Screening of 5- and 6-Substituted Amiloride Libraries Identifies Dual-uPA/NHE1 Active and Single Target-Selective Inhibitors.

The K+-sparing diuretic amiloride shows off-target anti-cancer effects in multiple rodent models. These effects arise from the inhibition of two distinct cancer targets: the trypsin-like serine protease urokinase-type plasminogen activator (uPA), a cell-surface mediator of matrix degradation and tumor cell invasiveness, and the sodium-hydrogen exchanger isoform-1 (NHE1), a central regulator of transmembrane pH that supports carcinogenic progression. In this study, we co-screened our library of 5- and 6-substituted amilorides against these two targets, aiming to identify single-target selective and dual-targeting inhibitors for use as complementary pharmacological probes. Closely related analogs substituted at the 6-position with pyrimidines were identified as dual-targeting (pyrimidine 24 uPA IC50 = 175 nM, NHE1 IC50 = 266 nM, uPA selectivity ratio = 1.5) and uPA-selective (methoxypyrimidine 26 uPA IC50 = 86 nM, NHE1 IC50 = 12,290 nM, uPA selectivity ratio = 143) inhibitors, while high NHE1 potency and selectivity was seen with 5-morpholino (29 NHE1 IC50 = 129 nM, uPA IC50 = 10,949 nM; NHE1 selectivity ratio = 85) and 5-(1,4-oxazepine) (30 NHE1 IC50 = 85 nM, uPA IC50 = 5715 nM; NHE1 selectivity ratio = 67) analogs. Together, these amilorides comprise a new toolkit of chemotype-matched, non-cytotoxic probes for dissecting the pharmacological effects of selective uPA and NHE1 inhibition versus dual-uPA/NHE1 inhibition.

Nanomolar-Potency 1,2,4-Triazoloquinoxaline Inhibitors of the Kidney Urea Transporter UT-A1.

Urea transporter A (UT-A) isoforms encoded by the Slc14a2 gene are expressed in kidney tubule epithelial cells, where they facilitate urinary concentration. UT-A1 inhibition is predicted to produce a unique salt-sparing diuretic action in edema and hyponatremia. Here we report the discovery of 1,2,4-triazoloquinoxalines and the analysis of 37 synthesized analogues. The most potent compound, 8ay, containing 1,2,4-triazolo[4,3- a]quinoxaline-substituted benzenesulfonamide linked by an aryl ether, rapidly and reversibly inhibited UT-A1 urea transport by a noncompetitive mechanism with IC50 ≈ 150 nM; the IC50 was ∼2 µM for the related urea transporter UT-B encoded by the Slc14a1 gene. Molecular modeling suggested a putative binding site on the UT-A1 cytoplasmic domain. In vitro metabolism showing quinoxaline ring oxidation prompted the synthesis of metabolically stable 7,8-difluoroquinoxaline analogue 8bl, which when administered to rats produced marked diuresis and reduced urinary osmolality. 8bl has substantially improved UT-A1 inhibition potency and metabolic stability compared with prior compounds.

CRRL269: a novel designer and renal-enhancing pGC-A peptide activator.

The natriuretic peptides (NPs) B-type NP (BNP) and urodilatin (URO) exert renal protective properties via the particulate guanylyl cyclase A receptor (pGC-A). As a potential renal-enhancing strategy, we engineered a novel designer peptide that we call CRRL269. CRRL269 was investigated in human cell lines and in normal canines to define potential cardiorenal enhancing actions. The mechanism of its cardiorenal selective properties was also investigated. In vitro NP receptor activity was quantified with guanosine 3',5'-cyclic monophosphate generation. In vivo effects were determined in normal canine acute infusion studies. We observed that CRRL269 demonstrated enhanced pGC-A activity in renal compared with nonrenal cell lines. CRRL269 exerted enhanced resistance to neprilysin compared with URO. Importantly, CRRL269 exhibited significant and greater increases in urinary sodium excretion and diuresis, with less blood pressure reduction, than BNP or URO in normal canines. CRRL269 retained potent renin-angiotensin-aldosterone system (RAAS) suppressing properties shared by URO and BNP. Also, CRRL269 exerted less arterial relaxation and higher cAMP cardiomyocytes generation than BNP. CRRL269 possessed superior renal and pGC-A activating properties compared with BNP or URO in vitro. CRRL269 exerted enhanced renal actions while suppressing RAAS in vivo and with less hypotension compared with URO or BNP. Together, our study suggests that CRRL269 is a promising innovative renal-enhancing drug, with favorable protective actions targeting cardiorenal disease states through the pGC-A receptor.

Fungal biotransformation of diuretic and antihypertensive drug spironolactone with Gibberella fujikuroi, Curvularia lunata, Fusarium lini, and Aspergillus alliaceus.

Derivatives of spironolactone (1), a diuretic and antihypertensive drug, were synthesized by using fungal cells for the first time. Ten different fungi were screened for their ability to biotransform 1, four of which were able to produce metabolites 2-8. Gibberella fujikuroi produced canrenone (2), 1-dehydrocanrenone (3), Curvularia lunuta provided compound 2, and 7α-thio-spironolactone (4), Fusarium lini yielded compounds 2, 3, 1ß-hydroxycanrenone (5), 1α-hydroxycanrenone (6), 1-dehydro-15α-hydroxycanrenone (7), and 15α-hydroxycanrenone (8), while Aspergillus alliaceus was able to produce all the seven metabolites. Metabolites 5, 6, and 7 were identified as new compounds. Their structures were elucidated by using different spectroscopic techniques. Substrate 1 and its metabolites 2, 3, and 5-8 were also evaluated for α-glucosidase inhibitory activity in vitro. Substrate 1 was found to be strongly active with IC50 = 335 ±â€¯4.3 µM as compared to the standard drug acarbose IC50 = 840 ±â€¯1.73 µM, whereas all of resulting metabolites were found to be inactive.

Synthesis and in vivo diuretic activity of some new benzothiazole sulfonamides containing quinoxaline ring system.

A series of new 6-substituted-N-[3-{2-(substituted phenyl)-ethenyl} quinoxaline-2(1H)-ylidene]-1,3-benzothiazole-2-amine (4a-f) were designed and synthesized by condensing 2-amino-benzothiazole-6-sulfonic acid amide (1) with chalcones of quinoxaline-2-one (3a-f) in a hope to obtain promising and a new class of diuretic agents. Structures of all the newly synthesized compounds were characterized by spectral data and elemental analysis. The pharmacological studies in experimental rats indicates that compound 4c possesses excellent in vivo diuretic activity of 1.13 and appears to be a better diuretic agent than the reference drugs, acetazolamide (1.0) and urea (0.88). Insight of the binding mode of the synthesized compounds (ligand) into the binding sites of carbonic anhydrase enzyme (PDF code: 4KUV) was provided by docking studies, performed with the help of Maestro 9.0 docking software. Further pharmacokinetic and toxicological studies are needed to confirm the safety of compound 4c which emerged as a lead diuretic compound.

Macrocyclic analogues of the diuretic insect neuropeptide helicokinin I show strong receptor-binding.

Helicokinin I, a diuretic neuropeptide of the relevant cotton pest Helicoverpa zea represents a promising target for the design of insect neuropeptide mimetics. Using a ring-closing metathesis reaction, N-terminal bridged macrocyclic helicokinin I analogues with different rigidity were prepared and tested in a helicokinin receptor assay. A partially peptidomimetic helicokinin analogue, containing two structural modifications provides a deeper insight into the structural-requirements for receptor-binding.

[SEARCHING FOR NEW SYNTHETIC DIURETICS].

This study was aimed at estimation of the diuretic and saluretic activity of 4-nitrophenyl-O-ß-D-glucopyranoside and its aglicon 4-nitrophenol in rats. Test animals daily received 4-nitrophenyl-O-ß-D-glucopyranoside (group 1) and 4-nitrophenol (group 2) intragastrically in 2 mL of distilled water in a dose of 18 µmol/kg from 1st to 7th day and 54 µmol/kg from 8th to 14th days. During the experiment, the most pronounced diuretic activity was observed for 4-nitrophenyl-O-ß-D-glucopyranoside in a dose of 54 µmol/kg, which increased the diuresis in rats 2.5 times as compared to the control value.

Synthesis and characterization of quinazoline derivatives: search for hybrid molecule as diuretic and antihypertensive agents.

To explore the pharmacological and structure-activity relationship of a series of N-substituted-(4-oxo-2-substituted-phenylquinazolin-3-(4H)-yl), substituted benzene sulfonamide derivatives (1-25) were synthesized from substituted anthranilic acids derived amino quinazolines and substituted benzene sulphonamides. All the synthesized compounds were evaluated for their diuretic (by Lipschitz et al. method), antihypertensive activity by non-invasive blood pressure (NIBP) using the tail-cuff method and anti-diabetic potential in rats. Six compounds showing significantly excellent activity were compared with metolazone, prazosin and diazoxide as standards. Compound N-[7-chloro-2-(4-methoxyphenyl)-4-oxoquinazolin-3(4H)-yl]-4 nitrobenzenesulfonamide (20) exhibited most potent of the series.

Unequivocal role of pyrazine ring in medicinally important compounds: a review.

Pyrazine is one of the important class of heterocyclic compounds that can be obtained naturally or synthesized chemically. Pyrazine ring has got importance in exhibiting various biological activities in association with other scaffolds like pyrrole, pyrazole, imidazole, triazole, tetrazole, thiophene, oxazole, pyridine, piperidine and piperazine. Presence of pyrazine ring as a basic scaffold in various clinically used drugs exhibits its importance in drug design. In this review, attempt has been made to disclose various therapeutic applications of pyrazine derivatives reported during the last decade.

Synthesis and preliminary evaluation of amiloride analogs as inhibitors of the urokinase-type plasminogen activator (uPA).

A known side-activity of the oral potassium-sparing diuretic drug amiloride is inhibition of the enzyme urokinase-type plasminogen activator (uPA, K(i)=7 µM), a promising anticancer target. Several studies have demonstrated significant antitumor/metastasis properties for amiloride in animal cancer models and it would appear that these arise, at least in part, through inhibition of uPA. Selective optimization of amiloride's structure for more potent inhibition of uPA and loss of diuretic effects would thus appear as an attractive strategy towards novel anticancer agents. The following report is a preliminary structure-activity exploration of amiloride analogs as inhibitors of uPA. A key finding was that the well-studied 5-substituted analogs ethylisopropyl amiloride (EIPA) and hexamethylene amiloride (HMA) are approximately twofold more potent than amiloride as uPA inhibitors.

PtI2-catalyzed tandem 3,3-rearrangement/Nazarov reaction of arylpropargylic esters: synthesis of indanone derivatives.

An efficient PtI(2)-catalyzed tandem reaction of arylpropargylic esters, involving 3,3-rearrangement and Nazarov reaction, has been developed to produce 3-substituted and 3,3-disubstituted indanone derivatives. This approach provided a pathway to the synthesis of indanone skeletons in natural products.

Synthesis of new functionalized 3-substituted [1,2,4]triazolo [4,3-a]pyrimidine derivatives: potential antihypertensive agents.

A convenient synthesis of a series of thiosemicarbazide, 1,3,4-oxadiazole, 1,3,4-thiadiazole, thiazole, 1,2,4-triazole, pyrazole and dioxoisoindoline derivatives incorporating 1,2,4-triazolo[4,3-a]pyrimidine via the reaction of the readily accessible 1,5-dihydro-5-oxo-1.7-diphenyl-1,2.4-triazolo[4,3-a]pyrimidine-3-carbohydrazide (2) with the appropriate reagents is described. The newly synthesized compounds were found to possess antihypertensive and diuretic activities compared to captopril and furosemide as reference controls, respectively.

Synthesis and in vivo diuretic activity of biphenyl benzothiazole-2-carboxamide derivatives.

A series of N-{(substituted)1,3-benzothiazol-2-yl}-1,1 '-biphenyl-4-carboxamides was synthesized by reaction between biphenyl acid chloride and 2-aminobenzothiazole. The synthesized compounds were screened in vivo for diuretic activity. Among the series, N-(1,3-benzothiazol-2-yl)-1,1'-biphenyl-4-carboxamide (II) was found to be the most promising candidate.

Structure-activity relationships for in vitro diuretic activity of CAP2b in the housefly.

A series of truncated and Ala-replacement analogs of the peptide Manse-CAP2b (pELYAFPRV-NH(2)) were assayed for diuretic activity on Malpighian tubules of the housefly Musca domestica (M. domestica). The C-terminal hexapeptide proved to be the active core, the minimum sequence required to retain significant diuretic activity. However, full activity required the C-terminal heptapeptide, which was equipotent with the most active of the native housefly CAP2b peptides. Replacement of Arg(7) and Val(8) with Ala led to inactivity and a large 70-fold drop in potency, respectively, indicating that these were critical residues. The Leu(2) was semicritical, where a six-fold loss in potency was observed. Conversely, the replacement of all other residues with Ala led to much smaller effects on potency and these positions were considered to be noncritical. This structure-activity relationship data can aid in the design of mimetic agonist/antagonist analogs of this diuretic peptide family with enhanced biostability and bioavailability, as tools for arthropod endocrinologists and as potential pest management agents capable of disrupting the water balance in pest flies.

Design, synthesis and diuretic activity of some novel 2,4-diamino-6-aryl-7-arylaminopyrimido [4,5-d]pyrimidin-5(6H)-ones.

Synthesis and diuretic activity of some novel 2,4-diamino-6-aryl-7-arylaminopyrimidol4,5-d]pyrimidin-5(6H) -ones were described. The series was designed on the basis of structural similarity between pteridines and pyridopyrimidines. Designed molecules were synthesized by cyclo-condensation of 5-cyano-6-methylmercapto-3-aryl-2-arylaminopyrimidin-4(3H)-one with guanidine. All the compounds were screened for their diuretic activity using triamterene and furosemide as standard drugs. Compounds 2a, 2b, 2d, 2e and 2f were found more potent than triamterene. Compound 2e exhibited diuretic activity comparable to furosemide with greater natriuretic effect. It also exhibited significant antihypertensive activity.

Highly potent 1-aminocyclohexane-1-carboxylic acid substituted V2 agonists of arginine vasopressin.

The synthesis and some pharmacological properties of two sets of analogues, one consisting of six peptides with 1-aminocyclohexane-1-carboxylic acid (Acc) in position 2 and the other with the amino acid in position 3, have been described. All the peptides were tested for their pressor, antidiuretic, and uterotonic in vitro activities. The Acc(2) modification has been shown to selectively modulate the activities of the analogues. Four of the compounds were highly potent antidiuretic agonists with different pressor and uterotonic activities. On the other hand, the 3-substituted counterparts failed to exhibit any of the activities. One exception was provided by the [Mpa(1),Acc(3),Val(4),D-Arg(8)]VP analogue, which exhibited antidiuretic activity matching that of AVP, yet, unlike AVP, it was fairly selective.

Synthesis and evaluation of some novel quinazolinone derivatives as diuretic agents.

A new series of quinazolin-4(3H)-one derivatives containing either a thiazole or a 1, 3, 4-thiadiazole moiety were prepared in order to study the effect of such a heterocyclic combination on the expected diuretic activity. Synthesis of the target compounds (2, 4, and 6) has been achieved through an interaction of the starting 7-chloro-2-methyl-4H-3, 1-benzoxazin-4-one 1 with different heterocyclic amines. Alkylation of 3-(2-mercapto-1, 3, 4-thiadiazol-5-yl)quinazolin-4(3H)-one derivative 4 with different alkyl halides or chloroacetic acid afforded the corresponding thioethers 5 while interaction of 2-methyl-3-(1, 3, 4-thiadiazol-5-yl or thiazol-5-yl)quinazolin-4(3H)-ones (2 and 6) with various aromatic aldehydes resulted in the formation of the arylvinyl analogs 3 and 7, respectively. On the other hand, 2-morpholinomethyl-3-(2-sulfamoyl or mercapto-1, 3, 4-thiadiazol-5-yl)quinazolin-4(3H)-one derivatives 10 have also been synthesized through an interaction of the sulfonamide or thiol analog 9 with the appropriate amine. Biological evaluation of some of the target compounds as diuretic agents was carried out. The results showed that 2-[2-(4-chlorophenyl)vinyl]-7-chloro-3-(2-sulfamoyl-1, 3, 4-thiadiazol-5-yl)quinazolin-4(3H)-one 7b exhibited significant diuretic activity. The detailed synthesis, spectroscopic and biological data are reported.

Potent nonpeptide vasopressin receptor antagonists based on oxazino- and thiazinobenzodiazepine templates.

Vasopressin receptor antagonists can elicit ion-sparing diuretic effects (i.e., aquaresis) in vivo by blunting the action of the circulating hypophyseal hormone arginine vasopressin. We have identified two new series of basic tricyclic benzodiazepines, represented by general structure 1, which contain compounds that bind with high affinity to human V2 receptors. For example, (S)-(+)-8 and 5 are potent and selective V2 receptor antagonists with pronounced aquaretic activity in rats on oral administration.

Guanylin and its lysine-containing analogue in the isolated perfused rat kidney: interaction with chymotrypsin inhibitor.

Guanylin and uroguanylin are two novel peptides that activate membrane-bound guanylate cyclases found in the kidney and intestine, influencing fluid and electrolyte homeostasis by cyclic GMP. Their natriuretic and kaliuretic activities are well documented. Since guanylin is inactivated by chymotrypsin in vitro, experiments were designed to evaluate the role of chymotrypsin-like proteases in renal metabolism of guanylin. Using the isolated perfused rat kidney, guanylin and a recombinant derivative containing a lysine residue in the N-terminus of the native peptide was tested. There were three experimental groups. In the first group, lys-guanylin (0.1-2.5 microg/ml) was placed into perfusate reservoir. In the second group, chymostatin (6 microg/ml), a chymotrypsin inhibitor, was placed into solution. In the third group, after 30 min. of perfusion with chymostatin (6 microg/ml), guanylin (0.3 microg/ml) was placed into solution. A maximal decrease in fractional Na+ reabsorption (%TNa+) was achieved at 1.0 microg/ml of lys-guanylin (from 73.25+/-2.29 to 54.97+/-0.10, P<0.05). Lys-guanylin (1.0 microg/ml) also decreased fractional K+ reabsorption (%TK+) from 59.26+/-3.93 to 30.75+/-0.78 (P<0.05). Chymostatin had no detectable effects in electrolyte reabsorption in this assay. When introduced after chymostatin, guanylin lowered %TNa+ (from 81.2+/-1.86 to 72.6+/-2.45, P<0.05) and %TK+ (from 69.4+/-4.12 to 65.8+/-2.81, P<0.05). At this subthreshold concentration, guanylin alone lacks effects in %TNa+ or %TK+. Furthermore, the ability of both peptides to promote increases in intestinal fluid secretion was evaluated in the in vivo suckling mouse model. When administered per os, guanylin failed to stimulate intestinal secretion. When chymostatin was present in the test solution, guanylin induced intestinal secretion in this assay. In marked contrast, lys-guanylin alone induced diarrhoea in the suckling mouse. The present paper concludes that guanylin undergoes metabolism in target tissues such as the intestine and kidney and its lysine-containing analogue retains full biological activity.

The synthesis of an analogue of the locust CRF-like diuretic peptide, and the biological activities of this and some C-terminal fragments.

The synthesis is described of an analogue of the locust CRF-like diuretic peptide in which methionine in positions 1,3, and 13 is replaced by isosteric methyl-homoserine residues. This analogue has been tested for biological activity on Malpighian tubules in vitro, and feeding behavior in vivo. It is highly active in stimulating fluid secretion and accumulation of cAMP in tubules, and on increasing the latency to feed and reducing meal duration. A 15 residue fragment from the C-terminus of the CRF-like peptide, Locmi-DP(32-46), is fully active in the feeding assay, but has only weak ability to stimulate the accumulation of cAMP in tubules. Two smaller fragments, Locmi-DP(32-37) and Locmi-DP(41-46), were tested but neither had consistent biological activity in any of the assays used here. None of the peptides tested have any substantive activity in increasing cGMP in tubules.