Design and Synthesis of sEH/HDAC6 Dual-Targeting Inhibitors for the Treatment of Inflammatory Pain

Huashen Xu; Yuanguang Chen; Hua Tong; Lu Chen; Christophe Morisseau; Zijian Zhou; Junning Zhuang; Chuqiao Song; Pengcheng Cai; Zhongbo Liu; Bruce D Hammock; Guoliang Chen

doi:10.1021/acs.jmedchem.4c00847

. Author manuscript; available in PMC: 2025 Aug 8.

Published in final edited form as: J Med Chem. 2024 Jul 21;67(15):12887–12911. doi: 10.1021/acs.jmedchem.4c00847

Design and Synthesis of sEH/HDAC6 Dual-Targeting Inhibitors for the Treatment of Inflammatory Pain.

Huashen Xu ^a,^†, Yuanguang Chen ^a,^†, Hua Tong ^d, Lu Chen ^a, Christophe Morisseau ^b, Zijian Zhou ^d, Junning Zhuang ^a, Chuqiao Song ^a, Pengcheng Cai ^a, Zhongbo Liu ^c,^*, Bruce D Hammock ^b,^*, Guoliang Chen ^a,^*

^aKey Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China

^bDepartment of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, USA

^cSchool of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, China

^dLiaoning Key Laboratory of Targeting Drugs for Hematological Malignancies, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China

^†

Author Contributions

Huashen Xu and Yuanguang Chen made equal contributions to this work.

Authors

Huashen Xu - Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China

Yuanguang Chen - Key Laboratory of Structure-Based Drug Design & Discovery ofMinistry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China

Hua Tong - Liaoning Key Laboratory of Targeting Drugs for Hematological Malignancies, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.

Lu Chen - Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China.

Christophe Morisseau - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, USA.

Zijian Zhou - Liaoning Key Laboratory of Targeting Drugs for Hematological Malignancies, Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.

Junning Zhuang - Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China.

Chuqiao Song - Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China.

Pengcheng Cai - Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China.

Corresponding Author Zhongbo Liu - School of Pharmacy, Shenyang Pharmaceutical University, Shenyang 110016, PR China.; 546265581@qq.com.; Bruce D Hammock - Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, USA, Fax: 530-752-1537, bdhammock@ucdavis.edu.; Guoliang Chen - Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China; chenguoliang@syphu.edu.cn.

PMCID: PMC11412156 NIHMSID: NIHMS2016629 PMID: 39033411

Abstract

sEH and HDAC6 mediate the NF-κB pathway in inflammatory responses, and their inhibitors exhibit powerful anti-inflammatory and analgesic activities in treating both inflammation and pain. Therefore, a series of dual-targeting inhibitors containing urea or squaramide and hydroxamic acid moieties were designed and synthesized, and their role as a new sEH/HDAC6 dual-targeting inhibitor in the inflammatory pain was evaluated in formalin-induced mice model and the xylene-induced mouse ear swelling model. Among them, the compounds (28g and 28j) showed the best inhibitory and selectivity of sEH and HDAC6. The compound 28g had satisfactory pharmacokinetic characteristics in rat. Following administration at 30 mg/kg, compound 28g exhibited more effective analgesic activity than that of either a sEH inhibitor (GL-B437) or a HDAC6 inhibitor (Rocilinostat) alone, and that of co-administration of both inhibitors. Thus, these novel sEH/HDAC6 dual-targeting inhibitors exhibited powerful analgesic activity in a nociceptive behavior and are worthy of further development.

INTRODUCTION

Inflammation encompasses multiple physiological and pathological processes, triggered by converging pathways that generate a network of pro-inflammatory and pro-resolving mediators, involving different cell types and cellular responses.¹ The arachidonic acid (AA) cascade is a key biochemical pathway for pharmacologically targeting inflammatory diseases. AA cascade pathways play a key role in cardiovascular disease, cancer, and inflammatory processes associated with pain, asthma, and arthritis. ^2,3 AA is metabolized through three main enzyme-catalyzed pathways: cyclooxygenases (COXs), lipoxygenerases (LOXs) and cytochrome P450 (CYP) enzymes. The CYP pathway leads to the formation of 20-hydroxyeicosatetranoic acid (20-HETE)⁴ and arachidonic acid monoepoxides known as epoxy-eicosatrienoic acids (EETs).⁵ The soluble epoxide hydrolase (sEH) catalyzes the conversion of these EETs into the corresponding diols, or dihydroxyeicosatrienoic acids (DHETs). EETs are known to exhibit a variety of biological activities such as vasodilatory,⁶ cardioprotective,⁷ anti-inflammatory⁸ and antihyperalgesic⁹ properties, while the DHETs have inflammatory activities in most assays.¹⁰ Numerous studies have shown that stabilizing EETs by suppressing sEH can significantly reduce inflammation and pain.¹¹ Therefore, sEH inhibitors are considered a promising and effective method of treating a variety of diseases.

t-AUCB (Figure 1) shows significant anti-inflammatory activity as well as protective effects on the liver and cardiovascular system.¹² EC5026 (Figure 1) is a small-molecular candidate drug and is a very effective simulated inhibitor of sEH enzyme transition state.¹³ EC5026 suppresses neural inflammation of the central nervous system in mouse models induced by lipid polysaccharide.¹⁴ AR-9281, a potent and selective sEH inhibitor, was evaluated in a phase IIa for hypertension and improving insulin resistance in pre-diabetic patients.¹⁵

Multiple HDACs are highly expressed in inflammatory-related diseases such as HDAC1, HDAC2 and HDAC6. Compared with other HDAC subtypes, HDAC6 exhibits unique characteristics. HDAC6 can directly engage with a host of cytosolic proteins and substrates, including α- and β-tubulin, assembled microtubules, cortactin, and heat shock proteins.^16–18 Accumulated studies have demonstrated that HDAC6 is involved in various human diseases, such as tumors, nervous dysfunction, inflammation, and so on.^19–21 In the past two years, researchers have found that HDAC6 played an important role in assembling and activating the NLRP3 inflammasome in various human disorders. Rocilinostat is a selective HDAC6 inhibitor and is considered a potential anti-inflammatory drug.²² In IL-1β-induced osteoarthritis models, Rocilinostat (Figure 1) could prevent p65 from being phosphoridized to the cell nucleus, thus inhibiting the expression of inflammatory cell factors (IL-6, IL-1b, TNF-α, IL-17) by inhibition of the NF-κB pathway.²³ HDAC6 inhibitor CAY10603 (Figure 1) prevents LPS-induced pulmonary microtubular deacetylation, reducing the production of inflammatory cell factors TNF-α, IL-1β, and IL-6, and reducing white cell immersion. HDAC6 inhibitors block the activation of NF-κB by inhibiting IκB phosphoridation in LPS-induced acute lung injury, suggesting that selectively HDAC6 inhibits inflammatory signaling pathways and reduces LPS-induced acute lung injury..²⁴ At present, HDAC inhibitors are only approved as anti-tumor agents in clinic due to their adverse effects, but, highly selective HDAC6 inhibitors are believed to hardly have adverse effects.²⁵

sEH and HDAC6 affect the NF-κB signaling pathway together, and then regulate transcription of downstream pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-17). (Figure 2). Therefore, multiple ligands (DMLs) targeting sEH and HDAC6 are designed. The aims of DMLs are to enhance drug efficacy and improve drug safety by acting specifically on multiple targets (“targeted polypharmacology”), as opposed to drugs that address only a single target. DMLs have advantages over combination drugs or combination therapies because they circumvent the inherent problems associated with formulation of two or more drugs used for co-administration. To date, there is no example of a sEH/HDAC6 dual-targeting inhibitor reported in literatures. Therefore, we sought to develop a series of novel derivatives with conjugates of urea or squaramides and hydroxamic acid having both sEH inhibitory activities and HDAC6 selective inhibitory effect as a new class of DML.

Figure 2. — Role of sEH and HDAC6 inhibitors in inflammation.

RESULTS AND DISCUSSION

Rational Design of the sEH/HDAC Dual-targeting Inhibitors.

Besides conditioning therapeutic efficacy, the selected targets to be hit by a multi-targeting drug also drive its design, which is usually performed by joining with a linker, merging or overlapping in two or more pharmacophores. To hit targets with binding regions that are buried deep inside the protein, the most useful design approach is the linked-pharmacophore strategy.²⁶ Such proteins contain the main binding site at the end of a cavity and a secondary or peripheral site at the cavity entrance. In those cases, one scaffold is selected for each target to interact with the main binding site, whereas the second scaffold might interact with the peripheral site, provided that the linker that joins the two moieties affords the appropriate geometry and distance. The resulting dual-site binding usually leads to increased potencies as an additional benefit besides modulating two different targets.²⁷ Both sEH and HDAC6 belong to this type of protein. The active site of sEH is buried inside the protein core pocket, with the total length of the sEH active site is up to 25 Å (Figure 3A) (PDB ID: 3WKE).^28,29 The end of the left active cavity pocket is in the solvent area, which provides a theoretical basis for connecting another target. The Zn²⁺ catalytic site of HDAC6 is buried at the bottom of a narrow gorge of 10 Å depth. And there is an open groove with a width of about 14 Å at the entrance in the solvent area (Figure 3B) (PDB ID: 5WGL),³⁰ which also provides a theoretical basis for connecting another target.

Figure 3. — Active site cavities of sEH (PDB ID: 3WKE) (A) and HDAC6 (PDB ID: 5WGL) (B).

The general pharmacophore of HDAC inhibitors consists of three parts: Zinc-binding group (ZBG) that chelates and catalyzes zinc ions, hydrophobic linker that occupies the channel of active site, and surface recognition module (CAP) that interacts with amino acid residues around the entrance of active site.³¹ Most reported HDAC6 inhibitors have hydroxamic acid as ZBG, such as Rocilinostat and ACY10603 (Figure 1). Among them, Rocilinostat is in the phase II clinical trials for the treatment of neuropathic pain caused by diabetes. The N, N’-disubstituted ureas as well as their corresponding amides and carbamates have been thoroughly studied as sEH inhibitors.³² In the field of contemporary medicinal chemistry, bioisosteres are often used to improve selectivity and efficacy, and to improve absorption, metabolism and toxicity (ADMET) characteristics. Squaramides represent a class of vinylogous amides that are derived from the squarate oxocarbon dianion.³³ Because of their similar capacity toward hydrogen bonding and ability to reliably engender defined conformations in drug ligands, squaramides are ideal candidates for bioisosteric replacements of several heteroatomic functional groups, notably ureas, thioureas, guanidines and cyanoguanidines.³⁴ Therefore, we intend to develop the conjugates of urea or squaramides and hydroxamic acid having both sEH and HDAC6 selective inhibitory activities as a new class of DML. Taking the sEH inhibitor GL-B437 discovered by our research group³⁵ and the highly selective HDAC6 inhibitor Rocilinostat as the lead compounds, we designed a series of novel sEH/HDAC6 dual-targeting inhibitors containing urea or squaramides and hydroxamic acid structures by using the combination principles. As shown in Figure 4.

Figure 4. — Design principle of sEH/HDAC6 dual-targeting compounds.

Chemistry.

The synthetic route of compounds 7a-7d was shown in Scheme 1. Intermediate 2 was obtained by acylation of 3-fluoro-4-nitrobenzoic acid 1, and further reduced to intermediate 3 in the presence of Fe powder and NH₄Cl. Intermediate 3 was reacted with memantine to obtain intermediate 4. Intermediate 4 was hydrolyzed under alkaline conditions to give intermediate 5. Intermediate 5 was reacted with small molecular amine to produce amide intermediates 6a-6d. In the presence of hydroxylamine aqueous solution, intermediates 6a-6d were aminated to give compounds 7a-7d.

The synthetic route of compounds 16a-16d was depicted in Scheme 2. Intermediate 9 was obtained from 3-fluoro-4-nitrobenzoic acid 8 by borane reduction and was nucleophilically substituted by phosphorus tribromides to produce intermediate 10. Then intermediate 10 was nucleophilically substituted with ethyl (S)-3-piperidinecarboxylate to give intermediate 11. Subsequently, compounds 16a-16d were synthesized according to the method of synthesizing compounds 7a-7d.

The synthetic route of compounds 16a-16d was shown in Scheme 3. With 17a-17c as the raw material, the compound 23a-23c was obtained according to the synthesis method of Scheme 1.

The synthetic route of compounds 28a-28k was shown in Scheme 4. With 24 as the starting material, the compound 28a-28k was obtained according to the synthesis method of Scheme 1.

The synthetic route of compounds 34a-34d was shown in Scheme 5. The intermediate 30 was obtained by nucleophilic substitution reaction between compound 29 and diethyl squarate. Intermediate 30 and memantine undergo nucleophilic substitution reaction to give intermediate 31 and was further hydrolyzed to produce intermediate 32. Then intermediate 32 was acylated with small molecular amine to obtain intermediates 33a-33d. Aminolysis of intermediates 33a-33d with hydroxylamine aqueous solution gave compounds 34a-34d.

The synthetic route of compounds 40a-40d was depicted in Scheme 6. With 35 as the raw material, the compound 40a-40k was obtained according to the synthesis method of Scheme 5.

The structure-activity relationships of series derivatives.

The activities of target compounds against HsEH and MsEH were evaluated according to the method reported in literature with AR-9281 and GL-B437 as control.³³ The results were shown on Table 1 and Table 2. The compounds containing urea structure had good inhibitory effects on both HsEH and MsEH. Except for compound 28k, the IC₅₀ values of other compounds were at the nanomolar level. Compounds 16d, 28a and 28b had the strongest inhibitory activity on HsEH, with IC₅₀ of 0.2 nM.

Table 1.

In Vitro Biological Activities of the sEH/HDAC6 Dual-targeting Inhibitors toward HsEH and MsEH^a


Compound	R₁	R₂	R₃	n	HsEH IC₅₀ (nM)	MsEH IC₅₀ (nM)
7a		F	-CO-	3	0.3	1.5
7b		F	-CO-	4	0.7	1.3
7c		F	-CO-	5	0.4	0.6
7d		F	-CO-	6	0.3	1.0
16a		F	-CH₂-	3	1.0	0.8
16b		F	-CH₂-	4	2.6	2.2
16c		F	-CH₂-	5	0.7	2.5
16d		F	-CH₂-	6	0.2	0.7
23a		Cl	-CO-	6	0.5	1.7
23b		CH₃	-CO-	6	0.3	1.0
23c		H	-CO-	6	0.3	2.5
28a		F	-CO-	6	0.2	2.9
28b		F	-CO-	6	0.2	6.0
28c		F	-CO-	6	0.3	0.9
28d		F	-CO-	6	3.7	0.7
28e		F	-CO-	6	0.7	5.4
28f		F	-CO-	6	0.4	8.9
28g		F	-CO-	6	0.9	46.8
28h		F	-CO-	6	0.7	7.4
28i		F	-CO-	6	1.2	7.7
28j		F	-CO-	6	3.6	102.6
28k		F	-CO-	6	10.2	3057.7
AR-9281					13.8	1.7
GL-B437					0.06

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IC₅₀ values were recorded on recombinant HsEH and MsEH using PHOME as substrate at 50 µM concentration. IC₅₀ values are the mean of at least three experiments. Standard deviation is < 10 % of the IC₅₀.

Table 2.

In Vitro Biological Activities of the sEH/HDAC6 Dual-targeting Inhibitors toward HsEH and MsEH^a


Compound	R₄	R₅	n	HsEH IC₅₀ (nM)	MsEH IC₅₀ (nM)
34a	F	-CH₂-	3	37.7	2302.0
34b	F	-CH₂-	4	88.4	1286.5
34c	F	-CH₂-	5	157.3	1229.8
34d	F	-CH₂-	6	72.04	9795.4
40a	H	-CO-	3	269.2	21329.2
40b	H	-CO-	4	103.6	4518.8
40c	H	-CO-	5	409.5	16129.9
40d	H	-CO-	6	140.1	23154.0
AR-9281				13.8	1.7
GL-B437				0.06

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Through the structure-activity relationship analysis of Table 1, the effect of carbon chain length on the activity of compounds was investigated. When R₁ was substituted by memantine, there was no significant difference in the inhibitory effects of compounds 7a-7d on HsEH and MsEH with the extension of carbon chain. However, when R₃ was -CH₂- moiety, the inhibitory effects of compounds 16a-16d on HsEH were increased slowly (except compound 16b) with the extension of carbon chain. And when the carbon chain length was 6, the inhibitory activity of compound 16d on both HsEH and MsEH was reached the best (HsEH IC₅₀ = 0.2 nM, MsEH IC₅₀ = 0.7 nM). The effects of the electronegativity of R₂ in compounds 7d, 23a-23c on the inhibitory activities of HsEH and MsEH were investigated. The results showed that the electronegativity of substituent R₂ had little effect on the inhibitory activities of HsEH and MsEH. When R₁ was substituted by aromatic amine, the structure-activity relationship of compounds 28a-28k were analyzed. When the 4-position of aromatic amine was substituted by -OCF₃ moiety, the compound had the best inhibitory effect on HsEH (28a-28c). When the 4-position of aromatic amine was substituted by F atom, the inhibitory effects of the compounds on HsEH were decreased with the decrease of electronegativity of the 3-position substituent (HsEH IC₅₀: 28i > 28e > 28f). On the contrary, when the 3-position of aromatic amine was substituted by F atom, the inhibitory effects of the compound on HsEH and MsEH were decreased with the increase of electronegativity of the 4-position substituent (HsEH and MsEH IC₅₀: 28g > 28h). When the 4-position of aromatic amine was not substituted, the inhibitory effects of the compound on HsEH and MsEH were significantly reduced (28j; HsEH IC₅₀ = 3.6 nM, MsEH IC₅₀ = 102.6 nM). In order to investigate the effect of steric hindrance of R₁ on HsEH and MsEH, it was found that the inhibition effects of compounds on HsEH and MsEH decreased significantly with the increase of steric hindrance of R₁ (28k; HsEH IC₅₀ = 10.2 nM, MsEH IC₅₀ = 3057.7 nM). On Table 2, the urea moiety was replaced by the squaramide moiety by bioisosteres strategy. The results showed that the inhibitory effects of the compounds on HsEH and MsEH were significantly reduced (34a-34d, 40a-40d). And when R₅ was substituted by -CO- moiety, the inhibitory activities of the compounds on HsEH and MsEH were weaker than that of the corresponding -CH₂- moiety.

The inhibitory rate of the target compounds on HDAC1, 3 and 6 at the final concentration of 2 μM was preliminarily screened with Vorinostat as control. The inhibitory activities of the compounds were shown in Table S1 and Table S2 of supporting information. Through the structure-activity relationship analysis, it could be seen from Table S1 that when R₁ was substituted by memantine, the inhibitory activities of the compounds on HDAC6 were generally weak (7a-7d, 16a-16d, 23a-23c: HDAC6; inhibitor rate: 7%- 74%). And when R₃ was substituted by -CH₂- moiety, the inhibitory activities of the compounds on HDAC6 were weaker than that of the corresponding -CO- moiety. It was worth noting that among these compounds, when the number of carbon atoms was 6, the compounds had the best inhibitory effects on HDAC6 (7d inhibitor rate: 63%; 16d inhibitor rate: 63%). Therefore, when R₃ was substituted by the carbonyl moiety and the number of carbon atoms was 6, the inhibitor effects of R₁ on HDAC6 were investigated. From the activity results of 28a-28k, compounds 28a, 28g, 28h and 28j had the highest inhibitory effects on HDAC6 when the 3-position of aromatic amine was replaced by F atom (inhibitor rate > 91%). Among them, compounds 28g and 28j had the best inhibitory activities on HDAC6 (28g, inhibitor rate = 99.78%; 28j, inhibitor rate = 98.92%). In order to investigate the inhibition effects of steric hindrance of R₁ on HDAC6, it was found that the inhibitory effects of compounds on HDAC6 were significantly decreased with increasing steric hindrance of R₁ (28k, inhibitor rate = 63.58%). Based on the data in Table S2, the structure-activity relationship was analyzed, and it was found that the compounds containing memantine scaffold with urea moiety substituted by squaramide and R₅ substituted by -CH₂- moiety had weak inhibition effects on HDAC6. However, the compounds containing memantine scaffold with R₅ substituted by -CO- moiety were significantly improved the inhibition effect of HDAC6. Among them, compounds 40c and 40d had the best inhibitory effect on HDAC6 (40c, inhibitor rate = 98.49%; 40d, inhibitor rate = 99.66%). Finally, four compounds (28g, 28j, 40c, 40d) with the highest HDAC6 inhibition rate were selected for IC₅₀ test. Results as shown in Table 3, Compounds 28g and 28j showed significant HDAC6 inhibitory activity (IC₅₀=8 nM), which was stronger than the positive control drug Vorinostat. Compounds 40c and 40d also showed good HDAC6 inhibitory activity (IC₅₀=0.282 μM, 0.162 μM respectively). 28g and 28j had significant selectivity on the inhibitory activity of HDAC6 (Selectivity Index; 28g: HDAC1/HDAC6=144, HDAC2/HDAC6=62, HDAC3/HDAC6=160, HDAC8/HDAC6=8, HDAC11/HDAC6=58; 28j: HDAC1/HDAC6=129, HDAC2/HDAC6=134, HDAC3/HDAC6=327, HDAC8/HDAC6=9, HDAC11/HDAC6=132, respectively).

Table 3.

IC₅₀ Values of Compounds 28g, 28j, 40c, 40d toward HDAC^a

	IC₅₀ (μM)
Compounds	HDAC1	HDAC2	HDAC3	HDAC6	HDAC8	HDAC11
28g	1.151	0.492	1.276	0.008	0.062	0.464
28j	1.032	1.074	2.616	0.008	0.072	1.055
40c	1.032	3.095	4.152	0.282	0.520	0.084
40d	0.415	2.526	1.451	0.162	0.158	0.241
Vorinostat	0.055	0.136	0.047	0.014	-	>10
Rocilinostat	0.681	0.451	0.059	0.003	0.106	>1

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IC₅₀ values were the mean of at least three experiments, -: means no detection. Standard deviation was < 10 % of the IC₅₀.

Combined with the results of sEH and HDAC enzyme activities inhibition, when R₁ was substituted by memantine, the compounds with urea moiety had good inhibition effects on both HsEH and MsEH, but the inhibition effects on HDAC6 were poor. When R₁ was substituted by aromatic amine, the compounds with urea moiety still had good inhibitory effects on both HsEH and MsEH, and also showed excellent inhibitory effects on HDAC6. Then, the influence of steric hindrance of R₁ on the inhibition effects of HsEH, MsEH and HDAC6 was investigated. It was found that steric hindrance was not conducive to the inhibition effects of compounds on HsEH, MsEH and HDAC6. The compounds with squaramide moiety had poor inhibitory effects on HsEH and MsEH. At the same time, when R₅ was substituted by -CH₂- moiety, the compounds had weak inhibition on HDAC6. When R₅ was substituted by -CO- moiety, the inhibitory effects of the compounds on HDAC6 were significantly improved.

Acetylation of α-Tubulin and Histone H3.

Compound-28g, 28j, 40c and 40d-induced acetylation of α-tubulin and histone H3 was analyzed in THP-1 cell after incubation with 1 μM and 2 μM for 24 h. Results of the Western blot were shown in Figure 5. Vorinostat (pan-inhibitor) and Rocilinostat (HDAC6 selective HDACi) were used as controls. In THP-1 cell lines, 28g induced an increase in acetylation of α-tubulin without affecting histone H3, indicating the selective inhibition of HDAC6. The effects were more pronounced in THP-1 cell and were similar to the HDAC6 selective effect in the cellular environment.

Figure 5. — THP-1 cells were treated with indicated concentration(μM) of **28g**, **28j**, **40c**, **40d**, **Rocilinostat, Vorinostat** for 24 h, the relative levels of each protein were analyzed with western blotting using specific antibodies.

Molecular docking.

In the predicted binding complexes of the best bioactive compounds 28g and sEH (Figure 6A, B), the modified urea group formed two hydrogen bonds with TYR-383 and TYR-466, respectively, and the 3-carbonyl group of piperidine formed additional hydrogen bonds with SER-415. The link part and hydroxamic acid part in 28g structure extended into the solvent region, which would not significantly interfere with the sEH-28g complex, and was beneficial to the stability and effectiveness of 28g frame. At the same time, the binding posture of compound 28g in the cavity of sEH protein was consistent with that of lead compound GL-B437 and eutectic compound TUCB, and it was well accommodated in the cavity of sEH protein. In the predicted binding complexes of 28g and HDAC6, the urea moiety in 28g structure formed hydrogen bonds with ASP-460 and HIS-462, respectively, and the 3-position amide N atom of piperidine formed hydrogen bonds with SER-531. It was worthy of mentioning that the structure of hydroxamic acid formed key hydrogen bonds with HIS-573, HIS-574, TYR-745 and zinc ions, among which the chelation with zinc ions played a crucial role (Figure 6C, D). The binding posture of compound 28g in the cavity of HDAC6 protein was consistent with that of lead compound Rocilinostat and was well accommodated in the cavity of HDAC6 protein. Thus, these computational and experimental studies confirmed that the sEH/HDAC6 dual-targeting inhibitor 28g occupied the whole long cavities of both enzymes, with the original pharmacophore interacting at the main binding site, the linker helping to span the cavity, and the second pharmacophore contributing to additional interactions at a known or at a so far unknown secondary or peripheral site. At the same time, in order to verify the selectivity of compound 28g to HDAC6 from the side, we simulated the molecular docking of compound 28g with HDAC1 and HDAC3 proteins respectively. Although the crystal structure of HDAC1 has not been validated until now, its homology to HDAC1 was highly consistent with that to human HDAC2³⁶. Therefore, human HDAC2 was preferentially selected to model the 3D structure of HDAC1. and found that compound 28g could not enter the cavity of the active site of HDAC1 protein. At the same time, in the docking simulation of 28g to HDAC3 protein, 28g had no key chelation with zinc ion, and had less hydrogen bond interaction with other amino acid residues (Figure 7). Compound 28g had the selectivity of HDAC6 by the result of molecular docking simulation.

Figure 7. — (A) Predicted complex of **28g** (pink) to HDAC1. human HDAC2 (PDB ID: 4LXZ) was selected to model the 3D structure of HDAC1. (B) 2D diagram of predicted complex of **28g** (pink) to HDAC1. (C) Predicted complex of **28g** (pink) to HDAC3 (PDB ID: 4A69). (D) 2D diagram of predicted complex of **28g** to HDAC3.

Molecular dynamics simulation.

To assess the stability of binding mode of 28g with sEH and HDAC6 from molecular docking, MD simulations were performed. During the whole MD simulations, the root-mean-square deviation (RMSD) of compound 28g, and protein–ligand contacts were recorded in Figure 8 and Figure 9. In Figure 8, the results indicated that the compound 28g had good stability and mode of interaction with sEH apo protein. Crucial amino acids involved in molecular interactions of 28g and sEH protein during the MD simulation were also summarized. As shown in Figure 8B, the intermediate benzene moiety of 28g formed π-π stacking interaction with Phe267 and Trp336, which accounted for 29% and 69% during the whole MD simulations. The urea moiety interacted with Asp335, Tyr383 and Tyr466 through H-bond interactions, which accounted for 70%, 94% and 28% during the whole MD simulations, respectively. Some amino acids formed water bridge interactions with 28g, such as Tyr343 and Gln384.

Figure 8. — MD simulations analyses of **28g** in complex with sEH. (A) RMSD plots of compound **28g** (yellow). (B) Protein-ligand contacts of 3WKE/**28g** complex.

In Figure 9, the results indicated that the compound 28g had good stability and mode of interaction with HDAC6 apo protein. Crucial amino acids involved in molecular interactions of 28g and HDAC6 protein during the MD simulation were also summarized. As shown in Figure 9B, the hydroxamic acid moiety of 28g interacted with Asp612, Asp705, His614, Glu742, His573 and Tyr745 through ionic (salt bridge), H-bond and water bridge interactions, which accounted for 100%, 100%, 100%, 98%, 94% and 61% during the whole MD simulations, respectively.

In vitro metabolic stabilities of the sEH/HDAC6 dual-targeting inhibitors in RLM.

Based on the results of sEH and HDAC6 enzyme activity inhibition, we next assessed the in vitro metabolic stability of 16d, 28g, 28j and 34d in rat liver microsomes (RLM). The results were shown in Table4. The introduction of squaramide slightly improved the stability of liver microsomal enzymes (16d: 48.99 min vs 34d: 73.36 min). The half-life of compound 28g in rat liver microsomal enzyme was 136.75 min, and its metabolic stability was better than that of 16d, 28j and 34d (48.99 min, 56.35min and 73.36 min respectively).

Table 4.

In Vitro Metabolic Stabilities of 16d, 28g, 28j and 34d in RLM

	0	10	30	45	60	t_1/2(min)
16d	100.00	89.16	79.33	52.13	46.62	48.99
28g	100.00	91.61	83.80	78.76	70.64	136.75
28j	100.00	85.62	73.00	59.75	50.47	56.35
34d	100.00	82.82	73.29	66.27	50.51	73.36

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Plasma protein binding rate.

The plasma protein binding was measured by a classical equilibrium dialysis device. Compound 28g and 28j exhibited moderate plasma protein binding rates (28g = 88.71%, 28j = 88.12%).

Pharmacokinetic study in vivo.

Pharmacokinetic parameters of compounds 28g and 28j in rats were evaluated by oral (ig: 50 mg/kg) and intravenous (iv: 10 mg/kg) administration at a single dose, blood levels were determined for 12 h. The results were shown in Table5. The maximum concentration of 28g (6.63 μM) was reached at 0.5 h after oral administration; the area under the curve (AUC_0−12h) was 15.01 μM·h; 28g had a plasma t_1/2 of 3.55 h. The maximum concentration of 28g (7.87 μM) was reached at 0.08 h after iv administration; the area under the curve (AUC_0−12h) was 12.29 μM·h; 28g had a plasma t_1/2 of 5.43 h. It showed that compound 28g exhibited median bioavailability (F = 24.42 %).

Table 5.

Pharmacokinetics of 28g and 28j in Rats Following Intravenous and Oral Administration^a

Parameter	28g		28j
	iv (10 mg/kg) (n = 4)	ig (50 mg/kg) (n = 4)	iv (10 mg/kg) (n = 4)	ig (50 mg/kg) (n = 4)
T_max (h)	0.08	0.50	0.08	0.17
C_max (μM)	7.87	6.63	8.10	10.10
t_1/2 (h)	5.43	3.55	4.08	2.84
CL (L/h)	0.16	0.82	0.18	0.96
V_z (L)	1.28	4.20	1.10	3.95
AUC _(0–12h) (μM·h)	12.29	15.01	13.76	14.82
AUC _(0−∞) (μM·h)	21.16	21.22	20.03	18.67
F (%)	24.42		21.54

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Species: SD rats; after administration, blood samples were collected at different times (iv: 5, 15, 30, 60, 120, 240, 480, and 720 min; ig: 5, 15, 30, 60, 120, 240, 480, and 720 min); compounds formulated in 0.5% CMC-Na solution were administered orally at the indicated doses; the combination of compounds in a ratio of 5% DMSO, 5% Tween-80, and 90% normal saline was chosen for the intravenous injection formulation.

The maximum concentration of 28j (10.10 μM) was reached at 0.17 h after oral administration; the area under the curve (AUC_0−12h) was 14.82 μM·h; 28j had a plasma t_1/2 of 2.84 h. The maximum concentration of 28j (8.10 μM) was reached at 0.08 h after iv administration; the area under the curve (AUC_0−12h) was 13.76 μM·h; 28j had a plasma t_1/2 of 4.08 h. It showed that compound 28j exhibited similar bioavailability (F = 21.54 %).

The inhibitory effects of 28g in mice with xylene-induced ear swelling inflammation.

According to the pharmacokinetic results and safety evaluation (Figure S1 of supporting information) of compounds 28g and 28j, we selected compound 28g to further evaluate the inhibitory effect with xylene-induced ear swelling inflammation model. In order to verify that sEH inhibitor and HDAC6 inhibitor have synergistic inhibitory effect against xylene-induced ear swelling inflammation pain, the inhibitory effects of 28g, GL-B437, Rocilinostat and GL-B437 + Rocilinostat against xylene-induced ear swelling inflammation in mice were shown in Table 6 and Figure 10. The co-administration of GL-B437 and Rocilinostat exhibited a synergistic effect in relieving the inflammation of xylene-induced ear swelling. It is worth noting that 28g exhibited more effective anti-inflammatory activity than that of either a sEH inhibitor (GL-B437) or a HDAC6 inhibitor (Rocilinostat) alone, and that of co-administration of both inhibitors.

Table 6.

The Inhibitory Effects of 28g, GL-B437, Rocilinostat and GL-B437 + Rocilinostat against Xylene-induced Ear Swelling Inflammation in Mice

Groups	Dose (mg/kg)	Swelling degree (mg)	Swelling rate (%)	Inhibition rate (%)
Normal saline		13.3±1.3	105.05
28g	30mg/kg	5.1±1.1	37.76	62.05
GL-B437	30 mg/kg	7.0±1.8	50.76	47.19
Rocilinostat	30 mg/kg	10.0±1.4	78.42	24.97
GL-B437+Rocilinostat	30 mg/kg+30 mg/kg	5.7±1.2	45.18	56.93

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Figure 10. — The inhibitory effects of **28g**, **GL-B437**, **Rocilinostat** and **GL-B437** + **Rocilinostat** against xylene-induced ear swelling inflammation in mice. Significance: ****P < 0.0001 compared with the blank group.

In order to better evaluate the inhibitory effect of compound 28g against ear swelling inflammation, we compared the inhibitory degree of different doses of 28g and the anti-inflammatory drug Celecoxib against ear swelling inflammtory in mice. As shown in Table 7, the high dose group of 28g showed excellent inhibitory effect, and the inhibitory rate was as high as 67.17%. According to Figure 11, the degree of ear swelling in the high-dose 28g group was lower than that in the low-dose 28g group and the Celecoxib group, and the inhibition rate of ear swelling was much higher than that in other groups, with a dose-dependent.

Table 7.

The Inhibitory Effects of Different Doses of 28g and Celecoxib against Xylene-induced Ear Swelling Inflammation in Mice

Groups	Dose (mg/kg)	Swelling degree (mg)	Swelling rate (%)	Inhibition rate (%)
Normal saline		15.6±1.0	106.59
Celecoxib	10 mg/kg	10.5±1.5	74.21	32.83
low-dose 28g	10 mg/kg	8.7±1.7	69.39	44.39
High-dose 28g	30 mg/kg	5.1±0.9	39.50	67.17

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Figure 11. — The inhibitory effects of different doses of **28g** and **Celecoxib** against xylene-induced ear swelling inflammation in mice. Significance: ####P < 0.0001 compared with the control group; ****P < 0.0001 compared with the blank group.

Effects of 28g against formalin-induced inflammatory pain in vivo.

In order to verify that sEH inhibitor and HDAC6 inhibitor have synergistic inhibitory effect against formalin-induced inflammatory pain, the inhibitory effects of 28g, GL-B437, Rocilinostat and GL-B437 + Rocilinostat against inflammatory pain in mice were shown in Figure 12. By subcutaneous injection of 5% formalin solution into the right hind sole of mice, the licking and lifting time was recorded within 1 h with every five minutes as the time node. The co-administration of GL-B437 and Rocilinostat also exhibited a synergistic effect in relieving inflammatory pain. It is worth noting that 28g exhibited more effective analgesic activity than that of either a sEH inhibitor (GL-B437) or a HDAC6 inhibitor (Rocilinostat) alone, and that of co-administration of both inhibitors.

Figure 12. — The licking and lifting time course curve of **28g**, **GL-B437**, **Rocilinostat** and **GL-B437** + **Rocilinostat** against formalin-induced pain response in mice.

The response time of mice in the first phasic response and the second phasic response were counted. The results were shown in Figure 13. In the first phasic response, 28g did not significantly reduce the pain response time of mice. In the second phasic response, compared with the model group, 28g significantly reduced the pain response time of mice and was superior to the co-administration group of GL-B437 and Rocilinostat. This indicates that 28g had a good therapeutic effect against formalin-induced inflammatory pain.

In order to better evaluate the inhibitory effect of compound 28g against formalin-induced inflammatory pain, we compared the inhibitory degree of different doses of 28g against inflammatory pain in mice. Within 60 min, the pain responses of mice in each group were shown in Figure 14. The Licking and lifting time of mice in Celecoxib group and low and high dose groups of 28g were all reduced, and the inhibitory effects of low and high dose groups of 28g were better than that of Celecoxib group.

Figure 14. — The licking and lifting time course curve of different doses of **28g** and **Celecoxib** against formalin-induced pain response in mice.

The pain response times of mice in the first and second phasic response were counted, and the results were shown in Figure 15. In the first phasic response, Celecoxib group and 28g low and high dose groups did not significantly alleviate the pain reaction of mice. In the second phasic response, compared with the model group, Celecoxib group and 28g low-dose group reduced the pain response of mice, while 28g high-dose group significantly reduced the pain response of mice and a dose-dependent manner.

Effects of 28g on the levels of inflammatory factors in LPS-induced RAW264.7 cells.

To determine the effects of 28g on the production of inflammatory factors, RAW264.7 cells were preconditioned with 28g (1–100 µg/mL) for 12 h, then treated with LPS (1 µg/mL) for 12 h. As shown in Figure. 16, LPS enhanced the levels of IL-6, IL-1β, TNF-α and NO, while 28g treatment significantly reversed this change. In addition, levels of gene expression of iNOS was suppressed by treatment with 28g.

Figure 16. — Effects of **28g** on the expression of inflammatory factors and iNOS proteins in LPS-induced RAW264.7 cells. (A-D) Release in the culture medium of IL-6, IL-1β, TNF-α, and NO was detected by ELISA assay. (E) iNOS protein expression levels were detected by western blot assay. Relative protein expression was expressed as optical density value relative to the control group after normalizing to β-actin optical density value. The data are expressed as mean ± SD, n=3. #p< 0.05, ##p< 0.01, ###p< 0.001, ####p< 0.0001 vs LPS-induced group.

CONCLUSIONS

In this study, a series of novel sEH/HDAC6 dual-targeting inhibitors were designed and synthesized, which laid the foundation for the study of structure-activity relationship. As a DML compound synthesized, 28g has obvious selective inhibition on sEH and HDAC6. This study proved that compounds bearing urea and hydroxamic acid can improve the inhibitory activities of the obtained molecules on sEH and HDAC6 in vitro. Although the sEH inhibitory activity was decreased by replacing urea moiety with squaramide moiety by bioisosteric, the results of liver microsomal enzyme test showed that the compounds containing squaric acid structure could slightly prolong the half-life. This study showed that 28g, a dual-targeting inhibitor of sEH/HDAC6, had better in vivo efficacy than GL-B437 and Rocilinostat alone or in combination, and the therapeutic effect was better than that of Celecoxib. The acute toxicity test showed that 28g has good safety. This may indicate that dual-targeting inhibitors are expected to show better safety than combination therapy.

EXPERIMENTAL SECTION

Chemistry.

All reactions were carried out with magnetic stirring and in dried glassware. The reactions were monitored by thin-layer chromatography (TLC: HG/T2354-92, GF254), and compounds were visualized on TLC with UV light. All chemicals and solvents were purchased from commercial sources and used without purification treatment. Analytical thin-layer chromatography was carried out on 0.20 mm silica gel plates (Haiyang, Qingdao, Shandong, China) with the QF-254 UV indicator. Column chromatography was conducted using Haiyang silica gel 60 (300–400 mesh). Melting points were determined with an X-4 apparatus and were uncorrected. The nuclear magnetic resonance (NMR) spectra were recorded on Bruker Avance III 400 spectrometer in DMSO-d₆ using tetramethylsilane (TMS) as an internal standard. Peak multiplicity of NMR signals were as follows: s, singlet; brs, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Chemical shift (δ): ppm relative to Me₄Si (internal standard). Coupling constant: J (Hz). High-resolution mass spectra (HRMS) of all target compounds were performed by a Waters Q-TOF Premier spectrometer with acetonitrile and water as solvents. Electrospray ionization mass spectrometry (ESI-MS) analyses were recorded in an Agilent 1100 Series MSD Trap SL (Santa Clara, CA, USA). All final compounds are >95% pure by HPLC analysis.

General synthesis method 1 (Condensation of carboxylic acid with ethyl (S)- piperidine −3- carboxylate).

3-Fluoro-4-nitrobenzoic acid, 3-chloro-4-nitrobenzoic acid, 3-methyl-4-nitrobenzoic acid or 4-nitrobenzoic acid (1 eq) and dried tetrahydrofuran were added into a three-neck bottle. After it was dissolved, HATU (1.2 eq) and DIPEA (2.4 eq) were added, and the solution was light yellow after stirring for 60 min, and (S)- piperidine −3- carboxylic acid ethyl ester (1 eq) was added dropwise. And the mixture was stirred for 60 min. Then, the organic phase was evaporated in vacuum. The crude was dissolved in dichloromethane, and 1 N HCl solution was employed to wash the organic phase. The dichloromethane layer was then dried on anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure.

General synthesis method 2 (Reduction reaction of nitro group).

The corresponding nitro compound (1 eq) was added into a three-neck bottle, and dissolved by anhydrous ethanol. And iron powder (5 eq), ammonium chloride (5 eq) and water were slowly added. and the reaction mixture was stirred at 80 °C for 0.5 h. Then, the reaction mixture was filtered, the filtrate was evaporated in vacuum. The crude was dissolved in dichloromethane, and the organic phase was washed with water and dried on anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure.

General synthesis method 3 (Preparation of urea).

The corresponding amine (1 eq) and solid phosgene (0.5 eq) were added into a three-necked bottle, then were dissolved by dry dichloromethane and cooled it to −10 °C in ice salt bath. Subsequently, triethylamine (6 eq) was slowly dropped in dichloromethane. The corresponding mixed solution of amine (1 eq), triethylamine (3 eq) and dried dichloromethane were added to the prepared isocyanate. Then, the organic phase was washed by 1 N HCl solution, H₂O and brine respectively. The dichloromethane layer was then dried with anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure.

General synthesis method 4 (Hydrolysis reaction of ester).

The corresponding ester (1 eq), sodium hydroxide (5 eq), and water were added in the single-necked bottle, and the reaction was carried out at 50°C for 4 h. The solution was acidified to pH=1, resulting in precipitation, filtered, and dries it to obtain the target product.

General synthesis method 5 (Condensation of carboxylic acid and amine).

The corresponding carboxylic acid raw material (1 eq) was dissolved by anhydrous DCM in thethree-necked bottle. EDCI (1.5 eq), HOBt (1.5 eq), triethylamine (3 eq)，and corresponding amine (1.1 eq) were added sequentially. The reaction mixture was stirred at room temperature for 2 h. The residues were poured into water. The organic layer was separated and washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.

General synthesis method 6 (Synthesis of hydroxamic acid).

The appropriate corresponding raw material (1 eq), and 1M NaOH aqueous solution were dissolved in methanol, then the 50 % hydroxylamine aqueous solution (10 eq) was added dropwise with stirring. After stirring for 36 h further at room temperature, methanol was evaporated. The solution was acidified to pH=1, resulting in precipitation, filtered, and dried it to obtain the target product.

Ethyl (S)-1-(3-fluoro-4-nitrobenzoyl)piperidine-3-carboxylate (2).

According to general synthesis method 1, 3-fluoro-4-nitrobenzoic acid 1 was used as raw material. Yellow solid 4.1 g, yield 78.1 %. ESI-MS (m/z): 325.1 [M+H]⁺.

Ethyl (S)-1-(4-amino-3-fluorobenzoyl)piperidine-3-carboxylate (3).

According to general synthesis method 2, 2 was used as raw material. Yellow solid 3.5 g, yield 94.6 %. ESI-MS (m/z): 295.1 [M+H]⁺.

Ethyl (S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl) piperidine-3-carboxylate (4).

According to general synthesis method 3, 3 was used as raw material. Yellow solid 4.5 g, yield 75.8 %. m.p. 180–183 °C. ESI-MS (m/z): 500.4 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl) piperidine-3-carboxylic acid (5).

According to general synthesis method 4, 4 was used as raw material. Yellow solid 4.0 g, yield 94.3 %. m.p. 183–185 °C. ESI-MS (m/z): 472.3 [M+H]⁺.

Methyl 4-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)butanoate (6a).

According to general synthesis method 5, 5 and methyl 4-aminobutyrate hydrochloride were used as raw material. Yellow solid 0.35 g, yield 85.4 %. m.p. 167–170 °C.

Methyl 5-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)pentanoate (6b).

According to general synthesis method 5, 5 and pentanoic acid-5-amino-methyl este were used as raw material. Yellow solid 0.34 g, yield 80.9 %. m.p. 152–156 °C. ESI-MS (m/z): 585.3 [M+H]⁺.

Methyl 6-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)hexanoate (6c).

According to general synthesis method 5, 5 and methyl 6-aminocaproate hydrochloride were used as raw material. Yellow solid 0.37 g, yield 86.0 %. m.p. 164–167 °C. ESI-MS (m/z): 599.3 [M+H]⁺.

Methyl 7-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)heptanoate (6d).

According to general synthesis method 5, 5 and methyl 7-aminoheptanoate were used as raw material. Yellow solid 0.33 g, yield 75.0 %. m.p. 189–192 °C. ESI-MS (m/z): 635.2 [M+Na]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(4-(hydroxyamino)-4-oxobutyl)piperidine-3-carboxamide (7a).

According to general synthesis method 6, 6a was used as raw material. Light yellow solid 0.17 g, yield 75.0 %. m.p. 191–192 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.36 (s, 1H), 8.72−8.71 (m, 1H), 8.31 (s, 1H), 8.19 (t, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.20 (d, J = 11.7 Hz, 1H), 7.08 (q, J = 8.5 Hz, 1H), 6.60 (s, 1H), 3.02−3.01 (m, 4H), 2.29 (t, J = 10.7 Hz, 1H), 2.09 (s, 1H), 1.94−1.85 (m, 3H), 1.76−1.74 (m, 3H), 1.61−1.58 (m, 6H), 1.40−1.24 (m, 7H), 1.12 (s, 2H), 0.85 (s, 1H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.8, 168.4, 153.8, 152.2, 149.7, 130.3, 128.8, 123.8, 119.3, 114.4, 52.1, 50.7, 48.0, 42.8, 38.5, 32.4, 30.5, 30.2, 30.0, 28.2, 26.8, 25.7. ESI-MS (m/z): 572.39 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(5-(hydroxyamino)-5-oxopentyl)piperidine-3-carboxamide (7b).

According to general synthesis method 6, 6b was used as raw material. Light yellow solid 0.19 g, yield 65.5 %. m.p. 154–155 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.34 (s, 1H), 8.71 (s, 1H), 8.31 (s, 1H), 8.20 (t, J = 8.3 Hz, 1H), 7.88 (s, 1H), 7.20 (d, J = 11.7 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 6.59 (s, 1H), 3.02−3.01 (m, 4H), 2.32−2.27 (m, 1H), 2.09 (s, 1H), 1.94 (t, J = 6.7 Hz, 2H), 1.87−1.84 (m, 1H), 1.76 (s, 3H), 1.61−1.58 (m, 6H), 1.46−1.44 (m, 2H), 1.40 (s, 1H), 1.35−1.27 (m, 4H), 1.26−1.24 (m, 3H), 1.12 (s, 2H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.3, 153.8, 152.2, 149.7, 130.3, 128.8, 123.8, 119.3, 114.2, 52.1, 50.7, 47.9, 42.7, 38.5, 32.4, 30.5, 30.0, 29.1, 28.2, 26.8, 23.0. ESI-MS (m/z): 586.39 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(6-(hydroxyamino)-6-oxohexyl)piperidine-3-carboxamide (7c).

According to general synthesis method 6, 6c was used as raw material. Light yellow solid 0.18 g, yield 60.0 %. m.p. 160–162 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.40 (s, 1H), 8.37 (d, J = 2.3 Hz, 1H), 8.21 (t, J = 8.4 Hz, 1H), 7.92 (s, 1H), 7.19 (dd, J =11.6 Hz, 1.5 Hz, 1H), 7.08 (dd, J = 8.4 Hz, 1.2 Hz, 1H), 6.64 (s, 1H), 3.04−2.99 (m, 4H), 2.30 (t, J = 10.8 Hz, 1H), 2.09 (s, 1H), 1.95−1.85 (m, 3H), 1.76 (s, 2H), 1.73 (s, 1H), 1.61−1.58 (m, 8H), 1.40 (s, 1H), 1.34−1.32 (m, 3H), 1.280−1.25 (m, 4H), 1.12 (s, 2H), 0.87−0.86 (m, 2H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.8, 169.1, 168.4, 153.9, 152.2, 149.8, 130.4, 123.8, 119.4, 114.4, 52.1, 50.7, 48.0, 42.8, 32.4, 31.4, 30.5, 30.3, 30.0, 28.9, 28.2, 26.8, 25.7, 25.2, 23.8, 22.9, 22.5. ESI-MS (m/z): 622.2 [M+Na]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (7d).

According to general synthesis method 6, 6d was used as raw material. Light yellow solid 0.21 g, yield 67.7 %. m.p. 171–172 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.33 (s, 1H), 8.68 (s, 1H), 8.31 (s, 1H), 8.20 (t, J = 8.4 Hz, 1H), 7.85 (s, 1H), 7.20 (d, J = 11.7 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 6.59 (s, 1H), 3.01−2.89 (m, 4H), 2.32−2.27 (m, 1H), 2.09−2.08 (m, 1H), 1.93 (t, J = 7.2 Hz, 2H), 1.86−1.83 (m, 1H), 1.76 (s, 3H), 1.69−1.61 (m, 1H), 1.58 (s, 5H), 1.47−1.46 (m, 3H), 1.40 (s, 1H), 1.35−1.30 (m, 3H), 1.28−1.26 (m, 2H), 1.24−1.21 (m, 4H), 1.12 (s, 2H), 0.85 (s, 1H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.3, 153.8, 152.2, 149.8, 130.4, 128.8, 123.8, 119.3, 114.2, 52.1, 50.7, 47.9, 42.8, 38.7, 32.7, 32.3, 30.5, 30.0, 29.4, 28.9, 28.7, 28.2, 26.8, 26.5, 25.5, 22.9, 20.3, 19.7, 18.1, 14.6, 14.4, 11.7. ESI-MS (m/z): 614.46 [M+H]⁺.

3-Fluoro-4-nitrophenyl)methanol (9).

3- Fluoro −4- nitrobenzoic acid (20 g, 0.11 mol) and tetrahydrofuran (200 ml) were added into a three-necked bottle, cool to 0 °C, and borane solution in THF (160 mL) was added drop by drop. The mixture was stirred at 0 °C for 10 h. Then, 200 mL of a solution of MeOH was added and the organic phase was evaporated under reduced pressure. White solid 17.8 g, yield 95.0 %. m.p. 93–94 °C. ESI-MS (m/z): 194.1 [M+Na]⁺.

4-(Bromomethyl)-2-fluoro-1-nitrobenzene (10)

9 (17.0 g, 99.4 mmol), phosphorus tribromide (18.5 g, 50 mmol) and DCM (50 mL) were added into a three-necked bottle. The mixture was stirred at room temperature for 8 h. Subsequently, water was added and the organic phase was washed three times, dried with anhydrous Na₂SO₄, filtered, and evaporated. Yellow oil 17.4 g, yield 92.0 %. ESI-MS (m/z): 233.2 [M+H]⁺.

Ethyl (S)-1-(3-fluoro-4-nitrobenzyl)piperidine-3-carboxylate (11).

10 (4.0 g, 17.3 mmol) and dried acetonitrile (35 mL) were added into a three-necked bottle. Then, K₂CO₃ (2.87 g, 20.76 mmol), KI (0.20 g, 1.73 mmol) and (S)- piperidine −3- carboxylate (3.39 g, 20.76 mmol) were added. The mixture was stirred at 50 °C for 8 h. Subsequently, the organic phase was washed three times, dried with anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. Yellow oil 4.4 g, yield 82.0 %. ESI-MS (m/z): 311.1 [M+H]⁺.

Ethyl (S)-1-(4-amino-3-fluorobenzyl)piperidine-3-carboxylate (12).

According to general synthesis method 2, 11 was used as raw material. Yellow oil 3.1 g, yield 87.8 %. ESI-MS (m/z): 281.1 [M+H]⁺.

Ethyl (S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzyl) piperidine-3-carboxylate (13).

According to general synthesis method 3, 12 was used as raw material. Yellow solid 3.9 g, yield 72.2 %. ESI-MS (m/z): 486.33 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzyl) piper-idine-3-carboxylic acid (14).

According to general synthesis method 4, 13 was used as raw material. Yellow solid 3.2 g, yield 87.2 %. ESI-MS (m/z): 458.31 [M+H]⁺.

Methyl 4-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzyl)piperidine-3-carboxamido)butanoate (15a).

According to general synthesis method 5, 14 was used as raw material. Light yellow solid 0.32 g, yield 80.0 %. m.p. 148–151 °C. ESI-MS (m/z): 557.2 [M+H]⁺.

Methyl 5-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzyl)piperidine-3-carboxamido)pentanoate (15b).

According to general synthesis method 5, 14 was used as raw material. Light yellow solid 0.33 g, yield 80.5 %. m.p. 163–167 °C. ESI-MS (m/z): 571.3 [M+H]⁺.

Methyl 6-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzyl)piperidine-3-carboxamido)hexanoate (15c).

According to general synthesis method 5, 14 was used as raw material. Light yellow solid 0.30 g, yield 71.4 %. m.p. 185–188 °C. ESI-MS (m/z): 585.2 [M+H]⁺.

Methyl 7-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzyl)piperidine-3-carboxamido)heptanoate (15d).

According to general synthesis method 5, 14 was used as raw material. Light yellow solid 0.43 g, yield 83.7 %. m.p. 168–171 °C. ESI-MS (m/z): 599.2 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)-N-(4-(hydroxyamino)-4-oxobutyl)piperidine-3-carboxamide (16a).

According to general synthesis method 6, 15a was used as raw material. Light yellow solid 0.17 g, yield 61.2 %. m.p. 197–199 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.41 (s, 1H), 8.43 (s, 1H), 8.42 (s, 1H), 8.26−8.20 (m, 2H), 7.44 (d, J = 12.2 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 6.73 (s, 1H), 3.59−3.36 (m, 3H), 3.04−2.92 (m, 3H), 2.51 (s, 1H), 2.09−1.91 (m, 3H), 1.76 (s, 2H), 1.60−1.55 (m, 6H), 1.35−1.30 (m, 3H), 1.27−1.23 (m, 7H), 1.12 (s, 2H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 175.0, 171.1, 169.2, 154.0, 153.8, 130.9, 129.8, 119.4, 52.1, 52.1, 50.7, 48.0, 48.0, 42.8, 38.6, 38.5, 37.7, 32.4, 31.7, 31.6, 30.5, 30.3,30.0, 29.5, 29.2, 25.8, 25.7, 25.6, 25.0, 22.6, 19.6. ESI-MS (m/z): 585.3 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)-N-(5-(hydroxyamino)-5-oxopentyl)piperidine-3-carboxamide (15b).

According to general synthesis method 6, 15b was used as raw material. Light yellow solid 0.15 g, yield 52.6 %. m.p. 170–171 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.15 (s, 1H), 8.44 (s, 1H), 8.08 (s, 1H), 7.90 (s, 2H), 7.18−7.13 (m, 1H), 6.90 (s, 1H), 6.45 (s, 1H), 3.33−3.27 (m, 2H), 2.76−2.74 (m, 2H), 1.87 (s, 1H), 1.72−1.68 (m, 3H), 1.52 (s, 4H), 1.37−1.31 (m, 4H), 1.23−1.18 (m, 3H), 1.10−1.06 (m, 4H), 1.03−1.00 (m, 7H), 0.88 (s, 2H), 0.59 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 174.9, 172.5, 169.4, 154.0, 152.5, 150.1, 119.8, 52.1, 50.7, 48.0, 42.8, 38.6, 34.1, 32.4, 31.7, 31.6, 30.5, 30.3, 30.0, 29.5, 29.4, 29.2, 29.2, 29.0, 25.0, 23.0, 22.5, 21.5. ESI-MS (m/z): 572.3 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)-N-(6-(hydroxyamino)-6-oxohexyl)piperidine-3-carboxamide (16c).

According to general synthesis method 6, 15c was used as raw material. Light yellow solid 0.16 g, yield 55.0 %. m.p. 174–176 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.37 (s, 1H), 8.45 (s, 1H), 8.26−8.21 (m, 1H), 8.14−8.08 (m, 1H), 7.97 (s, 1H), 7.25 (d, J = 8.5 Hz, 1H), 7.08 (s, 1H), 6.76−6.65 (m, 1H), 3.58−3.49 (m, 3H), 3.06−2.92 (m, 3H), 2.09 (s, 1H), 1.94−1.91 (m, 3H), 1.76 (s, 3H), 1.61−1.54 (m, 4H), 1.50−1.44 (m, 2H), 1.39−1.30 (m, 5H), 1.27−1.15 (m, 8H), 1.11 (s, 2H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.5, 171.0, 169.5, 154.1, 153.9, 131.0, 129.8, 119.8, 52.2, 52.1, 50.7, 48.1, 48.0, 42.8, 37.7, 32.6, 32.4, 30.5, 30.0, 29.5, 29.2, 29.0, 27.2, 26.8, 26.4, 26.3, 25.9, 25.3, 25.2, 25.0, 22.5, 21.6, 19.6. ESI-MS (m/z): 586.39 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (16d).

According to general synthesis method 6, 15d was used as raw material. Light yellow solid 0.20 g, yield 66.9 %. m.p. 168–169 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.34 (s, 1H), 8.66 (s, 1H), 8.24 (s, 1H), 8.11−7.95 (m, 2H), 7.25−7.09 (m, 2H), 6.62 (s, 1H), 3.51−3.35 (m, 2H), 2.99−2.97 (m, 3H), 2.09 (s, 1H), 1.95−1.91 (m, 4H), 1.76 (s, 4H), 1.61−1.52 (m, 5H), 1.47−1.44 (m, 3H), 1.40−1.32 (m, 5H), 1.27−1.21 (m, 7H), 1.12 (s, 2H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.5, 169.6, 154.0, 119.8, 52.1, 50.7, 48.0, 42.8, 38.8, 32.7, 32.4, 31.4, 30.5, 30.0, 29.3, 29.2, 29.0, 28.7, 27.5, 26.8, 26.5, 25.5, 23.0, 22.9, 22.5, 21.5. HRMS (ESI) m/z: calcd for C₃₃H₅₀FN₅O₄ [M+H]⁺: 600.3847; found: 600.3913.

Ethyl (S)-1-(3-chloro-4-nitrobenzoyl)piperidine-3-carboxylate (18a).

According to general synthesis method 1, 17a was used as raw material. yellow oil 0.80 g, yield 78.4 %. ESI-MS (m/z): 341.13 [M+H]⁺.

Ethyl (S)-1-(3-methyl-4-nitrobenzoyl)piperidine-3-carboxylate (18b).

According to general synthesis method 1, 17b was used as raw material. yellow oil 0.83 g, yield 86.4 %.

Ethyl (S)-1-(4-nitrobenzoyl)piperidine-3-carboxylate (18c).

According to general synthesis method 1, 17c was used as raw material. yellow oil 0.76 g, yield 82.6 %. ESI-MS (m/z): 307.14 [M+H]⁺.

Ethyl (S)-1-(4-amino-3-chlorobenzoyl)piperidine-3-carboxylate (19a).

According to general synthesis method 2, 18a was used as raw material. yellow oil 0.68 g, yield 95.4 %. ESI-MS (m/z): 311.10 [M+H]⁺.

Ethyl (S)-1-(4-amino-3-methylbenzoyl)piperidine-3-carboxylate (19b).

According to general synthesis method 2, 18b was used as raw material. yellow oil 0.70 g, yield 93.3 %.

Ethyl (S)-1-(4-aminobenzoyl)piperidine-3-carboxylate (19c).

According to general synthesis method 2, 18c was used as raw material. yellow oil 0.64 g, yield 93.4 %. ESI-MS (m/z): 277.14 [M+H]⁺.

Ethyl (S)-1-(3-chloro-4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)benzoyl) piperidine-3-carboxylate (20a).

According to general synthesis method 3, 19a was used as raw material. White solid 0.76 g, yield 67.8 %. m.p. 155–158 °C.

Ethyl (S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-methylbenzoyl) piperidine-3-carboxylate (20b).

According to general synthesis method 3, 19b was used as raw material. White solid 0.92 g, yield 77.3 %. m.p. 131–133 °C.

Ethyl (S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)benzoyl)piperi-dine-3-carboxylate (20c).

According to general synthesis method 3, 19c was used as raw material. White solid 0.62 g, yield 92.5 %. m.p. 159–161 °C. ESI-MS (m/z): 453.3 [M+H]⁺.

(S)-1-(3-Chloro-4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)benzoyl)pip-eridine-3-carboxylic acid (21a).

According to general synthesis method 4, 20a was used as raw material. White solid 0.70 g, yield 97.2 %. m.p. 161–163 °C. ESI-MS (m/z): 488.1 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-methylbenzoyl)pip-eridine-3-carboxylic acid (21b).

According to general synthesis method 4, 20b was used as raw material. White solid 0.80 g, yield 91.9 %. m.p. 149–152 °C. ESI-MS (m/z): 468.31 [M+H]⁺.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)benzoyl)piperidine-3-carboxylic acid (21c).

According to general synthesis method 4, 20c was used as raw material. White solid 0.62 g, yield 92.5 %. m.p. 159–161 °C. ESI-MS (m/z): 453.3 [M+H]⁺.

Ethyl 7-((S)-1-(3-chloro-4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)ben-zoyl)piperidine-3-carboxamido)heptanoate (22a).

According to general synthesis method 5, 21a and ethyl 7-aminoheptanoate were used as raw material. Light yellow solid 0.36 g, yield 80.0 %. m.p. 160–163 °C.

Ethyl 7-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-methylb-enzoyl)piperidine-3-carboxamido)heptanoate (22b).

According to general synthesis method 5, 21b and ethyl 7-aminoheptanoate were used as raw material. Light yellow solid 0.34 g, yield 77.3 %. m.p. 141–144 °C.

Ethyl 7-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)benzoyl)pipe-ridine-3-carboxamido)heptanoate (22c).

According to general synthesis method 5, 21c and ethyl 7-aminoheptanoate were used as raw material. Light yellow solid 0.32 g, yield 74.4 %. m.p. 180–183 °C.

(S)-1-(3-Chloro-4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)benzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (23a).

According to general synthesis method 6, 22a was used as raw material. White solid 0.24 g, yield 77.4 %. m.p. 150–151 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.34 (s, 1H), 8.66 (s, 1H), 8.22 (d, J = 8.5 Hz, 1H), 8.05 (s, 1H), 7.85 (s, 1H), 7.40 (s, 1H), 7.23 (d, J = 8.3 Hz, 1H), 6.99 (s, 1H), 3.01 (s, 2H), 2.30 (s, 1H), 2.10 (s, 1H), 1.93−1.91 (m, 2H), 1.86−1.83 (m, 1H), 1.78 (s, 2H), 1.63−1.59 (m, 6H), 1.48 (s, 2H), 1.40−1.33 (m, 5H), 1.28−1.22 (m, 8H), 1.12 (s, 2H), 0.87−0.86 (m, 2H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.1, 153.7, 138.5, 129.7, 128.4, 126.7, 120.6, 119.8, 52.2, 50.7, 47.9, 42.8, 38.7, 32.7, 32.4, 31.4, 30.5, 30.0, 29.4, 28.9, 28.7, 28.2, 26.8, 26.5, 25.5, 25.2, 22.9, 22.5, 21.1, 20.3, 19.1. HRMS (ESI) m/z: calcd for C₃₃H₄₈ClN₅O₅ [M+H]⁺: 630.3344; found: 630.3417.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-methylbenzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (23b).

According to general synthesis method 6, 22b was used as raw material. White solid 0.19 g, yield 63.3 %. m.p. 128–130 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.36 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.87 (s, 1H), 7.71 (s, 1H), 7.13 (s, 1H), 7.08 (dd, J = 8.5 Hz, 1.5Hz, 1H), 6.66 (s, 1H), 3.01 (s, 2H), 2.31−2.26 (m, 1H), 2.18 (s, 3H), 2.09 (s, 1H), 1.93 (T, J = 6.8 Hz, 2H), 1.86−1.78 (m, 4H), 1.65−1.57 (m, 6H), 1.48−1.46 (m, 3H), 1.35−1.30 (m, 6H), 1.27−1.23 (m, 8H), 1.12 (s, 2H), 0.83 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.8, 169.8, 154.4, 140.4, 129.5, 128.7, 125.7, 125.5, 118.9, 118.8, 52.0, 50.8, 48.1, 42.8, 32.7, 32.4, 31.8, 31.6, 30.6, 30.3, 30.1, 29.4, 29.2, 28.7, 28.3, 26.7, 26.5, 25.5, 22.6, 18.4. HRMS (ESI) m/z: calcd for C₃₄H₅₁N₅O₅ [M+H]⁺: 610.3890; found: 610.3964.

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)benzoyl)-N-(7-(hydro-xyamino)-7-oxoheptyl)piperidine-3-carboxamide (23c).

According to general synthesis method 6, 22c was used as raw material. White solid 0.19 g, yield 63.3 %. m.p. 176–177 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.22 (s, 1H), 7.88 (s, 1H), 7.43 (d, J = 6.2 Hz, 2H), 7.21 (d, J = 7.2 Hz, 2H), 6.62 (s, 1H), 3.00 (s, 3H), 2.32−2.30 (m, 1H), 2.08 (s, 1H), 1.94 (t, J = 6.9 Hz, 1H), 1.86−1.83 (m, 1H), 1.77−1.72 (m, 4H), 1.62−1.55 (m, 6H), 1.48−1.46 (m, 2H), 1.40−1.31 (m, 6H), 1.26−1.23 (m, 7H), 1.10 (s, 2H), 0.82 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.8, 169.8, 154.6, 142.9, 128.3, 128.0, 117.0, 116.7, 51.9, 51.8, 50.8, 48.1, 42.8, 38.7, 32.4, 32.3, 31.4, 30.6, 30.1, 29.6, 29.4, 28.7, 28.3, 26.8, 26.5, 25.6, 24.7, 20.3. HRMS (ESI) m/z: calcd for C₃₃H₄₉N₅O₅ [M+H]⁺: 596.3734; found: 596.3806.

Ethyl (S)-1-(3-fluoro-4-(3-(3-fluoro-4-(trifluoromethoxy)phenyl)ureido)benzoyl)pipe-ridine-3-carboxylate (25a).

According to general synthesis method 3, 24 and 3-fluoro-4-(trifluoromethoxy)aniline were used as raw material. White solid 0.87 g, yield 67.4 %. m.p. 153–156 °C. ESI-MS (m/z): 516.21 [M+H]⁺.

Ethyl (S)-1-(3-fluoro-4-(3-(4-(trifluoromethoxy)phenyl)ureido)benzoyl)piperidine-3-carboxylate (25b).

According to general synthesis method 3, 24 and 4-(trifluoromethoxy)aniline were used as raw material. White solid 0.81 g, yield 65.2 %. m.p. 135–138 °C. ESI-MS (m/z): 498.20 [M+H]⁺.

Ethyl (S)-1-(4-(3-(3-chloro-4-(trifluoromethoxy)phenyl)ureido)-3-fluorobenzoyl)pipe-ridine-3-carboxylate (25c).

According to general synthesis method 3, 24 and 3-chloro-4-(trifluoromethoxy)aniline were used as raw material. White solid 0.92 g, yield 69.2 %. m.p. 156–158 °C.

Ethyl (3S)-1-(4-(3-(1-((3r,5r,7r)-adamantan-1-yl)ethyl)ureido)-3-fluorobenzoyl)pipe-ridine-3-carboxylate (25d).

According to general synthesis method 3, 24 and 1-((3r,5r,7r)-adamantan-1-yl)ethan-1-amine were used as raw material. White solid 0.88 g, yield 70.4 %. m.p. 124–126 °C.

Ethyl (S)-1-(4-(3-(3-chloro-4-fluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carb-oxylate (25e).

According to general synthesis method 3, 24 and 3-chloro-4-fluoroaniline were used as raw material. White solid 0.80 g, yield 68.9 %. m.p. 142–144 °C.

Ethyl (S)-1-(3-fluoro-4-(3-(4-fluoro-3-(trifluoromethyl)phenyl)ureido)benzoyl)pipe-ridine-3-carboxylate (25f).

According to general synthesis method 3, 24 and 4-fluoro-3-(trifluoromethyl)aniline were used as raw material. White solid 0.85 g, yield 68.0 %. m.p. 148–150 °C. ESI-MS (m/z): 500.19 [M+H]⁺.

Ethyl (S)-1-(4-(3-(4-chloro-3-fluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carb-oxylate (25g).

According to general synthesis method 3, 24 and 4-chloro-3-fluoroaniline were used as raw material. White solid 0.82 g, yield 70.7 %. m.p. 156–159 °C. ESI-MS (m/z): 464.0 [M-H]⁺.

Ethyl (S)-1-(3-fluoro-4-(3-(3-fluoro-4-methylphenyl)ureido)benzoyl)piperidine-3-car-boxylate (25h).

According to general synthesis method 3, 24 and 3-fluoro-4-methylaniline were used as raw material. White solid 0.76 g, yield 68.5 %. m.p. 152–154 °C. ESI-MS (m/z): 446.2 [M+H]⁺.

Ethyl (S)-1-(3-fluoro-4-(3-(4-fluoro-3-methylphenyl)ureido)benzoyl)piperidine-3-car-boxylate (25i).

According to general synthesis method 3, 24 and 4-fluoro-3-methylaniline were used as raw material. White solid 0.81 g, yield 72.9 %. m.p. 146–148 °C.

Ethyl (S)-1-(4-(3-(3,5-difluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxyla-te (25j).

According to general synthesis method 3, 24 and 3,5-difluoroaniline were used as raw material. White solid 0.76 g, yield 67.8 %. m.p. 166–168 °C.

Ethyl (S)-1-(4-(3-benzhydrylureido)-3-fluorobenzoyl)piperidine-3-carboxylate (25k).

According to general synthesis method 3, 24 and diphenylmethanamine were used as raw material. White solid 0.86 g, yield 68.2 %. m.p. 154–156 °C.

(S)-1-(3-Fluoro-4-(3-(3-fluoro-4-(trifluoromethoxy)phenyl)ureido)benzoyl)piperidine-3-carboxylic acid (26a).

According to general synthesis method 4, 25a was used as raw material. White solid 0.79 g, yield 96.3 %. m.p. 162–165 °C.

(S)-1-(3-Fluoro-4-(3-(4-(trifluoromethoxy)phenyl)ureido)benzoyl)piperidine-3-carbo-xylic acid (26b).

According to general synthesis method 4, 25b was used as raw material. White solid 0.71 g, yield 93.4 %. m.p. 147–149 °C.

(S)-1-(4-(3-(3-Chloro-4-(trifluoromethoxy)phenyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxylic acid (26c).

According to general synthesis method 4, 25c was used as raw material. White solid 0.84 g, yield 95.6 %. m.p. 167–169 °C.

(3S)-1-(4-(3-(1-((3r,5r,7r)-Adamantan-1-yl)ethyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxylic acid (26d).

According to general synthesis method 4, 25d was used as raw material. White solid 0.75 g, yield 90.4 %. m.p. 139–141 °C.

(S)-1-(4-(3-(3-Chloro-4-fluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxylic acid (26e).

According to general synthesis method 4, 25e was used as raw material. White solid 0.70 g, yield 92.7 %. m.p. 152–154 °C.

(S)-1-(3-Fluoro-4-(3-(4-fluoro-3-(trifluoromethyl)phenyl)ureido)benzoyl)piperidine-3-carboxylic acid (26f).

According to general synthesis method 4, 25f was used as raw material. White solid 0.70 g, yield 87.5 %. m.p. 161–163 °C.

(S)-1-(3-Fluoro-4-(3-(4-fluoro-3-(trifluoromethyl)phenyl)ureido)benzoyl)piperidine-3-carboxylic acid (26g).

According to general synthesis method 4, 25g was used as raw material. White solid 0.70 g, yield 90.9 %. m.p. 161–163 °C.

(S)-1-(3-Fluoro-4-(3-(3-fluoro-4-methylphenyl)ureido)benzoyl)piperidine-3-carbox-ylic acid (26h).

According to general synthesis method 4, 25h was used as raw material. White solid 0.65 g, yield 91.5 %. m.p. 150–152 °C.

(S)-1-(3-Fluoro-4-(3-(4-fluoro-3-methylphenyl)ureido)benzoyl)piperidine-3-carboxy-lic acid (26i).

According to general synthesis method 4, 25i was used as raw material. White solid 0.69 g, yield 90.7 %. m.p. 151–153 °C.

(S)-1-(4-(3-(3,5-Difluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxylic acid (26j).

According to general synthesis method 4, 25j was used as raw material. White solid 0.65 g, yield 91.5 %. m.p. 178–180 °C. ESI-MS (m/z): 442.1 [M+H]⁺.

(S)-1-(4-(3-Benzhydrylureido)-3-fluorobenzoyl)piperidine-3-carboxylic acid (26k).

According to general synthesis method 4, 25k was used as raw material. White solid 0.71 g, yield 88.7 %. m.p. 163–166 °C.

Methyl (S)-7-(1-(3-fluoro-4-(3-(3-fluoro-4-(trifluoromethoxy)phenyl)ureido)benzoyl) piperidine-3-carboxamido)heptanoate (27a).

According to general synthesis method 5, 26a was used as raw material. Light yellow solid 0.35 g, yield 77.8 %. m.p. 155–158 °C. ESI-MS (m/z): 643.3 [M+H]⁺.

Methyl (S)-7-(1-(3-fluoro-4-(3-(3-fluoro-4-methyl(S)-7-(1-(3-fluoro-4-(3-(4-(trifluoro-methoxy)phenyl)ureido)benzoyl)piperidine-3-carboxamido)heptanoate (27b).

According to general synthesis method 5, 26b was used as raw material. Light yellow solid 0.33 g, yield 75.0 %. m.p. 164–167 °C. ESI-MS (m/z): 625.2 [M+H]⁺.

Methyl (S)-7-(1-(3-fluoro-4-(3-(3-fluoro-4-methyl(S)-7-(1-(4-(3-(3-chloro-4-(trifluoro-methoxy)phenyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxamido)heptanoate (27c).

According to general synthesis method 5, 26c was used as raw material. Light yellow solid 0.35 g, yield 76.1 %. m.p. 165–168 °C.

Methyl (S)-7-(1-(3-fluoro-4-(3-(3-fluoro-4-methyl-7-((3S)-1-(4-(3-(1-((3r,5r,7r)-adam-antan-1-yl)ethyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxamido)heptanoate (27d).

According to general synthesis method 5, 26d was used as raw material. Light yellow solid 0.31 g, yield 70.4 %. m.p. 170–173 °C. ESI-MS (m/z): 627.3 [M+H]⁺.

Methyl (S)-7-(1-(4-(3-(3-chloro-4-fluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxamido)heptanoate (27e).

According to general synthesis method 5, 26e was used as raw material. Light yellow solid 0.28 g, yield 66.7 %. m.p. 165–168 °C. ESI-MS (m/z): 593.2 [M+H]⁺.

Methyl (S)-7-(1-(3-fluoro-4-(3-(4-fluoro-3-(trifluoromethyl)phenyl)ureido)benzoyl) piperidine-3-carboxamido)heptanoate (27f).

According to general synthesis method 5, 26f was used as raw material. Light yellow solid 0.31 g, yield 70.4 %. m.p. 181–184 °C. ESI-MS (m/z): 627.2 [M+H]⁺.

Methyl (S)-7-(1-(4-(3-(4-chloro-3-fluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carboxamido)heptanoate (27g).

According to general synthesis method 5, 26g was used as raw material. Light yellow solid 0.32 g, yield 76.2 %. m.p. 180–183 °C. ESI-MS (m/z): 593.2 [M+H]⁺.

Methyl (S)-7-(1-(3-fluoro-4-(3-(3-fluoro-4-methylphenyl)ureido)benzoyl)piperidine-3-carboxamido)heptanoate (27h).

According to general synthesis method 5, 26h was used as raw material. Light yellow solid 0.31 g, yield 77.5 %. m.p. 169–173 °C. ESI-MS (m/z): 573.3 [M+H]⁺.

Methyl (S)-7-(1-(3-fluoro-4-(3-(4-fluoro-3-methylphenyl)ureido)benzoyl)piperidine-3-carboxamido)heptanoate (27i).

According to general synthesis method 5, 26i was used as raw material. Light yellow solid 0.29 g, yield 72.5 %. m.p. 171–174 °C. ESI-MS (m/z): 573.3 [M+H]⁺.

Methyl (S)-7-(1-(4-(3-(3,5-difluorophenyl)ureido)-3-fluorobenzoyl)piperidine-3-carb-oxamido)heptanoate (27j).

According to general synthesis method 5, 26j was used as raw material. Light yellow solid 0.29 g, yield 72.5 %. m.p. 185–186 °C. ESI-MS (m/z): 577.3 [M+H]⁺.

Methyl (S)-7-(1-(4-(3-benzhydrylureido)-3-fluorobenzoyl)piperidine-3-carboxamido-)heptanoate (27k).

According to general synthesis method 5, 26k was used as raw material. Light yellow solid 0.31 g, yield 70.4 %. m.p. 170–173 °C.

(S)-1-(3-Fluoro-4-(3-(3-fluoro-4-(trifluoromethoxy)phenyl)ureido)benzoyl)-N-(7-(hyd-roxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28a).

According to general synthesis method 6, 27a was used as raw material. Light yellow solid 0.18 g, yield 58.1 %. m.p. 156–157 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.42 (s, 1H), 9.72 (s, 1H), 8.99−8.69 (m, 1H), 8.17 (s, 1H), 7.84−7.73 (m, 2H), 7.47 (s, 1H), 7.28−7.19 (m, 4H), 3.00 (s, 2H), 2.32 (s, 2H), 1.94−1.85 (m, 2H), 1.64−1.61 (m, 2H), 1.45−1.35 (m, 6H), 1.21 (s, 5H), 0.85−0.84 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.1, 155.4, 152.9, 152.4, 150.7, 140.6, 130.9, 129.8, 128.9, 125.0, 123.9, 121.9, 120.7, 119.4, 114.9, 107.0, 42.7, 38.7, 32.5, 29.3, 28.9, 28.9, 28.7, 28.1, 26.8, 26.5, 25.6. HRMS (ESI) m/z: calcd for C₂₈H₃₂F₅N₅O₆ [M+H]⁺: 630.2273; found: 630.2342.

(S)-1-(3-Fluoro-4-(3-(4-(trifluoromethoxy)phenyl)ureido)benzoyl)-N-(7-(hydroxyam-ino)-7-oxoheptyl)piperidine-3-carboxamide (28b).

According to general synthesis method 6, 27b was used as raw material. Light yellow solid 0.16 g, yield 51.6 %. m.p. 180–182 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.20 (s, 1H), 9.52 (s, 1H), 8.13 (t, J = 7.7 Hz, 1H), 7.88 (s, 1H), 7.60 (s, 2H), 7.28−7.25 (m, 3H), 7.17 (d, J = 7.8 Hz, 1H), 3.00 (s, 4H), 2.32 (s, 1H), 1.99−1.84 (m, 3H), 1.76 (s, 2H), 1.67−1.61 (m, 2H), 1.45−1.30 (m, 4H), 1.25−1.18 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.2, 153.3, 152.9, 150.9, 143.1, 139.5, 130.5, 129.4, 129.3, 123.8, 122.2, 121.9, 121.1, 119.8, 119.4, 116.9, 42.8, 38.7, 29.5, 29.4, 28.7, 28.1, 26.5, 25.6, 22.5, 21.2. HRMS (ESI) m/z: calcd for C₂₈H₃₃F₄N₅O₆ [M+H]⁺: 612.2367; found: 612.2438.

(S)-1-(4-(3-(3-Chloro-4-(trifluoromethoxy)phenyl)ureido)-3-fluorobenzoyl)-N-(7-(hy-droxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28c).

According to general synthesis method 6, 27c was used as raw material. Light yellow solid 0.18 g, yield 58.1 %. m.p. 156–157 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.42 (s, 1H), 9.72 (s, 1H), 8.99−8.69 (m, 1H), 8.17 (s, 1H), 7.84−7.73 (m, 2H), 7.47 (s, 1H), 7.28−7.19 (m, 4H), 3.00 (s, 2H), 2.32 (s, 2H), 1.94−1.85 (m, 2H), 1.64−1.61 (m, 2H), 1.45−1.35 (m, 6H), 1.21 (s, 5H), 0.85−0.84 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.1, 155.4, 152.9, 152.4, 150.7, 140.6, 130.9, 129.8, 128.9, 125.0, 123.9, 121.9, 120.7, 119.4, 114.9, 107.0, 42.7, 38.7, 32.5, 29.3, 28.9, 28.9, 28.7, 28.1, 26.8, 26.5, 25.6. HRMS (ESI) m/z: calcd for C₂₈H₃₂F₅N₅O₆ [M+H]⁺: 630.2273; found: 630.2342.

(3S)-1-(4-(3-(1-((3r,5r,7r)-Adamantan-1-yl)ethyl)ureido)-3-fluorobenzoyl)-N-(7-(hyd-roxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28d).

According to general synthesis method 6, 27d was used as raw material. Light yellow solid 0.15 g, yield 46.9 %. m.p. 134–135 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.63 (s, 1H), 9.78 (s, 1H), 8.08 (t, J = 7.6 Hz, 1H), 7.94 (s, 2H), 7.47 (s, 2H), 7.26 (d, J = 10.8 Hz, 1H), 7.17 (d, J = 7.9 Hz, 1H), 3.01 (s, 4H), 2.32 (s, 1H), 1.99−1.93 (m, 2H), 1.87−1.77 (m, 2H), 1.67−1.61 (m, 2H), 1.46−1.36 (m, 5H), 1.30−1.18 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 170.8, 168.1, 152.9, 151.1, 140.8, 138.5, 131.0, 129.1, 126.6, 124.1, 123.7, 121.9, 121.5, 119.6, 119.4, 118.4, 42.7, 38.7, 30.3, 29.4, 28.7, 28.1, 26.5, 25.6, 22.5, 21.2. HRMS (ESI) m/z: calcd for C₂₈H₃₂ClF₄N₅O₆ [M+H]⁺: 646.1977; found: 646.2047.

(S)-1-(4-(3-(3-Chloro-4-fluorophenyl)ureido)-3-fluorobenzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28e).

According to general synthesis method 6, 27e was used as raw material. Light yellow solid 0.17 g, yield 58.6 %. m.p. 154–155 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.38 (s, 1H), 9.65 (s, 1H), 8.09 (t, J = 8.1 Hz, 1H), 7.84 (d, J = 5.9 Hz, 2H), 7.39−7.30 (m, 2H), 7.25 (d, J = 11.0 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 3.00 (s, 4H), 2.33−2.32 (m, 1H), 1.99−1.93 (m, 2H), 1.87−1.84 (m, 1H), 1.76 (s, 2H), 1.70−1.61 (m, 1H), 1.45−1.30 (m, 5H), 1.26−1.22 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.2, 152.9, 151.6, 137.6, 129.3, 129.2, 123.7, 121.3, 119.8, 119.7, 119.5, 118.9, 118.8, 117.4, 117.2, 42.8, 38.7, 34.9, 31.6, 30.3, 29.3, 28.7, 28.1, 26.5, 25.8, 22.5. HRMS (ESI) m/z: calcd for C₂₇H₃₂ClF₂N₅O₅ [M+H]⁺: 580.2060; found: 580.2134.

(S)-1-(3-Fluoro-4-(3-(4-fluoro-3-(trifluoromethyl)phenyl)ureido)benzoyl)-N-(7-(hydr-oxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28f).

According to general synthesis method 6, 27f was used as raw material. Light yellow solid 0.18 g, yield 58.1 %. m.p. 176–178 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.38 (s, 1H), 9.52 (s, 1H), 8.11−8.06 (m, 2H), 7.88 (s, 1H), 7.68 (s, 1H), 7.43 (s, 1H), 7.27 (d, J = 10.6 Hz, 1H), 7.17 (d, J = 7.7 Hz, 1H), 3.00 (s, 4H), 2.32 (s, 1H), 1.99−1.84 (m, 2H), 1.75 (s, 2H), 1.66−1.61 (m, 2H), 1.45−1.36 (m, 5H), 1.30−1.22 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.1, 153.0, 137.1, 130.8, 130.8, 129.1, 129.0, 124.6, 124.5, 124.4, 123.8, 121.7, 121.3, 118.2, 118.0, 116.2, 40.6, 38.7, 31.6, 30.3, 29.5, 29.4, 28.7, 28.1, 26.5, 25.6, 21.2. HRMS (ESI) m/z: calcd for C₂₈H₃₂F₅N₅O₅ [M+H]⁺: 614.2324; found: 614.2391.

(S)-1-(4-(3-(4-Chloro-3-fluorophenyl)ureido)-3-fluorobenzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28g).

According to general synthesis method 6, 27g was used as raw material. Light yellow solid 0.13 g, yield 44.8 %. m.p. 212–214 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.23 (s, 1H), 10.34 (s, 1H), 8.01 (t, J = 7.4 Hz, 1H), 7.89−7.88 (m, 1H), 7.75−7.66 (m, 2H), 7.44−7.42 (m, 1H), 7.29 (s, 1H), 7.23 (d, J = 10.9 Hz, 1H), 7.16 (d, J = 7.8 Hz, 1H), 3.00 (s, 4H), 2.32 (s, 1H), 1.93−1.84 (m, 2H), 1.74 (s, 4H), 1.45−1.35 (m, 5H), 1.26−1.21 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.2, 158.8, 156.4, 153.9, 153.2, 151.4, 141.6, 141.5, 132.0, 130.7, 123.6, 122.1, 115.5, 106.6, 106.4, 46.2, 38.7, 31.6, 30.3, 29.3, 28.7, 28.1, 26.5, 22.5, 19.1. HRMS (ESI) m/z: calcd for C₂₇H₃₂ClF₂N₅O₅ [M+H]⁺: 580.2060; found: 580.2148.

(S)-1-(3-Fluoro-4-(3-(3-fluoro-4-methylphenyl)ureido)benzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28h).

According to general synthesis method 6, 27h was used as raw material. Light yellow solid 0.15 g, yield 53.6 %. m.p. 171–173 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.22 (s, 1H), 9.60 (s, 1H), 8.12 (s, 1H), 7.89 (s, 1H), 7.70−7.67 (m, 1H), 7.47 (d, J = 12.1 Hz, 1H), 7.25 (d, J = 10.5 Hz, 1H), 7.17−7.10 (m, 3H), 3.01 (s, 4H), 2.32 (s, 1H), 2.16 (s, 3H), 1.93 (s, 2H), 1.84 (s, 2H), 1.66−1.61 (m, 2H), 1.45−1.36 (m, 5H), 1.30−1.22 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.2, 162.1, 159.8, 152.9, 139.8, 131.8, 131.8, 123.7, 121.1, 117.3, 117.1, 114.7, 114.3, 105.4, 105.1, 42.8, 38.7, 32.6, 31.6, 30.3, 29.4, 28.7, 28.1, 26.5, 25.6, 22.5, 14.0. HRMS (ESI) m/z: calcd for C₂₈H₃₅F₂N₅O₅ [M+H]⁺: 560.2606; found: 560.2675.

(S)-1-(3-Fluoro-4-(3-(4-fluoro-3-methylphenyl)ureido)benzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28i).

According to general synthesis method 6, 27i was used as raw material. Light yellow solid 0.15 g, yield 53.6 %. m.p. 184–186 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.27 (s, 1H), 9.74 (s, 1H), 8.10 (s, 1H), 7.90 (s, 1H), 7.71−7.66 (m, 1H), 7.42 (s, 1H), 7.37−7.33 (m, 1H), 7.23 (d, J = 10.9 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.03 (t, J = 8.4 Hz, 1H), 3.00−2.88 (m, 4H), 2.33 (s, 1H), 2.21 (s, 1H), 1.93−1.84 (m, 2H), 1.73−1.64 (m, 4H), 1.44−1.35 (m, 5H), 1.26−1.22 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.2, 157.5, 155.2, 153.1, 136.4, 129.7, 124.6, 123.7, 121.5, 121.2, 117.7, 115.4, 115.2, 114.7, 114.4, 42.8, 38.7, 34.9, 34.7, 31.7, 31.6, 30.3, 29.5, 28.1, 26.5, 22.5, 14.8. HRMS (ESI) m/z: calcd for C₂₈H₃₅F₂N₅O₅ [M+H]⁺: 560.2606; found: 560.2680.

(S)-1-(4-(3-(3,5-Difluorophenyl)ureido)-3-fluorobenzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (28j).

According to general synthesis method 6, 27j was used as raw material. Light yellow solid 0.13 g, yield 46.4 %. m.p. 203–204 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.31 (s, 1H), 10.25 (s, 1H), 8.02−7.90 (m, 2H), 7.67 (s, 1H), 7.25−7.16 (m, 5H), 6.74 (s, 1H), 6.74 (s, 1H), 3.00 (s, 4H), 2.33 (s, 1H), 1.73−1.66 (m, 4H), 1.43−1.35 (m, 5H), 1.26−1.22 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.2, 164.3, 164.2, 161.9, 161.8, 153.1, 129.1, 124.8, 123.9, 123.6, 114.6, 101.4, 101.0, 38.7, 34.9, 31.7, 31.6, 30.5, 30.3, 29.5, 29.2, 28.1, 26.5, 22.5. HRMS (ESI) m/z: calcd for C₂₇H₃₂F₃N₅O₅ [M+H]⁺: 564.2356; found: 564.2442.

(S)-1-(4-(3-Benzhydrylureido)-3-fluorobenzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl) piperidine-3-carboxamide (28k).

According to general synthesis method 6, 27k was used as raw material. Light yellow solid 0.16 g, yield 51.6 %. m.p. 143–144 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.44 (s, 1H), 8.79 (s, 1H), 8.20 (t, J = 8.3 Hz, 1H), 7.92−7.90 (m, 2H), 7.37−7.32 (m, 9H), 7.27−7.22 (m, 3H), 7.11 (d, J = 8.5 Hz, 1H), 5.98 (d, J = 7.9 Hz, 1H), 3.01 (s, 4H), 2.33−2.30 (m, 1H), 1.94 (s, 2H), 1.86−1.83 (m, 2H), 1.66−1.60 (m, 3H), 1.47−1.34 (m, 5H), 1.26−1.23 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 172.7, 168.2, 154.3, 152.3, 149.9, 143.5, 143.5, 130.1, 130.0, 129.4, 129.3, 129.0, 127.4, 127.3, 123.9, 119.4, 114.5, 114.3, 42.8, 38.7, 31.6, 30.0, 29.4, 28.7, 28.2, 26.7, 26.5, 25.6, 22.5. HRMS (ESI) m/z: calcd for C₃₄H₄₀FN₅O₅ [M+H]⁺: 618.3013; found: 618.3086.

Ethyl (S)-1-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)-3-fluorobenzyl)piperid-ine-3-carboxylate (30).

29 (2.14 g, 7.63 mmol), diethyl squaric acid (1.29 g, 7.63 mmol), TEA (0.93 g, 9.16 mmol) and EtOH (0.93g, 9.16mmol) were added into a three-necked bottle. The reaction mixture was stirred at 40 °C for 3 h. Then, filtering, the filtrate was evaporated reduced pressure. The crude was dissolved in dichloromethane, and H₂O was employed to wash the organic phase. The dichloromethane layer was then dried with anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure.

Light yellow solid 2.5 g, yield 81.2 %. m.p. 153–156 °C.

Ethyl (S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-dioxocycl-obut-1-en-1-yl)amino)-3-fluorobenzyl)piperidine-3-carboxylate (31).

30 (2.5 g, 6.19 mmol), memantine (1.11 g, 6.19 mmol), TEA (1.25 g, 12.4 mmol) and EtOH (30 mL) were added into a three-necked bottle. and the mixture was warmed to reflux for 8 h. Then, there is solid precipitation in the reaction solution, filtering. Light yellow solid 2.9 g, yield 87.9 %. m.p. 123–124 °C.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-fluorobenzyl)piperidine-3-carboxylic acid (32).

According to general synthesis method 4, 31 was used as raw material. Light yellow solid 2.4 g, yield 87.3 %. m.p. 130–133 °C.

Methyl 4-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-dioxo-cyclobut-1-en-1-yl)amino)benzyl)piperidine-3-carboxamido)butanoate (33a).

According to general synthesis method 5, 32 and methyl 4-aminobutyrate hydrochloride were used as raw material. Light yellow solid 0.36 g, yield 81.8 %. m.p. 126–129 °C.

Methyl 5-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-diox-ocyclobut-1-en-1-yl)amino)-3-fluorobenzyl)piperidine-3-carboxamido)pentanoate (33b).

According to general synthesis method 5, 32 and pentanoic acid-5-amino-methyl ester were used as raw material. Light yellow solid 0.35 g, yield 77.8 %. m.p. 139–143 °C.

Methyl 6-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-dioxo-cyclobut-1-en-1-yl)amino)-3-fluorobenzyl)piperidine-3-carboxamido)hexanoate (33c).

According to general synthesis method 5, 32 and methyl 6-aminocaproate hydrochloride were used as raw material. Light yellow solid 0.36 g, yield 78.3 %. m.p. 133–135 °C.

Methyl 7-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-dioxo-cyclobut-1-en-1-yl)amino)-3-fluorobenzyl)piperidine-3-carboxamido)heptanoate (33d).

According to general synthesis method 5, 32 and ethyl 7-aminoheptanoate were used as raw material. Light yellow solid 0.35 g, yield 74.5 %. m.p. 141–145 °C.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)benzyl)-N-(4-(hydroxyamino)-4-oxobutyl)piperidine-3-carboxamide (34a).

According to general synthesis method 6, 33a was used as raw material. Light yellow solid 0.19 g, yield 63.3 %. m.p. 146–148 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.32 (s, 1H), 9.36 (s, 1H), 8.25 (s, 1H), 7.82 (t, 1H, J = 8.6 Hz), 7.63 (d, 1H, J = 11.7 Hz), 7.40−7.35 (m, 2H), 7.25−7.23 (m, 1H), 3.05−3.00 (m, 3H), 2.89−2.87 (m, 3H), 2.19−2.15 (m, 2H), 1.84 (s, 2H), 1.69−1.66 (m, 2H), 1.61−1.58 (m, 2H), 1.46−1.33 (m, 2H), 1.28 (s, 3H), 1.24−1.20 (m, 7H), 1.14 (s, 2H), 0.85 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 183.0, 180.3, 172.8, 169.9, 169.3, 164.6, 140.7, 130.3, 128.9, 177.9, 54.9, 50.1, 49.1, 42.1, 41.4, 41.2, 40.6, 38.5, 34.6, 32.8, 31.4, 30.3, 30.2, 28.9, 28.2, 26.8, 25.7, 25.2, 22.9, 22.5. HRMS (ESI) m/z: calcd for C₃₃H₄₄FN₅O₅ [M+H]⁺: 610.3326; found: 610.3404.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-fluorobenzyl)-N-(5-(hydroxyamino)-5-oxopentyl)piperidine-3-carboxamide (34b).

According to general synthesis method 6, 33b was used as raw material. Light yellow solid 0.17 g, yield 54.8 %. m.p. 120–121 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.33 (s, 1H), 9.22 (s, 1H), 8.21 (s, 1H), 7.69−7.67 (m, 1H), 7.57 (s, 1H), 7.42−7.40 (m, 1H), 7.35 (s, 1H), 7.18−7.12 (m, 1H), 3.21 (s, 2H), 3.05−3.03 (m, 3H), 2.87−2.81 (m, 2H), 2.71 (s, 1H), 2.18−2.15 (m, 1H), 2.02−2.00 (m, 2H), 1.84 (s, 2H), 1.69−1.66 (m, 2H), 1.61−1.58 (m, 2H), 1.51−1.44 (m, 2H), 1.34−1.28 (m, 2H), 1.24−1.18 (m, 8H), 1.34−1.10 (m, 2H), 0.84 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 183.8, 180.1, 170.4, 164.5, 143.8, 133.7, 133.1, 129.6, 129.1, 128.9, 126.4, 124.8, 55.1, 50.1, 49.0, 45.8, 42.1, 33.7, 32.7, 32.4, 31.7, 30.4, 30.2, 30.0, 29.5, 29.1, 28.8, 26.9, 25.1, 22.5, 22.3. ESI-MS (m/z): 624.3 [M+H]⁺.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-fluorobenzyl)-N-(6-(hydroxyamino)-6-oxohexyl)piperidine-3-carboxamide (34c).

According to general synthesis method 5, 33c was used as raw material. Light yellow solid 0.18 g, yield 56.3 %. m.p. 128–129 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.09 (s, 1H), 9.82 (s, 1H), 8.56 (s, 1H), 7.55 (t, 1H, J = 5.04 Hz), 7.50 (t, 1H, J = 8.52 Hz), 6.90 (d, 1H, J = 11.56 Hz), 6.81 (d, 1H, J = 11.56 Hz), 3.17 (s, 2H), 2.73 (s, 2H), 2.65 (s, 1H), 2.45−2.44 (m, 2H), 2.08 (s, 1H), 1.91−1.90 (m, 1H), 1.67−1.64 (m, 2H), 1.60 (s, 2H), 1.45−1.34 (m, 4H), 1.23−1.18 (m, 4H), 1.15−1.10 (m, 4H), 1.04−0.98 (m, 6H), 0.94−0.90 (m, 3H), 0.60 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 183.5, 180.2, 173.6, 170.1, 164.8, 154.1, 151.7, 135.4, 135.3, 125.3, 122.1, 115.9, 61.8, 56.2, 54.9, 53.5, 52.7, 50.1, 49.1, 46.4, 43.1, 42.1, 41.9, 41.4, 32.7, 32.4, 30.4, 30.2, 30.0, 29.3, 26.5, 25.3, 21.9. HRMS (ESI) m/z: calcd for C₃₅H₄₈FN₅O₅ [M+H]⁺: 638.3639; found: 638.3713.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-fluorobenzyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carb-oxamide (34d).

According to general synthesis method 6, 33d was used as raw material. Light yellow solid 0.16 g, yield 48.5 %. m.p. 156–158 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.43 (s, 1H), 10.34 (s, 1H), 9.21 (s, 1H), 8.18 (s, 1H), 7.83 (t, 1H, J = 8.48 Hz), 7.63 (d, 1H, J=11.92 Hz), 7.39 (d, 2H, J = 7.76 Hz), 3.20 (s, 3H), 2.99−2.98 (m, 2H), 2.89 (s, 4H), 2.17−2.15 (m, 1H), 1.93 (t, 2H, J = 7.24 Hz), 1.89−1.85 (m, 2H), 1.70−1.67 (m, 2H), 1.61−1.58 (m, 2H), 1.45(s, 4H), 1.37−1.34 (m, 4H), 1.29 (s, 2H), 1.25−1.20 (m, 6H), 1.14 (s, 2H), 0.85 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 183.8, 180.1, 170.4, 169.6, 164.5, 153.7, 151.3, 128.3, 125.7, 122.3, 119.1, 118.8, 55.1, 52.8, 50.1, 49.0, 46.3, 45.8, 42.1, 41.4, 34.1, 32.7, 32.7, 32.4, 31.6, 30.4, 30.2, 29.4, 29.2, 28.7, 26.5, 25.5, 24.9, 22.5. HRMS (ESI) m/z: calcd for C₃₆H₅₀FN₅O₅ [M+H]⁺: 652.3796; found: 652.3868.

Ethyl (S)-1-(4-((2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino)benzoyl)piperidine-3-carboxylate (36).

35 (2.02 g, 7.63 mmol), diethyl squaric acid (1.29 g, 7.63 mmol), TEA (0.93 g, 9.16 mmol) and EtOH (30 mL) were added into a three-necked bottle. The reaction mixture was stirred at 40 °C for 3 h. Then, filtering, the filtrate was evaporated reduced pressure. The crude was dissolved in dichloromethane, and H₂O was employed to wash the organic phase. The dichloromethane layer was then dried on anhydrous Na₂SO₄, filtered, and evaporated under reduced pressure. Light yellow solid 2.8 g, yield 91.8 %. m.p. 153–155 °C.

Ethyl (S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-dioxocyc-lobut-1-en-1-yl)amino)benzoyl)piperidine-3-carboxylate (37).

114 (2.80 g, 7.00 mmol), memantine (1.26 g, 7.00 mmol), TEA (1.41 g, 14.00 mmol) and EtOH (30 mL) were added into a 100 mL tthree-necked bottle. And the mixture was warmed to reflux for 8 h. Then, there is solid precipitation in the reaction solution, filtering. White solid 3.4 g, yield 83.7 %. m.p. 164–167 °C.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)benzoyl)piperidine-3-carboxylic acid (38).

According to general synthesis method 4, 37 was used as raw material. White solid 2.6 g, yield 85.8 %. m.p. 155–158 °C.

Methyl 4-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-dioxo-cyclobut-1-en-1-yl)amino)-3-fluorobenzoyl)piperidine-3-carboxamido)butanoate (39a).

According to general synthesis method 5, 38 and methyl 4-aminobutyrate hydrochloride were used as raw material. Light yellow solid 0.34 g, yield 79.1 %. m.p. 175–178 °C.

Methyl 5-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-diox-ocyclobut-1-en-1-yl)amino)benzoyl)piperidine-3-carboxamido)pentanoate (39b).

According to General synthesis method 5, 38 and pentanoic acid-5-amino-methyl ester are used as raw material. Light yellow solid 0.35 g, yield 79.5 %. m.p. 182–185 °C.

Methyl 6-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-dioxo cyclobut-1-en-1-yl)amino)benzoyl)piperidine-3-carboxamido)hexanoate (39c).

According to general synthesis method 5, 38 and methyl 6-aminocaproate hydrochloride were used as raw material. Light yellow solid 0.34 g, yield 75.5 %. m.p. 169–172 °C.

Methyl 7-((S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)amino)-3,4-diox-ocyclobut-1-en-1-yl)amino)benzoyl)piperidine-3-carboxamido)heptanoate (39d).

According to general synthesis method 5, 38 and ethyl 7-aminoheptanoate were used as raw material. Light yellow solid 0.37 g, yield 79.7 %. m.p. 140–143 °C.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-fluorobenzoyl)-N-(4-(hydroxyamino)-4-oxobutyl)piperidine-3-carboxamide (40a).

According to general synthesis method 6, 39a was used as raw material. Light yellow solid 0.21 g, yield 63.3 %. m.p. 201–202 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.14 (s, 1H), 9.76 (s, 1H), 8.18 (s, 1H), 7.90 (s, 1H), 7.55 (d, 2H, J = 8.16 Hz), 7.55 (d, 2H, J = 8.28 Hz), 3.02−3.01 (m, 3H), 2.33−2.30 (m, 1H), 2.28 (s, 1H), 2.16 (s, 2H), 1.94 (s, 3H), 1.84−1.65 (m, 7H), 1.62−1.47 (m, 4H), 1.39−1.27 (m, 5H), 1.26 (s, 2H), 0.86 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 183.0, 180.3, 172.8, 169.9, 169.3, 164.6, 140.7, 130.3, 128.9, 177.9, 54.9, 50.1, 49.1, 42.1, 41.4, 41.2, 40.6, 38.5, 34.6, 32.8, 31.4, 30.3, 30.2, 28.9, 28.2, 26.8, 25.7, 25.2, 22.9, 22.5. HRMS (ESI) m/z: calcd for C₃₃H₄₂N₅O₆ [M+H]⁺: 606.3213; found: 606.3275.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)benzoyl)-N-(5-(hydroxyamino)-5-oxopentyl)piperidine-3-carbo-xamide (40b).

According to general synthesis method 6, 39b was used as raw material. Light yellow solid 0.18 g, yield 58.1 %. m.p. 194–195 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.90 (s, 1H), 10.35 (s, 1H), 8.81 (s, 1H), 7.63 (s, 1H), 7.62 (d, 2H, J = 7.88 Hz), 7.34 (d, 2H, J = 8.08 Hz), 3.02 (s, 3H), 2.31−2.28 (m, 1H), 2.15 (s, 1H), 1.94−1.91 (m, 3H), 1.85−1.70 (m, 2H), 1.67−1.58 (m, 5H), 1.46 (s, 2H), 1.37−1.23 (m, 5H), 1.15 (s, 2H), 0.85 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 182.8, 180.1, 172.8, 172.5, 170.1, 169.5, 169.4, 164.8, 140.9, 130.1, 128.8, 117.8, 55.0, 50.1, 49.0, 42.1, 41.2, 38.5, 32.7, 32.4, 30.4, 30.2, 29.5, 29.2, 29.1, 28.2, 23.0, 21.5. HRMS (ESI) m/z: calcd for C₃₄H₄₄N₅O₆ [M+H]⁺: 620.3370; found: 620.3435.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)benzoyl)-N-(6-(hydroxyamino)-6-oxohexyl)piperidine-3-carbo-xamide (40c).

According to general synthesis method 6, 39c was used as raw material. Light yellow solid 0.16 g, yield 50.4 %. m.p. 211–213 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.72 (s, 1H), 10.34 (s, 1H), 8.67 (s, 1H), 7.88 (s, 1H), 7.60 (d, 2H, J = 7.76 Hz), 7.35 (d, 2H, J = 8.00 Hz), 3.01−3.00 (m, 3H), 2.33−2.31 (m, 1H), 2.16 (s, 1H), 1.94−1.91 (m, 2H), 1.85 (s, 3H), 1.70−1.59 (m, 6H), 1.50−1.45 (m, 2H), 1.39−1.35 (m, 5H), 1.29−1.26 (m, 6H), 1.15 (s, 3H), 0.86 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 182.9, 180.1, 172.7, 170.0, 169.5, 169.4, 164.7, 140.9, 130.1, 128.8, 117.8, 54.9, 50.1, 49.0, 42.1, 38.6, 32.7, 32.6, 30.4, 30.2, 29.3, 28.2, 26.8, 26.4, 25.3, 22.9, 22.5, 21.5. HRMS (ESI) m/z: calcd for C₃₅H₄₆N₅O₆ [M+H]⁺: 634.3526; found: 634.3600.

(S)-1-(4-((2-(((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)benzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (40d).

According to general synthesis method 6, 39d was used as raw material. Light yellow solid 0.18 g, yield 55.5 %. m.p. 149–151 °C. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 10.33 (s, 1H), 10.20 (s, 1H), 8.26 (s, 1H), 7.86 (s, 1H), 7.56 (d, 2H, J = 7.52 Hz), 7.36 (d, 2H, J = 7.96 Hz), 3.41 (s, 2H), 3.00 (s, 2H), 2.31 (s, 1H), 2.17 (s, 1H), 1.91 (s, 4H), 1.84 (s, 2H), 1.69−1.60 (m, 6H), 1.46(s, 2H), 1.38−1.35 (m, 4H), 1.30−1.27 (m, 3H), 1.23 (s, 5H), 1.16 (s, 2H), 0.86 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆): δ (ppm) 182.9, 180.2, 172.7, 172.5, 170.0, 169.6, 169.3, 164.6, 140.7, 130.2, 128.9, 117.9, 54.9, 50.1, 49.1, 42.1, 41.4, 38.7, 32.8, 32.7, 32.4, 30.3, 30.2, 29.5, 29.4, 29.2, 28.7, 28.2, 26.5, 25.5, 22.6, 21.6. HRMS (ESI) m/z: calcd for C₃₆H₄₈N₅O₆ [M+H]⁺: 648.3683; found: 648.3753.

Biological activity assays in vitro.

All HsEH and MsEH the IC₅₀ values were determined using a fluorescent-based assay [(3-phenyloxiranyl) acetic acid cyano(6-methoxynaphthalen-2-yl)methyl ester (PHOME) as the substrate.³⁷ The fluorescent assay was used with purified recombinant human, or mouse sEH proteins. The enzymes were incubated at 37 °C with the inhibitors ([I]_final = 0.4–100,000 nM) for 10 min in 25 mM Tris HCl buffer (pH = 7.4) containing 0.1 mg/mL of BSA and 1 % of DMSO. The substrate (PHOME) was then added ([S]_final = 50 µM). Activity was assessed by measuring the formation of the fluorescent 6-methoxynaphthaldehyde product (lex=330 nm, lem=465 nm) on a SpectraMax M2 (molecular devices). Results were obtained by regression analysis from a linear region of the curve.

The general procedure for the enzyme-based assay was carried out according to published protocols.³⁸ with slight modification. The recombinant human HDAC1, 3 and 6 were purchased from BPS Bioscience (USA). All reactions were performed in the black half area 96-well plates. A serial dilution of the inhibitors (5 μL/well) and enzymes (5 μL/well) were pre-incubated in HDAC buffer (10 μL/well) at 25 °C for 15 min, then fluorogenic substrate (5 μL/well) Boc-Lys(Ac)-AMC was added. After incubation at 37 °C for 60 min, the mixture was stopped by the addition of developer (25 μL/well) for 10 min. Fluorescence intensity was measured using the Thermo Scientific Varioskan Flash Station at excitation and emission wavelengths of 355 and 460 nm, respectively.

Microsomal stability.

SD rat hepatic microsome was purchased from Research Institute for Liver Diseases (Shanghai, China) Co., Ltd. The incubation mixture consisted of microsomal protein in PBS buffer (100 mM, pH = 7.4) and 100 µg / mL 16d, 28g, 28j and 34d in a final volume of 100 mL. The concentration of human hepatic microsomal protein was 0.5 mg/mL (0.53 mg / mL of SD rat). A 0.83 mg/mL NADPH solution was prepared and added to the PBS buffer. After the addition of the NADPH-generating system, the resulting mixture was incubated at 37 °C for 0, 10, 30, 45, 60 min. The reaction was terminated by the addition of methanol 300 µL containing diphenhydramine (0.02 ng/mL). The mixture was vortexed for 1 min, centrifuged at 13000 rpm for 10 min at 4 °C. After centrifugation, 200 µL of the supernatant was transferred to 96-well plate, and 400 mL of purified water was added, and the solution was mixing with shaker at 500 rpm for 5 min, and the final concentration of compound 16d, 28g, 28j and 34d were analyzed by HPLC.

Pharmacokinetics study in vivo.

SPF-grade healthy Sprague-Dawley Rat(4 females, 4 males, 8 weeks old), weighing 180 ± 20 g, were provided by Liaoning Changsheng Biotechnology Co., Ltd (Liaoning, China), license No. SCXK (Liao) 2015–0001. SD rats were housed under controlled environmental conditions at 22–24 °C, 50–60 % relative humidity, natural circadian rhythm, free access to water and food, and acclimatized for one week. The experiments were approved by the animal management and use committee of Shenyang Pharmaceutical University, and the experiments conformed to the relevant experimental animal research guidelines of the ethics committee. Eight SD rats were randomly divided into two groups (half male and half female). Weigh before each dose to adjust the required volume. Four SD rats were given 10 mg/kg of compound 28g or 28j by tail vein injection, and the other four SD rats were given 50 mg/kg of compound 28g or 28j by gavage. Blood samples (iv groups) were collected at different times (5 min, 15 min, 30 min, 60 min, 120 min, 240 min, 480 min, 720 min). Blood samples (ig groups) were collected at different times (5 min, 15 min, 30 min, 60 min, 120 min, 240 min, 480 min, 720 min). Blood was collected from the orbital vein on an Eppendorf tube (1.5 mL) containing EDTA and then centrifuged at 4800 rpm for 10 minutes. Each plasma sample (50 µL) was mixed with 150 µL tolbutamide in acetonitrile (5 ng/mL) and centrifuged at 13000 rpm for 10 min. Afterwards, 50 µL of supernatant was reconstituted with 200 µL of acetonitrile and the final concentration of compound 28g or 28j was analyzed by HPLC.

Plasma protein binding rate.

The detailed experimental procedure followed the procedures reported of our previously work.³⁹ The experimental assay was calibrated using warfarin of known plasma protein binding rate: rat plasma (99.5 %).

In Vivo Efficacy.

Kunming mice (males, body weight 20–22 g) used for pharmacological experiments were provided by Liaoning Changsheng Biotechnology Co., Ltd (Liaoning, China), License No. SCXK (Liao) 2015–0001. All mice were kept under controlled environmental conditions at 22–24 °C, 50–60 % relative humidity, natural circadian rhythm, free access to water and food, and acclimatized for one week. The experiments were approved by the Animal Management and use committee of Shenyang Pharmaceutical University, and the experiments conformed to the relevant experimental animal research guidelines of the ethics committee.

Evaluation of the efficacy of compound 28g in mice with xylene-induced ear edema inflammation model.

In order to evaluation the synergistic effect of sEH and HDAC6 against mice ear swelling, the following experimental scheme was designed. Thirty Kunming mice (male, weighing 20–22 g) were randomly divided into five groups: model group, GL-B437 (30 mg/kg) group, Rocilinostat (30 mg/kg) group, GL-B437 + Rocilinostat (30 mg/kg + 30 mg/kg) group and 28g (30 mg/kg) group. 28g, GL-B437 and Rocilinostat were suspended in 0.1% sodium carboxymethyl cellulose aqueous solution. 10 mL/kg/d was given by gavage for 7 days, and the model group was replaced with normal saline. After the last administration for 1 h, the right ear of each group of mice was evenly smeared with 20 μL xylene, and after 30 min, the mice were killed by taking off their necks, cutting off two ears, overlapping them and tapping the ear piece with a 6 mm puncher. The weight of both ears was recorded, and the swelling degree and swelling inhibition rate were calculated.

In order to evaluation the inhibitory effect of different doses of 28g against mice ear swelling, the following experimental scheme was designed. Twenty-four Kunming mice (male, weighing 20–22 g) were randomly divided into four groups: model group, 28g (10 mg/kg) group, 28g (30 mg/kg) group and Celecoxib (10 mg/kg) group. 28g and Celecoxib were suspended in 0.1% sodium carboxymethyl cellulose aqueous solution. 10 mL/kg/d was given by gavage for 7 days, and the model group was replaced with normal saline. After the last administration for 1 h, the right ear of each group of mice was evenly smeared with 20 μL xylene, and after 30 min, the mice were killed by taking off their necks, cutting off two ears, overlapping them and tapping the ear piece with a 6 mm puncher. The weight of both ears was recorded, and the swelling degree and swelling inhibition rate were calculated.

Evaluation of the efficacy of compound 28g on formalin-induced inflammatory pain model.

In order to evaluation the synergistic effect of sEH and HDAC6 against formalin-induced inflammatory pain, the following experimental scheme was designed. Thirty Kunming mice (male, weighing 20–22 g) were randomly divided into six groups: normal group, model group, GL-B437 (30 mg/kg) group, Rocilinostat (30 mg/kg) group, GL-B437 + Rocilinostat (30 mg/kg + 30 mg/kg) group and 28g (30 mg/kg) group. The corresponding compounds were injected intraperitoneally in each group, and normal group and model group were replaced with normal saline. After administration, put it into a transparent experimental box to adapt to the environment. After administration for 30 min, the 5% formalin solution was injected subcutaneously into the right hind sole of mice, and the injection dose was 10μL/10g. The normal control group was replaced by normal saline, and then it was put into the experimental box. The pain response of each mouse was recorded within 1 h (based on licking/lifting/shaking paw behavior).

In order to evaluation the inhibitory effect of different doses of 28g against formalin-induced inflammatory pain, the following experimental scheme was designed.

Thirty Kunming mice (male, weighing 20–22 g) were randomly divided into five groups: normal group, model group, 28g (10 mg/kg) group, 28g (30 mg/kg) group and Celecoxib (10 mg/kg) group. The corresponding compounds were injected intraperitoneally in each group, and normal group and model group were replaced with normal saline. After administration, put it into a transparent experimental box to adapt to the environment. After administration for 30 min, the 5% formalin solution was injected subcutaneously into the right hind sole of mice, and the injection dose was 10μL/10g. The normal control group was replaced by normal saline, and then it was put into the experimental box. The pain response of each mouse was recorded within 1 h (based on licking/lifting/shaking paw behavior).

Cell Culture and Western Blot Analysis.

RAW 264.7 macrophage cell line was obtained from American Type Culture Collection (ATCC) and cultured in high glucose Dulbecco’s Modified Eagle’s Medium supplemented with 100 units/Ml penicillin, 100 μg/mL streptomycin and 10(v/v) heat-inactivated fetal bovine serum (FBS). The cells were treated with 1μg/mL LPS-stimulation and 28g for 12 hr. Total protein extracts (40 μg) were prepared by lysing cells in RIPA buffer (50 mM Tris-HCl, pH 8.0, 0.5% sodium deoxycholate, 100 μM leupeptin, 2 μg/mL aprotinin, 150 mM NaCl, 1% NP-40, 0.1% SDS and 1 mM phenylmethylsulfonyl fluoride). Protein concentrations in the lysates were determined using a BCA Protein Assay Kit (Beyotime Biotechnology). The samples were separated on SDS polyacrylamide gels and then transferred to nitrocellulose membranes and blocked with 5% nonfat dried milk. The membranes were incubated with antibodies to iNOS (sc-7271) and β-actin (sc-8432) overnight at 4 °C, all purchased from Santa Cruz Biotechnology. The immune-complexes were visualized using enhanced chemiluminescence Western blot detection reagents (Amersham Biosciences).

ELISA

The secretion of pro-inflammatory cytokines, including IL-6 and TNF-α, was measured with an ELISA assay kit (Jianglai, China). RAW 264.7 macrophages were treated with 1μg/mL LPS-stimulation and 28g for 12 hr. Thereafter, the supernatant was harvested and pro-inflammatory cytokine levels were determined using ELISA kits. Absorbance was measured at 450 nm using a microplate spectrophotometer (Bio-Tek). The quantities of cytokines secreted by RAW 264.7 cells were calculated using standard curves.

Molecular Docking.

The X-ray crystal structures of sEH (PDB ID: 3WKE) and HDAC6 (PDB ID: 5WGL) were retrieved from the Protein Data Bank. The protein structures were prepared using the Protein Preparation Wizard module. Hydrogen atoms were added, water molecules in the entire system were removed, followed by energy minimization and optimization by the Discovery Studio 2016 software. Grids of she and HDAC6 were generated using receptor-ligand interactions, following the standard procedure recommended by Discovery Studio 2016 software. The small molecules was employed to prepare the compounds for molecular docking. To prepare ligand structures, hydrogens were added, and 3D geometries, ionization, and tautomeric states were generated. Finally, the ligand structures were minimized using the full minimization. The conformational ensembles were docked flexibly using libdock. Only poses with low energy conformations and good hydrogen-bond geometries were considered.

MD Calculation.

Use Desmond v 3.8 program to verify and analyze data. Establish a periodic solvent model using System Builder for the prepared ligand receptor complex, using OPLS3e for the force field, SPC for the water solvent model, and a cube box (10 Å) for the periodic box × (10 Å × 10 Å), add Na⁺ and Cl^- with the same amount and opposite charge as the system as counterions, maintain a salt concentration of 0.15 M in the periodic box, and prepare the kinetic structure. The prepared periodic box is optimized through the minimization module to eliminate adverse collisions. The maximum number of iterations is 2000, and the energy convergence value is 100 kcal/mol/Å. The optimization model uses the LBFGS method, where the convergence limit of the gradient conjugate method is 25 kcal/mol/Å. Afterward, it is converted to the steepest descent method, and the cutoff value of the short-range interaction is 9.0 Å. The optimized structure runs in the product dynamics stage using an NPT ensemble, with a temperature control of Nose Hoover hot bath, a reference temperature of 300 K, and a pressure control of Martyna Tobias Klein pressure bath, with a reference pressure of 1 atm. The simulation duration is 500 ns, and energy and coordinate information are output every 0.1 ns. The short-range interaction cutoff value is 9.0 Å, and random numbers are used as the initial velocity seeds. After the product dynamics operation is completed, the generated dynamics trajectory is analyzed using the Simulation Interaction Diagram module, providing information such as RMSD and RMSF. The Simulation Event Analysis module analyzes the atomic distance of the trajectory.

Supplementary Material

Supporting Info

NIHMS2016629-supplement-Supporting_Info.pdf^{(9MB, pdf)}

ACKNOWLEDGMENTS

This work was funded by the National Natural Science Foundation of China (No. 82273793), Key Research Project of Department of Education of Liaoning Province (JYTZD2023134), NIEHS RIVER Award R35ES030443, NINDS Counter Act Program U54NS127758, and NIEHS/Superfund Research Program P42 ES004699.

ABBREVIATIONS USED

AA: arachidonic acid
DMLs: designed multiple ligands
EETs: epoxy-eicosatrienoic acids
HDAC6: histone deacetylase6
HRMS: high resolution mass spectrum
HsEH: human soluble epoxide hydrolase
MsEH: murine soluble epoxide hydrolase
NF-κB: nuclear factor kappa-B
NLRP3: NOD-like receptor thermal protein domain associated protein 3
sEH: soluble epoxide hydrolase

Footnotes

ASSOCIATED CONTENT

Supporting Information

This material is available free of charge via the Internet at http://pubs.acs.org. Supplemental figures and tables; NMR, MS spectra; and HPLC purity traces for all final compounds demonstrating ≥ 95% purity (PDF), Molecular formula strings (CSV)

sEH – 28g complex (PDB)

sEH – GL-B437 complex (PDB)

sEH – t-AUCB complex (PDB)

HDAC6 – Rocilinostat complex (PDB)

HDAC6 – 28g complex (PDB)

The authors declare no competing financial interest

REFERENCES

1.Medzhitov R Origin and physiological roles of inflammation. Nature, 2008, 454, 428–435. [DOI] [PubMed] [Google Scholar]
2.Wang D; Dubois RN Eicosanoids and cancer. Nat. Rev. Cancer, 2010, 10, 181–193. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Grosser T; Ricciotti E; Fitzgerald GA The cardiovascular pharmacology of nonsteroidal anti-inflammatory drugs. Trends. Pharmacol. Sci 2017, 38, 733–748. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Liu JY; Li N; Yang J; Li N; Qiu H; Ai D; Chiamvimonvat N; Zhu Y; Hammock BD Metabolic profiling of murine plasma reveals an unexpected biomarker in rofecoxib-mediated cardiovascular events. Proc. Natl. Acad. Sci. U.S.A 2010, 107, 17017–17022. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Capdevila JH; Falck JR; Harris RC Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase. J. Lipid Res 2000, 41, 163–181. [PubMed] [Google Scholar]
6.Spector AA; Norris AW Action of epoxyeicosatrienoic acids on cellular function. Am. J. Physiol. Cell. Physiol 2007, 292, C996–C1012. [DOI] [PubMed] [Google Scholar]
7.(a) Chaudhary KR; Pharm B; Abukhashim M; Hwang SH; Hammock BD Inhibition of soluble epoxide hydrolase by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is protective against ischemia-reperfusion injury. J. Cardiovasc. Pharmacol 2010, 55, 67–73. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Li N; Liu JY; Timofeyev V; Qiu H; Hwang SH; Tuteja D; Lu L; Yang J; Mochida H; Low R; Hammock BD; Chiamvimonvat N Beneficial effects of soluble epoxide hydrolase inhibitors in myocardial infarction model: insight gained using metabolomic approaches. J. Mol. Cell. Cardiol 2009, 47, 835–845. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.(a) Node K; Huo Y; Ruan X; Yang B; Spiecker M; Ley K; Zeldin DC; Liao JK Anti-inflammatory properties of cytochrome p450 epoxygenase-derived eicosanoids. Science. 1999, 285, 1276–1279. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Campbell WB New role for epoxyeicosatrienoic acids as antiinflammatory mediators. Trends. Pharmacol. Sci 2000, 21, 125–127. [DOI] [PubMed] [Google Scholar]
9.Inceoglu B; Jinks SL; Ulu A; Hegedus CM; Georgi K; Schmelzer KR; Wagner K; Jones PD; Morisseau C; Hammock BD Soluble epoxide hydrolase and epoxyeicosatrienoic acids modulate two distinct analgesic pathways. Proc. Natl. Acad. Sci. U.S.A 2008, 105, 18901–18906. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Spector AA; Fang X; Snyder GD; Weintraub NL Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog. Lipid Res 2004, 43, 55–90. [DOI] [PubMed] [Google Scholar]
11.Iyer MR; Kundu B; Wood CM Soluble epoxide hydrolase inhibitors: an overview and patent review from the last decade. Expert Opin Ther Pat 2022, 32, 629–647. [DOI] [PubMed] [Google Scholar]
12.Gui. Y; Li D; Chen J; Wang Y; Hu J; Liao C; Deng L; Xiang Q; Yang T; Du X; Zhang S; Xu D Soluble epoxide hydrolase inhibitors, t-AUCB, downregulated miR-133 in a mouse model of myocardial infarction. Lipids Health Dis 2018, 17, 129. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Hammock BD; McReynolds CB; Wagner K; Buckpitt A; Cortes-Puch I; Croston G; Lee KSS; Yang J; Schmidt WK; Hwang SH Movement to the clinic of soluble epoxide hydrolase inhibitor EC5026 as an analgesic for neuropathic pain and for use as a nonaddictive opioid alternative. J. Med. Chem 2021, 64, 1856–1872. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Manickam M; Meenakshisundaram S; Pillaiyar T Activating endogenous resolution pathways by soluble epoxide hydrolase inhibitors for the management of COVID-19. Arch. Pharm 2022, 355, 2100367. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Anandan S-K; Webb HK; Chen D; Wang Y-X; Aavula BR; Cases S; Cheng Y; Do ZN; Mehra U; Tran V 1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia. Bioorg. Med. Chem. Lett 2011, 21, 983–988. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Zhang X; Yuan Z; Zhang Y; Yong S; Salas-Burgos A; Koomen J; Olashaw N; Parsons JT; Yang XJ; Dent SR; Yao TP; Lane WS; Seto E HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol. Cell. 2007, 27, 197–213. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Kovacs JJ; Murphy PJ; Gaillard S; Zhao X; Wu JT; Nicchitta CV; Yoshida M; Toft DO; Pratt WB; Yao TP HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol. Cell. 2005, 18, 601–607. [DOI] [PubMed] [Google Scholar]
18.Hubbert C; Guardiola A; Shao R; Kawaguchi Y; Ito A; Nixon A; Yoshida M; Wang XF; Yao TP HDAC6 is a microtubule-associated deacetylase. Nature. 2002, 417, 455–458. [DOI] [PubMed] [Google Scholar]
19.Kalin JH; Bergman JA Development and therapeutic implications of selective histone deacetylase 6 inhibitors. J. Med. Chem 2013, 56, 6297–6313. [DOI] [PubMed] [Google Scholar]
20.Seidel C; Schnekenburger M; Dicato M; Diederich M Histone deacetylase 6 in health and disease. Epigenomics. 2015, 7, 103–118. [DOI] [PubMed] [Google Scholar]
21.Aldana-Masangkay GI; Sakamoto KM The role of HDAC6 in cancer. J. Biomed. Biotechnol 2011, 2011, 875824–875834. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Cheng C; Shan W; Huang W; Ding Z; Cui G; Liu F; Lu W; Xu J; He W; Yin Z ACY-1215 exhibits anti-inflammatory and chondroprotective effects in human osteoarthritis chondrocytes via inhibition of STAT3 and NF-κB signaling pathways. Biomed. Pharmacother 2019, 109, 2464–2471. [DOI] [PubMed] [Google Scholar]
23.Zhang WB; Yang F; Wang Y; Jiao FZ; Zhang HY; Wang LW; Gong ZJ Inhibition of HDAC6 attenuates LPS-induced inflammation in macrophages by regulating oxidative stress and suppressing the TLR4-MAPK/NF-κB pathways. Biomed. Pharmacother 2019, 117, 109166. [DOI] [PubMed] [Google Scholar]
24.Liu L; Zhou X; Shetty S; Hou G; Wang Q; Fu J HDAC6 inhibition blocks inflammatory signaling and caspase-1 activation in LPS-induced acute lung injury. Toxicol. Appl. Pharmacol 2019, 370, 178–183. [DOI] [PubMed] [Google Scholar]
25.Kalin JH; Bergman JA Development and therapeutic implications of selective histone deacetylase 6 inhibitors. J. Med. Chem 2013, 56, 6297–6313. [DOI] [PubMed] [Google Scholar]
26.Li X; Li X; Liu F; Li S; Shi D Rational multitargeted drug design strategy from the perspective of a medicinal chemist. J. Med. Chem 2021, 64, 10581–10605. [DOI] [PubMed] [Google Scholar]
27.Codony S; Pont C; Griñán-Ferré C; Di Pede-Mattatelli A; Calvó-Tusell C; Feixas F; Osuna S; Jarné-Ferrer J; Naldi M; Bartolini M; Loza MI; Brea J; Pérez B; Bartra C; Sanfeliu C; Juárez-Jiménez J; Morisseau C; Hammock BD; Pallàs M; Vázquez S; Muñoz-Torrero D Discovery and in vivo proof of concept of a highly potent dual inhibitor of soluble epoxide hydrolase and acetylcholinesterase for the treatment of alzheimer’s disease. J. Med. Chem 2022, 65, 4909–4925 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Morisseau C; Hammock BD Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu. Rev. Pharmacol. Toxicol 2005, 45, 311–333. [DOI] [PubMed] [Google Scholar]
29.Bzówka M; Mitusińska K; Hopko K; Góra A Computational insights into the known inhibitors of human soluble epoxide hydrolase. Drug. Discov. Today. 2021, 26, 1914–1921. [DOI] [PubMed] [Google Scholar]
30.Miyake Y; Keusch JJ; Wang L; Saito M; Hess D; Wang X; Melancon BJ; Helquist P; Gut H; Matthias P Structural insights into HDAC6 tubulin deacetylation and its selective inhibition. Nat. Chem. Biol 2016, 12, 748–754. [DOI] [PubMed] [Google Scholar]
31.Pulya S; Amin SA; Adhikari N; Biswas S; Jha T; Ghosh B HDAC6 as privileged target in drug discovery: a perspective. Pharmacol. Res, 2021, 163, 105274. [DOI] [PubMed] [Google Scholar]
32.Marino JP Soluble epoxide hydrolase, a Target with multiple opportunities for cardiovascular drug discovery. Curr. Top. Med. Chem 2009, 9, 452–463. [DOI] [PubMed] [Google Scholar]
33.Kylie AA-F; Craig MW Squaramides as Bioisosteres in Contemporary Drug Design. Chem Rev 2020, 120, 11616–11650. [DOI] [PubMed] [Google Scholar]
34.Agnew-Francis KA; Williams CM Squaramides as bioisosteres in contemporary drug design. Chem. Rev, 2020, 120, 11616–11650. [DOI] [PubMed] [Google Scholar]
35.Du FY; Cao RL; Chen L; Sun JW; Shi YJ; Fu Y; Hammock BD; Zheng ZH; Liu ZB; Chen GL Structure-guided discovery of potent and oral soluble epoxide hydrolase inhibitors for the treatment of neuropathic pain. Acta. Pharm. Sin. B 2022, 12, 1377–1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Wang MJ; Tang TK; Li RX; Huang ZH; Ling DZ; Zheng LL; Ding Y; Liu TP; Xu WY; Zhu F; Min H; Boonhok R; Mao; Zhu J; Li XK; Jiang LB; Li J Drug repurposing of quisinostat to discover novel plasmodium falciparum HDAC1 inhibitors with enhanced triple-stage antimalarial activity and improved safety. J. Med. Chem 2022, 65, 4156–4181. [DOI] [PubMed] [Google Scholar]
37.Inceoglu B; Jinks SL; Ulu A; Hegedus CM; Georgi K; Schmelzer KR; Wagner K; Jones PD; Morisseau C; Hammock BD Soluble epoxide hydrolase and epoxyeicosatrienoic acids modulate two distinct analgesic pathways. Proc. Natl. Acad. Sci. USA. 2008, 105, 18901–18906. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Wegener D; Wirsching F; Riester D; Schwienhorst A A fluorogenic histone deacetylase assay well suited for high-throughput activity screening. Chem. Biol 2003, 10, 61–68. [DOI] [PubMed] [Google Scholar]
39.Du F; Sun W; Morisseau C; Hammock BD; Bao X; Liu Q; Wang C; Zhang T; Yang H; Zhou J; Xiao W; Liu Z; Chen G Discovery of memantyl urea derivatives as potent soluble epoxide hydrolase inhibitors against lipopolysaccharide-induced sepsis. Eur. J. Med. Chem 2021, 223, 113678–113692. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Info

NIHMS2016629-supplement-Supporting_Info.pdf^{(9MB, pdf)}

[R1] 1.Medzhitov R Origin and physiological roles of inflammation. Nature, 2008, 454, 428–435. [DOI] [PubMed] [Google Scholar]

[R2] 2.Wang D; Dubois RN Eicosanoids and cancer. Nat. Rev. Cancer, 2010, 10, 181–193. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Grosser T; Ricciotti E; Fitzgerald GA The cardiovascular pharmacology of nonsteroidal anti-inflammatory drugs. Trends. Pharmacol. Sci 2017, 38, 733–748. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Liu JY; Li N; Yang J; Li N; Qiu H; Ai D; Chiamvimonvat N; Zhu Y; Hammock BD Metabolic profiling of murine plasma reveals an unexpected biomarker in rofecoxib-mediated cardiovascular events. Proc. Natl. Acad. Sci. U.S.A 2010, 107, 17017–17022. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Capdevila JH; Falck JR; Harris RC Cytochrome P450 and arachidonic acid bioactivation: molecular and functional properties of the arachidonate monooxygenase. J. Lipid Res 2000, 41, 163–181. [PubMed] [Google Scholar]

[R6] 6.Spector AA; Norris AW Action of epoxyeicosatrienoic acids on cellular function. Am. J. Physiol. Cell. Physiol 2007, 292, C996–C1012. [DOI] [PubMed] [Google Scholar]

[R7] 7.(a) Chaudhary KR; Pharm B; Abukhashim M; Hwang SH; Hammock BD Inhibition of soluble epoxide hydrolase by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is protective against ischemia-reperfusion injury. J. Cardiovasc. Pharmacol 2010, 55, 67–73. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Li N; Liu JY; Timofeyev V; Qiu H; Hwang SH; Tuteja D; Lu L; Yang J; Mochida H; Low R; Hammock BD; Chiamvimonvat N Beneficial effects of soluble epoxide hydrolase inhibitors in myocardial infarction model: insight gained using metabolomic approaches. J. Mol. Cell. Cardiol 2009, 47, 835–845. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] 8.(a) Node K; Huo Y; Ruan X; Yang B; Spiecker M; Ley K; Zeldin DC; Liao JK Anti-inflammatory properties of cytochrome p450 epoxygenase-derived eicosanoids. Science. 1999, 285, 1276–1279. [DOI] [PMC free article] [PubMed] [Google Scholar]; (b) Campbell WB New role for epoxyeicosatrienoic acids as antiinflammatory mediators. Trends. Pharmacol. Sci 2000, 21, 125–127. [DOI] [PubMed] [Google Scholar]

[R9] 9.Inceoglu B; Jinks SL; Ulu A; Hegedus CM; Georgi K; Schmelzer KR; Wagner K; Jones PD; Morisseau C; Hammock BD Soluble epoxide hydrolase and epoxyeicosatrienoic acids modulate two distinct analgesic pathways. Proc. Natl. Acad. Sci. U.S.A 2008, 105, 18901–18906. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Spector AA; Fang X; Snyder GD; Weintraub NL Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog. Lipid Res 2004, 43, 55–90. [DOI] [PubMed] [Google Scholar]

[R11] 11.Iyer MR; Kundu B; Wood CM Soluble epoxide hydrolase inhibitors: an overview and patent review from the last decade. Expert Opin Ther Pat 2022, 32, 629–647. [DOI] [PubMed] [Google Scholar]

[R12] 12.Gui. Y; Li D; Chen J; Wang Y; Hu J; Liao C; Deng L; Xiang Q; Yang T; Du X; Zhang S; Xu D Soluble epoxide hydrolase inhibitors, t-AUCB, downregulated miR-133 in a mouse model of myocardial infarction. Lipids Health Dis 2018, 17, 129. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] 13.Hammock BD; McReynolds CB; Wagner K; Buckpitt A; Cortes-Puch I; Croston G; Lee KSS; Yang J; Schmidt WK; Hwang SH Movement to the clinic of soluble epoxide hydrolase inhibitor EC5026 as an analgesic for neuropathic pain and for use as a nonaddictive opioid alternative. J. Med. Chem 2021, 64, 1856–1872. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] 14.Manickam M; Meenakshisundaram S; Pillaiyar T Activating endogenous resolution pathways by soluble epoxide hydrolase inhibitors for the management of COVID-19. Arch. Pharm 2022, 355, 2100367. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] 15.Anandan S-K; Webb HK; Chen D; Wang Y-X; Aavula BR; Cases S; Cheng Y; Do ZN; Mehra U; Tran V 1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia. Bioorg. Med. Chem. Lett 2011, 21, 983–988. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R16] 16.Zhang X; Yuan Z; Zhang Y; Yong S; Salas-Burgos A; Koomen J; Olashaw N; Parsons JT; Yang XJ; Dent SR; Yao TP; Lane WS; Seto E HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol. Cell. 2007, 27, 197–213. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] 17.Kovacs JJ; Murphy PJ; Gaillard S; Zhao X; Wu JT; Nicchitta CV; Yoshida M; Toft DO; Pratt WB; Yao TP HDAC6 regulates Hsp90 acetylation and chaperone-dependent activation of glucocorticoid receptor. Mol. Cell. 2005, 18, 601–607. [DOI] [PubMed] [Google Scholar]

[R18] 18.Hubbert C; Guardiola A; Shao R; Kawaguchi Y; Ito A; Nixon A; Yoshida M; Wang XF; Yao TP HDAC6 is a microtubule-associated deacetylase. Nature. 2002, 417, 455–458. [DOI] [PubMed] [Google Scholar]

[R19] 19.Kalin JH; Bergman JA Development and therapeutic implications of selective histone deacetylase 6 inhibitors. J. Med. Chem 2013, 56, 6297–6313. [DOI] [PubMed] [Google Scholar]

[R20] 20.Seidel C; Schnekenburger M; Dicato M; Diederich M Histone deacetylase 6 in health and disease. Epigenomics. 2015, 7, 103–118. [DOI] [PubMed] [Google Scholar]

[R21] 21.Aldana-Masangkay GI; Sakamoto KM The role of HDAC6 in cancer. J. Biomed. Biotechnol 2011, 2011, 875824–875834. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] 22.Cheng C; Shan W; Huang W; Ding Z; Cui G; Liu F; Lu W; Xu J; He W; Yin Z ACY-1215 exhibits anti-inflammatory and chondroprotective effects in human osteoarthritis chondrocytes via inhibition of STAT3 and NF-κB signaling pathways. Biomed. Pharmacother 2019, 109, 2464–2471. [DOI] [PubMed] [Google Scholar]

[R23] 23.Zhang WB; Yang F; Wang Y; Jiao FZ; Zhang HY; Wang LW; Gong ZJ Inhibition of HDAC6 attenuates LPS-induced inflammation in macrophages by regulating oxidative stress and suppressing the TLR4-MAPK/NF-κB pathways. Biomed. Pharmacother 2019, 117, 109166. [DOI] [PubMed] [Google Scholar]

[R24] 24.Liu L; Zhou X; Shetty S; Hou G; Wang Q; Fu J HDAC6 inhibition blocks inflammatory signaling and caspase-1 activation in LPS-induced acute lung injury. Toxicol. Appl. Pharmacol 2019, 370, 178–183. [DOI] [PubMed] [Google Scholar]

[R25] 25.Kalin JH; Bergman JA Development and therapeutic implications of selective histone deacetylase 6 inhibitors. J. Med. Chem 2013, 56, 6297–6313. [DOI] [PubMed] [Google Scholar]

[R26] 26.Li X; Li X; Liu F; Li S; Shi D Rational multitargeted drug design strategy from the perspective of a medicinal chemist. J. Med. Chem 2021, 64, 10581–10605. [DOI] [PubMed] [Google Scholar]

[R27] 27.Codony S; Pont C; Griñán-Ferré C; Di Pede-Mattatelli A; Calvó-Tusell C; Feixas F; Osuna S; Jarné-Ferrer J; Naldi M; Bartolini M; Loza MI; Brea J; Pérez B; Bartra C; Sanfeliu C; Juárez-Jiménez J; Morisseau C; Hammock BD; Pallàs M; Vázquez S; Muñoz-Torrero D Discovery and in vivo proof of concept of a highly potent dual inhibitor of soluble epoxide hydrolase and acetylcholinesterase for the treatment of alzheimer’s disease. J. Med. Chem 2022, 65, 4909–4925 [DOI] [PMC free article] [PubMed] [Google Scholar]

[R28] 28.Morisseau C; Hammock BD Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu. Rev. Pharmacol. Toxicol 2005, 45, 311–333. [DOI] [PubMed] [Google Scholar]

[R29] 29.Bzówka M; Mitusińska K; Hopko K; Góra A Computational insights into the known inhibitors of human soluble epoxide hydrolase. Drug. Discov. Today. 2021, 26, 1914–1921. [DOI] [PubMed] [Google Scholar]

[R30] 30.Miyake Y; Keusch JJ; Wang L; Saito M; Hess D; Wang X; Melancon BJ; Helquist P; Gut H; Matthias P Structural insights into HDAC6 tubulin deacetylation and its selective inhibition. Nat. Chem. Biol 2016, 12, 748–754. [DOI] [PubMed] [Google Scholar]

[R31] 31.Pulya S; Amin SA; Adhikari N; Biswas S; Jha T; Ghosh B HDAC6 as privileged target in drug discovery: a perspective. Pharmacol. Res, 2021, 163, 105274. [DOI] [PubMed] [Google Scholar]

[R32] 32.Marino JP Soluble epoxide hydrolase, a Target with multiple opportunities for cardiovascular drug discovery. Curr. Top. Med. Chem 2009, 9, 452–463. [DOI] [PubMed] [Google Scholar]

[R33] 33.Kylie AA-F; Craig MW Squaramides as Bioisosteres in Contemporary Drug Design. Chem Rev 2020, 120, 11616–11650. [DOI] [PubMed] [Google Scholar]

[R34] 34.Agnew-Francis KA; Williams CM Squaramides as bioisosteres in contemporary drug design. Chem. Rev, 2020, 120, 11616–11650. [DOI] [PubMed] [Google Scholar]

[R35] 35.Du FY; Cao RL; Chen L; Sun JW; Shi YJ; Fu Y; Hammock BD; Zheng ZH; Liu ZB; Chen GL Structure-guided discovery of potent and oral soluble epoxide hydrolase inhibitors for the treatment of neuropathic pain. Acta. Pharm. Sin. B 2022, 12, 1377–1389. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Wang MJ; Tang TK; Li RX; Huang ZH; Ling DZ; Zheng LL; Ding Y; Liu TP; Xu WY; Zhu F; Min H; Boonhok R; Mao; Zhu J; Li XK; Jiang LB; Li J Drug repurposing of quisinostat to discover novel plasmodium falciparum HDAC1 inhibitors with enhanced triple-stage antimalarial activity and improved safety. J. Med. Chem 2022, 65, 4156–4181. [DOI] [PubMed] [Google Scholar]

[R37] 37.Inceoglu B; Jinks SL; Ulu A; Hegedus CM; Georgi K; Schmelzer KR; Wagner K; Jones PD; Morisseau C; Hammock BD Soluble epoxide hydrolase and epoxyeicosatrienoic acids modulate two distinct analgesic pathways. Proc. Natl. Acad. Sci. USA. 2008, 105, 18901–18906. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R38] 38.Wegener D; Wirsching F; Riester D; Schwienhorst A A fluorogenic histone deacetylase assay well suited for high-throughput activity screening. Chem. Biol 2003, 10, 61–68. [DOI] [PubMed] [Google Scholar]

[R39] 39.Du F; Sun W; Morisseau C; Hammock BD; Bao X; Liu Q; Wang C; Zhang T; Yang H; Zhou J; Xiao W; Liu Z; Chen G Discovery of memantyl urea derivatives as potent soluble epoxide hydrolase inhibitors against lipopolysaccharide-induced sepsis. Eur. J. Med. Chem 2021, 223, 113678–113692. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Design and Synthesis of sEH/HDAC6 Dual-Targeting Inhibitors for the Treatment of Inflammatory Pain.

Huashen Xu

Yuanguang Chen

Hua Tong

Lu Chen

Christophe Morisseau

Zijian Zhou

Junning Zhuang

Chuqiao Song

Pengcheng Cai

Zhongbo Liu

Bruce D Hammock

Guoliang Chen

Abstract

INTRODUCTION

Figure 1.

Figure 2.

RESULTS AND DISCUSSION

Rational Design of the sEH/HDAC Dual-targeting Inhibitors.

Figure 3.

Figure 4.

Chemistry.

Scheme 1.

Scheme 2.

Scheme 3.

Scheme 4.

Scheme 5.

Scheme 6.

The structure-activity relationships of series derivatives.

Table 1.

Table 2.

Table 3.

Acetylation of α-Tubulin and Histone H3.

Figure 5.

Molecular docking.

Figure 6.

Figure 7.

Molecular dynamics simulation.

Figure 8.

Figure 9.

In vitro metabolic stabilities of the sEH/HDAC6 dual-targeting inhibitors in RLM.

Table 4.

Plasma protein binding rate.

Pharmacokinetic study in vivo.

Table 5.

The inhibitory effects of 28g in mice with xylene-induced ear swelling inflammation.

Table 6.

Figure 10.

Table 7.

Figure 11.

Effects of 28g against formalin-induced inflammatory pain in vivo.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Effects of 28g on the levels of inflammatory factors in LPS-induced RAW264.7 cells.

Figure 16.

CONCLUSIONS

EXPERIMENTAL SECTION

Chemistry.

General synthesis method 1 (Condensation of carboxylic acid with ethyl (S)- piperidine −3- carboxylate).

General synthesis method 2 (Reduction reaction of nitro group).

General synthesis method 3 (Preparation of urea).

General synthesis method 4 (Hydrolysis reaction of ester).

General synthesis method 5 (Condensation of carboxylic acid and amine).

General synthesis method 6 (Synthesis of hydroxamic acid).

Ethyl (S)-1-(3-fluoro-4-nitrobenzoyl)piperidine-3-carboxylate (2).

Ethyl (S)-1-(4-amino-3-fluorobenzoyl)piperidine-3-carboxylate (3).

Ethyl (S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl) piperidine-3-carboxylate (4).

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl) piperidine-3-carboxylic acid (5).

Methyl 4-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)butanoate (6a).

Methyl 5-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)pentanoate (6b).

Methyl 6-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)hexanoate (6c).

Methyl 7-((S)-1-(4-(3-((1r,3R,5S,7S)-3,5-dimethyladamantan-1-yl)ureido)-3-fluorobe-nzoyl)piperidine-3-carboxamido)heptanoate (6d).

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(4-(hydroxyamino)-4-oxobutyl)piperidine-3-carboxamide (7a).

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(5-(hydroxyamino)-5-oxopentyl)piperidine-3-carboxamide (7b).

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(6-(hydroxyamino)-6-oxohexyl)piperidine-3-carboxamide (7c).

(S)-1-(4-(3-((1r,3R,5S,7S)-3,5-Dimethyladamantan-1-yl)ureido)-3-fluorobenzoyl)-N-(7-(hydroxyamino)-7-oxoheptyl)piperidine-3-carboxamide (7d).

3-Fluoro-4-nitrophenyl)methanol (9).