Identification of imidazo[1,2-b]pyridazine TYK2 pseudokinase ligands as potent and selective allosteric inhibitors of TYK2 signalling.

As a member of the Janus (JAK) family of non-receptor tyrosine kinases, TYK2 mediates the signaling of pro-inflammatory cytokines including IL-12, IL-23 and type 1 interferon (IFN), and therefore represents an attractive potential target for treating the various immuno-inflammatory diseases in which these cytokines have been shown to play a role. Following up on our previous report that ligands to the pseudokinase domain (JH2) of TYK2 suppress cytokine-mediated receptor activation of the catalytic (JH1) domain, the imidazo[1,2-b]pyridazine (IZP) 7 was identified as a promising hit compound. Through iterative modification of each of the substituents of the IZP scaffold, the cellular potency was improved while maintaining selectivity over the JH1 domain. These studies led to the discovery of the JH2-selective TYK2 inhibitor 29, which provided encouraging systemic exposures after oral dosing in mice. Phosphodiesterase 4 (PDE4) was identified as an off-target and potential liability of the IZP ligands, and selectivity for TYK2 JH2 over this enzyme was obtained by elaborating along selectivity vectors determined from analyses of X-ray co-crystal structures of representative ligands of the IZP class bound to both proteins.

Angiotensin II receptor antagonists and receptor subtypes.

Recently discovered nonpeptide angiotensin II receptor antagonists represent a new class of potential drugs for the treatment of hypertension and congestive heart failure. Further, these antagonists have been successfully used as selective research tools for physiologic studies of angiotensin H and defining angiotensin II receptor subtypes.

Nonpeptide angiotensin II receptor antagonists. Studies with EXP9270 and DuP 753.

A series of nonpeptide angiotensin II (Ang II) receptor antagonists was evaluated in rat adrenal cortical microsomes for their inhibitory effects on the specific binding of [3H]Ang II, in the isolated rabbit aorta bioassay for their functional antagonism of contractile response to Ang II, and in high renin, renal-hypertensive rats for their intravenous antihypertensive effects, expressed as IC50, pA2, and intravenous ED30, respectively. Highly significant linear correlations were found between IC50 and pA2 (r = -0.88), between IC50 and intravenous ED30 (r = 0.79), and between pA2 and intravenous ED30 (r = -0.93). In both in vitro and in vivo functional assays, none of these antagonists exhibited agonistic effects. The orally active nonpeptide Ang II receptor antagonists EXP9270 and DuP 753 (oral ED30 = 3.6 and 0.59 mg/kg, respectively) were selected for further characterization. These antagonists exhibited selective and competitive Ang II antagonism in rabbit aorta and guinea pig ileum. In conscious normotensive rats, DuP 753 abolished the pressor response to saralasin, suggesting that the pressor effect of saralasin is attributed to its Ang II-like activity. In addition, DuP 753 also blocked the Ang II-induced drinking response and aldosterone release in rats. These results suggest that Ang II receptor blockade is the primary mechanism of the antihypertensive effect of these nonpeptide Ang II receptor antagonists. Further, the specificity and lack of partial agonistic effects of these molecules make them potentially useful physiological probes and therapeutic agents.

Hypotensive action of DuP 753, an angiotensin II antagonist, in spontaneously hypertensive rats. Nonpeptide angiotensin II receptor antagonists: X.

In conscious 18-21-week-old spontaneously hypertensive rats, DuP 753, a nonpeptide angiotensin II receptor antagonist, given orally at 3 and 10 mg/kg or intravenously at 3, 10, and 30 mg/kg, reduced blood pressure dose dependently. It did not alter heart rate at these doses. At 10 mg/kg i.v., DuP 753 decreased blood pressure significantly for at least 24 hours, suggesting a long duration of the antihypertensive effect. Unlike saralasin, DuP 753 did not cause a transient increase in blood pressure. The acute antihypertensive efficacy of DuP 753 was greater than that of captopril. Our data indicate that, for captopril to reduce blood pressure to a similar extent as that of DuP 753, it would need to be supplemented by a diuretic. DuP 753 did not have an acute diuretic effect. Bilateral nephrectomy, but not inhibition of prostaglandin synthesis, abolished the antihypertensive effect of DuP 753, suggesting that the antihypertensive effect of DuP 753 is dependent on an active renin-angiotensin system. Furthermore, DuP 753 inhibited the pressor response to angiotensin II but not the responses to norepinephrine, vasopressin, and Bay K 8644 (a calcium agonist). As neither DuP 753 nor captopril decreased blood pressure acutely in Wistar-Kyoto normotensive rats, our results suggest that the renin-angiotensin system plays a significant role in the control of blood pressure in spontaneously hypertensive rats.

Nonpeptide angiotensin II receptor antagonists. IV. EXP6155 and EXP6803.

EXP6155 (2-n-butyl-1-[4-carboxybenzyl]-4-chloroimidazole-5-acetic acid) and EXP6803 (methyl 2-n-butyl-1-[4-(2-carboxybenzamido)benzyl]-4-chloroimidazole -5-acetate, sodium salt) are shown to be novel, nonpeptide, antihypertensive, specific angiotensin II receptor antagonists. In rabbit aorta, they competitively inhibited the contractile response to angiotensin II with pA2 values of 6.54 and 7.20 and did not alter the response to norepinephrine or KCl. In guinea pig ileum, both agents blocked the responses to angiotensin I and II and did not alter the responses to bradykinin and acetylcholine. A similar specific angiotensin II antagonism was shown in vivo in the spinal pithed rat model. In renal artery-ligated rats, a high renin hypertensive model, EXP6155 and EXP6803 given intravenously, decreased blood pressure with ED30 of 10 and 11 mg/kg, respectively. Both compounds did not alter blood pressure when given orally at 100 mg/kg. Unlike saralasin, EXP6155 and EXP6803 given intravenously did not cause a transient increase in blood pressure in the renal artery-ligated and normotensive rats. Our results indicate that EXP6155 and EXP6803 are selective angiotensin II receptor antagonists and antihypertensive agents. Since neither compound had partial agonist activities or bradykinin potentiation effects, unlike the existing peptide angiotensin II receptor antagonists and angiotensin converting enzyme inhibitors, respectively, they may represent preferred probes for studying the physiological roles of angiotensin II.

Angiotensin II receptor antagonists. From discovery to antihypertensive drugs.

Some simple N-benzylimidazoles, originally described by Takeda Chemical Industries (Osaka, Japan), were characterized to be very weak but selective nonpeptide angiotensin II (Ang II) receptor antagonists with a competitive mode of action. Chemical modifications of these led to EXP6155 and EXP6803, which showed approximately 10- and 100-fold higher affinity, respectively, but were orally ineffective. Oral activity was obtained for the biphenyl carboxylic acid derivatives EXP7711 and especially EXP9654. A further advance in the design of nonpeptide Ang II receptor antagonists was provided by DuP 753, an analogue of EXP7711 in which the carboxylic acid function is replaced by its tetrazol-5-yl equivalent. DuP 753 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bi phe nyl-4- yl)methyl]imidazole, potassium salt) displaces radiolabeled Ang II from its specific binding sites in various tissues, affording IC50 values of approximately 20 nM. DuP 753 competitively antagonizes Ang II-induced responses in various in vitro and in vivo preparations but does not influence those to KCl, norepinephrine, vasopressin, and others, nor does it affect converting enzyme and renin. In high renin animal models of elevated arterial blood pressure, intravenous and oral administrations of DuP 753 produce a sustained decrease in pressure without influencing heart rate. Marked antihypertensive effects are observed in spontaneously hypertensive rats, but no efficacy is noticed in deoxycorticosterone acetate hypertensive animals. DuP 753 showed no agonistic properties in any of the above test systems and has been chosen to undergo clinical trials for the treatment of hypertension. In rats, the 5-carboxylic acid (EXP3174) represents a major metabolite of DuP 753.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonpeptide angiotensin II receptor antagonists: N-[(benzyloxy)benzyl]imidazoles and related compounds as potent antihypertensives.

A series of compounds has been synthesized and demonstrated to be antagonists of the angiotensin II (AII) receptor. These compounds are structurally related to the N-(benzamidobenzyl)imidazoles and extend the scope of this new class of nonpeptide AII antagonists. It has been found that the amide linkage (X = NHCO) in the N-(benzamidobenzyl)imidazoles can be replaced successfully by a variety of groups (X = single bond, O, S, CO, OCH2, CH = CH, NHCONH); linkers of 0-1 atoms in length are most effective. When administered intravenously to awake renal hypertensive rats, these compounds are potent antihypertensives.

Nonpeptide angiotensin II receptor antagonists: the discovery of a series of N-(biphenylylmethyl)imidazoles as potent, orally active antihypertensives.

A new series of nonpeptide angiotensin II (AII) receptor antagonists has been prepared. These N-(biphenylyl-methyl)imidazoles, e.g. 2-butyl-1-[(2'-carboxybiphenyl-4-yl)methyl]-4-chloro-5- (hydroxymethyl)imidazole, differ from the previously reported N-(benzamidobenzyl)imidazoles and related compounds in that they produce a potent antihypertensive effect upon oral administration; the earlier series generally were active only when administered intravenously. It has been found that the acidic group at the 2'-position of the biphenyl is essential. Only ortho-substituted acids possess both high affinity for the AII receptor and good oral antihypertensive potency. The carboxylic acid group has been replaced with a variety of acidic isosteres, and the tetrazole ring has been found to be the most effective. The tetrazole derivative, DuP 753, is currently in development for the treatment of hypertension.

The discovery of potent nonpeptide angiotensin II receptor antagonists: a new class of potent antihypertensives.

A new class of potent antihypertensives has been discovered that exert their effect through blockade of the angiotensin II (AII) receptor. Most AII antagonists reported so far are peptide mimics of the endogenous vasoconstrictor octapeptide angiotensin II. The compounds of this paper are nonpeptides and therefore constitute a new class of potent AII receptor antagonists. Based on the overlap of a conformation of AII with literature lead 3, a hypothesis was developed suggesting the need for an additional acidic functionality to increase the lead's potency. The substitution of an additional carboxylic acid resulted in a 10-fold increase in binding affinity observed for diacid 4. The binding affinities for subsequent compounds were eventually increased 1000-fold over that of the literature leads through a systematic SAR study. Thus the AII receptor binding affinity [IC50 (microM)] of 15 microM for literature lead 1, for example, was increased to 0.018 and 0.012 microM for compounds 33 and 53. A structure-affinity relationship has been found requiring the presence of four key elements for good activity: (1) an additional phenyl ring at the N-benzyl para position of the benzylimidazole nucleus, (2) an acidic functionality at the ortho position of the terminal aromatic ring, (3) a lipophilic side chain at the imidazole 2-position of three to five carbon atoms in length, and (4) a group at the imidazole 5-position capable of hydrogen bonding. The synthesis as well as the pharmacological activity of the compounds in this new series of AII receptor antagonists are presented.

In vivo pharmacology of DuP 753.

A review of the in vivo pharmacology of DuP 753 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl)biphen yl-4- yl)methyl]imidazole, potassium salt) is presented. In the pithed rat, DuP 753 exerted a selective and competitive inhibition of the pressor response to angiotensin II (AII). In conscious normotensive rats, DuP 753 inhibited the AII-induced aldosterone secretion and drinking response. DuP 753 lowered blood pressure in conscious normotensive rats pretreated with furosemide but not in untreated normotensive rats. Unlike saralasin, DuP 753 given intravenously did not cause pressor response. In conscious renal hypertensive rats (RHRs), a high renin model, DuP 753 decreased blood pressure with an intravenous ED30 of 0.78 mg/kg and an oral ED30 of 0.59 mg/kg. The antihypertensive efficacy of DuP 753 in RHRs was similar to that of captopril. In DOCA hypertensive rats, a low renin model, DuP 753 did not lower blood pressure. In conscious 18- to 21-week-old spontaneously hypertensive rats (SHRs), DuP 753 given orally or intravenously reduced blood pressure dose-dependently and did not alter heart rate at these doses. The acute antihypertensive efficacy of DuP 753 was greater than that of captopril in SHRs. In contrast, DuP 753 and captopril given orally at 10 mg/kg/day for 15 days in SHRs caused a similar decrease in blood pressure. Bilateral nephrectomy but not inhibition of prostaglandin synthesis abolished the antihypertensive effect of DuP 753 in SHRs. Our study, therefore, indicates that DuP 753 is an orally active, nonpeptide, selective, and competitive AII receptor antagonist lacking agonism. It appears that there is a relationship between basal renin level and the acute antihypertensive effect of DuP 753 in rats. Further, our results suggest that the renin-angiotensin system plays a significant role in the control of blood pressure in conscious SHRs.

Rationale for the chemical development of angiotensin II receptor antagonists.

The renin-angiotensin system (RAS) has been demonstrated to be a key element in blood pressure regulation and fluid volume homeostasis. Since angiotensin II (AII) is the effector molecule of the RAS, the most direct approach to block this system is to antagonize AII at the level of its receptor. Therefore, at Du Pont Merck the working hypothesis has been that the identification of metabolically stable and orally effective AII-receptor antagonists would constitute a new and superior class of agents useful in treating hypertension and congestive heart failure. Our program began with a detailed pharmacologic evaluation of some simple N-benzylimidazoles, originally described by Takeda Chemical Industries in Osaka, Japan. They were found to be a series of weak but selective AII-receptor antagonists with a competitive mode of action. We embarked on a program aimed to design and synthesize more potent and orally effective nonpeptide antagonists, while attempting to preserve their selective affinity for the AII receptor. The first major breakthrough in our efforts to increase the potency of these compounds came with the development of a series of N-benzylimidazole phthalamic acid derivatives. Although effective at lowering blood pressure when administered intravenously, the phthalamic acids were devoid of oral activity. The first orally active AII antagonists came with the discovery of the biphenyl carboxylic acids. Although these compounds are absorbed after oral dosing, their bioavailability was less than desired. In the hope of improving the oral absorption of these biphenyls, we investigated a variety of acidic groups as bioisosteric replacements for the carboxylic acid. The key to the discovery of nonpeptide AII-receptor antagonists with improved oral activity and duration of action resulted from replacing the carboxylic acid group with the isosteric but more lipophilic tetrazole ring. Hence, our efforts culminated in the discovery of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl) biphenyl-4-yl)methyl]imidazole, potassium salt), a highly potent angiotensin type 1 (AT1) selective receptor antagonist with a long duration of action. Losartan is currently undergoing clinical investigation for the treatment of hypertension. The history, including the rationale for the design of the compounds, and ensuing structure-activity relationships of losartan and related analogs will be described. Many of the newer compounds exceed the potency of losartan, and the best compounds in the series rival the affinity of the endogenous ligand, AII, for its receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

The discovery of a new class of highly specific nonpeptide angiotensin II receptor antagonists.

Since angiotensin II (AII) is the effector molecule of the renin angiotensin system, the most direct approach to interfere with this system would be to antagonize AII at the level of its receptor. AII receptor antagonists would represent an ideal species, for regardless of how and where AII is produced, its function could be specifically turned off. However, the AII receptor antagonists currently available have been limited to AII-like peptides and their usefulness as therapeutics and pharmacologic tools has been hampered by their lack of oral bioavailability, metabolic instability, and partial agonistic activity. A detailed pharmacologic characterization of some simple N-benzylimidazoles, originally described by Takeda Chemical Industries (Osaka, Japan), identified this class of compound as very weak but selective AII receptor antagonists with a competitive mode of action. Encouraged by the quality of these lead compounds, we embarked on a synthetic program aimed at designing more potent and orally effective antagonists, while preserving their selectivity for the AII receptor. Our efforts have culminated in the discovery of DuP 753, 2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl)biph eny l-4-yl) methyl]imidazole, potassium salt, a potent, nonpeptide AII receptor antagonist.

In vitro pharmacology of DuP 753.

DuP 753, 2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl) biphenyl-4-yl)methyl]imidazole, potassium salt, was characterized in vitro with respect to its affinity and specificity for functional antagonism of angiotensin II (AII) receptors. In rat adrenal cortical microsomes and cultured aortic smooth muscle cells DuP 753 inhibited the specific binding of [125I]AII in a concentration-dependent manner yielding IC50 values of 1.7 and 2.0 x 10(-8) mol/L, respectively. In contrast, DuP 753 had no appreciable affinity for other receptor systems as well as for Ca2+ channels. Functional antagonism was demonstrated by its blockade of AII-induced 45Ca2+ efflux in rat smooth muscle cells. The AII-induced contraction of rabbit aortic strips was competitively antagonized by DuP 753 resulting in a pA2 value of 8.48. Responses induced by other agonists, such as norepinephrine and KCl, were not altered. No partial agonistic effect was noted in any of the in vitro assays. In addition, DuP 753 (10(-5) mol/L) had no effect on rabbit lung converting enzyme or rat/human renin activity. These data demonstrate that DuP 753 is a highly potent and specific AII receptor antagonist. DuP 753 represents a useful experimental tool for dissecting the role of the renin-angiotensin system.

Angiotensin II receptor heterogeneity.

The possibility of receptor heterogeneity in the angiotensin II (AII) system has been suggested previously, based on differences in Kd values or sensitivity to thiol reagents. One of our earliest indications was the frequent observation of incomplete inhibition of the binding of AII to adrenal cortical membranes. Autoradiographic studies demonstrated that all of the labeling of the rat adrenal was blocked by unlabeled AII or saralasin, but not by DuP 753. The predominant receptor in the rat adrenal cortex (80%) is sensitive to dithiothreitol (DTT) and DuP 753, and is designated AII-1. The residual sites in the adrenal cortex and almost all of the sites in the rat adrenal medulla are insensitive to both DTT and DuP 753, but were blocked by EXP655. These sites have been confirmed by ligand binding studies and are designated AII-2. The rabbit adrenal cortex is unique in yielding a nonuniform distribution of AII-2 sites around the outer layer of glomerulosa cells. In the rabbit kidney, the sites on the glomeruli are AII-1, but the sites on the kidney capsule are AII-2. Angiotensin III appears to have a higher affinity for AII-2 sites since it inhibits the binding to the rabbit kidney capsule but not the glomeruli. Elucidation of the distribution and function of these diverse sites should permit the development of more selective and specific therapeutic strategies.

Nonpeptide angiotensin II receptor antagonists.

Although the most direct way to interfere with the renin-angiotensin system (RAS) is at the level of the angiotensin II (AII) receptor, the currently available AII receptor antagonists are peptides still retaining significant agonistic properties with the obvious drawbacks of limited stability and lack of oral activity. We have characterized simple N-benzylimidazoles as weak, but selective AII receptor antagonists with a competitive mode of action. Chemical modification of these early leads led to EXP6155 and EXP6803, which show approximately 10- and 100-fold higher affinity. Oral activity was obtained for EXP7711, and in particular for EXP9654. This class of compounds displaces 3H-AII from its specific binding sites in various tissues. They competitively antagonize AII-induced responses in various in vitro and in vivo preparations, but do not influence AII-induced responses to KCl, norepinephrine, and vasopressin, nor do they affect converting enzyme or renin. In high renin models of elevated blood pressure, such as the renal hypertensive rat and sodium-depleted dog, these substances produce a sustained decrease in arterial pressure without changing heart rate after intravenous and oral (EXP7711 and EXP9654) administration. None of these compounds showed agonistic activity in any of the above test systems. In conclusion, the nonpeptide structures described herein are selective and competitive AII receptor antagonists and add another dimension to the arsenal of drugs manipulating the RAS.

Monoclonal antibodies to the nonpeptide angiotensin II receptor antagonist, losartan.

Two murine monoclonal antibodies were produced to losartan (DuP 753), a nonpeptide angiotensin II receptor antagonist. Using a solid phase competitive enzyme-linked immunosorbent assay (ELISA), each antibody was examined for its ability to bind to a set of losartan analogs that differ structurally in varying degrees. Both antibodies distinguished fine structural changes in the analogs, particularly at the R5 position of the imidazole ring. No cross-reactivity towards either antibody was observed with the natural ligand angiotensin II, the peptide antagonist saralasin, or the AT2 selective nonpeptide antagonist PD123177.

Nonpeptide angiotensin II receptor antagonists. Studies with DuP 753 and EXP3174 in dogs.

DuP 753 (or EXP3174) and PD123177 are nonpeptide angiotensin (AII)-specific ligands, which show high affinities for two AII receptor subtypes, i.e. AT1 and AT2 sites, respectively. In furosemide-treated conscious dogs with high renin, DuP 753 and EXP3714, but not PD123177, were as effective as captopril in lowering blood pressure. Both DuP 753 and EXP3174 exhibited selective vascular antagonism of AII. In conscious dogs with normal renin, DuP 753, but not captopril or EXP3174, caused a dose-dependent but transient decrease in blood pressure. In anesthetized dogs, DuP 753 and captopril caused similar renal vasodilatation and natriuresis. The renal hemodynamic effects of DuP 753 and captopril were more pronounced in dogs with sodium depletion. These results suggest that the AT1 receptor mediates the pressor and renal effects of AII in dogs. The acute transient hypotensive effect of DuP 753 in normal-renin conscious dogs is probably unrelated to AII antagonism.

EXP597, a nonpeptide angiotensin II receptor antagonist with high affinities for the angiotensin AT1 and AT2 receptor subtypes.

AT1 and AT2 are the two major receptor subtypes for angiotensin II that have been pharmacologically defined by using the selective ligands losartan and PD123177, respectively. EXP597 (4-[(5-(2-benzoyl)benzyloxycarbonyl-4-ethyl-2-n-propylimidazole-1- yl)methyl]-3-fluoro-2'-isoamyloxycarbonylaminosulfonyl-[1,1']-biph enyl, potassium salt) is a nonpeptide angiotensin II receptor ligand which in the rat adrenal exhibits binding affinities (IC50) of 0.5 and 0.7 nM for angiotensin AT1 and AT2 receptor subtypes, respectively. Further, EXP597 is an insurmountable angiotensin II receptor antagonist in the isolated rabbit aorta and lowers blood pressure in renal hypertensive rats with i.v. and p.o. ED30 values of 0.05 and 0.9 mg/kg, respectively.

Discovery of losartan, the first angiotensin II receptor antagonist.

The 'discovery' of losartan represents three separate discoveries: (1) losartan as the unique biphenyltetrazole molecule and the first of a new chemical class; (2) losartan as a tool to identify AT1-subtype receptors; and (3) losartan as a specific probe for exploring the multiple roles of angiotensin II (Ang II) in normal physiology and pathologic states. Losartan is the first nonpeptide orally active Ang II receptor antagonist to reach clinical trials. Losartan was selected for its affinity for Ang II receptors, functional antagonism of Ang II, lack of agonist properties, and oral anti-hypertensive effects. Losartan has been widely used to define the distribution and function of AT receptor subtypes. Although possible roles of the AT2 subtype have been reported, virtually all of the known effects of Ang II are blocked by losartan. Specific AT1 receptor blockade has been broadly compared with ACE inhibition. Possible differences on the basis of AT1 selectivity, bradykinin potentiating effects and Ang II formed by non-ACE pathways are discussed. Losartan blocks the vascular constrictor effect of Ang II, the Ang II-induced aldosterone synthesis and/or release, and the Ang II-induced cardiovascular 'growth' in vitro and in vivo. In various models of experimental hypertension, losartan prevents or reverses the elevated blood pressure and the associated cardiovascular hypertrophy similar to ACE inhibitors. Likewise, in models of renal failure (for example reduced renal mass, puromycin, ochratoxin), losartan, like ACE inhibition, markedly reduced the elevation in blood pressure, proteinuria or sclerosis. In aortocaval shunt, coronary ligation and ventricular pacing models of heart failure, losartan demonstrated a pathological role for Ang II by reversing the associated haemodynamic findings. In SHR-stroke prone, losartan dramatically increased survival while having a limited effect on blood pressure, suggesting a non-pressure dependent effect of Ang II. These collective data show that Ang II exerts complex pathological effects in experimental models of vascular, cardiac, renal and cerebral disease. The effectiveness of losartan in experimental models of heart failure supports its evaluation in clinical trials with patients with heart failure.