Synthesis and biological evaluation of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold

Najah Albadari; Yang Xie; Tao Liu; Rui Wang; Lubing Gu; Muxiang Zhou; Zhongzhi Wu; Wei Li

doi:10.1016/j.ejmech.2023.115423

. Author manuscript; available in PMC: 2024 Jul 5.

Published in final edited form as: Eur J Med Chem. 2023 Apr 26;255:115423. doi: 10.1016/j.ejmech.2023.115423

Synthesis and biological evaluation of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold

Najah Albadari ¹, Yang Xie ¹, Tao Liu ², Rui Wang ¹, Lubing Gu ², Muxiang Zhou ^2,^*, Zhongzhi Wu ^1,^*, Wei Li ^1,^*

PMCID: PMC10246915 NIHMSID: NIHMS1898856 PMID: 37130471

Abstract

Overexpression of both human murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) is detected in tumor cells from several cancer types, including childhood acute leukemia lymphoma (ALL), neuroblastoma (NB), and prostate cancer, and is associated with disease progression and treatment resistance. In this report, we described the design and syntheses of a series of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold from our previously reported lead compound JW-2-107 and tested their cytotoxicity in a panel of human cancer cell lines. The best compound identified in this study is compound 3e. Western blot analyses demonstrated that treatments with 3e decreased MDM2 and XIAP protein levels and increased expression of p53, resulting in cancer cell growth inhibition and cell death. Furthermore, compound 3e effectively inhibited tumor growth in vivo when tested using a human 22Rv1 prostate cancer xenograft model. Collectively, results in this study strongly suggest that the tetrahydroquinoline scaffold, represented by 3e and our earlier lead compound JW-2-107, has abilities to dual target MDM2 and XIAP and is promising for further preclinical development.

Keywords: MDM2, XIAP, dual MDM2/XIAP inhibitors, structure-activity relationships, antiproliferative activities, human 22Rv1 prostate xenograft model

Graphical Abstract

graphic file with name nihms-1898856-f0001.jpg

1. Introduction

The MDM2 oncogene is one of the highly amplified or overexpressed genes in the cellular genomes of human malignancies [1,2]. MDM2 overexpression can be detected in various cancers, including prostate, lung, breast, liver, esophagogastric, colorectal cancers, neuroblastoma (NB), and leukemia [3-6]. Overexpression of MDM2 has been associated with cancer progression, treatment assistance, metastasis, and poor prognosis [7-10]. MDM2 is a bona fide human oncogene, and it is the primary negative regulatory factor for the tumor suppressor p53 [11]. MDM2 binds to p53 through its N-terminal domain, forms the MDM2-p53 complex, and inhibits the binding of p53 to its targeted DNA, rendering p53 ineffective as a transcription factor [12, 13]. MDM2 also reduces the transcriptional ability of p53 via promoting p53 nuclear export in cells, leaving p53 inaccessible to targeted DNA [14]. Furthermore, the C-terminal RING domain of MDM2 can act as an E3 ligase to ubiquitinate and degrade p53 [15], resulting in cancer cell proliferation and growth [16]. Thus, blockade of the MDM2-p53 protein-protein interaction would liberate p53 from MDM2 and restore the tumor suppressor function of wild-type p53 [17]. Intense efforts have been made to develop small-molecule inhibitors of the MDM2-p53 interaction for the treatment of human cancers retaining wild-type p53 [18-25]. Till now, nine MDM2 inhibitors have been advanced into clinical trials [26]. In addition to the oncogenic role of MDM2 in the inhibition of p53, MDM2 involves in cancer progression and chemotherapeutic resistance through a p53-independent pathway [27-29]. The MDM2 RING domain binds to mRNAs of other molecules involved in oncogenesis, such as VEGF [30]. MYCN [31], and Slug [32] to regulate their translation in cancer cells, thus MDM2 plays several p53-independent roles in cancer pathogenesis. In addition, MDM2 can act as an RNA-binding protein to upregulate the translation of anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP); The C-terminal RING domain of MDM2 binds to the internal ribosome entry site (IRES) of the XIAP mRNA and enhances the IRES-dependent -translation of XIAP [33]. XIAP is a member of the inhibitor of apoptosis protein (IAP) family that specifically binds to and inhibits the activated forms of caspases 3, 7, and 9, which are major initiators and effectors of the intrinsic (mitochondrial) apoptotic pathway, and thereby prevent cell death [34, 35]. The increased expression of XIAP is detected in most cancer cells, and it selects for tumor cell survival following treatment with various chemotherapy drugs [36-39]. On the other hand, binding of XIAP IRES to the MDM2 RING domain decreases the E3 ubiquitin-ligase activity of MDM2 and inhibits the capacity of the MDM2 RING domain E3 ligase to target itself for ubiquitination, resulting in stabilization and elevated expression of MDM2 protein [40]. This concomitant increase in the expression of both MDM2 and XIAP contributes to cancer progression and drug resistance. Accordingly, simultaneous inhibition of MDM2 and XIAP using small-molecule inhibitors to block the molecular interaction between MDM2 RING domain and XIAP IRES could result in inducing apoptosis in p53-deficient cancer cells that are dependent on the expression of high levels of both MDM2 and XIAP. Furthermore, dual inhibition of MDM2 and XIAP should result in the activation of p53 in p53 wild-type (p53-wt) tumor cells, similar to MDM2 inhibitors that work by disrupting the binding of MDM2 and p53, but has the added advantage of inducing caspases 3, 7, and 9 that is independent of the p53 status. Thus, dual inhibition of MDM2 and XIAP could be a promising strategy for developing targeted agents for cancer treatment.

We have previously reported the discovery of JW-2-107 [N-(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c] quinoline-8-sulfonamide, compound 14 [41]] (Fig. 1), which showed significant MDM2/XIAP inhibition and increased expression of p53 [41]. The results of the structure-activity relationship (SAR) study of the JW-2-107 scaffold showed that the tetrahydroquinoline moiety is essential for its antiproliferative activities (A and B rings, shown in Fig. 1). Furthermore, the sulfonamide NH between the B/C rings and the other NH in tetrahydroquinoline moiety of JW-2-107 have limited tolerance for large substitutions. Thus, substitution at either NH is highly detrimental to the antiproliferative potency. Moreover, substitutions on the tetrahydrofuran E-ring led to a significant loss of potency. To further optimize the JW-2-107 tetrahydroquinoline scaffold and thoroughly determine the SAR of this scaffold towards the development of a viable future clinical candidate targeting MDM2/XIAP, herein, we designed three focused sets of new JW-2-107 analogs (Fig. 1). First, we modified the phenyl C-ring by substituting the dimethyl with bulkier alkyl groups or replacing the phenyl C-ring with other ring systems. Second, we modified the phenyl D-ring by substituting the para-isobutyryl with different functional groups or replacing the phenyl D-ring with aromatic or aliphatic ring systems. Finally, we made reversed sulfonamide analogs of JW-2-107. This combined approach led to producing thirty-one new JW-2-107 analogs, which we evaluated in vitro and in vivo to identify compound 3e as the overall best analog as a new lead compound for future preclinical studies.

Figure 1. — Targeted modifications used to produce new JW-2-107 analogs.

2. Chemistry

The synthetic routes of JW-2-107 analogs were shown in Schemes 1-5. As shown in Scheme 1, compounds 3a-3i were synthesized from the commercially available 4-nitrobenzenesulfonyl chloride and substituted anilines. The substitution reaction of 4-nitrobenzenesulfonyl chloride and substituted anilines in the presence of pyridine in CH₂Cl₂ gave the intermediate sulfonamides 1a-1i. Then the palladium-catalyzed hydrogenation reaction of intermediate sulfonamides 1a-1i gave corresponding amines 2a-2i. Finally, target compounds 3a-3i were obtained by coupling amines 2a-2i, 4-isobutyrylbenzaldehyde, and 2,3-dihydrofuran in the presence of Lewis acid, scandium triflate in CH3CN. In the synthesis of compounds 4a-4d or 7a, 7b, and 8 (Scheme 2 and Scheme 3), coupling of N-(3-acetylphenyl)-4-aminobenzenesulfonamide (compound 2e) or 4-Amino-N-(3,4 dimethylphenyl)benzenesulfonamide (compound 6), substituted aldehydes, and 2,3-dihydrofuran in the presence of scandium triflate in CH3CN obtained compounds 4a-4d or 7a, 7b, and 8. The final compound 9 was obtained by removing the tert-butyloxycarbonyl (BOC) protecting group with trifluoroacetic acid (TFA) in CH₂Cl₂. As shown in Scheme 4, compounds 12a-12m were obtained by mixing anilines 6, 2d, 2e, or 11b-11i, substituted aldehydes, and 2,3-dihydrofuran in the presence of scandium triflate in CH₃CN. Scheme 5 shows the synthesis of compounds 14a and 14b. The substitution reaction of commercially available benzene-1,4-diamine and 3,4-dimethylbenzenesulfonyl chloride or 3-acetylbenzenesulfonyl chloride in dimethylformamide (DMF) gave 13a or 13b. Coupling of 13a or 13b, 4-isobutyrylbenzaldehyde, and 2,3-dihydrofuran in the presence of scandium triflate in CH₃CN gave compounds 14a or 14b.

Scheme 5. — Reagents and conditions: (a) Dry DMF, r.t; (b) Sc(OTf)₃, 4Å molecular sieves, CH₃CN, r.t

Scheme 2. — Reagents and conditions: (a) Pyridine, CH₂Cl₂, r.t; (b) Pd/C, H₂, MeOH/EtOAc (1:1), r.t; (c) Sc(OTf)₃, 4Å molecular sieves, CH₃CN, r.t

Scheme 3. — Reagents and conditions: (a) Pyridine, CH₂Cl₂, r.t; (b) Pd/C, H₂, MeOH/EtOAc (1:1), r.t; (c) Sc(OTf)₃, 4Å molecular sieves, CH₃CN, r.t; (d) TFA, CH₂Cl₂, r.t

Scheme 4. — Reagents and conditions: (a) Pyridine, CH₂Cl₂, r.t; (b) Pd/C, H₂, MeOH/EtOAc (1:1), r.t; (c) Sc(OTf)₃, 4Å molecular sieves, CH₃CN, r.t

3. Results and discussion

3.1. In vitro antiproliferative assay in a panel of human cancer cell lines.

All the new JW-2-107 analogs were tested for their antiproliferative activities, as stereoisomeric mixtures as synthesized, against a panel of human cancer cell lines including three p53-wt cell lines (leukemia EU-1 cell line, melanoma cancer A375 cell line and prostate cancer 22Rv1 cell line) and one p53 mutant cell line (breast cancer MDA-MB-231 cell line). Compound JW-2-107 was used as the reference for comparison. We also included the MDM2-p53 inhibitor AMG-232 and the pan-IAP inhibitor GDC-0152 in our assay. These in vitro biological results are summarized in Table 1

Table 1.

In vitro growth inhibitory effects of JW-2-107 analogs in a panel of human cancer cell lines (IC₅₀ ± standard error of the mean (SEM) [μM], n = 3)

Compound	EU-1 (μM)	A375 (μM)	MDA-MB-231 (μM)	22Rv1 (μM)
3a	2.1±0.4	3.2±0.8	1.5±0.5	1.1±1.7
3b	6.5±1.2	2.2±0.5	1.1±0.3	2.1±1.9
3c	6.2±1.1	5.1±1.2	2.0±0.4	1.3±2.4
3d	7.5±1.9	0.9±0.3	1.6±0.2	0.7±1.6
3e	0.2±0.0	1.0±0.2	1.5±0.3	5.3±2.6
3f	1.5±0.4	2.0±0.4	2.1±0.4	3.3±5.3
3g	1.5±0.6	1.3±0.6	1.9±0.3	3.5±2.3
3h	3.8±0.8	>10	3.8±0.7	nd^a
3i	5.3±1.2	5.9±1.4	3.3±1.0	7.9±3.1
4a	1.3±0.6	0.9±0.1	0.9±0.2	>10
4b	7.2±1.5	1.3±0.2	1.1±0.3	6.3±6.8
4c	1.0±0.3	2.5±0.4	2.2±0.3	>10
4d	0.6±0.2	1.9±0.3	0.6±0.2	>10
7a	>10	4.6±0.9	3.2±0.6	>10
7b	4.5±0.8	8.1±0.7	5.1±0.9	>10
9	9.2±2.3	>10	>10	nd^a
12a	0.8±0.2	9.8±1.7	5.7±0.4	>10
12b	2.6±0.7	>10	>10	nd^a
12c	1.8±0.3	>10	>10	nd^a
12d	5.1±1.3	>10	>10	nd^a
12e	>10	>10	>10	nd^a
12f	3.3±0.9	>10	>10	nd^a
12g	4.0±1.1	1.8±0.3	1.1±0.4	>10
12h	1.9±0.7	7.5±1.9	2.4±0.4	>10
12i	2.3±1.1	>10	2.2±0.6	>10
12j	1.5±0.4	1.1±0.3	1.0±0.2	>10
12k	1.6±0.6	12.1±2.4	3.0±0.4	>10
12l	>10	>10	>10	nd^a
12m	1.4±0.5	0.7±0.2	1.1±0.2	>10
14a	0.6±0.2	2.5±0.9	1.3±0.4	4.0±2.8
14b	0.7±0.2	4.7±0.8	2.0±0.3	7.7±4.7
AMG-232	0.9±0.2	0.6±0.1	>10	0.3±0.0
GDC-0152	0.8±0.2	nd^a	nd^a	nd^a
JW-2-107	0.5±0.3	2.2±0.4	2.2±0.5	5.3±2.2

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Not determined.

In the first phase of our SAR analysis, modifications in the C-ring were designed by substituting the dimethyl of the C-ring of JW-2-107 with larger substitutions. Most compounds exhibited better antiproliferative activity against EU-1 cells than A375, MDA-MB-231, and 22Rv1 cells. Therefore, we used the results of antiproliferative activity against EU-1 cells to discuss the structure-activity relationships. We replaced the dimethyl of the C-ring with bulkier moieties such as 3-ethyl (compound 3a), 3-isopropyl (compound 3b), and 3-tert-butyl (compound 3c). Unfortunately, all these compounds showed lower potency than JW-2-107 (3a, IC₅₀ = 2.1 μM; 3b, IC₅₀ = 6.5 μM; 3c, IC₅₀ = 6.2 μM vs 0.5 μM for JW-2-107). However, substituting the C-ring with 3-acetyl (compound 3e) improved the antiproliferative activity with an IC₅₀ of 0.2 μM. Moving the acetyl group from meta- to para- position afforded 3f (IC₅₀ =1.5 μM) resulted in 7.5- fold drops in potency compared to 3e, which suggests that the meta-position substitution is preferred. Similarly, compound 3g with 3-propionyl substitution has lower potency than compound 3e (3g, IC₅₀ = 1.5 μM vs. 0.2 μM for 3e). Also, replacing the phenyl C-ring with 1-oxo-dihydroisobenzofuran (3h) or benzodioxole (3i) resulted in a significant reduction in potency. Based on the results of 3a and 3g, we found that both the length and the geometry of the substitute groups are important for the activity. Taken together, the acetyl group was considered the optimal substituent at the meta-position of the C-ring.

Since compound 3e bearing the meta-acetyl group phenyl C-ring displayed the highest potency, this scaffold was used for subsequent SAR exploration. We investigated the effect of D-ring substitution to improve the antiproliferative activity of 3e further. Removing the isobutyryl group (4a, IC₅₀ = 1.3 μM) caused an approximately 6.5-fold potency loss compared to 3e. Replacing the para-isobutyryl group with a fused ring, 4b, did not help either. Replacing the para-isobutyryl moiety with an acetyl group at the meta position (compound 4c, IC₅₀ = 1 μM) showed decreased potency compared with 3e. Likewise, replacement of the para-isobutyryl group by isopropyl moiety (4d, IC₅₀ = 0.6 μM) did not exhibit improved potency compared to 3e but was equipotent to JW-2-107. Collectively, these results showed that the para-isobutyryl phenyl D-ring is better for the antiproliferative activity. To further confirm the beneficial effect of the hydrophobic moiety para-isobutyryl phenyl D-ring on the cytotoxic efficacy, we first designed and synthesized 7a, 7b, and 9 based on compound JW-2-107 structure where we kept the 3,4-dimethyl phenyl C-ring and introduced various D-rings. Like 4b, compound 7a with fused D-ring resulted in a significant drop in potency. Substitution with polar functional groups in D-ring, such as acetamide in compound 7b and indole in 9, led to less potency than JW-2-107 with the hydrophobic para isobutyryl phenyl D-ring.

Then, we synthesized a series of compounds with cyclohexyl D-rings 12a-12j in order to explore the aliphatic D-ring system (Scheme 4). Interestingly, compound 12a with cyclohexyl D-ring showed slightly reduced potency compared to JW-2-107 with an IC₅₀ value of 0.8 μM. However, removing the two methyl substituents in parent compound 12a (12b, IC₅₀ = 2.6 μM) reduced antiproliferative potency. Also, bioisosteric replacement of the methyl group(s) with halogens atoms, chlorine (12c, IC₅₀ =1.8 μM), and bromines (12d, IC₅₀ = 5.1 μM) resulted in lower potency. Substitutions of one (12f, IC₅₀ = 3.3 μM) or both methyl substitutions with trifluoromethyl groups (12e) did not increase the potency. Further analyses of this SAR indicated that substituting the C-ring with strong electron-withdrawing substituents dramatically lowered the antiproliferative activity. Furthermore, substituting the C-ring with bulkier electron-donating groups such as dimethylamino moiety (12g, IC₅₀ = 4 μM) gave a 5-fold reduction in potency compared to 12a. Based on these results, it is clearly demonstrated that the antiproliferative activity of compound JW-2-107 analogs is affected by both the size and electronic effects of the C-ring substituents. Encouraged by the antiproliferative activity of 12a, we replaced the C-ring dimethyl substitution of 12a with 3-acetyl and obtained 12j. Unfortunately, 12j showed a 7.5-fold decrease in cytotoxicity compared to 3e (IC₅₀ = 1.5 μM vs. 0.2 μM for 3e). Next, we made compounds 12k-12m by replacing the cyclohexyl D-ring of 12a with other aliphatic heterocycles. Compound 12k with tetrahydropyran D-ring, 12l with 1-acetylpiperidine, and 12m with Boc-piperidine have less activity than 12a.

With the currently most potent compound 3e in hand, we then asked whether a reversed sulfonamide could achieve better efficacy. Thus, we designed and synthesized reversed sulfonamide compounds 14a and 14b based on compound JW-2-107 and its most potent analog so far, compound 3e, respectively (Scheme 5). Compounds 14a and 14b were similar in potency to JW-2-107 but less potent than 3e with IC₅₀ values of 0.6 and 0.7 μM, respectively.

All the current JW-2-107 analogs contain three chiral centers, with two chiral centers in the E-ring restricted to the same geometry due to the structural constriction during the synthesis. Thus, the most potent compound 3e is a mixture of four stereoisomers. Since different stereoisomers often have different biological activities, we isolated the four stereoisomers of 3e using a chiral HPLC column and methanol as the mobile phase (3e peaks 1–4 shown in Fig. S1). We subsequently tested the antiproliferative activity of these four isomers in EU-1 and MDA-MB-231 cell lines. While peak 4 showed slightly better potency compared to the other three peaks, the difference is not significant (Table S1). Therefore, all the subsequent experiments were performed using 3e as a mixture of the four stereoisomers.

We compared the anticancer activity of 3e with the MDM2-p53 inhibitor AMG-232. AMG-232 outperformed 3e in p53-wt solid tumor cell lines A375 and 22Rv1, whereas 3e showed better antiproliferative activity than AMG-232 in p53-wt leukemia cell line EU-1 (Table 1), suggesting a cell type-dependent activity of 3e and AMG-232. 3e also showed better potency than the pan-IAP inhibitor, GDC-0152 in leukemia cell line EU-1. As expected, AMG-232 was not active in p53-mutant MDA-MB-231 cell line while 3e exhibited cytotoxicity to this cell line, which supports our notion that the MDM2/XIAP inhibitor is superior to the MDM2/p53 inhibitor in inhibition of both p53-wt and p53-mutant cancer cells.

3.2. Treatment with compound 3e significantly reduced colony formation by A375 melanoma cells and 22Rv1 prostate cancer cells.

Based on the in vitro cytotoxicity results of all newly synthesized JW-2-107 analogs against four different cancer cell lines and the strong anti-proliferating effect of 3e, compound 3e was prioritized for subsequent biological evaluations. First, we performed clonogenic assay for the anti-proliferative activity of 3e in two p53-wt cell lines A375 and 22Rv1 as previously characterized [42]. As shown in Figure 2A and B, 3e exhibited more potent inhibitory effect on colony formation in A375 at doses of 5 and 10 μM, as compared to JW-2-107.

Figure 2. — Representative pictures of a colony formation assay using (A) A375 cells treated with JW-2-107 and 3e at increasing concentrations (1, 5, or 10 μM). (B) Quantification of colony formation area (A375 cell) is expressed as the grand mean ± SEM compared with vehicle control (set to 100%) of three biological replicate experiments. (C) 22Rv1 cells treated with JW-2-107 and 3e at increasing concentrations (1, 5, or 10 μM). (D) Quantification of colony formation area (22Rv1 cell) is expressed as the grand mean ± SEM compared with vehicle control (set to 100%) of three biological replicate experiments.

Similarly, for 22Rv1 cells (Fig. 2C), treatments with 3e and JW-2-107 also reduced colony formation dose-dependently in this cell line (Fig. 2C). Though 3e showed less potency in 22Rv1 cell line comparing to A3 75 in the cell viability assay, it showed similar potency towards both cell lines in the clonogenic assay. This is partly because the metabolic activity of live cells measured by the cell viability assay and the clonogenic potential measured by colony formation are not necessarily parallel events. Also, unlike the cell viability assay in which cells were treated with compounds for no more than 3 days and were given only treatment once, cells in colony formation assay were treated much longer and the culture medium or medium containing the indicated compounds are replaced every 3 days.

3.3. Compound 3e retains dual inhibition for MDM2 and XIAP

We have previously demonstrated that JW-2-107 simultaneously downregulated the expression of MDM2 and XIAP in cancer cells [41]. To determine whether compound 3e maintained its mechanism of action as JW-2-107 to inhibit MDM2 and XIAP, we performed Western blot assays for the effect of 3e on MDM2 and XIAP expression in a leukemia cell line EU-1. Results showed that 3e induced a significant downregulation of MDM2 and XIAP in a dose and time-dependent manner (Fig. 3A). As also shown in Fig. 3A, downregulation of MDM2 was accompanied by increased expression of p53 and its transcriptional targets p21 and PUMA. Inhibition of XIAP by 3e resulted in activation of the XIAP’s downstream effectors caspases −3 and −9, as well as cleavage of the death substrate PARP (Fig. 3B). We also tested the effects of 3e on two solid tumor cell lines 22Rv1 and A375. Similar to the leukemia cell line EU-1, inhibition of MDM2 and XIAP as well as activation of p53 and cleavage of PARP were detected in these lines treated by 3e, although a higher doses of 3e was required in the solid tumor lines as compared to the leukemia cell line EU-1 (Fig. 3C and D).

We compared the effects of 3e and the MDM2-p53 interaction inhibitor AMG-232 [43] on MDM2 expression in both A375 and 22Rv1 cells. As shown in Figure 3C and D, 3e inhibited expression of MDM2, whereas AMG-232 remarkably induced MDM2 expression. In contrast, a much stronger activation of p53 was induced in 22Rv1 cell treated with AMG-232 than treated with 3e, although both compounds similarly induced cleavage of death substrate PARP (Figure 3C). This suggests that inhibition of XIAP by 3e, which results in activation of caspases and cleavage of its substrate PARP, plays a critical role in 3e-induced cell death.

3.4. In vivo assessment of antitumor efficacy

3.4.1. Antitumor activity of compound 3e in a 22Rv1 subcutaneous xenograft mouse model

As one of the most aggressive androgen-dependent prostate cancer cell lines, 22Rv1 was used to further evaluate the antitumor efficacy of 3e in vivo. 22Rv1 prostate cancer cells were implanted in the flank of NOD scid gamma (NSG) mice and allowed to grow for 14 days until the average tumor volume reached around 100 mm³ prior to treatment. Two groups of mice (n = 5/group) were treated with vehicle alone or with 60 mg/kg dose of 3e by intraperitoneal (IP) administration 7 days/week for 10 days. Tumor size (volume) and body weight were measured every 3 days. As shown in Figure 4A, compound 3e significantly inhibited tumor growth. All the mice were stable, and no significant body weight loss was observed, suggesting that a dose of 60 mg/kg was well tolerated (Fig. 4B). These results demonstrated that treatment of mice with compound 3e attenuated the progression of 22Rv1 tumors at a safe dosage as well.

Figure 4. — The antitumor efficacy of compound 3e against the growth of 22Rv1 xenograft tumors in NSG mice. 22Rv1 cells were subcutaneously injected into the dorsal right flank of NSG mice. When average tumor volume reached around 100 mm³, mice were randomized into 2 groups (n=5/group) and treated with vehicle control or with 60 mg/kg doses of compound 3e (every day, i.p) (A) Tumor volume growth curves with 60 mg/kg compound 3e versus vehicle. Tumor volumes were monitored every 3 days. (B) Mouse body weight was recorded every 3 days throughout the 10 days treatment period. (C) Immunostaining for MDM2 (red fluorescence) was performed to indicate MDM2 levels; c-PARP and c-Cas3 (red fluorescence) were performed to indicate apoptosis; Ki-67 (green fluorescence) was performed to identify proliferating cells; CD31 (PECAM-1, green fluorescence) was utilized to indicate the microvessel density in subcutaneous 22Rv1 xenograft tumors from mice treated by vehicle and compound 3e. Nuclei within each tumor were counterstained with DAPI (blue fluorescence). Scale bar = 50 μm. (D) The relative protein level was quantified by analyzing the fluorescence intensity using ImageJ. Data were presented as mean ± SEM.

3.4.2. 3e decreases MDM2 expression, induces anti-angiogenesis and apoptosis in 22Rv1 xenograft model

Subcutaneous human 22Rv1 prostate cancer tumors were harvested and sectioned (10 μm) using an HM525 NX Cryostat. Tumor sections were then stained with MDM2, cleaved caspase-3 (c-Cas3), c-PARP, Ki67, and CD31 by immunofluorescence (IF). Immunostaining results further revealed that the 22Rv1 xenograft model treated with 3e decreased the MDM2 expression (Fig. 4C). It also revealed that levels of apoptotic markers c-Cas3 and c-PARP (Fig. 4C) were increased in 3e treated xenograft animal models, which is consistent with we have shown in 22Rv1 cells in western blots (Fig. 2C). It indicates that an apoptotic mechanism might be involved in suppressive effects of 3e on prostate cancer either in vitro or in vivo. Moreover, 3e also inhibited the proliferation and disrupted the tumor vasculature of the 22Rv1 xenograft animal model (Fig. 4C).

4. Conclusion

In summary, we have reported the design and synthesis of new analogs of our previously reported lead compound JW-2-107, a dual MDM2 and XIAP inhibitor and obtained compound 3e that showed improved potency over JW-2-107 against a panel of human cancer cell lines, including EU-1 (ALL), A375 (melanoma cancer), MDA-MB-231 (breast cancer), and 22Rv1 (prostate carcinoma). We demonstrated that 3e maintains the on-target of inhibition of MDM2 and XIAP activity and induces apoptosis. The in vivo efficacy of compound 3e was determined in an aggressive 22Rv1 prostate cancer xenograft model, where it exhibited a significant inhibitory effect of tumor growth without acute toxicities. Further optimization of this tetrahydroquinoline scaffold will likely generate a unique dual MDM2 and XIAP inhibitor which will be promising for further preclinical development.

5. Experimental section

5.1. Chemistry

Chemical reagents and solvents were purchased from commercial sources and used without further purification. Glassware was oven-dried before use. All reactions were performed under an argon atmosphere. Aluminum-backed uniplates (Analtech, Newark, DE) were used for standard thin layer chromatography (TLC), which was monitored using ultraviolet (UV) light. Silica gel (230-400 mesh; Fisher Scientific, Pittsburgh, PA) was used for flash chromatography. A Bruker Ascend 400 (Billerica, MA) spectrometer was used to obtain NMR spectra. Coupling constants (J) are provided in Hertz (Hz). Chemical shifts are reported as parts per million (ppm) relative to DMSO-d₆ or tetramethylsilane (TMS) in CDCl₃. The purity of all final compounds (as stereoisomers not separable on the UPLC column used) was ≥95% as determined by UPLC and further confirmed by proton NMR (details given in the Supporting Information). High-resolution mass spectrometry data (HRMS) were obtained on a Waters Acquity UPLC linked to a Waters Acquity Photodiode Array Detector and a Waters qTof mass detector. UPLC analyses were performed using a BEH C18 (2.1 × 50 mm, 1.7μm) column and a mixture of solvent methanol/water at a flow rate of 0.3 mL/min, monitored by UV absorption at the appropriate wavelength.

5.1.1. General procedure for preparation of compounds 1a-1i.

4-nitrobenzenesulfonyl chloride (26.58 mmol) and substituted aniline (29.23 mmol) were dissolved in anhydrous methylene chloride (100 mL) at room temperature under argon. Pyridine (2.31 g, 29.23 mmol) was added dropwise via a syringe at room temperature. The reaction mixture was stirred at room temperature overnight under an argon atmosphere. Then, the volatile was removed under reduced pressure. The yellow solid residues were subjected to flash column chromatography (silica gel, CH₂Cl₂) to afford compounds 1a-1i.

5.1.1.1. N-(3-ethylphenyl)-4-nitrobenzenesulfonamide (1a).

97.1% yield; ¹H NMR (400 MHz, DMSO) δ 10.54 (s, 1H), 8.36 (d, J = 8.9 Hz, 2H), 7.98 (d, J = 8.9 Hz, 2H), 7.14 (s, 1H), 6.94 – 6.86 (m, 3H), 2.48 (q, J = 6.7 Hz, 2H), 1.06 (t, J = 7.6 Hz, 3H).

5.1.1.2. N-(3-isopropylphenyl)-4-nitrobenzenesulfonamide (1b).

97.9% yield; ¹H NMR (400 MHz, DMSO) δ 10.54 (s, 1H), 8.38 (d, J = 8.9 Hz, 2H), 8.00 (d, J = 9.0 Hz, 2H), 7.16 (s, 1H), 6.99 – 6.88 (m, 3H), 2.77 (p, J = 6.9 Hz, 1H), 1.10 (d, J = 7.0 Hz, 6H).

5.1.1.3. N-(3-(tert-butyl)phenyl)-4-nitrobenzenesulfonamide (1c).

97.3% yield; ¹H NMR (400 MHz, DMSO) δ 10.49 (s, 1H), 8.37 (d, J = 8.9 Hz, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.16 (s, 1H), 7.08 (d, J = 15.8 Hz, 2H), 6.89 (d, J = 7.9 Hz, 1H), 1.15 (s, 9H).

5.1.1.4. N-(3-(dimethylamino)phenyl)-4-nitrobenzenesulfonamide (1d).

93.8% yield; ¹H NMR (400 MHz, DMSO) δ 10.40 (s, 1H), 8.38 (d, J = 8.9 Hz, 2H), 8.01 (d, J = 8.9 Hz, 2H), 7.01 (s, 1H), 6.46 – 6.35 (m, 3H), 2.80 (s, 6H).

5.1.1.5. N-(3-acetylphenyl)-4-nitrobenzenesulfonamide (1e).

98% yield; ¹H NMR (400 MHz, CDCl₃) δ 8.29 (d, J = 8.9 Hz, 2H), 7.97 (d, J = 8.8 Hz, 2H), 7.75 (dt, J = 6.5, 1.8 Hz, 1H), 7.66 (d, J = 2.2 Hz, 1H), 7.45 (d, J = 1.9 Hz, 1H), 7.43 (s, 1H), 7.27 (s, 1H), 2.59 (s, 3H).

5.1.1.6. N-(4-acetylphenyl)-4-nitrobenzenesulfonamide (1f).

95.5% yield; ¹H NMR (400 MHz, DMSO) δ 11.20 (s, 1H), 8.39 (d, J = 7.8 Hz, 2H), 8.08 (d, J = 7.9 Hz, 2H), 7.87 (d, J = 8.3 Hz, 2H), 7.24 (d, J = 7.7 Hz, 2H), 2.48 (s, 3H).

5.1.1.7. 4-nitro-N-(3-propionylphenyl)benzenesulfonamide (1g).

96.4% yield; ¹H NMR (400 MHz, DMSO) δ 10.88 (s, 1H), 8.42 – 8.34 (m, 2H), 8.05 – 7.97 (m, 2H), 7.74 – 7.64 (m, 2H), 7.43 (t, J = 7.8 Hz, 1H), 7.35 (ddd, J = 8.1, 2.3, 1.1 Hz, 1H), 2.97 (q, J = 7.1 Hz, 2H), 1.04 (t, J = 7.1 Hz, 3H).

5.1.1.8. 4-nitro-N-(1-oxo-1,3-dihydroisobenzofuran-4-yl)benzenesulfonamide (1h).

94.6% yield; ¹H NMR (400 MHz, DMSO) δ 10.86 (s, 1H), 8.37 (d, J = 8.9 Hz, 2H), 7.99 (d, J = 8.9 Hz, 2H), 7.65 (dd, J = 7.6, 0.9 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.39 (dd, J = 8.0, 0.9 Hz, 1H), 5.26 (s, 2H).

5.1.1.9. N-(benzo[d][1,3]dioxol-5-yl)-4-nitrobenzenesulfonamide (1i).

97% yield; ¹H NMR (400 MHz, DMSO) δ 10.32 (s, 1H), 8.36 (d, J = 8.8 Hz, 2H), 7.92 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 8.3 Hz, 1H), 6.66 (d, J = 2.1 Hz, 1H), 6.46 (dd, J = 8.3, 2.2 Hz, 1H), 5.96 (s, 2H).

5.1.2. General procedure for preparation of compounds 2a-2i.

These compounds were derived from compounds 1a-1i and were synthesized individually in parallel. Sulfonamide 1a-1i (16.32 mmol) and Pd/C (10% Pd base) were mixed in 1:1 of MeOH: EtOAc (50 mL) at room temperature. Hydrogen gas was introduced via an H₂ balloon. The reaction mixture was stirred under an H₂ atmosphere overnight at room temperature. The reaction mixture was filtered to remove the solid. The solution was concentrated under reduced pressure, and the resulting residues were purified by column chromatography (silica gel, Hexane: EtOAc = 2/1 v/v) to give compounds 2a-2i.

5.1.2.1. 4-amino-N-(3-ethylphenyl)benzenesulfonamide (2a).

93.4% yield; ¹H NMR (400 MHz, DMSO) δ 9.79 (s, 1H), 7.37 (d, J = 8.8 Hz, 2H), 7.07 (s, 1H), 6.87 (t, J = 1.9 Hz, 1H), 6.85 (d, J = 7.8 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.51 (d, J = 8.8 Hz, 2H), 5.96 (s, 2H), 2.46 (q, J = 7.6 Hz, 2H), 1.07 (t, J = 7.6 Hz, 3H).

5.1.2.2. 4-amino-N-(3-isopropylphenyl)benzenesulfonamide (2b).

91.9% yield; ¹H NMR (400 MHz, DMSO) δ 9.78 (s, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.09 (t, J = 7.8 Hz, 1H), 6.93 (t, J = 1.9 Hz, 1H), 6.89 – 6.80 (m, 2H), 6.52 (d, J = 8.7 Hz, 2H), 5.97 (s, 2H), 2.75 (hept, J = 6.9 Hz, 1H), 1.10 (d, J = 6.9 Hz, 6H).

5.1.2.3. 4-amino-N-(3-(tert-butyl)phenyl)benzenesulfonamide (2c).

96.7% yield; ¹H NMR (400 MHz, DMSO) δ 9.76 (s, 1H), 7.38 (d, J = 8.7 Hz, 2H), 7.15 – 7.05 (m, 2H), 6.99 (ddd, J = 7.9, 1.9, 1.1 Hz, 1H), 6.84 (ddd, J = 7.9, 2.2, 1.0 Hz, 1H), 6.52 (d, J = 8.8 Hz, 2H), 5.97 (s, 2H), 1.17 (s, 9H).

5.1.2.4. 4-amino-N-(3-(dimethylamino)phenyl)benzenesulfonamide (2d).

95.9% yield; ¹H NMR (400 MHz, DMSO) δ 9.65 (s, 1H), 7.39 (d, J = 8.7 Hz, 2H), 6.95 (t, J = 8.1 Hz, 1H), 6.52 (d, J = 8.7 Hz, 2H), 6.42 (t, J = 2.2 Hz, 1H), 6.34 (ddd, J = 18.2, 7.9, 2.2 Hz, 2H), 5.95 (s, 2H), 2.79 (s, 6H).

5.1.2.5. N-(3-acetylphenyl)-4-aminobenzenesulfonamide (2e).

61.8% yield; ¹H NMR (400 MHz, DMSO) δ 10.13 (s, 1H), 7.63 – 7.51 (m, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 7.6 Hz, 1H), 7.33 – 7.27 (m, 1H), 6.55 – 6.47 (m, 2H), 6.00 (s, 2H), 2.49 (s, 3H).

5.1.2.6. N-(4-acetylphenyl)-4-aminobenzenesulfonamide (2f).

79.2% yield; ¹H NMR (400 MHz, DMSO) δ 10.50 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.17 (d, J = 8.3 Hz, 2H), 6.58 – 6.52 (m, 2H), 6.07 (s, 2H), 2.46 (s, 3H).

5.1.2.7. 4-amino-N-(3-propionylphenyl)benzenesulfonamide (2g).

93.4% yield; ¹H NMR (400 MHz, DMSO) δ 10.12 (s, 1H), 7.65 – 7.56 (m, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 7.6 Hz, 1H), 7.30 (ddd, J = 8.1, 2.2, 1.2 Hz, 1H), 6.52 (d, J = 8.8 Hz, 2H), 6.01 (s, 2H), 2.94 (q, J = 7.1 Hz, 2H), 1.04 (t, J = 7.2 Hz, 3H).

5.1.2.8. 4-amino-N-(1-oxo-1,3-dihydroisobenzofuran-4-yl)benzenesulfonamide (2h).

93.6% yield; ¹H NMR (400 MHz, DMSO) δ 10.01 (s, 1H), 7.56 (dd, J = 7.4, 1.0 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.41 (dd, J = 7.9, 1.1 Hz, 1H), 7.35 (d, J = 8.8 Hz, 2H), 6.53 (d, J = 8.8 Hz, 2H), 6.07 (s, 2H), 5.17 (s, 2H).

5.1.2.9. 4-amino-N-(benzo[d][1,3]dioxol-5-yl)benzenesulfonamide (2i).

89.8% yield; ¹H NMR (400 MHz, DMSO) δ 9.56 (s, 1H), 7.32 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.3 Hz, 1H), 6.62 (d, J = 2.1 Hz, 1H), 6.52 (d, J = 8.8 Hz, 2H), 6.46 (dd, J = 8.3, 2.2 Hz, 1H), 5.95 (d, J = 11.4 Hz, 4H).

5.1.3. General procedure for preparation of compounds 3a-3i.

These compounds were derived from compounds 2a-2i and were synthesized individually in parallel. Aniline 2a-2i (0.27 g, 0.87 mmol), 4-Isobutyrylbenzaldehyde (0.87 mmol) [41], and Sc(OTf)₃ (0.17 mmol) were mixed together and dissolved in anhydrous CH₃CN (10 mL) at room temperature under argon. The reaction mixture was stirred for 1 h at room temperature. 2,3-Dihydrofuran (1.74 mmol) was then added via a syringe. The resulting mixture was stirred at room temperature under argon overnight. The reaction was quenched by adding 50 mL of water at room temperature and neutralized by adding NaHCO₃. The solution was extracted with EtOAc (3 × 50 mL). The extracts were dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure to dryness. The solid residues were purified by column chromatography (silica gel, CH₂Cl₂/acetone = 9/1 v/v) to afford the desired compounds 3a-3i.

5.1.3.1. N-(3-ethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3a).

73.5% yield; ¹H NMR (400 MHz, DMSO) δ 9.89 (s, 1H), 7.98 (d, J = 8.1 Hz, 2H), 7.62 – 7.52 (m, 3H), 7.34 (dd, J = 8.7, 2.3 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 6.95 – 6.79 (m, 4H), 6.71 (t, J = 8.2 Hz, 1H), 5.09 (d, J = 7.4 Hz, 1H), 4.83 (d, J = 3.2 Hz, 1H), 3.72 – 3.53 (m, 2H), 3.48 (td, J = 8.7, 3.5 Hz, 1H), 2.68 (s, 1H), 2.46 (d, J = 7.6 Hz, 2H), 2.27 (s, 1H), 1.93 (s, 1H), 1.76 (t, J = 11.1 Hz, 1H), 1.36 – 1.23 (m, 1H), 1.10 (dd, J = 2.6, 1.4 Hz, 3H), 1.07 (d, J = 7.6 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 204.52, 149.62, 149.12, 147.20, 146.95, 145.22, 145.18, 138.53, 135.65, 135.18, 131.02, 129.44, 129.40, 129.13, 128.89, 128.81, 128.11, 127.38, 126.77, 125.89, 123.59, 123.53, 120.91, 119.24, 119.10, 118.35, 117.37, 117.09, 114.65, 114.33, 74.87, 74.43, 66.09, 64.63, 55.74, 54.94, 43.80, 41.87, 35.12, 35.08, 28.52, 24.44, 19.41, 19.37, 15.80. HRMS (ESI): calcd for C₂₉H₃₂N₂O₄S, 505.2161 [M + H]⁺; found, 505.2152 (mass error = −1.8 ppm).

5.1.3.2. 4-(4-isobutyrylphenyl)-N-(3-isopropylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3b).

67.6% yield; ¹H NMR (400 MHz, DMSO) δ 9.89 (s, 1H), 7.96 (d, J = 6.5 Hz, 2H), 7.56 (ddd, J = 14.0, 5.0, 2.2 Hz, 3H), 7.36 (ddd, J = 25.3, 8.6, 2.3 Hz, 1H), 7.14 (s, 1H), 7.10 (t, J = 7.8 Hz, 1H), 6.95 (p, J = 1.9 Hz, 1H), 6.85 (d, J = 8.0 Hz, 2H), 6.71 (dd, J = 8.7, 5.6 Hz, 1H), 5.07 (d, J = 7.5 Hz, 1H), 4.39 (d, J = 4.9 Hz, 1H), 3.91 – 3.73 (m, 4H), 3.46 (qd, J = 7.0, 3.4 Hz, 1H), 2.81 – 2.60 (m, 2H), 2.31 – 2.20 (m, 1H), 1.93 (dq, J = 16.4, 7.8 Hz, 1H), 1.85 – 1.66 (m, 1H), 1.52 (p, J = 6.4 Hz, 1H), 1.27 (dt, J = 13.5, 5.2 Hz, 1H), 1.09 (dd, J = 6.8, 4.8 Hz, 12H). ¹³C NMR (101 MHz, DMSO) δ 204.22, 204.19, 149.77, 149.73, 149.65, 149.13, 147.22, 146.99, 138.64, 135.68, 135.23, 131.05, 129.42, 129.38, 129.34, 129.15, 128.88, 128.80, 128.10, 127.41, 127.38, 126.87, 125.96, 122.04, 121.99, 120.92, 118.38, 117.77, 117.65, 117.55, 117.29, 114.62, 114.29, 74.92, 74.45, 66.11, 64.63, 55.76, 54.97, 43.86, 41.96, 35.08, 35.04, 33.75, 28.47, 24.47, 24.17, 24.13, 19.45, 19.41. HRMS (ESI): calcd for C₃₀H₃₄N₂O₄S, 541.2137 [M + Na]⁺; found, 541.2137 (mass error = 0 ppm).

5.1.3.3. N-(3-(tert-butyl)phenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3c).

75.9% yield; ¹H NMR (400 MHz, DMSO) δ 9.86 (d, J = 9.2 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.62 – 7.53 (m, 3H), 7.44 – 7.32 (m, 1H), 7.36 – 7.27 (m, 1H), 7.16 – 7.06 (m, 2H), 7.00 (dt, J = 8.0, 1.3 Hz, 1H), 6.96 – 6.77 (m, 2H), 6.72 (t, J = 8.2 Hz, 1H), 5.11 – 4.99 (m, 1H), 4.83 (d, J = 3.2 Hz, 1H), 3.92 – 3.82 (m, 1H), 3.79 (d, J = 10.5 Hz, 1H), 3.76 – 3.65 (m, 1H), 3.69 – 3.59 (m, 1H), 3.63 – 3.43 (m, 1H), 2.67 (d, J = 8.2 Hz, 1H), 1.87 – 1.73 (m, 1H), 1.76 – 1.66 (m, 1H), 1.19 (s, 9H), 1.09 (d, J = 6.8 Hz, 6H), 1.05 (d, J = 1.2 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 203.97, 203.93, 151.96, 151.92, 149.68, 149.13, 147.25, 147.05, 138.50, 135.72, 135.28, 131.08, 129.47, 129.18, 129.06, 129.02, 128.94, 128.88, 128.81, 128.10, 127.44, 127.39, 126.97, 126.05, 120.93, 120.73, 118.38, 117.27, 117.04, 116.94, 116.85, 114.61, 114.29, 103.49, 74.98, 74.50, 66.56, 66.13, 66.01, 64.64, 55.77, 55.01, 43.92, 42.03, 35.05, 35.02, 34.79, 34.62, 32.28, 32.18, 31.42, 31.31, 28.53, 24.49, 23.50, 23.42, 19.49, 19.44. HRMS (ESI): calcd for C₃₁H₃₆N₂O₄S, 533.2474 [M + H]⁺; found, 533.2460 (mass error = −2.6 ppm).

5.1.3.4. N-(3-(dimethylamino)phenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3d).

84.3% yield; ¹H NMR (400 MHz, DMSO) δ 9.77 (d, J = 11.3 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.64 – 7.56 (m, 3H), 7.36 (dd, J = 8.6, 2.2 Hz, 1H), 6.97 (t, J = 8.1 Hz, 1H), 6.90 (s, 1H), 6.73 (t, J = 8.1 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 6.38 (ddd, J = 7.6, 5.4, 1.9 Hz, 1H), 6.33 (dd, J = 8.4, 2.5 Hz, 1H), 5.10 (d, J = 7.4 Hz, 1H), 4.84 (d, J = 3.2 Hz, 1H), 3.89 (q, J = 7.9 Hz, 1H), 3.79 (d, J = 10.7 Hz, 1H), 3.75 – 3.45 (m, 4H), 2.80 (s, 6H), 2.74 – 2.64 (m, 1H), 2.00 – 1.87 (m, 1H), 1.77 (ddd, J = 20.1, 11.8, 8.9 Hz, 1H), 1.54 (p, J = 6.8 Hz, 1H), 1.36 – 1.26 (m, 1H), 1.10 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 203.97, 203.93, 151.23, 149.65, 149.10, 147.29, 147.04, 139.53, 135.73, 135.27, 131.11, 129.73, 129.69, 129.51, 129.24, 128.87, 128.81, 128.14, 127.46, 127.41, 127.21, 126.29, 120.96, 118.34, 114.63, 114.32, 108.06, 108.01, 107.76, 107.55, 103.55, 103.40, 75.07, 74.54, 66.15, 64.62, 55.80, 55.05, 43.98, 41.99, 35.06, 35.02, 28.52, 24.53, 19.50, 19.44. HRMS (ESI): calcd for C₂₉H₃₃N₃O₄S, 520.2270 [M + H]⁺; found, 520.2271 (mass error = 0.2 ppm).

5.1.3.5. N-(3-acetylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3e).

49.5% yield; ¹H NMR (400 MHz, DMSO) δ 10.23 (s, 1H), 7.98 (d, J = 7.0 Hz, 2H), 7.60 (dddd, J = 15.6, 8.9, 6.2, 1.8 Hz, 5H), 7.46 – 7.29 (m, 3H), 7.23 (s, 1H), 6.72 (t, J = 8.4 Hz, 1H), 4.42 (d, J = 4.8 Hz, 1H), 3.87 (q, J = 7.9 Hz, 1H), 3.78 (d, J = 10.7 Hz, 1H), 3.74 – 3.52 (m, 2H), 2.27 (dt, J = 10.7, 5.5 Hz, 1H), 1.93 (dq, J = 15.7, 7.8 Hz, 1H), 1.74 (p, J = 9.5 Hz, 1H), 1.52 (p, J = 6.9 Hz, 1H), 1.34 – 1.20 (m, 1H), 1.09 (d, J = 6.8 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 204.25, 204.22, 198.12, 198.08, 149.83, 149.31, 147.16, 146.90, 139.19, 137.93, 137.90, 135.69, 135.23, 131.05, 130.10, 130.05, 129.40, 129.18, 128.88, 128.80, 128.09, 127.41, 127.36, 126.35, 125.47, 124.38, 124.14, 124.10, 121.05, 118.64, 118.44, 114.71, 114.42, 74.89, 74.39, 66.07, 64.61, 55.71, 54.92, 43.79, 41.83, 35.09, 35.05, 29.93, 28.43, 27.11, 27.08, 24.46, 19.45, 19.40. HRMS (ESI): calcd for C₂₉H₃₀N₂O₅S, 519.1954 [M + H]⁺; found, 519.1965 (mass error = 2.1 ppm).

5.1.3.6. N-(4-acetylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3f).

50.6% yield; ¹H NMR (400 MHz, DMSO) δ 10.58 (s, 1H), 7.98 (d, J = 7.0 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 2.3 Hz, 1H), 7.63 – 7.53 (m, 3H), 7.49 (dd, J = 8.7, 2.3 Hz, 1H), 7.43 (dd, J = 8.6, 2.3 Hz, 1H), 7.28 (s, 1H), 7.19 (dd, J = 8.8, 3.0 Hz, 2H), 6.99 (s, 1H), 6.74 (t, J = 8.3 Hz, 1H), 5.11 (d, J = 7.4 Hz, 1H), 4.84 (d, J = 3.2 Hz, 1H), 4.44 (d, J = 4.8 Hz, 1H), 3.89 (td, J = 8.2, 6.2 Hz, 1H), 3.78 (d, J = 10.7 Hz, 1H), 3.75 – 3.53 (m, 2H), 3.49 (td, J = 8.6, 3.3 Hz, 1H), 2.65 (t, J = 12.3 Hz, 1H), 2.46 (s, 3H), 2.32 – 2.22 (m, 1H), 1.93 (ddd, J = 17.2, 13.6, 8.0 Hz, 1H), 1.75 (p, J = 9.4 Hz, 1H), 1.59 – 1.47 (m, 1H), 1.36 – 1.19 (m, 1H), 1.09 (d, J = 6.1 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 203.98, 203.94, 196.91, 150.02, 149.50, 147.15, 146.89, 143.35, 135.76, 135.29, 131.86, 131.81, 131.06, 130.25, 130.22, 129.37, 129.25, 128.88, 128.82, 128.18, 127.46, 126.42, 125.46, 121.23, 118.47, 117.89, 117.74, 114.72, 114.46, 74.97, 74.43, 68.97, 66.17, 64.60, 56.26, 55.71, 54.97, 43.85, 41.83, 35.06, 35.02, 32.59, 30.05, 28.48, 26.85, 24.55, 19.49, 19.45, 19.43. HRMS (ESI): calcd for C₂₉H₃₀N₂O₅S, 519.1954 [M + H]⁺; found, 519.1961 (mass error = 1.3 ppm).

5.1.3.7. 4-(4-isobutyrylphenyl)-N-(3-propionylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3g).

51.7% yield; ¹H NMR (400 MHz, DMSO) δ 10.23 (d, J = 14.0 Hz, 1H), 7.98 (d, J = 6.8 Hz, 2H), 7.66 (t, J = 1.9 Hz, 1H), 7.62 (dd, J = 7.1, 1.9 Hz, 1H), 7.62 – 1.53 (m, 4H), 7.43 (dd, J = 8.7, 2.3 Hz, 1H), 7.42 – 7.26 (m, 3H), 7.24 (s, 1H), 6.95 (s, 1H), 6.73 (t, J = 8.4 Hz, 1H), 5.09 (d, J = 7.4 Hz, 1H), 4.83 (d, J = 3.2 Hz, 1H), 4.42 (d, J = 4.8 Hz, 1H), 3.87 (td, J = 8.2, 6.2 Hz, 1H), 3.78 (d, J = 10.6 Hz, 1H), 3.74 – 3.51 (m, 2H), 3.44 (td, J = 8.6, 3.3 Hz, 1H), 2.95 (q, J = 7.1 Hz, 2H), 2.69 – 2.60 (m, 1H), 2.27 (dddd, J = 10.5, 7.3, 4.8, 1.9 Hz, 1H), 1.97 – 1.86 (m, 1H), 1.74 (tt, J = 11.8, 9.2 Hz, 1H), 1.58 – 1.47 (m, 1H), 1.29 (qd, J = 7.9, 4.0 Hz, 1H), 1.09 (d, J = 6.8 Hz, 6H), 1.04 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 203.96, 203.93, 200.32, 200.29, 149.87, 149.31, 147.19, 146.94, 139.30, 137.76, 137.73, 135.74, 135.28, 131.07, 130.00, 129.96, 129.41, 129.22, 128.87, 128.81, 128.08, 127.45, 127.37, 126.48, 125.58, 124.13, 123.89, 123.50, 123.45, 121.06, 118.53, 118.44, 118.35, 114.70, 114.43, 74.97, 74.45, 66.08, 64.62, 55.74, 54.97, 43.86, 41.89, 35.06, 35.02, 31.72, 28.49, 24.49, 19.48, 19.45, 19.43, 8.50. HRMS (ESI): calcd for C₃₀H₃₂N₂O₅S, 533.2110 [M + H]⁺; found, 533.2130 (mass error = 3.8 ppm).

5.1.3.8. 4-(4-isobutyrylphenyl)-N-(1-oxo-1,3-dihydroisobenzofuran-4-yl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3h).

51.7% yield; ¹H NMR (400 MHz, DMSO) δ 10.11 (s, 1H), 7.99 (d, J = 8.0 Hz, 2H), 7.63 – 7.54 (m, 4H), 7.54 – 7.50 (m, 1H), 7.50 – 7.45 (m, 2H), 7.39 (tt, J = 7.9, 4.0 Hz, 1H), 7.36 – 7.27 (m, 1H), 7.01 (s, 1H), 6.74 (t, J = 8.9 Hz, 1H), 5.21 (s, 1H), 5.18 – 5.12 (m, 1H), 5.11 – 5.06 (m, 1H), 4.85 (d, J = 3.2 Hz, 1H), 4.42 (d, J = 5.0 Hz, 1H), 3.93 – 3.79 (m, 1H), 3.74 – 3.52 (m, 3H), 3.44 (td, J = 8.6, 3.3 Hz, 1H), 2.66 (d, J = 7.9 Hz, 1H), 2.34 – 2.23 (m, 1H), 2.00 – 1.88 (m, 1H), 1.75 (p, J = 9.7 Hz, 1H), 1.59 – 1.49 (m, 1H), 1.31 (dt, J = 7.9, 4.0 Hz, 1H), 1.10 (d, J = 6.7 Hz, 6H), 1.06 (dt, J = 6.8, 2.0 Hz, 1H).¹³C NMR (101 MHz, DMSO) δ 203.98, 203.94, 170.55, 149.96, 149.47, 147.12, 146.99, 140.35, 139.96, 135.73, 135.30, 133.32, 133.24, 130.93, 130.72, 129.24, 129.19, 128.89, 128.83, 128.23, 128.07, 127.48, 127.43, 127.39, 126.92, 126.83, 126.35, 125.41, 121.85, 121.58, 121.26, 118.60, 114.63, 114.42, 74.81, 74.41, 69.27, 69.21, 66.12, 64.65, 55.67, 54.97, 43.81, 41.87, 35.06, 35.03, 28.52, 24.56, 19.49, 19.46, 19.43. HRMS (ESI): calcd for C₂₉H₂₈N₂O₆S, 533.1746 [M + H]⁺; found, 533.1755 (mass error =1.7 ppm).

5.1.3.9. N-(benzo[d][1,3]dioxol-5-yl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3i).

61.8% yield; ¹H NMR (400 MHz, DMSO) δ 9.67 (d, J = 19.0 Hz, 1H), 8.02 – 7.95 (m, 2H), 7.64 – 7.53 (m, 3H), 7.48 (d, J = 2.2 Hz, 1H), 7.37 – 7.25 (m, 2H), 6.91 (s, 1H), 6.78 – 6.69 (m, 2H), 6.64 (dd, J = 6.7, 2.1 Hz, 1H), 6.61 (d, J = 2.1 Hz, 1H), 6.54 – 6.49 (m, 1H), 5.96 (s, 1H), 5.93 (d, J = 3.9 Hz, 2H), 5.10 (d, J = 7.4 Hz, 1H), 4.85 (d, J = 3.2 Hz, 1H), 3.89 (td, J = 8.2, 6.2 Hz, 1H), 3.75 – 3.62 (m, 2H), 3.58 (t, J = 7.9 Hz, 1H), 3.49 (td, J = 8.7, 3.5 Hz, 1H), 2.67 (dtd, J = 11.2, 7.6, 2.8 Hz, 1H), 1.93 (dq, J = 13.7, 8.6 Hz, 1H), 1.78 (tt, J = 11.8, 9.1 Hz, 1H), 1.31 (dtt, J = 11.8, 7.7, 4.1 Hz, 1H), 1.10 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 203.99, 203.95, 153.20, 149.66, 149.12, 147.76, 147.73, 147.69, 147.29, 147.03, 144.46, 144.39, 144.28, 135.74, 135.28, 132.86, 132.69, 132.65, 130.95, 129.32, 129.25, 129.16, 128.88, 128.82, 128.05, 127.46, 127.35, 126.70, 125.84, 124.57, 121.00, 118.40, 114.83, 114.55, 114.50, 114.46, 114.30, 112.97, 108.69, 108.64, 108.60, 103.60, 103.35, 103.31, 101.63, 101.58, 75.03, 74.52, 66.05, 64.62, 55.79, 55.02, 46.05, 43.93, 41.95, 35.06, 35.03, 28.50, 24.49, 19.49, 19.46, 19.43. HRMS (ESI): calcd for C₂₈H₂₈N₂O₆S, 521.1746 [M + H]⁺; found 521.1749 (mass error = 0.6 ppm).

5.1.4. General procedure for preparation of compounds 4a-4d.

N-(3-acetylphenyl)-4-aminobenzenesulfonamide (2e) (0.27 g, 0.87 mmol), substituted aldehydes (0.87 mmol), and Sc(OTf)₃ (0.17 mmol) were mixed together and dissolved in anhydrous CH₃CN (10 mL) at room temperature under argon. The reaction mixture was stirred for 1 h at room temperature. 2,3-Dihydrofuran (1.74 mmol) was then added via a syringe. The resulting mixture was stirred at room temperature under argon overnight. The reaction was quenched by adding 50 mL of water at room temperature and neutralized by adding NaHCO₃. The solution was extracted with EtOAc (3 × 50 mL). The extracts were dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure to dryness. The solid residues were purified by column chromatography (silica gel, CH₂Cl₂/acetone = 9/1 v/v) to afford the desired compounds 4a-4d.

5.1.4.1. N-(3-acetylphenyl)-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4a).

68.7% yield; ¹H NMR (400 MHz, DMSO) δ 10.24 (d, J = 14.9 Hz, 2H), 7.66 (p, J = 1.6 Hz, 2H), 7.63 (d, J = 2.3 Hz, 1H), 7.62 – 7.56 (m, 3H), 7.42 (tt, J = 5.9, 1.6 Hz, 6H), 7.39 – 7.34 (m, 9H), 7.32 – 7.24 (m, 2H), 7.19 (s, 1H), 6.88 (s, 1H), 6.73 (dd, J = 13.5, 8.7 Hz, 2H), 5.09 (d, J = 7.4 Hz, 1H), 4.74 (d, J = 3.2 Hz, 1H), 4.41 (d, J = 4.8 Hz, 1H), 3.86 (td, J = 8.3, 6.4 Hz, 1H), 3.73 – 3.63 (m, 2H), 3.62 – 3.52 (m, 1H), 3.46 (td, J = 8.7, 3.3 Hz, 1H), 2.67 – 2.55 (m, 1H), 2.49 (s, 3H), 2.29 – 2.18 (m, 1H), 1.98 – 1.84 (m, 1H), 1.77 (tt, J = 11.8, 9.1 Hz, 1H), 1.52 (dddd, J = 12.8, 8.0, 5.7, 1.8 Hz, 1H), 1.31 (dtd, J = 11.7, 7.4, 3.4 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 197.87, 197.83, 150.12, 149.57, 141.94, 141.71, 139.34, 137.97, 137.95, 131.11, 130.02, 129.98, 129.43, 128.94, 128.82, 128.38, 128.03, 127.87, 127.31, 127.06, 126.21, 125.29, 124.23, 124.01, 123.97, 121.08, 118.63, 118.44, 118.40, 114.60, 114.37, 75.14, 74.54, 66.14, 64.56, 56.07, 55.18, 44.30, 41.97, 28.57, 27.15, 27.12, 24.56. HRMS (ESI): calcd for C₂₅H₂₄N₂O₄S, 449.1535 [M + H]⁺; found 449.1538 (mass error = 0.7 ppm).

5.1.4.2. N-(3-acetylphenyl)-4-(2,3-dihydrobenzofuran-5-yl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4b).

66.4% yield; ¹H NMR (400 MHz, DMSO) δ 10.21 (d, J = 14.7 Hz, 2H), 7.72 – 7.51 (m, 6H), 7.43 – 7.29 (m, 7H), 7.27 (d, J = 7.5 Hz, 2H), 7.11 (dt, J = 8.6, 2.5 Hz, 2H), 7.07 (s, 1H), 6.81 – 6.64 (m, 5H), 5.05 (d, J = 7.4 Hz, 1H), 4.65 (d, J = 3.1 Hz, 1H), 4.50 (td, J = 8.7, 3.2 Hz, 4H), 4.38 (d, J = 4.7 Hz, 1H), 3.84 (q, J = 7.9 Hz, 1H), 3.67 (td, J = 8.9, 5.4 Hz, 1H), 3.62 – 3.52 (m, 2H), 3.47 (td, J = 8.7, 3.2 Hz, 1H), 3.18 – 3.11 (m, 5H), 2.50 (s, 3H), 2.19 (dt, J = 11.5, 5.5 Hz, 1H), 1.92 (dq, J = 16.5, 7.8 Hz, 1H), 1.83 – 1.68 (m, 1H), 1.52 (dt, J = 13.6, 7.3 Hz, 1H), 1.38 (ddd, J = 11.7, 7.6, 3.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 197.88, 197.84, 159.91, 159.47, 150.19, 149.65, 139.35, 137.96, 137.93, 133.79, 133.43, 131.13, 130.01, 129.96, 129.43, 128.54, 128.03, 128.00, 127.74, 127.26, 126.59, 126.04, 125.32, 125.10, 124.22, 123.96, 123.78, 121.06, 118.62, 118.41, 118.32, 114.48, 114.28, 108.98, 75.30, 74.59, 71.49, 71.40, 66.18, 64.50, 61.09, 55.69, 54.87, 49.06, 45.82, 45.06, 44.64, 41.98, 29.57, 29.52, 28.65, 27.13, 27.11, 24.62, 24.18, 8.96. HRMS (ESI): calcd for C₂₇H₂₆N₂O₅S, 491.1641 [M + H]⁺; found 491.1643 ( mass error = 0.4 ppm).

5.1.4.3. N,4-bis(3-acetylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4c).

77.4% yield; ¹H NMR (400 MHz, DMSO) δ 10.24 (s, 2H), 8.00 (dt, J = 5.7, 1.9 Hz, 2H), 7.95 – 7.87 (m, 2H), 7.69 (ddd, J = 8.4, 6.7, 1.6 Hz, 2H), 7.64 (dt, J = 5.6, 2.2 Hz, 4H), 7.62 – 7.58 (m, 3H), 7.54 (td, J = 7.7, 2.7 Hz, 2H), 7.47 – 7.39 (m, 2H), 7.36 (ddd, J = 8.4, 5.6, 1.6 Hz, 5H), 7.22 (s, 1H), 6.95 (s, 1H), 6.74 (dd, J = 8.7, 4.7 Hz, 2H), 5.10 (d, J = 7.4 Hz, 1H), 4.85 (d, J = 3.2 Hz, 1H), 4.42 (d, J = 4.7 Hz, 1H), 3.89 (td, J = 8.2, 6.1 Hz, 1H), 3.79 (d, J = 10.8 Hz, 1H), 3.69 (td, J = 8.9, 5.5 Hz, 1H), 3.57 (q, J = 8.0 Hz, 1H), 3.45 (td, J = 8.7, 3.4 Hz, 1H), 2.72 – 2.61 (m, 1H), 2.58 (s, 6H), 2.30 (dtd, J = 10.7, 5.2, 2.4 Hz, 1H), 1.99 – 1.85 (m, 1H), 1.74 (tt, J = 11.7, 9.1 Hz, 1H), 1.57 – 1.46 (m, 1H), 1.34 – 1.18 (m, 1H).¹³C NMR (101 MHz, DMSO) δ 198.38, 197.89, 197.84, 149.95, 149.40, 142.63, 142.44, 139.31, 137.97, 137.95, 137.53, 137.32, 133.66, 131.86, 131.09, 130.03, 129.99, 129.42, 129.38, 129.29, 128.35, 128.08, 127.97, 127.35, 126.52, 126.43, 125.52, 124.27, 124.04, 124.00, 121.11, 118.65, 118.48, 118.45, 114.71, 114.44, 75.07, 74.46, 68.98, 66.08, 64.62, 56.25, 55.77, 54.88, 44.06, 41.87, 30.04, 28.54, 27.30, 27.27, 27.15, 27.13, 24.42. HRMS (ESI): calcd for C₂₇H₂₆N₂O₅S, 489.1484 [M − H]⁻; found, 489.1488 (mass error = 0.8 ppm).

5.1.4.4. N-(3-acetylphenyl)-4-(4-isopropylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4d).

73.6% yield; ¹H NMR (400 MHz, DMSO) δ 10.21 (d, J = 15.2 Hz, 2H), 7.67 – 7.53 (m, 6H), 7.44 – 7.29 (m, 11H), 7.24 (dd, J = 8.1, 5.8 Hz, 4H), 7.15 (s, 1H), 6.84 (s, 1H), 6.69 (dd, J = 18.9, 8.7 Hz, 2H), 5.07 (d, J = 7.4 Hz, 1H), 4.68 (d, J = 3.1 Hz, 1H), 4.39 (d, J = 4.7 Hz, 1H), 3.85 (q, J = 8.2 Hz, 1H), 3.73 – 3.52 (m, 3H), 3.47 (td, J = 8.7, 3.3 Hz, 1H), 2.87 (pd, J = 6.9, 5.0 Hz, 2H), 2.58 (q, J = 8.8 Hz, 1H), 2.50 (s, 6H), 2.27 – 2.17 (m, 1H), 1.98 (s, 1H), 1.95 – 1.86 (m, 1H), 1.85 – 1.73 (m, 1H), 1.52 (dt, J = 12.9, 6.4 Hz, 1H), 1.37 (ddd, J = 11.9, 7.7, 3.8 Hz, 1H), 1.18 (dd, J = 6.9, 2.9 Hz, 12H).¹³C NMR (101 MHz, DMSO) δ 197.86, 197.82, 150.14, 149.60, 148.59, 148.09, 139.34, 139.07, 137.97, 137.94, 131.12, 130.01, 129.97, 129.43, 128.80, 128.01, 127.28, 127.17, 126.86, 126.71, 126.08, 125.14, 124.22, 123.99, 123.95, 121.08, 118.62, 118.42, 118.29, 114.47, 114.27, 75.21, 74.60, 66.19, 64.54, 60.23, 55.80, 55.04, 44.28, 41.83, 33.66, 33.60, 28.66, 27.16, 27.13, 24.58, 24.45, 24.39, 24.34, 21.23, 14.54. HRMS (ESI): calcd for C₂₈H₃₀N₂O₄S, 491.2005 [M + H]⁺; found, 491.2000 (mass error = −1.0 ppm).

5.1.5. General procedure for preparation of compounds 7a,7b and 8.

These compounds were derived from 4-Amino-N-(3,4 dimethylphenyl)benzenesulfonamide (compound 6) which was synthesized from N-(3,4-Dimethylphenyl)-4-nitrobenzenesulfonamide (compound 5). Both compounds were described and characterized in the experimental section of our previous paper [41]. 4-amino-N-(3,4-dimethylphenyl)benzenesulfonamide (compound 6) (0.87 mmol), substituted aldehydes (0.87 mmol), and Sc(OTf)₃ (0.17 mmol) were mixed together, and dissolved in anhydrous CH₃CN (10 mL) at room temperature under argon. The reaction mixture was stirred for 1 h at room temperature. 2,3-Dihydrofuran (1.74 mmol) was then added via a syringe. The resulting mixture was stirred at room temperature under argon overnight. The reaction was quenched by adding 50 mL of water at room temperature and neutralized by adding NaHCO₃. The solution was extracted with EtOAc (3 × 50 mL). The extracts were dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure to dryness. The solid residues were purified by column chromatography (silica gel, CH₂Cl₂/acetone = 9/1 v/v) to afford the desired compounds 7a, 7b, and 8.

5.1.5.1. 4-(2,3-dihydrobenzofuran-5-yl)-N-(3,4-dimethylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (7a).

74.1% yield; ¹H NMR (400 MHz, DMSO) δ (d, J = 15.0 Hz, 1H), 7.60 (dd, J = 19.3, 2.3 Hz, 1H), 7.40 (dd, J = 8.7, 2.2 Hz, 1H), 7.34 (dd, J = 8.6, 2.3 Hz, 1H), 7.28 (d, J = 3.2 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 6.91 – 6.82 (m, 2H), 6.78 – 6.68 (m, 3H), 5.07 (d, J = 7.5 Hz, 1H), 4.64 (d, J = 3.1 Hz, 1H), 4.50 (td, J = 8.7, 3.2 Hz, 2H), 4.39 (d, J = 4.7 Hz, 1H), 3.85 (q, J = 8.0 Hz, 1H), 3.66 (ddd, J = 13.5, 9.9, 5.5 Hz, 1H), 3.60 (d, J = 10.6 Hz, 1H), 3.52 (td, J = 8.5, 3.2 Hz, 1H), 3.15 (t, J = 8.7 Hz, 2H), 2.56 (qd, J = 7.8, 3.1 Hz, 1H), 2.22 – 2.13 (m, 1H), 2.08 (d, J = 9.6 Hz, 6H), 1.96 – 1.73 (m, 1H), 1.53 (qd, J = 7.1, 2.7 Hz, 1H), 1.43 – 1.32 (m, 1H). ¹³C NMR (101 MHz, DMSO) δ 159.93, 159.48, 149.96, 149.41, 137.18, 137.13, 136.45, 136.42, 133.89, 133.51, 131.87, 131.79, 131.08, 130.32, 130.27, 129.41, 128.54, 128.04, 127.97, 127.74, 127.24, 126.80, 126.60, 125.84, 125.31, 123.78, 121.67, 121.47, 121.02, 118.27, 117.89, 117.57, 114.41, 114.20, 108.99, 75.39, 74.71, 71.50, 71.41, 66.18, 64.53, 55.81, 55.00, 44.77, 42.10, 29.60, 29.54, 28.67, 24.64, 19.99, 19.06. HRMS (ESI): calcd for C₂₇H₂₈N₂O₄S, 477.1848 [M + H]⁺; found, 477.1861 (mass error = 2.7 ppm).

5.1.5.2. N-(4-(8-(N-(3,4-dimethylphenyl)sulfamoyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin- 4-yl)phenyl)acetamide (7b).

65.2% yield; ¹H NMR (400 MHz, DMSO) δ 9.97 (d, J = 9.3 Hz, 1H), 9.72 (d, J = 14.8 Hz, 1H), 7.87 – 7.75 (m, 2H), 7.60 – 7.53 (m, 3H), 7.51 (d, J = 2.2 Hz, 1H), 7.41 – 7.28 (m, 3H), 6.94 (d, J = 8.1 Hz, 1H), 6.87 – 6.65 (m, 4H), 5.07 (d, J = 7.5 Hz, 1H), 4.67 (d, J = 3.1 Hz, 1H), 3.85 (q, J = 7.8 Hz, 1H), 3.76 – 3.44 (m, 4H), 2.58 (d, J = 8.6 Hz, 1H), 2.19 (s, 1H), 2.08 (d, J = 5.8 Hz, 6H), 2.02 (d, J = 2.1 Hz, 3H), 2.00 – 1.95 (m, 1H), 1.91 (d, J = 7.3 Hz, 1H), 1.77 (t, J = 10.8 Hz, 2H), 1.57 – 1.42 (m, 1H), 1.35 (ddd, J = 11.6, 7.6, 3.5 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 192.00, 169.60, 168.72, 168.68, 149.87, 149.33, 145.29, 139.43, 138.96, 137.15, 137.09, 136.46, 136.41, 136.38, 136.12, 131.85, 131.77, 131.54, 131.31, 131.01, 130.31, 130.27, 129.34, 129.06, 127.96, 127.38, 127.31, 127.23, 126.80, 125.86, 121.66, 121.46, 120.96, 119.40, 119.30, 118.98, 118.89, 118.26, 117.89, 117.57, 114.43, 114.21, 75.26, 74.60, 66.11, 64.53, 55.67, 54.84, 52.78, 44.47, 42.00, 28.60, 24.69, 24.44, 20.03, 19.10. HRMS (ESI): calcd for C₂₇H₂₉N₃O₄S, 492.1957 [M + H]⁺; found, 492.1978 (mass error = 4.3 ppm).

5.1.5.3. tert-butyl 3-(8-(N-(3,4-dimethylphenyl)sulfamoyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-4-yl)-1H-indole-1-carboxylate (8).

42.2% yield; ¹H NMR (400 MHz, CDCl₃) δ 8.18 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 2.2 Hz, 1H), 7.68 – 7.58 (m, 2H), 7.46 (dt, J = 8.3, 1.8 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.32 – 7.24 (m, 3H), 7.01 (d, J = 8.1 Hz, 1H), 6.91 (s, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.57 (dd, J = 8.6, 1.2 Hz, 1H), 6.33 (s, 1H), 5.25 (d, J = 7.5 Hz, 1H), 5.10 (d, J = 3.0 Hz, 1H), 4.32 (s, 1H), 3.75 (q, J = 7.9 Hz, 1H), 3.67 (td, J = 8.7, 4.0 Hz, 1H), 3.01 (q, J = 8.9 Hz, 1H), 2.20 (t, J = 1.8 Hz, 9H), 2.15 – 2.03 (m, 1H), 1.66 (dd, J = 8.3, 4.2 Hz, 1H), 1.60 (d, J = 1.0 Hz, 6H).

5.1.6. Preparation of N-(3,4-dimethylphenyl)-4-(1H-indol-3-yl)-2,3,3a,4,5,9b hexahydrofuro[3,2-c]quinoline-8-sulfonamide (9).

To a solution of tert-butyl 3-(8-(N-(3,4-dimethylphenyl)sulfamoyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-4-yl)-1H-indole-1-carboxylate (compound 8) (500 mg, 0.9 mmol) in 10 mL of CH₂Cl₂ was added TFA (2 mL) at 0 °C. The mixture was stirred at RT for 1 h. Upon completion, the mixture was poured into NaHCO₃ solution and extracted three times with CH₂Cl₂ (30 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, and evaporated to dryness. The crude product was purified by column chromatography (silica gel, (Hexane: EtOAc = 2/1 v/v) to yield target compound 9 as a pale-yellow solid (Yield: 81.8 %).¹H NMR (400 MHz, DMSO) δ 11.05 (s, 1H), 9.71 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 2.3 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.29 (dd, J = 8.7, 2.3 Hz, 1H), 7.09 (t, J = 7.6 Hz, 1H), 6.97 (dd, J = 17.2, 8.2 Hz, 2H), 6.86 (d, J = 2.3 Hz, 1H), 6.81 (dd, J = 8.1, 2.4 Hz, 1H), 6.69 (d, J = 8.5 Hz, 2H), 5.15 (d, J = 7.5 Hz, 1H), 5.04 (d, J = 3.0 Hz, 1H), 3.60 (q, J = 7.9 Hz, 1H), 3.50 (td, J = 8.6, 3.6 Hz, 1H), 2.79 (q, J = 7.8 Hz, 1H), 2.09 (d, J = 6.6 Hz, 6H), 1.96 – 1.81 (m, 1H), 1.46 (ddt, J = 14.4, 10.4, 4.9 Hz, 1H). ¹³C NMR (101 MHz, DMSO) δ 149.77, 137.10, 136.62, 136.43, 131.82, 130.28, 129.50, 127.16, 126.54, 126.00, 122.79, 121.67, 121.64, 121.25, 119.15, 118.98, 117.86, 115.38, 114.26, 112.02, 74.57, 66.06, 48.61, 43.72, 25.46, 20.04, 19.11. HRMS (ESI): calcd for C₂₇H₂₇N₃O₃S, 472.1695 [M − H]⁻; found 472.1679 (mass error = −3.4 ppm).

5.1.7. General procedure for preparation of compounds 10b-10f, 10h, and 10i

The method used to synthesize compounds 10b-10f, 10h, and 10i was used to synthesize compounds 1a-1i. Compounds 10b, 10c, 10d, and 10f were characterized in the experimental section of our previous paper [41].

5.1.7.1. N-(3,4-bis(trifluoromethyl)phenyl)-4-nitrobenzenesulfonamide (10e).

91.9% yield; ¹H NMR (400 MHz, CDCl₃) δ 8.41 – 8.34 (m, 2H), 8.11 – 8.04 (m, 2H), 7.78 (d, J = 8.7 Hz, 1H), 7.53 (d, J = 2.4 Hz, 1H), 7.47 (dd, J = 8.7, 2.3 Hz, 1H), 7.31 (s, 1H).

5.1.7.2. N-(4-methylcyclohexyl)-4-nitrobenzenesulfonamide (10h).

87.7% yield; ¹H NMR (400 MHz, DMSO) δ 8.41 (d, J = 8.5 Hz, 2H), 8.05 (dd, J = 8.1, 4.4 Hz, 3H), 2.93 (dtt, J = 11.5, 7.7, 3.9 Hz, 1H), 1.63 – 1.50 (m, 4H), 1.15 (qd, J = 13.1, 3.9 Hz, 3H), 0.90 – 0.80 (m, 2H), 0.78 (d, J = 6.4 Hz, 3H).

5.1.7.3. 4-nitro-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide (10i).

98.8% yield; ¹H NMR (400 MHz, DMSO) δ 10.37 (s, 1H), 8.41 – 8.33 (m, 2H), 8.02 – 7.94 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 6.84 – 6.76 (m, 2H), 2.59 (q, J = 4.3 Hz, 4H), 1.65 (p, J = 3.2 Hz, 4H).

5.1.8. General procedure for preparation of compounds 11b-11f, 11h, and 11i

These compounds were derived from compounds 10b-10f, 10h, or 10i and were synthesized individually in parallel. The method used to synthesize compounds 11b-11f, 11h and 11i was used to synthesize compounds 2a-2i. Compounds 11b, 11c, 11d, and 11f were characterized in the experimental section of our previous paper [41].

5.1.8.1. 4-amino-N-(3,4-bis(trifluoromethyl)phenyl)benzenesulfonamide (11e).

93.5% yield; ¹H NMR (400 MHz, DMSO) δ 10.97 (s, 1H), 7.91 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 2.3 Hz, 1H), 7.50 (dq, J = 9.7, 2.5 Hz, 3H), 6.62 – 6.54 (m, 2H), 6.17 (s, 2H).

5.1.8.2. 4-amino-N-(4-methylcyclohexyl)benzenesulfonamide (11h).

86.7%% yield; ¹H NMR (400 MHz, DMSO) δ 7.45 – 7.38 (m, 2H), 7.10 (d, J = 7.2 Hz, 1H), 6.62 – 6.55 (m, 2H), 5.89 (s, 2H), 2.71 (dtt, J = 11.5, 7.6, 3.8 Hz, 1H), 1.62– 1.49 (m, 4H), 1.21 – 1.15 (m, 1H), 1.08 (qd, J = 13.2, 3.7 Hz, 2H), 0.82 (d, J = 10.4 Hz, 1H), 0.78 (d, J = 6.5 Hz, 3H).

5.1.8.3. 4-amino-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide (11i).

92.6% yield; ¹H NMR (400 MHz, DMSO) δ 9.62 (s, 1H), 7.37 (d, J = 8.3 Hz, 2H), 6.85 (d, J = 8.2 Hz, 1H), 6.79 (dd, J = 8.2, 2.3 Hz, 1H), 6.73 (d, J = 2.2 Hz, 1H), 6.52 (d, J = 8.5 Hz, 2H), 5.93 (s, 2H), 2.61 – 2.53 (m, 4H), 1.65 (p, J = 3.2 Hz, 4H).

5.1.9. General procedure for preparation of compounds 12a-12m.

These compounds were derived from compounds 6, 11b-11f, 2d, 11h, 11i, or 2e and were synthesized individually in parallel. Aniline 6, 11b-11f, 2d, 11h, 11i, or 2e (0.27 g, 0.87 mmol), substituted aldehyde (0.87 mmol), and Sc(OTf)₃ (0.17 mmol) were mixed together and dissolved in anhydrous CH₃CN (10 mL) at room temperature under argon. The reaction mixture was stirred for 1 h at room temperature. 2,3-Dihydrofuran (1.74 mmol) was then added via a syringe. The resulting mixture was stirred at room temperature under argon overnight. The reaction was quenched by adding 50 mL of water at room temperature and neutralized by adding NaHCO₃. The solution was extracted with EtOAc (3 × 50 mL). The extracts were dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure to dryness. The solid residues were purified by column chromatography (silica gel, CH₂Cl₂/acetone = 9/1 v/v) to afford the desired compounds 12a-12m.

5.1.9.1. 4-cyclohexyl-N-(3,4-dimethylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12a).

76% yield; ¹H NMR (400 MHz, DMSO) δ 9.65 (s, 1H), 7.44 (s, 1H), 7.22 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.80 (s, 1H), 6.76 (d, J = 8.1 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.05 (s, 1H), 4.92 (d, J = 7.4 Hz, 1H), 3.67 (q, J = 7.9 Hz, 1H), 3.14 (d, J = 6.1 Hz, 1H), 2.06 (d, J = 3.7 Hz, 6H), 1.74 (dd, J = 28.1, 12.1 Hz, 4H), 1.58 (dd, J = 22.0, 10.9 Hz, 2H), 1.36 – 1.20 (m, 2H), 1.18 (s, 1H), 1.15 (d, J = 9.0 Hz, 2H), 0.93 (dq, J = 23.4, 11.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 149.11, 137.06, 136.38, 131.81, 130.23, 129.13, 127.08, 126.11, 121.65, 120.89, 117.88, 113.93, 74.79, 66.01, 55.72, 29.97, 28.68, 26.37, 26.17, 25.92, 23.65, 20.00, 19.08. HRMS (ESI): calcd for C₂₅H₃₂N₂O₃S, 441.2212 [M + H]⁺; found, 441.2210 (mass error = −0.5 ppm).

5.1.9.2. 4-cyclohexyl-N-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12b).

90.4% yield; ¹H NMR (400 MHz, DMSO) δ 9.88 (s, 1H), 7.46 (s, 1H), 7.28 – 7.14 (m, 3H), 7.04 (d, J = 7.9 Hz, 2H), 6.95 (t, J = 7.3 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 6.08 (s, 1H), 4.92 (d, J = 7.4 Hz, 1H), 4.01 (q, J = 7.1 Hz, 0H), 3.66 (q, J = 7.9 Hz, 1H), 3.44 (td, J = 8.8, 3.4 Hz, 1H), 3.14 (d, J = 5.8 Hz, 1H), 2.09 (d, J = 12.7 Hz, 1H), 1.97 (s, 0H), 1.78 (d, J = 8.1 Hz, 2H), 1.71 (d, J = 11.2 Hz, 2H), 1.57 (dt, J = 20.3, 10.3 Hz, 2H), 1.34 – 1.23 (m, 1H), 1.22 – 1.07 (m, 3H), 0.93 (dq, J = 22.2, 11.3 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 149.22, 138.84, 129.39, 129.09, 127.10, 125.94, 123.79, 120.95, 119.92, 113.95, 74.76, 66.07, 60.23, 55.69, 40.51, 39.23, 29.97, 28.68, 26.37, 26.17, 25.92, 23.65, 21.22, 14.54. HRMS (ESI): calcd for C₂₃H₂₈N₂O₃S, 413.1899 [M + H]⁺; found 413.1891 (mass error = −1.9 ppm).

5.1.9.3. 4-cyclohexyl-N-(3,4-dichlorophenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12c).

68.4% yield; ¹H NMR (400 MHz, DMSO) δ 10.29 (s, 1H), 7.49 – 7.42 (m, 2H), 7.28 (dd, J = 8.7, 2.4 Hz, 1H), 7.21 (d, J = 2.5 Hz, 1H), 7.05 (dd, J = 8.8, 2.6 Hz, 1H), 6.73 (d, J = 8.7 Hz, 1H), 6.18 (s, 1H), 4.93 (d, J = 7.4 Hz, 1H), 3.67 (q, J = 8.0 Hz, 1H), 3.43 (dd, J = 9.1, 3.6 Hz, 1H), 3.16 (d, J = 5.8 Hz, 1H), 2.08 (d, J = 12.3 Hz, 1H), 1.76 (dd, J = 24.5, 12.0 Hz, 3H), 1.68 (s, 1H), 1.64 – 1.48 (m, 2H), 1.35 – 1.02 (m, 5H), 0.92 (h, J = 12.5 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 149.55, 139.14, 131.69, 131.46, 129.15, 127.12, 125.63, 124.87, 121.02, 120.70, 119.66, 114.11, 74.69, 66.02, 55.61, 40.48, 39.12, 29.96, 28.67, 26.36, 26.16, 25.91, 23.63. HRMS (ESI): calcd for C₂₃H₂₆Cl₂N₂O₃S, 479.0963 [M − H]⁻; found 479.0974 (mass error = 2.3 ppm).

5.1.9.4. 4-cyclohexyl-N-(3,4-dibromophenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12d).

78.3% yield; ¹H NMR (400 MHz, DMSO) δ 10.27 (s, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.34 (d, J = 2.5 Hz, 1H), 7.27 (dd, J = 8.7, 2.4 Hz, 1H), 7.01 (dd, J = 8.8, 2.6 Hz, 1H), 6.73 (d, J = 8.7 Hz, 1H), 6.20 (s, 1H), 4.93 (d, J = 7.4 Hz, 1H), 3.68 (q, J = 7.9 Hz, 1H), 3.43 (td, J = 8.9, 3.7 Hz, 1H), 3.17 (dd, J = 8.6, 2.6 Hz, 1H), 2.53 (d, J = 8.4 Hz, 1H), 2.09 (d, J = 12.7 Hz, 1H), 1.80 (qt, J = 7.1, 3.4 Hz, 2H), 1.71 (d, J = 11.4 Hz, 2H), 1.65 – 1.49 (m, 2H), 1.35 – 1.08 (m, 4H), 0.93 (dq, J = 24.7, 13.0 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 149.55, 139.61, 134.51, 129.14, 127.12, 124.84, 124.41, 123.78, 121.03, 120.25, 117.76, 114.11, 74.68, 66.06, 55.60, 40.50, 39.12, 29.97, 28.68, 26.37, 26.17, 25.91, 23.63. HRMS (ESI): calcd for C₂₅H₃₃N₃O₃S, 566.9953 [M − H]⁻; found 566.9974 (mass error = 3.7 ppm).

5.1.9.5. N-(3,4-bis(trifluoromethyl)phenyl)-4-cyclohexyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12e).

89.8% yield; ¹H NMR (400 MHz, DMSO) δ 10.95 (s, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.62 (s, 1H), 7.55 – 7.47 (m, 2H), 7.37 (dd, J = 8.7, 2.4 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.26 (s, 1H), 4.93 (d, J = 7.4 Hz, 1H), 3.65 (q, J = 8.1 Hz, 1H), 3.38 (td, J = 8.8, 3.6 Hz, 1H), 3.15 (d, J = 5.9 Hz, 1H), 2.08 (d, J = 12.6 Hz, 1H), 1.78 – 1.68 (m, 4H), 1.57 (dd, J = 17.5, 9.6 Hz, 2H), 1.31 – 1.08 (m, 5H), 0.91 (h, J = 12.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 149.81, 143.33, 130.47, 130.41, 130.35, 130.28, 129.25, 128.20, 127.87, 127.55, 127.42, 127.23, 126.99, 124.72, 124.43, 124.27, 123.40, 122.01, 121.56, 121.09, 120.98, 120.72, 120.25, 119.92, 119.59, 119.31, 118.84, 118.61, 117.01, 116.95, 116.88, 116.82, 114.24, 74.62, 65.97, 55.58, 29.94, 28.64, 26.32, 26.11, 25.86, 23.57. HRMS (ESI): calcd for C₂₅H₂₆F₆N₂O₃S, 549.1647 [M + H]⁺; found 549.1650 (mass error = 0.5 ppm).

5.1.9.6. 4-cyclohexyl-N-(3-methyl-4-(trifluoromethyl)phenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12f).

81.5% yield; ¹H NMR (400 MHz, DMSO) δ 10.41 (s, 1H), 7.55 – 7.45 (m, 2H), 7.34 (dd, J = 8.7, 2.3 Hz, 1H), 7.03 (dd, J = 6.1, 2.3 Hz, 2H), 6.74 (d, J = 8.7 Hz, 1H), 6.18 (s, 1H), 4.94 (d, J = 7.4 Hz, 1H), 3.67 (q, J = 7.9 Hz, 1H), 3.41 (td, J = 8.8, 3.6 Hz, 1H), 3.16 (dd, J = 8.5, 2.7 Hz, 1H), 2.30 (t, J = 1.9 Hz, 3H), 2.09 (d, J = 12.6 Hz, 1H), 1.85 – 1.67 (m, 4H), 1.65 – 1.57 (m, 1H), 1.57 – 1.49 (m, 1H), 1.34 – 1.24 (m, 1H), 1.23 – 1.07 (m, 3H), 0.94 (dt, J = 24.5, 12.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 149.54, 142.44, 137.66, 129.25, 129.15, 127.41, 127.35, 127.30, 127.17, 126.43, 125.28, 123.73, 122.38, 122.08, 121.78, 121.49, 121.21, 121.02, 115.49, 114.14, 74.71, 66.03, 55.65, 40.48, 39.16, 29.97, 28.67, 26.36, 26.15, 25.90, 23.64, 19.42. HRMS (ESI): calcd for C₂₅H₂₉F₃N₂O₃S, 495.1929 [M + H]⁺; found 495.1927 (mass error = −0.4 ppm).

5.1.9.7. 4-cyclohexyl-N-(3-(dimethylamino)phenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12g).

74.4% yield; ¹H NMR (400 MHz, DMSO) δ 9.68 (s, 1H), 7.50 (d, J = 2.2 Hz, 1H), 7.26 (dd, J = 8.7, 2.3 Hz, 1H), 6.94 (t, J = 8.1 Hz, 1H), 6.70 (d, J = 8.7 Hz, 1H), 6.41 (t, J = 2.2 Hz, 1H), 6.33 (ddd, J = 15.6, 8.0, 2.2 Hz, 2H), 6.07 (s, 1H), 4.93 (d, J = 7.5 Hz, 1H), 3.67 (q, J = 7.9 Hz, 1H), 3.46 (dd, J = 9.0, 3.5 Hz, 1H), 3.15 (dd, J = 8.6, 2.6 Hz, 1H), 2.78 (s, 6H), 2.09 (d, J = 12.7 Hz, 1H), 1.78 (d, J = 12.0 Hz, 2H), 1.73 (s, 1H), 1.70 (s, 1H), 1.58 (dt, J = 20.0, 10.1 Hz, 2H), 1.21 (qd, J = 20.6, 10.3 Hz, 4H), 0.93 (dq, J = 22.2, 12.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 151.21, 149.14, 139.56, 129.63, 129.31, 127.18, 126.20, 120.85, 113.97, 107.98, 107.72, 103.52, 74.80, 66.09, 55.74, 29.98, 28.68, 26.38, 26.17, 25.92, 23.66. HRMS (ESI): calcd for C₂₅H₃₃N₃O₃S, 456.2321 [M − H]⁻; found 456.2323 (mass error = 0.4 ppm).

5.1.9.8. 4-cyclohexyl-N-(4-methylcyclohexyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12h).

79.6% yield; ¹H NMR (400 MHz, DMSO) δ 7.47 (s, 1H), 7.29 (dd, J = 8.7, 2.3 Hz, 1H), 7.14 (d, J = 7.3 Hz, 1H), 6.75 (d, J = 8.6 Hz, 1H), 6.02 (s, 1H), 5.00 (d, J = 7.6 Hz, 1H), 3.71 (q, J = 7.9 Hz, 1H), 3.51 (td, J = 8.6, 3.6 Hz, 1H), 3.19 (dd, J = 8.7, 2.5 Hz, 1H), 2.70 (dt, J = 7.6, 3.9 Hz, 1H), 2.56 (d, J = 8.9 Hz, 1H), 2.13 (d, J = 12.6 Hz, 1H), 1.82 (d, J = 12.6 Hz, 2H), 1.74 (d, J = 11.9 Hz, 2H), 1.64 (q, J = 11.7 Hz, 3H), 1.53 (d, J = 13.8 Hz, 4H), 1.33 (d, J = 9.8 Hz, 1H), 1.19 (p, J = 9.8 Hz, 4H), 1.01 (dq, J = 34.7, 11.9 Hz, 4H), 0.81 (d,J = 12.2 Hz, 1H), 0.77 (d, J = 6.5 Hz, 3H). ¹³C NMR (101 MHz, DMSO) δ 148.72, 128.82, 128.55, 126.71, 120.99, 114.08, 74.98, 66.03, 55.88, 52.58, 39.36, 34.03, 33.97, 33.59, 33.54, 31.62, 30.02, 28.73, 26.41, 26.21, 25.97, 23.72, 22.50. HRMS (ESI): calcd for C₂₄H₃₆N₂O₃S, 433.2525 [M + H]⁺; found 433.2518 (mass error =−1.6 ppm).

5.1.9.9. 4-cyclohexyl-N-(5,6,7,8-tetrahydronaphthalen-2-yl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12i).

49.4% yield; ¹H NMR (400 MHz, DMSO) δ 9.66 (s, 1H), 7.45 (d, J = 2.2 Hz, 1H), 7.23 (dd, J = 8.7, 2.3 Hz, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.76 (dd, J = 8.2, 2.3 Hz, 1H), 6.73 – 6.66 (m, 2H), 6.07 (s, 1H), 4.93 (d, J = 7.5 Hz, 1H), 3.68 (q, J = 8.0 Hz, 1H), 3.44 (td, J = 8.8, 3.6 Hz, 1H), 3.19 – 3.12 (m, 1H), 2.56 (t, J = 5.1 Hz, 4H), 2.10 (d, J = 12.7 Hz, 1H), 1.83 – 1.76 (m, 2H), 1.72 (d, J = 11.8 Hz, 2H), 1.63 (p, J = 3.2 Hz, 5H), 1.59– 1.50 (m, 1H), 1.25 (dd, J = 36.0, 10.7 Hz, 3H), 1.16 (d, J = 9.3 Hz, 2H), 0.94 (dq, J = 23.8, 11.6 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 149.12, 137.41, 136.08, 132.21, 129.75, 129.16, 127.08, 126.13, 120.88, 120.49, 117.87, 113.97, 74.79, 66.03, 55.73, 39.25, 29.99, 29.29, 28.69, 28.52, 26.38, 26.18, 25.92, 23.66, 23.16, 23.02. HRMS (ESI): calcd for C₂₇H₃₄N₂O₃S, 465.2212 [M − H]⁻; found 465.2227 (mass error = 3.2 ppm).

5.1.9.10. N-(3-acetylphenyl)-4-cyclohexyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12j).

75.8% yield; ¹H NMR (400 MHz, DMSO) δ 10.10 (s, 1H), 7.57 – 7.51 (m, 3H), 7.43 (d, J = 2.3 Hz, 1H), 7.34 – 7.18 (m, 4H), 6.65 (d, J = 8.7 Hz, 1H), 6.08 (s, 1H), 4.87 (d, J = 7.4 Hz, 1H), 3.70 – 3.53 (m, 2H), 3.35 (td, J = 8.7, 3.5 Hz, 2H), 3.09 (dd, J = 8.6, 2.6 Hz, 1H), 2.44 (d, J = 2.2 Hz, 6H), 2.06 – 1.99 (m, 1H), 1.78 – 1.62 (m, 5H), 1.60 – 1.29 (m, 5H), 1.26 – 1.04 (m, 6H), 0.96 – 0.76 (m, 2H). ¹³C NMR (101 MHz, DMSO) δ 197.81, 149.36, 139.38, 137.90, 129.96, 129.92, 129.19, 127.73, 127.13, 125.45, 124.19, 123.90, 120.97, 118.60, 114.04, 74.71, 66.04, 55.65, 40.49, 39.17, 36.93, 29.97, 28.67, 27.12, 26.37, 26.16, 25.91, 23.62. HRMS (ESI): calcd for C₂₅H₃₀N₂O₄S, 455.2005 [M + H]⁺; found, 455.2003 (mass error = −0.4 ppm).

5.1.9.11. N-(3,4-dimethylphenyl)-4-(tetrahydro-2H-pyran-4-yl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12k).

86.4% yield; ¹H NMR (400 MHz, DMSO) δ 9.67 (s, 1H), 7.48 (d, J = 2.3 Hz, 1H), 7.25 (dd, J = 8.7, 2.3 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.78 (dd, J = 8.1, 2.4 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 6.07 (s, 1H), 4.93 (d, J = 7.5 Hz, 1H), 3.93 – 3.82 (m, 2H), 3.68 (q, J = 7.9 Hz, 1H), 3.47 (dd, J = 9.1, 3.6 Hz, 1H), 3.28 (t, J = 10.8 Hz, 2H), 3.16 (dd, J = 8.7, 2.7 Hz, 1H), 2.07 (d, J = 5.5 Hz, 6H), 2.01 (d, J = 12.9 Hz, 1H), 1.80 (qd, J = 8.0, 3.9 Hz, 1H), 1.65 – 1.58 (m, 2H), 1.57 – 1.47 (m, 1H), 1.23 (dtd, J = 26.5, 12.4, 4.3 Hz, 2H). ¹³C NMR (101 MHz, DMSO) δ 148.93, 137.06, 136.38, 131.82, 130.22, 129.16, 127.13, 126.39, 121.68, 121.02, 117.90, 114.02, 74.64, 67.30, 67.18, 66.01, 55.58, 38.96, 38.01, 31.13, 30.25, 30.03, 28.84, 23.53, 19.98, 19.07. HRMS (ESI): calcd for C₂₄H₃₀N₂O₄S, 443.2005 [M + H]⁺; found, 443.2007 (mass error = 0.5 ppm).

5.1.9.12. 4-(1-acetylpiperidin-4-yl)-N-(3,4-dimethylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (12l).

86% yield; ¹H NMR (400 MHz, DMSO) δ 9.67 (s, 1H), 7.47 (d, J = 2.2 Hz, 1H), 7.24 (dd, J = 8.6, 2.3 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.82 (d, J = 2.4 Hz, 1H), 6.77 (dd, J = 8.1, 2.3 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 6.12 (d, J = 7.0 Hz, 1H), 4.93 (d, J = 7.4 Hz, 1H), 4.42 (d, J = 12.6 Hz, 1H), 3.83 (t, J = 11.3 Hz, 1H), 3.69 (q, J = 8.0 Hz, 2H), 3.46 (td, J = 8.7, 3.5 Hz, 2H), 3.18 (d, J = 8.4 Hz, 1H), 2.98 (t, J = 12.1 Hz, 1H), 2.50 – 2.42 (m, 2H), 2.07 (d, J = 4.7 Hz, 6H), 1.98 (d, J = 2.6 Hz, 3H), 1.87 – 1.79 (m, 1H), 1.58 (dp, J = 23.6, 10.4 Hz, 3H), 1.24 – 0.91 (m, 2H). ¹³C NMR (101 MHz, DMSO) δ 168.30, 148.94, 137.06, 136.37, 131.81, 130.23, 129.14, 127.14, 126.38, 121.66, 120.97, 117.89, 114.03, 113.97, 74.70, 66.05, 66.02, 55.17, 46.11, 45.99, 41.18, 41.05, 39.00, 29.77, 29.05, 28.63, 27.94, 23.61, 21.78, 21.75, 19.99, 19.07. HRMS (ESI): calcd for C₂₆H₃₃N₃O₄S, 482.2114 [M − H]⁻; found, 482.2098 (mass error = −3.3 ppm).

5.1.9.13. tert-butyl 4-(8-(N-(3,4-dimethylphenyl)sulfamoyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-4-yl)piperidine-1-carboxylate (12m).

63.7% yield; ¹H NMR (400 MHz, DMSO) δ 9.66 (s, 1H), 7.45 (d, J = 2.2 Hz, 1H), 7.23 (dd, J = 8.7, 2.3 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 6.76 (dd, J = 8.1, 2.3 Hz, 1H), 6.67 (d, J = 8.7 Hz, 1H), 6.10 (s, 1H), 5.74 (s, 1H), 4.92 (d, J = 7.4 Hz, 1H), 3.98 (s, 1H), 3.68 (q, J = 7.9 Hz, 1H), 3.45 (t, J = 4.5 Hz, 2H), 3.18 (dd, J = 8.6, 2.7 Hz, 1H), 2.68 (s, 2H), 2.07 (d, J = 3.7 Hz, 6H), 1.81 (dt, J = 8.0, 4.0 Hz, 1H), 1.71 (d, J = 12.8 Hz, 1H), 1.58 (p, J = 9.2 Hz, 2H), 1.47 (d, J = 9.0 Hz, 1H), 1.38 (s, 9H), 1.14 – 0.95 (m, 2H). ¹³C NMR (101 MHz, DMSO) δ 154.24, 148.96, 137.06, 136.36, 131.83, 130.23, 129.13, 127.13, 126.36, 121.68, 120.97, 117.90, 114.01, 79.00, 74.69, 66.02, 55.17, 39.19, 38.91, 28.54, 23.60, 20.00, 19.08. HRMS (ESI): calcd for C₂₉H₃₉N₃O₅S, 564.2508 [M + Na]⁺; found, 564.2508 (mass error = 0.0 ppm).

5.1.10. General procedure for preparation of compounds 13a and 13b.

Benzene-1,4-diamine (46.24 mmol) was dissolved in dry DMF (100 mL) at room temperature. Substituted benzenesulfonyl chloride (46.24 mmol) was added in portions with vigorous stirring at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction mixture was hydrolyzed by the addition of 100 mL of saturated NaHCO₃ solution, extracted with EtOAc, washed with brine, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel, CH₂Cl₂/MeOH = 9/1 v/v) to give compounds 13a and 13b.

5.1.10.1. N-(4-aminophenyl)-3,4-dimethylbenzenesulfonamide (13a).

94.8% yield; ¹H NMR (400 MHz, DMSO) δ 9.38 (s, 1H), 7.42 (s, 1H), 7.33 (dd, J = 7.9, 2.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 6.71 – 6.63 (m, 2H), 6.41 – 6.33 (m, 2H), 4.95 (s, 2H), 2.23 (d, J = 4.6 Hz, 6H).

5.1.10.2. 3-acetyl-N-(4-aminophenyl)benzenesulfonamide (13b).

92% yield; ¹H NMR (400 MHz, DMSO) δ 9.63 (s, 1H), 8.16 (dt, J = 6.5, 1.5 Hz, 2H), 7.87 – 7.80 (m, 1H), 7.68 (t,J = 8.1 Hz, 1H), 6.71 – 6.63 (m, 2H), 6.42 – 6.34 (m, 2H), 5.01 (s, 2H), 2.59 (s, 3H).

5.1.11. General procedure for preparation of compounds 14a and 14b.

These compounds were derived from compounds 13a and 13b and were synthesized individually in parallel. The method used to synthesize compounds 14a and 14b was used to synthesize compounds 3a-3i.

5.1.11.1. N-(4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-8-yl)-3,4-dimethylbenzenesulfonamide (14a).

60.4% yield; ¹H NMR (400 MHz, DMSO) δ 9.58 (d, J = 17.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.47 (d, J = 10.0 Hz, 1H), 7.40 (ddd, J = 6.7, 4.4, 2.0 Hz, 1H), 7.26 (d, J = 8.1 Hz, 1H), 6.93 (d, J = 2.5 Hz, 1H), 6.76 (ddd, J = 13.5, 8.5, 2.5 Hz, 1H), 6.56 (dd, J = 8.7, 5.5 Hz, 1H), 6.21 (s, 1H), 4.29 (d, J = 5.1 Hz, 1H), 3.84 (q, J = 8.1 Hz, 1H), 3.69 – 3.57 (m, 2H), 3.53 (q, J = 8.0 Hz, 1H), 3.44 (dd, J = 8.4, 3.6 Hz, 1H), 2.23 (s, 6H), 1.91 – 1.78 (m, 1H), 1.49 (p, J = 6.1 Hz, 1H), 1.30 – 1.20 (m, 1H), 1.10 (d, J = 6.8 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 203.98, 203.95, 148.19, 147.82, 143.93, 143.38, 142.03, 142.00, 137.73, 137.64, 137.60, 137.53, 135.54, 135.06, 130.30, 130.25, 129.20, 128.75, 127.97, 127.87, 127.40, 127.07, 125.60, 124.79, 124.76, 124.09, 123.82, 123.31, 122.29, 119.91, 115.74, 115.29, 75.50, 75.22, 65.96, 64.67, 56.79, 56.03, 44.68, 42.78, 35.03, 34.99, 28.61, 24.42, 19.84, 19.50, 19.46. HRMS (ESI): calcd for C₂₉H₃₂N₂O₄S, 505.2161 [M + H]⁺; found 505.2173 (mass error = 2.4 ppm).

5.1.11.2. 3-acetyl-N-(4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-8-yl)benzenesulfonamide (14b).

49.5% yield; ¹H NMR (400 MHz, DMSO) δ 9.80 (s, 1H), 8.18 (tt, J = 7.2, 2.3 Hz, 2H), 7.96 (d, J = 7.7 Hz, 2H), 7.88 (ddt, J = 7.8, 4.7, 1.5 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.61 – 7.54 (m, 2H), 6.89 (d, J = 2.4 Hz, 1H), 6.76 (ddd, J = 16.8, 8.4, 2.5 Hz, 2H), 6.57 (dd, J = 8.7, 4.0 Hz, 1H), 6.28 (s, 1H), 6.00 (s, 1H), 4.98 (d, J = 7.7 Hz, 1H), 4.63 (d, J = 3.0 Hz, 1H), 4.28 (d, J = 5.1 Hz, 1H), 3.86 – 3.78 (m, 1H), 3.69 – 3.57 (m, 2H), 3.50 (q, J = 7.9 Hz, 1H), 2.63 (t, J = 9.7 Hz, 1H), 2.59 (d, J = 1.7 Hz, 3H), 2.23 (dddd, J = 10.6, 7.7, 5.2, 2.2 Hz, 1H), 1.92 – 1.73 (m, 1H), 1.57 – 1.43 (m, 1H), 1.24 (dtd, J = 11.9, 7.6, 3.5 Hz, 1H), 1.09 (dd, J = 6.8, 1.7 Hz, 6H). ¹³C NMR (101 MHz, DMSO) δ 203.98, 203.95, 197.31, 197.28, 148.10, 147.75, 144.37, 143.81, 140.69, 140.62, 137.54, 137.46, 135.55, 135.07, 132.72, 132.69, 131.34, 131.29, 130.26, 130.22, 129.20, 128.76, 128.73, 127.40, 127.19, 126.49, 126.33, 126.27, 124.78, 124.59, 124.06, 122.33, 119.94, 115.80, 115.37, 75.39, 75.12, 65.97, 64.65, 56.68, 55.94, 44.58, 42.71, 35.03, 34.99, 28.59, 27.23, 24.40, 19.50, 19.46. HRMS (ESI): calcd for C₂₉H₃₀N₂O₅S, 519.1954 [M + H]⁺; found 519.1966 (mass error = 2.3 ppm).

5.2. Cell culture and reagents

Human melanoma A375, human breast cancer MDA-MB-231, and human prostate cancer 22Rv1 cell lines were purchased from ATCC. A375 and MDA-MB231 cell lines were cultured in DMEM (Mediatech, Inc) supplemented with 10 % FBS (Atlanta Biologicals) and 1% antibiotic-antimycotic solution (Sigma-Aldrich) at 37 °C in a humidified incubator containing 5% carbon dioxide (CO2). 22Rv1 cell line was cultured in RPMI 1640 medium (Gibco, Carlsbad, CA) containing a mixture of 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville, GA) and 1% antibiotic/antimycotic solution (Sigma-Aldrich, St. Louis, MO). EU-1 was established from a pediatric ALL patient and was well characterized for its expression of MDM2 and p53 status, as reported previously [44]. EU-1 was cultured in a standard culture medium (RPMI 1640 containing 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 50 U penicillin, and 50 μg/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were maintained to 80–90% confluency at 37 °C with 5% CO₂ in a humidified atmosphere.

5.3. Cytotoxicity assay

5.3.1. Leukemia EU-1 cell line

The cytotoxic effect of the new analogs of JW-2-107 on EU-1 cells was determined using the water-soluble tetrazolium salt (WST) assay. Briefly, cells cultured in 96-well microtiter plates were given different concentrations of test compounds for a 44-h period. Following this, WST (25 μg/well) was added, and incubation continued for an additional 4 h before the optical density (OD) of the wells was read with a microplate reader (set at a test wavelength of 450 nm and a reference wavelength of 620 nm). Appropriate controls lacking cells were included to determine background absorbance.

5.3.2. Melanoma A375, breast cancer MDA-MB-231 and prostate cancer 22Rv1 cancer cell lines

The human melanoma A375, human breast cancer MDA-MB-231, and human prostate cancer 22Rv1 cell lines were plated into 96-well plates at a density of 3,000 to 8,000 cells per well, depending on the proliferation rate of each cell line. After overnight incubation, cells were treated with the test compounds (concentration range: 3 nM-100 μM) for 72 h. Each compound treatment was carried out in four replicates. At the endpoint, the MTS reagent (Promega) was added to the cells, they were incubated in the dark at 37 °C for 1.5 h, and cell viability was determined by the absorbance at a wavelength of 490 nm using a plate reader (BioTek Instruments Inc., Winooski, VT). IC₅₀ values were normalized against untreated cells and calculated using GraphPad Prism 7 software using nonlinear regression.

5.4. Colony formation.

A375 cells (250 cells per well) at the logarithmic phase were seeded in 12-well plates and incubated for 48 h. The cells were treated with 1, 5, and 10 μM of 3e or JW-2-107 for 10 days. 22Rv1 cells (2,000 cells per well) at the logarithmic phase were seeded in 12-well plates and incubated for 72 h. The cells were treated with 1, 5, and 10 μM of 3e or JW-2-107 for 14 days. The culture medium or medium containing the indicated compounds 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 in triplicates.

5.5. Western blotting

5.5.1. EU-1 cell line

Cells were lysed for 30 min at 40 °C in a lysis buffer composed of 150 mM NaCl, 50 mM Tris (pH 8.0), 5mM EDTA, 1% (v/v) Nonidet p-40, 1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin and 25 μg/ml leupeptin. Equal amounts of protein extracts were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Following transfer to a nitrocellulose filter, it was blocked for 1 h at room temperature with buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% non-fat milk; incubated with specific antibodies for 3 h at room temperature; washed,and incubated with an HRP-labeled secondary antibody for 1 h. Finally, the blots were developed using a chemiluminescent detection system (ECL, Amersham Life Science, Buckinghamshire, England). 3e and JW-2-107 were prepared in DMSO (ATCC) at a stock concentration of 20 mM and stored at −20 °C in a freezer. Prior to experiments, the stock was diluted with the proper culture medium, and the final concentration of DMSO in drug solution was maintained below 1%.

5.5.2. A375 and 22Rv1 cell lines

A375 and 22Rv1 cells were seeded into 6 well plates at a density of either 2.5×10⁵ or 3×10⁵ cells/plate and allowed to reach the confluency of 80%. The medium was replaced with either fresh complete DMEM medium or DMEM containing the desired concentration (1, 5, and 10 μM) of 3e or compound AMG-232 (100 nM-1 μM). After 24 h of incubation, cells were washed with PBS, lysed with RIPA buffer (Thermo Fisher Scientific) containing Halt protease and phosphatase inhibitor (Thermo Fisher Scientific), and centrifuged at 13,000 rpm at 4 °C for 15 min. The BCA method was used to quantify the protein in each sample. 30 μg of each sample were subjected to Western blot analysis as described. Briefly, the protein samples were separated via SDS-PAGE, transferred to PVDF membranes, and blocked with 5 % nonfat milk for 1h at RT, followed by overnight incubation at 4 °C with primary antibodies, which included MDM2 (Millipore Sigma, #OP115), XIAP (Cell Signaling, #2045), p53 (Santa Cruz, #FL393), p21 (Cell Signaling, #2947S), PUMA (Cell Signaling, #98672S), c-PARP (Cell Signaling, #5625S) and GAPDH (Cell Signaling, #3683). Primary antibodies were detected with HRP-conjugated secondary antibodies (Cell Signaling Technology, #7074, #7076). and immunoreactive bands were visualized on film using an enhanced chemiluminescent substrate (ECL, Thermo Fisher Scientific).

5.6. In vivo efficacy of compound 3e on 22Rv1 subcutaneous prostate xenograft model in mice

The animal study was performed under NIH Principles of Laboratory Animal Care guidelines and protocols approved by the UTHSC Institutional Animal Care and Use Committee (IACUC) (protocol #20-0166). Animals were housed under a 12:12 h light/dark cycle at 20-26 °C with 30-70 % humidity. 8-9-week-old male humanized NSG mice (Jackson Laboratory) were used to implant 22Rv1 prostate tumors. Two million 22Rv1 logarithmically growing cells in 50 μL of phenol red-free, FBS-free RPMI 1640 medium were mixed with 50 μL Matrigel and subcutaneously injected into the dorsal right flank of each NSG mouse. 22Rv1 tumors were allowed to grow for 14 days until the average tumor volume reached around 100 mm³. Tumor volume was measured using a caliper and calculated as a × b² ×0.5, where a and b represented the larger and smaller dimensions of the tumors, respectively. The mice were randomly assigned into 2 groups (n=5/group): vehicle (5% DMSO, 65% PEG400, 30% saline), 60 mg/kg of 3e with the same formulation for the 22Rv1 xenograft model. Vehicle or 3e treatments were administrated intraperitoneally (IP) 7 times/week for 10 consecutive days. During the treatment, tumor volume and body weight were recorded every 3 days. All mice were sacrificed at the conclusion of the treatment, and tumors were collected, weighed, and photographed.

5.7. Immunofluorescence assay

Subcutaneous tumors were harvested and sectioned (10 μm) using an HM525 NX Cryostat (Thermo Scientific). The tumor tissue sections were fixed with 4% paraformaldehyde (PFA) at room temperature for 15 min and permeabilized with 0.25% Triton X-100 at room temperature for 10 mins. After blocking in 10% goat serum in PBS at room temperature for 1 h, the sections were incubated in primary antibodies at 4°C overnight: rabbit anti-MDM2 (1:200, Cell Signaling, #86934); rabbit anti-Ki-67 (1:200, Biocare Medical, #325); rat anti-cluster of differentiation 31 (CD31) (1:200, eBioscience, #14-0311-82); rabbit anti-cleaved caspase-3 (1:200, Cell Signaling Technology, #9664T); rabbit anti-cleaved-PARP (1:200, Cell Signaling Technology, #5625S). The sections were washed with PBS and incubated with secondary antibodies: goat anti-rabbit IgG-Alexa Fluor 488 (1:1,000, Invitrogen, #A-11034), goat anti-rabbit IgG-Alexa Fluor 546 (1:1,000, Invitrogen, #A11035), at room temperature for 2 h and DAPI (1 μg/mL in PBS) for 10 min. Stained sections were mounted on slides using DPX Mountant (Electron Microscopy Sciences). Images were obtained using a fluorescence microscope BZ-X800 (Keyence). The fluorescence intensity was quantified using ImageJ software (NIH).

5.8. Statistical analysis

All data were analyzed using GraphPad Prism 9.0. In vitro experiments were repeated using at least three technical replicates per group, and each assay was performed over three biological replicates. One-way or two-way ANOVA tests were first employed for experiments comparing more than two groups/conditions, followed a Dunnett multiple comparison test. Significance levels are defined as, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Supplementary Material

NIHMS1898856-supplement-1.pdf^{(10.9MB, pdf)}

Highlights.

The tetrahydroquinoline scaffold, represented by 3e, dually targets MDM2 and XIAP.
Treatments with 3e decreased both MDM2 and XIAP protein levels and increased expressions of p53 in EU-1 leukemia cells and 22Rv1 prostate cancer cells.
3e inhibited prostate tumor growth in a human 22Rv1 prostate xenograft model.

Acknowledgments

This work was supported by NIH grants R01CA193609, 1S10OD010678-01, and 1S10RR026377-01 to W.L., and R01CA240447 to W.L. and M.Z. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. We thank Dr. Dejian Ma for his assistance in the separation of the four stereoisomers for compound 3e.

Abbreviations

ALL: acute leukemia lymphoma
BOC: tert-butyloxycarbonyl
c-PARP: cleaved parp
c-Cas3: cleaved caspase-3
HRMS: high-resolution mass spectrometry
IAP: inhibitor of apoptosis protein
IRES: internal ribosome entry site
MDM2: murine double minute 2
NB: neuroblastoma
NSG: NOD scid gamma
OD: optical density
PFA: paraformaldehyde
ppm: parts per million
SAR: structure-activity relationship
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SEM: standard error of the mean
TFA: trifluoroacetic acid
TLC: thin layer chromatography
TMS: tetramethylsilane
WST: water-soluble tetrazolium salt
XIAP: X-linked inhibitor of apoptosis protein

Footnotes

Declaration of competing interest

The authors declare the following competing financial interest(s): ZW, LG, MZ, and WL are listed as inventors for the compounds reported in this paper. WL is the founder of SEAK Therapeutics LLC, who holds an exclusive optional licensing agreement with interests to license the reported compounds in this report in the future. However, SEAK Therapeutics LLC does not provide any financial support or has any influence on the work reported in this study.

Declaration of Interest Statement:

The authors declare the following competing financial interest(s): LG, MZ, ZW and WL are listed as inventors for the new compounds reported in this paper. WL is the founder and owner of SEAK Therapeutics LLC, who has signed an exclusive optional licensing agreement with interests to license the reported compounds in this report in the future. However, SEAK Therapeutics LLC does not provide any financial support or has any influence on the work reported in this study.

Appendix A. Supplementary Material

Supplementary data related to this article can be found online at

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References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS1898856-supplement-1.pdf^{(10.9MB, pdf)}

[R1] [1].Momand J, Jung D, Wilczynski S, Niland J, The MDM2 gene amplification database, Nucleic Acids Res, 26 (1998) 3453–3459. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R2] [2].Zhang H. Wang, MDM2 oncogene as a novel target for human cancer therapy, Curr Pharm Des, 6 (2000) 393–416. [DOI] [PubMed] [Google Scholar]

[R3] [3].Mendoza M, Mandani G, Momand J, The MDM2 gene family, Biomol Concepts, 5 (2014) 9–19. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] [4].Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B, Amplification of a gene encoding a p53-associated protein in human sarcomas, Nature, 358 (1992) 80–83. [DOI] [PubMed] [Google Scholar]

[R5] [5].Corvi R, Savelyeva L, Breit S, Wenzel A, Handgretinger R, Barak J, Oren M, Amler L, Schwab M, Non-syntenic amplification of MDM2 and MYCN in human neuroblastoma, Oncogene, 10 (1995) 1081–1086. [PubMed] [Google Scholar]

[R6] [6].Oliner JD, Saiki AY, Caenepeel S, The Role of MDM2 Amplification and Overexpression in Tumorigenesis, Cold Spring Harb Perspect Med, 6 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R7] [7].Rayburn E, Zhang R, He J, Wang H, MDM2 and human malignancies: expression, clinical pathology, prognostic markers, and implications for chemotherapy, Curr Cancer Drug Targets, 5 (2005) 27–41. [DOI] [PubMed] [Google Scholar]

[R8] [8].Bond GL, Hu W, Levine AJ, MDM2 is a central node in the p53 pathway: 12 years and counting, Curr Cancer Drug Targets, 5 (2005) 3–8. [DOI] [PubMed] [Google Scholar]

[R9] [9].Capoulade C, Bressac-de Paillerets B, Lefrere I, Ronsin M, Feunteun J, Tursz T, Wiels J, Overexpression of MDM2, due to enhanced translation, results in inactivation of wild-type p53 in Burkitt's lymphoma cells, Oncogene, 16 (1998) 1603–1610. [DOI] [PubMed] [Google Scholar]

[R10] [10].Momand J, Wu HH, Dasgupta G, MDM2--master regulator of the p53 tumor suppressor protein, Gene, 242 (2000) 15–29. [DOI] [PubMed] [Google Scholar]

[R11] [11].Momand J, Zambetti GP, Olson DC, George D, Levine AJ, The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation, Cell, 69 (1992) 1237–1245. [DOI] [PubMed] [Google Scholar]

[R12] [12].Wu X, Bayle JH, Olson D, Levine AJ, The p53-mdm-2 autoregulatory feedback loop, Genes Dev, 7 (1993) 1126–1132. [DOI] [PubMed] [Google Scholar]

[R13] [13].Juven-Gershon T, Oren M, Mdm2: the ups and downs, Mol Med, 5 (1999) 71–83. [PMC free article] [PubMed] [Google Scholar]

[R14] [14].Freedman DA, Wu L, Levine AJ, Functions of the MDM2 oncoprotein, Cell Mol Life Sci, 55 (1999) 96–107. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] [15].Haupt Y, Maya R, Kazaz A, Oren M, Mdm2 promotes the rapid degradation of p53, Nature, 387 (1997) 296–299. [DOI] [PubMed] [Google Scholar]

[R16] [16].Bose I, Ghosh B, The p53-MDM2 network: from oscillations to apoptosis, J Biosci, 32 (2007) 991–997. [DOI] [PubMed] [Google Scholar]

[R17] [17].Wang W, Qin JJ, Rajaei M, Li X, Yu X, Hunt C, Zhang R, Targeting MDM2 for novel molecular therapy: Beyond oncology, Med Res Rev, 40 (2020) 856–880. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] [18].Ding Q, Zhang Z, Liu JJ, Jiang N, Zhang J, Ross TM, Chu XJ, Bartkovitz D, Podlaski F, Janson C, Tovar C, Filipovic ZM, Higgins B, Glenn K, Packman K, Vassilev LT, Graves B, Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in clinical development, J Med Chem, 56 (2013) 5979–5983. [DOI] [PubMed] [Google Scholar]

[R19] [19].Holzer P, Masuya K, Furet P, Kallen J, Valat-Stachyra T, Ferretti S, Berghausen J, Bouisset-Leonard M, Buschmann N, Pissot-Soldermann C, Rynn C, Ruetz S, Stutz S, Chene P, Jeay S, Gessier F, Discovery of a Dihydroisoquinolinone Derivative (NVP-CGM097): A Highly Potent and Selective MDM2 Inhibitor Undergoing Phase 1 Clinical Trials in p53wt Tumors, J Med Chem, 58 (2015) 6348–6358. [DOI] [PubMed] [Google Scholar]

[R20] [20].Aguilar A, Lu J, Liu L, Du D, Bernard D, McEachern D, Przybranowski S, Li X, Luo R, Wen B, Sun D, Wang H, Wen J, Wang G, Zhai Y, Guo M, Yang D, Wang S, Discovery of 4-((3'R,4'S,5'R)-6"-Chloro-4'-(3-chloro-2-fluorophenyl)-1'-ethyl-2"-oxodispiro[cyclohexane-1,2'-pyrrolidine-3',3"-indoline]-5'-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-115): A Potent and Orally Active Murine Double Minute 2 (MDM2) Inhibitor in Clinical Development, J Med Chem, 60 (2017) 2819–2839. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] [21].Vu B, Wovkulich P, Pizzolato G, Lovey A, Ding Q, Jiang N, Liu JJ, Zhao C, Glenn K, Wen Y, Tovar C, Packman K, Vassilev L, Graves B, Discovery of RG7112: A Small-Molecule MDM2 Inhibitor in Clinical Development, ACS Med Chem Lett, 4 (2013) 466–469. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] [22].Wang S, Sun W, Zhao Y, McEachern D, Meaux I, Barriere C, Stuckey JA, Meagher JL, Bai L, Liu L, Hoffman-Luca CG, Lu J, Shangary S, Yu S, Bernard D, Aguilar A, Dos-Santos O, Besret L, Guerif S, Pannier P, Gorge-Bernat D, Debussche L, SAR405838: an optimized inhibitor of MDM2-p53 interaction that induces complete and durable tumor regression, Cancer Res, 74 (2014) 5855–5865. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R23] [23].Sun D, Li Z, Rew Y, Gribble M, Bartberger MD, Beck HP, Canon J, Chen A, Chen X, Chow D, Deignan J, Duquette J, Eksterowicz J, Fisher B, Fox BM, Fu J, Gonzalez AZ, Gonzalez-Lopez De Turiso F, Houze JB, Huang X, Jiang M, Jin L, Kayser F, Liu JJ, Lo MC, Long AM, Lucas B, McGee LR, McIntosh J, Mihalic J, Oliner JD, Osgood T, Peterson ML, Roveto P, Saiki AY, Shaffer P, Toteva M, Wang Y, Wang YC, Wortman S, Yakowec P, Yan X, Ye Q, Yu D, Yu M, Zhao X, Zhou J, Zhu J, Olson SH, Medina JC, Discovery of AMG 232, a potent, selective, and orally bioavailable MDM2-p53 inhibitor in clinical development, J Med Chem, 57 (2014) 1454–1472. [DOI] [PubMed] [Google Scholar]

[R24] [24].Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA, In vivo activation of the p53 pathway by small-molecule antagonists of MDM2, Science, 303 (2004) 844–848. [DOI] [PubMed] [Google Scholar]

[R25] [25].Zhao Y, Aguilar A, Bernard D, Wang S, Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 Inhibitors) in clinical trials for cancer treatment, J Med Chem, 58 (2015) 1038–1052. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] [26].Zhu H, Gao H, Ji Y, Zhou Q, Du Z, Tian L, Jiang Y, Yao K, Zhou Z, Targeting p53-MDM2 interaction by small-molecule inhibitors: learning from MDM2 inhibitors in clinical trials, J Hematol Oncol, 15 (2022) 91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R27] [27].Zhang Z, Zhang R, p53-independent activities of MDM2 and their relevance to cancer therapy, Curr Cancer Drug Targets, 5 (2005) 9–20. [DOI] [PubMed] [Google Scholar]

[R28] [28].Arena G, Cisse MY, Pyrdziak S, Chatre L, Riscal R, Fuentes M, Arnold JJ, Kastner M, Gayte L, Bertrand-Gaday C, Nay K, Angebault-Prouteau C, Murray K, Chabi B, Koechlin-Ramonatxo C, Orsetti B, Vincent C, Casas F, Marine JC, Etienne-Manneville S, Bernex F, Lombes A, Cameron CE, Dubouchaud H, Ricchetti M, Linares LK, Le Cam L, Mitochondrial MDM2 Regulates Respiratory Complex I Activity Independently of p53, Mol Cell, 69 (2018) 594–609. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R29] [29].Todoric J, Antonucci L, Di Caro G, Li N, Wu X, Lytle NK, Dhar D, Banerjee S, Fagman JB, Browne CD, Umemura A, Valasek MA, Kessler H, Tarin D, Goggins M, Reya T, Diaz-Meco M, Moscat J, Karin M, Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas, Cancer Cell, 32 (2017) 824–839. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] [30].Zhou S, Gu L, He J, Zhang H, Zhou M, MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia, Mol Cell Biol, 31 (2011) 4928–4937. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] [31].Gu L, Zhang H, He J, Li J, Huang M, Zhou M, MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells, Oncogene, 31 (2012) 1342–1353. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R32] [32].Jung CH, Kim J, Park JK, Hwang SG, Moon SK, Kim WJ, Um HD, Mdm2 increases cellular invasiveness by binding to and stabilizing the Slug mRNA, Cancer Lett, 335 (2013) 270–277. [DOI] [PubMed] [Google Scholar]

[R33] [33].Gu L, Zhu N, Zhang H, Durden DL, Feng Y, Zhou M, Regulation of XIAP translation and induction by MDM2 following irradiation, Cancer Cell, 15 (2009) 363–375. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] [34].Schimmer AD, Inhibitor of apoptosis proteins: translating basic knowledge into clinical practice, Cancer Res, 64 (2004) 7183–7190. [DOI] [PubMed] [Google Scholar]

[R35] [35].Deveraux QL, Reed JC, IAP family proteins--suppressors of apoptosis, Genes Dev, 13 (1999) 239–252. [DOI] [PubMed] [Google Scholar]

[R36] [36].Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S, Scudiero DA, Tudor G, Qui YH, Monks A, Andreeff M, Reed JC, Expression and prognostic significance of IAP-family genes in human cancers and myeloid leukemias, Clin Cancer Res, 6 (2000) 1796–1803. [PubMed] [Google Scholar]

[R37] [37].Tamm I, Richter S, Oltersdorf D, Creutzig U, Harbott J, Scholz F, Karawajew L, Ludwig WD, Wuchter C, High expression levels of x-linked inhibitor of apoptosis protein and survivin correlate with poor overall survival in childhood de novo acute myeloid leukemia, Clin Cancer Res, 10 (2004) 3737–3744. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Synthesis and biological evaluation of dual MDM2/XIAP inhibitors based on the tetrahydroquinoline scaffold

Najah Albadari

Yang Xie

Tao Liu

Rui Wang

Lubing Gu

Muxiang Zhou

Zhongzhi Wu

Wei Li

Abstract

Graphical Abstract

1. Introduction

Figure 1.

2. Chemistry

Scheme 1. Synthesis of compounds 3a-3i.

Scheme 5. Synthesis of compounds 14a and 14b.

Scheme 2. Synthesis of compounds 4a-4d.

Scheme 3. Synthesis of compounds 7a, 7b, and 9.

Scheme 4. Synthesis of compounds 12a-12m.

3. Results and discussion

3.1. In vitro antiproliferative assay in a panel of human cancer cell lines.

Table 1.

3.2. Treatment with compound 3e significantly reduced colony formation by A375 melanoma cells and 22Rv1 prostate cancer cells.

Figure 2.

3.3. Compound 3e retains dual inhibition for MDM2 and XIAP

Figure 3.

3.4. In vivo assessment of antitumor efficacy

3.4.1. Antitumor activity of compound 3e in a 22Rv1 subcutaneous xenograft mouse model

Figure 4.

3.4.2. 3e decreases MDM2 expression, induces anti-angiogenesis and apoptosis in 22Rv1 xenograft model

4. Conclusion

5. Experimental section

5.1. Chemistry

5.1.1. General procedure for preparation of compounds 1a-1i.

5.1.1.1. N-(3-ethylphenyl)-4-nitrobenzenesulfonamide (1a).

5.1.1.2. N-(3-isopropylphenyl)-4-nitrobenzenesulfonamide (1b).

5.1.1.3. N-(3-(tert-butyl)phenyl)-4-nitrobenzenesulfonamide (1c).

5.1.1.4. N-(3-(dimethylamino)phenyl)-4-nitrobenzenesulfonamide (1d).

5.1.1.5. N-(3-acetylphenyl)-4-nitrobenzenesulfonamide (1e).

5.1.1.6. N-(4-acetylphenyl)-4-nitrobenzenesulfonamide (1f).

5.1.1.7. 4-nitro-N-(3-propionylphenyl)benzenesulfonamide (1g).

5.1.1.8. 4-nitro-N-(1-oxo-1,3-dihydroisobenzofuran-4-yl)benzenesulfonamide (1h).

5.1.1.9. N-(benzo[d][1,3]dioxol-5-yl)-4-nitrobenzenesulfonamide (1i).

5.1.2. General procedure for preparation of compounds 2a-2i.

5.1.2.1. 4-amino-N-(3-ethylphenyl)benzenesulfonamide (2a).

5.1.2.2. 4-amino-N-(3-isopropylphenyl)benzenesulfonamide (2b).

5.1.2.3. 4-amino-N-(3-(tert-butyl)phenyl)benzenesulfonamide (2c).

5.1.2.4. 4-amino-N-(3-(dimethylamino)phenyl)benzenesulfonamide (2d).

5.1.2.5. N-(3-acetylphenyl)-4-aminobenzenesulfonamide (2e).

5.1.2.6. N-(4-acetylphenyl)-4-aminobenzenesulfonamide (2f).

5.1.2.7. 4-amino-N-(3-propionylphenyl)benzenesulfonamide (2g).

5.1.2.8. 4-amino-N-(1-oxo-1,3-dihydroisobenzofuran-4-yl)benzenesulfonamide (2h).

5.1.2.9. 4-amino-N-(benzo[d][1,3]dioxol-5-yl)benzenesulfonamide (2i).

5.1.3. General procedure for preparation of compounds 3a-3i.

5.1.3.1. N-(3-ethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3a).

5.1.3.2. 4-(4-isobutyrylphenyl)-N-(3-isopropylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3b).

5.1.3.3. N-(3-(tert-butyl)phenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3c).

5.1.3.4. N-(3-(dimethylamino)phenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3d).

5.1.3.5. N-(3-acetylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3e).

5.1.3.6. N-(4-acetylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3f).

5.1.3.7. 4-(4-isobutyrylphenyl)-N-(3-propionylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3g).

5.1.3.8. 4-(4-isobutyrylphenyl)-N-(1-oxo-1,3-dihydroisobenzofuran-4-yl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3h).

5.1.3.9. N-(benzo[d][1,3]dioxol-5-yl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (3i).

5.1.4. General procedure for preparation of compounds 4a-4d.

5.1.4.1. N-(3-acetylphenyl)-4-phenyl-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4a).

5.1.4.2. N-(3-acetylphenyl)-4-(2,3-dihydrobenzofuran-5-yl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4b).

5.1.4.3. N,4-bis(3-acetylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4c).

5.1.4.4. N-(3-acetylphenyl)-4-(4-isopropylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (4d).

5.1.5. General procedure for preparation of compounds 7a,7b and 8.

5.1.5.1. 4-(2,3-dihydrobenzofuran-5-yl)-N-(3,4-dimethylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide (7a).

5.1.5.2. N-(4-(8-(N-(3,4-dimethylphenyl)sulfamoyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin- 4-yl)phenyl)acetamide (7b).

5.1.5.3. tert-butyl 3-(8-(N-(3,4-dimethylphenyl)sulfamoyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinolin-4-yl)-1H-indole-1-carboxylate (8).

5.1.6. Preparation of N-(3,4-dimethylphenyl)-4-(1H-indol-3-yl)-2,3,3a,4,5,9b hexahydrofuro[3,2-c]quinoline-8-sulfonamide (9).

5.1.7. General procedure for preparation of compounds 10b-10f, 10h, and 10i

5.1.7.1. N-(3,4-bis(trifluoromethyl)phenyl)-4-nitrobenzenesulfonamide (10e).

5.1.7.2. N-(4-methylcyclohexyl)-4-nitrobenzenesulfonamide (10h).

5.1.7.3. 4-nitro-N-(5,6,7,8-tetrahydronaphthalen-2-yl)benzenesulfonamide (10i).

5.1.8. General procedure for preparation of compounds 11b-11f, 11h, and 11i

5.1.8.1. 4-amino-N-(3,4-bis(trifluoromethyl)phenyl)benzenesulfonamide (11e).