Macrocyclic Small Molecules Research Articles

Discovery of novel Macrocyclic small molecules Based on 2-Amino-4-thiazolylpyridineas selective EGFR inhibitors with high Blood-Brain barrier penetration for the treatment of glioblastoma

Because epidermal growth factor receptor (EGFR) is the most commonly mutated oncogene in glioblastoma (GBM), the development of EGFR inhibitors has become a promising direction for the treatment of GBM. However, due to factors such as limited blood–brain barrier (BBB) permeability and pathway compensation mechanisms, current EGFR inhibitors targeting GBM are not satisfactory. In the previous study, we obtained compound 10c with strong anti-cell proliferation activity. Since macrocyclization can effectively change the physical and chemical properties of molecules, and optimize their selectivity. Therefore, a series of 2-amino-4-thiazolyl pyridine scaffold macrocyclic derivatives were designed and synthesized using compound 10c as the lead compound in this study. Compound 3a, which inhibited the growth of glioblastoma cell lines U87MG and U87-EGFRVIII, had average IC50 values of 4.69 µM and 4.98 µM, respectively. Compound 3a was highly selective to 9 kinases in the ErbB family, including ErbB2 and ErbB4. In addition, compound 3a demonstrated good blood–brain barrier permeability in mice, the blood–brain concentration of the drug remained above 20 % within 5–60 min following intravenous administration in mice. In conclusion, compound 3a is a promising candidate for novel EGFR inhibitors targeting GBM.

Discovery of novel resorcinol biphenyl ether-based macrocyclic small molecules as PD-1/PD-L1 inhibitors with favorable pharmacokinetics for cancer immunotherapy

Programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) is one of the most promising immune checkpoints (ICs) in tumor immunology and has been actively pursued as a target for anticancer drug discovery. Based on our previous research in small molecule PD-1/PD-L1 modulators, we designed and synthesized a series of resorcinol biphenyl ether-bearing macrocyclic compounds and evaluated their anti-PD-1/PD-L1 activities. Among them, compound 8d exhibited the highest inhibitory activity against PD-1/PD-L1 interaction with IC50 of 259.7 nM in the homogenous time-resolved fluorescence (HTRF) assay. In addition, 8d displayed in vitro immunomodulatory effects by promoting HepG2 cell death in a HepG2/Jurkat cell co-culture model. Furthermore, 8d effectively inhibited tumor growth (TGI = 74.6% at 40 mg/kg) in a melanoma tumor model in mice without causing obvious toxicity. Moreover, 8d exhibited favorable pharmacokinetics [e.g. high stability, reasonable half-life, and good oral bioavailability (F = 21.5%)]. Finally, molecular modeling studies showed that 8d bound to PD-L1 with high affinity. These results suggest that 8d may serve as a starting point for further development of macrocyclic small molecule-based PD-1/PD-L1 inhibitors for cancer treatment.

Scaffold Hopping Transformations Using Auxiliary Restraints for Calculating Accurate Relative Binding Free Energies.

In silico screening of drug-target interactions is a key part of the drug discovery process. Changes in the drug scaffold via contraction or expansion of rings, the breaking of rings, and the introduction of cyclic structures from acyclic structures are commonly applied by medicinal chemists to improve binding affinity and enhance favorable properties of candidate compounds. These processes, commonly referred to as scaffold hopping, are challenging to model computationally. Although relative binding free energy (RBFE) calculations have shown success in predicting binding affinity changes caused by perturbing R-groups attached to a common scaffold, applications of RBFE calculations to modeling scaffold hopping are relatively limited. Scaffold hopping inevitably involves breaking and forming bond interactions of quadratic functional forms, which is highly challenging. A novel method for handling ring opening/closure/contraction/expansion and linker contraction/expansion is presented here. To the best of our knowledge, RBFE calculations on linker contraction/expansion have not been previously reported. The method uses auxiliary restraints to hold the atoms at the ends of a bond in place during the breaking and forming of the bonds. The broad applicability of the method was demonstrated by examining perturbations involving small-molecule macrocycles and mutations of proline in proteins. High accuracy was obtained using the method for most of the perturbations studied. The rigor of the method was isolated from the force field by validating the method using relative and absolute hydration free energy calculations compared to standard simulation results. Unlike other methods that rely on λ-dependent functional forms for bond interactions, the method presented here can be employed using modern molecular dynamics software without modification of codes or force field functions.

Omipalisib inspired macrocycles as dual PI3K/mTOR inhibitors.

Activation of the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway occurs frequently in a wide range of human cancers and is a main driver of cell growth, proliferation, survival, and chemoresistance of cancer cells. Compounds targeting this pathway are under active development as anticancer therapeutics and some of them have reached advanced clinical trials or been approved by the FDA. Dual PI3K/mTOR inhibitors combine multiple therapeutic efficacies in a single molecule by inhibiting the pathway both upstream and downstream of AKT. Herein, we report our efforts on the exploration of novel small molecule macrocycles (MCXs) as dual PI3K/mTOR inhibitors. Macrocyclization is an attractive approach used in drug discovery, as the semi-rigid character of these structures could provide improved potency, selectivity and favorable pharmacokinetic properties. Importantly, this strategy allows access to new chemical space thus obtaining a better intellectual property position. A series of MCXs based on GSK-2126458, a known clinical PI3K/mTOR inhibitor is described. These molecules showed potent biochemical and cellular dual PI3K/mTOR inhibition, demonstrated strong antitumoral effects in human cancer cell lines, and displayed good drug-like properties. Among them, MCX 83 presented remarkable selectivity against a panel of 468 kinases, high invitro metabolic stability, and favorable pharmacokinetic parameters without significant CYP450 and h-ERG binding inhibition. This profile qualified this compound as a suitable candidate for future invivo PK-PD and efficacy studies in mouse cancer models.

Scope of 3D Shape-Based Approaches in Predicting the Macromolecular Targets of Structurally Complex Small Molecules Including Natural Products and Macrocyclic Ligands.

A plethora of similarity-based, network-based, machine learning, docking and hybrid approaches for predicting the macromolecular targets of small molecules are available today and recognized as valuable tools for providing guidance in early drug discovery. With the increasing maturity of target prediction methods, researchers have started to explore ways to expand their scope to more challenging molecules such as structurally complex natural products and macrocyclic small molecules. In this work, we systematically explore the capacity of an alignment-based approach to identify the targets of structurally complex small molecules (including large and flexible natural products and macrocyclic compounds) based on the similarity of their 3D molecular shape to noncomplex molecules (i.e., more conventional, “drug-like”, synthetic compounds). For this analysis, query sets of 10 representative, structurally complex molecules were compiled for each of the 28 pharmaceutically relevant proteins. Subsequently, ROCS, a leading shape-based screening engine, was utilized to generate rank-ordered lists of the potential targets of the 28 × 10 queries according to the similarity of their 3D molecular shapes with those of compounds from a knowledge base of 272 640 noncomplex small molecules active on a total of 3642 different proteins. Four of the scores implemented in ROCS were explored for target ranking, with the TanimotoCombo score consistently outperforming all others. The score successfully recovered the targets of 30% and 41% of the 280 queries among the top-5 and top-20 positions, respectively. For 24 out of the 28 investigated targets (86%), the method correctly assigned the first rank (out of 3642) to the target of interest for at least one of the 10 queries. The shape-based target prediction approach showed remarkable robustness, with good success rates obtained even for compounds that are clearly distinct from any of the ligands present in the knowledge base. However, complex natural products and macrocyclic compounds proved to be challenging even with this approach, although cases of complete failure were recorded only for a small number of targets.

Development of Macrocycle Kinase Inhibitors for ALK2 Using Fibrodysplasia Ossificans Progressiva-Derived Endothelial Cells.

ABSTRACTFibrodysplasia ossificans progressiva (FOP) is an extremely rare congenital form of heterotopic ossification (HO), caused by heterozygous mutations in the activin A type I receptor (ACVR1), that encodes the bone morphogenetic protein (BMP) type I receptor ALK2. These mutations enable ALK2 to induce downstream signaling in response to activins, thereby turning them into bone‐inducing agents. To date, there is no cure for FOP. The further development of FOP patient‐derived models may contribute to the discovery of novel biomarkers and therapeutic approaches. Nevertheless, this has traditionally been a challenge, as biopsy sampling often triggers HO. We have characterized peripheral blood‐derived endothelial colony‐forming cells (ECFCs) from three independent FOP donors as a new model for FOP. FOP ECFCs are prone to undergo endothelial‐to‐mesenchymal transition and exhibit increased ALK2 downstream signaling and subsequent osteogenic differentiation upon stimulation with activin A. Moreover, we have identified a new class of small molecule macrocycles with potential activity against ALK2 kinase. Finally, using FOP ECFCs, we have selected OD36 and OD52 as potent inhibitors with excellent kinase selectivity profiles that potently antagonize mutant ALK2 signaling and osteogenic differentiation. We expect that these results will contribute to the development of novel ALK2 clinical candidates for the treatment of FOP. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Development of novel macrocyclic small molecules that target CTG trinucleotide repeats

We describe the molecular design, synthesis, and investigation of a series of acridine-triaminotriazine macrocycles that selectively bind to CTG trinucleotide repeats in DNA with minimal nonspecific binding. The limited conformational flexibility enforces the stacking of the triaminotriazine and acridine units. Isothermal titration calorimetry studies and Job plot analyses revealed that the ligands bound to d(CTG) mismatched sites. The acridine and triaminotriazine units were shown to intramolecularly π-stack in aqueous solutions. Compared to a noncyclic analog, the macrocycles showed an almost 10-fold lower cytotoxicity in HeLa cells and up to 4-fold higher transcription inhibition of d(CTG·CAG)74.

Structure-Based Macrocycle Design in Small-Molecule Drug Discovery and Simple Metrics To Identify Opportunities for Macrocyclization of Small-Molecule Ligands.

Interest is growing in the use of macrocycles in pharmaceutical discovery. Macrocylization may provide a gateway to an expanded chemical space for small-molecule drug discovery, and this could be beneficial in prosecuting difficult targets, e.g., protein-protein interactions. Most, but not all, macrocycle drugs are derived from natural products. Studies on synthetic drug-like small-molecule macrocycles are limited, and our current understanding of macrocycle drugs is similarly limited. Following some background discussion, we review several examples of the structure-based design of synthetic macrocycles. Our opinion is that in conformationally suitable systems macrocycles are an analog class worthy of consideration. We then summarize an approach for the initial evaluation of molecules as candidates for macrocyclization.

Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules

DNA-encoded libraries have emerged as a widely used resource for discovery of bioactive small molecules and offer substantial advantages compared to conventional small-molecule libraries. Here we developed and streamlined multiple fundamental aspects of DNA-encoded and DNA-templated library synthesis methodology, including computational identification and experimental validation of a 20×20×20×80 set of orthogonal codons, chemical and computational tools for enhancing the structural diversity and drug-likeness of library members, a highly efficient polymerase-mediated template library assembly strategy, and library isolation and purification methods. We integrated these improved methods to produce a second-generation DNA-templated library of 256,000 small-molecule macrocycles with improved drug-like physical properties. In vitro selection of this library for insulin-degrading enzyme (IDE) affinity resulted in novel IDE inhibitors including one of unusual potency and novel macrocycle stereochemistry (IC50 = 40 nM). Collectively, these developments enable DNA-templated small-molecule libraries to serve as more powerful, accessible, streamlined, and cost-effective tools for bioactive small-molecule discovery.

Rapid synthesis of cyclic oligomeric depsipeptides with positional, stereochemical, and macrocycle size distribution control

Macrocyclic small molecules are attractive tools in the development of sensors, new materials, and therapeutics. Within early-stage drug discovery, they are increasingly sought for their potential to interact with broad surfaces of peptidic receptors rather than within their narrow folds and pockets. Cyclization of linear small molecule precursors is a straightforward strategy to constrain conformationally mobile motifs, but forging a macrocycle bond typically becomes more difficult at larger ring sizes. We report the development of a general approach to discrete collections of oligomeric macrocyclic depsipeptides using an oligomerization/macrocyclization process governed by a series of Mitsunobu reactions of hydroxy acid monomers. Ring sizes of 18, 24, 30, and 36 are formed in a single reaction from a didepsipeptide, whereas sizes of 24, 36, and 60 result from a tetradepsipeptide. The ring-size selectivity inherent to the approach can be modulated by salt additives that enhance the formation of specific ring sizes. Use of chemical synthesis to prepare the monomers suggests broad access to functionally and stereochemically diverse collections of natural product-like oligodepsipeptide macrocycles. Two cyclodepsipeptide natural products were prepared along with numerous unnatural oligomeric congeners to provide rapid access to discrete collections of complex macrocyclic small molecules from medium (18) to large (60) ring sizes.

Practical stereoselective synthesis of eribulin fragment toward building a hybrid macrocyclic toolbox.

A practical stereoselective synthesis to obtain the substituted furan ring as the substructure of eribulin is developed. An asymmetric syn-aldol and intramolecular oxy-Michael were two key steps in our approach. The functionalized furan derivatives were then utilized further to build the 14- and 12-membered macrocyclic diversity as trans- and cis-fused (C-29 and C-30) compounds. This is the first report of building a chemical toolbox with macrocyclic small molecules having trans- or cis-fused 14- or 12-membered rings containing the substructure of eribulin and its diastereomer.

Abstract SY05-04: Biological discovery and therapeutic insight from DNA-templated macrocycle libraries

Abstract DNA-templated organic synthesis enables the translation of libraries of DNA templates into corresponding libraries of synthetic small molecules. The in vitro selection of a library of DNA-templated small-molecule macrocycles has resulted in the discovery of unusually selective and potent inhibitors of kinase, protease, and protein-protein interface targets. Using this approach, we recently discovered the first physiologically active inhibitor of an enzyme implicated in type-2 diabetes. In a series of biochemical, structural biological, and in vivo physiology experiments, we elucidated the molecular basis of target inhibition by this macrocycle, validated a new target for the potential treatment of type-2 diabetes, and discovered previously unknown and therapeutically relevant physiological roles for this enzyme. Citation Format: David R. Liu. Biological discovery and therapeutic insight from DNA-templated macrocycle libraries. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY05-04. doi:10.1158/1538-7445.AM2014-SY05-04

Prevention of Mitochondrial Membrane Permeabilization and Pancreatic β‐Cell Death by an Enantioenriched, Macrocyclic Small Molecule

AbstractMitochondria produce the majority of cellular energy through the process of oxidative phosphorylation and play a central role in regulating the functionality and survival of eukaryotic cells. Under physiological stress, mitochondrial membrane permeabilization results in the release of apoptogenic material such as cytochrome c in the cytoplasm, which thereby initiates caspase activation and the consequent cell death. In our present study, we screened a series of compounds for their ability to inhibit mitochondrial membrane permeabilization and to prevent cytochrome c release during the endoplasmic reticulum stress in cultured pancreatic β‐cells. Three benzofuran‐based macrocyclic small molecules, that is, 2.4c, c104, and c108, were found to restore the depolarization of mitochondrial membrane potential and to prevent the release of cytochrome c from mitochondria. Interestingly, the acyclic precursor of 2.4c (i.e., 2.3c) did not show any effect, whereas the macrocyclic derivative obtained by utilizing ring‐closing metathesis as the “stitching technology” led to this function. The macrocyclic architecture seems to play a crucial role in presenting various functional moieties in the right orientation to observe this effect.

Discovery of the Macrocycle (9E)-15-(2-(Pyrrolidin-1-yl)ethoxy)-7,12,25-trioxa-19,21,24-triaza-tetracyclo[18.3.1.1(2,5).1(14,18)]hexacosa-1(24),2,4,9,14(26),15,17,20,22-nonaene (SB1578), a Potent Inhibitor of Janus Kinase 2/Fms-LikeTyrosine Kinase-3 (JAK2/FLT3) for the Treatment of Rheumatoid Arthritis

Herein, we describe the synthesis and SAR of a series of small molecule macrocycles that selectively inhibit JAK2 kinase within the JAK family and FLT3 kinase. Following a multiparameter optimization of a key aryl ring of the previously described SB1518 (pacritinib), the highly soluble 14l was selected as the optimal compound. Oral efficacy in the murine collagen-induced arthritis (CIA) model for rheumatoid arthritis (RA) supported 14l as a potential treatment for autoimmune diseases and inflammatory disorders such as psoriasis and RA. Compound 14l (SB1578) was progressed into development and is currently undergoing phase 1 clinical trials in healthy volunteers.

Discovery of Kinase Spectrum Selective Macrocycle (16E)-14-Methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (SB1317/TG02), a Potent Inhibitor of Cyclin Dependent Kinases (CDKs), Janus Kinase 2 (JAK2), and Fms-like Tyrosine Kinase-3 (FLT3) for the Treatment of Cancer

Herein, we describe the design, synthesis, and SAR of a series of unique small molecule macrocycles that show spectrum selective kinase inhibition of CDKs, JAK2, and FLT3. The most promising leads were assessed in vitro for their inhibition of cancer cell proliferation, solubility, CYP450 inhibition, and microsomal stability. This screening cascade revealed 26 h as a preferred compound with target IC(50) of 13, 73, and 56 nM for CDK2, JAK2 and FLT3, respectively. Pharmacokinetic (PK) studies of 26 h in preclinical species showed good oral exposures. Oral efficacy was observed in colon (HCT-116) and lymphoma (Ramos) xenograft studies, in line with the observed PK/PD correlation. 26h (SB1317/TG02) was progressed into development in 2010 and is currently undergoing phase 1 clinical trials in advanced leukemias and multiple myeloma.

Abstract 3564: Design, synthesis and SAR studies leading to SB1518, a novel macrocyclic JAK2/FLT3 inhibitor in phase 2 clinical trials for myelofibrosis and lymphoma

Abstract Introduction: Here we present the unique chemical structure of SB1518 and SAR studies leading to the discovery of this exciting novel JAK2/FLT3 inhibitor, currently in clinical trials for myelofibrosis and lymphoma. Methods/Results: As part of our innovative chemical strategy towards creating novel kinase scaffolds we discovered a series of unusual small molecular weight macrocycles which were optimized for selective JAK2 inhibitory potency culminating in the discovery of SB1518, which is selective for JAK2 (IC50 = 22nM) and its V617F mutant (IC50 = 19nM) over JAK1 (IC50 > 1µM) and JAK3 (IC50 > 0.5µM). FLT3 was identified as an additional kinase target (IC50 = 22nM and 6nM for its D835Y mutant) giving a broader potential in other hematological malignancies. The exciting and novel chemical structures of these compounds led us into a full lead optimization program to take full advantage of the unique nature of these molecules. SAR studies modifying the macrocyclic linker allowed unwanted protein kinase activity to be tuned out, whilst retaining a narrow spectrum of desired JAK2 kinase activity and selectivity over JAK1 and JAK3, a distinguishing feature. SB1518 and analogues were synthesized via a ring-closing metathesis (RCM) strategy: coupling of the two halves of the molecule provided a diene which was closed by RCM using either Grubbs or Zhan catalysts. SAR investigations, led by structure-based design, influenced the choice and position of a critical solubilising side-chain. Preferred compounds had improved aqueous solubility and high permeability. SB1518 is very stable both chemically and metabolically with a long half life in human microsomal preparations. With the desired balance of potency and oral pharmacokinetics, SB1518 was selected for further profiling. SB1518 demonstrated potent activity in cell lines driven by mutant JAK2 or FLT3 and was efficacious at very well tolerated oral doses in tumor models generated with these cell lines. Based on its attractive preclinical profile, SB1518 was selected for clinical development. Conclusions: Discovery of a new series of small molecule macrocycles of a unique and highly novel chemical structure are presented as inhibitors of JAK2 and FLT3 kinases. SAR studies directed us towards an optimal linker and solubilising side chain fine-tuning the molecular structure. SB1518 was selected as a development candidate with an attractive and distinguishing JAK2 selective inhibitory profile and additional activity against FLT3. SB1518 has shown encouraging clinical activity demonstrating that its attractive preclinical profile translates to the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3564. doi:10.1158/1538-7445.AM2011-3564

Translation of DNA into a Library of 13 000 Synthetic Small-Molecule Macrocycles Suitable for in Vitro Selection

DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in vitro selection of a pilot library of 65 macrocycles. In this work, we report several key developments that enable the DNA-templated synthesis of much larger (>10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from 4 to 12, selected 8 different starting scaffolds which result in 4 macrocycle ring sizes and 2 building-block orientations, and confirmed the ability of the 36 building blocks and 8 scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. Integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (>90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles.

Exploiting Ligand Conformation in Selective Inhibition of Non-Ribosomal Peptide Synthetase Amino Acid Adenylation with Designed Macrocyclic Small Molecules

Macrocyclic aminoacyl-AMP analogs have been developed to inhibit non-ribosomal peptide synthetase amino acid adenylation domains selectively by mimicking a cisoid ligand binding conformation observed in crystal structures. In contrast, these macrocycles do not inhibit aminoacyl-tRNA synthetases, which are mechanistically closely related, but bind their ligands in a distinct transoid conformation. The macrocycles contain a two- or three-carbon linker between Cβ of the amino acid moiety and C8 of the adenine ring and a sulfamate in place of the phosphate group. These compounds are potent inhibitors of the cysteine adenylation domain activity of the yersiniabactin siderophore synthetase HMWP2 and, unlike the corresponding linear aminoacyl-AMP analogs, do not inhibit protein translation in vitro. Selective small molecule inhibitors of non-ribosomal peptide synthesis should provide a powerful means to study the biological functions of non-ribosomal peptide natural products and a potential avenue to develop novel antibiotics.

Macrocycloadditions Leading to Conformationally Restricted Small Molecules

[reaction: see text] The Cu(I)-catalyzed cycloaddition of alkynes and azides (click reaction) provides a robust method for the construction of macrocyclic small molecules via an intramolecular macrocycloaddition. A three-subunit system has been used to explore the tolerance of this macrocycloaddition to variations of stereochemistries and substituents.

DNA-templated organic synthesis and selection of a library of macrocycles.

The translation of nucleic acid libraries into corresponding synthetic compounds would enable selection and amplification principles to be applied to man-made molecules. We used multistep DNA-templated organic synthesis to translate libraries of DNA sequences, each containing three "codons," into libraries of sequence-programmed synthetic small-molecule macrocycles. The resulting DNA-macrocycle conjugates were subjected to in vitro selections for protein affinity. The identity of a single macrocycle possessing known target protein affinity was inferred through the sequence of the amplified DNA template surviving the selection. This work represents the translation, selection, and amplification of libraries of nucleic acids encoding synthetic small molecules rather than biological macromolecules.