Aldosterone Synthase Inhibitors as Promising Treatments for ...

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Jan 14, 2014 - Qingzhong Hu, Lina Yin, and Rolf W. Hartmann*. Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for ...
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Aldosterone Synthase Inhibitors as Promising Treatments for Mineralocorticoid Dependent Cardiovascular and Renal Diseases Miniperspective Qingzhong Hu, Lina Yin, and Rolf W. Hartmann* Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C2.3, D-66123 Saarbrücken, Germany

ABSTRACT: Besides the well-known roles of aldosterone as a mineralocorticoid in regulating homeostasis of electrolytes and volume, recent studies revealed that it is also a potent proinflammation factor inducing reactive oxygen species and up-regulating a panel of fibrosis related genes. Under pathological circumstances, excessive aldosterone is involved in a lot of chronic diseases, including hypertension, cardiac fibrosis, congestive heart failure, ventricular remodeling, and diabetic nephropathy. Therefore, the inhibition of aldosterone synthase (CYP11B2), which is the pivotal enzyme in aldosterone biosynthesis, was proposed as a superior approach. Expected pharmacodynamic effects have been demonstrated in both animal models and clinical trials after the application of CYP11B2 inhibitors. The importance of selectivity over other steroidogenic CYP enzymes, in particular 11βhydroxylase (CYP11B1), was also revealed. Recently, much more selective CYP11B2 inhibitors have been reported, which could be promising drug candidates for the treatment of aldosterone related diseases. exorbitant aldosterone levels lead to vascular fibrosis and vascular endothelium stiffening, which are direct causes and symptoms of atherosclerosis. Aldosterone also up-regulates a panel of fibrosis related genes such as tenascin X (TNX), urokinase plasminogen activator receptor (UPAR), and a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS1).2 Together with inflammation, they cause cardiac myocyte necrosis, collagen synthesis, and fibroblast proliferation leading to cardiac fibrosis. Subsequently, these actions lead to ventricular remodeling and cardiac hypertrophy as further structural deterioration accompanied with functional degradations. The ventricular remodeling reduces stroke volume, diminishes contractile capability, causes diastolic dysfunction, and ultimately results in heart failure. Similarly, aldosterone upregulates the plasminogen activator inhibitor 1 (PAI-1) and the transforming growth factor β1 (TGF-β1), both of which are prosclerotic growth factors. This up-regulation together with the chronic inflammation induced by aldosterone leads to glomerular injury, tubular damage, and interstitial fibrosis.4

1. FUNCTIONS OF ALDOSTERONE AND ITS ASSOCIATION WITH DISEASES Aldosterone is the major mineralocorticoid with well-known functions of regulating the homeostasis of electrolytes and blood volume. This is achieved via its actions on epithelial cells in the distal nephron, where aldosterone binds to mineralocorticoid receptors (MRs), leading to translocation of the bound receptors into the nucleus. These activated MRs bind to specific gene response elements to either initiate or repress target gene transcriptions and ultimately modulate epithelial sodium channels to retain sodium and water but excrete potassium. Blood volume and pressure are thus increased. However, recent studies unveiled that aldosterone has many other functions, which are rapid nongenomic effects and mediated via either MR or some membrane bound aldosterone receptors (yet to be identified).1 Aldosterone increases intracellular calcium concentrations of vascular smooth muscle cells and up-regulates the expression of adrenomedullin and regulator of G protein signaling 2 (RGS2), leading to vasoconstriction, which may exacerbate chronic hypertension. Furthermore, aldosterone is a potent proinflammation factor2 and induces reactive oxygen species (ROS).3 Via these mechanisms, © 2014 American Chemical Society

Received: September 16, 2013 Published: January 14, 2014 5011

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Aldosterone also decreases the expression of heparan sulfate,5 which is a component of glomerular basement membrane, and thus results in proteinuria. These deleterious effects on kidney are particularly severe in cases of diabetic nephropathy, which is a complication developed in up to 40% of diabetic patients. Interestingly, although the role of aldosterone in glucose metabolism still remains unclear, aldosterone excess has been demonstrated to impair the first phase insulin secretion6a and induce insulin resistance.6b Moreover, exorbitant aldosterone levels not only promote calcium secretion via urine and feces but also up-regulate the parathyroid hormone, leading to reductions in plasma calcium levels and bone mineral density.7 The diagnostic finding of elevated aldosterone levels in circulation has been termed as primary aldosteronism (PA). Under these circumstances the production of aldosterone is no longer under the control of the renin−angiotensin system. Although PA was first identified as a rare disease originating from adrenal adenoma, its prevalence increased recently with a possible association with obesity.8 Currently, PA is the most frequent cause of secondary hypertension, accounting for up to 10% of hypertensive cases, in particular resistant hypertension. On the other hand, aldosterone overproduction can be only local. It has been demonstrated that in patients with heart failure, aldosterone levels in failing hearts are much higher than those in peripheral plasma.9a Local production of aldosterone in kidneys also contributes to inflammation and matrix formation in diabetic rats.9b These findings indicate the potential involvement of aldosterone in some severe diseases even if no significant elevation in circulation is observed. The important pathological roles of aldosterone are also confirmed by the fact that aldosterone deficiency induced by aldosterone synthase (CYP11B2) knockout prevents angiotensin II induced cardiac, renal, and vascular injury in mice.10 Application of steroidal MR antagonists like spironolactone or eplerenone decreases the mortality in patients with chronic congestive heart failure (CHF) or left ventricular dysfunction after myocardial infarction.11a,b However, both drugs lead to severe hyperkalemia,11b and spironolactone also exhibits affinity to progesterone and androgen receptors, resulting in side effects like gynaecomastia.11c More importantly, these MR antagonists lead to an accumulation of renin and aldosterone and thus may amplify the MR independent effects. In contrast, the inhibition of CYP11B2, which is the crucial enzyme in aldosterone biosynthesis, is a promising treatment for these aldosterone related diseases.

Chart 1. CYP11B2 in the Biosynthesis of Aldosterone

17-position by the 17α-hydroxylase function of CYP17, leading to the corresponding 17α-hydroxyprogestogens. After hydroxylation at the 21-position, 11-deoxycortisol is afforded, which is further oxidized by 11β-hydroxylase (CYP11B1) to the principal human glucocorticoid cortisol. Moreover, in the zona reticularis the 17α-hydroxyprogestogens can be converted into dehydroepiandrosterone (DHEA) and androstenedione catalyzed by the C17-20 lyase function of CYP17. These two steroids are the precursors of more potent androgens, such as testosterone and dihydrotestosterone, and estrogens, which are produced from androgens under the catalysis of aromatase (CYP19). It is apparent that a safe CYP11B2 inhibitor should not inhibit other important enzymes of steroid biosynthesis. CYP11B2 is a member of the large cytochrome P450 (CYP) superfamily, in which all enzymes consist of a prosthetic moiety containing an iron(III) porphyrin group called heme. This moiety is covalently linked to the protein by a proximal cysteine ligand and is the reactive center to activate molecular oxygen for the metabolism of both endogenous and exogenous substrates. CYP11B2 is integrated into the inner mitochondrial membrane. CYP11B2 depends on two other proteins: adrenodoxin and adrenodoxin reductase, which is a flavoprotein containing flavin adenine dinucleotide. They are essential for electron transport to reduce the iron (and subsequently molecular oxygen). Adrenodoxin is an iron−sulfur protein of the [2Fe−2S] ferredoxin type, which functions as a soluble electron carrier between NADPH, adrenodoxin reductase, and CYP11B2. Steroid 11β-hydroxylase (CYP11B1) is another isoform of CYP11B2. Although CYP11B enzymes share a relatively low

2. ROLE OF CYP11B2 IN THE BIOSYNTHESIS OF ALDOSTERONE The initial step in the biosynthesis of all steroidal hormones is the conversion of cholesterol to pregnenolone, mediated by cholesterol desmolase CYP11A1 (side chain cleavage enzyme, Chart 1). Because of the absence of 17α-hydroxylase-17,20-lyase (CYP17) in the zona glomerulosa of the adrenal glands, where mineralocorticoids are predominately produced, pregnenolone is converted into progesterone and subsequently into 11deoxycorticosterone under the catalysis of 3β-hydroxysteroid dehydrogenase (3β-HSD) and steroid 21-hydroxylase (CYP21), respectively. 11-Deoxycorticosterone (DOC) is a substrate of CYP11B2, which is first hydroxylated at the 11β-position to afford corticosterone. Subsequently, two sequential oxidations at C18 also catalyzed by CYP11B2 and water release yield the major mineralocorticoid aldosterone. In contrast, in the other adrenal zones pregnenolone and progesterone can be hydroxylated at the 5012

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the structure, the resulting compound 3 was not confirmed to inhibit aldosterone synthase when evaluated in hCYP11B2expressing fission yeast.18b Screening of fungicides containing an azole moiety led to the identification of inhibitors showing IC50 values for hCYP11B2 in the low micromolar range. As an example, the unselective CYP inhibitor ketoconazole (Chart 3) exhibits an IC50 value of 3.5 μM in the yeast assay mentioned above. In cells originating from Chinese hamster lung fibroblasts (V79 MZ cells), which stably express hCYP11B2, an IC50 value of 0.65 μM was observed.15 Not surprisingly, ketoconazole was also active in an identical assay with V79 MZ cells expressing hCYP11B1, thus revealing only low selectivity. The calculated selectivity factor (SF, IC50 CYP11B1/IC50 CYP11B2) is as low as 20. Further compounds originally developed for other targets were subsequently found to be potent but also unselective inhibitors of CYP11B2. For example, the pan-kinase inhibitor staurosporine (Chart 3) showed potent inhibition of aldosterone biosynthesis (IC50 = 11 nM).19a The CYP11B1 inhibitor metyrapone (Chart 3), which is still in use for the treatment of Cushing’s syndrome (IC50 CYP11B2 = 208 nM, IC50 CYP11B1 = 46 nM),19b and the anesthetic R-etomidate (ETO, Chart 3) (IC50 CYP11B2 = 1.7 nM, IC50 CYP11B1 = 0.5 nM)19b are other examples. While the exact mode of action of staurosporine is not known, it is notable that compounds with a heterocycle containing a sp2 hybridized N, such as ketoconazole, ETO, and metyrapone, coordinate to the catalytic center heme iron20 and thus reversibly inhibit CYP11B2. This mechanism of inhibition not only is employed for the design of CYP11B2 inhibitors but also has been applied for inhibitors of other steroidogenic CYP enzymes like CYP1721 and CYP19.22 This strategy of efficiently inhibiting steroidogenic CYP enzymes is advantageous with regard to another aspect: in our experience the introduction of a heme-binding heterocycle largely reduces the possibility of the corresponding compounds binding to steroidal receptors. 3.3. Fadrozole and Related Derivatives. 3.3.1. Fadrozole. Fadrozole was published in 1987 as a CYP19 inhibitor showing an IC50 value of 4.5 nM (human placental microsomes as enzyme source).23 A few years later, fadrozole was identified as a potent inhibitor of corticoid biosynthesis in vitro and in vivo.24 Separation of the enantiomers revealed that R-fadrozole (FAD286, compound 4, Chart 4) inhibits predominantly

sequence identity with other CYP enzymes (500 nmol/L, indicating a normal response. Furthermore, significant reductions in 24 h ambulatory blood pressure and clinic systolic blood pressure were observed with all doses of compound 5. However, only application of compound 5 at 1.0 mg q.d. significantly decreased clinic diastolic blood pressure by 7.1 mmHg, which was comparable to that achieved by eplerenone at 50 mg b.i.d. (−7.9 mmHg).37 In contrast, results from another clinical trial in patients with resistant hypertension revealed that the reduction of systolic blood pressure caused by compound 5 (dose of 0.5 or 1 mg b.i.d.) was smaller than that caused by eplerenone and was not statistically significant compared to placebo.38 The reason that compound 5 showed different efficacy in primary and resistant hypertension remains unclear. Speculations could be that MR in resistant hypertension might be mutated and is thus more easily activated by the elevated DOC or that the CYP11B2 expression in patients with resistant hypertension is much higher than the one in primary hypertension, which could be compensated by higher doses of CYP11B2 inhibitors. Interestingly, compound 5 was also evaluated as a CYP11B1 inhibitor in patients with Cushing’s disease39 and showed a normalization of urinary free cortisol and a reduction of systolic blood pressure.

die within 7 or 8 weeks because of hypertension and severe damage of myocard and kidney. Treatment with compound 4 significantly reduced mortality, alleviated myocardial fibrosis, and improved renal function. Although circulating and cardiac aldosterone levels were reduced, compound 4 showed no significant influence on blood pressure or on concentrations of corticosterone and renin. In spontaneously hypertensive rats, treatment with R-fadrozole (po) decreased free urinary aldosterone levels dose-dependently in two treatment groups receiving different diets.26 Moreover, both enantiomers of fadrozole were tested in spontaneously hypertensive heart failure rats for their ability to reverse an existing myocardial fibrosis.27 Although both enantiomers led to a similar decrease of plasma aldosterone levels, only the R-enantiomer was able to reverse myocardial fibrosis by 50% at a dose of 1.2 mg kg−1 d−1). Furthermore, compound 4 was administered to rats with congestive heart failure (CHF) in a dose of 4 mg kg−1 d−1 after induction of a myocardial infarction.28 The compound not only increased cardiac output and decreased total peripheral resistance but also reversed left ventricular remodeling, improved its function, and reduced collagen accumulation. Another study employed uninephrectomized rats, which were treated with angiotensin II (Ang II) and fed with a high salt diet to induce renal fibrosis. Compound 4 (4 mg kg−1 d−1, po) reduced circulating aldosterone, led to a slight increase in corticosterone levels, but did not affect potassium and sodium concentrations.29 It also prevented glomerular injury, cardiac interstitial fibrosis, as well as cardiac and aortic hypertrophy. R-Fadrozole was also evaluated in comparison to metyrapone in a rat hyperaldosteronism model, in which the animals were infused with either Ang II or adrenocorticotropic hormone (ACTH) to increase the plasma concentrations of aldosterone and DOC, respectively.30 Both drugs dose-dependently reduced aldosterone and DOC levels. Importantly, compound 4 was around 50fold more potent in reducing aldosterone levels compared to its effects on DOC. Furthermore, in a recent study in apolipoprotein E-deficient mice, compound 4 (po applied) reduced atherosclerosis and inflammation without changing plasma aldosterone concentrations in the applied dose.31 Although showing promising results in experimental models, compound 4 was never evaluated in a clinical trial. 3.3.2. (R)-4-(6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3fluorobenzonitrile. In contrast, (R)-4-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluorobenzonitrile (LCI699, compound 5, Chart 4),32 a close analogue of fadrozole, has been tested clinically. The evaluation of this drug candidate in animal models is not reported in the literature. In our own lab we found, not surprisingly, some hCYP19 inhibition (IC50 CYP19 = 856 nM) and little selectivity (SF = 15) over hCYP11B1 (IC50 CYP11B2 = 0.2 nM, IC50 CYP11B1 = 2.9 nM, tested in V79MZ cells stably expressing hCYP11B1 and hCYP11B2). In a phase I trial with sodium depleted healthy persons,32 compound 5 exhibited rapid oral absorption, reaching maximum plasma concentration within 1 h, but also showed a relatively short elimination half-life of 4 h, which made twice daily application (b.i.d.) necessary. Daily one dose application of compound 5 for 7 days significantly reduced urinary and plasma aldosterone levels by 53% and 36%, respectively. In contrast, eplerenone elevated aldosterone concentrations by 88% and 38%, respectively. Compound 5 did not inhibit cortisol biosynthesis at a dose of 0.5 mg once daily (q.d.) with or without ACTH stimulation. However, at doses of more than 3 mg q.d., plasma cortisol concentrations were reduced and cortisol response to ACTH was blocked.33 As 5014

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Chart 5. Diverse Modifications of Fadrozolea

a

IC50 values against hCYP11B2 and selectivity factors between the inhibion of hCYP11B2 and hCYP11B1 (SF = IC50 CYP11B1/IC50 CYP11B2) are presented.

3.3.3. Derivatives of Fadrozole. Many modifications have been performed with fadrozole as the parent compound (Chart 5), and most of them are reported in patents.40−63 Optimizations were carried out on all moieties of fadrozole, namely, the tetrahydropyridine and imidazole rings, as well as the phenyl substituent. The tetrahydropyridine cycle was aromatized to form imidazo[1,5-a]pyridine, e.g. compound 6, which showed a strong inhibition of hCYP11B2 (IC50 = 50 nM) in homogenized rat glomerulosa tissue.40 Contraction or expansion of this ring was also attempted leading to compound 7 as a potent inhibitor of hCYP11B2 (IC50 = 2 nM).41 Omitting this cycle but introducing substituents at the 5-position of imidazole resulted in another series of CYP11B2 inhibitors. Among them, compound 8 is the most potent (IC50 = 1.7 nM) and selective (SF = 16.5) one evaluated in assays using V79MZ cells stably expressing hCYP11B2 or hCYP11B1.42 Insertion of heteroatoms, amide groups, or sulfonamide groups into this ring also led to potent inhibitors. Some sulfonamide analogues even showed significant improvement of the selectivity over hCYP11B1, such as compound 9 (IC50 = 0.3 nM, SF = 606).43 However, these compounds also exhibited a marked inhibition of CYP3A4 (IC50 < 68 nM).41 The imidazole group was exchanged by other nitrogen containing heterocycles, such as pyridine, oxazole, and thiazole. The position of the bridgehead nitrogen was also changed, leading to compounds like 10, which reduced the aldosterone plasma levels by 62% after 2 h in rats when applied 4 mg/kg perorally (p.o.).44 Via connecting the tetrahydropyridine ore and its phenyl substituent by an additional chain, chiral spiro compounds were obtained. Such

modifications were mostly performed in combination with other modifications on the tetrahydropyridine ring and/or the Ncontaining heterocycle. For example, compound 11 is the result of O insertion into the tetrahydropyridine cycle and fusion of the c-hexyl to the benzene moiety. Reduction of plasma aldosterone (−82%) and corticosterone (−15%) levels were observed in rats 2 h after a 4 mg/kg po application.45 Another ring fusion was also performed with the phenyl group together with an omission of the tetrahydropyridine cycle, leading to compounds that are also analogues of etomidate. Compound 12 showed an IC50 value of 3 nM toward hCYP11B2 but was not very selective regarding hCYP11B1 (94% inhibition at an inhibitor concentration of 100 nM, data determined in NCI-H295R cells).46 3.4. Heterocycle Substituted Indoles, Benzimidazoles, and Related Derivatives. Recently, a series of heterocycle substituted indoles were reported as CYP11B2 inhibitors (Chart 6). Evaluation in NCI-H295R cells revealed that some compounds inhibited aldosterone synthesis with IC50 values below 10 nM but showed minimal influence on cortisol formation (e.g., 13: IC50 = 5 nM, SF = 322).64 Introduction of additional nitrogens led to inhibitors with benzimidazole, imidazopyridine, pyrazolopyridine, and triazolopyridine cores (Chart 6). A typical compound of these series, 14, showed a potent inhibition of hCYP11B2 with an IC50 of 0.15 nM and excellent selectivity over CYP11B1 (SF > 1000) tested with V79MZ cells expressing hCYP11B1 or hCYP11B2.65 Replacement of the pyridine moiety of the azolopyridines by lactams retained inhibitory potency but slightly reduced selectivity as observed for compound 15 (IC50 = 2.2 nM, SF = 128).66 5015

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CYP3A4 and CYP2D6.69 However, they turned out to be potent inhibitors of CYP1A2 and could not be considered as drug candidates (compounds 19 and 20). Introduction of a methyl group next to the double bond together with the exchange of 3pyridine by 4-isoquinoline (compound 22) sustained the high inhibitory potency and CYP11B1 selectivity (IC50 = 0.5 nM, SF = 128) but also dramatically reduced the inhibition of CYP1A2.70 Another strategy to decrease CYP1A2 inhibition was the reduction of aromaticity and planarity of the naphthalene core. Saturation of the left-side benzene ring and introduction of polar groups resulted in tetralone 23 (IC50 = 7.8 nM, SF = 496) with a significantly reduced CYP1A2 inhibition (IC50 CYP1A2 = 1.55 μM).71 Full saturation of the core results in complete loss of CYP1A2 inhibition (compound 24, IC50 CYP1A2 > 100 μM). However, selectivity over CYP11B1 was reduced (IC50 CYP11B2 = 21 nM, SF = 50) and druglike properties were compromised (high log P value).72 After the combination of a ligand-based (pharmacophore model) and structure-based (docking simulations) approach had led to the identification of a new hydrophobic pocket,73 a para-substituted benzyl moiety attached to the naphthalene core was introduced to occupy this pocket. A series of highly potent and selective CYP11B2 inhibitor were thus obtained, such as compound 25 (IC50 = 3.9 nM, SF = 913).73 3.6. Heterocycle Substituted Dihydroquinolinones, Dihydropyrroloquinolinones, Dihydrotriazoloquinolines, and Related Derivatives. Despite of the good activity and selectivity profiles of tetralone 23, this compound was not considered for further development, as it showed cytotoxic effects in human U-937 cells at concentrations above 100 μM.71 The bioisosteric dihydroquinolinone 26 (Chart 8) showed no such effects at concentrations up to 200 μM (for solubility reasons higher concentrations could not be tested), while the good inhibitory potency and selectivity were sustained (IC50 = 28 nM, SF = 241).71 It also showed a reduced inhibition of CYP1A2 (IC50 = 1.95 μM). Exchange of 3-pyridine by 4-isoquinoline yielded a more potent hCYP11B2 inhibitor, compound 27 (IC50 = 0.2 nM, SF = 187) showing no inhibition of CYP17, CYP19, and hepatic CYP1A2 (IC50 > 150 μM).71 Furthermore, compound 27 significantly reduced aldosterone plasma levels by around 50% in ACTH stimulated rats after being intravenously applied at a dose of 20 mg/kg. Similar results were also obtained after po application (25 mg/kg). More modifications were performed on the 3-pyridyl substituted dihydroquinolinone scaffold (Chart 8). Alkylation of the lactam N was found to increase potency. However, steric restrictions at this region of the binding site limited the bulkiness of the substituents. A five- or six-membered alkyl ring fused to the core leading to 5,6-dihydro1H-pyrrolo[3,2,1-ij]quinolin-4(2H)-one analogues was discovered as an optimal structural modification.74 The resulting compound 28 was more potent and selective (IC50 = 1.1 nM, SF = 650) compared to its parent compound 26 and showed no inhibition toward CYP17 and CYP19. Furthermore, an expansion of the above-mentioned pharmacophore model led to the identification of another hydrophobic pocket near the heme. Substituted benzyl groups were hence introduced into the corresponding m-position of the pyridine moiety to exploit this pocket. Although the resulting compound 29 retained inhibitory potency (IC50 = 4.6 nM), its selectivity was decreased (SF = 299) compared to compound 28.74 Interestingly, the insertion of a methylene bridge between the core and heme-complexing heterocycle significantly increased inhibition of the CYP11B1 isozyme. In the subsequent studies it was found that heme-

Chart 6. Structures of Heterocycle Substituted Indoles, Benzimidazoles, and Related Derivativesa

a

IC50 values against hCYP11B2 and selectivity factors between the inhibion of hCYP11B2 and hCYP11B1 (SF = IC 50 CYP11B1 / IC50 CYP11B2) are presented.

3.5. Heterocycle Substituted Indanes, (Semisaturated) Naphthalenes, and Related Derivatives. After screening of a library consisting of about 500 compounds previously synthesized as potential inhibitors of different CYP enzymes, two hits were identified (compounds 16 and 17, Chart 7). Although they were potent with IC50 values around 50 nM, their selectivity was poor (SF < 5).67 Further modifications led to heterocycle substituted methylenetetrahydronaphthalenes and -indanes.68 These compounds are very potent with hCYP11B2 IC50 values lower than 100 nM tested in an assay using V79MZ cells stably expressing hCYP11B2. However, the imidazole analogues exhibited no improvement of selectivity against hCYP11B1. In contrast, compounds with 3-pyridine, 4-isoquinoline, or 5pyrimidine as heme-complexing groups were highly selective. Compound 18 was very potent toward hCYP11B2 (IC50 = 6 nM) and achieved a SF of 120.68b Ring manipulations in this class of inhibitors by incorporating their exocyclic double bond into a new cycle condensed to the benzene nucleus and removing the original aliphatic ring (Chart 7) led to naphthalenes69a and dihydronaphthalenes69b as new classes of hCYP11B2 inhibitors. Investigation of different heme-iron-coordinating heterocycles revealed that 3-pyridine was superior to 4-pyridine, 1-imidazole, 5-oxazole, or 5-pyrimidine. Introduction of small alkoxy (i.e., methoxy, ethoxy) or cyano substituents further improved activity and, importantly, selectivity over hCYP11B1, e.g., the 6-cyano substituted naphthalene 19 (IC50 = 3 nM, SF = 238) and the 6methoxy substituted dihydronaphthalene 20 (IC50 = 2 nM, SF = 275). Employment of an indene core instead of naphthalene actually resulted in the most selective compound of this series, compound 21 (IC50 = 4 nM, SF = 1421).69b Further biological evaluation of substances in this class regarding their selectivity toward hepatic CYP enzymes showed favorable profiles against 5016

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Chart 7. Structures of Heterocycle Substituted Indanes, (Semisaturated) Naphthalenes, and Related Derivativesa

a

IC50 values against hCYP11B2 and selectivity factors between the inhibion of hCYP11B2 and hCYP11B1 (SF = IC50 CYP11B1/IC50 CYP11B2) are presented.

no inhibition toward hCYP17, hCYP19, and a panel of hepatic CYP enzymes.78 3.7. Further hCYP11B2 Inhibitors. There are also patents focusing on the variation of the core structures while certain substituents at the pyridyl moiety, such as ether79a or sulfonamide groups,79b are kept constant (Chart 9). For example, compounds 35 and 36 are potent and selective in assays using NCI-H295R cells. Another class of hCYP11B2 inhibitors is also based on the lactam core; however, different from the previous class of compounds, the heme-complexing heterocycle is attached to the amide group directly (Chart 9).80 An IC50 value of 2.8 nM against hCYP11B2 and a SF of 589 are reported for compound 37 using G402 cells stably expressing hCYP11B1 and hCYP11B2. Similarly, a series of N-(pyridin-3-yl)benzamides is also potent and selective hCYP11B2 inhibitors were tested in assays using V79MZ cells, e.g., compound 38 (IC50 = 53 nM, SF = 111).81 In contrast, xanthone 39 showed a potent inhibition of hCYP11B2 (IC50 = 19 nM) but a poor selectivity over hCYP11B1(SF = 4.8).82

complexing heterocycle and substituents on the methylene bridge also exhibited profound influence on the inhibitory profile. Imidazole substituted compounds were found to be hCYP11B1 inhibitors (e.g., compound 30, IC50 CYP11B1 = 2.2 nM and IC50 CYP11B2 = 24 nM),12b whereas 4-pyridine led to dual inhibitors of hCYP19 and hCYP11B2 (e.g., compound 31, IC50 CYP19 = 32 nM and IC50 CYP11B2 = 41 nM).75 Furthermore, the replacement of the dihydroquinolinone core by benzoxazolone resulted in another class of potent hCYP11B2 inhibitors. Compound 32 strongly inhibited aldosterone formation in NCIH295R cells with an IC50 of 1.4 nM.76 Introduction of N atoms into the aromatic part of the core sustained inhibitory potency, while further substitution at the pyridyl ring was important to strongly increase selectivity. Compound 33 with an acetamido substituted tetrahydroisoquinoline as the heme-coordinating group exhibited an IC50 of 3 nM and a SF of 1228 when tested with G402 cells stably expressing hCYP11B1 and hCYP11B2, respectively.77 Moreover, bioisosteric exchange of the lactam O by N and subsequent ring closure resulted in a series of 3-pyridyl or 4-isoquinoline substituted 4,5-dihydro[1,2,4]triazolo[4,3a]quinolines. The best compound of this class (34) not only exhibited potent inhibition of hCYP11B2 (IC50 = 4.2 nM) and excellent selectivity over hCYP11B1 (SF = 421) but also showed

4. DUAL INHIBITION OF HCYP19/HCYP11B2 AND HCYP17/HCYP11B2 Inhibitors of hCYP1983 and hCYP1784 are successfully used for treating patients with breast and prostate cancers, respectively. 5017

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Chart 8. Structures of Heterocycle Substituted Dihydroquinolinones, Dihydropyrroloquinolinones, Dihydrotriazoloquinolines, and Related Derivativesa

a

IC50 values against hCYP11B2 and selectivity factors between the inhibion of hCYP11B2 and hCYP11B1 (SF = IC50 CYP11B1/IC50 CYP11B2) are presented.

Chart 9. Structures of Other hCYP11B2 Inhibitorsa

a

IC50 values against hCYP11B2 and selectivity factors between the inhibion of hCYP11B2 and hCYP11B1 (SF = IC50 CYP11B1/IC50 CYP11B2) are presented.

hCYP17 inhibition, respectively.75,85 Sex hormone deficiency is proposed to activate the renin−angiotensin−aldosterone system directly but also to increase potassium plasma concentrations, accumulate progesterone, and interfere with lipoprotein

However, the incidence of cardiovascular diseases in patients under these therapies is increased. This is probably due to the excessive aldosterone levels, which have been regarded as a result of estrogen or androgen deficiency caused by hCYP19 or 5018

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recently, it will be exciting to see whether they will result in better clinical effects and enlighten further development. Furthermore, the effects of CYP11B2 inhibitors in further diseases like CHF and diabetic nephropathy have not been clinically investigated yet, although positive results have been observed in animal models. The successful translation into patients would be very exciting because there is a real unmet medical need for these diseases.

metabolism, thus increasing aldosterone levels. Therefore, dual inhibitors of hCYP19/hCYP11B2 and hCYP17/hCYP11B2 were proposed as novel treatments for breast and prostate cancers to reduce the risk of cardiovascular diseases.75,85 This multitargeting strategy was also previously employed in the development of dual inhibitors of hCYP17 and hCYP11B186 to delay the relapse in prostate cancer treatment. The advantages of such multitargeting agents are apparently a reduced risk of drug− drug interactions and a better patient compliance. The first dual inhibitors for reducing cardiovascular risks for breast and prostate cancer patients with selectivity over other CYP enzymes were compounds 40 (IC50 CYP19 = 49 nM and IC50 CYP11B2 = 19 nM, Chart 10)85a and 41 (IC50 CYP17 = 11 nM and IC50 CYP11B2 =



AUTHOR INFORMATION

Corresponding Author

*Phone: +(49) 681 302 70300. Fax: +(49) 681 302 70308. Email: [email protected]. Homepage: http:// www.helmholtz-hzi.de/?id=3897.

Chart 10. Structures of the First Selective Dual Inhibitors of hCYP19/hCYP11B2 and hCYP17/hCYP11B2

Notes

The authors declare no competing financial interest. Biographies Qingzhong Hu studied pharmaceutical and medicinal chemistry in Shenyang Pharmaceutical University, China, where he obtained B.Sc. and M.Sc. degrees. After 1 year working in a CRO (Wuxi Apptech) as a chemist for custom synthesis, he joined Prof. Hartmann’s group in 2005 at Saarland University, Germany. He designed and synthesized selective inhibitors of CYP17 and CYP11B2 for the treatment of prostate cancer and aldosterone related cardiovascular diseases, respectively. After having received his Ph.D. in 2010, he stayed at the University of Saarland, working as a group leader.

13 nM, Chart 10).85b Currently, these proposed strategies are still in a very early stage; further studies in cells and animal models have to be performed to prove the concept.

Lina Yin received her B.Eng. and M.Sc. degrees from Shenyang Pharmaceutical University, China, in 2000 and 2004, respectively. She then spent 2 years working as a scientist in Aobo Pharmaceuticals in Shanghai, China, before joining Prof. Hartmann’s group in 2006. She completed her dissertation on the design and synthesis of selective CYP11B2 inhibitors and obtained her Ph.D. degree in 2011.

5. SUMMARY AND OUTLOOK Recent studies unveiled the roles of aldosterone as a potent proinflammation and profibrosis factor as well as ROS inducer. These nonclassic functions are rapid nongenomic effects and are mediated by MR or the hypothesized membrane-associated aldosterone receptors. The association of excessive aldosterone with various cardiovascular and renal diseases made hCYP11B2 very attractive as a drug target because the MR antagonists in use cause adverse effects and, more importantly, accumulation of aldosterone, which could enhance deleterious effects via MR independent pathways. Following the success of compound 4 in various animal disease models, the structurally related compound 5 was clinically evaluated in patients with PA and hypertension. However, the pharmacological effects on reducing blood pressure and elevating plasma concentrations of potassium and renin were only moderate. This is probably due to the fact that low doses (maximally 1 mg per day) had to be applied, as higher doses were expected to reduce cortisol biosynthesis as a result of the poor selectivity of compound 5 toward hCYP11B1 (SF = 15). However, it is not an easy task to estimate how much of selectivity is needed to avoid the unwanted impairment of cortisol secretion when CYP11B2 inhibitors in adequate doses are applied for curative effects, as the clinical outcome is determined by not only the in vitro inhibitory potency but also the expression levels and renewal rates of enzymes and the amount of applied compounds present in different layers of the adrenal glands. Nevertheless, on the basis of our experience, selectivity of more than 100-fold over CYP11B1 and other steroidogenic CYP enzymes should largely enhance the possibility of obtaining the desired clinical benefits without eliciting side effects due to inhibition of other CYP enzymes. Since many more selective hCYP11B2 inhibitors were identified

Rolf W. Hartmann is the head of the Department of Medicinal Chemistry at Saarland University, Germany, and Department of Drug Design and Optimization at the Helmholtz Institute in Saarbrücken, Germany. He received a Ph.D. in Pharmaceutical Chemistry from the University of Regensburg, Germany, working with Professor Schönenberger on the development of nonsteroidal antiestrogens. After postdoctoral studies at the Max Planck Institute for Endocrinology in Hannover, Germany, he received a Habilitation at the University of Regensburg for his work on the development of nonsteroidal aromatase inhibitors. He became Professor of Pharmaceutical Chemistry at the Free University of Berlin, Germany, in 1988, and in 1989 he moved to Saarland University. He has published more than 260 papers, mostly on the design, synthesis, and biological evaluation of selective inhibitors of steroidogenic enzymes.



ABBREVIATIONS USED MR, mineralocorticoid receptor; RGS2, regulator of G protein signaling 2; ROS, reactive oxygen species; TNX, tenascin X; UPAR, urokinase plasminogen activator receptor; ADAMTS1, a disintegrin and metalloprotease with thrombospondin motifs; PAI-1, plasminogen activator inhibitor 1; TGF-β1, transforming growth factor β1; PA, primary aldosteronism; CYP11B2, aldosterone synthase; CHF, congestive heart failure; CYP11A1, side chain cleavage enzyme; CYP17, 17α-hydroxylase-17,20-lyase; 3β-HSD, 3β-hydroxysteroid dehydrogenase; CYP21, steroid 21-hydroxylase; DOC, 11-deoxycorticosterone; CYP11B1, 11β-hydroxylase; DHEA, dehydroepiandrosterone; CYP19, aromatase; CYP, cytochrome P450; SF, selectivity factor; ETO, R-etomidate; MTD, maximally tolerated dose; 5019

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ACTH, adrenocorticotropic hormone; Ang II, angiotensin II; b.i.d, twice daily application; q.d, once daily



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Journal of Medicinal Chemistry

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dx.doi.org/10.1021/jm401430e | J. Med. Chem. 2014, 57, 5011−5022