Design and optimization of new piperidines as renin inhibitors

The discovery of a new series of piperidine-based renin inhibitors is described herein. SAR optimization upon the P3 renin sub-pocket is described, leading to the discovery of 9 and 41, two bioavailable renin inhibitors orally active at low doses in a transgenic rat model of hypertension.     

Identification of a new biaryl scaffold generating potent renin inhibitors

The discovery and SAR of a series of potent renin inhibitors possessing a novel biaryl scaffold are described herein. Molecular modeling revealed that the cyclopropylamide spacer present in 1 can be replaced by a simple, substituted aromatic ring such as a toluene in 2. The resulting compounds exhibit subnanomolar renin IC₅₀ and good oral bioavailability in rats.     

Renin inhibitors for the treatment of hypertension: design and optimization of a novel series of pyridone-substituted piperidines

An SAR campaign aimed at decreasing the overall lipophilicity of renin inhibitors such as 1 is described herein. It was found that replacement of the northern appendage in 1 with an N-methyl pyridone and subsequent re-optimization of the benzyl amide handle afforded compounds with in vitro and in vivo profiles suitable for further profiling. An unexpected CV toxicity in dogs observed with compound 20 led to the employment of a time and resource sparing rodent model for in vivo screening of key compounds. This culminated in the identification of compound 31 as an optimized renin inhibitor.     

Structure-based design of a new series of N-(piperidin-3-yl)pyrimidine-5-carboxamides as renin inhibitors

The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5–14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 (‘Leu-in’ to ‘Leu-out’) by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats.     

Design and optimization of a substituted amino propanamide series of renin inhibitors for the treatment of hypertension

The discovery and SAR of a new series of substituted amino propanamide renin inhibitors are herein described. This work has led to the preparation of compounds with in vitro and in vivo profiles suitable for further development. Specifically, challenges pertaining to oral bioavailability, covalent binding and time-dependent CYP 3A4 inhibition were overcome thereby culminating in the identification of compound 50 as an optimized renin inhibitor with good efficacy in the hypertensive double-transgenic rat model.     

Measurement of renin and substrate concentrations in human serum.

Methods for the measurement of renin and renin substrate by radioimmunoassay have been described. One method of measuring renin is based on the zero-order reaction velocity of angiotensin I formation when serum is incubated with an excess of hog substrate. This method was compared with a bioassay which has been described previously (A. B. Gould, L. T. Skeggs, and J. R. Kahn, 1966, Lab. Invest.15, 1802–1813) and with another radioimmunoassay which determines renin concentration from the rate of angiotensin I formation with endogenous substrate by using the integrated form of the Michaelis-Menten equation and the kinetic constants. Similar results were obtained by these three methods when 30 samples of serum from 15 normotensive people were assayed. No evidence was found to suggest any interference by activators or inhibitors in human serum. The mean recovery of human renin added to serum in 27 experiments was 93.5 ± 10.7% (SD). In addition, the kinetic analysis of human serum showed no difference in the rate of angiotensin formation, at comparable substrate levels, in sera from normotensive people (including women taking oral contraceptives) and patients with essential hypertension.     

Mineralocorticoid Receptor Antagonists Decrease the Rates of Positive Screening for Primary Aldosteronism

Objective: Mineralocorticoid receptor antagonists (MRAs) are effective in patients with resistant hypertension and/or primary aldosteronism (PA). Screening for PA should ideally be conducted after stopping medications that might interfere with the renin-angiotensin-aldosterone system, but this is challenging in patients with recalcitrant hypertension or hypokalemia. Herein, we aimed to evaluate the impact of MRAs on PA screening in clinical practice.Methods: We conducted a retrospective cohort study of patients with hypertension who had plasma aldosterone and renin measurements before and after MRA use in a tertiary referral center, over 19 years.Results: A total of 146 patients, 91 with PA, were included and followed for up to 18 months. Overall, both plasma renin and aldosterone increased after MRA initiation (from median, interquartile range: 0.5 [0.1, 0.8] to 1.2 [0.6, 4.8] ng/mL/hour and from 19.1 [12.9, 27.7] to 26.4 [17.1, 42.3] ng/dL, respectively; P<.0001 for both), while the aldosterone/renin ratio (ARR) decreased from 40.3 (18.5, 102.7) to 23.1 (8.6, 58.7) ng/dL per ng/mL/hour (P<.0001). Similar changes occurred irrespective of the MRA treatment duration and other antihypertensives used. Positive PA screening abrogation after MRA initiation was found in 45/94 (48%) patients. Conversely, 17% of patients had positive PA screening only after MRA treatment, mostly due to correction of hypokalemia. An initially positive screening test was more likely altered by high MRA doses and more likely persistent in patients with confirmed PA or taking beta-blockers.Conclusion: MRAs commonly reduce ARR and the proportion of positive PA screening results. When PA is suspected, screening should be repeated off MRAs.Abbreviations: ACEI = angiotensin-converting enzyme inhibitor; ARB = angiotensin receptor blocker; ARR = aldosterone/renin ratio; DRC = direct renin concentration; MRA = mineralocorticoid receptor antagonist; PA = primary aldosteronism; PAC = plasma aldosterone concentration; PRA = plasma renin activity; RAAS = renin-angiotensin-aldosterone system     

Discovery of benzimidazole derivatives as orally active renin inhibitors: Optimization of 3,5-disubstituted piperidine to improve pharmacokinetic profile

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.     

Prevelence of Primary Aldosteronism in an Urban Hypertensive Population

Objective: To determine the prevalence of primary aldosteronism (PA) in hypertensive patients presenting to the primary care clinic at The Mount Sinai Hospital, regardless of the degree of hypertension and to identify clinical criteria that should prompt screening for PA.Methods: An aldosterone:renin ratio (ARR, cutoff ≥20, with plasma aldosterone concentration [PAC] ≥10 and suppressed renin) was used to prospectively screen 296 hypertensive patients (blood pressure [BP] ≥140/90) over the age of 18 from August 2012 through May 2013. Subjects who screened positive then underwent confirmatory oral salt load testing (OSLT).Results: Of the 296 patients, 14 screened positive for PA, an overall prevalence of 4.7%. Six of the 14 cases underwent confirmatory OSLT, upon which 2 were confirmed positive, for a prevalence of 0.7%. Overall, patients with confirmed PA were more likely to have resistant hypertension (42.9% vs. 18.1% (P =.0334)) and require more antihypertensive agents (2.8 ± 1.2 agents vs. 2.1 ± 1.1 agents, P =.0213). There was a trend toward lower potassium values in the cases.Conclusion: The prevalence of PA in our clinic is much lower than in reports from certain “at-risk” populations. PA screening is indicated in patients with resistant hypertension, regardless of serum potassium levels.Abbreviations:ARR = aldosterone:renin ratioACTH = adrenocorticotropic hormoneAVS = adrenal venous samplingBP = blood pressureMRA = mineralocorticoid receptor antagonistOSLT = oral salt load confirmatory testPA = primary aldosteronismPAC = plasma aldosterone concentrationPCP = primary care providerPRA = plasma renin activity     

The P1 N-isopropyl motif bearing hydroxyethylene dipeptide isostere analogues of aliskiren are in vitro potent inhibitors of the human aspartyl protease renin

Novel nonpeptide small molecule renin inhibitors bearing an N-isopropyl P₁ motif were designed based on initial lead structures 1 and aliskiren (2). (P₃–P₁)-Benzamide derivatives such as 9a and 34, as well as the corresponding P₁ basic tertiary amine derivatives 10 and 35 were found to display low nanomolar inhibition against human renin in vitro.     

Discovery and optimization of a new class of potent and non-chiral indole-3-carboxamide-based renin inhibitors

Selective inhibition of the aspartyl protease renin has gained attraction as an interesting approach to control hypertension and associated cardiovascular risk factors given its unique position in the renin–angiotensin system. Using a combination of high-throughput screening, parallel synthesis, X-ray crystallography and structure-based design, we identified and optimized a novel series of potent and non-chiral indole-3-carboxamides with remarkable potency for renin. The most potent compound 5k displays an IC₅₀ value of 2 nM.     

Genetics of Primary Aldosteronism

Objective: The American Association of Clinical Endocrinologists Adrenal Scientific Committee has developed a series of articles to update members on the genetics of adrenal diseases.Methods: Case presentation, discussion of literature, table, and bullet point conclusions.Results: Primary aldosteronism (PA) is the most common form of secondary hypertension. Early detection, surveillance, and treatment of PA may mitigate future cardiovascular risk. The genetics of PA are rapidly evolving, and the consideration for genetic causes of PA are growing. Three inheritable forms of PA are now recognized: familial hyperaldosteronism type I (glucocorticoidremediable aldosteronism), familial hyperaldosteronism type II, and familial hyperaldosteronism type III. The recent discovery of familial hyperaldosteornism type III spurred a flurry of international and collaborative research that is identifying more genetic and molecular causes of PA that relate to mutations in membrane electrolyte transport channels of zona glomerulosa cells.Conclusion: This article reviews the various genetic forms of PA, including a focus on the molecular mechanisms involved, diagnosis, and treatment.Abbreviations: ACTH = adrenocorticotropic hormone ARR = aldosterone to renin ratio FH-I = familial hyperaldosteronism type I FH-II = familial hyperaldosteronism type II FH-III = familial hyperaldosteronism type III GRA = glucocorticoidremediable aldosteronism PA = primary aldosteronism PRA = plasma renin activity     

Optimization of orally bioavailable alkyl amine renin inhibitors

Structure-guided drug design led to new alkylamine renin inhibitors with improved in vitro and in vivo potency. Lead compound 21a, has an IC₅₀ of 0.83 nM for the inhibition of human renin in plasma (PRA). Oral administration of 21a at 10 mg/kg resulted in >20 h reduction of blood pressure in a double transgenic rat model of hypertension.     

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: Discovery of DS-8108b,an orally active renin inhibitor

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure–activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.     

Exploration Of The Seated Saline Suppression Test For The Diagnosis Of Primary Aldosteronism In The Chinese Population

Objective: We prospectively investigated the accuracy of the seated saline suppression test (SSST) in 113 patients with hypertension (including 93 primary aldosteronism [PA] and 20 essential hypertension patients) in the Department of Endocrinology and Metabolism.Methods: Each patient underwent a recumbent saline suppression test (RSST) and SSST. The accuracy of the SSST for a confirmative PA diagnosis and subtype classification was evaluated and compared with the RSST.Results: The area under the receiver operating characteristic (ROC) curve of plasma aldosterone concentration (PAC) for the SSST was significantly greater than that for the RSST (0.945 ± 0.0199 vs. 0.828 ± 0.0404; P<.05). The ROC analysis showed that the optimal PAC cut-off values were 12.94 ng/dL for the SSST (sensitivity 86.02%, specificity 95%; Youden index [YI] 0.810) and 12.04 ng/dL for the RSST (sensitivity 83.15%, specificity 57%; YI 0.401). The optimal PAC cut-off value for classifying aldosterone-producing adenoma and idiopathic hyperaldosteronism was 18.12 ng/dL for the SSST (sensitivity 73.5%, specificity 79.5%). No patients experienced adverse events during the SSST.Conclusion: The SSST is safe and convenient for PA diagnosis. The accuracy of the SSST for a confirmatory diagnosis of PA was better than that of the RSST. The SSST is a reliable alternative for PA confirmation in Chinese individuals.Abbreviations: APA = aldosterone-producing adenoma; ARR = aldosterone to renin ratio; AVS = adrenal vein sampling; CT = computed tomography; EH = essential hypertension; IHA = idiopathic hyperaldosteronism; MRI = magnetic resonance imaging; PA = primary aldosteronism; PAC = plasma aldosterone concentration; PRA = plasma renin activity; ROC = receiver operating characteristic; RSST = recumbent saline suppression test; SSST = seated saline suppression test; YI = Youden index     

Renal Function and Plasma Renin activity as Potential Factors Causing Hyperkalemia in Patients with Thyroid Carcinoma Undergoing Thyroid Hormone Withdrawal For Radioactive Iodine Therapy

Objective: Hypothyroidism is not commonly considered a cause of hyperkalemia. We previously reported that hyperkalemia was observed mainly in elderly patients treated with renin-angiotensin-aldosterone system (RAS) inhibitors when levothyroxine treatment was withdrawn for the thyroidectomized patients with thyroid carcinoma to undergo radioactive iodine treatment. Here, we investigated whether acute hypothyroidism causes hyperkalemia in patients who were not treated with RAS inhibitors. We also investigated factors influencing potassium metabolism in hypothyroid patients.Methods: We conducted a single-center, prospective cohort study of 46 Japanese patients with thyroid carcinoma undergoing levothyroxine withdrawal prior to radioiodine therapy. All patients were normokalemic before levothyroxine withdrawal. Blood samples were analyzed 3 times: before, and at 3 and 4 weeks after levothyroxine withdrawal. We investigated factors that may be associated with the elevation of serum potassium levels from a euthyroid state to a hypothyroid state.Results: None of the patients developed symptomatic hyperkalemia. The mean serum potassium level was significantly higher at 4 weeks after levothyroxine withdrawal compared to baseline. The serum sodium levels, the estimated glomerular filtration rate (eGFR), and the plasma renin activity (PRA) decreased significantly as hypothyroidism advanced. In contrast, the plasma levels of adrenocorticotropic hormone, cortisol, aldosterone, and antidiuretic hormone were not changed, while serum thyroid hormone decreased. At 4 weeks after their levothyroxine withdrawal, the patients' serum potassium values were significantly correlated with the eGFR and the PRA.Conclusion: Acute hypothyroidism can cause a significant increase in the serum potassium level, which may be associated with a decreased eGFR and decreased circulating RAS.Abbreviations: ACTH = adrenocorticotropic hormone; ADH = antidiuretic hormone; ATPase = adenosine triphosphatase; eGFR = estimated glomerular filtration rate; HbA1c = glycated hemoglobin; K⁺ = potassium; Na⁺ = sodium; PRA = plasma renin activity; RAS = renin-angiotensin-aldosterone system; T4 = thyroxine; TSH = thyroid-stimulating hormone     

Design and synthesis of potent macrocyclic renin inhibitors

Two types of P1-P3-linked macrocyclic renin inhibitors containing the hydroxyethylene isostere (HE) scaffold just outside the macrocyclic ring have been synthesized. An aromatic or aliphatic substituent (P3sp) was introduced in the macrocyclic ring aiming at the S3 subpocket (S3sp) in order to optimize the potency. A 5-6-fold improvement in both the K_i and the human plasma renin activity (HPRA)IC₅₀ was observed when moving from the starting linear peptidomimetic compound 1 to the most potent macrocycle 42 (K_i = 3.3 nM and HPRA IC₅₀ = 7 nM). Truncation of the prime side of 42 led to 8-10-fold loss of inhibitory activity in macrocycle 43 (K_i = 34 nM and HPRA IC₅₀ = 56 nM). All macrocycles were epimeric mixtures in regard to the P3sp substituent and X-ray crystallographic data of the representative renin macrocycle 43 complex showed that only the S-isomer buried the substituent into the S3sp. Inhibitory selectivity over cathepsin D (Cat-D) and BACE-1 was also investigated for all the macrocycles and showed that truncation of the prime side increased selectivity of inhibition in favor of renin.     

Addressing time-dependent CYP 3A4 inhibition observed in a novel series of substituted amino propanamide renin inhibitors,a case study

Time-dependent inhibitors of CYPs have the potential to perpetrate drug–drug interactions in the clinical setting. After finding that several leading compounds in a novel series of substituted amino propanamide renin inhibitors inactivated CYP3A4 in an NADPH-dependent and time-dependent manner, a search to identify the cause of this liability was initiated. Extensive SAR revealed that the amide bridge present in compound 1 as a possible culprit. Through the installation of a metabolic soft spot distal to this moiety, potent renin inhibitors with improved CYP profile were identified.     

Design and discovery of new (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-3-carboxamides as potent renin inhibitors

Utilizing X-ray crystal structure analysis, (3S,5R)-5-[4-(2-chlorophenyl)-2,2-dimethyl-5-oxopiperazin-1-yl]piperidine-3-carboxamides were designed and identified as renin inhibitors. The most potent compound 15 demonstrated favorable pharmacokinetic and pharmacodynamic profiles in rat.     

A DNA polymorphism,consistent with gene duplication,correlates with high renin levels in the mouse submaxillary gland

The renin regulatory locus (Rnr) is a genetic element governing mouse submaxillary gland (SMG) renin levels. A 45,000 dalton polypeptide detectable after in vitro translation of mouse SMG mRNA has been identified by genetic and physical criteria as SMG renin. A cDNA recombinant clone specific for SMG renin has been isolated and used to demonstrate that the previously described genetic regulation of SMG renin levels is manifest at the level of renin mRNA concentration. The renin cDNA clone has also been used in Southern blot analyses to study the organization of homologous DNA sequences in strains carrying different alleles at the Rnr locus. Restriction digest patterns of high renin strains (Rnr^s) are characteristically distinct from patterns observed for low renin strains (Rnr^b) and are suggestive of a structural gene duplication at the chromosome 1 locus in high renin strains. However, gene dosage cannot account for the increased levels in high renin strains, since SMG renin levels in Rnr^s and those in Rnr^b may differ up to 100-fold.