^CIs selected from indolyl, pyridinyl, pyrrolyl and thienyl, each of which is optionally substituted with 1 or 2 substituents independently selected from: methyl, trifluoromethyl, methoxy, fluorine, chlorine, bromine, CN, NO₂Amino, dimethylamino, NHC (O) CH₃And NHS (O)₂CH₃。

41. The compound of claim 1, wherein the compound is selected from any one of the following compounds:

or a pharmaceutically acceptable salt thereof.

42. A pharmaceutical composition comprising a compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

43. A method, the method comprising:

inhibition of phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) in cancer cells; and/or

Induction of cytoplasmic vacuolization in cancer cells; and/or

Blocking IL12/23 secretion in cells;

the method comprising contacting the cell with an effective amount of a compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof.

44. A method, the method comprising:

inhibition of phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) in an individual; and/or

Inducing cytoplasmic vacuolization in cancer cells of the individual; and/or

Treating cancer in the individual; and/or

Blocking secretion of IL12/23 in an individual; and/or

Treating an inflammatory disease or condition in an individual;

the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 42.

45. The method of claim 43 or claim 44, wherein the cancer is selected from lymphoblastic leukemia (lymphoblastic leukemia), lymphoma, colorectal cancer, glioblastoma multiforme (GBM), medulloblastoma, colorectal carcinoma, osteosarcoma and pancreatic cancer.

46. The method of claim 44 or claim 45, wherein the subject is administered a compound of any one of claims 1-41, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 42 in combination with a BRAF-V600E inhibitor, or a pharmaceutically acceptable salt thereof.

47. The method of claim 46, wherein the BRAF-V600E inhibitor is vemurafenib (vemurafenib) or a pharmaceutically acceptable salt thereof.

48. A pharmaceutical composition comprising a compound:

or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

49. A method, the method comprising:

inhibition of phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) in cancer cells; and/or

Induction of cytoplasmic vacuolization in cancer cells; and/or

Blocking IL12/23 secretion in cells;

the method comprises contacting the cell with an effective amount of a compound of:

or a pharmaceutically acceptable salt thereof.

50. A method, the method comprising:

inhibition of phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) in cancer cells of the individual; and/or

Inducing cytoplasmic vacuolization in cancer cells of the individual; and/or

Treating cancer in the individual; and/or

Blocking secretion of IL12/23 in an individual; and/or

Treating an inflammatory disease or condition in an individual;

the method comprises administering to a subject in need thereof a therapeutically effective amount of a compound:

or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 48.

51. The method of claim 49 or claim 50, wherein the cancer is selected from lymphoblastic leukemia, lymphoma, colorectal cancer, glioblastoma multiforme (GBM), medulloblastoma, colorectal carcinoma, osteosarcoma, and pancreatic cancer.

52. The method of claim 50 or claim 51, wherein the compound or pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 48, in combination with a BRAF-V600E inhibitor or pharmaceutically acceptable salt thereof, is administered to the subject.

53. The method of claim 52, wherein the BRAF-V600E inhibitor is vemurafenib or a pharmaceutically acceptable salt thereof.

54. The method of claim 44 or claim 50, wherein the inflammatory disease or condition is selected from: psoriasis, Crohn's disease and rheumatoid arthritis.

Technical Field

The present invention relates to compounds, in particular to compounds capable of inhibiting PIKfyve kinase and useful in the treatment of cancer such as lymphoblastic leukemia (lymphoblastic leukemia), lymphoma, colorectal cancer, medulloblastoma, osteosarcoma, pancreatic cancer or glioblastoma multiforme (GBM).

Background

Phosphorylated derivatives of Phosphatidylinositol (PI) modulate cytoskeletal function, membrane trafficking, and receptor signaling by recruiting protein complexes to the cell and endosomal membranes. Humans have a variety of phosphatidylinositol proteins that differ by the degree and location of phosphorylation of the inositol ring. PIKfyve (also known as phosphatidylinositol-3-phosphate 5-kinase type III or pipkii) is an enzyme that phosphorylates phosphatidylinositol 3-phosphate (PI3P) to produce phosphatidylinositol 3, 5-bisphosphate (PI (3,5) P2). PIKfyve activity is responsible for the production of PI (3,5) P2 and phosphatidylinositol 5-phosphate (PI5P) (see fig. 1), which regulate endosomal manipulations (division and fusion) that maintain the proper performance of the inner membrane stability and transport pathway.

PIKfyve is a larger protein that contains multiple functional domains and is expressed in several spliced forms. The full-length mouse and human cDNA clones reported encoded proteins of 2052 and 2098 amino acid residues, respectively. By binding directly to membrane PI3P, the FYVE finger domain of PIKfyve is critical in localizing proteins to the cytoplasmic lobules of endosomes. Impaired PIKfyve enzymatic activity causes increased endosome and cytoplasmic vacuolization due to impaired PI (3,5) P2 synthesis.

Disclosure of Invention

In a first general aspect, the present application provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein Y, Ar, X¹、X²、X³、R¹、R²、R³、R⁴、R⁵And R⁶As described herein.

In a second general aspect, the present application provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In a third general aspect, the present disclosure provides a method of inhibiting phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) in a cancer cell, the method comprising contacting the cell with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In a fourth general aspect, the present application provides a method of inducing cytoplasmic vacuolization in a cancer cell, the method comprising contacting the cell with an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In a fifth general aspect, the present application provides a method of inhibiting phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In a sixth general aspect, the present application provides a method of inducing cytoplasmic vacuolization in a cancer cell in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In a seventh general aspect, the present application provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In an eighth general aspect, the present application provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with a BRAF-V600E inhibitor (e.g., vemurafenib), or a pharmaceutically acceptable salt thereof.

In a ninth general aspect, the present application provides a method of blocking the secretion of IL12/23 in a cell, the method comprising contacting the cell with an effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In a tenth general aspect, the present application provides a method of blocking secretion of IL12/23 in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.

In an eleventh general aspect, the present application provides a method of treating an inflammatory disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof.

Certain embodiments of these general aspects are described herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials for use in the present application are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the application will be apparent from the following detailed description and drawings, and from the claims.

Drawings

Figure 1 is a scheme showing the PIKfyve enzymatic process.

Figure 2 shows an electron micrograph of murine lymphoma cells treated with vehicle only (left) or example 1 (right).

Figure 3 contains a line graph showing glioblastoma multiforme and medulloblastoma cell viability assessed by cell titer-Glo luminescence (ATP level) analysis using the compound of example 1.

Figure 4 contains a line graph showing killing by example 1 of a colorectal carcinoma cell line. (ATP activity is defined by the use of cell-titer glow luminescence analysis).

Figure 5 contains a line graph showing killing by osteosarcoma and example 1 of the pancreatic cell line.

Fig. 6 contains a line graph showing the killing of murine lymphoma cells (E2409, EC + F) by example 1.

Figure 7 contains a line graph showing the killing of human lymphoma cell lines by example 1.

Fig. 8 contains a line graph showing PK/PD testing of the compound of example 1 in mice following intraperitoneal, intravenous, or oral administration.

Figure 9 is a line graph showing the activity of example 2 (a biotin-labeled derivative of the example compound).

Fig. 10 is a line graph showing the activity of example 3.

Fig. 11 contains a line graph showing that glioblastoma (T98G, U251) or colorectal cancer cell lines (HT29, RKO, HCT116) are sensitive to killing by the compound of example 3.

Figure 12 is a dendrogram showing selective inhibition of PIKfyve by example 1, example 3, and inhibition of PIKfyve by apilimod (apilimod).

Fig. 13 is a line graph showing U251 glioblastoma cells killed by example 1 alone or in the presence of 10 μ M vemurafenib.

Figure 14 contains line graphs showing synergistic cell killing by vemurafenib in combination with various concentrations of example 1.

Figure 15 contains line graphs showing synergistic cell killing by vemurafenib in combination with various concentrations of example 1.

Fig. 16 is a line graph showing synergy of example 3 with vemurafenib in kill U25.

Fig. 17 is a line graph showing the mean plasma concentrations of example 1 after intravenous, intraperitoneal, and oral administration in mice.

Fig. 18 is a line graph showing plasma concentrations of example 1 after intravenous administration at 5 mg/kg.

Fig. 19 is a line graph showing the plasma concentration of example 1 after intraperitoneal administration at 30mg/kg in mice.

Fig. 20 is a line graph showing the plasma concentration of example 1 after oral administration at 30mg/kg in mice.

FIG. 21 is a line graph showing plasma concentrations (ng/mL) of example 3 in male C57 mice after intravenous administration of 5 mg/kg.

FIG. 22 is a line graph showing plasma concentrations (ng/mL) of example 3 in male C57 mice after intraperitoneal administration of 30 mg/kg.

Figure 23 contains images showing induction of autophagy of example 1 as evidenced by accumulation of LC3 over time.

Figure 24 contains western blot images showing accumulation of LC3 in U251 (glioblastoma) and RKO (colon cancer) cells treated with 1 μ M of example 1.

Fig. 25 contains images showing the accumulation of uncleaved precursor cathepsin D ("CTSD") and the relative loss of mature cleaved cathepsin by example 1.

Fig. 26 contains a line graph showing cytotoxicity of example 3 on MPNST cell line (S462, S462TY, 26T, T265, SNF96.2), colorectal carcinoma cell line (HCT116) and GBM cell line (U251).

Detailed Description

PIKfyve is a lipid kinase that catalyzes the addition of phosphate to the 5 position of Phosphatidylinositol (PI) or to phosphatidyl3-phosphate (PI3P) to produce PI5P and PI (3,5) P2, respectively. During the introduction of nutrients and other molecules by macroendocytosis, these minor lipids are important for intracellular vesicle trafficking and also for proper functioning of lysosomes and autophagy. It was previously shown that blocking gene expression or mutation of PIKfyve causes massive cytoplasmic vacuolization. The present application provides therapeutic compounds capable of inhibiting PIKfyve and inducing cellular vacuolization and suitable for treating cancer. Pharmaceutical compositions and methods of making the compounds are also provided.

Therapeutic compounds

In one general aspect, the present application provides compounds of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

is shown asEach bond of (a) is a single bond or a double bond, provided that it is represented byAre not simultaneously double bonds;

X¹is selected from N and CR^A；

X²Is selected from N and CR^A；

X³Is selected from N and CR^A；

Each R^AIndependently selected from H, halo, C_1-6Alkyl radical, C_1-6Haloalkyl, C_1-6Alkoxy and C_1-6A haloalkoxy group;

ar is selected from C_6-10Aryl and 5-10 membered heteroaryl, each of which is optionally selected from R through 1,2,3,4 or 5 independently⁷Substituted with the substituent(s);

R²Is C optionally substituted with 1,2 or 3 substituents independently selected from_1-6Alkyl groups: halogen radical, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2、NR^c2R^d2 NR^c2C(O)R^b2、NR^c2C(O)OR^a2、NR^c2C(O)NR^c2R^d2、NR^c2S(O)₂R^b2、NR^c2S(O)₂NR^c2R^d2、S(O)₂R^b2And S (O)₂NR^c2R^d2(ii) a Or

R³Is selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

R⁴is selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

y is selected from N, C and CR^A；

When R is⁵When the bond to Y is a single bond, R⁵Is a 5-10 membered heteroaryl, optionally substituted with 1,2,3,4 or 5 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3(ii) a Wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Each alkynyl is optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3；

When R is⁵When the bond between Y and R is a double bond⁵Is CR^BR^C；

R^BIs selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

R⁴And R⁵And R⁴Y and N attached together form a 5-14 membered heteroaryl group, optionally selected from R through 1,2,3,4 or 5⁹Substituted with the substituent(s);

R⁶Is selected from H, C_1-6Alkyl and C_1-6A haloalkyl group; or R⁶Is absent;

each R^c1、R^d1、R^c2、R^d2、R^c3And R^d3Independently selected from H, C_1-6Alkyl radical, C_1-4Haloalkyl, C_2-6Alkenyl radical, C_2-6Alkynyl, C (O) R^b7、C(O)NR^c7R^d7、C(O)OR^a7、NR^c7R^d7、S(O)R^b7、S(O)NR^c7R^d7、S(O)₂R^b7And S (O)₂NR^c7R^d7(ii) a Wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_2-6Alkynyl is optionally selected from 1,2,3,4 or 5 independentlyR^gSubstituted with the substituent(s);

each R^gIndependently selected from OH, NO₂CN, halogen radical, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-4Haloalkyl, C_1-6Alkoxy radical, C_1-6Haloalkoxy, cyano-C_1-3Alkylene, HO-C_1-3Alkylene, amino, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, thio, C_1-6Alkylthio radical, C_1-6Alkylsulfinyl radical, C_1-6Alkylsulfonyl, carbamoyl, C_1-6Alkylcarbamoyl, di (C)_1-6Alkyl) carbamoyl, carboxy, C_1-6Alkylcarbonyl group, C_1-6Alkoxycarbonyl, C_1-6Alkylcarbonylamino, C_1-6Alkanesulfonylamino, aminosulfonyl, C_1-6Alkylaminosulfonyl, di (C)_1-6Alkyl) aminosulfonyl, aminosulfonylamino, C_1-6Alkylamino sulfonylamino, di (C)_1-6Alkyl) ammoniaArylsulfonylamino, aminocarbonylamino, C_1-6Alkylamino carbonylamino and di (C)_1-6Alkyl) aminocarbonylamino;

in some embodiments, it is represented asBoth bonds of (2) are single bonds.

In some embodiments, it is represented asIs a single bond and is represented byIs a double bond.

In some embodiments, X¹Is N. In some embodiments, X¹Is CR^A. In some embodiments, X²Is N. In some embodiments, X²Is CR^A. In some embodiments, X³Is N. In some embodiments, X³Is CR^A。

In some embodiments, R^AIndependently selected from H, halo, C_1-6Alkyl and C_1-6A haloalkyl group. In some embodiments, R^AIndependently selected from H, C_1-6Alkyl and C_1-6A haloalkyl group. In some embodiments, R^AIs H.

In some embodiments, Ar is optionally selected from R via 1,2 or 3 independently⁷C substituted by a substituent of_6-10And (4) an aryl group. In some embodiments, Ar is phenyl or naphthyl.

In some embodiments, Ar is optionally selected from R via 1,2 or 3 independently⁷A 5-10 membered heteroaryl group substituted with the substituent(s) of (a). In some embodiments, Ar is pyridinyl (e.g., pyridin-2-yl).

In some embodiments, R⁷Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a1、C(O)R^b1、C(O)NR^c1R^d1、C(O)OR^a1、NR^c1R^d1、NR^c1C(O)R^b1、NR^c1C(O)OR^a1、NR^c1S(O)₂R^b1、S(O)₂R^b1And S (O)₂NR^c1R^d1。

In some embodiments, R⁷Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a1、NR^c1R^d1、NR^c1C(O)R^b1And NR^c1S(O)₂R^b1；

In some embodiments, R⁷Is selected from methyl, trifluoromethyl, methoxy, fluorine, chlorine, bromine, CN, NO₂Amino, dimethylamino, NHC (O) CH₃And NHS (O)₂CH₃. In some embodiments, Y is N. In some aspects of these embodiments, are denoted asOne of the bonds of (A) is a double bond, and R⁶Is absent. In other aspects of these embodiments, are represented asBoth bonds of (A) are single bonds, and R⁶Exist and are as described herein.

In some embodiments, it is represented asOne of the bonds of (a) is a double bond, and Y is C. In other embodiments, it is represented asAre both single bonds and Y is CR^A。

In some embodiments, R¹Is H. In some embodiments, R¹Is C optionally substituted with 1,2 or 3 substituents independently selected from_1-6Alkyl groups: halogenRadical, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2And NR^c2R^d2. In some embodiments, R¹Is optionally via OR^a2Substituted C_1-6An alkyl group.

In some embodiments, R²Is H. In some embodiments, R²Is C optionally substituted with 1,2 or 3 substituents independently selected from_1-6Alkyl groups: halogen radical, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2And NR^c2R^d2. In some embodiments, R²Is optionally via OR^a2Substituted C_1-6An alkyl group.

In some embodiments, R¹Is H and R²Is optionally via OR^a2Substituted C_1-6An alkyl group. In some embodiments, R¹And R²Each independently is optionally OR^a2Substituted C_1-6An alkyl group.

In some embodiments, R¹And R²Together form optionally 1,2 or 3 independently selected R⁸A 4-7 membered heterocycloalkyl substituted with a substituent.

In some embodiments, R¹And R²Together with the N to which it is attached, form a ring selected from: morpholinyl, thiomorpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, azetidinyl and 3-oxa-8-azabicyclo [3.2.1]Octyl, each of which is optionally substituted with 1,2 or 3 independently selected R⁸And (4) substitution.

In some embodiments, R¹And R²Together with the N to which it is attached, form a ring of the formula:

in some embodiments, R⁸Is selected from halo, C_1-6Alkyl radical, C_1-6Haloalkyl, CN, NO₂、OR^a2、C(O)R^b2、C(O)NR^c2R^d2、C(O)OR^a2、NR^c2R^d2、NR^c2C(O)R^b2、NR^c2S(O)₂R^b2、S(O)₂R^b2And S (O)₂NR^c2R^d2。

In some embodiments, R⁸Is selected from C_1-6Alkyl and OR^a2. In some embodiments, R⁸Is selected from C_1-6An alkyl group. In some embodiments, R⁸Is OH. In some embodiments, R⁸Is C_1-6An alkoxy group.

In some embodiments, R³Is selected from H and C_1-6An alkyl group. In some embodiments, R⁴Is selected from H and C_1-6An alkyl group. In some embodiments, R³And R⁴Each is H. In some embodiments, R³And R⁴One of which is H and the other is C_1-6An alkyl group.

In some embodiments, R⁵The bond between Y and R being a single bond⁵Is a 5-10 membered heteroaryl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3。

In some embodiments, R⁵The bond between Y and R being a double bond⁵Is CR^BR^C。

In some embodiments, R^BIs selected from H and C_1-6An alkyl group.

In some embodiments, R^BIs H.

In some embodiments, R^CIs a 5-10 membered heteroaryl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3。

In some embodiments, R^CIs selected from indolyl, pyridinyl, pyrrolyl and thienyl, each of which is optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3. In some embodiments, R^CIs selected from indolyl, pyridinyl, pyrrolyl and thienyl, each of which is optionally substituted with 1,2 or 3 substituents independently selected from: methyl, trifluoromethyl, methoxy, fluorine, chlorine, bromine, CN, NO₂Amino, dimethylamino, NHC (O) CH₃And NHS (O)₂CH₃。

In some embodiments, R^CIs indolyl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3。

In some embodiments, R^CIs indolyl, optionally substituted with 1,2 or 3 substituents independently selected from: methyl, trifluoromethylMethoxy, fluoro, chloro, bromo, CN, NO₂Amino, dimethylamino, NHC (O) CH₃And NHS (O)₂CH₃。

In some embodiments, R^CIs C_6-10Aryl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3. In some embodiments, R^CIs phenyl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3。

In some embodiments, R^CIs phenyl, optionally substituted with 1,2 or 3 substituents independently selected from: methyl, trifluoromethyl, methoxy, fluorine, chlorine, bromine, CN, NO₂Amino, dimethylamino, NHC (O) CH₃And NHS (O)₂CH₃。

In some embodiments, R⁴And R⁵And R⁴Y and N attached together form a 5-14 membered heteroaryl group, optionally with 1,2 or 3 independently selected from R⁹Is substituted with the substituent(s). In some embodiments, and R⁵And R⁴Y and N connected together form a pyridazinyl or pyrazolyl ring, each of which may be optionally fused to other rings, and optionally 1,2 or 3 independently selected from R⁹Is substituted with the substituent(s).

In some embodiments, R⁹Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_1-6Haloalkyl, OR^a3、SR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、OC(O)R^b3、OC(O)NR^c3R^d3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3C(O)NR^c3R^d3、NR^c3S(O)₂R^b3、NR^c3S(O)₂NR^c3R^d3、S(O)₂R^b3And S (O)₂NR^c3R^d3。

In some embodiments, R⁹Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、C(O)R^b3、C(O)NR^c3R^d3、C(O)OR^a3、NR^c3R^d3、NR^c3C(O)R^b3、NR^c3C(O)OR^a3、NR^c3S(O)₂R^b3、S(O)₂R^b3And S (O)₂NR^c3R^d3。

In some embodiments, R⁹Is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3。

In some embodiments, R^a1、R^c1And R^d1Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group. In some embodiments, each R is^b1Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group.

In some embodiments, R^a2、R^c2And R^d2Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group. In some embodiments, each R is^b2Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group. In some embodiments, each R is^a2Is selected from H and C_1-6An alkyl group.

In some embodiments, R^a3、R^c3And R^d3Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group. In some embodiments, each R is^b3Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group.

In some embodiments:

R¹、R³and R⁴Each is H;

R²is optionally via OR^a2Substituted C_1-6An alkyl group;

R^a2is selected from H and C_1-6An alkyl group.

R⁵The bond between Y and Y is a double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^c；

R⁷Is C_1-6An alkyl group;

R^Bis H; and is

R^CIs indolyl.

In some embodiments:

R¹and R²Each independently selected from optionally via OR^a2Substituted C_1-6An alkyl group;

R³and R⁴Each is H;

R^a2is selected from H and C_1-6An alkyl group;

R⁵the bond between Y and Y is a double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^c；

R⁷Is C_1-6An alkyl group;

R^Bis H; and is

R^CIs indolyl.

In some embodiments:

R¹and R²Together form a 4-7 membered heterocycloalkyl group, optionally substituted with 1,2 or 3 independently selected R⁸Substituent group substitution;

each R⁸Is selected from C_1-6Alkyl and OR^a2；

R^a2Is selected from H and C_1-6An alkyl group;

R³and R⁴Each is H;

R⁵the bond between Y and Y is a double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^C；

R^a1、R^c1And R^d1Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group;

each R^b1Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group;

R^Ais selected from H, C_1-6Alkyl and C_1-6A haloalkyl group;

R^Bis H.

R^CIs a 5-10 membered heteroaryl, optionally substituted with 1,2 or 3 substituents independently selected from: halogen radical, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a3、NR^c3R^d3、NR^c3C(O)R^b3And NR^c3S(O)₂R^b3；

R^a3、R^c3And R^d3Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group; and is

Each R^b3Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group.

In some embodiments:

R¹and R²Together with the N to which it is attached, form a ring selected from: morpholinyl, thiomorpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, azetidinyl and 3-oxa-8-azabicyclo [3.2.1]Octyl, each of which is optionally substituted with 1,2 or 3 independently selected R⁸And (4) substitution.

R⁸Is selected from C_1-6Alkyl and OR^a2；

R^a2Is selected from H and C_1-6An alkyl group;

R³and R⁴Each is H;

R⁷is selected from halo, CN, NO₂、C_1-6Alkyl radical, C_1-6Haloalkyl, OR^a1、NR^c1R^d1、NR^c1C(O)R^b1And NR^c1S(O)₂R^b1；

R^a1、R^c1And R^d1Each independently selected from H, C_1-6Alkyl and C_1-4A haloalkyl group;

each R^b1Independently selected from C_1-6Alkyl and C_1-4A haloalkyl group;

R⁵the bond between Y and Y is a double bond;

y is N;

R⁶is absent;

R⁵is CR^BR^C；

R^AIs H;

R^Bis H; and is

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is not:

in some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is any one of the compounds provided in table 8, or a pharmaceutically acceptable salt thereof.

In some embodiments, the salt of the compound of formula I is formed between an acid group and a base of the compound, such as an amino functional group, or between a base and an acid group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.

In some embodiments, acids commonly used to form pharmaceutically acceptable salts of compounds of formula I include inorganic acids such as hydrogen disulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid; and organic acids such as p-toluenesulfonic acid, salicylic acid, tartaric acid, ditartaric acid, ascorbic acid, maleic acid, benzenesulfonic acid (besylic acid), fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid (benzanesulfonic acid), lactic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid, and related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, decanoate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, iodide, acetate, propionate, caprate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, suberate, sebacate, cap, Phenylpropionates, phenylbutyrates, citrates, lactates, beta-hydroxybutyrate, glycolates, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, mandelates, and others. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids (e.g., hydrochloric acid and hydrobromic acid), and especially those formed with organic acids (e.g., maleic acid).

In some embodiments, bases commonly used to form pharmaceutically acceptable salts of compounds of formula I include alkali metal hydroxides, including sodium, potassium, and lithium; alkaline earth metal hydroxides, such as calcium and magnesium; other metal hydroxides, such as aluminum and zinc; ammonia, organic amines, such as unsubstituted or hydroxy-substituted monoalkylamines, dialkylamines or trialkylamines, dicyclohexylamines; tributylamine; pyridine; n-methyl, N-ethylamine; diethylamine; triethylamine; mono- (2-OH- (C1-C6) -alkylamine), bis- (2-OH- (C1-C6) -alkylamine), or tris- (2-OH- (C1-C6) -alkylamine), such as N, N-dimethyl-N- (2-hydroxyethyl) amine or tris- (2-hydroxyethyl) amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; a pyrrolidine; and amino acids such as arginine, lysine, and the like.

In some embodiments, the compound of formula I, or a pharmaceutically acceptable salt thereof, is substantially isolated.

Preparation method

The compounds of formula (I), including salts thereof, may be prepared using known organic synthesis techniques and may be synthesized according to any of a number of possible synthetic routes. Those skilled in the art know how to select and implement appropriate synthetic schemes and understand that a wide suite of synthetic organic reactions can potentially be employed in the synthesis of the compounds provided herein.

Suitable synthetic methods for starting materials, intermediates and products can be identified by reference, including reference sources such as: heterocyclic Chemistry development (Advances in Heterocyclic Chemistry), Vol.1-107 (Elsevier, 1963-; journal of Heterocyclic chemicals (Journal of Heterocyclic Chemistry) volume 1-49 (Journal of Heterocyclic chemicals, 1964-; carreira et al, (ed) Science of Synthesis, Vol.1-48 (2001-; 2011/1-4; 2012/1-2(Thieme, 2001-; katritzky et al, (ed.) synthetic Organic Functional Group Transformations (synthetic Organic Functional Group Transformations), (Pagamon Press, 1996); Katritzky et al, (ed.) synthetic Organic Functional Group Transformations II (Elsevier, 2 nd edition, 2004); Katritzky et al (edited.) synthetic Heterocyclic chemicals (synthetic Heterocyclic Chemistry) (Pagamon Press, 1984); Katritzky et al, synthetic Heterocyclic chemical II, (Pagamon Press, 1996); Smith et al, McHigh Organic Chemistry: Reactions, Mechanisms and structures (March's Advanced Organic Chemistry: Reactions, Mechanisms, mechanics, and structures), 6 th edition (Wiley, 2007); Trost et al (synthetic Organic Synthesis, 1991); synthetic Organic Synthesis (synthetic).

The reactions used to prepare the compounds provided herein can be carried out in suitable solvents that can be readily selected by those skilled in the art of organic synthesis. Suitable solvents may be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, for example, at a temperature in the range from the freezing temperature of the solvent to the boiling temperature of the solvent. A given reaction may be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, a suitable solvent for the particular reaction step may be selected by the skilled artisan.

The preparation of the compounds provided herein may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the choice of an appropriate protecting group, can be readily determined by one skilled in the art. Protecting group chemistries can be found, for example, in: p.g.m.wuts and t.w.greene, Protective Groups in Organic Synthesis (Protective Groups in Organic Synthesis), 4 th edition, willy & Sons, Inc., New York (2006).

Application method

Cancer treatment

In some embodiments, the compounds of the present disclosure inhibit phosphatidylinositol-3-phosphate 5-kinase type III (PIKfyve) in vitro, in vivo, or ex vivo. Inhibition of PIKfyve in cells induces cytoplasmic vacuolization and subsequent cell death. The role of cytoplasmic vacuolization in cell death and survival is described, for example, in Oncotarget,2016,7(34), 55863-. That is, the compounds can be used to induce cancer cell death. Accordingly, in some embodiments, the present application provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes the step of identifying an individual in need of cancer treatment. This step may include diagnosing the individual with cancer by a treating physician.

In some embodiments, the cancer is selected from the group consisting of: sarcoma, angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma, lipoma, teratoma (teratoma), lung cancer, breast cancer, squamous cell bronchial carcinoma, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, alveolar bronchiolar carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma (chondromatous hamartoma), mesothelioma, gastrointestinal cancer, esophageal cancer, squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma, gastric cancer, carcinoma, lymphoma, leiomyosarcoma, pancreatic cancer, pancreatic ductal adenocarcinoma (ductal adenocarcinomas), insulinoma, glucagon tumor, gastrinoma, carcinoid-like tumor (carcinomatoid), vasoactive intestinal peptide tumor, small intestinal cancer, adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, adenofibroma, adenocarcinoma, adenofibroma, adenocarcinoma, and adenocarcinoma, Fibroids, large or colon carcinoma, tubular adenomas, villous adenomas, hamartoma, leiomyoma, genitourinary tract carcinoma (genic cervical tract carcinoma), renal carcinoma, adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia, bladder carcinoma, urethral carcinoma, squamous cell carcinoma, transitional cell carcinoma, prostate carcinoma, testicular carcinoma, seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumor (enomatoid tumor), lipoma, hepatoma, hepatocellular carcinoma (hepatoma hepatocytology), cholangiocarcinoma, hepatoblastoma, hemangioma, osteocarcinoma, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrocytoma, chondrocytoma, Ewing's sarcoma (Ewing sarcoma), hemangioma, Ewing's sarcoma, fibroblastic sarcoma, fibroadenoma, fibroblastic sarcoma, chondroblastic sarcoma, Ewing's sarcoma, and Ewing's sarcoma, Malignant lymphoma (reticulosarcoma), multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma (osteochondroblastoma) (osteochondral exostoses), benign chondroblastoma, chondrmucomyxoid fibroma, osteoid osteoma giant cell tumor, nervous system cancer, cranial cancer, osteoma, hemangioma, granulation tumor, xanthoma, osteitis deformans (osteitis deformans), meningioma cancer, meningiosarcoma (meniacoma), glioma, brain cancer, astrocytoma, medulloblastoma, glioma, ependymoma (ependomoma), blastoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwanoma, retinoblastoma, congenital tumor, spinal cord cancer, neurofibroma, meningioma, glioma, endometrial cancer, uterine cancer, cervical cancer, Cervical cancer, pre-neoplastic cervical dysplasia (pre-cervical dysplasia), ovarian cancer, ovarian carcinoma, serous cystadenocarcinoma (serous cystadenocarcinomas), mucinous cystadenocarcinoma, unclassified carcinoma, granulosa-thecal cell tumor (granulosa-theca cell tumor), sertraline leiomycosis (Sertoli Leydig cell tumor), dysgerminoma, malignant teratoma, vulvar cancer, squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma, vaginal cancer, clear cell carcinoma (clear cell carcinoma), squamous cell carcinoma, botryoid sarcoma, embryonal rhabdomyosarcoma, fallopian tube carcinoma, hematological cancer, lymphoblastic leukemia, acute myelogenous leukemia (acute myelogenous leukemia; chronic myelogenous leukemia), chronic myelogenous leukemia (chronic myelogenous leukemia), acute myelogenous leukemia (leukemia; acute myelogenous leukemia; CML lymphoma; acute myelogenous leukemia; CML leukemia; acute myelogenous leukemia; myelogenous leukemia, etc.) Chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome (myelodysplastic syndrome), lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (malignant lymphoma), Waldenstrom's macroglobulinemia, skin cancer, malignant melanoma, basal cell carcinoma, colorectal cancer, squamous cell carcinoma, kaposi's sarcoma, nevus dysplastic nevus, lipoma, colorectal carcinoma, osteosarcoma, pancreatic cancer, hemangioma (angioma) skin fibroma, keloid, adrenal cancer, and neuroblastoma. In some embodiments, the cancer is lymphoblastic leukemia, lymphoma, colorectal cancer, glioblastoma multiforme (GBM), medulloblastoma, colorectal carcinoma, osteosarcoma, or pancreatic cancer.

Inflammation of

In some embodiments, a compound of the invention suppresses IL12 and/or IL23 production in vitro, in vivo, or ex vivo. Inhibition of IL12/23 in cells reduces the pathology of inflammatory diseases or conditions. Thus, the compounds of the present application are useful for treating or preventing inflammatory disorders or alleviating the symptoms associated with such disorders. Such conditions include sepsis (e.g., acute sepsis), alopecia, hearing loss syndrome, gout, arthritis, rheumatoid arthritis, cirrhosis, inflammatory bowel disease, Ankylosing Spondylitis (AS), antiphospholipid antibody syndrome (APS), myositis, scleroderma, Sjogren's syndrome, generalized lupus erythematosus, vasculitis, familial mediterranean fever, neonatal onset multiple system inflammatory disease (neonatal onset multiple system inflammation disease), Bellevis' disease, skin disease, type 1 diabetes, autoimmune disease, psoriasis, psoriatic arthritis, multiple sclerosis, Edison's disease, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, acute and chronic mastitis, acute and chronic mastitis, AS well AS acute and chronic inflammatory diseases, and inflammatory diseases, including Alzheimer's, psoriasis, encephalomyelitis, postinfectious small encephalitis (postinfectious cerebellis), neuromyelitis optica (e.g., Devick disease), encephalitis, metabolic encephalopathy, asthma, periodontitis, ulcerative colitis, Crohn's disease, sinusitis, and peptic ulcer. Symptoms associated with inflammatory conditions typically include chronic pain, redness, swelling of joints and other tissues, stiffness, heat, accumulation of blood proteins in organs, hair loss, fatigue, and damage to normal tissues. The compounds of the present application are useful for alleviating these symptoms.

In one example, the IL12/23-T helper 17 pathway acts as a mediator of psoriatic inflammation. Apilimod is a small molecule inhibitor of the lipid kinase PIKfyve originally found in high throughput screening for inhibitors of IL-12:

apilimod suppressed IL12 and IL23 produced by bone marrow cells, and oral administration suppressed TH1 but not TH2 responses in mice. Oral administration to mice significantly reduced inflammatory histopathological skin changes and reduced IFN- γ production by ex vivo cells. Clinical trials in humans with stable psoriasis vulgaris skin plaques (see Wada, y. et al, Apilimod inhibits IL-12 and IL-23 production and reduces dendritic cell infiltration of psoriasis (Apilimod inhibition of the production of IL-12 and IL-23 and recycling dendritic cell infiltration in psoriasis.) public science library-integrated (PLoS One),2012.7(4): page e 35069) found considerable improvement in the histological structure and clinical measurements of the oral administration of Apilimod causing disease activity. IL12 and IL23 expression was significantly reduced in skin lesions. At the highest dose level (70 mg once daily), 47% of patients had histological improvement and a PASI (psoriasis area and severity index) score with an average improvement of 46%. However, the cut-off value for the shift to the new phase II trial did not fit and the authors attributed an inadequate response (which was superior to the intravenous agent eurekinumab) to the inability to reach adequate drug levels. Apilimod has dose-limiting toxicity (due to headache, flushing, dysesthesia, dizziness, paresthesia) at 105mg twice daily. As shown in this application, example 3 more specifically targets PIKfyve even more than apilimod, and is more effective and less toxic than apilimod in blocking IL12/23 secretion. Thus, the compounds of the present disclosure may be an effective oral treatment of humans suffering from psoriasis and other inflammatory conditions (e.g., where IL12/23 is implicated).

Combination therapy

In some embodiments, the method of treating a cancer or inflammatory disease or condition in a subject further comprises administering to the subject an additional therapeutic agent, or a pharmaceutically acceptable salt thereof. In this method, the compound of formula (I) and the additional therapeutic agent may be administered to the subject simultaneously (e.g., in the same dosage form or in separate dosage forms) or sequentially (e.g., the additional therapeutic agent may be administered before or after the compound of formula (I) or pharmaceutically acceptable salt thereof).

Suitable examples of additional therapeutic agents include anti-HER 2 agents (e.g., trastuzumab (trastuzumab), pertuzumab (pertuzumab), lapatinib (lapatinib)), pain relief agents (e.g., non-steroidal anti-inflammatory drugs such as celecoxib (celecoxib) or rofecoxib (rofecoxib)), anti-emetic agents or additional anti-cancer agents (e.g., paclitaxel, european paclitaxel, daunorubicin (daunorubicin), epirubicin (epirubicin), fluorouracil, melphalan (melphalan), cisplatin, carboplatin, cyclophosphamide, mitomycin (mitomycin), methotrexate, mitoxantrone, etc.), anti-HER 2 agents (e.g., trastuzumab (trastuzumab), pertuzumab (pertuzumab), pain relief agents (e.g., non-steroidal anti-inflammatory drugs such as celecoxib (celecoxib) or rofecoxib), antiQuinone, vinblastine, vincristine, ifosfamide, teniposide (teniposide), etoposide (etoposide), bleomycin (bleomycin), leucovorin, paclitaxel, herceptin (herceptin), avastin (avastin), cytarabine (cytarabine), actinomycin (dactinomycin), interferon alpha, streptozotocin (streptozocin), prednisolone (prednisolone), irinotecan (irinotecan), sulindac (sulindac), 5-fluorouracil, capecitabine (capecitabine), oxaliplatin (oxalipiptin)/5 FU, abiraterone (abiraterone), letrozole (letrozole), 5-aza/romidepsin (romidepsin), or procarbazine). Other examples of additional therapeutic agents include polygamide (nexavar), sunitinib (sutent), torisel (torsel), femitor (afinitor) (everolimus), axitinib (axitinib), pazopanib (pazopanib), lenvatinib (levatinib), interleukin-2, and combinations thereof. In some embodiments, the method of treating cancer in a subject further comprises administering to the subject a proteasome inhibitor. Exemplary proteasome inhibitors include lactacystin (lactocystin), bortezomib (bortezomib), disulfiram, salinosporamide A, carfilzomib (carfilzomib), ONX0912, CEP-18770, MLN9708, epoxymicin (epoxymicin), and MG 132. Non-limiting examples of proteasome inhibitors include marezomib (NPI-0052), bortezomibAnd carfilzomibIn some embodiments, the additional therapeutic agent is a BRAF-V600E inhibitor (e.g., vemurafenib).

In some embodiments, the additional therapeutic agent comprises an anti-inflammatory agent. Suitable examples include non-steroidal anti-inflammatory drugs (NSAIDs) such as celecoxib, rofecoxib, ibuprofen, naproxen, aspirin, diclofenac, sulindac, oxaprozin, piroxicam (piroxicam), indomethacin (indomethacin), meloxicam (meloxicam), fenoprofen (fenoprofen), diflunisal (diflunisal), methotrexate, BAY 11-7082, or pharmaceutically acceptable salts thereof. Suitable examples of steroidal anti-inflammatory agents include cortisol, corticosterone, hydrocortisone, aldosterone, deoxycorticosterone, triamcinolone acetonide (triamcinolone), bardoxolone (bardoxolone), methylprednisolone, triamcinolone acetonide, cortisone (cortisone), prednisone (prednisone), and methylprednisolone, or a pharmaceutically acceptable salt thereof. In some embodiments, the anti-inflammatory agent is selected from: itumumab (ixekizumab), secukinumab (anti-IL 17A), guseculizumab (anti-IL 23), TNF inhibitors (adalimumab), etanercept (etaneracypt), infliximab (infliximab), and ustekumab (anti-IL 12/23.) the compounds of the present application can also be used in combination with non-specific immunosuppressive agents, including phototherapy, laser exposure, cyclosporine, and methotrexate.

Pharmaceutical composition

The present application also provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) as disclosed herein or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise any of the additional therapeutic agents described herein (e.g., vemurafenib). In certain embodiments, the present application also provides pharmaceutical compositions and dosage forms comprising any of the additional therapeutic agents described herein. A carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, is not deleterious to the recipient thereof in the amount used in the medicament.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of the present application include (but are not limited to): ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (e.g. human serum albumin), buffer substances (e.g. phosphate esters), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat).

The composition or dosage form may contain in the range of 0.005% to 100% of any of the compounds and therapeutic agents described herein, with the remainder made up of suitable pharmaceutically acceptable excipients. It is contemplated that the compositions may contain from 0.001% to 100% of any of the compounds and therapeutic agents provided herein, in one embodiment, from 0.1% to 95%; in another embodiment, 75% -85%; in another embodiment, 20% -80%, wherein the remainder can be made of any pharmaceutically acceptable excipient or any combination of such excipients described herein.

Routes of administration and dosage forms

The pharmaceutical compositions of the present application include those suitable for any acceptable route of administration. Acceptable routes of administration include (but are not limited to): buccal, dermal, endocervical, intranasal, intraparanasal, intratracheal, enteral, peridural, interstitial, intraabdominal, intraarterial, intrabronchial, intracapsular, intracerebral, intracisternal, intracoronary, intracutaneous, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, ileocecal, intralymphatic, intramedullary, intracerebroventricular, intramuscular, intranasal, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinus, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal feeding, oral, parenteral, transdermal, epidural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, tracheal, ureteral, transurethral, and vaginal.

The compositions and formulations described herein may be presented in unit dosage forms, such as tablets, sustained release capsules, and liposomes, and may be prepared by any of the methods well known in the pharmaceutical arts. See, for example, remington: pharmaceutical sciences and practices (Remington: The Science and Practice of Pharmacy), Lepidote Williams and Wilkins Press (Lippincott Williams & Wilkins), Ballmore, Maryland (Baltimore, MD) (20 th edition 2000). Such preparation methods include the step of bringing into association the ingredients, such as the carrier constituting one or more accessory ingredients, with the molecule to be administered. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, liposomes or finely divided solid carriers or both, and then, if necessary, shaping the product.

In some embodiments, any of the compounds and therapeutic agents disclosed herein are administered orally. Compositions of the present application suitable for oral administration may be presented as discrete units, such as capsules, sachets (sachets), granules or tablets, each containing a predetermined (e.g., effective) amount of the active ingredient; a powder or granules; solutions or suspensions in aqueous or non-aqueous liquids; an oil-in-water liquid emulsion; a water-in-oil type liquid emulsion; filling liposome; or as a bolus, etc. Soft gelatin capsules may be suitable to contain such suspensions, which may advantageously increase the rate of absorption of the compound. In the case of tablets for oral use, commonly used carriers include lactose, sucrose, glucose, mannitol, as well as silicic acid and starch. Other acceptable excipients may include: a) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; c) humectants, such as glycerol; d) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents, such as paraffin; f) absorption accelerators, such as quaternary ammonium compounds; g) wetting agents, such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. For oral administration in capsule form, suitable diluents include lactose and anhydrous corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added. Compositions suitable for oral administration include buccal tablets comprising a flavoured base, typically sucrose and acacia or tragacanth; and tablets comprising the active ingredient on an inert basis, such as gelatin and glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include: aqueous and non-aqueous sterile injection or infusion solutions that may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injection, physiological saline (for example, 0.9% physiological saline solution), or 5% dextrose solution, immediately prior to use. Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules and tablets. Injectable solutions may be in the form of, for example, sterile injectable aqueous or oleaginous suspensions. Such suspensions may be formulated according to the techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1.3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant.

For rectal administration, the pharmaceutical compositions of the present application may be administered in the form of suppositories. These compositions can be prepared by mixing the compounds of the present application with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the active components. Such materials include (but are not limited to): cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the present application may be administered by nasal spray or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in physiological saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, U.S. patent No. 6,803,031. Additional formulations and methods for intranasal administration are found in Ilium, L., J.Pharmacol (JPharm Pharmacol),56:3-17,2004 and Ilium, L., European J.Pharm Sci 11:1-18,2000.

The topical compositions of the present disclosure may be prepared and used in the form of spray sprays, creams, emulsions, solids, liquids, dispersions, foams, oils, gels, hydrogels, lotions, mousses, ointments, powders, patches, pomades, solutions, pump sprays, sticks, towelettes, soaps, or other forms commonly used in the field of topical administration and/or cosmetic and skin care formulations. The topical composition may be in the form of an emulsion. Topical administration of the pharmaceutical compositions of the present application is particularly useful when the desired treatment involves areas or organs that are easily accessible by topical administration. In some embodiments, the topical composition comprises a combination of any of the compounds disclosed herein and a therapeutic agent, and one or more additional ingredients, carriers, excipients, or diluents, including (but not limited to): absorbents, anti-irritants, anti-acne agents, preservatives, antioxidants, colorants/pigments, lubricants (moisturizers), emulsifiers, film forming/retaining agents, fragrances, leave-on exfoliants, prescription medications, preservatives, detergents, silicones, skin-friendly/restoratives, slip agents, sunscreen actives, surfactants/cleansers, penetration enhancers, and thickeners.

The compounds and therapeutic agents of the present application can be incorporated into compositions for coating implantable medical devices, such as prostheses, prosthetic valves, vascular grafts, stents, or catheters. General preparation of suitable coatings and coated implantable devices are known in the art and described in U.S. patent No. 6,099,562; 5,886,026, and 5,304,121. The coating is typically a biocompatible polymeric material such as hydrogel polymers, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coating may also optionally be covered by an outermost coating of a suitable fluorosilicone, polysaccharide, polyethylene glycol, phospholipid, or combinations thereof, to impart a controlled release profile in the composition. Coatings for use in an invasive device will be included within the definition of a pharmaceutically acceptable carrier, adjuvant or vehicle, as those terms are used herein.

According to another embodiment, the present application provides an implantable drug delivery device impregnated with or containing a compound or therapeutic agent, or a composition comprising a compound or therapeutic agent of the present application, such that the compound or therapeutic agent is released from the device and is therapeutically active.

Dosage and regimen

In the pharmaceutical compositions of the present application, the compound of formula (I) is present in an effective amount (e.g., a therapeutically effective amount).

The effective dosage may vary depending on the disease to be treated; the severity of the disease; the route of administration; sex, age and general health of the individual; the excipient is used; possibility of co-use with other therapeutic treatments (e.g. use of other agents); and the judgment of the treating physician.

In some embodiments, an effective amount of a compound of formula (I) may be, for example, from about 0.001mg/kg to about 500mg/kg (e.g., from about 0.001mg/kg to about 200 mg/kg; from about 0.01mg/kg to about 150 mg/kg; from about 0.01mg/kg to about 100 mg/kg; from about 0.01mg/kg to about 50 mg/kg; from about 0.01mg/kg to about 10 mg/kg; from about 0.01mg/kg to about 5 mg/kg; from about 0.01mg/kg to about 1 mg/kg; from about 0.01mg/kg to about 0.5 mg/kg; from about 0.01mg/kg to about 0.1 mg/kg; from about 0.1mg/kg to about 200 mg/kg; from about 0.1mg/kg to about 150 mg/kg; from about 0.1mg/kg to about 100 mg/kg; from about 0.1mg/kg to about 10 mg/kg; from about 0.1 mg/kg; and about 1 mg/kg; from about 1 mg/kg; and about 1.1 mg/ To about 5 mg/kg; about 0.1mg/kg to about 2 mg/kg; about 0.1mg/kg to about 1 mg/kg; or about 0.1mg/kg to about 0.5 mg/kg).

In some embodiments, the effective amount of a compound of formula (I) is about 0.1mg/kg, about 0.5mg/kg, about 1mg/kg, about 2mg/kg, or about 5 mg/kg.

The aforementioned dose can be administered daily (e.g., in a single dose or in divided doses of two or more times, e.g., once daily, twice daily, three times daily) or non-daily (e.g., every other day, every second day, every third day, once weekly, twice weekly, biweekly, monthly).

Reagent kit

The invention also includes pharmaceutical kits suitable, for example, for the treatment of the disorders, diseases, and conditions mentioned herein, comprising one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure. Such kits may further include one or more of a variety of conventional pharmaceutical kit components, such as containers with one or more pharmaceutically acceptable carriers, additional containers, and the like, if desired. Instructions may also be included in the kit as inserts or labels indicating the amounts of the components to be administered, the instructions for administration and/or the instructions for mixing the components. The kit can optionally include any of the additional therapeutic agents described herein (e.g., vemurafenib) or any of the pharmaceutically acceptable salts thereof in any of the amounts and dosage forms described herein.

Definition of

As used herein, the term "EC₅₀By (μ M) "is meant the μ M concentration of compound required for 50% activity in the vacuolar assay.

As used herein, the term "about" means "approximately" (e.g., plus or minus approximately 10% of the indicated value).

In various places in the specification, substituents of the compounds of the invention are disclosed in groups or ranges. It is specifically contemplated that the invention includes each and every individual subcombination of the members of such groups and ranges. For example, the term "C_1-6Alkyl is particularly desirably methyl, ethyl, C individually disclosed₃Alkyl radical, C₄Alkyl radical, C₅Alkyl and C₆An alkyl group.

Various aryl, heteroaryl, cycloalkyl and heterocycloalkyl rings are described at various places in this specification. Unless otherwise specified, these rings may be attached to the rest of the molecule at any ring member as permitted by valency. For example, the term "pyridine ring" or "pyridyl" may refer to a pyridin-2-yl, pyridin-3-yl, or pyridin-4-yl ring.

In addition, it is to be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

The term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more multiply unsaturated rings of aromatic character (i.e., having (4n +2) delocalized n (pi) electrons, where n is an integer).

The term "n-member" (where n is an integer) generally describes the number of ring-forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridinyl is an example of a 6-membered heteroaryl ring, and 1,2,3, 4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl ring.

As used herein, the phrase "optionally substituted" means unsubstituted or substituted. The substituents are independently selected and may be in any chemically accessible position. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced with a substituent. A single divalent substituent (e.g., pendant oxy group) may replace two hydrogen atoms. It is understood that substitution at a given atom is limited by valence.

Throughout the definitions, the term "Cn-m" indicates a range including endpoints, where n and m are integers and indicate the number of carbon atoms. Examples include C_1-4、C_1-6And the like.

As used herein, the term "Cn-m alkyl" used alone or in combination with other terms refers to a saturated hydrocarbon group having n to m carbon atoms, which may be straight or branched. Examples of alkyl moieties include (but are not limited to): chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher carbon number homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2, 2-trimethylpropyl and the like. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

As used herein, the term "C" used alone or in combination with other terms_n-mHaloalkyl "refers to an alkyl group having one halogen atom to 2s + l halogen atoms, which may be the same or different, wherein" s "is the number of carbon atoms in the alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is fluorinated only. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, "C" is_n-mAlkenyl "refers to an alkyl group having one or more double carbon-carbon bonds and having n to m carbon atoms. Example alkenyl groups include (but are not limited to): ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, "C" is_n-mAlkynyl "refers to an alkyl group having one or more three carbon-carbon bonds and having n to m carbon atoms. Example alkynyl groups include (but are not limited to): ethynyl, propyn-1-yl, propyn-2-yl and the like. In some embodiments, an alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, the term "C" used alone or in combination with other terms_n-mAlkylene "refers to a divalent alkyl linking group having n to m carbon atoms. Examples of alkylene groups include (but are not limited to): ethyl-1, 1-diyl, ethyl-1, 2-diyl, propyl-1, 1-diyl, propyl-1, 3-diyl, propyl-1, 2-diyl, butyl-1, 4-diyl, butyl-1, 3-diyl, butyl-1, 2-diyl, 2-methyl-propyl-1, 3-diyl, and the like. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4,2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

As used herein, aThe term "C" used alone or in combination with other terms_n-mAlkoxy "refers to a group of the formula-O-alkyl, wherein the alkyl group has n to m carbon atoms. Example alkoxy groups include (but are not limited to): methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, "C" is_n-mHaloalkoxy "refers to a group of the formula-O-haloalkyl having n to m carbon atoms. An example haloalkoxy is OCF₃. In some embodiments, the haloalkoxy group is only fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "amino" refers to the formula-NH₂A group of (1).

As used herein, the term "C_n-mAlkylamino "refers to a group of the formula-NH (alkyl), wherein alkyl has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylamino groups include (but are not limited to): n-methylamino, N-ethylamino, N-propylamino (e.g., N- (N-propyl) amino and N-isopropylamino), N-butylamino (e.g., N- (N-butyl) amino and N- (tert-butyl) amino), and the like.

As used herein, the term "di (C)_n-mAlkyl) amino "refers to the formula-N (alkyl)₂Wherein the two alkyl groups each independently have n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "C_n-mAlkoxycarbonyl "refers to a group of the formula-C (O) O-alkyl, wherein the alkyl group has from n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkoxycarbonyl groups include (but are not limited to): methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl (e.g., n-propoxycarbonyl and isopropoxycarbonyl), butoxycarbonyl (e.g., n-butoxycarbonyl and tert-butoxycarbonyl), and the like.

As used herein, the term "C_n-mAlkylcarbonyl "refers to a group of the formula-c (o) -alkyl, wherein alkyl has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylcarbonyl include (but are not limited to): methylcarbonyl, ethylcarbonyl, propylcarbonyl (e.g., n-propylcarbonyl and isopropylcarbonyl), butylcarbonyl (e.g., n-butylcarbonyl and t-butylcarbonyl), and the like.

As used herein, the term "C_n-mAlkylcarbonylamino "refers to a group of the formula-nhc (o) -alkyl, wherein alkyl has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "C_n-mAlkanesulfonylamino is of the formula-NHS (O)₂-a radical of an alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "aminosulfonyl" refers to the formula S (O)₂NH₂A group of (1).

As used herein, the term "C_n-mAlkylaminosulfonyl "is defined as having the formula-S (O)₂A group of NH (alkyl), wherein the alkyl has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "di (C)_n-mAlkyl) aminosulfonyl "refers to the formula-S (O)₂N (alkyl)₂Wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "aminosulfonylamino" refers to the formula-NHS (O)₂NH₂A group of (1).

As used herein, the term "C_n-mAlkylaminosulfonylamino is of the formula-NHS (O)₂A group of NH (alkyl), wherein the alkyl has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used hereinBy the term "di (C)_n-mAlkyl) aminosulfonylamino "is intended to mean a compound of formula-NHS (O)₂N (alkyl)₂Wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "aminocarbonylamino", used alone or in combination with other terms, refers to the formula-NHC (O) NH₂A group of (1).

As used herein, the term "C_n-mAlkylaminocarbonylamino "refers to a group of the formula-NHC (O) NH (alkyl), wherein alkyl has from n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "di (C)_n-mAlkyl) aminocarbonylamino "means a compound of the formula-NHC (O) N (alkyl)₂Wherein each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "carbamoyl" refers to a compound of the formula C (O) NH₂A group of (1).

As used herein, the term "C_n-mAlkylcarbamoyl "refers to a group of the formula-c (o) -NH (alkyl), wherein alkyl has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "di (C)_n-mAlkyl) carbamoyl "means a compound of formula-C (O) N (alkyl)₂Wherein the two alkyl groups each independently have n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "thio" refers to a group of the formula-SH.

As used herein, the term "C_n-mAlkylthio "refers to a group of the formula-S-alkyl, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As herein describedAs used, the term "C_n-mAlkylsulfinyl "refers to a group of the formula-s (o) -alkyl, wherein alkyl has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "C_n-mAlkylsulfonyl "means a group of the formula-S (O)₂-a radical of an alkyl group, wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "carbonyl", used alone or in combination with other terms, refers to a-C (═ O) -group, which can also be written as C (O).

As used herein, the term "carboxy" refers to a-C (O) OH group.

As used herein, the term "cyano-C_1-3Alkyl "means a group of the formula- (C)_1-3Alkylene) -CN.

As used herein, the term "HO-C_1-3Alkyl "means a group of the formula- (C)_1-3Alkylene) -OH groups.

As used herein, "halo" refers to F, Cl, Br, or I. In some embodiments, the halo is F, Cl or Br.

As used herein, the term "aryl", used alone or in combination with other terms, refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2,3, or 4 fused rings). The term "C_n-mAryl "refers to an aryl group having n to m ring carbon atoms. Aryl groups include, for example, phenyl, naphthyl, anthryl, phenanthryl, indanyl, indenyl, and the like. In some embodiments, the aryl group has 6 to 10 carbon atoms. In some embodiments, aryl is phenyl or naphthyl.

As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1,2,3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety can be an N-oxide. In some embodiments, heteroaryl is a 5-10 membered monocyclic or bicyclic heteroaryl having 1,2,3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, heteroaryl is a 5-6 membered monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, heteroaryl is a five or six membered heteroaryl ring. A five-membered heteroaryl ring is a heteroaryl having a ring of five ring atoms, wherein one or more (e.g., 1,2, or 3) ring atoms are independently selected from N, O and S. Exemplary five-membered ring heteroaryl groups are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2, 3-triazolyl, tetrazolyl, 1,2, 3-thiadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-triazolyl, 1,2, 4-thiadiazolyl, 1,2, 4-oxadiazolyl, 1,3, 4-triazolyl, 1,3, 4-thiadiazolyl, and 1,3, 4-oxadiazolyl. A six membered heteroaryl ring is a heteroaryl group having a ring with six ring atoms, wherein one or more (e.g., 1,2, or 3) ring atoms are independently selected from N, O and S. Exemplary six-membered ring heteroaryl groups are pyridyl, pyrazinyl, pyrimidinyl, triazinyl and pyridazinyl.

As used herein, "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N or S. Included among heterocycloalkyl groups are monocyclic 4-, 5-, 6-, 7-, 8-, 9-or 10-membered heterocycloalkyl groups. Heterocycloalkyl groups may also include spiro rings. Example heterocycloalkyl groups include pyrrolidin-2-one, 1, 3-isoxazolidin-2-one, pyranyl, tetrahydropyranyl, oxetanyl, azetidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazepine, and the like. The ring-forming carbon atoms and heteroatoms of heterocycloalkyl can optionally be interrupted by 1 or 2 independently selected pendant oxy or sulfide groups (e.g., C (O), S (O), C (S), or S (O))₂Etc.) are substituted. The heterocycloalkyl group may be attached through a ring-forming carbon atom or a ring-forming heteroatom. In some embodiments, the heterocycloalkyl group contains 0 to 3 double bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double bonds. Also included in the definition of heterocycloalkyl are those having a ring fused to a cycloalkyl ring(i.e., having a common bond) to one or more aromatic rings, such as benzo or thienyl derivatives of piperidine, morpholine, azepine, and the like. The heterocycloalkyl group containing a fused aromatic ring can be connected through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. In some embodiments, heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxygenated ring members. In some embodiments, heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1,2,3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxygenated ring members.

In certain places, definitions or embodiments refer to specific rings (e.g., azetidine rings, pyridine rings, etc.). Unless otherwise indicated, these rings may be attached to any ring member, provided that the valency of the atoms is not exceeded. For example, the azetidine ring may be attached at any position on the ring, while the pyridin-3-yl ring is attached at the 3-position.

As used herein, the term "pendant oxy" refers to an oxygen atom as a divalent substituent that when attached to a carbon (e.g., C ═ O) or to a heteroatom that forms a sulfoxide or sulfone group, forms a carbonyl group.

The term "compound" as used herein is meant to include all stereoisomers, geometric isomers, tautomers and isotopes of the depicted structures. Unless otherwise specified, a compound identified herein by name or structure as one particular tautomeric form is intended to include other tautomeric forms.

The compounds described herein can be asymmetric (e.g., have one or more stereocenters). Unless otherwise indicated, all stereoisomers (e.g., enantiomers and diastereomers) are desired. The compounds of the present invention containing asymmetrically substituted carbon atoms may be isolated in optically active or racemic forms. Methods for how to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C ═ N double bonds, N ═ N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as mixtures of isomers or as separate isomeric forms. In some embodiments, the compound has the (R) -configuration. In some embodiments, the compound has a reconfigured form.

The compounds provided herein also include tautomeric forms. The tautomeric forms result from the exchange of a single bond with an adjacent double bond and the concomitant migration of protons. Tautomeric forms include proton transfer tautomers in an isomeric protonated state with the same empirical formula and overall charge. Example proton transfer tautomers include keto-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which the proton may occupy two or more positions of the heterocyclic ring system, such as 1H-imidazole and 3H-imidazole, 1H-1,2, 4-triazole, 2H-1,2, 4-triazole and 4H-1,2, 4-triazole, 1H-isoindole and 2H-isoindole, and 1H-pyrazole and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

The term "cell" as used herein means a cell in vitro, ex vivo or in vivo. In some embodiments, the ex vivo cell may be a portion of a tissue sample excised from an organism (e.g., a mammal). In some embodiments, the in vitro cell can be a cell in a cell culture. In some embodiments, the in vivo cell is a living cell in an organism (e.g., a mammal).

As used herein, the term "contacting" refers to bringing together specified portions in an in vitro system or in an in vivo system. For example, "contacting" PIKfyve with a compound of the invention includes administering a compound of the invention to an individual or patient (e.g., a human) having PIKfyve, and, for example, introducing a compound of the invention into a sample containing cells or a purified preparation containing PIKfyve.

As used herein, the terms "individual (subject)", "patient" are used interchangeably and refer to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses or primates, and most preferably humans.

As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual, or human that is being sought by a researcher, veterinarian, medical doctor, or other clinician.

As used herein, the term "treating" refers to 1) inhibiting a disease; for example, inhibiting a disease, condition, or disorder in an individual who is experiencing or presenting with a pathology or symptom of the disease, condition, or disorder (i.e., arresting further development of the pathology and/or symptom); or 2) alleviating the disease; for example, alleviating (i.e., reversing) a disease, condition, or disorder in an individual who is experiencing or presenting with a pathology or symptom of the disease, condition, or disorder.

Examples of the invention

Example 1- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino-6-morpholinyl-N- (p-tolyl) -1,3, 5-triazin-2-amine

Physical Properties

The molecular weight of the compound is 428.5. It is soluble in DMSO. Additionally, a mixture of DMSO (10%), toluene (20%), and water (70%) can be used to prepare a solution of at least 10 mg/ml.

Chemical synthesis

Step 1

To 2,4, 6-trichloro- [ l,3,5]]Triazine (1.84g, 10mmol) and K₂CO₃(1.38g, 10mmol) to a cold (0-5 ℃ C.) stirred suspension in THF (30mL) was added dropwise a solution of p-toluidine (1.07g, 10mmol) in dry THF (20 mL). Then stirringThe mixture was stirred for 3 hours while slowly warming to ambient temperature. After stirring at ambient temperature for 14 hours, the reaction was monitored by LCMS. The mixture was diluted with EA (200mL) and then acidified with 1N aqueous hydrochloric acid to pH 6-7. The organic phase was separated, washed sequentially with water, saturated aqueous sodium bicarbonate and brine (about 20mL each) and passed over anhydrous Mg₂SO₄And (5) drying. The solution was filtered and concentrated in vacuo on a rotary evaporator to give (4, 6-dichloro- [ l,3, 5) as a yellow solid]Triazin-2-yl) -p-tolyl-amine (2.33g, yield: 91%).

¹H NMR(400MHz,DMSO-d6):δ＝11.05(s,1H),7.47(d,J＝8.4Hz,2H),7.20(d,J＝8.0Hz,2H),2.29(s,3H)。

Step 2

To a stirred solution of (4, 6-dichloro- [ l,3,5] triazin-2-yl) -p-tolyl-amine (2.33g, 9.13mmol) in dioxane (50mL) was added DIEA (1.17g, 9.13mmol) and morpholine (795mg, 9.13mmol), and the resulting mixture was heated at 85 ℃ for 12 hours. The reaction mixture was monitored by LCMS. The reaction mixture was cooled to ambient temperature, diluted with water (100mL) and stirred for 1 hour. The white precipitate was filtered and dried in vacuo to give (4-chloro-6-morpholin-4-yl- [1,3,5] triazin-2-yl) -p-tolyl-amine (1.94g, yield: 69%) as a white solid.

¹H NMR(400MHz,DMSO-d6):δ＝10.03(s,1H),7.50(d,J＝6.4Hz,2H),7.12(d,J＝8.0Hz,2H),2.85-3.60(m,8H).2.26(s,3H)。

Step 3

To a solution of (4-chloro-6-morpholin-4-yl- [1,3,5] triazin-2-yl) -p-tolyl-amine (300mg, 0.98mmol) in dioxane (20mL) was added hydrazine hydrate (2mL) and the mixture was stirred at room temperature for 30 min. The reaction was monitored by LCMS. The mixture was concentrated in vacuo to give (4-hydrazino-6-morpholin-4-yl- [1,3,5] triazin-2-yl) -p-tolyl-amine (252mg, yield: 85%) as a white solid.

¹H NMR(400MHz,DMSO-d6):δ＝8.97(s,1H),7.96(s,1H),7.59(d,J＝8.4Hz,2H),7.03(d,J＝8.0Hz,2H),4.18(s,2H),3.77-3.58(m,8H),2.22(s,3H)。

Step 4

To a solution of (4-hydrazino-6-morpholin-4-yl- [1,3,5] triazin-2-yl) -p-tolyl-amine (100mg, 0.33mmol) in EtOH (20mL) was added 1H-indole-3-carbaldehyde (95.8mg, 0.66mmol) and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated in vacuo to give a residue which was pre-purified by column chromatography followed by preparative HPLC to give {4- [ N' - (1H-indol-3-ylmethylene) -hydrazino ] -6-morpholin-4-yl- [ l,3,5] triazin-2-yl } -p-tolyl-amine (40mg, yield: 28%) as a white solid.

¹H NMR(400MHz,CD₃OD):δ＝8.45-8.34(m,1H),8.27(s,1H),7.71-7.52(m,3H),7.40(d,J＝8.0Hz,1H),7.26-7.08(m,4H).3.92-3.70(m,8H),2.32(s,3H).MS:m/z 429.0(M+H⁺)。

Biological activity

Example 1 at 50nM caused greater than 99% inhibition of PIKfyve. To this extent, no other kinase was blocked by 50nM of example 1, indicating a high selectivity.

Glioblastoma and medulloblastoma

Human glioblastoma multiforme cells (T98G, G22VF, U251) and human medulloblastoma cells (p16, p9) were incubated with various concentrations of example 1 for 72 hours, and then cell viability was assessed by cell titer-Glo luminescence (ATP level) analysis. Depending on the cell line, cells were effectively killed by a certain concentration, as shown in fig. 3.

The method comprises the following steps: example 1 was dissolved in dmso (atcc) at a concentration of 40mM, and this stock was further diluted to 2 x of the highest final working concentration into complete medium consisting of dmem (Life Technologies), penicillin/streptomycin antibiotic solution (Life Technologies) and 10% fbs (seradigm). This stock was dispensed into a white-walled, flat-bottomed 96-well pan (Corning) final column. Serial dilutions (1.5 x or 2 x depending on the assay) were then prepared using complete medium down to the lowest assay concentration of example 1. The final column of the 96-well plate was saved as vehicle only control and DMSO diluted 1:250 was included in the complete medium.

When the confluency of cells was 50% -85%, standard tissue culture methods were used at 5% CO₂Cells grown in 37 ℃ incubator were collected in logarithmic growth phase. To do so, it was washed with 1 × PBS pH 7.4(Life Technologies) at room temperature, then incubated with 0.05% trypsin-EDTA solution (Life technology) until the cells detached clearly from the culture dish. The cell suspension was then pipetted back and forth several times to disperse the cell clusters and then immediately diluted 1:5 into complete medium. Small aliquots of this cell suspension were used for counting on an Accuri C6 cytometer (BD Biosciences) using forward and side scatter to gate live cell populations. The remainder of the suspension was centrifuged at 180 × g, the supernatant removed, and the pellet resuspended in complete medium at a final concentration of 100 cells/μ Ι _. 50 μ L of this suspension was then inoculated into each well of a 96-well plate containing the examples (prepared as described above) such that each well contained 100 μ L of complete medium, 5000 cells,<2 μ L DMSO and example 1 at the indicated concentration.

These plates were placed in 5% CO₂Incubator at 37 ℃. After 72 hours of incubation, the relative ATP content was measured as an indirect indication of the number of viable cells per well. To each well, 100 μ L of cell titer Glo reagent (Promega) that had been prepared according to the manufacturer's instructions was added. This reaction was protected from light and incubated at room temperature for 10 minutes, then the total luminescence was read using a Synergy HI microplate reader (Biotek).

The original luminescence number or relative ATP concentration (reported as the percentage of vehicle-treated controls) is shown. All data points represent the mean of 2-3 independent experimental replicates and error bars represent standard deviations.

Colorectal carcinoma

Human colorectal cancer tumor cell lines were similarly tested for killing by compounds. (ATP activity is defined by the use of cell-titer glow luminescence analysis). As shown in figure 4, some of the colorectal cancer cells (HCT116, HT29) were more sensitive to compound-induced death than brain tumor cell lines. All experiments were performed using RPMI medium (Life Technologies) as the base formulation for "complete" medium. HT29 cells required physical scraping in addition to trypsin to dissociate from tissue culture plastic.

Osteosarcoma

Human osteosarcoma cells (U20S, 143B, MG63 and SAOS2) and human pancreatic cancer cells (Panc-1, CFPAC) were similarly tested for killing by the compounds, as shown in fig. 5. For experiments with 143B and MG63 cells, all experiments in fig. 5 were performed using DMEM: F12 medium (Life Technologies) as the base formulation for "complete" medium. In addition, Alamar Blue was used to analyze the relative number of viable cells per well after 72 hours of growth. For these assays, growth medium was removed from the cells and replaced with fresh ("whole") medium containing 1:10 diluted Alamar Blue reagent (Life Technologies). The plates were incubated in 5% CO₂Incubate at 37 ℃ for 40 minutes, then remove from the incubator and measure fluorescence using a Synergy HI microplate reader (excitation: 560nm emission: 590 nm). Values represent raw light counts minus the value of wells with Alamar Blue reagent (1:10 in medium) but without cells.

Lymphoma (lymphoma)

Murine lymphoma cells (E2409, EC + F) were killed by relatively low concentrations of the compound in the 48 hour assay, with viability determined by cell count, as shown in fig. 6. Murine lymphoma cells (E2409, EC + F) were killed by relatively low concentrations of example 1 in the 48 hour assay. All experiments in fig. 6 were performed using RPMI medium (Life Technologies) as the base formulation, and the medium also contained 1mM sodium pyruvate, 100mM HEPES, 2mM additional L-glutamine, 55 μ M2-mercaptoethanol, and 1 xmem NEAA solution (all from Life Technologies). For these non-adherent cells, trypsin is not necessary. Presto Blue reagent (Life Technologies) was used as an endpoint assay at 30 min incubation time using the same procedure as previously described for Alamar Blue.

Human lymphoma cell lines were incubated with various concentrations of compounds for 72 hours and viability determined by cell counting or by ATP analysis, as shown in figure 7. In lymphoma experiments, RPMI medium (Life Technologies) was used as the base formulation for "complete" medium. For these non-adherent cell lines, trypsin is not required. For "cell count" data, cells were pipetted back and forth to distribute evenly, and then 10 μ Ι _ of suspension was counted using forward and side scatter using an Accuri C6 cytometer to gate live populations.

The vacuolization due to treatment with the compound of example 1 was extremely rapid and became visible by light microscopy in 30 minutes or less. Electron micrographs of murine lymphoma cells treated with vehicle only (left) or example 1 (right) are shown in figure 2.

Autophagy inhibition

Macroautophagy (hereinafter "autophagy") is a process that requires precise fusion of endosomes, autophagosomes, and lysosomes. Lysates from cells treated with example 1 were prepared and probed for LC3-I and LC3-II by Western blotting. It was found that increased levels of LC3-II and increased p62 (chelators) demonstrated increased driving for autophagy in the event that the process could not be completed (i.e., ineffective autophagy). Lysosomes are the ultimate target for proteins intended to be degraded by autophagy, and proteolysis in this organelle depends on cathepsins as well as other enzymes. Cathepsin levels were measured by western blotting and it was found that the mature form became less abundant in glioblastoma cells treated with example 1, while the precursor form (pro-cathepsin) accumulated, indicating that these degraded enzymes could not be delivered to the appropriate low pH compartment.

Figure 23 contains images showing induction of autophagy of example 1 as evidenced by accumulation of LC3 over time. U251 glioblastoma cells were treated with 25, 500 or 20,000nM MC042 for the indicated time periods, and then lysates of LC3 and tubulin were prepared and blotted (internal controls). Starting at 6 hours and becoming more apparent by 24 hours LC3-II (lower band labeled LC3) accumulation, indicating activation of autophagy. However, p62 did not degrade, thus indicating that autophagy was not effective in removing proteins from cells.

Figure 24 shows that in a similar manner, U251 (glioblastoma) and RKO (colon cancer) cells were treated with 1 μ M of example 1 for a specified period of time and then analyzed by western blot for LC3 accumulation. RKO cells had significant and rapid induction of LC3, whereas the induction of U251 was less robust.

Figure 25 shows that example 1 caused the accumulation of uncleaved precursor cathepsin D ("CTSD") and the relative loss of mature cleaved cathepsin. U251 or RKO cells were treated with example 1 at the indicated concentrations for 18 hours and then analyzed by western blotting for cathepsin D and actin (internal controls).

U251 or RKO cells were seeded onto 10cm tissue culture disks and grown under the previously described conditions. When cells were grown in log phase and reached about 60% confluence, example 1 was diluted into growth medium at the indicated concentrations. Cells were in 5% CO₂Incubation at 37 ℃ for 18 hours, then washing and collection as previously described. Cells were pelleted by centrifugation at 180 Xg, resuspended in 20mL of 1 XPBS pH 7.4, and centrifuged again. The supernatant was removed and the pellet was resuspended in 100. mu.L of RIPA buffer containing a mixture of IX protease and phosphatase inhibitors (EMD Millipore) and incubated on ice for 1 hour. After the incubation, the remaining cell debris was pelleted by centrifugation at 17,000 × g, and the lysate was transferred to a clean tube on ice and 100 μ L of Laemlli buffer (BioRad) prepared according to the manufacturer's instructions was added. The tubes were incubated at 100 ℃ for 5 minutes, then vortexed and transferred back onto ice. Proteins from 50. mu.L of each sample were separated by electrophoresis on a 15% Tris-HCl acrylamide gel (Bio Rad) and then transferred to a PVDF-FL membrane (EMD-Millipore). Membranes were incubated at room temperature in SeaBlock reagent (EMD-Millipore) for 30 min, then at 4 ℃ in a medium containing goat-anti-hCathepsin D (1:1000, R)&D systems) and mouse-anti-actin (1:5000, Chemicon) antibodies were diluted in SeaBlock solution and incubated overnight. The membrane was washed 4 times with an excess of 1 × TBS + 1% Tween-20. Then incubated for 2 hours at room temperature in SeaBlock containing IR-dye-binding secondary antibody (Li-Cor) recognizing goats and mice (1:35,000 dilution). After 4 washes with TBS + Tween, an Odyssey swipe was usedThe film was imaged with a scanner (Li-Cor).

Example 2- (E) -N- (4- ((4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-l, 3, 5-triazin-2-yl) amino) phenyl) -5- (2-oxohexahydro-lH-thieno [3,4-d [ imidazol-4-yl) pentanamide

The title compound was prepared using methods and procedures analogous to those described for example 34.

¹H NMR(400MHz,DMSO-d₆):δ＝11.44(brs,1H),10.64(brs,1H),9.80(brs,1H),9.19-9.16(m,1H),8.45-8.44(m,1H),8.32(s,1H),8.07-8.00(m,1H),7.70-7.37(m,5H),7.23-7.15(m,1H),6.48(s,1H),6.38(s,1H),4.32-4.29(m,1H),4.15-4.14(m,1H),3.84-3.83(m,8H),3.16-3.10(m,1H),2.85-2.80(m,1H),2.59-2.57(m,2H),2.39-2.32(m,2H),1.64-1.45(m,4H),1.40-1.33(m,2H)。MS:m/z 656.1(M+H⁺)

Biological activity

Example 2 is a biotin-labeled derivative of the compound of example 1. This compound demonstrates vacuolization activity in cells in culture, its EC₅₀It was 0.008. mu.M. See fig. 9.

Example 3- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl N- (p-tolyl) pyrimidin-2-amine

Chemical synthesis

Step 1

To a solution of 2,4, 6-trichloropyrimidine (1.0g, 5.5mmol) and morpholine (479mg, 5.5mmol) in EtOH (20mL) was added DIPEA (710mg, 5.5mmol) and the reaction stirred at room temperature for 16 h. The reaction solution was concentrated to dryness in vacuo. The residue was purified by means of a silica gel column (PE/EA ═ 50/1) to give 4- (2, 6-dichloropyrimidin-4-yl) morpholine as a white solid (923mg, yield: 72%). MS M/z 233.9(M + H)⁺)。

Step 2

To a solution of 4- (2, 6-dichloropyrimidin-4-yl) morpholine (500mg, 2.2mmol) and p-toluidine (275mg, 2.6mmol) in 1, 4-dioxane (10mL) was added concentrated HCl (6 drops) and the reaction was heated in a microwave for 1 hour at 110 ℃. The reaction mixture was diluted with DCM (20mL) and the mixture was saturated with Na₂CO₃(20mL) washed. The organic phase was concentrated to dryness in vacuo to give crude 4-chloro-6-morpholinyl-N- (p-tolyl) pyrimidin-2-amine (760mg, crude material) as a white solid. MS M/z 305.0(M + H)⁺)。

Step 3

A mixed solution of 4-chloro-6-morpholinyl-N- (p-tolyl) pyrimidin-2-amine (260mg, 0.85mmol) in hydrazine hydrate (10mL) was stirred at 100 ℃ for 16 hours. The reaction mixture is poured into H₂O (10 mL). The mixture was extracted by EA (10 mL. times.2). The combined organic layers were washed with brine (10mL) and Na₂SO₄Dried and concentrated to give crude 4-hydrazino-6-morpholinyl-N- (p-tolyl) pyrimidin-2-amine (230mg, crude material) as a white solid.

Step 4

To a solution of 4-hydrazino-6-morpholinyl-N- (p-tolyl) pyrimidin-2-amine (230mg, 0.85mmol) in MeOH (20mL) was added lH-indole-3-carbaldehyde (123mg, 0.85mmol) and the mixture was stirred at 80 ℃ for 16 h. The mixture was concentrated to dryness in vacuo, and the residue was purified by preparative HPLC to give (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-N- (p-tolyl) pyrimidin-2-amine (17mg, yield: 5%) as a white solid.

¹H NMR(400MHz,CDCl₃):δ＝8.35-8.24(m,3H),7.94(s,1H),7.44(d,J＝8.4Hz,2H),7.39(d,J＝7.1Hz,1H),7.32-7.23(m,3H),7.10(d,J＝8.4Hz,2H),6.82(s,1H),6.05(s,1H),3.81(t,J＝4.4Hz,4H),3.65(t,J＝4.8Hz,4H),2.30(s,3H)。MS:m/z 428.0(M+H⁺)。

Biological activity

See fig. 10. The ECso was 0.004. mu.M.

Glioblastoma and colorectal carcinoma

Glioblastomas (T98G, U251) or colorectal cancer cell lines (HT29, RKO, HCT116) are also sensitive to killing compounds. See fig. 11. Experiments were performed using DMEM-based medium for glioblastoma lines and RMPI-based medium for colon rectal lines.

Malignant peripheral nerve sheath tumor

Example 3 demonstrates increased efficacy against a tumor type known as malignant peripheral schwannoma. The form of cell death caused by the compounds conforms to a mechanism known as "macrofoam death," a non-apoptotic programmed cell death process that has been shown to result from the high activation of oncogenic RAS. For this reason, it is hypothesized that tumor cells of the RAS that have been activated will have increased sensitivity to this compound. Malignant Peripheral Nerve Sheath Tumors (MPNST) occur sporadically in the population and have a very high frequency in patients with the NF1 mutation. NF1 is a RAS inhibitor, and thus tumors that develop in a NF 1-mutant background have prior activation of the RAS signaling pathway. MPNST cell lines (S462, S462TY, 26T, T265, SNF96.2) were tested in DMEM-based medium and were highly sensitive to compound-induced death. See fig. 26.

PK/PD characterization

The compounds were further characterized for PK/PD profile. The half-life after intraperitoneal injection was about 5.5 hours, and the peak concentration after a single 30mg/kg dose was 3268 ng/ml.

Example 4 comparative study on apilimod

Examples 1 and 3 are more selective for PIKfyve than the previously characterized PIKfyve inhibitor apilimod (3-methylbenzaldehyde 2- [6- (4-morpholinyl) -2- [2- (2-pyridinyl) ethoxy-4-pyrimidinyl ] hydrazine, CAS registry No. 541550-19-0):

at a concentration of 50nM, example 1 blocked only 1 out of 468 kinases to less than 1% of the control. It also does not block other kinases to 10% or to 35% of the control activity. In contrast, 50nM apilimod inhibited both kinases to 1% or 10% and 3 kinases to 35% of controls. To check for non-specific binding at higher concentrations, example 1, example 3 and apilimod were tested at 5000 nM. Under these conditions, example 1 blocked 2,4 or 11 kinases to 1%, 10% or 35% activity levels, while apilimod again had lower specificity for blocking 4, 9 and 15 kinases to those three levels. Example 3 blocked only 1,1 and 4 kinases to cutoff levels of 1%, 10% and 35% at 5000nM, indicating that it has the highest selectivity for PIKfyve for all three compounds. Example 3 high selectivity at 5000nM concentration was demonstrated graphically by Treespot kinase dendrograms, with larger spots indicating higher degrees of inhibition. PIKfyve is represented at the lower left of each dendrogram. See fig. 12.

Example 5-synergistic action with Verofinib

General procedure

Immediately prior to seeding the cell suspension onto pre-diluted example 1 or example 3, 25mM vemurafenib stock solution (from seleckchem) in DMSO was diluted 2-fold of the indicated concentration to yield the final IX concentrations for both compounds.

The B-RAF inhibitor vemurafenib (N- (3- (5- (4-chlorophenyl) -lH-pyrrolo [2,3-B ] pyridine-3-carbonyl) -2, 4-difluorophenyl) propane-l-sulfonamide; CAS registry No. 918504-65-1):

reflectively activating ras protooncogenes. Vemurafenib was combined with either example 1 or example 3 treatments. Due to the combination of compounds, a significant increase in cell death was observed, far beyond either.

Results

Referring to fig. 13, U251 glioblastoma cell killing by example 1 alone or in the presence of 10 μ M vemurafenib over a 72 hour time course is shown. It should be noted that at 0 μ M example 1 (leftmost point), vemurafenib had little effect, but at low concentrations of example 1 (e.g., 0.5 μ M), the addition of vemurafenib significantly improved cell killing.

Further examples of synergistic cell killing by vemurafenib in combination with various concentrations of example 1 are shown in fig. 14 and 15 for the indicated cell types.

As shown in fig. 16, example 3 also synergized with vemurafenib in killing U251.

EXAMPLE 6 pharmacokinetic and pharmacodynamic Studies of the Compound of example 1

Species: fasted male C57BL/6 mice.

PK/PD testing of compounds was performed in mice following intraperitoneal, intravenous, or oral administration. See fig. 8. Intraperitoneal administration gives kinetics in which the half-life is 10.1 hours and the maximum concentration after a single 30mg/kg dose is 534 ng/ml.

1 million E2409 murine lymphoma cells were injected into the flank of nude mice and allowed to grow for 4 days, when a small detectable mass was observed on each mouse. Mice were injected daily by intraperitoneal injection of 100 μ l vehicle (10% dmso, 20% cremaphor, 70% water) with or without 1mg of test compound. When the tumor size exceeds 1500mm³Mice were euthanized at time. The treated mice had prolonged survival (p) compared to vehicle-only treated mice<0.02)。

Table l. administration dose

	Intravenous administration of drugs	Intraperitoneal cavity	Through the mouth
				Nominal dose (mg/kg)	5.00	30.0	30.0
Administration dose (mg/kg)	4.75	28.5	28.5

TABLE 2 composition of the doses

Intravenous administration of drugs	In 5mg/mL clear solution of 75% PEG 400/25% water
		Intraperitoneal cavity	6mg/mL of opaque homogeneous suspension in water containing 0.5% MC/0.2% Tween80
Through the mouth	6mg/mL of opaque homogeneous suspension in water containing 0.5% MC/0.2% Tween80

Figure 17 shows the mean plasma concentrations of example 1 after intravenous, intraperitoneal and oral administration. FIGS. 18, 19 and 20 show plasma concentrations of example 1 after intravenous administration at 5mg/kg, intraperitoneal administration at 30mg/kg and oral administration at 30mg/kg, respectively.

TABLE 3 bioavailability of example 1 in mice, 5mg/kg intravenously, plasma (ng/mL).

TABLE 4 bioavailability of example 1 in mice, 30mg/kg intraperitoneally, plasma (ng/mL).

TABLE 5 bioavailability of example 1 in mice, oral 30mg/kg, plasma (ng/mL).

ND ═ undetermined (a parameter undetermined due to an insufficiently defined terminal elimination period). BQL ═ below the lower limit of quantitation (LLOQ). If the adjusted rsq (linear regression coefficient of concentration values over the end period) is less than 0.9, then T1/2 may not be accurately estimated. If AUC_Extra％>20%, then the AUC may not be accurately estimated_0-inf、Cl、MRT_0-infAnd Vd_ss. If AUMC is used_Extra％>20%, then the MRT may not be accurately estimated_0-infAnd Vd_ss. The adjusted linear regression coefficient for the concentration values in the terminal phase is less than 0.9, and it may not be possible to accurately estimate T1/2, a: use of AUC with nominal dose_0-inf(AUC_Extra％<20%) or AUC_0-Iast(AUC_Extra％>20%) the bioavailability (%) was calculated.

Example 7-pharmacokinetic and pharmacodynamic Studies of the example 3 Compound

Species: c57 mice. The administration route is as follows: intravenous and intraperitoneal. Preparing a medium: intravenous and intraperitoneal: 10% DMSO/30% PEG 400/60% saline. Dose level: intravenous (5mg/kg) and intraperitoneal (30 mg/kg). Dose capacity: intravenous (5mL/kg) and intraperitoneal (10 mL/kg). The concentration of the formulation: intravenous (1mg/mL) and intraperitoneal (3 mg/mL). LLOQ: intravenous (2ng/mL) and intraperitoneal (10 ng/mL).

FIGS. 21 and 22 show plasma concentrations (ng/mL) of example 3 in male C57 mice after intravenous administration of 5mg/kg and intraperitoneal administration of 30mg/kg IP, respectively.

TABLE 6 plasma concentration (ng/mL) and PK parameters for example 3 in male C57 mice after intravenous administration of 5 mg/kg.

TABLE 7 plasma concentration (ng/mL) and PK parameters for example 3 in male C57 mice after intraperitoneal administration of 30 mg/kg.

Example 8-N- (lH-indol-3-ylmethyl) -6-morpholin-4-yl-N' -p-tolyl- [1,3,5[ triazine-2, 4-diamine

To a solution of (4-chloro-6-morpholin-4-yl- [1,3,5] triazin-2-yl) -p-tolyl-amine (100mg, 0.32mmol) in dioxane (20mL) was added (1H-indol-3-yl) methylamine (187mg, 1.28mmol) and DIEA (82.56mg, 0.64mmol) and the mixture was stirred at 90 ℃ for 12 hours. The reaction was monitored by LCMS. The mixture was concentrated in vacuo to give a residue, which was purified by column chromatography followed by preparative HPLC to give N- (lH-indol-3-ylmethyl) -6-morpholin-4-yl-N' -p-tolyl- [1,3,5] triazine-2, 4-diamine as a white solid (37mg, yield: 27%).

¹H NMR(400MHz,DMSO-d6):δ＝10.83(s,1H),8.99-8.74(m,1H),7.68-7.55(m,3H),7.33(d,J＝8.0Hz,1H),7.27-7.14(m,2H),7.09-6.93(m,4H),4.62(s,2H),3.81-3.55(m,8H),2.21(s,3H)。MS:m/z 416.0(M+H⁺)。

Example 9- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-morpholin-4-yl- [1,3,5] triazin-2-yl } -m-tolyl-amine

Step 1

At 0 deg.C, adding 2,4, 6-trichloro- [ l,3,5]]Triazine (700mg, 3.8mmol) and K₂CO₃(528mg, 3.8mmol) in THF (15mL) was slowly added dropwise a solution of m-toluidine (407mg, 3.8mmol) in THF (5 mL). After the addition, the resulting mixture was stirred for 12 hours, and the temperature was slowly raised to room temperature. The solvent was removed and the residue was purified by silica gel column chromatography (PE/EA ═ 20/1 to 10/1) to give (4, 6-dichloro- [ l,3, 5) as a yellow solid]Triazin-2-yl) -m-tolyl-amine (200mg, yield: 21%). The reaction was monitored by TLC and the ionic strength of the molecule of the product was weaker on LCMS spectrum. Note that: the dimer \ trimer by-product increase can be significantly reduced and the yield increased if the reaction is carried out at-20 ℃.

Step 2

At 0 deg.C, to (4, 6-dichloro- [ l,3, 5)]To a mixture of triazin-2-yl) -m-tolyl-amine (200mg, 0.787mmol) and DIEA (103mg, 0.787mmol) in THF (10mL) was added morpholine (69mg, 0.787 mmol).The resulting mixture was stirred at room temperature for an additional 2 hours. Hydrazine monohydrate (0.5mL, 10mmol) was then added to the above mixture. After the addition, the reaction mixture was heated at 50 ℃ for 3 hours. The volatiles were removed in vacuo to give crude (4-hydrazino-6-morpholin-4-yl- [1,3, 5) as a white solid]Triazin-2-yl) -m-tolyl-amine (250mg, crude). MS M/z 301.9(M + H)⁺)。

Step 3

Heating (4-hydrazino-6-morpholin-4-yl- [1,3,5] at 70 deg.C]Triazin-2-yl) -m-tolyl-amine (95mg, crude, 0.31mmol)) and 1H-indole-3-carbaldehyde (46mg, 0.31mmol) in MeOH (5mL, one drop of HOAc added as catalyst) for 2 hours. The reaction mixture was filtered and purified by preparative HPLC (NH)₄CO₃System) the filtrate was purified to give {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino group as a white solid]-6-morpholin-4-yl- [ l,3,5]Triazin-2-yl } -m-tolyl-amine (60mg, yield: 45% through 2 steps).

¹H NMR(400MHz,DMSO-d6):δ＝11.42(brs,1H),10.57(brs,1H),9.08(brs,1H),8.42(d,J＝8.0Hz,1H),8.33(s,1H),7.69(s,1H),7.54-7.39(m,2H),7.25-7.07(m,3H),6.82-6.77(m,1H),3.89-3.59(m,8H),2.27(s,3H)。MS:m/z 429.0(M+H⁺)。

Example 10- (4-fluoro-phenyl) - {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-morpholin-4-yl- [ l,3,5] triazin-2-yl } -amine

Step 1

In N₂Under the atmosphere, 2,4, 6-trichloro- [1,3,5] at-50 DEG C]To a solution of triazine (500mg, 2.71mmol) and DIEA (350mg, 2.71mmol) in THF (25mL) was slowly added a solution of 4-fluoro-aniline (301mg, 2.711mmol) in THF (5 mL). The resulting mixture was stirred at-50 ℃ for 2 hours. THF was removed and H was added₂O (50 mL). The mixture was extracted with EA (50 mL. times.3). The combined organic layers were washed with brine (150mL × 3). The separated organic layer was then concentrated to give a residue. Residue removerPurification on silica gel column (PE/EA 50/1) to give (4, 6-dichloro- [ l,3, 5) as a white solid]Triazin-2-yl) - (4-fluoro-phenyl) -amine (524mg, yield: 75%).

Step 2

In N₂Under the atmosphere, at 0 deg.C to (4, 6-dichloro- [1,3, 5)]To a solution of triazin-2-yl) - (4-fluoro-phenyl) -amine (300mg, 1.16mmol) and DIEA (150mg, 1.16mmol) in THF (20mL) was added a solution of morpholine (101mg, 1.16mmol) in THF (5 mL). The resulting mixture was stirred at 0 ℃ for 1 hour. Then hydrazine monohydrate (0.6mL) was added. The reaction was continued with stirring at 50 ℃ for 30 minutes more. THF was removed and H was added₂O (30 mL). The mixture was extracted with EA (30 mL. times.3). The combined organic layers were washed with brine (90 mL. times.3). The separated organic layer was then concentrated to give crude (4-fluoro-phenyl) - (4-hydrazino-6-morpholin-4-yl- [1,3, 5)]Triazin-2-yl) -amine (353mg), which was used directly in the next step.

Step 3

In N₂(4-fluoro-phenyl) - (4-hydrazino-6-morpholin-4-yl- [1,3,5] stirring at 70 ℃ under an atmosphere]Triazin-2-yl) -amine (353mg, 1.16mmol), 1H-indole-3-carbaldehyde (169mg, 1.16mmol) and a mixture of AcOH (4 drops) in MeOH (20mL) for 1H. MeOH was removed and H was added₂O (10 mL). The mixture was extracted with EA (10 mL. times.3). The combined organic layers were washed with brine (30mL × 3). The separated organic layer was concentrated to give a residue. The residue was purified by preparative HPLC to give (4-fluoro-phenyl) - {4- [ N' - (1H-indol-3-ylmethylene) -hydrazino]-6-morpholin-4-yl- [ l,3,5]Triazin-2-yl } -amine (354mg, yield: 71% for the three steps).

¹H NMR(400MHz,DMSO-de):δ＝11.44(brs,1H),10.62(brs,1H),9.29(brs,1H),8.42(d,J＝7.6Hz,1H),8.33(s,1H),8.07(brs,1H),7.87-7.69(m,2H),7.43(d,J＝7.6Hz,1H),7.26-7.16(m,1H),7.25-7.06(m,3H),3.76-3.67(m,8H)。MS:m/z 433.0(M+H⁺)。

Example 11- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -N- (4-chlorophenyl) -6-morpholinyl-l, 3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described in example 10, using p-chloroaniline as the starting material.

¹H NMR(400MHz,DMSO-d₆):δ＝11.45(s,1H),10.70-10.63(m,1H),9.39-9.37(m,1H),8.42(d,J＝7.6Hz,1H),8.33(s,1H),8.15-8.05(m,1H),7.87-7.82(m,1H),7.71(d,J＝2Hz,1H),7.45-7.43(m,1H),7.37-7.26(m,2H),7.23-7.17(m,1H),7.13-7.09(m,1H),3.87-3.61(m,8H)。

MS:m/z 448.9(M+H⁺)

Example 12- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-morpholin-4-yl- [1,3,5] triazin-2-yl } - (4-methoxy-phenyl) -amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):5＝11.42(brs,1H),10.56(brs,1H),9.06(brs,1H),8.44(d,J＝8.4Hz,1H),8.32(s,1H),8.01(s,1H),7.80-7.54(m,2H),7.42(d,J＝8.4Hz,1H),7.26-7.17(m,1H),7.12(t,J＝7.2Hz,1H),6.89(d,J＝8.8Hz,2H),4.07-3.49(m,11H)。MS:m/z 445.0(M+H⁺)

Example 13-2- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-p-toluidino- [1,3,5] triazin-2-ylamino } -ethanol

Step 1

At-40 deg.C, adding 2,4, 6-trichloro- [1,3,5]]Triazine (3g, 16.3mmoL) and K₂CO₃(1.12g, 8.15mmoL) to a solution in THF (30mL) was added dropwise a solution of p-toluidine (0.87g, 8.15mmoL) in THF (20 mL). Stirring at-40 deg.CThe mixture was allowed to stand for 0.5 hour. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by silica flash column (PE containing 10% EA) to give (4, 6-dichloro- [ l,3, 5) as a white solid]Triazin-2-yl) -p-tolyl-amine (1.1g, yield: 55%).

¹H NMR(400MHz,DMSO-d6):δ＝11.05(s,1H),7.46(d,J＝8.4Hz,2H),7.20(d,J＝8.8Hz,2H),2.29(s,3H)。

Step 2

Stirring (4, 6-dichloro- [1,3,5] at 35 deg.C]A solution of triazin-2-yl) -p-tolyl-amine (800mg,3.1mmoL), DIEA (0.7mL,3.9mmoL) and 2-amino-ethanol (230mg,3.8mmoL) in dioxane (7mL) for 1.5 hours. Subjecting the reaction mixture to hydrogenation with H₂O (10mL) was diluted and the mixture was stirred at room temperature for 1 hour. The suspension was then filtered and the collected filter cake was air-dried to give 2- (4-chloro-6-p-tolylamino- [1,3,5] as a white solid]Triazin-2-ylamino) -ethanol (860mg, yield: 98%). MS M/z 280.0(M + H)⁺)。

Step 3

2- (4-chloro-6-p-toluylamino- [1,3,5] is stirred at room temperature]Triazin-2-ylamino) -ethanol (400mg, 1.43mmoL) and n2h4.h₂A solution of O (1mL) in dioxane (6mL) for 1 hour. The mixture was coevaporated with MeOH (5 mL. times.4) to give 2- (4-hydrazino-6-p-toluidino- [1,3,5] as a white solid]Triazin-2-ylamino) -ethanol (crude). MS M/z 276.0(M + H)⁺)。

Step 4

Stirring 2- (4-hydrazino-6-p-toluylamino- [1,3,5] at 75 deg.C]Triazin-2-ylamino) -ethanol (380mg, 1.38mmoL), 1H-indole-3-carbaldehyde (391mg, 2.69mmoL) and two drops of a solution of HOAc in MeOH (16mL) for 19 hours. The reaction mixture was concentrated and purified by preparative HPLC (NH)₃.H₂O) purification of the residue to give 2- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino as a white solid]-6-p-toluylamino- [ l,3,5]Triazin-2-ylamino } -ethanol (18mg, yield: 7%).

¹H NMR(400MHz,DMSO-d6):δ＝11.42(brs,1H),10.52-10.37(m,1H),8.97-8.95(m,1H),8.45-8.43(m,1H),8.33(s,1H),8.14-8.68(m,3H),7.42(d,J＝8.0Hz,1H),7.23-7.09(m,4H),6.62-6.60(m,1H),4.69(s,1H),3.55-3.52(m,2H),3.40-3.36(m,2H),2.28(s,3H)。

MS:m/z 403.0(M+H⁺)。

Example 14-2- ((2-hydroxy-ethyl) - {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-p-toluylamino- [ l,3,5] triazin-2-yl } -amino) -ethanol

The title compound was prepared using methods and procedures analogous to those used in example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.40(brs,1H),10.48(brs,1H),8.98(brs,1H),8.44(d,J＝8.4Hz,1H),8.34(s,1H),7.90-7.68(m,3H),7.41(d,J＝7.2Hz,1H),7.24-6.70(m,4H),5.01-4.74(m,2H),3.69-3.66(m,8H),2.28(s,3H)。

MS:m/z 447.0(M+H⁺)。

Example 15- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-piperidin-l-yl- [ l,3,5] triazin-2-yl } -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those used in example 13.

¹H NMR(300MHz,DMSO-d6):δ＝11.42(brs,1H),10.51(brs,1H),9.01(brs,1H),8.48-8.45(m,1H),8.32(s,1H),8.05-7.66(m,3H),7.45-7.39(m,1H),7.26-7.09(m,4H),3.76-3.75(m,4H),2.28(s,3H),1.68-1.52(m,6H)。

MS:m/z 427.0(M+H⁺)。

Example 16- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-pyrrolidin-l-yl- [ l,3,5] triazin-2-yl } -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those used in example 13.

¹H NMR(300MHz,DMSO-d6):δ＝11.41(brs,1H),10.54(brs,1H),9.02(brs,1H),8.49-8.46(m,1H),8.33(s,1H),7.93-7.66(m,2H),7.43-7.41(m,1H),7.26-7.22(m,1H),7.12-7.09(m,4H),3.56-3.53(m,4H),2.28(s,3H),1.92-1.90(m,4H)。

MS:m/z 413.0(M+H⁺)。

Example 17- [4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6- (4-methyl-piperazin-1-yl) - [1,3,5] triazin-2-yl ] -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those used in example 13.

¹H NMR(300MHz,DMSO-d6):5＝11.42(brs,1H),10.55(brs,1H),9.06(brs,1H),8.45(d,J＝7.2Hz,1H),8.31(s,1H),7.97-7.68(m,3H),7.42(d,J＝8.1Hz,1H),7.27-7.09(m,4H),3.77-3.76(m,4H),2.36-2.35(m,4H),2.28(s,3H),2.22(s,3H)。

MS:m/z 442.0(M+H⁺)。

Example 18- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-piperazin-l-yl- [ l,3,5] triazin-2-yl } -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those used in example 13.

¹H NMR(300MHz,DMSO-d6):5＝11.41(brs,1H),10.51(brs,1H),9.03(brs,1H),8.45(d,J＝7.2Hz,1H),8.31(s,1H),8.0-7.91(m,3H),7.68-7.41(m,1H),7.21-7.8(m,4H),3.73-3.67(m,4H),2.74-2.72(m,4H),2.28(s,3H)。

MS:m/z 428.3(M+H⁺)。

Example 19- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-morpholin-4-yl- [ l,3,5] triazin-2-yl } -o-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):5＝11.36(brs,1H),10.50(brs,1H),8.57-8.00(m,3H),7.85-7.44(m,2H),7.39(d,J＝8.0Hz,1H),7.29-7.13(m,3H),7.13-6.96(m,2H),4.05-3.50(m,8H),2.27(s,3H)。

MS:m/z 429.2(M+H⁺)。

Example 20- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-N- (4- (trifluoromethyl) phenyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):δ＝11.45(brs,1H),10.76(brs,1H),9.67(m,1H),8.45(d,J＝8.0Hz,1H),8.35-8.27(m,3H),7.89(s,1H),7.73-7.60(m,2H),7.45-7.42(m,1H),7.24-7.21(m,1H),7.11-7.07(m,1H),3.85-3.62(m,8H)。

MS:m/z 483.2(M+H⁺)

Example 21- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -N- (4-bromophenyl) -6-morpholinyl-l, 3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):δ＝11.45(brs,1H),10.68(brs,1H),9.25(brs,1H),8.42(d,J＝7.6Hz,1H),8.33(s,1H),8.06-8.05(m,1H),7.71-7.63(m,2H),7.46-7.41(m,3H),7.23-7.20(m,1H),7.13-7.09(m,1H),3.80-3.60(m,8H)。

MS:m/z 495.1(M+H⁺)。

Example 22- (E) -4- ((4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-l, 3, 5-triazin-2-yl) amino) benzonitrile

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):δ＝11.48(brs,1H),10.75(brs,1H),9.72(brs,1H),8.43(d,J＝7.6Hz,1H),8.39(s,1H),8.35-8.30(m,1H),8.18-7.90(m,1H),7.74-7.63(m,3H),7.73-7.65(m,1H),7.25-7.07(m,2H),4.02-3.83(m,8H)。

MS:m/z 440.2(M+H⁺)

Example 23- [ 4-Morpholin-4-yl-6- (N' -pyridin-3-ylmethylene-hydrazino) - [1,3,5] triazin-2-yl ] p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d6):δ＝11.06(s,1H),9.23(s,1H),8.82(s,1H),8.58-8.53(dd,J＝4.8,1.6Hz,1H),8.17(s,1H).8.08-8.03(m,1H),7.68(s,2H),7.50-7.44(m,1H),7.08(d,J＝8.4Hz,2H),3.81-3.62(m,8H),2.25(s,3H)。

MS:m/z 391.0(M+H⁺)。

Example 24- { 4-Morpholin-4-yl-6- [ N' - (lH-pyrrol-3-ylmethylene) -hydrazino [ - [ l,3,5] triazin-2-yl } -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d6):δ＝11.02(s,1H),10.37(s,1H),9.14(s,1H),8.04(s,1H),7.70-7.66(m,2H),7.07(d,J＝2.0Hz,1H),7.06(d,J＝8.8Hz,2H),6.80(d,J＝2.0Hz,1H),6.36(s,1H),3.78-3.59(m,8H),2.24(s,3H)。

MS:m/z 379.0(M+H⁺)。

Example 25- (3-chloro-phenyl) - {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-morpholin-4-yl- [ l,3,5] triazin-2-yl } -amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):v＝11.44(brs,1H),10.67(brs,1H),9.60-9.11(m,1H),8.41(s,1H),8.35(s,1H),8.16-7.57(m,3H),7.42(d,J＝8.0Hz,1H),7.37-7.24(m,1H),7.20(t,J＝7.2Hz,1H),7.12(t,J＝7.2Hz,1H),7.07-6.94(m,1H),3.96-3.55(m,8H)。

MS:m/z 448.9(M+H⁺)。

Example 26- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-N- (4-nitrophenyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):δ＝11.49(brs,1H),10.85(brs,1H),10.04(brs,1H),8.46(d,J＝7.8Hz,1H),8.43(s,2H),8.37-8.16(m,2H),8.08-8.07(m,1H),7.75(s,1H),7.47-7.43(m,1H),7.26-7.20(m,2H),3.91-3.62(m,8H)。

MS:m/z 460.0(M+H⁺)。

Example 27-Nl- (4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-1, 3, 5-triazin-2-yl) benzene-l, 4-diamine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(800MHz,DMSO-d₆):δ＝11.38(brs,1H),10.46(brs,1H),8.86-8.75(m,1H),8.43-8.41(m,1H),8.29(s,1H),7.73-7.69(m,1H),7.66-7.64(m,1H),7.43-7.38(m,1H),7.34-7.14(m,3H),6.59-6.48(m,2H),4.84-4.74(m,2H),3.82-3.59(m,8H)。

MS:m/z 430.2(M+H⁺)

Example 28- (E) -N- (4- ((4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-l, 3, 5-triazin-2-yl) amino) phenyl) acetamide

To a solution of (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazine) -N- (4-aminophenyl) -6-morpholinyl-1, 3, 5-triazin-2-amine (100mg, 0.2mmol) and AcOH (14mg, 0.2mmol) in DMF (15mL) was added DIEA (90.3mg, 0.6mmol) and HATU (132.8mg, 0.3 mmol). At room temperature under N₂The solution was stirred under atmosphere for 2 hours. The solution was concentrated to give a residue. By preparative HPLC (NH)₄HCO₃) The residue was purified to give (E) -N- (4- ((4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-l, 3, 5-triazin-2-yl) amino) phenyl) acetamide (15mg, yield: 13.6%).

¹H NMR(400MHz,DMSO-d₆):δ＝11.40(brs,1H),10.60(brs,1H),9.85(brs,1H),9.12(brs,1H),8.45-8.32(m,2H),8.05-8.00(m,1H),7.69-7.41(m,5H),7.22-7.19(m,2H),3.81-3.63(m,8H),2.07-2.04(m,3H)。

MS:m/z 472.0(M+H⁺)

Example 29- (E) -6- (2- ((lH-indol-3-yl) methylene) hydrazino) -N2, N2-bis (2-methoxyethyl) -N4- (p-tolyl) -1,3, 5-triazine-2, 4-diamine

The title compound was prepared using methods and procedures analogous to those described for example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.40(brs,1H),10.49(brs,1H),9.13-8.81(m,1H),8.46(d,J＝4.8Hz,1H),8.30(d,J＝6.4Hz,1H),7.67-7.63(m,3H),7.42(d,J＝7.6Hz,1H),7.21-7.10(m,4H),3.76-3.72(m,4H),3.61-3.50(m,4H),3.29(s,6H),2.28(s,3H)。

MS:m/z 475.0(M+H⁺)。

Example 30- (E) -6- (2- ((lH-indol-3-yl) methylene) hydrazino) -N2, N2-dimethyl-N4- (p-tolyl) -l,3, 5-triazine-2, 4-diamine

The title compound was prepared using methods and procedures analogous to those described for example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.41(brs,1H),10.50(brs,1H),9.01(brs,1H),8.46(d,J＝8.0Hz,1H),8.33(s,1H),8.10-7.65(m,3H),7.42(d,J＝7.6Hz,1H),7.25-7.03(m,4H),3.13(s,6H),2.28(s,3H)。

MS:m/z 387.0(M+H⁺)。

Example 31- [4- (N' -benzo [ b ] thiophen-3-ylmethylene-hydrazino) -6-morpholin-4-yl- [ l,3,5] triazin-2-yl ] -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d6):δ＝10.91(brs,1H),9.29-8.88(m,2H),8.42(s,1H),8.13-7.96(m,2H),7.88-7.55(m,2H),7.52-7.34(m,2H),7.09(d,J＝8.0Hz,2H),4.5-3.47(m,8H),2.28(s,3H)。

MS:m/z 446.0(M+H⁺)。

Example 32- [ 4-Morpholin-4-yl-6- (N' -pyridin-4-ylmethylene-hydrazino) - [1,3,5] triazin-2-yl ] -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d6):δ＝11.19(brs,1H),9.27(brs,1H),8.42(s,1H),8.62(d,J＝6.0Hz 2H),8.10(s,1H),7.79-7.63(m,2H),7.60(d,J＝5.6Hz,2H),7.09(d,J＝8.4Hz,2H),3.84-3.61(m,8H),2.26(s,3H)。

MS:m/z 391.0(M+H⁺)。

Example 33- [4- (N' -benzylidene-hydrazino) -6-morpholin-4-yl- [ l,3,5] triazin-2-yl ] -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d6):δ＝10.87(s,1H),9.21(s,1H),8.14(s,1H),7.78-7.56(m,4H),7.48-7.32(m,3H),7.08(d,J＝8.4Hz,2H),3.87-3.57(m,8H),2.25(s,3H)。

MS:m/z 390.0(M+H⁺)。

Example 34- (E) -Nl- (4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-1, 3, 5-triazin-2-yl) -N4, N4-xylene-l, 4-diamine

Step 1

In N₂Under the atmosphere, 2,4, 6-trichloro- [ l,3,5] at-50 DEG C]Triazine (6.7g, 36.2mmol) and K₂CO₃(2.6g, 18.8mmol) in THF (160mL) was slowly added a solution of tert-butyl (4-aminophenyl) carbamate (5.2mg, 25.0mmol) in THF (40 mL). The resulting mixture was stirred at-50 ℃ for 2 hours. THF was removed and H was added₂O (150 mL). The mixture was extracted with EA (150 mL. times.3). The combined organic layers were washed with brine (150mL) and concentrated to give a residue. The residue was purified by a silica gel column (PE/EA ═ 10/1) to give tert-butyl (4- ((4, 6-dichloro-l, 3, 5-triazin-2-yl) amino) phenyl) carbamate as a white solid (4.4g, yield: 50%).

¹H NMR(400MHz,CDCl₃):δ＝7.57(s,1H),7.42(d,J＝8.8Hz,2H),7.38(d,J＝8.8Hz,2H),6.54(s,1H),1.53(s,9H)。

Step 2

In N₂To a solution of (4- ((4, 6-dichloro-l, 3, 5-triazin-2-yl) amino) phenyl) carbamate (2g, 5.6mmol) and DIEA (726mg, 5.6mmol) in THF (50mL) at 0 ℃ under an atmosphere was added a solution of morpholine (489mg, 5.6mmol) in THF (15 mL). The resulting mixture was stirred at 0 ℃ for 2 hours. The solution was then concentrated and purified by a silica gel column (PE/EA ═ 8/1) to give tert-butyl (4- ((4-chloro-6-morpholinyl-l, 3, 5-triazin-2-yl) amino) phenyl) carbamate as a white solid (1.8g, yield: 80%).

Step 3

A solution of tert-butyl (4- ((4-chloro-6-morpholinyl-l, 3, 5-triazin-2-yl) amino) phenyl) carbamate (1g, 2.5mmol) in DCM/TFA (5/1) (20mL) was stirred at room temperatureOvernight. The reaction was concentrated and the residue partitioned between NaHCO₃(aqueous solution) and EA (50mL each). The organic phase is then passed over Na₂SO₄Dried and concentrated to give Nl- (4-chloro-6-morpholinyl-1, 3, 5-triazin-2-yl) benzene-1, 4-diamine as a grey solid (crude material).

Step 4

To a solution of Nl- (4-chloro-6-morpholinyl-1, 3, 5-triazin-2-yl) benzene-1, 4-diamine (900mg,2.9mmol) in MeCN (40mL) was added HCHO (37%) (2.38g, 29.3mmol), NaBH₃CN (553.5mg,8.9mmol) and HOAc (616.5mg,10.3mmol), and the solution was stirred at room temperature overnight. The reaction was concentrated and the residue was purified by silica gel column (PE/EA ═ 5/1) to give Nl- (4-chloro-6-morpholinyl-1, 3, 5-triazin-2-yl) -N4, N4-dimethylbenzene-1, 4-diamine (216.7g, yield: 31%) as a yellow solid.

Step 5

To a solution of Nl- (4-chloro-6-morpholinyl-1, 3, 5-triazin-2-yl) -N4, N4-dimethylbenzene-1, 4-diamine (200mg,0.6mmol) and THF (10mL) was added N₂H₄.H₂O (0.5 mL). The reaction was stirred at 50 ℃ for a further 4 hours. THF was removed and H was added₂O (30 mL). The product was collected by filtration to give Nl- (4-hydrazino-6-morpholinyl-1, 3, 5-triazin-2-yl) -N4, N4-dimethylbenzene-1, 4-diamine (190mg, 85% yield).

Step 6

The procedure of step 6 was similar to that used in step 4 of example 10.

¹H NMR(400MHz,DMSO-d₆):δ＝11.40(brs,1H),10.50(brs,1H),8.90(s,1H),8.45-8.45(m,1H),8.31(s,1H),7.91-7.90(m,1H),7.68(s,1H),7.48-7.41(m,2H),7.22-7.11(m,2H),6.73-6.71(m,2H),3.80-3.62(m,8H),2.86-2.82(m,6H)。

MS:m/z 458.0(M+H⁺)

Example 35- (E) -6- (2- ((lH-indol-3-yl) methylene) hydrazino) -N2-ethyl-N2-methyl-N4- (p-tolyl) -l,3, 5-triazine-2, 4-diamine

The title compound was prepared using methods and procedures analogous to those described for example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.40(brs,1H),10.49(brs,1H),8.98(brs,1H),8.48-8.40(m,1H),8.33(s,1H),8.08-7.67(m,3H),7.42(d,J＝8.4Hz,1H),7.26-7.9(m,4H),3.64-3.65(m,2H),3.10(s,3H),2.28(s,3H),1.17-1.14(m,3H)。

MS:m/z 401.0(M+H⁺)。

Example 36- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino-6- (azetidin-l-yl) -N- (p-tolyl) -1,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described for example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.43(brs,1H),10.75(brs,1H),9.18(brs,1H),8.46(d,J＝8.0Hz,1H),8.30(d,J＝6.4Hz,1H),7.99-7.96(m,2H),7.68(s,1H),7.43(d,J＝6.4Hz,1H),7.25-7.12(m,4H),4.06-4.00(m,4H),2.30-2.29(m,5H)。

MS:m/z 399.0(M+H⁺)。

Example 37- (E) -l- (4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6- (p-toluylamino) -l,3, 5-triazin-2-yl) piperidin-4-ol

The title compound was prepared using methods and procedures analogous to those described for example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.42(brs,1H),10.53(brs,1H),9.05(brs,1H),8.47(d,J＝7.2Hz,1H),8.32(s,1H),8.07-7.68(m,3H),7.43(d,J＝8.4Hz,1H),7.22-7.10(m,4H),4.73(d,J＝4.4Hz,1H),4.33-4.29(m,2H),3.75(d,J＝3.6Hz,1H),3.28-3.27(m,2H),2.28(s,3H),1.80-1.78(m,2H),1.35-1.33(m,2H)。

MS:m/z 443.0(M+H⁺)。

Example 38- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-thiomorpholinyl-N- (p-tolyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described for example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.42(brs,1H),10.60(brs,1H),9.13(brs,1H),8.46(d,J＝6.0Hz,1H),8.32(s,1H),8.08-7.60(m,3H),7.43(d,J＝8.0Hz,1H),7.22-7.12(m,4H),4.12-4.08(m,4H),2.64-2.59(m,4H),2.29(s,3H)。

MS:m/z 445.0(M+H⁺)。

Example 39- {3- [ N' - (lH-indol-3-ylmethylene-hydrazino [ -5-morpholin-4-yl-phenyl } -p-tolyl-amine

Step 1

To a solution of 2,4, 6-trichloro-pyrimidine (1g, 5.5mmol) and p-toluidine (588.5mg, 5.5mmol) in EtOH (15mL) was added TEA (555.5mg, 5.5 mmol). The reaction was stirred at room temperature overnight. The reaction solution was partitioned between EA (60mL) and water (60 mL). The organic layer was washed with water (60mL) and brine (60mL) and washed with Na₂SO₄Dried and concentrated in vacuo to give (2, 6-dichloro-pyrimidin-4-yl) -p-tolyl-amine (1.4g, crude) as a white solid. MS M/z 253.8(M + H)⁺。

Step 2

To a solution of (2, 6-dichloro-pyrimidin-4-yl) -p-tolyl-amine (1.4g, 5.5mmol) and morpholine (478.5mg, 5.5mmol) in EtOH (15mL) was added TEA (555.5mg, 5.5 mmol). The reaction was stirred at 80 ℃ for 4 hours. Will be reversedThe solution was partitioned between EA (100mL) and water (100 mL). The organic layer was washed with water (100mL) and brine (100mL) and washed with Na₂SO₄Dried and concentrated in vacuo. The residue was purified by preparative TLC (PE/EA ═ 15/1) to give (6-chloro-2-morpholin-4-yl-pyrimidin-4-yl) -p-tolyl-amine (900mg, yield: 54%) as a white solid.

¹H NMR(400MHz,CDCl₃):δ＝7.17(s,4H),6.50(brs,1H),5.95(s,1H),3.77-3.72(m,8H),2.35(s,3H).MS:m/z 304.8(M+H)⁺。

Step 3

A solution of (6-chloro-2-morpholin-4-yl-pyrimidin-4-yl) -p-tolyl-amine (250mg, 0.82mmol) and hydrazine monohydrate (82mg, 1.64mmol) in dioxane (8mL) was stirred at 80 ℃ for 4 hours. The reaction was taken up in water (100mL) and filtered. The filter cake was dried to give (6-hydrazino-2-morpholin-4-yl-pyrimidin-4-yl) -p-tolyl-amine as a white solid (240mg, yield: 97.6%). MS M/z 300.9(M + H)⁺。

Step 4

A solution of {3- [ N' - (lH-indol-3-ylmethylene) -hydrazino ] -5-morpholin-4-yl-phenyl } -p-tolyl-amine (240mg, 0.8mmol) and lH-indole-3-carbaldehyde (116mg, 0.8mmol) in dioxane (10mL) was stirred at 100 ℃ for 2 hours. The reaction was taken up in water (100mL) and filtered. The filter cake was dried to give {3- [ N' - (1H-indol-3-ylmethylidene) -hydrazino ] -5-morpholin-4-yl-phenyl } -p-tolyl-amine (198mg, yield: 58%) as a red solid.

¹H NMR(400MHz,DMSO-d6):δ＝11.40(d,J＝2.0Hz,1H),10.21(s,1H),8.81(s,1H),8.22-8.19(m,2H),7.66(d,J＝1.6Hz,1H),7.47-7.41(m,3H),7.21(t,J＝7.2Hz,1H),7.19-7.10(m,3H),6.01(s,1H),3.65(s,8H),2.28(s,3H)。MS:m/z 428.0(M+H)⁺。

Example 40- [ 2-Morpholin-4-yl-6- (8H-pyrazolo [3,4-b ] indol-1-yl) -pyrimidin-4-yl ] -p-tolyl-amine

Step 1

A solution of (6-chloro-2-morpholin-4-yl-pyrimidin-4-yl) -p-tolyl-amine (156mg, crude material) in hydrazine monohydrate (5mL) was stirred at 100 ℃ overnight. The reaction solution was partitioned between EA (60mL) and water (60 mL). The organic layer was washed with water (60mL) and brine (60mL) and washed with Na₂SO₄Dried and concentrated in vacuo to give (6-hydrazino-2-morpholin-4-yl-pyrimidin-4-yl) -p-tolyl-amine (74mg, crude). MS M/z 300.9(M + H)⁺。

Step 2

Stirring {3- [ N' - (lH-indol-3-ylmethylene) -hydrazino group at 78 deg.C]-5-morpholin-4-yl-phenyl } -p-tolyl-amine (74mg, 0.25mmol) and a solution of lH-indole-3-carbaldehyde (36mg, 0.25mmol) in MeOH (5mL) overnight. The reaction solution was partitioned between EA (60mL) and water (60 mL). The organic layer was washed with water (60mL) and brine (60mL) and washed with Na₂SO₄Dried and concentrated in vacuo. By preparative HPLC (NH)₃.H₂O as an additive) to give [ 2-morpholin-4-yl-6- (8H-pyrazolo [3, 4-b) as a yellow solid]Indol-1-yl) -pyrimidin-4-yl]P-tolyl-amine (9.6mg, yield: 4.3%).

¹H NMR(400MHz,DMSO-d6):δ＝12.86(s,1H),11.54(s,1H),7.77-7.75(m,2H),7.60(s,1H),7.53(d,7＝8.4Hz,1H),7.25-7.21(m,3H),7.13-7.11(m,1H),7.09-7.06(m,2H),3.72-3.67(m,8H),2.33(s,3H)。

MS:m/z 426.0(M+H)⁺。

Example 41- {2- [ N' - (lH-indol-3-ylmethylene) -hydrazino [ -6-morpholin-4-yl-pyrimidin-4-yl } -p-tolyl-amine

Step 1

A solution of (2, 6-dichloro-pyrimidin-4-yl) -p-tolyl-amine (200mg, 0.79mmol) and hydrazine hydrate (39mL, 0.79mmol) in dioxane (5mL) was stirred at 100 ℃ for 2 hours. The reaction was wet-milled with water (100mL) and filtered. The filter cake was dried to give (6-chloro-2-hydrazino-pyrimidin-4-yl) -p-tolyl-amine (140mg, crude material) as a white solid.

¹H NMR(400MHz,DMSO-d6):δ＝9.31(s,1H),8.21(s,1H),7.56(d,J＝8.0Hz,2H),7.10(d,J＝8.4Hz,2H),5.98(s,1H),4.22(s,2H),2.25(s,3H)。MS:m/z 249.9(M+H)⁺。

Step 2

A solution of (6-chloro-2-hydrazino-pyrimidin-4-yl) -p-tolyl-amine (140mg, 0.56mmol) and lH-indole-3-carbaldehyde (81.2mg, 0.56mmol) in dioxane (10mL) was stirred at 100 ℃ for 3 hours. The reaction was treated with water (100mL) and filtered. Drying the filter cake to obtain { 6-chloro-2- [ N' - (lH-indol-3-ylmethylene) -hydrazino group]Pyrimidin-4-yl } -p-tolyl-amine (164mg, crude). MS M/z 376.8(M + H)⁺。

Step 3

Stirring { 6-chloro-2- [ N' - (lH-indol-3-ylmethylidene) -hydrazino ] group at 100 deg.C]-pyrimidin-4-yl } -p-tolyl-amine (164mg, 0.436mmol) and morpholine (42mg, 0.48mmol) in dioxane (5mL) for 3 hours. The reaction was treated with water (100mL) and filtered to obtain a solid. Then, by preparative HPLC (NH)₃.H₂O as additive) to give {2- [ N' - (lH-indol-3-ylmethylene) -hydrazino) -as a white solid]-6-morpholin-4-yl-pyrimidin-4-yl } -p-tolyl-amine (8.8mg, yield: 4.7%).

¹H NMR(400MHz,DMSO-d6):δ＝11.34(s,1H),10.15(s,1H),8.82(s,1H),8.49(d,J＝8.0Hz,1H),8.27(s,1H),7.82-7.81(m,2H),7.63(s,1H),7.42(d,J＝7.6Hz,1H),7.21(t,J＝7.6Hz,1H),7.12-7.08(m,3H),5.45(s,1H),3.71-3.69(m,4H),3.47(s,4H),2.29(s,3H)。

MS:m/z 427.8(M+H)⁺。

Example 42- (2-chloro-phenyl) - {4- [ N' - (lH indol-3-ylmethylene) -hydrazino [ -6-morpholin-4-yl- [1,3,5] triazin-2-yl ] -amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d₆):δ＝11.40(brs,1H),10.68(brs,1H),8.62-8.21(m,3H),8.15(s,1H),7.68(d,J＝2.4Hz,1H),7.50(d,J＝8.0Hz,1H),7.42-7.32(m,2H),7.18(t,J＝7.2Hz,2H),7.09(t,J＝7.2Hz,1H),4.05-3.56(m,8H)。MS:m/z 448.9(M+H⁺)

Example 43- [ 4-Morpholin-4-yl-6- (N' -pyridin-2-ylmethylene-hydrazino) - [1,3,5] triazin-2-yl ] -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d6):δ＝11.12(brs,1H),9.26(brs,1H),8.56(d,J＝4.4Hz,1H),8.18(s,1H).8.02-7.83(m,2H),7.78-7.54(m,2H),7.36(d,J＝5.4Hz,1H),7.9(d,J＝8.8Hz,2H),3.86-3.58(m,8H),2.25(s,3H)。

MS:m/z 391.0(M+H⁺)。

Example 44- {4- [ N' - (lH-indol-2-ylmethylene) -hydrazino [ -6-morpholin-4-yl- [ l,3,5] triazin-2-yl } -p-tolyl-amine

The title compound was prepared using methods and procedures analogous to those described for example 10.

¹H NMR(400MHz,DMSO-d6):δ＝11.22(s,1H),10.85(s,1H),9.12(s,1H),8.21(s,1H),7.64(d,J＝8.4Hz,2H),7.53(d,J＝7.6Hz,1H),7.43(d,J＝8.0Hz,1H),7.16-7.05(m,3H),6.99(t,J＝6.8Hz,1H),6.71(s,1H),3.86-3.62(m,8H),2.25(s,3H)。

MS:m/z 429.0(M+H⁺)。

Example 45- {4- [ N' - (lH-indol-3-ylmethylene) -hydrazino-6-morpholin-4-yl- [1,3,5] triazin-2-yl } - (5-methyl-pyridin-2-yl) -amine

The title compound was prepared using methods and procedures analogous to those described in example 10.

¹H NMR(400MHz,DMSO-d₆):δ＝11.45(brs,1H),10.73(brs,1H),9.07(s,1H),8.45(d,J＝8.0Hz,1H),8.33(s,1H),8.12(s,1H),7.71(s,1H),7.59(d,J＝9.2Hz,1H),7.43(d,J＝9.2Hz,1H),7.30-7.04(s,2H),3.85-3.61(m,8H),3.29(s,1H),2.28(s,3H)。

MS:m/z 430.0(M+H⁺)。

Example 46- (E) -N- (4- ((4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-l, 3, 5-triazin-2-yl) amino) phenyl) methanesulfonamide

The title compound was prepared using methods and procedures analogous to those described in example 34.

¹H NMR(400MHz,DMSO-d₆):δ＝11.43(brs,1H),10.62(brs,1H),9.49-9.47(m,1H),9.23(s,1H),8.45-8.42(m,1H),8.33-8.32(m,1H),8.12-8.10(m,1H),7.72-7.68(m,2H),7.44-7.42(m,1H),7.25-7.14(m,4H),3.84-3.62(m,8H),2.91(s,3H)。

MS:m/z 508.0(M+H⁺)

Example 47- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6- (3, 4-dihydroisoquinolin-2 (lH) -yl) -N- (p-tolyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described in example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.44(brs,1H),10.71(brs,1H),9.33-9.16(m,1H),8.57-8.45(m,1H),8.34(s,1H),8.00-7.70(m,3H),7.44(d,J＝8.4Hz,1H),7.23-7.10(m,8H),4.92-4.89(m,2H),4.02(s,2H),2.90(t,J＝1.6Hz,2H),2.30(s,3H)。

MS:m/z 475.0(M+H⁺)。

Example 48- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6- (2H-benzo [ b [ [ l,4[ oxazin-4 (3H) -yl) -N- (p-tolyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described in example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.47(d,J＝2.0Hz,1H),10.80-10.71(m,1H),9.33-9.16(m,1H),8.57-8.38(m,2H),8.19-8.02(m,1H),7.72-7.64(m,2H),7.43(d,J＝6.4Hz,1H),7.24-6.90(m,8H),4.31-4.21(m,4H),2.28(s,3H)。

MS:m/z 477.0(M+H⁺)。

Example 49- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6- (4-methoxypiperidin-l-yl) -N- (p-tolyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described in example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.42(brs,1H),10.47(brs,1H),9.06(brs,1H),8.47(d,J＝7.2Hz,1H),8.32(s,1H),7.95-7.68(m,3H),7.43(d,J＝7.6Hz,1H),7.21-7.10(m,4H),4.22-4.19(m,2H),3.45-3.39(m,3H),3.30(s,3H),2.29(s,3H),1.90-1.88(m,2H),1.42-1.40(m,2H)。

MS:m/z 457.3(M+H⁺)。

Example 50- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6- (3-methylmorpholinyl) -N- (p-tolyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described in example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.41(brs,1H),10.56(brs,1H),9.08(m,1H),8.46(d,J＝7.2Hz,1H),8.33(s,1H),8.07-7.68(m,3H),7.42(d,J＝8.0Hz,1H),7.23-7.12(m,4H),4.69(s,1H),4.32(s,1H),3.93-3.91(m,1H),3.73-3.71(m,1H),3.60-3.57(m,1H),3.46-3.40(m,1H),3.18-3.17(m,1H),2.28(s,3H),1.26-1.25(m,3H)。

MS:m/z 443.0(M+H⁺)。

Example 51- (E) -6- (2- ((lH-indol-3-yl) methylene) hydrazino) -N2-isopropyl-N2- (2-methoxyethyl) -N4- (p-tolyl) -l,3, 5-triazine-2, 4-diamine

The title compound was prepared using methods and procedures analogous to those described in example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.40(brs,1H),10.47(brs,1H),8.99-8.98(m,1H),8.46(d,J＝4.4Hz,1H),8.33(s,1H),7.97-7.67(m,3H),7.42(d,J＝7.6Hz,1H),7.21-7.09(m,4H),4.99-4.98(m,1H),3.71-3.48(m,4H),3.31(s,3H),2.28(s,3H),1.23-1.17(m,6H)。

MS:m/z 459.1(M+H⁺)。

Example 52- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6- (3-oxa-8-azabicyclo [3.2.1] oct-8-yl) -N- (p-tolyl) -l,3, 5-triazin-2-amine

The title compound was prepared using methods and procedures analogous to those described in example 13.

¹H NMR(400MHz,DMSO-d6):δ＝11.43(d,J＝2.0Hz,1H),10.64(brs,1H),9.15(brs,1H),8.45(d,J＝6.4Hz,1H),8.32(s,1H),8.10-7.60(m,3H),7.43(d,J＝8.4Hz,1H),7.23-7.13(m,4H),4.60-4.59(m,2H),3.67-3.59(m,4H),2.29(s,3H),1.99-1.92(m,4H)。

MS:m/z 455.0(M+H⁺)。

Example 53-4- (2- (benzofuran-3-ylmethylene) hydrazino) -6-morpholinyl-N- (p-tolyl) -1,3, 5-triazin-2-amine

The title compound was prepared according to methods and procedures analogous to those described herein for example 10.

¹H NMR(400MHz,DMSO-dd):δ＝11.07(s,1H),9.44(m,1H),8.16(s,1H),7.70-7.57(m,4H),7.37(t,J＝8.4Hz,1H),7.28(t,J＝1.6Hz,1H),7.19(s,1H),7.09(d,J＝8.4Hz,2H),3.80-3.60(m,8H),2.25(s,3H)。

MS:m/z 430.2(M+H⁺)。

Example 54-N2- (2- (lH-indol-3-yl) ethyl) -6-morpholinyl-N4- (p-tolyl) -l,3, 5-triazine-2, 4-diamine

The title compound was prepared according to methods and procedures analogous to those described herein for example 1.

¹H NMR(400MHz,DMSO-d6):δ＝10.82(d,J＝8.0Hz,1H),8.86(s,1H),7.65-7.57(m,2H),7.54(t,J＝8.0Hz,1H),7.33(d,J＝8.0Hz,1H),7.16(s,1H),7.10-6.91(m,5H),3.75-3.46(m,10H),2.93(t,J＝8.0Hz,2H),2.22(s,3H)。

MS:m/z 430.0(M+H⁺)。

Example 55- (E) -4- (2- ((lH-indol-3-yl) methylene) hydrazino) -6-morpholinyl-N- (naphthalen-l-yl) -l,3, 5-triazin-2-amine

The title compound was prepared according to methods and procedures analogous to those described herein for examples 1-52.

Example 56- (E) -4- (azepan-l-yl) -6- (2- ((2-methyl-lH-indol-3-yl) methylene) hydrazino) -N- (4-nitrophenyl) -l,3, 5-triazin-2-amine

The title compound was prepared according to methods and procedures analogous to those described herein for examples 1-52.

Example a-U251 vacuolation analysis data (24 hours) for the compounds of examples 8-56 table 8

+EC₅₀<1μM。

++EC₅₀Is 1<10μM

+++EC₅₀Is 10<50μM

++++EC₅₀Is composed of>50μM

OTHER EMBODIMENTS

It is to be understood that while the application has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the application, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

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