Synthesis of a novel series of L-isoserine derivatives as aminopeptidase N inhibitors

Abstract

A series of novel L-isoserine derivatives were synthesised and evaluated for their ability to inhibit aminopeptidase N (APN)/CD13. In our preliminary biological results, some of these compounds possessed a potent inhibitory activity against the APN. Within this series, compound 14b not only showed similar enzyme inhibition (IC₅₀ of 12.2 μM) compared with the positive control bestatin (half maximal inhibitory concentration (IC₅₀) of 7.3 μM), but also had a potent antiproliferative activity against human cancer cell lines cells.

Keywords::

• Aminopeptidase N inhibitor
• L-isoserine
• synthesis

Introduction

Aminopeptidase N (APN EC 3.4.11.2), also known as CD13, is a zinc-dependent membrane metalloprotease which belongs to the M1 family of ectoenzymes [1]. It usually forms a noncovalently bound homodimer on the cell membrane of monocytes, myeloid, epithelial cells of the intestine and kidney, fibroblasts and tumour cells [2–3]. This enzyme is able to cleave the N-terminal residues from polypeptide chains, as well as play an important role in modulating bioactive peptides or protein responses such as modification, activation, and degradation. APN is also involved in various physiological processes, such as cell proliferation, secretion, invasion and angiogenesis [4–5]. For example, APN is over-expressed on the tumour cell surface for regulating ECM degradation and the invasion of tumour cells [6]. Therefore, inhibition of APN/CD13 could prevent the spread and metastasis of cancer cells since several APN inhibitors have been well studied clinically over the last two decades. Among the APN inhibitors, bestatin is a representative molecule for the treatment of adult acute nonlymphocytic leukemia [7].

So far, X-ray crystal structures for APN for both the holo- and apo-forms [8] have been resolved along with a number of different ligands, such as bestatin [9–10], arginine and lysine [11]. According to the literature for binding modes, our group has developed various APN inhibitors, including L-lysine derivates [12] and AHP A(α-Amino-β-Hydroxyl-phenylbutanoic acid) derivates, [13] which both showed potent APN inhibitory activities. In our recent screening studies, L-isoserine showed the ability to inhibit APN at an half maximal inhibitory concentration (IC₅₀ ) value of 563 μM, which could serve as a new lead compound for further chemical modification and optimisation.

It has been reported that L-isoserine-L-leucine dipeptide shows moderate aminopeptidase B inhibitory activity (IC₅₀ of 140 μM) [14]. This evidence indicates that the incorporation of an amino acid to L-isoserine may contribute to its inhibitory activity against APN. Therefore in our current work, different amino acids have been coupled with L-isoserine by forming dipeptides or tripeptides. This article will describe the synthesis, preliminary biological evaluation and structure-activity relationship (SAR) study of such a novel series of L-isoserine derivatives as potent APN inhibitors.

Materials and methods

Chemistry

The starting material L-Isoserine (7), is a white solid, (mp 199-201°C, [α]²⁵_D = -32.5° (c 1, H₂O)), was purchased from JiaDing District, Shanghai, China. Unless otherwise specified, all reagents and buffer salts were purchased from commercial vendors and used without further purification. The solvents were distilled prior to use and flash chromatography was performed using silica gel (60 Å, 200 ± 300 mesh). Melting points were determined on an electrothermal melting point apparatus in an uncorrected form. Proton nuclear magnetic resonance (¹H NMR) spectra were determined on a Brucker Avance 600 spectrometer using TMS as an internal standard in DMSO-d6 solutions. Chemical shifts were reported in delta (δ) units, parts per million (ppm) downfield from trimethylsilane. High-resolution mass spectral (HRMS) data are reported as m/e (relative intensity). The optical rotation was determined on a GYROMAT-HP Digital Automatic Polarimeter. All reported yields correspond to purified products.

General procedure for the synthesis of 2a, 2b, 4a and 4b

The title compound L-leucine methyl ester hydrochloride (2a) and L-phenylalanine methyl ester hydrochloride (2b) were prepared from L-leucine and L-phenylalanine according to the methods described in the literature [15]. Using the same materials, the title compound L-leucine benzyl ester hydrochloride (4a) and L-phenylalanine benzyl ester hydrochloride (4b) were prepared as described by Kim et al. [16].

General procedure for the synthesis of 6a, 6b, 6c and 6d

To a 200 mL solution of compound 3a (0.92 g, 4 mmol), 2b (0.88 g, 4.4 mmol), and hydroxybenzotriazole (HOBt) (0.65 g, 4.8 mmol), 4-Dimethylaminopyridine (DMAP) (0.09 g, 0.8 mmol) in dry DCM, was added TEA (1.33 g, 13.2 mmol). The reaction mixture was gently cooled to 0°C in an ice bath. To the reaction mixture was added dropwise a solution of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (1.53 g, 8 mmol) in DCM for 1 h. After removal of the ice bath, the reaction mixture was stirred at room temperature for 12h and then filtered to remove the precipitate. The filtrate was washed with a 1 mol/L citric acid solution, saturated NaHCO₃ and brine, dried over Na₂SO₄, and evaporated in vacuo. The residue was purified by flash column chromatography (EtOAc/PE, 1:3(V/V)) to give the desired compound 5a as a white solid (1.48 g), yield: 94.2%, mp 70–73°C. ¹H-NMR: (DMSO-d₆) δ 7.919 (d, J = 7.8 Hz, 1H, -CONH-), 7.287–7.214 (m, 5H, Ar-H), 5.807 (d, J = 5.4 Hz, 1H, -CONH-), 4.474–4.459 (m, 1H, CH), 4.346–4.311 (m, 1H, CH), 3.568 (s, 3H, -OCH₃), 3.102–3.089 (m, 1H, CH₂), 2.787–2.75 (m, 1H, CH₂), 1.526–1.501 (m, 1H, CH), 1.435 (s, 9H, CH₃), 1.475–1.328 (m, 2H, CH₂), 0.863 (d, J = 6.6 Hz, 3H, CH₃), 0.828 (d, J = 6.6 Hz, 3H, CH₃); ESI-MS m/z: 393.2 [M+H]⁺.

To a solution of compound 5a (0.78 g, 2 mmol) in dry EtOAc at 0°C was added dropwise a solution of EtOAc (10 mL) saturated by dry HCl gas. The reaction solution was stirred at 0°C for 2 h, and then the temperature was raised to room temperature and the reaction proceeded for 5 h before being concentrated in vacuo. The residue was recrystallised with MeOH and ether to give 6a (0.56 g) as a white crystals, yield: 85.3%, mp 190–193°C, [α]²⁵_D = +14.2° (c 1, MeOH). ¹H-NMR: (DMSO-d₆) δ 8.403 (d, J = 7.8 Hz, 1H, -CONH-), 8.054 (s, 3H, NH₃), 7.285–7.202 (m, 5H, Ar-H), 4.482–4.472 (m, 1H, CH), 4.351–4.318 (m, 1H, CH), 3.573 (s, 3H, -OCH₃), 3.11–3.092 (m, 1H, CH₂), 2.791–2.751 (m, 1H, CH₂), 1.527–1.495 (m, 1H, CH), 1.486–1.351 (m, 2H, CH₂), 0.861 (d, J = 6.6 Hz, 3H, CH₃), 0.831 (d, J = 6.6 Hz, 3H, CH₃); ESI-MS m/z: 293.1 [M+H]⁺.

Compounds 6b, 6c and 6d were prepared following the general procedure as described above.

L-phenylalanyl-L-leucine methyl ester hydrochloride (6b)

White crystals, yield: 79.4%, mp 141–143°C; [α]²⁵_D = +17.5° (c 1, MeOH); ¹H-NMR: (DMSO-d₆) δ 8.578 (d, J = 7.8 Hz, 1H, -CONH-), 8.108 (s, 3H, NH₃), 7.28–7.182 (m, 5H, Ar-H), 4.622–4.587 (m, 1H, CH), 4.325–4.198 (m, 1H, CH), 3.628 (s, 3H, -OCH₃), 2.793–2.605 (m, 1H, CH₂), 2.787–2.52 (m, 1H, CH₂), 1.652–1.604 (m, 1H, CH), 1.541–1.479 (m, 2H, CH₂), 0.893 (d, J = 6.6 Hz, 3H, CH₃), 0.842 (d, J = 6.6 Hz, 3H, CH₃); ESI-MS m/z: 293.2 [M+H]⁺.

L-leucyl-L-phenylalanine benzyl ester hydrochloride (6c)

A white solid, yield: 83.9%, mp 160–162°C; [α]²⁵_D = +6.3° (c 1, H₂O); ¹H-NMR: (DMSO-d₆) δ 9.203 (d, J = 7.8 Hz, 1H, -CONH-), 8.345 (s, 3H, NH₃), 7.363–7.226 (m, 10H, Ar-H), 5.09–5.04 (m, 2H, Ar-CH₂-O-), 4.612–4.576 (m, 1H, CH), 3.805–3.781 (m, 1H, CH), 3.102–3.032 (m, 2H, Ar-CH₂-),1.671–1.627 (m, 1H, CH), 1.522–1.499 (m, 2H, CH₂), 0.85 (d, J = 6.6 Hz, 3H, CH₃), 0.829 (d, J = 6.6 Hz, 3H, CH₃); ESI-MS m/z: 369.3 [M+H]⁺.

L-phenylalanyl-L-leucine benzyl ester hydrochloride (6d)

A white solid, yield: 81.8%, mp 161–163°C; [α]²⁵_D = +5.6° (c 1, H₂O); ¹H-NMR: (DMSO-d₆) δ 9.187 (d, J = 7.8, 1H, -CONH-), 8.32 (s, 3H, NH₃), 7.389–7.24 (m, 10H, Ar-H), 5.168–5.105 (m, 2H, Ar-CH₂-O-), 4.398–4.36 (m, 1H, CH), 4.133–4.111 (m, 1H, CH), 3.162–3.13 (m, 1H, Ar-CH₂-), 2.953–2.917 (m, 1H, Ar-CH₂-), 1.72–1.674 (m, 1H, CH), 1.678–1.526 (m, 2H, CH₂), 0.896 (d, J = 6.6 Hz, 3H, CH₃), 0.848 (d, J = 6.6 Hz, 3H, CH₃); ESI-MS m/z: 369.2 [M+H]⁺.

Synthesis of (S)-3-(tert-butoxycarbonylamino)-2-hydroxypropanoic acid (8)

Compound 8 was synthesised from L-Isoserine following the general procedure as described above (preparation of 3a). a colourless solid, yield: 93.5%, mp 85–88°C; [α]²⁵_D = +6.7° (c 1, MeOH); ¹H-NMR: (DMSO-d₆) δ 7.934 (d, J = 7.8 Hz, 1H, -CONH-), 6.54 (s, 1H, OH), 4.674–4.651 (m, 1H, CH), 3.457 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 3.241 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 1.435 (s, 9H, CH₃); ESI-MS m/z: 206.1 [M+H]⁺.

General procedure for the synthesis of 11a–11d and 13a–13d

Compound 9a was synthesised from N protected L-Isoserine (8) and L-phenylalanine methyl ester hydrochloride (2b) following the general procedure as described above (preparation of 5a), yield: 84.5%, mp 95–97°C; [α]²⁵_D = -9.72° (c 1, MeOH); ¹H-NMR (DMSO-d₆) δ 7.921 (d, J = 7.8 Hz, 1H, -CONH-), 7.287–7.362 (m, 2H, Ar-H), 7.177–7.166 (m, 3H, NH₃), 6.526 (s, 1H, OH), 5.801 (d, J = 5.4 Hz, 1H, -CONH-), 4.604–4.567 (m, 1H, CH), 3.889–3.869 (m, 1H, CH), 3.627 (s, 3H, -OCH₃), 3.173–3.135 (m, 1H, Ar-CH₂- ), 3.075 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 3.032 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.817–2.77 (m, 1H, Ar-CH₂-),1.435 (s, 9m, CH₃); HRMS (FAB) calcd for C₁₈H₂₆N₂O₆ 389.1683; found: 389.167 [M+Na]⁺.

Compound 11a was obtained through the deprotection of compound 9a in the saturated HCl/EtOAc solution as the preparation of 6a, yield: 77.7%, mp 138–140°C; [α]²⁵_D = -19.8° (c 1, H₂O). ¹H-NMR (DMSO-d₆) δ 8.25 (d, J = 7.8 Hz, 1H, -CONH-), 8.094 (s, 3H, NH₃), 7.294–7.27 (m, 2H, Ar-H), 7.23–7.197 (m, 3H, Ar-H), 6.541 (s, 1H, OH), 4.6–4.563 (m, 1H, CH), 4.203–4.173 (m, 1H, CH), 3.107 (s, 3H, -OCH₃), 3.101 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 3.05 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.927 (dd, J = 13.2 Hz, J = 9 Hz, 1H, Ar-CH₂-),2.569 (dd, J = 13.2 Hz, J = 9 Hz, 1H, Ar-CH₂-); HRMS (FAB) calcd for C₁₃H₁₈N₂O₄ 267.1339; found: 267.1335 [M+H]⁺.

Compounds 9c–9d were prepared from N protected L-Isoserine and amino acid methyl/benzyl ester hydrochlorides (2a,4a and 4b) and compounds 10a–10d were prepared from N protected L-Isoserine and dipeptide methyl/benzyl ester hydrochlorides(6a, 6b, 6c and 6d). Compounds 11c–11d and 13a–13d were obtained through the deprotection of compounds 9c–9d and 10a–10d.

L-Isoseryl-L-leucine methyl ester hydrochloride (11b)

A white solid, yield: 63.1%, mp 120–123°C; [α]²⁵_D = -36.1° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 8.054 (d, J = 7.8 Hz, 1H, -CONH-), 7.903 (s, 3H, NH₃), 6.427 (s, 1H, OH), 4.607–4.593 (m, 1H, CH), 4.186–4.152 (m, 1H, CH), 3.218 (s, 3H, -OCH₃), 3.106 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 3.023 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 1.523–1.704 (m, 3H, CH₂CH), 0.926–0.947 (m, 6H, CH₃); HRMS (FAB) calcd for C₁₀H₂₀N₂O₄ 233.1496; found: 233.1493 [M+H]⁺.

L-Isoseryl-L- phenylalanine benzyl ester hydrochloride (11c)

A white solid, yield: 76.6%, mp 177–179°C; [α]²⁵_D = -23.3° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 8.285 (d, J = 7.8 Hz, 1H, -CONH-), 8.032 (s, 3H, NH₃), 7.391–7.303 (m, 5H, Ar-H), 7.274–7.177 (m, 5H, Ar-H), 6.511 (s, 1H, OH), 5.14–5.097 (m, 2H, Ar-CH₂-O-), 4.644–4.607 (m, 1H, CH), 4.195–4.164 (m, 1H, CH), 3.126 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 3.028 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.95–2.93 (m, 1H, Ar-CH₂-), 2.601–2.566 (m, 1H, Ar-CH₂-); HRMS (FAB) calcd for C₁₉H₂₂N₂O₄ 343.1652; found: 343.1650 (M+H)⁺.

L-Isoseryl-L-leucine benzyl ester hydrochloride (11d)

A white solid, yield: 84.6%, mp 98–103°C; [α]²⁵_D = -25.7° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 8.387 (d, J = 7.8 Hz, 1H, -CONH-), 8.189 (s, 3H, NH₃), 7.405–7.324 (m, 5H, Ar-H), 6.48 (s, 1H, OH), 5.187–5.13 (m, 2H, Ar-CH₂-O-), 4.372–4.335 (m, 1H, CH), 4.287–4.256 (m, 1H, CH), 3.088 (dd, J = 12.6 Hz, J = 8.4 Hz, 1H, CH), 2.778 (dd, J = 12.6 Hz, J = 8.4 Hz, 1H, CH), 1.7–1.594 (m, 2H, CH₂), 1.567–1.522 (m, 1H, CH), 0.882 (d, J = 6.6 Hz, 3H, CH₃), 0.839 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₁₆H₂₄N₂O₄ 309.1809; found: 309.1807 (M+H)⁺.

L-Isoseryl-L-leucyl-L-phenylalanine methyl ester hydrochloride (13a)

A white solid, yield: 64.3%, mp 65–68°C; [α]²⁵_D = -33.2° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 8.588 (d, J = 7.8 Hz, 1H, -CONH-), 8.12 (s, 3H, NH₃), 7.843 (d, J = 7.8 Hz, 1H, -CONH-), 7.298–7.2 (m, 5H, Ar-H), 6.485 (s, 1H, OH), 4.486–4.499 (m, 1H, CH), 4.368–4.329 (m, 1H, CH), 4.12–4.199 (m, 1H, CH), 3.579 (s, 3H, -OCH₃), 3.102–3.093 (m, 1H, Ar-CH₂-), 3.055 (dd, J = 13.80 Hz, J = 8.4 Hz, 1H, CH₂), 2.977 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.798–2.752 (m, 1H, Ar-CH₂-), 1.528–1.494 (m, 1H, CH), 1.483–1.359 (m, 2H, CH₂), 0.861 (d, J = 6.6 Hz, 3H, CH₃), 0.831 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₁₉H₂₉N₃O₅ 380.2180; found: 380.2174 (M+H)⁺.

L-Isoseryl-L-phenylalanyl-L-leucine methyl ester hydrochloride (13b)

A white solid, yield: 50.9%, mp 77–80°C; [α]²⁵_D = -26.7° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 8.631(d, J = 7.8 Hz, 1H, -CONH-), 8.1 (s, 3H, NH₃), 7.862 (d, J = 7.8 Hz, 1H, -CONH-), 7.298–7.195 (m, 5H, Ar-H), 6.512 (s, 1H, OH), 4.633–4.596 (m, 1H, CH), 4.33–4.269 (m, 1H, CH), 4.166–4.145 (m, 1H, CH), 3.639 (s, 3H, -OCH₃), 3.077 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.918 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.895–2.55 (m, 1H, Ar-CH₂-), 2.568–2.521 (m, 1H, Ar-CH₂-), 1.656–1.597 (m, 2H, CH₂), 1.544–1.497 (m, 1H, CH), 0.894 (d, J = 6.6 Hz, 3H, CH₃), 0.847 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₁₉H₂₉N₃O₅ 380.2180; found: 380.2179 (M+H)⁺.

L-Isoseryl-L-leucyl-L-phenylalanine benzyl ester hydrochloride (13c)

A white solid, yield: 52.2%, mp 135–137°C; [α]²⁵_D = -30.5° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 8.631(d, J = 7.8 Hz, 1H, -CONH-), 8.134 (s, 3H, NH₃), 7.827 (d, J = 7.8 Hz, 1H, -CONH-), 7.368–7.311 (m, 3H, Ar-H), 7.27–7.202 (m, 7H, Ar-H), 6.493 (s, 1H, -OH), 5.088–5.033 (m, 2H, Ar-CH₂-O-), 4.547–4.51 (m, 1H, CH), 4.381–4.341 (m, 1H, CH), 4.216–4.201 (m, 1H, CH), 3.084–3.074 (m, 1H, Ar-CH₂-), 3.067 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.993 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.782–2.63 (m, 1H, Ar-CH₂-),1.512-1.35 (m, 3H, CH₂CH), 0.844 (d, J = 6.6 Hz, 3H, CH₃), 0.797 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₂₅H₃₃N₃O₅ 456.2493; found: 456.2490 (M+H)⁺.

L-Isoseryl-L-phenylalanyl-L-leucine benzyl ester hydrochloride (13d)

A white solid, yield: 58.1%, mp 140–142°C; [α]²⁵_D = -28.6° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 8.644 (d, J = 7.8 Hz, 1H, -CONH-), 8.037 (s, 3H, NH₃), 7.852 (d, J = 7.8 Hz, 1H, -CONH-), 7.377–7.319 (m, 3H, Ar-H), 7.242–7.171 (m, 5H, Ar-H), 6.494 (s, 1H, OH), 5.166–5.121 (m, 2H, Ar-CH₂-O-), 4.64–4.603 (m, 1H, CH), 4.385–4.348 (m, 1H, CH), 4.142–4.126 (m, 1H, CH), 3.028 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.912–2.892 (m, 1H, Ar-CH₂-), 2.851 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.54–2.504 (m, 1H, Ar-CH₂-),1.657–1.529 (m, 3H, CH₂CH), 0.906 (d, J = 6.6 Hz, 3H, CH₃), 0.846 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₂₅H₃₃N₃O₅ 456.2493; found: 456.2496 (M+H)⁺.

General procedure for the synthesis of 12a–12b and 14a–14b

Compound 9c (1.77 g, 4 mmol) was hydrogenated in absolute MeOH (30 mL) with a catalytic quantity of 10% Pd/C (0.17 g). The mixture was stirred for 10 h under normal pressure then filtered. The filtrate was evaporated to give a white solid. The solid was dissolved in dry EtOAc (5 mL) at 0°C and added dropwise to a solution of EtOAc (10 mL) saturated by dry HCl gas. The reaction solution was stirred at 0°C for 12 h to provide a white solid. The solution was removed by filtration, and the remaining solid was recrystallised with MeOH and Et₂O to give 12a white crystals (0.91 g), yield: 78.9%, mp 187–189°C; [α]²⁵_D = -13.4° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 12.964 (s, 1H, COOH), 8.081 (s, 3H, NH₃), 8.07 (d, J = 8.4 Hz, 1H, -CONH-), 7.275 (t, J = 7.8 Hz, 2H, Ar-H), 7.223–7201 (m, 3H, Ar-H), 6.527 (s, 1H, OH), 4.543–4.507 (m, 1H, CH), 4.183–4.169 (m, 1H, CH), 3.113 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 3.031 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.957–2.938 (m, 1H, Ar-CH₂-), 2.62–2.561 (m, 1H, Ar-CH₂-); HRMS (FAB) calcd for C₁₂H₁₆N₂O₄ 253.1183; found: 253.1180 (M+H)⁺.

L-Isoseryl-L-leucine hydrochloride (12b)

A white solid, yield: 88.3%, mp 213–215°C; [α]²⁵_D = -44.5° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 12.713 (s, 1H, COOH), 8.102 (s, 3H, NH₃), 8.088 (d, J = 8.4 Hz, 1H, -CONH-), 6.441 (s, 1H, OH), 4.236–4.229 (m, 1H, CH), 4.274–4.251 (m, 1H, CH), 3.086 (dd, J = 12.6 Hz, J = 7.2 Hz, 1H, CH₂), 2.784 (dd, J = 12.6 Hz, J = 7.2 Hz, 1H, CH₂), 1.681–1.585 (m, 2H, CH₂), 1.556–1.513 (m, 1H, CH), 0.893 (d, J = 6.6 Hz, 3H, CH₃), 0.854 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₉H₁₈N₂O₄ 219.1339; found: 219.1340 (M+H)⁺.

L-Isoseryl-L-leucyl-L-phenylalanine hydrochloride (14a)

A white solid, yield: 56.9%, mp 90–92°C; [α]²⁵_D = -34.7° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 12.69 (s, 1H, COOH), 8.468 (d, J = 7.8 Hz, 1H, -CONH-), 8.119 (s, 3H, NH₃), 7.836 (d, J = 7.8 Hz, 1H, -CONH-), 7.245–7.168 (m, 5H, Ar-H), 6.52 (s, 1H, OH), 4.652–4.573 (m, 1H, CH), 4.263–4.225 (m, 1H, CH), 4.176–4.084 (m, 1H, CH), 3.986 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.887 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.812–2.798 (m, 1H, Ar-CH₂), 2.51–2.439 (m, 1H, Ar-CH₂), 1.502–1.414 (m, 3H, CH₂CH), 0.862 (d, J = 6.6 Hz, 3H, CH₃), 0.813 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₁₈H₂₇N₃O₅ 366.2023; found: 366.2026 (M+H)⁺.

L-Isoseryl-L-phenylalanyl-L-leucine hydrochloride (14b)

A white solid, yield: 53.6%, mp 87–89°C; [α]²⁵_D = -35.1° (c 1, H₂O); ¹H-NMR (DMSO-d₆) δ 12.647 (s, 1H, COOH), 8.475 (d, J = 7.8 Hz, 1H, -CONH-), 8.109 (s, 3H, NH₃), 7.847 (d, J = 7.8 Hz, 1H, -CONH-), 7.265–7.176 (m, 5H, Ar-H), 6.524 (s, 1H, OH), 4.637–4.6 (m, 1H, CH), 4.274–4.236 (m, 1H, CH), 4.159–4.095 (m, 1H, CH), 3.09 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.905 (dd, J = 13.8 Hz, J = 8.4 Hz, 1H, CH₂), 2.896–2.882 (m, 1H, Ar-CH₂), 2.539–2.505 (m, 1H, Ar-CH₂), 1.72–1.526 (m, 3H, CH₂CH), 0.907 (d, J = 6.6 Hz, 3H, CH₃), 0.853 (d, J = 6.6 Hz, 3H, CH₃); HRMS (FAB) calcd for C₁₈H₂₇N₃O₅ 366.2023; found: 366.2027 (M+H)⁺.

In vitro APN inhibition assay

The IC₅₀ values against APN were determined using L-Leu-p-nitroanilide as the substrate and microsomal aminopeptidase from porcine kidney microsomes (Sigma) in 50 mM PBS, pH 7.2, at 37°C. The hydrolysis of the substrate was monitored by following the change in the absorbance measured at 405 nm with a UV-Vis spectrophotometer Pharmacia LKB, Biochrom 4060. All solutions of inhibitors were prepared in the assay buffer, and the pH was neutralised to 7.5 by the addition of either 0.1 M HCl or 0.1 M NaOH.

The samples and positive controls were serial diluted to various concentrations: 1280 μg/mL, 320 μg/mL, 80 μg/mL, and 20 μg/mL, 5 μg/mL and 1.25 μg/mL. All compounds were pre-incubated with APN on a 96-well plate for 30 min. The assay mixture, including the compound solution, the enzyme solution (5 μg/mL final concentration) and the assay buffer, was adjusted to give a total volume of 200 μL. Fifteen minutes later, the absorbance (OD405) of the wells was recorded on a microplate reader. The percentage inhibition was calculated according to the absorbance of the assay wells relative to those of the control wells. Finally, the IC₅₀ values were determined using a linear regression analysis of the concentration/inhibition data.

In vitro MMP-2 inhibition assay

Gelatinase A (MMP-2) and picrylsulphonic acid (TNBS) were purchased from Sigma. Succinylated gelatin was synthesised as described by Baragi et al. [17]. The compound samples were assayed for inhibitory activity against MMP-2 in 96-well microplates using succinylated gelatin as the substrate. The compounds and gelatinase were dissolved in sodium borate buffer (pH 8.5, 50 mM), and incubated at 37°C for 30 min. The substrate was added and incubated at 37°C for another 60 min. Then 0.03% picrylsulphonic acid solution was added and incubated at room temperature for an additional 20 min. The resulting solutions were detected under 450 nm wavelength to gain absorbance (OD₄₅₀). MMP-2 inhibition activity was measured by the following formula [17]: Inhibition ration (%) = [OD₄₅₀(100%)- OD₄₅₀(compound)]/[OD₄₅₀(100%)- OD₄₅₀(blank)]×100%. The IC₅₀ values were obtained from the above inhibitory rates using the OriginPro 7.5 software.

In vitro HL-60 and SKOV3 cells viability assay

The HL-60 and SKOV3 Cells were grown in RPMI1640 medium with 10% fetal bovine serum at 37°C in a 5% CO₂ humidified incubator. Cell proliferation was determined by the MTT (3-[4, 5- dimethyl-2-thiazolyl]-2.5-diphenyl-2H-tetrazolium bromide) assay. Briefly, 10,000 HL-60 cells (or 5,000 SKOV3 cells) per well were plated in a 96-well plate, cultured for 4 h in complete growth medium, then treated with 400 μg/mL, 200 μg/mL, 100 μg/mL, 50 μg/mL and 25 μg/mL of compounds for 48 h, respectively. An aliquot of 0.5% MTT (10 μL/well) was then added to each well. After further incubation for 4h, the formazan formed from the MTT was extracted by adding DMSO and mixing for 10 min. The optical absorbance was read with an micoplate reader at 550 nm, and the IC₅₀ values were calculated according to a regression analysis of the concentration/inhibition data.

Results and discussion

Chemistry

L-leucine (1a) and L-phenylalanine (1b) were used as the starting materials to yield their methyl esters 2a and 2b, and then protected by (Boc)₂O to obtain 3a and 3b. The carboxylic acids of 3a–3b were protected using benzyl chloride, and then the Boc group was deprotected with 3 N HCl in ethyl acetate to give 4a and 4b. N-Boc-protected amino acids (3a and 3b) were coupled with intermediates (2a, 2b, 4a and 4b) by a classical EDCI/HOBt method, and then converted into 6a–6d by deprotecting the Boc group (Scheme 1). Using the same procedure, Boc-protected L-isoserine 8 was coupled with the intermediate (2a, 2b, 4a and 4b) and 6a–6d to yield the dipeptide 9a–9d and tripeptide 10a–10d containing protecting group. Finally, target compounds 11a–11d and 12a–12d were obtained by easily cleaving Boc, while the target compounds 13a–13b and 14a–14b were obtained from 9c–9d and 10c–10d by first reacting in a hydrogen atmosphere with 15% Pd/C and then deprotecting Boc (Scheme 2).

Scheme 1.  Reagents and conditions: a. CH₃OH, HCl; b. (Boc)₂O, CH₃OH, NaOH; c. i) K₂CO₃, KI, TBAI, benzyl chloride, CH₃COCH₃; ii) HCl/anhydrous EtOAc; d. EDCI, HOBt/anhydrous, DCM, 2a∼2b,4a∼4b, 0°C to room temperature; e. HCl/anhydrous EtOAc.

Scheme 2.  Reagents and conditions: a. (Boc)₂O, CH₃OH, NaOH; b. EDCI, HOBt/anhydrous, DCM, 2a∼2b,4a∼4b,6a∼6d, 0°C to room temperature; c. HCl/anhydrous EtOAc; d. i) 15% Pd/C, CH₃OH. ii) HCl/anhydrous EtOAc.

In vitro APN and MMP assays

All the target compounds were tested for their inhibitory potential against APN and MMP-2 and the results are shown in Table 1. The matrix metalloproteinase-2 (MMP-2) also belongs to the zinc-dependent metalloproteinase family and is associated with tumour invasion and metastasis. These inhibitory studies were performed on both APN and MMP-2 so as to identify the selectivity of the target compounds.

Table 1.  The structures and IC₅₀ values of target compounds against APN and MMP-2.

Compound	R1	R2	APN/IC50^a (μM)	MMP/IC50^a (μM)
11a		-CH3	301.9 ± 1.3	>1000
11b		CH3	229.2 ± 0.6	681.7 ± 2.8
11c		-Bn	99.5 ± 2.1	328.5 ± 1.9
11d		-Bn	195.1 ± 0.7	>1000
12a		-H	249.1 ± 0.9	710.3 ± 2.3
12b		-H	199.2 ± 3.1	364.2 ± 4.0

Table

Compound	R3	R4	R5	APN /IC50 (μM)	MMP/IC50 (μM)
13a			-CH3	139.6 ± 0.5	125.6 ± 2.4
13b			-Bn	65.1 ± 0.7	225.4 ± 1.1
13c			-CH3	69.1 ± 1.4	236..6 ± 3.3
13d			-Bn	32.2 ± 2.3	333.7 ± 2.7
14a			-H	20.9 ± 0.8	115.0 ± 0.9
14b			-H	12.2 ± 1.6	203.4 ± 2.9
Bestatin				7.3 ± 0.2	167 ± 2.8

a. Each value represents the mean of three experiments, standard deviation is given.

From the results of the enzyme assay, compound 14b (IC₅₀ of 12.2 μM) is the most potent APN inhibitor of all the target compounds, and showed an inhibitory activity similar to that of bestatin (IC₅₀ of 7.3 μM). In addition, the dipeptides derivatives (11a, 11b, 11c, 11d, 12a and 12b) showed less inhibition than their tripeptide analogues (13a, 13b, 13c, 13d, 14a and 14b). This phenomenon suggests that the third amino acid residue helps to increase the interaction with the APN binding site. One the other hand, the tripeptide compounds (13c, 13d and 14b) with a scaffold of isoser-L-Phe-L-Leu exhibited a better inhibitory activity than that of the compounds (13a, 13b and 14a) with an isoser-L-Leu-L-Phe scaffold. As for the compounds (13c, 13d and 14b) with a substituted R₅, the hydrogen seems to be the best substitution for this position. A possible reason may be that the free terminal carboxylate group could enhance the hydrogen bond interaction with the active sites of APN.

In our previous studies, most of the compounds tested showed better inhibition on MMP-2 than APN [18]. However, most of these L-isoserine dipeptides and tripeptides derivatives exhibited a better inhibitory activity on APN than MMP-2. This result may be due to the differences between the 3D structure of the active sites of APN and MMP-2. According to the binding site of APN (PDB code: 1HS6) and MMP-2 (PDB code: 1HOV, 1CK7, 1QIB), the active site of APN was much deeper than the active site of MMP-2 [17].The scaffold of our L-isoserine derivatives are linear structures, and therefore there is easier access to the APN active site.

Antiproliferative activity assays on HL-60 and SKOV3 cells

The human promyelocytic leukaemia cell line (HL-60) was used to evaluate antiproliferative activity with the MTT assay due to APN being over-expressed. In addition, the human ovarian carcinoma cell line (SKOV3) was chosen as an additional model for MTT assays. According to the results (Table 2), all the test compounds showed cytostatic activity except for compound 13d. It should also be pointed out that compound 14b not only displays the most potent inhibition on APN, but also has a higher antiproliferative activity than bestatin.

Table 2.  In vitro antiproliferative inhibiting potency of compounds 11c, 13c, 13d and 14b.

Compound	HL-60 IC50^a (mM)	SKOV3 IC50 ^a(mM)
11c	0.33 ± 0.06	0.28 ± 0.03
13c	0.31 ± 0.05	0.07 ± 0.02
13d	>1000	>1000
14b	0.17 ± 0.03	0.38 ± 0.07
Bestatin	0.87 ± 0.07	1.98 ± 0.09

Each value represents the mean values with S.E values for 5 independent experiments.

In order to determine the interaction between our target compounds with APN, the most potent compound, 14b, was constructed with a Sybyl/Sketch module and optimised using the Tripos force field. The docking study performed with the Sybyl/FlexX module was based on the active site of the APN co-crystal structure with bestatin (PDB code: 2DQM). The docking results showed that the carbonyl group and hydroxyl group of compound 14b could chelate with the zinc ion in APN (Figures 1 and 2). The terminal amino group of 14b could form a hydrogen bond with Glu²⁶⁴ and the carboxylic group of 14b is able to interact with Arg⁸²⁵. In addition, the phenylalanine residue of 14b can insert an S1′ pocket which is surrounded by Gly²⁶¹, Ala²⁶² and Glu²⁹⁸, while the leucine residue in 14b interacts with the S2′ pocket containing Met²⁶³ and Tyr³⁷⁶

Figure 1.  The docking result for 14b with the active site of APN (PDB: 2DQM).

Figure 2.  The docking results for 14b with APN showed by LIGPLOT.

Conclusion

In summary, we have described the synthesis and performed a SAR study for a novel series of L-isoserine derivatives as inhibitors of APN. Most of these compounds possessed potent activity toward APN. Among them, compound 14b exhibited a potent inhibitory activity and a significant selectivity for APN over MMP-2. Furthermore, this compound also showed good cell-based inhibitory activity, and could be used as a potential lead compound for new anti-cancer agents in the future.

Declaration of interest

This work was supported by National Natural Foundation Research Grant (Grant No. 9071304; No 30772654) and National High Technology Research and Development Program of China (863 proJect; Grant No 2007AA02Z314) and Doctoral Foundation of Ministry of Education of the People’s Republic of China (Grant No 20060422029).