Aldo-Keto Reductase 1C3 Inhibitor Prodrug Improves Pharmacokinetic Profile and Demonstrates In Vivo Efficacy in a Prostate Cancer Xenograft Model.

The replacement of the prenyl side chain of the lead compound KV49g with aromatic and heteroaromatic groups was accomplished through the route depicted in Scheme 1 . Treatment of 3-bromo-5-iodobenzoic acid ( 1 ) with SOCl 2 under reflux afforded the acid chloride, which was subsequently coupled with 4-methylbenzylamine in the presence of Et 3 N at room temperature to yield amide 2 in excellent yield. The amide underwent selective Heck coupling with methyl acrylate in the presence of Pd(OAc) 2 and PPh 3 to afford methyl ester 3 . Suzuki coupling with various functionalized aryl boronic acids or esters mediated by Pd(dppf)Cl 2 –CH 2 Cl 2 and Cs 2 CO 3 afforded access to the corresponding arylsubstituted esters ( 4a–ab ) in good yields. The methyl ester was hydrolyzed with aqueous 1 N NaOH in a mixture of THF/MeOH (4:1) at 60 °C to yield the target trans -cinnamic acids ( 5a–ab ). To determine the effect of the olefin on activity, 5r was reduced in the presence of Pd/C and H 2 to obtain saturated carboxylic acid 5r-H 2 ( Scheme 1 ). The O -prenylated cinnamic acid analog was obtained as depicted in Scheme 2 . Commercially available 3-bromo-5-hydroxybenzoic acid ( 6 ) underwent a sequence of Heck coupling conditions as described in Scheme 1 , followed by amide formation with 4-methylbenzylamine in the presence of EDC, HOBt, and Et 3 N at room temperature. The resultant phenol 8 was O -prenylated with prenyl bromide, and a base. The resulting methyl ester ( 9 ) was hydrolyzed with 1 N NaOH under the same reaction condition as described in Scheme 1 to afford the target acid ( 10 ) in good yield. Carboxamide analogs of the carboxylic acid were synthesized as depicted in Scheme 3 . ( E )-3-(3-bromo-5-((4-methylbenzyl)carbamoyl)phenyl)acrylic acid 11 was obtained by hydrolysis of the methyl ester with 1 N NaOH. The acid was converted to carboxamide by reaction with SOCl 2 to afford the corresponding acid chloride, which was then treated with 30% aqueous NH 4 OH to afford bromide 12 . The phenoxyphenyl substituted analogue 13 was accessed by applying the same reaction conditions to starting material 5n ( Scheme 3 ). Nitrile analogs of the carboxylic acid were accessed as depicted in Scheme 4 . The previously obtained intermediate 3-bromo-5-iodo- N -(4-methylbenzyl)benzamide ( 2 ) underwent selective Heck coupling with N , N -dimethylacrylamide or acrylonitrile at the iodo position, under the same reaction conditions as described in Scheme 1 . Bromides 14 and 15 were then exposed to Suzuki conditions with (4-phenoxyphenyl)-boronic acid or (3-methylbut-2-en-1-yl)boronic acid to yield the target compounds 16a and 16b . To investigate substituent effects at the benzylic position with the intent to improve metabolic stability, an ethyl group was installed as depicted in Scheme 5 . Commercially available 3-bromo-5-iodobenzoic acid was treated with thionyl chloride and reacted with ( R )-1-phenylpropan-1-amine to yield amide 17 . The amide was then subjected to consecutive Heck and Suzuki reactions with methyl acrylate and the corresponding aromatic boronic acid respectively, followed by hydrolysis as described in Scheme 1 , to afford target analogues 20a and 20b . The ability of the compounds to inhibit AKR1C3 in the first instance were determined by measuring the NADP + dependent oxidation of S -tetralol catalyzed by recombinant enzyme as described in the Methods section. Compounds that showed potent inhibition (IC 50 values < 100 nM) of AKR1C3 were counter-screened against AKR1C2 to determine selectivity. Replacement of the prenyl chain of parent AKR1C3 inhibitor KV49g with an unsubstituted phenyl ring ( 5a ) afforded a compound with increased inhibition potency with an IC 50 = 40 nM but with slightly reduced selectivity over AKR1C2 (2500 compared to >2850) ( Table 1 ). A halogen scan at the para position revealed no correlation with electron-withdrawing effect and activity with a sequence of fluorine ( 5d ) < chlorine ( 5c ) < bromine ( 5b ) with IC 50 values = 60, 60, and 90 nM, respectively. The same sequence correlation was apparent in the selectivity over AKR1C2, all of which were substantially decreased compared with KV49g . A positional sweep using fluorine revealed that meta -fluoro 5f was slightly more potent than para -fluoro 5d , with both being slightly more potent than ortho -fluoro 5e , with IC 50 values = 50, 60, and 70 nM, respectively. Interestingly ortho -F 5e possessed greater selectivity over AKR1C2 (>1430-fold), while meta -F 5f possessed 960-fold selectivity. 2,4-Diflouro substitution ( 5g ) resulted in reduced potency with an IC 50 value = 130 nM. Substitution with the highly electronegative para -CF 3 moiety ( 5h ) resulted in attenuated potency with an IC 50 value = 200 nM. Electron-donating substituents generally produced less potent AKR1C3 inhibitors with para -methyl ( 5i ), ethyl ( 5j ), and isopropyl ( 5k ) possessing values of 100, 190, and 190 nM, respectively. The para -methoxy derivative ( 5l ) possessed an IC 50 = 170 nM, which was equipotent with para -cyano 5m (IC 50 = 170 nM), while para -phenoxy ( 5n ) afforded an IC 50 = 94 nM with just 51-fold selectivity over AKR1C2. To further expand into the open pocket within the AKR1C3 steroid binding pocket ( Figure 2 ), a series of fused bicyclic compounds were synthesized. Naphthyl 5o possessed an IC 50 = 100 nM with 140-fold selectivity over AKR1C2. A nitrogen screen within the fused ring systems afforded an equipotent compound with quinoline 5p , while quinoxaline 5q possessed slightly increased potency with an IC 50 = 94 nM. [1,2,4]-Triazolo[1,5- a ]pyridine ( 5r ) provided a compound with enhanced AKR1C3 inhibition over the KV49g parent, yielding an IC 50 = 51 nM and 1216-fold selectivity over AKR1C2, with similar selectivity over AKR1C4, and no effect versus AKR1C1 (8% inhibition at 100 μ M concentration). Saturation of the double bond to form 5r-H 2 reduced the AKR1C3 IC 50 value to 205 nM, a fourfold loss of potency, indicating a preference for the carboxylic acid to be positioned in a rigid trans conformation. Addition of oxygen atoms to the bicyclic ring in the form of 2,3-dihydrobenzo[ b ][1,4]dioxine 5s afforded a compound with approximately twofold less potency than 5r with an IC 50 = 110 nM and reduced 200-fold selectivity over AKR1C2. Contraction of the ring to benzo[ d ][1,3]dioxole 5t afforded a compound with similar potency (IC 50 = 110 nM with 327-fold selectivity over AKR1C2). Increasing the length of the aromatic moiety with biphenyl 5u restored activity with an IC 50 = 50 nM but with just 580-fold selectivity over AKR1C2. Substantially increasing the bulk of the aromatic moiety with 9-phenyl-9 H -carbazole 5v resulted in attenuation of potency with an IC 50 = 140 nM and just 221-fold selectivity over AKR1C2. Introduction of the ethynylbenzene ring 5w achieved elongation and rotational constriction of the aromatic moiety and reduced potency and selectivity with an IC 50 = 180 nM and a much reduced 61-fold selectivity over AKR1C2. A cumene analogue 5x attenuated the potency against AKR1C3 (IC 50 = 880 nM) and exhibited 177-fold selectivity over AKR1C2. Addition of a 4-(2-fluorobenzyl)-morpholine moiety ( 5y ) considerably ameliorates potency and selectivity (IC 50 = 330 nM and 16-fold selectivity over AKR1C2). Removal of one of the terminal methyl groups of the prenyl side chain affords ( E )-but-2-ene 5z , with an IC 50 = 110 nM and 354-fold selectivity over AKR1C2. Interestingly, the introduction of the geometric isomer ( Z )-but-2-ene 5aa afforded a compound with an IC 50 = 80 nM, similar to the prenyl side chain-containing parent compound KV49g , with 200-fold selectivity over AKR1C2, which is less than that seen with KV49g . These data indicate that both terminal methyl groups are required for selective inhibition of AKR1C3. Introduction of ethynylcyclopropane 5ab resulted in attenuated inhibition potency (IC 50 = 500 nM) and selectivity (74-fold). Prenyl ether 10 possessed essentially equipotent activity to inhibit AKR1C3 (IC 50 = 80 nM) to KV49g but attenuated selectivity over AKR1C2 (187-fold). This derivative is similar to the far more selective compound reported by Endo et al. 38 but with a metabolically stable retroamide bond, 47 over a metabolically labile ester bond and had a different substitution pattern of the central phenyl ring. A simple bromine substituent at the prenyl position ( 11 ) ameliorates activity for AKR1C3, yielding an IC 50 = 300 nM. We next investigated analogues of the carboxylic acid ( Table 2 ). Our previous work has revealed that esters and a boronic acid bioisostere suffer from completely ablated AKR1C3 inhibition activity. 46 Interestingly, when bromo-functionalized derivative 11 is protected as its methyl ester ( 3 ), its potency improves, with the IC 50 value reducing from 300 to 200 nM. While still much less active than many other derivatives, this led to the enticing possibility of identifying more active carboxylic acid derivatives. Substitution of the carboxylic acid with a terminal amide ( 12 ) effectively inactivated the compound with IC 50 = 28 μ M. This reduction in potency was confirmed with p -phenoxyphenyl derivative 13 possessing an IC 50 = 2.7 μ M compared with the direct carboxylic acid bioisostere 5n (IC 50 = 94 nM). Replacement of the carboxylic acid with a terminal N -dimethyl amide ( 14 ) increased potency >66-fold (IC 50 = 400 nM) over terminal primary amide 12 , while a terminal cyano compound ( 15 ) retained some AKR1C3 inhibition potency with an IC 50 = 170 nM for the bromo-substituted derivative. However, this observation did not hold true for the prenyl-substituted terminal cyano compound 16a , which possessed an IC 50 = 1.4 μ M compared with its carboxylic acid counterpart KV49g (IC 50 = 70 nM) nor for p -phenoxylphenyl substituted terminal cyano compound 16b , which possessed an IC 50 = 2.7 μ M compared with its carboxylic acid counterpart 5n (IC 50 = 94 nM). Thus, replacement of the carboxylic acid was deemed to be untenable for the identification of AKR1C3 inhibitors. Finally, we investigated the substitution of the benzylic position with an ethyl moiety alpha to the amide bond with the intention to further increase the metabolic stability of this moiety ( Table 3 ). The p -phenoxylphenyl-substituted compound 20a possessed attenuated potency (IC 50 = 120 nM) over its non-benzylic substituted counterpart 5n (IC 50 = 94 nM) but identical selectivity over AKR1C2. Modification to the benzylic position of p -isopropyl phenyl ( 20b ) similarly reduced potency (IC 50 = 260 nM) compared with its non-benzylic-substituted counterpart 5k (IC 50 = 190 nM). It is possible that the ethyl moiety is hindering crucial hydrogen bond formation between the amide carbonyl and AKR1C3 binding site amino acid residues. The results demonstrate that unsubstituted benzylic amides are more beneficial for AKR1C3 enzyme inhibition activity. In silico docking studies were employed to understand the predicted binding interactions between inhibitors ( 5r and 5a ) and the AKR1C3 protein (PDB ID: 3UG8, AKR1C3·NADP + indomethacin) using Schrödinger-2022-3 software ( Figure 2 ). These studies predict key hydrogen bonding interactions between the amide carbonyl oxygen ( 5r ) and TYR55 and HIS117 residues. This carbonyl group directly attached to the central phenyl ring forms stronger hydrogen bonds within the oxyanion site and shorter hydrogen bond distance with the amino acid residues, which results in superior selectivity and inhibitory potency as we have previously reported. 46 Most AKR1C3 ligands anchor to the oxyanion site via the presence of either a carbonyl or a carboxylate group forming strong hydrogen bonds with TYR55 and HIS117, bringing the ligands in close proximity to the nicotinamide head group of the cofactor. 20 The carboxylic acid side chain is predicted to bind in the sub-pocket 3 (SP3) region forming hydrogen bonding interactions with TYR24 and a salt bridge with ARG226 ( Figure 2A , B ). The [1,2,4]triazolo[1,5- a ]pyridin-6-yl moiety of 5r is predicted to occupy the hydrophobic region of sub-pocket 1 (SP1) and surrounding residues (PHE311, TYR317, PRO318, and TYR319), forming strong hydrophobic interactions accounting for selectivity over the closely related AKR1C1, 2, and 4 isoforms. In each of these isoforms, the amino acid residues of the SP1 pocket collapse inward, forming a much smaller pocket. 20 , 48 This would exclude the 5r chemotype from entering the SP1 pocket of AKR1C1, AKR1C2, or AKR1C4 and therefore increase selectivity. The phenyl derivative’s ( 5a ) amide carbonyl group is predicted to engage in the same hydrogen bond interactions within the oxyanion site of AKR1C3, and the carboxylate ion is predicted to form hydrogen bond interactions with TYR24 and a salt bridge with the ARG226 residue. Additionally, this compound’s phenyl group has been predicted to have very strong hydrophobic interactions with TRP227, PHE306, and PHE311 in the SP2 region, which contributes to greater selectivity (>2500-fold) for AKR1C3 over AKR1C2 ( Figure 2C , D ). Compound 5r , possessing the best combination of potency, selectivity, and favorable metabolic structure (i.e., no exposed phenyl ring) was chosen to be taken forward to determine plasma stability. We also wished to investigate a potential prodrug strategy to overcome the inherent absorption, distribution, metabolism, and excretion (ADME) limitations often encountered with free carboxylic acid drugs. 49 Thus, we employed methyl ester intermediate 4r as our prodrug of choice. Gastrointestinal (GI) fluid stability studies were performed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Compounds 4r and 5r were stable in SGF for 1 h and in SIF for 2 h, which suggested that these compounds were favorable for oral administration ( Figure 3A , B ). Moreover, the methyl ester 4r was unstable in mouse plasma, being rapidly converted into 5r . The ester was reduced to below detectable limits within 30 min of exposure to mouse plasma, while the free acid 5r was stable for over 240 min ( Figure 3A , B ). In vitro metabolic stability of 4r and 5r was investigated using mouse liver microsomes (MLM) and human liver microsomes (HLM). The result of the metabolic stability study was expressed as the % parent compound remaining and % formation of free acid 5r at different time points relative to the parent at 0 min (100% parent) ( Figure 4A - C ). The free acid 5r was stable in MLM, HLM, and negative control (NC), indicating no or limited non-P450 metabolism ( Figure 4A ). Compound 4r was moderately degraded (approximately 25%) in MLM, HLM, as well as NC, indicating non-P450-mediated metabolism ( Figure 4B ). Ester 4r was found to be hydrolyzed to the free acid 5r by MLM, HLM, and NC at a similar rate over 60 min ( Figure 4C ). Thus, the methyl ester 4r possessed good potential for use as a prodrug AKR1C3 inhibitor in both mouse and human. Given the favorable activity, selectivity, in vitro stability, and hydrolysis data of compounds 4r and 5r , a pharmacokinetic study was performed ( Table 4 ). The plasma concentration vs time profile for 4r and the metabolite ( 5r meta ) is shown in Figure 5A , B following oral administration (10 mg/kg) of 5r or 4r in male Balb/C mice (mean ± SD, n = 5). The absorption of 5r from the gastrointestinal tract was rapid as it could be detected in plasma within 5 min and 5r metab was rapidly formed from the methyl ester prodrug 4r . The prodrug 4r was undetectable in systemic circulation 1 h post administration but 5r metab was detected at the last study point (24 h) ( Figure 5A ). Pharmacokinetic parameters for 4r and 5r are shown in Table 4 . Following administration of 4r , the metabolite 5r metab has significantly greater exposure (AUC o– t and AUC o–INF , 17-fold and 13-fold higher, respectively) than direct administration of 5r and an increase in peak plasma concentration (C max > 66-fold higher than following 5r administration). Prodrug 4r was rapidly converted to 5r metab and resulted in greater drug exposure following a near equivalent oral dose of 5r . These parameters validate our prodrug approach with the released active AKR1C3 inhibitor metabolite 5r metab possessing improved pharmacokinetic characteristics compared to the directly administered compound 5r . We next evaluated the in vivo efficacy of AKR1C3 inhibitor prodrug 4r in a 22Rv1 tumor xenograft model of PCa, which is reported to be resistant to the standard treatment of care enzalutamide. 50 , 51 Five-week-old NSG mice were implanted with 22Rv1 cells, and treatment with 4r was initiated on day 11 when tumors reached a mean volume of 125 mm 3 . Doses of 25 and 50 mg/kg of 4r were administered once a day intraperitoneally (IP) for a total of 26 days ( Figure 6 ). A clear dose-dependent relationship was observed, with a 25 mg/kg dose of 4r significantly reducing tumor volume by approximately 30%. Gratifyingly, a 50 mg/kg dose of 4r significantly reduced the tumor burden by approximately 45% ( Figure 6A ). This in vivo efficacy confirms effective conversion of the inactive prodrug 4r into the active AKR1C3 inhibitor 5r in mice. Furthermore, reduction of the tumor burden was combined with no observed loss of mouse body weight at either dose; indeed, the mice continued to grow normally ( Figure 6B ). Excised tumors supported a reduction of tumor mass ( Figure 6C , D ).

In this study, we have identified a number of prenyl side chain derivatives of our previous lead AKR1C3 inhibitor, which possess superior activity and/or selectivity via rational drug design and detailed structure–activity relationship studies. Our efforts identified 5r possessing a [1,2,4]triazolo[1,5- a ]pyridine moiety that exploits the open pocket present in AKR1C3, but absent in 1C1 and 1C2, to afford a highly active and selective AKR1C3 inhibitor. Wishing to subvert potential pharmacokinetic issues inherent with carboxylic acid-based drugs, we identified an inactive methyl ester 4r as a suitable prodrug that rapidly released the active AKR1C3 inhibitor 5r in both human and mouse liver microsomes. The prodrug achieved 17-fold greater exposure and far greater peak plasma concentration than direct administration of 5r in in vivo pharmacokinetic studies. The prodrug demonstrated dose-dependent reduction of tumor volume in a 22Rv1 PCa xenograft model. Taken together, this study both identifies new AKR1C3 inhibitors for further development and suggests that the general scaffold is suitable for prodrug design to optimize new compounds for the treatment of prostate and other cancers.

All reactions were carried out in oven- or flame-dried glassware under a nitrogen atmosphere unless otherwise noted. The reaction progress was monitored by thin-layer chromatography carried out on silica gel plates (2.5 cm × 7.5 cm, 200 μ m thick, 60 F254) and visualized using UV (254 nm) or by potassium permanganate and/or phosphomolybdic acid solution and/or ninhydrin as an indicator. Flash column chromatography was performed with silica gel (40–63 μ m, 60 Å) using the mobile phase indicated or on a Biotage Selekt (Rf 200 UV/vis). Solvents and reagents were purchased from Fisher Scientific, Sigma-Aldrich, and used without further purification, except as indicated. 1 H and 13 C NMR spectra were recorded on Bruker 600, 500, or 400 MHz spectrometers. The chemical shifts of 1 H NMR are reported in parts per million (ppm) relative to the internal standard tetramethylsilane or residual solvent peak. 13 C NMR chemical shifts are reported in ppm with the solvents (CDCl 3 : 77.23 ppm, CD 3 OD: 49.15 ppm, DMSO- d 6 : 39.51 ppm). Multiplicities are indicated by s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and br (broad). Chemical shifts ( δ ) are reported in parts per million (ppm), and coupling constants ( J ) are reported in hertz. High-resolution mass spectra (HRMS) were recorded with an Agilent 6230 LC/TOF spectrometer using an ESI source coupled to an Agilent Infinity 1260 system running in reverse phase with a ZORBAX RRHT Extend-C18 (80 Å, 2.1 × 50 mm, 1.8 μ m) column using solvent A (water with 0.1% formic acid), solvent B (acetonitrile with 0.1% formic acid), and a flow rate of 0.6 mL/min starting with a mixture of 95% A and 5% B. Solvent B was gradually increased to 95% at 5 min, held at 95% until 6 min, then gradually ramped back down to 5% at 8.0 min. The purity analysis of the final compounds was determined to be ≥95% pure (except 16a , which also contains reverse prenylated compound) using a Waters ACQUITY ultra-performance liquid chromatography (UPLC) H-Class System with a TUV (254 nm) detector and Empower 2 software (Milford, MA, USA) using an Agilent Eclipse plus C18 5 μ column (4.6 × 150 mm). Chromatography was performed using solvent A (water with 0.1% trifluoroacetic acid), solvent B (methanol with 0.1% trifluoroacetic acid), and a flow rate of 1.0 mL/min for 20 min with an isocratic system (20:80, A/B) (traces and purity analysis can be viewed in the Supporting Information ). To a stirred solution of aryl bromide 3 (0.20 g, 0.50 mmol) and appropriately substituted arylboronic acids or esters (0.21 g, 1.0 mmol) in anhydrous DMF (10 mL) were added Pd(dppf)Cl 2 ·CH 2 Cl 2 (40 mg, 0.05 mmol) and CS 2 CO 3 (0.50 g, 1.5 mmol). The mixture was stirred at 100 °C for 12 h under a N 2 atmosphere. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL). The filtrate was washed with 1 N HCl (30 mL) and brine 50 (mL). The aqueous layer was extracted with EtOAc (2 × 50 mL), and the combined organic layers were dried over Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography or Biotage Selekt flash column chromatography using EtOAc/hexanes as the eluent to afford the pure esters. To a stirred solution of methyl esters 4a–ab (0.2 g, 0.42 mmol) in a mixture of THF/MeOH (10 mL, 4:1) was added aqueous 1 N NaOH (1.3 mL, 1.3 mmol). The mixture was stirred at 60 °C for 3 h. The solvent was evaporated in vacuo, and the pH of the reaction mixture was adjusted to 2–4 with 1 N HCl. The mixture was extracted with CH 2 Cl 2 (3 × 50 mL) and washed with brine (50 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography or Biotage Selekt flash column chromatography using CH 2 Cl 2 /MeOH as the eluent to afford the pure acids. White solid (120 mg, yield 60%). 1 H NMR (400 MHz, CDCl 3 ): δ 8.01 (1H, s, ArCH), 7.91 (1H, s, ArCH), 7.79 (1H, s, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.57 (2H, d, J = 7.2 Hz, ArCH), 7.47–7.39 (3H, m, ArCH), 7.26 (2H, d, J = 8.0 Hz, ArCH), 7.16 (2H, d, J = 8.0 Hz, ArCH), 6.93 (1H, t, J = 5.2 Hz, NH), 6.52 (1H, d, J = 16.0 Hz, CH), 4.61 (2H, d, J = 5.6 Hz, CH 2 ), 3.80 (3H, s, OCH 3 ), 2.35 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 167.10, 166.77, 143.74, 142.41, 139.46, 137.37, 135.85, 135.28, 135.00, 129.52, 129.46, 128.99, 128.14, 127.99, 127.45, 127.16, 125.20, 119.29, 51.82, 44.05, 21.12. White solid (68 mg, yield 71%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.18 (1H, t, J = 5.6 Hz, NH), 8.20 (2H, s, ArCH), 8.15 (1H, s, ArCH), 7.81 (2H, d, J = 7.2 Hz, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.52 (2H, t, J = 7.6 Hz, ArCH), 7.43 (1H, t, J = 7.2 Hz, ArCH), 7.25 (2H, d, J = 8.0 Hz, ArCH), 7.15 (2H, d, J = 8.0 Hz), 6.74 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, d, J = 5.6 Hz, CH 2 ), 2.29 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 168.01, 165.90, 143.45, 141.43, 139.40, 137.03, 136.88, 136.35, 135.95, 135,74, 129.76, 129.46, 129.34, 128.53, 127.81, 127.46, 125.79, 121.45, 42.99, 21.14. HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 22 NO 3 , 372.1594; found, 372.1595. White solid (54 mg, yield 48%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.95 (1H, s, ArCH), 7.88 (1H, s, ArCH), 7.74 (1H, s, ArCH), 7.70 (1H, d, J = 16.0 Hz, CH), 7.56 (2H, d, J = 8.0 Hz, ArCH), 7.43 (2H, d, J = 8.0 Hz, ArCH), 7.25 (2H, d, J = 8.0 Hz, ArCH), 7.16 (2H, d, J = 8.0 Hz, ArCH), 6.64 (1H, t, J = 5.5 Hz, NH), 6.52 (1H, d, J = 16.0 Hz, CH), 4.61 (2H, d, J = 5.5 Hz, CH 2 ), 3.80 (3H, s, OCH 3 ), 2.24 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 166.99, 166.56, 143.44, 141.19, 138.32, 137.46, 135.99, 135.46, 134.90, 132.13, 129.49, 129.23, 128.70, 128.01, 127.26, 125.36, 122.55, 119.56, 51.88, 44.10, 21.13. White solid (28 mg, yield 73%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.18 (1H, t, J = 5.6 Hz, NH), 8.21–8.17 (3H, m, ArCH), 7.79 (2H, d, J = 8.4 Hz, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.70 (2H, d, J = 7.6 Hz, ArCH), 7.25 (2H, d, J = 7.6 Hz, ArCH), 7.15 (2H, d, J = 7.6 Hz, ArCH), 6.76 (1H, d, J = 16.0 Hz, CH), 4.50 (2H, d, J = 5.6 Hz, CH 2 ), 2.29 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 167.92, 165.78, 143.48, 140.09, 138.54, 136.84, 136.36, 136.06, 135.82, 132.34, 129.60, 129.56, 129.35, 127.81, 127.28, 126.26, 122.10, 121.39, 43.01, 21.15. HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 21 BrNO 3 , 450.0699; found, 450.0673. White solid (125 mg, yield 58%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.97 (1H, s, ArCH), 7.89 (1H, s, ArCH), 7.75 (1H, s, ArCH), 7.70 (1H, d, J = 16.0 Hz, CH), 7.50 (2H, d, J = 8.0 Hz, ArCH), 7.42 (2H, d, J = 7.6 Hz, ArCH), 7.26 (2H, d, J = 7.6 Hz, ArCH), 7.17 (2H, d, J = 7.6 Hz, ArCH), 6.85 (1H, br s, NH), 6.52 (1H, d, J = 16.0 Hz, CH), 4.61 (2H, d, J = 4.8 Hz, CH 2 ), 3.81 (3H, s, OCH 3 ), 2.35 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 166.99, 166.58, 143.45, 141.19, 137.86, 137.47, 135.97, 135.44, 134.89, 134.37, 129.49, 129.28, 129.18, 128.40, 128.02, 127.30, 125.31, 119.55, 51.87, 44.10, 21.12. White solid (57 mg, yield 59%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.22 (1H, t, J = 5.6 Hz, NH), 8.22 (2H, s, ArCH), 8.15 (1H, s, ArCH), 7.86 (2H, d, J = 8.4 Hz, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.55 (2H, d, J = 8.4 Hz, ArCH), 7.25 (2H, d, J = 7.6 Hz, ArCH), 7.14 (2H, d, J = 7.6 Hz, ArCH), 6.77 (1H, d, J = 16.0 Hz, CH), 4.50 (2H, d, J = 5.6 Hz, CH 2 ), 2.27 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 168.16, 165.82, 143.18, 140.04, 138.20, 136.87, 136.34, 136.03, 135.90, 133.47, 129.60, 129.39, 129.33, 129.22, 127.82, 127.28, 126.18, 121.81, 43.03, 21.13. HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 21 ClNO 3 , 406.1204; found, 406.1205. White solid (160 mg, yield 80%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.94 (1H, s, ArCH), 7.86 (1H, s, ArCH), 7.69 (1H, s, ArCH), 7.66 (1H, d, J = 16.0 Hz, CH), 7.50–7.48 (2H, m, ArCH), 7.22 (2H, d, J = 7.5 Hz, ArCH), 7.13–7.08 (4H, m, ArCH), 6.99 (1H, t, J = 5.0 Hz, NH), 6.48 (1H, d, J = 16.0 Hz, CH), 4.56 (2H, d, J = 5.0 Hz, CH 2 ), 3.77 (3H, s, OCH 3 ), 2.31 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 167.03, 166.68, 162.88 (d, J = 246.3 Hz), 143.56, 141.36, 137.38, 135.90, 135.56 (d, J = 2.5 Hz), 135.33, 134.96, 129.45, 129.30, 128.78 (d, J = 7.5 Hz), 127.97, 127.37, 125.07, 119.40, 115.9 (d, J = 21.3 Hz), 51.83, 44.04, 21.10. White solid (73 mg, yield 76%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 9.22 (1H, t, J = 6.0 Hz, NH), 8.20 (2H, s, ArCH), 8.14 (1H, s, ArCH), 7.88–7.85 (2H, m, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.34 (2H, d, J = 9.0 Hz, ArCH), 7.25 (2H, d, J = 8.0 Hz, ArCH), 7.15 (2H, d, J = 8.0 Hz, ArCH), 6.76 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, d, J = 6.0 Hz, CH 2 ), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.93, 165.85, 162.71 (d, J = 243.7 Hz), 143.58, 140.36, 136.88, 136.34, 135.98, 135.85 (d, J = 1.3 Hz), 135.70, 129.65, 129.58, 129.55 (d, J = 7.5 Hz), 129.33, 127.82, 127.38, 125.88, 121.26, 116.26 (d, J = 21.6 Hz), 42.99, 21.14. 19 F NMR (376 MHz, DMSO- d 6 ): −114.58. HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 21 FNO 3 , 390.1500; found, 390.1511. White solid (165 mg, yield 79%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.96 (2H, s, ArCH), 7.76 (1H, s, ArCH), 7.68 (1H, d, J = 16.0 Hz, CH), 7.32–7.43 (2H, m, ArCH), 7.24–7.18 (4H, m, ArCH), 7.15–7.11 (3H, m, 2 ArCH, 1 NH, overlapped), 6.49 (1H, d, J = 16.0 Hz, CH), 4.58 (2H, d, J = 5.6 Hz, CH 2 ), 3.78 (3H, s, OCH 3 ), 2.32 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 167.08, 166.64, 159.64 (d, J = 247.0 Hz), 143.60, 137.26, 136.91, 135.60, 135.01 (d, J = 5.0 Hz), 131.44 (d, J = 4.0 Hz), 130.61, 130.58, 129.90 (d, J = 8.0 Hz), 129.40, 129. 13 (d, J = 2.0 Hz), 127.93, 127.37 (d, J = 13.0 Hz), 125.75, 124.62 (d, J = 4.0 Hz), 119.35, 116.24 (d, J = 22 Hz), 51.79, 43.99, 21.09. White solid (75 mg, yield 78%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.35 (1H, t, J = 5.6 Hz, NH), 8.17 (1H, s, ArCH), 7.98 (1H, s, ArCH), 7.75 (1H, s, ArCH), 7.62 (1H, t, J = 8.0 Hz, ArCH), 7.48–7.43 (1H, m, ArCH), 7.36–7.23 (5H, m, 4 ArCH, 1CH, overlapped), 7.13 (2H, d, J = 7.6 Hz, ArCH), 6.63 (1H, d, J = 16.0 Hz, CH), 4.48 (2H, d, J = 5.6 Hz, CH 2 ), 2.27 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 171.17, 166.02, 159.57 (d, J = 245.0 Hz), 137.78, 137.08, 136.23, 136.06, 135.47, 135.24, 131.97, 131.40 (d, J = 3.0 Hz), 130.64, 130.44 (d, J = 9.0 Hz), 129.28, 128.02 (d, J = 13.0 Hz), 127.81, 125.46 (d, J = 4.0 Hz), 125.05, 116.58 (d, J = 22.0 Hz), 42.96, 21.13. 19 F NMR (376 MHz, DMSO- d 6 ): −118.24. HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 21 FNO 3 , 390.1500; found, 390.1503. White solid (120 mg, yield 58%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.99 (1H, s, ArCH), 7.92 (1H, s, ArCH), 7.75 (1H, s, ArCH), 7.69 (1H, d, J = 16.0 Hz, CH), 7.43–7.38 (1H, m, ArCH), 7.34 (2H, d, J = 8.0 Hz, ArCH), 7.28–7.24 (3H, m, ArCH), 7.15 (2H, d, J = 8.0 Hz, ArCH), 7.08 (1H, dt, J = 8.0, 1.6 Hz, ArCH), 7.02 (1H, t, J = 5.6 Hz, NH), 6.51 (1H, d, J = 16.0 Hz, CH), 4.60 (2H, d, J = 5.6 Hz, CH 2 ), 3.80 (3H, s, OCH 3 ), 2.33 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 167.00, 166.59, 163.18 (d, J = 245.0 Hz), 143.44, 141.39 (d, J = 7.0 Hz), 141.06 (d, J = 2.0 Hz), 137.39, 135.98, 135.42, 134.93, 130.53 (d, J = 8.0 Hz), 129.45, 129. 38, 127.99, 127.40, 125.68, 122.81 (d, J = 3.0 Hz), 119.53, 114.98 (d, J = 21.0 Hz), 114.10 (d, J = 22.0),51.84, 44.07, 21.10. White solid (55 mg, yield 57%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.38 (1H, br s, NH), 8.15 (1H, s, ArCH), 8.12 (1H, s, ArCH), 7.96 (1H, s, ArCH), 7.69–7.64 (2H, m, ArCH), 7.56–7.50 (1H, m, ArCH), 7.33 (1H, d, J = 16.0 Hz, CH), 7.27–7.23 (3H, m, ArCH), 7.14 (2H, d, J = 7.6 Hz, ArCH), 6.69 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, d, J = 5.2 Hz, CH 2 ), 2.27 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 171.39, 166.09, 163.19 (d, J = 242.0 Hz), 142.31 (d, J = 8.0 Hz), 139.69, 138.17, 137.08, 136.23, 135.81, 135.58, 131.70, 131.34 (d, J = 8.0 Hz), 129.29, 128.60, 127.81, 125.61, 123.48, 115.01 (d, J = 20.0 Hz), 114.16 (d, J = 22 Hz), 42.95, 21.14. 19 F NMR (376 MHz, DMSO- d 6 ): −112.71 to −112.77 (m). HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 21 FNO 3 , 390.1500; found, 390.1503. White solid (160 mg, yield 74%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.94 (1H, s, ArCH), 7.91 (1H, s, ArCH), 7.71 (1H, s, ArCH), 7.67 (1H, d, J = 16.0 Hz, CH), 7.40–7.35 (1H, m, ArCH), 7.23 (2H, d, J = 8.0 Hz, ArCH), 7.13 (2H, d, J = 8.0 Hz, ArCH), 7.12 (1H, br s, NH), 6.96–6.89 (2H, m, ArCH), 6.49 (1H, d, J = 16.0 Hz, CH), 4.57 (2H, d, J = 5.2 Hz, CH 2 ), 3.78 (3H, s, OCH 3 ), 2.32 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 167.01, 166.53, 162.73 (dd, J = 250.0, 11.5 Hz), 159.70 (dd, J = 250.0, 11.5 Hz), 143.42, 137.30, 136.07, 135.69, 135.08, 134.97, 131.40 (dd, J = 9.0, 4.0 Hz), 131.29 (d, J = 3.0 Hz), 129.41, 129.09, 127.92, 125.69, 123.69 (dd, J = 13.0, 4.0 Hz), 119.49, 119. 90 (t, J = 25.5 Hz), 51.81, 44.01, 21.08. White solid (72 mg, yield 74%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 8.16 (1H, s, ArCH), 7.96 (1H, s, ArCH), 7.70 (1H, s, ArCH), 7.67–7.63 (1H, m, ArCH), 7.39–7.34 (1H, m, ArCH), 7.28 (1H, d, J = 16.0 Hz, CH), 7.25–7.17 (3H, m, ArCH), 7.11 (2H, d, J = 8.0 Hz, ArCH), 6.11 (1H, d, J = 16.0 Hz, CH), 4.46 (2H, s, CH 2 ), 2.26 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 171.30, 166.01, 162.34 (dd, J = 245.5, 11.5 Hz), 159.62 (dd, J = 247.5, 12.5 Hz), 137.65, 137.58, 136.28, 136.02, 135.43, 135.80, 132.55 (dd, J = 10.5, 4.5 Hz), 131.73, 130.23, 129.20, 127.84, 125.25, 124.87, 124.80 (d, J = 14.0 Hz), 122.71, 112.54 (d, J = 24.0 Hz), 104. 96 (t, J = 26.0 Hz), 43.53, 21.12. 19 F NMR (376 MHz, DMSO- d 6 ): −110.86, 113.83 (m). HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 20 F 2 NO 3 , 408.1406; found, 408.1405. White solid (165 mg, yield 71%). 1 H NMR (500 MHz, CDCl 3 ): δ 8.03 (1H, s, ArCH), 7.94 (1H, s, ArCH), 7.81 (1H, s, ArCH), 7.74 (1H, d, J = 16.0 Hz, CH), 7.72–7.68 (4H, m, ArCH), 7.27 (2H, d, J = 8.0 Hz, ArCH), 7.18 (2H, d, J = 8.0 Hz, ArCH), 6.75 (1H, t, J = 5.0 Hz, NH), 6.55 (1H, d, J = 16.0 Hz, CH), 4.63 (2H, d, J = 5.0 Hz, CH 2 ), 3.83 (3H, s, OCH 3 ), 2.36 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.93, 166.45, 143.24, 142.93, 141.05, 137.58, 136.08, 135.63, 134.77, 129.58, 129.54, 128.04, 127.59, 127.52, 125.97, 125.95, 125.76, 119.82, 51.90, 44.17, 21.11. White solid (56 mg, yield 58%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 9.21 (1H, t, J = 6.0 Hz, NH), 8.26 (2H, s, ArCH), 8.24 (1H, s, ArCH), 8.05 (2H, d, J = 16. 0 Hz, ArCH), 7.86 (1H, d, J = 8.0 Hz, ArCH), 7.74 (1H, d, J = 16.0 Hz, CH), 7.25 (2H, d, J = 8.0 Hz, ArCH), 7.15 (2H, d, J = 8.0 Hz, ArCH), 6.78 (1H, d, J = 16.0 Hz, CH), 4.50 (2H, d, J = 6.0 Hz, CH 2 ), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.89, 165.73, 143.58, 139.83, 136.80, 136.38, 136.13, 135.90, 130.08, 129.34, 128.99, 128.87 (q, J = 23.8 Hz), 128.30, 127.82, 127.76, 126.78, 126.30, 125.87, 123.70, 121. 50, 43.02,21.13. 21.14. 19 F NMR (376 MHz, DMSO- d 6 ): −60.93. HRMS–ESI ( m/z ): [M + H] + calcd for C 25 H 21 F 3 NO 3 , 440.1468; found, 440.1469. White solid (150 mg, yield 73%). 1 H NMR (500 MHz, CDCl 3 ): δ 8.05 (1H, s, ArCH), 7.95 (1H, s, ArCH), 7.87 (1H, s, ArCH), 7.81 (1H, d, J = 16.0 Hz, CH), 7.56 (2H, d, J = 8.0 Hz, ArCH), 7.34 (4H, m, ArCH), 7.25 (2H, d, J = 8.0 Hz, ArCH), 6.68 (1H, br s, NH), 6.59 (1H, d, J = 16.0 Hz, CH), 4.70 (2H, d, J = 5.5 Hz, CH 2 ), 3.89 (3H, s, OCH 3 ), 2.49 (3H, s, CH 3 ), 2.43 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 167.10, 166.76, 143.79, 142.43, 138.11, 137.48, 136.56, 135.81, 135.31, 134.93, 129.73, 129.52, 129.36, 128.04, 127.17, 126.99, 124.84, 119.29, 51.83, 44.11, 21.14, 21.12. White solid (65 mg, yield 70%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 9.24 (1H, t, J = 6.0 Hz, NH), 8.20 (1H, s, ArCH), 8.19 (1H, s, ArCH), 8.11 (1H, s, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.71 (2H, d, J = 8.0 Hz, ArCH), 7.31 (2H, d, J = 8.0 Hz, ArCH), 7.24 (2H, d, J = 8.0 Hz, ArCH), 7.14 (2H, d, J = 8.0 Hz, ArCH), 6.75 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, d, J = 6.0 Hz, CH 2 ), 2.36 (3H, s, CH 3 ), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 168.00, 165.93, 143.56, 141.29, 137.94, 136.93, 136.46, 136.31, 135.91, 135.66, 130.05, 129.45, 129.32, 127.81, 127.25, 127.14, 125.52, 121.30, 42.97, 21.16, 21.14. HRMS–ESI ( m/z ): [M + H] + calcd for C 25 H 24 NO 3 , 386.1751; found, 386.1753. White solid (130 mg, yield 61%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.99 (1H, s, ArCH), 7.89 (1H, s, ArCH), 7.82 (1H, s, ArCH), 7.74 (1H, d, J = 16.0 Hz, CH), 7.52 (2H, d, J = 8.0 Hz, ArCH), 7.31–7.27 (4H, m, ArCH), 7.18 (2H, d, J = 8.0 Hz, ArCH), 6.64 (1H, br s, NH), 6.54 (1H, d, J = 16.0 Hz, CH), 4.64 (2H, d, J = 5.0 Hz, CH 2 ), 3.83 (3H, s, OCH 3 ), 2.72 (2H, q, J = 7.5 Hz, CH 2 ), 2.36 (3H, s, CH 3 ), 1.30 (3H, t, J = 7.5 Hz, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 167.10, 166.75, 144.47, 143.81, 142.46, 137.47, 136.81, 135.81, 135.30, 134.94, 129.51, 129.40, 128.54, 128.03, 127.20, 127.09, 124.86, 119.28, 51.83, 44.10, 28.54, 21.12, 15.54. White solid (56 mg, yield 58%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 9.18 (1H, t, J = 6.0 Hz, NH), 8.18 (1H, s, ArCH), 8.17 (1H, s, ArCH), 8.11 (1H, s, ArCH), 7.72 (1H, d, J = 8.0 Hz, CH), 7.70 (1H, d, J = 16.0 Hz, CH), 7.34 (2H, d, J = 8.0 Hz), 7.25 (2H, d, J = 8.0 Hz), 7.15 (2H, d, J = 8.0 Hz), 6.74 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, d, J = 6.0 Hz, CH 2 ), 2.66 (2H, q, J = 7.5 Hz, CH 2 ), 2.27 (3H, s, CH 3 ), 1.22 (3H, t, J = 7.5 Hz, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 168.14, 165.95, 144.25, 143.40, 141.37, 136.91, 136.78, 136.33, 135.91, 135.71, 129.51, 129.33, 128.87, 127.80, 127.36, 127.18, 125.46, 121.51, 42.98, 28.30, 21.14, 16.04. HRMS–ESI ( m/z ): [M + H] + calcd for C 26 H 26 NO 3 , 400.1907; found, 400.1908. White solid (0.17 g, yield 77%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.98 (1H, s, ArCH), 7.89 (1H, s, ArCH), 7.83 (1H, s, ArCH), 7.77 (1H, d, J = 16.0 Hz, CH), 7.54 (2H, d, J = 8.5 Hz, ArCH), 7.35 (2H, d, J = 8.0 Hz, ArCH), 7.29 (2H, d, J = 7.5 Hz, ArCH), 7.20 (2H, d, J = 7.5 Hz, ArCH), 6.57 (1H, d, J = 16.0 Hz, CH), 6.46 (1H, br t, J = 5.5 Hz, NH), 4.66 (2H, d, J = 5.5 Hz, CH 2 ), 3.84 (3H, s, OCH 3 ), 3.00 (1H, m, CH), 2.38 (3H, s, CH 3 ), 1.31 (6H, d, J = 7.0 Hz, (CH 3 )2). 13 C NMR (125 MHz, CDCl 3 ): δ 167.15, 166.94, 149.00, 143.90, 142.26, 137.24, 136.90, 135.79, 135.16, 135.10, 129.41, 129.35, 127.94, 127.38, 127.07, 124.97, 119.10, 51.78, 44.00, 33.83, 23.96, 21.11. White solid (0.12 g, yield 82%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 12.52 (1H, br s, COOH), 9.20 (1H, t, J = 5.5 Hz, NH), 8.19 (2H, s, ArCH), 8.11 (1H, s, ArCH), 7.73 (1H, d, J = 16.0 Hz, CH), 7.72 (2H, d, J = 8.0 Hz, ArCH), 7.37 (2H, d, J = 8.0 Hz, ArCH), 7.25 (2H, d, J = 8.0 Hz, ArCH), 7.14 (2H, d, J = 7.5 Hz, ArCH), 6.75 (1H, d, J = 16.0 Hz, CH), 4.50 (2H, d, J = 5.5 Hz, CH 2 ), 2.94 (1H, m, CH), 2.28 (3H, s, CH 3 ), 1.24 (6H, d, J = 7.0 Hz, (CH 3 )2). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.94, 165.93, 148.83, 143.76, 141.43, 136.96, 136.91, 136.32, 135.92, 135.60, 129.62, 129.32, 127.81, 127.41, 127.39, 127.34, 125.48, 121.07, 42.99, 33.61, 24.28, 28.14. HRMS–ESI ( m/z ): [M + H] + calcd for C 27 H 28 NO 3 , 414.2064; found, 414.2063. White solid (130 mg, yield 61%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.97 (1H, s, ArCH), 7.85 (1H, s, ArCH), 7.75 (1H, s, ArCH), 7.71 (1H, d, J = 16.0 Hz, CH), 7.51 (2H, d, J = 8.8 Hz, ArCH), 7.27 (2H, d, J = 8.0 Hz, ArCH), 7.17 (2H, d, J = 8.0 Hz, ArCH), 6.99 (2H, d, J = 8.8 Hz, ArCH), 6.83 (1H, J = 5.6 Hz, NH), 6.52 (1H, d, J = 16.0 Hz, CH), 4.61 (2H, d, J = 5.2 Hz, CH 2 ), 3.86 (3H, s, OCH 3 ), 3.81 (3H, s, OCH 3 ), 2.34 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 167.13, 166.85, 159.78, 143.85, 142.00, 137.39, 135.79, 135.22, 135.00, 131.86, 129.47, 129.07, 128.23, 128.00, 127.00, 124.49, 119.16, 114.42, 55.37, 51.81, 44.05, 21.12. White solid (55 mg, yield 57%). 1 H NMR (600 MHz, CD 3 OD): δ 8.12 (1H, ArCH), 8.04 (1H, ArCH), 7.97 (1H, ArCH), 7.78 (1H, d, J = 16.2 Hz, CH), 2ArCH, 1CH), 7.68 (2H, d, J = 8.4 Hz, ArCH), 7.28 (2H, d, J = 7.8 Hz, ArCH), 7.18 (2H, d, J = 7.8 Hz, ArCH), 7.06 (2H, d, J = 8.4 Hz, ArCH), 6.66 (1H, d, J = 16.2 Hz, CH), 4.59 (2H, s, CH 2 ), 3.87 (3H, s, OCH 3 ), 2.34 (3H, s, CH 3 )· 13 C NMR (100 MHz, DMSO- d 6 ): δ 168.02, 165.98, 159.84, 143.67, 141.04, 136.97, 136.31, 135.90, 135.62, 131.68, 129.32, 129.23, 128.61, 127.83, 126.92, 125.12, 121.21, 114.89, 55.72, 42.97, 21.15. HRMS–ESI ( m/z ): [M + H] + calcd for C 25 H 24 NO 4 , 402.1700; found, 402.1705. White solid (70 mg, yield 70%). 1 H NMR (500 MHz, CDCl 3 ): δ 8.03 (1H, s, ArCH), 7.95 (1H, s, ArCH), 7.82 (1H, s, ArCH), 7.77–7.72 (4H, m, ArCH), 7.71 (1H, d, J = 16.0 Hz, CH), 7.28 (2H, d, J = 8.0 Hz, ArCH), 7.19 (2H, d, J = 8.0 Hz, ArCH), 6.63 (1H, t, J = 5.5 Hz, NH), 6.57 (1H, d, J = 16.0 Hz, CH), 4.64 (2H, d, J = 5.5 Hz, CH 2 ), 3.83 (3H, s, OCH 3 ), 2.36 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.84, 166.25, 143.90, 143.04, 140.47, 137.57, 136.21, 135.76, 134.76, 132.79, 129.52, 129.48, 128.04, 127.95, 127.85, 127.60, 126.13, 120.02, 118.56, 111.89, 51.94, 44.16, 21.12. White solid (45 mg, yield 67%). 1 H NMR (500 MHz, CDCl 3 +CD 3 OD): δ 8.48 (1H, t, J = 6.0 Hz, NH), 7.99 (1H, s, ArCH), 7.95 (1H, s, ArCH), 7.52–7.46 (5H, m, ArCH), 7.41 (1H, d, J = 16.0 Hz, CH), 6.97 (2H, d, J = 8.0 Hz, ArCH), 6.84 (2H, d, J = 8.0 Hz, ArCH), 6.34 (1H, d, J = 16.0 Hz, CH), 4.29 (2H, d, J = 6.0 Hz, CH 2 ), 2.03 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 +CD 3 OD): δ 172.90, 170.83, 148.98, 144.46, 141.41, 140.77, 140.66, 140.42, 137.47, 137.40, 137.35, 134.34, 134.21, 133.88, 132.72, 132.66, 132.61, 132.53, 125.77, 123.43, 116.12, 48.24, 25.82. HRMS–ESI ( m/z ): [M + H] + calcd for C 25 H 21 N 2 O 3 , 397.1547; found, 397.1549. White solid (0.35 g, yield 71%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.98 (1H, s, ArCH), 7.87 (1H, s, ArCH), 7.81 (1H, s, ArCH), 7.77 (1H, d, J = 16.0 Hz, CH), 7.57 (2H, d, J = 8.5 Hz, ArCH), 7.39 (2H, t, J = 8.0 Hz, ArCH), 7.29 (2H, d, J = 7.5 Hz, ArCH), 7.21–7.16 (3H, m, ArCH), 7.12–7.06 (4H, m, ArCH), 6.56 (1H, d, J = 16.0 Hz, CH), 6.49 (1H, t, J = 5.5 Hz, NH), 4.65 (2H, d, J = 5.5 Hz, CH 2 ), 3.84 (3H, s, OCH 3 ), 2.38 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 167.04, 166.63, 157.70, 156.80, 143.66, 141.91, 137.57, 135.89, 135.42, 134.86, 134.36, 129.88, 129.55, 129.29, 128.56, 128.07, 127.11, 124.79, 123.68, 119.45, 119.21, 119.08, 51.86, 44.15, 21.13. White solid (0.17 g, yield 58%). 1 H NMR (500 MHz, CDCl 3 + CD 3 OD): δ 8.07 (1H, s, ArCH), 7.99 (1H, s, ArCH), 7.83 (1H, s, ArCH), 7.73 (1H, d, J = 16.0 Hz, CH), 7.59 (2H, d, J = 8.0 Hz, ArCH), 7.34 (2H, t, J = 8.0 Hz, ArCH), 7.23 (2H, d, J = 7.5 Hz, ArCH), 7.12–7.11 (3H, m, ArCH), 7.06–7.01 (4H, m, ArCH), 6.56 (1H, d, J = 16.0 Hz, CH), 4.55 (2H, s, CH 2 ), 2.29 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 +CD 3 OD): δ 172.78, 171.76, 161.56, 160.79, 148.04, 145.54, 140.71, 139.51, 139.30, 138.40, 133.64, 133.13, 132.98, 132.35, 131.44, 131.30, 128.89, 127.45, 123.64, 122.93, 122.77, 47.36, 24.41. HRMS–ESI ( m/z ): [M + H] + calcd for C 30 H 26 NO 4 , 464.1856; found, 464.1859. White solid (160 mg, yield 71%). 1 H NMR (400 MHz, CDCl 3 ): δ 8.14 (1H, s, ArCH), 8.04 (1H, s, ArCH), 7.94–7.87 (5H, m, ArCH), 7.77 (1H, d, J = 16.0 Hz, CH), 7.71 (1H, dd, J = 8.4, 1.6 Hz, ArCH), 7.56–7.51 (2H, m, ArCH), 7.30 (2H, d, J = 7.6 Hz, ArCH), 7.19 (2H, d, J = 7.6 Hz, ArCH), 6.75 (1H, t, J = 5.2 Hz, NH), 6.76 (1H, d, J = 16.0 Hz, CH), 4.65 (2H, d, J = 5.6 Hz, CH 2 ), 3.83 (3H, s, OCH 3 ), 2.36 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 167.07, 166.72, 143.70, 142.36, 137.49, 136.68, 135.93, 135.42, 134.95, 133.54, 132.93, 129.74, 129.52, 128.80, 128.28, 128.06, 127.70, 127.64, 126.62, 126.47, 126.19, 125.13, 125.04, 119.42, 51.86, 44.13, 21.13. White solid (62 mg, yield 64%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 12.51 (1H, br s, OH), 9.26 (1H, t, J = 6.0 Hz, NH), 8.39 (2H, d, J = 7.6 Hz, ArCH), 8.32 (1H, s, ArCH), 8.24 (1H, s, ArCH), 8.07–7.99 (4H, m, ArCH), 7.77 (1H, d, J = 16.0 Hz, CH), 7.60–7.54 (2H, m, ArCH), 7.27 (2H, d, J = 8.0 Hz, ArCH), 7.16 (2H, d, J = 8.0 Hz, ArCH), 6.80 (1H, d, J = 16.0 Hz, CH), 4.52 (2H, d, J = 5.6 Hz, CH 2 ), 2.29 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 167.95, 165.93, 143.68, 141.22, 136.91, 136.66, 136.35, 136.06, 135.77, 133.73, 132.98, 129.98, 129.34, 129.03, 128.74, 128.01, 127.84, 127.71, 127.02, 126.90, 126.27, 126.00, 125.55, 121.25, 43.02, 21.15. HRMS–ESI ( m/z ): [M + H] + calcd for C 28 H 24 NO 3 , 422.1751; found, 422.1752. White solid (0.11 g, yield 69%). 1 H NMR (500 MHz, CDCl 3 ): δ 8.83 (1H, s, ArCH), 8.25–8.13 (3H, m, ArCH), 7.96 (1H, s, ArCH), 7.88 (2H, br s, ArCH), 7.77 (1H, s, ArCH), 7.69 (1H, d, J = 16.0 Hz, CH), 7.43 (1H, s, ArCH), 7.29 (2H, br s, ArCH), 7.16–7.12 (3H, m, ArCH), 6.50 (1H, d, J = 16.0 Hz, CH), 4.65 (2H, s, CH 2 ), 3.81 (3H, s, OCH 3 ), 2.33 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.98, 166.59, 150.74, 147.86, 143.41, 141.27, 140.63, 137.40, 136.26, 136.16, 135.56, 134.97, 129.66, 129.49, 128.62, 128.09, 127.76, 127.60, 127.00, 125.95, 121.43, 119.58, 51.85, 44.14, 21.11. White solid (70 mg, yield 73%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 12.54 (1H, br s, OH), 9.35 (1H, br s, NH), 8.98 (1H, s, ArCH), 8.50 (1H, s, ArCH), 8.46–8.38 (3H, m, ArCH), 8.28 (1H, s, ArCH), 8.13 (2H, s, ArCH), 7.78 (1H, d, J = 16.0 Hz, CH), 7.58 (1H, dd, J = 4.0, 8.0 Hz, ArCH), 7.27 (2H, d, J = 7.5 Hz, ArCH), 7.15 (2H, d, J = 7.5 Hz, ArCH), 6.83 (1H, d, J = 16.0 Hz, CH), 4.51 (2H, d, J = 5.5 Hz, CH 2 ), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.94, 165.80, 151. 53, 148.21, 143.57, 140.54, 140.41, 136.91, 136.56, 136.33, 136.11, 135.94, 130.19, 129.34, 127.93, 127.83, 126.88, 126.64, 126.33, 122.21, 121.42, 43.01, 21.15. HRMS–ESI ( m/z ): [M + H] + calcd for C 27 H 23 N 2 O 3 , 423.1703; found, 423.1707. White solid (0.09 g, yield 82%). 1 H NMR (500 MHz, CDCl 3 ): δ 8.85 (2H, d, J = 7.5 Hz, ArCH), 8.25 (1H, s, ArCH), 8.15 (2H, d, J = 4.5 Hz, ArCH), 7.98 (2H, d, J = 4.5 Hz, ArCH), 7.92 (1H, s, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.27 (2H, d, J = 8.0 Hz, ArCH), 7.16 (2H, d, J = 8.0 Hz, ArCH), 6.97 (1H, br s, NH), 6.54 (1H, d, J = 16.0 Hz, CH), 4.64 (2H, d, J = 5.5 Hz, CH 2 ), 3.81 (3H, s, OCH 3 ), 2.33 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.92, 166.43, 145.67, 145.22, 143.25, 143.07, 142.57, 141.02, 140.76, 137.48, 136.24, 135.71, 134.84, 130.20, 129.74, 129.51, 129.31, 128.07, 127.71, 127.36, 126.06, 119.82, 51.89, 44.18, 21.11. White solid (65 mg, yield 75%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 9.31 (1H, t, J = 6.0 Hz, NH), 9.02 (1H, d, J = 1.5 Hz, ArCH), 8.98 (1H, d, J = 1.5 Hz, ArCH), 8.58 (1H, d, J = 1.5 Hz, ArCH), 8.43 (2H, d, J = 5.5 Hz, ArCH), 8.37 (1H, dd, J = 9.0, 1.5 Hz, ArCH), 8.28 (1H, s, ArCH), 8.23 (1H, d, J = 7.5 Hz, ArCH), 7.78 (1H, d, J = 16.0 Hz, CH), 7.27 (2H, d, J = 8.0 Hz, ArCH), 7.16 (2H, d, J = 8.0 Hz, ArCH), 6.84 (1H, d, J = 16.0 Hz, CH), 4.52 (2H, d, J = 6.0 Hz, CH 2 ), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.93, 165.74, 146.83, 146.38, 143.47, 142.99, 142.36, 140.90, 139.84, 136.86, 136.36, 136.16, 136.01, 130.29, 130.21, 129.93, 129.35, 127.90, 127.83, 127.26, 126.99, 121.53, 43.02, 21.15. HRMS–ESI ( m/z ): [M + H] + calcd for C 26 H 22 N 3 O 3 , 424.1656; found, 424.1659. White solid (110 mg, yield 50%). 1 H NMR (400 MHz, CDCl 3 ): δ 8.84 (1H, s, ArCH), 8.39 (1H, s, ArCH), 8.08 (1H, s, ArCH), 7.99 (1H, s, ArCH), 7.81 (3H, br s, ArCH), 7.73 (1H, d, J = 16.0 Hz, CH), 7.28 (2H, d, J = 7.6 Hz, ArCH), 7.17 (2H, d, J = 7.6 Hz, ArCH), 6.96 (1H, br s, NH), 6.56 (1H, d, J = 16.0 Hz, CH), 4.64 (2H, d, J = 0.4 Hz, CH 2 ), 3.83 (3H, s, OCH 3 ), 2.35 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.78, 166.07, 154.02, 142.84, 137.58, 137.30, 136.52, 136.03, 134.77, 130.15, 129.53, 129.25, 128.08, 127.53, 127.45, 126.32, 126.12, 120.27, 116.75, 51.98, 44.19, 21.13. HRMS–ESI ( m/z ): [M + H] + calcd for C 25 H 23 N 4 O 3 , 427.1765; found, 427.1768. White solid (60 mg, yield 75%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 9.52 (1H, s, ArCH), 9.19 (1H, t, J = 6.0 Hz, NH), 8.58 (1H, s, ArCH), 8.36 (1H, s, ArCH), 8.32 (1H, s, ArCH), 8.21–8.19 (2H, m, ArCH), 7.99 (1H, d, J = 9.5 Hz), 7.69 (1H, d, J = 16.0 Hz, CH), 7.26 (2H, d, J = 8.0 Hz, ArCH), 7.26 (2H, d, J = 8.0 Hz, ArCH), 6.83 (1H, d, J = 16.0 Hz, CH), 4.51 (2H, s, CH 2 ), 2.23 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.93, 165.73, 155.01, 143.34, 136.80, 136.40, 136.20, 135.98, 130.48, 129.51, 129.37, 127.82, 127.45, 127.38, 127.15, 126.78, 121.63, 116.62, 43.02, 21.14. HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 21 N 4 O 3 , 413.1608; found, 413.1617. To a solution of 5r (75 mg, 0.18 mmol) in EtOAc (5 mL), 10% Pd/C (19 mg, 0.018 mmol) was added, and the flask was degassed and backfilled with H 2 gas using a balloon. The reaction was stirred at room temperature for 6 h, and the reaction mixture was filtered, washed with EtOAc, and the organic layer was evaporated under vacuum to obtain the saturated carboxylic acid 5r-H 2 . White solid (20 mg, yield 27%). 1 H NMR (600 MHz, DMSO- d 6 ): δ 12.19 (1H, br s, OH), 9.42 (1H, s, ArCH), 9.09 (1H, t, J = 5.4 Hz, NH), 8.56 (1H, s, ArCH), 8.14–8.11 (2H, m, ArCH), 7.98 (1H, d, J = 9.0 Hz, ArCH), 7.89 (1H, s, ArCH), 7.82 (1H, s, ArCH), 7.25 (2H, d, J = 7.8 Hz, ArCH), 7.15 (2H, d, J = 7.2 Hz, ArCH), 4.49 (2H, d, J = 4.8 Hz, CH 2 ), 2.97 (2H, t, J = 7.5 Hz, CH 2 ), 2.68 (2H, t, J = 7.5 Hz, CH 2 ), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 174.2, 166.3, 154.9, 149.7, 142.7, 137.0, 136.3, 136.0, 135.7, 130.5, 130.2, 129.3, 127.8, 127.4, 127.0, 123.7, 116.6, 42.9, 35.4, 30.8, 21.2. HRMS–ESI ( m/z ): [M + H] + calcd for C 24 H 23 N 4 O 3 , 415.1765; found 415.1760. White solid (0.17 g, yield 77%). 1 H NMR (500 MHz, CDCl 3 + CD 3 OD): δ 7.90 (1H, s, ArCH), 7.83 (1H, s, ArCH), 7.70 (1H, s, ArCH), 7.68 (1H, d, J = 16.0 Hz, CH), 7.22 (2H, d, J = 8.0 Hz, ArCH), 7.12 (2H, d, J = 8.0 Hz, ArCH), 7.07 (1H, d, J = 1.5 Hz, ArCH), 7.04 (1H, dd, J = 8.5, 1.6 Hz, ArCH), 6.89 (1H, d, J = 8.0 Hz, ArCH), 6.48 (1H, d, J = 16.0 Hz, CH), 4.56 (2H, s, CH 2 ), 4.25 (4H, s, (OCH 2 )2), 3.77 (3H, s, OCH 3 ), 2.30 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 + CD 3 OD): δ 167.31, 166.93, 143.94, 143.86, 143.82, 141.77, 137.35, 135.65, 135.19, 134.94, 132.90, 129.42, 129.11, 127.95, 127.02, 124.79, 120.16, 119.11, 117.77, 115.89, 64.46, 64.39, 51.83, 43.89, 21.05. White solid (85 mg, yield 77%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 12.49 (1H, br s, OH), 9.18 (1H, t, J = 5.5 Hz), NH), 8.14 (2H, s, ArCH), 8.08 (1H, s, ArCH), 7.71 (1H, d, J = 16.0 Hz, CH), 7.35 (1H, d, J = 1.5 Hz, ArCH), 7.29 (1H, dd, J = 1.5, 8.0 Hz, ArCH), 7.24 (2H, d, J = 8.0 Hz, ArCH), 7.14 (2H, d, J = 8.0 Hz, ArCH), 6.97 (1H, d, J = 8.0 Hz, ArCH), 6.74 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, d, J = 5.5 Hz, CH 2 ), 4.29 (4H, s, (OCH 2 )2), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.96, 165.91, 144. 23, 144.09, 143.77, 140.81, 136.91, 136.33, 135.83, 135.58, 132.57, 129.33, 129.25, 127.78, 126.88, 125.42, 121.04, 120.33, 117.99, 115.98, 64.68, 64. 60, 42.97, 21.14. HRMS–ESI ( m/z ): [M + H] + calcd for C 26 H 24 NO 5 , 430.1649; found, 430.1648. White solid (130 mg, yield 59%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.92 (1H, d, J = 1.6 Hz, ArCH), 7.86 (1H, s, ArCH), 7.72 (1H, d, J = 1.6 Hz, ArCH), 7.71 (1H, d, J = 16.0 Hz, CH), 7.27 (2H, d, J = 8.0 Hz, ArCH), 7.17 (2H, d, J = 8.0 Hz, ArCH), 7.06–7.04 (2H, m, ArCH), 6.88 (1H, d, J = 8.0 Hz, ArCH), 6.73 (1H, t, J = 5.6 Hz, NH), 6.52 (1H, d, J = 16.0 Hz, CH), 6.01 (2H, s, CH 2 ), 4.62 (2H, d, J = 5.6 Hz, CH 2 ), 3.82 (3H, s, OCH 3 ), 2.34 (3H, s, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 167.08, 166.71, 148.36, 147.78, 143.70, 142.12, 137.45, 135.82, 135.28, 134.94, 133.71, 129.49, 129.22, 128.02, 127.10, 124.76, 120.88, 119.30, 108.73, 107.56, 101.38, 51.83, 44.08, 21.12. White solid (62 mg, yield 64%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.16 (1H, t, J = 5.6 Hz, NH), 8.12 (2H, s, ArCH), 8.08 (1H, s, ArCH), 7.67 (1H, d, J = 16.0 Hz, CH), 7.43 (1H, s, ArCH), 7.31 (1H, d, J = 8.0 Hz, ArCH), 7.24 (2H, d, J = 7.6 Hz, ArCH), 7.15 (2H, d, J = 8.0 Hz, ArCH), 7.04 (1H, d, J = 8.0 Hz, ArCH), 6.75 (1H, d, J = 16.0 Hz, CH), 6.09 (2H, s, CH 2 ), 4.49 (2H, d, J = 5.6 Hz, CH 2 ), 2.28 (3H, s, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 168.14, 165.94, 148.54, 147.79, 143.10, 141.04, 136.90, 136.33, 135.84, 135.76, 133.59, 129.33, 127.79, 126.91, 125.47, 121.91, 121.19, 109.15, 107.85, 101.75, 42.97, 21.14. HRMS–ESI ( m/z ): [M + H] + calcd for C 25 H 22 NO 5 , 416.1492; found, 416.1492. A transparent oil (74%). 1 H NMR (400 MHz; CDCl 3 ): δ 7.88 (1H, s, CH), 7.74 (1H, s, CH), 7.38–7.50 (6H, m, ArCH and CH), 7.14–7.21 (8H, m, ArCH), 6.26 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, s, CH 2 ), 3.89 (3H, s, CH 3 ), 2.32 (3H, s, CH 3 ). 13 C NMR (100 MHz; CDCl 3 ): δ 141.09, 141.07, 139.61, 139.28, 137.29, 135.00, 133.98. 133.32, 132.95, 130.64, 128.71, 128.39, 128.11, 127.22, 126.22, 126.00, 125.62, 124.90, 123.04, 118.00, 41.41, 18.23. A white solid (71%). 1 H NMR (400 MHz; CD 3 OD): δ 7.88 (1H, s, CH), 7.74 (1H, s, CH), 7.38–7.50 (6H, m, ArCH and CH), 7.14–7.21 (8H, m, ArCH), 6.26 (1H, d, J = 16.00 Hz, CH), 4.49 (2H, s, CH 2 ). 13 C NMR (100 MHz; CD 3 OD): δ 167.00, 165.00, 141.09, 141.07, 139.61, 139.28, 137.29, 135.00, 133.98, 133.32, 132.95, 130.64, 128.71, 128.39, 128.11, 127.22, 126.22, 126.00, 125.62, 124.90, 123.04, 118.00, 41.41, 18.23. HRMS–ESI ( m/z ): [M + H] + calcd for C 30 H 26 NO 3 , 448.1907; found, 448.1918. A transparent oil (74%). 1 H NMR (400 MHz; CDCl 3 ): δ 8.17 (1H, s, ArCH). 7.88 (1H, s, ArCH),7.74 (1H, s, CH), 7.38–7.62 (10H, m, ArCH), 7.14–7.21 (7H, m, ArCH), 6.26 (1H, d, J = 16.0 Hz, CH), 4.49 (2H, s, CH 2 ), 3.89 (3H, s, CH 3 ), 2.32 (3H, s, CH 3 ). 13 C NMR (100 MHz; CDCl 3 ): δ 141.09, 141.07, 139.61, 139.28, 137.29, 135.00, 133.98, 133.32, 132.95, 130.64, 128.71, 128.39, 128.11, 127.22, 126.22, 126.00, 125.62, 124.90, 123.04, 118.00, 41.41, 18.23. A white solid (71%). 1 H NMR (400 MHz; CD 3 OD): δ 2.32 (3H, s, CH 3 ), 4.49 (2H, s, CH 2 ), 6.26 (1H, d, J = 16.0 Hz, CH), 7.14–7.21 (7H, m, ArCH), 7.38–7.62 (10H, m, ArCH), 7.74 (1H, s, CH), 7.88 (1H, s, ArCH), 8.17 (1H, s, ArCH). 13 C NMR (100 MHz; CD 3 OD): δ 18.23, 41.41, 118.00, 123.04, 124.90, 125.62, 126.00, 126.22, 127.22, 128.11, 128.39.128.71, 130.64, 132.95, 133.32, 133.98. 135.00, 137.29, 139.28, 139.61, 141.07, 141.09, 166.71. 167.07. HRMS–ESI ( m/z ): [M + H] + calcd for C 36 H 29 N 2 O 3 , 537.2173; found, 537.2188. A transparent oil (72%). 1 H NMR (400 MHz; CDCl 3 ): δ 2.32 (3H, s, CH 3 ), 3.89 (3H, s, CH 3 ), 4.64 (2H, d, J = 7.4 Hz, CH 2 ), 6.54 (1H, s, CH), 7.15 (2H, d, J = 7.8 Hz, ArCH), 7.24 (2H, d, J = 8.0 Hz, ArCH), 7.28–7.39 (5H, m, ArCH), 7.53–7.54 (4H, m, CH), 7.63 (1H, s, ArCH), 7.83 (1H, s, ArCH), 7.98 (1H, s, ArCH). 8.01 (1H, s, ArCH). 13 C NMR (100 MHz; CDCl 3 ): δ 63.7, 69.9, 114.4, 118.6, 119.3, 122.1, 122.1, 124.6, 128.9, 129.2, 133.6, 137.8, 142.9, 159.5, 166.5. A yellow solid (72%). 1 H NMR (400 MHz; CD 3 OD): δ 2.32 (3H, s, CH 3 ), 4.64 (2H, d, J = 7.4 Hz, CH 2 ), 6.54 (1H, s, CH), 7.15 (2H, d, J = 7.8 Hz, ArCH), 7.24 (2H, d, J = 8.0 Hz, ArCH), 7.28–7.39 (5H, m, ArCH), 7.53–7.54 (4H, m, ArCH), 7.63 (1H, s, CH), 7.83 (1H, s, ArCH), 7.98 (1H, s, ArCH), 8.01 (1H, s, ArCH), 13 C NMR (100 MHz; CD 3 OD): δ 63.7, 69.9, 114.4, 118.6, 119.3, 122.1, 122.1, 124.6, 128.9, 129.2, 133.6, 137.8, 142.9, 159.5, 166.5. HRMS–ESI ( m/z ): [M + H] + calcd for C 26 H 22 NO 3 , 396.1600; found, 396.1603. A transparent oil (82%). 1 H NMR (400 MHz; CDCl 3 ): δ 1.20 (3H, s, CH 3 ), 1.28–1.32 (2H, m, J = 7.4 Hz, CH 2 ), 1.40 (1H, d, J = 7.4 Hz, CH), 2.39 (3H, s, CH 3 ), 3.89 (3H, s, CH 3 ), 4.64 (2H, d, J = 7.4 Hz, CH 2 ), 6.54 (1H, s, CH), 7.19–7.28 (7H, m, ArCH), 7.50–7.76 (7H, m, ArCH and CH), 7.02 (1H, s, ArCH), 7.08 (1H, s, ArCH), 7.89 (1H, s, ArCH). 13 C NMR (100 MHz; CDCl 3 ): δ 29.2, 33.4, 36.0, 61.9, 87.8, 127.3, 128.9, 132.5, 135.6, 136.1, 136.3, 138.9, 139.2, 145.6, 148.7, 150.1, 150.5, 151.5, 152.1, 153.1, 163.3, 180.2. A yellow solid (57%). 1 H NMR (400 MHz; CD 3 OD): δ 1.18 (3H, s, CH 3 ), 1.26–1.30 (2H, m, J = 7.4 Hz, CH 2 ), 1.38 (1H, d, J = 7.4 Hz, CH), 2.37 (3H, s, CH 3 ), 3.87 (3H, s, CH 3 ), 4.62 (2H, d, J = 7.4 Hz, CH 2 ), 6.52 (1H, s, CH), 7.17–7.26 (7H, m, ArCH), 7.48–7.74 (7H, m, ArCH and CH), 7.02 (1H, s, ArCH), 7.08 (1H, s, ArCH), 7.89 (1H, s, ArCH). 13 C NMR (100 MHz; CD 3 OD): δ 29.2, 33.4, 36.0, 61.9, 87.8, 127.3, 128.9, 132.5, 135.6, 136.1, 136.3, 138.9, 139.2, 145.6, 148.7, 150.1, 150.5, 151.5, 152.1, 153.1, 163.3, 180.2. HRMS–ESI ( m/z ): [M + H] + calcd for C 27 H 28 NO 3 , 414, 2069; found, 414.2073. A yellow solid (78%). 1 H NMR (400 MHz; CDCl 3 ): δ 2.32 (3H, s, CH 3 ), 2.64 (2H, t, J = 7.4 Hz, CH 2 ), 3.68–3.87 (8H, m, CH 2 ), 3.89 (3H, s, CH 3 ), 4.58 (2H, t, J = 7.4 Hz, CH 2 ), 6.65 (1H, d, J = 15.9 Hz, CH), 7.15–7.28 (4H, m, ArCH), 7.50–7.76 (5H, m, ArCH and CH), 8.02 (1H, s, ArCH), 8.08 (1H, s, ArCH), 8.89 (1H, s, CH). 13 C NMR (100 MHz; CDCl 3 ): δ 28.2, 31.4, 39.0, 64.9, 80.8, 120.3, 120.9, 122.5, 125.6, 126.1, 126.3, 128.9, 129.2, 135.6, 138.7, 140.1, 140.5, 141.5, 142.1, 143.1, 166.3, 181.2. A yellow solid (60%). 1 H NMR (400 MHz; CD 3 OD): δ 2.32 (3H, s, CH 3 ), 2.64 (2H, t, J = 7.4 Hz, CH 2 ), 3.68–3.87 (8H, m, CH 2 ), 4.58 (2H, t, J = 7.4 Hz, CH 2 ), 6.65 (1H, d, J = 15.9 Hz, CH), 7.15–7.28 (4H, m, ArCH), 7.50–7.76 (5H, m, ArCH and CH), 8.02 (1H, s, ArCH), 8.08 (1H, s, ArCH), 8.89 (1H, s, CH). 13 C NMR (100 MHz; CD 3 OD): δ 28.2, 31.4, 39.0, 64.9, 80.8, 120.3, 120.9, 122.5, 125.6, 126.1, 126.3, 128.9, 129.2, 135.6, 138.7, 140.1, 140.5, 141.5, 142.1, 143.1, 166.3, 181.2. ESI-HRMS ( m/z ): [M + H] + calcd for C 29 H 30 FN 2 O 4 , 489.2184; found, 489.2138. A transparent oil (75%). 1 H NMR (400 MHz; CDCl 3 ): δ 1.72 (3H, s, CH 3 ), 2.34 (3H, s, CH 3 ), 2.96 (2H, t, J = 6.9 Hz, CH 2 ), 3.76 (2H, d, J = 6.6 Hz, CH 2 ), 3.89 (3H, d, s, CH 3 ), 5.09–5.15 (1H, m, CH), 5.89–5.99 (1H, m, CH), 6.40 (1H, d, J = 15.9 Hz, CH), 7.25–7.30 (2H, m, ArCH), 7.34–7.38 (2H, m, ArCH), 7.44 (1H, s, ArCH), 7.52 (1H, s, ArCH), 7.56 (1H, d, J = 16.0 Hz, CH), 7.63 (1H, s, ArCH). 13 C NMR (100 MHz; CDCl 3 ): δ 28.1, 35.6, 39.8, 41.7, 80.7, 116.9, 121.3, 126.6, 128.5, 128.7, 128.8, 129.6, 130.8, 135.2, 135.5, 136.2, 138.8, 141.3, 142.4, 165.9, 167.0. A white solid (83%). 1 H NMR (400 MHz; CD 3 OD): δ 1.72 (3H, s, CH 3 ), 2.34 (3H, s, CH 3 ), 2.97 (2H, t, J = 6.9 Hz, CH 2 ), 3.77 (2H, d, J = 6.6 Hz, CH 2 ), 5.09–5.15 (1H, m, CH), 5.89–5.99 (1H, m, CH), 6.40 (1H, d, J = 15.9 Hz, CH), 7.25–7.30 (2H, m, ArCH), 7.34–7.38 (2H, m, ArCH), 7.44 (1H, s, ArCH), 7.52 (1H, s, ArCH), 7.56 (1H, d, J = 16.0 Hz, CH), 7.63 (1H, s, ArCH). 13 C NMR (100 MHz; CD 3 OD): δ 28.1, 35.6, 39.8, 41.7, 80.7, 116.9, 121.3, 126.6, 128.5, 128.7, 128.8, 129.6, 130.8, 135.2, 135.5, 136.2, 138.8, 141.3, 142.4, 165.9, 167.0. HRMS–ESI ( m/z ): [M + H] + calcd for C 22 H 24 NO 3 , 350.1756; found, 350.1762. A transparent oil (75%). 1 H NMR (400 MHz; CDCl 3 ): δ 1.77 (3H, s, CH 3 ), 2.34 (3H, s, CH 3 ), 2.96 (2H, t, J = 6.9 Hz, CH 2 ), 3.76 (2H, d, J = 6.6 Hz, CH 2 ), 3.89 (3H, d, s, CH 3 ), 5.09–5.15 (1H, m, CH), 5.89–5.99 (1H, m, CH), 6.40 (1H, d, J = 15.97.6 Hz, CH), 7.25–7.30 (2H, m, ArCH), 7.34–7.38 (2H, m, ArCH), 7.44 (1H, s, ArCH), 7.52 (1H, s, ArCH), 7.56 (1H, d, J = 16.07.6 Hz, CH), 7.63 (1H, s, ArCH). 13 C NMR (100 MHz; CDCl 3 ): δ 28.1, 35.6, 39.8, 41.7, 80.7, 116.9, 121.3, 126.6, 128.5, 128.7, 128.8, 129.6, 130.8, 135.2, 135.5, 136.2, 138.8, 141.3, 142.4, 165.9, 167.0. White solid (87%). 1 H NMR (400 MHz; CD 3 OD): δ 1.77 (3H, s, CH 3 ), 2.34 (3H, s, CH 3 ), 2.96 (2H, t, J = 6.9 Hz, CH 2 ), 3.76 (2H, d, J = 6.6 Hz, CH 2 ), 5.09–5.15 (1H, m, CH), 5.89–5.99 (1H, m, CH), 6.40 (1H, d, J = 15.97.6 Hz, CH), 7.25–7.30 (2H, m, ArCH), 7.34–7.38 (2H, m, ArCH), 7.44 (1H, s, ArCH), 7.52 (1H, s, ArCH), 7.56 (1H, d, J = 16.07.6 Hz, CH), 7.63 (1H, s, ArCH). 13 C NMR (100 MHz; CD 3 OD): δ 28.1, 35.6, 39.8, 41.7, 80.7, 116.9, 121.3, 126.6, 128.5, 128.7, 128.8, 129.6, 130.8, 135.2, 135.5, 136.2, 138.8, 141.3, 142.4, 165.9, 167.0. HRMS (ESI): ( m / z ): [M + H] + calcd for C 22 H 24 NO 3 , 350.1756; found, 350.1769. A transparent oil (79%). 1 H NMR (400 MHz; CDCl 3 ): δ 0.89 (2H, m, J = 7.4 Hz, CH 2 ), 1.28 (3H, s, J = 7.4 Hz, CH 2 and CH), 2.32 (3H, s, CH 3 ), 3.89 (3H, s, CH 3 ), 4.64 (2H, d, J = 7.4 Hz, CH 2 ), 6.54 (1H, s, CH), 7.15–7.22 (2H, d, J = 7.8 Hz, ArCH), 7.34 (2H, d, J = 8.0 Hz, ArCH), 7.56 (1H, d, J = 8.0, CH), 7.73 (1H, s, ArCH), 7.83 (1H, s, ArCH), 7.98 (1H, d, J = 8.0, ArCH). 13 C NMR (100 MHz; CDCl 3 ): δ 16.5, 24.4, 33.4, 42.4, 119.3, 119.3, 120.7, 122.0, 123.8, 128.7, 128.8, 130.7, 133.1, 134.8, 134.9, 138.1, 143.3, 143.8, 148.1, 168.2. A white solid (71%). 1 H NMR (400 MHz; CD 3 OD): δ 0.89 (2H, m, J = 7.4 Hz, CH 2 ), 1.28 (3H, s, J = 7.4 Hz, CH 2 and CH), 2.32 (3H, s, CH 3 ), 4.64 (2H, d, J = 7.4 Hz, CH 2 ), 6.54 (1H, s, CH), 7.15 (2H, d, J = 7.8 Hz, ArCH), 7.24 (2H, d, J = 8.0 Hz, ArCH), 7.56 (1H, d, J = 8.0, CH), 7.78 (1H, s, ArCH), 7.83 (1H, s, ArCH), 7.98 (1H, d, J = 8.0, ArCH). 13 C NMR (100 MHz; CD 3 OD): δ 16.5, 24.4, 33.4, 42.4, 119.3, 119.3, 120.7, 122.0, 123.8, 128.7, 128.8, 130.7, 133.1, 134.8, 134.9, 138.1, 143.3, 143.8, 148.1, 168.2. HRMS–ESI ( m/z ): [M + H] + calcd for C 23 H 22 NO 3 , 360.1600; found, 360.3633. A 250 mL round bottom flask was charged with 3-bromo-5-hydroxybenzoic acid 6 (5 g, 23.0 mmol), PPh 3 (1.21 g, 4.6 mmol), and Pd(OAc) 2 (258.63 mg, 1.2 mmol); the flask was flushed with N 2 , and 100 mL toluene was added to the flask, followed by the addition of Et 3 N (9.6 mL, 69.1 mmol) and methyl acrylate (3.1 mL, 34.6 mmol). The reaction mixture was heated at 110 °C overnight, and the contents were filtered on celite, the filtrate was evaporated under vacuum, and the crude product was purified via column chromatography (40% EtOAc in hexanes) to obtain ( E )-3-hydroxy-5-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid 7 in 68% yield. To a solution of compound 7 (5.1 g, 22.9 mmol) in 50 mL DCM/DMF (1:1), EDC (4.28 g, 27.5 mmol) and HOBt (3.72 g, 27.5 mmol) was added. The reaction mixture was stirred at room temperature for 15 min, followed by the addition of p -tolylmethanamine (3.5 mL, 27.5 mmol) and Et 3 N (9.6 mL, 68.9 mmol). The reaction was stirred at room temperature overnight, and the contents were evaporated under vacuum and resuspended in water, filtered, and washed with water and hexanes. The solid obtained was purified via column chromatography (50% EtOAc/Hexanes) to get methyl ( E )-3-(3-hydroxy-5-((4-methylbenzyl)carbamoyl)phenyl)acrylate 8 in 79% yield. To a solution of 8 (500 mg, 1.54 mmol) in 20 mL acetonitrile, 1-bromo-3-methylbut-2-ene (0.36 mL, 3.1 mmol) was added, followed by K 2 CO 3 (424.8 mg, 3.1 mmol). The reaction was stirred at reflux overnight, allowed to cool, and filtered and evaporated under vacuum. The crude product was purified via column chromatography (50% EtOAc/hexanes) to obtain methyl ( E )-3-(3-((4-methylbenzyl)carbamoyl)-5-((3-methylbut-2-en-1-yl)oxy)-phenyl)acrylate 9 . To a solution of compound 9 in 10 mL MeOH-THF (1:1), NaOH (91.5 mg, 2.3 mmol) was added, and the reaction was stirred at 60 °C for 4 h. The reaction was quenched in 6N HCl, and the resulting solid was filtered and washed with water and hexanes. The crude product was purified via column chromatography using MeOH-DCM to obtain ( E )-3-(3-((4-methylbenzyl)-carbamoyl)-5-((3-methylbut-2-en-1-yl)oxy)phenyl)acrylic acid 10 as a white solid (312 mg, yield 52%). 1 H NMR (400 MHz, DMSO- d 6 ): δ 12.52 (1H, s, OH), 9.08 (1H, t, J = 4.9 Hz, NH), 7.81 (1H, s, ArCH), 7.62–7.57 (1H, m, ArCH), 7.49 (1H, s, ArCH), 7.42 (1H, s, ArCH), 7.23 (2H, d, J = 6.3 Hz, ArCH), 7.14 (2H, d, J = 6.7 Hz, ArCH), 6.65 (1H, dd, J = 2.3, 16.0 Hz, CH), 5.45 (1H, t, J = 5.4 Hz, CH), 4.62 (2H, d, J = 6.5 Hz, CH 2 ), 4.45 (2H, d, J = 5.2 Hz, CH 2 ), 2.27 (3H, s, CH 3 ), 1.74 (6H, d, J = 8.9 Hz, CH 3 ). 13 C NMR (100 MHz, DMSO- d 6 ): δ 167.9, 165.7, 159.3, 143.7, 138.0, 136.9, 136.5, 136.3, 136.2, 129.3, 127.8, 121.0, 120.0, 119.2, 117.6, 115.7, 65.2, 42.9, 25.9, 21.1, 18.5. HRMS–ESI ( m/z ): [M + H] + calcd for C 23 H 25 NO 4 , 380.1857; found, 380.1846. To a stirred solution of ester 3 (0.33 g, 0.85 mmol) in a mixture of THF/MeOH (4:1, 20 mL) was added aqueous 1 N NaOH (2.5 mL, 2.5 mmol). The mixture was stirred at 60 °C for 3 h. The solvent was evaporated in vacuo, and the pH of the reaction mixture was adjusted to 2–4 with 1 N HCl. The mixture was extracted with CH 2 Cl 2 (3 × 50 mL). The combined organic layers were washed with brine (40 mL), dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using CH 2 Cl 2 /MeOH as the eluent to afford the titled compound as a white solid (0.25 g, yield 78%). 1 H NMR (500 MHz, DMSO- d 6 ): δ 12.58 (1H, br s, OH), 9.13 (1H, t, J = 6.0 Hz, NH), 8.22 (1H, s, ArCH), 8.10 (1H, s, ArCH), 8.06 (1H, s, ArCH), 7.61 (1H, d, J = 16.0 Hz, CH), 7.22 (2H, d, J = 8.0 Hz, ArCH), 7.14 (2H, d, J = 8.0 Hz, ArCH), 7.69 (1H, d, J = 16.0 Hz, CH), 4.45 (2H, d, J = 6.0 Hz, CH 2 ), 2.28 (3H, s, CH 3 ). 13 C NMR (125 MHz, DMSO- d 6 ): δ 167.68, 164.57, 141.98, 137.36, 137.16, 136.60, 136.41, 133.94, 131.73, 129.34, 127.83, 125.66, 122.80, 122.50, 43.06, 21.14. HRMS–ESI ( m/z ): [M + H] + calcd for C 18 H 17 BrNO 3 , 374.0386; found, 374.0390. To a stirred solution of acid 11 (0.1 g, 0.27 mmol) in an anhydrous THF (8 mL) was added thionyl chloride (78 μ L, 1.07 mmol). The mixture was stirred at 70 °C for 3 h. The solvent was evaporated in vacuo, and the residue was dissolved in anhydrous CH 2 Cl 2 (10 mL) and treated with 30% aq ammonium hydroxide solution (140 μ L, 1.07 mmol) at 0 °C. The pH of the reaction mixture was adjusted to 5–7 with 1N HCl and extracted with CH 2 Cl 2 (3 × 30 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure to afford a crude compound. The residue was purified by silica gel column chromatography using CH 2 Cl 2 /MeOH as the eluent to afford the titled compound as a white solid (65 mg, yield 65%). 1 H NMR (500 MHz, CD 3 OD): δ 8.12 (1H, s, ArCH), 7.94 (2H, d, J = 1.5 Hz, ArCH), 7.56 (1H, d, J = 16.0 Hz, CH), 7.36 (2H, d, J = 8.5 Hz, ArCH), 7.27 (2H, d, J = 8.5 Hz, ArCH), 6.84 (1H, d, J = 16.0 Hz, 1H), 4.66 (2H, s, CH 2 ), 2.38 (3H, s, CH 3 ). 13 C NMR (125 MHz, CD 3 OD): δ 168.30, 162.85, 138.19, 138.13, 137.71, 134.47, 131.64, 131.20, 131.05, 129.33, 127.71, 126.06, 124.14, 123.02, 46.4, 19.78. HRMS–ESI ( m/z ): [M + H] + calcd for C 18 H 18 BrN 2 O 2 , 373.0546; found, 373.0748. To a stirred solution of acid 5n (0.05 g, 0.1 mmol) in THF (5 mL) was added thionyl chloride (30 μ L, 0.4 mmol). The mixture was stirred at 70 °C for 3 h. After being cooled to room temperature, the solvent was evaporated in vacuo. The residue was dissolved in anhydrous CH 2 Cl 2 (10 mL). The mixture was cooled to 0 °C and 30% aqueous ammonium hydroxide (60 μ L, 0.4 mmol). The reaction mixture was stirred at 0 °C for 1 h, and the pH of the reaction mixture was adjusted to 5–7 with 1N HCl. The mixture was extracted with CH 2 Cl 2 (3 × 25 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using CH 2 Cl 2 /MeOH as the eluent to afford the titled compound as a white solid (28 mg, yield 56%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.88 (1H, s, ArCH), 7.84 (1H, s, ArCH), 7.55 (1H, s, ArCH), 7.52 (1H, d, J = 16.0 Hz, CH), 7.43 (1H, br s, NH), 7.39 (2H, d, J = 8.5 Hz, ArCH), 7.32 (2H, t, J = 8.0 Hz, ArCH), 7.21 (2H, d, J = 7.5 Hz, ArCH), 7.11 (1H, t, J = 7.5 Hz, ArCH), 7.07 (2H, d, J = 7.5 Hz, ArCH), 6.99 (2H, d, J = 8.0 Hz, ArCH), 6.96 (2H, d, J = 8.0 Hz, ArCH), 6.52 (1H, d, J = 16.0 Hz, CH), 6.43 (1H, br s, NH), 6.24 (1H, br s, NH), 4.55 (2H, d, J = 4.5 Hz, CH 2 ), 2.26 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 168.00, 167.13, 157.50, 156.77, 141.44, 141.10, 137.27, 135.64, 135.45, 135.18, 134.29, 129.87, 129.42, 128.43, 127.99, 126.78, 126.78, 124.52, 123.65, 119.14, 118.99, 43.97, 21.09. HRMS–ESI ( m/z ): [M + H] + calcd for C 30 H 27 N 2 O 3 , 463.2016; found, 463.2018. To a stirred suspension of 3-bromo-5-iodo- N -(4-methylbenzyl)benzamide 2 (0.15 g, 0.35 mmol) in anhydrous toluene (15 mL) were added N , N -dimethylacrylamide (50 μ L, 0.45 mmol), Pd(OAc) 2 (8.0 mg. 0.035 mmol), and triethylamine (0.1 mL, 0.7 mmol). The mixture was heated at 90 °C for 12 under a N 2 atmosphere. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL). The filtrate was washed with 1N HCl (25 mL) and brine (25 mL). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by Biotage select flash column chromatography using EtOAc/hexanes as the eluent to afford the titled compound as an off-white solid (90 mg, yield 50%). 1 H NMR (400 MHz, CD 3 OD): δ 8.01 (1H, s, ArCH), 7.96 (1H, s, ArCH), 7.86 (1H, s, ArCH), 7.44 (1H, d, J = 15.2 Hz, CH), 7.21 (2H, d, J = 8.0 Hz, ArCH), 7.15 (1H, d, J = 15.2 Hz, CH), 7.10 (2H, d, J = 8.0 Hz, ArCH), 4.51 (2H, s, CH 2 ), 3.17 (3H, s, NCH 3 ), 3.01 (3H, s, NCH 3 ), 2.28 (3H, s, CH 3 ). 13 C NMR (100 MHz, MeOH- d 4 ): δ 165.26, 164.70, 138.10, 136.12, 135.12, 135.10, 133.91, 131.58, 129.44, 127.31, 125.80, 123.44, 121.17, 118.54, 41.65, 35.03, 33.44, 18.35. HRMS–ESI ( m/z ): [M + H] + calcd for C 20 H 22 BrN 2 O 2 , 401.0859; found, 401.0858. To a stirred suspension of 3-bromo-5-iodo- N -(4-methylbenzyl)-benzamide 2 (1.5 g, 3.48 mmol) in anhydrous toluene (30 mL) were added acrylonitrile (0.34 mL, 5.25 mmol), Pd(OAc) 2 (78 mg. 0.35 mmol), triphenylphosphine (91 mg, 0.35 mmol), and triethylamine (1.46 mL, 10.5 mmol). The mixture was heated at 90 °C for 12 under a N 2 atmosphere. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL). The filtrate was washed with 1 N HCl (50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using EtOAc/hexanes as the eluent to afford the titled compound as a white solid (0.62 g, yield 50%). 1 H NMR (400 MHz, CDCl 3 ): δ 7.91 (1H, d, J = 1.2 Hz, ArCH), 7.84 (1H, d, J = 1.2 Hz, ArCH), 7.71 (1H, d, J = 1.2 Hz, ArCH), 7.35 (1H, d, J = 16.8 Hz, CH), 7.26 (2H, d, J = 7.6 Hz, ArCH), 7.20 (2H, d, J = 7.6 Hz, ArCH), 6.41 (1H, br s, NH), 5.97 (1H, dd, J = 16.8, 1.2 Hz, CH), 4.61 (2H, d, J = 5.2 Hz, CH 2 ), 2.38 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): 164.76, 147.88, 137.80, 137.22, 135.80, 134.42, 132.68, 131.88, 129.61, 128.09, 124.89, 123.47, 117.17, 99.45, 44.27, 21.14. HRMS–ESI ( m / z ): [M + H] + calcd for C 18 H 16 BrN 2 O, 355.0441; found, 355.0444. To a stirred solution of aryl bromide 15 (0.20 g, 0.56 mmol) and (4-phenoxyphenyl)boronic acid (0.24 g, 1.1 mmol) in anhydrous DMF (10 mL) were added Pd(dppf)Cl 2 · CH 2 Cl 2 (46 mg, 0.056 mmol), and CS 2 CO 3 (0.55 g, 1.7 mmol). The mixture was stirred at 100 °C for 12 h under a N 2 atmosphere. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL). The filtrate was washed with 1 N HCl (30 mL) and brine (50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using EtOAc/hexanes as the eluent to afford the titled compound as a white solid (0.17 g, yield 67%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.98 (1H, s, ArCH), 7.86 (1H, s, ArCH), 7.73 (1H, s, ArCH), 7.55 (2H, d, J = 8.5 Hz, ArCH), 7.48 (1H, d, J = 17.0 Hz, CH), 7.41–7.38 (3H, m, ArCH), 7.29 (2H, d, J = 8.0 Hz, ArCH), 7.20 (2H, d, J = 8.0 Hz, ArCH), 7.18 (1H, t, J = 7.0 Hz, ArCH), 7.11 (2H, d, J = 8.5 Hz, ArCH), 7.08 (2H, d, J = 8.5 Hz, ArCH), 6.48 (1H, br s, NH), 6.03 (1H, d, J = 17.0 Hz, CH), 4.65 (2H, d, J = 5.5 Hz, CH 2 ), 2.38 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.24, 157.94, 156.68, 149.47, 142.27, 137.68, 136.13, 134.73, 134.53, 133.92, 129.91, 129.58, 128.58, 128.10, 127.70, 124.31, 123.80, 119.27, 119.10, 117.67, 98.19, 44.20, 21.13. HRMS–ESI ( m/z ): [M + H] + calcd for C 30 H 25 NO 2 , 445.1911; found, 445.1911. To a stirred solution of aryl bromide 15 (0.3 g, 0.84 mmol) in anhydrous DMF (10 mL) were added prenyltributyltin (0.55 mL, 1.63 mmol), and Pd(Ph 3 ) 4 (98 mg, 0.084 mmol). The mixture was heated at 100 °C for 24 h under a N 2 atmosphere. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL. The filtrate was washed with a 10% aqueous KF solution (2 × 50 mL) and 1 N NaOH three times (50 mL). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using EtOAc/hexanes as the eluent to afford the titled compound as a white solid (0.14 g, yield 48%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.74 (1H, s, ArCH), 7.68 (1H, s, ArCH), 7.34 (1H, s, ArCH), 7.23 (1H, d, J = 16.5 Hz, CH), 7.22 (2H, d, J = 7.5 Hz, ArCH), 7.14 (3H, m, ArCH), 5.86 (1H, d, J = 16.5 Hz, CH), 5.28 (1H, t, J = 7.5 Hz, NH), 4.55 (2H, d, J = 6.0 Hz, CH 2 ), 3.37 (2H, d, J = 7.5 Hz, CH 2 ), 2.34 (3H, s, CH 3 ), 1.77 (3H, s, CH 3 ), 1.72 (3H, s, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.65, 149.86, 143.55, 137.28, 135.48, 135.09, 134.08, 133.92, 130.20, 129.72, 129.41, 127.89, 123.53, 121.70, 117.98, 97.27, 43.89, 34.00, 25.77, 21.12, 17.96. HRMS–ESI ( m/z ): [M + H] + calcd for C 23 H 25 N 2 O, 345.1961; found, 345.1965. A mixture of 3-bromo-5-iodobenzoic acid 1 (3.2 g, 9.79 mmol) and thionyl chloride (15 mL) was refluxed for 3 h. After being cooled to room temperature, the solvent was evaporated to dryness under reduced pressure. The residue was dissolved in anhydrous CH 2 Cl 2 (30 mL) and cooled to 0°, followed by addition of triethylamine (4.1 mL, 29.7 mmol), and ( R )-1-phenylpropan-1-amine (1.6 g, 11.88 mmol). The reaction mixture was stirred at room temperature for 8 h. The reaction mixture was washed with aqueous saturated NaHCO 3 (2 × 50 mL) and 1 N HCl (2 × 50 mL). The CH 2 Cl 2 solution was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure to afford the tilted compound as a white solid (3.52 g, yield 81%). 1 H NMR (500 MHz, CDCl 3 + CD 3 OD): δ 7.98 (1H, d, J = 4.0 Hz, ArCH), 7.86 (1H, d, J = 3.5 Hz, ArCH), 7.82 (1H, d, J = 3.5 Hz, ArCH), 7.28–7.25 (4H, m, ArCH), 7.21–7.18 (1H, m, ArCH), 4.91 (1H, t, J = 7.5 Hz, CH), 1.90–1.78 (2H, m, CH 2 ), 0.90–0.87 (3H, m, CH 3 ). 13 C NMR (125 MHz, CDCl 3 + CD 3 OD): δ 164.58, 142.06, 141.92, 137.88, 134.91, 129.76, 128.53, 127.31, 126.69, 122.93, 94.21, 55.87, 28.77, 10.97. To a stirred suspension of 17 (1.0 g, 2.25 mmol) in anhydrous toluene (30 mL) were added methyl acrylate (260 μ L, 2.92 mmol), Pd(OAc) 2 (50 mg, 0.23 mmol), triphenylphosphine (60 mg, 0.23 mmol), and triethylamine (1.0 mL, 6.75 mmol). The mixture was stirred at 90 °C for 12 h. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL). The filtrate was washed with aqueous 1 N HCl (50 mL) and brine (50 mL). The aqueous layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using EtOAc/hexanes as the eluent to afford the titled compound as an off-white solid (0.58 g, yield 64%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.84 (1H, s, ArCH), 7.77 (1H, s, ArCH), 7.62 (1H, s, ArCH), 7.46 (1H, d, J = 16.0 Hz, CH), 7.39 (1H, br d, J = 8.0 Hz, NH), 7.33–7.28 (4H, m, ArCH), 7.25–7.22 (1H, m, ArCH), 6.36 (1H, d, J = 16. 0 Hz, CH), 5.01 (1H, m, CH), 3.78 (3H, s, OCH 3 ), 1.98–1.86 (2H, m, CH 2 ), 0.93 (3H, t, J = 7.5 Hz, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 166.71, 165.19, 142.17, 141.92, 137.21, 136.45, 133.08, 131.55, 128.64, 127.43, 126.78, 125.45, 123.01, 120.18, 55.94, 51.94, 28.99, 11.05. To a stirred solution of 18 (0.20 g, 0.50 mmol) and (4-isopropylphenyl)boronic acid (0.16 g, 1.0 mmol) in anhydrous DMF (10 mL) were added Pd(dppf)Cl 2 ·CH 2 Cl 2 (40 mg, 0.05 mmol) and CS 2 CO 3 (0.50 g, 1.5 mmol). The mixture was stirred at 100 °C for 12 under a N 2 atmosphere. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL). The filtrate was washed with 1 N HCl (30 mL) and brine 50 (mL). The aqueous layer was extracted with EtOAc (2 × 50 mL), and the combined organic layers were dried over Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using EtOAc/hexanes as the eluent to afford the titled compound as a white solid (0.16 g, yield 72%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.98 (1H, s, ArCH), 7.86 (1H, s, ArCH), 7.79 (1H, s, ArCH), 7.73 (1H, d, J = 16.0 Hz, CH), 7.51 (2H, d, J = 8.5 Hz, ArCH), 7.40–7.28 (7H, m, ArCH), 6.72 (1H, d, J = 8.0 Hz, NH), 6.53 (1H, d, J = 16.0 Hz, CH), 5.13 (1H, m, CH), 3.82 (3H, s, OCH 3 ), 2.98 (1H, m, CH), 2.04–1.94 (2H, m, CH 2 ), 1.31 (6H, d, J = 6.5 Hz, (CH 3 )2), 0.99 (3H, t, J = 7.5 Hz, CH 3 ). 13 C NMR (100 MHz, CDCl 3 ): δ 166.69, 148.93, 142.45, 141.99, 141.44, 137.51, 135.97, 133.06, 132.84, 129.55, 129.03, 128.76, 127.59, 127.48, 127.28, 127.08, 126.81, 124.93, 124.46, 55.68, 33.86, 29.72, 29.14, 23.99, 10.98. To a stirred solution of 18 (0.20 g, 0.50 mmol) and (4-phenoxyphenyl)boronic acid (0.21 g, 1.0 mmol) in anhydrous DMF (10 mL) were added Pd(dppf)Cl 2 ·CH 2 Cl 2 (40 mg, 0.05 mmol) and CS 2 CO 3 (0.50 g, 1.5 mmol). The mixture was stirred at 100 °C for 12 under a N 2 atmosphere. After being cooled to room temperature, the reaction mixture was filtered through celite and washed with EtOAc (50 mL). The filtrate was washed with 1 N HCl (30 mL) and brine 50 (mL). The aqueous layer was extracted with EtOAc (2 × 50 mL), and the combined organic layers were dried over Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using EtOAc/hexanes as the eluent to afford the titled compound as a white solid (0.21 g, yield 86%). 1 H NMR (500 MHz, CDCl 3 ): δ 7.97 (1H, s, ArCH), 7.85 (1H, s, ArCH), 7.77 (1H, s, ArCH), 7.72 (1H, d, J = 16.0 Hz, CH), 7.52 (2H, d, J = 9.0 Hz, ArCH), 7.41–7.35 (6H, m, ArCH), 7.29 (1H, t, J = 7.0 Hz, ArCH), 7.17 (1H, t, J = 7.5 Hz, ArCH), 7.09–707 (4H, m, ArCH), 6.72 (1H, d, J = 8.0 Hz, NH), 6.52 (1H, d, J = 16.0 Hz, CH), 5.13 (1H, m, CH), 3.82 (3H, s, OCH 3 ), 2.06–1.94 (2H, m, CH 2 ), 0.99 (3H, t, J = 7.5 Hz, CH 3 ). 13 C NMR (125 MHz, CDCl 3 ): δ 167.07, 166.25, 157.68, 156.79, 143.72, 141.93, 141.79, 136.13, 135.29, 134.34, 129.89, 129.11, 128.76, 128.53, 127.53, 127.23, 126.79, 124.81, 123.70, 119.32, 119.22, 119.05, 55.70, 51.85, 29.08, 10.96. To a stirred solution of methyl ester 19a (0.13 g, 0.29 mmol) in a mixture of THF/MeOH (10 mL, 4:1) was added aqueous 1 N NaOH (0.9 mL 0.88 mmol). The mixture was stirred at 60 °C for 3 h. The solvent was evaporated in vacuo, and the pH of the reaction mixture was adjusted to 2–4 with 1 N HCl. The mixture was extracted with CH 2 Cl 2 (3 × 40 mL) and washed with brine (30 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using CH 2 Cl 2 /MeOH as the eluent to afford the titled compound as a white solid (90 mg, yield 72%). 1 H NMR (500 MHz, CDCl 3 + CD 3 OD): δ 7.89 (1H, s, ArCH), 7.88 (1H, s, ArCH), 7.67 (1H, s, ArCH), 7.62 (1H, d, J = 16.0 Hz, CH), 7.41 (2H, d, J = 8.0 Hz, ArCH), 7.26–7.14 (6H, m, ArCH), 7.12 (1H, t, J = 7.5 Hz, ArCH), 6.42 (1H, d, J = 16.0 Hz, CH), 4.93 (1H, t, J = 7.5 Hz, CH), 2.85–2.80 (1H, m, CH), 1.86–1.77 (2H, m, CH 2 ), 1.15 (6H, d, J = 6.5 Hz, (CH 3 )2), 0.85 (3H, t, J = 7.5 Hz, CH 3 ). 13 C NMR (125 MHz, CDCl 3 + CD 3 OD): δ 168.80, 167.01, 148.94, 144.24, 142.26, 142.12, 136.93, 135.71, 135.14, 129.31, 128.50, 127.46, 127.23, 126.97, 126.62, 124.78, 119.56, 55.65, 33.73, 28.85, 23.74, 10.85. HRMS–ESI ( m/z ): [M + H] + calcd for C 28 H 30 NO 3 , 428.2220; found, 428.2225. To a stirred solution of methyl ester 19b (0.15 g, 0.31 mmol) in a mixture of THF/MeOH (10 mL, 4:1) was added aqueous 1 N NaOH (0.9 mL 0.92 mmol). The mixture was stirred at 60 °C for 3 h. The solvent was evaporated in vacuo, and the pH of the reaction mixture was adjusted to 2–4 with 1 N HCl. The mixture was extracted with CH 2 Cl 2 (3 × 40 mL) and washed with brine (30 mL). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using CH 2 Cl 2 /MeOH as the eluent to afford the titled compound as a white solid (0.12 g, yield 86%). 1 H NMR (500 MHz, CDCl 3 + CD 3 OD): δ 7.87 (1H, s, ArCH), 7.80 (1H, s, ArCH), 7.64 (1H, s, ArCH), 7.56 (1H, d, J = 16.0 Hz, CH), 7.42 (2H, d, J = 8.5 Hz, ArCH), 7.26–7.14 (6H, m, ArCH), 7.07 (1H, t, J = 7.5 Hz, ArCH), 6.96 (1H, t, J = 7.5 Hz, ArCH), 6.91–6.86 (4H, m, ArCH), 6.38 (1H, d, J = 16.0 Hz, CH), 4.86 (1H, t, J = 7.5 Hz, CH), 1.84–1.73 (2H, m, CH 2 ), 0.81 (3H, t, J = 7.5 Hz, CH 3 ). 13 C NMR (125 MHz, CDCl 3 + CD 3 OD): δ 172.81, 171.22, 161.50, 160.72, 148.12, 146.33, 145.53, 139.75, 139.19, 138.42, 133.72, 133.11, 132.40, 131.46, 131.10, 130.60, 128.86, 127.53, 123.64, 123.00, 122.86, 59.79, 32.79, 14.86. HRMS–ESI ( m/z ): [M + H] + calcd for C 31 H 28 NO 4 , 478.2013; found, 478.2014. ( S )-(+)-1,2,3,4-tetrahydro-1-naphthol ( S -tetralol) was purchased from Sigma-Aldrich (St. Louis, MO). Nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP + ) were purchased from Roche Diagnostics (Indianapolis, IN). Homogeneous recombinant enzymes AKR1C1, AKR1C2, AKR1C3, and AKR1C4 were prepared and purified as previously described. 11 The specific activities of the enzymes were as follows: AKR1C1, AKR1C2, and AKR1C3 for the oxidation of S -tetralol are 1.6, 1.5, and 3.5 μ mol min −1 mg −1 , respectively; and for the oxidation of androsterone, AKR1C4 had a specific activity of 0.32 μ moles/min/mg. Assay of enzyme activity: The dehydrogenase activities of AKR1C1, AKR1C2, and AKR1C3 were determined by measuring the NADH formation at 340 nm using a Beckman DU640 spectrophotometer. A typical assay solution contained 100 mM potassium phosphate pH 7.0, 2.3 mM NAD + , 3.0 mM ( S )-(+)-1,2,3,4-tetrahydro-1-naphthol ( S -tetralol), 4% acetonitrile (v/v). The mixtures were incubated at 37 °C for 3 min followed by addition of a serial dilution of AKR1C1, AKR1C2, or AKR1C3 solution to obtain a final volume of 1 mL to initiate the reaction. For AKR1C4, 75 μ M androsterone was substituted for S -tetralol. After continuously monitoring for 5 min, the increase in UV absorption using different concentrations of enzyme were recorded to calculate the initial velocity and determine the enzyme specific activity. The inhibitory potency for each compound was represented by the IC 50 value and measured as described before. 52 The IC 50 value of coumarin analogues was determined by measuring their inhibition of the NADP + dependent oxidation of S -tetralol catalyzed by AKR1C1, AKR1C2, and AKR1C3 in a 96-well plate format, and the reaction was measured fluorometrically with a BIOTEK Synergy 2 Multimode plate reader (exc/emi, 340/460 nm). The assay mixture consisted of 100 mM phosphate buffer, pH 7.0, S -tetralol (in DMSO), inhibitor (in DMSO), 200 μ M NADP + , and purified recombinant enzyme to give a total volume of 200 μ L, and 4% DMSO. The concentration of S -tetralol used in the assays for each AKR1C isoform was equal to the K m value for the respective enzyme so that IC 50 values could be directly compared assuming a competitive pattern of inhibition. The K m value obtained for S -tetralol for AKR1C1, AKR1C2, AKR1C3, and AKR1C4 under the same experimental conditions are 8, 15, 165, and 25 μ M, respectively. The IC 50 value of each compound was acquired from a single experiment, with each inhibitor concentration run in quadruplicate and directly calculated by fitting the inhibition data to an equation [ y = (range)/[1 + ( I /IC 50 ) S ] + background] using Grafit 5.0 software. In this equation, the “range” is the fitted uninhibited value minus the “background,” and “ S ” is a slope factor. “ I ” is the concentration of inhibitor. The equation assumes that y falls with increasing “ I .” Molecular docking studies of AKR1C3 inhibitors to the AKR1C3·NADP + complex was carried out using Schrödinger Maestro 13.3. The AKR1C3 crystal structure was retrieved from RCSB protein data bank (PDB: 3UG8) and processed by default with the Protein Preparation Workflow panel (Schrödinger, 2022-3 version), and the prepared protein–ligand complex was defined as the binding site. The size of the docking Glide grid box was 20 Å × 20 Å × 20 Å. Based on the OPLS4 force field, the grid of the AKR1C3 crystal structure was generated. As per default options, the ligands were prepared with LigPrep tool, and docked using extra precision (XP) mode without constrained binding. The ultra-performance liquid chromatography mass spectrometry system (UPLC–MS/MS) consisted of a Shimadzu 8060NX mass spectrometer and Nexera Series UPLC (Shimadzu Scientific Instruments, Columbia, MD). Analyte separation was achieved utilizing an Acquity UPLCBEH column (C18, 2.1 × 100 mm, 1.7 μ m) equipped with an Acquity UPLC C18 guard column (Waters, Inc. Milford MA). The mobile phase consisted of water containing 0.1% formic acid (mobile phase A) and methanol (mobile phase B) at a flow rate of 0.25 mL/min operated at room temperature. The total run time was set to last 7.5 min with a gradient elution as follows: 35% B, increasing to 95% B over 3.5 min, then held constant for 3.0 min, and finally brought back to the initial condition of 35% B in 0.20 min followed by 1-min re-equilibration. The injection volume (2 μ L) was consistent for all samples. The auto-sampler chamber was maintained at 4 °C throughout the analysis. The MS/MS system was operated at unit resolution in the multiple reaction monitoring (MRM) in positive ESI mode, using precursor ion > product ion combinations of 427.20 > 105.10 for 5r and 413.15 > 265.10 m / z for 4r . The mass spectrometer source settings were optimized to the following: nebulizer gas: 2.0 L/min; heating gas: 10 L/min; drying gas: 10 L/min; interface temperature: 300 °C; desolvation line temperature: 250 °C; heat block temperature: 400 °C. Data acquisition and quantitation were performed using LabSolutions software Ver.5.99 (Shimadzu Scientific Inc, Columbia, MD). Plasma samples were prepared by spiking 5 μ L of the appropriate calibration (CC) and quality (QCs) control working stock into a 45 μ L blank mouse plasma. The concentration of the CC ranged from 0.2 to 1000 ng/mL with the final concentrations of 0.2, 0.5, 1, 5, 10, 50, 100, 500, and 1000 ng/mL. A simple protein precipitation technique using a Phree 96-well phospholipid elimination plate (Phenomenex Inc, Torrance CA.) was utilized to isolate the analyte from the plasma matrix. The CC, QCs, and study plasma samples were added to the Phree 96-well plate and spiked with 10 μ L of IS, a working solution. The precipitation of the matrix proteins was carried out using 300 μ L of ice-cold acetonitrile. The plate was again vortexed on MixMate at 950 rpm for 2 min followed by applied 5 psi positive pressure for 10 min (Resprep VM-96 Vacuum Manifold for 96-Well Plates, Catalog # 25858) (Restek; Bellefonte, PA) to collect the supernatant. Two microliters of the reconstituted sample were injected into the liquid chromatography mass spectrometry system (LC–MS/MS) for analysis. Gastrointestinal (GI) fluid stability studies were performed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). All the required media were prepared according to the USP guidelines. Mouse plasma stability studies were performed for neat-spiked and plasma spiked samples (pre-extraction) at 37 °C for 4 h. In addition to assessing the analyte stability in matrix, at 48 h, the stability study of the extracted samples was performed at 4 °C in the autosampler. Metabolic stability was assessed using mouse and human liver microsomes, (XenoTech, LLC, Lenexa, KS). Incubation of 5r and 4r with the microsome fractions was performed in triplicate utilizing a concentration of 1 μ g/mL as previously described. 53 , 54 The reaction media contained MLM and HLM (protein content, 0.5 mg/mL), 1.0 mM nicotinamide adenine dinucleotide phosphate (NADPH), 10 mM MgCl 2 , and phosphate buffer (100 mM, pH 7.4). After 5 min pre-incubation with NADPH at 37 °C, 5r and 4r were spiked at 1 μ g/mL final concentration. Incubation without the addition of NADPH was used as negative control. Serial samples (40 μ L) were collected at selected time intervals and quenched with 300 μ L of acetonitrile and then spiked with 10 μ L of internal standard (0.5 μ g/mL). All the samples were vortexed and centrifuged at 13,000 g for 15 min, and the supernatant was collected and transferred to an autosampler vial and injected (2 μ L) onto the LC–MS/MS system. Testosterone, 7-HC, and diclofenac were used as positive controls to ensure that the microsomes and incubation conditions were appropriate to conduct metabolism studies. Animal studies were approved by the University of Nebraska Medical Center (UNMC) Institutional Animal Care and Use Committee (IACUC protocol number 17-046-06-FC). BALB/c mice were purchased from Charles River Labs. Pharmacokinetic (PK) studies of 5r and 4r were conducted in BALB/c mice. Animals were housed in the University of Nebraska Medical Center animal facility, for at least 7 days prior to the experiments, in order to acclimatize the animals to the laboratory conditions, at a temperature of 23–24 °C, relative humidity of 40–70%, and 12/12 h light/dark cycles with free access to food and water. The dosing solution was made up of DMSO-Polyethylene glycol 400 (PEG400)-Propylene glycol (PG)-EtOH-Cremophor-PBS (2/20/10/10/5/53% v/v). Compound 5r or 4r (10 mg/kg) was administered separately by oral gavage. After dosing, approximately 50 μ L of blood was collected from the maxillary vein at 5, 15, and 30 min and 1, 2, 7, and 24 h (5 mice/group/per time point). A total of three blood samples were collected, with the third blood sample being a terminal collection. Plasma was separated by centrifugation at 4000 g at 4 °C for 10 min. The collected plasma samples were stored at −80 °C until analysis. A non-compartmental analysis (NCA) was performed to estimate PK parameters using Phoenix WinNonlin 8.2 (Certara Corporation, Mountain View, CA, USA). All animal experiments were carried out according to approved Institutional Animal Care and Use Committee protocols at the University of Texas Southwestern Medical Center (Dallas, TX). Five-week-old female NSG mice (low circulating testosterone) were implanted with 3 × 10 6 22Rv1 cells (ATCC, mycoplasma negative) in Matrigel. When the tumor volume reached approximately 125 mm 3 (day 11), the mice were randomly divided into three groups of six mice. One group was treated with vehicle control PO (0.5% Methocel A4M + 0.1% Tween 80) + vehicle IP (10% DMSO/10% Cremophor EL/80% D5W). One group was treated with vehicle PO + IP 25 mg/kg/day 4r in 10% DMSO/10% Cremophor EL/80% D5W. One group was treated with vehicle PO + IP 50 mg/kg/day 4r in 10% DMSO/10% Cremophor EL/80% D5W. Dosing was continued for a total of 26 days. Tumor volumes were measured twice a week with Vernier calipers, and tumor volume was calculated as (L × W 2 ) x 3.14)/6 as previously reported. 39 2–3 h after a final IP dose, the animals were humanely euthanized, and tumors were collected, weighed, and frozen in liquid nitrogen after taking pictures.

Prostate cancer (PCa) is the second leading cause of mortality in males, with the highest incidence rate of all cancers reported in the U.S. 1 The development of PCa is androgen-dependent, and androgen-deprivation therapy, achieved through chemical or surgical castration, is a mainstay of the treatment for locally advanced or metastatic disease. 2 However, within 2–3 years, castration-resistant prostate cancer (CRPC), which possess greater metastatic potential, develops in 10–20% of patients, despite castrate levels of circulating androgens. 3 CRPC is marked by molecular changes including increases in the expression of androgen synthesizing enzymes and androgen receptor splice variant formation. 4 Such adaptations lead to increased intratumoral androgen biosynthesis, along with an increase in tumor responsiveness to circulating castrate levels of androgens leading to disease progression. 5 , 6 Abiraterone acetate, which acts by inhibiting CYP17A1, an upstream enzyme in the steroid biosynthetic pathway, was approved by the FDA for CRPC treatment in 2011. 7 Enzalutamide (ENZ), is a clinically approved chemotherapeutic that exerts potent androgen receptor (AR) antagonistic activity that is an effective treatment up to stage 3 CRPC. 8 While the initial response is promising, resistance to abiraterone acetate, ENZ, 9 and related AR antagonist analogues 10 develops rapidly. Thus, the prognosis for patients with advanced PCa to CRPC is bleak. Aldo-keto reductase (AKR) 1C3, also known as type 5 17 β -hydroxysteroid dehydrogenase (17 β -HSD) and prostaglandin [PG] F2 α synthase, is a member of the AKR1C subfamily, a member of a superfamily of NAD(P)H-linked oxidoreductases 11 , 12 that reduce ketones on steroid and prostaglandin substrates to their corresponding secondary alcohols. 13 , 14 Three highly homologous isoforms also participate in steroid hormone synthesis and metabolism; AKR1C1, AKR1C2, and AKR1C4, with varied stereo- and positional specificities for steroid substrates. 15 , 16 Inhibition of androgen signaling and cell proliferation is mediated by the enzymes AKR1C1 and AKR1C2 (possessing >84% sequence homology to AKR1C3), which function to reduce the potent androgen 5 α -dihydrotestosterone to the corresponding products 5 α -androstane-3 β ,17 β -diol and 5 α -androstane-3 α ,17 β -diol, respectively. 17 – 19 In the prostate, AKR1C3 converts 4-androstene-3,17-dione and 5 α -androstane-3,17-dione to testosterone (T) and dihydrotestosterone (DHT), respectively, which are potent ligands for the androgen receptor (AR), a driving force for PCa development and progression. 20 The enzyme is overexpressed at both the mRNA and protein levels in prostate tumors from castration-resistant prostate cancer (CRPC) patients but has low or undetectable expression in healthy prostate tissue. 21 , 22 Reduction of AKR1C3 expression or pharmacological inhibition significantly decreases the levels of T and DHT and androgen-dependent gene expression [prostate specific antigen (PSA)]. 22 AKR1C3 has been shown to contribute to chemotherapeutic resistance in CRPC since its expression is increased in enzalutamide and abiraterone acetate-resistant PCa cell lines. 23 – 25 An emerging role of AKR1C3 to promote the stabilization of AR splice variant 7 (ARv7), 26 a major determinant of enzalutamide resistance, 27 further delineates its role as a central mediator of drug resistance in advanced PCa. 28 Thus, selective inhibition of AKR1C3 has emerged as a promising strategy for the discovery of potential therapeutics to directly treat PCa 29 and to counter AR antagonist drug resistance. 23 The literature reports a wide variety of synthetic and natural inhibitors of AKR1C3. 20 , 29 , 30 Given the structural similarities between AKR1C isoforms that perform different physiological roles, it is of great interest to develop selective inhibitors for AKR1C3. Many nonsteroidal anti-inflammatory drug (NSAID) analogues have been reported ( Figure 1 ) with activity and selectivity for AKR1C3 over other AKR1C isoforms, 31 including analogues of naproxen, 32 indomethacin, 33 , 34 and flufenamic acid. 35 However, despite these advances, no selective AKR1C3 inhibitor has progressed to the clinic. This can be attributed, at least in part, to the poor pharmacokinetics of published inhibitors. Endo et al. recently reported the development of 8-hydroxy-2-imino-2 H -chromene-3-carboxamide derivatives as AKR1C3 inhibitors, with the most active compound possessing an IC 50 = 27 nM and >370-fold selectivity to other isoforms. 36 The cinnamic acid natural product baccharin has been reported as a potent and selective inhibitor of AKR1C3. 37 The same group reported a variety of ether analogues at the prenyl chain substitution position of the central phenyl ring of baccharin with one analogue possessing an IC 50 for AKR1C3 of 20 nM and >4000-fold selectivity over AKR1C1, >5000-fold over AKR1C2, and >1000-fold over AKR1C4. 38 However, these would likely suffer from metabolic instability of the ester. 39 Two designed AKR1C3 inhibitors have progressed to clinical trials (ASP9521 and BAY1128688), with a third using indomethacin as a “non-designed” AKR1C3 inhibitor. The ASP9521 inhibitor possessed an IC 50 value of approximately 10 nM, depending on the mammalian AKR1C3 source, and >100-fold selectivity for AKR1C3 over AKR1C2. 40 In a small (13 patients) phase I–IIb clinical trial, ASP9521 exhibited good tolerability but “no relevant evidence of clinical activity” in CRPC patients. 41 The BAY1128688 inhibitor, which had been developed for the treatment of endometriosis, had its clinical trial terminated in 2019 due to observed hepatotoxicity ( NCT03373422 ). 42 A phase Ib/II clinical trial of indomethacin (a “non-designed” AKR1C3 inhibitor) to counter enzalutamide resistance in CRPC patients is actively recruiting ( NCT02935205 ). There is a clear need for the continued development of potent, selective, and “drug-like” AKR1C3 inhibitors to combat CRPC and other hormone and hematological cancers for both a stand-alone chemotherapeutic effect and to address drug resistance development against multiple frontline chemotherapeutic agents. 23 Our ongoing efforts in this area have developed AKR1C3 inhibitors with enhanced potency, selectivity, 43 – 45 in vitro and in vivo “drug-like” properties, 39 and the ability to counter drug resistance against multiple CRPC clinically approved chemotherapeutics. 10 Our recently reported lead, KV49g ( Figure 1 ), possesses an AKR1C3 IC 50 value of 70 nM with >2800-fold selectivity. 46 Based on molecular modeling data of KV49g in the AKR1C3 binding pocket, 46 we identified a large pocket that is absent in AKR1C1, 2, and 4, occupied by the prenyl side chain. We report herein the structure–activity relationship of filling this pocket with various aromatic, heteroaromatic, alkyl, and alkoxy analogs. Cognizant of the poor pharmacokinetics of free carboxylic acids in in vivo systems, we report a methyl ester prodrug of one of our lead compounds, providing proof-of-principle conversion of the inactive ester prodrug to the active AKR1C3 inhibitor acid to attenuate tumor volume in PCa 22Rv1 xenografts with no observed toxicity.