Vicinal Diaryl-Substituted Isoxazole and Pyrazole Derivatives with In Vitro Growth Inhibitory and In Vivo Antitumor Activity

Abstract

The vicinal diaryl heterocyclic framework has been widely used for the development of compounds with significant bioactivities. In this study, a series of diaryl heterocycles were designed and synthesized based on an in-house diaryl isoxazole derivative (9), and most of the newly synthesized derivatives demonstrated moderate to good antiproliferative activities against a panel of hepatocellular carcinoma and breast cancer cells, exemplified with the diaryl isoxazole 11 and the diaryl pyrazole 85 with IC₅₀ values in the range of 0.7–9.5 μM. Treatments with both 11 and 85 induced apoptosis in these tumor cells, and they displayed antitumor activity in vivo in the Mahlavu hepatocellular carcinoma and the MDA-MB-231 breast cancer xenograft models, indicating that these compounds could be considered as leads for further development of antitumor agents. Important structural features of this compound class for the antitumor activity have also been proposed, which warrant further exploration to guide the design of new and more potent diaryl heterocycles.

1. Introduction

Vicinal diaryl-substituted heterocycles can be considered privileged scaffolds as they form the core structure of a number of compounds with diverse biological properties such as anticancer, antiviral, and anti-inflammatory activities.¹ Therefore, the use of this scaffold incorporating different central heterocycles could be pivotal in the drug development practice since they exist in the structures of various clinical therapeutics or compounds with therapeutic potential, which establishes their value as chemical tools in the field of medicinal chemistry (Figure 1). For example, the first COX-2 selective nonsteroidal anti-inflammatory drug celecoxib (1) was a member of vicinal diaryl pyrazole derivatives,² which stimulated the development of latter COX-2 inhibitors belonging to the vicinal diaryl heterocyclic class such as rofecoxib and valdecoxib.³ Another vicinal diaryl pyrazole derivative developed as an antiobesity drug was rimonabant (2),⁴ which continues to be an inspiration for further development of cannabinoid CB1 receptor antagonists, i.e., surinabant.⁵ Oxaprozin (3) and licofelone (4) are anti-inflammatory drugs for the treatment of osteo- and rheumatoid arthritis, also belonging to a vicinal diaryl oxazole and pyrrolizidine class, respectively.^6,7

Figure 1.

Examples of clinical drugs and experimental compounds with therapeutic potential having the vicinal diaryl heterocyclic motif.

Furthermore, a considerable number of vicinal diaryl heterocyclic motifs spread throughout the literature with various biological activities.¹ For example, a 4,5-diaryl isoxazole derivative BRP-187 (5) was reported to be a 5-lipoxygenase-activating protein (FLAP) inhibitor with potent anti-inflammatory activity.^8,9 The 3,4-diarylpyrazole derivative mardepodect (6, PF-2545920) was discovered as a phosphodiesterase (PDE) 10A inhibitor,¹⁰ which progressed through Phase-II clinical trials for Schizophrenia treatment, and was used as a model compound leading to a considerable number of follow-up PDE10A inhibitors having vicinal diaryl heterocyclic framework.¹¹ In addition, a good number of vicinal diaryl-substituted heterocycles were studied as anticancer therapeutics in which the vicinal diaryl scaffold was shown to be a simple pharmacophore for tubulin polymerization inhibitors mimicking the features of combretastatin A4 with potent anticancer activity as exemplified with combretatiophene (7).¹² In addition, 4,5-diarylisoxazole luminespib (8, NVP-AUY922) was developed as an Hsp90 inhibitor for the treatment of several cancer types and reached Phase-II clinical trials.¹³

Breast cancer (BC) is the most commonly occurring cancer in women and the most common cancer overall, while hepatocellular carcinoma (HCC) is the sixth most common and the second most lethal cancer.¹⁴ A large number of treatment options including localized and/or systematic therapies are available for both cancer types. Despite the initial effectiveness in controlling tumor growth and prolonging patient survival, nearly all current treatments result in resistance to therapies. This necessitates the development of novel approaches as well as effective chemotherapeutic agents for cancer treatments. We have long been working with the vicinal diaryl heterocyclic skeleton toward novel anti-inflammatory, antiplatelet, and anticancer agents.^8,9,15−20 In this context, inspired by the therapeutic potential of the vicinal diaryl motif around a central heterocycle, our compiled library of compounds belonging to this promising class was screened for their potential cytotoxic activity against an HCC cell line. Compound 9 with 3,4-diaryl isoxazole motif with synthetic accessibility for compound library generation was identified, although the cytotoxicity against HCC cells was negligible (IC₅₀ ≥ 20 μM) (Figure 1, Table 1). However, encouraged by the anticancer potential of vicinal diaryl heterocyclic scaffold and the synthetic feasibility of compound 9 skeleton, a focused library of compounds around compound 9 was prepared by decorating the vicinal diaryl rings and by incorporating various heterocyclic rings as the central core, which resulted in new analogues with improved cytotoxicity against both HCC and BC cancer cell lines. Here, we report the biological assessment of a new set of compounds that we have developed against cell lines derived from BC and HCC, which would be valuable for the understanding of the main features of these new vicinal diaryl heterocyclic analogues as anticancer therapeutic leads.

Table 1. In Vitro Cell Growth Inhibitory Activity against Hepatocellular Carcinoma and Breast Cancer Cell Lines^a.

^aIC₅₀ values were determined at least with five different concentrations of the compounds from the cell growth inhibition percentages.

Results and Discussion

Chemistry

Based on the developable potential of compound 9, we developed additional analogues to establish structure–activity relationships (SAR). New 3,4-diaryl-5-methylisoxazole derivatives 11-42 were synthesized according to the literature,²¹ and synthesis procedures and schemes of the relevant intermediates are given in the Supporting Information. In brief, hydrogenation of 9 to remove the benzyl group produced the key intermediate 10, which was subsequently used to generate desired final compounds 11–42 through alkylation of the phenolic hydroxyl (Scheme 1). The similar procedures in Scheme 1 were successfully applied to the synthesis of 44–57 with modifications at the middle phenyl and 59 with amine linker, following the reaction conditions in Schemes 2 and 3. The synthesis of 4,5-diaryl-3-methylisoxazole analogue 60 (Table 3) was achieved as we previously reported.⁸

Scheme 1. Reaction Conditions and Reagents: (i) Pd/C (10% w/w), HCl, EtOH, rt; (ii) Aryl/Alkyl Halide Derivative, K₂CO₃, MeCN, Δ; (iii) LiOH·H₂O, THF:water, Δ.

Scheme 2. Reaction Conditions and Reagents: (i) TEA, Diethyl Ether, 0 °C; (ii) Phenyl/pyridyl Acetone Derivative, NaH, THF, 0 °C.

Scheme 3. Reaction Conditions and Reagents: (i) 2-Methylbenzyl Bromide, DIEA, DMF, 65 °C.

Table 3. In Vitro Cell Growth Inhibitory Activity of Compounds with Distinct Central Heterocycles against Hepatocellular Carcinoma and Breast Cancer Cell Lines^a.

The preparation of 4,5-diarylisoxazole derivative 63 was achieved starting with readily available starting materials as illustrated in Scheme 4. Compound 61 was obtained according to the corresponding procedures.^10,22 The obtained 4,5-diarylisoxazole framework 62 was subsequently used to produce the desired 63 through first the hydrolysis of the methoxy and then the alkylation of the phenolic hydroxyl group (Scheme 4).

Scheme 4. Reaction Conditions and Reagents: (i) BBr₃, DCM, 0 °C; (ii) 2-Methylbenzyl Bromide, K₂CO₃, MeCN, MWI, 120 °C, 20 min.

The 3,4-diaryl-5-aminoisoxazole congener 65 was synthesized as shown in Scheme 5. The 3,4-diaryl-5-aminoisoxazole 65 was synthesized by cyclization of the β-cyanoketone 64, which was obtained according to the literature procedure.^23,24 The synthesis of 3,4-diaryl-5-methylisoxazole 67 where the 2-methylbenzyloxy arm was relocated on the 4-aryl ring was analogous to the similar chemistry that has been described in Scheme 1 (Scheme 6).

Scheme 5. Reaction Conditions and Reagents: (i) (4-Chlorophenyl)acetonitrile, LHMDS, THF, −70 °C; (vi) NH₂OH·HCl, EtOH, Δ.

Scheme 6. Reaction Conditions and Reagents: (i) TEA, Diethyl Ether, 0 °C; (ii) 4-((2-Methylbenzyl)oxy)phenylacetone, NaH, THF, 0 °C; (iii) Na₂CO₃, MeOH, H₂O, Δ.

The preparation of the 1,5-diaryl-3-methylpyrazole derivative 69 carrying the 2-methylbenzyloxy arm on 5-aryl group is outlined in Scheme 7. The synthesis of the β-diketone 68 was achieved by Claisen–Schmidt condensation.²⁵ The regioselective synthesis of the 1,5-diaryl-3-methylpyrazole 69 was then conveniently achieved by condensation of the diketone 68 with the hydrochloride salt of the 4-chlorophenylhydrazine in methanol/triethylamine.¹⁵

Scheme 7. Reaction Conditions and Reagents: (i) 4-Chlorophenylhydrazine·HCl, TEA, MeOH, Δ.

The central pyrazole series continued with the synthesis of 4,5-diaryl-1H-pyrazole 71 and 3,4-diaryl-1-methylpyrazole 72. Briefly, the in situ formed enaminone intermediate, obtained by the treatment of 70 with DMFDMA,¹⁰ was cyclized by hydrazine or methylhydrazine to produce the desired pyrazole compounds 71 and 72, respectively (Scheme 8). For the synthesis of the vicinal diaryl 2-methylthiazole 75, hydrolysis of 73 to remove the methyl group furnished the intermediate 74, which was utilized to generate the desired 4,5-diarylthiazole compound 75 through the benzylation of the phenolic hydroxyl (Scheme 9).

Scheme 8. Reaction Conditions and Reagents: (i) DMFDMA, DMF; (ii) NH₂·H₂O, MeOH, Δ; (iii) Methylhydrazine, MeOH, Δ.

Scheme 9. Reaction Conditions and Reagents: (i) BBr₃, DCM, 0 °C; (ii) 2-Methylbenzyl Bromide, K₂CO₃, MeCN, Δ.

1,5-Diarylpyrazole scaffolds 78 and 79 were produced according to previously developed procedures (Scheme 10).²⁶

Scheme 10. Reaction Conditions and Reagents: (i) 2-Methylbenzyl Bromide, K₂CO₃, MeCN, Δ.

1,5-Diarylpyrazole analogues 85–89 bearing the benzyloxy fragment on the 5-aryl as opposed to that of 78 were obtained through the cyclization of enaminones 80–84 with the hydrochloride salt of the 4-chlorophenylhydrazine in ethanol (Scheme 11). In addition, analogous compounds where the ether bridge in 85 exchanged with amide 91 or amine 92 were accomplished starting from 90 and following the standard reaction steps outlined in Scheme 12.

Scheme 11. Reaction Conditions and Reagents: (i) 4-Chlorophenylhydrazine·HCl, EtOH, Δ.

Scheme 12. Reaction Conditions and Reagents: (i) 2-Methylbenzoylchloride, DIEA, DCM, rt for Compound 91; 2-Methylbenzyl Bromide, DIEA, DMF, 65 °C for Compound 92.

Bioactivity Studies and SAR

To deduce SARs, several specific areas were focused on compound 9. Hence, it was aimed to explore (i) the influence of differently positioned substituents on the benzyloxy arm and the replacement of the phenyl ring of the benzyl group by heteroaryl moieties, (ii) the incorporation of different substituents at the isoxazole-4-phenyl group, (iii) the modification of the middle phenyl, and (iv) the replacement of central isoxazole with isosteric heterocycles. Collectively, a total of seventy vicinal diaryl heterocyclic compounds were synthesized to screen their inhibitory effects on cancer cell proliferation against Huh7 and MCF7 cells by introducing different chemical functionalities with the aforementioned modifications (Tables 1–4). Initially, the substitution pattern at the benzyl functionality was scrutinized by comparison of the differently substituted analogues (11–42) as illustrated in Table 1. Methylation of the aromatic ring of the benzyl at 2-position (11) caused a sudden increase in the potency (IC₅₀ = 1.3 μM for Huh7 and 3.8 μM for MCF7) versus compound 9 (IC₅₀ ≥ 20 μM for both Huh7 and MCF7) with unsubstituted benzyl arm. However, relocation of the methyl group to 3- or 4-position (19 and 24) led to a significant activity loss (IC₅₀ ≥ 20 μM). Substitutions at 2-position other than methyl, i.e., electron-donating (12-14) and electron-withdrawing groups (15–18), were undesirable resulting in an activity decrease against both cell lines (IC₅₀ values of 8.5 to >20 μM). Seemingly, voluminous substituents than methyl at 2-position were not well tolerated and impaired the efficiency of the compounds. Benzyl analogues differently substituted at 3- (19-23) or 4-position (24–29) were not chased further as they demonstrated a significant loss of inhibitory activity (IC₅₀ values of 12 to >20 μM) for both cell lines except for 20 with 3-methoxy, which showed comparable activity to 11 for the Huh7 cell line (IC₅₀ = 1.3 vs 3.6 μM). Compounds with dimethyl substitution pattern, i.e., 2,3-diMe (30) or 2,4-diMe (31), suggested that additional methyl group other than 2-methyl was not well tolerated on the benzyl moiety.

Table 4. In Vitro Growth Cell Inhibitory Activity of 85 Analogues against Hepatocellular Carcinoma and Breast Cancer Cell Lines^a.

Next, it was explored the impact of installing a heteroaromatic group in place of the phenyl ring of the benzyl functionality (32–42). When the 2-methylphenyl was replaced by 3-pyridinyl (32), 4-pyridinyl (33), or a voluminous 2-quinonyl (34), these compounds showed decreased cytotoxic activity, while compound 35 with the 3-pyridine ring having a methyl at a topologically equivalent position as in 11 retained the potency with IC₅₀ values of 3.9 μM for Huh7 and 6.1 μM for MCF7. A similar analogue 36 with a 2-pyridine ring having a methyl at the same position partially restored the potency against Huh7 (IC₅₀ = 9.4 μM), while the activity loss toward MCF7 was more pronounced (IC₅₀ ≥ 20 μM). Of interest, several five-membered heteroaromatic counterparts with alkyl substitutions such as 3,5-dimethylisoxazole (39), 1,3-dimethylpyrazole (40), and 1-isopropylimidazole (41) were well tolerated for their inhibition potential against Huh7 with IC₅₀ values of 3.7, 0.9, and 2.5 μM, respectively, while these three compounds appeared to be less effective against MCF7 cells (IC₅₀ values of 12 to >20 μM). From this point of view, the 2-methylbenzyl group remains the best choice for the 3,4-diaryl-3-methylisoxazole core, i.e., compound 11, for cytotoxic potency albeit several five-membered heteroaryl moieties exemplified with compounds 39–41 are also conceivable. Apparently, a consistent SAR for cytotoxic activity at this part was not accessible since small structural differences on the benzyl arm were not well tolerated.

Then, it was investigated if the 4-chloro substituent on the phenyl ring at C(4)-isoxazole was replaceable with different atoms or groups, while keeping the 2-methylbenzyl unit due to its good impact on the activity (compounds 44–55 in Table 2). Moving the 4-chloro in 11 to 2-position (44) or replacing it with a smaller fluoro (45) or larger methoxy (46) group resulted in a decreased potency against both cell lines (IC₅₀ = 9.9 to >20 μM). The amino replacement of 4-chloro (51) further reduced the potency for both cell types, similar to 45 and 46. On the other hand, substitution of the 4-chloro with a methyl group (47), which is about the same size as a chlorine atom, was pertinent and regained the bioactivity in both cell lines (IC₅₀ = 1.8 and 4.7 μM, respectively), whereas with a linear and less voluminous nitrile group in this position (48), the efficiency again dropped, especially against MCF7 cells (IC₅₀ ≥ 20 μM). Introducing a nitrogen atom to the 4-methylphenyl ring in 47 also caused an activity loss against MCF7 as exemplified with 54. Finally, compounds with substitutions at 3-position (49-50) or with 2,4-dihalogen substitutions (52–53) as well as with 4-pyridyl (55) were also significantly less effective indicating that the phenyl ring of C4-isoxazole may require substitution at 4-position with a similar steric size such as methyl or chlorine (Table 2). The next goal was to explore the substituent effect on the middle phenyl group, and this was briefly examined by fluoro substitution at 2- (56) and 3- (57) positions (Table 2). Although the 2-F substituted 56 preserved the potency against Huh7 and MCF7 cells with IC₅₀ of 2.25 and 9.53 μM, respectively, 3-F substitution in the compound 57 hampered the potency against both cell lines, implying that 2-substitution on the middle phenyl ring may be allowable for retaining or even improving the activity. Lastly, our efforts at the benzyloxyphenyl component included an isosteric exchange of the ether bridge with an amino linker (59), which was found tolerable for the Huh7 potency (IC₅₀ = 4 μM) with a concomitant loss of the activity against the MCF7 cell line (IC₅₀ ≥ 20 μM) (Table 2).

Table 2. In Vitro Cell Growth Inhibitory Activity against Hepatocellular Carcinoma and Breast Cancer Cell Lines^a.

^aIC₅₀ values were determined at least with five different concentrations of the compounds from the cell growth inhibition percentages.

After the biological confidence was rationalized with 3,4-diarylisoxazole derivatives with respect to C3-benzyloxyphenyl and C4-phenyl pendants, the next area of interest was to explore the central isoxazole with a series of common five-membered heterocycles to broaden the SAR investigation. To this end, eleven heterocyclic congeners with different heteroatom counts and orders were synthesized to probe the effect on the cytotoxic activity against HCC (Huh7 and Mahlavu) and BC (MCF7) cells and to search for the optimal central core for further SAR studies (Table 3). The position exchange of nitrogen and oxygen atoms in 11 to produce 60 resulted in a significant drop of activity against MCF7 cells (IC₅₀ ≈ 19 μM), while the potency toward Huh7 cells was still preserved (IC₅₀ = 3.8 μM). Furthermore, removal of 3-methyl of isoxazole in 60 to afford 63 appeared to diminish the potency against Mahlavu and MCF7 cells (IC₅₀ ≥ 20 μM) but improved the cytotoxic activity for Huh7 (IC₅₀ = 1.5 μM). Based on this result, the methyl in 11 was replaced with a polar amino group to further explore the hydrophobic effect and procured 65 with comparable potency to parent 11 against all cell lines with IC₅₀ = 2.0-3.9 μM. Interestingly, switching the sites of C3-benzyloxyphenyl unit and C4-phenyl in 11 to afford 67 appeared to diminish the cytotoxic activity for all three cell lines (IC₅₀ ≥ 20 μM). Meanwhile, other combinations of heteroatoms such as a group of pyrazoles as well as a thiazole core were also evaluated for their potential to replace the isoxazole core in 11. While thiazole 75 and pyrazole 78 did not produce the desired activity enhancement, other pyrazole derivatives with different orders of nitrogens and methyl substitutions (69, 71, 72, 79, and 85) maintained adequate cytotoxicity against all cell lines with IC₅₀ values in the range of 0.7 to 14.4 μM. Gratifyingly, the reversal of benzyloxyphenyl and 4-chlorophenyl components in 78 to produce 85 displayed a potency boost for Huh7 and MCF7 with IC₅₀ values of <1 μM, while still maintaining a decent activity against Mahlavu cells (IC₅₀ = 3.7 μM) compared with the parent 11. Based on the promising potential of 85 as an anticancer agent, the SAR around the middle phenyl and the ether linker was briefly examined, while preserving the 2-methylphenyl and 4-chlorophenyl units, which had been found optimal for the potency (Table 4). The introduction of a nitrogen atom into the middle phenyl in 85 resulted in isosteric pyridine mimics²⁷86 and 87 in which the 2-pyridyl regioisomer (86) exhibited higher cytotoxicity toward Huh7 and Mahlavu cells (IC₅₀ = 2.0 and 7.1 μM, respectively), although this was accompanied by a reduction in the potency against MCF7 cells (IC₅₀ ≥ 20 μM). The bioisosterism between the azine C-N and the aryl C-F bond in compounds 88 and 89 was also examined.²⁸ The activity results indicated an efficient bioisosterism of the C-F bond with the pyridine N atom in this context because the replacement of the pyridine N atom with the C-F moiety led to improved potency in 88 (IC₅₀ = 1.6–6.4 μM). This was also in good correlation with its isoxazole congener 56 with the 2-F substituted middle phenyl group (Table 2). Lastly, the role of ether linker was studied in 91 and 92. As seen, while the amide linker was not tolerated for any cell line, the amine replacement of the ether oxygen (92) was tolerable for Huh7 and Mahlavu cells (IC₅₀ = 3.6 and 4.5 μM, respectively) with a diminished activity against MCF7 cells (Table 4), again in good agreement with its isoxazole counterpart 59 (Table 2). According to the SAR results, compound 11 with 3,4-diarylisoxazole and compound 85 with 1,5-diarylpyrazole frameworks were selected for further analysis, and unambiguous structural elucidation of 11 and 85 using the single-crystal X-ray diffraction method was accomplished showing the accurate rearrangement of aromatic rings and atoms in 3D-shape (Figure S1).^29,30

Next, judged by the cellular activity of 11 and 85, both compounds in addition to Huh7, Mahlavu, and MCF7 cell lines were further screened against a panel of the hepatocellular carcinoma (HepG2, SNU475, Hep3B, FOCUS, Hep40, and PLC-PRF-5) and breast cancer (MDA-MB-231, MDA-MB-468, SKBR3, and ZR-75) cell lines along with the non-tumorigenic immortalized breast epithelial cells MCF10A (Table 5). The results demonstrated that both compounds are endowed with potent antiproliferative activity against all cancer cells with IC₅₀ values in the range of 1.3–9.5 μM for 11 and 0.77–7.8 μM for 85 for hepatocellular carcinoma and breast cancer cell lines, while found less toxic to the MCF10A immortalized normal breast epithelial cells (Table 5).

Table 5. In Vitro Growth Inhibitory Activity of 11 and 85 Analogues against Hepatocellular Carcinoma and Breast Cancer Cell Line Panel^a.

		IC50 (μM)
	hepatocellular carcinoma								breast
#	Huh7	HepG2	SNU475	Hep3B	FOCUS	Hep40	PLC-PRF-5	Mahlavu	MCF7	MDA-MB231	MDA-MB468	SKBR3	ZR75	MCF10A
11	1.3	2.1	1.7	3.0	2.1	8.6	9.5	3.2	3.4	2.0	2.8	3.5	7.6	12.1
85	0.7	1.4	1.5	7.9	2.4	5.2	6.5	3.7	0.9	0.9	1.0	1.8	5.5	7.6

^aIC₅₀ values were determined at least with five different concentrations of the compounds from the cell growth inhibition percentages.

Moreover, in vitro single-dose anticancer screening of 11 and 85 at 10 μM was performed utilizing the stable panel of 60 cell lines comprising 9 different cancer types at the National Cancer Institute (NCI) under the Developmental Therapeutics Program (Table S1).³¹ Compounds 11 and 85 demonstrated variations in sensitivity and selectivity against individual cell lines in the panel, as illustrated in Table S1. Collectively, both compounds exhibited similar antiproliferative activity against 22 cancer cells with growth inhibition (GI) values in the range of 45–100% at 10 μM. The NCI panel results revealed that both compounds displayed a good preference for leukemia such as CCRF-CEM, HL-60, K-562, MOLT-4, and SR, and colon cancer including HCT-116, HCT-15, HT-29, KM-12, and SW-620 cell lines, in addition to hepatocellular carcinoma and breast cancer cells of this work.

Based on the encouraging potency of 85 in cellular assays against hepatocellular carcinoma and breast cancer as well as in the NCI-60 panel, we decided to evaluate in vitro ADME and pharmacokinetic (PK) properties in mice (Figure S2). Metabolic stability of 85 in human microsomes was moderate (55% remaining after 45 min incubation). The compound is highly lipophilic with logD_7.4 of 6.64, sparingly soluble at the physiological pH, and highly plasma protein-bound (99.9%). The capacity of 85 to inhibit human CYP isoforms 2C9, 2D6, and 3A4 was insignificant (7.3, 22.2, and 0% at 10 μM, respectively) implying a safe window for clinical drug interactions.

The PK parameters after intravenous (iv) administration of 85 constitute a moderate volume of distribution, low total plasma clearance, and a moderate iv half-life of 3.85 h resulting in mice with low oral bioavailability (Table 6). Although PK parameters are nonoptimal, this may be counterbalanced by the potent in vitro antiproliferative activity spectrum of 85 and is not considered limiting for further progression of 85 to in vivo antitumor efficacy studies.

Table 6. PK Properties of Compound 85^a.

Mouse IV PK, 1 mpk					Mouse PO PK, 10 mpk
t1/2	Vd	Co	Clp	AUC	Cmax	AUC	t1/2	F%
3.85	13.2	393	2.33	436	19.3	192	6.64	5

^at_1/2 = h, V_d = (L/kg), C_o, C_max = (ng/mL), Clp = (L/h/kg), AUC = (h*ng/mL).

Real-Time Cellular Response of Cells with Compound 11 and 85 Treatment

Time- and dose-dependent effects of compounds 11 and 85 on Mahlavu, Huh7, MDA-MB-231, and MCF-7 were evaluated with the use of real-time cell electronic sensing by monitoring dynamic cell proliferation. Both 11 and 85 caused inhibition in the growth of both breast and hepatocellular carcinoma cell lines compared to the control (DMSO) group, whereas there was no difference in the growth of MCF10A immortalized normal breast epithelial cells upon treatment. This assay further confirmed that both compounds displayed time and dose-dependent growth inhibitory effects. Cytotoxic effects of 11 and 85 on cell lines could be observed after 24 h of compound treatment and reached their highest values after 72 h (Figure 2).

Figure 2.

Real-time cell growth response of Mahlavu, Huh7, MCF-7, and MDA-MB-231 cancer cells and MCF10A immortalized normal breast epithelial cells treated with compounds 11 and 85 and control (DMSO) for 96 h. The experiment was done in triplicate, and results were normalized to controls of 10-5-2.5 μM concentrations 11 and 85.

Characterization of Cell Death Mechanism Induced by 11 and 85

To control the cell death mechanism triggered by 11 and 85, Huh7 and Mahlavu hepatocellular carcinoma cells and MDA-MB-231 and MCF-7 breast cancer cells were cultured according to their cell growth rate and were treated with both 11 and 85 for 48 h. The apoptotic morphological changes were observed in both breast cancer cells and hepatocellular carcinoma cells upon treatment by nuclear staining with Hoechst compared to the control group (Figure 3A). The apoptotic cell populations were further examined with Annexin V staining using flow cytometry. Compared to the control group, the percentage of apoptotic populations in both breast cancer cell lines and hepatocellular carcinoma cell lines treated with 11 or 85 was increased after 24 h (Figure 3B). For further investigation of apoptosis activation through 11 and 85 treatments, apoptosis-associated PARP protein levels were assessed using western blotting. Except for 11 treated MCF7 and Huh7 cells, both 11 and 85 compounds caused the increase in PARP cleavage in both breast cancer cells (MCF7 and MDA-MB-231) and hepatocellular carcinoma cells (Mahlavu) (Figure 3C). These results further supported the increased cytotoxic effects of compounds on breast cancer and hepatocellular carcinoma cancer cells. We next investigated in vivo antitumor efficacy of 11 and 85 on nude mice tumor xenografts.

Figure 3.

Compounds 11 and 85 induced cell death. (A) Cells were treated with compounds 11 and 85 using their IC₁₀₀ concentrations or DMSO as a negative control for 48 h. Nuclear morphology was revealed by Hoechst staining under fluorescent microscopy. Apoptotic bodies were detectable in 11- and 85-treated cells after 48 h. Arrows show apoptotic cells. Scale bar: 20 μM. (B) HCC cells and breast cancer cells were treated with 5 μM compounds 11 and 85 and DMSO as control and were analyzed after 48 h. (C) PARP in Huh7, Mahlavu, MDA-MB-231, and MCF-7 cells treated with 11 and 85 for 48h. Calnexin was used for equal loading control. (D) Bar graph represents relative band intensities of Cleaved PARP/Total PARP, which were normalized with their calnexin loading controls. Statistical analysis was performed using one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant.

In Vivo Antitumor Effects of 11 and 85 in Mice Xenograft Models

The antitumor effects of 11 and 85 in the hepatocellular carcinoma (Mahlavu cells) and breast (MDA-MB-231 cells) xenograft models were assessed twice a week by oral administration of 11 and 85 at 40 mg/kg for 4 weeks. Both compounds conferred a sustained antitumor efficiency (Figures 4 and S3). In the Mahlavu xenografts, mice administered with compounds 11 and 85 had a significant reduction in tumor volume following 4 weeks of treatment, i.e., 85 and 40% reductions in tumor volumes, respectively. Moreover, for MDA-MB-231 xenografts, mice treated with both compounds resulted in about a 50% decrease in tumor volumes as compared to the control group (Figure 4). In all studies, the administered dose was well tolerated and neither significant bodyweight loss nor toxic effects or mortality were observed.

Figure 4.

Compounds 11 and 85 reduce tumor growth. Mahlavu and MDA-MB-231 xenograft nude mice were treated with 40 mg/kg 11 and 85 prepared in 0.5% hydroxypropyl methyl cellulose plus 1% Tween 80 or with vehicle only twice a week once the tumor size reached 100 mm³. Tumor volumes were recorded twice a week during the experiment. Each group contained six mice (n = 6). Statistical analysis was performed using one-way ANOVA. **p < 0.05, ****p < 0.00001.

Conclusions

We synthesized a series of vicinal diaryl isoxazole and pyrazole derivatives as putative anticancer agents and checked their growth inhibitory activity against human hepatocellular carcinoma and breast cancer cell lines. Most of the derivatives represented moderate cytotoxicity in the selected cancer cells. In general, the substitution type and pattern on the benzyloxyphenyl group linked to the central heterocycle had a significant effect on the potency and the selectivity of the compounds in the tested cancer cell lines. Following a comprehensive evaluation of twelve central heterocyclics comprising a combination of different heteroatom positions and numbers, we observed that the type of the central heterocycle, i.e., isoxazole and pyrazole, was also crucial for the observed anticancer potency. Subsequently, two analogues, the 3,4-diarylisoxazole derivative 11 and 1,5-diarylpyrazole 85, stand out as developable anticancer compounds based on their significant in vitro antiproliferative activities toward the 13 hepatocellular and breast cancer cell lines with IC₅₀ values in the range of 0.77 to 9.53 μM. We also demonstrated that both compounds displayed dose- and time-dependent growth inhibition through the RT-CES system, which was also correlated to initial SRB screening results. Further analysis showed that both compounds induced apoptotic cell death in both hepatocellular carcinoma and breast cancer cell lines and demonstrated antitumor activity in vivo in the mouse hepatocellular and breast tumor xenografts with inhibition rates between 40% and 85%, and with insignificant effect on the mice bodyweight.

In conclusion, our results revealed that these diaryl-isoxazole and -pyrazole derivatives exemplified with 11 and 85 show promise as leads for further development of improved anticancer compounds against hepatocellular carcinoma and breast cancers. Hence, further investigation of novel analogues that could be integral to the current SAR in this study would be of value to identify novel diaryl heterocycles with strong anticancer and druglike properties. Elucidation of detailed molecular mechanisms associated with 11 and 85 such as direct profiling of compounds using NanoString analysis as well as target fishing experiments with biotin-labeled conjugates to identify potential molecular targets are under investigation and will be reported in due time.

Experimental Section

Chemistry

All chemicals were purchased from different suppliers such as Sigma-Aldrich Chemicals (Sigma-Aldrich Corp., St. Louis, MO), Merck Chemicals (Merck KGaA, Darmstadt, Germany), and ABCR (abcr GmbH, Karlsruhe, Germany). THF was dried from benzophenone-sodium. ¹H and ¹³C NMR spectra were recorded in CDCl₃ or DMSO-d₆ on a Varian Mercury 400 MHz spectrometer using tetramethylsilane as the internal standard. Coupling constants were reported as Hertz and all chemical shifts were recorded as δ (ppm). High-resolution mass spectra data (HRMS) were collected using Waters LCT Premier XE Mass Spectrometer operating in ESI(+) or ESI(−) method, also coupled to an AQUITY Ultra Performance Liquid Chromatography system (Waters Corporation) using a UV detector monitoring at 254 nm. Purity for all final compounds was >95%, according to the UPLC-MS method using a water/MeCN solvent gradient containing 0.1% formic acid (1%/90%); flow rate: 0.3 mL/min, column: Aquity BEH C18 column (2.1 × 100 mm², 1.7 mm). All microwave irradiation reactions were carried out in a Biotage Initiator + microwave apparatus with Biotage sealed microwave vials. Flash chromatography was performed on a Combiflash Rf Automatic Flash Chromatography System with RediSep silica gel columns (12, 24, and 40 g) (Teledyne-Isco, Lincoln, NE) or Reveleris PREP Purification System (Buchi, New Castle, DE). Preparative chromatography was performed on Buchi US15C18HQ-250/212-C18 silica gel columns with the following devices; Reveleris PREP Purification System or PuriFlash 4250 System (Interchim, Montluçon, France). Melting points of the compounds were determined using the SMP50 automated melting point apparatus (Stuart, Staffordshire, ST15 OSA, U.K.). The in vitro ADME and in vivo mouse PK studies were conducted at Syngene International Ltd., Bangalore, India. General synthetic procedures (Methods 1–9) and experimental data for all intermediate compounds can be found in the Supporting Information.

3-(4-(Benzyloxy)phenyl)-4-(4-chlorophenyl)-5-methylisoxazole (9)

It was synthesized according to method 4 by the reaction of 4-chlorophenylacetone and the 1-(benzyloxy)-4-(nitrosomethyl)benzene unstable intermediate obtained in situ following the literature as shown in Scheme S1.²¹ The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 50.0%; mp 114.7–115.7 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.06 (2H, s), 6.93 (2H, d, J = 8.8 Hz), 7.12 (2H, d, J = 8.8 Hz), 7.32–7.43 (9H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 69.99, 114.53, 114.91, 121.41, 127.49, 128.61, 129.01, 129.09, 129.75, 131.11, 133.69, 136.58, 159.78, 160.60, 166.54. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₉ClNO₂: 376.1104, found: 376.1106.

4-(4-Chlorophenyl)-5-methyl-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (11)

It was synthesized from compound 10 using 2-methylbenzyl bromide according to synthesis method 1a. Yield 77.0%; mp 114.1–115.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.43 (3H, s), 5.03 (2H, s), 6.94 (2H, d, J = 8.4 Hz), 7.13 (2H, d, J = 8.8 Hz), 7.19–7.28 (3H, m), 7.35–7.40 (5H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.62, 18.89, 68.60, 114.57, 114.84, 121.41, 126.06, 128.39, 128.63, 129.02, 129.10, 129.78, 130.44, 131.15, 133.71, 134.39, 136.69, 159.91, 160.62, 166.58. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁NO₂Cl: 390.1261, found: 390.1263. CAS: 2758520-84-0.

4-(4-Chlorophenyl)-3-(4-((2-methoxybenzyl)oxy)phenyl)-5-methylisoxazole (12)

It was synthesized from compound 10 using 2-methoxybenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 75.0%; mp 156.8–157.7 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 3.85 (3H, s), 5.11 (2H, s), 6.89-6.99 (4H, m), 7.12 (2H, d, J = 8.4 Hz), 7.28–7.32 (1H, m), 7.33–7.36 (4H, m), 7.43 (1H, d, J = 7.6 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 55.37, 65.06, 110.27, 114.53, 114.95, 120.58, 121.17, 124.94, 128.59, 128.99, 129.04, 129.15, 129.68, 131.12, 133.67, 156.81, 160.00, 160.68, 166.48. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁NO₃Cl: 406.1210, found: 406.1212.

4-(4-Chlorophenyl)-5-methyl-3-(4-((2-(trifluoromethoxy)benzyl)oxy)phenyl)isoxazole (13)

It was synthesized from compound 10 using 2-trifluoromethoxybenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 46.0%; mp 85.8–86.7 °C. ¹H NMR (400 MHz, DMSO-d₆): δ_H 2.39 (3H, s), 5.13 (2H, s), 7.04 (2H, d, J = 8.8 Hz), 7.23 (2H, d, J = 8.4 Hz), 7.28 (2H, d, J = 8.8 Hz), 7.40–7.52 (5H, m), 7.64 (1H, d, J = 7.6 Hz). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 11.20, 64.22, 113.96, 114.91, 120.12 (q, ¹J_C-F = 255.2 Hz), 120.57, 121.14, 127.56, 128.79, 128.83, 128.97, 129.48, 130.29, 130.88, 131.41, 132.56, 146.65, 159.11, 160.03, 166.94. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₁₈NO₃ClF₃: 460.0927, found: 460.0928.

4-(4-Chlorophenyl)-3-(4-((2-(difluoromethoxy)benzyl)oxy)phenyl)-5-methylisoxazole (14)

It was synthesized from compound 10 using 2-difluoromethoxybenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 86.0%; mp 95.5–96.9 °C. ¹H NMR (400 MHz, DMSO-d₆): δ_H 2.42 (3H, s), 5.12 (2H, s), 7.06 (2H, d, J = 8.8 Hz), 7.24–7.27 (5H, m), 7.70 (2H, d, J = 8.8 Hz), 7.42–7.47 (1H, m), 7.49 (2H, d, J = 8.4. Hz), 7.58 (1H, dd, J = 7.6 Hz, 1.2 Hz). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 11.20, 64.44, 113.95, 114.90, 116.60 (t, ¹J_C-F = 256.5 Hz), 118.52, 121.01, 125.40, 127.56, 128.81, 128.84, 129.47, 129.89, 130.37, 131.40, 132.55, 149.13 (t, ³J_C-F = 3.2 Hz), 159.24, 160.05, 166.92. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₁₉NO₃ClF₂: 442.1022, found: 442.1016.

4-(4-Chlorophenyl)-3-(4-((2-fluorobenzyl)oxy)phenyl)-5-methylisoxazole (15)

It was synthesized from compound 10 using 2-fluorobenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 91.0%; mp 119.5–120.9 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.13 (2H, s), 6.94 (2H, d, J = 8.4 Hz), 7.06–7.08 (1H, m), 7.12 (2H, d, J = 8.8 Hz), 7.16 (1H, td, J = 7.6 Hz, 1.2 Hz), 7.29–7.33 (1H, m), 7.35 (2H, d, J = 8.4 Hz), 7.36 (2H, d, J = 8.8 Hz), 7.49 (1H, td, J = 7.4, 1.6 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 63.68 (d, ³J_C-F = 3.8 Hz), 114.54, 114.86, 115.38 (d, ²J_C-F = 20.6 Hz), 121.63, 123.76 (d, ²J_C-F = 13.7 Hz), 124.28 (d, ³J_C-F = 3.1 Hz), 129.01, 129.06, 129.69 (d, ³J_C-F = 3.8 Hz), 129.79, 129.87, 131.09, 133.71, 159.52, 160.44 (d, ¹J_C-F = 245.4 Hz), 160.58, 166.55. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₈NO₂ClF: 394.1010, found: 394.1008.

4-(4-Chlorophenyl)-3-(4-((2-chlorobenzyl)oxy)phenyl)-5-methylisoxazole (16)

It was synthesized from compound 10 using 2-chlorobenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 91.0%; mp 105.2–106.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.43 (3H, s), 5.17 (2H, s), 6.95 (2H, d, J = 9.2 Hz), 7.13 (2H, d, J = 8.8 Hz), 7.27–7.32 (2H, m), 7.35–7.41 (5H, m), 7.53–7.55 (1H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 67.11, 114.54, 114.93, 121.70, 126.98, 128.74, 129.03, 129.07, 129.08, 129.41, 129.81, 131.11, 132.58, 133.74, 134.32, 159.49, 160.56, 166.58. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₈NO₂Cl₂: 410.0715, found: 410.0717.

2-((4-(4-(4-Chlorophenyl)-5-methylisoxazol-3-yl)phenoxy)methyl)benzonitrile (17)

It was synthesized from compound 10 using α-bromo-o-tolunitrile according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 77.0%; mp 111.2–112.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.26 (2H, s), 6.95 (2H, d, J = 8.8 Hz), 7.12 (2H, d, J = 8.8 Hz), 7.35–7.39 (4H, m), 7.44 (1H, td, J = 7.4, 1.6 Hz), 7.61–7.71 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 67.51, 111.17, 114.57, 114.96, 116.96, 122.14, 128.44, 128.52, 128.97, 129.05, 129.88, 131.09, 132.91, 133.09, 133.77, 140.16, 159.09, 160.48, 166.61. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₁₈N₂O₂Cl: 401.1057, found: 401.1054.

4-(4-Chlorophenyl)-5-methyl-3-(4-((2-(trifluoromethyl)benzyl)oxy)phenyl)isoxazole (18)

It was synthesized from compound 10 using 2-trifluoromethylbenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 70.0%; mp 132.8–134.4 °C. ¹H NMR (400 MHz, DMSO-d₆): δ_H 2.42 (3H, s), 5.24 (2H, s), 7.06 (2H, d, J = 8.4 Hz), 7.26 (2H, d, J = 8.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.49 (2H, d, J = 8.2 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.73 (1H, dd, J = 7.6, 7.2 Hz), 7.77 (1H, d, J = 7.2 Hz), 7.80 (1H, d, J = 7.6 Hz). ¹³C NMR (100 MHz, DMSO-d₆): δ_C 11.20, 66.26 (q, ⁴J_C-F = 2.6 Hz), 113.96, 114.92, 121.23, 124.24 (q, ¹J_C-F = 271.9 Hz), 126.13 (q, ³J_C-F = 5.8 Hz), 126.96 (q, ²J_C-F = 30.1 Hz), 128.79, 128.84, 129.52, 130.66, 131.41, 132.56, 132.80, 134.49 (q, ⁴J_C-F = 1.3 Hz), 159.03, 160.01, 166.96. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₁₈NO₂ClF₃: 444.0978, found: 444.0973.

4-(4-Chlorophenyl)-5-methyl-3-(4-((3-methylbenzyl)oxy)phenyl)isoxazole (19)

It was synthesized from compound 10 using 3-methylbenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 90.0%; mp 119.9–121.0 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.42 (3H, s), 5.02 (2H, s), 6.93 (2H, d, J = 8.8 Hz), 7.12 (2H, d, J = 8.4 Hz), 7.13–7.15 (1H, m), 7.20–7.29 (3H, m), 7.34–7.36 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 21.40, 70.07, 114.53, 114.91, 121.36, 124.61, 128.25, 128.52, 128.85, 129.01, 129.11, 129.74, 131.11, 133.69, 136.47, 138.33, 159.85, 160.62, 166.52. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁NO₂Cl: 390.1261, found: 390.1260.

4-(4-Chlorophenyl)-3-(4-((3-methoxybenzyl)oxy)phenyl)-5-methylisoxazole (20)

It was synthesized from compound 10 using 3-methoxybenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 95.0%; mp 105.1–106.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 3.81 (3H, s), 5.04 (2H, s), 6.85-6.88 (1H, m), 6.92 (2H, d, J = 8.8 Hz), 6.97-6.99 (2H, m), 7.11 (2H, d, J = 8.4 Hz), 7.29 (1H, t, J = 8.0 Hz), 7.34–7.37 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 55.24, 69.86, 112.91, 113.56, 114.53, 114.92, 119.62, 121.44, 129.01, 129.09, 129.67, 129.74, 131.11, 133.70, 138.17, 159.73, 159.85, 160.59, 166.53. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁NO₃Cl: 406.1210, found: 406.1191.

4-(4-Chlorophenyl)-3-(4-((3-fluorobenzyl)oxy)phenyl)-5-methylisoxazole (21)

It was synthesized from compound 10 using 3-fluorobenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 90.0%; mp 98.1–99.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.06 (2H, s), 6.91 (2H, d, J = 8.8 Hz), 6.98–7.03 (1H, m), 7.11 (2H, d, J = 8.4 Hz), 7.13–7.19 (2H, m), 7.32–7.36 (5H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 69.14 (d, ⁴J_C-F = 2.0 Hz), 114.22 (d, ²J_C-F = 22.5 Hz), 114.53, 114.89, 114.91 (d, ²J_C-F = 21.1 Hz), 121.69, 122.69 (d, ⁴J_C-F = 3.2 Hz), 129.02, 129.05, 129.79, 130.16 (d, ³J_C-F = 8.3 Hz), 131.09, 133.72, 139.19 (d, ³J_C-F = 7.0 Hz), 159.46, 160.53, 162.98 (d, ¹J_C-F = 244.9 Hz), 166.58. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₈NO₂ClF: 394.1010, found: 394.1011.

3-((4-(4-(4-Chlorophenyl)-5-methylisoxazol-3-yl)phenoxy)methyl)benzonitrile (22)

It was synthesized from compound 10 using α-bromo-m-tolunitrile according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 50% EtOAc in Hexane). Yield 97.0%; mp 114.4–115.6 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.08 (2H, s), 6.91 (2H, d, J = 9.2 Hz), 7.11 (2H, d, J = 8.4 Hz), 7.36 (2H, d, J = 8.4 Hz), 7.37 (2H, d, J = 9.2 Hz), 7.50 (1H, dd, J = 8.0, 7.2 Hz), 7.61–7.66 (2H, m), 7.73 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 68.64, 112.83, 114.53, 114.84, 118.55, 122.04, 128.99, 129.04, 129.43, 129.89, 130.68, 131.09, 131.44, 131.66, 133.76, 138.26, 159.13, 160.44, 166.65. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₁₈N₂O₂Cl: 401.1057, found: 401.1059.

Methyl-3-((4-(4-(4-chlorophenyl)-5-methylisoxazol-3-yl)phenoxy)methyl)benzoate (23)

It was synthesized from compound 10 using methyl-3-(bromomethyl) benzoate according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 93.0%; mp 121.5–122.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 3.92 (3H, s), 5.10 (2H, s), 6.92 (2H, d, J = 8.4 Hz), 7.11 (2H, d, J = 8.4 Hz), 7.33–7.37 (4H, m), 7.46 (1H, t, J = 7.8 Hz), 7.62 (1H, d, J = 7.8 Hz), 8.00 (1H, d, J = 7.8 Hz), 8.09 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 52.20, 69.42, 114.53, 114.91, 121.66, 128.53, 128.75, 129.01, 129.04, 129.26, 129.80, 130.55, 131.09, 131.84, 133.71, 137.06, 159.52, 160.55, 166.57, 166.79. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₁NO₄Cl: 434.1159, found: 434.1165.

4-(4-Chlorophenyl)-5-methyl-3-(4-((4-methylbenzyl)oxy)phenyl)isoxazole (24)

It was synthesized from compound 10 using 4-methylbenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 82.0%; mp 116.9–118.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.36 (3H, s), 2.42 (3H, s), 5.02 (2H, s), 6.92 (2H, d, J = 9.2 Hz), 7.12 (2H, d, J = 8.8 Hz), 7.20 (2H, d, J = 7.8 Hz), 7.30 (2H, d, J = 7.8 Hz), 7.33–7.37 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 21.19, 69.94, 114.53, 114.92, 121.32, 127.63, 128.99, 129.11, 129.29, 129.72, 131.11, 133.53, 133.68, 137.89, 159.86, 160.62, 166.51. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁NO₂Cl: 390.1261, found: 390.1259.

4-(4-Chlorophenyl)-3-(4-((4-methoxybenzyl)oxy)phenyl)-5-methylisoxazole (25)

It was synthesized from compound 10 using 4-methoxybenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 93.0%; mp 126.1–127.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 3.81 (3H, s), 4.98 (2H, s), 6.91 (4H, m), 7.12 (2H, d, J = 8.8 Hz), 7.33–7.37 (6H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 55.29, 69.80, 114.03,114.53, 114.91, 121.31, 128.59, 128.99, 129.11, 129.26, 129.72, 131.11, 133.68, 159.55, 159.84, 160.62, 166.51. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁NO₃Cl: 406.1210, found: 406.1226.

4-(4-Chlorophenyl)-3-(4-((4-fluorobenzyl)oxy)phenyl)-5-methylisoxazole (26)

It was synthesized from compound 10 using 4-fluorobenzyl chloride according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 88.0%; mp 99.3–100.7 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.01 (2H, s), 6.91 (2H, d, J = 8.8 Hz), 7.06 (2H, t, J = 8.8 Hz), 7.11 (2H, d, J = 8.8 Hz), 7.34–7.41 (6H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.55, 69.32, 114.53, 114.88, 115.53 (d, ²J_C-F = 21.3 Hz), 121.59, 129.01, 129.07, 129.35 (d, ³J_C-F = 7.6 Hz), 129.78, 131.10, 132.33 (d, ⁴J_C-F = 3.8 Hz), 133.71, 159.59, 160.55, 162.56 (d, ¹J_C-F = 244.6 Hz), 166.57. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₈NO₂ClF: 394.1010, found: 394.1009.

4-((4-(4-(4-Chlorophenyl)-5-methylisoxazol-3-yl)phenoxy)methyl)benzonitrile (27)

It was synthesized from compound 10 using α-bromo-p-tolunitrile according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 50% EtOAc in Hexane). Yield 77.0%; mp 138.5–139.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.12 (2H, s), 6.90 (2H, d, J = 8.8 Hz), 7.11 (2H, d, J = 8.4 Hz), 7.34–7.37 (4H, m), 7.53 (2H, d, J = 8.2 Hz), 7.68 (2H, d, J = 8.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.55, 68.87, 111.86, 114.52, 114.84, 118.60, 122.04, 127.54, 128.99, 129.03, 129.87, 131.09, 132.43, 133.76, 142.03, 159.14, 160.42, 166.66. C₂₄H₁₈N₂O₂Cl for; HRMS (m/z) [M + H]⁺ calcd for 401.1057, found:401.1070.

Methyl-4-((4-(4-(4-chlorophenyl)-5-methylisoxazol-3-yl)phenoxy)methyl)benzoate (28)

It was synthesized from compound 10 using methyl-4-(bromomethyl) benzoate according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 86.0%; mp 110.7–111.9 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 3.92 (3H, s), 5.12 (2H, s), 6.92 (2H, d, J = 9.2 Hz), 7.11 (2H, d, J = 8.8 Hz), 7.34–7.37 (4H, m), 7.49 (2H, d, J = 8.4 Hz), 8.06 (2H, d, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 52.15, 69.32, 114.53, 114.91, 121.74, 126.97, 129.02, 129.04, 129.81, 129.90, 131.10, 133.73, 141.75, 159.45, 160.51, 166.59, 166.77. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₁NO₄Cl: 434.1159, found: 434.1161.

4-((4-(4-(4-Chlorophenyl)-5-methylisoxazol-3-yl)phenoxy)methyl)benzoic acid (29)

Compound 28 (1.0 equiv) was stirred with LiOH.H₂O (3.0 eq) in a THF:water solvent system under reflux for 3 h. At the end of the period, the reaction mixture was acidified with concentrated HCl and the precipitate formed was filtered under vacuum by washing with water. Yield 89.0%; mp 210.8–212.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.43 (3H, s), 5.15 (2H, s), 6.93 (2H, d, J = 8.4 Hz), 7.12 (2H, d, J = 8.8 Hz), 7.35–7.38 (4H, m), 7.53 (2H, d, J = 8.8 Hz), 8.14 (2H, d, J = 8.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 69.26, 114.56, 114.91, 121.77, 127.03, 128.91, 129.04, 129.84, 130.55, 131.11, 133.75, 142.77, 159.41, 160.50, 166.63, 171.42. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₁₉NO₄Cl: 420.1003, found: 420.1005.

4-(4-Chlorophenyl)-3-(4-((2,3-dimethylbenzyl)oxy)phenyl)-5-methylisoxazole (30)

It was synthesized from compound 10 using 2,3-dimethylbenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 87.0%; mp 167.2–168.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.26 (3H, s), 2.32 (3H, s), 2.43 (3H, s), 5.04 (2H, s), 6.94 (2H, d, J = 8.4 Hz), 7.10–7.18 (4H, m), 7.24 (1H, d, J = 6.8 Hz), 7.35–7.38 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 14.88, 20.34, 69.22, 114.53, 114.83, 121.36, 125.58, 126.87, 129.01, 129.12, 129.74, 130.21, 131.13, 133.70, 134.22, 135.50, 137.29, 159.94, 160.62, 166.54. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₃NO₂Cl: 404.1417, found: 404.1414.

4-(4-Chlorophenyl)-3-(4-((2,4-dimethylbenzyl)oxy)phenyl)-5-methylisoxazole (31)

It was synthesized from compound 10 using 2,4-dimethylbenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 77.0%; mp 119.7–122.1 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.33 (3H, s), 2.34 (3H, s), 2.42 (3H, s), 4.99 (2H, s), 6.93 (2H, d, J = 8.8 Hz), 7.02 (1H, d, J = 7.4 Hz), 7.05 (1H, s), 7.13 (2H, d, J = 8.8 Hz), 7.26 (1H, d, J = 7.4 Hz), 7.35–7.37 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 18.82, 21.09, 68.52, 114.53, 114.83, 121.30, 126.64, 128.94, 129.01, 129.13, 129.72, 131.13, 131.32, 131.39, 133.69, 136.74, 138.26, 159.99, 160.63, 166.53. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₃NO₂Cl: 404.1417, found: 404.1414.

4-(4-Chlorophenyl)-5-methyl-3-(4-(pyridin-3-ylmethoxy)phenyl)isoxazole (32)

It was synthesized from compound 10 using 3-(chloromethyl)pyridine.HCl according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 60% EtOAc in Hexane). Yield 53.0%; mp 115.5–116.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.14 (2H, s), 6.92 (2H, d, J = 8.4 Hz), 7.11 (2H, d, J = 8.4 Hz), 7.34–7.38 (4H, m), 7.52 (1H, dd, J = 8.0 Hz, 4.8 Hz), 7.98 (1H, d, J = 8.0 Hz), 8.64 (1H, d, J = 4.8 Hz), 8.77 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 66.90, 114.55, 114.83, 122.26, 124.53, 128.93, 129.05, 129.94, 131.07, 133.79, 133.94, 137.83, 145.99, 146.58, 158.93, 160.40, 166.68. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₁₈N₂O₂Cl: 377.1057, found: 377.1063.

4-(4-Chlorophenyl)-5-methyl-3-(4-(pyridin-4-ylmethoxy)phenyl)isoxazole (33)

It was synthesized from compound 10 using 4-(chloromethyl)pyridine.HCl according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 70% EtOAc in Hexane). Yield 25.0%; mp 124.2–125.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.41 (3H, s), 5.19 (2H, s), 6.91 (2H, d, J = 8.8 Hz), 7.11 (2H, d, J = 8.4 Hz), 7.35 (2H, d, J = 8.4 Hz), 7.38 (2H, d, J = 8.8 Hz), 7.61 (2H, d, J = 6.4 Hz), 8.69 (2H, d, J = 6.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.49, 67.77, 114.54, 114.85, 122.49, 122.62, 128.95, 129.05, 129.97, 131.07, 133.84, 146.27, 150.53, 158.65, 160.29, 166.71. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₁₈N₂O₂Cl: 377.1057, found: 377.1049.

4-(4-Chlorophenyl)-5-methyl-3-(4-(quinolin-2-ylmethoxy)phenyl)isoxazole (34)

It was synthesized from compound 10 using 2-(chloromethyl)quinoline.HCl according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 60% EtOAc in Hexane). Yield 49.0%; mp 164.7–165.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.41 (3H, s), 5.38 (2H, s), 6.97 (2H, d, J = 8.4 Hz), 7.10 (2H, d, J = 8.4 Hz), 7.32–7.36 (4H, m), 7.53–7.56 (1H, m), 7.64 (1H, d, J = 7.8 Hz), 7.71–7.75 (1H, m), 7.82 (1H, d, J = 8.8 Hz), 8.07 (1H, d, J = 8.8 Hz), 8.19 (1H, d, J = 8.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.55, 71.27, 114.53, 115.02, 119.08, 121.76, 126.64, 127.59, 127.70, 128.88, 129.01, 129.83, 129.89, 131.07, 133.71, 137.14, 147.44, 157.40, 159.42, 160.52, 166.55. HRMS (m/z) [M + H]⁺ calcd for C₂₆H₂₀N₂O₂Cl: 427.1213, found: 427.1205.

4-(4-Chlorophenyl)-5-methyl-3-(4-((2-methylpyridin-3-yl)methoxy)phenyl)isoxazole (35)

It was synthesized from compound 10 using 3-(chloromethyl)-2-methylpyridine according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 20% MeOH in DCM). Yield 80.0%; mp 107.2–108.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 2.58 (3H, s), 5.04 (2H, s), 6.93 (2H, d, J = 8.8 Hz), 7.12 (2H, d, J = 8.8 Hz), 7.16 (1H, dd, J = 8.0, 5.2 Hz), 7.34–7.39 (4H, m), 7.71 (1H, dd, J = 8.0 Hz, 1.2 Hz), 8.47 (1H, dd, J = 5.2 Hz, 1.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.56, 21.90, 67.36, 114.54, 114.81, 121.32, 121.90, 129.03, 129.86, 130.03, 131.10, 133.75, 135.84, 148.51, 156.58, 159.41, 160.48, 166.63. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₀N₂O₂Cl: 391.1213, found: 391.1205.

4-(4-Chlorophenyl)-5-methyl-3-(4-((3-methylpyridin-2-yl)methoxy)phenyl)isoxazole (36)

It was synthesized from compound 10 using 2-(chloromethyl)-3-methylpyridine according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 60% EtOAc in Hexane). Yield 62.0%; mp 138.7–141.2 °C. ¹H NMR (400 MHz, DMSO): δ_H 2.33 (3H, s), 2.39 (3H, s), 5.17 (2H, s), 7.05 (2H, d, J = 8.8 Hz), 7.22–7.30 (5H, m), 7.46 (2H, d, J = 8.0 Hz), 7.63 (1H, d, J = 7.6, 1.2 Hz) 8.36 (1H, dd, J = 4.8 Hz, 1.2 Hz). ¹³C NMR (100 MHz, DMSO): δ_C 11.20, 17.47, 70.32, 113.94, 114.94, 120.88, 123.64, 128.82, 128.84, 129.39, 131.42, 132.54, 132.98, 138.24, 146.21, 153.72, 159.50, 160.05, 166.92. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₀N₂O₂Cl: 391.1213, found: 391.1201.

4-(4-Chlorophenyl)-5-methyl-3-(4-((6-methylpyridin-2-yl)methoxy)phenyl)isoxazole (37)

It was synthesized from compound 10 using 2-(bromomethyl)-6-methylpyridine according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 60% EtOAc in Hexane). Yield 61.0%; mp 126.1–127.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.41 (3H, s), 2.57 (3H, s), 5.16 (2H, s), 6.92 (2H, d, J = 8.4 Hz), 7.08 (1H, d, J = 8.0 Hz), 7.10 (2H, d, J = 8.4 Hz), 7.29 (1H, d, J = 8.0 Hz), 7.34 (4H, d, J = 8.4 Hz), 7.59 (1H, t, J = 8.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.50, 24.20, 70.52, 114.53, 114.96, 118.28, 121.69, 122.42, 128.99, 129.08, 129.77, 131.07, 133.73, 137.20, 156.05, 157.94, 159.48, 160.55, 166.49. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₀N₂O₂Cl: 391.1213, found: 391.1205.

4-(4-Chlorophenyl)-3-(4-((6-chloropyridin-3-yl)methoxy)phenyl)-5-methylisoxazole (38)

It was synthesized from compound 10 using 2-chloro-(5-chloromethyl)pyridine according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 60% EtOAc in Hexane). Yield 70.0%; mp 168.2–170.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.05 (2H, s), 6.90 (2H, d, J = 8.4 Hz), 7.10 (2H, d, J = 8.4 Hz), 7.34–7.38 (5H, m), 7.73 (1H, dd, J = 8.0 Hz, 2.4 Hz), 8.44 (1H, d, J = 2.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.51, 66.72, 114.54, 114.85, 122.18, 124.29, 128.99, 129.03, 129.89, 131.08, 131.18, 133.79, 138.04, 148.67, 151.27, 159.09, 160.41, 166.63. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₁₇N₂O₂Cl₂: 411.0667, found: 411.0681.

4-(4-Chlorophenyl)-3-(4-((3,5-dimethylisoxazol-4-yl)methoxy)phenyl)-5-methylisoxazole (39)

It was synthesized from compound 10 using 4-(chloromethyl)-3,5-dimethylisoxazole according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 60% EtOAc in Hexane). Yield 87.0%; mp 138.9–141.3 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.28 (3H, s), 2.40 (3H, s), 2.42 (3H, s), 4.79 (2H, s), 6.89 (2H, d, J = 8.4 Hz), 7.12 (2H, d, J = 8.8 Hz), 7.35–7.38 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 10.14, 11.15, 11.56, 59.47, 109.99, 114.53, 114.79, 121.96, 129.01, 129.04, 129.84, 131.10, 133.76, 159.30, 159.68, 160.46, 166.66, 167.56. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₂₀N₂O₃Cl: 395.1162, found: 395.1153.

4-(4-Chlorophenyl)-3-(4-((1,3-dimethyl-1H-pyrazol-5-yl)methoxy)phenyl)-5-methylisoxazole (40)

It was synthesized from compound 10 using 5-(chloromethyl)-1,3-dimethylisoxazole according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 70% EtOAc in Hexane). Yield 73.0%; mp 156.4–157.9 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.25 (3H, s), 2.42 (3H, s), 3.83 (3H, s), 4.98 (2H, s), 6.09 (1H, s), 6.90 (2H, d, J = 9.2 Hz), 7.11 (2H, d, J = 8.8 Hz), 7.34–7.37 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.55, 13.35, 36.44, 60.48, 106.82, 114.53, 114.79, 122.07, 128.99, 129.03, 129.83, 131.09, 133.76, 137.51, 147.29, 158.99, 160.44, 166.64. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₂₁N₃O₂Cl: 394.1322, found: 394.1320.

4-(4-Chlorophenyl)-3-(4-((1-isopropyl-1H-imidazol-5-yl)methoxy)phenyl)-5-methylisoxazole (41)

It was synthesized from compound 10 using 2-(chloromethyl)-1-isopropyl-1H-imidazole according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 15% MeOH in DCM). Yield 69.0%; mp 168.4–170.7 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 1.44 (6H, d, J = 6.8 Hz), 2.41 (3H, s), 4.56-4.59 (1H, m), 5.18 (2H, s), 6.98 (2H, d, J = 8.4 Hz), 7.03 (1H, d, J = 1.2 Hz), 7.06 (1H, d, J = 1.2 Hz), 7.10 (2H, d, J = 8.4 Hz) 7.34–7.36 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.54, 23.76, 48.12, 62.29, 114.54, 114.90, 116.85, 121.95, 127.87, 128.99, 129.03, 129.80, 131.09, 133.76, 141.78, 158.99, 160.46, 166.59. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₃N₃O₂Cl: 408.1479, found: 408.1469.

4-(4-Chlorophenyl)-5-methyl-3-(4-((5-(trifluoromethyl)furan-2-yl)methoxy)phenyl) isoxazole (42)

It was synthesized from compound 10 using 2-(bromomethyl)-5-(trifluoromethyl)furan according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 97.0%; mp 105.7–106.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.42 (3H, s), 5.03 (2H, s), 6.49 (1H, d, J = 3.6 Hz), 6.78 (1H, d, J = 3.6 Hz), 6.91 (2H, d, J = 9.2 Hz), 7.11 (2H, d, J = 8.4 Hz), 7.34–7.38 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.50, 62.04, 110.38, 112.34 (q, ³J_C-F = 3.2 Hz), 114.56, 114.89, 118.88 (q, ¹J_C-F = 265.4 Hz), 122.21, 129.02, 129.84, 131.07, 133.78, 138.43, 142.19 (q, ²J_C-F = 42.3 Hz), 152.75, 158.96, 160.45, 166.59. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₁₆NO₃ClF₃: 434.0771, found: 434.0741.

5-Methyl-4-(2-chlorophenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (44)

It was synthesized from compound 43a using 2-chlorophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 42.0%; mp 103.8–104.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.33 (3H, s), 2.35 (3H, s), 5.00 (2H, s), 6.89 (2H, d, J = 8.8 Hz), 7.18–7.38 (9H, m), 7.50 (1H, dd, J = 8.4, 0.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.68, 18.87, 68.53, 113.13, 114.79, 121.92, 126.03, 127.05, 128.34, 128.59, 129.01, 129.75, 129.82, 129.98, 130.40, 132.58, 134.45, 134.98, 136.66, 159.84, 160.70, 167.46. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁ClNO₂: 390.1261, found: 390.1249.

5-Methyl-4-(4-fluorophenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (45)

It was synthesized from compound 43a using 4-fluorophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 31.0%; mp 119.0–120.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.42 (3H, s), 5.03 (2H, s), 6.93 (2H, d, J = 8.8 Hz), 7.08 (2H, t, J = 8.8 Hz), 7.14–7.28 (5H, m), 7.35–7.39 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.50, 18.87, 68.57, 114.65, 114.79, 115.82 (d, ²J_C-F = 21.4 Hz), 121.55, 126.05, 126.55 (d, ⁴J_C-F = 3.8 Hz), 128.38, 128.62, 129.71, 130.43, 131.55 (d, ³J_C-F = 8.4 Hz), 134.41, 136.69, 159.86, 160.64, 162.28 (d, ¹J_C-F = 245.4 Hz), 166.44. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁FNO₂: 374.1556, found: 374.1559.

5-Methyl-4-(4-methoxyphenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (46)

It was synthesized from compound 43a using 4-methoxyphenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 43.0%; mp 121.3–122.7 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.36 (3H, s), 2.41 (3H, s), 3.84 (3H, s), 5.02 (2H, s), 6.92 (2H, d, J = 9.2 Hz), 6.93 (2H, d, J = 8.8 Hz), 7.11 (2H, d, J = 8.8 Hz), 7.19–7.26 (3H, m), 7.38–7.42 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 15.44, 22.81, 59.17, 72.49, 118.12, 118.64, 110.09, 125.85, 126.65, 129.98, 132.29, 132.55, 133.65, 134.35, 134.96, 138.42, 140.62, 163.00, 163.69, 164.62, 170.13. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₄NO₃: 386.1756, found: 386.1753.

5-Methyl-4-(4-methylphenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (47)

It was synthesized from compound 43a using 4-methylphenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 34.0%; mp 130.8–131.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.39 (3H, s), 2.42 (3H; s), 5.03 (2H, s), 6.93 (2H, d, J = 8.8 Hz), 7.08 (2H, d, J = 8.4 Hz), 7.18–7.28 (5H, m), 7.38–7.43 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.53, 18.88, 21.26, 68.55, 114.69, 115.44, 121.90, 126.04, 127.53, 128.35, 128.62, 129.42, 129.71, 129.74, 130.41, 134.49, 136.68, 137.36, 159.76, 160.69, 166.28. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₄NO₂: 370.1807, found: 370.1796.

5-Methyl-4-(4-cyanophenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (48)

It was synthesized from compound 43a using 4-cyanophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 34.0%; mp 135.3–137.1 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.47 (3H, s), 5.04 (2H, s), 6.95 (2H, d, J = 8.8 Hz), 7.19–7.33 (7H, m), 7.33–7.39 (1H, m), 7.67 (2H, d, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.74, 18.89, 68.63, 111.46, 114.28, 114.98, 118.53, 120.87, 126.07, 128.45, 128.62, 129.83, 130.35, 130.47, 132.50, 134.29, 135.68, 136.69, 160.10, 160.60, 167.08. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₁N₂O₂: 381.1603, found: 381.1600.

5-Methyl-4-(3-trifluoromethylphenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (49)

It was synthesized from compound 43a using 3-(trifluoromethyl)phenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 41.0%; mp 120.7–122.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.36 (3H, s), 2.46 (3H, s), 5.03 (2H, s), 6.94 (2H, d, J = 8.8 Hz), 7.19–7.28 (3H, m), 7.33–7.39 (4H, m), 7.48–7.52 (2H, m), 7.62 (1H, d, J = 8.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.60, 18.85, 68.61, 114.42, 114.90, 121.15, 123.84 (q, ¹J_C-F = 271.2 Hz), 124.43 (q, ³J_C-F = 3.6 Hz), 126.05, 126.41 (q, ³J_C-F = 3.8 Hz), 128.39, 128.60, 129.23, 129.75, 130.43, 131.23 (q, ²J_C-F = 32.1 Hz), 131.60, 133.20, 134.37, 136.68, 160.00, 160.60, 166.88. HRMS (m/z) [M + H]⁺ calcd for C₂₅H₂₁F₃NO₂: 424.1524, found: 424.1523.

5-Methyl-4-(3-fluoromethylphenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (50)

It was synthesized from compound 43a using 3-fluorophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 43.0%; mp 89.3–91.2 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.44 (3H, s), 5.03 (2H, s), 6.89-6.99 (4H, m), 7.05 (1H, td, J = 8.4, 2.4 Hz), 7.20–7.28 (3H, m), 7.32–7.40 (4H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.58, 18.88, 68.58, 114.66 (d, ²J_C-F = 20.5 Hz), 114.83, 116.74 (d, ²J_C-F = 21.4 Hz), 121.35, 125.63, 125.64, 126.05, 128.38, 128.64, 129.74, 130.27 (d, ³J_C-F = 8.3 Hz), 130.42, 132.81 (d, ³J_C-F = 8.4 Hz), 134.41, 136.70, 159.93, 160.61, 162.79 (d, ¹J_C-F = 245.4 Hz), 166.70. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁FNO₂: 374.1556, found: 374.1563.

5-Methyl-4-(4-aminophenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (51)

5-Methyl-4-(4-nitrophenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole was synthesized from compound 43a using 4-nitrophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 24.0%; mp 119.4–121.1 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.29 (3H, s), 2.46 (3H, s), 5.08 (2H, s), 7.06 (2H, d, J = 8.4 Hz), 7.16–7.29 (5H, m), 7.38 (1H, d, J = 7.2 Hz), 7.50 (2H, d, J = 8.8 Hz), 8.25 (2H, d, J = 8.8 Hz). HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁N₂O₄: 401.1501, found: 401.1493. In the next step, compound 51 was synthesized from the intermediate nitro product according to synthesis method 5. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 71.0%; mp 149.5–151.3 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.41 (3H, s), 5.03 (2H, s), 6.74 (2H, d, J = 8.8 Hz), 6.93 (2H, d, J = 8.8 Hz), 7.00 (2H, d, J = 8.8 Hz), 7.21–7.27 (3H, m), 7.39–7.42 (1H, m), 7.43 (2H, d, J = 8.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.51, 18.89, 68.55, 114.66, 115.38, 115.68, 120.97, 122.07, 126.05, 128.35, 128.63, 129.71, 130.42, 130.90, 134.51, 136.69, 144.98, 159.71, 160.70, 166.04. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₃N₂O₂: 371.1760, found: 371.1759.

5-Methyl-4-(2,4-difluoromethylphenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (52)

It was synthesized from compound 43a using 2,4-difluorophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 34.0%; mp 116.1–117.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.39 (3H, s), 5.03 (2H, s), 6.90-6.96 (4H, m), 7.10–7.15 (1H, m), 7.20–7.27 (3H, m), 7.35–7.40 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.61 (d, ⁵J_C-F = 1.9 Hz), 18.88, 68.58, 104.63 (t, ²J_C-F = 25.3 Hz), 108.60, 111.83 (dd, ²J_C-F = 21.8, ⁴J_C-F = 3.8 Hz), 114.43 (dd, ²J_C-F = 21.8, ⁴J_C-F = 3.8 Hz), 114.86, 121.58, 126.06, 128.40, 128.63, 129.21, 130.44, 133.01 (dd, ³J_C-F = 9.6, ³J_C-F = 3.8 Hz), 134.40, 136.69, 159.94, 160.29 (d, ¹J_C-F = 248.4 Hz, ³J_C-F = 11.8 Hz), 160.94, 162.84 (dd, ¹J_C-F = 248.8, ³J_C-F = 11.5 Hz), 167.74. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₀FNO₂: 392.1462, found: 392.1468.

5-Methyl-4-(2,4-dichlorophenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (53)

It was synthesized from compound 43a using 2,4-dichlorophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 40.0%; mp 98.0–98.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.33, (3H, s), 2.35, (3H, s), 5.01 (2H, s), 6.91, (2H, d, J = 8.8 Hz), 7.13 (1H, d, J = 8.0 Hz), 7.18–7.26 (3H, m), 7.28 (1H, dd, J = 8.0, 2.4 Hz), 7.32 (2H, d, J = 8.8 Hz), 7.36–7.38 (1H, m), 7.53 (1H, d, J = 2.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.66, 18.87, 68.55, 112.15, 114.89, 121.61, 126.05, 127.52, 128.37, 128.45, 128.59, 128.98, 129.93, 130.42, 133.25, 134.39, 135.04, 135.74, 136.67, 159.94, 160.65, 167.63. HRMS (m/z) [M + H]⁺ calc for C₂₄H₂₀Cl₂NO₂: 424.0871, found: 424.0870.

5-Methyl-3-(4-((2-methylbenzyl)oxy)phenyl)-4-(5-methylpyridin-2-yl)isoxazole (54)

It was synthesized from compound 43a using 1-(5-methylpyridin-2-yl)acetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 10% MeOH in DCM). Yield 52.0%; mp 121.4–122.3 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (6H, s), 2.57 (3H, s), 5.04 (2H, s), 6.94-6.99 (3H, m), 7.20–7.28 (3H, m), 7.38–7.44 (4H, m), 8.52 (1H, d, J = 1.6 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 12.09, 18.26, 18.88, 68.60, 114.80, 115.01, 121.71, 124.19, 126.05, 128.38, 128.63, 129.98, 130.43, 131.76, 134.45, 136.69, 137.06, 147.64, 150.11, 156.91, 160.81, 168.61. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₃N₂O₂: 371.1760, found: 371.1769.

5-Methyl-3-(4-((2-methylbenzyl)oxy)phenyl)-4-(pyridin-4-yl)isoxazole (55)

It was synthesized from compound 43a using 4-(pyridyl)acetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 30.0%; mp 128.5–129.3 °C. ¹H NMR (CDCl₃): δ_H 2.37 (3H, s), 2.50 (3H, s), 5.04 (2H, s), 6.96 (2H, d, J = 8.6 Hz), 7.13 (2H, d, J = 6.2 Hz), 7.19–7.28 (3H, m), 7.34 (2H, d, J = 8.6 Hz), 7.38–7.40 (1H, m), 8.62 (2H, d, J = 6.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.79, 18.88, 68.63, 113.36, 114.99, 120.81, 124.35, 126.07, 128.44, 128.64, 129.86, 130.46, 134.29, 136.70, 139.25, 149.96, 160.13, 160.62, 167.40. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₁N₂O₂: 357.1603, found: 357.1593.

4-(4-Chlorophenyl)-3-(2-fluoro-4-((2-methylbenzyl)oxy)phenyl)-5-methylisoxazole (56)

It was synthesized from compound 43b using 4-chlorophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 20.0%; mp 96.1–98.3 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.49 (3H, s), 5.03 (2H, s), 6.68 (1H, dd, J = 11.6 Hz, 2.4 Hz), 6.82 (1H, dd, J = 8.4 Hz, 2.4 Hz), 7.07 (2H, d, J = 8.4 Hz), 7.35–7.38 (7H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.68, 18.88, 69.02, 102.95 (d, ²J_C-F = 25.1 Hz), 109.43 (d, ²J_C-F = 15.3 Hz), 111.07 (d, ⁴J_C-F = 3.0 Hz), 115.93, 126.12, 128.61, 128.71, 128.88, 130.04, 130.52, 131.69 (d, ³J_C- F = 4.6 Hz), 133.45, 133.83, 136.76, 157.60, 160.67 (d, ¹J_C-F = 249.9 Hz), 161.46 (d, ³J_C-F = 10.7 Hz), 166.13. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₀ClNO₂F: 408.1167, found: 408.1180.

4-(4-Chlorophenyl)-3-(3-fluoro-4-((2-methylbenzyl)oxy)phenyl)-5-methylisoxazole (57)

It was synthesized from compound 43c using 4-chlorophenylacetone according to synthesis method 4. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 24.0%; mp 108.0–110.2 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.39 (3H, s), 2.43 (3H, s), 5.12 (2H, s), 6.96–7.00 (1H, m), 7.10–7.14 (3H, m), 7.22–7.29 (4H, m), 7.37–7.41 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.53, 18.88, 69.88, 114.54, 115.19 (d, ⁴J_C-F = 1.9 Hz), 116.29 (d, ²J_C-F = 20.5 Hz), 122.13 (d, ³J_C-F = 7.1 Hz), 124.54 (d, ³J_C-F = 3.9 Hz), 126.06, 128.53, 128.59, 128.70, 129.16, 130.47, 131.10, 133.93, 133.98, 136.72, 147.94 (d, ²J_C-F = 10.2 Hz), 152.54 (d, ¹J_C-F = 245.6 Hz), 159.69 (d, ⁴J_C-F = 1.9 Hz), 166.95. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₀ClNO₂F: 408.1167, found: 408.1169.

4-(4-(4-Chlorophenyl)-5-methylisoxazol-3-yl)-N-(2-methylbenzyl)aniline (59)

It was synthesized from compound 58 according to synthesis method 1c. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 31.0%; mp 134.9–137.0 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.35 (3H, s), 2.40 (3H, s), 4.27 (2H, s), 6.58 (2H, d, J = 8.8 Hz), 7.14 (2H, d, J = 8.4 Hz), 7.17–7.30 (6H, m), 7.35 (2H, d, J = 8.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.54, 18.94, 46.33, 112.74, 114.35, 126.21, 127.69, 128.39, 128.93, 129.46, 129.52, 130.49, 131.18, 133.55, 136.01, 136.40, 148.68, 160.84, 157.60, 166.27. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₂N₂OCl: 389.1421, found: 389.1418.

4-(4-Chlorophenyl)-3-methyl-5-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (60)

Compound 60 was prepared as described previously.⁸ mp 153.8-154.9 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.23, (3H, s),2.37 (3H, s), 5.04 (2H, s), 6.94 (2H, d, J = 8.8 Hz), 7.20–7.29 (5H, m), 7.37–7.39 (1H, m), 7.42 (2H, d, J = 8.0 Hz), 7.47 (2H, d, J = 8.8 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 10.62, 18.89, 68.65, 113.80, 114.97, 120.49, 126.09, 128.44, 128.49, 128.62, 129.37, 129.41, 130.49, 131.25, 134.06, 134.20, 136.70, 159.80, 160.10, 164.59. CAS: 1884212-37-6.

4-(4-Chlorophenyl)-5-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (63)

It was synthesized from compound 62 using 2-methylbenzyl bromide according to synthesis method 1b. The resulting crude product was purified by flash column chromatography (0% → 20% EtOAc in Hexane). Yield 47.0%; mp 115.3–117.3 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.38 (3H, s), 5.07 (2H, s), 7.00 (2H, d, J = 8.8 Hz), 7.23–7.28 (3H, m), 7.32 (2H, d, J = 8.8 Hz), 7.36–7.40 (3H, m), 7.56 (2H, d, J = 8.8 Hz), 8.31 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.89, 68.70, 113.93, 115.11, 120.10, 126.11, 128.53, 128.65, 128.85, 128.88, 129.26, 129.93, 130.51, 133.91, 134.15, 136.72, 151.55, 160.37, 164.36. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₉ClNO₂: 376.1104, found: 376.1102.

4-(4-Chlorophenyl)-3-(4-((2-methylbenzyl)oxy)phenyl)isoxazole-5-amine (65)

Compound 64 (1.0 equiv) and NH₂OH.HCl (1.5 equiv) were dissolved in ethanol and heated under reflux for 4.5 h. The reaction mixture was cooled, and the precipitate was filtered under vacuum and dried. The resulting crude product was purified by flash column chromatography (0% → 50% EtOAc in Hexane). Yield 75.0%; mp 163.1–165.6 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 5.03 (2H, s), 6.94 (2H, d, J = 8.8 Hz), 7.14 (2H, d, J = 8.4 Hz), 7.19–7.29 (3H, m), 7.33 (2H, d, J = 8.4 Hz), 7.36–7.39 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.88, 68.59, 93.16, 114.80, 121.57, 126.05, 128.39, 128.64, 129.25, 129.32, 129.70, 130.43, 130.54, 132.91, 134.40, 136.70, 159.98, 161.38, 165.48. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₀ClN₂O₂: 391.1213, found: 391.1207.

3-(4-Chlorophenyl)-5-methyl-4-(4-((2-methylbenzyl)oxy)phenyl)isoxazole (67)

3-(4-Chlorophenyl)-5-methyl-4-(4-((2-methylbenzyl)oxy)-phenyl)-4,5-dihydroisoxazol-5-ol was synthesized from compound 66 using 4-chlorophenylacetone according to method 4. Yield 18.0%. HRMS (m/z) [M + H]+ calcd for C₂₄H₂₃ClNO₃: 408.1366, found: 408.1360. To the solution of obtained isoxazoline intermediate (0.2 mmol) in methanol:water (4 mL:2 mL) was added Na₂CO₃ (0.41 mmol) and stirred at 90 °C under reflux for 2.5 h. At the end of the period, methanol was evaporated and extracted with ethyl acetate, and the organic layer was dried with anhydrous Na₂SO₄. Ethyl acetate was evaporated under reduced pressure. The resulting crude product was purified by flash column chromatography (0% → 10% EtOAc in Hexane). Yield 76.0%; mp 84.5–86.5 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.41 (3H, s), 2.43 (3H, s), 5.06 (2H, s), 7.01 (2H, d, J = 8.8 Hz), 7.09 (2H, d, J = 8.8 Hz), 7.22–7.32 (5H, m), 7.38–7.44 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 11.52, 18.91, 68.65, 115.13, 115.28, 122.39, 126.08, 127.78, 128.43, 128.70, 128.76, 129.68, 130.47, 130.96, 134.49, 135.42, 136.73, 158.57, 160.16, 166.65. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁ClNO₂: 390.1261, found: 390.1251.

1-(4-Chlorophenyl)-3-methyl-5-(4-((2-methylbenzyl)oxy)phenyl)-1H-pyrazole (69)

It was synthesized from compound 68 using 4-chlorophenylhydrazine.HCl according to synthesis method 8c. The resulting crude product was purified by preparative column chromatography (0% → 60% ACN in Water). Yield 54.0%; mp 111.8–113.6 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.38 (3H, s), 5.03 (2H, s), 6.25 (1H, s), 6.93 (2H, d, J = 8.8 Hz), 7.14 (2H, d, J = 8.8 Hz), 7.21–7.30 (7H, m), 7.38–7.41 (1H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 13.52, 18.89, 68.63, 107.68, 114.83, 123.04, 126.06, 126.16, 128.43, 128.65, 128.98, 129.96, 130.46, 132.59, 134.38, 136.70, 138.68, 143.61, 149.71, 158.96. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₂ClN₂O: 389.1421, found: 389.1413.

4-(4-Chlorophenyl)-5-(4-((2-methylbenzyl)oxy)phenyl)-1H-pyrazole (71)

It was synthesized from compound 70 using hydrazine hydrate according to synthesis method 8b. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 88.0%; mp 127.8–129.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.38 (3H, s), 5.05 (2H, s), 6.98 (2H, d, J = 8.4 Hz), 7.22–7.30 (7H, m), 7.36–7.41 (3H, m), 7.65 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.90, 68.65, 115.10, 118.41, 123.08, 126.07, 128.42, 128.64, 128.97, 129.48, 129.58, 130.46, 131.51, 132.50, 134.42, 135.57, 136.69, 142.98, 159.24. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₀ClN₂O: 375.1264, found: 375.1266.

4-(4-Chlorophenyl)-1-methyl-3-(4-((2-methylbenzyl)oxy)phenyl)-1H-pyrazole (72)

It was synthesized from compound 70 using methylhydrazine according to synthesis method 8b. The resulting crude product was purified by flash column chromatography (0% → 50% EtOAc in Hexane). Yield 56.0%; mp 142.2–143.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.41 (3H, s), 3.78 (3H, s), 5.08 (2H, s), 7.06 (2H, d, J = 8.8 Hz), 7.11 (2H, d, J = 8.8 Hz), 7.18 (2H, d, J = 8.8 Hz), 7.21 (2H, d, J = 8.8 Hz), 7.23–7.31 (3H, m), 7.42–7.44 (1H, m), 7.70 (1H, s). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.92, 37.21, 68.70, 115.28, 119.81, 122.30, 126.10, 128.52, 128.59, 128.72, 130.50, 131.36, 131.76, 131.77, 134.32, 136.75, 137.19, 139.89, 159.41. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₂ClN₂O: 389.1421, found: 389.1429.

5-(4-Chlorophenyl)-2-methyl-4-(4-((2-methylbenzyl)oxy)phenyl)thiazole (75)

It was synthesized from compound 74 using 2-methylbenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 52.0%; mp 90.5–92.1 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37, (3H, s), 2.73 (3H, m), 5.03 (2H, s), 6.91 (2H, d, J = 8.8 Hz), 7.21–7.30 (7H, m), 7.39–7.43 (3H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.89, 19.20, 68.55, 114.70, 126.01, 127.37, 128.28, 128.61, 128.93, 129.75, 130.25, 130.38, 130.77, 130.91, 133.77, 134.65, 136.65, 149.57, 158.65, 163.98. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₁ClNOS: 406.1032, found: 406.1028.

5-(4-Chlorophenyl)-1-(4-((2-methylbenyl)oxy)phenyl)-1H-pyrazole (78)

It was synthesized from compound 76 according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 85.0%; mp 145.2–146.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.38 (3H, s), 5.04 (2H, s), 6.50 (1H, d, J = 2.0 Hz), 6.96 (2H, d, J = 9.2 Hz), 7.17 (2H, d, J = 8.8 Hz), 7.20–7.26 (5H, m), 7.28 (2H, d, J = 8.8 Hz), 7.39–7.41 (1H, m), 7.70 (1H, d, J = 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.89, 68.87, 107.44, 115.11, 126.06, 126.66, 128.43, 128.63, 128.74, 128.95, 129.92, 130.45, 133.14, 134.23, 134.32, 136.68, 139.91, 141.81, 158.31. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₀ClN₂O: 375.1264, found: 375.1262.

5-(4-Chlorophenyl)-3-methyl-1-(4-((2-methylbenyl)oxy)phenyl)-1H-pyrazole (79)

It was synthesized from compound 77 using 2-methylbenzyl bromide according to synthesis method 1a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 83.0%; mp 103.4–105.2 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.37 (3H, s), 2.38 (3H, s), 5.03 (2H, s), 6.29 (1H, s), 6.94 (2H, d, J = 8.4 Hz), 7.15 (2H, d, J = 8.4 Hz), 7.18 (2H, d, J = 8.4 Hz), 7.21–7.28 (5H, m), 7.39 (1H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 13.48, 18.89, 68.85, 107.25, 115.11, 126.04, 126.63, 128.38, 128.59, 128.68, 129.10, 129.78, 130.43, 133.24, 134.06, 134.38, 136.65, 142.49, 149.13, 158.10. HRMS (m/z) [M + H]⁺ calcd for C₂₄H₂₂ClN₂O: 389.1421,found: 389.1416.

1-(4-Chlorophenyl)-5-(4-((2-methylbenzyl)oxy)phenyl)-1H-pyrazole (85)

It was synthesized from compound 80 using 4-chlorophenylhydrazine.HCl according to synthesis method 8a. The resulting crude product was purified by flash column chromatography (0% → 20% EtOAc in Hexane). Yield 61.0%; mp 146.0–147.9 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.38 (3H, s), 5.04 (2H, s), 6.45 (1H, d, J = 2.0 Hz), 6.94 (2H, d, J = 8.8 Hz), 7.16 (2H, d, J = 8.8 Hz), 7.21–7.29 (5H, m), 7.32 (2H, d, J = 8.8 Hz), 7.38–7.41 (1H, m), 7.71 (1H, d, J = 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.90, 68.65, 107.72, 114.88, 122.87, 126.08, 126.27, 128.45, 128.66, 129.05, 130.08, 130.47, 132.98, 134.36, 136.70, 138.68, 140.49, 142.91, 159.04. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₀ClN₂O: 375.1264, found: 375.1263. CAS: 2750233-50-0.

2-(1-(4-Chlorophenyl)-1H-pyrazol-5-yl)-5-((2-methylbenzyl)oxy)pyridine (86)

It was synthesized from compound 81 using 4-chlorophenylhydrazine.HCl according to synthesis method 8a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 84.0%; mp 108.4–110.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.38, (3H, s), 5.09 (2H, s), 6.68 (1H, d, J = 1.4 Hz), 7.17 (1H, d, J = 8.8 Hz), 7.21–7.34 (8H, m), 7.38 (1H, d, J = 7.6 Hz), 7.72 (1H, d, J = 1.4 Hz), 8.34 (1H, d, J = 2.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.90, 69.11, 108.55, 121.68, 124.11, 126.16, 126.38, 128.72, 128.80, 128.97, 130.61, 133.12, 133.58, 136.76, 137.99, 139.06, 140.53, 141.91, 142.00, 154.36. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₁₉ClN₃O: 376.1217, found: 376.1207.

5-(1-(4-Chlorophenyl)-1H-pyrazol-5-yl)-2-((2-methylbenzyl)oxy)pyridine (87)

It was synthesized from compound 82 using 4-chlorophenylhydrazine.HCl according to synthesis method 8a. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 37.0%; mp 126.5–127.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.41 (3H, s), 5.38 (2H, s), 6.50 (1H, d, J = 1.8 Hz), 6.74 (1H, d, J = 8.8 Hz), 7.22- 7.28 (5H, m), 7.33–7.37 (3H, m), 7.42–7.44 (1H, m), 7.73 (1H, d, J = 1.8 Hz), 8.14 (1H, d, J = 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.98, 66.45, 108.07, 111.13, 119.83, 125.96, 126.33, 128.32, 129.09, 129.31, 130.35, 133.44, 134.77, 137.01, 138.35, 138.77, 139.90, 140.74, 146.58, 163.49. HRMS (m/z) [M + H]⁺ calcd for C₂₂H₁₉ClN₃O: 376.1217, found: 376.1213.

1-(4-Chlorophenyl)-5-(2-fluoro-4-((2-methylbenzyl)oxy)phenyl)-1H-pyrazole (88)

It was synthesized from compound 83 using 4-chlorophenylhydrazine.HCl according to synthesis method 8a. The resulting crude product was purified by flash column chromatography (0% → 50% EtOAc in Hexane). Yield 66.0%; mp 131.8–132.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.39 (3H, s), 5.03 (2H, s), 6.51 (1H, d, J = 2.0 Hz), 6.71 (1H, dd, J = 11.6 Hz, 2.4 Hz), 6.78 (1H, dd, J = 8.4 Hz, 2.4 Hz), 7.13 (1H, dd, J = 8.8, 8.4 Hz), 7.22–7.32 (7H, m), 7.40 (1H, m), 7.75 (1H, d, J = 2.0 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 19.07, 69.23, 103.11 (d, ²J_C-F = 25.1 Hz), 109.51 (d, ⁴J_C-F = 1.5 Hz), 111.12 (d, ²J_C-F = 15.3 Hz), 111.24 (d, ³J_C-F = 3.1 Hz), 125.57, 126.32, 128.84, 128.89, 129.22, 130.74, 131.90 (d, ³J_C-F = 4.6 Hz), 133.12, 133.98, 136.94, 137.06, 139.05, 140.68, 160.23 (d, ¹J_C-F = 248.4 Hz), 160.98 (d, ²J_C-F = 10.6 Hz). HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₉ClFN₂O: 393.1170, found: 393.1172.

1-(4-Chlorophenyl)-5-(3-fluoro-4-((2-methylbenzyl)oxy)phenyl)-1H-pyrazole (89)

It was synthesized from compound 84 using 4-chlorophenylhydrazine.HCl according to synthesis method 8a. The resulting crude product was purified by flash column chromatography (0% → 30% EtOAc in Hexane). Yield 43.0%; mp 95.0–95.4 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.40 (3H, s), 5.12 (2H, s), 6.46 (1H, d, J = 1.6 Hz), 6.88-6.91 (1H, m), 6.96–7.02 (2H, m), 7.20–7.27 (5H, m), 7.32–7.35 (2H, m), 7.40–7.41 (1H, m), 7.71 (1H, d, J = 1.6 Hz).¹³C NMR (100 MHz, CDCl₃): δ_C 19.07, 70.17, 108.23, 115.62 (d, ³J_C-F = 2.0 Hz), 116.93 (d, ²J_C-F = 19.2 Hz), 123.75 (d, ³J_C-F = 7.1 Hz), 125.02 (d, ⁴J_C-F = 3.8 Hz), 126.24, 126.46, 128.76, 128.82, 129.35, 130.68, 133.46, 134.09, 136.92, 138.62, 140.77, 141.91 (d, ⁴J_C-F = 1.9 Hz), 147.18 (d, ²J_C-F = 10.3 Hz), 152.67 (d, ¹J_C-F = 246.2 Hz). HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₉ClFN₂O: 393.1170, found: 393.1173.

N-(4-(1-(4-Chlorophenyl)-1H-pyrazol-5-yl)phenyl)-2-methylbenzamide (91)

o-Toluic acid was dissolved in dichloromethane, then oxalyl chloride and a catalytic amount of DMF were added, and it was stirred for 1 h at rt in a nitrogen atmosphere. At the end of the reaction, the reaction mixture was evaporated. The residue was dissolved in dichloromethane and then DIEA, and compound 90 was added and stirred at rt overnight. It was extracted with dichloromethane; the organic layer was dried with anhydrous Na₂SO₄ and evaporated. The resulting crude product was purified by flash column chromatography (0% → 40% EtOAc in Hexane). Yield 63.0%; mp 179.1–180.0 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.48 (3H, s), 6.48 (1H, d, J = 1.6 Hz), 7.18–7.37 (9H, m), 7.44 (1H, d, J = 7.6 Hz), 7.57 (2H, d, J = 8.4 Hz), 7.69 (1H, d, J = 1.6 Hz), 7.70 (1H, m). ¹³C NMR (100 MHz, CDCl₃): δ_C 19.82, 108.02, 119.74, 125.92, 126.22, 126.31, 126.59, 129.12, 129.48, 130.47, 131.34, 133.15, 136.05, 136.52, 138.22, 138.57, 140.60, 142.55, 168.14. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₁₉ClN₃O: 388.1217, found: 388.1213.

4-(1-(4-Chlorophenyl)-1H-pyrazol-5-yl)-N-(2-methylbenzyl)aniline (92)

It was synthesized from compound 90 according to synthesis method 1c. The resulting crude product was purified by preparative column chromatography (0% → 20% ACN in Water). Yield 32.0%; mp 141.5–142.8 °C. ¹H NMR (400 MHz, CDCl₃): δ_H 2.36 (3H, s), 4.27 (2H, s), 6.39 (1H, d, J = 1.2 Hz), 6.56 (2H, d, J = 8.4 Hz), 7.03 (2H, d, J = 8.8 Hz), 7.18–7.32 (8H, m), 7.67 (1H, d, J = 1.2 Hz). ¹³C NMR (100 MHz, CDCl₃): δ_C 18.93, 46.23, 107.13, 112.54, 119.15, 126.21, 126.25, 127.66, 128.29, 128.94, 129.85, 130.52, 132.69, 136.29, 136.35, 139.00, 140.49, 143.55, 148.07. HRMS (m/z) [M + H]⁺ calcd for C₂₃H₂₁ClN₃: 374.1424, found: 374.1425.

Biological Studies

Cell Culture

HepG2, FOCUS, Hep3B, Mahlavu, Huh7, Hep40, and PLC-PRF-5 hepatocellular carcinoma cells; MCF-7, MDA-MB-231, MDA-MB-468, SKBR3, and ZR-75 breast cancer cells; and normal-like epithelial MCF-10A breast cells were cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Biological Industries-BI) supplemented with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific), 2 mM l-Glutamine (Gibco/Thermo Fisher Scientific), 100 Units/mL Penicillin/Streptomycin, and 0.1 mM non-essential amino acid (Gibco/Thermo Fisher Scientific). SNU475 hepatocellular carcinoma cells were maintained in RPMI (Biological Industries-BI) supplemented with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific), l-Glutamine (Gibco/Thermo Fisher Scientific), and 100 Units/mL Penicillin/Streptomycin, while ZR-75 breast cancer cells were cultured RPMI (Biological Industries-BI) supplemented with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific), 1× sodium pyruvate, and %4.5 glucose. Normal-like MCF-10A breast cells were sustained in DMEM/HAM’S F12 (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Gibco/Thermo Fisher Scientific), EGF (20 ng/mL), Hydrocortisone (0.5 mg/mL), cholera toxin (100 ng/mL), insulin (10 μg/mL), and 100 Units/mL Penicillin/Streptomycin. The cells were cultivated at 37 °C in a humidified incubator under 5% CO₂.

NCI-60 Sulforhodamine B (SRB) Cytotoxicity Assay

Mahlavu, FOCUS, SNU475 (1000 cell/well in 150 μL/well); Huh7, MCF7 (2500 cell/well in 150 μL/well); HepG2, Hep3B, SKBR3 (3000 cell/well in 150 μL/well); PLC-PRF-5, Hep40 (5000 cell/well in 150 μL/well); MDA-MB-231, MDA-MB-468, MCF10A (6000 cell/well in 150 μL/well); and ZR-75 (7500 cell/well in 150 μL/well) were cultured in 96-well plates and were inoculated in an incubator for 24 hours. The compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma, St Louis, MO) as a 20 mM stock solution. The treatment of cells with the compounds was done in a concentration range starting from 40 to 2.5 μM. The compounds which were below 2.5 μM were tested in a concentration range from 2.5 to 0.15 μM. End of the 72h compound treatment, the cells were fixed using 10% (v/v) trichloroacetic acid (MERCK) for an hour and washed with ddH₂O, and left for air-drying. The fixed plates were stained with 50 μL of sulforhodamine B (SRB) solution (Sigma) at RT for 10 min. To remove unbound SRB dye, acetic acid was used to wash cells three times and left for drying. The protein-bound SRB was solubilized with 10 mM Tris-base (Sigma) and their absorbance was measured with a 96-well plate reader at 515 nm wavelength (ELx800, Biotek). The IC₅₀ values of compounds were calculated in comparison with control group DMSO. Data with R² values larger than 0.9 are considered significant.

Real-Time Cell Growth Surveillance by Electronic Sensing (RT-CES)

To monitor the real-time cell growth of cells and cytotoxicity of compounds, the xCELLigence system (Roche Applied Sciences) was used. The cells were seeded onto 96-well E-Plate Mahlavu (1500 cell/well), MCF7, Huh7 (2500 cell/well), MDA-MB-231 (5000 cell/well), and MCF10A (6000 cell/well) in 100 μL of the medium/well. To examine the proliferation of cells, the cellular growth was monitored as cell index (CI) every 30 min for 24 h. When the cells were in a log growth phase, treatment with compounds 11 and 85 in 2.5, 5, and 10 μM concentrations was done, the CI values were recorded every 10 min for 24 h for controlling fast drug response and then every 30 min for the remaining 48 h for long-term dose response. After 72 h of incubation, the cellular growth ratios were calculated by CI_Drug/CI_DMSO.

Cell Death Analysis with Flow Cytometry

Mahlavu, Huh7, MCF7, and MDA-MB-231 were seeded onto six-well plates. After 24 h, the cells were incubated with 5 μM compounds 11 and 85 by keeping DMSO as a negative control. After 48 h incubation, the cells were collected to identify programmed cellular death with Annexin V assay. PI and Annexin V dyes were added onto cell lysates for 15 min at room temperature in the dark to stain the cells for detecting apoptotic cell populations. The cells were analyzed by flow cytometry.

Immunofluorescence Staining for the Detection of Cell Death

Mahlavu, Huh7, MCF7, and MDA-MB-231 cells were cultured onto cover slides in six-well plates for 24 h according to their cell growth rate. The cells were treated with compounds 11 and 85 with their IC₁₀₀ concentrations or DMSO as a negative control for 48 h. The 1× PBS was used to wash the cells three times, and the cells were fixed with 100% ice-cold methanol. Then, the cells were stained with 1 μg/mL Hoechst (#33258, Sigma). The cells were analyzed under the fluorescence microscope (Nikon Eclipse 50i).

Western Blotting

Cells were treated for 5 μM compounds 11 and 85 or control (DMSO). The treated cells were collected with a scraper at end of 48 h incubation and lysates were prepared. Protein concentration was calculated with a BCA assay. The protein levels were analyzed using protein electrophoresis according to the manufacturer’s protocol for near-infrared (NIR) Western Blot analysis (Mini-PROTEAN Tetra Cell Systems, Bio-Rad). The samples (40 μg /well) were loaded onto TGX precast gels, and protein transfer from gel to an LF-PVDF membrane was done using Trans-Blot Turbo System (Bio-Rad). Primary antibodies (PARP, CST, #9532S) and Calnexin (CST, #2679) and secondary antibodies IRDye 680RD Goat Anti Rabbit (Li-Cor, # 92668071) and IRDye- 800CW Goat Anti Rabbit (Licor, # 926e32211) were used for western blotting, and the proteins were monitored in fluorescence system using Odyssey CLx- LICOR imaging system.³²

In Vivo Mouse Xenograft Experiments

Mahlavu cells (10 × 10⁶ cells/mouse) prepared in 150 μL of DMEM were injected subcutaneously to the flank of 6–8 weeks old male athymic nude mice, while MDA-MB-231 cells (2 × 10⁶cells/mouse) in 1:1 DMEM and matrigel were injected into mammary fat pad (MFP) of 6–8 weeks old female athymic nude mice. Tumor volume and mouse weight were measured twice a week using calipers. When the tumor volume reached ∼150 mm³, xenografts were randomized into groups (6 mice per group), and the mice were administered subsequently with 11 or 85 (40 mg/kg in 0.5% hydroxypropyl methylcellulose plus 1% Tween 80) or vehicle by gavage feeding (3–4 days/week) for 4 weeks. The mice were sacrificed 30 days after initiation of the compound treatment and tumors were collected. Two-way ANOVA was performed for statistical significance by GraphPad Prism. All of the mice were maintained under a temperature-controlled environment with a 12 h light/dark cycle and received a standard diet and water ad libitum.

Statistical Analysis

All data were obtained from three independent experiments and standard deviation (S.D) values were accessed. All experiments except western blotting were done two times with n ≥ 3 biological replicates. One-way ANOVA and two-way ANOVA were applied using GraphPad (Prism) for statistical analysis. Results were shown as follows: ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c03405.

• Chemical procedures and experimental data of intermediates, NCI-60 cancer cell panel results for compounds 11 and 85, X-ray data, PK and in vivo test data, and copies of ¹H and ¹³C NMR spectra of final compounds (PDF)

Author Present Address

^∥ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adıyaman University, Adıyaman, Turkey

Author Present Address

^⊥ Department of Medical Biology, Hacettepe University, Ankara 06800, Turkey.

Author Present Address

^# Center for Genomics and Rare Diseases & Biobank for Rare Diseases, Hacettepe University, Ankara 06800, Turkey.

Author Present Address

^∇ Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois 60637, United States.

Author Contributions

The manuscript was written with the contribution of all authors. All authors have approved the final version of the manuscript.

The authors declare no competing financial interest.