Iwan de Esch Henk Timmerman and Rob Leurs

Asthma Free Forever

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17.1 Introduction 283

17.2 The Histamine H1 Receptor: Molecular Aspects and Selective Ligands 284

17.2.1 Molecular Aspects of the Histamine Hj Receptor Protein 284

17.2.2 H1 Receptor Agonists 284

17.2.3 H1 Receptor Antagonists 285

17.2.4 Therapeutic Use of H1 Receptor Ligands 287

17.3 The Histamine H2 Receptor: Molecular Aspects and Selective Ligands 287

17.3.1 Molecular Aspects of the Histamine H2 Receptor Protein 287

17.3.2 H2 Receptor Agonists 287

17.3.3 H2 Receptor Antagonists 288

17.3.4 Therapeutic Use of H2 Receptor Ligands 289

17.4 The Histamine H3 Receptor: Molecular Aspects and Selective Ligands 290

17.4.1 Molecular Aspects of the Histamine H3 Receptor Protein 290

17.4.2 Histamine H3 Receptor Agonists 291

17.4.3 Histamine H3 Receptor Antagonists 291

17.4.4 Therapeutic Use of Histamine H3 Receptor Ligands 294

17.5 The Histamine H4 Receptor: Molecular Aspects and Selective Ligands 294

17.5.1 Molecular Aspects of the Histamine H4 Receptor Protein 294

17.5.2 Histamine H4 Receptor Agonists 294

17.5.3 Histamine H4 Receptor Antagonists 295

17.5.4 Therapeutic Use of Histamine H4 Receptor Ligands 296

17.6 Concluding Remarks 296

Further Readings 297


Histamine is both an aminergic neurotransmitter and a local hormone and plays major roles in the regulation of several (patho) physiological processes. In biological systems histamine is synthesized from L-histidine by histidine-decarboxylase (HDC, Scheme 17.1). In the brain (where histamine acts as a neurotransmitter) the synthesis takes place in restricted populations of neurons that are located in the tuberomammillary nucleus of the posterior hypothalamus. These neurons project to most cerebral areas and have been implicated in several brain functions (e.g., sleep/wakefulness, hormonal secretion, cardiovascular control, thermoregulation, food intake, and memory formation). In peripheral tissues (where histamine acts as a local hormone), the compound is stored in mast cells, eosinophils, basophils, enterochromaffin cells, and probably also in some specific neurons. Once released, histamine is rapidly metabolized via N-methylation of the imidazole ring by the enzyme histamine N-methyltransferase (HMT) and by the oxidation of the amine function by diamine oxidase (DAO, Scheme 17.1).

\=n T-Methylhistamine

SCHEME 17.1 Synthesis and metabolism of histamine. HDC, histidine decarboxylase; DAO, diamine oxidase; HMT, histamine N-methyltransferase.

Some of the symptoms of allergic conditions in the skin and the airway system are known to result from histamine release after mast cell degranulation. In 1937, Bovet and Staub discovered the first compounds that antagonize these effects of histamine. From henceforth, there has been intense research devoted toward finding novel ligands with (anti)histaminergic activity.

As more (patho) physiological processes that are mediated by histamine (e.g., stomach acid secretion and neurotransmitter release) were studied, it became apparent that the action of hista-mine is mediated by several subtype receptors. This resulted in the identification of the histamine H2 receptor in 1966, the histamine H3 receptor in 1983, and the histamine H4 receptor in 2000 (note the interval of 17 years). The first three histamine receptor subtypes were discovered by classical pharmacological means, i.e., using subtype selective ligands that were developed by medicinal chemists. The histamine H4 receptor was discovered using the human genome, nicely illustrating the impact of molecular biology and genomics in drug discovery.

In this chapter we will describe in detail the state-of-the-art knowledge on the molecular features of the histamine receptor proteins, the medicinal chemistry of the four histamine receptors, and the (potential) therapeutic applications of selective receptor ligands.


17.2.1 Molecular Aspects of the Histamine H1 Receptor Protein

The Hj receptor belongs to the large family of rhodopsin-like, G-protein coupled receptors (GPCRs). In 1991, the cDNA encoding a bovine H1 receptor protein was cloned after an expression cloning strategy in Xenopus oocytes by Fukui and coworkers. The human H1 receptor gene resides on chromosome three and the deduced amino acid sequence revealed a 491 amino acid protein of 56 kDa. Using the cDNA sequence encoding the bovine H1 receptor, the cDNA sequences and intronless genes encoding the rat, guinea pig, human, and mouse H1 receptor proteins were cloned soon thereafter. These receptor proteins are slightly different in length, highly homologous, and do not show major pharmacological differences. The stimulation of the H1 receptor leads to the phospholipase C-catalyzed formation of the second messengers inositol 1,4,5-triphosphate (IP3) and 1,2-diacylglycerol (DAG), which in turn lead to the mobilization of intracellular calcium and the activation of protein kinase C, respectively.

17.2.2 H-, Receptor Agonists

The modification of the imidazole moiety of histamine has been the most successful approach for obtaining selective H1 agonists (Figure 17.1). The presence of the tautomeric Nn-N system of the


Br nh2

Histamine 2-(3-Bromophenyl)

(endogenous ligand) 2-Pyridylethylamine 2-Thiazolylethylamine histamine

Histamine 2-(3-Bromophenyl)

(endogenous ligand) 2-Pyridylethylamine 2-Thiazolylethylamine histamine

Methylhistaprodifen Suprahistaprodifen Lisuride

FIGURE 17.1 Histamine H1 receptor agonists.

Methylhistaprodifen Suprahistaprodifen Lisuride

FIGURE 17.1 Histamine H1 receptor agonists.

imidazole ring is not obligatory, as reflected by the selective H1 agonists 2-pyridylethylamine and 2-thiazolylethylamine. Substitution of the imidazole ring at the 2-position will lead to relatively selective H1 agonists. For example, 2-(3-bromophenyl)histamine is a relatively potent H1 receptor agonist. Schunack and colleagues subsequently developed a series of so-called histaprodifens on the basis of the hypothesis that the introduction of a diphenylalkyl substituent on the 2-position of the imidazole ring yields high affinity agonists. This hypothesis was based on the realization that a diphenylmethyl group is a common feature of high-affinity H1 antagonists (see Section 17.2.3). The introduction of the diphenylpropyl substituent at the 2-position of the imidazole ring and N-methylation of the ethylamine side chain results in the high potency agonist N-methyl-histaprodifen. Further modifications of the diphenylmethyl moiety were unsuccessful and indicated a considerable difference in structure-activity relationship (SAR) (and most likely receptor-binding site) of the diphenyl moieties of the histaprodifens and the structurally related H1 antagonists. A further increase in H1 receptor agonist potency was obtained by a bivalent ligand approach. Suprahistaprodifen, a dimer of histaprodifen and histamine is currently one of the most potent H1 receptor agonists available. Surprisingly, recent high-throughput screening (HTS) of CNS-active drugs at the histamine H1 receptor has identified the nonimidazole ergot derivative lisuride as another high affinity H1 receptor agonist.

17.2.3 H-j Receptor Antagonists

The first antihistamines were identified and optimized by exclusively studying in vivo activities. This might be the explanation why several compounds originally reported as antihistamines were later on developed for other applications; e.g., the first so-called tricyclic antidepressants (e.g., doxepin) are often also very potent H1 antagonists. More modern approaches, using genetically modified cells expressing the human H1 receptor, currently provide more in-depth information on the molecular mechanism of actions. All therapeutically used H1 antagonists, in fact, act as inverse agonists (see Chapter 12) and favor an inactive conformation of the GPCR protein. In view of the detectable level




Cetirizine ci

Diphenhydramine y

F AstemizoIe






Fexofenadine COOH


-O Doxepin

Fexofenadine COOH

-O HY-2901

FIGURE 17.2 Histamine Hj receptor antagonists (inverse agonists).

of spontaneous activity of the Hj receptor (i.e., receptor signaling without agonist, also known as constitutive GPCR activity), all the Hj antagonists tested so far inhibit the constitutive activation of e.g., nuclear factor-KB (NF-kB).

Of the many first generation histamine blockers, diphenhydramine (Figure 17.2) is considered as the archetype. The compound is known in medicine as Benadryl®, the first antihistamine successfully used in man. Other compounds of this class are, e.g., mepyramine and triprolidine. These compounds are highly potent H1 antagonists and very useful both for pharmacological investigations and medicinal use. The so-called classical "antihistamines" easily penetrate the brain and are therefore also useful for in vivo CNS studies.

The early antihistamines had two major drawbacks: they were ligands for several targets; especially the antimuscarinic effects caused unpleasant side effects (e.g., dry mouth). By careful structural modifications, it is possible to obtain selective antihistamines. Another drawback of the first generation H1 antagonists was that the compounds show strong sedating effects, to such a level that some of them are still used as sleeping aids.

The notion that sedation is caused by a blockade of H1 receptors in the brain, sparked the search for nonbrain penetrating compounds. Minor structural modifications resulted in a number of new, nonsedating H1 antagonists (e.g., cetirizine, astemizole, fexofenadine, and desloratidine), also referred to as the second-generation H1 blockers. Interestingly, the first of such compounds were more or less found by chance and it took quite some time to understand why the compounds did not manifest CNS effects. It is now understood that these compounds act as substrates of the P-glycoprotein (PgP) transport system in the blood-brain barrier. Consequently, these compounds are actively transported out of the brain and thereby are not able to occupy significant amounts of brain H1 receptors. The new class of compounds, including terfenadine (later on due to HERG-blockade replaced by its active metabolite fexofenadine), cetirizine (now replaced by the L-enantiomer), and loratidine (now replaced by its desoxy-active metabolite, desloratidine) reached as antiallergics the blockbuster status.

Although effective in treating allergic reactions, the second-generation H1 antagonists do not display significant antiinflammatory effects. Currently, research focuses on compounds also targeting inflammation; compounds having besides Hj blocking properties antagonizing also, e.g., LTB4 or blocking the synthesis of leukotrienes, have the interest of pharmaceutical companies. Recently, the combined blockade of H1 and H4 receptors (see below) has also been indicated as an attractive new approach. Interestingly, since the turn of the century the interest in the sleep promoting effects of histamine H1 receptor antagonists has increased. Especially, the "old" derivative doxepin, a compound that blocks the H1 receptor and also the H2 receptor is used as a sleep inducer. An analog of doxepin, HY-2901, has been shown to have interesting properties for use as a sleep inducer and is currently under clinical evaluation as sleep aid.

17.2.4 Therapeutic Use of Ht Receptor Ligands

The histamine Hj receptor is a well-established drug target and has been thoroughly studied for decades. The first- but especially the second-generation antihistamines are clinically very successful and are widely available drugs. The main indications are hay fever, allergic rhinitis, and conjunctivitis as well as comparable allergic affections; the application for asthmatic conditions does not seem to be of much use. The first generation antihistamines are still used as in over the counter (OTC) sleep aids or antiflu combination pills. As indicated before, currently interest in sleep aids is increasing and new molecules are being developed (e.g., HY-2901).


17.3.1 Molecular Aspects of the Histamine H2 Receptor Protein

The fact that the "antihistamines" did not antagonize histamine-induced effects at the stomach and the heart, led in 1966 to the proposal by Ash and Schild of two distinct histamine receptors: the H1 and H2 receptors. This hypothesis became generally accepted in 1972 when Black and his coworkers at Smith, Kline & Beecham presented burimamide and related compounds. These ligands antagonize the effects of histamine on the stomach and the heart. Nowadays, the H2 receptor is (as all histamine receptor subtypes) known to belong to the rhodopsin-like family of GPCRs. Using a polymerase chain reaction (PCR)-based method, based on the known sequence similarity of various GPCRs and gastric parietal mRNA, the H2 receptor nucleotide sequence was elucidated. This DNA sequence encodes for a 359 amino acid GPCR receptor protein. Soon thereafter, the intronless genes encoding the rat, human, guinea pig, and mouse H2 receptor were cloned by means of homology. The H2 receptor proteins are slightly different in length, highly homologous, and do not show major pharmacological differences. Interestingly, several polymorphisms have been found in the human H2 receptor gene and one of the mutations might be linked to schizophrenia.

The histamine H2 receptor is positively coupled to the adenylate cyclase system via Gs proteins in a variety of tissues (e.g., brain, stomach, heart, gastric mucosa, and lungs). Moreover, cell lines recom-binantly expressing the H2 receptor show increases in cAMP following H2 receptor activation.

17.3.2 H2 Receptor Agonists

A simple modification of the histamine molecule has not been very successful to obtain selective and potent H2 receptor agonists. A first step toward an H2 receptor agonist was made with the





FIGURE 17.3 Reference histamine H2 receptor agonists.


discovery of dimaprit (Figure 17.3), which was found during a search for H2 receptor antagonists in a series of isothiourea derivatives. Dimaprit is an H2 receptor agonist that is almost as active as histamine at the H2 receptor, but hardly displays any H1 receptor agonism. Later it was found that dimaprit is also a moderate H3 receptor antagonist and a moderate H4 receptor agonist. Using dimaprit as a template, amthamine (2-amino-5-(2-aminoethyl)-4-methylthiazole) was designed as a rigid dimaprit analog. Following the original suggestion of Green et al. that the sulfur atom of dimaprit might act as a proton acceptor in a hydrogen bonding network with the H2 receptor (in analogy to the idea of the interaction of the imidazole ring with the receptor protein), quantum chemical calculations by and synthesis of 2-aminothiazole analogs confirmed this idea. Amthamine combines a high H2 receptor selectivity with a potency, which is slightly higher compared to histamine, both in vitro and in vivo. An H2 receptor agonist that is more potent than histamine is the guanidine derivative impromidine. This ligand actually combines a rather high H2 receptor affinity with a reduced efficacy. Impromidine also shows moderate H1- and potent H3-receptor antagonistic and H4-receptor agonistic activity. Interestingly, replacement of the propyl-imidazole moiety of impromidine with an a-methyl-ethylimidazole group results in the chiral analog (Figure 17.3). The P(-)-isomer, sopromidine, acts as a potent H2 agonist, whereas the S(+)-isomer is a weak H2 antagonist. Both compounds posses only weak H3 and H4 antagonistic activities, making P(-)-sopromodine one of the most potent and selective H2 agonist to date.

17.3.3 H2 Receptor Antagonists

The identification of Na-guanylhistamine as a partial H2 agonist in a gastric acid secretion model led to the development of the relatively weak H2 antagonist burimamide (Scheme 17.2) following the replacement of the strong basic guanidine group by the noncharged, polar thiourea, and side chain elongation. Years later, it was shown that burimamide is also an H3 and H4 receptor partial agonist. As H2 receptor antagonist, burimamide lacked oral activity in man most likely due to its moderate potency. Nevertheless, burimamide was the lead for the development of selective and clinically useful H2 receptor antagonists, such as cimetidine. Over time, many H2 antagonists have been described; almost all of them possess two planar n-electron systems connected by a flexible chain. The 4-methylimidazole moiety of cimetidine can easily be replaced by other heterocyclic groups. Replacement by a substituted furan (e.g., ranitidine) or thiazole ring (e.g., famotidine) leads to compounds that are usually more potent at the H2 receptor than cimetidine. Moreover, the replacement of the imidazole moiety also eliminates the undesired inhibition of cytochrome P-450. Most H2 antagonists are rather polar compounds, which do not readily cross the blood-brain barrier.



N a-Guanylhistamine (pA2 = 3.88)

Famotidine (pA2=7.8)

Famotidine (pA2=7.8)


hnach3 s


Zolantidine (pA2=7.2)

Zolantidine (pA2=7.2)

SCHEME 17.2 Stepwise structural modifications leading to the development of histamine H2 receptor drugs to treat gastric ulcers.

The brain-penetrating H2 antagonist zolantidine represents a rather nonclassical structure with the oxygen atom of the furan ring in ranitidine placed outside the aromatic ring and replacement of the polar group with a benzothiazole group (Scheme 17.2) and has become an important tool for in vivo CNS studies.

Like the H1 receptor, the H2 receptor was reported to be spontaneously active in transfected CHO cells. Based on this concept, many H2 antagonists were reclassified: cimetidine, ranitidine, and famotidine are in fact inverse agonists, whereas burimamide acts in this model system as a neutral antagonist. This difference in pharmacological profile was also seen in a differential effect on H2 receptor regulation; whereas long-term treatment with inverse agonists, like cime-tidine, resulted in H2 receptor upregulation, exposure to the neutral antagonist burimamide did not affect the receptor expression. Such receptor upregulation was also observed in rabbit parietal cells, resulting in acid hypersecretion after H2 antagonist withdrawal. These data present a mechanistic explanation for the known tolerance induction by H2 antagonist that sometimes occurs in man.

17.3.4 Therapeutic Use of H2 Receptor Ligands

At the moment there is no clinical application of H2 agonists, although sometimes histamine is used as a diagnostic aid in patients with stomach problems. In contrast, H2 antagonists have proven to be very effective drugs to alleviate the symptoms of duodenal ulcers, stomach ulcers, and reflux oesophagitits. Nowadays, the blockbuster status of the H2 antagonists has been strongly reduced with the introduction of the proton pump inhibitors, like omeprazole, to directly inhibit the gastric acid secretion and the eradication of Helicobacter pylori with antibiotics as actual cure, instead of symptomatic treatment.


Molecular Aspects of the Histamine H3 Receptor Protein

The physiological role of histamine as a neurotransmitter became apparent in 1983, when Arrang and coworkers discovered the inhibitory effect of histamine on its own release and synthesis in the brain. This effect was not mediated by the known Hj and H2 receptor subtypes as no correlation with either the H1 or the H2 receptor activity of known histaminergic ligands was observed. Soon thereafter, the H3 receptor agonist ^-a-methylhistamine and the antagonist thioperamide (see Figures 17.4 and 17.5, respectively) were developed, thereby confirming that a new receptor


N -Methylhistamine

(fi)-a-Methylhistamine (eutomer)


(S)-a-Methylhistamine (distomer)


VNH2 tl

N -Methylhistamine

i HN

(fi)-a-Methylhistamine (eutomer)

i HN

Immepip Immethridine

FIGURE 17.4 Reference histamine H3 receptor agonists.




(S)-a-Methylhistamine (distomer)




R = H: Impentamine



R = H: Impentamine





A-423579 GSK-189254

FIGURE 17.5 Histamine H3 receptor antagonists and inverse agonists.

o subtype regulates the release and synthesis of histamine. In addition, the H3 receptor regulates the release of other important neurotransmitters, such as acetylcholine, serotonin, noradrenalin, and dopamine. Next to its high expression in certain regions of the human brain (for example, the basal ganglia, hippocampus, and cortical areas, i.e., the parts of the brain that are associated with cognition) the H3 receptor is also present to some extent in the peripheral nervous system, e.g., in the gastrointestinal tract, the airways, and the cardiovascular system.

Initial efforts to identify the H3 receptor gene, using the anticipated homology with the previously identified H1 and H2 receptor genes ended in vain. Eventually, the human H3 receptor cDNA was identified by Lovenberg and his coworkers at Johnson & Johnson in 1999. In search for novel GPCR proteins using a homology search of commercial genome databases, a receptor with high similarity to the M2 muscarinic acetylcholine receptor and high brain expression was identified. Expression of the gene and full pharmacological characterization established this protein as the histamine H3 receptor. Cloning of the H3 receptor genes of other species, including rat, guinea pig, and mouse, soon followed, and important H3 receptor species differences have been identified. The H3 receptor mRNA undergoes extensive alternative splicing, resulting in many H3 receptor isoforms that have different signaling properties and expression profiles. Moreover, the H3 receptor displays particularly high constitutive activity, which can also be observed in vivo, again leading to a reclassification of existing ligands into agonists, neutral antagonists, and inverse agonists.

The H3 receptor signals via Gi/o proteins as shown by the pertussis toxin sensitive stimulation of [35S]-GTPyS binding in rat cortical membranes. The inhibition of adenylyl cyclase after stimulation of the H3 receptor results in lowering of cellular cAMP levels and modulation of cAMP responsive element-binding protein (CREB) dependent gene transcription.

17.4.2 Histamine H3 Receptor Agonists

At the H3 receptor, histamine itself is an highly active agonist. Methylation of the amino function results in N a-methylhistamine (Figure 17.4), a compound that is H3 selective and even more active than histamine. Methylation of the a-carbon atom of the ethylamine side chain also increases the potency at the H3 receptor. This increased activity resides completely in the ^-isomer; the corresponding S-isomer is approximately 100-fold less potent. Since the methylation leads to highly reduced activity at both the H1 and H2 receptor, but still substantial activity at the H4 receptor, ^-(a)-methylhistamine is a moderately selective agonist at the H3 receptor. In combination with its less active S-isomer, this compound has proven to be highly useful for the pharmacological characterization of H3 receptor-mediated effects. For potent H3 agonism, the amine function of histamine can be replaced by an isothiourea group, as in imetit. This compound is also very active in vitro and in vivo, as is ^-(a)-methylhistamine. The basic group in the imidazole side chain can also be incorporated in ring structures. For example, immepip is a potent H3 agonist that is effective in vitro and in vivo. Although the described first generation H3 agonists were intensively used as reference ligands to study the H3 receptor, all of them proved to have considerable activity for the recently discovered H4 receptor. Therefore, a new generation of potent and selective H3 agonists has been developed, most notably immethridine (pEC50 = 9.8; 300-fold selectivity over the H4 receptor) and methimepip (pEC50 = 9.5; >10,000-fold selectivity over the H4 receptor). These latter compounds are devoid of high H4 receptor activity.

17.4.3 Histamine H3 Receptor Antagonists

As with the first generation H3 agonists, the first generation H3 antagonists (all of them possessing an imidazole heterocycle) have considerable affinity for the more recently discovered histamine H4 receptor. The first potent H3 receptor antagonist (later reclassified as an inverse agonist) that was devoid of H1 receptor and H2 receptor activity was thioperamide (Figure 17.5). This compound has been used in many H3 receptor studies as reference ligand and is active in vitro and in vivo



f 1000-

15 400200-

FIGURE 17.6 Alkylation of the primary amine function of impentamine leads to ligands that cover the complete spectrum of functional activity, i.e., agonism, neutral antagonism, and inverse agonism.

(the compound is able to penetrate the CNS). However, thioperamide displays some 5-HT3 receptor antagonism and also is an inverse agonist at the H4 receptor. Moreover, a remarkable H3 receptor species differences can be demonstrated with thioperamide, as the compound has a 10-fold higher affinity for the rat H3 receptor than for the human H3 receptor. Based on the H3 receptor agonist imetit, the highly potent H3 inverse agonist clobenpropit was developed (pA2 = 9.9). This compound also has some 5-HT3 receptor activity and displays partial agonist activity at H4 receptors. Impentamine is a potent histamine H3 receptor partial agonist in SK-N-MC cells expressing human H3 receptors. It has also been shown that small structural modifications of impentamine, i.e., alkylation of the primary amine moiety of impentamine with, e.g., methyl-, isopropyl-, and p-chlorobenzyl-groups results in ligands that cover the complete spectrum of functional activity, i.e., agonism, neutral antagonism, and inverse agonism (Figure 17.6). The compound VUF5681 (Figure 17.5) was reported as a neutral H3 antagonist, not affecting the basal signaling of the histamine H3 receptor. As such, it has proven to be a useful molecular tool in H3 receptor studies, for example, when studying H3 constitutive activity in the rat brain.

The imidazole-containing compounds have been very important in characterizing the H3 receptor. Furthermore, similarity studies resulted in pharmacophore models (Figure 17.7) that explain the SAR of the different classes of imidazole-containing ligands and indirectly describe the ligand-binding site of the receptor.

Imidazole-containing ligands are associated with inhibition of cytochrome P-450 enzymes. Via this mechanism, the clearance of coadministrated drugs can be compromised, leading to severe drug-drug interactions and extrapyramidal symptoms. Classic medicinal chemistry work, elegantly conducted by the team of Ganellin (already involved in the development of the H2 antagonist cimetidine, vide supra) at University College London led to a first breakthrough in the search of nonimidazole H3 antagonists, as illustrated in Scheme 17.3. The endogenous agonist histamine was once again taken as a lead structure. Attachment of a lipophilic group to the amine moiety led to

FIGURE 17.7 Pharmacophore model for imidazole-containing H3 antagonists. Superposed are 10 different compounds. Carbon atoms in green, nitrogen atoms in blue, sulfur atoms in yellow, and hydrogen atoms in white. All imidazole rings are perfectly superposed. The aromatic heterocycle and the basic groups in the imidazole side chain can interact with a total of four predicted H-bonding groups of the receptor site (yellow sphere indicating a H-bonding donor atom of the site and purple indicating H-bonding acceptor atoms of the site. The lipophilic groups at the terminus of the side chain of the ligands are located in two distinct positions, suggesting that the H3 receptor has two lipophilic pockets for ligand binding. These findings were later validated by several groups using receptor homology modeling.

FIGURE 17.7 Pharmacophore model for imidazole-containing H3 antagonists. Superposed are 10 different compounds. Carbon atoms in green, nitrogen atoms in blue, sulfur atoms in yellow, and hydrogen atoms in white. All imidazole rings are perfectly superposed. The aromatic heterocycle and the basic groups in the imidazole side chain can interact with a total of four predicted H-bonding groups of the receptor site (yellow sphere indicating a H-bonding donor atom of the site and purple indicating H-bonding acceptor atoms of the site. The lipophilic groups at the terminus of the side chain of the ligands are located in two distinct positions, suggesting that the H3 receptor has two lipophilic pockets for ligand binding. These findings were later validated by several groups using receptor homology modeling.


Na-(4-Phenylbutyl)histamine K = 0.63 |M

SCHEME 17.3 Illustration of the stepwise development of UCL 2190 as one of the first potent nonimidazole H3 receptor antagonists.

N a-(4-phenylbutyl)histamine, a compound with H3 antagonist activity. Replacement of the imidazole heterocycle, initially deemed essential for H3 affinity led to N-ethyl-N-(4-phenylbutyl) amine with merely a twofold drop in affinity. Subsequent stepwise optimization of the structure for H3 affinity, by systemically modifying the basic group, the linker and the aromatic moiety ultimately led to UCL 2190, a potent nonimidazole H3 antagonist. Structural features of this compound, e.g., the amino-proxyphenyl substructure, reoccur in most H3 medicinal chemistry programs that have been reported since.

Especially since the cloning of the H3 receptor gene in 1999, the pharmaceutical industry has been actively exploring the potential of H3 receptor ligands and many new antagonists/inverse agonists have been described. Typical examples are the inverse agonist ABT-239 and the neutral antagonist JNJ-5207852 (Figure 17.5). Interestingly, this latter compound is active in several models for cognition, but does not act as an appetite suppressant and has no effect on food intake. Other compounds, such as Abbott's A-423579, have good efficacy in obesity models, but lack clear procognitive effects. At present the differences in efficacy for distinct clinical applications of the different classes of H3 ligands is not understood (e.g., involvement of different H3 receptor isoforms) and subject of intense research.

17.4.4 Therapeutic Use of Histamine H3 Receptor Ligands

Multiple lines of evidence indicate that the H3 receptor is involved in numerous physiological processes and that this receptor bears potential as a promising drug target. A handful of applications for H3 agonists has emerged from preclinical studies in the areas of migraine (modulating release of neurogenic peptides) and ischemic arrhythmias (modulating noradrenaline release). In migraine, the H3 agonistic properties of N a-methylhistamine have been reported to be beneficial in a Phase II trial. Intriguingly, both H3 agonists and H3 inverse agonist are claimed to have a beneficial activity when studied in pre-clinical obesity models. The full spectrum of diseases, where H3 receptor mediated treatment might be applicable, is striking. H3 antagonists and inverse agonists have been successfully used in animal models for narcolepsy, cognitive disorders, neuropathic pain, and others. In this respect, GSK-189254 is a remarkable H3 ligand as it is in trials for three different diseases: neuropathic pain, narcolepsy, and dementia. It has proven a challenging task to predict in what specific preclinical model(s) a given structural series of H3 antagonist or inverse agonist will be useful. Moreover, a full clinical validation of the promising role of H3 receptor antagonists is still awaited.


17.5.1 Molecular Aspects of the Histamine H4 Receptor Protein

Immediately following the cloning of the H3 receptor gene, several groups identified the homologous H4 receptor sequence in the human genome databases. Indeed, the H4 receptor has high sequence identity with the H3 receptor (31% at the protein level, 54% in the transmembrane domains). The H3 and H4 receptors are also similar in gene structure. The human H4 receptor gene is present on chromosome 18q11.2 and the gene contains three exons that are interrupted by two large introns (like the H3 receptor gene). To date, two H4 receptor isoforms have been identified, but no functional role has been reported so far. Cloning of the genes that encode the mouse, rat, guinea pig, and pig H4 receptors reveal only limited sequence homology with the human H4 receptor. The H4 receptor is mainly expressed in bone marrow and peripheral leukocytes and mRNA of the human H4 receptor is detected in, e.g., mast cells, dentritic cells, spleen, and eosinophils. The H4 receptor has a pronounced effect on the chemotaxis of several cell types that are associated with immune and inflammatory responses.

The H4 receptor couples to Gi/o proteins, thereby leading to a decrease in cAMP production and the regulation of CREB gene transcription. Furthermore, H4 receptor stimulation affects the Gi/o protein mediated activation of mitogen-activitated protein (MAP) kinase. Studying the increased [35S]GTPyS levels in H4 transfected cells, it has been shown that also the H4 receptor is constitutively active.

17.5.2 Histamine H4 Receptor Agonists

Most of the first generation imidazole-containing H3 ligands have reasonable affinity for the H4 receptor as well. The first imidazole-containing ligand that was reported to have some selectivity for the hn'5^ v^n n-hn—//


hn hn nh2

VsN 4-Methylhistamine nh2

VsN 4-Methylhistamine nh




FIGURE 17.8 Histamine H4 receptor agonists.

H4 (40-fold) over the H3 receptor is OUP-16 (Figure 17.8). This compound acts as a full H4 agonist. More recently, the potent H4 agonist 4-methylhistamine was discovered after the screening of a large number of histaminergic compounds. This compound was originally developed for an H2 research program, but appears to be more than 100-fold more potent on the H4 receptor than on any other histamine receptor subtype, including the H2 receptor. VUF8430 was also reported as a potent H4 agonist (pEC50 = 7.3) with a complimentary selectivity profile, being 33-fold selective over the H3 receptor. Again, VUF8430 was originally developed as a dimaprit analog in an H2 research program. VUF6884 was developed as a clozapine analog with optimized histamine H4 receptor affinity. This rigid compound is particularly useful for pharmacophore modeling studies (see below). Interestingly, VUF6884 is a full agonist on histamine H4 receptors and an even more potent histamine H1 receptor inverse agonist. Clozapine derivatives are well known as promiscuous GPCR ligands.

Histamine H4 Receptor Antagonists

Potent and selective H4 receptor antagonists are also emerging. For this histamine receptor subtype, the first nonimidazole ligands were found by successful HTS campaigns. The first reported neutral antagonist was derived from an indole-containing hit structure that was efficiently converted into JNJ7777120 (Scheme 17.4). This compound can currently be considered as an H4 receptor reference ligand. Unfortunately, the compound has a poor stability in human and rat liver microsomes and

HTS hit

JNJ7777i20 K =4 nM

JNJ7777i20 K =4 nM


VUF6002 K =26 nM

SCHEME 17.4 Illustration of two histamine H4 receptor HTS hits (A and B) and subsequent hit optimization.

FIGURE 17.9 Pharmacophore modeling leading to the design of new ligands. Two reference histamine H4 ligands (VUF6884 and JNJ7777120) were used to construct a pharmacophore model. This model indirectly describes the histamine H4 receptor-binding site. Based on this model, novel and potent ligands that fit the binding pocket could be designed, e.g., VUF10148). Carbon atoms in green, nitrogen atoms in blue, oxygen atoms in red, and hydrogen atoms in white. Color coding surface: hydrogen-bonding region in purple, hydro-phobic regions in yellow, and mild polar regions in blue.

FIGURE 17.9 Pharmacophore modeling leading to the design of new ligands. Two reference histamine H4 ligands (VUF6884 and JNJ7777120) were used to construct a pharmacophore model. This model indirectly describes the histamine H4 receptor-binding site. Based on this model, novel and potent ligands that fit the binding pocket could be designed, e.g., VUF10148). Carbon atoms in green, nitrogen atoms in blue, oxygen atoms in red, and hydrogen atoms in white. Color coding surface: hydrogen-bonding region in purple, hydro-phobic regions in yellow, and mild polar regions in blue.

a half-life of only 2 h in rats. The subsequently developed benzimidazole derivative JNJ10191584 is also a neutral H4 antagonist. This compound is orally active in vivo and has improved liver microsomes stability but still a limited half-life. Also derived from a HTS hit, a series of 2-arylbenzimidazoles have been described as ligands with low nanomolar affinity for the H4 receptor. In addition, rational approaches like pharmacophore modeling and subsequent ligand design (Figure 17.9) are being used to develop novel H4 receptor ligands. Considering the number of H4 receptor related patent applications that have recently been disclosed, it can be anticipated that many new H4 ligands will be described in scientific literature in the near future.

17.5.4 Therapeutic Use of Histamine H4 Receptor Ligands

The presence of the H4 receptor on immunocompetent cells and cells of hematopoietic lineage suggests that this new histamine receptor subtype plays an important role in the immune system. This hypothesis is supported by the fact that IL-10 and IL-13 modulate H4 receptor expression and that binding sites for cytokine-regulated transcription factors, like interferon-stimulated response element (ISRE), interferon regulatory factor-1 (IRF-1), NF-kB, and nuclear factor-IL6 (NF-L6), are present upstream of the H4 gene. Considering the physiological role of the H4 receptor, several applications are currently under preclinical investigation, including allergy and asthma, chronic inflammations such as inflammatory bowel disease (IBD) and rheumatoid arthritis. The H4 receptor is also being associated with pruritus (itch) and is involved in the progression of colon cancer. At the moment, therapeutic applications are clearly anticipated for H4 receptor antagonists (inverse agonists). In view of the strong interests of pharmaceutical industries more evidence for a therapeutic role of H4 receptor ligands is soon to be expected.


The medicinal chemistry of histamine receptors has so far been a very rewarding arena. Major blockbuster drugs have been developed on the basis of H1 and H2 receptor targeting. Expectations for ligands targeting the two latest additions to the histamine receptor family are currently also very high. Interestingly, for each of these receptor subtypes highly selective agonists and antagonists have been developed. The wide chemical diversity of the various selective receptor ligands reflects the relatively low homology between the various receptors (only the H3 and H4 receptors resemble each other to some extent). Moreover, it offers today's medicinal chemists an attractive arena for highly effective drug discovery efforts. This will be further aided by the recent elucidation of the x-ray structure of the beta2 receptor, hopefully allowing future structure-based drug design.


Hancock, A.A. 2006. The challenge of drug discovery of a GPCR target: Analysis of preclinical pharmacology of histamine H3 antagonists/inverse agonists. Biochem. Pharmacol. 71:1103-1113.

Hill, S.J., Ganellin, C.R., Timmerman, H., Schwartz, J.C., Shankley, N.P., Young, J.M., Schunack, W., Levi, R., and Haas, H.L. 1997. International Union of Pharmacology. XIII. Classification of histamine receptors. Pharmacol. Rev. 49:253-278.

Leurs, R., Bakker, R.A., Timmerman, H., and de Esch, I.J. 2005. The histamine H3 receptor: From gene cloning to H3 receptor drugs. Nat. Rev. Drug Discovery 4:107-120.

Thurmond, R.L., Gelfand, E.W., and Dunford, P.J. 2008. The role of histamine H1 and H4 receptors in allergic inflammation: The search for new antihistamines. Nat. Rev. Drug Discovery 7:41-53.

Zhang, M.Q., Leurs, R., and Timmerman, H. 1997. Histamine Hrreceptor antagonists. In M.E. Wolff (ed.), Burger's Medicinal Chemistry and Drug Discovery, 5th edn. New York: John Wiley & Sons, Inc., p. 495.

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