Asymmetric Synthesis of Derivatives of Alanine via Michael Addition Reaction and their Biological Study

Anna F. Mkrtchyan; Anna S. Tovmasyan; Ani M. Paloyan; Armen S. Sargsyan; Hayarpi M. Simonyan; Lusine Yu. Sahakyan; Satenik Gh. Petrosyan; Liana A. Hayriyan; Tatev H. Sargsyan

doi:10.1055/a-1941-2068

Synlett, Table of Contents

Synlett 2022; 33(20): 2013-2018
DOI: 10.1055/a-1941-2068

letter

Asymmetric Synthesis of Derivatives of Alanine via Michael Addition Reaction and their Biological Study

Anna F. Mkrtchyan ∗

^aScientific and Production Center ‘Armbiotechnology’ of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia

^bYerevan State University, 1 Alex Manoogian Str., 0025 Yerevan, Armenia

,

Anna S. Tovmasyan

^aScientific and Production Center ‘Armbiotechnology’ of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia

,

Ani M. Paloyan

^aScientific and Production Center ‘Armbiotechnology’ of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia

,

Armen S. Sargsyan

^aScientific and Production Center ‘Armbiotechnology’ of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia

,

Hayarpi M. Simonyan

^bYerevan State University, 1 Alex Manoogian Str., 0025 Yerevan, Armenia

,

Lusine Yu. Sahakyan

^bYerevan State University, 1 Alex Manoogian Str., 0025 Yerevan, Armenia

,

Satenik Gh. Petrosyan

^bYerevan State University, 1 Alex Manoogian Str., 0025 Yerevan, Armenia

,

Liana A. Hayriyan

^aScientific and Production Center ‘Armbiotechnology’ of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia

,

Tatev H. Sargsyan

^aScientific and Production Center ‘Armbiotechnology’ of NAS RA, 14 Gyurjyan Str., 0056 Yerevan, Armenia

^bYerevan State University, 1 Alex Manoogian Str., 0025 Yerevan, Armenia

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Abstract

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Abstract

Ni(II) complex of the Schiff base of the chiral auxiliary (S)-2-N-(N′-benzylprolyl)aminobenzophenone (BPB) and dehydroalanine as the initial complex in the addition reaction was investigated. The obtained four new derivatives of α-alanine were investigated as inhibitors of aldose reductase. Only one of them: (S)-2-amino-3-[(4-methylbenzyl)amino]propanoic acid showed activity. It becomes a reason for studying the patterns of biological activity of the structure of α-amino acids. The results of docking analysis indicated that (S)-2-amino-3-[(4-methylbenzyl)amino]propanoic acid demonstrated the ability to form bonds with different functional groups of the enzyme which let us assume that some amino acids of nonfunctional groups, such as Trp²⁰ of ALR2, can play a key role in inhibitor–enzyme interactions.

Key words

unnatural amino acids - asymmetric synthesis - Michael addition reaction - docking analysis - inhibitor - aldose reductase

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References

References and Notes
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26 Chemistry: General Procedures All nucleophiles were obtained from commercial sources and used without further purification. The initial complexes 1 and 2 were prepared following literature protocols.¹⁸ TLC analyses were performed on glass plates coated with silica gel 60 F₂₅₄. Column chromatography was performed on silica gel (60 × 120 mesh) on a glass column. Melting points (mp) were determined by Electrothermal. ¹H and ¹³C NMR spectra (Mercury-300 Varian 300 MHz, respectively) were recorded using TMS as an internal standard (0 ppm). Elemental analyses were done by elemental analyzer EURO EA 3000. The enantiomeric purity of the amino acids was determined by HPLC (Waters Alliance 2695 HPLC System) on the chiral phase Diaspher-110-Chirasel-E-PA 6:0 mkm 4.0 × 250 mm, and a mixture of 20% MeOH and 80% 0:1 M aqueous solution NaH₂PO₄ x 2H₂O was used as the eluent. The optical rotation was measured on a PerkinElmer-341 polarimeter. Compound Characterization Complete details of the synthesis and physicochemical characterization of all compounds are included in the Supporting Information. Structures of all reported compounds were confirmed by ¹H and ¹³C NMR and their mass confirmed by elemental analysis. Enantiomeric purity of amino acids was determined by HPLC analysis using a Waters 2690 Alliance system with an inline photodiode array detector. All reported compounds had ≥95% chemical and >99% enantiomeric purities. General Procedure A: Asymmetric Nucleophilic Addition to the C=C Double Bond of the Initial Complex 2 To a solution of complex 2 (1 equiv) in MeCN, the nucleophile (2 equiv) was added at room temperature with stirring. The reaction was monitored by TLC (SiO₂, CHCl₃/ethyl acetate = 1:3) following the disappearance of the spot of the initial complex. Upon completion of the reaction, the mixture was precipitated by H₂O. The products were crystallized from methanol. General Procedure B: Isolation of Amino Acids Isolation of amino acids from the complexes was carried out according to literature.¹⁸ Complexes 3–6 were dissolved in 50 mL MeOH and then slowly added to 50 mL of 2 M HCl solution, and the mixture was heated at 50 °C. After disappearance of the red color of the metal complex, the solution was concentrated in vacuum, then 50 mL of water was added, and the precipitated (S)-BPB × HCl was filtered. The optically active amino acid was isolated from the water layer by ion-exchange sorption and desorption using Ku-2 × 8H⁺ for cation exchange and an aq. solution of NH₄OH (5%) as eluent (1 g/500 mL). The eluate was concentrated in vacuum, and the amino acid was recrystallized from a mixture of water and EtOH (1:2). All compounds were isolated in analytically pure form. Complexes 1 and 2 were obtained by the following literature.¹⁸ Complex 3 Following General Procedure A: To a solution of compound 2 (0.5 g, 0.98 mmol) in MeCN (15 mL), 4-methylbenzylamine (0.278 g, 1.116 mmol) was added. The resulting mixture was kept under stirring at 25 °C for 5 h. The purified product was obtained as a red powder (50% yield); mp 210 °C; [α]_D ²⁰ +2314.0 (c = 0.15, MeOH). Anal. Calcd for C₃₆H₃₆N₄NiO₃ (631.4): C, 68.48; H, 5.75; N, 8.87. Found: C, 68.35; H, 5.35; N, 8.36. ¹H NMR (300 MHz, CDCl₃): δ = 1.94–2.12 (3 H, m, N–H and Pro), 2.25 (s, 3 H, CH₃), 2.36–2.53 (1 H, m, Pro), 2.60–2.79 (1 H, m, Pro), 2.92 (2 H, d, J = 4.3 Hz, CH₂Ph), 3.46 (3 H, dt, J = 10.9, 6.1 Hz,), 3.59 (2 H, d, J = 12.6 Hz, CH₂NH), 3.88 (1 H, d, J = 13.3 Hz, CH₂C₆H₄), 3.98 (1 H, t, J = 4.8 Hz, CH₂C₆H₄), 4.43 (1 H, d, J = 12.7 Hz, CH), 6.42 (1 H, d, J = 7.7 Hz, Ar), 6.57 (1 H, dd, J = 8.2, 1.8 Hz, Ar), 6.61–6.70 (1 H, m, Ar), 6.99 (2 H, d, J = 7.9 Hz, Ar), 7.12–7.19 (2 H, m, Ar), 7.22 (4 H, t, J = 3.0 Hz, Ar), 7.36 (2 H, t, J = 7.6 Hz, Ar), 7.43–7.50 (2 H, m, Ar), 8.06 (2 H, d, J = 7.7 Hz, Ar), 8.23 (1 H, d, J = 7.7 Hz, Ar). ¹³C NMR (75 MHz, CDCl₃): δ = 21.08 (CH₃), 23.49 (Pro), 30.88 (Pro), 53.00 (CNH), 57.17 (CNH), 63.14 (CH), 70.40 (Pro), 96.24, 120.63 (Ar), 123.86, 127.21, 127.85, 128.30, 128.93, 129.13, 129.66, 131.72, 132.18, 133.23, 133.36, 136.63, 142.71, 170.59 (C=N), 178.81 (C=O), 180.51 (C=O). Complex 4 Following General Procedure A: To a solution of compound 2 (0.5 g, 0.98 mmol) in MeCN (1.5 mL), hexamethyleneimine (0.291 g, 2.45 mmol) was added. The resulting mixture was kept under stirring at 25 °C for 15 min. The purified product was obtained as a red powder (62% yield); mp 186–188 °C; [α]_D ²⁰ +2438.0 (c = 0.05, MeOH). Anal. Calcd for C₃₄H₃₈N₄NiO₃ (609.7): C, 67.01; H, 6.29; N, 9.19. Found: C, 67.15; H, 6.35; N, 9.26. ¹H NMR (400 MHz, CDCl₃): δ = 1.50–1.64 (8 H, m, C₆H₁₂N), 2.02–2.12(1 H, m, δ-H_a Pro), 2.12–2.22 (1 H, m, γ-H_a, Pro), 2.47–2.62 (1 H, m, β-H_a, Pro), 2.73–2.95 (5 H, m, β-H_b, Pro and α,α′-CH₂, C₆H₁₂N), 3.12 (1 H, dd, J = 14.2, 5.3 Hz, NCH ₂CH), 3.22 (1 H, dd, J = 14.2, 4.6 Hz, NCH ₂CH), 3.44 (1 H, dd, J = 10.8, 6.1 Hz, α-H Pro), 3.52–3.59 (1 H, m, δ-H_b, Pro), 3.59 (1 H, d, J = 12.7 Hz CH ₂Ph), 3.67–3.84 (1 H, m, γ-H_b, Pro), 3.97 (1 H, dd, J = 5.3, 4.6 Hz, CH), 4.45 (1 H, d, J = 12.7 Hz, CH ₂Ph), 6.60–6.68 (2 H, m, H-3,4, C₆H₄), 6.91–6.95 (1 H, m, H-2, C₆H₅), 7.12 (1 H, ddd, J = 8.6, 6.2, 2.5 Hz, H-5, C₆H₄), 7.18 (1 H, tt, J = 7.5, 1.2 Hz, para-Ph), 7.24–7.28 (1 H, m, Ar), 7.31–7.37 (2 H, m, Ar), 7.41–7.53 (3 H, m, Ar), 8.02–8.07 (2 H, m, ortho-Ph), 8.21 (1 H, dd, J = 8.6, 1.2 Hz, H-6 C₆H). ¹³C (75.5 MHz, CDCl₃): δ = 23.6 (γ-CH₂, Pro), 27.5 (2 CH₂, C₆H₁₂N), 27.5 (2 CH₂, C₆H₁₂N), 31.0 (β-CH₂, Pro), 56.1 (α,α′-CH₂, C₆H₁₂N), 57.1 (δ-CH₂, Pro), 59.2 (NCH₂), 63.1 (CH₂Ph), 70.5 (α-CH, Pro), 71.5 (CHCH₂N), 120.7 (4-CH, C₆H₄), 123.7 (6-CH, C₆H₄), 126.7, 127.6 (CH), 128.5 (CH), 128.7 (CH), 128.9 (CH), 128.9 (meta-CH, Ph), 129.0 (CH), 129.6 (CH), 131.7 (ortho-CHPh), 132.1 (CH), 133.3, 133.4 (CH), 134.4, 142.8, 170.2, 178.8 180.4. Complex 5 Following General Procedure A: To a solution of compound 2 (0.5 g, 0.98 mmol) in MeCN (2.5 mL), morpholine (0.17 g, 1.96 mmol) was added. The resulting mixture was kept under stirring at 25 °C for 1 h. The purified product was obtained as a red powder (68% yield); mp 204–205 °C, [α]_D ²⁰ +24.76 (c = 0.05, MeOH). Anal. Calcd for C₃₂H₃₄N₄NiO₄ (596.19): C, 64.34; H, 5.74; N, 9.38; Ni, 9.83. Found: C, 64.30; H, 5.69; N, 9.29. ¹H NMR (400 MHz, CDCl₃): δ = 2.05–2.24 (2 H, m, γ-H_a, δ-H_a), 2.32–2.49 (4 H, m, N(CH₂)₂ morph.), 2.52–2.63 (1 H, m, β-H_a pro), 2.73–2.85 (1 H, m, β-H_b pro), 2.93 (1 H, dd, J = 13.5, 5.6, CH ₂CH), 2.99 (1 H, dd, J = 13.5, 5.2 Hz, CH ₂CH), 3.47 (1 H, dd, J = 10.8, 6.0 Hz, α-H, Pro), 3.53–3.63 (1 H, m, γ-H_b, Pro), 3.60 (1 H, d, J = 12.6 Hz, CH ₂Ph), 3.64–3.78 (5 H, m, δ-H_b, Pro and O(CH₂)₂ morph.), 4.04 (1 H, dd, J = 5.6, 5.2 Hz, CHCH₂), 4.45 (1 H, d, J = 12.6 Hz, CH ₂Ph), 6.60–6.70 (2 H, m, H-3,4, C₆H₄), 6.97–7.02 (1 H, m, C₆H₅), 7.14 (1 H, ddd, J = 8.6, 6.5, 2.2 Hz, 5-H, C₆H₄), 7.18 (1 H, tt, J = 7.5, 1.3 Hz, para-Ph), 7.26–7.37 (3 H, m, Ar), 7.43–7.55 (3 H, m, Ar), 8.01–8.06 (2 H, m, ortho-Ph), 8.21 (1 H, dd, J = 8.6, 1.1 Hz, 6-H C₆H₄). ¹³C (75.5 MHz, CDCl₃): δ = 24.1 (γ-CH₂), 31.0 (β-CH₂), 54.8 (NCH₂ morph.), 57.2 (δ-CH₂), 63.2 (CH₂CH), 63.7 (CH₂Ph), 66.8 (OCH₂ morph.), 69.7 (CHCH₂), 70.4 (α-CH), 120.8, 123.6, 126.4, 127.5, 128.8, 128.9, 129.0 (meta-Ph), 129.0, 129.8, 131.7 (ortho-Ph), 132.4, 133.3, 133.5, 134.2, 142.8, 170.9, 178.3, 180.3. Complex 6 Following General Procedure A: To a solution of compound 2 (0.5 g, 0.98 mmol) in MeCN (2.5 mL), 6-[(3,5-dimethyl-1H-pyrazol-4-yl)oxy]-2-phenylpyridazin-3(2H)-one (0.55 g, 1.96 mmol) and potassium (0.677 g, 4.9 mmol) were added. The resulting mixture was kept under stirring at 50 °C for 6 h. The purified product was obtained as a red powder (56% yield); mp 220–222 °C; [α]_D ²⁰ +766.0 (c = 0.05, MeOH). Anal. Calcd for C₄₃H₃₉N₇NiO₅ (792.51): C, 65.17; H, 4.96; N, 12.37. Found: C, 67.15; H, 6.35; N 9.26. ¹H NMR (400 MHz, CDCl₃): δ = 1.81–2.01 (2 H, m, γ-H_a, δ-H_a Pro), 2.09 (3 H, s, CH₃), 2.12 (3 H, s, CH₃), 2.36–2.49 (1 H, m, β-H_a Pro), 2.56–2.68 (1 H, m, β-H_b Pro), 2.87–3.00 (1 H, m, γ-H_b, Pro), 3.22–3.29 (1 H, m, δ-H_b, Pro), 3.36 (1 H, dd, J = 10.3, 6.8 Hz, α-H, Pro), 3.58 (1 H, d, J = 12.6 Hz CH ₂Ph), 4.08 (1 H, dd, J = 14.4, 4.7 Hz, CH ₂CH), 4.19 (1 H, dd, J = 4.7, 4.0 Hz CH₂CH), 4.30 (1 H, d, J = 12.6 Hz, CH ₂Ph), 4.39 (1 H, dd, J = 14.4, 4.0 Hz, CH ₂CH), 6.60–6.70 (2 H, m, H-3,4 C₆H₄), 7.09 (1 H, d, J = 9.8 Hz, CH=CH), 7.09–7.29 (1 H, Ar), 7.20 (1 H, d, J = 9.8 Hz, CH=CH), 7.30–7.41 (5 H, m, Ar), 7.45–7.55 (4 H, m, Ar), 7.96–8.00 (2 H, m, ortho-Ph), 8.31 (1 H, dd, J = 8.7, 1.0 Hz, H-6 C₆H₄). ¹³C (75.5 MHz, CDCl₃): δ = 8.9 (CH₃), 11.4 (CH₃), 23.3 (γ-CH₂, Pro), 30.7 (β-CH₂, Pro), 51.4 (CH₂CH), 57.0 (δ-CH₂, Pro), 63.4 (CH₂Ph), 70.1 (CH), 70.5 (α-CH Pro), 120.6, 123.6, 125.1 (2 CH), 125.8, 126.2, 126.9, 127.8, 128.7, 128.8 (2 CH), 128.9 (2 CH), 129.0, 129.0, 129.8, 131.3, 131.7 (2 CH), 132.6, 133.2, 133.3, 133.7, 134.4, 134.9, 140.4, 141.2, 143.2, 152.1, 158.8, 172.5, 177.5, 180.1. (S)-2-Amino-3-[(4-methylbenzyl)amino]propanoic Acid (7) Following General Procedure B from 3: White powder; yield 25%; mp 237 °C (decomposed and became black); [α]_D ²⁰ +5.00 (c = 1.5; 6 M HCl). Anal. Calcd for C₁₁H₁₆N₂O₂ (208.3): C, 63.44; H, 7.74; N, 13.45. Found: C, 63.14; H, 7.39; N, 13.14. ¹H NMR (300 MHz, DMSO): δ = 2.34 (3 H, s, CH₃), 2.50 (1 H, dt, J = 3.6, 1.8 Hz, N H), 3.43 (1 H, dd, J = 13.6, 8.0 Hz, CH ₂), 3.60 (1 H, dd, J = 13.6, 3.8 Hz CH ₂), 4.17 (2 H, q, J = 12.8 Hz, CH₂), 4.42 (1 H, dd, J = 7.9, 3.8 Hz, C-H), 7.17 (2 H, d, J = 7.9 Hz, Ar), 7.42 (2 H, d, J = 8.0 Hz, Ar). ¹³C NMR (75.5 MHz, DMSO): δ = 7.43, 7.40, 7.19, 7.16, 4.44, 4.41, 4.40, 4.23, 4.18, 4.15, 4.11, 3.62, 3.61, 3.58, 3.57, 3.46, 3.44, 3.42, 2.51, 2.50, 2.34. (S)-2-Amino-3-(azepan-1-yl)propanoic Acid (8) Following General Procedure B from 4: White powder; yield 25 %; mp 224–226 °C (decomposed and became black); [α]_D ²⁰ +15.43 (c = 1.6; 6 M HCl). Anal. Calcd for C₉H₁₈N₂O₂ (246): C, 58.04; H, 9.74; N, 15.04. Found: C, 58.14; H, 9.79; N, 15.14. ¹H NMR (300 MHz, DMSO-d₆/CCl₄ + CF₃COOD): δ = 1.64–1.74 (4 H, m, 2 CH₂, C₆H₁₂N), 1.84–1.92 (4 H, m, 2 CH₂, C₆H₁₂N), 3.32–3.47 (4 H, m, α,α′-CH₂C₆H₁₂N), 3.59 (1 H, dd, J = 14.2, 6.6 Hz, CH ₂CH), 3.73 (1 H, dd, J = 14.2, 4.6 Hz, CH ₂CH), 4.50 (1 H dd, J = 6.6, 4.6 Hz, CH). ¹³C NMR (75.5 MHz, DMSO/CCl₄ = 1/3): δ = 22.983, 25.830, 38.950, 39.225, 39.508, 39.783, 40.058, 48.317, 54.844, 56.341, 95.717, 168.306. (S)-2-Amino-3-morpholinopropanoic Acid (9) Following General Procedure B from 5: White powder; yield 25%; mp 207.6 °C (decomposed and became black); [α]_D ²⁰ +0.016 (c = 0.327; 2 M HCl). Anal. Calcd for C₇H₁₄N₂O₃ (174.2): C, 48.26; H, 8.10; N, 16.08. Found: C, 48.20; H, 8.08; N, 16.04. ¹H NMR (300 MHz, D₂O): δ = 2.56–2.65 (2 H, m), 2.70–2.79 (2 H, m, N(CH₂)₂, morph.), 2.87 (1 H, dd, J = 13.9, 9.0 Hz), 2.93 (1 H, dd, J = 13.9, 4.6 Hz, CH₂), 3.77–3.89 (4 H, m, O(CH₂)₂, morph.), 3.94 (1 H, dd, J = 9.0, 4.6 Hz, CH). ¹³C NMR (75.5 MHz, D₂O): δ = 51.3 (CH₂), 52.1 (N(CH₂)₂), 56.9 (CH), 65.8 (O(CH₂)₂), 172.7 (CO). (S)-2-Amino-3-{3,5-dimethyl-4-[(6-oxo-1-phenyl-1,6-dihydropyridazin-3-yl)oxy]-1H-pyrazol-1-yl}propanoic Acid (10) Following General Procedure B from 6: White powder; yield 24%; mp 198–199 °C (decomposed and became black); [α]_D ²⁰ +11.44 (c = 1.8; 6 M HCl). Anal. Calcd for C₁₈H₁₉N₅O₄ (369.37): C, 58.53; H, 5.18; N, 18.96. Found: C, 58.04; H, 9.74; N, 15.04. ¹H NMR (300 MHz, D₂O): δ = 2.02 (3 H, s, CH₃), 2.10 (3 H, s, CH₃), 4.02 (1 H, dd, J = 6.8, 4.5 Hz CH), 4.33 (1 H, dd J = 15.3, 6.8 Hz, CH₂), 4.37 (1 H, dd, J = 15.3, 4.5 Hz, CH₂), 7.17 (1 H d, J = 9.8 Hz, CH=CH), 7.29–7.34 (2 H, m, ortho-Ph), 7.38–7.47 (3 H, m, meta,para-Ph), 7.46 (1 H d, J = 9.8 Hz, CH=CH). ¹³C NMR (75.5 MHz, D₂O): δ = 7.2 (CH₃), 9.4 (CH₃), 47.7 (CH₂), 54.1 (CH), 125.6 (2 CH), 127.6 (CH), 129.0, 129.1 (CH), 129.1 (2 CH), 132.4, 133.4 (CH), 139.7, 141.0, 153.4, 160.6, 170.5. N-tert-Butoxycarbonyl-(S)-Ala Succinimide Ester (13) 0.218 g (1.058 mmol) of dicyclohexylcarbodiimide, preliminary dissolved in 3 mL of 1,4-dioxane, were added at 0 °C to 0.189 g (1.0 mmol) of N-tert-butyloxycarbonylglycine (11) and 0.127 g (1.104 mmol) of N-hydroxysuccinimide (13) in a mixture of 6 mL of 1,4-dioxane and 3 mL of methylene chloride. The reaction mixture was stirred for ca. 2 h at 0 °C and left overnight in a refrigerator. The analysis was performed by TLC [SiO₂, CHCl₃/ethyl acetate/MeOH (4:2:1); developer: chlorotoluidine]. The precipitate formed was filtered off, the solvent distilled off on a rotary evaporator, and the precipitate crystallized from a mixture of ethyl acetate hexane (1:2). Yield: 0.25 g (75%). For the analytical data please see the following.²⁰ N-tert-Butoxycarbonyl-(S)-Ala-(S)-β-morpholino-α-Ala·HCl (14) The resulted succinimide ether 13 was used at the next stage of dipeptide synthesis. In a flat-bottomed flask with a magnetic stirrer, 0.078 g (0.448 mmol) of (S)-β-morpholino-α-Ala (9), 1.25 mL (0.63 mmol) of 0.5 M sodium hydroxide solution, and 0.016 (0.19 mmol) of baking soda were placed. At room temperature, 0.2 g (0.699 mmol) of N-Boc-(S)-Ala-OSu (13) was added to 2 mL of 1,4-dioxane, and the reaction mixture was stirred for 3 h. The next day, 5 mL of ethyl acetate and 1.45 mL of 10% citric acid were added to the flask contents. After vigorous stirring, the organic layer was separated, and the aqueous layer was extracted twice with ethyl acetate (5 mL each). The organic layer was dried with anhydrous sodium sulfate, then the solvent was evaporated to dryness. The product was isolated by column chromatography using SiO₂ L-40/100 silica gel. The analysis was performed by TLC [SiO₂, CHCl₃/ethyl acetate/MeOH (4: 2: 2); developer: chlorotoluidine]. The product yield per succinimide ester was 75%, a viscous transparent substance with a yellowish tint. Anal. Calcd for C₁₅H₂₇N₃O₆ (345.40): C, 52.16; H, 7.88; N, 12.17. Found: C, 52.36; H, 7.92; N, 12. ¹H NMR (300 MHz, DMSO): δ = 1.23 (d, 3 H, J = 7.0, CH₃), 1.41 (s, 9 H, t-C₄H₉), 2.46–2.54 (m, 4 H, C₄H₈NO), 2.62 and 2.73 (d, 1 H & 1 H, J = 15.9 Hz, NCH₂), 3.52–3.61 (m, 4 H, C₄H₈NO), 3.95–4.11 (m, 1 H, NCH₂), 4.39 (q, 1 H J = 7.4, CHCH₃), 7.49 (br, 1 H, COOH). ¹³C NMR (75.5 MHz, DMSO): δ = 25.0 (CH₃), 28.1 (3×CH₃), 42.74 (NCH₂), 49.5 (NCH), 53.0 (NCH₂), 58.4 (NCH), 65.9 (OCH₂), 72.3 (C*), 171.0 (C=O), 171.0 (C=O), 174.6 (C=O). Isolation and Purification of Enzymes ALR2 and ALR1 were obtained from bovine lenses and kidney, respectively. ALR1 and ALR2 were purified from bovine kidney using extraction method previously described.¹⁶ Enzyme Assays Measurement of enzymes activity was performed at 37 °C in a reaction mixture containing 0.2 mM NADPH, 0.1 M sodium phosphate buffer, pH 6.2, 10 mM of substrate (d,l-glyceraldehyde for ALR2 or sodium d-glucuronate for ALR1) and needed quantity of enzyme preparation in a final volume of 1.5 mL. The reaction mixture, except for substrates, was incubated at 37 °C for 10 min. The substrate was then added to start the reaction, which was monitored for 15 min. The inhibitory activity of the amino acid compounds against ALR2 and ALR1 was assayed in the presence of compounds of 1 mM final concentration. To ensure the solubility, all amino acid compounds were dissolved in 30% dimethyl sulfoxide (DMSO). The compounds found to be active were tested at additional final concentrations between 0.1–2.0 mM. To correct the nonenzymatic oxidation of NADPH, the rate of NADPH oxidation in the presence of all the reaction mixture components, except the substrate, was subtracted from each experimental rate. Enzyme activity was assayed spectrophotometrically on a Shimadzu UV-1800 spectrofotometer by measuring the decrease in absorption of NADPH at λ 340 nm. IC50 values of compounds were determined by fitting the inhibition data using GraphPad Prism 8.1.2 software. Molecular Docking Studied compounds and known inhibitors structures were built by ChemBioOffice 2010 (ChemBio3D Ultra12.0). Ligand free energy was minimized using MM2 force field and truncated Newton–Raphson method. Crystallographic structures of enzymes were taken from the Protein Data Bank of Research Collaboratory for Structural Bioinformatics (http://www.rcsb.org). Due to the lack of a bovine enzymes structure in the data bank, structures of human enzymes were chosen for investigation. Such a manipulation is also justified by a high (more than 80%) homology between the animal and human aldose reductases.^21–23 The APO R268A human aldose reductase conformation of ALR2 (PDB-ID: 1XGD) was chosen and used for the docking study. The docking behavior of human ALR1 (PDB-ID: 2ALR) with studied compounds was also investigated. Docking of ligand to enzyme has been done by AutoGrid 4, AutoDock Vina software.²⁴ AutoDock uses the Lamarckian genetic algorithm by alternating local search with selection and crossover.²⁵ The ligands were ranked using an energy-based scoring function and a grid-based protein–ligand interaction was used to speed up the score calculation.

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