Design, Synthesis, and Biological Evaluation of Stable Colchicine-Binding Site Tubulin Inhibitors 6-Aryl-2-benzoyl-pyridines as Potential Anticancer Agents

Abstract

We previously reported a potent tubulin inhibitor CH-2-77. In this study, we optimized the structure of CH-2-77 by blocking metabolically labile sites and synthesized a series of CH-2-77 analogues. Two compounds, 40a and 60c, preserved the potency while improving the metabolic stability over CH-2-77 by 3- to 4-fold (46.8 and 29.4 vs 10.8 min in human microsomes). We determined the high-resolution X-ray crystal structures of 40a (resolution 2.3 Å) and 60c (resolution 2.6 Å) in complex with tubulin and confirmed their direct binding at the colchicine-binding site. In vitro, 60c maintained its mode of action by inhibiting tubulin polymerization and was effective against P-glycoprotein-mediated multiple drug resistance and taxol resistance. In vivo, 60c exhibited a strong inhibitory effect on tumor growth and metastasis in a taxol-resistant A375/TxR xenograft model without obvious toxicity. Collectively, this work showed that 60c is a promising lead compound for further development as a potential anticancer agent.

Graphical Abstract

INTRODUCTION

Microtubule-targeting agents (MTAs) are classified into two major categories: microtubule-stabilizing agents (e.g., taxanes and epothilones) and microtubule-destabilizing agents (e.g., vinca alkaloids and colchicine).^1,2 MTAs bind to tubulin via three major binding sites, taxane-, vinca alkaloid-, and colchicine-binding sites, and disrupt microtubule dynamics.^3,4 MTAs interacting with the taxane- or vinca alkaloid-binding sites in tubulin are widely used in cancer therapy, but their clinical efficacy is often limited by the development of acquired drug resistance, most notably the overexpression of ABC transporters.^5–8 In contrast, extensive preclinical studies have indicated that MTAs targeting the colchicine-binding site are significantly less susceptible to transporter-mediated drug resistance.^9–12 We have previously reported 6-aryl-2-benzoylpyridines (ABPs, Figure 1) as a new generation of MTAs that target the colchicine-binding site. These compounds showed low nanomolar potency against a panel of human melanoma and breast cancer cell lines and can effectively overcome taxol resistance.¹³ However, further evaluations of these compounds have shown that they have low oral bioavailability and low metabolic stability. The half-life time of CH-2–1, the prototype of this class of compounds, is 4.2 min in human liver microsomes (HLMs), while the half-life time of CH-2–77, the most potent compound in this class (average IC₅₀ = 2.5 nM), is 10.8 min in HLM (Figure 1).

Figure 1.

Recently developed ABPs that target the colchicine-binding site.

The short half-life time of these compounds might be due to the reduction of the carbonyl linker and demethylation in the trimethoxyphenyl (TMP) group by HLM. The centrally located carbonyl moiety in the ABP scaffold is important for forming hydrogen-bonding interactions with the β-D249.¹³ However, this moiety is susceptible to nucleophilic attack, which leads to a change in the geometry and thus loss of potency of the compounds. Therefore, we attempted to cyclize the carbonyl with the “B” ring to generate a new “D” ring to block metabolic reduction of the ketone moiety to a secondary alcohol functional group, thereby improving the metabolic stability while retaining efficacy of ABPs. The TMP group in the “C” ring of ABPs is a common moiety shared by many colchicine-binding site inhibitors (CBSIs) and is experimentally important in maintaining the potency of CBSIs.^14–16 However, the methoxy group undergoes O-demethylation in vivo, producing a phenol moiety that is less active and more toxic.^17,18 Thus, we hypothesize that replacing the methoxy moiety with more stable functional groups will result in enhanced metabolic stability and improved safety profiles.

In this report, we describe strategies to reduce the metabolic lability of ABPs while maintaining or improving their antiproliferative activities. Among all analogues synthesized, compound 60c, which contains a 3-hydroxy-4-methyl moiety in the “A” ring, was most active, with an average IC₅₀ value of 2.4 nM against all cancer cell lines tested. In addition, its half-life time in HLM was ~30 min. In vitro tubulin polymerization assay confirmed that 60c inhibited tubulin polymerization. Furthermore, 60c could overcome P-glycoprotein (P-gp)-mediated multiple drug resistance and paclitaxel resistance. It also significantly inhibited the proliferation and colony formation of A375/TxR cells in a dose-dependent manner, arrested cells in the G2/M phase, and induced cellular apoptosis. Our in vivo studies demonstrated that 60c suppressed the growth and metastasis of A375/TxR xenografts with limited systemic toxicity. Collectively, our results substantiated that compound 60c is a potent CBSI with low systemic toxicity that may serve as a promising lead compound for further development as a potential anticancer agent.

RESULTS AND DISCUSSION

Antiproliferative Activities of ABP Analogues in a Panel of Melanoma and Breast Cancer Cell Lines.

We focused our efforts on preparing four series of modifications to the ABP scaffold. The first three series of compounds were designed in an attempt to overcome the major metabolism-related labilities: methyl oxidation in the “A” ring, ketone reduction, and demethylation in the “C” ring (Figure 1). The last series of compounds were obtained by replacing the pyridine “B” ring of the ABP scaffold with imidazole, pyrazine, pyrimidine, and substituted pyridine (Figure 1).

First, we designed and synthesized compound 4a by replacing the methyl group in the “A” ring with difluoromethyl in CH-2–1 (Figure 1 and Scheme 1A). With an average IC₅₀ value of 3.8 nM, compound 4a exhibited comparable potency to CH-2–1 with an IC₅₀ of 3.7 nM (Table 1) and similar metabolic stability to that of CH-2–1 (4a, HLM t_1/2 = 5.4 min; CH-2–1, HLM t_1/2 = 4.2 min) (Table 2). We continued this series of comparisons by synthesizing compound 4b (Scheme 1A) with an indoline in the “A” ring and tested its antiproliferative activity. Compound 4b showed similar cytotoxic activity against cancer cell lines with an average IC₅₀ value of 5.5 nM. Interestingly, the metabolic stability of compound 4b is slightly improved compared to that of CH-2–1 (4b, HLM t_1/2 = 15 min; CH-2–1, HLM t_1/2 = 4.2 min) (Table 2). We replaced the para-methyl moiety in the “A” phenyl ring with an N,N-dimethyl group to generate compound 4c (Scheme 1A). This modification led to a 13-fold reduction in antiproliferative activity (an average IC₅₀ value of 48.7 nM) compared to that of CH-2–1 (Table 1).

Scheme 1.

Synthesis of 4a or 4v Analogues (4a–d, 7, 10, and 13) with Modifications in Rings A and C^a

^aReagents and conditions: (a) n-BuLi, THF; (b) Dess–Martin periodinane, CH₂Cl₂; (c) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (d) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (e) AlCl₃, CH₂Cl₂; (f) DMF, Cs₂CO₃; (g) n-BuLi, THF; (h) Dess–Martin periodinane, CH₂Cl₂; (i) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (j) n-BuLi, THF; (k) Dess–Martin periodinane, CH₂Cl₂; and (l) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O.

Table 1.

Cytotoxic Effects of ABP Analogues against Three Human Malignant Melanoma Cell Lines and Three Breast Cancer Cell Lines (IC₅₀ ± SEM, n = 4)

	breast cancer cell lines			melanoma cell lines
compound	MDA-MB-231	MDA-MB-453	SKBR3	A375	M14	RPMI7951	average IC50 (nM)
4a	4.7 ± 0.5	4.0 ± 0.9	3.8 ± 0.8	3.0 ± 0.6	4.7 ± 0.8	2.8 ± 0.5	3.8
4b	5.3 ± 1.0	5.9 ± 1.4	5.5 ± 1.2	5.0 ± 1.0	5.6 ± 1.4	5.9 ± 1.5	5.5
4c	42.7 ± 8.5	33.7 ± 9.2	54.5 ± 13.7	53.8 ± 10.4	63.5 ± 12.1	44.0 ± 10.9	48.7
4d	33.4 ± 7.2	19.3 ± 4.1	15.2 ± 2.8	28.8 ± 4.2	12.6 ± 1.5	21.2 ± 4.2	20.1
7	6.6 ± 1.6	2.1 ± 0.3	4.2 ± 0.7	3.7 ± 0.7	2.4 ± 0.6	5.0 ± 1.0	4
10	5.3 ± 1.0	3.2 ± 0.7	3.2 ± 0.9	3.6 ± 0.5	4.5 ± 0.6	3.1 ± 0.8	3.8
13	37.4 ± 9.2	8.7 ± 2.3	10.1 ± 2.1	15.1 ± 2.5	24.8 ± 4.4	13.9 ± 2.1	18.3
16	514.7 ± 66.0	139.3 ± 35.5	82.9 ± 26.1	174.5 ± 19.6	76.5 ± 15.9	204.9 ± 22.1	198.8
22	22.3 ± 4.4	54.9 ± 10.6	65.6 ± 9.7	21.1 ± 2.8	18.5 ± 2.8	22.9 ± 2.5	34.1
28a	24.3 ± 4.3	23.2 ± 4.0	57.6 ± 10.1	41.2 ± 6.8	32.4 ± 4.3	78.4 ± 9.1	42.9
28b	112.4 ± 27.3	28.1 ± 4.5	25.6 ± 4.8	52.3 ± 7.1	46.6 ± 7.8	56.0 ± 10.1	53.5
29	70.9 ± 12.5	68.1 ± 19.0	119.3 ± 29.1	85.0 ± 19.0	76.3 ± 16.3	132.6 ± 25.2	80.5
35	4.8 ± 1.2	6.7 ± 2.2	7.1 ± 1.9	2.5 ± 0.4	1.7 ± 0.3	2.2 ± 0.7	4.2
40a	8.0 ± 1.7	4.0 ± 0.7	26.7 ± 5.5	5.9 ± 0.8	5.2 ± 1.0	5.2 ± 0.9	9.2
40b	12.4 ± 1.8	13.2 ± 3.4	35.0 ± 8.9	16.7 ± 2.5	19.2 ± 2.6	14.4 ± 3.5	16.8
40c	38.0 ± 7.8	51.5 ± 11.1	46.1 ± 14.3	20.8 ± 4.2	21.9 ± 4.0	23.8 ± 5.8	33.7
46a	15.1 ± 2.5	2.8 ± 0.4	12.0 ± 2.1	6.8 ± 1.4	5.4 ± 1.1	7.2 ± 1.0	8.2
46b	31.6 ± 5.3	14.2 ± 3.5	17.1 ± 6.8	36.1 ± 5.4	29.1 ± 4.4	29.7 ± 5.6	26.3
50a	16.0 ± 3.1	9.7 ± 2.0	5.5 ± 1.1	4.3 ± 0.9	4.4 ± 0.9	6.4 ± 1.9	7.7
50b	5.5 ± 1.2	5.5 ± 1.5	3.5 ± 0.8	8.8 ± 1.7	5.9 ± 1.3	6.3 ± 1.5	5.8
54a	11.1 ± 2.0	5.0 ± 1.2	4.5 ± 1.0	7.9 ± 1.5	7.7 ± 1.7	6.8 ± 1.4	7
55b	17.6 ± 3.7	14.7 ± 4.0	16.9 ± 4.4	22.2 ± 5.7	17.5 ± 4.1	17.2 ± 4.1	17.7
59a	2.3 ± 0.4	2.5 ± 0.4	1.90 ± 0.5	3.7 ± 0.6	2.6 ± 0.4	3.4 ± 0.6	2.7
59b	4.3 ± 0.8	4.2 ± 0.9	2.4 ± 0.5	2.6 ± 0.4	3.2 ± 0.5	2.1 ± 0.4	3.1
60c	2.2 ± 0.5	1.4 ± 0.3	4.8 ± 1.0	1.8 ± 0.4	1.9 ± 0.4	2.4 ± 0.5	2.4
64	45 ± 123.9	89.7 ± 16.0	52.3 ± 12.9	80.9 ± 15.0	56.3 ± 8.8	40.8 ± 11.3	60.8
CH-2–1	5.3 ± 1.2	3.9 ± 1.2	4.2 ± 1.4	2.4 ± 0.4	3.1 ± 0.4	3.5 ± 0.7	3.7
CH-2–77	3.3 ± 0.7	1.1 ± 0.4	4.1 ± 0.7	2.0 ± 0.4	2.0 ± 0.5	2.8 ± 0.5	2.5

Table 2.

In Vitro Microsomal Stability of Tested Compounds in Liver Microsome of Different Species

	metabolic stability in human liver microsome		metabolic stability in mouse liver microsome
compounds	t1/2 (min)	Clint (mL/(min kg))	t1/2 (min)	Clint (mL/(min kg))
CH-2–1	4.2 ± 0.00	317.4	3 ± 0.6	1050.4
CH-2–77	10.8 ± 0.6	116.4	60 ± 1.8	57.3
4a	5.4 ± 0.6	240.1	12.6 ± 1.2	270.6
4b	15 ± 1.2	83.6	4.2 ± 0.00	818.3
7	9 ± 0.6	135.6	8.4 ± 0.6	397.1
10	7.2 ± −0.6	172.2	1.8 ± 0.00	1900.5
13	54.6 ± 3	22.9	211.8 ± 27	16.2
22	205.2 ± 16.2	6.1	167.4 ± 11.4	20.5
28a	142.2 ± 15.6	8.8	84.6 ± 7.2	40.6
35	20.4 ± 1.2	169.5	42.6 ± 1.2	65.8
40a	46.8 ± 1.2	26.6	119.4 ± 7.8	28.7
46a	9 ± 1.2	142.2	78.6 ± 3.6	43.8
50a	43.2 ± 1.8	28.8	4.2 ± 0.00	824.2
54a	27.6± 1.8	45.0	>4 h	13.0
55b	42.6 ± 0.6	29.2	45 ± 1.2	76.7
59a	9.6 ± 0.6	133.4	12.6 ± 0.6	272.8
59b	33 ± 1.2	38.1	90 ± 2.4	38.2
60c	29.4 ± 2.4	42.3	>4 h	4.6
verapamil	37.2 ± 1.2	33.3	23.4 ± 1.8	145.8

Second, we modified the para-position on the phenyl ring in the “C” ring (Figure 1 and Scheme 1B). One purpose of this approach was to avoid the potential metabolic instability caused by O-demethylation. Our previous attempts to modify the TMP moiety showed that modifying the para-position of the “C” ring or linking the two adjacent methoxy moieties into a conformationally restricted ring system exhibited comparable or improved cytotoxic activity relative to the parent compound.^18,19 Based on our extensive studies on the trimethoxy template, we modified the “C” ring and synthesized compounds 7 (para-2-methoxyethoxy) and 10 (3-methoxybenzo[4,5]-dioxane) (Scheme 1B). Compounds 7 and 10 displayed similar activities to the parent compound CH-2–1, with average IC₅₀ values of 4 and 3.8 nM, respectively (Table 1). However, compared with the CH-2–1 (HLM t_1/2 = 4.2 min) parent compound, neither compound 7 nor 10 exhibited improved metabolic stability in human liver microsomes (t_1/2 = 9 and 7.2 min, respectively; Table 2). Introducing an ethyl group at the para-position generated compound 13 (Scheme 1B), which exhibited moderate activity with an average IC₅₀ value of 18.3 nM, but this derivative demonstrated enhanced metabolic stability in human liver microsomes with t_1/2 = 54.6 min compared to 10.8 min for CH-2–77 (Table 2). This result is consistent with our hypothesis that the para-methoxy moiety may be unstable on exposure to liver microsomes.

Third, we blocked the ketone reduction by introducing a new “D” ring. We replaced the carbonyl linker between the “B” and “C” rings in compound CH-2–1 with a pyridine fused to pyrrole to generate compound 16 (Figure 1 and Scheme 2). Compound 16 did not exhibit cytotoxicity in any examined cancer cell line. Further modification of the linker by introducing a group comprised pyridine fused to pyrazole generated compound 22 (Scheme 2). Compound 22 demonstrated a 19-fold improved half-life in the human liver (22, HLM t_1/2 = 205.2 min) (Table 2), indicating that the metabolic stability of ABPs can be extended by blocking ketone reduction. However, these modifications resulted in decreased antiproliferative activities.

Scheme 2.

Synthesis of CH-2–1 and CH-2–77 Analogues 16 and 22 with a Cyclized Carbonyl Moiety^a

^aReagents and conditions: (a) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (b) Pd(pddf)₂Cl₂·CH₂Cl₂, NaOBu-t; (c) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (d) NBS, THF; (e) SEMCl, NaH, THF; (f) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; and (g) TFA, CH₂Cl₂.

Finally, we modified the “B” ring of the ABP template since the 2-aryl-4-benzoyl-imidazole (ABI) template exhibited improved metabolic stability along with its high potency.²⁰ We integrated the imidazole ring into the ABP template to generate compounds 28a, 28b, and 29 (Scheme 3A). These compounds exhibited a decreased antiproliferative activity, 28a (IC₅₀ = 42.9 nM), 28b (IC₅₀ = 53.5 nM), and 29 (IC₅₀ = 80.5 nM), relative to the lead compound CH-2–77 (IC₅₀ = 2.5 nM) but showed significantly improved metabolic stability in human liver microsomes (for example, compound 28a, HLM t_1/2 = 142.2 min; CH-2–77, HLM t_1/2 = 10.8 min). Further modification of the “B” ring of the ABP template by inserting pyrazine generated compounds 35 and 40a–c (Scheme 3B). Compounds 35 and 40a displayed similar antiproliferative activities with average IC₅₀ values of 4.2 and 9.2 nM, respectively, compared to the parent compounds CH-2–1 and CH-2–77. It is interesting to note that compound 40a, which contains a pyrazine heteroaromatic ring, exhibited a significant improvement in the metabolic stability in human liver microsomes (40a, HLM t_1/2 = 46.8 min; CH-2–77, HLM t_1/2 = 10.8 min) (Table 2). Inspired by these observations, we continued this series of comparisons by adding pyrimidine to the “B” ring to generate compounds 46a and 46b (Scheme 4A). Compound 46a, which contains 3-hydroxy-4-methyl in the “A” phenyl ring, showed comparable activity to its parent with an average IC₅₀ of 8.2 nM, while compound 46b, which contains the 3-indole “A” ring, was less potent, with an average IC₅₀ of 26.3 nM compared to CH-2–77 (Table 1). Compound 46a showed comparable metabolic stability in human liver microsomes, with a t_1/2 value of 9 min compared to 10.8 min for CH-2–77 (Table 2).

Scheme 3.

Synthesis of ABP Analogues with Imidazole and Pyrazine in Ring B^a

^aReagents and conditions: (a) NaH, SEMCl, THF; (b) trimethyl(2-((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methoxy)-ethyl)silane, Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (c) 2-PrMgBr.LiCl, 3,4,5-trimethoxybenzoyl chloride; (d) (i): Pd/C, H₂; (ii): methylboronic acid, Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (e) TFA, CH₂Cl₂; (f) TFA, CH₂Cl₂; (g) n-BuLi, 1,3-dithiane, THF; (h) MeI, Ca₂CO₃, CH₃CN/H₂O; (i) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (j) n-BuLi, THF; (k) Dess–Martin periodinane, CH₂Cl₂; (l) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (m) MeI, CaCO₃, CH₃CN/H₂O; (n) n-BuLi, THF; (o) Dess–Martin periodinane, CH₂Cl₂; and (q) (i): TFA, CH₂Cl₂; (ii): KOH, MeOH, reflux.

Scheme 4.

Synthesis of ABP Analogues with Pyrimidine in Ring B and Modified Pyridines^a

^aReagents and conditions: (a) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (b) SeO₂, dioxane; (c) n-BuLi, THF; (d) Dess–Martin periodinane, CH₂Cl₂; (e) (i): TFA, CH₂Cl₂; (ii): KOH, MeOH, reflux, (f) n-BuLi, THF; (g) Dess–Martin periodinane, CH₂Cl₂; (h) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (i) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (j) n-BuLi, THF; (k) Dess–Martin periodinane, CH₂Cl₂; and (l) TFA, CH₂Cl₂.

To investigate the effects on metabolic stability and antiproliferative potency of substitutions at the meta- and para-positions of the ABP pyridine “B” ring, we prepared and evaluated analogues 50a, 50b, 54a, and 55b with various substituents such as −OMe and -Me at para-positions of the “B” pyridine ring (Tables 1 and 2 and Scheme 4B). Compounds 50a and 50b containing a para-methyl group in the “B” pyridine ring displayed similar activities, with average IC₅₀ values of 7.7 and 5.8 nM, respectively. The para-methyl moiety in the “B” ring of compound 50a was replaced by a para-methoxy group to generate compound 54a, which was comparable in potency to 50a (54a: average IC₅₀ = 7.0 nM) (Table 1). However, compound 55b, which contains one more hydroxyl group in the “A” ring of compound 54a, showed reduced antiproliferative activity with an average IC₅₀ value of 17.7 nM. Interestingly, compounds 50a, 54a, and 55b demonstrated 3-fold improvements in their half-life in the human liver (50a, 54a, and 55b; HLM t_1/2 = 43.2, 27.6, and 42.6 min, respectively) (Table 2). Compounds with fluorine substitution at the site of metabolic attack can prevent oxidative metabolism since the C–F bond is more resistant to attack than the C–H bond. Compounds 59a, 59b, and 60c (Scheme 5), which contain meta-F in the “B” ring, showed comparable activities when they were tested against human melanoma and breast cancer cell lines, with average IC₅₀ values ranging from 1 to 5 nM (Table 1). Compound 60c, which contains 3-hydroxy-4-methyl in the “A” phenyl ring, was the most potent compound, with an average IC₅₀ value of 2.4 nM against all cancer cell lines tested. The metabolic stability in human liver microsomes for 59a, 59b, and 60c is 9.6, 33, and 29.4 min, respectively (Table 2). To investigate whether the “B” pyridine ring played an indispensable role in the antiproliferative potency, we replaced the pyridine group with a phenyl group to produce compound 64 (Scheme 5). Compound 64 was much less active against the tested cancer cell lines, with an average IC₅₀ value of 60.8 nM. These results suggested that the pyridine moiety of the “B” ring played an important role in improving its cytotoxicity.

Scheme 5.

Synthesis of Ring B-Modified 6-Aryl-2-Benzoyl-Pyridine Analogues^a

^aReagents and conditions: (a) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O; (b) n-BuLi, THF; (c) Dess–Martin periodinane, CH₂Cl₂; (d) TFA, CH₂Cl_2; (e) n-BuLi, THF; (f) Dess–Martin periodinane, CH₂Cl₂; and (g) Pd(PPh₃)₄, Na₂CO₃, dioxane/H₂O.

In Vitro Microsomal Stability.

Compounds built on the ABP template were anticipated to be deactivated metabolically by methyl oxidation, O-demethylation, and/or ketone reduction. To improve the therapeutic indexes of ABPs, we blocked their metabolism labile sites and designed the novel ABPs analogues described above. We first measured the metabolic stability for two compounds that were demethylated at the “A” ring (4a and 4b). These modifications preserved the potency of the compounds but did not result in improved metabolic stability. To bypass the potential metabolic instability (reduced half-life) in vivo caused by O-demethylation, we designed and synthesized two potent compounds, 7 and 10, that contained an extended methoxyethoxy group in the para-position of the “C” ring (7) and linked two adjacent methoxy moieties into a conformationally restricted 3-methoxybenzo[4,5]-dioxane ring (10). However, compounds 7 and 10 failed to show improvement in the metabolic stability in human liver microsomes. We introduced an ethyl group in the “C” ring at the para-position to generate compound 13, which resulted in improved metabolic stability but reduced potency relative to its parent compound, CH-2–77. Incorporating a metabolically liable ketone into a new metabolically stable “D” ring, as exemplified in compound 22, enhanced metabolic stability. However, compound 22 exhibited reduced antiproliferative activity, possibly due to the cyclization of the carbonyl moiety resulting in a change in the compound’s geometry, leading to loss of its bioactivity. These approaches to block the metabolism of labile sites were expected to impede the potential phase I metabolic reactions but they were not successful. Compounds based on these modifications either lost cytotoxic activity against cancer cells and/or maintained unchanged metabolic stability.

To understand the metabolic patterns and major sites of biological lability exhibited by ABP analogues in liver microsomes, we performed an additional metabolite identity study for parent compounds CH-2–1 and CH-2–77 (Figure 2). For compound CH-2–1, three metabolic pathways were observed when incubated with human liver microsomes. The major metabolic products were M2, which was derived from monohydroxylation of CH-2–1, M1, which was derived from ketone reduction of CH-2–1, and M3, a hydroxylated form of M1. For compound CH-2–77, the major metabolites were M4 derived from monohydroxylation and M5 derived from ketone reduction (Figure 2). These results are consistent with the short half-lives (<10 min) of the parental compounds CH-2–1 and CH-2–77 since ketones are easily reduced and the pyridine “B” ring is prone to be hydroxylated on the nucleophilic attack due to the electron-deficiency effect.

Figure 2.

Proposed metabolites and metabolic pathways of CH-2–1 and CH-2–77.

We next resorted to modifying the “B” ring of the ABP template to adjust for the effects of electron push–pull and thereby to improve metabolic stability while preserving potency. To test this hypothesis, we first synthesized and measured compound 28a, which replaced the pyridine group with an imidazole group, resulting in improved metabolic stability but reduced potency. Strategies to further replace the pyridine “B” ring with a pyrazine ring or to introduce meta-F in the “B” ring resulted in several compounds, including 40a and 60c, which preserved the potency while simultaneously improving the metabolic stability about 3- to 4-fold in human liver microsomes compared to the parent compound CH-2–77. The metabolic stability-enhancing effect of 60c might be attributable to the intermolecular interactions between the fluorine and the ketone moieties, which induces either steric hindrance of metabolism at the ketone moiety or changes the conformation of the molecule and prevents the interaction between the carbonyl group and cytochrome P450 enzymes. These results confirmed our hypothesis that the pyridine “B” ring was subject to modifications that would improve metabolic stability. However, the selection of functional groups to place into the “B” ring was critically important. Our results suggest that it is worth further developing the ABP scaffold as the basis of tubulin inhibitors for anticancer therapy.

In Vitro ADME Toxicity and In Vivo Pharmacokinetic (PK) Study.

We next evaluated the drug-like properties of compound 60c for its aqueous solubility, hERG, and major CYP inhibitions. These studies were performed by Eurofins Panlabs (St. Charles, MO. Study ID: US034–0012825), following vendor’s standard protocols. As shown in Table 3, although compound 60c has a low aqueous solubility of 100 nM, it is not unexpected based on its chemical structure and it is still about 40-fold of its IC₅₀ values in cancer cells (2.4 nM, Table 1). It has essentially no inhibition to the hERG channel at 10 μM, suggesting a good cardiac safety profile. At 10 μM, 60c showed inhibition for several major CYPs, especially for CYP2C8 (92.5%). However, such a high concentration is unlikely physiologically relevant since it is over 4000-fold higher than the antiproliferative IC₅₀ values of 60c. The extent of CYP inhibition is expected to be negligible at nanomolar concentrations. Consistent with the result from the hERG assay, AMES assay supports a good safety profile of 60c since no mutagenic potential was detected in any of the four strains of bacteria. Finally, we evaluated the pharmacokinetic parameters of 60c in mice upon dosing at 10 mg/kg intraperitoneally (Table 3). The terminal elimination half-life (t_1/2) was calculated to be 4.39 h, and the area under the curve from the time of dosing (dosing_time) to the last measurable (positive) concentration (AUClast) was at 4872.07 (h ng)/mL. Moreover, compound 60c displayed a total body clearance for extravascular administration (Cl_F_obs) at 2045.22 (mL h)/kg.

Table 3.

Aqueous Solubility, hERG, CYP, AMES, and Pharmacokinetic Assays of Compound 60c

assay	results
aqueous solubility (PBS, pH 7.4)	0.10 μM
hERG inhibition (at 10 μM)	5.14%
CYP inhibition (at 10 μM)	CYP1A (HLM, phenacetin substrate): 50.9%
	CYP2B6 (HLM, bupropion substrate): 12%
	CYP2C8 (HLM, amodiaquine substrate): 92.5%
	CYP2C9 (HLM, diclofenac substrate): 54.7%
	CYP2C19 (HLM, omeprazole substrate): 49%
	CYP2D6 (HLM, dextromethorphan substrate): 11.9%
	CYP3A (HLM, midazolam substrate): 34.7%
	CYP3A (HLM, testosterone substrate): 51.8%
AMES fluctuation test	TA98: no mutation
	A100: no mutation
	TA1535: no mutation
	TA1537: no mutation
pharmacokinetic parameters of 60c with i.p. injection (10 mg/kg)	t1/2: 4.39 h
	tmax: 0.17 h
	Cmax: 6213.52 ng/mL
	AUClast: 4872.07 (h ng)/mL
	Vz_F_obs: 12 347.41 mL/kg
	Cl_F_obs: 2045.22 (mL h)/kg

Crystallographic Analyses of the 40a- and 60c-Complexes with Tubulin.

To characterize how compounds 40a and 60c bind to the tubulin polymer, we generated the complexes using the tubulin/RB3-SLD assembly (sT2R) known to generate excellent crystals (Figure 3A).²¹ We previously used the T2R-TTL assembly to characterize colchicine-binding-site inhibitors (CBSIs)^13,22 but switched to sT2R, with which we typically obtain higher-resolution structures. We first confirmed that colchicine binds identically into the two assemblies and obtained crystals of the complex that diffracted to 2.5 Å (PDB code: 6XER). As shown in Figure 3B, the sT2R complex is almost identical to the T2R-TTL complex that was determined at 2.9 Å (PDB code: 5XIW). Colchicine occupies its binding pocket within β-tubulin at the interface of the α- and β-tubulin subunits, opposite the GTP molecule bound within the α-subunit (Figures 3C and S1A and Table S1).

Figure 3.

Crystal structures of tubulin–RB3-SLD (sT2R) complexes with colchicine, compound 40a, and compound 60c. Panel A shows the overall crystal structure of the tubulin–RB3-SLD (sT2R) complex with GTP (red), GDP (magenta), and ligand (green). The tubulin α-monomer, β-monomer, and RB3-SLD are shown in cyan, gold, and salmon, respectively. Panel B shows colchicine bound in T2R-TTL (gray, PDB 5XIW) and sT2R (magenta) complexes. Panels C–E show sT2R complexes with colchicine (panel C, resolution 2.5 Å, PDB code: 6XER), compound 40a (panel D, resolution 2.3 Å, PDB code: 6XES), and compound 60c (panel E, resolution 2.6 Å, PDB code: 6XET). Panel F shows superimposed tubulin complexes with colchicine (tubulin in gray and colchicine in magenta) and compound 60c bound (tubulin in cyan and gold and 60c in green). Hydrogen bonds with colchicine and 60c are shown as magenta and green dashed lines, respectively. Panel G shows superimposed compound 40a (blue stick) and compound 60c (green stick). Panel H shows superimposed CH-2–77 (orange, PDB code: 6AGK), 40a, and 60c.

We then crystallized and determined the structures of compounds 40a and 60c bound to sT2R at 2.3 Å (PDB code: 6XES) and 2.6 Å (PDB code: 6XET), respectively. Both compounds bound at the colchicine site (Figures 3D–F and S1B,C and Table S1) and in a very similar fashion (Figure 3G). The key interactions are as follows: the “C” ring that contains the three methoxy groups engaged a hydrophobic pocket comprising residues Cys239, Leu240, Leu246, Ala248, Leu253, Ala314, Ile316, Ala352, and Ile368. Two methoxy oxygen atoms formed hydrogen-bonding interactions with the main-chain carbonyl oxygen of Gly235 and amide nitrogen of Cys239 via a water molecule at the distal end of this pocket. The “A” ring at the opposite end of the molecules also engaged a hydrophobic pocket composed of residues Leu253, Asn256, Met257, Ala314, and Lys350. In addition, Ala180 and Val181 from the α-subunit engage the “A” ring and the hydroxyl substituent forms two hydrogen bond interactions with the main-chain carbonyl oxygen of Asn347 (β-subunit) and the main-chain amide nitrogen of Val181 (α-subunit). Note that Leu253 and Ala314 were able to engage both “A” and “C” rings because compounds 40a and 60c adopted a bent conformation. The central “B” ring (pyrazine in 40a and fluoropyridine in 60c) occupies a pocket bound by residues Leu246, Lys252, Leu253, Asn256, and Lys350 from the β-subunit and residues Asn101, Thr179, and Ala180 from the α-subunit. Finally, the carbonyl group that links rings “B” and “C” forms a hydrogen bond interaction with the main-chain amide nitrogen of Asp249. Compared to the binding mode of CH-2–77 that we previously characterized (PDB code: 6AGK, 2.8 Å),¹³ compounds 40a and 60c bind in very similar ways. The major difference is that the “C” ring is shifted by ~1.3 Å in 40a and 60c, which allows distal water-mediated hydrogen bonds to Gly235 and Cys239 that are not present in the CH-2–77 structure (Figure 3H). This shift may be caused by the interactions of the additional nitrogen in the “B” ring of 40a with Asn101 and the additional fluorine in the “B” ring of 60c with Leu246, Ala248, and Lys252. Thus, the tight interaction between tubulin and compound 40a or 60c mediated by water molecules might contribute to the enhanced metabolic stability of 40a or 60c as compared to that of CH-2–77.

Effects of the Most Potent Compound 60c on Paclitaxel-Resistant Cell Lines.

Recently, we generated two new drug-selected paclitaxel-resistant cell lines by incubating parental A375 or MDA-MB-231 cancer cells with paclitaxel gradually and continually until the cells were stable in growth media containing 100 nM of paclitaxel. These paclitaxel-resistant cells (A375/TxR and MDA-MB-231/TxR) were confirmed by their overexpression of the resistance marker p-glycoprotein (P-gp; encoded by the MDR1gene) by Western blot (Figure S2). The parental A375 and MDA-MB-231 cells and their paclitaxel-resistant daughter cell lines were used to evaluate the effect of the most potent compound 60c on paclitaxel resistance. As shown in Table 4, after 72 h of treatment, A375/TxR and MDA-MB-231/TxR cells were resistant to paclitaxel as expected, with resistance indices of 70 and 131, respectively. In contrast, compound 60c maintained an equivalent potency on A375/TxR or MDA-MB-231/TxR cells, with IC₅₀ values in the low nanomolar range and the resistance indices of 1.04 and 0.36, respectively. Similar results were also observed with 60c treatment of M14 cells and its MDR1-overexpressing daughter cell line M14/LCC6MDR1, both of which are highly resistant to paclitaxel and colchicine. These data demonstrated that 60c was not a substrate of P-gp and thus could overcome paclitaxel resistance, which suggested the potential roles of newly synthesized ABPs in circumventing P-gp-mediated taxane resistance.

Table 4.

In Vitro Cytotoxic Effects of Compounds 60c, Colchicine, and Paclitaxel on Three Parental or Paclitaxel-Resistant Cell Lines^a

	IC50 ± SEM (nM)
cancer cell lines	60c	colchicine	paclitaxel
A375	5.1 ± 0.9	16.8 ± 2.6	1.6 ± 0.2
A375/TxR	5.3 ± 1.2	29.6 ± 4.3	112.1 ± 15.7
RI*	1.04	1.76	70.06
MDA-MB-231	5.8 ± 1.2	24.3 ± 4.2	0.8 ± 0.1
MDA-MB-231/TxR	2.1 ± 0.4	24.0 ± 2.1	105.0 ± 18.2
RI*	0.36	0.99	131.25
M14	2.8 ± 0.4	10.6 ± 1.5	0.5 ± 0.1
M14/LCC6MDR1	4.9 ± 1.0	>1μM	186.3 ± 24.1
RI*	1.8	>94.3	372.6

^aRI*: resistance index is calculated by dividing the IC₅₀ value of the resistant cell lines by the IC₅₀ value of their respective parental cell lines.

Inhibition of Tubulin Polymerization In Vitro by Compound 60c.

Many antimitotic agents bind to microtubules and influence their polymerization and dynamics in multiple ways. We therefore evaluated the tubulin polymerization inhibitory effects of the new ABPs.²³ The most potent compound, 60c, was selected to evaluate the tubulin assembly in a cell-free tubulin polymerization assay with the parental compound CH-2–77 as the reference control. Colchicine and paclitaxel were employed as positive and negative assay controls, respectively. As shown in Figure 4, 10 μM of paclitaxel treatment led to an increased rate of tubulin polymerization by bypassing the nucleation phase, growing microtubules rapidly in the growth phase, and reaching the steady-state phase at a higher OD number at 340 nm, compared to the control group that progressed through three normal phases. However, the presence of 10 μM colchicine or CH-2–77 lengthened the nucleation phase, slowed microtubule growth in the growth state phase, and reduced the final tubulin polymer mass with a significantly low OD number in the steady-state phase, suggesting that they interfered with tubulin polymerization. Compound 60c proved to be a tubulin depolymerizing agent that strongly inhibited microtubule polymerization by the mode of action similar to that of CH-2–77 and colchicine.

Figure 4.

Inhibition of tubulin polymerization by compound 60c at 10 μM. The effect of 60c on an in vitro tubulin polymerization assay was investigated in a tubulin buffer containing a purified porcine brain tubulin (3 mg/mL) and GTP (1 mM) in the presence of DMSO, colchicine, paclitaxel, CH-2–77, or 60c at a concentration of 10 μM each. Colchicine and paclitaxel were used as known inhibitors that enhance tubulin depolymerization and polymerization, respectively. The polymerization of tubulin was monitored at 340 nm for 1 h at 30 s intervals. The curve represents the average of two experiments.

Treatment with Compound 60c Inhibited Colony Formation by Paclitaxel-Resistant Melanoma Cells.

Based on the in vitro cytotoxicity results of all newly synthesized ABPs against six different cancer cell lines and the strong antiproliferating effect of 60c on a panel of paclitaxel-resistant cancer cell lines, compound 60c was prioritized for mechanistic studies in A375/TxR cells. First, we investigated the effect of 60c on the inhibition of cell growth and colony formation of A375/TxR cells by performing a clonogenic assay. As shown in Figure 5A, A375/TxR cells treated with 60c at varying concentrations (2.5, 5, and 10 nM) formed fewer colonies compared with that of the control, colchicine, or paclitaxel groups. Also, treatment with 5 nM compound 60c significantly reduced the size of the colony formation area by 95% compared with that of the control group, indicating that 60c remarkably inhibited the proliferation and colony formation of A375/TxR cells in a concentration-dependent manner (Figure 5B). Treatment of these cells with 60c exhibited similar results as CH-2–77 treatment, suggesting that both compound 60c and CH-2–77 possessed comparable anticolony formation activities.

Figure 5.

Colony formation assay displaying A375/TxR cell colonies after treatment with colchicine, paclitaxel, CH-2–77, or compound 60c. Each seeded single well was split into four wells, and cells were treated for 7 days with 2.5, 5, or 10 nM of each drug. Panel A, representative images of colonies after staining with crystal violet. Panel B, relative colony formation (%) is expressed as the percentage of colony area density in triplicate. ****p < 0.0001 versus control cells of each drug group.

Treatment with Compound 60c Induced G2/M Phase Arrest in A375/TxR Cells.

Antimitotic agents usually affect the progression of the cell cycle and induce cell cycle arrest, thereby influencing cell division and the proliferation of cancer cells. Because compound 60c exhibited a strong antiproliferating activity against a panel of paclitaxel-sensitive and paclitaxel-resistant cancer cell lines, its effect on cell cycle distribution of A375/TxR cells was tested using flow cytometry after the cells were stained with propidium iodide. Cells were treated with 2.5, 5, and 10 nM of compound 60c in complete culture medium for 24 h with 10 nM of colchicine, paclitaxel, or CH-2–77 as positive controls. The distribution of cell cycle phases (G1, S, and G2/M) was compared among the cells from each group. A375/TxR cells treated with colchicine or paclitaxel displayed the normal cell cycle progression in untreated cells as expected since A375/TxR cells overexpressed P-gp, which would expel colchicine or paclitaxel from the cells, thereby negating their effect (Figure 6A,B). In contrast, treatment of cells with 60c resulted in a pronounced dose-dependent accumulation of cells in the G2/M phase and concomitant decrease of cells in G1 and S phases. Cell cycle analysis of A375/TxR cells treated with 10 nM compound 60c showed that major populations of the cells arrested at the G2/M phase (62%), which was comparable to those treated with 10 nM of CH-2–77 (G2/M phase: 60%), while only 12% of untreated cells arrested at the G2/M phase (Figure 6). Together, these results demonstrate that compound 60c potently induced the G2/M phase arrest of paclitaxel-resistant melanoma cells.

Figure 6.

Effect of compound 60c on cell cycle distribution of A375/TxR cells by flow cytometry. (A) Representative images of A375/TxR cells treated with the indicated concentrations of 60c for 24 h followed by cell cycle analysis using propidium iodide (PI) staining. Colchicine, paclitaxel, and CH-2–77 were used as the positive controls. (B) Bar graph shows the percentage of A375/TxR cells in each phase of the cell cycle after treatment with the different inhibitors.

Treatment with Compound 60c Caused Apoptosis in A375/TxR Cells.

The cell cycle analyses showed that compound 60c caused arrest in the G2/M phase. To determine whether the continued 60c treatment would eventually induce cellular apoptosis, we used Annexin V-FITC/PI staining to investigate the effect of 60c on A375/TxR cells. As shown in Figure 7A, both CH-2–77 (10 nM) and 60c (5 or 10 nM) induced apoptosis of A375/TxR cells in comparison with the control, colchicine (10 nM), or paclitaxel (10 nM) groups. Untreated control cells exhibited 8.8% apoptotic cells in necrosis, early apoptosis, and late apoptosis, while cells treated with 10 nM colchicine or 10 nM paclitaxel exhibited 10.6 and 11.8% of apoptotic cells, respectively (Figure 7A,B). However, treatment of the cells with 10 nM CH-2–77 or 10 nM 60c led to a significant increase in the proportion of apoptotic cells (from 8.8 to 51.1 and 54.3%, respectively), demonstrating that compound 60c induced apoptosis with a similar potency to CH-2–77. Moreover, 60c treatment resulted in 14.5, 45.4, and 54.3% apoptotic cells at concentrations of 2.5, 5, and 10 nM, respectively, showing that 60c induced apoptosis of A375/TxR cells in a dose-dependent manner.

Figure 7.

Induction of cellular apoptosis after treatment with compound 60c. (A) Representative flow cytometry images showing A375/TxR cells treated with colchicine, paclitaxel, CH-2–77, or 60c at the indicated concentrations for 24 h followed by staining with Annexin V-FITC and PI. (B) Percentage of apoptotic cells in each group was shown by adding the proportion of cells in early apoptosis (AV⁺PI⁻), later apoptosis (AV⁺PI⁺), and necrosis (AV⁺PI⁻). ****p < 0.0001 significant as compared with control.

Antitumor Activity of 60c in A375/TxR Xenografts.

Considering the potent cytotoxic and apoptotic properties of compound 60c in vitro, its antitumor activity was further assessed in an A375/TxR xenograft model,²⁴ which we have been used extensively in this project with consistent tumor formation and metastasis.^19,22,25 Based on the A375 cell line, we generated a paclitaxel-resistant A375/TxR cell subline and used A375/TxR xenograft model here to evaluate the efficacy of compound 60c in overcoming paclitaxel resistance in vivo. The A375/TxR xenograft model was established by subcutaneously inoculating A375/TxR cells in the logarithmic phase into the right flank of immunodeficient NOD scid γ (NSG) mice. When tumors had grown to approximately 100 mm³, mice were randomly assigned to four groups based on their body weight and tumor volume. Compound 60c was intraperitoneally (IP) administered at a dose of 10 or 20 mg/kg to the mice 3 times/week for 18 days, and paclitaxel was intraperitoneally administered in parallel at a dose of 10 mg/kg to a separate group of the mice. It is evident from Figure 8A,B that 60c treatment showed dose-dependently tumor growth inhibition without weight loss for the entire drug therapy period. At the end of the study, the tumors were excised from the mice, weighed, and imaged. As shown in Figure 8C, the 10 and 20 mg/kg 60c treatment groups exhibited 57.6 and 72.9% reductions in the average tumor volume compared with the vehicle group, respectively, while paclitaxel treatment increased the tumor volume by approximately 5.3%. Similarly, the average tumor weights of 10 mg/kg 60c-treated and 20 mg/kg 60c-treated groups were 1.13 g (tumor growth inhibitory rate 44.4%) and 0.76 g (tumor growth inhibitory rate 62.4%), respectively, which were much less than those of the vehicle-treated mice (average tumor weight 2.0 g) or 10 mg/kg paclitaxel-treated mice (average tumor weight: 2.1 g) (Figure 8D). Imaging of tumors excised from all groups further confirmed the strong antitumor activity of 60c in suppressing A375/TxR tumor growth (Figure 8E). These results indicate that compound 60c could inhibit tumor growth effectively.

Figure 8.

Compound 60c suppressed the growth of melanoma grafts in vivo. (A) Changes in the tumor volume of mice in each group were recorded three times per week during the treatment. Data are shown as mean tumor volume ± SEM. (B) Relative body weight change was monitored three times per week and calculated based on the body weight of each mouse at the time of initiating drug treatment. Dashed lines denote weights relative to the starting point (y-axis = 0) and 20% of weight gain (y-axis = 20). (C, D) At the humane end point of the experiment, the final tumor volume (C) and tumor weight (D) were measured and plotted for each group of mice. Each dot indicates the tumor volume or tumor weight of each tumor, while the error bars represent the mean ± SEM. (E) Representative photograph of tumors excised from each group. (F) Representative H&E staining of tumor sections. Blue arrows indicate necrotic tumor cells. Images were acquired at 20× magnification; scale bar: 50 μm. (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

We next analyzed tumor necrosis in the excised tumor tissue stained with hematoxylin/eosin (H&E) and found that 60c-treated tumors were highly necrotic. As shown in Figure 8F, a number of necrotic tumor cells were observed in both the 10 and 20 mg/kg 60c treatment groups, which was consistent with the in vitro findings, while the cells in vehicle or paclitaxel-treated tumors were all viable. Representative images of whole tumor sections further confirmed that the necrotic area of tumors increased upon 60c treatment as compared to vehicle or paclitaxel treatment (Figure S3A).

Treatment with Compound 60c Attenuated Spontaneous Metastasis to Major Organs without Appreciable Toxicity.

While removing organs from xenograft mice, we detected axillary lymph node metastases and the presence of multiple tumor nodules in lungs or livers from mice treated with vehicle or paclitaxel, suggesting that A375/TxR cells were highly metastatic. We evaluated the antimetastasis effect of compound 60c by performing H&E staining to detect metastasis in lungs and livers. As shown in Figure 9A,B, treatment with 60c significantly inhibited the spontaneous melanoma metastasis to lungs and livers and suppressed liver metastases in a dose-dependent manner. Although all of the mice in the study have spontaneous lung metastasis, large lung metastatic nodules and extensive metastatic burden were observed only in the lungs of the vehicle- and paclitaxel-treated groups and lung nodules were markedly sparse and of reduced size in the animals treated with 60c (Figures 9C and S3B). In this model, 83% of vehicle-treated mice and 83% of paclitaxel-treated mice formed developed liver metastases. Only 33% of 10 mg/kg 60c-treated mice and none of the 20 mg/kg 60c-treated mice developed liver metastases. These results indicate that compared with the vehicle or paclitaxel groups, 60c treatment strikingly reduced the incidence of A375/TxR liver metastasis in the mice (Figure 9B). In addition, the liver metastases detected in mice treated with 10 mg/kg 60c were of significantly reduced size and number compared to those observed in mice treated with vehicle or paclitaxel, as exhibited in Figures 9C and S3B.

Figure 9.

Compound 60c inhibited lung and liver spontaneous metastases in the A375/TxR xenograft model. (A, B) At the study’s humane end point, lung and liver tissues from all of the mice in the study were collected and stained with H&E. Lung (A) and liver (B) metastases were manually scored by counting the number of metastases present in each whole tissue section. Data shown represent the number of metastatic foci present in six mice from each treatment group. (C) Representative images showing metastatic nodules from lungs (top) or livers (bottom) in each group. Lung metastases are indicated by blue arrows, while liver metastases are circled with blue dotted lines. Scale bar: 500 μm.

We evaluated systemic toxicity of 60c by comparing histological images of major organs (lung, liver, kidney, heart, and spleen) collected in this experiment by staining with H&E. Since lung and liver tissues from these mice contain many metastatic nodules, we acquired representative images from areas outside of the metastatic area. H&E-stained tissue sections exhibited no apparent pathological changes in the major organs between groups treated with vehicle, paclitaxel, 10 mg/kg 60c, or 20 mg/kg 60c (Figure 10). In short, these results indicated that compound 60c was efficacious in suppressing the growth and metastasis of A375/TxR xenografts with low systemic toxicity in vivo.

Figure 10.

Representative H&E-stained images of several healthy organ tissue sections (heart, lung, liver, kidney, and spleen) for monitoring systemic toxicity of compound 60c in A375/TxR tumor-bearing mice. Scale bar: 50 μm.

CONCLUSIONS

In summary, we have reported the design and synthesis of four series of new ABP derivatives by blocking the methyl oxidation in the “A” ring, ketone reduction, demethylation in the “C” ring, or modifications of the pyridine “B” ring of the ABP template. Structure–activity relationships (SARs) of these compounds led to the identification of lead analogue compound 60c, which showed improved metabolic stability on human liver microsomes while maintaining potent antiproliferative activity with IC₅₀ values of 1–3 nM against a panel of human melanoma and breast cancer cell lines. Here, we also described the high-resolution crystal structures of representative compounds 40a and 60c in complex with tubulin, confirming their direct binding to the colchicine-binding site in tubulin and providing further insight into their molecular interactions with tubulin. Moreover, compound 60c induced tubulin depolymerization, arrested the cell cycle in the G2/M phase, and induced apoptosis. Most interestingly, compound 60c effectively overcame resistance to paclitaxel and inhibited the growth and metastasis of A375/TxR xenografts in mice in vivo and thus represents a promising new generation of tubulin inhibitor that merits further investigation.

EXPERIMENTAL SECTION

Chemistry.

Moisture-sensitive reactions were performed in oven-dried glassware under an inert atmosphere of argon. All chemical reagents and solvents were purchased from Fisher Scientific (Pittsburgh, PA), Sigma-Aldrich Chemical Co. (St. Louis, MO), Ark Pharm (Libertyville, IL), or Oakwood Products (West Columbia, SC) and were used directly without further purification. Analytical thin-layer chromatography was performed on silica gel 60 GF254 (Sigma-Aldrich) and was visualized by fluorescence quenching under UV light. A Biotage (Charlotte, NC) SP1 flash chromatography purification system (Biotage SNAP cartridges, silica, 4, 12, 25, 40, and 80 g) was used to purify the compounds. Melting points were recorded on a Fisher–Johns melting point apparatus (uncorrected). All of the tested compounds were characterized and confirmed by traditional analytical chemistry methods, including ¹H NMR, ¹³C NMR, HRMS, and UPLC. ¹H NMR and ¹³C NMR spectra were collected on a Bruker Ascend 400 instrument (¹H, 400 MHz; ¹³C, 101 MHz). Chemical shift values are reported in ppm on the δ scale, and the following abbreviations are used: singlet (s), doublet (d), doublet of doublet (dd), triplet (t), quadruplet (q), and multiplet (m). High-resolution mass spectrometry (HRMS) data were obtained using a Waters Xevo G2-S qTOF mass spectrometer (Waters, Milford, MA) equipped with a Waters Acquity M-Class UPLC system. The purity of all tested compounds was determined to be ≥95% by ¹H NMR and UPLC. The UPLC method used to determine purity is as follows: analytical acquity ultraperformance liquid chromatography (UPLC) on a BEH C18 (2.1 50 mm, 1.7 μm) column using a mixture of solvent acetonitrile/water (with 0.1% formic acid) at a flow rate of 0.9 mL/min and monitored by a photodiode assay detector, which acquired UV absorption from 210 to 400 nm. All reported yields were not optimized.

(6-Bromopyridin-2-yl)(3,4,5-trimethoxyphenyl)methanol (2) and (6-Bromopyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (3).

Compounds 2 and 3 were prepared following the same procedures described in the previously published literature.¹³

General Procedure for the Synthesis of 6-Aryl-2-benzoyl-pyridines (4a–d).

To a solution of (6-bromopyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (0.2 mmol) and different aryl boronic acid or aryl boronic acid pinacol (BPin) ester reagents (0.2 mmol) in 1,4-dioxane/H₂O (60 mL, v/v = 2/1) were added Pd(PPh₃)₄ and Na₂CO₃ (0.4 mmol). The reaction mixture was refluxed overnight under an argon atmosphere. The solvent was removed, 50 mL of water was added, and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure to yield a crude product, which was purified by column chromatography to obtain pure compounds 4a–d.

(6-(4-(Difluoromethyl) phenyl) pyridin-2-yl) (3,4,5-trimethoxyphenyl)methanone (4a).

Light-yellow solid; 47% yield. Mp: 131–133 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.19 (d, J = 8.0 Hz, 2H), 8.02 (ddd, J = 14.8, 4.8, 1.7 Hz, 3H), 7.71−7.42 (m, 4H), 6.71 (t, J = 56.4 Hz, 1H), 3.98 (d, J = 0.9 Hz, 3H), 3.88 (d, J = 0.9 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 191.51, 155.27, 154.50, 152.67, 142.75, 140.61, 138.17, 135.54, 135.32, 135.10, 131.09, 127.11, 126.19, 126.13, 123.70, 122.55, 116.81, 114.43, 112.06, 109.13, 61.01, 56.27. HRMS [C₂₂H₂₀F₂NO₄⁺]: calcd 400.1360, found 400.1356 (mass error −1.0 ppm). UPLC purity 98.6% (t_R = 1.09 min).

(6-(Indolin-6-yl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (4b).

Light-yellow solid; 67% yield. Mp: 86–88 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.01−7.80 (m, 3H), 7.65 (d, J = 1.3 Hz, 2H), 7.42 (d, J = 7.7 Hz, 1H), 7.36 (s, 1H), 7.18 (d, J = 7.6 Hz, 1H), 3.97 (d, J = 1.3 Hz, 3H), 3.89 (d, J = 1.3 Hz, 7H), 3.59 (t, J = 8.3 Hz, 2H), 3.06 (t, J = 8.4 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 191.86, 156.09, 154.74, 152.54, 152.49, 142.35, 137.73, 131.31, 131.22, 124.75, 122.71, 122.22, 117.59, 109.04, 107.26, 60.98, 56.22, 47.53, 29.60. HRMS [C₂₃H₂₃N₂O₄⁺]: calcd 391.1658, found 391.1656 (mass error −0.5 ppm). UPLC purity 95% (t_R = 0.67 min).

(6-(4-(Dimethylamino) phenyl) pyridin-2-yl) (3,4,5-trimethoxyphenyl)methanone (4c).

Off-white solid; 72% yield. Mp: 151–153 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.17−7.93 (m, 2H), 7.86 (d, J = 1.2 Hz, 3H), 7.67 (d, J = 0.9 Hz, 2H), 6.76 (d, J = 8.7 Hz, 2H), 3.98 (d, J = 0.9 Hz, 3H), 3.90 (d, J = 0.9 Hz, 6H), 3.02 (d, J = 0.9 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 192.09, 155.80, 154.67, 152.53, 151.31, 142.26, 137.61, 131.49, 127.73, 125.95, 121.52, 120.81, 112.04, 109.11, 61.02, 56.25, 40.32. HRMS [C₂₃H₂₅N₂O₄⁺]: calcd 393.1814, found 393.1804 (mass error −2.5 ppm). UPLC purity 98.1% (t_R = 1.13 min).

N-(2-Methyl-5-(6-(3,4,5-trimethoxybenzoyl)pyridin-2-yl)phenyl)-acetamide (4d).

Off-white solid; 77% yield. Mp: 190–192 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.50−8.29 (m, 1H), 8.00−7.85 (m, 4H), 7.62 (s, 2H), 7.29 (s, 1H), 7.17 (s, 1H), 3.97 (s, 3H), 3.88 (s, 6H), 2.30 (s, 3H), 2.23 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 191.86, 168.59, 155.23, 154.92, 152.61, 142.42, 137.94, 137.07, 136.15, 131.37, 131.25, 131.09, 123.98, 123.12, 122.50, 122.03, 109.00, 61.01, 56.25, 29.71, 24.29, 17.79. HRMS [C₂₄H₂₅N₂O₅⁺]: calcd 421.1763, found 421.1764 (mass error 0.2 ppm). UPLC purity 97.8% (t_R = 0.8 min).

(6-(p-Tolyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (5).

Compound 5 was prepared from (6-bromopyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (compound 3; 1.2 g, 3.4 mmol) and p-tolylboronic acid (0.56 g, 4.1 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 1 g (81% yield) of pure product 5 was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 8.05−7.89 (m, 5H), 7.68 (s, 2H), 7.34−7.21 (m, 2H), 3.97 (s, 3H), 3.89 (s, 6H), 2.41 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 191.67, 155.60, 154.97, 152.59, 142.51, 139.67, 137.86, 135.54, 131.29, 129.58, 126.67, 122.82, 121.97, 109.17, 61.00, 56.25, 21.31.

(4-Hydroxy-3,5-dimethoxyphenyl)(6-(p-tolyl)pyridin-2-yl)-methanone (6).

To a solution of compound 5 (1 g, 2.8 mmol) in anhydrous CH₂Cl₂ (100 mL) was added AlCl₃ (0.75 g, 5.6 mmol). The reaction mixture was stirred at room temperature until the starting materials disappeared on TLC and then was quenched with water (30 mL). The resulting mixture was extracted with CH₂Cl₂ (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to produce a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 2:1) to yield key intermediate 6 as a white solid (0.6 g, 61% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.01−7.79 (m, 5H), 7.67 (s, 2H), 7.26−7.09 (m, 2H), 3.86 (s, 6H), 2.34 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 190.26, 154.39, 154.23, 145.33, 138.65, 138.61, 136.84, 134.52, 128.54, 126.48, 125.59, 121.81, 120.75, 108.04, 55.38, 20.29.

(3,5-Dimethoxy-4-(2-methoxyethoxy)phenyl)(6-(p-tolyl)pyridin-2-yl)methanone (7).

To a solution of compound 6 (0.1 g, 0.28 mmol) in DMF (100 mL) was added Cs₂CO₃ (0.18 g, 0.56 mmol). The mixture was stirred for 1 h at room temperature, and then 1-bromo-2-methoxyethane (78 mg, 0.56 mmol) was added. The reaction mixture was stirred at room temperature until starting materials disappeared on TLC and was then quenched with water (30 mL). The resulting mixture was extracted with CH₂Cl₂ (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to produce compound 7 as a white solid (80 mg, 70% yield). Mp: 63–65 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.07−7.82 (m, 5H), 7.65 (d, J = 1.7 Hz, 2H), 7.28 (s, 2H), 4.26 (t, J = 4.7 Hz, 2H), 3.88 (d, J = 1.6 Hz, 6H), 3.80−3.63 (m, 2H), 3.46 (d, J = 1.6 Hz, 3H), 2.41 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 191.83, 155.58, 154.92, 152.82, 141.38, 139.70, 137.91, 135.51, 131.47, 129.60, 126.66, 122.85, 122.00, 109.00, 72.12, 71.82, 59.09, 56.23, 21.34. HRMS [C₂₄H₂₆NO⁺₅]: calcd 408.1811, found 408.1805 (mass error −1.5 ppm). UPLC purity 96.1% (t_R = 1.18 min).

(6-Bromopyridin-2-yl)(8-methoxy-2,3-dihydrobenzo[b][1,4]-dioxin-6-yl)methanol (8).

Compound 8 was prepared from 2,6-dibromopyridine (1.0 g, 4.3 mmol) and 8-methoxy-2,3-dihydrobenzo-[b][1,4]dioxine-6-carbaldehyde (0.83 g, 4.3 mmol) following the same procedure as described in the preparation of compound 2. The crude compound was used for the next step without further purification.

(6-Bromopyridin-2-yl)(8-methoxy-2,3-dihydrobenzo[b][1,4]-dioxin-6-yl)methanone (9).

Compound 9 was prepared from (6-bromopyridin-2-yl)(8-methoxy-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanol (0.68 g, 1.9 mmol) and Dess–Martin periodinane (DMP) (1.6 g, 3.8 mmol) following the same procedure as described in the preparation of compound 3. The crude compound was used for the next step without further purification.

(8-Methoxy-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)(6-(p-tolyl)-pyridin-2-yl)methanone (10).

Compound 10 was prepared from (6-bromopyridin-2-yl)(8-methoxy-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)methanone (0.1 g, 0.3 mmol) and p-tolylboronic acid (49 mg, 0.36 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 73 mg (67% yield) of pure product 10 was obtained as a white solid upon purification. Mp: 133–135 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, J = 8.0 Hz, 2H), 7.93−7.84 (m, 3H), 7.57 (s, 2H), 7.28 (d, J = 8.0 Hz, 2H), 4.47−4.34 (m, 2H), 4.34−4.22 (m, 2H), 3.93 (s, 3H), 2.41 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 191.69, 155.80, 155.29, 148.51, 143.26, 139.49, 137.85, 137.71, 135.73, 129.57, 128.42, 126.83, 122.55, 121.89, 115.30, 106.95, 64.99, 64.00, 56.29, 21.31. HRMS [C₂₂H₂₀NO₄⁺]: calcd 362.1392, found 362.1392 (mass error 0 ppm). UPLC purity 98.3% (t_R = 1.12 min).

(6-Bromopyridin-2-yl)(4-ethyl-3,5-dimethoxyphenyl)methanol (11).

Compound 11 was prepared from 2,6-dibromopyridine 1 (1.0 g, 4.3 mmol) and 4-ethyl-3,5-dimethoxybenzaldehyde (0.83 g, 4.3 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.5 g (33% yield) of pure product 11 was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 8.47 (dt, J = 4.9, 1.3 Hz, 1H), 7.54 (td, J = 7.7, 1.8 Hz, 1H), 7.12−7.03 (m, 1H), 6.48 (s, 2H), 5.62 (s, 1H), 3.69 (s, 6H), 2.53 (q, J = 7.5 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 159.92, 157.06, 146.70, 140.61, 135.88, 121.41, 120.28, 119.21, 101.50, 74.35, 54.67, 15.24, 12.69.

(6-Bromopyridin-2-yl)(4-ethyl-3,5-dimethoxyphenyl)methanone (12).

Compound 12 was prepared from (6-bromopyridin-2-yl)(4-ethyl-3,5-dimethoxyphenyl)methanol (0.5 g, 1.4 mmol) and DMP (1.2 g, 2.8 mmol) following the same procedure as described in the preparation of compound 3. The crude compound was used for the next step without further purification.

(4-Ethyl-3,5-dimethoxyphenyl)(6-(3-hydroxy-4-methylphenyl)-pyridin-2-yl)methanone (13).

Compound 13 was prepared from (6-bromopyridin-2-yl)(4-ethyl-3,5-dimethoxyphenyl)methanone (0.1 g, 0.3 mmol) and 3-hydroxy-4-methylbenzeneboronic acid (54.7 mg, 0.36 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 40 mg (35% yield) of pure product 13 was obtained as a white solid upon purification. Mp: 110–112 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.97−7.71 (m, 3H), 7.60−7.33 (m, 4H), 7.18 (d, J = 7.8 Hz, 1H), 5.79 (s, 1H), 3.81 (s, 6H), 2.69 (q, J = 7.4 Hz, 2H), 2.27 (s, 3H), 1.08 (t, J = 7.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 193.03, 157.55, 155.44, 154.96, 154.58, 137.77, 137.35, 134.46, 131.32, 127.11, 125.86, 122.99, 122.10, 118.80, 113.20, 107.15, 55.89, 16.77, 15.80, 13.37. HRMS [C₂₃H₂₄NO₄⁺]: calcd 378.1705, found 378.1713 (mass error 2.1 ppm). UPLC purity 97.4% (t_R = 1.26 min).

6-(p-Tolyl)-1H-pyrrolo[2,3-b]pyridine (15).

To a solution of 6-bromo-1H-pyrrolo[2,3-b]pyridine (1.0 g, 5.1 mmol) and p-tolylboronic acid (0.83 g, 6.1 mmol) in DME/H₂O (60 mL, v/v = 2/1) were added Pd(PPh₃)₄ and Cs₂CO₃ (3.3 g, 10 mmol). The reaction mixture was refluxed overnight under an argon atmosphere. The solvent was removed, 50 mL of water was added, and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to give the crude intermediate 15 as a white solid (0.42 g, 40% yield). The crude compound was used for the next step without further purification.

6-(p-Tolyl)-1-(3,4,5-trimethoxyphenyl)-1H-pyrrolo[2,3-b]pyridine (16).

To a solution of 6-(p-tolyl)-1H-pyrrolo[2,3-b]pyridine (0.2 g, 1 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.37 g, 1.5 mmol) in toluene/H₂O (60 mL, v/v = 10/1) were added Pd(dppf)Cl₂·CH₂Cl₂ and sodium tert-butoxide (0.38 g, 4 mmol). The reaction mixture was refluxed overnight under an argon atmosphere. The solvent was removed, 50 mL of water was added, and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to give compound 16 as a white solid (97 mg, 26% yield). Mp: 108–110 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (dd, J = 18.8, 8.1 Hz, 3H), 7.62 (d, J = 8.1 Hz, 1H), 7.52 (d, J = 3.7 Hz, 1H), 7.23 (d, J = 5.4 Hz, 4H), 6.69−6.47 (m, 1H), 3.95 (s, 6H), 3.91 (s, 3H), 2.38 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 153.48, 151.33, 147.51, 138.27, 137.37, 136.05, 134.65, 129.65, 129.38, 127.66, 126.71, 120.36, 113.66, 109.24, 101.75, 101.09, 61.07, 56.30, 21.26. HRMS [C₂₃H₂₃N₂O₃⁺]: calcd 375.1709, found 375.1714 (mass error 1.3 ppm). UPLC purity 95.2% (t_R = 1.36 min).

5-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-1H-pyrazolo[4,3-b]pyridine (18).

Compound 18 was prepared from 5-bromo-1H-pyrazolo[4,3-b]pyridine (1 g, 5.1 mmol) and trimethyl(2-((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-methoxy)ethyl)silane (2.2 g, 6.1 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.63 g (35% yield) of product 18 was obtained as a white solid. The crude compound was used for the next step without further purification.

3-Bromo-5-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)-1H-pyrazolo[4,3-b]pyridine (19).²⁶

To a solution of compound 18 (0.63 g, 1.8 mmol) in THF (100 mL) was added NBS (0.36 g, 2 mmol). The reaction mixture was stirred at room temperature until the starting materials disappeared on TLC and was then quenched with water (30 mL). The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was used for the next step without further purification.

3-Bromo-5-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)-1-((2(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]-pyridine (20).

To a solution of compound 19 (0.3 g, 0.5 mmol) in THF (50 mL) was added NaH (40 mg, 1 mmol). The mixture was stirred for 1 h at 0 °C, and then SEMCl (0.17 g, 1 mmol) was added. The reaction mixture was stirred at room temperature until the starting materials disappeared on TLC and was then quenched with water (30 mL). The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to produce the crude intermediate 20 as a white solid (0.23 g, 80% yield). The crude compound was used for the next step without further purification.

5-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[4,3-b]pyridine (21).

Compound 21 was prepared from compound 20 (0.23 g, 0.4 mmol) and (3,4,5-trimethoxyphenyl)-boronic acid (0.1 g, 0.48 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.13 g (51% yield) of pure product 21 was obtained as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (s, 2H), 7.96 (d, J = 8.8 Hz, 1H), 7.89−7.75 (m, 2H), 7.73−7.46 (m, 1H), 7.38−7.14 (m, 1H), 5.81 (d, J = 13.9 Hz, 2H), 5.36 (d, J = 4.1 Hz, 2H), 4.05 (s, 6H), 3.94 (s, 3H), 3.88−3.75 (m, 2H), 3.69−3.38 (m, 2H), 2.31 (d, J = 7.3 Hz, 3H), 1.13−0.78 (m, 4H), 0.00 (s, 9H), −0.04 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 157.40, 155.01, 154.74, 144.64, 141.95, 140.10, 139.53, 134.61, 132.45, 129.91, 129.37, 121.69, 120.35, 119.73, 113.80, 108.68, 105.67, 94.26, 79.72, 68.07, 67.74, 62.40, 57.60, 19.47, 19.11, 17.72.

2-Methyl-5-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[4,3-b]-pyridin-5-yl)phenol (22).

To a solution of compound 21 (0.13 g, 0.2 mmol) in CH₂Cl₂ (10 mL) was added TFA (2 mL). The reaction mixture was stirred at room temperature until the starting materials disappeared on TLC and was then quenched with water (30 mL). The resulting mixture was extracted with CH₂Cl₂ (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to produce compound 22 as a white solid (51.6 mg, 66% yield). Mp: 170–172 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.40 (s, 1H), 9.51 (d, J = 3.6 Hz, 1H), 8.46−7.81 (m, 4H), 7.67 (dd, J = 4.1, 1.8 Hz, 1H), 7.54 (ddd, J = 7.7, 5.3, 1.8 Hz, 1H), 7.32−7.14 (m, 1H), 3.95 (s, 6H), 3.76 (s, 3H), 2.20 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 155.80, 153.04, 152.11, 141.51, 141.31, 138.23, 138.10, 137.21, 133.15, 130.92, 128.35, 124.90, 119.71, 118.20, 117.40, 112.80, 103.39, 60.09, 54.89, 15.86. HRMS [C₂₂H₂₂N₃O₄⁺]: calcd 392.1610, found 392.1610 (mass error 0 ppm). UPLC purity 95.6% (t_R = 0.83 min).

2,4,5-Tribromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole (24).

To a solution of compound 23 (3.0 g, 9.9 mmol) in THF (150 mL) was added NaH (0.6 g, 15.0 mmol). The mixture was stirred for 1 h at 0 °C, and then SEMCl (2.5 g, 15.0 mmol) was added. The reaction mixture was stirred at room temperature until the starting materials disappeared on TLC and was then quenched with water (30 mL). The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to produce the key intermediate 24 as a white solid (3.7 g, 87% yield). ¹H NMR (400 MHz, CDCl₃) δ 5.33 (d, J = 1.3 Hz, 2H), 3.71−3.35 (m, 2H), 1.04−0.79 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 120.42, 118.89, 107.05, 77.19, 68.57, 19.09.

4,5-Dibromo-2-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)-1-((2(trimethylsilyl)ethoxy)methyl)-1H-imidazole (25).

Compound 25 was prepared from compound 24 (3.7 g, 8.6 mmol) and trimethyl(2-((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methoxy)ethyl)silane (3.1 g, 8.6 mmol) following the same procedure as described in the preparation of compound 4a. The regioisomers at the 4 or 5 positions of 2,4,5-tribromo-1-[[2-(trimethylsilyl)ethoxy]methyl]-1H-imidazole were detected as minor byproducts.¹⁹ An amount of 1.6 g (32% yield) of product 25 was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(5-Bromo-2-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-(3,4,5-trimethoxyphenyl)methanone (26).

To a solution of compound 25 (1.6 g, 2.7 mmol) in THF (100 mL) was added iPrMgBr dropwise (2.5 M, 3.2 mmol) at room temperature. The mixture was stirred for 1 h, and then 3,4,5-trimethoxybenzoyl chloride (0.8 g, 3.5 mmol) in THF was added dropwise. The reaction mixture was stirred at room temperature for 1 h and then refluxed for 30 min. The mixture was quenched with water (30 mL), and the resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to produce the key intermediate 26 as a white solid (0.76 g, 40% yield). ¹H NMR(400 MHz, CDCl₃) δ 7.49 (s, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.32−7.25 (m, 1H), 7.22 (d, J = 1.3 Hz, 2H), 5.61 (s, 2H), 5.32 (s, 2H), 3.96 (s, 3H), 3.93 (s, 6H), 3.77 (t, J = 8.0 Hz, 2H), 3.45 (t, J = 8.4 Hz, 2H), 2.31 (s, 3H), 0.96 (t, J = 8.5 Hz, 2H), 0.81 (t, J = 8.4 Hz, 2H), −0.08 (s, 9H), −0.14 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 187.38, 157.01, 154.57, 154.24, 144.60, 133.59, 132.57, 131.64, 129.72, 128.34, 124.43, 124.01, 116.09, 109.33, 94.26, 75.30, 68.18, 67.96, 62.66, 57.89, 19.63, 19.45, 18.01.

(5-Bromo-2-(3-hydroxy-4-methylphenyl)-1H-imidazol-4-yl)-(3,4,5-trimethoxyphenyl)methanone (29).

Compound 29 was prepared from compound 26 (0.1 g, 0.14 mmol) and TFA (2 mL) following the same procedure as described in the preparation of compound 22. An amount of 43.7 mg (70% yield) of pure product 29 was obtained as a white solid upon purification. Mp: 230–232 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.56 (s, 1H), 9.64 (s, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.0 Hz, 1H), 7.13 (s, 2H), 3.86 (s, 6H), 3.78 (s, 3H), 2.17 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 183.46, 155.57, 152.56, 149.27, 141.37, 132.13, 130.96, 128.40, 126.99, 126.53, 122.64, 116.80, 112.45, 107.00, 60.17, 55.99, 15.97. HRMS [C₂₀H₂₀BrN₂O₅⁺]: calcd 447.0556, found 447.0549 (mass error −1.6 ppm). UPLC purity 96.7% (t_R = 0.76 min).

(2-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-1-((2-(trimethylsilyl)ethoxy) methyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (27a).

To a solution of compound 26 (0.3 g, 0.42 mmol) in MeOH (100 mL) was added Pd/C (20 mg). The reaction mixture was stirred at room temperature under H₂ until the starting materials disappeared on TLC and then was filtered and evaporated to yield a crude product, which was used for the next step without further purification.

(2-(3-Hydroxy-4-methylphenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)methanone (28a).

Compound 28a was prepared from compound 27a (0.19 g, 0.3 mmol) and TFA (2 mL) following the same procedure as described in the preparation of compound 22. An amount of 76 mg (69% yield) of pure product 28a was obtained as a white solid upon purification. Mp: 241–243 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.30 (d, J = 79.3 Hz, 1H), 9.61 (d, J = 12.0 Hz, 1H), 8.19−7.05 (m, 6H), 3.89 (s, 6H), 3.78 (s, 3H), 2.17 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 184.90, 182.28, 155.69, 155.48, 152.74, 152.32, 150.94, 146.81, 140.95, 140.92, 139.18, 133.15, 132.98, 131.43, 130.89, 130.86, 128.52, 127.92, 126.21, 126.13, 125.33, 117.95, 117.10, 115.89, 112.76, 111.54, 107.55, 106.01, 60.10, 55.94, 55.88, 15.98, 15.93. HRMS [C₂₀H₂₁N₂O₅⁺]: calcd 369.1450, found 369.1456 (mass error 1.6 ppm). UPLC purity 96.5% (t_R = 0.56 min).

(5-Methyl-2-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-(3,4,5-trimethoxyphenyl)methanone (27b).

To a solution of compound 26 (0.3 g, 0.42 mmol) and methylboronic acid (75.6 mg, 1.26 mmol) in dioxane/H₂O (60 mL, v/v = 10/1) were added Pd(dppf)Cl₂·CH₂Cl₂ and Cs₂CO₃ (0.41 g, 1.26 mmol). The reaction mixture was refluxed overnight under an argon atmosphere. The solvent was removed, 50 mL of water was added, and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 3:1) to produce the crude intermediate 27b as a white solid (0.11 g, 41% yield). The crude compound was used for the next step without further purification.

(2-(3-Hydroxy-4-methylphenyl)-5-methyl-1H-imidazol-4-yl)-(3,4,5-trimethoxyphenyl)methanone (28b).

Compound 28b was prepared from compound 27b (0.11 g, 0.17 mmol) and TFA (2 mL) following the same procedure as described in the preparation of compound 22. An amount of 50 mg (77% yield) of pure product 28b was obtained as a white solid upon purification. Mp: 190–191 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.52−12.73 (m, 1H), 9.60 (d, J = 11.4 Hz, 1H), 8.23−7.05 (m, 5H), 3.87 (dd, J = 6.6, 1.3 Hz, 6H), 3.77 (d, J = 1.6 Hz, 3H), 2.5 (s, 3H), 2.16 (d, J = 4.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 185.80, 155.69, 152.74, 152.32, 152.06, 146.81, 143.81, 140.94, 140.61, 138.40, 135.98, 133.51, 130.88, 128.59, 125.03, 115.90, 115.60, 111.54, 111.13, 107.93, 107.54, 106.01, 60.10, 55.94, 55.88, 55.82, 15.93, 11.91. HRMS [C₂₁H₂₃N₂O₅ ⁺]: calcd 383.1607, found 383.1600 (mass error −1.8 ppm). UPLC purity 97.3% (t_R = 0.55 min).

2-Chloro-6-(1,3-dithian-2-yl)pyrazine (31).

To a solution of 1,3-dithiane (3.65 g, 19 mmol) in THF (150 mL) was added BuLi (2.5 M, 9 mL). The mixture was stirred for 1 h at −78 °C, and then compound 30 (3 g, 20 mmol) in THF (20 mL) was added dropwise. The reaction mixture was warmed to room temperature and stirred until the starting materials disappeared on TLC and then was quenched with NH₄Cl solution (30 mL). The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 4:1) to produce the key intermediate 31 as a white solid (3.5 g, 80% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, 1H), 8.53 (s, 1H), 5.25 (s, 1H), 3.21−2.79 (m, 4H), 2.20 (dtt, J = 14.2, 5.7, 2.8 Hz, 1H), 2.05 (dtt, J = 14.1, 10.7, 3.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 154.23, 148.53, 143.86, 141.36, 48.30, 30.54, 24.83.

6-Chloropyrazine-2-carbaldehyde (32).²⁷

To a solution of compound 31 (3.5 g, 15 mmol) in CH₃CN/H₂O (150 mL, v/v = 2/1) were added Ca₂CO₃ (15.4 g, 60 mmol) and MeI (10.7 g, 75 mmol). The mixture was stirred at 60 °C until the starting materials disappeared on TLC, and then CH₃CN was removed. The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 4:1) to produce the key intermediate 32 as a white solid (0.64 g, 30% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.09 (d, J = 0.8 Hz, 1H), 9.07 (s, 1H), 8.84 (s, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 190.89, 149.78, 148.89, 145.98, 140.73.

6-(p-Tolyl)pyrazine-2-carbaldehyde (33).

Compound 33 was prepared from compound 32 (0.64 g, 4.5 mmol) and p-tolylboronic acid (0.73 g, 5.4 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.18 g (20% yield) of pure product 33 was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 10.22 (s, 1H), 9.20 (s, 1H), 9.05 (s, 1H), 8.01 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 2.45 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 193.11, 152.76, 146.03, 145.35, 141.08, 140.76, 132.32, 130.02, 126.98, 21.44.

(6-(p-Tolyl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanol (34).

Compound 34 was prepared from compound 33 (0.18 g, 0.9 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.22 g, 0.9 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.18 g (55% yield) of pure product 34 was obtained as a white solid upon purification (2.6 g, 87% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.90 (s, 1H), 8.44 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 6.68 (s, 2H), 5.82 (d, J = 3.7 Hz, 1H), 4.92 (d, J = 4.0 Hz, 1H), 3.83 (dd, J = 5.1, 0.8 Hz, 9H), 2.44 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 155.38, 153.53, 150.51, 140.87, 140.55, 140.23, 137.83, 137.65, 137.48, 132.92, 129.87, 126.85, 103.94, 74.02, 60.84, 56.16, 21.40.

(6-(p-Tolyl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanone (35).

Compound 35 was prepared from compound 34 (0.18 g, 0.5 mmol) and DMP (0.42 g, 1 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.13 g (70% yield) of pure product 35 was obtained as a white solid upon purification. Mp: 162–163 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.21 (s, 1H), 9.13 (s, 1H), 8.02 (d, J = 7.9 Hz, 2H), 7.62 (d, J = 1.0 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 3.99 (s, 3H), 3.90 (s, 6H), 2.44 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 190.37, 152.78, 150.24, 149.13, 143.65, 143.21, 143.10, 140.90, 132.62, 130.61, 129.96, 126.77, 108.89, 61.04, 56.28, 21.41. HRMS [C₂₁H₂₁N₂O₄⁺]: calcd 365.1501, found 365.1499 (mass error −0.5 ppm). UPLC purity 97.5% (t_R = 1.08 min).

2-(1,3-Dithian-2-yl)-6-(4-methyl-3-((2-(trimethylsilyl)ethoxy)-methoxy)phenyl)pyrazine (36a).

Compound 36a was prepared from compound 31 (1 g, 4.3 mmol) and trimethyl(2-((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methoxy)ethyl)silane (1.9 g, 5.2 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 1.3 g (70% yield) of pure product 36a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 8.90 (d, J = 1.3 Hz, 1H), 8.62 (d, J = 1.3 Hz, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 7.3 Hz, 1H), 5.47−5.17 (m, 3H), 3.91−3.72 (m, 2H), 3.15 (ddd, J = 14.3, 5.6, 2.9 Hz, 2H), 3.09−2.89 (m, 2H), 2.29 (s, 3H), 2.19 (tt, J = 6.0, 2.7 Hz, 1H), 2.12−1.95 (m, 1H), 1.11−0.89 (m, 2H) 0.0 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 157.49, 155.01, 152.87, 142.70, 142.11, 136.21, 132.62, 130.75, 121.54, 113.69, 94.39, 67.69, 50.53, 31.86, 26.51, 19.44, 17.78.

3-(6-(1,3-Dithian-2-yl)pyrazin-2-yl)-1-(phenylsulfonyl)-1H-indole (36b).

Compound 36b was prepared from compound 31 (1 g, 4.3 mmol) and (1-(phenylsulfonyl)-1H-indol-3-yl)boronic acid (1.6 g, 5.2 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 1.4 g (72% yield) of crude product 36b was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

4-(6-(1,3-Dithian-2-yl)pyrazin-2-yl)-1-(phenylsulfonyl)-1H-indazole (36c).

Compound 36c was prepared from compound 31 (1 g, 4.3 mmol) and 1-(phenylsulfonyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (2 g, 5.2 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 1.3 g (68% yield) of crude product 36c was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

6-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-pyrazine-2-carbaldehyde (37a).

Compound 37a was prepared from compound 36a (1.3 g, 3 mmol) and MeI (2.1 g, 15 mmol) following the same procedure as described in the preparation of compound 32. An amount of 0.58 g (56% yield) of crude product 37a was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

6-(1-(Phenylsulfonyl)-1H-indol-3-yl)pyrazine-2-carbaldehyde (37b).

Compound 37b was prepared from compound 36b (1.4 g, 3.1 mmol) and MeI (2.2 g, 15.5 mmol) following the same procedure as described in the preparation of compound 32. An amount of 0.68 g (60% yield) of pure product 37b was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 10.26 (s, 1H), 9.21 (s, 1H), 9.04 (s, 1H), 8.55−8.38 (m, 1H), 8.30 (s, 1H), 8.08 (dt, J = 8.5, 0.9 Hz, 1H), 8.01−7.89 (m, 2H), 7.63−7.30 (m, 5H). ¹³C NMR (101 MHz, CDCl₃) δ 192.67, 149.36, 145.99, 145.88, 140.42, 137.74, 135.55, 134.42, 129.58, 127.88, 127.03, 126.32, 125.91, 124.51, 122.34, 118.35, 113.66.

6-(1-(Phenylsulfonyl)-1H-indazol-4-yl)pyrazine-2-carbaldehyde (37c).

Compound 37c was prepared from compound 36c (1.3 g, 2.9 mmol) and MeI (2.1 g, 14.5 mmol) following the same procedure as described in the preparation of compound 32. An amount of 0.74 g (70% yield) of crude product 37c was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(6-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanol (38a).

Compound 38a was prepared from compound 37a (0.58 g, 1.7 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0. 42 g, 1.7 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.44 g (50% yield) of pure product 38a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 8.91 (s, 1H), 8.41 (s, 1H), 7.77 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 6.67 (d, J = 1.3 Hz, 2H), 5.81 (d, J = 4.0 Hz, 1H), 5.36 (d, J = 1.4 Hz, 2H), 4.81 (dd, J = 4.1, 1.4 Hz, 1H), 3.99−3.70 (m, 11H), 2.32 (s, 3H), 0.98 (t, J = 8.2 Hz, 2H), 0.0 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 157.65, 156.45, 154.96, 151.86, 142.49, 141.95, 139.19, 138.79, 135.97, 132.79, 131.50, 121.50, 113.57, 105.30, 94.36, 75.24, 67.83, 62.26, 57.56, 19.46, 17.83.

(6-(1-(Phenylsulfonyl)-1H-indol-3-yl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanol (38b).

Compound 38b was prepared from compound 37b (0.68 g, 1.9 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.47 g, 1.9 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.52 g (52% yield) of pure product 38b was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 8.90 (s, 1H), 8.53 (s, 1H), 8.33−8.15 (m, 2H), 8.15−8.02 (m, 1H), 8.02−7.90 (m, 2H), 7.57 (ddt, J = 8.7, 6.8, 1.4 Hz, 1H), 7.44 (dddd, J = 22.6, 8.5, 7.0, 1.5 Hz, 3H), 7.33 (ddd, J = 8.2, 7.3, 1.1 Hz, 1H), 6.72 (s, 2H), 5.87 (d, J = 3.5 Hz, 1H), 4.42−4.27 (m, 1H), 3.83 (d, J = 6.4 Hz, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 171.20, 155.92, 153.55, 147.12, 147.06, 140.97, 140.31, 137.83, 137.78, 137.52, 135.53, 134.34, 129.54, 128.05, 126.98, 125.78, 125.67, 124.24, 122.01, 119.11, 113.70, 103.63, 74.52, 60.85, 56.16.

(6-(1-(Phenylsulfonyl)-1H-indazol-4-yl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanol (38c).

Compound 38c was prepared from compound 37c (0.74 g, 2 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.49 g, 2 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.59 g (55% yield) of crude product 38c was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(6-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanone (39a).

Compound 39a was prepared from compound 38a (0.44 g, 0.85 mmol) and DMP (0.72 g, 1.7 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.33 g (75% yield) of pure product 39a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 9.21 (d, J = 1.4 Hz, 1H), 9.13 (d, J = 1.4 Hz, 1H), 7.80 (s, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 1.4 Hz, 2H), 7.30 (d, J = 8 Hz, 1H), 5.34 (s, 2H), 4.00 (s, 3H), 3.91 (s, 6H), 3.85−3.53 (m, 2H), 2.33 (s, 3H), 1.20−0.82 (m, 2H), 0 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 192.04, 157.77, 154.21, 151.77, 150.62, 145.11, 144.85, 144.48, 135.73, 132.92, 132.02, 131.89, 121.60, 113.54, 110.24, 94.44, 67.87, 62.49, 57.76, 19.44, 17.89.

(6-(1-(Phenylsulfonyl)-1H-indol-3-yl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanone (39b).

Compound 39b was prepared from compound 38b (0.52 g, 1 mmol) and DMP (0.85 g, 2 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.37 g (70% yield) of crude product 39b was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(6-(1-(Phenylsulfonyl)-1H-indazol-4-yl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanone (39c).

Compound 39c was prepared from compound 38c (0.59 g, 1.1 mmol) and DMP (0.93 g, 2.2 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.41 g (71% yield) of crude product 39c was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(6-(3-Hydroxy-4-methylphenyl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)methanone (40a).

Compound 40a was prepared from compound 39a (100 mg, 0.2 mmol) and TFA (2 mL) following the same procedure as described in the preparation of compound 22. An amount of 0.62 mg (82% yield) of pure product 40a was obtained as a white solid upon purification. Mp: 199–201 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.71 (s, 1H), 9.42 (d, J = 1.4 Hz, 1H), 9.01 (d, J = 1.4 Hz, 1H), 7.72−7.53 (m, 2H), 7.48 (d, J = 1.4 Hz, 2H), 7.25 (d, J = 7.8 Hz, 1H), 3.82 (d, J = 1.6 Hz, 9H), 2.18 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 190.55, 156.15, 152.52, 149.43, 148.63, 143.54, 143.01, 142.39, 133.68, 131.26, 130.17, 127.08, 117.47, 112.35, 108.42, 60.26, 55.93, 15.95. HRMS [C₂₁H₂₁N₂O₅⁺]: calcd 381.1450, found 381.1440 (mass error −2.6 ppm). UPLC purity 99.1% (t_R = 0.88 min).

(6-(1H-Indol-3-yl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)-methanone (40b).

To a solution of compound 39b (100 mg, 0.19 mmol) in MeOH (100 mL) was added KOH (21.3 mg, 0.38 mmol). The mixture was stirred under reflux until the starting materials disappeared on TLC, and then MeOH was removed. The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 4:1) to produce the key intermediate 40b as a white solid (64 mg, 87% yield). Mp: 178–180 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.15 (s, 1H), 8.96 (s, 1H), 8.88 (s, 1H), 8.40 (dd, J = 8.0, 1.1 Hz, 1H), 7.95 (d, J = 2.8 Hz, 1H), 7.58 (s, 2H), 7.45 (dt, J = 8.2, 1.0 Hz, 1H), 7.28 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.19 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 4.00 (s, 3H), 3.84 (s, 6H). ¹³C NMR (101 MHz, chloroform-d) δ 191.47, 152.82, 149.37, 149.08, 143.14, 142.92, 141.09, 136.89, 130.99, 125.35, 125.19, 123.53, 121.90, 121.72, 113.63, 111.65, 108.85, 61.08, 56.29. HRMS [C₂₂H₂₀N₃O₄⁺]: calcd 390.1454, found 390.1449 (mass error −1.3 ppm). UPLC purity 97.3% (t_R = 0.86 min).

(6-(1H-Indazol-4-yl)pyrazin-2-yl)(3,4,5-trimethoxyphenyl)-methanone (40c).

Compound 40c was prepared from compound 39c (100 mg, 0.19 mmol) and KOH (21.3 mg, 0.38 mmol) following the same procedure as described in the preparation of compound 40b. An amount of 63 mg (85% yield) of pure product 40c was obtained as a white solid upon purification. Mp: 215–217 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.42 (s, 1H), 9.19 (s, 1H), 8.67 (s, 1H), 8.09−7.78 (m, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.65−7.49 (m, 3H), 3.99 (s, 3H), 3.81 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 190.21, 152.93, 150.37, 149.53, 144.47, 143.91, 143.42, 130.55, 126.84, 120.83, 108.89, 61.09, 56.29. HRMS [C₂₁H₁₉N₄O₄⁺]: calcd 391.1406, found 391.1413 (mass error 1.8 ppm). UPLC purity 98.4% (t_R = 0.72 min).

4-Methyl-2-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)pyrimidine (42a).

Compound 42a was prepared from compound 41 (1 g, 7.8 mmol) and trimethyl(2-((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methoxy)-ethyl)silane (2.8 g, 7.8 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 1.3 g (51% yield) of crude product 42a was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

3-(4-Methylpyrimidin-2-yl)-1-(phenylsulfonyl)-1H-indole (42b).

Compound 42b was prepared from compound 41 (1 g, 7.8 mmol) and (1-(phenylsulfonyl)-1H-indol-3-yl)boronic acid (2.3 g, 7.8 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 1.5 g (55% yield) of pure product 42b was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 8.72−8.65 (m, 1H), 8.59 (d, J = 5.1 Hz, 1H), 8.55 (s, 1H), 8.06−8.01 (m, 1H), 7.99−7.92 (m, 2H), 7.53−7.46 (m, 1H), 7.43−7.33 (m, 4H), 7.01−6.95 (m, 1H), 2.56 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.01, 161.99, 156.59, 138.09, 135.74, 134.05, 129.37, 128.95, 128.59, 126.99, 125.06, 124.05, 123.45, 121.79, 118.06, 113.33, 24.37.

2-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-pyrimidine-4-carbaldehyde (43a).

To a solution of compound 42a (1.3 g, 3.9 mmol) in dioxane (100 mL) was added SeO₂ (0.87 g, 7.8 mmol). The mixture was stirred under reflux until the starting materials disappeared on TLC, and then dioxane was removed. The resulting mixture was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine, dried over Na₂SO₄, filtered, and evaporated to yield a crude product, which was purified by flash column chromatography (hexane/ethyl acetate 4:1) to produce the key intermediate 43a as a white solid (0.47 g, 35% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.12 (s, 1H), 9.02 (d, J = 4.8 Hz, 1H), 8.23 (s, 1H), 8.11 (dd, J = 7.8, 1.7 Hz, 1H), 7.64 (dd, J = 4.9, 1.3 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 5.39 (d, J = 1.2 Hz, 2H), 4.08−3.72 (m, 2H), 2.33 (s, 3H), 1.00 (t, J = 8.0 Hz, 2H), 0.11 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 195.05, 167.06, 160.64, 159.41, 157.27, 136.91, 132.81, 132.47, 122.96, 115.35, 114.68, 94.27, 67.77, 19.40, 17.97.

2-(1-(Phenylsulfonyl)-1H-indol-3-yl)pyrimidine-4-carbaldehyde (43b).

Compound 43b was prepared from compound 42b (1.5 g, 4.3 mmol) and SeO₂ (0.95 g, 8.6 mmol) following the same procedure as described in the preparation of compound 43a. An amount of 0.72 g (46% yield) of product 43b was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(2-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-pyrimidin-4-yl)(3,4,5-trimethoxyphenyl)methanol (44a).

Compound 44a was prepared from compound 43a (0.47 g, 1.4 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.34 g, 1.4 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.31 g (43% yield) of pure product 44a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 8.68 (dd, J = 5.1, 1.4 Hz, 1H), 8.18 (d, J = 1.7 Hz, 1H), 8.06 (dd, J = 7.8, 1.6 Hz, 1H), 7.28 (t, J = 8.7 Hz, 1H), 7.02 (d, J = 5.1 Hz, 1H), 6.64 (s, 2H), 5.66 (d, J = 2.7 Hz, 1H), 5.38 (s, 2H), 5.23 (d, J = 3.5 Hz, 1H), 3.84 (dd, J = 3.9, 1.5 Hz, 11H), 2.33 (s, 3H), 0.99 (t, J = 8.2 Hz, 2H), 0.07 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 170.36, 164.82, 158.87, 157.20, 155.12, 154.93, 153.96, 139.24, 138.57, 137.26, 133.10, 132.59, 132.42, 122.89, 117.26, 114.69, 105.53, 94.30, 94.23, 76.13, 67.81, 62.46, 62.27, 57.56, 57.32, 19.45, 17.97.

(2-(1-(Phenylsulfonyl)-1H-indol-3-yl)pyrimidin-4-yl)(3,4,5-trimethoxyphenyl)methanol (44b).

Compound 44b was prepared from compound 43b (0.72 g, 2.0 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.49 g, 2.0 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.6 g (55% yield) of crude product 44b was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(2-(4-Methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)phenyl)-pyrimidin-4-yl)(3,4,5-trimethoxyphenyl)methanone (45a).

Compound 45a was prepared from compound 44a (0.31 g, 0.6 mmol) and DMP (0.51 g, 1.2 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.25 g (82% yield) of pure product 45a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 9.05 (dd, J = 4.9, 1.4 Hz, 1H), 8.22 (s, 1H), 8.09 (d, J = 7.9 Hz, 1H), 7.73 (dd, J = 5.0, 1.3 Hz, 1H), 7.62 (d, J = 1.4 Hz, 2H), 7.27 (d, J = 6.7 Hz, 1H), 5.37 (d, J = 1.4 Hz, 2H), 4.00 (s, 3H), 3.91 (s, 6H), 3.84−3.62 (m, 2H), 2.34 (s, 3H), 0.98 (t, J = 8.3 Hz, 2H), −0.15 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 192.27, 165.02, 163.26, 160.63, 157.35, 154.25, 144.71, 137.39, 132.82, 132.46, 131.31, 122.92, 119.28, 114.80, 110.27, 94.36, 67.84, 62.51, 57.75, 19.43, 18.02.

(2-(1-(Phenylsulfonyl)-1H-indol-3-yl)pyrimidin-4-yl)(3,4,5-trimethoxyphenyl)methanone (45b).

Compound 45b was prepared from compound 44b (0.44 g, 0.8 mmol) and DMP (0.67 g, 1.6 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.34 g (80% yield) of pure product 45b was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 9.04 (d, J = 5.0 Hz, 1H), 8.62 (s, 1H), 8.58 (dt, J = 7.8, 1.0 Hz, 1H), 8.04 (dt, J = 8.4, 0.9 Hz, 1H), 8.01−7.92 (m, 2H), 7.70 (d, J = 5.0 Hz, 1H), 7.64−7.54 (m, 3H), 7.51−7.44 (m, 2H), 7.38 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H), 7.30 (ddd, J = 8.2, 7.3, 1.1 Hz, 1H), 4.00 (s, 3H), 3.85 (s, 6H). ¹³C NMR (101 MHz, chloroform-d) δ 190.80, 161.75, 159.06, 152.95, 137.98, 135.74, 134.24, 129.81, 129.63, 129.49, 128.11, 127.04, 125.42, 124.24, 123.28, 121.02, 117.44, 113.41, 108.86, 61.11, 56.35.

(2-(3-Hydroxy-4-methylphenyl)pyrimidin-4-yl)(3,4,5-trimethoxyphenyl)methanone (46a).

Compound 46a was prepared from compound 45a (0.1 g, 0.2 mmol) and TFA (2 mL) following the same procedure as described in the preparation of compound 22. An amount of 57 mg (75% yield) of pure product 46a was obtained as a white solid upon purification. Mp: 189–191 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.67 (s, 1H), 9.15 (dd, J = 4.9, 1.5 Hz, 1H), 7.92 (s, 1H), 7.86−7.65 (m, 2H), 7.49 (d, J = 1.4 Hz, 2H), 7.23 (d, J = 7.8 Hz, 1H), 3.91−3.61 (m, 9H), 2.19 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 190.73, 162.68, 161.42, 159.81, 155.83, 152.59, 142.70, 135.34, 130.97, 129.42, 127.87, 118.59, 117.94, 113.54, 108.40, 60.28, 55.96, 16.04. HRMS [C₂₁H₂₁N₂O₅⁺]: calcd 381.1450, found 381.1443 (mass error −1.8 ppm). UPLC purity 98.4% (t_R = 0.91 min).

(2-(1H-Indol-3-yl)pyrimidin-4-yl)(3,4,5-trimethoxyphenyl)-methanone (46b).

Compound 46b was prepared from compound 45b (0.1 g, 0.19 mmol) and KOH (21 mg, 0.38 mmol) following the same procedure as described in the preparation of compound 40b. An amount of 52 mg (70% yield) of pure product 46b was obtained as a white solid upon purification. Mp: 189–191 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.97 (dd, J = 4.9, 2.4 Hz, 1H), 8.64−8.46 (m, 2H), 8.26 (d, J = 2.8 Hz, 1H), 7.60 (d, J = 2.3 Hz, 2H), 7.57 (dd, J = 4.9, 2.3 Hz, 1H), 7.45 (dq, J = 8.1, 1.1 Hz, 1H), 7.30−7.27 (m, 1H), 7.22 (ddt, J = 8.1, 7.1, 1.2 Hz, 1H), 4.00 (s, 3H), 3.85 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 191.57, 163.16, 161.55, 158.88, 152.84, 143.20, 136.92, 130.13, 128.68, 125.74, 123.18, 122.63, 121.69, 116.38, 115.78, 111.38, 108.88, 61.07, 56.29. HRMS [C₂₂H₂₀N₃O₄⁺]: calcd 390.1454, found 390.1452 (mass error −0.5 ppm). UPLC purity 96.9% (t_R = 0.92 min).

(6-Bromo-4-methylpyridin-2-yl)(3,4,5-trimethoxyphenyl)-methanol (48).

Compound 48 was prepared from compound 47 (1 g, 4 mmol) and 3,4,5-trimethoxybenzaldehyde (0.78 g, 4 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.46 g (31% yield) of crude product 48 was obtained as a white solid. The crude compound was used for the next step without further purification.

(6-Bromo-4-methylpyridin-2-yl)(3,4,5-trimethoxyphenyl)-methanone (49).

Compound 49 was prepared from compound 48 (0.46 g, 1.3 mmol) and DMP (1.1 g, 2.6 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.35 g (73% yield) of crude product 49 was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(4-Methyl-6-(p-tolyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)-methanone (50a).

Compound 50a was prepared from compound 49 (0.1 g, 0.3 mmol) and p-tolylboronic acid (49 mg, 0.36 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 59 mg (52% yield) of pure product 50a was obtained as a white solid upon purification. Mp: 117–119 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 8.0 Hz, 2H), 7.80 (s, 1H), 7.67 (s, 2H), 7.26 (d, J = 8.1 Hz, 2H), 3.97 (s, 3H), 3.89 (s, 6H), 2.52 (s, 3H), 2.40 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 192.03, 155.63, 154.92, 152.58, 149.04, 142.49, 139.47, 135.70, 131.47, 129.51, 126.70, 123.75, 122.87, 109.20, 60.98, 56.25, 21.41, 21.29. HRMS [C₂₃H₂₄NO₄+]: calcd 378.1705, found 378.1703 (mass error −0.5 ppm). UPLC purity 96% (t_R = 1.28 min).

(6-(3-Amino-4-methylphenyl)-4-methylpyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (50b).

Compound 50b was prepared from compound 49 (0.1 g, 0.3 mmol) and (3-amino-4-methylphenyl)boronic acid (54 mg, 0.36 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 42 mg (36% yield) of pure product 50b was obtained as a white solid upon purification. Mp: 91–93 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.78 (s, 1H), 7.73 (s, 1H), 7.64 (s, 2H), 7.46 (d, J = 1.7 Hz, 1H), 7.40 (dd, J = 7.8, 1.7 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 6H), 3.78−3.61 (m, 2H), 2.51 (s, 3H), 2.22 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 192.25, 155.70, 154.73, 152.56, 148.94, 145.07, 142.36, 137.28, 131.47, 130.87, 123.88, 123.75, 122.96, 117.00, 113.03, 109.12, 61.02, 56.26, 21.44, 17.31. HRMS [C₂₃H₂₅N₂O₄⁺]: calcd 393.1814, found 393.1810 (mass error −1.0 ppm). UPLC purity 96.8% (t_R = 0.96 min).

2-Bromo-4-methoxy-6-(p-tolyl)pyridine (52a).

Compound 52a was prepared from compound 51 (1 g, 3.8 mmol) and p-tolylboronic acid (0.52 g, 3.8 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.43 g (41% yield) of crude product 52a was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

2-Bromo-4-methoxy-6-(4-methyl-3-((2-(trimethylsilyl)ethoxy)-methoxy)phenyl)pyridine (52b).

Compound 52b was prepared from compound 51 (1 g, 3.8 mmol) and trimethyl(2-((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)methoxy)ethyl)silane (1.4 g, 3.8 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.48 g (30% yield) of crude product 52b was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(4-Methoxy-6-(p-tolyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)-methanol (53a).

Compound 53a was prepared from compound 52a (0.43 g, 1.6 mmol) and 3,4,5-trimethoxybenzaldehyde (0.31 g, 1.6 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.3 g (47% yield) of pure product 53a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 7.14 (d, J = 2.1 Hz, 1H), 6.65 (s, 2H), 6.56−6.48 (m, 1H), 5.86 (s, 1H), 5.62 (s, 1H), 3.83 (d, J = 3.2 Hz, 12H), 2.42 (s, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 167.23, 161.95, 156.91, 153.38, 139.51, 138.94, 137.63, 135.85, 129.49, 126.83, 105.48, 104.95, 104.31, 75.00, 60.82, 56.16, 55.35, 21.31.

(4-Methoxy-6-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)methanol (53b).

Compound 53b was prepared from compound 52b (0.48 g, 1.1 mmol) and 3,4,5-trimethoxybenzaldehyde (0.22 g, 1.1 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.29 g (48% yield) of pure product 53b was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.22−7.07 (m, 1H), 6.65 (s, 2H), 6.52 (d, J = 2.1 Hz, 1H), 5.87 (s, 1H), 5.61 (s, 1H), 5.35 (s, 2H), 3.84 (d, J = 4.9 Hz, 14H), 2.31 (s, 3H), 1.05−0.92 (m, 2H), 0.0 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 168.55, 163.26, 158.30, 157.32, 154.74, 140.33, 139.04, 138.88, 132.38, 130.39, 121.46, 113.73, 107.23, 106.32, 105.58, 94.40, 76.35, 67.73, 62.22, 57.52, 56.77, 19.44, 17.73.

(4-Methoxy-6-(p-tolyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)-methanone (54a).

Compound 54a was prepared from compound 53a (0.3 g, 0.8 mmol) and DMP (0.68 g, 1.6 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.22 g (70% yield) of pure product 54a was obtained as a white solid upon purification. Mp: 161–163 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.96 (d, J = 7.9 Hz, 2H), 7.69 (s, 2H), 7.53 (d, J = 2.2 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.26 (d, J = 8.1 Hz, 2H), 3.99 (s, 3H), 3.97 (d, J = 0.8 Hz, 3H), 3.90 (s, 6H), 2.40 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 191.61, 167.44, 157.41, 156.75, 152.57, 142.56, 139.63, 135.65, 131.33, 129.49, 126.73, 109.25, 108.78, 108.06, 60.99, 56.25, 55.59, 21.29. HRMS [C₂₃H₂₄NO₅⁺]: calcd 394.1654, found 394.1664 (mass error 2.5 ppm). UPLC purity 97.9% (t_R = 1.22 min).

(4-Methoxy-6-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (54b).

Compound 54b was prepared from compound 53b (0.22 g, 0.4 mmol) and DMP (0.34 g, 0.8 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.15 g (68% yield) of pure product 54b was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 7.71 (s, 1H), 7.66 (d, J = 1.9 Hz, 3H), 7.52 (d, J = 2.1 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 5.31 (d, J = 1.9 Hz, 2H), 4.00 (d, J = 1.8 Hz, 3H), 3.98 (d, J = 1.8 Hz, 3H), 3.90 (d, J = 1.8 Hz, 6H), 3.84−3.74 (m, 2H), 2.30 (s, 3H), 1.02−0.84 (m, 2H), 0.10 (d, J = 1.8 Hz, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 193.29, 168.81, 158.79, 158.10, 157.37, 153.96, 143.87, 138.82, 132.74, 132.43, 130.58, 121.54, 113.60, 110.55, 110.52, 109.39, 94.39, 67.74, 62.40, 57.70, 57.04, 19.41, 17.77.

(6-(3-Hydroxy-4-methylphenyl)-4-methoxypyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (55b).

Compound 55b was prepared from compound 54b (0.15 g, 0.27 mmol) and TFA (2 mL) following the same procedure as described in the preparation of compound 22. An amount of 71.2 mg (65% yield) of pure product 55b was obtained as a white solid upon purification. Mp: 172–174 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.63 (d, J = 1.4 Hz, 2H), 7.57−7.44 (m, 3H), 7.38 (t, J = 1.9 Hz, 1H), 7.19 (d, J = 7.7 Hz, 1H), 5.11 (s, 1H), 3.98 (s, 3H), 3.96 (s, 3H), 3.89 (s, 6H), 2.29 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 191.83, 167.38, 156.95, 156.60, 154.32, 152.54, 142.47, 137.54, 131.29, 131.22, 125.57, 118.91, 113.23, 109.11, 108.82, 108.27, 60.99, 56.25, 55.61, 15.72. HRMS [C₂₃H₂₄NO₆⁺]: calcd 410.1604, found 410.1606 (mass error 0.5 ppm). UPLC purity 98.4% (t_R = 0.99 min).

3-Fluoro-6-(p-tolyl)picolinaldehyde (57a).

Compound 57a was prepared from 6-bromo-3-fluoropicolinaldehyde (0.5 g, 2.5 mmol) and p-tolylboronic acid (0.4 g, 3 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.37 g (68% yield) of pure product 57a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 10.23 (s, 1H), 7.92 (dd, J = 8.6, 3.6 Hz, 3H), 7.58 (t, J = 9.2 Hz, 1H), 7.30 (d, J = 7.9 Hz, 2H), 2.41 (s, 3H). ¹³C NMR (101 MHz, chloroform-d) δ 190.46, 159.87, 157.15, 154.03, 139.84, 139.76, 134.37, 129.71, 126.78, 126.33, 126.14, 125.75, 125.70, 21.30.

6-(4-(Difluoromethyl)phenyl)-3-fluoropicolinaldehyde (57b).

Compound 57b was prepared from 6-bromo-3-fluoropicolinaldehyde (0.5 g, 2.5 mmol) and (4-(difluoromethyl)phenyl)boronic acid (0.52 g, 3 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.44 g (70% yield) of crude product 57b was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

3-Fluoro-6-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)picolinaldehyde (57c).

Compound 57c was prepared from 6-bromo-3-fluoropicolinaldehyde (0.5 g, 2.5 mmol) and trimethyl(2-((2-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-methoxy)ethyl) silane (1.1 g, 3 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.54 g (60% yield) of pure product 57c was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 10.23 (s, 1H), 8.12−7.88 (m, 1H), 7.76 (s, 1H), 7.66−7.47 (m, 2H), 7.41−7.05 (m, 1H), 5.36 (d, J = 1.2 Hz, 2H), 4.22−3.74 (m, 2H), 2.30 (s, 3H), 1.07−0.87 (m, 2H), 0.0 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 192.13, 161.11, 158.38, 157.53, 155.31, 155.26, 141.07, 141.01, 137.42, 132.59, 130.68, 127.69, 127.50, 127.34, 127.29, 121.26, 113.56, 94.35, 67.75, 19.42, 17.71.

(3-Fluoro-6-(p-tolyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)-methanol (58a).

Compound 58a was prepared from 57a (0.37 g, 1.7 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.42 g, 1.7 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.36 g (55% yield) of pure product 58a was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 7.89 (d, J = 8.0 Hz, 2H), 7.65 (dd, J = 8.6, 3.6 Hz, 1H), 7.41 (t, J = 8.7 Hz, 1H), 7.33−7.24 (m, 2H), 6.68 (s, 2H), 5.95 (dd, J = 6.6, 1.8 Hz, 1H), 5.53 (d, J = 6.6 Hz, 1H), 3.82 (d, J = 7.7 Hz, 9H), 2.42 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 156.70, 154.15, 153.29, 151.99, 151.95, 147.92, 147.75, 139.42, 137.86, 137.66, 134.94, 129.64, 126.64, 124.67, 124.48, 120.64, 120.60, 104.70, 103.92, 70.26, 70.24, 60.78, 56.13, 21.27.

(6-(4-(Difluoromethyl)phenyl)-3-fluoropyridin-2-yl)(3,4,5-trimethoxyphenyl)methanol (58b).

Compound 58b was prepared from compound 57b (0.44 g, 1.8 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.44 g, 1.8 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.2 g (60% yield) of crude product 58b was obtained as a yellow solid upon purification. The crude compound was used for the next step without further purification.

(3-Fluoro-6-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)methanol (58c).

Compound 58c was prepared from compound 57c (0.54 g, 1.5 mmol) and 5-bromo-1,2,3-trimethoxybenzene (0.37 g, 1.5 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.4 g (50% yield) of pure product 58c was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 7.74−7.61 (m, 2H), 7.54 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 8.7 Hz, 1H), 7.26 (d, J = 8 Hz, 1H), 6.67 (s, 2H), 5.94 (d, J = 6.7 Hz, 1H), 5.54 (dd, J = 6.8, 1.3 Hz, 1H), 5.35 (s, 2H), 3.89−3.72 (m, 11H), 2.31 (s, 3H), 0.98 (t, J = 8.3 Hz, 2H), 0.0 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 157.50, 154.66, 153.40, 153.35, 149.25, 149.08, 139.27, 138.88, 138.10, 132.55, 130.29, 126.05, 125.86, 122.34, 122.30, 121.27, 113.56, 105.14, 94.39, 71.64, 71.61, 67.76, 62.22, 57.51, 19.49, 17.72.

(3-Fluoro-6-(p-tolyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)-methanone (59a).

Compound 59a was prepared from compound 58a (0.1 g, 0.26 mmol) and DMP (0.22 g, 0.52 mmol) following the same procedure as described in the preparation of compound 3. An amount of 69 mg (70% yield) of pure product 59a was obtained as a white solid upon purification. Mp: 138–140 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.90 (dd, J = 8.6, 3.0 Hz, 3H), 7.62 (t, J = 8.9 Hz, 1H), 7.37 (s, 2H), 7.26 (d, J = 8.0 Hz, 2H), 3.96 (s, 3H), 3.85 (s, 6H), 2.40 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 189.59, 158.55, 155.92, 152.92, 152.28, 152.23, 143.63, 143.51, 143.29, 139.55, 134.64, 130.91, 129.64, 126.61, 125.66, 125.47, 122.83, 122.78, 108.40, 61.02, 56.30, 21.27. HRMS [C₂₂H₂₁FNO₄⁺]: calcd 382.1455, found 382.1447 (mass error −2.1 ppm). UPLC purity 95.7% (t_R = 1.16 min).

(6-(4-(Difluoromethyl)phenyl)-3-fluoropyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (59b).

Compound 59b was prepared from compound 58b (0.1 g, 0.23 mmol) and DMP (0.2 g, 0.46 mmol) following the same procedure as described in the preparation of compound 3. An amount of 68 mg (71% yield) of pure product 59b was obtained as a white solid upon purification. Mp: 186–188 °C. 1H NMR (400 MHz, CDCl₃) δ 8.10 (d, J = 8.1 Hz, 2H), 7.97 (dd, J = 8.8, 3.5 Hz, 1H), 7.68 (t, J = 8.8 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.33 (s, 2H), 6.70 (t, J = 56.3 Hz, 1H), 3.97 (d, J = 0.8 Hz, 3H), 3.85 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 189.76, 152.99, 143.49, 139.59, 130.68, 127.04, 126.26, 126.19, 126.13, 125.87, 125.67, 123.41, 123.37, 114.39, 108.32, 61.05, 56.33, 29.71. HRMS [C₂₂H₁₉F₃NO₄⁺]: calcd 418.1266, found 418.1267 (mass error 0.2 ppm). UPLC purity 97.3% (t_R = 1.08 min).

(3-Fluoro-6-(4-methyl-3-((2-(trimethylsilyl)ethoxy)methoxy)-phenyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (59c).

Compound 59c was prepared from compound 58c (0.4 g, 0.76 mmol) and DMP (0.64 g, 1.52 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.32 g (80% yield) of pure product 59c was obtained as a white solid upon purification. ¹H NMR (400 MHz, CDCl₃) δ 7.91 (ddd, J = 9.0, 3.8, 1.5 Hz, 1H), 7.69 (s, 1H), 7.67−7.53 (m, 2H), 7.37 (d, J = 1.5 Hz, 2H), 7.28 (d, J = 1.5 Hz, 1H), 5.32 (d, J = 1.7 Hz, 2H), 3.98 (d, J = 1.5 Hz, 3H), 3.88 (d, J = 1.6 Hz, 6H), 3.82−3.67 (m, 2H), 2.30 (d, J = 1.6 Hz, 3H), 1.04−0.90 (m, 2H), 0.0 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 191.14, 157.48, 154.33, 153.72, 153.67, 145.03, 144.91, 144.60, 137.79, 132.59, 132.32, 130.49, 127.02, 126.83, 124.51, 124.46, 121.35, 113.49, 109.68, 94.39, 67.76, 62.45, 57.73, 19.43, 17.74.

(3-Fluoro-6-(3-hydroxy-4-methylphenyl)pyridin-2-yl)(3,4,5-trimethoxyphenyl)methanone (60c).

Compound 60c was prepared from compound 59c (0.32 g, 0.6 mmol) and TFA (2 mL) following the same procedure as described in the preparation of compound 22. An amount of 0.21 g (88% yield) of pure product 60c was obtained as a white solid upon purification. Mp: 160–162 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.88−7.73 (m, 1H), 7.57 (td, J = 8.9, 1.5 Hz, 1H), 7.48−7.36 (m, 2H), 7.32 (d, J = 1.6 Hz, 2H), 7.17 (d, J = 7.7 Hz, 1H), 5.67 (d, J = 1.6 Hz, 1H), 3.94 (s, 3H), 3.83 (s, 6H), 2.26 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 189.92, 158.49, 155.86, 154.57, 152.85, 151.96, 151.91, 143.39, 143.27, 143.21, 136.32, 131.39, 130.78, 125.85, 125.67, 125.48, 123.05, 123.01, 118.63, 113.10, 108.27, 61.01, 56.26, 15.73. HRMS [C₂₂H₂₁FNO₅⁺]: calcd 398.1404, found 398.1403 (mass error −0.3 ppm). UPLC purity 99.5% (t_R = 0.95 min).

(3-Bromo-5-fluorophenyl)(3,4,5-trimethoxyphenyl)methanol (62).

Compound 62 was prepared from 5-bromo-1,2,3-trimethoxybenzene (1 g, 4 mmol) and 3-bromo-5-fluorobenzaldehyde (0.8 g, 4 mmol) following the same procedure as described in the preparation of compound 2. An amount of 0.83 g (56% yield) of crude product 62 was obtained as a white solid upon purification. The crude compound was used for the next step without further purification.

(3-Bromo-5-fluorophenyl)(3,4,5-trimethoxyphenyl)methanone (63).

Compound 63 was prepared from compound 62 (0.83 g, 2.2 mmol) and DMP (1.9 g, 4.4 mmol) following the same procedure as described in the preparation of compound 3. An amount of 0.57 g (70% yield) of crude product 63 was obtained as a yellow solid upon purification. The crude compound was used for the next step without further purification.

(4′-(Difluoromethyl)-5-fluoro-[1,1′-biphenyl]-3-yl)(3,4,5-trimethoxyphenyl)methanone (64).

Compound 64 was prepared from compound 63 (0.1 g, 0.27 mmol) and (4-(difluoromethyl)-phenyl)boronic acid (51.6 mg, 0.3 mmol) following the same procedure as described in the preparation of compound 4a. An amount of 0.1 g (89% yield) of pure product 64 was obtained as a white solid upon purification. Mp: 173–175 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.81 (q, J = 1.4 Hz, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.62 (d, J = 8.0 Hz, 2H), 7.51 (dddt, J = 16.0, 8.5, 2.4, 1.3 Hz, 2H), 7.10 (d, J = 1.1 Hz, 2H), 6.71 (t, J = 56.4 Hz, 1H), 3.96 (s, 3H), 3.89 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 194.15, 163.95, 161.48, 153.04, 142.65, 141.28, 140.50, 134.60, 131.82, 127.48, 126.46, 124.23, 117.93, 117.71, 116.78, 116.09, 115.86, 114.41, 112.03, 107.71, 61.06, 56.40. HRMS [C₂₃H₂₀F₃O₄⁺]: calcd 417.1314, found 417.1312 (mass error −0.5 ppm). UPLC purity 98.4% (t_R = 1.21 min).

Cell Culture.

Human breast cancer cell lines (MDA-MB-231, MDA-MB-453, and SKBR3) and human malignant melanoma cell lines (A375, RPMI7951, and M14) were purchased from the American Type Culture Collection (Manassas, VA). The cancer cell lines were cultured in Dulbecco’s modified Eagle’s medium (Mediatech, Inc., Manassas, VA) supplemented with 10% FBS (Atlanta Biologicals, Minneapolis, MN) and 1% antibiotic–anti-mycotic solution (Sigma-Aldrich, St. Louis, MO) at 37 °C in a humidified atmosphere containing 5% CO₂. A375/TxR and MDA-MB-231/TxR paclitaxel-resistant cancer cells were developed by the gradual continuous exposure of parental cells to increasing concentrations of paclitaxel. Paclitaxel was removed from the media for paclitaxel-resistant A375/TxR and MDA-MB-231/TxR cells for at least 1 week before in vitro or in vivo testing.

Antiproliferative Activity.

The cytotoxic effects of the newly synthesized ABP compounds against a panel of cancer cell lines were evaluated by an MTS cell proliferation assay as previously described.¹³ Briefly, cells were seeded in 96-well plates at a density of 3500–5000 cells per well depending on the doubling time of each cell line and incubated for 24 h. Compounds were dissolved in DMSO (ATCC) to obtain 20 mM stock solutions, diluted with cell culture medium, added to the appropriate well at 10 different concentrations ranging from 0.1 nM to 3 μM, and incubated for 72 h in four replicates. Cells were then treated with MTS for 1–1.5 h in the dark. The absorbance was recorded at 490 nm using a plate reader (BioTek Instruments Inc., Winooski, VT). Cell growth inhibition of 50% (IC₅₀) was calculated from concentration–response curves using GraphPad Prism 7 software (San Diego, CA). The resistance index (RI) was calculated by dividing the IC₅₀ of 60c obtained on the resistant cells by that of parent cells.

Liver Microsome Stability Assay.^28,29

This assay provides important information on the metabolic liability of early drug discovery compounds based on liver microsomes. Human and mouse liver microsomal degradation are determined using multiple time points to monitor the rate of disappearance of the parent compound during incubation. NADPH regenerating agent solutions A and B were obtained from BD Gentest (Woburn, MA). Pooled human liver microsomes were purchased from XenoTech (Lenexa, KS). Pooled mouse (CD-1) liver microsomes were purchased from Gibco (Ottawa, Ontario). Ninety-six-well deep well plates were obtained from Midsci (St. Louis, MO). Ninety-six well analytical plates were obtained from Corning Incorporated (Acton, MA).

Sample preparation for microsomal stability was modified from Dr. Di’s publications.^30,31 A set of incubation times of 0, 15, 30, 60, 120, and 240 min were used. DMSO stock solutions of test compounds and verapamil (as a system control) were prepared at a 10 mM concentration. Concentrated human or mouse liver microsomes (20 mg/mL protein concentration) and 0.5 M EDTA were diluted into a 0.1 M potassium phosphate buffer (pH 7.4), mixed well, and spiked with the indicated compound solutions. After mixing, 90 μL of this solution was transferred to six time-point plates (each in triplicate wells). For the time 0 h plate, a 3-fold excess volume of cold acetonitrile containing an internal standard (4 μg/mL warfarin) was added to each well, followed by the addition of NADPH regenerating agent (mixing NADPH solutions A and B in PBS, pH 7.4), and samples were processed without incubation. For plates from the other five-time points, NADPH regenerating agent was added to each well to initiate the reaction, the plate was incubated at 37 °C for the required time, followed by quenching of the reaction by adding a 3-fold excess volume of cold acetonitrile with warfarin internal standard to each well. The final concentration of each component applied in this reaction was as follows: 0.5 mg/mL of liver microsome protein, 1 mM of EDTA, 10 μM of the indicated compound, 1.3 mM of NADPH solution A, and 0.4 U/mL of NADPH solution B. All of the plates were sealed and mixed well at 600 rpm for 10 min after quenching and were centrifuged at 4000 rpm for 20 min. The supernatants were transferred to analytical plates for analysis by UPLC–MS. The metabolic stability was evaluated via the half-life from the least-squares fit of the multiple time points based on first-order kinetics.

UPLC/MS System.

LC-MS chromasolv-grade acetonitrile (ACN) was purchased from Fisher Scientific (Loughborough, UK). LC-MS chromasolv-grade and formic acid were obtained from Sigma-Aldrich (St. Louis, MO). Milli-Q water as ultrapure laboratory-grade water was used in the aqueous mobile phase.

Chromatographic separation was performed on an Acquity UPLC BEH C18 1.7 m, 2.1 × 50 mm column (Waters Corporation, Milford, MA) using an Acquity ultraperformance liquid chromatography system. Data were acquired using Masslynx v. 4.1 and analyzed using the Quanlynx software suite. This was coupled to an SQ mass spectrometer. The total flow rate was 1.0 mL/min. The UPLC column was maintained at 65 °C. Solvent A was 0.1% formic acid in Milli-Q H₂O and solvent B was 0.1% formic acid in ACN. Samples were eluted from the column under a gradient condition. The mass spectrometer was operated in positive-ion mode with electrospray ionization. The conditions were as follows: capillary voltage 3.4 kV, cone voltage 30 V, source temperature 150 °C, desolvation temperature 350 °C, desolvation gas 750 L/h, cone gas 25 L/h. A full scan range from m/z 110 to 1000 in 0.2 s was used to acquire MS data. A single ion recording mass spectrometry for each compound was used to determine the quantification of the samples.

Protein Expression and Purification.

The double mutant (Cys14Ala and Phe20Trp) stathmin-like domain of the RB3 (RB3-SLD) gene was kindly provided by Dr. Benoît Gigant (Université Paris-Saclay, France). RB3-SLD was expressed and purified as described previously.^21,32,33 Briefly, RB3-SLD was overexpressed in Escherichia coli and purified by thermal denaturation, nucleic acid precipitation, anion-exchange chromatography, and gel filtration chromatography. The purified protein was concentrated to 12 mg/mL in 10 mM HEPES, pH 7.2, 150 mM NaCl, and stored at −80 °C until use. Subtilisin-treated tubulin (sT) was generated as described.^21,34 Briefly, porcine brain tubulin (catalogue # T-240, Cytoskeleton, Inc.) was resuspended at 50 mg/mL in MAB (MES assembly buffer: 100 mM MES pH 6.9, 1 mM MgCl₂, 1 mM EGTA) and diluted to 5 mg/mL in distilled water, and then GTP was added to a final concentration of 1 mM. After preincubation of the sample at 25 °C for 5 min, subtilisin (P8038, Sigma-Aldrich) was added in a weight ratio of 1/100. After 45 min of incubation at 25 °C, the reaction was stopped by adding PMSF (final concentration 0.05%). MES, MgCl₂, and EGTA were added to the sample to final concentrations of 100, 1, and 1 mM, respectively. The sample was incubated on ice for 40 min, and the supernatant containing subtilisin-treated tubulin was collected after centrifugation.

Crystallization, Data Collection, and Structure Determination.

The tubulin/RB3-SLD complex (sT2R) was prepared as described previously.^21,33,35 Subtilisin-treated tubulin and RB3-SLD were mixed at molar ratios of 2:1.3 (tubulin: RB3-SLD) and concentrated to 15 mg/mL at 4 °C. To obtain sT2R–colchicine, sT2R–40a, and sT2R–60c complex structures, the complexes were pre-formed in solution and then crystallized. The compounds were dissolved in DMSO at 100 mM concentration, the sT2R complex was incubated with 1 mM of each compound, and the undissolved compound was removed by centrifugation. Crystals were grown using the sitting drop vapor diffusion method at 20 °C with a reservoir solution of 0.1 M trisodium citrate, pH 5.6, 0.2 M ammonium sulfate, and 12–15% PEG 4000. For data collection, crystals were immersed in a cryoprotectant (0.1 M trisodium citrate, pH 5.6, 0.2 M ammonium sulfate, 10 mM MgCl₂, 15% PEG 4000, 0.2 mM compound, and 30% glycerol) and flash-frozen in liquid nitrogen. For the sT2R–colchicine complex structure, diffraction data were collected at the beamline 8.2.1 at the Advanced Light Source (ALS) at the Lawrence Berkeley National Laboratory (Berkeley, CA). For the sT2R–40a and sT2R–60c complex structures, diffraction data were collected at the beamline 22-BM at the Advanced Photon Source at the Argonne National Laboratory (Lemont, IL). All diffraction data were processed using HKL2000 software (HKL Research, Inc., Charlottesville, VA).³⁶ Structures were solved by molecular replacement using the tubulin–RB3 of the T2R-TTL–colchicine complex (PDB 5XIW) as the search model. Structures were refined and optimized using PHENIX (Python-based Hierarchical Environment for Integrated Xtallography) and COOT (Crystallographic Object-Oriented Toolkit) software packages, respectively.^37,38 The atomic coordinates and structure factors have been deposited in the Protein Data Bank. Data collection and refinement statistics are summarized in Table S1 together with the PDB accession codes. The electron density maps are shown in Figure S1. For the figures, all structures were rendered using the PyMOL version 2.3.0 molecular graphics system (Schrödinger, LLC).

Western Blot Analysis of P-gp.

Parental A375 and MDA-MB-231 cells and paclitaxel-resistant cell lines A375/TxR and MDA-MB-231/TxR were washed with ice-cold PBS and lysed for 30 min in RIPA lysis buffer (25 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing Halt protease and phosphatase inhibitors (Thermo Fisher Scientific). After centrifugation, supernatants were collected, and the protein concentrations were determined by the Bradford method. Denatured protein samples were separated by 8% SDS-PAGE electrophoresis and subsequently transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with anti-P-gp (MA1–26528, Thermo Fisher Scientific) and anti-β-actin antibodies (#3700, Cell Signaling Technology) at 4 °C overnight. The next day, after incubation with the HRP-conjugated antimouse IgG secondary antibody (#7076, Cell Signaling Technology) for 1 h, the proteins were detected by treating the membranes with Clarity Western ECL Substrate (Bio-Rad, #1705060) and exposing them to X-ray film.

Tubulin Polymerization Assay.

The cell-free tubulin polymerization assay was performed using a tubulin polymerization assay kit (Cytoskeleton, Denver, CO) according to the manufacturer’s protocol. Assay mixtures containing tubulin protein (3 mg/mL) and 1 mM GTP were prepared in general tubulin buffer on ice, and the polymerization reactions were initiated at 37 °C by adding 10 μM of the respective test compounds into the assay mixtures in a 96-well plate. The absorbance kinetics for each sample were recorded using a SYNERGY 4 Microplate Reader (BioTek Instruments, Inc., Winooski, VT) by measuring the turbidity at a wavelength of 350 nm every 30 s for 1 h.

Colony Formation.

A375/TxR cells (1000 cells per well) at the logarithmic phase were seeded in 6-well plates and incubated for 24 h. The cells were treated with 2.5, 5, and 10 nM of colchicine, paclitaxel, CH-2–77, or 60c, respectively, for 1 week. The culture medium or medium containing the indicated drugs was changed every 3 days. At the end of the experiment, colonies resulting from the surviving cells were washed with PBS, fixed with cold methanol, stained with 0.5% crystal violet, and counted using a Keyence BZ-X700 microscope (Itasca, IL). The assay was performed once in triplicate.

Cell Cycle Analysis.

The DNA contents of A375/TxR cells treated with colchicine (10 nM), paclitaxel (10 nM), CH-2–77 (10 nM), and 60c (2.5, 5, and 10 nM) in complete medium were analyzed to study the cell cycle distribution by staining the cells with propidium iodide (PI). Cells (2 × 10⁶) per dish were seeded in 100 mm of dishes and incubated overnight. Following treatment with the listed drugs for 24 h, cells were washed with PBS, fixed in ice-cold 70% ethanol, incubated with 100 μg/mL of RNase A for 1 h at 37 °C, and exposed to PI for 5 min in the dark. Analysis of cell cycle population was performed using a ZE5 Cell Analyzer (Bio-Rad Laboratories, Inc., Hercules. CA) in the University of Tennessee Health Science Center (UTHSC) Flow Cytometry and Cell Sorting Core facility, and the results were analyzed using ModFit LT software (Verity Software House, Topsham, ME). The experiment was carried out once in triplicate.

Detection of Cellular Apoptosis.

The effect of 60c on apoptosis was analyzed using an FITC Annexin V apoptosis detection kit (eBioscience, San Diego, CA). Following the same compound treatments as described for the cell cycle analysis, 10⁵ cells were harvested and stained with anti-Annexin V-FITC and PI according to the manufacturers’ instructions. After 10 min of incubation in the dark, the samples were diluted with a 1× binding buffer, and the analysis was performed on the Bio-Rad ZE5 Cell Analyzer. Data were processed, and graphs were generated using FlowJo software (Becton, Dickinson, and Co., Ashland, OR).

Pharmacokinetic Assessments.

All animal studies were performed in adherence to the NIH Principles of Laboratory Animal Care and were approved by the Institutional Animal Care and Use Committee of the University of Tennessee Health Science Center. Male NSG mice (6–7 weeks; n = 24) maintained at a 12 h light/dark cycle were allowed free access to food and water before experiments. The animals were dosed via intraperitoneal (IP) injection with 10 mg/kg of 60c. Blood samples were collected at 0.17, 0.5, 1, 2, 4, 6, 8, and 24 h post dose using three mice per time point, and a single blood sample was collected from a single mouse. All samples were transferred to heparinized tubes and centrifuged at 10 000 rpm for 10 min. The resulting plasma was separated and kept frozen at −80 °C before analysis. For quantification of compound 60c concentrations in plasma, samples were processed by protein precipitation with 4 volumes of methanol and analyzed by LC-MS/MS. Chromatographic separations were carried out on a Zorbax SB-C18 3.5 μm, 150 × 4.6 mm column (Agilent Technologies, Santa Clara, CA) using a Nexera XR liquid chromatography (Shimadzu Corporation, Columbia, MD). The mobile phase consisted of water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) and was used in gradient mode at a flow rate of 0.5 mL/min. The eluate was led directly into an API 5500 triple quadruple mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a turbospray ion source, which was operated in the positive-ion mode. Multiple reaction monitoring was used to track the characteristic mass transitions of compound 60c (m/z 397.1326) and the internal standard VERU-111 (m/z 377.1376). Data were acquired and processed with Analyst software version 1.6.2 (Applied Biosystems, Foster City, CA). The obtained plasma concentration–time profiles were analyzed by the standard noncompartmental pharmacokinetic analysis using the software package WinNonlin 8.0 (Certara, Princeton, NJ).

In Vivo Antitumor Efficacy and Systemic Toxicity Studies in NSG Mice.

Five to six week old immunodeficient NOD scid γ (NSG) male and female mice were obtained from Jackson Laboratories (Bar Harbor, ME) and used for these studies. All animals’ treatments were performed according to the rules of the NIH Principles of Laboratory Animal Care and used under a protocol (protocol #17–056) approved by the University of Tennessee Health Science Center (UTHSC) Institutional Animal Care and Use Committee (IACUC). A375/TxR cells (2.5 × 10⁶) grown to the logarithmic growth phase were resuspended in a 1:1 (v:v) mixture of FBS-free DMEM medium and Matrigel matrix (Corning Life Sciences, Glendale, AZ) and injected subcutaneously into the right flank of the NSG mice. After implantation, the tumor growth was monitored with a caliper and calculated as tumor volume (mm³) = (length × width²)/2, where “length” is the largest diameter and “width” is the smallest diameter. When the tumor volume reached ~100 mm³, mice were randomized into four groups, each containing three male mice and three female mice. Group 1 is the vehicle group treated with a vehicle (3:1:6 ratio of PEG300: Tween 80: saline). Group 2 is the 10 mg/kg 60c treatment group, and group 3 is the 20 mg/kg 60c treatment group. Group 4 is the 10 mg/kg paclitaxel group (1:1:18 ratio of ethanol:cremophor EL:saline). The mice were injected intraperitoneally (IP) with the indicated compound and dosed 3 times per week, and the tumor volume and body weight of each mouse were recorded before the treatment. The experiment was terminated at the humane end point when the xenograft in the vehicle group approached about 1000 mm³. Tumor volumes and tumor weights of the treatment groups were compared with that of the vehicle group using one-way ANOVA followed by multiple comparisons. The mouse tumors and principal organs were collected, fixed in 4% paraformaldehyde, embedded in paraffin wax, and used for hematoxylin–eosin (H&E) staining for histological examination. H&E slides were digitally scanned by a Panoramic FLASH III system (3DHistech Ltd, Budapest, Hungary), and tissue images were captured using CaseViewer digital microscopy software (3DHistech). Metastatic burden was manually quantified by counting the number of metastatic lesions present in the whole tissue section.

Statistical Analysis.

All experimental data were expressed as the mean ± SEM and analyzed by one-way or two-way ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 7 software (San Diego, CA). Statistical significance is presented as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

ABBREVIATIONS USED

ABPs

6-aryl-2-benzoyl-pyridine

CBSIs

colchicine-binding site inhibitors

DMP

Dess–Martin periodinane

EtOAc

ethyl acetate

H&E

hematoxylin/eosin

HLMs

human liver microsomes

IHC

immunohistochemistry

IP

intraperitoneally

MTAs

microtubule-targeting agents

P-gp

P-glycoprotein

PK

pharmacokinetic

PI

propidium iodide

RI

resistance index

SARs

structure–activity relationships

SEMCl

2-(trimethylsilyl)ethoxymethyl chloride

sT

subtilisin-treated tubulin

TFA

trifluoroacetic acid

TMP

trimethoxyphenyl