Design, Synthesis And Preliminary Evaluation Of Matrix Metalloproteinase-2 Inhibitors Based On Pyrrolidine Scaffold

Malignant tumor is one of the diseases that seriously threaten peoples' health and the search of the selective antitumor agents with high potency and low toxicity is urgent. The distinct character of malignant tumor is its invasion and metastasis,which causes most of clinic death.The conventional therapies such as radiotherapy,chemotherapy and surgical therapy mainly aim at the primary tumor,and are not efficient for the metastasis tumor.So the metastasis tumor is also the challenge for the antitumor therapy.The matrix metalloproteinases(MMPs)are a family of zinc-dependent endopeptidases that are involved in the degradation of all components of the extracellular matrix.MMPs are highly involved in the process of tumor invasion and metastasis.MMPs inhibition might remarkably block the invasion of primary or metastasis tumor,thus acting as tumor therapy.The expression and activation of MMPs is a multi-step process,including transcription of MMP genes,the entry of MMP zymogen into the matrix,and the activation of zymogen,and so on.Blocking anyone of these steps might inhibit the activity of MMPs.Currently drug design is mainly based on the following:(1)blockage of the transcription of MMP genes;(2)blockage of the translation of MMP mRNA;(3)blockage of the activation of MMP zymogen;(4) blockage of MMPs proteolytic activities.MMP inhibitors can block MMPs proteolytic activities,protect the extracellular matrix,inhibit the tumor growth,and reduce the occurrence of tumor metastasis.It has been reported that besides the catalytic activity center zinc(â…¡)ion of MMPs, there are two hydrophobic domains,which are called S1' pocket and S2' pocket, respectively.S1' pocket,the key domain of MMPs,is deep and narrow,and S2' pocket is solvent exposed.Currently identified MMP-2 inhibitors shared the following structural character and binding mode:(1)a zinc binding group(ZBG,such as hydroxamate and carboxylate)capable of chelating the active site zinc ion;(2)one at least functional group which provides a hydrogen bond interaction with the enzyme backbone;(3)one or more side chains which undergo effective interactions with the enzyme subsites,such as S1' pocket and S2' pocket.Our group has been developing pyrrolidine scaffold-based MMP inhibitors for a number of years.Further studies found that one of these compounds,LY52 could significantly block the proteolytic activity of MMP-2(IC₅₀=9.0nM).LY52 also suppressed human ovarian carcinoma cell line SKOV₃ invasion in vitro.Furthermore,a significant inhibition of pulmonary metastasis of Lewis lung carcinoma cells was observed in LY52-administrated mice.We might conclude from these results that LY52 might suppress invasion and metastasis of carcinoma cells via inhibition of MMP-2 proteolytic activities.In order to identify more potent MMP-2 inhibitors,based on our previous work and the structural character of MMP inhibitors,we use LY52 as the lead compound to perform the structural optimization.Three series of compounds(A,B,C,71 compounds in all)were synthesized,among which there were 29 compounds in A series,18 compounds in B series and 24 compounds in C series.Starting from trans-4-hydroxy-L-proline(1)as a chiral template,the important intermediate(2S,4S)-methyl 1-acyl-4-aminopyrrolidine-2-carboxylate(A6a-c)were prepared via the route of esterification,acylation,sulphonation,SN₂ nucleophilic substitution and catalytic hydrogenation.In the nucleophilic substitution step, (3R,5S)-5-(methoxycarbonyl)-1-acylpyrrolidin-3-yl sulfonate(A4a-b)reacted with sodium azide to produce configurationally reversed azide(A5a-b),which were then catalytically hydrogenated using 5%Pd-CaCO₃ to afford the intermediate A6a-c. Acylation of A6a-c with various acyl chlorides provided compounds(2S,4S)-methyl 4-acylamido-1-acylpyrrolidine-2-carboxylate(A7a-m),some of which were further ammonolyzed or hydrolyzed to obtain the target compounds(2S,4S)-1-acyl-4-(acyl amido)-N-hydroxypyrrolidine-2-carboxamide(A8a-e)and (2S,4S)-4-(3,4,5-trimethoxybenzamido)-1-acetylpyrrolidine-2-carboxylic acid(A9). Further coupling of compound A9 with glycine methyl ester gave methyl 2-((2S,4S)-4-(3,4,5-trimethoxybenzamido)-1-acetylpyrrolidine-2-carboxamido)acetate (A10). Starting from trans-4-hydroxy-L-proline(1)as a chiral template,the important intermediate 1-sulfonyl-4-oxo-pyrrolidine-2-carboxylic acid methyl ester(B4a-c)was prepared via the route of esterification,sulphonation and Jones oxidation.Addition reaction of B4a-c with various diols produced the ketal compounds B5a-f.In this step, the p-toluenesulfonic acid acted as catalyst and the Dean-Stark trap was used to remove the water product.Ammonolysis of B5a-f with NH₂OK provided the final spiro hydroxamate compounds(B6a-f).Starting from trans-4-hydroxy-L-proline(1)as a chiral template,the intermediate trans-4-hydroxy-L-proline methyl ester hydrochloride(2)was prepared according to the literature.1-Sulfonyl-4-hydroxy-pyrrolidine-2-carboxylic acid methyl ester(C3a-c)was prepared by sulphonation of compound 2 with various sulfonyl chlorides in the presence of 4-(dimethylamino)pyridine as catalyst.Esterification of C3a-c afforded the target compounds C4a-r and ammonolysis of C3a-c with NH₂OK provided the target compounds C5a-c.The chemical structures of the target compounds were confirmed by IR,¹H NMR and ESI-MS.The newly synthesized pyrrolidine derivatives were assayed for the inhibitory activities on MMP-2 and AP-N.The results showed that these pyrrolidine derivatives exhibited highly selective inhibition against MMP-2 as compared with AP-N.The compounds A8a-c,B6a-d,C4c,C4j,C5a and C5b were more potent MMP-2 inhibitors than the positive control LY52.The FlexX docking of these compounds with MMP-2 was done using Sybyl 7.0 of Tripos Incorporation.The side chain at N₁ position or C₄ position of pyrrolidine ring occupied the deep S1' pocket of MMP-2,and the ZBG at C₂ position of pyrrolidine ring chelated the active site zinc ion 166.The structure activity relationship studies of A series compounds indicated that compounds containing hydroxamate ZBG were more potent than the compounds containing carboxylate ZBG.Moderately long side chain at C₄ position of pyrrolidine ring and short side chain at N₁ position of pyrrolidine ring favored the inhibitory activity against MMP-2.The structure activity relationship studies of B series compounds indicated that compounds containing hydroxamate ZBG were more potent than the compounds containing carboxylate ZBG.Introduction of toluene-4-sulfonyl group at N₁ position and five-member-ring ketal group at C₄ position at pyrrolidine ring favored the inhibitory activity against MMP-2.The structure activity relationship studies of C series compounds indicated that compounds containing hydroxamate ZBG showed excellent potency,and the compounds containing butterfly like symmetrical structure and carboxylate ZBG also displayed high potency.Introduction of toluene-4-sulfonyl group at N₁ position of pyrrolidine ring favored the inhibitory activity against MMP-2.B and C series of compounds are both sulfonyl pyrrolidine derivatives and there are some SAR differences from A series compounds.This might be due to the introduction of sulfonyl group at N₁ position of pyrrolidine ring,which altered the binding mode of compound with the enzyme.Hereby,the binding mode of the compounds with MMP-2 was proposed.In conclusion,three series of novel pyrrolidine scaffold based MMP inhibitors were designed and synthesized.The compounds A8a-c,B6a-d,C4c,C4j,C5a and C5b were more potent MMP-2 inhibitors than the positive control LY52.These compounds are promising lead compounds for developing new generation of MMP inhibitors as antitumor agents.