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Home > Targeted Synthesis of Complex Spiro[3H-indole-3,2’- pyrrolidin]-2(1H)-ones by Intramolecular Cyclization of Azomethine Ylides: Highly Potent MDM2–p53 Inhibitors
Targeted Synthesis of Complex Spiro[3H-indole-3,2’- pyrrolidin]-2(1H)-ones by Intramolecular Cyclization of Azomethine Ylides: Highly Potent MDM2–p53 Inhibitors
Andreas Gollner+,* Harald Weinstabl+,* Julian E. Fuchs, Dorothea Rudolph, Geraldine Garavel, Karin S. Hofbauer, Jale Karolyi-Oezguer, Gerhard Gmaschitz, Wolfgang Hela, Nina Kerres, Elisabeth Grondal, Patrick Werni, Juergen Ramharter, Joachim Broeker, and Darryl B. McConnell
The transcription factor tumor protein p53 (TP53), frequently referred to as the “guardian of the genome”, is a pivotal tumor suppressor protein and mainstay of the body’s cellular anti- cancer defense system.[1] TP53 is activated following cellular stress and regulates multiple downstream target genes impli- cated in cell-cycle control, apoptosis, senescence and DNA repair.[2] The TP53 gene is mutated in about 50 % of all human cancers whereas the other 50% have tumors with TP53 wild- type status.[3] However, the function of TP53 is frequently atte- nuated in these TP53 wild-type cancers by other mechanisms, including overexpression of its key negative regulator HDM2, which is the human homologue of mouse double minute 2 (MDM2). Stabilization and activation of TP53 by the inhibition of TP53 binding to its negative regulator MDM2 has been ex- plored as a novel approach to cancer therapy in patients with TP53 wild-type tumors.[4] These research efforts have yielded several MDM2–p53 protein–protein interaction (PPI) inhibitors, which have been or are currently still being evaluated in early clinical development.[5]
High-grade thrombocytopenia was reported for several MDM2–p53 inhibitors as a dose-limiting toxicity (DLT) in the clinic, in particular when testing continuous dose schedule- s.[5a,6] To clinically manage thrombocytopenia, a next genera- tion of MDM2–p53 inhibitors with the potency and pharmaco- kinetic properties to allow less-frequent dose schedules,[7] is needed. Our recently reported MDM2–p53 inhibitor BI-0252 (1) resulted in tumor regressions in all animals of a mouse SJSA-1 xenograft study with a single, but high oral dose of 100 mgkg@1. To deliver a compound suitable to test less-fre- quent dose schedules in the clinic, we strived for further im- provements in potency and pharmacokinetic properties of our MDM2–p53 inhibitors to decrease the required human dose on a less-frequent dose schedule. Herein we report the targeted syntheses of structurally complex and highly potent MDM2– p53 inhibitors with modified spiro-oxindole core structures, which were made accessible by employing unprecedented 1,3- dipolar cycloaddition chemistry.
BI-0252 (1) is as a chemically stable and orally active inhibi- tor of the MDM2–p53 interaction which bears a spiro[3H- indole-3,2’-pyrrolidin]-2(1H)-one core structure.[8] In contrast to the pioneering spiro[3H-indole-3,3’-pyrrolidin]-2(1H)-ones ini- tially reported by Wang et al. (Scheme 1 A)[9] and later by other groups,[10] which can undergo epimerization to four diastereo- mers via a retro-Mannich/Mannich reaction in solution.[11] Addi- tional spiro-oxindole MDM2 inhibitors include clinical candi- date DS-3032b[12] and others.[13] The new class of spiro[3H- indole-3,2’-pyrrolidin]-2(1H)-ones is not prone to this epimerization.[8] The problem of epimerization was also recently ad- dressed by Aguilar et al. leading to chemically stable inhibitors and the clinical candidate AA-115/APG-115, Scheme 1 B.[14]
The X-ray co-crystal structure of 1 in MDM2 (PDB ID: 5LAZ) revealed a hydrogen bond between the basic secondary nitro- gen of 1 and the side chain of His96 of the MDM2 protein as being important for the binding of 1 to MDM2 (Scheme 1 C, Figure 2 B).[8] In contrast many other MDM2–p53 inhibitors ad- dress His96 with a carbonyl oxygen functioning as hydrogen bond acceptor.[11b] Striving for further potency optimization we aimed at evaluating the influence of replacing the secondary amine (hydrogen bond donor) by a carbonyl group oxygen (hydrogen bond acceptor) in our lead series (Scheme 1 D). To test this hypothesis we designed the five-membered lactam analogue 2 (NH to C=O) and the six-membered lactam ana- logue 3 as close analogues of 1.
1,3-Dipolar cyclodadditions of azomethine ylides are a versa- tile tool for the generation of highly substituted pyrrolidines with dense stereochemistry.[15] We took advantage of this pow- erful method in our earlier study to generate the core structure of 1 by reacting 6-chloroisatin, 1-(2-fluoro-3-chlorophenyl)-2-ni- troethene and l-homoserine in a three-component reaction.[8] For the synthesis of the new core structures the use of an in- termolecular cycloaddition seemed less attractive, as published reports suggest that the outcome of such reactions using sub- stituted styrene analogues as dipolarophiles with electron- withdrawing groups other than a nitro group would favor an undesired regioisomer.[16] We therefore envisioned an intramo- lecular cycloaddition which should favor the desired regioiso- mer.
Intramolecular cyclodadditions of azomethine ylides have been used successfully to generate complex fused pyrrolidine, and therein describes the reaction of isatin with 2-(methylami- no)-N-phenyl-N-(prop-2-en-1-yl)acetamide which yielded a oc- tahydro-1’H-spiro[indole-3,2’-pyrrolo[2,3-c]pyrrole]-2,6’-dione compound with undetermined stereochemistry in moderate yield.[18] To access the desired unprecedented octahydro-1’H- spiro[indole-3,2’-pyrrolo[2,3-c]pyrrole]2,4’-dione core structure we planned to modify the amine components to 2-amino-3- (prop-2-enamido)propanoic acids and 2-amino-3-(prop-2-en- amido)butanoic acids (Scheme 2 B), which should readily gen- erate the azomethine ylide after imine formation with an isatin analogue followed by subsequent decarboxylation. The ylide was expected to react with the remaining double bond to yield the desired product.
We started the synthesis by preparing the amino acid cycli- zation precursors 9 and 10 (Scheme 3) from commercially available 3-amino-N-(tert-butoxycarbonyl)-l-alanine tert-butyl ester (5) and (S)-2-tert-butoxycarbonylamino-4-aminobutyric acid tert-butyl ester (6) by amide coupling with (2E)-3-(3- chloro-2-fluorophenyl)prop-2-enoic acid (4). Both reactions proceeded in close to quantitative yields to give compounds 7 and 8. To remove the Boc and tert-butyl protecting groups, compounds 7 and 8 were treated with trifluoroacetic acid in CH2Cl2 at RT, and after completion of the reactions amino acids 9 and 10, respectively, were precipitated from water at pH 6–7 in excellent yields. With the cyclization precursors 9 and 10 now in hand, we performed the first decarboxylative cycloaddi- tion by heating 9 with one equivalent of 6-chlorisatin (11) in methanol at 100 8C for 30 min in a microwave reactor.[19] We were able to isolate an inseparable mixture of the diastereo- mers rac-12 a and rac-12 b in 23 % yield and a diastereomeric ratio of 1:3. After reductive amination of this mixture with cy- clopropylcarboxaldehyde we were able to isolate rac-13 in quantitative yield based on the content of rac-12 a which was separated by chiral SFC to obtain enantiomerically pure 13.
Buchwald coupling with methyl 4-bromobenzoate (14) and subsequent saponification delivered target compound 2. De- spite the low yield and unfavorable selectivity in the cycloaddi- tion step we were able to obtain the complex polycyclic struc- ture of 2 in only six synthetic steps to obtain sufficient material for biological testing.
For the synthesis of the six-membered lactam analogue 3 we reacted the cyclization precursor 10 with one equivalent of 11 under the same conditions and isolated the two diastereo- mers rac-15 a and rac-15 in a yield of 61 % favoring the desired compound rac-15 a (d.r. = 1.7:1). Reductive amination of rac- 15 a with cyclopropylcarboxaldehyde and subsequent chiral SFC separation gave compound 16. Buchwald coupling of lactam 16 with methyl 4-bromobenzoate and saponification delivered compound 3 in good overall yields (Scheme 3).
To test whether the higher yield for the ylide intermediate 10 (n = 2) versus 9 (n = 1) is due to pre-organization we ex- plored their conformational ensembles using a fine-grained systematic conformational search in MOE 2016.0802 (Figure 1). We enumerated accessible conformations in an energy window of 10 kcal mol@1 with a minimum pair-wise RMSD of
0.1 a using the default AMBER10 :EHT force field along with a dielectric constant of 32.7 resembling methanol. Resulting con- formations were subsequently energy minimized on B3LYP-D3/6-31G* level in implicit methanol solvation using Gaussi- an 09.[20] Resulting conformations were analyzed with respect to their compactness using the radius of gyration rgyr as well as with respect to their RMSD to modelled intermediate confor- mations leading to desired and undesired reaction products.
For both linker lengths we found a large set of collapsed conformations within the respective ensemble that are pre-or-
ganized for an intramolecular cycloaddition. For n = 1 we found 60 of 175 total conformations (34 %) with rgyr < 4 a which typically indicates stacking interactions between both p-systems in the ylide intermediate. For the molecule with linker length n = 2, we similarly found 57 of 148 conformations with rgyr < 4 a (39 %). For compounds with n = 1 collapsed confor- mations do not directly correspond to the minimum energy conformations but are very close with a strain energy of only+ 0.7 kcal mol@1. Elongating the linkage to n = 2 focusses the conformational ensemble specifically around the collapsed form. The lowest non-collapsed conformation is strongly unfav- orable and only found at + 6.5 kcal mol@1 in the latter case. These findings are consistent with the higher reaction yields obtained for educts with linker length n = 2. In addition to the formation of pre-organized collapsed conformations of the ylide intermediates, we found a focusing of the conformations around structures leading to specific diastereomeric reaction products. Amongst all conformations with rgyr < 4 a precursor conformations locking the lactam 5-ring in the unde- sired direction are found more often (60 %) than in the desired conformation (40 %). For the larger intermediates leading to the lactam 6-ring product we found only 51 % of conformations pre-organized to form the undesired diastereomer versus 49% for the desired one. Both, dominance of the precursor to the undesired product for the 5-ring lactam as well as an almost equal distribution of precursor confor- mations for the 6-ring lactam are in line with the observed syntheses yields.
To evaluate the influence of replacing the secon- dary amine (hydrogen bond donor) by a carbonyl group (hydrogen bond acceptor) we measured bio- chemical (IC50 (MDM2–p53)) and also the cellular po- tency in the p53 wild-type osteosarcoma SJSA-1 cell Figure 2. A) X-ray co-crystal structure of 13 (magenta) in MDM2. (The racemic compound rac-13 was used for crystallization, only the eutomer 13 was found in the co-crystal structures (PDB ID: 6I3S). B) Overlay of X-ray co-crystal structure of 13 (magenta) in MDM2 with X-ray co-crystal structure of compound 1 (yellow) as observed in PDB ID 5LAZ.
An X-ray crystal structure of compound 13 in MDM2 (Fig- ure 1 A) showed that the lactam carbonyl indeed forms a hy- drogen bond interaction with the side chain of His96MDM2. The overlay of the structure with the X-ray crystal structure of com- pound 1 (PDB ID: 5LAZ) shows a very similar binding mode with compound 13 slightly shifted to enable hydrogen bond formation with His96MDM2 (Figure 2).
In conclusion, we have developed an unprecedented intra- molecular cyclization of azomethine ylides which enabled access to octahydropyrrolo[2,3-c]pyrrol-4-ones and octahydro- pyrrolo[2,3-c]pyridine-4-ones and allowed the targeted synthe- sis of structurally complex and highly functionalized spiro-oxin- doles 2 and 3 in highly efficient 6-steps sequences. We investi- gated the pre-organization of the ylide intermediates of the cy- cloaddition reaction to rationalize the outcome of the experi- ments by computational methods which could facilitate future synthesis planning of intramolecular cycloadditions. The com- pounds were prepared to investigate the effect of introducing a hydrogen bond acceptor (lactam carbonyl oxygen) to our spiro-oxindole MDM2–p53 PPI inhibitors to address the side- chain of His96MDM2 in comparison with the earlier compound BI-0252 (1) which carried a hydrogen bond donor at an equiva- lent position. Compound 2 displayed a threefold improvement in potency in the p53 wild-type osteosarcoma SJSA-1 cell line proliferation assay relative to 1. This finding guided our optimi- zation efforts toward hydrogen bond acceptors at this position and was an important milestone toward a MDM2–p53 PPI inhibitor suitable to test less frequent dose schedules with the intention to manage thrombocytopenia in the clinic. In vivo profiling of 2 and additional compounds will be reported in due course.
Acknowledgements
Boehringer Ingelheim is grateful for financial support by the Aus- trian Research Promotion Agency FFG (grants 832260, 837815, and 842856). We thank Christoph Albrecht, Heribert Arnhof, Christian Aumeyer, Gerd Bader, Alexandra Beran, Helmut Berger, David Covini, Maike Dettling, Sophia M. Blake, Guido Boehmelt, Xiao-Ling Cockcroft, Ida Dinold, Nora Derkop, Norbert Eidkum, Wolfgang Egermann, Peter Ettmayer, Gerlinde Flotzinger, Thomas Gerstberger, Gregor Grotemeier, Eric Haaksma, Petra Handler, Daniela H-ring, Matthew Kennedy, Dirk Kessler, Matthias Klemen- cic, Michael Kulhanek, Roland Kousek, Norbert Kraut, Moriz Mayer, Katharina Mayr, Sabine Olt, Christina Puri, Christoph Pein- sipp, Oliver Petermann, Jens Quant, Carlos Roberto Ramirez- Santa Cruz, Maria Rieger, Genther Ross, Christian Salamon, Alexander Savchenko, Otmar Schaaf, Yvonne Scherbantin, Renate Schnitzer, Andreas Schrenk, Joshua D. Sieber, Norbert Schweifer, Anita Seebçck, Gorana Sijan, Steffen Steurer, Michaela Streicher, Harald Studensky, Steffen Weik, Irene Weiner, Anika Weiss, An- dreas Wernitznig, Ulrike Weyer-Czernilofsky, Alexander Wlachos, Susanne Wollner-Gaida, Tobias Wunberg, Markus Zeeb, and An- dreas Zçphel.
Conflict of interest
All authors were full-time employees of Boehringer Ingelheim RCV GmbH & Co. KG when this study was performed.
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