Discovery of Irbesartan Derivatives as BLT2 Agonists by Virtual Screening

Abstract

Leuktriene B4 receptor 2 (BLT2) is a G-protein coupled receptor modulation of which is discussed to be a therapeutic option for healing of intestinal lesions. In this work, new BLT2 agonists were identified by a virtual screening of a repurposing library and in vitro assay of the most promising compounds. Irbesartan, an approved type-1 angiotensin II receptor (AT1) antagonist, was identified as a moderate BLT2 agonist. An initial SAR study on the irbesartan scaffold was performed resulting in the discovery of a new potent BLT2 agonist (8f, EC₅₀ = 67.6 nM). Irbesartan and 8f were shown to promote proliferation of epithelial colon cells, an effect which was reversible by a BLT2 antagonist.

The leukotriene B4 receptor 2 (BLT2), first described in 2000 by Yokomizo et al.,¹ is primarily known as the low affinity receptor for leukotriene B₄ (LTB₄, Figure 1A) in contrast to BLT1, which is the high affinity receptor for LTB₄.² Human BLT2 is expressed in a variety of tissues including spleen, blood leukocytes, liver, ovary, pancreas, heart, prostate gland, testes, small intestine, kidney, lung, colon, thymus, muscle, placenta, and skin, where BLT2 is detected in high concentrations.³ However, studies performed by Okuno et al. demonstrated that 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT, Figure 1B), which is a byproduct of thromboxane A₂ biosynthesis, can be considered as a potent endogenous agonist of BLT2.⁴ BLT2 modulation has been considered as a potential therapeutic strategy for a variety of pathologies including diabetic wound healing,⁵ neuropathic pain,⁶ carcinogenesis,⁷ and inflammatory arthritis.⁸ Very recently, Matsumoto et al. demonstrated that BLT2 activation accelerates the healing of intestinal lesions by promoting epithelial cell proliferation.⁹ Intestinal lesions are formed in different pathologies, including inflammatory diseases of the intestine and cancer, but may also be induced by nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, BLT2 agonists may serve as a therapeutic option to heal intestinal lesions.

Figure 1.

Structures of LTB₄ (A), 12-HHT (B), and CAY10583 (C).

Our group has recently described the first structure–activity relationship (SAR) study for a BLT2 agonist based on the CAY10583 scaffold.¹⁰ However, there is still a need to discover new scaffolds to be used as starting points for the development of novel potent BLT2 agonists. One of the most convenient approaches to find new hits is the use of repurposing drug libraries. The use of this strategy presents some advantages because it involves the use of well-studied compounds with potentially lower development costs and shorter development time.¹¹ The Repurposing Hub Database can be considered as one of the most complete resources for in silico repurposing and screening of approved and clinical compounds.¹² We followed a virtual screening approach to identify BLT2 agonists using the MOE software suite (Chemical Computing Group, Montreal, Canada). After downloading the database, all compounds with a molecular weight below 250 Da and above 650 Da were removed. Furthermore, only compounds exhibiting an acidic group (indicated by the MOE descriptor a_acid > 0) were considered for virtual screening. For the remaining compounds, adjustment of the protonation state (MOE wash routine) and energy minimization using default settings were performed. Multiple low energy conformations were generated using default settings of the LowModeMD routine, setting the energy window to 5 kJ/mol and the RMSD cutoff to 0.75 A.

For the generation of the pharmacophore model, three known active BLT2 agonists CAY10583, 12-(S)-HHT, and LTB₄ were pasted into the MOE database. Subsequently, adjustment of the protonation state (MOE wash routine) and energy minimization using default settings was performed. The initial alignment of the active compounds was performed using the Pharmacophore Elucidation routine with default settings, while the conformers were generated during the search by bond rotation. The top rated superposition was examined manually and subsequently refined using the Flexible Alignment routine. A previous publication on the conformation of 12-(S)-HHT in the receptor bound state by Giusti et al. demonstrated the importance of the interactions of the carboxylic group and the −OH group which is supposed to act as a H-bond acceptor.¹³ These observations were in perfect agreement with the SAR of CAY10583, where the removal of the acidic or the H-bond acceptor group resulted in fully inactive derivatives. Therefore, we used default settings for refinement of the existing alignment, while weights for H-Bond acceptor and carboxylate (O₂)-type Centroid were set to 10. The resulting molecular alignment was used to generate a pharmacophore model manually, considering the knowledge of SAR from our previous publication (Figure 2A).¹⁰ The pharmacophore model consisted of six features; the importance of the anionic group and the H-bond acceptor group (F4, cyan) was mentioned above, while three hydrophobic/aromatic features (F2, F3, F6) as well as a hydrophobic feature F5 were added.

Figure 2.

(A) Pharmacophore model based on CAY10583 (purple sticks), 12-(S)-HHT (pink sticks), and LTB₄ (cyan sticks). The anionic group (F1, red), the H-bond acceptor group (F4, cyan), and four hydrophobic features (F2, F3, F5, F6) are represented by spheres. (B) Overlay of irbesartan (orange sticks) and CAY10583 (purple lines) with the pharmacophore model.

The pharmacophore search was performed on the prepared multiconformer database. For this search, a partial match of at least 5 features was required. Features F1, F4, and F5 were considered to be essential due to our prior knowledge of the SAR (vide supra). The pharmacophore search delivered 54 hits, which were inspected manually. The full list of the potential hits is enclosed in the Supporting Information (SI). The manual inspection revealed that most of the hits do not superpose well with the shape of the active ligands, and these were discarded. The 10 most promising hits were purchased and tested for their potency in a functional cell-based assay with detection of accumulation of inositol monophosphate (IP-1) as a measure for activation of BLT2 (Table 1).

Table 1. Selected Virtual Screening Hits for BLT2 Activation.

compd	BLT2 EC50 ± SEM (nM)
irbesartan (1)	410 ± 34
candesartan	∼15 μM
valsartan	∼16 μM
azilsartan	inactive
setipiprant	inactive
elafibranor	inactive
DCPIB	inactive
zaltoprofen	inactive
tienilic acid	inactive
chenodeoxycholic acid	inactive

Of the 10 compounds selected, 3 compounds from the “sartan” family showed BLT2 activation while the other 7 were inactive. Candesartan and valsartan showed weak activation with EC₅₀’s around 15–16 μM, while irbesartan (1), which overlays very nicely with the pharmacophore model and with the reference agonist CAY10583 (Figure 2B), was confirmed as a moderate BLT2 activator with EC₅₀ = 410 nM. Therefore, the irbesartan scaffold was selected to perform an initial SAR study.

Most irbesartan derivatives were prepared according to the synthetic route depicted in Scheme 1.

Scheme 1.

Conditions: (a) (1) Et₃N, DCM; (2) KOH, MeOH, 64–95%. (b) NaH, DMF, 24–87%. (c) LiOH, THF/MeOH/H₂O, 60 °C, 32–91%. (d) nBu₃SnN₃, p-xylene, 160 °C, 50–54%.

In the first step of the synthesis, the imidazolone ring was built in a one-pot procedure by condensation reaction between the corresponding substituted 2-aminoacetamide (2) and acid chloride (3) in the presence of triethylamine, followed by treatment with potassium hydroxide in methanol to give intermediates 6a–g. In the next step, the amide was deprotonated with sodium hydride and reacted with benzylic bromides (5, 6) to yield compounds 7a–g. In the case that R₄ is a methyl ester group, hydrolysis of the ester under alkaline conditions provided the final products 8a–8e. In the case that R₄ is a nitrile group, the final product containing a tetrazole ring was obtained by reaction of the nitrile precursors (7f,g) with azidotributyltin (8f,g).

Alternatively, the synthetic routes depicted in Scheme 2 were used to obtain products 13–15. In this case, an imidazolinidone ring was formed in the first step by reaction of substituted 2-aminoacetamide (2) and valeraldehyde (9) in the presence of PTSA. Intermediate 10 can react chemoselectively with 5 either through the amino or amide functional groups. Thus, compound 10a reacted with 5 in the presence of potassium carbonate in DMF to give tertiary amine 11. On the contrary, deprotonation of 10b with sodium hydride in DMF and subsequent nucleophilic attack to 5 gave intermediate 12. Hydrolysis of the ester under alkaline conditions provided the final products. In the case of the hydrolysis of ester 12, also the elimination product 15 was obtained as a side product, which was also isolated and tested.

Scheme 2.

Conditions: (a) PTSA, MeOH, reflux, 84%. (b) K₂CO₃, DMF, 34%. (c) NaH, DMF, 27%. (d) LiOH, THF/MeOH/H₂O, 60 °C, 96%. (e) nBu₃SnN₃, p-xylene, 160 °C, 62% for 14 and 6.4% for 15.

Finally, bicylclic imidazolidinones 19a,b were prepared using the same route as for compound 14. Chirally pure 2-aminoacetamides 18a,b were selected as starting materials. The condensation reaction of 18a,b with valeraldheyde (9) took place diastereoselectively, providing only one of the possible diastereoisomers of intermediate 17 in each case (Scheme 3).

Scheme 3.

Conditions: (a) PTSA, MeOH, reflux, 36–44%. (b) NaH, DMF, 27–32%. (c) nBu₃SnN₃, p-xylene, 160 °C, 34–39%.

For EC₅₀ determination, the compounds were tested for their potency in a functional cell-based assay with the detection of accumulation of inositol monophosphate (IP-1) as a measure for activation of BLT2. Efficacy was compared to that of the reference agonist CAY10583. Compounds were also tested for off-target activity on BLT1 using the same assay principle and LTB₄ as the reference agonist as a positive control for BLT1. Irbesartan is a known antagonist for the type 1 angiotensin II (AT1) receptor. Hence, off-target activity on AT1 was tested in the IP-One assay in antagonist mode in the presence of the AT1 agonist [Val⁵]-angiotensin II. The data are summarized in Table 2.

Table 2. Activation of Gq Pathway and Cellular Toxicity^a.

compd	R1	R2	R3	R4	BLT2 EC50 ± SEM (nM)	efficacy rel. to CAY10583% ± SEM	AT1 IC50 ± SEM (nM)	BLT1	live rate (%) after 3 days at 50 μM
1	Cyp		nBu	tetrazole	410 ± 34	69 ± 2	6 ± 0.6**	inactive	unaffected
8a	Cyp		nBu	COOH	1490 ± 168	95 ± 3	190 ± 6.6**	inactive	92
8b	Cyp		Ph	COOH	inactive		>10 μM	inactive	unaffected
8c	Cyp		Bn	COOH	6580 ± 956	88 ± 4	8650 ± 620	inactive	79
8d	Cyp		CH2CH2Ph	COOH	inactive	inactive	inactive	inactive	unaffected
8e	Cy		nBu	COOH	860 ± 88	64 ± 8	155 ± 2.2**	inactive	90
8f	CH3	iPr	nBu	tetrazole	67.6 ± 4	42 ± 4	15 ± 1.7**	inactive	unaffected
8g	CH3	Ph	nBu	tetrazole	550 ± 88	11 ± 4	25 ± 2.1**	inactive	56
13					24200 ± 3400	31 ± 1	7750 ± 660	inactive	unaffected
14					6390 ± 358	36 ± 4	390 ± 74**	inactive	77
15	CH3	CH3	nBu	tetrazole	8140 ± 815	36 ± 4	32 ± 8.9**	inactive	56
19a					inactive		1010 ± 34**	inactive	89
19b					7500 ± 1100	25 ± 2	∼10 μM	inactive	88

^aCompounds were tested for activation of BLT2 or BLT1 in the IP-One assay in agonist mode. For BLT2, efficacy was determined by comparison to reference agonist CAY10583 at 20 μM each. For BLT1, the reference agonist LTB₄ served as a positive control. Inhibition of the second off-target AT1 was tested in the IP-One assay in antagonist mode with [Val⁵-angiotensin II being present at a constant concentration of 10 nM. **Indicates that the respective compound is a full agonist with efficacy comparible to irbesartan (1). IP-One assays for the determination of EC₅₀ or IC₅₀ with SEM were conducted at least two times (n = 2). Cellular toxicity was tested on Hep-G2 cells treated for 3 days. ATP content as a measure for overall metabolic activity and cell survival was determined using the CellTiter-Glo assay. For 50 μM of compound, the live rate is reported as percent of DMSO control.

As in the reference agonist CAY10583, a first series of irbesartan derivatives was prepared bearing a carboxylic group instead of tetrazole and different substituents in R₃ (8a–e). Contrary to the SAR observed with CAY10583,¹⁵ aromatic substituents were not well tolerated in this position (8b–d) and the carboxylic acid brought a significant decrease in activity (8a E₅₀ = 1490 nM vs 1 EC₅₀ = 410 nM, Table 2). Variations in the substitution or R₁ and R₂ were studied next. Exchange of the original cyclopentyl group for cyclohexyl brought an increase in activity (8e EC₅₀ = 860 nM vs 8a EC₅₀ = 1490 nM, Table 2). Then, three more examples were prepared by keeping R₁ = CH₃ and varying R₂. Compound 8f, with R₂ = iPr, resulted in a very active compound with an EC₅₀ = 67.6 nM, while compounds 8g and 15 performed worse than irbesartan, especially considering maximum efficacy.

Other structural modifications like the substitution inversion in 13, the imidazolidine ring instead of imidazolone ring as in 14, or the fused rings in 19a,b were not well tolerated and resulted in weakly active or inactive compounds (Table 2).

Compounds were also tested for activity as AT1 inhibitors. Most of the irbesartan derivatives, which displayed BLT2 activity, were also potent as AT1 antagonists (Table 2). Several structural features correlate with both BLT2 and AT1 activity, suggesting similar binding site properties of these receptors. Kim et al. described a binding mode of the reference agonist CAY10583 in the binding site of BLT2 built by homology modeling.¹⁴ It shows an interaction of the carboxylate with Arg301. Arg301 resembles Arg167 in AT1, which interacts with the tetrazole moiety of the AT1 antagonist olmesartan as described by Zhang et al.¹⁵ Furthermore, Tyr129 in BLT1 is supposed to interact with the carbonyl moiety and is also present in AT1 as Tyr35, which exhibits a H-bond donor interaction toward the imidazole nitrogen of olmesartan. The pharmacophore model (Figure 2) assumes that interactions of the carboxylate and the carbonyl moieties are essential pharmacophore features, suggesting a certain degree of similarity between the pharmacophores of AT1 and BLT2. Notably, the most potent BLT2 agonist 8f displays low nanomolar inhibitory activity toward AT1 (IC₅₀ = 15 nM). In contrast, regarding BLT1 activity, all compounds resulted in being inactive. All in all, a series of 12 irbesartan (1) derivatives was investigated and the first key structural features for activity have been identified. The initial SARs of the irbesartan scaffold are summarized in Figure 3.

Figure 3.

SAR summary of the irbesartan scaffold for BLT2 activation.

In order to investigate the potential of irbesartan and the most potent derivative of the series, 8f, as well as BLT2 agonist CAY10583 to promote epithelial cell proliferation, the cell line HCT116 was treated with indicated concentrations of the compounds alone or in combination with the BLT2 antagonist Ly255283 (1 μM). As depicted in Figure 4, we demonstrated that the compound 8f similarly to CAY10583 and irbesartan significantly induced colon epithelial cell proliferation. Of note, at 0.03 μM, this effect was stronger in 8f-treated cells compared to cells that were exposed to 0.03 μM irbesartan or the BLT2 agonist CAY10583. Importantly, BLT2 agonist-induced effects on epithelial colon cell proliferation were abrogated by the BLT2 antagonist Ly255283 and this effect was significant for irbesartan and 8f (0.1, 0.3, and 1 μM). The application of the antagonist alone did not affect HCT116 cell proliferation.

Figure 4.

Proliferation of HCT116 colon epithelial cells induced by BLT2 agonists. The cells were seeded at a density of 5 × 10³ cells per well in 96-well plates. After 72 h of incubation, the cells were treated with indicated concentrations of CAY10583, irbesartan, and 8f alone or in combination with the BLT2 antagonist Ly255283 (1 μM) for 24 h. After 20 h, CellTiterBlue reagent was added to the cells for 4 h. Fluorescent resorufin was measured using a plate reader. Data are expressed as mean ± SEM; n = 3; ns = not significant; *p ≤ 0.05 vs ctrl; ^#p ≤ 0.05 vs CAY10583/irbesartan/8f alone.

In conclusion, we were able to identify AT1 receptor antagonists as BLT2 agonists using virtual screening of the repurposing library. Among them, irbesartan showed the highest potency. Preliminary SAR investigations led to the identification of more potent irbesartan derivatives, which displayed good activity in a functional assay. Furthermore, irbesartan and 8f were able to promote the proliferation of colon epithelial cells, which makes them an interesting option for further investigation in the context of intestinal lesions.

Glossary

Abbreviations

12-HHT

12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid

AT1

angiotensin II receptor type 1

bFGF

basic fibroblast growth factor

BLT1

leukotriene B₄ receptor type 1

BLT2

leukotriene B4 receptor type 2

CAY10583

4′-((N-phenylpentanamido)methyl)-[1,1′-biphenyl]-2-carboxylic acid

IP-1

inositol monophosphate

LTB₄

leukotriene B₄

TGF-β1

transforming growth factor beta 1.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00240.

• Synthesis methods and characterization of intermediate and final compounds; IP-One assay; proliferation assay (PDF)

• Full list of virtual screening hits (PDF)

Author Contributions

^∥ V. H.-O. and J.H. contributed equally. All authors have given approval to the final version of the manuscript.

This research was supported by the Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz (LOEWE) Research Centre for Translational Medicine and Pharmacology of the State of Hessen, Germany and Deutsche Forschungsgemeinschaft (DFG, DFG-RBFR joint project PR1405/8-1, Heisenberg-Professur PR1405/7-1; SFB 1039 TP A02 and TP A07).

The authors declare the following competing financial interest(s): V.H.-O., J.H., D.S., and E.P have a patent application that covers the composition of matter for BLT2 agonists.