the transcription factor p53 
 plays vital roles in the regulation of cellular processes and suppression 
 of tumor development .
mice lacking p53 develop normally but are prone to the development 
 of a variety of tumors.tp53 , the gene encoding the p53 protein , is mutated or deleted in nearly 
 50% of human cancers , rendering p53 nonfunctional as a tumor suppressor .
although p53 retains wild - type status in the 
 remaining 50% of human cancers , its function is effectively inhibited 
 by a variety of mechanisms .
one major inhibitory mechanism is through 
 overexpression of mdm2 ( also called hdm2 in humans ) . the role of mdm2 
 as a primary negative endogenous regulator of p53
is supported by 
 evidence that mdm2-null is embryonically lethal in 
 mice which can only be rescued by concurrent deletion of the tp53 gene .
mdm2 and p53 regulate 
 each other mutually through the 
 autoregulatory feedback loop shown in figure 1 . the autoregulatory feedback loop of the 
 p53mdm2 interplay operates as follows : upon activation , p53 
 transcribes mdm2 , leading to an increase of mdm2 mrna and protein 
 and in turn , mdm2 protein binds to p53 directly through their n - termini 
 and inhibits p53 function through three major mechanisms : ( 1 ) upon 
 binding , mdm2 ubiquitinates p53 by functioning as an e3 ligase promoting 
 proteasomal degradation of p53 ; ( 2 ) interaction of mdm2 with p53 blocks 
 the binding of p53 to its targeted dna , rendering p53 ineffective 
 as a transcription factor ; ( 3 ) mdm2 promotes export of p53 out of 
 the cell nucleus , leaving p53 inaccessible to targeted dna and reducing 
 its transcriptional ability . through these three inhibitory 
 mechanisms , mdm2 functions as an effective antagonist of wild - type 
 p53 .
consistent with its role as an efficient inhibitor of p53 
 tumor 
 suppressor , mdm2 in cells , when overexpressed , is oncogenic .
an analysis of 28 different types of cancers 
 involving nearly 4000 human tumor samples shows that in 7% of human 
 cancers , the mdm2 gene has been amplified .
in addition to mdm2 gene amplification , mdm2 
 overexpression can be caused by a variety of mechanisms , such as single 
 nucleotide polymorphism at nucleotide 309 ( snp309 ) in its gene promoter , 
 enhanced transcription , or increased translation .
mdm2 overexpression correlates 
 with poor clinical prognosis and poor response to current cancer therapies . in supporting its powerful inhibitory role of p53 tumor suppressor 
 function , mdm2 gene amplification and tp53 gene mutation 

are mutually exclusive in human cancers . because mdm2 plays a 
 primary role in inhibition of the p53 tumor suppressor function and 
 antagonizes p53 through their direct interaction , blockade of the 
 mdm2p53 protein
 protein interaction would liberate 
 p53 from mdm2 , thus restoring the tumor suppressor function of wild - type 
 p53 .
agents designed to block the mdm2p53 interaction may 
 have a therapeutic potential for the treatment of human cancers retaining 
 wild - type p53 .
the mdm2p53 interaction has been mapped 
 to the first 120 n - terminal amino acid residues of mdm2 and 
 the first 30 n - terminal residues of p53 . in 1996 ,
a high - resolution cocrystal structure of mdm2 with a p53 
 peptide ( residues 1529 ) was reported ( figure 2 , pdb code 1ycr ) .
the cocrystal structure showed that 
 the mdm2-bound p53 peptide adopts an -helical conformation 
 and interacts with mdm2 primarily through three hydrophobic residues , 
 phe19 , trp23 , and leu26 . in this review
, these p53 binding pockets 
 on the mdm2 surface will be referred to as the phe19 , trp23 , and leu26 
 pockets .
these three p53 binding pockets in mdm2 are compact and well - defined 
 and suggest the feasibility of the design of high - affinity , non - peptide 
 inhibitors that bind in the mdm2 pockets ( mdm2 inhibitors ) and block 
 the mdm2p53 interaction .
several reviews have summarized the progress in the design 
 of mdm2 
 inhibitors . in this review , we will focus on those mdm2 inhibitors that have 
 been advanced into human clinical trials and discuss the outlook of 
 mdm2 inhibitors as a new class of anticancer agents .
currently , seven small molecule mdm2 inhibitors 
 have advanced into 
 clinical trials for treatment of human cancers ( table 1 ) . of these clinical - stage compounds ,
the chemical structures 
 for rg7112 ( 3 ) , rg7388 ( 6 ) , mi-77301/sar405838 ( 10 ) , and amg 232 ( 14 ) have been disclosed 
 but no structural information is available for the other three compounds . the 
 first mdm2 inhibitor to enter phase i clinical trials was 3 ( figure 3 ) , discovered at hoffmann - la roche 
 and based on a class of compounds called nutlins ( figure 3 ) .
cis - diphenyl 
 substituted imidazoline - containing 
 compounds , called the  nutlins  , were reported as a 
 first class of potent , specific , and orally active small molecule 
 mdm2 inhibitors .
one of the most potent 
 compounds in the initial report was nutlin 3a ( 1 ) , which 
 binds to mdm2 protein with ic50 = 90 nm .
a cocrystal structure 
 of mdm2 complexed with nutlin 2 ( 2 , figure 4 , pdb code 1rv1 ) was obtained at 
 a resolution of 2.3  , which provided for the first time structural 
 information for a non - peptide , small - molecule inhibitor bound to mdm2 . 

superimposition of the cocrystal structures of mdm2/2 and mdm2/p53 peptide ( figure 4 ) shows that 
 two 4-bromophenyl rings and ethoxy substituent of 2 occupy 
 the trp23 , leu26 , and phe19 pockets of mdm2 , respectively .
this study 
 provides an elegant example of a non - peptide , druglike , small molecule 
 mimicking the -helical p53 peptide structure in its binding 
 to mdm2 .
compound 1 activates 
 wild - type p53 in cancer and normal 
 cells but selectively kills tumor cells .
it inhibits cell growth with 
 ic50 of 12 m in human cancer cell lines 
 with wild - type p53 ( sjsa-1 , hct116 , and rko ) and demonstrates approximately 
 10-fold selectivity over cancer cells harboring mutated p53 ( mda - mb-435 
 and sw480 ) .
these observations are consistent 
 with the hypothesis that the antitumor activity of mdm2 inhibitors 
 should be p53-dependent . in vivo , oral administration of 1 dose - dependently activates p53 and induces expression of p53 targeted 
 genes , including p21 and mdm2 .
importantly , 
 90% inhibition of tumor growth is achieved in the sjsa-1 tumor xenograft 
 model in mice upon treatment with nutlin 3 ( the racemic form of 1 ) at 200 mg / kg orally twice a day for 20 days with no sign 
 of toxicity in treated animals .
further optimization of 1 to improve its binding affinity 
 to mdm2 , cellular potency , pharmacokinetics , and chemical stability 
 ultimately yielded 3 ( figure 3 ) , the first mdm2 inhibitor to advance 
 into clinical trials .
four major modifications were 
 made to 1 to yield 3 : ( i ) dimethyl groups 
 on the imidazoline ring were added to prevent oxidation of the imidazoline 
 ring ; ( ii ) an ethyl ether group replaced the isopropyl ether in 1 , reducing the molecular weight slightly while retaining 
 good mdm2 binding affinity ; ( iii ) a tert - butyl group 
 was used to replace the methoxyl group in 1 , which is 
 a metabolic  soft spot  ; ( iv ) a 3-(methylsulfonyl)propyl 
 group was introduced into the piperidine ring to enhance mdm2 binding 
 and improve pharmacokinetic ( pk ) properties . notwithstanding these 
 substantial modifications to the 1 structure , 3 and 1 interact with mdm2 very similarly based upon 
 superposition of their cocrystal structures ( figure 5 , pbd codes 4ipf and 4j3e ) . 

the two 4-chlorophenyl groups occupy the trp23 and leu26 pockets of 
 mdm2 , and the ethoxyl group is in the phe19 pocket .
the methylsulfonyl 
 moiety of 3 extends out of the binding pocket and is 
 exposed to solvent .
superposition of cocrystal structures of mdm2 to 3 ( cyan ) and 1 ( orange ) . compared with 1
it effectively 
 inhibits cell growth in cancer cell lines with wild - type p53 ( ic50 = 0.182.2 m ) and demonstrates good selectivity 
 over cancer cell lines with a p53 mutation ( ic50 = 5.720.3 
 m ) .
3 robustly activates wild - type p53 in vitro and in vivo , and upon oral administration at 50 mg / kg in mice , good 
 systemic exposure is achieved with a high auc ( 251.2 gh / ml ) 
 and cmax ( 15.5 g / ml ) and slow clearance 
 ( t1/2 = 8.8 h ) . in two xenograft models 
 of sjsa1 and mhm osteosarcoma cell lines with mdm2 gene amplification 
 and overexpression of mdm2 protein , 3 dose - dependently 

inhibits tumor growth and achieves partial tumor regression at 100 
 mg / kg daily , oral administration without signs of toxicity in mice .
compound 3 has been tested in clinical trials 
 against a wide range of cancers , including sarcoma , myelogenous leukemia , 
 and hematologic neoplasms , and results of phase 
 i studies of 3 in chemotherapy - naive patients with mdm2-amplified 
 liposarcoma have recently been reported . upon treatment with 3 , a good human pk profile was 
 achieved and a steady state was reached on day 8 .
clear activation 
 of p53 , increase in p21 protein , and apoptosis induction in tumors 
 were observed .
treatment with 3 showed signs of antitumor 
 activity in patients ; post - treatment , 14 patients were found to have 
 stable disease and one had a confirmed partial response .
in addition , 
 16 out of 20 patients were treated with 3 for more than 
 6 months .
all patients treated with 3 had at least one 
 adverse event , and 12 serious adverse events were observed in eight 
 patients , including neutropenia ( six patients ) and thrombocytopenia 
 ( three patients ) .
it was found that exposure to 3 was 
 correlated with hematological toxicity . on the basis of the clinical 
 data for 3 ,
it was concluded that the potential for late 
 hematological toxicity , particularly thrombocytopenia , should be considered 
 in future clinical trials of mdm2 inhibitors .
the 
 synthesis of 3 , based on published reports , is summarized 
 in scheme 1 .
reagents and reaction 
 conditions : 
 ( a ) alme3 , toluene , reflux ; ( b ) phosgene , et3n ; ( c ) et3n ; ( d ) resolution by chiral chromatography . on the 
 basis of the structures of mi-219 ( 8 , figure 8) and 3 , the hoffmann - la roche group designed and synthesized the pyrrolidine - containing 
 compound 4(33 ) as an mdm2 inhibitor . 

the two phenyl groups in 4 adopt a trans configuration , 
 in contrast to 3 and 8 in which the two 
 substituted phenyl groups are in a cis configuration .
compound 4 binds to mdm2 with a good affinity ( ic50 = 196 
 nm ) but is weaker than 3 .
the cocrystal structure of mdm2/4 ( figure 7 , pbd code 4jrg ) shows that the 4-chlorophenyl group and neopentyl 
 group of 4 occupy the trp23 and phe19 pockets , respectively , 
 while the 2-chlorophenyl group occupies the leu26 pocket and partakes 
 in a  interaction with the his96 residue . additionally , 
 the carbonyl group of 4 forms a hydrogen bond with nh 
 of his96 .
pharmacokinetic studies showed that 4 has a high clearance 
 rate and poor oral bioavailability , issues that were addressed in 
 further modification efforts .
activity 
 relationship ( sar ) analyses show that addition of fluorine atoms to 
 the two phenyl rings of 5(33 ) enhances the mdm2 binding affinity .
the dihydroxybutyl side chain 
 of 5 was replaced with a variety of groups with the goal 
 of improving the mdm2 binding affinity , cellular potency , microsomal 
 stability , and pk properties .
this led to the identification of m - methoxybenzoic acid group as an optimal group at this 
 site , which ultimately resulted in the discovery of 6 ( also known as ro5503781 , figure 6 ) for clinical 
 development .
it displays potent 
 cell growth inhibitory activity in cancer cell lines containing wild - type 
 p53 ( average ic50 = 30 nm ) and displays > 100-fold selectivity 
 over cancer cell lines containing mutated p53 .
furthermore , it has 
 good microsomal stability and good pk properties . in mice , it has 
 high oral bioavailability ( 80% ) , moderate clearance ( t1/2 = 1.6 h ) , and good systemic exposure and metabolic 
 stability and is more potent than 3 and 1 in induction of p53 activation in vivo . with oral daily administration , 

it achieves tumor regression in the sjsa-1 osteosarcoma xenograft 
 model in mice at 25 mg / kg . it is currently in three phase i clinical 
 trials for treatment of patients with solid tumors , acute myelogenous 
 leukemia , or advanced malignancies as a single agent and in combination 
 with chemotherapeutics .
the maximum tolerated 
 dose for 6 is 500 mg in a daily dosing ( q.d . ) , 5 day 
 schedule , 500 mg in a twice - daily ( b.i.d . ) , 3-day schedule and 1600 
 mg in a b.i.d . , weekly schedule .
activation of p53 , as measured by 
 mic-1 concentration in plasma , was observed either with daily dosing 
 for 5 days or with b.i.d .
dosing for 3 days , but it is stronger with 
 daily , 5-day dosing . interestingly ,
thrombocytopenia , neutropenia , febrile 
 neutropenia , and diarrhea were the dose - limiting toxicities .
reagents and reaction conditions : 
 ( a ) agf , et3n , ch2cl2 , rt , 18 h ; 
 ( b ) cf3co2h , ch2cl2 , rt , 
 18 h ; ( c ) resolution by chiral chromatography ; ( d ) ph2pocl , 
 ( i - pr)2net , ch2cl2 , rt ; ( e ) aq naoh , thf , methanol , 40 c .
the 
 small molecule mdm2 inhibitor 10 ( figure 8) , discovered at the 
 university of michigan , was advanced into phase i clinical trials 
 by sanofi in 2012 .
compound 10 was obtained through extensive optimization of a class of spirooxindoles , 
 such as 7 , first reported 
 in 2005 from our laboratory .
compound 7 was designed 
 using a structure - based approach to mimic three key p53 binding residues 
 ( phe19 , trp23 , and leu26 ) but has weak mdm2 binding affinity ( ki = 8.46 m ) .
subsequent modifications of the two phenyl rings and the aliphatic 
 and the amide side chain were carried out to improve its mdm2 binding 
 affinity , cellular potency , and in vivo pk properties .
addition of 
 a 3-chloro substituent atom on the phenyl ring and replacement of 
 the isopropyl group with neopentyl improves the mdm2 protein binding 
 affinity , as does replacement of the dimethylamide with a monosubstituted 
 amine .
the addition of a fluorine atom next to the chlorine on the 
 oxindole ring generally improves the pk profile of spiro - oxindole - containing 
 mdm2 inhibitors . following these modifications , a potent compound , 8(41 )
this compound binds to mdm2 with ki = 23.5 nm and activates wild - type p53 in cancer cells .
8 shows fairly potent cell growth inhibitory activity ( ic50  1 m ) in cancer cell lines with wild - type 
 p53 , and it is more than 10-fold less potent in cancer cells harboring 
 mutated or deleted p53 .
oral administration of 8 at a dose of 300 
 mg / kg twice daily completely inhibits tumor growth inhibition but 
 fails to achieve tumor regression in the sjsa-1 xenograft tumor model .
further optimization 
 of 8 led to 9(44 ) ( mi-888 ) , in which the spiro - oxindole scaffold 
 has a different stereochemistry .
the trans conformation of two substituted 
 phenyl rings in 9 was found to be critical for improved 
 mdm2 binding affinity .
in addition , removal of the fluorine atom from 
 the oxindole ring is beneficial to mdm2 binding affinity and addition 
 of a single fluoro substituent on the phenyl ring improves the pk 
 profile .
finally , the soluble tail , the cis-3-hydroxy-3-methyl - cyclobutylamino 
 group , also contributes to the improved mdm2 binding affinity and 
 a good in vivo pk profile .
9 has an excellent mdm2 binding 
 affinity ( ki = 0.44 nm ) and shows potent 
 cell growth inhibition potency in sjsa-1 and rs4;11 cell lines ( ic50 of 80 and 60 nm , respectively ) .
it displays > 100-fold selectivity for cancer cell lines with 
 wild - type tp53 over those with mutated or deleted tp53 .
it is stable in mouse , rat , and human microsomes and 
 has high systemic 
 exposure in a mouse pk study .
oral daily administration of 9 at 100 mg / kg achieves complete and durable tumor regression in both 
 the sjsa-1 osteosarcoma and acute leukemia rs4;11 xenograft models 
 in mice without signs of toxicity .
replacement of the cis-3-hydroxy-3-methylcyclobutylamino 
 group of 9 with a trans-4-hydroxycyclohexylamino 
 group led to clinical compound 10 .
compound 10 binds 
 to mdm2 with a ki value of 0.88 nm . a cocrystal structure of the mdm2/10 complex ( figure 9 ) was determined and provided 
 a structural basis 
 for its high binding affinity to mdm2 .
10 mimics the 
 three key p53 binding residues ( phe19 , trp23 , and leu26 ) in hydrophobic 
 contacts and hydrogen bonding in its interaction with mdm2 but captures 
 additional interactions .
the cl atom in the oxindole group of 10 has extensive hydrophobic contacts with mdm2 .
there is 
  stacking between the his96 of mdm2 and the 
 2-fluoro-3-chlorophenyl in 10 , and a hydrogen bond between 
 the imidazole side chain of his96 and the carbonyl group in 10 .
interestingly , the n - terminus of mdm2 ( residues 1018 ) 
 refolds and enjoys extensive interactions with 10 through 
 val14 and thr16 , which contributes 25-fold to the binding affinity . 

in contrast , the n - terminus of mdm2 ( residues 118 ) has no 
 significant contribution to the binding of p53 peptides and 1 .
compound 10 inhibits cell growth with 
 ic50 values of 100200 nm in sjsa-1 , rs4;11 , lncap , 
 and hct116 cancer cell lines containing wild - type p53 and demonstrates 
 > 100-fold selectivity over cancer cells lines with mutated or deleted 
 p53 .
compound 10 effectively 
 activates wild - type p53 in cancer cell lines at concentrations as 
 low as 30 nm in vitro and in xenograft tumor tissue of leukemia and 
 solid tumors , leading to p53-dependent cell cycle arrest and/or apoptosis 
 in tumor cells . with daily oral administration at 100 mg / kg , it achieves 
 complete and durable tumor regression in mouse xenograft models of 
 sjsa-1 osteosarcoma and rs4;11 acute leukemia , partial ( 80% ) regression 
 in the lncap prostate cancer xenograft model , and complete tumor growth 
 inhibition in the hct116 colon cancer xenograft model . a single oral 
 dose of compound 10 at 200 mg / kg induces strong apoptosis 
 in the sjsa-1 tumor tissue with the effect lasting for > 3 days 
 and 
 complete tumor regression in 100% of animals .
mechanistically , robust 
 transcriptional up - regulation of puma induced by 10 is 
 associated with robust apoptosis in cancer cell lines in vitro , tumor 
 tissue in vivo , and complete tumor regression in xenograft models 
 in mice .
these preclinical data suggest the strong therapeutic potential 
 of compound 10 for the treatment of different types of 
 human cancer .
sanofi initiated a phase 
 i clinical trial of compound 10 to assess its safety , 
 tolerability , pharmacokinetics , and 
 biological activity in patients with advanced cancer .
a second phase i trial was initiated in 2013 to evaluate 10 in combination with pimasertib , an allosteric inhibitor 
 of mek1/2 in patients with solid tumors .
( a ) toluene , molecular sieves 4  , reflux overnight ; ( b ) trans-4-aminohexanol , thf , rt , 2 days ; ( c ) can , acetonitrile  h2o 
compound 14 ( figure 10 ) , discovered 
 and developed 
 by amgen , entered phase i clinical trials for the treatment of human 
 cancer in 2012 .
this compound was obtained through 
 structure - based design and extensive optimization of a new class of 
 mdm2 inhibitors containing piperidin-2-one .
structures of piperidinone - containing compounds . the piperidinone - containing compound 11(48 ) ( figure 10 ) was designed 
 as an mdm2 inhibitor based upon a structural analysis of known mdm2 
 inhibitors , such as 1 , 8 , and a previously 
 reported mdm2 inhibitor by amgen , to mimic three key binding residues 
 in p53 .
compound 11 binds to mdm2 with ic50 = 34 nm and served as an excellent lead compound for further optimization . 
 upon the basis of the nmr and cocrystal structures of 11 or its analogues complexed with mdm2 ( figure 11 , pdb codes 4hmb and 2lzg ) , further optimization of two sites of 11 yielded 12 ( am 8553 ) , which 
 has an ic50 value of 1.1 nm to mdm2 ( kd = 0.4 nm to mdm2 ) .
compound 12 effectively 
 activates wild - type p53 in vitro and in vivo and dose - dependently 
 inhibits tumor growth in the sjsa-1 xenograft model in mice . at 150 
 and 200 mg / kg daily oral dosing for 2 weeks , it achieves partial tumor 
 regression ( 27% ) after 2 weeks of treatment .
it was further optimized 
 for potency , pharmacokinetic properties , and in vivo efficacy .
this 
 yielded a series of potent and orally active mdm2 inhibitors including 13(38 ) and 14 , whereas 
 the latter was selected for clinical development .
nmr structure of mdm2/11 ( left ) and x - ray crystal 
 structures of mdm2/11 analogue ( middle and right ) .
several high - resolution 
 cocrystal structures of mdm2 complexed 
 with members of this class of mdm2 inhibitors have been determined and provide structural insights 
 into their high affinity binding . in the mdm2/13 cocrystal 
 structure ( figure 12 , pdb code 4oas ) , the 3-chlorophenyl 
 and 4-chlorophenyl groups project into the leu26 and trp23 pockets , 
 respectively .
the sulfonyl tert - butyl group 
 resides in the small pocket around g58 , and the carboxylic acid forms 
 a salt bridge with the his96 residue of mdm2 .
the cocrystal structure 
 for mdm2/14 has not been reported , but amgen scientists 
 predict that 14 has a similar binding model as 13 . furthermore , binding between 
 inhibitors in this class ( e.g. , 11 ) and mdm2 induces 
 reorganization of the extreme n - terminus of the mdm2 protein and promotes 
 additional hydrophobic contacts between the m - chlorophenyl 
 moiety in the inhibitors and the val14 and thr16 residues in mdm2 
 ( figure 11 , right ) .
hence , 14 not only mimics the three key p53 residues 
 but captures additional interactions not observed between p53 and 
 the refolded mdm2 to achieve a very high binding affinity with mdm2 .
compound 14 binds to mdm2 with ic50 = 0.6 nm in a competitive binding assay and has a kd value of 0.045 nm with mdm2 , determined by 
 surface plasmon resonance ( spr ) .
it potently 
 inhibits cell proliferation with ic50 values of 9.1 and 
 10 nm in an brdu proliferation assay in the sjsa-1 and hct-116 cell 
 lines , respectively , and demonstrates over > 1000-fold selectivity 
 over the hct-116 p53 knockout ( p53 ) cell 
 line . in efficacy studies using human tumor xenograft models in mice
, 
 it effectively inhibits tumor growth in the sjsa-1 osteosarcoma model 
 with an ed50 of 9.1 mg / kg with daily oral administration 
 and demonstrates complete tumor regression in 10 of 12 animals treated 
 with 60 mg / kg daily .
it also effectively and dose - dependently inhibits 
 tumor growth in the hct-116 xenograft model with twice daily dosing 
 with an ed50 of 16 mg / kg and achieves tumor stasis ( 100% 
 tumor growth inhibition ) without tumor regression . in preclinical 
 safety evaluations ,
no significant liability was found for 14 to preclude it from clinical development ; it was projected to have 
 a low clearance rate and consequently a long half - life in humans .
reagents and reaction conditions : 
 ( a ) 10% t - buok , thf , rt , 2 d ; ( b ) 0.2% rucl2[(s - xylbinap)(s - daipen ) ] , 40% t - buok , i - proh , h2 ( 50 psi ) , 
 rt , 65 h ; ( c ) lioh , thf / meoh / h2o ; ( d ) ppts , toluene , reflux ; 
 ( e ) lihmds , allyl bromide , thf ; ( f ) neat , 100 c , overnight ; 
 ( g ) tf2o , 2,6-lutidine , ch2cl2 , 78 
 to 0 c ; ( h ) aq nahco3 ; ( i ) tf2o , 2,6-lutidine . 

the mdm2 inhibitor 
 mk-8242 , also known 
 as sch 900242 , developed by merck has 
 been tested since 2011 as a single agent or in combination with cytarabine 
 in patients with advanced solid tumors or leukemia .
the mdm2 inhibitors , 
 cgm097 ( 35 ) developed by 
 novartis international ag and ds-3032b ( 36 ) developed by daiichi sankyo inc . , have also 
 entered phase i clinical trials in 2013 .
compound 35 is 
 to be tested in selected patients who have advanced solid tumors with 
 p53 wild status .
compound 36 will be tested in patients 
 who have advanced solid tumors or lymphomas .
the chemical structures 
 and preclinical data for these three clinical - stage mdm2 inhibitors 
 have not been disclosed .
in addition to the seven compounds that 
 have been advanced into 
 clinical development , there are several additional classes of mdm2 
 inhibitors that have been reported by industrial and academic groups . 

johnson & johnson reported a class of benzodiazepine - containing 
 compounds as mdm2 inhibitors .
one of 
 the better compounds they reported was 15 ( figure 13 and table 2 ) , which had 
 mdm2 binding affinity ( kd = 80 nm ) , while 
 its corresponding enantiomer was 50 times less potent .
compound 15 has low cell permeability , rapid in vivo 
 clearance , and low bioavailability .
subsequent 
 modifications led to 16 , in which soluble tails were 
 added and the carboxylic acid moiety was replaced by a substituted 
 benzyl group .
compound 16 shows mdm2 binding ( ic50 = 394 nm ) and brdu incorporation 
 ( ic50 = 1.1 m ) in the mcf-7 cell line .
examples 
 of reported mdm2 inhibitors in the literature . binding affinity was measured as kd ; binding affinity 
 was measured as ki average of three cell lines sjsa-1 , 
 hct116 , and rko researchers 
 at the university of newcastle reported isoindolinone - containing 
 compounds as mdm2 inhibitors .
subsequent optimization efforts guided by nmr analysis led to the 
 identification of compound 17 ( figure 8) , which has binding affinity 
 to mdm2 ( ic50 = 0.17 m ) in an elisa assay and cell 
 growth inhibition ( gi50 = 5.2 m ) in the sjsa-1 cell 
 line .
in addition to 14 , scientists from amgen 
 reported 
 a class of chromenotriazolopyrimidine compounds as mdm2 inhibitors .
extensive modifications of these compounds 
 led to 18 , which binds to 
 mdm2 with ic50 = 350 nm and shows moderate cellular activity 
 and microsomal stability , high oral bioavailability ( 54% ) , and slow 
 clearance in rodents . in 2010 ,
chemical modifications led to 19 , which has cellular growth inhibition 
 activity > 20 times that of nutlin-3 in a selected subset of cancer 
 cell lines .
researchers from daiichi sankyo reported a class 
 of 5,6-dihydroimidazo[2,1-b]thiazol - containing 
 compounds ( 20 , 21 ) as potent mdm2 inhibitors.20 has high mdm2 binding affinity 
 ( ic50 = 
 9.2 nm ) but poor metabolic stability , resulting in weak in vivo antitumor 
 efficacy in an mv4 - 11 xenograft model .
after extensive modifications , 21 was obtained and shows high mdm2 binding potency ( ic50 = 58 nm ) , moderate solubility ( 25 g / ml ) in ph 6.8 
 phosphate buffer , good hepatic microsomal stability , and high systemic 
 exposure in mice .
it also showed 50-fold selectivity in tp53 wild type over tp53-mutated cell lines 
 ( gi50 = 0.22 m in mv4;11 vs gi50 = 10 
 m in dld-1 ) . upon oral administration of 21 at 
 a daily dose of 200 mg / kg ,
76% tgi was achieved at the end of treatment 
 in mouse mv4;11 tumor xenograft model with no sign of acute toxicity .
a class of compounds featuring a 3-(1h - imidazol-5-yl)-1h - indole-2-carboxylic acid structure was reported independently 
 by two groups .
scientists from novartis reported 22 , 
 which has an mdm2 binding affinity of ic50 = 30 nm .
in addition , 23 was synthesized 
 by dmling s group at the university of pittsburgh by 
 means of a three - component reaction and 
 was found to have mdm2 binding affinity ( ki = 916 nm ) in a fluorescent polarization 
 assay .
another class of 2-indolecarboxylic acid containing mdm2 inhibitors 
 also has three hydrophobic groups converging on a sp carbon 
 and 24 ; the best of these compounds binds to mdm2 with ki = 130 nm . in 2012 , 1h - pyrrolone - containing compounds , such 
 as 25 ,
25 has mdm2 binding ( ki = 0.15 m ) 
 and cell growth inhibition ( ic50  1.9728.11 
 m ) in saoc-2 , u-2os , a549 , and nci - h1299 cell lines . using pharmacophore - based virtual screening , 
 wang and co - workers identified thiophene - containing compounds as mdm2 
 inhibitors .
one of the most potent of 
 these compounds , mcl0527 - 3 ( 26 ) , shows mdm2 binding affinity 
 ( ic50 = 0.90 m ) and cell growth inhibition ( ic50 of 2.41 , 0.59 , and 102.3 m in a549 , hct116 , and sjsa-1 
 cell lines , respectively ) .
a dihydroimidazole - containing compound 
 pb11 ( 27 ) , synthesized from a multiple component reaction 
 followed by amidation , has mdm2 binding affinity ( kd = 0.8 m ) .
merck scientists recently reported a class of piperidine - containing 
 compounds ( 28 , 29 ) with mdm2 binding ( ic50 of 169 and 41 nm , respectively ) .
detailed sar studies and computational calculations 
 suggested that substituents on the 2-position of the piperidine ring , 
 for example the allyl moiety of 28 , can stabilize the 
 binding conformation of the piperidine ring and are compatible with 
 both polar and nonpolar functional groups .
however , both compounds 28 and 29 inhibit cyp 3a4 ( ic50 < 
 5 m ) , a liability that may need to be addressed for further 
 development for this class of compounds . a class of morpholinone - containing 
 mdm2 inhibitors , such as 30 , was recently reported by 
 amgen scientists .
compound 30 has mdm2 binding ( ic50 = 0.4  0.1 nm ) and brdu 
 incorporation ( ic50 = 25  9 nm ) in the sjsa-1 cell 
 line . with oral administration 
 of 30 at 100 mg / kg daily ,
partial tumor regression was 
 achieved at the end of treatment in an sjsa-1 female athymic nude 
 mice xenograft .
most interestingly , 30 has slower clearance 
 in rats and higher hepatocyte stability than the corresponding piperidinone - containing 
 analogue .
metabolite profiling showed that metabolic oxidation and 
 glucuronidation of 30 are significantly slower than those 
 of its corresponding piperidinone - containing analogue .
these data 
 indicate that morpholinone - containing mdm2 inhibitors such as 30 do have advantages in their dmpk profile relative to piperidinone - containing 
 mdm2 inhibitors such as 14 .
additional modification of 14 focused on replacement of its carboxylic acid moiety with 
 corresponding isosteres .
it was interesting 
 to find that heterocycles such as a thiazole group are a good replacement 
 for the carboxylic acid group and forms hydrogen bonds with the imidazole 
 nh of his96 through the nitrogen atom .
this modification led to identification 
 of 31 ( am 6761 ) , which has good mdm2 binding ( ic50 = 0.1 nm ) , good cellular potency ( sjsa-1 ic50 = 16 nm ) , and favorable pharmacokinetic properties . upon oral administration 
 of 31 at a daily dose of 50 mg / kg , 5.6% tumor regression 
 was achieved in sjsa-1 female athymic nude mice xenografts after 12 
 days of treatment .
the metabolite profiles of 31 in mouse , 
 rat , dog , monkey , and human hepatocytes suggest that 31 is primarily cleared through oxidative pathways and thus is distinct 
 from the main metabolism of 14 which is through glucuronidation .
very recently , zhang and coauthors reported 32 ( ro2468 ) 
 and 33 ( ro5353 ) as potent mdm2 inhibitors with useful 
 in vivo antitumor activity .
the medicinal 
 chemistry optimization was based on 34 ( ro8994 ) and the 
 clinical drug 6 and featured incorporation of a pyridine 
 or thiophene ring as a replacement of one of the phenyl rings of 34 .
compounds 32 and 33 have mdm2 
 binding in the low nanomolar region , with average cellular ic50 values in three cancer cell lines of 15 and 7 nm , and good 
 oral pharmacokinetic profiles comparable to that of 6 . upon oral administration of 32 at 10 mg / kg daily dose , 
 complete tumor regression is achieved in sjsa-1 nude mice xenograft 
 at the end of a 14-day treatment . in the same mouse tumor model , oral 
 administration of 33 at a daily dose of 10 mg / kg also 
 achieves significant tumor regression .
intense research efforts in the past decade have led to the discovery 
 of a number of classes of highly potent and selective mdm2 inhibitors , 
 and seven such compounds are now in clinical trials .
several of these 
 clinically tested mdm2 inhibitors bind to mdm2 with a very high affinity 
 ( kd or ki of 
 < 1 nm ) and demonstrate significant selectivity over mdmx , a homologue 
 of mdm2 , and other proteins tested .
cocrystal structures of a large 
 number of high - affinity inhibitors in complex with mdm2 show that 
 these mdm2 inhibitors not only mimic the three key p53 binding residues 
 ( phe19 , trp23 , and leu26 ) but also capture additional interactions 
 not observed between p53 and mdm2 . in particular , both 10 and 14 have  stacking with his96 
 and induce refolding of the extreme n - terminus of mdm2 , gaining additional 
 hydrophobic interactions , with residues thr12 and val14 .
furthermore , 14 also forms a surface electrostatic 
 interaction with the g58  shelf  region in mdm2 . as 
 a result
, these small - molecule mdm2 inhibitors can achieve binding 
 affinities to mdm2 that are > 1000 times better than the affinities 
 of p53 peptides .
pharmacokinetic properties , including oral bioavailability , 
 have also been extensively optimized for these clinical - stage mdm2 
 inhibitors .
the successful discovery of these highly potent and selective 
 mdm2 inhibitors with optimized pharmacokinetic properties is a success 
 of modern medicinal chemistry in targeting protein 
protein 
 interactions . when the interaction of mdm2 with p53 is blocked , 
 these potent 
 mdm2 inhibitors inhibit mdm2-mediated p53 ubiquitination and degradation , 
 leading to accumulation and activation of p53 protein in cells with 
 wild - type p53 .
activation of p53 results in transcription of p53-targeted 
 genes , including p21 , a cell cycle regulator , 
 and puma , a proapoptotic protein , leading 
 to cell cycle arrest and/or apoptosis in tumor cells .
since mdm2 is 
 also a p53-targeted gene , mdm2 inhibitors also induce up - regulation 
 of mdm2 mrna and protein .
as expected , these 
 potent and selective mdm2 inhibitors dose - dependently and effectively 
 activate p53 in tumor cells with wild - type p53 but not in tumor cells 
 harboring mutated or deleted p53 .
they potently inhibit cell growth 
 in cancer cell lines with wild - type p53 and show high selectivity 
 over cancer cell lines with mutated or deleted p53 .
interestingly , 
 while mdm2 inhibitors can inhibit cell cycle progression in all tumor 
 cell lines with wild - type p53 , they effectively induce apoptosis in 
 only some cancer cell lines .
analysis 
 of p53-regulated proapoptotic genes , puma , noxa , and bax , in a number 
 of cancer cell lines revealed that puma , but not noxa and bax , was 
 induced by mdm2 inhibitors and the magnitude of puma induction correlates 
 with the degree of apoptosis induction . although earlier generations of mdm2 inhibitors such as 1 and 8 are capable of inhibiting 100% tumor 
 growth in 
 xenograft models of human cancer , they are unable to achieve complete 
 tumor regression
. however , recently discovered mdm2 inhibitors such 
 as 10 and 14 , with improved binding affinities 
 to mdm2 , cellular potencies , and significantly optimized pharmacokinetic 
 properties , can effectively induce complete tumor regression in the 
 sjsa-1 osteosarcoma xenograft model in mice .
the sjsa-1 osteosarcoma 
 cell line has mdm2 gene amplification and is sensitive to mdm2 inhibitors 
 in apoptosis induction .
although only 7% of all human cancers 
 have an amplified mdm2 gene , a higher 
 frequency of mdm2 gene amplification occurs in certain tumor types , 
 including well - differentiated liposarcomas ( > 80% ) , soft tissue 
 tumors 
 ( 20% ) , osteosarcomas ( 16% ) , and esophageal carcinomas ( 13% ) .
the complete tumor regression achieved by these new mdm2 inhibitors 
 in the sjsa-1 xenograft model suggests their therapeutic potential 
 as single agents for the treatment of human cancers with mdm2 gene 
 amplification .
additionally , mdm2 inhibitors such as 6 and 10 have been shown to achieve tumor regression 
 in leukemia xenograft models in mice .
in other xenograft 
 models , such as the xenograft model of the hct-116 colon cancer cell 
 line , mdm2 inhibitors effectively inhibit tumor growth ( tumor stasis ) 
 but fail to achieve tumor regression .
this is consistent with their ability to effectively inhibit cell 
 cycle progression and their inability to induce apoptosis in the hct-116 
 cell line . taken together , these in vivo data suggest that mdm2 inhibitors 
 may have a strong therapeutic potential for the treatment of a subset 
 of human solid tumors and leukemia .
in addition to the development 
 of mdm2 inhibitors as single agents , 
 mdm2 inhibitors have been evaluated in combination with both traditional 
 chemotherapeutic agents and molecularly targeted agents .
these potent 
 mdm2 inhibitors in clinical development are highly selective for mdm2 , 
 but they fail to target mdmx , which directly interacts with p53 and 
 inhibits its function
. since a number of chemotherapeutic agents such 
 as irinotecan and doxorubicin can effectively down - regulate mdmx , 
 their combination with mdm2 inhibitors can be very effective for the 
 treatment of human cancers with high expression of both mdm2 and mdmx 
 proteins .
in addition to these traditional chemotherapeutic agents , 
 mek inhibitors such as trametinib are very effective in induction 
 of down - regulation of mdmx .
indeed , mek 
 inhibitors such as trametinib and pimasertib are currently being evaluated 
 in combination with either 10 or 14 in clinical 
 trials for the treatment of patients with acute myeloid leukemia or 
 solid tumors .
acquired resistance of 
 tumor cells to cancer drugs is a major clinical 
 challenge in drug development . because mdm2 inhibitors are only effective 
 in targeting tumor cells with wild - type p53 , they may select resistant 
 tumor cells that acquire p53 mutation(s ) that escape mdm2 control . 
 indeed ,
treatment of sjsa-1 cells with nutlin 3 in cell cultures has 
 resulted in selection of colonies that contain p53 mutations in dna 
 binding domain and become highly resistant to nutlin 3 .
therefore , combinations of mdm2 inhibitors with 
 agents that are effective against tumor cells with p53 mutations should 
 be tested to prevent or delay acquired resistance .
since mdm2 
 inhibitors also activate p53 in normal cells and normal 
 tissues , there is a concern that activation of p53 can cause toxicity 
 in those normal tissues sensitive to p53 activation , such as bone 
 marrow , spleen , and small intestines .
data from phase i clinical trials 
 of 3(40 ) and 6(34 ) showed that these mdm2 inhibitors cause thrombocytopenia 
 ( decrease of platelets in blood ) , which is a major dose - limiting toxicity . 

mechanistic studies showed that 3 promoted apoptosis of megakaryocyte ( mk ) progenitor cells , 
 resulting in a reduction of their numbers and affected mature mk cells 
 by blocking dna synthesis during endomitosis and impairing platelet 
 production .
therefore , determination of appropriate dose schedules 
 that can achieve robust p53 activation and strong antitumor activity 
 but have manageable on - target toxicity will be another critical task 
 for the successful development of these potent mdm2 inhibitors .
 design of small - molecule inhibitors 
 ( mdm2 inhibitors ) to block 
 the mdm2p53 protein  
 protein interaction has been pursued 
 as a new cancer therapeutic strategy . in recent years 
 , potent , selective , 
 and efficacious mdm2 inhibitors have been successfully obtained and 
 seven such compounds have been advanced into early phase clinical 
 trials for the treatment of human cancers . here , we review the design , 
 synthesis , properties , preclinical , and clinical studies of these 
 clinical - stage mdm2 inhibitors . 
Introduction
MDM2 Inhibitors in Clinical Trials for Treatment
of Human Cancer
Other MDM2 Inhibitors Reported
by Industrial
and Academic Groups
Concluding
Remarks and Outlook for Small-Molecule
MDM2 Inhibitors as a New Class of Drugs for Cancer Treatment