Monophosphate Decarboxylase Inhibitors as Anticancer Agents

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J. Med. Chem. 2009, 52, 1648–1658

Structure-Activity Relationships of Orotidine-5′-Monophosphate Decarboxylase Inhibitors as Anticancer Agents Angelica M. Bello,†,‡ Danijela Konforte,†,§ Ewa Poduch,‡ Caren Furlonger,§ Lianhu Wei,‡ Yan Liu,⊥ Melissa Lewis,‡ Emil F. Pai,§,⊥ Christopher J. Paige,§,| and Lakshmi P. Kotra*,‡,#,∇,O Center for Molecular Design and Preformulations and DiVision of Cellular and Molecular Biology, Toronto General Research Institute, Toronto General Hospital, Toronto, Ontario M5G 2C4, Canada, Department of Medical Biophysics, Ontario Cancer Institute, Princess Margaret Hospital, 620 UniVersity AVenue, Toronto, Ontario M5G 2M9, Canada, Department of Immunology, UniVersity of Toronto, Toronto, Ontario, Canada, Departments of Biochemistry and Molecular Genetics, UniVersity of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada, Departments of Pharmaceutical Sciences and Chemistry, UniVersity of Toronto, Toronto, Ontario, Canada, McLaughlin Center for Molecular Medicine, UniVersity of Toronto, Toronto, Ontario, Canada, Department of Chemistry & Biochemistry, The UniVersity of North Carolina at Greensboro, Greensboro, North Carolina 27412 ReceiVed September 27, 2008

A series of 6-substituted and 5-fluoro-6-substituted uridine derivatives were synthesized and evaluated for their potential as anticancer agents. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. The mononucleotide derivatives were used for enzyme inhibition investigations against ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. 5-Fluoro-UMP was a very weak inhibitor of ODCase. 6-Azido-5-fluoro and 5-fluoro-6-iodo derivatives are covalent inhibitors of ODCase, and the active site Lys145 residue covalently binds to the ligand after the elimination of the 6-substitution. Among the synthesized nucleoside derivatives, 6-azido-5-fluoro, 6-amino-5-fluoro, and 6-carbaldehyde-5-fluoro derivatives showed potent anticancer activities in cell-based assays against various leukemia cell lines. On the basis of the overall profile, 6-azido-5-fluoro and 6-amino-5-fluoro uridine derivatives exhibited potential for further investigations. Introduction Antimetabolites have been a useful tool in the fight against cancer over the past several decades.1 Most of these drugs such as 5-fluorouracil and methotrexate impair the synthesis of nucleotides in the cancer cells, thus seriously impairing the uncontrolled proliferation of these cells.2 In humans, these nucleosides, in particular the pyrimidine mononucleotides, are recruited into the nucleic acid biosynthesis either via the de novo pathway or the salvage pathway. During the uncontrolled and fast replication of cells, de novo pathway must also contribute toward supplying nucleotides despite the existence of salvage pathway because of the higher demand for nucleotides in a short period of time. Orotidine-5′-monophosphate decarboxylase (ODCase,a EC 4.1.1.23) plays a central role in the de novo * Corresponding Author: Phone: (416) 581-7601or (336) 334-9862. Fax: (336) 334-5402. E-mail: [email protected] or [email protected]. Mailing address: #5-356, Toronto Medical Discoveries Tower/MaRS Center, 101 College Street, Toronto, Ontario M5G 1L7, Canada. † Authors contributed equally. ‡ Center for Molecular Design and Preformulations and Division of Cellular and Molecular Biology, Toronto General Research Institute, Toronto General Hospital. § Department of Medical Biophysics, Ontario Cancer Institute, Princess Margaret Hospital. | Department of Immunology, University of Toronto. ⊥ Departments of Biochemistry and Molecular Genetics, University of Toronto. # Departments of Pharmaceutical Sciences and Chemistry, University of Toronto. ∇ McLaughlin Center for Molecular Medicine, University of Toronto. O Department of Chemistry & Biochemistry, The University of North Carolina at Greensboro. a Abbreviations: ODCase, orotidine 5′-monophosphate decarboxylase; OMP, orotidine 5′-monophosphate; UMP, uridine 5′-monophosphate; Hs, Homo sapiens; Mt, Methanobacterium thermoautotrophicum; 5-FU, 5-fluorouracil; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffer saline.

synthesis of the ribonucleotide, uridine-5′-O-monophosphate (UMP, 2), from orotidine-5′-O-monophosphate (OMP, 1). This enzyme catalyzes the decarboxylation of OMP (1) to UMP (2) (Figure 1). UMP, synthesized de novo from aspartic acid, is a building block for the synthesis of pyrimidine nucleotides such as uridine-5′-triphosphate (UTP), cytidine-5′-triphosphate (CTP), thymidine-5′-triphosphate (TTP), and 2′-deoxycytidine-5′-triphosphate (dCTP). During the decarboxylation reaction, ODCase exhibits an extraordinary level of catalytic rate enhancement of over 17 orders of magnitude compared to the uncatalyzed decarboxylation reaction in water at pH 7.0 at 25 °C.3-6 One interesting difference when one looks at this enzyme across the species is that in certain higher level organisms such as human, rat, or mouse, ODCase is a part of the bifunctional enzyme, UMP synthase.7 A handful of investigations in the past focused on developing ODCase inhibitors against cancer, malaria, and RNA viral infections.8 6-Aza-UMP (8), 6-hydroxy-UMP (or BMP, 9), Pyrazofurin-5′-monophosphate (12), xanthosine-5′-monophosphate (XMP, 13), and 6-thiocarboxamido-UMP (10) are some of the potent inhibitors that were studied against ODCase (Figure 2).9-11 Although, ODCase did not gain much traction in the 1980s and 1990s as a drug target, its inhibitors were explored as potential drugs due to its essential role in the de novo synthetic pathway. For example, Plasmodia species such as Plasmodium falciparum and Plasmodium ViVax are dependent on their own de novo synthesis of pyrimidine nucleotides due to the absence of the salvage pathway in these parasites.12-19 Similarly, ODCase inhibitors were also proposed as potential antiviral agents, although very limited studies have been carried out. Inhibitors of ODCase such as 6-aza-uridine (7) and Pyrazofurin (11) (Figure 2) exhibited good anticancer activities against

10.1021/jm801224t CCC: $40.75  2009 American Chemical Society Published on Web 03/04/2009

Orotidine-5′-Monophosphate Decarboxylase Inhibitors

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1649

Figure 1. De novo synthesis of UMP from OMP by ODCase.

Figure 2. Various nucleosides and nucleotides with anticancer and ODCase inhibition activities.

a number of clinical tumor models.20,21 6-Aza-uridine (7) is transformed into its mononucleotide form, 6-aza-uridine-5′-Omonophosphate (8), in vivo and the resulting mononucleotide inhibits ODCase, impairing the de novo production of pyrimidine nucleotides.22 Compound 8 inhibits yeast and Mt ODCases with the inhibition constants (Ki) of 64 nM and 11 µM, respectively.23,34 Another potent inhibitor of ODCase, Pyrazofurin (11), has been investigated as an anticancer agent including in clinical trials. Pyrazofurin-5′-O-monophosphate (12), which is a C-nucleotide, inhibits ODCase with a Ki of 5 nM and its nucleoside analogue is readily taken up by the cells and transformed into its monophosphate form.24 In a separate study, measurement of levels of pyrimidine and purine intermediates in cultured mouse L1210 leukemia cells showed that 25 µM Pyrazofurin induced an 8-fold increase in the accumulation of OMP and an abrupt decrease in the pyrimidine ribosyl mononucleotides.25 Pyrazofurin, however, was not clinically developed further due to its toxicity to patients in phase I studies. With the above studies available on ODCase that illustrate the role and relevance of this target in anticancer drug development, we became interested in exploring novel ODCase inhibitors carrying C6 substitutions on the pyrimidine nucleosides as anticancer agents. As alluded above, ODCase carries out the decarboxylation of OMP very efficientlysits only but essential function in the cell. A number of mechanisms for the decarboxylation of OMP by ODCase were proposed prior to and after the availability of its X-ray crystal structures.26-32 Our groups were interested in a series of C6-substituted pyrimidine nucleotides to investigate the mechanism of ODCase. These probe molecules present small substitutions at the C6 position of the uridine moiety, such that they can sterically and/or electronically mimic the carboxyl group present on the substrate, OMP.33,34 For example, 6-cyanoUMP (34) was designed as a potential bioisosteric inhibitor of ODCase and its interactions with the enzyme were investigated

using X-ray crystallography and enzymology.33,34 ODCase catalyzed the surprising conversion of the chemically stable 6-cyano-UMP (34) into 6-hydroxy-UMP (BMP, 9), albeit slowly with a half-life of 5 h, in what can be categorized as a “pseudohydrolysis” process.33 The conversion of compound 34 into 9 by ODCase established the catalytic promiscuity for this fascinating decarboxylase.33,34 Compound 34 also exhibited noncovalent, competitive inhibition of ODCase activity with a Ki of 29 ( 2 µM.34,35 We also revealed two covalent inhibitors to ODCase and the potential of these compounds in antimalarial drug development.35,36 As part of an ongoing program exploring ODCase inhibitors, we synthesized a series of 6-substituted and 5-fluoro-6-substituted uridine derivatives and evaluated their potential as anticancer agents. Here, we reveal the medicinal chemistry of these novel compounds, their anticancer activities, their ODCase inhibition activities, and the structural determinants for their binding to ODCase in the context of the potential of ODCase inhibitors for targeting various types of cancer. Results and Discussion Nucleosides have played a key role in cancer chemotherapy during the past four decades and continue to provide therapeutic options for hard-to-treat neoplastic diseases.37 Most of these nucleoside analogues mimic various metabolites or inhibit an essential enzyme, ultimately affecting the synthesis of nucleic acids in the fast replicating cancer cells. Nucleotides are essential molecules for the synthesis of ribo and deoxyribo nucleic acids during replication, and thus this is a very attractive pathway for anticancer drug design. The recent discovery of novel mechanism-based inhibitors targeting ODCase inspired us to evaluate the potential of these compounds as anticancer agents. The designed compounds are various 6-substituted pyrimidine nucleosides with substitutions such as 6-cyano, 6-azido, 6-amino, 6-iodo, 6-N-methylamino, 6-N,N-dimethylamino, 6-aldehyde, 6-ethyl ester, and 6-hydroxyl amino moieties (Figure 3).

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Bello et al.

Scheme 1. Synthesis of Compounds 26-28 and 40-42a

Figure 3. Various C6-substituted pyrimidine derivatives.

Additionally, we designed derivatives carrying 5-fluoro moiety along with the above 6-substitutions to study its effect on binding to human ODCase and potentially their anticancer activities (Figure 3). An introduction of the fluorine moiety at C5 position would have stabilizing effects on the C6 substitutions due to the electronegativity of the fluorine moiety. We also envisioned that the C6 center could be more electrophilic due to the 5-fluoro substitution. The designed molecules were synthesized from either fully protected uridine or the corresponding 5-fluorouridine derivatives. Synthesis of uridine derivatives 14-18, 21, 22, and 34-38 was already published by our group earlier.36 5-Fluorouridine derivatives were synthesized according to Schemes 1 and 2. 5-Fluorouridine (25) was fully protected and treated with LDA followed by iodine to obtain 6-iodo derivative 45. This compound 45 served as a key intermediate to synthesize other C6 derivatives. Thus 6-azido derivative 46 was synthesized by the treatment of 45 with sodium azide. Deprotection of the silyl and isopropylidene moieties on 45 and 46 led to the corresponding nucleoside derivatives 26 and 27, respectively. Monophosphorylation of the nucleosides was accomplished using the standard conditions of phosphorus chloride/pyridine. 6-Amino derivatives 28 and 42 were synthesized by the reduction of corresponding azido derivatives 27 and 41 (Scheme 1). When compound 44 was treated with LDA and methyl iodide, 6-ethyl derivative was obtained due to the additional methylation of the 6-monomethylated 5-fluorouridine derivative. We did not observe the formation of any monomethylation at C6 position, and 6-ethyl derivative 47 was isolated as the only product. Treatment of 44 with methyl formate led to the synthesis of 6-aldehyde derivative 48. Compounds 47 and 48 were fully deprotected under acidic conditions, leading to the nucleoside derivatives 29 and 30, respectively (Scheme 2). Monophosphorylation of compound 29 generated the mononucleotide derivative of 6-ethyl-5fluorouridine derivative (Scheme 2). The mononucleotide derivatives were all transformed into the corresponding ammonium salt forms for enzyme inhibition investigations against human ODCase. Anticancer activities of all the synthesized derivatives were evaluated using the nucleoside forms of the inhibitors. First, mononucleotide derivatives 34-38 and 39-43 were evaluated as potential inhibitors of Hs ODCase and/or Mt ODCase (Table 1). Compounds 39, 42, and 43 exhibited an interesting pattern of inhibition of Hs ODCase (Figure 5).

a Reagents: (a) acetone, H+; (b) TBDMSiCl, imidazole, CH2Cl2; (c) LDA, I2, THF, -78 °C; (d) NaN3, DMF; (e) 50% TFA/H2O; (f) POCl3, Py, CH3CN, 0 °C; (g) NH4OH, 0 °C; (h) Pd/C, H2.

Compound 39, which is a 5-fluorouridine derivative with no substitution at C6 position, inhibited Hs ODCase weakly, with a Ki of 98 µM, and this inhibition was much worse against that of Mt, with a Ki of 645 µM. This perhaps is anticipated because 5-fluorouracil derivative 39 does not carry any substitution at C6 position, and although 5-fluoro moiety fits well within the binding pocket, it may not enhance the binding affinity significantly. The 5-fluoro-6-amino derivative 42 is a moderate inhibitor of both Hs and Mt ODCases, with Kis of 16.6 and 11.4 µM, respectively. The most potent inhibitor of Hs ODCase is the 5-fluoro-6-ethyl derivative 43 and inhibited Hs ODCase with a Ki of 0.35 µM. However, compound 43 is approximately 2 orders of magnitude weaker against Mt ODCase. In light of the differences in inhibition patterns that exist between ODCases from different species for the same inhibitor,39 the differences of up to 2 orders of magnitude in the inhibition constants for compounds 39 and 43 is not surprising in this case. Compounds 40 and 41 exhibited potent inhibition of Hs ODCase as covalent inhibitors (Table 2). The 5-fluoro-6-azido derivative 41 exhibited interesting results, where it did not show competitive inhibition with Hs ODCase, but with Mt ODCase, we were able to derive a competitive inhibition constant by measuring the initial rate constants. This again is further confirmation of the species-related differences for the inhibition of ODCases by the same inhibitor. Compound 40 inhibits human ODCase with an equilibrium inhibition constant (KI) of 1.1 µM, where as compound 41 inhibited with a KI of 18.2 µM (Figure 6). The second-order rate constant (kobs/[I]) for the inactivation of Hs ODCase by compound 40 is estimated to be 1.9 M-1 s-1 (Table 2). On the basis of the enzymatic studies, compounds 40, 41, and 43 are good inhibitors of Hs ODCase among 5-fluoro-6-substituted uridine mononucleotide derivatives. Mass



Scheme 2. Synthesis of Compounds 29-30 and 43a

a Reagents: (a) LDA, methyl iodide, THF, -78 °C; (b) 50% TFA/H2O; (c) POCl3, Py, CH3CN, 0 °C; (d) NH4OH, 0 °C; (e) LDA, methyl formate, THF, -78 °C.

Figure 4. X-ray crystal structure of the complex of 41 and Hs ODCase. (A) Active site region with the ligand 41 bound covalently to Lys125 residue. The enzyme is represented by the secondary structural elements. The critical residues Asp128, Lys125, Asp123, and Lys92 and the ligands 41 are shown as a capped stick model, and the atoms are color-coded (C, gray; N, blue; O, red; P, orange; F, turquoise). (B) Hydrogen bonding network between the ligand 41 and various residues in the active site of Hs ODCase. Table 1. Enzyme Inhibition Studies on ODCases Using Various Inhibitors Ki (µM) compd 34,35

34 3634,35 3734,35 3834,35 39 41 42 43

Hs

Mt

ND ND ND ND 98 ( 6 NDa 16.6 ( 0.7 0.35 ( 0.01

29 ( 2 0.2 ( 0.1 0.84 ( 0.02 134 ( 5 645 ( 15 0.36 ( 0.03 11.4 ( 0.6 29.0 ( 0.3

a Note: Compound 41 did not exhibit reversible inhibition in a competitive assay with Hs ODCase.

spectral analyses of the samples of Hs ODCase treated with compound 40 and 41 revealed that these compounds bind covalently to the enzyme after the elimination of the C6substitutions (iodo and azido moieties, respectively), further supporting the enzyme kinetics analyses (see Supporting Information).

To understand the molecular interactions of compounds 40 and 41 with ODCase, this compound was cocrystallized with Hs ODCase (Table 3). Compounds 40 and 41 generated identical complex structures with Hs ODCase, and here the discussion is thus facilitated with the structure of compound 41 bound in the active site of Hs ODCase (Figure 4). Compound 41 (similar to compound 40) is a covalent inhibitor of Hs ODCase, and this is clearly seen in the cocrystal structure, supporting the enzyme kinetics (Table 2). The ligand is bound in the catalytic pocket formed by the TIM barrel similar to other ligands. The Nε of Lys125 in Hs ODCase is clearly seen bound to C6 atom of the inhibitor 41 (Figure 4A). The 5′-monophosphate group engaged in a tight hydrogen bonding network with residues Asn241, Tyr243, Arg262, Gln261, and three water molecules providing the most binding energy for the inhibitor, as expected (Figure 4B). The 2′- and 3′-hydroxyl moieties as well as the urea portion of the nucleic base are interacting with the enzyme with residues Ser68, Asp70, Lys92, Thr132, Asp128, Ser183, and Asn241, as was seen in previous cocrystal structures with


Figure 5. Reversible inhibition of Hs ODCase by the compounds 39 (A), 42 (B), and 43 (C) shown in double reciprocal plots. All experiments are conducted in triplicate. Table 2. Kinetic Characterization of ODCase in the Presence of Irreversible Inhibitors coinjection assay compd

KI (µM)

35 36

ND 0.6 ( 0.1

40 41

1.1 ( 0.4 18.2 ( 3.6

kinact (h-1)

time-dependent assay kobs/[I] (M-1 s-1)

Mt ODCase ND 61.2

260000 ND

Hs ODCase 158 ( 18 41 ( 3

1.9 NA

ligands.34-36 The 5-fluoro moiety due to its small size fits into the enzyme pocket well and does not specifically engage in any direct interactions with the enzyme, as seen from this structure (Figure 4B). Among other compounds, 34-38 showed a range of inhibition potencies against Mt ODCase and were revealed in previously published reports from our group.34-36 Compounds 34, 37, and 38 are competitive inhibitors of ODCase, while compounds 35 and 36 inhibited ODCase covalently. Compounds 34 and 37 inhibited Mt ODCase with a Ki of 29.2 ( 2 and 0.84

Bello et al.

( 0.02 µM, respectively. Compound 36 and 38 inhibited Mt ODCase (considering initial rates of inhibition for the former compound) with Ki of 0.19 ( 0.1 and 134 ( 5 µM, respectively. Compounds 36 and 37 have exhibited submicromolar inhibition against Mt ODCase, and are the most potent among the nonfluorinated compounds, next to compound 35. Compound 35 inhibited Mt ODCase with a second-order rate constant (kobs/ [I]) of 260000 M-1 s-1. Compound 36, on the other hand, is a relatively poor inactivator, with kinact of 61.2 h-1 and inactivation constant of 0.6 ( 0.1 µM.36 The general trend of the covalent inhibition with 6-iodo and 6-azido moieties at C6 position was observed in all cases. On the basis of the in vitro cell-based assays (vide infra), we did not further evaluate compounds 34-38 against human ODCase because their nucleoside forms did not exhibit any significant anticancer activities. The nucleoside derivatives of compounds 8, 9, and 14-33 were screened against various cancer cell lines in order to test their potential to inhibit cancer cell growth in vitro (Table 4). The evaluation of all these compounds in WST.1 assays revealed that 27, 28, 29, and 30 strongly inhibited cell lines of hematopoietic origin, including leukemias (acute myelogenous leukemia lines OCI-AML-1 and OCI-AML-2, and T-cell leukemia line SKW3), lymphomas (non-Hodgkin’s B cell lymphoma-OCI-Ly-7), and multiple myeloma (OCI-My-2). The inhibitory concentrations (IC50) for these compounds were in the range of 0.3-2.1 µM (Table 4). Expansion of adherent cancer cell lines including breast cancer lines (MCF7 and MDA468) were generally less sensitive to the above compounds, with IC50 values ranging between 1-31 µM. None of the other compounds showed inhibitory activities against the adherent cancer lines up to 50 µM. Melanoma cell line Lox was highly sensitive to compound 30, with an IC50 of 300 nM. In further evaluations, compounds 27 and 28 revealed the inhibitory concentrations (IC50) against healthy human PBMCs stimulated with concanavalin A were greater than 10 µM. Following this encouraging outcome, compounds 27, 28, and 30 were further evaluated for their ability to induce apoptosis in different leukemia and lymphoma cell lines. To determine the relationships between the cell survival and the reduced expansion of different cell lines in the presence of 27, 28, and 30, the cell lines were treated with Annexin V and PI. Four cell lines (OCI-AML1, OCI-Ly7, OCI-My2, and SKW3) were observed for 3 days in the presence and absence of the above inhibitors. The results clearly showed an increase in the percentage of apoptotic cells in the presence of the inhibitors, 27, 28, and 30 compared to the control cells (Figure 7). Increased cell death was first observed on day 2 after exposing the cancer cells to the compounds. While all three compounds induced apoptosis in these cell lines, compound 27 was relatively more effective than 28 and 30 (Figure 7). It occurred to us that 5-fluoro-uridine derivatives such as 25-30 might inhibit thymidylate synthase, similar to 5-fluoro2′-deoxyuridine. However, thymidylate synthase accepts 2′deoxynucleosides as substrates, and the nucleosides evaluated in this report are ribonucleosides. In fact, we evaluated 5-fluoroUMP (39) against Hs and Mt ODCases for its inhibitory potential. This compound was not a potent inhibitor of ODCases and did not indicate that the 5-fluoro-UMP has favorable effect without a C6-substitution. The other possibility could be that the above nucleosides, including compound 25, are degraded into their nucleic bases in the cells, which then are further transformed into the corresponding 2′-deoxynucleosides and exhibit anticancer activities through the inhibition of thymidylate synthase. Thus, there may potentially be multiple mechanisms,



Figure 6. Inactivation of Hs ODCase by 40 and 41 (A and B, respectively) following the simultaneous addition of substrate mixed with the inhibitor. Nonlinear least-squares fit of kobs at different inhibitor concentrations (eq 3) was used to determine the inactivation rate (kinact) and inactivation constant (KI). All experiments are conducted in triplicate. Table 3. Crystallographic Parameters for the Structure of the Complex of Human ODCase with Compounds 40 and 41 Hs ODCase + 40 Hs ODCase + 41 Diffraction Data resolution (Å)a measured reflections (n) unique reflections (n) completeness (%)a Rsym (%)a apace group unit cell axes (Å) a b c unit cell angle (deg) β molecules in asymmetric unit (n)

1.40 (1.45-1.40) 321875 52359 93.1 (60.7) 5.7 (21.3) C2221

1.70 (1.73-1.70) 205710 58035 94.9 (61.3) 7.4 (49.3) P21

78.3 116.6 62.1

69.7 61.9 70.3

1

112.7 2

Refinement Statistics resolution (Å)

a

protein atoms (n) water molecules (n) reflections used for Rfree (n)a Rwork (%)a Rfree (%)a root mean square deviation bond length (Å) root mean square deviation bond angle (deg) average B factor (Å2)

64.96-1.40 (1.436-1.400) 2261 273 2643 (122) 16.5 (27.6) 18.6 (29.2) 0.010

28.72-1.70 (1.70-1.74) 4286 333 2927 (159) 17.5 (30.1) 21.0 (37.8) 0.010

1.55

1.54

15.0

23.9

Numbers in brackets are for the highest resolution shells. Rsym ) ∑|I 〈I〉|/∑I, where I is the observed intensity and 〈I〉 is the average intensity from multiple observations of symmetry-related reflections. a

however, based on the enzymatic data ODCase inhibition, certainly is one of the predominant means of activities for these compounds such as 27 and 28. Here, we reveal novel ODCase inhibitors with potent anticancer activities. Although at the enzymatic level, several compounds inhibited ODCase, at the cellular level, only three compounds 27, 28, and 30 showed potential as anticancer agents against various leukemias. Among these compounds, compound 27 is the best compound for further evaluation based on the in vitro data. Experimental Section General. All anhydrous reactions were performed under the nitrogen atmosphere. All solvents and reagents were obtained from commercial sources. Column chromatography was performed using silica gel (60 Å, 70-230 mesh). The NMR spectra were recorded on a Varian spectrometer (300 and 400 MHz for 1H, 75 and 100

MHz for 13C). The chemical shifts are reported in δ ppm using tetramethylsilane as the reference for the 1H and 13C NMR spectra. Mass spectra were obtained on a Q-Star mass spectrometer using either ESI or EI techniques. The monophosphate compounds (free acid forms) were transformed into the corresponding ammonium salts by neutralization with 0.5 M NH4OH solution at 0 °C, followed by lyophilization to obtain the corresponding ammonium salts. ODCase enzyme assays were performed at 55 or 37 °C using a VP-ITC microcalorimeter (MicroCal, Northampton, MA) using previously published procedures.34 Mass spectra for the enzymes and the complexes were obtained in a Q-TOF mass spectrometer with MassLynx software for data analysis (Waters Micromass, Manchester, UK) at the Mass Spectrometry Facility, Advanced Protein Technology Centre, The Hospital for Sick Children, Toronto, Canada, and an AB/Sciex QStar mass spectrometer with an ESI source and on a an Agilent 1100 capillary LC attached (MDS Sciex, USA) at the Mass Spectrometry-AIMS Laboratory, Department of Chemistry, University of Toronto. Synthesis of uridine derivatives 14-18, 21, 22, and 34-38 was already published by our group earlier.36 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluorouridine (44). A stirred suspension of 5-fluorouridine 25 (1 g, 3.8 mmol) in anhydrous acetone (70 mL) was treated with H2SO4 (0.5 mL) dropwise at 0 °C. The reaction mixture was then stirred at rt for an hour. The reaction was then neutralized with triethylamine and was concentrated. The crude mixture was purified by column chromatography (5-8% MeOH:CHCl3) to afford 2′,3′-O-isopropylidene-5-fluorouridine (1.15 g, quantitative) as a white solid. A solution of this isopropylidene-protected derivative (0.5 g, 1.7 mmol) in anhydrous CH2Cl2 (20 mL) was treated with imidazole (225 mg, 3.3 mmol) and TBDMSCl (250 mg, 1.7 mmol) at 0 °C. The reaction mixture was brought to rt and was stirred for an hour. The solvent was evaporated under vacuum, and the crude was dissolved in ethyl acetate (30 mL), washed with water (15 mL), brine (15 mL), and dried (Na2SO4). Evaporation of the solvent and purification of the crude by column chromatography (30% EtOAc: Hx) yielded compound 44 (674 mg, 98%) as a foam. 1H NMR (CDCl3) 9.54 (s, 1H), 7.78 (d, J ) 6 Hz, 1H), 5.91 (d, J ) 1.6 Hz, 1H), 4.66 (dd, 2.4, 5.6 Hz, 1H), 5.56 (dd, J ) 2.8, 5.6 Hz), 4.27 (m, 1H), 3.86 (dd, J ) 1.6, 11.6 Hz), 3.72 (dd, J ) 2, 11.6 Hz, 1H), 1.50 (s, 3H), 1.27 (s, 3H), 0.82 (s, 9 H), 0.02 (s, 6H). 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6iodouridine (45).38 Compound 44 (250 mg, 0.6 mmol) was dissolved in 3 mL anhyd THF at -78 °C and was treated with LDA (0.9 mL, 1.8 mmol, 2.0 M solution in THF). After stirring for an hour, iodine (152 mg, 0.6 mmol) dissolved in anhyd THF (2 mL) was added and the mixture was stirred for an additional 5 h in dark. The reaction was quenched with water (0.5 mL) and then brought to rt and dissolved in ethyl acetate (25 mL). The organic layer was washed with water (10 mL), brine (10 mL), and dried (Na2SO4). Evaporation of the solvent and purification of the crude by column chromatography (30% EtOAc:Hx) provided compound 45 (310 mg, 95%) as an yellow foam. 1H NMR (CDCl3) 9.47 (s, 1H), 6.09 (d, J ) 1.6 Hz, 1H), 5.20 (dd, J ) 1.6, 6.8 Hz,


Bello et al.

Table 4. Anticancer Activities (IC50) for Compounds 8, 9, and 14-30 in Various Cell Linesa IC50 (µM) compd

OCI-AML-1

OCI-AML-2

OCI-Ly-7

OCI-My-2

SKW3

MCF7

LOX

MDA 468

OVCR 4

COLO 205

8 9 14 15 16 17 18 19 20 21 22 23 24 26 27 28 29 30 31 32 33

6.1 >50 >50 15.3 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 0.6 0.7 2.1 0.3 >50 15.2 >50

16 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 0.7 0.9 ND 0.7 >50 11.7 >50

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 0.7 1.3 1.9 0.6 >50 36.2 >50

18.7 >50 >50 27.8 >50 >50 >50 42.3 >50 >50 >50 >50 >50 >50 0.6 0.9 1.4 0.4 >50 1.3 >50

2.8 >50 >50 18.9 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 0.4 0.6 1.2 0.3 >50 1.6 >50

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 1.0 1.4 31.4 23.7 >50 4.4 >50

6.7 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 2.3 3.4 4.5 0.4 >50 1.1 >50

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 1.8 2.6 5.3 1.9 >50 >50 >50

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 NA NA >50 1.5 2.0 ND ND >50 >50 >50

>50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 NA NA >50 3.8 3.3 ND ND >50 >50 >50

a The details of the cell lines used in this study are detailed in Supporting Information Table S-1. There is no activity detected in CCD 967, HS58T, T47D, PC3, and SNB19 cell lines (for compounds 27, 28 up to 4 µM, and for all other compounds up to 50 µM).

Figure 7. Apoptosis as a result of treatment with compounds 27, 28, and 30 in various cancer cell lines. The increase in apoptotic cells as seen through the annexin V+/PI- stain in comparison to control cells when treated with each compound on days 1, 2, and 3.

1H), 4.81 (dd, J ) 4.4, 6.4 Hz, 1H), 4.18 (m, 1H), 3.79 (dd, J ) 2.4, 6 Hz, 2H), 1.56 (s, 3H), 1.35 (s, 3H), 0.89 (s, 9H), 0.05 (s, 6H). 5-Fluoro-6-iodouridine (26).38 A stirred suspension of compound 45 (300 mg, 0.55 mmol) in water (2 mL) was treated with 50% aqueous TFA (2 mL) at 0 °C, brought to room temperature, and stirred for an additional 2 h, in dark. The reaction mixture was concentrated under vacuum and was purified by column chromatography (10-15% EtOH:CHCl3) to afford compound 26 (193 mg, 90%) as a light-yellow solid. UV (H2O) λmax ) 272 nm; ε272 7440. 1 H NMR (CD3OD) 5.95 (d, J ) 3.2 Hz, 1H), 4.73 (dd, 3.2, 6.4 Hz, 1H), 4.33 (t, 6.4 Hz, 1H), 3.89 (m, 1H), 3.80 (dd, J ) 2.8, 12 Hz, 1H), 3.67 (dd, 6, 12 Hz). 13C NMR (CD3OD) δ 154.8 (2JCF ) 30.3 Hz), 147.5, 144.0 (1JCF ) 228.4 Hz), 103.9 (2JCF ) 39.0 Hz), 102.0, 85.1, 72.1, 70.1, 62.5. HRMS (ESI, +ve): calculated (M + Na+) C9H10N2O6FNaI, 410.9459; found, 410.9448. 5-Fluoro-6-iodouridine-5′-O-monophosphate (40). A stirred solution of H2O (34 mg, 1.9 mmol) and POCl3 (0.3 mL, 3 mmol) in anhydrous acetonitrile (3 mL) was treated with pyridine (0.3 mL, 3 mmol) at 0 °C and stirred for 10 min. Compound 26 (260 mg, 0.7 mmol) was then added and the mixture was stirred for an additional 5 h at 0 °C in dark. The reaction mixture was then

quenched with 25 mL of cold water and stirring was continued for an additional hour. The evaporation of the solvent and purification of the crude by column chromatography (Dowex ion-exchange basic resin, 0.1 M formic acid) afforded compound 40 as syrup. Free acid form was transformed into the corresponding ammonium salt by neutralization with 0.5 M NH4OH solution at 0 °C to yield compound 40 as powder (202 mg, 60%). UV (H2O) λmax ) 271 nm; ε271 ) 6400. 1H NMR (D2O) 5.89 (d, J ) 3.1 Hz, 1H), 4.7 (m, 1H), 4.31 (t, J ) 6.9 Hz, 1H), 4.01-3.42 (m, 3H). HRMS (ESI, +ve): calculated (M + H+) C9H10N2O9FPI, 466.9158; found, 466.9145. 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6azido Uridine (46). Compound 45 (260 mg, 0.5 mmol) was dissolved in anhydrous DMF (3 mL) and NaN3 (34 mg, 0.5 mmol) was added. The reaction mixture was stirred at room temperature for two hours in dark. The reaction mixture was concentrated under vacuum at room temperature, and the crude was dissolved in ethyl acetate (15 mL), washed with brine, and dried (Na2SO4). The combined organic layers were evaporated, and the residue was purified by silica gel column chromatography (1% MeOH:CHCl3) to yield compound 46 (201 mg, 92%) as a light-yellow solid. 1H NMR (CDCl3) 9. 47 (s, 1H), 6.07 (d, J ) 1.6 Hz, 1H), 5.13 (dd, J ) 1.2, 6.8 Hz, 1H), 4.78 (dd, J ) 84.8, 6.4 Hz, 1H), 4.12 (m, 1H), 3.79 (m, 2H), 1.55 (s, 3H), 1.34 (s, 3H), 0.89 (s, 9H), 0.06 (s, 6H). 9. 47 (s, 1H), 6.07 (d, J ) 1.6 Hz, 1H), 5.13 (dd, J ) 1.2, 6.8 Hz, 1H), 4.78 (dd, J ) 84.8, 6.4 Hz, 1H), 4.12 (m, 1H), 3.79 (m, 2H), 1.55 (s, 3H), 1.34 (s, 3H), 0.89 (s, 9H), 0.06 (s, 6H). 5-Fluoro-6-azido Uridine (27). A stirred solution of compound 46 (200 mg, 0.4 mmol) was treated with 50% aqueous trifluoroacetic acid (4 mL) at 0 °C. The reaction mixture was then brought to room temperature and was stirred for an additional two hours in the dark. Evaporation of the solvent and purification of the crude by silica gel column chromatography (10-15% MeOH: CHCl3) afforded the product 27 (121 mg, 89%) as a light-yellow solid. UV (H2O) λmax ) 291 nm; ε291 ) 8905. 1H NMR (CD3OD) 5.88 (d, J ) 2.8 Hz, 1H), 4.58 (dd, J ) 4, 6 Hz, 1H), 4.28 (t, J ) 6.8 Hz, 1H), 4.23 (dd, J ) 3.2, 6.4 Hz, 1H), 4.00 (m, 1H), 3.79 (m, 2H). 13C NMR (CD3OD) δ 156.4 (2JCF ) 23.4 Hz), 148.0, 137.1 (1JCF ) 212.7 Hz), 132.8, 91.3, 84.5, 71.9, 69.3, 61.7. HRMS (ESI, +ve): calculated (M + Na+) C9H10N5O6FNa, 326.0507; found, 326.0519. 5-Fluoro-6-azidouridine-5′-O-monophosphate (41). A stirred solution of water (30 mg, 1.9 mmol) and POCl3 (0.3 mL, 3 mmol) in anhydrous acetonitrile (3 mL) was treated with pyridine (0.3 mL, 3.2 mmol) at 0 °C and was stirred for 10 min. Compound 27


was then added (206 mg, 0.7 mmol) to the reaction mixture and was stirred for an additional 5 h at 0 °C in dark. The reaction mixture was quenched with 2.5 mL of cold water. Evaporation of the solvent and the purification of the crude by column chromatography (Dowex ion-exchange basic resin, 0.1 M formic acid) gave the mononucleotide as a syrup. This was transformed into the corresponding ammonium salt by neutralization with 0.5 M NH4OH solution at 0 °C and freeze-dried to yield compound 41 as a white powder (0.199 mg, 70%). UV (H2O) λmax ) 286 nm; ε286 ) 1585. 1 H NMR (D2O) 5.01 (bs, 1H), 4.42 (m, 1H), 4.08 (m, 2H), 3.95 (m, 1H), 3.51 (m, 1H). HRMS (ESI, +ve): calculated (M + H+) C9H10N5O9FP, 382.0205; found, 382.0196. 5-Fluoro-6-aminouridine (28). Compound (27) (200 mg, 0.66 mmol) was dissolved in methanol and 10% Pd/C (10 mg) was added. The reaction mixture was stirred for 3 h in dark under a hydrogen atmosphere at rt. The mixture was filtered through celite, and the solvent was evaporated to dryness to give compound 28 as a white solid (165 mg, 90%). UV (H2O) λmax ) 281 nm; ε281 ) 17806. 1H NMR (CD3OD) 6.36 (dd, J ) 1.2, 7.6 Hz, 1H), 4.61 (t, J ) 4, 6 Hz, 1H), 4.28 (t, 6 Hz, 1H), 3.77 (m, 2H), 3.62 (dd, J ) 6, 12.8 Hz, 1H). 13C NMR (CD3OD) δ 156.2, 149.6, 125.8, 123.1, 89.2, 85.5, 70.1, 69.8, 60.5. HRMS (ESI, +ve): calculated (M + Na+) C9H12N3O6FNa, 300.0602; found, 300.0608. 5-Fluoro-6-aminouridine-5′-O-monophosphate (42). The mononucleotide 41 (R1 ) -PO42-) (120 mg, 0.29 mmol) was dissolved in 50% aqueous methanol and 10% Pd/C (10 mg) was added. A procedure similar to that for 41 was used to obtain compound 42 as a white solid (101 mg, 90%). 1H NMR (D2O) δ 5.92 (bs, 1H), 4.53 (m, 1H), 4.12 (m, 2H), 3.86 (m, 2H). HRMS (ESI, +ve): calculated (M + H+) C9H12N3O9FP, 356.0300; found, 56.0301. 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6ethyluridine (47). Compound 44 (250 mg, 0.6 mmol) was dissolved in 3 mL of anhyd THF at -78 °C and was treated with a solution of LDA (1.5 mL, 3.0 mmol, 2.0 M solution in THF). After stirring for 1 h, CH3I (225 mg, 1.8 mmol) in anhyd THF (1 mL) was added, and the mixture was stirred for an additional 5 h at the same temperature. The reaction was quenched with water (0.5 mL) and then brought to room temperature and dissolved in ethyl acetate (25 mL). The organic layer was washed with water (10 mL), brine (10 mL), and dried (Na2SO4). Evaporation of the solvent and purification of the crude by column chromatography (hexanes: EtOAc, 70:30) yielded compound 47 (187 mg, 70%) as a white foam. 1H NMR (CDCl3) 9.60 (s, 1H), 5.60 (d, J ) 1.3 Hz, 1H), 5.20 (dd, J ) 1.4, 6.5 Hz, 1H), 4.81 (dd, J ) 4.5, 6.4 Hz, 1H), 4.16 (m, 1H), 3.81 (m, 2H), 2.86-2.67 (m, 2H), 1.55 (s, 3H), 1.345 (s, 3H), 1.31 (t, J ) 8.3 Hz, 3H), 0.88 (s, 9H), 0.04 (s, 6H). 5-Fluoro-6-ethyluridine (29). A stirred suspension of compound 47 (0.200 g, 0.45 mmol) in water (2 mL) was treated with 50% aqueous TFA (2 mL) at 0 °C and the reaction mixture was stirred for 2 h at rt. Evaporation of the solvent and purification of the crude by column chromatography (10-15% MeOH:CHCl3) afforded compound 29 (118 mg, 94%) as a light-yellow solid. UV (H2O) λmax ) 269 nm; ε269 ) 8205. 1H NMR (CD3OD) δ 5.45 (d, J ) 3.6 Hz, 1H), 4.77 (dd, J ) 4.1, 6.3 Hz, 1H), 4.31 (t, J ) 6.1 Hz, 1H), 3. 90 (m, 1H), 3.80 (dd, J ) 3, 11.9, 1H), 3.66 (dd, J ) 5.4, 11.9 1H), 2.78 (m, 2H), 1.29 (t, J ) 7.3 Hz, 3H). 13C NMR (CD3OD) δ 157.8, 149.7, 143.8 (2JCF ) 24.3 Hz), 137.8 (1JCF ) 226.8 Hz), 92.9, 85.2, 71.6, 70.2, 62.5, 18.5, 11.4. HRMS (ESI, +ve): calculated (M + H+) C11H16N2O6F, 291.0986; found, 291.0986. 5-Fluoro-6-ethyluridine-5′-O-monophosphate (43). A stirred solution of H2O (34 mg, 1.9 mmol) and POCl3 (0.3 mL, 3 mmol) in anhyd acetonitrile (3 mL) was treated with pyridine (0.3 mL, 3.3 mmol) at 0 °C and stirred for 10 min. Compound 29 (195 mg, 0.7 mmol) was added, and the mixture was stirred for an additional 5 h at 0 °C. The reaction mixture was then quenched with cold water (25 mL) and stirring was continued for an additional hour. Evaporation of the solvent and purification of the crude by column chromatography (Dowex ion-exchange basic resin, 0.1 M formic acid) afforded the free acid form of 43 as a syrup. The product was transformed into the corresponding ammonium salt to afford


a white powder (222 mg, 82%). UV (H2O) λmax ) 265 nm; ε265 ) 3018. 1H NMR (D2O) 5.39 (bs, 1H), 4.73 (m, 1H), 4.30 (t, J ) 6.0 Hz, 1H), 3.89 (m, 2H), 3.82 (m, 1H), 2.77 (m, 2H), 1.30 (t, J ) 7.1 Hz, 3H). HRMS (ESI, +ve): calculated (M + H+) C11H15N2O9FP, 369.0504; found, 369.0505. 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-5-fluoro-6carbaldehydeuridine (48). Compound 44 (250 mg, 0.6 mmol) dissolved in 3 mL of anhyd THF at -78 °C and was treated with a solution of LDA (1.5 mL, 3.0 mmol, 2.0 M solution in THF). After stirring for an hour, methyl formate (54 mg, 0.9 mmol) in anhyd THF (1 mL) was added and the mixture was stirred for an additional 5 h at the same temperature. The reaction was quenched with water (0.5 mL), brought to room temperature, and dissolved in ethyl acetate (25 mL). The organic layer was washed with water (10 mL), brine (10 mL), and dried (Na2SO4). Evaporation of the solvent and purification of the crude by column chromatography (MeOH:CHCl3, 5:95) gave 48 (0.187 g, 70%) as a brown foam. 1 H NMR (CDCl3) δ 10.1 (d, J ) 0.8 Hz, 1H), 6.25 (d, J ) 1.6 Hz, 1H), 5.10 (dd, J ) 4, 8 Hz, 1H), 4.74 (dd, J ) 4, 8 Hz, 1H), 4.09 (m, 1H), 3.82 (dd, J ) 4, 12 Hz, 1H), 3.75 (dd, J ) 8, 12 Hz, 1H), 1.56 (s, 3H), 1.36 (s, 3H), 0.90 (s, 9H), 0.07 (s, 6H). 5-Fluoro-6-carbaldehyde Uridine (30). A stirred suspension of compound 48 (200 mg, 0.5 mmol) in water (2 mL) was treated with 50% aqueous TFA (2 mL) at 0 °C and was stirred at room temperature for 2 h. Evaporation of the solvent and the purification of the crude by silica gel column chromatography (10-15% MeOH: CHCl3) afforded compound 30 (115 mg, 88%) as a light-brown solid. UV (H2O) λmax ) 269 nm; ε269 ) 5734. 1H NMR (D2O) 6.26 (d, 1H), 6.92 (dd, 1H), 6.58 (m, 1H), 6.38 (t, 1H), 3.79 (m, 2H), 3.69 (m, 1H). 13C NMR (D2O) δ 159.1, 149.8, 138.7, 137.4 (1JCF ) 225.0 Hz), 93.5, 89.0, 83.3, 72.1, 69.2, 61.5. HRMS (ESI, +ve): calculated (M + H+) C10H12N2O7F, 291.0623; found, 291.0600. Enzymology. ODCase from human (Hs) or from Methanobacterium thermoautotrophicum (Mt) was used in enzyme activity studies. Hs ODCase is a C-terminal portion of the bifunctional enzyme UMP synthase. Reversible Inhibition. The inhibition of Mt ODCase by various inhibitors was evaluated in a competitive inhibition assay as described previously.34 The activity of 20 nM ODCase was monitored at 55 °C. The substrate concentration was 40 µM. The concentrations of 42 were 0, 10, 20, 35, and 50 µM and for 41 were 0, 0.25, 0.50, 0.75, and 1.0 µM. The concentration of 43 in the assay samples was 0, 20, 40, 100, and 200 µM. The concentration of 39 was 0, 0.5, 1.0, 2.5, and 4.0 mM and was evaluated as a competitive inhibitor of Mt ODCase. The reversible inhibition of human ODCase was studied at 37 °C, as described previously.39 The enzyme stock was diluted with 50 mM Tris buffer containing 1 mM DTT to prepare 60 nM enzyme assay samples. The final substrate concentration was either 15 or 20 µM. Concentrated inhibitors stock solutions were prepared in 50 mM Tris (pH 7.5). All four compounds were tested in a competitive inhibition assay where enzyme was mixed with the inhibitor and the reaction was initiated by the substrate addition. The final assay concentrations of 41 were 0, 0.25, 0.50, 0.75, and 1.0 µM, for 42 were 0, 10, 25, 50, and 120 µM, for 43, they were 0, 0.2, 0.4, 1.0, and 2.0 µM and for 39, 0, 50, 100, 250, and 500 µM. Time-Dependent Inhibition. The inactivation of human ODCase by 40 was monitored at 37 °C following the coinjection of the substrate and inhibitor into the calorimetric cell containing the enzyme. Concentrated enzyme sample (60 µM) was prepared in 50 mM Tris (pH 7.5), 20 mM DTT, and 40 mM NaCl and incubated overnight at 4 °C. The assay samples were prepared in 50 mM Tris, 1 mM DTT to obtain 60 nM enzyme concentration. Substrate and the inhibitor were mixed and loaded into the automatic injection syringe. The substrate concentration was kept constant while the final concentration of the inhibitor was varied. A single, 5.7 µL injection of the ligands’ mixture resulted in 0.5, 1.0, 1.5, 1.8, or 2.5 µM concentration of 40, and 20 µM OMP.


Bello et al.

Second-order rate of inactivation of Hs ODCase by 40 was studied in a time-dependent assay. The incubation samples of the enzyme (control) or the enzyme with inhibitor were set up at room temperature, and the remaining enzymatic activity of ODCase was monitored for up to 48 h. All samples were prepared in the buffer (pH 7.5) consisted of 50 mM Tris, 20 mM DTT, and 40 mM NaCl. The control reaction contained 60 µM Hs ODCase. The initial concentration of the enzyme in the inactivation samples was also 60 µM, and of 40 was varied from 60, 120, 150, or 300 µM. All samples were incubated for 0.25, 0.5, 1, 2, 4, 6, 24, and 48 h. At each incubation time point, 2.5 µL aliquots were removed from the concentrated samples and diluted to 2.5 mL in 50 mM Tris, 1 mM DTT. The remaining activity of ODCase was measured after 5.7 µL injection of 5 mM OMP that resulted in 20 µM final substrate concentration. Inactivation of Hs ODCase was investigated in the presence of 41 by coinjecting the substrate OMP and the inhibitor. Final substrate concentration was 20 µM in all assays. The concentrations of the inhibitor 41 after the 5.7 µL injection (containing the substrate and inhibitor) in the reaction cell were (control), 25, 35, 50, and 75 µM. Data Analysis. The initial data analyses for the reversible inhibition were performed using Origin 7.0 software. The raw data representing the heat changes over time were converted into the reaction rate at each recorded time point. The raw data representing the rate of decarboxylation at several substrate concentrations in the absence or presence of the inhibitors were directly fitted to eq 1 to calculate the reversible inhibition constant, Ki.

V) KM

(

Vmax[S] [I] 1+ + [S] Ki

)

(1)

To determine the inactivation of Hs ODCase following the coinjection of the substrate and the inhibitor, kobs was first computed from each progress curve. The value (Power, µcal/s) at each inflection point was normalized by setting the inflection point value to zero. The data representing the change in thermal power over time were fitted to the eq 2 to calculate kobs.

[P] )

Vi (1 - exp(-kobst)) kobs

(2)

where [P] represents the product concentration, Vi is the initial reaction rate, and t is time. The calculated kobs and the inhibitor concentrations [I] were used to calculate the inactivation constant KI and the rate of inactivation kinact from the equation:

kobs )

kinact[I] KI + [I]

(3)

To analyze the time-dependent inactivation of Hs ODCase, kobs was first determined at each inhibitor concentration. The rate constant for loss of enzyme activity, kobs, was determined from the slope of ln(% remaining enzyme activity) versus time. The kobs values were then plotted against the inhibitor concentration in the incubation samples ([I]). The potency of each inhibitor in terms of the second-order rate constant, kobs/[I] (M-1 s-1), was calculated from the slope of the linear fit of data. Anticancer Activity Evaluation. Cell Lines and Cell Culture Conditions. All cell lines were grown in Iscove’s MDM (GIBCO no. 12440) supplemented with 5% fetal calf serum (FCS), 50 µM 2-mercaptoethanol, 100 µg/mL penicillin, and 100 µg/mL streptomycin. Cells lines growing in suspension were maintained at 0.5-1.5 × 106 cell/mL, while adherent cell lines were maintained subconfluent. Cells were kept in culture for ca. 10 weeks. Isolation and Culture of Human Peripheral Blood Mononuclear Cells. The protocol for informed consent and the use of human blood was approved by the Ethics Review Committees at both the University Health Network and St. Michael’s Hospital in

Toronto, Canada. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by differential density centrifugation over Ficoll-Hypaque (Amersham Biosciences) according to the manufacturer’s instructions. The blood was collected from two healthy donors at the Ontario Cancer Institute, University Health Network. Briefly, collected blood was diluted in phosphate buffered saline (PBS) (1:1). A layer of Ficoll-Hypaque was carefully overlaid with diluted blood to a final concentration of 1:1. Cells were spun at 2000 rpm at room temperature for 30 min. Buffy coat, containing PBMCs, was removed and washed twice in PBS. PBMCs were then stimulated with 3 µg/mL of Concanavalin A and seeded to be used in WST.1 assay as described below. In Vitro Cell Proliferation WST.1 Assay. Cell lines were seeded in triplicate at 104 cells/100 µL in 96-well plates. Peripheral blood mononuclear cells were seeded in triplicate at 105 cells/100 µL in 96-well plates. Inhibitors were added immediately, at final concentrations ranging between 0 to 100 µM. All compounds were dissolved in sterile deionized water. On day 3, WST.1 assay kit (Roche Biosciences) was used to measure the effect of inhibitors on the cell growth. WST.1 assay relies on the cleavage of WST.1 (water soluble tetrazolium salt: 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) to formazan by cellular enzymes. The amount of formazan dye formed correlates to the number of metabolically active cells in the culture and was read in an ELISA microplate reader at 440 nm. These OD values were plotted against concentrations of different compounds and the inhibition constants (IC50) were determined for each compound. Apoptosis Assay. Cells were seeded in duplicate at 105 cells/ mL in 24-well plates. Immediately after seeding, the inhibitors (27, 28, and 30) were added to obtain the final concentration of 4 µM. Water was added to the control wells. Cells were harvested every 24 h, washed in PBS, and stained with Annexin V/propidium iodide (PI) (1:200) using Annexin-V-FLUOS staining kit (Roche). Annexin V is a Ca2+-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS). It is used as a probe to detect PS exposure on the outer leaflet of the cell membrane and is therefore widely used for detection of early apoptotic cells. Unlike apoptotic cells (Annexin V+/PI-), necrotic cells are Annexin V+/ PI+. FACS was used to analyze the staining due to Annexin V and PI. X-ray Crystallography. All Hs ODCase concentrations were determined using a BioRAD protein assay kit and BSA as a standard. ODCase protein was dissolved in 20 mM Tris (pH 8.4), 10 mM NaCl, and 1 mM TCEP-HCl. Hs ODCase complex crystals were grown in hanging drops using 2.4 M ammonium sulfate, pH 8.4, as the main precipitant. To obtain larger single crystals of the enzyme complexes, microseeding was performed the following day. Crystals of the complexes with compound 41 grew in space group P21 with unit cell dimensions of close to a ) 69.6 Å, b ) 61.7 Å, c ) 70.7 Å, β ) 112.2°. The crystals of the complex of compound 40 with the enzyme adopted space group C2221. For data collection, the crystals were cryo-protected by Parotone-N oil before being flash-frozen in a stream of boiling nitrogen. Diffraction data for the crystals of HsODCase cocrystallized with compound 41 were collected at 100 K and λ ) 1.10 Å on beamline X08C, Brookhaven National Laboratory; those for the other two complexes were from beamline 08ID-1 at the Canadian Macromolecular Crystallography Facility, Canadian Light Source, Inc., collected also at 100 K but at λ ) 0.97034 Å. All data were reduced and scaled using HKL2000.40 The structures of all complexes were determined using molecular replacement techniques with the help of the program package MOLPREP;41 subsequent refinements were done with Refmac-5.2,42 and model building used COOT.43 Data collection and refinement statistics are given in Table 3. Atomic coordinates and structure factors have been deposited into the Protein Data Bank (PDB IDs: 3G3M, and 3G3D). Mass Spectral Analyses. The concentrations of 40 and 41 for incubation with Hs ODCase for mass spectral analyses were 22.6 and 80 mM, respectively. The controls and the samples containing the inhibitors were analyzed 2 h after the onset of incubation at room temperature and then again after 24 h incubation. The samples


with Mt ODCase were diluted in water from a 2 mM stock of the enzyme. The initial enzyme concentration in all samples, including control, was 300 µM. The concentration of 41 was 170 mM, while 40 was added to the enzyme samples to obtain final concentration of 42.5 mM. These samples were incubated at room temperature for 24 h before the mass spectral analyses were performed.

Acknowledgment. This work was supported by grant no. 200611PPP from the Canadian Institutes of Health Research, Proof-of-Principle program (L.P.K., C.P.J., and E.F.P.). L.P.K. is a recipient of an Rx&D HRF-CIHR research career award. E.F.P. thanks the Canada Research Chair Program for support. We thank staff at the NSLS beamline X8C and the Canadian Macromolecular Crystallography Facility for their time commitments and expert help. A joint grant from the Canadian Institutes of Health Research and the National Sciences and Engineering Research Council of Canada enabled the use of beamline X8C at the National Synchrotron Light Source, Brookhaven National Laboratory. Part of the research described in this paper was performed at the Canadian Light Source, which is supported by NSERC, NRC, CIHR, and the University of Saskatchewan. Supporting Information Available: Purity data, details of the cancer cell lines, thermograms/enzyme inhibition kinetics profiles, and mass spectral analyses of the enzyme-inhibitor covalent complexes. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Galmarini, C. M.; Popowickz, F.; Joseph, B. Cytotoxic nucleoside analogs: different strategies to improve their clinical efficacy. Curr. Med. Chem. 2008, 15, 1072–1082. (2) Uga, H.; Kuramori, C.; Ohta, A.; Tsuboi, Y.; Tanaka, H.; Hatakeyama, M.; Yamaguchi, Y.; Takahashi, T.; Kizaki, M.; Handa, H. A new mechanism of methotrexate action revealed by target screening with affinity beads. Mol. Pharmacol. 2006, 70, 1832–1839. (3) Warshel, A.; Florian, J. Computer simulations of enzyme catalysis: finding out what has been optimized by evolution. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5950–5955. (4) Miller, B. G.; Wolfenden, R. Catalytic proficiency: The unusual case of OMP decarboxylase. Annu. ReV. Biochem. 2002, 71, 847–885. (5) Sievers, A.; Wolfenden, R. Equilibrium of formation of the 6-carbanion of UMP, a potential intermediate in the action of OMP decarboxylase. J. Am. Chem. Soc. 2002, 124, 13986–13987. (6) Snider, M. J.; Wolfenden, R. The rate of spontaneous decarboxylation of amino acids. J. Am. Chem. Soc. 2000, 122, 11507–11508. (7) Richard, P. The enzymatic synthesis of pyrimidines. AdV. Enzymol. Mol. Biol. 1959, 21, 263–294. (8) Meza-Avina, M. E.; Wei, L.; Buhendwa, M. G.; Poduch, E.; Bello, A. M.; Pai, E. F.; Kotra, L. P. Inhibition of orotidine 5′-monophosphate decarboxylase and its therapeutic potential. Mini-ReV. Med. Chem. 2008, 8, 239–247. (9) Christopherson, R. I.; Lyons, S. D.; Wilson, P. K. Inhibitors of de novo nucleotide biosynthesis as drugs. Acc. Chem. Res. 2002, 35, 961– 971. (10) Scott, H. V.; Gero, A. M.; O’Sullivan, W. J. In vitro inhibition of Plasmodium falciparum by Pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo. Mol. Biochem. Parasitol. 1986, 18, 3–15. (11) Levine, H. L.; Brody, R. S.; Westheimer, F. H. Inhibition of orotidine5′-phosphate decarboxylase by 1-(5′-phospho-b-D-ribofuranosyl)barbituric acid, 6-azauridine-5′-phosphate, and uridine 5′-phosphate. Biochemistry 1980, 19, 4993–4999. (12) Gero, A. M.; O’Sullivan, W. J. Purines and pyrimidines in malarial parasites. Blood Cells 1990, 16, 467–484. (13) Seymour, K. K.; Lyons, S. D.; Phillips, L.; Rieckmann, K. H.; Christopherson, R. I. Cytotoxic effects of inhibitors of de novo pyrimidine biosynthesis upon Plasmodium falciparum. Biochemistry 1994, 33, 5268–5274. (14) Krungkrai, J.; Krungkrai, S. R.; Phakanont, K. Antimalarial activity of orotate analogs that inhibit dihydroorotase and dihydroorotate dehydrogenase. Biochem. Pharmacol. 1992, 43, 1295–1301. (15) Smiley, J. A.; Saleh, L. Active site probes for yeast OMP decarboxylase: Inhibition constants of UMP and thio-substituted UMP analogues and greatly reduced activity toward CMP-6-carboxylate. Bioorg. Chem. 1999, 27, 297–306.

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1657 (16) Gabrielsen, B.; Kirsi, J. J.; Kwong, C. D.; Carter, D. A.; Krauth, C. A.; Hanna, L. K.; Huggins, J. W.; Monath, T. P.; Kefauver, D. F.; Blough, H. A.; Rankin, J. T.; Bartz, C. M.; Huffman, J. H.; Smee, D. F.; Sidwell, R. W.; Shannon, W. M.; Secrist, J. A. In vitro and in vivo antiviral (RNA) evaluation of orotidine 5′-monophosphate decarboxylase inhibitors and analogs including 6-azauridine-5′-(ethyl methoxyalaninyl)phosphate (a 5′-monophosphate prodrug). AntiViral Chem. Chemother. 1994, 5, 209–220. (17) Nord, L. D.; Willis, R. C.; Smee, D. F.; Riley, T. A.; Revankar, G. R.; Robins, R. K. Inhibition of orotidylate decarboxylase by 4(5H)-oxo1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidine-3-thiocarboxamide (APR-TC) in B lymphoblasts. Activation by adenosine kinase. Biochem. Pharmacol. 1988, 37, 4697–4705. (18) Smee, D. F.; McKernan, P. A.; Nord, L. D.; Willis, R. C.; Petrie, C. R.; Riley, T. M.; Revankar, G. R.; Robins, R. K.; Smith, R. A. Novel pyrazolo[3,4-d]pyrimidine nucleoside analog with broadspectrum antiviral activity. Antimicrob. Agents Chemother. 1987, 31, 1535–1541. (19) Jones, M. E. Pyrimidine nucleotide biosynthesis in animals: Genes, enzymes, and regulation of UMP biosynthesis. Annu. ReV. Biochem. 1980, 49, 253–279. (20) Chen, J. J.; Jones, M. E. Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. Orotate inhibition of dihydrorotase and dihydroorotase dehydrogenase. J. Biol. Chem. 1979, 254, 4908–4914. (21) Cadman, E. C.; Dix, D. E.; Handschumacher, R. E. Clinical, biological and biochemical effect of Pyrazofurin. Cancer Res. 1978, 38, 682– 698. (22) Cihak, A.; Veseley, J.; Skoda, J. Azapyrimidine nucleosides: metabolism and inhibitory mechanism. AdV. Enzyme Regul. 1985, 24, 235– 254. (23) Miller, B. G.; Hassell, A. M.; Wolfenden, R.; Milburn, M. V.; Short, S. A. Anatomy of a proficient enzyme: The structure of orotidine 5′monophosphate decarboxylase in the presence and absence of a potential transition-state analog. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2011–2016. (24) Dix, D. E.; Lehman, C. P.; Jakubowski, A.; Moyer, J. D.; Handschumacher, R. E. Pyrazofurin metabolism, enzyme inhibition, and resistance in L5178Y cells. Cancer Res. 1979, 39, 4485–4490. (25) Sant, M. E.; Lyons, S. D.; Kemp, A. J.; McClure, L. K.; Szabados, E.; Christopherson, R. I. Dual effects of Pyrazofurin and 3-deazauridine upon pyrimidine and purine biosynthesis in mouse L1210 leukemia. Cancer. Res. 1989, 49, 2645–2650. (26) Harris, P.; Poulsen, J. C. N.; Jensen, K. F.; Larsen, S. Substrate binding induces domain movements in orotidine 5′-monophosphate decarboxylase. J. Mol. Biol. 2002, 318, 1019–1029. (27) Wu, N.; Mo, Y.; Gao, J.; Pai, E. F. Electrostatic stress in catalysis: structure and mechanism of the enzyme orotidine monophosphate decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2017–2022. (28) Appleby, T. C.; Kinsland, C.; Begley, T. P.; Ealick, S. E. The crystal structure and mechanism of orotidine 5′-monophosphate decarboxylase. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2005–2010. (29) Miller, B. G.; Hassell, A. M.; Wolfenden, R.; Milburn, M. V.; Short, S. A. Anatomy of a proficient enzyme: the structure of orotidine 5′monophosphate decarboxylase in the presence and absence of a potential transition state analog. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2011–2016. (30) Lee, T. S.; Chong, L. T.; Chodera, J. D.; Kollman, P. A. An alternative explanation for the catalytic proficiency of orotidine 5′-phosphate decarboxylase. J. Am. Chem. Soc. 2001, 123, 12837–12848. (31) Miller, B. G.; Butterfoss, G. L.; Short, S. A.; Wolfenden, R. Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5′-phosphate decarboxylase: a role for substrate destabilization. Biochemistry 2001, 40, 6227–6232. (32) Warshel, A.; Strajbl, M.; Villa, J.; Florian, J. Remarkable rate enhancement of orotidine 5′-monophosphate decarboxylase is due to transition-state stabilization rather than to ground-state destabilization. Biochemistry 2000, 39, 14728–14738. (33) Fujihashi, M.; Bello, A. M.; Poduch, E.; Wei, L.; Annedi, S. C.; Pai, E. F.; Kotra, L. P. An unprecedented twist to ODCase catalytic activity. J. Am. Chem. Soc. 2005, 127, 15048–15050. (34) Poduch, E.; Bello, A. M.; Tang, S.; Fujihashi, M.; Pai, E. F.; Kotra, L. P. Design of inhibitors of orotidine monophosphate decarboxylase using bioisosteric replacement and determination of inhibition kinetics. J. Med. Chem. 2006, 49, 4937–4945. (35) Bello, A. M.; Poduch, E.; Fujihashi, M.; Amani, M.; Li, Y.; Crandall, I.; Hui, R.; Lee, P. I.; Kain, K. C.; Pai, E. F.; Kotra, L. P. A potent, covalent inhibitor of ODCase with antimalarial activity. J. Med. Chem. 2007, 50, 915–921. (36) Bello, A. M.; Poduch, E.; Liu, Y.; Wei, L.; Crandall, I.; Wang, X.; Dyanand, C.; Kain, K. C.; Pai, E. F.; Kotra, L. P. Structure-Activity Relationships of C6-Uridine Derivatives Targeting Plasmodia Orotidine Monophosphate Decarboxylase. J. Med. Chem. 2008, 51, 439–448.

1658 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 (37) Damaraju, D.; Damaraju, V. L.; Brun, M.; Mowles, D.; Kuzma, M.; Berendt, R. C.; Sawyer, M. B.; Cass, C. E. Cytotoxic activities of nucleoside and nucleobase analog drugs in malignant mesothelioma: characterization of a novel nucleobase transport activity. Biochem. Pharmacol. 2008, 15, 1901–1911. (38) Tanaka, H.; Matsuda, A.; Iijima, S.; Hayakawa, H.; Miyasaka, T. Synthesis and biological activities of 5-substituted 6-phenylthio and 6-iodouridines, a new class of antileukemic nucleosides. Chem. Pharm. Bull. 1983, 31, 2164–2167. (39) Poduch, E.; Wei, L.; Pai, E. F.; Kotra, L. P. Structural diversity and plasticity associated with nucleotides targeting orotidine monophosphate decarboxylase. J. Med. Chem. 2008, 51, 432–438.

Bello et al. (40) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307– 326. (41) Vagin, A.; Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 1997, 30, 1022–1025. (42) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of macromolecular structures by the maximum-likehood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240–255. (43) Emsley, P.; Cowtan, K. Model building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, D60, 2126–2132.

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