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Inhibition of the MDM2-p53 interactions has been demonstrated through in vitro HTRF assays (IC50 up to 3.1 nM), while Western blot analysis showed act...
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Brief Article Cite This: J. Med. Chem. 2018, 61, 9386−9392

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Inhibition of p53-Murine Double Minute 2 (MDM2) Interactions with 3,3′-Spirocyclopentene Oxindole Derivatives Maxime Gicquel, Catherine Gomez, Maria Concepcion Garcia Alvarez, Olivier Pamlard, Vincent Gueŕ ineau, Eric Jacquet, Jeŕ ôme Bignon,* Arnaud Voituriez,* and Angela Marinetti* Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Sud, Université Paris-Saclay, 1, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France

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S Supporting Information *

ABSTRACT: 3,3′-Spirocyclopentene oxindoles structurally related to Wang’s spiropyrrolidine oxindoles have been highlighted as a new class of antiproliferative agents against cancer cell lines with wild-type p53 status (IC50 up to 0.96 μM on SJSA-1 and 2.9 μM in HCT116 p53-wt). Inhibition of the MDM2-p53 interactions has been demonstrated through in vitro HTRF assays (IC50 up to 3.1 nM), while Western blot analysis showed activation of p53 selectively in HCT116 cancer cell lines with wild-type p53.

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INTRODUCTION The tumor suppressor protein p53 is known to be inhibited in many human cancers by the murine double minute 2 oncoprotein (MDM2). Accordingly, restoration of p53 activity has become an appealing therapeutic strategy for which several small molecules have been designed during the past 15 years, notably in the nutlin family.1−5 The 2-oxospiro[indoline-3,3′pyrrolidine] 1 (Figure 1) typifies a well-known series of inhibitors of the MDM2-p53 interaction initially reported by Shaomeng Wang and co-workers and developed then by the same authors in collaboration with Sanofi.6−11 Compounds 1

bind to MDM2 with low nanomolar affinity, reactivate p53 in tumor cells, and induce strong tumor growth inhibition. Wang’s pioneering work on this class of compounds has founded an innovative strategy that ends up to early stage clinical trials for the treatment of human cancers retaining wild-type p53 with overexpressed MDM2, such as myeloid leukemia or solid tumors.12,13 A known shortcoming of compounds 1 is that they isomerize via reversible ring-opening retro-Mannich reactions leading to equilibrium mixtures of less active stereoisomers (Figure 1a).10,14 Indeed, stereoisomers of 1 have been isolated and clear correlations have been established between the relative configurations of the stereogenic centers, the consequent geometry of the pyrrolidine unit and the binding affinity. The cis−cis stereoisomer (R,R,R,S) shown in Figure 1 proved to be the most active compound. To circumvent this stability issue, several strategies have been applied. Thus, for instance, spirooxindoles with two identical substituents on the pyrrolidine C2 carbon have been designed that force the isomerization process toward a single, well-defined diastereomer. In this series, compound 2 (Figure 1b) has entered clinical trials recently.15,16 Alternatively, Gollner designed another series of spirooxindoles typified by 3 in Figure 1b, in which the C2 carbon of a pyrrolidine constitutes the quaternary center at the ring junction instead of the C3 carbon. The 3,2′-spiropyrrolidinyl units ensure the configurational stability of these compounds since the retroMannich reaction cannot occur here. Compound 3 was tested in the SJSA-1 osteosarcoma xenograft model by oral

Figure 1. (a) Wang’s 2-oxospiro[indoline-3,3′-pyrrolidine] 1 and its epimerization via retro-Mannich/Mannich reactions. (b) Compounds designed to prevent epimerization: 2 (see ref 16) and 3 (see ref 17). © 2018 American Chemical Society

Received: July 19, 2018 Published: September 17, 2018 9386

DOI: 10.1021/acs.jmedchem.8b01137 J. Med. Chem. 2018, 61, 9386−9392

Journal of Medicinal Chemistry

Brief Article

administration. Findings confirmed on-target activity, i.e., MDM2 inhibition as the mode of action.17 Herein we report a new strategy to avoid the undesired epimerization process above, that is the use of spiro-oxindole derivatives, structurally related to 1, in which the pyrrolidine unit of the spiranic scaffold is replaced by a cyclopentene ring (Figure 2).

Figure 3. Synthesis of spirocyclopenteneoxindoles via phosphine catalyzed [3 + 2] cyclizations.

Figure 2. Carbocyclic analogues of the MDM2-p53 inhibitors 1 (this work).

In our preliminary study,25 the method has been illustrated notably by the synthesis of esters 7a from the N-acetyl 3-(3chloro-2-fluorobenzylidene)oxindole (E)-5 and allenoate 6a (R = Et) in the presence of PPh3 or PMe2Ph as the catalyst (Scheme 1, entries 1, 2). Unfortunately, these [3 + 2] cyclization reactions produced the cis−cis stereoisomer 7a′ as a minor product only (98% enantiomeric excess ([α]D25 = +68 (c = 0.1, CHCl3) for the first eluted isomer). The other diastereomers 4a″−4a⁗ of the same spirooxindole-amide have been prepared as reported previously.25 With S. Wang having demonstrated that the nature of the amide side chain modulates significantly the MDM2 affinity of the spirooxindole derivatives, we have prepared also spirooxindoles 4b′−4d′ displaying various side chains, for comparison purposes. Driven by the nature of some of the most active compounds 1, we have targeted especially carboxamides with R = 3-hydroxy-3-methylcyclobutyl,10,11 4-hydroxycyclohexyl,12,26,27 and 4-(hydroxycarbonyl)-phenyl.15

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RESULTS AND DISCUSSION Structure−activity relationship studies have demonstrated that in the spiro-pyrrolidine oxindoles 1, the NH group in the 3position is not crucial to their antitumor activity related to inhibition of the MDM2-p53 interaction. It has been shown, for instance, that analogues in which the NH function occupies the 2-position and a carbon atom takes the 3-position may be strong inhibitors as well.17 Also, analogues of 1 displaying bicyclic pyrrolidine units with substituted N atoms have demonstrated good activity.18 To the best of our knowledge, however, analogues of 1 displaying an all-carbon ring as the spirocyclic component instead of a pyrrolidinyl unit have not been considered so far in the literature, although they will have the main advantage of being chemically and configurationally stable. In this context, the main aim of this study has been to establish if carbon analogues of the spiro-pyrrolidineoxindoles 1 might display analogous MDM2-p53 inhibition. For these initial studies we have targeted the spiro-3,3′cyclopenteneoxindoles 4 in Figure 2. For comparison purposes, the targeted carbocyclic analogues of 1 display the same substitution pattern on the oxindole (6-chlorooxindole), as well as the same 3-chloro-2-fluorophenyl substituent on the αcarbon. Compared to 1, compounds 4 lack the stereogenic center in C4, which should simplify the synthetic approach and reduce the number of possible diastereomers. Synthesis. For building the spirocyclic scaffold of 4, we take advantage of the so-called Lu’s reaction, a phosphine promoted [3 + 2] cyclization between electron-poor allenes and electron-poor olefins leading to highly functionalized cyclopentenes.19,20 We and others have demonstrated previously that this general method can be conveniently applied to the cyclization between 3-arylidene oxindoles I and allenoates II to afford spirooxindole derivatives III or IV in racemic and enantioenriched forms (Figure 3).21−24 Starting from nonsubstituted allenoates (R′ = H), the reaction affords spirooxindoles III. With γ-substituted allenoates (R′ ≠ H) the reaction takes place regioselectively via α-addition of the allenoate−phosphine zwitterionic adduct and ends up with spirocyclopent-3-enes IV.25 With these efficient synthetic tools in hand, we have envisioned access to spirooxindole esters of the general formula IV (for R′ = CH2tBu) as precursors for the target spirooxindole carboxamides 4. 9387

DOI: 10.1021/acs.jmedchem.8b01137 J. Med. Chem. 2018, 61, 9386−9392

Journal of Medicinal Chemistry

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Scheme 1. Optimization of the Phosphine-Promoted [3 + 2] Cyclization Reactions toward the Synthesis of the cis−cis Spirooxindoles 7′a

a

Conditions: PR3 (20 mol %), rt, 12−24 h.

Scheme 2. (a) Synthesis of the Spirooxindole cis−cis 4a′ and (b) Molecular Structure of Diastereomers 4a″−4a⁗

Carboxamides 4b′−4d′ have been obtained from cis−cis 7b′ in two steps, by a saponification−amidation sequence under the conditions shown in Scheme 3. For the synthesis of 4b′,c′, the amidation reaction was carried out on the corresponding O-TBS protected amines. The silyl group was removed by following classical procedures. Pure enantiomers of these carboxamides have been obtained by HPLC separation on Chiracel ID columns.

Thus, we have demonstrated that phosphine-promoted [3 + 2] cyclization reactions, combined with a few, subsequent functional group transformation steps, provide a flexible access to carbocyclic analogues of 1 from easily available starting materials. The method complements other cycloaddition reactions currently used for the synthesis of MDM2-p53 inhibitors.28 Biological Results. Compounds 4a′ and the reference compound 1 display an overall analogous spiranic scaffold and 9388

DOI: 10.1021/acs.jmedchem.8b01137 J. Med. Chem. 2018, 61, 9386−9392

Journal of Medicinal Chemistry

Brief Article

Scheme 3. Synthesis of the Spirooxindole Amides 4b′−4d′a

a

Conditions. For 4b′ and 4c′: (a) RNH2, HATU, DIPEA, CH2Cl2, rt, 16 h; (b) KOH in THF (for 4b′) or HCl 2 M in ether (for 4c′). For 4d′: (a) SOCl2, CH2Cl2, 0 °C to rt; (b) p-NH2C6H4CO2tBu, pyridine, rt, 16 h; (c) TFA, CH2Cl2, 1 h, rt.

Table 1. Inhibitory Activities of Compounds 4 and 8′ against the p53-MDM2 Interaction, Using the HTRF Assaya

a

All the experiments were done in triplicate. IC50 values were calculated using the GraphPad Prism software from polynomial curves. Data are presented as the mean ± SD.

cyclopentene unit, without significant loss of MDM2 affinity. Thus, the pyrrolidine/cyclopentene core units can be viewed here as simple platforms giving appropriate distribution of the key substituents. Most notably, the highest IC50 value (33 nM) was obtained with the cis−cis isomer 4a′. Thus, the relationships between the stereochemistry of the spiranic scaffold and the binding affinity follow an overall analogous trend30 as for the Wang’s spiro-pyrrolidine-3,3′-indolines.10 Variation of the amide side-chain of cis−cis 4 showed that a 3-hydroxy-3-methylcyclobutyl chain gives moderate affinity (4b′). The 4-hydroxycyclohexyl (4c′) and 4(hydroxycarbonyl)phenyl (4d′) chains afford good binding affinity to MDM2 (IC50 = 619 and 349 nM respectively). Nevertheless compounds 4c′ and 4d′ do not attain better affinity levels than the 2-morpholinoethyl-substituted compound 4a′. These few examples suggest that SARs may not follow exactly the same trends in the Wang’s pyrrolidine series and in their cyclopentene analogues 4. Therefore, further SAR studies, involving variations of the substitution pattern, might allow further optimization of the binding affinity in this new cyclopentene series.

the same substitution pattern. However, the three-dimensional arrangement and the related spatial distribution of the substituents may differ significantly in the two series, due to the presence of the planar olefinic unit in 4′. Thus, in vitro binding affinity studies on MDM2 are crucial here to establish the possible analogy between the two series with respect to their biological behavior. We have investigated the biochemical binding affinity of compounds 4a′−a⁗ and 4b′−d′ to MDM2, using an HTRF assay29 to determine the drug concentration required to inhibit by 50% (IC50) the interaction between MDM2 and p53. Nutlin-3a was used as the reference (IC50 = 3.5 ± 2.65 nM). Results are shown in Table 1. At first, the binding affinities of the four diastereomers 4a have been measured showing that the affinity strongly depends on the relative configuration of the stereogenic centers of the spiranic scaffold. Although the trans−cis (4a‴) and trans−trans (4a″) isomers displayed IC50 values in the low μM range only, the cis−trans (4a⁗) and cis−cis (4a′) derivatives displayed significant in vitro affinity, with IC50 values of 739 and 33 nM, respectively. These initial assays strongly support our working hypothesis, i.e., the feasibility of replacing the pyrrolidine ring of 1 by a 9389

DOI: 10.1021/acs.jmedchem.8b01137 J. Med. Chem. 2018, 61, 9386−9392

Journal of Medicinal Chemistry

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Table 2. Binding Affinities to MDM2 and Anti-Proliferative Activities Against Cancer Cell Lines cell growth inhibition, IC50 (μM)a

HTRF assay IC50 (nM) rac-4a′ (+)-4a′ rac-8′ (+)-8′ nutlin-3a

33.03 ± 4.9 23.7 ± 3.1 10.1 ± 2.8 3.1 ± 1.9 3.5 ± 2.6

SJSA-1 p53-wt 7.5 ± 0. 34 0.96 ± 0.01 7.44 ± 0.45 7.8 ± 0.47 1.44 ± 0.30

HCT116

HCT116

p53-wt

p53 −/−

p53 −/−

± ± ± ± ±

>20 >20 >20 >20 >20

>20 9.73 ± 1.18 >20 >20 10.4 ± 0.10

4.84 2.92 10.5 11.6 1.30

0.17 0.35 0.40 4.80 0.5

H1299

a IC50 for 72 h cell treatment with the tested drug (values represent the average and standard deviations of three experiments). IC50 values were calculated, using the GraphPad Prism software, from polynomial curves.

Finally, the two enantiomers of cis−cis 4a′, 4b′, 4c′, and 4d′ have been tested separately (Table 1). Most notably, the (+) enantiomer of 4a′ displayed high affinity (23.7 nM) while its (−) antipode led to an only moderate affinity (IC50 = 1144 nM). The same trend has been observed for the cis−cis 4b′, 4c′, and 4d′ derivatives. During these studies, having samples of the intermediate spirocyclic acid cis−cis 8′, the binding affinity of this compound was evaluated also. Acid 8′ displayed an MDM2 affinity even higher than that of the targeted amides 4, with an IC50 value as high as 10.1 nM. Here also, the (+) antipode proved to be the most potent enantiomer of 8′ (IC50 = 3.1 nM), with affinity level as high as that of the reference compound, nutlin-3a. From the literature, it can be noticed that the analogous cis−cis-acid in the spiro-3,3′-pyrrolidineoxindole series also displays high MDM2 inhibition (IC50 = 55.1 ± 5.2 nM).10,14 In further experiments, we have measured the antiproliferative activity of all compounds, including nutlin-3a as a reference, against both wild type (SJSA-1 and HCT116 p53 +/+) and p53 null cancer cell lines (Table 2). Compounds 4a′ and 8′ exhibit an antiproliferative activity against p53 wild type cell lines with IC50 ranging from 0.96 to 11.6 μM. (+)-4a′ proved to be the most active, with IC50 in the same range as nutlin-3a. More interestingly, we also observed that the IC50 values of 4a′ and 8′ were above 20 μM on HCT116 isogenic p53 knockout (HCT116 p53 −/−) and p53 null cell lines (H1299 p53 −/− have a homozygous partial deletion of the p53 gene and lack expression of p53 protein), which highlights a selective antiproliferative activity, depending on the p53 status of the treated cells. From Table 2 it may be noticed that, in terms of activity level, 8′ outperforms (+)-4a′ in HTRF assays, while (+)-4a′ displays higher antiproliferative activity. This effect might result from the different physicochemical properties of the two compounds (e.g., solubility, polarity, etc.) due to the presence of an amide and an acid functions, respectively. We further tested the ability of (+)-4a′ and (+)-8′ to activate p53 in HCT116 p53 +/+ and −/− cells by Western blot, by analyzing the levels of p53, MDM2, and p21 proteins. Consistent with the results of the HTRF assay, we observed that (+)-4a′ and (+)-8′ activate p53 and induce an increase of MDM2 and p21 proteins in HCT116 cancer cell line with wild-type p53 but not in the isogenic HCT116 cell line with deleted p53 (Figure 4), clearly indicating their high cellular specificity. Taken together, these results confirm that 4a′ and 8′ relieve p53 from MDM2 ubiquitination and degradation. It must be noted here that compounds 4 display an enamide function that might act as a Michael acceptor. Therefore, the

Figure 4. Accumulation of products of the p53 gene and p53regulated genes upon treatment of cells with 4a′ and 8′ (10 μM) for 6 h. The actin level served as a protein loading control.

observed antiproliferative activity might be due to an irreversible binding to MDM2 via this function. Some evidence against this hypothesis has been afforded by MALDI-TOF mass spectrometry analysis showing that the protein is unchanged after incubation with rac-4a′ under the conditions of the HTRF assays (see Supporting Information).

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CONCLUSIONS We have demonstrated that 4a′ and 8′ display antiproliferative activity in the μM range against SJSA-1 osteosarcoma and HCT116 p53-wt cell lines. HTRF experiments and Western blot analysis of the levels of p53, MDM2, and p21 proteins afford evidence for inhibition of the MDM2-p53 interaction by these compounds, as expected based on their structural analogy to Wang’s spiropyrrolidine oxindoles 1. We thus show that, in this series, bioactivity tolerates the presence of an unsaturated five-membered ring in the spiranic scaffold instead of the saturated ring of 1, as well as that the sp3 N atom of 1 can be suitably replaced by a sp2 carbon center. Further SAR studies will be carried out on these spiro-cyclopentene derivatives and their cyclopentane analogues.31 Overall, these preliminary results establish an unprecedented bioactive scaffold and outline a new playground for the development of MDM2-P53 inhibitors.

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EXPERIMENTAL SECTION

Chemical purities were established by HPLC (CHIRACEL ID) and NMR analyses. All final compounds exhibited purity greater than 95%. Benzyl 1′-Acetyl-6′-chloro-2-(3-chloro-2-fluorophenyl)-5neopentyl-2′-oxospiro[cyclopent[3]ene-1,3′-indoline]-3-carboxylate (cis−cis 7b′). To a solution of arylidene oxindole 5a (500 mg, 1.4 mmol) and PPh3 (75 mg, 0.28 mmol, 20 mol %) in anhydrous CH2Cl2 (15 mL), under argon, was added allenoate 6b (520 mg, 2.1 9390

DOI: 10.1021/acs.jmedchem.8b01137 J. Med. Chem. 2018, 61, 9386−9392

Journal of Medicinal Chemistry

Brief Article

*A.V.: e-mail, [email protected]. *A.M.: e-mail, [email protected].

mmol, 1.5 equiv). The solution was stirred overnight at room temperature. 1H NMR monitoring showed a trans:trans/cis:cis ratio of 63/37. The desired cis−cis 7b′ was isolated by flash chromatography on silica gel with a toluene/heptanes gradient (297 mg, 35%): pale yellow solid, Rf = 0.38 (15% EtOAc/petroleum ether); 1H NMR (500 MHz, CDCl3) δ 8.14 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.29−7.24 (m, 5H), 7.20 (t, J = 7.5 Hz, 1H), 7.16−7.06 (m, 3H), 6.89−6.85 (m, 1H), 5.13 (d, J = 12.0 Hz, 1H), 5.02 (d, J = 12.0 Hz, 1H), 4.87 (bs, 1H), 3.32 (d, J = 9.5 Hz, 1H), 2.26 (s, 3H), 1.70−1.63 (m, 1H), 0.88 (d, J = 14.0 Hz, 1H), 0.81 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 175.4 (C), 170.4 (C), 163.7 (C), 156.3 (d, JC−F = 248.3 Hz, C), 148.4 (CH), 140.6 (C), 135.5 (C), 134.8 (C), 133.5 (C), 129.9 (CH), 128.8 (CH), 128.6 (CH), 128.3 (CH), 128.2 (CH), 127.8 (C), 125.9 (CH), 125.5 (d, JC−F = 14.2 Hz, C), 124.0 (d, JC−F = 5.2 Hz, CH), 123.4 (CH), 121.2 (d, JC−F = 19.5 Hz, C), 116.9 (CH), 66.5 (CH2), 65.4 (C), 53.5 (CH), 50.3 (CH), 42.8 (CH2), 30.8 (C), 29.4 (CH3), 26.3 (CH3); HRMS (ESI) calcd. For C33H31Cl2FNO4 [M + H]+: 594.1614, found: 594.1647. The two enantiomers were further separated by semipreparative HPLC on ©CHIRACEL ID (25 °C, 10% EtOH/n-heptane, 5 mL/min, retention times: 6.5 and 12.3 min). For (+)-7b′: [α]D25 = +78 (c = 0.1, CHCl3) 6′-Chloro-2-(3-chloro-2-fluorophenyl)-N-(2-morpholinoethyl)-5-neopentyl-2′-oxospiro[cyclopent[3]ene-1,3′-indoline]-3-carboxamide (4a′). 4-(2-Aminoethyl)-morpholine (110 μL, 0.84 mmol, 10 equiv) was dissolved in toluene (0.5 mL), and a 2 M solution of AlMe3 in toluene (0.42 mL, 0.84 mmol, 10 equiv) was added dropwise at 0 °C. The mixture was stirred for 30 min, then a solution of 7b′ (50 mg, 0.084 mmol) in toluene (0.5 mL) was added. After 22 h stirring at 100 °C, the solution was treated with citric acid 10% (10 mL) and extracted with EtOAc. The organic extracts were washed with H2O, dried over MgSO4, and concentrated under reduced pressure. The crude mixture was purified by flash chromatography on silica gel (CH2Cl2/MeOH: 0 to 6%) to give cis−cis 4a′ (38 mg, 79%, white powder); Rf = 0.23 (CH2Cl2/MeOH 5%); 1H NMR (500 MHz, MeOD) δ 7.60 (d, J = 8.0 Hz, 1H), 7.25 (td, J = 8.0, 1.5 Hz, 1H), 7.12 (dd, J = 8.0, 2.0 Hz, 1H), 7.05 (bs, 1H), 6.96 (t, J = 8.0 Hz, 1H), 6.76 (d, J = 2.0 Hz, 1H), 6.70 (t, J = 2.0 Hz, 1H), 5.04 (bs, 1H), 3.68 (t, J = 4.5 Hz, 4H), 3.46 (d, J = 9.5 Hz, 1H), 3.36 (t, J = 6.5 Hz, 2H), 2.58−2.45 (m, 6H), 1.67 (dd, J = 14.0, 9.5 Hz, 1H), 0.89 (d, J = 14.0 Hz, 1H), 0.84 (s, 9H); 13C NMR (75 MHz, MeOD) δ 179.1 (C), 168.0 (C), 157.7 (d, JC−F = 247.7 Hz, C), 144.5 (C), 142.7 (CH), 138.9 (C), 135.2 (C), 130.6 (CH), 130.4 (CH), 127.6 (d, JC−F = 14.1 Hz, C), 126.1 (CH), 125.0 (d, JC−F = 4.4 Hz, CH), 123.5 (CH), 121.5 (d, JC−F = 17.7 Hz, C), 110.8 (CH), 67.5 (CH2), 66.8 (C), 58.5 (CH2), 54.5 (CH2), 53.3 (CH), 50.3 (CH), 44.1 (CH2), 37.0 (CH2), 31.4 (C), 29.8 (CH3); HRMS (ESI) calcd. For C30H35Cl2FN3O3 [M + H]+: 574.2040, found: 574.2067. The two enantiomers were further separated using semipreparative HPLC [©CHIRACEL ID, 25 °C, 20% iPrOH/n-heptane (0.1% Et3N), 5 mL/min, r.t. 8.5 min [(+)-4a′] and 11.0 min]. (+)-4a′ > 99.5% ee, [α]D25 = +68 (c = 0.1, CHCl3)

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ORCID

Catherine Gomez: 0000-0002-3680-1235 Arnaud Voituriez: 0000-0002-7330-0819 Angela Marinetti: 0000-0002-4414-073X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Laure Eloy for her valuable technical assistance. M.G. thanks ICSN for a Ph.D. grant.

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ABBREVIATIONS USED MDM2,murine double-minute 2; TBS,tert-butyldimethylsilyl; HATU,1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DIPEA,diisopropylethylamine; TFA,trifluoroacetic acid

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(1) Vassilev, L. T.; Vu, B. T.; Graves, B.; Carvajal, D.; Podlaski, F.; Filipovic, Z.; Kong, N.; Kammlott, U.; Lukacs, C.; Klein, C.; Fotouhi, N.; Liu, E. A. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004, 303, 844−848. (2) Vassilev, L. T. MDM2 inhibitors for cancer therapy. Trends Mol. Med. 2007, 13, 23−31. (3) Wang, S.; Zhao, Y.; Bernard, D.; Aguilar, A.; Kumar, S. Targeting the MDM2-p53 protein-protein interaction for new cancer therapeutics. Top. Med. Chem. 2012, 8, 57−80. (4) Wade, M.; Li, Y. C.; Wahl, G. M. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat. Rev. Cancer 2013, 13, 83−96. (5) Burgess, A.; Chia, K. M.; Haupt, S.; Thomas, D.; Haupt, Y.; Lim, E. Clinical overview of MDM2/X-targeted therapies. Front. Oncol. 2016, 6, 7 DOI: 10.3389/fonc.2016.00007. (6) Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Qiu, S.; Ding, Y.; Gao, W.; Stuckey, J.; Krajewski, K.; Roller, P. P.; Tomita, Y.; Parrish, D. A.; Deschamps, J. R.; Wang, S. Structure-based design of potent nonpeptide MDM2 inhibitors. J. Am. Chem. Soc. 2005, 127, 10130− 10131. (7) Ding, K.; Lu, Y.; Nikolovska-Coleska, Z.; Wang, G.; Qiu, S.; Shangary, S.; Gao, W.; Qin, D.; Stuckey, J.; Krajewski, K.; Roller, P. P.; Wang, C. Structure-based design of spiro-oxindoles as potent, specific small-molecule inhibitors of the MDM2-p53 interaction. J. Med. Chem. 2006, 49, 3432−3435. (8) Shangary, S.; Qin, D.; McEachern, D.; Liu, M.; Miller, R. S.; Qiu, S.; Nikolovska-Coleska, Z.; Ding, K.; Wang, G.; Chen, J.; Bernard, D.; Zhang, J.; Lu, Y.; Gu, Q.; Shah, R. B.; Pienta, K. J.; Ling, X.; Kang, S.; Guo, M.; Sun, Y.; Yang, D.; Wang, S. Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3933−3938. (9) Yu, S.; Qin, D.; Shangary, S.; Chen, J.; Wang, G.; Ding, K.; McEachern, D.; Qiu, S.; Nikolovska-Coleska, Z.; Miller, R.; Kang, S.; Yang, D.; Wang, S. Potent and orally active small-molecule inhibitors of the MDM2-p53 interaction. J. Med. Chem. 2009, 52, 7970−7973. (10) Zhao, Y.; Liu, L.; Sun, W.; Lu, J.; McEachern, D.; Li, X.; Yu, S.; Bernard, D.; Ochsenbein, P.; Ferey, V.; Carry, J.-C.; Deschamps, J. R.; Sun, D.; Wang, S. Diastereomeric spirooxindoles as highly potent and efficacious MDM2 inhibitors. J. Am. Chem. Soc. 2013, 135, 7223− 7234. (11) Zhao, Y.; Yu, S.; Sun, W.; Liu, L.; Lu, J.; McEachern, D.; Shargary, S.; Bernard, D.; Li, X.; Zhao, T.; Zou, P.; Sun, D.; Wang, S. A potent small-molecule inhibitor of the MDM2-p53 Iinteraction (MI-888) achieved complete and durable tumor regression in mice. J. Med. Chem. 2013, 56, 5553−5561.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b01137.

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REFERENCES

Molecular formula strings (CSV) Synthetic procedures, 1H NMR and 13C NMR spectra and HPLC chromatograms of the final compounds, HTRF assays, cell culture and proliferation assays, SDS− PAGE and Western blot analysis, MALDI-TOF experiments (PDF)

AUTHOR INFORMATION

Corresponding Authors

*J.B.: e-mail, [email protected]. 9391

DOI: 10.1021/acs.jmedchem.8b01137 J. Med. Chem. 2018, 61, 9386−9392

Journal of Medicinal Chemistry

Brief Article

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DOI: 10.1021/acs.jmedchem.8b01137 J. Med. Chem. 2018, 61, 9386−9392