Discovery and Optimization of LATS1 and LATS2 Kinase Inhibitors for Use in Regenerative Medicine

Abstract

The Large Tumor Suppressor Kinases 1 and 2 (LATS1/2) are serine/threonine kinases that play an essential role in Hippo pathway activation and influence multiple physiological events ranging from organ growth to tissue regeneration. As a result, pharmacological inhibition of LATS1/2 represents a promising strategy for therapeutic intervention in multiple indications. Within, we present the discovery of potent and selective inhibitors of the kinases LATS1 and LATS2. Using a scaffold hopping strategy from the reported AKT inhibitor AT-7867 (1) we pursued a series of structure activity relationship (SAR) studies that dramatically improved potency versus LATS1 and LATS2, while concurrently improving kinome-wide selectivity. ADME properties were further optimized via introduction of conformational restriction into the target molecule, to lead to compound 27 which possesses potent inhibitory activity against both LATS1 and LATS2 and proof of concept activity in wound healing models.

Introduction

The Hippo pathway is a highly conserved signaling pathway that regulates cell growth and proliferation and acts as a central regulator of organ size control and tissue homeostasis during development. The pathway responds to a wide range of external and internal cellular signaling elements, including cell polarity, cell–cell contact, mechanical stresses, hormonal factors, and signals from Notch and Wnt, such that when the pathway is activated, it greatly limits cell proliferation.

The core of the canonical Hippo pathway is a kinase cascade whereby cellular signaling mechanisms feed into the Mammalian sterile 20-like kinases 1 and 2 (MST1/2). MST1/2 in turn phosphorylate and activate two central kinases, the Large Tumor Suppressor Kinases 1 and 2 (LATS1 and LATS2). Activated LATS1 and LATS2 (LATS1/2) then phosphorylate the Yes-associated protein 1 (YAP1) at two key sites, serine 397 and serine 127. Phosphorylation at these two sites promotes YAP1 proteasomal degradation and 14–3–3 dependent cytoplasmic sequestration, respectively.

However, when LATS1/2 are inactivated, such as under conditions of low cell density, YAP1 is allowed to translocate into the nucleus and associate with the transcriptional enhanced associate domain (TEAD). This triggers a gene expression program responsible for cell growth and proliferation. Inhibiting LATS1/2 provides a pharmacological means to activate the cellular machinery that promotes cell growth and proliferation for a variety of medical indications, including regenerative medicine.

Regenerative medicine is a broad field of study with remarkable potential to positively impact multiple states of disease, injury and aging. Utilizing LATS1/2 inhibition to inactivate the Hippo pathway and promote cell growth and proliferation is a promising strategy for a variety of concepts within the broader regenerative medicine field. LATS inhibitors could have impact on indications including skin wounds, burns, cardiac repair, and liver regeneration. Many of these indications represent areas of large unmet medical need including burn wounds, where 180,000 patients are estimated to die worldwide every year and diabetic foot ulcers, which has a patient population of 1.6 million people in the United States and a 5-year mortality rate of 30%. Unfortunately, limited treatment options are currently available for the affected individuals.

Alternate use of LATS inhibitors may impact more varied and unconventional areas. Rudolf et al. showed that conditional deletion of LATS1/2 can mediate hair cell regeneration. Several groups have demonstrated that LATS inhibition can enhance the activity of other chemotherapeutics to treat selected cancers. − Finally, aberrant LATS activation has been implicated in the pathology of Huntington’s disease. ,

Given the potential therapeutic value, there is expanded interest and progress in the development of LATS1/2 inhibitors (Figure ). Kastan et al. have developed both TRULI and its improved analog TDI-011536 (2) which has demonstrated in vivo phospho-YAP1 reduction at 200 mg/kg administration. Ma and co-workers have disclosed VT02956 (3) with a reported cellular IC₅₀ of 0.16 μM in HEK293A cells to reduce downstream YAP phosphorylation. Aihara et al. and Namoto et al. have more recently developed GA-017 and NIBR LATSi respectively, as small molecule inhibitors of LATS1/2. Finally, a number of pharmaceutical companies have patented LATS1/2 inhibitors, including Niven Therapeutics (4), Novartis Pharmaceuticals (5) and Genentech, Inc. (6).

1.

Chemical starting point and known LATS inhibitors.

Aiming to establish additional agents for the study of this important target, we report here the discovery of a series of potent and selective LATS1/2 inhibitors developed using a scaffold hopping strategy from the reported AKT inhibitor AT-7867 (1).

Results and Discussion

Chemical Starting Point and Initial SAR

Most kinase inhibitors target the highly conserved ATP-binding pocket and consequently target promiscuity is a common feature even for clinical stage kinase inhibitors. Off-target activity can confound phenotypic understanding of these agents and be a causative feature of drug toxicity. Target promiscuity can, however, be exploited to establish novel inhibitors of underrepresented kinase targets using a strategy referred to as scaffold hopping. We utilized an internal selectivity profile of approved and investigational kinase inhibitors as a starting point to select a lead for LATS1/2 optimization. Several identified compounds had notable activity against LATS1/2 (Supporting Table 1). These compounds were evaluated for LATS1/2 activity, kinase promiscuity, molecular weight, lipophilicity, number of rotatable bonds, total polar surface area and fraction sp³, in addition to synthetic tractability. From this profile, the AKT inhibitor 1 represented an attractive starting place for our SAR campaign based on multiple factors, but most notably a lack of kinase promiscuity, synthetic tractability and relative molecular simplicity.

Initial structure–activity relationship studies (Table ) focused on the hinge binding element of compound 1, the pyrazole functionality. Previous crystallographic studies of 1 bound to AKT identified the pyrazole as the hinge binder. To identify an optimized hinge binding moiety for possible improvements to LATS1/2 potency, we synthesized and screened alternative hinge binding heterocycles (Supporting Table 2). Ultimately, the pyrrolopyridine in compound 7 was identified as the only group that significantly increased the inhibitory activity of the compound against LATS1/2. This unfortunately came at the cost of also increasing activity against AKT. Next, the 4-chlorophenyl group was examined. Ultimately, its removal was found necessary to gain selectivity over AKT, giving compound 8. This is consistent with the observation that the 4-chloro group forms a key binding interaction with AKT. The pyrrolopyridine was then re-examined and it was found that a wide range of substituents at the 3-position were tolerated. Ultimately, the fluorine demonstrated the best increase in activity, while also maintaining a degree of metabolic stability, leading to compound 9. Finally, re-examination of the benzylic position revealed that the installation of a hydroxyl group (analog 10) gave a modest boost in activity, while not adversely affecting selectivity. Analogs with larger alkyl, aryl, or heterocyclic groups reintroduced into this position led to a significant increase in AKT activity (data not shown). Notably, there was a large difference between the observed biochemical activity and observed cellular activity, especially for compound 8. One potential reason for this would be poor compound permeability. However, when permeability was assessed via PAMPA (Table ), it became apparent that low permeability was not the root cause for the difference between the cellular and biochemical potencies.

1. Initial SAR Development.

^aBiochemical IC₅₀ determined via ϒ-³³P functional assay.

^bCellular IC₅₀ determined by NanoBRET assay.

^cFold change in CYR61 mRNA expression after treatment of 5 μM compound for 4 h in HepG2 cells, relative to DMSO control.

^dApparent permeability in a parallel artificial membrane permeability assay, in units of 1 × 10^–6 cm/s.

^eAverage 3 replicates.

^fAverage 2 replicates.

A Shift to Cellular Assays as a Primary SAR Readout

While the successful establishment of highly potent LATS1/2 inhibitors from these early SAR examinations was gratifying, we realized that the lower limit of quantitation for the biochemical assay system would be restrictive. Thus, we integrated two cellular assay systems as the primary target informing readouts. First, a NanoBRET cellular assay was utilized to directly measure the displacement of a cellular probe from target enzymes LATS1 and AKT, to provide more informative IC₅₀ values and demonstrate target engagement in a cellular environment. Second, quantitative real-time polymerase chain reaction (qPCR) data was generated for three target genes downstream of LATS1/2 (AMOTL2, CTGF and CYR61) as a proxy for the effect of LATS1/2 inhibition on gene expression. Testing the analogs listed in Table with these cellular assays demonstrated none had a cellular IC₅₀ of less than 1 μM against LATS1. Additionally, apart from compound 7, it was observed that even at 5 μM, none of the compounds elicited a significant increase in CYR61 expression by qPCR. Notably, the starting point 1 had a similar cellular AKT activity in the NanoBRET assay to the reported values measured by phosphorylation of GSK3β.

Modification of the Piperidine Domain

With cellular assays established, we sought to further improve the inhibition of our target small molecules against LATS1/2. We examined the impact of amine positioning with the 3-piperidine (11), 3-pyrrolidine (12), 3-azetidine (13) and 4-azepane (14) compounds (Table ). Interestingly, only relatively modest changes in activity were observed. Removing entropic degrees of freedom can increase the relative activity of a molecule. Therefore, we incorporated more rigidified versions of the piperidine scaffold. Our initial attempts included bridgehead system 15, as well as the spiro[3.3]­heptane analog 16, which often lowers lipophilicity relative to piperidine analogs. Unfortunately, a modest loss of activity was observed in both cases.

2. Modification of the Piperidine Domain.

^aBiochemical IC₅₀ determined via ϒ-³³P functional assay.

^bCellular IC₅₀ determined by NanoBRET assay.

^cFold change in CYR61 mRNA expression after treatment of 5 μM compound for 4 h in HepG2 cells, relative to DMSO control.

^dAverage 2 replicates.

We next installed methyl groups on the piperidine ring to alter ring conformation equilibrium. Encouragingly, analog 17 (single diastereomer, of undefined relative stereochemistry) did result in a significant gain of activity.

Installation of two methyl groups afforded compound 18 (stereochemistry defined as shown) which demonstrated even greater potency improvements, reaching submicromolar IC₅₀ values in the LATS1 NanoBRET assay and greatly improved CYR61 expression as judged by qPCR. From the assessment of this data, it appeared that we needed to pass a threshold on the NanoBRET cellular activity in order to observe meaningful qPCR induction.

Our hypothesis was that syn-methyl groups on the piperidine would shift the piperidine ring strongly toward a single chair conformation, due to the unfavorable syn-pentane interaction in its alternate conformation. Alternative explanations include the possibility that the methyl groups are interacting via hydrophobic interactions with the enzyme, or potentially helping to lock the aryl ring in a single conformation.

Assessment of the Central Phenyl Group

To further improve the potency of these compounds, the central phenyl group was explored (Table ). Installation of a single fluorine (19) afforded modest improvements in NanoBRET potency and in activity in the qPCR assay at 1 μM. Replacement of the fluorine with a chlorine (20) gave a modest improvement in activity in the qPCR assay, with methyl substitution (21) being even more advantageous in both the NanoBRET and qPCR assays. This, however, came at the cost of lower microsomal stability (mouse). To improve metabolic stability while maintaining a good activity profile, both methyl and fluoro substituents were added to the central phenyl ring to afford analog 22. This compound maintained moderate metabolic stability, while demonstrating good activity in the qPCR assay, and submicromolar activity in the NanoBRET assay. Within this series (18 to 22) we observed a good correlation between the cellular NanoBRET activity and the qPCR fold-change induction, while steadily improving the permeability.

3. Piperidine Functionalization.

^aBiochemical IC₅₀ determined via ϒ-³³P functional assay.

^bCellular IC₅₀ determined by NanoBRET assay.

^cFold change in CYR61 mRNA expression after treatment of 1 μM compound for 4 h in HepG2 cells, relative to DMSO control.

^dMouse liver microsomal stability, half-life.

^eApparent permeability in a parallel artificial membrane permeability assay (PAMPA), in units of 1 × 10^–6 cm/s.

^fAverage 3 replicates.

^gAverage 2 replicates.

Functionalization of the Piperidine Nitrogen

We next sought to further optimize cellular potency and especially the ADME properties in this series via modification of the piperidine NH substituent (Table ). N-Methyl compound 23 led to a slight drop in potency, and was less metabolically stable, as was the isopropyl-modified compound 24. Interestingly, we observed that the basicity of the nitrogen could be reduced with compound 25 or eliminated entirely with the amide 26 and activity could be retained, albeit at lower levels than the N-methyl compound 23. However, we observed that these groups provided minimal improvements to metabolic stability. Examination of a cyclic sulfone analog (27) and a glycine-modified analog (28) proved beneficial as both agents possessed good on-target activity profiles (biochemical and cell-based) with reasonable selectivity over AKT. Both agents also displayed good metabolic stability (MLM T _1/2 of 45.9 and 84.1 min respectively for 27 and 28). Sulfone 27 exhibited high membrane permeability while the glycine 28 was acceptable. One potential concern during this series of modifications (23 to 28) was that the observed cellular activity and qPCR fold induction were not as well correlated. There are potential reasons for this, including the fact that these two orthogonal assays were in different cell lines, and that the qPCR induction can be affected by alternate biochemical inputs and not just LATS1/2. Ultimately the goals of this series; increasing microsomal stability and PAMPA, while maintaining activity; were achieved. Given these promising outcomes, we advanced both 27 and 28 into a wider panel of assays and phenotypic assessments.

Validation of Target Engagement and Biophysical Parameters

Expanding our assessment of compounds 27 and 28, we next examined their activity versus LATS2 and AKT utilizing NanoBRET. We also examined their activity in time-resolved fluorescence energy transfer (TR-Fret) assays for LATS1, LATS2 and AKT to determine K _d values and residence times. We also profiled 27 and 28 in additional ADME examinations including aqueous solubility, Caco-2 permeability, and protein plasma binding. Gratifyingly, 27 and 28 both exhibited single digit nanomolar biochemical IC₅₀ values against LATS1 and LATS2 with good selectivity relative to AKT. This accomplished the desired goal, going from a compound which was more active against AKT1 compared to LATS1 (1, Table ), to a compound which had >50:1 selectivity for LATS1 versus AKT1 (27, 28, Table ). The LATS2 NanoBRET assays confirmed good activity for both 27 and 28. The TR-Fret binding assays revealed binding constants in the low nanomolar to subnanomolar range and residence times varying from 7 to 27 min for both compounds versus LATS1 and LATS2. Both agents possessed good aqueous solubility and reasonable membrane permeability as judged by the Caco2 assay. Both agents had limited, but acceptable, free fraction (FU) levels in mouse plasma (Table ).

4. Selected Activity and ADME Properties for Compounds 27 and 28 .

assay	compound 27	compound 28
biochemical LATS1 IC50 (nM)	4.4 (1.7)	1.8 (1.2)
biochemical LATS2 IC50 (nM)	5.5 (3.6)	2.6 (0.8)
biochemical AKT1 IC50 (nM)	346 (102)	165 (45)
cellular LATS1 IC50 (nM)	136	6.1
cellular LATS2 IC50 (nM)	36.0	5.1
cellular AKT1 IC50 (nM)	31,900	3440
TR-Fret LATS1 K d (nM)	2.5	0.9
TR-Fret LATS1 τ (min)	9.1	7.3
TR-Fret LATS2 K d (nM)	1.7	0.49
TR-Fret LATS2 τ (min)	16	27
TR-Fret AKT K d (nM)	724	244
TR-Fret AKT τ (Min)	0.56	1.0
solubility (μg/mL)	>50	11.9
Caco2 permeability	14.8	5.2
PPB (FU)	3.1%	1.4%

^aBiochemical IC₅₀ determined via ϒ-³³P functional assay.

^bCellular IC₅₀ determined by NanoBRET assay.

^cBinding constant or resonance time in a TR-Fret assay.

^dAqueous kinetic solubility.

^ePermeability in units of 1 × 10^–6 cm/s.

^fFree fraction in a protein plasma binding assay in mouse plasma.

^gAverage of 3 different batches.

^hAverage of two different batches.

ⁱStandard deviation.

Pharmacokinetic Analysis

Key compounds 27 and 28 exhibited good pharmacokinetic profiles when dosed in mice via IV, PO, or IP administration. (Table ). Both compounds demonstrated low plasma clearance when dosed intravenously at 1 mg/kg, and acceptable oral bioavailability at 3 mg/kg. In both cases AUC values were in an acceptable range, while terminal half-lives were longer than 2 h in all cases. Compound 28 displayed a better overall pharmacokinetic profile when dosed IV and PO. This may be due to its better stability in mouse liver microsomes compared to compound 27, (Table ) or potentially due to its higher protein plasma binding (Table ). However, compound 27 displayed a better pharmacokinetic profile when dosed interperitoneally (IP) at 30 mg/kg, with a longer T _1/2 and higher AUC than compound 28.

5. Pharmacokinetic Parameters,

	compound 27			compound 28
	IV (1 mg/kg)	PO (3 mg/kg)	IP (30 mg/kg)	IV (1 mg/kg)	PO (3 mg/kg)	IP (30 mg/kg)
F%	NA	37.7%	NA	NA	30.7%	NA
Clp_Obs	5.86	NA	NA	0.775	NA	NA
AUC(0–24h) d	3023	3224	119,816	18,433	16,857	71,745
cMax	1620	750	15,900	1975	1260	13,000
V ss obs	0.935	NA	NA	0.529	NA	NA
t 1/2	2.6	2.5	24.3	9.0	8.5	6.7

^aDosing in male mice at the indicated route of administration and dosing. Formulation and other details available in SI.

^bOral Bioavailability.

^cObserved plasma clearance in mL/min/kg.

^dArea under the curve from 0–24 h, in h* ng/mL.

^eMaximum plasma concentration in ng/mL.

^fObserved volume at steady state in L/kg.

^gTerminal half-life, in hours.

Selectivity and Kinome Profiling

To fully assess the kinase selectivity of compounds 27 and 28, the two compounds were tested in two orthogonal selectivity assays. The first was a biochemical panel of 370 wild-type kinases via a functional ϒ-³³P ATP phosphorylation assay at two compound concentrations, 100 and 15 nM (Figure ). This assay revealed that both compounds had off-target activities primarily within the AGC subcluster. Notable off-targets included ROCK1/2, NDR1/2, PKG1, and DMPK2.

2.

Kinome selectivity for compounds 27 and 28 via both a functional biochemical kinase panel at 15 and 100 nM (A) and a KiNativ assessment at 1 μM compound concentration (B).

Compounds 27 and 28 were also examined using a previously published in situ assessment of target selectivity (i.e., the KiNativ assay), at three concentrations (10, 1, 0.1 μM) utilizing HEK 293T lysate. The KiNativ assessment is more physiologically relevant as it examines kinome selectivity in the cellular context involving physiological levels of ATP and ADP, as well as the various full length kinases. Within this assay, both compounds demonstrated exceptional selectivity for LATS1/2 (Figure ). Notably at 1 μM, compound 27 was observed to have five off-target kinases at >35% inhibition, while compound 28 was observed to have just two off-target kinases at >35% inhibition, out of approximately 210 kinases expressed and identified in the HEK293T lysates (see Supporting Information). Off targets included AMPK and CDK8/CDK11 for compound 27, and MSPK1 and JAK1 for compound 28. Interestingly, while both ROCK1/2 and NDR1/2 were present and captured using this methodology, neither 27 nor 28 demonstrated significant binding affinity.

In Vivo Pharmacodynamic Biomarker Assessment

Given the good in vitro and cellular activities observed for both compounds 27 and 28, as well as their solid pharmacokinetic properties, both compounds were assessed for their activity against LATS1 and LATS2 in CD-1 mice. Since qPCR induction models demonstrated good efficacy after a 4 h treatment, our pharmacodynamic assessment was also done at 4 h. Both compounds, 27 and 28 were dosed intraperitoneal in male CD-1 mice 30 mg/kg. Four hours post dose, the mice were euthanized, and homogenized liver samples were analyzed by Western blot to assess the levels of phospho-YAP1 (Figure ). Gratifyingly, both 27 and 28 demonstrated marked reduction in phospho-YAP1 (S397). At serine 127, compound 28 also demonstrated marked reduction in phosphorylation of YAP1. By contrast, compound 27 demonstrated modest reduction, at best, with respect to a reduction in phospho-YAP1 (S127). One potential reason for the apparent superiority of 28 within this assessment is the superior NanoBRET activity for 28 against LATS1. Total YAP1 was also examined, but no major differences were found between the control groups and treatment groups.

3.

Phosphorylation status of YAP1 at serine-397 and serine-127 and total YAP1 in the liver of CD-1 mice 4 h after treatment with either Compound 27 (30 mg/kg) or Compound 28 (30 mg/kg) compared to the saline control.

Wound Healing–Cell-Based Assays

A scratch-wound assay was developed to quantify the relative regenerative capabilities of compounds 27 and 28 (Figure and Supporting Figure 3). In this assay, a scratch is created in a two-dimensional monolayer of HT-1080 fibrosarcoma cells. This scratch-wound assay has also been utilized with alternate cell lines and in conjunction with shLATS1 and shLATS2 constructs to demonstrate accelerated wound healing. As the scratch progressively closes over time, the regenerative potential of compounds of interest can be assessed in comparison to the DMSO treated control cells. Immediately after scratching, Compounds 27 and 28 were dosed at concentrations ranging from 4 to 500 nM, and phase imaging was performed every 2 h to monitor wound closure over time. Notably, every examined concentration of the LATS inhibitors accelerated wound closure, including the most modest 4 nM dosing. This relative wound-closing was often most pronounced at 8 h, with the treated groups demonstrating 10–20% acceleration over the DMSO control (Supporting Figure 3). While both agents promoted wound healing, compound 27 demonstrated superior potency, speed and range of healing over compound 28 at each concentration utilized.

4.

Relative wound healing in a scratch assay in HT1080 cells for compound 27 and compound 28 at multiple doses over 16 h.

Wound Healing–In Vivo

Given the success in this cell-based model, we sought to examine these agents in an animal dermal wound healing model. The relative speed of wound closure in skin provided a straightforward measure of our compounds’ potential in regenerative medicine. While the pharmacokinetic parameters of 27 and 28 provided information regarding systemic dosing, topical treatment could directly deliver the compound to the site of injury. Ultimately, a topical treatment was selected, as it closely matched most treatments currently available for wound healing. − In considering the choice of compound for these initial studies, compound 27 was utilized, as it had superior permeability properties, and we considered this of importance for a topical treatment.

Additionally, 27 demonstrated superiority in the cellular models of wound healing (Figure ). Compound 27 was formulated using a PF-127 thermal hydrogel. This allowed for accurate dosing of the compound in its sol phase, while readily gelling on the skin of the subjects, due to the body temperature. Following confirmation that 27 was stable over time within the hydrogel, we assessed this agent’s activity in a full thickness punch biopsy model in hairless SKH1 mice. Topical administration of 0.1 mL of the gel was dosed directly to the wound site, every other day, for 3 weeks or until the wound closed. Two different concentrations of compound 27 were utilized, 0.01 and 1.0 mg/mL, to ensure testing over a wide range of concentrations. Wound photographs were taken every other day and analyzed for relative rates of wound closure (Figure ). Notably, both concentrations of compound 27 promoted accelerated wound closure relative to vehicle (Figure ). At the interim time-point of day 8, both the 0.01 and 1.0 mg/mL gel concentrations demonstrated notable accelerations in wound closure, with an average surface area of approximately 5 mm² compared to the control at approximately 8 mm². This carried over to the completion mark, with the wounds for both concentrations being completely healed in an average of 12–13 days, compared to the control (the PF-127 hydrogel without compound) at almost 17 days (Figure ).

5.

Wound healing photographs for compound 27.

6.

Wound healing for compound 27.

Compound Synthesis

Initial compound synthesis typically involved a Grignard reaction between the piperidinone and the central phenyl group, followed by a Suzuki-type coupling reaction to append the pyrrolopyridine hinge-binding domain (Scheme ). However, the synthesis of the more advanced compounds required some development, especially the more sterically hindered dimethylpiperidine substrates. Starting with compound 18, more forcing conditions were required for the Grignard addition. Initially, using 4-chlorophenylmagnesium bromide and adding it to Boc-protected dimethyl piperidone in THF at elevated temperatures (60 °C), was sufficient. Diversification of the central aryl moiety with the 2-fluoro (19), and 2-chloro (20), substituted systems proceeded well by use of the Knochel conditions utilizing isopropylmagnesium chloride lithium chloride complex (‘Turbo Grignard’) to selectively form the desired organomagnesium compound, which performed well in the subsequent addition to the carbonyl, again at 60 °C. However, when additional steric bulk was presented with the 2-methyl (21), or 2-fluoro-4-methyl (22) systems, yields were substantially diminished, on occasion leading to no product formation at all, presumably due to the sterically congested reaction center. In order to counteract this issue, initially an organolanthanium system was utilized, which brought modest improvements to the yield. Ultimately, the organolithium system was utilized, as this proved to be significantly more reactive, even with the more sterically congested 2-fluoro-4-methyl system. Utilizing the organolithium system, we were able to achieve the nucleophilic addition, even at −78 °C. The relative stereochemistry of the isolated major product was demonstrated through NMR NOE experiments and was later confirmed by single crystal X-ray crystallography (Supporting Information). Significant care needed to be taken with the lithium halogen exchange on the aryl ring, however, especially with compound 30, as a major side product was discovered where direct lithiation of the aryl ring via deprotonation was competing with lithium–bromine exchange. This side product formation was effectively reduced by using the corresponding aryl iodide for the lithium-halogen exchange: the faster rate of Li–I exchange compared to Li–Br exchange allowed for reduction this side reaction.

1. Synthesis of Lead Compounds.

Following the organometallic addition, a trifluoroacetic acid–mediated deprotection of the piperidine nitrogen, followed by nitrogen functionalization through either an amide coupling or reductive amination, then introduction of the azaindole via a Suzuki reaction gave the desired products 27 and 28 (Scheme ). This procedure is readily scalable.

Conclusions

We have reported the discovery of a series of potent and selective inhibitors of LATS1 and LATS2 developed via scaffold hopping from a published AKT inhibitor. Key compounds in this series displayed good in vitro ADME and in vivo PK properties, as well as promising activity in multiple cellular models and in vivo models of wound healing. Cellular assays of on-target activity included both NanoBRET and qPCR assays which proved critical in the development of SAR to deliver compounds 27 and 28 which possessed good LATS1/2 activity and limited AKT activity. Further, both 27 and 28 possessed excellent ADME properties and relatively long half-lives in vivo. Using phospho-YAP as an in vivo pharmacodynamic marker, it was demonstrated that both 27 and 28 have significant on-target activity in mice at reasonable dosing levels. Overall kinase selectivity for both agents was good, and ultimately 27 was utilized in an in vivo wound healing model, demonstrating efficacy when dermally administered. Further development in this series, including exploration of additional animal models of tissue regeneration, is currently ongoing, and will be reported in due course.

Experimental Methods

Chemistry: General Synthetic Methods

All compounds are >95% pure by HPLC analysis. All commercially available reagents and solvents were purchased and used without further purification. All microwave reactions were carried out in a sealed microwave vial equipped with a magnetic stir bar and heated in a Biotage Initiator Microwave Synthesizer. ¹H NMR and ¹³C NMR spectra were recorded on Varian 400 MHz or Varian 600 MHz spectrometers in CD₃OD, CDCl₃, or DMSO-d ₆ as indicated. For spectra recorded in CD₃OD, chemical shifts are reported in ppm with CD₃OD (3.31 ppm) as reference for ¹H NMR spectra and CD₃OD (49.0 ppm) for ¹³C NMR spectra. For spectra recorded in CDCl₃, chemical shifts are reported in ppm relative to deuterochloroform (7.26 ppm for ¹H NMR, 77.23 ppm for ¹³C NMR). For spectra in DMSO-d ₆ chemical shifts are reported in ppm relative to DMSO-d ₆ (2.50 ppm for ¹H NMR, 39.5 ppm for ¹³C NMR). The coupling constants (J value) are reported as Hertz (Hz). The splitting patterns of the peaks were described as singlet (s); broad singlet (bs); doublet (d); triplet (t); quartet (q); multiplet (m) and septet (septet). Coupling constants in ¹³C NMR refer to ¹⁹F–¹³C coupling. Samples were analyzed for purity and low resolution mass spectrometry on an Agilent 1200 series LC/MS equipped with a Luna C18 (3 mm × 75 mm, 3 μm) reversed-phase column with UV detection at λ = 220 nm and λ = 254 nm. The mobile phase consisted of water containing 0.05% trifluoroacetic acid as component A and acetonitrile containing 0.025% trifluoroacetic acid as component B. A linear gradient was run as follows: 0 min 4% B; 7 min 100% B; 8 min 100% B at a flow rate of 0.8 mL/min. The HRMS was recorded on an Agilent 6230 Time-of-Flight (TOF) MS system (Agilent Technologies, Wilmington, DE) equipped with a DUAL AJS ESI source operating in positive ion mode, 1290 series LC, binary pump, autosampler, and diode array detector (DAD).

General Synthetic Protocol A: Grignard Reaction

(4-Chlorophenyl)­magnesium bromide (2.5 mL, 2.5 mmol, 1.0 M in diethyl ether, 1.0 equiv) was added to a solution of the desired ketone (2.5 mmol, 1.0 equiv) in tetrahydrofuran (15.0 mL) within a sealed round-bottom flask under a nitrogen atmosphere at 0 °C. The reaction was stirred for 2 h at 0 °C, then allowed to warm to 20 °C and stirred for an hour. The reaction was quenched by being poured into aqueous saturated sodium bicarbonate. It was then extracted into ethyl acetate. The organic phase was taken, and the solvent removed by rotary evaporation to give the crude product. Purification by silica gel chromatography (0 to 50% ethyl acetate in hexanes) gave the title product in the reported yield.

General Synthetic Protocol B: Knochel-Type Grignard Reaction

The desired aryl halide (2.0 mmol, 1.0 equiv) was added to a round-bottom flask containing a stir bar. The flask was sealed, evacuated, and backfilled with nitrogen. Tetrahydrofuran (10.0 mL) was then added, and the flask was cooled to 0 °C using an ice bath. Then a solution of isopropylmagnesium chloride lithium chloride (1.5 mL, 2.0 mmol, 1.0 equiv 1.3 M in tetrahydrofuran) was added to the reaction mixture via syringe. The reaction was stirred for 1 h at 0 °C. In a separate 20 mL microwave vial with a stir bar, tert-butyl (3S,5R)-3,5-dimethyl-4-oxopiperidine-1-carboxylate (0.45 g, 2.0 mmol, 1.0 equiv) was placed. The vial was sealed, evacuated, and backfilled with nitrogen. Tetrahydrofuran (5.0 mL) was added to the vial. Then the organomagnesium solution was added to the microwave vial via syringe. This reaction was heated via microwave irradiation to 60 °C for 2 h. The reaction was cooled, then poured into a saturated solution of aqueous sodium bicarbonate. The aqueous phase was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation. Purification by silica gel chromatography (0 to 50% ethyl acetate in hexanes) gave the title product in the reported yield.

General Synthetic Protocol C: Aryl Lithium Formation and Addition

The desired aryl halide (7.3 mmol, 1.0 equiv) was added to a round-bottom flask containing a stir bar. The flask was sealed, evacuated, and backfilled with nitrogen. Tetrahydrofuran (10.0 mL) was added to the reaction via syringe. The reaction was then cooled to −78 °C using a dry ice–acetone bath. Then n-butyllithium in hexanes (3.2 mL, 2.5 M, 1.1 equiv) was added via syringe. The reaction was stirred for 5 min at −78 °C. In a separate flask, tert-butyl (3R,5S)-3,5-dimethyl-4-oxopiperidine-1-carboxylate (1.7 g, 7.3 mmol, 1.0 equiv) was dissolved in tetrahydrofuran (10.0 mL). This solution was then slowly added via syringe to the organolithium solution at −78 °C. The reaction was allowed to stir for 2 h and slowly raised to 0 °C. The reaction was then quenched by being poured into an aqueous saturated solution of sodium bicarbonate. The aqueous phase was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation. Purification by silica gel chromatography (10 to 50% ethyl acetate in hexanes) gave the desired product in the reported yield.

General Synthetic Protocol D: Suzuki Reaction

The desired aryl halide (0.15 mmol, 1.0 equiv) was added to a 2.0 mL microwave vial containing the boronic ester (0.20 mmol, 1.3 equiv), potassium phosphate tribasic (0.45 mmol, 3.0 equiv), chloro­(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)­[2-(2′-amino-1,1′-biphenyl)]­palladium­(II) (0.015 mmol, 0.1 equiv, Xphos Pd G2) and a stir bar. The vial was sealed, evacuated, and backfilled with nitrogen. Then water (1.0 mL) and tetrahydrofuran (1.0 mL) were added to the vial, and it was degassed by nitrogen bubbling for 5 min. The vial was then heated via microwave irradiation to 100 °C for 0.5 h. The vial was cooled and poured into an aqueous saturated solution of sodium bicarbonate. The aqueous phase was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation to give the crude product. The residue was suspended in methanol (∼1.0 mL) filtered and purified by reverse phase high pressure chromatography (5 to 95% acetonitrile in water with 0.1% TFA) to give the title product in the reported yield.

General Synthetic Protocol E: Suzuki Reaction followed by TFA-Mediated BOC Deprotection

The desired aryl halide (0.15 mmol, 1.0 equiv) was added to a 2.0 mL microwave vial containing the boronic ester (0.20 mmol, 1.3 equiv), potassium phosphate tribasic (0.45 mmol, 3.0 equiv), chloro­(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)­[2-(2′-amino-1,1′-biphenyl)]­palladium­(II) (0.015 mmol, 0.1 equiv, Xphos Pd G2) and a stir bar. The vial was sealed, evacuated, and backfilled with nitrogen. Then water (1.0 mL) and tetrahydrofuran (1.0 mL) were added to the vial, and it was degassed by nitrogen bubbling for 5 min. The vial was then heated via microwave irradiation to 100 °C for 0.5 h. The vial was cooled and poured into an aqueous saturated solution of sodium bicarbonate. The aqueous phase was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation to give the crude intermediate. The intermediate was then suspended/dissolved in dichloromethane (1.0 mL) and trifluoroacetic acid (1.0 mL) was added. The reaction was stirred at 23 °C for 0.5 h. The solvent and trifluoroacetic acid were then removed by rotary evaporation. The residue was suspended in methanol (∼1.0 mL) filtered and purified by reverse phase high pressure chromatography (5 to 95% acetonitrile in water with 0.1% TFA) to give the title product in the reported yield.

Synthetic Procedures and Analytical Data for Reported Compounds

4-(4-(4-(4-Chlorophenyl)­piperidin-4-yl)­phenyl)-1H-pyrrolo­[2,3-b]­pyridine (7)

Palladium tetrakis­(tri­phenyl­phosphine(0)) (6.0 mg, 0.005 mmol) was added to a 2 mL microwave vial containing 4-(4-bromophenyl)-4-(4-chlorophenyl)­piperidine hydrochloride (40 mg, 0.10 mmol), 4-(4,4,5,5-tetramethyl-13,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine (38 mg, 0.15 mmol), potassium carbonate (43 mg, 0.31 mmol), and a stir bar. The vial was sealed, evacuated, and backfilled with nitrogen. 1,4-Dioxane (0.52 mL) and water (0.17 mL) were then added via syringe, and the solvent was degassed by nitrogen bubbling for 5 min. The reaction was than heated to 150 °C for 30 min via microwave irradiation. The vial was cooled, and the solvent removed by rotary evaporation. Purification by high pressure liquid chromatography (5 to 100% MeCN in water with 0.1% TFA) gave the title product in 1.6% yield, (1.0 mg) as the di TFA salt. ¹H NMR (400 MHz, CD₃OD) δ 8.42 (d, J = 6.2 Hz, 1H), 7.97–7.81 (m, 2H), 7.73 (d, J = 3.6 Hz, 1H), 7.71–7.56 (m, 3H), 7.51–7.33 (m, 4H), 6.98 (d, J = 3.6 Hz, 1H), 3.25 (dd, J = 13.5, 7.3 Hz, 4H), 2.76 (t, J = 5.7 Hz, 4H). ¹³C NMR (101 MHz, CD₃OD) δ 149.50, 134.53, 133.36, 132.50, 129.42, 129.34, 128.80, 128.12, 127.34, 123.05, 117.03, 114.58, 114.17, 111.79, 102.10, 56.90, 43.39, 41.08, 32.03, 16.93. m/s ES+ [M + H]⁺: 388.1. HRMS (ESI+): Expected 388.1575 [M + H⁺] (C₂₄H₂₂ClN₃). Observed 388.1595.

4-(4-(Piperidin-4-yl)­phenyl)-1H-pyrrolo­[2,3-b]­pyridine (8)

Utilizing General Synthetic Protocol E tert-butyl 4-(4-bromophenyl)­piperidine-1-carboxylate and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 34% yield as the di-TFA salt. NMR spectra are on the free base. ¹H NMR (400 MHz, CD₃OD) δ 8.44 (d, J = 6.2 Hz, 1H), 7.99–7.79 (m, 2H), 7.74 (d, J = 3.6 Hz, 1H), 7.65 (d, J = 6.2 Hz, 1H), 7.62–7.50 (m, 2H), 6.99 (d, J = 3.6 Hz, 1H), 3.63–3.41 (m, 2H), 3.19 (td, J = 12.9, 3.0 Hz, 2H), 3.06 (tt, J = 12.1, 3.7 Hz, 1H), 2.23–2.11 (m, 2H), 2.11–1.88 (m, 2H). ¹³C NMR (151 MHz, DMSO-d ₆) δ 148.02, 145.10, 141.90, 140.94, 136.50, 128.58, 127.24, 127.01, 117.74, 114.12, 99.33, 43.64, 38.72, 29.38. m/s ES+ [M + H]⁺: 278.1 HRMS (ESI+): Expected 278.1652 [M + H⁺] (C₁₈H₁₉N₃). Observed 278.1656.

3-Fluoro-4-(4-(piperidin-4-yl)­phenyl)-1H-pyrrolo­[2,3-b]­pyridine (9)

Utilizing General Synthetic Protocol E tert-butyl 4-(4-bromophenyl)­piperidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 24% overall yield. ¹H NMR (400 MHz, CD₃OD) δ 8.44 (d, J = 6.2 Hz, 1H), 7.99–7.79 (m, 2H), 7.74 (d, J = 3.6 Hz, 1H), 7.65 (d, J = 6.2 Hz, 1H), 7.62–7.50 (m, 2H), 6.99 (d, J = 3.6 Hz, 1H), 3.63–3.41 (m, 2H), 3.19 (td, J = 12.9, 3.0 Hz, 2H), 3.06 (tt, J = 12.1, 3.7 Hz, 1H), 2.23–2.11 (m, 2H), 2.11–1.88 (m, 2H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.78, −164.98 (d, J = 2.7 Hz). ¹³C NMR (DMSO-d ₆, 151 MHz): 144.94, 144.30 (d, J = 3.5 Hz), 143.96, 141.99, (d, J = 243.1 Hz), 139.96, (d, J = 3.0 Hz), 135.71, 129.10 (d, J = 3.3 Hz), 126.68, 115.79, 109.29 (d, J = 27.8 Hz), 105.50 (d, J = 11.5 Hz), 43.65, 38.67, 29.37. m/s ES+ [M + H]⁺: 296.1. HRMS (ESI+): Expected 296.1558 [M + H⁺] (C₁₈H₁₈FN₃). Observed 296.1550.

4-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)­piperidin-4-ol (10)

Utilizing General Synthetic Protocol D 4-(4-bromophenyl)­piperidin-4-ol and (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 37% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.64 (s, 1H), 8.38 (s, 1H), 8.31 (d, J = 4.9 Hz, 1H), 7.70–7.53 (m, 4H), 7.52 (s, 1H), 7.14 (d, J = 4.9 Hz, 1H), 3.21–2.92 (m, 4H), 2.05 (dd, J = 13.1, 8.7 Hz, 2H), 1.72 (d, J = 13.5 Hz, 2H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.41, −164.92 (t, J = 2.7 Hz). ¹³C NMR (DMSO-d ₆, 151 MHz): 149.28, 144.36, 144.34, 142.00, (d, J = 242.9 Hz), 139.96, 135.74, 128.59 (d, J = 3.3 Hz), 124.80, 115.83, 109.26, (d, J = 27.6 Hz), 105.51 (d, J = 11.6 Hz), 68.88, 40.06, 35.44. m/s ES+ [M + H]⁺: 312.1. HRMS (ESI+): Expected 312.1507 [M + H⁺] (C₁₈H₁₈FN₃O). Observed 312.1501.

tert-Butyl 3-(4-Chlorophenyl)-3-hydroxypiperidine-1-carboxylate (Int-11)

Following General Synthetic Protocol A tert-butyl 3-oxopiperidine-1-carboxylate and 4-(chlorophenyl)­magnesium chloride were combined to give the title product in 19% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.47–7.39 (m, 2H), 7.35–7.27 (m, 2H), 4.09–4.01 (m, 1H), 3.99–3.81 (m, 1H), 3.13 (d, J = 13.7 Hz, 1H), 2.85 (ddd, J = 13.8, 10.9, 3.3 Hz, 1H), 1.99–1.84 (m, 3H), 1.62–1.54 (m, 1H), 1.46 (s, 9H). m/s ES+ [M-OC­(CH₃)₃]+: 237.9.

3-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)­piperidin-3-ol (11)

Using General Synthetic Protocol E, tert-butyl 3-(4-chlorophenyl)-3-hydroxypiperidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 54% yield. NMR spectra are on the free base. ¹H NMR (400 MHz, CD₃OD) δ 8.50 (d, J = 6.0 Hz, 1H), 7.85 (dd, J = 8.5, 2.8 Hz, 2H), 7.80 (d, J = 8.6 Hz, 2H), 7.64 (d, J = 2.4 Hz, 1H), 7.61 (d, J = 6.1 Hz, 1H), 3.42 (t, J = 11.6 Hz, 2H), 3.23 (d, J = 12.7 Hz, 1H), 3.18–3.06 (m, 1H), 2.38–2.17 (m, 2H), 2.03–1.85 (m, 2H). ¹⁹F NMR (376 MHz, CD₃OD) δ −164.99 (q, J = 2.9 Hz). ¹³C NMR (100 MHz, CD₃OD): 151.16 (d, J = 4.2 Hz), 148.87, 144.81 (d, J = 246.0 Hz), 136.80, 135.98, 130.88, 130.84, 126.62, 117.46, 113.37 (d, J = 29.4 Hz), 112.85 (d, J = 15.2 Hz), 70.86, 53.69, 44.75, 35.36, 19.84. m/s ES+ [M + H]⁺: 312.0. HRMS (ESI+): Expected [M + H⁺] 312.1507 (C₁₈H₁₈FN₃O). Observed 312.1516.

tert-Butyl 3-(4-Chlorophenyl)-3-hydroxypyrrolidine-1-carboxylate (Int-12)

Utilizing General Synthetic Protocol A tert-butyl 3-oxopyrrolidine-1-carboxylate and 4-(chlorophenyl)­magnesium chloride were combined to give the title product in 29% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.43–7.36 (m, 2H), 7.35–7.28 (m, 2H), 3.43–3.39 (m, 3H), 2.68 (bs, 1H), 2.34–2.10 (m, 2H), 1.44 (s, 9H). m/s ES+ [M-OC­(CH₃)₃]+: 223.9.

3-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)­pyrrolidin-3-ol (12)

Using General Synthetic Protocol E, tert-butyl 3-(4-chlorophenyl)-3-hydroxypyrrolidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 28% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.30 (d, J = 5.1 Hz, 1H), 7.76 (dd, J = 8.5, 2.6 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 2.2 Hz, 1H), 7.19 (d, J = 5.0 Hz, 1H), 3.74–3.62 (m, 2H), 3.61–3.45 (m, 2H), 2.60–2.47 (m, 1H), 2.46–2.39 (m, 1H). ¹⁹F NMR (376 MHz, CD₃OD) δ −77.10, −166.95 (q, J = 2.5 Hz). ¹³C NMR (151 MHz, DMSO-d ₆): 144.31 (d, J = 3.5 Hz), 144.08, 142.10, 141.99 (d, J = 243.0 Hz), 139.71 (d, J = 3.5 Hz), 136.70, 128.75 (d, J = 3.2 Hz), 125.84, 115.87, 109.39 (d, J = 27.3 Hz), 105.51 (d, J = 11.3 Hz), 79.38, 56.35, 42.12, 38.53. m/s ES+ [M + H]⁺: 298.1. HRMS (ESI+): Expected 298.1350 [M + H⁺] (C₁₇H₁₆FN₃O). Observed 298.1342.

3-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)­azetidin-3-ol (13)

Using General Synthetic Protocol D, 3-(4-chlorophenyl)­azetidin-3-ol hydrochloride and fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 30% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.68 (s, 1H), 8.32 (d, J = 5.0 Hz, 2H), 7.77–7.64 (m, 4H), 7.53 (d, J = 2.0 Hz, 1H), 7.16 (dd, J = 4.7, 3.3 Hz, 1H), 4.34 (d, J = 10.9 Hz, 2H), 4.08 (d, J = 10.8 Hz, 2H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −77.0, −165.00 (q, J = 2.7 Hz). ¹³C NMR (101 MHz, CD₃OD) δ 144.20 (d, J = 243.2 Hz), 114.10, 143.92, 143.52, 143.21, 138.90, 130.68 (d, J = 3.4 Hz), 126.53, 117.22, 110.56 (d, J = 27.8 Hz), 74.62, 60.95. m/s ES+ [M + H]⁺: 283.9. HRMS (ESI+): Expected 284.1194 [M + H⁺] (C₁₆H₁₄FN₃O). Observed 284.1189.

tert-Butyl 4-(4-Chlorophenyl)-4-hydroxyazepane-1-carboxylate (Int-14)

Utilizing General Synthetic Protocol A tert-butyl 4-oxoazepane-1-carboxylate and 4-(chlorophenyl)­magnesium chloride were combined to give the title product in 36% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.42–7.35 (m, 2H), 7.29 (d, J = 8.1 Hz, 2H), 3.88–3.49 (m, 3H), 3.42–3.23 (m, 2H), 2.66–2.60 (m, 1H), 2.24–2.07 (m, 1H), 2.07–1.70 (m, 3H), 1.47 (s, 9H). m/s ES+ [M-OC­(CH₃)₃]+: 251.9.

4-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)­azepan-4-ol (14)

Using General Synthetic Protocol E, tert-butyl 4-(4-chlorophenyl)-4-hydroxyazepane-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 52% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.28 (d, J = 5.1 Hz, 1H), 7.69 (dd, J = 8.5, 2.6 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 2.1 Hz, 1H), 7.19 (d, J = 5.1 Hz, 1H), 3.61 (t, J = 12.5 Hz, 1H), 3.50–3.40 (m, 1H), 3.29–3.23 (m, 2H), 2.46 (ddd, J = 14.3, 11.4, 2.8 Hz, 1H), 2.33–2.20 (m, 1H), 2.20–2.01 (m, 3H), 1.98–1.85 (m, 1H). ¹⁹F NMR (376 MHz, CD₃OD) δ −77.25, −166.66 (q, J = 2.6 Hz). ¹³C NMR (151 MHz, DMSO-d ₆) δ 150.73, 144.31 (d, J = 3.3 Hz), 144.02, 142.01 (d, J = 243.0 Hz), 140.03 (d, J = 3.5 Hz), 135.51, 128.49 (d, J = 3.3 Hz), 124.61, 115.83, 109.27 (d, J = 27.6 Hz), 105.54 (d, J = 11.6 Hz), 73.06, 48.60, 44.87, 40.62, 37.81, 19.22. m/s ES+ [M + H]⁺: 326.2. HRMS (ESI+): Expected 326.1663 [M + H⁺] (C₁₈H₁₈FN₃O). Observed 326.1667.

(1R,5S)-3-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)-8-azabicyclo­[3.2.1]­octan-3-ol (15)

Utilizing General Synthetic Protocol D, (1R,5S)-3-(4-chlorophenyl)-8-azabicyclo­[3.2.1]­octan-3-ol hydrochloride and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 4% yield. Note, formic acid was used as a modifier instead of TFA in the HPLC elution of this sample. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.63 (s, 1H), 8.39 (s, 1H), 8.30 (d, J = 4.9 Hz, 1H), 7.68–7.58 (m, 4H), 7.51 (s, 1H), 7.14 (d, J = 4.9 Hz, 1H), 3.89 (s, 2H), 2.38 (d, J = 7.4 Hz, 1H), 2.35–2.25 (m, 2H), 1.91 (d, J = 14.5 Hz, 2H) 1.86–1.79 (m, 3H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −164.94 (t, J = 2.7 Hz). ¹³C NMR (151 MHz, DMSO-d ₆) δ 150.50, 144.34 (d, J = 3.5 Hz), 144.04, 142.00 (d, J = 243.0 Hz), 140.00 (d, J = 3.6 Hz), 135.45, 128.30 (d, J = 3.3 Hz), 125.07, 115.82, 109.23 (d, J = 27.4 Hz), 105.51 (d, J = 11.4 Hz), 71.00, 53.61, 42.59, 26.33. m/s ES+ [M + H]⁺: 338.1. HRMS (ESI+): Expected 338.1663 [M + H⁺] (C₂₀H₂₀FN₃O). Observed 338.1651.

tert-Butyl 6-(4-Chlorophenyl)-6-hydroxy-2-azaspiro[3.3]­heptane-2-carboxylate (Int-16)

Utilizing General Synthetic Protocol A tert-butyl 6-oxo-2-azaspiro[3.3]­heptane-2-carboxylate and 4-(chlorophenyl)magnesium chloride were combined to give the title product in 79% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.18 (m, 4H), 3.99 (s, 2H), 3.73 (s, 2H), 2.70–2.56 (m, 2H), 2.52–2.36 (m, 2H), 1.41 (s, 9H). m/s ES+ [M-C­(CH3)­3]+2H: 268.0

6-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)-2-azaspiro­[3.3]­heptan-6-ol (16)

Utilizing General Synthetic Protocol E, tert-butyl 6-(4-chlorophenyl)-6-hydroxy-2-azaspiro[3.3]­heptane-2-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 38% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.26 (d, J = 5.0 Hz, 1H), 7.68 (dd, J = 8.4, 2.8 Hz, 2H), 7.64–7.49 (m, 2H), 7.26 (d, J = 2.1 Hz, 1H), 7.15 (d, J = 5.0 Hz, 1H), 4.20 (s, 2H), 3.98 (s, 2H), 2.93–2.84 (m, 2H), 2.66–2.52 (m, 2H). ¹⁹F NMR (376 MHz, CD₃OD) δ −166.73 (q, J = 2.8 Hz). ¹³C NMR (151 MHz, DMSO-d ₆) δ 147.34, 144.34 (d, J = 3.5 Hz), 144.06, 142.00 (d, J = 243.0 Hz), 140.01 (d, J = 3.6 Hz), 135.66, 128.48 (d, J = 3.3 Hz), 125.14, 115.80, 109.25 (d, J = 27.4 Hz), 105.51 (d, J = 11.5 Hz), 71.70, 57.77, 55.75, 46.99, 34.64. m/s ES+ [M + H]+: 324.2. HRMS (ESI+): Expected 324.1507 [M + H⁺] (C₁₉H₁₈FN₃O). Observed 325.1500.

tert-Butyl 4-(4-Chlorophenyl)-4-hydroxy-3-methylpiperidine-1-carboxylate (Int-17)

Utilizing General Synthetic Protocol A tert-butyl 3-methyl-4-oxopiperidine-1-carboxylate and 4-(chlorophenyl)magnesium chloride were combined to give the title product in 25% yield. Relative stereochemistry between the methyl group and alcohol was not determined ¹H NMR (400 MHz, CDCl₃) δ 7.42–7.26 (m, 4H), 4.08–3.81 (m, 2H), 3.24–3.04 (m, 1H), 2.93–2.73 (m, 1H), 2.11–1.99 (m, 1H), 1.96–1.86 (m, 1H), 1.63 (dt, J = 14.0, 2.4 Hz, 1H), 1.49 (s, 9H), 0.61 (d, J = 6.9 Hz, 3H). m/s ES+ [M-OC­(CH₃)₃]+: 251.9

4-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)-3-methylpiperidin-4-ol (17)

Using General Synthetic Protocol E, tert-butyl 4-(4-chlorophenyl)-4-hydroxy-3-methylpiperidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 66% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.48 (d, J = 6.0 Hz, 1H), 7.84 (dd, J = 8.5, 3.1 Hz, 2H), 7.72 (d, J = 8.3 Hz, 2H), 7.66 – 7.56 (m, 2H), 3.50 – 3.34 (m, 2H), 3.29 – 3.10 (m, 2H), 2.47 – 2.33 (m, 2H), 1.95 (dt, J = 14.9, 2.4 Hz, 1H), 0.73 (d, J = 6.8 Hz, 3H). ¹⁹F NMR (376 MHz, CD₃OD) δ −164.93 (q, J = 3.1 Hz). ¹³C NMR (151 MHz, DMSO-d ₆) δ 147.52, 142.89, 142.78, 142.00 (d, J = 236.0 Hz), 141.27, 135.25, 128.65 (d, J = 3.5 Hz) 125.07, 115.87, 109.81 (d, J = 27.6 Hz), 106.27 (d, J = 12.2 Hz) 71.28, 56.01, 45.08, 36.35, 35.71, 11.79. m/s ES+ [M + H]⁺: 326.0. HRMS (ESI+): Expected 326.1663 [M + H⁺] (C₁₉H₂₀FN₃O). Observed 326.1647.

tert-Butyl (3R,4s,5S)-4-(4-Chlorophenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate (Int-18)

Utilizing a modification of General Synthetic Protocol A, tert-butyl (3S,5R)-3,5-dimethyl-4-oxopiperidine-1-carboxylate and 4-(chlorophenyl)magnesium chloride were combined to give the title product in 25% yield. The reaction was modified by the temperature of the reaction was 60 °C for 1 h, instead of room temperature for 1 h. Relative stereochemistry was determined by analogy to compound 31 and its X-ray crystal structure. ¹H NMR (400 MHz, CDCl₃) δ 7.37–7.27 (m, 4H), 4.01–3.86 (m, 2H), 2.83 (t, J = 12.5 Hz, 2H), 2.09–1.94 (m, 2H), 1.49 (d, J = 0.5 Hz, 9H), 0.58 (d, J = 6.8 Hz, 6H). m/s ES+ [M-OC­(CH₃)₃]+2H: 266.1

(3R,4s,5S)-4-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)-3,5-dimethylpiperidin-4-ol (18)

Using General Synthetic Protocol E, tert-butyl (3R,4s,5S)-4-(4-chlorophenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 10% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.66 (s, 1H), 8.74–8.42 (m, 2H), 8.32 (d, J = 5.0 Hz, 1H), 7.70 (s, 3H), 7.53 (d, J = 2.9 Hz, 1H), 7.30 (bs, 1H), 7.18 (d, J = 4.9 Hz, 1H), 5.16 (s, 1H), 3.32–3.11 (m, 2H), 2.93 (q, J = 11.7 Hz, 2H), 2.31 (dd, J = 12.1, 5.4 Hz, 2H), 0.58 (d, J = 6.7 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −74.24, −164.76. ¹³C NMR (151 MHz, DMSO-d ₆) δ 144.78, 144.34 (d, J = 3.5 Hz), 144.02, 141.98 (d, J = 243.0 Hz), 139.91 (d, J = 3.5 Hz), 135.57, 128.98, 128.13, 126.59, 123.84, 115.82, 109.27 (d, J = 27.6 Hz) 105.52 (d, J = 11.4 Hz), 74.40, 44.90, 42.41, 40.43, 37.74, 11.86. m/s ES+ [M + H]⁺: 339.9. HRMS (ESI+): Expected 340.1820 [M + H⁺] (C₂₀H₂₂FN₃O). Observed 340.1813.

tert-Butyl (3R,4s,5S)-4-(4-Chloro-2-fluorophenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate (Int-19)

Utilizing General Synthetic Protocol B 4-chloro-2-fluoro-1-iodobenzene and tert-butyl (3S,5R)-3,5-dimethyl-4-oxopiperidine-1-carboxylate were combined to give the title product in 18% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.51 (bs, 1H), 7.13 (dt, J = 8.4, 2.3 Hz, 1H), 7.04 (dt, J = 11.8, 2.2 Hz, 1H), 3.90 (d, J = 49.2 Hz, 2H), 2.82 (bs, 2H), 2.41 (bs, 2H), 1.48 (t, J = 2.2 Hz, 9H), 0.68 – 0.52 (m, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −109.42. m/s ES+ [M-C­(CH₃)₃] + H: 302.1.

(3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)-3,5-dimethylpiperidin-4-ol (19)

Utilizing General Synthetic Protocol E, tert-butyl (3R,4s,5S)-4-(4-chloro-2-fluorophenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 8% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.70 (d, J = 2.8 Hz, 1H), 8.64 (s, 2H), 8.32 (d, J = 4.9 Hz, 1H), 7.71 (t, J = 8.3 Hz, 1H), 7.60–7.51 (m, 2H), 7.47 (d, J = 13.3 Hz, 1H), 7.21 (d, J = 4.9 Hz, 1H), 5.40 (s, 1H), 3.12 (d, J = 12.0 Hz, 2H), 2.93 (d, J = 12.1 Hz, 3H), 2.61 (dq, J = 11.8, 6.8, 4.8 Hz, 2H), 0.62 (d, J = 6.8 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.90 (s), −112.60 (s), −164.92 (s) (TFA salt). 13C NMR (151 MHz, DMSO-d ₆) δ 158.02 (d, J = 243.9 Hz), 144.35 (d, J = 3.3 Hz), 144.08, 141.82 (d, J = 242.5 Hz), 138.58 (d, J = 8.6 Hz), 138.19, 130.53 (d, J = 12.8 Hz), 129.67 (d, J = 5.4 Hz), 124.44, 116.24 (d, J = 29.3 Hz), 115.86, 109.67 (d, J = 27.6 Hz), 105.30 (d, J = 11.4 Hz), 74.21 (d, J = 5.9 Hz), 44.52, 35.38 (d, J = 4.8 Hz), 12.25. m/s ES+ [M + H]⁺: 358.2. HRMS (ESI+): Expected 358.1725 [M + H⁺] (C₂₀H₂₁F₂N₃O). Observed 358.1703.

tert-Butyl (3R,4s,5S)-4-(4-Bromo-2-chlorophenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate (Int-20)

Utilizing General Synthetic Protocol B 4-bromo-2-chloro-1-iodobenzene and tert-butyl (3S,5R)-3,5-dimethyl-4-oxopiperidine-1-carboxylate were combined to give the title product in 49% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.64 (d, J = 8.6 Hz, 1H), 7.50 (d, J = 2.1 Hz, 1H), 7.39 (dd, J = 8.6, 2.1 Hz, 1H), 3.91 (dd, J = 12.8, 4.4 Hz, 2H), 2.97 (tt, J = 11.3, 6.8 Hz, 2H), 2.90–2.72 (m, 2H), 1.48 (s, 9H), 0.57 (d, J = 6.8 Hz, 6H). m/s ES+ [M-C­(CH₃)₃]+: 363.9.

(3S,4s,5R)-4-(2-Chloro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)­phenyl)-3,5-dimethylpiperidin-4-ol (20)

Utilizing general synthetic protocol E tert-butyl (3R,4s,5S)-4-(4-bromo-2-chlorophenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 53% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.72 (s, 1H), 8.33 (d, J = 4.9 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.67 (dd, J = 8.1, 5.3 Hz, 2H), 7.56 (d, J = 1.9 Hz, 1H), 7.23 (d, J = 4.9 Hz, 1H), 5.12 (s, 1H), 3.08–2.99 (m, 2H), 2.95 (dd, J = 12.4, 4.0 Hz, 2H), 2.85 (t, J = 12.0 Hz, 2H), 0.55 (d, J = 6.7 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.47, −165.07 (t, J = 2.7 Hz). ¹³C NMR (151 MHz, DMSO-d ₆) δ 147.43 (d, J = 3.3 Hz), 147.22, 144.92, (d, J = 242.2 Hz), 144.44, 141.01 (d, J = 3.3 Hz), 140.75, 134.31 (d, J = 3.6 Hz), 133.53, 132.16, 130.14 (d, J = 3.5 Hz), 118.94, 112.79 (d, J = 27.3 Hz), 108.38 (d, J = 11.5 Hz), 79.52, 49.25, 37.76, 15.44. m/s ES+ [M + H]⁺: 374.1. HRMS (ESI+): Expected 374.1430 [M + H⁺] (C₂₀H₂ClFN₃O). Observed 374.1416.

tert-Butyl (3S,4s,5R)-4-(4-Chloro-2-methylphenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate (Int-21)

Utilizing General Synthetic Protocol C, 1-bromo-4-chloro-2-methylbenzene and tert-butyl (3R,5S)-3,5-dimethyl-4-oxopiperidine-1-carboxylate were combined to give the title product in 62% yield. Note, ¹H NMR is a mixture of rotamers at ∼1:2 ratio for this compound. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 8.6 Hz, 0.7H), 7.16 (dd, J = 8.4, 2.4 Hz, 0.7H), 7.13–6.98 (m, 1.6H), 4.05–3.73 (m, 2H), 2.95–2.74 (bs, 2H), 2.68–2.60 (bs, 1H), 2.57 (s, 1H), 2.49–2.43 (bs, 1H), 2.42 (s, 2H), 1.49 (d, J = 2.0 Hz, 9H), 0.67 (d, J = 6.8 Hz, 2H), 0.58 (d, J = 6.8 Hz, 4H). m/s ES+ [M-OC­(CH₃)₃]+: 280.0.

(3S,4s,5R)-4-(4-(3-Fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-2-methylphenyl)-3,5-dimethylpiperidin-4-ol (21)

Utilizing General Synthetic Protocol E tert-butyl (3R,4s,5S)-4-(4-chloro-2-methylphenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 4% yield. ¹H NMR (600 MHz, DMSO-d ₆, approximate 2:1 mixture of rotamer 1 to rotamer 2) δ 11.65 (d, J = 3.0 Hz, 1H), 8.75–8.57 (m, 2H), 8.30 (dd, J = 4.9, 2.7 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.53–7.48 (m, 2H), 7.44 (dt, J = 4.7, 2.6 Hz, 1H), 7.17 (d, J = 4.9 Hz, 1H), 5.15 (s, 0.66H, rotamer 1), 4.75 (s, 0.33H, rotamer 2) 3.15–3.07 (m, 2H), 3.05–2.93 (m, 2H), 2.75–2.68 (m, 1H), 2.66 (s, 1H, rotamer 2), 2.55 (s, 2H, rotamer 2), 2.42–2.35 (m, 1H), 0.70 (d, J = 6.9 Hz, 2H, rotamer 2), 0.59 (d, J = 6.8 Hz, 4H, rotamer 1). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.43, −164.7. ¹³C NMR (151 MHz, DMSO-d ₆, approximate 2:1 mixture of rotamer 1 to rotamer 2) δ 144.34 (d, J = 3.5 Hz), 144.33, 144.00, 141.99 (d, J = 242.7 Hz), 141.92, 139.74 (d, J = 3.3 Hz), 137.83 (rotamer 2), 135.80 (rotamer 1), 132.88 (rotamer 1), 132.81 (d, J = 3.2 Hz, rotamer 2), 132.80, 128.46, 125.76 (d, J = 3.9 Hz, rotamer 1), 125.71 (rotamer 2), 115.72 (rotamer 1), 115.69 (rotamer 2), 109.24 (d, J = 27.5 Hz), 105.49 (d, J = 11.6 Hz), 77.86 (rotamer 2), 75.35 (rotamer 1), 44.91 (rotamer 1), 44.59 (rotamer 2), 34.32, 23.46 (rotamer 2), 22.32 (rotamer 1), 12.14 (rotamer 1), 11.86 (rotamer 2). m/s ES+ [M + H]+: 354.3 HRMS (ESI+): Expected 354.1976 [M + H⁺] (C₂₁H₂₄FN₃O). Observed 354.1968.

tert-Butyl (3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate (30)

Utilizing General Synthetic Protocol C, 5-chloro-1-fluoro-2-iodo-3-methylbenzene and tert-butyl (3R,5S)-3,5-dimethyl-4-oxopiperidine-1-carboxylate were combined to give the title product in 41% yield. ¹H NMR (400 MHz, Chloroform-d) δ 6.95–6.82 (m, 2H), 4.35 (bs, 1H), 3.84 (bs, 2H), 2.76 (bs, 2H), 2.59 (s, 3H), 2.57–2.43 (m, 2H), 1.47 (s, 9H), 0.70 (d, J = 6.9 Hz, 6H). ¹⁹F NMR (376 MHz, cdcl₃) δ −103.59. m/s ES+ [M-CO₂C­(CH₃)₃]+ 2H: 272.1.

(3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (31)

tert-Butyl (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidine-1-carboxylate (0.500 g, 1.35 mmol) was added to a vial containing a stirbar. Dichloromethane (2.0 mL), was added followed by trifluoroacetic acid (2.96 g, 26.0 mmol, 2.0 mL). The vial was loosely capped, and the reaction was stirred at ambient temperature for 0.5 h. The solvent and trifluoroacetic acid were then removed by rotary evaporation. The residue was treated with aqueous saturated sodium bicarbonate and then extracted into ethyl acetate. The organic phase was taken, and the solvent removed by rotary evaporation to give the title product in 99% yield (0.362 g) without further purification. Relative stereochemistry was proven by single crystal X-ray crystallography (available in the Supporting Information). ¹H NMR (400 MHz, Methanol-d ₄) δ 6.99 – 6.93 (m, 2H), 2.89 (td, J = 12.2, 3.0 Hz, 2H), 2.80 (dd, J = 12.5, 4.4 Hz, 2H), 2.64 (s, 3H), 2.57 (dtd, J = 11.4, 6.9, 4.5 Hz, 2H), 0.72 (d, J = 6.9 Hz, 6H). ¹⁹F NMR (376 MHz, Methanol-d ₄) δ −105.34 (d, J _F–H = 12.9 Hz). m/s ES+ [M + H]⁺: 272.1.

(3S,4s,5R)-4-(2-Fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (22)

Utilizing General Synthetic Protocol D, (3S,4s,5R)-4-(4-chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 12% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.71 (s, 1H), 8.62 (s, 2H), 8.35 (dd, J = 22.3, 4.8 Hz, 1H), 7.56 (t, J = 2.5 Hz, 1H), 7.32 (d, J = 9.5 Hz, 2H), 7.27–7.06 (m, 1H), 4.94 (s, 1H), 3.09 (d, J = 11.9 Hz, 2H), 3.02–2.97 (m, 2H), 2.78–2.71 (m, 2H), 2.71 (s, 3H), 0.77 (d, J = 6.8 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −74.05, −107.14 (d, J = 14.5 Hz), −164.86. ¹³C NMR (151 MHz, DMSO-d ₆) δ 159.92 (d, J = 241.9 Hz), 144.31 (d, J = 3.5 Hz), 144.01, 41.80 (d, J = 242.5 Hz) 141.09 (d, J = 4.8 Hz), 138.06, 137.15 (d, J = 10.9 Hz), 129.35, 128.57 (d, J = 7.7 Hz), 115.68, 114.42 (d, J = 28.8 Hz), 109.63 (d, J = 27.5 Hz), 105.25 (d, J = 11.3 Hz), 77.89 (d, J = 6.3 Hz), 44.14, 36.02 (d, J = 7.9 Hz), 24.13 (d, J = 2.9 Hz), 12.27. m/s ES+ [M + H]⁺: 372.2. HRMS (ESI+): Expected 372.1882 [M + H⁺] (C₂₁H₂₃F₂N₃O). Observed 372.1874.

(3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-1,3,5-trimethylpiperidin-4-ol (Int-23)

Formaldehyde (0.065 g, 0.80 mmol, 37% solution in water) was added to a vial containing a solution of (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (31) (0.054 g, 0.20 mmol) in tetrahydrofuran (5.0 mL). The reaction was stirred at room temperature, and sodium triacetoxyborohydride (0.127 g, 0.60 mmol) was added portion wise. The reaction was stirred at 23 °C for 2 h. The reaction was then quenched by being poured into an aqueous saturated solution of sodium bicarbonate. The mixture was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation to give the title product in 71% yield, which was used without further purification. ¹H NMR (400 MHz, CD₃OD) δ 6.95 (d, J = 12.4 Hz, 2H), 2.72–2.65 (m, 2H), 2.64 (s, 3H), 2.54 (dd, J = 11.3, 4.1 Hz, 2H), 2.32–2.22 (m, 2H), 2.28 (s, 3H), 0.72 (d, J = 7.0 Hz, 6H). ¹⁹F NMR (376 MHz, CD₃OD) δ −104.90. m/s ES+ [M + H]⁺: 286.1.

(3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-6-methylphenyl)-1,3,5-trimethylpiperidin-4-ol (23)

Utilizing General Synthetic Protocol D (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-1,3,5-trimethylpiperidin-4-ol and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 35% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.70 (s, 1H), 8.32 (d, J = 4.9 Hz, 1H), 7.55 (d, J = 2.8 Hz, 1H), 7.30 (d, J = 8.3 Hz, 2H), 7.21 (d, J = 4.9 Hz, 1H), 4.98 (s, 1H), 3.29–3.21 (m, 2H), 3.06–2.96 (m, 2H), 2.82 (d, J = 4.5 Hz, 3H), 2.81–2.72 (m, 2H), 2.70 (s, 3H), 0.77 (d, J = 6.8 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.96, −107.06 (d, J = 14.0 Hz), −164.87. ¹³C NMR (151 MHz, DMSO-d6) 158.89 (d, J = 241.8 Hz), 144.32 (d, J = 3.3 Hz), 114.03, 141.81 (d, J = 242.63 Hz), 141.28 (d, J = 4.8 Hz), 138.02, 137.26 (d, J = 11.0 Hz), 129.44, 128.15 (d, J = 11.4 Hz), 115.69, 114.46 (dd, J = 29.3, 4.2 Hz), 109.65 (d, J = 27.6 Hz), 105.24 (d, J = 11.4 Hz), 77.07 (d, J = 6.3 Hz), 54.34, 42.76, 36.67, 24.06 (d, J = 2.9 Hz), 12.10. TFA salt: 158.11 (q, J = 33.6 Hz), 116.46 (q, J = 296.3 Hz). m/s ES+ [M + H]⁺: 386.1. HRMS (ESI+): Expected 386.2038 [M + H⁺] (C₂₂H₂₅F₂N₃O). Observed 386.2043.

(3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-1-isopropyl-3,5-dimethylpiperidin-4-ol (Int-24)

Acetone (0.16 g, 2.7 mmol, 0.20 mL) was added to a 5 mL microwave vial containing a stir bar. To this was added a solution of (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (32) (0.10 g, 0.37 mmol) in tetrahydrofuran (4.0 mL). Then acetic acid (2.2 mg, 0.04 mmol, 2.1 μL) was added, followed by sodium triacetoxyborohydride (0.234 g, 1.1 mmol) as a solid. The vial was sealed and was then heated to 60 °C for 1 h. The reaction was then allowed to cool and was quenched by being poured into an aqueous saturated solution of sodium bicarbonate. The mixture was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation to give the title product in 94% yield (0.11 g). For analytical purposes, a portion of the material was purified by reverse phase medium pressure liquid chromatography (5 to 90% acetonitrile in water, with 0.1% trifluoroacetic acid), and the analytical data is reported as the TFA salt. ¹H NMR (400 MHz, CDCl₃) δ 10.95 (s, 1H), 7.12–6.83 (m, 2H), 3.65–3.40 (m, 1H), 3.29–2.87 (m, 6H), 2.58 (s, 3H), 1.38 (d, J = 6.7 Hz, 6H), 0.80 (d, J = 6.8 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −75.93, −103.03, −103.06. m/s ES+ [M + H]⁺: 314.2.

(3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-6-methylphenyl)-1-isopropyl-3,5-dimethylpiperidin-4-ol (24)

Utilizing General Synthetic Protocol D, (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-1-isopropyl-3,5-dimethylpiperidin-4-ol and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 17% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.71 (s, 1H), 9.26 (s, 1H), 8.32 (d, J = 4.9 Hz, 1H), 7.56 (t, J = 2.4 Hz, 1H), 7.33 (d, J = 8.5 Hz, 2H), 7.22 (d, J = 4.9 Hz, 1H), 5.02 (s, 1H), 3.56–3.54 (m, 2H), 3.32–3.14 (m, 2H), 3.02 (q, J = 11.7 Hz, 2H), 2.87–2.75 (m, 2H), 2.71 (s, 3H), 1.31 (d, J = 6.6 Hz, 6H), 0.80 (d, J = 6.8 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.88, −107.28 (d, J = 14.8 Hz), −164.84. ¹³C NMR (151 MHz, DMSO-d ₆) δ 158.95 (d, J = 242.1 Hz), 144.33 (d, J = 3.5 Hz), 144.04, 141.80 (d, J = 242.8 Hz), 141.23 (d, J = 4.7 Hz), 138.02, 137.24 (d, J = 10.9 Hz), 129.47, 128.21 (d, J = 11.8 Hz), 115.70, 114.44 (dd, J = 28.7, 3.9 Hz), 109.65 (d, J = 27.6 Hz), 105.24 (d, J = 11.5 Hz), 77.56 (d, J = 6.5 Hz), 57.28, 48.33, 36.68 (d, J = 7.5 Hz),24.20, 16.38, 12.29. m/s ES+ [M + H]⁺: 414.1. HRMS (ESI+): Expected 414.2351 [M + H⁺] (C₂₄H₂₉F₂N₃O). Observed 414.2349.

(3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-3,5-dimethyl-1-(oxetan-3-yl)­piperidin-4-ol (Int-25)

Oxetan-3-one (0.044 g, 0.61 mmol) was added to a vial containing a stir bar and a solution of (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (32) (0.055 g, 0.20 mmol) in 1,2-dichloroethane (2.5 mL) and tetrahydrofuran (2.5 mL). Then sodium triacetoxyborohydride (0.21 g, 1.0 mmol) was added portion wise as a solid to the reaction. The vial was capped and the reaction was stirred for 2 h at 23 °C. The reaction was then quenched by being poured into an aqueous saturated solution of sodium bicarbonate. The mixture was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation to give the title product in 80% yield (0.053 g) which was used without further purification. ¹H NMR (400 MHz, CDCl₃) δ 6.98–6.83 (m, 2H), 4.66 (dd, J = 6.6, 1.3 Hz, 4H), 3.52 (p, J = 6.6 Hz, 1H), 2.71 (ddt, J = 11.3, 7.0, 3.6 Hz, 2H), 2.58 (s, 3H), 2.47 (dd, J = 11.2, 4.2 Hz, 2H), 1.96 (td, J = 11.4, 3.2 Hz, 2H), 0.71 (d, J = 6.9 Hz, 6H). ¹⁹F NMR (376 MHz, CDCl₃) δ −103.19 (d, J = 12.8 Hz). m/s ES+ [M + H]⁺: 328.0.

(3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-6-methylphenyl)-3,5-dimethyl-1-(oxetan-3-yl)­piperidin-4-ol (25)

Utilizing General Synthetic Protocol D, (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-3,5-dimethyl-1-(oxetan-3-yl)­piperidin-4-ol and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 24% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.67 (s, 1H), 8.31 (d, J = 4.9 Hz, 1H), 7.54 (s, 1H), 7.25 (d, J = 6.8 Hz, 3H), 7.21 (dd, J = 4.9, 1.2 Hz, 1H), 4.58–4.52 (m, 2H), 4.52–4.45 (m, 3H), 4.24–4.16 (m, 1H), 3.49–3.38 (m, 1H), 2.70 (s, 3H), 2.63–2.54 (m, 1H), 2.44–2.32 (m, 2H), 2.07–1.97 (m, 2H), 0.70 (d, J = 6.8 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.42, −106.68 (d, J = 14.3 Hz), −164.86. ¹³C NMR (151 MHz, DMSO-d ₆): 159.12 (d, J = 241.4 Hz), 144.35 (d, J = 3.5 Hz), 144.03, 141.84 (d, J = 242.8 Hz), 141.20 (d, J = 4.8 Hz), 138.29, 136.44 (d, J = 11.0 Hz), 130.75 (d, J = 10.4 Hz), 129.07, 115.66, 114.38 (dd, J = 29.4, 4.1 Hz), 109.51 (d, J = 27.6 Hz), 105.24 (d, J = 11.2 Hz), 79.11 (d, J = 6.0 Hz), 74.67, 58.42, 51.39, 37.94 (d, J = 7.4 Hz), 24.53, 12.96. m/s ES+ [M + H]+: 428.0. HRMS (ESI+): Expected 428.2144 [M + H⁺] (C₂₄H₂₇F₂N₃O₂). Observed 428.2131.

1-((3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)­ethan-1-one (Int-26)

Acetic anhydride (0.079 g, 0.77 mmol, 0.073 mL) was added to a vial containing a stir bar and a solution of (3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (31) (0.070 g, 0.258 mmol) in tetrahydrofuran (5.0 mL). Then diisopropylethylamine (0.100 g, 0.773 mmol, 0.135 mL) was added via syringe. The vial was capped and stirred for 3 h at 23 °C. The reaction was then quenched by being poured into a saturated aqueous solution of sodium bicarbonate. The mixture was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation. The product was then dried under vacuum (<1 Torr) to give the title product in 100% yield (82 mg) without further purification. ¹H NMR (400 MHz, CDCl₃) δ 6.98 – 6.79 (m, 2H), 4.43 (ddd, J = 12.4, 4.0, 1.9 Hz, 1H), 3.48 (ddd, J = 13.3, 4.6, 1.9 Hz, 1H), 3.18 (ddd, J = 13.4, 11.6, 3.0 Hz, 1H), 2.60 (s, 3H), 2.59–2.45 (m, 2H), 2.31–2.19 (m, 1H), 2.13 (s, 3H), 0.75 (dd, J = 10.2, 6.7 Hz, 6H). m/s ES+ [M + H]⁺: 314.0.

1-((3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)­ethan-1-one (26)

Utilizing General Synthetic Protocol D, 1-((3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)­ethan-1-one and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 22% yield. ¹H NMR (400 MHz, DMSO-d ₆) δ 11.76 (s, 1H), 8.34 (d, J = 4.9 Hz, 1H), 7.78–7.54 (m, 1H), 7.35 (dd, J = 12.3, 2.6 Hz, 2H), 7.26 (d, J = 4.9 Hz, 1H), 4.99 (s, 1H), 4.19 (d, J = 10.3 Hz, 1H), 3.60–3.47 (m, 1H), 3.16 (t, J = 12.4 Hz, 1H), 2.64 (t, J = 12.1 Hz, 2H), 2.39–2.28 (m, 1H), 2.27–2.17 (m, 1H), 2.05 (s, 3H), 0.80 (t, J = 7.4 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −73.41, −106.88 (d, J = 14.1 Hz), −164.76. ¹³C NMR (101 MHz, CD₃OD) δ 171.35, 160.94 (d, J = 240.2 Hz), 145.46 (d, J = 3.6 Hz), 144.83, 144.12 (d, J = 242.6 Hz), 142.76 (d, J = 4.7 Hz), 141.50 (d, J = 2.9 Hz), 138.60 (d, J = 10.4 Hz), 131.31 (d, J = 10.8 Hz), 130.58 (dd, J = 2.7, 2.8 Hz), 116.93 (d, J = 1.6 Hz), 115.83 (dd, J = 29.6, 4.1 Hz), 110.06 (d, J = 28.0 Hz), 107.90 (d, J = 11.8 Hz), 81.06 (d, J = 6.7 Hz), 40.46 (dd, J = 83.4, 7.7 Hz), 24.67 (d, J = 3.2 Hz), 21.26, 12.85 (d, J = 8.8 Hz). m/s ES+ [M + H]⁺: 414.2. HRMS (ESI+): Expected 414.1988 [M + H⁺] (C₂₃H₂₅F₂N₃O₂). Observed 414.1997.

4-((3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)­tetrahydro-2H-thiopyran 1,1-dioxide (32)

(3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (31) (0.55 g, 2.0 mmol) was added to a 20 mL microwave vial with a stir bar. Tetrahydro-4H-thiopyran-4-one 1,1-dioxide (0.39 g, 2.6 mmol) was then added, followed by 1,2-dichloroethane (15.0 mL). The reaction was stirred and acetic acid (0.012 g, 0.20 mmol, 0.012 mL) was added, followed by portion-wise addition of sodium triacetoxyborohydride (1.5 g, 7.1 mmol). The vial was sealed, and then heated via microwave irradiation to 50 °C for 5 h. The reaction was cooled, and then poured into an aqueous saturated solution of sodium bicarbonate. The mixture was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation to give the crude product. Purification by silica gel chromatography (40 to 100% ethyl acetate in hexanes) gave the title product in 54% yield (0.44 g). ¹H NMR (400 MHz, CDCl₃) δ 6.90 (dd, J = 10.8, 2.4 Hz, 2H), 3.42–3.35 (m, 1H), 3.22 (ddd, J = 11.1, 7.3, 3.4 Hz, 2H), 3.01–2.90 (m, 3H), 2.68–2.61 (m, 1H), 2.58 (s, 3H), 2.57–2.52 (m, 2H), 2.35–2.23 (m, 5H), 2.23–2.12 (m, 1H), 0.71 (d, J = 6.8 Hz, 6H). m/s ES+ [M + H]⁺: 404.1.

tert-Butyl (2-((3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)-2-oxoethyl)­carbamate (33)

(3R,4s,5S)-4-(4-Chloro-2-fluoro-6-methylphenyl)-3,5-dimethylpiperidin-4-ol (31) (0.82 g, 3.0 mmol) was added to a vial containing a stir bar, followed by (tert-butoxycarbonyl)­glycine (0.60 g, 3.4 mmol) and 1-[bis­(dimethylamino)­methylene]-1H-1,2,3-triazolo­[4,5-b]­pyridinium 3-oxide hexafluorophosphate (1.3 g, 3.4 mmol, HATU). Then tetrahydrofuran (10.0 mL) was added to the vial, and the reaction was stirred at 23 °C. Diisopropylethylamine (0.97 g, 7.5 mmol, 1.3 mL) was then added to the reaction, the vial was capped, and the reaction stirred for 3 h at 23 °C. The reaction was then quenched by being poured into an aqueous saturated solution of sodium bicarbonate. The mixture was extracted with ethyl acetate, the organic phase taken, and the solvent removed by rotary evaporation to give the crude product. Purification by silica gel chromatography (0 to 100% ethyl acetate in hexanes) gave the title product in 65% yield (0.84 g). ¹H NMR (400 MHz, CDCl₃) δ 6.90 (d, J = 12.7 Hz, 2H), 5.58 (s, 1H), 4.38 (ddd, J = 12.9, 4.9, 1.6 Hz, 1H), 4.00 (qd, J = 16.7, 4.3 Hz, 2H), 3.46–3.26 (m, 1H), 3.14 (td, J = 13.4, 12.6, 3.0 Hz, 1H), 2.77–2.65 (m, 1H), 2.60 (s, 3H), 2.57–2.46 (m, 2H), 1.46 (s, 9H), 0.75 (d, J = 6.8 Hz, 6H). m/s ES+ [M + H]⁺: 429.2.

4-((3R,4s,5S)-4-(2-Fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)­tetrahydro-2H-thiopyran 1,1-dioxide (27)

Utilizing General Synthetic Protocol D, 4-((3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)­tetrahydro-2H-thiopyran 1,1-dioxide (32) and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 20% yield. Note the compound was formulated as the hydrochloride salt by treatment of the free base with excess hydrogen chloride in diethyl ether, and removal of the solvent. ¹H NMR (600 MHz, DMSO-d ₆) δ 11.87 (s, 1H), 11.53 (s, 1H), 8.34 (d, J = 5.0 Hz, 1H), 7.59 (t, J = 2.4 Hz, 1H), 7.38–7.16 (m, 3H), 3.64–3.53 (m, 1H), 3.37–3.24 (m, 4H), 3.16–3.04 (m, 6H), 2.71 (s, 3H), 2.67–2.59 (m, 2H), 2.19 (td, J = 11.8, 8.4 Hz, 2H), 0.77 (d, J = 6.5 Hz, 6H). ¹⁹F NMR (376 MHz, DMSO-d ₆) δ −106.60 (d, J = 14.4 Hz), −164.69 (d, J = 3.3 Hz). ¹³C NMR: (151 MHz, DMSO-d ₆): 158.94 (d, J = 243.6 Hz), 143.43, 143.23, 141.86 (d, J = 242.8 Hz), 141.28 (d, J = 4.7 Hz), 138.87, 136.90 (d, J = 10.4 Hz), 129.32, 128.51 (d, J = 11.6 Hz), 115.71, 114.45 (dd, J = 27.9, 4.4 Hz), 109.95 (d, J = 27.6 Hz), 105.74 (d, J = 11.6 Hz), 77.65 (d, J = 6.3 Hz), 60.50, 49.03, 48.70, 36.21 (d, J = 8.0 Hz), 24.28 (d, J = 2.9 Hz), 23.94, 12.28. m/s ES+ [M + H]⁺: 504.2. HRMS (ESI+): Expected 504.2127 [M + H⁺] (C₂₆H₃₁F₂N₃O₃S). Observed 504.2132.

2-Amino-1-((3R,4s,5S)-4-(2-fluoro-4-(3-fluoro-1H-pyrrolo­[2,3-b]­pyridin-4-yl)-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)­ethan-1-one (28)

Utilizing general synthetic protocol E, tert-butyl (2-((3R,4s,5S)-4-(4-chloro-2-fluoro-6-methylphenyl)-4-hydroxy-3,5-dimethylpiperidin-1-yl)-2-oxoethyl)­carbamate (33) and 3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo­[2,3-b]­pyridine were combined to give the title product in 30% yield. ¹H NMR (400 MHz, CD₃OD) δ 8.26 (d, J = 5.0 Hz, 1H), 7.32–7.27 (m, 2H), 7.25–7.13 (m, 2H), 4.30 (dd, J = 13.1, 4.4 Hz, 1H), 3.62–3.45 (m, 3H), 3.24 (t, J = 12.1 Hz, 1H), 2.92–2.78 (m, 1H), 2.76 (s, 3H), 2.59 (qt, J = 12.4, 5.0 Hz, 2H), 0.83 (dd, J = 7.1, 3.9 Hz, 6H). ¹⁹F NMR (376 MHz, CD₃OD) δ −76.95, −108.34 (d, J = 14.2 Hz), −166.58 (d, J = 3.3 Hz). ¹³C NMR (CD₃OD, 100 MHz) 165.14, 161.02 (d, J = 242 Hz), 149.73, 144.71 (d, J = 246.2 Hz), 143.76 (d, J = 5.0 Hz), 137.05, 136.64, 136.05 (d, J = 11.3 Hz), 133.62 (d, J = 9.6 Hz), 131.07, 117.27, 116.34 (d, J = 26.2 Hz), 113.65 (d, J = 28.4 Hz), 112.84 (d, J = 14.0 Hz), 81.22 (d, J = 7.2 Hz), 47.56, 44.93, 41.06, 40.70 (d, J = 7.2 Hz), 39.96 (d, J = 8.0 Hz), 24.62 (d, J = 3.0 Hz), 12.84, 12.73. m/s ES+ [M + H]⁺: 429.2 HRMS (ESI+): Expected 429.2097 [M + H⁺] (C₂₃H₂₆F₂N₄O₂). Observed 429.2092.

Animal Ethics Statement

The wound healing studies were performed in compliance with the ethical rules on animal experimentation in accordance with the European Directive of 22 September 2010 concerning animal experimentation (2010/63/EU) and in accordance with the protocol described in the request of the project authorization using animals for scientific purposes subjected to the approval of the Ministry of Higher Education and Research (France). The mice used in this study were treated in accordance with the ethical rules edicted by the ASAB (ASAB Ethical Committee, 2012) and the Canadian Council on Animal Care (2003). Test facility authorization D 54–547–01 (France). Government authorization for live animal experiments 54–85/2012. The protocol was also submitted to the expertise of the local animal ethic committee (Comite Ethique Lorrain en Matiere d Experimentation Animale = CELMEA) via the APAFiS Platform and received a favorable opinion (Project authorization number APAFiS#35475) (France).

Glossary

Abbreviations Used

AcOH

acetic acid

ADME

adsorption distribution metabolism and excretion

AKT

serine-threonine protein kinase AKT

AMOTL2

angiomotin-like protein 2

AMPK

AMP-activated protein kinase

ATP

adenosine triphosphate

AUC

area under the curve

CDK8/11

cyclin-dependent kinase 8 or 11

Cl_p Obs

observed plasma clearance

CTGF

connective tissue growth factor

CYR61

cysteine-rich angiogenic inducer 61

DMPK2

CDC42 binding protein kinase γ

cMax

maximum concentration

DCM

dichloromethane

DIPEA

diisopropylethylamine

DMSO

dimethyl sulfoxide

F%

oral bioavailability

FU

fraction unbound

GSK3β

glycogen synthase kinase 3 β

HATU

hexafluorophosphate azabenzotriazole tetramethyl uronium

IC₅₀

concentration at which 50% of the enzymatic activity is inhibited

IP

intraperitoneal

IV

intravenous

K _d

dissociation constant

JAK1

Janus kinase 1

LATS1

large tumor suppressor kinase 1

LATS2

large tumor suppressor kinase 2

MLM

mouse liver microsomes

MPSK1

myristoylated and palmitoylated serine/threonine protein kinase

mRNA

mRNA

MST1/2

mammalian ST20-like kinase 1 or 2

nBuLi

n-butyl lithium

NDR1/2

nuclear dbf2-related kinase 1 or 2

nM

nanomolar

PAMPA

parallel artificial membrane permeability assay

PKG1

protein kinase G 1

PO

oral administration

PPB

plasma protein binding

qPCR

quantitative polymerase chain reaction

ROCK1/2

Rho-associated, coiled-coil-containing protein kinase 1 or 2

SAR

structure–activity relationship

t _1/2

terminal half-life

TEAD

transcriptional enhanced associated domain

TFA

trifluoroacetic acid

THF

tetrahydrofuran

TR-FRET

time-resolved fluorescence energy transfer

μM

micromolar

Vss obs

observed volume of distribution at steady state

YAP1

Yes-associated protein 1

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c01027.

• Selected potential leads from initial scaffold hopping; hinge binding domain screen; cellular scratch-wound assay8 h time point; in vivo wound healing assayselected time points and time to wound closure; experimental procedures and characterization for compounds in S2; experimental procedures, ethical statements and processes for biological assays; statistical analysis for biological assays; complete kinase selectivity tables for 27 and 28; synthetic schemes for compounds 7–26, analytical data and HPLC data for compounds evaluated in vivo; PDB structure of compound 31 (PDF)

• Molecular formula strings (CSV)

• X-ray structure of 31 (CIF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

This work was supported by the Intramural Research Program of the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, 1ZIATR000472. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The authors declare the following competing financial interest(s): The Authors declare the following competing financial interests. PM, DD, MC, CT, and SH are inventors on the patent Synthesis of pyrrolopyridine dual Lats /AKT inhibitors treating wounds, cancers, and heavy metal poisoning" WO2023239727. Their rights have been assigned to the US Government, but they may receive royalties on the patent.