Recent Small-Molecule Inhibitors of the P53–MDM2 Protein–Protein Interaction

molecules

Review Recent Small-Molecule Inhibitors of the p53–MDM2 Protein–Protein Interaction

Anastasia Beloglazkina, Nikolai Zyk, Alexander Majouga and Elena Beloglazkina * M.V. Lomonosov Moscow State University, Leninskie gory, 1-3, 119991 Moscow, Russia; [email protected] (A.B.); [email protected] (N.Z.); [email protected] (A.M.) * Correspondence: [email protected]

Received: 15 February 2020; Accepted: 6 March 2020; Published: 7 March 2020

Abstract: This review presents the last decade of studies on the synthesis of various types of small-molecule inhibitors of the p53– Mouse double minute 2 homolog (MDM2) protein–protein interaction. The main focus is placed on synthetic approaches to such molecules, their cytotoxicity, and MDM2 binding characteristics.

Keywords: p53–MDM2 inhibitors; anticancer drugs; cytotoxicity

1. Introduction In recent years, a number of reviews on p53–MDM2 interaction inhibitors have been published [1–5]. These reviews indicate continuing research interest on the topic. We note, however, that none of the reviews referenced above cover all aspects of p53–MDM2 interaction inhibition: biological, chemical, and medical. Sanz et al. [1] described the biomedical aspects of the p53–MDM2 interaction inhibitors, Nayak et al. [2] focused on the study of the structural aspects of p53–MDM2 interaction inhibitors, and the review by Estrada-Ortiz and co-authors [3] is devoted to an analysis of the crystal structure of complexes of inhibitors with the indicated proteins. Since the writing of the reviews by Khoury and Domling [4] and Popowicz, Domling and Holak [5], a signiﬁcant number of new works have appeared. A feature of the present review is a more detailed description of synthetic approaches to known inhibitor compounds and the possibility of synthetic scheme changes, allowing the key reaction steps to be realized in an enantioselective version. Some attention is also given to the biotesting results and biomedical data of the obtained compounds. The p53 protein, which is a tumor suppressor, is one of the potential targets of antitumor therapy. The role of this protein in living organisms is very large, and includes participation in the processes of DNA repair, cell cycle arrest, apoptosis, and aging; in this regard, it has received the name “guardian of the genome.” In more than 50% of tumor cell cultures, the p53 protein is mutated [6], which makes it possible to use a fairly wide range of biological and chemical methods for its activation or restoration of its function. Activation of the p53 protein leads to one of two functions: initiating apoptosis or arresting cell growth [7]. p53-induced apoptosis occurs via the mitochondrial pathway through transcription-dependent or transcription-independent mechanisms and by the death receptor pathway through the transcriptional activation of FAS (a membrane dound proteine) and KILLER/DR5 (dead receptor) [8]. p53 is also able to transcriptionally suppress cell survival genes such as Bcl-2 (B-cell lymphoma 2), survivin, IGFR (insulin-like growth factor 1), Mcl-1 (induced myeloid leukemia cell diﬀerentiation protein), and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase), through various mechanisms [9]. On the other hand, p53-induced cell cycle arrest is mainly due to an increase in the concentration of proapoptotic proteins p21 (cyclin-dependent kinase inhibitor 1), Gadd45 (Growth arrest and DNA-damage-inducible protein), 14-3-3σ, and PTGFβ, by direct binding to DNA or its

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or its transactivation [10]. Other p53-dependent anti-cancer mechanisms include the activation of transactivationaccelerated cell [10 aging]. Other [11], p53-dependent inhibition of angiogenesis anti-cancer mechanisms [12], and regulation include the of activationautophagy of [13]. accelerated cell agingp53 [[14,15]11], inhibition triggers of the angiogenesis expression [12of], proteins and regulation with the of autophagyfollowing functions: [13]. a) the proteins involvedp53 [ 14in, 15control] triggers of the the cell expression cycle; b) the of proteins proteins involved with the in following DNA repair functions: processes; (a) c) the the proteins proteins involvedthat prevent in control angiogenesis; of the cell d) cycle; the (b) proteins the proteins with involvedantioxidant in DNA action; repair e) the processes; proteins (c) that the proteinsregulate thatmetabolism; prevent angiogenesis;and f) inducers (d) of theapop proteinstosis. The with most antioxidant important action;is the last (e) group the proteins of proteins, that which regulate are metabolism;capable of triggering and (f) inducers various of pathways apoptosis. of The cell most death. important This group is the also last in groupcludes of the proteins, proapoptotic which areproteins capable Bax of (also triggering known various as bcl- pathways2-like protein of cell 4), death. NOXA This (phorbol-12-myristate-13-acetate-induced group also includes the proapoptotic proteinsprotein 1), Bax and (also PUMA known (p53 as upregulated bcl-2-like protein modulator 4), NOXA of apoptosis) (phorbol-12-myristate-13-acetate-induced [14]. The main pathway of cell death proteincontrolled 1), and by PUMAthe p53 (p53protein upregulated is the mitochondrial modulator of apoptosis apoptosis) mechanism [14]. The main [16]. pathwayProapoptotic of cell proteins death controlled(Bax, NOXA, by theand p53 PUMA), protein as iswell the as mitochondrial the p53 protein apoptosis itself, interact mechanism with the [16 mitochondrial]. Proapoptotic membrane, proteins (Bax,causing NOXA, a decrease and PUMA), in its wall as well thickness. as the p53This protein leads to itself, the release interact of with cytochrome the mitochondrial C from mitochondria membrane, causinginto the a cytoplasm. decrease in Cytochrome its wall thickness. C interacts This leadswith prot to theeins release of the of cell cytochrome cytoplasm, C triggering from mitochondria a cascade intoprocess the cytoplasm. for the activation Cytochrome of various C interacts caspases with [17] proteins. Caspases of the ruin cell the cytoplasm, cytoskeleton, triggering which a cascadeleads to processinevitable for cell the death. activation Therefore, of various there caspases is a comple [17x]. system Caspases of interactions ruin the cytoskeleton, between proteins which and leads DNA to inevitablethat controls cell life death. processes Therefore, and therecell death. is a complex system of interactions between proteins and DNA that controlsIn the lifeoccurrence processes of and the cell cellular death. stress response, the level of p53 increases via the post- translationalIn the occurrence mechanism, of the which cellular ultimately stress response, leads to the ce levelll cycle of p53 arrest increases or apoptosis. via the post-translational In the absence of mechanism,cellular stress, which the amount ultimately of p53 leads in tothe cell cell cycle is controlled arrest or by apoptosis. a negative In theregulator absence of ofthe cellular p53 protein— stress, thethe amount MDM2 ofprotein. p53 in theThe cell MDM2 is controlled protein bybinds a negative to p53 regulatoramino acid of theresidues, p53 protein—the causing ubiquitin- MDM2 protein.dependent The p53 MDM2 degradation protein [18]. binds The to p53 p53 and amino MDM2 acid pr residues,oteins are causing linked to ubiquitin-dependent each other by a feedback p53 degradationmechanism, [ 18i.e.,]. Thewith p53 an and increase MDM2 in proteins the concen are linkedtration to eachof the other p53 by protein, a feedback a mechanism,decrease in i.e.,the withconcentration an increase of in the the MDM2 concentration protein is of observed, the p53 protein, and vice adecrease versa. Therefore, in the concentration there is negative of the feedback MDM2 proteinbetween is observed,these proteins and vice[14]. versa. Therefore, there is negative feedback between these proteins [14].

2.2. p53–MDM2p53–MDM2 Inhibitors Inhibitors AmongAmong the the attempts attempts to disruptto disrupt the regulation the regulation of the functioning of the functioning of the p53-dependent of the p53-dependent mechanism aremechanism the following are approaches:the following the approaches: synthesis of the small-molecule synthesis of inhibitors small-molecule of the p53–MDM2 inhibitors of interaction, the p53– p53-dependentMDM2 interaction, gene p53-dependent therapy, and the gene use therapy, of compounds and the use that of can compounds bind to mutant that can p53 bind and to restoremutant itsp53 function. and restore its function.

2.1.2.1. NaturalNatural Compounds Compounds AboutAbout 60%60% ofof drugsdrugs on the market market today today (excluding (excluding biologics) biologics) have have a natural a natural origin origin [19,20]. [19,20 To]. Todate, date, three three natural-based natural-based compounds compounds have have been been desc describedribed as exhibiting inhibitoryinhibitory activityactivity towardstowards thethe p53–MDM2 p53–MDM2 interaction. interaction. Chalcone-basedChalcone-based inhibitors inhibitors were were the the ﬁrst first reported reported compounds compounds of thisof this type type and and they they have have been been the µ mostthe most extensively extensively studied studied [21–24 [21–24].]. Chalcone Chalcone1 (Figure 1 (Figure1) had an1) ELISAhad an IC ELISA50 value IC50 of value 206 ofM 206 and µM caused and 1 15 15 acaused shift in a theshift pattern in the ofpattern the H- of theN HSQC1H-15N NMRHSQC spectrum NMR spectrum of N-enriched of 15N-enriched MDM2, MDM2, consistent consistent with chalconewith chalcone binding binding in the in tryptophan the tryptophan pocket. pocket. A gel A shift gel assayshift assay revealed revealed that thethat p53 the releasedp53 released from from the p53the/ MDM2p53/MDM2 complex complex by treatment by treatment with 1 waswith unable 1 was tounable bind DNA,to bind suggesting DNA, suggesting an additional an additional inﬂuence ofinfluence the chalcone of the on chalcone the p53 on protein. the p53 protein.

. Figure 1. 1 and 2 as p53–MDM2 antagonists [21–24]. Figure 1. 1 and 2 as p53–MDM2 antagonists [21–24].

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A series of boronic chalcone analogues were prepared by Khan and co-workers to address the lack A series of boronic chalcone analogues were prepared by Khan and co-workers to address the of specificity in the carboxylic acid-containing chalcones [23]. They reported a modest improvement; lack of specificity in the carboxylic acid-containing chalcones [23]. They reported a modest compound 2 was 2.5- to 10-fold more toxic to human breast cancer cell lines than to a normal breast improvement; compound 2 was 2.5- to 10-fold more toxic to human breast cancer cell lines than to a epithelial cell line at 10–40 µM. Isoliquiritigenin (4,2 ,4 -trihydroxychalcone), which is a natural normal breast epithelial cell line at 10–40 µM. Isoliquiritigenin0 0 (4,2′,4′-trihydroxychalcone), which is chalcone that is isolated from licorice root and shallot, has been shown to induce cell cycle arrest and a natural chalcone that is isolated from licorice root and shallot, has been shown to induce cell cycle apoptosis in liver cancer cells via the p53 pathway at 10–20 µg/mL, but its binding to MDM2 was not arrest and apoptosis in liver cancer cells via the p53 pathway at 10–20 µg/mL, but its binding to characterized [24]. MDM2 was not characterized [24]. Chlorofusin was the second natural product inhibitor of p53/MDM2 to be reported [25]. Williams Chlorofusin was the second natural product inhibitor of p53/MDM2 to be reported [25]. and co-workers identified chlorofusin as an inhibitor of p53/MDM2 binding (3, Figure2) after testing Williams and co-workers identified chlorofusin as an inhibitor of p53/MDM2 binding (3, Figure 2) over 53,000 extracts from the fermentation of a diverse collection of microorganisms for this activity [25]. after testing over 53,000 extracts from the fermentation of a diverse collection of microorganisms for This novel metabolite from the fungus Microdochium caespitosum had an IC50 of 4.6 µM in a p53/MDM2 this activity [25]. This novel metabolite from the fungus Microdochium caespitosum had an IC50 of 4.6 ELISA. Further studies using SPR (surface plasmon resonance) confirmed that chlorofusin binds to the µM in a p53/MDM2 ELISA. Further studies using SPR (surface plasmon resonance) confirmed that N-terminal region of MDM2 (Kd = 4.7 lM) [26]. chlorofusin binds to the N-terminal region of MDM2 (Kd = 4.7 lM) [26].

Figure 2. Structures of chlorofusin (3) and hexylitaconic acid (4) [25–27]. Figure 2. Structures of chlorofusin (3) and hexylitaconic acid (4) [25–27]. Recently, another natural product inhibitor of the p53/MDM2 interaction, (-)-hexylitaconic acid Recently, another natural product inhibitor of the p53/MDM2 interaction, (-)-hexylitaconic acid (4, Figure2), was reported [ 27]. Isolated from the fermentation culture of a Arthrinium sp. Fungus, (4, Figure 2), was reported [27]. Isolated from the fermentation culture of a Arthrinium sp. fungus, which was isolated from a marine sponge, (-)-hexylitaconic acid had an IC50 of 50 µg/mL (~230 µM) which was isolated from a marine sponge, (-)-hexylitaconic acid had an IC50 of 50 µg/mL (~230 µM) for p53/MDM2. The inhibition of the p53–MDM2 interaction was tested by ELISA, according to for p53/MDM2. The inhibition of the p53–MDM2 interaction was tested by ELISA, according to the the standard procedure, using puriﬁed recombinant p53 and HDM2 (human homologue of MDM2) standard procedure, using purified recombinant p53 and HDM2 (human homologue of MDM2) proteins, and the following primary anti-MDM2 antibody. Other derivatives of 4, including the proteins, and the following primary anti-MDM2 antibody. Other derivatives of 4, including the monomethyl ester, a dihydro derivative, and a dihydro derivative of the monomethyl ester, as well monomethyl ester, a dihydro derivative, and a dihydro derivative of the monomethyl ester, as well as two commercially available dicarboxylic acids (itaconic acid and succinic acid) did not inhibit the as two commercially available dicarboxylic acids (itaconic acid and succinic acid) did not inhibit the interaction at all at the concentration of 50 µg/mL. interaction at all at the concentration of 50 μg/mL. 2.2. Nutlin Analogs 2.2. Nutlin Analogs The most important push for the development of small-molecule inhibitors of the p53–MDM2 interactionThe most was important the development push for of 4,5-dihydroimidazolinethe development of small-molecule (Nutlin). In 2004 inhibitors [28], based of the on p53–MDM2 molecular modelinginteraction data, was it the was development shown that of the 4,5-dihydroimidazoline Nutlin-3 molecule is able (Nutlin). to integrate In 2004 into [28], a smallbased hydrophobicon molecular pocketmodeling of the data, MDM2 it was protein, shown simulatingthat the Nutlin-3 three aminomolecule acid is residuesable to integrate in the p53 into protein a small (Phe19, hydrophobic Trp23, andpocket Leu26), of the which MDM2 are protein, the most simulating important three binding amin fragments.o acid residues The crystal in the structure p53 protein of one (Phe19, of Nutlin’s Trp23, isomersand Leu26), (Nutlin-3a) which are in thethe ﬁrstmost binding important site binding to MDM2 fragments. is currently The usedcrystal as structure a model of for one creating of Nutlin’s new inhibitorsisomers (Nutlin-3a) of the p53–MDM2 in the first protein–protein binding site to interaction MDM2 is [ 29currently] (Figure used3). as a model for creating new inhibitors of the p53–MDM2 protein–protein interaction [29] (Figure 3). Nutlin-3 (Scheme 1, compound 11), as a racemic mixture, demonstrates a cytotoxicity value on p53-expressing cell lines, with an IC50 value of about of 100–300 nm [4]. The enantiomers were separated on a chiral column, and when studying enantiomerically pure preparations, it was shown that (-)-Nutlin-3 (also called Nutlin-3a) is a 150 times more effective inhibitor compared to (+)-Nutlin- 3. The synthesis of Nutlin by the pharmaceutical company Roche includes eight stages, with separation on a chiral chromatographic column (Scheme 1): initial bromination of 3-methoxyphenol

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(5), subsequent alkylation (6) to obtain isopropyl ether (7), and palladium-catalyzed cross-coupling with the formation of imine (8), which then reacts with meso- (4-chlorophenyl)ethane-1,2-diamine (9) to form imidazoline (10). Compound 10 reacts with phosgene to give carbamoyl chloride, which is then sequentially treated with piperazine and a solution of hydrogen chloride in ether, resulting in racemica Nutlin 3 (11). The separationb of the latter on a chiral chromatographicc column yields the Nutlin-3a active enantiomer [30]. FigureFigure 3. (a) 3. (a)MDM2 MDM2 protein protein fragmentfragment with with the the Nutlin-3a Nutlin-3a inhibitor inhibitor located located at the p53 at the binding p53 bindingsite. (b) site. (b) Nutlin-3Nutlin-3 overlay (carbon (carbon atoms atoms are are marked marked in whit in white,e, nitrogen nitrogen atoms atomsin blue, inoxygen blue, atoms oxygen in red, atoms in red, andand bromine in in brown) brown) and and amino amino acid acid fragments fragments of Phe19, of Phe19, Trp23, Trp23, and Leu26 and of Leu26 the p53 of theprotein. p53 ( protein.c) (c) TheThe surface surface of the p53– p53– MDM2 MDM2 binding binding site site(hollows (hollows are marked are marked in green, in and green, convex and sections convex in sections red), in red),showing showing one one bromophenyl bromophenyl group group located located deep deepin the inTrp the pocket. Trp pocket.

Nutlin-3 (Scheme1, compound 11), as a racemic mixture, demonstrates a cytotoxicity value on p53-expressing cell lines, with an IC50 value of about of 100–300 nm [4]. The enantiomers were separated on a chiral column, and when studying enantiomerically pure preparations, it was shown that (-)-Nutlin-3 (also called Nutlin-3a) is a 150 times more eﬀective inhibitor compared to (+)-Nutlin-3. The synthesis of Nutlin by the pharmaceutical company Roche includes eight stages, with separation a b c on a chiral chromatographic column (Scheme1): initial bromination of 3-methoxyphenol ( 5), subsequent alkylation (6) toFigure obtain 3. isopropyl (a) MDM2 ether protein (7), fragment and palladium-catalyzed with the Nutlin-3a cross-coupling inhibitor located with at the the p53 formation binding ofsite. (b) imine (8), whichNutlin-3 then reacts overlay with (carbonmeso- atoms (4-chlorophenyl)ethane-1,2-diamine are marked in white, nitrogen atoms (9) to in form blue, imidazoline oxygen atoms (10 in). red, Compound 10 reactsand bromine with phosgene in brown) toand give amino carbamoyl acid fragments chloride, of whichPhe19, isTrp23, then and sequentially Leu26 of the treated p53 protein. with (c) piperazine andThe a solution surface ofof hydrogenthe p53– MDM2 chloride binding in ether, site (hollows resulting are in racemicmarked in Nutlin green, 3 and (11 ).convex The separation sections in red), of the latter onshowing a chiral chromatographicone bromophenyl group column located yields deep the Nutlin-3ain the Trp pocket. active enantiomer [30].

Scheme 1. Nutlin-3 (11) synthesis as proposed by the pharmaceutical company Roche [30].

An alternative enantioselective method for Nutlin-3a synthesis, which includes only six stages (Scheme 2), was proposed by a group of researchers from Vanderbild University [31]. Initially, by diastereo- and enatioselective cross-coupling of a para-chloronitrobenzyl derivative 12 and the Boc- protected imine 13 in the presence of a chiral catalyst 14, the nitro-substituted cis-stilbene 15 was obtained, which was reduced to amine using generated in situ cobalt boride; the amine was then acylated to obtain a Boc-protected amino amide 16. After removal of the Boc-protection with trifluoroacetic acid, the resulting amine was acylated using carbonyldiimidazole, whereby an isocyanate was subsequently obtained, which was then treated with piperazinone and cyclized in the

Scheme 1. Nutlin-3 (11) synthesis as proposed by the pharmaceutical company Roche [30]. Scheme 1. Nutlin-3 (11) synthesis as proposed by the pharmaceutical company Roche [30]. An alternative enantioselective method for Nutlin-3a synthesis, which includes only six stages (Scheme2), wasAn proposed alternative by enantioselective a group of researchers method for from Nutlin Vanderbild-3a synthesis, University which [ includes31]. Initially, only six stages by diastereo-(Scheme and enatioselective2), was proposed cross-coupling by a group of of researchers a para-chloronitrobenzyl from Vanderbild derivative University12 and [31]. the Initially, by Boc-protecteddiastereo- imine and13 in enatioselective the presence cross-coupling of a chiral catalyst of a para14, the-chloronitrobenzyl nitro-substituted derivativecis-stilbene 12 and15 the Boc- protected imine 13 in the presence of a chiral catalyst 14, the nitro-substituted cis-stilbene 15 was obtained, which was reduced to amine using generated in situ cobalt boride; the amine was then acylated to obtain a Boc-protected amino amide 16. After removal of the Boc-protection with trifluoroacetic acid, the resulting amine was acylated using carbonyldiimidazole, whereby an isocyanate was subsequently obtained, which was then treated with piperazinone and cyclized in the

Molecules 2020, 25, 1211 5 of 17 was obtained, which was reduced to amine using generated in situ cobalt boride; the amine was then acylated to obtain a Boc-protected amino amide 16. After removal of the Boc-protection with triﬂuoroacetic acid, the resulting amine was acylated using carbonyldiimidazole, whereby an isocyanate was subsequentlyMolecules 2020 obtained,, 25, x FOR PEER which REVIEW was then treated with piperazinone and cyclized in5 of the 19 presence of triphenylphosphine oxide in Tf2O to form the desired Nutlin-3a. This method allowed the total number ofpresence stages toof betriphenylphosphine reduced, and theoxide stage in Tf2 ofO to separation form the desired on a chiralNutlin-3a. column This method to be allowed avoided. the total number of stages to be reduced, and the stage of separation on a chiral column to be avoided.

Scheme 2.SchemeNutlin-3a 2. Nutlin-3a synthesis synthesis proposed proposed by by the the researchersresearchers from from Vanderbild Vanderbild University University [31]. [31].

ScientistsScientists from Daiichi from Daiichi Sankyo Sankyo reported reported the thesynthesis synthesis of of compounds compounds 24 and24 25and using25 prolineusing as proline as the startingthe material starting material (Scheme (Scheme3)[ 32 3)]. [32]. Firstly, Firstly, the the reaction reaction with with alkyl alkyl lithium lithium was carried was carriedout with outthe with the previous protection of amide and carboxylic groups. Then, the racemic pyrrolidine 22 was obtained previous protectionin three stages. of amide and carboxylic groups. Then, the racemic pyrrolidine 22 was obtained in three stages. Molecules 2020, 25, x FOR PEER REVIEW 6 of 19

Scheme 3. Pyrrolidine-based inhibitors synthesized by Daiichi Sankyo [32]. Scheme 3. Pyrrolidine-based inhibitors synthesized by Daiichi Sankyo [32].

Compound 24 (Protein Data Bank ID: 3W69), with an IC50 value of 59 nm (homogeneous time resolved fluorescence), also exhibited a good pharmacokinetic profile and significant antitumor efficacy via oral administration in a mouse xenograft model using MV4-11 cells bearing wild type (WT) p53. On the basis of the Nutlin-3a compound, the pyrrolidine-containing compound 32 was synthesized [33]. Starting from the condensation of benzyl cyanide derivatives 26 with aromatic aldehydes 27 in the presence of sodium methylate, and further imine addition, the racemic pyrrolidine derivative 31 was obtained. After Boc-deprotection by triflic acid and amine addition, the racemic amide 32 was formed and then separated by a chiral supercritical fluid chromatography (). The most potent compound 35 is shown in Scheme 4 [34,35].

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Compound 24 (Protein Data Bank ID: 3W69), with an IC50 value of 59 nm (homogeneous time resolved fluorescence), also exhibited a good pharmacokinetic profile and significant antitumor efficacy via oral administration in a mouse xenograft model using MV4-11 cells bearing wild type (WT) p53. On the basis of the Nutlin-3a compound, the pyrrolidine-containing compound 32 was synthesized [33]. Starting from the condensation of benzyl cyanide derivatives 26 with aromatic aldehydes 27 in the presence of sodium methylate, and further imine addition, the racemic pyrrolidine derivative 31 was obtained. After Boc-deprotection by triflic acid and amine addition, the racemic amide 32 was formed and then separated by a chiral supercritical fluid chromatography (). The most potentMolecules compound 2020, 25, x FOR35 PEERis shown REVIEW in Scheme4[34,35]. 7 of 19

SchemeScheme 4.4.Synthesis Synthesis of of RG RG 7388, 7388, a potent a potent and and selective selective p53 p53MDM2−MDM2 inhibitor inhibitor in clinical in clinical development development [34,35]. − [34,35]. In 2014, Furet and co-workers published a new class of tetra-substituted imidazoles as a newIn class 2014, of Furet inhibitors and co-workers of the p53–MDM2 published interaction a new class (Scheme of tetra-substituted5)[ 36]. Commercially imidazoles availableas a new 2-fluoro-3-chloro-anilineclass of inhibitors of the p53–MDM2 was subjected interaction to iodination (Scheme with 5) [36]. N-Iodosuccinimide Commercially available (NIS), providing 2-fluoro-3- a regioisomericchloro-aniline mixturewas subjected of 4- and to iodination 6-iodo products, with N-Iodosuccinimide which were separable (NIS), byproviding flash chromatography. a regioisomeric Themixture desired of 4- 6-iodo and regioisomer6-iodo products, was reactedwhich were with cyclohexaneseparable by carbonitrile flash chromatography. in the presence The of desired trimethyl 6- aluminumiodo regioisomer to form was the reacted corresponding with cyclohexane benzamidine carb36onitrile, which in underwentthe presence smooth of trimethyl cyclization aluminum with ethylbromo-pyruvateto form the corresponding under benzamidine mild basic conditions 36, which (NaHCO underwent3; room smooth temperature). cyclization Waterwith ethylbromo- elimination waspyruvate effected under by mild the basic addition conditions of p-toluene (NaHCO sulfonic3; room acidtemperature). and heating Water to elimination 120 ◦C to was furnish effected the imidazoleby the addition core. Selective of p-toluene Sonogashira sulfonic coupling acid and of heating the iodine to with120 °C trimethyl to furnish silyl the acetylene imidazole provided core. intermediateSelective Sonogashira37. Conversion coupling of the the iodine acetylene with sidetrimethyl chain silyl to theacetylene desired provided acid was intermediate achieved by37. hydroborationConversion of (cyclohexenethe acetylene/borane-dimethylsulfide side chain to the desired complex) acid and was oxidative achieved workup. by hydroboration Efficient and selective(cyclohexene/borane-dimethylsulfide bromination of the imidazole complex) core was and effected oxidative by treatment workup. with Efficient N-Bromosuccinimide and selective (NBS)bromination in acetonitrile of the imidazole at room temperature, core was effected providing by treatment the suitable with substrate N-Bromosuccinimide for Suzuki coupling (NBS) with in acetonitrile at room temperature, providing the suitable substrate for Suzuki coupling with commercially available 3-chloro-4-fluoro boronic acid. Orthogonal ester protection/deprotection steps provided acid 38, which was converted to the 2-amino-oxadiazole in a two-step sequence (HATU ((1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, Hexafluorophosphate Azabenzotriazole Tetramethy Uronium) promoted hydrazone formation and ring closure with BrCN). Finally, deprotection of the tert-butyl ester 39 liberated carboxylic acid, which was converted to the corresponding carboxamide 40 using Propsal™ as a coupling reagent.

Molecules 2020, 25, 1211 7 of 17 commercially available 3-chloro-4-fluoro boronic acid. Orthogonal ester protection/deprotection steps provided acid 38, which was converted to the 2-amino-oxadiazole in a two-step sequence (HATU ((1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, Hexafluorophosphate Azabenzotriazole Tetramethy Uronium) promoted hydrazone formation and ring closure with BrCN). Finally, deprotection of the tert-butyl ester 39 liberated carboxylic acid, which was convertedMolecules 2020 to, 25 the, x FOR corresponding PEER REVIEW carboxamide 40 using Propsal™ as a coupling reagent.8 of 19

SchemeScheme 5. 5.Syntheses Syntheses of of inhibitor inhibitor 4040 [36].[36 ].

The mostThe most potent potent compounds compounds of of the the series series show show significant significant and specific specific anti-proliferative anti-proliferative activity activity in culturesin cultures of p53-dependent of p53-dependent cancer cancer cells. Thesecells. Thes resultse results warrant warrant further further evaluations evaluations of the of new the inhibitorsnew towardsinhibitors the goal towards of developing the goal of anti-cancer developing agents anti-cancer to fight agents tumors to harboringfight tumors an harboring overexpressed an or amplifiedoverexpressed MDM2 geneor amplified [36]. MDM2 gene [36].

2.3. Spirooxindole2.3. Spirooxindole Derivatives Derivatives The most promising MDM2 inhibitor sare possibly the compounds obtained by Shaomeng The most promising MDM2 inhibitor sare possibly the compounds obtained by Shaomeng Wang’s Wang’s group from the University of Michigan [37]. Based on molecular docking data for these group from the University of Michigan [37]. Based on molecular docking data for these structures, structures, the group proposed modeling the tryptophan fragment in the p53 protein with a the groupspirooxindole proposed fragment, modeling which, the due tryptophan to its greater fragment conformational in the rigidity, p53 protein is able to with provide a spirooxindole a better fragment,affinity which, for the dueMDM2 to protein. its greater This conformationalclass of compounds rigidity, can be obtained is able to as provideshown in a Scheme better 6, affi withnity for the MDM2high stereoselectivity. protein. This The class condensation of compounds reaction can of be the obtained aromatic as aldehydes shown inwith Scheme oxindoles6, with was high stereoselectivity.initially carried The out condensation either in the presence reaction of ofa base the aromaticduring boiling aldehydes or using with microwave oxindoles radiation, was initially to carriedobtain out either(E)-3-aryl-1,3-dihydroindol-2-ones in the presence of a base during41, which boiling were or further using microwaveintroduced into radiation, 1,3-dipolar to obtain E-3-aryl-1,3-dihydroindol-2-onescycloaddition reaction with 41aliphatic, which werealdehydes further 42 introducedand optically into 1,3-dipolaractive (5R,6S cycloaddition) -5,6- reactiondiphenylmorpholin-2-one with aliphatic aldehydes 43 in42 toluene.and optically The resulting active (5R,6S product) -5,6-diphenylmorpholin-2-one 44 was purified by column 43 in chromatography and then treated with a solution of dimethylamine in tetrahydrofuran and lead(IV) toluene. The resulting product 44 was purified by column chromatography and then treated with a acetate to give amide 45 in a quantitative yield. The absolute configuration of the products was solution of dimethylamine in tetrahydrofuran and lead(IV) acetate to give amide 45 in a quantitative confirmed by X-ray diffraction data [38]. yield. The absolute configuration of the products was confirmed by X-ray diffraction data [38].

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Scheme 6. TheThe general general scheme scheme for for the the synthesis synthesis of of compounds compounds of of the the MI MI series, series, proposed proposed by a group of researchers from the UniversityUniversity of Michigan [[38].38]. The synthesized compounds showed a significant cytotoxic effect on the prostate cancer cell line The synthesized compounds showed a significant cytotoxictoxic effecteffect onon thethe prostateprostate cancercancer cellcell lineline LNCap, with with IC IC5050 = =8686 nM, nM, and and on on the the colorectal colorectal cancer cancer cell cell line line HCTwt, HCTwt, with with IC IC50 =50 22= µM.22 µ M.Based Based on LNCap, with IC50 = 86 nM, and on the colorectal cancer cell line HCTwt, with IC50 = 22 µM. Based on on this method, a large library of spiro derivatives was synthesized, the most active of which are thisthis method,method, aa largelarge librarylibrary ofof spirospiro derivativesderivatives waswas synthesized,synthesized, thethe mostmost activeactive ofof whichwhich areare shownshown shown in Figure4. Compounds MI-43 ( 46), MI-63 (47), and MI-219 (48) showed excellent binding to inin FigureFigure 4.4. CompoundsCompounds MI-43MI-43 ((46),), MI-63MI-63 ((47),), andand MI-219MI-219 ((48)) showedshowed excellentexcellent bindingbinding toto thethe the MDM2 protein (Ki~5.7 nM), and compound MI-219 (48) was recognized as a selective inhibitor of MDM2 protein (Ki~5.7 nM), and compound MI-219 (48)) waswas recognizedrecognized asas aa selectiveselective inhibitorinhibitor ofof thethe the p53–MDM2 interaction due to its ability to induce cell apoptosis in tumor cells without affecting p53–MDM2 interaction due to its ability to induce cell apoptosis in tumor cells without affecting healthy ones [39]. Upon an oral injection of compound 48 in xenograft models with the SJSA-1 grafted healthy ones [39]. Upon an oral injection of compound 48 inin xenograftxenograft modelsmodels wiwithth thethe SJSA-1SJSA-1 graftedgrafted tumor line, the inhibition of tumor growth was 86% at a double dose of 200 mg/kg [40]. tumortumor line,line, thethe inhibitioninhibition ofof tumortumor growthgrowth waswas 86%86% atat aa doubledouble dosedose ofof 200200 mg/kgmg/kg [40].[40].

Figure 4. Most active compound-leaders of the MI series, synthesized by a team of researchers from Figure 4. Most active compound-leaders of the MI series, synthesized by a team of researchers from Figurethe University 4. Most ofactive Michigan compound-leaders [39,40]. of the MI series, synthesized by a team of researchers from thethe UniversityUniversity ofof MichiganMichigan [39,40].[39,40]. In 2019, Barakata and co-workers [41] synthesized a series of spiroindolinones using an asymmetric In 2019, Barakata and co-workers [41] synthesized a series of spiroindolinones using an 1,3-dipolarIn 2019, reaction Barakata as the and key co-workers step (Figure 5[41]). Their synthe investigationsized a series involved of spiroindolinones the design and synthesis using ofan asymmetric 1,3-dipolar reaction as the key step (Figure 5). Their investigation involved the design asymmetricsubstituted spirooxindoles1,3-dipolar reaction as potent as the MDM2 key step inhibitors, (Figure using5). Their an einvestigationfficient 1,3-dipolar involved cycloaddition the design and synthesis of substituted spirooxindoles as potent MDM2 inhibitors, using an efficient 1,3-dipolar andreaction synthesis [42,43 of]. Thesubstituted one-pot spirooxindoles multi-component as potent reactions MDM2 of a α inhibitors,,β-unsaturated using dienone an efficient derivative 1,3-dipolar with cycloaddition reaction [42,43]. The one-pot multi-component reactions of a α,β-unsaturated dienone cycloadditionsubstituted isatines reaction and [42,43]. amino The acid on derivativee-pot multi-component (l-4-thiazolidinecarboxylic reactions of a acid), α,β-unsaturated and underheating dienone in derivative with substituted isatines and amino acid derivative (L-4-thiazolidinecarboxylic acid), and derivativeMeOH at 60 with◦C forsubstituted 1.5–2.0 h, isatines were conducted and amino to acid generate derivative the focused (L-4-thiazolidinecarboxylic cycloadducts library, whichacid), and had underheating in MeOH at 60 °C for 1.5–2.0 h, were conducted to generate the focused cycloadducts underheatingfour stereogeneric in MeOH centers at with60 °C an for excellent 1.5–2.0 yieldh, were (69–89%). conducted to generate the focused cycloadducts library, which had four stereogeneric centers with an excellent yield (69–89%). library,The which anticancer had four activities stereogeneric of thesynthesized centers with compounds an excellent wereyield tested(69–89%). against colon (HCT-116), prostate (PC-3), and hepatocellular (HepG-2) cancer cell lines. The mechanism underlying the anti-cancer activity of the obtained spiroindolinones was further evaluated, and the study showed that these compounds inhibited colony formation and cell migration, arrested cancer cell growth at G2/M, and induced apoptosis through intrinsic and extrinsic pathways. The transactivation of p53 was confirmed using flow cytometry, where tested compounds increased the level of activated p53 and induced mRNA levels of cell cycle inhibitor p21.

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Scheme 6. The general scheme for the synthesis of compounds of the MI series, proposed by a group of researchers from the University of Michigan [38].

The synthesized compounds showed a significant cytotoxic effect on the prostate cancer cell line LNCap, with IC50 = 86 nM, and on the colorectal cancer cell line HCTwt, with IC50 = 22 µM. Based on this method, a large library of spiro derivatives was synthesized, the most active of which are shown in Figure 4. Compounds MI-43 (46), MI-63 (47), and MI-219 (48) showed excellent binding to the MDM2 protein (Ki~5.7 nM), and compound MI-219 (48) was recognized as a selective inhibitor of the p53–MDM2 interaction due to its ability to induce cell apoptosis in tumor cells without affecting healthy ones [39]. Upon an oral injection of compound 48 in xenograft models with the SJSA-1 grafted tumor line, the inhibition of tumor growth was 86% at a double dose of 200 mg/kg [40].

Figure 4. Most active compound-leaders of the MI series, synthesized by a team of researchers from the University of Michigan [39,40].

In 2019, Barakata and co-workers [41] synthesized a series of spiroindolinones using an asymmetric 1,3-dipolar reaction as the key step (Figure 5). Their investigation involved the design and synthesis of substituted spirooxindoles as potent MDM2 inhibitors, using an efficient 1,3-dipolar cycloaddition reaction [42,43]. The one-pot multi-component reactions of a α,β-unsaturated dienone derivative with substituted isatines and amino acid derivative (L-4-thiazolidinecarboxylic acid), and MoleculesMoleculesunderheating 20202020,, 2525 ,,in x 1211x FORFORMeOH PEERPEER at REVIEWREVIEW 60 °C for 1.5–2.0 h, were conducted to generate the focused cycloadducts10109 of of 19 1719 library, which had four stereogeneric centers with an excellent yield (69–89%). FigureFigure 5.5. SpiroindolinonesSpiroindolinones designdesign inin thethe workwork [41].[41].

TheThe anticanceranticancer activitiesactivities ofof thethe synthesizedsynthesized compoundscompounds werewere testedtested againstagainst coloncolon (HCT-116),(HCT-116), prostateprostate (PC-3),(PC-3), andand hepatocellularhepatocellular (HepG-2)(HepG-2) cancercancer cellcell lines.lines. TheThe mechanismmechanism underlyingunderlying thethe anti-anti- cancercancer activityactivity ofof thethe obtainedobtained spiroindolinonesspiroindolinones waswas furtherfurther evaluated,evaluated, andand thethe studystudy showedshowed thatthat thesethese compoundscompounds inhibitedinhibited colonycolony formationformation andand cellcell migration,migration, arrestedarrested cancercancer cellcell growthgrowth atat G2/M,G2/M, andand inducedinduced apoptosisapoptosis throughthrough intrinsicintrinsic andand extrinsicextrinsic pathways.pathways. TheThe transactivationtransactivation ofof p53p53 waswas confirmedconfirmed usingusing flowflow cytometry,cytometry,Figure 5.wherewhereSpiroindolinones testedtested cocompoundsmpounds design in increasedincreased the work [ 41thethe]. levellevel ofof activatedactivated p53p53 andand inducedinduced mRNAmRNA levelslevels ofof cellcell cyclecycle inhibitorinhibitor p21.p21. AA series series of of compounds compounds using using the the four-component four-component UgiUgi reactionreaction (Scheme(Scheme 77)7))[ [44][44]44] werewere obtained.obtained. TheThe four-componentfour-component UgiUgi Ugi reactionreaction reaction waswas was carriedcarried carried ouou outtt byby by mixingmixing mixing equivalentequivalent equivalent amountsamounts amounts ofof ofethylethyl ethyl 6-6- chloroindole-2-carboxylate6-chloroindole-2-carboxylatechloroindole-2-carboxylate 494949,, benzylaminebenzylamine, benzylamine 505050,, ,formicformic formic acidacid acid 515151,, ,andand and thethe the correspondingcorresponding corresponding isocyanideisocyanide 5252 inin methanol. methanol. TheThe resulting resulting esters esters 5353 werewere hydrolyzed hydrolyzed toto givegive thethe acidacid 5454..

SchemeScheme 7.7. GeneralGeneral schemeschemescheme for forfor the thethe synthesis synthesisynthesis ofs ofof inhibitors inhibitorsinhibitors of ofof the thethe p53–MDM2 p53–MDM2p53–MDM2 interaction interactioninteraction obtained obtainedobtained by the byby theUgithe UgiUgi reaction reactionreaction [44]. [44].[44]. Using molecular docking results, it was found that in the co-crystalline structure of molecule 54 UsingUsing molecularmolecular dockingdocking results,results, itit waswas foundfound thatthat inin thethe co-crystallineco-crystalline structurestructure ofof moleculemolecule 5454 with the MDM2 protein, there is a π–π-stacking interaction between the benzylamine fragment in the withwith thethe MDM2MDM2 protein,protein, therethere isis aa ππ––ππ-stacking-stacking interactioninteraction betweenbetween thethe benzylaminebenzylamine fragmentfragment inin thethe inhibitor molecule and the His96 protein fragment. Based on these data, a library of 19 possible isomers inhibitorinhibitor moleculemolecule andand thethe His96His96 proteinprotein fragmentfragment.. BasedBased onon thesethese data,data, aa librarylibrary ofof 1919 possiblepossible of compound 54, with different positions of the fluorine atom in the benzyl position, was obtained [44]. isomersisomers ofof compoundcompound 5454,, withwith differentdifferent positionspositions ofof thethe fluorinefluorine atomatom inin thethe benzylbenzyl position,position, waswas These compounds were tested for binding to the MDM2 protein in the form of racemic mixtures; obtainedobtained [44].[44]. TheseThese compoundscompounds werewere testedtested forfor bindbindinging toto thethe MDM2MDM2 proteinprotein inin thethe formform ofof racemicracemic however,mixtures; onlyhowever, one of only the synthesized one of the molecules synthesize withd molecules a 3,4,5-trifluorobenzylamine with a 3,4,5-trifluorobenzylamine substituent was mixtures; however, only one of the synthesize1 d molecules with a 3,4,5-trifluorobenzylamine active and showed a binding constant of 130 nM . −1 substituentsubstituent waswas activeactive andand showedshowed aa bindingbinding constantconstant− ofof 130130 nMnM−1.. In 2014, the dispiro-compound 58, as a potential p53–MDM2 inhibitor, was proposed [45,46]. InIn 2014,2014, thethe dispiro-compounddispiro-compound 5858,, asas aa potentialpotential p53–MDM2p53–MDM2 inhibitor,inhibitor, waswas proposedproposed [45,46].[45,46]. This compound was obtained via a 1,3-dipolar cycloaddition reaction with isatine 57, sarcosine 56, ThisThis compoundcompound waswas obtainedobtained viavia aa 1,3-dipolar1,3-dipolar cycloadditioncycloaddition reactionreaction withwith isatineisatine 5757,, sarcosinesarcosine 5656,, and thiohydantoin-based dipolarophiles 55 (Scheme8). andand thiohydantoin-bathiohydantoin-basedsed dipolarophilesdipolarophiles 5555 (Scheme(Scheme 8).8).

SchemeScheme 8. 8. GeneralGeneral schemescheme for for the the synthesis synthesis of of dispiroindolinone dispiroindolinone 5858 [45,46].[[45,46].45,46].

The most potent compound hadhad ICIC5050 =1.08=1.08 ± 0.960.96 µMµM on on LNCap LNCap cell cell lines. lines. The most potent compound had IC50 =1.08 ±± 0.96 µM on LNCap cell lines.

2.4.2.4. Benzodiazepine-2,5-dionesBenzodiazepine-2,5-diones (BDP)(BDP)

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2.4.Molecules Benzodiazepine-2,5-diones 2020, 25, x FOR PEER REVIEW (BDP) 11 of 19

The pharmaceutical company Johnson and Johnson screened a library of 1,4-benzodiazepine- 2,5-dione (BDP)(BDP) capablecapable ofof binding binding to to the the p53 p53 domain domain of of the the MDM2 MDM2 protein. protein. These These compounds compounds can can be obtainedbe obtained in two in stagestwo stages (Scheme (Scheme9), with 9), the with initial the multi-component initial multi-component Ugi reaction Ugi between reaction equimolar between amountsequimolar of amounts aldehyde 59of, aminealdehyde60, Boc59-protected, amine 60 anthranilic, Boc-protected acid 61 ,anthranilic and 1-isocyanidecyclohexane acid 61, and 1- 62isocyanidecyclohexanein methanol, to obtain 62 in the methanol, product 63to, obtain which the then product forms target63, which cyclic then product forms64 targetunder cyclic the actionproduct of 64 acetyl under chloride the action [47]. of The acetyl most chloride active compound [47]. The most of this active group compound is the enantiomer of this group containing is the 1 2 3 theenantiomerpara-chlorophenyl containing groupthe para at-chlorophenyl R , an α-carboxybenzyl group at R1, groupan α-carboxybenzyl at R , and an group iodine at atom R2, and at Ran; thisiodine compound atom at R has3; this a binding compound constant has a with binding the MDM2 constant protein with the of KMDM2d = 80 nM.protein of Kd = 80 nM.

Scheme 9. General scheme for the synthesis of compounds of the benzodiazepine (BDP) series [47]. Scheme 9. General scheme for the synthesis of compounds of the benzodiazepine (BDP) series [47]. In the next investigation step, Johnson and Johnson undertook an enantioselective synthesis of the In the next investigation step, Johnson and Johnson undertook an enantioselective synthesis of benzodiazepine compounds of the BDP series (Scheme 10). Compound 65 was initially introduced into the benzodiazepine compounds of the BDP series (Scheme 10). Compound 65 was initially an alkylation reaction with methyl lithium to form a racemic secondary alcohol 66, which reacted with introduced into an alkylation reaction with methyl lithium to form a racemic secondary alcohol 66, camphoric acid chloride to form a mixture of two diastereomers. This mixture was then recrystallized which reacted with camphoric acid chloride to form a mixture of two diastereomers. This mixture to isolate ester 68 as a single stereoisomer, which was then hydrolyzed and introduced into a Mitsunobu was then recrystallized to isolate ester 68 as a single stereoisomer, which was then hydrolyzed and reaction with phthalimide, and the phthalimide protection was then removed with hydrazine to form introduced into a Mitsunobu reaction with phthalimide, and the phthalimide protection was then amine 70. This amine was used in the Ugi reaction, followed by cyclization, described above, and the removed with hydrazine to form amine 70. This amine was used in the Ugi reaction, followed by mixture of diastereomers was then separated by column chromatography, after which alkylation at cyclization, described above, and the mixture of diastereomers was then separated by column the nitrogen atom yielded the necessary compound 71 [48], the structure of which was confirmed by chromatography, after which alkylation at the nitrogen atom yielded the necessary compound 71 X-ray diffraction. [48], the structure of which was confirmed by X-ray diffraction. Compound 71 was further modified by introducing carboxyalkyl groups at the unsubstituted Compound 71 was further modified by introducing carboxyalkyl groups at the unsubstituted amide nitrogen atom, as well as by replacing the nitro group with an amino group. The obtained amide nitrogen atom, as well as by replacing the nitro group with an amino group. The obtained compound showed higher than initial activity in a polarization fluorescence immunoassay (0.25 µM compound showed higher than initial activity in a polarization fluorescence immunoassay (0.25 µM compared to 0.85 µM), since the presence of the amino group ensured binding to the Val93 carbonyl compared to 0.85 µM), since the presence of the amino group ensured binding to the Val93 carbonyl atom in the MDM2 protein, but it showed less activity in cytotoxicity experiments, possibly due to its atom in the MDM2 protein, but it showed less activity in cytotoxicity experiments, possibly due to zwitterionic structure, which limited the ability to penetrate the cell membrane. The authors also made its zwitterionic structure, which limited the ability to penetrate the cell membrane. The authors also attempts to introduce morpholine and isopropyloxy groups into the target molecule; however, these made attempts to introduce morpholine and isopropyloxy groups into the target molecule; however, substitutions led to a decrease in cytotoxicity and loss of activity. these substitutions led to a decrease in cytotoxicity and loss of activity. A group of scientists led by Alexander Domling of the University of Pittsburgh, together with A group of scientists led by Alexander Domling of the University of Pittsburgh, together with Thad Holak of the Max Planck Institute, also proposed an interesting approach to creating p53–MDM2 Thad Holak of the Max Planck Institute, also proposed an interesting approach to creating p53– interaction inhibitors using the multi-component Ugi reaction [49,50]. MDM2 interaction inhibitors using the multi-component Ugi reaction [49,50].

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SchemeScheme 10.10. GeneralGeneral schemescheme ofof thethe enantioselectiveenantioselective pathwaypathway for thethe synthesissynthesis ofof compoundscompounds ofof thethe BDPBDP seriesseries [[48]48] (DIAD(DIAD isis diisopropyldiisopropyl azodicarboxylate).azodicarboxylate).

2.5. Chromenotriazole Pyrimidines 2.5. Chromenotriazole Pyrimidines AnotherAnother interestinginteresting exampleexample ofof thethe developmentdevelopment ofof p53–MDM2p53–MDM2 interactioninteraction inhibitorsinhibitors isis thethe synthesissynthesis ofof chromenotriazolechromenotriazole pyrimidinepyrimidine derivativesderivatives (Scheme(Scheme 1111))[ [51].51]. InitialInitial aldolaldol condensationcondensation ofof 78 79 80 aldehydesaldehydes 78 andand methylmethyl ketones 79 inin an alkaline medium aaffordsﬀords hydroxychalcones 80,, whichwhich thenthen 81 condensatecondensate withwith 4H-1,2,4-triazole-3-amine4H-1,2,4-triazole-3-amine by heating to give compounds 81 as thethe mixturesmixtures ofof twotwo tautomers.tautomers. TheThe desired desired enamine enamine tautomer tautomer can be can isolated be byisolated mashing by inmashing chloroform. in Thechloroform. condensation The condensation of compound 81 with various aldehydes by heating or in an acidic medium with phenols allows the condensation products 82 to be obtained as mixtures of stereoisomers. After the

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of compound 81 with various aldehydes by heating or in an acidic medium with phenols allows Moleculesthe condensation 2020, 25, x FOR productsPEER REVIEW82 to be obtained as mixtures of stereoisomers. After the methylation13 of 19 of compounds 82 at the nitrogen atom, 11-methylchromenotriazolepyrimidines 83 and 84 were formed as methylation of compounds 82 at the nitrogen atom, 11-methylchromenotriazolepyrimidines 83 and the mixture of the racemic anti- and syn-isomers, which could be separated by column chromatography, 84 were formed as the mixture of the racemic anti- and syn-isomers, which could be separated by giving the target compound 85. column chromatography, giving the target compound 85.

Scheme 11. General scheme for the synthesis of inhibitors of the p53–MDM2 interaction based on Schemechromenotriazolepyrimidines 11. General scheme for [the51]. synthesis of inhibitors of the p53–MDM2 interaction based on chromenotriazolepyrimidines [51]. The best cytotoxicity values in the analysis using the method of homogeneous ﬂuorescence with timeThe resolution best cytotoxicity (HTRF assay) values showed in the analysis the compound using the with method R1 = Br of andhomogeneous R2 = Cl (IC fluorescence= 0.89 0.20 withµM). 50 ± time resolution (HTRF assay) showed the compound with R1 = Br and R2 = Cl (IC50 = 0.89 ± 0.20 µM). 2.6. Piperidinones 2.6. PiperidinonesA piperidinone compound AMG 232 (93) was synthesized using the Michael condensation of ketoneA piperidinone86 and methacrylic compound acid AMG ester, 232 to (93 get) was compound synthesized87. Then,using the reduction Michael and condensation ester formation of ketonewith 86 further and methacrylic cyclization atacid base ester, conditions to get compound allowed cyclic 87. Then, lactone reduction89 to be and obtained. ester formation The subsequent with furtheralkylation cyclization with allyl at base bromide conditions and the allowed reaction ofcyclic the lactonelactone opening 89 to be by obtained. an amine derivativeThe subsequent gave the alkylationchiral amide with 91allyl, which bromide was and then the cyclized reaction to of epypiperidinone the lactone opening92 (Scheme by an amine 12)[52 derivative,53]. gave the chiral amideIn 2015, 91, Gessierwhich was and then co-workers cyclized published to epypiperidinone a synthesis 92 of (Scheme a new class 12) [52,53]. of the piperidinone-base small-moleculeIn 2015, Gessier inhibitors and co-workers of the p53–MDM2 publishedprotein–protein a synthesis of a interactionnew class of (Scheme the piperidinone-base 13). Starting from small-moleculean initial hit identifiedinhibitors by of virtualthe p53–MDM2 screening, protein–protein a derivatization programinteraction resulted (Scheme in compound 13). Starting101 from, which an isinitial a low hit nanomolar identified inhibitor by virtual of the screening, p53–MDM2 a derivatization interaction showing program significant resulted cellularin compound activity 101[54,,55 ]. whichThis is compound a low nanomolar reached inhibitor a low nanomolar of the p53–MDM2 biochemical interaction potency, accompaniedshowing significant by significant cellular and activity specific [54,55].inhibition This of compound the proliferation reached of the a low p53-dependent nanomolar SJSA-1 biochemical cells in thepotency, low micromolar accompanied range. by The significant production andof specific compound inhibition101 allowed of the an proliferation interesting level of th ofe cellularp53-dependent potency toSJSA-1 be reached, cells in which the low was micromolar comparable to range.that The of the production reference p53–MDM2 of compound inhibitor 101 allowed Nutlin-3a an interesting (IC50 = 1.9 levelµM in of the cellular SJSA-1 potency assay). to be reached, which wasA representative comparable to synthesis that of the for reference compound p53–MDM2101 is described inhibitor in Nutlin-3a Scheme (IC13.50 The= 1.9 commercially µM in the SJSA-1available assay). methyl 2-(4-hydroxy-3-methoxyphenyl)acetate was subjected to a Mitsunobu reaction with (S)-butan-2-ol, using diethyl azodicarboxilate (DEAD) and triphenylphosphine as the reagents, to deliver the sec-butyl ether 95 as its pure (R)-enantiomer. The methyl ester 95 was hydrolyzed with

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LiOH into the corresponding carboxylic acid 96, and then reacted with oxalyl chloride to give the acyl chloride 97 in quantitative yields. The cycloaddition reaction of 97 with the imine 98, obtained from the condensation of the commercially available 4-chlorobenzaldehyde and 4-nitroaniline, led to the dihydroisoquinolinone analog 99, with no stereo selectivity observed for the newly created asymmetric center. The nitro group of 99 was reduced with iron following classical reaction conditions, to give the corresponding aniline 100 in nearly quantitative yields. Finally, the aniline functionality underwent two consecutive reductive aminations with isonicotinaldehyde and formaldehyde, respectively, using NaBH(Oac)3 as a reducing agent, to deliver compound 101 as a mixture of two diastereoisomers. At that point, the separation of the diastereoisomeric pair could not be achieved by classical normal phase or reversed phase chromatography and required the use of chiral chromatography conditions. Therefore, this potent inhibitor has been the starting point of another round of medicinal chemistry optimization that has culminated in NVP-CGM097 (highly potent and selective MDM2 inhibitor), which is a compound currently under evaluation in a phase I clinical trial for cancer patients [54,55].

COOMe O O 10% t-BuOK, THF, 0.2% RuCl2[(S xylBINAP)(S-DAIPEN)], COOMe rt, 2 d 40% t-BuOK, i-PrOH, + Cl Cl H2 (50 PSI),rt, 65 h; Cl Cl 86 87 O O

COOi-Pr O O OH Br 1. LiOH, THF/MeOH/H2O Cl 2. PPTS, toluene, reflux LiHMDS, THF Cl Cl Cl Cl Cl 88 89 9 0

HO O 1. Tf O, 2,6-lutidine, OH 2 HN CH2Cl2, -78 to 0°C N OH 2. aq NaHCO Tf O, H N 3 2 2 HOO 2,6-lutidine neat, 100 °C, overnight Cl Cl Cl Cl 91 92

O O S OTf O 1. Method A: i-PrSNa, DMF O N Method B: i-PrSO2Na, acetonitrile N COOH Method C:i-PrSH, base, DMF

2. RuCl3, NaIO4, acetonitrile/CCl /H O. Cl 4 2 Cl Cl Cl 93

SchemeScheme 12. 12.The The synthesis synthesis of of piperidinone piperidinone 93 [52].93 [52].

A representative synthesis for compound 101 is described in Scheme 13. The commercially available methyl 2-(4-hydroxy-3-methoxyphenyl)acetate was subjected to a Mitsunobu reaction with (S)-butan-2-ol, using diethyl azodicarboxilate (DEAD) and triphenylphosphine as the reagents, to deliver the sec-butyl ether 95 as its pure (R)-enantiomer. The methyl ester 95 was hydrolyzed with LiOH into the corresponding carboxylic acid 96, and then reacted with oxalyl chloride to give the acyl chloride 97 in quantitative yields. The cycloaddition reaction of 97 with the imine 98, obtained from the condensation of the commercially available 4-chlorobenzaldehyde and 4-nitroaniline, led to the dihydroisoquinolinone analog 99, with no stereo selectivity observed for the newly created asymmetric center. The nitro group of 99 was reduced with iron following classical reaction conditions, to give the corresponding aniline 100 in nearly quantitative yields. Finally, the aniline functionality underwent two consecutive reductive aminations with isonicotinaldehyde and formaldehyde, respectively, using NaBH(OAc)3 as a reducing agent, to deliver compound 101 as a mixture of two diastereoisomers. At that point, the separation of the diastereoisomeric pair could not be achieved by classical normal phase or reversed phase chromatography and required the use of chiral chromatography conditions. Therefore, this potent inhibitor has been the starting point of another round of medicinal chemistry optimization that has culminated in NVP-CGM097 (highly potent and selective MDM2 inhibitor), which is a compound currently under evaluation in a phase I clinical trial for cancer patients [54,55].

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Molecules 2020, 25, x FOR PEER REVIEWSchemeScheme 13. 13. TheThe synthesis of of piperidinone piperidinone 101101 [54].[54 ]. 16 of 19

2.7. Peptide-Based Compounds Peptides and peptide derivatives can be designed to become potent p53–MDM2/X interaction inhibitors. The Novartis group mapped the MDM2-binding site on p53 using synthetic peptide libraries derived from the N-terminal region of p53. The active peptides defined the consensus MDM2-binding site on p53 to be Thr18-Phe-Ser-Asp-Leu-Trp23. This hexapeptide, however, was only a weak inhibitor, with an IC50 value, or concentration required for the inhibition of 50% of p53 binding to MDM2, of 700 µM in an ELISA format [60]. The most active peptide obtained in this way (Figure 6) showed a 28-fold greater inhibition of the p53/MDM2 interaction than the wild-type p53- derived peptide. (Peptide 102 was effective at inhibiting the p53/MDM2 interaction in cells [56]; in addition, this sequence was active when expressed either with a glutathione S-transferase tag51 or in the active-site loop of thioredoxin [57]. Figure 6. Peptide 102 [57]. Figure 6. Peptide 102 [57]. 3. Conclusions 3. Conclusions Fifteen years after the discovery of Nutlin, a number of new potential inhibitors of the p53–MDM2 interactionFifteen have years been after proposed, the discovery some of of Nutlin, which havea number shown of anew high potential cytotoxicity inhibitors and encouraging of the p53– resultsMDM2 ininteraction preclinical have and clinicalbeen proposed, studies. Over some the of past which decade, have some shown attention a high has cytotoxicity been paid bothand toencouraging the search forresults new in small preclinical molecules and clinical and the studie improvements. Over the of past the biomedical decade, some properties attention of has already been knownpaid both classes to the of search compounds, for new as small well asmolecules to the modiﬁcation and the improvement of existing of synthesis the biomedical schemes properties to obtain enantiomericallyof already known pureclasses compounds. of compounds, Further as well directions as to the of modification research in thisof existing area will synthesis depend schemes on the resultsto obtain of ongoingenantiomerically clinical trials. pure Amongcompounds. the most Further promising directions classes of research of small in molecules this area tested will depend for the on the results of ongoing clinical trials. Among the most promising classes of small molecules tested for the inhibition of p53–MDM2 protein–protein interactions are Nutlin’s analogues, as well as spirooxindoles and benzodiazepine-2,5-diones, whose rigid polycyclic framework allows one to achieve the optimal arrangement of substituents in the molecule necessary for interactions with the MDM2 binding site.

Funding: This research was funded by the Russian Foundation for Basic Research, grant number 19-03-00201.

Conflicts of Interest: The authors declare no conflicts of interest.

References

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Molecules 2020, 25, 1211 15 of 17 inhibition of p53–MDM2 protein–protein interactions are Nutlin’s analogues, as well as spirooxindoles and benzodiazepine-2,5-diones, whose rigid polycyclic framework allows one to achieve the optimal arrangement of substituents in the molecule necessary for interactions with the MDM2 binding site.

Author Contributions: A.B. and E.B. wrote the manuscript; N.Z. and A.M. contributed to the manuscript writing; E.B. supervised the study. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Russian Foundation for Basic Research, grant number 19-03-00201. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

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