Synthesis, flow cytometric evaluation, and identification of highly potent dipyridamole analogues as equilibrative nucleoside transporter 1 inhibitors.

Dipyridamole (Persantine) is a clinically used vasodilator with equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2) inhibitory activity albeit less potent than the prototype ENT1 inhibitor nitrobenzylmercaptopurine riboside (NBMPR). Dipyridamole is a good candidate for further exploration because it is a non-nucleoside and has a proven record of safe use in humans. A series of dipyridamole analogues were synthesized with systematic modification and evaluated as ENT1 inhibitors by flow cytometry. Compounds with much higher potency were identified, the best being 2,6-bis(diethanolamino)-4,8-diheptamethyleneiminopyrimido[5,4-d]pyrimidine (13) with a K(i) of 0.49 nM compared to a K(i) of 308 nM for dipyridamole. Compound 13 is similar in potency to the prototype potent ENT1 inhibitor NBMPR (0.43 nM). For the first time, a dipyridamole analogue has been identified that is equipotent with NBMPR. The SAR indicated that diethanolamine substituted analogues were more active than monoethanolamine compounds. Also, free hydroxyl groups are not essential for activity.

Quenching of singlet molecular oxygen, O2(1Deltag), by dipyridamole and derivatives.

Dipyridamole (DIP) is known for its vasodilating and antiplatelet activity, exhibiting also a potent antioxidant effect, strongly inhibiting lipid peroxidation. This effect has been studied in mitochondria and a correlation between the DIP derivatives' structure, the ability to bind to micelles and biological activity has been suggested. In the present work, the quenching of singlet molecular oxygen, O(2)((1)Delta(g)), by DIP and RA47 and RA25 derivatives was analyzed in acetonitrile (ACN) and aqueous acid solutions. Laser flash photolysis excitation of methylene blue (MB) was made at 532 nm and monomol light emission of O(2)((1)Delta(g)) was monitored at 1270 nm. Bimolecular quenching constants in ACN are consistent with an efficient physical quenching, presenting values a bit lower than the diffusion limit (k(t) = 3.4-6.8 x 10(8) M(-1 )s(-1)). The quenching process probably occurs via reversible charge transfer with the formation of an exciplex. Calculation of DeltaG(et) associated with O(2)((1)Delta(g)) quenching corroborates with uncompleted electron transfer. In aqueous acid solutions (pH = 3.0), the k(t) values for DIP and derivatives are 20-fold smaller when compared with ACN. The electrochemical properties of DIP in ACN are characterized by two consecutive one-electron processes with half-wave oxidation potentials of 0.30 and 0.67 V vs saturated calomel electrode (SCE). However, in an aqueous acid medium, a single oxidation wave is observed involving a two-electron process (0.80 V vs SCE). Therefore, O(2)((1)Delta(g)) quenching is consistent with electrochemical data.

BIBW 22, a dipyridamole analogue, acts as a bifunctional modulator on tumor cells by influencing both P-glycoprotein and nucleoside transport.

BIBW 22, a phenylpteridine analogue of dipyridamole (DPM), enhanced vincristine cytotoxicity approximately 10 times more than DPM in a multidrug-resistant (MDR) KB V20C cell line. Using rhodamine 123 accumulation in KB V20C cells as an indicator of MDR phenotype, BIBW 22 was shown to be about 100 times more potent than DPM in inhibiting the MDR-associated efflux of rhodamine 123. Photolabeling of P-glycoprotein in KB V20C plasma membranes with 0.2 microM [3H]azidopine was strongly inhibited by 1 microM BIBW 22, indicating that this compound reverses the MDR phenotype by interfering with MDR-associated P-glycoprotein. In addition, BIBW 22 at 1 microM could also enhance the cytotoxicity of 5-fluorouracil in KB cells about 20-fold. Its potency in inhibiting nucleoside transport is 7-fold more potent than that of DPM. These results suggest that BIBW 22 is a potent bifunctional modulator which influences both P-glycoprotein and nucleoside transport in tumor cells. Potential use of this compound as a modulator of combination chemotherapy involving antimetabolites and drugs affected by MDR should be explored.

Chemometrics Tools in Detection and Quantitation of the Main Impurities Present in Aspirin/Dipyridamole Extended-Release Capsules.

Aspirin (ASP) and dipyridamole (DIP) in combination is widely used in the prevention of secondary events after stroke and transient ischemic attack. Salicylic acid is a well-known impurity of ASP, and the DIP extended-release formulation may contain ester impurities originating from the reaction with tartaric acid. UV spectral data analysis of the active ingredients in the presence of their main impurities is presented using multivariate approaches. Four chemometric-assisted spectrophotometric methods, namely, partial least-squares, concentration residuals augmented classical least-squares (CRACLS), multivariate curve resolution (MCR) alternating least-squares (ALS), and artificial neural networks, were developed and validated. The quantitative analyses of all the proposed calibrations were compared by percentage recoveries, root mean square error of prediction, and standard error of prediction. In addition, r(2) values between the pure and estimated spectral profiles were used to evaluate the qualitative analysis of CRACLS and MCR-ALS. The lowest error was obtained by the CRACLS model, whereas the best correlation was achieved using MCR-ALS. The four multivariate calibration methods could successfully be applied for the extended-release formulation analysis. The application results were also validated by analysis of the stored dosage-form solution, which showed a susceptibility of DIP esterification in the extended-release formulation. Statistical comparison between the proposed and official methods showed no significant difference.

Simultaneous Determination of Aspirin, Dipyridamole and Two of Their Related Impurities in Capsules by Validated TLC-Densitometric and HPLC Methods.

Aspirin (ASP) and dipyridamole (DIP) are widely used as a combination in pharmaceutical formulations for treatment of strokes. Many of these formulations are containing tartaric acid as an excipient (in DIP pellets formulation for sustained release), which increases the probability of formation of dipyridamole tartaric acid ester impurity (DIP-I). On the other hand, salicylic acid (SAL) is considered to be one of the synthesis impurities and a degradation product of ASP. In this work, two chromatographic methods, namely, TLC-densitometry and HPLC, have been established and validated for simultaneous determination of ASP, DIP, SAL and DIP-I. Good separation was achieved by using silica gel as stationary phase and toluene-methanol-ethyl acetate (2:3:5, by volume) as mobile phase in the case of TLC-densitometry and Zorbax ODS column with mobile phase consisting of phosphate buffer (pH 3.3)-acetonitrile-triethylamine (40:60:0.03, by volume) for HPLC. Influence of different organic solvents in mobile phase composition has been studied to optimize the separation efficiency in TLC densitometry. Moreover, factors affecting the efficiency of HPLC, like pH of the buffer used, organic solvent ratio in the mobile phase and flow rate, have been carefully studied using one variable at a time approach. Finally, the proposed methods were validated as per ICH guidelines.

Polymeric inhibitors of platelet aggregation. II. Copolymers of dipyridamole and related drugs with N-vinylpyrrolidone.

A study has been made of the behaviour of the drugs dipyridamole and mopidamol (RA 233) attached to poly(N-vinylpyrrolidone). The inhibitory activities towards platelet aggregation induced by ADP or platelet activation factor (PAF) in sheep plasma have been examined and found to exceed the activities of the parent drugs, by factors up to 20. At the same time the abilities of the polymer-bound drugs in potentiating prostaglandin-type anti-aggregatory activities are much lower than those of the unattached drugs. The observations are explained in terms of polymer adsorption on to the platelet membranes, producing a high surface concentration of the drugs with consequent high inhibitory action. Intracellular action, such as the inhibition of phosphodiesterase, is reduced by the difficulty experienced by the polymeric drug in passing through the membrane, so that potentiation of prostaglandin activity, particularly against PAF, becomes small. A terpolymer containing units of dipyridamole and the prostaglandin analogue 5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)hydantoin (245C) shows a degree of 'self-potentiation'.

Antioxidant effect of dipyridamole and its derivative RA-25 in mitochondria: correlation of activity and location in the membrane.

Dipyridamole (DIP), a coronary vasodilator, presents coactivator activity for a number of antitumor drugs as well as antioxidant activity in membrane systems. DIP and derivatives interact with membrane systems such as micelles, phospholipid monolayers and vesicles. The antioxidant effect of DIP and several derivatives upon iron-induced lipoperoxidation on mitochondria has been reported and a good correlation between the hydrophobicity and their protective effect was found (M.F. Nepomuceno et al., Free Radic. Biol. Med., 23 (1997) 1046-1054). In the present work an effort is made to better understand the role of DIP as inhibitor of Fe2+-induced lipid peroxidation in mitochondria. At low concentration, no significant effect on either state IV or state III respiration was found, discarding a possible direct interaction of DIP or RA-25 with the peripheral benzodiazepine receptor. The association constants for DIP and RA-25 in mitochondria were estimated, being 0.7 (mg/ml)-1 for DIP and 0.2 (mg/ml)-1 for RA-25. Oxygen consumption studies in the presence of FeSO4 showed that the antioxidant effect of DIP or RA-25 did not involved the initial step of Fe2+ oxidation. Our data strongly support the hypothesis that the antioxidant effect of both DIP and RA-25 is related to their partition in the lipid phase of the mitochondrial membrane and not to a specific interaction with membrane proteins. This protection may be due either to a direct inhibition of the propagation steps or a scavenger effect on the radicular species that would trigger the peroxidative process.

Binding of dipyridamole to phospholipid vesicles: a fluorescence study.

Binding and localization of the vasodilator and antitumor drug coactivator dipyridamole (DIP) and one of its derivatives, RA25, to phospholipid vesicles of DMPC (dimyristoylphosphatidylcholine) and DPPC (dipalmitoylphosphatidylcholine) was studied using fluorescence spectroscopy as well as quenching of fluorescence. The analysis of fluorescence data indicates that neutral dipyridamole binds to the phospholipids in their liquid crystalline phase with an association constant of 950 M(-1) and 1150 M(-1) to DMPC and DPPC, respectively. Protonation of DIP leads to a 3-fold reduction of the association constant. For the gel phospholipid phase, the binding is smaller (a factor of 2), independently of pH, suggesting that the more flexible lipid packing in the liquid crystalline phase facilitates the binding of the drug. The association constant of RA25 neutral form is considerably lower than for DIP, being around 295 M(-1). Fluorescence quenching with nitroxides TEMPO and stearic acid doxyl derivatives suggests the localization of DIP to be closer to the 5th carbon of alkyl chain. The quenching effect of 5-DSA below the lipid phase transition suggests that a strong static quenching may be operative. The quenching effect of 16-DSA is almost as great as that for 5-DSA below the phase transition, being even higher above the phase transition. This effect is probably due to the trans-gauche isomerization of the stearic acid nitroxide, making the encounter of its paramagnetic fragment with the DIP chromophore possible. Our data are consistent with DIP location close to the bilayer surface in the border of hydrophobic-polar heads interface which is similar to the data in micellar systems. In the case of RA25, the drug is in the outer part of the head group interface being much exposed to the aqueous phase and being significantly less accessible to the membrane nitroxide quenchers.

Adenosine transporters in chromaffin cells. Quantification by dipyridamol monoacetate.

Chromaffin cells from bovine adrenal medulla are a useful model to approach adenosine transport and metabolism in neural cells. Dipyridamol has been shown to be an adenosine transport inhibitor with high affinity. To quantify the adenosine transporters a labelled dipyridamol analogue, [14C]dipyridamol acetate, was synthesized. This compound had a Ki = 5.3 +/- 0.43 nM according to the Dixon method, and 4.58 +/- 0.46 nM when the receptor number molarity was taken into account showing, like dipyridamol, a non-competitive mechanism. The high-affinity receptors present in chromaffin cells showed a Kd = 6.8 +/- 0.8 nM and the receptor number was 630 000 +/- 40 000 per cell.

The effect of intravenous adenosine diphosphate on the number of circulating platelets in experimental animals: inhibition by prostaglandin E1, dipyridamole, SH-869 and VK-774.

The number of circulating platelets was monitored in anaesthetized animals by a continuous flow technique, using a Technicon Autocounter. Intravenous infusions of adenosine diphosphate (ADP) produced transient, dose-dependent falls in circulating platelet numbers in rabbits, dogs, rats, pigs and squirrel monkeys. The rat was the most sensitive of the species investigated. In the rabbit, the effect of a submaximal dose of ADP was inhibited in a dose-dependent manner by intravenous infusions of prostaglandin E1 (PGE1), dipyridamole, and two derivatives of dipyridamole (SH-869 and VK-774). The dose-response curves for PGE1, SH-869 and VK-774 were approximately parallel, whereas that for dipyridamole was considerably less steep. PGE1 was the most potent inhibitor, but the duration of action was very short. Dipyridamole and SH-869 produced inhibition of long duration. The duration of action of VK-774 was intermediate. All inhibitors produced marked and often long-lasting hypotension. The fact no inhibition of ADP effects could be demonstrated with dibenzyline and hexamethonium, which also produced marked hypotension of long duration, indicated that inhibition of the ADP effect by the four antagonists studied was not due to changes in blood pressure.

Effect of dipyridamole-like compound (R-E 244) on aggregation and cyclic AMP accumulation in human platelets.

The aim of this in vitro study was to evaluate the effect of a clinical concentration (2 microM) of dipyridamole alone or in combination with adenosine, 5'-N-ethyl-carboxamido-adenosine (NECA), or prostaglandin E2 on ADP-induced whole blood aggregability. Cyclic AMP accumulation in platelet-rich plasma was also evaluated. For comparison, R-E 244 (a dipyridamole analogue with low phosphodiesterase inhibition) was examined. In whole blood, dipyridamole (2 microM), but not R-E 244 (2 microM), had a small inhibitory effect (16% +/- 5%, p less than 0.01) on aggregation. Adenosine (1 or 5 microM) had an inhibitory effect that was enhanced by the combination with dipyridamole or R-E 244. Adenosine + dipyridamole produced an inhibition almost equal to that of adenosine + R-E 244. Dipyridamole and R-E 244 had no influence on the antiaggregatory effect of NECA and prostaglandin E2. In platelet-rich plasma, dipyridamole and R-E 244 did not enhance cyclic AMP, nor did they reinforce the cyclic AMP production during treatment with adenosine, NECA, and prostaglandin E2. Our results suggest that inhibition of the uptake of adenosine into red blood cells may play a more important role than the inhibition of phosphodiesterase as the pharmacological mechanism for the antiaggregatory effect of dipyridamole in clinical treatment.

Antitumor activity of an alkylating derivative of dipyridamole.

Synthesis of 2,6-Bis[bis(2-chloroethyl)amino]-4,8-dipiperidino-pyrimido [5,4-d]pyrimidine (DIP-C1) was carried out, and the new derivative showed cytotoxic activity comparable to other alkylating drugs on cultured P388 leukaemia cells and HeLa cells. The present paper reports the effects of DIP-C1 on respiration of Ehrlich ascites tumor cells and on survival of the mice implanted with Ehrlich ascites tumor cells. The compound showed a significant activity in both experimental models.

Synthesis and cytotoxic activity of a new alkylating derivative of dipyridamole.

Synthesis of 2,6-Bis[bis(2-chloroethyl) amino]-4,8-dipiperidinopyrimido[5,4-d] pyrimidine, a derivative of dipyridamole, was carried out by treating dipyridamole with thionyl chloride. Cytotoxic activity of this compound was assessed using cultured P388 leukaemia cells and HeLa cells. The compound inhibited the colony-forming ability of HeLa cells and showed a cytotoxicity on P388 cells comparable to that of other alkylating drugs (chlorambucil and CCNU).

Synthesis, crystal structure, and biological activity of a Pt-dipyridamole salt.

In the present paper we present data on the synthesis, crystal structure and biological activity of bis(dipyridamole) tetrachloroplatinate(II).dipyridamole.dihydrate, [dpmH]2 PtCl4.dpm.2H2O. The crystals are Triclinic P1 with a = 11.490(2) A, b = 13.630(2) A, c = 15.81(1) A, a = 100.97(2) degrees, beta = 100.89(3) degrees, gamma = 112.35(1) degrees, Z = 1, M = 1885.9, Dx = 1.46 g/cm3, MoK alpha (lambda = 0.71069 A), mu = 0.0184 mm-1, R = 4.4%, Rw = 5.0%, 3231 (1 > 2 sigma (I)). The structure is stabilized by a hydrogen-bonding network. It was observed that although dpm alone is not able to alter the electrophoretic mobility of pUC8 DNA forms, the synthesized Pt-dpm compound substantially modifies the DNA conformation since it significantly alters the electrophoretic mobility of nicked and closed circular forms of pUC8 DNA. However, the alteration in mobility of pUC8 DNA induced by this compound upon binding is lower than that induced by cis-DDP. The analysis of the antiproliferative activity of the Pt-dpm salt against MDA-MB 468 (breast carcinoma) and HL-60 (leukemia) human cancer cells showed that this compound has ID50 values of 0.87 microM and 0.65 microM, respectively. Interestingly, it was found out that although the dpm molecule does not present any significant antiproliferative activity, the ID50 values of Pt-dpm are about 3-fold and 7-fold lower than those of cis-DDP and K2PtCl4, respectively. Altogether the biological data suggest that in Pt-dpm a synergic effect between cation and anion is produced.

Dipyridamole and its derivatives modify the kinetics of the electron transport in reaction centers from Rhodobacter sphaeroides.

A well known vasodilator dipyridamole (DIP), 2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido[5,4-d]pyrim idine, and its derivatives have recently been shown as potential co-activators (modulators) in the phenomenon of multidrug resistance (MDR) in cancer therapy. They inhibit the specific function of a transmembrane P-glycoprotein responsible for the ex-flux of anti-cancer drugs from tumor cells. To clarify molecular mechanisms of the anti-MDR activity of DIP and its two derivatives, RA25 and RA47, we have studied their effects on electron transport in reaction centers (RC) from purple photosynthetic bacteria Rb. sphaeroides, using RC as a model system. Increasing concentrations of DIP and RA47 progressively accelerate the back electron transfer from the primary quinone acceptor QA to the bacteriochlorophyll dimer Bchl2 (Bchl2+ -QA- recombination). In the absence of o-phenantroline, when both quinone acceptors QA and QB are involved in the electron transport, RA47 is more effective than DIP. DIP stabilizes the electron on the secondary quinone acceptor QB, the effect manifested as the retardation of Bchl2+ -QB- recombination. Effects of RA25 are negligible in all cases. The drugs are proposed to change the electron transport affecting the RC structural dynamics and the stabilization of the electron on quinone acceptors through modification of H-bonds in the system.

Binding and location of dipyridamole derivatives in micelles: the role of drug molecular structure and charge.

Binding and localization of the vasodilator and antitumor drug coactivator dipyridamole (DIP) and of its three derivatives, RA14, RA47 and RA25 (DIPD), to cationic (cetyltrimethylammonium chloride), anionic (sodium dodecylsulfate), zwitterionic (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), and neutral (t-octylphenoxypolyethoxyethanol) micelles was studied using fluorescence, optical absorption and 1H NMR spectroscopy. The analysis of NMR, optical absorption and fluorescence data indicates that the depth of localization of the drugs in the micelles from the surface decreased in the order DIP > RA14 > RA47 > RA25. The binding constants for the neutral drug forms change in the same order in the range of 1400-3100 M-1 for DIP to 80-300 M-1 for RA25. This order is identical with the reported biological activity of DIPD. For the protonated drugs in zwitterionic or neutral micelles the binding constants are reduced by a factor of 20-75.

Antiviral action of dipyridamole and its derivatives against influenza virus A.

Dipyridamole proved to be active against influenza viruses A/England 42/72, A/Bangkok 1/79 and A/fowl plague (FPV). The antiviral activities assayed by various methods varied from 90-99 per cent. No inhibition was found against influenza virus B/Leningrad 235/74 in vitro. Three dipyridamole derivatives were significantly active in tissue cultures against influenza virus A/England 42/72 and A/FPV. In white mice infected with influenza virus A/England 42/72 dipyridamole administered orally showed a protection rate of 62.5 per cent.

Antiviral activity of dipyridamole derivatives.

Among 46 novel pyrimido [5.4-d] pyrimidine derivatives, 26 compounds were found to exhibit antiviral activity as revealed in a test programme against Mengo, Coxsackie B1, fowl plague, vaccinia and pseudorabies viruses, as concerns inhibition of plaque formation and of infectious virus yield. Attempts to disclose structure-activity relationships by discriminant analysis pointed to a possible importance of hydrophobic substitution for the antiviral effectiveness against Mengo virus of the derivatives investigated.

Blood tritium level as an estimate of soft tissue dose.

To test the blood tritium level as an estimate of free water tritium (FWT) and organically bound tritium (OBT) in human soft tissues, a tritiated amino acid mixture (TAA) and tritiated water (HTO) were injected or orally administered to mice. Relatively high tritium retention in the blood was found after TAA injection. This observation was explained by a low rate of clearance of non-volatile tritium components. The dose estimated for the soft tissues after TAA injection was larger than that estimated on the basis of tritium concentration in urine by a factor of 1.8-2.3. In both HTO and TAA injections, the blood tritium level gave a conservative estimate of the dose to soft tissues including active bone marrow. The accumulated dose to soft tissues after oral administration tended to be lower than that for intraperitoneal injection.

Antioxidant effect of dipyridamole (DIP) and its derivative RA 25 upon lipid peroxidation and hemolysis in red blood cells.

The antioxidant effects of dipyridamol (DIP), a coronary vasodilator, and its derivative RA-25 were compared in intact red blood cells (RBC) and in isolated ghost membranes. Both compounds are quite effective antioxidants in cumene hydroperoxide-induced lipid peroxidation of RBC, showing a much smaller effect for hydrogen peroxide oxidation. The antioxidant effect of DIP was considerably higher than that of RA25. For isolated ghost membranes, the apparent IC50 (the drug concentration that produces 50% inhibition of lipid peroxidation) in cumene hydroperoxide-induced peroxidation was 25 microM, while the maximum protective effect of RA-25 was around 30% in the drug concentration range of 50-100 microM. The drugs can protect the oxidative hemolysis induced by cumene hydroperoxide with a lower effect when the hemolysis is induced by H2O2. The significant antioxidant effect against damages induced by cumene hydroperoxide suggests that DIP, due to its lipophilic character, can interact with RBC membranes, and the protective effect is associated with the binding of the drug to the membrane. On the other hand, RA-25 is more hydrophilic than DIP, binds to the membrane to a smaller extent, and, for this reason, has a lower antioxidant effect.