Discovery of 1,5-Dihydro-4H-imidazol-4-one Derivatives as Potent, Selective Antagonists of CXC Chemokine Receptor 2

Abstract

CXC chemokine receptors 1 (CXCR1) and 2 (CXCR2) have been demonstrated to have critical roles in cancer metastasis. Because they share high homology sequences, it is still unclear how to design selective CXCR1 or CXCR2 antagonists. Based on a pharmacophore model we built, compound 2 bearing a 1,5-dihydro-4H-imidazol-4-one scaffold was identified as a selective CXCR2 antagonist with a low CXCR1 antagonism preference. Further optimization and structure–activity relationship studies led to compound C5 that overcame the disadvantages of compound 2 and performed with higher selectivity. It showed excellent oral bioavailability and in vitro anticancer metastasis activity. Further dynamic simulation of the molecular protein complex showed that the amino acid residue K320 of CXCR2 contributed most to the selectivity of C5. This study provides important clues for the design of new CXCR2 selective antagonists, and C5 can be a molecular tool for investigating the difference in the biological function of CXCR1 and CXCR2.

Chemokines are intercellular signaling proteins involved in different biological processes, including immunoreaction, hematopoiesis, and angiogenesis.¹⁻³ Based on the distance between the essential cysteine residues, they can be classified into the C, CC, CXC, and CX₃C classes. The CXC chemokine receptors (CXCRs), CXCR1 and CXCR2, contain seven transmembrane domains and function as G-protein-coupled receptors (GPCRs). The two proteins share 76% sequence homology and similar binding features of ligands.^4,5 Both of the receptors exhibit high binding affinity with CXCL6 and CXCL8, but CXCR2 also binds to other chemokines, including CXCL1–7.⁶

CXCR1 and CXCR2 have been demonstrated to play an essential role in cancer development by mediating cancer metastasis or assisting cancer cells in immune evasion.⁷ In recent years, CXCR1/2 antagonists with diverse structural features have been reported, including N,N′-diarylureas analogues and arylamide derivatives.⁷⁻⁹ Among them, some CXCR1/2 antagonists have been tested in clinical trials to treat metastatic cancer (Figure 1). Navarixin (also known as MK-7123 or SCH527123) is a CXCR1/2 antagonist bearing N,N′-diarylsquaramide scaffold,¹⁰ and a clinical trial has starting in combination with pembrolizumab (MK-3475) for treating programmed death-ligand 1 (PD-L1) positive refractory solid tumors (NCT03473925). AZD5069 and SX-682 are arylamide derivatives showing CXCR1/2 antagonistic activity.¹¹⁻¹⁶ A multicenter proof of concept Phase I/II clinical trial of AZD5069 combined with enzalutamide to treat metastatic castration-resistant prostate cancer (mCRPC) is now ongoing (NCT03177187). Additionally, SX-682 is currently in combination with other drugs in clinical trials for the treatment of metastatic cancer, including metastatic melanoma (NCT03161431), metastatic colorectal cancer (NCT04599140), metastatic pancreatic ductal adenocarcinoma (NCT04477343), and others. The outcomes of these clinical trials suggest a promising application of CXCR1/2 antagonists.

Figure 1.

Representative CXCR2 antagonists.

Among the compounds in clinical trials, the antagonistic activity of navarixin against CXCR1 is 10-fold less potent than CXCR2;¹⁰ AZD5069 displays 100-fold greater affinity for CXCR2 than CXCR1,¹⁷ and SX-682 is an antagonist that showed no selectivity against CXCR1/2.¹⁵ Because of the similar distribution and functions of CXCR1 and CXCR2, it is hard to tell the difference between the biological function of the two targets. There is still a need to explore highly selective CXCR1 or CXCR2 antagonists and illustrate the essential amino acid residues involved in achieving the selectivity of antagonists, thereby providing tools for biological research and clues for drug design. As a continuation of our interests in discovering selective CXCR1/2 antagonists,^7,18,19 we report a new series of CXCR2 selective antagonists.

In our previous study, compound 1 was identified as a CXCR2 antagonist with low activity (Figure 2A), exhibiting an IC₅₀ value of 15 μM. However, its further development was halted due to its low activity.¹⁸ After mapping the structure with the pharmacophore model that we built, we found that the nitrogen at 1-position of the triazole core did not match well with the pharmacophore region of hydrogen bond receptor. Therefore, a carbonyl was designed to replace the nitrogen and gave the 1,5-dihydro-4H-imidazol-4-one derivative compound 2. Then, compound 2 was synthesized and subjected to CXCR1 and CXCR2 antagonism tests. The compound showed a highly potent IC₅₀ value of 6 nM against CXCR2 and was 778-fold more potent against CXCR1.

Figure 2.

Discovery of 1,5-dihydro-4H-imidazol-4-one derivatives as CXCR2 antagonists. (A) Design rationale of compound 2. (B) Result of chiral HPLC of (S)-2. (C) Aromatization mechanism of 1,5-dihydro-4H-imidazol-4-one scaffold. (D) SIF and SGF stability of compound 2. (E) Further structure optimization of 1,5-dihydro-4H-imidazol-4-one derivatives. Results are shown as mean ± SD ****p < 0.0001 vs the remaining percentage at 0 min.

We noticed that the 5-position of 1,5-dihydro-4H-imidazol-4-one scaffold is a chiral center, which led to a 1:1 ratio of the two isomers using chiral high performance liquid chromatography (HPLC) analysis (Figure S1). To fully understand the difference of activity and selectivity of the two isomers, we tried to synthesize one of the isomers (S)-2 by employing (S)-2-amino-2-phenylacetamide as the starting material. However, chiral HPLC analysis also indicated racemization of (S)-2 (Figure 2B). It was hypothesized that this might function through an aromatization mechanism. Once scaffold I formed, a stable π-system can be induced by 5-phenyl of the scaffold to generate the intermediate II, which subsequently turns to a racemic scaffold III (Figure 2C).

Moreover, although compound 2 was stable in simulated gastric fluid (SGF), it was significantly unstable in simulated intestinal fluid (SIF), which showed a remaining percentage of 45.2% after 45 min incubation (Figure 2D). Therefore, a focused structure–activity relationship (SAR) effort was needed to increase its structural and metabolic stability according to the above results of the preliminary assessment of compound 2 (Figure 2E). Considering the importance of the phenol and the two urea-like -NH groups in maintaining the compounds’ antagonistic activity, these groups were retained during the following optimization. Moreover, the sulfonyl was designed to be replaced with other electron-withdrawing groups because electron-donating groups at this position showed disadvantages in contributing to CXCR2 antagonism of compounds.⁷ Aliphatic groups were introduced to the 5-positon of the scaffold to investigate whether it can prevent racemization of compounds.

All compounds disclosed herein can be prepared as outlined in Schemes 1 and 2. The synthetic route for the key intermediates 9a–9f is shown in Scheme 1. The thiophenol 3 was prepared as previously reported,²⁰ and the corresponding thioethers 4a–4c were afforded through nucleophilic reaction with 3 utilizing bromo- or mesylate-substituted aliphatic chains. Further oxidation with m-CPBA resulted in the sulphones 5a–5c. Deprotection of 5a and 5b under concentrated HCl give intermediates 6a and 6b. Compound 5c was treated with concentrated HCl, and then the resulting intermediate was reprotected with Boc to give compound 6c. Compound 7a was prepared as previously reported;¹⁹ compounds 7b and 7c were commercially available. The aminophenols 6a–6c and 7a–7c were protected by allyl to yield 8a–8f that were then treated with thiophosgene to afford the thio-isocyanates 9a–9f.

Scheme 1. Preparation of Compounds.

Reagents and conditions: (a) mesylate group or bromine substituted aliphatic chains, K₂CO₃, DMF, RT, 12 h; (b) m-CPBA, DCM, RT, 5 h, 53–95% over two steps; (c) concentrated HCl, MeOH, reflux, 12 h; (d) Boc₂O, TEA, DCM, RT, 6 h; (e) 3-bromoprop-1-ene, K₂CO₃, DMF, RT, 8 h, 26-36%; (f) thiophosgene, NaHCO₃, DCM, RT, 3 h, 83–96%.

Scheme 2. Preparation of Compounds.

Reagents and conditions: (a) DCM, RT, 6 h, 83-98%; (b) O,O-di(pyridin-2-yl) carbonothioate, DMAP, DCM, RT, 3 h, 63–93%; (c) Pd(PPh₃)₄, DCM, RT, 2 h, 34–80%.

The thio-isocyanates 9a–9f were nucleophilic and reacted with different α-amino acetamides, including racemic or chiral starting materials (10, 13a–13r), to give thiourea 11a–11f and 14a–14r. Treatment of the thioureas with di-2-pyridyl thionocarbonate via a cyclization reaction can yield the 1,5-dihydro-4H-imidazol-4-one products 12a–12f and 15a–15r,²¹ which is then followed by deprotection with Pd(PPh₃)₄ to afford the target compounds.

At first, our SAR efforts involved the electron-withdrawing groups of the left phenol moiety of compound 2 (Tables 1 and S1). Introducing extended chain groups such as methoxyethyl (A1) or N-Boc aminopropyl (A2) to the sulfonyl can improve the compounds’ selectivity, while the CXCR2 activity was slightly affected, indicating that this position was well-tolerated. However, compounds with replacement of the sulfonyl with carbamoyl (A3), nitryl (A4), or cyano (A5) showed a dramatic loss of CXCR1/2 inhibitory activities. Based on this observation, a follow-up SAR investigation of the right phenyl was performed.

Table 1. CXCR1/2 Antagonistic Activity for Compounds A1–A5.

^aThe IC₅₀ values are an average of triplicate determinations.

^bRatio = CXCR1 IC₅₀/CXCR2 IC₅₀.

As shown in Tables 2 and S1, removing the phenyl of compound 2 (B1) led to a significant decrease of CXCR2 antagonistic activity. All of the compounds with halogen-substituted phenyl displayed a drop in activity. The compound with 4-Cl substitution (compound B2) showed activity twofold higher than that of the compound with 4-F substitution (compound B5). The compounds’ antagonistic activity increased when moving the 4-Cl substitution to the 3-position (compound B3) and 2-position (compound B2). Moreover, introducing hydrophobic chains to the 5-position of 1,5-dihydro-4H-imidazol-4-one scaffold can improve the activity of compounds (B6–B9 vs B1). The longer the chain, the better the compound’s activity, and compound B9 with n-butyl substitution exhibited the best IC₅₀ value of 43 nM and 1270-fold selectivity against CXCR2. However, the activity of the compound suffered a sevenfold drop after replacing n-butyl with isobutyl (B10). The combined result indicated that the hydrophobic groups of the 5-position of 1,5-dihydro-4H-imidazol-4-one scaffold were essential for antagonistic activity maintenance of compounds.

Table 2. CXCR1/2 Antagonistic Activity for Compounds B1–B10.

	R1	CXCR1 IC50 (μM)a	CXCR2 IC50 (μM)a	ratiob
2	Ph	4.7	0.006	778
B1	H	>100	29.7	3
B2	4-Cl-Ph	38.1	0.915	42
B3	3-Cl-Ph	47.8	0.305	157
B4	2-Cl-Ph	21.7	0.054	402
B5	4-F-Ph	∼100	2.19	∼46
B6	methyl	>100	0.269	>372
B7	ethyl	∼100	0.140	∼714
B8	propyl	14.7	0.068	216
B9	n-butyl	54.6	0.043	1270
B10	isobutyl	41.8	0.305	137
SCH527123		0.16	0.01	16

^aThe IC₅₀ values are an average of triplicate determinations.

^bRatio = CXCR1 IC₅₀/CXCR2 IC₅₀.

According to the above SAR study, the phenyl of compound 2 was replaced with the aliphatic substituents to get the stable chiral product. As shown in Tables 3 and S1, the resulting chiral product was stable as expected, and the activity of compounds with R conformation was better than that of compounds with S conformation (C1 vs C2, C3 vs C4, C5 vs C6, C7 vs C8). Consistent with the above SAR, the activity of R-propyl substituted C3 was better than that of R-methyl substituted C1. Compared with compound 2, compound C5 showed comparable inhibitory activity and selectivity against CXCR2. Whereas, the compound with benzyl substitution (C7) displayed inhibitory activity against CXCR2 lower than that with phenyl substitution (compound 2), which indicated that the limited tolerance of the hydrophobic region.

Table 3. CXCR1/2 Antagonistic Activity for Compounds C1–C8.

	R1	CXCR1 IC50 (μM)a	CXCR2 IC50 (μM)a	ratiob
C1	methyl	>100	0.47	>212
C2	methyl	>100	3.1	>32
C3	propyl	20.3	0.029	700
C4	propyl	>100	2.8	>36
C5	cyclohexyl	9.1	0.01	910
C6	cyclohexyl	∼100	4.0	∼25
C7	benzyl	>100	0.098	>1020
C8	benzyl	>100	25.0	>4
SCH527123		0.16	0.01	16

^aThe IC₅₀ values are an average of triplicate determinations.

^bRatio = CXCR1 IC₅₀/CXCR2 IC₅₀.

Based on the above SAR studies, compound C5 was selected and evaluated for its pharmacokinetic properties. C5 was stable in SIF and SGF, which was better than compound 2 (Table 4). However, the stability of C5 in rat or human liver microsomes was lower than that in mouse liver microsomes, and it was also slightly dropped in rat or human plasma. Moreover, we found that the absorption of C5 was excellent (Table 5), exhibiting a bioavailability value of 90.2% after 10 mg/kg oral dosing in rats. C5 displayed a low human ether-a-go-go-related gene (hERG) blockage, showing an IC₅₀ value over 50 μM (Figure S2). Therefore, C5 is suitable for further in vivo efficacy studies.

Table 4. In Vitro Stability Evaluation of C5.

	remaining percent (%)a
SIF stability (at 45 min)	>99
SGF stability (at 45 min)	>99
liver microsome stability (at 1.5 h; rat, mouse, human)	21.5, 71.4, 9.0
plasma stability (at 1.5 h; rat, human)	72.9, 67.3

^aThe values are an average of triplicate determinations.

Table 5. Pharmacokinetic Profiles of C5 in Rats.

parameter	p.o. (10 mg/kg)	i.v. (2 mg/kg)
Kel (h–1)	0.232 ± 0.031	0.981 ± 0.263
Tmax (h)	2.17 ± 3.3
T1/2 (h)	3.02 ± 0.38	0.74 ± 0.193
Cmax (ng/mL)	4499 ± 1754
AUC0–t (ng/mL·h)	20 742 ± 4166	4601 ± 1684
AUC0–∞ (ng/mL·h)	26 943 ± 2245	4617 ± 1688
MRT (h)	5.58 ± 1.36	0.73 ± 0.21
F (%)	90.2 ± 18.1

To further understand the effectiveness of metastatic inhibition of C5 against cancer cells, wound healing and transwell analysis were subsequently performed. Cancer cells such as A375, A549, CNE, MDA-MB-231, and PC3 were treated with compound C5, and cell viability was detected. The result indicated that C5 showed low toxicity against the cells dose- or time-dependently (Figure 3A and B). Further, a wound healing assay was performed to test the migration inhibitory capability of C5 against A375 cells. It was found that the wound closure was inhibited dose-dependently in cells treated with C5 (Figure 3C). However, the migration inhibitory activity of C5 was inferior to that of SCH527123 (Figure 3D). We next verified the invasive inhibition of C5 using transwell assay. CXCL8 or C5 was added to the lower chamber of the transwell to evaluate whether C5 can inhibit the invasion of A375 cells. As shown in Figure 3E and F, CXCL8 markedly induced the invasion of cells. Compound C5 can significantly decrease the number of invasive cells dose-dependently, which showed comparable invasive inhibitory activity with SCH527123 at 10 μM. Therefore, compound C5 is effective in inhibiting metastasis of cancer cells induced by CXCL8.

Figure 3.

Compound C5 exhibits low cytotoxicity against cancer cells dose-dependently (A) or time-dependently (B). (C) Confluent A375 cells were wounded with a pipet tip, and the scraped area of cell migration was analyzed (D). The cell migration rate was calculated as = (C – D)/(A – B) × 100% (A = blank area of control 0 h; B = blank area of control 42 h; C = blank area of compound 0 h; D = blank area of compound 42 h). (E) Schematic diagram of transwell assay, representative images, and (F) statistical analysis of the invasion of A375 cells treated with CXCL8 or compound C5. SCH represents SCH527123. Scale bars: 100 μm. Results are shown as mean ± SD *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Although CXCR1 and CXCR2 share 76% homology, compound C5 exhibited a 910-fold higher antagonistic activity against CXCR2 than that of CXCR1. For exploring the binding features of C5 against CXCR1 and CXCR2, computational docking and molecular dynamics (MD) were employed. CXCR proteins belong to the GPCR family that transmit signals mainly through Gi proteins. First, we analyzed the stable CXCR2-CXCL8-Gi complexes (PDB ID: 6LFM).²² After CXCL8 binds and activates CXCR2, the receptor interacts with the Gα_i subunit through some key amino acid residues, including L348, K349, L353 and F354 of Gα_i subunit, and R144, A241, I317, and Q319 of the receptor (Figure 4A). The key residues of the receptor belong to the TM3, TM5, and TM6 interaction interface which lie in the cytoplasmic part of CXCR2 and have been identified as allosteric sites for CXCR2 antagonists. CXCR2 antagonists function mainly through disturbing the interaction between CXCR2 and Gα_i and subsequently preventing the conformational changes of TM3, TM6, and TM7 when CXCR2 is activated. A similar pocket was identified in CXCR1 (PDB ID: 2LNL) for the following docking study.

Figure 4.

Compound C5 displayed stronger binding interactions against CXCR2 than CXCR1. (A) Structure of CXCR2-CXCL8-Gi complexes. Interaction forces’ contribution of amino acids of C5-CXCR1 (B) or C5-CXCR2 complexes (C). The binding pattern of C5 with CXCR1 (D) or CXCR2 (E). (F) Comparison of the CXCR1 and CXCR2 amino acid sequences.

Compound C5 was docked with CXCR1 and CXCR2 (PDB ID: 6LFL), respectively. The well-prepared CXCR structures were embedded into a pre-equilibrated lipid bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) solvated in 0.15 M NaCl. Desmond was used to performed all molecular dynamics simulations. The complexes were then simulated for 15 ns. Simulations were performed on membrane-containing systems with the OPLS3e force field of Desmond. After the dynamic equilibrium was attained, the root-mean-square deviation (RMSD) became stable (Figure S3). The Glide_SP score and MM_GBSA score of the C5-CXCR2 complex were lower than that of C5-CXCR1 complex (Table S2), consistent with the antagonistic activity of C5 against CXCR1 or CXCR2. For the C5-CXCR1 complex, the residues such as I64, R68, G70, R71, and D75 mainly contributed to the interaction (Figure 4B). For the C5-CXCR2 complex, the residues such as S81, V82, T83, D84, D143, R144, and K320 mainly contributed to the binding (Figure 4C). According to the relatively stable conformation, C5 formed hydrogen bonds with CXCR1 residues I64, R71, and D75 (Figure 4D), which indicated a weak interaction of C5 against CXCR1. The result of interaction exhibits a lack of conformational constraints on TM6 and TM7; thus, C5 cannot prevent the conformational changes of TM6 and TM7 when CXCR1 is activated. Whereas, C5 interacted well with CXCR2. The sulfonyl of C5 was hydrogen-bonded with R144 and interacted with V82, T83, and D143 through a water bridge. The phenol of C5 was hydrogen-bonded with S81 and K320. The 1,5-dihydro-4H-imidazol-4-one scaffold was hydrogen-bonded with D84 and interacted with K320 through a water bridge. Moreover, the cyclohexyl of C5 was inserted into a hydrophobic pocket formed by V69, V72, I73, and L88 (Figure 4E). These strong interactions of C5 against CXCR2 can prevent the conformational changes of TM3, TM6, and TM7 when CXCR2 is activated.

Further, we compared the amino acid sequences between CXCR1 and CXCR2. The residues’ significant difference that C5 interacted with CXCR1 or CXCR2 was the amino acid residue at the 320-position (Figure 4F). The residue at the 320-position of CXCR2 is lysine located on TM7 and can form strong hydrogen bonds with phenol of C5, thus preventing the conformational changes of TM7. However, the residue of CXCR1 at the same position is asparagine, which showed no interaction with C5. Therefore, K320 of CXCR2 may be a critical amino acid residue for the high selectivity of compound C5.

In conclusion, the results reported here describe the optimization and SAR studies of a novel series of derivatives as CXCR1/2 antagonists. Based on an established pharmacophore model and rational design, 1,5-dihydro-4H-imidazol-4-one compound 2 resulting from a triazole CXCR2 antagonist was identified as a selective CXCR2 antagonist with a low CXCR1 antagonism preference. However, it exhibited some disadvantages, including easily being metabolized in SIF and aromatization-induced racemization. Further optimization and SAR studies led to compound C5 that overcame the disadvantages of compound 2 and performed with excellent oral bioavailability.

Compound C5 displayed good inhibitory activity against metastasis of cancer cells, indicating that there may be no significant difference between CXCR2 selective and nonselective antagonists in antitumor metastasis. Further dynamic simulation of molecular protein complexes disclosed that K320 of CXCR2 may contribute most to the selectivity of compound C5, providing important clues for the design of new CXCR2 selective antagonists. Because CXCR1 and CXCR2 share high sequence homology, efforts are still needed to investigate the different biologic functions between these two proteins. Further biological studies using the selective CXCR2 antagonist, compound C5, are ongoing in our lab.

Glossary

Abbreviations

CXCR

CXC chemokine receptor

GPCRs

G-protein-coupled receptors

CXCL8

interleukin-8

PD-L1

programmed death-ligand 1

mCRPC

metastatic castration resistant prostate cancer

RT

room temperature

DMF

N,N′-dimethylformamide

DCM

dichloromethane

m-CPBA

3-chloroperbenzoic acid

DMAP

4-dimethylaminopyridine

TEA

triethylamine

SAR

structure–activity relationship

HPLC

high performance liquid chromatography

SGF

simulated gastric fluid

SIF

simulated intestinal fluid

hERG

human ether-a-go-go-related gene

MRT

mean retention time

MD

molecular dynamics

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

RMSD

root-mean-square deviation

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00113.

• Details for the synthesis and characterization of target compounds; biological experimental methods; Figures S1–S3; Tables S1 and S2; NMR and HPLC spectra (PDF)

The authors declare no competing financial interest.