Harish Nagaraj

Discovery of the Macrocycle 11-(2-Pyrrolidin-1-yl-ethoxy)-14, 19-dioxa-5,7,26-triaza-tetracyclo[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8,10,12(27),16,21,23-decaene (SB1518), a Potent Janus Kinase 2/Fms-Like Tyrosine Kinase-3 (JAK2/FLT3) Inhibitor for the Treatment of Myelofibrosis and Lymphoma

Discovery of the activating mutation V617F in Janus Kinase 2 (JAK2(V617F)), a tyrosine kinase critically involved in receptor signaling, recently ignited interest in JAK2 inhibitor therapy as a treatment for myelofibrosis (MF). Herein, we describe the design and synthesis of a series of small molecule 4-aryl-2-aminopyrimidine macrocycles and their biological evaluation against the JAK family of kinase enzymes and FLT3. The most promising leads were assessed for their in vitro ADME properties culminating in the discovery of 21c, a potent JAK2 (IC(50) = 23 and 19 nM for JAK2(WT) and JAK2(V617F), respectively) and FLT3 (IC(50) = 22 nM) inhibitor with selectivity against JAK1 and JAK3 (IC(50) = 1280 and 520 nM, respectively). Further profiling of 21c in preclinical species and mouse xenograft and allograft models is described. Compound 21c (SB1518) was selected as a development candidate and progressed into clinical trials where it is currently in phase 2 for MF and lymphoma.

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.

VS-5584, a Novel and Highly Selective PI3K/mTOR Kinase Inhibitor for the Treatment of Cancer

Dysregulation of the PI3K/mTOR pathway, either through amplifications, deletions, or as a direct result of mutations, has been closely linked to the development and progression of a wide range of cancers. Moreover, this pathway activation is a poor prognostic marker for many tumor types and confers resistance to various cancer therapies. Here, we describe VS-5584, a novel, low-molecular weight compound with equivalent potent activity against mTOR (IC50 = 37 nmol/L) and all class I phosphoinositide 3-kinase (PI3K) isoforms IC50: PI3Kα = 16 nmol/L; PI3Kβ = 68 nmol/L; PI3Kγ = 25 nmol/L; PI3Kδ = 42 nmol/L, without relevant activity on 400 lipid and protein kinases. VS-5584 shows robust modulation of cellular PI3K/mTOR pathways, inhibiting phosphorylation of substrates downstream of PI3K and mTORC1/2. A large human cancer cell line panel screen (436 lines) revealed broad antiproliferative sensitivity and that cells harboring mutations in PI3KCA are generally more sensitive toward VS-5584 treatment. VS-5584 exhibits favorable pharmacokinetic properties after oral dosing in mice and is well tolerated. VS-5584 induces long-lasting and dose-dependent inhibition of PI3K/mTOR signaling in tumor tissue, leading to tumor growth inhibition in various rapalog-sensitive and -resistant human xenograft models. Furthermore, VS-5584 is synergistic with an EGF receptor inhibitor in a gastric tumor model. The unique selectivity profile and favorable pharmacologic and pharmaceutical properties of VS-5584 and its efficacy in a wide range of human tumor models supports further investigations of VS-5584 in clinical trials. Mol Cancer Ther; 12(2); 151–61. ©2012 AACR.

Structure-based design of oxygen-linked macrocyclic kinase inhibitors: discovery of SB1518 and SB1578, potent inhibitors of Janus kinase 2 (JAK2) and Fms-like tyrosine kinase-3 (FLT3)

Macrocycles from our Aurora project were screened in a kinase panel and were found to be active on other kinase targets, mainly JAKs, FLT3 and CDKs. Subsequently these compounds became leads in our JAK2 project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. This residue is conserved in most CDKs resulting in potent pan CDK inhibition. One of the main project objectives was to achieve JAK2 potency with 100-fold selectivity against CDKs. Macrocycles with an ether linker have potent JAK2 activity with the ether oxygen forming a hydrogen bond to Ser936. A hydrogen bond to the equivalent residues of JAK3 and most CDKs cannot be formed resulting in good selectivity for JAK2 over JAK3 and CDKs. Further optimization of the macrocyclic linker and side chain increased JAK2 and FLT3 activity as well as improving DMPK properties. The selective JAK2/FLT3 inhibitor 11 (Pacritinib, SB1518) has successfully finished phase 2 clinical trials for myelofibrosis and lymphoma. Another selective JAK2/FLT3 inhibitor, 33 (SB1578), has entered phase 1 clinical development for the non-oncology indication rheumatoid arthritis.

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.

Structure-based design of nitrogen-linked macrocyclic kinase inhibitors leading to the clinical candidate SB1317/TG02, a potent inhibitor of cyclin dependant kinases (CDKs), Janus kinase 2 (JAK2), and Fms-like tyrosine kinase-3 (FLT3)

AbstractA high-throughput screen against Aurora A kinase revealed several promising submicromolar pyrimidine-aniline leads. The bioactive conformation found by docking these leads into the Aurora A ATP-binding site had a semicircular shape. Macrocycle formation was proposed to achieve novelty and selectivity via ring-closing metathesis of a diene precursor. The nature of the optimal linker and its size was directed by docking. In a kinase panel screen, selected macrocycles were active on other kinase targets, mainly FLT3, JAK2, and CDKs. These compounds then became leads in a CDK/FLT3/JAK2 inhibitor project. Macrocycles with a basic nitrogen in the linker form a salt bridge with Asp86 in CDK2 and Asp698 in FLT3. Interaction with this residue explains the observed selectivity. The Asp86 residue is conserved in most CDKs, resulting in potent pan-CDK inhibition by these compounds. Optimized macrocycles generally have good DMPK properties, and are efficacious in mouse models of cancer. Compound 5 (SB1317/TG02), a pan-CDK/FLT3/JAK2 inhibitor, was selected for preclinical development, and is now in phase 1 clinical trials. FigureStructure of SB1317 (left). SB1317 docked into CDK2 (right)

Structure-based optimization of morpholino-triazines as PI3K and mTOR inhibitors

A virtual screen of our in-house database using various fingerprint techniques returned several triazine hits which were found to be mTOR inhibitors with a slight selectivity over PI3Kα. Using structure-guided lead optimization the inhibitory activity towards mTOR and PI3Kα was increased to the low nanomolar range. Exploiting shape differences in the binding-site allowed for the design of mTOR selective inhibitors. Focus on ligand efficiency ensured the inhibitors retained a low molecular weight and desirable drug-like properties.

Structure and ligand-based design of mTOR and PI3-kinase inhibitors leading to the clinical candidates VS-5584 (SB2343) and SB2602.

Phosphoinositide 3-kinases (PI3Ks) and the mammalian target of rapamycin (mTOR) act as critical effectors in a commonly deregulated cell signaling pathway in human cancers. The abnormal activation of the PI3K/mTOR pathway has been shown to play a role in initiation, progression, and metastasis of human tumors. Being one of the most frequently activated pathways in cancer, much effort has been directed toward inhibition of the PI3K/mTOR pathway as a novel oncology therapy. Previous work by a number of groups has revealed several selective PI3K and dual mTOR/PI3K inhibitors. However, there are few reports of therapeutic agents with a pan-PI3K/mTOR inhibitory profile within a narrow concentration range. We therefore initiated a drug discovery project with the aim of discovering dual mTOR/PI3K inhibitors which would equipotently inhibit the 4 isoforms of PI3K, α, β, γ, and δ, and mTOR a compelling profile for powerful blockage of the PI3K/mTOR pathway. A pharmacophore model was generated and used for designing a series of novel compounds, based on a purine scaffold, which potently inhibited mTOR and PI3Ks. These compounds contained a phenol headgroup essential for binding to the target proteins. Early efforts concentrated on finding replacements for the phenol as it was rapidly conjugated resulting in a short half-life in vivo. Compounds with a variety of headgroups were docked into the PI3Kα and mTOR ATP-binding sites, and aminopyrimidine and aminopyrazine were found to make excellent phenol replacements. Further structure guided optimization of side chains in the 8- and 9-positions of the purine resulted in potent inhibitors with good PKDM properties. As the PI3 kinases play a role in insulin signaling, it is believed that targeting mTOR selectively may give the benefit of blocking the AKT-pathway while avoiding the potential side effects associated with PI3K inhibition. As a result we designed a further series of selective mTOR kinase inhibitors. The project was successfully concluded by progressing both a dual mTOR/PI3K inhibitor, SB2343, and a selective mTOR inhibitor, SB2602, into preclinical development. SB2343 has since entered phase 1 clinical development as VS-5584.

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

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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

Abstract 3591: Discovery of SB1317: A novel small molecule macrocycle with a unique kinase inhibitory spectrum in phase 1 clinical trials for hematological malignancies

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: During our quest to find novel kinase inhibitor motifs for our protein kinase research projects we discovered a novel series of macrocyclic structures which possess unique but tunable kinase inhibitory profiles. Here we present the chemical structure of SB1317 and the SAR studies leading to the discovery of this novel CDK/FLT3/JAK2 inhibitor, currently in clinical trials for hematological malignancies. Methods/Results: The unusual structure of SB1317 is characterized by a biaryl connection to the hinge-binding 2-anilinopyrimidine and an allylic tertiary amine – homo allylic ether linker forming an 18-membered macrocyclic ring. Combination of the biaryl motif and unsaturation in the linker confers limited conformational freedom and may explain the specific biological activity observed with this series. Following extensive SAR studies a single preferred compound, SB1317, emerged with potent CDK (IC50 against CDKs 1, 2 and 9 = 9, 5 and 3 nM, respectively), FLT3 (IC50 = 19nM) and JAK2 (IC50 = 19nM) potency. SB1317 has low molecular weight and good physicochemical properties conferring good oral exposure and prompting its further evaluation in vitro and in in vivo cancer models. Following promising efficacy data in these studies, SB1317 was nominated as the preferred candidate for progression to the clinic. SB1317 was synthesized via a ring-closing metathesis (RCM) strategy where the macrocycle was formed from coupling the two halves of the molecule to provide a diene which was then closed by RCM using either Grubbs or Zhan catalysts. Crucially the RCM reaction requires acidic conditions to neutralize the basic centre to allow the metathesis to proceed. Conclusions: SB1317 is a low molecular weight compact macrocyclic structure with a unique spectrum of kinase activity suggesting it may have particular utility in the treatment of hematological malignancies and potential for solid tumor therapy as well. SB1317 is currently being developed in the clinic by Tragara Pharmaceuticals (San Diego, CA) under the name TG02. 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 3591. doi:10.1158/1538-7445.AM2011-3591