Poly(amidoamine) Dendrimers with Carbonic Anhydrase Inhibitory

Apr 7, 2015 - ... Science, King Saud University, P.O. Box 2455 Riyadh 11451, Saudi Arabia ... carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action wi...
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Poly(amidoamine) dendrimers with carbonic anhydrase inhibitory activity and antiglaucoma action Fabrizio Carta, Sameh M Osman, Daniela Vullo, Antonella Gullotto, JeanYves Winum, Zeid Abdullah Alothman, Emanuela Masini, and Claudiu T Supuran J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b00383 • Publication Date (Web): 07 Apr 2015 Downloaded from http://pubs.acs.org on April 12, 2015

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Poly(amidoamine) dendrimers with carbonic anhydrase inhibitory activity and antiglaucoma action

Fabrizio Carta,a Sameh M. Osman,b Daniela Vullo, a Antonella Gullotto,a Jean-Yves Winum, c Zeid AlOthman,b Emanuela Masini,d and Claudiu T. Supuran*b,e

a

Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della

Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy. b

Department of Chemistry, College of Science, King Saud University, P.O. Box 2455 Riyadh

11451, Saudi Arabia. c

Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS-ENSCM-Université de

Montpellier, Bâtiment de Recherche Max Mousseron, Ecole Nationale Supérieure de Chimie de Montpellier, 8 rue de l’Ecole Normale, 34296 Montpellier Cedex, France. d

Università degli Studi di Firenze, Dipartimento NEIROFABA; Sezione di Farmacologia, Via

G. Pierracini 6, Firenze, Italy. e

Università degli Studi di Firenze, Polo Scientifico, Dipartimento NEUROFARBA; Sezione di

Scienze Farmaceutiche e Nutraceutiche, Via Ugo Schiff 6, 50019 Sesto Fiorentino (Firenze), Italy.

Keywords: PAMAM dendrimers, sulfonamide; carbonic anhydrase, glaucoma.

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Abstract: Four generations of poly(amidoamine) (PAMAM) dendrimers decorated with benzenesulfonamide moieties were prepared by derivatizing the amino groups of the dendrimer with 4-carboxy-benzenesulfonamide functionalities. Compounds incorporating 4, 8, 16 and 32 sulfonamide moieties were thus obtained which showed an increasing carbonic anhydrase (CA, EC 4.2.1.1) inhibitory action with the increase of the number of sulfamoyl groups in the dendrimer. Best inhibitory activity (in the low nanomolar – subnanomolar range) was observed for isoforms CA II and XII, involved among others in glaucoma. In an animal model of this disease, the chronic administration of such dendrimers for 5 days led to a much more efficient drop of intraocular pressure compared to the standard drug dorzolamide.

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Introduction Dendrimers are repetitively branched molecules possessing symmetry around a central core and often adopting a spherical three-dimensional morphology, which makes them highly attractive for a large number of biotechnological and biomedical applications, such as catalysis, preparation of synthetic enzymes, drug delivery and gene transfection systems, contrast agents for magnetic resonance imaging or as optical sensors.1-9 The poly(amidoamine) (PAMAM) dendrimers, consisting of repetitively branched subunits of amide and amine functionalities, are the first dendrimer family to be prepared,1,10 thoroughly characterized and commercialized. Due to their interesting physico-chemical properties, versatility and ease of derivatization, they are widely used for many biomedical applications.10-13 Dendrimer-based drugs, as well as diagnostic/imaging agents, are emerging as promising candidates for various nanomedicine applications, mainly in oncology and as anti-inflammatory agents.12,13 The branched tree-like concentric layers of the dendrimers, referred to as 'generations', allow a precise number of various functional groups to be incorporated in the macromolecule, which thereafter may act as a platform for controlling the interactions with the receptor, enzyme or tissue. In addition, the particular three-dimensional architecture that the functionalized dendrimer generations adopt may be also exploited both for targeting nano-drugs to different tissues or cell compartments as well as for enhancing bioavailability of some drugs.10-13 Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes involved in many crucial physiologic processes, as they catalyze a simple but fundamental reaction, the reversible hydration of carbon dioxide to bicarbonate and protons.14-16 CAs are widespread in organisms all over the phylogenetic tree, with six genetic families encoding them, the α-, β-, γ-, δ-, ζ- and η-CA classes, an interesting example of convergent evolution at molecular level.16-21 In vertebrates, including humans, at least 15 different α-CA isoforms are known,14,15 which play various physiologic functions, such as pH

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and CO2 homeostasis, respiration and transport of CO2/bicarbonate, electrolyte secretion in many tissues/organs, biosynthetic reactions (e.g., gluconeogenesis, lipogenesis and ureagenesis in which bicarbonate not CO2 acts as a substrate for the carboxylation reaction), bone resorption, calcification, tumorigenicity, etc.14-20 In algae, plants and cyanobacteria CAs play an important role in photosynthesis, by concentrating CO2/bicarbonate nearby the RUBISCO enzyme complex, as well as in several other biosynthetic reactions.21 In diatoms δ- and ζ-CAs also play a crucial role in CO2 fixation but probably also in the SiO2 cycle.22 The 12 catalytically active human (h) isoforms (hCAs) can be grouped in four different subclasses depending on their subcellular localization: hCA I, II, III, VII and XIII are located in the cytosol, hCA IV, IX, XII and XIV are membrane-associated, hCAs VA and VB are found in mitochondria, whereas hCA VI is secreted in saliva and milk.14-16,20 The dysregulated activity of some of these enzymes leads to a variety of diseases, such as retinal/cerebral oedema (in which hCA I is involved); glaucoma, epilepsy, oedema, high altitude sickness (hCA II seems to be the main, but not the only isoform involved in these conditions); oxidative stress (hCA III); retinitis pigmentosa (hCA IV); obesity (hCA VA/VB); cariogenesis (hCA VI); epilepsy (hCA VII); tumorigenesis (hCA IX and XII; but hCA XII is also implicated in glaucoma); sterility (hCA XIII) and various retinopathies (in which hCA XIV is the main isoform involved).14-20 Thus, many CA isoforms are established drug targets, with their inhibitors having a range of diverse pharmacological applications. Indeed, sulfonamide CA inhibitors (CAIs) are in clinical use for the treatment of some of these conditions for decades, with the best known representatives being acetazolamide (AAZ) and dorzolamide (DRZ).14,15 Indisulam (IND) arrived in Phase II clinical trials as an anticancer agent to treat solid tumors,23 but its development has been stopped. More recently SLC-0111, another sulfonamide CAI, entered Phase I clinical trials as an antitumor/antimetastatic agent.24 Although sulfonamide CAIs are effective drugs for the management of many such conditions/diseases mentioned above, the large number of isoforms and their high affinity for the

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classical inhibitors of the AAZ/DRZ type, lead to side effects due to the inhibition of CAs in other tissues/ organs than the targeted one.14-18,25,26 This is the reason why various new approaches have been investigated for designing different CAIs, for example of the nanoparticle (NP) type.27 In fact the Au(0) NPs reported earlier by us which were decorated with aromatic sulfonamide functionalities, showed very interesting properties in inhibiting in vitro and in vivo the tumor/associated CA isoforms hCA IX and XII.27 Poly(amidoamine) (PAMAM)-type dendrimers have been particularly considered for their bioadhesive properties on the corneal surface as well as for their effects in prolonging the release of Pilocarpine for the treatment of glaucomatous rabbits.28 However such dendrimers were not yet investigated for the treatment of the same disease using CAIs, for which the isoforms hCA II and XII present in the ciliary processes within the eye are the main targets. This is the reason we underwent the drug design study herein reported.

Results and Discussion Compounds design and synthesis. As reported in Scheme 1 we investigated the use of the PAMAM cores for the designing of four generations of sulfonamide/based CAIs.

Scheme 1. here

4−Carboxybenzensulfonamide was converted to the activated ester 1 by coupling reaction with N−hydroxysuccinimide.29 This key intermediate was then reacted with the commercially available PAMAM aminoethyl derivatives (G0− −G3), leading thus to four generations of dendrimers incorporating a geometrically increasing number of benzenesulfonamide functionalities (Scheme 1). The dendrimers were purified by HPLC or GFC and characterized by MS techniques.

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Carbonic anhydrase inhibition. The key intermediate 1 and the four generation of dendrimers were assayed in vitro for the inhibition against the four physiologically relevant CA isoforms, the cytosolic hCA I and II as well as the transmembrane hCA IX and XII. Table 1 here The data obtained showed that whereas 1 is a rather ineffective CAI against all isoforms with KIs in the micromolar range (380-7800 nM), the dendrimeric derivatives G0− −G3 possess highly effective inhibitory properties against all enzymatic isoforms. In particular the inhibitory power against the hCA I showed a slight tendency to increase with the generation, from 24.1 nM of G0 to 10.8 − 10.5 nM for G2 and G3 respectively. A more evident correlation of the inhibitory properties with the increase of the PAMAM-CAI based generation was observed for hCA II: the first two generations were highly effective, low nanomolar inhibitors (KIs of 10.4 nM and similar to those of AAZ or DRZ for G0, and of 3.1 nM for G1). The inhibition pattern for hCA II continued thus making G2 a subnanomolar inhibitor (KI 0.93 nM) and G3 picomolar (KI 0.07 nM) (Table 1). In analogy the tumor associated hCA IX showed a similar inhibition profile to that of hCA I, being effectively inhibited in the low nanomolar range by the various generations of dendrimers reported. Moreover hCA XII, similarly to hCA II, showed higher affinity for G0-G3, being inhibited in the subnanomolar and picomolar range by the late generations G2 and G3 (Table 1). Multivalent effects were clearly observed for the generations of dendrimers explored in comparison with the monovalent inhibitor 1 (Table 1). This was particularly the case with hCA II and hCA XII where the decrease of the inhibition constants (KIs) from G0 to G3 registered potency improvement (rp) of 9142 for hCA II and of 6333 for hCA XII respectively. Normalized to the sulfonamide units,

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the relative potency (rp/n) values were of 285.7 for hCA II and of 197.9 for hCA XII respectively, thus indicating significant multivalency effects. Antiglaucoma activity.

The freshly prepared CAI-functionalized dendrimers G0-G3 were investigated for their ability to reduce the intraocular pressure (IOP) in a glaucomatous animal model25 by inhibiting the hCA II and XII enzymes present within the eye.20 As described in detail in the experimental section, a panel of anaesthetized rabbits was treated by injection in the vitreous of both eyes with a 0.1 mL hypertonic saline solution (5% in distilled water), followed by direct instillation in the conjuntival pocket of one drop of a 2 % solution/suspension of CAI of the classical type (DRZ) and dendrimer type. Then IOPs were constantly monitored for several days, by using a digital tonometer. (Figure 1).

Figure 1 here

The results reported in Figure 1 are expressed as ∆IOPs (obtained from the difference of IOPs between the drug-treated and non-treated eye) and normalized to the basal IOP values measured. It may be observed that G2 was the most effective IOP lowering agent among the investigated CAIs, having a potent lowering effect (6 mm Hg) just after 2 h post-administration, which is 33 % better than DRZ at the same time (4 mm Hg), and maintenance of the activity for the entire treatment period. On the other hand, dendrimers G1 and G3 were less effective than G2 or DRZ in lowering the IOPs at short times post-administration, but instead they showed to be quite effective (as much as G2) in the chronic treatment. G0 revealed to possess scarce ability in IOP lowering (2 mm Hg maximum for the first days), being ineffective at 3 days post-administration. Rationalizing these in vivo data is not straightforward but probably the penetration of the different dendrimers to the eye tissue is quite different, and this may be a plausible explanation of the observed differences. ACS Paragon Plus Environment

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Conclusions

In conclusion, this is the first study reporting dendrimers with CA inhibitory properties. By using the classical PAMAM core we prepared four generations (G0− −G3) of dendrimers incorporating benzenesulfonamide functionalities by a relatively facile chemistry approach. The obtained dendrimers showed excellent enzyme inhibitory properties and multivalent effects against four physiologically relevant CA isoforms, the human hCA I, II, IX and XII, some of which are established drug targets for antiglaucoma/antitumor agents. In vivo, in an animal model of glaucoma, some of the dendrimers were more effective compared to the standard drug dorzolamide in lowering the elevated intraocular pressure, which is feature of this disease. Among the tested compounds, derivative G2 showed the best performance in IOP lowering and maintenance over the time of this effect, which are important characteristics for a candidate drug for the treatment of glaucoma, a chronic disease. Such a behavior it is the result of an equilibrium between the physico-chemical and pharmacologic properties of this compound, thus confirming the additional value in using properly functionalized dendrimeric species for drug design purposes. As CAs are ubiquitous enzymes with many biomedical applications, our findings may be extended for targeting different other conditions in which these enzymes are involved, such as for example obesity, epilepsy, cancer, etc.

Experimental protocols

General. Anhydrous solvents and all reagents were purchased from Sigma-Aldrich, Alfa-Aesar and TCI. HPLC-grade water was prepared by purifying demineralized water in a Milli-Q filtration system (Millipore). All reactions involving air- or moisture-sensitive compounds were performed under a nitrogen atmosphere using dried glassware and syringes techniques to transfer solutions. ACS Paragon Plus Environment

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Nuclear magnetic resonance (1H-NMR,

13

C-NMR, DEPT-135, HSQC, HMBC) spectra were

recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d6. Chemical shifts are reported in parts per million (ppm) and the coupling constants (J) are expressed in Hertz (Hz). Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; brs, broad singlet; dd, double of doublets. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D2O. Analytical thin-layer chromatography (TLC) was carried out on Merck silica gel F-254 plates. HRMS data were obtained with a Q-TOF micro highresolution mass spectrometer with ESI (ESI+/ESI–). HPLC was performed by using a Waters 2690 Separation Module coupled with a PhotodiodeArray Dectector (PDA Waters 996) and as column a Nova-Pak C18 4µm 3.9x150 mm (Waters), silica-based reverse phase column. Sample was dissolved in acetonitrile 10% and an injection volume of 45µl was used. The mobile phase, at a flow rate of 1ml/min, was a gradient of water + trifluoroacetic acid (TFA) 0.1% (A) and acetonitrile + TFA 0.1% (B), with steps as follows (A % : B %): 0-10 min 90:10, 10-25 min gradient to 60:40, 26:28 min isocratic 20:80, 29-35 min isocratic 90:10. TFA 0,1% in water as well in acetonitrile was used as counter ion. Fractions of 0.5 ml each tube were collected, monitoring at 210nm. For GFC a Waters 600 pump and PDA as detector were used. 802A, dissolved with acetonitrile 25%, was loaded onto a XK column 16/40 (GE Healthcare), packed with Superdex 75 prep grad (GE Healthcare). Column was equilibrated with acetonitrile 25%, and eluted with the same solution at a flow rate of 0.5 ml/min. Fractions of 1.0 ml each tube were collected, monitoring at 210nm. All compounds obtained from HPLC and GFC methods previously described were >95 % pure.

Synthesis of 4-sulfamoyl-benzoic acid 2,5-dioxo-pyrrolidin-1-yl ester (1).29

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SO2NH2 O

OH N

SO2NH2 O

DCC

DMA dry HO

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O O

O N

O O

1

A solution of 4-sulfamoyl-benzoic acid (1.0g, 1.0eq) was dissolved in dry DMA (13 ml) and the solution was treated at -10°C with NHS (0.6g, 1.05 eq) and DCC (1.23g, 1.2 eq). The reaction was stirred under a nitrogen atmosphere and at the same temperature for 1 h and then 2 hrs at r.t. The precipitate formed (the urea derivative) was collected by filtration and the filtrated was concentrated under vacuo to give a residue that was purified by silica gel column chromatography eluting with 70% ethyl acetate/n-hexane v/v to afford the title compound as a white solid. 4-Sulfamoyl-benzoic acid 2,5-dioxo-pyrrolidin-1-yl ester (1): 69 % yield; silica gel TLC Rf 0.38 (Ethyl acetate/n-hexane 70 % v/v); m.p. 152 °C; δH (400 MHz, DMSO-d6): 3.00 (s, 4H), 7.72 (s, 2H, exchange with D2O, SO2NH2), 8.10 (2H, d, J= 8.7, Ar-H), 8.32 (2H, d, J= 8.7, Ar-H); δC (100 MHz, DMSO-d6) 26.5, 127.6, 128.1, 131.9, 150.8, 161.9, 171.1; m/z (ESI positive) 299.03 [M+H]+. Experimental data are in agreement with reported data.29

General procedure for the synthesis of sulfonamide containing dendrimers (G0-G3).

SO 2NH2

O O O N O

NH2 n PAMAM G0-G3

H N O

1

DMA, dry

SO2NH2

n

PAMAM G0-G3 n= 4; G0 = 8; G1 = 16; G2 = 32; G3

A solution of the commercially available poly(amidoamine)-dendrimer (G0-G3; 1.0 eq) in dry DMA (3.0 ml) was treated under a nitrogen atmosphere with 4-sulfamoyl-benzoic acid 2,5-dioxo-

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pyrrolidin-1-yl ester 1 (4.1 eq. for G0; 8.4 eq. for G1; 17.0 eq. for G2; 33.0 eq for G3). The solution was stirred at r.t. 24hrs for G0, 48hrs for G1-G3 then the solvent was evaporated under vacuo to give a residue that was triturated with Et2O and dried under vacuo to afford the desired compounds.

Mass Spectrometry ESI-MS spectra were recorded by direct introduction at 5µl/min flow rate in an LTQ linear ion trap (Thermo, San Jose, CA, USA), equipped with a conventional ESI source. The spectra were acquired in both positive and negative ion mode; the specific conditions used for these experiments were as follows: the spray voltage was 5 kV in both polarity; the capillary voltage were 49V in positiv ion mode and -15V in negative ion mode; the capillary temperature was kept at 280C. The sheath gas was set at 10 (arbitrary units), the sweep gas and auxiliary gas were kept at 5 (arbitrary units). Scan Time was 2 microscans and the maximun injection time was 50ms; ESI spectra were acquired using Xcalibur 2.0 (Thermo) the spectrum range was 150-500 m/z. Synthesis of derivative G0.

H2NO2S

NH O

O HN

H2NO2S

N 2

HN O NH O

G0

The title compound was obtained according to the general procedure previously reported as a light brown solid in 65 % yield. The crude compound was lyophilized previous purification by means of HPLC, monitoring at 210 nm. The major peak at 19 minutes was confirmed by GC-MS analysis. MS is reported within the supporting information section. ACS Paragon Plus Environment

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Synthesis of derivative G1.

O NH

SO2NH2

HN O

NH HN

O

SO2NH2 O

NH

N

O

NH

N

O

N 2 O

O HN O

SO2NH2 NH

HN NH

SO2NH2

O

G1

The title compound was obtained according to the general procedure previously reported as a light brown solid in 59 % yield. The crude compound was purified by several triturations from diethyl ether. MS is reported within the supporting information section.

Synthesis of derivative G2.

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SO2NH2

SO2NH2 O NH

HN

SO2NH2

O O N H

HN

HN

N

O

O

O NH

NH N

O

SO2NH2

NH

HN N

O

O

O

O

O

N H

O NH

N 2

NH O N

H N

HN O

NH

N NH NH O

O

SO2NH2

O O

N

HN H N

HN O O HN

SO2NH2

NH O SO2NH2

SO2NH2

G2

The title compound was obtained according to the general procedure previously reported as a pale yellow solid in 55 % yield. The crude compound was lyophilized previous purification by means of GFC, monitoring at 210 nm. The major peak at 70 minutes was confirmed by GC-MS analysis. MS is reported within the supporting information section.

Synthesis of derivative G3.

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H 2NO2S O H2NO 2S

H N

HN

O

H N

O

NH

NH

HN

O

O O

O

N N

H N

N O

O

N H

O

NH O

O

HN

O

HN

SO 2NH2

HN

N H H2NO2 S

N H

O

SO 2NH 2

NH O O

N O

H2NO2 S

N

HN O

NH N

HN

HN

HN

O

NH

O O

N O

NH

O

SO2NH 2

NH

N SO 2NH 2 2 NH

O H2NO 2S

N

SO2 NH 2

O O O HN

H 2NO2S

HN

O

O HN

NH O

HN

O

O

O NH HN

O

HN

O SO 2NH2

N H NH

HN H2NO2 S

HN

O

NH

O

N H

O

O H2NO 2S

H N O

N

N

SO2 NH 2

N H N

NH

HN

O

O

NH

O

H N

N

N

N

NH O

HN

O

O

SO2NH2

G3

The title compound was obtained according to the general procedure previously reported as a light brown solid in 42 % yield. The crude compound was purified by several triturations from diethyl ether. MS is reported within the supporting information section.

CA Enzyme Assay An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalysed CO2 hydration activity.30 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were ACS Paragon Plus Environment

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preincubated together for 1 h – 18 h (15 min for time drive assay in Hepes) at room temperature prior to assay, in order to allow for the formation of the E-I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3, as reported earlier,30and represent the mean from at least three different determinations. All CA isoforms were recombinant ones obtained in-house as reported earlier.31

Normotensive Rabbit IOP Lowering Studies. Male white New Zealand rabbits weighing 1500−2000 g were used in these studies. Animals were anaesthetized using Zoletil (Tiletamine chloride plus Zolazepam chloride, 3 mg/kg body wt, im) and injected with 0.1 mL hypertonic saline solution (5% in distilled water) into the vitreous of both eyes. IOP was determined using a tonometer (Tono-pen Avia Tonometer, Reichhert Inc. Depew, NY 14043, USA) prior to hypertonic saline injection (basal) at 0.5, 1.0, 1.5, 3, and 6 h thereafter. Vehicle (phosphate buffer 7.00 plus DMSO 2%) or drugs were instilled immediately after the injection of hypertonic saline. Eyes were randomly assigned to different groups. Vehicle or drug (0.50 mL) were directly instilled into the conjunctival pocket at the desired doses (2%).32

Acknowledgements. We thank The Distinguished Scientist Fellowship Program (DSFP) at KSU for funding this project. This work was also supported by an EU FP7 ITN Project (Dynano). The authors also express their gratitude to Prof. Alberto Mariani at Dipartimento di Chimica e Farmacia, Università di Sassari (Italy) for helpful discussion regarding this project.

Nonstandard abbreviations. CA, carbonic anhydrase; CAI, CA inhibitor; KI, inhibition constant; IOP, intraocular pressure; PAMAM, poly(amido)amine. Corresponding

Author.

Phone:

+39-055-4573005;

fax:

[email protected] (CTS).

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E-mail:

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References. 1. a) Tomalia, DA.; Naylor, AM.; Goddard, WA. Starburst Dendrimers: Molecular-Level Control of Size, Shape, Surface Chemistry, Topology, and Flexibility from Atoms to Macroscopic Matter. Angew. Chem., Int. Ed. Engl. 1990, 29, 138-175; b) Newkome, GR.; Moorefield, CN.; Vögtle, F. Dendritic molecules: Concepts, syntheses, perspectives, WILEY-VCH, Verlag GmbH, 1997. 2. Cai, X.; Hu, J.; Xiao, J.; Cheng, Y. Dendrimer and cancer: a patent review (2006-present). Expert Opin. Ther. Pat. 2013, 23, 515-529. 3. Soršak, E.; Valh, JV.; Urek, ŠK.; Lobnik, A. Application of PAMAM dendrimers in optical sensing. Analyst, 2015, 140, 976-989. 4. Bagul, RS.; Jayaraman, N. Multivalent dendritic catalysts in organometallic catalysis. Inorg. Chim. Acta, 2014, 409, 34-52. 5. Smith, DK.; Diederich, F. Functional Dendrimers: Unique Biological Mimics. Chem. Eur. J, 1998, 4, 1353-1361. 6. Baussanne, I.; Benito, JM.; Mellet, CO.; Fernandez, JMG.; Law, H.; Defaye, J. Synthesis and comparative lectin-binding affinity of mannosyl-coated β-cyclodextrin-dendrimer constructs. Chem. Commun., 2000, 1489-1490. 7. Roy, R.; Zanini, D.; Meunier, SJ.; Romanowska, A. Solid-phase synthesis of dendritic sialoside inhibitors of influenza A virus haemagglutinin. J. Chem. Soc., Chem. Commun., 1993, 1869-1872. 8. Wiener, EC.; Brechbiel, MW.; Brothers, H.; Magin, RL.; Gansow, OA.; Tomalia, DA.; Lauterbur, PC. Dendrimer-based metal chelates: a new class of magnetic resonance imaging contrast agents. Magn. Reson. Med., 1994, 31, 1-8. 9. Albertazzi, L.; Storti, B.; Marchetti, L.; Beltram, F. Delivery and subcellular targeting of dendrimer-based fluorescent pH sensors in living cells. J. Am. Chem. Soc., 2010, 132, 1815818167. 10. Esfand, R.; Tomalia, DA. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov. Today, 2001, 6, 427-436. ACS Paragon Plus Environment

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11. Svenson, S.; Tomalia, DA. Dendrimers in biomedical applications-reflections on the field. Adv. Drug Deliv. Rev. 2005, 57, 2106-2129. 12. Tomalia, DA.; Reyna, LA.; Svenson, S. Dendrimers as multi-purpose nanodevices for oncology drug delivery and diagnostic imaging. Biochem. Soc. Trans. 2007, 35, 61-67. 13. Kannan, RM.; Nance, E.; Kannan, S.; Tomalia, DA. Emerging concepts in dendrimer-based nanomedicine: from design principles to clinical applications. J. Intern. Med. 2014, 276, 579-617. 14. a) Alterio, V.; Di Fiore, A.; D'Ambrosio, K.; Supuran, CT.; De Simone, G. Multiple binding modes of inhibitors to carbonic anhydrases: how to design specific drugs targeting 15 different isoforms? Chem Rev. 2012, 112, 4421-4468; b) Neri, D.; Supuran, CT. Interfering with pH regulation in tumours as a therapeutic strategy. Nat. Rev. Drug Discov. 2011, 10, 767-777; c) Supuran, CT. Carbonic anhydrases: from biomedical applications of the inhibitors and activators to biotechnological use for CO(2) capture. J. Enzyme Inhib. Med. Chem. 2013, 28, 229-230. 15. a) Supuran, CT. Carbonic anhydrases: novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discov. 2008, 7, 168-181; b) Supuran, CT. Carbonic anhydrases as drug targets. Curr. Pharm. Des. 2008, 14, 601-602; c) Supuran, CT. Structure-based drug discovery of carbonic anhydrase inhibitors. J. Enzyme Inhib. Med. Chem. 2012, 27, 759-772; d) De Simone, G.; Supuran, CT. (In)organic anions as carbonic anhydrase inhibitors. J. Inorg. Biochem. 2012, 111, 117-129. 16. a) Harju, AK.; Bootorabi, F.; Kuuslahti, M.; Supuran, CT.; Parkkila, S. Carbonic anhydrase III: a neglected isozyme is stepping into the limelight. J. Enzyme Inhib. Med. Chem. 2013, 28, 231-239; b) Supuran, CT. Bacterial carbonic anhydrases as drug targets: toward novel antibiotics? Front. Pharmacol. 2011, 2, 34. 17. a) Smith, KS.; Jakubzick, C.; Whittam, TS.; Ferry, JG. Carbonic anhydrase is an ancient enzyme widespread in prokaryotes. Proc. Natl. Acad. Sci. U S A, 1999, 96, 15184-15189; b); Capasso, C.; Supuran, CT. Anti-infective carbonic anhydrase inhibitors: a patent and literature review. Expert Opin. Ther. Pat. 2013, 23, 693-704; c) Aggarwal, M.; Kondeti, B.; McKenna, R. ACS Paragon Plus Environment

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Anticonvulsant/antiepileptic carbonic anhydrase inhibitors: a patent review. Expert Opin. Ther. Pat. 2013, 23, 717-724. 18. a) Del Prete, S., De Luca, V.; Vullo, D.; Scozzafava, A.; Carginale, V.; Supuran, CT.; Capasso, C. Biochemical characterization of the γ-carbonic anhydrase from the oral pathogen Porphyromonas gingivalis, PgiCA. J. Enzyme Inhib. Med. Chem. 2014, 29, 532-537; b) Vullo, D.; Del Prete, S., Osman, SM., De Luca, V.; Scozzafava, A.; AlOthman, Z.; Supuran, CT.; Capasso, C. Sulfonamide inhibition studies of the δ-carbonic anhydrase from the diatom Thalassiosira weissflogii. Bioorg Med. Chem. Lett. 2014, 24, 275-279; c) Del Prete, S.; Vullo, D.; Osman, SM.; Scozzafava, A.; AlOthman, Z.; Capasso, C.; Supuran, CT. Sulfonamide inhibition study of the carbonic anhydrases from the bacterial pathogen Porphyromonas gingivalis: the β-class (PgiCAb) versus the γ-class (PgiCA) enzymes. Bioorg. Med. Chem. 2014, 22, 4537-4543; d) Del Prete, S.; Vullo, D.; Fisher, GM.; Andrews, KT.; Poulsen, SA.; Capasso, C.; Supuran, CT. Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum--the η-carbonic anhydrases Bioorg. Med. Chem. Lett. 2014, 24, 4389-4396. 19. a) Winum, J-Y.; Maresca, A.; Carta, F.; Scozzafava, A.; Supuran, CT. Polypharmacology of sulfonamides: pazopanib, a multitargeted receptor tyrosine kinase inhibitor in clinical use, potently inhibits several mammalian carbonic anhydrases. Chem. Commun. 2012, 48, 8177-8179; b) Di Fiore, A.; Maresca, A.; Supuran, CT.; De Simone, G. Hydroxamate represents a versatile zinc binding group for the development of new carbonic anhydrase inhibitors. Chem. Commun. 2012, 48, 8838-8840; c) Lock, FE.; McDonald, PC.; Lou, Y.; Serrano, I.; Chafe, SC.; Ostlund, C.; Aparicio, S.; Winum, J-Y.; Supuran, CT.; Dedhar, S. Targeting carbonic anhydrase IX depletes breast cancer stem cells within the hypoxic niche. Oncogene, 2013, 32, 5210-5219. 20. a) Carta, F.; Supuran, CT. Diuretics with carbonic anhydrase inhibitory action: a patent and literature review (2005 - 2013). Expert Opin. Ther. Pat. 2013, 23, 681-691; b) Supuran, CT. Carbonic anhydrase inhibitors: an editorial. Expert Opin. Ther. Pat. 2013, 23, 677-679; c) Scozzafava, A.; Supuran, CT.; Carta, F. Antiobesity carbonic anhydrase inhibitors: a literature and ACS Paragon Plus Environment

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patent review. Expert Opin. Ther. Pat. 2013, 23, 725-735; d) Masini, E.; Carta, F.; Scozzafava, A.; Supuran, CT. Antiglaucoma carbonic anhydrase inhibitors: a patent review. Expert Opin. Ther. Pat. 2013, 23, 705-716. 21. a) Moya, A.; Tambutté, S.; Bertucci, A.; Tambutté, E.; Lotto, S.; Vullo, D.; Supuran, CT.; Allemand, D.; Zoccola, D. Carbonic anhydrase in the scleractinian coral Stylophora pistillata: characterization, localization, and role in biomineralization. J. Biol. Chem. 2008, 283, 2547525484; b) Bertucci, A.; Moya, A.; Tambutté, S.; Allemand, D.; Supuran, CT.; Zoccola, D. Carbonic anhydrases in anthozoan corals-A review. Bioorg. Med. Chem. 2013, 21, 1437-1450. 22. Xu, Y.; Feng, L.; Jeffrey, PD.; Shi, Y.; Morel, FM. Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms. Nature, 2008, 452, 56-61. 23. a) Supuran, C.T. Indisulam – Eisai. IDrugs, 2002, 5, 1075-1079; b) Supuran, C.T. Indisulam an anticancer sulfonamide in clinical development. Expert Opin. Investig. Drugs 2003, 12, 283287. 24. https://clinicaltrials.gov/ct2/show/NCT02215850?term=SLC_0111&rank=1 25. Fabrizi, F.; Mincione, F.; Somma, T.; Scozzafava, G.; Galassi, F.; Masini, E.; Impagnatiello, F.; Supuran, CT. A new approach to antiglaucoma drugs: carbonic anhydrase inhibitors with or without NO donating moieties. Mechanism of action and preliminary pharmacology. J. Enzyme Inhib. Med. Chem. 2012, 27, 138-147. 26. Stiti, M.; Cecchi, A.; Rami, M.; Abdaoui, M.; Scozzafava, A.; Guari, Y.; Winum, J-Y.; Supuran, CT. Carbonic anhydrase inhibitor coated gold nanoparticles selectively inhibit the tumor-associated isoform IX over the cytosolic isozymes I and II. J. Am. Chem. Soc. 2008, 130, 16130-16131. 27. Ratto, F.; Witort, E.; Tatini, F.; Centi, S.; Lazzeri, L.; Carta, F.; Lulli, M.; Vullo, D.; Fusi, F.; Supuran, CT.; Scozzafava, A.; Capaccioli, S.; Pini, R. Plasmonic Particles that Hit Hypoxic Cells. Adv. Funct. Mater. 2015, 25, 316-323. 28. Vandamme, T. F.; Brobeck, L. Poly(amidoamine) Dendrimers as Ophthalmic Vehicles for Ocular Delivery of Pilocarpine Nitrate and Tropicamide. J. Controlled Release 2005, 102, 23–38. ACS Paragon Plus Environment

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29) Yeo, W-S.; Min, D-H.; Hsieh, RW.; Greene, GL.; Mrksich, M. Label-free detection of proteinprotein interactions on biochips. Angew. Chem. Int. Ed. 2005, 44, 5480–5483. 30. Khalifah, R.G. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow kinetic studies on the native human isoenzymes B and C J. Biol. Chem. 1971, 246, 2561-2573. 31. a) Maresca, A.; Temperini, C.; Vu, H.; Pham, N.B.; Poulsen, S.A.; Scozzafava, A.; Quinn, R.J.; Supuran, C.T. Non-zinc mediated inhibition of carbonic anhydrases: coumarins are a new class of suicide inhibitors. J. Am. Chem. Soc. 2009, 131, 3057-3062; b) Maresca, A.; Temperini, C.; Pochet, L.; Masereel, B.; Scozzafava, A.; Supuran, C.T. Deciphering the mechanism of carbonic anhydrase inhibition with coumarins and thiocoumarins. J. Med. Chem. 2010, 53, 335–344. 32. Krauss, AHP.; Impagnatiello, F.; Toris, CB.; Gale, DC.; Prasanna, G.; Borghi, V.; Chiroli, V.; Chong, WKM.; Carreiro, ST.; Ongini, E. Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2α agonist, in preclinical models. Exp. Eye Res. 2011, 93, 250−255.

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SO2NH2 SO2NH2

NHS, DCC DMA dry HO

O

O

O

O

N

O

1 O NH

H 2NO 2S

SO 2NH2

HN O

NH HN

SO2 NH 2

O

SO 2NH 2

NH O

O

O

NH

N

NH

N

O

HN PAMAM-G0 H 2NO2S

PAMAM-G1

N

N O N

DMA, dry

2

HN

O

O

O

DMA, dry

2

O

O

O

NH

O HN

O

SO 2NH2 NH

O

1

HN

G0

SO2NH 2

NH O

G1 SO 2NH2

SO2 NH 2 O NH

HN

SO 2NH2

O O N H

HN

HN

N

O

O

O NH

O

NH N

O

N SO2NH2

SO2NH2

NH

HN N H

O

O

O NH PAMAM-G2 N

O

O N

DMA, dry

O

2

O

NH O N

1

O

O

H N

HN O

NH

N NH

SO 2NH 2

O

NH O O

O

N

HN H N

HN O O HN

SO2 NH 2

NH O SO 2NH2

SO2NH 2

G2 H 2NO2S O H2NO 2S

H N

HN

O

O

H N

O

NH

NH

HN

O

O

O

O

N N

H N

NH O

O

HN

N

SO 2NH2

HN

N H H 2NO2 S

N H

SO 2NH 2

O

HN O

O

N H

O

NH O O

N O

H2NO2S

N

HN O

NH N

HN

HN

HN O

N SO 2NH2

O

NH

O O

NH

O

SO2NH 2

NH PAMAM-G3 N

O

O N

O

SO 2NH 2

DMA, dry 2

O NH

O

1

H2 NO 2S

N

SO2NH 2

O O O HN

H 2NO2 S

HN

O

O HN

NH O

HN

O

O

O

N

N

O NH HN

O NH

N H

O

O HN

O

O SO 2NH2

N H

HN NH

HN H2NO2 S

H N O

O H2 NO 2S

SO2NH 2

N H N

NH

HN

O

NH

O

H N

N

N

N

NH O

HN

O

O

SO 2NH2

G3

Scheme 1. Synthesis of activated ester 1: NHS 1.05 eq, DCC 1.2 eq, dry DMA, 2 hrs r.t.; and dendrimers G0-G3.

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Table 1: CA inhibition data against isoforms hCA I, II, IX and XII with compounds G0-G3 and acetazolamide (AAZ) and dorzolamide (DRZ) as standards, by a stopped-flow CO2 hydrase assay.30 _________________________________________________________ Compound KI (nM)* hCA I hCA II hCA IX hCA XII _________________________________________________________ 1 7800 640 475 380 G0 rp rp/n

24.1 323 80.9

10.4 61 15.3

34.7 13 3.4

9.3 40 10.2

G1 rp rp/n

12.0 650 81.2

3.1 206 25.8

20.5 23 2.8

1.1 345 43.1

G2 rp rp/n

10.8 722 45.13

0.93 688 43

8.6 55 3.45

0.94 404 25;26

G3 rp rp/n

10.5 742 23.21

0.07 9142 285.7

5.1 93 2.9

0.06 6333 197.9

AAZ 250 12 25 5.7 DRZ 50000 9 52 3.5 _________________________________________________________ * Errors in the range of ±5 % of the reported values, from three different assays. rp: relative potency=KI(1)/KI(Gn). rp/n: relative potency/number of sulfonamide units.

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Figure 1. Intraocular pressure (IOP) lowering with dorzolamide (DRZ) as standard drug and dendrimers G0-G3 at 2% concentration in a normotensive rabbit model after chronic administration for 5 days (four animals treated in each group< mean ±SD).25

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Table of Contents Graphic

SO 2NH2

O O O N O

NH2 n PAMAM G0-G3

H N O

1

DMA, dry

SO2NH2

n

PAMAM G0-G3

n= 4; G0 = 8; G1 = 16; G2 = 32; G3

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