Abstract
The salt-inducible kinases (SIKs) SIK1, SIK2, and SIK3 belong to the adenosine monophosphate-activated protein kinase (AMPK) family of serine/threonine kinases. SIK inhibition represents a new therapeutic approach modulating pro-inflammatory and immunoregulatory pathways that holds potential for the treatment of inflammatory diseases. Here, we describe the identification of GLPG3970 (32), a first-in-class dual SIK2/SIK3 inhibitor with selectivity against SIK1 (IC50 of 282.8 nM on SIK1, 7.8 nM on SIK2 and 3.8 nM on SIK3). We outline efforts made to increase selectivity against SIK1 and improve CYP time-dependent inhibition properties through the structure–activity relationship. The dual activity of 32 in modulating the pro-inflammatory cytokine TNFα and the immunoregulatory cytokine IL-10 is demonstrated in vitro in human primary myeloid cells and human whole blood, and in vivo in mice stimulated with lipopolysaccharide. Compound 32 shows dose-dependent activity in disease-relevant mouse pharmacological models.
Introduction
Salt-inducible kinases (SIKs) belong to the adenosine monophosphate-activated protein kinase (AMPK) family of serine/threonine kinases.1 The first member of the SIK family (SIK1) was identified in 1999 when rats fed on a high-salt diet were found to induce a protein kinase in adrenal cortical tissue.2 Later, two other protein kinases with closely related catalytic domains were identified and are now called SIK2 and SIK3. The three SIK isoforms have very closely related kinase domain structures, making identification of selective compounds highly challenging, and are expressed broadly in tissues. The mRNA encoding SIK1 is regulated by multiple stimuli, including high dietary salt intake, adrenocorticotropic hormone signaling, glucagon signaling, circadian rhythms, and membrane depolarization. In contrast, the expression of SIK2 and SIK3 is constitutive in tissues, and little knowledge is available about ligands that increase or decrease their level of expression.
Activity of SIKs is induced by phosphorylation of their activation loop by the liver kinase B1 (LKB1) and is inhibited by cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent phosphorylation leading to increased binding to 14-3-3 proteins (Figure 1). Two important groups of substrates for the SIKs have been identified, namely the cAMP-response element binding protein (CREB)-regulated transcriptional coactivators (CRTCs: CRTC1, CRTC2, and CRTC3) and the Class 2a histone deacetylases (HDACs: HDAC4, HDAC5, HDAC7, and HDAC9). Phosphorylation of CRTC and HDAC family members by SIKs induces their binding to 14-3-3 proteins and retention in the cytosol. The inhibition of SIKs, either by cAMP/PKA pathway-dependent phosphorylation or by direct pharmacological inhibition, allows the dephosphorylation of CRTCs and HDACs, and their respective translocation to the nucleus, where they promote or repress gene transcription, respectively.3
Figure 1.
Signaling pathway of SIKs. (A) SIKs are activated. (B) SIKs are inactivated. Abbreviations: cAMP, cyclic adenosine monophosphate; CREB, cAMP-response element binding protein; CRTC, CREB-regulated transcription coactivators; GPCR, G protein coupled receptor; HDAC, histone deacetylases; IL-10, interleukin-10; IL-12, interleukin-12; IL-1R, interleukin-1 receptor; LKB1, liver kinase B1; NFκB, nuclear factor kappa B; PKA, protein kinase A; SIK, salt-inducible kinases; TLR, Toll-like receptor; TNFα, tumor necrosis factor alpha.
The effect of SIK inhibition on immune responses has been explored in innate and adaptive immune cells. Investigation of pan-SIK inhibitors in mouse bone marrow-derived dendritic cells (BMDCs) stimulated with zymosan has demonstrated increased production of the immunoregulatory cytokines interleukin-10 (IL-10) associated with decreased secretion of the pro-inflammatory cytokines tumor necrosis factor alpha (TNFα), IL-12, IL-1β, and IL-6.4 Furthermore, SIK inhibition in human macrophages stimulated with lipopolysaccharide (LPS) or IL-1β markedly reduced the phosphorylation of CRTC3 and HDAC4 substrates, resulting in modulation of pro- and anti-inflammatory cytokines.5 SIK inhibition during mouse macrophage differentiation induced a phenotypic switch to regulatory macrophages characterized by a reduced capacity to produce pro-inflammatory cytokines coupled with enhanced IL-10 production upon stimulation with LPS.6
SIK inhibition represents a promising novel approach for the treatment of inflammatory diseases, specifically for those that are characterized by an imbalance of the immune system such as inflammatory bowel disease (IBD), where a dysregulation of pro- and anti-inflammatory pathways in intestinal tissues defines the disease pathogenesis.7
The enzymatic activity of endogenous SIK isoforms was determined following immunoprecipitation from mouse fetal liver-derived macrophages (FLDMs) and bone marrow-derived macrophages (BMDMs); SIK2 showed higher enzymatic activity than SIK3, and SIK1 had the lowest contribution. Investigation of the role of SIK isoforms using catalytically inactive mutants and combination with pan-SIK inhibitors led to the conclusion that all three isoforms, in particular SIK2 and SIK3, contribute toward the overall phenotype of macrophages. SIK2 and SIK3 were also found to be essential for the secretion of inflammatory mediators in mouse bone marrow-derived mast cells (BMMCs) stimulated with IL-33.8 Exploration of the role of SIK isoforms on T cell development in mice suggested that SIK2 and SIK3 are the major functional isoforms in T cells, with combined knockout of SIK2 and SIK3 resulting in a severe block in thymic T cell development, consistent with a defect in the positive or negative selection of double-positive thymocytes.9 Consequently, SIK1 appears to have a minor contribution to the activity in immune cells. In contrast, SIK1 was reported to play a key role in blood pressure regulation and vascular remodeling.10−13 Dual SIK2/SIK3 inhibition with selectivity against SIK1 could therefore offer a better safety profile with a lower impact on the cardiovascular system than pan-SIK inhibition, while retaining the desired activity on the immune system.
An increasing number of chemotypes inhibiting the kinase activity of SIKs have been described in the literature. Examples of small-molecule SIK inhibitors are shown in Figure 2, and their associated potency on SIKs is reported in Table 1. Improvement of selectivity against other kinases and between SIK isoforms is a key objective in the development of new SIK inhibitors to provide tools for interrogating the pharmacology of SIKs. We previously disclosed the identification of the potent and selective pan-SIK inhibitor GLPG3312 (7), a type 1 kinase inhibitor, and the first human SIK3 crystal structure.14 In parallel to the efforts that led to GLPG3312, we took advantage of the knowledge gained from the crystal structure of SIK3 to explore the possibility to achieve potent dual SIK2 and SIK3 inhibition with selectivity against SIK1 to avoid SIK1-related side effects mentioned above.
Figure 2.
Structures of selected small-molecule SIK inhibitors described in the literature and of GLPG3970 (32).4,5,14−20
Table 1. Reported SIK Inhibitory Activity of Compounds from Figure 2.
Here, we describe the discovery of GLPG3970, a first-in-class dual SIK2/SIK3 inhibitor evaluated in clinical trials for the treatment of autoimmune and inflammatory diseases. Efforts to increase selectivity against SIK1 and improve CYP time-dependent inhibition (TDI) properties through modulation of structural properties will be described. The dual mechanism of GLPG3970 in modulating pro- and anti-inflammatory cytokines is demonstrated in immune-mediated in vitro and in vivo models.
Results and Discussion
Strategy and Hit Identification
We previously described the identification of a 4-(5-substituted-benzimidazol-1-yl)benzamide chemical series displaying pan-SIK inhibition following an HTS campaign.14 The first crystal structure of SIK3 in complex with compound 8 (Table 2) was elucidated, allowing a better understanding of the binding mode and of the selectivity of the chemical series.
Table 2. Compounds in Cocrystal Structure and Early Structure–Activity Relationship.
Sequence alignment of the three SIK isoforms showed that there are only five non-conserved residues in the ATP-binding site, pointing at the high similarity between isoforms (Figure 3). Sequence alignment and visual inspection of the ligand binding site in the X-ray structure were used to predict how different residues could have an impact on SIK selectivity (Figure 4). Most of the residues differing between the isoforms are located close to the hinge and in the top part of the pocket, while the residues in the rest of the binding site are all conserved.
Figure 3.
Sequence alignment between the kinase domain of SIK3 (top), SIK2 (middle), and SIK1 (bottom). The residues that form the different binding site regions are highlighted in different colors: P-loop in green, catalytic center in orange, hinge in red, bottom part in blue, and DFG motif in yellow. Additionally, non-conserved residues in these regions are indicated in black bold, showing the high similarity between the binding sites of isoforms.
Figure 4.
X-ray structure of SIK3 in complex with 8 (PDB: 8OKU). Non-conserved residues between SIK1, SIK2, and SIK3 that are located close to the binding site are displayed in sticks.
Structure–activity relationship (SAR) optimization described previously led to the identification of the alkyl pyrazole and second methoxy group on the phenyl ring as important features for high potency on SIKs. As can be seen in Figure 4, residues are very similar between isoforms around the dimethoxybenzamide moiety of compound 8. In contrast, different residues between SIK isoforms are present around the pyrazole substituent in position 5 of the benzimidazole scaffold of compound 8. In particular, a tyrosine residue in SIK2 (Tyr98) and SIK3 (Tyr144) is replaced by a phenylalanine residue in SIK1 (Phe105). The pyrazole moiety in compound 8 is coplanar with the benzimidazole scaffold allowing a displaced π–π interaction with tyrosine or phenylalanine and a weak hydrogen bond between the slightly polarized C–H group of the pyrazole and the carbonyl moiety of alanine in the hinge, which resulted in pan-SIK inhibition. We hypothesized that exploration of structurally diverse substituents in position 5 of the benzimidazole scaffold could allow escape from flatland, achieving selectivity against SIK1 through interactions with the side chain of residues in an area of the active site where non-conserved residues between isoforms are present. For example, H-bond interaction with the tyrosine residue in SIK2 (Tyr98) and SIK3 (Tyr144) would not be feasible with the phenylalanine residue in SIK1 (Phe105). The possibility that more distal, non-conserved residues could lead to subtle differences in the conformation of the active site between isoforms and impact the binding of compounds was not excluded and could result in isoform selectivity. Therefore, a broad exploration including modification of substituents and scaffold was envisaged.
Our goal was to achieve single-digit nanomolar potency on SIK2 and SIK3 and at least 30-fold selectivity against SIK1. We decided to start from a moderately active hit compound on SIK isoforms, trying to improve potency on SIK2 and SIK3 only.
As shown in Table 2, replacement of the ethyl pyrazole moiety in position 5 of the benzimidazole scaffold in compound 9 by a non-aromatic morpholine substituent (10) led to at least a 25-fold drop of activity on SIKs. Interestingly, hit compound 10 displayed a trend of selectivity against SIK1.
SAR Exploration
Starting from the moderately active hit compound 10, scaffold hopping, replacement of morpholine, and modification of benzamide moiety were explored to improve potency on SIKs and selectivity against SIK1. Unexpectedly, scaffold hopping from benzimidazole in 10 to imidazo[1,2-a]pyridine in 11 resulted in increased potency on SIK2 and SIK3, and improved selectivity against SIK1 (Table 3). The bottom part of the molecule was also modified, as previously reported,14 by replacing one of the methyl groups on the dimethoxyphenyl portion with a difluoromethyl group and replacing the trifluoroethyl group on the carboxamide moiety with a cyclopropyl group to produce analog 12. These changes further increased potency on SIK2 but also on SIK1. Analogs with other amine substituents on the imidazo[1,2-a]pyridine scaffold were tested pairwise using the bottom parts of molecules 11 and 12. The substituents examined were difluoroazetidine (13 and 14) and N-methylpiperazine (15 and 16), which showed improved potency, in particular on SIK2, for the bottom part containing the difluoromethoxy group and cyclopropyl amide (Table 3). The difluoroazetidine derivative 14 exhibited greater inhibition of SIK1, SIK2, and SIK3 compared with analog 13, as was similarly observed for the N-methylpiperazine derivatives 16 and 15.
Table 3. Matched Pairs SAR on Bottom Parta.
Abbreviations: ND, not determined; TDI, time-dependent inhibition.
Despite potent activity on SIK2 and SIK3 and encouraging selectivity against SIK1, progression of N-methylpiperazine derivative 16 was stopped owing to IC50 shift in a CYP TDI assay in human liver microsomes. This property is undesirable because it may lead to drug–drug interaction (DDI) or idiosyncratic drug toxicity.21,22
Further exploration of the amine substituent in position 7 of the imidazo[1,2-a]pyridine scaffold to increase selectivity against SIK1 was carried out with cyclopropylamide bottom part as summarized in Table 4.
Table 4. SAR Exploration in Position 7 of the Imidazo[1,2-a]pyridine Scaffolda.
Abbreviations: ND, not determined; TDI, time-dependent inhibition.
Replacement of the N-methyl basic center from 16 with a polar non-basic acetamide group in 17 retained comparable potency on SIKs (Table 4), as did extending the chain on the piperazine nitrogen with a hydroxyethyl group in 18.
Introduction of a basic dimethyl amino group on position 4 of a piperidine ring substituent (19) maintained potency on SIK2 and SIK3, but it reduced the selectivity against SIK1. In contrast, analog 20 with a dimethyl amino group on an azetidine substituent retained comparable potency and selectivity as a methyl piperazine derivative 16. These data suggested the position of the basic group was important for selectivity against SIK1. However, compound 20 turned out to be positive in the CYP TDI assay and so was not further progressed.
Substitution of the imidazo[1,2-a]pyridine scaffold by an aniline moiety in 21 or a heteroaromatic amine in 22 led to potent activity on SIKs with no isoform selectivity. Compounds 23 and 24 bearing an amide substituent could maintain potent activity on SIK3 with selectivity against SIK1 but resulted in a drop of activity on SIK2.
Incorporating a flexible acyclic linker between the imidazo[1,2-a]pyridine scaffold and the basic group was tolerated, as illustrated with compound 25 bearing a 2-morpholinoethylamine substituent, which displayed low nanomolar potency on SIK2 and SIK3, and some selectivity against SIK1. Replacing the nitrogen in the linker by oxygen in the ether analog 26 led to single-digit nanomolar activity on SIK2 and SIK3, and 10-fold/30-fold selectivity against SIK1, respectively. Similarly, replacing the morpholine moiety by a tetrahydropyran in derivative 27 led to a drop of selectivity against SIK1 to 4-fold/10-fold.
An important observation was made upon assessment in the CYP TDI assay; although the N-linked derivative 25 was positive in the CYP TDI assay, the O-linked analog 26 was negative, and the tetrahydropyran analog 27 was positive. Although the mechanism that led to CYP TDI was not investigated, we speculated that the imidazo[1,2-a]pyridine scaffold could be involved, as it is known to be a structural alert for risk of formation of reactive metabolites and occurrence of CYP TDI.23−25 Strategies to remove CYP TDI within a chemical series include the modification of physicochemical properties, the modulation of stereoelectronic properties of substituents, and the introduction of soft spots to redirect metabolism to another site.26 We hypothesized that the absence of CYP TDI for O-linked compound 26 resulted from a decreased electron density in the imidazo[1,2-a]pyridine ring system compared with the N-linked substituent in compound 25. Data also suggest that the increase of lipophilicity in compound 27 (CLogP = 4.4) compared with 26 (CLogP = 3.8) restored CYP TDI activity. Since the ether linker could improve DDI properties, it was kept for further investigations, and lipophilicity of compounds was monitored.
Although residues are well conserved between SIK isoforms in the active site around the dimethoxybenzamide moiety, the possibility that more distal, non-conserved residues could lead to subtle differences in the conformation of the active site between isoforms and contribute to isoform selectivity was not excluded. Therefore, we explored the impact of modifying the benzamide part of ether derivative 26 on potency on SIKs and selectivity against SIK1 (Table 5). Replacing the phenyl ring with a pyridine ring which could form an intramolecular hydrogen bond was found to be detrimental to potency. Compounds 28 and 29 with a cyclopropyl amide and compound 30 with a trifluoroethyl amide displayed IC50 higher than 50 nM on the three SIK isoforms with some selectivity against SIK1 (Table 5). Cyclization of the amide with one of the meta positions to form a lactam ring substituted by a cyclopropyl group in derivative 31 led to an improvement of potency and selectivity compared with pyridine analog 28. Finally, introduction of a trifluoroethyl group instead of a cyclopropyl group on the lactam ring gave GLPG3970 (32) which displayed single digit nanomolar potency on SIK2 and SIK3 with IC50 of 7.8 and 3.8 nM, and 36-fold/74-fold selectivity against SIK1, respectively (IC50 of 282.8 nM). Compound 32 displayed comparable lipophilicity to compound 26 and was negative in the CYP TDI assay.
Table 5. Bottom Part Exploration with Ether Substitution in Position 7 of the Imidazo[1,2-a]pyridine Scaffold.
In summary, consideration of the cocrystal structure of compound 8 in SIK3 and the non-conserved residues in the different SIK isoforms around the pyrazole group bound to the benzimidazole scaffold prompted us to investigate structural modifications in this area to achieve selectivity against SIK1. Exploration started from hit compound 10 bearing a morpholine substituent on the benzimidazole scaffold instead of a pyrazole group, which displayed moderate activity on SIKs. Scaffold hopping from benzimidazole to imidazo[1,2-a]pyridine and changing the benzamide moiety, as in our previous investigation,14 boosted potency on SIKs. Replacing the morpholine substituent by an N-linked cyclic or acyclic substituent bearing a basic group could display potent activity on SIK2 and SIK3, and improve selectivity against SIK1. Oxygen-linked substitution as in analog 26 improved CYP TDI properties and displayed potent activity on SIK2 and SIK3, but better selectivity against SIK1 was desired. Finally, cyclization of the benzamide moiety into a lactam alkylated by a trifluoroethyl group led to ether analog 32 (GLPG3970), which displayed the desired levels of potency on SIKs and of selectivity against SIK1, and was negative in the CYP TDI assay.
Molecular Modeling
In order to understand the selectivity of compound 32, docking and molecular dynamics simulations were carried out in SIK3. Compound 32 binds in the ATP site with the protein, adopting an active-like conformation as observed for compound 8, and as such can be classed as a type 1 kinase inhibitor. In the simulations, the morpholine ring of 32 was occupying the solvent-exposed area while being able to maintain an H-bond interaction with Tyr144 (Figure 5). As shown in Figure 4, this tyrosine residue is conserved in SIK2 but not in SIK1, suggesting that interacting with this residue could favor a dual SIK2/SIK3 selectivity profile. In the molecular dynamics simulations of compound 32, the carbonyl of the lactam was able to establish an H-bond interaction with the catalytic Lys95 (Figure 5). Considering that all the residues are conserved in the region where the lactam ring is hypothesized to be, molecular modeling investigations using homology modeling, docking, and molecular dynamics simulation did not provide an unambiguous rationale as to why the lactam ring is better tolerated in SIK2 and SIK3 than in SIK1.
Figure 5.
Representative frame from molecular dynamics simulations of compound 32 (orange sticks) in SIK3 (shown in white ribbons and gray carbons). The morpholine ring is facing Tyr144, present in SIK3 and SIK2. H-bond interactions are highlighted in yellow.
Chemistry
Benzimidazole derivative 10 was prepared from intermediate 33 described previously14 via a palladium-catalyzed Buchwald–Hartwig cross-coupling reaction with morpholine (Scheme 1). The preparation of imidazo[1,2-a]pyridine analogs 11–32 used 7-substituted or 3,7-disubstituted imidazo[1,2-a]pyridine building blocks and palladium-catalyzed cross-coupling, C–H insertion, amide coupling, or aromatic nucleophilic substitution reactions to introduce the desired substituents on positions 3 and 7 of the scaffold.
Scheme 1. Preparation of Compound 10.
Reagents and conditions: (a) morpholine, Pd2dba3, tBuXPhos, tBuOK, dioxane, 110 °C, overnight, 17%.
The preparation of imidazo[1,2-a]pyridine analogs with 2,6-dimethoxy-N-(2,2,2-trifluoroethyl)benzamide moiety started with a Suzuki cross-coupling reaction between 7-bromo-3-iodo-imidazo[1,2-a]pyridine 34 and commercially available boronate 35 to obtain ester 36 (Scheme 2). Saponification followed by amide coupling with 2,2,2-trifluoroethylamine led to amide 38. The Buchwald–Hartwig cross-coupling reaction with morpholine, 3,3-difluoroazetidine, and N-methylpiperazine afforded compounds 11, 13, and 15, respectively.
Scheme 2. Preparation of Compounds 11, 13, and 15.
Reagents and conditions: (a) Pd(dppf)Cl2, Cs2CO3, dioxane, water 90 °C, 1 h, 68%; (b) NaOH 2N, THF, MeOH, 70 °C, 18 h, 96%; (c) HATU, DIPEA, 2,2,2-trifluoroethylamine, DMF, rt, 18 h, 57%; (d) amine, RuPhos Pd G3, RuPhos, Cs2CO3, dioxane, 90 °C, 5 h to overnight, 36–83%.
The exploration of analogs bearing a difluoromethoxy group on the phenyl ring required the preparation of the corresponding pinacol boronate (Scheme 3). Commercially available 2-hydroxy-6-methoxybenzoic acid 39 was transformed into the corresponding N-cyclopropyl-benzamide 40 via amide coupling. Difluoromethylation of the phenol group was performed using bromodifluoromethyl diethylphosphonate27 to generate the difluoromethoxy intermediate 41. Direct and regioselective C–H borylation in the para position of the carboxamide moiety afforded the pinacol boronate building block 42,28 which was engaged in Suzuki–Miyaura coupling with 3-halo-imidazo[1,2-a]pyridine scaffolds to obtain intermediates 43a–c. Intermediate 43b was reacted with 3-methoxypropionic acid activated with Ghosez’s reagent29 to give compound 24. Nucleophilic aromatic substitution (SNAr) on fluoro derivative 43c with the anion of 2-morpholinoethanol or 2-tetrahydropyran-4-ylethanol led to ether analogs 26 and 27, respectively. Bromo intermediate 43a was engaged in Buchwald–Hartwig cross-coupling reactions with amines, 3-morpholinoaniline, or 1-methyl-1H-imidazol-4-amine, to obtain compounds 12, 14, 16–23, and 25.
Scheme 3. Preparation of Compounds 12, 14, 16–27.
Reagents and conditions: (a) cyclopropylamine, HATU, DIPEA, DMF, rt, 3 d, 28%; (b) bromodifluoromethyl diethylphosphonate, KOH, ACN/water, −20 °C then rt, 1 h, 79%; (c) bis(pinacolato)diboron, [Ir(OCH3)(COD)]2, dtbpy, THF, 70 °C then rt, overnight, 49%; (d) 7-bromo-3-iodo-imidazo[1,2-a]pyridine (34), Pd(PPh3)4, Cs2CO3, dioxane, water, 90 °C, overnight, 96% (43a); (e) 3-bromoimidazo[1,2-a]pyridin-7-amine or 7-fluoro-3-iodoimidazo[1,2-a]pyridine, Pd(dppf)Cl2, Cs2CO3 or KOAc, dioxane/water or DMAC, 90 °C, 1 or 4 h, 65% (43b), 77% (43c); (f) 3-methoxypropionic acid, Ghosez’s reagent, DCM, rt, 4 d, 38%; (g) 2-morpholinoethanol or 2-tetrahydropyran-4-ylethanol, NaH, DMF, rt, overnight, 60–74%; (h) amine, RuPhos, RuPhos Pd G3, Cs2CO3, dioxane, 90 °C, overnight, 14–48%; (i) amine, RuPhos, RuPhos Pd G2, K3PO4, dioxane, 90 °C, 7 h, 19–24%; (j) 2-morpholinoethan-1-amine, 1-methyl-1H-imidazol-4-amine or 3-morpholinoaniline, Pd2(dba)3, XantPhos, Cs2CO3, dioxane, 90 °C, 2 h to overnight, 25–43%; (k) tetrahydropyran-4-carboxamide, Pd(OAc)2, Xantphos, dioxane, 150 °C (microwave irradiation), 1 h, 45% (23).
In order to explore replacement of the benzamide part by heteroaryl groups, intermediate 45 was prepared by SNAr on 7-fluoro-imidazo[1,2-a]pyridine 44 with the anion of 2-morpholinoethanol (Scheme 4). 5-Bromo-picolinamide intermediates 47a–c were prepared from picolinic acid reagents 46 and 50 via amide coupling with the corresponding amines. Picolinic acid reagent 50 was synthesized in 2 steps from commercially available methyl 5-bromo-3-hydroxypicolinate 48 by difluoromethylation (49) followed by saponification. Imidazo[1,2-a]pyridine intermediate 45 was reacted with 5-bromo-picolinamide intermediates 47a–c using a palladium-catalyzed direct C–H arylation30 to deliver 28–30.
Scheme 4. Preparation of Compounds 28–30.
Reagents and conditions: (a) 2-morpholinoethanol, NaH, DMF, rt, 20 h, 100%; (b) amine, HATU, DIPEA, DMF, rt, 17–20 h, 54–83%; (c) KOAc, Pd(dppf)Cl2·DCM, DMAC, 110 °C, 2 h to overnight, 23–55%; (d) K2CO3, sodium chlorodifluoroacetate, acetonitrile, reflux, 2 h, 56%; (e) LiOH, THF, water, rt, 2 h, 85%.
Introduction of 3,4-dihydroisoquinolin-1(2H)-one moiety started from commercially available building blocks 51 and 54 (Scheme 5). First, a Chan–Lam coupling31 on 51 was performed to install a cyclopropyl ring (52), then SNAr reaction with sodium methoxylate led to intermediate 53a. The 2,2,2-trifluoroethyl chain of 53b was introduced on commercially available 54 by N-alkylation of the lactam ring using 2,2,2-trifluoroethyl trifluoro-methanesulfonate. Palladium-catalyzed direct C–H arylation on 7-fluoroimidazo[1,2-a]pyridine with 53a,b led to intermediates 55a,b which underwent SNAr with the anion of 2-morpholinoethanol to give compounds 31 and 32.
Scheme 5. Preparation of Compounds 31 and 32.
Reagents and conditions: (a) cyclopropylboronic acid, Cu(OAc)2, TEA, pyridine, THF, 70 °C, 18 h, 36%; (b) MeONa, MeOH, THF, rt, 1.5 h, 77%; (c) 2,2,2-trifluoroethyl trifluoromethanesulfonate, LiHMDS, THF, 0 °C to rt, 22 h, 76%; (d) 7-fluoroimidazo[1,2-a]pyridine (44), KOAc, Pd(dppf)Cl2·DCM, DMAC, 110 °C, 2 h, 77–93%; (e) 2-morpholinoethanol, NaH, DMF, rt, 5 h, 47–90%.
Kinase Selectivity Profiling
In addition to potent dual SIK2/SIK3 inhibition and selectivity against SIK1, we aimed to identify a compound with good kinome selectivity to explore the therapeutic potential of dual SIK2 and SIK3 inhibition only. The inhibition of enzymatic activity by compound 32 at a concentration of 1 μM was assessed against a panel of 372 kinases and is represented in Figure 6. Apart from SIKs, 32 had a percentage of inhibition (PIN) of kinase activity above 50% on only three other kinases: RIPK2 (79%), ABL1 (58%), and MKNK2 (54%). Further characterization led to IC50 values of 78.4 nM (RIPK2), 1,095 nM (ABL1), and 1,074 nM (MKNK2). 32 shows 10-fold/20-fold selectivity against RIPK2 based on potency in biochemical assays on SIK2 and SIK3, respectively. Since RIPK2 has been explored for the treatment of inflammatory diseases including IBD, RIPK2 inhibition was not considered as incompatible with further progression of 32.32,33
Figure 6.
Kinome tree of 32 at 1 μM.
In summary, profiling of compound 32 against a panel of 372 kinases at 1 μM showed excellent selectivity, with RIPK2 identified as the only off-target with an IC50 below 1 μM. Interestingly RIPK2 is also the main off-target for GLPG3312.14 Compound 32 is approximately 10-fold more potent on SIK2 and 20-fold more potent on SIK3 than on RIPK2. Compound 32 is, therefore, a highly selective dual SIK2/SIK3 inhibitor.
In Vitro Pharmacology Profiling of 32
Activity of 32 was explored in cellular assays to confirm the isoforms’ selectivity, monitor activity on target substrate, and assess phenotypic activity on immune cells.
SIK isoform selectivity of 32 in the cellular context was determined using NanoBRET binding assays in HEK293 cells and resulted in IC50 values of >17,500 nM, 254 nM, and 79 nM on SIK1, SIK2, and SIK3, respectively (Table 6). The NanoBRET kinase assays confirmed 32 to be a potent SIK2 and SIK3 binder highly selective against SIK1.
Table 6. Activity of 32 in Target-Based and Phenotypic Cellular Assays.
| Target-Based Cellular Assays | ||
|---|---|---|
| Assay Model | IC50/EC50 (nM) | |
| NanoBRET assay (HEK293 cells) | SIK1 | >17,500 |
| SIK2 | 254 | |
| SIK3 | 79 | |
| CRTC3 translocation assay (U2OS cells) | 1,703 | |
| Phenotypic Cellular and Whole Blood Assays | |||
|---|---|---|---|
| Assay Model | Trigger/Time | Cytokine Inhibition IC50 (nM)a | Maximum Fold Change Increase of IL-10b |
| Human monocytes | LPS/4 h | TNFα: 231 (5) | 13.8 ± 1.9 (4) |
| Human monocytes | LPS/20 h | IL-12: 67 (5) | ND |
| Human monocyte-derived macrophages | LPS/20 h | TNFα: 365 (6) | ND |
| LPS/2 h | ND | 2.7 ± 0.4 (5) | |
| Human whole blood | LPS/2 h | TNFα: 1,000 (52) | 5.83 ± 0.32 (52) |
Number of replicates/donors for each readout indicated in parentheses.
Compared with LPS stimulation with no compound treatment at the same time point. Abbreviations: IL-12, interleukin-12; ND, not determined; U2OS, human bone osteosarcoma cells.
SIK inhibition has demonstrated the capacity to skew pro-inflammatory macrophages to an immunoregulatory phenotype with a mechanism that involves phosphorylation of CRTC3 by SIKs.15 As shown in Figure 1, inhibition of SIKs leads to dephosphorylation of CRTC3 and subsequent translocation to the nucleus. Compound 32 induced a concentration-dependent nuclear translocation of CRTC3 in U2OS cells with an EC50 of 1,703 nM, as measured by high-content imaging technology (Table 6). The anti-inflammatory and immunoregulatory activities of 32 were examined in phenotypic assays using human primary myeloid cell types and whole blood from healthy donors stimulated with LPS. In LPS-stimulated monocytes, 32 suppressed TNFα and IL-12 at IC50s of 231 and 67 nM, respectively. In monocyte-derived macrophages (MdMs), 32 inhibited TNFα at an IC50 of 365 nM. In both assays, 32 dose-dependently increased the production of IL-10 (Table 6, Figure 7). Data on IL-10 are expressed as fold-induction versus LPS trigger at the top concentration of 20 μM evaluated in the assay as the clear dose response obtained did not however allow accurate curve fitting on IL-10 induction for robust EC50 determination across different experiments. In human whole blood from healthy donors stimulated with LPS, compound 32 confirms its dual activity inhibiting TNFα production with an IC50 of 1,000 nM coupled with a dose-dependent IL-10 increase (Table 6, Figure 7).
Figure 7.
Representative graphs for inhibition of TNFα and induction of IL-10 by 32 in LPS-stimulated human monocytes (A), human monocyte-differentiated macrophages (B), and human whole blood (C). Data are represented as mean values of percentage of inhibition of TNFα and fold change increase of IL-10 levels. Abbreviations: IL, interleukin; LPS, lipopolysaccharide; TNFα, tumor necrosis factor alpha.
Overall, compound 32 displayed high selectivity against SIK1 in cellular context. Nuclear translocation of CRTC3 was observed in the presence of 32, indicating SIK1 is dispensable for this activity. Compound 32 inhibited the production of TNFα and IL-12, and increased the release of IL-10 by primary human myeloid cells and in human whole blood stimulated by LPS. Dual SIK2/SIK3 inhibition as with compound 32 therefore leads to both anti-inflammatory and immunoregulatory properties in vitro.
Pharmacokinetic Properties of 32
Pharmacokinetic properties of 32 were evaluated in mice, rats, and dogs. Mice models were used to assess the in vivo pharmacological activity of 32. Rats and dogs are the preferred species for in vivo toxicology investigations in preclinical development, and pharmacokinetic properties from several preclinical species are generally used to make a prediction of human pharmacokinetic properties. Pharmacokinetic properties of 32 were evaluated in male mice, rats, and dogs with doses of 1 mg/kg and 5 mg/kg administered iv and po, respectively (Table 7). Following iv dosing in CD1 mice, 32 was characterized by moderate total and low unbound plasma clearances, a large steady-state volume of distribution, and a short apparent terminal half-life. Absolute oral bioavailability of 68.7% was observed in mice. In Sprague–Dawley rats, 32 was characterized by moderate total blood clearance, low unbound plasma clearance, large steady-state volume of distribution, and moderate apparent terminal half-life. Absolute oral bioavailability was 55.9% in rats. Following iv administration in beagle dogs, 32 was characterized by moderate total blood clearance, low unbound plasma clearance, large steady-state volume of distribution, and long apparent terminal half-life. Following po administration at 5 mg/kg in beagle dogs, absolute oral bioavailability was 41.0%.
Table 7. Pharmacokinetic Properties of 32a.
| Species |
|||
|---|---|---|---|
| Mouse | Rat | Dog | |
| Strain | CD1 | Sprague–Dawley | Beagle |
| Doses (mg/kg) | 1 iv/5 po | 1 iv/5 po | 1 iv/5 po |
| CLb (L/h/kg) | 2.48b | 1.89 (21) | 0.767 (26) |
| CLu (L/h/kg)c | 5.57 | 7.64 | 1.57 |
| Vss (L/kg) | 2.51 | 2.82 (27) | 2.89 (22) |
| Half-life, iv (h) | 0.555 | 1.42 (8.1) | 4.28 (11) |
| Half-life, po (h) | 1.28 (34) | 3.61 (31) | 4.63 (26) |
| AUC0–∞, iv (ng·h/mL) | 402 | 759 (21) | 897 (7.0) |
| AUC0–∞, po (ng·h/mL) | 1,380 (54) | 2,120 (45) | 1,840 (33) |
| F (%) | 68.7 | 55.9 | 41.0 |
Mean values; % coefficient of variation indicated in parentheses.
Assuming blood-to-plasma ratio equals 1.
Fraction unbound in plasma is 0.445, 0.246, and 0.507 in mouse, rat, and dog, respectively. Abbreviations: CLb, blood clearance; CLu, unbound plasma clearance; Vss, apparent volume of distribution at steady state.
Overall, 32 displayed moderate total clearance, low unbound plasma clearance, and moderate to high oral bioavailability across species. The pharmacokinetic properties of 32 made it suitable for further development.
In Vivo Pharmacology Activity of 32
For in vivo translation of the observed in vitro effect on TNFα and IL-10, the activity of 32 was explored in an acute LPS challenge model in mice. In this model, stimulation by LPS triggers an immune response with increased levels of TNFα and IL-10 circulating in blood. LPS was injected intraperitoneally 15 min after oral administration of 32 at doses of 1, 3, 5, 10, 30, and 60 mg/kg or corresponding vehicle (Figure 8A). Blood was collected 1.5 h post LPS stimulation, and levels of TNFα and IL-10 in plasma were quantified. As shown in Figure 8, compound 32 dose-dependently reduced the release of TNFα with significant inhibition higher than 85% at doses of 10, 30, and 60 mg/kg compared with vehicle group in mice stimulated with LPS, despite high inter-individual variability seen in the vehicle group (Table 8). Analysis of the exposure–response curve for 32 led to an IC50 of 705 nM on TNFα production. Compound 32 also dose-dependently increased IL-10 levels with a significant induction of 3.0-fold and above, starting at the dose of 3 mg/kg onward, relative to vehicle in mice stimulated with LPS. No EC50 was determined on IL-10 as the exposure–response curve did not reach a plateau.
Figure 8.
Plasma levels of TNFα and IL-10 after oral administration of 32 and in vivo LPS challenge in mice. (A) Study schematic illustrating administration of 32 (n = 6 mice/group) followed by administration of LPS before collecting plasma for exposure of 32 and cytokine levels. Data are presented as mean value levels of TNFα (B) and IL-10 (C) ± SEM for each group. Statistical analysis of plasma TNFα levels versus LPS + vehicle was performed with a Kruskal–Wallis and Dunn’s post-test: *p < 0.05; ***p < 0.001. Statistical significance of plasma IL-10 levels was calculated using ANOVA and Dunnett’s multiple comparison test: ***p < 0.001. Exposure–response curves for TNFα (D) and IL-10 (E) are shown using mean values for concentrations (ng/mL) and cytokine levels (±SEM). Abbreviations: ANOVA, analysis of variance; IL-10, interleukin-10.
Table 8. Plasma Exposure of 32 and Levels of TNFα and IL-10 in LPS Challenge in Micea.
| compound 32 dose | Cplasma at 1.75 h (nM) | TNFα (pg/mL) | % inhibition TNFα vs LPS + vehicle | IL-10 (pg/mL) | fold-induction IL-10 vs LPS + vehicle |
|---|---|---|---|---|---|
| 1 mg/kg po | 129 ± 21 | 1,981 ± 426 | 44 ± 12 | 1,217 ± 149 | 1.3 ± 0.2 |
| 3 mg/kg po | 705 ± 63 | 1,328 ± 220 | 63 ± 6 | 2,856 ± 226 | 3.0 ± 0.2 |
| 5 mg/kg po | 1,216 ± 107 | 821 ± 96 | 77 ± 3 | 3,596 ± 628 | 3.8 ± 0.7 |
| 10 mg/kg po | 2,729 ± 186 | 562 ± 107 | 85 ± 3 | 5,814 ± 767 | 6.2 ± 0.8 |
| 30 mg/kg po | 13,871 ± 1,235 | 438 ± 30 | 88 ± 1 | 9,198 ± 997 | 9.8 ± 1.1 |
| 60 mg/kg po | 17,578 ± 1,126 | 473 ± 23 | 87 ± 1 | 14,457 ± 626 | 15.3 ± 0.7 |
Mean values with standard error of mean (SEM).
Efficacy of 32 in the Murine DSS-Induced Colitis Model
In vivo efficacy of compound 32 was tested in a mouse DSS-induced colitis model to evaluate its therapeutic potential for IBD.34 In this model, ulcerative colitis-like inflammation is induced by 2 cycles of 4 days 4% DSS in drinking water (Figure 9A). Disease evolution is recorded as disease activity index (DAI), a composite score of body weight (BW) loss, stool consistency, and fecal blood over a course of 12 days. Efficacy was further assessed by histological examination of colonic tissues collected at study termination using a mouse colitis histology index (MCHI) score. MCHI is a composite scoring system of eight histological components (inflammatory infiltrate, goblet cell loss, crypt hyperplasia, crypt density, muscle thickness, submucosal infiltration, ulcerations, and crypt abscesses), categorized from 0 to 3 and summed into a total MCHI score. The MCHIs were optimized as valid and reliable measures of intestinal inflammation in mice, responsive to treatment effects in preclinical studies, and relevant to clinical IBD where histopathologic indices are routinely used as outcome measures in controlled clinical trials.35 Activity of 32 was assessed by prophylactic treatment at doses of 3, 10, and 30 mg/kg (twice daily, po). As seen for vehicle groups, the DSS treatment significantly increased the area under the curve (AUC) of DAI score (sum of scores for BW loss, stool consistency, and fecal blood), reflecting active colitis. In this study, oral treatment with 32 at 3, 10, or 30 mg/kg twice daily reduced dose-dependently and significantly the AUC of DAI score by 29, 33, and 59%, respectively, compared with mice treated with vehicle only (Figure 9B). In addition to reduced DAI score, treatment with 32 dose-dependently and significantly reduced histological end points as recorded by the MCHI (Figure 9C). Representative images of periodic acid–Schiff (PAS) staining visualizing mucin (in pink) produced by goblet cells and hematoxylin counter-staining for mucosal leukocyte infiltration are shown in Figure 9D. Oral treatment with 32 at 30 and 10 mg/kg twice daily clearly demonstrated prevention of goblet cell loss, reduced inflammatory infiltrates, decreased crypt density, and reduced mucosal erosion.
Figure 9.
Activity of compound 32 in the DSS-induced colitis model. (A) Schematic setup of the mouse DSS-induced colitis model. (B) Activity of 32 on the AUC of DAI score (composite score of body weight loss, stool consistency, and fecal blood). Data are presented as mean values ± SEM (n = 10 mice/group). Statistical analysis of Log(Y) AUC DAI data transformation without SHAM – vehicle group was calculated using one-way ANOVA and Dunnett’s post-test analysis vs DSS 4% disease vehicle group: ###p < 0.001; *p < 0.05; **p < 0.01; ***p < 0.001. (C) Activity of 32 on MCHI score (mouse colitis histology index, a composite histological score of eight subscores following the published methodology).35 Data are presented as mean values ± SEM (n = 10 mice/group). Statistical analysis was performed with a one-way ANOVA without SHAM – vehicle group and Dunnett’s post hoc multiple comparison test vs DSS 4% disease vehicle group: ###p < 0.001; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Representative images (scale 100 μm) of PAS-stained (and hematoxylin counter-stained) colonic tissues collected at study termination from each treatment group: “SHAM – vehicle”, “DSS 4% disease – vehicle”, and “DSS 4% disease – compound 32” treated with 3, 10, and 30 mg/kg (b.i.d. po). (E) Plasma exposure of compound 32 in the mouse DSS-induced colitis model on day 9 (n = 2 or 3 mice/time point between 0 and 6 h). The curve of the second daily dose was extrapolated and indicated by a dashed line for each group. Abbreviations: ANOVA, analysis of variance; AUC, area under the curve; DSS, dextran sulfate sodium; DAI, disease activity index; MCHI, mouse colitis histology index; PAS, periodic acid–Schiff; SEM, standard error of the mean.
Analysis of plasma exposure of 32 showed more than dose-proportional exposure increase in the study as highlighted by the AUC/dose ratio (Table 9). As shown in Figure 9E, the dose of 3 mg/kg twice daily led to only a short coverage of the IC50 on TNFα from in vivo LPS challenge. In contrast, exposure at 30 mg/kg twice daily was above the IC50 on TNFα for approximately 16 h. The significant activity at 3 mg/kg twice daily suggests that this colitis model is sensitive to local SIK inhibition in the gut such that local exposure in the gut contributes to efficacy. Overall compound 32 dose-dependently reduced disease activity in a mouse DSS-induced colitis model.
Table 9. Plasma Exposure of 32 in a DSS-Induced Colitis Model on Day 9.
| Dosing
Regimen |
|||
|---|---|---|---|
| 3 mg/kg po b.i.d. | 10 mg/kg po b.i.d. | 30 mg/kg po b.i.d. | |
| Cmax (ng/mL) | 705 | 3,580 | 21,300 |
| AUC0–24 (ng·h/mL) | 2,490 | 13,900 | 86,800 |
| AUC/dose | 415 | 695 | 1,447 |
In summary, the dual SIK2/SIK3 inhibitor 32 decreased the production of TNFα and increased the release of IL-10 in mice stimulated with LPS. These results suggest SIK1 inhibition is dispensable to exert a dual mechanism of immunomodulatory activity by compound 32in vivo in a short LPS challenge model along with robust preclinical activity in a mouse model of DSS-induced colitis.
Drug–Drug Interaction and In Vitro Safety Properties
Further in vitro properties of 32 were evaluated to assess the risk of DDI and potential safety concerns. Reversible inhibition of CYP isoforms by 32 was determined in human liver microsomes (HLMs) and led to IC50 > 33 μM for CYP2C19 and CYP2C9, and IC50 > 100 μM for CYP3A4, CYP2D6, and CYP1A2 (Table 10). As mentioned previously, 32 was negative in the CYP3A4 TDI assay in HLMs using midazolam and testosterone as probe substrates. The compound also did not show any significant induction of CYP3A4 mRNA at 10 μM in primary human hepatocytes. 32 inhibited hERG channel in a manual patch clamp assay with an IC50 of 15.3 μM. Finally, the compound was negative in in vitro assays for genotoxicity with and without S9 metabolic activation. Overall properties of 32 were considered suitable for further development.
Table 10. DDI and In Vitro Safety Properties of 32a.
| CYP450: IC50 (μM) in HLMs | >33: 2C19, 2C9 |
| >100: 3A4, 2D6, 1A2 | |
| CYP3A4 HLM-TDI midazolam/testosterone: IC50 (μM) initial, change (fold shift) | >33, no shift/>100, no shift |
| CYP3A4 mRNA induction at 10 μM in hepatocytes: fold increase vs vehicle, % increase vs rifampicin | <2-fold |
| <20% | |
| hERG: IC50 (μM) (manual patch clamp assay) | 15.3 |
| Genotoxicity Ames/micronucleus +/– S9 | Negative/negative |
Abbreviations: HLM, human liver microsome; TDI, time-dependent inhibition.
Conclusion
Starting from the same hit series that led to the pan-SIK inhibitor GLPG3312 and taking advantage of the cocrystal structure obtained previously, structural modifications of the chemotype were made in areas where differences of residues were identified between SIK isoforms. SAR optimization of amine substituents was performed, aiming at increasing potency on SIKs and selectivity against SIK1. Introduction of an oxygen linker improved the CYP TDI properties. Unexpectedly, scaffold modification and cyclization of the benzamide moiety into a lactam ring positively impacted potency and selectivity and allowed identification of GLPG3970 (32), a potent dual SIK2/SIK3 inhibitor showing high selectivity against SIK1 and high kinome selectivity. In vitro analysis showed that 32 had an immunomodulatory effect by inhibiting the production of TNFα and stimulating the release of IL-10 from LPS-stimulated human monocytes and MdMs, with similar effects seen in human whole blood from healthy volunteers. Compound 32 showed concordant results on TNFα and IL-10 in vivo in mice challenged with LPS. 32 displayed significant efficacy in a mouse DSS colitis model, suggesting that SIK1 inhibition is dispensable for immunoregulatory properties and activity in IBD models. Finally, 32 showed suitable pharmacokinetics, ADMET, and in vitro safety properties for further development. The results of clinical investigations with 32 will be described in a dedicated report.
Experimental Section
All reagents were of commercial grade and were used as received, without further purification. Full-length SIK3 protein and compounds 8 and 9 were prepared as described previously.14 Testing of 32 on 372 kinases panel and follow-up IC50 determination were performed at Eurofins (Eurofins Cerep, Le Bois l’Evêque, France). Homo sapiens SIK1 (full length, reference 02-131), was purchased from Carna Biosciences. Homo sapiens SIK2 (full length, reference PR8353A), was purchased from Invitrogen. AMARA peptide (AMARAASAAALARRR, A11-58) was obtained from SignalChem. NanoBRET Kinase Tracer-04 (CS181041), Extracellullar NanoLuc inhibitor (CS181048), NanoBRET Nano-Glo substrate (CS181063A or CS181046) were purchased from Promega. Alexa Fluor 594-labeled goat anti-rabbit secondary antibody (A-11012) was obtained from ThermoFisher Scientific. Rabbit monoclonal antibody to human CRTC3 (AB91654) was purchased from Abcam. JetPEI (101-40) was obtained from PolyPlus. HLM were obtained from BD Gentest. GraphPad Prism software was used for generation and fitting of graphs, IC50 determination, and statistical analysis. Pharmacokinetic parameters were calculated using Phoenix software (Certara, version 6.4.0.768). Commercially available anhydrous solvents were used for reactions conducted under an inert atmosphere. Reagent-grade solvents were used in all other cases, unless otherwise specified. Column chromatography was performed on silica gel 60 (thickness: 35–70 μm). 1H NMR spectra were recorded on a 400 MHz Bruker Avance spectrometer (SEI probe) or a 300 MHz DPX Bruker spectrometer (QNP probe). Chemical shifts (δ) for 1H NMR spectra are reported in ppm relative to tetramethylsilane (δ 0.00) or the appropriate residual solvent peak (i.e., CHCl3 [δ 7.27], as internal reference). Multiplicities are given as singlet (s), doublet (d), doublet of doublets (dd), doublet of doublet of doublets (ddd), doublet of quartets (dq), doublet of triplets (dt), doublet of triplet of doublets (dtd), triplet (t), quartet (q), quintuplet (quin), multiplet (m), and broad (br). Electrospray MS spectra were obtained with a Waters Acquity UPLC instrument equipped with a Waters Acquity photodiode array detector and single quad detector mass spectrometer. Columns used were UPLC bridged-ethylene hybrid (BEH) C18 1.7 μm, 2.1 × 5 mm VanGuard precolumn with Acquity UPLC BEH C18 1.7 μm, 2.1 × 30 mm column, or Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm column. All of the methods used MeCN/H2O gradients. MeCN and H2O contained either 0.1% formic acid or 0.05% NH3. As needed an autopurification system from Waters was used for LC-MS purification. LC-MS columns used were Waters XBridge Prep C18 5 μm, ODB 30 mm inner diameter (ID) × 100 mm length (L) (preparative column); and Waters XBridge C18 5 μm, 4.6 mm ID × 100 mm L (analytical column). All of the methods used MeCN/H2O gradients. MeCN and H2O contained either 0.1% formic acid or 0.1% diethylamine. All final compounds reported were analyzed using these analytical methods, and purities were greater than 95% unless otherwise indicated.
Chemistry
General Procedure A for Buchwald–Hartwig Cross-Coupling Reaction
In a microwave vial under inert atmosphere, to a solution of 7-bromoimidazo[1,2-a]pyridine derivative (1.0 equiv) in dioxane (10 mL per mmol) were added amine (1.5–2 equiv), Cs2CO3 (2–3 equiv), RuPhos Pd G3 (0.1–0.2 equiv), and RuPhos (0.1–0.2 equiv). The reaction mixture was degassed and stirred at 90 °C until completion of the reaction (monitored by HPLC). The mixture was quenched with saturated aqueous NaHCO3 and extracted with AcOEt. The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by preparative LC-MS to afford the desired compound.
2,6-Dimethoxy-4-(5-morpholinobenzimidazol-1-yl)-N-(2,2,2-trifluoroethyl) benzamide, (10)
In a sealed tube containing a previously degassed solution of 33 (50 mg, 0.1 mmol, 1 equiv) and morpholine (13 μL, 0.15 mmol, 1.5 equiv) in dioxane (0.7 mL) under nitrogen atmosphere were added potassium tert-butoxide (34 mg, 0.3 mmol, 3 equiv), tBuXPhos (8.5 mg, 0.02 mmol, 0.2 equiv), and catalyst Pd2dba3 (9.1 mg, 0.01 mmol, 0.1 equiv). The vial was sealed, and the resulting solution was heated to 110 °C overnight. The reaction mixture was then diluted with DCM and water. The organic layer was separated, washed with water and brine, dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by flash chromatography on silica gel (gradient DCM/MeOH 100/0 to 90/10) to deliver 10 (8 mg, 17%). 1H NMR (400 MHz, CDCl3) δ ppm 1H NMR (400 MHz, DMSO-d6) δ 8.85 (t, J = 6.4 Hz, 1H), 8.53 (s, 1H), 7.62 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 2.3 Hz, 1H), 7.12 (dd, J = 9.0, 2.3 Hz, 1H), 6.97 (s, 2H), 4.01 (qd, J = 9.7, 6.3 Hz, 2H), 3.83 (s, 6H), 3.81–3.73 (m, 4H), 3.16–3.09 (m, 4H). LC-MS: m/z = 465.2 [M+H].
Methyl 4-(7-bromoimidazo[1,2-a]pyridin-3-yl)-2,6-dimethoxy-benzoate (36)
To 7-bromo-3-iodo-imidazo[1,2-a] pyridine 34 (6.97 g, 21.58 mmol, 1 equiv), methyl 2,6-dimethoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 35 (8.6 g, 25.90 mmol, 1.2 equiv), Cs2CO3 (16 g, 43.16 mmol, 2 equiv), and dioxane/water solvent mixture (4/1, 210 mL) degassed with N2, Pd(dppf)Cl2 (1.77 g, 2.16 mmol, 0.1 equiv) was added, and the mixture was purged with N2 then stirred at 90 °C for 1 h. Dioxane was evaporated, saturated aqueous NaHCO3 was added, and the mixture was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude was purified by flash chromatography on silica gel (eluting with a gradient of 10% to 30% EtOAc in CH2Cl2) to afford 36 (5.78 g, 68%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.66 (dd, J = 7.4, 0.8 Hz, 1H), 8.03–7.98 (m, 1H), 7.88 (s, 1H), 7.10 (dd, J = 7.4, 2.1 Hz, 1H), 6.98 (s, 2H), 3.85 (s, 6H), 3.79 (s, 3H). LC-MS: m/z = 391.4–393.4 [M+H].
4-(7-Bromoimidazo[1,2-a]pyridin-3-yl)-2,6-dimethoxy-benzoic acid (37)
Intermediate 36 (2.4 g, 6.13 mmol, 1 equiv) was dissolved in MeOH (35 mL) and THF (35 mL). NaOH 2N (12.3 mL, 24.54 mmol, 4 equiv) was added. The mixture was stirred at 70 °C for 18 h. Another portion of NaOH 2N (6.15 mL, 12.27 mmol, 2 equiv) was added. The mixture was stirred at 70 °C for 18 h and acidified with HCl 2N to pH = 4. Organic solvents were removed by evaporation under reduced pressure, and the precipitate in the remaining water was filtered to afford 37 (2.2 g, 96%). 1H NMR (400 MHz, DMSO-d6) δ ppm 12.87 (s, 1H), 8.63 (d, J = 7.4 Hz, 1H), 8.00 (d, J = 2.0 Hz, 1H), 7.86 (s, 1H), 7.10 (dd, J = 7.4, 2.1 Hz, 1H), 6.95 (s, 2H), 3.85 (s, 6H). LC-MS: m/z = 377.4–379.3 [M+H].
4-(7-Bromoimidazo[1,2-a]pyridin-3-yl)-2,6-dimethoxy-N-(2,2,2-trifluoroethyl)benzamide (38)
To 37 (5.0 g, 13.25 mmol, 1 equiv) in DMF (98 mL) were added DIPEA (6.6 mL, 39.76 mmol, 3 equiv) and HATU (5.0 g, 13.25 mmol, 1 equiv). The mixture was stirred at room temperature for 20 min. 2,2,2-Trifluoroethylamine hydrochloride (3.6 g, 26.51 mmol, 2 equiv) was added. The mixture was stirred at room temperature for 18 h. The reaction mixture was concentrated in vacuo. Saturated aqueous NaHCO3 was added, and the mixture was extracted with CH2Cl2. The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was recrystallized in CH3CN twice. The solid was filtered and was then triturated in aqueous NaHCO3 and filtered to afford 38 (3.47 g, 57%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.82 (t, J = 6.4 Hz, 1H), 8.59 (d, J = 7.4 Hz, 1H), 8.03–7.98 (m, 1H), 7.86 (s, 1H), 7.12 (dd, J = 7.4, 2.1 Hz, 1H), 6.95 (s, 2H), 4.07–3.94 (m, 2H), 3.82 (s, 6H). LC-MS: m/z = 458.5–460.4 [M+H].
2,6-Dimethoxy-4-(7-morpholinoimidazo[1,2-a]pyridin-3-yl)-N-(2,2,2-trifluoroethyl)benzamide (11)
Intermediate 38 was reacted with morpholine according to general procedure A, to afford 11 (18 mg, 36%). 1H NMR (400 MHz, CDCl3) δ 8.07 (d, J = 0.6 Hz, 1H), 7.43 (s, 1H), 6.77 (d, J = 2.4 Hz, 1H), 6.61 (s, 2H), 6.59–6.54 (m, 1H), 6.23 (t, J = 6.4 Hz, 1H), 4.14–4.01 (m, 2H), 3.85–3.76 (m, 10H), 3.20–3.13 (m, 4H). LC-MS: m/z = 465.3 [M+H].
4-[7-(3,3-Difluoroazetidin-1-yl)imidazo[1,2-a]pyridin-3-yl]-2,6-dimethoxy-N-(2,2,2-trifluoroethyl)benzamide (13)
Intermediate 38 was reacted with difluoroazetidine hydrochloride salt according to general procedure A, to afford 13 (43 mg, 83%). 1H NMR (400 MHz, CDCl3) δ ppm 8.16 (d, J = 0.7 Hz, 1H), 7.54 (s, 1H), 6.71–6.64 (m, 3H), 6.35 (dd, J = 7.5, 2.4 Hz, 1H), 6.23 (t, J = 6.4 Hz, 1H), 4.39 (t, J = 11.6 Hz, 4H), 4.22–4.09 (m, 2H), 3.88 (s, 6H). LC-MS: m/z = 471.3 [M+H].
2,6-Dimethoxy-4-[7-(4-methylpiperazin-1-yl)imidazo[1,2-a]pyridin-3-yl]-N-(2,2,2-trifluoroethyl)benzamide (15)
Intermediate 38 was reacted with 1-methylpiperazine according to general procedure A, to afford 15 (37 mg, 71%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.48–8.40 (m, 2H), 7.71 (s, 1H), 7.11 (dd, J = 7.9, 2.5 Hz, 1H), 6.92 (s, 2H), 6.89 (d, J = 2.5 Hz, 1H), 4.08 (q, J = 9.3 Hz, 2H), 3.89 (s, 6H), 3.55 (t, J = 5.1 Hz, 4H), 2.83 (t, J = 5.1 Hz, 4H), 2.53 (s, 3H). LC-MS: m/z = 478.3 [M+H].
N-Cyclopropyl-2-hydroxy-6-methoxy-benzamide (40)
At room temperature, 6-methoxysalicylic acid 39 (10 g, 59.5 mmol, 1 equiv) was dissolved in DMF (50 mL), HATU (33.93 g, 89.2 mmol, 1.5 equiv) was added, followed 15 min later by cyclopropylamine (10.18 g, 178.6 mmol, 3 equiv) and DIPEA (34.55 g, 260 mmol, 4.3 equiv). The reaction mixture was stirred at room temperature for 18 h. HATU (22.63 g, 59.5 mmol, 1 equiv), cyclopropylamine (6.74 g, 118.4 mmol, 2 equiv), and DIPEA (15.73 g, 118.4 mmol, 2 equiv) were added. The reaction mixture was stirred at room temperature for 48 h. The reaction mixture was concentrated in vacuo. Purification by flash chromatography on silica gel (eluting with heptane/EtOAc 100/0 to 50/50) and then trituration in MeOH/diethyl ether to get rid of an insoluble byproduct (repeated twice) were done. The filtrate was concentrated in vacuo to afford 40 (3.5 g, 28%). 1H NMR (400 MHz, CDCl3) δ ppm 8.32 (s, 1H), 7.21 (t, J = 8.4 Hz, 1H), 6.57 (dd, J = 8.4, 1.0 Hz, 1H), 6.34 (dd, J = 8.4, 1.1 Hz, 1H), 3.87 (s, 3H), 2.84 (tq, J = 7.3, 3.8 Hz, 1H), 0.87–0.81 (m, 2H), 0.61–0.55 (m, 2H). LC-MS: m/z = 208.4 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-benzamide (41)
Under an inert atmosphere, 40 (2.80 g, 13.5 mmol, 1 equiv) was dissolved in acetonitrile (20 mL) and cooled to −20 °C. KOH (7.57 g, 135 mmol, 10 equiv) solubilized with water (20 mL) was added, and the mixture was stirred for 10 min, then bromodifluoromethyl diethylphosphonate (10.9 g, 40 mmol, 3 equiv) was added slowly. The reaction mixture was stirred at −20 °C for 30 min then at room temperature for an additional 30 min. Water was added, and 3 extractions with EtOAc were performed. The organic layers were dried with Na2SO4, filtered, and concentrated in vacuo. Purification by flash chromatography on silica gel (eluting with a gradient heptane/EtOAc 100/0 to 0/100) afforded 41 (2.86 g, 79%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.26 (d, J = 4.5 Hz, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.05 (t, J = 72.0 Hz, 1H), 6.97–6.92 (m, 1H), 6.78 (dd, J = 8.4, 0.9 Hz, 1H), 3.77 (s, 3H), 2.76 (ddt, J = 11.4, 7.3, 4.0 Hz, 1H), 0.65 (td, J = 7.0, 4.7 Hz, 2H), 0.46–0.41 (m, 2H). LC-MS: m/z = 258.4 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (42)
At room temperature, 41 (47 g, 183 mmol, 1 equiv) and bis(pinacolato)diboron (96 g, 366 mmol, 2 equiv) were dissolved in dry THF (400 mL). The mixture was purged with N2 for 15 min. (1,5-Cyclooctadiene)(methoxy)iridium(I) dimer (6.2 g, 9.15 mmol, 0.05 equiv) was added, followed by addition of 2-(4-tert-butyl)bipyridyl (2.6 g, 9.15 mmol, 0.05 equiv). The mixture was refluxed and stirred for 6 h then left overnight at room temperature. The solvent was removed under reduced pressure. The residue was dissolved in DCM (300 mL) and quenched slowly with water (300 mL, Caution: exothermic). The organic layer was separated and concentrated to dryness under reduced pressure. The crude residue was purified by flash chromatography on silica gel (eluting with cyclohexane/EtOAc 80/20 to 60/40). The collected fractions were combined and concentrated in vacuo. The solid obtained was further triturated in diisopropyl ether, stirred for 45 min, filtered, and dried in vacuo to afford 42. (34.1 g, 49%). 1H NMR (400 MHz, CDCl3) δ ppm 7.26–7.18 (m, 2H), 6.61 (t, J = 74.5 Hz, 1H), 5.88 (s, 1H), 3.90 (s, 3H), 2.92 (tq, J = 7.1, 3.6 Hz, 1H), 1.37 (s, 12H), 0.94–0.80 (m, 2H), 0.69–0.60 (m, 2H). LC-MS: m/z = 384.2 (M+H of boronic ester), m/z = 302.1 (M+H of boronic acid).
4-(7-Bromoimidazo[1,2-a]pyridin-3-yl)-N-cyclopropyl-2-(difluoromethoxy)-6-methoxy-benzamide (43a)
A flask was charged with 7-bromo-3-iodo-imidazo[1,2-a]pyridine 34 (5.0 g, 15.4 mmol, 1 equiv), 42 (6.5 g, 17.0 mmol, 1.1 equiv), Cs2CO3 (11.0 g, 30.9 mmol, 2 equiv), and degassed with N2 in dioxane/water solvent mixture (4/1, 80 mL). Then, Pd(PPh3)4 (1.8 g, 0.77 mmol, 0.05 equiv) was added. The mixture was purged with N2 and stirred at 90 °C for 20 h. Dioxane was evaporated, aqueous NaHCO3 sat was added, and the mixture was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The solid obtained was triturated with CH3CN and filtered to afford 43a (7 g, 96%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.56 (d, J = 7.3 Hz, 1H), 8.36 (d, J = 4.5 Hz, 1H), 8.01 (d, J = 2.0 Hz, 1H), 7.88 (s, 1H), 7.22 (t, J = 72.0 Hz, 1H), 7.21 (s, 1H), 7.15 (dd, J = 7.3, 2.1 Hz, 1H), 7.06 (s, 1H), 3.87 (s, 3H), 2.79 (td, J = 7.3, 3.7 Hz, 1H), 0.68 (td, J = 7.0, 4.7 Hz, 2H), 0.50–0.44 (m, 2H). LC-MS: m/z = 450.1–452.1 [M - H].
4-(7-Aminoimidazo[1,2-a]pyridin-3-yl)-N-cyclopropyl-2-(difluoromethoxy)-6-methoxy-benzamide (43b)
To 3-bromoimidazo[1,2-a]pyridin-7-amine (200 mg, 0.94 mmol, 1 equiv), 42 (397 mg, 1.03 mmol, 1.1 equiv), Cs2CO3 (717 mg, 1.88 mmol, 2 equiv) in dioxane/water solvent mixture (4/1, 5 mL) degassed with N2 was added Pd(dppf)Cl2 (77 mg, 0.1 mmol, 0.1 equiv), and the mixture was purged with N2 then stirred at 90 °C for 1 h. Dioxane was evaporated, aqueous NaHCO3 sat was added, and the mixture was extracted with EtOAc. The combined organic layers were washed with water and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by flash chromatography on silica gel (eluting with a gradient of 0 to 10% MeOH in CH2Cl2) to afford 43b (240 mg, 65%). LC-MS: m/z = 389.3 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-4-(7-fluoroimidazo[1,2-a]pyridin-3-yl)-6-methoxy-benzamide (43c)
A solution of 7-fluoroimidazo[1,2-a]pyridine (2.0 g, 14.7 mmol, 1 equiv) in dry DMAC (40 mL) was degassed with N2, and then 42 (6.0 g, 17.6 mmol, 1.2 equiv), KOAc (4.3 g, 44.1 mmol, 3 equiv), and Pd(dppf)Cl2·DCM (600 mg, 0.73 mmol, 0.05 equiv) were added. The mixture was degassed with N2 for 10 min and was then stirred at 110 °C for 4 h. The reaction mixture was filtered, and the filtrate was concentrated. The crude material was purified by flash chromatography on silica gel (eluting with a gradient of 0 to 2% MeOH in EtOAc) to give a solid that was triturated in EtOH to afford 43c (4.4 g, 77%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.67 (ddd, J = 7.6, 5.8, 0.7 Hz, 1H), 8.35 (d, J = 4.5 Hz, 1H), 7.84 (s, 1H), 7.57–7.53 (m, 1H), 7.23 (t, J = 72.0 Hz, 1H), 7.21 (d, J = 1.3 Hz, 1H), 7.07–7.03 (m, 2H), 3.87 (s, 3H), 2.83–2.75 (m, 1H), 0.68 (td, J = 7.0, 4.8 Hz, 2H), 0.49–0.44 (m, 2H). LC-MS: m/z = 392.2 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(7-morpholinoimidazo[1,2-a]pyridin-3-yl)benzamide (12)
Intermediate 43a was reacted with morpholine according to general procedure A, to afford 12 (29 mg, 48%). 1H NMR (400 MHz, CDCl3) δ ppm 8.41 (d, J = 4.2 Hz, 1H), 7.68 (s, 1H), 7.17 (d, J = 1.3 Hz, 1H), 7.06 (dd, J = 9.0, 1.8 Hz, 2H), 6.91 (t, J = 73.7 Hz, 1H), 6.84 (d, J = 2.6 Hz, 1H), 3.94 (s, 3H), 3.91–3.82 (m, 4H), 3.39 (dd, J = 5.9, 3.9 Hz, 4H), 2.87 (tt, J = 7.4, 3.9 Hz, 1H), 0.92–0.76 (m, 2H), 0.63 (ddd, J = 7.0, 5.1, 3.9 Hz, 2H). LC-MS: m/z = 459.2 [M+H].
N-Cyclopropyl-4-(7-(3,3-difluoroazetidin-1-yl)imidazo[1,2-a]pyridin-3-yl)-2-(difluoromethoxy)-6-methoxybenzamide (14)
Intermediate 43a was reacted with 3,3-difluoroazetidine hydrochloride according to general procedure A, to afford 14 (18 mg, 35%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.50 (d, J = 7.4 Hz, 1H), 8.34 (d, J = 4.5 Hz, 1H), 8.14 (s, 1H), 7.68 (s, 1H), 7.22 (t, J = 74 Hz, 1H), 7.13 (d, J = 1.3 Hz, 1H), 6.97 (s, 1H), 6.60–6.51 (m, 2H), 4.43 (t, J = 12.2 Hz, 4H), 3.87 (s, 3H), 2.84–2.73 (m, 1H), 0.73–0.61 (m, 2H), 0.50–0.38 (m, 2H). LC-MS: m/z = 465.4 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(7-(4-methylpiperazin-1-yl)imidazo[1,2-a]pyridin-3-yl)benzamide (16)
Intermediate 43a was reacted with N-methylpiperazine according to general procedure A, to afford 16 (20 mg, 19%). 1H NMR (400 MHz, methanol-d4) δ 8.37 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.13 (d, J = 1.3 Hz, 1H), 7.04–7.00 (m, 1H), 6.97 (dd, J = 7.8, 2.5 Hz, 1H), 6.91 (t, J = 75 Hz, 1H), 6.80 (d, J = 2.5 Hz, 1H), 3.93 (s, 3H), 3.40 (t, J = 5.1 Hz, 4H), 2.86 (hept, J = 3.9 Hz, 1H), 2.67 (t, J = 5.1 Hz, 4H), 2.40 (s, 3H), 0.87–0.76 (m, 2H), 0.67–0.58 (m, 2H). LC-MS: m/z = 472.4 [M+H].
4-(7-(4-Acetylpiperazin-1-yl)imidazo[1,2-a]pyridin-3-yl)-N-cyclopropyl-2-(difluoromethoxy)-6-methoxybenzamide (17)
To a solution of 43a (50 mg, 0.11 mmol, 1 equiv) in dioxane (1 mL) was added 1-piperazin-1-ylethanone (17 mg, 0.133 mmol, 1.2 equiv), RuPhos (10 mg, 0.011 mmol, 0.1 equiv), RuPhos Pd G2 (13 mg, 0.022 mmol, 0.2 equiv), and K3PO4 (117 mg, 0.553 mmol, 5 equiv). The reaction mixture was stirred at 90 °C for 7 h then filtered, and the filtrate was purified by preparative LC-MS. The recovered material was crystallized in hot ACN then rinsed with ACN and Et2O to give 17 (20 mg, 19%). 1H NMR (400 MHz, CDCl3) δ ppm 8.12 (d, J = 7.7 Hz, 1H), 7.55 (s, 1H), 6.98–6.94 (m, 1H), 6.92–6.88 (m, 2H), 6.73–6.67 (m, 1H), 6.63 (t, J = 74.3 Hz, 1H), 6.10 (d, J = 3.1 Hz, 1H), 3.89 (s, 3H), 3.80 (t, J = 5.2 Hz, 2H), 3.66 (t, J = 5.2 Hz, 2H), 3.33–3.24 (m, 4H), 2.93 (tq, J = 7.2, 3.7 Hz, 1H), 2.16 (s, 3H), 0.91–0.84 (m, 2H), 0.68–0.60 (m, 2H). LC-MS: m/z = 500.5 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-4-(7-(4-(2-hydroxyethyl)piperazin-1-yl)imidazo[1,2-a]pyridin-3-yl)-6-methoxybenzamide (18)
To a solution of 43a (50 mg, 0.11 mmol, 1 equiv) in dioxane (1 mL) was added 1-2-(piperazin-1-yl)ethan-1-ol (17 mg, 0.133 mmol, 1.2 equiv), RuPhos (10 mg, 0.011 mmol, 0.1 equiv), RuPhos Pd G2 (13 mg, 0.022 mmol, 0.2 equiv), and K3PO4 (117 mg, 0.553 mmol, 5 equiv). The reaction mixture was stirred at 90 °C for 7 h then filtered, and the filtrate was purified by preparative HPLC. The recovered material was crystallized in hot ACN then rinsed with ACN and Et2O to give 18 (13 mg, 24%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.39 (d, J = 7.8 Hz, 1H), 8.33 (d, J = 4.5 Hz, 1H), 7.64 (s, 1H), 7.22 (t, J = 74.0 Hz, 1H), 7.12 (d, J = 1.3 Hz, 1H), 6.97 (d, J = 1.3 Hz, 1H), 6.93 (dd, J = 7.8, 2.6 Hz, 1H), 6.77 (d, J = 2.5 Hz, 1H), 4.45 (t, J = 5.3 Hz, 1H), 3.86 (s, 3H), 3.54 (q, J = 5.9 Hz, 2H), 3.29–3.23 (m, 4H), 2.80–2.74 (m, 1H), 2.57 (t, J = 5.1 Hz, 4H), 2.44 (t, J = 6.2 Hz, 2H), 0.70–0.64 (m, 2H), 0.50–0.42 (m, 2H). LC-MS: m/z = 502.3 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-4-[7-[4-(dimethylamino)-1-piperidyl]imidazo[1,2-a]pyridin-3-yl]-6-methoxy-benzamide (19)
Intermediate 43a was reacted with N,N-dimethylpiperidin-4-amine according to general procedure A, to afford 19 (7 mg, 14%). 1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 7.8 Hz, 1H), 8.33 (d, J = 4.5 Hz, 1H), 7.63 (s, 1H), 7.22 (t, J = 74.0 Hz, 1H), 7.13 (d, J = 1.3 Hz, 1H), 6.97 (d, J = 1.2 Hz, 1H), 6.92 (dd, J = 7.8, 2.6 Hz, 1H), 6.77 (d, J = 2.4 Hz, 1H), 3.87 (s, 5H), 2.80 (ddt, J = 11.2, 7.2, 2.7 Hz, 3H), 2.28 (tt, J = 10.9, 3.6 Hz, 1H), 2.19 (s, 6H), 1.90–1.81 (m, 2H), 1.47 (qd, J = 12.0, 3.8 Hz, 2H), 0.68 (td, J = 7.1, 4.8 Hz, 2H), 0.50–0.43 (m, 2H). LC-MS: m/z = 500.4 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-4-(7-(3-(dimethylamino)azetidin-1-yl)imidazo[1,2-a]pyridin-3-yl)-6-methoxybenzamide (20)
Intermediate 43a was reacted with N,N-dimethylazetidin-3-amine hydrochloride according to general procedure A, to afford 20 (22 mg, 42%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.32 (dd, J = 7.5, 0.7 Hz, 1H), 7.50 (s, 1H), 7.09 (d, J = 1.3 Hz, 1H), 6.97 (q, J = 1.1 Hz, 1H), 6.97 (d, J = 74.5 Hz, 1H), 6.48 (dd, J = 7.5, 2.4 Hz, 1H), 6.32 (d, J = 2.3 Hz, 1H), 4.16–4.08 (m, 2H), 3.91 (s, 3H), 3.81 (dd, J = 8.0, 5.3 Hz, 2H), 3.37–3.32 (m, 1H), 2.89–2.82 (m, 1H), 2.26 (s, 6H), 0.85–0.77 (m, 2H), 0.63–0.58 (m, 2H). LC-MS: m/z = 472.5 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(7-((3-morpholinophenyl)amino)imidazo[1,2-a]pyridin-3-yl)benzamide (21)
Under inert atmosphere, to a solution of 43a (50 mg, 0.11 mmol, 1 equiv) in dioxane (1.5 mL) was added 3-morpholinoaniline (30 mg, 0.166 mmol, 1.5 equiv), Xantphos (19 mg, 0.033 mmol, 0.3 equiv), Pd2(dba)3 (10 mg, 0.011 mmol, 0.1 equiv), and Cs2CO3 (108 mg, 0.332 mmol, 3 equiv). The reaction mixture was stirred at 90 °C for 3 h in a sealed vial, then quenched with water, extracted with AcOEt (twice). The combined layers were passed on a phase separator and concentrated under reduced pressure. The residue was purified by preparative HPLC to give 21 (26 mg, 43%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.58 (s, 1H), 8.47 (d, J = 7.5 Hz, 1H), 8.33 (d, J = 4.5 Hz, 1H), 7.62 (s, 1H), 7.24 (t, J = 73.9 Hz, 1H), 7.20 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 1.3 Hz, 1H), 6.99–6.95 (m, 2H), 6.77 (dd, J = 7.6, 2.4 Hz, 1H), 6.75–6.69 (m, 2H), 6.66–6.60 (m, 1H), 3.88 (s, 3H), 3.77–3.71 (m, 4H), 3.14–3.07 (m, 4H), 2.82–2.74 (m, 1H), 0.70–0.64 (m, 2H), 0.48–0.43 (m, 2H). LC-MS: m/z = 550.4 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(7-((1-methyl-1H-imidazol-4-yl)amino)imidazo[1,2-a]pyridin-3-yl)benzamide (22)
Under inert atmosphere, to a solution of 35a (50 mg, 0.11 mmol, 1 equiv) in dioxane(1.5 mL) was added 1-methylimidazol-4-amine (16 mg, 0.166 mmol, 1.5 equiv), Xantphos (19 mg, 0.033 mmol, 0.3 equiv), Pd2(dba)3 (10 mg, 0.011 mmol, 0.1 equiv), and Cs2CO3 (108 mg, 0.33 mmol, 3 equiv). The mixture was stirred at 90 °C for 3 h in a sealed vial, then quenched with water, extracted with AcOEt (twice). The combined layers were passed on a phase separator and concentrated under reduced pressure. The residue was purified by preparative HPLC to give 22 as formic acid salt (18 mg, 35%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.81 (s, 1H), 8.40 (d, J = 7.5 Hz, 1H), 8.33 (d, J = 4.5 Hz, 1H), 8.14 (s, 1H), 7.60 (s, 1H), 7.46 (d, J = 1.4 Hz, 1H), 7.22 (t, J = 74.0 Hz, 1H), 7.11 (d, J = 1.3 Hz, 1H), 7.06 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = 5.0, 1.3 Hz, 2H), 6.83 (dd, J = 7.6, 2.4 Hz, 1H), 3.87 (s, 3H), 3.66 (s, 3H), 2.81–2.75 (m, 1H), 0.72–0.64 (m, 2H), 0.50–0.43 (m, 2H). LC-MS: m/z = 469.3 [M+H].
N-(3-(4-(Cyclopropylcarbamoyl)-3-(difluoromethoxy)-5-methoxyphenyl)imidazo[1,2-a]pyridin-7-yl)tetrahydro-2H-pyran-4-carboxamide (23)
To a solution of 43a (60 mg, 0.13 mmol, 1 equiv) in dioxane (1 mL) was added tetrahydropyran-4-carboxamide (34 mg, 0.26 mmol, 2 equiv) Pd(OAc)2 (3 mg, 0.013 mmol, 0.1 equiv), Xantphos (15 mg, 0.026 mmol, 0.2 equiv), and Cs2CO3 (100 mg, 0.26 mmol, 2 equiv). The reaction mixture was heated in a microwave oven at 150 °C for 1 h, then quenched with saturated NaHCO3, extracted with AcOEt (3 times). The combined layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC to give 23 (30 mg, 45%). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.42 (s, 1H), 7.74 (dd, J = 7.5, 0.9 Hz, 1H), 7.52 (d, J = 4.5 Hz, 1H), 7.25 (dd, J = 2.2, 0.8 Hz, 1H), 6.92 (s, 1H), 6.40 (t, J = 73.9 Hz, 1H), 6.32 (d, J = 1.3 Hz, 1H), 6.28 (dd, J = 7.6, 2.2 Hz, 1H), 6.17 (d, J = 1.2 Hz, 1H), 3.13–3.06 (m, 2H), 3.04 (s, 3H), 2.58–2.52 (m, 2H), 1.99–1.90 (m, 1H), 1.85–1.76 (m, 1H), 0.95–0.78 (m, 4H), −0.13 – −0.21 (m, 2H), −0.34 – −0.41 (m, 2H). LC-MS: m/z = 501.3 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(7-(3-methoxypropanamido)imidazo[1,2-a]pyridin-3-yl)benzamide (24)
To a solution of 43b (30 mg, 0.07 mmol, 1 equiv) in DCM (1 mL) were added 3-methoxypropionic acid (16 mg, 0.15 mmol, 2 equiv) and Ghosez’s reagent (31 mg, 0.23 mmol, 3 equiv). The mixture was stirred at room temperature for 4 days. The mixture was quenched with NaHCO3 sat and extracted with DCM. The combined layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with a gradient DCM/MeOH (99/1 to 96/4) to afford 24 (14 mg, 38%). 1H NMR (400 MHz, methanol-d4) δ ppm δ 8.48 (d, J = 7.6 Hz, 1H), 8.17–8.11 (m, 1H), 7.69 (s, 1H), 7.18 (dd, J = 7.5, 1.8 Hz, 1H), 7.15 (d, J = 1.3 Hz, 1H), 7.03 (q, J = 1.0 Hz, 1H), 6.91 (t, J = 73.7 Hz, 1H), 3.93 (s, 3H), 3.74 (t, J = 6.0 Hz, 2H), 3.37 (s, 3H), 2.85 (tt, J = 7.3, 3.8 Hz, 1H), 2.67 (t, J = 6.0 Hz, 2H), 0.82 (td, J = 7.1, 5.1 Hz, 2H), 0.64–0.57 (m, 2H). LC-MS: m/z = 475.3 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(7-((2-morpholinoethyl)amino)imidazo[1,2-a]pyridin-3-yl)benzamide (25)
In a sealed tube under inert atmosphere, to a solution of 43a (50 mg, 0.11 mmol, 1 equiv) in dioxane (1.5 mL) were added 2-morpholinoethanamine (22 mg, 0.17 mmol, 1.5 equiv), Cs2CO3 (108 mg, 0.33 mmol, 3 equiv), Pd2(dba)3 (10 mg, 0.011 mmol, 0.1 equiv), and XantPhos (19 mg, 0.033 mmol, 0.3 equiv). The reaction mixture was stirred at 90 °C until completion of the reaction (monitored by HPLC). The reaction mixture was evaporated and partitioned between DCM and water, the aqueous phase was extracted with DCM on a phase separator, and the organic layer was concentrated under reduced pressure. The residue was purified by preparative HPLC to afford 25 (14 mg, 25%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.33–8.26 (m, 2H), 7.51 (s, 1H), 7.21 (t, J = 74.0 Hz, 1H), 7.07 (d, J = 1.3 Hz, 1H), 6.91 (d, J = 1.2 Hz, 1H), 6.58 (dd, J = 7.6, 2.3 Hz, 1H), 6.34 (d, J = 2.3 Hz, 1H), 6.26 (t, J = 5.3 Hz, 1H), 3.86 (s, 3H), 3.60 (t, J = 4.6 Hz, 4H), 3.23–3.18 (m, 2H), 2.81–2.73 (m, 1H), 2.54 (t, J = 6.6 Hz, 2H), 2.44 (t, J = 4.6 Hz, 4H), 0.70–0.63 (m, 2H), 0.48–0.43 (m, 2H). LC-MS: m/z = 502.5 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-(7-(2-morpholinoethoxy)imidazo[1,2-a]pyridin-3-yl)benzamide (26)
To a solution of 2-morpholin-4-ylethanol (150 μL, 1.02 mmol, 10 equiv) in DMF (1 mL) was added NaH 60% in oil (16 mg, 0.41 mmol, 4 equiv). The reaction was stirred for 10 min at room temperature then 43c (40 mg, 0.10 mmol, 1 equiv) was added. The mixture was stirred at room temperature overnight. The solvent was evaporated. The residue was purified by flash chromatography on silica gel eluting with a gradient DCM/MeOH (99/1 to 93/7 to afford 26 (38 mg, 74%). 1H NMR (400 MHz, CDCl3) δ ppm 8.10 (d, J = 7.6 Hz, 1H), 7.56 (s, 1H), 6.99–6.95 (m, 2H), 6.91–6.89 (m, 1H), 6.66–6.63 (m, 1H), 6.63 (d, J = 74.0 Hz, 1H), 6.06 (d, J = 3.2 Hz, 1H), 4.20 (t, J = 5.5 Hz, 2H), 3.89 (s, 3H), 3.79–3.72 (m, 4H), 2.97–2.90 (m, 1H), 2.87 (t, J = 5.5 Hz, 2H), 2.64–2.58 (m, 4H), 0.92–0.83 (m, 2H), 0.69–0.60 (m, 2H). LC-MS: m/z = 503.4 [M+H].
N-Cyclopropyl-2-(difluoromethoxy)-6-methoxy-4-[7-(2-tetrahydropyran-4-ylethoxy)imidazo[1,2-a]pyridin-3-yl]benzamide (27)
To a solution of 2-tetrahydropyran-4-ylethanol (50 mg, 0.38 mmol, 5 equiv) in DMF (1 mL) was added NaH 60% in oil (15 mg, 0.38 mmol, 5 equiv). The reaction was stirred for 10 min at room temperature then 43c (30 mg, 0.077 mmol, 1 equiv) was added. The mixture was stirred at room temperature overnight. The solvent was evaporated. The residue was purified by preparative HPLC to afford 27 (23 mg, 60%). 1H NMR (400 MHz, DMSO-d6) δ 8.85 (t, J = 6.4 Hz, 1H), 8.53 (s, 1H), 7.62 (d, J = 8.9 Hz, 1H), 7.26 (d, J = 2.3 Hz, 1H), 7.12 (dd, J = 9.0, 2.3 Hz, 1H), 6.97 (s, 2H), 4.01 (qd, J = 9.7, 6.3 Hz, 2H), 3.83 (s, 6H), 3.81–3.73 (m, 4H), 3.16–3.09 (m, 4H). LC-MS: m/z = 502.5 [M+H].
4-(2-Imidazo[1,2-a]pyridin-7-yloxyethyl)morpholine (45)
To a solution of 2-morpholinoethanol (1.6 mL, 13.2 mmol, 6 equiv) in DMF (22 mL) was added NaH (60% dispersion in mineral oil, 529 mg, 13.2 mmol, 6 equiv) portionwise. After stirring for 30 min at room temperature, 7-fluoroimidazo[1,2-a]pyridine (44, 300 mg, 2.2 mmol, 1 equiv) was added, and the mixture was stirred at room temperature for 20 h. The reaction mixture was quenched with a saturated aqueous NaHCO3 solution, diluted with EtOAc, and stirred at room temperature for 15 min. The solid was filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was triturated with pentane 3 times. The solid obtained was purified by flash chromatography on silica gel (eluting with a gradient MeOH: 0 to 10% in DCM) to afford 45 (545 mg, 100%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.27 (dd, J = 7.5, 0.7 Hz, 1H), 7.65 (dd, J = 1.5, 0.7 Hz, 1H), 7.40 (d, J = 1.5 Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.66 (dd, J = 7.4, 2.5 Hz, 1H), 4.23 (t, J = 5.4 Hz, 2H), 3.77–3.66 (m, 4H), 2.87 (t, J = 5.4 Hz, 2H), 2.66–2.59 (m, 4H). LC-MS: m/z = 248.2 [M+H].
5-Bromo-N-cyclopropyl-3-methoxy-pyridine-2-carboxamide (47a)
To a solution of 5-bromo-3-methoxypicolinic acid 46 (60 mg, 0.26 mmol, 1 equiv) in anhydrous DMF (1 mL) were added DIPEA (68 μL, 0.39 mmol, 1.5 equiv) and HATU (108 mg, 0.28 mmol, 1.1 equiv). The reaction medium was stirred at room temperature for 1 h, and cyclopropylamine (22 μL, 0.31 mmol, 1.2 equiv) was added. The reaction mixture was stirred at room temperature for 20 h, then solvents were evaporated. The residue was diluted with DCM, washed with a 1N aqueous solution of NaOH, and passed through a phase separator. The filtrate was concentrated in vacuo, and the crude was purified by flash chromatography on silica gel (eluting with heptane/EtOAc 8/2 to 1/9) to afford 47a (46 mg, 65%). LC-MS m/z = 271.1–273.2 [M+H].
Methyl 5-bromo-3-(difluoromethoxy)picolinate (49)
Methyl 5-bromo-3-hydroxypicolinate 48 (50 mg, 0.215 mmol, 1 equiv), sodium chlorodifluoroacetate (35 mg, 0.26 mmol, 1.2 equiv), and K2CO3 (60 mg, 0.63 mmol, 3 equiv) were mixed in CH3CN (1.5 mL), and the reaction mixture was stirred at reflux for 2 h. The reaction mixture was quenched with a saturated aqueous NaHCO3 solution and ice. The mixture was extracted with DCM and then EtOAc. The combined organic layers are dried over Na2SO4, filtered, and concentrated. The crude material was purified by flash chromatography on silica gel (eluting with EtOAc in heptane) to afford 49 (34 mg, 56%). 1H NMR (400 MHz, CDCl3) δ ppm 8.65 (d, J = 1.9 Hz, 1H), 7.87 (dt, J = 1.9, 1.0 Hz, 1H), 6.65 (t, J = 73.1 Hz, 1H), 4.00 (s, 3H). LC-MS m/z = 282.1–284.1 [M+H].
5-Bromo-3-(difluoromethoxy)pyridine-2-carboxylic acid (50)
To a solution of intermediate 49 (30 mg, 0.11 mmol, 1 equiv) in THF (1 mL) and water (1 mL) was added LiOH·H2O (14 mg, 0.32 mmol, 3 equiv), and the reaction mixture was stirred at room temperature for 2 h. THF was evaporated, and the suspension was diluted with water. The mixture was acidified to pH 2 with a 2N HCl aqueous solution and was then extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated to afford 50 (24 mg, 85%). 1H NMR (400 MHz, CDCl3) δ ppm 8.60 (d, J = 1.8 Hz, 1H), 7.99 (dt, J = 1.9, 0.9 Hz, 1H), 6.79 (t, J = 73.0 Hz, 1H). LC-MS m/z = 268.0–270.0 [M+H].
5-Bromo-N-cyclopropyl-3-(difluoromethoxy)pyridine-2-carboxamide (47b)
To a solution of 50 (24 mg, 0.09 mmol, 1 equiv) in DMF (0.5 mL) were added HATU (38 mg, 0.10 mmol, 1.1 equiv) and DIPEA (22 μL, 0.13 mmol, 1.5 equiv), and the mixture was stirred at room temperature for 15 min. Then, cyclopropylamine (6 mg, 0.11 mmol, 1.2 equiv) was added, and the reaction mixture was stirred at room temperature for 17 h. The reaction was hydrolyzed with a saturated aqueous NaHCO3 solution and extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by flash chromatography on silica gel (eluting with heptane/EtOAc 9/1 to 7/3) to give 47b (15 mg, 54%). 1H NMR (400 MHz, CDCl3) δ ppm 8.49 (d, J = 1.9 Hz, 1H), 7.84 (dt, J = 1.8, 0.8 Hz, 1H), 7.74 (s, 1H), 6.86 (t, J = 75.4 Hz, 1H), 2.88 (tq, J = 7.4, 3.8 Hz, 1H), 0.95–0.79 (m, 2H), 0.73–0.57 (m, 2H). LC-MS m/z = 307.1–309.1 [M+H].
5-Bromo-3-methoxy-N-(2,2,2-trifluoroethyl)pyridine-2-carboxamide (47c)
To a solution of 46 (500 mg, 2.15 mmol, 1 equiv) in anhydrous DMF (8.3 mL) were added DIPEA (563 μL, 3.23 mmol, 1.5 equiv) and HATU (901 mg, 2.37 mmol, 1.1 equiv). The mixture was stirred at room temperature for 30 min, and 2,2,2-trifluoroethanamine hydrochloride (350 mg, 2.59 mmol) was added. The reaction mixture was stirred at room temperature for 20 h and was then evaporated to dryness. The residue was diluted with DCM, and a precipitate was formed. The solid was filtered, and the filtrate was concentrated in vacuo and purified by flash chromatography on silica gel (eluting with heptane/EtOAc 1/0 to 1/1) to afford 47c (562 mg, 83%). 1H NMR (400 MHz, CDCl3) δ ppm 8.27 (d, J = 1.7 Hz, 1H), 8.07 (s, 1H), 7.58 (d, J = 1.7 Hz, 1H), 4.11 (qd, J = 9.1, 6.6 Hz, 2H), 4.00 (s, 3H). LC-MS: m/z = 313.2–315.2 [M+H].
N-Cyclopropyl-3-methoxy-5-[7-(2-morpholinoethoxy)imidazo[1,2-a]pyridin-3-yl]pyridine-2-carboxamide (28)
Under an inert atmosphere, 45 (45 mg, 0.18 mmol, 1.1 equiv), 47a (45 mg, 0.17 mmol, 1 equiv), KOAc (33 mg, 0.33 mmol, 2 equiv), and Pd(dppf)Cl2·DCM adduct (7 mg, 0.008 mmol, 0.05 equiv) were suspended in dry DMAC (1.7 mL). The mixture was degassed with N2 and stirred at 110 °C for 1.5 h. Then, additional Pd(dppf)Cl2·DCM adduct (7 mg, 0.008 mmol, 0.05 equiv) was added, and the reaction mixture was stirred at 110 °C overnight. The reaction mixture was concentrated, and the crude residue was purified by flash chromatography on silica gel (eluting with MeOH, 0 to 10% in AcOEt) and preparative HPLC to afford 28 (17 mg, 23%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.48 (dd, J = 7.6, 0.7 Hz, 1H), 8.41 (d, J = 1.7 Hz, 1H), 7.79 (d, J = 1.7 Hz, 1H), 7.73 (s, 1H), 7.00 (dd, J = 2.6, 0.7 Hz, 1H), 6.79 (dd, J = 7.6, 2.5 Hz, 1H), 4.27 (t, J = 5.4 Hz, 2H), 4.01 (s, 3H), 3.76–3.69 (m, 4H), 2.95–2.84 (m, 3H), 2.65–2.60 (m, 4H), 0.89–0.81 (m, 2H), 0.69–0.62 (m, 2H). LC-MS m/z = 438.3 [M+H].
N-Cyclopropyl-3-(difluoromethoxy)-5-[7-(2-morpholinoethoxy)imidazo[1,2-a]pyridin-3-yl]pyridine-2-carboxamide (29)
Under an inert atmosphere, 45 (12 mg, 0.018 mmol, 1 equiv) and 47b (15 mg, 0.048 mmol, 1 equiv) were suspended in dry DMAC (0.5 mL), the mixture was degassed under N2, and KOAc (14 mg, 0.14 mmol, 3 equiv) and Pd(dppf)Cl2·DCM adduct (2 mg, 0.0024 mmol, 0.05 equiv) were added. Then, the medium was degassed again with N2, and the mixture was stirred at 110 °C for 2 h. The reaction medium was concentrated, and the crude residue was purified by flash chromatography on silica gel (eluting with AcOEt/MeOH 99/1 to 80/20) to afford 29 (12 mg, 55%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.75 (d, J = 1.8 Hz, 1H), 8.46 (d, J = 7.6 Hz, 1H), 8.02–7.95 (m, 1H), 7.77 (s, 1H), 7.04 (t, J = 74.1 Hz, 1H), 7.02 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 7.6, 2.5 Hz, 1H), 4.28 (t, J = 5.4 Hz, 2H), 3.77–3.70 (m, 4H), 2.93–2.85 (m, 3H), 2.70–2.61 (m, 4H), 0.91–0.83 (m, 2H), 0.73–0.64 (m, 2H). LC-MS m/z = 474.2 [M+H].
3-Methoxy-5-[7-(2-morpholinoethoxy)imidazo[1,2-a]pyridin-3-yl]-N-(2,2,2-trifluoroethyl)pyridine-2-carboxamide (30)
Under an inert atmosphere, 45 (32 mg, 0.13 mmol, 1 equiv), 47c (40 mg, 0.13 mmol, 1 equiv), KOAc (25 mg, 0.26 mmol, 2 equiv), and Pd(dppf)Cl2·DCM adduct (10 mg, 0.013 mmol, 0.1 equiv) were suspended in dry DMAC (1.3 mL). The mixture was degassed with N2 and was stirred at 110 °C overnight. The reaction mixture was concentrated, and the crude residue was purified by preparative HPLC and by flash chromatography on silica gel (eluting with MeOH, 0 to 3% in DCM) to afford 30 (20 mg, 33%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.51 (d, J = 7.6 Hz, 1H), 8.48 (d, J = 1.7 Hz, 1H), 7.84 (d, J = 1.7 Hz, 1H), 7.77 (s, 1H), 7.01 (d, J = 2.5 Hz, 1H), 6.80 (dd, J = 7.5, 2.5 Hz, 1H), 4.27 (t, J = 5.4 Hz, 2H), 4.14 (q, J = 9.3 Hz, 2H), 4.03 (s, 3H), 3.76–3.70 (m, 4H), 2.88 (t, J = 5.4 Hz, 2H), 2.67–2.59 (m, 4H). LC-MS m/z = 480.4 [M+H].
6-Bromo-2-cyclopropyl-8-fluoro-3,4-dihydroisoquinolin-1-one (52)
To a solution of 6-bromo-8-fluoro-3,4-dihydroisoquinolin-1(2H)-one 51 (500 mg, 2.05 mmol, 1 equiv) in THF (20.5 mL) were added TEA (1.43 mL, 10.24 mmol, 5 equiv), pyridine (1.33 mL, 16.39 mmol, 8 equiv) Cu(OAc)2 (818 mg, 4.10 mmol, 2 equiv), and cyclopropyl boronic acid (528 mg, 6.15 mmol, 3 equiv). The reaction mixture was stirred at 70 °C for 18 h. Cyclopropyl boronic acid (409 mg, 2.05 mmol, 1 equiv) was added, and the mixture was stirred at 70 °C for 2 h. The mixture was quenched with a saturated aqueous NaHCO3 solution and extracted with AcOEt. The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The crude was purified by flash chromatography gradient heptane/AcOEt (1/0 to 4/6) to afford 52 (207 mg, 36%). LC-MS m/z = 284.2–286.2 [M+H].
6-Bromo-2-cyclopropyl-8-methoxy-3,4-dihydroisoquinolin-1-one (53a)
To a solution of 52 (58 mg, 0.25 mmol, 1 equiv) in THF (0.58 mL) at room temperature was added dropwise MeONa (25% in MeOH, 65 μL, 0.29 mmol, 1.2 equiv), and the suspension was stirred for 1.5 h. The reaction was quenched with a saturated aqueous NH4Cl solution, and the THF was evaporated in vacuo. The aqueous phase was extracted with CH2Cl2, the organic layer was dried over MgSO4, filtered, and concentrated to afford 53a (54 mg, 77%). 1H NMR (400 MHz, methanol-d4) δ ppm 7.17 (d, J = 1.8 Hz, 1H), 7.06 (dt, J = 1.7, 0.8 Hz, 1H), 3.87 (s, 3H), 3.50 (dd, J = 7.0, 5.8 Hz, 2H), 2.88 (dtd, J = 8.1, 6.7, 3.9 Hz, 3H), 0.89 (tdd, J = 6.7, 4.8, 0.7 Hz, 2H), 0.87–0.69 (m, 2H). LC-MS m/z = 296.3–298.2 [M+H].
6-Bromo-8-methoxy-2-(2,2,2-trifluoroethyl)-3,4-dihydroisoquinolin-1-one (53b)
To a stirred solution of 6-bromo-8-methoxy-3,4-dihydroisoquinolin-1(2H)-one 54 (10 g, 39.05 mmol, 1 equiv) in THF (240 mL) at 0 °C was added dropwise a solution of LiHMDS 1N in THF (59 mL, 58.57 mmol, 1.5 equiv). The resulting mixture was stirred for 45 min at 0 °C, and 2,2,2-trifluoroethyl trifluoromethanesulfonate (8.44 mL, 58.57 mmol, 1.5 equiv) was added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 22 h. The reaction mixture was quenched with water, THF was evaporated, and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified by flash chromatography on silica gel (eluting with DCM/MeOH 100/0 to 99/1) to afford 53b (9.98 g, 76%). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.20 (d, J = 1.8 Hz, 1H), 7.13 (d, J = 1.9 Hz, 1H), 4.30 (q, J = 9.7 Hz, 2H), 3.80 (s, 3H), 3.56 (t, J = 6.2 Hz, 2H), 2.90 (t, J = 6.1 Hz, 2H). LC-MS m/z = 338.2–340.2 [M+H].
2-Cyclopropyl-6-(7-fluoroimidazo[1,2-a]pyridin-3-yl)-8-methoxy-3,4-dihydroisoquinolin-1-one (55a)
Under inert atmosphere, 7-fluoroimidazo[1,2-a]pyridine 44 (30 mg, 0.22 mmol, 1.3 equiv), 53a (50 mg, 0.17 mmol, 1 equiv), KOAc (33 mg, 0.34 mmol, 2 equiv), and Pd(dppf)Cl2·DCM adduct (7 mg, 0.008 mmol, 0.05 equiv) were suspended in dry DMAC (1.7 mL). The mixture was degassed with N2 and stirred at 120 °C for 2 h. The reaction mixture was concentrated, and the crude residue was purified by flash chromatography on silica gel (0 to 10% MeOH in DCM) to afford 55a (55 mg, 93%). LC-MS m/z = 352.4 [M+H].
6-(7-Fluoroimidazo[1,2-a]pyridin-3-yl)-8-methoxy-2-(2,2,2-trifluoroethyl)-3,4-dihydroisoquinolin-1-one (55b)
To a solution of 53b (16.6 g, 49 mmol, 1 equiv) in dry and degassed DMAC (225 mL) were added 7-fluoroimidazo[1,2-a]pyridine 44 (7 g, 51.42 mmol, 1.05 equiv), KOAc (12 g, 122.5 mmol, 2 equiv), and Pd(dppf)Cl2·DCM (2.8 g, 3.43 mmol, 0.07 equiv). The reaction mixture was placed in a preheated bath at 125 °C and was stirred at this temperature for 3 h. The solvent was concentrated in vacuo, and the crude material was purified by flash chromatography on silica gel (eluting with heptane/EtOAc 7/3 to 0/1 then EtOAc/MeOH 100/0 to 95/5) to afford 55b (14.9 g, 77%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.85–8.77 (m, 1H), 7.93 (s, 1H), 7.63–7.55 (m, 1H), 7.27 (d, J = 1.6 Hz, 1H), 7.21 (d, J = 1.5 Hz, 1H), 7.13–7.04 (m, 1H), 4.44–4.32 (m, 2H), 3.92 (s, 3H), 3.66 (t, J = 6.1 Hz, 2H), 3.02 (t, J = 6.1 Hz, 2H). LC-MS m/z = 394.2 [M+H].
2-Cyclopropyl-8-methoxy-6-[7-(2-morpholinoethoxy)imidazo[1,2-a]pyridin-3-yl]-3,4-dihydroisoquinolin-1-one (31)
To a solution of 2-morpholinoethanol (95 μL, 0.78 mmol, 5 equiv) in dry DMF (2.1 mL) was added NaH (60% dispersion in mineral oil, 19 mg, 0.78 mmol, 5 equiv), and the mixture was stirred at room temperature for 5 min. Then, 55a (55 mg, 0.16 mmol, 1 equiv) was added, and the reaction mixture was stirred at room temperature for 3 h. Then, 2-morpholinoethanol (95 μL, 0.78 mmol, 5 equiv) and NaH (60% dispersion in mineral oil, 19 mg, 0.78 mmol, 5 equiv) were added, and the reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with a saturated aqueous NaHCO3 solution and extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered, and concentrated. The crude material was purified by flash chromatography on silica gel (eluting with a gradient of 0 to 10% MeOH in DCM). The obtained residue was dissolved in DCM and washed with a 1N aqueous NaOH solution. The organic layer was passed through a phase separator and concentrated to afford 31 (34 mg, 47%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.48 (dd, J = 7.6, 0.7 Hz, 1H), 7.64 (s, 1H), 7.16 (d, J = 1.6 Hz, 1H), 7.09 (d, J = 1.5 Hz, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.75 (dd, J = 7.6, 2.5 Hz, 1H), 4.26 (t, J = 5.4 Hz, 2H), 3.93 (s, 3H), 3.78–3.71 (m, 4H), 3.55 (t, J = 6.2 Hz, 2H), 2.98 (t, J = 6.3 Hz, 2H), 2.94–2.83 (m, 3H), 2.68–2.58 (m, 4H), 0.94–0.87 (m, 2H), 0.81–0.70 (m, 2H). LC-MS m/z = 463.7 [M+H].
8-Methoxy-6-[7-(2-morpholinoethoxy)imidazo[1,2-a]pyridin-3-yl]-2-(2,2,2-trifluoroethyl)-3,4-dihydroisoquinolin-1-one (32)
To a solution of 2-morpholinoethanol (7.80 mL, 69.55 mmol, 5 equiv) in dry DMF (200 mL) at 0 °C was added NaH (60% dispersion in mineral oil, 2.00 g, 50.84 mmol, 4 equiv) portionwise, and the mixture was stirred at 0 °C for 10 min. At this temperature, 55b (5.00 g, 12.71 mmol, 1 equiv) was added, and the reaction mixture was warmed up to room temperature and stirred for 3 h. The reaction mixture was cooled down to 0 °C and quenched with a saturated aqueous NaHCO3 solution and water. The mixture was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by flash chromatography on silica gel (eluting with a gradient of 0 to 8% MeOH in DCM). The obtained material was dissolved in DCM and washed with a 2N aqueous NaOH solution twice. The phases were passed through a phase separator, and the filtrate was concentrated to afford 32 (5.77 g, 90%). 1H NMR (400 MHz, methanol-d4) δ ppm 8.51 (dd, J = 7.6, 0.7 Hz, 1H), 7.67 (s, 1H), 7.21 (d, J = 1.6 Hz, 1H), 7.13 (d, J = 1.5 Hz, 1H), 6.98 (d, J = 2.5 Hz, 1H), 6.76 (dd, J = 7.6, 2.5 Hz, 1H), 4.34 (q, J = 9.3 Hz, 2H), 4.26 (t, J = 5.4 Hz, 2H), 3.94 (s, 3H), 3.76–3.69 (m, 6H), 3.07 (t, J = 6.2 Hz, 2H), 2.88 (t, J = 5.4 Hz, 2H), 2.66–2.59 (m, 4H). LC-MS m/z = 505.4 [M+H].
Methodology
ADP-Glo Kinase Assay with SIKs
1.11 to 2.23 nM of SIK1, 0.11 to 0.48 nM of SIK2, or 0.45 nM of SIK3 was incubated with 45 μM AMARA peptide and 5 μM ATP in 25 mM Tris pH 7.5, 0.5 mM EGTA, 0.01% Triton X-100, 5 mM MgCl2, and 2.5 mM DTT at rt for 120 min in the presence or absence of compound. To determine IC50 values, compounds were tested in a 10-point dose–response with 1/5 serial dilution starting from a top concentration of 20 μM. The kinase reaction was stopped after addition of an equal volume of ADP-Glo reagent and was incubated at rt for 40 min to remove all the remaining ATP. Afterward, a double volume of kinase detection reagent was added and incubated for a minimum of 30 min at rt before luminescence signal was measured with an Envision PerkinElmer plate reader.
SIK1, SIK2, SIK3 NanoBRET Assay
Briefly, 14 million HEK293 cells were transiently transfected with 70 μL JetPEI, 2.33 μg NanoLuc-SIK1 Fusion DNA (or NanoLuc-SIK2 or NanoLuc-SIK3), and 21 μg pBlueScript and reseeded in a T175 culture flask. One day later, the transfected HEK293 cells were harvested by trypsinization and resuspended in Opti-MEM without phenol red containing NanoBRET Kinase Tracer-04 (0.5 μM for SIK1 and SIK2, 1 μM for SIK3) and 30 μM Extracellullar NanoLuc inhibitor. Serial dilutions of test compounds and references (staurosporin) were prepared in PBS to obtain final highest concentrations of 30 μM in 0.3% DMSO. Cells were seeded on top of compound at 8,000 cells per 384 well. After 2 h incubation at 37 °C, 5% CO2, NanoBRET Nano-Glo substrate was added, and BRET readout was done with an Envision PerkinElmer plate reader within 10 min after addition of the substrate by recording donor (450 nm) and acceptor (610–630 nm) emissions. The ratio of the acceptor/donor emission was calculated and multiplied by 1,000 using the Envision software to obtain data in mBRET units.
CRTC3 Translocation Assay
Briefly, 1 million U2OS cells (ATCC, HTB-96) were thawed in a T175 culture flask in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin and incubated at 37 °C, 5% CO2. Three days after thawing, U2OS cells were reseeded in the same medium in the 384-well plate at a cell density of 2,500 cells per well and incubated for 16 to 20 h at 37 °C, 5% CO2. One day after seeding, serial dilutions of test compounds and references (forskolin) were prepared in DMEM and added on top of the cells to obtain final highest concentrations of 20 μM in 0.2% DMSO. After 1 h of compound incubation on the cells at 37 °C, 5% CO2, cells were fixed with 4% formaldehyde, followed by CRTC3 immunostaining using a rabbit monoclonal antibody to human CRTC3 and an Alexa Fluor 594-labeled goat anti-rabbit secondary antibody and by nuclei staining using Hoechst 33342. Stained cells were imaged on the IN Cell Analyzer 2200, and nuclear translocation was calculated using a specific Translocation Algorithm defined in the specific High-Content Imaging Software. At the same time, nuclei staining was analyzed.
In Vitro LPS-Triggered Human Primary Monocytes Assay
Activity of 32 was evaluated on LPS-stimulated cytokine release in monocytes. Peripheral blood mononuclear cells (PBMCs) were first isolated from blood using lymphoprep-based separation, a method which is based on the lower buoyant density of mononuclear cells (monocytes and lymphocytes) compared to other blood cell types such as erythrocytes and polymorphonuclear leukocytes (granulocytes). From these PBMCs, CD14+ monocytes were selected using antibody-coated magnetic beads (Miltenyi Biotec). CD14+ monocytes were seeded in 96-well plates and preincubated with a serial dilution of 32 for 1 h before LPS triggering (Sigma-Aldrich; 100 ng/mL final concentration). TNFα (4 h), IL-12 (20 h), and IL-10 (4 h) were measured in the supernatant at indicated time points after LPS triggering using standard enzyme-linked immunosorbent assay (ELISA)-based read-outs.
In Vitro LPS-Triggered Human Primary MdM Assay
To evaluate 32 on MdMs, CD14+ monocytes (isolated as described above) were differentiated toward macrophages using macrophage-colony stimulating factor (M-CSF [Immunotools]); 100 ng/mL final concentration) during 10 days. Differentiated MdMs were preincubated with a serial dilution of 32 for 1 h before LPS triggering (100 ng/mL final concentration). Supernatant was collected at 2 h for IL-10 and 20 h for TNFα after LPS triggering and measured using ELISA-based read-outs.
Human Whole Blood Activity
Blood was collected from healthy volunteers into lithium heparin tubes. 200 μL of blood was dispensed into a polypropylene 96-well microplate and preincubated in duplicate with DMSO 0.1% or 32 at different concentrations (from 30 to 0.003 μM, 3-fold dilutions to get 0.1% DMSO at the final) for 30 min at 37 °C. After this preincubation, blood was triggered with LPS (100 ng/mL) or vehicle (RPMI) for 2 h at 37 °C. Plates were centrifuged at 750g for 10 min at 4 °C, and around 100 μL of plasma was collected and frozen at −80 °C within 30 min. The quantification of TNFα was performed on plasma diluted 6 times using the human TNF-alpha Quantikine according to the manufacturer’s instructions. The optical density (OD) was determined at 450 nm on the Spectramax i3 or Ensight. The quantification of IL-10 was done without any dilution using the V-PLEX Human IL-10 Kit, and the electrochemiluminescence (ECL) was determined on the MESO QUICKPLEX SQ 120.
Mouse Pharmacokinetics
One group of six male CD1 mice was dosed intravenously with a dose level of 1 mg/kg, and one group of three male CD1 mice was dosed orally via a single gavage with a dose level of 5 mg/kg. The mice were fasted before the oral administration. For the iv route, compound was formulated as a solution in polyethylene glycol (PEG) 200 and water for injection (60/40; v/v). For the oral route, compound was formulated as a homogeneous suspension in Solutol/methyl cellulose (MC) 0.5% (2/98; v/v). Blood was sampled by retro-orbital puncture under light gaseous anesthesia into polypropylene tubes containing lithium heparin, and plasma was prepared. 32 was quantified in plasma using LC-MS/MS.
Rat Pharmacokinetics
One group of three male Sprague–Dawley rats was dosed intravenously with 32 at a dose level of 1 mg/kg, and one group of three male Sprague–Dawley rats was dosed orally via a single gavage with a dose level of 5 mg/kg. The rats were fasted before the oral administration. For the iv route, the compound was used as dihydrochloride salt formulated in saline NaCl 0.9%. For the oral route, the compound was formulated in Solutol/MC 0.5% (2/98; v/v). Blood was sampled under light gaseous anesthesia into polypropylene tubes containing lithium heparin, and an aliquot was used to prepare plasma. 32 was quantified in blood and in plasma using LC-MS/MS.
Dog Pharmacokinetics
One group of three male beagle dogs was dosed intravenously as a slow bolus with 32 at a dose level of 1 mg/kg, and one group of three male beagle dogs was dosed orally via a single gavage with a dose level of 5 mg/kg. The dogs were fasted before the intravenous and the oral administrations. For the iv route, the compound was used as dihydrochloride salt formulated in saline NaCl 0.9%. For the oral route, the compound was formulated in Solutol/MC 0.5% (2/98; v/v). Blood was sampled from the jugular vein into polypropylene tubes containing lithium heparin, and an aliquot was used to prepare plasma. 32 was quantified in blood and in plasma using LC-MS/MS.
In Vivo Mouse LPS Challenge
32 was prepared in MC 0.5% the day before administration and gently mixed overnight at room temperature in the dark. The next day, 32 was administered orally to Balb/cN female mice at 1, 3, 5, 10, 30, and 60 mg/kg. Fifteen minutes later (corresponding to the Tmax of the pharmacokinetic of 32), 100 μg of LPS (in 0.2 mL of H2O) was injected intraperitoneally to mice. A control group was included with MC 0.5% po without LPS challenge. Mice were sacrificed 1.5 h after LPS challenge, and blood was collected by carotid exsanguination in EDTA tubes. Plasma samples were obtained by centrifugation for 15 min, 2,000g at +4 °C and frozen at −80 °C before cytokine quantifications and bioanalysis. IL-10 and TNFα were quantified by alphaLISA according to the manufacturer’s instructions. Optical densities were determined using Ensight (PerkinElmer). Statistical analysis was performed on raw data or log transformed data (a group with another compound was removed from the graph and for statistical analysis). The normality of residuals and equality of variances for a parametric analysis were checked. Statistical analysis of plasma TNFα levels was performed with a Kruskal–Wallis and Dunn’s post-test: *p < 0.05; ***p < 0.001. Statistical significance of plasma IL-10 levels was calculated using ANOVA and Dunnett’s multiple comparison test: ***p < 0.001. Statistics were done versus LPS + vehicle group (***: p < 0.001; **: p < 0.01; *: p < 0.05).
Dextran Sodium Sulfate (DSS)-Induced Colitis Model
Balb/cJ female mice (Janvier, Le Genest-Sant-Isle, France) were housed in a dedicated in-house animal facility under specific pathogen-free conditions according to the Federation for Laboratory Animal Science Association guidelines. The study as performed according to ethical guidelines approved by the animal Institutional Care and Use Committee of Galapagos controlled by the French Authorities (Agreement No. 93-063-06, DDPP, Seine Sant Denis). Animals were housed in filter top cages, provided with filtered tap water and standard chow ad libitum, and maintained at 22 ± 2 °C in 55 ± 10% humidity on a 12 h light/dark cycle. The randomization of mice in the different groups was based on BW at study start. A model of chronic colitis was induced in 6- to 8-week-old mice with two DSS cycles, consisting of 4% DSS administered in drinking water for 4 days followed by 3 days of regular drinking water followed by 4 days of DSS 4%. The mice (n = 10 per group) were orally administered vehicle (methyl cellulose 0.5%) or 3, 10, or 30 mg/kg of 32 b.i.d. from day 1 of DSS administration and throughout the dosing period. Steady-state PK was performed on day 9. For each group treated with 32, each mouse was sampled once at one of the selected time points: 0 h (n = 3), 0.25 h (n = 3), 1.5 h (n = 2), 6 h (n = 3); then the average plasma concentration at each time point was used to perform PK analysis. Clinical parameters were measured every other day. DAI (a combined score of BW loss, stool consistency, and fecal blood loss) was recorded daily (except on weekends). Each criterion was graded from 0 to 4. Animals were sacrificed by cervical dislocation after isoflurane anesthesia at day 12 of the experiment. For histological analysis, colon tissues were harvested, fixed in buffered formalin, and embedded in paraffin. Sections of 4 μm were cut and stained with PAS using a standard procedure. To evaluate colon lesion by histology, MCHI scoring was performed. Eight histological components were assessed: inflammatory infiltrate, goblet cell loss, crypt hyperplasia, crypt density, muscle thickness, submucosal infiltration, ulcerations, and crypt abscesses (all categorized from 0 to 3). A total MCHI score was obtained by summing up the scores from the eight different components.35 Statistical analysis was done with a one-way ANOVA performed on the Log(Y) AUC of DAI data transformation without intact vehicle group. Dunnett’s post hoc multiplicity correction procedure was applied to the AUC of DAI data. For MCHI, statistical analysis was performed using a one-way ANOVA without intact vehicle group followed by a Dunnett’s posthoc multiplicity correction procedure.
CYP Inhibition Assay
A 5 mM stock solution of 32 was prepared in methanol. This stock was further serially diluted 1:3 in methanol and then added to a mixture containing 50 mM potassium phosphate buffer pH 7.4, human liver microsomes, and probe substrate. After prewarming for 5 min at 37 °C, the reaction was started by adding cofactor mix (7.65 mg/mL glucose-6-phosphate, 1.7 mg/mL NADP, 6 U/mL of glucose-6-phosphate dehydrogenase), resulting in seven final concentrations of 32 in the range 0.137–100 μM (2% methanol). Final concentrations of cofactor mix components were as follows: 1.56 mg/mL glucose-6-phosphate, 0.34 mg/mL NADP, 1.2 U/mL of glucose-6-phosphate dehydrogenase. After incubation at 37 °C for 5–15 min, the reaction (aliquot of 50 μL) was terminated with 150 μL acetonitrile/methanol (2/1) solution with internal standard (warfarin for 2C9, diclofenac for all other tested isoforms). Samples were centrifuged and the supernatant fractions analyzed by LC-MS/MS. The instrument responses (ratio of probe substrate and internal standard peak areas) were referenced to those for solvent controls (assumed as 100%) in order to determine the percentage reduction in probe metabolism.
CYP Time-Dependent Inhibition
A 5 mM stock solution of compound to be assessed was prepared in methanol. This stock was serially diluted 1:3 in methanol and then added in duplicates to mixture containing 50 mM potassium phosphate buffer pH 7.4 and human liver microsomes. A total of 7 different concentrations (0.14–100 μM in final reaction mixture; 2% methanol) of compound were prepared in duplicates. After prewarming for 5 min at 37 °C, the 20 min preincubation was started by adding a probe substrate (midazolam or testosterone) to the first concentration range (first replica) and a cofactor mix (7.65 mg/mL glucose-6-phosphate, 1.7 mg/mL NADP, 6 U/mL of glucose-6-phosphate dehydrogenase) to the second replica. Final concentrations of cofactor mix components were as follows: 1.56 mg/mL glucose-6-phosphate, 0.34 mg/mL NADP, 1.2 U/mL of glucose-6-phosphate dehydrogenase. After preincubation completion, the reaction was finally started by adding the cofactor mix to the first replica and probe substrate to second replica (opposite to preincubation step). After incubation at 37 °C for 5–15 min, the reaction (aliquot of 50 μL) was terminated with 150 μL acetonitrile/methanol (2/1) solution with internal standard (diclofenac). Samples were centrifuged and the supernatant fractions analyzed by LC-MS/MS.
The instrument responses (ratio of probe substrate and internal standard peak areas) were referenced to those for solvent controls (assumed as 100%) in order to determine the percentage reduction in probe metabolism.
CYP Induction
Cryopreserved human hepatocytes from a single donor are seeded on a 96-well collagen coated plate so that the final seeding density is 0.1 × 106 cells/well (final volume per well 0.1 mL). The cells are then incubated in seeding medium at 37 °C, 95% humidity, 5% CO2 to allow the cells to attach. After 4 h, the seeding medium is replaced with 0.1 mL of prewarmed serum-free Williams E medium (William’s E containing 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 μg/mL insulin, 2 mM l-glutamine, and 0.1 μM hydrocortisone). The next day, cells are dosed with test compound in assay medium (final test compound concentration 10 μM; final DMSO concentration 0.1%). Positive control inducer, rifampicin for CYP3A4, is incubated alongside the test compound. Negative control wells are included where the test compound is replaced by vehicle solvent (typically 0.1% DMSO in assay medium). Each test or control compound is dosed in triplicate at a single concentration. The cells are exposed to the solutions for 72 h with fresh solution added every 24 h. For mRNA assessment, all media is removed from each of the wells, and the cells were washed once with 0.1 mL of prewarmed assay medium. The cells are lysed by adding 100 μL of lysis buffer to each well. Total RNA is then isolated from the hepatocyte lysates. Reverse transcription is performed, and quantitative PCR analysis is performed on the resulting cDNA, using gene-specific primer probe sets for CYP3A4 target cDNA and endogenous control. Samples are analyzed using an ABI 7900 HT real-time PCR system. For mRNA assessment, relative fold mRNA expression is determined based on the threshold cycle (CT) data of target gene relative to endogenous control for each reaction and normalized to negative control using the 2-ΔΔCT. Data are expressed as fold activation relative to the vehicle control and, as a percent, to the 10 μM rifampicin using the following formula:
 |
hERG Channel Test (Manual Patch Clamp Assay)
The effects of 32 at the nominal concentrations of 0.1, 1, 10, and 100 μM on the delayed rectifier potassium current (IKr) encoded by hERG were studied at a stimulation frequency of 6 pulses/min (0.1 Hz) in 6 different stably transfected HEK293 cells. A vehicle group (four superfusion periods with 0.3% DMSO in extracellular solution) was included in the study for comparison, and terfenadine (nominal concentration of 1 μM) was used as reference substance.
Mutagenicity Test
Compound 32 was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537, and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver postmitochondrial fraction (S9), in a single experiment. Treatments of all tested strains were performed using final concentrations of 32 of 5, 16, 50, 160, 500, 1,600, and 5,000 μg/plate, plus vehicle and positive controls.
Without metabolic activation: the following procedure was followed for each strain used in the test. Each bacterial suspension was from a culture that was agitated overnight at ca. 37 °C. Five to 20 μL of the test item at the relevant concentrations, 0.1 mL of the bacterial suspension, and 95 μL of PBS were successively added. The plates were incubated at ca. 37 °C under stirring for 90 min. Then, the content of each well was transferred in 2 mL of top agar, supplemented with 10% of 0.5 mM biotin histidine solution, maintained in a state of superfusion at ca. 45 °C. The contents of each tube were agitated, and then spread out in Petri plates containing 20 mL of minimum agar. For each strain and incubation condition, each dose was tested in three plates: after incubation at ca. 37 °C for about 68 h, the number of revertant colonies was determined for each plate.
With metabolic activation: The method was the same as described above, except that the PBS was replaced by 95 μL of the S9-mix metabolic activation system.
Modeling
All calculations were carried out using Schrödinger software suite, release 2017–3.36
Ligand Docking
All docked compounds were built and protonated using LigPrep software,37 whereas ionization states at pH 7 ± 2 were calculated with Epik.38,39
For the internal SIK3 X-ray structure, hydrogen atoms were added to the protein through the Protein Preparation Wizard tool.40 In order to optimize the H-bond network, the most probable protonation state of the residues was carefully selected by visual inspection, and hydrogen atoms were minimized using OPLS3 force field.41 Before running the docking procedure, all water molecules present in the structure were removed.
Docking of the ligands was carried out with Glide.42−44 A docking grid was generated using SIK3 prepared structure. The cocrystallized ligand was selected as the center of the grid, a H-bond constraint with the hinge H-bond donor (Ala145 NH for SIK3) was created, whereas the rest of the settings were kept as default. For the docking run, the flexible docking standard precision (SP) option was selected, together with an enhanced sampling protocol (four times) of the ligands. The constraint was applied to all docking runs, whereas the number of poses to return was set as three for each ligand.
The binding modes were then selected based on spatial geometries of the ligand within the binding cavity, complementarity with the pocket (shape and electrostatic complementarity), H-bond geometries of the protein–ligand interactions, and docking score.
Homology Models
Homology models for SIK1 and SIK2 were built using the kinase domain of the internal X-ray structure of SIK3 as template (residues 59 to 339) using Prime45 as part of the Schrödinger suite. The native ligand was kept in the active site, and only the side chains of non-matching residues were rebuilt. A single knowledge-based model was requested. The resulting structure was further refined using Prime’s Refine Protein–Ligand Complex tool: the residues within 5 Å of the ligand and the ligand were submitted to an implicit-solvent minimization using a local optimization sampling algorithm. The variable-dielectric generalized Born model (VSGB) for water was selected together with a dielectric constant of 80. The OPLS3 force field was used.
Molecular Dynamics Simulations
Molecular dynamics simulations were run using the Desmond46 package included in the Schrödinger suite. The UBA domain of the internal SIK3 X-ray structure was removed to keep only the kinase domain (residues 59 to 339). The protein structures obtained by homology modeling were used for SIK1 and SIK2. Ligand geometries were obtained from the docking studies and were parametrized using OPLS3 force field. The complexes generated were initially solvated in a cubic box with SPC water molecules leaving at least 10 Å between the solute atoms and the border of the box. The systems were then neutralized with 7 Cl– counterions (8 in the case of positively charged ligands), and NaCl salt concentration of 0.15 M was added. The systems generated were equilibrated and gradually heated from 0 to 300 K using Desmond default equilibration protocol. After the equilibration, the systems were subjected to 50 ns MD simulations in the NPT ensemble at 1.01325 bar (by Martyna–Tobias–Klein barostat) and 300 K (by Nosé–Hoover chain thermostat), setting a cutoff of 9 Å for the short-range non-bonded interactions. Trajectories were saved every 100 ps, and for each system two replicas, with different starting velocities (random), were run. Analysis of the simulation was based on protein and ligands RMSD values, H-bond occupancy along 100 ns simulation, and visual inspection of the binding geometries.
Acknowledgments
The authors would like to thank Nicolas Houvenaghel and Nele Vandervoort who actively contributed to the investigation, formal analysis, and validation of data, and Philippe Clement-Lacroix and Thierry Christophe who actively contributed to data curation, formal analysis, visualization, and validation. Editorial support was provided by Aaron Borg, Ph.D., of PharmaGenesis London, London, UK, funded by Galapagos NV (Mechelen, Belgium). Publications management was provided by John Gonzalez, Ph.D., a consultant funded by Galapagos NV.
Glossary
Abbreviations Used
- AMPK
adenosine monophosphate-activated protein kinase
- BMDC
bone marrow-derived dendritic cell
- BMDM
bone marrow-derived macrophage
- BMMC
bone marrow-derived mast cell
- BW
body weight
- CV
coefficient of variation
- CREB
cAMP-response element binding protein
- CRTC
CREB-regulated transcriptional coactivator
- DAI
disease activity index
- dd
doublet of doublets
- ddd
doublet of doublet of doublets
- DMAC
N,N-dimethylacetamide
- dq
doublet of quartets
- DSS
dextran sodium sulfate
- dt
doublet of triplets
- dtd
doublet of triplet of doublets
- dioxane
1,4-dioxane
- DIPEA
N,N-diisopropylethylamine
- DDI
drug–drug interaction
- Et2O
diethyl ether
- EtOAc
ethyl acetate
- EtOH
ethanol
- FLDM
fetal liver-derived macrophage
- HATU
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
- HDAC
histone deacetylase
- IBD
inflammatory bowel disease
- IL
interleukin
- LKB1
liver kinase B1
- LPS
lipopolysaccharide
- MC
methyl cellulose
- MCHI
mouse colitis histology index
- MdM
monocyte-derived macrophage
- MeCN
acetonitrile
- MeOH
methanol
- PAS
periodic acid–Schiff
- PBMC
peripheral blood mononuclear cell
- ND
not determined
- Pd(PPh3)4
tetrakis(triphenylphosphine)palladium(0)
- Pd(dppf)Cl2·DCM
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1:1)
- PEG
polyethylene glycol
- PKA
protein kinase A
- RuPhos
dicyclohexyl(2′,6′-diisopropoxy-[1,1′-biphenyl]-2-yl)phosphine
- SEM
standard error of the mean
- SIK
salt-inducible kinase
- SNAr
nucleophilic aromatic substitution
- tBuXPhos
2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl
- TDI
time-dependent inhibition
- TNFα
tumor necrosis factor alpha
- Xantphos
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
Supporting Information Available
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c02246.
Author Contributions
All authors contributed to the design of the studies and the acquisition, analysis, or interpretation of the data. All authors contributed to manuscript development and approved the final version for submission.
This work was completed and funded by Galapagos.
The authors declare the following competing financial interest(s): All authors were employees of Galapagos at the time of the work. W.L., A.P.F., M.D., M.L.R., E.W., C.R.M., S.D.V., M.A., D.A., and N.D. are employees of Galapagos. C.P., A.J., T.T.L., A.T., A.D.S., H.J., S.L., C.D., D.M., C.S., and S.D. were employees of Galapagos at the time of the work, and are employees of NovAliX. A.M., D.B., and M.B. were employees of Galapagos at the time of the work. R.B. was an employee of Galapagos at the time of the work and is an employee of Agomab Therapeutics. J.-M.J. was an employee of Galapagos at the time of the work and is an employee of The Janssen Pharmaceutical Companies of Johnson & Johnson.
Supplementary Material
References
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