Design, Synthesis, and Pharmacological Evaluation of Fluorinated

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Design, Synthesis, and Pharmacological Evaluation of Fluorinated Tetrahydrouridine Derivatives as Inhibitors of Cytidine Deaminase Dana Ferraris,†,§ Bridget Duvall,†,§ Greg Delahanty,†,§ Bipin Mistry,† Jesse Alt,†,§ Camilo Rojas,†,§ Christopher Rowbottom,‡ Kristen Sanders,‡ Edgar Schuck,‡ Kuan-Chun Huang,‡ Sanjeev Redkar,*,∥ Barbara B. Slusher,†,§ and Takashi Tsukamoto*,†,§ †

Eisai Inc., Baltimore, Maryland 21224, United States Eisai Inc., Andover, Massachusetts 01810, United States § Brain Science Institute and Department of Neurology, Johns Hopkins University, 855 North Wolfe Street, Suite 231, Baltimore, Maryland 21205, United States ∥ Astex Pharmaceuticals, Inc., Dublin, California 94568, United States ‡

ABSTRACT: Several 2′-fluorinated tetrahydrouridine derivatives were synthesized as inhibitors of cytidine deaminase (CDA). (4R)2′-Deoxy-2′,2′-difluoro-3,4,5,6-tetrahydrouridine (7a) showed enhanced acid stability over tetrahydrouridine (THU) 5 at its Nglycosyl bond. As a result, compound 7a showed an improved oral pharmacokinetic profile with a higher and more reproducible plasma exposure in rhesus monkeys compared to 5. Co-administration of 7a with decitabine, a CDA substrate, boosted the plasma levels of decitabine in rhesus monkeys. These results demonstrate that compound 7a can serve as an acid-stable alternative to 5 as a pharmacoenhancer of drugs subject to CDA-mediated metabolism.

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INTRODUCTION Cytidine deaminase (CDA, EC 3.5.4.5) catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively.1 Because of the structural similarity to cytidine, several nucleoside-based drugs are also subject to deamination by CDA. These drugs include not only cytarabine 1 and gemcitabine 2 bearing a cytosine ring but also decitabine 3 and azacitidine 4 containing an azacytosine ring (Figure 1).

Compound 2 exhibited poor oral availability due to its extensive first-pass metabolism by CDA when given to patients with advanced or metastatic cancer refractory to standard therapy.5 One of the options to improve the oral bioavailability of these cytidine-based drugs is to cotreat with a CDA inhibitor which blocks the deamination of the active drugs in the intestine, liver, blood, and kidneys, resulting in increased plasma exposure. The early efforts on this approach have utilized tetrahydrouridine 5 (THU, Figure 2), one of the first potent CDA inhibitors identified by a group at the Upjohn Company.6 Oral coadministration of 1 and 5 resulted in increased plasma levels of 1 and enhanced therapeutic effect in a murine L1210 leukemia model compared to oral administration of compound 1 alone.7 Similarly, oral coadministration

Figure 1. Nucleoside-based drugs that are metabolized by cytidine deaminase (CDA).

Because CDA is present in the liver and kidneys of humans,2 its enzymatic action has significant effects on the pharmacokinetics of these drugs. For example, intravenously administered 1 is rapidly metabolized by CDA to uracil arabinoside, a biologically inert product.3 Effects of CDA are even more significant when the drugs are given orally and undergo first-pass metabolism. For instance, in a phase I study of oral 4, all pharmacokinetic parameters displayed a high degree of interpatient variability and the mean AUC value following the highest oral dose (600 mg) was less than half of the mean AUC value achieved by subcutaneous dose (75 mg/m2) currently used clinically.4 © 2014 American Chemical Society

Figure 2. Structures of tetrahydrouridine (THU) 5 and its degradant, β-ribopyranosyl form. Received: December 3, 2013 Published: February 12, 2014 2582

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Scheme 1. Synthesis of Fluorinated Derivatives of 5a

a

Reagents and conditions: (a) 5% Rh/C, H2 (40 psi) H2O, 61−100%; (b) NaBH4, MeOH, 35−77% combined yield.

Figure 3. The ORTEP drawing of compound 7a showing the thermal ellipsoids at the 50% probability level.

only a minor role in the interaction made by 5 with the active site of CDA. Furthermore, 2′-deoxytetrahydrouridine was recently reported to be a more potent inhibitor than 5 in the human CDA assay.16 Herein, we describe 2′-fluorinated tetrahydrouridine derivatives as a new series of CDA inhibitors that exhibit improved acid stability over 5. One of the fluorinated derivatives was also tested for its ability to enhance oral bioavailability of decitabine in primates by oral coadministration.

of 5 and 4 resulted in improved oral activity of 4 compared to efficacy of oral administration of 4 alone.8 Furthermore, recent pharmacokinetic studies in mice showed that 5 is orally available9 and increases oral bioavailability of coadministered 2.10 Despite the promising therapeutic utility of the oral form of 5 demonstrated in mice, oral administration of 5 (up to 1000 mg/kg) did not increase the oral availability of 4 in rhesus monkeys.11 This is considered primarily due to the poor aqueous stability of 5 at low pH as described by the Marquez laboratory, whose model experiments indicated that compound 5 undergoes a rapid, acid-catalyzed isomerization to the more stable β-ribopyranosyl form (Figure 2), resulting in a 100-fold reduction in potency.12 They speculated that this isomerization is attenuated in mice as their gastric acidity is somewhat suppressed compared to that of primates. Access to orally available CDA inhibitors would expand the therapeutic options available to patients currently receiving intravenous treatment. Although CDA is specific to cytosine-bearing nucleosides, both cytidine and 2-deoxycytidine are equally potent CDA substrates, suggesting that the 2′-hydroxy group of cytidine does not play a crucial role in its binding affinity to CDA.13,14 This is consistent with findings from the cocrystal structure of mouse CDA with 5.15 The structure reveals that while the sugar 3′-OH and 5′-OH groups form critical hydrogen bonds with the key residues of the CDA active site, the 2′-OH group plays

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CHEMISTRY For the preparation of fluorinated analogues of 5, we used a procedure similar to the one described previously by the Sturla group for the synthesis of 2′-deoxytetrahydrouridine.17 As illustrated in Scheme 1, compound 2 served as a starting material for the synthesis of 2′-deoxy-2′,2′-difluorotetrahydrouridine. Rhodium-catalyzed hydrogenation of 2 gave 2′deoxy-2′,2′-difluorodihydrouridine 6. Reduction of 6 with sodium borohydride afforded a mixture of difluorinated tetrahydrouridine epimers 7a and 7b, differing in the stereochemistry of the hydroxyl group at the 4-position. The mixture also contained the 4-deoxy byproduct 8. To our surprise, our attempt to remove the byproduct 8 by preparative HPLC also led to the complete separation of the two epimers with a retention time difference of nearly 5 min, providing three pure products, 7a, 7b, and 8. The two epimers differed 2583

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each fluorinated series exhibited significantly higher inhibitory potency over the other epimers as exemplified by compounds 7a (IC50 = 400 nM), 11a (IC50 = 200 nM), and 15a (IC50 = 400 nM). The other epimers, 7b (IC50 = 5.0 μM), 11b (IC50 = 4.0 μM), and 15b (IC50 = 5.0 μM), are 13−20-fold less potent in inhibiting CDA. A similar trend was observed in the two epimers of 5 which exhibited 13-fold difference in Ki values for mouse CDA.18 Interestingly, compound 7a, the more potent epimer of 2′-deoxy-2′,2′-difluorotetrahydrouridine, has C4 stereochemistry (4R) analogous to the epimer of 5 bound to the active site of mouse CDA in the X-ray crystal structure analysis.15 The epimers of 5 bound to CDA has the 4-hydroxyl group oriented toward the active site zinc, playing a critical role in the potent inhibition of CDA. This interaction cannot be established by the other epimer and may explain the difference in potency between the two epimers of 5. Given the similar trend observed across all fluorinated analogues of 5, it is conceivable that the fluorinated analogues retain the same mode of binding as 5, owing to the minimal steric aberrations caused by fluorine introduction. This is also consistent with the negligible difference in inhibitory potencies among the three fluorinated analogues 7a, 11a, and 15a. These findings also reaffirm the notion that the 2′-hydroxy group of 5 plays an insignificant role in its interaction with the CDA active site. Although the stereochemistry of monofluorinated epimers 11a and 15a is not definitive, it was tentatively assigned in Scheme 1 by assuming that the more potent epimers have the absolute configuration at C4 corresponding to compound 7a. Dihydrouridine intermediates 6, 10, and 14 exhibited some degree of inhibitory activity, with IC50 values ranging from 13 to 77 μM. Meanwhile, nearly complete loss of potency was seen for the fully reduced tetrahydropyrimidinone-based byproducts 8 and 12. The results are in a good agreement with the previous reports on the CDA inhibitory potency of nonfluorinated counterparts, dihydrouridine (Ki = 40 μM)1 and the 4-deoxy derivative of 5 (Ki = 440 μM).16 These findings also underscore the critical role played by the 4-hydroxy group of 5 and its fluorinated analogues in the interaction with the active site zinc atom. Acid stability of compound 7a was compared to that of 5 in simulated gastric fluid (pH 1.2) at 37 °C. As shown in Figure 4, nearly 50% of 5 (two epimers combined) was found to be degraded in 10 min and with less than 15% remaining intact after 2 h of incubation. This is consistent with the welldocumented instability of 5 in acidic medium.12,18 Dissolution of compound 7a in simulated gastric fluid immediately produced an approximately 1:1 mixture of epimers 7a and 7b. However, total concentrations of the two epimers remained largely unchanged over a period of 6 h. These results clearly demonstrate the superior acid stability of compounds 7a and 7b relative to 5. Further investigation into the acid-mediated degradation of 5 and compound 7a was conducted using 1H NMR in D2O. Figure 5A shows a doublet peak at 5.8 ppm, which represents the anomeric proton (at C1′) of both epimers of 5. This is consistent with the previous observation that the two epimers of 5 share nearly identical chemical shift for this proton.18 Upon addition of TFA-d, the intensity of this peak gradually decreased while two new doublet peaks emerged at 5.4 ppm. The upfield chemical shift indicates the anomeric proton of the ribopyranosyl derivative in its two epimeric forms. Nearly 50% of 5 was converted into the ribopyranosyl derivative in 4 h (Figure 5B) and 80% in 72 h (Figure 5C). As shown in Figure

markedly in their melting points, 162−165 °C for 7a and 59− 62 °C for 7b, respectively. Indeed, compound 7a was found to be stable enough for X-ray crystallographic analysis. As shown in Figure 3, compound 7a has the (R) configuration at the 4position (C1 in Figure 3). As shown in Scheme 1, the two stereoisomers of 2′-deoxy-2′fluorouridines, 9 and 13, served as starting materials for the synthesis of 2′-monoflurinated analogues. In a manner similar to the synthesis of the difluorinated derivatives, two epimers 11a and 11b along with the 4-deoxy derivative 12 were obtained from 9 through the intermediate 10. Similarly, two epimers 15a and 15b were prepared from 13 in two steps, although the 4-deoxy derivative was not successfully isolated in this particular case.

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RESULTS AND DISCUSSION The synthetically obtained fluorinated analogues were tested for their ability to inhibit human recombinant CDA using cytidine as a substrate. The results are summarized in Table 1. In our assay, compound 5 (a mixture of two epimers) inhibited CDA with an IC50 value of 340 nM. One of the epimers from Table 1. Inhibition of CDA by Fluorinated Tetrahydrouridines and Other Derivatives

Values are the means ± SD of three or more independent experiments.

a

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stomach.20 This is particularly important in assessing the pharmacological advantages of acid-stable CDA inhibitors such as compound 7a. Figure 6 shows the plasma concentrations of compound 7a and 5 (two epimers combined) over time following oral

Figure 4. Stability of 5 (two epimers combined) and a mixture of 7a and 7b in simulated gastric fluid at 37 °C. The percent of the CDA inhibitors remaining was plotted as a function of time. Data points collected in the first 2 h are mean values of triplicate preparations, while the remaining points collected were from duplicate preparations. HPLC was used for the quantification of the remaining CDA inhibitors.

Figure 6. Plasma concentration versus time profiles of 5 and compound 7a in rhesus monkeys following oral administration.

administration (10 mg/kg) in rhesus monkeys. Compound 7a displayed higher (3−4-fold) Cmax and AUC values compared to 5. It is also apparent that the variability in plasma levels is larger for 5, presumably due to the interindividual variation in gastric acidity. This presents another advantage of compound 7a in that it allows more precise control of CDA inhibition and, as a result, the pharmacokinetics of coadministered cytidine-based drugs. Subsequently, we assessed the effects of compound 7a (0.1, 1, and 10 mg/kg, po) on plasma levels of coadministered decitabine 3 (10 mg/kg, po) in rhesus monkeys (Figure 7). Decitabine was administered 2 h after compound 7a was given so that the maximal CDA inhibition is achieved during

Figure 5. 1H NMR spectra in parts per million (ppm) of 5 (two epimers combined) and compound 7a in D2O. (A) Compound 5 prior to addition of TFA-d. (B) Compound 5 at 4 h after addition of TFA-d. (C) Compound 5 at 72 h after addition of TFA-d. (D) Compound 7a prior to addition of TFA-d. (E) Compound 7a immediately after addition of TFA-d. (F) Compound 7a at 72 h after addition of TFA-d.

5D, the anomeric proton of 7a is found at 6.0 ppm (dd). Immediately after the addition of TFA-d, a new peak, representing the anomeric proton of 7b (dd), appears at a slightly lower chemical shift (Figure 5E). This indicates rapid epimerization at C4 of compound 7a in acidic conditions. However, no peak indicative of ribopyranosyl derivative formation emerged even after 72 h (Figure 5F). The data demonstrates that compound 7a does not undergo acidmediated conversion to the ribopyranosyl form. With the more stable CDA inhibitor 7a in hand, its effects on the oral pharmacokinetics of decitabine were investigated in vivo. Rhesus monkeys were chosen for this study for two key reasons. First, like humans, rhesus monkeys have substantial levels of CDA in both the liver and the kidneys.2 Mice express a significant amount of CDA only in kidneys, while rats have an appreciable amount of CDA in neither liver nor kidneys. With their resemblance to humans in CDA tissue distribution, rhesus monkeys appear to be a more appropriate model to predict human pharmacokinetics of drugs metabolized by CDA. Second, the gastric pH of rhesus monkeys can drop to as low as 2.019 and better simulates that of humans, whereas the gastric pH of mice is higher than the pH of the human

Figure 7. Effects of compound 7a on plasma pharmacokinetics of decitabine 3 (10 mg/kg) in rhesus monkeys. 2585

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(Milwaukee, WI). Preparative HPLC was performed using a Jasco HPLC system equipped with a Jasco U-987 pump and a Jasco RI-2031 refractive index detector. The instrument was fitted with a Phenomenex Luna C18 column (10 μm, 250 mm × 21.2 mm, Torrance, CA). An isocratic flow (10 mL/min) of 10% CH3CN in water was utilized. Melting points were obtained on a Mel-Temp apparatus and are uncorrected. 1H NMR spectra were recorded at 400 MHz. 13C NMR spectra were recorded at 100 MHz. Elemental analyses were obtained from Atlantic Microlabs, Norcross, GA. The purity of test compounds was determined by elemental analysis (within ±0.4% of the calculated value). 2′-Deoxy-2′,2′-difluoro-5,6-dihydrouridine (6). To a solution of 2 (3.0 g, 11.4 mmol) in H2O (50 mL) was added rhodium on carbon (5 wt %, 900 mg), and the suspension was hydrogenated at 40 psi in a Parr hydrogenator at rt overnight. The reaction was incomplete, and the catalyst was removed by filtration. A fresh batch of catalyst (900 mg) was added to the filtrate, which was hydrogenated at 40 psi overnight. The catalyst was removed by filtration, and the filtrate was concentrated in vacuo. The residual material was purified by preparative HPLC to give 1.84 g of 6 as a white solid (61% yield): mp 59−62 °C. 1H NMR (DMSO-d6) δ 2.39−2.63 (m, 2H), 3.09−3.30 (m, 1H), 3.48−3.79 (m, 4H), 4.06 (q, J = 8.8 Hz, 1H), 5.09 (brs, 1H), 5.87 (dd, J = 5.1, 13.6 Hz, 1H), 6.19 (brs, 1H), 10.49 (brs, 1H). 13C NMR (DMSO-d6) δ 25.89, 36.86, 59.30, 69.51 (dd, J = 19.0, 25.6 Hz), 78.87 (d, J = 8.8 Hz), 83.94 (dd, J = 22.69, 40.3 Hz), 124.02 (dd, J = 253, 259 Hz), 158.65, 169.72. Anal. Calcd for C9H12N2O5F2·0.25H2O: C, 39.93; H, 4.65; N, 10.35. Found: C, 39.87; H, 4.61; N, 10.26. (4R)-2′-Deoxy-2′,2′-difluoro-3,4,5,6-tetrahydrouridine (7a). To a solution of 6 (1.2 g, 4.9 mmol) in methanol (30 mL) was added sodium borohydride (540 mg, 14.3 mmol) portionwise at 0 °C. The reaction mixture was slowly warmed to rt. After 4 h of stirring at rt, the mixture was concentrated in vacuo and the residue was dissolved in H2O (15 mL). The solution was neutralized with 2.0 N HCl to pH 7 and purified by preparative HPLC. Epimer 7a eluted at 9.5 min and was obtained as a white solid after freeze-drying (350 mg, 29% yield): mp 162−165 °C. 1H NMR (DMSO-d6) δ 1.52−1.77 (m, 2H), 3.14−3.31 (m, 1H), 3.42−3.76 (m, 4H), 3.91−4.13 (m, 1H), 4.79 (brs, 1H), 5.02 (t, J = 5.6 Hz, 1H), 5.66 (d, J = 4.6 Hz, 1H), 5.93−6.03 (dd, J = 6.1, 14.4 Hz, 1H), 6.07 (brs, 1H), 7.35 (d, J = 4.0 Hz, 1H). 13C NMR (DMSO-d6) δ 29.15, 34.06, 59.29, 69.33 (dd, J = 18.3, 25.6 Hz), 71.43, 78.80 (d, J = 8.8 Hz), 82.85 (dd, J = 22.0, 40.98 Hz), 124.07 (dd, J = 253.2, 259.1, Hz), 153.34. Anal. Calcd for C9H14N2O5F2·0.15H2O: C, 39.90; H, 5.32; N, 10.34. Found: C, 39.87; H, 5.41; N, 10.26. (4S)-2′-Deoxy-2′,2′-difluoro-3,4,5,6-tetrahydrouridine (7b). Epimer 7b eluted at 14.3 min and was obtained as a white solid after freeze-drying (370 mg, 31% yield): mp 59−62 °C. 1H NMR (DMSO-d6) δ 1.66−1.78 (m, 2H), 2.99 (dt, J = 12.1, 4.3 Hz, 1H), 3.43 (dt, J = 7.8, 4.2 Hz, 1H), 3.48−3.58 (m, 2H), 3.69 (d, J = 10.9 Hz, 1H), 3.95−4.04 (m, 1H), 4.79 (d, J = 3.3 Hz, 1H), 4.97 (brs, 1H), 5.61−5.75 (m, 1H), 5.98 (dd, J = 5.2, 16.0 Hz, 1H), 6.04−6.13 (m, 1H), 7.31 (d, J = 3.8 Hz, 1H). 13C NMR (DMSO-d6) δ 29.15, 34.03, 59.58, 69.51 (dd, J = 19.0, 24.9 Hz), 71.75, 78.66 (d, J = 8.8 Hz), 83.39 (dd, J = 23.4, 39.5 Hz), 124.20 (dd, J = 252.5, 259.1 Hz), 153.76. Anal. Calcd for C9H14N2O5F2·0.6H2O: C, 38.74; H, 5.49; N, 10.04. Found: C, 38.55; H, 5.36; N, 9.87. 1-(2-Deoxy-2,2-difluoro-β- D -erythro-pentofuranosyl)tetrahydro-2(1H)-pyrimidinone (8). Compound 8 eluted at 17 min and was obtained as a white foam after freeze-drying (200 mg, 17% yield). 1H NMR (DMSO-d6) δ 1.59−1.92 (m, 2H), 3.01 (ddd, J = 3.5, 8.7, 11.8 Hz, 1H), 3.11 (dt, J = 2.4, 5.9 Hz, 2H), 3.39−3.59 (m, 3H), 3.69 (dd, J = 4.3, 9.9 Hz, 1H), 3.89−4.15 (m, 1H), 4.98 (t, J = 5.1 Hz, 1H), 6.00 (dd, J = 5.8, 15.7 Hz, 1H), 6.04 (d, J = 6.8 Hz, 1H), 6.74 (s, 1H). 13C NMR (DMSO-d6) δ 21.65, 39.42, 39.76, 59.38, 69.58 (dd, J = 25.6, 18.3 Hz), 78.60 (d, J = 9.51 Hz), 83.25 (dd, J = 40.98, 21.96 Hz), 124.08 (dd, J = 259.8, 251.8 Hz), 154.37. Anal. Calcd for C9H14N2O4F2·0.2H2O: C, 42.56; H, 5.63; N, 11.03. Found: C, 42.43; H, 5.69; N, 11.00. 2′-Deoxy-2′-fluoro-5,6-dihydrouridine (10). The fluorodeoxyuridine 9 (1.2 g, 4.9 mmol) was dissolved in water with a few drops of

decitabine absorption. With the lowest dose of compound 7a (0.1 mg/kg), the decitabine plasma profile showed an AUC value of 138 ng·h/mL, slightly higher than that of decitabine treatment alone (AUC = 74 ng·h/mL) conducted in a separate experiment (results not shown). Plasma decitabine levels were significantly higher when coadministered with 1 or 10 mg/kg of compound 7a with AUC values of 1030 and 1130 ng·h/mL, respectively. The results clearly demonstrate the ability of compound 7a to boost the decitabine pharmacokinetics through CDA inhibition. While 1 mg/kg of compound 7a appears sufficient to achieve the maximal effect, 10 mg/kg of compound 7a provided notably less variability in decitabine levels compared to 1 mg/kg of compound 7a. Recent studies have shown that oral coadministration of 5 (20 mg/kg) increases oral bioavailability of 3 (5 mg/kg) in baboons although the effects of 5 vary in each individual.21 Although the lack of plasma pharmacokinetics data for 5 in the baboon studies precludes a firm conclusion, this could at least in part be due to inconsistent plasma exposure to 5 as a result of its poor acid stability. Therefore, compound 7a may produce more robust effects on pharmacokinetics of oral decitabine with less interindividual variation.

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CONCLUSIONS Despite the successful clinical development of carbidopa− levodopa decades ago, a combination of an active drug with an inhibitor of its metabolism has not been actively pursued as an option to develop metabolically unstable drugs. The complexity of dealing with a mixture of two drugs is probably one of the major reasons for the lack of interest in this approach. However, the increasing efforts on the use of CYP3A4 inhibitors as boosters for anti-HIV drugs set an encouraging precedent for pharmaceutical combination therapy. It is of particular importance that CYP3A4, one of the most common causes of drug−drug interactions for a broad range of oral drugs, can be targeted in a clinically viable manner. Given the much narrower substrate specificity of CDA relative to CYP3A4, inhibition of CDA presents an attractive approach for delivering cytidine-based drugs prone to CDA-mediated deamination. Compound 7a exhibited superior acid stability and, as a result, showed an improved oral pharmacokinetic profile with a higher and more reproducible plasma exposure in rhesus monkeys compared to 5. Furthermore, coadministration of compound 7a with 3, a CDA substrate, boosted the plasma levels of decitabine in rhesus monkeys in a reproducible manner, demonstrating the pharmacological advantage of an acid-stable CDA inhibitor as opposed to 5. It is worth noting that the pharmacological benefit of compound 7a is not exclusive to decitabine but can be extended to other cytidine-based drugs that are currently used clinically. A combination with 2 is particularly attractive as oral form of this widely used chemotherapeutic agent might broaden its therapeutic utility in the treatment of cancer.

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EXPERIMENTAL SECTION

General. All solvents were reagent grade or HPLC grade. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Gemcitabine 2 and 2′-deoxy-2′-fluorouridines (compounds 9 and 13) were purchased from TCI America (Portland, OR), Carbosynth (Compton, Berkshire, UK), and Ark Pharm (Libertyville, IL), respectively. Rhodium on carbon (Degussa type, 5 wt %) was purchased from Sigma-Aldrich 2586

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(4S)-1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)tetrahydro4-hydroxy-2(1H)-pyrimidinone (15b). Epimer 15b eluted at 13.4 min and was obtained as a white solid after freeze-drying (236 mg, 21%): mp 47−50 °C. 1H NMR (DMSO-d6) δ 1.62−1.75 (m, 2H), 3.28 (m, 1H), 3.45−3.62 (m, 4H), 4.05 (dq, J = 21.7, 4.3 Hz, 1H), 4.75−4.90 (m, 3H), 5.58 (d, J = 5.1 Hz, 1H), 5.66 (d, J = 4.6 Hz, 1H), 6.05 (dd, J = 5.1, 18.2 Hz, 1H), 7.12 (d, J = 3.5 Hz, 1H). 13C NMR (DMSO-d6) δ 29.17, 37.20 (d, J = 6.6 Hz), 60.78, 71.85, 73.11 (d, J = 24.2 Hz), 81.10 (d, J = 13.2 Hz), 81.72 (d, J = 1.5 Hz), 97.21 (d, J = 192.5 Hz), 153.84. Anal. Calcd for C9H15N2O5F·0.4H2O: C, 41.96; H, 6.19; N, 10.87. Found: C, 41.99; H, 6.15; N, 10.91. X-ray Structure Analysis of 7a. Measurement of the crystal structure of compound 7a was performed on a Rigaku RAXIS-SPIDER diffractometer with graphite monochromated Cu Kα (λ = 1.54182 Å) at 150 K. The structure was solved by direct methods22 and expanded using Fourier techniques. The structure was refined by full-matrix least-squares on F2. Crystal data: C9H14N2O5F2, FW 268.22, trigonal, space group P3121 (no. 152), a = 9.78961(18) Å, c = 20.4588(7) Å, V = 1698.02(7) Å3, Z = 6, R1 = 0.0303, wR2 = 0.0733, GOF = 1.099. The supplementary crystallographic data for this paper has been deposited with the Cambridge Crystallographic Data Centre as CCDC-974853. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/ cif. CDA Assay. CDA assay was carried out in potassium phosphate buffer (pH 7.4, 20 mM, containing 1 mM DTT) in a total volume of 200 μL in a 96-well plate format. The final reaction mixture contains cytidine (50 μM) and purified human recombinant CDA. 23 Approximately 500 μg protein/mL of purified enzyme was diluted so as to produce an absorbance change of approximately 2 milliabsorbance units/min. After CDA addition, the reaction was monitored for 20−30 min by following the absorbance decrease at 286 nm due to the CDA-catalyzed deamination of cytidine to uridine. Stability Studies in Simulated Gastric Fluid. The initial (t = 0) samples were prepared by diluting compound 7a in DMSO or 5 in water. All other samples were prepared by dissolving ∼1.5 mg of sample in ∼1.0 mL of simulated gastric fluid (Ricca Chemical Company, Arlington, TX) at 37 °C. HPLC analyses were conducted on an Atlantis T3 column (4.6 mm × 250 mm, 5 μm, Waters) with a gradient of acetonitrile (2−30%) in water. Pharmacokinetic Studies in Rhesus Monkeys. The rhesus monkey pharmacokinetic studies were conducted at Covance Laboratories Inc. (Madison, WI). Blood samples (approximately 1 mL each) were collected from the femoral vein from each animal at predose and various postdose time points into tubes containing sodium heparin. Plasma was harvested within 1 h of collection and stored at −70 °C or lower until analysis, which was performed using an API 4000 LC-MS/MS system (Applied Biosystems, Inc.). Details of the pharmacokinetics study will be reported elsewhere.

concentrated ammonium hydroxide. Rhodium on carbon (5 wt %, 300 mg) was added to the solution, and the mixture was hydrogenated at 40 psi overnight. The mixture was filtered and concentrated. The residual material was purified by preparatory HPLC to give 780 mg of 10 as a colorless oil (64% yield). 1H NMR (DMSO-d6) δ 2.45−2.61 (m, 2H), 3.24−3.34 (m, 1H), 3.38−3.54 (m, 1H), 3.57−3.76 (m, 3H), 4.02 (dq, J = 17.6, 5.9 Hz, 1H), 4.88−4.95 (m, 1H), 5.06 (dd, J = 3.4, 4.9 Hz, 1H), 5.51 (d, J = 6.3 Hz, 1H), 5.87 (dd, J = 3.0, 20.5 Hz, 1H), 10.36 (brs, 1H). 13C NMR (DMSO-d6) δ 30.70, 36.34, 60.32, 68.48 (d, J = 16.1 Hz), 82.35, 86.36 (d, J = 32.9 Hz), 92.11 (d, J = 184.4 Hz), 152.86, 170.37. Anal. Calcd for C9H13FN2O5·0.35H2O: C, 42.47; H, 5.43; N, 11.01. Found: C, 42.25; H, 5.42; N, 11.06. (4R)-2′-Deoxy-2′-fluoro-3,4,5,6-tetrahydrouridine (11a). Compound 11a was prepared as described for the preparation of 7a except 10 was used in place of 6. Epimer 11a eluted at 7.2 min and was obtained as a white solid after freeze-drying (275 mg, 35% yield): mp 48−52 °C. 1H NMR (DMSO-d6) δ 1.58−1.80 (m, 2H), 3.19 - 3.32 (m, 2H), 3.40−3.52 (m, 1H), 3.55−3.70 (m, 2H), 3.97 (dq, J = 5.3, 16.4 Hz, 1H), 4.70−4.90 (m, 2H), 4.97 (dd, J = 3.8, 4.8 Hz, 1H), 5.41 (d, J = 6.1 Hz, 1H), 5.59 (d, J = 5.1 Hz, 1H), 5.93 (dd, J = 3.5, 21.2 Hz, 1H), 7.22 (d, J = 4.3 Hz, 1H). 13C NMR (DMSO-d6) δ 28.97, 34.40, 60.73, 68.60 (d, J = 16.1 Hz), 71.59, 81.68 (d, J = 1.5 Hz), 86.20 (d, J = 32.9 Hz), 91.54 (d, J = 183.7 Hz), 153.45. Anal. Calcd for C9H15N2O5F·0.5H2O: C, 41.70; H, 6.22; N, 10.81. Found: C, 41.67; H, 6.26; N, 10.76. (4S)-2′-Deoxy-2′-fluoro-3,4,5,6-tetrahydrouridine (11b). Epimer 11b eluted at 8.6 min and was obtained as a white solid after freeze-drying (125 mg, 16% yield): mp 47−51 °C. 1H NMR (DMSOd6) δ 1.59−1.85 (m, 2H), 2.97 (dt, J = 11.9, 4.8 Hz, 1H), 3.37−3.51 (m, 2H), 3.53−3.66 (m, 2H), 3.94 (dq, J = 11.6, 5.6 Hz, 1H), 4.70− 5.03 (m, 3H), 5.40 (d, J = 6.3 Hz, 1H), 5.60 (d, J = 4.6 Hz, 1H), 5.97 (dd, J = 3.3, 23.0 Hz, 1H), 7.16 (d, J = 3.5 Hz, 1H). 13C NMR (DMSO-d6) δ 29.03, 35.12, 60.94, 68.93 (d, J = 16.1 Hz), 71.76, 81.67, 86.66 (d, J = 33.7 Hz), 92.38 (d, J = 182.96 Hz), 154.12. Anal. Calcd for C9H15FN2O5·0.4H2O: C, 41.99; H, 6.19; N, 10.88. Found: C, 41.83; H, 6.22; N, 10.76. 1-(2-Deoxy-2-fluoro-β- D -ribofuranosyl)tetrahydro-2(1H)pyrimidinone (12). Compound 12 eluted at 14.9 min and was obtained as a white foam after freeze-drying (50 mg, 7% yield). 1H NMR (DMSO-d6) δ 1.65−1.75 (m, 2H), 3.11 (m, 1H), 3.35−3.6 (m, 6H), 4.12 (m, 1H), 4.90 (t, J = 4.0 Hz, 1H), 4.95 (t, J = 5.6 Hz, 1H), 5.78 (d, J = 3.8 Hz, 1H), 5.97 (dd, J = 5.1, 16.9 Hz, 1H), 10.35 (brs, 1H). Anal. Calcd for C9H15FN2O4·0.4H2O: C, 45.80; H, 6.49; N, 11.87. Found: C, 45.83; H, 6.56; N, 11.89. 1-(2-Deoxy-2-fluoro-β- D -arabinofuranosyl)dihydro-2,4(1H,3H)-pyrimidinedione (14). Compound 14 was prepared as described for the preparation of 10 except 13 was used in place of 9: white solid (76% yield); mp 94−97 °C. 1H NMR (DMSO-d6) δ 2.40− 2.58 (m, 2H), 3.20−3.32 (m, 1H), 3.44−3.66 (m, 3H), 4.05−4.19 (m, 1H), 4.90 (dd, J = 3.9, 4.9 Hz, 1H), 4.95 (t, J = 5.6 Hz, 1H), 5.03 (dd, J = 3.9, 4.9 Hz, 1H), 5.77 (d, J = 5.1 Hz, 1H), 5.97 (dd, J = 4.9, 16.8 Hz, 1H), 10.34 (brs, 1H). 13C NMR (DMSO-d6) δ 30.99, 37.16 (d, J = 5.9 Hz), 60.14, 73.09 (d, J = 23.4 Hz), 81.55 (d, J = 11.0 Hz), 81.67, 97.11 (d, J = 189.6 Hz), 152.66, 170.57. Anal. Calcd for C9H13FN2O5· 0.5H2O: C, 42.03; H, 5.49; N, 10.89. Found: C, 42.20; H, 5.55; N, 11.00. (4R)-1-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)tetrahydro4-hydroxy-2(1H)-pyrimidinone (15a). Compound 15a was prepared as described for the preparation of 7a, except 14 was used in place of 6. Epimer 15a eluted at 9.3 min and was obtained as a white solid after freeze-drying (163 mg, 14%): mp 49−52 °C. 1H NMR (DMSO-d6) δ 1.50−1.73 (m, 2H), 3.29 (tt, J = 3.0, 12.1 Hz, 1H), 3.42−3.62 (m, 4H), 4.06 (dq, J = 20.5, 3.8 Hz, 1H), 4.75−4.90 (m, 3H), 5.56 (d, J = 4.3 Hz, 1H), 5.66 (d, J = 4.8 Hz, 1H), 6.06 (dd, J = 4.8, 20.2 Hz, 1H), 7.16 (d, J = 4.0 Hz, 1H). 13C NMR (DMSO-d6) δ 29.41, 34.90 (d, J = 6.6 Hz), 60.59, 71.48, 73.54 (d, J = 23.4 Hz), 81.23 (d, J = 13.2 Hz), 81.34 (d, J = 1.5 Hz), 97.57 (d, J = 189.6 Hz), 153.45. Anal. Calcd for C9H15N2O5F·0.25H2O: C, 42.44; H, 6.13; N, 11.00. Found: C, 42.49; H, 6.09; N, 10.82.

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AUTHOR INFORMATION

Corresponding Authors

*For S.R.: phone, (925) 560-0100; fax, (925) 560-0101; E-mail, [email protected]. *For T.T.: phone, (410) 614-0982; fax, (410) 614-0659; Email, [email protected]. Author Contributions

With the exception of Sanjeev Redkar, the authors’ contribution to this publication was within their capacity as former or current employees of Eisai Inc. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Hyeong-Wook Choi for providing additional supply of compound 7a and Veronique Marceau for technical assistance in analyzing plasma samples. 2587

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Journal of Medicinal Chemistry

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Article

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ABBREVIATIONS USED CDA, cytidine deaminase; THU, tetrahydrouridine; AUC, area under the curve

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REFERENCES

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