Abstract
The retinoic acid receptor alpha (RARα) has emerged as a compelling genetically and pharmacologically validated target for nonhormonal male contraception due to its essential role in spermatogenesis. In the present study, a search for specific inhibitors of RARα utilized systematic linker bioisosterism, hydrophobic core modification, and iterative structure–activity relationship refinement, identified the acid 9, a pyrrole-linked analog that potently inhibits RARα (IC50 = 1.2 nM) with >300-fold selectivity over RARβ and RARγ. Sprague–Dawley rat studies with the sodium salt of 9, YCT-529, showed good oral bioavailability and dose-proportional pharmacokinetics without drug accumulation after 28 days of dosing. Once-daily oral administration (0.75 mg/kg for 28 days) reversibly suppressed epididymal sperm counts and fertility in rats, with a no-observed-adverse-effect level at 30 mg/kg (highest dose tested), affording a ≥40-fold therapeutic window. These findings validated aromatic linker substitution as a powerful design strategy for identifying RARα antagonists and led to the clinical advancement of YCT-529 as a nonhormonal male contraceptive.
Introduction
Modern birth control methods have transformed family planning and reduced pregnancy-related health risks, yet the heavy reliance on female contraceptives highlights an unmet need for male options. Globally, over 40% of pregnancies are unintended, primarily due to factors such as limited access, improper use, and inherent failure rates of current methods. , Current male contraceptive options are limited to condoms, which exhibit a 13% annual failure rate under typical use, and vasectomy, a surgical solution that is difficult to reverse. , The imbalance in contraceptive responsibilities and limited male contraceptive options has spurred interest in developing effective, reversible male contraceptives that could enhance reproductive autonomy for men and contribute to a more equitable sharing of contraceptive responsibility.
Male contraceptive research has pursued both hormonal and nonhormonal strategies. Hormonal approaches, typically involving testosterone or testosterone/progestin combinations, aim to reduce sperm production but often encounter issues such as suboptimal efficacy and side effects, including acne, weight gain, and mood changes. − Recently, a transdermal gel delivering nestorone and testosterone that is well-tolerated has emerged as the most advanced hormonal male contraceptive in clinical development. An alternative approach involving nonhormonal targeting of proteins uniquely essential for sperm production and function could thereby avoid the systemic side effects associated with hormonal treatments. Promising targets in this realm include the ion channels Na, K-ATPase α4, CatSper, and Slo3, enzymes such as soluble adenylyl cyclase, − cyclin-dependent kinase 2, serine/threonine kinases 33, structural and signaling molecule junction plakoglobin, key transcriptional regulatory proteins like BRDT, and the Retinoic acid (RA) receptor alpha (RARα), which play crucial roles in spermatogenesis.
All-trans RA (ATRA), an endogenous metabolite of vitamin A and β-carotene, is crucial for spermatogenesis, spermatogonial differentiation, and spermiation. An enzyme involved in this metabolic conversion, retinaldehyde dehydrogenases (RAD2), was shown to be inhibited by the drug WIN 18,446 (2, Figure ), resulting in a reduction in sperm counts in men. However, this drug was not pursued for its potential as a male contraceptive because of concomitant severe disulfiram reactions caused by nonselective inhibition of liver aldehyde dehydrogenase-2 (ALDH2) in addition to testes-specific ALDH1A2. RA exerts its effects through two nuclear-receptor families: RA receptors (RARs) and retinoid X receptors (RXRs). RARs are primarily activated through the binding of all-trans-retinoic acid (ATRA) (1, Figure ), while RXRs are exclusively activated by 9-cis RA. Among the three RAR isoforms (α, β, γ), RARα and RARγ are indispensable for male reproductive function. Rara –/– and Rarg –/– knockout mice are sterile and otherwise normal, whereas Rarb –/– male mice retain fertility, highlighting RARα and RARγ as compelling targets for nonhormonal male contraception. − Efforts to inhibit RARs led to the development of a series of RAR inhibitors that function by antagonizing ATRA. BMS-189453 (3, Figure ), a nonselective RAR antagonist that inhibits both RARα and RARγ while exhibiting partial agonist activity at RARβ, was shown to induce reversible sterility in rodents after oral administration. However, α-selective antagonists such as BMS-189532 (4) and BMS-189614 (5) had limited effectiveness in vivo due to poor oral bioavailability and testicular permeability. BMS-189453 (3), BMS-189532 (4), and BMS-189614 (5) all share a dihydronaphthalene scaffold. In contrast, analog 6 in Figure , a novel RARα-selective antagonist previously reported by our group, features a chromene scaffold. This compound exhibits potent antagonistic activity and induces distinct, yet modest, effects on spermatogenesis. Further optimizations based on the chromene scaffold yielded YCT-529 (the sodium salt of analog 9,Table ), the first orally bioavailable, effective, reversible, and well-tolerated selective RARα antagonist. , The first-in-human Phase I clinical trial for YCT-529, initiated in December 2023, was completed in June 2024 (NCT06094283). This was followed by a Phase 1b/2a clinical trial (NCT06542237), which commenced in September 2024 to evaluate its safety and efficacy. Here, we detail the design, synthesis, and structure–activity relationship (SAR) campaigns that led to the discovery of YCT-529.
1.
Structures of RAR pathway inhibitors.
1. In Vitro Antagonism Activity of RAR Antagonists with Linker Derivatizations.
Results and Discussion
Design of Analogs
Analog 6 is composed of three essential structural elements that mimic key features of ATRA and are crucial for high-affinity binding and proper orientation within the RAR ligand binding domain (LBD). , These elements include (Figure ): (1) a bicyclic hydrophobic core that engages in van der Waals interactions with a hydrophobic cavity in the LBD; (2) a terminal benzoic acid group that forms polar contacts with Ser287 and Arg276; and (3) a linker region that maintains the optimal spatial arrangement between these functional groups. Previous studies have demonstrated that the nature of the linker significantly influences subtype selectivity among synthetic retinoids. Notably, hydrogen bond-donating linkers, such as an amide, can engage the hydroxyl group of Ser232, a residue unique to the RARα LBD, via the amide NH group. In contrast, the corresponding position in RARβ and RARγ features alanine, which lacks hydrogen-bonding capability through the side chain. , This structural difference has facilitated the development of several amide-linked RARα-selective ligands with diverse agonist and antagonist profiles. − Structural analysis further revealed that agonist binding stabilizes the active conformation of helix 12 (H12), promoting coactivator recruitment and subsequent transcriptional activation. , In contrast, antagonists that bear sterically demanding substituents on the hydrophobic core shift H12 into an inactive conformation, thereby hindering coactivator binding and suppressing gene expression.
2.
SAR summary for amide analog 6.
As shown in Figure , comparison of the in vivo active antagonist BMS-189453 (3) with the in vivo inactive analogs BMS-189532 (4), BMS-189614 (5), and amide analog 6 reveals a key difference in linker composition. The orally active BMS-189453 (3) contains a hydrophobic alkene linker, whereas the antagonists with limited oral activities possess polar amide linkers. We hypothesize that the limited in vivo efficacy associated with amide-containing antagonists, such as analog 6, results from unfavorable physicochemical properties that restrict their uptake into testicular tissue. Thus, replacement of the amide linker with bioisosteric linkers shown in Figure , capable of hydrogen bonding with the key residue Ser232 to maintain RARα selectivity, could improve testicular exposure and therefore confer in vivo efficacy.
3.
Bioisosteric replacement for the amide group of analog 6.
In Vitro Testing of Analogs
We evaluated the agonist and antagonist potencies of new compounds against human RAR subtypes (RARα, β, γ) using GeneBLAzer reporter cell assays. These reporter cells express the recombinant RAR LBDs fused to a Gal4 DNA-binding domain, driving β-lactamase gene expression upon agonist binding. β-Lactamase concentration was quantified using the cell-permeable fluorescence resonance energy transfer (FRET) substrate CCF4-AM. Increased agonist concentrations enhance β-lactamase expression, thereby reducing the FRET signal and allowing determination of agonist EC50 values. For antagonist studies, cells were stimulated with the natural ligand ATRA at its EC80 concentration (0.8 nM), along with varying concentrations of test compounds. The assay setup we employed allowed antagonist testing at a maximum concentration of 430 nM, which is sufficient to assess RARα selectivity over RARβ and RARγ.
The in vitro antagonism activity of RAR antagonists with various linker derivatizations is summarized in Table . We evaluated analog 6 for antagonism activity against RARα, β, and γ, confirming its potency and selectivity as a benchmark compound. Analog 6 exhibited an IC50 of 0.54 nM against RARα and demonstrated >790-fold and 37-fold selectivity over RARβ and RARγ, respectively. These results differ from our previous findings using the INDIGO cell-based luciferase reporter assay (RARα: 4.6 nM, RARβ: >3300 nM, RARγ: 310 nM), which is likely due to the increased sensitivity of the GeneBLAzer assay. The GeneBLAzer system requires a lower concentration of the agonist (ATRA), leading to lower IC50 values for the tested RAR antagonists. Our data show that the GeneBLAzer assay decreased the IC50 value for RARα of analog 6 8-fold and 16-fold for RARγ, compared to the INDIGO assay, while maintaining selectivity. Similarly, the data obtained with the INDIGO cell-based luciferase reporter assay for compound 9, the free acid of YCT-529 (RARα: 6.8 nM, RARβ: >3750 nM, RARγ: >3750 nM) led to a 6-fold lower IC50 value of 1.2 nM for RARα inhibition in the GeneBLAzer assay. We had previously shown that analog 6 has excellent metabolic stability in male mouse liver microsomes (100% remaining after 60 min) compared to BMS189532 (4) (49% remaining after 60 min), which encouraged us to continue the exploration of chromene analogs. Analog 6 served as the reference compound for comparison with its derivatives.
Linker Modifications
We initially explored modifications to the linker region of analog 6 using a bioisosteric approach, replacing the amide moiety with alternative hydrogen-bond donor or acceptor bioisosteres that are known to enhance potency and selectivity, and investigated novel chemical space. Two structurally related sulfonamide analogs, 7 and 8 (Table ), lacked antagonist activity toward RARα, β, and γ. This inactivity suggested that the tetrahedral geometry of the sulfonyl group is incompatible with the planar RAR LBD, likely causing steric clashes. Subsequently, we designed and synthesized compound 9 (Table ), featuring a planar pyrrole linker wherein the NH could act as a hydrogen-bond donor that could interact with Ser232. Compound 9 displayed potent antagonist activity against RARα (IC50 = 1.2 nM) with substantially improved selectivity compared to analog 6, exhibiting greater than 358-fold and 308-fold selectivity over RARβ and RARγ, respectively. However, replacing the pyrrole ring with an imidazole ring, as seen for 10 (Table ), resulted in a marked reduction in antagonist activity.
We then investigated whether relocating the NH group in the pyrrole linker could induce a different binding orientation and affect potency and selectivity. This was tested with the 2,4-substituted pyrrole 11 (Table ), but the modification reduced RARα potency 10-fold and was accompanied by a loss of selectivity. Introducing an additional nitrogen in imidazole 10 abolished activity, whereas the 3,5-disubstituted 1H pyrazole 12 retained significant activity at RARα, but lost selectivity. Analogs 11 and 12 showed significantly reduced selectivity over RARβ and RARγ, indicating that the NH position is critical for RARα selectivity, with compound 9’s NH position being optimal.
Recognizing the critical role of the H-bond interaction with the hydroxyl group in Ser232 for RARα selectivity, we explored the possibility of forming this interaction through H-bond acceptor linkers. Since the hydroxyl hydrogen of Ser232 can act as an H-bond donor, we designed and synthesized compounds with H-bond acceptor linkers, including oxadiazole (13 and 14), tetrazole (15), furan (16), and triazole (17) (Table ). However, these compounds exhibited significantly reduced potency and selectivity over RARβ and RARγ. Moreover, compounds 13 and 15 even showed a slight preference for RARβ antagonism. This suggests that these H-bond acceptors do not engage in the desired interaction with Ser232 and fail to confer RARα selectivity. Analog 16, where the pyrrole was substituted with a furan moiety, lost potency across all subtypes, demonstrating the need for the NH of the pyrrole for optimal binding affinity.
Next, we evaluated analog 18 (Table ), featuring an alkene (isopropylene) linker present in RA, to mimic the natural ligand and to validate the importance of the H-bond interaction formed by the linker moiety in conferring RARα selectivity. As anticipated, analog 18 exhibited activity as an antagonist but lacked selectivity, displaying similar single-digit nanomolar IC50 values across all three RAR subtypes.
Given the excellent activity and selectivity demonstrated by compound 9, we utilized the induced fit docking function in the Schrödinger Small Molecule Drug Discovery Platform Maestro to predict the binding mode of 9 with RARα. As shown in Figure , compound 9 is expected to form a critical hydrogen bond with the unique Ser232 residue in RARα, conferring its selectivity for this receptor. Additionally, compound 9 establishes another hydrogen bond with Ser287 via its carboxylic acid group and forms a salt bridge with Arg276. Pi–pi stacking interactions between the chromene bicyclic core and Phe228, as well as between the benzoic acid ring and Phe286, further contribute to its high binding affinity with RARα. Based on this predicted binding mode, we pursued further derivatization of 9, focusing on modifications to the toluene moiety, chromene core, and benzoic acid group.
4.
Predicted binding pose of compound 9 with RARα (PDB: 1DKF). Polar interactions and pi–pi stacking interactions are shown as blue dashed lines. The hydrophobic pocket is shown in green. Helix 12 is shown in yellow. Compound 9 forms hydrogen bonds with Ser232 through the pyrrole NH. Superimposed at the same position, alanine of RARβ (PDB: 4JYI) and γ (PDB: 3LBD) are shown in magenta and cyan, respectively, demonstrating a lack of H-bond formation.
RAR Antagonists with Antagonism Moiety Derivatizations
Sterically demanding substituents, such as the toluene moiety in compounds 9 and MN-256 (6), are critical for inducing antagonism in retinoids by sterically disrupting the H12 helix and locking H12 in an inactive conformation. Various functional groups, such as phenyl, toluene, and quinoline, have been reported to induce antagonism. Further optimization efforts to improve the physicochemical properties of retinoids involved incorporating hydrophilic groups like morpholine, piperidine, and piperazine onto the phenyl ring, as demonstrated for the derivatization of ER-50891 and BMS-185411. , The antagonist region of the pocket is solvent-exposed in the antagonist-bound conformation and therefore should be able to tolerate hydrophilic substitutions. However, these modifications often resulted in significantly reduced RAR antagonistic activity. Consequently, our efforts prioritized introducing minimal structural changes to the antagonism pharmacophore to maintain biological activity while concurrently exploring structural modifications to enhance physicochemical properties. Using computational binding predictions with the Glide docking function from the Schrödinger Maestro suite, minor changes to the methyl group of the toluene moiety were designed and prioritized based on predicted binding affinities.
We first explored the optimal size of the antagonism moiety’s phenyl group by substituting the toluene methyl group with larger groups, including ethyl (19), isopropyl (20), and tert-butyl (21) (Table ). These modifications resulted in a ∼ 5-fold reduction in RARα activity for each additional methyl group, indicating that the toluene group in 9 provides the best fit within the RARα LBD. Next, we examined smaller substituents, such as a fluorine group. The resulting analog, 22 (Table ), improved antagonism activity against RARα with an IC50 in the subnanomolar range. Compound 22 also maintained high selectivity for RARα, showing no measurable antagonism against RARβ or RARγ at concentrations up to 430 nM.
2. In Vitro Antagonism Activity of RAR Antagonists with Antagonism Moiety Derivatizations.
We further investigated trifluoromethyl substitution (23, Table ), as the methyl group in the toluene moiety of 9 was predicted to be a metabolic soft spot by SMARTCyp calculations. The trifluoromethyl group could potentially block metabolism while preserving structural similarity. Compound 23 exhibited an IC50 of 3.2 nM against RARα and retained high selectivity for RARβ and RARγ.
In addition, we explored polar functional group substitutions on the toluene ring for potential interactions with nearby residues Ser229 and Thr233, while also reducing lipophilicity to improve drug-likeness. This effort included hydroxyethyl 24, methoxy 25, and nitro 26 (Table ). Among these, the methoxy analog 24 yielded the best results, with an IC50 of 1.9 nM against RARα and preserved selectivity over RARβ and RARγ. However, the other polar derivatives 24 and 26 exhibited reduced potency compared to compound 9, likely due to suboptimal binding of polar groups or their high desolvation penalty.
Analogs with Chromene Derivatizations
Next, we examined derivatizations for the chromene scaffold, as summarized in Table . Initially, we designed and synthesized compounds with substitutions aimed at filling an empty pocket near the chromene C8 position observed in the crystal structure of RAR (PDB 1DKF). Furthermore, the introduction of a bromo substituent at this position has previously been shown to enhance potency. We therefore introduced bromo, chloro, and methyl groups. The resulting analogs, 27, 28, and 29 (Table ), exhibited slightly reduced activity against RARα while maintaining high selectivity over RARβ and RARγ compared with 9. We also introduced hydroxy substitution at the chromene C7 position, yielding 30 (Table ), because similar modifications were reported to improve the potency of RAR agonists AGN 194078 and AGN 195183, presumably through the additive effects of the interaction of both the amide and the hydroxy substitution with S232 of RARα. However, introducing a hydroxy group to the C7 position led to a significant decrease in RARα antagonism, with the IC50 increasing approximately 40-fold. These results indicate that derivatizations at the chromene C8 and C7 positions were not beneficial for potency.
3. In Vitro Antagonism Activity of RAR Antagonists with Chromene Derivatizations.
Next, we investigated the substitution of the gem-dimethyl group in compound 9. Replacing the gem-dimethyl group with a gem-diethyl group, as seen with 31 (Table ), resulted in a ∼10-fold reduction in RARα activity, while complete removal of the gem-dimethyl group rendered compound 32 (Table ) inactive. These findings underscore the importance of the gem-dimethyl group for optimal binding affinity of the chromene scaffold.
We then explored a ring-opening strategy to modify the chromene core. The benzophenone derivative 33 retained RARα antagonism potency, whereas the diphenylethylene derivative 34 was inactive (Table ). Interestingly, introducing an ortho-hydroxy substitution to the benzophenone core, as seen with 35 (Table ), slightly improved potency. However, incorporating alkoxy groups led to a 5- to 10-fold reduction in activity, as observed with 36 and 37 (Table ). Docking studies suggest that the phenolic hydroxy group in compound 35 may form an intramolecular hydrogen bond with the carbonyl group of the benzophenone (Figure S1), which could stabilize the binding conformation and account for its superior activity compared to compounds 36 and 37.
Compound 38 (Table ), featuring a dihydronaphthalene core in place of the chromene core, was also designed and synthesized. The dihydronaphthalene motif is commonly utilized in known retinoids with amide or alkene linkers to interact with the hydrophobic pocket effectively. However, compound 38 was found to be inactive against all three RAR receptors. This lack of activity may be attributed to the structural differences between the linear amide and alkene linkers and the cyclic pyrrole linker, which alter the overall angle and distance between the hydrophobic core and the benzoic acid ring. These changes likely render the dihydronaphthalene core incompatible with the pyrrole-based compounds.
RAR Antagonists with Carboxylic Acid Derivatizations
The terminal carboxylic acid group is critical for the binding efficiency of RAR agonists and antagonists, as it establishes a salt bridge with Arg276 and a hydrogen bond with Ser287. , However, due to the potential liabilities associated with free carboxylic acids in drug development, such as permeability issues and fast metabolism by glucuronidation, we designed and synthesized two derivatives, analog 39, featuring a terminal cyanide group, and analog 40, incorporating an amide group (Table ). These modifications aimed to retain the ability to form polar interactions with Arg276 and Ser287 while eliminating ionizability. Both analogs failed to exhibit RAR activity, further underscoring the indispensable role of the terminal carboxylic acid group in maintaining RAR activity.
4. In Vitro Antagonism Activity of RAR Antagonists with Carboxylic Acid Derivatizations.
Compound 9 was selected for further development and tested in vivo as it was the first potent and highly selective compound that we discovered during our SAR campaign. As shown above in Table , several other closely related analogs, such as the 4-fluor-derivative 22, the 4-CF3-derivative 23, and the 4-OMe-derivative 25, are also potent and selective antagonists. However, since it was found that the tolyl group in YCT-529 (9) is not a metabolic liability, analogs 22, 23, and 25 do not offer any advantage from a metabolism perspective. Analog 22 is 2.6-fold more potent than 9, but this was not considered a significant advantage over 9, which we had already selected as a promising candidate to take forward by the time we prepared and tested analog 22. The benzophenone analogs 33, 35, 36, and 37 (Table ) represent a new scaffold for retinoids. While a limited number of marketed drugs feature this moiety, benzophenone is a phototoxic compound, and several of its analogs have been found to have carcinogenic, endocrine, reproductive, and developmental effects. Furthermore, benzophenone was removed by the FDA in 2018 from the list of approved food additives because it was shown to be carcinogenic at high doses in animals. However, an in vitro study of benzophenone analogs showed that 2-hydroxybenzophenone is not phototoxic and has a better safety profile than other benzophenones. Therefore, analog 35, which potently and selectively inhibits RARα with an IC50 of 0.69 nM and carries a 2-hydroxy-benzophenone moiety, could be a potential back-up compound for YCT-529, but its potential liabilities would have to be monitored closely.
In Vivo Evaluation of Compound YCT-529
For the in vivo evaluation, we converted the carboxylic acid 9 into its sodium salt to improve solubility, yielding YCT-529.We previously reported that the orally active RARα-selective antagonist YCT-529 effectively reduced sperm counts in male mice and nonhuman primates and was 99% effective in inducing infertility in mouse mating studies at a dose of 10 mg/kg which, importantly, was reversible. YCT-529 further showed an excellent safety profile in humans in a Phase I clinical trial. To investigate the effects of YCT-529 in an additional animal species and because rats are commonly used for preclinical toxicological studies to obtain regulatory approval for conducting clinical trials with new drug candidates, we evaluated the pharmacokinetic (PK) properties, efficacy in lowering sperm counts, and contraceptive efficacy in mating studies in sexually mature Sprague–Dawley rats.
Rat Pharmacokinetics and Efficacy Study after 28 Day Repeat Dose Administration
Pharmacokinetics (PK) parameters were assessed in animals from the efficacy study at dose levels between 0.25 and 1 mg/kg/day (Figure A,B, Tables S1 and S2) and the safety study at dose levels between 3 and 30 mg/kg/day (Figure C,D, Tables S1 and S2). These results indicate that YCT-529 became readily bioavailable at all dose levels (0.25–30 mg/kg/day) without drug accumulation over 28 day dose administration, which was a good starting position for subsequent efficacy and safety studies.
5.
Plasma concentration time-curves of YCT-529 in rat plasma after repeat-dose administration for 28 days. Blood was drawn from all animals that received YCT-529 at the indicated time points to assess 24 h PK profiles on Day 1 and Day 28. Concentrations of YCT-529 were assessed with LC–MS/MS. Shown are means ± SD of 3 animals per time point of the efficacy study on Day 1 (A) and Day 28 (B) and of the safety study on Day 1 (C) and Day 28 (D).
In the animals of the efficacy study, maximum plasma concentrations (C max) on Day 1 were 50.7 ng/mL (0.25 mg/kg/day), 124 ng/mL (0.5 mg/kg/day), 177 ng/mL (0.75 mg/kg/day), and 244 ng/mL (1 mg/kg/day) (Figure A and Table S1). On Day 28, C max values were very similar: 80.2 ng/mL (0.25 mg/kg/day), 139 ng/mL (0.5 mg/kg/day), 180 ng/mL (0.75 mg/kg/day), and 281 ng/mL (1 mg/kg/day) (Figure B and Table S1). The time to reach C max (T max) on Day 1 was 8 h (0.25, 0.5, and 0.75 mg/kg/day), and 2 h (1 mg/kg/day) after administration of YCT-529 (Figure A and Table S1). On Day 28, T max values were 2 h (0.25 and 0.5 mg/kg/day) and 8 h (0.75 and 1 mg/kg/day) after administration of YCT-529 (Figure B and Table S1). The calculated systemic half-life (T 1/2) on Day 28 was 12.6 h (0.25 mg/kg/day) and 10 h (0.5 mg/kg/day). T 1/2 could not be calculated for 0.75 and 1.0 mg/kg/day due to fewer than 3 quantitative time points. T max, absorption (C max) and systemic exposure (area under the plasma concentration–time curve from time zero to 24 h post dose [AUC0–24h]) increased dose-proportionally on Day 1 and Day 28 as the dose was increased from 0.25 to 1.0 mg/kg/day. No marked drug accumulation in systemic exposure was observed at any dose level (Figure A,B, Table S2).
Rat Safety Study
In the animals from the safety study, C max on Day 1 was 693 ng/mL (3 mg/kg/day), 3150 ng/mL (10 mg/kg/day), and 9310 ng/mL (30 mg/kg/day) (Figure C and Table S1). C max on Day 28 was 1040 ng/mL (3 mg/kg/day), 3060 ng/mL (10 mg/kg/day), and 6770 ng/mL (30 mg/kg/day) (Figure D and Table S1). T max was reached at 4 h (3 mg/kg/day) and 8 h (10 and 30 mg/kg/day) on Day 1 (Figure C and Table S1) and at 4 h (3 and 30 mg/kg/day) and 8 h (10 mg/kg/day) after administration of YCT-529 on Day 28 (Figure D and Table S1). T 1/2 was not calculated due to fewer than 3 quantitative time points after T max. C max and AUC0–24h increased dose-proportionally on Day 1 and Day 28 as the dose was increased from 3 to 30 mg/kg/day. No marked drug accumulation in systemic exposure was observed at any dose level (Figure C,D and Table S2).
Efficacy of YCT-529 in Affecting Sperm Counts and Sperm Motility
Having demonstrated its bioavailability, we next determined whether administration of YCT-529 (0, 0.25, 0.5, 0.75, or 1 mg/kg/day) for 28 days affected the levels of epididymal sperm concentration and motility and the histopathology of testes and epididymides. These end points were evaluated over a 14 day mating period that followed the 28 day dosing period, and over a 14 day mating period that followed the 180 day recovery period to determine reversibility. At the end of the dosing phase, decreased sperm concentration (Figure A) and sperm motility (Figure B) were noted at ≥0.75 mg/kg/day (light gray bars in Figure ). Vitamin A is important for spermatogenesis and also known to play a role in the rat epididymis. Although the exact role of vitamin A in the epididymis remains unknown, it is assumed that it plays an important role for epididymal sperm maturation and sperm motility. Study results point to a region-specific role in the proximal caput and the distal cauda. ,
6.
Rat sperm parameters after 28 day repeat dosing with YCT-529 at 0.25–1 mg/kg/day and after 180 day recovery. Shown are means ± SD per dose group from 10 animals per time point for sperm concentrations (A) and the percentage of immotile sperm cells (B). Light and dark gray bars represent assessments at the end of the dosing period and at the end of the recovery period, respectively. ***p ≤ 0.001 (Kruskal–Wallis and Dunnet on Ranks).
Compared to control, sperm concentrations were reduced from 3.78 ± 1.09 × 108/g to 0.98 ± 0.80 × 108/g (0.75 mg/kg/day) and 0.51 ± 0.46 × 108/g (1 mg/kg/day). Conversely, the percentage of immotile sperm cells increased from 25 ± 7% to 79 ± 13% (0.75 mg/kg/day) and 90 ± 11% (1 mg/kg/day). These changes in sperm concentration and sperm motility were statistically significant with a respective p value of ≤0.001.
At the end of the recovery phase, there were no significant changes in sperm concentration or sperm motility compared to control (dark gray bars in Figure ).
Changes in sperm concentration correlated with gross pathology and histopathology. At the end of the dosing phase, bilateral decreased mean testicular and epididymal weights were noted in animals that received YCT-529 at doses of ≥0.75 mg/kg/day (Table S3). Microscopically, minimal to marked degeneration/atrophy in the seminiferous tubules and/or spermatid retention was observed in the testes of animals dosed at ≥0.75 mg/kg/day. In the epididymis, minimal to moderate cellular debris was observed with dosing at ≥ 0.5 mg/kg/day, along with minimal to severe decreased sperm at ≥0.75 mg/kg/day (data not shown as images were not taken by the contracted study pathologist as per standard operating procedure). At the end of the recovery phase, testes and epididymides looked normal, indicating full recovery at 0.5 and 0.75 mg/kg/day. At the time of necropsy, 9 out of 10 animals of the 1.0 mg/kg/day group also displayed normal spermatogenesis (data not shown as images were not taken by the contracted study pathologist as per standard operating procedure).
Efficacy of YCT-529 in Affecting Male Fertility
To determine if decreased levels of sperm concentration and aberrant histopathological findings at the dosing regimen of ≥0.75 mg/kg/day for 28 days correlated with decreased male fertility. The male fertility index ([number of males impregnating a female/number of males cohabitating] × 100) was assessed and shown to decrease to 40% as compared to 100% in controls, 40% (0.75 mg/kg/day) and 30% (1.0 mg/kg/day) (Table ). These changes were statistically significant with respective p values of ≤0.05 and ≤0.01. The female fecundity index ([number of pregnant females/number of females with confirmed matings] × 100) was decreased to 40% (0.75 mg/kg/day) and 42.9% (1 mg/kg/day) compared to control (100%) (Table ). These changes were also statistically significant with a respective p-value of ≤0.05. In the high dose group (1 mg/kg/day), the number of viable embryos was significantly reduced compared to the control, with a p value of ≤0.05 (Table ).
5. Summary of Mating Data at the End of the Two-Week Dosing Period (Day 28) and at the End of the Recovery Period (Day 180).
| dose level (mg/kg/day) | 0 | 0.25 | 0.5 | 0.75 | 1.0 | |
|---|---|---|---|---|---|---|
| number of males with confirmed mating | day 28 | 10 | 9 | 10 | 10 | 7 |
| day 180 | 10 | 9 | 10 | 10 | 9 | |
| number of males cohabited | day 28 | 10 | 10 | 10 | 10 | 10 |
| day 180 | 10 | 10 | 10 | 10 | 10 | |
| number of males impregnating a female | day 28 | 10 | 7 | 8 | 4 | 3 |
| day 180 | 10 | 8 | 8 | 10 | 7 | |
| number of females with confirmed mating | day 28 | 10 | 9 | 10 | 10 | 7 |
| day 180 | 10 | 9 | 10 | 10 | 9 | |
| number of females cohabiting | day 28 | 10 | 10 | 10 | 10 | 10 |
| day 180 | 10 | 10 | 10 | 10 | 10 | |
| number of pregnant females | day 28 | 10 | 7 | 8 | 4 | 3 |
| day 180 | 10 | 8 | 8 | 10 | 7 | |
| male mating index | day 28 | 100 | 90 | 100 | 100 | 70 |
| day 180 | 100 | 90 | 100 | 100 | 90 | |
| male fertility index | day 28 | 100 | 70 | 80 | 40* | 30** |
| day 180 | 100 | 80 | 80 | 100 | 70 | |
| female fecundity index | day 28 | 100 | 77.8 | 80 | 40* | 42.9* |
| day 180 | 100 | 88.9 | 80 | 100 | 77.8 | |
Male mating index = (no. of males with confirmed mating/no. of males cohabitated) × 100.
Male fertility index = (no. of males impregnating a female/no. of males cohabitating) × 100.
Female fecundity index = (no. of pregnant females/no. of females with confirmed mating) × 100. * = p < 0.05. ** = p < 0.01 (Fisher’s Exact test).
6. Summary of Litter Data at the End of the Two-Week Dosing Period (Day 28) and at the End of the Recovery Period (Day 180).
| 0 | 0.25 | 0.5 | 0.75 | 1.0 | ||
|---|---|---|---|---|---|---|
| dose level (mg/kg/day) | mean ± SD (n) | |||||
| number of corpora lutea | day 28 | 20.4 ± 4.1 (10) | 20.4 ± 3.8 (7) | 16.1 ± 4.7 (8) | 17.0 ± 4.1 (4) | 20.5 ± 0.7(2) |
| day 180 | 16.9 ± 2.9 (10) | 18.8 ± 4.6 (8) | 17.5 ± 2.6 (8) | 17.8 ± 3.3 (10) | 20.7 ± 4.3 (7) | |
| number of implants | day 28 | 17.9 ± 2.5 (10) | 17.6 ± 2.8 (7) | 13.1 ± 5.7 (8) | 14.3 ± 5.1 (4) | 10.3 ± 8.1 (3) |
| day 180 | 14.4 ± 4.1 (10) | 16.5 ± 2.6 (8) | 16.3 ± 1.9 (8) | 14.9 ± 3.4 (10) | 16.1 ± 1.5 (7) | |
| number of viable embryos | day 28 | 17.3 ± 2.4 (10) | 16.0 ± 2.4 (7) | 12.4 ± 5.9 (8) | 12.8 ± 3.9 (4) | 9.0 ± 7.9* (3) |
| day 180 | 12.2 ± 6.0 (10) | 15.0 ± 3.3 (8) | 14.4 ± 5.0 (8) | 13.9 ± 4.0 (10) | 14.3 ± 2.5 (7) | |
| number of nonviable embryos | day 28 | 0.6 ± 1.0 (10) | 1.6 ± 1.3 (7) | 0.8 ± 1.4 (8) | 1.5 ± 2.4 (4) | 1.3 ± 0.6 (3) |
| day 180 | 0.7 ± 0.7 (10) | 1.5 ± 1.6 (8) | 1.9 ± 3.4 (8) | 1.0 ± 0.9 (10) | 1.9 ± 1.7 (7) | |
Inappropriate for statistics as per analysis of variance (ANOVA) and Dunnett. * p < 0.05 (Kruskal–Wallis and Dunnett on Ranks).
At the end of the recovery phase, male fertility indices and female fecundity recovered (Tables and ) and correlated with sperm counts that had returned to normal. These results demonstrated that YCT-529 efficaciously and reversibly impaired male fertility in Sprague–Dawley rats at a lowest observed effect dosing level of (LOEL) of 0.75 mg/kg/day.
Determination of Safety Parameters of YCT-529 in the Sprague–Dawley Rat Model
We next sought to assess key safety parameters of YCT-529 to determine its safety profile upon repeat-dose administration. Sexually mature male Sprague–Dawley rats received either saline or YCT-529 in saline at 3, 10, or 30 mg/kg/day once daily for 42 days. Safety end points were assessed on Day 43 (24 h after administration of the last dose). These standard assessments included viability (morbidity/mortality), clinical observations, food consumption, body weight, clinical pathology (e.g., hematology, serum chemistry, and coagulation), gross observations (from carcass and musculoskeletal system, all external surfaces and orifices, cranial cavity and external surface of the brain, thoracic, abdominal, and pelvic cavities with their associated organs and tissues, and gross lesions), and histopathology. Given that RARα plays a role in the immune system, we specifically examined neutrophil granulocyte maturation, but also T cell modulation, and evaluated white blood cell (WBC) subtype counts with immunophenotyping.
All animals survived to their scheduled necropsy; there were no apparent changes in behavior, and there were no gross or histopathological findings. All assessments of the animals of the 3 and 10 mg/kg/day groups were unremarkable. Some clinical pathology parameters were significantly changed from control at the end of the dosing phase (Table ): Chloride was decreased in the 3 mg/kg/day and 30 mg/kg/day groups (103 ± 1 vs 104 ± 1 mmol/L). Increases were noted in leukocyte counts (11.83 ± 2.90 vs 9.00 ± 2.79 × 103/μL), lymphocyte counts (9.31 ± 1.93 vs 7.46 ± 2.44 × 103/μL), activated partial thromboplastin time (APTT; 22.6 ± 2.0 vs 19.4 ± 2.0 s), and alkaline phosphatase (ALP, 132 ± 24 vs 105 ± 12 U/L) in the 30 mg/kg/day group. Although statistically significant, these changes were also considered nonadverse as they were small in magnitude and lacked histopathological correlations. As for neutrophil granulocytes (assessed with standard hematology), and total T cells, total B cells, cytotoxic T cells, T helper cells, and natural killer cells (assessed with immunophenotyping), there were no changes in any of the dose groups compared to control (Figure ). Hematology, serum chemistry, and coagulation parameters were also assessed in all the males from the efficacy studies (Figure , Table , and Table ), where sperm parameters and male fertility were assessed. No changes related to YCT-529 were noted (data not shown).
7. Clinical Pathology Parameters at the End of the Two-Week Dosing Period (Only Values of Parameters That Were Significantly Different from Control in at Least One Dose Group are Shown) .
| hematology | coagulation | serum chemistry | |||
|---|---|---|---|---|---|
| WBC (103/μL) | #LYMP (103/μL) | APTT (s) | ALP (U/L) | Cl (mmol/L) | |
| mean ± SD (n) | |||||
| 0mg/kg/day | 9.00 ± 2.79 (15) | 7.46 ± 2.44 (15) | 19.4 ± 2.0 (14) | 105 ± 12 (16) | 104 ± 1 (16) |
| 3mg/kg/day | 9.68 ± 1.69 (16) | 7.79 ± 1.41 (16) | 20.0 ± 2.4 (15) | 108 ± 18 (16) | 103 ± 1** (16) |
| 10mg/kg/day | 9.34 ± 2.53 (16) | 7.39 ± 2.09 (16) | 19.9 ± 2.3 (16) | 114 ± 19 (16) | 103 ± 1 (16) |
| 30mg/kg/day | 11.83 ± 2.90** (16) | 9.31 ± 1.93* (16) | 22.6 ± 2.0*** (16) | 132 ± 24*** (16) | 103 ± 1* (16) |
*p ≤ 0.05 (ANOVA and Dunnett). ** p ≤ 0.01 (ANOVA & Dunnett). ** p ≤ 0.001 (ANOVA and Dunnett). Hematology parameters that were assessed in total: #BASO = basophils absolute, % BASO = basophils percent, #EOS = eosinophils absolute, % EOS = eosinophils percent, HCT = hematocrit, HGB = hemoglobin, #LYMP = lymphocytes absolute, % LYMP = lymphocytes percent, MCH = mean corpuscular hemoglobin, MCHC = mean corpuscular hemoglobin concentration, MCV = mean corpuscular volume, #MONO = monocytes absolute, %MONO = monocytes percent, MPV = mean platelet volume, #NEUT = neutrophils absolute, % NEUT = neutrophils percent, PLT = platelet Count, RBC = red blood cells/erythrocyte count, RDW = RBC distribution width, #RET = reticulocytes, absolute, % RET = reticulocytes, percent, WBC = white blood cells/leukocyte count. Coagulation parameters that were assessed in total: APTT = activated partial thromboplastin time, FIB = fibrinogen, and PT = prothrombin time. Serum chemistry parameters that were assessed in total: A/G = albumin/globulin ratio, ALB = albumin, ALP = alkaline phosphatase, ALT = alanine aminotransferase, AST = aspartate aminotransferase, BIL-T = total bilirubin, Ca = calcium, CK = creatine kinase, Cl = chloride, CRE = creatinine, GGT = gamma-glutamyltransferase, GLB = globulin, GLU = glucose, K = potassium, Na = sodium, P = inorganic phosphorus, TCHO = total cholesterol, TG = triglyceride, TP = total protein, UREA = urea.
7.
WBC subtype counts after 42 day repeat dosing with YCT-529 at 10–30 mg/kg/day. Shown are means ± SD of various leukocyte subtypes at the end of the dosing phase from 10 animals per time point of the control group (white bars), the 3 mg/kg/day group (light gray bars), the 10 mg/kg/day group (dark gray bars), and the 30 mg/kg/day group (black bars).
These results demonstrate that YCT-529 at doses up to 30 mg/kg/day (the highest dose tested) did not significantly affect standard safety parameters and was well-tolerated by male Sprague–Dawley rats upon once daily administration for 42 days, and the no-observed-adverse-effect level (NOAEL) was ≥30 mg/kg/day. With the lowest efficacious dose of 0.75 mg/kg/day, the therapeutic window is therefore at least 40×.
Chemistry
Compounds 6–8 are described in Al Noman., et al. YCT-529 and compound 9 are described in Mannowetz et al.
Compound 10 was synthesized as described in Scheme . Chromanone 41 was converted into triflate 42 using Comins’ reagent and lithium bis(trimethylsilyl)amide. Subsequently, a Suzuki coupling reaction was utilized to cross-couple triflate 42 with p-tolylboronic acid pinacol ester, yielding substituted 6-bromo-chromene 43. Intermediate 43 was then converted into acetophenone 44 through Stille coupling and subsequent acidic hydrolysis of the vinyl ether. Bromination at the C2 position of the acetyl group also resulted in the bromination of the chromene ring, resulting in intermediate 45. Condensation of 45 with methyl 4-carbamimidoylbenzoate yielded the imidazole ring in intermediate 46. At this stage, the undesired vinyl bromide was dehalogenated using sodium formate as the hydride source in the presence of Pd(PPh3)4 to prepare intermediate 47. Saponification of the methyl ester furnished compound 10 as the desired product.
1. Synthesis of Compound 10 .
Compound 11 was synthesized as described in Scheme . The synthesis started with a Suzuki coupling reaction between N-Boc-pyrrole-2-boronate 48 and methyl 4-bromobenzoate to give intermediate 49. Intermediate 49 was then converted into 2,4-disubstituted pyrrole 50 with iridium-catalyzed borylation at the C4 position of the N-Boc pyrrole in 49. Intermediate 50 was then coupled with chromene 43 by Suzuki reaction to give intermediate 51. Subsequent Boc-deprotection and saponification of the methyl ester yielded the desired 2,4-disubstituted pyrrole 11.
2. Synthesis of Compound 11 .
Compound 12 was synthesized as described in Scheme . The pyrazole linker synthesis commenced with enolate formation of acetophenone 44, followed by nucleophilic addition of the enolate to the acid chloride of methyl 4-(chlorocarbonyl)benzoate, yielding 1,3-diketone 52. Condensation of hydrazine hydrate with the 1,3-diketone formed the pyrazole ring in 53, and subsequent hydrolysis provided the acid 12.
3. Synthesis of Compound 12 .
Compound 13 was synthesized as outlined in Scheme . 3-Acetyl-4-hydroxybenzonitrile (54) was first condensed with acetone to afford chromanone 55. Grignard addition of p-tolylmagnesium bromide to intermediate 55, followed by dehydration, yielded chromene 56. The cyano group of 56 then underwent nucleophilic addition with hydroxylamine and subsequent condensation with methyl 4-(chlorocarbonyl)benzoate to form 1,2,4-oxadiazole 57. The final saponification of 57 afforded compound 13.
4. Synthesis of Compound 13 .
Compound 14 was synthesized as outlined in Scheme . Chromene nitrile 56 was first hydrolyzed under aqueous basic conditions to yield carboxylic acid 58. In parallel, 4-(methoxycarbonyl)benzoic acid (59) was reacted with hydrazine to afford acyl hydrazide 60. Intermediates 58 and 60 were then subjected to a one-pot condensation to generate 1,3,4-oxadiazole 61. Final saponification of 61 furnished compound 14.
5. Synthesis of Compound 14 .
Compound 15 was synthesized as outlined in Scheme . The nitrile group of compound 56 was first converted with sodium azide to tetrazole 62. This intermediate was then coupled with (4-(methoxycarbonyl)phenyl)boronic acid to yield 63, which subsequently underwent saponification to provide the final compound 15.
6. Synthesis of Compound 15 .
Compound 16 was synthesized as outlined in Scheme . The synthesis began with a Fries rearrangement of the starting ketoester 64 to obtain diketone 65. Cyclization of diketone 65, mediated by pyrrolidine, yielded chromene 66. Chromene 66 was then reacted with ethyl 4-(2-bromoacetyl)benzoate, forming 1,4-diketone 67, which subsequently underwent acid-mediated cyclization to generate furan intermediate 68. Intermediate 68 was then converted to triflate 69 using Comins’ reagent. Suzuki coupling of intermediate 69 with p-tolyl boronic acid pinacol ester provided intermediate 70, which was subjected to saponification to afford the final acid 16.
7. Synthesis of Compound 16 .
Compound 17 was synthesized as outlined in Scheme . The 1,2,4-triazole linker was synthesized by condensing aryl nitrile 56 with methyl 4-carbamimidoylbenzoate using copper(I) cyanide as the catalyst to give intermediate 71. Subsequent hydrolysis of the methyl ester yielded the free carboxylic acid 17.
8. Synthesis of Compound 17 .
Compound 18 was synthesized as outlined in Scheme . The synthesis began with a Wittig olefination of the acetophenone moiety in compound 44, followed by rhodium-catalyzed dehydrogenative borylation to form vinyl boronate 73. Suzuki reaction of vinyl boronate with methyl 4-bromobenzoate and hydrolysis of the methyl esters afforded the desired acid 18 featuring the propenyl linker.
9. Synthesis of Compound 18 .
Compounds 19–21 were synthesized as outlined in Scheme . The synthetic route began with mild Boc deprotection of intermediate 49 with sodium methoxide to give pyrrole 75. Subsequently, iridium-catalyzed C–H activation was used to achieve C5 borylation of pyrrole 75, forming 2,5-disubstituted pyrrole 76. The synthesis of the chromene components began with Suzuki coupling reactions to cross-couple triflate 42 with the corresponding aromatic boronic acid pinacol esters, yielding substituted 6-bromo-chromenes 77a–77c, which underwent a subsequent Suzuki reaction with pyrrole boronic acid pinacol ester 76, furnishing the conjugated compounds 78a–78c. These were then subjected to saponification to produce the final carboxylic acid compounds 19–21.
10. Synthesis of Compounds 19-21 .
Compounds 22–26 were synthesized as outlined in Scheme . Boc protection was accomplished using Boc anhydride and a catalytic amount of DMAP to give intermediate 79. Then, compound 79 was coupled with bromochromone 41 using the Suzuki reaction to give compound 80. Subsequently, ketone 80 was converted to vinyl triflate 81, followed by a second Suzuki coupling to introduce phenyl derivatives. Boc deprotection at this stage was challenging because neither mild acidic nor basic conditions could remove the Boc group, possibly due to the steric hindrance created by 2,5-diaryl substitutions. Although harsh acidic conditions were able to remove the Boc group, the compound decomposed significantly under those conditions due to the susceptibility of the pyrrole to acidic degradation. Here, thermal deprotection proved to be effective without significant decomposition. Finally, the methyl ester was hydrolyzed to reveal the free carboxylic acids 22–26.
11. Synthesis of Compounds 22–26 .
Compounds 27–29 were synthesized as outlined in Scheme , starting with the condensation of 2-bromo-4-iodophenol (83) and 2-methyl-3-buten-2-ol, resulting in the formation of the chroman ring in 84. Subsequent benzylic oxidation of 84 using chromium(VI) oxide yielded 85. This intermediate was then subjected to a Grignard reaction and dehydration, introducing a tolyl substitution on the chromene ring and generating 86. Suzuki coupling between chromene 86 and 76 achieved regioselective coupling product 87. Hydrolysis of the methyl ester yielded the desired bromo-substituted acid 27. Following a similar approach, methyl-substituted compound 29 was prepared by alkyl Suzuki coupling of 87 with trimethylboroxine, followed by ester hydrolysis. Additionally, a copper-catalyzed halogen exchange reaction afforded chloro-substituted compound 28 after ester hydrolysis of the methyl ester.
12. Synthesis of Compounds 27–29 .
Compound 30 was synthesized as outlined in Scheme , starting with Eaton’s reagent-mediated condensation (10:1 mixture of methanesulfonic acid and phosphorus pentoxide) between 3,3-dimethylacrylic acid and 4-bromoresorcinol (88), yielding chromone 89. Subsequent Grignard reaction followed by dehydration produced 90. Then, 91 with a pyrrole linker was synthesized via Suzuki coupling between bromochromene 90 and pyrrole boronate 76, followed by hydrolysis of the methyl ester of the coupling product 91 to provide acid 30.
13. Synthesis of Compound 30 .
Compound 31 was synthesized as outlined in Scheme , starting with a pyrrolidine-catalyzed aldol condensation, followed by cyclization to form the chromone ring in compound 93. A subsequent Grignard reaction and dehydration introduced the tolyl substitution in 94, which was coupled with 76 under Suzuki reaction conditions, followed by saponification to reveal the carboxylic acid in compound 31.
14. Synthesis of Compound 31 .
Compound 32 was synthesized as outlined in Scheme , starting with a Suzuki coupling between 6-bromochroman-4-one (95) and 79, yielding the coupling product 96. The benzylic ketone in 96 was converted to vinyl triflate 97 using triflic anhydride. Next, vinyl triflate 97 was coupled with p-tolylboronic acid using Suzuki reaction conditions to introduce the tolyl group in 98. Thermal Boc deprotection of 98 followed by saponification of the methyl ester afforded the desired acid 32.
15. Synthesis of Compound 32 .
Compounds 33 and 34 were synthesized as outlined in Scheme , starting with a Suzuki reaction between (3-bromophenyl)(p-tolyl) methanone (100) and 76, yielding the coupled product 101. Subsequent hydrolysis of the methyl ester produced the carboxylic acid 33. To synthesize compound 34, the carbonyl group in 101 was converted to a vinyl group via Witting olefination, followed by the hydrolysis of the methyl ester to the free carboxylic acid 34.
16. Synthesis of Compounds 33 and 34 .
Compounds 35–37 were synthesized as outlined in Scheme , starting with the benzoylation of 4-bromophenol (103) to form ester 104, which underwent Fries rearrangement with aluminum chloride, resulting in benzophenone 105. Suzuki coupling between 105 and pyrrole boronate 76 furnished 106, which was converted to the desired acid 35 through direct hydrolysis. Alternatively, the phenolic hydroxyl group of 106 was alkylated with methyl and ethyl iodide, and then the terminal ester was hydrolyzed to prepare acids 36 and 37, respectively.
17. Synthesis of Compounds 35–37 .
Compound 38 was synthesized as outlined in Scheme , starting with a Suzuki coupling between bromotetralene 107 and 76, followed by saponification of the methyl ester to reveal the carboxylic acid in final compound 38.
18. Synthesis of Compound 38 .
Compound 39 was synthesized as outlined in Scheme , starting with a Suzuki coupling between 4-bromobenzonitrile and 1-Boc-pyrrole-2-boronic acid (48), followed by Boc-deprotection to prepare nitrile 109. Iridium-catalyzed C5 borylation of the pyrrole afforded 110, which served as a substrate for a Suzuki reaction with bromochromene 43, preparing the desired nitrile analog 39.
19. Synthesis of Compound 39 .
Compound 40 was synthesized as outlined in Scheme . The carboxamide analog 40 was synthesized from compound 9 via amide coupling between the carboxylic acid and an aqueous ammonium hydroxide solution.
20. Synthesis of Compound 40 .
Conclusions
In this study, strategic linker modifications transformed the chromene amide RARα antagonist MN-256 (6) to analog 9 that resolved the parent compound’s in vivo liabilities, a pyrrole-linked antagonist that inhibits RARα with an IC50 of 1.2 nM while maintaining >300-fold selectivity over RARβ and RARγ. Modifications of the hydrophobic antagonism moiety, the chromene core, and the benzoic acid group were explored. Several closely related analogs, modified at the tolyl group, retained excellent potencies and selectivities, like 9. The 4-fluoro analog 22 was a 2.6-fold more potent RARα antagonist than 9. The benzophenone analogs 33, 35, 36, and 37 (Table ) represent a new scaffold for antagonist design but need more extensive evaluation to assess potential liabilities.
The extensive in vivo analyses in a distinct animal heretofore not studied confirmed the oral bioavailability of YCT-529, the sodium salt of 9, and further showed dose-proportional exposure and a 10–13 h half-life in rats without drug accumulation after 28 days of daily dosing. Once-daily oral administration (0.75 mg/kg) for 28 days reversibly suppressed sperm counts, sperm motility, and normal spermatogenesis and induced male infertility that, importantly, was reversible, while remaining well tolerated up to 30 mg/kg (NOAEL), affording a ≥40-fold therapeutic window.
Previous cross-species validation by Mannowetz et al. demonstrated YCT-529’s translational potential as a male contraceptive. In male CD-1 mice, dosed with 10 mg/kg/day for 4 weeks, achieved 99% contraception with full fertility recovery 6 weeks after withdrawal. In cynomolgus macaques, daily oral dosing (0.5–7.5 mg/kg) induced severe oligospermia/azoospermia within 2 weeks, with complete recovery 10–15 weeks after cessation and no adverse effects on body weight, clinical chemistry, or reproductive hormones.
Collectively, these data establish the pyrrole as a superior linker moiety for RARα antagonists and position YCT-529 as a highly selective and fully reversible oral RARα antagonist poised to become the first-in-class nonhormonal male contraceptive with potential to reach the market.
Experimental Section
Docking Studies
An SD file containing the structures of the retinoid analogs was generated in ChemDraw and imported into the Maestro, Schrödinger molecular modeling suite. Within the project workspace, bond orders were assigned, hydrogens were added, and all ionization states were generated using Epik (pH 5.0–9.0), allowing desalting and tautomerization while retaining chiralities (maximum of 32 states per ligand). Only the lowest energy ring conformation per ligand was retained for docking. For protein preparation, the PDB file (1DKF) was imported with mixed hydrogen display. Preprocessing involved assigning bond orders, adding hydrogens, creating zero-order bonds to metals, forming disulfide bonds, and removing water molecules beyond 5 Å from the ligand-binding site. Epik was used to generate protonation states, and the sample water orientation was optimized. A restrained minimization was performed using the OPLS 2001 force field. Next, Receptor Grid Generation was performed by selecting the cocrystallized ligand 2b as the reference binding site. Glide Receptor Grid Generation was executed with a van der Waals scaling factor of 1.0 and a partial charge cutoff of 0.25, softening the potential for nonpolar receptor regions. Finally, Ligand Docking was conducted using Glide in standard precision mode, with nitrogen inversion and ring conformation sampling enabled. Nonplanar amide conformations were biased with penalties applied, and Epik state penalties were incorporated. A maximum of 10 poses per ligand were allowed, followed by postdocking minimization.”
RAR Transactivation Assay
The antagonism and agonism potency of test compounds of RARα, RARβ, and RARγ was evaluated using the GeneBLAzer RAR-UAS-bla HEK 293T cell-based reporter assay (Thermo Scientific, Waltham, MA). These HEK 293T cells stably express a recombinant human RAR subtype fused to a GAL4 DNA-binding domain, along with a beta-lactamase reporter gene under the control of an upstream activator sequence. When an agonist binds to the ligand-binding domain (LBD) of the GAL4-RAR fusion protein, it activates transcription of the beta-lactamase reporter gene. The level of beta-lactamase expression was quantified by measuring cleavage of the FRET substrate CCF4-AM. For antagonist testing, 0.8 nM of ATRA in DMSO (final 0.1%) was added to control and test compound wells using an Echo acoustic nanoliter dispenser (Labcyte, San Jose, CA). Test and reference compounds were added in an 8-point dose–response format in triplicate. For agonist testing, compounds were added in an 8-point dose–response format using an Echo without ATRA. RARα, RARβ, and RARγ cells were serum-starved for 16–24 h before assay initiation, and 40 μL of cell suspension (2.5 × 105 cells/mL) was added per well. Plates were incubated at 37 °C in a 5% CO2 incubator for 16–24 h. Following incubation, 8 μL of beta-lactamase detection reagent containing CCF4-AM was added, and plates were incubated at room temperature for 2 h. Fluorescence was measured using a SpectraMax M2e plate reader (Molecular Devices, San Jose, CA) with excitation at 410 nm and emission at 460 and 520 nm. After background subtraction, the 460/520 emission ratio was calculated, and IC50 and EC50 values were determined using a four-parameter logistic equation in GraphPad Prism 8.0. Mean ± SEM values were calculated from the geometric mean of the log IC50 and EC50 values. Three independent measurements were performed for the transactivation assays. All other measurements were taken from distinct samples, and no sample was measured repeatedly.
Animal Studies
The protocol and any amendments or procedures involving the care or use of animals on this study had been reviewed and approved by WuXi AppTec Institutional Animal Care and Use Committee (IACUC) prior to the initiation of such procedures. The IACUC approval number for the rat safety study is #SZ20220711-Rats-D. The IACUC approval number for the rat efficacy study is #SZ20231207-Rats-B. A staff veterinarian monitored the study for animal welfare issues.
Animal Studies
Animals and Husbandry
In the efficacy study, 127 sexually mature, male Sprague–Dawley rats (10–11 weeks of age at dosing initiation) and 100 sexually mature, nulliparous, female Sprague–Dawley rats (11–13 weeks of age at dosing initiation) were used. In the safety study, 85 sexually mature, male Sprague–Dawley rats (at least 10 weeks of age at dosing initiation) were used. Upon arrival, all animals were examined by the veterinary staff and were quarantined/acclimated for 6 days before dosing initiation. A staff veterinarian constantly monitored the animals’ welfare. All animals were individually housed in humidity- and temperature-controlled rooms with a 12 h light/12 h dark diurnal cycle. The animals had access to water and food ad libitum (except for presampling fasting periods). During the prepairing period, the rats were group housed (up to 3 animals of the same sex and same dosing group). During the mating periods, the rats were housed based on one male to one female, and after mating was confirmed, male and female rats were single-housed.
Dosing Regimens and Study Designs
YCT-529 was suspended in 0.9% saline to prepare working solutions of desired concentrations, which were administered to the animals via oral gavage at a dosing volume of 10 mL/kg. The formulation (YCT-529 in saline) was prepared freshly for each day of dosing. The first day of dosing was designated as Day 1. Efficacy study: For the main study, 100 male rats were randomly assigned to 5 groups of 20 animals each. For the pharmacokinetic (PK) part of the study, 3 control animals and 6 animals per treated group were used. The animals received either vehicle (saline) or YCT-529 in saline at 0.25, 0.5, 0.75, or 1.0 mg/kg/day once daily via oral gavage for 28 days. The dosing period was followed by a 180 day recovery period. The first 10 male animals per group were necropsied on Day 43 after completion of the 28 day dosing period, followed by a 2 week dosing-free mating period. The last 10 male animals per group entered the 180 day recovery period on Day 29, followed by a 14 day mating period, and were necropsied on Day 223. Safety study: For the main study, 64 male rats were randomly assigned to 4 groups of 16 animals each. The animals received either vehicle (saline) or YCT-529 in saline at 3, 10, or 30 mg/kg/day once daily via oral gavage for 42 days and were necropsied on Day 43 (24 h after administration of the last dose).
Sperm Collection and Sperm Parameter Assessment
Sperm samples were collected from the left cauda epididymis from all main study males at their scheduled necropsy on Day 43 (after dosing and subsequent mating phase) or Day 223 (after recovery and subsequent mating phase). Sperm parameters were evaluated with an automated Sperm Analysis System. The epididymal sperm concentration was calculated as follows: sperm concentration = sperm count per cauda (×108)/weight of cauda epididymis (g). No data point was excluded from the analysis.
Mating, Pregnancy Assessments, and Fertility Assessments
The respective 14 day mating periods started at the end of the dosing phase (on Day 28) and at the end of the recovery phase (on Day 108). In this study, 1:1 mating (one male to one female in the same group) was used. One female rat was introduced to one male in the afternoon on Days 28 and 108. Thereafter, the females were checked for evidence of mating (presence of vaginal plug or sperm in vaginal smear) each morning for a maximum of 14 days. The day when mating was confirmed was defined as gestational day 0 (GD 0). Following mating, the females were single-housed until they were euthanized and necropsied on GD 15 to assess the number of corpora lutea, implantation sites, viable and nonviable embryos. Mating, fertility, and fecundity indices were calculated as follows: Male mating index = (number of males with confirmed mating/number of males cohabitated) × 100, Male fertility index = (number of males impregnating a female/number of males cohabitated) × 100, and Fecundity index = (number of pregnant females/number of females with confirmed mating) × 100.
Clinical Pathology
Blood samples were collected from all main study male animals of the efficacy and safety studies to assess hematology, coagulation, and clinical chemistry parameters. The animals were fasted overnight before blood collection. Blood samples were obtained from the abdominal aorta at the respective necropsy on Days 43 or 223 (efficacy study) and on Day 43 (safety study) as a terminal procedure after isoflurane anesthesia. Whole blood was collected into K2EDTA anticoagulant tubes for complete blood count analysis, into serum separator tubes for serum chemistry analysis, and into sodium citrate tubes for coagulation analysis. The serum was separated by centrifugation at 3,500g for 10 min at 4 °C and stored frozen at −80 °C until analysis. No data point was excluded from the analysis.
Blood Sampling and Plasma Preparation for PK Analysis
In the efficacy study, 27 male Sprague–Dawley rats were randomly assigned to 5 groups. The animals received either vehicle (saline) or YCT-529 in saline at 0.25, 0.5, 0.75, or 1 mg/kg/day once daily via oral gavage for 28 days. The control group consisted of 3 animals, and the groups receiving YCT-529 consisted of 6 animals each. In the safety study, 21 male Sprague–Dawley rats were randomly assigned to 4 groups. The animals received either vehicle (saline) or YCT-529 in saline at 3, 10, or 30 mg/kg/day once daily via oral gavage for 28 days. The control group consisted of 3 animals, and the groups receiving YCT-529 consisted of 6 animals each. On Days 1 and 28, blood samples (approximately 0.3 mL) were collected from animals of both studies into K2EDTA anticoagulant tubes at t = 0 (predose), 0.5, 1, 2, 4, 8, and 24 h postdose from alternating sets of 3 animals/YCT-529 group/time point. Blood samples were collected at t = 0 (predose) and 1 h postdose from all 3 control animals/time point of the efficacy study and at t = 8 and 24 h postdose from all 3 control animals/time point of the safety study. Plasma was obtained within 2 h of collection by centrifugation at 3200g for 10 min at 4 °C and stored frozen at −80 °C until liquid chromatography with tandem mass spectrometry (LC/MS/MS) analysis.
LC/MS/MS Analysis
A protein precipitation procedure was used to extract YCT-529 from 20 μL of the study samples. LC/MS/MS analysis was performed using a LC-30AD liquid chromatograph (Shimadzu, Kyoto, Japan) coupled with an Acquity UPLC BEH Phenyl 1.7 μm, 50 × 2.1 mm analytical column (Waters, Milford, MA, USA) and an API 4000 mass spectrometer (AB Sciex, Framingham, MA, USA). Elution was isocratic using 0.1% acetic acid in water/acetonitrile (95:5, v/v) and 0.1% acetic acid in water/acetonitrile (5:95, v/v), flowing at 5 μL/s. The total run time was 4.0 min. YCT-529 eluted at 1.22 min, and diclofenac sodium salt (internal standard) at 0.63 min. MS/MS was performed with the instrument operating in electrospray positive ion mode (ES+). The lower limit of quantification (LLOQ) for YCT-529 was 30.0 ng/mL, and the upper limit of quantification was 30,000 ng/mL.
PK Analysis
Mean plasma concentration–time profiles of YCT-529 were analyzed using a noncompartmental model of WinNonlin, version 8.3.5 (Certara, Radnor, PA, USA). Maximum plasma concentration (C max) and time to reach C max (T max) were taken directly from the mean plasma concentration versus time profiles. The area under the plasma concentration–time curve (AUC) from time zero to 24 h post dose (AUC0–24h) was calculated using the linear up/log down trapezoidal rule. Mean plasma concentrations below the LLOQ were set to zero for the analysis. AUC0–24h and C max ratios were used to evaluate dose proportionality and accumulation index. Generally, differences of more than 0.5-fold and less than 2-fold in PK parameters per group were not considered to indicate a significant difference.
Statistics and Reproducibility
All measurements were taken from distinct samples, and no sample was measured repeatedly. Males and females were analyzed separately. Overall testing and follow-up pairwise testing were conducted for the control and all treatment groups with a sufficient sample size (n ≥ 3). All pairwise group comparisons of interest were performed via a two-sided test at the 5% significance level. Significant results were reported as either p ≤ 0.001, p ≤ 0.01, or p ≤ 0.05, where p represents the observed probability. All end points were analyzed using two-tailed tests unless indicated otherwise.
Data consisting of percent values (sperm motility) were first transformed using the arcsin of the square root. The homogeneity of the group variances of the transformed percentage values or from data sets from more than two groups was evaluated using Levene’s test at the 0.05 significance level. If differences between group variances were not significant (p > 0.05), then a parametric one-way ANOVA was performed. When significant differences among the means were indicated by ANOVA (p ≤ 0.05), the Dunnett’s test was used to perform the group mean comparisons between the control group and each treated group.
When Levene’s test indicated heterogeneous group variances (p ≤ 0.05) and the data set contained only positive values, a log transformation was performed. If transformed data still failed the test for homogeneity of variance (p ≤ 0.05) or where the data contained zero and/or negative values, then the nonparametric Kruskal–Wallis test was used to compare all considered groups. When the Kruskal–Wallis test was significant (p ≤ 0.05), the Dunnett’s test on ranks was used to perform the pairwise group comparisons of interest.
An overall test for association between response and treatment for fertility data (mating, fertility, and fecundity indices) was done using Fisher’s exact test. When no results could be obtained by Fisher’s exact test due to an oversized sample, a chi-square test was used. When the overall test was significant (p ≤ 0.05) and there were more than two groups, a follow-up analysis was conducted to compare each treatment group to the control group.
General Chemistry
All solvents and reagents that were obtained from commercial sources were used as received unless otherwise specified. Metal-catalyzed reactions were carried out in anhydrous solvents, which were degassed with nitrogen and passed through activated alumina or molecular sieves. All reactions that required anhydrous conditions were run under a nitrogen atmosphere. Reaction progress was monitored by thin-layer chromatography (TLC) using silica gel 60 F254 plates and visualized under UV light at 254 and 365 nm. Compounds were purified by silica gel flash column chromatography. The purity of final compounds was determined by HPLC/MS (solvent system from Fisher Scientific International, Inc.). All tested compounds are >95% pure by HPLC analysis, except MN-533 (94%) and MN-437 (94%). Mass spectrometric analysis was performed on an Agilent InfinityLab LC/MSD iQ instrument in positive ion mode. Microwave-assisted reactions were conducted using an Initiator+ (Biotage, San Jose, CA) with Biotage microwave reaction vials. All NMR spectroscopy experiments were performed on a Bruker Avance II 400/100 MHz instrument equipped with a BBO broadband probe, and spectra were processed using MestReNova software (Mestrelab Research S.L.). Chemical shifts are reported in ppm and referenced to residual solvent peaks: Acetone in acetone-d 6 at 2.05 ppm for 1H NMR and 29.9 ppm for 13C NMR; CHCl3 in CDCl3 at 7.26 ppm for 1H NMR and 77.16 ppm for 13C NMR; DMSO in DMSO-d 6 at 2.50 ppm for 1H NMR and 39.52 ppm for 13C NMR. Coupling constants (J) are given in Hertz (Hz), and splitting patterns are designated as singlet (s), doublet (d), triplet (t), quartet (q), and broad singlet (br s).
Synthesis of Compounds
Synthesis of Compound 10
6-Bromo-2,2-dimethyl-2H-chromen-4-yl Trifluoromethanesulfonate (42)
6-Bromo-2,2-dimethylchroman-4-one (41) (600 mg, 2.4 mmol, 1.0 equiv) and N-(5-chloro-2 pyridyl)bis(trifluoromethanesulfonimide) (Comins’ reagent, 1.0 g, 2.6 mmol, 1.2 equiv) were dissolved in degassed anhydrous THF (10 mL) and cooled to −78 °C. Lithium bis(trimethylsilyl)amide 1.0 M in THF (2.8 mL, 2.8 mmol, 1.2 equiv) was added. The reaction mixture was stirred at −78 °C for 3 h and then left overnight to warm to room temperature. The reaction was quenched by adding water and extracted with EtOAc. The organic layer was dried over anhydrous MgSO4 and then purified by flash chromatography using mixtures of hexanes and EtOAc to afford 6-bromo-2,2-dimethyl-2H-chromen-4-yl trifluoromethanesulfonate (0.82 g, 90% yield) as a brown oil. 1H NMR (400 MHz, CDCl3): δ 7.33 (dq, J = 4.3, 2.3 Hz, 2H), 6.77–6.68 (m, 1H), 5.67 (s, 1H), 1.51 (s, 6H).
6-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (43)
6-Bromo-2,2-dimethyl-2H-chromen-4-yl trifluoromethanesulfonate (42) (800 mg, 2.07 mmol, 1.0 equiv), 4,4,5,5-tetramethyl-2-(p-tolyl)-1,3,2-dioxaborolane (451 mg, 2.07 mmol, 1.0 equiv), and K2CO3 (714 mg, 5.17 mmol, 2.5 equiv) were dissolved in degassed 1,4-dioxane (10 mL). [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (151 mg, 0.21 mmol, 0.1 equiv) was added, and the reaction mixture was stirred at 60 °C for 5 h under a nitrogen gas atmosphere. The solvent was evaporated, and the crude product was then purified by flash chromatography using hexanes and EtOAc to afford 6-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (0.37 g, 55% yield) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 7.26 (s, 1H), 7.25–7.21 (m, 4H), 7.11 (d, J = 2.5 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 5.61 (s, 1H), 2.40 (s, 3H), 1.47 (s, 6H).
1-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)ethan-1-one (44)
In a dry 20 mL thick-walled pressure vial, a mixture of 6-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (43) (1.00 g, 3.04 mmol, 1.0 equiv), tributyl(1-ethoxyvinyl)stannane (1.67 g, 4.62 mmol, 1.5 equiv), and bis(triphenylphosphine)palladium(II) dichloride (0.107 g, 0.153 mmol, 0.05 equiv) was dissolved in anhydrous toluene (12 mL). The reaction mixture was degassed and purged with nitrogen gas for 5 min. The vial was sealed and heated at 100 °C for 24 h. After the reaction was complete, the vial was cooled to room temperature, the reaction mixture was filtered through Celite, and toluene was evaporated under reduced pressure. The residue was dissolved in hydrochloric acid (2 N) in 1,4-dioxane (20 mL) and stirred for an additional 1 h. The 1,4-dioxane was evaporated, and the semisolid residue was purified by flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) to obtain 1-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)ethan-1-one (44) as a white solid (570 mg, 64%). mp 91–93 °C. 1H NMR (400 MHz, CDCl3): δ 7.80 (dd, J = 8.5, 2.2 Hz, 1H), 7.68 (d, J = 2.2 Hz, 1H), 7.23 (s, 4H), 6.90 (d, J = 8.4 Hz, 1H), 5.64 (s, 1H), 2.45 (s, 3H), 2.41 (s, 3H), 1.51 (s, 6H).
Methyl 4-(5-(3-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate (46)
1-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)ethan-1-one (44) (570 mg, 1.95 mmol, 1.0 equiv) was dissolved in EtOAc (15 mL), and copper(II) bromide (900 mg, 4.03 mmol, 2.07 equiv) was added to the reaction mixture and refluxed for 6 h. After the reaction mixture was cooled to room temperature, water was added and extracted with EtOAc (3 × 10 mL), washed with saturated brine solution, and dried over anhydrous MgSO4. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain a mixture of 2-bromo-1-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)ethan-1-one and 2-bromo-1-(3-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)ethan-1-one (45) as a colorless liquid (425 mg, estimated as a 1:1 mixture by NMR). The mixture was used in the next step without purification.
To a refluxing solution of amino(4-(methoxycarbonyl)phenyl)methaniminium acetate (225 mg, 0.94 mmol, 1.0 equiv) and sodium bicarbonate (160 mg, 1.90 mmol, 2.0 equiv) in THF (15 mL) and water (3 mL), the mixture from the preceding reaction (425 mg, 1.0 equiv) dissolved in THF (12 mL) was added dropwise over 30 min. The reaction mixture was refluxed for 22 h and cooled to room temperature. THF was removed under reduced pressure, and water (20 mL) was added to the residue. The mixture was extracted with EtOAc (10 mL × 3), washed with brine, and dried over anhydrous MgSO4. The solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 60% EtOAc in hexanes) to obtain a mixture of methyl 4-(5-(3-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate and methyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate (46) as a white solid (388 mg). 1H NMR (400 MHz, CDCl3): δ 11.17 (s, 1H), 7.83 (d, J = 8.1 Hz, 2H), 7.78–7.66 (m, 2H), 7.48 (s, 1H), 7.10 (d, J = 8.1 Hz, 3H), 7.00 (d, J = 7.6 Hz, 2H), 6.87 (t, J = 11.6 Hz, 2H), 3.86 (s, 3H), 2.25 (s, 3H), 1.61 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 166.8, 151.1, 146.0, 137.6, 135.1, 134.6, 134.1, 130.1, 130.0, 129.5, 129.3, 129.2, 128.5, 126.8, 125.2, 124.3, 123.1, 117.1, 80.3, 52.3, 27.0, 21.4. Three 13C were not observed, possibly due to peak broadening by tautomerism.
Methyl 4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate (47)
Methyl 4-(5-(3-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate and methyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate (46) (200 mg, 1.0 equiv) and anhydrous DMF (8 mL) were degassed by bubbling nitrogen through the solution for 10 min. Then, tetrakis(triphenylphosphine)palladium(0) (44 mg, 0.038 mmol, 0.10 equiv) and sodium formate (100 mg, 1.5 mmol, 3.9 equiv) were added. The resulting mixture was heated at 60 °C under argon for 16 h. The mixture was diluted with brine and extracted with EtOAc (10 mL × 3). The combined organic layers were dried over anhydrous MgSO4, and the solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 60% EtOAc in hexanes) to obtain methyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate (47) as a white solid (152 mg, 28% over three steps). 1H NMR (400 MHz, CDCl3): δ 10.74 (s, 1H), 8.01–7.94 (m, 3H), 7.88 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 7.9 Hz, 1H), 7.27 (s, 1H), 7.24 (d, J = 7.6 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 8.3 Hz, 1H), 5.61 (s, 1H), 3.89 (s, 3H), 2.35 (s, 3H), 1.48 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 166.9, 145.7, 134.4, 130.2, 129.5, 129.2, 128.6, 126.4, 125.1, 122.5, 117.4, 77.4, 52.3, 27.7, 21.3. Ten 13C peaks were missing, possibly due to tautomerism.
4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoic Acid (10)
To a solution of methyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoate (100 mg, 0.22 mmol, 1.0 equiv) in THF (1 mL), water (1 mL), and MeOH (1 mL), was added lithium hydroxide hydrate (140 mg, 3.34 mmol, 15 equiv) and the resulting mixture was stirred for 15 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 5.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, and dried over anhydrous MgSO4. The solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 100% EtOAc in hexanes) to obtain 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-imidazole-2-yl)benzoic acid (10) (77 mg, 79%) as a white solid. mp 285–287 °C. 1H NMR (400 MHz, THF-d 8): δ 11.69 (s, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.98 (d, J = 8.6 Hz, 2H), 7.72 (s, 1H), 7.49 (d, J = 17.4 Hz, 1H), 7.29 (d, J = 8.1 Hz, 2H), 7.25 (s, 1H), 7.22 (d, J = 7.9 Hz, 2H), 6.85 (d, J = 8.3 Hz, 1H), 5.66 (s, 1H), 2.38 (s, 3H), 1.46 (s, 6H). LRMS calcd m/z: [C28H25N2O3]+, 437.19; found, 437.27. 98% pure by UPLC analysis.
Synthesis of Compound 11
tert-Butyl 2-(4-(Methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (49)
Methyl 4-bromobenzoate (48, 17.00 g, 79.05 mmol, 1.0 equiv) and (1-(tert-butoxycarbonyl)-1H-pyrrol-2-yl)boronic acid (20.00 g, 94.78 mmol, 1.2 equiv) were combined in a round-bottom flask, followed by the addition of THF (190 mL), and 0.5 M aqueous solution of tribasic potassium phosphate (380 mL, 2.4 equiv). Nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of XPhos Pd G2 (1.50 g, 1.91 mmol, 0.024 equiv). Then the flask was placed in a preheated heating block at 45 °C and stirred for 5 h. After the reaction was complete, brine was added to the reaction mixture, extracted with EtOAc (50 mL × 3), and dried over anhydrous MgSO4. The solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (49) as a white solid (22.14 g, 92%). mp 72–74 °C. 1H NMR (400 MHz, CDCl3): δ 8.06–7.98 (m, 2H), 7.45–7.35 (m, 3H), 6.29–6.21 (m, 2H), 3.93 (s, 3H), 1.37 (s, 9H). 13C NMR (100 MHz, CDCl3): δ 167.1, 149.3, 139.1, 134.1, 129.1, 128.7, 123.6, 115.6, 111.0, 84.2, 52.2, 27.8.
tert-Butyl 2-(4-(Methoxycarbonyl)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (50)
tert-Butyl 2-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (49) (870 mg, 2.9 mmol, 1.0 equiv), bispinacolatodiboron (585 mg, 2.3 mmol, 0.80 equiv), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (60 mg, 0.01 mmol, 0.03 equiv), and 4,4′-di-tert-butyl-2,2′-bipyridine (25 mg, 0.01 mmol, 0.03 equiv) were added to a round-bottom flask and evacuated with a vacuum pump and purged with nitrogen gas. This process was repeated three times, followed by mixing with hexanes (20 mL). The suspension was refluxed under a nitrogen gas atmosphere for 6 h. After cooling to room temperature, the reaction mixture was solubilized in DCM and a silica gel slurry was prepared for flash chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(4-(methoxycarbonyl)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (50) as a colorless viscous oil (740 mg, 60%). 1H NMR (400 MHz, CDCl3): δ 8.01 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 1.7 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 6.53 (d, J = 1.7 Hz, 1H), 3.92 (s, 3H), 1.40 (s, 9H), 1.33 (s, 12H). 13C NMR (101 MHz, CDCl3): δ 167.1, 148.9, 138.7, 135.0, 132.4, 129.1, 128.9, 128.7, 119.9, 84.5, 83.6, 52.2, 27.8, 24.9. 13C–B was not observed.
tert-Butyl 4-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-2-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (51)
6-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (43) (115 mg, 0.35 mmol, 1.0 equiv) and tert-butyl 2-(4-(methoxycarbonyl)phenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (50) (150 mg, 0.35 mmol, 1.0 equiv) were added to a vial and dissolved in THF (1.0 mL), and then a 0.5 M aqueous solution of tribasic potassium phosphate (2 mL, 2.8 equiv) was added to the solution. Then nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of XPhos Pd G2 (0.015 g, 0.02 mmol, 0.05 equiv). Then the vial was placed in a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, brine was added to the reaction mixture, and it was extracted with EtOAc (50 mL × 3), and dried over anhydrous MgSO4. The solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 40% EtOAc in hexanes) to obtain tert-butyl 4-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-2-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (51) as a bright yellow oil (181 mg, 94%). 1H NMR (400 MHz, CDCl3): δ 8.07–7.99 (m, 2H), 7.45 (d, J = 2.0 Hz, 1H), 7.44–7.40 (m, 2H), 7.34 (dd, J = 8.3, 2.1 Hz, 1H), 7.28 (d, J = 1.9 Hz, 2H), 7.21 (dd, J = 7.4, 2.1 Hz, 3H), 6.90 (d, J = 8.3 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 5.62 (s, 1H), 3.94 (d, J = 12.7 Hz, 3H), 2.40 (d, J = 6.1 Hz, 3H), 1.50 (s, 6H), 1.34 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 152.7, 149.3, 139.0, 137.7, 135.5, 134.7, 134.6, 129.33, 129.30, 129.1, 129.1, 128.7, 126.6, 126.50, 126.46, 122.9, 122.8, 118.4, 117.3, 114.1, 84.3, 76.0, 52.3, 27.8, 21.4.
4-(4-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (11)
tert-Butyl 4-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-2-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (51) (150 mg, 0.27 mmol, 1.0 equiv) was dissolved in THF (3 mL), and sodium methoxide (1 mL, 25 wt % in MeOH, 6 equiv) was added to the reaction mixture. The reaction mixture was stirred for 5 min and quenched with saturated ammonium chloride solution. The reaction mixture was extracted with DCM (3 mL × 3), and the combined organic layers were dried over anhydrous MgSO4 and evaporated to dryness. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 40% EtOAc in hexanes) to methyl 4-(4-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate as a bright yellow solid (112 mg, 91%). mp could not be determined because the compound degraded at 187 °C. 1H NMR (400 MHz, CDCl3): δ 8.52 (s, 1H), 8.01 (d, J = 8.5 Hz, 2H), 7.54–7.47 (m, 2H), 7.36 (dt, J = 8.4, 2.4 Hz, 1H), 7.32–7.28 (m, 2H), 7.25–7.21 (m, 3H), 7.02–6.99 (m, 1H), 6.90 (s, 1H), 6.76 (dd, J = 2.8, 1.7 Hz, 1H), 5.63 (s, 1H), 3.91 (s, 3H), 2.42 (s, 3H), 1.50 (d, J = 3.0 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 167.0, 152.1, 137.6, 136.7, 135.7, 134.8, 131.7, 130.5, 129.3, 129.2, 128.7, 127.9, 127.6, 127.2, 126.3, 123.2, 122.7, 122.6, 117.3, 116.5, 105.8, 75.9, 52.2, 27.7, 21.4.
To a solution of methyl 4-(4-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (100 mg, 0.22 mmol, 1.0 equiv) in THF (2 mL) and MeOH (2 mL) was added a 2 N aqueous sodium hydroxide solution (2.0 mL, 2.0 mmol, 18 equiv) and the resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (5 mL × 3), washed with brine, and dried over anhydrous MgSO4. The solvent was removed under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(4-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (11) (65 mg, 67%) as a yellow solid. mp could not be determined because the compound degraded at 235 °C. 1H NMR (400 MHz, THF-d8): δ 10.59 (s, 1H), 7.99–7.92 (m, 2H), 7.63–7.57 (m, 2H), 7.37 (dd, J = 8.3, 2.2 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H), 7.25–7.19 (m, 3H), 7.01 (dd, J = 2.8, 1.7 Hz, 1H), 6.84–6.75 (m, 2H), 5.64 (s, 1H), 2.38 (s, 3H), 1.45 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.2, 152.5, 137.9, 136.6, 135.8, 132.5, 132.4, 130.8, 129.6, 129.5, 129.5, 129.2, 128.3, 127.2, 126.6, 123.4, 123.0, 122.6, 117.5, 117.3, 105.7, 75.9, 27.5, 21.1. LRMS calcd m/z [C29H26NO3]+, 436.19; found, 436.25. 97% pure by UPLC analysis.
Synthesis of Compound 12
Methyl 4-(3-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-3-oxopropanoyl)benzoate (52)
To a stirred solution of 1-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)ethan-1-one (44) (183 mg, 0.63 mmol, 1.0 equiv) in anhydrous toluene (5 mL) at 0 °C, lithium hexamethyldisilazide (1.5 mL 1.0 M in THF, 1.5 mmol, 2.4 equiv) was added dropwise. The reaction mixture was stirred for 15 min, followed by the addition of methyl 4-(chlorocarbonyl)benzoate (140 mg, 0.71 mmol, 1.1 equiv) dissolved in toluene (1 mL). The reaction mixture was slowly warmed to room temperature and quenched with a half-saturated ammonium chloride solution and extracted with EtOAc (10 mL × 3). The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 100% hexanes to 40% EtOAc in hexanes) to obtain methyl 4-(3-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-3-oxopropanoyl)benzoate (52) (263 mg, 92%) as a white solid. mp 159–161 °C. 1H NMR (400 MHz, CDCl3): δ 16.80 (s, 1H), 8.14–8.08 (m, 2H), 7.95 (d, J = 8.4 Hz, 2H), 7.86–7.80 (m, 1H), 7.26 (s, 5H), 6.96 (d, J = 8.5 Hz, 1H), 6.69 (s, 1H), 5.66 (s, 1H), 3.95 (s, 3H), 2.42 (s, 3H), 1.53 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 187.8, 181.5, 166.5, 158.2, 139.5, 138.1, 134.8, 134.2, 133.1, 129.9, 129.5, 129.4, 129.2, 128.6, 128.2, 127.0, 125.4, 122.5, 117.2, 93.4, 77.4, 52.5, 28.2, 21.4.
4-(3-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrazol-5-yl)benzoic Acid (12)
A solution of methyl 4-(3-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-3-oxopropanoyl)benzoate (52) (80 mg, 0.18 mmol, 1.0 equiv) in EtOH (5 mL) was mixed with hydrazine hydrate (0.1 mL 70% in water, 1.4 mmol, 7.9 equiv), and the mixture was heated under reflux for 30 min and then cooled to ambient temperature. After leaving the mixture sit overnight at room temperature, the product crystallized and was filtered to afford methyl 4-(3-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrazol-5-yl)benzoate (53) (72 mg of the crude reaction product) as yellow needles. The crystals were used in the next step without further purification and characterization.
To a solution of methyl 4-(3-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrazol-5-yl)benzoate (53) (63 mg, 0.14 mmol, 1.0 equiv) in THF (2 mL) and MeOH (2 mL) was added lithium hydroxide hydrate (100 mg, 2.38 mmol, 17 equiv) dissolved in water (2 mL), and the resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (3 mL × 3), washed with brine, and dried over anhydrous MgSO4. The extract was concentrated under reduced pressure and purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(3-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrazol-5-yl)benzoic acid (12) (51 mg, 84%) as a yellow solid. mp 242–244 °C. 1H NMR (400 MHz, THF-d 8): δ 8.01 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.3 Hz, 2H), 7.57 (dd, J = 8.3, 2.2 Hz, 1H), 7.43 (d, J = 2.2 Hz, 1H), 7.28 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.3 Hz, 1H), 6.83 (s, 1H), 5.70 (s, 1H), 2.38 (s, 3H), 1.48 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 154.4, 149.5, 147.0, 138.1, 138.0, 136.2, 135.3, 130.6, 130.0, 129.7, 129.2, 127.1, 125.5, 124.5, 123.3, 123.0, 117.9, 108.2, 99.8, 76.5, 27.6, 21.1. LRMS calcd m/z: [C28H25N2O3]+, 437.19; found, 437.25. 97% pure by UPLC analysis.
Synthesis of Compound 13
2,2-Dimethyl-4-oxochromane-6-carbonitrile (55)
3-Acetyl-4-hydroxybenzonitrile (54) (3.25 g, 20.2 mmol), acetone (1.48 mL, 20.2 mmol), and pyrrolidine (1.72 mL, 20.6 mmol) in MeOH (60 mL) were stirred for 48 h at room temperature. The volatiles were evaporated under reduced pressure, and the resulting residue was treated with HCl (1 N, aqueous solution) to adjust the pH to 1 and then extracted with diethyl ether (3 × 60 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was purified on flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 70:30) to give the ketone 55 (2.81 g, 69%) as a brown solid; mp 128–129 °C; 1H NMR (400 MHz, CDCl3): δ 8.11 (d, J = 2.2 Hz, 1H), 7.65 (dd, J = 8.7, 2.2 Hz, 1H), 7.00 (d, J = 8.6 Hz, 1H), 2.74 (s, 2H), 1.46 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 190.4, 162.7, 138.5, 131.9, 120.5, 120.0, 118.3, 104.6, 80.9, 48.5, 26.7.
2,2-Dimethyl-4-(p-tolyl)-2H-chromene-6-carbonitrile (56)
Ketone 55 (2.00 g, 9.95 mmol) was taken into dry THF (60 mL) and cooled to 0 °C. A solution of p-tolylmagnesium bromide (19.9 mL, 1.0 M solution in THF, 19.9 mmol) was added dropwise at 0 °C under a nitrogen gas atmosphere. The reaction was warmed to room temperature and stirred for 48 h as progress was monitored by TLC. A saturated aqueous ammonium chloride solution (30 mL) was added to quench the reaction. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3 × 60 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, and evaporated under reduced pressure to afford 4-hydroxy-2,2-dimethyl-4-(p-tolyl)chromane-6-carbonitrile as a yellow oil (3.42 g). The crude mixture 4-hydroxy-2,2-dimethyl-4-(p-tolyl)chromane-6-carbonitrile was directly used for the next step without purification.
To 4-hydroxy-2,2-dimethyl-4-(p-tolyl)chromane-6-carbonitrile (3.42 g, 9.95 mmol, calculated based on the starting material used in the previous step) in MeOH (60 mL), pyridinium p-toluenesulfonate (PPTS, 0.500 g, 1.99 mmol) was added and refluxed for 8 h. Volatiles were evaporated under reduced pressure, and water (50 mL) was added to the residue. The aqueous solution was extracted with EtOAc (3 × 50 mL). The combined organic extracts were washed with brine, dried over Na2SO4, and evaporated under reduced pressure. Purification of the resulting residue by flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 70:30) gave chromene 56 (1.40 g, 51%, for two steps) as an off-white solid; mp 135–137 °C; 1H NMR (400 MHz, CDCl3): δ 7.43 (dd, J = 8.4, 2.0 Hz, 1H), 7.31 (d, J = 2.0 Hz, 1H), 7.28–7.23 (m, 2H), 7.20 (d, J = 8.2 Hz, 2H), 6.92 (d, J = 8.4 Hz, 1H), 5.67 (s, 1H), 2.43 (s, 3H), 1.53 (s, 6H). 13C NMR (100 MHz, CDCl3): δ 157.3, 138.3, 134.1, 133.3 (2C), 129.9, 129.7, 129.5, 128.5, 123.2, 119.4, 117.8, 103.8, 77.6, 28.1, 21.3.
Methyl 4-(3-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1,2,4-oxadiazol-5-yl)benzoate (57)
To a mixture of cyano compound 56 (0.402 g, 1.45 mmol) and hydroxylamine hydrochloride (0.151 g, 2.18 mmol) in ethanol (10 mL), DIPEA (0.400 mL, 2.32 mmol) was added at room temperature under a nitrogen gas atmosphere. The solution was stirred at reflux for 10 h until the reaction was complete, as monitored by TLC. Volatiles were evaporated under reduced pressure, water (30 mL) was added to the residue, which was extracted with EtOAc (3 × 25 mL). The organic fractions were combined, washed with brine, dried over Na2SO4, filtered, and evaporated under reduced pressure to give a white foam (0.405 g), which was used for the next step without purification. This crude white foam (0.390 g, 1.26 mmol) was suspended in anhydrous toluene (10 mL). Anhydrous pyridine (3 mL) and methyl 4-(chlorocarbonyl)benzoate (0.251 g, 1.26 mmol) were added under a nitrogen gas atmosphere. Then the solution was stirred for 12 h at room temperature and heated at 110 °C for 8 h until the complete consumption of the starting material. The reaction mixture was cooled to room temperature and neutralized with HCl (1 N, aqueous solution). Then extracted with CH2Cl2 (3 × 20 mL). The combined organic fractions were washed with brine, dried over Na2SO4, filtered, evaporated under reduced pressure and purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 70:30) to give ester 57 (0.170 g, 25% for two steps) as an off-white solid; mp 239–240 °C; 1H NMR (400 MHz, CDCl3): δ 8.21 (d, J = 8.5 Hz, 2H), 8.16 (d, J = 8.5 Hz, 2H), 7.96 (dd, J = 8.3, 2.1 Hz, 1H), 7.84 (d, J = 2.1 Hz, 1H), 7.27 (d, J = 8.1 Hz, 2H), 7.25–7.20 (m, 2H), 6.99 (d, J = 8.4 Hz, 1H), 5.65 (s, 1H), 3.94 (s, 3H), 2.40 (s, 3H), 1.51 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 174.5, 169.1, 166.1, 156.4, 137.9, 135.0, 134.3, 133.6, 130.3, 129.4, 129.4, 129.0, 128.6, 128.2, 128.1, 124.9, 122.9, 119.1, 117.5, 77.3, 52.6, 27.9, 21.4.
4-(3-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1,2,4-oxadiazol-5-yl)benzoic Acid (13)
To ester 57 (0.090 g, 0.175 mmol) in ethanol (4.0 mL), aqueous NaOH (10%, 1.0 mL) was added at room temperature and stirred for 48 h until the reaction was complete, as monitored by TLC. Then the reaction was acidified with HCl (1 N, 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, and then filtered. The solvent was removed under reduced pressure, and the residue was purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 00:100) to give 13 (65 mg, 74%) as an off-white solid; mp 247–248 °C; with a purity of >99%; 1H NMR (400 MHz, DMSO-d 6): δ 13.45 (s, 1H), 8.22 (d, J = 8.5 Hz, 2H), 8.15 (d, J = 8.4 Hz, 2H), 7.92 (dd, J = 8.3, 2.1 Hz, 1H), 7.69 (d, J = 2.1 Hz, 1H), 7.35–7.24 (m, 4H), 7.07 (d, J = 8.4 Hz, 1H), 5.84 (s, 1H), 2.38 (s, 3H), 1.49 (s, 6H); 13C NMR (100 MHz, DMSO-d 6): δ 174.4, 168.0, 166.3, 155.8, 137.5, 134.7, 134.2, 132.6, 130.2 (2C) 129.3, 128.6, 128.2, 128.1, 126.7, 123.7, 122.2, 118.3, 117.6, 76.8, 27.4, 20.8; UPLC–MS calcd m/z: [C27H22N2O4]+, 438.16, found (ES+) (M + H)+, 439.22.
Synthesis of Compound 14
2,2-Dimethyl-4-(p-tolyl)-2H-chromene-6-carboxylic Acid (58)
Compound 56 (0.250 g, 0.908 mmol) was dissolved in ethanol (6.0 mL), then a NaOH solution (5 N aqueous solution, 2.5 mL) was added, and the solution was heated at 90 °C for 9 h until the consumption of the starting material as monitored by TLC analysis. The reaction was cooled to room temperature. The volatiles were evaporated under reduced pressure. The residue was acidified with HCl (1 N, aqueous solution) to pH 1 and then extracted with EtOAc (3 × 15 mL). The combined organic extracts were washed with water (20 mL) and brine, dried over Na2SO4, filtered, evaporated under reduced pressure, and purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 00:100) to provide acid 58 (0.173 g, 65%) as an off-white solid; mp 204–206 °C; 1H NMR (400 MHz, DMSO-d 6): δ 12.62 (s, 1H), 7.75 (dd, J = 8.4, 2.1 Hz, 1H), 7.55 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 7.9 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 8.4 Hz, 1H), 5.78 (s, 1H), 2.36 (s, 3H), 1.46 (s, 6H); 13C NMR (100 MHz, DMSO-d 6): δ 166.8, 156.8, 137.4, 134.2, 132.7, 130.9, 129.5, 129.2, 128.2, 126.4, 123.0, 121.3, 116.6, 76.9, 27.5, 20.7.
Methyl 4-(Hydrazinecarbonyl)benzoate (60)
4-(Methoxycarbonyl)benzoic acid (59) (2.00 g, 11.1 mmol) was suspended in dry CH2Cl2 (40 mL) and cooled to 0 °C. N-Methylmorpholine (NMM) (1.50 mL, 13.3 mmol) and ethyl chloroformate (ClCO2Et) (0.951 mL, 10.0 mmol) were added under a nitrogen gas atmosphere and stirred for 1 h. Then, anhydrous hydrazine (2 mL, 91.7 mmol) was added at 0 °C and stirred for 4 h at room temperature. Saturated aqueous ammonium chloride (25 mL) was added to the reaction and stirred for 30 min. A white precipitation appeared, which was collected by filtration to provide compound 60 (0.980 g, 45%) as a white foam; 1H NMR (400 MHz, DMSO-d 6): δ 9.96 (s, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.93 (d, J = 8.2 Hz, 2H), 4.58 (s, 2H), 3.87 (s, 3H); 13C NMR (100 MHz, DMSO-d 6) 165.7, 164.8, 137.4, 131.6, 129.1, 127.3, 52.3.
Methyl 4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1,3,4-oxadiazol-2-yl)benzoate (61)
Acid 58 (0.120 g, 0.400 mmol) was dissolved in dry THF (6 mL), and then hydrazide 60 (0.079 g, 0.400 mmol), DIPEA (0.142 mL, 0.815 mmol), and HATU (0.155 g, 0.400 mmol) were added at room temperature under a nitrogen gas atmosphere. The solution was stirred for 1 h. Then, methyl N-(triethylammoniosulfonyl)carbamate (0.242 g, 1.02 mmol) was added and stirred for 16 h at the same temperature. Water (20 mL) was added to the reaction mixture and extracted with EtOAc (3 × 15 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, evaporated under reduced pressure, and purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 50:50) to furnish the compound 61 (0.132 g, 71%) as a brown solid; 1H NMR (400 MHz, CDCl3): δ 8.22–8.11 (m, 4H), 7.94 (dd, J = 8.4, 2.2 Hz, 1H), 7.86 (d, J = 2.3 Hz, 1H), 7.25–7.30 (m, 4H), 7.03 (d, J = 8.4 Hz, 1H), 5.70 (s, 1H), 3.98 (s, 3H), 2.44 (s, 3H), 1.55 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 166.3, 165.2, 163.4, 157.0, 138.1, 134.7, 134.0, 132.7, 130.3, 129.7, 129.5, 128.6, 128.4, 128.0, 126.8, 124.7, 123.1, 117.8, 116.1, 77.3, 52.5, 28.0, 21.4.
4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1,3,4-oxadiazol-2-yl)benzoic Acid (14)
To ester 61 (122 mg, 0.270 mmol) in ethanol (4.0 mL), aqueous NaOH (20%, 1.0 mL) was added at room temperature and stirred for 16 h. Then the reaction was acidified with HCl (1 N, aqueous solution) to pH 1. The 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, evaporated under reduced pressure, and purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 00:100) to give acid 14 (55 mg, 46%) as a white solid; mp 269–271 °C; with a purity of >98%; 1H NMR (400 MHz, DMSO-d 6): δ 13.28 (s, 1H), 8.22–8.08 (m, 4H), 7.96 (dd, J = 8.5, 2.1 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.37–7.21 (m, 4H), 7.11 (d, J = 8.4 Hz, 1H), 5.88 (s, 1H), 2.39 (s, 3H), 1.50 (s, 6H); 13C NMR (100 MHz, DMSO-d 6): δ 166.5, 164.2, 163.0, 156.2, 137.6, 133.9, 133.4, 132.4, 130.3, 130.2, 129.3, 128.3, 128.2, 127.0, 126.7, 123.4, 122.4, 117.7, 115.6, 77.1, 27.5, 20.8; UPLC–MS calcd m/z: [C27H22N2O4]+, 438.16; found, (M + H)+, 439.22.
Synthesis of Compound 15
5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-2H-tetrazole (62)
In a sealed tube (15 mL), dry DMF (6 mL) was added, and then nitrile 56 (0.503 g, 1.82 mmol), sodium azide (0.474 g, 7.29 mmol), and triethylammonium chloride (1.00 g, 7.29 mmol) were added. The sealed tube was closed tightly and heated at 100 °C for 24 h. The reaction was cooled to room temperature, diluted with water (20 mL), adjusted to pH 1 by adding HCl (1 N, aqueous solution), and then extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, evaporated under reduced pressure, and purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 00:100) to provide the tetrazole compound 62 (0.402 g, 86%) as a white solid; mp 244–245 °C; 1H NMR (400 MHz, CD3OD): δ 7.80 (dd, J = 8.5, 2.2 Hz, 1H), 7.66 (d, J = 2.2 Hz, 1H), 7.29–7.19 (m, 4H), 7.01 (d, J = 8.4 Hz, 1H), 5.74 (s, 1H), 2.38 (s, 3H), 1.50 (s, 6H).
4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-2H-tetrazol-2-yl)benzoic Acid (15)
Tetrazole 62 (0.320 g, 1.01 mmol), (4-(methoxycarbonyl)phenyl)boronic acid (0.361 g, 2.00 mmol), and Cu2O (0.014 g, 0.100 mmol) were added to a two-neck flask (25 mL) under positive nitrogen gas pressure. Dry DMSO (10 mL) was added, and the reaction was stirred at 100 °C under an oxygen atmosphere (balloon) for 6 h. The reaction mixture was cooled to room temperature and stirred for an additional 16 h. Water (40 mL) was added to the reaction mixture and then extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, evaporated under reduced pressure, and purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 70:30) to give an inseparable mixture of 63 along with impurities (0.210 g) as a viscous oil, which was taken for the next step without further purification. To mixture 63 (201 mg) in ethanol (8.0 mL), aqueous NaOH (20%, 2.0 mL) was added at room temperature and stirred for 48 h until completion of the reaction, as monitored by TLC. Then the reaction was acidified with HCl (1 N, aqueous solution) to pH 1. Volatiles were evaporated under reduced pressure. The resulting mixture was diluted with water (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4, filtered, evaporated under reduced pressure, and purified using flash column chromatography (silica gel, hexanes/EtOAc, 100:00 to 00:100) to give 15 (0.115 mg, 25% for two steps) as a white solid; mp 213–215 °C; with a purity of >97%; 1H NMR (400 MHz, DMSO-d 6): δ 13.36 (s, 1H), 8.22–817 (m, 4H), 7.99 (dd, J = 8.3, 2.1 Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.32–7.27 (m, 4H), 7.09 (d, J = 8.4 Hz, 1H), 5.84 (s, 1H), 2.38 (s, 3H), 1.49 (s, 6H); 13C NMR (100 MHz, DMSO-d 6): δ 166.2, 164.5, 155.3, 138.7, 137.5, 134.2, 132.7, 131.9, 131.2, 130.1, 129.3, 128.2, 128.0, 123.3, 122.3, 119.8, 118.6, 117.7, 76.7, 27.4, 20.8; UPLC–MS calcd m/z: [C26H22N4O3]+, 438.17; found, (M + H)+, 439.22.
Synthesis of Compound 16
1,1′-(4-Hydroxy-1,3-phenylene)bis(ethan-1-one) (65)
4-Acetylphenyl acetate (1.5 g, 8.4 mmol) was mixed with aluminum chloride (4.5 g, 34 mmol) and then placed into an oil bath that was preheated to 140 °C. The reaction was stirred for 4 h at 140 °C. Ice and 15% HCl were added. The mixture was then diluted with EtOAc, washed to neutral pH with water, dried over MgSO4, and evaporated under reduced pressure. The residue was purified by flash chromatography using hexanes and EtOAc to afford 1,1’-(4-hydroxy-1,3-phenylene)bis(ethan-1-one) (65) as a yellow solid (1050 mg, 70% yield). 1H NMR (400 MHz, CDCl3): δ 12.69 (s, 1H), 8.44 (d, J = 2.2 Hz, 1H), 8.07 (dd, J = 8.8, 2.2 Hz, 1H), 7.03 (d, J = 8.8 Hz, 1H), 2.72 (s, 3H), 2.59 (s, 3H).
6-Acetyl-2,2-dimethylchroman-4-one (66)
To 1,1′-(4-hydroxy-1,3-phenylene)bis(ethan-1-one) (690 mg, 3.87 mmol) in toluene (10 mL) was added acetone (284 μL, 3.87 mmol) and pyrrolidine (127 μL, 1.55 mmol). The reaction mixture was left for 1 h at room temperature and then refluxed for 24 h using a Dean–Stark apparatus to remove water. Then the reaction mixture was washed with a 15% HCl solution, followed by water and EtOAc. The organic layer was separated, dried over MgSO4, and evaporated under reduced pressure. The residue was purified by flash chromatography using hexanes and EtOAc to afford 6-acetyl-2,2-dimethylchroman-4-one (66) as a yellow solid (422 mg, 50%). 1H NMR (400 MHz, CDCl3): δ 8.44 (d, J = 2.3 Hz, 1H), 8.13 (dd, J = 8.8, 2.3 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 2.77 (s, 2H), 2.59 (s, 3H), 1.49 (s, 6H).
Ethyl 4-(4-(2,2-Dimethyl-4-oxochroman-6-yl)-4-oxobutanoyl)benzoate (67)
In a sealed microwave vial, zinc chloride (250 mg, 1.83 mmol) was heated under reduced pressure with a heat gun for 30 min to remove any water. Anhydrous toluene (2.5 mL), anhydrous diisopropylamine (239 μL, 1.37 mmol), and anhydrous tert-butanol (131 μL, 1.37 mmol) were added. After 24 h of stirring at room temperature, zinc chloride was completely dissolved. 6-Acetyl-2,2-dimethylchroman-4-one (200 mg, 916 μmol) and ethyl 4-(2-bromoacetyl)benzoate (373 mg, 1.37 mmol) were dissolved in anhydrous toluene (1 mL) and added to the microwave vial. The mixture was left in the microwave for 4 h at 140 °C. Then the reaction mixture was washed with a 15% HCl solution, followed by extraction with water and EtOAc. The organic layer was separated, dried over MgSO4, and evaporated under reduced pressure. The residue was purified by flash chromatography using hexanes and EtOAc to afford ethyl 4-(4-(2,2-dimethyl-4-oxochroman-6-yl)-4-oxobutanoyl)benzoate (67) as a yellow solid (215 mg, 58%). 1H NMR (400 MHz, CDCl3): δ 8.58 (d, J = 2.3 Hz, 1H), 8.19–8.06 (m, 5H), 7.01 (d, J = 8.8 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 3.46 (d, J = 2.2 Hz, 4H), 2.78 (s, 2H), 1.50 (s, 6H), 1.42 (t, J = 7.1 Hz, 3H).
Ethyl 4-(5-(2,2-Dimethyl-4-oxochroman-6-yl)furan-2-yl)benzoate (68)
To an oven-dried flask, ethyl 4-(4-(2,2-dimethyl-4-oxochroman-6-yl)-4-oxobutanoyl)benzoate (100 mg, 245 μmol), acetonitrile (2.5 mL), and trifluoromethanesulfonic acid (0.11 mL, 0.25 mmol) were added. The reaction mixture was stirred at 85 °C for 16 h. Then, the reaction mixture was quenched with a saturated NaHCO3 solution and extracted with EtOAc. The organic layer was separated, dried over MgSO4, and evaporated under reduced pressure. The residue was purified by flash chromatography using hexanes and EtOAc to afford ethyl 4-(5-(2,2-dimethyl-4-oxochroman-6-yl)furan-2-yl)benzoate (68) as an off-white solid (89 mg, 93% yield). 1H NMR (400 MHz, CDCl3): δ 8.22 (d, J = 2.3 Hz, 1H), 8.12–8.04 (m, 2H), 7.88 (dd, J = 8.7, 2.3 Hz, 1H), 7.83–7.76 (m, 2H), 7.01 (d, J = 8.7 Hz, 1H), 6.87 (d, J = 3.5 Hz, 1H), 6.72 (d, J = 3.5 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 2.78 (s, 2H), 1.50 (s, 6H), 1.42 (t, J = 7.2 Hz, 3H). LRMS calcd m/z: [C24H22O5]+, 390.15, found [M + H]+, 391.10.
Ethyl 4-(5-(2,2-Dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)furan-2-yl)benzoate (69)
Ethyl 4-(5-(2,2-dimethyl-4-oxochroman-6-yl)furan-2-yl)benzoate (102 mg, 0.261 mmol) and Comins’ reagent (123 mg, 0.313 mmol) were dissolved in dry THF (10 mL) and cooled to −78 °C. Lithium bis(trimethylsilyl)amide 1.0 M in THF (0.392 mL, 0.392 mmol) was added. The reaction mixture was stirred at – 78 °C for 3 h and then left overnight to warm to room temperature. The reaction was quenched by adding water and extracted with EtOAc. The organic layer was dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and purified by flash chromatography using mixtures of hexanes and EtOAc to afford ethyl 4-(5-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)furan-2-yl)benzoate (69) (111 mg, 81% yield) as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.12–8.03 (m, 2H), 7.79–7.72 (m, 2H), 7.68–7.61 (m, 2H), 6.91 (dd, J = 8.1, 0.8 Hz, 1H), 6.86 (d, J = 3.5 Hz, 1H), 6.67 (d, J = 3.5 Hz, 1H), 5.71 (s, 1H), 4.40 (q, J = 7.1 Hz, 2H), 1.56 (s, 6H), 1.42 (t, J = 7.2 Hz, 3H). LRMS calcd m/z: [C25H21F3O7S]+, 522.10; found, [M + H]+ 523.10.
Ethyl 4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)furan-2-yl)benzoate (70)
Ethyl 4-(5-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)furan-2-yl)benzoate (69) (111 mg, 0.212 mmol), 4,4,5,5-tetramethyl-2-(p-tolyl)-1,3,2-dioxaborolane (56 mg, 0.255 mmol), Pd(Amphos)Cl2 (15 mg, 21 μmol), and K2CO3 (59 mg, 0.43 mmol) were dissolved in 1,4-dioxane/water (10/1, 5 mL). This mixture was stirred at 100 °C for 12 h under a nitrogen gas atmosphere. The solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and then purified by flash chromatography using mixtures of hexanes and EtOAc to afford ethyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)furan-2-yl)benzoate (70) as an off-white solid (94 mg, 95% yield). 1H NMR (400 MHz, CDCl3): δ 8.06–7.99 (m, 2H), 7.71–7.66 (m, 2H), 7.59 (dd, J = 8.4, 2.2 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.30 (d, J = 8.0 Hz, 2H), 7.25 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 3.5 Hz, 1H), 6.50 (d, J = 3.5 Hz, 1H), 5.66 (s, 1H), 4.39 (q, J = 7.1 Hz, 2H), 2.44 (s, 3H), 1.51 (s, 6H), 1.41 (t, J = 7.1 Hz, 3H). LRMS calcd m/z: [C31H28O4]+, 464.20; found, (ES+) [M + H]+ 465.20.
4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)furan-2-yl)benzoic Acid (16)
A mixture of lithium hydroxide hydrate (0.17 g, 4.0 mmol) and ethyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)furan-2-yl)benzoate (70) (94 mg, 0.20 mmol) in THF (8 mL) and water (2 mL) was stirred at 40 °C for 12 h. The resulting mixture was cooled to room temperature, acidified with HCl (1 M) to pH = 1, followed by extraction with EtOAc. The organic layer was washed with brine and dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and then purified by flash chromatography using mixtures of hexanes and EtOAc to afford 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)furan-2-yl)benzoic acid (16) as an off-white solid (84 mg, 96% yield). 1H NMR (400 MHz, DMSO-d 6): δ 12.96 (s, 1H), 8.00–7.92 (m, 2H), 7.77 (dd, J = 8.6, 1.8 Hz, 2H), 7.67 (dd, J = 8.4, 2.2 Hz, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.30 (s, 4H), 7.18 (d, J = 3.5 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.80 (d, J = 3.5 Hz, 1H), 5.82 (s, 1H), 2.39 (s, 3H), 1.47 (s, 6H). 13C NMR (101 MHz, DMSO-d 6): δ 167.4, 154.2, 153.5, 151.4, 137.9, 134.8, 134.3, 133.5, 130.5, 130.3, 129.7, 129.4, 128.7, 125.7, 123.4, 123.2, 122.5, 120.9, 117.8, 111.1, 107.4, 76.7, 27.8, 21.3. LRMS calcd m/z: [C29H24O4]+, 436.17, found [M + H]+ 437.2.
Synthesis of Compound 17
Methyl 4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-1,2,4-triazol-3-yl)benzoate (71)
To a solution of 2,2-dimethyl-4-(p-tolyl)-2H-chromene-6-carbonitrile (56) (100 mg, 0.36 mmol, 1.0 equiv), amino (4-(methoxycarbonyl)phenyl)methaniminium acetate (102 mg, 0.43 mmol, 1.2 equiv), and copper(I) cyanide (9.2 mg, 0.06 mmol, 0.2 equiv) in DMSO (2 mL) were added to a reaction vial (20 mL). The vial was heated at 120 °C for 24 h, open to the air. The reaction mixture was cooled to room temperature, and water (20 mL) was added to the reaction. The mixture was extracted with EtOAc (10 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 80% EtOAc in hexanes) to obtain methyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-1,2,4-triazol-3-yl)benzoate (71) as a white solid (18 mg, 11%). mp 209–211 °C. 1H NMR (400 MHz, CDCl3): δ 8.06 (t, J = 6.7 Hz, 4H), 7.77 (dd, J = 8.4, 2.2 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.21 (t, J = 6.2 Hz, 4H), 6.89 (d, J = 8.4 Hz, 1H), 5.64 (s, 1H), 3.94 (s, 3H), 2.38 (s, 3H), 1.50 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 166.4, 148.2, 146.7, 140.9, 138.2, 130.2, 130.0, 129.5, 128.5, 126.8, 124.3, 123.2, 118.1, 77.5, 52.5, 28.1, 21.3. (Six peaks missing due to tautomerism).
4-(3-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-1,2,4-triazol-5-yl)benzoic Acid (17)
To a solution of methyl 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-1,2,4-triazol-3-yl)benzoate (71) (5 mg, 0.01 mmol, 1.0 equiv) in THF (1 mL), water (1 mL), and MeOH (1 mL) was added lithium hydroxide hydrate (10 mg, 0.34 mmol, 23 equiv). The resulting mixture was stirred for 15 h at room temperature. The organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 5.0. The product precipitated and was collected by filtration, followed by washing with distilled water to obtain 4-(3-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-1,2,4-triazol-5-yl)benzoic acid (17) (3.8 mg, 79%) as a white residue. 1H NMR (400 MHz, THF-d 8): δ 8.17 (dd, J = 8.3, 1.6 Hz, 2H), 8.07 (dd, J = 8.3, 1.5 Hz, 2H), 7.95 (dd, J = 8.4, 2.1 Hz, 1H), 7.74 (t, J = 2.0 Hz, 1H), 7.30 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H), 6.97 (d, J = 8.4 Hz, 1H), 5.72 (s, 1H), 2.39 (s, 3H), 1.49 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.9, 156.0, 138.2, 136.1, 135.6, 135.1, 132.5, 130.5, 130.1, 129.8, 129.2, 128.4, 126.5, 123.8, 123.2, 117.9, 76.8, 27.7, 21.1. Three 13C peaks could not be observed, possibly due to tautomerism. LRMS calcd m/z: [C27H24N3O3]+, 438.18; found, 438.27. 99% pure by UPLC analysis.
Synthesis of Compound 18
2,2-Dimethyl-6-(prop-1-en-2-yl)-4-(p-tolyl)-2H-chromene (72)
A solution of methyltriphenylphosphonium bromide (700 mg, 1.96 mmol, 1.9 equiv) in THF (10 mL) was cooled to 0 °C, and a solution of potassium tert-butoxide in THF (1 M, 1.9 mL, 1.9 mmol, 1.9 equiv) was added to the solution dropwise. The reaction mixture was stirred for 1 h at 0 °C, followed by the addition of 1-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)ethan-1-one (44) (300 mg, 1.03 mmol, 1.0 equiv) dissolved in THF (5 mL). The resulting mixture was slowly warmed to room temperature and stirred for 20 h. The reaction was quenched with saturated ammonium chloride solution and extracted with EtOAc (10 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 5% EtOAc in hexanes) to obtain 2,2-dimethyl-6-(prop-1-en-2-yl)-4-(p-tolyl)-2H-chromene (72) (241 mg, 81%) as a white solid. mp 70–72 °C. 1H NMR (400 MHz, CDCl3): δ 7.27–7.21 (m, 3H), 7.18 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 2.3 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 5.57 (s, 1H), 5.13 (d, J = 1.6 Hz, 1H), 4.88 (t, J = 1.5 Hz, 1H), 2.37 (s, 3H), 2.00 (s, 3H), 1.46 (s, 6H).
(E)-2-(2-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (73)
A mixture of bis(pinacolato)diboron (453 mg, 1.78 mmol, 2.6 equiv) and 2,2-dimethyl-6-(prop-1-en-2-yl)-4-(p-tolyl)-2H-chromene (72) (200 mg, 0.69 mmol, 1.0 equiv) was added to a solution of trans-bis(triphenylphosphine)rhodium carbonyl chloride (25 mg, 36 μmol, 0.05 equiv) in benzene (3 mL) and acetonitrile (1 mL) in a microwave vial. The vial was sealed and heated in the microwave reactor at 150 °C for 2 h. The solvent was evaporated under reduced pressure, and the residue was purified by flash column chromatography (silica gel, 100% hexanes to 5% EtOAc in hexanes) to obtain (E)-2-(2-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (73) (191 mg, 66%) as a white solid. mp 168–170 °C. 1H NMR (400 MHz, CDCl3): δ 7.34 (d, J = 8.4 Hz, 1H), 7.27–7.22 (m, 5H), 6.85 (d, J = 8.4 Hz, 1H), 5.59 (s, 1H), 5.57 (d, J = 0.9 Hz, 1H), 2.43 (s, 3H), 2.32 (d, J = 0.9 Hz, 3H), 1.50 (s, 6H), 1.30 (d, J = 1.6 Hz, 12H).
Methyl (E)-4-(2-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)benzoate (74)
(E)-2-(2-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (73) (100 mg, 0.24 mmol, 1.0 equiv), methyl 4-bromobenzoate (80 mg, 0.37 mmol, 1.5 equiv), and potassium carbonate (150 mg, 1.09 mmol, 4.5 equiv) were added to a heavy-walled pressure flask, followed by the addition of 1,2-dimethoxyethane (5 mL) and water (1 mL). After sparging of the mixture with nitrogen gas for 10 min, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (18 mg, 0.025 mmol, 0.1 equiv) was added in one portion, and the flask was sealed and heated at 90 °C for 5 h. After cooling to room temperature, the layers were separated, and the aqueous layer was extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with brine and dried over anhydrous MgSO4. The resulting solution was first absorbed on silica gel, then the solvent was removed under reduced pressure and then the compounds was purified by flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) to obtain methyl (E)-4-(2-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)benzoate (74) (61 mg, 60%) as an amorphous white solid. mp 217–219 °C. 1H NMR (400 MHz, CDCl3): δ 7.90 (d, J = 8.4 Hz, 2H), 7.28–7.22 (m, 3H), 7.18 (d, J = 8.1 Hz, 2H), 7.14–7.10 (m, 3H), 6.79 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 1.8 Hz, 1H), 5.53 (s, 1H), 3.81 (s, 3H), 2.30 (s, 3H), 2.08 (d, J = 1.3 Hz, 3H), 1.41 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 167.1, 153.4, 143.5, 139.4, 137.7, 135.9, 135.4, 134.7, 129.5, 129.3 (two 13C), 129.1, 128.7, 127.8, 126.9, 125.4, 123.4, 122.2, 116.8, 76.2, 52.1, 27.8, 21.4, 17.8.
(E)-4-(2-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)benzoic Acid (18)
To a solution of methyl (E)-4-(2-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)benzoate (74) (70 mg, 0.16 mmol, 1.0 equiv) in THF (2 mL) and MeOH (2 mL) was added an aqueous sodium hydroxide solution (2 N, 1.0 mL, 2.0 mmol, 12 equiv). The resulting mixture was stirred for 22 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (3 × 10 mL), washed with brine, dried over anhydrous MgSO4, evaporated under reduced pressure, and purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain (E)-4-(2-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)prop-1-en-1-yl)benzoic acid (18) (36 mg, 53%) as a yellow solid. mp 211–213 °C. 1H NMR (400 MHz, THF-d 8): δ 7.97 (d, J = 8.4 Hz, 2H), 7.40–7.33 (m, 3H), 7.30–7.17 (m, 5H), 6.84 (d, J = 8.4 Hz, 1H), 6.70 (d, J = 1.7 Hz, 1H), 5.66 (s, 1H), 2.35 (s, 3H), 2.17 (d, J = 1.4 Hz, 3H), 1.46 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 154.2, 143.6, 139.6, 138.1, 136.6, 136.2, 135.5, 130.1, 129.7, 129.6, 129.5, 129.4, 129.1, 127.5, 125.9, 123.8, 122.7, 117.3, 76.4, 27.7, 21.1, 17.6. LRMS calcd m/z: [C28H27O3]+, 411.20; found, 411.25. 96% pure by UPLC analysis.
Synthesis of Compound 19–21
Methyl 4-(1H-Pyrrol-2-yl)benzoate (75)
To a solution of tert-butyl 2-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (49) (5.6 g, 19 mmol) in THF (100 mL) was added a sodium methoxide solution (25 wt %, 6.4 mL, 28 mmol) and stirred at room temperature for 30 min. The mixture was quenched with water and extracted with EtOAc. The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash chromatography using mixtures of hexanes and EtOAc to afford methyl 4-(1H-pyrrol-2-yl)benzoate as a white solid (3.3 g, 87%). 1H NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 8.06–7.98 (m, 2H), 7.57–7.44 (m, 2H), 6.93 (td, J = 2.7, 1.4 Hz, 1H), 6.66 (ddd, J = 3.9, 2.7, 1.4 Hz, 1H), 6.33 (dt, J = 3.5, 2.5 Hz, 1H), 3.92 (s, 3H).
Methyl 4-(5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76)
Methyl 4-(1H-pyrrol-2-yl)benzoate (75) (3.7 g, 18 mmol), 4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (2.6 g, 10 mmol), [Ir(OMe)(COD)]2 (0.37 g, 0.55 mmol), and 4,4′-di-tert-butyl-2,2′-bipyridine (0.30 g, 1.1 mmol) were dissolved in dry THF (100 mL) and then stirred for 8 h at room temperature. The solvent was removed under reduced pressure, and the residue was partitioned between EtOAc and water. The resulting organic layer was dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and purified by flash chromatography using mixtures of hexanes and EtOAc to afford methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) as a white solid (4.8 g, 79%). 1H NMR (400 MHz, CDCl3): δ 8.96 (s, 1H), 8.08–7.98 (m, 2H), 7.65–7.56 (m, 2H), 6.89 (dd, J = 3.7, 2.4 Hz, 1H), 6.69 (dd, J = 3.7, 2.5 Hz, 1H), 3.92 (s, 3H), 1.35 (s, 12H).
General Synthetic Method for 77a–77c
6-Bromo-2,2-dimethyl-2H-chromen-4-yl trifluoromethanesulfonate (42) (1.0 equiv), substituted phenyl boronic acid pinacol esters (1.0 equiv), and K2CO3 (2.5 equiv) were dissolved in deoxygenated 1,4-dioxane (0.2 M). [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (0.1 equiv) was added, and the reaction mixture was stirred at 60 °C for 5 h under a nitrogen gas atmosphere. The solvent was evaporated under reduced pressure, and the residue was then purified by flash chromatography using hexanes and EtOAc to afford 77a–77c.
6-Bromo-4-(4-ethylphenyl)-2,2-Dimethyl-2H-chromene (77a)
Colorless oil (55% yield). 1H NMR (400 MHz, CDCl3): δ 7.28 (s, 4H), 7.27–7.23 (m, 1H), 7.15 (d, J = 2.4 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 5.63 (s, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.49 (s, 6H), 1.31 (t, J = 7.6 Hz, 3H).
6-Bromo-4-(4-isopropylphenyl)-2,2-Dimethyl-2H-chromene (77b)
Colorless oil (43% yield). 1H NMR (400 MHz, CDCl3): δ 7.28–7.23 (m, 4H), 7.22 (t, J = 2.0 Hz, 1H), 7.14 (d, J = 2.4 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 5.61 (s, 1H), 2.95 (p, J = 6.9 Hz, 1H), 1.47 (s, 6H), 1.30 (d, J = 6.9 Hz, 6H).
6-Bromo-4-(4-(tert-butyl)phenyl)-2,2-Dimethyl-2H-chromene (77c)
Colorless oil (62% yield). 1H NMR (400 MHz, CDCl3): δ 7.44–7.40 (m, 2H), 7.26–7.21 (m, 3H), 7.15 (d, J = 2.4 Hz, 1H), 6.76 (d, J = 8.5 Hz, 1H), 5.62 (s, 1H), 1.47 (s, 6H), 1.36 (s, 9H).
General Synthetic Method for 78a–78c
Compounds 77a–77c (1.0 equiv), methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) (1.1 equiv), Pd(PPh3)4 (0.1 equiv), and K2CO3 (3.0 equiv) were dissolved in 1,4-dioxane/water (10/1, 0.2 M). This mixture was stirred at 100 °C for 8 h under a nitrogen gas atmosphere, cooled to room temperature, acidified with HCl (1 M) to pH = 1, and then extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and purified by flash chromatography using mixtures of hexanes and EtOAc to afford the desired products.
Methyl 4-(5-(4-(4-Ethylphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (78a)
Yellow solid (74% yield). 1H NMR (400 MHz, CDCl3): δ 8.44 (s, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.1 Hz, 2H), 7.38–7.29 (m, 3H), 7.26 (d, J = 8.3 Hz, 2H), 7.21 (s, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.65 (d, J = 3.4 Hz, 1H), 6.38 (s, 1H), 5.66 (s, 1H), 3.91 (s, 3H), 2.72 (q, J = 7.6 Hz, 2H), 1.51 (s, 6H), 1.30 (t, J = 7.6 Hz, 3H).
Methyl 4-(5-(4-(4-Isopropylphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (78b)
Yellow solid (76% yield). 1H NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.37–7.21 (m, 6H), 6.93 (d, J = 8.3 Hz, 1H), 6.65 (t, J = 3.2 Hz, 1H), 6.39 (t, J = 3.1 Hz, 1H), 5.67 (s, 1H), 3.91 (s, 3H), 2.97 (p, J = 6.9 Hz, 1H), 1.51 (s, 6H), 1.31 (d, J = 6.9 Hz, 6H).
Methyl 4-(5-(4-(4-(tert-Butyl)phenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (78c)
Yellow solid (80% yield). 1H NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 7.23 (s, 2H), 6.94 (d, J = 8.3 Hz, 1H), 6.65 (s, 1H), 6.39 (s, 1H), 5.67 (s, 1H), 3.91 (s, 3H), 1.51 (s, 6H), 1.38 (s, 9H).
General Synthetic Method for 19–21
A mixture of lithium hydroxide hydrate (10 equiv) and 78a–78c (1.0 equiv) in THF (10 mL) and water (2 mL) was stirred at 40 °C for 18 h. The resulting mixture was cooled to room temperature and acidified with HCl (1 M) to pH = 1 and then extracted with EtOAc. The organic layer was washed with brine and dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and then purified by flash chromatography using mixtures of hexanes and EtOAc to afford the final compounds.
4-(5-(4-(4-Ethylphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (19)
Yellow solid (92% yield). 1H NMR (400 MHz, DMSO-d 6): δ 12.73 (s, 1H), 11.33 (t, J = 2.4 Hz, 1H), 7.97–7.86 (m, 2H), 7.84–7.76 (m, 2H), 7.60 (dd, J = 8.4, 2.2 Hz, 1H), 7.31 (d, J = 6.9 Hz, 5H), 6.95 (d, J = 8.4 Hz, 1H), 6.70 (dd, J = 3.7, 2.4 Hz, 1H), 6.27 (dd, J = 3.7, 2.3 Hz, 1H), 5.80 (s, 1H), 2.67 (q, J = 7.6 Hz, 2H), 1.46 (s, 6H), 1.23 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d 6O): δ 167.1, 151.9, 143.5, 136.6, 134.9, 134.7, 133.4, 131.2, 129.8, 129.8, 128.2, 128.0, 127.0, 126.0, 125.3, 123.1, 121.9, 121.7, 116.9, 109.6, 107.2, 75.7, 27.9, 27.1, 15.4. LRMS calcd m/z: [C30H27NO3]+, 449.20; found, (ES+) [M + H]+ 450.20.
4-(5-(4-(4-Isopropylphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (20)
Yellow solid (98% yield). 1H NMR (400 MHz, DMSO-d 6): δ 12.73 (s, 1H), 11.33 (t, J = 2.4 Hz, 1H), 7.97–7.86 (m, 2H), 7.84–7.76 (m, 2H), 7.60 (dd, J = 8.4, 2.2 Hz, 1H), 7.33 (d, J = 3.6 Hz, 5H), 6.95 (d, J = 8.4 Hz, 1H), 6.70 (dd, J = 3.7, 2.4 Hz, 1H), 6.27 (dd, J = 3.7, 2.3 Hz, 1H), 5.80 (s, 1H), 2.95 (hept, J = 6.9 Hz, 1H), 1.46 (s, 6H), 1.25 (d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, DMSO-d 6): δ 167.1, 151.9, 148.1, 136.6, 135.0, 134.8, 133.4, 131.2, 129.9, 129.8, 128.2, 127.0, 126.5, 126.0, 125.3, 123.4, 123.1, 121.9, 121.7, 116.9, 109.6, 107.2, 75.7, 33.1, 27.1, 23.8. LRMS calcd m/z: [C31H29NO3]+, 463.21, found [M + H]+ 464.20.
4-(5-(4-(4-(tert-Butyl)phenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (21)
Yellow solid (91% yield). 1H NMR (400 MHz, DMSO-d 6): δ 12.74 (s, 1H), 11.34 (t, J = 2.4 Hz, 1H), 7.89 (dd, J = 9.3, 2.5 Hz, 2H), 7.83–7.76 (m, 2H), 7.60 (dd, J = 8.4, 2.2 Hz, 1H), 7.52–7.44 (m, 2H), 7.44–7.29 (m, 3H), 6.95 (d, J = 8.4 Hz, 1H), 6.70 (dd, J = 3.7, 2.4 Hz, 1H), 6.27 (dd, J = 3.7, 2.3 Hz, 1H), 5.80 (s, 1H), 1.46 (s, 6H), 1.33 (s, 9H). 13C NMR (101 MHz, DMSO-d 6): δ 167.1, 151.9, 150.4, 136.6, 134.8, 134.7, 133.3, 131.2, 130.0, 129.8, 128.6, 127.9, 127.0, 126.1, 125.4, 123.1, 121.8, 121.7, 116.9, 109.5, 107.2, 75.7, 34.3, 31.1, 27.1. LRMS calcd m/z: [C32H31NO3]+, 477.23, found [M + H]+ 478.20.
Synthesis of 22
tert-Butyl 2-(4-(Methoxycarbonyl)phenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (79)
Under a nitrogen atmosphere, di-tert-butyl dicarbonate (20.0 mL 1 M in DCM, 2.1 equiv) was added to a solution of a methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)cyclohexa-1,5-diene-1-carboxylate (76) (3.20 g, 9.72 mmol, 1.0 equiv) and 4-dimethylaminopyridine (60 mg, 0.49 mmol, 0.05 equiv) in anhydrous acetonitrile (5 mL) at room temperature. The mixture was stirred for 52 h until the starting material disappeared completely, as monitored by TLC. After water was added, the reaction mixture was extracted with DCM (15 mL × 3). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) to obtain tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (79) (4.00 g, 96%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.04–7.97 (m, 2H), 7.40–7.34 (m, 2H), 6.64 (d, J = 3.3 Hz, 1H), 6.25 (d, J = 3.3 Hz, 1H), 3.92 (s, 3H), 1.35 (s, 12H), 1.32 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 150.4, 139.0, 137.2, 129.1, 128.8, 128.8, 121.1, 114.7, 84.5, 84.0, 52.2, 27.5, 24.9. One 13C signal for C-Bpin could not be observed.
tert-Butyl 2-(2,2-Dimethyl-4-oxochroman-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (80)
6-Bromo-2,2-dimethylchroman-4-one (550 mg, 2.16 mmol, 1.1 equiv), tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (79) (822 mg, 1.92 mmol, 1.0 equiv), and potassium carbonate (830 mg, 6.01 mmol, 3.1 equiv) were added to a vial followed by addition of 1,2-dimethoxyethane (15 mL) and water (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (160 mg, 0.196 mmol, 0.1 equiv). The vial was sealed and placed in a preheated heating block at 90 °C and refluxed for 6 h. After the reaction was complete, as monitored by TLC, brine was added to the reaction mixture, extracted with EtOAc (20 mL × 3), and dried over anhydrous MgSO4, and the solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(2,2-dimethyl-4-oxochroman-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (80) as a white solid (765 mg, 84%). mp 129–131 °C. 1H NMR (400 MHz, CDCl3): δ 8.08–8.01 (m, 2H), 7.88 (d, J = 2.3 Hz, 1H), 7.53 (dd, J = 8.5, 2.3 Hz, 1H), 7.48–7.41 (m, 2H), 7.03–6.92 (m, 1H), 6.30 (d, J = 3.4 Hz, 1H), 6.23 (d, J = 3.4 Hz, 1H), 3.93 (s, 3H), 2.74 (s, 2H), 1.48 (s, 6H), 1.19 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 192.3, 167.0, 159.5, 149.7, 138.9, 137.1, 136.4, 135.5, 129.3, 128.6, 128.4, 126.8, 126.6, 119.6, 117.9, 113.7, 112.9, 84.6, 79.6, 52.2, 49.0, 27.3, 26.8.
tert-Butyl 2-(2,2-Dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (81)
To a stirred solution of tert-butyl 2-(2,2-dimethyl-4-oxochroman-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (80) (583 mg, 1.23 mmol, 1.0 equiv), 2,6-lutidine (925 mg, 8.63 mmol, 7.0 equiv), and 4-dimethylaminopyridine (41 mg, 0.34 mmol, 0.3 equiv) in anhydrous DCM (10 mL) at 0 °C was added triflic anhydride (1.00 g, 3.54 mmol, 2.9 equiv) dropwise. The reaction mixture was slowly warmed to room temperature with continuous stirring. Stirring was continued overnight at room temperature. The reaction was quenched with saturated sodium bicarbonate solution, and the reaction mixture was then extracted with DCM (20 mL × 3). The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to yield a crude brown oil. The crude product was purified by flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) to obtain tert-butyl 2-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (81) (550 mg, 74%) as a colorless liquid. 1H NMR (400 MHz, CDCl3): δ 8.09–8.02 (m, 2H), 7.50–7.42 (m, 2H), 7.32–7.23 (m, 2H), 6.86 (d, J = 8.9 Hz, 1H), 6.31 (d, J = 3.4 Hz, 1H), 6.21 (d, J = 3.4 Hz, 1H), 5.66 (s, 1H), 3.93 (s, 3H), 1.55 (s, 6H), 1.19 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 153.2, 149.6, 142.4, 138.9, 136.6, 135.4, 132.6, 129.4, 128.7, 128.4, 127.3, 122.2, 118.5, 117.3 (q, CF3) 116.4, 115.6, 113.6, 112.9, 84.6, 78.1, 52.3, 28.2, 27.3.
tert-Butyl 2-(4-(4-Fluorophenyl)-2,2-dimethyl-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82a)
tert-Butyl 2-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (200 mg, 0.33 mmol, 1.0 equiv), (4-fluorophenyl)boronic acid (68 mg, 0.49 mmol, 1.5 equiv), and an aqueous solution of tribasic potassium phosphate (0.5 M, 4.0 mL, 2.0 mmol, 6.1 equiv) were added to a reaction vial, followed by the addition of THF (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of XPhos Pd G2 (60 mg, 0.076 mmol, 0.2 equiv). The vial was sealed and placed into a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, as monitored by TLC, brine was added to the reaction mixture, extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(4-(4-fluorophenyl)-2,2-dimethyl-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82a) as a white solid (132 mg, 72%). mp could not be determined because the compound degraded at 160 °C. 1H NMR (400 MHz, CDCl3): δ 8.09–7.99 (m, 2H), 7.45–7.38 (m, 2H), 7.37–7.28 (m, 2H), 7.21 (dd, J = 8.3, 2.2 Hz, 1H), 7.13–7.02 (m, 2H), 7.01 (d, J = 2.1 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.27 (d, J = 3.4 Hz, 1H), 6.14 (d, J = 3.4 Hz, 1H), 5.62 (s, 1H), 3.93 (s, 3H), 1.52 (s, 6H), 1.16 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 162.56 (d, J = 245 Hz), 153.1, 149.7, 138.8, 137.5, 134.8, 134.2 (d, J = 3 Hz), 133.8, 130.5(d, J = 7 Hz), 130.4, 129.7, 129.3, 128.5, 128.2, 126.3, 126.2, 121.6, 116.5, 115.5 (d, J = 22 Hz), 113.4, 112.2, 84.3, 76.1, 52.2, 27.8, 27.3.
4-(5-(4-(4-Fluorophenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (22)
tert-Butyl 2-(4-(4-fluorophenyl)-2,2-dimethyl-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82a) (100 mg, 0.18 mmol, 1.0 equiv) was heated without solvent at 180 °C under nitrogen gas for 30 min. The dark residue was dissolved in THF (1 mL) and MeOH (1 mL), and lithium hydroxide (50 mg, 1.2 mmol, 6.7 equiv) was dissolved in water (1 mL) and added to the reaction mixture, which was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, dried over anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc, and 2% formic acid in hexanes) to obtain 4-(5-(4-(4-fluorophenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (22) (36 mg, 74%) as a yellow solid. mp 247–249 °C. 1H NMR (400 MHz, THF-d 8): δ 10.40 (s, 1H), 7.96–7.89 (m, 2H), 7.64–7.57 (m, 2H), 7.50–7.36 (m, 3H), 7.27 (d, J = 2.2 Hz, 1H), 7.21–7.12 (m, 2H), 6.86 (d, J = 8.4 Hz, 1H), 6.60 (dd, J = 3.7, 2.5 Hz, 1H), 6.27 (dd, J = 3.7, 2.4 Hz, 1H), 5.72 (s, 1H), 1.46 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.2, 163.4(d, J = 243 Hz), 153.2, 137.9, 135.8, 135.4 (d, J = 3 Hz), 134.7, 132.6, 131.2 (d, J = 8 Hz), 130.8, 130.5, 128.1, 126.8, 126.4, 123.5, 122.9, 122.3, 117.8, 115.8 (d, J = 21 Hz), 109.9, 107.8, 76.3, 27.5. LRMS calcd m/z: [C28H23FNO3]+, 440.17; found, 440.22. 95% pure by UPLC analysis.
Synthesis of 23
tert-Butyl 2-(2,2-Dimethyl-4-(4-(trifluoromethyl)phenyl)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82b)
tert-Butyl 2-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-5-(4-methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (81) (167 mg, 0.28 mmol, 1.0 equiv), (4-(trifluoromethyl)phenyl)boronic acid (90 mg, 0.47 mmol, 1.7 equiv), and an aqueous solution of tribasic potassium phosphate (0.5 M, 2.2 mL, 1.1 mmol, 4.0 equiv) were added to a reaction vial followed by addition of THF (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of XPhos Pd G2 (60 mg, 0.076 mmol, 0.3 equiv). The vial was sealed and placed in a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, brine was added to the reaction mixture and extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(2,2-dimethyl-4-(4-(trifluoromethyl)phenyl)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82b) as a white solid (138 mg, 83%). mp could not be determined because the compound degraded at 160 °C. 1H NMR (400 MHz, CDCl3): δ 8.05–7.99 (m, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.50–7.45 (m, 2H), 7.44–7.37 (m, 2H), 7.22 (dd, J = 8.3, 2.2 Hz, 1H), 6.96 (d, J = 2.1 Hz, 1H), 6.93 (s, 1H), 6.26 (d, J = 3.4 Hz, 1H), 6.13 (d, J = 3.4 Hz, 1H), 5.67 (s, 1H), 3.92 (s, 3H), 1.53 (s, 6H), 1.14 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 153.1, 149.7, 142.1, 138.9, 137.3, 134.9, 133.9, 130.7, 130.6, 130.1 (q, J = 32 Hz) 129.4, 129.2, 128.5, 128.3, 126.4, 126.1, 125.6 (q, J = 3.7 Hz), 121.1, 116.7, 113.4, 112.3, 84.3, 76.2, 52.2, 27.8, 27.3. (CF3 could not be seen).
4-(5-(2,2-Dimethyl-4-(4-(trifluoromethyl)phenyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (23)
tert-Butyl 2-(2,2-dimethyl-4-(4-(trifluoromethyl)phenyl)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82b) (100 mg, 0.17 mmol, 1.0 equiv) was heated without solvent at 180 °C under a nitrogen gas atmosphere for 30 min. The dark residue was dissolved in THF (1 mL) and MeOH (1 mL). Lithium hydroxide (50 mg, 1.2 mmol, 6.7 equiv) was then dissolved in water (1 mL) and added to the reaction mixture, which was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, dried over anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(2,2-dimethyl-4-(4-(trifluoromethyl)phenyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (23) (46 mg, 57%) as a bright yellow solid. mp 246–248 °C. 1H NMR (400 MHz, THF-d 8): δ 10.42 (s, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 8.1 Hz, 2H), 7.64–7.58 (m, 4H), 7.54–7.48 (m, 1H), 7.28 (s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.64–6.58 (m, 1H), 6.32–6.27 (m, 1H), 5.82 (s, 1H), 1.49 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 153.1, 143.3, 137.9, 135.6, 134.6, 132.7, 131.8, 130.8, 130.3 (q, J = 33 Hz), 129.9, 128.2, 127.0, 126.6, 126.1, (q, J = 4 Hz), 123.5, 122.4, 122.0, 117.9, 110.0, 107.9, 76.3, 27.4. (13C of CF3 was not observed). LRMS calcd m/z: [C29H23F3NO3]+, 490.17; found, 490.23. 95% pure by UPLC analysis.
Synthesis of 24
tert-Butyl 2-(4-(4-(2-Hydroxyethyl)phenyl)-2,2-dimethyl-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82c)
tert-Butyl 2-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (81) (167 mg, 0.28 mmol, 1.0 equiv), (4-(2-hydroxyethyl)phenyl)boronic acid (80 mg, 0.48 mmol, 1.8 equiv), and an aqueous solution of tribasic potassium phosphate (0.5 M, 2.2 mL, 1.1 mmol, 4.0 equiv) were added to a reaction vial followed by addition of THF (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of XPhos Pd G2 (60 mg, 0.076 mmol, 0.3 equiv). The vial was sealed and placed in a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, monitored by TLC, brine was added to the reaction mixture, and extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(4-(4-(2-hydroxyethyl)phenyl)-2,2-dimethyl-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82c) as a white solid (135 mg, 85%). mp could not be determined because the compound degraded at 160 °C. 1H NMR (400 MHz, CDCl3): δ 8.05–7.98 (m, 2H), 7.44–7.37 (m, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.24 (d, J = Hz, 2H), 7.19 (dd, J = 8.2, 2.2 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 6.26 (d, J = 3.4 Hz, 1H), 6.13 (d, J = 3.4 Hz, 1H), 5.62 (s, 1H), 3.91 (d, J = 9.9 Hz, 5H), 2.90 (t, J = 6.5 Hz, 2H), 1.51 (s, 6H), 1.15 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 153.2, 149.7, 138.8, 138.2, 137.5, 136.5, 134.7, 134.5, 130.4, 129.6, 129.4, 129.2, 129.0, 128.5, 128.2, 126.4, 126.3, 121.8, 116.5, 113.3, 112.2, 84.3, 76.2, 63.7, 52.2, 39.1, 27.9, 27.3.
4-(5-(4-(4-(2-Hydroxyethyl)phenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (24)
tert-Butyl 2-(4-(4-(2-hydroxyethyl)phenyl)-2,2-dimethyl-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82c) (100 mg, 0.17 mmol, 1.0 equiv) was heated without solvent at 180 °C under a nitrogen gas atmosphere for 30 min. The dark residue was dissolved in THF (1 mL) and MeOH (1 mL). Lithium hydroxide (50 mg, 1.2 mmol, 6.7 equiv) was then dissolved in water (1 mL) and added to the reaction mixture, which was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, and dried over anhydrous MgSO4 and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(4-(4-(2-hydroxyethyl)phenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (24) (47 mg, 58% over two steps) as a yellow solid. mp 267–269 °C. 1H NMR (400 MHz, THF-d 8): δ 10.49 (s, 1H), 7.98–7.91 (m, 2H), 7.66–7.59 (m, 2H), 7.47 (dd, J = 8.4, 2.2 Hz, 1H), 7.33 (dd, J = 27.4, 2.4 Hz, 5H), 6.86 (d, J = 8.4 Hz, 1H), 6.61 (dd, J = 3.7, 2.4 Hz, 1H), 6.29 (dd, J = 3.7, 2.4 Hz, 1H), 5.68 (s, 1H), 3.74 (t, J = 6.9 Hz, 2H), 2.82 (t, J = 6.9 Hz, 2H), 1.46 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.8, 153.3, 140.4, 137.9, 136.8, 135.9, 135.5, 132.5, 130.7, 130.0, 129.7, 129.1, 128.3, 126.7, 126.3, 123.5, 123.1, 122.4, 117.7, 109.9, 107.8, 76.2, 63.7, 40.1, 27.5. LRMS calcd m/z: [C30H28NO4]+, 466.21; found, 466.27. 94% pure by UPLC analysis.
Synthesis of 25
tert-Butyl 2-(4-(Methoxycarbonyl)phenyl)-5-(4-(4-methoxyphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (82d)
tert-Butyl 2-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (81) (167 mg, 0.28 mmol, 1.0 equiv), 4-methoxyphenylboronic acid MIDA ester (120 mg, 0.46 mmol, 1.7 equiv), and an aqueous solution of tribasic potassium phosphate (0.5 M, 2.2 mL, 1.1 mmol, 4.0 equiv) were added to a reaction vial, followed by the addition of THF (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of XPhos Pd G2 (60 mg, 0.076 mmol, 0.3 equiv). The vial was sealed and placed in a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, brine was added to the reaction mixture and extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-(4-methoxyphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (82d) as a white solid (146 mg, 94%). mp could not be determined because the compound degraded at 160 °C. 1H NMR (400 MHz, CDCl3): δ 8.07–8.00 (m, 2H), 7.46–7.40 (m, 2H), 7.31–7.27 (m, 2H), 7.20 (dd, J = 8.2, 2.2 Hz, 1H), 7.08 (s, 1H), 6.91 (dd, J = 8.5, 2.9 Hz, 3H), 6.27 (s, 1H), 6.15 (s, 1H), 5.61 (s, 1H), 3.93 (s, 3H), 3.83 (s, 3H), 1.52 (s, 6H), 1.17 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.0, 159.4, 153.2, 149.7, 138.8, 137.6, 134.7, 134.2, 130.6, 130.1, 129.9, 129.3, 129.0, 128.4, 128.2, 126.4, 126.2, 122.0, 116.4, 113.9, 113.3, 112.1, 84.2, 76.1, 55.4, 52.2, 27.8, 27.3.
4-(5-(4-(4-Methoxyphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (25)
tert-Butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-(4-methoxyphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (82d) (100 mg, 0.17 mmol, 1.0 equiv) was heated without solvent at 180 °C under a nitrogen gas atmosphere for 30 min. The dark residue was dissolved in THF (1 mL) and MeOH (1 mL). Lithium hydroxide (50 mg, 1.2 mmol, 6.7 equiv) was then dissolved in water (1 mL) and added to the reaction mixture, which was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, and dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(4-(4-methoxyphenyl)-2,2-dimethyl-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (25) (40 mg, 51%) as a bright yellow solid. mp 242–244 °C. 1H NMR (400 MHz, THF-d 8): δ 8.72 (s, 1H), 6.26–6.19 (m, 2H), 5.94–5.87 (m, 2H), 5.74 (dd, J = 8.3, 2.2 Hz, 1H), 5.63 (d, J = 2.2 Hz, 1H), 5.61–5.56 (m, 2H), 5.27–5.21 (m, 2H), 5.14 (d, J = 8.4 Hz, 1H), 4.89 (dd, J = 3.7, 2.5 Hz, 1H), 4.57 (dd, J = 3.7, 2.4 Hz, 1H), 3.94 (s, 1H), 2.08 (s, 3H), −0.26 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 160.5, 153.3, 137.9, 136.0, 135.2, 132.5, 131.3, 130.8, 130.3, 129.5, 128.0, 126.7, 126.2, 123.5, 123.3, 122.5, 117.7, 114.4, 109.9, 107.8, 76.2, 55.3, 27.6. LRMS calcd m/z: [C29H26NO4]+, 451.19; found, 452.26. 98% pure by UPLC analysis.
Synthesis of 26
tert-Butyl 2-(2,2-Dimethyl-4-(4-nitrophenyl)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82e)
tert-Butyl 2-(2,2-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (81) (167 mg, 0.28 mmol, 1.0 equiv), (4-nitrophenyl)boronic acid (70 mg, 0.42 mmol, 1.5 equiv), and an aqueous solution of tribasic potassium phosphate (0.5 M, 2.2 mL, 1.1 mmol, 4.0 equiv) were added to a vial followed by addition of THF (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of XPhos Pd G2 (60 mg, 0.076 mmol, 0.3 equiv). The vial was sealed and placed in a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, brine was added to the reaction mixture and extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(2,2-dimethyl-4-(4-nitrophenyl)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82e) as a yellow solid (76 mg, 48%). mp could not be determined; the compound degraded at 160 °C. 1H NMR (400 MHz, CDCl3): δ 8.28–8.21 (m, 2H), 8.05–7.99 (m, 2H), 7.58–7.51 (m, 2H), 7.44–7.37 (m, 2H), 7.24 (dd, J = 8.3, 2.2 Hz, 1H), 6.99–6.90 (m, 2H), 6.26 (d, J = 3.4 Hz, 1H), 6.13 (d, J = 3.4 Hz, 1H), 5.73 (s, 1H), 3.92 (s, 3H), 1.54 (s, 6H), 1.15 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.0, 153.0, 149.6, 147.5, 145.1, 138.8, 137.2, 135.0, 133.4, 131.3, 131.0, 129.7, 129.3, 128.5, 128.2, 126.5, 126.1, 123.9, 120.6, 116.8, 113.4, 112.4, 84.3, 76.1, 52.2, 27.6, 27.3.
4-(5-(2,2-Dimethyl-4-(4-nitrophenyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (26)
tert-Butyl 2-(2,2-dimethyl-4-(4-nitrophenyl)-2H-chromen-6-yl)-5-(4-(methoxycarbonyl)phenyl)-1H-pyrrole-1-carboxylate (82e) (50 mg, 0.09 mmol, 1.0 equiv) was heated without solvent at 180 °C under a nitrogen gas atmosphere for 30 min. The dark residue was dissolved in THF (1 mL) and MeOH (1 mL). Lithium hydroxide hydrate (50 mg, 1.2 mmol, 6.7 equiv) was then dissolved in water (1 mL) and added to the reaction mixture, which was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, and dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(2,2-dimethyl-4-(4-nitrophenyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (26) (26 mg, 65%) as an off-white solid. mp 259–261 °C. 1H NMR (400 MHz, THF-d 8): δ 10.40 (s, 1H), 8.34–8.27 (m, 2H), 7.97–7.90 (m, 2H), 7.68–7.63 (m, 2H), 7.63–7.58 (m, 2H), 7.52 (dd, J = 8.4, 2.2 Hz, 1H), 7.25 (d, J = 2.2 Hz, 1H), 6.91 (d, J = 8.4 Hz, 1H), 6.60 (dd, J = 3.7, 2.5 Hz, 1H), 6.29 (dd, J = 3.7, 2.4 Hz, 1H), 5.90 (s, 1H), 1.50 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 153.1, 148.5, 145.8, 137.8, 135.5, 134.2, 132.7, 132.4, 130.8, 130.3, 128.3, 127.1, 126.7, 124.3, 123.5, 122.2, 122.0, 118.0, 109.9, 107.9, 76.4, 27.3. LRMS calcd m/z: [C28H23N2O5]+, 467.16; found, 467.21. 95% pure by UPLC analysis.
Synthesis of 27
8-Bromo-6-iodo-2,2-dimethylchromane (84)
To a refluxing solution of 2-bromo-4-iodophenol (10.0 g, 33.0 mmol, 1 equiv) and bismuth trifluoromethanesulfonate (3.00 g, 5 mmol, 0.1 equiv) in 1,2-dichloroethane (150 mL), 2-methyl-3-buten-2-ol (15 mL, 140 mmol, 4.3 equiv) was added using a syringe pump over 36 h (5 mL over 5 h, next 5 mL over 16 h, then 5 mL over 8 h). Then the dark reaction mixture was cooled to room temperature and quenched with a saturated solution of sodium carbonate. The mixture was extracted with DCM (3 × 100 mL), dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to obtain a dark, viscous residue. Purifications using flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) afforded 8-bromo-6-iodo-2,2-dimethylchromane (84) (5.68 g, 46%) as a golden viscous liquid. 1H NMR (400 MHz, CDCl3): δ 7.64 (s, 1H), 7.32 (s, 1H), 2.76 (t, J = 6.8 Hz), 1.79 (td, J = 6.8, 1.8 Hz, 2H), 1.37 (d, J = 1.8 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 151.0, 138.7, 137.2, 125.1, 112.8, 80.8, 76.2, 32.5, 26.9, 22.6.
8-Bromo-6-iodo-2,2-dimethylchroman-4-one (85)
Acetic anhydride (50 mL, 450 mmol, 30 equiv) was added slowly to acetic acid (25 mL, 450 mmol, 30 equiv) at 0 °C, and then chromium(VI) oxide (12.00 g, 120 mmol, 8.2 equiv) was added slowly to that mixture with continuous stirring and stirred at 0 °C for 15 min. Then anhydrous benzene (15 mL) was added to that reaction to dilute the mixture. 8-Bromo-6-iodo-2,2-dimethylchromane (84) (5.41 g, 15 mmol, 1 equiv) was dissolved in benzene (15 mL) and added slowly to the reaction over 15 min. The mixture was run overnight at 0 °C. Then, cold water was added to the flask to quench the reaction. The crude reaction mixture was extracted with EtOAc (3 × 50 mL), and the organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The compound was purified by flash column chromatography (silica gel, 100% hexanes to 30% EtOAc in hexanes) to obtain 8-bromo-6-iodo-2,2-dimethylchroman-4-one (85) as a brown viscous liquid (3.81 g, 68%). 1H NMR (400 MHz, CDCl3): δ 8.05 (s, 1H), 7.96 (s, 1H), 2.71 (d, J = 1.9 Hz, 2H), 1.48 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 190.3, 156.2, 146.5, 134.6, 122.7, 113.6, 82.4, 80.9, 48.2, 26.6.
8-Bromo-6-iodo-2,2-Dimethyl-4-(p-tolyl)-2H-chromene (86)
To a solution of 8-bromo-6-iodo-2,2-dimethylchroman-4-one (85) (4.94 g, 13.0 mmol, 1.0 equiv) in anhydrous THF (20 mL), p-tolylmagnesium bromide (30 mL, 0.5 M in Et2O, 2.3 equiv) was added dropwise at −78 °C. The reaction mixture was allowed to reach room temperature with stirring. After 3 h, the reaction was again cooled to −78 °C, and a second charge of p-tolylmagnesium bromide (30 mL, 0.5 M in Et2O, 2.3 equiv) was added gradually. The reaction was warmed to room temperature, stirred for 15 h, and then quenched with a saturated ammonium chloride solution. The aqueous layer was extracted with EtOAc (2 × 20 mL), washed with brine, dried over anhydrous Na2SO4, filtered, and evaporated to dryness. Without further purification, the dried residue (4.38 g) was dissolved in MeOH (50 mL), mixed with pyridinium p-toluenesulfonate (2.0 g, 8.0 mmol), and refluxed for 4 h. The reaction mixture was cooled to room temperature, and MeOH was evaporated under reduced pressure. The residue was dissolved in EtOAc and water and extracted with EtOAc (50 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, evaporated under reduced pressure, and purified using flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) to obtain 8-bromo-6-iodo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (86) as a colorless viscous oil (2.25 g, 38% over two steps). 1H NMR (400 MHz, CDCl3): δ 7.69 (t, J = 1.8 Hz, 1H), 7.25–7.16 (m, 5H), 5.64 (s, 1H), 2.41 (s, 3H), 1.53 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 150.5, 140.2, 138.1, 134.4, 133.6, 133.3, 130.5, 129.4, 128.6, 126.2, 112.4, 82.3, 77.6, 27.7, 21.4.
Methyl 4-(5-(8-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (87)
8-Bromo-6-iodo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (86) (1.08 g, 2.37 mmol, 1.1 equiv), methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) (740 mg, 2.26 mmol, 1.0 equiv), and potassium carbonate (1000 mg, 7.23 mmol, 3.2 equiv) were added to a vial and dissolved in 1,2-dimethoxyethane (20 mL) and water (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (198 mg, 0.27 mmol, 0.1 equiv). Then the vial containing the reaction mixtures was placed in a preheated heating block at 50 °C and stirred for 3 h. After the reaction was complete, as monitored by TLC, brine was added, and the reaction mixture was extracted with EtOAc (20 mL × 3), dried over anhydrous MgSO4, filtered, and evaporated to dryness. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain methyl 4-(5-(8-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (87) as a bright yellow solid (0.96 g, 80%). mp 185–187 °C. 1H NMR (400 MHz, CDCl3): δ 10.50 (s, 1H), 8.11–8.03 (m, 2H), 7.68 (d, J = 2.3 Hz, 1H), 7.58–7.51 (m, 2H), 7.23 (s, 4H), 6.97 (d, J = 2.3 Hz, 1H), 6.77–6.68 (m, 2H), 5.74 (s, 1H), 3.93 (s, 3H), 2.42 (s, 3H), 1.65 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 167.0, 147.3, 138.2, 136.8, 134.8, 134.7, 131.1, 130.9, 130.7, 129.4, 129.3, 128.7, 128.4, 127.4, 126.4, 125.7, 122.8, 122.1, 114.1, 109.2, 109.0, 77.6, 52.2, 28.0, 21.4.
4-(5-(8-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (27)
To a solution of methyl 4-(5-(8-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (87) (50 mg, 0.1 mmol, 1.0 equiv) in THF (1 mL) and MeOH (1 mL) was added lithium hydroxide hydrate (80 mg, 1.9 mmol, 20 equiv) dissolved in water (1 mL) and the resulting mixture was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (10 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(8-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (27) (37 mg, 76%) as a yellow solid. mp 218–220 °C. 1H NMR (400 MHz, THF-d 8): δ 10.59 (s, 1H), 8.05–7.97 (m, 2H), 7.76 (d, J = 2.4 Hz, 1H), 7.72–7.66 (m, 2H), 7.23 (s, 4H), 6.92 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 3.8, 2.4 Hz, 1H), 6.73 (dd, J = 3.9, 2.6 Hz, 1H), 5.81 (s, 1H), 2.38 (s, 3H), 1.60 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.2, 148.8, 138.4, 137.4, 135.8, 135.1, 132.5, 131.0, 130.47, 130.48, 129.8, 129.2, 128.7, 128.7, 126.2, 126.1, 124.0, 123.6, 113.8, 112.3, 109.6, 77.7, 27.5, 21.0. LRMS calcd m/z: [C29H25BrNO3]+, 514.10; found, 514.13. 99% pure by UPLC analysis.
Synthesis of 28
4-(5-(8-Chloro-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (28)
Methyl 4-(5-(8-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (87) (100 mg, 0.19 mmol, 1.0 equiv) and copper(I) chloride (93 mg, 0.25 mmol, 5.0 equiv) were mixed in anhydrous DMSO (5 mL) and heated in a sealed vial at 180 °C for 12 h. The solvent was removed under reduced pressure while purging nitrogen gas into the vial. The crude reaction mixture was carried over to the next step without further purification. The crude reaction mixture from the last step was dissolved in THF (1 mL), water (1 mL), and MeOH (1 mL), and lithium hydroxide hydrate (100 mg, 2.4 mmol, 11.0 equiv) was added to the resulting mixture and stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(8-chloro-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (28) (13 mg, 67% over two steps) as a yellow solid. mp 253–255 °C. 1H NMR (400 MHz, THF-d 8): δ 10.59 (s, 1H), 8.02 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H), 7.63 (s, 1H), 7.23 (s, 4H), 6.89 (dd, J = 3.8, 2.4 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 6.73 (dd, J = 3.9, 2.6 Hz, 1H), 5.82 (s, 1H), 2.38 (s, 3H), 1.60 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 148.3, 138.4, 137.4, 135.8, 135.2, 132.5, 131.0, 130.6, 130.5, 129.8, 129.2, 128.7, 126.4, 125.74, 125.71, 123.57, 123.55, 123.3, 112.2, 109.6, 77.7, 27.4, 21.1. LRMS calcd m/z: [C29H25ClNO3]+, 470.16; found, 470.21. 99% pure by UPLC analysis.
Synthesis of 29
4-(5-(2,2,8-Trimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (29)
In a vial charged with methyl 4-(5-(8-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (87) (100 mg, 0.19 mmol, 1.0 equiv), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (15 mg, 0.02 mmol, 0.1 equiv), and potassium carbonate (138 mg, 1.0 mmol, 5.3 equiv) in DMF (5 mL), trimethylboroxine (120 mg, 0.96 mmol, 5.0 equiv) was added and the mixture was heated at 160 °C for 6 h and then allowed to cool, filtered through Celite, washed with brine, and extracted with EtOAc (5 mL × 3). The organic extracts were dried over anhydrous MgSO4, filtered, concentrated under reduced pressure, and carried to the next step without further purification.
The crude reaction mixture from the last step was dissolved in THF (1 mL), water (1 mL), and MeOH (1 mL), and lithium hydroxide hydrate (100 mg, 2.4 mmol, 11.0 equiv) was added to the resulting mixture and stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, and dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(2,2,8-trimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (29) (15 mg, 18% over two steps) as a yellow solid. mp 259–261 °C. 1H NMR (400 MHz, THF-d 8): δ 10.59 (s, 1H), 8.04–7.98 (m, 2H), 7.67–7.61 (m, 2H), 7.44 (d, J = 2.1 Hz, 1H), 7.27–7.17 (m, 4H), 6.79–6.66 (m, 3H), 5.74 (s, 1H), 2.37 (s, 3H), 2.21 (s, 3H), 1.59 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 147.5, 138.0, 137.6, 136.6, 136.1, 132.5, 131.4, 131.0, 130.3, 129.5, 129.3, 129.32, 128.25, 127.1, 125.0, 124.1, 123.1, 121.2, 110.2, 109.3, 77.0, 27.5, 21.0, 20.7. LRMS calcd m/z: [C30H28NO3]+, 450.21; found, 450.27. 95% pure by UPLC analysis.
Synthesis of 30
6-Bromo-7-hydroxy-2,2-dimethylchroman-4-one (89)
A solution of 4-bromoresorcinol (7.80 g, 41.3 mmol, 1.0 equiv) and 3-methylcrotonic acid (5.00 g, 50 mmol, 1.2 equiv) was dissolved in Eaton’s reagent (20 mL) and heated at 70 °C for 5 h. Then the dark reaction mixture was cooled to room temperature and quenched with a saturated solution of sodium carbonate. The mixture was extracted three times with DCM (3 × 100 mL), dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to obtain a dark viscous residue. Then flash column chromatography (silica gel, 100% hexanes to 50% EtOAc in hexanes) afforded 6-bromo-7-hydroxy-2,2-dimethylchroman-4-one (89) (8.27 g, 74%) as a viscous golden liquid. 1H NMR (400 MHz, CDCl3): δ 8.01 (s, 1H), 6.56 (s, 1H), 2.67 (s, 2H), 1.45 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 190.4, 161.3, 158.5, 130.4, 115.8, 104.7, 103.7, 80.1, 48.5, 26.8.
6-Bromo-2,2-Dimethyl-4-(p-tolyl)-2H-chromen-7-ol (90)
In an oven-dried round-bottom flask, 6-bromo-7-hydroxy-2,2-dimethylchroman-4-one (89) (2.00 g, 7.38 mmol, 1.0 equiv) was dissolved in anhydrous THF (20 mL). The solution was placed in an ice bath at 0 °C, and 4-tolylmagnesium bromide (30 mL, 1 M, in THF, 22 mmol, 4.1 equiv) was added to the flask dropwise, and the mixture was slowly warmed to room temperature with continuous stirring for 16 h. The reaction was quenched with a saturated ammonium chloride solution and extracted with EtOAc (50 mL × 3). The organic extract was washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure to obtain a semisolid residue. The residue was dissolved in anhydrous MeOH (40 mL), and pyridinium-p-toluenesulfonate (700 mg, 20% w/w of residue) was added to the reaction mixture and refluxed for 4 h. The MeOH was evaporated under reduced pressure, and the dried residue was purified by flash column chromatography (silica gel, 100% hexanes to 5% EtOAc in hexanes) to obtain 6-bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-7-ol (90) as a golden oil (1.65 g, 65%). 1H NMR (400 MHz, CDCl3): δ 7.21 (s, 4H), 7.07 (s, 1H), 6.59 (s, 1H), 5.49 (s, 1H), 5.44 (s, 1H), 2.40 (s, 3H), 1.47 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 154.6, 152.8, 137.8, 135.2, 133.7, 129.3, 128.6, 128.2, 127.4, 117.9, 104.9, 101.0, 76.7, 27.7, 21.4.
Methyl 4-(5-(7-Hydroxy-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (91)
6-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromen-7-ol (90) (150 mg, 0.43 mmol, 1.0 equiv) and methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) (150 mg, 0.46 mmol, 1.1 equiv) were added to a vial and dissolved in THF (0.9 mL), and an aqueous solution of tribasic potassium phosphate (0.5 M, 1.7 mL, 2.0 equiv) was added to the solution. Then nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of XPhos Pd G2 (35 mg, 0.04 mmol, 0.1 equiv). Then the vial was placed in a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, brine was added to the reaction mixture and extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, filtered, and the solvent was evaporated to dryness. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain methyl 4-(5-(7-hydroxy-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (91) as a golden-brown oil (141 mg, 70%). 1H NMR (400 MHz, acetone-d 6): δ 7.98–7.92 (m, 2H), 7.68 (d, J = 8.5 Hz, 2H), 7.33–7.25 (m, 5H), 6.71 (d, J = 3.8 Hz, 1H), 6.57 (s, 1H), 6.32 (d, J = 3.8 Hz, 1H), 5.60 (s, 1H), 3.86 (s, 3H), 2.82 (s, 1H), 2.39 (s, 3H), 1.47 (s, 6H). 13C NMR (101 MHz, acetone-d 6): δ 167.0, 163.2 154.8, 154.4, 138.8, 138.3, 136.5, 135.1, 130.9, 130.0, 129.3, 127.4, 124.8, 123.4, 116.4, 109.7, 109.1, 108.4, 107.7, 106.8, 105.4, 77.0, 52.1, 27.8, 21.2.
4-(5-(7-Hydroxy-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (30)
To a solution of methyl 4-(5-(7-hydroxy-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (91) (90 mg, 0.19 mmol, 1.0 equiv) in THF (1 mL) and MeOH (1 mL) was added lithium hydroxide hydrate (82 mg, 1.95 mmol, 10 equiv) dissolved in water (1 mL) and the resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(7-hydroxy-2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (30) (59 mg, 68%) as a yellow solid. mp could not be determined because the compound degraded at 270 °C. 1H NMR (400 MHz, THF-d 8): δ 10.48 (s, 1H), 7.97–7.90 (m, 2H), 7.59–7.51 (m, 2H), 7.26 (dd, J = 6.0, 2.2 Hz, 3H), 7.21 (d, J = 7.9 Hz, 2H), 6.59 (dd, J = 3.8, 2.7 Hz, 1H), 6.40 (s, 1H), 6.25 (dd, J = 3.8, 2.4 Hz, 1H), 5.51 (s, 1H), 2.37 (s, 3H), 1.93 (s, 1H), 1.45 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 154.9, 154.2, 137.9, 137.9, 136.6, 135.4, 133.5, 130.9, 130.6, 129.6, 129.1, 127.8, 126.7, 124.6, 122.9, 116.0, 113.3, 109.0, 107.9, 105.0, 76.5, 27.7, 21.1. LRMS calcd m/z: [C29H26NO4]+, 452.19; found, 452.22. 96% pure by UPLC analysis.
Synthesis of 31
6-Bromo-2,2-diethylchroman-4-one (93)
To a solution of 1-(5-bromo-2-hydroxyphenyl)ethan-1-one (5.00 g, 23.3 mmol, 1.0 equiv) in EtOH (20 mL), pyrrolidine (7.0 mL, 66 mmol, 3.7 equiv), and 3-pentanone (7.0 mL, 85 mmol, 3.7 equiv) were added sequentially, and the reaction mixture was heated to reflux for 6 h. The crude reaction mixture was washed with hydrochloric acid (1 N) and extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, and filtered, and the solvent was evaporated under reduced pressure. The compound was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain 6-bromo-2,2-diethylchroman-4-one (93) as a brown viscous liquid (5.05 g, 77%). 1H NMR (400 MHz, CDCl3): δ 7.93 (d, J = 2.6 Hz, 1H), 7.52 (dd, J = 8.8, 2.6 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 2.70 (s, 2H), 1.75 (ddq, J = 27.0, 14.5, 7.3 Hz, 4H), 0.91 (t, J = 7.5 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 191.8, 158.9, 138.8, 129.0, 122.0, 120.5, 113.2, 84.3, 44.6, 28.3, 7.8.
6-Bromo-2,2-diethyl-4-(p-tolyl)-2H-chromene (94)
In an oven-dried round-bottom flask, 6-bromo-2,2-diethylchroman-4-one (93) (2.00 g, 7.07 mmol, 1.0 equiv) was dissolved in anhydrous THF (20 mL), and the solution was placed in an ice bath at 0 °C. p-Tolylmagnesium bromide (48 mL 0.5 M, 24 mmol, 3.4 equiv) was added to the flask dropwise, and the mixture was slowly warmed to room temperature with continuous stirring for 16 h. The reaction was quenched with a saturated ammonium chloride solution and extracted with EtOAc (50 mL × 3). The combined organic extracts were washed with brine, dried over anhydrous MgSO4, and filtered, and the solvent was evaporated under reduced pressure to obtain a semisolid residue. The residue was dissolved in anhydrous MeOH (40 mL), and pyridinium-p-toluenesulfonate (700 mg, 20% w/w of residue) was added to the reaction mixture and refluxed for 4 h. After the completion of reaction, as monitored by TLC, the MeOH was evaporated under reduced pressure and the residue was purified by flash column chromatography (silica gel, 100% hexanes to 5% EtOAc in hexanes) to obtain 6-bromo-2,2-diethyl-4-(p-tolyl)-2H-chromene (94) as a yellow solid (0.82 g, 32%). mp 75–77 °C. 1H NMR (400 MHz, CDCl3): δ 7.24 (m, 5H), 7.13 (d, J = 2.4 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 5.53 (s, 1H), 2.44 (s, 3H), 1.90–1.66 (m, 4H), 1.01 (t, J = 7.5 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 153.4, 137.8, 135.3, 135.1, 131.8, 129.4, 128.6, 128.1, 127.8, 124.3, 118.2, 112.3, 81.8, 31.9, 21.4, 8.2.
4-(5-(2,2-Diethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (31)
6-Bromo-2,2-diethyl-4-(p-tolyl)-2H-chromene (94) (350 mg, 1.17 mmol, 1.0 equiv), methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) (370 mg, 1.13 mmol, 1.2 equiv), and potassium carbonate (470 mg, 3.40 mmol, 3.5 equiv) were added to a vial and dissolved in 1,2-dimethoxyethane (4 mL) and water (1 mL). Then nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (85 mg, 0.12 mmol, 0.1 equiv). Then the vial was placed in a preheated heating block at 90 °C and stirred for 5 h. After the reaction was complete, as determined by TLC, brine was added to the reaction mixture, extracted with EtOAc (20 mL × 3), dried over anhydrous MgSO4, and filtered, and the solvent was evaporated to dryness under reduced pressure, yielding approximately 95% of the ester. Then, 18 mg of the crude mixture was carried over to the next step without purification.
To a solution of the crude product (18 mg, 0.04 mmol, 1.0 equiv) in THF (1 mL) and MeOH (1 mL) was added lithium hydroxide (10 mg, 0.24 mmol, 6.3 equiv) dissolved in water (1 mL), and the resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. Then the reaction mixture was extracted with EtOAc (3 mL × 3), washed with brine, and dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(2,2-diethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (31) (13 mg, 74% over two steps) as a yellow solid. mp 226–228 °C. 1H NMR (400 MHz, THF-d 8): δ 10.42 (s, 1H), 7.95–7.89 (m, 2H), 7.63–7.58 (m, 2H), 7.44 (dd, J = 8.4, 2.3 Hz, 1H), 7.37–7.27 (m, 3H), 7.23 (d, J = 7.9 Hz, 2H), 6.85 (d, J = 8.3 Hz, 1H), 6.63–6.55 (m, 1H), 6.25 (dd, J = 3.7, 2.4 Hz, 1H), 5.58 (s, 1H), 2.38 (s, 3H), 1.86–1.72 (m, 7H), 0.98 (t, J = 7.5 Hz, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 154.0, 138.1, 137.9, 137.0, 136.5, 136.0, 132.4, 130.7, 129.7, 129.2, 128.0, 127.8, 126.34, 126.30, 123.4, 122.9, 122.5, 117.1, 109.9, 107.7, 81.7, 32.2, 21.0, 8.2. LRMS calcd m/z: [C31H30NO3]+, 464.22; found, 464.24. 95% pure by UPLC analysis.
Synthesis of 32
tert-Butyl 2-(4-(Methoxycarbonyl)phenyl)-5-(4-oxochroman-6-yl)-1H-pyrrole-1-carboxylate (96)
6-Bromochroman-4-one (95, 700 mg, 3.08 mmol, 1.0 equiv), tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrole-1-carboxylate (79) (1.29 g, 3.02 mmol, 1.0 equiv), and potassium carbonate (1.26 g, 9.12 mmol, 3.0 equiv) were added to a vial followed by addition of 1,2-dimethoxyethane (15 mL) and water (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II)-dichloromethane adduct (250 mg, 0.31 mmol, 0.1 equiv). The vial was sealed and placed in a preheated heating block at 90 °C and refluxed for 6 h. After the reaction was complete, brine was added to the reaction mixture and extracted with EtOAc (20 mL × 3), dried over anhydrous MgSO4, and filtered, and the solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-oxochroman-6-yl)-1H-pyrrole-1-carboxylate (96) as a colorless viscous liquid (0.94 g, 70%). 1H NMR (400 MHz, CDCl3): δ 8.08–8.02 (m, 2H), 7.92 (d, J = 2.3 Hz, 1H), 7.55 (dd, J = 8.6, 2.3 Hz, 1H), 7.49–7.42 (m, 2H), 7.00 (d, J = 8.6 Hz, 1H), 6.31 (d, J = 3.4 Hz, 1H), 6.24 (d, J = 3.5 Hz, 1H), 4.58 (t, J = 6.5 Hz, 2H), 3.94 (s, 3H), 2.84 (t, J = 6.5 Hz, 2H), 1.19 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 191.6, 167.1, 161.4, 149.7, 139.0, 136.9, 136.3, 135.5, 129.4, 128.7, 128.5, 127.4, 127.3, 120.8, 117.6, 113.7, 113.0, 84.8, 67.3, 52.3, 37.9, 27.4.
tert-Butyl 2-(4-(Methoxycarbonyl)phenyl)-5-(4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (97)
To a stirred solution of tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-oxochroman-6-yl)-1H-pyrrole-1-carboxylate (96) (300 mg, 0.67 mmol, 1.0 equiv) and 2,6-di-tert-butylpyridine (1.00 mL, 4.63 mmol, 6.7 equiv) in anhydrous DCM (3 mL) at 0 °C was added triflic anhydride (0.50 mL, 3.0 mmol, 4.4 equiv) dropwise. The reaction mixture was slowly warmed to room temperature with continuous stirring and stirred for 16 h at room temperature. The reaction was quenched with saturated sodium bicarbonate solution and extracted with DCM (5 mL × 3). The combined organic layers were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to yield a crude brown oil. The crude product was purified by flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) to obtain tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (97) (267 mg, 69%) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 8.06 (dd, J = 8.3, 1.3 Hz, 2H), 7.49–7.43 (m, 2H), 7.28 (d, J = 1.3 Hz, 2H), 6.91–6.84 (m, 1H), 6.34–6.28 (m, 1H), 6.22 (dd, J = 3.4, 1.1 Hz, 1H), 5.80 (dd, J = 4.5, 3.4 Hz, 1H), 5.03 (dd, J = 3.8, 1.2 Hz, 2H), 3.94 (d, J = 1.1 Hz, 3H), 1.19 (d, J = 1.2 Hz, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 154.6, 149.6, 143.0, 139.0, 136.4, 135.4, 132.5, 129.3, 128.7, 128.5, 127.8, 122.4, 118.6 (q, J = 321 Hz), 116.9, 115.9, 113.6, 112.9, 110.2, 84.7, 65.4, 52.2, 27.3.
tert-Butyl 2-(4-(Methoxycarbonyl)phenyl)-5-(4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (98)
tert-Butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-(((trifluoromethyl)sulfonyl)oxy)-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (97) (200 mg, 0.35 mmol, 1.0 equiv), p-tolylboronic acid (100 mg, 0.74 mmol, 2.1 equiv), and an aqueous solution of tribasic potassium phosphate (0.5 M, 2.2 mL, 1.1 mmol, 3.2 equiv) were added to a vial followed by addition of THF (2 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of XPhos Pd G2 (30 mg, 0.04 mmol, 0.1 equiv). The vial was sealed and placed in a preheated heating block at 45 °C and stirred for 30 min. After the reaction was complete, as determined by TLC, brine was added to the reaction mixture and extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, and filtered. The solvent was evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain tert-butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (98) as a white solid (163 mg, 91%). mp could not be determined because the compound degraded at 160 °C. 1H NMR (400 MHz, CDCl3): δ 8.02 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.1 Hz, 2H), 7.19 (td, J = 6.1, 2.9 Hz, 3H), 7.06 (s, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.26 (dd, J = 3.3, 0.8 Hz, 1H), 6.13 (dd, J = 3.5, 0.8 Hz, 1H), 5.80 (t, J = 3.9 Hz, 1H), 4.89 (d, J = 3.9 Hz, 2H), 3.93 (s, 3H), 2.37 (s, 3H), 1.15 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 167.1, 154.5, 149.7, 138.9, 137.7, 137.4, 137.0, 135.2, 134.8, 130.2, 129.33, 129.29, 128.6, 128.5, 128.3, 126.8, 126.6, 123.3, 120.1, 115.8, 113.4, 112.2, 84.3, 65.6, 52.2, 27.3, 21.3.
Methyl 4-(5-(4-(p-Tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (99)
tert-Butyl 2-(4-(methoxycarbonyl)phenyl)-5-(4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrole-1-carboxylate (98) (143 mg, 0.27 mmol, 1 equiv) was heated at 180 °C under a nitrogen gas atmosphere for 30 min. The dark residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc in hexanes) to obtain methyl 4-(5-(4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (99) (55 mg, 48%) as a yellow semisolid. 1H NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 8.04–7.97 (m, 2H), 7.49 (s, 2H), 7.35 (dd, J = 8.3, 2.2 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 2.2 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.64 (dd, J = 3.7, 2.6 Hz, 1H), 6.39 (dd, J = 3.7, 2.5 Hz, 1H), 5.84 (t, J = 4.0 Hz, 1H), 4.87 (d, J = 4.0 Hz, 2H), 3.91 (s, 3H), 2.42 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 167.0, 154.2, 138.0, 136.9, 136.7, 135.2, 134.8, 131.4, 130.5, 129.4, 128.6, 127.3, 125.7, 125.3, 124.4, 123.0, 121.9, 120.4, 116.9, 109.9, 107.8, 65.5, 52.2, 21.4.
4-(5-(4-(p-Tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic Acid (32)
To a solution of methyl 4-(5-(4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoate (99) (50 mg, 0.12 mmol, 1.0 equiv) in THF (2 mL), water (2 mL), and MeOH (2 mL) was added lithium hydroxide hydrate (50 mg, 1.2 mmol, 10 equiv). The resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzoic acid (32) (35 mg, 72%) as a bright yellow solid. mp 194–196 °C. 1H NMR (400 MHz, THF-d 8): δ 10.49 (s, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 7.47 (dd, J = 8.4, 2.2 Hz, 1H), 7.35 (d, J = 2.2 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 6.88 (d, J = 8.3 Hz, 1H), 6.64–6.59 (m, 1H), 6.30 (d, J = 1.3 Hz, 1H), 5.85 (d, J = 4.1 Hz, 1H), 4.79 (d, J = 4.0 Hz, 2H), 2.37 (s, 3H). 13C NMR (101 MHz, THF-d 8): δ 167.3, 154.8, 138.2, 137.9, 137.6, 136.2, 135.7, 132.6, 130.7, 129.8, 129.0, 128.1, 127.1, 126.0, 124.5, 123.5, 122.5, 121.0, 117.0, 109.9, 107.9, 65.7, 21.1. LRMS calcd m/z: [C27H22NO3]+, 408.16; found, 408.25. 97% pure by UPLC analysis.
Synthesis of 33
Methyl 4-(5-(3-(4-Methylbenzoyl)phenyl)-1H-pyrrol-2-yl)benzoate (101)
(3-Bromophenyl)(p-tolyl)methanone (275 mg, 1.0 mmol, 1.0 equiv), methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) (350 mg, 1.1 mmol, 1.1 equiv), and potassium carbonate (414 mg, 3.0 mmol, 4.0 equiv) were added to a mixture of 1,2-dimethoxyethane:water (5.0 mL/1.0 mL) at room temperature in a heavy-walled pressure vial. Following sparging of the mixture with nitrogen gas for 10 min, [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (140 mg, 0.2 mmol, 0.2 equiv) was added in one portion, and the flask was sealed and heated at 90 °C for 5 h. After cooling to room temperature, the layers were separated, and the aqueous layer was extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with brine and dried over anhydrous MgSO4. The resulting solution was absorbed on silica gel, and then the solvent was removed under reduced pressure and then purified by flash column chromatography (silica gel, 100% hexanes to 10% EtOAc in hexanes) to obtain methyl 4-(5-(3-(4-methylbenzoyl)phenyl)-1H-pyrrol-2-yl)benzoate (101) (218 mg, 55%) as a yellow solid. mp 159–161 °C. 1H NMR (400 MHz, CDCl3): δ 9.06 (s, 1H), 8.05–7.96 (m, 3H), 7.81–7.70 (m, 3H), 7.63–7.53 (m, 3H), 7.46 (t, J = Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 6.72 (dd, J = 3.8, 2.6 Hz, 1H), 6.66 (dd, J = 3.8, 2.5 Hz, 1H), 3.91 (s, 3H), 2.44 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 196.8, 167.1, 143.8, 138.8, 136.5, 134.8, 133.7, 132.8, 132.6, 130.6 (two 13C), 129.3, 128.9, 128.4, 128.0, 127.7, 124.9, 123.5, 110.1, 109.4, 52.3, 21.9.
4-(5-(3-(4-Methylbenzoyl)phenyl)-1H-pyrrol-2-yl)benzoic Acid (33)
To a solution of methyl 4-(5-(3-(4-methylbenzoyl)phenyl)-1H-pyrrol-2-yl)benzoate (101) (50 mg, 0.13 mmol, 1.0 equiv) in THF (10 mL) and MeOH (5 mL) was added an aqueous sodium hydroxide solution (2 N, 1.0 mL, 2.0 mmol, 16 equiv), and the resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (10 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to 4-(5-(3-(4-methylbenzoyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (33) (37 mg, 71%) as a yellow solid. mp 209–211 °C. 1H NMR (400 MHz, THF-d 8): δ 10.80 (s, 1H), 8.11 (t, J = 1.8 Hz, 1H), 8.01–7.97 (m, 2H), 7.90 (dt, J = 7.6, 1.6 Hz, 1H), 7.74 (dd, J = 6.4, 1.9 Hz, 3H), 7.62–7.56 (m, 1H), 7.52 (dt, J = 7.6, 1.5 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 6.72 (dd, J = 3.7, 2.4 Hz, 1H), 6.66 (dd, J = 3.8, 2.4 Hz, 1H), 2.43 (s, 3H). 13C NMR (101 MHz, THF-d 8): δ 195.5, 167.2, 143.7, 139.6, 135.9, 134.6, 133.95, 133.92, 130.9, 130.80, 130.77, 129.6, 129.0, 128.2, 128.0, 125.3, 123.9, 123.3, 110.2, 109.4, 21.4. LRMS calcd m/z: [C25H20NO3]+, 382.15; found, 382.22. 79% pure by UPLC analysis.
Synthesis of 34
Methyl 4-(5-(3-(1-(p-Tolyl)vinyl)phenyl)-1H-pyrrol-2-yl)benzoate (102)
A solution of methyltriphenylphosphonium bromide (200 mg, 0.56 mmol, 3.2 equiv) in THF (5 mL) was cooled to 0 °C, and a solution of sodium bis(trimethylsilyl)amide in THF (1 M, 0.50 mL, 2.8 equiv) was added to the solution dropwise. The reaction mixture was stirred for 1 h, followed by the addition of methyl 4-(5-(3-(4-methylbenzoyl)phenyl)-1H-pyrrol-2-yl)benzoate (101) (70 mg, 0.18 mmol, 1.0 equiv) dissolved in THF (2 mL). The resulting mixture was slowly warmed to room temperature and stirred for 1 h. The reaction was quenched with saturated ammonium chloride solution and extracted with EtOAc (10 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc in hexanes) to obtain methyl 4-(5-(3-(1-(p-tolyl)vinyl)phenyl)-1H-pyrrol-2-yl)benzoate (102) (65 mg, 93%) as a yellow solid. mp 179–181 °C. 1H NMR (400 MHz, CDCl3): δ 8.66 (s, 1H), 8.05–7.97 (m, 2H), 7.57–7.51 (m, 2H), 7.49 (dt, J = 7.0, 1.6 Hz, 2H), 7.39–7.31 (m, 1H), 7.24 (d, J = 2.6 Hz, 2H), 7.20 (dt, J = 7.7, 1.5 Hz, 1H), 7.14 (d, J = 8.0 Hz, 2H), 6.71–6.66 (m, 1H), 6.60–6.55 (m, 1H), 5.49 (d, J = 1.2 Hz, 1H), 5.42 (d, J = 1.2 Hz, 1H), 3.89 (s, 3H), 2.36 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 167.0, 149.8, 142.8, 138.4, 137.9, 136.6, 134.5, 132.13, 132.06, 130.5, 129.1, 129.0, 128.2, 127.5, 127.2, 124.0, 123.6, 123.2, 114.2, 110.0, 108.8, 52.2, 21.3.
4-(5-(3-(1-(p-Tolyl)vinyl)phenyl)-1H-pyrrol-2-yl)benzoic Acid (34)
To a solution of methyl 4-(5-(3-(1-(p-tolyl)vinyl)phenyl)-1H-pyrrol-2-yl)benzoate (102) (50 mg, 0.13 mmol, 1.0 equiv) in THF (10 mL) and MeOH (5 mL) was added an aqueous sodium hydroxide solution (2 N, 1.0 mL, 2.0 mmol, 16 equiv), and the resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (10 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(3-(1-(p-tolyl)vinyl)phenyl)-1H-pyrrol-2-yl)benzoic acid (34) (31 mg, 64%) as a yellow solid. mp 197–199 °C. 1H NMR (400 MHz, THF-d 8): δ 10.63 (s, 1H), 8.00–7.93 (m, 2H), 7.73–7.65 (m, 3H), 7.63 (dt, J = 7.8, 1.5 Hz, 1H), 7.31 (t, J = 7.7 Hz, 1H), 7.28–7.23 (m, 2H), 7.17–7.07 (m, 3H), 6.68 (dd, J = 3.7, 2.4 Hz, 1H), 6.56 (dd, J = 3.8, 2.4 Hz, 1H), 5.48 (d, J = 1.5 Hz, 1H), 5.42 (d, J = 1.5 Hz, 1H), 2.34 (s, 3H). 13C NMR (101 MHz, THF-d 8): δ 167.2, 151.2, 143.1, 139.3, 138.1, 137.8, 135.4, 133.7, 133.3, 130.8, 129.5, 129.0, 128.6, 128.4, 127.0, 124.5, 124.4, 123.7, 113.5, 110.1, 108.9, 21.0. LRMS calcd m/z: [C26H21NO2]+, 380.17; found, 380.19. 99% pure by UPLC analysis.
Synthesis of 35
4-Bromophenyl Benzoate (104)
To a solution of 4-bromophenol (103, 6.00 g, 34.7 mmol, 1.0 equiv) in pyridine (10.0 mL), benzoyl chloride (5.20 g, 37.0 mmol, 1.1 equiv) was added slowly at 0 °C, and the resulting mixture was slowly warmed to room temperature and quenched with a saturated sodium bicarbonate solution. The reaction mixture was extracted with DCM (10 mL × 3), washed with brine, dried over anhydrous MgSO4, and filtered. The solvent was evaporated under reduced pressure to obtain 4-bromophenyl benzoate (104) (9.52 g, 99%) as a white solid. mp 136–138 °C. 1H NMR (400 MHz, CDCl3): δ 8.23–8.16 (m, 2H), 7.70–7.61 (m, 1H), 7.59–7.48 (m, 4H), 7.16–7.08 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 165.0, 150.1, 133.9, 132.7, 130.3, 129.3, 128.8, 123.7, 119.1.
(5-Bromo-2-hydroxyphenyl)(phenyl)methanone (105)
4-Bromophenyl benzoate (104) (5.00 g, 18.0 mmol, 1.0 equiv) was mixed well with aluminum trichloride (2.50 g, 18.8 mmol, 1.0 equiv), and the mixture was heated at 130 °C without solvent for 2 h. After cooling, the solid was crushed and added to cold water (20 mL) with stirring. Then, hydrochloric acid (12 N, 5 mL) was added, and the reaction mixture was stirred for 30 min, extracted with EtOAc (20 mL × 3), washed with brine, dried over anhydrous MgSO4, and filtered. The solvent was evaporated to dryness under reduced pressure, and the residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc in hexanes) to obtain (5-bromo-2-hydroxyphenyl)(phenyl)methanone (105) as a white solid (3.15 g, 63%). mp 177–179 °C. 1H NMR (400 MHz, CDCl3): δ 11.92 (s, 1H), 7.73–7.65 (m, 3H), 7.65–7.57 (m, 2H), 7.57–7.50 (m, 2H), 6.99 (d, J = 8.9 Hz, 1H).
Methyl 4-(5-(3-Benzoyl-4-hydroxyphenyl)-1H-pyrrol-2-yl)benzoate (106)
(5-Bromo-2-hydroxyphenyl)(phenyl)methanone (105) (1.53 g, 5.52 mmol, 1.0 equiv) and methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) (2.00 g, 6.11 mmol, 1.1 equiv) were added to a vial and dissolved in THF (10.5 mL). An aqueous solution of tribasic potassium phosphate (0.5 M, 21 mL, 2.0 equiv) was added to the solution. Then nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of XPhos Pd G2 (0.083 g, 0.11 mmol, 0.02 equiv). Then the vial was placed into a preheated heating block at 45 °C, and the reaction mixture was stirred for 3 h. After the reaction was complete, as determined by TLC, brine was added to the reaction mixture, extracted with EtOAc (50 mL × 3), dried over anhydrous MgSO4, and filtered, and the solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 40% EtOAc in hexanes) to obtain methyl 4-(5-(3-benzoyl-4-hydroxyphenyl)-1H-pyrrol-2-yl)benzoate (106) as a bright yellow solid (1.93 g, 88%). mp 159–161 °C. 1H NMR (400 MHz, CDCl3): δ 11.94 (s, 1H), 8.48 (s, 1H), 8.05–7.99 (m, 2H), 7.78–7.70 (m, 4H), 7.64 (d, J = 7.5 Hz, 1H), 7.59–7.54 (m, 2H), 7.54–7.49 (m, 2H), 7.15 (d, J = 8.6 Hz, 1H), 6.67 (s, 1H), 6.42 (s, 1H), 3.92 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 201.5, 167.0, 162.3, 137.8, 136.4, 133.6, 132.54, 132.50, 131.9, 130.6, 129.5, 129.0, 128.7, 127.6, 123.7, 123.2, 119.5, 119.4, 110.0, 108.2, 52.2.
4-(5-(3-Benzoyl-4-hydroxyphenyl)-1H-pyrrol-2-yl)benzoic Acid (35)
To a solution of methyl 4-(5-(3-benzoyl-4-hydroxyphenyl)-1H-pyrrol-2-yl)benzoate (106) (100 mg, 0.25 mmol, 1.0 equiv) in THF (1 mL), water (1 mL), and MeOH (1 mL) was added lithium hydroxide hydrate (140 mg, 3.34 mmol, 13 equiv). The resulting mixture was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, dried over anhydrous MgSO4, and filtered. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 100% EtOAc in hexanes) to obtain 4-(5-(3-benzoyl-4-hydroxyphenyl)-1H-pyrrol-2-yl)benzoic acid (35) (65 mg, 67%) as a bright yellow solid. mp 1H NMR (400 MHz, THF-d 8): δ 10.56 (s, 1H), 7.97–7.92 (m, 2H), 7.89 (d, J = 2.3 Hz, 1H), 7.86 (dd, J = 8.6, 2.3 Hz, 1H), 7.82–7.78 (m, 2H), 7.66–7.60 (m, 3H), 7.58–7.53 (m, 2H), 7.07 (d, J = 8.7 Hz, 1H), 6.64 (dd, J = 3.7, 2.4 Hz, 1H), 6.39 (dd, J = 3.7, 2.4 Hz, 1H). 13C NMR (101 MHz, THF-d 8): δ 201.4, 167.2, 162.1, 138.8, 137.7, 134.6, 133.10, 133.07, 132.9, 130.8, 130.2, 129.1, 129.0, 128.4, 125.2, 123.6, 120.5, 119.1, 110.0, 108.2. LRMS calcd m/z: [C24H18NO4]+, 384.13; found, 384.19. 98% pure by UPLC analysis.
Synthesis of 36
4-(5-(3-Benzoyl-4-methoxyphenyl)-1H-pyrrol-2-yl)benzoic Acid (36)
In a dry vial, a solution of methyl 4-(5-(3-benzoyl-4-hydroxyphenyl)-1H-pyrrol-2-yl)benzoate (106, 200 mg, 0.50 mmol, 1.0 equiv) in anhydrous DMF (5 mL) was mixed with potassium carbonate (200 mg, 1.455 mmol, 2.9 equiv) and iodomethane (456 mg, 3.21 mmol, 6.4 equiv). The vial was sealed, and the mixture was stirred at 60 °C for 30 min. The reaction was quenched with the addition of water. The mixture was extracted with EtOAc (5 mL × 4). The organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 40% EtOAc in hexanes) to obtain methyl 4-(5-(3-benzoyl-4-methoxyphenyl)-1H-pyrrol-2-yl)benzoate (198 mg, 96%) as a yellow solid. mp 131–131 °C. 1H NMR (400 MHz, CDCl3): δ 9.63 (s, 1H), 7.99–7.93 (m, 2H), 7.84–7.78 (m, 2H), 7.65–7.60 (m, 2H), 7.57 (td, J = 8.0, 1.8 Hz, 2H), 7.49 (d, J = 2.3 Hz, 1H), 7.42 (t, J = 7.7 Hz, 2H), 6.85 (d, J = 8.7 Hz, 1H), 6.68 (dd, J = 3.8, 2.4 Hz, 1H), 6.46 (dd, J = 3.8, 2.4 Hz, 1H), 3.88 (s, 3H), 3.52 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 197.0, 167.2, 156.2, 137.4, 136.8, 133.9, 133.5, 132.1, 130.4, 130.2, 128.8, 128.5, 128.2, 127.0, 125.3, 125.1, 123.2, 112.0, 109.7, 107.9, 55.5, 52.1.
To a solution of methyl 4-(5-(3-benzoyl-4-methoxyphenyl)-1H-pyrrol-2-yl)benzoate (100 mg, 0.24 mmol, 1.0 equiv) in THF (1 mL), water (1 mL), and MeOH (1 mL) was added lithium hydroxide hydrate (140 mg, 3.34 mmol, 14 equiv), and the resulting mixture was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, and dried over anhydrous MgSO4. The extract was purified by flash column chromatography (silica gel, 100% hexanes to 100% EtOAc in hexanes) to obtain 4-(5-(3-benzoyl-4-methoxyphenyl)-1H-pyrrol-2-yl)benzoic acid (36) (81 mg, 84%) as a bright yellow solid. mp 198–200 °C. 1H NMR (400 MHz, THF-d 8): δ 10.61 (s, 1H), 8.00–7.93 (m, 2H), 7.79 (dt, J = 8.7, 1.9 Hz, 3H), 7.73–7.65 (m, 3H), 7.54 (t, J = 7.4 Hz, 1H), 7.47–7.39 (m, 2H), 7.10 (s, 1H), 6.71–6.65 (m, 1H), 6.54–6.49 (m, 1H), 3.69 (s, 3H). 13C NMR (101 MHz, THF-d 8): δ 195.6, 167.3, 156.6, 138.9, 137.8, 134.8, 133.2, 133.0, 130.8, 130.5, 130.1, 128.8, 128.3, 128.1, 126.9, 125.4, 123.6, 112.5, 110.0, 108.2, 55.7. LRMS calcd m/z: [C25H20NO4]+, 398.14; found, 398.17. 99% pure by UPLC analysis.
Synthesis of 37
4-(5-(3-Benzoyl-4-ethoxyphenyl)-1H-pyrrol-2-yl)benzoic Acid (37)
In a dry vial, a solution of methyl 4-(5-(3-benzoyl-4-hydroxyphenyl)-1H-pyrrol-2-yl)benzoate (106) (200 mg, 0.50 mmol, 1.0 equiv) in anhydrous DMF (5 mL) was mixed with potassium carbonate (200 mg, 1.455 mmol, 2.9 equiv) and iodoethane (388 mg, 2.49 mmol, 5.0 equiv). The vial was sealed, and the resulting mixture was stirred at 60 °C for 30 min. The reaction was quenched with the addition of water. The mixture was extracted with EtOAc (5 mL × 4), and the organic layer was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 40% EtOAc in hexanes) to obtain methyl 4-(5-(3-benzoyl-4-ethoxyphenyl)-1H-pyrrol-2-yl)benzoate (177 mg, 83%) as a yellow solid. mp 137–139 °C. 1H NMR (400 MHz, CDCl3): δ 9.60 (s, 1H), 8.00–7.91 (m, 3H), 7.82–7.75 (m, 2H), 7.68–7.57 (m, 4H), 7.57–7.49 (m, 1H), 7.40 (t, J = Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 3.91–3.85 (m, 5H), 1.00 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 197.2, 167.2, 155.7, 138.0, 136.8, 134.0, 133.1, 132.0, 130.3, 129.8, 129.3, 128.3, 128.2, 127.0, 125.4, 125.3, 123.2, 113.0, 109.8, 107.9, 64.3, 52.1, 14.3.
To a solution of methyl 4-(5-(3-benzoyl-4-ethoxyphenyl)-1H-pyrrol-2-yl)benzoate (100 mg, 0.24 mmol, 1.0 equiv) in THF (1 mL), water (1 mL), and MeOH (1 mL) was added lithium hydroxide hydrate (140 mg, 3.34 mmol, 14 equiv), and the resulting mixture was stirred overnight at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (2 mL × 3), washed with brine, dried over anhydrous MgSO4, and filtered. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 100% EtOAc in hexanes) to obtain 4-(5-(3-benzoyl-4-ethoxyphenyl)-1H-pyrrol-2-yl)benzoic acid (37) (58 mg, 60%) as a bright yellow solid. mp 208–210 °C. 1H NMR (400 MHz, THF-d 8): δ 10.62 (s, 1H), 7.99–7.93 (m, 2H), 7.80–7.74 (m, 3H), 7.73–7.67 (m, 3H), 7.57–7.49 (m, 1H), 7.46–7.38 (m, 2H), 7.08 (d, J = 8.6 Hz, 1H), 6.68 (dd, J = 3.7, 2.4 Hz, 1H), 6.52 (dd, J = 3.7, 2.4 Hz, 1H), 3.97 (q, J = 7.0 Hz, 2H), 1.01 (t, J = 6.9 Hz, 3H). 13C NMR (101 MHz, THF-d 8): δ 195.8, 167.3, 156.1, 139.4, 137.8, 134.8, 132.99, 132.98, 130.8, 130.5, 129.9, 128.7, 128.4, 128.3, 126.8, 125.6, 123.6, 113.5, 110.0, 108.1, 64.6, 14.4. LRMS calcd m/z: [C26H22NO4]+, 412.16; found, 412.25. 100% pure by UPLC analysis.
Synthesis of 38
Methyl 4-(5-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)-1H-pyrrol-2-yl)benzoate (108)
6-Bromo-1,1-dimethyl-4-(p-tolyl)-1,2-dihydronaphthalene (107) (350 mg, 1.07 mmol, 1.0 equiv) and methyl 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzoate (76) (352 mg, 1.08 mmol, 1.0 equiv) were added to a vial and dissolved in THF (2.5 mL). An aqueous solution of tribasic potassium phosphate (0.5 M, 5.0 mL, 2.3 equiv) was added to the solution. Then nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of XPhos Pd G2 (0.040 g, 0.05 mmol, 0.05 equiv). The reaction mixture was placed into a preheated heating block at 45 °C and stirred for 3 h. After the reaction was complete, brine was added to the reaction mixture, extracted with EtOAc (5 mL × 3), dried over anhydrous MgSO4, and filtered, and the solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain methyl 4-(5-(5,5-dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)-1H-pyrrol-2-yl)benzoate (108) as a bright yellow solid (681 mg, 86%). mp 168–170 °C. 1H NMR (400 MHz, CDCl3): δ 8.53 (s, 1H), 8.02 (d, J = 1.8 Hz, 1H), 8.00 (d, J = 1.9 Hz, 1H), 7.53–7.46 (m, 2H), 7.40 (d, J = 1.3 Hz, 2H), 7.32–7.27 (m, 2H), 7.25–7.19 (m, 3H), 6.64 (dd, J = 3.7, 2.6 Hz, 1H), 6.43 (dd, J = 3.8, 2.5 Hz, 1H), 6.02 (t, J = 4.7 Hz, 1H), 3.91 (s, 3H), 2.42 (s, 3H), 2.36 (d, J = 4.7 Hz, 2H), 1.36 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 167.0, 144.4, 139.1, 137.9, 137.1, 136.7, 134.9, 134.7, 131.6, 130.5, 129.9, 129.2, 128.7, 127.3, 127.1, 124.6, 123.4, 123.1, 122.1, 109.9, 108.3, 52.2, 39.1, 33.8, 28.3, 21.4.
4-(5-(5,5-Dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)-1H-pyrrol-2-yl)benzoic Acid (38)
To a solution of methyl 4-(5-(5,5-dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)-1H-pyrrol-2-yl)benzoate (108) (120 mg, 0.27 mmol, 1.0 equiv) in THF (2 mL) and MeOH (2 mL) was added an aqueous sodium hydroxide solution (2 N, 2.0 mL, 2.0 mmol, 15 equiv), and the resulting mixture was stirred for 20 h at room temperature. Then the organic solvents were evaporated under reduced pressure, and the aqueous suspension was acidified with hydrochloric acid (2 N) to reach pH 1.0. The reaction mixture was extracted with EtOAc (10 mL × 3), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 50% EtOAc and 2% formic acid in hexanes) to obtain 4-(5-(5,5-dimethyl-8-(p-tolyl)-5,6-dihydronaphthalen-2-yl)-1H-pyrrol-2-yl)benzoic acid (38) (87 mg, 75%) as a yellow solid. mp 123–125 °C. 1H NMR (400 MHz, THF-d 8): δ 10.51 (s, 1H), 7.97–7.90 (m, 2H), 7.61 (s, 2H), 7.52 (dd, J = 8.1, 2.0 Hz, 1H), 7.41–7.33 (m, 2H), 7.30–7.24 (m, 2H), 7.19 (d, J = 7.8 Hz, 2H), 6.60 (dd, J = 3.7, 2.5 Hz, 1H), 6.33–6.28 (m, 1H), 5.97 (d, J = 4.7 Hz, 1H), 2.39–2.31 (m, 5H), 1.34 (s, 6H). 13C NMR (101 MHz, THF-d 8): δ 167.2, 144.1, 140.4, 138.8, 137.9, 137.3, 135.9, 134.8, 132.9, 131.4, 130.7, 129.5, 129.1, 128.2, 126.6, 124.6, 124.3, 123.6, 123.1, 110.0, 108.4, 39.6, 34.1, 28.3, 21.0. LRMS calcd m/z: [C30H28NO2]+, 434.22; found, 434.27. 99% pure by UPLC analysis.
Synthesis of 39
4-(1H-Pyrrol-2-yl)benzonitrile (109)
4-Bromobenzonitrile (1.82 g, 10.0 mmol, 1.0 equiv), 1-Boc-pyrrole-5-boronic acid (48, 2.53 g, 12 mmol, 1.2 equiv), and tribasic potassium phosphate (5.50 g, 26 mmol, 2.6 equiv) were added to a vial, followed by the addition of THF (20 mL) and water (40 mL). Then nitrogen gas was bubbled through the reaction mixture for 10 min, followed by the addition of XPhos Pd G2 (230 mg, 0.29 mmol, 0.03 equiv). The vial was sealed and placed in a preheated heating block at 45 °C and stirred for 6 h. After the reaction was complete, as determined by TLC, brine was added to the reaction mixture, extracted with EtOAc (10 mL × 3), dried over anhydrous MgSO4, and filtered, and the solvent was evaporated. The crude reaction mixture was used in the next step without further purification.
The crude reaction mixture from the previous step was dissolved in THF (20 mL), and sodium methoxide (25 mL, 25 wt % in MeOH) was added to the reaction mixture. The reaction mixture was stirred for 5 min and quenched with a saturated ammonium chloride solution. The reaction mixture was extracted with DCM (20 mL × 3), and the combined organic layers were dried over anhydrous MgSO4, filtered, and evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 30% EtOAc in hexanes) to obtain 4-(1H-pyrrol-2-yl)benzonitrile (109) as a yellow solid (1.11 g, 66% over two steps). 1H NMR (400 MHz, CDCl3): δ 8.61 (s, 1H), 7.65–7.59 (m, 2H), 7.57–7.50 (m, 2H), 6.95 (td, J = 2.7, 1.4 Hz, 1H), 6.68 (td, J = 2.6, 1.4 Hz, 1H), 6.37–6.31 (m, 1H). 13C NMR (101 MHz, CDCl3): δ 136.9, 132.9, 130.2, 123.8, 121.1, 120.5, 119.3, 111.1, 108.9.
4-(5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzonitrile (110)
4-(1H-Pyrrol-2-yl)benzonitrile (109) (750 mg, 4.46 mmol, 1.0 equiv), bispinacolatodiboron (650 mg, 2.56 mmol, 0.57 equiv), (1,5-cyclooctadiene)(methoxy)iridium(I) dimer (0.045 g, 0.068 mmol, 0.015 equiv), and 4,4′-di-tert-butyl-2,2′-bipyridine (0.036 g, 0.13 mmol, 0.03 equiv) were added to a round-bottom flask and evacuated and purged with nitrogen. This process was repeated three times, followed by mixing with hexane (20 mL). The suspension was refluxed under a nitrogen gas atmosphere for 6 h. After cooling to room temperature, the reaction mixture was solubilized in DCM, and a silica gel slurry was prepared for flash chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzonitrile (110) as an off-white solid (1.08 g, 82%). 1H NMR (400 MHz, CDCl3): δ 8.99 (s, 1H), 7.68–7.57 (m, 4H), 6.91–6.86 (m, 1H), 6.69 (dd, J = 3.7, 2.5 Hz, 1H), 1.34 (s, 12H). 13C NMR (101 MHz, CDCl3): δ 136.4, 132.94, 132.91, 124.6, 123.8, 122.2, 121.1, 119.1, 110.0, 109.8, 84.2, 24.9.
4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzonitrile (39)
6-Bromo-2,2-dimethyl-4-(p-tolyl)-2H-chromene (43) (350 mg, 1.06 mmol, 1.0 equiv) and 4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrol-2-yl)benzonitrile (110) (350 mg, 1.2 mmol, 1.1 equiv) were added to a vial and dissolved into THF (2.5 mL), and 0.5 M aqueous solution of tribasic potassium phosphate (5.0 mL, 2.3 equiv) was added to the solution. Then nitrogen gas was bubbled through the reaction mixture for 15 min, followed by the addition of XPhos Pd G2 (0.040 g, 0.05 mmol, 0.05 equiv). The reaction mixture was placed in a preheated block at 45 °C and stirred for 3 h. After the reaction was complete, brine was added to the reaction mixture, extracted with EtOAc (5 mL × 3), and dried over anhydrous MgSO4, and the solvent was evaporated to dryness under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 20% EtOAc in hexanes) to obtain 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzonitrile (39) as a bright yellow solid (238 mg, 54%). mp 1H NMR (400 MHz, CDCl3): δ 8.49 (s, 1H), 7.63–7.56 (m, 2H), 7.54–7.47 (m, 2H), 7.35 (dd, J = 8.3, 2.3 Hz, 1H), 7.29 (d, J = 8.1 Hz, 2H), 7.26–7.20 (m, 3H), 6.94 (d, J = 8.3 Hz, 1H), 6.65 (dd, J = 3.8, 2.6 Hz, 1H), 6.39 (dd, J = 3.7, 2.5 Hz, 1H), 5.66 (s, 1H), 2.42 (s, 3H), 1.52 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 153.1, 137.9, 136.6, 135.8, 135.3, 134.4, 132.9, 130.4, 129.8, 129.4, 128.7, 125.5, 124.8, 123.5, 123.1, 121.8, 119.4, 117.6, 110.7, 108.5, 107.9, 76.3, 27.7, 21.4. LRMS calcd m/z: [C29H25N2O]+, 417.20; found, 417.26. 98% pure by UPLC analysis.
Synthesis of 40
4-(5-(2,2-Dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzamide (40)
To a solution of 9 (100 mg, 0.23 mmol, 1.0 equiv) and hydroxybenzotriazole (60 mg, 0.44 mmol, 1.9 mmol) in DMF (3 mL) was added 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (88 mg, 0.46 mmol, 1.0 equiv). The mixture was stirred at room temperature for 2 h. The resulting solution was cooled to 0 °C, and then an ammonia solution (25%, 0.2 mL) was added. The mixture was warmed to room temperature and stirred for 12 h. The reaction mixture was poured into cold water, extracted with EtOAc (3 × 5 mL), washed with brine, dried over anhydrous MgSO4, filtered, and evaporated under reduced pressure. The residue was purified by flash column chromatography (silica gel, 100% hexanes to 100% EtOAc in hexanes) to obtain 4-(5-(2,2-dimethyl-4-(p-tolyl)-2H-chromen-6-yl)-1H-pyrrol-2-yl)benzamide (40) (81 mg, 81%) as a white solid. 1H NMR (400 MHz, CDCl3): δ 8.62 (s, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.54–7.48 (m, 2H), 7.40–7.33 (m, 1H), 7.27 (s, 2H), 7.25–7.20 (m, 3H), 6.93 (d, J = 8.3 Hz, 1H), 6.62 (dd, J = 3.7, 2.6 Hz, 1H), 6.37 (dd, J = 3.6, 2.5 Hz, 1H), (s, br, 2H), 5.65 (s, 1H), 2.41 (s, 3H), 1.51 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 169.0, 152.9, 137.9, 136.1, 135.3, 135.0, 134.5, 131.2, 130.1, 129.7, 129.4, 128.7, 128.3, 125.4, 125.2, 123.3, 123.0, 121.8, 117.6, 109.6, 107.6, 76.2, 27.7, 21.4. LRMS calcd m/z: [C29H27N2O2]+, 435.21; found, 435.28. 97% pure by UPLC analysis.
Supplementary Material
Acknowledgments
This project was funded by NIH/NICHD: HHSN275201300017C (G.I.G.), 5P50HD093540 (G.I.G. and D.J.W.), and YourChoice Therapeutics. M.A.A.N. and E.H. were supported by predoctoral fellowships from the Male Contraceptive Initiative.
Glossary
Abbreviations Used
- ALB
albumin
- A/G
albumin/globulin ratio
- ALDH
aldehyde dehydrogenase
- ALP
alkaline phosphatase
- ALT
alanine aminotransferase
- APTT
activated partial thromboplastin time
- AST
aspartate aminotransferase, ATRA, all-trans-retinoic acid
- AUC
area under the curve
- BASO
basophil
- BIL-T
total bilirubin
- BRDT
testis-specific bromodomain
- CatSper
sperm-specific calcium channel
- CBC
complete blood count
- CK
creatine kinase
- CRE
creatinine
- EOS
eosinophil
- FIB
fibrinogen
- FRET
fluorescence resonance energy transfer
- GD
gestational day
- GGT
gamma-glutamyltransferase
- GLB
globulin
- GLU
glucose
- HCT
hematocrit
- HGB
hemoglobin
- HPLC/MS
high performance liquid chromatography/mass spectrometry
- LBD
ligand binding domain
- LC/MSD iQ
liquid chromatography/mass spectrometry detector intelligence quotient
- LLOQ
lower limit of quantification
- LOEL
lowest observed effect level
- LYMP
lymphocyte
- MCH
mean corpuscular hemoglobin
- MCHC
mean corpuscular hemoglobin concentration
- MCV
mean corpuscular volume
- MONO
monocyte
- MPV
mean platelet volume
- NEUT
neutrophil
- Na
K-ATPase α4, sodium, potassium ATPase alpha 4
- ON
overnight
- NOAEL
no-observed-adverse-effect level
- PK
pharmacokinetic
- PLT
platelet
- PT
prothrombin time
- RA
retinoic acid
- RARα
retinoic acid receptor alpha
- RARβ
retinoic acid receptor beta
- RARγ
retinoic acid receptor gamma
- RBC
red blood cell
- RET
reticulocyte
- RXR
retinoid X receptor
- SAR
structure–activity relationship
- TCHO
total cholesterol
- TG
triglyceride
- T|LC
thin-layer chromatography
- TP
total protein, ULOQ, upper limit of quantification
- UV
ultraviolet
- WBC
white blood cell
All inquiries and requests should be sent to Dr. Gunda I. Georg at georg@umn.edu.
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c03051.
¶.
R.S. and M.A.A.N. contributed equally. R.S., M.A.A.N., N.C., E.H., S.M., and T.N. designed experiments, acquired data, and analyzed data for this work. N.M., S.S.W.C., A.B., J.E.H., H.L.W., D.J.W., and G.I.G. helped conceive and design experiments and analyze data. R.S., M.A.A.N., N.M., A.B., D.J.W. and G.I.G. wrote, edited, and reviewed the manuscript. E.H., S.M., N.C., T.N., S.S.W.C., J.E.H., and H.L.W. reviewed the manuscript.
The authors declare the following competing financial interest(s): The Regents of the University of Minnesota hold a patent related to this work (Publication No. US-2022-0388993-A1, Publication Date: 12/08/2022); G.I.G. and N.C. are listed as inventors; N.M. is co-founder and CSO of YourChoice Therapeutics; A.B. is co-founder and CEO of YourChoice Therapeutics; YourChoice Therapeutics holds the exclusive license for the IP owned by the University of Minnesota; G.I.G. is a consultant with YourChoice Therapeutics.
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Data Availability Statement
All inquiries and requests should be sent to Dr. Gunda I. Georg at georg@umn.edu.