Discovery of Focal Adhesion Kinase (FAK) activators to promote intestinal mucosal healing

Abstract

Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) causes injuries in the gastrointestinal mucosa. Proton pump inhibitors are the most common drugs prescribed to decrease upper gastrointestinal side effects caused by NSAIDs. However, they worsen the lower GI injuries caused by NSAIDs and do not directly promote restitution of the epithelial mucosa. Focal adhesion kinase (FAK) is essential in wound healing and epithelial sheet migration, making it an attractive pharmacological target. We have designed, synthesized, and evaluated a series of novel, specific FAK activators that increase FAK phosphorylation at Tyr-397 and promote mucosal healing in a Caco-2 model. We found that novel analogs 10bHCl, 10kHCl, and our original lead 5aHCl are preclinical candidates for further studies towards mucosal wound healing. For lead 5aHCl, which had previously shown healing in a mouse model, we performed a pharmacokinetic study. There are currently no therapeutics that promote mucosal healing, and no specific FAK activators beyond our work.

Graphical Abstract

1. Introduction

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most used drugs [1]. However, 10–40% of NSAID users and 50% of chronic users develop injuries in the gastrointestinal (GI) mucosa, thus developing upper or lower GI ulcers at increased rates [2–4]. The medical costs of adverse GI events associated with NSAID use likely exceed $4 billion per year [5]. NSAIDs injure the mucosa by two different mechanisms. Proximal GI injury reflects COX-1 inhibition [6]. However, distal small bowel injury is caused by NSAIDs complexing to bile acids, potentiating bile acid toxicity, as well as by changes in the numbers and types of enteric bacteria [7, 8].

Proton pump inhibitors (PPIs) are the drugs that are most commonly co-prescribed with NSAIDs to ameliorate proximal GI NSAID injury [9]. However, PPIs potentiate distal small bowel injury, perhaps by altering the intestinal microbiome [10], and are no longer recommended for prophylactic use in this context unless the patient is at particularly high risk for an upper GI injury [11]. Since many patients depend upon NSAIDs for pain relief or cardiovascular risk reduction, we must find a way to ameliorate their mucosal complications. Misoprostol, a prostaglandin analog, has been shown to reduce the risk of both upper and small intestinal injuries associated with the use of NSAIDs [12]. Unfortunately, misoprostol causes undesired secondary side effects such as abdominal pain, nausea or vomiting, or diarrhea in some patients [13]. It is therefore poorly tolerated, limiting its clinical utility for long-term prophylaxis [14]. Thus, a novel therapeutic agent that promotes proximal and distal GI mucosal healing is a significant unmet medical need that could affect millions of patients.

Mucosal healing represents a complex equilibrium between the processes that injure the bowel mucosa (inflammation, ischemia, and luminal agents) and the epithelial sheet migration and proliferation required to resurface the injured gut. This process involves restitution, proliferation, and angiogenesis, but its sine qua non is epithelial sheet migration [14]. Focal adhesion kinase (FAK), a 125 kDa non-receptor tyrosine kinase, is a key regulator of several cell processes, including cell adhesion, migration, and proliferation. FAK is overexpressed in many tumor lines, making it a novel target for anticancer therapy [15, 16]. Most anticancer compounds target the FAK kinase domain. In contrast, we have focused on the FERM FAK domain, which is involved in cell motility [17, 18].

Our innovative approach builds on the observation that FAK is activated by specific small molecules that mimic the tertiary structure of the FAK FERM subdomain [19]. FAK activation begins with a conformational change releasing the FAK kinase domain from the (inhibitory) FAK FERM domain. This frees the FAK kinase domain to autophosphorylate FAK at Tyr-397 [20]. FAK-FAK dimerization via the interaction of the FAT and FERM domains may also alter FAK conformation, allowing FAK autophosphorylation [21]. In either case, this initial Tyr-397 phosphorylation indicates FAK activation and triggers additional conformational changes that enable further activation of FAK by other kinases, such as Src [22].

A previous set of studies aimed to modulate FAK by blocking AKT1-FAK interactions [23–25]. A virtual screen based on the mutant peptide yielded effective molecules. However, molecules based on the actual FAK sequence activated FAK [19, 26–29]. Since FAK activation is critical for epithelial restitution [17, 30–32], we, therefore, tested whether these molecules might not only activate FAK but also promote epithelial monolayer wound closure. To show that faster wound closure reflects cell motility (not proliferation), we demonstrated that the most potent molecule from our selection stimulates wound closure and confirmed that it is not mitogenic [33].

Based on that, we performed structure-activity relationship (SAR) studies on 13 structurally similar compounds with predicted in silico drug-like properties using industry-standard Schrödinger software. Several of these molecules exhibit activity at low concentrations. Our further studies, including synthesizing a library of novel FAK small-molecule activators that activate FAK and accelerate in vitro wound closure at concentrations as low as 100 pM [34], suggested that the 1-(2-morpholino-5-(trifluoromethyl)phenyl)urea moiety connected to an aromatic ring via a short linear or cyclic hydrocarbon chain is essential. Members of this library also promote small-bowel mucosal wound healing in mice over 3 days without apparent toxicity [34]. In further SAR studies, we designed a novel third-generation series that resulted in a lead molecule 5a (Figure 1), with promising drug-like properties, which activates FAK in Caco-2 cells at 100 pM but does not activate Src or Pyk2 at 10 μM and induces epithelial sheet migration in vitro and stimulates gut mucosal wound healing in vivo [35]. Our present study aimed to screen a series of molecules to identify new candidates for further evaluation. This work describes the design, chemical synthesis, biological screening, and molecular modeling of novel FAK activators.

Fig. 1.

FAK activators identified in the previous screening campaign [34].

2. Results and discussion

Previously, we described a novel class of FAK activators that activate FAK at concentrations as low as 10 nM in human Caco-2 intestinal epithelial cells [34, 35]. Compounds 1–5aHCl increased FAK phosphorylation at Tyr-397, thereby activating FAK and promoting the healing of intestinal epithelial wounds in vitro. Further analysis showed that compounds 1–5aHCl also promoted mucosal healing by stimulating wound closure in Caco-2 cells by approximately 10–26% over baseline wound closure rates (Figure 1). Among these compounds, in vitro studies of 4 and 5aHCl demonstrated superior FAK activation and monolayer wound closure when compared to 1–3 [34, 35].

Compounds 1–5aHCl have a common central 2-morpholino-5-(trifluoromethyl)aniline core, which is required for their biological activity. In our continuing efforts to explore structure-activity relationships for this series, we focused our attention on further developing compounds 4 and 5aHCl. For example, compound 4 features a novel, ubiquitous aliphatic linker bearing a terminal methoxyphenyl ring and compound 5aHCl bearing a 4-pyridyl moiety. In comparison, less active compounds such as 1–3 bear a pyrrolidine moiety as shown in Figure 1.

Structure Activity Relationship (SAR) Studies

In the present study, we continued our structure-activity studies by exploring the influence of aromatic substitution by introducing pyridines, modifying linkers, and modifying the morpholine ring to probe the impact of these structural modifications on FAK activation (Figure 2).

Fig. 2.

Novel FAK activators.

The computed physicochemical properties of 4 suggested a potential toxicity with borderline hERG liability. Therefore, we replaced the terminal phenyl ring with pyridines to address hERG liability by pursuing a nitrogen walk on the pyridine ring, as shown in compounds 5a-5c and their salts 5aHCl-5cHCl. In addition, a metabolic prediction, performed in SMARTCyp [36], suggested that the N,N-dimethylamino moiety might be prone to metabolism. Therefore, we introduced modifications to the linker to improve metabolic stability and the pharmacological profile of the new FAK activators. We replaced the N,N-dimethyl-1-(pyridin-4-yl)ethane-1,2-diamine moiety with different linkers such as 2-methyl-2-(pyridin-4-yl)propan-1-amine, compound 6, 2-(pyridin-4-yl)ethan-1-amine, compound 7, and their respective salts, 6HCl and 7HCl. Similarly, a pyrazine moiety was introduced at positions 8 and 8HCl to improve metabolic stability and reduce the ring’s basicity. Finally, we further explored variations of the side chain with methyl 2-amino-3-(pyridin-4-yl)propanoate (9a) and 2-amino-N-(2-methoxyethyl)-3-(pyridin-4-yl)propenamide (9b). We also sought to examine the influence of the aniline moiety on activity by replacing the CF₃ group with both electron-withdrawing and electron-donating substituents. Finally, substitution at the 2-position of the morpholine ring was pursued.

The synthesis of the novel analogs of 4 and 5aHCl was performed by coupling phenyl carbamate 14, prepared from 2-morpholino-5-(trifluoromethyl)aniline, with commercially available aliphatic amines or their 2-amino-3-(pyridin-4-yl)propanoic acid derivatives to obtain the desired ureas 5-9 (Scheme 1). Hydrochloric salts of selected compounds, in addition to acetate and phosphate salts, were also synthesized as previously described [34].

Scheme 1.

Synthesis of novel analogs of 4 as FAK activators. Reagents and conditions: a) Phenyl chloroformate, Et₃N, DCM, 0 °C, overnight; b) appropriate amine, py, 80 °C, overnight; c) 2N HCl in diethyl ether, rt, overnight; d) 2N AcOH in diethyl ether, overnight; f) 2N H₃PO₄ in diethyl ether, rt, overnight.

The synthesis of morpholine analogs is shown in Scheme 2. Alkylation of commercially available 4-fluoro-3-nitrophenyl provided intermediate ethers 15. The synthesis of methoxy and PEG-like analogs was performed by alkylation of 4-fluoro-3-nitrophenol with the respective bromo- or chloroalkyl derivatives, affording intermediates 16a-16f, following previously reported procedures [37]. Intermediates 15 and 16 underwent S_NAr reaction with commercially available morpholines, followed by reduction of the nitro groups to obtain the corresponding aniline derivatives 17–19. The anilines were converted to their respective carbamates, followed by the addition of N,N-dimethyl-1-(pyridin-4-yl)ethane-1,2-diamine to obtain the corresponding ureas 10-11, which were converted to the corresponding salts 10HCl and 11HCl. The synthesis of analogs 12 and 13, in which the aromatic substitution pattern on the aniline moiety was modified, is shown in Scheme 3. Anilines 20 and 21 were converted to intermediates 22 and 23 by reaction with phenyl chloroformate. Subsequent reaction with N,N-dimethyl-1-(pyridin-4-yl)ethane-1,2-diamine provided analog 12 with a meta-substituted morpholine and analog 13 with a para-CF₃ moiety. Both analogs were converted to the corresponding salts 12HCl and 13HCl.

Scheme 2.

Synthesis of morpholine analogs of 5. Reagents and conditions: a) appropriate bromoalkyl, K2CO3, acetone, reflux, overnight; b) appropriate amine, K2CO3, DMF, 60 °C, overnight; c) H2, Pd/C in MeOH or hydrazine, Pd/C in EtOH, rt, 3 h; d) Phenyl chloroformate, Et3N, DCM, 0 °C, overnight, or K2CO3, acetone, rt, overnight; e) N,N-dimethyl-1-(pyridin-4-yl)ethane-1,2-diamine, py, 80 °C, overnight; f) 2N HCl in diethyl ether, rt, overnight.

Scheme 3.

Synthesis of morpholine analogs of 5. Reagents and conditions: a) Phenyl chloroformate, Et₃N, DCM, 0 °C, overnight; b) N,N-dimethyl-1-(pyridin-4-yl)ethane-1,2-diamine, py, 80 °C, overnight; c) 2N HCl in diethyl ether, rt, overnight.

Biological evaluation of the novel analog library was performed (Tables 1–5) to assess their potency to stimulate HIEC-6 FAK activation (using commercially available ELISA for FAK-397) at 100 nM and 10 nM, respectively, for those compounds with high activation. The ability of the most potent analogs was examined for wound closure (Table 6, Figure 3) in Caco-2 cells (FAK-397 by western blotting).

Table 1.

Activity at 100 nM concentration^a

Compound	R	R1	R2	R3	R4	FAK-Tyr-397/FAK fold change, ± SEM
5a	4-pyridinyl	N(CH3)2	H	H	H	0.82±0.06
5b	3-pyridinyl	N(CH3)2	H	H	H	1.0±0.04
5c	2-pyridinyl	N(CH3)2	H	H	H	0.84±0.11
5aHCl	4-pyridinyl	N(CH3)2	H	H	H	1.25±0.08
5bHCl	3-pyridinyl	N(CH3)2	H	H	H	1.05±0.06
5cHCl	2-pyridinyl	N(CH3)2	H	H	H	0.98±0.07
6	4-pyridinyl	CH3	CH3	H	H	1.09±0.12
7	4-pyridinyl	H	H	H	H	0.98±0.07
8	Pyrazine	H	H	H	H	0.93±0.08
6HCl	4-pyridinyl	CH3	CH3	H	H	1.11±0.12
7HCl	4-pyridinyl	H	H	H	H	0.91±0.13
8HCl	Pyrazine	H	H	H	H	1.05±0.07
9a	4-pyridinyl	N(CH3)2	H	H	CO2CH3	1.03±0.08
9b	4-pyridinyl	N(CH3)2	H	H	CON(CH2)2OCH3	0.97±0.08

^an = 8 measurements

Table 5.

^aActivity^a at 100 nM, activity^b at 10 nM^c

Compound	R1	R2	R3	R4	FAK-Tyr-397/FAK fold change, ± SEM
5a	CF3	H	H	morpholine	0.82a±0.06
5aHCl	CF3	H	H	morpholine	1.25a±0.08; 1.25b ±0.08
11	CF3	H	H	3-methylmorpholine	1.2a±0.08; 1.06b±0.05
11HCl	CF3	H	H	3-methylmorpholine	1.04a±0.04; N/Ab
12	CF3	H	morpholine	H	0.93a±0.06; N/Ab
13	H	CF3	H	morpholine	1.03a±0.10; N/Ab
12HCl	CF3	H	morpholine	H	0.98a±0.14; N/Ab
13HCl	H	CF3	H	morpholine	1.05a±0.20; N/Ab

^cn = 8 measurements

Table 6.

EC₅₀ of lead compounds

Compound	EC50 (nM), ± SEM
5aHCl	3.01±0.52
10bHCl	2.36±0.58
10kHCl	3.01±0.51

Fig. 3.

(a) Typical wound images after treatment with vehicle control (H₂O), compounds 5aHCl, 10bHCl, and 10kHCl at 100 nM with the addition of 4 mM hydroxyurea to all conditions. 0- and 24-h images were obtained after wounding Caco-2 monolayers. (b) Although cellular proliferation is inhibited by 4 mM hydroxyurea, wound closure is nevertheless accelerated by compounds 5aHCl, 10bHCl, and 10kHCl (n=33–35, pooled from 2 different studies with similar results; *p<0.05).

As shown in Table 1, compounds 5a-9b were tested at 100 nM for FAK activation. FAK activation was assessed as the ratio of FAK-Y-397 phosphorylation to total FAK protein by Western blot, and the ratio was expressed as the ratio of that measurement in cells treated with the experimental agent to that in cells exposed only to the relevant vehicle control. Among all analogs, our previously reported activator 5aHCl [35], the hydrochloride salt of 5a, displayed the best FAK activation across this series. Compound 5aHCl bears the nitrogen in the para position of the pyridine ring. The 3-pyridyl and 2-pyridyl analogs 5bHCl and 5cHCl had lower activities. A comparison of FAK activation of the parent compounds 5a-c, with 6, 7, and 8, which bear modified linkers lacking the dimethylamine moiety, showed reduced activity. Compounds 6HCl, 7HCl, and 8HCl, the hydrochloric salts of 6, 7, and 8, respectively, also did not show enhanced FAK activation when compared to 5aHCl. We posit that the lack of the dimethylamine side chain in compounds 7 and 8 caused the decreased FAK activation as compared to compound 5aHCl. Previously, we reported that the hydrochloride salt enhances activation [34]. Therefore, we synthesized both the acetate and phosphate salt derivatives of 5a (Scheme 2) to compare FAK activation with the corresponding hydrochloric salt. As shown in Table 2, when comparing the analogs side by side, 5aHCl showed slightly superior activity to both 5aAcetate and 5aPhosphate. With these results in hand, we proceeded further to screen hydrochloric salts of the most promising compounds. Quantitative ¹H NMR was performed with hydrochloric acid analogs, and it was determined that the number of HCl equivalents is 1–1.5. Compounds 9a and 9b, bearing a methyl ester and a methoxyethyl amino moiety, respectively, showed modest FAK activation compared to 5aHCl. These R⁴ side-chain modifications offer some room to accommodate longer aliphatic chains at this position for future designs.

Table 2.

Activity at 100 nM concentration^a

Compound	FAK-Tyr-397/FAK fold change, ± SEM
5aHCl	1.25±0.08
5aAcetate	1.18±0.05
5aPhosphate	0.96±0.07

^an = 8 measurements

In continuation of our exploration of novel analogs of 5a, we replaced the CF₃ group with both electron-withdrawing and electron-donating moieties, while maintaining both the morpholine and the N,N-dimethyl-1-(pyridin-4-yl)ethane-1,2-diamine side-chain constant. As shown in Table 3, compounds 10a and 10b, bearing H and F, respectively, showed greater FAK phosphorylation activity than compound 5a. We further explored the substitution of CF₃ with other fluorinated groups, such as CHF₂ (compound 10f) and OCF₃ (compound 10g). We found that substituting CF₃ with F, as shown for compound 10b, and compounds 10f and 10g, displayed enhanced FAK activation compared to parent compound 5a. In contrast, those analogs bearing a CH₃ (compound 10c), a CN (compound 10d), or a Cl (compound 10e) did not show enhanced FAK activation compared to compound 5a.

Table 3.

Activity at 100 nM concentration^a

Compound	R	FAK-Tyr-397/FAK fold change, ± SEM
5a	CF3	0.82±0.06
10a	H	1.23±0.07
10b	F	1.12±0.05
10c	CH3	0.89±0.05
10d	CN	0.83±0.07
10e	Cl	0.82±0.06
10f	CHF2	1.2±0.08
10g	OCF3	1.12±0.03
10h	OCH3	1.29±0.07
10i	OMOM	1.28±0.1
10j	O(CH2)2OCH3	1.05±0.1
10k	O(CH2)2O(CH2)2OCH3	1.32±0.03
10l	O(CH2)2O(CH2)2O(CH2)2OCH3	1.13±0.1
10m	O(CH2)2O(CH2)2OH	0.93±0.1

^an = 8 measurements

Similarly, we also pursued the substitution of the CF₃ group with the OMe group (compound 10h) and the MOM group (compound 10i). As shown in Table 2, the new analogs with PEG-like moieties showed greater FAK activation than compound 5a. Based on these encouraging results, we pursued the synthesis of new analogs with longer PEG-like side chains, including O(CH₂)₂O CH₃ (10j), O(CH₂)₂O(CH₂)₂O CH₃ (10k), O(CH₂)₂O(CH₂)₂O(CH₂)₂O CH₃ (10l), and O(CH₂)₂O(CH₂)₂OH (10m). All the PEG-like derivatives showed greater FAK activation than compound 5a. Among them, compound 10k had a slightly higher FAK activation than compounds 10h and 10i. From these results, we concluded that replacing the CF₃ group with fluorinated or PEG-like side-chain moieties directly affects FAK activation, depending on the substituent’s size and chemical nature.

The encouraging data for compounds 10a, 10b, 10f-10i, and 10k, showing enhanced FAK activation compared to compound 5a (Table 3), prompted us to pursue the synthesis of their respective hydrochloric salts and compare them side-by-side with compound 5aHCl for FAK activation. These novel compounds were tested at 100 nM; some were also tested at 10 nM. As shown in Table 4, among the hydrochlorides, 10bHCl showed a 1.39-fold increase in FAK activation at 100 nM, compared to its parent compound, 10b, which showed a 1.12-fold increase at 100 nM. Analog 10bHCl was equipotent with compound 5aHCl at 10 nM, resulting in a 1.25-fold increase in FAK activation. In contrast, compounds 10fHCl and 10gHCl showed decreased FAK activation compared to their parent compounds 10f and 10g in the fluorine series. A similar trend was found in the PEG-like series. The hydrochloric salts of 10h and 10i, 10hHCl and 10iHCl, were less effective as FAK activators than their parent compounds.

Table 4.

Activity^a at 100 nM, activity^b at 10 nM^c

Compound	R	FAK-Tyr-397/FAK fold change, ± SEM
5aHCl	CF3	1.25±0.08a; 1.25±0.08b
10aHCl	H	1.06a±0.06; N/Ab
10bHCl	F	1.39a±0.12; 1.25b±0.05
10fHCl	CHF2	1.09a±0.03; N/Ab
10gHCl	OCF3	1.17a±0.06; N/Ab
10hHCl	OCH3	1.06a±0.08; N/Ab
10iHCl	OMOM	1.19a±0.05; N/Ab
10kHCl	O(CH2)2O(CH2)2OCH3	1.19a±0.09; N/Ab

^cn = 8 measurements

We next tested the influence of morpholine modifications on FAK activation (Table 5). Our previous studies showed that replacing the morpholine ring ablated FAK activation [34]. Compound 11, bearing a methyl group on the morpholine ring, displayed FAK activation similar to that of compound 5aHCl at 100 nM. However, the relative phosphorylation activity decreased at 10 nM. Compound 11HCl retained FAK activation, but it was lower than that of compounds 5aHCl, 10bHCl, and 10kHCl. Likewise, we investigated analogs in which the placement of CF₃ and morpholine groups was changed, as demonstrated by compounds 12 and 13, together with their hydrochloric salts, 12HCl and 13HCl, respectively (see Table 5). The analog with morpholine in the meta position (12) and its corresponding hydrochloric salt (12HCl) showed decreased activity towards FAK activation. When the CF₃ moiety was placed in the para position, as shown in compound 13, while maintaining the morpholine in the ortho position, it and its hydrochloric salt (13HCl) did not show any enhanced activity towards FAK phosphorylation. From these results, we hypothesize that the addition of methyl groups to the morpholine ring was detrimental to activity.

Wound healing and PK of lead compounds

Testing of the active analogs 5aHCl and 10kHCl for wound healing, shown in Table 6, demonstrates that both compounds are equipotent in the Caco-2 assay, with EC₅₀s of 3.01 nM. In the case of 10bHCl, it showed a slightly better EC₅₀ of 2.36 nM, compared to 5aHCl and 10kHCl.

Since enhanced monolayer wound closure could reflect more rapid cell motility or increased cell proliferation, we repeated these studies in the presence of 4 mM hydroxyurea to prevent proliferation [35]. Wound closure was somewhat slower in the presence of hydroxyurea, as expected, but we nevertheless observed comparable stimulation of wound closure by all three compounds (Figure 3). Therefore, compounds 10bHCl and 10kHCl are potent new FAK activators that promote epithelial wound closure by promoting epithelial cell migration.

In addition to wound closure studies, in vitro pharmacokinetic (PK) parameters of our lead compounds 5aHCl, 10bHCl, and 10kHCl were also measured. As shown in Table 7, the lead compounds showed low hERG inhibition at 30 μM. The mouse liver microsome (MLM) and the hepatocyte half-life were both below 15 min. Their intrinsic clearance values for both 5aHCl and 10bHCl are high and agree with their half-lives, as they are metabolized faster than 10kHCl. Lastly, all the compounds are very soluble in PBS at pH = 7.4.

Table 7.

In vitro pharmacokinetic parameters

Compound	hERG Inhibition % (at 30 μM)	MLM t1/2 (min)	Hepatocytes t1/2 (min)	Clint (uL/min/mg protein)	Solubility (μg/mL)
5aHCl	4.87	< 4.51	7.85	> 307	1074
10bHCl	15.2	<4.51	7.27	> 307	985
10kHCl	0.608	14.9	64.8	93.0	1018

We previously showed that 5aHCl has drug-like properties, is water-soluble, orally bioavailable, shows no in vitro cytotoxicity at concentrations 10,000x higher than the effective dose, and has no in vivo toxicity after four days of continuous administration [35]. We are now reporting the pharmacokinetics (PK) study performed with compound 5aHCl. A single dose via oral gavage at 10 mg/kg of 5aHCl, in saline solution, was given to two-year-old male Fischer (CDF) rats. Blood samples were collected from the tail immediately before dosing and at 0.5, 1, 1.5, 2, 4, 6, and 10 h post-dosing. The resulting plasma concentrations were analyzed by UPLC-MS. As shown in Figure 4, the maximum concentration (C_max) was 967 ng/mL at 1 h, and the area under the curve was 3149 mg·h/L. The half-life is approximately 3 h. These findings suggest that 5aHCl is a good preclinical candidate for further studies towards mucosal wound healing.

Fig 4.

Pharmacokinetics of 5aHCl in rats (mean ± SD, n = 4).

Molecular Modeling

We also performed molecular modeling to identify possible binding sites of our small library targeting the FERM domain of Focal Adhesion Kinase (FAK). The 1.97 Å crystal structure of FAK FERM (PDB ID: 6CB0), of Gallus gallus, which has 96% sequence identity and 99% homology to the human protein, was used in this study (Figure 5) [38]. Binding site identification was performed using the Maestro’s SiteMap tool (Schrödinger, LLC, New York, NY, 2023). Five possible binding sites were found by SiteMap. To discriminate among these sites, we visually inspected each possible binding cavity and compared Sitescore values for each site. Among these binding sites, Site 1, the gray surface with magenta dots, had the highest Sitescore and is located on the dimerization interface, W266, of the monomers of the FERM domain (Figure 5) and (Figure S1).

Fig 5.

Cartoon representation of binding Sites 1–5. Site 1 is shown with magenta dots, on the FAK FERM domain (PDB 6CB0).

To understand key interactions between the potential binding site, Site 1, and the FAK activator library, we docked several compounds using Glide SP precision. For example, we compared the docking pose of protonated 5a and its analogs 5b and 5c. As shown in Figure 6, there are subtle changes in the position of the terminal pyridine ring when comparing 5a, 5b, and 5c within the binding site. Although the docking score is very similar for these compounds (Table 1, Supplementary Information), FAK activity is dictated by the interaction patterns with the residues around the pocket (Figure S2). For example, the terminal pyridine ring in 5a is in close contact with Lys259. In the case of 5b, the pyridine ring has close contact with Ala288 and Asp285; meanwhile, the pyridine ring of 5c interacts with Cys260 and Pro322. In addition, it is possible that the nitrogen in the para position, within the pyridine ring, could be better solvated and thus enhance phosphorylation activity compared to the 5b, meta analog, and 5c, ortho-analog.

Fig. 6.

A) Pose of compound 5a in the binding site; B) Pose of compound 5b in the binding site; C) Pose of compound 5c in the binding site

We also analyzed the docking pose of the N,N-dimethylamine moiety’s position. As shown in Figure 7, we compared the binding poses of protonated 5a and the analogs 7 and 8, which differ in the aliphatic moiety, an ethylene chain, connected to the 4-pyridinyl and pyrazine terminal rings, and do not carry the N,N-dimethylamine moiety. The N,N-dimethylamine moiety in 5a is in close contact with key residues, such as Lys259 and Ser269, in FAK. In contrast, the terminal side chains of analogs 7 and 8 point in a different direction than that of compound 5a. The terminal pyridine ring in 7 is proximal to Ala261. In the case of 8, the pyrazine ring is in close contact with Lys259 on the back side. In addition, the poses of these molecules differ within the pocket. The results demonstrate that the N,N-dimethylamine moiety is essential for the superior activity of 5a compared to analogs without this side chain.

Fig. 7.

A) Pose of compound 5a in the binding site; B) Pose of compound 7 in the binding site; C) Pose of compound 8 in the binding site.

Site 1, the possible binding site for FAK activators within the FAK FERM domain, as shown with magenta dots in Figure 5, is located at the interface between the two chains and is solvent-exposed. We hypothesize that the pocket would be flexible enough to accommodate different compounds and to accommodate large PEG-like side chains, such as those in compounds 10j-10m For instance, the docking pose for protonated compounds 10i and 10k shows that the MOM protrudes behind Lys286 and Asp285; meanwhile, the O(CH₂)₂O(CH₂)₂OCH₃ group in compound 10k protrudes beyond Glu271. Surprisingly, the orientation of compounds 10i and 10k is reversed. Thus, the MOM group in compound 10i interacts with Lys286 and Asp285, the identical residues as the terminal pyridine ring in compound 10k. Similarly, the O(CH₂)₂O(CH₂)₂OCH₃ group in compound 10k interacts with Lys259, in proximity to the terminal pyridine ring of compound 10i. (Figure 8).

Fig. 8.

A) Pose of compound 10i in the binding site; B) Pose of compound 10k in the binding site.

Lastly, we analyzed the protonated docking poses for the most promising compounds 5a, 10b, and 10k (Figure 9). The morpholine ring in compound 5a forms an H-bond with Ly286 (not shown for clarity), while Glu271 and Thr284 are proximal. The CF₃ group is in proximity to Leu283 and Tyr282. The N,N-dimethylamino side chain, with its terminal pyridine ring, interacts with Cys260 and Lys259. In the case of 10b, Cys260 and Lys259 are in the vicinity of the fluorine moiety, while Leu283 and Thr284 are proximal to the N,N-dimethylamino side chain, as well as Tyr282 and Thr284, respectively (Figure S2). The docking pose of 10k shows an H-bonding interaction between Thr284 and the carbamate NH, not shown for clarity, as well as between Glu285 and Leu283 (Figure S3). Meanwhile, the OEtOEtOME chain interacts with Ser269 by H-bonding and is close to Val270; meanwhile, the morpholine ring is in proximity to Leu283 and Thr284, chain A. In summary, 5a, 10b, and 10k interact with the same residues but adopt different orientations within Site1, the possible binding pocket.

Fig. 9.

A) Pose of compound 5a in the binding site; B) Pose of compound 10b in the binding site; C) Pose of compound 10k in the binding site.

Conclusions

Patients taking NSAIDs chronically develop both upper and lower gastrointestinal mucosal injury over time. Because no therapeutic modality currently exists, there is a medical need to develop a new pharmacological agent that can directly promote both upper and lower GI mucosal healing. Our structure-activity relationship studies based on previously reported compound 5aHCl showed that its 4-pyridyl moiety is superior to a 2- and 3-pyridyl moiety in the phosphorylation activity. We found that 5aHCl had superior activity compared to its salts 5aAcetate and 5aPhosphate. Side chain modified analogs 6HCl-8HCl that lack the terminal dimethylamino group showed activity but were not superior to 5aHCl. Compounds 9a and 9b, modified at R⁴ with ester and amid groups, retained activity, and related analogs could be explored further. Removal of the CF₃ group (10a) increased activity, as did replacement with CHF₂ (10b) and OCF₃ (10g), OMe (10h), the ether analogs MOM (10i), −(CH₂)₂OCH₃ (5a), and the PEG derivatives 10k-10m. Methyl (10c), cyano (10d), and Cl (10e) substituents had the same activity as 5a. However, of the 10HCl salts, only 10bHCl had superior activation activity compared to 5aHCl. Introduction of a 3-methyl group to the morpholine moiety (compound 11HCl) and a meta-morpholino group (12HCl) did not increase activation compared to 5aHCl. Computational docking studies were also performed to better understand the interactions between the synthesized library of FAK activators and the FAK FERM domain. These studies helped provide a qualitative understanding of the importance of key interactions between the morpholine ring, the N,N-dimethylamino moiety, the PEG derivative side chains, and the pocket residues (Site 1). Similarly, the absence of the N,N-dimethylamino moiety, as shown in compounds 7 and 8, did not improve activity. Lastly, the PEG derivatives 10k-10m showed enhanced activity compared to 5a, and the CF₃ group can be substituted. Even though computational docking scores can help guide the synthesis of improved FAK activators, it is necessary to have a crystallographic structure of the FAK FERM domain bound to 5aHCl to fully understand the molecular-level interactions. To date, co-crystallization has remained elusive and is beyond the scope of this work. Lead compounds 5aHCl, 10bHCl, and 10kHCl were tested for wound healing activity. Compounds 5aHCl, 10bHCl, and 10kHCl showed EC_50s of 3.01 nM in the Caco-2 assay. These results were confirmed by testing the analogs in the presence of 4 mM hydroxyurea to rule out rapid cell motility or increased cell proliferation as the source of wound healing. Wound closure was somewhat slower in the presence of hydroxyurea, as expected, but comparable stimulation of wound closure by all three compounds (Figure 9) was observed. As a result, compounds 10bHCl and 10kHCl were found to be potent new FAK activators that promote epithelial wound closure by promoting epithelial cell migration. Lastly, we performed a pharmacokinetic study with 5aHCl. The C_max was 967 ng/mL at 1 h, and the half-life was 3 h. In summary, our findings suggest that 5aHCl, 10bHCl, and 10kHCl are promising preclinical leads for further studies towards mucosal wound healing. The next generation of analogs, based on compounds 5aHCl and 10kHCl, with improved pharmacokinetics, has the potential to become a first-in-class therapy for NSAID-associated GI mucosal injury, promoting both proximal and distal mucosal healing. Although this work is focused on NSAID injury, a first-in-class drug that promotes epithelial healing could also be used to treat peptic ulcer disease or inflammatory bowel disease, and may apply to other wounds, such as diabetic foot ulcers or corneal ulcerations.

3. Experimental Section

3.1. Chemistry

All commercially available reagents and solvents were used without further purification. All yields reported were not optimized. ¹H NMR spectra were recorded on a Bruker 400/100 MHz spectrometer, and chemical shifts are reported in parts per million. Except for compound 10i (purity = 90%), all final compounds are ≥95% pure as determined by LC-MS analysis. Compounds were analyzed using a quadrupole/time-of-flight MS (G2S, Waters) coupled to a UPLC binary pump system (Acquity, Waters). Compounds were resolved on an ACQUITY UPLC HSS T3 column (1.8 μM, 100 Å pore diameter, 2.1 × 150 mm; Waters). Data were collected with the MS^E method, and MS was operated in sensitivity mode. Compounds were ionized by positive electrospray ionization. Preparative HPLC isolations were performed on an ACCQPrep HP150 (Kinetex C18, 250 × 21.2 mm, 5 μm, flow rate 20.0 mL/min, detection at 254 nm and 214 nm). Melting points were recorded on OptiMelt MPA100 series digital melting point apparatus.

All chiral compounds are racemic mixtures. Intermediates 17a-g, 18a, and 21 are commercially available.

3.1.1. General Procedure

3.1.1.1. Synthesis of 1-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea (5a).

Synthesis of Phenyl (2-morpholino-5-(trifluoromethyl)phenyl)carbamate. To a stirred solution of 2-morpholino-5-trifluoromethyl aniline (0.8 g, 3.2 mmol) in dry DCM (25 mL), under N₂ atmosphere at 0 °C, was added a solution of phenyl chloroformate (0.6 mL, 4.8 mmol) in dry DCM (5 mL), followed by Et₃N (0.65 mL, 8.10 mmol). The crude reaction was allowed to warm up at room temperature and stirred overnight. The crude reaction was concentrated in vacuo, and the product was purified by column chromatography using hexanes:EtOAc (70:30) to afford the pure product as a clear oil in 85% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.47 (s, 1H), 8.19 (s, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 4.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 3.91 (t, J = 4.0 Hz, 4 H), 2.92 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₈H₁₇F₃N₂O₃ [M+1]⁺: 367.3, observed: 367.3.

To a stirred solution of phenyl (2-morpholino-5-(trifluoromethyl)phenyl)carbamate (0.81 g, 2.21 mmol) in dry pyridine (5 mL) was added N,N-dimethyl-1-(pyridin-4-yl)ethane-1,2-diamine (0.44 g, 2.21 mmol), and the solution was heated at 80 °C for 16 hrs. The crude reaction was cooled down at room temperature and concentrated in vacuo. The product was purified by column chromatography using DCM:EtOH (95:5) to afford the pure product as an orange solid in 60% yield. M.p. 181–183 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.45 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.23–7.18 (m, 2H), 7.12 (d, J = 8.0 Hz, 1H), 6.26 (t, J = 8.0 Hz, 1H), 4.08–4.02 (m, 1H), 3.58–3.53 (m, 2H), 3.47–3.38 (m, 2H), 3.36-3-31 (m, 2H), 2.86–2.80 (m, 2H), 2.69–2.64 (m, 2H), 2.22 (s, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 155.3 149.6, 149.3, 145.3, 135.0, 128.0 127.7, 127.4 127.1, 125.5, 124.1 122.8, 120.4, 119.2, 119.2, 119.2, 119.1, 116.3, 116.3, 116.2, 69.12, 66.9, 52.0, 43.0, 42.5.

HRMS (ESI⁺) calculated for C₂₁H₂₆F₃N₅O₂ [M+1]⁺: 438.2111, observed: 438.2130.

Compounds 5b, 5c, 6–8, 9a-10l, 10m*, 11–13 were synthesized following the general procedure for the synthesis of 5a, and their respective spectroscopic characterization data can be found in the Supporting Information. *TBS deprotection was carried out with a 1M TBAF solution in THF to afford the crude product.

1-(2-(Dimethylamino)-2-(pyridin-3-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea (5b).

Obtained as a white solid in 50% yield. M.p. 146–148 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.61 (d, J = 4.0 Hz, 1H), 8.54 (s, 1H), 8.35 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.55 (s, br, 1H), 7.34 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 1H), 3.89–3.85 (m, 6H), 3.67–3.64 (m, 1H), 2.87 (t, J = 4.0 Hz, 4H), 2.37 (s, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 154.7, 150.1, 150.0, 143.5, 136.1, 134.6, 127.6, 127.3, 123.6, 120.4, 119.4, 116.1, 77.2, 67.2, 52.0, 41.6, 41.2. HRMS (ESI⁺) calculated for C₂₁H₂₆F₃N₅O₂ [M+1]⁺: 438.2111, observed: 438.2130.

1-(2-(Dimethylamino)-2-(pyridin-2-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea (5c).

Obtained as a white solid in 50% yield. M.p. 112–115 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.57 (d, J = 4.0 Hz, 1H), 8.31 (s, 1H), 7.95 (s, 1H), 7.76 (td, J = 8.0 Hz,4.0 Hz 1H), 7.34 (d, J = 8.0 Hz 1H), 7.29 (td, J = 8.0 Hz, 4.0 Hz, 1H), 7.23 (s, 1H), 7.16 (s, br, 1H), 3.78 (t, J = 4.0 Hz, 4H), 3.69–3.63 (m, 2H), 3.58–3.51 (m, 1H), 2.77 (t, J = 4.0 Hz, 4H), 2.16 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 154.9, 150.0, 143.2, 137.3, 135.2. 131.6, 126.9, 124.9, 120.6, 118.8, 109.4, 101.6, 77.2, 69.0, 67.2, 52.7, 40.4. HRMS (ESI⁺) calculated for C₂₁H₂₆F₃N₅O₂ [M+1]⁺: 438.2111, observed: 438.2130.

1-(2-Methyl-2-(pyridin-4-yl)propyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea (6).

Obtained as a white solid in 50% yield. M.p. 213–216 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.48 (d, J = 4.0 Hz, 2H), 8.19 (s, 1H), 7.84 (s, 1H), 7.38 (dd, J = 8.0 Hz, 4.0 Hz 2H), 7.23 (s, 2H), 7.05 (t, J = 4.0 Hz, 1H), 3.78 (t, J = 4.0 Hz, 4H), 3.35 (s, 2H), 2.76 (t, J = 4.0 Hz, 4H), 1.27 (s, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 156.1, 155.0, 149.4, 144.5, 134.5. 121.5, 120.0, 118.3, 116.2, 116.1, 65.8, 51.1, 49.5, 39.2, 25.6. HRMS (ESI⁺) calculated for C₂₁H₂₅F₃N₄O₂ [M+1]⁺: 423.2002, observed: 423.2012.

1-(2-Morpholino-5-(trifluoromethyl)phenyl)-3-(2-(pyridin-4-yl)ethyl)urea (7).

Obtained as a white solid in 50% yield. M.p. 121–124 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.59 (d, J = 4.0 Hz, 2H), 8.29 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.37 (dd, J = 4.0 Hz, 2H), 4.14 (s, 4H), 3.73 (t, J = 4.0 Hz, 2H), 3.31 (s, 4H), 3.22 (t, J = 4.0 Hz, 2H). ¹³C NMR (CDCl₃, 400 MHz): δ 148.4, 141.6, 134.6, 120.6, 119.7, 115.6, 118.8, 111.7, 77.3, 67.6, 67.3, 52.2, 51.1. HRMS (ESI⁺) calculated for C₁₉H₂₁F₃N₄O₂ [M+1]⁺: 395.1689, observed: 395.1698.

1-(2-Morpholino-5-(trifluoromethyl)phenyl)-3-(2-(pyrazin-2-yl)ethyl)urea (8).

Obtained as a white-cream solid in 40% yield. M.p. 315–317 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.58 (s, 1H), 8.54–8.51 (m, 2H), 8.26 (m, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 4.07 (s, 4H), 3.76 (t, J = 4.0 Hz, 2H), 3.23 (s, 4H), 3.16 (t, J = 4.0 Hz, 2H). ¹³C NMR (acetone-d₆, 400 MHz): δ 156.3, 146.0, 145.0, 143.5, 121.3, 119.0, 111.1, 107.9, 79.8, 67.3, 62.8, 53.8, 52.7, 36.2, 30.4. HRMS (ESI+) calculated for C₁₈H₂₀F₃N₅O₂ [M+1]⁺: 396.1641, observed: 396.1636.

Methyl 2-(3-(2-Morpholino-5-(trifluoromethyl)phenyl)ureido)-3-(pyridin-4-yl)propanoate (9a).

Obtained as a white solid in 40% yield. M.p. 157–158 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.48 (d, J = 8.0 Hz, 2H), 8.37 (s, 1H), 7.92 (s, 1H), 7.47 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.48 (s, br, 1H), 5.01 (q, J = 4.0 Hz, 1H), 3.94–3.87 (m, 4H), 3.81 (s, 3H), 3.37 (dq, J = 12.0 Hz, 4.0 Hz, 2H), 2.93–2.82 (m, 4H). ¹³C NMR (acetone-d₆, 400 MHz): δ 172.7, 168.0, 146.5, 133.3, 127.1, 121.4, 107.7, 96.8, 67.1, 53.9, 52.6, 44.1, 38.0. HRMS (ESI+) calculated for C₂₁H₂₃F₃N₄O₄ [M+1]⁺: 453.1744, observed: 453.1741.

N-(2-Methoxyethyl)-2-(3-(2-morpholino-5-(trifluoromethyl)phenyl)ureido)-3-(pyridin-4-yl)propanamide (9b).

Obtained as a white oil in 30% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.47 (s, br, 1H), 8.39 (s, 1H), 7.77 (s, 1H), 7.31 (s, br, 2H), 7.24 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.48 (s, br, 1H), 6.39 (d, J = 8.0 Hz, 1H), 4.74 (q, J = 4.0 Hz, 1H), 3.82 (t, J = 4.0 Hz, 4H), 3.45–3.35 (m, 4H), 3.29 (s, 3H), 3.18 (dq, J = 12.0 Hz, 4.0 Hz, 2H), 2.83 (t, J = 4.0 Hz, 4H). ¹³C NMR (CDCl₃, 400 MHz): δ 176.4, 170.7, 154.3, 147.3, 143.3, 137.0, 134.1, 124.8, 119.2, 115.9, 110.3, 70.2, 66.0, 57.8, 53.2, 51.1. HRMS (ESI+) calculated for C₂₃H₂₈F₃N₅O₄ [M+1]⁺: 496.2166, observed: 496.2178.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholinophenyl)urea (10a).

Obtained as a white solid in 30% yield. M.p. 157–158 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.50 (d, J = 4.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.17 (d, J = 4.0 Hz, 2H), 7.09 (d, J = 4.0 Hz, 2H), 7.02–6.98 (m, 1H), 5.41 (d, J = 4.0 Hz, 1H), 3.88–3.80 (m, 1H), 3.77–3.67 (m, 4H), 3.51–3.44 (m, 2H), 2.86–2.76 (m, 4H), 2.22 (s, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 155.5, 149.7, 148.3, 141.0, 134.3, 125.6, 123.9, 122.8, 120.4, 119.7, 68.9, 67.3, 52.3, 42.8, 42.2. HRMS (ESI⁺) calculated for C₂₀H₂₇N₅O₂ [M+1]⁺: 370.2237, observed: 370.2247.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-fluoro-2-morpholinophenyl)urea (10b).

Obtained as a white solid in 30% yield. M.p. 191–193 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.53 (d, J = 8.0 Hz, 2H), 7.87 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 7.70 (s, 1H), 7.19 (d, J = 4.0 Hz, 2H), 7.07–7.04 (m, 1H), 6.68–6.63 (m, 1H), 5.47 (s, br, 1H), 3.88–3.83 (m, 1H), 3.82–3.70 (m, 4H), 3.50–3.47 (m, 1H), 2.81–71 (m, 4H), 2.24 (s, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 154.9, 150.0, 136.2, 136.1, 123.9, 121.9, 121.8, 108.6, 108.3, 106.4, 106.1, 77.3, 68.6, 67.5, 52.9, 42.5, 41.8. HRMS (ESI⁺) calculated for C₂₀H₂₆FN₅O₂ [M+1]⁺: 388.2143, observed: 388.2158.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-methyl-2-morpholinophenyl)urea (10c).

Obtained as a white solid in 30% yield. M.p. 136–139 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.55 (d, J = 8.0 Hz, 2H), 7.71 (s, 1H), 7.39 (s, br, 1H), 7.21 (d, J = 4.0 Hz, 2H), 6.99 (d, J = 4.0 Hz, 1H), 6.81 (d, J = 4.0 Hz, 1H), 3.89–3.83 (m, 1H), 3.80–3.71 (m, 4H), 3.57–3.49 (m, 2H), 2.83–2.73 (m, 4H), 2.29 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 155.5, 149.8, 148.0, 138.6, 135.5, 134.0, 123.9, 123.5, 120.3, 120.2, 68.7, 67.4, 52.5, 42.7, 42.1, 21.4. HRMS (ESI⁺) calculated for C₂₁H₂₉N₅O₂ [M+1]⁺: 384.2393, observed: 384.2401.

1-(5-Cyano-2-morpholinophenyl)-3-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)urea (10d).

Obtained as a white solid in 30% yield. M.p. 189–191 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.68 (d, J = 4.0 Hz, 2H), 8.45 (s, 1H), 7.68 (s, br, 1H), 7.30 (t, J = 4.0 Hz, 2H), 7.27 (d, J = 4.0 Hz, 2H), 7.14 (d, J = 4.0 Hz, 1H), 3.92 (s, 4H), 3.74–3.64 (m, 2H), 2.89–2.86 (m, 5H), 2.52 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 154.7, 150.1, 144.6, 134.9, 126.8, 123.9, 122.7, 120.8, 108.9, 68.5, 67.1, 51.9, 42.3, 41.6, 29.8. HRMS (ESI⁺) calculated for C₂₁H₂₆N₆O₂ [M+1]⁺: 395.2189, observed: 395.2205.

1-(5-Chloro-2-morpholinophenyl)-3-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)urea (10e).

Obtained as a white solid in 30% yield. M.p. 284–286 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.64 (d, J = 4.0 Hz, 2H), 8.11 (d, J = 4.0 Hz, 1H), 7.73 (s, br, 1H), 7.27 (d, J = 4.0 Hz, 2H), 7.04 (d, J = 4.0 Hz, 1H), 6.95 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 3.86 (s, 4H), 3.68–3.64 (m, 2H), 2.84–2.76 (m, 5H), 2.44 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 154.8, 150.1, 139.1, 135.5, 131.3, 123.8, 122.5, 121.7, 119.3, 68.4, 67.4, 52.5, 42.3, 29.8. HRMS (ESI⁺) calculated for C₂₀H₂₆ClN₅O₂ [M+1]⁺: 404.1853, observed: 404.1870.

1-(5-(Difluoromethyl)-2-morpholinophenyl)-3-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)urea (10f).

Obtained as a white solid in 30% yield. M.p. 136–138 °C. ¹H NMR (CDCl₃, 400 MHz): δ 9.91 (s, 1H), 8.70 (d, J = 4.0 Hz, 1H), 8.57 (s, 1H), 7.70 (s, br, 1H), 7.55 (d, J = 4.0 Hz, 1H), 7.36 (d, J = 4.0 Hz, 1H), 7.22 (d, J = 40.0 Hz, 1H), 4.06–4.00 (m, 6H), 3.81–3.78 (m, 1H), 2.92 (s, 4H), 2.60 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 155.0, 150.0, 146.5, 142.8, 134.5, 131.8, 123.8, 120.7, 117.0, 68.4, 67.3, 52.2, 42.3, 41.7. HRMS (ESI⁺) calculated for C₂₁H₂₇F₂N₅O₂ [M+1]⁺: 420.2205, observed: 420.2215.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethoxy)phenyl)urea (10g).

Obtained as a white solid in 25% yield. M.p. 178–180 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.78 (d, J = 4.0 Hz, 2H), 8.34 (s, br, 1H), 7.40 (d, J = 4.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 6.81 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.34–4.30 (m, 1H), 4.02 (s, 4H), 3.93–3.90 (m, 2H), 2.86 (t, J = 4.0 Hz, 4H), 2.76 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 154.8, 150.1, 146.8, 138.7, 135.9, 123.9, 121.9, 121.5, 114.5, 112.1, 77.3, 68.5, 67.4, 52.6, 42.3, 41.6. HRMS (ESI+) calculated for C₂₁H₂₆F₃N₅O₃ [M+1]⁺: 454.2060, observed: 454.2063.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-methoxy-2-morpholinophenyl)urea (10h).

Obtained as a white solid in 25% yield. M.p. 209–212 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.51 (d, J = 4.0 Hz, 2H), 7.87 (s, 1H), 7.61 (d, J = 4.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.13 (t, J = 4.0 Hz, 1H), 7.02 (d, J = 8.0 Hz, 1H), 6.44 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 3.75 (t, J = 4.0 Hz, 4H), 3. 68 (s, 3H), 3.66–3.59 (m, 1H), 3.51–3.42 (m, 2H), 2.67–2.61 (m, 4H), 2.12 (s, 6H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 154.1, 154.9, 149.3, 135.7, 133.9, 124.0, 120.7, 106.0, 104.9, 67.9, 66.2, 55.0, 52.3, 42.0. HRMS (ESI⁺) calculated for C₂₁H₂₉N₅O₃ [M+1]⁺: 400.2343, observed: 400.2351.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-(methoxymethoxy)-2-morpholinophenyl)urea (10i).

Obtained as a white solid in 45% yield. M.p. 116–118 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.67 (d, J = 4.0 Hz, 2H), 7.88 (s, 1H), 7.84 (d, J = 4.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.05 (d, J = 8.0 Hz, 1H), 6.64 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 5.14 (s, 2H), 3.98–3.94 (m, 2H), 3.88 (q, J = 4.0 Hz, 4H), 3.72–3.69 (m, 1H), 3.46 (s, 3H), 2.79 (s, br, 4H), 2.49 (s, 6H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 160.8, 134.8, 133.6, 133.5, 130.2, 130.1, 119.0, 118.1, 38.9, 36.9, 28.9, 28.7, 28.6, 28.5, 28.0, 26.2. HRMS (ESI⁺) calculated for C₂₂H₃₁N₅O₄ [M+1]⁺: 430.2454, observed: 430.2478.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-(2-methoxyethoxy)-2-morpholinophenyl)urea (10j).

Obtained as a white solid in 25% yield. M.p. 174–175 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.52 (d, J = 4.0 Hz, 2H), 7.87 (s, 1H), 7.77 (s, br, 1H), 7.61 (d, J = 4.0 Hz, 1H), 7.28 (d, J = 4.0 Hz, 2H), 7.16 (t, J = 8.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.44 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 3.98–3.96 (m, 2H), 3.75 (t, J = 4.0 Hz, 4H), 3.64–3.59 (m, 2H), 3.46 (t, J = 4.0 Hz, 1H), 3.42–3.37 (m, 2H), 3.29 (s, 3H), 2.12 (s, 6H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 155.3, 154.9, 149.2, 147.0, 135.7, 134.0, 123.9, 120.6, 106.5, 105.6, 70.4, 67.9, 66.8, 66.2, 58.1, 52.3, 42.1, 40.7. HRMS (ESI⁺) calculated for C₂₃H₃₃N₅O₄ [M+1]⁺: 444.2605, observed: 444.2603.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-(2-(2-methoxyethoxy)ethoxy)-2-morpholinophenyl)urea (10k).

Obtained as a white solid in 25% yield. M.p. 104–105 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.58 (d, J = 4.0 Hz, 2H), 7.70 (s, br, 1H), 7.68 (d, J = 4.0 Hz, 1H), 7.23 (d, J = 4.0 Hz, 2H), 7.03 (d, J = 4.0 Hz, 1H), 6.54 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.12 (t, J = 4.0 Hz, 2H), 3.86–3.80 (m, 3H), 3.77–3.76 (m, 4H), 3.72–3.70 (m, 3H), 3.58– 3.55 (m, 2H), 3.38 (s, 3H), 2.81–2.71 (m, 4H), 2.33 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 156.7, 155.1, 150.2, 135.5, 133.8, 123.9, 121.7, 108.8, 105.1, 72.1, 70.8, 69.9, 68.6, 67.7, 67.6, 59.2, 53.0, 42.3, 41.5. HRMS (ESI⁺) calculated for C₂₅H₃₇N₅O₅ [M+1]⁺: 488.2867, observed: 488.2858.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2-morpholinophenyl)urea (10l).

Obtained as a white solid in 25% yield. M.p. 85–87 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.61 (d, J = 4.0 Hz, 2H), 7.80 (s, br, 1H), 7.69 (d, J = 4.0 Hz, 1H), 7.03 (d, J = 4.0 Hz, 2H), 6.54 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.10 (t, J = 4.0 Hz, 2H), 3.86–3.78 (m, 6H), 3.74–3.71 (m, 2H), 3.68–3.62 (m, 7H), 3.56– 3.53 (m, 2H), 3.37 (s, 3H), 2.77 (d, J = 4.0 Hz, 4H), 2.41 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 156.7, 155.2, 150.1, 135.5, 133.8, 123.9, 121.6, 108.9, 105.1, 77.3, 72.0, 70.9, 70.8, 70.7, 69.8, 68.6, 67.8, 67.6, 59.1, 53.0, 42.3, 41.6. HRMS (ESI⁺) calculated for C₂₇H₄₁N₅O₆ [M+1]⁺: 532.3135, observed: 532.3150.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-(2-(2-hydroxyethoxy)ethoxy)-2-morpholinophenyl)urea (10m).

Obtained as a white solid in 25% yield. M.p. 133–135 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.58 (d, J = 4.0 Hz, 2H), 7.80 (s, br, 1H), 7.23 (d, J = 4.0 Hz, 1H), 7.03 (d, J = 4.0 Hz, 2H), 6.54 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 5.64 (s, br, 1 H), 4.13 (t, J = 4.0 Hz, 2H), 3.84–3.82 (m, 3H), 3.79–3.75 (m, 4H), 3.66–3.64 (m, 2H), 3.59– 3.52 (m, 1H), 3.38–3.34 (m, 1H), 2.77 (t, J = 4.0 Hz, 4H), 2.32 (s, br, 6H). ¹³C NMR (CDCl₃, 400 MHz): δ 156.7, 155.3, 149.9, 147.4, 135.5, 134.1, 123.9, 121.6, 109.2, 105.5, 73.0, 70.1, 68.6, 68.1, 67.6, 61.9, 59.1, 52.9, 42.6, 42.0. HRMS (ESI⁺) calculated for C₂₄H₃₅N₅O₅ [M+1]⁺: 474.2716, observed: 474.2730.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-(3-methylmorpholino)-5-(trifluoromethyl)phenyl)urea (11).

Obtained as a white solid in 25% yield. M.p. 190–193 °C.¹H NMR (DMSO-d₆, 400 MHz): δ 8.54 (s, 2H), 8.43 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 4.0 Hz, 1H), 7.39 (t, J = 4.0 Hz, 1H), 7.32 (s, br, 2H), 7.23 (d, J = 8.0 Hz, 1H), 3.84–3.77 (m, 4H), 3.68 (s, br, 1H), 3.39–3.34 (m, 1H), 3.13– 3.08 (m, 1H), 2.66 (s, br, 3H), 2.15 (s, br, 6H), 0.65 (t, J = 4.0 Hz, 3H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 163.9, 155.7, 141.3, 137.8, 131.7, 123.6, 117.4, 114.1, 71.8, 66.4, 53.3, 16.6, 13.9. HRMS (ESI⁺) calculated for C₂₂H₂₈F₃N₅O₂ [M+1]⁺: 452.2273, observed: 452.2250.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(3-morpholino-5-(trifluoromethyl)phenyl)urea (12).

Obtained as a white solid in 25% yield. M.p. 354–357 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.58 (d, J = 4.0 Hz, 2H), 7.63 (s, br, 1H), 7.33 (s, 1H), 7.25 (d, J = 4.0 Hz, 2H), 6.93 (s, 1H), 6.75 (s, 1H), 5.87 (s, br, 1H), 3.96–3.89 (m, 1H), 3.83 (t, J = 4.0 Hz, 4H), 3.74 (t, J = 4.0 Hz, 1H), 3.59–3.52 (m, 1H), 3.18 (t, J = 4.0 Hz, 4H), 2.40 (s, 3H). ¹³C NMR (CDCl₃, 400 MHz): δ 155.5, 152.4, 150.1, 124.7, 124.0, 106.5, 97.1, 77.3, 66.8, 48.8, 42.2. HRMS (ESI⁺) calculated for C₂₁H₂₆F₃N₅O₂ [M+1]⁺: 438.2111, observed: 438.2128.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-4-(trifluoromethyl)phenyl)urea (13).

Obtained as a white solid in 25% yield. M.p. 208–210 °C. ¹H NMR (CDCl₃, 400 MHz): δ 8.57 (s, 2H), 8.14 (d, J = 8.0 Hz, 1H), 7.86 (s, br, 1H), 7.27 (d, J = 4.0 Hz, 3H), 7.22 (s, 1H), 3.83 (s, 4H), 3.64–3.52 (m, 3H), 2.78 (s, 4H), 2.37 (s, br, 3H). ¹³C NMR (CDCl₃, 400 MHz): δ 166.3, 155.2, 150.3, 150.2, 146.1, 140.5, 125.6, 124.0, 122.4, 120.5, 118.8, 66.8, 67.0, 60.9, 52.2, 47.7. HRMS (ESI⁺) calculated for C₂₁H₂₆F₃N₅O₂ [M+1]⁺: 438.2111, observed: 438.2135.

3.1.1.2. Synthesis of 1-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea hydrochloric salt (5aHCl).

1-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea (0.438 g, 1.0 mmol) was stirred in 6 mL of a 3N solution of HCl in diethyl ether at room temperature overnight. The crude product was concentrated in vacuo and lyophilized to yield a pure, orange solid in quantitative yield. M.p. 183–185 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 12.13 (s, br, 1H), 9.04 (d, J = 4.0 Hz, 2H), 8.32 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 8.0 Hz, 2H), 7.25 (s, 2H), 4.87 (s, br, 1H), 4.08–4.04 (m, 1H), 3.85–3.80 (m, 5H), 2.98 (s, 2H), 2.74 (s, 6H), 2.62 (s, 2H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 154.7, 150.7, 144.3, 143.4, 134.4, 127.5, 125.7, 124.4, 124.1, 123.1, 120.4, 118.5, 115.4, 67.2, 65.7, 51.2. HRMS (ESI⁺) calculated for C₂₁H₂₇ClF₃N₅O₂ [M+1]⁺: 474.1883, observed: 438.2117 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea acetate salt (5aAcetate).

1-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea (0.100 g, 0.22 mmol) was stirred in 2 mL of a 2N solution of AcOH in diethyl ether at room temperature overnight. The crude product was concentrated in vacuo and lyophilized to yield a pure, orange solid in quantitative yield. M.p. 77–78 °C. ¹H NMR (D₂O, 400 MHz): δ 8.70 (d, J = 4.0 Hz, 2H), 7.74 (s, 1H), 7.67 (d, J = 4.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 4.65–4.62 (m, 1H), 4.06–3.96 (m, 4H), 3.74 (t, J = 4.0 Hz, 4H), 2.91 (s, 6H), 2.77 (t, J = 4.0 Hz, 4H), 2.04 (s). ¹³C NMR (D₂O, 400 MHz): δ 176.8, 157.0, 148.4, 146.6, 142.6, 131.9, 128.0, 125.5, 125.4, 125.3, 125.2, 122.6, 121.5, 120.5, 119.1, 67.9, 66.6, 50.7, 41.6, 38.8, 20.4. HRMS (ESI⁺) calculated for C₂₃H₃₀F₃N₅O₄ [M+1]⁺: 498.2308, observed: 438.2130 - AcOH

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea phosphate salt (5aPhosphate).

1-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea (0.100 g, 0.22 mmol) was stirred in 2 mL of a 2N solution of H₃PO₄ in diethyl ether at room temperature overnight. The crude product was concentrated in vacuo and lyophilized to yield a pure, orange oil in quantitative yield. ¹H NMR (D₂O, 400 MHz): δ 8.91 (d, J = 4.0 Hz, 2H),8.21 (d, J = 4.0 Hz, 2H), 7.67 (d, J = 4.0 Hz, 1H), 7.41 (d, J = 4.0 Hz, 1H), 7.29 (d, J = 4.0 Hz, 1H), 4.05–3.99 (m, 2H), 3.80 (t, J = 4.0 Hz, 4H), 2.93 (s, 4H), 2.92 (s, 6H). ¹³C NMR (D₂O, 400 MHz): δ 157.4, 150.9, 145.2, 142.6, 131.7, 128.0, 126.4, 126.1, 125.8, 125.1, 122.4, 120.9, 120.1, 68.0, 66.2, 51.3, 42.0, 39.3. HRMS (ESI⁺) calculated for C₂₁H₂₉F₃N₅O₆P [M+1]⁺: 536.1886, observed: 438.2135 – H₃PO₄.

Compounds 5bHCl, 5cHCl, 6HCl-8HCl, 10aHCl-10kHCl, 11HCl-13HCl were synthesized following the general procedure for the synthesis of 5aHCl, and their respective NMR and LC/MS can be found in the Supplementary Information.

1-(2-(Dimethylamino)-2-(pyridin-3-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea hydrochloric salt (5bHCl).

Obtained as a brown solid in quantitative yield. M.p. 185–187 °C. ¹H NMR (D₂O, 400 MHz): δ 9.05 (s, 1H), 8.95 (s, 1H), 8.74 (d, J = 8.0 Hz, 1H), 8.17 (t, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 4.90 (t, J = 8.0 Hz, 1H), 4.06 (dq, J = 12.0 Hz, 4.0 Hz, 2H), 3.83 (s, 4H), 2.94 (s, 6H), 2.89 (s, 4H). ¹³C NMR (D₂O, 400 MHz): δ 157.3, 146.4, 146.2, 144.6, 144.0, 131.8, 122.0, 120.7, 119.7, 66.6, 66.1, 51.0, 38.9. HRMS (ESI⁺) calculated for C₂₁H₂₇ClF₃N₅O₂ [M+1]⁺: 474.1883, observed: 438.2130 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-2-yl)ethyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea hydrochloric salt (5cHCl).

Obtained as a yellow solid in quantitative yield. M.p. 120–122 °C. ¹H NMR (D₂O, 400 MHz): δ 8.61 (d, J = 4.0 Hz, 1H), 7.92 (t, J = 8.0 Hz, 1H), 7.73 (s, 1H), 7.51 (dd, J = 8.0 Hz, 4.0 Hz, 2H), 7.43 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 4.0 Hz, 1H), 4.39 (s, 1H), 3.93 (dq, J = 12.0 Hz, 4.0 Hz, 2H), 3.76 (s, 4H), 2.83 (d, J = 4.0 Hz, 4H), 2.74 (s, 5H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 152.3, 139.8, 134.2, 127.2, 126.7, 122.2, 118.2, 68.0, 52.4, 43.1. MS (ESI⁺) calculated for C₂₁H₂₇ClF₃N₅O₂ [M+1]⁺: 474.1883, observed: 438.2125 – HCl.

1-(2-Methyl-2-(pyridin-4-yl)propyl)-3-(2-morpholino-5-(trifluoromethyl)phenyl)urea hydrochloric salt (6HCl).

Obtained as a yellow solid in quantitative yield. M.p. 219–224 °C. ¹H NMR (D₂O, 400 MHz): δ 8.67 (d, J = 4.0 Hz, 2H), 8.11 (d, J = 8.0 Hz, 2H), 7.66 (s, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 3.84 (s, 4H), 3.54 (s, 2H), 2.87 (s, 4H), 1.44 (s, 6H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 159.1, 148.8, 141.8, 140.0, 132.2, 123.1, 112.5, 105.8, 101.8, 100.4, 70.3, 68.2, 62.0, 58.0, 52.5, 26.0. HRMS (ESI⁺) calculated for C₂₁H₂₆ClF₃N₅O₂ [M+1]⁺: 459.1774, observed: 423.2010 – HCl.

1-(2-Morpholino-5-(trifluoromethyl)phenyl)-3-(2-(pyridin-4-yl)ethyl)urea hydrochloric salt (7HCl).

Obtained as a yellow solid in quantitative yield. M.p. 133–134 °C. ¹H NMR (D₂O, 400 MHz): δ 8.65 (d, J = 4.0 Hz, 2H), 7.95 (d, J = 4.0 Hz, 2H), 7.69 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H), 3.85 (s, 4H), 3.63 (t, J = 8.0 Hz, 2H), 3.17 (t, J = 8.0 Hz, 2H), 2.92 (s, 4H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 141.9, 133.6, 129.4, 122.1, 107.1, 68.3, 52.5, 37.8 HRMS (ESI⁺) calculated for C₁₉H₂₂ClF₃N₅O₂ [M+1]⁺: 431.1461, observed: 395.1698 – HCl.

1-(2-Morpholino-5-(trifluoromethyl)phenyl)-3-(2-(pyrazin-2-yl)ethyl)urea hydrochloric salt (8HCl).

Obtained as a clear oil in quantitative yield. ¹H NMR (D₂O, 400 MHz): δ 8.58 (s, 2H), 8.51 (s, 1H), 7.64 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 3.87 (t, J = 4.0 Hz, 4H), 3.63 (t, J = 8.0 Hz, 2H), 3.09 (t, J = 8.0 Hz, 2H), 3.03 (t, J = 4.0 Hz, 4H). ¹³C NMR (DMSO-d₆400 MHz): δ 156.0, 153.9, 144.6, 141.7, 135.4, 128.3, 109.0, 83.0, 77.3, 66.4, 52.1, 35.3. HRMS (ESI⁺) calculated for C₁₈H₂₁ClF₃N₅O₂ [M+1]⁺: 432.1414, observed: 396.1634 – HCl.

1-2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholinophenyl)urea hydrochloric salt (10aHCl).

Obtained as a white solid in quantitative yield. M.p. 134–137 °C. ¹H NMR (D₂O, 400 MHz): δ 9.01 (d, J = 8.0 Hz, 2H), 8.30 (d, J = 8.0 Hz, 2H), 7.66–7.63 (m, 1H), 7.49–7.46 (m, 2H), 7.37–7.34 (m, 1H), 4.97 (t, J = 8.0 Hz, 1H), 4.12 (d, J = 8.0 Hz, 2H), 4.08 (t, J = 4.0 Hz, 4H), 3.56 (t, J = 4.0 Hz, 4H), 3.02 (s, 6H). ¹³C NMR (D₂O, 400 MHz): δ 158.7, 150.3, 142.9, 136.9, 130.5, 129.8, 128.1, 127.8, 126.6, 121.3, 67.9, 64.8, 53.7, 42.0, 39.6. HRMS (ESI⁺) calculated for C₂₀H₁₈ClN₅O₂ [M+1]⁺: 406.201, observed: 370.2245 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-fluoro-2-morpholinophenyl)urea hydrochloric salt (10bHCl).

Obtained as a white solid in quantitative yield. M.p. 169–171 °C. ¹H NMR (DMSO-d₆, 400 MHz): δ 8.99 (d, J = 8.0 Hz, 2H), 8.23 (s, 2H), 8.08 (s, 1H), 7.98 (s, br, 2H), 7.79 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 7.15 (dd, J = 8.0 Hz, 4.0 HZ, 1H), 6.70 (td, J = 8.0 Hz, 4.0 Hz, 1H), 4.81 (t, J = 4.0 Hz, 1H), 4.06–4.00 (m, 1H), 3.85–3.82 (m, 1H), 3.78 (t, J = 4.0 Hz, 4H), 2.97 (s, br, 6 H), 2.66 (t, J = 4.0 Hz, 4H). ¹³C NMR (D₂O, 400 MHz): δ 160.9, 158.5, 157.2, 148.8,144.0, 137.4, 133.7, 133.6, 127.4, 122.3, 122.3, 110.8, 110.5, 108.6, 108.4, 68.0, 66.5, 52.0, 41.9, 39.1. HRMS (ESI⁺) calculated for C₂₀H₂₇ClFN₅O₂ [M+1]⁺: 424.1915, observed: 388.2155 – HCl.

1-(5-(Difluoromethyl)-2-morpholinophenyl)-3-(2-(dimethylamino)-2-(pyridin-4-yl)ethyl)urea hydrochloric salt (10fHCl).

Obtained as a white solid in 95% yield. M.p. 268–270 °C. ¹H NMR (D₂O, 400 MHz): δ 8.98 (d, J = 4.0 Hz, 2H), 8.21 (d, J = 4.0 Hz, 2H), 7.69 (s, 1H), 7.40 (d, J = 4.0 Hz, 1H), 7.35 (d, J = 4.0 Hz, 1H), 6.80 (t, J = 56 Hz, 1H), 4.94–4.91 (m, 1H), 4.10 (d, J = 8.0 Hz, 2H), 3.88 (t, J = 4.0 Hz, 4H), 3.03 (s, 6H), 2.96 (t, J = 4.0 Hz, 4H). ¹³C NMR (D₂O, 400 MHz): δ 194.9, 157.4, 151.0, 150.2, 142.6, 131.3, 131.1, 128.9, 128.0, 124.6, 120.2, 68.1, 66.4, 50.6, 42.1, 39.4. HRMS (ESI⁺) calculated for C₂₁H₂₈ClF₂N₅O₂ [M+1]⁺: 456.1978, observed: 420.2218 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-5-(trifluoromethoxy)phenyl)urea hydrochloric salt (10gHCl).

Obtained as a yellow solid in 95% yield. M.p. 308–310 °C. ¹H NMR (D₂O, 400 MHz): δ 8.68 (s, br, 2H), 7.57 (s, 2H), 7.49 (s, 1H), 7.20 (s, 1H), 7.04 (s, 1H), 4.60–4.59 (m, 1H), 4.02–3.96 (m, 2H), 3.75 (s, 4H), 2.89 (s, 6H), 2.73 (s, 4H). ¹³C NMR (D₂O, 400 MHz): δ 156.9, 149.5, 145.1, 133.2, 121.4, 116.6, 114.4, 67.8, 66.7, 51.2, 41.5. HRMS (ESI⁺) calculated for C₂₁H₂₇ClF₃N₅O₃ [M+1]⁺: 490.1883, observed: 454.2060 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-methoxy-2-morpholinophenyl)urea hydrochloric salt (10hHCl).

Obtained as a white solid in 95% yield. M.p. 175–178 °C. ¹H NMR (D₂O, 400 MHz): δ 8.94 (s, 2H), 8.16 (d, J = 4.0 Hz, 2H), 7.45 (d, J = 4.0 Hz, 1H), 7.03 (d, J = 4.0 Hz, 1H), 6.92 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.89–4.86 (m, 1H), 4.07 (d, J = 8.0 Hz, 2H), 3.97 (t, J = 4.0 Hz, 4H), 3.82 (s, 3H), 3.27 (t, J = 4.0 Hz, 4H), 2.98 (s, 6H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 161.2, 158.6, 152.8, 143.4, 133.9, 129.5, 127.2, 124.9, 111.6, 69.0, 65.4, 57.2, 56.0, 43.6. HRMS (ESI⁺) calculated for C₂₁H₃₀ClN₅O₃ [M+1]⁺: 436.2115, observed: 400.2354 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-(methoxymethoxy)-2-morpholinophenyl)urea hydrochloric salt (10iHCl).

Obtained as a white solid in 95% yield. M.p. 220–223 °C. ¹H NMR (D₂O, 400 MHz): δ 9.00 (d, J = 8.0 Hz, 2H), 8.31 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 4.0 Hz, 1H), 6.94 (dd, J = 8.0 HZ, 4.0 Hz, 1H), 6.74 (d, J = 4.0 Hz, 1H), 4.97 (t, J = 4.0 Hz, 1H), 4.11–4.09 (m, 7H), 3.66 (t, J = 4.0 Hz, 4H), 3.00 (s, 6H). ¹³C NMR (D₂O, 400 MHz): δ 15.8, 150.4, 142.7, 131.6, 128.2, 127.8, 122.8, 115.0, 67.9, 64.4, 63.5, 54.5, 43.1, 42.0, 39.7. HRMS (ESI⁺) calculated for C₂₂H₃₂ClN₅O₄ [M+1]⁺: 466.2221, observed: 430.2450. – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(5-(2-(2-methoxyethoxy)ethoxy)-2-morpholinophenyl)urea hydrochloric salt (10kHCl).

Obtained as an orange solid in 95% yield. M.p. 159–161 °C. ¹H NMR (D₂O, 400 MHz): δ 8.74 (d, J = 8.0 Hz, 2H), 7.73 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 4.0 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H), 6.77 (dd, J = 4.0 Hz, 1H), 4.69–4.65 (m, 1H), 4.17–4.14 (m, 2H), 4.07–3.94 (dq, J = 12.0 Hz, 8.0 Hz, 2H), 3.87–3.85 (m, 2H), 3.78 (t, J = 4.0 Hz, 4H), 3.73–3.72 (m, 2H), 3.62–3.60 (m, 2H), 3.35 (s, 3H), 2.92 (s, 6H), 2.80 (t, J = 4.0 Hz, 4H). ¹³C NMR (DMSO-d₆, 400 MHz): δ 155.5, 154.9, 144.9, 135.3, 126.9, 121.1, 106.9, 105.4, 71.3, 69.7, 68.9, 67.4, 67.1, 66.1, 58.1, 52.4, 25.4. HRMS (ESI⁺) calculated for C₂₅H₃₈ClN₅O₅ [M+1]⁺: 524.2639, observed: 488.2856 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-(3-methylmorpholino)-5-(trifluoromethyl)phenyl)urea hydrochloric salt (11HCl).

Obtained as an orange solid in 95% yield. M.p. 187–190 °C. ¹H NMR (D₂O, 400 MHz): δ 8.96 (d, J = 4.0 Hz, 2H), 8.26 (d, J = 4.0 Hz, 2H), 7.88 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 8.0 Hz, 2H), 4.92–4.91 (m, 1H), 4.06 (d, J = 4.0 Hz, 2H), 3.96–3.87 (m, 4H), 3.58 (t, J = 12.0 Hz, 1H), 3.43 (s, 1H), 3.13 (t, J = 12.0 Hz, 1H), 2.98 (s, 3H), 0.72 (t, J = 4.0 Hz, 3H). ¹³C NMR (D₂O, 400 MHz): δ 157.3, 150.8, 142.7, 141.5, 134.7, 128.1, 128.0, 123.9, 121.5, 71.5, 68.0, 66.3, 55.6, 52.8, 42.1, 39.9, 12.7. HRMS (ESI⁺) calculated for C₂₂H₂₉ClF₃N₅O₂ [M+1]⁺: 488.2040, observed: 452.2270 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(3-morpholino-5-(trifluoromethyl)phenyl)urea hydrochloric salt (12HCl).

Obtained as a dark yellow oil in 95% yield. ¹H NMR (D₂O, 400 MHz): δ 8.96 (d, J = 8.0 Hz, 2H), 8.25 (d, J = 8.0 Hz, 2H), 7.39 (s, 1H), 7.35 (s, 1H), 7.28 (s, 1H), 4.91–4.90 (m, 2H), 4.04 (d, J = 8.0 Hz, 2H), 3.97 (s, 4H), 3.39 (s, 4H), 2.99 (s, 6H). ¹³C NMR (D₂O, 400 MHz): δ 157.0, 150.5, 142.8, 139.8, 127.9, 124.5, 68.2, 66.4, 51.1, 42.1, 39.3. MS (ESI⁺) calculated for C₂₁H₂₇Cl₂F₃N₅O₂ [M+1]⁺: 474.1883, observed: 438.2129 – HCl.

1-(2-(Dimethylamino)-2-(pyridin-4-yl)ethyl)-3-(2-morpholino-4-(trifluoromethyl)phenyl)urea hydrochloric salt (13HCl).

Obtained as a yellow solid in 95% yield. M.p. 170–173 °C. ¹H NMR (D₂O, 400 MHz): δ 8.97 (d, J = 4.0 Hz, 2H), 8.26 (d, J = 4.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 1H), 7.57 (s, 1H), 7.45 (d, J = 8.0 Hz, 1H), 4.93–4.91 (m, 1H), 4.06 (d, J = 8.0 Hz, 2H), 3.87 (s, 4H), 2.99 (s, 6H), 2.93 (s, 4H). ¹³C NMR (D₂O, 400 MHz): δ 157.8, 150.7, 142.5, 139.9, 128.2, 125.5, 124.3, 119.0, 67.9, 65.4, 53.0, 42.0, 39.5. HRMS (ESI⁺) calculated for C₂₁H₂₇Cl₂F₃N₅O₂ [M+1]⁺: 474.1883, observed: 438.2134 – HCl.

3.1.1.3. Synthesis of Phenyl (2-morpholino-5-(trifluoromethyl)phenyl)carbamate (14)

To a stirred solution of 2-morpholino-5-trifluoromethyl aniline (0.8 g, 3.2 mmol) in dry DCM (25 mL), under an N₂ atmosphere at 0 °C, was added a solution of phenyl chloroformate (0.6 mL, 4.8 mmol) in dry DCM (5 mL), followed by Et₃N (0.65 mL, 8.10 mmol). The crude reaction was allowed to warm up at room temperature and stirred overnight. The crude reaction was concentrated in vacuo, and the product was purified by column chromatography using hexanes:EtOAc (70:30) to afford the pure product as a clear oil in 85% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.47 (s, 1H), 8.19 (s, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 4.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 2H), 3.91 (t, J = 4.0 Hz, 4 H), 2.92 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₈H₁₇F₃N₂O₃ [M+1]⁺: 367.3, observed: 367.3

The following compounds were synthesized following the general procedure for the synthesis of 14

Phenyl (2-morpholinophenyl)carbamate was obtained as a white solid with an 80% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.28 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.24 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.08 (t, J = 8.0 Hz, 1H), 3.91 (t, J = 4.0 Hz, 4H), 2.92 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₇H₁₈N₂O₃ [M+1]⁺: 299.3, observed: 299.3

Phenyl (3-morpholino-5-(trifluoromethyl)phenyl)carbamate (22)was obtained as a cream solid in 80% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.52 (s, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.26 (d, J = 16.0 Hz, 1H), 7.18 (d, J = 8.0 Hz, 2H), 7.07 (s, 1H), 6.90 (s, 2H), 3.88 (t, J = 4.0 Hz, 4H), 3.24 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₈H₁₇F₃N₂O₃ [M+1]⁺: 367.3, observed: 367.3

Phenyl (2-morpholino-4-(trifluoromethyl)phenyl)carbamate (23) was obtained as a cream solid in 80% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.39 (s, 1H), 7.47–7.41 (m, 3H), 7.29–7.21 (m, 4H), 6.93 (t, J = 8.0 Hz, 1H), 3.94 (t, J = 4.0 Hz, 4H), 2.95 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₈H₁₇F₃N₂O₃ [M+1]⁺: 367.3, observed: 367.3

Phenyl (2-(3-methylmorpholino)-5-(trifluoromethyl)phenyl)carbamate was obtained as a cream solid in 80% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.55 (s, 1H), 7.43–7.41 (m, 2H), 7.35 (s, 1H), 7.25–7.21 (m, 4H), 6.83 (d, J = 8.0 Hz, 1H), 4.00–3.94 (m, 2H), 3.84 (t, J = 4.0 Hz, 1H), 3.45 (t, J = 8.0 Hz, 1H), 3.24–3.20 (m, 1H), 2.98–2.88 (m, 1H), 2.81 (d, J = 12.0 Hz, 1H), 0.8 (d, J = 4.0 Hz, 3H). MS (ESI⁺) calculated for C₁₉H₁₉F₃N₂O₃ [M+1]⁺: 381.3, observed: 381.3

Phenyl (2-(3,5-dimethylmorpholino)-5-(trifluoromethyl)phenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.62 (s, 1H), 8.13 (s, 1H), 7.45–7.39 (m, 2H), 7.24–7.17 (m, 3H), 6.93 (d, J = 8.0 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 3.99–3.92 (m, 2H), 3.85 (d, J = 8.0 Hz, 1H), 3.71–3.63 (m, 1H), 3.35 (t, J = 8.0 Hz, 1H), 3.04 (d, J = 8.0 Hz, 1H), 1.18 (d, J = 4.0 Hz, 3H), 0.81 (d, J = 4.0 Hz, 3H). MS (ESI⁺) calculated for C₂₀H₂₁F₃N₂O₃ [M+1]⁺: 395.3, observed: 395.3

Phenyl (5-cyano-2-morpholinophenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.49 (s, 1H), 8.09 (s, 1H), 7.44–7.37 (m, 2H), 7.30–7.28 (m, 2H), 7.23–7.30 (m, 3H), 3.94 (t, J = 4.0 Hz, 4H), 2.96 (t, J = 4.0 Hz, 4H). MS (ESI+) calculated for C₁₈H₁₇N₃O₃ [M+1]+: 324.3, observed: 324.3.

Phenyl (5-fluoro-2-morpholinophenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.41 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 2H), 7.28–7.25 (m, 2H), 7.23–7.15 (m, 3H), 6.75 (td, J = 4.0 Hz, 2.0 Hz, 1H), 3.90 (t, J = 4.0 Hz, 4H), 2.88 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₇H₁₇FN₂O₃ [M+1]⁺: 317.3, observed: 317.3.

Phenyl (5-(difluoromethyl)-2-morpholinophenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.35 (s, 2H), 7.7.44–7.40 (m, 2H), 7.28 (s, 2H), 7.22 (d, J = 8.0 Hz, 2H), 6.59 (t, J = 56.0 Hz, 1H), 3.95 (t, J = 4.0 Hz, 4H), 2.98 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₈H₁₈F₂N₂O₃ [M+1]⁺: 349.3, observed: 349.3.

Phenyl (5-methoxy-2-morpholinophenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.48 (s, 1H), 7.84 (m, 1H), 7.44–7.40 (m, 2H), 7.28–7.21 (m, 3H), 7.14 (d, J = 12.0 Hz, 1H), 6.61 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 3.90 (t, J = 4.0 Hz, 4H), 3,78 (s, 3H), 2.87 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₈H₂₀N₂O₄ [M+1]⁺: 329.3, observed: 329.3.

Phenyl (5-(methoxymethoxy)-2-morpholinophenyl)carbamate was obtained as a yellow-orange solid in 60% yield ¹H NMR (CDCl₃, 400 MHz): δ 8.44 (s, br, 1H), 7.92 (s, 1H), 7.40 (t, J = 4.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 6.74 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 5.16 (s, 2H), 3.81 (t, J = 4.0 Hz, 4H), 3.47 (s, 3H), 2.97 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₉H₂₂N₂O₅ [M+1]⁺: 359.3, observed: 359.3.

Phenyl (5-(2-methoxyethoxy)-2-morpholinophenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.58 (s, br, 1H), 7.83 (s, 1H), 7.43–7.39 (m, 3H), 7.23 (d, J = 8.0 Hz, 2H), 7.6 (d, J = 8.0 Hz, 1H), 6.68 (dd, J = 4.0 Hz, 2.0 Hz, 1H), 4.11 (t, J = 4.0 Hz, 2H), 3.92 (s, br, 4H), 3.71 (t, J = 4.0 Hz, 2H), 3.42 (s, 3H), 2.91 (s, br, 4H). MS (ESI⁺) calculated for C₂₀H₂₄N₂O₅ [M+1]⁺: 373.4, observed: 373.4.

Phenyl (5-methyl-2-morpholinophenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.45 (s, br, 1H), 7.99 (s, 1H), 7.41 (t, J = 4.0 Hz, 2H), 7.27–7.25 (m, 1H), 7.12 (dd, J = 8.0 Hz, 4.0 Hz, 2H), 6.81 (dd, J = 4.0 Hz, 2.0 Hz, 1H), 3.94 (s, 3H), 2.94 (s, 4H), 2.33 (s, 3H). MS (ESI⁺) calculated for C₁₈H₂₀N₂O₃ [M+1]⁺: 313.3, observed: 313.3.

Phenyl (5-(2-(2-((tert-butyldimethylsilyl)oxy)ethoxy)ethoxy)-2-morpholinophenyl)carbamate was obtained as a cream solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.44 (s, br, 1H), 7.84 (s, 1H), 7.42 (t, J = 4.0 Hz, 2H), 7.30–7.27 (m, 2H), 7.12 (dd, J = 8.0 Hz, 4.0 Hz, 2H), 6.64 (dd, J = 4.0 Hz, 2.0 Hz, 1H), 4.09 (t, J = 4.0 Hz, 2H), 3.89 (t, J = 4.0 Hz, 4H), 3.82 (t, J = 4.0 Hz, 2H), 3.77 (t, J = 4.0 Hz, 2H), 3.60 (t, J = 4.0 Hz, 2H), 2.86 (t, J = 4.0 Hz, 4H), 0.88 (s, 9H), 0.06 (s, 6H). MS (ESI⁺) calculated for C₂₇H₄₀N₂O₆Si [M+1]⁺: 517.7, observed: 517.7.

3.1.1.4. Synthesis of Phenyl (5-(2-(2-methoxyethoxy)ethoxy)-2-morpholinophenyl)carbamate

To a stirred solution of 5-(2-(2-methoxyethoxy)ethoxy)-2-morpholinoaniline (0.36 g, 1.21 mmol) and K₂CO₃ (0.33 g, 2.42 mmol) in acetone (10 mL), at 0 °C, phenyl chloroformate (0.16 mL, 1.33 mmol) was added dropwise. The crude reaction was allowed to warm to room temperature, stirred overnight, then concentrated in vacuo, dissolved in DCM (15 mL), and washed with water (10 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated. The crude product was taken to the following reaction without further purification. ¹H NMR (CDCl₃, 400 MHz): δ 8.54 (s, br, 1H), 7.82 (s, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H), 6.65 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.12 (t, J = 4.0 Hz, 2H), 3.92 (s, 4 H), 3.83 (t, J = 4.0 Hz, 2H), 3.70 (t, J = 4.0 Hz, 2H), 3.56 (t, J = 4.0 Hz, 2H), 3.38 (s, 3H), 2.91 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₂₂H₂₈N₂O₆ [M+1]⁺: 417.4, observed: 417.4.

The following compounds were synthesized following the general procedure for the synthesis of Phenyl (5-(2-(2-methoxyethoxy)ethoxy)-2-morpholinophenyl)carbamate.

Phenyl (5-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2-morpholinophenyl)carbamate was obtained as a white solid in 40% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.43 (s, 1H), 7.83 (s, 1H), 7.42 (t, J = 4.0 Hz, 2H), 7.28–7.26 (m,1 H), 7.23 (d, J = 8.0 Hz, 2H), 713 (d, J = 8.0 Hz, 1H), 6.63 (dd, J = 4.0 Hz, 2.0 Hz, 1H), 4.13 (t, J = 4.0 Hz, 2H), 3.89 (t, J = 4.0 Hz, 4H), 3.82 (t, J = 4.0 Hz, 2H), 3.73–3.70 (m, 2H), 3.66–3.63 (m, 4H), 3.55.−3.53 (m, 2H), 3.37 (s, 3H), 2.86 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₂₄H₃₂N₂O₇ [M+1]⁺: 461.5, observed: 461.5.

Phenyl (5-chloro-2-morpholinophenyl)carbamate was obtained as a white solid in 50% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.59 (s, 1H), 8.21 (s, 1H), 7.42 (t, J = 4.0 Hz, 2H), 7.28–7.26 (m, 1H), 7.22 (d, J = 8.0 Hz, 2H), 7.15 (d, J = 8.0 Hz, 1H), 7.07 (dd, J = 4.0 Hz, 2.0 Hz, 1H), 3.97 (t, J = 4.0 Hz, 2H), 2.98 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₇H₁₇ClN₂O₃ [M+1]⁺: 333.7, observed: 333.7.

Phenyl (2-morpholino-5-(trifluoromethoxy)phenyl)carbamate was obtained as an orange solid in 60% yield. ¹H NMR (CDCl₃, 400 MHz): δ 8.41 (s, 1H), 8.13 (s, 1H), 7.42 (t, J = 4.0 Hz, 2H), 7.27–7.25 (m, 2H), 7.22 (d, J = 8.0 Hz, 2H), 6.93 (dd, J = 4.0 Hz, 2.0 Hz, 1H), 3.93 (t, J = 4.0 Hz, 2H), 2.93 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₈H₁₇F₃N₂O₄ [M+1]⁺: 383.3, observed: 383.3.

3.1.1.5. Synthesis of 2-morpholino-4-(trifluoromethyl)aniline (21)

To a stirred solution of 2-fluoro-1-nitro-4-(trifluoromethyl)benzene (0.95 g, 4.7 mmol) in dry DMF (10 mL) was added morpholine (0.45 mL, 5.2 mmol), followed by K₂CO₃ (0.97 g, 7.05 mmol). The crude reaction was stirred under heating at 60 °C overnight. After cooling at room temperature, the crude reaction was diluted with DCM and washed with brine several times. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (50:50) to afford a red oil in 76% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.83 (d, J = 8.0 Hz, 1H), 7.35 (s, 1H), 7.30 (d, J = 8.0 Hz, 1H), 3.84 (t, J = 8.0 Hz, 4H), 3.10 (t, J = 8.0 Hz, 4H). MS (ESI⁺) calculated for C₁₁H₁₁F₃N₂O₃ [M+1]⁺: 277.2, observed: 277.2.

To a stirred solution of 4-(2-nitro-5-(trifluoromethyl)phenyl)morpholine (0.98 g, 3.55 mmol) in EtOH (15 mL), purged under nitrogen, was added Pd/C (0.1 g). Then, hydrazine monohydrate (2.8 mL, 57.6 mmol) was added dropwise, and the mixture was stirred at room temperature for 3 hrs. Crude was filtered and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (30:70) to afford a white solid in 70% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.39 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 1H), 7.23 (m, 1H), 6.78(d, J = 12.0 Hz, 1H), 3.86 (t, J = 4.0 Hz, 4H), 2.92 (d, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₁H₁₃F₃N₂O [M+1]⁺: 247.2, observed: 247.2.

3.1.1.6. Synthesis of 1-fluoro-4-methoxy-2-nitrobenzene (15a)

To a stirred solution of 4-fluoro-3-nitrophenol (1.0 g, 6.36 mmol) in acetone (10 mL) was added K₂CO₃ (4.4 g, 31.8 mmol), followed by CH₃I (0.80 mL, 12.72 mmol). The crude reaction was stirred under heating at 50 °C overnight. After cooling at room temperature, the crude reaction was diluted with DCM and filtered. The crude was concentrated in vacuo, redissolved in EtOAc (20 mL), and washed with 1N NaOH (5 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to afford a red oil with a 57% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.54–7.51 (m, 1H), 7.23–7.15 (m, 2H), 3.86 (s, 3H). MS (ESI+) calculated for C₇H₆FNO₃ [M+1]+: 172.2, observed: 172.2.

The following compounds were synthesized according to the synthetic procedure for intermediate 15a

1-Fluoro-4-(methoxymethoxy)-2-nitrobenzene (15b) was obtained as a red oil in 63% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.73–7.71 (m, 1H), 7.30–.7.28 (m, 1H), 7.20 (t, J = 8.0 Hz, 1H), 5.19 (s, 2H), 3.49 (s, 3H). MS (ESI⁺) calculated for C₈H₈FNO₄ [M+1]⁺: 202.1, observed: 202.1.

1-Fluoro-4-(2-methoxyethoxy)-2-nitrobenzene (15c) was obtained as a red oil in 50% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.54 (d, J = 4.0 Hz, 1H), 7.19 (d, J = 4.0 Hz, 1H), 7.18 (d, J = 4.0 Hz, 1H), 4.14 (t, J = 4.0 Hz, 2H), 3.75 (t, J = 4.0 Hz, 2H), 3.44 (s, 3H). MS (ESI⁺) calculated for C₉H₁₀FNO₄ [M+1]⁺: 216.05, observed: 216.3.

1-Fluoro-4-(2-(2-methoxyethoxy)ethoxy)-2-nitrobenzene (15d) was obtained as a red oil in 63% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.58 (d, J = 4.0 Hz, 1H), 7.18 (d, J = 4.0 Hz, 2H), 4.17 (t, J = 4.0 Hz, 2H), 3.87 (t, J = 4.0 Hz, 2H), 3.71 (m, 2H), 3.59–3.58 (m, 2H), 3.39 (s, 3H). MS (ESI⁺) calculated for C₁₁H₁₄FN₂O₅ [M+1]⁺: 260.3, observed: 260.3.

1-Fluoro-4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-2-nitrobenzene (15e) was obtained as a yellow-brown oil in 87% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.56 (dd, J = 4.0 Hz, 1H), 7.19 (d, J = 4.0 Hz, 2H), 4.16 (t, J = 4.0 Hz, 1H), 3.87 (t, J = 4.0 Hz, 1H), 3.81 (t, J = 8.0 Hz, 1H), 3.75–3.72 (m, 1H), 3.70–3.64 (m, 5H), 3.57–3.54 (m, 2H), 3.47 (t, J = 8.0 Hz, 1H), 3.38 (s, 3H). MS (ESI⁺) calculated for C₁₃H₁₈FN₂O₆ [M+1]⁺: 304.3, observed: 304.3.

tert-Butyl(2-(2-(4-fluoro-3-nitrophenoxy)ethoxy)ethoxy)dimethylsilane (15f): To a stirred solution of 4-fluoro-3-nitrophenol (0.77 g, 4.93 mmol) in dry acetonitrile (10 mL) was added K₂CO₃ (1.3 g, 9.86 mmol), followed by 2-(2-bromoethoxy)ethan-1-ol (1.34 mL, 12.7 mmol). The crude reaction was stirred under heating at 80 °C overnight. After cooling at room temperature, the crude reaction was diluted with DCM and filtered. The crude was concentrated in vacuo, redissolved in EtOAc (20 mL), and washed with 1N NaOH (5 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo to afford a red oil 87% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.60 (dd, J = 4.0 Hz, 1H), 7.21–7.19 (d, J = 4.0 Hz, 2H), 4.18 (t, J = 4.0 Hz, 2H), 3.89 (t, J = 4.0 Hz, 2H), 3.78 (t, J = 4.0 Hz, 2H), 3.68 (t, J = 4.0 Hz, 2H), 1.97 (s, br, 1H). MS (ESI⁺) calculated for C₁₀H₁₂FNO₅ [M+1]⁺: 246.2, observed: 246.2.

To a stirred solution of 2-(2-(4-fluoro-3-nitrophenoxy)ethoxy)ethan-1-ol (0.48 g, 1.86 mmol) in dry DCM (10 mL) was added 2,6-Lutidine (0.47 mL, 4.09 mmol), followed by TBSOTf (0.51 mL, 2.23 mmol). The crude reaction was stirred at room temperature overnight. Crude reaction was diluted with DCM (20 mL) and washed with brine (15 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (80:20) to afford a clear oil in 71% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.55 (d, J = 4.0 Hz, 1H), 7.20–7.18 (m, 2H), 4.15 (t, J = 4.0 Hz, 2H), 3.86 (t, J = 4.0 Hz, 2H), 3.78 (t, J = 4.0 Hz, 2H), 3.63 (t, J = 4.0 Hz, 2H), 0.89 (s, 9H), 0.07 (s, 6H). MS (ESI⁺) calculated for C₁₆H₂₆FNO₅Si [M+1]⁺: 360.4, observed: 360.4.

3.1.1.7. Synthesis of 5-fluoro-2-morpholinoaniline (17b)

To a stirred solution of 4-(4-fluoro-2-nitrophenyl)morpholine (0.40 g, 1.76 mmol) in EtOH (5 mL), purged under nitrogen, was added Pd/C (0.1 g), and hydrazine monohydrate (1.5 mL, 30.4 mmol) was added dropwise. The crude reaction was stirred at room temperature for 2 h. Crude was filtered and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (30:70) to afford a white solid in 40% yield. ¹H NMR (acetone-d₆, 400 MHz): δ 6.90 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 6.49 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 6.30 (td, J = 8.0 Hz, 4.0 Hz, 1H), 3.78 (t, J = 4.0 Hz, 4H), 2.81 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₀H₁₃FN₂O [M+1]⁺: 197.2, observed: 197.2

The following compounds were synthesized according to the synthetic procedure for intermediate 17b

5-Methyl-2-morpholinoaniline (17c) was obtained as a white solid in 90% yield. ¹H NMR (CDCl₃, 400 MHz): δ 6.92 (d, J = 8.0 Hz, 1H), 6.58 (d, J = 8.0 Hz, 2H), 3.90 (s, 4H), 2.97 (s, 4H), 2.24 (s, 3H). MS (ESI⁺) calculated for C₁₁H₁₆N₂O [M+1]⁺: 193.2, observed: 193.2.

3-Amino-4-morpholinobenzonitrile (17d) was obtained as a white solid in 50% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.04 (t, J = 4.0 Hz, 1H), 6.98–6.94 (m, 2H), 3.84 (t, J = 4.0 Hz, 4H), 2.95 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₁H₁₃N₃O [M+1]⁺: 204.2, observed: 204.2.

5-Chloro-2-morpholinoaniline (17e) was obtained as a white solid in 50% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.08 (t, J = 4.0 Hz, 1H), 6.90 (t, J = 8.0 Hz, 2H), 6.72 (d, J = 4.0 Hz, 1H), 3.87 (t, J = 4.0 Hz, 4H), 2.92 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₀H₁₃ClN₂O [M+1]⁺: 213.6, observed: 213.6.

5-(Difluoromethyl)-2-morpholinoaniline (17f) was obtained as a clear oil in 44% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.13 (d, J = 8.0 Hz, 1H), 6.94 (s, 2H), 6.54 (t, J = 56.0 Hz, 1H), 4.06 (s, 4H), 3.19 (s, 4H). MS (ESI⁺) calculated for C₁₁H₁₄F₂N₂O [M+1]⁺: 229.2, observed: 229.2.

2-Morpholino-5-(trifluoromethoxy)aniline (17g) was obtained as a clear oil with a 44% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.01 (d, J = 8.0 Hz, 1H), 6.60 (s, 2H), 3.96 (s, 4H), 3.06 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₁H₁₃F₃N₂O₂ [M+1]⁺: 263.2, observed: 263.2.

3.1.1.8. Synthesis of 5-methoxy-2-morpholinoaniline (17h)

To a stirred solution of 1-fluoro-4-methoxy-2-nitrobenzene (0.62 g, 3.62 mmol) in dry DMF (5 mL) was added morpholine (0.35 mL, 4.0 mmol), followed by K₂CO₃ (1.0 g, 7.2 mmol). The crude reaction was stirred at 60 °C overnight. After cooling at room temperature, the crude reaction was diluted with DCM and washed with brine several times. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (50:50) to afford a yellow solid in 80% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.20 (s, 1H), 7.09 (d, J = 4.0 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 3.82 (s, 3H), 3.82 (t, J = 4.0 Hz, 4H), 2.98 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₁H₁₄N₂O₄ [M+1]⁺: 239.2, observed: 239.2.

To a stirred solution of 4-(4-methoxy-2-nitrophenyl)morpholine (0.69 g, 2.89 mmol) in MeOH (10 mL), purged under nitrogen, was added Pd/C (0.1 g) and then a balloon filled with H₂. The crude reaction was stirred at room temperature for 1 h. Crude was filtered and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (30:70) to afford a white solid in 80% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.20 (s, 1H), 7.09–7.07 (dd, J = 4.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 6.32 (t, J = 4.0 Hz, 1H), 3.82 (s, 3H), 3.82 (t, J = 4.0 Hz, 4H), 2.87 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₁H₁₆N₂O₂ [M+1]⁺: 209.2, observed: 209.2.

The following compounds were synthesized according to the synthetic procedure for intermediate 17h

5-(Methoxymethoxy)-2-morpholinoaniline (17i) was obtained as a white solid in 90% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.43 (d, J = 4.0 Hz, 1H), 7.20 (d, J = 4.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.95 (d, J = 4.0 Hz, 1H), 6.48 (d, J = 4.0 Hz, 1H), 5.16 (s, 2H), 3.82 (t, J = 4.0 Hz, 4H), 3.48 (s, 3H), 2.98 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₂H₁₈N₂O₃ [M+1]⁺: 239.1, observed: 239.1.

5-(2-Methoxyethoxy)-2-morpholinoaniline (17j)was obtained as a white solid in 90% yield. ¹H NMR (CDCl₃, 400 MHz): δ 6.93 (d, J = 4.0 Hz, 1H), 6.35 (d, J = 4.0 Hz, 1H), 6.32 (dd, J = 4.0 Hz, 1H), 4.06 (t, J = 4.0 Hz, 2H), 3.86 (s, br, 4H), 3.70 (m, 2H), 3.49 (s, 3H), 2.84 (s, br, 4H). MS (ESI⁺) calculated for C₁₃H₂₀N₂O₃ [M+1]⁺: 253.3, observed: 253.3

5-(2-(2-Methoxyethoxy)ethoxy)-2-morpholinoaniline (17k) was obtained as a white solid in 81% yield. ¹H NMR (CDCl₃, 400 MHz): δ 6.93 (d, J = 4.0 Hz, 1H), 6.34 (d, J = 4.0 Hz, 1H), 6.30 (dd, J = 12.0 Hz, 4.0 Hz, 1H), 4.08 (t, J = 4.0 Hz, 2H), 3.89 (s, br, 4H), 3.82 (t, J = 4.0 Hz, 2H), 3.70 (t, J = 4.0 Hz, 2H), 3.57 (t, J = 4.0 Hz, 2H), 3.39 (s, 3H), 2.94 (s, br, 4H). MS (ESI⁺) calculated for C₁₅H₂₄N₂O₄ [M+1]⁺: 297.3, observed: 297.3.

5-(2-(2-(2-Methoxyethoxy)ethoxy)ethoxy)-2-morpholinoaniline (17l) was obtained as a clear oil in 81% yield. ¹H NMR (CDCl₃, 400 MHz): δ 6.91 (d, J = 8.0 Hz, 1H), 6.34 (d, J = 4.0 Hz, 1H), 6.29 (dd, J = 12.0 Hz, 4.0 Hz, 1H), 4.07–4.05 (m, 2H), 3.82–3.80 (m, 6H), 3.74–3.71 (m, 2H), 3.69–3.64 (m, 4H), 3.56–3.54 (m, 2H), 3.38 (s, 3H), 2.85 (t, J = 4.0 Hz, 4H). MS (ESI⁺) calculated for C₁₇H₂₈N₂O₅ [M+1]⁺: 341.4, observed: 341.4.

5-(2-(2-((tert-Butyldimethylsilyl)oxy)ethoxy)ethoxy)-2-morpholinoaniline (17m) was obtained as a clear oil in 81% yield. ¹H NMR (CDCl₃, 400 MHz): δ 6.92 (d, J = 8.0 Hz, 1H), 6.34 (d, J = 4.0 Hz, 1H), 6.30 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 4.05 (t, J = 4.0 Hz, 4H), 3.82 (t, J = 4.0 Hz, 4H), 3.78 (t, J = 4.0 Hz, 2H), 3.62 (t, J = 4.0 Hz, 2H), 2.85 (t, J = 4.0 Hz, 4H), 0.89 (s, 9H), 0.07 (s, 6H). MS (ESI⁺) calculated for C₂₀H₃₆N₂O₄Si [M+1]⁺: 397.6, observed: 397.6.

3.1.1.9. Synthesis of 2-morpholino-5-(trifluoromethoxy)aniline (18).

To a stirred solution of 1-fluoro-2-nitro-4-(trifluoromethyl)benzene (0.50 g, 2.39 mmol) in dry DMF (5 mL) was added 3-methylmorpholine (0.3 g, 2.87 mmol), followed by K₂CO₃ (0.66 g, 4.78 mmol). The crude reaction was stirred at 60 °C overnight. After cooling at room temperature, the crude reaction was diluted with DCM and washed with brine several times. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (50:50) to afford a red-orange oil in 95% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.97 (s, 1H), 7.12 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 3.82–3.77 (m, 3H), 3.50 (dd, J = 8.0 Hz, J = 4.0 Hz, 1H), 3.42–3.33 (m, 2H), 2.85–2.83 (m, 1H), 1.02 (d, J = 8.0 Hz, 3H). MS (ESI⁺) calculated for C₁₂H₁₃F₃N₂O₃ [M+1]⁺: 291.2, observed: 291.2.

To a stirred solution of 3-methyl-4-(2-nitro-4-(trifluoromethyl)phenyl)morpholine (0.68 g, 2.3 mmol) in EtOH (10 mL), purged under nitrogen, was added Pd/C (0.05 g), and hydrazine monohydrate (1.9 mL, 38.9 mmol) was added dropwise. The crude reaction was stirred at room temperature for 2 hrs. Crude was filtered and concentrated in vacuo. The product was purified by column chromatography using hexanes:EtOAc (30:70) to afford a white solid in 87% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.80 (s, 1H), 7.50 (s, 1H), 7.19 (d, J = 4.0 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 3.93–3.85 (m, 2H), 3.79–3.72 (m, 1H), 3.39–3.33 (m, 1H), 3.29–3.22 (m, 1H), 2.92–2.88 (m, 1H), 2.75–2.68 (m, 1H), 0.8 (d, J = 8.0 Hz, 3H). MS (ESI⁺) calculated for C₁₂H₁₅F₃N₂O [M+1]⁺: 261.2, observed: 261.2.

Compound 19 was synthesized using the same procedures as compound 18.

2-(3,5-Dimethylmorpholino)-5-(trifluoromethyl)aniline (19) was obtained as a white solid in 30% yield. ¹H NMR (CDCl₃, 400 MHz): δ 7.77 (s, 1H), 7.51 (d, J = 4.0 Hz, 1H), 7.17 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 7.01 (d, J = 4.0 Hz, 1H), 3.92–3.85 (m, 2H), 3.68–3.64 (m, 2H), 3.24 (t, J = 8.0 Hz, 1H), 3.02 (dd, J = 8.0 Hz, 4.0 Hz, 1H), 1.06 (d, J = 8.0 Hz, 3H), 0.75 (d, J = 8.0 Hz, 3H). MS (ESI⁺) calculated for C₁₃H₁₇F₃N₂O [M+1]⁺: 275.3, observed: 274.3.

3.2. Computer Simulation

The molecular modeling studies were done using Schrödinger Maestro. Protein was prepared using Maestro’s Protein Preparation Wizard with default settings. During pre-processing, heteroatom states were generated and selected at pH 7.4 with Epik. Hydrogen bond assignments were performed with default settings at pH 7.4 using PROPKA. Minimization was performed with default settings using the OPLS3 force field. Possible binding sites of the crystal structure FAK FERM domain (PDB ID: 6BC0), available from the ProteinData Bank (PDB) (http://www.rcsb.org), were searched with Maestro’s SiteMap tool using default settings. The resulting sites were visually inspected and compared based on their calculated SiteScore. Ligands were prepared for docking from their 2D structures using LigPrep with the OPLS3 force field. Docking was performed using Glide at SP precision, with a van der Waals radii scaling factor of 0.7. The glide docking grid was generated with the grid center at Leu262 and a box size of 20 Å. Docking was done using Maestro’s Structure-Based Virtual Docking protocol.

3.3. Biological Assays

3.3.1. FAK activation

For studies of FAK activation in suspended Caco-2 cells, 1 × 10⁶ cells were seeded into each well of 12-well plates pacificated to prevent adhesion. After 1 h of incubation at 37 °C with either the vehicle control (DMSO unless the molecule was water-soluble) or one of the small molecule FAK activators, cells were lysed with 100 μl of protein lysis buffer to extract protein. 30–35 μg of protein (12–20μl of protein lysate) was loaded per lane onto SDS-PAGE gels for resolution and Western blotting.

3.3.2. Cell culture

Human Caco-2 cells were from ATCC (Manassas, VA) and maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS, as previously described [35]. 80–90% confluent Caco-2 cells were seeded into cell culture plates pacificated with 1% heat-inactivated bovine serum albumin to prevent adhesion and avoid adhesion-associated background FAK activation. Suspended cells were then treated with DMSO (0.1%) or small molecules for 1 h before harvesting for western blotting.

3.3.3. Western blotting

Cells were lysed, and protein concentration was determined via bicinchoninic acid protein assay (Thermo Fisher, Rockford, IL). The proteins were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with antibodies to FAK-Tyr-397 (ab81298, 1:1000 dilution), or FAK (Anti-FAK, clone 4.47, 05–537, 1:1000 dilution EMD Millipore, Temecula, CA). Images were taken using a LI-COR–Odyssey-Fc imaging system (LI-COR Biosciences, Lincoln, NE). Densitometry was conducted on exposures within the linear range, and FAK phosphorylation was calculated as the ratio of FAK-Tyr-397 intensity to FAK intensity.

3.3.4. Monolayer wound closure assays

Caco-2 cells were seeded at 75–80% confluence into collagen I-coated 6-well plates. When the cells reached 100% confluence, wounds were created in the monolayers using non-barrier, autoclaved tips. Wound images were captured using an inverted light microscope (OLYMPUS CK2, Center Valley, PA) at 0 h and 24 h after treatment with DMSO or small molecules. In some studies, proliferation was inhibited by simultaneous treatment with 4 mM hydroxyurea. Wound areas were measured using ImageJ.

3.3.5. Statistical analysis

Data are depicted graphically as mean ± standard error and were analyzed by t-test or ANOVA, as appropriate, with 95% confidence.

3.3.6. Rat Pharmacokinetic Studies

A plasma pharmacokinetic study of a single dose of compound 5aHCl was conducted using two-year-old male Fischer (CDF) rats (420–460 grams) (Charles River Laboratories, Wilmington, MA). The animals were housed under standard conditions, including a 12-h light–dark cycle, a temperature of 22 ± 2 °C, and a relative humidity of 50 ± 10%. Animals had free access to tap water and standard commercial chow. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Northeast Ohio Medical University (IACUC Protocol #23-04-366).

A dose of 10 mg/kg of 5aHCl (dissolved in saline, administered in a volume of 1 mL) was given via oral gavage using Instech feed tubing (Catalogue#: FTP-13-150, Instech, Plymouth Meeting, PA, USA). Blood samples (approximately 120 μL) were collected from the tail immediately before dosing and at 0.5, 1, 1.5, 2, 4, 6, and 10 h post-dosing using Microvette^® 200 Heparin Capillary Blood Collection Tubes (catalogue #: 20.1292.100, Sarstedt, Nümbrecht, Germany). Samples were centrifuged at 2000×g for 15 min at 4 °C. The resulting plasma was stored at −80 °C and analyzed by ultra-performance liquid chromatography–mass spectrometry (UPLC-MS).

Supplementary Material

Supplementary data

Supplementary data to this article, including residue interaction of protonated compounds 5a, 5b, 5c, 10b, and 10k, site1 binding pocket residues, docking score of the selected compound, predicted properties of 5aHCl, 10bHCl, and 10kHCl using QikProp, and experimental procedures and spectroscopic data, can be found online at https://

Highlights.

• A series of small drug-like highly specific FAK activators were designed and tested.

• Lead molecules activate FAK and promote epithelial motility in vitro and in vivo.

• The most promising lead is water soluble and has a 3 h t_1/2 after enteral dosing.