Synthesis and Anti-HSV-1 In Vitro Activity of New

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Letters in Organic Chemistry, 2008, 5, 644-650

Synthesis and Anti-HSV-1 In Vitro Activity of New Phosphoramidates with 4-oxoquinoline and Phtalimidic Nuclei Thiago S. Torres, William P. de Macedo, Leandro F. Pedrosa, Maria Cecília B. V. de Souza, Vítor F. Ferreira, Anna Cláudia Cunha, Tamara Fogel, Fernanda da C. Santos, Isakelly P. Marques, Izabel Cristina de P. Paixão and Marcos C. de Souza* Universidade Federal Fluminense, Instituto de Química, Departamento de Química Orgânica. Programa de PG em Química Orgânica, Outeiro de São João Batista, s/no, Centro. Niterói, Rio de Janeiro, Brazil Received May 21, 2008: Revised July 22, 2008: Accepted July 22, 2008

Abstract: Aminoalkyl phosphoramidates were obtained by either direct phosphorylation of symmetric diamines or a three steps method analogue to Gabriel’s synthesis and coupled to a 4-oxoquinoline acyclonucleoside, in order to synthesize 4oxoquinolone phosphoramidates. Two unpublished compounds demonstrated low cytotoxity in comparison to Acyclovir and good HSV-1 cytophatic effects on Acyclovir resistant strains.

Keywords: Aminoalkyl phosphoramidates, 4-oxoquinoline acyclonucleoside, nucleotide analogues, Herpes Simplex Virus type 1 (HSV-1). 1. INTRODUCTION Herpes Simplex Virus type 1 (HSV-1) is a large, enveloped DNA containing virus with a genome of approximately 152 kb. In humans infection usually begins on the skin or mucosal epithelium and subsequently spreads to the sensory ganglia whose nerve processes contact the primary site of infection. Once inside these neurons the virus can enter a dormant state characterized by the absence of lytic gene transcription [1]. Periodic reactivation of the dormant virus can lead to the development of infective and painful facial lesions. Acyclovir (ACV) is used clinically as an anti-herpes drug. However, with increasing use of antiviral drugs there is an increased risk that resistant strains may develop. The herpes viruses, for example, have been shown to acquire resistance to ACV in immunocompromised patients [2]. The development of new antivirals that have a wide range of efficacy against pre-existing and resistant strains, but that lack serious adverse effects, would clearly be advantageous. However, only a few compounds have reached prominence at the clinical level so far. A large number of compounds within the group of acyclic nucleosides demonstrate antiviral activity [3-7] and as such have prompted recent research interest due to their clinical potential. Most current anti-herpes drugs are inactive until phosphorylated by viral timidine kinase (TK) inside the target cell so that viruses lacking TK are at an advantage. Phosphorylated nucleoside analogues lack the ability to cross the plasma membrane due to the high anionic charge of the phosphate groups. This unsuitability for therapeutic use is compounded by the action of non-specific stearases in the cytoplasm which cleave and render them inactive [8-9]. To overcome these obstacles phosphonates have been developed *Address correspondence to this author at the Universidade Federal Fluminense, Instituto de Química, Departamento de Química Orgânica. Programa de PG em Química Orgânica, Outeiro de São João Batista, s/no, Centro. Niterói, Rio de Janeiro, CEP 24020-150, Brazil; Tel: (021)26292230; Fax: (021)2629-2135; E-mail: [email protected]

1570-1786/08 $55.00+.00

where the P-C bond is resistant to hydrolysis [10]. As conversion to acyclic nucleoside phosphonates (ANP) does not depend on the virus induced TK, they should exert activity against a broader range of DNA viruses. In addition ANP metabolites have a long intracellular lifetime, offering a longer antiviral response. They have been approved for several clinical applications: Cyclovir for cytomegalovirus in Aids patients; Adefovir in HBV infections; Tenofovir in HIV infection. Tenofovir has been used in the treatment of HBV and Cyclovir has been used for mucocutaneous HSV-1 and HSV-2 infections [11]. Several phosphoramidate nucleoside analogues with antiviral activity in vitro were synthesized and some of them are now in clinical research [10, 12]. Phosphoramidates derived from ACV are not hydrolyzed rapidly due to the P-N bond. The 5´-phosphorylated nucleoside analogue metabolite 1 (Fig. 1) was more active than Valacyclovir, showing HSV1 inhibition similar to ACV [13]. Based on the search for new anti-HSV-1 drug candidates, convergent synthesis of two new phosphoramidic acid systems, bearing 4-oxoquinoline and phtalimide nuclei, is introduced herein. Synthesis of relatively few 4-oxoquinoline acyclonucleoside derivatives have been reported so far [14]. Recently, the new quinolones 2a and 2b (Fig. 1) have shown good HSV-1 inhibition in vitro. In relation to ACV they presented a 1.5- and 1.3-fold increase in their antiviral activity respectively [15]. N-substituted phtalimides have also been developed for antiviral purposes, with targets such as the inhibition of HIV integrase (3) and reverse transcriptase (4) (Fig. 1) [16, 17]. (Figure 1) 2. RESULTS AND DISCUSSION In recent work we described an efficient method in which aliphatic diamines were phosphorylated selectively by diiso-

© 2008 Bentham Science Publishers Ltd.

Synthesis and Anti-HSV-1 In Vitro Activity of New Phosphoramidates

Letters in Organic Chemistry, 2008, Vol. 5, No. 8

645

O N

HN

6 H2N O P HO O HN

N

3 R

7

2 N1

8 O O

HO

(2a): 6-Cl; R= OEt (2b): 7-Cl; R= OH

(1)

R O

Cl

N

O

O 4

5

O

OH

O

O

N N

N

Cl

H N

O

N

S O

OH

O

Cl

(3)

NO2

(4)

Fig. (1). Nucleoside analogues (1), 4-oxo-quinolines (2) and phtalimidic compounds (3 and 4) with antiviral activity. O

O P

O

O

H

+

i

n NH2

H2N

O

excess

P O

N H

n NH2 (6a): n = 2 (50%) (6b): n = 3 (53%) (6c): n = 4 (50%) (6d): n = 6 (52%)

(5)

Scheme 1. Direct monophosphorylation of diamines. (i) EtOH, CCl4, 15-20 min.

propyl phosphonate (5) generating the aminoalkyl phosphoramidate series 6a-d in moderate yields (Scheme 1) [18,19]. The method was adapted from industrial patents reported first in the 1970s [20]. Experimental difficulties in eliminating a diamine excess from the purification step of our previously reported methodology prompted us to introduce an alternative procedure to synthesize 6 (Scheme 2). Chloroalkyl phosphoramidic ester intermediates (7a, b) were first obtained with a yield of 73-

83% by using the biphasic phosphorylation method of Zhao & col. [21]. The same reaction using 2-bromoethylamine as a starting material gave 7c with a yield of 40%. This was lower than the previously reported yield [22], probably because of concurrent nucleophilic substitution reactions promoted by the alkali medium or intramolecular displacement where bromine acts as a more efficient leaving group than chlorine [22]. To circumvent this problem diisopropyl chlorophosphonate (8) [20] was used under dry conditions as a O

O O

P

+ H2N

Y

O

ii

nX

O

P O

N H

(5): Y=H (8): Y=Cl iii O O

P O

N H

n NH2

iv

(6a): n = 2 (87%) (6b) : n = 3 (86%)

O P O N O H

nX (7a): n = 2; X = Cl (73%) (7b): n = 3; X = Cl (83%) (7c): n = 2; X = Br (40%) O NK

O

O

nN O (9a): n = 2; from 7a (40%) (9a): n = 2; from 7c (40%) (9b): n = 3; from 7b (75%)

Scheme 2. Synthesis of mono-phosphorylated diamines 6a,b by analogue Gabriel´s Reaction. ii) 2 NaOH, CCl4, EtOH, H2O or CHCl3, Et3N; o o o iii) 160-176 C, 4h or 130 C, DMF, 3h; iv) N2H4 80%, EtOH, 125 C, 48h.

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Torres et al.

O O

O

O

Cl + N

Cl

O

N H

O

OEt

v

P

OEt

nX N

(10)

O

H

O

O

O O

O

Cl OEt +

O

P O

N H

OEt

n NH2

vi

N

MsO

NH

(11a): n=2 not obtained (11b): n=3, 55%

O

Cl

HO

n

P

(7a): n = 2; X=Cl (7b): n = 3; X = Cl (7c): n = 2; X = Br O

O

N

O

O (2a)

O P N O H

(6a): n = 2 (6b): n = 3

nN H

O (12a): n = 2 (12b): n = 3

TsO Cl

Scheme 3. Synthesis of 4-oxoquinoline phosphoramidates (11a,b) and attempts to phosphoryl acyclonucleosides (12a,b). v) Na2CO3, DMF, 70-80 oC. vi) EtOH, reflux.

phosphorylating agent. A mixture of triethylamine and chloroform was used as the solvent. The second step was analogous to the classic Gabriel´s synthesis of amines, where potassium phtalimide was the nitrogen source for the final product [23]. Dimethylformamide (DMF) was used as the solvent [24]. Compound 9b (3[(1,3-dioxo-1,3-dihydro-isoindol-2-yl)propyl] phosphoramidic acid diisopropyl ester), with a longer propylene chain, was produced with a yield of 75% while 9a (2-[(1,3-dioxo1,3-dihydro-isoindol-2-yl)ethyl] phosphoramidic acid diisopropyl ester), from both 7a and 7c, was obtained in lower yields (35-40%). The latter low yields probably due to steric hindrance between the bulky phtalimide anion and the halide. The performance of the leaving group seemed not to interfere in this case. The N-Phosphorylphtalimidic intermediates 9a, b are unreported so far and their preliminary antiviral activity is described below. The last step consists in the hydrazinolisys of the phtalimidic intermediate 9. Monophosphoryl diamines 6a,b were obtained in an 86-87% yield in this step [25]. One of our research goals was to develop a general procedure to synthesize new phosphoramidates bearing a 4oxoquinoline nucleus. As representative 4-oxoquinoline derivatives we chose the well known acyclonucleoside 1-[(2hydroxyethoxy) methyl]-3-carbethoxy-6-chloro-1, 4-dihidro4-oxoquinoline (2a) and its precursor 3-carbethoxy-6-chloro1, 4-dihidro-4-oxoquinoline (10). They were prepared as described in reference 26. Initially we attempted a coupling reaction between 2a and the nucleophilic aminoalkyl phosphoramidates 6a, b using either chlorine, mesylate or tosylate as the leaving group [27]. However, these reactions did not yield 12a or 12b. Therefore an alternative reaction, between 4-oxoquinoline 10 and the haloalkyl phosphoramidic ester derivative 7b was used. This reaction required a weakly basic medium of DMF at 70-80oC (Scheme 3) [28]. The desired 3-carbethoxy-6-chloro-1-{3-[(diisopropoxyphospho-

ryll) amino] propyl}-1, 4-dihydro-4-oxoquinoline (11b) was obtained with a yield of 55%. The same reaction using substrates 7a or 7c was unsuccessful, again for steric reasons. Stronger bases such as sodium hydride [29] or triphenylphosphine [30] were not useful for production of 11a while 11b was obtained in similar yield. We assume that the bulky 4-oxoquinolone nucleophile was unlikely to approach the substrate center with the carbon chain shorter than three methylene groups. The reaction follows a typical bimolecular nucleophilic substitution mechanism independent of the nucleophile strength or the nature of the leaving group. 3. BIOLOGICAL TESTS We investigated the antiviral effect of the new 4oxoquinoline and phtalimidic derivatives of phosphoramidic acid on HSV-1 virus replication. As presented in Table 1, both derivatives 9b and 11b showed a satisfactory HSV-1 cytophatic inhibition in ACV resistant strains. Data from ACV and compound 2a were obtained in non-mutated virus. 9b and 11b showed low cytotoxity, better than commercial ACV, as determined by the MTT (3-(4,5-dimethylthiazol2yl)-2-5 diphenyltetrazolium bromide) colorimetric method (Table 2). In order to calculate the Selective Index (SI) of 9b Table 1.

a

Data of Biological Tests for the New Compounds 9b and 11b and Reference Compounds Acyclovir and 2a

Compound

% of inhibition of virus yield (HSV-1)a

9b

68.1 %

11b

74.5%

acyclovir

96%b

2a

98%b

50 μM of the drugs were used for the test and 10 μM for ACV. bVirus strain used was not resistant to acyclovir. Results are presented as the mean of triplicate experiments. Acyclovir has been included for comparison purposes.


and 11b, we infected Vero cells with HSV-1 and determined their respective EC50s. As shown in Table 2, the EC50 value of compounds 9b and 11b were 40±7.4 M and 25 ±3.5 M, respectively. The SI (ratio of CC50 to EC50) was 37 and 60, respectively. These derivatives were inhibitory on the ACVresistant strain of HSV, suggesting that its antiviral activity may involve a mode of action different from ACV. Their mechanism of action is currently under investigation in our laboratory. Table 2.

Anti-HSV-1 Activity, Cytotoxicity and Selectivity Index in Vero Cells for 9b and 11b

Compound

EC50 a (μM)

CC50 b (μM)

S.I.c

9b

40±6.4

>1500

37

11b

25±3.5

>1500

60

ACV

1.09± 0.25

960 ± 156

880

a

50% Effective concentration or concentration required to inhibit HSV-1 virus yield. 50% Cytotoxic concentration or concentration required to reduce the viability of host cells by 50%. cSelective index (CC50/EC50). Results are presented as means of triplicate experiments. ACV-acyclovir has been included for comparison purposes.

b

4. CONCLUSION Aminoalkyl phosphoramidates could be obtained in comparable yields by either direct phosphorylation of symmetric diamines or a three step method analogous to Gabriel’s synthesis of amines. Despite a greater number of steps, this second method generated the desired monophosphorylated diamines without any risk of contamination with the bis-phosphorylated product. The synthesis of 4oxoquinolonic phosphoramidates followed a bimolecular mechanism, with the possibility that the size of both the nucleophile and the substrate was critical to a successful reaction. Both classes of compound, N-phosphorylphtalimide and 4-oxoquinoline phosphoramidate are unreported so far. The low cytotoxity and satisfactory HSV-1 cytophatic effects against Acyclovir resistant strains are promising. They represent potentially useful candidates as anti-HSV-1 agents. 5. EXPERIMENTAL General Remarks 1

H, 13C and 31P NMR spectra were recorded on a Varian UP-300 spectrometer at 299.95, 75.42 and 121.42 MHz, respectively, with TMS as internal standard or 85% H3PO4 as external standard. Signals multiplicity was assigned as: s, singlet; d, doublet; t, triplet; m, multiplet; dt, doublet of triplet and dhep, doublet of heptet. Infrared spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer; High resolution mass spectra (EI- 70eV) were recorded on a VARIAN MAT CH7 8500 direct inlet instrument; Melting points were uncorrected. Solvents were fractionally distilled before use. Diisopropyl phosphonate was prepared as described in reference [31]. Phosphorylation of Haloamines General Procedure for (Chloroalkyl) Phosphoramidic Acid Diisopropyl Esters (7a, b) To a stirred solution of chloroalkylamine hydrochloride (20 mmol) and NaOH (40 mmol) in water (6 mL) and etha-


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nol (6,5 mL), cooled to 0 ºC, a solution of recently distilled diisopropyl phosphonate (20 mmol) in CCl4 (10 mL) was dropped slowly, keeping the temperature at 0 ºC. The mixture was stirred for 24 h adding more aqueous NaOH to keep the basic pH if necessary. Water (3 mL) was added to the final mixture and the product extracted three times with hexane, dried over anhydrous MgSO4 and evaporated to giving an oily product. 7a. (2-chloroethyl) Phosphoramidic Acid Diisopropyl Ester Following the general procedure, 7a was obtained with a 73% yield. IR (film): 1234 (P=O); 988 (P-O-C); 3232 (NH); 1641 (NH). 31P-NMR (CDCl3) : 7.2. 1H NMR (CDCl3) : 1.30 and 1.32 (2d, JHCCH= 5.7, 12H, CH3); 4.59 (dhep, JHCCH= 5.7 and JPOCH= 7.5, 2H, CH); 3.22 (m, 2H, CH2NHP); 3.58 (t, JHCCH= 6.0, 2H, CH2 Cl);. 13C NMR (CDCl3) : 23.6 (d, JCCOP= 4.7, CH3); 70.8 (d, JCOP= 5.0, CH); 43.0 (CH2NP); 45.1 (d, JCCNP= 5.2, CH2 CH2NP). MS: C8H19NO3PCl calculated (M+1) 243.66950; found 243.66951. 7b. (3-chloropropyl) Phosphoramidic Acid Diisopropyl Ester Following the general procedure, 7b was obtained with an 83% yield. IR (film): 1230 (P=O); 980 (P-O-C); 3233 (NH); 1639 (NH). 31P NMR (CDCl3) : 7.7. 1H NMR (CDCl3) : 1.32 and 1.34 (2d, JHCCH= 6.3, 12H, CH3); 4.60 (dhep, JHCCH= 6.3 and JPOCH= 7.5, 2H, CH); 1.96 (quint, JHCCH= 6.6 and JHCCH= 6.0, 2H, CH2CH2CH2); 3.07 (m, 2H, CH2NH); 3.64 (t, JHCCH= 6.0, 2H, CH2Cl);. 3.55 (s, broad, 1H, NH); 13C NMR (CDCl3) : 23.5 (d, JCCOP= 5.2, CH3); 70.5 (d, JCOP= 4.7, CH); 41.9 (CH2Cl); 38.2 (CH2NP); 33.8 (d, JCCNP= 5.6, CH2CH2NHP). MS: C9H21NO3PCl calculated (M+1) 257.69630; found 257.69632. 7c. (2-bromoethyl) Phosphoramidic Acid Diisopropyl Ester 2-bromoethylamine hydrobromide (15 mmol) and diisopropyl chlorophosphonate (15 mmol) in dry CHCl3 (23 mL) were maintained under nitrogen in a three-necked roundbottomed flask. Triethylamine (2 mL) in dry CHCl3 (3 mL) was dropped over the reaction mixture. The mixture was stirred for 1 h at room temperature. The final solution was washed with water (20 mL), and then the organic layer dried over Na2SO4 and evaporated to resolve a crude oil. The product was dissolved in ethyl ether and washed twice with a 5% aqueous NaOH solution (10 mL), giving an oily product. Yield was 40%. IR (film): 1238 (P=O); 990 (P-O-C); 3240 and 3434 (NH); 1647 (NH); 31P NMR (CDCl3) : 7.6. 1 H NMR (CDCl3) : 1.31 and 1.34 (2d, JHCCH= 5.7, 12H, CH3); 4.65 (dhep, JHCCH= 5.7 and JHCCH= 7.5, 2H, CH); 3.35 (m, 2H, CH2NHP); 3.43 (t, JHCCH= 6.0, 2H, CH2Br);. 13C NMR (CDCl3) : 23.4 (d, JCCOP= 4.6, CH3); 70.7 (d, JCOP= 5.1, CH); 43.0 (CH2NHP); 45.1 (CH2CH2 Br). MS: C8H19NO3PBr calculated (M+1) 288.12080; found 288.12078. 9a. 2-[(1, 3-dioxo-1,3-dihydro-isoindol-2-yl)ethyl] Phosphoramidic Acid Diisopropyl Ester 7a or 7c (20 mmol) and potassium phtalimide (20 mmol) were heated at 165°C for 3 h in a round bottomed flask. The crude solid was washed with aqueous KOH 30% solution and the product extracted three times with CHCl3 (40 mL each). The organic layer was then treated with anhydrous

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MgSO4 giving an oily product after evaporation. A white solid was isolated by crystallization from a mixture of hexane and isopropanol (5:2) with a 40% yield for both 7a and 7c. MP= 85°C. IR (KBr disk): 1708 (C=O); 1229 (P=O); 982 (P-O-C). 31P NMR (CDCl3): : 7.3. 1H NMR (CDCl3 ) : 1.17 and 1.23 (d, JHCCH= 6.0, 12H, CH3); 4.50 (dhep, JHCCH= 6.0 and JPOCH= 7.5, 2H, CH); 3.21 (m, 2H, CH2NHP); 3.81 (t, JHCCH= 6.0, 2H, CH2NCO); 2.76 (m, broad, 1H, NH); 7.87 (m, 2H, CCH arom.); 7.72 (m, 2H, CCHCH arom.). 13C NMR (CDCl3) : 23.5 (d, JCCOP= 5.2, CH3); 70.7 (d, JCOP= 5.8, CH); 40.0 (d, JCCNP= 6.0, CH2CH2NP); 38.8 (CH2NP); 123.2 (CCHCH arom.); 131.0 (CCH arom.); 133.9 (CCH arom.); 168.3 (C=O). C16H23N2O5P: calculated (M+1) 354.12331; found 354.12333. 9b. 3-[(1,3-dioxo-1,3-dihydro-isoindol-2-yl)propyl] Phosphoramidic Acid Diisopropyl Ester Potassium phtalimide (3.17 mmol) and 7b (3.17 mmol) and were dissolved in DMF (15 mL) and heated in a round bottomed flask for 4h at 130 ºC. Water (100 mL) and CHCl3 (30 mL) were added to the reaction mixture and the product extracted from aqueous phase twice with CHCl3 (10 mL each). The organic layer was washed first with NaOH 0.2 N (20 mL) and then with water (20 mL). After evaporation, a white solid remained giving a yield of 75%. MP = 84 ºC. IR (KBr disk): 1718 (C=O); 1227 (P=O); 983 (P-O-C). 31P NMR (CDCl3) : 7.6. 1H NMR (CDCl3): : 1.30 and 1.32 (d, JHCCH= 6.3, 12H, CH3); 4.61 (dhep, JHCCH= 6.3 and JPOCH= 7.8, 2H, CH); 1.85 (quint., JHCCH= 6.6 and JHCCH= 6.3, 2H, CH2CH2 CH2); 2.92 (m, 2H, CH2NH); 3.79 (t, JHCCH= 6.6, 2H, CH2NCO); 2.76 (m, broad, 1H, NH); 7.85 (m, 2H,CCH arom.); 7.72 (m, 2H, CCHCH arom.). 13C NMR (CDCl3): : 23.7 (d, JCCOP= 5.2, CH3); 70.6 (d, JCOP= 4.7, CH); 42.0 (CH2NCO); 38.2 (CH2NP); 34.7 (d, JCCNP= 5.6, NHCH2CH2); 123.2 (CCHCH arom.); 125.5 (CCH arom.); 133.9 (CCH arom.); 168.5 (CO). C17H25N2O5P: calculated (M+1) 368.15011; found 368.15010. General Procedure for (Aminoalkyl) Phosphoramidic Acid Diisopropyl Ester (6a,b): HYDRAZINOLISYS In a three-necked round-bottomed flask adapted with a reflux condenser 9a or 9b (2.2 mmol) were dissolved in ethanol (150 mL). Two molar equivalents of hydrazine hydrate (85%) were added to the reaction. The temperature was maintained at 125 °C for 47 h. The solvent was evaporated and CHCl3 (30 mL) added. The chloroform suspension was extracted three times with cold aqueous KOH solution (40%). The product was extracted with CHCl3 (30 mL) and dried over Na2SO4 giving a crude oil after evaporation.

Torres et al.

6b. (3-aminopropyl) Phosphoramidic Acid Diisopropyl Ester Following the general procedure, 6b was obtained with an 86% yield. IR (film): 3234 (NH); 1603 (NH); 1232 (P=O); 985 (P-O-C). 31P NMR (CDCl3) : 8.1, 1H NMR (CDCl3) : 1.32, 1.30 (2d, JHCCH= 6.3, 12H, CH3); 4.57 (dhep, JHCCH= 6.3 and JPOCH= 7.8, 2H, CH); 3.00 (m. broad, 3H, CH2NHP); 1.68 (quint., JHCCH= 6.0, 2H, CH2CH2 CH2); 2.82 (t, JHCCH= 6.0, 2H, CH2NH2). 13C NMR (CDCl3): : 23.5 (2d, JCCOP= 5.0, CH3); 70.3 (d, JCOP= 5.5, CH); 39.2 (CH2NH2); 39.0 (CH2NHP); 34.1 (d, JCCNP= 6.3, CH2CH2NP). MS: C9H23N2O3P calculated (M+1) 238.14463; found 238.14460. General Procedure for 6-cloro-1-[(diisopropoxyphosphoryll)amino]alkyl]-4-oxo-1,4-dihydro-3-quinoline Ethyl Carboxylate (11a,b) In a three-necked round-bottomed flask, 3-carbethoxy-6chloro-1, 4-dihidro-4-oxoquinoline (10) (6.22 mmol) and K2CO3 (49,8 mmol) dissolved in dry DMF (20 mL) and were stirred for 1 h under a nitrogen atmosphere. 7a, 7b or 7c (12.44 mmol) in dry DMF (10 mL) was added and the solution stirred for 30 h at 80 ºC. The product was isolated from cold water (250 mL) by filtration. 11b..3-carbethoxy-6-chloro-1-{3-[(diisopropoxyphosphoryll) amino]propyl}-1,4-dihydro-4-oxoquinoline Following the general procedure, 11b was obtained as a yellow solid with a 55% yield. MP = dec.> 200ºC. IR (film): 1234 (P=O); 991 (P-O-C); 3468 (NH); 1712 (C=O). 31P NMR (CDCl3): : 8.5. 1H NMR (CDCl3) : 1.33 e 1.31 (d, JHCCH= 6.0, 12H, POCHCH3); 1.41 (t, JHCCH= 7.2, 3H, OCH2CH3); 2.07 (d, JHCCH= 6.6 and JPOCH= 7.5, 2H, CH2CH2 CH2); 4.32 (t, J= 7.5, 2H, NCH2); 3.17 (m, 2H, PNHCH2); 4.39 (quart, JHCCH= 7.2, 2H, OCH2CH3); 4.62 (dhep, JHCCH= 6.0 and JPOCH= 7.5, 2H, OCHCH3); 7.49 (d, JHCCCH= 9.0, 1H, CCHCCl); 7.61 (2d, JHCCH= 2.4 and JHCCCH= 9.0, 1H, NCCHCH); 8.46 (d, JHCCH= 2.4, 1H, NCCH); 8.49 (s, 1H, NCHC); 3.07 (s broad, 1H, NH). 13C NMR (CDCl3) : 14.3 (CH3); 23.7 (d, JCCOP= 5.2, POCHCH3); 30.6 (d, JCCNP= 5.6, CH2CH2CH2); 38.3 (PNHCH2); 51.4 (NCH2); 60.9 (OCH2 CH3); 71.1 (d, JCOP= 4.7, POCHCH3); 117.4 (CCHCCl); 127.3 (NCCH); 132.7 (CCHCH); 148.9 (NCH); 111.1 (OCCCO); 130.0 (OCCCH); 131.3 (CCl); 137.7 (NC); 165,2 (C=O ester); 172.9 (C=O ring). C81H30N2O6P: calculated (M+1) 251.13441; found 251.1206. 11a and 11c were not obtained by this method.

6a. (2-aminoethyl) Phosphoramidic Acid Diisopropyl Ester Following the general procedure, 6a was obtained with an 87% yield. IR (film): 3232 (NH); 1602 (NH); 1230 (P=O); 986 (P-O-C). 31P NMR (CDCl3) : 8.2. 1H NMR (CDCl3) : 1.31 and 1.33 (2d, JHCCH= 5.9, 12H, CH3); 4.55 (dhep, JHCCH= 5.9 and JPOCH= 7.8, 2H, CH); 2.79 (m, 2H, CH2NH2); 2.95 (t, JHCCH= 5.9, 2H, CH2NHP); 3.50 (m. broad, 1H, NH). 13C NMR (CDCl3) : 23.2 (2d, JCCOP= 4.8, CH3); 70.1 (d, JCOP= 5.6, CH); 42.2 (d, CH2CH2NP); 43.1 (CH2NP). MS: C8H22N2O3P calculated (M+1) 225.13681; found 225.13680.

For the Biological Assays Vero, African green monkey kidney cells (obtained from the American Type Culture Collection), were grown in Dulbecco’s modified Eagle’s medium (DMEM-Gibco Laboratories) supplemented with 2% heat-inactivated fetal bovine serum (purchased from Fazenda Pig), 8% calf serum (purchased from Centro Pan-Americano de Febre Aftosa), 2.25% sodium bicarbonate, 500 U/mL penicillin, 100 μg/mL streptomycin and 2.5g/mL amphotericin B. HSV-1, strain ACR2915, was kindly provided by Marcia Wigg (Universidade


Federal do Rio de Janeiro–Brasil) and was routinely propagated in the Vero cell line. Virus stocks were stored at -70 ºC until use. Virus infectivity was measured using a dilution method to determine the amount of specimen required to infect 50% of the cultured cells: the 50% tissue culture infections dose (TCID50) after Reed and Muench [32]. Cells grown in 96-well microtitre plates were inoculated with virus at 1 PFU (plaque forming unit)/cell for 2 h at 37ºC. After virus adsorption, the inocculum was replaced by a culture medium containing the tested phosphoramidates at the concentration of 50 M. The medium used to inoculate control cultures was devoid of the experimental compounds. After 3 days at 37 ºC in 5% CO2 atmosphere, the culture medium was harvested and the TCID50 was determined for each sample by endpoint dilution. The cytotoxicity of experimental compounds was tested in Vero cells using two methods, MTT (3-(4,5diethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and Trypan Blue dye exclusion assays. A monolayer of uninfected cells was incubated with culture medium containing different concentrations of compounds for 72 h at 37 ºC. The medium was then removed, the cells trypsinized and viable cells counted by the trypan blue dye exclusion test [33]. The 50% cytotoxic concentration (CC50) was calculated by regression analysis of the dose-response curves generated from these data. In the second method, a micro titre plate with cells containing a monolayer of Vero cells was incubated with MTT (5g/mL) at 37 ºC for 4 h. After this period, SDS 10% and HCl (0.01 N) was added to each well and incubated overnight. The plates were read using an automatic plate reader with a 540 nm test wavelength and a 690 nm reference wavelength [34]. A plaque reduction assay was performed utilizing Vero cells with a density of 3 x 105 cells per well, infected with various dilutions of the supernatant from the yield reduction assay for 1 h at 37 ºC and 5% CO2. After adsorption, the plates were washed with DMEM containing methylcellulose (1 %) and fetal bovine serum (5%). After 72h at 37 ºC, the monolayers were fixed with 1 % formaldehyde in PBS, the methylcellulose removed, and the cells stained with a 0.1% solution of crystal violet in 70% methanol. The virus yield assay was performed as follows. Confluent Vero cells were washed with PBS and infected with HSV-1 at 1 PFU/cell for 1 h at 37 ºC. The infected cells were washed with PBS and incubated with culture medium containing either no compounds or different concentrations of the experimental compounds. 20 h after adsorption, cells were lysed by three consecutive rounds freeze thawing. The supernatant was obtained by centrifugation at 400 x g for 10 min at 4 ºC. Virus titer was determined by a plaque reduction assay in Vero cells as described above. Data were statistically analyzed using Student’s t-test for significance at P < 0.05.


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