`
`J. Med. Chem. 2006, 49, 452-455
`
`masking group on the phosphate moiety, which has led to a
`further increase in cytostatic activity against a panel of cancer
`cell lines.7a,b
`
`The present work describes the synthesis and biological
`evaluation of a new family of BVdU ProTides, combining the
`modifications on the ester and amino acid moieties with the
`use of the new aryl group on the phosphate, naphthyl. The main
`objectives of this study were (i) to enhance the cytostatic potency
`and (ii) to further investigate the structure-activity relationship
`within BVdU phosphoramidates.
`The compounds involved with our study are shown in Scheme
`1. The target ProTides were all prepared in one step from BVdU
`using the phosphorochloridate chemistry we have extensively
`described.8,9
`The synthesis involves phosphorylation of 1-naphthol with
`phosphorus oxychloride, followed by the coupling with different
`esterified amino acid salts to give naphthyloxy-phosphorochlo-
`ridates, which were generally purified by flash chromatography
`and then coupled with BVdU in the presence of 1-methylimid-
`azole (NMI). Yield of the final purified compounds resulted in
`a 6.5-49.7% range. The general synthetic route is reported in
`Scheme 2.
`Due to the stereochemistry at the phosphorus center, the final
`compounds isolated from the coupling to BVdU are diastereo-
`isomers. The confirmation of the presence of two diastereo-
`isomers is shown by P-31 (two signals, 1:1 ratio), H-1, and C-13
`NMR.
`Compounds 1-13 were evaluated for their cytostatic activity
`against a panel of different tumor cell lines in vitro: MDA
`
`Scheme 1. Structures of 1-13
`
`Novel Potential Anticancer Naphthyl
`Phosphoramidates of BVdU: Separation of
`Diastereoisomers and Assignment of the Absolute
`Configuration of the Phosphorus Center
`
`Costantino Congiatu,† Andrea Brancale,†
`Malcolm D. Mason,‡ Wen G. Jiang,‡ and
`Christopher McGuigan*,†
`Welsh School of Pharmacy, Cardiff UniVersity,
`King Edward VII AVenue, Cardiff CF10 3XF, UK, and
`Wales College of Medicine, Cardiff UniVersity,
`Heath Park, Cardiff CF14 4XN, UK
`ReceiVed October 5, 2005
`
`Abstract: We have previously reported our SAR optimization of the
`anticancer agent thymectacin. Tuning of the parent ProTide structure
`initially involved the amino acid and, subsequently, the aromatic
`masking group on the phosphate moiety. Herein, derivatives bearing
`the combined modifications are reported and biological evaluation is
`described. Moreover, separation of the diastereoisomeric final product
`mixture shows a different cytostatic activity for the two diastereoiso-
`mers. Through computational and NMR studies, the absolute stereo-
`chemistry of the phosphorus center of the two diastereoisomers has
`been suggested.
`Nucleoside analogues represent an extremely effective tool
`for the treatment of cancer and viral infections. For these
`compounds, the action of kinases is required to convert the
`inactive nucleoside into biologically active nucleotide (mono-,
`di-, and triphosphate). Unfortunately, the dependence on kinases
`significantly limits the biological profile of nucleoside analogues
`because of their high specificity toward substrates and, no less
`important, a lower expression of these enzymes often leads to
`emergence of resistance to the nucleoside treatment. Due to the
`polarity of nucleotides themselves, circumventing kinase activa-
`tion problems cannot be achieved with direct administration of
`the preformed free phosphate, as the resulting cell penetration
`would be too poor to show any significant therapeutic effect.
`The phosphoramidate approach was introduced by McGuigan
`et al. in 19921 as a means to improve cellular penetration of
`nucleotides and to bypass the first step of kinase-mediated
`activation of nucleosides. Our method has been applied by both
`our lab and others to a wide variety of nucleosides and,
`nowadays, is recognized as one of the most successful ap-
`proaches for the delivery of nucleoside monophosphates inside
`cells.2
`Our technology has recently led NewBiotics to NB10113
`(thymectacin), an aryloxy phosphoramidate derivative of BVdU
`(brivudin), which entered clinical evaluation against colon
`cancer.4 On the basis of our experience of widespread phos-
`phoramidate modifications, we first prepared a new series of
`phenyl phosphoramidates related to thymectacin, tuning the
`phenyl, ester, and amino acid regions, observing significant
`enhancements in activity versus three different tumor cell lines.5
`Because lipophilicity might play a crucial role for the delivery
`of ProTides inside cells,6 new analogues have been designed
`focusing on the introduction of naphthyl as new aromatic
`
`* To whom correspondence should be addressed. Direct dial and Fax
`+44 029 20874537. Email: mcguigan@cardiff.ac.uk.
`† Welsh School of Pharmacy.
`‡ Wales College of Medicine.
`
`10.1021/jm0509896 CCC: $33.50 © 2006 American Chemical Society
`Published on Web 12/17/2005
`
`IPR2018-00126
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`
`Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 453
`
`Scheme 2. Synthesis of Naphthyl Phosphoramidates
`
`Table 1. Cytostatic Effect of Test Compounds (EC50/(cid:237)M)
`breast
`prostate
`MDAMB231
`PC-3
`
`compound
`
`amino
`
`ester
`
`bladder
`T24
`-
`-
`-
`-
`-
`-
`-
`43.5
`12.7
`5.3
`269
`2.8
`19.6
`4
`0.4
`
`NB1011
`CPF98
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11
`12
`13
`
`Me
`L-Ala
`Bn
`L-Ala
`Me2Gly
`Me
`Me2Gly
`Bn
`Me
`L-Ile
`Bn
`L-Ile
`L-PhGly Me
`L-Val
`Me
`L-Val
`Bn
`L-Phe
`Me
`L-Phe
`Bn
`D-Ala
`Bn
`L-Met
`Me
`L-Ala
`tBu
`L-Pro
`Me
`
`79
`15.2
`0.32
`2.7
`1.5
`130
`105
`14.8
`5.9
`8.5
`1.96
`6.3
`28.1
`4.8
`6.5
`
`155
`1.7
`65.9
`1.5
`6.9
`1.4
`1.7
`15.8
`8.3
`10.2
`5.8
`6.1
`44.6
`11
`10.5
`
`MB231 (breast cancer) and PC-3 (prostate cancer) for entries
`1-5 and thymectacin; T24 (bladder cancer) was also considered
`for entries 6-13. Data are reported in Table 1.
`According to the data shown in Table 1, most of the naphthyl
`phosphoramidates synthesized appear to be significantly more
`active than thymectacin, which displays only a moderate activity
`in our in vitro evaluations. Moreover, the L-alaninyl benzyl ester
`naphthyloxy-phosphoramidate (CPF98) which has been previ-
`ously reported by us has been added to Table 1.7a-b
`For the prostate cancer cell line, lipophilicity seems to play
`a fundamental role for the biological activity of these Pro-
`Tides: the activity of compounds bearing the same amino acid
`moiety (dimethylglycine: 1, 2; L-isoleucine 3, 4; L-valine: 6,
`7) is boosted every time the ester group is changed from methyl
`to benzyl, leading to a 100-fold increase versus thymectacin
`(entries 2 and 4). Not surprisingly, also the presence of a highly
`lipophilic amino acid as phenylglycine leads to a significant
`activity (compound 5).
`On the other hand, this trend is not applicable to the breast
`cancer cell
`line. Activation of phosphoramidates has been
`reported to be dependent on the action of esterases and
`phosphoramidase activities;8-10 it is possible that a different
`enzymatic activity in the two cell lines is responsible for the
`differences observed in the biological data. Further assays are
`underway to probe this aspect. Nevertheless, naphthyl phos-
`phoramidates did show noteworthy activities also versus the
`breast cancer cell line, in particular compounds 2, 3, and 9 have
`achieved between 30- and 50-fold increase in potency versus
`thymectacin. Moreover, compound 1, showing a 250-fold boost
`versus this particular cancer cell line, becomes the most active
`BVdU-related phosphoramidate with cytostatic activity against
`a breast cancer cell line reported. Significantly, the correspond-
`ing ‘phenyl’ ProTide reported previously by us displays an EC50
`of 41.1 (cid:237)M in the same assay.5
`Last, tuning the structure of the lead ProTide by introducing
`the naphthyl moiety and subsequently modifying the amino acid
`core has led to an enhancement of potency in all the three cell
`lines, such as compound 1 for the breast cancer, 4 and 5 for the
`prostate, and 13 for the bladder cancer cell line.
`As formerly mentioned, all
`the compounds have been
`synthesized and tested as mixtures of two phosphate diastereo-
`
`isomers (1:1 ratio). Previous work carried out by Saboulard et
`al.11 in 1999 indicates that carboxyl ester cleavage is a
`fundamental step for the activation of phosphoramidates.
`Enzymatic stability in the extracellular environment (i.e. plasma)
`and in different cellular preparations was found to be stereospe-
`cific with large and unpredictable differences in stereoselective
`metabolic rate noted by Siccardi et al.12 Separation of phos-
`phoramidate diastereoisomers by column chromatography has
`been shown to be problematic and even by using HPLC
`it remains a hard task to achieve.13
`preparative methods,
`Furthermore, when single diastereoisomers have been isolated,
`identification of the corresponding absolute stereochemistry has
`never been elucidated, leaving HPLC retention time and 31P
`NMR chemical shift as the only parameters to discriminate
`between the two isomers.
`In the case of compound 10, the mixture has been reasonably
`separated on reverse phase and the two diastereoisomers were
`tested against the MDA MB231 cell line (breast cancer). The
`fast eluting diastereoisomer (fe) emerged as slightly less active
`than the mixture (compd 10), with an EC50 of 7.4 (cid:237)M. On the
`other hand, the slow eluting diastereoisomer (se) is about 10
`times more active than the mixture, showing an EC50 of 0.5
`(cid:237)M. The higher activity could be due to a better diffusion
`through cell membranes,14 the slow eluting diastereoisomer
`being the more lipophilic, or to a more efficient stereoselective
`metabolism of the se diastereoisomer. Biological evaluation
`against the other different cancer cell lines is in progress.
`Given this striking result, our major interest has been to find
`out a method to attribute the corresponding absolute stereo-
`chemistry to each of the two diastereoisomers.
`The slow eluting (se) diastereoisomer shows downfield shifts
`(between 0.1 and 0.2 ppm) on H NMR for the H-5b, H-6, and
`H-2¢ protons compared to the fast eluting (fe) isomer. Owing
`to the presence of three aromatic systems (nucleoside analogue
`base, phenyl and naphthyl), the former protons’ chemical shifts
`might be perturbed by an anisotropic effect. The methylene
`protons of the benzylic ester display a more striking differ-
`ence: for the fe diastereoisomer, a double doublet results as
`the main feature of their signal (traces of the other isomer are
`present) while, for the se diastereoisomer, the two protons couple
`with each other and show an AB-system (Figure 1).
`Conformational studies were performed using the Sybyl 7.0
`software package,15 which allowed the identification of a series
`of distinct
`low energy conformations. The lowest energy
`conformation found for each diastereoisomer is shown in Figure
`2. Interestingly, when the computational and NMR data were
`compared, particular features were recognized.
`
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`Letters
`
`Figure 2. Lowest energy conformations of the two diastereoisomers
`of compound 10.
`
`anticancer ProTide for the first time, by a combination of NMR
`and conformational studies.
`
`Supporting Information Available: Synthesis, NMR, HPLC,
`low resolution mass, elemental analysis data, and conformational
`and biological evaluation methods’ descriptions. This material is
`available free of charge via the Internet at http://pubs.acs.org.
`
`References
`(1) McGuigan, C.; Pathirana, R. N.; Mahmood, N.; Hay, A. Aryl
`phosphate derivatives of AZT inhibit HIV replication in cells where
`the nucleoside is poorly active. J. Bioorg. Med. Chem. Lett. 1992, 2,
`701-704.
`(2) Meier, C. Pro-Nucleotides - Recent advances in the design of
`efficient tools for the delivery of biologically active nucleoside
`monophosphate. Synlett 1998, 233-242.
`(3) Lackey, D. B.; Groziak, M. P.; Sergeeva, M.; Beryt, M.; Boyer, C.;
`Stroud, R. M.; Sayre, P.; Park, J. W.; Johnston, P.; Slamon, D.;
`Shepard, H. M.; Pegram, M. Enzime-catalysed therapeutic agent
`(ECTA) design: activation of the antitumor ECTA compound
`NB1011 by thymidylate synthase. Biochem. Pharmacol. 2001, 61,
`179-189.
`(4) Pegram, M.; Ku, N.; Shepard, M.; Speid, L.; Lenz, H. L. Enzyme-
`catalysed therapeutic activation (ECTA) NB1011 (Thymectacin)
`selectively targets thymidilate synthase (TS) - overexpressing tumor
`cells: preclinical and phase I clinical results. Eur. J. Cancer 2002,
`38(Suppl. 7), S34.
`(5) McGuigan, C.; Thiery, J. C.; Daverio, F.; Jiang, W. G.; Davies, G.;
`Mason, M. Anti-cancer ProTides:
`tuning the activity of BVDU
`phosphoramidates related to thymectacin. Bioorg. Med. Chem. 2005,
`13, 3219-3227.
`(6) Knaggs, M. H.; McGuigan, C.; Harris, S. A.; Gilbert, I. H., Balzarini,
`J. A QSAR study investigating the effect of L-alanine ester variation
`on the anti-HIV activity of some novel phosphoramidate derivatives
`of d4T. Bioorg. Med. Chem. Lett. 2000, 10, 2075-2078.
`(7) Congiatu, C.; McGuigan, C.; Jiang, W. G.; Davies, G.; Mason, M.
`D. Naphthyl phosphoramidates derivates of BVdU as potential
`anticancer agents: design, synthesis and biological evaluation.
`Presented at the XVI International Roundtable of the International
`Society for Nucleosides, Nucleotides & Nucleic Acids, Minneapolis,
`MN, Sept 12-16, 2004, poster IS3NA. (b) Congiatu, C.; McGuigan,
`C.; Jiang, W. G.; Davies, G.; Mason, M. D. Naphthyl phosphor-
`amidate derivates of BVdU as potential anticancer agents: design,
`synthesis and biological evaluation. Nucleosides, Nucleotides Nucleic
`Acids, in press.
`(8) McGuigan, C.; Cahard, D.; Sheeka, H. M.; De Clercq, E.; Balzarini,
`J. Aryl phosphoramidate derivatives of d4T have improved anti-HIV
`efficacy in tissue culture and may act by the generation of a novel
`intracellular metabolite. J. Med. Chem. 1996, 39, 1748-1753.
`(9) Cahard, D.; McGuigan, C.; Balzarini, J. Aryloxy phosphoramidate
`triesters as Pro-Tides. Mini-ReV. Med. Chem. 2004, 4, 371-382.
`(10) McGuigan, C.; Sutton, P. W.; Cahard, D.; Turner, K.; O’Leary, G.;
`Wang, Y.; Gumbleton, M.; De Clercq, E.; Balzarini, J. Synthesis,
`anti-human immunodeficiency virus activity and esterase lability of
`some novel carboxylic ester-modified phosphoramidate derivatives
`of stavudine (d4T). AntiViral. Chem. Chemother. 1998, 9, 473-479.
`(11) Saboulard, D.; Naesens, L.; Cahard, D.; Salgado, A.; Pathirana, R.;
`Velazquez, S.; McGuigan, C.; De Clercq, E.; Balzarini, J. Charac-
`terization of the activation pathway of phosphoramidate triester
`prodrugs of stavudine (d4T) and zidovudine (AZT). Mol. Pharmacol.
`1999, 693-704.
`(12) Siccardi, D.; Gumbleton, M.; Omidi, Y.; McGuigan, C. Stereospecific
`chemical and enzymatic stability of phosphoramidate triester prodrugs
`of d4T in vitro. J. Pharm. Sci. 2004, 22, 25-31.
`
`Figure 1. H NMR spectra of the methylene protons of the benzylic
`ester moiety of the isolated diastereoisomers of compound 10.
`
`For the S diastereoisomer (for clarity the two diastereoisomers
`are named after the absolute stereochemistry of the correspond-
`ing phosphorus center) the three aromatic moieties are stacked
`in pi-pi interactions where the naphthyl lays between the phenyl
`group and the nucleoside base. In this case, chemical shift
`changes are reasonable to appear for the protons closer to such
`an extended pi-electron cloud (e.g. H-5b, H-6 and H-2¢).
`Furthermore, the apparent rigidity of this conformation, con-
`ferred by the described aromatic interactions,
`justifies the
`observed NMR pattern for the methylene hydrogens of the
`benzylic ester, which became nonequivalent.
`The R conformation does not show any pi-pi interaction
`among the aromatic rings as in the case of the S diastereoisomer,
`and the greater flexibility around the methylene group of the
`ester moiety reduces the magnetic differences between the two
`diastereotopic protons. Therefore, by combining the NMR and
`the conformational data, we can propose the S phosphorus
`absolute configuration to the slow eluting (more lipophilic)
`diastereoisomer and, consequently, the R configuration to the
`fast eluting one.
`The lowest energy conformation values generated by the
`Genetic Algorithm search used are -10.55 kcal mol-1 for the
`se diastereoisomer (suggested S phosphorus configuration) and
`-6.45 kcal mol-1 for the fe diastereoisomer (suggested R
`phosphorus configuration).
`In summary, a new series of naphthyl phosphoramidates of
`BVdU has led to a significant improvement in the cytostatic
`activity of the parent lead compound thymectacin, against a
`panel of different cancer cell lines. Separation of the diastereo-
`isomeric mixture of compound 10 has shown to be a useful
`approach to further enhance the anticancer effect of phosphor-
`amidate ProTides.
`the absolute
`Moreover, we have been able to suggest
`stereochemistry of
`the phosphorus center
`for a potential
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`Journal of Medicinal Chemistry, 2006, Vol. 49, No. 2 455
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`(13) Allender, C. J.; Brain, K. R.; Ballatore, C.; Cahard, D.; Siddiqui,
`A.; McGuigam, C. Separation of individual antiviral nucleotide
`prodrugs from synthetic mixtures using cross-reactivity of a mo-
`lecularly imprinted stationary phase. Anal. Chim. Acta 2001, 435,
`107-113.
`(14) Siccardi, D.; Kandalaft, L. E.; Gumbleton, M.; McGuigan, C.
`Stereoselective and concentration-dependent polarized epithelial
`
`permeability of a series of phosphoramidate triester prodrugs of
`d4T: an in vitro study in Caco-2 and Madin-Darby canine kidney
`cell monoayers. J. Pharmacol. Exp. Ther. 2003, 3, 1112-1119.
`(15) Tripos SYBYL 7.0; Tripos Inc., 1699 South Hanley Rd., St. Louis,
`MO 63144. http://www.tripos.com.
`
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