(3CTAs) and Other Boronated Nucleosides for Boron Neutron Capture ...

Anti-Cancer Agents in Medicinal Chemistry, 2006, 6, 127-144

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3-Carboranyl Thymidine Analogues (3CTAs) and Other Boronated Nucleosides for Boron Neutron Capture Therapy Youngjoo Byun1, Sureshbabu Narayanasamy1, Jayaseharan Johnsamuel1, Achintya K. Bandyopadhyaya1, Rohit Tiwari1, Ashraf S. Al-Madhoun2, Rolf F. Barth3, Staffan Eriksson4 and Werner Tjarks1,* 1

College of Pharmacy, The Ohio State University, Columbus, OH, USA; 2Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Canada; 3Department of Pathology, The Ohio State University; 4 Department of Molecular Biosciences, Division of Veterinary Medical Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden Abstract: One category of boron neutron capture therapy (BNCT) agents that has received extensive attention during recent years is 3-carboranyl thymidine analogues (3CTAs). These molecules are phosphorylated to the corresponding 5´monophosphates by human thymidine kinase 1 (TK1), an enzyme that is up-regulated in dividing malignant cells. Thus, these phosphorylated molecules are selectively entrapped in tumor cells due to the acquired negative charge. This review will analyze design strategies applied for the synthesis of boron-containing nucleosides in general and in particular reference to 3CTAs. Results of biological studies with these molecules will be discussed.

Key Words: Boron neutron capture therapy, carboranes, nucleosides, thymidine kinase, 3-carboranyl thymidine analogues, 3CTAs, N5-2OH. INTRODUCTION Boron neutron capture therapy (BNCT) is a binary chemotherapeutic method for the treatment of cancers [1]. BNCT is based on the nuclear reaction between boron atoms and low energy thermal neutrons. Natural occurring elemental boron has two stable isotopes, namely boron-10 (10B) and boron-11 (11B). The abundant isotope is 11B (around 80%), however the most distinguishing property of 10B is its high neutron capture cross-section for thermal neutrons. Hence the reaction of neutrons with 10B yields two charged particles, a 4He nucleus and a 7Li nucleus, each of which is able to kill tumor cells due to its high linear energy transfer. For successful BNCT, a minimum of 20 µg of nonradioactive 10B per gram of tumor tissue is required. Another key requirement for the success of BNCT is the selective delivery of high concentrations of boronated compounds to tumor cells, while at the same time the boron concentration in the cells of surrounding normal tissue should be kept low to minimize the damage to normal tissue. NUCLEOSIDES AS BNCT AGENTS Boronated nucleoside prodrugs may be good candidates for BNCT because of their potential to be retained in rapidly dividing tumor cells after 5´-monophosphorylation by phosphorylating enzymes [2]. Cellular efflux of such 5´-monophosphates would be retarded due to the negatively charged phosphate moiety [2]. Cytosolic thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) are crucial in the biosynthesis of DNA precursors through the salvage pathways, and both *Address correspondence to this author at the College of Pharmacy, The Ohio State University, Columbus, OH, USA; Tel: +1 614 292-7624; Fax: +1 614 292-2435; E-mail: [email protected] 1871-5206/06 $50.00+.00

enzymes could be particularly suited to act as phosphorylating enzymes of boronated nucleoside prodrugs [3]. TK1 is pyrimidine specific, phosphorylating thymidine (Thd) and 2´deoxyuridine (dUrd) to the 5´-monophosphate derivatives, whereas dCK has a broad substrate specificity, phosphorylating 2´-deoxycytidine (dCyd), 2´-deoxyadenosine (dAdo) and 2´deoxyguanosine (dGuo) [3]. TK1 activity is very low in G1 and G0 cells, increases at the G1/early S boundary and reaches its maximum in late S phase/G2 [4, 5]. Consequently, TK1 activity is only found in proliferating cells, and thus it is widely distributed and expressed in malignant tumors [3, 6, 7], including those of the brain [8-11], pancreas [12], lung [13], head and neck [14], ovary [15, 16], cervix [17], colon [18], stomach [19], breast [20, 21], as well as in melanoma [22] and leukemia [23, 24]. TK1 activity has proven to be of prognostic value as a marker for proliferation in various types of cancer [25-27]. In contrast, dCK activity does not appear to be tightly cell-cycle regulated [3, 7], although significantly increased dCK activity has been demonstrated in cancerous S-phase cells after exposure to the antifolate pemetrexate [28, 29]. The activation of dCK appears to be associated with its major role in DNA repair-dependent nucleotide synthesis [28-31]. dCK is widely expressed both in normal tissues and tumors [3, 7, 12, 3235]. In various malignancies including brain tumors [8, 32], pancreatic tumors [12] and liver metastasis of colorectal cancer origin [35], increased dCK levels were observed compared with contiguous normal tissues. Several other nucleoside-phosphorylating enzymes are expressed in cancerous tissue [3, 7, 36, 37], but not much is known about their activity levels in tumor versus normal tissue. It should be noted that activity of nucleoside membrane transport is positively coupled with the degree of cell proliferation, activity levels of nucleoside kinases and inhibition of de novo pathways of nucleic acid synthesis [38-48]. This may also benefit the intracellular accumulation of therapeutic and diagnostic nucleoside © 2006 Bentham Science Publishers Ltd.

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prodrugs in addition to their entrapment as their monophosphates. TK1 and dCK play pivotal roles in antiviral and anticancer chemotherapy [2, 49-55]. The anti-HIV prodrugs zidovudine (AZT) and stavudine (d4T) [51, 52] as well as the anticancer prodrugs gemcitabine, cytarabine, clofarabine, cladribine, and fludarabine [2, 50, 53-55] undergo a stepwise activation via mono-, di-, and triphosphorylation by human nucleoside and nucleotide kinases. With the exception of AZT, the initial 5´-monophosphorylation by TK1 (AZT, d4T) and dCK (anticancer prodrugs) is the rate-limiting step in this activation process [2, 49-55]. The specific toxic effects towards lytic viruses or proliferative cancer cells are predominantly exerted by the active triphosphate forms of these prodrugs, which block DNA synthesis by inhibition of DNA polymerases or HIV reverse transcriptase and/or incorporation at 3´-terminal ends of DNA chains [2, 49-55]. In general, BNCT agents may have to function at significantly higher concentrations than conventional pharmaceuticals [56]. However, this principle probably does not apply to boronated nucleoside prodrugs. In clinical trials of leukemia using gemcitabine, cladribine, and cytarabine [57-62], combined intracellular concentrations of the corresponding mono-, di-, and triphosphates in leukemia cells were several hundredfold higher than plasma concentration of the parent drug, attaining intracellular levels of ~1 mM in the case of cytarabine triphospate [58]. Assuming that a cytarabine triphosphate derivative, substituted with a carborane cluster consisting of ten boron atoms, would show a similar biodistribution pattern, a highly selective accumulation of ~ 100 µg 10B per 109 or 1 gram of leukemia cells could be theoretically achieved. Such a level would be much higher than the required BNCT concentration minimum of 20 µg of 10B/g tumor. Similar observations have been made with AZT, which is effectively monophosphorylated by TK1. The second phosphorylation step, catalyzed by thymidine monophosphate kinase (TMPK), has been proved to be the rate-limiting step in its activation pathway [63]. AZTmonophosphate accumulates intracellularly in millimolar concentrations while AZT-triphosphate concentration levels only reach about 0.5% that of AZT-monophosphate [64, 65]. It has been suggested that boron-containing nucleosides and nucleotides require access to cell nuclei to be active as BNCT agents [56], and the hypothetical incorporation of boron-containing nucleoside triphosphates into DNA has spurred early efforts to synthesize boron-containing nucleoside prodrugs as BNCT agents [66, 67]. However, in antimetabolite chemotherapy, active triphosphates are also responsible for most of the severe toxic side-effects, due to cytotoxicity to proliferating cells mainly in bone-marrow and intestinal epithelium [2, 50, 53-55]. Triphosphates of boron-containing nucleoside prodrugs could exert a similar toxicity profile without neutron radiation when administered systemically at high doses, and it is uncertain if this toxicity can be tolerated. In recent years, several radiolabeled nucleosides, including FLT (3´-deoxy-3´-[18F]fluorothymidine), FMAU [1(2´-deoxy-2´- fluoro - β- D - arabinofuranosyl) - 5 - methyluracil ] and FIAU [1-(2´-deoxy-2´-fluoro-β-D-arabinofuranosyl)-5iodouracil] have been extensively evaluated as potential tumor-imaging agents [12, 68-79]. All of these agents are substrates of TK1. FLT is retained intracellularly predominantly

Byun et al.

in its mono- and triphosphate forms [72]. The latter is not a suitable substrate for DNA polymerase and less than 1% is incorporated into DNA [69, 72], while FMAU- and FIAU triphosphates are more effectively incorporated into DNA [73, 80-82]. Grierson et al. have evaluated the intracellular uptake profiles of FLT, FMAU, and FIAU, and they found that FLT was better retained in A549 cells than FMAU and FIAU. Further, retention of all three agents was predominantly dependent on TK1 activity and the capacity of TK1 to phosphorylate these nucleosides and not on incorporation of their triphosphates into DNA [72]. On the other hand, an in vitro study of various halogenated Thd analogues suitable for imaging demonstrated highest cytotoxicity for FIAU, which coincided with incorporation into DNA [73]. Other labeled Thd derivatives that were only phosphorylated without further incorporation into DNA were less toxic than FIAU [73]. The aforementioned studies suggest that design strategies for BNCT nucleoside prodrugs should focus on structures which enter tumor cells either by passive diffusion or via nucleoside membrane transporter and are selectively trapped intracellularly as anabolically and catabolically stable nontoxic 5´-monophosphates and/or 5´-diphosphates. The formation of 5´-triphosphates that could be incorporated into DNA is not necessary and may be associated with unacceptable systemic toxicity. NUCLEOSIDES SUBSTITUTED WITH CARBORANE CLUSTERS Carboranes Carboranes (C 2B10H12) have been considered as efficient boron moieties for BNCT agents because of their high boron content and stability [66, 83]. Nucleosides substituted with a carborane cluster are more advantageous for BNCT than those with one single boron atom due to the 10-fold increase in boron atoms. Synthetic versatility of carboranes is another benefit for their use as boron moieties. As shown in Fig. (1), carboranes are available in form of three geometrical isomers (o-, m- and p-carborane) based on the position of the two carbon atoms in the cage scaffold, and they can be utilized either as neutral closo carborane (C2B10H12) species or anionic nido carborane (C2B9H12-) species [84, 85]. Alkylation at carbon atoms in carborane cages can be accomplished easily via formation of reactive C-lithium salts using nbutyllithium [85]. Generation of the closo o-carborane can also be achieved by reacting acetylenic groups with decarborane (B10H14) [85, 86]. Unfortunately, the extreme hydrophobicity of neutral closo carboranes, which is similar to that of the adamantyl group [87], and hydrophilicity of anionic nido carboranes [66, 88], have limited the use of carboranyl nucleosides for preclinical or clinical applications. Among other aspects, this review will address to some extent recent approaches to improve the physicochemical properties of carboranyl nucleosides, in particular, the 3-carboranyl thymidine analogues (3CTAs). Nucleosides Substituted with a Carborane Cage at the Carbohydrate Portion Table 1 summarizes Thd and dUrd analogues modified with a carborane cage at the sugar portion. All compounds,

3CTAs and Other Boronated Nucleosides

Anti-Cancer Agents in Medicinal Chemistry, 2006, Vol. 6, No. 2 129

Fig. (1). Closo and nido structures of o-, m-, and p-carborane.

except 8-10, are 2´-, 3´, or 5´-O-linked nucleosides. The first nucleosides substituted at the ribose portion were reported by Soloway and Anisuzzaman [89]. However, O-linked nucleosides were found to be slightly unstable during reaction, workup and phosphoryl transfer assays [90]. Yan et al. recently reported carborane-containing Thds, in which ocarborane cage and sugar moiety were linked via carboncarbon bonds to improve hydrolytic stability (8-10) [90]. Biological evaluation of compounds 5-10 in phosphoryl transfer assays with recombinant TK1 showed that introduction of the carborane cluster at the 3´-position of Thd is tolerated by this enzyme. Recently, Lesnikowski et al. reported a general method for the synthesis of pyrimidine and purine nucleosides substituted with the carborane cluster at the 2´-position (compounds 11-12 in Table 1 and compounds 13-15 in Fig. 2) [91, 92]. These nucleosides were developed as building blocks for carborane-containing oligonucleotides in antisense oligonucleotide technology (AOT) and as boron carriers for BNCT [91, 92]. Pyrimidine Nucleosides Substituted with a Carborane Cage at the C-5 Position The first 5-substituted carboranyl nucleoside, 5-(1-ocarboranyl)-2´-deoxyuridine (16, CDU), was reported by Yamamoto et al. [93]. A major motivation for the strategy to introduce the carborane cluster at the 5-position of dUrd certainly stems from the fact that substitution of the 5-methyl group in Thd with e.g. iodine and bromine resulted in nucleoside prodrugs, such as 5-iodo- and 5-bromo-2´-deoxyuridine, which are excellent substrates for enzymes involved in DNA synthesis. Indeed, both compounds are able to substitute Thd during DNA synthesis to a high extent [94]. Another motivation was that the carborane cluster could deliver a high number of boron atoms to the tumor site for BNCT. CDU was synthesized from 5-iodo-2´-deoxyuridine in 5 steps as shown in Scheme 1. Briefly, the 3´-, and 5´-hydroxyl groups of 5-iodo-2´-deoxyuridine were protected with benzoyl groups. Reaction of the protected 5-iodo-2´-deoxyuridine with trimethylsilyl acetylene in the presence of palladium catalyst, followed by deprotection of trimethylsilyl group with tetrabutylammonium fluoride (TBAF), gave the 3´,5´-bis-O-benzoyl-5-ethynyl-2´-deoxyuridine. Reaction of the acetylenic group with decarborane (B10H14), followed by debenzoylation using sodium methoxide in methanol, provided 5-(1-o-carboranyl)-2´-deoxyuridine (CDU).

Schinazi et al. synthesized and evaluated a variety of CDU analogues with modified carbohydrate portion in form of D- and L-nucleosides and/or the α- and β-anomers (Table 2, 17-24) [95-99]. These carbohydrates included natural types [ribose (17), arabinose (18), xylose (19) and 2´,3´dideoxyribose (20)] as well as unnatural types [2´-deoxy-2´fluoro-arabinose ( 21), dioxolane (23) and oxathiolane (24)]. Rong et al. synthesized compound 25 (Table 3), containing a dihydroxypropyl group linked to the carborane cage, to improve the water-solubility of CDU [100]. CDU was evaluated as a potential candidate for AOT as well as for BNCT by Schinazi and his coworkers [67, 95-97, 101-105]. Conversion of CDU to its 5´-monophosphate form was found to be catalyzed by thymidine kinase 2 (TK2) but not by TK1 [106]. Phosphorylation of CDU in vitro was detected in CEM and PBM cells [67]. Cell culture studies have shown that CDU has a low toxicity in human CEM cells, human U-251 glioma cells and rat 9L glioma cells [67]. In CEM cells, cellular uptake of CDU appeared to be mediated by a nucleobase transporter [107]. Retention studies revealed that CDU accumulated in CEM cells in non-phosphorylated, monophosphorylated and in a nido-carboranyl form [107]. Treatment of 9L rat brain tumors in Fischer rats by neutron capture therapy after i.p. application of CDU was reported [105]. CDU was not toxic in this tumor/rodent model after i.p. administration of 150 mg/Kg. Two hours post i.p. administration of 30 mg/Kg and 150 mg/Kg of CDU, respectively, tumor boron concentrations of 2.3 µg (0.43 µg 10B) boron/g tissue and 7.4 µg (1.38 µg 10B) boron/g tissue, respectively, were found. Tumor to normal brain ratios were 11.5 and 6.8, respectively. Neutron irradiation was carried out 2 h after compound administration (i.p.). All animals of the untreated control group died within 28 days. About 50% of the animals survived for 32 days in the group receiving neutron radiation alone, whereas the CDU treatment increased the life span of the animals substantially to 55 days in the group receiving neutron irradiation and 30 mg/Kg of CDU, and to 38 days in the group receiving neutron irradiation and 150 mg/Kg of CDU. Recently, the possibility of treating prostate cancer with CDU by BNCT was evaluated [101]. In vitro cellular accumulation of CDU was studied in LNCaP human prostate tumor cells, and biodistribution profiles were investigated in male nude mice bearing LNCaP and 9479 human prostate tumor xenografts. CDU achieved high cellular concentrations in LNCaP cells, and up to 2.5% of the total cellular compound was recovered in the 5´-

130


Table 1.

Byun et al.

Nucleosides Substituted with a Carborane Cage at the Carbohydrate Portion O R1

HN

Z

O

O

Y #

R1

1

H

N O

X

X

Y

Z

OH

H

OH

H

CH2O B10H10

CH2O 2

H

B9H10 CH2O

3

H

OH

H B10H10

4

H

OH

OH B10H10 CH2O

5

CH3

H

H B10H10

CH2O 6

CH3

H

H

B9H10 B5H5

7

CH3

(CH2)3O

H

H

B5H5

8

CH3

H

H B10H10

9

CH3

H

H B10H10

10

CH3

H

H B9H10

(CH2)3OCH2O

11

H

OH

H

OH

H

B10H10 B5H5

12

(CH2)3OCH2O

H B5H5



Base

HO

O B5H5 OH O

O B5H5 NH2

NH2 N

N N

13

N

N

N

O

O NH

N

N

N

14

NH2

15

Fig. (2). Nucleosides substituted with a carborane cluster at the 2´-position.

O

O HN

HN O HO

N

a

O BzO

O

BzO

O

b

BzO

H

N

c

O

OBz O

O HN

HN N

O

N

OBz

HN O

HN

O

OH

O

Si(CH3) 3

O I

I

B10H10

d

O BzO

N O

B10H10 e

O HO

N O CDU

OBz a

OBz

OH

Reagents: (a) BzCl, pyridine; (b) HC≡CSi(CH3)3; (Ph3P)2PdCl2, CuI, Et3N; (c) TBAF, THF; (d) B10H14, CH3CN, toluene; (e) NaOCH3, CH3OH.

Scheme 1. Synthesis of CDU by Yamomoto et al.a

monophosphorylated form [101]. In vivo concentrations of CDU were similar in both LNCaP- and 9479 tumor xenografts [101]. The synthesis and biological evaluation of 5-tethered carboranyl pyrimidine nucleosides was mainly pursued by Soloway, Rong, and their associates [100, 108-110]. The rationale for the design of these compounds (Table 3, 26-30) was based on the observation that during the purification of thymidine kinase by affinity chromatography, increased binding affinity of the enzyme to the column matrix was achieved by introducing polymethylene spacers (~ 8-10 Å) between matrix and Thd [111]. To increase the water solubility of 5-carboranylalkyl-2´-deoxyuridine analogues, compounds 31-33 (Table 3), which contain a dihydroxypropyl group linked to the carborane cage, were synthesized [110]. Another group of 5-tethered carboranyl 2´-deoxyuridines are

5-carboranylalkylmercaptopyrimidines (34-37) [100]. Compound 38, in which the carborane cluster is linked through an acetylenic spacer to the 5-position of 2´-deoxyuridine, was synthesized by Kabalka et al. [112]. Three-Carboranyl Thymidine Analogues (3CTAs) Lunato was the first to attach a carborane cluster through a spacer to the N3-position of Thd, based on his conclusion that Thd was most likely linked through this position to the stationary phase in aforementioned affinity chromatography studies [106]. This novel nucleoside prodrug class was termed 3CTAs (3-carboranyl nucleoside analogues). Since the first report of 3CTAs in 1996, 3CTAs have been extensively studied as boron delivery agents for BNCT by Tjarks, Eriksson, Barth, and their coworkers [6, 8, 49, 108, 113119].

132


Table 2.

Sugar-Modified Derivatives of CDU O

O

HN

HN B10H10

O

B9H10

N

O

R

#

R

R HO

N

#

R HO

O

16 (CDU)

O F

21 OH

OH

HO HO

O

O

17

22

O

OH OH

HO

18

O HO

HO O

OH

HO

19

O OH

HO

HO

O

24

OH

20

O

23

S

O

Unmodified 3CTAs (Library A) The first reported library of 3CTAs is shown in Table 4 (Library A: 39-45) [108, 113]. In the 3CTAs of library A, the o-carborane cluster is linked through alkylene spacers of various lengths (n = 1-7) to N3 of Thd. Phosphoryl transfer assays with recombinant TK1 showed that 3CTAs with ethylene (40) and pentylene spacers (43) between carborane and Thd were better substrates for TK1 than homologues with methylene, propylene, butylene, hexylene, and heptylene spacers [114, 116]. Dihydroxypropyl-Substituted 3CTAs (Library B) Although 3CTAs of library A were good substrates for TK1, lack of water solubility due to the lipophilic closocarborane cage and the alkyl tethers prevented further evaluation as boron carriers for BNCT. Lunato, Ji, and their coworkers introduced a hydrophilicity-enhancing dihydroxypropyl group at the carborane cages of library A (Table 5, Library B: 46-51) [114]. The synthetic procedure for compound 49 (N5-2OH), which is representative for all 3CTAs of this library, is shown in Scheme 2. Acetic acid hept-6ynyl ester was reacted with decarborane (B10H14) to provide

Byun et al.

the corresponding carboranylpentyl acetate. The allyl group was then introduced via palladium-catalyzed reaction and subsequently dihydroxylated using osmium tetraoxide and 4methylmorpholine N-oxide (NMO). The vicinal diol was isopropylidene-protected, followed by removal of the acetate group using potassium carbonate in aqueous methanolic solution and subsequent tosylation of the hydroxyl group with p-toluenesulfonyl chloride. The carboranylpentyl tosylate was then reacted with Thd under basic condition followed by acidic deprotection to yield N5-2OH. Selective N-3 alkylation over O-4 alkylation or alkylation at hydroxyl groups of the 2´-deoxyribose moiety was accomplished under mild basic conditions (K2CO3, acetone/DMF, 50 oC, and 48 hrs). In analogy to library A, 3CTAs with ethylene (46) and pentylene (49, N5-2OH) tethers showed higher relative TK1 phosphorylation rates than their homologues. Overall, 3CTAs of library B exhibited higher TK1 phosphorylation and improved water solubility compared to those of library A [49]. Based on a detailed physicochemical and biological evaluation of 3CTAs of libraries A and B, N5-2OH was selected for extensive in vivo studies. Results of in vitro and in vivo studies of N5-2OH and other 3CTAs will be discussed in a subsequent chapter. Ethyleneoxide-Modified 3CTAs (Libraries C and D) Motivated by the biological results obtained for the 3CTAs in library B, Johnsamuel et al. designed and synthesized two libraries of hydrophilicity-enhanced ethyleneoxide-modified 3CTAs [118]. The first group of 3CTAs (Table 6, library C: 52-56) has ethyleneoxide spacers between the Thd scaffold and carborane cage, while the second group (Table 6, library D: 57-61) has a pentylene spacer between Thd and a carborane and ethyleneoxide units attached to the second carbon atom of a carborane cluster. All 3CTAs of libraries C and D were phosphorylated by TK1. Overall, 3CTAs of library C showed ~50% higher relative phosphorylation rates by TK1 compared with 3CTAs of library D. However, the lengths of the ethyleneoxide spacer did not significantly change the rate of phosphorylation by TK1 in either library, suggesting that a substantial portion of the side chains of ethyleneoxide-modified 3CTAs may be located outside of the active site of TK1 (Fig. 4). Zwitterionic Nido m-Carboranyl 3CTAs (Libraries E and F) As already indicated, the low water-solubility of several closo carboranyl Thd analogues, caused by the extreme lipophilicity of the carborane cage, has limited further evaluation as BNCT agents. The most positively-charged boron atom in the closo o- and m-carborane cluster can be removed under basic conditions to generate the corresponding hydrophilic nido carborane cages [84, 88, 120, 121]. However, anionic nido carboranyl nucleosides appeared to be too hydrophilic to cross various lipophilic membranes by passive diffusion [83]. Recently, Byun et al. reported the synthesis and biological evaluation of 3CTAs containing zwitterionic NH3+nido-m-carborane (Table 7, Library E: 62-67) and the corresponding NH2-closo-m-carborane (Table 7, Library F: 6873) [115, 116]. The rationale for the design and synthesis of the charge-compensated zwitterionic 3CTAs was to achieve


Table 3.


Pyrimidine Nucleosides Substituted with a Carborane Cage at the C-5 Position O R

HN O HO

N O

OH

#

R

#

R O

16

32

HO

B10H10

HO

25

33 OH

O OH

B10H10

O

HO OH

B10H10

O

B10H10

O S

26

34

O

B10H10

B10H10 O

27

S

35

O

B10H10

B10H10

S

O 28

36

O

B10H10

B10H10

O

S

29

37

O

B10H10

B10H10 H N

30

38 B10H10

O

B10H10 O

31

HO

O OH

B10H10

a balance between closo-carborane associated hydrophobicity and nido carborane associated hydrophilicity and, thereby, a balance between passive diffusion through lipophilic cell membranes and water-solubility. Overall, the zwitterionic nido 3CTAs of library E showed 5-30% higher relative phosphorylation rates by TK1 than the corresponding neutral closo 3CTAs of library F.

Miscellaneous 3CTAs (Library G) Library G contains hydrophilicity-enhanced 3CTAs with structural features that differ from those of 3CTAs in libraries A-F. Compounds 74-75 of library G are substituted with three or four hydroxyl groups (Fig. 3) and showed TK1 substrate characteristics similar to N5-2OH [122]. The anionic (76-77) and zwitterionic (78) 3CTAs of library G were found

134


Table 4.

Byun et al.

Unmodified 3CTAs (Library A)a

Dihydroxypropyl-Substituted 3CTAs (library B) a

Table 5.

O

O N

HO

N

N

n B10H10

O

N

HO

O

n

OH B10H10

O

OH

O

OH

OH

#

n

PRb

kcat/KMc

#

n

PRb

kcat/KMc

39

1

10 ± 2

NA

46

2

45 ± 3

31.9 ± 14.1

40

2

39 ± 3

27.4 ± 3.8

47

3

40 ± 4

16.4 ± 0.2

41

3

30 ± 5

13.1 ± 3.8

48 (N4-2OH)

4

21 ± 1

14.4 ± 6.0

42

4

13 ± 4

8.5 ± 0.5

43

5

41 ± 6

26.7 ± 0.6

49 (N5-2OH)

5

41 ± 5

35.8 ± 2.6

44

6

28 ± 5

5.2 ± 1.9

50

6

32 ± 8

24.9 ± 0.6

45

7

11 ± 3

1.8 ± 0.9

51

7

13 ± 7

1.7 ± 0.5

a

10 µM concentrations of 3CTAs were used. b PR (Phosphorylation Rate). Expressed relative to the rate of Thd, which was set to 100. c Thd was set to 100.

a

AcO

a

10 µM concentrations of 3CTAs were used. PR (Phosphorylation Rate). Expressed relative to the rate of Thd, which was set to 100. c Thd was set to 100. b

AcO

b B10H10

AcO

c

AcO

OH

B10H10

B10H10

d, e, f

OH

O N TsO

O B10H10

O

O

g N HO

O

B 10H10

O

O

OH O N

h N HO

OH B 10H10

O

OH

O

OH a

N5-2OH

Reagents: (a) B 10H14, CH3CN, toluene; (b) Tris(dibenzylideneacetone)dipalladium, bis(diphenylphosphino)ethane, allylethyl carbonate, THF; (c) OsO4, NMO, THF; (d) p-TsOH, CH 3C(OCH3)2CH3; (e) K2CO3, acetone, H2O; (f) p-TsCl, Et3N, DMAP, CH2Cl2, (g) Thd, K2CO3, DMF, acetone; (h) 17% HCl, CH3OH.

Scheme 2. Synthesis of N5-2OH by Lunato et al.a


Table 6.


Ethyleneoxide-Modified 3CTAs (Libraries C & D)a O

O

HO

O

O

N

n

N

R

N

R

O

N

HO

O

OCH3

n

O

O D

C OH

OH

#

Group

n

52

C

1

PRb

#

Group

n

B10H10

39 ± 5

57

D

2

B10H10

38 ± 6

B10H10

42 ± 4

B10H10

37 ± 7

R

R

PRb

B5H5

19 ± 3 B5H5 B5H5

53c

C

2

58

D

21 ± 5

3 B5H5 B5H5

54d

C

3

59

D

17 ± 2

4 B5H5

BH

55e

C

4

60

D

26 ± 3

3 B9H9

B5H5

56f

C

40 ± 13

3

61

D

3

B10H10

B5H5

26 ± 3

a

10 µM concentrations of 3CTAs were used. PR (Phosphorylation Rate). Expressed relative to the rate of Thd, which was set to 100. c kcat/KM value relative to Thd was 0.78. d kcat/KM value relative to Thd was 0.64. e kcat/KM value relative to Thd was 1.15. f kcat/KM value relative to Thd was 1.52. b

Table 7.

Zwitterionic Nido m-Carboranyl 3CTAs (Libraries E & F) a O

O BH

X

X NH3

N

NH2

B8H9 HO

N

B9H9

O

HO

O

N

b

O

O F

E OH

a

BH

N

OH

#

Group

X

PRb

#

Group

X

PR

62

E

-(CH2)2-

89 ± 1

68

F

-(CH2)2-

72 ± 5

63

E

-(CH2)3-

65 ± 4

69

F

-(CH2)3-

58 ± 3

64

E

-(CH2)4-

64 ± 3

70

F

-(CH2)4-

58 ± 5

65

E

-(CH2)5-

51 ± 2

71

F

-(CH2)5-

45 ± 3

66

E

-(CH2)6-

45 ± 2

72

F

-(CH2)6-

14 ± 2

67

E

-(CH2)2O(CH2)2-

75 ± 1

73

F

-(CH2)2O(CH2)2-

48 ± 2

10 µM concentrations of 3CTAs were used. PR (Phosphorylation Rate). Expressed relative to the rate of Thd, which was set to 100.

136


Byun et al.

O HO

O

HO

B5H5

OH B5H5

B5H5

N

B5H5 N N

OH

OH

OH

N

HO

O

O

OH

OH

O

O OH OH

75

74 (N3-PC-4OH) O

O N

HO

n

N

O

B9H10

B9H10

N

HO

O

OH

76: n=1 77: n=4 a

NH3 N O

O

OH

78

The TK1 phosphorylation rate of N3-PC-4OH relative to that of Thd was 50.

Fig. (3). Miscellaneous 3CTAs (library G).a

A.

B.

A. Subunit structure of tetrameric hTK1 (PDB ID: 1W4R) with TTP. B. Subunit structure of a hTK1 homology model with TTP. The homology model of hTK1 was constructed using Clostridium acetobutylicum thymidine kinase (PDB ID: 1XX6) [137] as a template with SWISS-MODEL (Version 36.0003). a: lasso domain, b: loop connecting the β2 and β3 strands, c: extended gap between lasso domain and the loop connecting the β2 and β3 strands.

Fig. (4). Crystal structure [135] and homology model of human TK1 with TTP in the active site.


to be poor substrates for TK1 compared with zwitterionic 3CTAs of library E [115]. Biological Evaluation of 3CTAs As shown in Tables 4-7, most of the synthesized 3CTAs have been screened enzymatically in phosphoryl transfer assays using recombinant human TK1 [100, 108, 113-119, 122-126]. Among the 3CTAs in libraries A and B, there is a good correlation between phosphorylation rates, which were measured with fixed 3CTA concentrations, and kcat/KM values, both of which are relative to those of Thd [49]. The kcat/KM value is an established measure of the efficiency of an enzyme for a specific substrate and is often referred to as the efficiency coefficient. In both libraries, 3CTAs having ethylene and pentylene spacers showed consistently better properties as enzyme substrates than those with methylene, propylene, butylene, hexylene, and heptylene spacers. The TK1 phosphorylation rates of 3CTAs in libraries C-F appeared to correlate proportionally with the hydrophilicity of these compounds, measured as log P values [49, 118] and HPLC RP18 retention times [116], rather than with structural features. N3-PC4OH (library G), which is substituted with four hydroxyl groups at the side chain of p-carborane, showed a comparable phosphorylation rate to N5-2OH [122]. In the case of ethyleneoxidemodified 3CTAs (libraries C and D) [118, 123], there was apparently a significant discrepancy between relative phosphorylation rates and kcat/KM values (Al-Madhoun et al. unpublished results). However, the enzymatic analysis of 3CTAs in libraries C-G is not complete and, therefore, a definitive conclusion of the enzymatic characteristics of 3CTAs in these libraries is currently not possible. N5-2OH had the highest relative kcat/KM value of all 3CTAs with 35.8% [49]. Thus, in the absence of endogenous Thd, N5-2OH appears to be a better substrate of TK1 than FLT (kcat/KM = 7.6% that of Thd) [127] and comparable to AZT (kcat/KM = 43.7% that of Thd) [127] as substrate for TK1. N5-2OH and other 3CTAs were not substrates of TK2 [49, 108, 113-119, 122-126], Drosophila melanogaster deoxynucleoside kinase (Dm-dNK) [6] and dCK [122]. Therefore, the selectivity of N5-2OH for TK1 may be comparable with that of acyclovir and ganciclovir for human herpes simplex virus thymidine kinase (HSV-TK) [51, 52, 128, 129]. The remarkable selectivity of ganciclovir for HSV-TK has, in part, been the basis for many preclinical studies and clinical trials of HSV-TK/ganciclovir suicide gene therapy [130]. N5-2OH was a moderate inhibitor of Thd phosphorylation with an IC 50 of 9.3 µmol/L (µmol N5-2OH/L per 0.5 µmol/L Thd) [49]. The kinetic pattern of inhibition was very complex, indicating negative cooperativity, and therefore the IC 50 of inhibition rather than KI was determined [49]. N5-2OH and its monophosphate were not substrates for thymidine phosphorylase (TPase) and deoxynucleotidase-1 (dNT-1) [49], respectively, which are principal intracellular deoxynucleoside and deoxynucleotide catabolizing enzymes. Compound 106 (Fig. 9) was effectively dephosphorylated by calf alkaline phosphatase [108]. TK1 has been identified more than 25 years ago as a potential target for therapeutic anti-cancer drugs [131, 132]. So far, however, it has mainly been utilized as a HIV prodrugactivating enzyme (AZT, d4T) [51, 52] which is probably due to the fact that, for a long time, TK1 was thought to have the


most stringent substrate specificity among all human and viral nucleoside kinases allowing only phosphorylation of native Thd/dUrd and, to a limited extent, analogues with minor modifications either at the 5-position (Cl, Br, I) or at the 3´-position (N3, F) [3, 6, 7, 133, 134]. Also, only very recently two crystal structures of human TK1 (hTK1) [135, 136], one of Ureaplasma urealyticum TK (UuTK) [136] and one of Clostridium acetobutylicum TK (CaTK) [137] have become available for computational structure-based drug design. The crystal structures of human hTK1 and UuTK were resolved as tetramers containing thymidine triphosphate (TTP) in the active site while the CaTK was crystallized as a dimer with ADP. The hTK1s and UuTK apparently represent “closed” TK forms while CaTK may constitute an “open” or “semi-open” TK form (Fig. 4). As in case of many other nucleoside- and nucleotide kinases [138-157], binding of substrates and the phosphate donor (ATP) in TKs appears to be associated with a large conformational change from an open unoccupied form, over a partially closed form involving substrate- or ATP binding, to a closed form binding both substrate and ATP. The amino acid sequence identities of both UuTK and CaTK with hTK1 are 36% and 39%, respectively (http://www.ncbi.nlm.nih.gov/blast/bl2seq/). Thus, a homology model of hTK1, presumably representing an “open” or “semi-open” form of hTK1, was generated using CaTK as a template. As shown in Figs. 4A and 4B, the difference in the distance between the loop connecting the β2 and β3 strands (b) and the “lasso” domain (a) in the crystal structure of hTK1 and the homology model of hTK1 is substantial. The coordinates of TTP bound to the active site of the hTK1 crystal structure were incorporated into the homology model of hTK1. In the homology model, N3 of TPP is clearly oriented to the huge gap (c) between the loop connecting the β2 and β3 strands and the “lasso” domain (a). We hypothesize that this gap allows effective binding of Thd derivatives which are substituted at the N3-position, such as 3CTAs, to the active site of TK1. A detailed study of the exact binding mode of 3CTAs to TK1 is, however, by no means trivial because of the large conformational changes that this enzyme undergoes upon substrate/ATP binding. Such a study will require computational molecular dynamics techniques. N5-2OH and other 3CTAs were taken up and retained in four different TK1-containing wild-type cell lines (F98 [8], L929 [8, 49], MRA 27 [8] and CEM) but only to a significantly lesser degree in the TK1 (-) deficient counterparts (L929 [8, 49], CEM [122]). In contrast, boronophenylalanine (BPA), currently used in clinical BNCT, accumulated in L929 wild-type and L929 TK1 (-) cells to the same extent [116]. Boron concentration levels in TK1-expressing wildtype cell lines after exposure to N5-2OH exceeded significantly those necessary for BNCT. The in vitro toxicity of 3CTAs was generally moderate to low [8, 49, 122]. N5-2OH was noticeably more toxic to TK1 expressing L929 wild-type cells than to the L929 TK1 (-) counterparts [8, 49, 122]. Significant differences in cytotoxicity between wild-type and TK1 (-) cell lines could not be observed for most other tested 3CTAs, indicating a significant metabolic activity of N5-2OH. Overall, the moderate in vitro toxicity of 3CTAs appeared to correlate with carborane-dependent lipo-

138


philicity rather than with nucleoside metabolism [8, 49, 122]. In previous studies of carboranyl polyamines, carboranedependent lipophilicity has been identified as the source of substantial in vitro and in vivo toxicity [158, 159]. The cause of this toxicity is still unknown, but in the case of 3CTAs of libraries B and G, it could be partially reduced by the attachment of hydrophilicity-enhancing hydroxyl groups [114, 122]. Subcellular boron distributions after incubation of T98G human glioma cells with N4-2OH (Library B) were analyzed by Secondary Ion Mass Spectrometry (SIMS) [8]. Depending on the incubation time (0.5, 2, and 8h), boron concentrations in the nucleus and in the cytoplasm ranged from 311 to 415 µg boron/g cells [8] with no significant difference between both cellular compartments. In a previous SIMS experiment, exposure of T98G glioma cells to 5BrdUrd resulted in the almost exclusive uptake of this nucleoside in the nucleus, indicating that 5-BrdUrd is converted to its triphosphate form and subsequently incorporated into DNA [160]. The homogenous subcellular boron distribution in T98G human glioma cells after exposure to N4-2OH appears to be consistent with the low to moderate in vitro toxicity observed for 3CTAs. This is an indication that it may only be phosphorylated to relative non-toxic phosphate metabolites, which are not incorporated into DNA. The first biodistribution studies with 3CTAs were carried out by Barth, Yang and their associates [8]. After intratumoral injection or convection enhanced delivery (CED), N52OH showed high and selective tumoral uptake and retention in three different TK1 expressing tumor/rodent models (intracerebral F98, intracerebral L929 and subcutaneous L929) as compared to significantly lower tumoral uptake and retention in the TK1 (-) counterparts (intracerebral and subcutaneous L929) [8]. When administered via intratumoral injection, N5-2OH did not show any significant toxicity in rats with intracerebral F98 glioma [8]. Following intratumoral injection, compound 63 (Library E) also showed high and selective tumoral uptake in mice with subcutaneous wildtype L929 tumors and significantly lower tumoral uptake in mice with subcutaneous L929 TK1(-) tumors [116]. Compound 63 showed significantly increased water-solubility compared with N5-2OH [116]. Both in vitro and in vivo results suggest that nucleoside phosphorylation by TK1 is a main factor for the uptake and retention of N5-2OH and other 3CTAs in tumor cells. However, we cannot exclude the possibility that cellular efflux mechanisms specific to wild-type and TK1 (-) tumor cells contribute to the observed uptake patterns.

Byun et al.

Recently, pilot neutron irradiation experiments with 10Benriched N5-2OH were carried out at the MIT nuclear research facility in three different tumor/rodent models, two with TK1-expressing wild-type tumors and one with a TK1 (-) tumor. Significant therapeutic responses were only observed in both models with TK1-expressing tumors (Barth et al. unpublished results). Miscellaneous Carborane-Substituted Nucleosides Carboranyl nucleosides that have not been addressed in the previous chapters include compounds 79 and 80 (Fig. 5). Compound 79, reported by Palmisano and Santagostino [161], is the only carboranyl nucleoside having a carborane cage at C-6 position of a pyrimidine base. The carboranyl purine nucleoside ( 80), having a carborane linked to the C-2 position of guanosine, was synthesized by Yamamoto et al. [162]. OTHER BORANATED NUCLEOTIDES

The first boron-containing nucleoside prodrug, 5(dihydroxyboryl)-2´-deoxyuridine (81, DBDU), shown in Fig. (5), was synthesized as a potential BNCT and anticancer agent by Schinazi et al. [163]. This compound contains a single boron atom in form of a boronic acid moiety. The synthesis of DBDU is shown in Scheme 3. The three oxygen atoms at the 4-, 3´-, and 5´-positions of 5-bromo-2´-deoxyuridine were protected with trimethylsilane. Selective hydrolysis of the 4-O-trimethylsilyl group was achieved by evaporation in hexane and, subsequently, in chloroform. Treatment with n-butyllithium and hexamethylphosphoric triamide (HMPT), followed by addition of excess tri-n-butyl borate, provided DBDU. DBDU has undergone evaluation as a potential BNCT agent [102]. About 1% replacement of Thd by DBDU was observed in cellular DNA in vitro. In vivo studies using a murine melanoma model showed ~ 1.8 % Thd replacement in the DNA of the tumor. Yamamoto et al. explored a more readily applicable bond formation between carbon and boron for the synthesis of dihydroxyboryl-substituted nucleosides using palladium catalyzed coupling reactions (82-83, Fig. 6) [162, 164, 165]. Introduction of a single boron atom to nucleosides was also accomplished in form of cyanoborane adducts with pyrimidine- (84-85) and purine nucleosides (86-87), as

OMe N

HN O O

N

N

AcO

O

N N

O

B10H10

OH

B10H10 OAc OAc

79

Fig. (5). Miscellaneous carboranyl nucleosides.

AND

Nucleoside Analogues Containing One Boron Atom

O

MeO

NUCLEOSIDES

80



OH O

O

OH

OH

OH

B OH

HN

O

B HN

HN

B OH

O HO

O

N

HO

O

O

N

HO

O

81 (DBDU)

82

83 NH2

N

N

HO

O

N

O

84

NH

N

HO

O

N

O

OH

NH2BH2CN

O

NCH2B

BH2CN

N

HN O

OH

OH OH

O

HO

O

OH

OH

N

NH2

OH 85

86

NH2 N

N

N

HO

BH2CN

Si O

O

N

O

O O

O

N N Me

OH

B 88

87

OH

Fig. (6). Nucleoside analogues containing a single boron atom.

O HN O HO

OTMS Br

N

N O

a

TMSO

O

OH

O Br

N

O

b

TMSO

O

OTMS

OLi

c

O TMSO

O Li

N

OH B

HN

O

N O

OTMS

N

Br

HN

OH

d, e, f O HO

N O DBDU

OTMS a

OH

Reagents: (a) HMDS, TMSCl; (b) hexane, followed by CHCl3; (c) n-BuLi, THF, followed by HMPT; (d) B(OBu)3; (e) CH3OH, H2O; (f) HCl.

Scheme 3. Synthesis of DBDU by Schinazi et al.a

140


reported by Spielvogel et al. [166, 167]. The cyanoborane adducts are hydrolytically stable due to charge compensation between the positively charged nitrogen atom and the negatively charged boron atom. Compound 88, a ribose nucleoside containing an unnatural benzoborauracil base, was synthesized by Soloway et al. [168]. Even though compound 88 is a protected nucleoside with silyl and isopropylidene groups at the sugar moiety, it is the only boronated nucleoside in which the carbonyl function in the Thd ring is replaced with a B-OH group. Nucleosides Containing more than Ten Boron Atoms Lesnikowski et al. reported the synthesis and biological evaluation of the first metallocarboranyl Thd analogues (8990, Fig. 7), which consist of eighteen boron atoms [169, 170]. The lipophilicities of compounds 89-90 were comparable with that of CDU. Thd monophosphate analogues substituted with four carborane clusters were reported by Wiessler et al. [171]. It was suggested that compound 93, a precursor of compounds 91-92, can be easily conjugated to a wide variety of biologically active molecules to yield potential BNCT agents. Boronated Nucleotides Nucleotides shown in Figs. 8 and 9 were mainly synthesized and evaluated as precursors for oligonucleotide synthesis or as test compounds for metabolic enzyme studies. They are strictly nucleotides and not nucleosides. However, in form of appropriate phosphate esters, all of the structures displayed in Figs. 8 and 9 have the potential to enter tumor cells and, in analogy to nucleosides, become entrapped through catabolic and anabolic enzymatic activities. It is for this reason why these structures are also presented in this review of boronated nucleosides.

Fig. (7). Nucleosides containing more than ten boron atoms.

Byun et al.

Hosmane et al. synthesized cyanoborane adducts of pyrimidine and purine nucleoside monophosphates (94-97, Fig. 7) [172]. Nucleoside boranomono-, di-, and triphosphates 98-100 have been studied extensively by Shaw and Spielvogel and their associates [173-179]. They were synthesized as probes for enzyme reaction involved in purine nucleotide synthesis and as potential boron carriers for BNCT [175]. Recently, Li and Shaw reported the synthesis of compounds 101-102, which are nucleoside boranomono-phosphoramidates containing the amino acids such as D-alanine (101) and D-tryptophan (102) [180, 181]. Both compounds were designed as nucleoside prodrugs for cancer chemotherapy and as potential BNCT agents. Most of the carboranyl nucleotides shown in Fig. (9) were synthesized for metabolic or stability studies. Compound 103 was identified as a major metabolite of CDU in cell culture experiments [182] and exposure of compound 106 to alkaline phosphatase led to the formation of its parental 3CTA (compound 42, Table 4) [108], while exposure of compounds 107 and 108 to 5´-deoxynucleotidase 1 did not lead to dephosphorylation [49]. Hosmane et al. reported the synthesis of a carborane cluster encompassed with two 2´-deoxyadenosine diphosphate units (compound 109, Fig. 9) for potential use as a BNCT agent [183]. SUMMARY AND FUTURE WORK Most 3CTAs are excellent substrates for TK1, a prime molecular target for BNCT as well as conventional chemotherapy of cancer and viral diseases. From a library of over forty 3CTAs, N5-2OH was selected as the current lead compound because of its superior biological properties. To our knowledge, N5-2OH is the first drug that exploits TK1 as a molecular target for cancer therapy. Major disadvantages of



O BH2CN EtO

P

NHBz NH

Base

O

N OR

O

N

R=Ac

O

O P

Base

O

HO

95

96

O

O

P

OH

BH3

HO

O

BH3

NHiBu

N R=Ac 97

O

Base

O

NH

N

N

R=Bz

O

P

N

O

N

N

R=Bz

94

HO

N

N

O

OEt

O

NHBz

O

P

O

OH

P

O O

P

OH

Base

O

O

BH3

OH

OH 98

OH

99

100

O NH O

R

P MeO

N

O

O

101: R=CH3 102: R=3-indolyl-CH2

O

BH3 O OAc

Fig. (8). Nucleotides containing one boron atom. O O

HN O

HN B10H10

O HO

P

O

O

O

HO

P

N

O

O

HN O

N O2N

O

O

OH

O

P

O

O

N O

OH

OH

OH O

OH O

OH B10H10

104

103

O

O N O HO

P

N

O

N

n O

B10H10

O

HO

O

P

OH

N

O

B10H10

O

O

OH

OH OH

OH

106: n=4 107: n=5

108 NHBz

OBz

N O

O N N

B10H10

105

N

O

P

O

OMe

O

O

P

P

OMe

B10H10

N NHBz

Fig. (9). Nucleotides containing a carborane cluster.

109

O O

OMe

P

N O

O

OMe OBz

N N

OH

142


Byun et al.

N5-2OH are its low water solubility, lack of information on its intracellular metabolism both in vitro and in vivo and its moderate capacity to compete with endogenous Thd at the active site of TK1. These shortcomings should be the basis for future drug design and drug evaluation studies. dCK is also a promising target for BNCT nucleoside prodrugs and increased efforts in the rational design, synthesis and evaluation of potential dCK substrates are highly desirable.

[25]

ACKNOWLEDGEMENTS

[30]

Y. Byun gratefully thanks Proctor & Gamble for financial support in form of fellowship. We thank Dr. Larry W. Robertson for helpful comments. This work was supported by the U.S. Department of Energy grant DE-FG0290ER60972 (W. Tjarks), the National Institutes of Health grant 1R01 CA098945-01 (R.F. Barth), and The Swedish Research Council (S. Eriksson).

[31]

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Received: August 24, 2005

Accepted: October 12, 2005

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