Substituted 4-oxo-crotonic acid derivatives as a new class of protein kinase B (PknB) inhibitors: synthesis and SAR study

Abstract

Protein kinase B (PknB) is an essential serine/threonine protein kinase required for Mycobacterium tuberculosis (M. tb) cell division and cell-wall biosynthesis. A high throughput screen using PknB identified a (E)-4-oxo-crotonic acid inhibitor, named YH-8, which was used as a scaffold for SAR investigations. A significant improvement in enzyme affinity was achieved. The results indicated that the α,β-unsaturated ketone scaffold and “trans-” configuration are essential for the activity against PknB. And compounds with an aryl group, especially with electron-withdrawing substituents on benzene ring, exhibited four fold potency than that of YH-8.

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1. Introduction

As an essential Mycobacterium tuberculosis (Mtb) serine/threonine protein kinase (STPK), protein kinase B (PknB) is highly conserved in Gram-positive bacteria and apparently required for mycobacterial growth.^1–4 The knockout and overexpression of PknB can lead to alteration of growth rate and cell morphology of TB.^5–8 The crystal structure of the kinase domain of PknB in complexes with an ATP analogue⁹ exhibits less than 30% similarity with eukaryotic and prokaryotic STPKs, which suggests that PknB may be a potential drug target for the tuberculosis kinases and not those of the host. Previously many high affinity inhibitors have also been reported for PknB.¹⁰

YH-8, namely (E)-methyl-4-(4-methoxyphenyl)-4-oxobut-2-enoate (Fig. 1), was identified as a PknB inhibitor from a high throughput screen (HTS) of our compounds collection. Its majority anti-TB activity of minimum inhibitory concentrations (MICs) is falling in the 0.625–1.250 μmol L⁻¹ range in vitro, which is significantly higher than other reported PknB inhibitors, such as amminopyrimidines, aminoguanidines and anthraquinones classes.¹¹ Stability assay revealed that YH-8 was stable over 12 h in rat plasma samples, and the acute toxicity for the LD₅₀ values in rat were 600 mg kg⁻¹ (orally administered) and 200 mg kg⁻¹ (vein injected).¹² As a new unsaturated crotonic acid scaffold of YH-8 from all reported anti-TB chemical scaffolds,^10,13 the previously results formed the starting point for our chemistry programme. In this study, we reported on the synthesis and structure–activity relationship (SAR) study of series of YH-8 derivatives as potential PknB inhibitors.

Fig. 1 The HTS hit YH-8.

2. Chemical synthesis

In Scheme 1, 7 compounds were firstly designed and synthesized based on the scaffold A ((E)-α,β-unsaturated ketone, Fig. 1) of YH-8 for SAR investigations. γ-Oxobenzenebutanoic acid 2a was prepared using Friedel–Crafts acylation of anisole with succinic anhydride catalyzed by Lewis acid of aluminum chloride in 80% yield.¹⁴ Reduction of 2a by triethylsilane in trifluoroacetic acid obtained 3a in 72% yield. Methyl esters 1b, 2b and 3b were synthesized from corresponding acids 1a, 2a and 3a in methanol catalyzed by concentrated sulfuric acid in 92–95% yields.¹⁵ According to literature method,¹⁶ the carboxylic acid group of 1a, 2a and 3a was firstly activated by treatment with isobutylchloroformate (IBCF), the product of which was then treated in situ with ammonia gas at −15 °C to give corresponding amides 1c, 2c and 3c in 75–85% yields. In Scheme 2, the 20 aromatic E-3-acylacrylic acid derivatives 7a–t were directly prepared from corresponding ketones 6a–t and glyoxylic acid in 50–75% yields.¹⁷ Esters 8a–t, 9a–t and 10a–t were obtained using the same method of 2a in around 40% yield. The “cis-” configuration (4, (Z)-methyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate) of YH-8 (8a) was separated from a mixture of cis- and trans-products in 48% yield. Similarly, the carboxylic acid group of 7a–t was firstly activated by treatment with IBCF, the product of which was then treated in situ with N-Boc-ethanolamine to afford compounds 11a–t in 47–84% yields.

Scheme 1 (a–c) Synthesis of compounds 1a–c, 2a–c, 3a–c and 4. Regents and conditions: (i) MeOH, sulfuric acid (Cat.), reflux, overnight; (ii) IBCF, TEA, anhydrous THF, −15 °C, 1 h, then NH₃ (g), 0 °C, overnight; (iii) succinic anhydride, AlCl₃, nitrobenzene, 0 °C to rt, 1 h, then 60 °C, 3 h; (iv) triethylsilane, TFA, 50 °C, 5 h; (v) acetic acid, reflux, overnight.

Scheme 2 Synthesis of compounds 8a–t, 9a–t, 10a–t, and 11a–t. Regents and conditions: (i) glyoxylic acid, acetic acid, reflux, overnight, for 7a–r and 7t; glyoxylic acid, morpholine hydrochloride (Cat.), 120 °C, overnight, for 7s; (ii) R′OH, sulfuric acid (Cat.), reflux, overnight, for 8a–t, 9a–t, 10a–t; IBCF, TEA, anh. DCM, −15 °C, 8 h, then N-Boc-ethanolamine, rt, overnight, for 11a–t.

3. Results and discussion

As a promising PknB inhibitor, the structure of YH-8 is markedly different from the reported PknB inhibitors. As shown in Fig. 1, YH-8 was characterized as a substituted (E)-4-oxo-crotonic acid scaffold with three parts, namely A ((E)-α,β-unsaturated ketone), B (ester) and C (aryl). We presumed each part should play a different role of the interactions between YH-8 and PknB ATP-binding site. In other words, firstly we should clarify which part is essential for the activity and which part is allowed to be modified for increasing potency. So, initial SAR study was performed mainly around the scaffold A ((E)-α,β-unsaturated ketone). As shown in Scheme 1-a, scaffold A was firstly deduced to methyl ester 1b and amide 1c from corresponding acid 1a, which lacks a carbonyl (ketone) group comparing with the α,β-unsaturated ketone of A. In Scheme 1-b, the carbon–carbon double bond of scaffold A was deduced to an alkane acid 2a by a Friedel–Crafts acylation reaction from anisole. The carbonyl (ketone) group of 2a was sequentially reduced to methylene of 3a. Both acids of 2a and 3a were also converted to corresponding methyl esters 2b & 3b and amides 2c & 3c. In Scheme 1-c, the “cis-” configuration (4) of YH-8, (Z)-methyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate, was separated from a mixture of cis- and trans-products and characterized by ¹H NMR. The J_HH of “cis-” configuration is 3.6 Hz less than its “trans-” configuration. And the “cis-” configuration (4) is a yellow oil, whereas YH-8, namely 8a in this study, is a yellow solid.

According to preliminary measurements, inhibitor concentration of 20 μM in the assay of percent inhibition of PknB was selected mainly because this concentration can cover and reflect the inhibitory rates rightly for all test compounds. Including carboxylic acid, esters and amides, ten compounds in Scheme 1 were tested for inhibitory activity against PknB at 20 μM along with YH-8. As shown in Table 1, eight compounds exhibit less than 10% inhibition against PknB comparing with YH-8 (52.1%). Only compounds 1a & 4 have a moderate inhibition (30.7% & 24.7%). The results show the α,β-unsaturated ketone scaffold and “trans-” configuration are essential for the activity of YH-8 against PknB.

Table 1 Inhibitory activity of 1a–c, 2a–c, 3a–c and 4 against PknB

Compd.	Inhibition^a (%)	Compd.	Inhibition^a (%)	Compd.	Inhibition^a (%)
a Values represent the percent inhibition of PknB at 20 μM of the test compounds and are means of three independent experiments.
1a	30.7 ± 1.6	2a	3.3 ± 0.2	3a	7.0 ± 0.5
1b	1.7 ± 0.2	2b	5.1 ± 1.6	3b	5.6 ± 0.5
1c	5.4 ± 0.6	2c	8.7 ± 0.6	3c	8.0 ± 0.7
YH-8 (8a)	52.1 ± 1.8	4	24.7 ± 1.5

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Based on the findings above, the subsequent SAR study of YH-8 was carried out on modification of part B and C while maintaining the α,β-unsaturated ketone scaffold and “trans-” configuration. As shown in Scheme 2, series of compounds were synthesized from corresponding methyl ketones (6a–t). The structures of 8a–t, 9a–t, 10a–t, and 11a–t were shown in Table 2 and their PknB inhibitory activities were listed in Table 3. The data indicated that almost half of the tested compounds have comparable activity against PknB comparing with YH-8 (52.1%). Particularly, compounds 11a–t bearing a tert-butoxycarbonyl-aminoethyl group on part B generally show higher potency than that of other series. Among them, 11n–p exhibited much better activity (87.1%, 87.9% and 87.6%, respectively). Mostly, compounds bearing an aryl group on part C generally show higher potency than that substituted with alkyl analogues (8r–t, 9r–t, 10r–t and 11r–t). It indicated that the alkyl group on part C would be detrimental to the activity. For the aryl substituted compounds, 8–11g (61.4–70.7%) with 2,5-dimethoxyphenyl, 8–11h (62.1–68.3%) with 3,4-dimethoxyphenyl, 8–11m (61.3–73.6%) with naphthyl, 8–11n (61.3–73.6%) except 9n (49.0%) with 2,4-dichlorophenyl, 8–11o (59.1–87.9%) with 4-nitrophenyl, 8–11p (64.5–87.6%) with 4-bromophenyl and 8–11q (60.8–67.0%) with furyl exhibit higher activity than YH-8 of 52.1%. We speculated that the position of the substituted group on phenyl ring should be one of the factors that influence the binding between inhibitor and PknB ATP-binding site. Meanwhile, electron-withdrawing groups on phenyl ring should be beneficial to the potency. It maybe through the electron-withdrawing effect influence on the conjugation of scaffold A, which is known to be essential for the activity of YH-8 against PknB above.

Table 2 The structures of 8a–t, 9a–t, 10a–t & 11a–t

R	R′
	MeO	EtO	i-PrO	BocNH(CH2)2O
	YH-8 (8a)	9a	10a	11a
	8b	9b	10b	11b
	8c	9c	10c	11c
	8d	9d	10d	11d
	8e	9e	10e	11e
	8f	9f	10f	11f
	8g	9g	10g	11g
	8h	9h	10h	11h
	8i	9i	10i	11i
	8j	9j	10j	11j
	8k	9k	10k	11k
	8l	9l	10l	11l
	8m	9m	10m	11m
	8n	9n	10n	11n
	8o	9o	10o	11o
	8p	9p	10p	11p
	8q	9q	10q	11q
Me	8r	9r	10r	11r
	8s	9s	10s	11s
	8t	9t	10t	11t

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Table 3 Inhibitory activity of 8a–t, 9a–t, 10a–t and 11a–t against PknB

Compd.	Inhibition^a (%)	Compd.	Inhibition^a (%)	Compd.	Inhibition^a (%)	Compd.	Inhibition^a (%)
a Values represent the percent inhibition of PknB at 20 μM of the test compounds and are means of three independent experiments.
YH-8 (8a)	52.1 ± 1.8	9a	48.7 ± 4.9	10a	41.1 ± 3.4	11a	61.6 ± 2.8
8b	47.8 ± 4.1	9b	64.3 ± 7.7	10b	11.2 ± 1.7	11b	69.1 ± 4.5
8c	60.5 ± 5.5	9c	15.5 ± 2.3	10c	72.8 ± 7.4	11c	65.8 ± 5.5
8d	54.2 ± 4.8	9d	47.8 ± 7.1	10d	38.0 ± 4.2	11d	55.6 ± 2.7
8e	62.1 ± 5.0	9e	45.3 ± 3.7	10e	56.2 ± 5.0	11e	54.3 ± 3.7
8f	63.1 ± 6.8	9f	55.7 ± 7.0	10f	42.6 ± 2.9	11f	66.2 ± 4.9
8g	70.7 ± 4.0	9g	67.7 ± 5.0	10g	61.4 ± 4.7	11g	67.1 ± 5.4
8h	65.6 ± 8.3	9h	68.3 ± 5.4	10h	63.9 ± 7.6	11h	62.1 ± 4.8
8i	45.4 ± 2.8	9i	51.2 ± 1.4	10i	60.6 ± 1.7	11i	59.0 ± 4.5
8j	39.0 ± 2.7	9j	45.9 ± 3.2	10j	43.1 ± 5.9	11j	53.8 ± 5.3
8k	48.6 ± 5.4	9k	45.9 ± 6.1	10k	19.5 ± 1.9	11k	50.6 ± 5.6
8l	59.1 ± 4.9	9l	51.6 ± 7.8	10l	45.5 ± 5.8	11l	46.0 ± 2.6
8m	62.8 ± 6.1	9m	61.3 ± 4.6	10m	61.6 ± 3.8	11m	73.6 ± 4.0
8n	57.0 ± 6.0	9n	49.0 ± 3.9	10n	57.1 ± 5.1	11n	87.1 ± 4.3
8o	65.1 ± 7.6	9o	65.3 ± 8.5	10o	59.1 ± 4.3	11o	87.9 ± 5.3
8p	66.7 ± 5.8	9p	67.5 ± 5.7	10p	64.5 ± 4.7	11p	87.6 ± 5.8
8q	63.2 ± 3.7	9q	63.8 ± 6.2	10q	60.8 ± 5.3	11q	67.0 ± 4.6
8r	32.6 ± 3.7	9r	40.4 ± 4.2	10r	38.3 ± 1.5	11r	46.2 ± 5.4
8s	33.2 ± 2.7	9s	53.5 ± 6.8	10s	42.7 ± 3.2	11s	54.3 ± 4.0
8t	36.9 ± 4.0	9t	33.4 ± 5.8	10t	43.7 ± 2.9	11t	40.0 ± 3.4

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Finally, the 50% inhibition concentrations (IC₅₀) of compounds 11a–t were tested in Table 4. Among them, compounds 11a–c, f, g, i, m–q exhibit apparently higher IC₅₀ activities than YH-8. Particularly, compounds 11n, 11o and 11p (IC₅₀: 5.6 μM, 4.4 μM and 5.4 μM, respectively) with electron-withdrawing substituents on benzene ring show about fourfold more potent than that of YH-8 (IC₅₀: 20.2 μM). Additionally, most IC₅₀ values of compounds 11a–t in Table 4 consistently match with the corresponding inhibitory rates in Table 3. For example, the inhibitory rates of compounds 11o, 11p, 11n, 11f, 11a, 11e and 11t are 87.9%, 87.6%, 87.1%, 66.2%, 61.6%, 54.3% and 40.0%, respectively. And their corresponding IC₅₀ values are 4.4, 5.4, 5.6, 10.5, 14.1, 19.1 and 38.4 μM, respectively.

Table 4 The IC₅₀ values of 11a–t against PknB

Compd.	IC50^a (μM)	Compd.	IC50^a (μM)	Compd.	IC50^a (μM)
a Values are means of three independent experiments. b Not detected.
11a	14.1 ± 2.7	11h	17.7 ± 1.1	11o	4.4 ± 0.6
11b	10.2 ± 1.7	11i	12.6 ± 2.7	11p	5.4 ± 0.5
11c	7.9 ± 1.4	11j	17.3 ± 2.7	11q	11.9 ± 1.7
11d	19.6 ± 3.8	11k	18.9 ± 2.4	11r	45.5 ± 5.2
11e	19.1 ± 1.1	11l	ND^b	11s	27.9 ± 3.0
11f	10.5 ± 1.7	11m	10.6 ± 1.4	11t	38.4 ± 3.3
11g	8.1 ± 0.4	11n	5.6 ± 0.7	YH-8	20.2 ± 0.2

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4. Conclusions

In summary, the starting HTS hit of YH-8 was optimized for potency against PknB, and total 87 compounds were synthesized for SAR investigations. The initial SAR study of YH-8 indicated the α,β-unsaturated ketone scaffold and “trans-” configuration are essential for the activity of YH-8 against PknB. According to this finding, other 80 YH-8 derivatives were synthesized and evaluated. The results showed that the compounds bearing an aryl group on part C and a tert-butoxycarbonyl-aminoethyl group on part B generally showed higher potency than other compounds and YH-8. Among these compounds, 11n–o and 11p with electron-withdrawing substituents on benzene ring exhibited about fourfold more potent than that of YH-8.

5. Experimental section

Methods and materials

The chemicals were purchased from Aldrich Chemical Co., Sigma or Chemical Co. THF was distilled under argon from sodium-benzophenone ketyl and CH₂Cl₂ was distilled under argon from calcium hydride. The reaction products were purified by crystallization or flash column chromatography using a mixture of petroleum ether and ethyl acetate as the eluent. All melting points were obtained on a Mettler Toledo Melting Point MP70 apparatus (Mettler Toledo, Zurich, Switzerland) and are uncorrected. ¹H-NMR spectra were recorded on Varian Inova-400 MHz, Varian Inova-500 MHz and SYS-600 MHz instruments (Varian, Palo Alto, CA, USA). The chemical shifts (δ) are reported in ppm relative to the internal reference standard tetramethylsilane (TMS) and the coupling constants (J values) have been reported in Hertz (Hz). MS data were obtained using time-of-flight mass spectrometer (TOF-MS) or Bruker microTOF-Q instrument (Bruker, Billerica, MA, USA). High resolution mass spectra (HRMS) were obtained on a Q-TOF Ultima ESI instrument (micrOTOF-Q II, Bruker Daltonics, Leipzig, Germany). Analysis by thin layer chromatography (TLC) was performed on silica gel plates (Merck, Billerica, MA, USA). Automated column chromatography was conducted over silica gel using a Companion Rf 200 automated chromatography system (Teledyne ISCO, Lincoln, NE, USA).

General synthetic procedures

Method A: general procedure for the preparation of esters (1b, 2b, 3b, 8a–t, 9a–t and 10a–t) from corresponding acids¹⁵. To a solution of the corresponding acid (1 equiv.) in corresponding alcohol (2 mL mmol⁻¹) was added concentrated sulfuric acid (25 μL mmol⁻¹). The reaction mixture was refluxed overnight, then cooled, concentrated, diluted with sat. NaHCO₃ solution, extracted with DCM, dried over Na₂SO₄ and concentrated in vacuum. The residue was purified by flash column chromatography on silica with an elution of hexanes/EtOAc 4 : 1 to give the corresponding ester.

Method B: general procedure for the preparation of amides (1c, 2c and 3c) from corresponding acids¹⁶. To a solution of the corresponding acid (1 equiv.) in anhydrous THF (5 mL mmol⁻¹) was added triethylamine (2 equiv.) under argon atmosphere, and cooled to −15 °C. Isobutylchloroformate (IBCF, 2 equiv.) was added dropwise at a rate so as to not exceed an internal temperature of −10 °C. After stirring for 1 h at −15 °C, ammonia gas was slowly bubbled into the reaction mixture. During the bubbling, the reaction temperature rose to 0 °C and kept the reaction temperature at 0 °C overnight. Then the reaction mixture was diluted with EtOAc (10 mL mmol⁻¹) and washed with 10% aqueous sodium chloride solution, followed by saturated brine. The organic layer was dried over Na₂SO₄. After filtration, the solvent was removed under reduced pressure to give the corresponding amide.

Method C: general procedure for the preparation of E-γ-oxo-α,β-alkenyl acids (7a–t)¹⁷. To a solution of glyoxylic acid (5, 1 equiv.) in acetic acid (2 mL mmol⁻¹) was added corresponding methyl ketone (1 equiv.). The resulting mixture was refluxed for overnight and monitored by TLC using ethyl acetate/petroleum ether (2 : 1) as a solvent system. After cooling, the solvent was evaporated. The residue was washed with ice-cold water by decantation or on a filter. The crude product was dried in air at 40 °C and recrystallized from ethyl acetate or purified by column chromatography over silica gel with elution of a mixture of petroleum ether and ethyl acetate (2 : 1) to give the corresponding acid. For the synthesis of compound 7s, catalytic amount of morpholine hydrochloride was used as a catalyst without acetic acid as a solvent at 120 °C for overnight.

Method D: general procedure for the preparation of E-γ-oxo-α,β-alkenyl acids (11a–t). To a solution of 7a–t (1 equiv.) in anhydrous dichloromethane (10 mL mmol⁻¹) was added triethylamine (TEA, 2 equiv.), followed by the addition dropwise of isobutylchloroformate (IBCF, 1.5 equiv.) at −15 °C in nitrogen atmosphere. After the addition done, the resulting mixture was stirred at −15 °C for further 8 h. N-Boc-ethanolamine (0.8 equiv.) was added and the reaction mixture was stirred for overnight and allowed to warm to room temperature. The reaction mixture was washed sequentially by saturated aqueous ammonium chloride and saturated brine, dried over Na₂SO₄. After filtration, the solvent was removed under reduced pressure. The residue was purified by flash column chromatography on silica with an elution of hexanes/EtOAc 4 : 1 to give the corresponding ester.
(E)-Methyl 3-(4-methoxyphenyl) acrylate (1b). Pale yellow solid (92%) yield, mp: 89.0–90.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.65 (d, J = 15.6 Hz, 1H), 7.49–7.47 (m, 2H), 6.92–6.89 (m, 2H), 6.31 (d, J = 15.6 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H); HRMS (ESI) m/z calcd for C₁₁H₁₂O₃Na [M + Na] 215.0684, found 215.0672.
(E)-3-(4-Methoxyphenyl) acrylamide (1c). Yellow solid (75%), mp: 188.2–189.7 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.61 (d, J = 15.5 Hz, 1H), 7.48–7.46 (m, 2H), 6.91–6.89 (m, 2H), 6.33 (d, J = 15.5 Hz, 1H), 5.54 (m, 2H), 3.84 (s, 3H); HRMS (ESI) m/z calcd for C₁₀H₁₁NO₂Na [M + Na] 200.0687, found 200.0684.
4-(4-Methoxyphenyl)-4-oxobutanoic acid (2a)¹⁴. To a mixture of anisole (10.8 g, 100 mmol) and anhydrous aluminum chloride (32.0 g, 240 mmol) in nitrobenzene (150 mL) at 0–5 °C was added dropwise a solution of succinic anhydride (12.0 g, 120 mmol) in nitrobenzene (150 mL) and maintained the same temperature. After addition completed, the reaction mixture was stirred at room temperature for 1 h and then heated up to 60 °C for a further 3 h. The reaction mixture was cooled, poured into ice-cold water (800 mL) and the resulting precipitate was filtered, washed with water and hexane to give a crude, which was crystallized from methanol/ethyl acetate (1 : 9) to give a colorless crystal of 2a (16.7 g, 80%), mp: 150–151 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.98–7.96 (m, 2H), 6.96–6.94 (m, 2H), 3.88 (s, 3H), 3.29 (t, J = 6.8 Hz, 2H), 2.81 (t, J = 6.8 Hz, 2H); HRMS (ESI) m/z calcd for C₁₁H₁₂O₄Na [M + Na] 231.0633, found 231.0625.
Methyl 4-(4-methoxyphenyl)-4-oxobutanoate (2b). White solid (95%), mp: 48.1–49.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.98–7.96 (m, 2H), 6.95–6.93 (m, 2H), 3.87 (s, 3H), 3.71 (s, 3H), 3.28 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.8 Hz, 2H); HRMS (ESI) m/z calcd for C₁₁H₁₄O₄Na [M + Na] 245.0790, found 245.0779.
4-(4-Methoxyphenyl)-4-oxobutanamide (2c). White solid (80%), mp: 134.6–136.3 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.94–7.93 (m, 2H), 7.30 (s, 1H), 7.04–7.02 (m, 2H), 6.72 (s, 1H), 3.83 (s, 3H), 3.14 (t, J = 6.5 Hz, 2H), 2.41 (t, J = 6.5 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 197.26, 173.36, 163.00, 130.10, 129.61, 113.83, 55.50, 32.85, 28.97; HRMS (ESI) m/z calcd for C₁₁H₁₄NO₃Na [M + Na] 230.0793, found 230.0779.
4-(4-Methoxyphenyl) butanoic acid (3a). A mixture of 2a (3.12 g, 15.00 mmol) and triethylsilane (10.5 g, 90.0 mmol) in trifluoroacetic acid (20 mL) was heated to 50 °C under argon atmosphere for 5 h. After cooling, the solvent was evaporated in vacuum, then the reaction mass was diluted with water (100 mL) and EtOAc (100 mL) and stirred for 5 min. The aqueous layer was extracted with EtOAc (100 mL × 2) and the combined organic extracts were washed with saturated brine and dried over Na₂SO₄. After filtration, the solvent was removed under reduced pressure and then co-evaporated with hexanes (50 mL). The residue was treated by flash column chromatography on silica with a elution of hexanes/EtOAc 1 : 1 to give 3a as a colorless solid (2.10 g, 72%): mp 62.5–63.5 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.11–7.09 (m, 2H), 6.84–6.82 (m, 2H), 3.79 (s, 3H), 2.62 (t, J = 7.6 Hz, 2H), 2.36 (t, J = 7.6 Hz, 2H), 1.97–1.92 (m, 2H); HRMS (ESI) m/z calcd for C₁₁H₁₄O₃Na [M + Na] 217.0841, found 217.0830.
Methyl 4-(4-methoxyphenyl) butanoate (3b). Pale yellow oil (94%). ¹H NMR (400 MHz, CDCl₃) δ 7.10–7.08 (m, 2H), 6.84–6.82 (m, 2H), 3.78 (s, 3H), 3.66 (s, 3H), 2.59 (t, J = 7.6 Hz, 2H), 2.32 (t, J = 7.6 Hz, 2H), 1.96–1.91 (m, 2H); HRMS (ESI) m/z calcd for C₁₂H₁₆O₃Na [M + Na] 231.0997, found 231.0983.
4-(4-Methoxyphenyl) butanamide (3c). White solid (85%), mp: 121.6–123.3 °C. ¹H NMR (500 MHz, CDCl₃): δ 7.10–7.08 (m, 2H), 6.83–6.82 (m, 2H), 5.57 (s, 1H), 5.41 (s, 1H), 3.78 (s, 3H), 2.61 (t, J = 7.5 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2H), 1.96–1.91 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 174.04, 157.39, 133.67, 129.21, 113.68, 54.95, 34.49, 33.79, 27.11; HRMS (ESI) m/z calcd for C₁₁H₁₅NO₂Na [M + Na] 216.1000, found 216.0987.
(Z)-Methyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate (4) and (E)-methyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate (8a or YH-8). To a solution of 7.40 g of glyoxylic acid (5.56 mL, 100 mmol) in acetic acid (100 mL) was added 5 (15.0 g, 100 mmol), and the resulting mixture was stirred at 120 °C overnight. After cooling, the solvent was evaporated. The residue was washed with ice-cold water (50 mL) by decantation or on a filter. The crude product was dried in air at 50 °C and recrystallized from ethyl acetate to give 7a ((E)-4-(4-methoxyphenyl)-4-oxobut-2-enoic acid) as a pale yellow powder (10.7 g, 52%), mp: 139.2–140.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.03–7.97 (m, 3H), 7.01–6.98 (m, 2H), 6.89 (d, J = 15.6 Hz, 1H), 3.90 (s, 3H); HRMS (ESI) m/z calcd for C₁₁H₁₀O₄Na [M + H] 207.0657, found 207.0649 [M + H]. To a stirring solution of 7a (1.03 g, 5.00 mol) in methanol (10 mL) was added concentrated sulfuric acid (125 μL). The reaction mixture was refluxed overnight, then cooled, concentrated, diluted with sat. NaHCO₃ solution, extracted with DCM, dried over Na₂SO₄ and concentrated in vacuum. The residue was purified by flash column chromatography on silica with an elution of hexanes/EtOAc 4 : 1 to give 4a and 8a, respectively. 4a as a yellow oil (0.53 g, 48%); ¹H NMR (400 MHz, CDCl₃) δ 7.92–7.90 (m, 2H), 6.96–6.94 (m, 2H), 6.89 (d, J = 12.0 Hz, 1H), 6.26 (d, J = 12.0 Hz, 1H), 3.87 (s, 3H), 3.61 (s, 3H); 8a as a yellow solid (0.53 g, 48%), mp: 72.9–73.7 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.02–8.00 (m, 2H), 7.93 (d, J = 15.6 Hz, 1H), 6.99–6.97 (m, 2H), 6.88 (d, J = 15.6 Hz, 1H), 3.84 (s, 3H), 3.89 (s, 3H); HRMS (ESI) m/z calcd for C₁₂H₁₂O₄Na [M + Na] 243.0633, found 243.0627 [M + Na].
(E)-4-Oxo-4-phenylbut-2-enoic acid (7b). Yellow solid (74%), mp: 88.6–90.2 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.17 (s, 1H), 8.07–8.01 (m, 2H), 7.89 (d, J = 15.6 Hz, 1H), 7.74–7.70 (m, 1H), 7.61–7.57 (m, 2H), 6.69 (d, J = 15.6 Hz, 1H); HRMS (ESI) m/z calcd for C₁₀H₇O₃ [M − H] 175.0395, found 175.0393 [M − H].
(E)-4-Oxo-4-(p-tolyl)but-2-enoic acid (7c). Yellow solid (76%), mp: 141.4–142.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.15 (s, 1H), 7.96–7.94 (m, 2H), 7.88 (d, J = 15.6 Hz, 1H), 7.40–7.38 (m, 2H), 6.67 (d, J = 15.6 Hz, 1H), 2.41 (s, 3H); HRMS (ESI) m/z calcd for C₁₁H₉O₃ [M − H] 189.0552, found 189.0559 [M − H].
(E)-4-(4-Ethoxyphenyl)-4-oxobut-2-enoic acid (7d). Yellow solid (72%), mp: 150.3–151.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.09 (s, 1H), 8.04–8.01 (m, 2H), 7.89 (d, J = 15.6 Hz, 1H), 7.09–7.06 (m, 2H), 6.66 (d, J = 15.6 Hz, 1H), 4.15 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H); HRMS (ESI) m/z calcd for C₁₂H₁₁O₄ [M − H] 219.0657, found 219.0651 [M − H].
(E)-4-(2-Methoxyphenyl)-4-oxobut-2-enoic acid (7e). Yellow solid (73%), mp: 151.8–153.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 13.05 (s, 1H), 7.66–7.52 (m, 3H), 7.23–7.21 (m, 1H), 7.12–7.05 (m, 1H), 6.51 (d, J = 15.5 Hz, 1H), 3.88 (s, 3H); HRMS (ESI) m/z calcd for C₁₁H₁₀O₄Na [M + Na] 229.0477, found 229.0467 [M + Na].
(E)-4-(3-Methoxyphenyl)-4-oxobut-2-enoic acid (7f). Yellow solid (71%), mp: 114.5–115.8 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 13.17 (s, 1H), 7.86 (d, J = 15.5 Hz, 1H), 7.66–7.61 (m, 1H), 7.53–7.46 (m, 2H), 7.30–7.27 (m, 1H), 6.68 (d, J = 15.5 Hz, 1H), 3.84 (s, 3H); HRMS (ESI) m/z calcd for C₁₁H₁₀O₄Na [M + Na] 229.0477, found 229.0466 [M + Na].
(E)-4-(2,5-Dimethoxyphenyl)-4-oxobut-2-enoic acid (7g). Yellow solid (73%). Mp: 152.4–153.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 13.06 (s, 1H), 7.60 (d, J = 15.5 Hz, 1H), 7.23–7.15 (m, 2H), 7.09–7.08 (m, 1H), 6.52 (d, J = 15.5 Hz, 1H), 3.83 (s, 3H), 3.75 (s, 3H); HRMS (ESI) m/z calcd for C₁₂H₁₁O₅ [M − H] 235.0606, found 235.0603 [M − H].
(E)-4-(3,4-Dimethoxyphenyl)-4-oxobut-2-enoic acid (7h). Yellow solid (75%). Mp: 183.1–184.3 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.11 (s, 1H), 7.93 (d, J = 15.6 Hz, 1H), 7.77–7.75 (m, 1H), 7.51–7.50 (m, 1H), 7.12–7.10 (m, 1H), 6.66 (d, J = 15.6 Hz, 1H), 3.87 (s, 3H), 3.84 (s, 3H); HRMS (ESI) m/z calcd for C₁₂H₁₁O₅ [M − H] 235.0606, found 235.0610 [M − H].
(E)-4-(Benzo[d][1,3]dioxol-5-yl)-4-oxobut-2-enoic acid (7i). Yellow solid (74%). Mp: 204.0–205.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.12 (s, 1H), 7.86 (d, J = 15.6 Hz, 1H), 7.74–7.72 (m, 1H), 7.50–7.49 (m, 1H), 7.09–7.07 (m, 1H), 6.65 (d, J = 15.6 Hz, 1H), 6.18 (s, 2H); MS-ESI (m/z): 219.06 [M − H].
(E)-4-(2,3-Dihydrobenzofuran-5-yl)-4-oxobut-2-enoic acid (7j). Yellow solid (73%). Mp: 168.6–170.2 °C. ¹H NMR (400 MHz, CD₃OD) δ 7.96–7.89 (m, 3H), 6.89–6.84 (m, 1H), 6.74 (d, J = 15.6 Hz, 1H), 4.68 (t, J = 8.8 Hz, 2H), 3.30 (t, J = 8.8 Hz, 2H); HRMS (ESI) m/z calcd for C₁₂H₁₀O₄Na [M + Na] 241.0477, found 241.0480 [M + Na].
(E)-4-([1,1′-Biphenyl]-4-yl)-4-oxobut-2-enoic acid (7k). Yellow solid (65%). Mp: 226.3–227.4 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.15 (s, 1H), 8.14–8.12 (m, 2H), 7.93 (d, J = 15.6 Hz, 1H), 7.90–7.88 (m, 2H), 7.79–7.77 (m, 2H), 7.54–7.50 (m, 2H), 7.46–7.43 (m, 1H), 6.76 (d, J = 15.6 Hz, 1H); HRMS (ESI) m/z calcd for C₁₆H₁₀O₃ [M − H] 251.0708, found 251.0714 [M − H].
(E)-4-Oxo-4-(4-phenoxyphenyl)but-2-enoic acid (7l). Yellow solid (50%). Mp: 123.2–125.6 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.15 (s, 1H), 8.09–8.07 (m, 2H), 7.87 (d, J = 15.6 Hz, 1H), 7.50–7.46 (m, 2H), 7.30–7.26 (m, 1H), 7.17–7.15 (m, 2H), 7.09–7.07 (m, 2H), 6.67 (d, J = 15.6 Hz, 1H); HRMS (ESI) m/z calcd for C₁₆H₁₁O₄ [M − H] 267.0657, found 267.0658 [M − H].
(E)-4-(Naphthalen-1-yl)-4-oxobut-2-enoic acid (7m). Yellow solid (55%). Mp: 150.1–151.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.21 (s, 1H), 8.47–8.45 (m, 1H), 8.23–8.21 (m, 1H), 8.08–8.04 (m, 2H), 7.70–7.60 (m, 4H), 6.61 (d, J = 15.6 Hz, 1H); HRMS (ESI) m/z calcd for C₁₄H₁₀O₃Na [M + Na] 249.0528, found 249.0515 [M + Na].
(E)-4-(2,4-Dichlorophenyl)-4-oxobut-2-enoic acid (7n). Yellow solid (58%). Mp: 201.3–202.8 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.30 (s, 1H), 7.81–7.80 (m, 1H), 7.70–7.68 (m, 1H), 7.61–7.58 (m, 1H), 7.32 (d, J = 16.0 Hz, 1H), 6.50 (d, J = 16.0 Hz, 1H); HRMS (ESI) m/z calcd for C₁₀H₆Cl₂O₃ [M − H] 242.9616, found 242.9612 [M − H].
(E)-4-(4-Nitrophenyl)-4-oxobut-2-enoic acid (7o). Yellow solid (60%). Mp: 172.8–174.4 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.26 (s, 1H), 8.37–8.34 (m, 2H), 8.25–8.23 (m, 2H), 7.85 (d, J = 15.6 Hz, 1H), 6.71 (d, J = 15.6 Hz, 1H); HRMS (ESI) m/z calcd for C₁₀H₆NO₅ [M − H] 220.0246, found 220.0261 [M − H].
(E)-4-(4-Bromophenyl)-4-oxobut-2-enoic acid (7p). Yellow solid (64%). Mp: 161.1–163.4 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.18 (s, 1H), 6.97 (d, J = 15.6 Hz, 1H), 7.84 (d, J = 15.6 Hz, 1H), 7.97–7.95 (m, 2H), 7.79–7.77 (m, 2H); HRMS (ESI) m/z calcd for C₁₀H₆BrO₃ [M − H] 253.9500, found 252.9489 [M − H].
(E)-4-(Furan-2-yl)-4-oxobut-2-enoic acid (7q). Brown solid (64%). Mp: 158.9–160.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.18 (s, 1H), 8.14 (s, 1H), 7.83–7.82 (m, 1H), 7.71 (d, J = 15.6 Hz, 1H), 6.85–6.78 (m, 1H), 6.74 (d, J = 15.6 Hz, 1H); HRMS (ESI) m/z calcd for C₈H₅O₄ [M − H] 165.0188, found 165.0193 [M − H].
(E)-4-Oxohex-2-enoic acid (7r). White solid (54%). Mp: 123.5–125.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.13 (s, 1H), 6.80 (d, J = 16.0 Hz, 1H), 6.66 (d, J = 16.0 Hz, 1H), 2.34 (s, 3H); HRMS (ESI) m/z calcd for C₅H₅O₃ [M − H] 113.0239, found 113.0244 [M − H].
(E)-4-Cyclohexyl-4-oxobut-2-enoic acid (7s). White solid (60%). Mp: 125.6–127.1 °C. ¹HNMR (400 MHz, DMSO-d₆) δ 13.08 (s, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.60 (d, J = 16.0 Hz, 1H), 2.82–2.75 (m, 1H), 1.80–1.60 (m, 5H), 1.37–1.12 (m, 5H); HRMS (ESI) m/z calcd for C₁₀H₁₃O₃ [M − H] 181.0865, found 181.0868 [M − H].
(E)-4-Cyclopropyl-4-oxobut-2-enoic acid (7t). White solid (65%), mp: 95.55–96.3 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 13.12 (s, 1H), 7.04 (d, J = 16.0 Hz, 1H), 6.70 (d, J = 16.0 Hz, 1H), 2.53–2.47 (m, 1H), 1.03–0.97 (m, 4H); MS-ESI (m/z): 163.04 [M + Na].
(E)-Methyl 4-oxo-4-phenylbut-2-enoate (8b). Yellow solid (45%), mp: 36.4–37.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.03–7.98 (m, 2H), 7.93 (d, J = 15.6 Hz, 1H), 7.66–7.59 (m, 1H), 7.54–7.50 (m, 2H), 6.90 (d, J = 15.6 Hz, 1H), 3.85 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.23, 165.38, 136.89, 136.01, 134.10, 131.21, 129.05, 128.84, 52.23; HRMS (ESI) m/z calcd for C₁₁H₁₀O₃Na [M + Na] 213.0528, found 213.0538 [M + Na].
(E)-Methyl 4-oxo-4-(p-tolyl) but-2-enoate (8c). Yellow solid (46%), mp: 48.7–49.6 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.94–7.90 (m, 3H), 7.32–7.30 (m, 2H), 6.88 (d, J = 15.6 Hz, 1H), 3.85 (s, 3H), 2.44 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.54, 165.43, 144.83, 136.93, 133.57, 130.94, 129.63, 128.99, 52.20, 21.25; HRMS (ESI) m/z calcd for C₁₂H₁₂O₃Na [M + Na] 227.0684, found 227.0691 [M + Na].
(E)-Methyl 4-(4-ethoxyphenyl)-4-oxobut-2-enoate (8d). Yellow solid (42%), mp: 81.7–83.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.03–7.97 (m, 2H), 7.94 (d, J = 15.6 Hz, 1H), 7.01–6.92 (m, 2H), 6.88 (d, J = 15.6 Hz, 1H), 4.13 (q, J = 7.2 Hz, 2H), 3.85 (s, 3H), 1.46 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.12, 165.51, 163.30, 137.03, 131.42, 130.53, 128.84, 114.72, 63.76, 52.19, 14.45; HRMS (ESI) m/z calcd for C₁₃H₁₄O₄Na [M + Na] 257.0790, found 257.0778 [M + Na].
(E)-Methyl 4-(2-methoxyphenyl)-4-oxobut-2-enoate (8e). Yellow solid (47%), mp: 68.3–69.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.67 (d, J = 15.5 Hz, 1H), 7.65–7.56 (m, 2H), 7.24–7.22 (m, 1H), 7.10–7.07 (m, 1H), 6.59 (d, J = 15.5 Hz, 1H), 3.89 (s, 3H), 3.76 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.57, 165.62, 158.68, 140.77, 134.81, 130.14, 128.93, 126.80, 120.83, 112.69, 56.01, 52.19; HRMS (ESI) m/z calcd for C₁₂H₁₂O₄Na [M + Na] 243.0633, found 243.0625 [M + Na].
(E)-Methyl 4-(3-methoxyphenyl)-4-oxobut-2-enoate (8f). Yellow solid (42%), mp: 58.4–60.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.95 (d, J = 15.5 Hz, 1H), 7.66–7.64 (m, 1H), 7.55–7.47 (m, 2H), 7.30–7.28 (m, 1H), 6.75 (d, J = 15.5 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.95, 165.38, 159.62, 137.41, 136.85, 131.28, 130.22, 121.53, 120.37, 112.82, 55.39, 52.21; HRMS (ESI) m/z calcd for C₁₂H₁₂O₄Na [M + Na] 243.0633, found 243.0616 [M + Na].
(E)-Methyl 4-(2,5-dimethoxyphenyl)-4-oxobut-2-enoate (8g). Yellow solid (43%). Mp: 74.4–75.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.68 (d, J = 15.5 Hz, 1H), 7.23–7.17 (m, 2H), 7.11–7.10 (m, 1H), 6.59 (d, J = 15.5 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 3H), 3.75 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.19, 165.61, 153.15, 153.04, 140.71, 128.99, 127.07, 120.84, 114.35, 113.78, 56.50, 55.60, 52.20; HRMS (ESI) m/z calcd for C₁₃H₁₄O₅Na [M + Na] 273.0739, found 273.0747 [M + Na].
(E)-Methyl 4-(3,4-dimethoxyphenyl)-4-oxobut-2-enoate (8h). Yellow solid (46%). Mp: 97.7–99.0 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.01 (d, J = 15.2 Hz, 1H), 7.79–7.77 (m, 1H), 7.52–7.51 (m, 1H), 7.12–7.10 (m, 1H), 6.73 (d, J = 15.2 Hz, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 3.78 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.98, 165.53, 154.07, 149.02, 136.77, 130.52, 129.02, 124.32, 110.95, 110.39, 55.85, 55.53, 52.16; HRMS (ESI) m/z calcd for C₁₃H₁₄O₅Na [M + Na] 273.0739, found 273.0733 [M + Na].
(E)-Methyl 4-(benzo[d][1,3] dioxol-5-yl)-4-oxobut-2-enoate (8i). White solid (46%). Mp: 132.1–133.6 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.95 (d, J = 15.5 Hz, 1H), 7.75–7.73 (m, 1H), 7.52–7.51 (m, 1H), 7.10–7.08 (m, 1H), 6.71 (d, J = 15.5 Hz, 1H), 6.18 (s, 2H), 3.78 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.89, 165.46, 152.47, 148.30, 136.91, 130.79, 130.76, 126.25, 108.32, 107.66, 102.36, 52.19; MS-ESI (m/z): 235.15 [M + Na].
(E)-Methyl 4-(2,3-dihydrobenzofuran-5-yl)-4-oxobut-2-enoate (8j). Yellow solid (45%). Mp: 83.3–84.5 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.94–7.86 (m, 3H), 6.89–6.84 (m, 2H), 4.69 (t, J = 8.8 Hz, 2H), 3.84 (s, 3H), 3.28 (t, J = 8.8 Hz, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.85, 165.53, 164.89, 137.22, 131.11, 130.35, 129.32, 128.88, 126.36, 109.21, 72.45, 52.15, 28.25; HRMS (ESI) m/z calcd for C₁₃H₁₂O₄Na [M + Na] 255.0633, found 255.0627 [M + Na].
(E)-Methyl 4-([1,1′-biphenyl]-4 yl)-4-oxobut-2-enoate (8k). Yellow solid (42%). Mp: 116.0–118.6 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.15–8.13 (m, 2H), 8.02 (d, J = 15.6 Hz, 1H), 7.90–7.88 (m, 2H), 7.81–7.75 (m, 2H), 7.55–7.51 (m, 2H), 7.47–7.43 (m, 1H), 6.78 (d, J = 15.6 Hz, 1H), 3.80 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.65, 165.43, 145.40, 138.65, 136.93, 134.85, 131.14, 129.64, 129.15, 128.64, 127.21, 127.07, 52.25; HRMS (ESI) m/z calcd for C₁₇H₁₄O₃Na [M + Na] 289.0841, found 289.0836 [M + Na].
(E)-Methyl 4-oxo-4-(4-phenoxyphenyl) but-2-enoate (8l). Yellow solid (44%). Mp: 92.0–93.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.12–8.07 (m, 2H), 7.96 (d, J = 15.6 Hz, 1H), 7.52–7.46 (m, 2H), 7.31–7.25 (m, 1H), 7.19–7.14 (m, 2H), 7.11–7.06 (m, 2H), 6.74 (d, J = 15.6 Hz, 1H), 3.78 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.48, 165.45, 162.17, 154.64, 136.87, 131.64, 130.93, 130.44, 130.40, 125.08, 120.27, 117.29, 52.23; MS-ESI (m/z): 305.13 [M + Na].
(E)-Methyl 4-(naphthalen-1-yl)-4-oxobut-2-enoate (8m). Yellow oil (42%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.51–8.49 (m, 1H), 8.23–8.22 (m, 1H), 8.10–8.04 (m, 2H), 7.76 (d, J = 15.5 Hz, 1H), 7.71–7.60 (m, 3H), 6.69 (d, J = 15.5 Hz, 1H), 3.79 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 192.55, 165.34, 139.95, 133.59, 133.50, 133.45, 131.52, 129.96, 129.74, 128.65, 128.12, 126.65, 125.12, 124.72, 52.16; HRMS (ESI) m/z calcd for C₁₅H₁₂O₃Na [M + Na] 263.0684, found 263.0674 [M + Na].
(E)-Methyl 4-(2,4-dichlorophenyl)-4-oxobut-2-enoate (8n). Yellow solid (44%). Mp: 98.7–100.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.54 (d, J = 15.6 Hz, 1H), 7.50–7.44 (m, 2H), 7.38–7.36 (m, 1H), 6.69 (d, J = 15.6 Hz, 1H), 3.83 (s, 3H); HRMS (ESI) m/z calcd for C₁₁H₇Cl₂O₃ [M − H] 256.9789, found 256.9756 [M − H].
(E)-Methyl 4-(4-nitrophenyl)-4-oxobut-2-enoate (8o). Yellow solid (45%). Mp: 105.9–107.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.38–8.35 (m, 2H), 8.16–8.14 (m, 2H), 7.88 (d, J = 15.6 Hz, 1H), 6.95 (d, J = 15.6 Hz, 1H), 3.87 (s, 3H); HRMS (ESI) m/z calcd for C₂₂H₁₇N₂O₁₀ [2M − H] 469.0883, found 469.0875 [2M − H].
(E)-Methyl 4-(4-bromophenyl)-4-oxobut-2-enoate (8p). Yellow solid (46%). Mp: 77.8–79.0 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.87 (d, J = 15.6 Hz, 1H), 7.87–7.85 (m, 2H), 7.67–7.65 (m, 2H), 6.90 (d, J = 15.6 Hz, 1H), 3.85 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.50, 165.33, 136.60, 135.01, 132.12, 131.50, 130.84, 128.36, 52.27; HRMS (ESI) m/z calcd for C₁₁H₉BrO₃Na [M + Na] 290.9633, found 290.9616 [M + Na].
(E)-Methyl 4-(furan-2-yl)-4-oxobut-2-enoate (8q). Yellow solid (44%). Mp: 90.7–92.8 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.77 (d, J = 15.6 Hz, 1H), 7.70–7.69 (m, 1H), 7.39–7.38 (m, 1H), 6.99 (d, J = 15.6 Hz, 1H), 6.63–6.62 (m, 1H), 3.85 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 175.46, 165.27, 151.97, 149.73, 136.10, 130.72, 121.68, 113.29, 52.24; HRMS (ESI) m/z calcd for C₉H₇O₄ [M − H] 179.0362, found 179.0350 [M − H].
(E)-Methyl 4-oxopent-2-enoate (8r). White solid (41%). Mp: 61.9–63.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.02 (d, J = 16.0 Hz, 1H), 6.65 (d, J = 16.0 Hz, 1H), 3.81 (s, 3H), 2.35 (s, 3H); HRMS (ESI) m/z calcd for C₆H₇O₃ [M − H] 127.0412, found 127.0392 [M − H].
(E)-Methyl 4-cyclohexyl-4-oxobut-2-enoate (8s). White solid (43%). Mp: 52.3–54.6 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.19 (d, J = 16.0 Hz, 1H), 6.71 (d, J = 16.0 Hz, 1H), 3.81 (s, 3H), 2.61–2.55 (m, 1H), 1.89–1.68 (m, 5H), 1.42–1.17 (m, 5H); ¹³C NMR (125 MHz, DMSO-d₆) δ 202.32, 165.59, 138.83, 129.68, 52.19, 47.97, 27.68, 25.37, 24.88; HRMS (ESI) m/z calcd for C₁₁H₁₆O₃Na [M + Na] 219.0997, found 219.1006 [M + Na].
(E)-Methyl 4-cyclopropyl-4-oxobut-2-enoate (8t). Yellow oil (37%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.13 (d, J = 16.0 Hz, 1H), 6.79 (d, J = 16.0 Hz, 1H), 3.76 (s, 3H), 2.56–2.51 (m, 1H), 1.06–0.95 (m, 4H); ¹³C NMR (125 MHz, DMSO-d₆) δ 199.42, 165.61, 139.62, 129.80, 52.15, 19.44, 11.76; HRMS (ESI) m/z calcd for C₈H₁₀O₃Na [M + Na] 177.0528, found 177.0520 [M + Na].
(E)-Ethyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate (9a). Yellow solid (46%), mp: 41.2–42.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.06–8.05 (m, 2H), 7.99 (d, J = 15.5 Hz, 1H), 7.11–7.10 (m, 2H), 7.05–7.03 (m, 1H), 6.69 (d, J = 15.5 Hz, 1H), 4.18–4.16 (m, 2H), 3.88 (s, 3H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.14, 165.00, 163.95, 136.81, 131.36, 130.91, 129.00, 114.33, 60.93, 55.66, 13.99; HRMS (ESI) m/z calcd for C₁₃H₁₄O₄Na [M + Na] 257.0790, found 257.0792 [M + Na].
(E)-Ethyl 4-oxo-4-phenylbut-2-enoate (9b). Yellow oil (46%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.05–8.03 (m, 2H), 7.95 (d, J = 15.5 Hz, 1H), 7.75–7.69 (m, 1H), 7.63–7.56 (m, 2H), 6.73 (d, J = 15.5 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 1.28 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.15, 164.85, 136.64, 136.02, 134.01, 131.57, 128.99, 128.78, 60.98, 13.95; HRMS (ESI) m/z calcd for C₁₁H₁₀O₃Na [M + Na] 213.0528, found 213.0538 [M + Na].
(E)-Ethyl 4-oxo-4-(p-tolyl) but-2-enoate (9c). Yellow oil (43%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.96–7.92 (m, 3H), 7.40–7.38 (m, 2H), 6.71 (d, J = 15.5 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 2.41 (s, 3H), 1.28 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.51, 164.92, 144.77, 136.74, 133.58, 131.30, 129.60, 128.96, 60.97, 21.23, 13.98; HRMS (ESI) m/z calcd for C₁₃H₁₄O₃Na [M + Na] 241.0841, found 241.0840 [M + Na].
(E)-Ethyl 4-(4-ethoxyphenyl)-4-oxobut-2-enoate (9d). Yellow solid (40%), mp: 54.4–55.7 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.03–7.97 (m, 2H), 7.92 (d, J = 15.6 Hz, 1H), 6.99–6.93 (m, 2H), 6.87 (d, J = 15.6 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 4.13 (q, J = 6.8 Hz, 2H), 1.46 (t, J = 6.8 Hz, 3H), 1.35 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.15, 165.02, 163.28, 136.90, 131.40, 130.88, 128.84, 114.72, 63.75, 60.94, 14.44, 14.02; HRMS (ESI) m/z calcd for C₁₄H₁₆O₄Na [M + Na] 271.0946, found 271.0953 [M + Na].
(E)-Ethyl 4-(2-methoxyphenyl)-4-oxobut-2-enoate (9e). Yellow solid (41%), mp: 50.1–51.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.66 (d, J = 15.5 Hz, 1H), 7.64–7.57 (m, 2H), 7.24–7.22 (m, 1H), 7.10–7.07 (m, 1H), 6.57 (d, J = 15.5 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H), 1.26 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.62, 165.11, 158.65, 140.68, 134.76, 130.10, 129.32, 126.81, 120.83, 112.70, 60.91, 56.00, 13.97; HRMS (ESI) m/z calcd for C₁₃H₁₄O₄Na [M + Na] 257.0790, found 257.0778 [M + Na].
(E)-Ethyl 4-(3-methoxyphenyl)-4-oxobut-2-enoate (9f). Yellow oil (40%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.93 (d, J = 15.5 Hz, 1H), 7.68–7.62 (m, 1H), 7.55–7.47 (m, 2H), 7.30–7.28 (m, 1H), 6.73 (d, J = 15.5 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 3.85 (s, 3H), 1.28 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.99, 164.88, 159.61, 137.42, 136.74, 131.65, 130.22, 121.52, 120.32, 112.82, 61.00, 55.39, 13.98; HRMS (ESI) m/z calcd for C₁₃H₁₄O₄Na [M + Na] 257.0790, found 257.0793 [M + Na].
(E)-Ethyl 4-(2,5-dimethoxyphenyl)-4-oxobut-2-enoate (9g). Yellow solid (42%). Mp: 66.2–67.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.68 (d, J = 15.5 Hz, 1H), 7.22–7.17 (m, 2H), 7.11–7.10 (m, 1H), 6.57 (d, J = 15.5 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H), 3.84 (s, 3H), 3.75 (s, 3H), 1.26 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.18, 165.10, 153.15, 153.02, 140.61, 129.35, 127.08, 120.76, 114.33, 113.77, 60.90, 56.46, 55.57, 13.96; HRMS (ESI) m/z calcd for C₁₄H₁₆O₅Na [M + Na] 287.0895, found 287.0895 [M + Na].
(E)-Ethyl 4-(3,4-dimethoxyphenyl)-4-oxobut-2-enoate (9h). Yellow solid (43%). Mp: 92.6–94.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.00 (d, J = 15.6 Hz, 1H), 7.79–7.77 (m, 1H), 7.52–7.51 (m, 1H), 7.13–7.10 (m, 1H), 6.72 (d, J = 15.6 Hz, 1H), 4.24 (q, J = 7.2 Hz, 2H), 3.88 (s, 3H), 3.84 (s, 3H), 1.28 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.09, 165.06, 154.08, 149.04, 136.75, 130.93, 129.04, 124.38, 111.01, 110.41, 60.95, 55.89, 55.57, 14.03; HRMS (ESI) m/z calcd for C₁₄H₁₆O₅Na [M + Na] 287.0895, found 287.0878 [M + Na].
(E)-Ethyl 4-(benzo[d][1,3] dioxol-5-yl)-4-oxobut-2-enoate (9i). Yellow solid (45%). Mp: 90.8–93.0 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.93 (d, J = 15.5 Hz, 1H), 7.75–7.73 (m, 1H), 7.51–7.50 (m, 1H), 7.09–7.08 (m, 1H), 6.69 (d, J = 15.5 Hz, 1H), 6.18 (s, 2H), 4.24 (q, J = 7.0 Hz, 2H), 1.28 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.88, 164.97, 152.45, 148.29, 136.74, 131.12, 130.79, 126.22, 108.30, 107.64, 102.36, 60.95, 14.01; MS-ESI (m/z): 249.13 [M + Na].
(E)-Ethyl 4-(2,3-dihydrobenzofuran-5-yl)-4-oxobut-2-enoate (9j). Yellow solid (44%). Mp: 57.7–59.4 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.96–7.89 (m, 3H), 6.92–6.90 (m, 1H), 6.69 (d, J = 15.5 Hz, 1H), 4.67 (t, J = 9.0 Hz, 2H), 4.24 (q, J = 7.0 Hz, 2H), 3.26 (t, J = 9.0 Hz, 2H), 1.28 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.83, 165.03, 164.87, 137.03, 131.08, 130.70, 129.32, 128.85, 126.32, 72.43, 60.91, 28.25, 14.01; HRMS (ESI) m/z calcd for C₁₄H₁₄O₄Na [M + Na] 269.0790, found 269.0791 [M + Na].
(E)-Ethyl 4-([1,1′-biphenyl]-4 yl)-4-oxobut-2-enoate (9k). Yellow solid (43%). Mp: 80.5–82.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.15–8.13 (m, 2H), 8.00 (d, J = 15.6 Hz, 1H), 7.90–7.88 (m, 2H), 7.78–7.76 (m, 2H), 7.55–7.51 (m, 2H), 7.47–7.43 (m, 1H), 6.76 (d, J = 15.6 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.63, 164.93, 145.38, 138.65, 136.74, 134.85, 131.51, 129.61, 129.13, 128.61, 127.19, 127.06, 61.02, 14.01; HRMS (ESI) m/z calcd for C₁₈H₁₆O₃Na [M + Na] 303.0997, found 303.0990 [M + Na].
(E)-Ethyl 4-oxo-4-(4-phenoxyphenyl) but-2-enoate (9l). Yellow solid (41%). Mp: 53.7–55.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.12–8.06 (m, 2H), 7.94 (d, J = 15.6 Hz, 1H), 7.52–7.45 (m, 2H), 7.31–7.25 (m, 1H), 7.19–7.14 (m, 2H), 7.11–7.06 (m, 2H), 6.72 (d, J = 15.6 Hz, 1H), 4.24 (q, J = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.46, 164.93, 162.12, 154.65, 136.69, 131.59, 131.27, 130.75, 130.40, 125.04, 120.23, 117.27, 60.98, 13.99; HRMS (ESI) m/z calcd for C₁₈H₁₆O₄Na [M + Na] 319.0946, found 319.0926 [M + Na].
(E)-Ethyl 4-(naphthalen-1-yl)-4-oxobut-2-enoate (9m). Yellow oil (42%). ¹H NMR (400 MHz, CDCl₃) δ 8.52–8.50 (m, 1H), 8.05–8.03 (m, 1H), 7.94–7.88 (m, 1H), 7.87–7.85 (m, 1H), 7.74 (d, J = 15.6 Hz, 1H), 7.64–7.51 (m, 3H), 6.78 (d, J = 15.6 Hz, 1H), 4.30 (q, J = 7.2 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 192.82, 164.88, 139.97, 133.66, 133.45, 132.00, 129.92, 129.72, 128.68, 128.15, 126.71, 125.10, 124.81, 61.03, 13.94; HRMS (ESI) m/z calcd for C₁₆H₁₄O₃Na [M + Na] 277.0841, found 277.0837 [M + Na].
(E)-Ethyl 4-(2,4-dichlorophenyl)-4-oxobut-2-enoate (9n). Yellow solid (43%). Mp: 67.3–69.2 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.81–7.80 (m, 1H), 7.70–7.69 (m, 1H), 7.61–7.59 (m, 1H), 7.39 (d, J = 16.0 Hz, 1H), 6.56 (d, J = 16.0 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H), 1.25 (t, J = 7.0 Hz, 3H); HRMS (ESI) m/z calcd for C₁₂H₁₀Cl₂O₃Na [M + Na] 294.9918, found 294.9918 [M + Na].
(E)-Ethyl 4-(4-nitrophenyl)-4-oxobut-2-enoate (9o). Yellow solid (42%). Mp: 69.4–71.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.38–8.35 (m, 2H), 8.27–8.25 (m, 2H), 7.93 (d, J = 15.5 Hz, 1H), 6.77 (d, J = 15.5 Hz, 1H), 4.26 (q, J = 7.0 Hz, 2H), 1.29 (t, J = 7.0 Hz, 3H); HRMS (ESI) m/z calcd for C₂₄H₂₁N₂O₁₀ [2M − H] 497.1196, found 497.1193 [2M − H].
(E)-Ethyl 4-(4-bromophenyl)-4-oxobut-2-enoate (9p). Yellow solid (44%). Mp: 62.3–64.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.99–7.97 (m, 2H), 7.91 (d, J = 15.5 Hz, 1H), 7.80–7.78 (m, 2H), 6.74 (d, J = 15.5 Hz, 1H), 4.25 (q, J = 7.0 Hz, 2H), 1.28 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.50, 164.83, 136.42, 135.01, 132.10, 130.82, 128.33, 61.05, 14.00; HRMS (ESI) m/z calcd for C₁₂H₁₁BrO₃Na [M + Na] 306.9769, found 306.9783 [M + Na].
(E)-Ethyl 4-(furan-2-yl)-4-oxobut-2-enoate (9q). Yellow solid (46%). Mp: 63.6–65.2 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.76 (d, J = 15.6 Hz, 1H), 7.71–7.67 (m, 1H), 7.38–7.37 (m, 1H), 6.98 (d, J = 15.6 Hz, 1H), 6.63–7.61 (m, 1H), 4.30 (q, J = 7.2 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 175.50, 164.77, 151.98, 149.68, 135.96, 131.08, 121.59, 113.27, 61.03, 13.98; HRMS (ESI) m/z calcd for C₁₀H₁₀O₄Na [M + Na] 217.0477, found 217.0456 [M + Na].
(E)-Ethyl 4-oxopent-2-enoate (9r). Yellow oil (40%). ¹H NMR (400 MHz, CDCl₃) δ 7.00 (d, J = 16.0 Hz, 1H), 6.63 (d, J = 16.0 Hz, 1H), 4.25 (q, J = 7.2 Hz, 2H), 2.34 (s, 3H), 1.31 (t, J = 7.2 Hz, 3H); MS-ESI (m/z): 165.09 [M + Na].
(E)-Ethyl 4-cyclohexyl-4-oxobut-2-enoate (9s). Yellow oil (39%). ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 16.0 Hz, 1H), 6.70 (d, J = 16.0 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 2.60–2.53 (m, 1H), 1.86–1.65 (m, 5H), 1.36–1.24 (m, 5H); ¹³C NMR (125 MHz, DMSO-d₆) δ 202.12, 165.01, 138.59, 130.00, 60.87, 47.89, 27.71, 25.37, 24.88, 13.88; HRMS (ESI) m/z calcd for C₁₂H₁₈O₃Na [M + Na] 233.1154, found 233.1162 [M + Na].
(E)-Ethyl 4-cyclopropyl-4-oxobut-2-enoate (9t). Yellow oil (40%). ¹H NMR (500 MHz, CDCl₃) δ 7.17 (d, J = 16.0 Hz, 1H), 6.71 (d, J = 16.0 Hz, 1H), 4.26 (q, J = 7.0 Hz, 2H), 2.22–2.17 (m, 1H), 1.32 (t, J = 7.0 Hz, 3H), 1.19–1.14 (m, 2H), 1.04–1.00 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 199.54, 165.16, 139.58, 130.26, 60.99, 19.37, 13.96, 11.77; MS-ESI (m/z): 191.22 [M + Na].
(E)-Isopropyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate (10a). Yellow solid (45%), mp: 43.3–44.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.06–8.04 (m, 2H), 7.93 (d, J = 15.5 Hz, 1H), 7.11–7.09 (m, 2H), 6.67 (d, J = 15.5 Hz, 1H), 5.09–5.00 (m, 1H), 3.87 (s, 3H), 1.29 (s, 3H), 1.28 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.13, 164.50, 163.94, 136.66, 131.32, 129.01, 114.33, 68.53, 55.65, 21.49; HRMS (ESI) m/z calcd for C₁₄H₁₆O₄Na [M + Na] 271.0946, found 271.0959 [M + Na].
(E)-Isopropyl 4-oxo-4-phenylbut-2-enoate (10b). Yellow oil (47%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.07–8.01 (m, 2H), 7.92 (d, J = 15.5 Hz, 1H), 7.74–7.70 (m, 1H), 7.62–7.57 (m, 2H), 6.70 (d, J = 15.5 Hz, 1H), 5.09–5.02 (m, 1H), 1.29 (s, 3H), 1.28 (s, 3H); HRMS (ESI) m/z calcd for C₁₃H₁₄O₃Na [M + Na] 241.0841, found 241.0837 [M + Na].
(E)-Isopropyl 4-oxo-4-(p-tolyl) but-2-enoate (10c). Yellow solid (44%), mp: 60.2–61.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.92–7.86 (m, 3H), 7.32–7.30 (m, 2H), 6.85 (d, J = 15.6 Hz, 1H), 5.20–5.12 (m, 1H), 2.44 (s, 3H), 1.33 (s, 3H), 1.32 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.56, 164.43, 144.76, 136.65, 133.58, 131.71, 129.61, 128.96, 68.58, 21.48, 21.24; HRMS (ESI) m/z calcd for C₁₄H₁₆O₃Na [M + Na] 255.0997, found 255.0982 [M + Na].
(E)-Isopropyl 4-(4-ethoxyphenyl)-4-oxobut-2-enoate (10d). Yellow solid (44%), mp: 38.6–40.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.04–8.02 (m, 2H), 7.93 (d, J = 15.5 Hz, 1H), 7.09–7.07 (m, 2H), 6.67 (d, J = 15.5 Hz, 1H), 5.11–4.99 (m, 1H), 4.15 (q, J = 7.0 Hz, 2H), 1.36 (t, J = 7.0 Hz, 3H), 1.29 (s, 3H), 1.28 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.07, 164.49, 163.25, 136.67, 131.34, 131.27, 128.84, 114.66, 68.51, 63.72, 21.48, 14.41; HRMS (ESI) m/z calcd for C₁₅H₁₈O₄Na [M + Na] 285.1103, found 285.1107 [M + Na].
(E)-Isopropyl 4-(2-methoxyphenyl)-4-oxobut-2-enoate (10e). Yellow oil (43%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.65 (d, J = 15.5 Hz, 1H), 7.62–7.57 (m, 2H), 7.24–7.22 (m, 1H), 7.10–7.07 (m, 1H), 6.53 (d, J = 15.5 Hz, 1H), 5.05–5.00 (m, 1H), 3.89 (s, 3H), 1.27 (s, 3H), 1.26 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.64, 164.58, 158.62, 140.58, 134.71, 130.06, 129.73, 126.81, 120.82, 112.70, 68.46, 55.97, 21.46; HRMS (ESI) m/z calcd for C₁₄H₁₆O₄Na [M + Na] 271.0946, found 271.0938 [M + Na].
(E)-Isopropyl 4-(3-methoxyphenyl)-4-oxobut-2-enoate (10f). Yellow oil (45%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.91 (d, J = 15.5 Hz, 1H), 7.67–7.61 (m, 1H), 7.54–7.47 (m, 2H), 7.30–7.28 (m, 1H), 6.70 (d, J = 15.5 Hz, 1H), 5.10–5.00 (m, 1H), 3.84 (s, 3H), 1.29 (s, 3H), 1.28 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.92, 164.34, 159.59, 137.42, 136.53, 132.05, 130.16, 121.47, 120.22, 112.82, 68.59, 55.34, 21.43; HRMS (ESI) m/z calcd for C₁₄H₁₆O₄Na [M + Na] 271.0946, found 271.0932 [M + Na].
(E)-Isopropyl 4-(2,5-dimethoxyphenyl)-4-oxobut-2-enoate (10g). Yellow solid (40%). Mp: 43.5–45.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.66 (d, J = 15.5 Hz, 1H), 7.24–7.16 (m, 2H), 7.11–7.10 (m, 1H), 6.53 (d, J = 15.5 Hz, 1H), 5.08–4.99 (m, 1H), 3.84 (s, 3H), 3.75 (s, 3H), 1.27 (s, 3H), 1.25 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.20, 164.58, 153.15, 153.00, 140.52, 129.77, 127.09, 120.71, 114.34, 113.75, 68.46, 56.44, 55.56, 21.45; HRMS (ESI) m/z calcd for C₁₅H₁₈O₅Na [M + Na] 301.1052, found 301.1062 [M + Na].
(E)-Isopropyl 4-(3,4-dimethoxyphenyl)-4-oxobut-2-enoate (10h). Yellow solid (41%). Mp: 65.4–66.8 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.97 (d, J = 15.5 Hz, 1H), 7.78–7.76 (m, 1H), 7.51–7.50 (m, 1H), 7.12–7.11 (m, 1H), 6.69 (d, J = 15.5 Hz, 1H), 5.12–4.98 (m, 1H), 3.88 (s, 3H), 3.84 (s, 3H), 1.29 (s, 3H), 1.28 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.05, 164.55, 154.06, 149.03, 136.59, 131.31, 129.04, 124.35, 110.97, 110.38, 68.53, 55.87, 55.54, 21.50; HRMS (ESI) m/z calcd for C₁₅H₁₈O₅Na [M + Na] 301.1052, found 301.1052 [M + Na].
(E)-Isopropyl 4-(Benzo[d][1,3] dioxol-5-yl)-4-oxobut-2-enoate (10i). Yellow solid (43%). Mp: 54.4–55.8 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.90 (d, J = 15.5 Hz, 1H), 7.74–7.72 (m, 1H), 7.51–7.50 (m, 1H), 7.09–7.08 (m, 1H), 6.66 (d, J = 15.5 Hz, 1H), 6.18 (s, 2H), 5.04 (m, 1H), 1.29 (s, 3H), 1.27 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.86, 164.46, 152.42, 148.27, 136.56, 131.51, 130.79, 126.17, 108.27, 107.62, 102.35, 68.53, 21.49; MS-ESI (m/z): 285.16 [M + Na].
(E)-Isopropyl 4-(2,3-dihydrobenzofuran-5-yl)-4-oxobut-2-enoate (10j). Yellow solid (41%). Mp: 65.6–66.9 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.97–7.86 (m, 3H), 6.91–6.89 (m, 1H), 6.64 (d, J = 15.5 Hz, 1H), 5.09–4.97 (m, 1H), 4.66 (t, J = 9.0 Hz, 2H), 3.25 (t, J = 9.0 Hz, 2H), 1.27 (s, 3H), 1.26 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.84, 164.85, 164.53, 136.88, 131.11, 131.07, 129.32, 128.84, 126.30, 109.18, 72.42, 68.48, 28.25, 21.49; HRMS (ESI) m/z calcd for C₁₅H₁₆O₄Na [M + Na] 283.0946, found 283.0954 [M + Na].
(E)-Isopropyl 4-([1,1′-biphenyl]-4-yl)-4-oxobut-2-enoate (10k). Yellow solid (40%). Mp: 117.9–119.0 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.97–7.95 (m, 2H), 7.87–7.85 (m, 2H), 7.78–7.73 (m, 2H), 7.54–7.50 (m, 2H), 7.46–7.42 (m, 1H), 7.25 (d, J = 15.6 Hz, 1H), 6.37 (d, J = 15.6 Hz, 1H), 4.80 (m, 1H), 1.04 (s, 3H), 1.03 (s, 3H); MS-ESI (m/z): 317.21 [M + Na].
(E)-Isopropyl 4-oxo-4-(4-phenoxyphenyl) but-2-enoate (10l). Yellow solid (42%). Mp: 74.5–76.0 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.11–8.05 (m, 2H), 7.91 (d, J = 15.5 Hz, 1H), 7.53–7.45 (m, 2H), 7.31–7.24 (m, 1H), 7.19–7.14 (m, 2H), 7.11–7.06 (m, 2H), 6.69 (d, J = 15.5 Hz, 1H), 5.06–5.04 (m, 1H), 1.29 (s, 3H), 1.27 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.51, 164.45, 162.12, 154.65, 136.60, 131.67, 131.60, 130.76, 130.41, 125.05, 120.24, 117.29, 68.60, 21.50; HRMS (ESI) m/z calcd for C₁₉H₁₈O₄Na [M + Na] 333.1103, found 333.1087 [M + Na].
(E)-Isopropyl 4-(naphthalen-1-yl)-4-oxobut-2-enoate (10m). Yellow oil (43%). ¹H NMR (400 MHz, CDCl₃) δ 8.51–8.49 (m, 1H), 8.05–8.03 (m, 1H), 7.92–7.85 (m, 2H), 7.71 (d, J = 15.6 Hz, 1H), 7.66–7.50 (m, 3H), 6.75 (d, J = 15.6 Hz, 1H), 5.18–5.12 (m, 1H), 1.32 (s, 3H), 1.30 (s, 3H); HRMS (ESI) m/z calcd for C₁₇H₁₆O₃Na [M + Na] 291.0997, found 291.1002 [M + Na].
(E)-Isopropyl 4-(2,4-dichlorophenyl)-4-oxobut-2-enoate (10n). Yellow solid (45%). Mp: 58.0–60.1 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.51–7.44 (m, 3H), 7.37–7.35 (m, 1H), 6.63 (d, J = 16.0 Hz, 1H), 5.18–5.08 (m, 1H), 1.31 (s, 6H), 1.29 (s, 3H); HRMS (ESI) m/z calcd for C₁₃H₁₂Cl₂O₃Na [M + Na] 309.0061, found 309.0079 [M + Na].
(E)-Isopropyl 4-(4-nitrophenyl)-4-oxobut-2-enoate (10o). Yellow solid (44%). Mp: 81.5–82.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.42–8.34 (m, 2H), 8.29–8.21 (m, 2H), 7.90 (d, J = 15.5 Hz, 1H), 6.74 (d, J = 15.5 Hz, 1H), 5.09–5.04 (m, 1H), 1.30 (s, 3H), 1.28 (s, 3H); HRMS (ESI) m/z calcd for C₁₃H₁₂NO₅ [M − H] 262.0715, found 262.0698 [M − H].
(E)-Isopropyl 4-(4-bromophenyl)-4-oxobut-2-enoate (10p). Yellow solid (41%). Mp: 101.7–102.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.98–7.96 (m, 2H), 7.89 (d, J = 15.5 Hz, 1H), 7.81–7.79 (m, 2H), 6.71 (d, J = 15.5 Hz, 1H), 5.10–4.99 (m, 1H), 1.29 (s, 3H), 1.28 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.54, 164.33, 136.32, 135.02, 132.10, 130.82, 128.30, 68.86, 21.49; HRMS (ESI) m/z calcd for C₁₃H₁₃BrO₃Na [M + Na] 318.9946, found 318.9951 [M + Na].
(E)-Isopropyl 4-(furan-2-yl)-4-oxobut-2-enoate (10q). Yellow solid (42%). Mp: 41.7–43.4 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 15.6 Hz, 1H), 7.70–7.69 (m, 1H), 7.38–7.37 (m, 1H), 6.96 (d, J = 15.6 Hz, 1H), 6.63–6.62 (m, 1H), 5.19–5.10 (m, 1H), 1.33 (s, 6H), 1.31 (s, 1H); ¹³C NMR (125 MHz, DMSO-d₆) δ 175.53, 164.27, 151.98, 149.63, 135.83, 131.48, 121.50, 113.27, 68.66, 21.46; HRMS (ESI) m/z calcd for C₁₀H₁₀O₄Na [M + Na] 231.0633, found 231.0632 [M + Na].
(E)-Isopropyl 4-oxopent-2-enoate (10r). Yellow oil (42%). ¹H NMR (400 MHz, CDCl₃) δ 6.98 (d, J = 16.0 Hz, 1H), 6.61 (d, J = 16.0 Hz, 1H), 5.14–5.08 (m, 1H), 2.34 (s, 3H); MS-ESI (m/z): 179.14 [M + Na].
(E)-Isopropyl 4-cyclohexyl-4-oxobut-2-enoate (10s). Yellow oil (37%). ¹H NMR (500 MHz, CDCl₃) δ 7.14 (d, J = 16.0 Hz, 1H), 6.67 (d, J = 16.0 Hz, 1H), 5.14–5.09 (m, 1H), 2.63–2.57 (m, 1H), 1.91–1.66 (m, 5H), 1.39–1.22 (m, 11H); ¹³C NMR (125 MHz, DMSO-d₆) δ 202.34, 164.57, 138.55, 130.46, 68.51, 47.74, 27.75, 25.37, 24.87, 21.44; MS-ESI (m/z): 247.20 [M + Na].
(E)-Isopropyl 4-cyclopropyl-4-oxobut-2-enoate (10t). Yellow oil (39%). ¹H NMR (500 MHz, CDCl₃) δ 7.14 (d, J = 16.0 Hz, 1H), 6.69 (d, J = 16.0 Hz, 1H), 5.14–5.09 (m, 1H), 2.22–2.17 (m, 1H), 1.29 (s, 3H), 1.28 (s, 3H), 1.18–1.13 (m, 2H), 1.03–0.99 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 199.58, 164.66, 139.51, 130.71, 68.60, 21.47, 19.29, 11.71; HRMS (ESI) m/z calcd for C₁₀H₁₄O₃Na [M + Na] 205.0841, found 205.0826 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate (11a). White solid (81%), mp: 93.5–94.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.06–8.05 (m, 2H), 7.99 (d, J = 15.5 Hz, 1H), 7.11–7.10 (m, 2H), 7.05–7.03 (m, 1H), 6.69 (d, J = 15.5 Hz, 1H), 4.18–4.16 (m, 2H), 3.88 (s, 3H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.19, 164.92, 164.00, 155.71, 136.85, 131.38, 130.93, 129.01, 114.37, 77.85, 64.07, 55.71, 38.82, 28.19; HRMS (ESI) m/z calcd for C₁₈H₂₃NO₆Na [M + Na] 372.1423, found 372.1441 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-oxo-4-phenylbut-2-enoate (11b). White solid (75%), mp: 95.5–96.8 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.08–8.02 (m, 2H), 7.97 (d, J = 15.6 Hz, 1H), 7.75–7.71 (m, 1H), 7.62–7.58 (m, 2H), 7.06–7.03 (m, 1H), 6.72 (d, J = 15.6 Hz, 1H), 4.18–4.16 (m, 2H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.33, 164.81, 155.71, 136.78, 136.02, 134.14, 131.59, 129.07, 128.82, 77.85, 64.14, 38.80, 28.19; HRMS (ESI) m/z calcd for C₁₇H₂₁NO₅Na [M + Na] 342.1317, found 342.1320 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-oxo-4-(p-tolyl) but-2-enoate (11c). White solid (84%), mp: 88.7–91.4 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.99–7.95 (m, 3H), 7.41–7.39 (m, 2H), 7.06–7.03 (m, 1H), 6.70 (d, J = 15.6 Hz, 1H), 4.18–4.15 (m, 2H), 3.28–3.24 (m, 2H), 2.41 (s, 3H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.62, 164.85, 155.71, 144.88, 136.80, 133.58, 131.32, 129.65, 128.98, 77.85, 64.12, 38.80, 28.19, 21.21; HRMS (ESI) m/z calcd for C₁₈H₂₃NO₅Na [M + Na] 356.1474, found 356.1469 [M + Na].
(E)-2-((tert-butoxycarbonyl) amino) ethyl 4-(4-ethoxyphenyl)-4-oxobut-2-enoate (11d). White solid (79%), mp: 96.5–97.6 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.05–8.03 (m, 2H), 7.98 (d, J = 15.5 Hz, 1H), 7.09–7.07 (m, 2H), 7.05–7.03 (m, 1H), 6.69 (d, J = 15.5 Hz, 1H), 4.22–4.10 (m, 4H), 3.28–3.25 (m, 2H), 1.42–1.31 (m, 12H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.13, 164.92, 163.31, 155.71, 136.86, 131.38, 130.87, 128.83, 114.71, 77.84, 64.07, 63.76, 38.81, 28.19, 14.43; HRMS (ESI) m/z calcd for C₁₉H₂₄NO₆ [M − H] 362.1604, found 362.1595 [M − H].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(2-methoxyphenyl)-4-oxobut-2-enoate (11e). White solid (84%), mp: 83.6–85.4 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.67 (d, J = 15.5 Hz, 1H), 7.64–7.55 (m, 2H), 7.24–7.22 (m, 1H), 7.11–7.06 (m, 1H), 7.01–7.99 (m, 1H), 6.55 (d, J = 15.5 Hz, 1H), 4.16–4.14 (m, 2H), 3.89 (s, 3H), 3.25–3.22 (m, 2H), 1.35 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.84, 165.05, 158.62, 155.67, 140.72, 134.69, 130.00, 129.37, 126.88, 120.81, 112.67, 77.78, 63.89, 55.98, 38.80, 28.15; HRMS (ESI) m/z calcd for C₁₈H₂₃NO₆Na [M + Na] 372.1423, found 372.1424 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(3-eethoxyphenyl)-4-oxobut-2-enoate (11f). White solid (81%), mp: 65.7–66.8 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.96 (d, J = 15.6 Hz, 1H), 7.66–7.64 (m, 1H), 7.55–7.48 (m, 2H), 7.31–7.28 (m, 1H), 7.06–7.03 (m, 1H), 6.71 (d, J = 15.6 Hz, 1H), 4.18–4.15 (m, 2H), 3.85 (s, 3H), 3.28–3.24 (m, 2H), 2.50 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 189.08, 164.79, 159.64, 155.71, 137.42, 136.78, 131.67, 130.26, 121.52, 120.37, 112.89, 77.85, 64.14, 55.43, 38.80, 28.18; MS-ESI (m/z): 372.20 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(2,5-dimethoxyphenyl)-4-oxobut-2-enoate (11g). Yellow oil (81%). ¹H NMR (500 MHz, DMSO-d₆) δ 7.68 (d, J = 15.5 Hz, 1H), 7.24–7.16 (m, 2H), 7.10–7.09 (m, 1H), 7.01–6.99 (m, 1H), 6.55 (d, J = 15.5 Hz, 1H), 4.16–4.14 (m, 2H), 3.84 (s, 3H), 3.75 (s, 3H), 3.25–3.22 (m, 2H), 1.36 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 190.44, 165.06, 155.69, 153.15, 152.99, 140.66, 129.44, 127.16, 120.69, 114.33, 113.73, 77.81, 63.89, 56.46, 55.60, 38.81, 28.16; HRMS (ESI) m/z calcd for C₁₉H₂₅NO₇Na [M + Na] 402.1529, found 402.1519 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(3,4-dimethoxyphenyl)-4-oxobut-2-enoate (11h). Orange solid (69%), mp: 100.0–101.7 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.02 (d, J = 15.6 Hz, 1H), 7.80–7.77 (m, 1H), 7.53–7.52 (m, 1H), 7.13–7.11 (m, 1H), 7.06–7.03 (m, 1H), 6.71 (d, J = 15.6 Hz, 1H), 4.18–4.16 (m, 2H), 3.88 (s, 3H), 3.85 (s, 3H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.08, 164.95, 155.71, 154.10, 149.05, 136.73, 130.91, 129.03, 124.35, 110.99, 110.42, 77.84, 64.05, 55.90, 55.56, 38.81, 28.19; HRMS (ESI) m/z calcd for C₁₉H₂₅NO₇Na [M + Na] 402.1529, found 402.1540 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(benzo[d][1,3] dioxol-5-yl)-4-oxobut-2-enoate (11i). Brown solid (68%), mp: 115.0–117.4 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.96 (d, J = 15.6 Hz, 1H), 7.76–7.73 (m, 1H), 7.52–7.51 (m, 1H), 7.11–7.09 (m, 1H), 7.06–7.03 (m, 1H), 6.68 (d, J = 15.6 Hz, 1H), 6.18 (s, 2H), 4.17–4.14 (m, 2H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); MS-ESI (m/z): 386.16 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(2,3-dihydrobenzofuran-5-yl)-4-oxobut-2-enoate (11j). White solid (67%), mp: 106.0–107.6 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.00–7.96 (m, 2H), 7.93–7.90 (m, 1H), 7.06–7.03 (m, 1H), 6.94–6.92 (m, 1H), 6.68 (d, J = 15.6 Hz, 1H), 4.68 (t, J = 8.8 Hz, 2H), 4.17–4.15 (m, 2H), 3.29–3.24 (m, 4H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 186.90, 164.95, 164.92, 155.71, 137.09, 131.12, 130.72, 129.32, 128.92, 126.33, 109.22, 77.84, 72.46, 64.06, 38.81, 28.26, 28.19; HRMS (ESI) m/z calcd for C₁₉H₂₃NO₆Na [M + Na] 384.1423, found 384.1416 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-([1,1′-biphenyl]-4-yl)-4-oxobut-2-enoate (11k). Yellow solid (62%), mp: 87.4–89.1 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.16–8.14 (m, 2H), 8.03 (d, J = 15.6 Hz, 1H), 7.91–7.89 (m, 2H), 7.81–7.76 (m, 2H), 7.57–7.51 (m, 2H), 7.51–7.44 (m, 1H), 7.08–7.05 (m, 1H), 6.75 (d, J = 15.6 Hz, 1H), 4.20–4.17 (m, 2H), 3.30–3.25 (m, 2H), 1.38 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.70, 164.85, 155.71, 145.43, 138.64, 136.78, 134.86, 131.52, 129.62, 129.16, 129.11, 128.65, 127.22, 127.08, 127.05, 77.86, 64.15, 38.82, 28.19; MS-ESI (m/z): 418.15 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-oxo-4-(4-phenoxyphenyl) but-2-enoate (11l). Brown solid (60%), mp: 76.4–78.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.10–8.08 (m, 2H), 7.97 (d, J = 15.5 Hz, 1H), 7.50–7.47 (m, 2H), 7.29–7.28 (m, 1H), 7.22–7.13 (m, 2H), 7.10–7.08 (m, 2H), 7.05–7.03 (m, 1H), 6.71 (d, J = 15.5 Hz, 1H), 4.18–4.16 (m, 2H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 187.50, 164.85, 162.16, 155.70, 154.64, 136.71, 131.59, 131.27, 130.75, 130.41, 125.06, 120.24, 117.29, 77.84, 64.10, 38.81, 28.18; MS-ESI (m/z): 434.19 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(naphthalen-1-yl)-4-oxobut-2-enoate (11m). Yellow solid (57%), mp: 74.7–76.0 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92–8.90 (m, 1H), 8.46–8.44 (m, 1H), 8.24–8.19 (m, 2H), 8.08–8.02 (m, 4H), 7.75 (d, J = 15.6 Hz, 1H), 7.72–7.58 (m, 6H), 7.42 (d, J = 12.0 Hz, 1H), 7.04–7.01 (m, 1H), 6.83–6.81 (m, 1H), 6.64 (d, J = 15.6 Hz, 1H), 6.37 (d, J = 12.0 Hz, 1H), 4.18–4.15 (m, 2H), 3.89–3.86 (m, 2H), 3.26–3.24 (m, 2H), 3.04–3.00 (m, 2H), 1.35 (s, 9H), 1.34 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 193.07, 164.83, 155.69, 140.06, 133.74, 133.45, 132.10, 129.81, 129.71, 128.73, 128.21, 126.77, 125.05, 124.84, 77.82, 64.17, 38.77, 28.16; MS-ESI (m/z): 392.20 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(2,4-dichlorophenyl)-4-oxobut-2-enoate (11n). Yellow solid (56%), mp: 54.6–56.3 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.82–7.81 (m, 1H), 7.69–7.67 (m, 1H), 7.62–7.59 (m, 1H), 7.41 (d, J = 16.0 Hz, 1H), 7.02–6.99 (m, 1H), 6.52 (d, J = 16.0 Hz, 1H), 4.16–4.13 (m, 2H), 3.24–3.20 (m, 2H), 1.35 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.53, 164.73, 155.69, 136.41, 135.02, 132.12, 130.79, 128.37, 77.85, 64.15, 38.79, 28.18; HRMS (ESI) m/z calcd for C₁₇H₁₉Cl₂NO₅Na [M + Na] 410.0538, found 410.0560 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(4-nitrophenyl)-4-oxobut-2-enoate (11o). Yellow solid (55%), mp: 86.6–88.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.43–8.34 (m, 2H), 8.27–8.25 (m, 2H), 7.96 (d, J = 15.6 Hz, 1H), 7.06–7.03 (m, 1H), 6.76 (d, J = 15.6 Hz, 1H), 4.19–4.17 (m, 2H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); HRMS (ESI) m/z calcd for C₁₇H₁₉N₂O₇ [M − H] 363.1193, found 363.1201 [M − H].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(4-bromophenyl)-4-oxobut-2-enoate (11p). Yellow solid (53%), mp: 86.6–88.5 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 7.99–7.97 (m, 2H), 7.94 (d, J = 15.5 Hz, 1H), 7.81–7.79 (m, 2H), 7.05–7.03 (m, 1H), 6.73 (d, J = 15.5 Hz, 1H), 4.18–4.16 (m, 2H), 3.28–3.24 (m, 2H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 188.53, 164.73, 155.69, 136.41, 135.02, 132.12, 130.79, 128.37, 77.85, 64.15, 38.79, 28.18; MS-ESI (m/z): 420.10 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-(furan-2-yl)-4-oxobut-2-enoate (11q). White solid (55%), mp: 86.6–88.5 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 8.16–8.15 (m, 1H), 7.84–7.83 (m, 1H), 7.78 (d, J = 15.6 Hz, 1H), 7.04–7.01 (m, 1H), 6.84–6.82 (m, 1H), 6.78 (d, J = 15.6 Hz, 1H), 4.18–4.15 (m, 2H), 3.27–3.23 (m, 2H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 175.54, 164.69, 155.70, 151.97, 149.77, 149.74, 136.01, 131.08, 121.66, 121.62, 113.29, 77.85, 64.10, 38.79, 28.18; HRMS (ESI) m/z calcd for C₁₅H₁₉NO₆Na [M + Na] 332.1110, found 332.1105 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-oxopent-2-enoate (11r). White solid (51%), mp: 96.7–98.6 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 7.02–7.00 (m, 1H), 6.90 (d, J = 16.0 Hz, 1H), 6.72 (d, J = 16.0 Hz, 1H), 4.15–4.12 (m, 2H), 3.24–3.20 (m, 2H), 2.35 (s, 3H), 1.37 (s, 9H); ¹³C NMR (125 MHz, DMSO-d₆) δ 198.33, 165.15, 155.67, 140.21, 131.14, 77.84, 63.99, 38.77, 28.19, 27.88; HRMS (ESI) m/z calcd for C₁₂H₁₉NO₅Na [M + Na] 280.1161, found 280.1162 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-cyclohexyl-4-oxobut-2-enoate (11s). Yellow solid (48%), mp: 70.1–72.8 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.19 (d, J = 16.0 Hz, 1H), 6.70 (d, J = 16.0 Hz, 1H), 4.79 (s, 1H), 4.27–4.25 (m, 2H), 3.45–3.44 (m, 2H), 2.61–2.56 (m, 1H), 1.88–1.79 (m, 5H), 1.44 (s, 9H), 1.40–1.19 (m, 5H); MS-ESI (m/z): 348.22 [M + Na].
(E)-2-((tert-Butoxycarbonyl) amino) ethyl 4-cyclopropyl-4-oxobut-2-enoate (11t). White solid (47%), mp: 70.1–73.4 °C. ¹H NMR (500 MHz, CDCl₃) δ 7.19 (d, J = 16.0 Hz, 1H), 6.72 (d, J = 16.0 Hz, 1H), 4.79 (s, 1H), 4.28–4.26 (m, 2H), 3.45–3.44 (m, 2H), 2.22–2.17 (m, 1H), 1.44 (s, 9H), 1.21–1.14 (m, 2H), 1.06–1.02 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 199.61, 165.08, 155.68, 139.64, 130.24, 77.83, 63.99, 38.77, 19.39, 11.74; HRMS (ESI) m/z calcd for C₁₄H₂₁NO₅Na [M + Na] 306.1317, found 306.1309 [M + Na].

In vitro protein kinase assay¹²

The inhibition rates and IC₅₀ values of target compounds were determined by a non-radioactive assay using Promega ‘Kinase Glo’ plus Luminescent Kinase assay kit. The assay was carried out in a 96-well plate and the reaction mixture was prepared: 3 μM PknB¹² in buffer (25 mL Tris–HCl pH 7.4, 5 mM MgCl₂, 2 mM MnCl₂) containing the compound at various concentrations. Following inculation at 4 °C for 30 min, ATP was added to the reaction buffer at the final concentration of 100 μM, and the plates were conducted at 37 °C for 3 hours. Multilabel Plate Reader (PE Envision) was used to measure the intensity of luminescence signal with addition of 50 μL Kinase Glo reagent. The inhibition rates were calculated by the following formula and the IC₅₀ values were calculated using the GraphPad Prism5 software.
ΔLN: luminescence intensity of negative control; ΔLS: luminescence intensity of sample; ΔLP: luminescence intensity of positive control.

Conflict of interest

The authors declare no competing financial interests.

Acknowledgements

This work was supported by the National Science and Technology Project of China (No. 2014ZX09201001-011), the National Natural Science Foundation of China (No. 81373452, 81302644, 81473098, 81473099 and 30970038), and the National Infrastructure of Microbial Resources (No. NIMR-2016-03). Liyan Yu is supported by Xiehe Scholar.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: NMR spectra of synthesized compounds. See DOI: 10.1039/c6ra24953a

‡ These authors contributed equally to this work.

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