The endogenous ligands at these receptors, PACAP (27 or 38 a.a.r.) and VIP (28 a.a.r.), share some common features with other peptide ligands of the secretin-glucagon family of GPCRs.

The endogenous ligands of Class B receptors have amino acid sequences in the range between 27 and 44 a.a.r. in mammalian. They are synthetized by endocrine cells, neurons or immune cells. They form α helices and contain an NCap structure in the N-terminal part [18] [48] [49] . PACAP has been recognized as a compound acting as hormone, neurohormone, neurotransmitter and neurotrophic factor [50] . It is found in the brain, cardiovascular system, thyroid, pituitary, adrenal gland and placenta [10] [13] [51] - [53] . Both, PACAP and VIP, are regarded as master switches of circadian rhythm [54] [55] . For VIP its role as regulator of growth of whole fetus and of the embryonic brain has been classified as another important long-term effect, while short- term activities with involvement of VIP include exocrine secretion, hormone release, muscle relaxation and metabolic actions [18] . Furthermore, VIP is involved in neuroprotective, antiinflammatory and immunmodulatory effects and influences cell proliferation in cancer cells [10] . The level of PACAP38 in adult brain of mice has been found in the range of 120 fmol/mg protein and the level of VIP at 500 fmol/mg protein [56] . The levels of both peptides are changing from embryonic to adult age in murine brain [57] . PACAP was found at 24 fmol/mg protein at postnatal day 1 and VIP at 9.4 fmol/mg protein [56] at the same stage of development. PACAP achieves levels of the adult brain some days earlier than VIP. It binds to PAC1 with 1000 fold higher affinity than VIP but shows similar affinities to VPAC receptors like VIP [58] .

The selectivity between the three relevant receptors has been investigated in models of SAR at first for VIP [59] [60] , and subsequently also for PACAP [25] [58] . The first concise pharmacophore model of VIP at VPAC1 and VPAC2 receptors with systematic description of functional relevance of all a.a.r.s was provided by Nicole et al. (2000) [59] . A characterization of PACAP at the PAC1 receptor was later presented by Sun et al. [58] and Kumar et al. [23] . Nicole et al. identified T11 and N28 by alanine scanning, energetic optimization as well as investigations of pharmacodynamics as pivotal structures for the binding of VIP to VPAC receptors. For PACAP38, it has been observed that the removal of the first five amino acids can transform the polypeptide from an agonist at the PAC1 receptor into an antagonist without alteration of the binding affinity [58] [61] .

Regarding the functional roles of the peptides, for PACAP essential part of attention has been paid to its contribution to stress response [24] , whereas the main fields for VIP investigation are inflammatory diseases [62] and its cytokine-like role. However, for both peptides have been shown influences of knock-out on behavioral functions and on early development in mammals too [24] [35] [63] , e.g. in PACAP and VIP deficient mice was demonstrated an increase of locomotor activity in open field [56] . On the other hand, VPAC2 deficient mice have been shown to be more vulnerable to inductors of experimental colitis than their wild-type littermates [64] . Recently, a potential suitability of PACAP as a predictor of outcome from acute intracerebral ischaemia has been suggested by clinical studies [65] [66] . PACAP38/PAC1 signalling was demonstrated to be an important factor in regulation of trafficking of bone marrow cells which could facilitate their travel to vascular niches after focal cerebral ischaemia [66] . Already 2011 Ressler et al. [67] demonstrated that single nucleotide polymorphisms in the genes of PACAP (ADCYAP1) and PAC1 receptors (ADCYAP1R1) or methylation of the receptors mRNA can result in postischaemic stress disorder. Moreover, altered VIP levels have been described in obese women (low levels) and patients with anorexia nervosa [68] [69] . Furthermore, VIP is suggested to be involved in circuits regulating stimulation of the dentate subgranular stem cell niche and it is co-expressed by GABAergic neurons located in hippocampal regions close to this proliferatively important region [11] . For PACAP and VIP have been discovered direct and indirect neuroprotection via microglia mediated mechanisms (RANTES; CC chemokine Regulated upon Activation, Normal T cell Expressed and Secreted; MIP; macrophage inflammatory protein; ADNF; activity dependent neurotrophic factor) and for PACAP has been discussed a stimulation of chemokine release by microglia for inhibition of gp120 co-repressors like CCR3, CCR5 and CXCR4-a mechanism possibly of relevance for the protection against HIV [26] [70] . High attention has been paid to the presence of VIP in lung and bronchial tissue [71] - [73] . A high VIP content in the lung [74] was one of the main triggers of the ligand research on VPAC receptors.

Most of the binding data on vasoactive intestinal peptide receptors were measured in cellular systems with overexpression of wild-type and mutated types of the three target receptors. For chicken PAC1 receptors Zawilska et al. [9] ranked the affinities of the endogenous peptides in the following order: PACAP38~PACAP27 > PACAP6-27~PACAP6-38 > chicken VIP (cVIP) > mammalian VIP and secretin (inactive) [9] . The highest density in chicken cortical membranes has been demonstrated with 457 fmol/mg for the PAC1 receptor [9] and in mouse whole brain membranes with 857 fmol/ mg protein [75] using [¹²⁵I]-PACAP27 as receptor ligand. In guinea pig cerebral cortex maximal binding of [¹²⁵I]VIP (human /rat/porcine) was reported by Zawilska et al. 2005 [76] with 77 fmol/mg protein without differentiation of the receptor subtypes and the authors provided relative rank order of potency: cVIP ≥ PACAP38~PACAP27~guinea pig VIP (gpVIP) > mammalian VIP (mVIP) > peptide-histidine-methionine (PHM) > peptide-histidine-isoleucine (PHI) > secretin [76] . Main localizations of PAC1 receptors are neurogenic regions like subventricular zone of olfactory bulb and dentate gyrus [10] . Immunohistochemical investigations suggest high levels of PAC1 receptors also in layer I of the cerebral cortex, very high density of the receptor in hypothalamus, brainstem, midbrain and hindbrain, and in cerebellar nuclei. [57] [58] [77] .

In turn, PAC1 has been reported to be of less importance in hippocampal regions than VPAC receptors [75] [78] .

Autoradiographic and membrane binding assay data as well as species differences have been described also for peripheral tissue [12] . Comparison of [¹²⁵I]VIP binding at receptors of the lung in human and guinea pig tissues reveals B_max values of 11.2 fmol/mg protein and 226 fmol/mg protein, respectively, for the high affinity binding site and 589 fmol/mg protein and 1730 fmol/mg protein at the low affinity binding sites. For the rat lung have been reported B_max of 584 fmol/mg protein [73] .

For PAC1 receptors in rodents (PACAP38, K_d 0.5 nM, for comparison VIP-affinity > 500 nM) have been identified six splice variants relevant for the third intracellular loop which is coupled to G-proteins [79] . The two exons hip and hop, spliced in or out determine different PAC1 receptor variants triggering different signalling pathways [9] [24] . Moreover, splice variants with change in the a.a.r. sequence of the N-ted in position 21 have been described [5] - [7] which bind PACAP27, PACAP38 and VIP with similar affinity [5] - [7] .

Activation pathways related to neuroproliferation are regulated via G_q and G_i proteins [80] and via interaction with mitogen-activated protein kinase MAP-Kinase and arrestins which provide the regulatory input to receptor internalization, desensitization and proteolytic processes as well as to heteromeric signal protein complex activation. The preferred binding sites of VIP are the N-teds of VPAC1 and VPAC2 receptors (K_D 1 nM) [81] , which mediate their intracellular actions via G_s proteins, but also via G_q and G_o proteins. They also trigger calcium release as well as interact with RAMP1-3 (receptor activity modifying protein) (cf. Table 1). A differentiation between the VPAC receptor subtypes predominantly became possible by the discovery of helodermin obtained from gila monster venom [9] [82] [83] . The peptide shows some homology to PACAP and VIP.

Whereas Nicole et al. [59] had supplied a first systematic functional classification of VIP and PACAP a.a.r. components, Laburthe et al. (2007) [83] discussed critically basing on these data two different models of the mechanism in which VPAC receptors and especially its N-ted can transfer its endogenous ligands within the transmembrane/juxtamembrane domain (J-domain) of the receptor as well as close to intracellular loops (here IL3) involved in G proteins activation [84] - [86] . Endogenous peptide ligands [35] [87] [88] bind by their C-terminal portion to the extracellular domain whereas the N-terminus of the endogenous ligands interacts with the 7TMD structures of the receptors. All Class-B receptors are distinct from Class A receptors by the typically large N-ted (100 - 160 a.a.r, [36] [47] ) with consensus repeat or Sushi domain core formed by two antiparallel β sheets connected via three disulfide bonds between six cysteine residues and a salt bridge. This feature is regarded as the signature of Class B receptors, even if a full length structure of Class B receptors is yet not available [61] [84] .

VPAC1 is expressed in lung, small intestine, thymus, kidney and brain [10] [63] [78] [89] [90] . In the brain VPAC receptors are expressed in piriform cortex, cerebral cortex, dentate gyrus, hippocampus, lateral amygdaloid nucleus, caudate putamen, nucleus supraopticus, thalamic nuclei, choroid plexus and pineal gland as well as cerebellar cortex and deep cerebellar nuclei [11] [77] . B_max values for the binding of [¹²⁵I]cVIP and [¹²⁵I]PACAP have been obtained for chicken hypothalamus and chicken cortical cerebral membranes respectively. [¹²⁵I]cVIP has been shown to bind with 167 fmol/mg protein to chicken hypothalamic membranes [8] ~1/3 of the B_max for [¹²⁵I] PACAP in cortical cerebral membranes of the same species [9] .

Also the main target receptors of VIP as there are VPAC1 and VPAC2 have different pharmacology and distributions [5] [91] . E.g. VIP had a 4 fold higher affinity for hVPAC1 than for VPAC2 [60] [61] .

The VPAC2 receptor is suggested to mediate activation of insulin secretion [91] [92] , while VPAC1 receptors according to their expression pattern are suggested to contribute to the increase of hepatic glucose production [93] - [95] but also to attenuation of diabetes related inflammation [96] .

On the other hand, [24] chromosomal site duplication can result in serious neuroanatomic retardation [24] and characteristic mental diseases, e.g. alterations of VPAC2 expression by chromosomal site duplication can carry the risk of schizophrenia and autism [97] . In turn, VPAC2 receptor-deficient mice showed growth retardation [98] .

PAC1 knock-out mice have been reported to show mortality close to 60% during the first four weeks after birth. Surviving mice are characterized by a accelerated decrease in social investigations and less aggressively male animals but higher sexual activity. The experiments suggested that PACAP could be a counterpart of the vasopressin-oxytocin system [99] . Furthermore, PAC1 deficient mice were shown to develop reduction of anxiety-like behaviour [100] .

Labelling of synthetic peptide ligands with PET and SPECT isotopes for recognition of VPAC and PAC receptors for diagnostic and therapeutic purposes include ^99mTc, ¹⁸F, ¹²³I, ⁶⁸Ga, ⁶⁴Cu and ¹¹¹In labelled compounds, which are prepared using distinct chelating agents or prosthetic groups [45] . Somatostatin and its labelled analogues (e.g. [¹¹¹In] octreotide) are the most frequently clinically used peptides in tumor diagnostics. However, also further peptide receptor ligands are more and more in the focus of tumor visualization. E.g. bombesin peptides labelled with ¹⁸F and ^99mTc have been already described in studies with VPAC receptor ligands in the early years of this century. The overexpression of VPAC receptors, however, also in normal tissues of some organs restricts the efficiency of such peptide probes as tools in tumor identification [45] . Other applications e.g. the imaging of brain receptors are limited by vulnerability of the peptides to enzymatic breakdown and low in vitro- stability. Some success could be achieved by reconstituted peptides containing mutations in selected parts of the molecule, intramolecular ring structures, introduction of lactam ringsor acylation of the N-terminus of VIP [101] . Furthermore, the connection with polyethylene glycol (PEG) was used to improve the pharmacokinetic properties.

There had been no reports on small molecule non-peptide ligands of the VPAC1 receptor until Harikrishnan et al. (Bristol-Myers Squibb) [90] published 2012 the results of a high throughput screening based on cAMP assays and determination of antiproliferative activity. Three series of potential lead structures were presented providing moderate binding affinities until 81 nM (biaryl compounds; Figure 3, compounds 1 - 3); 290 nM (cyanothiophenes; Figure 4, compounds 4 - 8) and 410 nM (cyanothiophene phenethylamide) compounds 9 and 10; Figure 5).

Robl et al. provided in 1990 [120] the synthetic route used by Harikrishnan et al. in a modified mode via formation of a lactone and its aromatization followed by introduction of triflic anhydride which gave via aryl triflate finally the biaryl. A series of substitutions at the 2-aryl ring resulted in the best IC₅₀ values for a 3-chloro compound (100 nM) and a 3,4 dimethyl compound (81 nM) regarding inhibition of cAMP release. Actions on cell proliferation could not be verified.

The cyanothiophenes stood less active in the same screening assays if various amides had been introduced into the molecules. In general, the introduction of amides coupled to cyclopentyl and hexyl residues resulted in better IC₅₀ (until 290 nM) than the small aliphatic or small cyclic motifs. Further modification of phenetylamide cyanothiophenes (Figure 5) could not further improve the inhibitory efficacy in cAMP assays additionally [90] .

Neither orthosterically nor allosterically binding small molecule ligands of VPAC2 receptors were presented until 2010 [47] . The first compound discovered by high throughput screening was a nitrophenyl sulfonamide pentanamide which showed moderate IC₅₀ values for inhibition of VPAC2 receptor-mediated cAMP accumulation (IC₅₀ 3.8 µM) and for ligand-activated β-arrestin2 binding (IC₅₀ 2.3 µM; β arrestin Pathhunter assay). Chu et al. (Novartis) pronounciate that only a single lead structure was detected among 1.67 million compounds of their data collection. This compound is a non-competitive ligand of the human VPAC2 receptor [47] (Figure 6, compound 11).

One of the main obstacles of the discovery of the small-molecule ligands of class B GPCRs is the absence of highly pecific antagonists at VPAC1 and VPAC2 receptors [47] . Peptide ligands typically bind to orthosteric binding sites of these receptors. However, usually the structural difference between such peptide ligands are not sufficient to discriminate very selectively between the two receptor subtypes. Chu et al. demonstrated for their compound in Schild-Plot analysis of cAMP accumulation typical features of non-competitive ligands as the mismatch of the slope with requirements for perfect linear regression which would have confirmed competitive interaction with the VPAC2 receptor.

In the β-arrestin assay compound 1 (here compound 11, cf. Figure 6) decreased EC₅₀ of VIP but reduced also the maximal level of arrestin binding what fulfills rather the requirements for an allosteric modulator than for a competitive antagonist. The compound interacts with a.a.r. in the TM7 region which are, however, not conserved between human and mouse [47] [84] .

Compound 2 (here compound 12, Figure 6) is presumed by Chu et al. [47] to activate the VPAC2 receptor rather than to act as an antagonist.

A first series of small-molecule antagonists of PAC1 receptors with affinities in the nM range was proposed in 2008 by Beebe et al. (Abbott Laboratories) [46] . Hydrazides were chosen as lead structures containing three cyclic moieties and the authors of the study supplied also first landmarks on potential pharmacophore contributions of the essential parts of these compounds [25] . Identification of two lead structures was performed by means of a compound library using [¹²⁵I]PACAP27 binding at PAC1 receptors expressed in human embryonic

kidney cells (HEK293f cells). Finally, the structures based on series of alkylidene hydrazides developed by Madsen, Ling et al. (Novo-Nordisk ,Pfizer) as glucagon receptor antagonists in 2002 [121] . Most effective binding potencies at the PAC1 receptor had been observed for a hydrazide (Figure 7, compound 13) with a K_i of 56 nM and a hydrazide indole (Figure 7, compound 16, K_i 72 nM) [25] [46] .

The log P of compounds 13 and 16 are with 5.36 and 6.12 not in an appropriate range for use as probes for brain imaging. Modifications of the structure with demethylation in the distal ring, at the middle ring and fluorination at the distal ring can bring the ratio closer to 3 (Figure 7, 15). Beebe et al. [46] revealed in investigations of structure-activity relationships that alterations in these regions will be only of minor consequences for the affinity at the PAC1 receptor.

Furthermore, the pharmacophore analysis of hydrazide indoles described by Beebe et al. demonstrates the indole moiety tolerates only poor structural modifications. The p-phenol connected by the hydrazide linker with the middle ring as well as its m-electron withdrawing group had been identified to be crucial for high potency of the compound at the target receptor [46] .

Currently, Wu et al. [25] investigated the potential interaction between the PAC1 receptor and its endogenous ligand PACAP38 as well as with the two hydrazides mentioned above (compounds 13 and 16; Figure 7). In docking studies of a 3D model of the receptor, the 7TMD of the PAC1 receptor was mimicked by a structure based on homology studies on the PTH1 receptor. The signal sequence of the N-terminal ectodomain (N-ted) had been removed in this model [25] [122] . Previous experiments with exon 2 -/- mice had demonstrated that animals with targeted deletion of the signal peptide are viable, fertile and without morphological differences to wild-type animals [122] [123] . 76 and 89 conformations were identified for the binding of compounds 13 and 16 at the receptor model. In most of them hydrogen bonds were created to the amino acids I63, S100 and G105. Best binding conditions were obtained for compound 13 via hydrogen bonds with S100 and E339. The amino acid residues 116 - 120 of the N-ted domain had been identified as preferred binding sites of PACAP38. Because the signal peptide containing the first 20 amino acids is absent in the model created by Wu et al., the S100 corresponds to S120 in the native receptor [125] . The authors predicted that the investigated hydrazide compounds can inhibit the interaction of PACAP with the N-ted and prevent also the normal conformational consequences for the 7TMD region. They suggested that the compounds produce a steric hindrance for the binding of PACAP and inhibit competitively the biological activity of the peptide ligand.