Design, Synthesis And Antitumor Activity Of Indoleamine 2,3-Dioxygenase 1-Based Multi-Target Inhibitors

Cancer is a multifactorial disease often requiring multiple therapeutic interventions for successful long term outcomes.Immune checkpoint therapy has been recognized as an important approach of cancer therapy besides surgery,radiation,chemotherapy and targeted therapy.Indoleamine 2,3-dioxygenase 1?IDO1?is a heme-containing enzyme that catalyzes the first and rate-limiting step of the kynurenine pathway?KP?.IDO1 inhibitors control and eradicate the growth of tumor cells by enhancing antitumor immune responses.As single agents,IDO1 inhibitors generally exhibited moderate antitumor efficacy in preclinical studies.Despite the synergistic effects observed by the combination of IDO1 inhibitors with other antitumor agents,drug combination strategies are always limited by complex pharmacokinetics and drug-drug interactions.Therefore,designing a single agent that simultaneously targets two or more synergistic mechanisms may overcome these inherently challenging problems.To improve the efficacy of small molecule cancer immunotherapy,this thesis introduced the alkylating pharmacophore N,N-bis?2-chloroethyl?amine to the C5-or C6-position of 1-MT using the pharmacophore fusion strategy.As a result,the first-in-class orally active dual IDO1 and DNA inhibitors were designed and synthesized.The two bifunctional anticancer agents A1 and A2 showed higher IDO1 inhibitory potency than IDO1 inhibitor 1-MT,and better antiproliferative activities than DNA alkylating agent melphalan.Particularly,compounds A1?TGI = 63.8%?and A2?TGI-73.5%?exhibited significantly higher antitumor potency than 1-MT?TGI = 24.2%?and melphalan?TGI = 36.5%?in a murine LLC tumor model.To the best our knowledge,the study represents the first case of proof-of-concept of dual targeting strategy in emerging cancer immunotherapy.Considering that 1-MT was a weak IDO1 inhibitor and its mechanisms were still poorly understood,we further chose IDO1 inhibitor INCB024360 as the starting point in the design of dual targeting molecules.Guided by the binding mode,new hybrids of IDO1 inhibitors and DNA alkylating nitrogen mustards targeting respective IDO1 and DNA were rationally designed,synthesized and evaluated.Among them,compound B1 showed the most potent IDO1 inhibitory activity(IC₅₀ = 0.13 ?M)and good cellular potency(EC₅₀ = 0.27 ?M).Pharmacodynamic studies indicated that compound B1 significantly inhibited IDO1 activity in tumor tissues and reduced Kyn level in plasma.Furthermore,importantly,it exhibited better in vivo antitumor efficacy than clinical candidate IDO1 inhibitor INCB024360?58.2%versus 47.5%?in the CT-26 allograft models without significant body weight loss and adverse effects.In order to take advantages of both immunotherapeutic and epigenetic antitumor agents,the first generation of dual IDO1 and histone deacetylase?HDAC?inhibitors were designed.The highly active dual inhibitor C1 showed excellent and balanced activity against both IDO1(IC₅₀ = 69.0 nM)and HDAC1(IC₅₀ = 66.5 nM),whose dual targeting mechanisms were validated in cancer cells.Compound C1 had good pharmacokinetic profiles as an orally active antitumor agent and significantly reduced the L-kynurenine level in plasma.In particular,it showed excellent in vivo antitumor efficacy?TGI = 54.3%?in the murine LLC tumor model with low toxicity.In summary,the present work performed design and synthesis of IDO1-based multi-target inhibitors using the pharmacophore fusion strategy.The novelty of this thesis can be summarized as follows:?1?Developing the first 1-MT-and INCB024360-based dual IDO1 and DNA inhibitors.?2?Comfirming the feasiblity and powerfulness of IDOl/DNA dual-targeting molecules in improving the efficacy of cancer immunotherapy.?3?Developing the first-in-class dual IDO1 and HD AC inhibitors.Compound C1 represents a promising lead compound for the development of novel antitumor agents and can also be used a valuable probe to clarify the relationships and mechanisms between cancer immunotherapy and epigenetics.These proof-of-concept has established a critical step toward developing novel and effective immunotherapy for the treatment of cancers.