Synthesis and Cytostatic Evaluation of 4-N-Alkanoyl and 4-N-Alkyl Gemcitabine Analogues

Abstract

Couplings of gemcitabine with the functionalized carboxylic acids (C9-C13) or reactions of 4-N-tosylgemcitabine with the corresponding alkyl amines afforded 4-N-alkanoyl and 4-N-alkyl gemcitabine derivatives. The analogues with a terminal hydroxyl group on the alkyl chain were efficiently fluorinated under conditions that are compatible with protocols for ¹⁸F labeling. The 4-N-alkanoylgemcitabines showed potent cytostatic activities in the low nM range against a panel of tumor cell lines while cytotoxicity of the 4-N-alkylgemcitabines were in the low μM range. The cytotoxicity for the 4-N-alkanoylgemcitabine analogues were reduced approximately by two orders of magnitude in the 2′-deoxycytidine kinase (dCK)-deficient CEM/dCK- cell line whereas cytotoxicity of the 4-N-alkylgemcitabines were only 2-5 times lower. None of the compounds acted as efficient substrates for cytosolic dCK, and therefore, the 4-N-alkanoyl analogues need to be converted first to gemcitabine to display a significant cytostatic potential, while 4-N-alkyl derivatives attain the modest activity without “measurable” conversion to gemcitabine.

Introduction

Gemcitabine (2′,2′-difluoro-2′-deoxycytidine, dFdC) is a chemotherapeutic nucleoside analogue used in the treatment of solid tumors in various cancers.^1,2 Synthesized first in 1988 by Hertel et al.,³ gemcitabine represents first-line therapy for pancreatic and non-small cell lung cancers.^4-6 Gemcitabine is hydrophilic by nature and cellular uptake is primarily facilitated by human equilibrative nucleoside transport protein 1 (hENT1).⁷ Gemcitabine is activated via phosphorylation to its 5′-monophosphate (dFdCMP) by deoxycytidine kinase (dCK).^8,9 The dFdCMP then undergoes subsequent phosphorylation by intracellular kinases to the diphosphate (dFdCDP) and triphosphate (dFdCTP) forms.^10,11 The dFdCTP can incorporate into DNA and inhibit DNA polymerases by chain termination during DNA replication and repair processes, invariably triggering apoptosis.^10-12 It can also participate in “self potentiation” by inhibiting CTP synthetase and depleting CTP pools available to compete with dFdCTP for incorporation into RNA.^12,13 Moreover, dFdCD(T)P inhibits both R1 and R2 subunits of ribonucleotide reductase (RNR),^14-20 depleting the deoxyribonucleotide pool available to compete with dFdCTP for incorporation into DNA.^15,16 Gemcitabine is therapeutically restricted by high toxicity to normal cells and rapid intracellular deamination into inactive 2′,2′-difluorouridine (dFdU) by cytidine deaminase (CDA).²¹

Since clinical studies have indicated that prolonged infusion times with lower doses of gemcitabine can be effective while reducing toxicity to normal cells,^22,23 various prodrug strategies have been developed featuring acyl modifications on either the exocyclic 4-N-amine or 5′-hydroxyl group.²⁴ The hydrolyzable amide modifications facilitate a slow release of gemcitabine, increasing its bioavailability and uptake while also providing resistance to enzymatic deamination.^25-32 In 2004, Immordono et al. reported the increased anticancer activity of 4-N-stearoyl gemcitabine which was stable in plasma and showed an improved resistance to deamination.²⁸ Couvreur and Cattel developed the 4-N-squalenoyl gemcitabine prodrug (SQgem) as a chemotherapeutic nanoassembly that accumulates in the cell membranes prior to releasing gemcitabine²⁶ (Figure 1). The SQgem overcomes the low efficacy of gemcitabine in chemoresistant pancreatic cell lines and is currently undergoing preclinical development.³¹ The orally active 4-N-valproylgemcitabine prodrug 1 (LY2334737) currently undergoing Phase I clinical trials,^30,32 was designed to be resistant to deamination by hydrolysis in acidic conditions similar to those found in the human digestive system^25,29 while systematically releasing gemcitabine upon action by carboxylesterase 2. Recently, the gemcitabine prodrug with Hoechst conjugate attached to the 4-amino group targeting extracellular DNA has been reported with low toxicity but high tumor efficacy.²⁷ Also, the lipophilic gemcitabine pro-drug CP-4126 with the 5′-OH group esterified with an elaidic fatty acid has shown antitumor activity in various xenograft models.³³ It remains active when orally administered,³³ however, has not yet met criteria for advancement to Phase II clinical trials.³⁴ Very recently, the 5′-acylated gemcitabine prodrugs with coumarin or boron-dipyrromethene/biotin conjugate have been reported for monitoring drug delivery at subcellular levels by fluorescence imaging.^35,36

Figure 1.

The 4-N-acylated gemcitabine pro-drugs.

The 1-(2′-deoxy-2′-¹⁸F-fluoro-β-D-arabinofuranosyl)cytosine ([¹⁸F]-FAC) was developed by Radu et. al.³⁷ as a PET tracer possessing a substrate affinity for dCK and CDA comparable to gemcitabine. Determination of [¹⁸F]-FAC uptake and pretreatment levels of dCK serve as a non-invasive prognosticator for gemcitabine chemotherapy response.^37-40 The dCK specific PET tracers such as 1-(2′-deoxy-2′-¹⁸F-fluoro-arabinofuranosyl)cytosine ([¹⁸F]-L-FAC) and 1-(2′-deoxy-2′-¹⁸F-fluoro-β-L-arabinofuranosyl)-5-methylcytosine (L-¹⁸F-FMAC), which possess no substrate affinity to CDA, were also developed to study uptake of [¹⁸F]-FAC serving as additional predictive tools for gemcitabine treatment response.^41,42

Herein we report synthesis and cytostatic activity of a series of gemcitabine analogues with 4-N-alkanoyl or 4-N-alkyl chains modified with a terminal hydroxyl, halide or alkene groups. The 4-N-alkyl analogues stable towards deamination were designed to examine their anticancer activities and also to explore their compatibility with radiofluorination protocols.

Chemistry

Condensation of gemcitabine (2a, dFdC) with undecanoic acid employing peptide coupling conditions^25,43 [N-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDCI)/1-hydroxybenzotriazole (HOBt)/N-methylmorpholine (NMM)] in DMF/DMSO (3:1) at 60 °C afforded the 4-N-undecanoylgemcitabine 3 (50%, Scheme 1). Analogous coupling of 2a with 8-nonenoic acid, 10-undecenoic acid, 12-tridecenoic acid, 11-fluoroundecanoic acid (S4; see Supporting Information) or 11-hydroxyundecanoic acid afforded the 4-N-acyl analogues 4-8 with 40% to 66% yields after silica gel purification. It is noteworthy that these couplings in the presence of HOBt typically progressed to >90% completion (TLC), while the 1,1′-carbonyldiimidazole(CDI)-mediated coupling⁴⁴ of 2a with the corresponding carboxylic acids in CH₃CN and pyridine without HOBt proceeded less efficiently.

Scheme 1.

Synthesis of the lipophilic 4-N-alkanoyl gemcitabine derivatives.

Reagents and conditions: (a) R′(CH₂)_nCOOH/NMM/HOBt/EDCI/DMF/DMSO/60 °C/overnight; (b) (i) TMSCl/Pyr/CH₃CN, 0 °C/2.5 h, (ii) BrCH₂CH₂(CH₂)₈COOH/CDI/CH₃CN/65 °C/overnight; (c) BrCH₂CH₂(CH₂)₈COOH/ClCOOEt/Et₃N/DMF (d) (Boc)₂O/KOH/1,4-dioxane; (e) BrCH₂CH₂(CH₂)₈COCl/NaHCO₃/CH₂Cl₂; (f) DAST/CH₂Cl₂; (g) TFA

Unexpectedly, the HOBt-promoted coupling of 2a with 11-bromoundecanoic acid led to the formation of the 4-N-[11-(1H-benzotriazol-1-yloxy)undecanoyl derivative 9 in a 53% yield rather than the expected 4-N-(11-bromoundecanoyl) derivative 12. Other attempts to synthesize the bromo derivative either by transiently protecting of 2a with a trimethylsilyl group⁴⁵ followed by treatment with 11-bromoundecanoic acid/CDI or by employing a mixed anhydride procedure²⁶ (11-bromoundecanoic acid/ethyl chloroformate/TEA) gave instead the chloro derivative 10. The labile nature of the terminal bromide necessitated an alternative approach for the preparation of 12. We found that condensation of 3′,5′-di-O-Boc-protected gemcitabine⁴⁶ 2b with 11-bromoundecanoyl chloride (S5; SI) provided the bromo derivative 11 in a 33% isolated yield. The deprotection of 11 with TFA gave desired 12 (86%). The coupling of 2b with 11-hydroxyundecanoic acid yielded the protected 4-N-(11-hydroxyundecanoyl) derivative 13 (37%), bearing the hydroxyl group at the alkyl chain suitable for further chemical modifications. Thus, fluorination of 13 with DAST afforded the 4-N-(11-fluoroundecanoyl) derivative 14 (40%). Deprotection of 13 or 14 with TFA gave 7 (87%) or 8 (82%), respectively.

Recently, Haufe et al. established the methodology for radiofluorination of terminal olefins employing the [¹⁸F]-HF/pyridine reagent.⁴⁷ We performed the model fluorination using this condition employing regular, non-radioactive HF/pyridine with the olefin 5. Thus, treatment of 5 with Olah’s Reagent (70% HF in pyridine) in an HDPE vessel at 0 °C for 2 h gave a regioisomeric mixture of 10-fluoro, 9-fluoro, and 8-fluoro derivatives 15-17 with an isomeric ratio of 75:20:5 (91%, Scheme 2). The ¹H and ¹⁹F NMR spectra were diagnostic for the regioisomeric composition [¹⁹F δ −179.79 (m, 0.05 F), −178.83 (m, 0.1 F) and −170.27 (m, 0.75 F)]. Fluorination involved Markovnikov addition of HF to double bond while generation of the minor isomers 16 and 17 is attributed to migration of the carbocation along the chain during the addition reaction as has been observed before.⁴⁸ Since addition of HF to 5 gave multiple products and radiolabeling with [¹⁸F]-HF was reported to proceed with low radiochemical efficiencies,⁴⁷ while other commonly used fluorination protocols required conditions⁴⁹ under which the 4-N-alkanoyl linkage might be cleaved, we turned our attention to developing 4-N-alkyl gemicitabine analogues.

Scheme 2.

Synthesis of the fluorinated 4-N-alkanoyl gemcitabine derivatives by the addition of HF to the olefin.

Synthesis of 4-N-alkyl gemcitabine derivatives, which to the best of our knowledge, are limited to short 4-N alkyl modifications and their anticancer activities have not been studied in depth.⁵⁰ The 4-N-alkyl analogues are expected to be resistant to chemical hydrolysis as well as CDA-catalyzed deamination.⁵¹ From the available methods for the N-alkylation of cytosine nucleosides,^52-55 we found that the alkylation of the 4-exocyclic amine group in gemcitabine was achieved efficiently by displacement of a 4-N-tosylamine group⁵⁴ with an aliphatic alkyl amine. Thus, reaction of 2b with TsCl in the presence of Et₃N in 1,4-dioxane afforded the protected 4-N-tosylgemcitabine 18 (45%, Scheme 3). Treatment of 18 with 10-undecenyl amine effected simultaneous displacement of the p-toluenesulfonamido group from the C4 position of the cytosine ring and deprotection to give the 4-N-(10-undecenyl) derivative 19. Analogous reaction of 18 with 11-aminoundecanol (S7; SI) gave the 5′ monoprotected 11-hydroxyundecanyl analogue 20 (47%) as the major product in addition to fully deprotected 21 (24%). Deprotection of 20 with TFA provided 21 with 38% overall yield from 18.

Scheme 3.

Synthesis of the 4-N-alkyl gemcitabine derivatives

Reagents and conditions: (a) TsCl/Et₃N/1,4-dioxane; (b) CH₂=CH(CH₂)₉NH₂ or HOCH₂CH₂(CH₂)₉NH₂; (c) TFA

Owing to instability of Boc protection group during displacement of the p-toluenesulfonamido group, we explored other protection strategies that would lead to gemcitabine derivatives suitable for the selective modification of the primary hydroxyl group on the 4-N-alkyl chain. Thus, transient protection of 2a with TMSCl and subsequent treatment with TsCl followed by methanolic ammonia afforded the 4-N-tosylgemcitabine 22 in 96% yield (Scheme 4). Displacement of the p-toluenesulfonamido group from 22 with O-benzyl protected 11-aminoundecanol (S11; SI) proceeded efficiently to give the 4-N-(11-benzyloxyundecanyl)gemcitabine 23 in 61% isolated yield. Subsequent treatment of 23 with BzCl yielded the fully protected analogue 24 (60%). The lengthy treatment of 24 with ceric ammonium nitrate (CAN) effected the selective removal of the benzyl group to give the 3′,5′-di-O-benzoyl protected 11-hydroxyundecanyl analogue 25 (70%). It is noteworthy that attempted hydrogenolysis of 24 (H₂/Pd-C/EtOH/24 h) produced 25 with the inconsistent yields of 5-50% in addition to substantial quantities of other byproducts including the 5,6-dihydro reduced derivative.

Scheme 4.

Synthesis of the 4-N-fluoroalkyl gemcitabine derivatives

Reagents and conditions: (a) (i) TMSCl/Pyr, (ii) TsCl, (iii) MeOH/NH₃; (b) BnO(CH₂)₁₁NH₂/Et₃N/1,4-dioxane; (c) 2,6-Lutidine/DMAP/BzCl/CH₂Cl₂; (d) CAN/CH₃CN; (e) DAST/CH₂Cl₂; (f) MeOH/NH_3/rt; (g) MsCl/Et₃N/CH₂Cl_2/0 °C; (h) KF/K₂CO_3/K₂₂₂/CH₃CN/110 °C, (ii) MeONa/MeOH/100 °C

Fluorination of 25 with DAST afforded the 4-N-(11-fluoroundecanyl) derivative 26 and subsequent deprotection with methanolic ammonia at room temperature gave the 4-N-fluoroalkyl analogue 27 (43% overall from 25). We also examined fluorination of 25 using conditions which are compatible with general radiosynthetic protocols for ¹⁸F labeling.^46,56 Thus, reaction of 25 with MsCl/Et₃N gave the mesylate precursor 28 (90%). Fluorination of the latter with KF/K₂CO₃/Kryptofix 2.2.2 in CH₃CN at 110 °C for 18 minutes yielded the protected fluoro analogue 26 and subsequent debenzoylation with 0.5 M MeONa/MeOH at 100 °C for 8 minutes and purification by HPLC afforded the desired 4-N-fluoroalkyl gemcitabine 27 (overall 62% from 28; in total of 50 min). This fluorination protocol meets criteria for working with 18-fluorine isotope which has limited availability and short half-life (110 min.) and as such is applicable for labeling studies.

Cytostatic Activity

The growth inhibitory activities of the 4-N-acyl (3-8) and 4-N-alkyl (19, 21, 27) gemcitabine analogues were assessed on a panel of murine and human tumor cell lines (Table 1). All 4-N-alkanoyl 3-8 analogues demonstrated potent antiproliferative activities with the IC₅₀ values in the range of low nM, similar to gemcitabine 2a acting probably as prodrugs as established before.²⁵ On the other hand, the 4-N-alkylgemcitabine derivatives 19, 21, and 27 showed cytostatic activities at IC₅₀ values in the low to modest μM range. It appears that the cytostatic activity only varies slightly between compounds with different chain lengths or functional groups. The activity for the 4-N-acyl gemcitabine derivatives were drastically diminished (almost by two orders of magnitude) in the dCK-deficient CEM/dCK^- cell line implying again the role for dCK in the metabolism of these compounds.²⁴ Interestingly, cytotoxicity of the 4-N-alkylgemcitabines 19, 21, and 27 in dCK-deficient CEM/dCK^- cells was only 2-5 times lower. It is noteworthy that 4-N-valproylgemcitabine 1 in its free-base form showed cytostatic activity in the low μM range more comparable to our 4-N-alkyl analogues than the 4-N-acyl counterparts. The inhibitory activities for 1 are in agreement with the cytotoxicity described by Pratt et al. on a NCI-60 panel, who reported IC₅₀ values of 1 being “80-fold more” than the value for gemcitabine on most cell lines.³⁰

Table 1.

In vitro cytostatic activity of representative 4-N-modified analogues on the tumor cell lines L1210, CEM/0, CEM/dCK⁻, HeLa and MCF-7

Compound	IC50 (μM)
	L1210	CEM/0	CEM/dCK−	HeLa	MCF-7
1a	1.1 ± 0.7	5.2 ± 2.3	161 ± 8	0.76 ± 0.30	0.55 ± 0.49
2a	0.013 ± 0.001	0.069 ± 0.002	7.6 ± 0.5	0.0099 ± 0.0041	0.0072 ± 0.0002
3	0.014 ± 0.002	0.060 ± 0.012	5.8 ± 0.5	0.0089 ± 0.0024	0.0053 ± 0.0023
4	0.024 ± 0.017	0.14 ± 0.00	20 ± 2	0.042 ± 0.005	0.0079 ± 0.0002
5	0.018 ± 0.016	0.071 ± 0.015	12 ± 9	0.012 ± 0.007	0.0062 ± 0.0029
6	0.021 ± 0.018	0.069 ± 0.002	6.8 ± 1.8	0.013 ± 0.007	0.0079 ± 0.0012
7	0.023 ± 0.003	0.24 ± 0.19	19 ± 6	0.049 ± 0.030	0.0081 ± 0.0005
8	0.053 ± 0.040	0.059 ± 0.009	7.2 ± 0.8	0.011 ± 0.004	0.0077 ± 0.0006
19	7.0 ± 3.0	13 ± 6	60 ± 15	3.4 ± 0.0	28 ± 14
21	29 ± 11	86 ± 10	140 ± 28	22 ± 4	27 ± 3
27	28 ± 2	28 ± 4	134 ± 18	17 ± 4	26 ± 7

^aIn free-base form.

The selected compounds were also investigated for their interaction with dCK or mitochondrial thymidine kinase (TK-2) that has also 2′-deoxycytidine kinase activity. None of the prodrug derivatives showed significant inhibition of the phosphorylation of dCyd or dThd by dCK and TK-2, respectively. When directly evaluated as a potential substrate for dCK, the 4-N-alkanoyl derivatives 6 and 8 displayed very poor substrate activity (< 1%), whereas the 4-N-alkyl analogues 11 and 12 displayed no measurable substrate activity under experimental conditions that converted gemcitabine to its 5′-monophosphate by at least 15%. Taken together, these findings suggest the 4-N-alkanoyl analogues first need to be converted to gemcitabine before acting as an efficient substrate for dCK. This release of gemcitabine from the 4-N-alkanoyl prodrugs seems to occur quite efficiently given their pronounced cytostatic potential in the cell cultures, and the marked loss of cytostatic activity in the dCK-deficient CEM tumor cell cultures. Instead, the rather modest antiproliferative activities of the 4-N-alkyl analogues may be attributed, at least to certain extent, to a poor cellular uptake and/or to an inefficient conversion to the parental gemcitabine which may fall outside the assay detection limits (<1%). Moreover, the fact that the cytostatic activity of the 4-N-alkyl analogues are only moderately decreased in dCK-deficient CEM tumor cell cultures may not only confirm a poor, if any intracellular conversion to gemcitabine but also point to a potential different mechanism of cytostatic activity of these prodrugs. To gain insight into the metabolism of the 4-N-alkylgemcitabine derivatives, the stabilities of representative 4-N-alkanoyl 7 and 4-N-alkyl 21 analogues towards hydrolysis and resistance to enzymatic deamination were evaluated in parallel with gemcitabine in human serum and in murine liver extract. Figure 2 showed that gemcitabine was deaminated to its inactive uracil derivative dFdU as a function of time (panel A), while the 4-N-alkanoylgemcitabine prodrug 7 was slowly converted to gemcitabine, which was then gradually deaminated to dFdU (panel B). On the other hand the 4-N-alkylgemcitabine derivative 21 was not deaminated nor was there any measurable conversion to gemcitabine observed (panel C). When 7 and 21 were exposed to the murine liver extract, 7 was rapidly converted to gemcitabine (and dFdU) whereas 21 was fully stable for at least 2 hours (Figure 3). These findings support again the assumption that 7 is enzymatically efficiently converted to gemcitabine whereas 21 is not, explaining the differences in the cytostatic activity of both compounds. Although the cellular target for the antiproliferative activity of the 4-N-alkyl analogues is currently unclear, it might well be different from inhibition of DNA synthesis.

Figure 2.

Time-point evaluation of the stability and resistance to deamination for gemcitabine (A), 4-N-alkanoylgemcitabine 7 (B) and 4-N-alkylgemcitabine 21 (C) in 50% human serum in PBS.

Figure 3.

Time-point evaluation of the stability of 4-N-alkanoylgemcitabine 7 and 4-N-alkylgemcitabine 21 in murine liver extract in PBS.

In conclusion, we have demonstrated that coupling of gemcitabine with various carboxylic acids or reaction of 3′,5′-di-O-Boc-protected gemcitabine with acyl halides gave 4-N-alkanoylgemcitabine analogues with a hydroxyl, fluoro, chloro, bromo or alkene functional group on the alkyl chain. Displacement of the p-toluenesulfonamido group from 4-N-tosylgemcitabine with alkyl amines provided 4-N-alkylgemcitabine analogues suitable for further chemical modifications including fluorination compatible with the synthetic protocols for ¹⁸F labeling. The 4-N-alkanoylgemcitabine analogues showed potent antiproliferative activities against the L1210, CEM, HeLa and MCF-7 cell lines with the IC₅₀ values in the range of low nM while the cytostatic activity of the 4-N-alkylgemcitabine derivatives was in the low to modest μM range. The 4-N-alkanoyl derivatives display significant cytostatic activity, acting as efficient prodrugs, while the 4-N-alkyl analogues appear to attain their modest activity without “measurable” conversion to gemcitabine. The cytostatic activity appears to be independent of the length of alkyl chain and varies slightly for the different functional groups present on the molecule.

Experimental Part

The ¹H (400 MHz), ¹³C (100 MHz), or ¹⁹F (376 MHz) NMR spectra were recorded at ambient temperature in solutions of CDCl₃ or MeOH-d₄ or DMSO-d₆, as noted. The reactions were followed by TLC with Merck Kieselgel 60-F₂₅₄ sheets and products were detected with a 254 nm light or with Hanessian’s stain. Column chromatography was performed using Merck Kieselgel 60 (230-400 mesh). Reagent grade chemicals were used and solvents were dried by reflux distillation over CaH₂ under nitrogen gas, unless otherwise specified, and reactions carried out under Ar atmosphere. The carboxylic acid and amine derivatives used for the coupling with gemcitabine were commercially available except for 11-fluoroundecanoic acid (S4), 11-bromoundecanoyl chloride (S5), 11-aminoundecanol (S7) and 11-benzyloxyundecan-1-amine (S11) which synthesis is described in Supporting Information. The purity of the synthesized compounds was determined to be ≥95% by elemental analysis (C, H, N) and/or HPLC on Phenomenex Gemini RP-C18 with isocratic mobile phase (50% CH₃CN/H₂O) and flow rate of 5 mL/min. Representative HPLC chromatograms are included in the Supporting Information Section.

Tumor cell and enzyme sources

Murine leukemia L1210, human lymphocyte CEM and human cervix carcinoma HeLa cell lines were obtained from ATCC, Rockville, MD. Human breast carcinoma MCF-7 cells were a kind gift from G. Peters (Amsterdam, The Netherlands). The dCK-deficient CEM cell line was obtained upon selection in the presence of araC and found to be deficient in cytosolic dCK activity.

General synthetic procedure for preparation of the 4-N-acyl gemcitabine derivatives (3-10). Procedure A

N-Methylmorpholine (1.1 eq.), 1-hydroxybenzotriazole (1.1 eq.), the appropriate carboxylic acid (1.1 eq.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.3 eq.) were sequentially added to a stirred solution of gemcitabine hydrochloride (2a, 1.0 eq.) in DMF/DMSO (3:1, 2 mL) at ambient temperature under Argon. The reaction mixture was then gradually heated to 65 °C (oil-bath) and kept stirring overnight. After the reaction was completed (TLC), the reaction mixture was cooled to 15 °C and partitioned between a small amount of brine and EtOAc. The organic phase was separated and the aqueous layer extracted with fresh portions of EtOAc (3 x 30 mL). The combined organic layers was then sequentially washed with 20% LiCl/H₂O, saturated NaHCO₃/H₂O, brine, dried over Na₂SO₄, and evaporated under reduced pressure to give the crude products 3-10.

4-N-(Undecanoyl)-2′-deoxy-2′,2′-difluorocytidine (3)

Treatment of 2a (34 mg, 0.11 mmol) with commercially available undecanoic acid (23.3 mg, 0.120 mmol) by Procedure A gave 45.7 mg of the crude product, which was then column chromatographed (5% MeOH/EtOAc) to give 3 (23.8 mg, 50%) as a white solid: ¹H NMR (CD₃OD) δ 0.90 (t, J = 6.9 Hz, 3H, CH₃), 1.27-1.39 (m, 14H, 7 x CH₂), 1.63-1.70 (m, 2H, CH₂), 2.45 (t, J = 7.4 Hz, 2H, CH₂), 3.79-3.83 (m, 1H, H5′), 3.94-3.99 (m, 2H, H5′, H4′), 4.31 (dt, J = 20.8, 10.5 Hz, 1H, H3′), 6.24-6.28 (m, 1H, H1′), 7.50 (d, J = 7.6 Hz, 1H, H5), 8.34 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) 14.41, 23.69, 25.93, 30.15, 30.40, 30.40, 30.56, 30.64, 33.03, 38.16, 60.29 (C5′), 70.21 (“t,” J = 23.1 Hz, C3′), 82.86 (d, J = 8.6 Hz, C4′), 86.44 (dd, J = 26.6, 38.3 Hz, C1′), 98.26 (C5′), 123.90 (t, J = 259.3 Hz, C2′), 145. 94 (C6), 157.65 (C2), 164.80 (C4), 175.97; ¹⁹F NMR (CD₃OD) δ −120.09 (br. d, J = 240.9 Hz, 1F), −119.14 (dd, J = 11.3, 240.9 Hz, 1F); HRMS (ESI⁺) m/z calcd for C₂₀H₃₁F₂N₃NaO₅ [M+Na]⁺ 454.2124; found 454.2136.

4-N-(8-Nonenoyl)-2′-deoxy-2′,2′-difluorocytidine (4)

Treatment of 2a (34 mg, 0.110 mmol) with commercially available 8-nonenoic acid (21 μL, 19.5 mg, 0.120 mmol) by Procedure A gave 29.0 mg of the crude product, which was then column chromatographed (70 → 100% EtOAc/hexane) to give 4 (20 mg, 45%) as a white solid: ¹H NMR (CD₃OD) δ 1.32-1.46 (br. s, 6H, 3 x CH₂), 1.65-1.69 (m, 2H, CH₂), 2.03-2.07 (m, 2H, CH₂), 2.45 (t, J = 7.4 Hz, 2H, CH₂), 3.81 (dd, J = 12.3, 2.8 Hz, 1H, H5′), 3.96-3.99 (m, 2H, H5″, H4′), 4.30 (td, J = 12.2, 8.6 Hz, 1H, H3′), 4.90-5.01 (m, 2H, CH₂), 5.81 (ddt, J = 16.9, 10.0, 3.4 Hz, 1H, CH), 6.24-6.28 (m, 1H, H1′), 7.50 (d, J = 7.6 Hz, 1H, H5), 8.34 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 25.90, 29.87, 29.90, 30.00, 34.79, 38.15, 60.31 (C5′), 70.23 (dd, J = 21.9, 23.4 Hz, C3′), 82.86 (C4′), 86.14 (d, J = 20.1 Hz, C1′), 98.28 (C5) , 114.83, 123.94 (t, J = 259.2 Hz, C2′), 140.03, 145.97 (C6) , 157.37 (C2), 164.84 (C4), 175.97; ¹⁹F NMR (CD₃OD) δ −120.13 (br. d, J = 242.5 Hz, 1F), −119.21 (dd, J = 11.4, 240.0 Hz, 1F); HRMS (ESI⁺) m/z calcd for C₁₈H₂₅F₂N₃NaO₅ [M+Na]⁺ 424.1654; found 424.1656.

4-N-(10-Undecenoyl)-2′-deoxy-2′,2′-difluorocytidine (5)

Treatment of 2a (40 mg, 0.134 mmol) with commercially available undecylenic acid (31 μL, 28 mg, 0.148 mmol) by Procedure A gave 114 mg of the crude product, which was then column chromatographed (80 → 100% EtOAc/hexane) to give 5 (38 mg, 66%) as a white solid: UV (CH₃OH) λ_max 252 nm (ε 15 150), 286 nm (ε 8950), λ_min 228 nm (ε 5900), 275 nm (ε 8650); ¹H NMR (DMSO-d₆) δ 1.23-1.29 (br. s, 8H, 4 × CH₂), 1.30-1.39 (m, 2H, CH₂), 1.50-1.57 (m, 2H, CH₂), 2.01 (q, J = 7.0 Hz, 2H, CH₂), 2.40 (t, J = 7.3 Hz, 2H, CH₂), 3.66 (“br. d,” J = 12.4 Hz, 1H, H5″), 3.81 (br. d, J = 12.4 Hz, 1H, H5′), 3.89 (dt, J = 8.5, 2.7 Hz, 1H, H4′), 4.19 (“q,” J = 10.6 Hz, 1H, H3′), 4.93 (“d. quin,” J = 10.1, 1.0 Hz, 1H, CH), 4.99 (“d. quin,” J = 17.2, 1.7 Hz, 1H, CH), 5.33 (br. t, J = 5.0 Hz, 1H, OH), 5.79 (tdd, J = 6.6, 10.3, 17.1 Hz, 1H, CH), 6.17 (t, J = 7.5 Hz, 1H, H1′), 6.35 (br. s, 1H, OH), 7.29 (d, J = 7.6 Hz, 1H, H5), 8.24 (d, J = 7.6 Hz, 1H, H6), 10.98 (br. s, 1, NH); ¹³C NMR (CD₃OD) δ 25.95, 30.08, 30.15 (2 x CH₂), 30.37, 30.39, 34.88, 38.18, 60.32 (C5′), 70.24 (dd, J = 21.9, 23.4 Hz, C3′), 82.89 (dd, J = 2.7, 5.2 Hz, C4′), 86.48 (dd, J = 25.8, 38.2 Hz, C1′), 98.28 (C5), 114.73, 123.93 (t, J = 259.2 Hz, C2′), 140.13, 145.97 (C6), 157.69 (C2), 164.83 (C4), 176.00; ¹⁹F NMR (CD₃OD) δ −120.09 (br. d, J = 239.6 Hz, 1F), −119.16 (dd, J = 10.9, 239.9 Hz, 1F); MS (ESI⁺) m/z 430 (100, [M+H]⁺). HRMS (ESI⁺) m/z calcd for C₂₀H₂₉F₂N₃NaO₅ [M+Na]⁺ 452.1967; found 452.1982. Elemental Anal. calcd for C₂₀H₂₉F₂N₃O₅•0.5H₂O (438.47): C, 54.79; H, 6.90; N, 9.58. Found: C, 54.48; H, 6.53; N, 9.21.

4-N-(12-Tridecenoyl)-2′-deoxy-2′,2′-difluorocytidine (6)

Treatment of 2a (30 mg, 0.1 mmol) acid (23 mg, 0.11 mmol) by Procedure A gave 43.1 mg of the crude product, which was then column chromatographed (70 → 80% EtOAc/hexane) to give 6 (20.1 mg, 44%) as a white solid: ¹H NMR (CD₃OD) δ 1.27-1.38 (m, 14H, 7 x CH₂), 1.66 (quin, J = 6.9 Hz, 2H, CH₂), 2.04 (dd, J = 14.3, 6.7 Hz, 2H, CH₂), 2.45 (t, J = 7.4 Hz, 2H, CH₂), 3.81 (dd, J = 12.4, 2.8 Hz, 1H, H5′), 4.07-3.88 (m, 2H, H5″, H4′), 4.31 (dt, J = 20.8, 10.4 Hz, 1H, H3′), 4.89-5.00 (m, 2H, CH₂), 5.80 (ddt, J = 17.0, 10.2, 6.7 Hz, 1H, CH), 6.26 (“t,” J = 7.2 Hz, 1H, H1′), 7.50 (d, J = 7.6 Hz, 1H, H5), 8.34 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 25.94, 30.11, 30.15, 30.20, 30.39, 30.53 (2 x CH₂), 30.62, 34.87, 38.16, 60.30, 70.24 (“t,” J = 23.1 Hz, C3′), 82.83 (C4′), 86.46 (“t,” J = 32.2 Hz, C1′), 98.26 (C5), 114.67, 123.1 (t, J = 260.1 Hz, C2′), 140.14, 145.95 (C6), 157.68 (C2), 164.82 (C4), 176.0; ¹⁹F NMR (CD₃OD) δ −120.13 (br. d, J = 239.4 Hz, 1F), −119.21 (dd, J = 9.3, 239.3 Hz, 1F); HRMS (ESI⁺) m/z calcd for C₂₂H₃₃F₂N₃NaO₅ [M+Na]⁺ 480.2280; found 480.2289.

4-N-(11-Hydroxyundecanoyl)- 2′-deoxy-2′,2′-difluorocytidine (7)

Treatment of 2a (58 mg, 0.194 mmol) with commercially available 11-hydroxyundecanoic acid (43 mg, 0.213 mmol) by Procedure A gave 75.5 mg of the crude product, which was then column chromatographed (7.5% MeOH/CHCl₃) to give 7 (35 mg, 40%) as a white solid: ¹H NMR (CD₃OD) δ 1.33 (br. s, 12H, 6 x CH₂), 1.49-1.54 (m, 2H, CH₂), 1.66 (quin, J = 7.2 Hz, 2H, CH₂), 2.45 (t, J = 7.4 Hz, 2H, CH₂), 3.53 (t, J = 6.6 Hz, 2H, CH₂), 3.81 (dd, J = 3.1, 12.8 Hz, 1H, H5′), 3.94-3.99 (m, 2H, H4′, H5′), 4.26-4.34 (m, 1H, H3′), 6.26 (“t,” J = 7.3 Hz, 1H, H1′), 7.49 (d, J = 7.6 Hz, 1H, H5), 8.33 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 25.93, 26.94, 30.13, 30.37, 30.48, 30.53, 30.64, 33.65, 38.17, 60.30 (C5′), 63.01, 70.23 (“t,” J = 23.0 Hz, C3′), 82.88 (“d,” J = 9.0 Hz, C4′), 86.47 (“dd,” J = 27.0, 37.6 Hz, C1′), 98.25 (C5), 123.91 (t, J = 258.9 Hz, C2′), 145.95 (C6), 157.67 (C2), 164.82 (C4), 176.00; ¹⁹F NMR (CD₃OD) δ −120.16 (“br. d,” J = 239.0 Hz, 1F), −119.21 (dd, J = 10.5, 242.6 Hz, 1F); HRMS (ESI⁺) m/z calcd for C₂₀H₃₁F₂N₃NaO₆ [M+Na]⁺ 470.2073; found 470.2073.

4-N-(11-Fluoroundecanoyl)-2′-deoxy-2′,2′-difluorocytidine (8)

Treatment of 2a (69.8 mg, 0.233 mmol) with 11-fluoroundecanoic acid (S4, 52 mg, 0.256 mmol) by Procedure A gave 82.7 mg of the crude product, which was then column chromatographed (70% EtOAc/hexane) to give 8 (42.1 mg, 41%) as a white solid: ¹H NMR (CD₃OD) δ 1.35 (br. s, 12H, 6 x CH₂), 1.62-1.74 (m, 4H, 2 x CH₂), 2.47 (t, J = 7.4 Hz, 2H, CH₂), 3.83 (dd, J = 3.0, 12.8 Hz, 1H, H5′), 3.96-4.02 (m, 2H, H5″, H4′), 4.32 (dt, J = 8.6, 12.2 Hz, 1H, H3′), 4.42 (dt, J = 6.1, 47.5 Hz, 2H, CH₂), 6.28 (t, J = 7.2 Hz, 1H, H1′), 7.51 (d, J = 7.6 Hz, 1H, H5), 8.35 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 25.95, 26.35, 30.16, 30.38, 30.48, 30.59, 31.50, 31.69, 38.17, 60.29(C5′), 70.20 (“t,” J = 23.0 Hz, C3′), 82.85 (“dd,” J = 2.3, 3.6 Hz, C4′), 84.89 (d, J = 163.8 Hz, CH₂F), 86.47 (dd, J = 29.6, 34.7 Hz, C1), 98.29 (C5), 123.94 (t, J = 259.2 Hz, C2′), 145.96 (C6), 157.69 (C2), 164.83 (C4), 176.01; ¹⁹F NMR (CD₃OD) δ −219.87 (tt, J = 24.7, 47.5 Hz, 1F), −120.09 (br. d, J = 239.0 Hz, 1F), −119.17 (br. dd, J = 10.2, 239.0Hz, 1F); MS (ESI) m/z 450 (100, [M+H]⁺); HRMS (+ESI) m/z calcd for C₂₀H₃₀F₃N₃Na₃O₅ [M+Na]⁺ 472.2023; found 472.2011.

4-N-[11-(1H-benzotriazol-1-yloxy)-undecanoyl]-2′-deoxy-2′,2′-difluorocytidine (9)

Treatment of 2a (50 mg, 0.167 mmol) with commercially available 11-bromoundecanoic acid (48.7 mg, 0.184 mmol) by Procedure A gave 85.5 mg of the crude product, which was then column chromatographed (5% MeOH/EtOAc) to give 9 (50 mg, 53%) as a white solid: ¹H NMR (DMSO-d₆) δ 1.28 (br. s, 10H, CH₂), 1.45-1.57 (m, 4H, CH₂), 1.73-1.80 (m, 2H, CH₂), 2.40 (t, J = 7.3 Hz, 2H, CH₂), 3.66 (“br. d,” J = 13.6 Hz, 1H, H5′), 3.80 (“br. d,” J = 13.6 Hz, 1H, H5″), 3.89 (dt, J = 2.7, 8.4 Hz, 1H, H4′) , 4.20 (“br. dt,” J = 9.1, 12.6 Hz, 1H, H3′), 4.55 (t, J = 6.5 Hz, 2H, CH₂), 5.35 (“br. t,” J = 4.6 Hz, 1H, OH), 6.17 (t, J = 7.5 Hz, 1H, H1′), 6.39 (br. s, 1H, OH), 7.28 (d, J = 7.6 Hz, 1H, H5), 7.48 (t, J = 7.6 Hz, 1H, Ar), 7.64 (t, J = 7.6 Hz, 1H, Ar), 7.82 (d, J = 8.4 Hz, 1H, Ar), 8.07 (d, J = 8.4 Hz, 1H, Ar), 8.25 (d, J = 7.6 Hz, 1H, H6), 10.99 (br. s, 1H, NH); ¹³C NMR (CD₃OD) 25.90, 26.64, 29.12, 30.06, 30.24, 30.28, 30.35, 30.40, 38.14, 58.32, 60.30, 70.23 (“t,” J = 23.1 Hz, C3′), 82.32, 82.89 (m, C4′), 98.25 (C5), 110.16, 120.50, 123.92, 126.38, 128.72, 129.55, 144.49, 145.95, 157.66, 164.81, 175.99; ¹⁹F NMR (CD₃OD) δ −120.09 (br. d, J = 239.0 Hz, 1F), −119.14 (dd, J = 243.7, 12.3 Hz, 1F); HRMS (+ESI) m/z calcd for C₂₆H₃₄F₃N₆NaO₆ [M+Na]⁺ 587.2406; found 587.2442.

4-N-(11-Chloroundecanoyl)-2′-deoxy-2′,2′-difluorocytidine (10).

Method A. TMSCl (79 μL, 68 mg, 0.630 mmol) was added to a suspension of 2a (150 mg, 0.500 mmol) in Pyr/MeCN (3:1, 2 mL) at 0 °C under Ar and stirred for 2.5 h, resulting in a clear solution. In a separate vessel, carbonyldiimidazole (CDI, 22.5 mg, 0.138 mmol) was added to a solution of 11-bromoundecanoic acid (36.5 mg, 0.138 mmol) in MeCN (1 mL) portion-wise and stirred at ambient temperature. After 30 minutes, the latter solution was combined with the previously prepared solution of transiently protected nucleoside and the new reaction mixture was stirred at 65 °C overnight. After 19 h, EtOH (2 mL) was added and mixture followed by H₂O (4 mL) and the solution stirred at 65 °C for 20 min. The volatiles were then evaporated under reduced pressure and the residue was partitioned between EtOAc and H₂O, the pH was adjusted to 2.0 with phosphoric acid, and the aqueous layer was extracted with EtOAc. The combined organic layer was washed with saturated NaHCO₃/H₂O, brine, dried over Na₂SO₄, evaporated under reduced pressure and the resulting residue (47.2 mg) was column chromatographed (70% EtOAc/hexane) to give 10 (11 mg, 5%) as a white solid: ¹H NMR (CD₃OD) δ 1.34 (br. s, 10H, 2 x CH₂), 1.41-1.49 (m, 2H, CH₂), 1.66-1.71 (m, 2H, CH₂), 1.73-1.82 (m, 2H, CH₂), 2.47 (t, J = 7.5 Hz, 2H, CH₂), 3.56 (t, J = 6.7 Hz, 2H, CH₂), 3.83 (“dd,” J = 12.7, 3.1 Hz, 1H, H5′), 3.96-4.03 (m, 2H, H5″, H4′), 4.27-4.37 (m, 1H, H3′), 6.28 (“t,” J = 7.2 Hz, 1H, H1′), 7.51 (d, J = 7.6 Hz, 1H, H5), 8.36 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 25.94, 27.93, 29.94, 30.13, 30.35, 30.43, 30.51, 33.83, 38.15, 45.74, 60.31 (C5′), 70.25 (C3′), 82.89 (C4′), 86.81 (C1′), 98.26 (C5), 123.93 (t, J = 258.0 Hz, C2′), 145.97 (C6), 157.71 (C2), 164.86 (C4), 176.02 (CO); ¹⁹F NMR (CD₃OD) δ −120.13 (br. d, J = 240.2 Hz, 1F), −119.2 (br. dd, J = 10.9, 240.2 Hz, 1F); MS (ESI⁺) m/z 466 (100, [M+H]⁺ for ³⁵Cl), 468 (100, [M+H]⁺ for ³⁷Cl); HRMS (ESI⁺) m/z calcd for C₂₀H₃₀³⁵ClF₂N₃NaO₅ [M+Na]⁺ 488.1734; found 488.1742.

Method B. Et₃N (28 μL, 0.200 mmol) was added to a mixture of 11-bromoundecanoic acid (26.6 mg, 0.100 mmol) in THF (1 mL) and stirred at ambient temperature under Ar. The reaction mixture was then cooled to −15 °C followed by the dropwise addition of a solution of ClCO₂Et (19 μL, 0.200 mmol) in THF (0.5 mL) with continued stirring. After 15 minutes, a solution of 2a (30 mg, 0.100 mmol) in DMF/DMSO (2.5 mL, 1.5:1) was added dropwise and the reaction mixture allowed to warm up to ambient and kept stirring overnight. After 24 h, the reaction was treated with NaHCO₃ and extracted with EtOAc (3x). The combined organic layer was washed with brine, dried over Na₂SO₄, evaporated under reduced pressure and the residue was column chromatographed (70% EtOAc/hexane) to give 10 (7 mg, 15%) with data as above.

4-N-(11-Bromoundecanoyl)-3′,5′-di-O-(tert-butoxycarbonyl)-2′-deoxy-2′,2′-difluorocytidine (11)

A solution of 2b⁴⁶ (35.5 mg, 0.077 mmol) and NaHCO₃ (400 mg, 4.76 mmol) in CH₂Cl₂ (0.5 mL) was added to a stirred solution of 11-bromoundecanoyl chloride (S5, 0.1 mL, 122 mg, 0.43 mmol) in CH₂Cl₂ (1 mL) at 0°C under Ar. After 15 minutes, the reaction mixture was allowed to warm up to ambient temperature and kept stirring for 3 h. The reaction mixture was quenched by addition of saturated NaHCO₃/H₂O, the mixture partitioned with water and the aqueous layer was extracted with CH₂Cl₂ (2 x 10 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, evaporated under reduced pressure and the resulting residue (141.0 mg) was chromatographed (25% EtOAc/hexane) to give 11 (18 mg, 33%) as a colorless oil: ¹H NMR (CDCl₃) δ 1.30 (br. s, 10H, 5 x CH₂), 1.40-1.45 (m, 2H, CH₂), 1.53 (s, 18H, 6 x CH₃), 1.68 (“quin,” J = 7.3 Hz, 2H, CH₂), 1.86 (“quin,” J = 7.3 Hz, 2H, CH₂), 2.48 (t, J = 7.5 Hz, 2H, CH₂), 3.42 (t, J = 6.9 Hz, 2H, CH₂), 4.37-4.50 (m, 3H, H4′, H5′,5″), 5.14 (“dt,” J = 4.5, 11.2 Hz, 1H, H3′), 6.46 (dd, J = 7.3, 9.5 Hz, 1H, H1′), 7.51 (d, J = 7.6 Hz, 1H, H5), 7.85 (d, J = 7.6 Hz, 1H, H6), 9.05 (br. s, 1H, NH); ¹³C NMR (CDCl₃) δ 24.77, 27.54, 27.70, 28.14, 28.72, 28.96, 29.22, 29.26, 29.33, 32.82, 34.07, 37.58, 63.87 (C5′), 72.64 (dd, J = 17.2, 34.0 Hz, C3′), 77.79 (C4′), 83.37, 84.21 (m, C1′), 84.83, 97.02 (C5), 120.40 (dd, J = 260.7, 267.3 Hz, C2′), 145.27 (C6), 151.42, 152.91, 153.94 (C2), 163.40 (C4), 174.17; ¹⁹F NMR (CDCl₃) δ −120.00 (br. d, J = 246.9 Hz, 1F), −115.57 (dt, J = 11.4, 246.9Hz, 1F); MS (ESI⁺) m/z 710 (100, [M+H]⁺ for ⁷⁹Br), 712 (100, [M+H]⁺ for ⁸¹Br).

4-N-(11-Bromoundecanoyl)-2′-deoxy-2′,2′-difluorocytidine (12)

Compound 11 (32 mg, 0.045 mmol) was dissolved in TFA (1.0 mL) and the mixture was stirred at 20 °C. After 4 h, the reaction mixture was diluted with toluene, the volatiles were evaporated, and the residue co-evaporated with a fresh portion of toluene. The resulting residue (32 mg) was column chromatographed (80 → 100% EtOAc/hexane) to give 12 (19.9 mg, 86%) as a colorless solid: ¹H NMR (CD₃OD): δ 1.31–1.41 (m, 10H, 5 x CH₂), 1.41-1.52 (m, 2H, CH₂), 1.63-1.73 (m, 2H, CH₂), 1.81-1.89 (m, 2H, CH₂), 2.47 (t, J = 7.4 Hz, 2H, CH₂), 3.45 (t, J = 6.7 Hz, 2H, CH₂), 3.75-3.89 (m, 1H, H5″), 3.93-4.05 (m, 2H, H4′, H5″), 4.32 (dt, J = 8.5, 12.2 Hz, 1H, H3′), 6.28 (“t,” J = 7.3 Hz, 1H, H1′), 7.51 (d, J = 7.6 Hz, 1H, H5), 8.35 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 25.94, 29.17, 29.80, 30.13, 30.34, 30.43, 30.48, 34.01, 34.42, 38.17, 60.32 (C5′), 70.25 (dd, J = 22.2, 23.6 Hz, C3′), 82.88 (‘d’, J = 8.6 Hz, C4′), 86.48 (dd, J = 26.6, 37.6 Hz, C1), 98.29 (C5), 123.93 (t, J = 259.9 Hz, C2′), 145.98 (C6), 157.69 (C2), 164.84 (C4), 176.03;¹⁹F NMR (CD₃OD) δ −120.10 (br. d, J = 240.0 Hz, 1F), −119.17 (ddd, J = 3.9, 12.1, 240.0 Hz, 1F); MS (ESI⁺) m/z 510 (100, [M+H]⁺ for ⁷⁹Br), 512 (100, [M+H]⁺ for ⁸¹Br); HRMS (ESI⁺) m/z calcd for C₂₀H₃₀⁷⁹BrF₂N₃NaO₅ [M+Na]⁺ 532.1229; found 532.1239.

4-N-(11-Hydroxyundecanoyl)-3′,5′-di-O-(tert-butoxycarbonyl)-2′-deoxy-2′,2′-difluorocytidine (13)

Treatment of 2b (39 mg, 0.084 mmol) with 11-hydroxyundecanoic acid (29 mg, 0.14 mmol) by Procedure A gave 102 mg of the crude product, which was then column chromatographed (55 → 65% EtOAc/hexane) to give 13 (20 mg, 37%) as a colorless oil: ¹H NMR (CDCl₃) δ 1.30 (br. s, 12H, 6 x CH₂), 1.53 (s, 18H, 6 x CH₃), 1.58 (“quin,” J = 6.9 Hz, 2H, CH₂), 1.69 (“quin,” J = 7.4 Hz, 2H, CH₂), 2.47 (t, J = 7.5 Hz, 2H, CH₂), 3.65 (t, J = 6.6 Hz, 2H, CH₂), 4.37-4.50 (m, 3H, H4′, H5′,5″), 5.14 (“dt,” J = 4.8, 11.1 Hz, 1H, H3′), 6.46 (dd, J = 7.2, 9.5 Hz, 1H, H1′), 7.51 (d, J = 7.6 Hz, 1H, H5), 7.85 (d, J = 7.6 Hz, 1H, H6), 9.08 (br. s, 1H, NH); ¹³C NMR (CDCl₃) δ 24.75, 25.65, 27.53, 27.69, 28.88, 29.11, 29.16, 29.27, 29.38, 32.75, 37.78, 62.99, 63.88 (C5′), 72.67 (dd, J = 17.0, 33.8 Hz, C3′), 77.73 (C4′), 83.33, 84.16 (dd, J = 18.2, 37.9 Hz, C1′), 84.77, 97.08 (C5), 120.42 (t, J = 263.8 Hz, C2′), 144.78 (C6), 151.44, 152.93, 154.67 (C2), 162.93 (C4), 173.46; ¹⁹F NMR (CDCl₃) δ −120.00 (br. d, J = 246.7 Hz, 1F), −115.58 (dt, J = 11.4, 246.7Hz, 1F); MS (ESI⁺) m/z 648 (100, [M+H]⁺).

Treatment of 13 (4.0 mg, 0.008 mmol) with TFA as descibed for 12 gave 7 (3.1 mg, 87%) with data sa reported above.

4-N-(11-Fluoroundecanoyl)-3′,5′-di-O-(tert-butoxycarbonyl)-2′-deoxy-2′,2′-difluorocytidine (14)

A chilled (−78°C) solution of DAST (6.2 μL, 7.6 mg, 0.048 mmol,) in CH₂Cl₂ (500 2μ) was added to a stirred solution of 13 (9.8 mg, 0.016 mmol) in CH₂Cl₂ (1.5 mL) at −78°C. After 30 minutes, the reaction mixture was allowed to warm up to ambient temperature and kept stirring. After 2 h, the reaction mixture was then poured into a separatory funnel containing a chilled solution of NaHCO₃/H₂O (10 mL, pH=8) and was then extracted with CHCl₃ (3 x 10 mL). The combined organic layer was washed with brine, dried over MgSO₄, evaporated under reduced pressure and the resulting residue (14 mg) was column chromatographed (5% MeOH/CHCl₃) to give 14 (4.2 mg, 40%) as a colorless oil: ¹H NMR (CDCl₃) δ 1.28 (br. s, 12H, 6 x CH₂), 1.51 (s, 9H, t-Bu), 1.52 (s, 9H, t-Bu), 1.60-1.78 (m, 4H, 2 x CH₂), 2.45 (t, J = 7.4 Hz, 2H, CH₂), 4.38-4.47 (m, 3H, H4′, H5′, H5″), 4.44 (dt, J = 6.2, 47.3 Hz, 2H, CH₂), 5.12-5.15 (m, 1H, H3′), 6.43 (t, J = 7.3 Hz, 1H, H1′), 7.51-7.54 (m, 1H, H5), 7.87 (d, J = 7.0 Hz, 1H, H6); ¹⁹F NMR (CDCl₃) δ −217.97 (dt, J = 25.0, 47.4 Hz, 2F), −120.27 (br. d, J = 240.7 Hz, 1F), −115.77 (dt, J = 10.9, 247.4 Hz, 1F); HRMS (ESI⁺) m/z calcd for C₃₀H₄₆F₃N₃NaO₉ [M+Na]⁺ 672.3078; found 672.3096.

Treatment of 14 (4.0 mg, 0.008 mmol) with TFA as descibed for 12 gave 8 (2.9 mg, 82%) with data sa reported above.

4-N-(10-Fluoroundecanoyl)-2′-deoxy-2′,2′-difluorocytidine (15)

Chilled hydrogen fluoride/pyridine (70%, 1.0 mL) was added to 5 (20 mg, 0.044 mmol) in an HDPE vessel at 0 °C and stirred. After 2 h, the reaction mixture was treated with saturated NaHCO₃/H₂O (10 mL) and extracted with EtOAc (3 x 10 mL). The combined organic layer was washed with brine, dried over Na₂SO₄, evaporated under reduced pressure and the resulting residue (24.6 mg) was then column chromatographed (70% EtOAc/hexane) to give 15 (19 mg, 91%; isomeric mixture of 15:16:17 in 75:20:5 ratio) as a white solid: UV (CH OH) λ_max 250 nm (ε 13 250), 298 nm (ε 5350), λ_min 226 nm (ε 4650), 279 nm (ε 4700); ¹H NMR (DMSO-d₆) δ 1.22 (br. d, J = 6.1 Hz, 2H, CH₂), 1.27 (br. s, 8H, 4 x CH₂), 1.29 (br. s, 2H, CH₂), 1.44-1.62 (m, 5H, CH₂, CH₃), 2.40 (t, J = 7.3 Hz, 2H, CH₂), 3.66 (dt, J = 12.5, 4.3 Hz, 1H, H5″), 3.81 (“br. d,” J = 12.0 Hz, 1H, H5′), 3.89 (“br. d,” J = 8.5 Hz, 1H, H4′), 4.19 (sep, J = 6.4 Hz, 1H, H3′), 4.64 (dsex, J = 49.0, 6.0 Hz, 1H, CH), 5.31 (t, J = 5.1 Hz, 1H, OH), 6.17 (t, J = 7.5 Hz, 1H, H1′), 6.33 (d, J = 5.8 Hz, 1H, OH), 7.29 (d, J = 7.6 Hz, 1H, H5), 8.24 (d, J = 7.6 Hz, 1H, H6), 10.98 (s, 1H, NH); ¹³C NMR (DMSO-d₆) δ 24.30, 24.42, 24.46, 28.37, 28.57, 28.71, 28.74, 36.13, 36.35, 58.78 (C5′), 68.37 (t, J = 22.5 Hz, C3′), 81.01 (t, J = 3.9 Hz, C4′), 84.50 (d, J = 82.2 Hz, C1′), 90.53 (d, J = 162.9 Hz), 95.87 (C5), 124.18 (d, J = 260.1 Hz, C2′), 144.68 (C6), 154.17 (C2), 162.85 (C4), 174.06; ¹⁹F NMR (DMSO-d₆) δ −170.27 (symmetric m, 0.75F), δ −116.91 (br. s, 2F); MS (ESI) m/z 450 (100, [M+H]⁺); HRMS (ESI⁺) m/z calcd for C₂₀H₃₀F₃N₃NaO₅ [M+Na]⁺ 472.2030; found 472.2048. Elemental Anal. Calcd for C₂₀H₃₀F₃N₃O₄•H₂O•0.33CH₃CN (481.03): C, 51.59; H, 6.91; N, 9.70. Found: C, 51.36; H, 6.89; N, 9.97.

Minor isomers 16 [4-N-(9-Fluoroundecanoyl)] and 17 [4-N-(8-Fluoroundecanoyl)] had the following distinguishable peaks: ¹H NMR (DMSO-d₆): δ 4.41 (d quin, J = 49.6, 5.8 Hz, 0.2, CHF); ¹⁹F NMR (DMSO-d₆) δ −179.79 (symmetric m, 0.15F), −178.83 (m, 0.1F), −116.91 (br. s, 2F).

4-N-(p-Toluenosulfonyl)-3′,5′-di-O-(tert-butoxycarbonyl)-2′-deoxy-2′,2′-difluorocytidine (18)

Et₃N (1.45 mL, 10.5 mmol) and TsCl (997 mg, 5.2 mmol) were added to a solution of 2b (242 mg, 0.52 mmol) in dry 1,4-dioxane (4.0 mL) and stirred at ambient temperature under Ar. The tightly sealed reaction mixture was then gradually heated to 65 °C and kept stirring. After 24 h, the reaction mixture was diluted with EtOAc, partitioned with saturated NaHCO₃/H₂O solution, and the aqueous layer was then extracted with EtOAc (2x). The combined organic layer was washed with brine, dried over Na₂SO₄, evaporated under reduced pressure and the resulting residue (403 mg) was then column chromatographed (35% EtOAc/hexane) to give 18 (146 mg, 45%) as a colorless, solidifying oil: ¹H NMR δ 1.49 (s, 9H, 3 x CH₃), 1.52 (s, 9H, 3 x CH₃), 2.43 (s, 3H, CH₃), 4.46–4.32 (m, 3H, H4′, H5′, 5″), 5.11 (ddd, J = 4.0, 5.3, 12.8 Hz, 1H, H3′), 5.80 (br. s, 1H, H5), 6.24 (dd, J = 6.6, 10.6 Hz, 1H, H1′), 7.31 (d, J = 8.1 Hz, 2H, Ar), 7.48 (dd, J = 1.9, 8.1, Hz, 1H, H6), 7.84 (d, J = 8.3 Hz, 2H, Ar), 10.96 (br. s, 1H, NH); ¹³C NMR δ 21.54, 27.51, 27.65, 63.80 (C5′), 72.40 (dd, J = 16.9, 33.8 Hz, C3′), 78.02 (dd, J = 2.2, 4.7 Hz, C4′), 83.31 (dd, J = 20.6, 38.7 Hz, C1′), 83.41, 84.99, 98.41 (C5), 120.38 (dd, J = 260.2, 266.5 Hz, C2′), 126.71 (Ar), 129.58 (Ar), 138.26 (d, J = 3.4 Hz, Ar), 139.88 (d, J = 2.2 Hz, C6), 143.74 (Ar), 147.16 (C2), 151.35, 152.82, 154.82 (C4); ¹⁹F NMR δ −120.59 (br. d, J = 247.6 Hz, 1F), −115.80 (br. d, J = 247.6 Hz, 1F); MS (ESI⁺) m/z 618 (100, [M+H]⁺); HRMS (ESI⁺) m/z calcd for C₂₆H₃₃ F₂N₃NaO₁₀S [M+Na]⁺ 640.1747; found 640.1754.

4-N-(10-Undecenyl)-2′-deoxy-2′,2′-difluorocytidine (19)

In a tightly sealed vessel, a mixture of 18 (40 mg, 0.065 mmol) and 1-amino-10-undecene (0.50 mL, 404 mg, 2.4 mmol) was stirred at 60 °C. After 30 h, the volatiles were evaporated the resulting residue was column chromatographed (8% MeOH/EtOAc) to give 19 (9.5 mg, 36%) as colorless viscous oil: UV (CH₃OH) λ_max 268 nm (ε 11 600), λ_min 228 nm (ε 7800); ¹H NMR (CD₃OD) δ 1.43-1.30 (m, 12H, 6 x CH₂), 1.65-1.56 (m, 2H, CH₂), 2.03-2.09 (m, 2H, CH₂), 3.39 (t, J = 7.1 Hz, 2H, CH₂), 3.80 (dd, J = 3.3, 12.6 Hz, 1H, H5′), 3.89 (td, J = 2.8, 8.3 Hz, 1H, H4′), 3.95 (d, J = 12.6 Hz, 1H, H5″), 4.26 (dt, J = 8.3, 12.1 Hz, 1H, H3′), 4.91-5.02 (m, 2H, CH₂), 5.82 (tdd, J = 6.7, 10.3, 17.0 Hz, 1H, CH), 5.87 (d, J = 7.6 Hz, 1H, H5), 6.23 (t, J = 8.0 Hz, 1H, H1′), 7.74 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 28.01, 29.98, 30.12, 30.19, 30.42, 30.51, 30.63, 34.88, 41.75, 60.56 (C5′), 70.67 (dd, J = 22.4, 23.8 Hz, C3′), 82.26 (dd, J = 3.6, 5.0 Hz, C4′), 85.94 (dd, J = 26.0, 38.0 Hz, C1), 97.33 (C5), 114.68, 124.05 (t, J = 258.4 Hz, C2′), 140.16, 140.77 (C6), 158.30 (C2), 165.37 (C4); ¹⁹F NMR (CD₃OD) δ −119.89 (br. d, J = 240.1 Hz, 1F), −118.80 (br. d, J = 240.1 Hz, 1F); MS (ESI⁺) m/z 416 (100, [M+H]⁺); HRMS (ESI⁺) m/z calcd for C₂₀H₃₁F₂N₃NaO₄ [M+Na]⁺ 438.2175; found 438.2178; Elemental Anal. Calcd for C₂₀H₃₁F₂N₃O₄•0.5H₂O•0.5CH₃CN (445.01): C, 56.68; H, 7.59; N, 11.02. Found: C, 56.93; H, 7.77; N, 10.76.

4-N-(11-Hydroxyundecanyl)-2′-deoxy-2′,2′-difluorocytidine (21)

11-Amino-1-undecanol (S7; 88 mg, 0.47 mmol) and Et₃N (0.5 mL) were added to a solution of 18 (23.2 mg, 0.038 mmol) in 1,4-dioxane (0.5 mL) and stirred at ambient temperature under Ar. The reaction mixture was then gradually heated to 65 °C (oil bath) and kept stirring overnight. After 40 h, the volatiles were evaporated and the residue (97 mg) was column chromatographed (1 → 3% MeOH/EtOAc) to give mono-protected product 20 [9.5 mg, 47%: ¹H NMR (CD₃OD) δ 1.32 (br. s, 12H, 6 x CH₂), 1.49 (s, 9H, t-Bu), 1.49-1.61 (m, 4H, 2 x CH₂), 3.37 (t, J = 7.1 Hz, 2H, CH₂), 3.49-3.62 (m, 2H, CH₂), 4.17 (dt, J = 9.9, 19.4 Hz, 1H, H4′), 4.02-4.09 (m, 1H, H3′), 4.48 (dd, J = 2.6, 12.4 Hz, 1H, H5′), 4.33 (dd, J = 4.3, 12.4 Hz, 1H, H5″), 5.86 (d, J = 7.6 Hz, 2H, H5), 6.25 (t, J = 8.2 Hz, 2H, H1′), 7.51 (d, J = 7.6 Hz, 2H, C6); MS (ESI⁺) m/z 534 (100, [M+H]⁺)] followed by 21 (4 mg, 24%) of 90% purity. Compound 20 (9.5 mg, 0.018 mmol) was dissolved in TFA (1.0 mL) and reaction mixture was stirred at 18 °C. After 5 h, the reaction mixture was diluted with toluene (2 mL), the volatiles were evaporated, and the residue was co-evaporated with a toluene (2 x 1 mL). The resulting residue (17 mg) was then column chromatographed (1% MeOH/EtOAc) to give 21 (2.2 mg, 29% from 20; 38% overall from 18) as a colorless oil: ¹H NMR (CD₃OD) δ 1.30-1.41 (m, 14H, 7 x CH₂), 1.50-1.57 (m, 2H, CH₂), 1.58-1.64 (m, 2H, CH₂), 3.39 (t, J = 7.1 Hz, 2H, CH₂), 3.55 (t, J = 6.6 Hz, 2H, CH₂), 3.80 (dd, J = 3.3, 12.6 Hz, 1H, H5′), 3.89 (td, J = 2.8, 8.3 Hz, 1H, H4′), 3.95 (br. dd, J = 2.0, 12.6, 1H, H5″), 4.26 (dt, J = 8.3, 12.1 Hz, 1H, H3′), 5.87 (d, J = 7.6 Hz, 1H, H5), 6.23 (“t,” J = 8.0 Hz, 1H, H1′), 7.74 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 26.94, 28.01, 29.97, 30.42, 30.58, 30.63, 30.66, 30.71, 33.67, 41.74, 60.56 (C5′), 63.03, 70.63 (dd, J = 22.0, 24.8 Hz, C3′), 82.23 (dd, J = 3.8, 5.0 Hz, C4′), 85.82 (C1), 97.32 (C5), 124.04 (t, J = 259.8 Hz, C2′), 140.77 (C6), 158.29 (C2), 165.37 (C4); ¹⁹F NMR (CD₃OD) δ −119.90 (br. d, J = 239.2 Hz, 1F), −118.83 (dd, J = 11.6, 239.2Hz, 1F); MS (ESI) m/z 434 (100, [M+H]⁺); HRMS (ESI⁺) m/z calcd for C₂₀H₃₃F₂N₃NaO₅ [M+Na]⁺ 456.2280; found 456.2287.

4-N-(p-Toluenosulfonyl)-2′-deoxy-2′,2′-difluorocytidine (22)

TMSCl (5.1 mL) was added to a suspension of 2a (600 mg, 2.0 mmol) in anhydrous pyridine (10 mL) and stirred at ambient temperature under Ar. After 2 h, TsCl (3.8 g, 20.027 mmol) was added and the reaction mixture gradually heated to 60 °C (oil-bath) and kept stirring. After 20 h, volatiles were evaporated under reduced pressure and the resulting residue was treated with MeOH/NH₃ (10 mL) and stirred at ambient temperature overnight. After 24 h, volatiles were evaporated under reduced pressure and the resulting residue was column chromatographed (90% EtOAc/hexane) to give 22 (808 mg, 96%) as a white solid: ¹H NMR (CD₃OD) δ 2.42 (s, 3H, CH₃), 3.78 (dd, J = 3.4, 12.8 Hz, 1H, H5′), 3.90-3.95 (m, 2H, H4′, H5″), 4.28 (dt, J = 8.4, 12.0 Hz, 1H, H3′), 6.13 (“dd,” J = 5.3, 9.5 Hz, 1H, H1′), 6.65 (d, J = 8.2 Hz, 1H, H5), 7.36 (d, J = 8.0 Hz, 2H, Ar), 7.79 (d, J = 8.3 Hz, 2H, Ar), 7.99 (d, J = 8.1 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 21.43, 60.34 (C5′), 70.21 (dd, J = 18.8, 27.2 Hz, C3′), 82.99 (d, J = 8.4, C4′), 85.46 (dd, J = 23.9, 41.3 Hz, C1′), 98.46 (C5), 123.84 (t, J = 258.7 Hz, C2′), 127.58 (Ar), 130.52 (Ar), 140.71 (Ar), 142.62 (C6), 144.66 (Ar), 150.21 (C2), 160.54 (C4); ¹⁹F NMR (CD₃OD) δ −120.17 (br. s, 1F), −119.41 (dd, J = 4.1, 12.7 Hz, 1F); MS (ESI⁺) m/z 440 (100, [M+Na]⁺. HRMS (ESI⁺) m/z calcd for C₁₆H₁₇F₂N₃NaO₆S [M+Na]⁺ 440.0698; found 440.0711.

4-N-(11-Benzyloxyundecanyl)-2′-deoxy-2′,2′-difluorocytidine (23)

In a tightly sealed container, a solution of 22 (158 mg, 0.383 mmol), 11-(benzyloxy)undecanyl amine (S11; 945 mg, 3.41 mmol) and TEA (2 mL) in 1,4-dioxane was stirred at 75 °C. After 96 h, the volatiles were evaporated under reduced pressure and the resulting residue was column chromatographed (1% MeOH/EtOAc) to give 23 (122 mg, 61%): ¹H NMR (CD₃OD) δ 1.28 (br. s, 10H, 5 x CH₂), 1.32 (br. s, 4H, 2 x CH₂), 1.54-1.61 (m, 4H, 2 x CH₂), 3.36 (t, J = 7.1 Hz, 2H, CH₂), 3.47 (t, J = 6.6 Hz, 2H, CH₂), 3.79 (dd, J = 3.3, 12.6, 1H, H5′), 3.88-3.96 (m, 2H, H4′, H5″), 4.25 (dt, J = 8.3, 12.0 Hz, 1H, H3′), 4.48 (s, 2H, CH₂), 5.89 (d, J = 7.6 Hz, 1H, H5), 6.21 (t, J = 8.0 Hz, 1H, H1′), 7.32 (“br. s,“ 5H, Ar), 7.70 (d, J = 7.6 Hz, 1H, H6); ¹³C NMR (CD₃OD) δ 27.23, 28.00, 29.96, 30.42, 30.48, 30.60, 30.63, 30.65, 30.71, 41.74, 60.54, 68.14 (C3′), 71.44, 73.86, 82.24 (“t,” J = 2.95 Hz, C4′), 85.92 (“dd,” J = 26.7, 37.7 Hz, C1′), 97.31 (C5), 124.04 (t, J = 258.7 Hz, C2′), 128.62, 128.84, 129.35, 139.87, 140.75, 158.27, 165.34; ¹⁹F NMR (CD₃OD) δ-119.47 (br. d, J = 236.7 Hz, 1F), −118.42 (dd, J = 8.6, 236.7 Hz, 1F); HRMS (ESI⁺) m/z calcd for C₂₇H₃₉F₂N₃NaO₅ [M+Na]⁺ 546.2750; found 546.2774.

4-N-(11-Benzyloxyundecanyl)-3′,5′-di-O-benzoyl-2′-deoxy-2′,2′-difluorocytidine (24)

BzCl (50 μL, 0.49 mmol) was added to a solution of 23 (117 mg, 0.22 mmol), 2,6-Lutidine (64 μL, 0.89 mmol) and 4-dimethylaminopyridine (27 mg, 0.22 mmol) in CH₂Cl₂ (10 mL) and stirred at 35 °C under Argon. After 20 h, the reaction mixture was diluted with CH₂Cl₂ (40 mL), partitioned with H₂O, and the aqueous layer extracted with CH₂Cl₂ (2 x 20 mL). The combined organic layer was sequentially washed with 1M HCl (20 mL), saturated NaHCO₃/H₂O (20 mL) and brine (20 mL), dried over Na₂SO₄, evaporated under reduced pressure and the resulting residue (157 mg) was column chromatographed (1% MeOH/CHCl₃) to give 24 (50.6 mg, 60%) as a mixture of rotamers (80:20). The major rotamer had the following peaks: ¹H NMR (CD₃OD) δ 1.24 (br. s, 12H, 6 x CH₂), 1.51-1.62 (m, 4H, 2 x CH₂), 3.20 (t, J = 7.1 Hz, 2H, CH₂), 3.39 (t, J = 12.3 Hz, 2H, CH₂), 4.48 (s, 2H, CH₂), 4.49-4.53 (m, 1H, H5′), 4.63-4.67 (m, 1H, H5′), 4.73-4.79 (m, 1H, H4′), 5.54 (d, J = 7.6 Hz, 1H, H5), 5.57-5.61 (m, 1H, H3′), 6.60-6.65 (m, 1H, H1′), 7.26-7.33 (m, 5H, Ar), 7.41-7.49 (m, 4H, Ar), 7.55-7.64 (m, 2H, Ar), 8.02-8.08 (m, 4H, Ar); ¹⁹F NMR (CD₃OD) δ −120.48 (br. d, J = 246.7 Hz, 1F), −115.34 (dt, J = 13.6, 246.7 Hz, 1F); MS (ESI⁺) m/z 754 (100, [M+Na]⁺. HRMS (ESI⁺) m/z calcd for C₄₁H₄₇F₂N₃NaO₇ [M+Na]⁺ 754.3274; found 754.3303.

Minor rotamer had the following distinguishable peaks: ¹H NMR (CD₃OD): δ 5.70 (d, J = 7.8, 1H, H5); ¹⁹F NMR (CD₃OD): δ −115.82 (dt, J = 13.4, 246.7, 1F).

4-N-(11-Hydroxyundecanyl)-3′,5′-di-O-benzoyl-2′-deoxy-2′,2′-difluorocytidine (25)

Ammonium cerium (IV) nitrate (63 mg, 0.115 mmol) was added to a solution of 24 (106 mg, 0.145 mmol) in CH₃CN:H₂O (9:1, 5 mL) and stirred at ambient temperature overnight. Additional portions of CAN (240 mg) were added to the reaction mixture every 24 h until no starting material could be detected by TLC. After 72 h, the reaction was quenched by the addition of saturated NaHSO₃ (20 mL), the volatiles evaporated under reduced pressure and the resulting aqueous residue was extracted with EtOAc (3 x 20 mL). The combined organic layer was washed with brine (20 mL), dried over Na₂SO₄, evaporated under reduced pressure and the resulting residue (103.5 mg) was then column chromatographed (1% MeOH/EtOAc) to give 25 (66 mg, 70%) as a mixture of rotamers (72:28). The major rotamer had the following peaks: ¹H NMR (CD₃OD) δ 1.24 (br. s, 14H, 7 x CH₂), 1.51-1.64 (m, 4H, 2 x CH₂), 3.20 (t, J = 7.0 Hz, 2H, CH₂), 3.63 (t, J = 6.6 Hz, 2H, CH₂), 4.50-4.58 (m, 1H, H4′), 4.64-4.71 (m, 1H, H5′), 4.75-4.82 (m, 1H, H5″), 5.57-5.62 (m, 1H, H3′) 5.60 (d, J = 7.6 Hz, 1H, H5), 6.58-6.61 (m, 1H, H1′), 7.31 (“d,” J = 7.6 Hz, 1H, H6), 7.41-7.65 (m, 6H, Ar), 8.02-8.11 (m, 4H, Ar); ¹³C NMR (CDCl₃) δ 25.79, 26.93, 29.20, 29.29, 29.44, 29.47, 29.53, 29.57, 32.78, 41.14, 62.90, 63.01, 71.86 (m, C3′), 77.74 (C4′), 83.51 (br. s, C1′), 96.55 (C5), 120.98 (t, J = 256.3 Hz, C2′), 128.10, 128.70, 128.81, 129.39, 129.81, 130.27, 133.60, 134.28, 139.86, 155.75, 163.59, 165.05, 166.09; ¹⁹F NMR δ −115.36 (dt, J = 13.7, 246.3 Hz, 1F), −120.50 (br. d, 1F); MS (ESI⁺) m/z 664 (100, [M+Na]⁺. HRMS (ESI⁺) m/z calcd for C₃₄H₄₁F₂N₃NaO₇ [M+Na]⁺ 664.2805; found 664.2837.

Minor rotamer had the following distinguishable peaks: ¹H NMR (400 MHz, CDCl₃) δ 3.40-3.41 (m, 2H, CH ), 5.71 (d, J = 7.8 Hz, 1H, H5); ¹⁹F NMR δ −115.85 (dt, J = 12.4, 246.0 Hz, 1F).

4-N-(11-Fluoroundecanyl)-3′,5′-di-O-benzoyl-2′-deoxy-2′,2′-difluorocytidine (26)

A chilled (−78 °C) solution of DAST (14 μL, 17.2 mg, 0.107 mmol) in CH₂Cl₂ (250 μL) was added to a stirred solution of 25 (21.7mg, 0.034 mmol) in CH₂Cl₂ (1 mL) at −78 °C. After 30 minutes, the reaction mixture was allowed to warm up to ambient temperature and kept stirring. After 3 h, the reaction mixture was poured into a separatory funnel containing a ice-cold solution of Na₂HCO₃ in H₂O (10 mL, pH = 8) and was extracted with CHCl₃ (3 x 10 mL). The combined organic layer was washed with brine, dried over MgSO₄, evaporated under reduced pressure and the resulting oily residue (20.5 mg) was then column chromatographed (40% EtOAc/hexane) to give 26 (10.6 mg, 48%) as a mixture of rotamers (76:24). The major rotamer had the following peaks: ¹H NMR (CDCl₃) δ 1.27 (br. s, 14H, 7 x CH₂), 1.55-1.69 (m, 4H, 2 x CH₂), 3.47-3.52 (m, 2H, CH₂), 4.43 (dt, J = 6.2, 47.4 Hz, 2H, CH₂), 4.51-4.55 (m, 1H, H4′), 4.67 (dd, J = 4.5, 12.3 Hz, 1H, H5′), 4.79 (dd, J = 3.2, 12.3 Hz, 1H, H5″), 5.54 (d, J = 7.6 Hz, 1H, H5), 5.58-5.63 (m, 1H, H3′), 6.61 (br.s, 1H, H1′), 7.32 (d, J = 7.5 Hz, 1H, H6), 7.42-7.66 (m, 6H, Ar), 8.03-8.16 (m, 4H, Ar);¹³C NMR (CDCl₃) δ 25.30, 27.01, 29.24, 29.32, 29.49, 29.56, 29.83, 30.44, 30.63, 41.28, 63.02 (C5′), 71.87 (br. s, C3′), 79.16 (br. s, C4′), 83.56 (br. s, C1′), 84.37 (d, J = 164.0 Hz'), 96.17 (C5), 121.88 (t, J = 255.7 Hz, C2′), 128.14, 128.73, 128.84, 129.43, 129.84, 130.31, 133.59, 134.29, 140.10, 155.46, 163.39, 165.06, 166.10; ¹⁹F NMR (CDCl₃) δ −217.94 (tt, J = 24.9, 47.3 Hz, 1F), −120.62 (br. d, J = 203.1, 1F), −115.40 (dt, J = 14.1, 247.3 Hz, 1F); MS (ESI) m/z 644 (100, [M+H]⁺); HRMS (ESI-TOF⁺) m/z calcd for C₃₄H₄₀F₃N₃NaO₆ [M+Na]⁺ 666.2761; found 666.2763.

Minor rotamer had the following distinguishable peaks: ¹H NMR (400 MHz, CDCl₃) δ 3.20-3.21 (m, 2H, CH N), 5.71 (d, J = 7.8 Hz, 1H, H5); ¹⁹F NMR δ −115.98 (dt, J = 12.9, 247.5 Hz, 1F)

4-N-(11-Fluoroundecanyl)-2′-deoxy-2′,2′-difluorocytidine (27)

Method A. Compound 26 (10.6 mg, 0.017 mmol) was dissolved in methanolic ammonia (2 mL) and stirred at ambient temperature. After 2 h, volatiles were evaporated under reduced pressure and the resulting residue was chromatographed (5% MeOH/EtOAc) to give 27 (6.5 mg, 90%) as a clear oil: ¹H NMR (CD₃OD) δ 1.32 (br.s, 14H, 7 x CH₂), 1.55-1.73 (m, 4H, 2 x CH₂), 3.37 (t, J = 7.1 Hz, 2H, CH₂), 3.78 (dd, J = 3.3, 12.6 Hz, 1H, H5′), 3,87 (dt, J = 3.0, 8.28 Hz, 1H, H3′), 3.93 (‘d’, J = 13.3 Hz, 1H, H5″), 4.20-4.28 (m, 1H, H4′), 4.40 (dt, J = 6.1, 47.6 Hz, 1H, CH₂), 5.85 (d, J = 7.6 Hz, 1H, H5), 6.21 (“t”, J = 7.96 Hz, H1′), 7.73 (d, J = 7.6 Hz, 1H, H6); ¹⁹F NMR δ −219.94 (tt, J = 25.5, 47.3 Hz, 1F, CH₂F), −119.60 (br. s, 1F), −119.14 (br. s, 1F); MS (ESI) m/z 436 (100, [M+H]⁺); HRMS (ESI⁺) m/z calcd for C₂₀H₃₂F₃N₃NaO₄ [M+Na]⁺ 458.2237; found 458.2256.

Method B. In a tightly sealed cylindrical pressure vessel with screw cap, a solution of KF (1.6 mg, 0.028 mmol), K₂CO₃ (3.8 mg, 0.028 mmol), Kryptofix 2.2.2 (10.5 mg, 0.028 mmol) and 28 (5.0 mg, 0.007 mmol) in CH₃CN (1 mL) was stirred at 110 °C. After 18 min, the reaction mixture was quickly cooled in a water bath and vacuum filtrated into another pressure vessel. The effluent containing crude 26 was concentrated under reduced pressure and the resulting residue treated with 0.5 CH₃ONa/MeOH (1 mL), then stirred and heated at 100 °C. After 8 min, the reaction mixture was neutralized with 1N HCl and evaporated under reduced pressure to dryness. The crude sample was then dissolved in 45% CH₃CN/H₂O to a total volume of 4.5 mL, passed through a 0.2 μM PTFE syringe filter and then injected into a semi-preparative HPLC column (Phenomenex Gemini RP-C18 column; 5μ, 25 cm X 1 cm) via 5 mL loop. The HPLC column was eluted with an isocratic mobile phase mixture 45% CH₃CN/H₂O at a flow rate = 5 mL/min to give 27 (1.9 mg, 62% overall yield from 28, t_R = 13.1 min) with spectral properties as above.

4-N-[11-(Methanesulfoxy)undecanyl]-3′,5′-di-O-benzoyl-2′-deoxy-2′,2′-difluorocytidine (28)

Et₃N (3.8 μL, 2.7 mg, 0.027 mmol) and MsCl (1.5 μL, 2.3 mg, 0.020 mmol) were sequentially added to a stirred solution of 25 (11.6 mg, 0.018 mmol) in CH₂Cl₂ at 0 °C. After 5 minutes, the reaction mixture was allowed to warm up to ambient temperature and kept stirring. After 3 h, the reaction mixture was then partitioned between H₂O and CH₂Cl₂, and the aqueous layer then extracted with CH₂Cl₂ (2 x 20 mL). The combined organic layer was washed with brine, dried over MgSO₄, evaporated under reduced pressure and the resulting residue (12.1 mg) was column chromatogramphed (50% EtOAc/hexane) to give 28 (11.7 mg, 90%) as a mixture of rotamers (71:29). The major rotamer had the following peaks: ¹H NMR (CDCl₃) δ 1.25 (br.s, 14H, 7 x CH₂), 1.55-1.77 (m, 4H, 2 x CH₂), 2.99 (s, 3H, CH₃), 3.46-3.52 (m, 2H, CH₂), 4.21 (t, J = 6.6 Hz, 2H, CH₂), 4.51-4.58 (m, 1H, H4′), 4.64-4.81 (m, 2H, H5′, H5″), 5.55 (d, J = 7.6 Hz, 1H, H5), 5.59-5.63 (m, 1H, H3′), 6.55-6.67 (m, 1H, H1′), 7.32 (dd, J = 1.6, 7.5 Hz, 1H, H6), 7.43-7.51 (m, 4H, Ar), 7.57-7.66 (m, 2H, Ar), 8.03-8.10 (m, 4H, Ar); ¹³C NMR (CDCl₃) δ 21.15, 25.47, 26.94, 29.00, 29.22, 29.30, 29.39, 29.46, 29.55, 37.50, 41.18, 63.02 (C5′), 70.39, 71.96 (“dd,” J = 17.3, 35.8 Hz, C3′), 77.36 (C4′), 84.00 (br. s, C1′), 96.22 (C5), 120.93 (t, J = 262.8 Hz, C2′), 128.13, 128.71, 128.82, 129.41, 129.82, 130.28, 133.61, 134.28, 139.97 (C6), 155.66, 163.55, 165.05, 166.08; ¹⁹F NMR δ −120.61 (br.d, J = 261.9 Hz, 1F), −115.38 (dt, J = 14.1, 246.7 Hz, 1F); MS (ESI) m/z 720 (100, [M+H]⁺); HRMS (ESI⁺) m/z calcd for C₃₅H₄₃F₂N₃NaO₉S [M+Na]⁺ 742.2580; found 742.2603.

Minor rotamer had the following distinguishable peaks: ¹H NMR (CDCl₃) δ 3.23-3.25 (m, CH₂N), 5.77 (d, J = 7.9 Hz, H5); ¹⁹F NMR δ −115.98 (dt, J = 13.1, 234.1 Hz, 1F).

Cytostatic Activity Assays.³³

The compounds tested were added to murine leukemia L1210, human T-lymphocyte CEM, human cervix carcinoma HeLa and human breast carcinoma MCF-7 cell cultures in 96-well microtiter plates. After two (L1210) or three (CEM) or four (HeLa, MCF-7) days incubation at 37°C, the number of living cells was determined by a Coulter counter. The 50% inhibitory concentration (IC₅₀) was defined as the compound concentration required to inhibit cell proliferation by 50%.

dCK and TK-2 Activity Assays

The activity of recombinant mitochondrial thymidine kinase (TK-2) and cytosolic 2′-deoxycytidine kinase (dCK), and the 50% inhibitory concentration of the test compounds were assayed in a 50 μL reaction mixture containing 50 mM Tris/HCl, pH 8.0, 2.5 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM CHAPS, 3 mg/mL bovine serum albumin, 2.5 mM ATP, 1 μM [5-³H]dCyd or [CH₃-³H]dThd and enzyme. The samples were incubated at 37 °C for 30 min in the presence or absence of different concentrations (5-fold dilutions) of the test compounds. Aliquots of 45 μL of the reaction mixtures were spotted on Whatman DE-81 filter paper disks. The filters were washed three times for 5 min each in 1 mM ammonium formate, once for 1 min in water, and once for 5 min in ethanol. The radioactivity retained on the filter discs was determined by scintillation counting. To evaluate substrate activity against TK-2 and dCK, the tested compounds were added to the enzyme reaction mixture at 100 μM and conversion to their 5′-monophosphates was monitored by HPLC on an anion exchange Partisil Sax column.

Human Serum and Murine Liver Extract Stability Assays

The compounds tested were exposed to 50% human serum in phosphate buffered saline (PBS) or murine liver extract in PBS at 100 μM concentrations and incubated for 0, 60 and 240 min (human serum) or 0, 30 and 120 min (murine liver extract) at 37 °C. At each time point (0, 60, 240 min) an aliquot was withdrawn and subjected to HPLC analysis on a reverse phase RP-18 column (mobile phase: acetonitrile/H₂O). Elution times were 13.2 min and 16.4 min for dFdU and gemcitabine, respectively, and 22.8 min and 22.7 min for compounds 7 and 21, respectively.

Abbreviations

dFdC

2′,2′-difluoro-2′-deoxycytidine (Gemcitabine)

hENT1

human equilibrative nucleoside transport protein 1

dCK

deoxycytidine kinase

RNR

ribonucleotide reductase

CTP

cytidine triphosphate

dFdU

2′,2′-difluorouridine

CDA

cytidine deaminase

SQgem

4-N-squalenoylgemcitabine

[¹⁸F]-FAC

1-(2′-deoxy-2′-¹⁸F-fluoro-β-D-arabinofuranosyl)cytosine)

L-¹⁸F-FMAC

1-(2′-deoxy-2′-¹⁸F-fluoro-β-L-arabinofuranosyl)-5-methylcytosine

EDCI

N-dimethylaminopropyl)-N′-ethyl-carbodiimide

HOBt

1-hydroxybenzotriazole

NMM

N-methylmorpholine

CDI

1,1′-carbonyldiimidazole

TEA

triethylamine

DAST

(diethylamino)sulfur trifluoride

HDPE

high-density polyethylene

TK-2

thymidine kinase