Development of activators for SERCA2a for heart failure treatments

Abstract

The sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) is a central regulator of cardiac Ca²⁺ handling and an emerging therapeutic target for heart failure. Here, we report a comprehensive structure–activity relationship (SAR) study around small-molecule activator compound 1, integrating Ca²⁺-ATPase and Ca²⁺-uptake assays, isoform selectivity profiling, and ADMET characterization across more than fifty analogues. Systematic modification of the left-hand aryl/heteroaryl region revealed a strong dependence of activity on aromaticity and lipophilicity, with CF₃- and Br-substituted analogues providing substantial gains in potency. Optimization of the central amide linker established the importance of N-alkyl chain length, subtle hydrogen-bonding capacity, and a bent ligand geometry for productive SERCA2a engagement. Electronic tuning of the right-hand benzyl group further modulated efficacy, highlighting the essential contribution of an ortho-donor substituent. Functional evaluation across multiple Ca²⁺ concentrations identified several analogues with ATPase activation but inhibitory Ca²⁺-uptake effects, underscoring the need for dual-assay assessment to ensure bona fide activation. Among the series, compound 25 emerged as a balanced lead, displaying micromolar potency, robust concordant enhancement of ATPase and Ca²⁺-uptake activity, favorable solubility, and improved cytotoxicity relative to compound 1. Collectively, these findings define key structural determinants governing SERCA2a activation and provide a rational framework for developing next-generation, drug-like cardiac SERCA2a modulators.

1. Introduction

The sarco/endoplasmic reticulum calcium ATPase (SERCA) is a transmembrane protein in the sarcoplasmic reticulum (SR, muscle) or endoplasmic reticulum (ER, non-muscle) membrane in most mammalian cells. SERCA uses the Ca²⁺-dependent hydrolysis of ATP to provide the energy for active transport of calcium ions from the cytoplasm into the SR lumen, to induce relaxation (diastole) of muscle (Figure 1A). SERCA acts in synchrony with the Ca²⁺ release channel, ryanodine receptor, which releases calcium from the SR lumen to induce contractility (systole) of myofibrils in muscle. Like its skeletal muscle counterpart (SERCA1a), cardiac SERCA2a is a multifunctional transmembrane protein with specific sites for nucleotide binding (N) and phosphorylation (P), which form the catalytic site that is influenced by the actuator (A) region (Figure 1A). Impairment of SERCA2a activity in the heart reduces the SR Ca²⁺ load and increases cytoplasmic [Ca²⁺], which are both associated with heart failure (HF) in humans. Disruption of Ca²⁺ homeostasis could be caused by diminished SERCA2a activity, either due to (1) decrease in gene or protein expression, (2) post-translational modifications to amino side chains (as in aging or diabetes), or (3) alterations in SERCA2a’s interactions with its regulatory peptides such as phospholamban and DWORF [1–3]. Increasing SERCA2a activity is a target for treating heart failure. Therapeutic agents for heart failure are urgently needed, as current therapies only assist in managing this condition, prolonging life by only up to 6 years [4]. Current SERCA2a activators demonstrate the therapeutic potential of targeting SERCA2a for HF, but these compounds are currently not viable for chronic usage. These SERCA2a activators, include CP-154526 [5], Ro 41–0960 [5], ellagic acid [6, 7], GM1869 [8], gingerol [6, 7], Yakuchinone A[9], Alpinoid D[9], and istaroxime [10, 11] and its derivative Compound 8 [12, 13] (Figure 1B). Though, reduced ATPase activity has been reported for CP-154526 and Ro 41–0960[14]. Very recently, Ard and colleagues reported using 6-paradol, Yakuchinone A and Alpinoid D to inform expert-guided SAR, as they improved compound activatory effects on SERCA2a ATPase activity[15]. As in the present study, Ard and colleagues defined three pharmacophoric regions for modification, and used an iterative medicinal chemistry campaign. Analogues of SERCA modulator CDN1163 have been reported to increase SERCA2a ATPase activity[16], but further evaluation in cardiomyocytes or on Ca²⁺ uptake is desirable[8]. Among SERCA activators, istaroxime is the only compound that has been in clinical trials for treatment of HF [10], but it was found to be readily and quickly metabolized (PST3093) so was deemed unsuitable for clinical use. Its primary metabolite, PST3093 was assessed for potential therapeutic use [11]. However, PST3093 also has a genotoxic oxime moiety that may render the compound unsuitable for chronic usage. Despite the more favorable therapeutic profile of PST 3093 vs. the parent drug, neither has been successfully developed for use as a SERCA2a activator in HF [11, 12]. Recently a derivative of istaroxime (“Compound 8”) that lacks the oxime moiety has been shown to activate SERCA2a, and it appears to be a favorable candidate for further development as a HF therapeutic [12, 17], which underscores the potential of SERCA as a molecular target.

Figure 1.

A. SERCA function: hydrolysis of ATP and transport of Ca²⁺. B. Known SERCA activators. C. Chemical structure of compound 1 showing left, central, and right moieties for rational design of analogues that are numbered after the arrows. D. Target product profile of desired parameters.

We have previously performed high-throughput screening (HTS) of a 46,000-compound ChemBridge DIVERSet library [18], a diverse collection of drug-like small molecules. In our ATPase-based, SERCA-targeted HTS assay, we discovered 19 promising compounds that activated SERCA with isoform-specific effects [18]. Here, we focused on an N-aryl-N-alkyl-thiophene-2-carboxamide (“compound 1”) for further development, as it showed promising characteristics for drugability [18]. Compound 1 activated SERCA2a function; ATPase activity by 58% and Ca²⁺-uptake by 24% [18], and increased Ca²⁺ dynamics and Ca²⁺ load without interfering effects from the Na⁺/Ca²⁺ exchanger or L-type Ca²⁺ channels [19].

Here, we report on the systematic investigation of compound 1 (Figure 1C), through chemical modification, aiming to improve targeted parameters as described in Figure 1D. The functional effects of these analogues on SERCA2a were determined by measuring the Ca²⁺-ATPase and Ca²⁺-uptake activities in porcine cardiac SR vesicles, accompanied by analysis of SAR. We also determined the solubility, intrinsic clearance (S9 stability), and cell viability (cytotoxicity) properties of selected analogues with improved potency on Ca²⁺-ATPase activity. Further, SERCA isoform selectivity was determined by testing this subset of analogues on Ca²⁺-ATPase activity using SERCA1a in skeletal SR vesicles.

2. Results

2.1. Chemistry

The stepwise synthesis of compound 1 (N-aryl-N-alkyl-thiophene-2-carboxamide) derivatives 2-54, presented in Figure 2, was carried out as described previously [20]. In brief, primary amines (Ia-l) were first treated with benzaldehyde (II) to give intermediate III (Figure 2A). The direct reductive amination reactions were conducted with sodium triacetoxyborohydride [NaBH(OAc)₃] in 1,2-dichloroethane (DCE) under a nitrogen atmosphere at room temperature. Subsequently, the desired compounds 1–54 were produced by amide coupling reactions between secondary amines and corresponding commercially available carboxylic acids (Figure 2B–D). Depending on which moiety was modified (left, central or right), various secondary amines (IV, VI, VII, respectively) were used. The reactions were carried in the presence of either 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCl) in dichloromethane (DCM) at room temperature or a mixture of N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt), and N,N-diisopropylethylamine (DIPEA) in dry dimethylformamide (DMF) at 70 °C in a microwave. The structures were characterized by ¹H NMR, ¹³C NMR and high-resolution mass spectrometry (HRMS).

Figure 2.

Reagents and conditions: A. Reductive amination: (a) NaBH(OAc)₃, AcOH, DCE, rt, 24h, >75%; B-D. Acid-amine coupling reaction for left, middle and right side derivatives: (b) EDCI, DCM, rt, 4 h, >80%; (c) HBTU, HOBt, DIPEA, DMF, MW, 70 °C, 10 min, >70%.

Tertiary aromatic benzamides with bulky substitution at positions ortho to the carbonyl carbon may, depending on the relative sizes of those substituents and the two groups on the amide nitrogen exist as atropisomers arising from the axial chirality (R_a and S_a) about the carbon-carbon bond between carbonyl and ipso-aryl carbon due to the high energy barrier of rotation. Additionally, tertiary amides may exist as a pair of rotamers due to hindered rotation about the carbon-nitrogen bond, which can further complicate the NMR spectra resulting in a pair of rotamers for each atropisomer. We observed this in our tertiary benzamides. Among them, the highest complexity was demonstrated by the ortho-methyl benzoyl (9), ortho-chloro benzoyl (15), ortho-methoxy benzoyl (18) and ortho-trifluoromethyl (20) analogues. We describe in Supplementary Material compound 18 as a representative example to explore this interesting phenomenon. Compound 18 exists as a racemic pair of atropisomers (Figure 3A). The axial chirality of the molecule renders all of the methylene protons in this molecule diastereotopic accounting for the observed geminal coupling. Each atropisomer can potentially exist as a pair of rotamers affording four diasteromers. In the one-dimensional ¹H NMR spectrum (Figure 3B) only two sets of distinct resonances for the benzylic protons are observed in a 1.05:1.00 ratio, corresponding to a single amide rotamer for each atropisomer. The chemical environments of the benzylic protons (vide infra) are vastly different: one displays an AB coupling pattern at 4.37 ppm while the other has pair of doublet of doublets at 4.62 and 5.04 ppm. For structural elucidation, we turned to 1D NOESY (nuclear Overhauser enhancement spectroscopy). It involves selectively irradiation of a single proton resonance (negative peak) and observing the effect on nearby proton resonances (positive peaks). However, for those protons undergoing chemical exchange faster than the ‘NMR NOE timescale’, the irradiated protons will appear in the same phase as the irradiated peak [21]. After selective excitation of the resonance at 3.02 ppm corresponding to the NCH_aH_bEt protons we observed two other negative peaks at 3.22 ppm and 3.65 ppm, indicating the exchange of those protons on the NOE timescale (Figure 3C). The spectrum also showed a number of positive enhancements; particularly diagnostic was the aromatic resonance at 7.46 ppm, the furthest downfield peak, which corresponds to the benzoyl proton at C6 (C₆H₄CON). This indicates the spatial relationship between protons labeled “a” and “b” in the s-trans amide structures. We did not observe a correlation between the propyl proton resonance at 3.24 ppm and benzoyl proton (7.46 ppm), indicating that it (and its geminal methylene partner) is remote from the benzoyl aryl ring. However, irradiation at 4.37 ppm for benzylic protons (CONCH_aH_bAr) resulted in enhancement at 3.78 ppm for the methoxy group on the benzoyl ring, consistent with its s-cis amide structure (Figure 3D). These correlations were absent when the peak at 4.62 ppm was irradiated.

Figure 3.

(A) Rotamers of 18 with particularly distinguishing NOE interactions. (B) ¹H NMR spectrum of 18 in CDCl₃, 900 MHz. (C) 1D NOESY of 18; irradiation of propyl CH₂ (3.02 ppm) in s-trans isomer. (D) 1D NOESY of 18; irradiation of benzylic CH₂ (4.37 ppm) in s-cis isomer.

The NOESY 2D spectrum also revealed diagnostic correlations (Figure S40, Supplementary Material). One type was the usual through-space interactions; the second type arose from protons that are dynamically exchanging places during the timeframe of the NOE relaxation (a so-called EXSY exchange spectroscopy effect). The two types can be distinguished by the phase of the off-diagonal spot. True NOESY correlations are of opposite color of the diagonal spots, whereas the EXSY (exchange) spots are of the same color as the diagonals.

In general, we observed the following patterns among our products: (1) the thiophene carboxamides generally did not display restricted rotation in CDCl₃ at room temperature, with the exceptions of thiophene-3-carboxamide (5), N-cyclopropyl (35) and N-3-fluoropropyl (39); (2) compounds with phenyl or pyridinyl rings on the left side molecule existed as a mixture of s-cis and s-trans atropisomeric rotomers in the same conditions, presumably as a result of steric effects.

2.2. Iterative medicinal chemistry campaign driven by analogue effect on SERCA2a activity

To assess the impact of the analogues (in a two-fold dilution series from 50 μM down to 0.048 μM) on SERCA2a function in cardiac SR, we used the NADH-coupled enzyme-linked ATPase activity assay at [Ca²⁺] where SERCA2a has no activity (basal, pCa 8.0), submaximal activity [Ca²⁺]_MID (pCa 6.2) and maximal activity [Ca²⁺]_MAX (pCa 5.4), as previously described [18, 22]. Effects of SERCA modulators are commonly assessed at [Ca²⁺]_MAX, including a recent SERCA activator, SAR-focused report by Ard and colleagues [15], so for comparison purposes we focused our evaluation of the analogue’s potency at this [Ca²⁺]. Acquiring activity at [Ca²⁺]_MID informs on the functional effect of these compounds at [Ca²⁺] more representative of cardiomyocyte cytoplasmic concentrations. To correct for activity not attributable to SERCA2a function, the Ca²⁺ ATPase activities at pCa 8 were subtracted from the values at [Ca²⁺]_MID and [Ca²⁺]_MAX. Typically, the concentration response curves (CRC) of the Ca²⁺-ATPase activity trended sigmoidal and the plots were fitted to the Hill’s equation (See Experimental Section), where the functional potency (1/EC₅₀, where EC₅₀ is the compound concentration at 50% of the maximum effect under [Ca²⁺]_MID conditions) and functional efficacy (maximal % change in the compound’s effect on function compared to function in the absence of the analogue under [Ca²⁺]_MAX conditions) were determined. For many of the analogues, the effect on ATPase activity did not saturate by 50 μM, which resulted in EC₅₀ values >50.

Initially, we explored conservative changes to the left-hand side of compound 1 through shifting, removal and substitution of the thiophene methyl groups (Table 1, 2–6) to determine the flexibility of this position to modification. This initial set of analogues afforded an increase of ATPase activity ranging from 29 to 47% with activity monotonically increasing with lipophilicity, which is consistent with the requirement to partition to the membrane in order to bind SERCA2a’s transmembrane domain. Guided by this hypothesis, benzannulation of 1 through cyclization of adjacent methyl groups afforded benzothiophene derivative 7 with cLogD_7.4 of 4.9 that realized a substantial boost in activity increasing ATPase activity by 76% among the highest of any reported small-molecule SERCA2a activator. The partially saturated tetrahydrobenzothiophene derivative 8 resulted in a significant loss of activity (45% stimulation) suggesting that pi-stacking may be important to obtain maximal activity. Thiophene and its derivatives are prone to cytochrome P450 (CYP450)-catalyzed metabolism that can lead to rapid clearance and generation of reactive metabolites resulting in toxicity. Hence, replacement of the 2,3-dimethylthiophene group containing seven heavy atoms with a bioisosteric tolyl group, also with seven heavy atoms, was next explored to eliminate this potential metabolic liability. The tolyl analogues 9–11 have a similar lipophilicity and both the meta- and para-derivatives 10 and 11 had nearly equivalent activity (59 and 61%) compared to 1 (57%), whereas the ortho-derivative 9 realized a dramatic loss of activity (29%). However, none of the tolyl analogues attained saturation of the SERCA2a receptor providing EC₅₀ values greater than 50 μM, thus efforts focused on optimizing the aryl group for potency and ATP stimulation while keeping the lipophilicity within a reasonable boundary. This significant challenge is underscored by the recent report from Ard and co-workers, whose most potent SERCA2a activator possessed an impressive EC₅₀ of 0.7 μM, but this came at the expense of both activity (25% ATPase stimulation) and lipophilicity (cLogD_7.4 = 7.1). Increasing lipophilicity beyond cLogD_7.4 of 5.0 was not pursued given the inherent challenges of compound development. To confirm the SAR trends of the tolyl analogues (cLogD_7.4 = 4.4) and investigate the impact of subtle changes to lipophilicity, we prepared a series of fluorophenyl 12–14 (cLogD_7.4 = 4.0) and chlorophenyl 15–17 (cLogD_7.4 = 4.6) analogues bearing substitution at the ortho, meta and para-positions. The resulting ATP stimulation mirrored the tolyl series with meta and para-substituted analogues displaying nearly equal ATP stimulation that was about twice the level of the ortho-derivative. Moreover, the activity correlated nicely with lipophilicity when comparing the average ATP activation of the meta- and para-analogues. The fluoro analogues 13–14 (cLogD_7.4 = 4.0, 54% average) predictably displayed decreased stimulation (54% average) while the chloro analogues 16–17 (cLogD_7.4 = 4.6, 63% average) demonstrated increased stimulation (63% average) relative to the tolyl analogues 10–11 (cLogD_7.4 = 4.3, 60% average). Unfortunately, neither the fluoro or chloro series achieved saturation of SERCA2a yielding EC₅₀ values greater than 50 μM.

Table 1.

Effects of the left-moiety modified analogues of compound 1 on SERCA2a Ca²⁺-ATPase activity.

Cpd	R	CRC shape	Max increase (%)	Max decrease (%)	EC50 (μM)	Cpd	R	CRC shape	Max increase (%)	Max decrease (%)	EC50 (μM)
1		Sigmoid	57±13	-	5.3±1.8	16		Sigmoid*	61±9.4	-	>50
2		Sigmoid	47±7.1	-	4.8±0.7	17		Sigmoid*	64±7.6	-	>50
3		Sigmoid*	45±9.2	-	>50	18		Sigmoid*	27±9.3	-	>50
4		Sigmoid	30±14	-	6.1±1.8	19		Sigmoid*	45±14	-	>50
5		Sigmoid*	29±8.4		>50	20		Sigmoid*	51±17	-	>50
6		Sigmoid	37±9.4	-	5.1±0.7	21		Sigmoid	40±14	-	5.1±2.1
7		Sigmoid	76±19	-	5.8±1.5	22		Sigmoid	46±5.5	-	1.5±0.3
8		Sigmoid	45±10	-	3.3±0.5	23		Bell	28±8.1	28±9.0	0.9±0.4
9		Sigmoid*	28±10	-	>50	24		Sigmoid	48±4.6	-	3.4±0.3
10		Sigmoid*	59±13	-	>50	25		Sigmoid	53±7.7	-	4.0±2.1
11		Sigmoid*	61±7.0	-	>50	26		Bell	33±6.5	-	2.2±0.3
12		Sigmoid*	40±10	-	>50	27		Sigmoid	46±7.8	-	>50
13		Sigmoid*	53±17	-	>50	28		Sigmoid*	28±5.7	-	>50
14		Sigmoid*	55±11	-	>50	29		Sigmoid*	21±4.9	-	>50
15		Sigmoid*	35±10	-	>50

^*Hill fit, but does not plateau at the higher concentrations tested. Mean±SD, n= 4–6 individual experiments.

Table 2.

Effects of the central-moiety modified analogues of compound 1 on SERCA2a Ca²⁺-ATPase activity.

Cpd	R	CRC shape	Max increase (%)	Max decrease (%)	EC50 (μM)	Cpd	R	CRC shape	Max increase (%)	Max decrease (%)	EC50 (μM)
30		Sigmoid*	24±9.1	-	>50	37		Sigmoid*	23±8.0	-	>50
31		Sigmoid*	52±9.2	-	>50	38		Sigmoid*	57±5.4	-	>50
32		Sigmoid	43±6.7	-	6.3±1.8	39		Sigmoid*	22±8.6	-	>50
33		Sigmoid	72±8.5	-	4.0±0.80	40		Sigmoid*	91±12.6	-	>50
34		Sigmoid	43±7.9	-	4.7±1.1	41		Sigmoid*	75±8.6	-	>50
35		Sigmoid*	46±8.9	-	>50	42		Sigmoid*	22±6.4	-	>50
36		Inactive	N/A	-	N/A	43		Sigmoid*	68±10	-	>50

^*Hill fit, but does not plateau at the higher concentrations tested. Mean±SD, n= 4–6 individual experiments.

Table 6.

Physicochemical properties, metabolism and cell viability evaluation of selected analogues compared to compound 1.

Cpd	Structure	MW (g/mol)	cLogD7.4	tPSA (Å2)	Solubility (μg/mL)	Thalf (min)	CLint * (μL/min/mg protein)	Cell viability IC50 (μM)
1		317	4.50	29.5	501±4	97.6	14±1.1	3.9±1.6
8		343	5.12	29.5	5393±35	20.1	73±9.3	29±18
21		351	4.79	29.5	900±7	10.3	133±11	31±16
22		362	4.71	29.5	36±1	23.8	56±9.0	9.2±4.9
23		441	5.40	29.5	2591±24	15.3	79±19	28±6.4
24		380	4.88	29.5	28±1	51.3	25±4.0	24±9.4
25		380	4.88	29.5	507±7	74.5	15±7.2	36±12
26		380	4.88	29.5	787±16	62.4	22±1.1	18+5.0
32		303	3.97	29.5	5922±60	34.7	37±6.2	20±3.8
33		331	5.03	29.5	165±1	16.6	83±4.2	24±13

^*The Intrinsic Clearance; CL_Int for a positive control Verapamil = 63±2.9 μL/min/mg protein. The extraction ratio (E) of 0.3 and 0.7 were respectively assumed as the low and high boundaries.

Mean±SD, n= 4–6 individual experiments.

We then extended our investigation to include methoxy analogues 18–19 (cLogD_7.4 = 4.0) and trifluoromethyl analogues 20–21 (cLogD_7.4 = 4.8) covering a broader range of lipophilicities. The SAR of the methoxy analogues was consistent with the aryl analogues 9–17; however, the trifluoromethyl analogues did not follow the SAR trends as the ortho-trifluoromethyl 20 showed greater ATP stimulation (51%) than the meta-trifluoromethyl 21. Moreover, the maximum ATP stimulation was substantially less than predicted by the SAR. Further confounding matters, 21 displayed more than an order-of-magnitude increase in potency (EC₅₀ = 5.1 μM over 9–20 (EC₅₀ > 50 μM)). Although an outlier, 21 yielded an attractive sigmoidal dose-response curve and the boost in potency is consistent with prior studies showing a positive correlation between potency and lipophilicity. Bioisosteric replacement of the trifluoromethyl group in 21 with a bromine afforded 22 that possessed similar lipophilicity (cLogD_7.4 = 4.7), but a more than three-fold enhancement in potency (EC₅₀ = 1.0 μM). Dibromo analogue 23 (cLogD_7.4 = 5.4) explored out of curiosity, yielded the first sub-micromolar analogue (EC₅₀ = 0.9 μM), but the modest increase in potency was overshadowed by the increased lipophilicity and liability of two adjacent bromine atoms. Returning to 22, the low solubility prompted efforts to modulate solubility through incorporation of fluorine in 24–26 or nitrogen in pyridyl analogues 27–29. The increased polarity of the pyridyl analogs 27–29 predictably enhanced solubility, but at the expense of potency (EC₅₀ >50 μM). Although not germane to our objective to increase potency, the series showed interesting SAR trends with respect to ATP stimulation and the 2-pyridyl group showed the highest ATP stimulation while the 4-pyridyl analogue had the lowest. On the other hand, fluorination was well tolerated and afforded analogues with increased ATP stimulation, potency and solubility. However, concurrent optimization of all three properties was only attained by 3-bromo-4-fluorophenyl 25, which displayed 53% maximum ATP stimulation with an EC₅₀ of 4 μM and a solubility in excess of 500 μg/mL. Compound 25 (cLogD_7.4 = 4.9) compares favorably with the optimized benzofuran SERCA2a inhibitor (cLogD_7.4 = 6.1, 9 rotatable bonds) recently reported by Ard and co-workers, whose maximal ATP stimulation is 57% with an EC₅₀ of 8.6 μM. The compact structure of 25 and limited conformational mobility along with its superior physicochemical properties may translate to enhanced selectivity, safety and pharmacokinetics.

Following preliminary optimization of the left-hand side of 1, we explored modifications of the central amide linker through a variety of alkyl and cycloalkyl N-substituents to define the steric and electronic requirements along with a couple of conformationally-restricted analogues to ascertain the ligand conformation required to bind SERCA2a. We did not evaluate classical amide bioisosteres developed to address the metabolic liability of amides, which are susceptible to hydrolysis by tissue amidases because tertiary amides as found in 1 are, with rare exception, not substrates for amidases. The impact of chain-length and alpha branching was examined with analogues 30–35. ATP stimulation was decreased in all truncated and alpha-branched analogues yielding activities ranging from 24–43% with the lowest activity arising from removal of the N-propyl substituent. Homologation by one carbon in n-butyl 33 significantly boosted activity yielding 72% ATP stimulation and enhanced potency with an EC₅₀ of 4.0 μM. However, the addition of one additional methylene in 33 also increased the lipophilicity (cLogD_7.4 = 5.0). Extension to an n-pentyl chain (cLogD_7.4 = 5.6) was predicted to exceed our threshold cLogD_7.4 of five while introducing two more rotational bonds, both deleterious to drug disposition properties, hence further homologs were not pursued.

To understand the electronic requirements of the n-alkyl chain of the amide, we prepared a few analogues incorporating oxygen, either at the terminal position in alcohol derivatives 36–37 or within the chain in methyl ether 38. Introduction of terminal alcohol was poorly tolerated and either abolished (36) or significantly attenuated (37) activity. Insertion of an oxygen atom was better tolerated and methyl ether 38 exhibited the same level of ATP activation as 1, but substantially lower potency (EC₅₀ >50 μM). The increased polarity of this set of analogues makes it challenging to disentangle the impact of hydrogen-bond donors and acceptors on activity. The data suggest that a threshold lipophilicity may be required (cLogD_7.4 > 3) given 36 (cLogD_7.4 = 3.0) failed to demonstrate any response. To unravel the effect of hydrogen-bonding versus lipophilicity on activity, we prepared mono-, di-, and trifluorinated analogues 39–41. The monofluoro 39 (cLogD_7.4 = 3.9; 22% activity) and trifluoro 41 (cLogD_7.4 = 4.2, 75% activity) derivative are both more lipophilic, yet exhibited lower activity than difluoro 40 (cLogD_7.4 = 3.6, 91% activity). While each of the terminal fluorine groups (-CH₂F, -CHF₂, -CF₃) can act as hydrogen-bond acceptors, only the difluoromethyl group is able to serve as hydrogen-bond donor, which is caused by polarization of the C-H bond adjacent to the fluorines. Taken together, these results suggest the N-alkyl side-chain is positioned in a lipophilic binding pocket with potentially multiple hydrogen-bond interactions. Notably, difluoromethyl derivative 40 displayed the highest stimulation of ATPase activity (91%) in this study, despite not being optimized for lipophilicity.

To begin to understand the conformational requirement of binding, we prepared two conformationally-constrained analogues 42 and 43. Tetrahydroisoquinoline 42 was designed by ligating the N-alkyl side chain onto the aryl ring of the right-hand side of the molecule while pyrrolidine 43 used a shorter tether and connected the N-alkyl chain to the closer benzylic position of the right-hand side. Tetrahydroisoquinoline enforcing a coplanar arrangement of the N-alkyl and N-benzyl side chains was nearly inactive with a meager 22% activation and weak potency (EC₅₀ > 50 μM). Pyrrolidine 43, which orients the left and right-hand sides of the molecule perpendicular to each other displayed enhanced activity (69%) relative to 1; although, it failed to reach saturation (EC₅₀ > 50 μM). These preliminary studies suggest that further exploration is warranted to illuminate the bioactive conformation.

Lastly, we examined the benzyl substituent on the right-hand side of the molecule. Removal of the methoxy group in 44 abolished activity, underscoring the importance of the ortho-methoxy group. To understand the impact of the methoxy group’s steric size and electron-donating ability, we prepared the meta-methoxy 45 and para-methoxy 46 regioisomers. The methoxy regioisomers afforded virtually identical ATP stimulation as 1, suggesting that electron-donating ability is more important that steric encumbrance at the ortho-position. Because the ortho-methoxy group is predicted to undergo CYP450 metabolism leading to O-demethylation, we prepared the putative metabolite des-methyl 47 as well ortho-fluoro 48 designed to block metabolism. Removal of the methyl group of the methoxy was well tolerated and the activity of 47 was commensurate with 1. Given the apparent requirement for an electron-donating substituent, we expected ortho-fluoro 48 to exhibit diminished activity. To our surprise, fluorination enhanced the maximal ATPase activity to 89%; although it was not saturable. Incorporation of a nitrogen atom in pyridyl analogues 49–50 ablated activity, but this is likely due to decreased lipophilicity (cLogD_7.4 ~ 3.4) of the molecules rather than a specific repulsive interaction.

To complete our initial investigation, we designed dual modified analogues containing an N-ethyl substituent in order to reduce lipophilicity along with a subset of bromophenyl and bromofluorophenyl substituents on the left-hand side of the molecule fixing the right-hand side to the parent ortho-methoxybenzyl group. The activity and potency of 51–53 were virtually indistinguishable, which deviated from the SAR of the parent N-propyl compounds that showed enhanced potency, but slightly diminished activity relative to 1 (Table 4). Continuing the discrepant behavior, 54 containing fluoro and bromo meta-substituents along with para-methyl group reversed the functional activity and showed 70% inhibition at 50 μM behaving as an inverse agonist. Additional data, including EC₅₀ values and % of activation for all final compounds, for Ca²⁺-ATPase at two various calcium concentrations, are presented in Table S1 (Supplementary Materials).

Table 4.

Effects of the double-modified analogues of compound 1 on Ca²⁺-ATPase activity.

Cpd	R1	CRC shape	Max increase (%)	Max decrease (%)	EC50 (μM)
51		Sigmoid	46±6.3	-	4.8±1.8
52		Sigmoid	46±7.4	-	5.9±1.3
53		Sigmoid	45±8.4	-	6.1±2.8
54		Sigmoid*	-	−70±3.4	>50

^*Hill fit, but does not plateau at the higher concentrations tested.

Mean±SD, n= 4–6 individual experiments.

2.3. Functional assays of selected analogues with improved function

Nine analogues that showed improved SERCA2a ATPase activity function were selected for further functional testing of their effects on Ca²⁺-uptake (Table 5). The range of effects is represented in Figure 4. These nine analogues were also tested for selectivity with the dominant SERCA isoform in skeletal muscle, SERCA1a (Table S2, Supplementary Materials) and some ADMET properties (Table 6). The analogues with modifications in the right moiety did not induce significant improvements in SERCA2a ATPase activity. Therefore, they were not selected for further testing. The analogues with modifications in the left moiety (8, 21, 22, 23, 24, 25, 26) and central moiety (32, 33) that improved SERCA2a activity were further tested for effects on Ca²⁺-uptake function (Table 5). The Ca²⁺ uptake assay using cardiac SR membranes is a good partner to the ATPase activity assay, especially to test if the compound is a true SERCA activator by increasing both ATPase and Ca²⁺ uptake activities. A slight limitation of using SR membranes is that it is less physiologically relevant than possible testing in cardiomyocytes. However, the higher throughput with using SR membranes is desirable for our SAR studies. Further, compound 1 has been confirmed to increase SR Ca²⁺ load in cardiomyocytes [19]. Several analogues (8, 22, 26) increased SERCA2a ATPase activities at both [Ca²⁺]_MAX and [Ca²⁺]_MID, though only one analogue (23) induced a divergent effect (inhibition) on SERCA2a ATPase activity under [Ca²⁺]_MID condition. At [Ca²⁺]_MAX, the Ca²⁺-uptake effects were more variable in that the EC₅₀ values were decreased by most of the analogues (22, 25, 26, 32), but some were either unchanged (8, 33) or increased (21). At [Ca²⁺]_MID, the analogues increased EC₅₀. Divergent in their effects on ATPase activity, several analogues (8, 22, 23, 26) reduced Ca²⁺-uptake at [Ca²⁺]_MID (Table 5). This would likely be undesirable in a physiological setting.

Table 5.

Functional effects of selected compound 1 analogues on SERCA2a activities in cardiac SR.

Cpd	Structure	Ca2+-ATPase				Ca2+-Uptake
		[Ca2+]MAX		[Ca2+]MID		[Ca2+]MAX		[Ca2+]MID
		EC50 (μM)	% effect	EC50 (μM)	% effect	EC50 (μM)	% effect	EC50 (μM)	% effect
1		5.3±1.8	57±13	6.0±1.2	25±3.5	1.1±0.73	17±4.5	0.46±.35	5.4±2.8
8		3.3±0.5	45±10	3.6±2.8	10±5.1	1.1±0.45	8.8±2.3	> 50	−30±6.1
21		5.1±2.1	40±14	5.3±1.3	25±7.4	1.5±0.33	15±5.9	1.0±0.86	7.0±3.7
22		1.5±0.31	46±5.5	1.4±0.8	22±5.6	0.36±0.14	11±3.8	12.4±8.4	−25±21
23		0.9±0.38	28±8.1	15.3±4.1	−36±10	>50	−68±12	9.2±1.5	−66±10
24		3.4±0.29	48±4.6	3.3±1.9	25±5.6	1.4±0.65	12±3.0	0.83±0.3	8.5±3.3
25		4.0±2.1	53±7.7	3.7±0.73	33±6.1	0.80±0.57	12±5.6	0.89±0.4	8.6±5.7
26		2.2±0.27	33±6.5	2.1±0.88	21±7.4	0.54±0.3	14±4.7	> 50	−45±12
32		6.3±1.8	43±6.7	4.6±2.0	24±6.0	0.77±0.57	13±5.7	0.91±0.85	6.0±1.6
33		4.0±0.80	72±8.5	3.0±1.0	23±6.0	1.1±0.35	22±5.6	0.63±0.21	15±7.0

Mean±SD, n= 4–6 individual experiments.

Figure 4: Representative effects of compound 1 analogues on SERCA2a functional assays:

Ca²⁺-ATPase (left) and Ca²⁺-uptake (right) at [Ca²⁺]_MID (red circle) and [Ca²⁺]_MAX (black square). All representative compounds increased Ca-ATPase activity at [Ca²⁺]_MAX, but had divergent effects at [Ca²⁺]_MID and/or Ca²⁺-uptake. A) Compound 1 increased both Ca²⁺-ATPase and Ca²⁺-uptake at both [Ca²⁺]. B) Compound 23 decreased Ca²⁺-ATPase at [Ca²⁺]_MID, and Ca²⁺-uptake at both [Ca²⁺]. C) Compound 26 increased Ca²⁺-ATPase activity at [Ca²⁺]_MID, and Ca-uptake at [Ca²⁺]_MAX, but decreased Ca²⁺-uptake at [Ca²⁺]_MID. Mean±SD, n= 4–6 individual experiments.

To gain further insight into the mode of action of these compounds, we compared CRCs acquired [Ca²⁺]_MID and [Ca²⁺]_MAX. We observed strong (>28%) Ca²⁺-ATPase activation at [Ca²⁺]_MAX for all nine selected analogues. However, when Ca²⁺-ATPase activity was measured at [Ca²⁺]_MID, analogue 8 did not reach 20% activation. A different pattern was found in the CRCs of Ca²⁺-uptake, where we observed much lower effect at both Ca²⁺ concentrations, unless the compound showed inhibitory effect, as in case of 8 (−30%) and 26 (−45%) at [Ca²⁺]_MID (Table 5, Figure 4). Compound 23 acted as an inhibitor not only in Ca²⁺-uptake assay, but also Ca²⁺-ATPase assay at [Ca²⁺]_MID (Table 5, Figure 4).

The primary SERCA isoform in skeletal muscle, SERCA1a, is structurally very similar to SERCA2a, despite only sharing 84% in sequence identity. In principle, studying SERCA isoform selectivity may inform on the mechanism of action. To determine isoform selectivity, we tested compound effects on SERCA1a in skeletal SR. By comparison between the effects on ATPase activity (Table 5 and Table S2 (Supplementary Materials)), most of the prioritized nine analogues displayed similar functional effects between isoforms at both [Ca²⁺]_MAX and [Ca²⁺]_MID. However, there was a clear trend that the maximal effect of the compounds on ATPase activity was greater on SERCA1a relative to effects on SERCA2a. Larger dissimilarities are noted for compounds 21, 23 and 32. Compound 21 displayed at least 8-fold greater selectivity for SERCA1a at [Ca²⁺]_MID. Compound 32 displayed the greater selectivity for SERCA2a relative to SERCA1a, with at least 8-fold higher efficacy at [Ca²⁺]_MAX.

2.4. ADMET

Determination of in vitro drug disposition profiles is a critical component of early drug discovery. To assess the preliminary absorption, distribution, metabolism, excretion, and toxicity (ADMET) characteristics of selected analogues, we evaluated solubility, metabolic stability in human liver S9 fractions and cytotoxicity in HEK-293 cells (Table 6). Compounds were quantified by LC–MS/MS using external calibration standards. As all analogues possessed comparable topological polar surface areas (tPSA = 29.5 Ų), differences in ADMET properties could be largely attributed to the molecular weight, lipophilicity and the nature of structural modifications.

Kinetic solubility exhibited a broad dynamic range (28–5922 μg/mL), reflecting the diverse physicochemical features of the series. Most analogues demonstrated solubility above 60 μg/mL, with compound 1 showing moderate solubility (501 μg/mL). The highest solubilities were observed for compounds 8 (5393 μg/mL) and 32 (5922 μg/mL), the latter representing the most polar analogue examined. Several structural trends emerged among closely related series. Solubility was inversely related to the N-alkyl chain length on the central amide, increasing nearly ten-fold when the chain was shortened from N-propyl in 1 to N-ethyl in 32, and decreasing more than three-fold when elongated to N-butyl in 33. Cyclization of the thiophene moiety to afford tetrahydrobenzothiophene 8 dramatically enhanced solubility despite the modest rise in lipophilicity. In contrast, bioisosteric replacement of the thiophene with bromophenyl (22) or fluorophenyl (24) rings markedly reduced solubility (36 and 28 μg/mL, respectively). This effect could be partially mitigated by repositioning fluorine, increasing halogen content, or substituting CF₃ for Br, although no simple predictive trend emerged. Collectively, these observations suggest solubility can be modulated through modifications to the left-hand aryl region; however, the relationship between structure and aqueous solubility is non-linear and context dependent.

Given their moderate lipophilicity (cLogD_7.4 = 3.97–5.40), hepatic oxidative metabolism was anticipated to be the primary clearance pathway for this class. Human liver S9 stability measurements revealed half-lives ranging from 10.3 to 97.6 min (Table 6). The parent compound 1 displayed the greatest stability (T_half = 97.6 min; CL_int = 14 μL/min/mg), falling near the low-clearance boundary and outperforming all analogues designed to address its putative metabolic liabilities. The meta-CF₃ analogue 21 exhibited the most rapid turnover (T_half = 10.3 min; CL_int = 133 μL/min/mg), consistent with high intrinsic clearance. Analogues 8, 23, and 33 also showed elevated clearance (73–83 μL/min/mg), indicating enhanced susceptibility to oxidative metabolism. In contrast, compounds 24, 25, and 26 demonstrated improved stability (T_half = 51–75 min; CL_int = 15–25 μL/min/mg), representing the most metabolically robust members aside from compound 1. Interestingly, the most polar analogue (32) showed increased clearance (CL_int = 37 μL/min/mg), suggesting that reducing lipophilicity does not uniformly improve metabolic stability and may enhance enzyme accessibility. Overall, these findings indicate that compound 1 and many of its analogues are subject to oxidative metabolism, and further structural refinement—particularly within the left-hand heteroaryl region—will be required to attenuate metabolic turnover.

Cytotoxicity was assessed in HEK-293 cells following 72 hours exposure, with viability measured by MTT assay. SERCA modulator CDN1163 (IC₅₀ = 63 μM) was included as a reference standard [23]. Compound 1 exhibited pronounced cytotoxicity (IC₅₀ = 3.9 μM). In contrast, all analogues showed reduced toxicity, with IC₅₀ values spanning 9.2–36 μM. The least cytotoxic derivatives were 25 (IC₅₀ = 36 μM) and 8/21 (~29–31 μM), representing nearly an order-of-magnitude improvement over the parent. Compound 22 displayed the lowest IC₅₀ among the analogues (9.2 μM), aligning with its poor solubility and moderate clearance profile.

Taken together, the ADMET evaluation highlights compounds 8, 25, 26, and 32 as the most promising members of the series. These analogues combine significantly enhanced solubility with moderate to improved metabolic stability and reduced cytotoxicity. Among them, 25 emerges as the most balanced candidate, exhibiting solubility of 507 μg/mL, a metabolic half-life of 74.5 min, and the highest cell viability (IC₅₀ = 36 μM). These properties support the advancement of compound 25 as a lead for further optimization.

Discussion

This study provides a comprehensive structure–activity relationship (SAR) analysis of compound 1, a previously reported small-molecule SERCA2a activator [18, 19], and highlights multiple structural determinants of functional efficacy, potency, and physicochemical parameters. Using a combination of Ca²⁺-ATPase and Ca²⁺-uptake assays in cardiac SR membranes at both maximal and submaximal Ca²⁺ concentrations, supported by SERCA1a selectivity and ADMET profiling, we identified several chemical features that strongly influence the activation profile of SERCA2a and uncovered both opportunities and liabilities for future optimization.

Modifications of the left-hand heteroaryl/aryl moiety established a strong dependence of activity on both aromaticity and lipophilicity. Conservative changes to the thiophene motif resulted in moderate stimulation and poor potency, whereas benzannulation markedly increased maximal ATPase activation, consistent with a beneficial role of extended aromatic surface. Replacement of thiophene with phenyl rings revealed that meta- and para-substitution patterns were preferred over ortho-substitution, and efficacy increased proportionally with lipophilicity. More pronounced improvements were achieved with CF₃- and Br-containing analogues, leading to several micromolar and sub-micromolar compounds. However, such potency gains were frequently accompanied by high lipophilicity and, in some cases, functional liabilities. Fluorinated analogues, particularly compound 25, demonstrated that balanced potency, efficacy, and solubility can be achieved without excessive lipophilicity, making this substitution pattern especially promising for further optimization.

Within the central amide region, both steric and electronic factors were found to be critical. Shorter or α-branched N-alkyl substituents diminished activity, while chain extension to n-butyl improved both efficacy and potency. Introduction of heteroatoms into the alkyl chain was generally unfavorable unless lipophilicity was maintained. Fluorinated N-alkyl analogues provided mechanistic insight: the difluoromethyl substituent preserved high activity despite moderate lipophilicity, suggesting a role for hydrogen-bond donation in stabilizing ligand–protein interactions. Conformationally restricted analogues further indicated that a bent or V-shaped ligand geometry is preferred within the SERCA2a binding site, while coplanar orientations were poorly tolerated.

The right-hand benzyl region also influenced activity through electronic and steric effects. Removal of the ortho-methoxy substituent abolished activity, underscoring its importance, while relocation of the methoxy group or substitution with fluorine maintained or enhanced efficacy to varying degrees. Pyridyl replacements were inactive, reinforcing the requirement for sufficient hydrophobicity in this region. Together, these findings emphasize the importance of maintaining lipophilicity within an optimal window (cLogD_7.4 approximately 3.5–5.0), as lower values reduce efficacy and higher values increase the risk of functional inversion or poor developability.

A key outcome of this work is the recognition that ATPase activation alone does not reliably predict enhanced Ca²⁺-uptake. Several potent ATPase stimulators produced neutral or inhibitory effects on Ca²⁺ transport at physiological Ca²⁺ concentrations, and some compounds behaved as partial or inverse agonists under submaximal Ca²⁺ conditions. This divergence highlights the need to evaluate both ATP turnover and Ca²⁺ translocation to identify compounds with true physiological relevance. Among the series, compound 25 consistently increased both ATPase activity and Ca²⁺-uptake at both Ca²⁺ concentrations tested, marking it as a bona fide SERCA2a activator and a leading candidate for further development.

Isoform selectivity studies revealed that most analogues displayed similar profiles across SERCA1a and SERCA2a, although select examples demonstrated modest isoform preferences, indicating that subtle structural adjustments can influence isoform bias. This suggests that the binding site is shared between SERCA isoforms. For most of the compounds, the maximal effect in increasing activity was higher for SERCA1a relative to SERCA2a, which has been observed previously [18]. Perhaps SERCA2a is more resistant to further activation than SERCA1a. ADMET profiling further supported the developability of selected analogues. While compound 1 showed the highest metabolic stability, several analogues, notably 25, achieved a favorable balance of solubility, stability, and reduced cytotoxicity relative to the parent compound. Compounds with excessive lipophilicity exhibited poor solubility, rapid clearance, or cytotoxicity, reaffirming the observed lipophilicity threshold.

It is unlikely that our activators are binding in the transmembrane domain, as drug-like compounds binding here are all inhibitors that interfere with the necessary structural transition for Ca²⁺ transport [24]. Computational models of SERCA propose that an activator could bind to the interface between the A and P domains, close to the ATP binding site, to potentially improve nucleotide binding or orient the nucleotide to facilitate hydrolysis [16]. Although we cannot be certain that this is an activator binding site until experimental structural evidence is available, our compounds may increase SERCA ATPase and Ca²⁺-uptake activities through such a binding site and mechanism.

Collectively, the SAR trends from this study establish clear design principles for next-generation SERCA2a activators: maintenance of an aromatic left-hand region with controlled lipophilicity; an N-alkyl chain of optimal length and polarity; an electron-donating substituent on the right-hand benzyl group; and overall ligand geometry that supports a bent conformation. Among all analogues evaluated, compound 25 emerges as the most balanced lead, combining micromolar potency, robust and concordant functional activation, favorable solubility, and improved cytotoxicity relative to the parent scaffold. These results provide a strong platform for further medicinal chemistry optimization and advance the development of selective SERCA2a activators with potential therapeutic relevance.

3. Conclusions

In summary, we have identified key structural determinants for small-molecule activation of SERCA2a, and through SAR optimization have produced compound 25 as a balanced lead exhibiting potent activation, favorable solubility, and promising metabolic stability. These findings open the pathway to the development of selective, potent and drug-like SERCA2a activators with potential for cardioprotective therapy. Future work will focus on enhancing isoform selectivity, in vivo translation, and detailed binding-site elucidation.

4. Experimental section

4.1. Chemistry

All of the chemicals and solvents used were obtained from commercial sources (Acros Organic, Millipore Sigma, Fisher Scientific, TCI America, VWR, Ambeed, Combi Blocks, Aurum Pharmatech, 1Click Chemistry, A2B, PharmaBlock) and were used without further purification. Neutral alumina-dried dichloromethane (CH₂Cl₂), tetrahydrofuran (tetrahydrofuran), and 4 Å molecular sieves-dried N,N’-dimethylformamide were dispensed by INERT PURESOLV solvent system under nitrogen. Reactions were performed under an inert atmosphere of argon in oven-dried (145 °C) glassware. Aluminum-backed silica gel 60 F254 plates were used for thin-layer chromatography and the compounds were visualized with ultraviolet light at wavelength 254 nm. Silica gel grade 60 (particle size 0.040–0.063 mm; Merck, Germany) was used for manual flash column chromatography. Additionally, CombiFlash Nextgen 300+ (Teledyne ISCO) was used for automatic flash column chromatography. All NMR spectra were recorded on a Varian 400 and Bruker 600 MHz spectrometers at 400 and 601 MHz for ¹H, 100 and 151 MHz for ¹³C. ¹H NMR spectra were referenced to residual CDCl₃ (7.26 ppm) or CD₃OD (3.31 ppm); ¹³C NMR, CDCl₃ (77.16 ppm) or CD₃OD (49.00 ppm). NMR chemical shift data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, p =quintet, sx = sextet, tt =triplet of triplets, m = multiplet), coupling constant, integration. Coupling constants are given in Hertz (Hz). 1D NOE data was collected on a Bruker Avance III 900 or 601 MHz NMR spectrometers. For 1D NOE spectra, 4 scans were collected using a standard selective NOESY pulse sequence (selnogpzs.2) over a 12 ppm spectral window with 2 s relaxation delay, 300 ms mixing time and 3 s acquisition time using a Gaussian selective pulse calibrated to the bandwidth of each signal. As detailed below in the NMR characterization of each compound, the NMR spectra of all compounds were consistent with the proposed structures and showed no significant extraneous peaks, qualitatively indicating acceptable chemical purity (>95%). High-resolution mass spectra (HRMS) were obtained with an Exactive Plus Orbitrap Mass Spectrometer instrument and an Agilent^® 1290 Infinity II mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA).

4.1.1. General Procedure for Synthesis of Secondary Amines

Secondary amines were prepared via reductive amine amination with aldehydes and sodium triacetoxyborohydride [20]. Primary alkylamine (1.0 equiv.) and aromatic aldehyde (1.0 equiv.) were mixed in 1,2-dichloroethane and then treated with sodium triacetoxyborohydride (1.4 equiv.) and acetic acid (1.0 equiv.). The mixture was stirred at room temperature for 24 h and quenched by adding 1N aqueous NaOH. The product was extracted with ether, washed with brine, and dried over MgSO₄. Removal of solvent yielded the crude free bases.

4.1.2. General Procedure for Coupling Reaction

Method A: To each scintillation vial with carboxylic acid (1.0 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.0 equiv.) in dichloromethane was added corresponding amine (1.2 equiv.). The mixtures were stirred for 4 hours at room temperature and concentrated with silica gel (1:1 w/w crude/sorbent). Subsequent column chromatography (ethyl acetate/hexanes 1:6 to 1:1) afforded products (1, 2, 5, 8-29, 31-41, 43-54).

Method B: In microwave vial, acid (1.0 equiv.), HBTU (1.5 equiv.), HOBt (1.5 equiv.) and DIPEA (3.0 equiv.) were dissolved in anhydrous N,N-dimethylformamide (2.2 mL/mmol). Amine (1.2 equiv.) was added, and the resulting solution was stirring under microwave conditions (70 °C, 10 min, 15 W, 0 – 5 psi). The reaction was quenched with 2 mL of saturated ammonium chloride. The reaction mixture was extracted three times with ethyl acetate (5 mL). The combined organic phases were washed with 15 mL of water, 15 mL of brine and dried over anhydrous sodium sulfate. Then the solvent was removed under reduced pressure. The obtained crude product was purified via flash chromatography using n-hexane/ethyl acetate (0−100%) as the eluent to yield the final amides (3, 4, 6, 7, 30 and 42).

4.1.2.1. N-(2-Methoxybenzyl)-4,5-dimethyl-N-propylthiophene-2-carboxamide (1): yellow transparent oil (65 mg, 69%); R_f = 0.85 (DCM/MeOH = 9/1); ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.9 Hz, 1H), 7.24 (s, 1H), 7.21 (d, J = 7.5 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 4.78 (s, 2H), 3.84 (s, 3H), 3.40 (t, J = 7.8 Hz, 2H), 2.31 (s, 3H), 2.05 (s, 3H), 1.68 (sx, J = 7.4 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 157.3, 137.4, 133.2, 133.0, 132.0, 128.5, 127.5, 125.5, 120.8, 110.3, 55.4, 48.8, 29.8, 20.8, 13.7, 13.3, 11.4 ppm; HRMS (ESI+) m/z calcd for C₁₈H₂₄NO₂S⁺ [(M + H)⁺] 318.1522, found 318.1519 (error −0.9 ppm).

4.1.2.2. N-(2-Methoxybenzyl)-3,4-dimethyl-N-propylthiophene-2-carboxamide (2): colorless oil (66 mg, 69%); R_f = 0.31 (ethyl acetate/hexanes = 1/4); ¹H NMR (400 MHz, CDCl₃) δ 7.25 (t, J = 7.5 Hz, 1H), 7.20–7.10 (m, 1H), 6.94 (t, J = 7.4 Hz, 1H), 6.88–6.83 (m, 2H), 4.66 (s, 2H), 3.81 (s, 3H), 3.30 (s, 2H), 2.15 (s, 3H), 2.13 (s, 3H), 1.62 (sx, J = 7.2 Hz, 2H), 0.86 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.8, 157.5, 137.4, 136.7, 131.2, 128.9, 128.7, 125.1, 120.8, 120.6, 110.3, 55.3, 48.0, 42.8, 21.0, 14.9, 13.4, 11.4; HRMS (ESI+) m/z calcd for C₁₈H₂₄NO₂S⁺ [(M + H)⁺] 318.1522, found 318.1517 (error −1.6 ppm).

4.1.2.3. N-(2-Methoxybenzyl)-4-methyl-N-propylthiophene-2-carboxamide (3): yellow solid (42 mg, 65%); R_f = 0.38 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.34–7.19 (m, 2H), 7.09–6.92 (m, 3H), 6.89 (d, J = 8.2 Hz, 1H), 4.78 (s, 2H), 3.83 (s, 3H), 3.41 (t, J = 7.5 Hz, 2H), 2.19 (s, 3H), 1.69 (sx, J = 7.5 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 165.0, 157.3, 137.3, 130.8, 130.2, 129.9, 129.0, 128.5, 125.3, 125.2, 124.2, 120.7, 110.3, 55.3, 39.8, 15.7, 11.3. HRMS (ESI+) m/z calcd for C₁₇H₂₂NO₂S⁺ [(M + H)⁺] 304.1366, found 304.1359 (error −2.3 ppm).

4.1.2.4 N-(2-Methoxybenzyl)-N-propylthiophene-2-carboxamide (4): transparent oil (36 mg, 68%); R_f = 0.36 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.39 (d, J = 5.1 Hz, 1H), 7.29 (t, J = 7.5 Hz, 1H), 7.24 (d, J = 7.6 Hz, 2H), 6.97 (t, J = 7.5 Hz, 2H), 6.90 (d, J = 8.2 Hz, 1H), 4.78 (s, 2H), 3.83 (s, 3H), 3.47–3.36 (m, 2H), 1.70 (sx, J = 7.5 Hz, 2H), 0.90 (t, J = 7.5 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 157.3, 128.9, 128.6, 128.4, 126.9, 125.3, 120.8 (2C), 110.3 (2C), 55.3, 48.8, 40.0, 20.7, 11.4 ppm; HRMS (ESI+) m/z calcd for C₁₆H₂₀NO₂S⁺ [(M + H)⁺] 290.1209, found 290.1204 (error −1.7 ppm).

4.1.2.5. N-(2-Methoxybenzyl)-N-propylthiophene-3-carboxamide (5): light yellow oil (39 mg, 59%); R_f = 0.23 (ethyl acetate/hexanes = 1/5); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.52–7.39 (bs, 1H), 7.33–7.19 (m, 4H), 6.98 (t, J = 7.5 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H), 4.85–4.70 (brs, 0.6H, minor), 4.69–4.54 (brs, 1.4H, major), 3.80 (s, 3H), 3.49–3.32 (brs, 1.4H, major), 3.31–3.17 (brs, 0.6H, minor), 1.71–1.54 (m, 2H), 0.92 (t, J = 7.4 Hz, 2.1H, major), 0.78 (t, J = 7.4 Hz, 0.9H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 167.5 (2C), 157.2 (2C), 137.2 (2C), 129.1 (2C), 128.6 (2C), 127.6 (2C), 127.2 (2C), 125.9 (2C), 125.5 (2C), 120.8 (2C), 110.4 (2C), 55.3 (2C), (50.3, 42.5), (48.1, 47.6), (22.0, 20.4), (11.5, 11.2); HRMS (ESI+) m/z calcd for C₁₆H₂₀NO₂S⁺ [(M + H)⁺] 290.1209, found 290.1204 (error −1.7 ppm).

4.1.2.6. 5-Chloro-N-(2-methoxybenzyl)-N-propylthiophene-2-carboxamide (6): transparent oil (34 mg, 67%); R_f = 0.46 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.29 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.03–6.84 (m, 3H), 6.75 (bs, 1H), 4.75 (s, 2H), 3.84 (s, 3H), 3.45–3.34 (m, 2H), 1.69 (sx, J = 7.5 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 163.6, 157.2, 134.2, 128.8, 128.2, 126.2 (2C), 125.0, 120.9, 110.4 (2C), 55.4, 49.4, 40.3, 20.9, 11.4 ppm; HRMS (ESI+) m/z calcd for C₁₆H₁₉ClNO₂S⁺ [(M + H)⁺] 324.0820, found 324.0815 (error −1.5 ppm).

4.1.2.7. N-(2-Methoxybenzyl)-N-propylbenzo[b]thiophene-2-carboxamide (7): transparent oil (40 mg, 70%); R_f = 0.41 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J = 7.2 Hz, 1H), 7.77–7.62 (m, 1H), 7.50–7.31 (m, 3H), 7.29 (d, J = 7.7 Hz, 2H), 6.99 (t, J = 7.5 Hz, 1H), 6.90 (d, J = 8.2 Hz, 1H), 4.81 (s, 2H), 3.83 (s, 3H), 3.48–3.40 (m, 2H), 1.71 (sx, J = 7.5 Hz, 2H), 0.91 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 165.5, 157.4, 140.4, 139.0, 128.8, 125.7 (2C), 125.1, 124.73, 124.68, 122.4, 120.9 (2C), 110.4 (2C), 55.4, 48.3, 40.0, 20.5, 11.5 ppm; HRMS (ESI+) m/z calcd for C₂₀H₂₂NO₂S⁺ [(M + H)⁺] 340.1366, found 340.1361 (error −1.5 ppm).

4.1.2.8. N-(2-Methoxybenzyl)-N-propyl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide (8): colorless oil (79 mg, 73%); R_f = 0.29 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.5 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 6.99–6.92 (m, 2H), 6.89 (d, J = 8.2 Hz, 1H), 4.78 (s, 2H), 3.84 (s, 3H), 3.40 (t, J = 7.8 Hz, 2H), 2.73 (t, J = 6.2 Hz, 2H), 2.52 (brs, 2H), 1.85–1.72 (m, 4H), 1.68 (sx, J = 7.5 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 165.4, 157.3, 140.0, 135.3, 133.9, 129.9, 128.5, 127.7, 125.5, 120.8, 110.3, 55.4, 49.1, 39.9, 25.6, 25.1, 23.5, 22.8, 20.9, 11.4; HRMS (ESI+) m/z calcd for C₂₀H₂₆NO₂S⁺ [(M + H)⁺] 344.1679, found 344.1673 (error −1.7 ppm).

4.1.2.9. N-(2-Methoxybenzyl)-2-methyl-N-propylbenzamide (9): colorless oil (39 mg, 73%); R_f = 0.32 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.43 (d, J = 7.4 Hz, 0.47H minor), 7.32–7.18 (m, 4H + 0.53H major), 7.15–7.10 (m, 1H), 7.00–6.94 (m, 1H), 6.92 (d, J = 8.2 Hz, 0.47H, minor), 6.82 (d, J = 8.2 Hz, 0.53H, major), 5.13–4.88 (brs, 0.45H, minor), 4.87–4.60 (brs, 0.45H, minor), 4.37 (s, 1.1H, major), 3.85 (s, 1.4H, minor), 3.75 (s, 1.6H, major), 3.96–3.62 (brs, 0.55H, overlap with -OCH₃, major), 3.30–3.06 (brs, 0.55H, major), 2.97 (t, J = 7.7 Hz, 0.9H, minor), 2.34 (s, 1.6H, major), 2.29 (s, 1.4H, minor), 1.77–1.65 (m, 1.1H, major), 1.55–1.42 (m, 0.9H, minor), 0.95 (t, J = 7.4 Hz, 1.6H, major), 0.67 (t, J = 7.4 Hz, 1.4H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (172.0, 171.7), (157.8, 157.2), (137.1, 137.0), (134.3, 134.2), (130.42, 130.37), 130.0 (2C), (128.7, 128.6), 128.0 (2C), 126.0 (2C), (125.78, 125.75), (125.5, 125.0), (120.8, 120.6), (110.4, 110.3), (55.4, 55.2), (47.0, 46.2), (49.4, 41.4), (21.3, 20.5), (19.2, 19.1), (11.6, 11.2); HRMS (ESI+) m/z calcd for C₁₉H₂₄NO₂⁺ ([M + H⁺]) 298.1802, found 298.1796 (error −2.0 ppm).

4.1.2.10. N-(2-Methoxybenzyl)-3-methyl-N-propylbenzamide (10): light yellow oil (35 mg, 69%); R_f = 0.15 (ethyl acetate/hexanes = 1/4); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.35–7.13 (m, 6H), 6.97 (t, J = 7.5 Hz, 1H), 6.92–6.79 (m, 1H), 4.81 (s, 0.85H, minor), 4.49 (s, 1.15H, major), 3.86 (s, 1.3H, minor), 3.75 (s, 1.7H, major), 3.41 (t, J = 7.6 Hz, 1.15H, major), 3.11 (t, J = 7.6 Hz, 0.85H, minor), 2.37 (s, 1.3H, minor), 2.32 (s, 1.7H, major), 1.67 (sx, J = 7.5 Hz, 1.15H, major), 1.46–1.48 (m, 0.85H, minor), 0.94 (t, J = 7.4 Hz, 1.7H, major), 0.71 (t, J = 7.4 Hz, 1.3H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 172.6 (2C), 157.3 (2C), (138.3, 138.2), (137.2, 137.0), (130.1, 130.0), (128.6, 128.5), (128.3, 128.2), 127.9 (2C), (127.6, 127.3), (125.6, 125.3), 123.6 (2C), (120.8, 120.7), 110.3 (2C), (55.4, 55.2), (48.0, 46.5), (50.3, 41.9), 21.5 (2C), (21.7, 20.5), (11.5, 11.2); HRMS (ESI+) m/z calcd for C₁₉H₂₄NO₂⁺ ([M + H⁺]) 298.1802, found 298.1796 (error −2.0 ppm).

4.1.2.11. N-(2-Methoxybenzyl)-4-methyl-N-propylbenzamide (11): colorless oil (39 mg, 78%); R_f = 0.19 (ethyl acetate/hexanes = 1/1); ¹H NMR (400 MHz, CD₃OD) δ (mixture of rotamers) 7.32–7.09 (m, 6H), 7.03–6.90 (m, 2H), 4.76 (s, 0.9H, minor), 4.51 (s, 1.1H, major), 3.87 (s, 1.3H, minor), 3.74 (s, 1.7H, major), 3.39 (t, J = 7.5 Hz, 1.1H, major), 3.15 (t, J = 7.4 Hz, 0.9H, minor), 2.38 (s, 1.3H, minor), 2.35 (s, 1.7H, major), 1.62 (sx, J = 7.5 Hz, 1.1H, major), 1.57–1.47 (m, 0.9H, minor), 0.91 (t, J = 7.5 Hz, 1.7H, major), 0.68 (t, J = 7.5 Hz, 1.3H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 174.8 (2C), 158.9 (2C), 141.2 (2C), 135.0 (2C), 130.2 (2C), 130.0 (2C), 129.9 (2C), 129.4 (2C), 127.8 (2C), 127.6 (2C), 125.7 (2C), 121.6 (2C), 111.6 (2C), (55.9, 55.6), (49.8, 47.8), (51.7, 43.7), 22.5 (2C), 21.4 (2C), (11.7, 11.2); HRMS (ESI+) m/z calcd for C₁₉H₂₄NO₂⁺ ([M + H⁺]) 298.1802, found 298.1796 (error −2.0 ppm).

4.1.2.12. 2-Fluoro-N-(2-methoxybenzyl)-N-propylbenzamide (12): colorless oil (21 mg, 59%); R_f = 0.21 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.40–7.30 (m, 2.5H), 7.23–7.17 (m, 1.5H), 7.14–7.04 (m, 2H), 6.97 (t, J = 7.5 Hz, 0.47H, minor), 6.92 (t, J = 7.4 Hz, 0.53H, major), 6.89 (d, J = 8.2 Hz, 0.47H, minor), 6.80 (d, J = 8.2 Hz, 0.53H, major), 4.93–4.71 (brs, 0.9H, minor), 4.44 (s, 1.1H, major), 3.85 (s, 1.4H, minor), 3.73 (s, 1.6H, major), 3.57–3.27 (brs, 1.1H, major), 3.07 (t, J = 7.4 Hz, 0.9H, minor), 1.66 (sx, J = 7.4 Hz, 1.1H, major), 1.50 (h, J = 7.4 Hz, 0.9H, minor), 0.94 (t, J = 7.4 Hz, 1.6H, major), 0.70 (t, J = 7.4 Hz, 1.4H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (167.4, 167.2), [158.1 (d, ¹J_C-F = 247.7 Hz), 158.3 (d, ¹J_C-F = 247.3 Hz)], (157.6, 157.5), (130.9, 130.8), (128.92, 128.88), (128.86, 128.83), (128.52, 128.48), (125.5, 125.3), (125.0, 124.7), [124.6 (d, ⁴J_C-F = 3.5 Hz), 124.4 (d, ⁴J_C-F = 3.5 Hz)], (120.8, 120.6), [115.91 (d, ²J_C-F = 21.6 Hz), 115.87 (d, ²J_C-F = 21.6 Hz)], (110.4, 110.3), (55.4, 55.2), (49.9, 47.5), (46.1, 42.1), (21.5, 20.4), (11.4, 11.1); HRMS (ESI+) m/z calcd for C₁₈H₂₁FNO₂⁺ [(M + H)⁺] 302.1551, found 302.1544 (error −2.3 ppm).

4.1.2.13. 3-Fluoro-N-(2-methoxybenzyl)-N-propylbenzamide (13): light yellow oil (38 mg, 81%); R_f = 0.35 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.46–7.00 (m, 6H), 6.96 (t, J = 7.4 Hz, 1H), 6.93–6.78 (m, 1H), 4.80 (s, 0.8H, minor), 4.47 (s, 1.2H, major), 3.85 (rotamer, s, 1.2H, minor), 3.75 (s, 1.8H, major), 3.41 (t, J = 7.6 Hz, 1.2H, major), 3.10 (t, J = 7.6 Hz, 0.8H, minor), 1.67 (sx, J = 7.6 Hz, 1.2H, major), 1.59–1.49 (m, 0.8H, minor), 0.93 (t, J = 7.4 Hz, 1.8H, major), 0.72 (t, J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (170.8, 170.6), 162.5 (d, ¹J_C-F = 247.8 Hz, 2C), (157.7, 157.3), 139.1 (2C), 130.3 (2C), (130.14, 130.06), (129.3, 128.7), (128.8, 127.8), (125.2, 124.8), (122.38, 122.35), (120.77, 120.67), [116.4 (d, ²J_C-F = 21.2 Hz), 114.1 (d, ²J_C-F = 21.2 Hz)], 110.4 (2C), (55.4, 55.2), (48.1, 46.6), (50.2, 42.1), (21.6, 20.4), (11.5, 11.1); HRMS (ESI+) m/z calcd for C₁₈H₂₁FNO₂⁺ [(M + H)⁺] 302.1551, found 302.1545 (error −2.0 ppm).

4.1.2.14. 4-Fluoro-N-(2-methoxybenzyl)-N-propylbenzamide (14): light yellow oil (75 mg, 79%); R_f = 0.38 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.45–7.36 (m, 2H), 7.26 (t, J = 7.5 Hz, 1H), 7.23–7.02 (m, 3H), 6.96 (t, J = 7.5 Hz, 1H), 6.91–6.81 (m, 1H), 4.79 (s, 0.8H, minor), 4.48 (s, 1.2H, major), 3.84 (s, 1.2H, minor), 3.75 (s, 1.8H, major), 3.41 (t, J = 7.4 Hz, 1.2H), 3.12 (t, J = 7.4 Hz, 0.8H), 1.70–1.62 (m, 1.2H, major), 1.57–1.51 (m, 0.8H, minor), 0.92 (t, J = 7.4 Hz, 1.8H, major), 0.72 (J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ 171.3 (2C), 163.2 (d, ¹J_C-F = 248.6 Hz, 2C), (157.7, 157.2), 133.1 (2C), 129.3 (2C), 128.9 (d, ³J_C-F = 8.4 Hz, 2C), 128.7 (2C), 127.6 (2C), 125.3 (2C), 125.1 (2C), 120.7 (2C), 115.40 (d, ²J_C-F = 21.7 Hz, 2C), 110.4 (2C), (55.3, 55.2), (48.3, 46.9), (50.3, 42.2), (21.7, 20.4), (11.5, 11.1); HRMS (ESI+) m/z calcd for C₁₈H₂₁FNO₂⁺ [(M + H)⁺] 302.1551, found 302.1545 (error −2.0 ppm).

4.1.2.15. 2-Chloro-N-(2-methoxybenzyl)-N-propylbenzamide (15): colorless oil (37 mg, 69%); R_f = 0.21 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.49–7.08 (m, 6H), 7.00–6.78 (m, 2H), 5.04 (d, J = 15.2 Hz, 0.3H, minor), 4.66 (d, J = 15.4 Hz, 0.3H, minor), 4.46–4.28 (m, 1.4H, major), 3.93–3.83 (m, 0.6H, minor, overlapped with peak at 3.85 ppm), 3.85 (s, 0.9H, minor), 3.74 (s, 2.1H, major), 3.04–2.94 (m, 1.4H, major), 1.71 (sx, J = 7.5 Hz, 1.4H, major), 1.58–1.42 (m, 0.6H, minor), 0.96 (t, J = 7.4 Hz, 2.1H, major), 0.68 (t, J = 7.4 Hz, 0.9H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (168.9, 168.8), (157.7, 157.4), (136.8, 136.7), (130.6, 130.5), (129.97, 129.96), (129.8, 129.7), (129.6, 128.9), (128.6, 128.1), (128.3, 128.0), (127.02, 126.97), (125.1, 124.7), (120.8, 120.6), (110.4, 110.3), (55.5, 55.2), (47.1, 46.3), (49.6, 41.6), (21.4, 20.3), (11.7, 11.3); HRMS (ESI+) m/z calcd for C₁₈H₂₁ClNO₂⁺ [(M + H)⁺] 318.1255, found 318.1248 (error −2.2 ppm).

4.1.2.16. 3-Chloro-N-(2-methoxybenzyl)-N-propylbenzamide (16): colorless oil (35 mg, 80%); R_f = 0.27 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.44–7.22 (m, 5H), 7.14 (d, J = 7.5 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 6.92–6.80 (m, 1H), 4.79 (s, 0.8H, minor), 4.46 (s, 1.2H, major), 3.86 (s, 1.2H, minor), 3.75 (s, 1.8H, major), 3.40 (t, J = 7.6 Hz, 1.2H, major), 3.10 (t, J = 7.6 Hz, 0.8H, minor), 1.66 (sx, J = 7.5 Hz, 1.2H, major), 1.54 (sx, J = 7.5 Hz, 0.8H, minor), 0.93 (t, J = 7.4 Hz, 1.8H, major), 0.72 (t, J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (170.7, 170.5), (157.7, 157.3), 138.8 (2C), 134.4 (2C), (129.9, 129.7), 129.4 (2C), (128.9, 128.7), 128.0 (2C), (127.2, 126.9), 125.2 (2C), 124.7 (2C), (120.8, 120.7), 110.4 (2C), (55.4, 55.2), (48.2, 46.6), (50.2, 42.1), (21.7, 20.4), (11.5, 11.1); HRMS (ESI+) m/z calcd for C₁₈H₂₁ClNO₂⁺ [(M + H)⁺] 318.1255, found 318.1248 (error −2.2 ppm).

4.1.2.17. 4-Chloro-N-(2-methoxybenzyl)-N-propylbenzamide (17): colorless oil (41mg, 76%); R_f = 0.27 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CD₃OD) δ (mixture of rotamers) 7.51–7.37 (m, 4H), 7.29 (t, J = 7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.03–6.90 (m, 2H), 4.77 (s, 0.8H, minor), 4.49 (s, 1.2H, major), 3.87 (s, 1.2H, minor), 3.74 (s, 1.8H, major), 3.36 (t, J = 7.5 Hz, 1.2H, major), 3.13 (t, J = 7.4 Hz, 0.8H, minor), 1.68–1.47 (m, 2H), 0.91 (t, J = 7.4 Hz, 1.8H, major), 0.69 (t, J = 7.5 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (173.3, 173.2), (159.1, 158.9), 136.7 (2C), 136.5 (2C), 130.4 (2C), 130.2 (2C), 129.9 (2C), 129.7 (2C), 129.5 (2C), 129.3 (2C), 125.5 (2C), 121.6 (2C), 111.7 (2C), (55.9, 55.7), (50.0, 47.9), (51.6, 43.8), (22.5, 21.3), (11.6, 11.2); HRMS (ESI+) m/z calcd for C₁₈H₂₁ClNO₂⁺ [(M + H)⁺] 318.1255, found 318.1249 (error −1.9 ppm).

4.1.2.18. 2-Methoxy-N-(2-methoxybenzyl)-N-propylbenzamide (18): colorless oil (37 mg, 78%); R_f = 0.29 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.46 (d, J = 7.4, 0.5H, trans), 7.34 (t, J = 7.6 Hz, 0.5H), 7.29–7.19 (m, 2.5H), 7.15 (d, J = 7.5 Hz, 0.5H, cis), 6.99 (t, J = 7.4 Hz, 0.5H, cis), 6.97 (t, J = 7.4 Hz, 0.5H, trans), 6.93–6.89 (m, 1H + 0.5H trans), 6.88 (d, J = 8.1, 0.5H, trans), 6.86 (d, J = 8.3, 0.5H, cis), 6.79 (d, J = 8.1 Hz, 0.5H, cis), 5.04 (d, J = 15.7 Hz, 0.5H, trans), 4.62 (d, J = 15.7 Hz, 0.5H, trans), 4.37 (d, J = 16.4 Hz, 1H, cis), 3.86 (s, 1.5H, trans), 3.85 (s, 1.5H, trans), 3.78 (s, 1.5H, cis), 3.73 (s, 1.5H, cis), 3.70–3.60 (brs, 0.5H, cis), 3.26–3.17 (brs, 0.5H, cis), 3.07–2.94 (m, 1H, trans), 1.72–1.61 (m, 1H, cis), 1.52–1.41 (m, 1H, trans), 0.95 (t, J = 7.4 Hz, 1.5H, cis), 0.67 (t, J = 7.4 Hz, 1.5H, trans); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (170.0, 169.9), (157.6, 157.3), (155.5, 155.3), (130.10, 130.08), (128.5, 128.4), (128.6, 128.1), (128.0, 127.9), (127.0, 126.9), (125.5, 125.3), (121.0, 120.8), (120.7, 120.5), (111.04, 111.00), 110.2 (2C), (55.64, 55.57), (55.4, 55.2), (46.9, 46.0), (49.8, 41.7), (21.5, 20.4), (11.4, 11.2); HRMS (ESI+) m/z calcd for C₁₉H₂₄NO₃⁺ ([M + H⁺]) 314.1751, found 314.1746 (error −1.6 ppm).

4.1.2.19. 3-Methoxy-N-(2-methoxybenzyl)-N-propylbenzamide (19): colorless oil (36 mg, 81%); R_f = 0.16 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.34–7.15 (m, 3H), 7.00–6.80 (m, 5H), 4.80 (s, 0.8H, minor), 4.49 (s, 1.2H, major), 3.89–3.79 (m, 3H, 2 rotamers overlapped), 3.74 (s, 1.8H, major), 3.68 (s, 1.2H, minor), 3.42 (t, J = 7.5 Hz, 1.2H, major), 3.12 (t, J = 7.5 Hz, 0.8H, minor), 1.68 (sx, J = 7.6 Hz, 1.2H, major), 1.60–1.48 (m, 0.8H, minor), 0.94 (t, J = 7.4 Hz, 1.8H, major), 0.72 (t, J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (172.1, 171.9), (159.6, 159.4), (157.7, 157.2), (138.5, 138.2), 129.2 (2C), (129.5, 128.6), 127.6 (2C), (125.4, 125.3), (120.8, 120.6), 118.9 (2C), (115.7, 115.0), (112.2, 111.8), 110.3 (2C), 55.4 (2C), (55.3, 55.2), (48.0, 46.8), (50.2, 42.0), (21.7, 20.4), (11.5, 11.2); HRMS (ESI+) m/z calcd for C₁₉H₂₄NO₃⁺ ([M + H⁺]) 314.1751, found 314.1746 (error −1.6 ppm).

4.1.2.20. N-(2-Methoxybenzyl)-N-propyl-2-(trifluoromethyl)benzamide (20): colorless oil (40 mg, 68%); R_f = 0.33 (ethyl acetate/hexanes = 1/4); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.71–7.65 (m, 1H), 7.58 (t, J = 7.4 Hz, 0.4H, minor), 7.53–7.31 (m, 3H), 7.23–7.30 (m, 1H), 7.14 (d, J = 7.4 Hz, 0.6H, major), 7.00–6.94 (m, 1H), 6.89 (d, J = 8.2 Hz, 0.4H, minor), 6.82 (d, J = 8.2 Hz, 0.6H, major), 5.00 (d, J = 15.0 Hz, 0.4H, minor), 4.71 (d, J = 15.0 Hz, 0.4H, minor), 4.36 (d, J = 16.6 Hz, 0.6H, major), 4.21 (d, J = 16.6 Hz, 0.6H, major), 4.01–3.91 (m, 0.8H, minor), 3.85 (s, 1.2H, minor), 3.74 (s, 1.8H, major), 2.98–2.81 (m, 1.2H, major), 1.68 (sx, J = 7.5 Hz, 1.2H, major), 1.60–1.38 (m, 0.8H, minor), 0.92 (t, J = 7.4 Hz, 1.8H, major), 0.66 (t, J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (169.2, 169.0), (157.7, 157.3), 132.0 (2C), 130.3 (2C), (128.93, 128.92), (128.84, 128.77), 127.9 (2C), 127.7 (2C), 127.3 (2C), [126.7 (q, ³J_C-F = 4.6 Hz), 126.6 (q, ³J_C-F = 4.6 Hz)], 125.1 (2C), 124.5 (2C), (120.9, 120.6), (110.4, 110.3), (55.4, 55.2), (47.6, 46.6), (49.9, 41.4), (21.1, 19.8), (11.5, 11.2); HRMS (ESI+) m/z calcd for C₁₉H₂₁F₃NO₂⁺ ([M + H⁺]) 352.1519, found 352.1518 (error −0.3 ppm).

4.1.2.21. N-(2-Methoxybenzyl)-N-propyl-3-(trifluoromethyl)benzamide (21): colorless oil (35 mg, 75%); R_f = 0.22 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.71–7.43 (m, 3H), 7.38–7.23 (m, 2H), 7.15 (d, J = 7.4 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 6.93–6.80 (m, 1H), 4.82 (s, 0.8H, minor), 4.45 (s, 1.2H, major), 3.86 (s, 1.2H, minor), 3.73 (s, 1.8H, major), 3.43 (t, J = 7.5 Hz, 1.2H, major), 3.08 (t, J = 7.5 Hz, 0.8H, minor), 1.68 (sx, J = 7.5 Hz, 1.2H, major), 1.60–1.50 (m, 0.8H, minor), 0.94 (t, J = 7.4 Hz, 1.8H, major), 0.72 (t, J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 170.7 (2C), 157.3 (2C), 137.9 (2C), 130.0 (2C), 129.5 (2C), (129.1, 128.9), 128.1 (2C), (126.11, 126.08), 125.2 (2C), 124.7 (2C), (124.1, 123.8), 122.5 (2C), (120.8, 120.7), 110.5 (2C), (55.4, 55.2), (48.5, 46.8), (50.2, 42.3), (21.7, 20.5), (11.5, 11.1); HRMS (ESI+) m/z calcd for C₁₉H₂₁F₃NO₂⁺ ([M + H⁺]) 352.1519, found 352.1513 (error −1.7 ppm).

4.1.2.22. 3-Bromo-N-(2-methoxybenzyl)-N-propylbenzamide (22): light yellow oil (43 mg, 76%); R_f = 0.17 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CD₃OD) δ (mixture of rotamers) 7.64–7.53 (m, 2H), 7.40–7.25 (m, 3H), 7.13 (d, J = 7.4 Hz, 1H), 7.01–6.90 (m, 2H), 4.76 (s, 0.8H, minor), 4.46 (s, 1.2H, major), 3.87 (s, 1.2H, minor), 3.74 (s, 1.8H, major), 3.36 (t, J = 7.5 Hz, 1.2H, major), 3.12 (t, J = 7.4 Hz, 0.8H, minor), 1.65–1.46 (m, 2H), 0.90 (t, J = 7.4 Hz, 1.8H, major), 0.70 (t, J = 7.5 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (172.5, 172.4), 158.9 (2C), 140.0 (2C), 133.7 (2C), (131.7, 131.3), 130.8 (2C), (130.5, 130.3), (130.0, 129.9), (126.4, 126.3), (125.8, 125.4), (123.5, 123.3), (121.7, 121.6), 111.7 (2C), (55.9, 55.7), (50.0, 47.9), (51.6, 43.8), (22.5, 21.3), (11.6, 11.2); HRMS (ESI+) m/z calcd for C₁₈H₂₁BrNO₂⁺ ([M + H⁺]) 362.0750, found 362.0743 (error −1.9 ppm).

4.1.2.23. 3,4-Dibromo-N-(2-methoxybenzyl)-N-propylbenzamide (23): colorless oil (126 mg, 85%); R_f = 0.29 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.67 (s, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.22–7.08 (m, 2H), 6.96 (t, J = 7.5 Hz, 1H), 6.92–6.79 (m, 1H), 4.77 (s, 0.7H, minor), 4.44 (s, 1.3H, major), 3.85 (s, 1.1H, minor), 3.76 (s, 1.9H, major), 3.39 (t, J = 7.5 Hz, 1.3H, major), 3.09 (t, J = 7.6 Hz, 0.7H, minor), 1.64 (sx, J = 7.5 Hz, 1.3H, major), 1.59–1.49 (m, 0.7H, minor), 0.92 (t, J = 7.5 Hz, 1.9H, major), 0.74 (t, J = 7.5 Hz, 1.1H, minor); ¹³C NMR (100 MHz, CDCl₃) δ 169.7 (2C), 157.3 (2C), 137.6 (2C), (133.8, 133.6), (132.3, 132.0), 129.5 (2C), (129.1, 128.8), 127.9 (2C), 126.7 (2C), 125.9 (2C), (125.0, 124.6), (120.8, 120.7), 110.5 (2C), (55.4, 55.2), (48.4, 46.8), (50.2, 42.2), (21.7, 20.4), (11.5, 11.2); HRMS (ESI+) m/z calcd for C₁₈H₂₀Br₂NO₂⁺ ([M + H⁺]) 439.9855, found 439.9847 (error −1.8 ppm).

4.1.2.24. 3-Bromo-2-fluoro-N-(2-methoxybenzyl)-N-propylbenzamide (24): light yellow oil (44 mg, 67%); R_f = 0.33 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.61–7.50 (m, 1H), 7.35–7.22 (m, 2H), 7.09 (t, J = 7.8 Hz, 1H), 7.04–6.96 (m, 2H), 6.81 (d, J = 8.2 Hz, 1H), 5.02–4.64 (brs, 0.9H, minor), 4.41 (s, 1.1H, major), 3.86 (s, 1.3H, minor), 3.74 (s, 1.7H, major), 3.60–3.25 (brs, 1.1H, major), 3.05 (t, J = 7.5 Hz, 0.9H, minor), 1.65 sx, J = 7.5 Hz, 1.1H, major), 1.51 (sx, J = 7.4 Hz, 0.9H, minor), 0.94 (t, J = 7.4 Hz, 1.7H, major), 0.71 (t, J = 7.4 Hz, 1.3H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (166.1, 166.0), (157.7, 157.5), [154.8 (d, ¹J_C-F = 248.1 Hz), 154.6 (d, ¹J_C-F = 248.0 Hz)], (134.31, 134.26), 129.1 (2C), (128.98, 128.97), (127.88, 127.85), [126.80 (d, ²J_C-F = 19.6 Hz), 126.75 (d, ²J_C-F = 19.8 Hz)], [125.7 (d, ⁴J_C-F = 4.2 Hz), 125.5 (d, ⁴J_C-F = 4.2 Hz)], (124.7, 124.3), (120.9, 120.6), (110.43, 110.36), [109.70 (d, ²J_C-F = 21.1 Hz), 109.62 (d, ²J_C-F = 21.1 Hz)], (55.4, 55.2), (49.8, 47.7), (46.2, 42.2), (21.4, 20.4), (11.4, 11.1); HRMS (ESI+) m/z calcd for C₁₈H₂₀BrFNO₂⁺ ([M + H⁺]) 380.0656, found 380.0650 (error −1.6 ppm).

4.1.2.25. 3-Bromo-4-fluoro-N-(2-methoxybenzyl)-N-propylbenzamide (25): colorless oil (85 mg, 78%); R_f = 0.27 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.67–7.61 (m, 1H), 7.38–7.23 (m, 2H), 7.19–7.01 (m, 2H), 6.97 (t, J = 7.5 Hz, 1H), 6.92–6.82 (m, 1H), 4.78 (s, 0.8H, minor), 4.46 (s, 1.2H, major), 3.85 (s, 1.2H, minor), 3.76 (s, 1.8H, major), 3.40 (t, J = 7.4 Hz, 1.2H, major), 3.11 (t, J = 7.5 Hz, 0.8H, minor), 1.74–1.60 (m, 1.2H, major), 1.60–1.48 (m, 0.8H, minor), 0.93 (t, J = 7.5 Hz, 1.8H, major), 0.74 (t, J = 7.5 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ 169.8 (2C), 159.6 (d, ¹J_C-F = 250.2 Hz, 2C), (157.7, 157.3), 134.5 (2C), (132.6, 132.3), (129.5, 129.0), 127.9 (2C), (127.6, 127.5), 124.7 (2C), 120.7 (2C), (116.5, 116.3), 110.5 (2C), 109.2 (d, ²J_C-F = 21.8 Hz, 2C), 55.3 (2C), (48.5, 46.9), (50.3, 42.3), (21.7, 20.4), (11.5, 11.2); HRMS (ESI+) m/z calcd for C₁₈H₂₀BrFNO₂⁺ ([M + H⁺]) 380.0656, found 380.0649 (error −1.8 ppm).

4.1.2.26. 3-Bromo-5-fluoro-N-(2-methoxybenzyl)-N-propylbenzamide (26): light yellow oil (32 mg, 66%); R_f = 0.21 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.34 (s, 1H), 7.32–7.21 (m, 2H), 7.14–7.02 (m, 2H), 6.97 (t, J = 7.4 Hz, 1H), 6.92–6.81 (m, 1H), 4.77 (s, 0.8H, minor), 4.44 (s, 1.2H, major), 3.86 (s, 1.2H, minor), 3.76 (s, 1.8H, major), 3.39 (t, J = 7.5 Hz, 1.2H, major), 3.08 (t, J = 7.5 Hz, 0.8H, minor), 1.63 (sx, J = 7.5 Hz, 1.2H, major), 1.59–1.47 (m, 0.8H, minor), 0.92 (t, J = 7.4 Hz, 1.8H, major), 0.75 (t, J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (169.2, 169.0), 162.26 (d, ¹J_C-F = 251.5 Hz, 2C), (157.7, 157.3), 140.4 (2C), (129.5, 128.9), (129.2, 128.2), (126.0, 125.7), (124.9, 124.4), 122.8 (2C), (120.9, 120.7), (120.1, 119.8), (113.1, 112.9), 110.5 (2C), (55.5, 55.2), (48.4, 46.7), (50.1, 42.3), (21.6, 20.4), (11.5, 11.2); HRMS HRMS (ESI+) m/z calcd for C₁₈H₂₀BrFNO₂⁺ ([M + H⁺]) 380.0656, found 380.0651 (error −1.3 ppm).

4.1.2.27. 6-Bromo-N-(2-methoxybenzyl)-N-propylpicolinamide (27): colorless oil (45mg, 62%); R_f = 0.31 (ethyl acetate/ hexanes = 1/3); ¹H NMR (400 MHz, CD₃OD) δ (mixture of rotamers) 7.85–7.73 (m, 1H), 7.71–7.51 (m, 2H), 7.32–7.17 (m, 2H), 7.02–6.87 (m, 2H), 4.78 (s, 0.9H, minor), 4.61 (s, 1.1H, major), 3.88 (s, 1.35H, minor), 3.73 (s, 1.65H, major), 3.39 (t, J = 7.6 Hz, 1.1H, major), 3.22 (t, J = 7.6 Hz, 0.9H, minor), 1.70–1.51 (m, 2H), 0.92 (t, J = 7.4 Hz, 1.65H, major), 0.75 (t, J = 7.4 Hz, 1.35H, minor); ¹³C NMR (100 MHz, CD₃OD) δ (mixture of rotamers) (169.7, 169.6), 159.0 (2C), (156.7, 156.4), (141.8, 141.6), (141.3, 141.0), (130.40, 130.39), (130.5, 130.2), (129.90, 129.87), (125.7, 125.5), 123.3 (2C), (121.7, 121.5), 111.6 (2C), (55.9, 55.6), (49.2, 48.4), (51.7, 44.5), (22.7, 21.4), (11.6, 11.3); HRMS (ESI+) m/z calcd for C₁₇H₂₀BrN₂O₂⁺ ([M + H⁺]) 363.0703, found 363.0699 (error −1.1 ppm).

4.1.2.28. 5-Bromo-N-(2-methoxybenzyl)-N-propylnicotinamide (28): light yellow oil (46 mg, 63%); R_f = 0.27 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CD₃OD) δ (mixture of rotamers) 8.77–8.68 (m, 1H), 8.58–8.52 (m, 1H), 8.09–8.00 (m, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.17 (d, J = 7.4 Hz, 1H), 7.04–6.91 (m, 2H), 4.79 (s, 0.6H, minor), 4.48 (s, 1.4H, major), 3.88 (s, 0.95H, minor), 3.74 (s, 2.05H, major), 3.40 (t, J = 7.5 Hz, 1.4H, major), 3.14 (t, J = 7.6 Hz, 0.6H, minor), 1.65–1.49 (m, 2H), 0.91 (t, J = 7.4 Hz, 2.05H, major), 0.72 (t, J = 7.5 Hz, 0.95H, minor); ¹³C NMR (100 MHz, CD₃OD) δ (mixture of rotamers) 169.6 (2C), 158.9 (2C), (152.5, 152.3), (146.6, 146.4), (138.6, 138.5), 135.8 (2C), 130.7 (2C), 130.3 (2C), (130.5, 130.2), 125.1 (2C), (121.7, 121.5), 111.8 (2C), (55.9, 55.7), (50.2, 48.3), (51.6, 44.0), (22.6, 21.4), (11.6, 11.2); HRMS (ESI+) m/z calcd for C₁₇H₂₀BrN₂O₂⁺ ([M + H⁺]) 363.0703, found 363.0690 (error −3.6 ppm).

4.1.2.29. 2-Bromo-N-(2-methoxybenzyl)-N-propylisonicotinamide (29): light yellow oil (14 mg, 37%); R_f = 0.29 (dichloromethane/ methanol = 10/1); ¹H NMR (400 MHz, CD₃OD) δ (mixture of rotamers) 8.54–8.37 (m, 1H), 7.63–7.57 (m, 1H), 7.42–7.36 (m, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.18–7.06 (m, 1H), 7.02–6.87 (m, 2H), 4.77 (s, 0.6H, minor), 4.44 (s, 1.4H, major), 3.88 (s, 0.9H, minor), 3.76 (s, 2.1H, major), 3.38 (t, J = 7.5 Hz, 1.4H, major), 3.18 (t, J = 7.6 Hz, 0.6H, minor), 1.65–1.49 (m, 2H), 0.91 (t, J = 7.5 Hz, 2.1H, major), 0.73 (t, J = 7.5 Hz, 0.9H, minor); ¹³C NMR (100 MHz, CD₃OD) δ (mixture of rotamers) 161.2 (2C), 159.3 (2C), 152.2 (2C), 143.5 (2C), 141.7 (2C), (132.7, 131.5), 130.1 (2C), 128.8 (2C), 127.2 (2C), 123.4 (2C), (121.3, 121.2), (111.7, 111.5), 58.1 (2C), 45.4 (2C), (39.1, 35.8), 29.0 (2C), (15.8, 12.2); HRMS (ESI+) m/z calcd for C₁₇H₂₀BrN₂O₂⁺ ([M + H⁺]) 363.0703, found 363.0689 (error −3.9 ppm).

4.1.2.30. N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (30): White solid (46 mg, 68%); R_f = 0.42 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ 7.32 (d, J = 7.5, 1H), 7.27 (t, J = 7.9, 1H), 7.19 (s, 1H), 6.92 (t, J = 7.6, 1H), 6.89 (d, J = 8.0, 1H), 6.37 (s, 1H), 4.58 (d, J = 5.8 Hz, 2H), 3.88 (s, 3H), 2.34 (s, 3H), 2.11 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 161.9, 157.7, 138.4, 134.2, 133.6, 131.2, 130.0, 129.0, 126.4, 120.9, 110.5, 55.5, 39.9, 13.7, 13.6 ppm; HRMS (ESI+) m/z calcd for C₁₅H₁₈NO₂S⁺ [(M + H)⁺] 276.1053, found 276.1049 (error −1.4 ppm).

4.1.2.31. N-(2-Methoxybenzyl)-N,4,5-trimethylthiophene-2-carboxamide (31): light yellow oil (95 mg, 79%); R_f = 0.32 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.28 (t, J = 7.5, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.07–6.92 (m, 2H), 6.89 (d, J = 8.1 Hz, 1H), 4.78 (s, 2H), 3.83 (s, 3H), 3.16–3.00 (brs, 3H), 2.32 (s, 3H), 2.06 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 157.5, 157.4, 137.6, 133.2, 132.8, 132.3, 129.0, 128.6, 125.1, 120.8, 110.4, 62.2, 55.4, 39.9, 13.7, 13.3; HRMS (ESI+) m/z calcd for C₁₆H₂₀NO₂S⁺ [(M + H)⁺] 290.1209, found 290.1205 (error −1.4 ppm).

4.1.2.32. N-Ethyl-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (32): colorless oil (27 mg, 45%); R_f = 0.27 (ethyl acetate/ hexanes = 1/4); ¹H NMR (400 MHz, CDCl₃) δ 7.29–7.20 (m, 2H), 7.00–6.92 (m, 2H), 6.89 (d, J = 8.2 Hz, 1H), 4.77 (s, 2H), 3.84 (s, 3H), 3.51 (q, J = 7.1 Hz, 2H), 2.31 (s, 3H), 2.05 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 157.3, 137.4, 133.2, 132.9, 132.0, 128.5, 127.8, 125.5, 120.8, 110.3, 55.4, 42.4, 29.7, 13.7, 13.3, 12.9; HRMS (ESI+) m/z calcd for C₁₇H₂₂NO₂S⁺ [(M + H)⁺] 304.1366, found 304.1360 (error −2.0 ppm).

4.1.2.33. N-Butyl-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (33): light yellow oil (46 mg, 69%); R_f = 0.17 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.4 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 7.00–6.91 (m, 2H), 6.88 (d, J = 8.1 Hz, 1H), 4.77 (s, 2H), 3.84 (s, 3H), 3.43 (t, J = 7.6 Hz, 2H), 2.31 (s, 3H), 2.05 (s, 3H), 1.64 (p, J = 7.6 Hz, 2H), 1.31 (sx, J = 7.5 Hz, 2H), 0.91 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 157.3, 137.4, 133.2, 133.0, 132.0, 128.5, 128.1, 125.5, 120.8, 110.3, 55.4, 47.2, 39.0, 29.8, 20.3, 14.0, 13.7, 13.3; HRMS (ESI+) m/z calcd for C₁₉H₂₆NO₂S⁺ [(M + H)⁺] 322.1679, found 322.1674 (error −1.6 ppm).

4.1.2.34. N-Isopropyl-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (34): light yellow oil (20 mg, 35%); R_f = 0.25 (ethyl acetate/ hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.29–7.20 (m, 2H), 6.99–6.90 (m, 2H), 6.87 (d, J = 8.1 Hz, 1H), 4.68 (s, 2H), 4.68–4.56 (m, 1H), 3.86 (s, 3H), 2.31 (s, 3H), 2.04 (s, 3H), 1.18 (d, J = 6.8 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 165.3, 156.5, 138.7, 133.7, 133.2, 131.6, 127.9, 127.6, 127.1, 120.6, 110.1, 55.4, 49.5, 20.7, 14.2, 13.72, 13.68, 13.3; HRMS (ESI+) m/z calcd for C₁₈H₂₄NO₂S⁺ [(M + H)⁺] 318.1522, found 318.1516 (error −1.9 ppm).

4.1.2.35. N-Cyclopropyl-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (35): light yellow oil (35 mg, 70%); R_f = 0.19 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.32 (s, 1H), 7.28–7.15 (m, 2H), 6.95–6.82 (m, 2H), 4.76 (s, 2H), 3.81 (s, 3H), 2.78 (dt, J = 6.7, 2.8 Hz, 1H), 2.34 (s, 3H), 2.11 (s, 3H), 0.80–0.69 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 165.8, 157.4, 138.5, 133.9, 133.6, 133.2, 128.9, 128.3, 126.2, 120.5, 110.3, 62.1, 55.4, 47.4, 31.4, 13.7, 13.4, 10.7; HRMS (ESI+) m/z calcd for C₁₈H₂₂NO₂S⁺ [(M + H)⁺] 316.1366, found 316.1360 (error −1.9 ppm).

4.1.2.36. N-(2-Hydroxyethyl)-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (36): colorless oil (48 mg, 69%); R_f = 0.23 (ethyl acetate/hexanes = 1/3); ¹H NMR (601 MHz, CDCl₃) δ 7.30 (t, J = 7.8 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.02–6.95 (m, 2H), 6.90 (d, J = 8.1 Hz, 1H), 4.84 (s, 2H), 3.82 (s, 3H), 3.78 (t, J = 5.0 Hz, 2H), 3.64 (t, J = 5.0 Hz, 2H), 3.52–3.24 (brs, 1H), 2.30 (s, 3H), 2.02 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 167.3, 157.2, 138.8, 133.6, 132.8, 131.9, 128.9, 127.4, 124.7, 120.9, 110.4, 61.9, 55.4, 50.9, 49.5, 13.7, 13.3; HRMS (ESI+) m/z calcd for C₁₇H₂₂NO₃S⁺ [(M + H)⁺] 320.1315, found 320.1309 (error −1.9 ppm).

4.1.2.37. N-(3-Hydroxypropyl)-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (37): colorless oil (43 mg, 73%); R_f = 0.27 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.30 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.00–6.93 (m, 2H), 6.91 (d, J = 8.1 Hz, 1H), 4.78 (s, 2H), 4.22–4.01 (brs, 1H), 3.83 (s, 3H), 3.63 (t, J = 6.2 Hz, 2H), 3.58 (q, J = 5.9 Hz, 2H), 2.30 (s, 3H), 2.01 (s, 3H), 1.74 (p, J = 5.8 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 166.3, 157.2, 138.5, 133.5, 132.6, 131.9, 128.8, 127.1, 124.6, 120.9, 110.4, 58.5, 55.3, 47.8, 42.5, 30.0, 13.7, 13.3; HRMS (ESI+) m/z calcd for C₁₈H₂₄NO₃S⁺ [(M + H)⁺] 334.1471, found 334.1465 (error −1.8 ppm).

4.1.2.38. N-(2-Methoxybenzyl)-N-(2-methoxyethyl)-4,5-dimethylthiophene-2-carboxamide (38): colorless oil (23 mg, 45%); R_f = 0.13 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.26 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.04–6.91 (m, 2H), 6.88 (d, J = 8.1 Hz, 1H), 4.87 (s, 2H), 3.82 (s, 3H), 3.69–3.58 (m, 4H), 3.32 (s, 3H), 2.30 (s, 3H), 2.03 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 165.3, 157.2, 137.7, 133.2, 132.7, 132.3, 128.5, 127.5, 125.4, 120.8, 110.3, 70.7, 62.2, 59.0, 55.3, 47.2, 13.7, 13.3; HRMS (ESI+) m/z calcd for C₁₈H₂₄NO₃S⁺ [(M + H)⁺] 334.1471, found 334.14657 (error −4.1 ppm).

4.1.2.39. N-(3-Fluoropropyl)-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (39): colorless oil (39 mg, 63%); R_f = 0.29 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.43 (s, 1H), 7.31–7.22 (m, 2H), 6.91 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 4.29 (t, J = 6.1 Hz, 1.8H, major), 4.15 (t, J = 6.2 Hz, 0.2H, minor), 3.94 (s, 2H), 3.84 (s, 2.7H, major), 3.76 (s, 0.3H, minor), 3.21 (s, 0.2H, minor), 2.82 (t, J = 7.2 Hz, 1.8H, major), 2.35 (s, 2.7H, major), 2.32 (s, 0.3H, minor), 2.12 (s, 2.7H, major), 2.08 (s, 0.3H, minor), 2.02 (p, J = 6.6 Hz, 1.8H, major), 1.98–1.92 (m, 0.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 162.5 (2C), 157.9 (2C), 142.1 (2C), 136.5 (2C), 134.7 (2C), 131.1 (2C), 130.2 (2C), 127.9 (2C), 122.6 (2C), 120.8 (2C), 110.5 (2C), (70.8, 62.2), (59.0, 55.5), (49.0, 48.6), 45.0, 27.1 (2C), 13.8 (2C), 13.6 (2C); HRMS (ESI+) m/z calcd for C₁₈H₂₃FNO₂S⁺ [(M + H)⁺] 336.1428, found 336.1423 (error −1.5 ppm).

4.1.2.40. N-(3,3-Difluoropropyl)-N-(2-methoxybenzyl)-4,5-dimethylthiophene-2-carboxamide (40): colorless oil (43 mg, 76%); R_f = 0.35 (ethyl acetate/hexanes = 1/1); ¹H NMR (400 MHz, CDCl₃) δ 7.30 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.00–6.94 (m, 2H), 6.90 (d, J = 8.1 Hz, 1H), 5.90 (tt, ²J_H-F = 56.4, ³J_H-H = 4.4 Hz, 1H), 4.80 (s, 2H), 3.83 (s, 3H), 3.57 (t, J = 7.3 Hz, 2H), 2.32 (s, 3H), 2.28–2.11 (m, 2H), 2.05 (s, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 165.4, 157.4, 138.1, 137.4, 133.4, 132.4, 129.0, 127.8, 124.8, 120.9, 116.2 (t, ¹J_C-F = 239.2 Hz), 110.5, 55.4, 48.8, 41.1, 32.6, 13.7, 13.3; HRMS (ESI+) m/z calcd for C₁₈H₂₂F₂NO₂S⁺ [(M + H)⁺] 354.1334, found 354.1327 (error −2.0 ppm).

4.1.2.41. N-(2-Methoxybenzyl)-4,5-dimethyl-N-(3,3,3-trifluoropropyl)thiophene-2-carboxamide (41): colorless oil (68 mg, 77%); R_f = 0.39 (ethyl acetate/hexanes = 1/5); ¹H NMR (400 MHz, CDCl₃) δ 7.31 (t, J = 7.5 Hz, 1H), 7.20 (d, J = 7.5 Hz, 1H), 7.00–6.94 (m, 2H), 6.91 (d, J = 8.2 Hz, 1H), 4.81 (s, 2H), 3.83 (s, 3H), 3.62 (t, J = 7.5 Hz, 2H), 2.56–2.40 (m, 2H), 2.32 (s, 3H), 2.06 (s, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 165.3, 157.3, 138.1, 133.4, 132.4, 132.1, 129.1, 127.9, 126.3 (q, ¹J_C-F = 276.8 Hz), 124.6, 120.9, 110.5, 55.3, 49.0, 41.0, 31.9, 13.6, 13.2; HRMS (ESI+) m/z calcd for C₁₈H₂₁F₃NO₂S⁺ [(M + H)⁺] 372.1240, found 372.1233 (error −1.9 ppm).

4.1.2.42. (4,5-diMethylthiophen-2-yl)(8-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone (42): yellow oil (42 mg, 69%); R_f = 0.42 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ 7.19–7.10 (m, 2H), 6.77 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 4.81 (s, 2H), 3.91 (t, J = 5.8 Hz, 2H), 3.82 (s, 3H), 2.93 (t, J = 5.8 Hz, 2H), 2.36 (s, 3H), 2.15 (s, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.1, 156.1, 137.5, 135.8, 133.3, 132.5, 132.4, 127.2, 122.0, 121.0, 107.6, 55.37, 55.35, 43.9, 29.2, 13.7, 13.3; HRMS (ESI+) m/z calcd for C₁₇H₂₀NO₂S⁺ [(M + H)⁺] 302.1209, found 302.1216 (error 2.3 ppm).

4.1.2.43 [2-(2-Methoxybenzyl)pyrrolidine-1-yl]-4,5-dimethylthiophene-2-carboxamide (43): colorless oil (37 mg, 63%); R_f = 0.37 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.38–7.13 (m, 1H), 7.11–6.98 (m, 1H), 6.97–6.80 (m, 2H), 6.74 (s, 1H), 5.61 (dd, J = 7.9, 2.4 Hz, 1H), 4.08–3.93 (m, 2H), 3.88 (s, 3H), 2.41–2.08 (m, 5H), 2.05–1.76 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (162.6, 161.6), (156.3, 155.5), 138.5 (2C), 133.8 (2C), 133.3 (2C), 132.9 (2C), (131.5, 131.1), (128.4, 127.7), (126.2, 125.6), (120.7, 120.4), (110.6, 110.4), (58.3, 58.0), 55.4 (2C), (50.0, 48.3), (34.1, 32.1), (25.0, 20.9), 13.6 (2C), 13.2 (2C); HRMS (ESI+) m/z calcd for C₁₈H₂₂NO₂S⁺ [(M + H)⁺] 316.1366, found 316.1360 (error −1.9 ppm).

4.1.2.44. N-Benzyl-4,5-dimethyl-N-propylthiophene-2-carboxamide (44): colorless oil (26 mg, 53%); R_f = 0.16 (ethyl acetate/hexanes = 1/2); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 8.32 (s, 0.4H, minor), 8.23 (s, 0.6H, major), 7.56 (s, 1H), 7.39–7.28 (m, 2H), 7.26 (t, J = 7.4 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H), 4.54 (s, 1.25H, major), 4.40 (s, 0.75H, minor), 3.20 (t, J = 7.5 Hz, 0.75H, minor), 3.11 (t, J = 7.4 Hz, 1.25H, major), 2.39 (s, 3H), 2.15 (s, 3H), 1.59–1.44 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 166.9 (2C), 163.4 (2C), 143.3 (2C), 137.5 (2C), (136.5, 136.2), 135.0 (2C), 129.0 (2C), 128.8 (2C), (128.3, 128.2), (127.8, 127.4), 127.6 (2C), (51.6, 48.8), (45.7, 43.9), (21.4, 20.3), 14.0 (2C), 13.7 (2C), (11.4, 11.0); HRMS (ESI+) m/z calcd for C₁₇H₂₂NOS⁺ [(M + H)⁺] 288.1417, found 288.1410 (error −2.4 ppm).

4.1.2.45. N-(3-Methoxybenzyl)-4,5-dimethyl-N-propylthiophene-2-carboxamide (45): light yellow oil (37 mg, 68%); R_f = 0.21 (ethyl acetate/hexanes = 1/4); ¹H NMR (400 MHz, CDCl₃) δ 7.27 (t, J = 7.5 Hz, 1H), 7.02 (s, 1H), 6.88–6.79 (m, 3H), 4.76 (s, 2H), 3.80 (s, 3H), 3.40 (t, J = 7.6 Hz, 2H), 2.33 (s, 3H), 2.07 (s, 3H), 1.66 (sx, J = 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 160.1, 139.2, 137.5, 133.3, 132.6, 132.2, 129.9 (2C), 119.6, 112.9, 55.4, 49.2, 29.8, 21.1, 13.7, 13.3, 11.3; HRMS (ESI+) m/z calcd for C₁₈H₂₄NO₂S⁺ [(M + H)⁺] 318.1522, found 318.1516 (error −1.9 ppm).

4.1.2.46. N-(4-Methoxybenzyl)-4,5-dimethyl-N-propylthiophene-2-carboxamide (46): yellow oil (41 mg, 61%); R_f = 0.17 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.20 (d, J = 8.2 Hz, 2H), 7.03 (s, 1H), 6.88 (d, J = 8.2 Hz, 2H), 4.71 (s, 2H), 3.80 (s, 3H), 3.37 (t, J = 7.6 Hz, 2H), 2.33 (s, 3H), 2.08 (s, 3H), 1.65 (sx, J = 7.4 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 164.8, 159.1, 137.4 (2C), 133.3, 132.7 (2C), 132.1, 129.4, 128.8, 114.2, 55.4, 48.9, 29.8, 21.2, 13.7, 13.3, 11.3; HRMS (ESI+) m/z calcd for C₁₈H₂₄NO₂S⁺ [(M + H)⁺] 318.1522, found 318.1517 (error −1.6 ppm).

4.1.2.47. N-(2-Hydroxybenzyl)-4,5-dimethyl-N-propylthiophene-2-carboxamide (47): light yellow oil (49 mg, 67%); R_f = 0.18 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 10.32–9.68 (brs, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.21 (s, 1H), 7.12 (d, J = 7.5 Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H), 6.81 (t, J = 7.5 Hz, 1H), 4.59 (s, 2H), 3.48 (t, J = 7.6 Hz, 2H), 2.36 (s, 3H), 2.13 (s, 3H), 1.85 (sx, J = 7.5 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 166.2, 156.8, 139.5, 134.4, 133.8, 132.0, 130.44, 130.41, 121.7, 119.1, 117.8, 49.9, 47.0, 21.7, 13.7, 13.4, 11.1; HRMS (ESI+) m/z calcd for C₁₇H₂₂NO₂S⁺ [(M + H)⁺] 304.1366, found 304.1360 (error −2.0 ppm).

4.1.2.48. N-(2-Fluorobenzyl)-4,5-dimethyl-N-propylthiophene-2-carboxamide (48): colorless oil (39 mg, 71%); R_f = 0.16 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 7.33 (t, J = 7.6 Hz, 1H), 7.30–7.23 (m, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.09–6.99 (m, 2H), 4.83 (s, 2H), 3.43 (t, J = 7.6 Hz, 2H), 2.33 (s, 3H), 2.09 (s, 3H), 1.69 (sx, J = 7.5 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H); ¹³C NMR (151 MHz, CDCl₃) δ 165.1, 161.0 (d, ¹J_C-F = 245.7 Hz), 137.7, 133.4, 132.4, 129.14 (d, ³J_C-F = 8.0 Hz), 128.3, 124.63, 124.57, 124.5, 115.5 (d, ²J_C-F = 21.9 Hz), 49.7, 38.7, 21.4, 13.7, 13.3, 11.3; HRMS (ESI+) m/z calcd for C₁₇H₂₁FNOS⁺ [(M + H)⁺] 306.1322, found 306.1318 (error −1.3 ppm).

4.1.2.49. N-((4-Methoxypyridin-3-yl)methyl)-4,5-dimethyl-N-propylthiophene-2-carboxamide (49): colorless oil (37 mg, 59%); R_f = 0.24 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 8.46 (d, J = 5.8 Hz, 1H), 8.33 (s, 1H), 7.04 (s, 1H), 6.86 (d, J = 5.8 Hz, 1H), 4.74 (s, 2H), 3.92 (s, 3H), 3.43 (t, J = 7.9 Hz, 1.8H, major), 3.36 (s, 0.3H, minor), 3.20 (dd, J = 13.6, 7.4 Hz, 0.2H, minor), 2.33 (s, 2.7H, major), 2.09 (s, 2.7H, major), 1.96 (s, 0.3H, minor), 1.66 (sx, J = 7.4 Hz, 1.8H, major), 1.51 (t, J = 7.4 Hz, 0.2H, minor), 0.91 (t, J = 7.4 Hz, 0.3H, minor), (0.89 (t, J = 7.4 Hz, 2.7H, major); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 165.1 (2C), 149.7 (2C), 148.1 (2C), 137.7 (2C), 133.3 (2C), 132.5 (2C), 132.2 (2C), 126.3 (2C), 122.1 (2C), 106.2 (2C), 55.8 (2C), 53.5 (2C), 41.5 (2C), (23.0, 21.5), 13.7 (2C), 13.3 (2C), (11.5, 11.3); HRMS (ESI+) m/z calcd for C₁₇H₂₃N₂O₂S⁺ [(M + H)⁺] 319.1475, found 319.1468 (error −2.2 ppm).

4.1.2.50. N-((3-Fluoropyridin-4-yl)methyl)-4,5-dimethyl-N-propylthiophene-2-carboxamide (50): colorless oil (41 mg, 67%); R_f = 0.30 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ 8.45 (d, J = 1.7 Hz, 1H), 8.40 (d, J = 4.9 Hz, 1H), 7.32–7.28 (m, 1H), 7.08 (s, 1H), 4.81 (s, 2H), 3.49 (t, J = 7.6 Hz, 2H), 2.34 (s, 3H), 2.11 (s, 3H), 1.71 (sx, J = 7.6 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 158.0 (d, ¹J_C-F = 255.6 Hz), 145.7, 138.4, 137.5 (d, ²J_C-F = 24.1 Hz), 134.5, 133.6, 133.0, 131.5, 123.6, 50.9, 44.1, 21.9, 13.7, 13.3, 11.2; HRMS (ESI+) m/z calcd for C₁₆H₂₀FN₂OS⁺ [(M + H)⁺] 307.1275, found 307.1269 (error −2.0 ppm).

4.1.2.51. 3-Bromo-N-ethyl-N-(2-methoxybenzyl)benzamide (51): colorless oil (43 mg, 62%); R_f = 0.18 (ethyl acetate/hexanes = 1/4); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.58 (s, 1H), 7.55–7.45 (m, 1H), 7.39–7.09 (m, 4H), 6.97 (t, J = 7.4 Hz, 1H), 6.92–6.81 (m, 1H), 4.80 (s, 0.8H, minor), 4.46 (s, 1.2H, major), 3.86 (s, 1.2H, minor), 3.76 (s, 1.8H, major), 3.50 (q, J = 7.4 Hz, 1.2H, major), 3.29–3.14 (m, 0.8H, minor), 1.18 (t, J = 7.4 Hz, 1.8H, major), 1.08 (t, J = 7.4 Hz, 1.2H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (170.3, 167.2), (157.7, 157.3), (139.0, 138.9), (135.9, 133.1), 132.4 (2C), (130.1, 129.9), (129.6, 129.3), (129.0, 128.7), (128.6, 128.0), (125.1, 124.7), 122.4 (2C), 120.7 (2C), 110.4 (2C), (55.4, 55.2), (43.3, 41.8), (47.9, 40.0), (13.9, 12.4); HRMS (ESI+) m/z calcd for C₁₇H₁₉BrNO₂⁺ [(M + H)⁺] 348.0594, found 348.0590 (error −1.1 ppm).

4.1.2.52. 3-Bromo-N-ethyl-2-fluoro-N-(2-methoxybenzyl)benzamide (52): colorless oil (39 mg, 69%); R_f = 0.17 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.62–7.51 (m, 1H), 7.35–7.22 (m, 2H), 7.09 (t, J = 7.8 Hz, 1H), 7.04–6.85 (m, 2H), 6.81 (d, J = 8.2 Hz, 1H), 4.92–4.73 (brs, 0.9H, minor), 4.41 (s, 1.1H, major), 3.86 (s, 1.4H, minor), 3.74 (s, 1.6H, major), 3.63–3.39 (m, 0.9H, minor), 3.15 (q, J = 7.2 Hz, 1.1H, major), 1.17 (t, J = 7.2 Hz, 1.6H, major), 1.05 (t, J = 7.2 Hz, 1.4H, minor); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (165.79, 165.76), (157.6, 157.5), [154.9 (d, ¹J_C-F = 248.3 Hz), 154.6 (d, ¹J_C-F = 247.6 Hz)], (134.29, 134.26), (129.2, 128.9), (128.7, 128.6), [127.71 (d, ²J_C-F = 19.4 Hz), 127.67 (d, ²J_C-F = 19.6 Hz)], (126.8, 126.6), [125.8 (d, ⁴J_C-F = 4.2 Hz), 125.4 (d, ⁴J_C-F = 4.3 Hz)], (124.8, 124.2), (120.9, 120.6), (110.42, 110.36), [109.7 (d, ²J_C-F = 21.3 Hz), 109.6 (d, ²J_C-F = 21.3 Hz)], (55.5, 55.2), (43.0, 41.9), (47.3, 39.7), (13.7, 12.4); HRMS (ESI+) m/z calcd for C₁₇H₁₈BrFNO₂⁺ [(M + H)⁺] 366.0499, found 366.0494 (error −1.4 ppm).

4.1.2.53. 3-Bromo-N-ethyl-4-fluoro-N-(2-methoxybenzyl)benzamide (53): colorless oil (47 mg, 61%); R_f = 0.17 (ethyl acetate/hexanes = 1/3); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.67 (d, J = 6.5 Hz, 1H), 7.34 (s, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.21–7.00 (m, 2H), 6.97 (t, J = 7.5 Hz, 1H), 6.91–6.82 (m, 1H), 4.78 (s, 0.7H, minor), 4.46 (s, 1.3H, major), 3.80 (s, 3H), 3.56–3.39 (m, 1.3H, major), 3.34–3.11 (m, 0.7H, minor), 1.25–1.04 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) (169.7, 166.8), 159.6 (d, ¹J_C-F = 250.2 Hz, 2C), (135.84, 135.83), (132.7, 132.2), 131.3 (d, ³J_C-F = 8.7 Hz, 2C), (129.4, 129.0), 127.9 (2C), (127.6, 127.5), 124.6 (2C), 120.8 (2C), (116.6, 116.4), 110.5 (2C), 109.3 (d, ²J_C-F = 21.6 Hz, 2C), 55.3 (2C), (43.4, 42.0), (48.1, 40.4), (13.9, 12.4); HRMS (ESI+) m/z calcd for C₁₇H₁₈BrFNO₂⁺ [(M + H)⁺] 366.0499, found 366.0494 (error −1.4 ppm).

4.1.2.54. 3-Bromo-N-ethyl-5-fluoro-N-(2-methoxybenzyl)-4-methylbenzamide (54): colorless oil (54 mg, 61%); R_f = 0.21 (ethyl acetate/hexanes = 1/7); ¹H NMR (400 MHz, CDCl₃) δ (mixture of rotamers) 7.42 (s, 1H), 7.27 (t, J = 7.5 Hz, 1H), 7.21–7.10 (m, 1H), 7.06 (d, J = 9.2 Hz, 1H), 6.96 (t, J = 7.6 Hz, 1H), 6.92–6.80 (m, 1H), 4.77 (s, 0.8H, minor), 4.47 (s, 1.2H, major), 3.84 (s, 1.2H, minor), 3.77 (s, 1.8H, major), 3.54–3.37 (m, 1.2H, major), 3.32–3.12 (m, 0.8H, minor), 2.32 (s, 3H), 1.14 (t, J =7.4 Hz, 3H); ¹³C NMR (100 MHz, CDCl₃) δ (mixture of rotamers) 169.1 (2C), 160.5 (d, ¹J_C-F = 250.7 Hz, 2C), (157.6, 157.4), 136.8 (2C), (129.3, 129.0), (128.8, 128.1), 127.6 (2C), 127.1 (d, ²J_C-F = 19.0 Hz, 2C), (126.5, 126.0), (125.1, 124.5), (120.7, 120.6), (113.0, 112.7), 110.4 (2C), (55.4, 55.2), (43.2, 41.8), (48.0, 40.0), (14.82, 14.78), (13.9, 12.4); HRMS (ESI+) m/z calcd for C₁₈H₂₀BrFNO₂⁺ [(M + H)⁺] 380.0656, found 380.0650 (error −1.4 ppm).

4.2. Biology

4.2.1. Cardiac and skeletal SR preparation

Fresh porcine left ventricular tissue was used to isolate cardiac SR vesicles, using differential centrifugation of the homogenized tissue [25]. Skeletal light SR was prepared from fresh rabbit skeletal white muscle [26, 27]using differential centrifugation followed by a discontinuous sucrose gradient. The skeletal SR contained predominantly skeletal Ca²⁺-ATPase (SERCA1a). The SR vesicles were flash-frozen and stored at −80 °C until needed.

4.2.2. Functional characterization of analogues by SERCA ATPase activity

We used an NADH-coupled ATPase assay to measure SERCA2a activity in 384-well microplates with porcine cardiac SR and rabbit skeletal SR vesicles [22, 25]. An assay buffer containing 50 mM MOPS (pH 7.0), 100 mM KCl, and 1 mM EGTA, 0.2 mM NADH, 1 mM phosphoenol pyruvate, 10 IU/mL of pyruvate kinase, 10 IU/mL of lactate dehydrogenase, 7 μM of the calcium ionophore A23187 (Sigma), and CaCl₂ set to desired free calcium conditions was added to each well along with either 10μg/mL cardiac SR or 4μg/mL skeletal SR [28]. SERCA ATPase activity was measured in [compound] (0.048 to 50μM) at three [Ca]: pCa 5.4, pCa 6.2, and pCa 8.0. Control samples without compound and known tool compounds were included on the assay plates. Plates were sealed and incubated for 20 min before starting the reaction with MgATP at a final concentration of 5mM and monitored at 340 nm in a SpectraMax Plus³⁸⁴ microplate spectrophotometer (Molecular Devices, Sunnyvale, CA).

4.2.3. Functional characterization of analogues by SERCA Ca²⁺-Uptake assays

To determine the effect of the compounds on SERCA2a Ca²⁺-transport activity in porcine cardiac SR, we used an oxalate-based assay where the change in fluorescence of a Ca²⁺-sensitive dye, Fluor-4 was determined as previously described [22, 28]. The assay buffer containing 50mM MOPS, 100mM KCl, 30 mg/mL sucrose, 10 mM potassium oxalate, 2 μM Fluo-4, 30 μg/mL porcine cardiac SR vesicles or 12μg/mL skeletal SR vesicles, CaCl₂ to a final concentration of free [Ca²⁺] at pCa 8.0, 6.2, and 5.4, and compound (0.048 to 50 μM) were dispensed into 384-well black walled, transparent bottomed plates (Greiner Bio-One), covered to protect from light, and incubated at 22 °C for 20 minutes. The reaction was started by the addition of MgATP to a final concentration of 5 mM and monitored by observing the decrease in 485-nm excited fluorescence of Fluo-4 at 520 nm for 15 min using a FLIPR Tetra (Molecular Devices, San Jose, CA). Similar controls as in the ATPase activity assays were also used.

4.2.4. Functional assay data analysis

The calculated SERCA2a functional activity, F (rate of ATPase hydrolysis or Ca²⁺-transport) at [Ca²⁺]_MAX (saturating, pCa 5.4) or [Ca²⁺]_MID (subsaturating, midpoint, pCa 6.2) was corrected by subtracting the basal rate at pCa 8.0). The % effect due to the presence of the compound relative to the absence of compound was plotted against [compound], and concentration response curves (CRC) were fitted using the Hill function in Origin2015 software to determine V_MAX (the activity or Ca²⁺-transport at saturating [compound]), and EC₅₀, the compound concentration at 50% V_MAX effect [5]. In the absence of compound saturation, the maximal change (Δ) in activity was determined, to yield % of maximal effect at the [Ca²⁺]_MAX and [Ca²⁺]_MID conditions, respectively (Figure 4, Tables 1–5, and Tables S1 and S2, Supplementary Materials). Percentage of maximal effect, and EC₅₀ are reported in Tables 1–5 and Tables S1 and S2.

4.2.6. LC–MS/MS Methods

Quantitative analyses were performed on a TSQ Quantiva Triple Quadrupole MS (Thermo Scientific, Waltham, MA, USA) equipped with an electrospray ion source (KQ Integrated Solutions, Santa Clara, CA, USA) and a Dionex UltiMate 3000 RSLC nanosystem (Thermo Scientific). Analytes were separated on a Zorbax Eclipse XDB-C8 reverse phase column (150 × 4.6 mm, 5 μm, Agilent, Santa Clara, CA, USA) using a linear gradient elution at 25 °C. Water with 0.1% (v/v) formic acid (mobile phase A) and acetonitrile with 0.1% (v/v) formic acid (mobile phase B) were used as mobile phase A and B, respectively. The injection volume was 1 μL unless otherwise specified. The gradient was as follows: 0–3.0 min, 50–95% B; 3.0–5.5 min, 95–95% B; 5.5–7.0 min, 95–50% B; 7.0–9.0 min, 50–50% B. Parameters of LC-MS were set as follows: flow rate (800 μL/min), ion source temperature (300 °C), vaporizer temperature (200 °C), ion spray voltage (4500 V), collision-induced dissociation gas (1.5 mTorr), sheath gas (40 arb.), auxiliary gas (0 arb.), and sweep gas (0 arb). Ion pairs of target analytes (collision energy, V; RF lens, V) were set at: m/z 318 to 121 (15,88) for 1; m/z 344 → 121 (30,96) for 8; m/z 352 → 121 (15,88) for 21; m/z 362 → 121 (15,91) for 22; m/z 440 → 121 (15,102) for 23; m/z 380 → 121 (13,90) for 24; m/z 380 → 121 (16,91) for 25; m/z 380 → 121 (15,92) for 26; m/z 304 → 121 (15,86) for 32; m/z 332 → 121 (16,92) for 33. Data was processed with Xcalibur software (Thermo Scientific).

4.2.7. Intrinsic Clearance (S9 Stability) assay

Selected compounds (1 μM) were pre-incubated with human liver S9 fraction (0.5 mg/mL, XenoTech) in buffer (50 mM phosphate, 3 mM MgCl₂, 1 mM EDTA, pH = 7.4) at 37 °C for 10 minutes. Nicotinamide adenine dinucleotide phosphate (NADPH, 1.0 mM) was added to initiate the reaction. The final incubation volume was 400 μL. Aliquots (40 μL) of the reaction mixture at 0, 5, 10, 15, 20, 30, 45, 60, and 90 min were added to acetonitrile (220 μL). The samples were vortexed, centrifuged at 3000g for 10 min, and filtered through 0.22 μm filtration plates prior to analysis. A negative control was prepared without NADPH while a positive control was performed with Verapamil. The compound residual (%) acquired by LC-MS/MS were plotted against time. Elimination rate constant (k), half-life (t_1/2), and intrinsic clearance (CL_int) were calculated with the following equations [29, 30]:

$$\text{Elimination rate constant}\left(\text{k}\right)=\left(-\text{slope}\right)$$

$$\text{Half life}({\text{t}}_{1/2},\text{min})=\text{ln}2/\text{k}$$

$$\text{V}(\mu \text{L}/\text{mg})=\text{incubation volume per mg protein incubated}$$

$$\text{Intrinsic clearance}\left({\text{CL}}_{\text{int}}\right)(\mu \text{L}/\text{min}/\text{mg protein})=\text{V}\times \text{k}$$

Compounds were classified into low (<4.5) and high (>24.6) clearance (CL_int), which corresponds to low (0.3) and high (0.7) liver extraction ratio (E).

4.2.8. Solubility assay

The selected compounds were incubated in 50 mM phosphate buffer solution (pH 7.4) at 37 °C for 12h. The suspensions were centrifuged, and the supernatants were filtered through 0.22 μm filtration plates. Filtrates were diluted with acetonitrile (50% v/v) by one thousand-fold and delivered by an autosampler to LCMS. The solubility was determined against a calibration curve over 0.0001–0.01 mg/mL. Three runs were performed for each compound.

4.2.9. Cell viability (Toxicity) assay

The effect of each compound on cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, similar to previous evaluation of SERCA modulators [31, 32]. HEK-293.2sus cells were cultured in a 96-well plate (9.6 × 10³ cells/well) and treated with a concentration gradient of compounds. After addition of compounds to the 96-well plate, cells were incubated for 72 hrs at 37 °C with 5% CO₂. After incubation, 20 μL of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenltetrazolium bromide (MTT reagent, 0.5 mg/mL from 5mg/mL stock in PBS) was added to each well, and plates were further incubated for 3–4 hours. After this incubation with MTT reagent, the media was gently aspirated off and 100 μL of DMSO was added. The reagent and cells were resuspended using an orbital shaker at 21 °C for 15 min. The plate was then read on a Molecular Devices Spectramax 384Plus plate reader, acquiring absorbance at 595nm. After removal of background absorbance (wells treated with MTT and then DMSO, but with no cells added), the compound effect was calculated relative to the DMSO controls on the same assay plates. For calculating IC₅₀, the data was fit to the following equation using a non-linear least squares method in Excel:

$$Y=\text{Bottom}+(\text{Top}-\text{Bottom})/(1+10^((\text{logIC}50-\text{x})*\text{HillSlope}\left)\right)$$

4.2.10. Statistical analysis of data

Data are presented as mean ± SD and are calculated from a minimum of three to six separate experiments (n = 3–6).

Supplementary Material

Available online

Table 3.

Effects of the right-moiety modified analogues of compound 1 on Ca²⁺-ATPase activity.

Cpd	R	CRC shape	Max increase (%)	Max decrease (%)	EC50 (μM)
44		Inactive	-	-	N/A
45		Sigmoid*	57±22	-	>50
46		Sigmoid	54±12	-	7.9±2.3
47		Sigmoid	45±15	-	6.6±1.8
48		Sigmoid*	89±24	-	>50
49		Sigmoid*	8±13	-	>50
50		Inactive	N/A	-	N/A

^*Hill fit, but does not plateau at the higher concentrations tested.

Mean±SD, n= 4–6 individual experiments.

Abbreviations:

SERCA

sarco/endoplasmic reticulum calcium ATPase

SR

sarco/endoplasmic reticulum

HTS

high throughput screening

SAR

structure-activity relationship

NMR

nuclear magnetic resonance

NOE

Nuclear Overhauser effect

HRMS

high resolution mass spectrometry

LC-MS

liquid chromatography-mass spectrometry

EC₅₀

half maximal effective concentration

DMSO

dimethyl sulfoxide

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

DCE

1,2-dichloroethane

DMF

N,N-dimethylformamide

ADMET

absorption, distribution, metabolism, excretion, and toxicity

CRC

concentration response curves

Data Availability

All data is provided in the manuscript or in the Supplementary Materials.