Conversion of 5α-Androstane-3α,17ß-diol to bis[(4-dimethylamino)phenyl carbamate] derivative for sensitive determination of its rat brain level by LC/ESI-MS/MS.

RATIONALE: 5α-Androstane-3α,17ß-diol (3α,5α-Adiol) is a testosterone-derived neurosteroid and has anxiolytic and analgesic effects via γ-aminobutyric acid type A receptors as with the progesterone-derived neurosteroid, allopregnanolone (AP). Although the psychotropic drug-evoked changes in the brain AP concentration have been intensively studied, those in the brain 3α,5α-Adiol concentration remain poorly understood. One of the causes for this is the limited availability of a validated method for quantifying the brain 3α,5α-Adiol with a sufficient sensitivity and specificity, which is described in this study. METHODS: To enhance the detectability of 3α,5α-Adiol by electrospray ionization-tandem mass spectrometry (ESI-MS/MS), derivatization with 4-dimethylaminobenzoyl azide was employed. The brain sample was purified by solid-phase extraction and the recovered 3α,5α-Adiol and the deuterated internal standard were derivatized, then measured by liquid chromatography (LC)/ESI-MS/MS with selected reaction monitoring. RESULTS: The derivatized 3α,5α-Adiol, i.e., the bis[(4-dimethylamino)phenyl carbamate] derivative, provided the intense doubly-protonated molecule as the precursor ion, then the specific product ion containing the 3α,5α-Adiol-skeleton by collision-induced dissociation. The detectability of 3α,5α-Adiol was eventually increased 1000-fold by derivatization. Separation of the derivatized 3α,5α-Adiol from its stereoisomers and interfering brain components was achieved using a SunShell Biphenyl column with an isopropyl alcohol-containing mobile phase. A good linearity in the sufficient concentration range, acceptable precision and accuracy, and negligible matrix effect were demonstrated by the validation tests. The animal (rat) study using this method revealed that the brain 3α,5α-Adiol levels were unaffected by the administration of fluoxetine (FLX) and clozapine (CLZ), in contrast to the significant increase of the AP levels. CONCLUSION: An LC/ESI-MS/MS method capable of quantifying 3α,5α-Adiol in the rat brain using a 20-mg tissue was developed and validated. The brain levels of 3α,5α-Adiol had an entirely different behavior from those of AP due to FLX and CLZ administration.

Formation of 5α-dihydrotestosterone from 5α-androstane-3α,17ß-diol in prostate cancer LAPC-4 cells - Identifying inhibitors of non-classical pathways producing the most potent androgen.

5α-Dihydrotestosterone (5α-DHT) possesses a great affinity for the androgen receptor (AR), and its binding to AR promotes the proliferation of prostate cancer (PC) cells in androgen-dependent PC. Primarily synthesized from testosterone (T) in testis, 5α-DHT could also be produced from 5α-androstane-3α,17ß-diol (3α-diol), an almost inactive androgen, following non-classical pathways. We reported the chemical synthesis of non-commercially available [4-14C]-3α-diol from [4-14C]-T, and the development of a biological assay to identify inhibitors of the 5α-DHT formation from radiolabeled 3α-diol in LAPC-4 cell PC model. We measured the inhibitory potency of 5α-androstane derivatives against the formation of 5α-DHT, and inhibition curves were obtained for the most potent compounds (IC50 = 1.2-14.1 µM). The most potent inhibitor 25 (IC50 = 1.2 µM) possesses a 4-(4-CF3-3-CH3O-benzyl)piperazinyl methyl side chain at C3ß and 17ß-OH/17α-CCH functionalities at C17 of a 5α-androstane core.

Synthesis of novel 1,2,3-triazolyl derivatives of pregnane, androstane and D-homoandrostane. Tandem "click" reaction/Cu-catalyzed D-homo rearrangement.

Copper-catalyzed 1,3-dipolar cycloaddition has been employed in the reaction of steroidal azides with various terminal alkynes. A number of novel 1,2,3-triazolyl derivatives of pregnane, androstane and D-homoandrostane were obtained in high yield (70-98%). The developed synthetic protocols allowed us to attach the triazolyl moiety to both the side chain and the steroidal backbone directly, despite the steric hindrance exerted by the polycyclic system. The presence of Cu(II) was shown to evoke d-homo rearrangement under mild conditions. A rational choice of the copper precatalyst permitted us to carry out the "click" reaction either along with tandem d-homo rearrangement or in the absence of this process. The tendency of 16-heterosubstituted steroids to undergo D-homo rearrangement under Cu(II) catalysis was studied.

Biological evaluation of a new family of aminosteroids that display a selective toxicity for various malignant cell lines.

This study investigated the antineoplasic potential of a new family of aminosteroids. The antiproliferative activity of seven 5α-androstane-3α,17ß-diol derivatives selected from a screening study was measured on nine cancerous cell lines (HL-60, K-562, LNCaP, PC-3, Shionogi, MCF-7, MDA-MB-231, BT-20, and OVCAR-3) and two normal cell lines (peripheral blood lymphocytes and WI-38). The aminosteroids efficiently inhibited the cell growth of seven cancer cell lines [inhibitory concentration (IC(50)) values=0.2-6.4 µmol/l] and showed weak toxicity on normal cell lines. Two representative aminosteroids were tested and found to induce apoptosis and a G0/G1 cell cycle block in HL-60-treated cells, but not terminal myeloid differentiation. By a nuclear morphology analysis with fluorescence microscopy, typical apoptotic morphological changes were exhibited by treated cells. One aminosteroid tested in vivo (xenograft model) reduced the breast cancer (MCF-7 cells) tumor growth induced in nude mice. Furthermore, the information gathered suggests that this family of aminosteroids induced growth inhibition cells by arresting the cell cycle and triggering apoptosis.

Impact of electro-acupuncture and physical exercise on hyperandrogenism and oligo/amenorrhea in women with polycystic ovary syndrome: a randomized controlled trial.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder in women of reproductive age, is characterized by hyperandrogenism, oligo/amenorrhea, and polycystic ovaries. We aimed to determine whether low-frequency electro-acupuncture (EA) would decrease hyperandrogenism and improve oligo/amenorrhea more effectively than physical exercise or no intervention. We randomized 84 women with PCOS, aged 18-37 yr, to 16 wk of low-frequency EA, physical exercise, or no intervention. The primary outcome measure changes in the concentration of total testosterone (T) at week 16 determined by gas and liquid chromatography-mass spectrometry was analyzed by intention to treat. Secondary outcome measures were changes in menstrual frequency; concentrations of androgens, estrogens, androgen precursors, and glucuronidated androgen metabolites; and acne and hirsutism. Outcomes were assessed at baseline, after 16 wk of intervention, and after a 16-wk follow-up. After 16 wk of intervention, circulating T decreased by -25%, androsterone glucuronide by -30%, and androstane-3α,17ß-diol-3-glucuronide by -28% in the EA group (P = 0.038, 0.030, and 0.047, respectively vs. exercise); menstrual frequency increased to 0.69/month from 0.28 at baseline in the EA group (P = 0.018 vs. exercise). After the 16-wk follow-up, the acne score decreased by -32% in the EA group (P = 0.006 vs. exercise). Both EA and exercise improved menstrual frequency and decreased the levels of several sex steroids at week 16 and at the 16-wk follow-up compared with no intervention. Low-frequency EA and physical exercise improved hyperandrogenism and menstrual frequency more effectively than no intervention in women with PCOS. Low-frequency EA was superior to physical exercise and may be useful for treating hyperandrogenism and oligo/amenorrhea.

Libraries of 2ß-(N-substituted piperazino)-5α-androstane-3α, 17ß-diols: chemical synthesis and cytotoxic effects on human leukemia HL-60 cells and on normal lymphocytes.

Libraries of steroid derivatives with two levels of molecular diversity were prepared to optimize the antiproliferative activity on leukemia HL-60 cells by first varying the amino acid (AA) at R(1) (libraries A, B, C, and D: with 45, 45, 20, and 20 members, respectively) and, subsequently, the capping group at R(2) (library E: 168 members). The screening of these aminosteroids revealed interesting structure-activity relationships. In library A, the compounds bearing a tetrahydroisoquinolone residue as the first element of diversity showed potent cytotoxicity, principally when isovaleric or cyclohexyl acetic acid was used as a capping group (>40% of cell growth inhibition at 1 µM). In library B, the phenylalanine (Phe) derivatives bearing a cyano group induced a higher growth inhibition than the other Phe derivatives. The screening of library C indicated the increase of hydrophobicity of proline (Pro) seems to preserve the cytotoxic effect achieved by the lead compound. However, the synthesis of structural Pro variants (library D) clearly shows weaker activities when compared to L-Pro building blocks. Finally, by incorporating some of the most active AA of libraries A-D in library E, we observed that the amide coupling functionality gave stronger cytotoxic activity compared to the corresponding sulfonamides or benzylamines. Six of the most active amide derivatives (E-37P, E-41P, E-42P, E-46P, E-48F, and E-12T) were selected and IC(50) determined on HL-60 cells as well as on normal human lymphocytes. Among this series of new anticancer agents, good to high selectivity indices (SI = IC(50) (lymphocytes)/IC(50) (HL-60 cells) = 5 - 55) were obtained.

CYP7B1-mediated metabolism of 5alpha-androstane-3alpha,17beta-diol (3alpha-Adiol): a novel pathway for potential regulation of the cellular levels of androgens and neurosteroids.

The current study presents data indicating that 5alpha-androstane-3alpha,17beta-diol (3alpha-Adiol) undergoes a previously unknown metabolism into hydroxymetabolites, catalyzed by CYP7B1. 3alpha-Adiol is an androgenic steroid which serves as a source for the potent androgen dihydrotestosterone and also can modulate gamma-amino butyric acid A (GABA(A)) receptor function in the brain. The steroid hydroxylase CYP7B1 is known to metabolize cholesterol derivatives, sex hormone precursors and certain estrogens, but has previously not been thought to act on androgens or 3alpha-hydroxylated steroids. 3alpha-Adiol was found to undergo NADPH-dependent metabolism into 6- and 7-hydroxymetabolites in incubations with porcine microsomes and human kidney-derived HEK293 cells, which are high in CYP7B1 content. This metabolism was suppressed by addition of steroids known to be metabolized by CYP7B1. In addition, 3alpha-Adiol significantly suppressed CYP7B1-mediated catalytic reactions, in a way as would be expected for substrates that compete for the same enzyme. Recombinant expression of human CYP7B1 in HEK293 cells significantly increased the rate of 3alpha-Adiol hydroxylation. Furthermore, the observed hydroxylase activity towards 3alpha-Adiol was very low or undetectable in livers of Cyp7b1(-/-) knockout mice. The present results indicate that CYP7B1-mediated catalysis may play a role for control of the cellular levels of androgens, not only of estrogens. These findings suggest a previously unknown mechanism for metabolic elimination of 3alpha-Adiol which may impact intracellular levels of dihydrotestosterone and GABA(A)-modulating steroids.

Chemical synthesis of 2beta-amino-5alpha-androstane-3alpha,17beta-diol N-derivatives and their antiproliferative effect on HL-60 human leukemia cells.

Even though few steroids are used for the treatment of leukemia, 2beta-(4-methylpiperazinyl)-5alpha-androstane-3alpha,17beta-diol (1) was recently reported for its ability to inhibit the proliferation of human leukemia HL-60 cells. With an efficient procedure that we had developed for the aminolysis of hindered steroidal epoxides, we synthesized a series of 2beta-amino-5alpha-androstane-3alpha,17beta-diol N-derivatives structurally similar to 1. Hence, the opening of 2,3alpha-epoxy-5alpha-androstan-17beta-diol with primary and secondary amines allowed the synthesis of aminosteroids with diverse length, ramification, and functionalization of the 2beta-side chain. Sixty-four steroid derivatives were tested for their capacity to inhibit the proliferation of HL-60 cells; thus obtaining first structure-activity relationship results. Ten aminosteroids with long alkyl chains (7-16 carbons) or bulky groups (diphenyl or adamantyl) have shown antiproliferative activity over 78% at 10microM and superior to that of the lead compound. The 3,3-diphenylpropylamino, 4-nonylpiperazinyl and octylamino derivatives of 5alpha-androstane-3alpha,17beta-diol inhibited the HL-60 cell growth with IC(50) of 3.1, 4.2 and 6.4microM, respectively. They were also found to induce the HL-60 cell differentiation.

5Beta,6beta-epoxy-17-oxoandrostan-3beta-yl acetate and 5beta,6beta-epoxy-20-oxopregnan-3beta-yl acetate.

In the title compounds, C(21)H(30)O(4), (I), and C(23)H(34)O(4), (II), respectively, which are valuable intermediates in the synthesis of important steroid derivatives, rings A and B are cis-(5beta,10beta)-fused. The two molecules have similar conformations of rings A, B and C. The presence of the 5beta,6beta-epoxide group induces a significant twist of the steroid nucleus and a strong flattening of the B ring. The different C17 substituents result in different conformations for ring D. Cohesion of the molecular packing is achieved in both compounds only by weak intermolecular interactions. The geometries of the molecules in the crystalline environment are compared with those of the free molecules as given by ab initio Roothan Hartree-Fock calculations. We show in this work that quantum mechanical ab initio methods reproduce well the details of the conformation of these molecules, including a large twist of the steroid nucleus. The calculated twist values are comparable, but are larger than the observed values, indicating a possible small effect of the crystal packing on the twist angles.

5Alpha-androstane-3beta,7alpha,17beta-triol and 5alpha-androstane-3beta,7beta,17beta-triol as substrates for the human 11beta-hydroxysteroid dehydrogenase type 1.

Several studies have shown that the native 7alpha-hydroxy-dehydroepiandrosterone (7alpha-hydroxy-DHEA) is a substrate for the human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) which converts the 7alpha- into the 7beta-epimer through an oxido-reduction process. Research on the 11beta-HSD1 has investigated its function and structure through using native glucocorticoid substrates and known inhibitors. Other steroid substrates are also of interest. Among testosterone metabolites, 5alpha-androstane-3beta,17beta-diol (Adiol) is a substrate for the cytochrome P450 7B1 which produces 5alpha-androstane-3beta,7alpha,17beta-triol (7alpha-Adiol). This steroid may be a substrate for the 11beta-HSD1. We used recombinant yeast-expressed 11beta-HSD1 with NADP(H)-regenerating systems for examining the products obtained after incubation with 7alpha-Adiol, 7beta-Adiol or 7-oxo-Adiol. Oxidative conditions for the 11beta-HSD1 provided no trace of 7-oxo-Adiol but the inter-conversion of 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) (pmol min(-1) microg(-1)/microM) values of 2 and 0.5, respectively. This state was maintained under reductive conditions. The use of a 7-oxo-Adiol substrate under reductive conditions led to the production of both 7alpha- and 7beta-hydroxy-Adiol with V(max)/K(M) values of 3.43 and 0.22, respectively. These findings support the hypothesis that the oxido-reductase and epimerase activities of 11beta-HSD1 depend on the positioning of the steroid substrates within the active site and may provide insight into its fine structure and mechanism of action.

DHRS7 (SDR34C1) - A new player in the regulation of androgen receptor function by inactivation of 5α-dihydrotestosterone?

DHRS7 (SDR34C1) has been associated with potential tumor suppressor effects in prostate cancer; however, its function remains largely unknown. Recent experiments using purified recombinant human DHRS7 suggested several potential substrates, including the steroids cortisone and Δ4-androstene-3,17-dione (androstenedione). However, the substrate and cofactor concentrations used in these experiments were very high and the physiological relevance of these observations needed to be further investigated. In the present study, recombinant human DHRS7 was expressed in intact HEK-293 cells in order to investigate whether glucocorticoids and androgens serve as substrates at sub-micromolar concentrations and at physiological cofactor concentrations. Furthermore, the membrane topology of DHRS7 was revisited using redox-sensitive green-fluorescent protein fusions in living cells. The results revealed that (1) cortisone is a substrate of DHRS7; however, it is not reduced to cortisol but to 20ß-dihydrocortisone, (2) androstenedione is not a relevant substrate of DHRS7, (3) DHRS7 catalyzes the oxoreduction of 5α-dihydrotestosterone (5αDHT) to 3α-androstanediol (3αAdiol), with a suppressive effect on androgen receptor (AR) transcriptional activity, and (4) DHRS7 is anchored in the endoplasmic reticulum membrane with a cytoplasmic orientation. Together, the results show that DHRS7 is a cytoplasmic oriented enzyme exhibiting 3α/20ß-hydroxysteroid dehydrogenase activity, with a possible role in the modulation of AR function. Further research needs to address the physiological relevance of DHRS7 in the inactivation of 5αDHT and AR regulation.

Role of the alternate pathway of dihydrotestosterone formation in virilization of the Wolffian ducts of the tammar wallaby, Macropus eugenii.

Dihydrotestosterone in androgen target tissues is formed under most circumstances by the 5alpha-reduction of testosterone, but an alternate pathway involves the oxidation of androstanediol to dihydrotestosterone. To investigate the mechanism by which androgens virilize the Wolffian ducts in the tammar wallaby, [(3)H]progesterone was incubated with testes from d 10 and 19 pouch young, and radioactivity was recovered in testosterone and androstanediol at both ages. Analysis of the intermediates indicates that androstanediol was formed both from testosterone via 5alpha-reduction and 3alpha-keto reduction and directly from 5alpha-reduced progestogens. 5alpha-Reductase activity was high in minces of mesonephros/epididymis from d 6-21 pouch young. When minces of urogenital tract tissues from d 19 pouch young were incubated with [(3)H]testosterone, [(3)H]dihydrotestosterone, and [(3)H]androstanediol, dihydrotestosterone was the principal androgen formed in the mesonephros/epididymis, urogenital sinus, and urogenital tubercle, whereas androstanediol was the principal androgen formed by the testis. In intact pouch young studied between d 10 and 34, administration of the 5alpha-reductase inhibitor, 17beta-(N,N-diethyl)carbamoyl-4-methyl-4-aza-5alpha-androstan-3-one, blocked virilization of the Wolffian ducts in males, and administration of androstanediol caused virilization of the Wolffian ducts in females. We conclude that dihydrotestosterone, largely formed in the tissue by the oxidation of androstanediol derived from the testes and also the 5alpha-reduction of testosterone, is responsible for Wolffian duct virilization in this species.

Synthesis of 5α-androstane-3α,17ß-diol 17-O-glucuronide histaminyl conjugate for immunoassays.

Simple method of preparation of 5α-androstane-3α,17ß-diol 17-O-glucuronide N-histaminyl amide was developed for the construction of immunoanalytical kit. Improved method of glucuronide derivative synthesis was used, followed by hydroxybenzotriazole-dicyclohexylcarbodiimide coupling with histamine.

Human type 3 3alpha-hydroxysteroid dehydrogenase (aldo-keto reductase 1C2) and androgen metabolism in prostate cells.

Human aldo-keto reductases (AKRs) of the AKR1C subfamily function in vitro as 3-keto-, 17-keto-, and 20-ketosteroid reductases or as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidases. These AKRs can convert potent sex hormones (androgens, estrogens, and progestins) into their cognate inactive metabolites or vice versa. By controlling local ligand concentration AKRs may regulate steroid hormone action at the prereceptor level. AKR1C2 is expressed in prostate, and in vitro it will catalyze the nicotinamide adenine dinucleotide (NAD(+))-dependent oxidation of 3alpha-androstanediol (3alpha-diol) to 5alpha-dihydrotestosterone (5alpha-DHT). This reaction is potently inhibited by reduced NAD phosphate (NADPH), indicating that the NAD(+): NADPH ratio in cells will determine whether AKR1C2 makes 5alpha-DHT. In transient COS-1-AKR1C2 and in stable PC-3-AKR1C2 transfectants, 5alpha-DHT was reduced by AKR1C2. However, the transfected AKR1C2 oxidase activity was insufficient to surmount the endogenous 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity, which eliminated 3alpha-diol as androsterone. PC-3 cells expressed retinol dehydrogenase/3alpha-HSD and 11-cis-retinol dehydrogenase, but these endogenous enzymes did not oxidize 3alpha-diol to 5alpha-DHT. In stable LNCaP-AKR1C2 transfectants, AKR1C2 did not alter androgen metabolism due to a high rate of glucuronidation. In primary cultures of epithelial cells, high levels of AKR1C2 transcripts were detected in prostate cancer, but not in cells from normal prostate. Thus, in prostate cells AKR1C2 acts as a 3-ketosteroid reductase to eliminate 5alpha-DHT and prevents activation of the androgen receptor. AKR1C2 does not act as an oxidase due to either potent product inhibition by NADPH or because it cannot surmount the oxidative 17beta-HSD present. Neither AKR1C2, retinol dehydrogenase/3alpha-HSD nor 11-cis-retinol dehydrogenase is a source of 5alpha-DHT in PC-3 cells.

Synthesis and quantitative structure-activity relationship of 17 beta-(hydrazonomethyl)-5 beta-androstane-3 beta,14 beta-diol derivatives that bind to Na+,K(+)-ATPase receptor.

A series of 17 beta-(hydrazonomethyl)-5 beta-androstane-3, beta,14 beta-diol derivatives was synthesized and evaluated in the displacement of [3H]ouabain binding from Na+,K(+)-ATPase. The data were explored with multiple linear regression and partial least-squares to find possible quantitatives structure-activity relationships. Good correlations were found between binding to the receptor and van der Waals volumes or molar refractivities of the 17 beta-hydrazonomethyl substituents and pKa values of the compounds. Equivalent results were obtained using the proton affinity (calculated using MOPAC) of the hydrazone residues instead of experimental pKa. As basicity or related electronic factors of the substituents explain a significant portion of the observed changes in the activity, an ion-pair interaction between a carboxylate residue of the enzyme and the protonated 17 beta-hydrazonomethyl group, as postulated by Thomas, plays an important role in the interaction of the ligand to the Na+,K(+)-ATPase receptor.

Sugar/steroid/sugar conjugates: sensitivity of lipid binding to sugar structure.

[structure: see text]. Three steroids, each bearing a sugar on rings A and D, have been synthesized. Their effect on the "melting" behavior of a lipid bilayer depends on whether the sugar is glucose, galactose, or mannose. Packing constraints dictate how the lipid bilayer responds to the sugars.

Synthesis of 3-methyl-3-hydroxy-6-oxo-androstane derivatives.

3alpha,17beta-Dihydroxy-3beta-methyl-5alpha-androstan-6-one (1) and 3beta,17beta-dihydroxy-3alpha-methyl-5alpha-androstan-6-one (13) were prepared by the reaction of methylmagnesium bromide with the 3-ketosteroids. Structures and configurations in position 3 were determined by NMR spectra. Substitution in the position 6 influences the ratio of the products.

A simple, differential extraction method for the simultaneous direct radioimmunoassay of androgens and androgen glucuronides in human serum.

A method is described for the differential extraction of unconjugated androgens (testosterone and dihydrotestosterone) and glucuronidated androgens (androstane-3 alpha,17 beta-diol glucuronide and androsterone glucuronide) from human serum using solid-phase, gravity-flow extraction columns. In this method, 100-microL aliquots of serum are loaded onto the normal-phase columns, unconjugated androgens are eluted with ethyl ether, and glucuronides are eluted with ethyl ether containing 2% acetic acid. Glucuronide eluates are washed with 1% aqueous acetic acid to remove cross-reacting steroid sulfates. Assays of sera for the four steroids were performed using standard radioimmunoassay methodology, except for androsterone glucuronide. This steroid was assayed with a novel radioimmunoassay method that employees a tritiated, unconjugated androsterone tracer and an anti-dehydroepiandrosterone sulfate antiserum. The new method is well suited for the assay of conjugated and unconjugated steroids in large numbers of specimens, particularly where the sample volume is limited.

[Mass spectrometry of pipecuronium bromide and some related compounds]. / Tömegspektrometriás vizsgálatok a pipecuronium bromid és rokon vegyületei körében.

The mass spectral behaviour of the bis-quaternary salt pipecuronium bromide and some related Cn+ A-n type ammonium salts (n = 1, 2, 3) has been studied and compared. Under EI-MS conditions the evaporation of the samples preceded by in situ dealkylation led to formation of alkyl bromide and amine bases. It has been demonstrated that this technique, completed with quantitation of the relative amount of the decomposition products is applicable to get structure information about the basis part as well as the number and positions of the quaternary centres and the substituents at these centres. The FAB mass spectra of these mono-, bis- and this quaternary ammonium salts were found to be very interesting and provide direct structural information about the salt. They exhibit primary Cn+ intact cations and (Cn+ A-n + 1)+ single charged cluster ions and also fragments characteristic of partial structures at the quaternary centres.

[Determination of the protonation constants of pipecuronium bromide on the basis of optical rotation as a function of pH values]. / A pipecuronium bromid protonálódási állandóinak meghatározása optikai forgatóképességének pH függése alapán.

Pipecuronium bromide molecule contains two slightly basic nitrogen atoms in its two piperazine rings. Their pK values are quite similar to each other in consequence of their almost equivalent surroundings. Although the potentiometric titration used mostly for determination of pK values of organic compounds can be performed in this case too, but the polarimetric titration applied by us is more advantageous. Change of pH considerably influences the values of optical rotation; consequently, the curve obtained in this way is more utilizable than that of potentiometric titration. Protonation of two nitrogen atoms one after the other changes the optical rotation of the molecule in opposite direction. Consequently, a minimum can be seen on the curve of optical rotation versus pH value. The exact pK values were determined on the basis of the estimated parameters of a curve established corresponding to an equation and fitted to the measuring points. The sequence of deprotonation of nitrogen atoms could only be ascertained on the basis of the curves of polarimetric titration of compounds containing one piperazine ring; on the basis of the pK values and of the changing direction of optical rotation.