Potent Heterocyclic Ligands for Human Complement C3a Receptor

Article pubs.acs.org/jmc

Potent Heterocyclic Ligands for Human Complement C3a Receptor Robert C. Reid,* Mei-Kwan Yau, Ranee Singh,† Johan K. Hamidon, Junxian Lim, Martin J. Stoermer, and David P. Fairlie* Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia S Supporting Information *

ABSTRACT: The G-protein coupled receptor (C3aR) for human inflammatory protein complement C3a is an important component of immune, inflammatory, and metabolic diseases. A flexible compound (N2-[(2,2-diphenylethoxy)acetyl]-Larginine, 4), known as a weak C3aR antagonist (IC50 μM), was transformed here into potent agonists (EC50 nM) of human macrophages (Ca2+ release in HMDM) by incorporating aromatic heterocycles. Antagonists were also identified. A linear correlation between binding affinity for C3aR and calculated hydrogen-bond interaction energy of the heteroatom indicated that its hydrogen-bonding capacity influenced ligand affinity and function mediated by C3aR. Hydrogen-bond accepting heterocycles (e.g., imidazole) conferred the highest affinity and agonist potency (e.g., 21, EC50 24 nM, Ca2+, HMDM) with comparable efficacy and immunostimulatory activity as that of C3a in activating human macrophages (Ca2+, IL1β, TNFα, CCL3). These potent and selective modulators of C3aR, inactivated by a C3aR antagonist, are stable C3a surrogates for interrogating roles for C3aR in physiology and disease.

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INTRODUCTION The human anaphylatoxin protein, C3a, has a sequence of 77 amino acids arranged in a helix bundle and is a product of activation of the complement cascade, an important network of plasma and membrane-bound proteins that regulate innate and adaptive immunity.1,2 This small protein binds to the surface of immune and other cells via a specific G-protein coupled receptor known as the C3a receptor (C3aR) and activates several intracellular G-protein signaling pathways that initiate multiple cell functions.3 This receptor is primarily expressed on myeloid cells such as monocytes/macrophages, dendritic cells, and granulocytes like neutrophils and mast cells.4−6 These cells play critical roles in human immunity, with some reports indicating that activation of C3aR by C3a protein can regulate cytokine release from monocytes/macrophages7 and can induce mast cell degranulation.8,9 Upon binding of C3a to C3aR, the activated receptor couples to pertussis toxin-sensitive G-protein Gαi in various immune cells, triggering intracellular calcium release,10,11 reduces intracellular cAMP levels in murine dendritic cells,12 and couples to Gα12 and/or Gα13 to activate ERK1/2 phosphorylation in endothelial cells.13 C3aR is also able to signal through β-arrestins, but the exact signaling mechanism remains unclear.14 Activation of C3aR has most often been reported as proinflammatory and has been associated with a number of inflammatory diseases, including arthritis,15,16 asthma,17−19 renal inflammation,20,21 obesity and metabolic dysfunction,22,23 bone inflammation,24 sepsis,25 psoriasis,26 and lupus erythematosus.27 In addition, the C3a protein (or C3aR agonist) has been shown to have antibacterial activities.28,29 However, C3a also has been reported to show anti-inflammatory properties and notably attenuates secretion of pro-inflammatory cytokines (e.g., TNFα, IL-6) from isolated © 2014 American Chemical Society

peripheral blood mononuclear cells (PMBC) and lymphocytes.30−32 C3aR knockout mouse studies have also suggested possible anti-inflammatory roles for C3a with production of LPS-induced pro-inflammatory cytokines being reduced.33 Activation of C3aR also reduced neutrophil mobilization and intestinal injury in vivo, suggesting that a C3aR agonist may be beneficial in intestinal ischemia−reperfusion injury.34 All of these findings have stimulated interest in developing potent and selective agonists and antagonists for C3aR. Biological evaluation of potent C3aR ligands can provide new molecular and mechanistic insights to the roles of C3a in inflammatory and metabolic disorders as well as infectious diseases. Previously, we reported a library of C3aR hexapeptides possessing different binding affinities and agonist/antagonist activities.35 These were the shortest peptide agonists reported to activate C3aR, with the most potent hexapeptide agonist being FLTLAR (1, Figure 1). It bound C3aR with IC50 42 nM (human PBMCs) and activated the receptor (EC50 0.37 μM, dU937 cells). Substitution of the fourth residue leucine of 1 with cyclohexylalanine (Cha) led to a peptide (FLTChaAR) with moderate antagonist activity (IC50 1.5 μM) of intracellular calcium (iCa2+) release but with ∼6-fold reduced binding affinity compared to that of 1. Potent peptidic agonists of 15 residues have also been reported,35−37 but such peptides are not so potent against wildtype cells expressing natural levels of C3a receptor and are also unstable in serum due to proteolytic cleavage by proteases. The C3a protein is rapidly cleaved by serum carboxypeptidases with loss of the C-terminal arginine residue to give C3a-desArg, which does not bind to C3aR and has different physiological Received: June 24, 2014 Published: September 26, 2014 8459

dx.doi.org/10.1021/jm500956p | J. Med. Chem. 2014, 57, 8459−8470

Journal of Medicinal Chemistry

Article

flexibility, and commonality of its diphenylmethane and arginine components with other GPCR-binding compounds.42 Compound 4 has poor oral bioavailability, with high doses by per oral (≥30 mg/kg)16 or by continuous s.c. infusion (60 mg/kg/day)27 necessary to elicit effects in vivo. Other weakly potent small molecule C3aR antagonists (5, 6; Figure 1) have been reported.43,44 Poor DMPK profiles (poor bioavailability, rapid elimination in vivo) were also observed for 3 and 6 that contain an aminopiperidine linker.39 The next milestone is to learn how to significantly increase affinity and potency of nonpeptidic ligands for human C3aR, before then optimizing for functional specificity and bioavailability. This article reports structure−activity relationships (SAR) for compounds based on 4 to first better understand determinants of affinity and functional potency, and then we report derivatizing it by incorporating heterocyclic linkers. C3aR ligands reported herein were assessed using extracellular 125 I-C3a competitive ligand binding assays (to measure affinity for C3aR), intracellular calcium mobilization (to measure agonist/ antagonist function), and then effects on a small set of inflammatory genes, all using a primary immune cell type, human monocyte-derived macrophages (HMDMs), relevant to a multitude of inflammatory diseases in man.

Figure 1. Selected small molecule peptidic and nonpeptidic C3aR ligands.

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functions from those of C3a.14 The instability of the C3a protein and C3aR-binding peptides has severely limited their uses for studying the in vivo roles of C3aR in disease. In late 2013, we reported a small molecule C3aR agonist (2, Figure 1) with comparable potency to human C3a in human monocyte-derived macrophages (HMDMs), as measured using an iCa2+ release assay. That small molecule agonist (2) exhibited similar functions as those of the C3a protein in HMDMs, and, importantly, it was serum-stable.38 Another small molecule agonist, 3 (Figure 1), has also been reported, but it has activity only at low micromolar concentrations and has uncertain target specificity.39 There have been few reports of small molecule nonpeptidic C3aR antagonists. The most significant nonpeptidic antagonist of C3aR was SB290157 (N2-[(2,2-diphenylethoxy)acetyl]-Larginine), designated here as compound 4 (Figure 1), discovered by high-throughput screening at SmithKline Beecham.15 It has been widely used to investigate physiological roles of C3a in in vitro assays and animal models,16,40 but it has limitations. Compound 4 displayed potent binding to C3aR (IC50 10 nM), but antagonism of C3a-mediated intracellular calcium (iCa2+) release was much weaker (IC50 1.3 μM), and agonist responses were also observed in some cell types.41 Polypharmacology due to off-target effects is not surprising due to its small size, high

RESULTS AND DISCUSSION Analogues of Antagonist 4. The arginine residue of 4 confers a substantial component of the binding affinity for C3aR.15,45 We therefore decided to retain it in derivatives and focus on other regions of the molecule to further improve affinity and investigate requirements for functional activity. We first hypothesized that the ether oxygen atom might be a hydrogenbond acceptor and thereby contribute to binding affinity or antagonist function through a hydrogen-bonding interaction with the receptor. To test this proposition, a series of analogues, 7−9, was constructed in order to vary the H-bond accepting property of the heteroatom (Table 1), and these analogues were examined in concentration−response profiles for affinity (Figure 2A), antagonism (Figure 2B), and agonist activity (not shown). Thioethers have a similar capacity to ethers46 for accepting Hbonds, so it was not surprising to find that 7 and 4 showed almost identical C3a receptor binding affinity and antagonist activity against human C3a in HMDMs. The two longer C−S bond lengths in 7 versus the corresponding C−O bonds in 4 evidently had no effect, probably because the high flexibility of the molecules allowed compound 7 to compensate for such changes

Table 1. Influence of a H-Bond Acceptor Atom on Ligand Binding Affinity and Antagonist Activity (Ca2+ Release) on Human Macrophages (HMDMs)

binding affinity

antagonist activity

compd

X

pIC50 ± SEM

IC50 (nM)

4 7 8 9

O S CH2 NH

8.0 ± 0.2 8.1 ± 0.3 7.0 ± 0.2 6.8 ± 0.3

10 8 96 150

a

pIC50 ± SEM

IC50 (μM)b

5.9 ± 0.2 6.1 ± 0.2 5.2 ± 0.3 5.4 ± 0.2

1.3 0.8 6.0 4.0

a

Concentration of ligand required to inhibit 50% of [125I]-C3a (80 pM) binding to HMDMs. All data were obtained from at least three independent experiments (n ≥ 3). bConcentration of ligand causing 50% inhibition of Ca2+ response induced by 100 nM C3a. 8460



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(4 and 7−9) were thus comparably weak antagonists and had no agonist activity up to 100 μM in HMDMs (data not shown). All of the analogues (7−9) of compound 4 were prepared as described in Scheme 1 (full experimental details and spectroscopic data of intermediates are presented in the Supporting Information). (2-((2,2-Diphenylethyl)thio)acetyl)-L-arginine (7) was synthesized by conversion of 2,2-diphenylethanol (7a) to 2,2-diphenylethyl iodide (7b) with triphenylphosphine and iodine. The resulting intermediate was reacted with 2-mercaptoacetic acid under basic conditions. 2-((2,2-Diphenylethyl)thio)acetic acid (7c) was then coupled to H-Arg-OEt followed by ester hydrolysis to give final product 7. Synthesis of (5,5-diphenylpentanoyl)-L-arginine (8) began with reduction of 3,3-diphenylpropanoic acid (8a) using LiAlH4. The resulting alcohol was converted to the tosylate followed by nucleophilic substitution (Finkelstein) to give the iodide intermediate (8b). Reaction with diethyl malonate gave the diester intermediate (8c), and hydrolysis/decarboxylation gave the free acid (8d). Coupling of the free acid with H-ArgOEt and hydrolysis of the ethyl ester gave the desired product 8. (2,2-Diphenylethyl)glycyl-L-arginine (9) was synthesized via reductive amination of diphenylacetaldehyde (9a) with glycine methyl ester followed by Boc-proctection of the secondary amine and methyl ester hydrolysis to afford the acid (9c). The resulting

Figure 2. Affinity and antagonist activity mediated by C3aR in human macrophages. (A) C3aR-binding affinities of compound 4 (▼, red) and analogues 7 (■, black), 8 (⧫, magenta), and 9 (▲, blue) measured by displacement of 125I-C3a (80 pM) on HMDMs (n ≥ 3). (B) Compounds 4 and 7−9 inhibit Ca2+ release induced by C3a (100 nM) on HMDMs, with 8 and 9 being the least potent antagonists. Each data point represents mean ± SEM.

relative to 4. Replacement of the ether oxygen atom with a methylene unit removed the potential for H-bond formation, and 8 consequently suffered a 10-fold loss in binding affinity but only a 5-fold loss in antagonist potency. On the other hand, the secondary amine of 9, which is protonated and positively charged at physiological pH and can function as a H-bond donor, suffered a 15-fold reduction in receptor binding affinity compared to that of 4 but retained antagonist potency. All of these compounds Scheme 1. Synthesis of Analogues of Compound 4a

a

Reagents and conditions: (a) PPh3, I2, imidazole, DCM; (b) NaOH/MeOH; (c) (i) H-Arg-OEt, BOP, DIPEA, (ii) NaOH. H2O, EtOH; (d) (i) LiAlH4, (ii) TsCl, pyridine, (iii) NaI, acetone; (e) CH2(CO2Et)2, NaH, DMF; (f) (i) NaOH, EtOH/H2O, (ii) 2 M H2SO4, 150 °C, 15 min; (g) NaBH(OAc)3, MeOH; (h) (i) Boc2O, Et3N, THF, (ii) NaOH, EtOH/H2O; (j) (i) H-Arg(Pbf)-Wang resin, HBTU, DIPEA, DMF, (ii) TFA/TIPS/ H2O, 95:2.5:2.5% v/v. 8461



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acid was coupled to H-Arg(Pbf)-Wang resin followed by resin cleavage with TFA, giving compound 9. Incorporation of Five-Membered Heterocycles. Different heterocycles were next introduced between the diphenylmethane and arginine components of 4 to produce analogues 10− 22. The objectives were to reduce the number of rotatable bonds to confer increased rigidity, to fix the location of prospective hydrogen-bond acceptors or donors to vary hydrogen-bonding potential of the ligands, and to control the relative positioning and orientation of the diphenylmethane and arginine components for variable interactions with the receptor. Heterocyclic compounds 11−22 were synthesized as shown in Scheme 2 (full experimental details and spectroscopic data of intermediates are presented in the Supporting Information). Furan 10 was prepared according to literature procedures.44 Oxazole 11, in which the positions of N and O atoms were transposed relative to those in 18, was prepared together in the same reaction with imidazole 21. Alkylation of diphenylacetic acid with ethyl 2-chloroacetoacetate gave ester 11a. On heating with NH4OAc/AcOH, cyclization occurred, giving a mixture of oxazole 11b and imidazole 21b in a ratio of 3:1 that was easily separable by flash chromatography. The imidazole esters were methylated to give 16a and 22a, and the isomers were isolated by flash chromatography. Ester 15b was synthesized similarly to that for 11b. Condensation of amidoxime 12a with ethyl 2chloro-2-oxoacetate and likewise condensation of diphenylacetyl chloride with ethyl 2-amino-2-(hydroxyimino)acetate afforded the isomeric oxadiazoles 12b and 13a. Heating amidoxime 12a with ethyl propiolate yielded imidazole 20b. The phenyl-substituted imidazole 19b was prepared from the reaction of 2,2-diphenylacetimidamide 19a with ethyl 2-chloro-3-oxo-3-phenylpropanoate. Oxazoles 17 and 18 were prepared by condensation of diphenylacetyl serine/threonine methyl ester with DAST followed by oxidation of the intermediate oxazolines with CCl3Br and DBU.47 The heterocycle-esters (11b, 15b, 16a, 17a, 18a, 19b, 20b, 21b, and 22a) were hydrolyzed to acids and coupled to H-Arg(Pbf)Wang resin. Cleavage with TFA and purification by rp-HPLC afforded the desired heterocyclic compounds (11, 15, and 16−22). Oxadiazoles 12−14 were prepared directly from the esters 12b, 13a, and 14a by amination with H-Arg-OEt, as the route via the carboxylic acid proved to be unsatisfactory. Furan derivatives such as 10 have been previously proposed as conformationally rigid forms of 4 and reportedly showed enhanced C3aR binding affinity, as determined by 125I-C3a radioligand binding assays with membranes prepared from human mast cell-1.44 However, it is well-known that ligand affinities for GPCRs, including C3aR, can be 10-fold or more higher in isolated membrane preparations than in isolated but intact whole cells, whereas the latter are much more relevant to human physiology. We therefore resynthesized compound 10 and measured its binding affinity for C3aR in whole intact HMDMs using the same 125 I-C3a radioligand assay, but we found that it had 5-fold lower affinity than 4 for binding to C3aR (Table 2), although it had similar antagonist potency (Table 3). It is known that a furan oxygen is a weak hydrogen-bond acceptor,48 much weaker than an ether oxygen as in 4; therefore, this property could potentially be enhanced through a better choice of hydrogen-bond acceptor. For example, oxazole should bind with greater affinity than the furan because it is known that oxazole forms a much stronger H-bond through N-3 than furan does through O-1.48 Here, we have performed ab initio calculations49 and determined that the interaction energy between a water molecule H−O−H···N in oxazole is −5.7 kcal mol−1 but that H−O−H···O in furan is only

Table 2. Influence of Heteroatoms in a Heterocyclic Linker between Diphenylmethane and Arginine on Ligand Binding Affinity for C3aR on Human Macrophages (HMDMs)

binding affinitya compd

X

Y

Z

pIC50 ± SEM

IC50 (nM)

10 11 12 13 14 15 16 17 18 19 20 21 22

O O N N O O N-Me N N N N N N

CH N N O N N N O O NH NH NH N-Me

CH C-Me O N N C-Ph C-Me CH C-Me C-Ph CH C-Me C-Me

7.3 ± 0.4 7.0 ± 0.2 7.9 ± 0.3 7.3 ± 0.3 6.0 ± 0.3 7.0 ± 0.3 5.9 ± 0.3 7.7 ± 0.3 8.0 ± 0.2 7.1 ± 0.3 8.2 ± 0.3 8.0 ± 0.2 7.5 ± 0.2

55 100 12 54 967 110 1144 19 11 87 7 10 31

a

Concentration of ligand required to inhibit 50% of [125I]-C3a (80 pM) binding to HMDMs. All data were obtained from at least three independent experiments (n ≥ 3).

Figure 3. Influence of heterocycle on affinity for C3aR on human macrophages. (A) Calculated H-bond interaction energy (kcal mol−1) between water and heteroatom of heterocycles, compared with the water dimer, determined using ab initio methods (MP2/6-311+ +G(3d,3p) and corrected for the basis set superimposition error within Gaussian 09).48,49 (B) The binding affinities of C3aR ligands containing different heterocycles (Table 2) show a linear correlation with the calculated H-bond interaction energy (kcal mol−1). Compounds 15 and 19 were excluded from this plot, as their low binding affinity was a consequence of the bulky phenyl group at position Z.

−3.2 kcal mol−1 (Figure 3A). New protein−ligand hydrogen bonds can contribute to binding affinity only if they are stronger than the competing interactions with bulk water, where the interaction energy for the hydrogen-bonded water dimer was −4.6 kcal mol−1; it is therefore unlikely that H-bonds to furan are relevant under physiological conditions.48 8462



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Scheme 2. Synthesis of Oxazole-, Imidazole-, and Oxadiazole-Containing Compoundsa

Reagents and conditions: (a) DIPEA, DMF RT; (b) NH4OAc 5 equiv, AcOH, 100 °C, 30 min; (c) NaOH, H2O, EtOH; (d) H-Arg(Pbf)-Wang resin, HBTU, DIPEA, DMF; (e) TFA/TIPS/H2O, 95:2.5:2.5; (f) NaH, MeI, THF; (g) NH2OH·HCl, K2CO3, EtOH, 90 °C; (h) AlMe3, NH4Cl; (i) pyridine 5 equiv, THF, RT; (j) (i) EtOH, Δ, (ii) diphenylether, microwave 180 °C, 15 min; (k) DIPEA, EtOH, 70 °C, 30−60 min; (l) H-ArgOEt.2HCl, DIPEA, EtOH, 120 °C, 1 h; (m) (i) DIPEA, DCM, (ii) tosyl chloride, DIPEA, DCM; (n) (i) DAST, DCM, −78 °C, (ii) K2CO3; (o) CCl3Br, DBU, DCM, 0 °C. a

Next, the potential contributions of other heterocyclic rings, such as imidazole, to receptor binding affinity were investigated, along with

the optimal location for a H-bond acceptor/donor atom in the heterocycle and effects of additional substituents appended to the ring. 8463



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Table 3. Agonist or Antagonist Activity of Heterocyclic Ligands on HMDMs Measured by Calcium Mobilization Assay

antagonist activitya

agonist activityb

compd

X

Z

pIC50 ± SEM

IC50 (μM)

10 11 12 13 14 15 16 C3a 17 18 19 20 21 22

O O N N O O N-Me

CH N N O N N N

CH C-Me O N N C-Ph C-Me

5.7 ± 0.4 5.4 ± 0.3 6.0 ± 0.2 6.0 ± 0.2 4.7 ± 0.2 5.7 ± 0.1

1.8 2.9 1.3 1.2 19 2.0 inactive

N N N N N N

O O NH NH NH N-Me

CH C-Me C-Ph CH C-Me C-Me

Y

pEC50 ± SEM

EC50 (μM)

inactive 7.4 ± 0.1

0.04

c

c

c

c

30 0.13 0.02 0.04

a

Concentration of ligand causing 50% inhibition of Ca2+ response induced by 100 nM C3a. bConcentration of ligand causing 50% of Ca2+ release in HMDMs relative to 100% induced by C3a at 1 μM. cPartial agonists. All values were obtained from at least three independent experiments (n ≥ 3)

We predicted that any compound with a heterocyclic ring containing a good H-bond accepting nitrogen atom in the position equivalent to the furan oxygen (position X, Table 2) could bind to C3aR with higher affinity than that of compound 10. This was found to be true, with oxadiazole 12, oxazoles 17 and 18, and imidazoles 20−22 all consistently showing 5−10 times greater C3a receptor binding affinity than that of the furan compound (10). We predicted that transposing the positions of the oxazole N and O atoms, such as in the regioisomer compound 11, would result in a significant decrease in receptor binding affinity, as the calculated H−O−H···O hydrogenbonding interaction to oxazole O-1 of −3.0 kcal mol−1 is even lower than that found for furan O-1 (Figure 3a). This was found to be the case, with a 10-fold reduction in receptor binding affinity of 11 (IC50 100 nM) compared to that of 18 and a 2-fold weaker binding affinity than that of furan 10. For this compound series, the calculated H-bond interaction energy of each heterocycle was compared with C3a receptor affinity, and there was a linear relationship with a correlation coefficient of R2 = 0.72 (Figure 3b). This was consistent with increasing H-bond interaction between position X of the heterocycle and the C3a receptor leading to enhanced receptor binding affinity. Agonist or Antagonist Functions of Five-Membered Heterocycle Analogues. A significant observation, common to some of the higher affinity compounds (Table 2), was that they showed varying degrees of agonist activity. Oxazoles 17 and 18 were partial agonists, eliciting up to 50% of the Ca2+ response of C3a, whereas imidazoles 20−22 were full agonists. Imidazoles 19−21 can offer either N-1 or N-3 as equipotent H-bond acceptors via tautomerization, as occurs for histidine residues in proteins. The imidazoles can therefore either have a H-bond acceptor at position Y or can alternatively mimic the agonists 17 and 18 with a H-bond acceptor at position X. Compounds 20 and 21 bound to the receptor with high affinity (IC50 7 and 10 nM; Table 2) and were potent full agonists (EC50 130 and

Figure 4. C3a receptor-mediated antagonists versus agonists on HMDMs. (A) Similar to compound 4 (▼, red), compounds 10 (■, magenta), 11 (▲, blue), 12 (⧫, brown), 13 (▼, black), 14 (□, cyan), and 15 (△, orange) inhibit the Ca2+ release induced by C3a (100 nM) on HMDMs with micromolar potency. (B) Compounds 20 (◇), 21 (∗), and 22 (○) are full agonists, but only compounds 21−22 activate C3aR with comparable potency to that of human native C3a. Compounds 17 (◐) and 18 (◓) are partial C3aR agonists, whereas compound 19 (▽) activates the receptor and triggers Ca2+ release only at concentrations >30 μM. Each data point represents mean ± SEM.

20 nM, respectively; Table 3), suggesting that the tautomeric form recognized by the receptor had the H-bond acceptor nitrogen at position X and the NH at position Y. To confirm this unequivocally, the nitrogen atoms at positions X and Y were individually methylated to remove the possibility of tautomerization. Consistent with the hypothesis, compound 22, having NMe at position Y, forces the nitrogen at position X to be a H-bond acceptor, leading it to be a potent full agonist. A modest 2-fold reduction in agonist potency could be the consequence of a steric clash between the methyl group and receptor residues. Compound 16, with N-Me at position X that forces the nitrogen at position Y to be a H-bond acceptor, had completely lost all agonist activity. It was also observed that 16 displayed very broad peaks in its proton NMR spectra, suggesting that a steric clash between the N-Me and adjacent carbonyl group may be restricting access to a biologically active conformation, and this may explain the unexpected loss of binding affinity also observed 8464



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Proposed Binding Mode for Imidazole 21 with C3aR. C3aR is a membrane-spanning protein with seven principal transmembrane helices, and, in common with other class A GPCRs, C3aR recognizes and attaches to two binding domains of its native ligand human C3a. The 72-residue N-terminal helix bundle of human C3a is a high affinity binding domain that interacts with the charged N-terminus and the 170 residue extracellular loop 2 of the receptor but does not activate it. The C-terminal pentapeptide sequence (LGLAR) of C3a is thought to interact with an effector binding site in the transmembrane region of the receptor near the plasma membrane, but without the high affinity anchoring domain of C3a, this pentapeptide has negligible agonist activity.2 Site-directed mutagenesis studies on C3aR have demonstrated that three charged residues (Arg161, Arg340, and Asp 417) in C3aR are pivotal for C3a binding and receptor activation.45,50,51 Previously, we proposed that agonist 2 mimicked the LAR sequence of C3a with its carboxylate and guanidinium termini interacting with receptor residues Arg161, Arg340, and Asp 417, as supported by our C3aR homology modeling.38 Compound 2 was competitively inhibited by antagonist 4, suggesting that these two compounds bound at the same site in the receptor.38 Similarly, agonist 21, which is more constrained than flexible antagonist 4, was also expected to interact with the receptor at the same binding site. To support this hypothesis, a competition study was conducted using the intracellular calcium release assay to test whether 4 was also a competitive antagonist of agonist 21. Compound 4 was indeed capable of inhibiting the intracellular calcium release induced by agonist 21 (Figure 6A,C), with a Schild

for 16 (Table 2). These results support a strong H-bond interaction between C3aR and heterocyclic agonist ligands through their nitrogen heteroatom at position X. The most effective agonist (21) gave a Ca2+ response at least as potent as human C3a itself (EC50 24 nM, Figure 4B). The 5-phenyl-imidazole 19, although having nitrogen at position X, showed low activity in the iCa2+ assay and weak affinity for C3aR. The loss in activity observed for 19 could be due to a steric effect caused by the bulky phenyl group at position 5. In contrast to agonist compounds 17−22, furan 10, oxazoles 11 and 15, and oxadiazoles 12 and 13, having a weaker H-bond acceptor at position X than agonist imidazoles (19−22) and oxazoles (17 and 18), behaved as antagonists with comparable antagonist potency to that of 4 (Figure 4A). C3aR Selectivity for Agonist 21. Structure−activity relationship studies on these heterocyclic compounds have led to the discovery of a potent small molecule C3aR agonist (21) containing an imidazole ring. Compound 21 showed comparable agonist potency to that of the native peptide ligand C3a in the calcium release assay and high affinity for C3aR in HMDMs. Receptor selectivity of 21 was examined in a limited way by using receptor desensitization (Figure 5A−C) and C5a binding affinity

Figure 5. Compound 21 is selective for C3aR over other Ca2+-releasing GPCRs on HMDMs. (A) HMDMs treated with 300 nM C3a at t0 are desentized to C3aR-mediated activation by C3a (up to 1 μM) for at least 920 s thereafter. (B) Cells desensitized by 300 nM C3a at t0 are still very sensitive to Ca2+ release induced by 300 nM C5a injected 920 s later. (C) Cells desensitized by 300 nM C3a at t0 are no longer sensitive to compound 21 (10 μM) after 920 s, as no calcium signal was observed, supporting selective action of 21 on C3aR to produce Ca2+ release. (D) Neither C3a nor 21 bind to C5aR, unlike C5a and C5a receptor antagonist (3D53), which compete with 125I-C5a for C5a receptor on HMDMs.

Figure 6. Antagonist 4 is competitive with C3a and agonist 21, suggesting that they interact with C3aR at the same receptor binding site. (A) Concentration−response for agonist 21 in the presence of various concentrations of antagonist 4 (zero, ●; 0.1 μM, ■; 0.3 μM, ▲; 1 μM, ▼; 2 μM, ⧫; 3 μM, ○; 4 μM, □; n ≥ 3). The agonist potency of 21 was reduced when the cells were treated with higher concentration of antagonist 4, as indicated by the horizontal shift of the agonist curve from left to right. (B) Schild plot with slope ∼1, suggesting compound 4 is a competitive antagonist against agonist 21. (C) Antagonist 4 (a selective C3aR antagonist) was able to inhibit iCa2+ release induced by both human native C3a (●) and agonist 21 (▲) with similar potency. Each data point represents mean ± SEM (n ≥ 3).

(Figure 5D) assays. To assess the selectivity of 21, the C3a receptors in HMDMs were first selectively desensitized by treatment with 300 nM human C3a. Following treatment 5 min later with 10 μM 21, there was no second Ca2+ response, indicating that the Ca2+ release induced by 21 alone (up to 10 μM, or 500× EC50, Figure 5C) was mediated by C3aR. Agonist 21 was also tested for C5a receptor binding by competition with 125Ilabeled C5a and, like C3a, did not compete with C5a for binding to the C5a receptor, indicating selectivity for C3aR over C5aR. By contrast, both C5a and a C5a receptor antagonist (3D53) did compete with 125I-C5a. Together, these data establish that 21 does not activate other human GPCRs (other than C3aR) that mediate intracellular Ca2+ release in this cell type.

plot (Figure 6B) revealing that the inhibition of 21 by 4 was competitive (slope = 0.93), supporting likely binding of 4 and 21 at the same or overlapping site in the receptor. Compound 21 Stimulates Inflammatory Gene Expression through C3aR. The inflammatory gene expression profiles 8465



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Figure 7. Upregulation of expression of inflammatory genes (e.g., IL-1β, TNFα, CCL3) in HMDMs by treatment with human C3a or 21 is inhibited by pretreatment with 4. HMDMs were pretreated with 4 (10 μM, 30 min) before the addition of C3a or 21 (0.3 μM, 30 min). Gene expression was measured using qRT-PCR. The relative gene expression data was normalized against 18S. Fold changes were calculated against controls (untreated). Error bars represent mean (±SEM) of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.005.

constrained than most ligands reported to date for this receptor, with the imidazole compound 21 being the most potent and selective agonist for human macrophage C3aR discovered to date from this series. These heterocycles could potentially be similarly incorporated into other GPCR peptidomimetics as a rigid scaffold within which H-bond interactions could be finetuned for modulating receptor binding and ligand function as illustrated here.

for 21 and C3a in HMDMs were compared using quantitative real-time polymerase chain reaction (qRT-PCR). In this study, treatment with 21 and C3a (300 nM, 30 min) significantly upregulated the expression of the inflammatory genes interleukin 1-beta (IL-1β) and tumor necrosis factor alpha (TNFα) and the chemotactic cytokine macrophage inflammatory protein 1-alpha (MIP-1α or CCL3) in HMDMs (Figure 7), as well as other inflammatory genes to be reported elsewhere. These three genes are established inflammatory mediators and highlight immunostimulatory properties for 21 that are similar to that of the native immunostimulant C3a. Compound 21 was not quite as effective in inducing inflammatory genes as C3a. It may be that other regions of C3a in addition to the C-terminus are important for eliciting these responses too, by perturbing the 7TM conformation for more efficient coupling to different G proteins, leading to stronger gene induction. Alternatively, these differences might reflect small differences in the binding orientation of the two agonist effector regions at the C3aR binding site. To investigate whether these measures of cell activation by C3a or 21 were caused by receptors other than C3aR, the C3aR antagonist 4 was examined for its capacity to inhibit gene expression. The results show that pretreatment with 4 (10 μM, 30 min) was able to significantly inhibit the gene expression induced by C3a and 21 (300 nM, 30 min) (Figure 7). These findings indicate that the gene upregulation induced by C3a and 21 was mediated by C3aR.

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EXPERIMENTAL METHODS

General. All reagents were purchased from Sigma-Aldrich or ChemImpex International Inc. All compounds were synthesized via solid- or solution-phase chemistry approaches. The C5a receptor antagonist (3D53)59,60 is Ac-cyclo-(2,6)-Phe[Orn-Pro-dCha-Trp-Arg], prepared as described.61 Electrospray ionization mass spectra (ESI-MS) measurements were obtained on Micromass LCT, and high-resolution mass spectra (ESI-HRMS) measurements were obtained on a Bruker microTOF mass spectrometer equipped with a Dionex LC system (Chromeleon) in positive ion mode by direct infusion in MeCN at 100 μL h−1 using sodium formate clusters as an internal calibrant. Data was processed using Bruker Daltonics DataAnalysis 3.4 software. Mass accuracy was consistently better than 1 ppm error. 1H and 13C NMR spectra were recorded on Bruker Avance 600 spectrometers at 298 K in the deuterated solvents indicated. 1H NMR spectra were referenced to the residual 1H signals: DMSO-d6, 2.50; CD3OD, 3.31 ppm; CDCl3 solutions were referenced to internal TMS. 13C NMR resonances were referenced to the residual solvent peak (DMSO-d6, 39.51; CDCl3, 77.0 ppm). The exact concentration of the compounds was determined by the quantitative NMR integration “PULCON” experiment.62 These settings were used for all PULCON experiments: relaxation delay of 30 s, 64 scans, 2 dummy scans, 90° pulse, and temperature at 298 K. All final compounds were analyzed by HPLC, HRMS, and NMR, and the purity was determined to be >95%. Preparative-scale rpHPLC separations were performed on a Phenomenex Luna C18 15 μm, 250 × 21.2 mm column. Standard conditions were used for elution of all compounds unless otherwise indicated at a flow rate of 20 mL min−1: 100% A to 100% B linear gradient over 15 min followed by a further 10 min at 100% B, where solvent B was 90% MeCN, 10% H2O, and 0.1% TFA and solvent A was H2O and 0.1% TFA. Detection was by UV, and pure fractions were lyophilized. Analytical rpHPLC was used to assess compound purity (Phenomenex Luna C18 column, 5 μm, 90 Å, 4.6 × 250 mm) at three different wavelengths (λ 214, 230, and 254 nm). Standard conditions (same as preparative-scale rpHPLC) were used for all compounds, unless otherwise indicated, at a flow rate of 1 mL/min. Solvent conditions were the same as those for preparative-scale rpHPLC. All final compounds were synthesized as summarized in Schemes 1 and 2. Full experimental details and spectroscopic data of intermediates are reported in the Supporting Information. The spectroscopic data for all assayed compounds is listed below. (2-(2,2-Diphenylethoxy)acetyl)-L-arginine (4). Synthesis of compound 4 has been described previously;15 however, NMR data are reported here for the first time. 1H NMR (600 MHz, DMSO-d6): 9.64 (br s, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.33−7.23 (m, 10H),

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CONCLUSIONS Heterocycles are important components in many drug-like compounds and natural products, restricting rotation and imparting stereoelectronic effects on molecular conformation, chemical reactivity, and biological properties.52,53 Aromatic heterocycles are electron-rich, possess unique electronic properties, and are able to form a variety of interaction patterns with proteins such as hydrophobic, polar, hydrogen bonding, cation−π,54−56 halogen−π,57 and π−stacking interactions.58 Although we have not specifically established what residues in the C3a receptor interact with the heterocycles in ligands reported herein, we have identified that the hydrogen-bond accepting property of the heteroatom is important for ligand affinity for C3aR. Furthermore, the heterocycle has also been shown to influence function, with imidazoles being the most effective heterocycles in this compound series for conferring affinity and agonist potency. Heterocycles containing a weak H-bond acceptor, such as oxygen, at position X of the heterocycle generally had low affinity for human C3aR. By contrast, replacing the oxygen at position X with nitrogen substantially increased binding affinity for C3aR, an effect attributed to its far superior hydrogenbond accepting capability. These heterocyclic ligands are more 8466



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7.19−7.15 (m, 2H), 4.34 (t, J = 7.4 Hz, 1H), 4.07 (dd, J = 9.8, 7.5 Hz, 1H), 4.00 (dd, J = 8.5, 7.7 Hz, 1H), 3.90 (m, 1H), 3.86 (s, 2H), 3.05− 2.98 (m, 2H), 1.67−1.54 (m, 2H), 1.42−1.33 (m, 2H). 13C NMR (100 MHz, DMSO-d6): δ174.9, 167.7, 157.3, 142.1, 142.0, 128.4, 128.1, 126.3, 73.6, 69.9, 52.8, 50.2, 40.3, 29.8, 25.2. (2-((2,2-Diphenylethyl)thio)acetyl)-L-arginine (7). tR = 11.9 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C22H29N4O3S+, 429.1955; found, 429.1955. 1H NMR (600 MHz, DMSO-d6): δ 8.32 (d, 1H, J = 7.9 Hz), 7.76 (t, 1H, J = 5.8 Hz), 7.25−7.33 (m, 10H), 7.16 (t, 2H, J = 7.2 Hz), 4.17−4.27 (m, 2H), 3.31 (d, 2H, J = 7.9 Hz), 3.17 (d, 2H, J = 2.0 Hz), 3.04−3.11 (m, 2H), 1.71−1.79 (m, 1H), 1.55−1.64 (m, 1H), 1.44−1.53 (m, 2H). 13C NMR (150 MHz, DMSO-d6), δ 173.7, 169.7, 157.3, 144.2, 128.8, 128.2, 128.1, 126.7, 52.2, 50.3, 40.7, 37.2, 34.6, 28.7, 25.6. (5,5-Diphenylpentanoyl)-L-arginine (8). HRMS: [MH]+ calcd for C23H31N4O3+, 411.2391; found, 411.2391. 1H NMR (600 MHz, DMSO-d6): δ 8.10 (d, J = 7.8 Hz, 1H), 7.58 (t, J = 5.6 Hz, 1H), 7.30− 7.24 (m, 8H), 7.17−7.13 (m, 2H), 4.16 (m, 1H), 3.92 (t, J = 7.9 Hz, 1H), 3.12−3.02 (m, 2H), 2.20−2.11 (m, 2H), 2.06−1.94 (m, 2H), 1.71 (m, 1H), 1.55 (m, 1H), 1.51−1.44 (m, 2H), 1.44−1.36 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 173.6, 172.2, 156.7, 145.14, 145.09, 128.4, 127.6, 126.0, 51.3, 50.3, 40.3, 34.8, 34.3, 28.1, 25.2, 23.9. (2,2-Diphenylethyl)glycyl-L-arginine (9). tR = 10.1 min (0−100% B 15 min gradient plus 100% B for 10 min). HRMS: [MH]+ calcd for C22H30N5O3+, 412.2343; found, 412.2342. 1H NMR (600 MHz, DMSO-d6): δ 8.80 (d, 1H, J = 7.52 and br s), 7.76 (t, 1H, J = 6.0 Hz), 7.29−7.37 (m, 9H), 7.22−7.27 (m, 2 H), 4.42 (t, 1H, J = 7.6 Hz), 4.19− 4.25 (m, 1H), 3.70−3.78 (m, 4H), 3.08 (q, 1H, J = 6.9 Hz), 1.70−1.77 (m, 1H), 1.54−1.62 (m, 1H), 1.44−1.51 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 172.8, 165.1, 156.8, 140.8, 140.7, 128.8, 127.8, 127.7, 127.1, 51.8, 50.4, 48.1, 47.6, 40.2, 28.1, 25.1. (5-Benzhydrylfuran-2-carbonyl)-L-arginine (10). tR = 11.8 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C24H27N4O4+, 435.2027; found, 435.2025. 1H NMR (600 MHz, DMSO-d6): δ 8.41 (d, J = 8.0 Hz, 1H), 7.51 (t, J = 5.6 Hz, 1H), 7.34 (t, J = 7.6 Hz, 4H), 7.28−7.21 (m, 6H), 7.18 (d, J = 3.4 Hz, 1H), 6.15 (d, J = 3.4 Hz, 1H), 5.65 (s, 1H), 4.33 (m, 1H), 3.14−3.04 (m, 2H), 1.84 (m, 1H), 1.71 (m, 1H), 1.57−1.43 (m, 2H). (2-Benzhydryl-4-methyloxazole-5-carbonyl)-L-arginine (11). tR = 11.5 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C24H28N5O4+, 450.2136; found, 450.2132. 1H NMR (600 MHz, DMSO-d6): δ 8.46 (d, J = 8.0 Hz, 1H), 7.52 (t, J = 5.6 Hz, 1H), 7.39−7.20 (m, 13H), 5.75 (s, 1H), 4.33 (m, 1H), 3.13−3.04 (m, 2H), 2.36 (s, 3H), 1.85 (m, 1H), 1.74 (m, 1H), 1.56−1.40 (m, 2H). (3-Benzhydryl-1,2,4-oxadiazole-5-carbonyl)-L-arginine (12). tR = 11.8 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C22H25N6O4+, 437.1932; found, 437.1932. 1H NMR (600 MHz, DMSO-d6): δ 9.62 (d, J = 7.9 Hz, 1H), 7.61 (t, J = 5.6 Hz, 1H), 7.38−7.33 (m, 8H), 7.31−7.26 (m, 2H), 5.88 (s, 1H), 4.37 (m, 1H), 3.15−3.04 (m, 2H), 1.89 (m, 1H), 1.78 (m, 1H), 1.59−1.46 (m, 2H). (5-Benzhydryl-1,2,4-oxadiazole-3-carbonyl)-L-arginine (13). tR = 11.7 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C22H25N6O4+, 437.1932; found, 437.1934. 1H NMR (600 MHz, DMSO-d6): δ 9.19 (d, J = 7.9 Hz, 1H), 7.58 (t, J = 5.6 Hz, 1H), 7.42− 7.29 (m, 10H), 6.19 (s, 1H), 4.38 (m, 1H), 3.14−3.05 (m, 2H), 1.88 (m, 1H), 1.77 (m, 1H), 1.58−1.47 (m, 2H). (5-Benzhydryl-1,3,4-oxadiazole-2-carbonyl)-L-arginine (14). tR = 11.4 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C22H25N6O4+, 437.1932; found, 437.1932. 1H NMR (600 MHz, DMSO-d6): δ 9.53 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 5.6 Hz, 1H), 7.41− 7.28 (m, 11H), 6.08 (s, 1H), 4.35 (m, 1H), 3.14−3.05 (m, 2H), 1.88 (m, 1H), 1.79 (m, 1H), 1.60−1.46 (m, 2H). (2-Benzhydryl-4-phenyloxazole-5-carbonyl)-L-arginine (15). tR = 12.9 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C29H30N5O4+, 512.2292; found, 512.2292. 1H NMR (600 MHz, DMSO-d6): δ 8.69 (d, J = 7.7 Hz, 1H), 8.11 (m, 2H), 7.57 (t, J = 5.6 Hz, 1H), 7.51−7.35 (m, 12H), 7.33−7.27 (m, 2H), 5.85 (s, 1H), 4.36 (m, 1H), 3.14−3.06 (m, 2H), 1.88 (m, 1H), 1.76 (m, 1H), 1.58−1.47 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 172.9, 163.5, 157.7, 156.7,

141.8, 139.6, 139.5, 138.8, 130.1, 129.2, 128.7, 128.5, 128.4, 128.1, 127.3, 127.3, 51.9, 49.6, 40.3, 27.5, 25.4. (2-Benzhydryl-1,4-dimethyl-1H-imidazole-5-carbonyl)-L-arginine (16). tR = 9.8 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C25H31N6O3+, 463.2452; found, 463.2447. 1H NMR (600 MHz, DMSO-d6): δ 7.72, (m, 1H); 7.43−7.19 (m, 10H); 6.08 (br s, 1H); 4.34 (m, 1H), 3.61 (br s, 3H), 3.17−3.06 (m, 2H), 2.31 (s, 3H), 1.85 (m, 1H), 1.70 (m, 1H), 1.62−1.51 (m, 2H). (2-Benzhydryloxazole-4-carbonyl)-L-arginine (17). tR = 11.5 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C23H26N5O4+, 436.1979; found, 436.1979. 1H NMR (600 MHz, DMSO-d6): δ 8.64 (s, 1H), 8.25 (d, J = 8.1 Hz, 1H), 7.51 (t, J = 5.6 Hz, 1H), 7.40−7.26 (m, 10H), 5.87 (s, 1H), 4.39 (m, 1H), 3.14−3.04 (m, 2H), 1.86 (m, 1H), 1.77 (m, 1H), 1.55−1.44 (m, 2H). (2-Benzhydryl-5-methyloxazole-4-carbonyl)-L-arginine (18). tR = 11.9 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C24H28N5O4+, 450.2136; found, 450.2136. 1H NMR (600 MHz, DMSO-d6): δ 8.02 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 5.6 Hz, 1H), 7.39− 7.55 (m, 10H), 5.79 (s, 1H), 4.38 (m, 1H), 3.10 (m, 1H), 2.54 (s, 3H), 1.86 (m, 1H), 1.77 (m, 1H), 1.54−1.44 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 173.1, 161.3, 161.1, 156.7, 153.1, 139.7, 139.7, 128.7, 128.7, 128.4, 128.4, 127.2, 51.2, 49.5, 40.3, 28.0, 25.4, 11.4. (2-Benzhydryl-4-phenyl-1H-imidazole-5-carbonyl)-L-arginine (19). tR = 10.8 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C29H31N6O3+, 511.2452; found, 511.2453. 1H NMR (600 MHz, DMSO-d6): δ 7.98 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 7.8 Hz, 2H), 7.55 (t, J = 5.6 Hz, 1H), 7.40 (m, 6H), 7.34 (m, 5H), 7.26 (m, 2H), 5.64 (s, 11H), 4.38 (m, 1H), 3.17−3.05 (m, 2H), 1.85 (m, 1H), 1.74 (m, 1H), 1.55−1.46 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 173.4, 162.1, 156.7, 148.2, 141.3, 141.3, 51.2, 49.6, 40.3, 28.6, 25.3. (2-Benzhydryl-1H-imidazole-5-carbonyl)-L-arginine (20). tR = 9.3 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C23H27N6O3+, 435.2139; found, 435.2139. 1H NMR (600 MHz, DMSO-d6): δ 8.35 (br s, 1H), 7.88 (s, 1H), 7.69 (t, 1H, J = 5.3 Hz), 7.33−7.36 (m, 5H), 7.24−7.29 (m, 7H), 5.78 (s, 1H), 4.36−4.41 (m, 2H), 3.06−3.14 (m, 2H), 1.81−1.88 (m, 1H), 1.68−1.76 (m, 1H), 1.47−1.55 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 173.2, 156.9, 149.3, 139.8, 128.7, 128.6, 127.3, 121.6, 51.5, 48.9, 40.3, 28.2, 25.4. (2-Benzhydryl-4-methyl-1H-imidazole-5-carbonyl)-L-arginine (21). tR = 9.5 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C24H29N6O3+, 449.2296; found, 449.2295. 1H NMR (600 MHz, DMSO-d6): δ 7.84 (d, 1H, J = 7.8 Hz, Arg-α-NH), 7.61 (t, 1H, J = 5.6 Hz, guanidine-NH), 7.33−7.36 (m, 4H, Ar−CH), 7.26− 7.28 (m, 6H, Ar−CH), 5.65 (s, 1H, Ph2CH), 4.37−4.41 (m, 1H, Arg-αCH), 3.06−3.15 (m, 2H, Arg-δ-CH2), 2.41 (s, 3H, imidazole-5-CH3), 1.81−1.87 (m, 1H, Arg-β-CH), 1.69−1.76 (m, 1H, Arg-β-CH), 1.48− 1.53 (m, 2H, Arg-γ-CH2). 13C NMR (150 MHz, DMSO-d6): δ 173.4, 162.1, 156.7, 146.6, 140.7, 132.3, 128.6, 127.0, 51.0, 49.6, 40.3, 36.5, 36.4, 28.6, 25.3, 10.6. (2-Benzhydryl-1,5-dimethyl-1H-imidazole-4-carbonyl)-L-arginine (22). tR = 10.3 min (0−100% B 15 min gradient). HRMS: [MH]+ calcd for C25H31N6O3+, 463.2452; found, 463.2447. 1H NMR (600 MHz, DMSO-d6): δ 7.60, (d, J = 7.9 Hz, 1H), 7.56 (t, J = 5.7 Hz, 1H), 7.35−7.28 (m, 8H), 7.25−7.20 (m, 2H), 5.79 (s, 1H), 4.39 (m, 1H), 3.39 (s, 3H), 3.17−3.05 (m, 2H), 2.45 (s, 3H), 1.83 (m, 1H), 1.71 (m, 1H), 1.53−1.42 (m, 2H). 13C NMR (150 MHz, DMSO-d6): δ 173.4, 162.9, 156.6, 146.7, 141.1, 141.0, 132.9, 128.8, 128.7, 128.4, 128.3, 126.7, 50.8, 47.5, 40.3, 30.2, 28.8, 25.2, 9.5. Calculations of H-Bond Interaction Energy. The geometry of the water-heterocycle hydrogen-bonded complexes was optimized at the MP2/6-311++G(3d,3p) level and corrected for the basis set superimposition error by the counterpoise method using Gaussian 09.49 All optimized structures were verified as minima by having all real frequencies. The H-bond interaction energy was the difference in energy of the complex from the sum of energies of water and heterocycle calculated in isolation by the same method. Isolation of Human Monocyte Derived Macrophages (HMDMs). HMDMs were isolated using Ficoll-paque density centrifugation (GE Healthcare Bio-Science, Uppsala, Sweden) from buffy coat of anonymous human donors provided by Australian Red Cross Blood 8467



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Service, Brisbane. CD14+ monocytes were positively selected using CD14+ MACS magnetic beads (Miltenyi Biotech, Auburn, CA, USA) after successive magnetic sorting and washings. The CD14+ monocytes were then cultured at 37 °C with 5% CO2 and differentiated to HMDMs using 100 ng mL−1 recombinant human macrophage colony stimulating factor (M-CSF) (PeptroTech Inc., Rocky Hill, NJ, USA) at 1.5 × 106 cells mL−1. HMDMs were kept in IMDM supplemented with 10% FBS, penicillin (10 U mL−1), streptomycin (10 U mL−1), and L-glutamine (2 mM) (Invitrogen). HMDMs were supplemented after 5 days with fresh medium containing 100 ng mL−1 M-CSF. HMDMs were harvested by gentle scraping in saline solution on day 7. 125 I-C3a Radioligand Binding Assay. Receptor binding was performed by ligand competition with 80 pM [125I]-C3a (2200 Ci mmol−1; PerkinElmer, Torrance, CA, USA), HMDMs (1.2 × 106 cells mL−1), and 80 pM [125I]-C3a with/without various concentrations of unlabeled C3a or C3a nonpeptidic agonist/antagonist, which were mixed with Tris buffer (50 mM Tris, 3 mM MgCl2, 0.1 mM CaCl2, 0.5% (w/v) bovine serum albumin, pH 7.4) for 60 min in a 96-well Nunc round-bottomed plate at room temperature. Unbound [125I]-C3a was removed by filtration through a glass microfiber filter GF/B (Whatman Iner. Ltd., England) which had been soaked in 0.6% polyethylenimine to reduce nonspecific binding. The filter was washed 3 times with cold buffer (50 mM Tris-HCl, pH 7.4), and bound [125I]-C3a was assessed by scintillation counting on Microbeta counter. Specific [125I]-C3a binding was defined as the difference between total binding and nonspecific binding as determined in the presence of 1 μM unlabeled C3a. The binding IC50 value was derived from the concentration of compound required to inhibit 50% of [125I]-C3a binding. Intracellular Calcium Release Assay. HMDMs were plated at 5 × 104 cells/well in a 96-well clear-bottomed black-wall assay plate (Corning) kept in IMDM supplemented with 10% FBS, penicillin (10 U mL−1), streptomycin (10 U mL−1), and L-glutamine (2 mM) (Invitrogen) and incubated overnight at 37 °C. Before assaying calcium release, the medium was removed, and cells were incubated with dyeloading buffer (12 mL HBSS buffer, 4 μM Fluo-3 AM, 25 μL Pluronic acid F-127, and 1% FBS) for an hour at 37 °C. After an hour, cells were washed once with the same HBSS assay buffer (HBSS supplemented with 2.5 mM probenecid and 20 mM HEPES, pH 7.4). Stock solutions of compounds in DMSO were diluted with HBSS buffer to give the concentrations required for the assay. To screen for antagonist activity of a test compound, a range of concentrations of the test compound (50 μL) was preincubated with the cells for 30 min prior to the addition of agonist C3a (50 μL, 100 nM). After addition of agonist, the intracellular release of Ca2+ was monitored on a fluorescence plate reader (FLIPRTETRA) for at least 60 s (excitation 495 nm, emission 520 nm). On the other hand, to screen for agonist activity of a test compound, a range of concentrations of the compound (50 μL) was added to the cells containing 50 μL of HBSS buffer. The intracellular Ca2+ release was monitored for at least 60 s (excitation 495 nm, emission 520 nm) immediately after the injection of the desired concentration of the compound. Duplicate measurements were made for each data point; mean ± SEM were reported from multiple experiments as indicated. Changes in fluorescence (% response) were plotted against logarithmic compound concentrations. The half maximal inhibitory (IC50) and effective concentration (EC50) values were derived from the concentration−response curve using nonlinear regression curve fitting in GraphPad Prism v6. Gene Analysis. HMDMs were seeded at 1 × 106 mL−1 and allowed to adhere overnight. For inhibition experiments, HMDMs were pretreated with C3aR antagonist 4 (10 μM, 30 min), Gαi inhibitor (200 ng/mL PTX, overnight), or PLCβ inhibitor (10 μM U73122, 30 min) prior to C3a or 21 (0.3 μM, 30 min). RNA was extracted from HMDMs according to ISOLATE II RNA Mini Kit (Bioline). Real-time PCR was run on ABI PRISM 7900HT (Applied Biosystems), target gene expression was normalized to housekeeping 18S rRNA, and fold change was calculated relative to untreated control. Primer sequences are shown in Supporting Information Table S1.

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ASSOCIATED CONTENT

S Supporting Information *

Synthetic procedures and characterization data of reaction intermediates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(R.C.R.) Tel: +61-733462991. Fax: +61-733462990. E-mail: [email protected]. *(D.P.F.) Tel: +61-733462989. Fax: +61-733462990. E-mail: [email protected]. Present Address †

(R.S.) Department of Pharmacology & Toxicology, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen 40002, Thailand. Author Contributions

R.C.R., M.-K.Y., and D.P.F. conceived the experiments, analyzed the results, and wrote the paper; R.C.R. and M.-K.Y. synthesized and characterized the compounds; M.-K.Y., R.S., J.K.H., and J.L. performed the pharmacological assays and analyzed results. Notes

The authors declare the following competing financial interest(s): The authors are named inventors on a patent owned by the University of Queensland.

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ACKNOWLEDGMENTS This study was supported by the Australian National Health and Medical Research Council through grant nos. APP1000745 and APP1028423 and an SPRF 1027369 fellowship to D.P.F.; by Australian Research Council grant nos. DP130100629 and DP1093245, an ARC Federation fellowship (FF0668733) to D.P.F., and an ARC Centre of Excellence grant (CE140100011) in Advanced Molecular Imaging; by the Queensland State Government (CIF grant); and by Khon Kaen University (Thailand) and an Endeavour Postgraduate award to R.S. We thank the Australian Red Cross for providing the buffy coat for human monocyte isolation.

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ABBREVIATIONS USED C3aR, C3a receptor; Cha, cyclohexylalanine; CXCR4, chemokine receptor 4; DMPK, drug metabolism/pharmacokinetics; H-bond, hydrogen bond; HMDMs, human monocyte-derived macrophages; iCa2+, intracellular calcium; IL-6, interleukin-6; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; RT, room temperature; SAR, structure−activity relationship; TNFα, tumor necrosis factor alpha

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REFERENCES

(1) Hugli, T. E.; Vallota, E. H.; Muller-Eberhard, H. J. Purification and partial characterization of human and porcine C3a anaphylatoxin. J. Biol. Chem. 1975, 250, 1472−1478. (2) Ember, J. A.; Hugli, T. E. Complement factors and their receptors. Immunopharmacology 1997, 38, 3−15. (3) Caporale, L. H.; Tippett, P. S.; Erickson, B. W. The active site of C3a anaphylatoxin. J. Biol. Chem. 1980, 255, 10758−10763. (4) Zwirner, J.; Gotze, O.; Begemann, G.; Kapp, A.; Kirchhoff, K.; Werfel, T. Evaluation of C3a receptor expression on human leucocytes by the use of novel monoclonal antibodies. Immunology 1999, 97, 166− 172. (5) Martin, U.; Bock, D.; Arseniev, L.; Tornetta, M. A.; Ames, R. S.; Bautsch, W.; Kohl, J.; Ganser, A.; Klos, A. The human C3a receptor is

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