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Article

Development of Hydroxamate Derivatives Containing a Pyrazoline Moiety as APN Inhibitors to Overcome Angiogenesis

by
Yangyang Liu
,
Dongsheng Zhao
,
Chenghua Zhang
,
Hui Fang
,
Qingsitong Shen
,
Zhixian Wang
and
Jiangying Cao
*
School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355, China
*
Author to whom correspondence should be addressed.
Molecules 2022, 27(23), 8339; https://doi.org/10.3390/molecules27238339
Submission received: 30 October 2022 / Revised: 12 November 2022 / Accepted: 24 November 2022 / Published: 29 November 2022
(This article belongs to the Special Issue Medicinal Chemistry in China II)
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Abstract

:
Aminopeptidase N (APN) was closely associated with cancer invasion, metastasis, and angiogenesis. Therefore, APN inhibitors have attracted more and more attention of scientists as antitumor agents. In the current study, we designed, synthesized, and evaluated one new series of pyrazoline-based hydroxamate derivatives as APN inhibitors. Moreover, the structure–activity relationships of those were discussed in detail. 2,6-Dichloro substituted compound 14o with R1 = CH3, showed the best capacity for inhibiting APN with an IC50 value of 0.0062 ± 0.0004 μM, which was three orders of magnitude better than that of the positive control bestatin. Compound 14o possessed both potent anti-proliferative activities against tumor cells and potent anti-angiogenic activity. At the same concentration of 50 μM, compound 14o exhibited much better capacity for inhibiting the micro-vessel growth relative to bestatin in the rat thoracic aorta ring model. Additionally, the putative interactions of 14o with the active site of APN are also discussed. The hydroxamate moiety chelated the zinc ion and formed four hydrogen bonds with His297, Glu298 and His301. Meanwhile, the terminal phenyl group and another phenyl group of 14o interacted with S2′ and S1 pockets via hydrophobic effects, respectively.

1. Introduction

Aminopeptidase N (APN, CD13, EC 3.4.11.2), a type II transmembrane glycoprotein, is a zinc-dependent exopeptidase belonging to the M1 superfamily of the MA clan of peptidases [1]. APN is widely expressed on the surfaces of diverse cells, such as liver, renal, intestinal, fibroblast, endothelial, tumor cells and so on. Due to its multiple functions in the hydrolysis of peptides, signal transduction and animal coronavirus infection, APN is also called the “moonlighting enzyme” [2]. As an exopeptidase, APN preferentially cleaves the hydrophobic and basic amino acid residues from the N-terminus of biologically active peptides with broad substrate specificity [3]. For example, in the central nervous system, APN and neutral endopeptidase (NEP) cooperatively hydrolyzes enkephalin and endorphin to regulate pain [4]. Moreover, APN catalyzes the conversion of angiotensin III to angiotensin IV to regulate the renin–angiotensin system [5].
Ongoing interest is focused on APN due to its significant role in the metastasis and angiogenesis of tumors. Metastasis is a complex biological process involving cell migration, cell invasion and angiogenesis, which leads to more than 90% of cancer related deaths [6,7]. APN can metabolize the extracellular matrix (ECM) to promote the process of invasion and metastasis of tumor cells [8,9]. Moreover, expression of APN had been observed on the surfaces of angiogenic blood vessels rather than normal ones [10]. Impaired neovascularization was found in APN knockout mice [11]. Furthermore, it was reported that APN on the surfaces of both tumor cells and nonmalignant stromal cells cooperatively induced angiogenesis [12]. In addition, a strong correlation between up-regulated APN expression and poor prognosis of tumors has been certified through much research [13]. A recent study showed that APN was over-expressed in semi-quiescent liver cancer cells and considered as one of surface markers of liver cancer stem cells (CSCs), which mainly led to tumor recurrence [14]. All of this evidence supports the rationale that APN is an effective target for the chemotherapy of tumors.
Structurally, APN is composed of 967 amino acids, contains a short intracellular tail, a transmembrane anchor, a small Ser-/Thr-rich extracellular stalk and a large ectodomain [15]. Domain II, one component of the ectodomain, contains one catalytic zinc ion and two characteristic motifs (HEXXHX18E zinc-binding motif and GXMEN substrate-recognition motif) from the M1 aminopeptidase family [16]. Around the zinc ion, there were three hydrophobic pockets S1, S1′ and S2′, which could bind the P1, P1′ and P2′ sites of the substrates, respectively. So far, many natural products and synthetic small molecules are reported to be APN inhibitors (APNIs). The natural APNIs are mostly peptides or peptidomimetics, such as bestatin 1, amastatin 2, probestin 3, AHPA-Val 4, and so on (Figure 1) [17,18]. The synthetic APNIs are structurally diverse, containing 1,2,3-triazol ureido peptidomimetics 5 [19], an aminophosphinic derivative 6 (PL253) [20], a flavone derivative 7 [21], a dicarbonyl derivative 8 [22], a chloramphenicol amine derivative 9 [23], an amino benzosuberone derivative 10 [24], a L-isoserine derivative 11 [25], an amide derivative 12 and so on (Figure 1) [26]. Among them, the dipeptide derivative bestatin is marketed as an immunomodulating agent for the adjuvant treatment of acute myeloid leukemia. Many studies have subsequently revealed that bestatin could prevent the invasion, metastasis and angiogenesis associated with tumors [27,28].
The structure–activity relationships (SARs) of APNIs revealed that the potent APNIs possessed zinc binding groups (ZBGs) and hydrophobic groups interacting with at least one of three hydrophobic pockets (S1, S1′ and S2′) of APN. In our previous work, pyrazoline-based hydroxamate derivatives 13a and 13b showed the capacity of inhibiting APN (Figure 2) [29]. Subsequently, optimization focused on the terminal phenyl group of the diphenyl pyrazoline core gave compound 13c with improved APN inhibitory activity relative to compounds 13a and 13b [30]. In this work, in order to extend the SARs of these series of compounds and find more potent APNIs, further optimization was focused on two phenyl groups of compounds 13a and 13b, leading to compound 14. The introduction of hydrophobic substituents on the phenyl groups of compound 14 or the replacement of the terminal phenyl group with a naphthyl moiety may strengthen the hydrophobic interactions with APN. Interestingly, among all the newly synthesized compounds, compound 14o presented the best APN inhibitory activity, being two orders of magnitude better than those of lead compounds 13a and 13b.

2. Results and Discussion

2.1. Chemistry

The target compounds were synthesized following the procedures shown in Scheme 1. Firstly, benzaldehyde derivatives 15a–15f reacted with the corresponding ketone to form the key chalcone intermediates 18a–18s via the Claisen–Schmidt reaction in the presence of potassium hydroxide. Additionally, 2-hydroxyacetophenone 16 reacted with benzyl bromide or substituted benzyl bromide to give compounds 17t–17w, which further reacted with 2-hydroxybenzaldehyde 15a to give chalcone intermediates 18t–18w, respectively, following the same procedure of compounds 18a–18s. Subsequently, compounds 18a–18w were cyclized with excess hydrazine hydrate or semicarbazide hydrochloride to give the pyrazoline derivatives 19a–19z and 19aa–19hh, which further underwent nucleophilic substitution reaction with methyl bromoacetate to yield compounds 20a–20z and 20aa–20hh, respectively. Ultimately, the freshly prepared methanolic solution of NH2OK transformed the methyl ester groups of 20a–20z and 20aa–20hh to hydroxamate groups to form the target compounds 14a–14z and 14aa–14hh, respectively.

2.2. Inhibitory Activities of Target Compounds against APN

All the synthesized pyrazoline derivatives were first evaluated for their APN inhibitory activities using bestatin and compounds 13a–13c as the positive controls. The results are listed in Table 1. Similar to SARs previously reported, the substituents on the terminal phenyl group of the diphenyl pyrazoline moiety could significantly impact the inhibitory activities against APN [30]. Comparing mono-substituted compounds 14a–14f with a methyl group as R1, it was easily found that compounds (14a, 14d) with ortho-substituents showed better inhibitory potencies against APN than their counterparts with meta-substituents (14b, 14e) or para-substituents (14c, 14f), respectively. Moreover, among ortho mono-substituted compounds 14a, 14d, 14g–14i and 14k, compound 14i containing an ortho-iodine substitution exhibited the best capacity of inhibiting APN, with an IC50 value of 0.015 ± 0.001 μM. The APN inhibitory order of ortho mono-substituted compounds with R1 = CH3, was 14i (2-I) > 14h (2-Cl) > 14a (2-Br) > 14k (2-CH3) > 14g (2-F) > 14d (2-OCH3). Among dual-substituted compounds (14l, 14m, 14o) with R1 = CH3, compound 14o with 2,6-dichlorine substitution was two orders of magnitude better against APN than the analogues with 2,4-dichlorine (14l) or 2,6-dimethoxy (14m) substitution, suggesting that the substituents and substituted position on the terminal phenyl group significantly affected the inhibitory activities against APN. Moreover, ortho dual-substituted compounds showed improved APN inhibitory activities relative to the corresponding ortho mono-substituted analogs (14m vs. 14d; 14o vs. 14h). In addition, the APN inhibitory activities of compounds 14j and 14n, with NH2 as R1, were inferior to those of counterparts 14i and 14m, with CH3 as R1, respectively. Replacement of the terminal phenyl group of compound 13a with a naphthyl group led to compound 14p, with comparable APN inhibitory activity to 13a.
Moreover, in order to explore the APN inhibitory effects of substituents on the other phenyl group, compounds 14q–14z were designed using 14i and 14j as lead compounds. Compound 14w, with R1 = CH3 and R6 = Br, exhibited better APN inhibitory activity than the counterparts 14q (R3 = Br), 14s (R4 = Br) and 14u (R5 = Br), respectively. The same inhibitory tendency was also found among compounds 14r, 14t, 14v and 14x, with R1 = NH2. Furthermore, compounds 14y (IC50 = 0.023 ± 0.001 μM) and 14z (IC50 = 0.070 ± 0.003 μM), with R6 = Cl, displayed comparable or improved APN inhibition relative to corresponding bromo-substituted compounds 14w and 14x, respectively. However, the APN inhibitory activities of compounds 14y and 14z were inferior to those of corresponding lead compounds 14i and 14j, respectively, indicating that the introduction of substituents on the other phenyl group was detrimental.
Furthermore, introduction of one large hydrophobic ortho-benzyloxyl group on the terminal phenyl group of compounds 13a and 13b led to compounds 14aa and 14bb, respectively, with one order of magnitude improved APN inhibitory activities. However, bromo-substitution of the benzyloxyl group of 14aa and 14bb led to decreased APN inhibition (14cc, 14ee, 14gg vs. 14aa; 14dd, 14ff, 14hh vs. 14bb). Moreover, meta bromo-substituted compounds presented better APN inhibitory activities than the counterparts with ortho or para bromo-substitution (14ee vs. 14cc, 14gg; 14ff vs. 14dd, 14hh).
Generally, compounds with R1 = CH3, exhibited superior or inferior APN inhibitory activities to their counterparts with R1 = NH2, which depended on the substituents of the two phenyl groups of the diphenyl pyrazoline moiety. Interestingly, the APN inhibitory activities of compounds 14a, 14h-14j, 14o, 14w, 14y, 14aa, 14bb and 14ee were better than those of lead compounds 13a-13c, supporting our design strategy. Notably, the most potent one, 14o (IC50 = 0.0062 ± 0.0004 μM) was over 9-fold more potent than lead compound 13c (IC50 = 0.059 ± 0.002 μM) and over 1419-fold more potent than positive control bestatin (IC50 = 8.8 ± 0.4 μM).

2.3. Anti-Proliferative Activities of Selected Compounds against Tumor Cells

Compound 14o, the most potent against APN, was selected to further evaluate the anti-proliferative activities against six tumor cell lines (U937, K562, PLC/PRF/5, PC-3, ES-2, HepG2). As results listed in Table 2, compound 14o exhibited much better anti-proliferative potencies than bestatin against all the tested tumor cell lines. Among them, K562 cells were most sensitive to compound 14o.

2.4. Ex Vivo Anti-Angiogenesis Assay

The anti-angiogenic potency of selected compound 14o was preliminarily measured using the rat thoracic aorta ring model. As the results in Figure 3 show, compound 14o could prevent the growth of micro-vessels derived from the thoracic aorta rings in a dose–dependent manner. Compared with the group treated with 1 μM of 14o, obviously reduced micro-vessels were observed in the group treated with 50 μM of 14o. Furthermore, at the same concentration of 50 μM, the anti-angiogenic activity of 14o was much better than that of bestatin, which was consistent with the enzymatic inhibitory activities against APN.

2.5. Docking Assay

In order to investigate its binding mode in APN, compound 14o was docked into the active site of APN (PDB code: 2DQM) using Sybyl 2.1. As depicted in Figure 4A, the terminal phenyl group with 2,6-diclorine substitution and another phenyl group occupied the S2′ and S1 hydrophobic pockets, respectively. Meanwhile, the hydroxamate moiety chelated the zinc ion. Moreover, one more detailed binding mode of compound 14o with APN was illustrated in Figure 4B, generated with LIGPLOT. The terminal phenyl group could form hydrophobic interactions with the Arg293, Tyr381 and Glu382. Meanwhile, another phenyl group could form hydrophobic interactions with Met260 and Tyr376. In addition, the CH3CO group hydrophobically interacted with Arg783 and Arg825. Except for chelating the zinc ion, the hydroxamate moiety could form four hydrogen bonds with His297, Glu298 and His301 to enhance the potency of the interaction with APN.

3. Materials and Methods

3.1. Chemistry: General Procedures

All the commercially available materials were used without further purification otherwise noted. Thin-layer chromatography (TLC) was used for the monitoring of all the reactions on 0.25 mm silica gel plates (60 GF-254) and the product spots were visualized by ferric chloride, iodine vapor and UV light. The purification of the product was carried out by column chromatography. Melting points were determined on an electrothermal melting point apparatus without correction. 1H-NMR spectra were conducted on a Brucker DRX spectrometer with TMS as an internal standard. The values of chemical shifts were described as δ in parts per million and J in Hertz. HRMS were conducted by the Shandong Analysis and Test Center.

3.1.1. General Procedure for Synthesis of Compounds 17t–17w 1-(2-(Benzyloxy)phenyl)ethan-1-one (17t)

To a solution of 2-hydroxyacetophenone 16 (10.88 g, 80.00 mmol) in DMF was added the 60% NaH (3.84 g, 96.00 mmol), gradually. After stirring at 0 °C for 5 min, benzyl bromide (20.52 g, 120.00 mmol) was added into the mixture. The mixture was stirred at 25 °C for 12 h, then poured into water, followed by extraction with EtOAc. The organic phase was washed with brine and dried over MgSO4. After filtration and concentration under vacuum, the residue was purified by column chromatography (PE/EtOAc = 10:1, v/v) to give 16.63 g of compound 17t as oil. Yield: 92%; ESI-MS m/z 227.1 [M+H]+.
Compounds 17u–17w were prepared following the procedure described for compound 17t.

3.1.2. General Procedure for Synthesis of Compounds 18i, 18l, 18o–18w

(E)-3-(5-Bromo-2-hydroxyphenyl)-1-(2-iodophenyl)prop-2-en-1-one (18q)
5-Bromo-2-hydroxybenzaldehyde 15d (10.00 g, 50.00 mmol) and 2-iodoacetophen- one (14.76 g, 60.00 mmol) were dissolved in ethanol (200 mL), followed by the addition of potassium hydroxide aqueous solution (25.20 g, 450.00 mmol, in 50 mL water) at 0 °C. After stirring at 25 °C for 48 h, water was added into the mixture and pH was adjusted to 7 by 1N HCl. The mixture stood for 6 h. After filtration the residue was further purified by column chromatograph (DCM/MeOH = 100:5, v/v) to give 11.77 g of compound 18q as a yellow solid. Yield: 55%, mp: 160–162 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.65 (s, 1H), 7.97 (d, J = 7.9 Hz, 1H), 7.94–7.92 (m, 1H), 7.56–7.54 (m, 1H), 7.52–7.45 (m, 2H), 7.42 (dd, J = 8.8 Hz, J = 2.5 Hz, 1H), 7.32–7.21 (m, 2H), 6.87 (d, J = 8.8 Hz, 1H). ESI-MS m/z 428.5 [M+H] +.
Compounds 18a–18h, 18j, 18k, 18m and 18n were reported before [30]. Compounds 18i, 18l, 18o, 18p and 18r–18w were prepared following the procedure described for the compound 18q.

3.1.3. General Procedure for Synthesis of Compounds 19a–19i, 19k–19m, 19o–19q, 19s, 19u, 19w, 19y, 19aa, 19cc, 19ee and 19gg

1-(5-(2-Hydroxyphenyl)-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (19d)
To a solution of 18d (5.04 g, 20.00 mmol) in hot acetic acid (25 mL) was added 80% hydrazine hydrate (5.00 g, 80.00 mmol). After stirring at 120 °C for 5 h, the mixture was poured into water and the pH was adjusted to 7 by sodium bicarbonate. The formed precipitate was filtered off, dried at 50 °C and washed with EtOAc to give 4.03 g of compound 19d as a white solid. Yield: 65%, mp: 206–208 °C. 1H NMR (400 MHz, DMSO-d6): δ 9.65 (s, 1H), 7.83 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.43 (td, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.11–6.99 (m, 3H), 6.81 (td, J = 7.8 Hz, J = 1.4 Hz, 2H), 6.73–6.69 (m, 1H), 5.59 (dd, J = 11.7 Hz, J = 4.2 Hz, 1H), 3.84 (dd, J = 18.4 Hz, J = 11.7 Hz, 1H), 3.78 (s, 3H), 3.02 (dd, J = 18.4 Hz, J = 4.2 Hz, 1H), 2.31 (s, 3H). ESI-MS m/z 311.2 [M+H] +.
Compounds 19a–19c, 19e–19i, 19k–19m, 19o–19q, 19s, 19u, 19w, 19y, 19aa, 19cc, 19ee and 19gg were prepared following the procedure described for the compound 19d.

3.1.4. General Procedure for Synthesis of Compounds 19j, 19n, 19r, 19t, 19v, 19x, 19z, 19bb, 19dd, 19ff and 19hh

5-(2-Hydroxyphenyl)-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (19j)
Semicarbazide hydrochloride (2.68 g, 24.00 mmol) and sodium hydroxide (3.60 g, 90.00 mmol) were added to the solution of 18i (7.00 g, 20.00 mmol) in ethanol (100 mL). After stirring at 78 °C for 5 h, the mixture was poured into water and the excess base was neutralized by 10% HCl. The formed precipitate was filtered off, dried at 50 °C and washed with EtOAc to give 5.54 g of compound 19j as a white solid. Yield: 68%, mp: 176–178 °C. ESI-MS m/z 408.3 [M+H]+.
Compounds 19n, 19r, 19t, 19v, 19x, 19z, 19bb, 19dd, 19ff and 19hh were prepared following the procedure described for the compound 19j.

3.1.5. General Procedure for Synthesis of Compounds 20a–20z and 20aa–20hh

Methyl 2-(2-(1-acetyl-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy) acetate (20d)
To a solution of compound 19d (1.50 g, 4.84 mmol) in DMF (20 mL) was added 60% NaH (0.23 g, 5.81 mmol) gradually. After stirring at 25 °C for 5 min, methyl bromoacetate (1.11 g, 7.26 mmol) was added. Then the mixture was stirred at 25 °C for 12 h, followed by the addition of water (100 mL). The formed precipitate was filtered off and further purified by column chromatography (DCM/MeOH = 50:1, v/v) to give 1.04 g of compound 20d as a white solid. Yield: 56%, mp: 126–128 °C. 1H NMR (400 MHz, DMSO-d6): δ 7.81 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.43 (td, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.22–7.18 (m, 1H), 7.10 (d, J = 8.3 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 6.91–6.90 (m, 2H), 5.67 (dd, J = 11.7 Hz, J = 4.2 Hz, 1H), 4.95–4.86 (m, 2H), 3.85 (dd, J = 18.5 Hz, J = 11.7 Hz, 1H), 3.78 (s, 3H), 3.69 (s, 3H), 3.15 (dd, J = 18.5 Hz, J = 4.2 Hz, 1H), 2.32 (s, 3H). ESI-MS m/z 383.5 [M+H] +.
Compounds 20a–20c, 20e–20z and 20aa–20hh were prepared following the procedure described for the compound 20d.

3.1.6. General Procedure for Synthesis of Compounds 14a–14z and 14aa–14hh

2-(2-(1-Acetyl-3-(2-bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14a)
A solution of potassium hydroxide (15.00 g, 267.86 mmol) in anhydrous methanol (50 mL) was dropwise added into a solution of hydroxylamine hydrochloride (12.54 g, 180.43 mmol) in anhydrous methanol (50 mL) at 0 °C. After stirring at 0 °C for 40 min, the mixture was filtered and the filtrate was used for the transformation of the methyl ester group to a hydroxamate group. Compound 20a (1.00 g, 2.32 mmol) was dissolved in the above solution (10 mL) and the mixture was stirred at 25 °C for 0.5 h. Then the mixture was poured into water (100 mL) and 10% H3PO4 solution was added to neutralize the excess base. The formed precipitate was filtered off and dried under vacuum to give 0.54 g of compound 14a as a white solid. Yield: 54%, mp: 162–164 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.04 (s, 1H), 7.81 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.45–7.41 (m, 1H), 7.24–7.20 (m, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.03–6.96 (m, 2H), 6.94–6.86 (m, 2H), 5.81 (dd, J = 11.7 Hz, J = 4.3 Hz, 1H), 4.62–4.54 (m, 2H), 3.88 (dd, J = 18.6 Hz, J = 11.7 Hz, 1H), 3.79 (s, 3H), 3.14 (dd, J = 18.6 Hz, J = 4.3 Hz, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.25, 164.76, 155.12, 154.77, 134.51, 132.62, 131.73, 131.47, 129.88, 128.89, 128.37, 126.02, 121.65, 121.14, 112.69, 66.53, 55.37, 44.23, 22.24; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H19BrN3O4: 432.0559, found: 432.0548.
Compounds 14b–14z and 14aa–14hh were prepared following the procedure described for the compound 14a.
2-(2-(1-Acetyl-3-(3-bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14b)
White solid, yield: 55%, mp: 152–154 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 9.04 (s, 1H), 7.91 (t, J = 1.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.66 (dd, J = 7.8 Hz, J = 2.0 Hz, 1H), 7.42 (t, J = 7.9 Hz, 1H), 7.26–7.20 (m, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 4.5 Hz, 2H), 5.85 (dd, J = 11.8 Hz, J = 4.6 Hz, 1H), 4.62 (d, J = 14.2 Hz, 1H), 4.55 (d, J = 14.2 Hz, 1H), 3.80 (dd, J = 18.2 Hz, J = 11.8 Hz, 1H), 3.21 (dd, J = 18.2 Hz, J = 4.6 Hz, 1H), 2.36 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.07, 164.78, 154.73, 154.28, 134.01, 133.32, 131.40, 129.95, 129.34, 128.88, 126.05, 125.84, 122.57, 121.63, 112.72, 66.67, 55.62, 41.37, 22.21; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H19BrN3O4: 432.0559, found: 432.0549.
2-(2-(1-Acetyl-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14c)
White solid, yield: 55%, mp: 202–204 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 9.04 (s, 1H), 7.71–7.65 (m, 4H), 7.26–7.20 (m, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 4.5 Hz, 2H), 5.85 (dd, J = 11.8 Hz, J = 4.6 Hz, 1H), 4.62 (d, J = 14.2 Hz, 1H), 4.54 (d, J = 14.2 Hz, 1H), 3.81 (dd, J = 18.1 Hz, J = 11.8 Hz, 1H), 3.19 (dd, J = 18.1 Hz, J = 4.6 Hz, 1H), 2.35 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.96, 164.75, 154.73, 154.66, 132.23, 130.89, 129.99, 128.97, 128.86, 125.87, 124.09, 121.63, 112.71, 66.61, 55.59, 41.41, 22.19; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H19BrN3O4: 432.0559, found: 432.0563.
2-(2-(1-Acetyl-3-(2-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14d)
White solid, yield: 52%, mp: 170–172 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.04 (s, 1H), 7.81 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.45–7.41 (m, 1H), 7.24–7.20 (m, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.03–6.96 (m, 2H), 6.94–6.86 (m, 2H), 5.81 (dd, J = 11.7 Hz, J = 4.3 Hz, 1H), 4.62–4.54 (m, 2H), 3.88 (dd, J = 18.6 Hz, J = 11.7 Hz, 1H), 3.79 (s, 3H), 3.14 (dd, J = 18.6 Hz, J = 4.3 Hz, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.82, 164.77, 158.45, 154.94, 154.64, 132.17, 130.42, 129.09, 128.72, 125.62, 121.68, 121.08, 120.57, 112.85, 112.62, 66.52, 56.18, 54.87, 44.94, 22.20; HRMS (AP-ESI) m/z [M+H]+ calcd for C20H22N3O5: 384.1559, found: 384.1552.
2-(2-(1-Acetyl-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14e)
White solid, yield: 57%, mp: 170–172 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.06 (s, 1H), 7.40–7.32 (m, 2H), 7.28–7.20 (m, 2H), 7.06–6.98 (m, 2H), 6.94–6.89 (m, 2H), 5.85 (dd, J = 11.7 Hz, J = 4.4 Hz, 1H), 4.63 (d, J = 14.3 Hz, 1H), 4.56 (d, J = 14.3 Hz, 1H), 3.84–3.77 (m, 4H), 3.20 (dd, J = 18.1 Hz, J = 4.4 Hz, 1H), 2.36 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.90, 164.78, 159.83, 155.47, 154.70, 132.98, 130.37, 130.12, 128.82, 125.74, 121.65, 119.51, 116.56, 112.69, 112.03, 66.65, 55.68, 55.30, 41.65, 22.17; HRMS (AP-ESI) m/z [M+H]+ calcd for C20H22N3O5: 384.1559, found: 384.1552.
2-(2-(1-Acetyl-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14f)
White solid, yield: 55%, mp: 120–122 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.06 (s, 1H), 7.70 (d, J = 8.6 Hz, 2H), 7.25–7.19 (m, 1H), 7.02–6.97 (m, 3H), 6.93–6.85 (m, 2H), 5.83 (dd, J = 11.6 Hz, J = 4.2 Hz, 1H), 4.63 (d, J = 14.3 Hz, 1H), 4.56 (d, J =14.3 Hz, 1H), 3.79 (s, 3H), 3.78 (dd, J = 18.2 Hz, J = 11.6 Hz, 1H), 3.15 (dd, J = 18.2 Hz, J = 4.2 Hz, 1H), 2.34 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.60, 164.79, 161.36, 155.35, 154.69, 130.23, 128.77, 128.71, 125.67, 124.12, 121.64, 114.65, 112.64, 66.62, 55.80, 55.04, 41.71, 22.17; HRMS (AP-ESI) m/z [M+H]+ calcd for C20H22N3O5: 384.1559, found: 384.1564.
2-(2-(1-Acetyl-3-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14g)
White solid, yield: 50%, mp: 176–178 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 9.03 (s, 1H), 7.90–7.85 (m, 1H), 7.54–7.48 (m, 1H), 7.33–7.29 (m, 2H), 7.27–7.20 (m, 1H), 6.99–6.91 (m, 3H), 5.84 (dd, J = 11.8 Hz, J = 4.5 Hz, 1H), 4.62–4.53 (m, 2H), 3.93–3.85 (m, 1H), 3.20–3.13 (m, 1H), 2.34 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.12, 164.79, 159.46, 154.73, 151.82, 132.70, 130.02, 129.53, 128.88, 125.83, 125.28, 121.66, 119.69, 117.18, 112.67, 66.51, 55.08, 43.74, 22.15; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H19FN3O4: 372.1360, found: 372.1365.
2-(2-(1-Acetyl-3-(2-chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14h)
White solid, yield: 54%, mp: 166–168 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 9.02 (s, 1H), 7.72 (dd, J = 7.5 Hz, J = 2.1 Hz, 1H), 7.55 (dd, J = 7.7 Hz, J = 1.6 Hz, 1H), 7.48–7.40 (m, 2H), 7.26–7.22 (m, 1H), 6.99–6.91 (m, 3H), 5.84 (dd, J = 11.8 Hz, J = 4.5 Hz, 1H), 4.62–4.53 (m, 2H), 3.95 (dd, J = 18.1 Hz, J = 11.8 Hz, 1H), 3.21 (dd, J = 18.1 Hz, J = 4.5 Hz, 1H), 2.32 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.25, 164.80, 154.78, 154.25, 132.12, 131.64, 131.27, 131.17, 130.66, 129.90, 128.92, 127.90, 125.98, 121.68, 112.70, 66.53, 55.37, 44.25, 22.20; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H19ClN3O4: 388.1064, found: 388.1072.
2-(2-(1-Acetyl-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14i)
White solid, yield: 58%, mp: 174–176 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.92 (s, 1H), 9.02 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.58–7.47 (m, 3H), 7.21–7.07 (m, 3H), 5.89 (dd, J = 12.0 Hz, J = 5.4 Hz, 1H), 4.64 (d, J = 13.2 Hz, 1H), 4.43 (d, J = 13.2 Hz, 1H), 3.94 (dd, J = 18.3 Hz, J = 12.0 Hz, 1H), 3.16 (dd, J = 18.3 Hz, J = 5.4 Hz, 1H), 2.31 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.05, 164.83, 156.19, 154.82, 137.70, 132.03, 130.45, 130.15, 129.88, 129.79, 128.82, 126.53, 125.79, 121.67, 112.70, 66.60, 54.37, 43.76, 23.37, 22.24; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H19IN3O4: 480.0420, found: 480.0418.
5-(2-(2-(Hydroxyamino)-2-oxoethoxy)phenyl)-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14j)
White solid, yield: 51%, mp: 164–166 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.93 (s, 1H), 9.01 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.57–7.52 (m, 2H), 7.50–7.46 (m, 1H), 7.23–7.11 (m, 3H), 6.48 (s, 2H), 5.77 (dd, J = 12.2 Hz, J = 6.2 Hz, 1H), 4.64 (d, J = 13.1 Hz, 1H), 4.41 (d, J = 13.1 Hz, 1H), 3.92 (dd, J = 18.1 Hz, J = 12.2 Hz, 1H), 3.12 (dd, J = 18.1 Hz, J = 6.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.84, 155.54, 154.59, 153.22, 141.08, 136.17, 131.32, 131.27, 130.80, 128.77, 128.72, 126.20, 121.66, 112.44, 95.70, 66.56, 55.10, 44.46; HRMS (AP-ESI) m/z [M+H]+ calcd for C18H18IN4O4: 481.0373, found: 481.0371.
2-(2-(1-Acetyl-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14k)
White solid, yield: 50%, mp: 170–172 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 9.03 (s, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 4.3 Hz, 2H), 7.29–7.21 (m, 2H), 6.99–6.92 (m, 3H), 5.78 (dd, J = 11.7 Hz, J = 4.4 Hz, 1H), 4.64–4.54 (m, 2H), 3.88 (dd, J = 17.8 Hz, J = 11.7 Hz, 1H), 3.22 (dd, J = 17.8 Hz, J = 4.4 Hz, 1H), 2.61 (s, 3H), 2.33 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.05, 164.83, 156.19, 154.82, 137.70, 132.03, 130.45, 130.15, 129.88, 129.79, 128.82, 126.53, 125.79, 121.67, 112.70, 66.60, 54.37, 43.76, 23.37, 22.24; HRMS (AP-ESI) m/z [M+H]+ calcd for C20H22N3O4: 368.1610, found: 368.1607.
2-(2-(1-Acetyl-3-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14l)
White solid, yield: 53%, mp: 128–130 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.67 (s, 1H), 8.95 (s, 1H), 7.68–7.64 (m, 2H), 7.43 (dd, J = 8.6 Hz, J = 2.1 Hz, 1H), 7.17–7.13 (m, 1H), 6.90–6.82 (m, 3H), 5.74 (dd, J = 11.7 Hz, J = 4.6 Hz, 1H), 4.52 (d, J = 14.2 Hz, 1H), 4.45 (d, J = 14.2 Hz, 1H), 3.86 (dd, J = 18.1 Hz, J = 11.7 Hz, 1H), 3.14 (dd, J = 18.1 Hz, J = 4.6 Hz, 1H), 2.24 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.28, 164.73, 154.78, 153.21, 135.30, 133.10, 132.40, 130.77, 129.76, 129.59, 128.93, 128.11, 126.10, 121.63, 112.70, 66.48, 55.57, 43.99, 22.20; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H18Cl2N3O4: 422.0674, found: 422.0675.
2-(2-(1-Acetyl-3-(2,6-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14m)
White solid, yield: 55%, mp: 178–180 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.70 (s, 1H), 8.98 (s, 1H), 7.36 (t, J = 8.4 Hz, 1H), 7.25–7.21 (m, 1H), 7.07 (d, J = 7.6 Hz, 1H), 7.01–6.95 (m, 2H), 6.71 (d, J = 8.4 Hz, 2H), 5.84 (dd, J = 11.7 Hz, J = 4.3 Hz, 1H), 4.62–4.53 (m, 2H), 3.73 (s, 3H), 3.66 (dd, J = 18.1 Hz, J = 11.7 Hz, 1H), 3.33 (s, 2H), 2.75 (dd, J = 18.1 Hz, J = 4.3 Hz, 1H), 2.21 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.69, 164.84, 158.58, 154.43, 153.17, 131.56, 130.84, 128.78, 125.93, 121.87, 112.46, 110.02, 104.78, 66.70, 56.45, 53.69, 45.71, 22.22; HRMS (AP-ESI) m/z [M+H]+ calcd for C21H24N3O6: 414.1665, found: 414.1661.
3-(2,6-Dimethoxyphenyl)-5-(2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14n)
White solid, yield: 52%, mp: 212–214 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 8.94 (s, 1H), 7.35 (t, J = 8.4 Hz, 1H), 7.24–7.17 (m, 2H), 7.00 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.70 (d, J = 8.5 Hz, 2H), 6.33 (s, 2H), 5.73 (dd, J = 11.9 Hz, J = 4.8 Hz, 1H), 4.61 (d, J = 14.4 Hz, 1H), 4.54 (d, J = 14.4 Hz, 1H), 3.73 (s, 6H), 3.63 (dd, J = 17.9 Hz, J = 11.9 Hz, 1H), 2.72 (dd, J = 17.9 Hz, J = 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.88, 158.65, 155.48, 154.25, 149.53, 132.23, 131.35, 128.59, 126.36, 121.80, 112.09, 110.34, 104.65, 66.56, 56.37, 53.38, 45.88; HRMS (AP-ESI) m/z [M+H]+ calcd for C20H23N4O6: 415.1618, found: 415.1622.
2-(2-(1-Acetyl-3-(2,6-dichlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14o)
White solid, yield: 54%, mp: 174–176 °C. 1H NMR (600 MHz, DMSO-d6): δ 10.73 (s, 1H), 9.02 (s, 1H), 7.61–7.57 (m, 1H), 7.52 (t, J = 8.1 Hz, 1H), 7.29–7.20 (m, 1H), 7.04 (d, J = 7.7 Hz, 1H), 7.02–6.95 (m, 2H), 5.98 (dd, J = 11.9 Hz, J = 4.7 Hz, 1H), 4.60 (d, J = 14.2 Hz, 1H), 4.55 (d, J = 14.2 Hz, 1H), 3.82 (dd, J = 18.6 Hz, J = 11.9 Hz, 1H), 2.91 (dd, J = 18.6 Hz, J = 4.7 Hz, 1H), 2.28 (s, 3H); 13C NMR (150 MHz, DMSO-d6): δ 168.21, 164.73, 154.49, 153.23, 134.25, 132.58, 130.55, 129.99, 129.04, 128.91, 125.53, 121.70, 112.68, 66.72, 54.86, 44.88, 22.17; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H18Cl2N3O4: 422.0674, found: 422.0668.
2-(2-(1-Acetyl-3-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxy acetamide (14p)
White solid, yield: 55%, mp: 198–200 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.81 (s, 1H), 9.25 (d, J = 8.7 Hz, 1H), 9.06 (s, 1H), 8.04–8.01 (m, 2H), 7.75 (d, J = 7.2 Hz, 1H), 7.72–7.68 (m, 1H), 7.63–7.60 (m, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.25 (td, J = 7.7 Hz, J = 1.8 Hz, 1H), 7.03–6.99 (m, 2H), 6.94 (t, J = 7.4 Hz, 1H), 5.86 (dd, J = 11.7 Hz, J = 4.3 Hz, 1H), 4.65 (d, J = 14.3 Hz, 1H), 4.58 (d, J = 14.3 Hz, 1H), 4.07 (dd, J = 17.8 Hz, J = 11.7 Hz, 1H), 3.42–3.38 (m, 1H), 2.45 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.05, 164.82, 155.98, 154.88, 134.09, 131.38, 130.32, 130.09, 129.41, 129.27, 128.84, 128.17, 127.90, 126.77, 125.89, 125.66, 121.66, 112.70, 66.58, 54.27, 44.11, 22.43; HRMS (AP-ESI) m/z [M+H]+ calcd for C23H22N3O4: 404.1610, found: 404.1605.
2-(2-(1-Acetyl-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-6-bromophenoxy)-N-hydroxyacetamide (14q)
White solid, yield: 50%, mp: 96–98 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.92 (s, 1H), 9.02 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.58–7.47 (m, 3H), 7.21–7.07 (m, 3H), 5.89 (dd, J = 12.0 Hz, J = 5.4 Hz, 1H), 4.64 (d, J = 13.2 Hz, 1H), 4.43 (d, J = 13.2 Hz, 1H), 3.94 (dd, J = 18.3 Hz, J = 12.0 Hz, 1H), 3.16 (dd, J = 18.3 Hz, J = 5.4 Hz, 1H), 2.31 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.41, 164.17, 155.77, 151.90, 141.24, 138.41, 135.59, 132.92, 131.68, 130.96, 128.86, 127.46, 126.00, 116.95, 95.70, 70.77, 54.74, 44.92, 22.40; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H18BrIN3O4: 557.9525, found: 557.9528.
5-(3-Bromo-2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14r)
White solid, yield: 52%, mp: 140–142 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.93 (s, 1H), 9.01 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.57–7.52 (m, 2H), 7.50–7.46 (m, 1H), 7.23–7.11 (m, 3H), 6.48 (s, 2H), 5.77 (dd, J = 12.2 Hz, J = 6.2 Hz, 1H), 4.64 (d, J = 13.1 Hz, 1H), 4.41 (d, J = 13.1 Hz, 1H), 3.92 (dd, J = 18.1 Hz, J = 12.2 Hz, 1H), 3.12 (dd, J = 18.1 Hz, J = 6.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.28, 155.45, 152.79, 151.72, 140.93, 139.58, 136.11, 132.66, 131.39, 130.94, 128.79, 127.34, 126.41, 116.93, 95.96, 70.81, 55.10, 45.27; HRMS (AP-ESI) m/z [M+H]+ calcd for C18H17BrIN4O4: 558.9478, found: 558.9482.
2-(2-(1-Acetyl-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-5-bromophenoxy)-N-hydroxyacetamide (14s)
White solid, yield: 54%, mp: 168–170 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 9.08 (s, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.54–7.43 (m, 2H), 7.19–7.10 (m, 3H), 6.96 (d, J = 8.2 Hz, 1H), 5.76 (dd, J = 11.8 Hz, J = 4.8 Hz, 1H), 4.68–4.58 (m, 2H), 3.88 (dd, J = 18.1 Hz, J = 11.8 Hz, 1H), 3.20 (dd, J = 18.1 Hz, J = 4.8 Hz, 1H), 2.34 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.42, 164.43, 156.35, 155.72, 141.31, 135.66, 131.60, 130.81, 129.41, 128.83, 128.08, 124.45, 121.20, 116.03, 95.54, 66.71, 55.28, 43.69, 22.40; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H18BrIN3O4: 557.9525, found: 557.9520.
5-(4-Bromo-2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14t)
White solid, yield: 57%, mp: 196–198 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.04 (s, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.50–7.44 (m, 2H), 7.18–7.13 (m, 3H), 7.01 (d, J = 8.5 Hz, 1H), 6.49 (s, 2H), 5.68 (dd, J = 12.0 Hz, J = 5.4 Hz, 1H), 4.69–4.58 (m, 2H), 3.87 (dd, J = 18.0 Hz, J = 12.0 Hz, 1H), 3.13 (dd, J = 18.0 Hz, J = 5.4 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.48, 155.51, 155.48, 153.43, 141.00, 136.14, 131.32, 130.82, 130.79, 128.77, 128.04, 124.47, 120.96, 115.78, 95.80, 66.73, 55.06, 44.12; HRMS (AP-ESI) m/z [M+H]+ calcd for C18H17BrIN4O4: 558.9478, found: 558.9482.
2-(2-(1-Acetyl-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-4-bromophenoxy)-N-hydroxyacetamide (14u)
White solid, yield: 51%, mp: 106–108 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.04 (s, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.53–7.42 (m, 3H), 7.18 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 2.5 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 5.79 (dd, J = 11.9 Hz, J = 4.9 Hz, 1H), 4.64–4.53 (m, 2H), 3.89 (dd, J = 18.2 Hz, J = 11.9 Hz, 1H), 3.23 (dd, J = 18.2 Hz, J = 4.9 Hz, 1H), 2.35 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 168.58, 164.46, 156.34, 154.25, 141.36, 135.52, 132.41, 131.65, 131.49, 130.85, 128.87, 115.21, 113.23, 95.47, 66.72, 55.30, 43.74, 22.41; HRMS (AP-ESI) m/z [M+H]+ calcd for C19H18BrIN3O4: 557.9525, found: 557.9522.
5-(5-Bromo-2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14v)
White solid, yield: 54%, mp: 202–204 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 9.02 (s, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.51–7.41 (m, 3H), 7.18–7.14 (m, 2H), 6.96 (d, J = 8.8 Hz, 1H), 6.53 (s, 2H), 5.71 (dd, J = 12.0 Hz, J = 5.4 Hz, 1H), 4.64–4.54 (m, 2H), 3.89 (dd, J = 18.0 Hz, J = 12.0 Hz, 1H), 3.15 (dd, J = 18.0 Hz, J = 5.4 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.48, 155.53, 154.04, 153.53, 141.04, 136.04, 133.76, 131.38, 131.23, 130.82, 128.81, 128.76, 115.01, 113.24, 95.72, 66.77, 55.16, 44.18; HRMS (AP-ESI) m/z [M+H]+ calcd for C18H17BrIN4O4: 558.9478, found: 558.9472.
2-(2-(1-Acetyl-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-bromophenoxy)-N-hydroxyacetamide (14w)
White solid, yield: 55%, mp: 152–154 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.65 (s, 1H), 8.98 (s, 1H), 8.08–8.02 (m, 1H), 7.61–7.59 (m, 1H), 7.53–7.45 (m, 1H), 7.24–7.06 (m, 3H), 6.93–6.82 (m, 1H), 6.28–5.86 (m, 1H), 4.66–4.45 (m, 2H), 3.93–3.73 (m, 1H), 3.61–3.23 (m, 1H), 2.26–2.20 (m, 3H); HRMS (AP-ESI) m/z [M+H]+ calcd for C19H18BrIN3O4: 557.9525, found: 557.9520.
5-(2-Bromo-6-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14x)
White solid, yield: 59%, mp: 132–134 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.62 (m, 1H), 8.99 (s, 1H), 8.04–8.00 (m, 1H), 7.61–7.57 (m, 1H), 7.51–7.44 (m, 1H), 7.28–6.93 (m, 4H), 6.21–5.79 (m, 3H), 4.72–4.43 (m, 2H), 3.88–3.70 (m, 1H), 3.64–3.28 (m, 1H); HRMS (AP-ESI) m/z [M+H]+ calcd for C18H17BrIN4O4: 558.9478, found: 558.9484.
2-(2-(1-Acetyl-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-3-chlorophenoxy)-N-hydroxyacetamide (14y)
White solid, yield: 56%, mp: 168–170 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.68–10.63 (m, 1H), 9.01 (s, 1H), 8.08–8.03 (m, 1H), 7.61–7.46 (m, 2H), 7.30–7.15 (m, 2H), 7.08–6.88 (m, 2H), 6.26–5.89 (m, 1H), 4.68–4.45 (m, 2H), 3.95–3.73 (m, 1H), 3.64–3.24 (m, 1H), 2.25–2.21 (m, 3H); HRMS (AP-ESI) m/z [M+H]+ calcd for C19H18ClIN3O4: 514.0031, found: 514.0035.
5-(2-Chloro-6-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-3-(2-iodophenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14z)
White solid, yield: 57%, mp: 190–192 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.62 (s, 1H), 8.97 (s, 1H), 8.04–8.00 (m, 1H), 7.59–7.56 (m, 1H), 7.51–7.44 (m, 1H), 7.28–7.00 (m, 4H), 6.19–5.83 (m, 3H), 4.73–4.44 (m, 2H), 3.90–3.70 (m, 1H), 3.67–3.28 (m, 1H); HRMS (AP-ESI) m/z [M+H]+ calcd for C18H17ClIN4O4: 514.9983, found: 514.9987.
2-(2-(1-Acetyl-3-(2-(benzyloxy)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14aa)
White solid, yield: 55%, mp: 162–164 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 9.02 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.44–7.39 (m, 3H), 7.36–7.28 (m, 3H), 7.24–7.20 (m, 2H), 7.02 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.92–6.86 (m, 2H), 5.79 (dd, J = 11.6 Hz, J = 4.2 Hz, 1H), 5.15 (s, 2H), 4.57 (d, J = 14.2 Hz, 1H), 4.47 (d, J = 14.2 Hz, 1H), 3.82 (dd, J = 18.5 Hz, J = 11.6 Hz, 1H), 3.18 (dd, J = 18.5 Hz, J = 4.2 Hz, 1H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.88, 164.76, 157.33, 154.92, 154.61, 136.97, 132.05, 130.28, 129.54, 128.90, 128.76, 128.34, 128.14, 125.58, 121.66, 121.32, 121.01, 114.02, 112.57, 70.37, 66.53, 54.82, 44.91, 22.19; HRMS (AP-ESI) m/z [M+H]+ calcd for C26H26N3O5: 460.1872, found: 460.1868.
3-(2-(Benzyloxy)phenyl)-5-(2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14bb)
White solid, yield: 52%, mp: 176–178 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 8.99 (s, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.40–7.28 (m, 6H), 7.22–7.17 (m, 2H), 7.02–6.88 (m, 4H), 6.47 (s, 2H), 5.70 (dd, J = 11.9 Hz, J = 4.8 Hz, 1H), 5.17–5.10 (m, 2H), 4.58 (d, J = 14.4 Hz, 1H), 4.47 (d, J = 14.4 Hz, 1H), 3.80 (dd, J = 18.2 Hz, J = 11.9 Hz, 1H), 3.15 (dd, J = 18.2 Hz, J = 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.79, 157.09, 155.48, 154.40, 151.32, 137.04, 131.75, 131.55, 129.58, 128.92, 128.55, 128.35, 128.15, 125.74, 121.64, 121.34, 121.23, 113.93, 112.29, 70.34, 66.48, 54.54, 45.22; HRMS (AP-ESI) m/z [M+H]+ calcd for C25H25N4O5: 461.1825, found: 461.1830.
2-(2-(1-Acetyl-3-(2-((2-bromobenzyl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14cc)
White solid, yield: 53%, mp: 82–84 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.68 (s, 1H), 9.04 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.6 Hz, 1H), 7.47–7.36 (m, 2H), 7.30 (t, J = 7.6 Hz, 1H), 7.23–7.19 (m, 2H), 7.06 (t, J = 7.5 Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 6.90–6.84 (m, 2H), 5.79 (dd, J = 11.7 Hz, J = 4.2 Hz, 1H), 5.20–5.12 (m, 2H), 4.56 (d, J = 14.3 Hz, 1H), 4.45 (d, J = 14.3 Hz, 1H), 3.78 (dd, J = 18.5 Hz, J = 11.7 Hz, 1H), 3.16 (dd, J = 18.5 Hz, J = 4.2 Hz, 1H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.82, 164.65, 157.14, 154.73, 154.54, 135.84, 133.12, 132.10, 130.92, 130.70, 130.18, 129.63, 128.70, 128.34, 125.51, 123.33, 121.59, 121.00, 113.91, 112.49, 70.31, 66.51, 54.73, 44.75, 22.15; HRMS (AP-ESI) m/z [M+H]+ calcd for C26H25BrN3O5: 538.0978, found: 538.0982.
3-(2-((2-Bromobenzyl)oxy)phenyl)-5-(2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14dd)
White solid, yield: 56%, mp: 92–94 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.67 (s, 1H), 9.03 (s, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.43–7.27 (m, 3H), 7.22–7.17 (m, 2H), 7.04 (t, J = 7.5 Hz, 1H), 6.94–6.87 (m, 3H), 6.49 (s, 2H), 5.70 (dd, J = 11.8 Hz, J = 4.8 Hz, 1H), 5.19–5.11 (m, 2H), 4.57 (d, J = 14.4 Hz, 1H), 4.45 (d, J = 14.4 Hz, 1H), 3.75 (dd, J = 18.4 Hz, J = 11.8 Hz, 1H), 3.13 (dd, J = 18.4 Hz, J = 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.68, 156.92, 155.41, 154.33, 151.15, 135.87, 133.14, 131.64, 131.60, 130.94, 130.72, 129.63, 128.51, 128.36, 125.69, 123.38, 121.57, 121.48, 121.34, 113.79, 112.19, 70.28, 66.44, 54.46, 45.04; HRMS (AP-ESI) m/z [M+H]+ calcd for C25H24BrN4O5: 539.0930, found: 539.0934.
2-(2-(1-Acetyl-3-(2-((3-bromobenzyl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14ee)
White solid, yield: 57%, mp: 164–166 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.73 (s, 1H), 9.01 (s, 1H), 7.74 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.66 (d, J = 2.2 Hz, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.43 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 7.8 Hz, 1H), 7.23–7.18 (m, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.89 (d, J = 7.4 Hz, 1H), 5.79 (dd, J = 11.6 Hz, J = 4.2 Hz, 1H), 5.17 (s, 2H), 4.58 (d, J = 14.4 Hz, 1H), 4.50 (d, J = 14.4 Hz, 1H), 3.85 (dd, J = 18.4 Hz, J = 11.6 Hz, 1H), 3.17 (dd, J = 18.4 Hz, J = 4.2 Hz, 1H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.87, 164.78, 157.01, 154.69, 154.63, 139.89, 132.01, 131.18, 131.14, 130.75, 130.24, 129.69, 128.77, 127.07, 125.61, 122.11, 121.65, 121.49, 121.06, 114.03, 112.57, 69.40, 66.51, 54.74, 44.81, 22.20; HRMS (AP-ESI) m/z [M+H]+ calcd for C26H25BrN3O5: 538.0978, found: 538.0986.
3-(2-((3-Bromobenzyl)oxy)phenyl)-5-(2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14ff)
White solid, yield: 59%, mp: 120–122 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.74 (s, 1H), 8.98 (s, 1H), 7.87 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.62 (t, J = 1.8 Hz, 1H), 7.51–7.49 (m, 1H), 7.41–7.36 (m, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.22–7.14 (m, 2H), 7.03–6.88 (m, 4H), 6.46 (s, 2H), 5.70 (dd, J = 11.9 Hz, J = 4.8 Hz, 1H), 5.16 (s, 2H), 4.59 (d, J = 14.4 Hz, 1H), 4.50 (d, J = 14.4 Hz, 1H), 3.81 (dd, J = 18.3 Hz, J = 11.9 Hz, 1H), 3.14 (dd, J = 18.3 Hz, J = 4.8 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.79, 156.81, 155.46, 154.40, 151.18, 139.92, 131.72, 131.54, 131.18, 130.80, 129.64, 128.56, 127.10, 125.77, 122.10, 121.64, 121.41, 113.97, 112.28, 69.36, 66.45, 54.53, 45.19; HRMS (AP-ESI) m/z [M+H]+ calcd for C25H24BrN4O5: 539.0930, found: 539.0936.
2-(2-(1-Acetyl-3-(2-((4-bromobenzyl)oxy)phenyl)-4,5-dihydro-1H-pyrazol-5-yl)phenoxy)-N-hydroxyacetamide (14gg)
White solid, yield: 53%, mp: 96–98 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.75 (s, 1H), 9.02 (s, 1H), 7.74 (dd, J = 7.7 Hz, J = 1.8 Hz, 1H), 7.55–7.52 (m, 2H), 7.44–7.36 (m, 3H), 7.24–7.17 (m, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.3 Hz, 1H), 6.91–6.85 (m, 2H), 5.78 (dd, J = 11.6 Hz, J = 4.2 Hz, 1H), 5.13 (d, J = 1.8 Hz, 2H), 4.58 (d, J = 14.2 Hz, 1H), 4.50 (d, J = 14.2 Hz, 1H), 3.82 (dd, J = 18.4 Hz, J = 11.6 Hz, 1H), 3.18 (dd, J = 18.4 Hz, J = 4.2 Hz, 1H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6): δ 167.86, 164.78, 157.11, 154.95, 154.64, 136.45, 132.03, 131.84, 130.37, 130.23, 129.62, 128.74, 125.63, 121.64, 121.54, 121.44, 121.10, 114.01, 112.54, 69.57, 66.48, 54.85, 44.81, 22.19; HRMS (AP-ESI) m/z [M+H]+ calcd for C26H25BrN3O5: 538.0978, found: 538.0980.
3-(2-((4-Bromobenzyl)oxy)phenyl)-5-(2-(2-(hydroxyamino)-2-oxoethoxy)phenyl)-4,5-dihydro-1H-pyrazole-1-carboxamide (14hh)
White solid, yield: 55%, mp: 96–98 °C. 1H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 8.99 (s, 1H), 7.85 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.40–7.35 (m, 3H), 7.23–7.14 (m, 2H), 7.02–6.88 (m, 4H), 6.47 (s, 2H), 5.69 (dd, J = 11.9 Hz, J = 4.7 Hz, 1H), 5.16–5.09 (m, 2H), 4.59 (d, J = 14.4 Hz, 1H), 4.50 (d, J = 14.4 Hz, 1H), 3.80 (dd, J = 18.3 Hz, J = 11.9 Hz, 1H), 3.14 (dd, J = 18.3 Hz, J = 4.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6): δ 164.80, 156.86, 155.44, 154.42, 151.32, 136.53, 131.85, 131.68, 131.52, 130.36, 129.63, 128.54, 125.76, 121.61, 121.53, 121.44, 121.34, 113.95, 112.25, 69.51, 66.43, 54.58, 45.12; HRMS (AP-ESI) m/z [M+H]+ calcd for C25H24BrN4O5: 539.0930, found: 539.0938.

3.2. Biological Evaluation

3.2.1. Enzymatic Inhibition Assay against APN In Vitro

The enzymatic inhibitory activities of target compounds were evaluated using L-Leu-p-nitroanilide (Sigma-Aldrich, St. Louis, MO, USA) as substrate and soluble APN from porcine kidney microsomal (Enzo Life Sciences, Farmingdale, NY, USA) as enzyme. Firstly, the substrate was dissolved in DMSO to a concentration of 16 mmol/L and the enzyme was dissolved in buffer solution of 50 mM PBS (pH = 7.2) for a concentration of 0.15 IU/L. Subsequently, inhibitors (40 μL) and PBS (145 μL) were added into the 96-well plates, followed by the addition of substrate (5 μL) and enzyme (10 μL). After incubation at 37 °C for 30 min, the absorbance values were measured at 405 nm with a plate reader (Varioskan, Thermo Fisher Scientific, Waltham, MA, USA).

3.2.2. Anti-Proliferative Assay

The MTT method was used for the determination of anti-proliferative activities of selected compounds against tumor cells. Human histiocytic lymphoma cells U937, human chronic myeloid leukemia cells K562, liver carcinoma alexander cells PLC/PRF/5, human prostate cancer cells PC-3, human ovarian clear cell carcinoma cells ES-2, human hepatocellular carcinoma cells HepG2 were selected as tested tumor cell lines. Firstly, the cells were cultured in RPMI 1640 medium with 10% FBS at 37 °C in 5% CO2 humidified incubator. Cells (100 μL) in logarithmic phase were inoculated in 96-well plates and allowed to grow for 4 h. Subsequently, inhibitors (100 μL) at the tested concentration were added, followed by incubation for 48 h. Then, to the mixture was added MTT solution (20 μL, 5 mg/mL) and further incubated for 4 h. The plates were centrifuged at 800 rpm for 3 min. The supernatant was poured off and the formed formazan was dissolved in DMSO (200 μL). Finally, the mixture was shaken for 15 min and the absorbance values of the formazan solution were measured at 570 nm with a plate reader (Varioskan, Thermo, Waltham, MA, USA). All the tumor cells were owned by our laboratory.

3.2.3. Rat Aortic Ring Assay

The thoracic aortas were separated from 8- to 10-week-old male Sprague Dawley rats and cut into rings with a 1-mm-long cross-section after removal of the connective tissues around the aortas. The fresh aortic ring was added into the 96-well plates pre-coated with matrigel (50 μL, BD bioscience), and covered with another 50 μL of matrigel. After incubation at 37 °C in 5% CO2 for 0.5 h, the tested compounds (100 μL) in M199 culture medium with 10% FBS was added to the mixture and incubated at 37 °C in 5% CO2 for 9 d. For the treatment group, the M199 culture medium with 10% FBS and inhibitors was changed every three days. For the control group, the M199 culture medium with 10% FBS and 0.5% DMSO was changed every three days. The formed micro-vessels were photographed by inverted microscope at 100× magnification. Experiments were repeated three times. All experiments involving laboratory animals were performed with the approval of the Laboratory Animal Welfare Review Committee of Shandong University of Traditional Chinese Medicine.

3.3. Molecular Docking Studies

In the APN-bestatin co-crystal structure (PDB code: 2DQM), the residues in a 10.0 Å radius circle around bestatin was selected as the active site. Compound 14o was docked into it using Sybyl 2.1. Before the docking studies, the protein structure was prepared by deleting water molecules, adding hydrogen atoms, modifying atom types, and assigning with AMBER7 FF99 charges, followed by a 100-step minimization process. Using the Sybyl/Sketch module, the small molecular structures were prepared and further optimized using Powell’s method with the Tripos force field with convergence criterion set at 0.05 kcal/Å mol and assigned using the Gasteiger–Hückel method. Other docking parameters implied in the program were default values. Molecular docking was carried out via the Sybyl/Surflex-Dock module. The top-scoring pose was selected for discussions.

4. Conclusions

Summarily, one series of pyrazoline-based hydroxamate derivatives was designed, synthesized and evaluated as APNIs. Interestingly, in the enzymatic assay against APN, some compounds exhibited improved APN inhibitory activities relative to the lead compounds 13a–13c and positive control bestatin. Among them, the 2,6-dichloro substituted compound 14o (IC50 = 0.0062 ± 0.0004 μM) was the best one, with three orders of magnitude better APN inhibitory activity than bestatin. The SARs revealed that the substituents on the two phenyl groups of the diphenyl pyrazoline scaffold significantly impacted the capacities of inhibiting APN. Compounds with an ortho mono-substituent on the terminal phenyl group presented better APN inhibitory activities than their counterparts with a meta or para mono-substituent. Compounds with an ortho-substituent, such as 2-F (14g), 2-Cl (14h), 2-I (14i), 2-CH3 (14k), showed improved capacities of APN inhibition relative to the unsubstituted lead compound 13a. However, introduction of substituents on another phenyl group resulted in decreased APN inhibitory activities. The putative interactions of 14o with the active site of APN was predicted using Surflex-Dock module of Sybyl 2.1., which further supported our design strategy. Moreover, compared with bestatin, compound 14o demonstrated superior anti-proliferative activities against tumor cells and anti-angiogenic activity, which confirmed the potency of 14o as an anti-tumor lead.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules27238339/s1, the spectra data of some of intermediates 18, 19 and 20; the 1HNMR and 13C NMR spectra of target compounds 14h–14j, 14o, 14aa–14cc, 14ee and 14ff.

Author Contributions

Conceptualization, J.C.; methodology, Y.L. and D.Z.; validation, C.Z.; formal analysis, H.F.; investigation, Y.L.; writing—original draft preparation, Y.L. and J.C.; writing—review and editing, Q.S. and Z.W.; supervision, J.C.; funding acquisition, J.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Natural Science Foundation of Shandong Province (Grant No. ZR2022QH186), Medical Science and Technology Development Project of Shandong Province (Grant No. 202013051070) and Strengthening Foundation Plan for Young Teachers of School of Pharmacy (Grant No. 2021-0013, No. 2021-009). The APC was funded by the Natural Science Foundation of Shandong Province (Grant No. ZR2022QH186).

Institutional Review Board Statement

The animal study protocol was approved by the Laboratory Animal Welfare Review Committee of Shandong University of Traditional Chinese Medicine (protocol code: SDUTCM20220303013). The date of approval was 3 March 2022.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds 14i and 14o are available from the authors.

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Molecules 27 08339 g001 550
Figure 1. The structures of representative aminopeptidase N inhibitors (APNIs).
Figure 1. The structures of representative aminopeptidase N inhibitors (APNIs).
Molecules 27 08339 g001
Molecules 27 08339 g002 550
Figure 2. Design strategy of APNIs containing pyrazoline moiety.
Figure 2. Design strategy of APNIs containing pyrazoline moiety.
Molecules 27 08339 g002
Molecules 27 08339 sch001 550
Scheme 1. Reagents and conditions: (a) substituted acetophenone for 18a–18m, 18o–18s, 1-(naphthalen-1-yl)ethan-1-one for 18n, KOH, 80% EtOH, 25 °C, 48 h; (b) benzyl bromide or substituted benzyl bromide, NaH, DMF, 25 °C, 12 h; (c) for 18t–18w, 15a, KOH, 80% EtOH, 25 °C, 48 h; (d) R1 = CH3, acetic acid, 80% hydrazine hydrate, 130 °C, 5 h or R1 = NH2, semicarbazide hydrochloride, NaOH, EtOH, 78 °C, 5 h; (e) methyl bromoacetate, NaH, DMF, 25 °C, 12 h; (f) NH2OK, MeOH, 25 °C, 0.5 h.
Scheme 1. Reagents and conditions: (a) substituted acetophenone for 18a–18m, 18o–18s, 1-(naphthalen-1-yl)ethan-1-one for 18n, KOH, 80% EtOH, 25 °C, 48 h; (b) benzyl bromide or substituted benzyl bromide, NaH, DMF, 25 °C, 12 h; (c) for 18t–18w, 15a, KOH, 80% EtOH, 25 °C, 48 h; (d) R1 = CH3, acetic acid, 80% hydrazine hydrate, 130 °C, 5 h or R1 = NH2, semicarbazide hydrochloride, NaOH, EtOH, 78 °C, 5 h; (e) methyl bromoacetate, NaH, DMF, 25 °C, 12 h; (f) NH2OK, MeOH, 25 °C, 0.5 h.
Molecules 27 08339 sch001
Molecules 27 08339 g003 550
Figure 3. Representative images of the rat thoracic aorta rings treated with DMSO (0.5%) or compounds.
Figure 3. Representative images of the rat thoracic aorta rings treated with DMSO (0.5%) or compounds.
Molecules 27 08339 g003
Molecules 27 08339 g004 550
Figure 4. (A) The FlexX docking result of 14o in the active site of APN (PDB code: 2DQM). The carbon atoms are marked in gray, oxygen atoms in red, hydrogen atoms in light blue, nitrogen atoms in blue and chlorine atoms in green. The green sphere represents zinc ion. S1 and S2′ represent two hydrophobic pockets. (B) The docking result shown by LIGPLOT. The carbon atoms, oxygen atoms, nitrogen atoms and chlorine atoms are marked in black, red, blue and green, respectively. The backbone of compound 14o is labeled violet and the zinc ion is shown as green sphere. The hydrogen bonds are shown as dashed lines (distance in Å).
Figure 4. (A) The FlexX docking result of 14o in the active site of APN (PDB code: 2DQM). The carbon atoms are marked in gray, oxygen atoms in red, hydrogen atoms in light blue, nitrogen atoms in blue and chlorine atoms in green. The green sphere represents zinc ion. S1 and S2′ represent two hydrophobic pockets. (B) The docking result shown by LIGPLOT. The carbon atoms, oxygen atoms, nitrogen atoms and chlorine atoms are marked in black, red, blue and green, respectively. The backbone of compound 14o is labeled violet and the zinc ion is shown as green sphere. The hydrogen bonds are shown as dashed lines (distance in Å).
Molecules 27 08339 g004
Table
Table 1. The structures and IC50 values of target compounds against porcine kidney aminopeptidase N (APN).
Table 1. The structures and IC50 values of target compounds against porcine kidney aminopeptidase N (APN).
Molecules 27 08339 i001
CompdR1R7APN
IC50 (μM) a		CompdR1R7APN
IC50 (μM) a
13aCH3H0.32 ± 0.0114qCH3--3.5 ± 0.2
13bNH2H0.16 ± 0.0114rNH2--3.4 ± 0.3
13cNH22,6-diCl0.059 ± 0.00214sCH3--2.1 ± 0.2
14aCH32-Br0.046 ± 0.00314tNH2--1.6 ± 0.1
14bCH33-Br0.32 ± 0.0314uCH3--0.37 ± 0.02
14cCH34-Br4.4 ± 0.314vNH2--0.54 ± 0.04
14dCH32-OCH30.32 ± 0.0114wCH3--0.024 ± 0.001
14eCH33-OCH30.37 ± 00314xNH2--0.090 ± 0.005
14fCH34-OCH33.8 ± 0.214yCH3--0.023 ± 0.001
14gCH32-F0.25 ± 0.0114zNH2--0.070 ± 0.003
14hCH32-Cl0.029 ± 0.00214aaCH3Molecules 27 08339 i0020.017 ± 0.001
14iCH32-I0.015 ± 0.00114bbNH2Molecules 27 08339 i0030.015 ± 0.001
14jNH22-I0.044 ± 0.00314ccCH3Molecules 27 08339 i0040.068 ± 0.004
14kCH32-CH30.21 ± 0.0114ddNH2Molecules 27 08339 i0050.23 ± 0.01
14lCH32,4-diCl0.58 ± 0.0414eeCH3Molecules 27 08339 i0060.031 ± 0.002
14mCH32,6-diOCH30.20 ± 0.0114ffNH2Molecules 27 08339 i0070.058 ± 0.003
14nNH22,6-diOCH30.44 ± 0.0314ggCH3Molecules 27 08339 i0080.56 ± 0.04
14oCH32,6-diCl0.0062 ± 0.000414hhNH2Molecules 27 08339 i0090.55 ± 0.04
14p----0.31 ± 0.02Bestatin----8.8 ± 0.4
a Assays were performed in triplicate; data are shown as mean ± SD.
Table
Table 2. In vitro anti-proliferative activities of selected compounds against tumor cells.
Table 2. In vitro anti-proliferative activities of selected compounds against tumor cells.
CompoundIC50 (μM) a
U937K562PLC/PRF/5PC-3ES-2HepG2
14o38.3 ± 3.610.4 ± 1.288.5 ± 7.445.8 ± 4.1114.6 ± 9.784.2 ± 7.6
Bestatin>500>500>500>500>500>500
a Assays were performed in triplicate; data are shown as mean ± SD.
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Liu, Y.; Zhao, D.; Zhang, C.; Fang, H.; Shen, Q.; Wang, Z.; Cao, J. Development of Hydroxamate Derivatives Containing a Pyrazoline Moiety as APN Inhibitors to Overcome Angiogenesis. Molecules 2022, 27, 8339. https://doi.org/10.3390/molecules27238339

AMA Style

Liu Y, Zhao D, Zhang C, Fang H, Shen Q, Wang Z, Cao J. Development of Hydroxamate Derivatives Containing a Pyrazoline Moiety as APN Inhibitors to Overcome Angiogenesis. Molecules. 2022; 27(23):8339. https://doi.org/10.3390/molecules27238339

Chicago/Turabian Style

Liu, Yangyang, Dongsheng Zhao, Chenghua Zhang, Hui Fang, Qingsitong Shen, Zhixian Wang, and Jiangying Cao. 2022. "Development of Hydroxamate Derivatives Containing a Pyrazoline Moiety as APN Inhibitors to Overcome Angiogenesis" Molecules 27, no. 23: 8339. https://doi.org/10.3390/molecules27238339

APA Style

Liu, Y., Zhao, D., Zhang, C., Fang, H., Shen, Q., Wang, Z., & Cao, J. (2022). Development of Hydroxamate Derivatives Containing a Pyrazoline Moiety as APN Inhibitors to Overcome Angiogenesis. Molecules, 27(23), 8339. https://doi.org/10.3390/molecules27238339

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