Strategies for developing retinoic acid receptor alpha-selective antagonists as novel agents for male contraception

Abstract

Reported here are the synthesis and in vitro evaluation of a series of 26 retinoic acid analogs based on dihydronaphthalene and chromene scaffolds using a transactivation assay. Chromene amide analog 21 was the most potent and selective retinoic acid receptor α antagonist identified from this series. In vitro evaluation indicated that 21 has favorable physicochemical properties and a favorable pharmacokinetic PK profile in vivo with significant oral bioavailability, metabolic stability, and testes exposure. Compound 21 was evaluated for its effects on spermatogenesis and disruption of fertility in a mouse model. Oral administration of compound 21 at low doses showed reproducibly characteristic albeit modest effects on spermatogenesis, but no effects on fertility were observed in mating studies. The inhibition of spermatogenesis could not be enhanced by raising the dose and lengthening the duration of dosing. Thus, 21 may not be a good candidate to pursue further for effects on male fertility.

Graphical abstract

1. Introduction

To lower the high levels of unintended pregnancies observed worldwide [1], additional effective contraceptive methods for women and men are desirable [2]. Multiple contraceptive methods are available for women, however, male contraception options are limited to condom use and vasectomy. A novel male birth control method would provide an alternate method for family planning and lessen the gender gap in contraceptive responsibility. The significant demand for male birth control methods among both men and women makes it a commercially viable option [3, 4]. Clinical trials are underway to investigate oral as well as topically applied hormones for male contraception [5], however, the discovery and clinical evaluation of non-hormonal contraceptives for men and women lag behind [6].

Metabolic, genetic, and pharmacologic approaches have suggested that interference with the retinoic acid synthesis and retinoic acid receptor (RAR) pathways could be exploited for the development of a non-hormonal male contraceptive agent [7]. Male mice placed on a vitamin A deficient diet are unable to produce retinoic acid and become infertile [8, 9]. Further, Rara^−/− male mice are viable and females are fertile, while males are completely infertile [10, 11]. Targeted deletion of Rara, the gene encoding RARα, resulted in a disruption in spermatogenesis similar to the effects of vitamin A deficiency (VAD) [10]. Deletion of Rara caused early postnatal lethality; the Rara^−/− mice that survived appeared normal, but were infertile with drastically reduced spermatogenesis [10]. RARα is expressed in both Sertoli cells and germ cells. To eliminate the developmental effects of genetic deletion of RARα, the phenotype of conditional knockout animals may more closely approximate the effects of selective RARα antagonists in the adult. Conditional knockout of RARα in germ cells indicate that RARα has a broad spectrum of functions throughout spermatogenesis, including the maintenance of seminiferous epithelium organization, integrity of the meiotic genome, and spermatogonial proliferation and differentiation. RARα also mediates crosstalk with Sertoli cells, controlling the cell junctional physiology for coordinating proper spatial and temporal development of germ cells during spermatogenesis [12]. These results suggest that RARα plays an important role during development, but is dispensable in the adult except for spermatogenesis.

RAR antagonism was shown to be a promising approach when BMS-189453 (1, Fig. 1), a RARα,γ antagonist and RARβ agonist was shown to produce significant defects in spermatogenesis that resulted in reversible infertility when administered orally to male mice [13, 14]. While 1 is efficacious and has a good safety profile at therapeutic doses, RAR subtype selectivity is desirable to reduce potential somatic side effects. The selective antagonist of RARα 2a (BMS-189532, AGN 193491) that we previously investigated, did not elicit the desired spermatogenic inhibition when administered orally [15]. Therefore, the aim of our ongoing research is to discover an orally bioavailable RARα-selective antagonist for male contraception.

Fig. 1. Retinoic acid receptor ligands.

Antagonist pocket-filler (black), hydrophobic ring (blue), liker (pink), conjugated acid (green).

Retinoic acid receptor ligands have been studied for a variety of indications over several decades, and structure-activity information has been summarized in several reviews [16, 17]. The general scaffold consists of a hydrophobic ring system (Fig. 1, blue) and a conjugated benzoic acid (green) which are connected by a linker moiety (pink). This linker has been shown to impart receptor subtype specificity, while modifications to the adornments on the hydrophobic ring (black) determine type of activity (agonism versus antagonism) with larger groups exhibiting antagonistic activity. Co-crystal structures have shown that agonist binding to the RAR ligand binding domain leads to a conformational change of the C-terminal helix 12 (H12) that allows co-activator recruitment, which is followed by initiation of gene expression [18, 19]. Antagonists, however, protrude from the RAR ligand binding domain and reposition H12 into an antagonist position that no longer allows co-activator recruitment and binding [19, 20]. To identify antagonists selectively targeting RARα, our initial efforts focused on modifying the amide linker, which is the distinguishing characteristic between the selective RARα antagonist 2a [21] and the more broadly antagonistic compound 1 [13]. Additionally, our prior work has shown differences in metabolism, plasma protein binding, and permeability that may have negatively impacted the bioavailability of the RARα-selective compounds when compared to compound 1 [15, 22]. We therefore explored how modifications to the linker and the other regions of the molecule would impact potency, selectivity, metabolic stability, and physicochemical properties of the newly developed RAR antagonists.

2. Results and discussion

2.1. Design of analogs

The availability of the co-crystal structure of the moderately potent, RARα/RARβ-preferring antagonist BMS-195614 (2b, Fig. 1) with RARα (PDB: 1dkf) allowed for a structure-based analysis of analogs. We designed compounds based on known published structure-activity relationships (SAR) but with different linker groups that could mimic the amide bond and provide RARα selective inhibitors [17]. In addition we designed and prepared analogs to expand the structure-activity relationship information for this class of compounds Table 1). The Schrödinger molecular modeling suite was used for docking experiments of analogs where we varied the four different structural areas of 2a. We selected modified amides (compounds 3 (BMS-185411 [23]), compounds 4-8, and 15 (AGN194202 [24]), inverse amides (compounds 12-14, 16-17), ureas (compounds 9-11), and sulfonamides (compounds 22-26) for the linker alterations. Moreover, modifications to the antagonist pocket focused on bulky, preferably aromatic groups that incorporated more basic, more polar, less lipophilic moieties such as aryl-morpholino derivatives (compounds 4, 5, 11-14, 16). Changing the naphthalene to a chromene moiety was also explored in light of work by Teng and collaborators (compounds 16-21) [21]. The designed analogs shown in Table 1 displaying docking scores similar to or better than the docking score of for the parent compound 2a (Table S1), indicating that the proposed linker changes could be tolerated. The calculated physicochemical properties of the antagonists (cLogP data shown in Table S1) [25] predicted that the majority of the designed analogs would be less lipophilic than parent compounds 1, 2a, and 2b. Examples include analogs that are carrying a solubility enhancing morpholino group (compounds 4, 5, 11-14 and 16, Table 1), urea analogs 9-11 and sulfonamides 22-26. Such solubilizing or solubility enhancing groups were predicted to improve the water solubility and bioavailability of the antagonists. The docking studies also predicted that appending various morpholino functionalities onto the C8 aryl ring could be beneficial to binding (Table 1, compounds 11-14; Fig. 2 and Table S1). Previous retinoid research has shown that strategic halogenation about the benzoic acid and hydrophobic ring has imparted additional RARα activity/selectivity [21]. Therefore, we wanted to determine if such halogenation or other modifications to the benzoic acid moiety could be tolerated or improve the RARα antagonism of some of our scaffolds. One novel modification included the use of cubane as a bioisostere for the benzene of the benzoic acid moiety, which was predicted to be one of the least lipophilic compounds in this series (Table S1) [26].Both changes were tolerated in the docking studies. Based on these results, the compounds in Table 1 were selected for synthesis and testing.

Table 1. Antagonism of RARα, RARβ and RARγ.

Antagonist potencies were determined by inhibition of an EC₈₀ concentration of 9-cis-retinoic acid (9-cis-RA) for RARα (EC₅₀ = 12.6 ± 1.8 nM) or ATRA for RARβ (EC₅₀ = 2.0 ± 0.74 nM) and RARγ (EC₅₀ = 7.2 ± 2.5 nM) in transactivation assays. for IC₅₀ values are expressed as the mean ± SEM of the number of independent experiments indicated

^aNo effect on RARα cellular viability, n = 1.

Fig. 2.

Predicted binding of RAR antagonist 2a (green) and compound 11 (gray) to RARα (PDB: 1dkf) in docking studies.

2.2. In vitro Transactivation Assay Testing of Analogs

The potency of compounds as antagonists of RARα, γ, and γ was determined using cellular transactivation assays (Table 1). Compounds with moderate to high RARα antagonist potency were tested for cellular viability to confirm that the antagonist activity observed was not confounded by cytotoxicity. Promising selective RARα antagonists and reference compounds were then evaluated for agonism at RARα, β, and γ (Table 2). Before assessing new compounds for potency and selectivity, we tested known RAR antagonists 1 and 2a to validate our assay and to compare our data with reports from the literature. We previously reported RAR antagonist 1 to be a pan-antagonist with estimated IC₅₀ values of ~200 nM for RARα, β, and γ in transactivation assays [14]. An earlier report [13] found that compound 1 was a more potent antagonist in transactivation assays with estimated IC₅₀ values of ~10 nM for RARα and ~3 nM for RARγ and also showed that compound 1 displayed low efficacy partial agonist/partial antagonist activity at RARβ with an EC₅₀ of ~3 nM and an IC₅₀ ~10 nM [13]. Consistent with this high potency in transactivation assays, another group reported high binding affinity for compound 1 with K_i values of 8, 17, and 17 nM for RARα, RARβ, and RARγ, respectively [27]. In agreement with these latter reports, we found that 1 is a potent antagonist of RARα (IC₅₀ 9.4 nM) and RARγ (IC₅₀ 7.5 nM) (Table 1) in transactivation assays. Interestingly, compound 1 was devoid of RARβ antagonist activity, but instead was a potent, pure agonist at RARβ (EC₅₀ 7.5 nM) with high efficacy (E_max 72% of all-trans retinoic acid, ATRA) (Table 2). Therefore, we found compound 1 to be a potent RARα/RARγ antagonist and potent, high efficacy RARβ agonist in our current transactivation assays.

Table 2. Compound agonism of RARα, RARβ and RARγ.

Agonist potencies were determined in transactivation assays. EC₅₀ values are expressed as the mean ± SEM of the number of independent experiments indicated. E_max values are expressed as a percent of the maximal response of 9-cis-RA for RARα or ATRA for RARβ and γ

Compound 2a has been described as an RARα-selective antagonist [15]. Our previous transactivation data indicated that compound 2a was a weak but selective RARα antagonist (IC₅₀ values of ~1, >10, and >10 μM at RARα, RARβ, and RARγ, respectively) [15]. Much higher potency RARα-selective antagonism was reported by Teng et al. in a transactivation assay (IC₅₀ values of ~4, ~100, and ~300 nM at RARα, RARβ, and RARγ, respectively [21]. High potency RARα-selective antagonism was consistent with their binding data (K_i values of 27, 1000, and 3100 nM for RARα, RARβ, and RARγ, respectively) [21]. Our current data indicate that compound 2a is a highly potent RARα-selective antagonist in transactivation assays (RARα IC₅₀ 29 nM) (Table 1). In contrast to previous reports, we also found compound 2a to be a potent, partial agonist of RARβ (Table 2). We previously reported that replacement of the phenyl group of compound 1 with a quinolone in compound 2b resulted in lower RARα-selectivity (IC₅₀ values of ~400, ~4000, and >10,000 nM at RARα, RARβ, and RARγ, respectively) [15]. Our current transactivation data indicate that compound 2b is a moderately potent RARα/RARβ antagonist, with ~10-fold lower potency at RARγ. Unlike compounds 1 and 2a, compound 2b is a pure antagonist devoid of agonist activity at any isoform, suggesting that the hydrophobic ring system imparting receptor antagonism must be rather bulky to ensure pure antagonism rather than partial agonism/antagonism. The differences in the pharmacological activity (agonism vs antagonism vs partial agonism/antagonism) for compounds 1 and 2a in the current and previously reported transactivation assays are most likely caused by differences in the reporter cells used, including transient vs. stable transfection as well as different expression vectors, levels of receptor expression, reporters used, and cell types. These differences likely result in variable levels of agonist-induced signal amplification and sensitivity to detect agonism and antagonism by modulators. Compound 2b has been shown to be a neutral antagonist of RARα as it destabilizes corepressor complexes but does not induce recruitment of coactivators [28].

We initially explored the influence of structural modification of the 5,6-dihydronaphthalene scaffold (analogs 1-20). Compound 3 (BMS-185411), in which the tolyl of compound 2a was replaced with a phenyl group, was highly selective for RARα, but was 5-fold less potent than compound 2a. However, as shown in Table 2, analog 3 is a potent RARβ agonist with high efficacy (E_max 150% of ATRA), which has been described previously [23]. We also found that 3 is a moderate potency, partial RARγ agonist. Compound 3 agonism at RARβ and RARγ is consistent with its small hydrophobic group as in compound 1. We modified position 8 of antagonist 2a and found that the introduction of a morpholinophenyl group (compound 4) greatly reduced inhibitory activity for RARα and RARγ. However, linking the morpholino group through a methylene group (compound 5) resulted in a RARβ-selective antagonist that retained moderate RARα activity. The introduction of an 8-thiophene moiety instead of a phenyl group (compound 6) retained RARα-selective antagonism with moderate potency. Interestingly, compound 6 was a high potency agonist at RARβ consistent with the relatively small thiophene group binding to the antagonist pocket. No inhibitory activity was found when the 4-aminobenzoic acid was replaced with 4-(aminomethyl)benzoic acid for tolyl compound 7 and phenyl analog 8. When the amide was replaced by a urea group (compounds 9-11), compounds 10 and 11 lost all activity, but the tolyl analog 9 retained moderate activity at RARβ. The reversed amides, analogs 12-14, 16, and 17 either had greatly reduced or no activity at the three receptors, although analog 12 retained moderate activity at RARβ. While introduction of 2-fluorobenzoic acid moieties increased potency and selectivity for some RARα antagonists [21], in the case of 2,6-difluoro analog 15 we noticed a 15-fold reduction for RARα potency compared to parent compound 2a. Tetrahydroisoquinoline 18 retaining the amide bond was completely inactive, but tetrahydroisoquinoline 19 linked directly to the dihydronaphthalene core surprisingly retained moderate activity at RARβ. Quinoline 20 was the most potent RARβ-selective antagonist identified (IC₅₀ 21 nM), which was 6-fold less potent at RARγ and inactive at RARα. Although it does not have high selectivity for RARβ over RARγ, compound 20 may be a useful pharmacological probe to antagonize RARβ without disrupting the function of RARα. Next, we explored the chromene scaffold (analogs 21-26). Chromene 21 was a selective antagonist of RARα with 6-fold higher potency than the parent dihydronaphthalene 2a. Analog 21 was also a potent RARβ agonist, and a moderately potent mixed RARγ agonist/antagonist. While this compound has not been tested before, 8-bromo-analogs of 21 have been tested in vitro for RAR selectivity. The best analog AGN 194574 (4-(8-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene-6-carboxamido)-2,6-difluorobenzoic acid) has K_D values at RARα, RARβ and RARγ of 1.5, 900, and 11,000 nM, respectively [21]. Isosteric replacement of the amide group with a sulfonamide [29, 30] (analogs 22-25) and the reversed sulfonamide 26 yielded only inactive analogs at all RAR isoforms. The results obtained from the transactivation assays did not support the anticipated activity of the compounds from the docking studies, which had predicted that most of the newly designed analogs would bind effectively to RARα. There are several reasons why docking scores obtained from a crystal structure may not be predictive and generate false positives due to the inherent limitations of the scoring algorithm that ignores the molecular dynamics of protein structures and imperfectly approximates the energetic contribution of each interaction [31]. Incongruity between in silico results and experimental data can originate from protein flexibility resulting in different adopted conformations depending on ligand binding, the solvation of the binding site may not be accurately represented in the crystal structure, or steric clashes and electrostatic repulsions could prevent binding despite favorable docking scores. Additional factors that could influence activity are desolvation penalties for a compound to move from the aqueous environment to the lipophilic binding pocket and poor cell permeability in our cell-based assay. Our goal for the docking and scoring was to assess the feasibility of compounds fitting in the binding pocket. Even in the absence of high-level computational tools such as free-energy perturbation to predict the binding affinity of analogs more accurately, we were still able to identify and study several molecules which helped to refine structure-activity relationships (SARs) of this class of retinoic acid antagonists.

2.3. Evaluation of in vitro physicochemical properties of compound 21.

Analog 21 was the most potent and selective RARα antagonist identified in our study. While its agonist activity is a potential concern, the agonist effect is about 16-fold (RARβ) and 113-fold (RARγ) weaker than its antagonist activity at RARα. We therefore carried out the assays shown in Table 3 for compound 21 to test for aqueous solubility, partition coefficient (log D), plasma protein binding, bi-directional permeability, and in vitro metabolic stability in hepatic and intestinal microsomes. For comparison, Table 3 also shows data for RAR antagonists that produce pronounced (compound 1) and marginal (compound 2a) disruption of in vivo spermatogenesis, both of which we had previously tested in the same assays [15]. From the data for compounds 1 and 2a we had concluded that a combination of relatively rapid hepatic metabolism and high protein binding for compound 2a were the reasons for poor activity in mice when administered orally [15]. Compound 21 showed low solubility in PBS buffer at pH 7.4 that is similar to compound 1, which is also poorly soluble . However, all three compounds 1, 2a and 21 possess favorable LogD partition coefficients of 3.3 to 3.8, which are within the Lipinski’s rule (<5) for oral bioavailability [32]. Also, plasma protein binding for compound 21, though extensive, is not expected to hamper in vivo efficacy, whereas the high plasma protein binding of compound 2a was considered less favorable. Caco-2 monolayer permeability assays were conducted to evaluate the ability of the compounds to be absorbed by the intestinal membrane. For compound 21 a low apical to basolateral permeability was observed. The basolateral to apical permeability (B-A) of was higher resulting in an elevated efflux ratio of 3.0, suggesting that compound 21 may be a substrate for a drug transporter. The most pronounced difference between compounds 2a and 21 is the high metabolic stability of 21. Compound 21 was not metabolized by liver and intestinal microsomes in 60 min, whereas compound 2a was substantially degraded in liver microsomes. Analog 21 also had higher microsomal stability than compound 1.

Table 3.

In vitro physicochemical properties of compounds 1, 2a, and 21

Assay (conditions)	Compound 1 [15]	Compound 2a [15]	Compound 21
Solubilitya	3.1 μM	136 μM	4.2 μM
LogD partition coefficientb	3.25	3.84	3.35
Plasma protein bindingc	95.3% bound 93.3% recovery	99.2% bound 99.8% recovery	97.0% bound 100% recovery
A-B permeabilityd	2.89 ×10−6 cm/s 88% recovery	3.23 ×10−6 cm/s 83% recovery	1.92 ×10−6 cm/s 90% recovery
B-A permeabilityd	1.51 ×10−6 cm/s 100% recovery	5.41 ×10−6 cm/s 95% recovery	5.83 ×10−6 cm/s 98% recovery
Metabolic stability (liver)e	79% parent remaining	49% parent remaining	100% parent remaining
Metabolic stability (intestine)f	Not determined	86% parent remaining	100% parent remaining

^aPBS, pH 7.4.

^blog D, 1-octanol/PBS, pH 7.4,

^cCD-1 mouse male and female, test at 1.0 10⁻⁵ M.

^dCaco-2, pH 7.4/7.4, 1.0 ×10⁻⁵ M in the presence of 2% BSA.

^eLiver microsomes with NADPH, CD-1 male mouse, 1.0 ×10⁻⁶ M, 60 min.

^fIntestinal microsomes with NADPH, CD-1 male mouse, 2.0 ×10⁻⁶ M, 60 min.

Data generated by Pharmaron.

2.3. Pharmacokinetic study of compound 21

As the in vitro ADMET studies suggested that compound 21 may have a favorable PK profile with significant oral bioavailability and metabolic stability, a comprehensive PK study was performed in male mice to test this hypothesis. Animals (CD-1, 6-8 weeks, n = 27) were dosed orally at 10 mg/kg and sets of 3 mice were then euthanized at 9 time points (5, 15, 30 min and 1, 2, 4, 8, 18, 24 h). Plasma and testes were collected from each animal and the levels of compound 21 in plasma and testes were determined by LC-MS/MS analysis. As can be seen in Fig. 3, levels of compound 21 quickly reached a steady-state level in the plasma within 30 min following oral administration, indicating significant oral bioavailability. The steady-state nature also indicates a high degree of metabolic stability consistent with the microsomal stability data and suggests that excretion by renal filtration is minimal. The sustained plasma levels averaged between 30 min and 24 h was 5,000 ± 1,900 ng/mL or 12 μM, which is approximately 2600-fold the in vitro RARα IC₅₀. More importantly, the levels in the testes also rapidly reached a steady-state and were sustained up to 24 h, mirroring the plasma levels. Due to the sustained plasma levels, standard PK parameters such as half-life and clearance could not be calculated, but estimation of exposure levels was possible, providing a testes to plasma exposure ratio of 0.13. Average testes levels were 610 ± 240 ng/mL or 1.5 μM, which translates to approximately 330-fold the IC₅₀. Although the absolute oral bioavailability was not determined, we conclude that compound 21 has high oral absorption, has a long half-life, and was suitable for evaluation in vivo to determine its effect on spermatogenesis and fertility in mice.

Fig. 3. Pharmacokinetic analysis of compound 21.

Data were obtained from plasma and testes from animals (n = 3) euthanized at specific times following oral bolus dosing of compound 21 (10 mg/kg). Concentrations in plasma are expressed as the mean (ng/mL) ± S.D. whereas concentrations in testes are expressed as the mean (ng/g) ± S.D. AUCs were calculated using Prism Graphpad v6; AUC_plasma = 109,100 h*ng/ml, AUC_Testes = 13,900 h*ng/ml.

2.4. In vivo evaluation of the effects of compound 21 on spermatogenesis and fertility

To determine if compound 21 elicited the characteristic effects on spermatogenesis that were observed in Rara−/− mice [10, 11] and in mice after treatment with compound 1 [13, 14], in an initial pilot experiment, compound 21 suspended in Avicel was administered orally at 5 mg/kg/day for 7 days (n = 10). One day after cessation of drug treatment (CDT), there was a slight but marginally statistically significant drop in testicular weight (Fig. 4A; 0.11 g ± 0.01 versus standard control, 0.12 g ± 0.01, p <0.01). We also observed a drop in sperm counts (Fig. 4B; 24.50 ± 5.08 x 10⁶ versus standard control 55.50 ± 8.18 x 10⁶). At the morphological level, in 9 out of the 10 samples, the most striking observation was that elongated spermatids reproducibly failed to be released and were retained in 15.9% of abnormal stage IX tubules (Fig. 5B–C, n = 4) as compared to normal testicular tubules from young adult wild-type males, in which 0% abnormal stage IX tubules and only tubules with normal spermatid alignment and sperm release were observed Fig. 5A. There were also fewer or no leptotene spermatocytes in 4.4% of these abnormal stage IX tubules (n = 4; Fig. 5C–D). The missing layer of specific cell types is indicated with an arrow and question mark, labeled “no L?”), presumably reflecting a delay of pre-leptotene spermatocytes to become leptotene spermatocytes in abnormal stage IX tubules. Abnormal tubules are indicated by the approximate stage followed by an asterisk in Fig. 5. At 4 weeks after CDT, normal testicular weight was observed (Fig. 4A; 0.12 g ± 0.01; n = 10) and the number of sperm had recovered (Fig. 4B; 54.90 ± 7.53 x 10⁶, n = 10). Tubules with full spermatogenesis were seen in all testes examined (Fig. 5E, n = 10) and the epididymal morphology was normal (Fig. 5F, n = 10). Proper spermatid translocation and spermatid release were consistently found in all treated testes (Fig. 5E, n = 10). From our previous observations using antagonist compound 1-treated mice [14]. we anticipated a more drastic disruption of spermatogenesis at the 4-week time point. Nonetheless, these abnormalities in stage VIII and IX tubules indeed resembled those quite readily and frequently observed in RARα-deficient and compound 1 antagonist-treated testes [11, 14, 33, 34].

Fig. 4.

Gonad weight, cauda epididymal sperm count and fertility assessment of mice treated with the dose of 5.0 mg/kg of compound 21 for 7 days. A. The testicular weight of compound 21-treated male mice at one day and 4 weeks after 7 days of dosing. The bars represent the mean ± SD of ten mice for each time point. Significant differences in weight between testes were observed between control and 1 day, 1 day and 4 weeks, but not between control and 4 weeks, as assessed by one-way ANOVA analysis; *, P < 0.01; **, P < 0.005. B. Cauda epididymal sperm counts of compound 21-treated male mice at one day and 4-week post-dose time points. The horizontal bars show the average number of sperm counts obtained at the assessed time point. Significant differences in sperm count were observed between control and 1 day, 1 day and 4 weeks, but not between control and 4 weeks, as assessed by one-way ANOVA analysis;***, P < 0.0001. Panel C. Fertility assessment of mice as revealed by the numbers of embryos (solid triangles) obtained from mating of each male treated with compound 21 at 2 weeks after cessation of drug treatment (n=10). The horizontal bars show the average number of embryos per litter obtained at the assessed time point.

Fig. 5. Effects of compound 21 on spermatogenesis.

A-F: Histological sections of mice treated with 5 mg/kg/day of compound 21 for 7 days and terminated one-day (B-D) and 4-week (E-F) post-treatment, as compared to young adult wild-type male (A). A-F: Magnification, 40x. Pl, preleptotene; L, leptotene; Z, zygotene; P, pachytene; and D, diplotene spermatocytes. The bracket in stage IX* tubules indicates retained spermatids. Arabic numerals indicate the step of spermatid differentiation; Roman numerals indicate tubule stage. Although abnormal cell associations complicate staging, an attempt was made to stage the drug-treated tubules using the acrosomal system [35], and tubules are labeled with a Roman numeral, with abnormal tubules designated with an asterisk (e.g., stage IX*). The missing layer or reduced number of specific cell types is indicated with an arrow and “no L?”.

A concomitant pilot fertility assessment was performed by mating the drug-treated males (n = 10) individually with two virgin females of the same strain for 2 weeks (during weeks 1 and 2 after CDT) as previously described [14]. No disruption of fertility was observed in the compound 21-treated males mated for two weeks after CDT as revealed by the size of embryonic litters produced (12.15 ± 1.83, n=10, Fig. 4C), which was comparable to that observed in our previous control studies with this strain of mice (for example, 11.90 ± 2.16 in reference [14]). This lack of effect on fertility was not surprising since mouse fertility is usually not affected unless sperm counts drop to 1/10 of the normal levels.

2.5. Effect of raising the dose and lengthening the duration of dosing period of compound 21

Although the drop by one half of sperm counts one day after CDT was not physiologically significant, we were encouraged by the characteristic morphological effects to examine whether a higher dose of compound 21 would trigger more drastic disruption of spermatogenesis. Specifically, we increased both the dose and the length of time of treatment. For this study, compound 21 was administered orally at 10 mg/kg for 2 weeks (n = 10 per time-point), along with controls that were administered aqueous 20% Kollisolv (vehicle only, n = 5 per time-point). Testes were collected, weighed, and fixed for morphological examination and caudal epididymal sperm counts were made at one day and 4 weeks post CDT. At both 1 day and 4 weeks after CDT, doubling the dose and the length of time of treatment did not yield a significant change in either the testicular weights (one-day after CDT, 0.12 g ± 0.01 vs control, 0.11 g ± 0.02; 4 weeks after CDT, 0.13 g ± 0.02 vs control, 0.13 g ± 0.00) or sperm counts (one-day after CDT, 37.20 ± 8.59 x 10⁶ vs control, 41.80 ± 7.92 x 10⁶; 4 weeks after CDT, 44.75 ± 9.87 x 10⁶ vs control, 50.30 ± 5.90 x 10⁶). In vehicle-only control males, tubules with full spermatogenesis were seen in all testes collected (n = 5) as were proper spermatid translocation and spermatid release. In drug-treated testes, similar to what was observed with the lower dosing regimen, a failure of sperm release was consistently found in ~14% of the abnormal stage IX tubules in testes examined one day post CDT (n = 6 counted), whereas all control testes exhibited normal stage IX tubules. At 4 weeks after CDT, tubules with full spermatogenesis and proper spermatid translocation and spermatid release were consistently found in all treated testes (n = 10). Although two different vehicles were used between the two sets of experiments, we found that the overall morphological observations were consistent. From these data we concluded that compound 21 exhibited reproducibly characteristic albeit modest effects on spermatogenesis as compared with RAR antagonist 1 [14]. Raising the dose and lengthening the duration of dosing period did not enhance the inhibition of spermatogenesis.

An alternative explanation for the failure to observe a contraceptive effect in these animals is the possibility that the use of different formulations in the mating (1.5% Avicel) and PK (80% Carbitol (diethylene glycol monoethyl ether, Sigma Aldrich)/20% saline) studies could result in different drug exposures and effectivenes. The Avicel formulation was a suspension and the Carbitol formulation was a complete solution which could have resulted in less drug exposure for the former. We tested this hypothesis by performing a sperm count study using the Carbitol formulation where animals were dosed (10 mg/kg) orally daily for 28 consecutive days. On day 29, mice were ethanized and epididymal sperm counts were performed. We observed an approximately 73% reduction in sperm count (Fig. SI.1), which essentially mirrored the results obtained in the Avicel study where sperm counts were reduced by approximately 56%.Once again, sperm counts did not reach the 90% threshhold for contraception. We conclude that the pilot study with 21 revealed characteristic albeit modest effects on spermatogenesis.

2.6. Synthesis of Analogs

Synthesis of amide analogs of 2a was accomplished (Scheme 1) by first installing the occupant of the antagonist pocket onto the naphthalene core scaffold. The substituents (4-tolyl, phenyl, 4-morpholinophenyl, and thiophene) were installed via Grignard addition to commercially available bromotetralone followed by dehydration. The substituted bromonaphthalenes were then converted to the corresponding acids by lithium-halogen exchange followed by trapping with carbon dioxide. The naphthoic acids were reacted with aminobenzoates by EDCI-mediated coupling. The resulting esters were hydrolyzed to the acids 2a-4, 6-8, and 15.

Scheme 1.

Synthesis of amide analogs 2a-4, 6, and 15

a) R¹MgBr, THF, 0 °C, 16 h; b) 4-TsOH, benzene, 80 °C, 4 h (30-87% over two steps); c) t-BuLi, THF, CO₂, −78 °C, 0.5 h then HCl, 32-49%; d) EDCI, HOBt, DMAP, DCM, 37 °C, 16 h; or oxalyl chloride, 0 °C to rt, 1 h; e) NaOH, H₂O, EtOH, DCM, 37 °C, 16 h, 18-99%.

The more basic substituent in compound 5, the 4-(methylmorpholino)phenyl substituent, needed to be installed via a Suzuki reaction as shown in Scheme 2. The ketal 41 was converted to benzoic acid 42, then to methyl ester 43, which was subsequently subjected to Comins’ reagent to furnish vinyl triflate 44. At that point, a palladium-catalyzed Suzuki reaction was used to introduce the desired aryl substituent from the requisite boronic acid to furnish intermediate 45. Following ester hydrolysis, amide formation and hydrolysis of ester 47 yielded desired morpholinomethyl analog 5.

Scheme 2.

Synthesis of amide 5

a) 1. t-BuLi, THF, −78 °C, 0.25 h; 2. CO₂, −78 °C, 0.25 h; 3. 1M HCl, EtOAc, rt, 0.25 h; b) K₂CO₃, MeI, DMSO, rt, 0.5 h, 64% (over two steps); c) NaHMDS, THF, −78 °C to rt, 16 h, 52%; d) Pd(PPh₃) ₄, NaHCO₃, 1,2-DME, rt, 3 h; e) NaOH EtOH, 37 °C, 16 h; f) EDCI, HOBt, DMAP, DCM, 37 °C, 16 h, 15% (over two steps); g) NaOH, EtOH, 37 °C, 16 h, 100%.

Analogs 7 and 8 (Scheme 3) were prepared by reaction of the naphthoic acids 33 and 36 with methyl 4-(aminomethyl)benzoate via EDCI-mediated coupling followed by hydrolysis to corresponding acids 7 and 8.

Scheme 3.

Synthesis of methylamino analogs 7 and 8

a) EDCI, HOBt, DMAP, DCM, 35 °C, 16 h, 84-91%; b) NaOH, H₂O, EtOH, DCM, rt, 16 h, 85-93%.

Urea-linked analogs 9-11 were prepared as shown in Scheme 4. The arylamines were accessed from the corresponding bromonaphthalenes (32, 50, and 30) using CuI and sodium azide. Intermediate anilines 51-53 were then reacted with ethyl 4-isocyanatobenzoate. Hydrolysis of the ethyl esters provided urea analogs 9-11.

Scheme 4.

Synthesis of urea-linked retinoids 9-11

a) NaN₃, NaOH, L-proline, CuI, DMSO, EtOH, 110 °C, 10 h, 60-83%; b) THF, rt, 24 h; c) LiOH, H₂O, THF, MeOH or NaOH, H₂O, EtOH, DCM, rt, 16 h, 33-88% (over two steps).

Reversed amides 12 and 16 were obtained from morpholinonaphthoic amine 53 by EDCI-mediated coupling with the respective benzoic acid methyl esters followed by ester hydrolysis (Scheme 5). Cubane analog 17 was prepared similarly from aniline 51.

Scheme 5.

Synthesis of reversed amides 12, 16 and 17

a) EDCI, HOBt, DMAP, DCM, 35 °C, 16 h, 64-86%; b) NaOH, EtOH, 37 °C, 16 h, 58-100%.

An alternative strategy was used for the preparation of reversed amides 13 and 14 (Scheme 6). In this case, the reversed amide moiety was introduced first, followed by preparation of the enol triflate, palladium catalyzed Suzuki reaction and methyl ester hydrolysis.

Scheme 6.

Synthesis of reversed amides 13 and 14

a) CuI, K₂CO₃, N,N’-dimethylethylenediamine, toluene, 120 °C, 40 h, 30%; b) Comins’ reagent, NaHMDS, THF, −78 °C, 16 h, 38%; c) R-Bpin, Pd(PPh₃)₄, NaHCO₃, 1,2-DME, 85 °C, 2 h; d) NaOH, H₂O, EtOH, DCM, rt, 16 h, 52-56% (over two steps).

Tetrahydroisoquinoline acid 18 was prepared (Scheme 7) by reaction between chromene acid 36 and methyl 1,2,3,4-tetrahydroisoquinoline-6-carboxylate and subsequent ester hydrolysis.

Scheme 7.

Synthesis of tetrahydroisoquinoline 18

a) EDCI, HOBt, DMAP, DCM, 3 °C, 16 h, 94%; b) NaOH, EtOH, rt, 16 h, 99%.

Isoquinoline analog 19 was obtained following a Buchwald-Hartwig amination reaction using Pd₂(dba)₃ and Xantphos to couple the tetrahydroisoquinolinecarboxylate and naphthalene bromide 32 (Scheme 8), and quinoline carboxylic acid 20 was obtained through a rhodium catalyzed direct arylation of naphthalene bromide 32 (Scheme 9).

Scheme 8.

Synthesis of tetrahydroisoquinoline 19

a) Pd2(dba)₂, Xantphos, NaOtBu, toluene, 110 °C, 36 h, 38%; b) 1. NaOH, H₂O/THF, 2. HCl, H₂O, pH 4, rt, 16 h, 45%.

Scheme 9.

Synthesis of quinoline 20

a) [Rh(CO)₂Cl]₂, 1,4-dioxane, 175 °C, 24 h, 61%; b) 1. NaOH, MeOH, 2. HCl, 65 °C, 2 h, 38%.

Synthesis of chromene amide 21 (Scheme 10) likewise began via a Grignard addition of 4-tolylmagnesium bromide to the commercially available starting material 6-bromo-2,2-dimethylchroman-4-one, followed by dehydration. Bromide 67 was then converted to carboxylic acid 68 using tert-butyllithium and trapping the lithiated species with carbon dioxide as previously described. Amide ester 69 was formed via an EDCI-mediated coupling reaction, followed by ester hydrolysis to furnish acid 21.

Scheme 10.

Synthesis of chromene amide 21

a) 4-tolyl-magnesium bromide, THF, rt, 16 h; b) p-TsOH, C₆H₆, 80°C, 4 h, 49% (over two steps); c) t-BuLi, THF, CO₂ then HCl, −78 °C, 1 h, 83%; d) ethyl 4-aminobenzoate, EDCI, HOBt, DMAP, DCM, 40 °C, 16 h, 52%; e) NaOH, H₂O, rt, 16 h, 63%.

Synthesis of the chromene sulfonamides began via cyclization of 1-(2-hydroxyphenyl-ethan-1-one) with acetone (Scheme 11). Grignard addition of the tolyl substituent and subsequent dehydration provided the chromene. Installation of the sulfonyl chloride proceeded utilizing the SO₃•DMF complex to provide the sulfate, which was then converted to the sulfonyl chloride with oxalyl chloride. The sulfonyl chloride was then coupled to 4-aminobenzoic acid for the synthesis of 22 and 23 and to ethyl 4-amino-2,6-difluorobenzoate, followed by ester hydrolysis to furnish sulfonamide acids 24 and 25.

Scheme 11.

Synthesis of chromene sulfonamides 22-25

a) acetone, pyrrolidine, MeOH, rt, 72 h, 82-85%; b) 4-tolylmagnesium bromide, THF, −78 °C to rt, 24 h; c) p-TsOH, MeOH, reflux, 14 h, 37-70% d) SO₃-DMF, DCE, 70 °C, 10 h; e) (COCl)₂, 0 to 65 °C, 4 h, 54-82%; f) 4-aminobenozic acid, pyridine, DCM, rt, 24 h, 60-80%; g) ethyl 4-aminobenzoate, pyridine, DCM, rt, 24 h, 60-75%; h) NaOH, EtOH, rt, 16 h, 53-60%.

Synthesis of the reverse sulfonamide used 2-hydroxy-5-iodoacetophenone for the reaction with acetone (Scheme 12). Following Grignard addition and dehydration, iodide 80 was converted to aniline 83 using aqueous ammonia and CuI, which was then coupled to chlorosulfonylbenzoic acid to provide target acid 26.

Scheme 12.

Synthesis of reversed chromene sulfonamide 26

a) acetone, pyrrolidine, MeOH, rt, 72 h, 81%; b) 4-tolylmagnesium bromide, THF, −78 °C to rt, 24 h; c) p-TsOH, MeOH, reflux, 14 h, 51% (over two steps); d) NH₃, CuI (20 mol%), L-proline, K₂CO₃, DMSO, rt, 48 h, 33%: e) pyridine, DCM, rt, 24 h, 55%.

3. Conclusion

We prepared and tested 26 retinoic acid antagonists for potency and selectivity (Fig. 6). Our structure-activity studies indicated that structural changes at the C8 phenyl group of the dihydronaphthalene core, which reaches into the antagonist pocket of RARα, showed reduced potency against RARα compared to parent compound 2a, while RARα selectivity was retained for analogs 3 and 6. Replacement of the 4-aminobenzoic acid moiety with a 4-methylamino acid moiety in compounds 7 and 8 furnished inactive analogs at all three receptors. Compounds 9-11 with a urea linker in place of the amide were inactive except for 8-tolyl analog 9, which retained moderate to weak activity at RARβ. Most of the analogs featuring a reversed amide moiety (12-14, 16, 17) were inactive at all three receptors, but C8 morpholino analog 12 showed moderate to weak activity at RARβ. Compound 15, the difluoro analog of compound 2a, retained RARα selectivity but had lower potency, which is about 10-fold less potent than had been reported previously [21]. Tetrahydroisoquinolines 18 and 19 and quinoline analog 20 were inactive at RARα. Direct attachment to the dihydronaphthalene core without a linker in compounds 19 and 20 resulted in RARβ-selective antagonists, with analog 20 having the highest RARβ potency of the compounds tested. Chromene analog 21 was the most potent and selective RARα antagonist identified. Chromenes 22-26 with a sulfonamide or reversed sulfonamide instead of an amide linker were found to be inactive at all RAR isoforms. Overall, our study indicates that small antagonist pocket rings (phenyl, tolyl) provide high potency full antagonists for RARα. However, these small antagonist pocket rings fail to reliably convert the agonist scaffold into pure antagonists for RARβ and RARγ, resulting in either partial agonist/antagonists or even pure agonists at these isoforms. Indeed, compounds 3 and 6 with small pocket groups (phenyl, thiophene) may be useful tool probe compounds as selective agonists for RARβ. Retinoid-like agonism activity has been reported for some (5,6)-dihydronaphthalene analogs [36] including compound 6, which was previously reported to be a selective RARβ selective agonist [37]. The agonists affect, however, appears to be dependent on C8 substitution. Compound 1 carrying a C8 phenyl group is a potent RARβ agonist, whereas compounds 2b and 5 with sterically demanding C8 substituents lack RARβ agonist activity. Whereas both amide and vinyl linkers impart agonism for RARβ and RARγ in compounds with small antagonist pocket fillers, cyclized linkers (quinolone) provide high antagonist potency at RARβ. The vinyl linker confers high antagonist potency for RARγ, although low efficacy agonism may be present. These findings indicate that it is difficult to predict the selectivity of even close analogs for the three RAR isoforms as well as their pharmacological activity in terms of their relative levels of agonism versus antagonism. In general, small antagonist pocket rings are sufficient to convert the agonist scaffold to pure antagonists for RARα, however larger pocket rings are required to impart pure antagonism at RARβ and RARγ.

Fig. 6.

Summary of structure-activity relationships.

Evaluation of the physicochemical properties and pharmacokinetics of compound 21 indicated that this compound is orally available, has a high degree of metabolic stability and reaches the testes. Compound 21 was therefore selected for in vivo evaluation for effects on spermatogenesis and fertility as an oral agent in a mouse model. Modest effects on the inhibition of spermatogenesis were observed, but 21 was not effective as a contraceptive agent in mating studies. The lack of contraceptive effect of compound 21 was surprising considering its high testes levels relative to its cellular potency and long duration of exposure. Another possibility for the observed modest effects could be related to fast clearance of the compound, rapidly reversing to normal sperm counts. In summary, despite its potency and relative RARα-selectivity, compound 21 may not be a good candidate to pursue further for effects on male fertility but underscores the importance of combining detailed synthetic and in vitro assessments with subsequent in vivo validation.

4. Experimental section

4.1. Cell viability assay

The cytotoxicity of the compounds was evaluated by Live Cell (LCMA) kit (Indigo Biosciences, State College, PA) following the instruction manual. This kit measures the conversion of calcein AM dye to fluorescent calcein by intracellular esterases of live cells. The cells were incubated with the test compound for 22 h and the media was discarded after the incubation. Following a buffer wash, cells were treated with the LCMA reagent containing calcein AM for 15 min at room temperature. After the incubation, fluorescence was measured with a plate reader (SpectraMax M2e, Molecular Devices, San Jose, CA) at 485 nm excitation and 535 nm emission wavelength. The percentage of live cells were calculated by comparing the fluorescence of untreated wells and compound-treated wells. After performing the fluorescence-based assay, the detection reagent was discarded, and the luminescence detection reagent was added to the same wells to obtain the luminescence-based readout for the RAR transactivation assay.

4.2. Transactivation Assay

The Human Retinoic Acid Receptor Reporter Assay System kits (Indigo Biosciences, State College, PA) were used to quantify the potency of test compounds as antagonists of the α, β and γ RAR subtypes. These proprietary mammalian reporter cells express a recombinant human RAR subtype, and a beetle luciferase reporter gene functionally linked to a RAR response element. A dose-response of agonist, 9-cis-retinoic acid (9-cis-RA) for RARα or ATRA for RARβ and γ was included on every 384-well plate. Reference compounds 2a, 2b, or 1 were for RARα, RARβ or RARγ, respectively, were also included on every plate. Control wells containing cells without added agonist defined the background signal values. An EC₈₀ concentration of agonist in DMSO (180 nM 9-cis-RA for RARα, 8 nM ATRA for RARβ and γ) was added to control and compound wells using the Echo acoustic nanoliter dispenser (Labcyte, San Jose, CA). Test and reference compounds in DMSO (final 0.4%) were added to plates in 8-point dose response in triplicate. Cell suspension (30 μL) was added to each well and the assay plate was incubated overnight in a 5% CO₂ incubator at 37 °C. Luciferase detection reagent (15 μL) was then added, and the plate was incubated at r.t. for 30 min. Luminescence was quantified using an EnSpire plate reader (PerkinElmer, Waltham, MA). IC₅₀ and EC₅₀ values were determined by fitting dose response data using the four-parameter logistic equation in GraphPad Prism 7.0. Mean ± SEM values were calculated from the geometric mean of the log IC₅₀ and EC₅₀ values.

4.3. Pharmacokinetic studies

All animal procedures for the pharmacokinetic study were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). Animals were allowed to acclimate in their cages after arrival for at least 72 h and were allowed access to rodent chow and water ad libitum. The in-life portion of the study consisted of dosing 27 male CD-1 mice (6-8 weeks, Envigo, Indianapolis, IN) PO with 10 mg/kg of compound 21. Compound was formulated with 80% Carbitol (diethylene glycol monoethyl ether, SigmaAldrich)/20% saline at a final concentration of 1.75 mg/ml. Animals were weighed and received a volume commensurate with the prescribed dose. At specified time points (5, 15, 30 min and 1, 2 4, 8, 16 and 24 h) sets of 3 animals were bled, euthanized and testes harvested. Resulting plasma and tissue were frozen at ≤ −20° C until processing and analysis by LC-MS/MS. Processing of testes consisted of weighing samples and adding 2x volume of cold H₂O. The tissue mixtures were then homogenized using a Polytron PT 2500E (Kinematica AG, Luzern, Switzerland) with a 5 mm probe (PT-DA 05/2 EC-E85, Kinematica AG). Homogenates were stored at ≤ −20 °C until analysis.

4.4. LC/MS/MS Quantitation of Samples

LC/MS/MS analysis was performed using a Quattro Ultima triple quadrupole mass spectrometer (Waters, Milford, MA) coupled with a Waters Acquity Ultra Performance Liquid Chromatography (UPLC) system by operating in electrospray in the positive ion mode (ES+). Mass-to-charge ratio (m/z) transitions for the analyte (compound 21, MW = 413.47) and internal standard (SM-1-194, MW = 449.55) were determined to be 413.9 > 276.9 and 450.2 > 141.97, respectively. For liquid chromatographic separation, an isocratic elution (35% H₂O/0.1% formic acid, 65% acetonitrile/0.1% formic acid, 0.5 mL/min) was performed using a Synergi Polar-RP column (75 × 2 mm, 4 μm; Phenomenex, Torrance, CA). The total run time was 4.0 minutes. Standard curve working solutions were prepared by serial dilution of stock solutions (made from 1 mg/mL master stock in DMSO) with 100% acetonitrile to obtain working concentrations at 0.21, 0.62, 1.86, 5.56, 16.67, 50, 150 and 450 ng/μL. Calibration standards were prepared by spiking 50 μL of blank with freshly prepared working solutions to achieve standards with concentrations of 0.23, 0.7, 2.1, 6.2, 18.6, 55.6, 166.7, 500, 1500 and 4500 ng/ml. Quality control (QC) samples were prepared by spiking 50 μL of blank with freshly prepared working solutions of 0.21, 0.62, 5.56 and 450 ng/μL to obtain the limit of quantitation (LoQ), low quality control (LQC), medium quality control (MQC) and high quality control (HQC). Plasma and tissue samples (50 μL) were placed in a 1.5-mL Eppendorf tube, 100 μL 100% acetonitrile was added with a spiked amount of internal standard, and the mixture was vortexed. Samples were left to stand at 4 °C for at least 30 min and then microcentrifuged at 16,000 x g for 5 min and the supernatant was recovered. Supernatants (100 μL) were transferred to a clean tube and evaporated to dryness under a N₂ stream at 25 °C for approximately 10-15 min. Samples were reconstituted with HPLC elution buffer (100 μL, 70% H₂O/0.1% formic acid, 30% acetonitrile/0.1% formic acid). Processed samples were then transferred to 12 x 32 mm glass vials with 400 μL inserts and loaded into 96-vial auto-sampler plates from where 7.5 μL was injected into the LC/MS/MS system. Sample analysis was performed using MassLynx Software (v4.1, Waters, Milford, MA). Data are expressed as the mean of the 3 replicate animals ± S.D. Relative Exposure of the compound in various tissues is defined as the fraction of exposure compared to the total exposure in plasma and is described by the following equation (1):

$$Rel.Exp.=AU{C}_{inf}(Tissue)/AU{C}_{inf}(Plasma)$$ (1)

4.5. Animal source and treatment of mice with the RARα-selective antagonist, compound 21

The use of animals was approved by Columbia University Irving Medical Center’s Animal Care and Use Committee. CD1 mice (8 weeks, 30 g body weight) were obtained from Charles River Laboratories (Wilmington, MA). Oral delivery of the drug followed our previously described protocols [14, 15, 34]. Briefly, 21 was initially suspended in a vehicle of aqueous 1.5% Avicel (CL-611, FMC BioPolymer) to obtain the desired concentrations and was administered to CD1 males (n = 10 per time-point; a sample size of 10 males was shown in our previous studies 1 to yield statistically significant and reproducible assessments) via oral gavage at a dose of 5 mg/kg/day for 7 days. Since mice treated with aqueous 1.5% Avicel only had previously been shown to not display any testicular abnormalities when examined at various time-points [14, 15], untreated young adult wild-type males were used as controls for the initial pilot experiments. Subsequent experiments to test the effect on spermatogenesis of a higher dose (10 mg/kg/day) for a longer period (2 weeks) used 20% Kollisolv/water as a vehicle because we found that it is a better solvent for 21 compared to aqueous Avicel. In brief, 21 was dissolved in 20% aqueous Kollisolv to obtain the desired concentration and was administered to CD1 males (n = 10 per time-point). For control, 20% aqueous Kollisolv only was administered to CD1 males (n = 5 per time-point). For both regimens, the mice were observed daily for changes in overall health and behavior. Body weights were recorded, and physical examinations were performed weekly.

4.6. Gross and histological assessment of testes

After CDT, the treated males were euthanized at two time points (one day and 4 weeks) for assessing the effect of the compound on spermatogenesis. At the specified time-points, testes were dissected from anesthetized mice (which were then euthanized) and weighed. Testicular weight and sperm number in the treatment group as compared to that in control was assessed by statistical analysis using GraphPad Prism version 9.2 via a one-way ANOVA followed by Tukey’s multiple comparisons test as previously described [34]. One testis was fixed with 4% paraformaldehyde in 1x Phosphate Buffered Saline (PBS) buffer and the second with Bouin’s fixative overnight at 4 °C. Fixed tissues were embedded in paraffin, sectioned, and mounted as previously described [14, 15]. For staging of testicular tubules [35], sections were stained for the Periodic acid-Schiff reaction before hematoxylin counterstaining as previously reported [11, 33] and examined by bright-field microscopy.

4.7. Sperm counts

Sperm were collected into modified Whitten’s medium at 37 °C from both cauda epididymides of the euthanized animals as described previously [14, 38]. Briefly, caudae were minced with small scissors to release the sperm, which were further extruded from the pieces by round forceps. Large cells, tissue fragments, and debris were filtered out via a 100-μm filter. A 1:10 dilution was made, and sperm were counted manually using a hemocytometer.

4.8. Assessment of fertility

Fertility was assessed using our well-established procedures in the laboratory [14]. Briefly, drug-treated males were placed in individual cages with two untreated virgin females of the same strain for 14 days (three estrus cycles elapsed). Females were checked daily for postcoital vaginal plugs. At 14 days, they were euthanized and numbers of pregnant females, implantation sites, viable fetuses, resorptions, and females with resorptions were recorded.

4.9. Docking studies

The docking studies consisted of 4 steps: 1) LigPrep, 2) Protein Prep, 3) Receptor Grid Generation, and 4) docking of the prepared ligands into the receptor/grid. An SD file containing the structures of the retinoid analogs shown in Table 1 was created in ChemDraw and imported into a new project workspace in Schrodinger’s molecular modeling suite Maestro. Within the project table, all scaffold entries were selected, bond orders were assigned, and hydrogens were added. Next the LigPrep function was performed with all ionization states generated with Epik from pH 5.0-9.0, desalting, and tautomerization allowed while retaining specified chiralities (max 32/ligand) and only 1 lower energy ring conformation/ligand. The Maestro output file was saved for docking after protein preparation. First the PDB file (1dkf) was imported with mixed hydrogens shown and pre-processing of assigning bond orders, adding hydrogens, creating zero-order bonds to metals, creating disulfide bonds, and deleting waters molecules that were more than 5 Å from the groups. After pre-process, Epik was run with states generated, and protein assignment was run by optimizing sample water orientation. For restrained minimization, force field OPLS 2001 was used. Next the protein grid was generated. To this end the prepped protein was loaded with only the co-crystallized ligand 2b selected in the workspace. Glide Receptor Grid Generation was run using Van der Waals scaling factor of 1.0 and partial charge cutoff of 0.25 to soften the potential for nonpolar parts of the receptor. To complete the actual docking, the MAEGZ file output file from LigPrep was loaded, Glide Ligand Docking was conducted using standard precision with sampling of nitrogen inversions, sampling of ring conformations, bias sampling with penalization of non-planar amide conformations, and Epik state penalties added. A maximum of 10 poses per ligand with post-docking minimization were allowed. The top docking score (i.e., the most negative score) of each ligand where the carboxylic acid and antagonist pocket moieties showed overlap with the experimental ligand 2b is reported in Table S1.

4.10. General chemistry

NMR spectroscopy was conducted using a 400 MHz instrument equipped with a BBO broadband probe. Chemical shifts are reported in ppm and referenced to residual solvent peaks: CHCl₃ in CDCl₃: 7.26 ppm for ¹H NMR spectroscopy, 77 ppm for ¹³C NMR spectroscopy. THF in THF-d₈: 3.58 & 1.72 ppm for ¹H NMR spectroscopy, 67.21 & 25.31 ppm for ¹³C NMR spectroscopy. DMSO in DMSO-d₆: 2.50 ppm for ¹H NMR spectroscopy, 39.52 ppm for ¹³C NMR spectroscopy. MeOH in MeOH-d₄: 3.31 ppm for ¹H NMR spectroscopy, 49.00 ppm for ¹³C NMR spectroscopy. Coupling constants are reported in Hertz. Splitting patterns are designated as singlet (s), doublet (d), triplet (t), quartet (q), and broad singlet (br s). All reactions were performed with anhydrous solvent unless noted. Anhydrous solvents were degassed with N₂ and passed through a column of activated alumina or molecular sieves. Purity of final compounds were determined by HPLC/MS. The analytical methods for purity determination by HPLC/MS are provided in the SI.

4.11. Materials

All starting materials were purchased from commercial vendors and used without further purification unless noted. Compounds 1 and 2b are commercially available. Compound 2a was prepared according to the published procedure [15]. The NMR data for 1, 2a and 2b were in agreement with their structures. HPLC analysis showed that the compound 2a was >99% pure. Compound 1 was 98% pure and compound 2b was 97% pure.

4.12. Synthesis of analogs

Synthesis of analogs 2a-4, 6-8, 15 from Scheme 1

Synthesis of compound 3

Ethyl 4-(5,5-Dimethyl-8-phenyl-5,6-dihydronaphthalene-2-carboxamido)benzoate (37).

To a vial with a stir bar was added 5,5-dimethyl-8-phenyl-5,6-dihydronaphthalene-2-carboxylic acid (33) [39, 40] (160 mg, 0.575 mmol, 1.0 equiv), ethyl 4-aminobenzoate (142 mg, 0.862 mmol, 1.5 equiv), EDCI-HCl (220 mg, 1.15 mmol, 2.0 equiv), HOBt (92 mg, 0.60 mmol, 1.05 equiv), and DMAP (176 mg, 1.44 mmol, 2.5 equiv). DCM (2 mL) was added, and the resultant solution was stirred for 16 h at 35 °C. The reaction was quenched with a saturated aqueous solution of NaHCO₃ and extracted 3 times with DCM. The combined organics were washed with an aqueous 1M solution of HCl, dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 7:3-1:1-3:7 to remove excess starting amine (yellow by TLC with anisaldehyde stain) and carried on to the final saponification without further purification. TLC R_f (hexanes:EtOAc, 87:3) 0.45. ¹H NMR (400 MHz, CDCl₃) δ 8.19-8.07 (m, 1H), 8.06-8.00 (m, 1H), 7.94-7.73 (m, 2H), 7.65 (d, J = 8.8 Hz, 1H), 7.61 (d, J = 8.1 Hz, 1H), 7.55-7.49 (m, 2H), 7.40-7.37 (m, 4H), 6.09 (t, J = 4.7 Hz, 1H), 4.37 (q, J = 7.1 Hz, 2H), 2.41 (d, J = 4.7 Hz, 2H), 1.56 (s, 1H), 1.41-1.37 (m, 9H).

4-(5,5-Dimethyl-8-phenyl-5,6-dihydronaphthalene-2-carboxamido)benzoic Acid (3).

To a vial with stir bar was added ethyl 4-(5,5-dimethyl-8-phenyl-5,6-dihydronaphthalene-2-carboxamido)benzoate (37) (180 mg, 0.423 mmol, 1.0 equiv) and EtOH (8 mL). NaOH (1 M in water, 2.12 mL, 2.12 mmol, 5.0 equiv) was added and the clear solution became cloudy. The mixture was stirred for 16 h at r.t. before acidifying with an aqueous 1 M solution of HCl. The biphasic mixture was extracted 3 times with EtOAc. The combined organics were dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified via silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (DCM:MeOH, 99:1-9:1) to yield 42 mg (18% over 2 steps) as a colorless solid; mp 227 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.20. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 8.7 Hz, 2H), 7.78 (s, 1H), 7.76-7.72 (m, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.54-7.33 (m, 7H), 6.08 (t, J = 4.5 Hz, 1H), 2.40 (d, J = 4.6 Hz, 2H), 1.38 (s, 6H), 0.88 (s, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 165.8, 149.9, 142.9, 140.2, 138.8, 134.6, 132.1, 131.6, 128.6, 128.6, 127.8, 127.6, 126.3, 124.5, 124.3, 119.1, 38.6, 34.0, 28.0. LRMS (m/z ESI⁺) calculated for C₂₆H₂₄NO₃ [M+H]⁺: 398.2, found, [M+H]⁺: 398.4. Purity >97%.

Synthesis of compound 4

7-Bromo-4,4-dimethyl-1-(4-morpholinophenyl)-1,2,3,4-tetrahydronaphthalen-1-ol (27).

A flask with stir bar and magnesium turnings (208 mg, 8.57 mmol, 1.35 equiv) was flame dried under reduced pressure. Two crystals of iodine were added and the flask was re-purged with N₂. A solution of 4-(4-bromophenyl)morpholine (2 g, 8 mmol, 1.30 equiv) in THF (18 mL) was added and the mixture was heated to ebullition before stirring at r.t. When only small, blackened magnesium remained (approximately 1 h), the solution was then chilled to 0 °C and a solution of 7-bromo-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one (1.61 g, 6.25 mmol, 1.0 equiv) in THF (6 mL) was added dropwise. The reaction was allowed to stir for 16 h at r.t. before quenching with saturated aqueous NH₄Cl. The remaining magnesium was dissolved with 1M HCl and the resulting biphasic solution was extracted 3 times with EtOAc. The combined organics were washed with brine, dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude yellow oil (a mixture of desired alcohol and the dehydrated product from the next step) was immediately taken on to complete dehydration. TLC R_f (hexanes:EtOAc, 7:3) 0.325.

4-(4-(7-Bromo-4,4-dimethyl-3,4-dihydronaphthalen-1-yl)phenyl)morpholine (30).

To the residue from above was added benzene (20 mL), p-toluenesulfonic acid monohydrate (157 mg, 0.826 mmol, 0.13 equiv), and one scoop of flame-dried, crushed 4 Å molecular sieves. The flask was equipped with a condenser, and the mixture was stirred at 80 °C for 4 h. After cooling to r.t., brine was added, and the biphasic mixture was extracted 3 times with EtOAc. The combined organics were washed with saturated aqueous NaHCO₃, dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified via silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (hexanes:EtOAc, 8:2-65:35) to yield 2.21 g (87%) as an oily colorless solid. TLC R_f (hexanes:EtOAc, 7:3) 0.40. ¹H NMR (400 MHz, CDCl₃) δ 7.38 (dd, J = 8.2, 2.1 Hz, 1H), 7.34-7.31 (m, 3H), 7.24 (d, J = 8.2 Hz, 1H), 6.96-6.94 (m, 2H), 6.02 (t, J = 4.7 Hz, 1H), 3.91-3.86 (m, 4H), 3.20-3.17 (m, 4H), 2.35 (d, J = 4.7 Hz, 2H), 1.36 (s, 6H).

5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalene-2-carboxylic Acid (34).

To a flame dried vial with stir bar was added bromide 30 (500 mg, 1.26 mmol, 1.0 equiv) and THF (5 mL) and purged with N₂. The solution was chilled to −78 °C before adding tert-butyllithium (1.7 M in pentane, 1.45 mL, 2.46 mmol, 1.96 equiv) and stirring for 20 min at −78 °C. Next CO₂ (from dry ice off-gassing through a Drierite drying tube) was bubbled under the surface of the solution (using 6-inch CO₂ inlet needle and ensuring vent needle outlet) for 10 min. The reaction was allowed to stir under a CO₂ atmosphere for 15 min at −78 °C before purging with N₂ and allowed to slowly warm to r.t. The solution was concentrated and acidified with 1 M HCl before extracting 3 times with EtOAc. The combined organics were dried over MgSO₄, filtered, and concentrated. The resultant residue was purified via silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (hexanes:EtOAc, 7:3) to yield the desired product (207 mg, 45%) as a light-yellow solid. TLC R_f (hexanes:EtOAc, 1:1) 0.075.

Methyl 4-(5,5-dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalene-2-carboxamido)benzoate (38).

To a stirring solution of acid 34 (542 mg, 1.49 mmol) dissolved in DCM (5 mL) was added oxalyl chloride (379 mg, 2.98 mmol, 2 equiv) at 0 °C followed by 2 drops of DMF. The resulting mixture was allowed to warm up to r.t. over 1 h. The reaction mixture was evaporated under reduced pressure and the crude reaction mixture was re-dissolved in in DCM (3 mL). The reaction mixture was cooled to 0 °C and Et₃N (0.41 mL, 2.98 mmol, 2 equiv) was added followed by the addition of methyl 4-aminobenzoate (225 mg, 1.49 mg, 1 equiv). The resulting reaction mixture was stirred at rt for 1 h. Upon the completion of the reaction as monitored by TLC, the reaction mixture was partitioned between an aqueous solution of 1M HCl (4 mL) and ethyl acetate (10 mL). The organic layer was washed with saturated NaHCO₃ (3 x 4 mL), water (4 mL) and brine (4 mL). The organic layer was dried over Na₂SO₄, and the solvent was removed under reduced pressure. The crude material was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography to obtain titled compound. Yield not determined; mp 226 – 229 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.97 -7.92 (m, 2H), 7.70 (s, 1H), 7.64 (dd, J = 8.0, 2.0 Hz, 1H), 7.61-7.55 (m, 2H), 7.50 (d, J = 2.0 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.23-7.19 (m, 2H), 6.93 −6.83 (m, 2H), 5.96 (t, J = 4.7 Hz, 1H), 3.83 (s, 3H), 3.82-3.78 (m, 4H), 3.14 (t, J = 4.0 Hz, 4H), 2.30 (d, J = 4.7 Hz, 2H), 1.29 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 166.7, 166.1, 149.4, 150.1, 142.3, 138.5, 134.9, 132.3, 131.7, 130.9, 129.5, 126.9, 126.2, 125.9, 124.6, 124.5, 119.3, 115.7, 113.9, 67.1, 52.2, 49.4, 38.7, 34.2, 28.1.

4-(5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalene-2-carboxamido)benzoic Acid (4).

To a stirring solution of methyl ester 38 (20.0 mg, 0.040 mmol) dissolved in anhydrous ethanol (0.49 mL) was added NaOH (1 M, 0.40 mL, 10 equiv) at r.t. and the resulting mixture was left stirring for 48 h until the disappearance of starting material as monitored by TLC. The crude reaction mixture was acidified to pH ~4 using 1M HCl solution. The solvent was then removed under reduced pressure and the crude product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (0-20% MeOH in DCM) to obtain the titled compound as a cream-colored solid (15 mg, 77%); mp ≥ 250 °C (compound started changing color after this temperature from cream-colorless to black); ¹H NMR (400 MHz, THF-d₈) δ 9.46 (s, 1H), 7.96- 7.91 (m, 2H), 7.80- 7.77 (m, 2H), 7.75 (dd, J = 8.1, 2.0 Hz, 1H), 7.67 (d, J = 1.9 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.25- 7.20 (m, 2H), 6.98 −6.92 (m, 2H), 5.99 (t, J = 4.7 Hz, 1H), 3.79- 3.74 (m, 4H), 3.17- 3.11 (m, 4H), 2.35 (d, J = 4.7 Hz, 2H), 1.35 (s, 6H). ¹³C NMR (101 MHz, THF-d₈) δ 167.5, 166.8, 152.1, 150.0, 144.8, 140.2, 135.2, 134.5, 132.4, 131.4, 130.0, 127.5, 126.7, 126.5, 126.2, 124.7, 119.8, 116.1, 50.2, 39.6, 34.8, 30.8, 28.4. LRMS m/z (ESI⁻) calculated for C₃₀H₂₉N₂O₄ [M-H]⁻: 481.2, found, [M-H]⁻: 481.3. Purity 96%.

Synthesis of compound 6

2-(7-Bromo-4,4-dimethyl-3,4-dihydronaphthalen-1-yl)thiophene (31).

Commercially available thiophen-2-ylmagnesium bromide (1 M in THF, 7.1 mmol, 7.1 mL, 1.2 equiv) was added to a flask and cooled to 0 °C. A solution of commercially available 7-bromo-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one (1.5 g, 5.9 mmol, 1 equiv) in anhydrous THF (15 mL) was added dropwise. The reaction mixture was allowed to warm to r.t. overnight. The solution was acidified to pH 3 using an aqueous 1 M solution of HCl, and the reaction mixture was extracted 3 times with EtOAc. The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The resulting oil was further dried via high vacuum. Alcohol intermediate 28 was combined with catalytic 4-toluenesulfonic acid (110 mg, 0.59 mmol, 0.1 equiv) and heat-dried 4 Å molecular sieves. Dry benzene (15 mL) was added to the mixture and heated to reflux overnight. After cooling the reaction mixture to r.t., it was rinsed with brine and extracted 3 times with EtOAc. The combined organic extracts were rinsed with a saturated aqueous solution of NaHCO₃ and dried using Na₂SO₄, before being condensed under reduced pressure. The product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 0:1-1:10), providing target thiophene 31 as a yellow solid in 30% yield over two steps (550 mg); mp 189.7 °C (decomp). ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J = 1.9 Hz, 1H), 8.00 (dd, J = 8.0, 1.9 Hz, 1H), 7.48 (d, J = 8.1 Hz, 1H), 7.31 (d, J = 1.6 Hz, 1H), 7.14-7.07 (m, 2H), 6.26 (t, J = 4.8 Hz, 1H), 2.39 (d, J = 4.8 Hz, 2H), 1.37 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 151.2, 141.9, 133.5, 131.9, 129.8, 128.6, 127.4, 127.3, 126.8, 125.7, 124.3, 124.2, 38.4, 34.0, 27.9.

5,5-Dimethyl-8-(thiophen-2-yl)-5,6-dihydronaphthalene-2-carboxylic Acid (35).

Bromo dihydronaphthalene 31 (320 mg, 1.0 mmol, 1 equiv) was dissolved in anhydrous THF (10 mL) and cooled to −78 °C in a dry ice/acetone bath. A solution of tert-butyllithium (1.1 mL, 2.0 mmol, 1.9 M in pentanes, 2 equiv) was added dropwise. The reaction mixture was allowed to stir for 15 min at −78 °C. CO₂ was bubbled through the solution through a drying tube for 1 h. The solution was then purged using N₂ and allowed to warm to r.t. overnight. The reaction mixture was then diluted with EtOAc. An aqueous solution of HCl (10%) was added to the solution, and the mixture was allowed to stir for 10 min. The reaction mixture was washed with a saturated aqueous solution of NH₄Cl and extracted 3 times with EtOAc. The combined organic fractions were dried over Na₂SO₄ and condensed under reduced pressure. The residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 1:1) to provide target acid 35 as a beige solid in 49% yield (140 mg); mp 208.3 – 209.8 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.89 (s, 1H), 7.95-7.80 (m, 2H), 7.65-7.48 (m, 2H), 7.24-7.00 (m, 2H), 6.26 (t, J = 4.8 Hz, 1H), 2.36 (d, J = 4.8 Hz, 2H), 1.30 (d, J = 4.3 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.1, 149.6, 141.2, 132.6, 131.2, 129.1, 128.7, 128.6, 127.6, 125.9, 125.8, 125.2, 124.4, 37.7, 33.4, 27.6.

Methyl 4-(5,5-Dimethyl-8-(thiophen-2-yl)-5,6-dihydronaphthalene-2-carboxamido)benzoate (39).

Carboxylic acid 35 (47 mg, 0.17 mmol, 1 equiv) was combined in with methyl 4-aminobenzaote (25 mg, 0.17 mmol, 1 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (63 mg, 0.33 mmol, 2 equiv), and DMAP (50 mg, 0.41 mmol, 2.5 equiv). dCm (5 mL) was added, and the reaction was stirred overnight at 37 °C. After cooling the reaction mixture to r.t., it was rinsed with a saturated aqueous solution of NaHCO₃ and extracted 3 times with EtOAc. The combined organic extracts were dried using Na₂SO₄ before being condensed under reduced pressure. The product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 1:1), providing target amide 39 as a yellow oil in 72% yield (49 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.03 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 1.9 Hz, 1H), 7.81 (s, 1H), 7.76 (dd, J = 8.0, 1.9 Hz, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.1 Hz, 1H), 7.31-7.28 (m, 1H), 7.12-7.05 (m, 2H), 6.27 (t, J = 4.8 Hz, 1H), 3.88 (s, 3H), 2.29 (d, J = 4.8 Hz, 2H), 1.27 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 166.6, 165.7, 149.6, 142.2, 141.8, 133.9, 132.3, 131.9, 130.9, 129.2, 127.4, 126.6, 125.8, 125.7, 124.6, 124.5, 124.2, 119.1, 52.0, 38.5, 33.9, 28.0.

4-(5,5-Dimethyl-8-(thiophen-2-yl)-5,6-dihydronaphthalene-2-carboxamido)benzoic Acid (6).

Amide 39 (48 mg, 0.12 mmol) was dissolved in minimal DCM and combined with NaOH (1 M, 1 mL). MeOH was added until the two layers were emulsified. The reaction was stirred overnight at 37 °C. The reaction mixture was condensed under reduced pressure and the residue was dissolved in DCM and extracted from water. The water layer was then acidified and extracted 3 times with DCM to obtain desired acid 6 as a beige solid in 73% yield (35 mg); mp 174 – 176 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.47 (s, 1H), 7.90 (m, 3H), 7.84 (dd, J = 5.4, 3.4 Hz, 3H), 7.60-7.50 (m, 2H), 7.18-7.13 (m, 2H), 6.29 (d, J = 4.7 Hz, 1H), 2.36 (d, J = 4.9 Hz, 2H), 1.32 (s, 6H). LRMS m/z (ESI⁺) calculated for C₂₄H₂₀NO₃S [M-H]⁻: 402.1, found, [M-H]⁻: 402.2. Purity 98%.

Synthesis of compound 15

Methyl 4-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carboxamido)-2,6-difluorobenzoate (40).

To a vial with stir bar was added 5,5-dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carboxylic acid 36 [39, 40] (52 mg, 0.18 mmol, 1.0 equiv), methyl 4-amino-2,6-difluorobenzoate (40. mg, 0.21 mmol, 1.2 equiv), EDCI-HCl (68 mg, 0.36 mmol, 2.0 equiv), HOBt (29 mg, 0.19 mmol, 1.05 equiv), and DMAP (54 mg, 0.44 mmol, 2.5 equiv). DCM (2 mL) was added, and the reaction mixture was stirred at 37 °C for 16 h before quenching with saturated aqueous NaHCO₃ and extracting 3 times with DCM. The combined organics were washed with an aqueous 1 M solution of HCl, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99.5:0.5) to reveal a clear, colorless oil of the desired product (20 mg, 25% yield). TLC R_f (hexanes:EtOAc, 1:1) 0.70.

4-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carboxamido)-2,6-difluorobenzoic Acid (15).

A vial equipped with stir bar was charged with methyl ester 40 (20 mg, 0.043 mmol, 1.0 equiv), EtOH (1 mL), DCM (1 mL), and 1 M NaOH (0.5 mL). The cloudy mixture was stirred at r.t. for 48 h before extracting 3 times with DCM. The aqueous layer was acidified to pH 1 with an aqueous 1 M solution of HCl before extracting 3 times with DCM. The combined organic fractions from the acidic extractions were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (DCM:MeOH, 98:2-9:1) to reveal a colorless solid as the desired product (17 mg, 88% yield); mp 195 – 196 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.50. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (dd, J = 8.1, 1.8 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 7.46-7.34 (m, 2H), 7.18-7.11 (m, 5H), 7.08-7.00 (m, 1H), 5.94 (t, J = 4.7 Hz, 1H), 2.33 (s, 3H), 2.29 (d, J = 4.7 Hz, 2H), 2.26 (s, 1H), 1.28 (s, 6H). LRMS m/z (ESI⁻) calculated for C₂₇H₂₂F₂NO₃ [M-H]⁻: 446.2, found, [M-H]⁻: 446.2. Purity 99%.

Scheme 2

Synthesis of compound 5

7-Bromo-4,4-dimethyl-3,4-dihydro-2H-spiro[naphthalene-1,2’-[1,3]dioxolane] (41).

To a dry vial with stir bar was added 7-bromo-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one (700 mg, 2.77 mmol, 1.0 equiv), triethyl orthoformate (0.92 ml, 5.5 mmol, 2.0 equiv), and ethylene glycol (1.0 ml, 19 mmol, 7.0 equiv) followed by 4-tosic acid monohydrate (53 mg, 0.28 mmol, 0.10 equiv) and 1 small scoop of activated 4 Å molecular sieves. The mixture was stirred at r.t. for 16 h before washing the organic layer with saturated aqueous NaHCO₃. The aqueous layer was extracted twice with DCM and once with EtOAc. The combined organics were dried over MgSO₄, filtered, and concentrated under vacuum. The residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 8:2). A clear, faintly yellow oil was isolated as desired product (696 mg, 85%). TLC R_f (hexanes:EtOAc, 7:3) 0.53. ¹H NMR (400 MHz, CDCl₃) δ 7.58 (d, J = 2.2 Hz, 1H), 7.39 (dd, J = 8.5, 2.2 Hz, 1H), 7.17 (d, J = 8.5 Hz, 1H), 4.24-4.17 (m, 2H), 4.15-4.05 (m, 2H), 2.02-1.95 (m, 2H), 1.87-1.76 (m, 2H), 1.28 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 145.8, 138.2, 131.9, 129.0, 128.3, 119.7, 106.6, 65.1, 35.7, 33.7, 31.0, 30.1.

5,5-Dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-2-carboxylic Acid (42).

To a flame dried vial with stir bar was added bromide 41 (230 mg, 0.774 mmol, 1.0 equiv) and THF (4 mL) and purged with N₂. The solution was chilled to −78 °C before adding tert-butyllithium (1.6 M in pentane, 0.95 ml, 1.5 mmol, 1.96 equiv) and stirring for 15 min at −78 °C. Next CO₂ (from dry ice off-gassing through Drierite drying tube) was bubbled under the surface of the solution (using 6 inches CO₂ inlet needle and ensuring vent needle outlet) for 10 min. The reaction was allowed to stir under a CO₂ atmosphere for 10 min at −78 °C before purging with N₂ and allowing the reaction mixture to slowly warm to r.t. The yellow solution was diluted with EtOAc (5 mL) before acidifying with a 1 M aqueous solution of HCl (~8 mL) and diluting further with EtOAc (25 mL). The biphasic mixture was stirred for 10 min at r.t. before extracting 3 times with EtOAc. The combined organics were dried over MgSO₄, filtered through Silica gel with 9:1 DCM:MeOH, and concentrated under reduced pressure. The resultant residue was purified via silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (DCM:MeOH, 99:1) to provide a colorless solid was that carried on immediately without further purification. TLC R_f (hexanes:EtOAc, 1:1) 0.15. ¹H NMR (400 MHz, CDCl₃) δ 8.74 (d, J = 1.9 Hz, 1H), 8.22 (dd, J = 8.3, 2.0 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 2.88-2.67 (m, 2H), 2.19-1.98 (m, 2H), 1.43 (s, 6H).

Methyl 5,5-Dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalene-2-carboxylate (43).

Acid 42 (0.26 g, 1.2 mmol, 1.0 equiv) was combined with K₂CO₃ (0.25 g, 1.8 mmol, 1.5 equiv) and iodomethane (0.089 mL, 1.4 mmol, 1.2 equiv) then dissolved in DMSO (10 mL). The reaction mixture was stirred for 30 min at r.t, then quenched with water. The reaction mixture was washed with a saturated aqueous solution of NaHCO₃ and extracted 3 times with EtOAc. The combined organic fractions were dried over Na₂SO₄ and condensed under reduced pressure. The residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 2:10) to provide 178 mg (64%) of methyl ester 43 as a colorless solid; mp 105.1 – 106.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.59 (d, J = 2.0 Hz, 1H), 8.10 (dd, J = 8.3, 2.0 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 3.86 (s, 3H), 2.69 (dd, J = 7.3, 6.3 Hz, 2H), 1.98 (dd, J = 7.4, 6.2 Hz, 2H), 1.35 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 197.4, 166.4, 156.9, 134.3, 131.2, 128.9, 128.5, 126.3, 52.2, 36.7, 35.0, 34.4, 29.6.

Methyl 5,5-Dimethyl-8-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydronaphthalene-2-carboxylate (44).

In a round bottom flask, sodium hexamethyldisilazide (0.90 mL, 1.9 mmol, 2.0 M in THF, 1.12 equiv) was combined with anhydrous THF (10 mL) and cooled to −78 °C. A solution of the tetralone ester methyl 43 (390 mg, 1.7 mmol, 1 equiv) in anhydrous THF (10 mL) was added slowly and the reaction mixture was allowed to stir for 30 min at −78 °C. A solution of Comins’ reagent (N,N-bis(trifluoromethylsulfonyl)-5-chloro-2-pyridylamine) (730 mg, 1.8 mmol, 1.1 equiv) in anhydrous THF (5 mL) was added slowly to the reaction mixture and allowed to stir for 45 min at −78 °C before being allowed to slowly warm to r.t. overnight. The reaction was quenched with a saturated aqueous solution of NH₄Cl and extracted 3 times with EtOAc. The combined organic fractions were washed with a 5% aqueous solution of NaOH, dried over Na₂SO₄, and condensed under reduced pressure. The product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 2:10) to provide triflate 44 as a colorless solid in 52% yield (316 mg); mp 277.8 °C (decomp). ¹H NMR (400 MHz, CDCl₃) δ 8.04-7.84 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 5.97 (t, J = 4.9 Hz, 1H), 3.86 (s, 3H), 2.38 (d, J = 4.8 Hz, 2H), 1.26 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 166.4, 149.7, 144.8, 131.0, 128.7, 127.6, 124.6, 122.6, 120.2 (q, J = 320 Hz), 117.5, 117.0 (q, J = 320 Hz), 52.3, 37.2, 34.2, 28.0. ¹⁹F NMR (376 MHz, CDCl₃) δ −73.45.

Methyl 5,5-Dimethyl-8-(4-(morpholinomethyl)phenyl)-5,6-dihydronaphthalene-2-carboxylate (45).

Triflate 44 (223 mg, 0.612 mmol) was combined in a vial with 4-(4-morpholinomethyl)phenylboronic acid pinacol ester (292 mg, 0.918 mmol, 1.5 equiv), K₃PO₄ (260 mg, 1.22 mmol, 2 equiv), and XPhos Pd G2 (48 mg, 0.061 mmol, 0.1 equiv). THF (2 mL) and degassed water (4 mL) were added to the solution in a 1:2 ratio. The reaction mixture was allowed to stir for 3 h at r.t. The reaction mixture was washed with a saturated aqueous solution of NaHCO₃ and extracted 3 times with EtOAc. The combined organic fractions were dried over Na₂SO₄ and condensed under reduced pressure. The residue was subjected to silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (MeOH). Since the boronic acid coeluted with product, the mixture was used in the next step without any further purification. ¹H NMR (400 MHz, CDCl₃) δ 7.91 (dd, J = 8.0, 1.9 Hz, 1H), 7.73 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.40-7.30 (m, 4H), 6.04 (t, J = 4.7 Hz, 1H), 3.84 (s, 3H), 3.78 (t, J = 4.3 Hz, 4H), 3.58 (s, 2H), 2.54 (d, J = 5.2 Hz, 4H), 2.38 (d, J = 4.8 Hz, 2H), 1.27 (s, 6H).

5,5-Dimethyl-8-(4-(morpholinomethyl)phenyl)-5,6-dihydronaphthalene-2-carboxylic Acid (46).

Methyl ester 45 (330 mg, 0.87 mmol, 1 equiv) was dissolved in minimal DCM and combined with NaOH (1 M, 2 mL). MeOH was added until the two layers were emulsified. The reaction was stirred overnight at 37 °C. The reaction mixture was condensed under reduced pressure and the residue was dissolved in DCM and extracted from water. The water layer was then acidified and extracted 3 times with DCM to obtain the desired acid product as a colorless solid (0.404 g) and was directly used in the next step; mp 155.4-161.3 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.77 (br s, 1H), 7.86-7.79 (m, 1H), 7.55-7.48 (m, 2H), 7.40-7.24 (m, 4H), 6.05 (t, J = 4.6 Hz, 1H), 3.60 (t, J = 4.7 Hz, 4H), 3.52 (s, 2H), 2.40 (s, 4H), 2.35 (d, J = 4.8 Hz, 2H), 1.32 (s, 6H).

Methyl 4-(5,5-dimethyl-8-(4-(morpholinomethyl)phenyl)-5,6-dihydronaphthalene-2-carboxamido)benzoate (47).

Acid intermediate 46 from above was combined with methyl 4-aminobenzoate (404 mg, 1.070 mmol, 1 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (410 mg, 2.14 mmol, 2 equiv), hydroxybenzotriazole (164 mg, 1.07 mmol, 1 equiv), and DMAP (327 mg, 2.68 mmol, 2.5 equiv). DCM (7 mL) was added, and the reaction was stirred overnight at 37 °C. After cooling the reaction mixture to r.t., it was rinsed with a saturated solution of NaHCO₃ and extracted 3 times with EtOAc. The combined organic extracts were dried using Na₂SO₄ before being condensed under reduced pressure. The product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (MeOH:DCM, 0:10-1:10), providing 80 mg (15%) of amide 47 as an oil over the two steps. ¹H NMR (400 MHz, CDCl₃ δ 7.99-7.91 (m, 2H), 7.69 (s, 1H), 7.64 (dd, J = 8.0, 2.1 Hz, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 2.1 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.32-7.22 (m, 4H), 6.00 (t, J = 4.7 Hz, 1H), 3.83 (s, 3H), 3.67 (d, J = 4.6 Hz, 4H), 3.47 (s, 2H), 2.43 (s, 4H), 2.32 (d, J = 4.7 Hz, 2H), 1.30 (s, 6H).

4-(5,5-Dimethyl-8-(4-(morpholinomethyl)phenyl)-5,6-dihydronaphthalene-2-carboxamido)benzoic Acid (5).

Methyl ester 47 (80 mg, 0.20 mmol) was dissolved in minimal DCM and combined with an aqueous NaOH solution (1 M, 1 mL). MeOH was added until the two layers were emulsified. The reaction was stirred overnight at 37 °C. The reaction mixture was condensed under reduced pressure and the residue was dissolved in DCM and extracted from water. The water layer was then acidified and extracted 3 times with DCM to obtain desired acid 5 as a colorless solid in quantitative yield (78 mg); mp 164 – 166 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (br s, 1H), 7.87 (m, 3H), 7.76 (d, J = 7.9 Hz, 2H), 7.55 (d, J = 8.1 Hz, 1H), 7.48 (s, 1H), 7.43-7.24 (m, 4H), 6.09 (t, J = 4.7 Hz, 1H), 3.59 (t, J = 4.5 Hz, 4H), 3.51 (s, 2H), 2.45-2.28 (m, 6H), 1.34 (s, 6H). LRMS m/z (ESI⁻) m/z calculated for C₃₁H₃₁N₂O₄ [M-H]⁻ 495.2, found 495.2. Purity 97%.

Scheme 3. Synthesis of compounds 7 and 8

Synthesis of compound 7

6-Bromo-1,1-dimethyl-4-(p-tolyl)-1,2-dihydronaphthalene (32).

Commercially available 4-tolylmagnesium bromide 32 (8.8 mmol, 11 mL, 0.8 M in THF, 1.2 equiv) was added to a flask and cooled to 0 °C. THF (10 mL) was added to the flask. A solution of 7-bromo-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one (1.8 g, 7.1 mmol) in dry THF (5 mL) was added dropwise. This reaction was allowed to warm to r.t. overnight. The solution was acidified to pH 3 with an aqueous 1 M solution of HCl, and the reaction mixture was extracted 3 times with EtOAc. The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The resulting oil was further dried via high vacuum. Alcohol intermediate 29 was combined with catalytic 4-toluenesulfonic acid (169 mg, 0.887 mmol, 0.13 equiv) and flame-dried 4 Å molecular sieves. Dry benzene (13 mL) was added to the mixture and heated to reflux overnight. After cooling the reaction mixture to r.t., it was washed with brine and extracted 3 times with EtOAc. The combined organic extracts were washed with a saturated solution of NaHCO₃ and dried using Na₂SO₄, before being condensed under reduced pressure. The product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 1:10) providing targeted tolyl intermediate 32 as a colorless solid in 42% yield over two steps (520 mg). ¹H NMR (400 MHz, CDCl₃) δ 7.34 (dd, J = 8.3, 2.0 Hz, 1H), 7.23 (m, 5H), 7.17 (d, J = 2.0 Hz, 1H), 5.99 (t, J = 4.7 Hz, 1H), 2.42 (s, 3H), 2.34 (d, J = 4.7 Hz, 2H), 1.33 (s, 6H). Consistent with published data [15].

5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carboxylic Acid (36).

Bromide 32 (620 mg, 1.9 mmol, 1 equiv) was dissolved in anhydrous THF (3 mL) and cooled to −78 °C in a dry ice/acetone bath. A solution of tert-butyllithium (1.6 M in pentanes, 2.3 mL, 2 equiv) was added dropwise. The reaction mixture was allowed to stir for 15 min at −78 °C. CO₂ was bubbled through the solution through a drying tube for 1 h. The solution was then purged using N₂ and allowed to warm to r.t. overnight. The reaction mixture was then diluted with EtOAc. An aqueous solution of HCl (10%) was added to the solution, and the mixture was allowed to stir for 10 min. The reaction mixture was washed with a saturated aqueous solution of NH₄Cl and extracted 3 times with EtOAc. The combined organic fractions were dried over Na₂SO₄ and condensed under reduced pressure. The residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 1:1) to provide a fluffy colorless solid in 32% yield (180 mg). ¹H NMR (400 MHz, DMSO-d₆) δ 12.77 (s, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.54-7.44 (m, 2H), 7.28-7.09 (m, 4H), 6.00 (t, J = 4.7 Hz, 1H), 2.36 (s, 3H), 2.33 (d, J = 4.8 Hz, 2H), 1.30 (s, 6H). Consistent with published data [15].

Methyl 4-((5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carboxamido)methyl)benzoate (49).

To a vial with stir bar was added acid 36 (212 mg, 0.725 mmol, 1.0 equiv), methyl 4-(aminomethyl)benzoate hydrochloride (220 mg, 1.09 mmol, 1.5 equiv), EDCI-HCl (278 mg, 1.45 mmol, 2.0 equiv), HOBt (117 mg, 0.761 mmol, 1.05 equiv), and DMAP (310 mg, 2.54 mmol, 3.5 equiv). DCM (3 mL) was added, and the resultant yellow solution was stirred for 16 h at 35 °C. The reaction was quenched with a saturated aqueous solution of NaHCO₃ and extracted 3 times with DCM. The combined organic fractions were washed with an aqueous 1 M HCl solution, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 6:4) to yield a colorless solid as desired product (268 mg, 84%). TLC R_f (hexanes:EtOAc 1:1) 0.55. ¹H NMR (400 MHz, CDCl₃) δ 8.01-7.94 (m, 2H), 7.68 (dd, J = 8.0, 1.9 Hz, 1H), 7.46 (d, J = 1.9 Hz, 1H), 7.43 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.2 Hz, 2H), 7.21 (q, J = 8.1 Hz, 4H), 6.31 (t, J = 5.5 Hz, 1H), 6.01 (t, J = 4.7 Hz, 1H), 4.63 (d, J = 6.0 Hz, 2H), 3.91 (s, 3H), 2.39 (s, 3H), 2.35 (d, J = 4.7 Hz, 2H), 1.34 (s, 6H).

4-((5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carboxamido)methyl)benzoic Acid (7).

To a vial with stir bar was added methyl ester 49 (100 mg, 0.228 mmol, 1.0 equiv) and EtOH (4 mL). An aqueous 1 M solution of NaOH (1.14 mL, 1.14 mmol, 5.0 equiv) was added. The solution was stirred for 16 h at r.t. before acidifying with an aqueous 1 M solution of HCl and extracting 3 times with EtOAc. The combined organics were washed with brine, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified via recrystallization from DCM:hexanes to yield 90 mg (93%) of a colorless solid as desired product; mp 216 °C (223 decomp). TLC R_f (hexanes:EtOAc, 1:1) 0.225. ¹H NMR (400 MHz, CDCl₃) δ 10.5 (br s, 1H), 8.03 (d, J = 8.2 Hz, 2H), 7.70 (dd, J = 8.0, 1.9 Hz, 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.26-7.18 (m, 4H), 6.44 (s, 1H), 6.03 (t, J = 4.7 Hz, 1H), 4.65 (d, J = 5.9 Hz, 2H), 2.39 (s, 3H), 2.36 (d, J = 4.7 Hz, 2H), 1.35 (s, 6H). LRMS m/z (ESI⁺) calculated for C₂₈H₂₈NO₃ [M+H]⁺: 426.2, found 426.3. Purity >99%.

Synthesis of compound 8

Methyl 4-((5,5-Dimethyl-8-phenyl-5,6-dihydronaphthalene-2-carboxamido)methyl)benzoate (48).

To a vial with stir bar was added carboxylic acid 33 (266 mg, 0.956 mmol, 1.0 equiv), methyl 4-aminomethyl)benzoate hydrochloride (288 mg, 1.43 mmol, 1.5 equiv), EDCI-HCl (366 mg, 1.91 mmol, 2.0 equiv), HOBt (153 mg, 1.00 mmol, 1.05 equiv), and DMAP (409 mg, 3.34 mmol, 3.5 equiv). DCM (4 mL) was added, and the resultant yellow solution was stirred for 16 h at 35 °C. The reaction was quenched with saturated aqueous NaHCO₃ and extracted 3 times with DCM. The combined organic fractions were washed with an aqueous 1 M solution of HCl, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 6:4) to yield a colorless solid as desired product (371 mg, 91%) mp 97-99 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.625. ¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J = 8.2 Hz, 2H), 7.68 (dd, J = 8.0, 1.9 Hz, 1H), 7.48 (d, J = 1.8 Hz, 7H), 7.41 (d, J = 8.0 Hz, 1H), 7.39-7.26 (m, 1H), 6.58 (s, 1H), 6.03 (t, J = 4.7 Hz, 1H), 4.57 (d, J = 5.8 Hz, 2H), 3.88 (s, 3H), 2.36 (d, J = 4.7 Hz, 2H), 1.35 (s, 6H).

4-((5,5-Dimethyl-8-phenyl-5,6-dihydronaphthalene-2-carboxamido)methyl)benzoic Acid (8).

To a vial with stir bar was added methyl ester 48 (180 mg, 0.423 mmol, 1.0 equiv) and EtOH (5 mL). NaOH (1 M in water, 2.1 mL, 2.1 mmol, 5.0 equiv) was added. The solution was stirred for 16 h at r.t. before acidifying with an aqueous 1 M solution of HCl and extracting 3 times with diethyl ether. The combined organics were washed with brine, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc 6:4-3:7) to yield 147 mg (85%) of a colorless solid as desired product; mp 162 – 164 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.21. ¹H NMR (400 MHz CDCl₃) δ 8.03 (m, 2H), 7.69 (dd, J = 8.0, 2.0 Hz, 1H), 7.48-7.39 (m, 2H), 7.43-7.29 (m, 7H), 6.32 (t, J = 5.7 Hz, 1H), 6.04 (t, J = 4.7 Hz, 1H), 4.65 (d, J = 6.0 Hz, 2H), 2.37 (d, J = 4.7 Hz, 2H), 1.35 (s, 6H). LRMS m/z (ESI⁻) calculated for C₂₇H₂₄NO₃ [M-H]⁻: 410.2, found 410.3. Purity >96%.

Scheme 4. Synthesis of compounds 9-11

Synthesis of Compound 9

5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-amine (51).

To a dry vial equipped with stir bar was added bromide 32 (104 mg, 0.319 mmol, 1.0 equiv), sodium azide (250 mg, 3.83 mmol, 12.0 equiv), L-proline (33 mg, 0.287 mmol, 0.90 equiv), sodium hydroxide (12 mg, 0.309 mmol, 0.97 equiv), and copper (I) iodide (55 mg, 0.287 mmol, 0.90 equiv). The vial was sealed with a cap with inlaid septum, purged with N₂, and the charged with DMSO (4.5 mL) and EtOH (2.25 mL). The resultant suspension was stirred at 110 °C for 10 h. The brown mixture was cooled to r.t. and water was added before extracting 3 times with EtOAc (avoiding the solids). The combined organics were dried over MgSO₄, filtered through cotton, and concentrated under reduced pressure. The brown oil was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (hexanes:EtOAc, 8:2) to yield 68 mg (81%) of desired product as a yellow oil. TLC R_f (hexanes:EtOAc, 7:3) 0.48, blue under UV light.¹H NMR (400 MHz, CDCl₃) δ 7.16-7.04 (m, 5H), 6.46 (dd, J = 8.1, 2.4 Hz, 1H), 6.29 (d, J = 2.3 Hz, 1H), 5.83 (t, J = 4.7 Hz, 1H), 3.35 (br s, 2H), 2.30 (s, 3H), 2.20 (d, J = 4.7 Hz, 2H), 1.20 (s, 6H).

Ethyl 4-(3-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)ureido)benzoate (54).

To aniline 51 (50 mg, 0.189 mmol, 1.0 equiv) was added THF (1 mL). The solution was chilled to 0 °C before adding dropwise a solution of ethyl 4-isocyanatebenzoate (37 mg, 0.194 mmol, 1.02 equiv in 1 ml of THF). The reaction was warmed to r.t. and stirred for 24 h. The solution was filtered through Silica gel and concentrated under vacuum. The residue was carried on without further purification. TLC R_f (hexanes:EtOAc, 7:3) 0.30; blue under UV light. ¹H NMR (400 MHz, CDCl₃) δ 7.66 (d, J = 8.2 Hz, 2H), 7.33 (s, 1H), 7.12-7.03 (m, 4H), 7.01 (s, 1H), 6.97 (d, J = 7.8 Hz, 2H), 6.87 (d, J = 7.6 Hz, 2H), 6.71 (s, 1H), 5.74 (t, J = 4.4 Hz, 1H), 3.57 (ddd, J = 6.6, 4.2, 2.5 Hz, 2H), 1.71-1.63 (m, 3H), 1.17 (t, J = 7.1 Hz, 3H), 1.09 (s, 6H).

4-(3-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)ureido)benzoic Acid (9).

Ethyl ester 54 was mixed with THF (1 mL), MeOH (1 mL), and water (1 mL). Lithium hydroxide monohydrate (16 mg, 0.378 mmol, 2.0 equiv) was added, and the mixture was stirred at r.t. for 16 h. The solution was then acidified to pH ~1 with 1M HCl and extracted 3 times with EtOAc. The combined organics were washed with brine, dried over MgSO₄, filtered, and concentrated. The residue was purified via silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (hexanes:EtOAc, 8:2 to 1:1 to 2:8) to yield 55 mg (88%) of the desired product as a cream-colored solid; mp 152 °C (decomp). TLC R_f (DCM:MeOH, 9:1) 0.375. ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J = 8.7 Hz, 2H), 7.39-7.29 (m, 4H), 7.19 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 7.01 (s, 1H), 6.90 (s, 1H), 6.84 (d, J = 2.2 Hz, 1H), 5.98 (t, J = 4.7 Hz, 1H), 2.37-2.30 (m, 5H), 1.32 (s, 6H), 0.92-0.82 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 175.7, 166.8, 153.2, 142.7, 138.6, 137.5, 137.0, 134.8, 130.8, 129.1, 128.5, 127.2, 125.1, 124.6, 118.5, 60.5, 52.0, 38.9, 33.4, 28.2, 21.1, 14.2. LRMS m/z (ESI⁺) calculated for C₂₇H₂₇N₂O₃ [M+H]⁺: 427.2, found 427.2, [M+H]⁺. Purity 80%.

Synthesis of Compound 10

6-Bromo-1,1-dimethyl-4-phenyl-1,2-dihydronaphthalene (50).

To a solution of 7-bromo-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one (1.5 g, 5.93 mmol) in THF (24 mL) under N₂ at 0 °C was added phenylmagnesium bromide (1 M solution in THF, 7.7 mL, 7.7 mmol, 1.3 equiv). After stirring at r.t. for 3 h, the reaction was quenched with a saturated NH₄Cl solution, and extracted 3 times with diethyl ether. The organic extract was washed with brine, then dried over MgSO₄, and then the diethyl ether was removed under reduced pressure. The resultant 7-bromo-4,4-dimethyl-1-phenyl-1,2,3,4-tetrahydronaphthalen-1-ol was obtained as a viscous yellow oil in quantitative yield and taken directly into the next step. 7-Bromo-4,4-dimethyl-1-phenyl-1,2,3,4-tetrahydronaphthalen-1-ol (1.96 g), 5.93 mmol) was dissolved in benzene (18 mL). After addition of a catalytic amount of 4-toluensulfonic acid, the reaction was heated to reflux for 4 h, and then cooled to rt. Brine was added to the mixture, the organic phase was separated, washed with dilute NaHCO₃ solution and then with brine. The biphasic mixture was extracted 3 times with EtOAc. The combined organics were washed with saturated aqueous NaHCO₃, dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified via silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (hexanes:EtOAc, 85:15) to yield 1.28 g (69%) of 50 as a slightly yellow oil.

5,5-Dimethyl-8-phenyl-5,6-dihydronaphthalen-2-amine (52).

Prepared from 6-bromo-1,1-dimethyl-4-phenyl-1,2-dihydronaphthalene according to the procedure for the synthesis of aniline 51. Yield 83%, obtained as a dark yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.26-7.20 (m, 5H), 7.04 (d, J = 8.0 Hz, 1H), 6.43 (d, J = 8.0 Hz, 1H), 6.25 (s, 1H), 5.84 (t, J = 4.7 Hz, 1H), 3.31 (br s, 2H), 2.20 (d, J = 3.4 Hz, 2H), 1.19 (s, 6H). ¹³C NMR (400 MHz, CDCl₃) δ 144.2, 141.3, 139.4, 135.6, 134.7, 128.9, 128.2, 126.9, 126.9, 124.6, 114.1, 113.5, 39.4, 33.0, 28.5.

Ethyl 4-(3-(5,5-Dimethyl-8-phenyl-5,6-dihydronaphthalen-2-yl)ureido)benzoate (55).

To a vial containing a stir bar and cap with inlaid septum was added aniline 52 (266 mg, 1.07 mmol, 1.0 equiv) and THF (5 mL). The solution was purged with N₂ and chilled to 0 °C. Next a solution of ethyl 4-isocyanatobenzoate (208 mg, 1.09 mmol, 1.02 equiv) in THF (5 mL) was added slowly. The reaction was allowed to warm to r.t. and stirred for 16 h. The solvent was removed under vacuum and the remaining residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 67:33) to yield a brown solid as desired product (372 mg, 79%) that was used in the next step without further characterization. TLC R_f (hexanes:EtOAc, 1:1) 0.55.

4-(3-(5,5-Dimethyl-8-phenyl-5,6-dihydronaphthalen-2-yl)ureido)benzoic Acid (10).

To a vial containing stir bar was added ethyl ester 55 (366 mg, 0.831 mmol, 1.0 equiv), EtOH (4.2 mL), and 1 M NaOH (4.2 ml). The suspension was stirred for 16 h at r.t. before extracting 3 times with DCM. The aqueous layer was acidified with 1 M HCl and extracted 3 times with EtOAc. The combined EtOAc extracts were dried over MgSO₄, filtered, and concentrated under vacuum. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (99:1 to 98:2 CH₂Cl₂:MeOH) to yield a tan solid as desired product (167 mg, 49%); mp 170 °C (237 °C decomp). TLC R_f (hexanes:EtOAc, 1:1) 0.10. ¹H NMR (400 MHz, MeOH-d₄) δ 7.96-7.92 (m, 2H), 7.51-7.46 (m, 3H), 7.40 (d, J = 6.4 Hz, 2H), 7.35 (d, J = 7.0 Hz, 4H), 6.91 (d, J = 2.3 Hz, 1H), 6.00 (t, J = 4.6 Hz, 1H), 3.37 (s, 1H), 3.36 (s, 1H), 2.36 (d, J = 4.6 Hz, 2H), 1.35 (s, 6H). LRMS m/z (ESI⁺) calculated for C₂₆H₂₅N₂O₃ [M+H]⁺: 413.2, found 413.2. Purity >99%.

Synthesis of Compound 11

5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalen-2-amine (53).

Arylbromide 30 (450 mg, 1.21 mmol, 1.0 equiv), NaN₃ (940 mg, 14.46 mmol, 12.0 equiv), L-proline (124 mg, 1.08 mmol, 0.90 equiv), sodium hydroxide (47 mg, 1.17 mmol, 0.97 equiv), and copper (I) iodide (206 mg, 1.08 mmol, 0.90 equiv). The vial was sealed with a cap with inlaid septum, purged with N₂, and the charged with DMSO (13 mL) and EtOH (6.5 mL). The resultant suspension was stirred at 110 °C for 10 h. The brown mixture was cooled to r.t. and brine added before extracting 3 times with EtOAc (avoiding the solids). The combined organics were dried over MgSO₄, filtered through cotton, and concentrated under reduced pressure. The resultant residue was purified via silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash chromatography (hexanes:EtOAc, 1:1) to yield 241 mg (60% over 2 steps) as a light yellow oil. TLC R_f (hexanes:EtOAc, 1:1) 0.20. ¹H NMR (400 MHz, CDCl₃) δ 7.31-7.25 (m, 1H), 7.26-7.24 (m, 1H), 7.14 (d, J = 8.1 Hz, 1H), 6.92 (d, J = 8.7 Hz, 2H), 6.56 (dd, J = 8.1, 2.5 Hz, 1H), 6.42 (d, J = 2.5 Hz, 1H), 5.91 (t, J = 4.7 Hz, 1H), 3.96-3.84 (m, 4H), 3.45 (s, 2H), 3.30-3.08 (m, 4H), 2.28 (d, J = 4.7 Hz, 2H), 1.29 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 150.2, 144.1, 138.8, 135.7, 134.9, 132.8, 129.6, 126.1, 124.6, 115.3, 113.9, 113.5, 67.0, 49.4, 39.3, 33.0, 28.4.

Ethyl 4-(3-(5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalen-2-yl)ureido)benzoate (56).

To a vial containing a stir bar and cap with inlaid septum was added aniline 53 (388 mg, 1.16 mmol, 1.0 equiv) and 5 mL THF. The solution was purged with N₂ and chilled to 0 °C. Next a solution of ethyl 4-isocyanatobenzoate (226 mg, 1.18 mmol, 1.02 equiv) in THF (5 mL) was added slowly. The reaction was allowed to warm to r.t. and stirred for 16 h. The solvent was removed under reduced pressure and the remaining residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 67:33) to yield a brown solid as desired product (383 mg, 63%); mp 236 – 238 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.18. ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J = 8.7 Hz, 2H), 7.55 (s, 1H), 7.46-7.27 (m, 5H), 7.21 (d, J = 8.6 Hz, 2H), 7.04-6.90 (m, 2H), 6.68 (s, 1H), 5.94 (t, J = 4.6 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.01-3.83 (m, 4H), 3.25-3.09 (m, 4H), 2.27 (d, J = 4.6 Hz, 2H), 1.37 (t, J = 7.1 Hz, 3H), 1.28 (s, 6H).

4-(3-(5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalen-2-yl)ureido)benzoic Acid (11).

To a vial containing stir bar was added ethyl ester 56 (383 mg, 0.729 mmol, 1.0 equiv), EtOH (3.6 mL), and 1 M aqueous NaOH (3.6 ml). The suspension was stirred for 16 h at r.t. before adding water and extracting 3 times with EtOAc. The aqueous layer was acidified with 1 M HCl and extracted 3 times with EtOAc. The combined EtOAc extracts were dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99:1-9:1) to yield a cream colored solid as desired product (118 mg, 33%); mp 226 °C (228 °C decomp). TLC R_f (hexanes:EtOAc, 1:1) 0.16. ¹H NMR (400 MHz, CDCl₃) δ 9.66 (br s, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 8.7 Hz, 2H), 7.38-7.27 (m, 2H), 7.23 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 8.6 Hz, 3H), 6.80 (s, 1H), 6.59 (s, 1H), 5.99 (t, J = 4.7 Hz, 1H), 3.88-3.75 (m, 4H), 3.19-3.02 (m, 4H), 2.34 (d, J = 4.7 Hz, 2H), 1.33 (s, 6H). HRMS m/z (ESI⁺) calculated for C₃₀H₃₂N₃O₄ [M+H]⁺: 498.2387, Found 498.2404. LRMS (ESI⁺) Calculated for C₃₀H₃₂N₃O₄ [M+H]⁺: 498.2, found 498.2. Purity 99%.

Scheme 5. Synthesis of compounds 12, 16 and 17

Synthesis of Compound 12

Methyl 4-((5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)benzoate (57).

To a vial with stir bar was added 4-(methoxycarbonyl)benzoic acid (37 mg, 0.21 mmol, 1.0 equiv), aniline 53 (76 mg, 0.227 mmol, 1.1 equiv), EDCI-HCl (60 mg, 0.32 mmol, 1.5 equiv), HOBt (37 mg, 0.239 mmol, 1.05 equiv), and DMAP (51 mg, 0.42 mmol, 2.0 equiv). DCM (1 mL) was added and the resultant yellow solution was stirred for 16 h at 35 °C. The reaction was quenched with saturated aqueous NaHCO₃ and extracted 3 times with DCM. The combined organics were washed with an aqueous 1 M solution of HCl, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99:1 to 97:3) to yield a yellow powder as desired product (96 mg, 86%); mp 229 – 231 °C. TLC Rf (hexanes:EtOAc, 1:1) 0.44. ¹H NMR (400 MHz, CDCl₃) δ 8.13-8.06 (m, 2H), 7.87-7.79 (m, 3H), 7.72 (s, 1H), 7.38 (d, J = 8.4 Hz, 1H), 7.28 (s, 2H), 6.96 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H), 5.96 (t, J = 4.7 Hz, 1H), 3.94 (s, 3H), 3.89-3.79 (m, 4H), 3.24-3.13 (m, 4H), 2.33 (d, J = 4.7 Hz, 2H), 1.34 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 166.2, 164.7, 150.4, 142.1, 138.9, 138.4, 135.3, 134.9, 132.9, 132.2, 129.9, 129.5, 127.1, 126.5, 124.7, 119.8, 118.0, 115.3, 66.9, 52.4, 49.2, 39.0, 33.4, 28.2.

4-((5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)benzoic Acid (12).

To a vial containing stir bar was added methyl ester 57 (66 mg, 0.133 mmol, 1.0 equiv), EtOH (6 mL), and 1 M NaOH (1.5 mL, 1.5 mmol, 11.3 equiv). The colorless suspension was stirred for 16 h at r.t. before acidifying with 1 M HCl and extracting 3 times with EtOAc. The combined organics were dried over MgSO₄, filtered, and concentrated under vacuum. The resultant residue was chromatographically purified via two silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 97:3-95:5) to yield 37.4 mg (58%) of a cream colored solid as desired product; mp 316 – 318 °C (decomp). TLC R_f (hexanes:EtOAc, 1:1) 0.10. ¹H NMR (400 MHz, THF-d₈) δ 9.37 (s, 1H), 8.10-8.01 (m, 3H), 7.97-7.90 (m, 2H), 7.33 (d, J = 8.4 Hz, 1H), 7.26-7.17 (m, 2H), 7.06 (d, J = 2.3 Hz, 1H), 6.98-6.89 (m, 2H), 5.88 (t, J = 4.7 Hz, 1H), 3.81-3.73 (m, 4H), 3.18-3.10 (m, 4H), 2.31 (d, J = 4.7 Hz, 2H), 1.33 (s, 6H). LRMS m/z (ESI⁺) calculated for C₃₀H₃₁N₂O₄ [M+H]⁺: 483.2, found 483.4. Purity 99%.

Compound 16

Methyl 4-((5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)-2-nitrobenzoate (58).

To a vial of aniline 53 (130 mg, 0.39 mmol, 1.0 equiv), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (150 mg, 0.78 mmol, 2.0 equiv), hydroxybenzotriazole (62 mg, 0.41 mmol, 1.05 equiv), DMAP (120 mg, 0.98 mmol, 2.5 equiv), and 4-(methoxycarbonyl)-3-nitrobenzoic acid (88 mg, 0.39 mmol, 1.0 equiv) were added. Anhydrous DCM (8 mL) was added, and the reaction mixture was heated to 37 °C overnight. After cooling the reaction mixture to r.t., it was rinsed with a saturated solution of NaHCO₃ and extracted 3 times with EtOAc. The combined organic extracts were dried over Na₂SO₄ before being condensed under reduced pressure. The product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (MeOH:DCM, 1:10), providing the target compound as an oil in 64% yield (130 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.25 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.73 (t, J = 7.9 Hz, 3H), 7.32 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H), 6.88 (dd, J = 17.7, 4.7 Hz, 3H), 5.90 (t, J = 4.7 Hz, 1H), 3.87 (s, 3H), 3.79 (t, J = 4.7 Hz, 4H), 3.11 (t, J = 4.8 Hz, 4H), 2.27 (d, J = 4.7 Hz, 2H), 1.27 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 165.1, 162.2, 148.2, 142.7, 138.4, 138.2, 135.0, 134.7, 131.4, 130.4, 129.9, 129.5, 128.5, 126.8, 124.8, 122.6, 119.9, 118.2, 115.4, 115.3, 66.8, 53.6, 49.2, 38.9, 33.5, 28.2.

4-((5,5-Dimethyl-8-(4-morpholinophenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)-2-nitrobenzoic Acid (16).

Methyl ester 58 (130 mg, 0.25 mmol, 1.0 equiv) was dissolved in minimal DCM in a vial. A 1 M aqueous solution of NaOH (2 mL) was added to the solution and MeOH was added until the solution was clear and did not separate. The reaction mixture was heated to 37 °C overnight and then condensed under reduced pressure and the residue was dissolved in DCM and extracted with water. The water layer was then acidified to pH 5 and extracted 3 times with DCM to obtain the targeted acid in quantitative yield (127 mg) as a brown solid; mp 176 °C (decomp). ¹H NMR (400 MHz, DMSO-d₆) δ 14.14 (br s, 1H), 10.46 (s, 1H), 8.44 (d, J = 1.7 Hz, 1H), 8.26 (dd, J = 8.0, 1.7 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.77 (dd, J = 8.4, 2.4 Hz, 1H), 7.43-7.30 (m, 2H), 7.20 (d, J = 8.3 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 5.93 (t, J = 4.7 Hz, 1H), 3.75 (t, J = 4.8 Hz, 4H), 3.14 (t, J = 4.8 Hz, 4H), 2.28 (d, J = 4.8 Hz, 2H), 1.28 (s, 6H). LRMS (ESI⁺) m/z calculated for C₃₀H₃₀N₃O₆ [M+H]⁺ 528.2, found 528.2. Purity 99%.

Compound 17

Methyl (1s,2R,3R,4r,5r,6S,7S,8s)-4-((5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)cubane-1-carboxylate (59).

To a dry vial with stir bar was added (2R,3R,4S,5S)-4-(methoxycarbonyl)cubane-1-carboxylic acid (63 mg, 0.31 mmol, 1.0 equiv) and aniline 51 (120 mg, 0.46 mmol, 1.5 equiv), EDCI-HCl (120 mg, 0.61 mmol, 2.0 equiv), HOBt (49 mg, 0.32 mmol, 1.05 equiv), and DMAP (37 mg, 0.31 mmol, 1.0 equiv). DCM (1.5 mL) was added, and the resultant yellow solution was stirred for 16 h at 40 °C. The reaction was quenched with saturated aqueous NaHCO₃ and extracted 3 times with DCM. The combined organics were washed with 1 M HCl, however a terrific emulsion formed. Copious EtOAc and water were added to obtain separation. The combined organics were dried over Na₂SO₄, filtered, under concentrated under reduced pressure. The crude NMR revealed a slight presence of DMAP, but the material was carried on to final saponification without further purification. TLC R_f (hexanes:EtOAc, 7:3) 0.33. ¹H NMR (400 MHz, MeOH-d₄) δ 7.74 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.25-7.18 (m, 4H), 6.99 (s, 1H), 6.83 (d, J = 1.8 Hz, 1H), 5.96 (t, J = 4.6 Hz, 1H), 4.23 (s, 6H), 3.71 (s, 3H), 2.40 (s, 3H), 2.32 (d, J = 4.6 Hz, 2H), 1.31 (s, 6H). ¹³C NMR (400 MHz, CD₃OD) δ 172.1, 169.4, 141.7, 138.9, 138.1, 137.0, 135.4, 134.7, 129.2, 128.8, 127.2, 124.8, 119.5, 117.4, 58.5, 55.9, 51.8, 47.3, 46.7, 39.1, 33.5, 28.4, 21.4.

(1s,2R,3R,4r,5r,6S,7S,8s)-4-((5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)cubane-1-carboxylic Acid (17).

To a vial with stir bar was added 59 (82 mg, 0.18 mmol, 1.0 equiv), EtOH (2 mL), DCM (2 mL), and 1M NaOH_(aq) (4 mL). The cloudy biphasic mixture was further diluted with DCM (2 mL) and EtOH (13 mL) to ensure a monophasic solution which was stirred for 16 h at r.t. The reaction was quenched with 1 M HCl (5 mL), which resulted in formation of a colorless precipitate. The mixture was extracted 4 times with EtOAc. The combined organics were dried over MgSO₄, filtered, and concentrated under reduced pressure. The residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 98:2-9:1) to reveal a colorless solid as desired product (77 mg, 97% yield); mp 139 – 141 °C. TLC R_f (hexanes:EtOAc, 7:3) 0.33. ¹H NMR (400 MHz, CDCl₃) δ 7.74 (dd, J = 8.4, 1.9 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.23 (m, 4H), 6.94 (s, 1H), 6.81 (d, J = 1.9 Hz, 1H), 5.96 (t, J = 4.6 Hz, 1H), 4.27 (s, 6H), 2.40 (s, 3H), 2.32 (d, J = 4.6 Hz, 2H), 1.31 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 175.6, 169.1, 141.6, 138.7, 137.9, 136.9, 135.1, 134.6, 129.1, 128.6, 127.1, 124.7, 58.4, 55.3, 47.2, 46.6, 39.0, 33.4, 28.2, 21.2. LRMS m/z (ESI⁺) calculated for C₂₉H₂₈NO₃ [M+H]⁺: 438.2, found 438.4. Purity 98%.

Scheme 6. Synthesis of reversed amides 13 and 14

Synthesis of Compound 13

Methyl 4-((5,5-Dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-2-yl)carbamoyl)benzoate (60).

To a flame dried microwave vial, 7-bromo-4,4-dimethyl-3,4-dihydronaphthalen-1(2H)-one (500 mg, 1.98 mmol, 1 equiv), methyl 4-carbamoylbenzoate (425 mg, 2.37 mmol, 1.2 equiv), copper (I) iodide (40 mg, 0.21 mmol, 0.11 equiv) was added and the vial was closed with a rubber septum and placed in high vacuum for 5 min using a needle and filled with N₂ afterwards. This process was repeated two more times and N,N’-dimethylethylenediamine (40 mg, 0.45 mmol, 0.21 equiv) and anhydrous toluene (4 mL) were added to the vial using a syringe under a positive pressure of N₂. The septum was carefully removed under a flow of N₂ and freshly dried 4 A molecular sieves (200 mg) were added to the vial, and it was capped immediately. The reaction mixture was heated at 120 °C for 40 h. The mixture was cooled to room temperature and filtered to remove the molecular sieves. The filtrate was evaporated to obtain a solid residue that was purified by silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography using EtOAc:hexanes as solvent using 0:1 to 2:5 gradient over 15 min to obtain a solid colorless crystal (208 mg, 30%); mp 196 – 198 °C. ¹H NMR (400 MHz, CDCl₃) δ 9.09 (s, 1H), 8.40 (dd, J = 8.6, 2.5 Hz, 1H), 8.14-8.07 (m, 3H), 8.07-8.00 (m, 2H), 7.44 (d, J = 8.6 Hz, 1H), 3.95 (s, 3H), 2.65 (t, J = 7.3 Hz, 2H), 2.03-1.95 (t, 7.3 Hz, 2H), 1.38 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 199.1, 166.4, 165.4, 148.8, 138.8, 136.8, 133.1, 131.5, 129.9, 127.6,7 127.1, 126.9, 118.7, 52.6, 37.1, 35.2, 33.8, 29.8.

Methyl 4-((5,5-Dimethyl-8-(((trifluoromethyl)sulfonyl)oxy)-5,6-dihydronaphthalen-2-yl)carbamoyl)benzoate (61).

To a flame-dried vial with stir bar was added NaHMDS (1.0 M in THF, 0.96 mL, 0.96 mmol, 2.0 equiv) and TH (1 mL). The vial was purged with N₂ and chilled to −78 °C before adding a solution of ketone 60 (168 mg, 0.478 mmol, 1.0 equiv) in THF (2 mL). The yellow solution turned dark orange and was stirred for 30 min at −78 °C before adding a solution of Comins’ reagent (207 mg, 0.526 mmol, 1.1 equiv) in THF (2 mL). The reaction turned dark red and was stirred for 45 min at −78 °C before warming to r.t. and stirring for 16 h. The reaction was quenched with saturated aqueous NH₄Cl and extracted 3 times with EtOAc. The combined organics were washed with 5% aqueous NaOH, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified via two silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:methanol, 99:1 and hexanes:EtOAc, 8:2). A light-yellow solid was isolated as desired product (87 mg, 38%); mp 152 – 154 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.63. ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.84-7.81 (m, 1H), 7.45 (d, J = 2.2 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 6.00 (t, J = 4.8 Hz, 1H), 3.97 (s, 3H), 2.43 (d, J = 4.8 Hz, 2H), 1.32 (s, 6H).

Methyl 4-((5,5-Dimethyl-8-(4-(morpholinomethyl)phenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)benzoate (62).

To a vial with stir bar was added triflate 61 (86 mg, 0.178 mmol, 1.0 equiv) and 1,2-dimethoxyethane (2.75 mL). Palladium tetrakis(triphenylphosphine) (31 mg, 0.027 mmol, 0.15 equiv), 4-(4-morpholinomethyl)phenyl-boron pinacol ester (65 mg, 0.21 mmol, 1.2 equiv), and saturated aqueous NaHCO₃ (1.7 mL) was added. The vessel was purged with N₂ and the mixture was stirred at 85 °C for 2 h over which time the biphasic colorless/yellow mixture changed to greenish-red with a colorless precipitate. The reaction was cooled to r.t., diluted with EtOAc and water, and extracted 3 times with EtOAc. The dark red solution was dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant material was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99:1-98:2) and a light, cream-colored foamy solid was obtained as desired product mixed with some residual Pd(PPh₃)₄ and carried on to the final step without further purification. TLC R_f (hexanes:EtOAc, 1:1) 0.18. ¹H NMR (400 MHz CDCl₃) δ 8.11 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 7.71-7.68 (m, 3H), 7.57-7.55 (m, 1H), 7.48-7.47 (m, 2H), 7.40 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 2.1 Hz, 1H), 6.02 (t, J = 4.7 Hz, 1H), 3.96 (s, 3H), 3.78-3.66 (m, 4H), 3.54 (s, 2H), 2.56-2.47 (m, 4H), 2.37 (d, J = 4.7 Hz, 2H), 1.35 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 173.6, 166.2, 155.0, 142.1, 139.6, 138.7, 136.7, 135.3, 134.6, 132.2, 132.1, 131.9, 130.0, 129.3, 128.6, 128.4, 127.4, 127.1, 124.8, 67.0, 63.3, 53.7, 52.4, 39.0, 33.5, 28.2, 24.9.

4-((5,5-Dimethyl-8-(4-(morpholinomethyl)phenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)benzoic Acid (13).

To a vial containing stir bar was added crude methyl ester 62, EtOH (3.5 mL), and 1 M NaOH (0.88 mL). The suspension was stirred for 16 h at r.t. before adding water and extracting 3 times with EtOAc. The aqueous layer was acidified with 1 M HCl and extracted 3 times with EtOAc. The combined organic extracts were dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 98:2:95:5-9:1) to yield a colorless/clear glassy solid as desired product (44 mg, 52% over 2 steps); mp 183 – 185 °C. TLC R_f (DCM:MeOH, 9:1) 0.08. ¹H NMR (400 MHz, MeOH-d₄) δ 8.06 (d, J = 8.3 Hz, 2H), 7.88 (d, J = 8.2 Hz, 2H), 7.55-7.37 (m, 7H), 6.03 (t, J = 4.6 Hz, 1H), 4.21 (s, 2H), 3.89-3.80 (m, 4H), 3.16 (m, 4H), 2.36 (d, J = 4.6 Hz, 2H), 1.33 (s, 6H). ¹³C NMR (400 MHz, MeOH-d₄) δ 169.5, 167.9, 143.8, 143.0, 140.2, 139.9, 137.5, 135.1, 132.2, 130.8, 130.5, 130.1, 128.7, 128.6, 125.4, 121.7, 120.2, 65.5, 62.2, 53.2, 49.9, 40.0, 34.5, 28.6. LRMS m/z (ESI⁻) calculated for C₃₁H₃₁N₂O₄ [M-H]⁻: 495.2, found, [M-H]⁻: 495.3. Purity 97%.

Synthesis of compound 14

Methyl 4-((5,5-Dimethyl-8-(4-(morpholine-4-carbonyl)phenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)benzoate (63).

To a vial with stir bar was added triflate 61 (122 mg, 0.252 mmol, 1.0 equiv), 4-(morpholino-4-carbonyl)phenyl boronic acid pinacol ester (96 mg, 0.30 mmol, 1.2 equiv), and 1,2-dimethoxyethane (3.9 mL). Palladium tetrakis(triphenylphosphine) (44 mg, 0.038 mmol, 0.15 equiv) and saturated aqueous NaHCO₃ (2.4 mL) was added, and the mixture was stirred at 85 °C for 2 h. The brown solution was diluted with water and EtOAc before extracting 3 times with EtOAc. The combined organics were dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was pushed through a plug of silica (DCM:MeOH, 99:1 −97:3) to reveal a light red foamy solid as desired product mixed with some remaining palladium. The mixture was carried on without further purification. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J = 8.3 Hz, 2H), 7.94 (s, 1H), 7.90 (d, J = 8.3 Hz, 3H), 7.71-7.67 (m, 2H), 7.56-7.53 (m, 1H), 7.47-7.45 (m, 2H), 6.84 (d, J = 2.0 Hz, 1H), 6.02 (t, J = 4.6 Hz, 1H), 3.94 (s, 3H), 3.88-3.55 (m, 8H), 2.36 (d, J = 4.6 Hz, 2H), 1.34 (s, 6H).

4-((5,5-Dimethyl-8-(4-(morpholine-4-carbonyl)phenyl)-5,6-dihydronaphthalen-2-yl)carbamoyl)benzoic Acid (14).

To a vial containing a stir bar was added crude methyl ester 63, EtOH (5 mL), and 1 M NaOH (1.26 mL). The suspension was stirred for 48 h at r.t. before acidifying with 1 M HCl and extracting 3 times with EtOAc. The combined organic extracts were dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified via two silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography columns (DCM: MeOH, 97:3 −95:5) to yield a cream colored solid as desired product (72 mg, 56% over 2 steps); mp 257 °C (275 °C decomp). TLC R_f (DCM:MeOH, 9:1) 0.18. ¹H NMR (400 MHz, CDCl₃) δ 9.89 (s, 1H), 8.14 (d, J = 8.5 Hz, 2H), 7.96-7.89 (m, 3H), 7.80 (s, 1H), 7.71-7.67 (m, 2H), 7.58-7.53 (m, 1H), 7.49-7.46 (m, 2H), 6.81 (d, J = 2.1 Hz, 1H), 6.03 (t, J = 4.6 Hz, 1H), 3.90-3.56 (m, 8H), 2.37 (d, J = 4.7 Hz, 2H), 1.35 (s, 6H). LRMS m/z (ESI⁺) calculated for C₃₁H₃₁N₂O₅ [M+H]⁺: 511.2, found 511.4. Purity 99%.

Scheme 7. Synthesis of tetrahydroisoquinoline 18

Compound 18

Methyl 2-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carbonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (64).

To a vial with stir bar was added carboxylic acid 36 (212 mg, 0.725 mmol, 1.0 equiv), methyl 1,2,3,4-tetrahydroisoquinoline-6-carboxylate hydrochloride (248 mg, 1.09 mmol, 1.5 equiv), EDCI-HCl (278 mg, 1.45 mmol, 2.0 equiv), HOBt (117 mg, 0.761 mmol, 1.05 equiv), and DMAP (310 mg, 2.54 mmol, 3.5 equiv). DCM (3 mL) was added, and the resultant yellow solution was stirred for 16 h at 35 °C. The reaction was quenched with saturated aqueous NaHCO₃ and extracted 3 times with DCM. The combined organics were washed with 1 M HCl_aq, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 1:1) to yield the desired product as colorless solid (amide rotamers) (318 mg, 94%); mp 67 – 69 °C. TLC R_f (hexanes:EtOAc 1:1) 0.55. ¹H NMR (400 MHz, CDCl₃) δ 7.83 (d, J = 13.0 Hz, 2H), 7.42 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.28-7.20 (m, 4H), 7.18 (d, J = 6.5 Hz, 2H), 7.11 (d, J = 1.6 Hz, 1H), 6.01 (t, J = 4.7 Hz, 1H), 3.92 (s, 3H), 3.64 (s, 1H), 2.89 (d, J = 79.9 Hz, 2H), 2.42-2.34 (m, 5H), 1.36 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 167.0, 147.3, 138.9, 137.6, 137.1, 134.3, 133.3, 129.3, 128.6, 127.0, 124.3, 77.5, 77.2, 76.8, 52.3, 38.9, 34.0, 28.2, 21.3.

2-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalene-2-carbonyl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic Acid (18).

To a vial with stir bar was added methyl ester 64 (100. mg, 0.215 mmol, 1.0 equiv) and EtOH (4 mL). NaOH (1 M in water, 1.1 mL, 1.1 mmol, 5.0 equiv) was added. The solution was stirred for 16 h at r.t. before acidifying with 1 M HCl_aq and extracting 3 times with EtOAc. The combined organic fractions were washed with brine, dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99:1-93:7) to yield 96 mg (99%) of a colorless solid as desired product; mp 201 – 203 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.21. ¹H NMR (400 MHz, THF-d₈) δ 7.79 (s, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.33 (dd, J = 7.9, 1.8 Hz, 1H), 7.25-7.13 (m, 5H), 7.09 (d, J = 1.8 Hz, 1H), 5.97 (t, J = 4.7 Hz, 1H), 4.73 (m, 2H), 3.84-3.59 (m, 2H), 2.85 (m, 2H), 2.36 (d, J = 4.7 Hz, 2H), 2.33 (s, 3H),1.35 (s, 6H). ¹³C NMR (101 MHz, THF-d₈) δ 170.4, 167.5, 147.5, 140.2, 139.4, 138.6, 137.6, 135.7, 135.0, 134.7, 131.0, 130.1, 129.8, 129.2, 128.2, 127.5, 127.2, 126.9, 125.7, 124.6, 45.8, 39.6, 34.5, 30.6, 28.6, 28.4, 21.20. LRMS m/z (ESI⁻) calculated for C₃₀H₂₈NO₃ [M-H]⁻: 450.2, found 450.3. Purity 98%.

Compound 19

Methyl 2-(5,5-dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylate (65).

To a two-neck round-bottom flask was added Xantphos (53 mg, 0.092 mmol, 10 mol%) and Pd₂(dba)₃ catalyst (42 mg, 0.046 mmol, 5 mol%) in toluene (2 mL). The reaction was purged with N₂ for 10-15 min then heated to 110 °C for 15 min. After the reaction vessel was cooled to r.t., NaOtBu (180 mg, 1.8 mmol) was added followed by bromide 32 (300 mg, 0.92 mmol), then methyl 1,2,3,4-tetrahydroisoquinoline-6-carboxylate (350 mg, 1.8 mmol). The reaction was purged again with N₂ for 10-15 min then heated to 110 °C and allowed to stir for 36 h. After the flask had cooled to r.t., the reaction was filtered through a Celite plug and the solvent was removed under reduced pressure. Silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 1:99) gave 153 mg (38%) of the target compound as a colorless solid; mp 187 – 190 °C. ¹H NMR (400 MHz, CDCl³) δ 7.77 (s, 1H), 7.26-7.12 (m, 6H), 7.10 (d, J = 8.4 Hz, 1H), 6.83 (dd, J = 8.4, 2.6 Hz, 1H), 6.70 (d, J = 2.6 Hz, 1H), 5.94 (t, J = 4.7 Hz, 1H), 4.25 (s, 2H), 3.87 (s, 3H), 3.37 (t, J = 5.8 Hz, 2H), 2.91 (t, J = 5.7 Hz, 2H), 2.38 (s, 3H), 2.28 (d, J = 4.7 Hz, 2H), 1.28 (s, 6H).

2-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)-1,2,3,4-tetrahydroisoquinoline-6-carboxylic Acid (19).

To a flask containing methyl ester 65 (100 mg, 0.12 mmol) in THF: H₂O: MeOH (2:1: 2 mL), was added 2 M NaOH (1 mL). The reaction was stirred at room temperature for 16 h. Then, the solvent was removed under reduced pressure. The reaction mixture was diluted with water and acidified to pH 4 with an aqueous 0.1 M solution of HCl. The product was extracted with EtOAc (3 × 20 mL) and dried with MgSO₄ and purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 8:2) to obtain 44 mg (45%) of the target compound as yellow solid; mp 243 – 244 °C; ¹H NMR (400 MHz, THF-d₈) δ 7.76 (d, J = 8.4 Hz, 2H), 7.25-7.20 (m, 3H), 7.19-7.12 (m, 3H), 6.87 (dd, J = 8.4, 2.6 Hz, 1H), 6.71 (d, J = 2.6 Hz, 1H), 5.91 (t, J = 4.7 Hz, 1H), 4.24 (d, J = 12.3 Hz, 2H), 3.37 (td, J = 6.0, 3.5 Hz, 2H), 2.91 (t, J = 5.8 Hz, 2H), 2.37 (s, 3H), 2.28 (d, J = 4.7 Hz, 2H), 1.28 (s, 6H). ¹³C NMR (101 MHz, THF-d₈) δ 167.3, 149.5, 140.8, 140.4, 139.1, 137.0, 136.8, 135.6, 135.0, 130.5, 129.7, 129.4, 129.1, 127.7, 127.1, 126.3, 124.9, 115.6, 115.2, 52.1, 47.7, 39.9, 33.6, 29.5, 28.5, 21.1. LRMS m/z (ESI⁻) calcd for C₂₉H₂₈NO₂ [M-H]⁻ 422.2, found 422.3. Purity 97%.

Scheme 9, Synthesis of Compound 20

Methyl 2-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)quinoline-6-carboxylate (66).

To a sealable microwave vial of bromodihydronaphthalene 32 (130 mg, 0.400 mmol) and methyl quinoline-6-carboxylate (450 mg, 0.240 mmol) in dioxane (2 mL) was added [Rh(CO)₂Cl]₂ catalyst (8 mg, 5 mol %). The reaction was heated to 175 °C in an oil bath for 24 h and then allowed to cool to r.t. The reaction was sent through a Celite plug and washed with methyl tert-butyl ether and then the solvent was removed under reduced pressure. The product was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (EtOAc:hexanes, 1:99-1:9) to provide 66 in 61% yield (105 mg) as a colorless solid; mp 190 – 191 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.54 (d, J = 1.5 Hz, 1H), 8.27 (dd, J = 8.9, 1.5 Hz, 1H), 8.20 (d, J = 8.6 Hz, 1H), 8.17-8.08 (m, 2H), 7.79 (d, J = 1.7 Hz, 1H), 7.64 (dd, J = 72.1, 8.3 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 6.05 (t, J = 4.7 Hz, 1H), 3.99 (s, 3H), 2.42 (s, 3H), 2.39 (d, J = 4.7 Hz, 2H), 1.39 (s, 6H); HRMS (ESI) m/z calcd for C₃₀H₂₇NO₂ [M+H]⁺ requires 434.2121, found 434.2132.

2-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)quinoline-6-carboxylic Acid (20).

To a flask containing methyl ester 66 (50.0 mg, 0.115 mmol) in MeOH (3 mL), was added aqueous 1 M NaOH (1 mL). The reaction was heated to 65 °C and stirred for 2 h. Then, the flask was allowed to cool to r.t. and the solvent was removed under reduced pressure. The reaction was diluted with water and acidified to pH 4 with aqueous 1 M HCl. The product precipitated and was collected via filtration and recrystallized with DCM:MeOH to provide the desired quinoline acid 20 in 38% yield (18.4 mg). Colorless solid mp 268 – 269 °C; ¹H NMR (400 MHz, CDCl₃): δ 8.56 (d, J = 8.5 Hz, 1H), 8.36-8.02 (m, 4H), 7.79-7.67 (m, 2H), 7.55 (d, J = 7.2 Hz, 1H), 7.30 (d, J = 7.6 Hz, 2H), 7.22 (d, J = 6.9 Hz, 2H), 6.08-5.98 (m, 1H), 2.39 (s, 3H), 2.37 (d, J = 3.9 Hz, 2H), 1.38 (s, 6H); LRMS m/z (ESI⁺) calcd for C₂₉H₂₆NO₂ [M+H]⁺ 420.2, found 420.2. Purity 99%.

Scheme 10. Synthesis of chromene amide 21

6-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (67).

A flask equipped with stir bar and magnesium shavings (430. mg, 17.6 mmol, 1.5 equiv) was flame dried before cooling under a N₂ stream, adding 1 crystal of iodine, and purging with N₂. A solution of 4-bromotoluene (2.61 g, 15.3 mmol, 1.3 equiv) in THF (7.5 mL) was added dropwise. The reaction did not self-initiate, so it was heated to ebullition with a heat gun before stirring approximately 3 h until magnesium was mostly consumed. Next a solution of 6-bromo-2,2-dimethylchroman-4-one (3 g, 10 mmol, 1.0 equiv) in THF (30 mL) was added before stirring at r.t. for 16 h. The reaction was quenched with saturated aqueous NH₄Cl and extracted 3 times with EtOAc. The combined organics were dried over MgSO₄, filtered, and concentrated under reduced pressure to reveal a viscous yellow oil as desired product. It was carried on to dehydration step without further purification. (Note: there is some presence of product dehydrated in situ, so chromatographic purification results in loss of dehydrated product.) TLC R_f (hexanes:EtOAc, 7:3) 0.73.) Next a flask equipped with stir bar and Dean-Stark trap was charged with the crude reaction product, benzene (40 mL), and 4-toluenesulfonic acid monohydrate (450 mg, 2.4 mmol, 0.2 equiv). The solution was stirred at reflux for 4 h before adding water and extracting 3 times with EtOAc. The combined organic fractions were dried over MgSO₄, filtered, and concentrated under reduced pressure. The resultant residue was subjected to silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99:1). Off-white solid, mp 64.8 – 65.5 (1.89 g, 49% yield). TLC R_f (hexanes:EtOAc, 7:3) 0.73. ¹H NMR (400 MHz, MeOH-d₄) δ 7.31-7.27 (m, 5H), 7.19 (d, J = 2.3 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 5.67 (s, 1H), 2.45 (s, 3H), 1.53 (s, 6H). ¹³C NMR (400 MHz, MeOH-d₄) δ 152.5, 137.8, 134.8, 133.9, 131.8, 129.8, 129.3, 128.5, 128.2, 124.5, 118.7, 112.8, 76.2, 27.6, 21.3.

2,2-Dimethyl-4-(p-tolyl)-2H-chromene-6-carboxylic Acid (68).

A flame-dried vial equipped with stir bar and screw cap with inlaid septum was purged with N₂ and charged with a solution of bromochromene 67 (200. mg, 0.607 mmol, 1.0 equiv) in THF (2.5 mL). The mixture was chilled to −78 °C before slowly adding a 1.9 M solution of tert-butyllithium in pentane (0.32 ml, 0.61 mmol, 1.0 equiv). The now reddish solution was stirred for 30 min at −78 °C. Dry ice was used to produce CO₂, which was pushed through a Drierite drying tube equipped with needle outlet. The CO₂ was bubbled below the surface of the reaction mixture for 10 min, and the reaction was stirred for an additional 20 min under a CO₂ atmosphere. The flask and solution were purged with N₂ while warming to r.t. Next the reaction was diluted with EtOAc before quenching with 1 M HCl. After stirring the biphasic mixture for 10 min, it was extracted 3 times with EtOAc. The combined organic extracts were dried over MgSO₄, filtered through a silica plug, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99:1-92:8) to furnish 68 as a colorless solid (149 mg, 83% yield); mp 196 – 198 °C. TLC R_f (hexanes:EtOAc, 1:1) 0.45. ¹H NMR (400 MHz, MeOH-d₄) δ 11.68 (br s, 1H), 7.92 (d, J = 3.4 Hz, 1H), 7.80 (d, J = 1.8 Hz, 1H), 7.24 (s, 4H), 6.93 (d, J = 8.5 Hz, 1H), 5.65 (s, 1H), 2.43 (s, 3H), 1.54 (s, 6H).

Ethyl 4-(2,2-Dimethyl-4-(p-tolyl)-2H-chromene-6-carboxamido)benzoate (69).

To a dry vial with stir bar was added 2,2-dimethyl-4-(p-tolyl)-2H-chromene-6-carboxylic acid (130 mg, 0.43 mmol, 1.0 equiv) and ethyl 4-aminobenzoate (71 mg, 0.43 mmol, 1.0 equiv), EDCI-HCl (160 mg, 0.86 mmol, 2.0 equiv), HOBt (69 mg, 0.45 mmol, 1.05 equiv), and DMAP (53 mg, 0.43 mmol, 1.0 equiv). DCM (1.6 mL) was added, and the resultant yellow solution was stirred for 16 h at 40 °C. The reaction was quenched with saturated aqueous NaHCO₃ and extracted 3 times with DCM. The combined organics were washed with 1 M HCl_aq, and re-extraction was conducted with EtOAc after diluting with water due to the resultant emulsion. The organics were dried over Na₂SO₄, filtered, concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99.5:0.5-97:3) to furnish the target compound as a pale-yellow solid (32 mg, 52%); TLC R_f (hexanes:EtOAc, 1:1) 0.80. ¹H NMR (400 MHz, MeOH-d₄) δ 8.01 (d, J = 8.7 Hz, 2H), 7.82 (s, 1H), 7.69-7.63 (m, 3H), 7.58 (d, J = 2.2 Hz, 1H), 7.24-7.19 (m, 4H), 6.94 (d, J = 8.4 Hz, 1H), 5.66 (s, 1H), 4.35 (q, J = 7.1 Hz, 2H), 2.39 (s, 3H), 1.51 (s, 6H), 1.38 (t, J = 7.1 Hz, 3H). ¹³C NMR (400 MHz, MeOH-d₄) δ 166.2, 165.4, 157.1, 142.2, 138.0, 134.6, 134.0, 130.8, 129.5, 129.4, 128.4 128.2, 126.7, 125.9, 124.9, 122.6, 119.0, 117.1, 77.2, 60.9, 27.9, 21.2, 14.4.

4-(2,2-Dimethyl-4-(p-tolyl)-2H-chromene-6-carboxamido)benzoic Acid (21).

To a vial equipped with stir bar was added ethyl ester 69 (33 mg, 0.075 mmol, 1.0 equiv), DCM (1 mL), EtOH (1 mL), and 1 M NaOH (1 mL). The cloudy solution was stirred for 16 h at r.t. before acidifying to pH 1 with 1 M HCl and extracting 3 times with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The resultant residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (DCM:MeOH, 99:1-9:1) followed by recrystallization from DCM/hexanes to furnish 21 as a colorless crystalline solid (19 mg, 63%); mp 262 – 264 °C. ¹H NMR (400 MHz, CDCl₃/ MeOH-d₄) δ 7.98 (d, J = 8.6 Hz, 2H), 7.70 (dd, J = 8.4 & 2.0 Hz, 1H), 7.66 (d, J = 8.6 Hz, 2H) ,7.56 (d, J = 2.1 Hz, 1H), 7.23-7.18 (m, 4H), 6.92 (d, J = 8.4 Hz, 1H), 5.63 (s, 1H), 2.36 (s, 3H), 1.48 (s, 6H). ¹³C NMR (400 MHz, CDCl₃/ MeOH-d₄) δ 168.4, 166.1, 156.9, 142.7, 137.9, 134.7, 134.0, 130.9, 129.4, 129.2, 128.6, 128.5, 126.7, 125.5, 125.0, 122.4, 119.3, 117.0, 77.0, 27.7, 21.1. LRMS m/z (ESI⁺) calculated for C₂₆H₂₄NO₄⁺ [M+H]⁺: 414.2, found 414.2. Purity 99%.

Scheme 11. Synthesis of chromene sulfonamides 22-25

Compound 22

2,2-Dimethylchroman-4-one (70).

A mixture of 1-(2-hydroxyphenyl)-ethan-1-one (3.74 g, 27.5 mmol), acetone (3.10 mL, 42.2 mmol), pyrrolidine (3.60 mL, 43.0 mmol, 1.02 equiv) in MeOH (100 mL) was stirred for 3 days at r.t. The volatiles were evaporated under reduced pressure, and the resulting residue was treated with a 1 M aqueous solution of HCl to achieve pH 1. The solution was extracted with Et₂O (3 x 60 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, filtered, and evaporated under reduced pressure. The resulting residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 1:0-7:3) to give chroman-4-one 70 (4.11 g, 85%) as an off-white solid; mp 95 – 97 °C. This compound is also commercially available. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (dd, J = 7.9, 1.8 Hz, 1H), 7.46 (ddd, J = 8.7, 7.2, 1.8 Hz, 1H), 7.02-6.88 (m, 2H), 2.72 (s, 2H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 192.6, 160.0, 136.2, 126.6, 120.8, 120.3, 118.4, 79.2, 49.0, 26.7.

2,2-Dimethyl-4-(p-tolyl)-2H-chromene (74).

2,2-Dimethylchroman-4-one 72 (1.50 g, 5.87 mmol) was taken into dry THF (45 mL) and cooled to −78 °C. p-Tolylmagnesium bromide (9.00 mL, 1.0 M solution in THF, 9.00 mmol, 1.53 equiv) was added dropwise to the solution at −78 °C under a N₂ atmosphere. The reaction was warmed to r.t. and stirred for 24 h. The reaction mixture was cooled again to −78 °C and a second portion of p-tolylmagnesium bromide (6.30 mL, 1.0 M, solution in THF, 6.30 mmol, 1.07 equiv) was added to the reaction mixture. The reaction mixture was allowed to warm to r.t. and stirred for an additional 24 h. Saturated aqueous NH₄Cl (30 mL) was added to quench the reaction. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, filtered and evaporated under reduced pressure to afford 2,2-dimethyl-4-(p-tolyl)chroman-4-ol (72) as yellow oil (1.97 g). The crude product was directly used for the next step without further purification. To intermediate 72 (1.90 g, 5.87 mmol, calculated based on the starting material used in the previous step, 1.00 equiv) in MeOH (60 mL), pyridinium p-toluenesulfonate (0.295 g, 1.17 mmol, 0.200) was added and refluxed for 14 h. Volatiles were evaporated under reduced pressure and water (50 mL) was added to the residue. The aqueous solution was extracted 3 times with EtOAc. The combined organic extracts were washed with brine, dried over Na₂SO₄, and evaporated under reduced pressure. Purification of the resulting residue using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 1:0-7:3) gave 2,2-dimethyl-4-(4-tolyl)-2H-chromene (74; 0.788 g, 37%, over 2 steps) as a yellow solid; mp 101 – 102 °C. Starting material 2,2-dimethylchroman-4-one (72; 0.540 g, 30%) was also recovered. ¹H NMR (400 MHz, CDCl₃) δ 7.28-7.20 (m, 4H), 7.20-7.13 (m, 1H), 7.05 (dd, J = 7.7, 1.6 Hz, 1H), 6.91 (dd, J = 8.1, 1.2 Hz, 1H), 6.83 (td, J = 7.4, 1.2 Hz, 1H), 5.61 (s, 1H), 2.42 (s, 3H), 1.51 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 153.5, 137.5, 135.6, 134.7, 129.2, 129.1, 128.8, 128.7, 125.7, 122.6, 120.6, 116.9, 75.8, 27.7, 21.3.

2,2-Dimethyl-4-(p-tolyl)-2H-chromene-6-sulfonyl Chloride (76).

2,2-Dimethyl-4-(p-tolyl)-2H-chromene (74; 250. mg, 1.00 mmol) was taken into anhydrous 1,2-dichloroethane (5 mL). Then SO₃·DMF complex (229 mg, 1.50 mmol) was added under N₂. The solution was stirred at 70 °C for 10 h, and then at r.t. for 12 h. To this solution, oxalyl chloride (0.135 mL, 1.60 mmol) was added at 0 °C and then stirred for 4 h at 65 °C. The reaction was quenched with water (10 mL). DCM (10 mL) was added, the organic layer was separated, and the aqueous phase was twice extracted with additional DCM. The combined organic fractions were washed with brine, dried over Na₂SO₄, filtered, and condensed under reduced pressure. The resulting residue was purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 1:0-7:3) to provide 76 (285 mg, 82%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 7.85 (dd, J = 8.7, 2.5 Hz, 1H), 7.75 (d, J = 2.5 Hz, 1H), 7.27 (q, J = 8.1 Hz, 4H), 7.03 (d, J = 8.7 Hz, 1H), 5.76 (s, 1H), 2.45 (s, 3H), 1.59 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 159.5, 138.5, 136.0, 133.6, 133.0, 130.3, 129.6, 128.7, 128.3, 124.9, 123.0, 117.8, 78.4, 28.3, 21.3.

4-((2,2-Dimethyl-4-(p-tolyl)-2H-chromene)-6-sulfonamido)benzoic Acid (22).

To a solution of 2,2-dimethyl-4-(p-tolyl)-2H-chromene-6-sulfonyl chloride (76; 80 mg, 0.23 mmol, 1.0 equiv) in pyridine/CH₂Cl₂ (1:2, 3 mL) was added 4-aminobenzoic acid (38 mg, 0.27 mmol, 1.2 eq) under N₂. The reaction mixture was stirred at room temperature for 24 h. HCl (1N, aqueous solution, 10 mL) and CH₂Cl₂ (15 mL) were added. The organic layer was separated, and the aqueous layer was extracted with additional DCM. Combined organic fractions were washed with brine, dried over Na₂SO₄, filtered, evaporated under reduced pressure, and purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 1:0-0:1) to furnish 62 mg (60%) of the targeted acid 22 as a colorless solid; mp 254 – 255 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.99 (d, J = 8.7 Hz, 2H), 7.66 (dd, J = 8.6, 2.4 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.18 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 7.05-6.97 (m, 3H), 6.89 (d, J = 8.6 Hz, 1H), 5.61 (s, 1H), 2.40 (s, 3H), 1.47 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 170.4, 158.0, 142.0, 138.3, 134.0, 133.4, 131.9, 130.06, 130.03, 129.5, 128.7, 128.3, 125.1, 125.0, 122.8, 118.9, 117.6, 77.7, 28.1, 21.3. LRMS m/z (ES⁺) (M + H)⁺ calculated for C₂₅H₂₄NO₅S [M+H]⁺: 450.1, found 450.0. Purity 99%.

Compound 23

8-Bromo-2,2-dimethylchroman-4-one (71).

8-Bromo-2,2-dimethylchroman-4-one (71) was prepared from 1-(3-bromo-2-hydroxyphenyl)ethan-1-one (0.952 g, 4.41 mmol, 1.00 equiv), acetone (0.500 mL, 6.84 mmol, 1.55 equiv) and pyrrolidine (0.580 mL, 6.93 mmol, 1.57 equiv) following the procedure described for the synthesis of 2,2-dimethylchroman-4-one (70) to yield 924 mg (82%) of the targeted compound as an off-white solid; mp 239 – 240 °C. This compound is also commercially available. ¹H NMR (400 MHz, CDCl₃) δ 7.80 (dd, J = 7.8, 1.6 Hz, 1H), 7.70 (dd, J = 7.8, 1.7 Hz, 1H), 6.84 (t, J = 7.8 Hz, 1H), 2.74 (s, 2H), 1.49 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 191.8, 156.5, 139.4, 125.9, 121.48, 121.44, 112.2, 80.5, 48.5, 26.6.

8-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (75).

8-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (75) was prepared from 71 (0.802 g, 3.13 mmol, 1.00 equiv), p-tolylmagnesium bromide (8.15 mL, 1.0 M solution in THF, 8.15 mmol, 2.50 equiv) and pyridinium p-toluenesulfonate (0.157 g, 0.626 mmol, 0.200 equiv) following the procedure described for the synthesis of compound 74 (724 mg, 70% for two steps, yellow solid; mp 120 – 121 °C). ¹H NMR (400 MHz, CDCl₃) δ 7.36 (dd, J = 8.0, 1.5 Hz, 1H), 7.19 (s, 4H), 6.94 (dd, J = 7.8, 1.5 Hz, 1H), 6.67 (t, J = 7.8 Hz, 1H), 5.61 (s, 1H), 2.38 (s, 3H), 1.52 (s, 6H). ¹³C NMR (101 MHz, CDCl³) δ 150.3, 137.7, 135.1, 134.5, 132.6, 129.5, 129.2, 128.6, 124.8, 124.3, 121.3, 111.2, 77.1, 27.7, 21.3.

8-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene-6-sulfonyl Chloride (77).

Compound 77 was prepared from 75 (0.500 g, 1.51 mmol, 1.00 equiv), SO₃·DMF (0.349 mg, 2.22 mmol, 1.50 equiv) and oxalyl chloride (0.205 mL, 2.42 mmol, 1.60 equiv) following the procedure described for compound 76 to yield 350 mg (54%) of the target compound 77 as a brown oil. ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J = 2.4 Hz, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.32-7.26 (m, 2H), 7.24-7.20 (m, 2H), 5.79 (s, 1H), 2.45 (s, 3H), 1.64 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 156.3, 138.8, 136.4, 133.2, 133.1, 131.5, 130.9, 129.8, 128.3, 124.2, 123.6, 111.7, 79.9, 28.3, 21.3.

4-((8-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene)-6-sulfonamido)benzoic Acid (23).

Acid 23 was prepared from 77 (0.10 g, 0.23 mmol, 1.0 equiv) and 4-aminobenzoic acid (0.035 g, 0.25 mmol, 1.1 equiv) following the procedure described for compound 22 to yield 98 mg (80%) of the target compound as a colorless solid; mp 192 – 194 °C, purity >97%. ¹H NMR (400 MHz, MeOH-d₄) δ 7.93 (s, 1H), 7.92-7.89 (m, 2H), 7.23-7.17 (m, 3H), 7.14 (d, J = 8.3 Hz, 2H), 6.93 (d, J = 7.7 Hz, 2H), 5.76 (s, 1H), 2.41 (s, 3H), 1.51 (s, 6H). ¹³C NMR (101 MHz, MeOH-d₄) δ 169.3, 155.4, 143.6, 139.6, 135.1, 134.6, 133.5, 132.4, 132.3, 131.9, 130.6, 129.4, 127.8, 125.1, 124.7, 120.8, 112.4, 80.3, 28.1, 21.4. LRMS m/z (ES⁺) calculated for C₂₅H₂₂BrNO₅S [M + Na]⁺: 551.0, found 551.9 Purity 98%.

Compound 24

Ethyl 4-((2,2-dimethyl-4-(p-tolyl)-2H-chromene)-6-sulfonamido)-2,6-difluorobenzoate (78).

Compound 78 was prepared from 76 (0.175 g, 0.501 mmol, 1.00 equiv) and ethyl 4-amino-2,6-difluorobenzoate (0.121 g, 0.601 mmol, 1.20 equiv) following the procedure described for compound 22 to yield 0.170 g (75%) of the target compound as an off-white solid; mp 138 – 139 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.64 (dd, J = 8.6, 2.4 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H), 7.22 (d, J = 7.8 Hz, 2H), 7.07 (dd, J = 7.6, 5.7 Hz, 3H), 6.91 (d, J = 8.6 Hz, 1H), 6.66 (d, J = 9.3 Hz, 2H), 5.64 (s, 1H), 4.38 (q, J = 7.1 Hz, 2H), 2.40 (s, 3H), 1.50 (s, 6H), 1.37 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.0, 161.2, 160.4, 158.3, 141.4, 138.4, 133.9, 133.3, 130.1, 129.6, 129.4, 128.7, 128.3, 125.2, 122.9, 106.6, 102.6, 102.3, 77.9, 62.0, 28.2, 21.3, 14.3.

4-((2,2-Dimethyl-4-(p-tolyl)-2H-chromene)-6-sulfonamido)-2,6-difluorobenzoic Acid (24).

To 78 (0.090 g, 0.17 mmol) in EtOH (4 mL) was added 20% NaOH (aqueous, 1.6 mL) at room temperature and then the reaction mixture was stirred for 16 h. Next, the reaction was acidified with HCl (1N, aqueous solution) to pH 1. Volatiles were evaporated under reduced pressure. The resulting mixture was diluted with water (15 mL) and extracted with EtOAc (3 × 15 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, filtered, and purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes:EtOAc, 1:0-0:1) to furnish targeted acid 24 (45 mg, 53%) as a colorless solid; mp 209 – 211 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.69 (dd, J = 8.5, 2.3 Hz, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.25 (d, J = 7.7 Hz, 2H), 7.09-7.03 (m, 2H), 6.97 (d, J = 8.6 Hz, 1H), 6.77-6.69 (m, 2H), 5.74 (s, 1H), 2.41 (s, 3H), 1.49 (s, 6H). ¹³C NMR (101 MHz, MeOH-d₄) δ 164.1, 161.4, 159.0, 144.1, 139.4, 135.4, 134.5, 132.0, 131.2, 130.5, 129.6, 129.3, 125.6, 123.9, 118.5, 103.3, 103.0, 78.8, 28.1, 21.2. LRMS m/z (ES⁺) calculated for [M + Na]⁺: 508.1, found 508.0. Purity 99%.

Compound 25

Ethyl 4-((8-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene)-6-sulfonamido)-2,6-difluorobenzoate (79).

Compound 79 was prepared from 77 (0.200 g, 0.467 mmol, 1.00 equiv) and 4-amino-2,6-difluorobenzoate (0.122 g, 0.608 mmol, 1.30 equiv) following the procedure described for compound 78 to furnish 0.160 g (60%) of target compound 79 as a colorless solid; mp 173 – 174 °C. ¹H NMR (400 MHz, CDCl³) δ 7.90 (d, J = 2.2 Hz, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.21 (d, J = 7.7 Hz, 2H), 7.05-6.99 (m, 3H), 6.66 (d, J = 9.5 Hz, 2H), 5.68 (s, 1H), 4.40 (q, J = 7.1 Hz, 2H), 2.40 (s, 3H), 1.55 (s, 6H), 1.38 (t, J = 7.1 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.1, 163.0, 161.2, 160.5, 160.4, 155.1, 141.1, 141.0, 140.9, 138.7, 133.5, 133.2, 131.6, 130.6, 130.3, 129.7, 128.3, 124.1, 123.9, 112.0, 107.1, 106.9, 106.7, 102.7, 102.4, 79.4, 77.3, 62.1, 29.8, 28.3, 21.3, 14.3.

4-((8-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene)-6-sulfonamido)-2,6-difluorobenzoic Acid (25).

Acid 25 (57 mg, 60%, off-white solid; mp 222 – 223 °C, purity >99%) was prepared from 79 (0.100 g, 0.169 mmol, 1.00 equiv) in EtOH (4 mL) and 20% NaOH (aqueous, 1.60 mL) following the procedure described for compound 24. ¹H NMR (400 MHz, MeOH-d₄) δ 7.91 (d, J = 2.2 Hz, 1H), 7.27-7.20 (m, 3H), 7.06-6.99 (m, 2H), 6.73 (d, J = 9.8 Hz, 2H), 5.79 (s, 1H), 2.40 (s, 3H), 1.53 (s, 6H). ¹³C NMR (101 MHz, MeOH-d₄) δ 164.0, 163.9, 161.49, 161.40, 155.6, 143.7, 139.6, 134.9, 134.4, 133.0, 132.2, 131.9, 130.5, 129.3, 125.2, 124.4, 112.4, 103.5, 103.2, 80.4, 28.1, 21.2. UPLC-MS m/z (ES⁺) calculated for C₂₅H₂₀BrF₂NO₅SNa (M + Na)⁺ 586.0, found 586.0. LRMS m/z (ES⁺) calculated for C₂₅H₂₁BrF₂NO₅S [M + H]⁺: 564.0, found 564.0. Purity 99%.

Scheme 12. Synthesis of sulfonamide 26

Compound 26

6-Iodo-2,2-dimethylchroman-4-one (80) [41].

Compound 80 (2.80 g, 81%, brown oil) was prepared from 1-(2-hydroxy-5-iodophenyl)ethan-1-one (2.97 g, 11.3 mmol, 1.00 equiv), acetone (1.27 mL, 17.3 mmol, 1.53 equiv) and pyrrolidine (1.47 mL, 17.7 mmol, 1.56 equiv) following the procedure described for compound 70. ¹H NMR (400 MHz, CDCl₃) δ 8.12 (d, J = 2.3 Hz, 1H), 7.68 (dd, J = 8.7, 2.3 Hz, 1H), 6.69 (d, J = 8.7 Hz, 1H), 2.69 (s, 2H), 1.43 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 191.1, 159.6, 144.4, 135.2, 122.1, 120.9, 82.9, 79.7, 48.5, 26.6.

6-Iodo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (82).

Compound 82 (1.79 g, 51%, off-white solid; mp 70 –71 °C) was prepared from 2,2-dimethylchroman-4-one 80 (1.70 g, 5.62 mmol, 1.00 equiv), p-tolylmagnesium bromide (11.2 mL, 1.0 M solution in THF, 11.2 mmol, 2.00 equiv) and pyridinium p-toluenesulfonate (0.282 g, 1.12 mmol, 0.200 equiv) following the procedure described for compound 74. ¹H NMR (400 MHz, CDCl₃) 7.47 (dd, J = 8.5, 2.2 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.26 (s, 4H), 6.70 (d, J = 8.5 Hz, 1H), 5.64 (s, 1H), 2.45 (s, 3H), 1.52 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) 153.4, 137.8, 134.8, 134.0, 133.8, 129.6, 129.3, 128.5, 125.1, 119.3, 82.8, 76.2, 27.7, 21.3.

2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-amine (83).

A mixture of compound 82 (0.300 g, 0.790 mmol), 28% NH₃ (aqueous, 0.0720 mL, 1.20 mmol), CuI (30.0 mg, 0.160 mmol) and L-proline (36.0 mg, 0.0320 mmol) in DMSO (5 mL) was stirred at r.t. under N₂ for 48 h. Then, water (25 mL) was added to the reaction mixture and the biphasic solution was extracted 3 times with EtOAc. The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, evaporated under reduced pressure, and purified using silica gel (RediSep Flash Columns, Teledyne ISCO, Lincoln, NE) flash column chromatography (hexanes) to give aniline 83 (70.0 mg, 33%) as a brown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.27-7.17 (m, 4H), 6.73 (d, J = 8.4 Hz, 1H), 6.56 (dd, J = 8.4, 2.8 Hz, 1H), 6.41 (d, J = 2.7 Hz, 1H), 5.60 (s, 1H), 3.50 (s, 2H), 2.41 (s, 3H), 1.46 (s, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 146.4, 139.3, 137.5, 135.7, 134.7, 129.7, 129.1, 128.8, 123.3, 117.5, 116.4, 113.0, 75.3, 27.3, 21.3.

4-(N-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)sulfamoyl)benzoic Acid (26).

Acid 26 was obtained as a colorless solid (56 mg, 55%); mp 221 – 222 °C by reaction of 83 (60 mg, 0.23 mmol, 1.0 equiv) and 4-(chlorosulfonyl)benzoic acid (50 mg, 0.22 mmol, 1.0 equiv) following the procedure described for compound 22. ¹H NMR (400 MHz, MeOH-d₄) δ 8.11 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 7.8 Hz, 2H), 6.99 (dd, J = 8.6, 2.6 Hz, 1H), 6.93-6.86 (m, 2H), 6.76 (d, J = 8.6 Hz, 1H), 6.41 (d, J = 2.5 Hz, 1H), 5.60 (s, 1H), 2.37 (s, 3H), 1.42 (s, 6H). ¹³C NMR (101 MHz, MeOH-d₄) δ 168.2, 153.0, 144.6, 138.7, 136.0, 135.9, 135.4, 131.2, 130.5, 130.4, 130.0, 129.3, 128.4, 127.0, 124.0, 122.5, 118.3, 77.1, 27.6, 21.2. LRMS m/z (ES⁺) calculated for C₂₅H₂₃NO₅SNa [M + Na]⁺: 472.1. Found 472.0. Purity 99%.

Highlights.

• Synthesis and evaluation of 26 retinoic acid receptor antagonists for potency and selectivity.

• Chromene analog 21 was the most potent and selective RARα antagonist identified.

• Compound 21 inhibited of spermatogenesis but was not effective in mating studies.

• Amides had best potency and selectivity. Reversed amides, urea and sulfonamides were inactive.

• Compounds 3 and 6 with small antagonist pocket groups (phenyl, thiophene) are selective agonists for RARβ.

Abbreviations

A-B

apical to basolateral

ATRA

all-trans retinoic acid

B-A

basolateral to apical

decomp

decomposition

EDCI

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

E_max

concentration required for the maximum activation

HOBT

hydroxybenzotriazole

K_d

dissociation constant

NaHMDS

sodium bis(trimethylsilyl)amide

RARα

retinoic acid receptor alpha

RARβ

retinoic acid receptor beta

RARγ

retinoic acid receptor gamma

TLC

thin layer chromatography