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Abstract

Background:

Following the example of L-valine prodrugs of antiviral nucleoside analogues, L-valine ester of cyclopropavir (valcyclopropavir) was synthesized.

Methods:

The known tetrahydropyranylcyclopropavir was transformed to N-(tert-butoxycarbonyl)-L-valine ester, which was deprotected to valcyclopropavir.

Results:

Stability of valcyclopropavir towards hydrolysis at pH 7.0 roughly corresponded to that of valganciclovir. Valcyclopropavir inhibited replication of human cytomegalovirus (HCMV, Towne and AD169 strains) to approximately the same extent as the parent drug cyclopropavir. Pharmacokinetic studies in mice established that the oral bioavailability of valcyclopropavir was 95%.

Conclusions:

The prodrug valcyclopropavir offers some improved therapeutic parameters over the parent compound cyclopropavir.

Introduction

Efficacy of antiviral nucleoside analogues is often hampered by their low solubility and bioavailability. To address this deficiency, several types of prodrugs have been synthesized [1]. Amino acid esters were found to be especially advantageous. Valine ester prodrugs of acyclovir (valacyclovir [Valtrex] (1)) [2 –4] and ganciclovir (valganciclovir [Valcyte] (2); Figure 1) [5,6] are already part of our armament for fighting infections caused by herpes simplex virus type-1 and type-2, varicella zoster virus and human cytomegalovirus (HCMV). An additional advantage of these prodrugs resides in their ability to facilitate the membrane transport by using the peptide transporters [6,7] PEPT1 and PEPT2. L-Valine ester prodrugs are readily hydrolysed by chemical or esterase-catalysed hydrolysis and/or a biphenyl hydrolase-like protein (valaciclovirase) [8]. More recently, this approach has been extended to the nucleoside analogue 2′-deoxy-L-cytidine (L-dC) [9], which is effective against hepatitis B virus and 2′-C-methylcytidine [10], which is an anti-hepatitis C virus agent. Both L-valine esters, Val-L-dC (valtorcitabine (3)) [11] and valopicitabine (4) [12] have improved pharmacokinetic profiles over their parent analogues.

Figure 1.

Valine ester prodrugs of some antiviral nucleoside analogues

In our laboratory, we have developed a new class of antiviral agents that are methylenecyclopropane analogues of nucleosides [13]. Among these analogues, cyclopropavir (5) exhibits antiviral parameters superior to ganciclovir [14 –16] and it is a subject of current preclinical studies. In order to improve therapeutic efficacy of cyclopropavir (5), we have turned our attention to the L-valine ester valcyclopropavir (6). The synthesis, antiviral activity and pharmacokinetic parameters of analogue 6 are the subject of this communication.

Methods

Synthesis

The mass spectra (MS) were determined in an electrospray ionization (ESI) mode. Valcyclopropavir (6) was synthesized as described below. Cyclopropavir (5) was synthesized by GLSynthesis, Inc. (Worcester, MA, USA) by a previously published method [14]. All other chemicals were of an analytical reagent grade and all solvents were of an HPLC grade.

(Z)-9-{[2-(N-tert-Butoxycarbonyl-L-valyloxymethyl)-2-(tetrahydropyranyloxymethyl)cyclopropylidene]-methyl}guanine (8)

A mixture of N-(tert-butoxycarbonyl)-L-valine (955 mg, 4.4 mmol) and dicyclohexylcarbodiimide (537 mg, 2.6 mmol) in dichloromethane (36 ml) was stirred at room temperature under nitrogen for 3 h. The insoluble portion was filtered off and solvent from the filtrate was evaporated in vacuo. The resultant N-(tert-butoxycarbonyl)-L-valine anhydride [(BOCVal)₂O] was dissolved in N,N-dimethylformamide (DMF; 50 ml) and compound 7 (0.7 g, 2.02 mmol) was added followed by 4-dimethylaminopyridine (DMAP; 25 mg, 0.3 mmol). The mixture was stirred at room temperature for 40 h, the solvent was removed in vacuo and the crude product was chromatographed on a silica gel column using CH₂Cl₂–MeOH (30:1 to 8:1) to give compound 8 (0.81 g, 73%) as a solid with a melting point (mp) of 228–230°C. UV spectrum λ_max (ethanol) 274 nm (ɛ 11,200), 231 nm (ɛ 27,100). ESI-MS (MeOH+NaCl) 547 (10.9, M+H), 569 (100.0, M+Na). The nuclear magnetic resonance (NMR) spectra could not be obtained because of poor solubility.

(Z)-9-{[2-Hydroxymethyl-2-(L-valyloxymethyl) cyclopropylidene]methyl}guanine hydrochloride (6)

A solution of compound 8 (0.5 g, 0.92 mmol) in 10% HCl/ethyl acetate (50 ml) was stirred at room temperature for 1.5 h. The volatile components were removed in vacuo and the crude product was chromatographed on a silica gel column using CH₂Cl₂–MeOH (10:1 to 5:1). Evaporation of solvents afforded product 6 as a white solid after trituration of the residue with methanol and ethyl acetate (280 mg, 76.4%), mp>300°C, [α]_D²⁰ 21.0°C (c 1.0, dimethyl sulfoxide [DMSO]). UV (ethanol) λ_max 273 nm (ɛ 12,600), 230 nm (ɛ 29,100). ¹H NMR (DMSO-d₆, 400 MHz) δ 10.92 (s, 1H, NH), 8.51 (bs, 3H, NH₃⁽⁺⁾ of Val), 8.22, 8.18 (2s, 1H, H₈), 7.16 (s, 1H, H_1′), 6.78 (s, 2H, NH₂), 5.36 (poorly resolved t, 1H, OH), 4.38–4.25 (m, 2H, H_5″), 3.82–3.73 (m, 2H, H_5′), 3.46–3.42 (m, 1H, CHα of Val, partly overlapped with H₂O), 2.15–2.12, 2.03–1.96/2m, 1H, CH(CH₃)₂/, 1.51–1.46 (m, 2H, H_3′), 0.94, 0.89 (2d, J=6.4, 6.8 Hz), 0.84–0.83 (poorly resolved apparent t, 6H, CH₃); ¹³C NMR 169.5, 169.4 (C=O), 157.1 (C₆), 155.0 (C₂), 150.4, 150.3 (C₄), 134.9, 134.8 (C₈), 116.9, 116.5 (C_2′), 116.4 (C_2′, C₅), 112.2 (C_1′), 67.5, 66.9 (C_5′), 63.2, 63.1 (C_5″), 58.0 (Cα of Val), 29.93, 29.88 (C_4′), 27.8, 27.7 (CH(CH₃)₂, 18.9, 18.7, 18.3, 18.2 (CH₃), 12.4, 12.1 (C_3′). ESI–MS (MeOH–KOAc) 363 (100.0, M+H), 401 (36.9, M+K). For analysis it was dried at 100°C in vacuo. Anal Calcd for C₁₆H₂₂N₆O₄ x 1.2 HCl x 1.4 H₂O: C, 44.55; H, 6.08; Cl, 9.86; N, 19.48. Found: C, 44.93; H, 5.87; Cl, 9.63; N, 19.13.

Kinetics of hydrolysis of valcyclopropavir (6) at pH 7.0 A solution of compound 6 (1 mg, 2.25 μmol) in 0.02 M Na₂HPO₄ (pH 7.0, 1 ml) was stirred at room temperature for 36 h. At selected time intervals, aliquots of the mixture were analysed by HPLC on DuPont Zorbax NH₂ column 250×4.6 mm (Wilmington, DE, USA). The mobile phase was 0.05 M KH₂PO₄–acetonitrile (3:1), flow rate at 1 ml/min and UV detection was at 273 nm (Figure 2).

Figure 2.

Kinetics of hydrolysis of prodrug (6) at pH 7.0 and room temperature

Antiviral assays

Plaque reduction assays in human foreskin fibroblasts were performed with HCMV Towne and AD169 strains as previously described [14,15].

Stability of the prodrug valcyclopropavir (6) in mouse plasma and whole blood

Male Swiss–Webster mice (Taconic Farms, Germantown, PA, USA) were anaesthetized and blood was collected (1 ml per animal) by cardiac puncture into Monoject® collection tubes (number 8881-311149; Tyco Healthcare, Mansfield, MA, USA), containing an anticoagulant (0.04 ml of 7.5% EDTA[K₃]). The blood was cooled in ice and used immediately for plasma preparation and stability studies. For plasma preparation, half of the collected blood was centrifuged for 10 min at 1,100 g in a refrigerated centrifuge (4°C). For stability at 0°C, ice cooled blood and plasma samples were spiked with a 10 mM solution of the prodrug (6) in DMSO to a final concentration of 50 μM. The samples were kept in ice water. For stability at 37°C, the plasma and blood samples were brought to 37°C by placing them in warm water, and then in a thermostatic water bath. Aliquots of 200 μl plasma and 400 μl blood were taken at the indicated times. The plasma samples were frozen immediately in a dry ice/acetone mixture. The blood samples were centrifuged in a refrigerated centrifuge (4°C) for 5 min at 2,200 g. Plasma (150 μl) was removed, frozen in a dry ice/acetone mixture and stored at −20°C until analysed. The samples were analysed for valcyclopropavir (6) and cyclopropavir (5) by the HPLC method describe below.

Bioavailability of cyclopropavir (5) after intravenous and oral dosing of the prodrug valcyclopropavir (6) to mice

Dosing and blood sampling of mice

Prodrug (6) was dissolved in physiological saline at 4.92 mg/ml. Male Swiss–Webster mice (n=3 per group per time point) received intravenous (IV) injection via the tail vein at a dose of 24.6 mg/kg (5 ml/kg, ca. 140 μl per animal), or given orally by gavage (IO) at a dose of 49.2 mg/kg (10 ml/kg, ca. 300 μl per animal). Mice given the IV dose had a mean body weight of ca. 28 g. Mice given the IO dose were fasted overnight before dosing and had a mean body weight on the day of dosing of ca. 30 g. At the indicated sampling times, (10, 20, 40, 80, 160 and 240 min for IV mice and 20, 40, 60, 120, 240 and 320 min for IO mice), animals were anaesthetized and sacrificed, and blood was collected by cardiac puncture. Blood samples were cooled on ice and immediately subjected to centrifugation at 4°C for plasma preparation. The plasma samples were frozen on dry ice and stored at −20°C until analysed.

Analytical methods

Preparation of standard solutions and spiked plasma calibration samples

Six standard solutions containing a mixture of prodrug (6) and cyclopropavir (5) at concentrations of 0.6, 2.0, 6.0, 20.0 and 60.0 μg/ml were prepared by diluting DMSO stock solutions (10 mg/ml) with acetonitrile. For spiked plasma calibration samples, aliquots of 250 μl of the standard solutions were mixed with equal volume of mouse plasma in polypropylene microfuge tubes. For extraction efficiency determination, another set of standard solutions (250 μl) were mixed with an equal volume of water. All tubes were vortexed for 30 s, maintained at room temperature for 30 min, vortexed again for 30 s and then centrifuged for 15 min at 13,000 rpm. The supernatants (375 μl each) were pipetted from each tube into a new microfuge tube and evaporated to dryness in a Speed Vac (4–5 h; Savant, Nassau-Suffolk, NY, USA). DMSO (250 μl) was added to each residue and the samples were vortexed for 60 min. The solutions were centrifuged for 15 min at 13,000 rpm, and transferred to HPLC vials. Each sample was analysed by HPLC by the method described below.

Preparation and extraction of plasma samples from dosed mice

The plasma samples were thawed on ice, and aliquots (250 μl) were immediately transferred to microfuge tubes and deproteinated by addition of acetonitrile (250 μl). The samples were further processed as described above for the spiked plasma standards.

HPLC method

The HPLC analysis was performed on a Varian ProStar® HPLC System (Varian Inc., Palo Alto, CA, USA) consisting of two pumps, a variable wavelength detector, an autosampler and a desktop computer for HPLC control, data acquisition and data analysis. The data analysis software was the Varian® Star Chromatography Workstation (version 6.0). Compound elution was performed on a 4.6×150 mm Ace 5 C18 HPLC column (Advanced Chromatography Technologies, Aberdeen, Scotland, UK). The mobile phase consisted of reservoir A, containing 100% water with 0.1% trifluoroacetic acid, and reservoir B, containing 100% methanol. Each solvent was filtered through a 0.45 μm nylon filter and degassed before use. Elution from the column was at a flow rate of 1 ml/min using the following gradient programme: a linear gradient of 0% B to 30% B from 0 to 18 min, followed by a linear gradient from 30% to 100% B for 1 min by a hold at 100% B for 2 min, followed by a linear gradient from 100% to 0% B for 1 min, and then equilibration by a hold at 0% B for 5 min. Compound elution was monitored by UV absorbance at 274 nm. Total analysis time was 27 min per sample. Retention times were 9.8 min for cyclopropavir (5) and 14.6 min for the prodrug (6).

Data analyses

Peak areas from HPLC analysis of plasma samples were measured and concentrations of cyclopropavir (5) and prodrug (6) were calculated based on the standard curves generated with the plasma spiked samples. The extraction efficiency for each compound was determined by comparing the peak areas for spiked plasma samples with those for standard samples processed with water instead of plasma. The average extraction efficiency was 67% for cyclopropavir (5) and 88% for the prodrug (6). The limit of detection and the limit of quantification, determined as 3x and 5x the intensity of the matrix peaks in the regions of detection of the analytes, were 0.24 μg/ml and 0.40 μg/ml for cyclopropavir (5) and 0.048 μg/ml and 0.080 μg/ml for the prodrug (6), respectively. The pharmacokinetic modelling and analysis was done using the pharmacokinetic modelling program WinNonLin® (Pharsight Corp., Mountain View, CA, USA).

Results

Antiviral activity

Prodrug (6) inhibited replication of HCMV in two strains of virus, Towne and AD169 (Table 1). These studies confirmed that its potency was comparable to ganciclovir, which was used as a control, and was also similar to cyclopropavir (5) as reported previously [14]. These results confirmed that valcyclopropavir (6) was readily hydrolysed to cyclopropavir (5) inside the virus-infected cells or in the culture medium that contained bovine plasma. However, its efficacy relative to cyclopropavir (5) cannot be discerned from these data.

Table 1.

Anti-HCMV activity of valcyclopropavir (6) in human foreskin fibroblasts^a

	HCMV Towne^b		HCMV AD169^c
Compound	Mean EC₅₀, μM (±SD)	CC₅₀, μM	Mean EC₅₀, μM (±SD)	CC₅₀, μM

Valcyclopropavir (6)^d	0.21	>100	2.9	>300
Ganciclovir	1.58 (1.53)	>100	3.6 (1.4)	>100
Cyclopropavir (5)^e	0.42 (0.23)	>100	1.2 (0.8)	>380

Efficacy and cytotoxicity in human foreskin fibroblasts (HFFs) as determined by plaque reduction assay.

Visual cytotoxicity in uninfected HFFs used in plaque reduction assays.

Cytotoxicity by neutral red assay.

Results from single assays.

The 50% effective concentration (EC₅₀) values for cyclopropavir (5) were reported previously for both strains of the virus [14]. CC₅₀, 50% cytotoxicity concentration; HCMV, human cytomegalovirus.

Conversion of valcyclopropavir (6) to cyclopropavir (5) in vitro in mouse plasma and whole blood

The prodrug valcyclopropavir (6) was converted into the parent drug, cyclopropavir (5) when incubated at 37°C in mouse blood or plasma with a half-life of 1 h 20 min and 40.4 min, respectively (Figure 3 and Table 2). At 0°C the conversion was more than an order of magnitude slower, with estimated half-lives in blood and plasma of 22 h and 12 h, respectively. In a control experiment, blood and plasma samples were processed immediately after spiking with the prodrug (no incubation). The level of the cyclopropavir peak detected in those samples corresponded to a 3.7% and 9.0% conversion of valcyclopropavir (6) to cyclopropavir (5) in plasma and blood, respectively. This is assumed to take place during the plasma preparation and sample processing, and should not affect the overall kinetic parameters observed for the in vitro or in vivo metabolism of the prodrug (6), as it is the same for all incubation/sampling times. In our opinion, this small conversion does not warrant correction of the dose-dependent pharmacokinetic parameters for the prodrug (6).

Table 2.

Conversion of valcyclopropavir (6) to cyclopropavir (5) in mouse whole blood and plasma at 0°C and 37°C

Incubation condition	First order rate constant, min⁻¹	Half-life, min	R²

Plasma 0°C	0.0010	727	0.9925
Plasma 37°C	0.0172	40.4	0.9998
Blood 0°C	0.0005	1310	0.9645
Blood 37°C	0.0087	80	0.9997

Figure 3.

Conversion of prodrug (6) into cyclopropavir (5) after incubation at 0°C and 37°C in mouse whole blood and plasma

Intravenous dosing

The average plasma concentration of the prodrug (6) and cyclopropavir (5) are plotted in Figure 4.

Figure 4.

Plasma concentrations of cyclopropavir (5), and prodrug (6) versus time after single intravenous bolus injection of 24.6 mg/kg prodrug (6) in mice

Prodrug (6)

The plasma concentration data for prodrug (6) were fitted into a single compartment bolus IV first order elimination model (Figure 5A).

Figure 5.

Single and two compartment bolus intravenous first order elimination models showing plasma concentration data for prodrug (6)

The best fit curve is presented in Figure 6A and the best fit pharmacokinetic parameters are presented in Table 3. Analysis of the plasma concentration profile suggested that the prodrug (6) is converted by a fast process to cyclopropavir (5). At the same time, some amount of the prodrug (6) remains in a depot compartment, and is released by a slow process. This results in a large half-life and volume of distribution parameter in the single compartment model (Table 3). Therefore, the data for the prodrug (6), was fitted to a two compartment bolus IV first order elimination model (Figure 5B). The best fit graph and corresponding parameters are presented in Figure 6B and Table 4. The parameters are more consistent with the properties expected of the prodrug (6). The lack of very early time points resulted in high coefficients of variability, especially of the parameters highly influenced by K₁₂, that is, the rate constant for transfer from compartment 1 to compartment 2.

Table 3.

Single compartment first order elimination pharmacokinetic parameters for prodrug (6) after intravenous dosing in mice

Parameter	Best fit	CV, %

V_D, l/kg	93.5	11.9
AUC, mg•min/l	61.2	7.33
K, min⁻¹	0.0043	14.8
T_1/2, min	161	23.9
C_max, mg/l	0.263	11.9
Cl, l/min	0.402	7.33

AUC, area under the curve; Cl, total plasma clearance; C_max, intercept (time zero concentration); CV, coefficient of variation; K, elimination rate constant; T_1/2, half-life; V_D, apparent volume of distribution.

Figure 6.

Pharmacokinetic modelling of the experimental data for prodrug (6) after intravenous bolus injection in mice

Cyclopropavir (5)

The plasma concentration of cyclopropavir (5) was first modelled using a single compartment first order elimination model, in which the last three data points were used to estimate the terminal elimination rate constant, β (Figure 7A). The pharmacokinetic parameters based on this analysis are presented in Table 4.

Table 4.

Two compartment first order distribution and elimination pharmacokinetic parameters for prodrug (6) after bolus intravenous dosing in mice

Parameter	Best fit	CV, %

V, l/kg	0.0753	14.9
AUC, mg•min/l	97.1	85.6
α, min⁻¹	0.25	536
T_1/2(α), min	2.73	535
β, min⁻¹	0.00263	114
T_1/2(β), min	301	114
C₀ (A), mg/l	36.5	1234
C₀ (B), mg/l	1.037	12.3
Cl, l/min	0.217	222

AUC, area under the curve; Cl, total plasma clearance; CV, coefficient of variation; C₀ (A), intercept (time zero concentration) of distribution phase; C₀ (B), intercept (time zero concentration) of elimination phase; T_1/2(α), half-life of distribution phase; T_1/2(β), half-life of elimination phase; V, volume of main compartment; α, rate constant of distribution phase; β, rate constant of elimination phase.

Figure 7.

Pharmacokinetic modelling of the experimental data for cyclopropavir (5) after intravenous bolus injection of prodrug (6) in mice

The time dependence of the cyclopropavir (5) plasma concentration was also assessed by using a two compartment model described in Figure 8.

Figure 8.

Two compartment model showing time dependence of the cyclopropavir (5) plasma concentration

The best fit graph and corresponding parameters are presented in Figure 7B and Table 5. The pharmacokinetic parameters obtained by this analysis are in good overall agreement with the corresponding pharmacokinetic parameters, obtained by the single compartment method (Table 6). However, because the elimination of the prodrug (6) does not follow first order kinetics, there is significant uncertainty in the estimation of the input rate constant K₀₁.

Table 5.

Two compartment analysis of cyclopropavir (5) plasma levels after intravenous bolus dosing of prodrug (6)

Parameter	Best fit	CV, %

V, l/kg	2.61	21.0
AUC, mg•min/l	874	22.3
K₀₁, min⁻¹	0.21	121
K₁₀, min⁻¹	0.0071	32.7
T_1/2K10, min	97	32.7
T_max, min	16.7	85.8
C_max, mg/l	5.52	15.0
Cl, l/min	0.0185	22.3

A complete conversion of the prodrug (6) to cyclopropavir (5) is assumed, which would result in a equivalent dose of cyclopropavir (5) of 16.2 mg/kg. AUC, area under the curve; Cl, total plasma clearance; C_max, maximum plasma concentration; CV, coefficient of variation; K₀₁, input rate constant; K₁₀, elimination rate constant; T_max, time of maximum plasma concentration; T_1/2K10, elimination half-life of K₁₀; V, volume of distribution.

Table 6.

Single compartment first order elimination pharmacokinetic parameters for cyclopropavir (5) after intravenous dosing of prodrug (6) in mice

Parameter	Best fit
β, min⁻¹	0.0092
T_1/2(β), min	75.5
T_max, min	10
C_max, mg/l	5.59
V_z, l	1.94
AUC_∞, mg•min/l	902
Cl, l/min.	0.018

The last three data points were used to model the elimination rate constant. In this analysis a complete conversion of the prodrug (6) to cyclopropavir (5) was assumed, which would result in an equivalent dose of cyclopropavir (5) of 16.2 mg/kg. AUC_∞, area under the curve at infinity; Cl, total plasma clearance; C_max, maximum plasma concentration; T_max, time of maximum plasma concentration; T_1/2(β), half-life based on terminal elimination phase; V_z, volume of distribution based on the terminal phase; β, rate constant of elimination phase.

Oral dosing

The results of the plasma sample analysis after a single oral dose of 49.2 mg of prodrug (6) in mice is presented in Figure 9. No prodrug (6) was detected in the oral samples (limit of detection 0.048 μg/ml). The absorption phase took <10 min and was not observed. The log₁₀ of the observed experimental cyclopropavir (5) concentrations demonstrated an overall linear decrease (Figure 9B, red line). The data was fitted into a first order elimination non-compartmental model. The model-predicted and experimental cyclopropavir concentrations are shown in Table 7, and are also shown plotted in semilog form in Figure 9B. The pharmacokinetic parameters derived from the above model are presented in Table 8. The prodrug (6) oral bioavailability, calculated according to the formula oral bioavailability %=(IV_AUC/oral_AUC)(IV dose/oral dose)x100, was found to be very high at 95.3%.

Table 7.

Experimental and model-predicted concentrations of cyclopropavir (5) and AUC for oral dosing of prodrug (6)

Time, min	Experimental concentration, mg/l	Predicted concentration mg/l	AUC, mg•min/l

20	15.71	–	157.1
40^a	14.39	14.75	458.1
60^a	10.17	11.94	703.7
120^a	5.10	6.330	1161.8
240^a	2.82	1.780	1637.0
320^a	0.71	0.764	1778.2

Time point included in modelling of the elimination phase. AUC, area under the curve.

Table 8.

Pharmacokinetic parameters of cyclopropavir (5) after oral dosing of prodrug (6)

Parameter	Best fit
λ_z, l/min	0.0106
T_1/2λz, min	65.6
T_max, min	≤20
C_max, mg/l	≥15.7
V_z, l	2.52
AUC_∞, mg•min/l	1,845
Cl, l/min	0.0267
Oral bioavailability, %	95.3

In this analysis, a complete conversion of the bioavailable prodrug (6) to cyclopropavir (5) was assumed, which would result in a dose equivalent to 32.4 mg/kg of cyclopropavir (5). AUC_∞, area under the curve at infinity; Cl, total plasma clearance; C_max, maximum plasma concentration; T_1/2λz, half-life of oral elimination phase; T_max, time of maximum plasma concentration; V_z, volume of distribution based on the terminal phase; λ_z, rate constant of oral elimination phase.

Figure 9.

Plot of plasma concentrations of cyclopropavir (5) versus time in mice after a single oral dose of 49.2 mg/kg of prodrug (6)

Discussion

Chemistry

The previously described [17] tetrahydropyranyl (THP) cyclopropavir (7), which is available from cyclopropavir in two steps (73% yield) served as a convenient starting material for synthesis of valcyclopropavir (6; Figure 1B). The method described [2] for synthesis of valacyclovir (1) was followed. Reaction of 7 with N-(t-butoxycarbonyl)-L-valine anhydride (BOCVal)₂O in DMF under catalysis with DMAP gave intermediate 8 in 73% yield. The BOC and THP group were removed in acid medium (10% HCl in ethyl acetate) to give valcyclopropavir (6) as a hydrochloride in 76% yield. Similar to valganciclovir (2) [3], prodrug (6) is a mixture of two stereoisomers. The stereoisomeric ratio of 2 determined from the ¹H NMR spectrum is 2:1.

As expected, prodrug (6) had a limited stability at pH 7.0 (Figure 2) with the half-life of 22.5 h at room temperature. For comparison, the half-life of valganciclovir (2) [18] at pH 7.08 and 37°C is 11 h.

Pharmacokinetic studies

Prodrug (6) is converted to cyclopropavir (5) in vitro in mouse whole blood and blood plasma, and in vivo after both IV and oral administration. Prodrug (6) is quickly and efficiently absorbed by the gastrointestinal tract, with oral bioavailability of 95%. The high apparent volume of distribution of 5 after both IV and oral dosing with prodrug (6) is suggestive of possible sequestering of either 6 or 5 in non-sampling compartments. The pharmacokinetic profile of prodrug (6) after IV dosing also supports the possibility of non-uniform distribution of 6 with sequestering outside of the plasma compartment.

Footnotes

Acknowledgements

The work described herein was supported by SBIR grant 5 R44 AI054135, contract NO1-AI30049 and research grant RO1-CA32779 from the National Institutes of Health, Bethesda, MD, USA. We thank LM Hrihorczuk (Central Instrumentation Facility, Department of Chemistry, Wayne State University, Detroit, MI, USA) for mass spectra, Kathy Borysko (University of Michigan, Ann Arbor, MI, USA) for plaque assays, and Sofya Dvoskin and George Wright (GLSynthesis, Worcester, MA, USA) for valuable assistance in bioavailability studies.

The authors declare no competing interests.

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L-Valine Ester of Cyclopropavir: A New Antiviral Prodrug

Abstract

Background:

Methods:

Results:

Conclusions:

Introduction

Methods

Synthesis

(Z)-9-{[2-(N-tert-Butoxycarbonyl-L-valyloxymethyl)-2-(tetrahydropyranyloxymethyl)cyclopropylidene]-methyl}guanine (8)

(Z)-9-{[2-Hydroxymethyl-2-(L-valyloxymethyl) cyclopropylidene]methyl}guanine hydrochloride (6)

Antiviral assays

Stability of the prodrug valcyclopropavir (6) in mouse plasma and whole blood

Bioavailability of cyclopropavir (5) after intravenous and oral dosing of the prodrug valcyclopropavir (6) to mice

Dosing and blood sampling of mice

Analytical methods

Preparation of standard solutions and spiked plasma calibration samples

Preparation and extraction of plasma samples from dosed mice

HPLC method

Data analyses

Results

Antiviral activity

Conversion of valcyclopropavir (6) to cyclopropavir (5) in vitro in mouse plasma and whole blood

Intravenous dosing

Prodrug (6)

Cyclopropavir (5)

Oral dosing

Discussion

Chemistry

Pharmacokinetic studies

Footnotes

Acknowledgements

References