Synthesis and Anti-HIV Properties of New Carbamate Prodrugs of AZT

Authors


Corresponding author: Marina K. Kukhanova, kukhan86@hotmail.com

Abstract

A series of new 5′-O-carbamate prodrugs of AZT have been prepared. The stability in biological media, anti-HIV properties and pharmacokinetic parameters in dogs were evaluated. The compounds display moderate anti-HIV activity in cell culture. After oral administration of carbamate IV in dogs, both intact prodrug IV and released AZT were discovered in dog blood. Pharmacokinetic parameters of the compound IV were estimated. Half-life (T1/2) of AZT released after oral administration of IV in dogs was close to that after administration of AZT itself, and time to the maximum concentration (Tmax) of AZT released from IV was two and three times longer compared with that of AZT and H-phosphonate AZT, respectively. Acute toxicity was more than five times less if compared with AZT. As a result, we consider this series of carbamate derivatives of AZT as perspective for development of anti-HIV agents.

Anti-HIV drug development is one of the leading trends in medicinal chemistry due to the increasing rate of patients with HIV-related infections. Nucleoside analogs targeting reverse transcriptase (RT), a key enzyme in HIV replicative cycle, remain important components in drug development. Among eight nucleoside RT inhibitors (NRTI) used in treatment of patients with HIV, the most extensively studied is 3′-azido-3′-deoxythymidine (AZT, zidovudine, retrovir®). Major clinical AZT usage restrictions are caused by toxicities that include bone marrow suppression (1), hepatic abnormalities and myopathy (2), limited brain uptake (3), a short half-life in body (4,5) and rapid development of HIV resistance (6,7). Numerous chemical strategies have been developed to overcome these problems, particularly by designing of AZT prodrugs (8,9). Most of AZT prodrugs are prepared by derivatization of 5′-O-position and some of them became drugs. The mechanism of action of depot forms is based on chemical hydrolysis and/or enzymatic cleavage of their 5′-O-bonds to give AZT or its 5′-O-monophosphate after penetration in cells. The prodrugs are expected to enhance anti-HIV activity, raise AZT intracellular uptake, intensify AZT brain delivery, decrease toxicity, or improve pharmacokinetics. For example, the depot form of AZT, H-phosphonate AZT (HpAZT, Nikavir®, phosphazide) displays some advantages over AZT and has been approved in Russia for treatment and prevention of AIDS (10–13).

Herein, we describe biological properties of new carbamate derivatives of AZT including their anti-HIV activity in cell culture, stability, intracellular transformation, pharmacokinetic, and toxicological data.

Methods and Materials

All reagents and solvents were purchased from Acros (Belgium). AZT was a gift from the ‘AZT Association’ (Moscow, Russia).

NMR spectra (δ, ppm; J, Hz) were registered on an AMX III-400 spectrometer (Bruker BioSpin Gmbh, Karlsruhe, Germany) with the working frequency of 400 MHz for 1H NMR (Me4Si as an internal standard for organic solvents) and 100.6 MHz for 13C NMR (with carbon-proton interaction decoupling) at 27 °C.

UV spectra were registered on a Shimadzu UV-2401PC spectrophotometer (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) in water in the range of 200–300 nm and agreed to those for thymidine derivatives (data not given).

Mass spectra (m/e) were registered on a Thermo Finnigan LCQ Advantage spectrometer (Thermo Fisher Scientific Corporation, Waltham, MA, USA) under the following conditions: ESI from the solution of a sample compound in acetonitrile, flow rate of 50 μL/second, voltage of 4.5 kV, the ion transfer capillary temperature of 250 °C.

Centrifugation was performed on an Eppendorf 5415 centrifuge (Eppendorf AG, Hamburg, Germany). An automatic Speed-Vac Concentrator AS260 (Savant, Osterville, MA, USA) was used for concentrating the sample.

HPLC analysis was performed using a Gilson chromatograph (Gilson Inc., Middleton, WI, USA) supplied with a digital GSIOC 506 controller and a Gilson-315 UV detector with varying wavelengths. Compounds were detected at λ 267 nm using a Nucleosil 100 C-18 column (5 μm, 4 × 150 mm) with a C-18 precolumn (10 μm, 4 × 8 mm); using mobile phase of 80% aqueous EtOH (A) in 5 mm aqueous phosphate buffer (pH 5.20). Gradient parameters: 0% A for 5 min; 0%→20% A for 13 min; 20%→50% A for 12 min; 50%→100% A for 15 min; the flow rate of 0.5 mL/min.

Synthesis

Standard procedure for synthesis of carbamates (I–V)

A solution of AZT (0.80 g, 3 mmol) and 1,1′-carbonyldiimidazole (CDI) (0.81 g, 5 mmol) in dimethylformamide (10 mL) stirred at 18 °C for 3 h, then amine (10–20 mmol) was added, and the solution was stirred for another 18 h. The reaction mixture was concentrated by evaporation under vacuum and chromatographed on a silica gel column (20 × 150 mm), eluted with a linear gradient of methanol (3%→10%) in chloroform. The fraction containing the target product was concentrated by evaporation under vacuum and freeze-dried.

5′-O-(Aminocarbonyl)-3′-azido-3′-deoxythymidine (I)

Prepared according to standard procedure, from AZT, CDI, and 32% aqueous ammonia (7 mL). Purification and freeze-drying gave 0.76 g (78%) of carbamate (I) as a white lyophilizate. 1H NMR (DMSO-d6): 11.3 s (1H, 3-NH), 7.43 s (1H, H-6), 6.62 br.s (2H, NH2), 6.12 t (1H, 3J1′, 2′ 6.5, H-1′), 4.40 m (1H, H-3′), 4.21 dd (1H, 2J 5′a, 5′b 11.8, 3J 5′a, 4′ 3.4, H-5′a), 4.08 dd (1H, 2J 5′b, 5′a 11.8, 3J 5′b, 4′ 5.2, H-5′b), 3.98 m (1H, H-4′), 2.47 m (1H, H-2′a), 2.30 m (1H, H-2′b), 1.79 s (3H, 5-CH3). 13C NMR (DMSO-d6): 163.8 s (C-4), 156.4 s (NC(O)O), 150.5 s (C-2), 135.9 s (C-6), 110.0 s (C-5), 83.8 s (C-1′), 81.4 s (C-4′), 63.5 s (C-3′), 60.9 s (C-5′), 35.8 s (C-2′), 12.2 s (5-CH3). MS (ESI): 311.3 (M + 1), 333.4 (M + Na).

5′-O-(Methylaminocarbonyl)-3′-azido-3′-deoxythymidine (II)

Prepared according to standard procedure, from AZT, CDI, and 40% aqueous methylamine (5 mL). Purification and freeze-drying gave 0.66 g (65%) of carbamate (II) as a white lyophilisate. 1H NMR (DMSO-d6): 11.34 s (1H, 3-NH), 7.43 q (1H, 4JH-6, 5­CH3 0.9, H-6), 7.20 q (2H, 3NH, CH3, 4.4, CH3NH), 6.12 t (1H, 3J1′, 2′ 6.8, H-1′), 4.42 m (1H, H-3′), 4.24 dd (1H, 2J 5′a, 5′b 11.8, 3J 5′a, 4′ 3.4, H-5′a), 4.10 dd (1H, 2J 5′b, 5′a 11.8, 3J 5′b, 4′ 5.2, H-5′b), 3.98 m (1H, H-4′), 2.59 d (2H, 3JCH3, NH 4.4, CH3NH), 2.45 m (1H, H-2′a), 2.29 m (1H, H­2′b), 1.78 d (3H, 4J 5­CH3, H-6 0.9, 5-CH3). 13C NMR (DMSO-d6): 163.7 s (C-4), 156.3 s (NC(O)O), 150.4 s (C-2), 135.9 s (C-6), 109.9 s (C-5), 83.6 s (C-1′), 81.2 s (C-4′), 63.8 s (C-3′), 60.8 s (C-5′), 35.8 s (C-2′), 27.0 s (CH3NH), 12.1 s (5-CH3). MS (ESI): 325.1 (M + 1), 347.3 (M + Na).

5′-O-(Ethylaminocarbonyl)-3′-azido-3′-deoxythymidine (III)

Prepared according to standard procedure, from AZT, CDI, and 70% aqueous ethylamine (5 mL). Purification and freeze-drying gave 0.76 g (72%) of carbamate (III) as a white lyophilisate. 1H NMR (DMSO-d6): 11.29 s (1H, 3-NH), 7.43 s (1H, H-6), 7.24 t (2H, 3J NH, CH2 5.3, EtNH), 6.12 dd (1H, 3J 1, 2′a 6.9, 3J 1′, 2′b 6.5, H-1′), 4.41 m (1H, H-3′), 4.23 dd (1H, 2J 5′a, 5′b 11.8, 3J 5′a, 4′ 3.7, H-5′a), 4.10 dd (1H, 2J 5′b, 5′a 11.8, 3J 5′b, 4′ 5.0, H-5′b), 3.99 m (1H, H-4′), 3.03 m (2H, CH2CH3), 2.48 m (1H, H-2′a), 2.30 m (1H, H-2′b), 1.79 s (3H, 5-CH3), 1.02 t (3H, 3J CH3, CH2 7.2, CH2CH3). 13C NMR (DMSO-d6): 163.6 s (C-4), 155.6 s (NC(O)O), 150.4 s (C-2), 135.8 s (C-6), 109.8 s (C-5), 83.7 s (C-1′), 81.3 s (C-4′), 63.7 s (C-3′), 60.8 s (C-5′), 35.6 s (C-2′), 35.1 s (CH2CH3), 14.9 s (CH2CH3), 12.1 (s, 5-CH3). MS (ESI): 339.4 (M + 1), 361.6 (M + Na), 362.6 (M + Na + 1).

5′-O-(N-Morpholinocarbonyl)-3′-azido-3′-deoxythymidine (IV)

Prepared according to standard procedure, from AZT, CDI, and morpholine (1.5 mL, 17 mmol). Purification and freeze-drying gave 0.91 g (82%) of carbamate (IV) as a white lyophilisate. 1H NMR (DMSO-d6): 11.23 s (1H, 3-NH), 7.40 s (1H, H-6), 6.10 dd (1H, 3J1′, 2′a 6.2, 3J1′, 2′b 6.9, H-1′), 4.45 m (1H, H-3′), 4.27 m (2H, H-5′), 3.98 m (1H, H-4′), 3.55 t (4H, 4JCH2O, CH2N 4.7, 2 × CH2O (morpholine)), 3.36 br.s (4H, 2 × CH2-N (morpholine)), 2.37 m (2H, H-2′), 1.79 s (3H, 5-CH3). 13C NMR (DMSO-d6): 163.6 s (C-4), 154.2 s (NC(O)O), 150.3 s (C-2), 135.9 s (C-6), 109.9 s (C-5), 83.9 s (C-1′), 80.9 s (C-4′), 65.7 s (2 × CH2O (morpholine)), 64.2 s (C-5′), 60.1 s (C-3′), 43.8 s (2 × CH2N (morpholine)), 35.7 s (C-2′), 11.9 s (5-CH3). MS (ESI): 403.5 (M + Na), 425.5 (M + 2 × Na), 805.2 (2 × M + 2 × Na – 1), 806.3 (2 × M + 2 × Na).

5′-O-(N-Piperazinocarbonyl)-3′-azido-3′-deoxythymidine (V)

A solution of 3′-azido-3′-deoxythymidine (1 g, 3.7 mmol) and CDI (1 g, 6 mmol) in dioxane (10 mL) was stirred at 18 °C for 1 h, then piperazine (0.95 g, 11 mmol) in dioxane (5 mL) was added, and the solution was stirred for another 18 h. The reaction mixture was concentrated by evaporation under vacuum and chromatographed on a reversed-phase silica gel column (RP-18, 25 × 250 mm) eluted with a linear gradient of acetonitrile (0→40%) in water. The fraction containing the target product was concentrated by evaporation under vacuum and freeze-dried from water resulting in 0.84 g (60%) of carbamate (V) as a white lyophilisate. 1H NMR (DMSO-d6): 7.42 q (1H, 4J H-6, 5-CH3 1.1, H-6), 6.10 dd (1H, 3J1′, 2′a 6.2, H-1′), 4.47 m (1H, H-3′), 4.27 dd (1H, 2J 5′a, 5′b 11.9, 3J 4′, 5′a 4.1, H-5′a), 4.20 dd (1H, 2J 5′a, 5′b 11.9, 3J 4′, 5′b 5.2, H-5′b), 3.97 m (1H, H-4′), 3.30 m (4H, 2 × CH2NCO (piperazine)), 2.90 br.s (4H, 2 × CH2NH (piperazine)), 2.39 m (2H, H-2′), 1.79 d (3H, 4J 5-CH3, H-6 1.1, 5-CH3). 13C NMR (DMSO-d6): 163.6 s (C-4), 154.1 s (NC(O)O), 150.3 s (C-2), 135.8 s (C-6), 109.8 s (C-5), 83.6 s (C-1′), 80.8 s (C-4′), 64.0 s (C-5′), 60.0 s (C-3′), 45.1 s (2 × CH2NH (piperazine)), 44.5 s (2 × CH2NCO (piperazine)), 35.6 s (C-2′), 12.0 s (5-CH3). MS (ESI): 380.5 (M + 1), 381.5 (M + 2), 402.5 (M + Na), 424.5 (M + 2 × Na – 1).

Anti-HIV activity

MT-4 cells were infected with HIV-1899A strain at the multiplicity of infection 0.2–0.5 units/cell. The antiviral activity was determined in the presence of the tested compounds (0.01–1000 μm, three replicates for each dose) and was assessed by the measuring of p24 antigen amount in 4 days. The cell concentration and viability were estimated using the calorimetric assay (14).

Cytotoxicity

The cells were cultured in the presence of various doses of the compounds (0.01–6000 μm) for 4 days. The cell concentration and viability were measured by the trypan blue-dye exclusion calorimetric assay, and the CD50 was calculated.

Stability in human blood serum

Water solutions of the tested compounds (500 μm) were incubated with human blood serum at 37 °C. After certain time intervals aliquots were taken, and the reaction was terminated by the addition of cool methanol up to 66% (v/v). The formed precipitate was pelleted by centrifugation for 10 min at 10 000 × g. The supernatant was dried in a Speed-Vac concentrator, the residue was dissolved in 5 mm aqueous phosphate buffer (pH 5.20), and the products were analyzed by HPLC under conditions described previously (see ‘Methods and Materials’ section). Retention times were as follows: AZT – 23.70 min; I– 24.75 min; II– 27.05 min; III– 29.30 min; IV – 30.00 min; V– 25.05 min.

Pharmacokinetic parameters in dogs

The studies were performed following standard protocols (15) with outbred dogs (males and females, 13.6 ± 2.6 kg body weight). The compound IV was administered orally in the fasting state; the animals were fed 3 h after dosing (580 mg/body weight). Ten blood samples (not <3 mL) were taken out from the femoral vein 0–24 h after administration and placed into tubes containing heparin (5 μL, 5000 U/mL) and centrifuged for 10 min at 1500 × g. The plasma samples were kept at −24 °C. The samples were treated as described in section ‘Stability in human blood serum’ and analyzed by HPLC using a Gynkotek chromatograph (Optimize Technologies Inc, Oregon City, OR, USA) on a column Ultrasphere ODC (Beckman Coulter, Inc., Brea, CA, USA), mobile phase: 6% acetonitrile in 0.1% H3PO4 in the presence of 0.15% triethylamine, detection at 265 nm, temperature 30 °C.

Pharmacokinetic parameters were calculated using the Thermo Kinetica 4.4.1 program (Thermo Electron Corporation, Marietta, OH, USA). Pharmacokinetic parameters following oral administration were studied by the extravascular non-compartmental model of the Thermo Kinetica program. The following parameters were determined: the total area under the plasma concentration-time curve (AUCtot), the apparent elimination half-life (T1/2), the maximum plasma concentration of the compound (Cmax), the time to Cmax (Tmax).

Acute toxicity was estimated according to the standard procedure (16) using BALB/c mice (males and females, 19 ± 1 g body weight). The mice were received from the laboratory animal hatchery of the Scientific Center of Biomedical Technologies of Russian Academy of Medical Sciences. The tested compounds dissolved in a sterile isotonic solution of sodium chloride were administered intraperitoneally in various doses. The control animals received the corresponding volume of the isotonic solution of sodium chloride. The animals were examined for 14 days.

Results

Structures of the new synthesized carbamate depot forms of AZT are presented in Figure 1.

Figure 1.

 Structures of the synthesized compounds.

The compounds were prepared according to scheme on Figure 2. AZT carbamates were obtained using a facile procedure of nucleoside treatment with CDI and the corresponding amine. The advantage of CDI condensation is providing smoother and safe-to-perform conditions instead of highly irritant phosgene or triphosgene treatment for preparing nucleoside carbamates (17,18).

Figure 2.

 General scheme of synthesis of AZT carbamates.

Antiviral properties of the (IV) were studied in MT-4 cell culture infected with HIV-1. The results given in Table 1 indicate that the compounds inhibited virus replication but the activity was lower than AZT and HpAZT taken as control samples. The carbamate I was the most active among synthesized compounds with SI close to that of AZT and HpAZT.

Table 1.   Anti-HIV activity AZT derivatives in MT-4 cell culture
CompoundCC50a, μmEC50b, μmSIc
  1. aCompound concentration required to reduce cell viability by 50%.

  2. bCompound concentration required to inhibit HIV replication by 50%.

  3. cSelectivity index SI = CC50/EC50.

I >60003.2>1875
II >600050>120
III >600045>133
IV >600035>171
V >600020>300
H-phosphonate AZT6480.292240
AZT800.0372300

Pharmacokinetic parameters of compound IV

Some pharmacokinetic parameters were evaluated in dogs after oral administration of 580 mg of compound IV. The analysis of dog blood by HPLC showed the presence of intact compound IV as well as the product of its hydrolysis AZT (Figure 3A).

Figure 3.

 (A) Time-concentration dependences of carbamate IV and its hydrolysis product AZT at a single oral dose IV (580 mg/body weight dose) in dog (n = 3); (B) Time-concentration dependences of AZT (680 mg/body weight dose) and AZT released from H-phosphonate AZT (at doses equivalent to AZT) (n = 6). The data in (B) were taken for comparison from our article published earlier (19).

It should be noted that only AZT was found in dog blood after oral administration of H-phosphonate AZT in dogs (Figure 3). Thus, the half-life of IV in dog blood is longer than that of H-phosphonate AZT. We estimated pharmacokinetic parameters of compound IV and compared them with that of AZT and H-phosphonate AZT (Table 2).

Table 2.   Pharmacokinetic parameters of IV, AZT released from IV (580 mg/body weight), AZT and H-phosphonate AZT (680 mg/body weight each) after oral administration in dogs
Compound C max, mg/L T max, h T 1/2, hAUC, mg/h × L
  1. C max– maximum plasma concentration; Tmax– time to maximum plasma concentration; T1/2– elimination half-life; AUC is the area under the plasma concentration-time curve.

IV 20.12 ± 2.803.03.31 ± 0.04114.60 ± 18.84
AZT released from IV1.07 ± 0.168.04.41 ± 0.567.76 ± 1.55
AZT released from H-phosphonate AZT1.94.07.216.6
AZT9.82.55.258.8

As one can see from the Table 2, half-life (T1/2) of AZT released after oral administration of IV in dogs was close to that after administration of AZT itself, and time to the maximum concentration (Tmax) of AZT released from IV was two and three times longer compared with that of AZT and H-phosphonate AZT, respectively. Acute toxicity was more than five times less if compared with AZT.

Acute toxicity

Acute toxicity was estimated in BALB/c mice after a single intraperitoneal dose of compound IV in the range of 3.5–7.1 g/kg. The results are shown in Table 3. As one can see the acute toxicity of IV was much less if compared with AZT and HpAZT.

Table 3.   Single dose toxicity in BALB/c mice (n = 10) of compound IV
CompoundLD16a, g/kgLD50b, g/kgLD84c, g/kg
  1. Data are presented as mean ± SE (p < 0.01).

  2. a–cLethal doses that cause death of 16% (LD16), 50% (LD50), 84% (LD84) mice, respectively.

IV >7.10  
AZT1.301.50 ± 0.071.70
H-phosphonate AZT2.002.30 ± 0.102.50

Discussion

A number of depot forms of AZT and 3TC based on carbamate, and carbonate derivatives were described earlier (17,18,20,21). Chang et al. synthesized amino acid carbamate derivatives of AZT and showed that anti-HIV activity was several orders of magnitude lower than that of parent AZT. These results can be explained by the high stability of the compounds and inability to release active drug AZT (17). 5′-O-3TC carbamates with alkyl- or ω-substituted fragments also displayed low anti-HIV activity (15–254 μm), and SI was not higher than 10 (18). Similar to 5′-O-AZT carbamates, 5′-O-3TC carbamates showed high stability in cell medium and did not generate 3TC. On the contrary, 3TC carbonate analogs were found to be sensitive to enzymatic hydrolysis and displayed high anti-HIV activity (20,21).

Herein, a series of new AZT carbamates was prepared and evaluated in cell culture infected with HIV-1. The compounds displayed moderate anti-HIV activity (3–40 μm) and low toxicity (>6000 μm). The compound I was the most active among synthesized AZT carbamates. Its selectivity index was similar to that of AZT and H-phosphonate AZT. All the synthesized depot forms of AZT were stable chemically and in human blood serum (T1/2 >> 24 h), but after oral administration in dogs carbamate IV released AZT, and its concentration was only twice lower compared with H-phosphonate AZT. The advantages of pharmacokinetic characteristics of compound IV compared with AZT and H-phosphonate AZT are the following: (i) the analysis of dog blood after administration IV showed the presence of both IV and the product of its hydrolysis AZT in contrast to H-phosphonate AZT which degraded very fast to AZT; (ii) blood half-life of AZT after administration of IV in dogs was twice longer than that of orally administered AZT; (iii) AZT released from IV could be detected in blood even after 24 h after administration, while orally administered AZT completely eliminates after 12 h; (iv) Tmax is three and two times longer than that of AZT and H-phosphonate AZT, respectively; (v) Cmax is 2 and 10 times lower compared with H-phosphonate AZT and AZT, respectively, and, as a result of a lower content of AZT released, the compound is less toxic. The amount of AZT generated by carbamate IV was only a half than AZT generated by H-phosphonate AZT. We compared acute toxicity of IV with that of AZT and H-phosphonate AZT, both approved for clinical trials. The results of determination of acute toxicity in mice showed considerably less toxicity of carbamate IV compared with AZT and H-phosphonate AZT and offered obvious advantages IV over AZT and H-phosphonate AZT. Indeed, we could not achieve even LD16 of IV at a peritoneal dose equal 7.1 g/kg while the same important parameters for AZT and H-phosphonate AZT were 1.3 and 2.0 g/kg, respectively. Further increasing of the dose was problematical due to solubility of IV in water. Thereby, we showed that carbamates IIV are depot forms of AZT, their anti-HIV activity is associated with the release of AZT, in contrast to amino acid carbamates of AZT that did not release AZT and had the amdigous mechanism of their anti-HIV action (17).

Acknowledgments

This work was supported by the Russian foundation for Basic Research, projects 12-04-00581; 11-04-12035-OFI; Presidium of Russian Academy of Sciences, project ‘Molecular and Cellular Biology.’

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