U.S. patent application number 17/453619 was filed with the patent office on 2022-02-24 for compositions and methods for treating cdk4/6-mediated cancer.
The applicant listed for this patent is Icahn School of Medicine at Mount Sinai. Invention is credited to Tamer Ahmed, Jian Jin, Zoi Karoulia, Jing Liu, Poulikos Poulikakos, Xuewei Wu, Yan Xiong, Xiaobao Yang.
Application Number | 20220054488 17/453619 |
Document ID | / |
Family ID | 1000005945529 |
Filed Date | 2022-02-24 |
United States Patent
Application |
20220054488 |
Kind Code |
A1 |
Jin; Jian ; et al. |
February 24, 2022 |
COMPOSITIONS AND METHODS FOR TREATING CDK4/6-MEDIATED CANCER
Abstract
Methods for designing heterobifunctional small molecules which
selectively degrade/disrupt CDK4/6 and compositions and methods of
using such degraders/disruptors to treat CDK4/6-mediated cancer are
provided.
Inventors: |
Jin; Jian; (New York,
NY) ; Yang; Xiaobao; (New York, NY) ; Liu;
Jing; (Oradell, NJ) ; Xiong; Yan; (New York,
NY) ; Poulikakos; Poulikos; (New York, NY) ;
Karoulia; Zoi; (New York, NY) ; Wu; Xuewei;
(New York, NY) ; Ahmed; Tamer; (Edison,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Icahn School of Medicine at Mount Sinai |
New York |
NY |
US |
|
|
Family ID: |
1000005945529 |
Appl. No.: |
17/453619 |
Filed: |
November 4, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16467888 |
Jun 7, 2019 |
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PCT/US2017/065027 |
Dec 7, 2017 |
|
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17453619 |
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62431806 |
Dec 8, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 35/00 20180101; A61K 31/519 20130101; A61K 31/506 20130101;
A61K 47/55 20170801 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 47/55 20060101 A61K047/55; A61P 35/00 20060101
A61P035/00; A61K 31/519 20060101 A61K031/519; A61K 45/06 20060101
A61K045/06 |
Claims
1. A bivalent compound comprising a cyclin-dependent kinase 4/6
(CDK4/6) ligand conjugated to a degradation/disruption tag.
2. The bivalent compound of claim 1, wherein the CDK4/6 ligand is a
CDK4/6 inhibitor.
3. The bivalent compound of claim 2, wherein the CDK4/6 ligand is
selected from the group consisting of abemaciclib, palbociclib,
ribociclib, trilaciclib, G1T38, SHR6390, and analogs thereof.
4. The bivalent compound of any one of claims 1-3, wherein the
CDK4/6 ligand is bound to CDK4/6.
5. The bivalent compound of any one of claims 1-4, wherein the
degradation/disruption tag is selected from the group consisting of
pomalidomide, thalidomide, lenalidomide, VHL-1, adamantane, and
analogs thereof.
6. The bivalent compound of any one of claims 1-5, wherein the
degradation/disruption tag binds to a ubiquitin ligase or serves as
a hydrophobic group that leads to CDK4 or CDK6 protein
misfolding.
7. The bivalent compound of any one of claims 1-6, wherein the
CDK4/6 ligand is conjugated to the degradation/disruptor tag
through a linker.
8. The bivalent compound of any one of claims 1-7, wherein the
bivalent compound has the structure of formula I: ##STR00290##
wherein PI comprises a CDK4/6 ligand and EL comprises a
degradation/disruption tag.
9. The bivalent compound of claim 8, wherein PI has the structure
of Formula II, ##STR00291## wherein X.sup.1, X.sup.2, and X.sup.3
are independently hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C1-C8 alkoxyalkyl, NR.sup.5R.sup.6, CN, NO.sub.2, COR.sup.5,
CO.sub.2R.sup.5, CONR.sup.5R.sup.6, or NR.sup.5COR.sup.6; R.sup.1
and R.sup.4 are independently hydrogen, halogen, C1-C8 alkyl, C1-C8
alkoxy, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl,
C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.5, SR.sup.5, NR.sup.5R.sup.6, CN, NO.sub.2,
(CR.sup.5R.sup.6)mNR.sup.7R.sup.8, (CR.sup.5R.sup.6)mC(O)R.sup.7,
COR.sup.5, CO.sub.2R.sup.5, CONR.sup.5R.sup.6, NR.sup.5COR.sup.6,
NR.sup.5SOR.sup.6, NR.sup.5SO.sub.2R.sup.6, SOR.sup.5,
SO.sub.2R.sup.5, SO.sub.2NR.sup.5R.sup.6, (CR.sup.5R.sup.6)m-aryl,
or (CR.sup.5R.sup.6)m-heteroaryl, wherein m is 0-8; R.sup.2 is
hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy, C3-C7 cycloalkyl, or
C3-C7 heterocyclyl; R.sup.3 is hydrogen, aryl, C1-C8 alkyl, C1-C8
alkoxy, C3-C7 cycloalkyl, or C3-C7 heterocyclyl; R.sup.5, R.sup.6,
R.sup.7, and R.sup.8 are independently hydrogen, C1-C8 alkyl, C2-C8
alkenyl, C2-C8 alkynyl, arylalkyl, cycloalkyl, heterocycloalkyl,
aryl, heteroaryl, or heteroarylalkyl; optionally, R.sup.1 and
R.sup.2, R.sup.5 and R.sup.6, or R.sup.7 and R.sup.8 independently
form 4-8 membered alkyl or heterocyclyl rings; and X and Y are
independently CR.sup.5R.sup.6 or N; the structure of Formula III,
##STR00292## wherein R.sup.1 is hydrogen, C1-C8 alkyl, C1-C8
alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl,
C3-C7 heterocyclyl, C2-C8 alkenyl, or C2-C8 alkynyl; R.sup.2 is
hydrogen, C1-C3 alkyl, or cyclopropyl; R.sup.3, R.sup.4, and
R.sup.5 are independently hydrogen, halogen, C1-C8 alkyl, C1-C8
alkoxy, C3-C7 cycloalkyl, or C3-C7 heterocyclyl; R.sup.6 is
hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkoxyalkyl,
C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl, OR.sup.7, SR.sup.7,
NR.sup.7R.sup.8, CN, NO.sub.2, (CR.sup.7R.sup.8)mNR.sup.9R.sup.10,
(CR.sup.7R.sup.8)mC(O)R.sup.9, COR.sup.7, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, NR.sup.7COR.sup.8, NR.sup.7SOR.sup.8,
NR.sup.7SO.sub.2R.sup.8, SORT, SO.sub.2R.sup.7,
SO.sub.2NR.sup.7R.sup.8, (CR.sup.7R.sup.8)m-aryl, or
(CR.sup.7R.sup.8)m-heteroaryl; wherein m is 0-8; R.sup.7, R.sup.8,
R.sup.9, and R.sup.10 are independently hydrogen, C1-C8 alkyl,
C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, or heteroarylalkyl; optionally,
R.sup.7 and R.sup.8 or R.sup.9 and R.sup.10 independently form 4-8
membered alkyl or heterocyclyl rings; and X and Y are independently
CR.sup.7R.sup.8, or N; the structure of Formula IV, ##STR00293##
wherein R.sup.1 is hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8
haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, or C2-C8 alkynyl; R.sup.2 is hydrogen,
C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8
hydroxyalkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl,
or C2-C8 alkynyl, CN, COR.sup.4, CO.sub.2R.sup.4, or
CONR.sup.4R.sup.5; R.sup.3 is hydrogen, halogen, C1-C8 alkyl, C1-C8
alkoxy, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl,
C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, CN, NO.sub.2,
(CR.sup.4R.sup.5)mNR.sup.6R.sup.7, (CR.sup.4R.sup.5)mC(O)R.sup.6,
COR.sup.4, CO.sub.2R.sup.4, CONR.sup.4R.sup.5, NR.sup.4COR.sup.5,
NR.sup.4SOR.sup.5, NR.sup.4SO.sub.2R.sup.5, SOR.sup.4,
SO.sub.2R.sup.4, SO.sub.2NR.sup.4R.sup.5, (CR.sup.4R.sup.5)m-aryl,
or (CR.sup.4R.sup.5)m-heteroaryl, wherein m is 0-8; R.sup.4,
R.sup.5, R.sup.6, and R.sup.7 are independently hydrogen, C1-C8
alkyl, C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl, cycloalkyl,
heterocycloalkyl, aryl, heteroaryl, or heteroarylalkyl; optionally,
R.sup.1 and R.sup.2, R.sup.4 and R.sup.5, or R.sup.6 and R.sup.7
independently form 4-8 membered alkyl or heterocyclyl rings; and V,
W, X, Y, and Z are independently CR.sup.4R.sup.5 or N; or the
structure of Formula VI, ##STR00294## wherein R.sup.1 and R.sup.2
are independently hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8
haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, or C2-C8 alkynyl; R.sup.3 is hydrogen,
C1-C6 alkyl, C1-C6 alkoxyalkyl, C1-C6 haloalkyl, C1-C6
hydroxyalkyl, C3-C6 cycloalkyl, C3-C6 heterocyclyl, C2-C6 alkenyl,
or C2-C6 alkynyl; R.sup.4 is hydrogen, halogen, C1-C8 alkyl, C1-C8
alkoxy, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl,
C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.5, SR.sup.5, NR.sup.5R.sup.6, CN, NO.sub.2,
(CR.sup.5R.sup.6)mNR.sup.7R.sup.8, (CR.sup.5R.sup.6)mC(O)R.sup.7,
COR.sup.5, CO.sub.2R.sup.5, CONR.sup.5R.sup.6, NR.sup.5COR.sup.6,
NR.sup.5SOR.sup.6, NR.sup.5SO.sub.2R.sup.6, SOR.sup.5,
SO.sub.2R.sup.5, SO.sub.2NR.sup.5R.sup.6, (CR.sup.5R.sup.6)m-aryl,
or (CR.sup.5R.sup.6)m-heteroaryl, wherein m is 0-8; n is 0-4;
optionally, R.sup.1 and R.sup.2, R.sup.5 and R.sup.6, or Rand
R.sup.8 independently form 4-8 membered alkyl or heterocyclyl
rings; and V, W, X, Y, and Z are independently CR.sup.5R.sup.6 or
N.
10. The bivalent compound of claim 8 or 9, wherein the CDK4/6
ligand is selected from the group consisting of abemaciclib,
palbociclib, ribociclib, trilaciclib (G1T28), G1T38, SHR6390, and
analogs thereof.
11. The bivalent compound of claim 8 or 9, wherein the CDK4/6
ligand is selected from the group consisting of: ##STR00295##
12. The bivalent compound of any one of claims 8-11, wherein the
CDK4/6 ligand is bound to CDK4/6.
13. The bivalent compound of any one of claims 8-12, wherein EL is
selected from: ##STR00296## wherein V, W, and X are independently
CR.sup.2 or N; Y is CO or CH.sub.2; Z is CH.sub.2, NH, or O;
R.sup.1 is hydrogen, methyl, or fluoro; and R.sup.2 is hydrogen,
halogen, or C1-C5 alkyl; ##STR00297## wherein R.sup.1 and R.sup.2
are independently hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8
haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, or C2-C8 alkynyl; or ##STR00298##
wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are independently
hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8
hydroxyalkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl,
or C2-C8 alkynyl; and V, W, X, and Z are independently CR.sup.4 or
N.
14. The bivalent compound of any one of claims 1-13, wherein the
degradation/disruption tag is selected from the group consisting of
pomalidomide, thalidomide, lenalidomide, VHL-1, adamantane,
1-((4,4,5,5,5-pentafluoropentyl) sulfinyl)nonane, nutlin-3a,
RG7112, RG7338, AMG 232, AA-115, bestatin, MV-1, LCL161, and
analogs thereof.
15. The bivalent compound of any one of claims 1-13, wherein the
degradation/disruption tag is selected from the group consisting
of: ##STR00299## ##STR00300## ##STR00301##
16. The bivalent compound of any one of claims 1-15, wherein the
degradation/disruption tag is bound to a ubiquitin ligase.
17. The bivalent compound of any one of claims 1-16, wherein the
degradation/disruption tag functions as a hydrophobic group that
leads to CDK4 or CDK6 protein misfolding.
18. The bivalent compound of any one of claims 8-17, wherein the
linker comprises acyclic or cyclic saturated or unsaturated carbon,
ethylene glycol, amide, amino, ether, urea, carbamate, aromatic,
heteroaromatic, heterocyclic or carbonyl containing groups with
different lengths.
19. The bivalent compound of any one of claims 8-17, wherein the
linker is selected from the group consisting of: ##STR00302##
wherein X is C.dbd.O or CH.sub.2; Y is C.dbd.O or CH.sub.2; and n
is 0-15; ##STR00303## wherein X is C.dbd.O or CH.sub.2; Y is
C.dbd.O or CH.sub.2; m is 0-15; n is 0-6; and o is 0-15; or
##STR00304## wherein X is C.dbd.O or CH.sub.2; Y is C.dbd.O or
CH.sub.2; R is --CH.sub.2--, --CF.sub.2--, --CH(C.sub.1-3 alkyl)-,
--C(C.sub.1-3 alkyl)(C.sub.1-3 alkyl)-, --CH.dbd.CH--,
--C(C.sub.1-3 alkyl).dbd.C(C.sub.1-3 alkyl)-, --C.dbd.C--, --O--,
--NH--, --N(C.sub.1-3 alkyl)-, --C(O)NH--, --C(O)N(C.sub.1-3
alkyl)-, a 3-13 membered ring, a 3-13 membered fused ring, a 3-13
membered bridged ring, or a 3-13 membered spiro ring; m is 0-15;
and n is 0-15.
20. The bivalent compound of claim 19, wherein the linker is
Formula C and R is selected from the group consisting of 3-13
membered rings, 3-13 membered fused rings, 3-13 membered bridged
rings, and 3-13 membered spiro rings, wherein R contains one or
more heteroatoms.
21. The bivalent compound of claim 19, wherein the linker is
Formula C and R is selected from the group consisting of:
##STR00305##
22. The bivalent compound of claim 1, wherein the CDK4/6 ligand is
selected from the group consisting of: ##STR00306##
23. The bivalent compound of any one of claims 6-22, wherein the
ubiquitin ligase is an E3 ligase.
24. The bivalent compound of claim 23, wherein the E3 ligase is
selected from the group consisting of cereblon E3 ligase, VHL E3
ligase, MDM2 ligase, TRIM24 ligase, TRIM21 ligase, and TAP
ligase.
25. The bivalent compound of any one of claims 1-8, wherein the
bivalent compound is selected from the group consisting of
XY028-082, XY028-003, XY028-004, XY028-005, XY019-098, XY028-006,
XY028-007, XY028-008, XY028-009, XY028-085, XY028-084, XY028-083,
XY028-132, XY028-133, XY019-106, XY028-162, XY028-163, XY028-002,
XY028-114, XY028-097, XY019-108, XY028-105, XY028-106, XY028-140,
XY028-141, XY028-142, XY028-143, XY028-144, XY028-145, YX26-56,
YX26-66, YX26-58, YX30-108, YX30-107, YX30-85, YX30-86, YX30-117,
YX30-118, YX30-126, YX30-125, XY028-186, YX33-29, YX33-31, YX33-74,
YX33-94, YX33-108, YX33-96, YX33-97, YX33-109, YX33-110, YX33-112,
YX33-123, YX35-48, YX39-47, YX39-48, YX39-56, YX39-65, YX39-74,
YX39-123, YX39-124, YX39-147, YX44-18, YX44-19, YX44-22, YX44-46,
YX44-48, YX44-78, YS36-95, YS36-60, YS36-61, YS36-62, YS36-63,
YS36-64, YS36-65, YS36-66, YS36-67, YS36-68, YS36-69, YS36-70,
YS36-71, compound examples 80-135, and analogs thereof.
26. A bivalent compound selected from the group consisting of
XY028-082, XY028-003, XY028-004, XY028-005, XY019-098, XY028-006,
XY028-007, XY028-008, XY028-009, XY028-085, XY028-084, XY028-083,
XY028-132, XY028-133, XY019-106, XY028-162, XY028-163, XY028-002,
XY028-114, XY028-097, XY019-108, XY028-105, XY028-106, XY028-140,
XY028-141, XY028-142, XY028-143, XY028-144, XY028-145, YX26-56,
YX26-66, YX26-58, YX30-108, YX30-107, YX30-85, YX30-86, YX30-117,
YX30-118, YX30-126, YX30-125, XY028-186, YX33-29, YX33-31, YX33-74,
YX33-94, YX33-108, YX33-96, YX33-97, YX33-109, YX33-110, YX33-112,
YX33-123, YX35-48, YX39-47, YX39-48, YX39-56, YX39-65, YX39-74,
YX39-123, YX39-124, YX39-147, YX44-18, YX44-19, YX44-22, YX44-46,
YX44-48, YX44-78, YS36-95, YS36-60, YS36-61, YS36-62, YS36-63,
YS36-64, YS36-65, YS36-66, YS36-67, YS36-68, YS36-69, YS36-70,
YS36-71, compound examples 80-135, and analogs thereof.
27. A method of treating a cyclin-dependent kinase 4/6
(CDK4/6)-mediated cancer, which comprises administering to a
subject in need thereof with a CDK4/6-mediated cancer a bivalent
compound comprising a CDK4/6 ligand conjugated to a
degradation/disruption tag.
28. The method of claim 27, wherein the CDK4/6-mediated cancer
overexpresses cyclin-dependent kinase 4 (CDK4) or cyclin-dependent
kinase 6 (CDK6) relative to a wild-type tissue of the same species
and tissue type.
29. The method of claim 27, wherein the CDK4/6-mediated cancer
comprises higher CDK4 or CDK6 enzymatic activity relative to a
wild-type tissue of the same species and tissue type.
30. The method of any one of claims 27-29, wherein at least one
bivalent compound is selected from the group consisting of
XY028-082, XY028-003, XY028-004, XY028-005, XY019-098, XY028-006,
XY028-007, XY028-008, XY028-009, XY028-085, XY028-084, XY028-083,
XY028-132, XY028-133, XY019-106, XY028-162, XY028-163, XY028-002,
XY028-114, XY028-097, XY019-108, XY028-105, XY028-106, XY028-140,
XY028-141, XY028-142, XY028-143, XY028-144, XY028-145, YX26-56,
YX26-66, YX26-58, YX30-108, YX30-107, YX30-85, YX30-86, YX30-117,
YX30-118, YX30-126, YX30-125, XY028-186, YX33-29, YX33-31, YX33-74,
YX33-94, YX33-108, YX33-96, YX33-97, YX33-109, YX33-110, YX33-112,
YX33-123, YX35-48, YX39-47, YX39-48, YX39-56, YX39-65, YX39-74,
YX39-123, YX39-124, YX39-147, YX44-18, YX44-19, YX44-22, YX44-46,
YX44-48, YX44-78, YS36-95, YS36-60, YS36-61, YS36-62, YS36-63,
YS36-64, YS36-65, YS36-66, YS36-67, YS36-68, YS36-69, YS36-70,
YS36-71, compound examples 80-135, and analogs thereof.
31. The method of any one of claims 27-30, wherein at least one
bivalent compound is administered orally, parenterally,
intradermally, subcutaneously, topically, or rectally.
32. The method of any one of claims 28-31, further comprising
treating the subject with one or more additional therapeutic
regimens for treating cancer.
33. The method of claim 32, wherein the one or more additional
therapeutic regimens are selected from the group consisting of
surgery, chemotherapy, radiation therapy, hormone therapy, and
immunotherapy.
34. The method of any of claims 27-33, wherein the CDK4/6-mediated
cancer is selected from the group consisting of mesothelioma,
hepatocellular cancer, central nervous system neoplasm, lung
cancer, bone cancer, pancreatic cancer, skin cancer, head and neck
cancer, melanoma, ovarian cancer, colon cancer, rectal cancer, anal
cancer, stomach cancer, gastrointestinal cancer, breast cancer,
uterine cancer, fallopian tube cancer, endometrial cancer, cervical
cancer, vaginal cancer, vulvar cancer, esophageal cancer,
gastrointestinal cancer, endocrine cancer, thyroid cancer,
parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral
cancer, penile cancer, prostate cancer, testicular cancer,
leukemia, lymphoma, bladder cancer, renal cell cancer, brain stem
glioma, pituitary cancer, adrenocortical cancer, gallbladder
cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma,
neuroblastoma, and retinoblastoma.
35. The method of claim 34, wherein the breast cancer is estrogen
receptor positive (ER+).
36. The method of any of claims 27-35, wherein the CDK4/6-mediated
cancer is a relapsed cancer.
37. The method of any of claims 27-36, wherein the CDK4/6-mediated
cancer was refractory to one or more previous treatments.
38. A method for identifying a bivalent compound which mediates
degradation/disruption of CDK4 or CDK6, the method comprising:
providing a heterobifunctional test compound comprising a CDK4/6
ligand conjugated to a degradation/disruption tag; contacting the
heterobifunctional test compound with a cell comprising a ubiquitin
ligase and at least one of CDK4 and CDK6; determining whether CDK4
or CDK6 levels decrease in the cell; and (i) identifying the
heterobifunctional test compound as a bivalent compound which
mediates degradation/reduction of CDK4 if CDK4 levels decrease in
the cell and CDK6 levels do not decrease in the cell, (ii)
identifying the heterobifunctional test compound as a bivalent
compound which mediates degradation/reduction of CDK6 if CDK6
levels decrease in the cell and CDK4 levels do not decrease in the
cell, or (iii) identifying the heterobifunctional test compound as
a bivalent compound which mediates degradation/reduction of CDK4
and CDK6 if both CDK4 and CDK6 levels decrease in the cell.
39. The method of claim 38, wherein the cell is a cancer cell.
40. The method of claim 39, wherein the cancer cell is a
CDK4/6-mediated cancer cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional, and claims priority, of
co-pending U.S. application Ser. No. 16/467,888, filed Jun. 7,
2019, which is a U.S. National Stage application, and claims
priority of International Application No. PCT/US2017/065027, filed
Dec. 7, 2017, which claims priority of U.S. Provisional Application
Ser. No. 62/431,806, filed Dec. 8, 2016. The contents of all of the
prior applications are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing that has been
submitted electronically as an ASCII text file named
27527-0152002SEQ.txt. The ASCII text file, created on Nov. 3, 2021,
is *4 kilobytes in size. The material in the ASCII text file is
hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] This disclosure relates to bivalent compounds (e.g.,
bi-functional small molecule compounds) which selectively degrade
and/or disrupt cyclin-dependent kinase (CDK) 4 and/or 6,
compositions comprising one or more of the bivalent compounds, and
to methods of use thereof for the treatment of CDK4/6-mediated
cancer in a subject in need thereof. The disclosure also relates to
methods for designing such bivalent compounds.
BACKGROUND OF THE INVENTION
[0004] Cyclin-dependent kinase 4 (CDK4, also known as CMM3 and
PSK-J3) (Matsushime et al., 1992) and cyclin-dependent kinase 6
(CDK6, also known as MCPH12 and PLSTIRE) (Meyerson and Harlow,
1994) are related serine/threonine kinases (referred to together as
"CDK4/6") that play a critical role in the cyclin D/CDK4/6/Rb/E2F
signaling pathway ("CDK4/6/Rb signaling") (Sherr et al., 2016).
CDK4/6/Rb signaling mediates physiological cell cycle progression
and cell proliferation. Dephosphorylated Rb binds transcription
factors of the E2F family, thus preventing transition through the S
phase. CDK4 and CDK6 promote cell cycle progression by
phosphorylating Rb. Phosphorylation of Rb allows E2F to dissociate
from Rb and promote transition through the S phase of the cell
cycle (Burkhart and Sage, 2008).
[0005] Multiple types of cancer, including breast cancer, have been
found to depend on aberrant activation of CDK4/6/Rb signaling for
their progression ("CDK4/6-mediated cancer") (Yu et al., 2006;
Hamilton and Infante, 2016; Lim et al., 2016). Conventional cancer
treatments include surgery, radiation therapy, chemotherapy (e.g.,
gemcitabine HCl and temozolomide, a cytotoxic DNA alkylating
agent), hormonal therapy, targeted antibody therapy, and
combinations thereof. Among women, breast cancer has a considerably
higher incident rate (43.3 per 100,000) than any other cancer. In
North America, breast cancer is one of the leading causes of cancer
death among women (about 15%), only second to lung cancer.
[0006] Significant effort has been spent on developing therapeutics
capable of inhibiting the activity of CDK4/6. Three CDK4/6
inhibitors (palbociclib (PD-0332991; Pfizer), abemaciclib
(LY2835219; Lilly), and ribociclib (LEE011; Novartis)) have been
approved. All three compounds have shown preclinical activity in a
range of tumor models, dependent on the expression of intact Rb in
the tumor. Recently, three additional CDK4/6 inhibitors,
trilaciclib (G1T28), G1T38, and SHR6390, have entered phase I
clinical trials.
[0007] Clinical results have shown that patients with estrogen
receptor (ER) positive (ER+) breast tumors show remarkable
responses and increased progression-free survival when treated with
CDK4/6 inhibitors coupled with endocrine therapy (i.e., treatment
with one or more aromatase inhibitors or ER antagonists) (Finn et
al., 2015; Turner et al., 2015). Despite the initial response to
such treatments, however, the majority of these patients eventually
develop resistance to such treatment within 14-28 months (Finn et
al., 2015). Preliminary data suggest that such acquired resistance
can arise from failure of inhibitor to suppress CDK4/6/Rb signaling
or from loss of Rb (Herrera-Abreu et al., 2016). Overall, the
clinical efficacy of CDK4/6 inhibitor monotherapy (i.e., CDK4/6
treatment alone without endocrine or other therapy) appears to be
modest (Sherr et al., 2016). Toxicity can limit the administration
of these inhibitors at a concentration high enough to sufficiently
inhibit Rb phosphorylation in the tumor.
SUMMARY
[0008] The present disclosure relates generally to bivalent
compounds (e.g., bi-functional small molecule compounds) which
selectively degrade and/or disrupt CDK4/6, and to methods for the
treatment of CDK4/6-mediated cancer (i.e., a cancer that
overexpresses CDK4, CDK6 or both; cancer which depends on CDK4,
CDK6, or both activity; or cancer having elevated levels of CDK4,
CDK6, or both, activity relative to a wild-type tissue of the same
species and tissue type). It is important to note, because the
CDK4/6 degraders/disruptors have dual functions (enzyme inhibition
plus protein degradation/disruption), the bivalent compounds
disclosed/claimed here can be significantly more effective
therapeutic agents than current CDK4/6 inhibitors, which inhibit
the enzymatic activity of CDK4/6 but do not affect CDK4/6 protein
levels. The present disclosure further provides methods for
identifying CDK4/6 degraders/disruptors as described herein.
[0009] More specifically, the present disclosure provides a
bivalent compound including a cyclin-dependent kinase 4/6 (CDK4/6)
ligand conjugated to a degradation/disruption tag.
[0010] In an aspect, the CDK4/6 degraders/disruptors have the form
"PI-linker-EL" as shown below:
##STR00001##
wherein PI comprises a CDK4/6 ligand (e.g., a CDK4/6 inhibitor) and
EL comprises a degradation/disruption tag (e.g., E3 ligase ligand).
Exemplary CDK4/6 ligands (PI) and exemplary degradation/disruption
tags (EL) are disclosed herein.
[0011] For example, PI can include, but is not limited to:
##STR00002##
[0012] wherein X.sup.1, X.sup.2, and X.sup.3 are independently
hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkoxyalkyl,
NR.sup.5R.sup.6, CN, NO.sub.2, COR.sup.5, CO.sub.2R.sup.5,
CONR.sup.5R.sup.6, or NR.sup.5COR.sup.6;
[0013] R.sup.1 and R.sup.4 are independently hydrogen, halogen,
C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkoxyalkyl, C1-C8 haloalkyl,
C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8
alkenyl, C2-C8 alkynyl, OR.sup.5, SR.sup.5, NR.sup.5R.sup.6, CN,
NO.sub.2, (CR.sup.5R.sup.6)mNR.sup.7R.sup.8,
(CR.sup.5R.sup.6)mC(O)R.sup.7, COR.sup.5, CO.sub.2R.sup.5,
CONR.sup.5R.sup.6, NR.sup.5COR.sup.6, NR.sup.5SOR.sup.6,
NR.sup.5SO.sub.2R.sup.6, SOR.sup.5, SO.sub.2R.sup.5,
SO.sub.2NR.sup.5R.sup.6, (CR.sup.5R.sup.6)m-aryl, or
(CR.sup.5R.sup.6)m-heteroaryl, wherein m is 0-8;
[0014] R.sup.2 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C3-C7 cycloalkyl, or C3-C7 heterocyclyl;
[0015] R.sup.3 is hydrogen, aryl, C1-C8 alkyl, C1-C8 alkoxy, C3-C7
cycloalkyl, or C3-C7 heterocyclyl;
[0016] R.sup.5, R.sup.6, R.sup.7, R.sup.8 are independently
hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or
heteroarylalkyl;
[0017] optionally, R.sup.1 and R.sup.2; R.sup.5 and R.sup.6; or
R.sup.7 and R.sup.8 independently form 4-8 membered alkyl or
heterocyclyl rings; and
[0018] X and Y are independently CR.sup.5R.sup.6 or N.
[0019] For example, PI can include:
##STR00003##
[0020] wherein R.sup.1 is independently hydrogen, C1-C8 alkyl,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, or C2-C8
alkynyl;
[0021] R.sup.2 is hydrogen, C1-C3 alkyl, or cyclopropyl;
[0022] R.sup.3, R.sup.4, and R.sup.5 are independently hydrogen,
halogen, C1-C8 alkyl, C1-C8 alkoxy, C3-C7 cycloalkyl, or C3-C7
heterocyclyl;
[0023] R.sup.6 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.7, SR.sup.7, NR.sup.7R.sup.8, CN, NO.sub.2,
(CR.sup.7R.sup.8)mNR.sup.9R.sup.10, (CR.sup.7R.sup.8)mC(O)R.sup.9,
COR.sup.7, CO.sub.2R.sup.7, CONR.sup.7R.sup.8, NR.sup.7COR.sup.8,
NR.sup.7SOR.sup.8, NR.sup.7SO.sub.2R.sup.8, SORT, SO.sub.2R.sup.7,
SO.sub.2NR.sup.7R.sup.8, (CR.sup.7R.sup.8)m-aryl, or
(CR.sup.7R.sup.8)m-heteroaryl, wherein m is 0-8;
[0024] R.sup.7, R.sup.8, R.sup.9, R.sup.10 are independently
hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or
heteroarylalkyl;
[0025] optionally, R.sup.7 and R.sup.8; R.sup.9 and R.sup.10
independently form 4-8 membered alkyl or heterocyclyl rings;
and
[0026] X and Y are independently CR.sup.7R.sup.8 or N.
[0027] For example, PI can include:
##STR00004##
[0028] wherein R.sup.1 is independently hydrogen, C1-C8 alkyl,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, or C2-C8
alkynyl;
[0029] R.sup.2 is hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8
haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl, CN, COR.sup.4,
CO.sub.2R.sup.4, or CONR.sup.4R.sup.5;
[0030] R.sup.3 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, CN, NO.sub.2,
(CR.sup.4R.sup.5)mNR.sup.6R.sup.7, (CR.sup.4R.sup.5)mC(O)R.sup.6,
COR.sup.4, CO.sub.2R.sup.4, CONR.sup.4R.sup.5, NR.sup.4COR.sup.5,
NR.sup.4SOR.sup.5, NR.sup.4SO.sub.2R.sup.5, SOR.sup.4,
SO.sub.2R.sup.4, SO.sub.2NR.sup.4R.sup.5, (CR.sup.4R.sup.5)m-aryl,
or (CR.sup.4R.sup.5)m-heteroaryl, wherein m is 0-8;
[0031] R.sup.4, R.sup.5, R.sup.6, R.sup.7 are independently
hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or
heteroarylalkyl;
[0032] optionally, R.sup.1 and R.sup.2; R.sup.4 and R.sup.5;
R.sup.6 and R.sup.7 independently form 4-8 membered alkyl or
heterocyclyl rings; and
[0033] V, W, X, Y, and Z are independently CR.sup.4R.sup.5 or
N.
[0034] For example, PI can include:
##STR00005##
wherein R.sup.1 and R.sup.2 are independently hydrogen, C1-C8
alkyl, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl,
C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, or C2-C8
alkynyl;
[0035] R.sup.3 is hydrogen, C1-C6 alkyl, C1-C6 alkoxyalkyl, C1-C6
haloalkyl, C1-C6 hydroxyalkyl, C3-C6 cycloalkyl, C3-C6
heterocyclyl, C2-C6 alkenyl, or C2-C6 alkynyl;
[0036] R.sup.4 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.5, SR.sup.5, NR.sup.5R.sup.6, CN, NO.sub.2,
(CR.sup.5R.sup.6)mNR.sup.7R.sup.8, (CR.sup.5R.sup.6)mC(O)R.sup.7,
COR.sup.5, CO.sub.2R.sup.5, CONR.sup.5R.sup.6, NR.sup.5COR.sup.6,
NR.sup.5SOR.sup.6, NR.sup.5SO.sub.2R.sup.6, SOR.sup.5,
SO.sub.2R.sup.5, SO.sub.2NR.sup.5R.sup.6, (CR.sup.5R.sup.6)m-aryl,
or (CR.sup.5R.sup.6)m-heteroaryl, wherein m is 0-8;
[0037] n is independently 0-4;
[0038] optionally, R.sup.1 and R.sup.2; R.sup.5 and R.sup.6;
R.sup.7 and R.sup.8 independently form 4-8 membered alkyl or
heterocyclyl rings; and
[0039] V, W, X, Y, and Z are independently CR.sup.5R.sup.6 or
N.
[0040] The CDK4/6 ligand can be a CDK4/6 inhibitor, such as, e.g.,
abemaciclib, palbociclib, ribociclib, trilaciclib (G1T28), G1T38,
SHR6390, and/or analogs thereof.
[0041] In some aspects, the CDK4/6 ligand can be, e.g.,
##STR00006##
The CDK4/6 ligand can be bound to CDK4/6.
[0042] EL includes, but is not limited to:
##STR00007##
[0043] wherein V, W, X are independently CR.sup.2 or N;
[0044] Y is CO or CH.sub.2;
[0045] Z is CH.sub.2, NH, or O;
[0046] R.sup.1 is hydrogen, methyl, or fluoro; and
[0047] R.sup.2 is hydrogen, halogen, or C1-C5 alkyl.
[0048] For example, EL can include:
##STR00008##
[0049] wherein R.sup.1 and R.sup.2 are independently hydrogen,
C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8
hydroxyalkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl,
or C2-C8 alkynyl.
[0050] For example, EL can include:
##STR00009##
[0051] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
independently hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8
haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, or C2-C8 alkynyl; and
[0052] V, W, X, Z are independently CR.sup.4 or N.
[0053] In some aspects, the degradation/disruption tag can be,
e.g., pomalidomide, thalidomide, lenalidomide, VHL-1, adamantane,
1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane, nutlin-3a, RG7112,
RG7338, AMG 232, AA-115, bestatin, MV-1, LCL161, and/or analogs
thereof.
[0054] In some aspects, the degradation/disruption tag can be,
e.g.,
##STR00010## ##STR00011## ##STR00012##
Formulas XIV-XXX (left to right, then top to bottom, starting with
Formula XIV at the top left corner and ending with Formula XXX at
the bottom right corner).
[0055] In some aspects, the degradation/disruption tag can bind to
a ubiquitin ligase (e.g., an E3 ligase such as a cereblon E3
ligase, a VHL E3 ligase, a MDM2 ligase, a TRIM21 ligase, a TRIM24
ligase, and/or a IAP ligase) and/or serve as a hydrophobic group
that leads to CDK4 or CDK6 protein misfolding.
[0056] In any of the above-described compounds, the CDK4/6 ligand
can be conjugated to the degradation/disruption tag through a
linker. The linker can include, for example, acyclic or cyclic
saturated or unsaturated carbon, ethylene glycol, amide, amino,
ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic
and/or carbonyl containing groups with different lengths.
[0057] In some embodiments, the linker can be a moiety of:
##STR00013##
wherein X is C.dbd.O or CH.sub.2,
Y is C.dbd.O or CH.sub.2, and
[0058] n is 0-15;
##STR00014##
wherein X is C.dbd.O or CH.sub.2,
Y is C.dbd.O or CH.sub.2,
[0059] m is 0-15, n is 0-6, and o is 0-15; or
##STR00015##
wherein
X is C.dbd.O or CH.sub.2,
Y is C.dbd.O or CH.sub.2,
[0060] R is --CH.sub.2--, --CF.sub.2--, --CH(C.sub.1-3 alkyl)-,
--C(C.sub.1-3 alkyl)(C.sub.1-3 alkyl)-, --CH.dbd.CH--,
--C(C.sub.1-3 alkyl).dbd.C(C.sub.1-3 alkyl)-, --C.dbd.C--, --O--,
--NH--, --N(C.sub.1-3 alkyl)-, --C(O)NH--, --C(O)N(C.sub.1-3
alkyl)-, a 3-13 membered ring, a 3-13 membered fused ring, a 3-13
membered bridged ring, and/or a 3-13 membered spiro ring, m is
0-15, and n is 0-15.
[0061] In some embodiments of Formula C, R is a 3-13 membered ring,
a 3-13 membered fused ring, a 3-13 membered bridged ring, and/or a
3-13 membered spiro ring, one or more of which can contain one or
more heteroatoms.
[0062] In some embodiments of Formula C, R has a structure of
##STR00016##
[0063] In some aspects, the bivalent compound is a compound
selected from XY028-082, XY028-003, XY028-004, XY028-005,
XY019-098, XY028-006, XY028-007, XY028-008, XY028-009, XY028-085,
XY028-084, XY028-083, XY028-132, XY028-133, XY019-106, XY028-162,
XY028-163, XY028-002, XY028-114, XY028-097, XY019-108, XY028-105,
XY028-106, XY028-140, XY028-141, XY028-142, XY028-143, XY028-144,
XY028-145, YX26-56, YX26-66, YX26-58, YX30-108, YX30-107, YX30-85,
YX30-86, YX30-117, YX30-118, YX30-126, YX30-125, XY028-186,
YX33-29, YX33-31, YX33-74, YX33-94, YX33-108, YX33-96, YX33-97,
YX33-109, YX33-110, YX33-112, YX33-123, YX35-48, YX39-47, YX39-48,
YX39-56, YX39-65, YX39-74, YX39-123, YX39-124, YX39-147, YX44-18,
YX44-19, YX44-22, YX44-46, YX44-48, YX44-78, YS36-95, YS36-60,
YS36-61, YS36-62, YS36-63, YS36-64, YS36-65, YS36-66, YS36-67,
YS36-68, YS36-69, YS36-70, YS36-71, and compound examples 80-135,
or analogs thereof.
[0064] In some aspects, the document provides a method of treating
a cyclin-dependent kinase 4/6 (CDK4/6)-mediated cancer, the method
including administering to a subject in need thereof with a
CDK4/6-mediated cancer one or more bivalent compounds including a
CDK4/6 ligand conjugated to a degradation/disruption tag. The
CDK4/6-mediated cancer may be a cancer which overexpresses
cyclin-dependent kinase 4 (CDK4) and/or cyclin-dependent kinase 6
(CDK6) relative to a wild-type tissue of the same species and
tissue type. The CDK4/6-mediated cancer can have elevated CDK4
and/or CDK6 enzymatic activity relative to a wild-type tissue of
the same species and tissue type. Non-limiting examples of
CDK4/6-mediated cancer include mesothelioma, hepatocellular cancer,
central nervous system neoplasm, lung cancer, bone cancer,
pancreatic cancer, skin cancer, head and neck cancer, melanoma,
ovarian cancer, colon cancer, rectal cancer, anal cancer, stomach
cancer, gastrointestinal cancer, breast cancer (e.g., estrogen
receptor positive (ER+) breast cancer), uterine cancer, fallopian
tube cancer, endometrial cancer, cervical cancer, vaginal cancer,
vulvar cancer, esophageal cancer, gastrointestinal cancer,
endocrine cancer, thyroid cancer, parathyroid cancer, adrenal
cancer, soft tissue sarcoma, urethral cancer, penile cancer,
prostate cancer, testicular cancer, leukemia, lymphoma, bladder
cancer, renal cell cancer, brain stem glioma, pituitary cancer,
adrenocortical cancer, gallbladder cancer, multiple myeloma,
cholangiocarcinoma, fibrosarcoma, neuroblastoma, and/or
retinoblastoma. The CDK4/6-mediated cancer can be a relapsed
cancer. The CDK4/6-mediated cancer can have been refractory to one
or more previous treatments.
[0065] In any of the above-described methods, the bivalent
compounds can be XY028-082, XY028-003, XY028-004, XY028-005,
XY019-098, XY028-006, XY028-007, XY028-008, XY028-009, XY028-085,
XY028-084, XY028-083, XY028-132, XY028-133, XY019-106, XY028-162,
XY028-163, XY028-002, XY028-114, XY028-097, XY019-108, XY028-105,
XY028-106, XY028-140, XY028-141, XY028-142, XY028-143, XY028-144,
XY028-145, YX26-56, YX26-66, YX26-58, YX30-108, YX30-107, YX30-85,
YX30-86, YX30-117, YX30-118, YX30-126, YX30-125, XY028-186,
YX33-29, YX33-31, YX33-74, YX33-94, YX33-108, YX33-96, YX33-97,
YX33-109, YX33-110, YX33-112, YX33-123, YX35-48, YX39-47, YX39-48,
YX39-56, YX39-65, YX39-74, YX39-123, YX39-124, YX39-147, YX44-18,
YX44-19, YX44-22, YX44-46, YX44-48, YX44-78, YS36-95, YS36-60,
YS36-61, YS36-62, YS36-63, YS36-64, YS36-65, YS36-66, YS36-67,
YS36-68, YS36-69, YS36-70, YS36-71, and compound examples 80-135,
or analogs thereof.
[0066] In some embodiments of the disclosed methods, the bivalent
compounds can be administered, e.g., orally, parenterally,
intradermally, subcutaneously, topically, and/or rectally.
[0067] Any of the above-described methods can further include
treating the subject with one or more additional therapeutic
regimens for treating cancer. The one or more additional
therapeutic regimens for treating cancer can be, e.g., one or more
of surgery, chemotherapy, radiation therapy, hormone therapy, or
immunotherapy.
[0068] The document additionally provides a method for identifying
a bivalent compound which mediates degradation/disruption of CDK4
and/or CDK6, the method including providing a heterobifunctional
test compound including a CDK4/6 ligand conjugated to a
degradation/disruption tag, contacting the heterobifunctional test
compound with a cell (e.g., a cancer cell such as a CDK4/6-mediated
cancer cell) including a ubiquitin ligase and at least one of CDK4
and CDK6. The method can include determining whether CDK4 or CDK6
levels decrease in the cell, and (i) identifying the
heterobifunctional test compound as a bivalent compound which
mediates degradation/reduction of CDK4 if CDK4 levels decrease in
the cell and CDK6 levels do not decrease in the cell, (ii)
identifying the heterobifunctional test compound as a bivalent
compound which mediates degradation/reduction of CDK6 if CDK6
levels decrease in the cell and CDK4 levels do not decrease in the
cell, or (iii) identifying the heterobifunctional test compound as
a bivalent compound which mediates degradation/reduction of CDK4
and CDK6 if both CDK4 and CDK6 levels decrease in the cell.
[0069] As used herein, the terms "about" and "approximately" are
defined as being within plus or minus 10% of a given value or
state, preferably within plus or minus 5% of said value or
state.
[0070] Unless otherwise defined, 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 invention belongs. Methods
and materials are described herein for use in the present
invention; other, suitable methods and materials known in the art
can 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.
[0071] Other features and advantages of the invention will be
apparent from the following detailed description and figures, and
from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0072] FIG. 1 is a series of Western blots showing the effect of
various CDK4/6 inhibitors on CDK4/6 activity (as evidenced by Rb
phosphorylation (pRb level)) and expression of Rb/E2F downstream
targets PLK1 and cyclin A at different concentrations. palbociclib
(PB)-treated breast cancer cells (A); abemaciclib (AB)-treated
breast cancer cells (B); PB-treated melanoma cells (C); AB-treated
melanoma cells (D); 219476-treated breast cancer cells (E).
[0073] FIG. 2 is a series of bar graphs showing the effect of
various CDK4/6 inhibitors on PLK1 and cyclin A (CCNA2) mRNA levels
in indicated breast cancer cells at different concentrations.
[0074] FIG. 3 is a series of bar graphs showing the effect of PB on
PLK1 and cyclin A (CCNA2) mRNA levels in melanoma cancer cells at
different concentrations.
[0075] FIG. 4 is a series of bar graphs showing the effect of
various CDK4/6 inhibitors on cell cycle progression at different
concentrations.
[0076] FIG. 5 is a series of Western blots showing the effect of
various CDK4/6 inhibitors on CDK4 or CDK6 expression at different
concentrations. PB- or AB-treated breast cancer cells (A);
PB-treated melanoma cells (B); AB-treated melanoma cells (C).
[0077] FIG. 6 is a series of bar graphs showing the effect of
various CDK4/6 inhibitors on CDK4 mRNA levels at different
concentrations.
[0078] FIG. 7 is a series of Western blots showing the effect of
various CDK4/6 inhibitors on ectopically expressed of VS-tagged
CDK4 or CDK6 at different concentrations.
[0079] FIG. 8 is a series of pull-down assays showing the effect of
various CDK4/6 inhibitors on the ubiquitination of either
ectopically expressed (A), or endogenous (B) CDK4 at different
concentrations.
[0080] FIG. 9 is a series of Western blots showing the efficacy of
palbociclib in inhibiting Rb phosphorylation in ER+ breast cancer
cells, palbociclib-resistant ER+ breast cancer cells (a) and
melanoma cells (b).
[0081] FIG. 10 is a series of Western blots showing the efficacy of
various CDK4/6 degraders in inhibiting CDK4/6 activity and
suppressing CDK4/6 expression in either MCF7 (A) or T47D (B) breast
cancer cells.
[0082] FIG. 11 is a series of Western blots (A, B, and C) showing
the efficacy of various CDK4/6 degraders with different linkers in
inhibiting CDK4/6 activity and suppressing CDK4/6 expression in
melanoma cells.
[0083] FIG. 12 is a series of Western blots (A, B, C, and D)
showing the efficacy of various CDK4/6 degraders with different
linkers in inhibiting CDK4/6 activity and suppressing CDK4/6
expression in melanoma cells.
[0084] FIG. 13 is a series of Western blots showing the effect of
various CDK4/6 degraders in inhibiting CDK4/6 activity and
suppressing CDK4/6 expression in melanoma cells.
[0085] FIG. 14 is a series of Western blots showing the effect of
various CDK4/6 degraders in inhibiting CDK4/6 activity and
suppressing CDK4/6 expression in melanoma cells.
[0086] FIG. 15 is a series of Western blots showing the effect of
various CDK4/6 degraders in inhibiting CDK4/6 activity and
suppressing CDK4/6 expression in breast cancer (A) and melanoma (B)
cells.
[0087] FIG. 16 is a series of Western blots (A and B) showing the
effect of various CDK4/6 degraders in inhibiting CDK4/6 activity
and suppressing CDK4/6 expression in melanoma cells.
[0088] FIG. 17 is a series of Western blots showing the effect of
various CDK4/6 degraders in inhibiting CDK4/6 activity and
suppressing CDK4/6 expression in breast cancer cells.
[0089] FIG. 18 is a series of Western blots showing the effect of
various CDK4/6 degraders in inhibiting CDK4/6 activity and
suppressing CDK4/6 expression in melanoma cells.
[0090] FIG. 19 is a series of Western blots showing the effect of
various CDK4/6 degraders in inhibiting CDK4/6 activity and
suppressing CDK4/6 expression in breast cancer cells.
[0091] FIG. 20 is a series of images showing the effect of the
CDK4/6 degrader XY028-133 in suppressing cell proliferation of
melanoma cells.
[0092] FIG. 21 is a series of clonogenic assays showing the effect
of the CDK4/6 degrader XY028-133 in suppressing cell proliferation
of melanoma cells.
[0093] FIG. 22 is a series of clonogenic assays showing the effect
of various CDK4/6 degraders in suppressing cell proliferation of
breast cancer cells.
[0094] FIG. 23 is a series of clonogenic assays showing the effect
of various CDK4/6 degraders in suppressing cell proliferation of
breast cancer cells.
DETAILED DESCRIPTION
[0095] The present disclosure is based, in part, on the discovery
that novel heterobifunctional small molecules which selectively
degrade CDK4, CDK6, or both CDK4 and CDK6 ("PROteolysis TArgeting
Chimeras" or "PROTACs") are useful in the treatment of
CDK4/6-mediated cancers, particularly estrogen receptor (ER)
positive (ER+) breast cancer.
[0096] Successful strategies for selective degradation/disruption
of the target protein induced by a small molecule include
recruiting an E3 ubiquitin ligase and mimicking protein misfolding
with a hydrophobic tag (Buckley and Crews, 2014). PROTACs are
bivalent inhibitors with one moiety that binds an E3 ubiquitin
ligase and another moiety that binds the protein target of interest
(Buckley and Crews, 2014). The induced proximity leads to
ubiquitination of the target followed by their degradation at
proteasome. Two types of high affinity small-molecule E3 ligase
ligands have been identified/developed: immunomodulatory drugs
(IMiDs) such as thalidomide and pomalidomide, which bind cereblon
(CRBN or CRL4CRBN), a component of a cullin-RING ubiquitin ligase
(CRL) complex (Ito et al., 2010; Chamberlain et al., 2014; Fischer
et al., 2014; Bondeson et al., 2015; Winter et al., 2015); and
VHL-1, a hydroxyproline-containing ligand, which binds van
Hippel-Lindau protein (VHL or CRL2VHL), a component of another CRL
complex (Buckley et al., 2012; Buckley et al., 2012; Galdeano et
al., 2014; Bondeson et al., 2015; Zengerle et al., 2015). The
PROTAC technology has been successfully applied to degradation of
multiple targets (Bondeson et al., 2015; Buckley et al., 2015; Lu
et al., 2015; Winter et al., 2015; Zengerle et al., 2015; Lai et
al., 2016), but not to degradation of CDK4/6. In addition, a
hydrophobic tagging approach, which utilizes a bulky and
hydrophobic adamantyl group, has been developed to mimic protein
misfolding, leading to the degradation of the target protein by
proteasome (Buckley and Crews, 2014). This approach has also been
successfully applied to selective degradation of the pseudokinase
Her3 (Xie et al., 2014), but not to degradation of CDK4/6.
[0097] As discussed in the following examples, this disclosure
provides specific examples of novel CDK4/6 degraders/disruptors,
and examined the effect of exemplary degraders/disruptors in
inhibiting/disrupting CDK4/6 activity, suppressing CDK4/6
expression, and inhibiting cancer cell proliferation. The results
indicated that these novel small molecules can be beneficial in
treating cancer, especially breast cancer, melanoma, and lung
cancer.
[0098] A number of selective small-molecule CDK4/6 catalytic
inhibitors, such as palbociclib, abemaciclib, ribociclib,
trilaciclib (G1T28), G1T38, and SHR6390, have recently been
discovered. Some of these inhibitors have been in clinical trials
for treating ER+ breast cancer. However, these inhibitors have
exhibited very limited success when administered alone; rather,
they must be co-administered with a second therapy such as
endocrine therapy, causing increased off-target effects and
toxicity. Further, even when co-administered with a second therapy,
the majority of patients treated in the trials have developed
resistance as early as 14 months.
[0099] Surprisingly, it was discovered that in addition to toxicity
issues, CDK4/6 inhibitors weren't even able to suppress CDK4/6
activity when they were administered at high concentrations
(Example 5, FIG. 1). In fact, there was a positive correlation
between palbociclib or abemaciclib concentration and expression of
the Rb/E2F downstream targets PLK1 and cyclin A (Example 6, FIGS. 2
and 3), and a corresponding inverse correlation between inhibitor
concentration and suppression of cell cycle progression (Example 7,
FIG. 4). It was found that this was because, although CDK4/6
inhibitors are able to inhibit the activity of CDK4 and CDK6, the
inhibitors actually (unexpectedly) upregulate the expression of
both CDK4 and CDK6, with a positive correlation between inhibitor
concentration and CDK4/6 expression (Example 8, FIG. 5). CDK4/6
inhibitors don't upregulate the expression of CDK4 and CDK6 at the
mRNA level (Example 8, FIGS. 6 and 7); rather, the inhibitors
protect CDK4/6 from degradation by blocking them from
ubiquitination (Example 9, FIG. 8). Consistent with the above
results, increased CDK4/6 expression was associated with decreased
CDK4/6 inhibitor efficacy (Example 10, FIG. 9). This indicates that
CDK4/6 inhibitors only have a narrow window of activity and
suggests that if cancer cells develop resistance to the inhibitors,
inhibitor dosage cannot simply be increased to overcome the
resistance.
[0100] Current drugs targeting CDK4/6 generally focus on inhibition
of its catalytic function. Here, a different approach was taken: to
develop compounds that directly and selectively target not only the
catalytic function of CDK4/6, but also their level of expression at
the protein level. Strategies for inducing protein degradation
include recruiting E3 ubiquitin ligases, mimicking protein
misfolding with hydrophobic tags, and inhibiting chaperones. For
example, a thalidomide-JQ1 bivalent compound has been used to
hijack the cereblon E3 ligase, inducing highly selective BET
protein degradation in vitro and in vivo and resulting in a
demonstrated delay in leukemia progression in mice (Winter et al.,
2015). Similarly, BET protein degradation has also been induced via
another E3 ligase, VHL (Zengerle et al., 2015). Partial degradation
of Her3 has been induced using an adamantane-modified compound (Xie
et al., 2014). Such an approach, based on the use of bivalent small
molecule compounds, permits more flexible regulation of protein
expression in vitro and in vivo compared with techniques such as
gene knockout or shRNA knockdown. Unlike gene knockout or shRNA
knockdown, a small molecule approach further provides an
opportunity to study dose and time dependency in a disease model
through varying the concentrations and frequencies of
administration of the relevant small molecule.
PROTACs
[0101] In some aspects, the present disclosure provides bivalent
compounds, also referred to herein as PROTACs, comprising a CDK4/6
ligand (or targeting moiety) conjugated to a degradation tag.
Linkage of the CDK4/6 ligand to the degradation tag can be direct,
or indirect via a linker.
[0102] As used herein, the terms "cyclin-dependent kinase 4/6
(CDK4/6) ligand" or "CDK4/6 ligand" or "CDK4/6 targeting moiety"
are to be construed broadly, and encompass a wide variety of
molecules ranging from small molecules to large proteins that
associates with or binds to CDK4, CDK6, or both CDK4 and CDK6. The
CDK4/6 ligand or targeting moiety can be, for example, a small
molecule compound (i.e., a molecule of molecular weight less than
about 1.5 kilodaltons (kDa)), a peptide or polypeptide, nucleic
acid or oligonucleotide, carbohydrate such as oligosaccharides, or
an antibody or fragment thereof.
[0103] The CDK4/6 ligand or targeting moiety can be a CDK4/6
inhibitor (e.g., abemaciclib, palbociclib, ribociclib, trilaciclib
(G1T28), G1T38, SHR6390, and analogs thereof) which is capable of
interfering with the enzymatic activity of CDK4, CDK6, or both CDK4
and CDK6. As used herein, an "inhibitor" refers to an agent that
restrains, retards, or otherwise causes inhibition of a
physiological, chemical or enzymatic action or function. An
inhibitor can cause an at least 5% decrease in enzyme activity. An
inhibitor can also or alternately refer to a drug, compound, or
agent that prevents or reduces the expression, transcription, or
translation of a gene or protein. An inhibitor can reduce or
prevent the function of a protein, e.g., by binding to or
activating/inactivating another protein or receptor.
[0104] Exemplary CDK4/6 ligands include, but are not limited to,
the compounds shown below.
##STR00017##
[0105] As used herein, the term "degradation/disruption tag" refers
to a compound which associates with or binds to a ubiquitin ligase
for recruitment of the corresponding ubiquitination machinery to
CDK4, CDK6, or both CDK4 and CDK6 or induces CDK4, CDK6, or both
CDK4 and CDK6 protein misfolding and subsequent degradation at the
proteasome or loss of function.
[0106] In some aspects, the degradation/disruption tags of the
present disclosure include, e.g., thalidomide, pomalidomide,
lenalidomide, VHL-1, adamantane,
1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane, nutlin-3a, RG7112,
RG7338, AMG 232, AA-115, bestatin, MV-1, LCL161, and/or analogs
thereof.
[0107] As used herein, a "linker" is a bond, molecule, or group of
molecules that binds two separate entities to one another. Linkers
can provide for optimal spacing of the two entities. The term
"linker" in some aspects refers to any agent or molecule that
bridges the CDK4/6 ligand to the degradation/disruption tag. One of
ordinary skill in the art recognizes that sites on the CDK4/6
ligand or the degradation/disruption tag, which are not necessary
for the function of the PROTACs of the present disclosure, are
ideal sites for attaching a linker, provided that the linker, once
attached to the conjugate of the present disclosures, does not
interfere with the function of the PROTAC, i.e., its ability to
target CDK4/6 and recruit a ubiquitin ligase.
[0108] The length of the linker of the bivalent compound can be
adjusted to minimize the molecular weight of the
disruptors/degraders and avoid the clash of the CDK4/6 ligand or
targeting moiety with the ubiquitin ligase or induce CDK4/6
misfolding by the hydrophobic tag at the same time.
[0109] In some embodiments, the degradation/disruption tags of the
present disclosure include, for example, thalidomide, pomalidomide,
lenalidomide, VHL-1, adamantane,
1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane, nutlin-3a, RG7112,
RG7338, AMG 232, AA-115, bestatin, MV-1, LCL161, and analogs
thereof. The degradation/disruption tags can be attached to each
portion of interest in the structure of a CDK4/6 ligand or
targeting moiety (e.g., abemaciclib, palbociclib, ribociclib,
trilaciclib (G1T28), G1T38, or SHR6390) with linkers of different
types and lengths in order to generate effective bivalent
compounds. In particular, attaching thalidomide to either portion
of the molecule can recruit the cereblon E3 ligase to CDK4/6
without causing destructive steric interactions with the
CDK4/6/HSP90/CDCl37 complex.
[0110] The bivalent compounds disclosed herein can selectively
affect CDK4/6-mediated cancer cells (e.g., ER+ cells) compared to
WT cells (i.e., a CDK4/6 degrader disruptor able to kill or inhibit
the growth of a CDK4/6-mediated cancer cell while also having a
relatively low ability to lyse or inhibit the growth of a WT cell),
e.g., possess a GI.sub.50 for one or more CDK4/6-mediated cancer
cells more than 1.5-fold lower, more than 2-fold lower, more than
2.5-fold lower, more than 3-fold lower, more than 4-fold lower,
more than 5-fold lower, more than 6-fold lower, more than 7-fold
lower, more than 8-fold lower, more than 9-fold lower, more than
10-fold lower, more than 15-fold lower, or more than 20-fold lower
than its GI.sub.50 for one or more WT cells, e.g., WT cells of the
same species and tissue type as the CDK4/6-mediated cancer
cells.
[0111] Additional bivalent compounds (i.e., CDK4/6
degraders/disruptors) can be developed using the principles and
methods disclosed herein. For example, other linkers, degradation
tags, and CDK4/6 binding/inhibiting moieties (not limited to
abemaciclib, palbociclib, ribociclib, trilaciclib (G1T28), G1T38,
and SHR6390) can be synthesized and tested. Non-limiting examples
of CDK4/6 disruptors/degraders (e.g., bivalent compounds) are shown
in Table 1 (below). The left portion of the CDK4/6
disruptors/degraders bind to CDK4/6 (as abemaciclib, palbociclib,
ribociclib, trilaciclib (G1T28), G1T38, and SHR6390 do), and the
right portion recruits for the ubiquitination machinery to CDK4/6,
which induces the poly-ubiquitination and degradation of CDK4 and
CDK6, or induces CDK4/6 misfolding and subsequent loss of function
or degradation at the proteasome.
[0112] Non-limiting examples of bivalent compounds are set forth in
Table 1, below.
TABLE-US-00001 TABLE 1 Cpd. Example Cpd. Number Code Structure
Chemical Name 1 XY028- 082 ##STR00018##
(2S,4R)-1-((S)-2-(2-(2-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-2-
oxoethoxy)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 2
XY028- 003 ##STR00019## (2S,4R)-1-((S)-2-(3-(3-(4-(6-
((6-acetyl-8-cyclopentyl-5- methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)propanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 3 XY028- 004 ##STR00020##
(2S,4R)-1-((S)-2-(2-(2-(2-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-2-
oxoethoxy)ethoxy)acetamido)- 3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 4 XY028- 005 ##STR00021##
(2S,4R)-1-((S)-2-(3-(2-(3-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)ethoxy)propanamido)- 3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 5 XY019- 098 ##STR00022## (2S,4R)-1-((S)-14-(4-(6-((6-
acetyl-8-cyclopentyl-5-methyl- 7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-2- yl)amino)pyridin-3- yl)piperazin-1-yl)-2-(tert-
butyl)-4,14-dioxo-6,9,12- trioxa-3-azatetradecanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 6 XY028- 006 ##STR00023## (2S,4R)-1-((S)-16-(4-(6-((6-
acetyl-8-cyclopentyl-5-methyl- 7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-2- yl)amino)pyridin-3- yl)piperazin-1-yl)-2-(tert-
butyl)-4,16-dioxo-7,10,13- trioxa-3-azahexadecanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 7 XY028- 007 ##STR00024## (2S,4R)-1-((S)-19-(4-(6-((6-
acetyl-8-cyclopentyl-5-methyl- 7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-2- yl)amino)pyridin-3- yl)piperazin-1-yl)-2-(tert-
butyl)-4,19-dioxo-7,10,13,16- tetraoxa-3-azanonadecanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 8 XY028- 008 ##STR00025## (2S,4R)-1-((S)-20-(4-(6-((6-
acetyl-8-cyclopentyl-5-methyl- 7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-2- yl)amino)pyridin-3- yl)piperazin-1-yl)-2-(tert-
butyl)-4,20-dioxo-6,9,12,15,18- pentaoxa-3-azaicosanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 9 XY028- 009 ##STR00026## (2S,4R)-1-((S)-22-(4-(6-((6-
acetyl-8-cyclopentyl-5-methyl- 7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-2- yl)amino)pyridin-3- yl)piperazin-1-yl)-2-(tert-
butyl)-4,22-dioxo- 7,10,13,16,19-pentaoxa-3-
azadocosanoyl)-4-hydroxy-N- (4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 10 XY028- 085 ##STR00027##
(2S,4R)-1-((S)-2-(3-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 11 XY028- 084 ##STR00028##
(2S,4R)-1-((S)-2-(4-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-4- oxobutanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 12 XY028- 083 ##STR00029##
(2S,4R)-1-((S)-2-(5-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-5- oxopentanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 13 XY028- 132 ##STR00030##
(2S,4R)-1-((S)-2-(6-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-6- oxohexanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 14 XY028- 133 ##STR00031##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7- oxoheptanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 15 XY019- 106 ##STR00032##
(2S,4R)-1-((S)-2-(8-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-8- oxooctanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 16 XY028- 162 ##STR00033##
(2S,4R)-1-((S)-2-(9-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-9- oxononanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 17 XY028- 163 ##STR00034##
(2S,4R)-1-((S)-2-(10-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-10- oxodecanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 18 XY028- 002 ##STR00035##
(2S,4R)-1-((S)-2-(11-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-11- oxoundecanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 19 XY028- 114 ##STR00036##
4-((2-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)ethyl)amino)-2-
(2,6-dioxopiperidin-3- yl)isoindoline-1,3-dione 20 XY028- 097
##STR00037## 4-((2-(2-(3-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)ethoxy)ethyl)amino)- 2-(2,6-dioxopiperidin-3-
yl)isoindoline-1,3-dione 21 XY019- 108 ##STR00038##
4-((2-(2-(2-(3-(4-(6-((6-acetyl- 8-cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)ethoxy)ethoxy)ethyl)
amino)-2-(2,6- dioxopiperidin-3- yl)isoindoline-1,3-dione 22 XY028-
105 ##STR00039## 4-((15-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-15-oxo- 3,6,9,12-
tetraoxapentadecyl)amino)-2- (2,6-dioxopiperidin-3-
yl)isoindoline-1,3-dione 23 XY028- 106 ##STR00040##
4-((18-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-18-oxo- 3,6,9,12,15- pentaoxaoctadecyl)amino)-2-
(2,6-dioxopiperidin-3- yl)isoindoline-1,3-dione 24 XY028- 140
##STR00041## 4-((2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethyl)amino)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 25 XY028- 141 ##STR00042##
4-((3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropyl)amino)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 26 XY028- 142 ##STR00043##
4-((4-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-4- oxobutyl)amino)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 27 XY028- 143 ##STR00044##
4-((6-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-6- oxohexyl)amino)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 28 XY028- 144 ##STR00045##
4-((7-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7- oxoheptyl)amino)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 29 XY028- 145 ##STR00046##
4-((8-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-8- oxooctyl)amino)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 30 YX26-56 ##STR00047##
2-(2,6-dioxopiperidin-3-yl)-4- ((2-(2-(2-(3-(4-((6-((5-fluoro-4-
(4-fluoro-1-isopropyl-2- methyl-1H-benzo[d]imidazol-
6-yl)pyrimidin-2- yl)amino)pyridin-3- yl)methyl)piperazin-1-yl)-3-
oxopropoxy)ethoxy)ethoxy)ethyl) amino)isoindoline-1,3-dione 31
YX26-66 ##STR00048## N-(2-(2,6-dioxopiperidin-3-yl)-
1,3-dioxoisoindolin-4-yl)-8-(4- ((6-((5-fluoro-4-(4-fluoro-1-
isopropyl-2-methyl-1H- benzo[d]imidazol-6- yl)pyrimidin-2-
yl)amino)pyridin-3- yl)methyl)piperazin-1-yl)-8- oxooctanamide 32
YX26-58 ##STR00049## (2S,4R)-1-((S)-2-(8-(4-((6-((5-
fluoro-4-(4-fluoro-1-isopropyl- 2-methyl-1H- benzo[d]imidazol-6-
yl)pyrimidin-2- yl)amino)pyridin-3- yl)methyl)piperazin-1-yl)-8-
oxooctanamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 33
YX30-108 ##STR00050## 7-cyclopentyl-2-((5-(4-(6-(((S)-
1-((2S,4R)-4-hydroxy-2-((4-(4- methylthiazol-5-
yl)benzyl)carbamoyl)pyrrolidin- 1-yl)-3,3-dimethyl-1-
oxobutan-2-yl)amino)-6- oxohexanoyl)piperazin-1-
yl)pyridin-2-yl)amino)-N,N- dimethyl-7H-pyrrolo[2,3-
d]pyrimidine-6-carboxamide 34 YX30-107 ##STR00051##
7-cyclopentyl-2-((5-(4-(7-(((S)- 1-((2S,4R)-4-hydroxy-2-((4-(4-
methylthiazol-5- yl)benzyl)carbamoyl)pyrrolidin-
1-yl)-3,3-dimethyl-1- oxobutan-2-yl)amino)-7-
oxoheptanoyl)piperazin-1- yl)pyridin-2-yl)amino)-N,N-
dimethyl-7H-pyrrolo[2,3- d]pyrimidine-6-carboxamide 35 YX30-85
##STR00052## 7-cyclopentyl-2-((5-(4-(8-(((S)-
1-((2S,4R)-4-hydroxy-2-((4-(4- methylthiazol-5-
yl)benzyl)carbamoyl)pyrrolidin- 1-yl)-3,3-dimethyl-1-
oxobutan-2-yl)amino)-8- oxooctanoyl)piperazin-1-
yl)pyridin-2-yl)amino)-N,N- dimethyl-7H-pyrrolo[2,3-
d]pyrimidine-6-carboxamide 36 YX30-86 ##STR00053##
7-cyclopentyl-2-((5-(4-(3-(3- (((S)-1-((2S,4R)-4-hydroxy-2-
((4-(4-methylthiazol-5- yl)benzyl)carbamoyl)pyrrolidin-
1-yl)-3,3-dimethyl-1- oxobutan-2-yl)amino)-3-
oxopropoxy)propanoyl)piperazin- 1-yl)pyridin-2-yl)amino)-
N,N-dimethyl-7H-pyrrolo[2,3- d]pyrimidine-6-carboxamide 37 YX30-117
##STR00054## (2S,4R)-1-((S)-2-(6-(4-((6-((5-
fluoro-4-(4-fluoro-1-isopropyl- 2-methyl-1H- benzo[d]imidazol-6-
yl)pyrimidin-2- yl)amino)pyridin-3- yl)methyl)piperazin-1-yl)-6-
oxohexanamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 38
YX30-118 ##STR00055## (2S,4R)-1-((S)-2-(7-(4-((6-((5-
fluoro-4-(4-fluoro-1-isopropyl- 2-methyl-1H- benzo[d]imidazol-6-
yl)pyrimidin-2- yl)amino)pyridin-3- yl)methyl)piperazin-1-yl)-7-
oxoheptanamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 39
YX30-126 ##STR00056## 7-cyclopentyl-2-((5-(4-((2-(2,6-
dioxopiperidin-3-yl)-1,3- dioxoisoindolin-4- yl)glycyl)piperazin-1-
yl)pyridin-2-yl)amino)-N,N- dimethyl-7H-pyrrolo[2,3-
d]pyrimidine-6-carboxamide 40 YX30-125 ##STR00057##
2-(2,6-dioxopiperidin-3-yl)-4- ((2-(4-((6-((5-fluoro-4-(4-
fluoro-1-isopropyl-2-methyl- 1H-benzo[d]imidazol-6- yl)pyrimidin-2-
yl)amino)pyridin-3- yl)methyl)piperazin-1-yl)-2-
oxoethyl)amino)isoindoline- 1,3-dione 41 XY028- 186 ##STR00058##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1- yl)heptanamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 42
YX33-29 ##STR00059## (2S,4R)-1-((S)-2-(8-(4-(6-((6-
acetyl-8-cyclopentyl-5-methyl- 7-oxo-7,8-dihydropyrido[2,3-
d]pyrimidin-2- yl)amino)pyridin-3- yl)piperazin-1-yl)octanamido)-
3,3-dimethylbutanoyl)-4- hydroxy-N-(4-(4- methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 43 YX33-31 ##STR00060##
(2S,4R)-1-((S)-2-(6-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)hexanamido)- 3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 44 YX33-74 ##STR00061## 4-((2-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)ethyl)amino)-
2-(2,6-dioxopiperidin-3- yl)isoindoline-1,3-dione 45 YX33-94
##STR00062## 4-(6-((6-acetyl-8-cyclopentyl- 5-methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-yl)-N-(2-
(2,6-dioxopiperidin-3-yl)-1,3- dioxoisoindolin-4-
yl)piperazine-1-carboxamide 46 YX33-108 ##STR00063##
(2S,4R)-1-((S)-2-(3-((3-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropyl)(methyl)amino)propan- amido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 47 YX33-96 ##STR00064##
(2S,4R)-1-((S)-2-(6-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazine-1- carbonyl)spiro[3.3]heptane-2- carboxamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 48 YX33-97 ##STR00065##
(2S,4R)-1-((S)-2-((E)-8-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-8-oxooct-4- enamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 49 YX33-109 ##STR00066##
(2S,4R)-1-((S)-2-(2-(4-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazine-1- carbonyl)piperidin-1-
yl)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 50
YX33-110 ##STR00067## (2S,4R)-1-((S)-2-(2-(4-(2-(4-(6-
((6-acetyl-8-cyclopentyl-5- methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethyl)phenyl)acetamido)-
3,3-dimethylbutanoyl)-4- hydroxy-N-(4-(4- methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 51 YX33-122 ##STR00068##
(2S,4R)-1-((S)-2-(2-(4-(2-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-2- oxoethyl)piperazin-1-
yl)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 52
YX33-123 ##STR00069## 1-(2-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-2-oxoethyl)-
N-((S)-1-((2S,4R)-4-hydroxy-2- ((4-(4-methylthiazol-5-
yl)benzyl)carbamoyl)pyrrolidin- 1-yl)-3,3-dimethyl-1-
oxobutan-2-yl)piperidine-4- carboxamide 53 YX35-48 ##STR00070##
2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-(2-(2,6- dioxopiperidin-3-yl)-1,3-
dioxoisoindolin-4-yl)acetamide 54 YX39-47 ##STR00071##
(E)-3-(7-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3-oxoprop-1- en-1-yl)-1-oxoisoindolin-2-
yl)piperidine-2,6-dione 55 YX39-48 ##STR00072##
4-(2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethoxy)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 56 YX39-56 ##STR00073##
3-(7-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropyl)-1-oxoisoindolin-2-
yl)piperidine-2,6-dione 57 YX39-65 ##STR00074##
3-(4-((2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethyl)amino)-1-
oxoisoindolin-2-yl)piperidine- 2,6-dione 58 YX39-74 ##STR00075##
5-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropyl)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 59 YX39-123 ##STR00076##
3-(4-(2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethoxy)-1-oxoisoindolin-2-
yl)piperidine-2,6-dione 60 YX39-124 ##STR00077##
3-(4-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropyl)-1-oxoisoindolin-2-
yl)piperidine-2,6-dione 61 YX39-147 ##STR00078##
4-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropyl)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 62 YX44-18 ##STR00079##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3-yl)-1,4- diazepan-1-yl)-7- oxoheptanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 63 YX44-19 ##STR00080##
4-((2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-yl)-1,4-
diazepan-1-yl)-2- oxoethyl)amino)-2-(2,6- dioxopiperidin-3-
yl)isoindoline-1,3-dione 64 YX44-22 ##STR00081##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7- oxoheptanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-((S)-1-(4-(4-methylthiazol-5-
yl)phenyl)ethyl)pyrrolidine-2- carboxamide 65 YX44-46 ##STR00082##
2-((3R,5R,6S)-1-((S)-1-((4-(5- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-5-
oxopentanoyl)piperazin-1- yl)sulfonyl)-3,3-dimethylbutan-
2-yl)-5-(3-chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-
oxopiperidin-3-yl)acetic acid 66 YX44-48 ##STR00083##
5-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-5- oxopentanamide 67
YS36-95 ##STR00084## N-(6-(3-(4-((2-(4-(6-((6-acetyl-
8-cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-2-
oxoethyl)(methyl)amino)butoxy)- 5-propoxyphenoxy)-1,3-
dimethyl-2-oxo-2,3-dihydro- 1H-benzo[d]imidazol-5-yl)- 3,4-
dimethoxybenzenesulfonamide 68 YS36-60 ##STR00085##
N-(2-(2-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)ethoxy)ethyl)-2- ((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 69 YS36-61
##STR00086## N-(15-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-15-oxo- 3,6,9,12-tetraoxapentadecyl)-2-
((4-(3-((6-((3,4- dimethoxyphenyl)sulfonamido)-
1,3-dimethyl-2-oxo-2,3- dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 70 YS36-62
##STR00087## N-(18-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-18-oxo- 3,6,9,12,15-
pentaoxaoctadecyl)-2-((4-(3- ((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 71 YS36-63
##STR00088## N-(2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-2-oxoethyl)- 2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 72 YS36-64
##STR00089## N-(4-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-4-oxobutyl)- 2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 73 YS36-65
##STR00090## N-(2-(3-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)ethyl)-2-((4-(3- ((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 74 YS36-66
##STR00091## N-(2-(2-(2-(3-(4-(6-((6-acetyl-
8-cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)ethoxy)ethoxy)ethyl)- 2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 75 YS 36-67
##STR00092## N-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropyl)-2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 76 YS36-68
##STR00093## N-(5-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-5- oxopentyl)-2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 77 YS36-69
##STR00094## N-(6-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-6-oxohexyl)- 2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 78 YS36-70
##STR00095## N-(7-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7- oxoheptyl)-2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 79 YS36-71
##STR00096## N-(8-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-8-oxooctyl)- 2-((4-(3-((6-((3,4-
dimethoxyphenyl)sulfonamido)- 1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol- 5-yl)oxy)-5-
propoxyphenoxy)butyl)(methyl) amino)acetamide 80 ##STR00097##
4-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-4- oxobutanamide 81
##STR00098## 6-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-6- oxohexanamide
82 ##STR00099## 7-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-7- oxoheptanamide 83
##STR00100## 8-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-8- oxooctanamide 84
##STR00101## 9-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-9- oxononanamide 85
##STR00102## 10-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-10- oxodecanamide 86
##STR00103## 11-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-N-((S)-1- (((S)-1-cyclohexyl-2-((S)-2-(4-
(4-fluorobenzoyl)thiazol-2- yl)pyrrolidin-1-yl)-2-
oxoethyl)amino)-1-oxopropan- 2-yl)-N-methyl-11- oxoundecanamide 87
##STR00104## 3-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)-N-((S)-1-(((S)-1-
cyclohexyl-2-((S)-2-(4-(4- fluorobenzoyl)thiazol-2-
yl)pyrrolidin-1-yl)-2- oxoethyl)amino)-1-oxopropan-
2-yl)-N-methylpropanamide 88 ##STR00105##
3-(2-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)ethoxy)-N-((S)-1-
(((S)-1-cyclohexyl-2-((S)-2-(4- (4-fluorobenzoyl)thiazol-2-
yl)pyrrolidin-1-yl)-2- oxoethyl)amino)-1-oxopropan-
2-yl)-N-methylpropanamide 89 ##STR00106##
3-(2-(2-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)ethoxy)ethoxy)-N-
((S)-1-(((S)-1-cyclohexyl-2- ((S)-2-(4-(4- fluorobenzoyl)thiazol-2-
yl)pyrrolidin-1-yl)-2- oxoethyl)amino)-1-oxopropan-
2-yl)-N-methylpropanamide 90 ##STR00107##
2-((3R,5R,6S)-1-((S)-1-((4-(4- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-4-
oxobutanoyl)piperazin-1- yl)sulfonyl)-3,3-dimethylbutan-
2-yl)-5-(3-chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-
oxopiperidin-3-yl)acetic acid 91 ##STR00108##
2-((3R,5R,6S)-1-((S)-1-((4-(6- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-6-
oxohexanoyl)piperazin-1- yl)sulfonyl)-3,3-dimethylbutan-
2-yl)-5-(3-chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-
oxopiperidin-3-yl)acetic acid 92 ##STR00109##
2-((3R,5R,6S)-1-((S)-1-((4-(7- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-7-
oxoheptanoyl)piperazin-1- yl)sulfonyl)-3,3-dimethylbutan-
2-yl)-5-(3-chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-
oxopiperidin-3-yl)acetic acid 93 ##STR00110##
2-((3R,5R,6S)-1-((S)-1-((4-(8- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-8- oxooctanoyl)piperazin-1-
yl)sulfonyl)-3,3-dimethylbutan- 2-yl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2- oxopiperidin-3-yl)acetic acid 94
##STR00111## 2-((3R,5R,6S)-1-((S)-1-((4-(9-
(4-(6-((6-acetyl-8-cyclopentyl- 5-methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-9- oxononanoyl)piperazin-1-
yl)sulfonyl)-3,3-dimethylbutan- 2-yl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2- oxopiperidin-3-yl)acetic acid 95
##STR00112## 2-((3R,5R,6S)-1-((S)-1-((4-(10-
(4-(6-((6-acetyl-8-cyclopentyl- 5-methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-10- oxodecanoyl)piperazin-1-
yl)sulfonyl)-3,3-dimethylbutan- 2-yl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2- oxopiperidin-3-yl)acetic acid 96
##STR00113## 2-((3R,5R,6S)-1-((S)-1-((4-(11-
(4-(6-((6-acetyl-8-cyclopentyl- 5-methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-11- oxoundecanoyl)piperazin-1-
yl)sulfonyl)-3,3-dimethylbutan- 2-yl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2- oxopiperidin-3-yl)acetic acid 97
##STR00114## 2-((3R,5R,6S)-1-((S)-1-((4-(3- (3-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)propanoyl)piperazin- 1-yl)sulfonyl)-3,3-
dimethylbutan-2-yl)-5-(3- chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2- oxopiperidin-3-yl)acetic acid 98
##STR00115## 2-((3R,5R,6S)-1-((S)-1-((4-(3-
(2-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)ethoxy)propanoyl)
piperazin-1-yl)sulfonyl)-3,3- dimethylbutan-2-yl)-5-(3-
chlorophenyl)-6-(4- chlorophenyl)-3-methyl-2-
oxopiperidin-3-yl)acetic acid 99 ##STR00116##
2-((3R,5R,6S)-1-((S)-1-((4-(3- (2-(2-(3-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)ethoxy)ethoxy)pro- panoyl)piperazin-1-
yl)sulfonyl)-3,3-dimethylbutan- 2-yl)-5-(3-chlorophenyl)-6-(4-
chlorophenyl)-3-methyl-2- oxopiperidin-3-yl)acetic acid 100
##STR00117## (S)-N-((S)-2-((S)-2-(4-(3-(2-(4-
(6-((6-acetyl-8-cyclopentyl-5- methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethoxy)benzoyl)thiazol-2-
yl)pyrrolidin-1-yl)-1- cyclohexyl-2-oxoethyl)-2-
(methylamino)propanamide 101 ##STR00118##
(S)-N-((S)-2-((S)-2-(4-(3-(3-(4- (6-((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)benzoyl)thiazol-2- yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 102 ##STR00119##
(S)-N-((S)-2-((S)-2-(4-(3-(4-(4- (6-((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-4-
oxobutoxy)benzoyl)thiazol-2- yl)pyrrolidin-1-yl)-1-cyclohexyl-
2-oxoethyl)-2- (methylamino)propanamide 103 ##STR00120##
(S)-N-((S)-2-((S)-2-(4-(3-((5- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-5-
oxopentyl)oxy)benzoyl)thiazol- 2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 104 ##STR00121##
(S)-N-((S)-2-((S)-2-(4-(3-((6- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-6-
oxohexyl)oxy)benzoyl)thiazol- 2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 105 ##STR00122##
(S)-N-((S)-2-((S)-2-(4-(3-((7- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dthydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-7-
oxoheptyl)oxy)benzoyl)thiazol- 2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 106 ##STR00123##
(S)-N-((S)-2-((S)-2-(4-(3-((8- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-8-
oxooctyl)oxy)benzoyl)thiazol- 2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 107 ##STR00124##
(S)-N-((S)-2-((S)-2-(4-(3-((9- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-9-
oxononyl)oxy)benzoyl)thiazol- 2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 108 ##STR00125##
(S)-N-((S)-2-((S)-2-(4-(3-((10- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-10-
oxodecyl)oxy)benzoyl)thiazol- 2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 109 ##STR00126##
(S)-N-((S)-2-((S)-2-(4-(3-(2-(3- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)ethoxy)benzoyl)thi- azol-2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 110 ##STR00127##
(S)-N-((S)-2-((S)-2-(4-(3-(2-(2- (3-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropoxy)ethoxy)ethoxy)ben- zoyl)thiazol-2-yl)pyrrolidin-1-
yl)-1-cyclohexyl-2-oxoethyl)- 2-(methylamino)propanamide 111
##STR00128## (S)-N-((S)-2-((S)-2-(4-(3-(2-(2-
(2-(3-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-3- oxopropoxy)ethoxy)ethoxy)eth-
oxy)benzoyl)thiazol-2- yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 112 ##STR00129##
(S)-N-((S)-2-((S)-2-(4-(3-((15- (4-(6-((6-acetyl-8-cyclopentyl-
5-methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-15-oxo- 3,6,9,12-
tetraoxapentadecyl)oxy)benzoyl) thiazol-2-yl)pyrrolidin-1-yl)-
1-cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 113
##STR00130## (S)-N-((S)-2-((S)-2-(4-(3-((18-
(4-(6-((6-acetyl-8-cyclopentyl- 5-methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-18-oxo- 3,6,9,12,15-
pentaoxaoctadecyl)oxy)benzoyl) thiazol-2-yl)pyrrolidin-1-yl)-1-
cyclohexyl-2-oxoethyl)-2- (methylamino)propanamide 114 ##STR00131##
N.sup.1-(2-(4-(6-((6-acetyl-8- cyclopentyl-5-methyl-7-oxo-
7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-2-oxoethyl)- N.sup.4-((S)-1-((2S,4R)-4-hydroxy-
2-((4-(4-methylthiazol-5- yl)benzyl)carbamoyl)pyrrolidin-
1-yl)-3,3-dimethyl-1- oxobutan-2-yl)succinamide 115 ##STR00132##
(2S,4R)-1-((S)-2-(2-(4-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-4-
oxobutanamido)acetamido)- 3,3-dimethylbutanoyl)-4- hydroxy-N-(4-(4-
methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 116
##STR00133## (2S,4R)-1-((S)-2-(2-(4-(2-(4-(6-
((6-acetyl-8-cyclopentyl-5- methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-2-oxoethyl)- 1H-1,2,3-triazol-1-
yl)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 117
##STR00134## (2S,4R)-1-((S)-2-(2-(1-(2-(4-(6-
((6-acetyl-8-cyclopentyl-5- methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-2-oxoethyl)- 1H-1,2,3-triazol-4-
yl)acetamido)-3,3- dimethylbutanoyl)-4-hydroxy-
N-(4-(4-methylthiazol-5- yl)benzyl)pyrrolidine-2- carboxamide 118
##STR00135## (2S,4R)-1-((S)-2-(3-(4-(4-(6-
((6-acetyl-8-cyclopentyl-5- methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazine-1-carbonyl)-1H- 1,2,3-triazol-1- yl)propanamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide
119 ##STR00136## 1-(3-(4-(6-((6-acetyl-8-
cyclopentyl-5-methyl-7-oxo- 7,8-dihydropyrido[2,3- d]pyrimidin-2-
yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropyl)-N-((S)-1-((2S,4R)- 4-hydroxy-2-((4-(4- methylthiazol-5-
yl)benzyl)carbamoyl)pyrrolidin- 1-yl)-3,3-dimethyl-1-
oxobutan-2-yl)-1H-1,2,3- triazole-4-carboxamide 120 ##STR00137##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7-oxohept-5- ynamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 121 ##STR00138##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7-oxohept-4- ynamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 122 ##STR00139##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7-oxohept-3- ynamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 123 ##STR00140##
(2S,4R)-1-((S)-2-(7-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-7-oxohept-2- ynamido)-3,3-
dimethylbutanoyl)-4-hydroxy- N-(4-(4-methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 124 ##STR00141##
(2S,4R)-1-((S)-2-(3-((3-(4-(6- ((6-acetyl-8-cyclopentyl-5-
methyl-7-oxo-7,8- dihydropyrido[2,3-d]pyrimidin-
2-yl)amino)pyridin-3- yl)piperazin-1-yl)-3-
oxopropyl)thio)propanamido)- 3,3-dimethylbutanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 125 ##STR00142## (2S,4R)-1-((S)-2-(2-(3-(2-(4-(6-
((6-acetyl-8-cyclopentyl-5- methyl-7-oxo-7,8-
dihydropyrido[2,3-d]pyrimidin- 2-yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethyl)cyclobutyl)acetamido)-
3,3-dimethylbutanoyl)-4- hydroxy-N-(4-(4- methylthiazol-5-
yl)benzyl)pyrrolidine-2- carboxamide 126 YX44-78 ##STR00143##
(2S,4R)-N-(2-(2-(4-(6-((6- acetyl-8-cyclopentyl-5-methyl-
7-oxo-7,8-dihydropyrido[2,3- d]pyrimidin-2- yl)amino)pyridin-3-
yl)piperazin-1-yl)-2- oxoethoxy)-4-(4-methylthiazol-
5-yl)benzyl)-4-hydroxy-1-((S)- 3-methyl-2-(1-oxoisoindolin-2-
yl)butanoyl)pyrrolidine carboxamide 127 ##STR00144##
2-(2,6-dioxopiperidin-3-yl)-4- ((2-oxo-2-(4-(6-((6'-oxo-7',8'-
dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)ethyl)amino)isoindoline-1,3-
dione 128 ##STR00145## 2-(2,6-dioxopiperidin-3-yl)-4-
((3-oxo-3-(4-(6-((6'-oxo-7',8'- dihydro-6'H-
spiro[cyclohexane-1,9'- pyrazino[1',2':1,5]pyrrolo[2,3-
d]pyrimidin]-2'- yl)amino)pyridin-3- yl)piperazin-1-
yl)propyl)amino)isoindoline- 1,3-dione 129 ##STR00146##
2-(2,6-dioxopiperidin-3-yl)-4- ((4-oxo-4-(4-(6-((6'-oxo-7',8'-
dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)butyl)amino)isoindoline-1,3-
dione 130 ##STR00147## (2S,4R)-1-((S)-3,3-dimethyl-2-
(7-oxo-7-(4-(6-((6'-oxo-7',8'- dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)heptanamido)butanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 131 ##STR00148## (2S,4R)-1-((S)-3,3-dimethyl-2-
(6-oxo-6-(4-(6-((6'-oxo-7',8'- dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)hexanamido)butanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 132 ##STR00149## (2S,4R)-1-((S)-3,3-dimethyl-2-
(8-oxo-8-(4-(6-((6'-oxo-7',8'- dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)octanamido)butanoyl)-4-
hydroxy-N-(4-(4- methylthiazol-5- yl)benzyl)pyrrolidine-2-
carboxamide 133 ##STR00150## (2S,4R)-1-((S)-3,3-dimethyl-2-
(7-oxo-7-(4-(6-((6'-oxo-7',8'- dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)heptanamido)butanoyl)-4-
hydroxy-N-((S)-1-(4-(4- methylthiazol-5-
yl)phenyl)ethyl)pyrrolidine-2- carboxamide 134 ##STR00151##
(2S,4R)-1-((S)-3,3-dimethyl-2- (6-oxo-6-(4-(6-((6'-oxo-7',8'-
dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)hexanamido)butanoyl)-4-
hydroxy-N-((S)-1-(4-(4- methylthiazol-5-
yl)phenyl)ethyl)pyrrolidine-2- carboxamide 135 ##STR00152##
(2S,4R)-1-((S)-3,3-dimethyl-2- (8-oxo-8-(4-(6-((6'-oxo-7',8'-
dihydro-6'H- spiro[cyclohexane-1,9'-
pyrazino[1',2':1,5]pyrrolo[2,3- d]pyrimidin]-2'-
yl)amino)pyridin-3- yl)piperazin-1- yl)octanamido)butanoyl)-4-
hydroxy-N-((S)-1-(4-(4- methylthiazol-5-
yl)phenyl)ethyl)pyrrolidine-2- carboxamide
[0113] In an aspect, the CDK4/6 degraders/disruptors have the form
"PI-linker-EL" as shown below:
##STR00153##
wherein PI comprises a CDK4/6 ligand (e.g., a CDK4/6 inhibitor) and
EL comprises a degradation/disruption tag (e.g., E3 ligase ligand).
Exemplary CDK4/6 ligands (PI) and exemplary degradation/disruption
tags (EL) are disclosed herein.
[0114] For example, PI can include, but is not limited to:
##STR00154##
[0115] wherein X.sup.1, X.sup.2, and X.sup.3 are independently
hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkoxyalkyl,
NR.sup.5R.sup.6, CN, NO.sub.2, COR.sup.5, CO.sub.2R.sup.5,
CONR.sup.5R.sup.6, or NR.sup.5COR.sup.6;
[0116] R.sup.1 and R.sup.4 are independently hydrogen, halogen,
C1-C8 alkyl, C1-C8 alkoxy, C1-C8 alkoxyalkyl, C1-C8 haloalkyl,
C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8
alkenyl, C2-C8 alkynyl, OR.sup.5, SR.sup.5, NR.sup.5R.sup.6, CN,
NO.sub.2, (CR.sup.5R.sup.6)mNR.sup.7R.sup.8,
(CR.sup.5R.sup.6)mC(O)R.sup.7, COR.sup.5, CO.sub.2R.sup.5,
CONR.sup.5R.sup.6, NR.sup.5COR.sup.6, NR.sup.5SOR.sup.6,
NR.sup.5SO.sub.2R.sup.6, SOR.sup.5, SO.sub.2R.sup.5,
SO.sub.2NR.sup.5R.sup.6, (CR.sup.5R.sup.6)m-aryl, or
(CR.sup.5R.sup.6)m-heteroaryl, wherein m is 0-8;
[0117] R.sup.2 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C3-C7 cycloalkyl, or C3-C7 heterocyclyl;
[0118] R.sup.3 is hydrogen, aryl, C1-C8 alkyl, C1-C8 alkoxy, C3-C7
cycloalkyl, or C3-C7 heterocyclyl;
[0119] R.sup.5, R.sup.6, R.sup.7, R.sup.8 are independently
hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or
heteroarylalkyl;
[0120] optionally, R.sup.1 and R.sup.2; R.sup.5 and R.sup.6; or
R.sup.7 and R.sup.8 independently form 4-8 membered alkyl or
heterocyclyl rings; and
[0121] X and Y are independently CR.sup.5R.sup.6 or N.
[0122] For example, PI can include:
##STR00155##
[0123] wherein R.sup.1 is independently hydrogen, C1-C8 alkyl,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, or C2-C8
alkynyl;
[0124] R.sup.2 is hydrogen, C1-C3 alkyl, or cyclopropyl;
[0125] R.sup.3, R.sup.4, and R.sup.5 are independently hydrogen,
halogen, C1-C8 alkyl, C1-C8 alkoxy, C3-C7 cycloalkyl, or C3-C7
heterocyclyl;
[0126] R.sup.6 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.7, SR.sup.7, NR.sup.7R.sup.8, CN, NO.sub.2,
(CR.sup.7R.sup.8)mNR.sup.9R.sup.10, (CR.sup.7R.sup.8)mC(O)R.sup.9,
COR.sup.7, CO.sub.2R.sup.7, CONR.sup.7R.sup.8, NR.sup.7COR.sup.8,
NR.sup.7SOR.sup.8, NR.sup.7SO.sub.2R.sup.8, SORT, SO.sub.2R.sup.7,
SO.sub.2NR.sup.7R.sup.8, (CR.sup.7R.sup.8)m-aryl, or
(CR.sup.7R.sup.8)m-heteroaryl, wherein m is 0-8;
[0127] R.sup.7, R.sup.8, R.sup.9, R.sup.10 are independently
hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or
heteroarylalkyl;
[0128] optionally, R.sup.7 and R.sup.8; R.sup.9 and R.sup.10
independently form 4-8 membered alkyl or heterocyclyl rings;
and
[0129] X and Y are independently CR.sup.7R.sup.8 or N.
[0130] For example, PI can include:
##STR00156##
[0131] wherein R.sup.1 is independently hydrogen, C1-C8 alkyl,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, or C2-C8
alkynyl;
[0132] R.sup.2 is hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8
haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl, CN, COR.sup.4,
CO.sub.2R.sup.4, or CONR.sup.4R.sup.5;
[0133] R.sup.3 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.4, SR.sup.4, NR.sup.4R.sup.5, CN, NO.sub.2,
(CR.sup.4R.sup.5)mNR.sup.6R.sup.7, (CR.sup.4R.sup.5)mC(O)R.sup.6,
COR.sup.4, CO.sub.2R.sup.4, CONR.sup.4R.sup.5, NR.sup.4COR.sup.5,
NR.sup.4SOR.sup.5, NR.sup.4SO.sub.2R.sup.5, SOR.sup.4,
SO.sub.2R.sup.4, SO.sub.2NR.sup.4R.sup.5, (CR.sup.4R.sup.5)m-aryl,
or (CR.sup.4R.sup.5)m-heteroaryl, wherein m is 0-8;
[0134] R.sup.4, R.sup.5, R.sup.6, R.sup.7 are independently
hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, arylalkyl,
cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or
heteroarylalkyl;
[0135] optionally, R.sup.1 and R.sup.2; R.sup.4 and R.sup.5;
R.sup.6 and R.sup.7 independently form 4-8 membered alkyl or
heterocyclyl rings; and
[0136] V, W, X, Y, and Z are independently CR.sup.4R.sup.5 or
N.
[0137] For example, PI can include:
##STR00157##
[0138] wherein R.sup.1 and R.sup.2 are independently hydrogen,
C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8
hydroxyalkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl,
or C2-C8 alkynyl;
[0139] R.sup.3 is hydrogen, C1-C6 alkyl, C1-C6 alkoxyalkyl, C1-C6
haloalkyl, C1-C6 hydroxyalkyl, C3-C6 cycloalkyl, C3-C6
heterocyclyl, C2-C6 alkenyl, or C2-C6 alkynyl;
[0140] R.sup.4 is hydrogen, halogen, C1-C8 alkyl, C1-C8 alkoxy,
C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8 hydroxyalkyl, C3-C7
cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl, C2-C8 alkynyl,
OR.sup.5, SR.sup.5, NR.sup.5R.sup.6, CN, NO.sub.2,
(CR.sup.5R.sup.6)mNR.sup.7R.sup.8, (CR.sup.5R.sup.6)mC(O)R.sup.7,
COR.sup.5, CO.sub.2R.sup.5, CONR.sup.5R.sup.6, NR.sup.5COR.sup.6,
NR.sup.5SOR.sup.6, NR.sup.5SO.sub.2R.sup.6, SOR.sup.5,
SO.sub.2R.sup.5, SO.sub.2NR.sup.5R.sup.6, (CR.sup.5R.sup.6)m-aryl,
or (CR.sup.5R.sup.6)m-heteroaryl, wherein m is 0-8;
[0141] n is 0-4;
[0142] optionally, R.sup.1 and R.sup.2; R.sup.5 and R.sup.6;
R.sup.7 and R.sup.8 independently form 4-8 membered alkyl or
heterocyclyl rings; and
[0143] V, W, X, Y, and Z are independently CR.sup.5R.sup.6 or
N.
[0144] The CDK4/6 ligand can be a CDK4/6 inhibitor, such as, for
example, abemaciclib, palbociclib, ribociclib, trilaciclib (G1T28),
G1T38, SHR6390, and/or analogs thereof.
[0145] In some aspects, the CDK4/6 ligand can be, e.g.,
##STR00158##
The CDK4/6 ligand can be bound to CDK4/6.
[0146] EL includes, but is not limited to:
##STR00159##
[0147] wherein V, W, X are independently CR.sup.2 or N;
[0148] Y is CO or CH.sub.2;
[0149] Z is CH.sub.2, NH, or O;
[0150] R.sup.1 is hydrogen, methyl, or fluoro; and
[0151] R.sup.2 is hydrogen, halogen, or C1-C5 alkyl.
[0152] For example, EL can include:
##STR00160##
[0153] wherein R.sup.1 and R.sup.2 are independently hydrogen,
C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8 haloalkyl, C1-C8
hydroxyalkyl, C3-C7 cycloalkyl, C3-C7 heterocyclyl, C2-C8 alkenyl,
or C2-C8 alkynyl.
[0154] For example, EL can include:
##STR00161##
[0155] wherein R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are
independently hydrogen, C1-C8 alkyl, C1-C8 alkoxyalkyl, C1-C8
haloalkyl, C1-C8 hydroxyalkyl, C3-C7 cycloalkyl, C3-C7
heterocyclyl, C2-C8 alkenyl, or C2-C8 alkynyl; and
[0156] V, W, X, Z are independently CR.sup.4 or N.
[0157] In some aspects, the degradation/disruption tag can be, for
example, pomalidomide, thalidomide, lenalidomide, VHL-1,
adamantane, 1-((4,4,5,5,5-pentafluoropentyl)sulfinyl)nonane,
nutlin-3a, RG7112, RG7338, AMG 232, AA-115, bestatin, MV-1, LCL161,
and/or analogs thereof.
[0158] In some aspects, the degradation/disruption tag can be,
e.g.,
##STR00162## ##STR00163## ##STR00164##
Formulas XIV-XXX (left to right, then top to bottom, starting with
Formula XIV at the top left corner and ending with Formula XXX at
the bottom right corner).
[0159] In some aspects, the degradation/disruption tag can bind to
a ubiquitin ligase, such as, for example, an E3 ligase. Exemplary
E3 ligases include, for example, a cereblon E3 ligase, a VHL E3
ligase, a MDM2 ligase, a TRIM21 ligase, a TRIM24 ligase, and/or a
IAP ligase. In some aspects, the degradation/disruption tag can
serve as a hydrophobic group that leads to CDK4 or CDK6 protein
misfolding.
[0160] In any of the above-described compounds, the CDK4/6 ligand
can be conjugated to the degradation/disruption tag through a
linker. The linker can include, for example, acyclic or cyclic
saturated or unsaturated carbon, ethylene glycol, amide, amino,
ether, urea, carbamate, aromatic, heteroaromatic, heterocyclic
and/or carbonyl containing groups with different lengths.
[0161] In some embodiments, the linker can be a moiety of:
##STR00165##
wherein X is C.dbd.O or CH.sub.2,
Y is C.dbd.O or CH.sub.2, and
[0162] n is 0-15;
##STR00166##
wherein X is C.dbd.O or CH.sub.2,
Y is C.dbd.O or CH.sub.2,
[0163] m is 0-15, n is 0-6, and o is 0-15; or
##STR00167##
wherein
X is C.dbd.O or CH.sub.2,
Y is C.dbd.O or CH.sub.2,
[0164] R is --CH.sub.2--, --CF.sub.2--, --CH(C.sub.1-3 alkyl)-,
--C(C.sub.1-3 alkyl)(C.sub.1-3 alkyl)-, --CH.dbd.CH--,
--C(C.sub.1-3 alkyl).dbd.C(C.sub.1-3 alkyl)-, --C.dbd.C--, --O--,
--NH--, --N(C.sub.1-3 alkyl)-, --C(O)NH--, --C(O)N(C.sub.1-3
alkyl)-, a 3-13 membered ring, a 3-13 membered fused ring, a 3-13
membered bridged ring, and/or a 3-13 membered spiro ring, m is
0-15, and n is 0-15.
[0165] In some embodiments of Formula C, R is a 3-13 membered ring,
a 3-13 membered fused ring, a 3-13 membered bridged ring, and/or a
3-13 membered spiro ring, one or more of which can contain one or
more heteroatoms.
[0166] In some embodiments of Formula C, R has a structure of
##STR00168##
Synthesis and Testing of Bivalent Compounds
[0167] The binding affinity of novel synthesized bivalent compounds
(i.e., CDK4/6 degraders/disruptors) can be assessed using standard
biophysical assays known in the art (e.g., ITC). Cellular assays
can then be used to assess the bivalent compound's ability to
induce CDK4/6 degradation and inhibit cancer cell proliferation.
Besides evaluating bivalent compound's induced changes in the
protein expression of CDK4/6, CDK4/6 enzymatic activity can also be
assessed. Multiple cycles of designs, syntheses, and biological
tests can be performed. Promising bivalent compounds can then be
further optimized using methods known in the art to improve their
pharmacokinetic/pharmacodynamic properties (e.g., absorption,
distribution, metabolism, and excretion (ADME) properties). Assays
suitable for use in any or all of these steps are known in the art,
and include, for example, Western blotting, quantitative mass
spectrometry (MS) analysis, flow cytometry, enzymatic inhibition,
isothermal titration calorimetry (ITC), surface plasmon resonance
(SPR), cell growth inhibition and xenograft and PDX models.
Suitable cell lines for use in any or all of these steps are known
in the art and include, for example, breast cancer cell lines
(e.g., ER+ breast cancer cell lines such as MCF7, T47D, and
ZR-75-1) and melanoma cell lines (e.g., A375, SK-MEL-2, SK-MEL-30,
and WM1382).
[0168] By way of non-limiting example, detailed synthesis protocols
are described in the Examples for specific exemplary CDK4/6
degraders/disruptors.
[0169] Pharmaceutically acceptable isotopic variations of the
compounds disclosed herein are contemplated and can be synthesized
using conventional methods known in the art or methods
corresponding to those described in the Examples (substituting
appropriate reagents with appropriate isotopic variations of those
reagents). Specifically, an isotopic variation is a compound in
which at least one atom is replaced by an atom having the same
atomic number, but an atomic mass different from the atomic mass
usually found in nature. Useful isotopes are known in the art and
include, for example, isotopes of hydrogen, carbon, nitrogen,
oxygen, phosphorus, sulfur, fluorine, and chlorine. Exemplary
isotopes thus include, for example, .sup.2H, .sup.3H, .sup.13C,
.sup.14C, .sup.15N, .sup.17O, .sup.18O, .sup.32P, .sup.35S, 18F and
.sup.36Cl.
[0170] Isotopic variations (e.g., isotopic variations containing
.sup.2H) can provide therapeutic advantages resulting from greater
metabolic stability, e.g., increased in vivo half-life or reduced
dosage requirements. In addition, certain isotopic variations
(particularly those containing a radioactive isotope) can be used
in drug or substrate tissue distribution studies. The radioactive
isotopes tritium (.sup.3H) and carbon-14 (.sup.14C) are
particularly useful for this purpose in view of their ease of
incorporation and ready means of detection.
[0171] Pharmaceutically acceptable solvates of the compounds
disclosed herein are contemplated. A solvate can be generated, by
substituting a solvent used to crystallize a compound disclosed
herein with an isotopic variation (e.g., D.sub.2O in place of
H.sub.2O, d.sub.6-acetone in place of acetone, or d.sub.6-DMSO in
place of DMSO).
[0172] Pharmaceutically acceptable fluorinated variations of the
compounds disclosed herein are contemplated and can be synthesized
using conventional methods known in the art or methods
corresponding to those described in the Examples (substituting
appropriate reagents with appropriate fluorinated variations of
those reagents). Specifically, a fluorinated variation is a
compound in which at least one hydrogen atom is replaced by a
fluoro atom. Fluorinated variations can provide therapeutic
advantages resulting from greater metabolic stability, e.g.,
increased in vivo half-life or reduced dosage requirements.
[0173] Characterization of Exemplary CDK4/6 Degraders/Disruptors
Specific exemplary abemaciclib-, ribociclib-, and palbociclib-based
CDK4/6 degraders/disruptors were characterized in various different
breast cancer and melanoma cells (Examples 12 and 13, FIGS. 10-23).
XY019-098, XY019-106, XY028-003, XY019-108, XY028-132, XY028-133,
YX26-66, XY028-140, XY028-144, YX039-48, YX039-124, YX039-123,
YX039-147, YX039-56, and YX039-65 in particular were found to be
especially effective in suppressing both CDK4/6 expression and
CDK4/6 activity. This efficacy in suppressing CDK4/6 expression and
CDK4/6 activity correlated with efficacy in inhibiting cancer cell
proliferation.
Pharmaceutical Compositions
[0174] In some aspects, the compositions and methods described
herein include the manufacture and use of pharmaceutical
compositions and medicaments that include one or more bivalent
compounds as disclosed herein. Also included are the pharmaceutical
compositions themselves.
[0175] In some aspects, the compositions disclosed herein can
include other compounds, drugs, or agents used for the treatment of
cancer. For example, in some instances, pharmaceutical compositions
disclosed herein can be combined with one or more (e.g., one, two,
three, four, five, or less than ten) compounds. Such additional
compounds can include, for example, conventional chemotherapeutic
agents known in the art (e.g., gemcitabine HCl and temozolomide).
When co-administered, CDK4/6 degraders/disruptors disclosed herein
can operate in conjunction with conventional chemotherapeutic
agents to produce mechanistically additive or synergistic
therapeutic effects.
[0176] In some aspects, the pH of the compositions disclosed herein
can be adjusted with pharmaceutically acceptable acids, bases, or
buffers to enhance the stability of the CDK4/6 degraders/disruptor
or its delivery form.
[0177] Pharmaceutical compositions typically include a
pharmaceutically acceptable carrier, adjuvant, or vehicle. As used
herein, the phrase "pharmaceutically acceptable" refers to
molecular entities and compositions that are generally believed to
be physiologically tolerable and do not typically produce an
allergic or similar untoward reaction, such as gastric upset,
dizziness and the like, when administered to a human. A
pharmaceutically acceptable carrier, adjuvant, or vehicle is a
composition that can be administered to a patient, together with a
compound of the invention, and which does not destroy the
pharmacological activity thereof and is nontoxic when administered
in doses sufficient to deliver a therapeutic amount of the
compound. Exemplary conventional nontoxic pharmaceutically
acceptable carriers, adjuvants, and vehicles include saline,
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like,
compatible with pharmaceutical administration.
[0178] In particular, pharmaceutically acceptable carriers,
adjuvants, and vehicles that can be used in the pharmaceutical
compositions of this invention include, but are not limited to, ion
exchangers, alumina, aluminum stearate, lecithin, self-emulsifying
drug delivery systems (SEDDS) such as d-.alpha.-tocopherol
polyethylene glycol 1000 succinate, surfactants used in
pharmaceutical dosage forms such as Tweens or other similar
polymeric delivery matrices, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
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. Cyclodextrins such as .alpha.-, .beta.-, and
.gamma.-cyclodextrin, may also be advantageously used to enhance
delivery of compounds of the formulae described herein.
[0179] As used herein, the CDK4/6 degraders/disruptors disclosed
herein are defined to include pharmaceutically acceptable
derivatives or prodrugs thereof. A "pharmaceutically acceptable
derivative" means any pharmaceutically acceptable salt, solvate, or
prodrug, e.g., carbamate, ester, phosphate ester, salt of an ester,
or other derivative of a compound or agent disclosed herein, which
upon administration to a recipient is capable of providing
(directly or indirectly) a compound described herein, or an active
metabolite or residue thereof. Particularly favored derivatives and
prodrugs are those that increase the bioavailability of the
compounds disclosed herein when such compounds are administered to
a mammal (e.g., by allowing an orally administered compound to be
more readily absorbed into the blood) or which enhance delivery of
the parent compound to a biological compartment (e.g., the brain or
lymphatic system) relative to the parent species. Preferred
prodrugs include derivatives where a group that enhances aqueous
solubility or active transport through the gut membrane is appended
to the structure of formulae described herein. Such derivatives are
recognizable to those skilled in the art without undue
experimentation. Nevertheless, reference is made to the teaching of
Burger's Medicinal Chemistry and Drug Discovery, 5.sup.th Edition,
Vol. 1: Principles and Practice, which is incorporated herein by
reference to the extent of teaching such derivatives.
[0180] The CDK4/6 degraders/disruptors disclosed herein include
pure enantiomers, mixtures of enantiomers, pure diastereoisomers,
mixtures of diastereoisomers, diastereoisomeric racemates, mixtures
of diastereoisomeric racemates and the meso-form and
pharmaceutically acceptable salts, solvent complexes, morphological
forms, or deuterated derivative thereof
[0181] In particular, pharmaceutically acceptable salts of the
CDK4/6 degraders/disruptors disclosed herein include, for example,
those derived from pharmaceutically acceptable inorganic and
organic acids and bases. Examples of suitable acid salts include
acetate, adipate, benzoate, benzenesulfonate, butyrate, citrate,
digluconate, dodecylsulfate, formate, fumarate, glycolate,
hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,
hydroiodide, lactate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate,
picrate, pivalate, propionate, salicylate, succinate, sulfate,
tartrate, tosylate, trifluoromethylsulfonate, and undecanoate.
Salts derived from appropriate bases include, for example, alkali
metal (e.g., sodium), alkaline earth metal (e.g., magnesium),
ammonium and N-(alkyl)4+ salts. The invention also envisions the
quaternization of any basic nitrogen-containing groups of the
CDK4/6 degraders/disruptors disclosed herein. Water or oil-soluble
or dispersible products can be obtained by such quaternization.
[0182] In some aspects, the pharmaceutical compositions disclosed
herein can include an effective amount of one or more CDK4/6
degraders/disruptors. The terms "effective amount" and "effective
to treat," as used herein, refer to an amount or a concentration of
one or more compounds or a pharmaceutical composition described
herein utilized for a period of time (including acute or chronic
administration and periodic or continuous administration) that is
effective within the context of its administration for causing an
intended effect or physiological outcome (e.g., treatment or
prevention of cell growth, cell proliferation, or cancer). In some
aspects, pharmaceutical compositions can further include one or
more additional compounds, drugs, or agents used for the treatment
of cancer (e.g., conventional chemotherapeutic agents) in amounts
effective for causing an intended effect or physiological outcome
(e.g., treatment or prevention of cell growth, cell proliferation,
or cancer).
[0183] In some aspects, the pharmaceutical compositions disclosed
herein can be formulated for sale in the United States, import into
the United States, or export from the United States.
Administration of Pharmaceutical Compositions
[0184] The pharmaceutical compositions disclosed herein can be
formulated or adapted for administration to a subject via any
route, for example, any route approved by the Food and Drug
Administration (FDA). Exemplary methods are described in the FDA
Data Standards Manual (DSM) (available at
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/Forms
SubmissionRequirements/ElectronicSubmissions/DataStandardsManualmonograph-
s). In particular, the pharmaceutical compositions can be
formulated for and administered via oral, parenteral, or
transdermal delivery. The term "parenteral" as used herein includes
subcutaneous, intracutaneous, intravenous, intramuscular,
intraperitoneal, intra-articular, intra-arterial, intrasynovial,
intrasternal, intrathecal, intralesional, and intracranial
injection or infusion techniques.
[0185] For example, the pharmaceutical compositions disclosed
herein can be administered, for example, topically, rectally,
nasally (e.g., by inhalation spray or nebulizer), buccally,
vaginally, subdermally (e.g., by injection or via an implanted
reservoir), or ophthalmically.
[0186] For example, pharmaceutical compositions of this invention
can be orally administered in any orally acceptable dosage form
including, but not limited to, capsules, tablets, emulsions and
aqueous suspensions, dispersions and solutions. In the case of
tablets for oral use, carriers which are commonly used include
lactose and corn starch. Lubricating agents, such as magnesium
stearate, are also typically added. For oral administration in a
capsule form, useful diluents include lactose and dried corn
starch. When aqueous suspensions or emulsions are administered
orally, the active ingredient may be suspended or dissolved in an
oily phase is combined with emulsifying or suspending agents. If
desired, certain sweetening, flavoring, or coloring agents can be
added.
[0187] For example, the pharmaceutical compositions of this
invention can be administered in the form of suppositories for
rectal administration. These compositions can be prepared by mixing
a compound of this invention with a suitable non-irritating
excipient which is solid at room temperature but liquid at the
rectal temperature and therefore will melt in the rectum to release
the active components. Such materials include, but are not limited
to, cocoa butter, beeswax, and polyethylene glycols.
[0188] For example, the pharmaceutical compositions of this
invention can be administered by nasal aerosol or inhalation. Such
compositions are prepared according to techniques well-known in the
art of pharmaceutical formulation and can be prepared as solutions
in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, or other solubilizing or dispersing agents known in
the art.
[0189] For example, the pharmaceutical compositions of this
invention can be administered by injection (e.g., as a solution or
powder). Such compositions can be formulated according to
techniques known in the art using suitable dispersing or wetting
agents (such as, for example, Tween 80) 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, e.g., 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 can 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, e.g., olive oil or castor
oil, especially in their polyoxyethylated versions. These oil
solutions or suspensions can also contain a long-chain alcohol
diluent or dispersant, or carboxymethyl cellulose or similar
dispersing agents which are commonly used in the formulation of
pharmaceutically acceptable dosage forms such as emulsions and or
suspensions. Other commonly used surfactants such as Tweens, Spans,
or other similar emulsifying agents or bioavailability enhancers
which are commonly used in the manufacture of pharmaceutically
acceptable solid, liquid, or other dosage forms can also be used
for the purposes of formulation.
[0190] In some aspects, an effective dose of a pharmaceutical
composition of this invention can include, but is not limited to
about 0.00001, 0.0001, 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,
0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5,
0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.25, 1.5,
1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70,
80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2500,
5000, or 10000 mg/kg/day, or according to the requirements of the
particular pharmaceutical composition.
[0191] When the pharmaceutical compositions disclosed herein
include a combination of a compound of the formulae described
herein (e.g., a CDK4/6 degraders/disruptors) and one or more
additional compounds (e.g., one or more additional compounds,
drugs, or agents used for the treatment of cancer or any other
condition or disease, including conditions or diseases known to be
associated with or caused by cancer), both the compound and the
additional compound should be present at dosage levels of between
about 1 to 100%, and more preferably between about 5 to 95% of the
dosage normally administered in a monotherapy regimen. The
additional agents can be administered separately, as part of a
multiple dose regimen, from the compounds of this invention.
Alternatively, those agents can be part of a single dosage form,
mixed together with the compounds of this invention in a single
composition.
[0192] In some aspects, the pharmaceutical compositions disclosed
herein can be included in a container, pack, or dispenser together
with instructions for administration.
Methods of Treatment
[0193] The methods disclosed herein contemplate administration of
an effective amount of a compound or composition to achieve the
desired or stated effect. Typically, the compounds or compositions
of the invention will be administered from about 1 to about 6 times
per day or, alternately or in addition, as a continuous infusion.
Such administration can be used as a chronic or acute therapy. The
amount of active ingredient that can be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. A
typical preparation will contain from about 5% to about 95% active
compound (w/w). Alternatively, such preparations can contain from
about 20% to about 80% active compound.
[0194] In some aspects, the present disclosure provides methods for
using a composition comprising a CDK4/6 degrader/disruptor,
including pharmaceutical compositions (indicated below as `X`)
disclosed herein in the following methods:
[0195] Substance X for use as a medicament in the treatment of one
or more diseases or conditions disclosed herein (e.g., cancer,
referred to in the following examples as `Y`). Use of substance X
for the manufacture of a medicament for the treatment of Y; and
substance X for use in the treatment of Y.
[0196] In some aspects, the methods disclosed include the
administration of a therapeutically effective amount of one or more
of the compounds or compositions described herein to a subject
(e.g., a mammalian subject, e.g., a human subject) who is in need
of, or who has been determined to be in need of, such treatment. In
some aspects, the methods disclosed include selecting a subject and
administering to the subject an effective amount of one or more of
the compounds or compositions described herein, and optionally
repeating administration as required for the prevention or
treatment of cancer.
[0197] In some aspects, subject selection can include obtaining a
sample from a subject (e.g., a candidate subject) and testing the
sample for an indication that the subject is suitable for
selection. In some aspects, the subject can be confirmed or
identified, e.g. by a health care professional, as having had or
having a condition or disease. In some aspects, suitable subjects
include, for example, subjects who have or had a condition or
disease but that resolved the disease or an aspect thereof, present
reduced symptoms of disease (e.g., relative to other subjects
(e.g., the majority of subjects) with the same condition or
disease), or that survive for extended periods of time with the
condition or disease (e.g., relative to other subjects (e.g., the
majority of subjects) with the same condition or disease), e.g., in
an asymptomatic state (e.g., relative to other subjects (e.g., the
majority of subjects) with the same condition or disease). In some
aspects, exhibition of a positive immune response towards a
condition or disease can be made from patient records, family
history, or detecting an indication of a positive immune response.
In some aspects, multiple parties can be included in subject
selection. For example, a first party can obtain a sample from a
candidate subject and a second party can test the sample. In some
aspects, subjects can be selected or referred by a medical
practitioner (e.g., a general practitioner). In some aspects,
subject selection can include obtaining a sample from a selected
subject and storing the sample or using the in the methods
disclosed herein. Samples can include, e.g., cells or populations
of cells.
[0198] In some aspects, methods of treatment can include a single
administration, multiple administrations, and repeating
administration of one or more compounds disclosed herein as
required for the prevention or treatment of the disease or
condition from which the subject is suffering (e.g., a
CDK4/6-mediated cancer, e.g., ER+ breast cancer). In some aspects,
methods of treatment can include assessing a level of disease in
the subject prior to treatment, during treatment, or after
treatment. In some aspects, treatment can continue until a decrease
in the level of disease in the subject is detected.
[0199] The term "subject," as used herein, refers to any animal. In
some instances, the subject is a mammal. In some instances, the
term "subject," as used herein, refers to a human (e.g., a man, a
woman, or a child).
[0200] The terms "administer," "administering," or
"administration," as used herein, refer to implanting, ingesting,
injecting, inhaling, or otherwise absorbing a compound or
composition, regardless of form. For example, the methods disclosed
herein include administration of an effective amount of a compound
or composition to achieve the desired or stated effect.
[0201] The terms "treat", "treating," or "treatment," as used
herein, refer to partially or completely alleviating, inhibiting,
ameliorating, or relieving the disease or condition from which the
subject is suffering. This means any manner in which one or more of
the symptoms of a disease or disorder (e.g., cancer) are
ameliorated or otherwise beneficially altered. As used herein,
amelioration of the symptoms of a particular disorder (e.g.,
cancer) refers to any lessening, whether permanent or temporary,
lasting or transient that can be attributed to or associated with
treatment by the compositions and methods of the present invention.
In some embodiments, treatment can promote or result in, for
example, a decrease in the number of tumor cells (e.g., in a
subject) relative to the number of tumor cells prior to treatment;
a decrease in the viability (e.g., the average/mean viability) of
tumor cells (e.g., in a subject) relative to the viability of tumor
cells prior to treatment; a decrease in the rate of growth of tumor
cells; a decrease in the rate of local or distant tumor metastasis;
or reductions in one or more symptoms associated with one or more
tumors in a subject relative to the subject's symptoms prior to
treatment.
[0202] As used herein, the term "treating cancer" means causing a
partial or complete decrease in the rate of growth of a tumor,
and/or in the size of the tumor and/or in the rate of local or
distant tumor metastasis, and/or the overall tumor burden in a
subject, and/or any decrease in tumor survival, in the presence of
a degrader/disruptor (e.g., an CDK4/6 degrader/disruptor) described
herein.
[0203] The terms "prevent," "preventing," and "prevention," as used
herein, shall refer to a decrease in the occurrence of a disease or
decrease in the risk of acquiring a disease or its associated
symptoms in a subject. The prevention may be complete, e.g., the
total absence of disease or pathological cells in a subject. The
prevention may also be partial, such that the occurrence of the
disease or pathological cells in a subject is less than, occurs
later than, or develops more slowly than that which would have
occurred without the present invention.
[0204] Exemplary CDK4/6-mediated cancers that can be treated with
CDK4/6 degraders/disruptors include, for example, solid tumors
(e.g., breast cancer (e.g., ER+ breast cancer) and prostate
cancer), leukemia (e.g., acute lymphoblastic leukemia, acute
myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous
leukemia, hairy cell leukemia, and myeloid leukemia), lymphoma
(e.g., Burkitt's lymphoma, cutaneous T-cell lymphoma, diffuse large
B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin's
lymphoma, mantel cell lymphoma, and non-Hodgkin's lymphoma (NHL)),
adrenocortical cancer, AIDS-related cancer, anal cancer,
astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), bile
duct cancer, bladder cancer, bone cancer (e.g.,
fibrosarcoma/osteosarcoma/malignant fibrous histiocytoma), brain
tumor (e.g., cerebral astrocytoma, ependymoma, glioma,
medulloblastoma, and supratentorial primitive neuroectodermal
tumors (PNETs)), brainstem glioma, bronchial adenomas/carcinoids,
carcinoid tumors, central nervous system neoplasms, cervical
cancer, cholangiocarcinoma, chronic myeloproliferative disorder,
colon cancer, endometrial cancer, esophageal cancer, melanoma
(e.g., cutaneous or intraocular), gallbladder cancer,
gastrointestinal cancer (e.g., colorectal, duodenal, and gastric
(stomach) cancer), germ cell tumors, head and neck cancer,
hepatocellular (liver) cancer, hypopharyngeal cancer, islet cell
carcinoma, Kaposi's sarcoma, kidney (renal cell) cancer, laryngeal
cancer, lip and oral cavity cancer, lung cancer (small cell and
non-small cell), Merkel cell carcinoma, mesothelioma, endocrine
cancer (e.g., multiple endocrine neoplasia syndrome), multiple
myeloma/plasma cell neoplasm mycosis fungoides, myelodysplastic
syndrome, myeloproliferative disorders, nasal cavity and paranasal
sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal
cancer, ovarian cancer, pancreatic cancer, parathyroid cancer,
penile cancer, pituitary cancer, pleuropulmonary blastoma, rectal
cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,
Ewing sarcoma, soft tissue sarcoma, Sezary syndrome, squamous cell
carcinoma, squamous neck cancer, synovial sarcoma, testicular
cancer, thymoma, thymic carcinoma, thyroid cancer, transitional
cell cancer, trophoblastic tumors, urethral cancer, uterine cancer,
fallopian tube cancer, vaginal cancer, visual pathway and
hypothalamic glioma, vulvar cancer, Waldenstrom's
macroglobulinemia, and Wilms' tumor.
[0205] As used herein, the term "preventing a disease" (e.g.,
preventing cancer) in a subject means for example, to stop the
development of one or more symptoms of a disease in a subject
before they occur or are detectable, e.g., by the patient or the
patient's doctor. Preferably, the disease (e.g., cancer) does not
develop at all, i.e., no symptoms of the disease are detectable.
However, it can also result in delaying or slowing of the
development of one or more symptoms of the disease. Alternatively,
or in addition, it can result in the decreasing of the severity of
one or more subsequently developed symptoms.
[0206] Specific dosage and treatment regimens for any particular
patient will depend upon a variety of factors, including the
activity of the specific compound employed, the age, body weight,
general health status, sex, diet, time of administration, rate of
excretion, drug combination, the severity and course of the
disease, condition or symptoms, the patient's disposition to the
disease, condition or symptoms, and the judgment of the treating
physician.
[0207] An effective amount can be administered in one or more
administrations, applications or dosages. A therapeutically
effective amount of a therapeutic compound (i.e., an effective
dosage) depends on the therapeutic compounds selected. Moreover,
treatment of a subject with a therapeutically effective amount of
the compounds or compositions described herein can include a single
treatment or a series of treatments. For example, effective amounts
can be administered at least once. The compositions can be
administered one from one or more times per day to one or more
times per week; including once every other day. The skilled artisan
will appreciate that certain factors can influence the dosage and
timing required to effectively treat a subject, including but not
limited to the severity of the disease or disorder, previous
treatments, the general health or age of the subject, and other
diseases present.
[0208] Following administration, the subject can be evaluated to
detect, assess, or determine their level of disease. In some
instances, treatment can continue until a change (e.g., reduction)
in the level of disease in the subject is detected. Upon
improvement of a patient's condition (e.g., a change (e.g.,
decrease) in the level of disease in the subject), a maintenance
dose of a compound, or composition disclosed herein can be
administered, if necessary. Subsequently, the dosage or frequency
of administration, or both, can be reduced, e.g., as a function of
the symptoms, to a level at which the improved condition is
retained. Patients may, however, require intermittent treatment on
a long-term basis upon any recurrence of disease symptoms.
EXAMPLES
Example 1
Procedures for the Synthesis of VHL-1 Alkyl Linkers
##STR00169##
[0210] To a solution of diacid (10 mmol) in DCM/THF (1:1, 200 ml)
was added VHL-1 (2 mmol), triethylamine (1 ml, 7.1 mmol), HOAt (300
mg, 2.2 mmol), and EDCI (420 mg, 2.2 mmol) sequentially at
0.degree. C. The resulting solution was stirred for 2 h at
0.degree. C., before being warmed to room temperature (RT). After
stirring overnight at RT, the reaction was quenched with water.
After concentration under reduced pressure, the resulting residue
was purified by reverse-phase chromatography to yield the desired
product.
##STR00170##
[0211] Linker 1:
4-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl-
)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutanoic
acid (810 mg, 85%) as white solid. .sup.1H NMR (600 MHz CD.sub.3OD)
.delta. 9.10 (s, 1H), 7.51 (d, J=7.8 Hz, 2H), 7.44 (d, J=8.4 Hz,
2H), 4.64 (s, 1H), 4.60-4.49 (m, 3H), 4.39 (d, J=15.6 Hz, 1H), 3.91
(d, J=10.8 Hz, 1H), 3.82 (dd, J=9.6, 3.6 Hz, 1H), 2.67-2.55 (m,
4H), 2.52 (s, 3H), 2.25-2.22 (m, 1H), 2.12-2.07 (m, 1H), 1.06 (s,
9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.26H.sub.35N.sub.4O.sub.6S, 531.2272, found 531.2280.
##STR00171##
[0212] Linker 2:
5-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl-
)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentanoic
acid (230 mg, 43%) as white solid. .sup.1H NMR (600 MHz CD.sub.3OD)
.delta. 9.14 (s, 1H), 7.51 (d, J=9.0 Hz, 2H), 7.46 (d, J=8.4 Hz,
2H), 4.65 (s, 1H), 4.60-4.57 (m, 1H), 4.56 (d, J=15.6 Hz, 1H),
4.53-4.50 (m, 1H), 4.38 (d, J=15.6 Hz, 1H), 3.94 (d, J=11.4 Hz,
1H), 3.82 (dd, J=11.4, 3.6 Hz, 1H), 2.52 (s, 3H), 2.40-2.30 (m,
4H), 2.26-2.22 (m, 1H), 2.12-2.08 (m, 1H), 1.91 (t, J=7.8 Hz, 2H),
1.06 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.27H.sub.37N.sub.4O.sub.6S, 545.2428, found 545.2432.
##STR00172##
[0213] Linker 3:
6-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl-
)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic
acid (700 mg, 63%) as white solid. .sup.1H NMR (600 MHz CD.sub.3OD)
.delta. 9.12 (s, 1H), 7.51 (d, J=9.0 Hz, 2H), 7.46 (d, J=8.4 Hz,
2H), 4.65 (s, 1H), 4.60-4.55 (m, 2H), 4.53-4.50 (m, 1H), 4.38 (d,
J=16.8 Hz, 1H), 3.93 (d, J=10.8 Hz, 1H), 3.82 (dd, J=11.4, 3.6 Hz,
1H), 2.52 (s, 3H), 2.38-2.21 (m, 5H), 2.12-2.08 (m, 1H), 1.71-1.62
(m, 4H), 1.06 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated
for C.sub.28H.sub.39N.sub.4O.sub.6S, 559.2585, found 559.2605.
##STR00173##
[0214] Linker 4:
7-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl-
)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptanoic
acid (810 mg, 79%) as white solid. .sup.1H NMR (600 MHz CD.sub.3OD)
.delta. 8.98 (s, 1H), 7.50 (d, J=8.4 Hz, 2H), 7.44 (d, J=9.0 Hz,
2H), 4.65 (s, 1H), 4.60-4.49 (m, 3H), 4.38 (d, J=15.6 Hz, 1H), 3.93
(d, J=10.8 Hz, 1H), 3.82 (dd, J=11.4, 3.6 Hz, 1H), 2.51 (s, 3H),
2.35-2.22 (m, 5H), 2.13-2.08 (m, 1H), 1.68-1.59 (m, 4H), 1.42-1.34
(m, 2H), 1.06 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated
for C.sub.29H.sub.41N.sub.4O.sub.6S, 573.2741, found 573.2754.
##STR00174##
[0215] Linker 5:
8-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl-
)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctanoic
acid (980 mg, 78%) as white solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 8.94 (s, 1H), 7.47 (d, J=8.1 Hz, 2H), 7.42 (d,
J=8.2 Hz, 2H), 4.63 (s, 1H), 4.59-4.47 (m, 3H), 4.35 (d, J=15.4 Hz,
1H), 3.90 (d, J=11.0 Hz, 1H), 3.80 (dd, J=10.9, 3.9 Hz, 1H), 2.48
(s, 3H), 2.32-2.17 (m, 5H), 2.08 (ddd, J=13.3, 9.1, 4.5 Hz, 1H),
1.67-1.55 (m, 4H), 1.40-1.28 (m, 4H), 1.03 (s, 9H). HRMS (ESI-TOF)
m/z: [M+H].sup.+ calculated for C.sub.30H.sub.43N.sub.4O.sub.6S,
587.2898; found: 587.2903.
##STR00175##
[0216] Linker 6:
9-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl-
)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononanoic
acid (750 mg, 66%) as white solid. .sup.1H NMR (600 MHz CD.sub.3OD)
.delta. 9.09 (s, 1H), 7.51 (d, J=9.0 Hz, 2H), 7.46 (d, J=8.4 Hz,
2H), 4.66 (s, 1H), 4.61-4.50 (m, 3H), 4.38 (d, J=15.6 Hz, 1H), 3.93
(d, J=10.8 Hz, 1H), 3.82 (dd, J=11.4, 3.6 Hz, 1H), 2.52 (s, 3H),
2.36-2.22 (m, 5H), 2.12-2.07 (m, 1H), 1.68-1.59 (m, 4H), 1.40-1.34
(m, 8H), 1.06 (s, 9H); HPLC 98%; t.sub.R=4.24 min; HRMS(TOF)
calculated for C.sub.31H.sub.45N.sub.4O.sub.6S [M+H].sup.+,
601.3054, found 601.3064.
##STR00176##
[0217] Linker 7:
10-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoy-
l)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10-oxodecanoic
acid (900 mg, 73%) as white solid. .sup.1H NMR (600 MHz CD.sub.3OD)
.delta. 8.98 (s, 1H), 7.50 (d, J=8.4 Hz, 2H), 7.45 (d, J=9.0 Hz,
2H), 4.66 (s, 1H), 4.61-4.50 (m, 3H), 4.38 (d, J=14.4 Hz, 1H), 3.93
(d, J=10.8 Hz, 1H), 3.83 (dd, J=11.4, 3.6 Hz, 1H), 2.51 (s, 3H),
2.35-2.22 (m, 5H), 2.13-2.08 (m, 1H), 1.66-1.58 (m, 4H), 1.38-1.32
(m, 10H), 1.06 (s, 9H). HRMS(TOF) calculated for
C.sub.32H.sub.47N.sub.4O.sub.6S [M+H].sup.+, 615.3211, found
615.3224.
##STR00177##
[0218] Linker 8:
11-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoy-
l)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecanoic
acid (930 mg, 78%) as white solid. .sup.1H NMR (600 MHz CD.sub.3OD)
.delta. 8.95 (s, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.44 (d, J=7.8 Hz,
2H), 4.66 (s, 1H), 4.61-4.50 (m, 3H), 4.38 (d, J=15.6 Hz, 1H), 3.93
(d, J=9.6 Hz, 1H), 3.82 (dd, J=11.4, 3.6 Hz, 1H), 2.50 (s, 3H),
2.35-2.21 (m, 5H), 2.12-2.07 (m, 1H), 1.66-1.57 (m, 4H), 1.37-1.29
(m, 12H), 1.06 (s, 9H). HRMS (ESI-TOF) calculated for
C.sub.33H.sub.49N.sub.4O.sub.6S, 629.3367, found 629.3368.
Example 2
Procedures for the Synthesis of VHL-1 PEG Linkers
##STR00178##
[0220] To a solution of diacid (4 mmol) in DMF (10 ml) and DCM (250
ml) was added NMM (10 mmol), VHL-1 (2 mmol), HOAt (2.4 mmol), and
EDCI (2.4 mmol) at 0.degree. C. The resulting reaction solution was
stirred at 0.degree. C. for 6 h and then at RT overnight. The
progress of the reaction was monitored by LC/MS. After VHL-1 was
totally consumed, the reaction was concentrated and the resulting
residue was purified by reverse-phase chromatography to yield the
product.
##STR00179##
[0221] Linker 9:
2-(2-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbam-
oyl)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)acet-
ic acid (810 mg, 69%) as white solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 8.97 (s, 1H), 7.47 (d, J=8.2 Hz, 2H), 7.43 (d,
J=8.1 Hz, 2H), 4.69 (s, 1H), 4.60-4.47 (m, 3H), 4.36 (d, J=15.5 Hz,
1H), 4.27-4.17 (m, 2H), 4.16-4.07 (m, 2H), 3.89 (d, J=11.0 Hz, 1H),
3.81 (dd, J=11.0, 3.8 Hz, 1H), 2.48 (s, 3H), 2.22 (dd, J=13.1, 7.6
Hz, 1H), 2.08 (ddd, J=13.3, 9.2, 4.5 Hz, 1H), 1.05 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.26H.sub.35N.sub.4O.sub.7S, 547.2221; found: 547.2230.
##STR00180##
[0222] Linker 10:
3-(3-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbam-
oyl)
pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)pro-
panoic acid (450 mg, 63%) as white solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.00 (s, 1H), 7.45 (d, J=22.1 Hz, 4H), 4.64 (s,
1H), 4.61-4.44 (m, 3H), 4.36 (d, J=15.4 Hz, 1H), 3.84 (dd, J=57.3,
10.5 Hz, 2H), 3.75-3.56 (m, 4H), 2.60-2.39 (m, 7H), 2.24-2.17 (m,
1H), 2.11-2.03 (m, 1H), 1.03 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.28H.sub.39N.sub.4O.sub.7S,
575.2534; found: 575.2543.
##STR00181##
[0223] Linker 11:
2-(2-(2-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)
carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethox-
y)ethoxy)acetic acid (680 mg, 54%) as white solid. .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 9.05 (s, 1H), 7.48 (d, J=8.1 Hz, 2H), 7.44
(d, J=8.2 Hz, 2H), 4.69 (s, 1H), 4.56 (dd, J=18.6, 12.1 Hz, 2H),
4.50 (s, 1H), 4.36 (d, J=15.5 Hz, 1H), 4.21 (d, J=16.8 Hz, 1H),
4.13 (d, J=16.9 Hz, 1H), 4.08 (d, J=15.6 Hz, 1H), 4.04 (d, J=15.7
Hz, 1H), 3.88 (d, J=11.0 Hz, 1H), 3.83-3.69 (m, 5H), 2.49 (s, 3H),
2.25-2.19 (m, 1H), 2.08 (ddd, J=13.3, 9.2, 4.4 Hz, 1H), 1.04 (s,
9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.28H.sub.39N.sub.4O.sub.8S, 591.2483; found: 591.2477.
##STR00182##
[0224] Linker 12:
3-(2-(3-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)
carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropo-
xy)ethoxy) propanoic acid (680 mg, 64%) as white solid. .sup.1H NMR
(600 MHz, CD.sub.3OD) .delta. 8.98 (d, J=20.1 Hz, 1H), 7.48 (d,
J=8.0 Hz, 2H), 7.43 (d, J=8.1 Hz, 2H), 4.64 (s, 1H), 4.59-4.51 (m,
2H), 4.49 (s, 1H), 4.35 (d, J=15.5 Hz, 1H), 3.89 (d, J=11.0 Hz,
1H), 3.80 (dd, J=10.9, 3.8 Hz, 1H), 3.76-3.67 (m, 4H), 3.63-3.55
(m, 4H), 2.60-2.43 (m, 7H), 2.21 (dd, J=13.1, 7.6 Hz, 1H), 2.08
(ddd, J=13.2, 9.1, 4.5 Hz, 1H), 1.04 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.30H.sub.43N.sub.4O.sub.8S,
619.2796; found: 619.2800.
##STR00183##
[0225] Linker 13:
(S)-13-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)
pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadec-
anoic acid (880 mg, 54%) as white solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.05 (s, 1H), 7.48 (d, J=8.2 Hz, 2H), 7.44 (d,
J=8.3 Hz, 2H), 4.69 (s, 1H), 4.60-4.51 (m, 2H), 4.50 (s, 1H), 4.36
(d, J=15.5 Hz, 1H), 4.10 (s, 1H), 4.07 (d, J=15.6 Hz, 1H), 4.03 (d,
J=15.6 Hz, 1H), 3.87 (d, J=11.0 Hz, 1H), 3.80 (dd, J=11.0, 3.8 Hz,
1H), 3.76-3.64 (m, 9H), 2.50 (s, 3H), 2.22 (dd, J=13.1, 7.6 Hz,
1H), 2.08 (ddd, J=13.3, 9.2, 4.4 Hz, 1H), 1.04 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.30H.sub.43N.sub.4O.sub.9S, 635.2745; found: 635.2751.
##STR00184##
[0226] Linker 14:
(S)-15-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)
pyrrolidine-1-carbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptade-
canoic acid (677 mg, 57%) as white solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 8.95 (s, 1H), 7.47 (d, J=8.1 Hz, 2H), 7.42 (d,
J=8.1 Hz, 2H), 4.65 (s, 1H), 4.59-4.51 (m, 2H), 4.49 (s, 1H), 4.35
(d, J=15.5 Hz, 1H), 3.89 (d, J=11.1 Hz, 1H), 3.80 (dd, J=10.9, 3.9
Hz, 1H), 3.76-3.67 (m, 4H), 3.66-3.54 (m, 8H), 2.60-2.50 (m, 3H),
2.50-2.43 (m, 4H), 2.21 (dd, J=13.1, 7.6 Hz, 1H), 2.08 (ddd,
J=13.3, 9.1, 4.5 Hz, 1H), 1.04 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.32H.sub.47N.sub.4O.sub.9S,
663.3058; found: 663.3059.
##STR00185##
[0227] Linker 15:
(S)-18-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)
pyrrolidine-1-carbonyl)-19,19-dimethyl-16-oxo-4,7,10,13-tetraoxa-17-azaic-
osanoic acid (590 mg, 65%) as white solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 8.99 (s, 1H), 7.48 (d, J=8.1 Hz, 2H), 7.42 (d,
J=8.2 Hz, 2H), 4.65 (s, 1H), 4.59-4.51 (m, 2H), 4.49 (s, 1H), 4.35
(d, J=15.5 Hz, 1H), 3.89 (d, J=11.0 Hz, 1H), 3.80 (dd, J=10.9, 3.8
Hz, 1H), 3.77-3.67 (m, 4H), 3.67-3.54 (m, 12H), 2.61-2.43 (m, 7H),
2.21 (dd, J=13.0, 7.6 Hz, 1H), 2.08 (ddd, J=13.2, 9.1, 4.4 Hz, 1H),
1.04 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.34H.sub.51N.sub.4O.sub.10S, 707.3320; found: 707.3321.
##STR00186##
[0228] Linker 16:
(S)-19-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)
pyrrolidine-1-carbonyl)-20,20-dimethyl-17-oxo-3,6,9,12,15-pentaoxa-18-aza-
henicosanoic acid (496 mg, 54%) as white solid. .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 8.89 (s, 1H), 7.47 (d, J=8.1 Hz, 2H), 7.42
(d, J=8.1 Hz, 2H), 4.69 (s, 1H), 4.59-4.46 (m, 3H), 4.36 (d, J=15.5
Hz, 1H), 4.16-4.00 (m, 4H), 3.87 (d, J=11.0 Hz, 1H), 3.80 (dd,
J=11.0, 3.7 Hz, 1H), 3.76-3.53 (m, 16H), 2.48 (s, 3H), 2.22 (dd,
J=13.1, 7.6 Hz, 1H), 2.08 (ddd, J=13.3, 9.2, 4.4 Hz, 1H), 1.04 (s,
7H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.34H.sub.51N.sub.4O.sub.11S, 723.3270; found: 723.3269.
##STR00187##
[0229] Linker 17:
(S)-21-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)
pyrrolidine-1-carbonyl)-22,22-dimethyl-19-oxo-4,7,10,13,16-pentaoxa-20-az-
atricosanoic acid (420 mg, 42%) as white solid. .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 8.89 (s, 1H), 7.47 (d, J=8.0 Hz, 2H), 7.42
(d, J=8.1 Hz, 2H), 4.65 (s, 1H), 4.59-4.51 (m, 2H), 4.49 (s, 1H),
4.35 (d, J=15.5 Hz, 1H), 3.89 (d, J=11.0 Hz, 1H), 3.80 (dd, J=10.9,
3.8 Hz, 1H), 3.77-3.67 (m, 4H), 3.67-3.51 (m, 16H), 2.61-2.42 (m,
7H), 2.24-2.18 (m, 1H), 2.08 (ddd, J=13.2, 9.1, 4.4 Hz, 1H), 1.02
(d, J=14.3 Hz, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.36H.sub.55N.sub.4O.sub.11S, 751.3583; found: 751.3589.
Example 3
Procedures for the Synthesis of Pomalidomide Linkers
##STR00188##
[0231] A solution of pomalidomide analogue (1 eq.), amine (1 eq.),
and N,N-diisopropylethylamine (1.5 eq.) in DMF (2.0 ml per mmol of
pomalidamide) was heated to 85.degree. C. in a microwave reactor
for 40 min. After cooling to RT, the reaction was quenched with
water and extracted with ethyl acetate (3.times.). The combined
organic phase was dried over anhydrous sodium sulfate and
concentrated under reduced pressure. The resulting residue was
purified by silica gel chromatography (eluted with hexanes/EtOAc:
0-100%) to give the desired t-Bu ester intermediate as oil. This
intermediate was treated with a solution of hydrogen chloride in
dioxane (4 M, 5 ml per mmol of pomalidamide) for overnight. After
concentration under reduced pressure, the desired acid product was
obtained as yellow oil.
##STR00189##
[0232] Linker 18:
3-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)
propanoic acid. tert-Butyl 3-(2-aminoethoxy)propanoate (1.0 g, 5.3
mmol) was used to prepare the title compound (500 mg, 24%)
according to the above procedures. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 7.54 (dd, J=8.3, 7.0, 1.2 Hz, 1H), 7.09 (d,
1H), 7.04 (d, J=7.0, 1.1 Hz, 1H), 5.05 (dd, J=12.5, 5.4, 1.2 Hz,
1H), 3.75 (t, J=6.2, 1.2 Hz, 2H), 3.65-3.69 (m, 2H), 3.45-3.49 (m,
2H), 2.88-2.82 (m, 1H), 2.76-2.70 (m, 2H), 2.56 (t, J=6.2, 1.2 Hz,
2H), 2.10 (ddt, J=14.9, 7.6, 3.7, 1.6 Hz, 1H). MS (ESI) ink 390.2
[M+H].sup.+.
##STR00190##
[0233] Linker 19:
3-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)etho-
xy) ethoxy)-propanoic acid. tert-Butyl
3-(2-(2-aminoethoxy)ethoxy)propanoate (0.70 g, 3.0 mmol) was used
to prepare tert-butyl
3-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-eth-
oxy)ethoxy)propanoate (575 mg, 39%) according to the above
procedures. .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 8.13 (s, 1H),
7.53-7.45 (m, 1H), 7.10 (d, J=7.1 Hz, 1H), 6.92 (d, J=8.5 Hz, 1H),
6.49 (t, J=5.6 Hz, 1H), 4.91 (dd, J=12.4, 5.3 Hz, 1H), 3.76-3.69
(m, 4H), 3.67-3.60 (m, 4H), 3.46 (q, J=5.5 Hz, 2H), 2.89 (dt,
J=16.8, 3.2 Hz, 1H), 2.84-2.69 (m, 2H), 2.51 (t, J=6.6 Hz, 2H),
2.16-2.08 (m, 1H), 1.44 (s, 9H). MS (ESI) m/z 490.2 [M+H].sup.+.
The t-Bu ester intermediate was dissolved in formic acid (10 ml)
and the resulting solution was stirred at RT overnight. After
removal of the solvent under reduced pressure, the tittle compound
(512 mg, 100%) was obtained and used for the following reactions
without further purification.
##STR00191##
[0234] Linker 20:
3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)e-
thoxy) ethoxy)ethoxy)propanoic acid. tert-Butyl
3-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)propanoate (1.0 g, 3.6 mmol)
was used to prepare the title compound (240 mg, 10%) according to
the above procedures. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
7.55 (dd, J=8.4, 7.2 Hz, 1H), 7.10 (d, J=8.6 Hz, 1H), 7.05 (d,
J=7.1 Hz, 1H), 5.05 (dd, J=12.4, 5.4 Hz, 1H), 3.71 (dt, J=9.4, 5.7
Hz, 4H), 3.66-3.63 (m, 4H), 3.62 (dd, J=6.0, 3.5 Hz, 2H), 3.58 (dd,
J=6.1, 3.5 Hz, 2H), 3.50 (t, J=5.3 Hz, 2H), 2.86 (ddd, J=19.1,
14.1, 5.3 Hz, 1H), 2.77-2.66 (m, 2H), 2.52 (t, J=6.3 Hz, 2H), 2.11
(ddt, J=10.3, 5.0 Hz, 1H). MS (ESI) ink 478.3 [M+H].sup.+.
##STR00192##
[0235] Linker 21:
1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12--
tetraoxapentadecan-15-oic acid. tert-Butyl
1-amino-3,6,9,12-tetraoxapentadecan-15-oate (0.96 g, 3.0 mmol) was
used to prepare the t-Bu ester intermediate according to the
general procedures. The t-Bu ester intermediate was dissolved in
formic acid (10 ml) and the resulting solution was stirred at RT
overnight. After removal of the solvent under reduced pressure, the
title compound (950 mg, 61%) was obtained and used for the
following reactions without further purification. .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 7.55 (t, J=7.8 Hz, 1H), 7.10 (d, J=8.5 Hz,
1H), 7.06 (d, J=7.0 Hz, 1H), 5.05 (dd, J=12.6, 5.3 Hz, 1H),
3.75-3.68 (m, 4H), 3.68-3.55 (m, 12H), 3.50 (t, J=4.9 Hz, 2H),
2.90-2.81 (m, 1H), 2.78-2.66 (m, 2H), 2.52 (t, J=6.0 Hz, 2H),
2.14-2.07 (m, 1H). MS (ESI) m/z 522.2 [M+H].sup.+.
##STR00193##
[0236] Linker 22:
1-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,-
15-pentaoxaoctadecan-18-oic acid. tert-Butyl
1-amino-3,6,9,12,15-pentaoxaoctadecan-18-oate (1.10 g, 3.0 mmol)
was used to prepare tert-butyl
1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,-
15-pentaoxaoctadecan-18-oate (1.35 g, 72%) according to the above
procedures. .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 8.32 (s, 1H),
7.48 (dd, J=8.5, 7.1 Hz, 1H), 7.10 (d, J=7.1 Hz, 1H), 6.91 (d,
J=8.6 Hz, 1H), 6.49 (t, J=5.7 Hz, 1H), 4.91 (dd, J=12.4, 5.3 Hz,
1H), 3.74-3.68 (m, 4H), 3.68-3.63 (m, 12H), 3.63-3.58 (m, 4H), 3.46
(q, J=5.6 Hz, 2H), 2.92-2.85 (m, 1H), 2.83-2.68 (m, 2H), 2.49 (t,
J=6.6 Hz, 2H), 2.15-2.08 (m, 1H), 1.43 (s, 9H). MS (ESI) m/z 622.2
[M+H].sup.+. The t-Bu ester intermediate was dissolved in formic
acid (10 ml) and the resulting solution was stirred at RT
overnight. After removal of the solvent under reduced pressure, the
tittle compound (1.23 g, 100%) was obtained and used for the
following reactions without further purification.
##STR00194##
[0237] Linker 23:
(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycine.
tert-Butyl glycinate (838 mg, 5.0 mmol) was used to prepare the
title compound (240 mg, 14%) according to the general procedures.
.sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 7.57 (dd, J=8.5, 7.1 Hz,
1H), 7.11 (d, J=7.1 Hz, 1H), 6.95 (d, J=8.5 Hz, 1H), 5.07 (dd,
J=12.6, 5.5 Hz, 1H), 4.12 (s, 2H), 2.86 (ddd, J=18.0, 14.4, 5.4 Hz,
1H), 2.74-2.67 (m, 2H), 2.15-2.08 (m, 1H). MS (ESI) ink 332.1
[M+H].sup.+.
##STR00195##
[0238] Linker 24:
3-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propanoic
acid. tert-Butyl 3-aminopropanoate HCl salt (1.0 g, 5.97 mmol) was
used to prepare the title compound (700 mg, 4%) according to the
above procedures. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 7.57
(dd, J=8.6, 7.1 Hz, 1H), 7.11 (d, J=8.6 Hz, 1H), 7.06 (d, J=7.1 Hz,
1H), 5.05 (dd, J=12.6, 5.5 Hz, 1H), 3.62 (t, J=6.5 Hz, 2H),
2.88-2.82 (m, 1H), 2.76-2.69 (m, 2H), 2.64 (t, J=6.5 Hz, 2H),
2.13-2.07 (m, 1H). MS (ESI) ink 346.2 [M+H].sup.+.
##STR00196##
[0239] Linker 25:
4-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butanoic
acid. tert-Butyl 4-aminobutanoate (1.0 g, 6.2 mmol) was used to
prepare the title compound (550 mg, 25%) according to the above
procedures. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 7.55 (dd,
J=8.6, 7.1 Hz, 1H), 7.10 (d, J=8.5 Hz, 1H), 7.04 (d, J=7.1 Hz, 1H),
5.05 (dd, J=12.4, 5.5 Hz, 1H), 3.39 (t, J=7.2 Hz, 2H), 2.85-2.82
(m, 1H), 2.76-2.69 (m, 2H), 2.42 (t, J=7.1 Hz, 2H), 2.10 (tq,
J=8.0, 3.8 Hz, 1H), 1.94 (dp, J=14.3, 7.0 Hz, 2H). MS (ESI) m/z
360.1 [M+H].sup.+.
##STR00197##
[0240] Linker 26:
6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexanoic
acid. tert-Butyl 6-aminohexanoate (1.0 g, 4.47 mmol) was used to
prepare the title compound (460 mg, 27%) according to the above
procedures. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 7.54 (dd,
J=8.6, 7.1 Hz, 1H), 7.03 (dd, J=7.8, 3.8 Hz, 2H), 5.05 (dd, J=12.5,
5.4 Hz, 1H), 3.33 (t, J=7.1 Hz, 2H), 2.88-2.82 (m, 1H), 2.75-2.67
(m, 2H), 2.31 (t, J=7.4 Hz, 2H), 2.10 (tdd, J=10.1, 5.3, 3.1 Hz,
1H), 1.70-1.64 (m, 4H), 1.46 (dddd, J=13.0, 8.9, 7.1, 4.2 Hz, 2H).
MS (ESI) m/z 388.1 [M+H].sup.+.
##STR00198##
[0241] Linker 27:
7-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)heptanoic
acid. tert-Butyl 7-aminoheptanoate (1.0 g, 4.96 mmol) was used to
prepare the title compound (500 mg, 25%) according to the above
procedures. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 7.54 (dd,
1H), 7.03 (dd, J=7.8, 3.7 Hz, 2H), 5.05 (dd, J=12.5, 5.5 Hz, 1H),
3.30-3.33 (m, 2H), 2.90-2.79 (m, 1H), 2.77-2.68 (m, 2H), 2.29 (t,
J=7.4 Hz, 2H), 2.13-2.07 (m, 1H), 1.68-1.61 (m, 4H), 1.46-1.40 (m,
4H). MS (ESI) m/z 402.3 [M+H].sup.+.
##STR00199##
[0242] Linker 28:
8-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)octanoic
acid. tert-Butyl 8-aminooctanoate (1.0 g, 4.6 mmol) was used to
prepare the title compound (620 mg, 32%) according to the above
procedures. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 7.53 (dd,
J=8.6, 7.0, 1.5 Hz, 1H), 7.08-6.93 (m, 2H), 5.05 (dd, J=12.5, 5.5,
1.5 Hz, 1H), 3.31 (t, 2H), 2.90-2.79 (m, 1H), 2.75-2.66 (m, 2H),
2.28 (t, J=7.5, 1.5 Hz, 2H), 2.13-2.07 (m, 1H), 1.66-1.51 (m, 4H),
1.43-1.33 (m, 6H). MS (ESI) ink 416.4[M+H].sup.+.
Example 4
Procedures for the Synthesis of Bivalent Compounds
##STR00200##
[0244]
2-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoethoxy)acet-
ic acid (Intermediate 1). To a solution of palbociclib (25 mg,
0.0559 mmol) in DMSO (1 ml) and DCM (10 ml) were added NMM (34 mg,
0.335 mmol), 2,2'-oxydiacetic acid (18 mg, 0.135 mmol), HOAt (20
mg, 0.134 mmol), and EDCI (28 mg, 0.134 mmol). The mixture was
allowed to stir at room temperature overnight. After palbociclib
was consumed, the reaction was concentrated under reduced pressure.
The resulting residue was purified by prep-HPLC to yield the
product (30 mg, 96%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.10 (s, 1H), 8.20 (d, J=9.5 Hz, 1H), 7.88 (s, 1H), 7.55 (d, J=9.5
Hz, 1H), 6.00 (p, J=8.9 Hz, 1H), 4.41 (s, 2H), 4.21 (s, 2H),
3.85-3.70 (m, 4H), 3.39-3.26 (m, 4H), 2.49 (s, 3H), 2.43 (s, 3H),
2.35-2.25 (m, 2H), 2.13-2.04 (m, 2H), 1.95-1.85 (m, 2H), 1.74-1.64
(m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.28H.sub.34N.sub.7O.sub.6, 564.2565; found: 564.2560.
[0245]
(2S,4R)-1-((S)-2-(2-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-ox-
o-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl-
)-2-oxoethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthi-
azol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound Example 1). To
a solution of intermediate 1 (30 mg, 0.0553 mmol) in DMSO (1 mL)
were added NMM (27 mg, 0.266 mmol), VHL-1 (30 mg, 0.0639 mmol),
HOAt (11 mg, 0.0799 mmol), and EDCI (15 mg, 0.0799 mmol). The
mixture was allowed to stir at room temperature overnight. After
VHL-1 was consumed, the reaction was concentrated under reduced
pressure. The resulting residue was purified by prep-HPLC to yield
the product (36 mg, 69%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.07 (s, 1H), 9.03 (s, 1H), 8.17 (dd, J=2.9,
9.6 Hz, 1H), 7.86 (d, J=2.9 Hz, 1H), 7.52 (d, J=9.6 Hz, 1H),
7.46-7.42 (m, 2H), 7.38 (d, J=8.3 Hz, 2H), 5.99 (p, J=8.9 Hz, 1H),
4.69 (s, 1H), 4.62-4.30 (m, 6H), 4.17 (d, J=15.1 Hz, 1H), 4.10 (d,
J=15.0 Hz, 1H), 3.90 (d, J=10.9 Hz, 1H), 3.85-3.74 (m, 3H),
3.72-3.62 (m, 2H), 3.37-3.25 (m, 4H), 2.49 (s, 3H), 2.47 (s, 3H),
2.41 (s, 3H), 2.35-2.21 (m, 3H), 2.14-2.04 (m, 3H), 1.94-1.85 (m,
2H), 1.75-1.63 (m, 2H), 1.06 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.50H.sub.62N.sub.11O.sub.8S,
976.4498; found: 976.4492.
##STR00201##
[0246]
(2S,4R)-1-((S)-2-(3-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-ox-
o-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl-
)-3-oxopropoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound Example 2).
To a solution of palbociclib (53 mg, 0.119 mmol) in DMSO (2 ml)
were added DIPEA (76 mg, 0.594 mmol), 3,3'-oxydipropionic acid (25
mg, 0.154 mmol), and TBTU (57 mg, 0.178 mmol). The mixture was
allowed to stir at RT overnight, at which time TBTU (57 mg, 0.178
mmol) and VHL-1 (55 mg, 0.119 mmol) were added. The reaction
mixture was allowed to stir at RT overnight. After VHL-1 was
consumed, the reaction was concentrated under reduced pressure. The
resulting residue was purified by prep-HPLC to yield the product
(22 mg, 18%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD)
.delta. 8.92 (s, 1H), 8.79 (s, 1H), 7.98 (s, 1H), 7.95 (d, J=8.9
Hz, 1H), 7.41 (d, J=8.1 Hz, 1H), 7.37 (d, J=8.0 Hz, 2H), 7.32 (d,
J=7.7 Hz, 2H), 5.88 (p, J=9.1 Hz, 1H), 4.66 (s, 1H), 4.56 (t, J=8.4
Hz, 1H), 4.49 (s, 1H), 4.45 (d, J=15.6 Hz, 1H), 4.33 (d, J=15.4 Hz,
1H), 3.88 (d, J=11.1 Hz, 1H), 3.80-3.70 (m, 9H), 3.15 (dt, J=5.1,
29.0 Hz, 4H), 2.73 (d, J=2.7 Hz, 2H), 2.57-2.49 (m, 2H), 2.47 (s,
3H), 2.39 (s, 3H), 2.34-2.25 (m, 5H), 2.24-2.18 (m, 1H), 2.12-2.03
(m, 1H), 2.03-1.95 (m, 2H), 1.89-1.80 (m, 2H), 1.71-1.61 (m, 2H),
1.03 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.52H.sub.66N.sub.11O.sub.8S, 1004.4811; found: 1004.4813. The
Example 3, 4, 5, 6, 7, 8, 9, and 18 compounds (below) were
synthesized according to the procedures for the preparation of the
Example 2 compound (above).
##STR00202##
[0247]
(2S,4R)-1-((S)-2-(2-(2-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-
-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-
-yl)-2-oxoethoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-
-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound
Example 3) (9 mg, 4%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.06 (s, 1H), 8.94 (s, 1H), 8.16 (dd, J=2.9,
9.6 Hz, 1H), 7.82 (d, J=2.9 Hz, 1H), 7.50 (d, J=9.6 Hz, 1H), 7.41
(d, J=8.2 Hz, 2H), 7.38 (d, J=8.2 Hz, 2H), 6.00 (p, J=8.8 Hz, 1H),
4.73 (d, J=9.5 Hz, 1H), 4.57 (dd, J=7.5, 9.5 Hz, 1H), 4.53-4.45 (m,
2H), 4.41-4.31 (m, 3H), 4.06 (d, J=8.3 Hz, 2H), 3.89 (d, J=11.1 Hz,
1H), 3.85-3.70 (m, 9H), 3.37-3.24 (m, 4H), 2.50 (s, 3H), 2.45 (s,
3H), 2.42 (s, 3H), 2.34-2.23 (m, 3H), 2.14-2.05 (m, 3H), 1.94-1.86
(m, 2H), 1.74-1.65 (m, 2H), 1.04 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.52H.sub.66N.sub.11O.sub.9S,
1020.4760; found: 1020.4769.
##STR00203##
[0248]
(2S,4R)-1-((S)-2-(3-(2-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-
-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-
-yl)-3-oxopropoxy)ethoxy)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound
Example 4) (13 mg, 11%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.09 (s, 1H), 8.98 (s, 1H), 8.20 (dd, J=3.1,
9.6 Hz, 1H), 7.85 (d, J=2.9 Hz, 1H), 7.53 (d, J=9.5 Hz, 1H), 7.46
(d, J=8.0 Hz, 2H), 7.40 (d, J=8.2 Hz, 2H), 6.00 (p, J=8.9 Hz, 1H),
4.64 (s, 1H), 4.60-4.47 (m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.88 (d,
J=11.0 Hz, 1H), 3.82-3.75 (m, 7H), 3.74-3.67 (m, 2H), 3.65-3.57 (m,
5H), 3.33 (t, J=5.4 Hz, 2H), 3.26 (t, J=5.3 Hz, 2H), 2.72 (t, J=6.1
Hz, 2H), 2.56-2.51 (m, 1H), 2.50 (s, 3H), 2.47 (s, 3H), 2.43 (s,
3H), 2.35-2.26 (m, 2H), 2.25-2.20 (m, 1H), 2.14-2.04 (m, 3H),
1.94-1.86 (m, 2H), 1.74-1.66 (m, 2H), 1.03 (s, 9H). HRMS (ESI-TOF)
m/z: [M+H].sup.+ calculated for C.sub.54H.sub.70N.sub.11O.sub.9S,
1048.5073; found: 1048.5069.
##STR00204##
[0249]
(2S,4R)-1-((S)-14-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-
-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-(-
tert-butyl)-4,14-dioxo-6,9,12-trioxa-3-azatetradecanoyl)-4-hydroxy-N-(4-(4-
-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound
Example 5) (15 mg, 17%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.08 (s, 1H), 9.07 (s, 1H), 8.18 (dd, J=3.0,
9.6 Hz, 1H), 7.85 (d, J=2.9 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.46
(d, J=8.1 Hz, 2H), 7.42 (d, J=8.1 Hz, 2H), 6.00 (p, J=8.9 Hz, 1H),
4.69 (s, 1H), 4.60-4.48 (m, 3H), 4.39-4.26 (m, 3H), 4.09-3.96 (m,
2H), 3.88 (d, J=10.9 Hz, 1H), 3.83-3.63 (m, 12H), 3.27 (t, J=5.0
Hz, 4H), 2.49 (s, 3H), 2.48 (s, 3H), 2.42 (s, 3H), 2.34-2.21 (m,
3H), 2.13-2.04 (m, 3H), 1.93-1.86 (m, 2H), 1.72-1.66 (m, 2H), 1.04
(s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.54H.sub.70N.sub.11O.sub.10S, 1064.5022; found: 1064.5026.
##STR00205##
[0250]
(2S,4R)-1-((S)-16-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-
-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-(-
tert-butyl)-4,16-dioxo-7,10,13-trioxa-3-azahexadecanoyl)-4-hydroxy-N-(4-(4-
-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound
Example 6) (13 mg, 13%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.09 (s, 1H), 8.97 (s, 1H), 8.20 (dd, J=3.0,
9.7 Hz, 1H), 7.86 (d, J=3.0 Hz, 1H), 7.53 (d, J=9.7 Hz, 1H), 7.45
(d, J=8.0 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H), 6.01 (p, J=8.8 Hz, 1H),
4.64 (s, 1H), 4.58-4.47 (m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.88 (d,
J=11.0 Hz, 1H), 3.83-3.75 (m, 7H), 3.74-3.65 (m, 2H), 3.64-3.55 (m,
8H), 3.33 (t, J=5.2 Hz, 2H), 3.27 (d, J=7.2 Hz, 2H), 2.71 (t, J=6.2
Hz, 2H), 2.59-2.52 (m, 1H), 2.50 (s, 3H), 2.47 (s, 3H), 2.43 (s,
3H), 2.34-2.26 (m, 2H), 2.24-2.19 (m, 1H), 2.13-2.04 (m, 3H),
1.94-1.86 (m, 2H), 1.74-1.65 (m, 2H), 1.03 (s, 9H). HRMS (ESI-TOF)
m/z: [M+H].sup.+ calculated for C.sub.56H.sub.74N.sub.11O.sub.10S,
1092.5335; found: 1092.5339.
##STR00206##
[0251]
(2S,4R)-1-((S)-19-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-
-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-(-
tert-butyl)-4,19-dioxo-7,10,13,16-tetraoxa-3-azanonadecanoyl)-4-hydroxy-N--
(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound
Example 7) (12 mg, 12%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.08 (s, 1H), 8.99 (s, 1H), 8.21 (dd, J=3.0,
9.5 Hz, 1H), 7.87 (d, J=2.9 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.45
(d, J=8.0 Hz, 2H), 7.40 (d, J=8.1 Hz, 2H), 6.00 (p, J=8.9 Hz, 1H),
4.64 (s, 1H), 4.59-4.47 (m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.88 (d,
J=11.0 Hz, 1H), 3.83-3.75 (m, 7H), 3.75-3.66 (m, 2H), 3.64-3.53 (m,
13H), 3.34 (t, J=5.2 Hz, 2H), 3.28 (t, J=5.3 Hz, 2H), 2.72 (t,
J=6.2 Hz, 2H), 2.59-2.52 (m, 1H), 2.50 (s, 3H), 2.47 (s, 3H), 2.43
(s, 3H), 2.35-2.26 (m, 2H), 2.24-2.19 (m, 1H), 2.13-2.04 (m, 3H),
1.94-1.86 (m, 2H), 1.73-1.64 (m, 2H), 1.03 (s, 9H). HRMS (ESI-TOF)
m/z: [M+H].sup.+ calculated for C.sub.58H.sub.78N.sub.11O.sub.11S,
1136.5597; found: 1136.5595.
##STR00207##
[0252]
(2S,4R)-1-((S)-20-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-
-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-(-
tert-butyl)-4,20-dioxo-6,9,12,15,18-pentaoxa-3-azaicosanoyl)-4-hydroxy-N-(-
4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound
Example 8) (6 mg, 7%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.08 (s, 1H), 8.93 (s, 1H), 8.20 (dd, J=3.0,
9.6 Hz, 1H), 7.87 (d, J=3.0 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.44
(d, J=8.0 Hz, 2H), 7.41 (d, J=8.0 Hz, 2H), 6.00 (p, J=8.7 Hz, 1H),
4.69 (s, 1H), 4.59-4.48 (m, 3H), 4.36 (d, J=15.7 Hz, 1H), 4.30 (s,
2H), 4.05 (d, J=15.8 Hz, 1H), 4.00 (d, J=15.5 Hz, 1H), 3.88 (d,
J=11.1 Hz, 1H), 3.83-3.72 (m, 5H), 3.72-3.58 (m, 16H), 3.36-3.25
(m, 4H), 2.50 (s, 3H), 2.47 (s, 3H), 2.43 (s, 3H), 2.34-2.27 (m,
2H), 2.26-2.21 (m, 1H), 2.15-2.05 (m, 3H), 1.94-1.86 (m, 2H),
1.73-1.65 (m, 2H), 1.03 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.58H.sub.78N.sub.11O.sub.12S, 1152.5547; found:
1152.5548.
##STR00208##
[0253]
(2S,4R)-1-((S)-22-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-
-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-(-
tert-butyl)-4,22-dioxo-7,10,13,16,19-pentaoxa-3-azadocosanoyl)-4-hydroxy-N-
-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(compound Example 9) (25 mg, 19%) as yellow solid. .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 9.09 (s, 1H), 9.06 (s, 1H), 8.21 (dd,
J=3.0, 9.6 Hz, 1H), 7.88 (d, J=3.0 Hz, 1H), 7.54 (d, J=9.6 Hz, 1H),
7.46 (d, J=8.1 Hz, 2H), 7.41 (d, J=8.2 Hz, 2H), 6.00 (p, J=8.9 Hz,
1H), 4.64 (s, 1H), 4.60-4.46 (m, 3H), 4.35 (d, J=15.6 Hz, 1H), 3.88
(d, J=10.8 Hz, 1H), 3.84-3.66 (m, 10H), 3.65-3.55 (m, 16H), 3.35
(t, J=5.1 Hz, 2H), 3.28 (t, J=5.3 Hz, 2H), 2.72 (t, J=6.1 Hz, 2H),
2.61-2.53 (m, 1H), 2.49 (s, 3H), 2.48 (s, 3H), 2.42 (s, 3H),
2.34-2.26 (m, 2H), 2.25-2.19 (m, 1H), 2.12-2.03 (m, 3H), 1.94-1.86
(m, 2H), 1.73-1.65 (m, 2H), 1.03 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.60H.sub.82N.sub.11O.sub.12S,
1180.5860; found: 1180.5859.
##STR00209##
[0254]
(2S,4R)-1-((S)-2-(11-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo--
7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)--
11-oxoundecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol--
5-yl)benzyl)pyrrolidine-2-carboxamide (compound Example 18) (17 mg,
13%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.10 (s, 1H), 8.94 (s, 1H), 8.20 (dd, J=2.9, 9.6 Hz, 1H), 7.86 (d,
J=3.0 Hz, 1H), 7.54 (d, J=9.6 Hz, 1H), 7.46 (d, J=8.2 Hz, 2H), 7.41
(d, J=8.1 Hz, 2H), 6.01 (p, J=8.9 Hz, 1H), 4.63 (s, 1H), 4.59-4.46
(m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H),
3.84-3.72 (m, 5H), 3.36-3.24 (m, 4H), 2.53-2.40 (m, 10H), 2.36-2.18
(m, 5H), 2.15-2.03 (m, 3H), 1.95-1.86 (m, 2H), 1.74-1.53 (m, 6H),
1.42-1.27 (m, 11H), 1.03 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.57H.sub.76N.sub.11O.sub.7S, 1058.5644; found:
1058.5640.
[0255] The Intermediate 2, 3, and 4 and Example 10, 11, and 12
compounds were synthesized according to the procedures for the
preparation of the Example 1 compound (above).
##STR00210##
[0256]
3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2-
,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropanoic
acid (Intermediate 2) (18 mg, 51%). .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.07 (s, 1H), 8.09 (d, J=10.2 Hz, 1H), 7.90 (s,
1H), 7.62 (d, J=9.5 Hz, 1H), 6.06-5.95 (m, 1H), 3.86-3.72 (m, 4H),
3.59 (d, J=9.0 Hz, 2H), 3.40-3.22 (m, 4H), 2.50 (s, 3H), 2.42 (s,
3H), 2.37-2.27 (m, 2H), 2.14-2.03 (m, 2H), 1.95-1.85 (m, 2H),
1.75-1.66 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.27H.sub.32N.sub.7O.sub.5, 534.2459; found: 534.2450.
##STR00211##
[0257]
6-Acetyl-8-cyclopentyl-N-(5-(4-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4--
(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxo-
butan-2-yl)amino)-3-oxopropanoyl)piperazin-1-yl)pyridin-2-yl)-5-methyl-7-o-
xo-7,8-dihydropyrido[2,3-d]pyrimidin-2-aminium (compound Example
10) (19 mg, 59%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD)
.delta. 9.10 (s, 1H), 9.00 (s, 1H), 8.20 (dd, J=3.0, 9.7 Hz, 1H),
7.86 (d, J=3.0 Hz, 1H), 7.53 (d, J=9.5 Hz, 1H), 7.47 (d, J=8.2 Hz,
2H), 7.41 (d, J=8.3 Hz, 2H), 6.00 (p, J=9.0 Hz, 1H), 4.63 (s, 1H),
4.59-4.43 (m, 4H), 4.37 (d, J=15.5 Hz, 1H), 3.94-3.84 (m, 2H),
3.84-3.68 (m, 5H), 3.41-3.20 (m, 4H), 2.49 (s, 3H), 2.48 (s, 3H),
2.42 (s, 3H), 2.34-2.27 (m, 2H), 2.24-2.18 (m, 1H), 2.13-2.04 (m,
3H), 1.93-1.86 (m, 2H), 1.73-1.66 (m, 2H), 1.06 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.49H.sub.60N.sub.11O.sub.7S, 946.4392; found: 946.4391.
##STR00212##
[0258]
4-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2-
,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-4-oxobutanoic
acid (Intermediate 3) (27 mg, 88%). .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.09 (s, 1H), 8.17 (d, J=9.5 Hz, 1H), 7.88 (s,
1H), 7.57 (d, J=9.6 Hz, 1H), 6.00 (p, J=9.0 Hz, 1H), 3.78 (t, J=5.8
Hz, 4H), 3.35 (t, J=4.9 Hz, 2H), 3.28 (t, J=4.9 Hz, 2H), 2.73 (t,
J=6.5 Hz, 2H), 2.64 (t, J=6.5 Hz, 2H), 2.49 (s, 3H), 2.42 (s, 3H),
2.36-2.27 (m, 2H), 2.13-2.04 (m, 2H), 1.95-1.86 (m, 2H), 1.75-1.64
(m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.28H.sub.34N.sub.7O.sub.5, 548.2616; found: 548.2612.
##STR00213##
[0259]
(2S,4R)-1-((S)-2-(4-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-4-
-oxobutanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl-
)benzyl)pyrrolidine-2-carboxamide (compound Example 11) (32 mg,
68%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.09 (s, 1H), 9.01 (s, 1H), 8.20 (dd, J=3.0, 9.6 Hz, 1H), 7.86 (d,
J=2.9 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.46 (d, J=8.1 Hz, 2H), 7.41
(d, J=8.2 Hz, 2H), 6.00 (p, J=8.8 Hz, 1H), 4.61 (s, 1H), 4.58-4.54
(m, 1H), 4.52 (d, J=15.4 Hz, 1H), 4.50-4.47 (m, 1H), 4.36 (d,
J=15.5 Hz, 1H), 3.88 (d, J=11.3 Hz, 1H), 3.84-3.70 (m, 5H), 3.34
(t, J=5.1 Hz, 2H), 3.27 (t, J=5.5 Hz, 2H), 2.80-2.62 (m, 3H),
2.60-2.54 (m, 1H), 2.49 (s, 3H), 2.48 (s, 3H), 2.42 (s, 3H),
2.35-2.26 (m, 2H), 2.24-2.19 (m, 1H), 2.13-2.05 (m, 3H), 1.93-1.86
(m, 2H), 1.73-1.66 (m, 2H), 1.03 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.50H.sub.62N.sub.11O.sub.7S,
960.4549; found: 960.4545.
##STR00214##
[0260]
5-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2-
,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-5-oxopentanoic
acid (Intermediate 4) (30 mg, 96%). .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.14 (s, 1H), 8.21 (d, J=9.4 Hz, 1H), 7.92 (s,
1H), 7.56 (d, J=9.3 Hz, 1H), 6.00 (p, J=8.9 Hz, 1H), 4.04 (d,
J=13.3 Hz, 2H), 3.77 (t, J=12.2 Hz, 2H), 3.44 (d, J=12.5 Hz, 2H),
3.15 (td, J=3.4, 12.3 Hz, 2H), 2.53 (t, J=7.5 Hz, 2H), 2.49 (s,
3H), 2.43 (s, 3H), 2.40 (t, J=7.3 Hz, 2H), 2.35-2.26 (m, 2H),
2.13-2.05 (m, 2H), 1.95-1.86 (m, 4H), 1.74-1.65 (m, 2H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.29H.sub.36N.sub.7O.sub.5, 562.2772; found: 562.2775.
##STR00215##
[0261]
(2S,4R)-1-((S)-2-(5-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-5-
-oxopentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-y-
l)benzyl)pyrrolidine-2-carboxamide (compound Example 12) (40 mg,
77%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.12 (s, 1H), 9.09 (s, 1H), 8.17 (dd, J=2.9, 9.6 Hz, 1H), 7.86 (d,
J=2.9 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.47 (d, J=7.9 Hz, 2H), 7.41
(d, J=8.3 Hz, 2H), 5.99 (p, J=8.8 Hz, 1H), 4.62 (s, 1H), 4.59-4.49
(m, 3H), 4.37 (d, J=15.6 Hz, 1H), 3.94 (d, J=11.1 Hz, 1H),
3.83-3.71 (m, 6H), 3.34-3.25 (m, 4H), 2.49 (s, 3H), 2.48 (s, 3H),
2.42 (s, 3H), 2.39-2.20 (m, 6H), 2.11-2.05 (m, 3H), 1.95-1.85 (m,
4H), 1.72-1.65 (m, 2H), 1.04 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.51H.sub.64N.sub.11O.sub.7S,
974.4705; found: 974.4703.
##STR00216##
[0262] Methyl
8-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]p-
yrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-8-oxooctanoate
(Intermediate 5). To a solution of palbociclib (50 mg, 0.112 mmol)
in DMF (5 ml) and DCM (20 ml) were added NMM (34 mg, 0.335 mmol),
8-methoxy-8-oxooctanoic acid (25 mg, 0.134 mmol), HOAt (20 mg,
0.146 mmol), and EDCI (28 mg, 0.146 mmol). The mixture was allowed
to stir at RT overnight. After palbociclib was consumed, the
reaction was concentrated under reduced pressure. The resulting
residue was purified by prep-HPLC to yield the product (69 mg, 98%)
as yellow solid. HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.33H.sub.44N.sub.7O.sub.5, 618.3398; found: 618.3401.
[0263]
(2S,4R)-1-((S)-2-(8-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-8-
-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl-
)benzyl)pyrrolidine-2-carboxamide (compound Example 15) To a
stirring solution of intermediate 5 (69 mg, 0.112 mmol) in
THF/H.sub.2O (20 ml/5 ml) was added anhydrous LiOH (6 mg, 0.223
mmol) and the resulting mixture was stirred overnight at RT. The
disappearance of starting material was monitored by LC/MS. The
reaction mixture was concentrated under reduced pressure and the
resulting residue was dissolved in DCM/DMSO (10 ml/3 ml). To the
resulting solution were added NMM (13 mg, 0.335 mmol), VHL-1 (57
mg, 0.123 mmol), HOAt (20 mg, 0.145 mmol) and EDCI (28 mg, 0.145
mmol). The mixture was allowed to stir at room temperature
overnight. After the starting materials were consumed, the reaction
was concentrated under reduced pressure. The resulting residue was
purified by prep-HPLC to yield the product (68 mg, 60%) as yellow
solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.10 (s, 1H), 9.05
(s, 1H), 8.21 (dd, J=3.0, 9.7 Hz, 1H), 7.86 (s, 1H), 7.53 (d, J=9.7
Hz, 1H), 7.47 (d, J=7.9 Hz, 2H), 7.42 (d, J=7.9 Hz, 2H), 6.01 (p,
J=8.9 Hz, 1H), 4.63 (s, 1H), 4.59-4.45 (m, 3H), 4.36 (d, J=15.5 Hz,
1H), 3.90 (d, J=11.0 Hz, 1H), 3.84-3.72 (m, 5H), 3.33-3.25 (m, 4H),
2.53-2.40 (m, 10H), 2.36-2.18 (m, 6H), 2.13-2.03 (m, 3H), 1.95-1.85
(m, 2H), 1.74-1.58 (m, 6H), 1.45-1.32 (m, 4H), 1.03 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.54H.sub.70N.sub.11O.sub.7S, 1016.5175; found: 1016.5180.
##STR00217##
[0264]
(2S,4R)-1-((S)-2-(6-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-6-
-oxohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl-
)benzyl)pyrrolidine-2-carboxamide (compound Example 13). To a
solution of palbociclib (12 mg, 0.0268 mmol) in DMSO (1 mL) and DCM
(5 mL) were added NMM (13.6 mg, 0.134 mmol), linker 4 (15 mg,
0.0268 mmol), HOAt (5.5 mg, 0.042 mmol) and EDCI (7.7 mg, 0.042
mmol). The mixture was allowed to stir at rt overnight. After the
starting materials were consumed, the reaction was concentrated
under reduced pressure. The resulting residue was purified by
prep-HPLC to yield the product (24 mg, 81%) as yellow solid.
.sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.09 (s, 1H), 8.95 (s,
1H), 8.19 (dd, J=9.6, 2.9 Hz, 1H), 7.86 (d, J=2.8 Hz, 1H), 7.54 (d,
J=9.6 Hz, 1H), 7.46 (d, J=8.2 Hz, 2H), 7.40 (d, J=8.2 Hz, 2H), 6.00
(p, J=8.9 Hz, 1H), 4.63 (s, 1H), 4.60-4.48 (m, 3H), 4.36 (d, J=15.6
Hz, 1H), 3.90 (d, J=11.1 Hz, 1H), 3.83-3.73 (m, 5H), 3.35-3.31 (m,
2H), 3.29-3.25 (m, 2H), 2.50 (s, 3H), 2.47 (s, 3H), 2.43 (s, 3H),
2.36-2.27 (m, 4H), 2.24-2.19 (m, 1H), 2.13-2.05 (m, 3H), 1.94-1.86
(m, 2H), 1.73-1.61 (m, 6H), 1.41-1.24 (m, 2H), 1.04 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.52H.sub.66N.sub.11O.sub.7S, 988.4862; found: 988.4871.
[0265] The Example 14, 16, and 17 compounds were synthesized using
the procedures for the preparation of the Example 13 compound
(above).
##STR00218##
[0266]
(2S,4R)-1-((S)-2-(7-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-7-
-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-y-
l)benzyl)pyrrolidine-2-carboxamide (compound Example 14) (20 mg,
68%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.09 (s, 1H), 8.93 (s, 1H), 8.20 (dd, J=9.6, 2.9 Hz, 1H), 7.86 (d,
J=2.9 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.46 (d, J=8.1 Hz, 2H), 7.40
(d, J=8.2 Hz, 2H), 6.01 (p, J=8.8 Hz, 1H), 4.63 (s, 1H), 4.58-4.48
(m, 3H), 4.36 (d, J=15.5 Hz, 1H), 3.90 (d, J=11.1 Hz, 1H),
3.83-3.72 (m, 5H), 3.32 (t, J=5.0 Hz, 2H), 3.28 (t, J=5.0 Hz, 2H),
2.50 (s, 3H), 2.47 (s, 3H), 2.48-2.44 (m, 2H), 2.43 (s, 3H),
2.35-2.24 (m, 4H), 2.24-2.19 (m, 1H), 2.13-2.05 (m, 3H), 1.94-1.86
(m, 2H), 1.74-1.60 (m, 6H), 1.40 (dt, J=8.7, 7.4 Hz, 2H), 1.03 (s,
9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.53H.sub.68N.sub.11O.sub.7S, 1002.5018; found: 1002.5019.
##STR00219##
[0267]
(2S,4R)-1-((S)-2-(9-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-9-
-oxononanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl-
)benzyl)pyrrolidine-2-carboxamide (compound Example 16) (18 mg,
63%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.10 (s, 1H), 9.00 (s, 1H), 8.20 (dd, J=9.6, 2.8 Hz, 1H), 7.86 (d,
J=2.8 Hz, 1H), 7.54 (d, J=9.6 Hz, 1H), 7.46 (d, J=8.1 Hz, 2H), 7.41
(d, J=8.1 Hz, 2H), 6.00 (p, J=8.8 Hz, 1H), 4.63 (s, 1H), 4.60-4.47
(m, 3H), 4.36 (d, J=15.5 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H),
3.82-3.72 (m, 5H), 3.34-3.23 (m, 4H), 2.50 (s, 3H), 2.48 (s, 3H),
2.48-2.44 (m, 2H), 2.43 (s, 3H), 2.34-2.18 (m, 5H), 2.13-2.04 (m,
3H), 1.94-1.85 (m, 2H), 1.73-1.57 (m, 6H), 1.41-1.30 (m, 6H), 1.03
(s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.55H.sub.72N.sub.11O.sub.7S, 1030.5331; found: 1030.5335.
##STR00220##
[0268]
(2S,4R)-1-((S)-2-(10-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo--
7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)--
10-oxodecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5--
yl)benzyl)pyrrolidine-2-carboxamide (compound Example 17) (18 mg,
64%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.09 (s, 1H), 8.93 (s, 1H), 8.19 (dd, J=9.6, 2.9 Hz, 1H), 7.87 (d,
J=2.9 Hz, 1H), 7.54 (d, J=9.6 Hz, 1H), 7.46 (d, J=8.1 Hz, 2H), 7.41
(d, J=8.2 Hz, 2H), 6.00 (p, J=8.8 Hz, 1H), 4.63 (s, 1H), 4.60-4.47
(m, 3H), 4.35 (d, J=15.5 Hz, 1H), 3.90 (d, J=11.0 Hz, 1H),
3.83-3.70 (m, 5H), 3.36-3.23 (m, 4H), 2.50 (s, 3H), 2.47 (s, 3H),
2.48-2.44 (m, 2H), 2.43 (s, 3H), 2.34-2.19 (m, 5H), 2.12-2.05 (m,
3H), 1.93-1.86 (m, 2H), 1.73-1.66 (m, 2H), 1.65-1.56 (m, 4H),
1.40-1.28 (m, 8H), 1.03 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.56H.sub.74N.sub.11O.sub.7S, 1044.5488; found:
1044.5487.
##STR00221##
[0269] tert-Butyl
3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)e-
thoxy) ethoxy)ethoxy)propanoate (Intermediate 6). A solution of
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (100 mg,
0.362 mmol), tert-butyl 3-(2-(2-(2-aminoethoxy)ethoxy)
ethoxy)propanoate (100 mg, 0.362 mmol) and DIPEA (0.5 ml) in DMF (5
ml) was heated at 100.degree. C. overnight. After the starting
materials were consumed, the reaction was concentrated under
reduced pressure. The resulting residue was purified by prep-HPLC
to yield the product (72 mg, 37%) as yellow solid. .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 7.55 (dd, J=7.1, 8.6 Hz, 1H), 7.10 (d,
J=8.5 Hz, 1H), 7.05 (d, J=7.0 Hz, 1H), 5.06 (dd, J=5.4, 12.4 Hz,
1H), 4.10 (q, J=7.1 Hz, 1H), 3.72 (t, J=5.3 Hz, 2H), 3.69-3.59 (m,
8H), 3.59-3.55 (m, 2H), 3.50 (t, J=5.3 Hz, 2H), 2.91-2.82 (m, 1H),
2.78-2.68 (m, 2H), 2.45 (t, J=6.2 Hz, 2H), 1.43 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.26H.sub.36N.sub.3O.sub.9, 534.2446; found: 534.2459.
[0270]
4-((2-(2-(2-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-di-
hydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxop-
ropoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1-
,3-dione (compound Example 21). A solution of intermediate 6 (20
mg, 0.0308 mmol) in HCO.sub.2H (5 mL) was stirred overnight at RT.
The reaction was concentrated under reduced pressure and the
resulting residue was dissolved in DCM/DMSO (10 ml/2 ml). To the
resulting solution were added palbociclib (13.7 mg, 0.0308 mmol),
NMM (16 mg, 0.154 mmol), HOAt (5.4 mg, 0.0399 mmol), and EDCI (7.6
mg, 0.0399 mmol). The reaction mixture was allowed to stir at RT
overnight. After palbociclib was consumed, the reaction was
concentrated under reduced pressure. The resulting residue was
purified by prep-HPLC to yield the product (25 mg, 90%) as yellow
solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 8.98 (s, 1H), 8.13
(d, J=9.7 Hz, 1H), 7.72 (d, J=2.9 Hz, 1H), 7.44-7.35 (m, 2H), 6.94
(d, J=8.6 Hz, 1H), 6.77 (d, J=7.1 Hz, 1H), 6.03-5.93 (m, 1H), 5.09
(dd, J=5.5, 12.7 Hz, 1H), 3.87-3.75 (m, 8H), 3.67 (t, J=5.2 Hz,
2H), 3.65-3.58 (m, 9H), 3.41-3.37 (m, 2H), 2.92-2.84 (m, 1H),
2.79-2.63 (m, 4H), 2.52 (s, 3H), 2.42 (s, 3H), 2.36-2.28 (m, 2H),
2.13-2.07 (m, 4H), 1.97-1.88 (m, 2H), 1.75-1.67 (m, 2H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.46H.sub.55N.sub.10O.sub.10, 907.4097; found: 907.4095.
[0271] The Example 19, 20, 22, 23, 24, 25, 26, 27, 28, and 29
compounds were synthesized according to the procedures for the
preparation of the Example 21 compound (above).
##STR00222##
[0272]
4-((2-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydrop-
yrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropoxy-
)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(compound Example 19) (28 mg, 88%) as yellow solid. .sup.1H NMR
(600 MHz, CD.sub.3OD) .delta. 9.08 (s, 1H), 7.99 (dd, J=9.6, 2.8
Hz, 1H), 7.64 (d, J=2.8 Hz, 1H), 7.44 (dd, J=8.3, 7.2 Hz, 1H), 7.30
(d, J=9.6 Hz, 1H), 6.96 (d, J=8.6 Hz, 1H), 6.80 (d, J=7.0 Hz, 1H),
6.02 (p, J=8.9 Hz, 1H), 5.07 (dd, J=12.8, 5.6 Hz, 1H), 3.99-3.79
(m, 4H), 3.79-3.61 (m, 4H), 3.48-3.36 (m, 2H), 3.30-3.24 (m, 1H),
3.23-3.13 (m, 2H), 3.13-3.05 (m, 1H), 2.90-2.77 (m, 2H), 2.76-2.70
(m, 1H), 2.70-2.60 (m, 2H), 2.51 (s, 3H), 2.45 (s, 3H), 2.37-2.27
(m, 2H), 2.15-2.04 (m, 3H), 1.96-1.87 (m, 2H), 1.75-1.66 (m, 2H).
HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.42H.sub.47N.sub.10O.sub.8, 819.3573; found: 819.3573.
##STR00223##
[0273]
4-((2-(2-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihyd-
ropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxoprop-
oxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
(compound Example 20) (22 mg, 73%) as yellow solid. .sup.1H NMR
(600 MHz, CD.sub.3OD) .delta. 8.99 (s, 1H), 8.08 (dd, J=9.6, 2.8
Hz, 1H), 7.68 (d, J=2.6 Hz, 1H), 7.40 (dd, J=8.4, 7.2 Hz, 1H), 7.37
(d, J=9.6 Hz, 1H), 6.94 (d, J=8.6 Hz, 1H), 6.72 (d, J=7.1 Hz, 1H),
5.99 (p, J=8.9 Hz, 1H), 5.08 (dd, J=12.8, 5.6 Hz, 1H), 3.86-3.70
(m, 6H), 3.69-3.61 (m, 6H), 3.41-3.31 (m, 4H), 3.26-3.17 (m, 2H),
2.92-2.82 (m, 1H), 2.78-2.63 (m, 4H), 2.51 (s, 3H), 2.43 (s, 3H),
2.36-2.28 (m, 2H), 2.13-2.07 (m, 3H), 1.96-1.87 (m, 2H), 1.74-1.66
(m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.44H.sub.51N.sub.10O.sub.9, 863.3835; found: 863.3842.
##STR00224##
[0274]
4-((15-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyr-
ido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-15-oxo-3,6,9,1-
2-tetraoxapentadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dio-
ne (compound Example 22) (34 mg, 85%) as yellow solid. .sup.1H NMR
(600 MHz, CD.sub.3OD) .delta. 8.97 (s, 1H), 8.17 (d, J=9.6 Hz, 1H),
7.75 (s, 1H), 7.44 (d, J=9.6 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 6.92
(d, J=8.5 Hz, 1H), 6.80 (d, J=7.0 Hz, 1H), 5.96 (p, J=8.7 Hz, 1H),
5.07 (dd, J=12.8, 5.4 Hz, 1H), 3.87-3.73 (m, 6H), 3.70-3.51 (m,
14H), 3.42-3.32 (m, 4H), 3.30-3.22 (m, 2H), 2.91-2.83 (m, 1H),
2.78-2.59 (m, 4H), 2.50 (s, 3H), 2.39 (s, 3H), 2.34-2.24 (m, 2H),
2.15-2.03 (m, 3H), 1.95-1.86 (m, 2H), 1.74-1.64 (m, 2H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.48H.sub.59N.sub.10O.sub.11, 951.4359; found: 951.4353.
##STR00225##
[0275]
4-((18-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyr-
ido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-18-oxo-3,6,9,1-
2,15-pentaoxaoctadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-d-
ione (compound Example 23) (18 mg, 51%) as yellow solid. .sup.1H
NMR (600 MHz, CD.sub.3OD) .delta. 9.03-8.97 (m, 1H), 8.20 (dd,
J=9.6, 2.9 Hz, 1H), 7.78 (d, J=2.9 Hz, 1H), 7.45 (d, J=9.7 Hz, 1H),
7.43 (dd, J=8.5, 7.2 Hz, 1H), 6.97 (d, J=8.6 Hz, 1H), 6.85 (d,
J=7.0 Hz, 1H), 5.97 (p, J=8.8 Hz, 1H), 5.06 (dd, J=12.8, 5.5 Hz,
1H), 3.84-3.78 (m, 4H), 3.77 (t, J=6.0 Hz, 2H), 3.68 (t, J=5.1 Hz,
2H), 3.66-3.53 (m, 16H), 3.44-3.38 (m, 2H), 3.37-3.32 (m, 2H),
3.29-3.22 (m, 2H), 2.90-2.82 (m, 1H), 2.77-2.62 (m, 4H), 2.51 (s,
3H), 2.41 (s, 3H), 2.34-2.26 (m, 2H), 2.13-2.05 (m, 3H), 1.95-1.87
(m, 2H), 1.74-1.65 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.50H.sub.63N.sub.10O.sub.12, 995.4621; found:
995.4628.
##STR00226##
[0276]
4-((2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoethyl)amin-
o)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound
Example 24) (28 mg, 72%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.08 (s, 1H), 8.21 (dd, J=9.6, 2.8 Hz, 1H),
7.86 (d, J=2.8 Hz, 1H), 7.55-7.50 (m, 2H), 7.03 (d, J=7.0 Hz, 1H),
6.99 (d, J=8.5 Hz, 1H), 6.04-5.97 (m, 1H), 5.08 (dd, J=12.7, 5.5
Hz, 1H), 4.24 (s, 2H), 3.88-3.73 (m, 4H), 3.42-3.31 (m, 4H),
2.92-2.82 (m, 1H), 2.80-2.67 (m, 2H), 2.50 (s, 3H), 2.43 (s, 3H),
2.35-2.27 (m, 2H), 2.15-2.06 (m, 3H), 1.94-1.87 (m, 2H), 1.73-1.66
(m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.39H.sub.41N.sub.10O.sub.7, 761.3154; found: 761.3150.
##STR00227##
[0277]
4-((3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropyl)ami-
no)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound
Example 25) (26 mg, 67%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.07 (s, 1H), 8.13 (dd, J=9.6, 2.9 Hz, 1H),
7.77 (d, J=2.9 Hz, 1H), 7.52 (dd, J=8.5, 7.1 Hz, 1H), 7.49 (d,
J=9.6 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 6.95 (d, J=7.0 Hz, 1H), 6.00
(p, J=8.9 Hz, 1H), 5.03 (dd, J=12.7, 5.5 Hz, 1H), 3.76 (dt, J=10.3,
4.7 Hz, 4H), 3.68 (t, J=6.1 Hz, 2H), 3.18 (br. s, 4H), 2.88-2.80
(m, 1H), 2.79 (td, J=5.9, 1.9 Hz, 2H), 2.75-2.62 (m, 2H), 2.50 (s,
3H), 2.42 (s, 3H), 2.34-2.26 (m, 2H), 2.13-2.03 (m, 3H), 1.94-1.86
(m, 2H), 1.73-1.64 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.40H.sub.43N.sub.10O.sub.7, 775.3311; found:
775.3316.
##STR00228##
[0278]
4-((4-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-4-oxobutyl)amin-
o)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound
Example 26) (17 mg, 45%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.09 (s, 1H), 8.13 (dd, J=9.6, 2.9 Hz, 1H),
7.79 (d, J=2.8 Hz, 1H), 7.54-7.45 (m, 2H), 7.08 (d, J=8.6 Hz, 1H),
6.92 (d, J=7.0 Hz, 1H), 6.00 (p, J=8.8 Hz, 1H), 5.05 (dd, J=12.7,
5.5 Hz, 1H), 3.80-3.70 (m, 4H), 3.41 (td, J=6.5, 1.3 Hz, 2H),
3.25-3.16 (m, 4H), 2.90-2.82 (m, 1H), 2.77-2.65 (m, 2H), 2.58 (t,
J=6.9 Hz, 2H), 2.50 (s, 3H), 2.43 (s, 3H), 2.35-2.27 (m, 2H),
2.13-2.06 (m, 3H), 2.00 (p, J=6.7 Hz, 2H), 1.95-1.86 (m, 2H), 1.70
(dt, J=10.1, 8.6 Hz, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.41H.sub.45N.sub.10O.sub.7, 789.3467; found:
789.3474.
##STR00229##
[0279]
4-((6-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-6-oxohexyl)amin-
o)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound
Example 27) (24 mg, 66%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.09 (s, 1H), 8.13 (dd, J=9.6, 2.9 Hz, 1H),
7.78 (d, J=2.8 Hz, 1H), 7.52-7.46 (m, 2H), 7.02 (d, J=8.6 Hz, 1H),
6.90 (d, J=7.0 Hz, 1H), 6.01 (p, J=8.8 Hz, 1H), 5.04 (dd, J=12.7,
5.5 Hz, 1H), 3.83-3.69 (m, 4H), 3.36-3.29 (m, 2H), 3.27-3.16 (m,
4H), 2.85 (ddd, J=17.5, 13.9, 5.4 Hz, 1H), 2.76-2.62 (m, 2H),
2.54-2.45 (m, 5H), 2.43 (s, 3H), 2.36-2.27 (m, 2H), 2.14-2.04 (m,
3H), 1.95-1.86 (m, 2H), 1.75-1.63 (m, 6H), 1.54-1.44 (m, 2H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.43H.sub.49N.sub.10O.sub.7, 817.3780; found: 817.3773.
##STR00230##
[0280]
4-((7-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-7-oxoheptyl)ami-
no)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound
Example 28) (26 mg, 74%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.07 (s, 1H), 8.16 (dd, J=9.7, 2.9 Hz, 1H),
7.80 (d, J=2.8 Hz, 1H), 7.55-7.45 (m, 2H), 7.00 (d, J=8.6 Hz, 1H),
6.93 (d, J=7.1 Hz, 1H), 6.00 (p, J=8.9 Hz, 1H), 5.04 (dd, J=12.8,
5.5 Hz, 1H), 3.82-3.69 (m, 4H), 3.32-3.20 (m, 6H), 2.84 (ddd,
J=17.8, 14.1, 5.3 Hz, 1H), 2.77-2.63 (m, 2H), 2.50 (s, 3H), 2.46
(t, J=7.4 Hz, 2H), 2.42 (s, 3H), 2.35-2.25 (m, 2H), 2.13-2.05 (m,
3H), 1.95-1.85 (m, 2H), 1.74-1.60 (m, 6H), 1.50-1.39 (m, 4H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.44H.sub.51N.sub.10O.sub.7, 831.3937; found: 831.3929.
##STR00231##
[0281]
4-((8-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-8-oxooctyl)amin-
o)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound
Example 29) (23 mg, 66%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.07 (s, 1H), 8.16 (dd, J=9.6, 2.9 Hz, 1H),
7.82 (d, J=2.8 Hz, 1H), 7.54-7.48 (m, 2H), 7.01 (d, J=8.6 Hz, 1H),
6.95 (d, J=7.1 Hz, 1H), 6.01 (p, J=8.8 Hz, 1H), 5.05 (dd, J=12.8,
5.5 Hz, 1H), 3.82-3.71 (m, 4H), 3.33-3.22 (m, 6H), 2.85 (ddd,
J=17.7, 14.1, 5.3 Hz, 1H), 2.76-2.64 (m, 2H), 2.50 (s, 3H), 2.46
(t, J=7.4 Hz, 2H), 2.43 (s, 3H), 2.32 (td, J=15.4, 7.8 Hz, 2H),
2.14-2.04 (m, 3H), 1.95-1.86 (m, 2H), 1.74-1.60 (m, 6H), 1.50-1.36
(m, 6H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.45H.sub.53N.sub.10O.sub.7, 845.4093; found: 845.4101.
##STR00232##
[0282]
2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-(2-(3-(4-((6-((5-fluoro-4-(4-f-
luoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)p-
yridin-3-yl)methyl)piperazin-1-yl)-3-oxopropoxy)ethoxy)ethoxy)ethyl)amino)-
isoindoline-1,3-dione (compound Example 30). To a solution of
linker 20 (32 mg, 0.07 mmol) and abemaiclib (24 mg, 0.05 mmol) in
CH.sub.2Cl.sub.2 (4 ml) and DMF (1 ml) were added DIEA (17 .mu.l,
0.1 mmol) and TBTU (22 mg, 0.07 mmol). The reaction was stirred at
RT for 1 h before being concentrated under reduced pressure. The
resulting residue was purified by prep-HPLC to provide the title
compound (37 mg, 80% over 2 steps). .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 8.52 (d, J=3.6 Hz, 1H), 8.32 (d, J=8.4 Hz, 1H),
8.28 (s, 1H), 8.24 (s, 1H), 7.80-7.78 (m, 2H), 7.49 (t, J=7.8 Hz,
1H), 7.02 (dd, J=6.6 Hz, J=3.0 Hz, 1H), 6.98 (d, J=8.4 Hz, 1H),
5.00 (dd, J=12.6 Hz, J=6.0 Hz, 1H), 3.75 (t, J=6.0 Hz, 2H), 3.71
(t, J=5.4 Hz, 2H), 3.66-3.60 (m, 14H), 3.46 (t, J=5.4 Hz, 2H),
2.88-2.73 (m, 4H), 2.70 (s, 3H), 2.64 (t, J=6.0 Hz, 2H), 2.65-2.50
(m, 4H), 2.12-2.10 (m, 1H), 1.74 (d, J=6.6 Hz, 6H). HRMS (ESI-TOF)
m/z: [M+H].sup.+ calculated for
C.sub.47H.sub.54F.sub.2N.sub.11O.sub.8, 938.4119; found:
938.4148.
##STR00233##
[0283]
N-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-8-(4-((6-(-
(5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimi-
din-2-yl)amino)pyridin-3-yl)methyl)piperazin-1-yl)-8-oxooctanamide
(compound Example 31). Methyl 8-chloro-8-oxooctanoate (30 mg, 0.15
mmol) and pomalidomide (40 mg, 0.15 mmol) were dissolved in THF (5
ml). The resulting solution was heated at reflux for 3 h before the
solvent was evaporated under reduced pressure. The residue was
purified by prep-HPLC to provide intermediate 7 (53 mg, 0.12 mmol)
as yellow oil. A solution of intermediate 7 (53 mg, 0.12 mmol) in
DMF (1 ml) was treated with LiCl (25 mg, 0.6 mmol) at 160.degree.
C. in microwave reactor for 2 h. After being cooled to RT, the
reaction mixture was poured into water (5 mL) and extracted with
DCM (3.times.5 ml). The organic phase was combined, dried over
Na.sub.2SO.sub.4, and concentrated. The resulting residue was
purified by prep-HPLC to provide intermediate 8 (21 mg, 40%) as
yellow oil. To a solution of intermediate 8 (21 mg, 0.05 mmol) and
abemaiclib (19 mg, 0.04 mmol) in CH.sub.2Cl.sub.2 (4 ml) and DMF (1
ml) were added DIEA (17 .mu.l, 0.1 mmol) and TBTU (15 mg, 0.05
mmol). The reaction was stirred at RT for 1 h before being
concentrated under reduced pressure. The resulting residue was
purified by prep-HPLC to provide the title compound (25 mg, 70%).
.sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 9.47 (s, 1H), 8.95 (s,
1H), 8.86 (d, J=8.4 Hz, 1H), 8.50-8.49 (m, 2H), 8.28 (s, 1H), 8.23
(s, 1H), 7.84 (d, J=12.0 Hz, 1H), 7.75-7.73 (m, 2H), 7.58 (d, J=7.2
Hz, 1H), 5.02-4.99 (m, 1H), 4.79-4.74 (m, 1H), 3.86-3.85 (m, 1H),
3.61 (d, J=12.6 Hz, 1H), 3.52-3.33 (m, 3H), 3.41-3.37 (m, 1H),
3.03-3.01 (m, 1H), 2.89-2.81 (m, 2H), 2.72 (s, 3H), 2.63-2.56 (m,
1H), 2.53-2.37 (m, 4H), 2.36-2.28 (m, 3H), 2.25-2.18 (m, 1H),
1.86-1.78 (m, 2H), 1.74 (d. J=6.6 Hz, 6H), 1.70-1.60 (m, 2H),
1.46-1.35 (5H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.46H.sub.50F.sub.2N.sub.11O.sub.6, 890.3908; found:
890.3931.
##STR00234##
[0284]
(2S,4R)-1-((S)-2-(8-(4-((6-((5-Fluoro-4-(4-fluoro-1-isopropyl-2-met-
hyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)pipe-
razin-1-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methy-
lthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound Example
32). To a solution of linker 5 (29 mg, 0.05 mmol) and abemaiclib
(19 mg, 0.04 mmol) in CH.sub.2Cl.sub.2 (4 ml) and DMF (1 ml) were
added DIEA (17 .mu.l, 0.1 mmol) and TBTU (15 mg, 0.05 mmol). The
reaction was stirred at RT for 1 h before being concentrated under
reduced pressure. The resulting residue was purified by prep-HPLC
to provide the Example 32 compound (31 mg, 76% over 2 steps).
.sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 8.88 (s, 1H), 8.55 (d,
J=3.6 Hz, 1H), 8.36-8.35 (m, 2H), 8.25 (s, 1H), 7.86 (d, J=9.0 Hz,
1H), 7.81-7.78 (m, 2H), 7.48-7.41 (m, 3H), 4.66-4.51 (m, 4H), 4.36
(d, J=8.4 Hz, 1H), 3.91 (d, J=10.8 Hz, 1H), 3.81 (dd, J=10.8 Hz,
3.6 Hz, 1H), 3.66-3.54 (m, 7H), 2.71 (s, 3H), 2.51-2.48 (m, 6H),
2.40 (t, J=7.8 Hz, 2H), 2.34-2.21 (m, 3H), 2.12-2.06 (m, 1H), 1.73
(d, J=6.6 Hz, 6H), 1.63-1.60 (m, 4H), 1.37-1.27 (m, 5H), 1.05 (s,
9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.55H.sub.69F.sub.2N.sub.12O.sub.5S, 1047.5197; found:
1047.5192.
##STR00235##
[0285]
7-Cyclopentyl-2-((5-(4-(6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methy-
lthiazol-5-yl)benzyl)
carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexan-
oyl)piperazin-1-yl)pyridin-2-yl)amino)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrim-
idine-6-carboxamide (compound Example 33). To a solution of
ribociclib (9 mg, 0.021 mmol) and linker 3 (14 mg, 0.025 mmol) in
DCM/DMF (1:1, 2 ml) were added triethylamine (17 .mu.l, 0.12 mmol)
and TBTU (8 mg, 0.025 mmol). The reaction was stirred at RT for 1 h
before being concentrated under reduced pressure. The resulting
residue was dissolved in MeOH, and purified by prep-HPLC to yield
the product (19 mg, 84%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) of major isomer (rotamer ratio 6:1) .delta. 9.02 (s,
1H), 8.97 (s, 1H), 8.08 (dd, J=9.6, 2.4 Hz, 1H), 7.87 (d, J=2.4 Hz,
1H), 7.48 (d, J=7.8 Hz, 2H), 7.45 (d, J=10.2 Hz, 1H), 7.43 (d,
J=8.4 Hz, 1H), 6.82 (s, 1H), 4.86-4.80 (m, 1H), 4.65 (s, 1H),
4.62-4.52 (m, 3H), 4.38 (d, J=16.8 Hz, 1H), 3.93 (d, J=10.8 Hz,
1H), 3.84-3.76 (m, 5H), 3.29 (t, J=4.8 Hz, 2H), 3.24 (t, J=4.8 Hz,
2H), 3.19 (s, 3H), 3.17 (s, 3H), 2.52-2.47 (m, 7H), 2.39-2.30 (m,
3H), 2.14-2.08 (m, 5H), 1.77-1.64 (m, 6H), 1.06 (s, 9H); HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.51H.sub.67N.sub.12O.sub.6S, 975.5022, found 975.5024.
[0286] The Example 34, 35, and 36 compounds were synthesized
according to the procedures for the preparation of the Example 33
compound (above).
##STR00236##
[0287]
7-Cyclopentyl-2-((5-(4-(7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methy-
lthiazol-5-yl)benzyl)
carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxohepta-
noyl)
piperazin-1-yl)pyridin-2-yl)amino)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyr-
imidine-6-carboxamide (compound Example 34) (17 mg, 83%) as yellow
solid. .sup.1H NMR (600 MHz, CD.sub.3OD) of major isomer (rotamer
ratio 7:2) .delta. 9.03 (s, 1H), 8.97 (s, 1H), 8.08 (dd, J=9.6, 2.4
Hz, 1H), 7.88 (d, J=2.4 Hz, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.46-7.43
(m, 3H), 6.82 (s, 1H), 4.86-4.80 (m, 1H), 4.66 (s, 1H), 4.61-4.51
(m, 3H), 4.39 (d, J=14.4 Hz, 1H), 3.93 (d, J=10.8 Hz, 1H),
3.84-3.77 (m, 5H), 3.29 (t, J=4.8 Hz, 2H), 3.24 (t, J=4.8 Hz, 2H),
3.19 (s, 3H), 3.17 (s, 3H), 2.51-2.47 (m, 7H), 2.37-2.23 (m, 3H),
2.16-2.08 (m, 5H), 1.79-1.73 (m, 2H), 1.70-1.63 (m, 4H), 1.45-1.39
(m, 2H), 1.06 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated
for C.sub.52H.sub.69N.sub.12O.sub.6S, 989.5178, found 989.5180.
##STR00237##
[0288]
7-Cyclopentyl-2-((5-(4-(8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methy-
lthiazol-5-yl)benzyl)
carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctan-
oyl)piperazin-1-yl)pyridin-2-yl)amino)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrim-
idine-6-carboxamide (compound Example 35) (25 mg, 98%) as yellow
solid. .sup.1H NMR (600 MHz, CD.sub.3OD) of major isomer rotamer
ration 8:1) .delta. 9.02 (s, 1H), 8.97 (s, 1H), 8.08 (dd, J=9.6,
2.4 Hz, 1H), 7.87 (d, J=2.4 Hz, 1H), 7.49 (d, J=8.4 Hz, 2H), 7.50
(d, J=11.2 Hz, 1H), 7.43 (d, J=8.4 Hz, 2H), 6.82 (s, 1H), 4.86-4.79
(m, 1H), 4.66 (s, 1H), 4.62-4.52 (m, 3H), 4.38 (d, J=15.6 Hz, 1H),
3.93 (d, J=10.8 Hz, 1H), 3.84-3.77 (m, 5H), 3.29-3.28 (m, 2H),
3.24-3.22 (m, 2H), 3.19 (s, 3H), 3.17 (s, 3H), 2.50 (s, 3H),
2.49-2.47 (m, 4H), 2.36-2.32 (m, 3H), 2.16-2.08 (m, 5H), 1.80-1.72
(m, 2H), 1.69-1.62 (m, 4H), 1.46-1.36 (m, 4H), 1.06 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.53H.sub.71N.sub.12O.sub.6S, 1003.5335, found 1003.5361.
##STR00238##
[0289]
7-Cyclopentyl-2-((5-(4-(3-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-me-
thylthiazol-5-yl)benzyl)
carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropo-
xy)
propanoyl)piperazin-1-yl)pyridin-2-yl)amino)-N,N-dimethyl-7H-pyrrolo[2-
,3-d]pyrimidine-6-carboxamide (compound Example 36) (17 mg, 74%) as
yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) of major isomer
(rotamer ration 7:2) .delta. 9.04 (s, 1H), 8.96 (s, 1H), 8.07 (dd,
J=9.6, 2.4 Hz, 1H), 7.85 (d, J=2.4 Hz, 1H), 7.45 (d, J=9.0 Hz, 2H),
7.43 (d, J=9.6 Hz, 1H), 7.40 (d, J=8.4 Hz, 2H), 6.81 (s, 1H),
4.85-4.79 (m, 1H), 4.70 (s, 1H), 4.58 (t, J=8.4 Hz, 1H), 4.54-4.51
(m, 1H), 4.49 (d, J=15.6 Hz, 1H), 4.38 (d, J=14.4 Hz, 1H), 3.93 (d,
J=10.8 Hz, 1H), 3.84-3.78 (m, 7H), 3.74 (t, J=6.0 Hz, 2H),
3.28-3.22 (m, 4H), 3.19 (s, 3H), 3.17 (s, 3H), 2.81-2.71 (m, 2H),
2.60-2.48 (m, 7H), 2.28-2.25 (m, 1H), 2.16-2.08 (m, 5H), 1.79-1.72
(m, 2H), 1.07 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated
for C.sub.51H.sub.67N.sub.12O.sub.7S, 991.4971, found 991.4975.
##STR00239##
[0290]
(2S,4R)-1-((S)-2-(6-(4-((6-((5-Fluoro-4-(4-fluoro-1-isopropyl-2-met-
hyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)pipe-
razin-1-yl)-6-oxohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methy-
lthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound Example
37). To a solution of abemaciclib (13 mg, 0.028 mmol) and linker 3
(18 mg, 0.032 mmol) in DCM/DMF (1:1, 2 ml) were added triethylamine
(17 .mu.l, 0.12 mmol) and TBTU (10 mg, 0.031 mmol). The reaction
was stirred at RT for 1 h before being concentrated under reduced
pressure. The resulting residue was dissolved in MeOH and purified
by prep-HPLC to yield the desired product (31 mg, 96%) as white
solid. .sup.1HNMR (CD.sub.3OD, 600 MHz) (1H buried in solvent peak)
9.13 (s, 1H), 8.85 (d, J=3.6 Hz, 1H), 8.57 (d, J=11.4 Hz, 1H), 8.36
(d, J=8.4 Hz, 1H), 8.14 (d, J=10.8 Hz, 1H), 7.86 (d, J=8.4 Hz, 1H),
7.50 (d, J=8.4 Hz, 2H), 7.44 (d, J=7.8 Hz, 2H), 5.15-5.10 (m, 1H),
4.65 (s, 1H), 4.62-4.51 (m, 5H), 4.39 (d, J=15.6 Hz, 1H), 3.93-3.82
(m, 6H), 3.41-3.33 (m, 4H), 2.94 (s, 3H), 2.51-2.45 (m, 5H),
2.39-2.29 (m, 2H), 2.26-2.23 (m, 1H), 2.12-2.08 (m, 1H), 1.82 (d,
J=6.0 Hz, 6H), 1.72-1.62 (m, 4H), 1.06 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.53H.sub.65F.sub.2N.sub.12O.sub.5S
[M+H].sup.+, 1019.4884, found 1019.4881. The Example 38 compound
was synthesized according to the procedures for the preparation of
the Example 37 compound (above).
##STR00240##
[0291]
(2S,4R)-1-((S)-2-(7-(4-((6-((5-Fluoro-4-(4-fluoro-1-isopropyl-2-met-
hyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)methyl)pipe-
razin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-meth-
ylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (compound Example
38) (24 mg, 98%) as white solid. .sup.1H NMR (CD.sub.3OD, 600 MHz)
(1H buried in solvent peak) .delta. 9.11 (s, 1H), 8.85 (d, J=3.6
Hz, 1H), 8.56 (d, J=9.6 Hz, 1H), 8.35 (d, J=8.4 Hz, 1H), 8.14 (d,
J=10.8 Hz, 1H), 7.87 (d, J=8.4 Hz, 1H), 7.50 (d, J=7.2 Hz, 2H),
7.44 (d, J=9.0 Hz, 2H), 5.14-5.10 (m, 1H), 4.66 (s, 1H), 4.62-4.51
(m, 5H), 4.39 (d, J=15.6 Hz, 1H), 3.94-3.82 (m, 6H), 3.46-3.30 (m,
4H), 2.95 (s, 3H), 2.51 (s, 3H), 2.47 (t, J=7.8 Hz, 2H), 2.36-2.23
(m, 3H), 2.13-2.08 (m, 1H), 1.82 (d, J=6.0 Hz, 6H), 1.69-1.62 (m,
4H), 1.41-1.39 (m, 2H), 1.06 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.54H.sub.67F.sub.2N.sub.12O.sub.5S,
1033.5041, found 1033.5053.
##STR00241##
[0292]
7-Cyclopentyl-2-((5-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoin-
dolin-4-yl)glycyl)
piperazin-1-yl)pyridin-2-yl)amino)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidi-
ne-6-carboxamide (compound Example 39). To a solution of ribociclib
(12 mg, 0.027 mmol) and linker 23 (11 mg, 0.034 mmol) in DCM/DMF
(1:1, 2 ml) were added triethylamine (17 .mu.l, 0.12 mmol) and TBTU
(12 mg, 0.038 mmol). The reaction solution was stirred at RT for 1
h before being concentrated under reduced pressure. The resulting
residue was dissolved in MeOH and purified by prep-HPLC to yield
the desired product (15 mg, 74%) as yellow solid. .sup.1H NMR
(CD.sub.3OD, 600 MHz) .delta. 8.86 (s, 1H), 7.89 (dd, J=8.4, 2.4
Hz, 1H), 7.82 (d, J=2.4 Hz, 1H), 7.52-7.49 (m, 2H), 7.08 (d, J=7.2
Hz, 1H), 6.90 (d, J=8.4 Hz, 1H), 6.65 (s, 1H), 4.96-4.93 (m, 1H),
4.78-4.72 (m, 1H), 4.17 (s, 2H), 3.85-3.83 (m, 2H), 3.72-3.72 (m,
2H), 3.30-3.28 (m, 2H), 3.23-3.21 (m, 2H), 3.18 (s, 3H), 3.15 (s,
3H), 2.84-2.73 (m, 3H), 2.46-2.40 (m, 2H), 2.15-2.02 (m, 5H),
1.75-1.67 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.38H.sub.41N.sub.11O.sub.6, 748.3314, found 748.3391.
##STR00242##
[0293]
2-(2,6-Dioxopiperidin-3-yl)-4-((2-(4-((6-((5-fluoro-4-(4-fluoro-1-i-
sopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3--
yl)methyl)piperazin-1-yl)-2-oxoethyl)amino)isoindoline-1,3-dione
(compound Example 40). To a solution of abemaciclib (12 mg, 0.025
mmol) and compound 23 (10 mg, 0.032 mmol) in DCM/DMF (1:1, 2 ml)
were added triethylamine (17 .mu.l, 0.12 mmol) and TBTU (11 mg,
0.034 mmol). The reaction was stirred at RT for 1 h before being
concentrated under reduced pressure. The resulting residue was
dissolved in MeOH and purified by prep-HPLC to yield the desired
product (16 mg, 81%) as yellow solid. .sup.1H NMR (CD.sub.3OD, 600
MHz) .delta. 8.59 (d, J=2.4 Hz, 1H), 8.44 (s, 1H), 8.30 (s, 1H),
8.13 (s, 2H), 7.89 (d, J=10.8 Hz, 1H), 7.36 (t, J=7.2 Hz, 1H), 6.98
(d, J=7.2 Hz, 1H), 6.75 (d, J=8.4 Hz, 1H), 4.95-4.92 (m, 1H),
4.89-4.84 (m, 1H), 4.29 (s, 2H), 4.04 (s, 2H), 3.98-3.74 (m, 4H),
3.31-3.20 (m, 4H), 3.18 (s, 3H), 2.85-2.69 (m, 6H), 2.13-2.11 (m,
1H), 1.73 (d, J=7.8 Hz, 6H); HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.38H.sub.41N.sub.11O.sub.6, 792.3176, found
792.3180.
##STR00243##
[0294] Methyl
7-(4-(6-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]p-
yrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)heptanoate
(Intermediate 9). A solution of palbociclib (50 mg, 0.112 mmol),
methyl 7-bromoheptanoate (75 mg, 0.335 mmol) and K.sub.2CO.sub.3
(46 mg, 0.335 mmol) in DMF (5 ml) was heated at 60.degree. C. in a
microwave reactor for 1 h. After being cooled to RT, the reaction
mixture was filtered and concentrated, and the resulting residue
was purified by prep-HPLC to yield the title compound (40 mg, 60%)
as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.10 (s,
1H), 8.21 (dd, J=9.6, 2.7 Hz, 1H), 7.98 (d, J=2.5 Hz, 1H), 7.59 (d,
J=9.6 Hz, 1H), 5.99 (p, J=8.8 Hz, 1H), 3.93 (s, 2H), 3.75 (s, 2H),
3.65 (s, 3H), 3.38-3.16 (m, 6H), 2.49 (s, 3H), 2.42 (s, 3H),
2.37-2.25 (m, 4H), 2.13-2.03 (m, 2H), 1.94-1.86 (m, 2H), 1.85-1.78
(m, 2H), 1.72-1.59 (m, 4H), 1.47-1.36 (m, 4H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.32H.sub.44N.sub.7O.sub.4,
590.3449; found: 590.3446.
[0295]
(2S,4R)-1-((S)-2-(7-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)he-
ptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benz-
yl)pyrrolidine-2-carboxamide (compound Example 41) To a stirring
solution of intermediate 9 (40 mg, 0.0678 mmol) in THF/H.sub.2O (10
ml/2 ml) was added anhydrous LiOH (3.3 mg, 0.136 mmol). After the
resulting mixture was stirred overnight at RT, the reaction mixture
was concentrated under reduced pressure and the resulting residue
was dissolved in DCM/DMSO (5 ml/1 ml). To the resulting solution
were added NMM (68 mg, 0.678 mmol), VHL-1 (32 mg, 0.0678 mmol),
HOAt (14 mg, 0.102 mmol), and EDCI (19.5 mg, 0.102 mmol). After the
mixture was stirred at RT overnight, the reaction was concentrated.
The resulting residue was purified by prep-HPLC to yield the title
compound (8 mg, 11%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.10 (s, 1H), 8.98 (s, 1H), 8.20 (dd, J=9.5,
2.6 Hz, 1H), 7.97 (d, J=2.4 Hz, 1H), 7.58 (d, J=9.5 Hz, 1H), 7.47
(d, J=8.2 Hz, 2H), 7.42 (d, J=8.0 Hz, 2H), 6.05-5.96 (m, 1H), 4.64
(s, 1H), 4.58-4.47 (m, 3H), 4.37 (d, J=15.5 Hz, 1H), 3.99-3.84 (m,
3H), 3.84-3.79 (m, 1H), 3.80-3.66 (m, 2H), 3.34-3.18 (m, 6H), 2.50
(s, 3H), 2.48 (s, 3H), 2.43 (s, 3H), 2.36-2.26 (m, 4H), 2.23 (dd,
J=13.1, 7.6 Hz, 1H), 2.14-2.05 (m, 3H), 1.94-1.86 (m, 2H),
1.84-1.75 (m, 2H), 1.74-1.61 (m, 4H), 1.48-1.38 (m, 4H), 1.04 (s,
9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.53H.sub.70N.sub.11O.sub.6S, 988.5226; found: 988.5226.
##STR00244##
[0296]
(2S,4R)-1-((S)-2-(8-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)oc-
tanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzy-
l)pyrrolidine-2-carboxamide (example 42) A solution of palbociclib
(45 mg, 0.1 mmol), 8-bromooctanoic acid (64 mg, 0.28 mmol) and TEA
(0.05 mL, 0.36 mmol) in i-PrOH (1 ml) was heated at 130.degree. C.
in a microwave reactor for 15 min. After being cooled to RT, the
reaction mixture was concentrated, and the resulting residue was
purified by prep-HPLC to yield the intermediate 10 (24 mg, 40%) as
yellow solid. To a stirring solution of intermediate 10 (24 mg,
0.04 mmol) in DCM/DMSO (5 ml/1 ml) were added TEA (0.05 mL, 0.36
mmol), VHL-1 (20 mg, 0.043 mmol), and TBTU (12 mg, 0.038 mmol).
After the mixture was stirred at RT overnight, the reaction was
concentrated. The resulting residue was purified by prep-HPLC to
yield the title compound (20 mg, 50%) as yellow solid. .sup.1H NMR
(600 MHz, CD.sub.3OD) .delta. 9.13 (s, 1H), 9.07 (s, 1H), 8.23 (dd,
J=9.6, 3.0 Hz, 1H), 8.00 (d, J=2.8 Hz, 1H), 7.60 (d, J=9.6 Hz, 1H),
7.50 (d, J=8.1 Hz, 2H), 7.45 (d, J=8.2 Hz, 2H), 6.03 (p, J=8.7 Hz,
1H), 4.67 (s, 1H), 4.60-4.53 (m, 3H), 4.40 (d, J=15.5 Hz, 1H),
3.96-3.71 (m, 6H), 3.30-3.20 (m, 6H), 2.52 (s, 3H), 2.51 (s, 3H),
2.45 (s, 3H), 2.37-2.24 (m, 5H), 2.17-2.03 (m, 3H), 1.97-1.87 (m,
2H), 1.84-1.80 (m, 2H), 1.77-1.60 (m, 4H), 1.50-1.33 (m, 6H), 1.05
(s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.54H.sub.72N.sub.11O.sub.6S, 1002.5389; found: 1002.5390.
##STR00245##
[0297]
(2S,4R)-1-((S)-2-(6-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)he-
xanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzy-
l)pyrrolidine-2-carboxamide (example 43) A solution of palbociclib
(40 mg, 0.09 mmol), 6-bromohexanoic acid (55 mg, 0.28 mmol) and TEA
(0.05 mL, 0.36 mmol) in i-PrOH (1 ml) was heated at 130.degree. C.
in a microwave reactor for 15 min. After being cooled to RT, the
reaction mixture was concentrated, and the resulting residue was
purified by prep-HPLC to yield the intermediate 11 (44 mg, 88%) as
yellow solid. To a stirring solution of intermediate 11 (44 mg,
0.065 mmol) in DCM/DMSO (5 ml/1 ml) were added TEA (0.05 mL, 0.36
mmol), VHL-1 (34 mg, 0.073 mmol), and TBTU (22 mg, 0.070 mmol).
After the mixture was stirred at RT overnight, the reaction was
concentrated. The resulting residue was purified by prep-HPLC to
yield the title compound (37 mg, 59%) as yellow solid. .sup.1H NMR
(600 MHz, CD.sub.3OD) .delta. 9.12 (s, 1H), 9.12 (s, 1H), 8.23 (dd,
J=9.8, 2.4 Hz, 1H), 8.00 (d, J=2.7 Hz, 1H), 7.60 (d, J=9.6 Hz, 1H),
7.49 (d, J=7.8 Hz, 2H), 7.44 (d, J=8.1 Hz, 2H), 6.02 (p, J=8.8 Hz,
1H), 4.67 (s, 1H), 4.62-4.49 (m, 3H), 4.40 (d, J=15.5 Hz, 1H),
3.96-3.71 (m, 6H), 3.30-3.20 (m, 6H), 2.52 (s, 3H), 2.51 (s, 3H),
2.45 (s, 3H), 2.40-2.21 (m, 5H), 2.16-2.05 (m, 3H), 1.97-1.88 (m,
2H), 1.88-1.78 (m, 2H), 1.72-1.67 (m 4H), 1.49-1.40 (m, 2H), 1.07
(s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.52H.sub.68N.sub.11O.sub.6S, 974.5069; found: 974.5069.
##STR00246##
[0298]
4-((2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)ethyl)amino)-2-(-
2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (example 44) A
solution of palbociclib (45 mg, 0.1 mmol), tert-butyl
(2-bromoethyl)carbamate (69 mg, 0.31 mmol) and TEA (0.05 mL, 0.36
mmol) in i-PrOH (1 ml) was heated at 130.degree. C. in a microwave
reactor for 15 min. After being cooled to RT, the reaction mixture
was concentrated. The resulting residue was purified by prep-HPLC
to yield the intermediate 12 (24 mg, 40%) as yellow solid. To a
stirring solution of intermediate 12 (24 mg, 0.04 mmol) in DCM (2
mL) was added TFA (1 mL). The mixture was stirred at RT and the
reaction progress was monitored by LC/MS. After completion of the
reaction, the reaction solution was concentrated, and the resulting
residue was dissolved in DMF (1 ml). To this solution were added
TEA (0.05 mL, 0.36 mmol) and pomalidomide analogue (17 mg, 0.06
mmol). The resulting mixture was heated at 90.degree. C. in a
microwave reactor for 45 min. After being cooled to RT, the
reaction mixture was concentrated, and the resulting residue was
purified by prep-HPLC to yield the title compound (4 mg, 13%) as
yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.10 (s,
1H), 8.14 (dd, J=9.5, 2.7 Hz, 1H), 8.01 (d, J=2.8 Hz, 1H),
7.70-7.65 (m, 2H), 7.22 (d, J=7.8 Hz, 1H), 7.21 (d, J=6.6 Hz, 1H),
6.02 (p, J=8.9 Hz, 1H), 5.11 (dd, J=12.5, 5.5 Hz, 1H), 3.90 (t,
J=6.2 Hz, 2H), 3.63 (br, 8H), 3.55 (t, J=6.1 Hz, 2H), 2.94-2.86 (m,
1H), 2.80-2.65 (m, 2H), 2.52 (s, 3H), 2.43 (s, 3H), 2.38-2.26 (m,
42H), 2.17-2.05 (m, 3H), 1.99-1.84 (m, 2H), 1.77-1.64 (m, 2H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.39H.sub.43N.sub.10O.sub.6, 747.3362; found: 747.3342.
##STR00247##
[0299]
4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3--
d]pyrimidin-2-yl)amino)pyridin-3-yl)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-di-
oxoisoindolin-4-yl)piperazine-1-carboxamide (example 45) A solution
of pomalidomide (278 mg, 1 mmol), 4-nitrophenyl carbonochloridate
(307 mg, 1.52 mmol) in THF (5 ml) was heated at reflux overnight.
After being cooled to RT, the reaction mixture was concentrated.
The resulting residue was washed with ethyl acetate, dried over
NaSO.sub.4, and concentrated to yield the intermediate 13 (240 mg,
55%) as yellow solid. To a stirring solution of intermediate 13 (19
mg, 0.043 mmol) in DMF (1 mL) were added DIEA (0.014 mL, 0.1 mmol)
and palbociclib (18 mg, 0.041 mmol). The mixture was stirred at RT
overnight, before being concentrated. The resulting residue was
purified by prep-HPLC to yield the title compound (14 mg, 46%) as
yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.07 (s,
1H), 8.56 (d, J=8.5 Hz, 1H), 8.10 (dd, J=9.6, 2.6 Hz, 1H),
7.78-7.70 (m, 1H), 7.64 (d, J=8.8 Hz, 1H), 7.51 (d, J=7.3 Hz, 1H),
5.97 (p, J=8.9 Hz, 1H), 5.07 (dd, J=12.5, 5.5 Hz, 1H), 3.86-3.79
(m, 4H), 3.45-3.40 (m, 4H), 2.89-2.72 (m, 3H), 2.54 (s, 3H), 2.44
(s, 3H), 2.33-2.27 (m, 2H), 2.22-2.15 (m, 1H), 2.11-2.06 (m, 2H),
1.96-1.86 (m, 2H), 1.73-1.59 (m, 2H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.38H.sub.39N.sub.10O.sub.7,
747.2998; found: 747.2970.
##STR00248##
[0300]
3-((3-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl-
)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropy-
l)(methyl)amino)propanoic acid (intermediate 14) To a solution of
3,3'-(methylazanediyl)dipropionic acid (700 mg, 4 mmol) in DMSO/THF
(1:1, 10 ml) were added VHL-1 (472 mg, 1 mmol), triethylamine (0.5
ml, 3.5 mmol), HOAt (173 mg, 1.3 mmol), and EDCI (242 mg, 1.3 mmol)
sequentially at 0.degree. C. The resulting solution was stirred for
2 h at 0.degree. C., before being warmed to room temperature (RT).
After being stirred overnight at RT, the reaction was quenched with
water, and concentrated under reduced pressure. The resulting
residue was purified by reverse-phase chromatography to yield the
title compound (470 mg, 80%) as white solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 8.93 (s, 1H), 7.49 (d, J=8.7 Hz, 2H), 7.44 (d,
J=8.1 Hz, 2H), 4.61-4.54 (m, 4H), 4.37 (d, J=15.4 Hz, 1H), 3.98 (d,
J=11.0 Hz, 1H), 3.82 (dd, J=10.9, 3.8 Hz, 1H), 3.37 (s, 4H), 2.92
(s, 3H), 2.87 (dt, J=18.1, 6.5 Hz, 4H), 2.50 (s, 3H), 2.28-2.22 (m,
1H), 2.16-2.09 (m, 1H), 1.08 (s, 9H).
[0301]
(2S,4R)-1-((S)-2-(3-((3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-o-
xo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-y-
l)-3-oxopropyl)(methyl)amino)propanamido)-3,3-dimethylbutanoyl)-4-hydroxy--
N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(example 46) To a stirring solution of intermediate 14 (22 mg,
0.031 mmol) in DMF (1 ml) were added TEA (0.015 mL, 0.11 mmol),
palbociclib (13.1 mg, 0.029 mmol), and TBTU (11.3 mg, 0.035 mmol).
The mixture was stirred at RT overnight before being concentrated.
The resulting residue was purified by prep-HPLC to yield the title
compound (22 mg, 75%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.11 (s, 1H), 8.92 (s, 1H), 8.19 (d, J=7.9 Hz,
1H), 7.93 (d, J=2.7 Hz, 1H), 7.59 (d, J=9.6 Hz, 1H), 7.47 (d, J=7.8
Hz, 2H), 7.42 (d, J=8.1 Hz, 2H), 6.03 (p, J=8.8 Hz, 1H), 4.65-4.49
(m, 4H), 4.38 (d, J=15.5 Hz, 1H), 3.97 (d, J=10.8 Hz, 1H),
3.90-3.64 (m, 5H), 3.64-3.51 (m, 2H), 3.43-3.36 (m, 4H), 3.10-3.02
(m, 2H), 2.93 (s, 3H), 2.93-2.85 (m, 4H), 2.52 (s, 3H), 2.49 (s,
3H), 2.45 (s, 3H), 2.37-2.29 (m, 2H), 2.29-2.20 (m, 1H), 2.16-2.07
(m, 3H), 1.97-1.88 (m, 2H), 1.76-1.66 (m, 2H), 1.07 (d, J=10.7 Hz,
9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.53H.sub.69N.sub.12O.sub.7S, 1017.5127; found: 1017.5013.
##STR00249##
[0302]
(2S,4R)-1-((S)-2-(6-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazine-1-carb-
onyl)spiro[3.3]heptane-2-carboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-
-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (example
47) To a solution of spiro[3.3]heptane-2,6-dicarboxylic acid (250
mg, 1.36 mmol) in DCM/THF (1:1, 5 ml) were added VHL-1 (218 mg,
0.47 mmol), triethylamine (0.21 ml, 1.4 mmol), and EDCI (112 mg,
0.59 mmol) sequentially at 0.degree. C. The resulting solution was
stirred for 2 h at 0.degree. C., before being warmed to room
temperature (RT). After stirring overnight at RT, the reaction was
quenched with water, and concentrated under reduced pressure. The
resulting residue was purified by reverse-phase chromatography to
yield the intermediate 15 (210 mg, 75%) as white solid. To a
stirring solution of intermediate 15 (19 mg, 0.031 mmol) in DMF (1
ml) were added TEA (0.01 mL, 0.07 mmol), palbociclib (12.7 mg,
0.028 mmol), and TBTU (14.2 mg, 0.044 mmol). The mixture was
stirred at RT overnight before being concentrated. The resulting
residue was purified by prep-HPLC to yield the title compound (26
mg, 90%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.11 (s, 1H), 8.97 (s, 1H), 8.21 (dd, J=9.6, 3.0 Hz, 1H), 7.88 (d,
J=2.4 Hz, 1H), 7.57 (d, J=9.6 Hz, 1H), 7.48 (dd, J=8.0, 1.6 Hz,
2H), 7.45-7.42 (m, 2H), 6.02 (p, J=8.8 Hz, 1H), 4.69-4.62 (m, 1H),
4.62-4.47 (m, 3H), 4.41-4.27 (m, 1H), 3.92 (dd, J=11.0, 4.4 Hz,
1H), 3.86-3.70 (m, 3H), 3.71-3.56 (m, 2H), 3.37-3.22 (m, 6H),
3.17-3.03 (m, 1H), 2.52 (s, 3H), 2.50 (s, 3H), 2.45 (s, 3H),
2.40-2.04 (m, 13H), 1.96-1.88 (m, 2H), 1.76-1.68 (m, 2H), 1.04 (d,
J=3.4 Hz, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.55H.sub.68N.sub.11O.sub.7S, 1026.5018; found: 1026.4985.
##STR00250##
[0303]
(2S,4R)-1-((S)-2-((E)-8-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-o-
xo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-y-
l)-8-oxooct-4-enamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiaz-
ol-5-yl)benzyl)pyrrolidine-2-carboxamide (example 48) To a solution
of (E)-oct-4-enedioic acid (250 mg, 1.45 mmol) in THF (5 ml) were
added VHL-1 (239 mg, 0.51 mmol), triethylamine (0.21 ml, 1.4 mmol),
and EDCI (104 mg, 0.54 mmol) sequentially at 0.degree. C. The
resulting solution was stirred for 2 h at 0.degree. C., before
being warmed to room temperature (RT). After stirring overnight at
RT, the reaction was quenched with water and concentrated under
reduced pressure. The resulting residue was purified by
reverse-phase chromatography to yield the intermediate 16 (208 mg,
70%) as white solid. To a stirring solution of intermediate 16 (19
mg, 0.032 mmol) in DMF (1 ml) were added TEA (0.01 mL, 0.07 mmol),
palbociclib (11.1 mg, 0.025 mmol), and TBTU (10.4 mg, 0.032 mmol).
The mixture was stirred at RT overnight before being concentrated.
The resulting residue was purified by prep-HPLC to yield the title
compound (24 mg, 95%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.12 (s, 1H), 9.00 (s, 1H), 8.21 (dd, J=9.6,
2.9 Hz, 1H), 7.88 (d, J=2.8 Hz, 1H), 7.56 (d, J=9.6 Hz, 1H), 7.49
(d, J=8.1 Hz, 2H), 7.43 (d, J=8.1 Hz, 2H), 6.03 (p, J=8.8 Hz, 1H),
5.61-5.50 (m, 2H), 4.66 (s, 1H), 4.60-4.48 (m, 3H), 4.37 (dd,
J=15.3, 8.6 Hz, 1H), 3.92 (d, J=11.0 Hz, 1H), 3.83-3.77 (m, 5H),
3.31-3.25 (m, 3H), 2.54 (t, J=7.8 Hz, 2H), 2.51 (s, 3H), 2.49 (s,
3H), 2.45 (s, 3H), 2.40-2.28 (m, 9H), 2.26-2.23 (m, 1H), 2.16-2.07
(m, 3H), 1.96-1.87 (m, 2H), 1.76-1.66 (m, 2H), 1.04 (s, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.54H.sub.68N.sub.11O.sub.7S, 1014.5018; found: 1014.5011.
##STR00251##
[0304]
(2S,4R)-1-((S)-2-(2-(4-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-ox-
o-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazine-1-c-
arbonyl)piperidin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4--
methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (example 49) To
a solution of 2-(4-(methoxycarbonyl)piperidin-1-yl)acetic acid (300
mg, 1.5 mmol) in DMF (10 ml) were added VHL-1 (715 mg, 1.5 mmol),
triethylamine (1.07 ml, 7.7 mmol), and TBTU (513 mg, 1.6 mmol)
sequentially at RT. After being stirred overnight at RT, the
reaction was quenched with water and concentrated under reduced
pressure, the resulting residue was purified by reverse-phase
chromatography to yield the intermediate 17 (552 mg, 90%) as white
solid. To a stirring solution of intermediate 17 (52 mg, 0.085
mmol) in THF/H.sub.2O (5:1, 3 ml) was added anhydrous LiOH (3.3 mg,
0.136 mmol). The resulting mixture was stirred overnight at RT
before being concentrated under reduced pressure. The resulting
residue was dissolved in DMF (1 ml). To the resulting solution, TEA
(0.015 mL, 0.11 mmol), palbociclib (13.3 mg, 0.030 mmol), and TBTU
(14 mg, 0.044 mmol) were added. After the mixture was stirred at RT
overnight, the reaction was concentrated. The resulting residue was
purified by prep-HPLC to yield the title compound (25 mg, 30%) as
yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.12 (s,
1H), 8.97 (s, 1H), 8.22 (dd, J=10.1, 3.0 Hz, 1H), 7.91 (d, J=3.0
Hz, 1H), 7.58 (dd, J=9.6, 5.5 Hz, 1H), 7.50 (d, J=8.0 Hz, 2H), 7.43
(d, J=8.2 Hz, 2H), 6.03 (p, J=8.8 Hz, 1H), 4.69-4.65 (m, 1H),
4.61-4.54 (m, 3H), 4.38 (d, J=15.5 Hz, 1H), 4.13-3.90 (m, 3H),
3.90-3.76 (m, 5H), 3.72-3.64 (m, 2H), 3.40-3.32 (m, 2H), 3.30-3.28
(m, 2H), 3.15 (br, 2H), 2.52 (s, 3H), 2.50 (s, 3H), 2.45 (s, 3H),
2.37-2.29 (m, 2H), 2.29-2.23 (m, 1H), 2.16-1.99 (m, 9H), 1.98-1.88
(m, 2H), 1.77-1.65 (m, 2H), 1.09 (s, 9H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.54H.sub.69N.sub.12O.sub.7S,
1029.5127; found: 1029.5048.
##STR00252##
[0305]
(2S,4R)-1-((S)-2-(2-(4-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-
-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-
-yl)-2-oxoethyl)phenyl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4--
methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (example 50) To
a solution of 2,2'-(1,4-phenylene)diacetic acid (414 mg, 2.13 mmol)
in THF (10 ml) were added VHL-1 (217 mg, 0.47 mmol), triethylamine
(0.21 ml, 1.4 mmol), and EDCI (110 mg, 0.58 mmol) sequentially at
0.degree. C. The resulting solution was stirred for 2 h at
0.degree. C., before being warmed to room temperature (RT). After
stirring overnight at RT, the reaction was quenched with water and
concentrated under reduced pressure. The resulting residue was
purified by reverse-phase chromatography to yield the intermediate
18 (170 mg, 60%) as white solid. To a stirring solution of
intermediate 18 (20 mg, 0.033 mmol) in DMF (1 ml) were added TEA
(0.015 mL, 0.11 mmol), palbociclib (12 mg, 0.027 mmol), and TBTU
(12.2 mg, 0.038 mmol). After the mixture was stirred at RT
overnight, the reaction was concentrated under reduced pressure.
The resulting residue was purified by prep-HPLC to yield the title
compound (9 mg, 32%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.08 (s, 1H), 8.95 (s, 1H), 8.15 (dd, J=9.7,
2.8 Hz, 1H), 7.78 (d, J=2.6 Hz, 1H), 7.51 (d, J=11.4 Hz, 1H),
7.46-7.38 (m, 4H), 7.34-7.25 (m, 4H), 6.06-5.97 (m, 1H), 4.66-4.61
(m, 1H), 4.59-4.46 (m, 3H), 4.38-4.32 (m, 1H), 3.92-3.83 (m, 3H),
3.81-3.70 (m, 4H), 3.64 (d, J=14.4 Hz, 1H), 3.58 (d, J=14.7 Hz,
1H), 3.24-3.18 (m, 1H), 3.16-3.09 (m, 1H), 3.08-3.02 (m, 1H), 2.52
(s, 3H), 2.49 (s, 3H), 2.44 (s, 3H), 2.37-2.31 (m, 2H), 2.26-2.20
(m, 1H), 2.15-2.08 (m, 3H), 1.96-1.87 (m, 2H), 1.76-1.68 (m, 2H),
0.97 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.56H.sub.66N.sub.11O.sub.7S, 1036.4862; found: 1036.4860.
##STR00253##
[0306]
(2S,4R)-1-((S)-2-(2-(4-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-
-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-
-yl)-2-oxoethyl)piperazin-1-yl)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy--
N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(example 51) To a solution of
2-(4-(2-(tert-butoxy)-2-oxoethyl)piperazin-1-yl)acetic acid (340
mg, 0.7 mmol) in DMF (5 ml) were added VHL-1 (277 mg, 0.6 mmol),
triethylamine (0.7 ml, 5 mmol), and EDCI (224 mg, 1.2 mmol)
sequentially at RT. After being stirred overnight at RT, the
reaction was quenched with water, and concentrated under reduced
pressure. The resulting residue was purified by reverse-phase
chromatography to yield the intermediate 17 (350 mg, 88%) as white
solid. To a stirring solution of intermediate 19 (15 mg, 0.023
mmol) in DCM (1 ml) was added TFA (1 mL). The resulting mixture was
stirred at RT. The disappearance of starting material was monitored
by LC/MS. The reaction mixture was concentrated under reduced
pressure and the resulting residue was dissolved in DMF (1 ml). To
the resulting solution, TEA (0.015 mL, 0.11 mmol), palbociclib (8.3
mg, 0.019 mmol), and TBTU (7 mg, 0.022 mmol) were added. The
mixture was stirred at RT overnight before being concentrated. The
resulting residue was purified by prep-HPLC to yield the title
compound (7 mg, 35%) as yellow solid. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.13 (s, 1H), 8.99 (s, 1H), 8.24 (dd, J=9.6,
2.4 Hz, 1H), 7.93 (s, 1H), 7.59 (d, J=9.6 Hz, 1H), 7.49 (d, J=8.4
Hz, 2H), 7.45 (d, J=8.2 Hz, 2H), 6.08-5.98 (m, 1H), 4.69 (s, 1H),
4.61-4.50 (m, 3H), 4.44-4.38 (m, 1H), 4.25 (s, 2H), 3.92 (d, J=11.1
Hz, 1H), 3.88-3.79 (m, 3H), 3.68 (br, 2H), 3.50-3.37 (m, 8H),
3.37-3.34 (m, 2H), 3.06 (br, 4H), 2.53 (s, 2H), 2.51 (s, 2H), 2.46
(s, 3H), 2.37-2.25 (m, 3H), 2.17-2.07 (m, 3H), 1.97-1.87 (m, 2H),
1.76-1.69 (m, 2H), 1.07 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.54H.sub.70N.sub.13O.sub.7S, 1044.5236; found:
1044.5226.
##STR00254##
[0307]
1-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoethyl)-N--(-
(S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrr-
olidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)piperidine-4-carboxamide
(example 52) A solution of palbociclib (43 mg, 0.096 mmol) in DMF
were added 2-(4-(methoxycarbonyl)piperidin-1-yl)acetic acid (20 mg,
0.1 mmol), TEA (0.035 mL, 0.25 mmol), and TBTU (42 mg, 0.13 mmol)
sequentially at RT. After being stirred overnight at RT, the
reaction was quenched with water. After concentration under reduced
pressure, the resulting residue was purified by reverse-phase
chromatography to yield the intermediate 20 (50 mg, 82%) as white
solid. To a stirring solution of intermediate 20 (50 mg, 0.079
mmol) in THF/H.sub.2O (5:1, 3 mL) was added anhydrous LiOH (6 mg,
0.025 mmol). The mixture was stirred at RT and the reaction
progress was monitored by LC/MS. After total consumption of
intermediate 20, the reaction was concentrated. The resulting
residue was dissolved in DMF (1 ml). To the solution were added TEA
(0.02 mL, 0.14 mmol), VHL-1 (26 mg, 0.055 mmol), and TBTU (16 mg,
0.05 mmol). The reaction mixture was stirred at RT overnight before
being concentrated. The resulting residue was purified by prep-HPLC
to yield the title compound (30 mg, 37%) as yellow solid. .sup.1H
NMR (600 MHz, CD.sub.3OD) .delta. 9.15 (s, 1H), 9.13 (s, 1H), 8.24
(dd, J=9.6, 2.7 Hz, 1H), 7.93 (d, J=2.2 Hz, 1H), 7.58 (d, J=9.6 Hz,
1H), 7.51 (d, J=8.1 Hz, 2H), 7.46 (d, J=8.2 Hz, 2H), 6.03 (p, J=8.8
Hz, 1H), 4.65 (d, J=8.3 Hz, 1H), 4.62-4.51 (m, 3H), 4.42-4.32 (m,
3H), 3.91 (d, J=9.6 Hz, 1H), 3.87-3.82 (m, 3H), 3.79-3.70 (m, 1H),
3.65 (br, 2H), 3.50 (br, 1H), 3.40 (br, 2H), 3.37-3.34 (m, 3H),
3.20-3.07 (m, 1H), 2.74 (br, 1H), 2.52 (s, 3H), 2.52 (s, 3H), 2.45
(s, 3H), 2.36-2.24 (m, 3H), 2.21-2.03 (m, 7H), 1.97-1.88 (m, 2H),
1.77-1.64 (m, 2H), 1.07 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.54H.sub.69N.sub.12O.sub.7S, 1029.5127; found:
1029.5106.
##STR00255##
[0308]
2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2-
,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-N-(2-(2,6-dioxopipe-
ridin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide (example 53) A
solution of pomalidomide (90 mg, 0.33 mmol) and 2-chloroacetyl
chloride (0.026 mL, 0.33 mmol) in THF (2 ml) was heated at reflux
overnight. After being cooled to RT, the reaction mixture was
concentrated, and the resulting residue was washed with ethyl
acetate to yield the intermediate 21 (75 mg, 65%) as yellow solid.
To a stirring solution of intermediate 13 (36 mg, 0.082 mmol) in
DMSO (1 mL) were added DIEA (0.043 mL, 0.24 mmol) and palbociclib
(39 mg, 0.087 mmol). The mixture was heated at 80.degree. C. After
the starting materials were consumed, the reaction was concentrated
under reduced pressure. The resulting residue was purified by
prep-HPLC to yield the title compound (36 mg, 54%) as yellow solid.
.sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 11.19 (s, 1H), 8.89 (d,
J=8.5 Hz, 1H), 8.81 (s, 1H), 8.20 (d, J=9.1 Hz, 1H), 8.14 (d, J=2.8
Hz, 1H), 7.71 (t, J=4.2 Hz, 1H), 7.55 (d, J=7.3 Hz, 1H), 7.38 (dd,
J=9.1, 2.9 Hz, 1H), 5.87 (p, J=9.0 Hz, 1H), 4.94 (dd, J=12.7, 5.3
Hz, 1H), 3.39 (br, 4H), 3.34 (AB, J.sub.ab=17.4 Hz, 1H), 3.27 (AB,
J.sub.ab=17.4 Hz, 1H), 2.92-2.73 (m, 8H), 2.54 (s, 3H), 2.40-2.29
(m, 4H), 2.14-2.09 (m, 1H), 2.08-2.00 (m, 2H), 1.90-1.81 (m, 2H),
1.70-1.59 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.39H.sub.41N.sub.10O.sub.7, 761.3154; found: 761.3157.
Synthesis of Various Linkers of Pomalidomide Analogues.
##STR00256##
[0310] 3-(7-Bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione
(intermediate 22) A solution of methyl
2-bromo-6-(bromomethyl)benzoate (970 mg, 3.16 mmol),
3-aminopiperidine-2,6-dione (667 mg, 4.06 mmol) and TEA (0.62 mL,
4.4 mmol) in CH.sub.3CN (10 mL) was heated at reflux for 16 h.
After being cooled to RT, the reaction was concentrated. To the
resulting residue were added ethyl acetate (10 mL) and H.sub.2O (10
mL). After filtration, the solid was collected and dried to yield
the title compound (534 mg, 54%) as purple solid.
[0311]
(E)-3-(2-(2,6-Dioxopiperidin-3-yl)-3-oxoisoindolin-4-yl)acrylic
acid (linker 29) A solution of intermediate 22 (46 mg, 0.14 mmol),
acrylic acid (0.02 mL, 0.29 mmol),
tetrakis(triphenylphosphine)palladium (23.3 mg, 0.02 mmol) and TEA
(0.02 mL, 0.14 mmol) in DMSO (1 mL) was heated in a microwave
reactor at 130.degree. C. for 30 min. After being cooled to RT, the
mixture was filtered. The solution was concentrated and purified by
prep-HPLC to yield the title compound (38 mg, 86%) as yellow solid.
.sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. 12.51 (sbr, 1H), 11.03
(s, 1H), 8.87 (d, J=16.2 Hz, 1H), 7.98 (t, J=6.0 Hz, 1H), 7.64 (d,
J=4.7 Hz, 2H), 6.69 (d, J=16.3 Hz, 1H), 5.10 (dd, J=13.3, 5.1 Hz,
1H), 4.47 (d, J=17.4 Hz, 1H), 4.35 (d, J=17.4 Hz, 1H), 2.98-2.86
(m, 1H), 2.62 (d, J=17.1 Hz, 1H), 2.42 (qd, J=13.2, 4.5 Hz, 1H),
2.07-1.99 (m, 1H).
[0312]
3-(2-(2,6-Dioxopiperidin-3-yl)-3-oxoisoindolin-4-yl)propanoic acid
(linker 30) A mixture of linker 29 (20 mg, 0.064 mmol) and Pd/C (5
mg) in MeOH/DMSO (1:1, 2 mL) was stirred for 16 h under H.sub.2
atmosphere. After removal of Pd/C through filtration, the solution
was concentrated under reduced pressure. The resulting residue was
purified by prep-HPLC to yield the title compound (11 mg, 50%) as
yellow solid. .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. 10.99
(br, 2H), 7.55-7.47 (m, 1H), 7.43 (d, J=7.5 Hz, 1H), 7.31 (d, J=7.7
Hz, 1H), 5.13-5.03 (m, 1H), 4.41 (dd, J=16.8, 8.8 Hz, 1H), 4.30
(dd, J=16.8, 8.8 Hz, 1H), 3.33-3.27 (m, 2H), 2.97-3.84 (m, 1H),
2.63-2.55 (m, 3H), 2.43-2.38 (m, 1H), 2.06-1.98 (m, 1H).
##STR00257##
[0313]
2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetic
acid (linker 31) A round bottom flash was charged with
2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (68 mg,
0.25 mmol), DMF (2 mL), tert-butyl-2-bromo acetate ester (0.045 mL,
0.3 mmol), sodium bicarbonate (49 mg, 0.058 mmol), and potassium
iodide (10 mg, 0.06 mmol). The reaction mixture was heated at
60.degree. C. overnight. After being cooled to RT, the insoluble
solid was removed by filtration. The solution was collected and
concentrated. The resulting residue was purified by reverse-ISCO to
yield intermediated 23 (83 mg, 86%). Intermediate 23 (83 mg, 0.21
mmol) was dissolved in DCM/TFA (2:1, 3 mL). After the reaction was
stirred for 3 h at RT, the solvent was removed to yield the title
compound (60 mg, 90%) as white solid. .sup.1H NMR (600 MHz,
DMSO-d.sub.6) .delta. 11.11 (br, 2H), 7.82-7.77 (m, 1H), 7.48 (d,
J=7.2 Hz, 1H), 7.40 (d, J=8.6 Hz, 1H), 5.11 (ddd, J=12.8, 5.4, 1.2
Hz, 1H), 5.00 (s, 2H), 2.95-2.80 (m, 1H), 2.63-2.53 (m, 2H),
2.08-2.00 (m, 1H).
##STR00258##
[0314] (2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)glycine
(linker 32) The title compound was synthesized using the same
procedures for the preparation of linker 31. .sup.1H NMR (600 MHz,
DMSO-d.sub.6) .delta. 12.66 (br, 1H), 11.03 (s, 1H), 7.29 (t, J=7.7
Hz, 1H), 6.99 (d, J=7.4 Hz, 1H), 6.66 (d, J=8.0 Hz, 1H), 5.13 (dd,
J=13.3, 4.9 Hz, 1H), 4.27 (d, J=17.0 Hz, 1H), 4.18 (d, J=17.4 Hz,
1H), 3.93 (s, 2H), 2.98-2.84 (m, 1H), 2.63 (d, J=17.0 Hz, 1H), 2.34
(qd, J=13.2, 4.1 Hz, 1H), 2.08-1.97 (m, 1H).
##STR00259##
[0315]
3-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)propanoic
acid (linker 33) The title compound was synthesized using the same
procedures for the preparation of linker 30. .sup.1H NMR (600 MHz,
DMSO-d.sub.6) .delta. 11.15 (br, 2H), 7.85 (d, J=7.7 Hz, 1H), 7.82
(s, 1H), 7.76 (d, J=7.7 Hz, 1H), 5.14 (dd, J=12.9, 5.4 Hz, 1H),
3.02 (t, J=7.4 Hz, 2H), 2.93-2.84 (m, 1H), 2.65 (t, J=7.5 Hz, 2H),
2.60-2.53 (m, 2H), 2.08-2.04 (m, 1H).
##STR00260##
[0316]
3-(4-((tert-Butyldimethylsilyl)oxy)-1-oxoisoindolin-2-yl)piperidine-
-2,6-dione (intermediate 26) The title compound was synthesized
using the same procedures for the preparation of intermediate 22
(38%). .sup.1H NMR (600 MHz, DMSO-d.sub.6) .delta. 10.98 (s, 1H),
7.44 (t, J=7.7 Hz, 1H), 7.36 (d, J=7.4 Hz, 1H), 7.11 (d, J=7.9 Hz,
1H), 5.11 (dd, J=13.2, 4.8 Hz, 1H), 4.34 (d, J=17.3 Hz, 1H), 4.25
(d, J=17.2 Hz, 1H), 2.96-2.82 (m, 1H), 2.66-2.53 (m, 2H), 2.02-1.96
(m, 1H), 0.99 (s, 9H), 0.26 (s, 6H).
[0317]
2-((2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)oxy)acetic
acid (linker 34) The title compound was synthesized using the same
procedures for the preparation of linker 31. .sup.1H NMR (600 MHz,
DMSO-d.sub.6) .delta. 11.00 (s, 1H), 7.47 (t, J=7.8 Hz, 1H), 7.36
(d, J=8.6 Hz, 1H), 7.17 (d, J=8.2 Hz, 1H), 5.19-5.05 (m, 2H), 4.84
(s, 2H), 4.41 (d, J=17.4 Hz, 1H), 4.28 (d, J=17.3 Hz, 1H),
3.00-2.84 (m, 1H), 2.63-2.58 (m, 1H), 2.49-2.38 (m, 1H), 2.04-1.92
(m, 1H).
##STR00261##
[0318]
3-(2-(2,6-Dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)propanoic acid
(linker 35) The title compound was synthesized using the same
procedures for the preparation of linker 30. .sup.1H NMR (600 MHz,
DMSO-d.sub.6) .delta. 12.23 (br, 1H), 11.01 (s, 1H), 7.59 (d, J=7.1
Hz, 1H), 7.51-7.46 (m, 2H), 5.14 (dd, J=13.3, 5.1 Hz, 1H), 4.51 (d,
J=17.0 Hz, 1H), 4.36 (d, J=17.1 Hz, 1H), 2.97-2.83 (m, 3H),
2.67-2.58 (m, 3H), 2.49-2.39 (m, 1H), 2.05-1.99 (m, 1H).
##STR00262##
[0319]
3-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)propanoic
acid (linker 36) The title compound was synthesized using the same
procedures for the preparation of linker 30. .sup.1H NMR (600 MHz,
DMSO-d.sub.6) .delta. 11.15 (br, 2H), 7.79-7.73 (m, 3H), 5.14 (dd,
J=12.9, 5.4 Hz, 1H), 3.27 (t, J=7.2 Hz, 2H), 2.95-2.83 (m, 1H),
2.67-2.52 (m, 4H), 2.12-2.01 (m, 1H).
[0320] The example 54-61 compounds were synthesized using the same
procedures for the preparation of the example 40 compound with
linkers 29-36.
##STR00263##
[0321]
(E)-3-(7-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihyd-
ropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxoprop-
-1-en-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (example 54)
(52 mg, 58%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD)
.delta. 9.08 (s, 1H), 8.74 (d, J=15.7 Hz, 1H), 8.17 (dd, J=9.7, 2.7
Hz, 1H), 7.90 (d, J=2.7 Hz, 1H), 7.84 (d, J=7.7 Hz, 1H), 7.62-7.54
(m, 2H), 7.51 (d, J=7.5 Hz, 1H), 7.27 (d, J=15.7 Hz, 1H), 5.98 (p,
J=8.8 Hz, 1H), 5.13 (dd, J=12.6, 5.4 Hz, 1H), 4.47 (AB,
J.sub.ab=16.8 Hz, 1H), 4.43 (AB, J.sub.ab=16.8 Hz, 1H), 3.97 (br,
2H), 3.90 (br, 2H), 3.28 (br, 2H), 3.34 (br, 2H), 2.87-2.75 (m,
1H), 2.52 (s, 3H), 2.50-2.44 (m, 1H), 2.43 (s, 3H), 2.35-2.25 (m,
2H), 2.25-2.15 (m, 2H), 2.14-2.03 (m, 3H), 1.94-1.86 (m, 2H),
1.73-1.64 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.40H.sub.42N.sub.9O.sub.6, 744.3253; found: 744.3274.
##STR00264##
[0322]
4-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoethoxy)-2-(-
2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (example 55) (27 mg,
51%) as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta.
9.11 (s, 1H), 8.19 (d, J=8.6 Hz, 1H), 7.88 (br, 1H), 7.76 (t, J=7.9
Hz, 1H), 7.57 (d, J=7.9 Hz, 1H), 7.49 (d, J=7.2 Hz, 1H), 7.41 (d,
J=8.5 Hz, 1H), 6.06-5.96 (m, 1H), 5.22-5.15 (m, 2H), 5.12 (dd,
J=12.7, 5.4 Hz, 1H), 3.93-3.76 (m, 4H), 3.45-3.24 (m, 4H),
2.95-2.69 (m, 3H), 2.53 (s, 3H), 2.45 (s, 3H), 2.37-2.27 (m, 2H),
2.20-2.05 (m, 3H), 1.96-1.87 (m, 2H), 1.77-1.62 (m, 2H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.39H.sub.40N.sub.9O.sub.8, 762.2994; found: 762.3008.
##STR00265##
[0323]
3-(7-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropy-
rido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropyl)--
1-oxoisoindolin-2-yl)piperidine-2,6-dione (example 56) (9 mg, 48%)
as yellow solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 8.81 (s,
1H), 7.73-7.65 (m, 2H), 7.63 (s, 1H), 7.31 (t, J=7.5 Hz, 1H), 7.18
(d, J=7.4 Hz, 1H), 7.13 (d, J=7.4 Hz, 1H), 5.75-5.70 (m, 1H), 4.93
(dd, J=13.2, 5.1 Hz, 1H), 3.69-3.48 (m, 4H), 3.31-3.21 (m, 1H),
3.21-3.09 (m, 3H), 3.07-3.00 (m, 2H), 2.99-2.77 (m, 2H), 2.76-2.63
(m, 3H), 2.63-2.54 (m, 1H), 2.34 (s, 3H), 2.28-2.16 (m, 4H),
2.15-2.02 (m, 3H), 1.94-1.85 (m, 2H), 1.75-1.65 (m, 2H), 1.55-1.44
(m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.40H.sub.44N.sub.9O.sub.6, 746.3409; found: 746.3427.
##STR00266##
[0324]
3-(4-((2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydrop-
yrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoethyl)a-
mino)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (example 57) (39
mg, 80%).sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.07 (s, 1H),
8.09 (d, J=9.5 Hz, 1H), 7.89 (s, 1H), 7.61 (d, J=8.7 Hz, 1H), 7.34
(t, J=7.6 Hz, 1H), 7.16 (d, J=7.4 Hz, 1H), 6.79 (d, J=7.9 Hz, 1H),
5.96 (p, J=8.7 Hz, 1H), 5.14 (dd, J=13.2, 4.1 Hz, 1H), 4.37 (s,
2H), 4.18-4.06 (m, 2H), 3.85-3.79 (m, 4H), 3.38-3.22 (m, 4H),
2.90-2.76 (m, 2H), 2.56-2.48 (m, 3H), 2.49-2.38 (m, 4H), 2.27 (t,
J=18.1 Hz, 2H), 2.24-2.14 (m, 1H), 2.09 (s, 2H), 1.90 (s, 2H), 1.68
(d, J=3.0 Hz, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.39H.sub.43N.sub.10O.sub.6, 747.3362; found: 747.3370.
##STR00267##
[0325]
5-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropyl)-2-(-
2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (example 58) (4 mg,
33%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.04 (s, 1H),
7.99-7.96 (m, 1H), 7.85 (s, 1H), 7.82-7.78 (m, 1H), 7.74-7.70 (m,
1H), 7.67-7.63 (m, 1H), 6.31 (s, 1H), 5.99-5.93 (m, 1H), 5.04 (dd,
J=11.8, 6.0 Hz, 1H), 3.87-3.63 (m, 4H), 3.26-3.01 (m, 5H),
2.88-2.72 (m, 4H), 2.54 (s, 3H), 2.43 (s, 3H), 2.35-2.22 (m, 2H),
2.19-2.10 (m, 1H), 2.08-2.04 (m, 2H), 1.92-1.87 (m, 2H), 1.74-1.63
(m, 2H), 1.30-1.26 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+
calculated for C.sub.40H.sub.42N.sub.9O.sub.7, 760.3202; found:
760.3226.
##STR00268##
[0326]
3-(4-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropy-
rido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoethoxy)--
1-oxoisoindolin-2-yl)piperidine-2,6-dione (example 59) (43 mg, 90%)
.sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.09 (s, 1H), 8.14 (dd,
J=9.7, 2.8 Hz, 1H), 7.89 (d, J=2.5 Hz, 1H), 7.59 (d, J=9.6 Hz, 1H),
7.48-7.40 (m, 2H), 7.15 (d, J=8.0 Hz, 1H), 5.98 (p, J=8.8 Hz, 1H),
5.15 (dd, J=13.3, 5.1 Hz, 1H), 5.03 (AB, J.sub.ab=14.4 Hz, 1H),
4.99 (AB, J.sub.ab=14.4 Hz, 1H), 4.53 (AB, J.sub.ab=17.4 Hz, 1H),
4.49 (AB, J.sub.ab=17.4 Hz, 1H), 3.90-3.67 (m, 4H), 3.42-3.20 (m,
4H), 2.96-2.73 (m, 2H), 2.54 (s, 3H), 2.52-2.47 (m, 1H), 2.44 (s,
3H), 2.34-2.28 (m, 2H), 2.26-2.16 (m, 1H), 2.12-2.05 (m, 2H),
1.96-1.83 (m, 2H), 1.74-1.59 (m, 2H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calculated for C.sub.39H.sub.49N.sub.9O.sub.7,
748.3202; found: 748.3236.
##STR00269##
[0327]
3-(4-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropy-
rido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropyl)--
1-oxoisoindolin-2-yl)piperidine-2,6-dione (example 60) (15 mg,
75%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.12 (s, 1H), 8.11
(dd, J=9.7, 2.9 Hz, 1H), 7.79 (d, J=2.8 Hz, 1H), 7.65 (d, J=4.8 Hz,
1H), 7.58-7.52 (m, 2H), 7.49 (t, J=7.5 Hz, 1H), 6.01 (p, J=8.9 Hz,
1H), 5.17 (dd, J=13.3, 5.2 Hz, 1H), 4.56 (q, J=16.9 Hz, 2H),
3.83-3.72 (m, 2H), 3.63 (t, J=5.1 Hz, 2H), 3.22-3.13 (m, 2H), 3.08
(t, J=7.1 Hz, 2H), 3.03-2.97 (m, 1H), 2.94-2.82 (m, 5H), 2.59-2.49
(m, 4H), 2.45 (s, 3H), 2.35-2.29 (m, 2H), 2.27-2.16 (m, 1H),
2.14-2.04 (m, 2H), 1.95-1.83 (m, 2H), 1.75-1.63 (m, 2H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.40H.sub.44N.sub.9O.sub.6, 746.3409; found: 746.3382.
##STR00270##
[0328]
4-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropyl)-2-(-
2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (example 61) (17 mg,
43%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.10 (s, 1H), 8.14
(d, J=9.4 Hz, 1H), 7.86 (s, 1H), 7.81-7.65 (m, 3H), 7.59 (d, J=9.5
Hz, 1H), 6.00 (p, J=8.8 Hz, 1H), 5.13 (dd, J=12.5, 5.3 Hz, 1H),
3.82-3.74 (m, 4H), 3.47-3.37 (m, 2H), 3.31-3.20 (m, 4H), 2.92-2.69
(m, 5H), 2.54 (s, 3H), 2.45 (s, 3H), 2.39-2.26 (m, 2H), 2.22-2.16
(m, 1H), 2.14-2.06 (m, 2H), 1.96-1.86 (m, 2H), 1.76-1.65 (m, 2H).
HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.40H.sub.42N.sub.9O.sub.7, 760.3202; found: 760.3210.
##STR00271##
[0329]
(2S,4R)-1-((S)-2-(7-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)-1,4-diazepan-1-y-
l)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-
-5-yl)benzyl)pyrrolidine-2-carboxamide (example 62) The title
compound was synthesized using the same procedures for the
preparation of the example 40 compound with linker 4 (9 mg, 74%) as
white solid. .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.10 (s,
1H), 9.01 (s, 1H), 8.06-8.01 (m, 1H), 7.80-7.75 (m, 1H), 7.55-7.41
(m, 5H), 6.06-5.98 (m, 1H), 4.66-4.49 (m, 5H), 4.38 (dd, J=15.4,
4.1 Hz, 1H), 3.93-3.77 (m, 5H), 3.77-3.54 (m, 4H), 2.52 (s, 3H),
2.50 (s, 3H), 2.45 (s, 3H), 2.40-2.05 (m, 9H), 2.00-1.88 (m, 4H),
1.76-1.46 (m, 6H), 1.40-1.23 (m, 3H), 1.06-1.01 (m, 9H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.54H.sub.70N.sub.11O.sub.7S, 1016.5175; found: 1016.5166.
##STR00272##
[0330]
4-((2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)-1,4-diazepan-1-yl)-2-oxoethyl)-
amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (example
63) The title compound was synthesized using the same procedures
for the preparation of the example 40 compound with linker 24 (4
mg, 44%) as yellow solid. Rotamer (3:2) major one .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 9.02 (s, 1H), 7.81 (dd, J=9.6, 3.0 Hz,
1H), 7.51 (d, J=2.9 Hz, 1H), 7.37-7.33 (m, 2H), 6.92 (d, J=8.6 Hz,
1H), 6.87 (d, J=7.8 Hz, 1H), 6.06-6.00 (m, 1H), 5.05 (dd, J=12.8,
5.5 Hz, 1H), 4.24 (d, J=16.2 Hz, 1H), 4.16-4.14 (m, 1H), 3.96-3.79
(m, 4H), 3.74-3.65 (m, 4H), 2.90-2.63 (m, 4H), 2.54 (s, 3H), 2.44
(s, 3H), 2.40-2.28 (m, 2H), 2.16-2.06 (m, 2H), 1.98-1.87 (m, 4H),
1.78-1.65 (m, 2H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.40H.sub.43N.sub.10O.sub.7, 775.3311; found: 775.3314.
##STR00273##
[0331]
7-(((S)-1-((2S,4R)-4-Hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phe-
nyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-
-oxoheptanoic acid (linker 37) The title compound was synthesis
using the same procedures for the preparation of linker 1 (22 mg,
64%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.23 (s, 1H), 7.48
(dd, J=12.0, 7.3 Hz, 4H), 5.06-5.00 (m, 1H), 4.64 (s, 1H), 4.59 (t,
J=8.4 Hz, 1H), 4.46-4.42 (m, 1H), 3.91 (d, J=11.1 Hz, 1H), 3.77
(dd, J=11.0, 4.0 Hz, 1H), 2.53 (s, 3H), 2.35-2.26 (m, 4H),
2.24-2.16 (m, 1H), 1.99-1.94 (m, 1H), 1.67-1.62 (m, 4H), 1.52 (d,
J=7.2 Hz, 3H), 1.45-1.34 (m, 2H), 1.06 (s, 9H).
[0332]
(2S,4R)-1-((S)-2-(7-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7-
,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-7-
-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N--((S)-1-(4-(4-methylthi-
azol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (example 64) the
title compound was synthesized using the same procedures for the
preparation of the example 40 compound with linker 37 (15 mg, 37%).
.sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.12 (s, 1H), 8.98 (s,
1H), 8.23 (dd, J=9.6, 2.9 Hz, 1H), 7.90 (d, J=2.6 Hz, 1H), 7.57 (d,
J=9.5 Hz, 1H), 7.45 (q, J=8.3 Hz, 4H), 6.03 (p, J=8.8 Hz, 1H), 5.02
(q, J=6.6 Hz, 1H), 4.66 (s, 1H), 4.60 (t, J=7.7 Hz, 1H), 4.46 (br,
1H), 3.90 (d, J=11.0 Hz, 1H), 3.85-3.74 (m, 5H), 3.39-3.34 (m, 2H),
3.33-3.30 (m, 2H), 2.52 (s, 3H), 2.50 (s, 3H), 2.45 (s, 3H),
2.39-2.26 (m, 5H), 2.26-2.18 (m, 1H), 2.14-2.09 (m, 2H), 2.01-1.88
(m, 3H), 1.76-1.62 (m, 7H), 1.51 (d, J=6.6 Hz, 3H), 1.46-1.41 (m,
2H), 1.06 (s, 9H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated for
C.sub.54H.sub.70N.sub.11O.sub.7S, 1016.5175; found: 1016.5154.
##STR00274##
[0333]
2-((3R,5R,6S)-1-((S)-1-((4-(5-(4-(6-((6-Acetyl-8-cyclopentyl-5-meth-
yl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperaz-
in-1-yl)-5-oxopentanoyl)piperazin-1-yl)sulfonyl)-3,3-dimethylbutan-2-yl)-5-
-(3-chlorophenyl)-6-(4-chlorophenyl)-3-methyl-2-oxopiperidin-3-yl)acetic
acid (example 65) The title compound was synthesized using the same
procedures for the preparation of the example 15 compound (4 mg,
7%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.13 (s, 1H), 8.24
(dd, J=9.6, 2.8 Hz, 1H), 7.89 (d, J=2.8 Hz, 1H), 7.79 (br, 1H),
7.57 (d, J=9.6 Hz, 1H), 7.47 (br, 1H), 7.19 (br, 1H), 7.16-7.07 (m,
3H), 7.07 (br, 1H), 7.02 (d, J=7.4 Hz, 1H), 6.08-5.98 (m, 1H), 5.09
(d, J=11.2 Hz, 1H), 3.96 (dd, J=13.6, 11.5 Hz, 1H), 3.87-3.67 (m,
8H), 3.55 (dd, J=11.4, 2.2 Hz, 1H), 3.43-3.36 (m, 9H), 3.03 (d,
J=13.4 Hz, 1H), 2.94 (dd, J=13.6, 2.2 Hz, 1H), 2.66 (d, J=13.5 Hz,
1H), 2.56 (dd, J=13.6, 7.0 Hz, 4H), 2.53 (s, 3H), 2.46 (s, 3H),
2.89-2.35 (m, 3H), 2.16-2.08 (m, 3H), 2.01-1.90 (m, 4H), 1.72 (dd,
J=10.5, 5.4 Hz, 2H), 1.40 (s, 3H), 0.73 (s, 9H). HRMS (ESI-TOF)
m/z: [M+H].sup.+ calculated for
C.sub.59H.sub.73Cl.sub.2N.sub.10O.sub.9S, 1167.4654; found:
1167.4653.
##STR00275##
[0334]
5-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2-
,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-N--((S)-1-(((S)-1-c-
yclohexyl-2-((S)-2-(4-(4-fluorobenzoyl)thiazol-2-yl)pyrrolidin-1-yl)-2-oxo-
ethyl)amino)-1-oxopropan-2-yl)-N-methyl-5-oxopentanamide (example
66) The title compound was synthesized using the same procedures
for the preparation of the example 15 compound (9 mg, 18%). Rotamer
(4:3) major one .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.10 (s,
1H), 8.41-8.15 (m, 3H), 7.88 (d, J=8.4 Hz, 1H), 7.83 (s, 1H),
7.56-7.50 (m, 1H), 7.30-7.20 (m, 2H), 6.08-6.00 (m, 1H), 5.46 (d,
J=7.8 Hz, 1H), 5.17-5.13 (m, 1H), 4.58-4.52 (m, 1H), 4.28-4.23 (m,
1H), 3.85-3.69 (m, 5H), 3.36-3.19 (m, 4H), 2.88 (s, 3H), 2.56-2.25
(m, 12H), 2.21-2.06 (m, 4H), 2.01-1.66 (m, 11H), 1.35 (d, J=7.1 Hz,
4H), 1.34-0.84 (m, 7H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calculated
for C.sub.55H.sub.67FN.sub.11O.sub.7S, 1044.4924; found:
1044.4940.
##STR00276##
[0335]
N-(6-(3-(4-((2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-di-
hydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoe-
thyl)(methyl)amino)butoxy)-5-propoxyphenoxy)-1,3-dimethyl-2-oxo-2,3-dihydr-
o-1H-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide
(example 67) To a solution of intermediate 27 (10 mg, 0.01 mmol),
HOAt (1-hydroxy-7-azabenzo-triazole) (4.3 mg, 0.03 mmol), and
intermediate 2 (5 mg, 0.01 mmol) in DMSO (1 mL) were added NMM (14
.mu.L, 0.13 mmol) and EDCI (6.05 mg, 0.03 mmol) at room
temperature. After being stirred overnight at room temperature, the
mixture was purified by preparative HPLC (10%-100% methanol/0.1%
TFA in H.sub.2O) to afford example 67 as white solid (6 mg, 54%).
.sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.07 (s, 1H), 8.15 (dd,
J=10.0, 2.7 Hz, 1H), 7.83 (s, 1H), 7.51 (d, J=9.6 Hz, 1H), 7.29 (s,
1H), 7.18 (dd, J=8.5, 2.2 Hz, 1H), 7.13 (d, J=2.1 Hz, 1H), 6.77 (d,
J=8.5 Hz, 1H), 6.59 (s, 1H), 6.14 (s, 1H), 6.04-5.97 (m, 1H), 5.72
(t, J=2.1 Hz, 1H), 5.61 (t, J=2.1 Hz, 1H), 4.52-4.25 (m, 2H),
3.97-3.83 (m, 4H), 3.82-3.78 (m, 3H), 3.75 (t, J=6.5 Hz, 2H), 3.60
(s, 6H), 3.39 (s, 5H), 3.24 (s, 6H), 2.99 (s, 3H), 2.50 (s, 3H),
2.43 (s, 3H), 2.31 (dt, J=15.7, 8.4 Hz, 2H), 2.09 (dq, J=12.3, 7.0
Hz, 2H), 2.02-1.87 (m, 4H), 1.83 (p, J=6.8 Hz, 2H), 1.76-1.65 (m,
4H), 1.00 (t, J=7.4 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd
for C.sub.57H.sub.70N.sub.11O.sub.11S.sup.+, 1116.4971; found,
1116.4961.
##STR00277##
[0336]
N-(2-(2-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydr-
opyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropo-
xy)ethoxy)ethyl)-2-((4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimet-
hyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl-
)(methyl)amino)acetamide (example 68) The title compound was
synthesized using the same procedures for the preparation of the
example 67 compound as white solid (7 mg, 55%). .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 9.05 (s, 1H), 8.16 (dd, J=9.7, 2.9 Hz,
1H), 7.84 (d, J=2.9 Hz, 1H), 7.52 (d, J=9.5 Hz, 1H), 7.29 (s, 1H),
7.17 (dd, J=8.5, 2.1 Hz, 1H), 7.12 (d, J=2.1 Hz, 1H), 6.78 (d,
J=8.5 Hz, 1H), 6.60 (s, 1H), 6.14 (d, J=2.7 Hz, 1H), 6.00 (p, J=8.9
Hz, 1H), 5.73 (d, J=2.3 Hz, 1H), 5.62 (d, J=2.4 Hz, 1H), 3.96 (d,
J=45.2 Hz, 2H), 3.86 (t, J=5.9 Hz, 2H), 3.82-3.71 (m, 14H), 3.59
(d, J=3.9 Hz, 7H), 3.54 (t, J=5.3 Hz, 2H), 3.45-3.37 (m, 5H),
3.28-3.22 (m, 6H), 2.95 (d, J=1.1 Hz, 3H), 2.72 (t, J=6.2 Hz, 2H),
2.50 (d, J=1.1 Hz, 3H), 2.42 (s, 3H), 2.31 (dt, J=15.2, 7.6 Hz,
2H), 2.09 (s, 2H), 1.92 (dt, J=15.9, 8.2 Hz, 4H), 1.82 (p, J=6.3
Hz, 2H), 1.71 (dq, J=20.6, 6.3, 5.6 Hz, 4H), 1.00 (dd, J=7.9, 6.8
Hz, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.64H.sub.83N.sub.12O.sub.14S.sup.+, 1275.5867; found,
1275.5887.
##STR00278##
[0337]
N-(15-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-15-oxo-3,6,9,12-
-tetraoxapentadecyl)-2-((4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-d-
imethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)b-
utyl)(methyl)amino)acetamide (example 69) The title compound was
synthesized using the same procedures for the preparation of the
example 67 compound as white solid (6 mg, 44%). .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 9.06 (d, J=1.2 Hz, 1H), 8.17 (dd, J=9.6,
3.0 Hz, 1H), 7.85 (d, J=2.9 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.31
(d, J=1.2 Hz, 1H), 7.18 (dd, J=8.5, 1.9 Hz, 1H), 7.13 (d, J=2.0 Hz,
1H), 6.78 (d, J=8.5 Hz, 1H), 6.60 (d, J=1.2 Hz, 1H), 6.20-6.11 (m,
1H), 6.05-5.92 (m, 1H), 5.75 (d, J=2.3 Hz, 1H), 5.63-5.41 (m, 1H),
3.97 (d, J=43.7 Hz, 2H), 3.88-3.84 (m, 2H), 3.83-3.68 (m, 13H),
3.62-3.56 (m, 14H), 3.54 (t, J=5.3 Hz, 2H), 3.45-3.38 (m, 5H),
3.35-3.19 (m, 8H), 2.95 (d, J=1.3 Hz, 3H), 2.72 (t, J=6.2 Hz, 2H),
2.50 (d, J=1.3 Hz, 3H), 2.42 (d, J=1.2 Hz, 3H), 2.30 (d, J=9.6 Hz,
2H), 2.09 (s, 2H), 1.95-1.87 (m, 4H), 1.82 (q, J=7.1, 6.7 Hz, 2H),
1.72 (dq, J=20.9, 7.2 Hz, 4H), 1.01 (td, J=7.4, 1.2 Hz, 3H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.68H.sub.91N.sub.12O.sub.16S.sup.+, 1363.6391; found,
1363.6387.
##STR00279##
[0338]
N-(18-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyri-
do[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-18-oxo-3,6,9,12-
,15-pentaoxaoctadecyl)-2-((4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-
-dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy-
)butyl)(methyl)amino)acetamide (example 70) The title compound was
synthesized using the same procedures for the preparation of the
example 67 compound as white solid (9 mg, 64%). .sup.1H NMR (600
MHz, CD.sub.3OD) .delta. 9.09-9.04 (m, 1H), 8.18 (dd, J=9.6, 2.9
Hz, 1H), 7.86 (d, J=2.9 Hz, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.31 (d,
J=0.9 Hz, 1H), 7.18 (dd, J=8.4, 1.9 Hz, 1H), 7.13 (d, J=2.2 Hz,
1H), 6.78 (d, J=8.5 Hz, 1H), 6.60 (d, J=0.9 Hz, 1H), 6.15 (d, J=2.3
Hz, 1H), 6.00 (p, J=8.9 Hz, 1H), 5.75 (d, J=2.3 Hz, 1H), 5.61 (d,
J=2.5 Hz, 1H), 3.97 (d, J=42.8 Hz, 2H), 3.86 (t, J=5.9 Hz, 2H),
3.83-3.73 (m, 13H), 3.63-3.56 (m, 18H), 3.54 (t, J=5.3 Hz, 2H),
3.48-3.38 (m, 5H), 3.36-3.21 (m, 8H), 2.95 (d, J=1.0 Hz, 3H), 2.72
(t, J=6.2 Hz, 2H), 2.50 (d, J=1.0 Hz, 3H), 2.42 (s, 3H), 2.31 (dd,
J=12.5, 7.3 Hz, 2H), 2.09 (s, 2H), 1.96-1.88 (m, 4H), 1.82 (q,
J=7.2, 6.6 Hz, 2H), 1.72 (dq, J=25.3, 6.3, 5.6 Hz, 4H), 1.07-0.88
(m, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.74H.sub.94N.sub.12O.sub.17S.sup.+, 1407.6653; found,
1407.6628.
##STR00280##
[0339]
N-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-oxoethyl)-2-((-
4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo-2,3-dihydro-
-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)amino)acetam-
ide (example 71) The title compound was synthesized using the same
procedures for the preparation of the example 67 compound as white
solid (7 mg, 60%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.08
(s, 1H), 8.13 (d, J=9.6 Hz, 1H), 7.84 (s, 1H), 7.54 (d, J=9.6 Hz,
1H), 7.32 (d, J=1.6 Hz, 1H), 7.18 (d, J=8.5 Hz, 1H), 7.13 (d, J=1.9
Hz, 1H), 6.78 (dd, J=8.6, 1.6 Hz, 1H), 6.62 (d, J=1.6 Hz, 1H), 6.14
(d, J=2.0 Hz, 1H), 6.01 (t, J=8.8 Hz, 1H), 5.73 (d, J=2.1 Hz, 1H),
5.64 (d, J=2.1 Hz, 1H), 4.23 (d, J=37.0 Hz, 2H), 4.08 (d, J=31.5
Hz, 2H), 3.87 (t, J=5.9 Hz, 2H), 3.82-3.68 (m, 10H), 3.60 (d, J=1.6
Hz, 3H), 3.41 (d, J=1.6 Hz, 3H), 3.25 (d, J=1.6 Hz, 8H), 2.98 (d,
J=1.6 Hz, 3H), 2.50 (d, J=1.6 Hz, 3H), 2.43 (d, J=1.7 Hz, 3H), 2.31
(d, J=9.0 Hz, 2H), 2.09 (s, 2H), 1.93 (dd, J=16.5, 9.0 Hz, 4H),
1.83 (d, J=7.2 Hz, 2H), 1.73 (dt, J=19.8, 9.9 Hz, 4H), 1.00 (td,
J=7.4, 1.7 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.59H.sub.73N.sub.12O.sub.12S.sup.+, 1173.5186; found,
1173.5195.
##STR00281##
[0340]
N-(4-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-4-oxobutyl)-2-((-
4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo-2,3-dihydro-
-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)amino)acetam-
ide (example 72) The title compound was synthesized using the same
procedures for the preparation of the example 67 compound as white
solid (7 mg, 58%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.07
(s, 1H), 8.14 (dd, J=9.5, 2.9 Hz, 1H), 7.84 (s, 1H), 7.54 (d, J=9.5
Hz, 1H), 7.31 (s, 1H), 7.18 (d, J=8.5 Hz, 1H), 7.13 (d, J=2.2 Hz,
1H), 6.78 (d, J=8.5 Hz, 1H), 6.61 (s, 1H), 6.14 (d, J=2.5 Hz, 1H),
6.00 (p, J=8.9 Hz, 1H), 5.74 (s, 1H), 5.62 (d, J=2.5 Hz, 1H), 3.98
(d, J=50.2 Hz, 2H), 3.86 (t, J=5.9 Hz, 2H), 3.80 (s, 3H), 3.78-3.66
(m, 7H), 3.61 (s, 3H), 3.41 (d, J=1.1 Hz, 3H), 3.31-3.18 (m, 10H),
2.95 (s, 3H), 2.52-2.46 (m, 5H), 2.42 (s, 3H), 2.31 (q, J=9.1, 7.5
Hz, 2H), 2.09 (s, 2H), 1.94-1.88 (m, 4H), 1.83 (dt, J=14.3, 7.4 Hz,
4H), 1.76-1.66 (m, 4H), 1.00 (t, J=7.4 Hz, 3H). HRMS (ESI-TOF) m/z:
[M+H].sup.+ calcd for C.sub.61H.sub.77N.sub.12O.sub.12S.sup.+,
1201.5499; found, 1201.5489.
##STR00282##
[0341]
N-(2-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropy-
rido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropoxy)-
ethyl)-2-((4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo--
2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)a-
mino)acetamide (example 73) The title compound was synthesized
using the same procedures for the preparation of the example 67
compound as white solid (8 mg, 65%). .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.05 (s, 1H), 8.10 (d, J=9.6 Hz, 1H), 7.84 (s,
1H), 7.56 (d, J=9.4 Hz, 1H), 7.34-7.26 (m, 1H), 7.18 (d, J=8.4 Hz,
1H), 7.13 (d, J=1.7 Hz, 1H), 6.84-6.72 (m, 1H), 6.67-6.50 (m, 1H),
6.14 (s, 1H), 6.00 (p, J=8.9 Hz, 1H), 5.74 (s, 1H), 5.63 (s, 1H),
3.99 (s, 2H), 3.86 (t, J=5.8 Hz, 2H), 3.83-3.72 (m, 15H), 3.60 (d,
J=1.1 Hz, 3H), 3.56 (t, J=5.4 Hz, 2H), 3.40 (d, J=1.1 Hz, 4H),
3.26-3.22 (m, 5H), 2.96 (d, J=1.2 Hz, 3H), 2.70 (t, J=5.9 Hz, 2H),
2.50 (d, J=1.2 Hz, 3H), 2.42 (d, J=1.2 Hz, 3H), 2.31 (dd, J=12.5,
7.3 Hz, 2H), 2.09 (s, 3H), 1.93 (dt, J=16.9, 8.3 Hz, 4H), 1.86-1.79
(m, 2H), 1.72 (dt, J=19.2, 9.6 Hz, 4H), 1.00 (td, J=7.5, 1.2 Hz,
3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.62H.sub.79N.sub.12O.sub.13S.sup.+, 1231.5605; found,
1231.5621.
##STR00283##
[0342]
N-(2-(2-(2-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dih-
ydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopr-
opoxy)ethoxy)ethoxy)ethyl)-2-((4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-
-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphe-
noxy)butyl)(methyl)amino)acetamide (example 74) The title compound
was synthesized using the same procedures for the preparation of
the example 67 compound as white solid (8 mg, 61%). .sup.1H NMR
(600 MHz, CD.sub.3OD) .delta. 9.06 (s, 1H), 8.16 (dd, J=9.6, 2.9
Hz, 1H), 7.85 (s, 1H), 7.53 (d, J=9.6 Hz, 1H), 7.30 (s, 1H), 7.18
(dd, J=8.5, 2.1 Hz, 1H), 7.12 (d, J=2.1 Hz, 1H), 6.78 (d, J=8.4 Hz,
1H), 6.60 (s, 1H), 6.14 (d, J=2.6 Hz, 1H), 6.00 (p, J=9.0 Hz, 1H),
5.74 (d, J=2.4 Hz, 1H), 5.61 (d, J=2.5 Hz, 1H), 3.98 (d, J=36.9 Hz,
2H), 3.85 (t, J=5.9 Hz, 2H), 3.82-3.70 (m, 13H), 3.63-3.52 (m,
15H), 3.45-3.36 (m, 5H), 3.29-3.19 (m, 5H), 2.95 (d, J=1.1 Hz, 3H),
2.72 (t, J=6.2 Hz, 2H), 2.50 (d, J=1.2 Hz, 3H), 2.42 (s, 3H), 2.30
(d, J=9.4 Hz, 2H), 2.09 (s, 2H), 1.92 (q, J=8.3, 7.9 Hz, 4H), 1.81
(p, J=6.3 Hz, 2H), 1.71 (ddd, J=23.2, 12.4, 6.2 Hz, 4H), 1.00 (t,
J=7.4 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.66H.sub.87N.sub.12O.sub.15S.sup.+, 1319.6129; found,
1319.6132.
##STR00284##
[0343]
N-(3-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-3-oxopropyl)-2-(-
(4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo-2,3-dihydr-
o-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)amino)aceta-
mide (example 75) The title compound was synthesized using the same
procedures for the preparation of the example 67 compound as white
solid (9 mg, 76%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.07
(s, 1H), 8.15 (dd, J=9.6, 2.9 Hz, 1H), 7.82 (d, J=2.9 Hz, 1H), 7.52
(d, J=9.5 Hz, 1H), 7.30 (d, J=1.2 Hz, 1H), 7.17 (dd, J=8.4, 2.0 Hz,
1H), 7.12 (d, J=2.2 Hz, 1H), 6.77 (d, J=8.4 Hz, 1H), 6.60 (d, J=1.2
Hz, 1H), 6.14 (t, J=1.9 Hz, 1H), 6.01 (p, J=8.9 Hz, 1H), 5.74 (t,
J=1.9 Hz, 1H), 5.61 (t, J=1.9 Hz, 1H), 3.96 (d, J=48.7 Hz, 2H),
3.86 (t, J=5.9 Hz, 2H), 3.80 (d, J=1.3 Hz, 3H), 3.76 (t, J=6.6 Hz,
6H), 3.69 (t, J=5.2 Hz, 2H), 3.60 (d, J=1.2 Hz, 5H), 3.40 (d, J=1.3
Hz, 3H), 3.30-3.21 (m, 7H), 2.95 (d, J=1.2 Hz, 3H), 2.68 (t, J=6.4
Hz, 2H), 2.51 (d, J=1.2 Hz, 3H), 2.43 (d, J=1.2 Hz, 3H), 2.37-2.27
(m, 2H), 2.10 (s, 2H), 1.91 (q, J=8.1 Hz, 4H), 1.86-1.79 (m, 2H),
1.72 (dq, J=14.0, 7.1 Hz, 4H), 1.00 (td, J=7.4, 1.2 Hz, 3H). HRMS
(ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.57H.sub.70N.sub.11O.sub.11S.sup.+, 1187.5343; found,
1187.5355.
##STR00285##
[0344]
N-(5-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-5-oxopentyl)-2-(-
(4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo-2,3-dihydr-
o-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)amino)aceta-
mide (example 76) The title compound was synthesized using the same
procedures for the preparation of the example 67 compound as white
solid (5 mg, 41%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.08
(d, J=1.2 Hz, 1H), 8.17 (dd, J=9.6, 2.9 Hz, 1H), 7.83 (d, J=2.9 Hz,
1H), 7.52 (d, J=9.5 Hz, 1H), 7.31 (d, J=1.2 Hz, 1H), 7.18 (dd,
J=8.5, 2.0 Hz, 1H), 7.13 (d, J=2.0 Hz, 1H), 6.78 (dd, J=8.5, 1.2
Hz, 1H), 6.61 (d, J=1.1 Hz, 1H), 6.14 (t, J=2.0 Hz, 1H), 6.01 (p,
J=8.9 Hz, 1H), 5.73 (t, J=2.0 Hz, 1H), 5.62 (t, J=2.0 Hz, 1H),
4.07-3.93 (m, 2H), 3.86 (t, J=5.9 Hz, 2H), 3.80 (d, J=1.2 Hz, 3H),
3.79-3.68 (m, 10H), 3.60 (d, J=1.2 Hz, 3H), 3.40 (d, J=1.2 Hz, 3H),
3.34-3.23 (m, 7H), 2.95 (d, J=1.3 Hz, 3H), 2.50 (d, J=1.3 Hz, 3H),
2.48-2.38 (m, 5H), 2.31 (q, J=9.0, 7.5 Hz, 2H), 2.10 (s, 2H), 1.92
(dt, J=15.5, 7.9 Hz, 4H), 1.82 (p, J=6.3 Hz, 2H), 1.72 (dq, J=20.1,
6.3, 5.5 Hz, 4H), 1.61 (dq, J=28.9, 7.8 Hz, 4H), 1.00 (td, J=7.5,
1.3 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.62H.sub.79N.sub.12O.sub.12S.sup.+, 1215.5656; found,
1215.5637.
##STR00286##
[0345]
N-(6-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-6-oxohexyl)-2-((-
4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo-2,3-dihydro-
-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)amino)acetam-
ide (example 77) The title compound was synthesized using the same
procedures for the preparation of the example 67 compound as white
solid (6 mg, 41%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.07
(s, 1H), 8.16 (d, J=9.6 Hz, 1H), 7.85 (s, 1H), 7.53 (d, J=9.5 Hz,
1H), 7.31 (d, J=1.2 Hz, 1H), 7.18 (dd, J=8.5, 1.9 Hz, 1H), 7.13 (d,
J=2.1 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 6.61 (d, J=1.2 Hz, 1H), 6.14
(t, J=1.9 Hz, 1H), 6.05-5.93 (m, 1H), 5.74 (t, J=1.9 Hz, 1H), 5.62
(t, J=1.9 Hz, 1H), 3.96 (d, J=46.5 Hz, 2H), 3.86 (t, J=5.9 Hz, 2H),
3.81 (d, J=1.3 Hz, 3H), 3.79-3.69 (m, 10H), 3.60 (d, J=1.3 Hz, 3H),
3.41 (d, J=1.3 Hz, 3H), 3.25 (d, J=1.3 Hz, 7H), 2.95 (d, J=1.3 Hz,
3H), 2.50 (d, J=1.3 Hz, 3H), 2.43 (d, J=6.8 Hz, 5H), 2.31 (q,
J=9.0, 7.5 Hz, 2H), 2.09 (s, 2H), 1.92 (dt, J=15.7, 8.0 Hz, 4H),
1.82 (q, J=7.1, 6.6 Hz, 2H), 1.73 (ddt, J=16.9, 10.7, 6.1 Hz, 4H),
1.59 (dp, J=38.0, 7.4 Hz, 4H), 1.38 (p, J=7.8 Hz, 2H), 1.01 (td,
J=7.4, 1.3 Hz, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.63H.sub.80N.sub.12O.sub.12S.sup.+, 1229.5812; found,
1229.5802.
##STR00287##
[0346]
N-(7-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-7-oxoheptyl)-2-(-
(4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo-2,3-dihydr-
o-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)amino)aceta-
mide (example 78) The title compound was synthesized using the same
procedures for the preparation of the example 67 compound as white
solid (8 mg, 64%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.08
(s, 1H), 8.18 (d, J=9.5 Hz, 1H), 7.85 (s, 1H), 7.53 (d, J=9.5 Hz,
1H), 7.32 (s, 1H), 7.22-7.16 (m, 1H), 7.13 (d, J=2.1 Hz, 1H), 6.78
(d, J=8.5 Hz, 1H), 6.61 (s, 1H), 6.14 (t, J=1.9 Hz, 1H), 6.00 (p,
J=8.9 Hz, 1H), 5.74 (t, J=1.9 Hz, 1H), 5.61 (d, J=1.8 Hz, 1H), 3.96
(d, J=44.8 Hz, 2H), 3.85 (t, J=5.8 Hz, 2H), 3.82-3.67 (m, 13H),
3.60 (d, J=1.3 Hz, 3H), 3.41 (d, J=1.2 Hz, 3H), 3.26 (dd, J=10.4,
3.4 Hz, 7H), 2.94 (d, J=1.3 Hz, 3H), 2.50 (d, J=1.3 Hz, 3H), 2.43
(d, J=5.7 Hz, 5H), 2.36-2.29 (m, 2H), 2.09 (s, 2H), 1.92 (p, J=7.6
Hz, 4H), 1.81 (p, J=6.4 Hz, 2H), 1.72 (dq, J=19.2, 6.7 Hz, 4H),
1.59 (d, J=6.9 Hz, 2H), 1.53 (t, J=6.9 Hz, 2H), 1.36 (s, 4H),
1.03-0.89 (m, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.64H.sub.83N.sub.12O.sub.12S.sup.+, 1243.5969; found,
1243.5967.
##STR00288##
[0347]
N-(8-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrid-
o[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-8-oxooctyl)-2-((-
4-(3-((6-((3,4-dimethoxyphenyl)sulfonamido)-1,3-dimethyl-2-oxo-2,3-dihydro-
-1H-benzo[d]imidazol-5-yl)oxy)-5-propoxyphenoxy)butyl)(methyl)amino)acetam-
ide (example 79) The title compound was synthesized using the same
procedures for the preparation of the example 67 compound as white
solid (8 mg, 64%). .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 9.08
(s, 1H), 8.19 (dd, J=9.6, 2.9 Hz, 1H), 7.85 (d, J=2.9 Hz, 1H), 7.52
(d, J=9.6 Hz, 1H), 7.32 (s, 1H), 7.18 (dd, J=8.4, 2.1 Hz, 1H), 7.14
(d, J=2.1 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 6.61 (s, 1H), 6.14 (d,
J=2.4 Hz, 1H), 6.01 (p, J=8.9 Hz, 1H), 5.75 (d, J=2.4 Hz, 1H), 5.60
(d, J=2.4 Hz, 1H), 3.96 (d, J=48.0 Hz, 2H), 3.85 (t, J=5.9 Hz, 2H),
3.83-3.69 (m, 13H), 3.61 (d, J=1.0 Hz, 3H), 3.41 (d, J=1.0 Hz, 3H),
3.28-3.21 (m, 7H), 2.94 (d, J=1.1 Hz, 3H), 2.50 (d, J=1.0 Hz, 3H),
2.43 (q, J=3.4 Hz, 5H), 2.31 (dd, J=12.5, 7.5 Hz, 2H), 2.10 (s,
2H), 1.96-1.87 (m, 4H), 1.82 (q, J=7.1, 6.6 Hz, 2H), 1.78-1.66 (m,
4H), 1.59 (d, J=7.4 Hz, 2H), 1.56-1.48 (m, 2H), 1.34 (s, 6H),
1.03-0.90 (m, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.65H.sub.85N.sub.12O.sub.12S.sup.+, 1257.6125; found,
1257.6103.
##STR00289##
[0348] tert-Butyl
2-(2-(42S,4R)-4-hydroxy-1-((S)-3-methyl-2-(1-oxoisoindolin-2-yl)butanoyl)-
pyrrolidine-2-carboxamido)methyl)-5-(4-methylthiazol-5-yl)phenoxy)acetate
(intermediate 28) To a solution of
(2S,4R)-4-hydroxy-N-(2-hydroxy-4-(4-methylthiazol-5-yl)benzyl)-1-((S)-3-m-
ethyl-2-(1-oxoisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide
(124 mg, 0.23 mmol) in DMF (2 mL) were added K.sub.2CO.sub.3 (130
mg, 0.94 mmol) and tert-butyl-2-bromo-acetate ester (0.037 mmL,
0.25 mmol). The mixture was stirred at 50.degree. C. for 18 h.
After cooling to RT, the mixture was filtered to remove the
unsolvable materials. The filtrate was concentrated under reduce
pressure. The resulting residue was purified by prep-HPLC to yield
the title compound (52 mg, 34%) as brown oil. .sup.1H NMR (600 MHz,
CD.sub.3OD) .delta. 9.10 (s, 1H), 7.80 (d, J=7.8 Hz, 1H), 7.67-7.57
(m, 2H), 7.56-7.47 (m, 2H), 7.11 (d, J=7.9 Hz, 1H), 6.98 (s, 1H),
4.88-4.85 (m, 1H), 4.78 (s, 2H), 4.68-4.46 (m, 6H), 3.99 (d, J=11.2
Hz, 1H), 3.91 (d, J=11.1 Hz, 1H), 2.53 (s, 3H), 2.50-2.40 (m, 1H),
2.27-2.18 (m, 1H), 2.15-2.05 (m, 1H), 1.50 (s, 9H), 1.04 (d, J=6.6
Hz, 3H), 0.84 (d, J=6.6 Hz, 3H).
[0349]
(2S,4R)--N-(2-(2-(4-(6-((6-Acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8--
dihydropyrido[2,3-d]pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-2-ox-
oethoxy)-4-(4-methylthiazol-5-yl)benzyl)-4-hydroxy-1-((S)-3-methyl-2-(1-ox-
oisoindolin-2-yl)butanoyl)pyrrolidine-2-carboxamide (example 126)
Intermediate 28 (52 mg, 0.078 mmol) was dissolved in DCM/TFA (2:1,
3 mL). The solution was stirred at RT for 3 h before being
concentrated. The residue was dissolved in DCM/DMF (3:1, 2 mL). To
the resulting solution were added palbociclib (33 mg, 0.074 mmol),
TBTU (25 mg, 0.076 mmol), and DIEA (0.04 mL, 0.24 mmol). The
reaction mixture was stirred at RT for 18 h. After concentration,
the resulting residue was purified by prep-HPLC to yield the title
compound (32 mg, 40%) as yellow solid. .sup.1H NMR (600 MHz,
Methanol-d.sub.4) .delta. 9.07 (s, 1H), 8.96 (s, 1H), 8.18 (dd,
J=9.7, 2.9 Hz, 1H), 7.89 (d, J=2.9 Hz, 1H), 7.74 (d, J=7.6 Hz, 1H),
7.58 (t, J=7.5 Hz, 1H), 7.55 (d, J=9.6 Hz, 1H), 7.51 (d, J=7.7 Hz,
1H), 7.48-7.44 (m, 2H), 7.12-7.07 (m, 2H), 5.99 (p, J=8.9 Hz, 1H),
5.14-5.00 (m, 2H), 4.88 (d, J=11.2 Hz, 1H), 4.62-4.41 (m, 6H), 4.02
(d, J=11.1 Hz, 1H), 3.96 (dd, J=11.1, 3.9 Hz, 1H), 3.91-3.83 (m,
4H), 3.39-3.30 (m, 4H), 2.53 (s, 3H), 2.52 (s, 3H), 2.44 (s, 3H),
2.43-2.38 (m, 1H), 2.37-2.23 (m, 3H), 2.16-2.05 (m, 3H), 1.98-1.85
(m, 2H), 1.76-1.66 (m, 2H), 1.03 (d, J=6.5 Hz, 3H), 0.83 (d, J=6.6
Hz, 3H). HRMS (ESI-TOF) m/z: [M+H].sup.+ calcd for
C.sub.55H.sub.62N.sub.11O.sub.8S.sup.+, 1036.4498; found,
1036.4508.
Materials and Methods:
General Chemistry Methods
[0350] HPLC spectra for all compounds were acquired using an
Agilent 1200 Series system with DAD detector. Chromatography was
performed on a 2.1.times.150 mm Zorbax 300SB-C18 5 .mu.m column
with water containing 0.1% formic acid as solvent A and
acetonitrile containing 0.1% formic acid as solvent B at a flow
rate of 0.4 ml/min. The gradient program was as follows: 1% B (0-1
min), 1-99% B (1-4 min), and 99% B (4-8 min). High-resolution mass
spectra (HRMS) data were acquired in positive ion mode using an
Agilent G1969A API-TOF with an electrospray ionization (ESI)
source. Nuclear Magnetic Resonance (NMR) spectra were acquired on a
Bruker DRX-600 spectrometer with 600 MHz for proton (.sup.1H NMR)
and 150 MHz for carbon (.sup.13C NMR); chemical shifts are reported
in (6). Preparative HPLC was performed on Agilent Prep 1200 series
with UV detector set to 254 nm. Samples were injected onto a
Phenomenex Luna 75.times.30 mm, 5 .mu.m, C.sub.18 column at room
temperature. The flow rate was 40 ml/min. A linear gradient was
used with 10% (or 50%) of MeOH (A) in H.sub.2O (with 0.1% TFA) (B)
to 100% of MeOH (A). HPLC was used to establish the purity of
target compounds. All final compounds had >95% purity using the
HPLC methods described above.
Cell Culture
[0351] Breast cancer cell lines (MCF7, T47D, and ZR-75-1) and
melanoma cell lines (A375, SK-MEL-2, SK-MEL-30, and WM1382) were
cultured in the presence of DMEM or RPMI supplemented with 10% FBS
in the presence of penicillin and streptomycin.
Cell Viability Assay
[0352] Cells were cultured for 7-11 days in the presence of
different compounds. Media with compound was replenished every two
days. At the end of the experiment, media was aspirated and viable
cells were stained with 0.5% crystal violet dye.
BrdU Cell Proliferation Assay
[0353] BrdU incorporation was measured using a BrdU cell
proliferation kit (Millipore) according to the manufacturer's
instructions. Briefly, ER+ or melanoma cells were seeded at
1*10.sup.5 cells/ml in 96-well plates. The following day, cultured
cells were treated with different concentrations of abemaciclib or
palbociclib for 12 h, followed by incubation with BrdU labeling
solution for 4 h. BrdU absorbance was measured with a
spectrophotometer microplate reader at a dual wavelength of 450/550
nm. The measured absorbance reflected the degree of cell
proliferation. Each group was cultured in triplicate.
Quantitative Real-Time PCR Analysis
[0354] Cells were treated with CDK4/6i for 24 h, and total RNA was
extracted using an RNeasy Mini kit (Qiagen). Complementary DNA
(cDNA) was synthesized with a SuperScript III First Strand
Synthesis System (Thermo Fisher). Quantitative real-time PCR was
performed using a Fast SYBR Green Master Mix with an ABI-7500 Fast
Real-Time PCR System. Samples were normalized using the
housekeeping gene GAPDH. Differences in expression were calculated
using the .DELTA..DELTA.CT method.
[0355] The following primer sequences were used in PCR
analyses:
TABLE-US-00002 GAPDH: F: (SEQ ID NO: 1)
5'-ACAACTTTGGTATCGTGGAAGG-3'; R: (SEQ ID NO: 2)
5'-GCCATCACGCCACAGTTTC-3' CDK4: F: (SEQ ID NO: 3)
5'-CTGGTGTTTGAGCATGTAGACC-3'; R: (SEQ ID NO: 4)
5'-GATCCTTGATCGTTTCGGCTG-3' CDK6: F: (SEQ ID NO: 5)
5'-TCTTCATTCACACCGAGTAGTGC-3'; R: (SEQ ID NO: 6)
5'-TGAGGTTAGAGCCATCTGGAAA-3' CCNA2: F: (SEQ ID NO: 7)
5'-CGCTGGCGGTACTGAAGTC-3'; R: (SEQ ID NO: 8)
5'-GAGGAACGGTGACATGCTCAT-3' PLK1: F: (SEQ ID NO: 9)
5'-CACCAGCACGTCGTAGGATTC-3'; R: (SEQ ID NO: 10)
5'-CCGTAGGTAGTATCGGGCCTC-3'
Western Blot Assay
[0356] Protein concentrations were determined by BCA from cell
lysates and equal amounts of protein are loaded to a Bis-Tris 4-12%
gel. Gels were run at 80 V for 4 h using 1.times. running buffer
(25 mM Tris, pH 8.3, 1.92 M glycine, 0.1% SDS). Proteins were
transferred to a nitrocellulose membrane for 2 h at 100 V using
transfer buffer (25 mM Tris, pH 8.3, 1.92 M glycine, 20% methanol).
Ponceau S staining (0.1% Ponceau S, 1% acetic acid) was used to
confirm transfer of proteins to the membrane followed by blocking
of the membrane for 30 min at room temperature using blocking
buffer (5% dry nonfat milk, 0.1% Tween, 0.02% sodium azide in TBS).
The blocked membrane is incubated with primary antibodies (1:1000
dilution) in blocking buffer at 4.degree. C. overnight. When
primary phosphor-antibodies were used, then the membrane is
incubated with 5% BSA instead of milk. The membrane was washed
three times, 10 min each, with wash buffer (0.1% Tween in TBS)
followed by incubation with secondary HRP conjugated antibody
(1:5000 dilution) in 2.5% dry nonfat milk, 0.1% Tween in TBS for 1
h at room temperature. The membrane was washed three times, 10 min
each, with wash buffer (0.1% Tween in TBS). Membranes are incubated
with ECL for 1 min and proteins are detected after exposure to a
blue autoradiographic film.
Example 5. CDK4/6 Inhibitors Fail to Suppress CDK4/6 Activity at
High Concentrations
[0357] MCF7, T47D, or ZR-75-1 breast cancer cells were treated with
0, 0.1, 0.3, 1, 3, 10, or 30 .mu.M palbociclib (PB) or abemaciclib
(AB) for 24 h, then lysed and immunoblotted with antibodies to
phospho-Rb, PLK1, cyclin A, and total Rb and actin (as controls)
(FIG. 1, panels A and B). Similarly, SK-MEL-2, SK-MEL-30, or MCF7
melanoma cells were treated with 0, 0.1, 0.3, 1, 3, 10, or 30 .mu.M
palbociclib (PB), abemaciclib (AB), or 219476 for 24 h, then lysed
and immunoblotted with antibodies to phospho-Rb, PLK1, cyclin A,
and total Rb and actin (as controls) (FIG. 1, panels C, D, and E).
The results indicated that all three CDK4/6 inhibitors tested only
had a narrow window of activity. As expected, they failed to
suppress CDK4/6 activity (as evidenced by Rb phosphorylation (pRb
level)) in breast cancer or melanoma cells when administered at low
concentrations; surprisingly, they also failed to suppress CDK4/6
activity when administered at high concentrations (e.g., a
concentration .gtoreq.about 10 .mu.M).
Example 6. CDK4/6 Inhibitors Increase Expression of PLK1 and Cyclin
A mRNA
[0358] MCF7, T47D, or ZR-75-1 breast cancer cells or SK-MEL-2 or
SK-MEL-30 melanoma cells were treated with 0, 1, 7.5, 15, or 30
.mu.M palbociclib or abemaciclib for 24 h, then lysed and subjected
to PCR analysis of expression of the Rb/E2F downstream targets PLK1
and cyclin A (CCNA2). Neither palbociclib nor abemaciclib
suppressed expression of PLK1 or cyclin A at the mRNA level; in
fact, there was a positive correlation between increased inhibitor
concentration and increased gene expression (FIGS. 2 and 3). This
data suggests a mechanistic explanation for the failure of CDK4/6
inhibitors to suppress CDK4/6 activity when administered at high
concentrations.
Example 7. CDK4/6 Inhibitors Suppress Cell Proliferation Less
Effectively at High Concentrations
[0359] MCF7, T47D, or ZR-75-1 breast cancer cells or SK-MEL-2 or
SK-MEL-30 melanoma cells were treated with 0, 0.3, 1, 3, 10, 20, or
30 .mu.M palbociclib or abemaciclib for 16 h; cell cycle
progression through S phase was determined using the BrdU
incorporation assay. Consistent with the results in Examples 5 and
6 (above), the results indicated that there was an inverse
correlation between increased inhibitor concentration and
suppression of cell cycle progression (FIG. 4).
Example 8. CDK4/6 Inhibitors Increase Expression of CDK4/6
[0360] MCF7, T47D, or ZR-75-1 breast cancer cells or SK-MEL-2
melanoma cells were treated with 0, 0.1, 0.3, 1, 3, 10, or 30 .mu.M
palbociclib or abemaciclib for 24 h, then lysed and immunoblotted
with antibodies to CDK4, CDK6, and actin. The results indicated
that, despite the CDK4/6 inhibitors' ability to inhibit the
activity of CDK4 and CDK6, the inhibitors actually upregulate the
expression of both CDK4 and CDK6, with a positive correlation
between increased inhibitor concentration and increased CDK4/6
expression (FIG. 5, panels A-C). This data suggests a mechanistic
explanation for the positive correlation between CDK4/6 inhibitor
concentration and increased expression of PLK1 and cyclin A
mRNA.
[0361] MCF7, T47D, or ZR-75-1 breast cancer cells were treated with
0, 1, 7.5, 15, or 30 .mu.M palbociclib or abemaciclib for 24 h,
then lysed and subjected to PCR analysis of expression of CDK4. The
CDK4/6 inhibitors did not upregulate the expression of CDK4 mRNA
(FIG. 6), indicating that the CDK4/6 inhibitors increase CDK4/6
protein expression by inhibiting protein degradation (instead of by
increasing transcription or extending mRNA half-life).
[0362] To confirm the above findings, 293H cells ectopically
expressing V5-tagged CDK4 or V5-tagged CDK6 were treated with 1, 3,
or 10 .mu.M palbociclib (PB), abemaciclib (AB), or ribociclib (RIB)
for 24 h, then lysed and immunoblotted with antibodies to V5 (to
detect V5-tagged CDK4 or CDK6) and actin. Consistent with FIG. 5,
all three CDK4/6 inhibitors examined upregulated the expression of
V5-tagged CDK4 and CDK6 (FIG. 7).
Example 9. CDK4/6 Inhibitors Prevent Ubiquitination of CDK4
[0363] 293H cells ectopically co-expressing V5-tagged CDK4 and
HA-tagged ubiquitin were treated with 1 or 10 .mu.M palbociclib or
abemaciclib for 24 h, then lysed and either 1) subjected to
immunoprecipitation with a V5 antibody (to pull down VS-tagged
CDK4) followed by immunoblotting for HA (to detect HA-tagged
ubiquitin), or 2) directly immunoblotted with antibodies to V5, HA,
and actin. The data indicated that CDK4/6 inhibitors inhibit the
ubiquitination of CDK4, thus protecting it from degradation and
inducing elevated cellular levels of CDK4 (FIG. 8, panel A).
[0364] T47D breast cancer cells were treated with the proteasome
inhibitor MG132 for 24 h in the presence or absence of 1 or 10
.mu.M abemaciclib, then lysed and either 1) subjected to
immunoprecipitation with a CDK4 antibody followed by immunoblotting
for ubiquitin, or 2) directly immunoblotted with antibodies to
ubiquitin, CDK4, and actin. Consistent with the results above for
VS-tagged CDK4, the data indicated that CDK4/6 inhibitors inhibit
the ubiquitination of native CDK4 (FIG. 8, panel B).
Example 10 Increased CDK4/6 Expression is Associated with Decreased
CDK4/6 Inhibitor Efficacy
[0365] MCF7 breast cancer cells resistant to palbociclib (MCF7-PBR)
were generated by treating MCF7 cells with 1 .mu.M palbociclib for
12 weeks. MCF7-PBR cells were then treated with 1, 3, or 10 .mu.M
palbociclib (PB) for 24 h, then lysed and immunoblotted with
antibodies to phospho-Rb (pRB), Rb (RB), PLK1, cyclin A, CDK2,
CDK6, and actin (FIG. 9). These results confirmed the resistance of
the MCF7-PBR cells to palbociclib and demonstrated that this
resistance is associated with elevated expression of CDK6 (compare
CDK6 levels in the MCF7 lane with CDK6 levels in any of the
MCF7-PBR lanes, noting that the actin control indicates equal
amounts of total protein in the MCF7 and MCF7-PBR lanes).
Example 11. Palbociclib Suppresses Rb Phosphorylation More
Effectively in ER+ Breast Cancer Cells
[0366] MCF7 or T47D breast cancer cells or SK-MEL-2, A375, or
WM1382 melanoma cells were treated with or without 0.5 .mu.M
palbociclib for 24 h, then lysed and immunoblotted with antibodies
to phospho-Rb (pRb), cyclin A, PLK1, CDK4, CDK6, and actin (FIG.
9). The results indicated that palbociclib suppresses Rb
phosphorylation more effectively in ER+ breast cancer cells than
melanoma cells.
Example 12. CDK4/6 Degraders/Disruptors Inhibit CDK4/6 Activity and
Suppress CDK4/6 Expression
[0367] MCF7 breast cancer cells were treated with 1 or 5 .mu.M
XY019-095, XY019-098, XY019-0100, XY019-105A, XY019-105B,
XY019-106, or XY019-108 for 24 h, then lysed and immunoblotted with
antibodies to CDK4, CDK6, and actin (FIG. 10, panel A). The results
indicated that XY019-098, XY019-106, and XY019-108 were especially
effective in suppressing CDK4/6 expression even at 1 .mu.M; this
effect did not diminish at 5 .mu.M. T47D breast cancer cells were
treated with 0.1, 0.3, 1, 3, 10, or 30 .mu.M XY019-106 for 24 h,
then lysed and immunoblotted with antibodies to phospho-Rb (pRB),
CDK4, and actin (FIG. 10, panel B). The results showed that
XY019-106 both suppresses CDK4 expression and inhibit CDK4 activity
(as evidenced by decreased Rb phosphorylation), with significant
effects at concentrations as low as 0.3 .mu.M. Further, unlike
CDK4/6 inhibitors such as palbociclib, XY019-106 inhibited Rb
phosphorylation even when administered at high concentrations
(e.g., at concentrations as high as 30 .mu.M).
[0368] SK-MEL-2 or A375 melanoma cells (FIG. 11, panels A and B)
were treated with 1 or 3 .mu.M XY019-106, XY028-002, XY028-003,
XY028-004, XY028-005, XY028-006, or XY028-007 for 24 h, then lysed
and immunoblotted with antibodies to CDK4, CDK6, phospho-Rb (pRb),
and actin. Similarly, A375 melanoma cells were treated with 1 or 3
.mu.M palbociclib (PB), XY019-106, XY028-003, XY028-082, XY028-083,
XY028-084, or XY028-085 for 24 h, then lysed and immunoblotted with
antibodies to CDK6, CDK4, phospho-Rb (pRb), cyclin A, PLK1, and
actin (FIG. 11, panel C).
[0369] The results confirmed the efficacy of CDK4/6
degraders/disruptors, particularly XY019-106 and XY028-003, in
inhibiting both CDK4/6 expression and CDK4/6 activity (as evidenced
by decreased Rb phosphorylation).
[0370] A375 melanoma cells (FIG. 12, panel A) were treated with 1
or 3 .mu.M palbociclib (PB), XY019-106, XY028-003, XY028-132,
XY028-133, XY028-114, XY028-097, XY028-105, or XY028-106 for 24 h,
then lysed and immunoblotted with antibodies to CDK4, CDK6,
phospho-Rb (pRb), and actin. Similarly, A375 melanoma cells were
treated with 1 or 3 .mu.M palbociclib (PB), XY019-106, XY028-003,
XY028-132, XY028-133, XY028-140, XY028-141, XY028-142, or XY028-143
for 24 h, then lysed and immunoblotted with antibodies to CDK6,
CDK4, phospho-Rb (pRb), cyclin A, PLK1, and actin (FIG. 12, panel
B). Similarly, A375 melanoma cells were treated with 1 or 3 .mu.M
XY028-132, XY028-133, XY028-140, XY028-141, XY028-142, XY028-143,
XY028-144, or XY028-145 for 24 h, then lysed and immunoblotted with
antibodies to CDK6, CDK4, cyclin A, PLK1, and actin (FIG. 12, panel
C). Similarly, A375 melanoma cells were treated with 1 or 3 .mu.M
palbociclib (PB), XY019-106, XY028-103, XY028-108, XY028-132,
XY028-133, or XY028-140 for 24 h, then lysed and immunoblotted with
antibodies to CDK6, CDK4, phospho-Rb (pRb), cyclin A, PLK1, and
actin (FIG. 12, panel D).
[0371] The results confirmed the efficacy of CDK4/6 degraders,
particularly XY028-133, XY028-140, XY019-106 and XY028-003, in
inhibiting both CDK4/6 expression and CDK4/6 activity (as evidenced
by decreased Rb phosphorylation).
[0372] A375 melanoma cells (FIG. 13) were treated with 1 or 3 .mu.M
Ribociclib (RB), YX030-126, YX030-108, YX030-107, YX030-086,
YX030-085 or XY028-133 for 24 h, then lysed and immunoblotted with
antibodies to CDK4, CDK6, cyclin A, PLK1, phospho-Rb (pRb), and
actin.
[0373] The results demonstrated that ribociclib-based CDK4/6
degraders, in particular YX-085, are likewise effective in
inhibiting both CDK4/6 expression and CDK4/6 activity (as evidenced
by decreased Rb phosphorylation).
[0374] A375 melanoma cells (FIG. 14) were treated with 1 or 3 .mu.M
Abemaciclib (AB), YX030-125, YX030-117, YX030-118 or XY028-133 for
24 h, then lysed and immunoblotted with antibodies to CDK4, CDK6,
cyclin A, PLK1, phospho-Rb (pRb), and actin.
[0375] ZR-75-1 breast cancer cells (FIG. 15, panel A) or SK-MEL-2
melanoma cells (FIG. 15, panel B) were treated with 1 or 5 .mu.M
abemaciclib (AB), YX26-56, YX26-58, or YX26-66 for 24 h, then lysed
and immunoblotted with antibodies to phospho-Rb (pRb), total Rb,
PLK1, cyclin A, CDK4, CDK6, and actin. The results demonstrated
that abemaciclib-based CDK4/6 degraders/disruptors are likewise
effective in inhibiting both CDK4/6 expression and CDK4/6 activity
(as evidenced by decreased Rb phosphorylation).
[0376] A375 melanoma cells (FIG. 16, panel A) were treated with 0.3
or 1 .mu.M XY028-140, YX039-65, YX039-48, YX039-123, YX039-56,
YX039-124 or YX039-147 for 24 h. A375 melanoma cells were treated
with 1 or 3 .mu.M XY028-140, YX039-56, YX039-65 or XY028-133 (FIG.
16, panel B). Cells were lysed and immunoblotted with antibodies to
phospho-Rb (pRb), total Rb, PLK1, cyclin A, CDK4, CDK6, and actin.
The results confirmed the efficacy of CDK4/6 degraders,
particularly XY028-133 in inhibiting both CDK4/6 expression and
CDK4/6 activity (as evidenced by decreased Rb phosphorylation).
[0377] T47D breast cancer cells (FIG. 17) were treated with 0.3 or
1 .mu.M XY028-140, YX039-48, YX039-123, YX039-56, YX039-124,
YX039-65, YX039-147 or YX039-74 for 24 h, then lysed and
immunoblotted with antibodies to phospho-Rb (pRb), total Rb, PLK1,
cyclin A, CDK4, CDK6, and actin. The results confirmed the efficacy
of CDK4/6 degraders, particularly XY028-140 and YX039-123, in
inhibiting both CDK4/6 expression and CDK4/6 activity (as evidenced
by decreased Rb phosphorylation).
[0378] A375 melanoma cells (FIG. 18) were treated with 1 or 3 .mu.M
XY028-133, YX044-18, YX044-22, XY028-140, YX044-19 or YX044-38 for
24 h, then lysed and immunoblotted with antibodies to phospho-Rb
(pRb), total Rb, PLK1, cyclin A, CDK4, CDK6, and actin. The results
confirmed the efficacy of CDK4/6 degraders, particularly XY028-133
and YX044-18, in inhibiting both CDK4/6 expression and CDK4/6
activity (as evidenced by decreased Rb phosphorylation).
[0379] T47D breast cancer cells (FIG. 19) were treated with 0.3 or
1 .mu.M XY028-133, YX044-18, YX044-22, XY028-140, YX044-19 or
YX044-38 for 24 h, then lysed and immunoblotted with antibodies to
phospho-Rb (pRb), total Rb, PLK1, cyclin A, CDK4, CDK6, and actin.
The results confirmed the efficacy of CDK4/6 degraders,
particularly XY028-140 and YX044-22 and YX044-19, in inhibiting
both CDK4/6 expression and CDK4/6 activity (as evidenced by
decreased Rb phosphorylation).
Example 13. CDK4/6 Degraders/Disruptors Inhibit Cancer Cell
Proliferation
[0380] A375 melanoma cells (FIG. 20) were treated with 1 or 2 .mu.M
palbociclib or XY028-133 for 7 days. Bright field imaging indicated
that XY028-133 was more effective in inhibiting cancer cell
proliferation than palbociclib at the same concentration in
melanoma cells.
[0381] A375 melanoma cells (FIG. 21) were treated with 0.03, 0.1,
0.3, 1, or 3 .mu.M palbociclib or XY028-133 for 11 days. Crystal
violet staining indicated that XY028-133 was more effective in
inhibiting cancer cell proliferation than palbociclib at 1 and 3
.mu.M in melanoma cells.
[0382] T47D breast cancer cells (FIG. 22) were treated with 0.03,
0.1, 0.3, 1, or 3 .mu.M palbociclib, XY028-140, YX039-48,
YX039-124, YX039-123, YX039-147, YX039-56, or YX039-65 for 11 days.
Crystal violet staining indicated that CDK4/6 degraders and
particularly XY028-140, YX039-123, YX039-56, and YX039-65 inhibited
cancer cell proliferation more effective that palbociclib at the
same concentration in breast cancer cells.
[0383] T47D breast cancer cells (FIG. 23) were treated with 0.003,
0.01, or 0.03 .mu.M palbociclib, XY028-140, YX039-123, YX039-56, or
YX039-65 for 11 days. Crystal violet staining indicated that CDK4/6
degraders inhibited cancer cell proliferation more effective that
palbociclib at the same concentration in breast cancer cells.
OTHER EMBODIMENTS
[0384] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, 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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Sequence CWU 1
1
10122DNAArtificial SequenceAmplification Primer 1acaactttgg
tatcgtggaa gg 22219DNAArtificial SequenceAmplification Primer
2gccatcacgc cacagtttc 19322DNAArtificial SequenceAmplification
Primer 3ctggtgtttg agcatgtaga cc 22421DNAArtificial
SequenceAmplification Primer 4gatccttgat cgtttcggct g
21523DNAArtificial SequenceAmplification Primer 5tcttcattca
caccgagtag tgc 23622DNAArtificial SequenceAmplification Primer
6tgaggttaga gccatctgga aa 22719DNAArtificial SequenceAmplification
Primer 7cgctggcggt actgaagtc 19821DNAArtificial
SequenceAmplification Primer 8gaggaacggt gacatgctca t
21921DNAArtificial SequenceAmplification Primer 9caccagcacg
tcgtaggatt c 211021DNAArtificial SequenceAmplification Primer
10ccgtaggtag tatcgggcct c 21
* * * * *
References