Hedgehog (Hh) pathway plays an essential role during vertebrate embryonic development, postnatal tissue regeneration and carcinogenesis, and the Smoothened receptor (Smo) is a key signal transducer in the canonical hedgehog pathway [1]. Aberrant Hh signaling leads to several malignancies, and three Smo antagonists have been approved, including vismodegib and sonidegib for the treatment of basal cell carcinoma and glasdegib for acute myeloid leukemia [2, 3]. Meanwhile, the activation of Hh pathway demonstrates many therapeutic potentials, such as cardiac repair and regeneration [4], neuroprotective effects [5–7], wound repair and fracture healing [8, 9].

Smo is the main target for the development of Hh pathway modulators and Smo antagonists have been thoroughly researched with several published reviews [1, 10–12]. But the development of Smo agonists is relatively lagging behind. The Smo agonists reported in literature (Scheme 1) can be integrated into two types according to the binding site: one is oxidized sterols [20(S)-OHC and Oxy34] that bind to the extracellular cysteine rich domain (CRD) of Smo, and the other type includes synthetic glucocorticoids (halcinonide), GSA-10, SAG, purmorphamine and their analogues, which binds to the seven transmembrane domains (TMD) of Smo [13–16].

Scheme
scheme 1

1.

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To date, several high-resolution structures of human Smo protein have been resolved, including TMD and CRD. Most of these Smo structures are bound to ligands, including antagonists (cyclopamine, SANT-1, LY0940280) and agonists (SAG) in traditional TMD and sterols in CRD, which provided an understanding of possible reasons for the opposite effect (antagonistic or agonistic) of compounds binding to the same pocket of Smo [17–21]. Besides, Rohatgi et al. reported that the transfer of Smo protein to primary cilia undergoes a two-step process: the first step is for non-activated Smo to be transferred to cilia, and the second step is for activation [22]. Compounds can bind to Smo in different states during this two-step process which could partly explain why small molecule compounds binding to TMD of Smo resulted in opposite biological effects [23].

In this article, we tried to discover potential Smo agonists with novel molecular scaffold different from that of reported Smo agonists (oxidized sterols, glucocorticoids, GSA-10, SAG and purmorphamine). And we also tried to interpretate the differences between them and antagonists in binding to Smo in order to provide useful information for the research and development of Smo modulators.

Additionally, considering previous experience [24, 25], the research work of this article was introduced into a medicinal chemistry experimental course to assist undergraduates in understanding the concept and core of medicinal chemistry, a chemistry-based discipline primarily concerning with the invention, discovery, design, preparation, identification and interpretation of biologically active compounds at the molecular level [26]. The course included molecular design, chemical synthesis, biochemical testing, and docking simulation. And the agonistic effect on Hh pathway of synthesized compounds were discussed on the basis of experimental and computational results to enable students to evaluate lead compounds from various aspects.

RESULTS AND DISCUSSION

Molecular design. In our previous work for the development of compounds targeting both Smo and HDAC, a series of derivatives of vismodegib containing hydroxamic acid motif and acetamide linker were obtained, and they differed from vismodegib and other compounds (containing formamide or sulfonamide linker) in downregulating Gli1 expression, and could upregulate the expression of Gli1, which indicated an agonistic effect of compounds containing acetamide linker on the Hh pathway (Scheme 2). We speculated that the acetamide linker was the key structural scaffold responsible for the agonistic effect. However, the hydroxamic acid motif is prone to mutagenicity and genotoxicity [27]. Therefore, in this article, we designed derivatives of vismodegib with acetamide linker and without hydroxamic acid fragments, which was substituted by various functional groups, such as alkoxy, alkyl, halogen, amino and hydroxyl. Benzimidazole is a bioisostere of pyridine and derivatives of vismodegib obtained by replacing the pyridine with benzimidazole could maintain or enhance their antagonistic effect on Smo [28]. Therefore, we introduced benzimidazole motif into target compounds. Meanwhile, alkyl groups were introduced at the α position of the carbonyl group to further investigate the agonistic effect of acetamide linker.

Scheme
scheme 2

2.

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Synthesis. The synthesis of target compounds was carried out via a reported method for the preparation of vismodegib [29]. 2-(2-Chloro-5-nitrophenyl)pyridine 5 was the key intermediate, which was converted to 2-(5-amino-2-chlorophenyl)pyridine 6 via reduction reaction, and then reacted with commercially available substituted phenylacetic acid 7 to afford target compounds (Scheme 3). 2-(2-Chloro-5-nitrophenyl)pyridine 5 was synthesized via the reaction of commercially available 2-chloro-5-nitroacetophenone 4 and 1,2-dihydro-1,3-dimethyl-2-oxopyrimidinium salt 3, which was prepared via the reported method employing 1,1,3,3-tetramethoxypropane and 1,3-dimethylurea 2 as starting materials [30, 31]. In terms of compounds containing benzimidazole motif, 2-(2-chloro-5-nitrophenyl)-1H-benzo[d]imidazole 10 was the key intermediate, which could be converted to the target compounds via the above method. The preparation of intermediate 10 was starting from 2-chloro-5-nitrobenzoic acid 8, which was converted to 2-chloro-5-nitrobenzoyl chloride and then reacted with o-phenylenediamine to afford N-(2-aminophenyl)-2-chloro-5-nitrobenzamide 9, which cyclized in acetic acid to furnish intermediate 10 (Scheme 3).

Scheme
scheme 3

3.

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The expression of Gli1. Initial evaluation of the target compounds focused on determining Hh agonist properties through up-regulation of known pathway target genes [glioma-associated oncogene (GLI1)] in the Hh-dependent NIH 3T3 cell line. Detecting the expression of Gli1 is a universal method for evaluating the effects of compounds on Smo proteins [32, 33]. For these studies, DMSO was used as a baseline control and values for target compounds (10 μM) were presented as a fold induction of mRNA expression over DMSO control. The q-PCR results revealed that many compounds could upregulate the expression of Gli1, but there was no significant difference compared with the control group (Fig. 1, Table 1). Vismodegib downregulated Gli1 expression by approximately 50%. XH-5, XH-7, XH-11 and XH-12 upregulate Gli1 expression by approximately 30%. XH-16 and XH-17, with the strongest agonistic effect, upregulated the expression of Gli1 by more than 50%.

Fig. 1.
figure 1

The expression of Gli1.

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Cytotoxicity. The target compounds, as Hh pathway agonists, should have low cytotoxicity due to their potential therapeutic applications including heart failure, chronic degenerative diseases, etc. In vitro cytotoxicity against A549 (lung cancer) and the MDA-MB-231 (breast cancer) cell lines was evaluated and shown in Table 1 with a standard vorinostat for comparison. Few compounds exhibited weak cellular inhibitory activity at a concentration of 50 μM and all compounds did not show cytotoxicity at a concentration of 5 μM. XH-2 and XH-4 exhibits comparable inhibitory ability to vorinostat on A549 at a concentration of 50 μM, but it could not inhibit the growth of A549 at 5 μM. As for MDA-MB-231, only XH-1 and XH-5 had a moderate inhibitory effect at 50 μM.

Table 1. Effects of target compounds on Gli1 expression, A549, MDA-MB-231 and its log P, TPSA and BBB permeant
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Molecular docking. The docking study revealed that XH-11, XH-16, XH-17 and vismodegib could bind to the TMD of Smo (PDB ID: 5L7I [34]), with docking scores of –8.83, –7.50, –7.68, and –7.97, respectively (Fig. 2). The 2-(2-chlorophenyl)pyridine fragment of four ligands almost completely overlaped and the amino acid residues involved in the interactions between Smo and ligands were very similar. These results confirmed that target compounds could bind to the binding site of vismodegib, which suggested that the upregulation of Gli1 was caused by the interaction of target compounds and Smo protein. However, the hydrogen bonds between vismodegib and ARG400 was missing in terms of XH-11, XH-16, and XH-17. The difference interactions of ARG400 may be a reason for different biological activities of vismodegib and XH-11, XH-16, XH-17 (antagonistic or agonistic). Besides the binding affinity, log P, TPSA and BBB permeant were determined using the online program Swiss ADME (Table 1). The logP value of target compounds was high, but only the value of XH-8, XH-9 and XH-17 were higher than 5. And The BBB permeant values indicated that some compounds had the potential to enter the central nervous system.

Fig. 2.
figure 2

Molecular docking results. (a) vismodegib (violet), XH-11(green), XH-16 (magenta) and XH-17 (faded red) binding to the TMD of Smo protein (5L7I); (b) 2D diagram of vismodegib–Smo complexes; (c) 2D diagram of XH-17–Smo complexes; (d) 2D diagram of XH-11–Smo complexes; (e) 2D diagram of XH-16–Smo complexes; (f) docking score and amino acids involved in interactions between ligands and Smo.

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Teaching practice experiences. Medicinal chemistry is a practical discipline and new experimental teaching is needed to stimulate interest of students in drug discovery and development. The scientific research ability training course (an open one-week experimental course) and the college students’ innovative entrepreneurial training plan program (an open one-year experimental course) offered for undergraduates in our school involves content related to medicinal chemistry. Thus, the discovery of novel Smo agonists was employed for the course and 5 undergraduates participated in the latter.

To ensure safety and cultivate students′ safety awareness, goggles, gloves and laboratory coat were required to wear. All of the students are required to review the MSDS (Material Safety Data Sheet) for materials and reagents used prior to conducting experiments. All materials were handled using the standard procedures described in references. And all chemical waste was disposed of in accordance with local regulations.

In the laboratory course, students′ experimental skills, such as the ability of analysing experimental results and making observation notes, were particularly important, and were the key point of course evaluation. At the end of the course, students could independently carry out a medicinal chemistry project involving literature retrieving, molecular design, chemical synthesis, biochemical testing, docking simulation and data analysis. We also launched anonymous surveys to know students′ evaluations to this course. All students indicated that they strengthened the understanding of medicinal chemistry and gained more experimental skills. Additionally, the process aimed to develop potential drugs, which was more conducive to highlighting intellectual property compared to traditional repetitive experiments. Besides, the course, as an exploratory experimental processes, was more interesting and challenging, which inspired students to investigate related career paths. And 3 students planned to conduct medicinal chemistry research in the future.

After the teaching practice, 3 experiences were summed up which might be useful in many exploratory and continuous experimental course.

1. The time for students to participate in this course is scattered because of many other courses which students also need to participate in. Therefore, the designed experiment must adapt to dispersed time to ensure the smooth implementation of this course.

2. Because this is an exploratory experiment, teachers must lead the whole experiment arrangement and assist students in designing the experiment plan, correcting experimental operations and resolving problems, etc. And increasing discussions between undergraduates and teachers, as well as between undergraduates and postgraduates, is an effective method to improve learning outcomes.

3. The course process assessment and teaching management suitable for specific experiment are important for improving course quality. Compared to a ′traditional, exactly repeating′ experiments, it is more important to make students not settle for what results they got, but reflect on how they can do it better next time.

CONCLUSIONS

In order to obtain Smo agonists with novel molecular scaffold, a series of vismodegib derivatives were designed and synthesized based on our previous work. And many target compounds could activate the Hh pathway manifested by up-regulation of known pathway target genes Gli1. XH-16 and XH-17, with the strongest agonistic effect, could upregulate the expression of Gli1 by more than 50% at a concentration of 10 μM and did not exhibit cytotoxicity against A549 and the MDA-MB-231 cells at a concentration of 50 μM. Molecular docking results confirmed that the target compounds could bind to Smo indicating that their agonistic effect on the Hh pathway may be due to the activation of Smo. Moreover, the research process was introduced in a medicinal chemistry experimental course and could build capabilities of undergraduates for independently carrying out a medicinal chemistry project involving literature retrieving, molecular design, chemical synthesis, biochemical testing, docking simulation and data analysis, etc. And the teaching practice experiences were summed up which might be useful in many exploratory and continuous experimental course.

EXPERIMENTAL

All the starting materials, reagents and solvents are commercially available and used without further purification. Melting points were determined with a X-4 apparatus and were uncorrected. The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Ascend 400 (Billerica, USA) using tetramethylsilane (TMS) as an internal standard. Electrospray ionization mass spectrometry (ESI-MS) analyses was recorded in an Agilent 1100 Series MSD Trap SL (Santa Clara, USA). The reaction progress was monitored by thin-layer chromatography (HG/T2354-92, GF254).

1,2-Dihydro-1,3-dimethyl-2-oxopyrimidinium hydrogen sulfate (3). 1,3-Dimethylurea (5 g, 60 mmol) in 50 mL of absolute ethanol was added to 1,1,3,3-tetraethoxypropane (9.3 g, 60 mmol) and the resulting mixture was cooled in ice water bath. Upon dropwise addition of 95% sulfuric acid (5 mL) below 10°C the stirred mixture became light yellow and deposited precipitates. The mixture was then heated at 50°C for 1.5 h. After cooling to room temperature, the crystals were filtered off, washed with absolute ethanol, and dried to afford 1,2-dihydro-1,3-dimethyl-2-oxopyrimidinium hydrogen sulfate, a pale white solid. Yield 73.8% (9.3 g), mp 201–203°C (mp 204–206°C [31]).

2-(2-Chloro-5-nitrophenyl)pyridine (5). 2-Chloro-5-nitroacetophenone (8 g, 40 mmol), 1,2-dihydro-1,3-dimethyl-2-oxopyrimidinium hydrogen sulfate (9.3 g, 42 mmol) and ammonium acetate (20 g) were added to acetic acid (53 mL), and the mixture was heated at 115°C for 7 h. Then water (53 mL) was added with cooling to 0°C and stirring for 10 min. The suspension was filtered and washed with water to afford title compound. Yield 88.4% (7.44 g), mp 133–136°C. 1H NMR spectrum (400 MHz, chloroform-d), δ, ppm: 8.77 d (1H, Ar-H, J = 4.8 Hz), 8.52 d (1H, Ar-H, J = 2.9 Hz), 8.20 d. d (1H, Ar-H, J = 8.8, 2.9 Hz), 7.84 t (1H, Ar-H, J = 7.6 Hz), 7.70 d (1H, Ar-H, J = 7.8 Hz), 7.66 d (1H, Ar-H, J = 8.8 Hz), 7.38 d. d (1H, Ar-H, J = 7.5, 4.8 Hz). Mass spectrum (ESI), m/z: 235.1 [M + H]+.

2-(5-Amino-2-chlorophenyl)pyridine (6). A mixture of Fe powder (10.9 g, 0.20 mol), NH4Cl (10.5 g, 0.20 mol) and H2O (230 mL) was refluxed for 10 min. Then 2-(2-chloro-5-nitrophenyl)pyridine (11.4 g, 0.05 mol) was added and the mixture was stirred under reflux for another 2.5 h. After cooling to room temperature, the mixture was diluted with 120 mL of ethyl acetate and filtered through a pad of diatomaceous earth which was washed with ethyl acetate. After separating the ethyl acetate layer from the filtrate, the water layer was extracted with ethyl acetate (2×50 mL). The combined extracts were washed with water (200 mL), dried over anhydrous sodium sulfate, and filtered, and the mixture was concentrated under vacuum to about 20 mL. Then petroleum ether (40 mL) was added to precipitate solid and the title compound was obtained via filtration. Yield 77.5% (7.6 g), mp 162–165°C. 1H NMR spectrum (400 MHz, chloroform-d), δ, ppm: 8.64 d. t (1H, Ar-H, J = 4.8, 1.5 Hz), 7.85 t. d (1H, Ar-H, J = 7.7, 1.9 Hz), 7.57 d. d (1H, Ar-H, J = 7.9, 1.2 Hz), 7.37 d. d. d (1H, Ar-H, J = 7.6, 4.8, 1.2 Hz), 7.14 d (1H, Ar-H, J = 8.6 Hz), 6.76 d (1H, Ar-H, J = 2.8 Hz), 6.61 d. d (1H, Ar-H, J = 8.6, 2.8 Hz), 5.35 s (2H, NH2). Mass spectrum (ESI), m/z: 205.2 [M + H]+.

N-(2-Aminophenyl)-2-chloro-5-nitrobenzamide (9). A mixture of 2-chloro-5-nitrobenzoic acid (10 g, 49.8 mmol), sulfoxide chloride (36 mL, 0.49 mol), and DMF (1 d) was refluxed for 2 h, and then evaporated under reduced pressure to afford 2-chloro-5-nitrobenzoyl chloride. Acetone (50 mL) was added to the mixture. O-Phenylenediamine (5.4 g, 49.8 mmol) and triethylamine (10.3 mL, 74.6 mmol) were dissolved in acetone (200 mL), then acyl chloride solution was dropped into the mixture below –20°C and the mixture was reacted at low temperature for 2.5 h. Next, the mixture was poured into 500 mL of water. The suspension was filtered and washed with water to afford N-(2-aminophenyl)-2-chloro-5-nitrobenzamide as a white solid. Yield 89.0% (12.9 g), mp 170–173°C.

2-(2-Chloro-5-nitrophenyl)-1H-benzo[d]imidazole (10). N-(2-Aminophenyl)-2-chloro-5-nitrobenzamide (10 g, 34.3 mmol) and glacial acetic acid (50 mL) were refluxed for 5 h. Then the mixture was poured into 200 mL of ice water. The suspension was filtered and washed with water to afford 2-(2-chloro-5-nitrophenyl)-1H-benzimidazole as a white solid. Yield 98.0% (9.2 g), mp 195–199°C (mp 198–199°C [35]).

3-(1H-Benzo[d]imidazol-2-yl)-4-chloroaniline (11). A mixture of Fe powder (5.3 g, 94.7 mmol), NH4Cl (5.1 g, 94.7 mmol) and H2O (70 mL) was heated (90°C) and stirred for 20 min. Then 2-(2-chloro-5-nitrophenyl)-1H-benzo[d]imidazole (7.4 g, 27.1 mmol) was added and the mixture was stirred for another 2 h. After cooling to room temperature, the mixture was diluted with 70 mL of ethyl acetate and filtered through a pad of diatomaceous earth which was washed with ethyl acetate. After separating the ethyl acetate layer from the filtrate, the water layer was then extracted with ethyl acetate (2×30 mL). The combined extracts were washed with water (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under vacuum to afford crude product which was purified via recrystallization from ethanol (22 mL) to afford 3-(1H-benzo[d]imidazol-2-yl)-4-chloroaniline. Yield 54.8% (3.6 g), mp 205–208°C (mp 209–210°C [35]). 1H NMR spectrum (400 MHz, chloroform-d), δ, ppm: 7.75 d (1H, Ar-H, J = 2.9 Hz), 7.68 d. d (2H, Ar-H, J = 6.1, 3.2 Hz), 7.31 d. d (2H, Ar-H, J = 6.1, 3.2 Hz), 7.23 d (1H, Ar-H, J = 8.6 Hz), 6.69 d. d (1H, Ar-H, J = 8.7, 2.9 Hz). Mass spectrum (ESI), m/z: 244.1 [M + H]+.

N-{3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl}-2-phenylacetamide (XH-1). A mixture of 2-phenylacetic acid (0.18 g, 1.3 mmol), 3-(1H-benzo[d]imidazol-2-yl)-4-chloroaniline (0.33 g, 1.3 mmol), EDCI (0.44 g, 2.3 mmol), HOBt (0.2 g, 1.3 mmol), and pyridine (5 mL) was stirred for 2 h at room temperature. After the completion of the reaction (monitored by TLC), the mixture was evaporated to dryness and saturated aqueous sodium carbonate (20 mL) and ethyl acetate (20 mL) were added. Then the oil layer was separated, washed with saturated aqueous sodium carbonate (2×10 mL) and brine (10 mL) in sequence, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, to afford 1 g of liquid. The crude product was purified by column chromatography to afford the pure product. Yield 68.2% (0.33 g), light yellow solid. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 12.66 s (1H, NH), 10.48 s (1H, NH), 8.24 d (1H, Ar-H, J = 2.6 Hz), 7.77 d. d (1H, Ar-H, J = 8.8, 2.7 Hz), 7.69 d (1H, Ar-H, J = 7.7 Hz), 7.57 d (2H, Ar-H, J = 8.7 Hz), 7.37–7.20 m (7H, Ar-H), 3.67 s (2H, CH2). Mass spectrum (ESI), m/z: 362.14 [M + H]+.

Other compounds were prepared similarly.

N-{3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl}-2-(4-fluorophenyl)acetamide (XH-2). Yield 54.3% (0.38 g), mp 229.8–231.9°C. 1H NMR spectrum (600 MHz, DMSO-d6), δ, ppm: 12.66 s (1H, NH), 10.48 s (1H, NH), 8.23 d (1H, Ar-H, J = 2.6 Hz), 7.76 d. d (1H, Ar-H, J = 8.8, 2.7 Hz), 7.72–7.55 m (3H, Ar-H), 7.40–7.34 m (2H, Ar-H), 7.26–7.19 m (2H, Ar-H), 7.19–7.12 m (2H, Ar-H), 3.67 s (2H, CH2). Mass spectrum (ESI), m/z: 380.1 [M + H]+ .

N-{3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl}-2-(2-fluorophenyl)acetamide (XH-3). Yield 66.9% (0.62 g), mp 281–285°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 12.67 s (1H, NH), 10.53 s (1H, NH), 8.25 d (1H, Ar-H, J = 2.7 Hz), 7.76 d. d (1H, Ar-H, J = 8.8, 2.7 Hz), 7.69 d (1H, Ar-H, J = 7.8 Hz), 7.58 d. d (2H, Ar-H, J = 8.3, 4.3 Hz), 7.46–7.37 m (1H, Ar-H), 7.37–7.29 m (1H, Ar-H), 7.29–7.11 m (4H, Ar-H), 3.77 s (2H, CH2). Mass spectrum (ESI), m/z: 380.1 [M + H]+ .

N-{3-(1H-benzo[d]imidazol-2-yl)-4-chlorophenyl}-2-(p-tolyl) acetamide (XH-4). Yield 64.9% (0.63 g), mp 222.0–225.8°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 12.66 s (1H, NH), 10.44 s (1H, NH), 8.23 d (1H, Ar-H, J = 2.6 Hz), 7.76 d. d (1H, Ar-H, J = 8.8, 2.6 Hz), 7.73–7.66 m (1H, Ar-H), 7.61–7.53 m (2H, Ar-H), 7.29–7.18 m (4H, Ar-H), 7.13 d (2H, Ar-H, J = 7.8 Hz), 3.61 s (2H, CH2), 2.27 s (3H, CH3). Mass spectrum (ESI), m/z: 376.3 [M + H]+ .

N-{3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl}-2-methyl-2-phenylpropanamide (XH-5). Yield 54.6% (0.57 g), mp 196.2–197.3°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 12.64 s (1H, NH), 9.44 s (1H, NH), 8.24 d (1H, Ar-H, J = 2.7 Hz), 7.88 d. d (1H, Ar-H, J = 8.8, 2.7 Hz), 7.79–7.47 m (3H, Ar-H), 7.37 d (4H, Ar-H, J = 4.3 Hz), 7.24 q. d (3H, Ar-H, J = 8.0, 4.9 Hz), 1.58 s (6H, CH3). Mass spectrum (ESI), m/z: 390.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(o-tolyl)acetamide (XH-6). Yield 64.9% (0.63 g), mp 148.5–150°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.37 s (1H, NH), 8.68 d t (1H, Ar-H, J = 4.8 Hz), 7.96–7.85 m (2H, Ar-H), 7.74–7.62 m (2H, Ar-H), 7.50 d (1H, Ar-H, J = 8.7 Hz), 7.45–7.39 m (1H, Ar-H), 7.27–7.21 m (1H, Ar-H), 7.15 q. d (3H, Ar-H, J = 4.9, 2.6 Hz), 3.69 s (2H, CH2), 2.29 s (3H, CH3). Mass spectrum (ESI), m/z: 391.1 [M + H]+, 393.1 [M + H]+ .

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-1-phenylcyclopropane-1-carboxamide (XH-7). Yield 45.5% (0.41 g), mp 135.8–137.6°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 9.30 s (1H, NH), 8.67 d. t (1H, Ar-H, J = 4.8, 1.3 Hz), 7.89 t. d (1H, Ar-H, J = 7.7, 1.8 Hz), 7.81 d (1H, Ar-H, J = 2.6 Hz), 7.69 d. d (1H, Ar-H, J = 8.8, 2.6 Hz), 7.63 d. t (1H, Ar-H, J = 8.0, 1.1 Hz), 7.45 d (1H, Ar-H, J = 8.8 Hz), 7.43–7.40 m (1H, Ar-H), 7.40–7.37 m (2H, Ar-H), 7.35 d. d (2H, Ar-H, J = 8.5, 6.5 Hz), 7.30–7.25 m (1H, Ar-H), 1.45 d. d (2H, CH2, J = 4.2 Hz), 1.13 d. d (2H, CH2, J = 8.2, 6.7 Hz). Mass spectrum (ESI), m/z: 349.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(4-isobutylphenyl)propenamide (XH-8). Yield 63.9% (0.52 g), mp 107.7–109.1°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.24 s (1H, NH), 8.68 d. d. d (1H, Ar-H, J = 4.8, 1.9, 1.0 Hz), 7.96–7.81 m (2H, Ar-H), 7.75–7.57 m (2H, Ar-H), 7.53–7.37 m (2H, Ar-H), 7.34–7.21 m (2H, Ar-H), 7.14–7.00 m (2H, Ar-H), 3.78 q (1H, CH, J = 6.9 Hz), 2.40 d (2H, CH2, J = 7.2 Hz), 1.79 d. t (1H, CH, J = 13.5, 6.8 Hz), 1.40 d (3H, CH3, J = 7.0 Hz), 0.84 d (6H, CH3, J = 6.6 Hz). Mass spectrum (ESI), m/z: 393.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(2-fluoro-[1,1′-biphenyl]-4-yl)propenamide (XH-9). Yield 58.1% (0.25 g), mp 120.6–122.9°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.35 s (1H, NH), 8.81–8.59 m (1H, Ar-H), 7.98–7.81 m (2H, Ar-H), 7.77–7.61 m (2H, Ar-H), 7.56–7.35 m (8H, Ar-H), 7.34–7.27 m (2H, Ar-H), 3.90 q (1H, CH, J = 6.9 Hz), 1.46 d (3H, CH3, J = 7.0 Hz). Mass spectrum (ESI), m/z: 431.1 [M + H]+, 433.1 [M + H]+.

2-[4-(Benzyloxy)phenyl]-N-[4-chloro-3-(pyridin-2-yl)phenyl]acetamide (XH-10). Yield 62.8% (0.27 g), mp 129.6–131.9°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.32 s (1H, NH), 8.68 d. d. d (1H, Ar-H, J = 4.8, 1.9, 1.0 Hz), 7.98–7.79 m (2H, Ar-H), 7.75–7.59 m (2H, Ar-H), 7.48 d (1H, Ar-H, J = 8.8 Hz), 7.46–7.35 m (5H, Ar-H), 7.35–7.28 m (1H, Ar-H), 7.27–7.19 m (2H, Ar-H), 7.06–6.84 m (2H, Ar-H), 5.08 s (2H, CH2), 3.57 s (2H, CH2). Mass spectrum (ESI), m/z: 429.1 [M + H]+, 430.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-[3-(trifluoromethyl)phenyl]acetamide (XH-11). Yield 68.9% (0.66 g), mp 115.3–117.1°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.45 s (1H, NH), 8.68 d. d. d (1H, Ar-H, J = 4.9, 1.8, 1.0 Hz), 7.97–7.80 m (2H, Ar-H), 7.74–7.60 m (5H, Ar-H), 7.60–7.53 m (1H, Ar-H), 7.50 d (1H, Ar-H, J = 8.7 Hz), 7.43 d. d. d (1H, Ar-H, J = 7.5, 4.8, 1.2 Hz), 3.80 s (2H, CH2). Mass spectrum (ESI), m/z: 391.1 [M + H]+, 393.1 [M + H]+.

2-[4-(tert-Butyl)phenyl]-N-[4-chloro-3-(pyridin-2-yl)phenyl]acetamide (XH-12). Yield 77.8% (0.14 g), mp 66.6–68.9°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.37 s (1H, NH), 8.68 d. d. d (1H, Ar-H, J = 4.9, 1.8, 0.9 Hz), 7.96–7.82 m (2H, Ar-H), 7.73–7.61 m (2H, Ar-H), 7.49 d (1H, Ar-H, J = 8.7 Hz), 7.42 d. d. d (1H, Ar-H, J = 7.6, 4.8, 1.1 Hz), 7.38–7.29 m (2H, Ar-H), 7.29–7.19 m (2H, Ar-H), 3.59 s (2H, CH2), 1.26 s (9H, CH3). Mass spectrum (ESI), m/z: 379.1 [M + H]+, 381.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-methyl-2-phenylpropanamide (XH-13). Yield 37.9% (0.27g), mp117.9–119.4°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 9.32 s (1H, NH), 8.67 d. d. d (1H, Ar-H, J = 4.9, 1.8, 1.0 Hz), 7.93–7.84 m (2H, Ar-H), 7.78 d. d (1H, Ar-H, J = 8.7, 2.7 Hz), 7.64 d. t (1H, Ar-H, J = 7.9, 1.1 Hz), 7.46 d (1H, Ar-H, J = 8.7 Hz), 7.41 d. d. d (1H, Ar-H, J = 7.6, 4.8, 1.2 Hz), 7.35 d (4H, Ar-H, J = 4.2 Hz), 7.28–7.21 m (1H, Ar-H), 1.56 s (6H, CH3). Mass spectrum (ESI), m/z: 351.1 [M + H]+, 353.1 [M + H]+.

2-(4-Aminophenyl)-N-[4-chloro-3-(pyridin-2-yl)phenyl]acetamide (XH-14). Yield 54.4% (0.59 g), mp 120.6–122.2°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.22 s (1H, NH), 8.68 d. d. d (1H, Ar-H, J = 4.8, 1.8, 0.9 Hz), 7.95–7.82 m (2H, Ar-H), 7.73–7.61 m (2H, Ar-H), 7.48 d (1H, Ar-H, J = 8.8 Hz), 7.42 d. d. d (1H, Ar-H, J = 7.6, 4.9, 1.1 Hz), 7.02–6.92 m (2H, Ar-H), 6.57–6.46 m (2H, Ar-H), 4.95 s (2H, NH2), 3.43 s (2H, CH2). Mass spectrum (ESI), m/z: 338.0 [M + H]+ .

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-[4-(methylthio)phenyl]acetamide (XH-15). Yield 68.3% (0.71 g), mp 138.2–140.5°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.35 s (1H, NH), 8.68 d. d. d (1H, Ar-H, J = 4.8, 1.9, 1.0 Hz), 7.94–7.84 m (2H, Ar-H), 7.72–7.61 m (2H, Ar-H), 7.49 d (1H, Ar-H, J = 8.8 Hz), 7.45–7.40 m (1H, Ar-H), 7.31–7.17 m (4H, Ar-H), 3.61 s (2H, CH2), 2.45 s (3H, CH3). Mass spectrum (ESI), m/z: 369.0 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(3-nitrophenyl)acetamide (XH-16). Yield 54.7% (0.52 g), mp 140.8–143.7°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.48 s (1H, NH), 8.68 d (1H, Ar-H, J = 4.4 Hz), 8.33–8.04 m (2H, Ar-H), 7.96–7.83 m (2H, Ar-H), 7.79 d (1H, Ar-H, J = 7.7 Hz), 7.67 d. d. t (4H, Ar-H, J = 16.1, 13.9, 5.1 Hz), 7.50 d (1H, Ar-H, J = 8.7 Hz), 7.43 d. d (1H, Ar-H, J = 7.5, 4.9 Hz), 3.86 s (2H, CH2). Mass spectrum (ESI), m/z: 368.1 [M + H]+.

(S)-N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(6-methoxynaphthalen-2-yl)propenamide (XH-17). Yield 63.7% (0.65 g), mp 79.8–81.3°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.30 s (1H, NH), 8.67 d. d. d (1H, Ar-H, J = 4.8, 1.8, 1.0 Hz), 7.97–7.84 m (2H, Ar-H), 7.84–7.73 m (3H, Ar-H), 7.69 d. d (1H, Ar-H, J = 8.8, 2.7 Hz), 7.64 d. t (1H, Ar-H, J = 7.9, 1.1 Hz), 7.53–7.44 m (2H, Ar-H), 7.42 d. d. d (1H, Ar-H, J = 7.6, 4.9, 1.2 Hz), 7.27 d (1H, Ar-H, J = 2.6 Hz), 7.14 d. d (1H, Ar-H, J = 8.9, 2.6 Hz), 3.95 d (1H, CH, J = 7.0 Hz), 3.86 s (3H, CH3), 1.49 d (3H, CH3, J = 7.0 Hz). Mass spectrum (ESI), m/z: 417.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(4-hydroxyphenyl)acetamide (XH-18). Yield 55.9% (0.53 g), mp 181.1–183.7°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.28 s (1H, NH), 9.24 s (1H, OH), 8.68 d. d. d (1H, Ar-H, J = 4.9, 1.8, 1.0 Hz), 7.95–7.83 m (2H, Ar-H), 7.74–7.61 m (2H, Ar-H), 7.51–7.40 m (2H, Ar-H), 7.14–7.07 m (2H, Ar-H), 6.73–6.66 m (2H, Ar-H), 3.50 s (2H, CH2). Mass spectrum (ESI), m/z: 339.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(4-chlorophenyl)acetamide (XH-19). Yield 81.7% (0.74 g), mp 149.7–150.9°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.39 s (1H, NH), 8.68 d. t (1H, Ar-H, J = 4.8, 1.3 Hz), 7.95–7.83 m (2H, Ar-H), 7.71–7.62 m (2H, Ar-H), 7.49 d (1H, Ar-H, J = 8.7 Hz), 7.42 d. d. d (1H, Ar-H, J = 7.5, 4.8, 1.1 Hz), 7.38 d (2H, Ar-H, J = 8.6 Hz), 7.34 d (2H, Ar-H, J = 8.6 Hz), 3.66 s (2H, CH2). Mass spectrum (ESI), m/z: 357.0 [M + H]+, 359.0 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-phenylpropanamide (XH-20). Yield 48.6% (0.4 g), mp 137.0–138.5°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.26 s (1H, NH), 8.76–8.60 m (1H, Ar-H), 7.96–7.81 m (2H, Ar-H), 7.76–7.59 m (2H, Ar-H), 7.47 d (1H, Ar-H, J = 8.7 Hz), 7.42 d. d (1H, Ar-H, J = 7.6, 4.8 Hz), 7.40–7.35 m (2H, Ar-H), 7.32 t (2H, Ar-H, J = 7.5 Hz), 7.26–7.20 m (1H, Ar-H), 3.82 d (1H, CH, J = 7.0 Hz), 1.41 d (3H, CH3, J = 7.0 Hz). Mass spectrum (ESI), m/z: 337.1 [M + H]+.

N-[4-Chloro-3-(pyridin-2-yl)phenyl]-2-(p-tolyl)propenamide (XH-21). Yield 73.3% (0.66 g), mp 149.7–152.1°C. 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 10.21 s (1H, NH), 8.68 d. d. d (1H, Ar-H, J = 4.8, 1.9, 1.0 Hz), 7.93–7.84 m (2H, Ar-H), 7.71–7.61 m (2H, Ar-H), 7.47 d (1H, Ar-H, , J = 8.8 Hz), 7.42 d. d. d (1H, Ar-H, J = 7.6, 4.8, 1.2 Hz), 7.29–7.23 m (2H, Ar-H), 7.12 d (2H, Ar-H, J = 7.8 Hz), 3.77 q (1H, CH, J = 6.9 Hz), 2.26 s (3H, CH3), 1.39 d (3H, CH3, J = 7.0 Hz). Mass spectrum (ESI), m/z: 351.1 [M + H]+.

The expression of Gli1. Magnetic bead method total RNA extraction kit (DP761, TIANGEN, Beijing, China) was used to extract total RNA from NIH 3T3 cells when cells reached 90% confluence. Reverse transcription of mRNA into cDNA was performed using a Hifair II 1st strand cDNA synthesis supermix (11120ES60, YesEN, Shanghai, China). Then, RT-qPCR was conducted to measure the relative levels of mRNA transcripts using SYBR Green Master Mix accoding to the manufacturer’s instructions (1184ES08, YesEN, Shanghai, China). The sequences of primers are shown as below: Gli1 forward 5′-GGGATGATCCCACATCCTCAGTC-3′, reverse5′-CTGGAGCAGCCCCCCC AGT-3′; GAPDH: forward 5′-TGAAGGTCGGTGTGAACGG-3′, Reverse 5′-GTGAGTGGAGTCATACTGGAA-3′. The 2-ΔΔCt method was used for the calculation of the relative expression of Gli1 gene mRNA transcripts with GAPDH as a housekeeping gene.

Cytotoxicity. The MTT assay technique [36] was used to investigate in vitro cytotoxicity against the A549 (Lung cancer) and the MDA-MB-231 (breast cancer) cell line. The cells were treated with compounds at various concentrations (50 and 5 μM) for 48 h, and then 0.25 mg/mL MTT staining solution was added to each well, and the plate was incubated at 37°C for 4 h. After removing the culture medium, 100 μL of DMSO was added to dissolve the crystals, and absorbance at 490 nm was reported. The following formula was used to calculate the percentage inhibition of target compounds against cell survival.

$${\rm{Inhibition\;}} = 1 - {\rm{\;}}{{{\rm{Mean\;OD\;of\;test\;compound}}} \over {{\rm{Mean\;OD\;of\;negative\;control}}}}\; \times 100.$$

Molecular docking studies. The Glide program of Schrodinger Suites (V2018-4) was employed for molecular docking and the crystal structure of Smo (ID: 5L7I) was obtained from the PDB database. The Protein Preparation Wizard was used for protein structure processing, and the Ligprep was used to prepare ligands. The Receiver Grid Generation was used to generate a receptor grid file and determine the docking pocket based on the ligand in the crystal structure. All parameters for the preparation of proteins and ligands, as well as docking, used default values. SP mode and docking score were used for the Glide docking. Using the online program Swiss ADME, logP, TPSA, bioavailability score, and BBB permeant were determined [37, 38].