Synthesis of an acridine orange sulfonamide derivative with potent carbonic anhydrase IX inhibitory action

Abstract Acridine orange (AO) a fluorescent cationic dye used for the management of human musculoskeletal sarcomas, due to its strong tumoricidal action and accumulation in the acidic environment typical of hypoxic tumors, was used for the preparation of a primary sulfonamide derivative. The rationale behind the drug design is the fact that hypoxic, acidic tumors overexpress carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA IX, which is involved in pH regulation, proliferation, cell migration and invasion, and this enzyme is strongly inhibited by primary sulfonamides. The AO-sulfonamide derivative was indeed a potent, low nanomolar CA IX inhibitor whereas its inhibition of the cytosolic isoforms CA I and II was in the micromolar range. A second transmembrane, tumor-associated isoform, CA XII, was also effectively inhibited by the AO-sulfonamide derivative, making this compound an interesting theranostic agent for the management of hypoxic tumors.


Introduction
Acridine orange (AO) is a heterocyclic derivative used as a nucleic acid-selective fluorescent cationic dye useful for cell cycle determination, as it interacts with DNA and RNA by intercalation within the double helix or by electrostatic attractions to the negatively charged phosphate groups, respectively. It also enters acidic compartments such as lysosomes, becoming protonated and sequestered inside that region of the cell/tissue. In such low pH conditions, the dye emits orange light when excited by blue light, being used to identify engulfed apoptotic cells 1-3 . In the last years, Kusuzaki's and Baldini's groups found that AO accumulates in the musculoskeletal sarcomas [1][2][3][4][5][6] . After illumination of the tumors loaded with AO with visible light (or irradiation with low-dose X-rays), the dye rapidly exerted a selective killing of the cancer cells [1][2][3][4][5][6] . Thus, AO in combination with surgery and photodynamic (PD) or radiodynamic (RD) therapies has been proposed as an alternative approach for the management of human musculoskeletal sarcomas, due to its strong tumoricidal action following excitation with a light source at 466 nm, with promising results being obtained mainly in Japan 1-6 .
Here, we report a study in which we designed a compound which might combine the affinity of AO for the tumors and the fact that many of them overexpress CA isoforms involved in tumorigenesis (e.g. CA IX and XII) 9,19 . The designed compound incorporates both AO and sulfonamide moieties, which have affinity for the CAs. The AO-sulfonamide agent reported here could represent a theranostic agent for the management of hypoxic tumors. atmosphere using dried glassware and syringes techniques to transfer solutions. Nuclear magnetic resonance ( 1 H NMR, 13 C NMR) spectra were recorded using a Bruker Advance III 400 MHz spectrometer in DMSO-d 6 . 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; triplet; q, quadruplet; dd, double of doublet. The assignment of exchangeable protons (OH and NH) was confirmed by the addition of D 2 O.
Synthesis of 3,6-bis(dimethylamino)-9-acridanthione 1 51 Acridine orange (3.0 g, 1.0 eq) and sulfur (0.43 g, 1.2 eq) were accurately mixed in a mortar and transferred to a pear shaped flask, which was preheated at 205 C in a sand bath. The flask was maintained open at this temperature for 30 min. The dark purple residue formed was cooled down to r.t. and re-crystallized from DMF to afford the title compound 1 as a dark brown solid.

Carbonic anhydrase assay
A stopped-flow method 53 has been used for assaying the CA catalyzed CO 2 hydration activity with Phenol red as indicator, working at the absorbance maximum of 557 nm, following the initial rates of the CA-catalyzed CO 2 hydration reaction for 10-100 s. 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 (0.01 mM) were prepared in distilleddeionized water with 5% DMSO and dilutions up to 0.1 nM were done thereafter with the assay buffer. The Inhibition constant (K I ) was obtained by considering the classical Michaelis-Menten equation which has been fitted by non-linear least squares by using PRISM 3. All CA isozymes used in the experiments were purified recombinant proteins obtained as reported earlier by our group [54][55][56][57][58][59][60][61][62][63][64] .

Results and discussion
The rationale of this work was to design a hybrid molecule which may show enhanced affinity for tumor cells due to the presence of both AO and sulfonamide moieties in its molecule. In addition, these hybrid compounds may retain the fluorescent properties of AO, and thus could be useful for PD and/or RD therapies, but these aspects are not investigated in this paper.
The synthetic procedure for obtaining the hybrid is shown in Scheme 1. Acridine was reacted with elemental sulfur, leading to the thiol/thione derivative 1, which was methylated at the sulfur atom with methyl iodide, leading to the key methylthio-intermediate 2 51,52 .
Reaction of 2 with amines, such as phenethylamine or 4-aminoethylbenzensulfonamide, led to the heterocyclic amines 3 and 4, one incorporating the primary sulfonamide moiety (compound 3) and the other one possessing exactly the same scaffold as 3, but without the sulfonamide group (compound 4), Scheme 1. Derivative 4 is in fact useful as a negative control in the enzyme inhibition experiments reported here (see later in the paper). Since all the purification procedures used to isolate the derivative 3 as the free base were unsuccessful, we converted the in situ formed free-base 3 (TLC monitoring) to its corresponding perchlorate salt, which precipitated at 0 C within 1 h to afford the desired compound in good yield and excellent purity. The use of perchlorate salts for the isolation as well as purification of small molecule compounds as CAIs is well reported, also in consideration that the inorganic counterion does not have any effect on the CAs activity [7][8][9] .

Carbonic anhydrase inhibition
We assessed the CA inhibitory activity of compounds 3 and 4, using the clinically used drug acetazolamide (5-acetamido-1,3,4thiadiazole-2-sulfonamide, AAZ) as positive control, for the inhibition of four human (h) isoforms, hCA I and II (cytosolic, widely distributed enzymes) as well as hCA IX and XII (transmembrane, tumor-associated enzymes) - Table 1.
Data of Table 1 show that AO and its non-sulfonamide derivative 4, did not inhibit any CA isoform investigated here, whereas sulfonamides 3 and AAZ acted as inhibitors. The AO-sulfonamide hybrid 3 was a micromolar inhibitor of the cytosolic isoforms hCA I and II, with inhibition constants of 1.65-7.68 mM, whereas the transmembrane, tumor-associated isoforms hCA IX and XII were much more effectively inhibited, with inhibition constants of 4.9-9.1 nM. Acetazolamide was a medium potency hCA I inhibitor and a highly effective one for the remaining three isoforms, with inhibition constants of 5.6-25.1 nM (Table 1). These data show that the AO-sulfonamide hybrid 3 is a tumor-associated CA isoforms selective inhibitor, making it a valuable candidate for theranostic applications in the field of hypoxic tumors.

Conclusions
We report here the synthesis and enzyme inhibition data of acridine orange, a fluorescent cationic dye used for the management of human musculoskeletal sarcomas, as well as those of a new compound based on the AO scaffold on which a sulfonamide zinc-binding moiety was introduced by using an original procedure. Due to the strong tumoricidal action and accumulation in the acidic environment (typical of hypoxic tumors) of AO, we designed the hybrid in such a way as to incorporate an additional functionality which may lead to interaction with hypoxic tumors, many of which overexpress CA IX and XII. Such enzymes are in fact involved in pH regulation, proliferation, cell migration and invasion in many cancer types. The reported AO-sulfonamide derivative was indeed a potent, low nanomolar CA IX inhibitor whereas its inhibition of the cytosolic isoforms CA I and II was in the micromolar range. A second transmembrane, tumor-associated isoform, CA XII, was also effectively inhibited by the AO-sulfonamide derivative, making this compound an interesting theranostic agent for the management of hypoxic tumors.