Multicomponent reactions, applications in medicinal chemistry & new modalities in drug
discovery
Konstantinidou, Markella
DOI:10.33612/diss.111908148
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date: 2020
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Konstantinidou, M. (2020). Multicomponent reactions, applications in medicinal chemistry & new modalities in drug discovery. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.111908148
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
CHAPTER
10
DISCOVERY OF PROTEOLYSIS TARGETING CHIMERAS
FOR THE CYCLIN-DEPENDENT KINASES 4 AND 6
(CDK4/6)
ABSTRACT
Here we show an application of proteolysis targeting chimeras (PROTACs) on the cyclin-dependent kinases 4 and 6 (CDK4/6). Currently, three small molecules have been approved by the FDA as dual inhibitors. The ultimate selectivity for those two kinases seems elusive with small molecules, due to the highly conserved ATP binding site. However, differences on the lysine residues that are recognised by the E3 ligase could lead to selectivity by converting a dual inhibitor into a selective degrader. The project focuses on abemaciclib, an inhibitor developed by Eli Lilly and we present the rational for the design, the synthesis of PROTACs, as well as preliminary biological data. Future perspectives are also discussed.
INTRODUCTION
The dysregulation of cell division, resulting in uncontrolled cell proliferation is one of the key hallmarks of cancer. Cancer treatments often rely on blocking the uncontrollable cell division. Under normal conditions, cell division is regulated by a complex network of regulatory factors, which strictly control the transition from one stage of the cell cycle to the next. The cell cycle is regulated by the interaction of cyclins with their partner serine/threonine cyclin-dependent kinases (CDKs). Due to their role, CDKs have been considered promising cancer targets.[1,2]
The CDK family in mammals is divided in three cell-cycle related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20).[3] In particular for
breast cancer, the cyclin-dependent kinases CDk4 and CDk6 have been proven to be a significant target. The cyclin-dependent kinases 4 and 6 (CDK4/6) form complexes with D-type cyclins and mediate the transition from G0/G1-phase to Sphase of the cell cycle by phosphorylating the tumor suppressor retinoblastoma (Rb).[4] In the clinic, the first-generation of non-selective CDK
inhibitors aiming to block cell proliferation was met with toxicity in non-cancer cells and lack of efficacy. Currently, three selective, orally bioavailable dual CDK4/6 inhibitors are approved for breast cancer; palbociclib, ribociclib and abemaciclib.[5]
Palbociclib (PD-0332991), developed by Pfizer, was the first CDK4/6 inhibitor to gain FDA
approval in 2015. Ribociclib (LEE 011), developed by Novartis and Astex Pharmaceuticals was approved in March 2017. The last compound to gain approval in September 2017 was abemaciclib
(LY2835219), developed by Eli Lilly.
Palbociclib
(PD-0332991) Ribociclib(LEE 011) Abemaciclib(LY2835219)
CDK1: >10 μM CDK1: >100 μM CDK1: >1 μM CDK2: >10 μM CDK2: >50 μM CDK2: >500 nM CDK4: 9–11 nM CDK4: 10 nM CDK4: 2 nM CDK5: >10 μM CDK5: ND CDK5: ND CDK6: 15 nM CDK6: 39 nM CDK6: 5 nM CDK7: ND CDK7: ND CDK7: 300 nM CDK9: ND CDK9: ND CDK9: 57 nM
Figure 1.Structures of selective CDK4/6 inhibitors and reported IC50 values in cell-free assays.[5,6]
All three compounds are ATP competitive inhibitors of CDK4 and CDK6 and are highly selective in comparison to CDK1 and CDK2. The predominant effect of those dual inhibitors in cytostatic, rather than cytotoxic.[5]
Due to the fact that the approved compounds are targeting the ATP binding site, which is highly conserved, achieving selectivity was not a trivial task. Moreover, there is always the risk in kinase inhibitors of developing mutations that could lead to resistance. As it was discussed in the previous chapter, proteolysis targeting chimeras (PROTACs) have been able to convert non-selective inhibitors to non-selective degraders. Furthermore, in the dual CDK4/6 inhibitors, based on the reported co-crystal structures, the piperazine component is solvent exposed and thus provides an excellent and obvious position to attach the linker. Indeed, in early 2019, four groups reported CDK4/6 degraders, aiming to achieve selective degradation of CDK4 and CDK6 (Figure 2). The latter, would be useful for developing tool compounds or probes in order to dissect pharmacologically the distinct biological functions.
The first report for CDK4/6 PROTACs was published in February 2019[7] and focused on palbociclib-
and ribociclib-derived PROTACs. Pomalidomide was used as the ligand targeting cereblon, whereas triazole linkers with varying length were studied. The palbociclib PROTACs led to more efficient degradation than ribociclib PROTACs, with pal-pom being the most potent degrader.
The same month, a detailed study was published by Gray et al.[8] The authors show both dual
degraders of CDK4/6, but also for the first time selective degraders targeting either CDK4 or CDK6. Pomalidomide-based PROTACs with palbociclib, ribociclib and abemaciclib as the kinase inhibitors were tested. The choice of the inhibitor, as well as the length and type of the linkers had an impact on the selectivity profile of the degraders. For example PROTAC BSJ-02-162 is a dual degrader, whereas BSJ-01-187, using the same linker but with ribociclib instead of palbociclib is a selective CDK4 degrader. Interestingly, PROTAC YKL-06-102 with a long polyethylene glycol (PEG) linker is selectively degrading CDK6.
Almost simultaneously, a report for selective degradation of CDK6 by a palbociclib based PROTAC was published by Natarajan et al.[9] The authors show that although palbociclib is a dual inhibitor
and the ATP binding site is highly conserved, there are differences in the distribution of surface exposed lysine residues, required for ubiquitination by an E3 ligase in CDK4 and CDK6. The most potent PROTAC (PROTAC 6) was indeed a selective CDK6 degrader and in good agreement with the work reported by Gray et al.,[8] it is also based on a long PEG linker. A few months later, Rao et
al.[10] reported selective CDK6 degraders. The study included ribociclib, palbociclib and abemaciclib
as CDK4/6 inhibitors and interestingly four E3 ligase ligands targeting cereblon (pomalidomide), von Hippel Lindau (VH032), MDM2 (nutlin-3b) and cIAP (bestatin). In in vitro assays, CDK6 was degraded only with cereblon-recruiting PROTACs. The degradation of CDK4 was in general less significant than CDK6. Among the CRBN-recruiting PROTACs, palbociclib-derived degraders were moderately superior to abemaciclib-derived, whereas ribociclib-derived PROTACs barely induced
CDK6 degradation. The most potent PROTAC from these series, after varying the linker size, length and orientation, was CP-10, a palbociclib-cereblon PROTAC with a triazole linker.
The available data clearly demonstrate the potential of CDK4/6 PROTACs in achieving selective degradation, being in this aspect superior to the approved inhibitors. However, the principles for this selectivity are not totally elucidated. The published reports demonstrate the degradation potential in different cell lines, but to the best of our knowledge, in vivo experiments have not been reported to date. Moreover, there are clearly differences depending on the linkers, as expected, but also depending on the chosen kinase inhibitor. Palbociclib and ribociclib are more studied in the concept of PROTACs, however abemaciclib has received considerably less attention, despite being the most active inhibitor, based on the published IC50 values. Our aim in this project was to design and synthesize abemaciclib-based PROTACs, varying not only the linker length, but also the rigidity, since this seems to have a great impact on the biological effect and selectivity. Furthermore, depending on the degradation potential of these series, we aim to include in this study in vivo experiments in mice. The ultimate goal is to elucidate the structural features that have an effect on the degradation potential and this could be achieved by the co-crystal structure of a ternary complex, which would clearly facilitate future design of CDK4/6 PROTACs.
Figure 2.Structures of reported CDK4/6 PROTACs.
RESULTS AND DISCUSSION
The first step in designing abemaciclib-based PROTACs was to study the interactions of the ligand with the target protein in order to determine the optimal position for structure modification and linker attachment. The structural analysis was performed on the co-crystal structure of abemaciclib with CDK6 [PDB 5L2S], using the software DesertScorpion (http://saas1.desertsci.com/).
Figure 3. Top left: Hydrogen bonds (red dashes) between abemaciclib (magenta sticks) and key aminoacids (blue sticks), top right: van der Waals interactions (yellow dashes) between abemaciclib (magenta sticks) and key aminoacids (blue sticks), bottom left: surface representation of CDK6 (blue surface) and abemaciclib as magenta sticks, bottom left: Scorpion scores for abemaciclib (red balls: most significant interacting atoms, purple balls: significant atoms, grey balls: atoms insignificant for the interaction).
The performed structural analysis clearly indicates that the ethyl-piperazine moiety, which is completely solvent exposed, does not contribute to the binding. It does not participate to any specific interactions. Moreover, the methylene group connecting the amino-pyridine ring to the piperazine ring seems also of minor significance. Thus, the hypothesis is that the necessary linker for connecting the E3 ligase ligand could be attached on the piperazine ring, replacing the ethyl-substituent, without compromising the affinity to the kinase.
Regarding the synthetic part of the project, abemaciclib was synthesized as a reference compound and a straightforward route was designed for abemaciclib PROTACs, using ligands targeting cereblon and with various linkers.
1. Synthetic route for abemaciclib and main intermediates.
Abemaciclib was synthesized in two steps with slight modifications on the known synthetic routes.[11-12]The first step was a Suzuki coupling with commercially available starting materials to
obtain the main intermediate (1). With the modified reaction conditions the yield was 98%.
Scheme 1. Suzuki coupling.
Intermediate (1) was then used in a Buchwald-Hartwig coupling, which could be performed under microwave irradiation in 1 h, instead of overnight reflux, with a yield of 60 % in the case of abemaciclib (2).
Scheme 2. Buchwald-Hartwig coupling for the synthesis of abemaciclib.
For the synthesis of abemaciclib-based PROTACs, different 2-aminopyridines with various substituents were used in the Buchwald-Hartwig coupling. However, this step is substrate dependent, and in some cases the yields were very low.
R-substituent Result
-OH Starting materials
-Br Starting materials
-I Starting materials
-CHO Traces of product
-COOMe Traces of product
N N Boc Yield 55% (3)
Scheme 3. Buchwald-Hartwig coupling for the synthesis of intermediates.
Compound (3), which was isolated with 55% yield, was deprotected with HCl in dioxane (3a) and used as the main intermediate for the PROTACs.
2. Synthetic routes for cereblon building blocks.
The first synthetic step is the condensation of 4-nitroisobenzofuran-1,3-dione with 3-aminopiperidine-2,6-dione to obtain the nitro-substituted imide (4), which is then reduced to the aniline group to obtain pomalidomide (5). Pomalidomide (5) was then used in anhydride opening reactions to obtain the carboxylic acids (6) and (7).
Scheme 4. Synthetic route for carboxylic acids starting from 4-nitroisobenzofuran-1,3-dione.
In a similar way, the condensation of 4-fluoroisobenzofuran-1,3-dione with 3-aminopiperidine-2,6-dione was performed to obtain the fluoro-substituted imide (8), which underwent a nucleophilic
aromatic substitution with Boc-protected piperazine to obtain intermediate (9). Intermediate (9) was deprotected with HCl in dioxane and was used directly in an anhydride opening to obtain the carboxylic acid (10).
Scheme 5. Synthetic route for the carboxylic acid starting from 4-fluoroisobenzofuran-1,3-dione.
Scheme 6. Synthetic route for the isocyanide.
The fluoro-substituted imide (8) underwent a nucleophilic aromatic substitution with the amine-substitued formamide (11), which was prepared in two steps from mono-Boc-protected propane-1,3-diamine. The formamide (12) was used in a dehydration step with phosphorus oxychloride to obtain isocyanide (13).
3. PROTACs synthesis.
Three abemaciclib-pomalidomide derived PROTACs were synthesized with amide coupling reactions.
Scheme 7. Amide coupling reactions for PROTACs.
One abemaciclib-pomalidomide PROTAC was synthesized using the Ugi-tetrazole reaction.
Scheme 8. Ugi-tetrazole reaction for a PROTAC.
CONCLUSION – FUTURE PERSPECTIVE
In this project, four abemaciclib – cereblon PROTACs were synthesized, as well as the original inhibitor, as a reference compound. Preliminary biological data show that the compounds are potent degraders in different cell lines. The aim is to verify the most potent degrader of these series and then test the protein degradation in an in vivo model. Lastly, solving a co-crystal structure of a ternary complex would be a very useful achievement for rational design and further optimization. The biological data will be reported in due course.
REFERENCES
1. M.B. Kastan, J. Bartek, Nature 2004, 432(7015), 316 – 323.
2. M. Malumbres, M. Barbacid, Nat. Rev. Cancer 2009, 9(3), 153 – 166. 3. M. Malumbres, Genome Biology 2014, 15, 122.
4. S. Pernas, M. Tolaney, E.P. Winer, S. Goel, Ther. Adv. Med. Oncol. 2018, https://doi. org/10.1177/1758835918786451
5. B. O’Leary, R.S. Finn, N.C. Turner, Nat. Rev. Clin. Oncol. 2016, 13(7), 417 – 430.
6. H. Xu, S. Yu, Q. Liu, X. Yuan, S. Mani, R.G. Pestell, K. Wu, J. Hematol. Oncol. 2017, 10(1), 97. 7. B. Zhao, K. Burgess, Chem. Commun. 2019, 55(18), 2704 – 2707.
8. B. Jiang, E.S. Wang, K.A. Donovan, Y. Liang, E.S. Fischer, T. Zhang, N.S. Gray, Angew. Chem. Int. Ed. Engl. 2019, 58(19), 6321 – 6326.
9. S. Rana, M. Bendjennat, S. Kour, H.M. King, S. Kizhake, M. Zahid, A. Natarajan, Bioorg. Med. Chem. Lett. 2019. 29(11), 1375 – 1379.
10. S. Su, Z. Yang, H. Gao, H. Yang, S. Zhu, Z. An, J. Wang, Q. Li, S. Chandarlapaty, H. Deng, W. Wu, Y. Ra, J. Med. Chem. 2019, 62(16), 7575 – 7582.
11. E.M. Chan, (Eli Lilly), WO2015130540A1, 2015.
12. M.O. Frederick, D.P. Kjell, Tetrahedron Letters 2015, 56(7), 949 – 951.
EXPERIMENTAL SECTION
Experimental procedures
Procedure A (Suzuki coupling): In a 3-neck round bottom flask 2,4-dichloro-5-fluoropyrimidine
(1.1 equiv, 8.6 mmol, 1.4 g) was dissolved in a 5:1 mixture toluene : EtOH (15ml : 3ml) and then
a saturated solution of NaHCO3 (18 ml) and
4-fluoro-1-isopropyl-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (1 equiv, 7.8 mmol, 2.5 g) were added. The white
suspension was degassed by nitrogen for 30 min under vigorous stirring. Then Pd(dppf)Cl2.DCM
complex (0.004 equiv) was added and the reaction mixture was heating overnight at 85oC under
N2 flow. The next day, the reaction mixture was allowed to reach rt and then H20 was added and the reaction mixture was extracted with EtOAc (x3). The combined organic phases were washed with Brine (x3), dried over MgS04, filtered and the solvents were removed under vacuum to obtain a brown solid. The residue was purified by column chromatography (DCM – MeOH, 0 – 5% MeOH in DCM) to obtain compound (1).
Procedure B (Buchwald – Hartwig coupling): In a microwave vial
6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazole (1) (1 equiv), the appropriate
2-amino-pyridine (1.25 equiv) and Cs2C03 (2.5 equiv) were suspended in t-AmylOH (0.2M). The reaction
mixture was degassed by nitrogen for 10 min under vigorous stirring and then DPEPhos (0.04
equiv) and PdCl2 (0.02 equiv) were added. The microwave vial was sealed and it was stirred for
10min at room temperature under N2. The reaction mixture was subjected to microwave irradiation
for 1h at 110 oC. Then, the dark brown reaction mixture was filtered over silica under vacuum with
the appropriate solvent as mentioned in each case and the solvents were removed. The obtained residue was purified by column chromatography.
Procedure C (Synthesis of substituted 2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-diones):
In a round-bottom flask, the appropriate 4-substituted-isobenzofuran-1,3-dione (1 equiv), 3-amino-piperidine-2,6-dione hydrochloride (1 equiv) and sodium acetate (1.2 equiv) were mixed in AcOH (20 ml for 5 mmol scale). The resulting mixture was heated at 120 oC overnight. After cooling to room
temperature, most of the AcOH was removed under reduced pressure and the residue was taken in water, filtered and washed with water and dried with vacuum to obtain the crude solid compound.
Procedure D (Reduction): To a solution of
2-(2,6-dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione (8 mmol, 1.0 equiv) in dry DMF (50 ml) was added the Pd/C (1.6mmol, 0.2 equiv) under N2.
The reaction mixture was hydrogenated with 3.0 atm H2 pressure at room temperature for 4 h. The
progress of reaction was monitored by TLC. The reaction mixture was filtered over a pad of celite. The filtrate was diluted with EtOAc and the organic phase was washed with H2O and Brine (x3) and
was dried over MgSO4. The solvent was removed under reduced pressure, to obtain a solid, which
Procedure E1 (Anhydride opening) with 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione: A mixture of 4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (7.3mmol, 1.0
equiv), potassium acetate (29.3 mmol, 4.0 equiv) and the appropriate anhydride (29.23 mmol, 4.0 equiv) in glacial AcOH (60 ml) was heated at reflux under nitrogen for 3h. After cooling down, acetic
acid was removed under reduced pressure and the residue was extracted with (EtOAc – H2O). The
organic phases were dried with MgSO4 and solvents were removed under reduced pressure. The
crude product was purified by column chromatography (DCM – MeOH, 0 – 10% MeOH in DCM).
Procedure E2 (Anhydride opening) with 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione: in a small round-bottom flask containing tert-butyl 4-(2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate (9) (1 mmol, 1 equiv), 4N HCl in dioxane (5 ml) was added. Stirring rt overnight. Solvent was removed under reduced pressure. Diethylether was added (x2) and was removed under reduced pressure. The HCl salt of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione (1mmol, 1 equiv) was suspended in 5 ml DCM. DIPEA (2 equiv) was added and after 10 min stirring at room temperature, glutaric anhydride (1.1 equiv) was added. The clear yellow solution was heated at 40 oC for 1h. The reaction was allowed to reach rt and it was extracted x2 (DCM – H2O). The aqua phase was acidified with 2N HCl and it was extracted with DCM (x3). The combined organic phases were dried over MgSO4, filtered and the solvent was removed under reduced pressure.
Procedure F (Nucleophilic aromatic substitutions with amines): The appropriate mono-Boc
protected diamine (1.13 mmol, 1.1 equiv) was added to a stirred solution of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (1.03 mmol, 1.0 equiv) in DMF (6.0 ml) and DIPEA (2.06 mmol, 2.0 equiv). The reaction mixture was stirred at 90 °C for 12 h. Then the mixture was cooled to room
temperature, poured into H2O, and extracted twice with EtOAc (3 x 50 ml). The combined organic
layers were washed with brine, dried over anhydrous Na2SO4 and filtered. The crude compound
was purified by column chromatography.
Procedure G (Formamide synthesis): tert-butyl (3-aminopropyl)carbamate (4.0 mmol) was
dissolved in ethyl formate (10 ml) and heated under reflux for 4 hours. The solvent was removed under reduced pressure. The obtained formamide was further used without further purification. The Boc-protected formamide was dissolved in 10 ml DCM and under stirring 4.4 ml of TFA were added and the reaction mixture was stirred at rt overnight. Then the solvents were removed under reduced pressure. To remove residual TFA, methanol was added and evaporated in vacuum. This procedure was repeated three times. The obtained salt was used directly in the nucleophilic aromatic substitution (procedure F) without further purification.
Procedure H (Isocyanide synthesis):
oxychloride (1.5 mmol, 1.5 equiv) was added dropwise over 15min. After the addition was completed, the reaction mixture was stirred at -10 oC for 30min and then it was allowed to reach
room temperature for overnight stirring. The reaction was monitored by TLC. When no more starting material was detected, the reaction mixture was poured slowly in an ice cold saturated
solution of NaHCO3 and after 30min of stirring, the reaction mixture was extracted with DCM
(50ml x3). The combined organic phases were dried over MgS04, filtered and the solvent was
removed under reduced pressure. The residue was filtered over silica, first with petroleum ether (PE) as eluent and gradually increasing to PE : EtOAc 1:1.
Procedure I (Amide coupling): in a small round-bottom flask containing tert-butyl
4-(6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino) pyridin-3-yl) piperazine-1-carboxylate (3) (1equiv) 4 N HCl in dioxane was added. Stirring rt overnight. The reaction mixture (orange suspension) was dried under reduced pressure. Diethylether was added (x2) and was removed under reduced pressure. The obtained HCl salt was used directly in the amide coupling. The HCl salt (3a) (1 equiv) was suspended in CHCl3 (0.1 M). Under stirring DIPEA (2 equiv) was added, followed by the addition of the carboxylic acid (1 equiv) and EEDQ (2 equiv). The reaction mixture was heated at reflux for 2h. Then it was allowed to reach rt and was purified directly by column chromatography (DCM – MeOH, 0 – 10% MeOH in DCM).
Procedure K (Ugi tetrazole reaction): in a small round-bottom flask containing tert-butyl
4-(6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2yl)amino) pyridin-3-yl)piperazine-1-carboxylate (3) (1equiv) 4 N HCl in dioxane was added. Stirring rt overnight. The reaction mixture (orange suspension) was dried under reduced pressure. Diethylether was added (x2) and was removed under reduced pressure. The obtained HCl was suspended in MeOH and a few drops of DIPEA were added. In the yellow solution, paraformaldehyde (1 equiv) was added and the mixture was stirred rt for 10min. Then, the isocyanide (1 equiv) was added,
followed by TMSN3 (1 equiv). The reaction mixture was sonicated for 3h and then stirring rt
overnight. Solvent was removed under reduced pressure and the residue was purified by column chromatography (DCM – MeOH, 0 – 10% MeOH in DCM).
Characterization data
6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazole (1)
Obtained using procedure A on 7.8 mmol scale; 2.5g, 7.76 mmol, yield 98%, white solid. 1H NMR (500 MHz, CDCl
3) δ 8.52 (d, J = 3.4 Hz, 1H), 8.16 (d, J = 1.4
Hz, 1H), 7.80 (d, J = 11.4 Hz, 1H), 4.75 (hept, J = 7.0 Hz, 1H), 2.70 (s, 3H), 1.70 (d, J = 7.0 Hz, 6H). 13C NMR has been reported previously. [12]
N-(5-((4-ethylpiperazin-1-yl)methyl)pyridin-2-yl)-5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-amine [abemaciclib] (2)
Obtained using procedure B on 1 mmol scale; 300mg, 0.6 mmol, yield 60%, yellow solid. The reaction mixture was filtered over silica first with DCM and then with a mixture of
DCM – MeOH – NH3 to obtain the crude compound. Solvents
were removed under reduced pressure and the crude was purified by column chromatography with DCM – MeOH – NH3 (85 : 10: 5) to obtain the pure product. 1H NMR (500 MHz, CDCl
3) δ 9.32 (s,
1H), 8.49 (d, J = 3.7 Hz, 1H), 8.40 (d, J = 8.5 Hz, 1H), 8.34 (d, J = 1.7 Hz, 1H), 8.17 (s, 1H), 7.76 (d, J = 11.6 Hz, 1H), 7.66 (dd, J = 8.6, 2.0 Hz, 1H), 4.71 (hept, J = 7.0 Hz, 1H), 3.48 (s, 2H), 2.66 (s, 3H), 2.55 – 2.46 (b, 8H), 2.43 (q, J = 7.0 Hz, 2H), 1.69 (d, J = 7.0 Hz, 6H), 1.08 (t, J = 7.0 Hz, 3H). 13C NMR has been reported
previously.[12] HRMS (ESI): m/z calcd for C
27H33N8F2 [M+H]+: 507.27908; found 507.27872.
tert-butyl 4-(6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)
pyrimidin-2-yl)amino)pyridin-3-yl)piperazine-1-carboxylate (3)
Obtained using procedure B on 1 mmol scale; 312 mg, 0.55 mmol, yield 55 %, yellow solid. The reaction mixture was filtered over silica with DCM. Solvents were removed under reduced pressure and the crude was purified by column chromatography with (DCM - MeOH, 0 - 5% MeOH in DCM).
1H NMR (500 MHz, CDCl 3) δ 8.39 (d, J = 3.7 Hz, 1H), 8.31 (d, J = 9.0 Hz, 1H), 8.17 (s, 1H), 8.02 (d, J = 2.8 Hz, 1H), 7.99 (s, 1H), 7.79 (d, J = 11.6 Hz, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 4.73 (hept, J = 7.0 Hz, 1H), 3.62 – 3.60 (m, 4H), 3.10 – 3.08 (m, 4H), 2.68 (s, 3H), 1.71 (d, J = 7.0 Hz, 6H), 1.49 (s, 9H). 13C NMR (126 MHz, CDCl 3) δ 155.4, 154.6, 153.5, 153.2 (d, J = 251.9 Hz), 151.2 (d, J = 8.4 Hz), 150.7 (d, J = 255.2 Hz), 147.2, 147.0, 146.9, 142.8, 137.4, 136.3 (d, J = 9.3 Hz), 133.9 (d, J = 17.1 Hz), 127.5, 127.1, 112.3, 108.7 (d, J = 8.8 Hz), 108.1 (d, J = 6.8 Hz), 107.9 (d, J = 7.1 Hz), 80.0, 50.0, 48.6, 28.4, 21.4, 15.0. 2-(2,6-dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione (4)
Obtained using procedure C on 10 mmol scale; 2.7 g, 8.9 mmol, yield 90%, purple solid. The crude product was used directly in the next step.1H NMR
(500 MHz, DMSO-d6) δ 11.20 (s, 1H), 8.36 (dd, J = 8.1, 0.7 Hz, 1H), 8.25 (dd, J = 7.5, 0.5 Hz, 1H), 8.14 – 8.11 (m, 1H), 5.21 (dd, J = 12.9, 5.4 Hz, 1H), 2.90 (ddd, J = 17.3, 14.0, 5.4 Hz, 1H), 2.64 – 2.61 (m, 1H), 2.56– 2.52 (m, 1H), 2.11 -2.06 (m, 1H).13C NMR (126 MHz, DMSO-d 6) δ 172.8, 169.6, 165.3, 162.6, 144.5, 136.9, 133.1, 129.0, 127.4, 122.6, 49.5, 30.9, 21.8.
10
4-amino-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (5)
Obtained using procedure D on 8 mmol scale; 2.0 g, 7.3 mmol, yield 92 %,
yellow solid. 1H NMR (500 MHz, DMSO-d
6) δ 11.09 (s, 1H), 7.46 (dd, J = 8.4,
7.0 Hz, 1H), 7.02 – 6.99 (m, 2H), 6.52 (b, 2H), 5.04 (dd, J = 12.7, 5.4 Hz, 1H), 2.88 (ddd, J = 17.0, 13.9, 5.5 Hz, 1H), 2.54 – 2.50 (m, 1H), 2.04 – 2.00 (m, 1H).13C NMR
(126 MHz, DMSO-d6) δ 172.9, 170.2, 168.6, 167.4, 146.8, 135.5, 132.0, 121.7, 111.0, 108.5, 48.5, 31.01, 22.2.
5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-5-oxopentanoic acid (6)
Obtained using procedure E1 on 7.3 mmol scale; 904 mg, 2.4
mmol, yield 32 %, white solid. 1H NMR (500 MHz, DMSO-d
6) δ 11.14 (s, 1H), 9.73 (s, 1H), 8.43 (d, J = 8.3 Hz, 1H), 7.84 – 7.81 (m, 1H), 7.61 (d, J = 7.3 Hz, 1H), 5.14 (dd, J = 12.9, 5.4 Hz, 1H), 2.92 – 2.85 (m, 1H), 2.62 – 2.50 (m, 4H), 2.30 (t, J = 7.3 Hz, 2H), 2.08 – 2.05 (m, 1H), 1.86 – 1.80 (m, 2H).13C NMR (126 MHz, DMSO-d 6) δ 174.0, 172.7, 171.5, 169.7, 167.5, 166.6, 136.3, 136.0, 131.4, 126.5, 118.3, 117.2, 48.8, 35.5, 32.7, 30.7, 21.9, 20.1.HRMS (ESI): m/z calcd for C22H25O7N4 [M+H]+: 457.17178; found 457.17175.
2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethoxy)acetic acid (7)
Obtained using procedure E1 on 7.3 mmol scale; 850 mg, 2.2 mmol,
yield 30 %, white solid. 1H NMR (500 MHz, DMSO-d
6) δ 11.15 (s, 1H),
10.38 (s, 1H), 8.71 (d, J = 8.4 Hz, 1H), 7.87 (t, J = 7.9 Hz, 1H), 7.64 (d, J = 7.3 Hz, 1H), 5.16 (dd, J = 12.9, 5.4 Hz, 1H), 4.29 (s, 2H), 4.26 (s, 2H), 2.91 – 2.85 (m, 1H), 2.63 – 2.50 (m, 2H), 2.08 – 2.05 (m, 1H).13C NMR
(126 MHz, DMSO-d6) δ 172.8, 171.1, 169.8, 168.9, 168.1, 166.7, 136.6, 135.9, 131.4, 124.5, 118.3, 116.2, 70.1, 67.9, 49.0, 31.0, 22.0.HRMS (ESI): m/z calcd for C17H16O8N3[M+H]+:
390.0932; found 390.0932.
2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8)
Obtained using procedure C on 5 mmol scale; 1.2 g, 4.3 mmol, yield 85 %, white solid. The crude product was purified by column chromatography
(DCM – MeOH, 0 – 5% MeOH in DCM). 1H NMR (500 MHz, DMSO-d
6) δ 11.16 (s, 1H), 7.96 – 7.92 (m, 1H), 7.79 (d, J = 7.3 Hz, 1H), 7.75 – 7.7.2 (m, 1H), 5.16 (dd, J = 13.0, 5.4 Hz, 1H), 2.91 – 2.85 (m, 1H), 2.63 – 2.58 (m, 1H), 2.53 – 2.51 (m, 1H), 2.07 – 2.04 (m, 1H). 13C NMR (126 MHz, DMSO-d 6) δ 172.8, 169.7, 166.1, 164.0, 156.8 (d, J = 262.3 Hz), 138.0 (d, J = 7.9 Hz), 133.5, 123.0 (d, J = 19.6 Hz), 120.1 (d, J = 3.2 Hz), 117.0 (d, J = 12.6 Hz), 49.1, 30.9, 21.9.
tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate
(9)
Obtained using procedure F on 1.03 mmol scale; 300mg, 0.67 mmol, yield 65%, yellow solid. The crude product was purified by column
chromatography (DCM - MeOH, 0 – 5% MeOH in DCM). 1H NMR (500 MHz,
CDCl3) δ 8.22 (s, 1H), 7.62 – 7.59 (m, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 4.96 (dd, J = 12.3, 5.4 Hz, 1H), 3.65 – 3.63 (m, 4H), 3.29 – 3.27 (m, 4H), 2.88 – 2.72 (m, 3H), 2.16 – 2.09 (m, 1H), 1.48 (s, 9H).13C NMR (126 MHz, CDCl 3) δ 170.9, 168.1, 167.2, 166.5, 154.7, 150.3, 135.7, 134.1, 123.4, 117.9, 116.2, 80.0, 49.1, 43.4, 31.4, 28.4, 22.6. 5-(4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazin-1-yl)-5-oxopentanoicacid (10)
Obtained using procedure E2 on 1 mmol scale; 270 mg, 0.60
mmol, yield 60 %, orange solid. 1H NMR (500 MHz, DMSO-d
6) δ 12.04 (b, 1H), 11.09 (s, 1H), 7.72 (dd, J = 8.2, 7.4 Hz, 1H), 7.39 (d, J = 7.1 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 5.11 (dd, J = 12.7, 5.5 Hz, 1H), 3.64 – 3.59 (m, 4H), 3.34 – 3.30 (m, 4H), 2.88 (ddd, J = 16.8, 13.9, 5.3 Hz, 1H), 2.61 – 2.53 (m, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.29 – 2.23 (m, 2H), 2.05 – 2.02 (m, 1H), 1.76 – 1.70 (m, 2H). 13C NMR (126 MHz, DMSO-d 6) δ 174.3, 172.8, 170.4, 170.0, 167.0, 166.4, 149.5, 136.1, 133.6, 123.9, 116.9, 115.3, 50.9, 50.3, 48.8, 44.9, 40.9, 33.0, 31.5, 31.0, 22.1, 20.3. HRMS (ESI): m/z calcd for C22H25O7N4 [M+H]+: 457.17178; found 457.17175.
N-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)formamide (12)
Obtained using procedure G on 4 mmol scale; 572 mg, 1.6 mmol, yield 40 %, yellow solid. The crude product was purified by column
chromatography (DCM - MeOH, 0 – 5% MeOH in DCM). 1H NMR (500
MHz, DMSO-d6) δ 11.09 (s, 1H), 8.09 (s, 1H), 8.03 (s, 1H),7.59 – 7.56 (m, 1H), 7.10 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.68 (t, J = 6.0 Hz, 1H), 5.75 (s, 1H), 5.04 (dd, J = 12.7, 5.4 Hz, 1H), 3.15 (q, J = 6.5 Hz, 2H), 2.91 – 2.84 (m, 1H), 2.65 – 2.55 (m, 1H), 2.05 – 2.01 (m, 2H), 1.70 – 1.67 (m, 3H).13C NMR (126 MHz, DMSO-d 6) δ 172.8, 170.1, 168.8, 167.3, 161.2, 146.3, 136.3, 132.3, 127.6, 117.1, 109.2, 48.5, 34.7, 31.0, 28.7, 28.6, 22.1.
10
2-(2,6-dioxopiperidin-3-yl)-4-((3-isocyanopropyl)amino)isoindoline-1,3-dione (13)
Obtained using procedure H on 1 mmol scale; 177 mg, 0.52 mmol, yield 52 %, yellow solid.1H NMR (500 MHz, CDCl 3) δ 8.27 (s, 1H), 7.53 (dd, J = 8.4, 7.3 Hz, 1H), 7.14 (d, J = 7.1 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.28 (t, J = 6.0 Hz, 1H), 4.92 (dd, J = 12.4, 5.4 Hz, 1H), 3.56 – 3.52 (m, 2H), 3.50 – 3. 48 (m, 2H), 2.90 – 2.81 (m, 1H), 2.80 – 2.70 (m, 2H), 2.15 – 2.11 (m, 1H), 2.06 – 2.01 (m, 2H).13C NMR (126 MHz, CDCl 3) δ 171.0, 169.5, 168.9, 168.3, 167.4, 157.3, 146.5, 136.4, 132.5, 116.4, 112.2, 110.6, 48.9, 39.0, 31.4, 28.7, 22.7. N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-5-(4-(6-((5-fluoro-4-(4-fluoro-1- isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-5-oxopentanamide (14)
Obtained using procedure I on 0.2 mmol scale; 122 mg, 0.15 mmol, yield 75%,
yellow solid. 1H NMR (500 MHz, CDCl 3) δ 9.64 (s, 1H), 9.42 (s, 1H), 8.80 (d, J = 8.5 Hz, 1H), 8.55 (s, 1H), 8.40 (d, J = 3.8 Hz, 1H), 8.33 (d, J = 9.1 Hz, 1H), 8.16 (s, 1H), 8.07 (d, J = 2.9 Hz, 1H), 7.78 (d, J = 11.6 Hz, 1H), 7.72 – 7.69 (m, 1H), 7.54 (d, J = 7.3 Hz, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 4.95 (dd, J = 12.3, 5.4 Hz, 1H), 4.72 (hept, J = 7.0 Hz, 1H), 3.80 – 3.78 (m, 2H), 3.65 – 3.63 (m, 2H), 3.14 – 3.09 (m, 4H), 2.94 – 2.91 (m, 1H), 2.78 (ddd, J = 10.7, 10.2, 3.9 Hz, 2H), 2.68 (s, 3H), 2.60 (td, J = 6.9, 1.8 Hz, 2H), 2.50 (t, J = 7.1 Hz, 2H), 2.18 – 2.09 (m, 3H), 1.70 (d, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 171.8, 171.4, 170.5, 169.1, 168.2, 166.7, 155.3, 153.5, 153.3 (d, J = 251 Hz), 151.9, 151.3, 149.8, 147.2, 147.0, 142.4, 137.7, 137.1, 136.4, 134.1, 131.2, 127.4, 125.3, 118.5, 115.4, 112.5, 108.7 (d, J = 6.3 Hz), 108.0 (d, J = 20.0 Hz), 50.3, 50.1, 49.3, 48.6, 45.3, 41.42, 36.8, 31.9, 31.4, 22.7, 21.5, 20.5, 15.0. HRMS (ESI): m/z calcd for C42H42O6N11F2 [M+H]+ : 834.32821; found 834.32819.
N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-2-(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)piperazin-1-yl)-2-oxoethoxy)acetamide (15)
Obtained using procedure I on 0.1 mmol scale; 52 mg, 0.062 mmol, yield 62%, yellow solid. The product precipates in the reaction mixture. Instead of column chromatography, solvent was removed under reduced
pressure and the remaining solid was suspended in diethylether. Et20 was decanted and then
the residue was treated with methanol, which was also decanted. The remaining solid was dried under vacuum to obtain product (15). 1H NMR (500 MHz, DMSO) δ 11.16 (s, 1H), 10.47 (s, 1H), 9.84 (s,
1H), 8.73 (d, J = 8.4 Hz, 1H), 8.63 (d, J = 3.8 Hz, 1H), 8.25 (s, 1H), 8.06 (dd, J = 14.1, 5.9 Hz, 2H), 7.88 – 7.85 (m, 1H), 7.67 – 7.62 (m, 2H), 7.43 (dd, J = 9.1, 2.9 Hz, 1H), 5.14 (dd, J = 12.9, 5.4 Hz, 1H), 4.82 (hept, J = 7.0 Hz, 1H), 4.53 (s, 2H), 4.25 (s, 2H), 3.62 – 3.53 (m, 5H), 3.15 – 3.11 (m, 5H), 2.90 – 2.83 (m, 1H), 2.63 (s, 3H), 2.08 – 2.05 (m, 1H), 1.61 (d, J = 6.9 Hz, 6H).13C NMR (126 MHz, DMSO) δ 172.8, 169.7, 170.0, 168.1, 166.9, 166.7, 155.7, 154.6, 152.3 (d, J = 248 Hz),150.5, 149.2, 147.7, 146.2, 142.3, 136.6, 136.0, 133.3, 131.4, 129.5, 128.9, 126.6, 125.8, 124.5, 121.5, 118.4, 116.1, 113.0, 108.9 (d, J = 7.4 Hz), 107.0, 70.2, 69.0, 53.3, 48.9, 48.1, 41.6, 30.9, 21.0, 18.0, 16.7, 14.6, 12.2. HRMS (ESI): m/z calcd forC41H40O7N11F2 [M+H]+ : 836.30748;
found 836.30756.
2-(2,6-dioxopiperidin-3-yl)-4-(4-(5-(4-(6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-5-oxopentanoyl) piperazin-1-yl)isoindoline-1,3-dione (16)
Obtained using procedure I on 0.1 mmol scale; 64 mg, 0.071 mmol, yield
71%, yellow solid. 1H NMR (500 MHz, CDCl3) δ 9.10 (s, 1H), 8.42– 8.38 (m, 2H), 8.34 (d, J = 9.0 Hz, 1H), 8.17 (d, J = 0.9 Hz, 1H), 8.06 (s, 1H), 7.79 (d, J = 11.5 Hz, 1H), 7.63 – 7.60 (m, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.36 – 7.35 (m, 1H), 7.15 (d, J = 8.4 Hz, 1H), 4.97 (dd, J = 12.4, 5.3 Hz, 1H), 4.76 – 4.70 (m, 1H), 3.82 – 3.68 (m, 8H), 3.37 – 3.33 (m, 2H), 3.28 (t, J = 4.9 Hz, 2H), 3.15 – 3.09 (m, 4H), 2.92 – 2.75 (m, 3H), 2.69 (s, 3H), 2.50 (t, J = 6.9 Hz, 4H), 2.15 – 2.10 (m, 1H), 2.03 – 2.00 (m, 2H), 1.70 (d, J = 7.0 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 171.4, 171.3, 171.1, 168.3, 167.2, 166.6, 155.3, 153.6, 153.3 (d, J = 252 Hz), 151.8, 151.4, 151.3, 150.0, 147.2, 147.0, 142.5, 137.2, 136.4, 135.8, 134.1, 127.4, 123.3, 118.1, 116.4, 112.4, 108.7 (d, J = 5.8 Hz), 108.1 (d, J = 20.4 Hz), 51.7, 50.5, 50.1, 49.2, 48.6, 45.6, 45.4, 41.4, 32.5, 32.4, 31.4, 22.7, 21.5, 20.7, 15.0. HRMS (ESI): m/z calcd for C H ON F [M+H]+: 903.38606; found 903.38599.
2-(2,6-dioxopiperidin-3-yl)-4-((3-(5-((4-(6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl- 1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)methyl)-1H-tetrazol-1-yl)propyl)amino)isoindoline-1,3-dione (17)
Obtained using procedure K on 0.1 mmol scale; 52 mg, 0.06 mmol, yield
60%, yellow solid. 1H NMR (500 MHz, CDCl3) δ 10.47 (s, 1H), 8.93 (s, 1H), 8.36 (d, J = 3.7 Hz, 1H), 8.25 (d, J = 9.0 Hz, 1H), 8.13 (s, 1H), 8.00 (s, 1H), 7.74 (d, J = 11.6 Hz, 1H), 7.47 – 7.44 (m, 1H), 7.06 (d, J = 7.1 Hz, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.32 – 6.30 (m, 1H), 4.87 – 4.85 (m, 1H), 4.71 – 4.68 (m, 1H), 4.60 – 4.55 (m, 2H), 3.84 (s, 2H), 3.39 (d, J = 6.0 Hz, 2H), 3.03 – 3.01 (m, 4H), 2.85 – 2.82 (m, 1H), 2.74 – 2.72 (m, 2H), 2.66 (s, 3H), 2.58 – 2.56 (m, 4H), 2.35 – 2.32 (m, 2H), 2.08 – 2.06 (m, 1H), 1.98 – 1.95 (m, 1H), 1.67 (d, J = 6.9 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ 172.3, 169.5, 169.0, 167.3, 155.3, 153.5, 153.2 (d, J = 252 Hz), 151.7, 149.7, 147.0, 146.5, 146.3, 142.4, 136.4, 135.9, 132.5, 127.6, 126.9, 116.4, 112.5, 112.2, 110.7, 108.8, 108.0, 53.0, 50.5, 49.4, 49.0, 48.6, 45.1, 39.4, 31.4, 29.0, 22.8, 21.4, 15.0. HRMS (ESI): m/z calcd for C42H44O4N15F2 [M+H]+ : 860.36633;
