J. Med. Chem. 2009, 52, 4107–4110 4107 DOI: 10.1021/jm900350f
*To whom corresponce should be addressed. Phone: 819-564-5409. Fax: 819-564-5442. E-mail:
[email protected]. a Abbreviations: PDT, photodynamic therapy; ROS, reactive oxygen species; AMD, age-related macular degeneration; BRB, blood-retinal barrier; VEGF, vascular endothelial growth factor; Pc, phthalocyanine; STZ, streptozotocin; des-Arg9-BK, a kinin B1 receptor selective agonist.
presence of fluid and intravascular material in the subretinal space ultimately leads to the loss of visual function. Kinins and vascular endothelial growth factor-165 (VEGF165) have been shown to be among the mediators of plasma extravasation in the pathological retina.9-13 It has been demonstrated that in the retina, kinin B2 receptors are constitutively expressed, whereas B1 receptors appear to be up-regulated early in the development of diabetes.11,12,14 Other studies show that VEGF165, originally known as vascular permeability factor, is a key element in the breakdown of the BRB under pathological conditions, including diabetic retinopathy and AMD.9,10,15 Photodynamic therapy, using a benzoporphyrin derivative (verteporfin) as a photosensitizer, has been shown to be an efficient inhibitor of plasma extravasation from abnormal vessels and has been accepted for the treatment of AMD.16 During the past decades phthalocyanines (Pc) have been studied extensively as photosensitizers for PDT.17 A mixture of water-soluble sulfonated AlPc has been used in the clinic for over a decade, and it has been recognized for some time that the degree of sulfonation and the balance between hydrophobic and hydrophilic properties of the Pc permit modulation of the photodynamic response.18 More specifically, adjacently substituted, disulfonated Pc have been shown to possess the appropriate amphiphilic properties for optimal cell membrane penetration, resulting in high photodynamic activity against tumor cells in culture and experimental animal tumors.19 Further modifications of the disulfonated Pc to increase its lipophilic properties reduce water solubility, which complicates formulation for biomedical applications. Also, inherent to their classical procedure of preparation, disulfonated Pc give complex isomeric mixtures that are difficult to purify.20 We previously investigated the synthesis and properties of trisulfonated Pc substituted with a lipophilic group on the fourth nonsulfonated benzyl group and showed that such photosensitizers have amphiphilic and cell penetrating properties similar to those of the disulfonated Pc.21 These monofunctionalized trisulfonated Pc exhibit a typical Q band near 680 nm. In a search for photosensitizers with a wider range of activation wavelengths, which allows for variations in the depth of penetration of the therapeutic light, we prepared a new series of water-soluble, amphiphilic naphthobenzoporphyrazine dyes. These trisulfobenzonaphtho compounds are hybrid structures resembling phthalo- and naphthalocyanine molecules, displaying broad activation bands at red-shifted wavelengths in the range 650-750 nm. Such hybrid structures, substituted with an iodo atom, can be modified using palladium-catalyzed cross-coupling reactions, as previously shown for the analogous Pc-based photosensitizers.22 On the basis of these considerations, we prepared a series of mononaphthotrisulfobenzoporphyrazines. The general synthetic procedure for the preparation of the mononaphthotrisulfobenzoporphyrazines (5) is shown in Scheme 1. To obtain the indole protected iodonaphthoporphyrazine precursor 3, we used the mixed condensation reaction of 5-iodo-2,3-dicyanonaphthalene (2) and the indole protected 3,4-dicyanophenylsulfonyl (1) in the presence of zinc acetate at elevated temperature. The reaction mixtures contain mono- through tetraiodoporphyrazines, from which
r 2009 American Chemical Society
Published on Web 06/10/2009
Trisulfonated Porphyrazines: New Photosensitizers for the Treatment of Retinal and Subretinal Edema Johan E. van Lier,*,† Hongjian Tian,† Hasrat Ali,† Nicole Cauchon,† and Haroutioun M. Hassessian§,‡ †
Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences, Universit e de Sherbrooke, Sherbrooke, QC, J1H 5N4, Canada, and ‡Department of Ophthalmology, Universit e de Montr eal, Centre de Recherche Guy-Bernier, 5415 Boulevard de l’Assomption, Montr eal, QC, H1T § 2M4, Canada. Present address: NorVision Therapeutics Inc., 1 Place Ville-Marie, Montreal, QC, H3B 2C4, Canada Received March 19, 2009 Abstract: A new series of water-soluble, mononaphthotrisulfobenzoporphyrazines, bearing an alkynyl side chain of varying lengths on the naphtho ring, were prepared and tested for their efficacy to inhibit plasma extravasation when used as photosensitizers during photodynamic therapy (PDT) of the retina in the rat. The hexynyl substituted photosensitizer was the most potent, and was able to produce complete inhibition, at low doses of photosensitizer and light.
Photodynamic therapy (PDTa) of various medical conditions involves intravenous (iv) administration of a photosensitizer and, after a strategic period of time, the illumination of the targeted tissue with red light.1 This results in activation of the photosensitizer, leading to the formation of reactive oxygen species (ROS) and notably singlet oxygen.2 The ensuing oxidative stress results in localized cell death involving apoptosis or necrosis and vascular collapse if blood vessels are targeted during PDT.3 Although PDT has been most widely used for the treatment of light-accessible cancers,4 a particularly interesting application involves the treatment of wet-age-related macular degeneration (wet-AMD).5 AMD is the leading cause of blindness for people over the age of 50. The wet form involves rapid growth of blood vessels under the central retina, which most often do not fully develop, and leads to plasma extravasation. The retina is normally protected by a blood-retinal barrier (BRB), consisting of two spatially distinct monolayers of cells. The tight junctions between retinal capillary endothelial cells forms the inner retinal barrier, and cells of the retinal pigment epithelium form the outer barrier.6 The BRB keeps the retina dry and preserves its ionic balance. In addition, some circulating factors may be toxic to the retina and are kept out by the BRB. Hence, an intact BRB is essential for the normal function of the retina. The BRB is breached in many retinopathies involving vascular disorders including wet-AMD, diabetic retinopathy, exudative retinal detachment, Coats’ disease, and various other forms of macular edema.7,8 The
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Scheme 1a
Figure 1. Structure-activity relationship. (Left) Streptozotocin (STZ) diabetic rat model. Rats that were not administered Evan’s Blue showed no detectable plasma extravasation, n=3 (a). When rats were administered Evan’s Blue (45 mg/kg, iv), a low level of background plasma extravasation could be measured, n=3 (b). Rats injected with 65 mg/kg (ip) STZ, which did not become diabetic, demonstrated a degree of plasma extravasation that was not significantly (P>0.05) different from the control rats not injected with STZ, n=3 (c). Rats injected with STZ and that became diabetic showed a degree of plasma extravasation that was significantly (P< 0.05) greater than nondiabetic rats, n=3 (d). (Right) Doses were 0-1 μmol/kg 5a, 5b, 5c, AIPcS2a, and AIPcS4 was used as photosensitizer, followed 6 h later by a 15 min exposure to red light to verify the ability of photosensitizer to inhibit plasma extravasation in STZ-diabetic rats (n = 3).
a Compound. a: R = H. b: R = (CH2)3CH3. c: R = (CH2)6CH3. d: R = (CH2)9CH3. e: R = (CH2)13CH3.
the monoiodo analogue 3 is readily purified by silica gel column chromatography. The MS-FAB spectrum gives the expected molecular ion, and its UV-vis spectrum in MeOH shows a split Q band due to the combined naphthalocyanine and phthalocyanine nature of the macrocycle. The indole protected iodonaphtho porphyrazine 3 is the key intermediate for the synthesis of novel mononaphtho substituted trisulfobenzoporphyrazines. The palladium-catalyzed cross-coupling reaction of terminal alkynes with the iodonaphtho moiety (Sonogashira condition) provides an effective method for introducing an alkynyl chain onto the porphyrazine macrocycle to yield the indole protected derivatives 4a-e. The corresponding water-soluble trisulfobenzoporphyrazines 5a-e are obtained in high yield upon mild basic hydrolysis in sodium methoxide/methanol. Analytically pure samples for bioassays are obtained by reversed phase, medium pressure liquid chromatography. The purity of all new compounds was established to be >95% by analytical HPLC. The new porphyrazines are characterized by MALDI-HRMS and UV-vis spectroscopy in MeOH. The molecular ions of 5a-e were obtained as the free acids. All compounds show a broad absorption band between 660 and 730 nm with major absorption peaks at 703 nm (ε= 1.12 105 M-1 cm-1) and 681 nm (ε=1.21105 M-1 cm-1), confirming the presence of naphthalene and benzene rings.
All derivatives 5a-e show three closely eluting peaks on reversed phase HPLC representing the presence of more than one constitutional isomer. The retention times of 5a-e vary with the length of the alkynyl side chain, reflecting differences in their hydrophobic properties. Varying the length of the alkynyl side chain allowed us to establish structure-activity relationships between lipophilicity and the capacity to inhibit plasma extravasation from leaking vessels in the retina of diabetic rats after 15 min exposure to red light using a diode laser (673 nm, 14.4 mW/cm2, 13 J/cm2). The streptozotocin (STZ) induced diabetic rat model was used to test 5a-e for their capacity to inhibit plasma extravasation from leaking vessels in the diseased retina (Figure 1, left panel).11,12 Compounds 5a-c and reference AlPcS2a produced a dose dependent inhibition of plasma extravasation, whereas the porphyrazines 5d and 5e, bearing the longer lipophilic 12- and 16-carbon side chains, and the reference AlPcS4 showed no effect (Figure 1, right panel). At a 0.5 μmol/kg dose, followed 6 h later by a 15 min exposure to red light, 5b was able to completely inhibit plasma extravasation in the STZ-diabetic rat. None of the other compounds tested were able to produce such complete inhibition even at a dose as high as 1 μmol/kg. Although the responses to 1.0 μmol/kg 5a or 5b are not significantly different (P > 0.05), the IC50 values for the two dose response curves (5b IC50 is 0.06 ( 0.03 μmol/kg, whereas 5a IC50 is 0.45 ( 0.13 μmol/kg) are significantly different (P < 0.05) and thus show that 5b is clearly the most potent to inhibit plasma extravasation. Under the experimental conditions of this study, verteporfin showed IC50 = 0.62 ( 0.10 μmol/kg, demonstrating a 10-fold lower efficacy to inhibit plasma extravasation than 5b (Figure 5, Supporting Information). However,
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administration of the photosensitizer (5b) was not significantly (P>0.05) different from that observed in non-VEGF165 injected controls. In conclusion, our data supported by three different rat models show that trisulfonated porphyrazines bearing a lipophilic side chain, in conjunction with red light, are capable of completely inhibiting plasma extravasation from retinal vessels. The strongest response was observed with the derivative substituted with a hexynyl side chain, rendering this analogue of particular interest as a photosensitizer for the photodynamic treatment of retinal and subretinal edema.
Figure 2. PDT of leaking retinal vessels. Treatment with photosensitizer (5b) and light is required to produce inhibition of plasma extravasation in STZ-diabetic rats and STZ-diabetic rats that received 0.1 nmol of des-Arg9-BK (n = 3).
Acknowledgment. This work was supported by the Canadian Institutes for Health Research (CIHR Grant MOP37768) and the Jeanne and J.-Louis Levesque Chair in Radiobiology. We thank Dr. G. Cordahi for comments on this manuscript and Dr. D. Houde for technical assistance. Supporting Information Available: Experimental details, characterization data for all compounds, and details for in vitro and in vivo assays. This material is available free of charge via the Internet at http://pubs.acs.org.
References
Figure 3. Time dependent inhibition of VEGF165-evoked plasma extravasation in rat retinal vessels by PDT. A 5-6 h time interval between photosensitizer (5b) administration and light is required to completely inhibit plasma extravasation from retinal vessels (n = 4).
these experimental conditions may not be optimal for verteporfin and additional studies will be required to permit a thorough comparison between these two different classes of photosensitizers. In STZ-diabetic rats, injection of 0.1 nmol of des-Arg9-BK (a kinin B1 receptor selective agonist) evoked plasma extravasation that was significantly greater than that observed in non-des-Arg9-BK injected controls (Figure 2). In the absence of red light, the amount of plasma extravasation was not different from des-Arg9-BK-injected, STZ-diabetic control rats, which received only photosensitizer (5b). Similarly, a 15 min exposure to red light without prior injection of 5b did not affect plasma extravasation evoked by des-Arg9-BK. However, when a combination of 0.5 μmol/kg 5b and a 15 min exposure to red light applied 6 h postinjection of 5b was used, des-Arg9-BK was not able to evoke significant (P> 0.05) plasma extravasation (Figure 2). Photodynamic therapy reduced VEGF165-evoked plasma extravasation in a time dependent manner such that the effect of an exposure to 15 min of red light was greater with increasing time interval between injection of 5b and application of red light (Figure 3). The amount of plasma extravasation measured following red light exposure 5 h after
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