SYNTHESIS AND EVALUATION OF NOVEL 6-(3,5-DIMETHYLBENZYL)URACIL ANALOGS AS POTENTIAL ANTI-HIV-1 AGENTS © 2014 Nagy M. Khalifa3, b, #, Erik B. Pedersenc, Claus Nielsend, and Mohamed A. Al-Omara
aPharmaceutical Chemistry Department, Drug Exploration & Development Chair, College of Pharmacy, King Saud University, Riyadh 11451, Saudia Arabia bTherapeutical Chemistry Department, Pharmaceutical and Drug Industries Division, National Research Centre, Dokki 12622, Cairo, Egypt cDepartment of Chemistry, University of Southern Denmark, Dk-5230 Odense M, Denmark dRetrovius Laboratory Department of Virology, State Serum Institute, DK-2300 Copenhagen, Denmark Received December 28, 2013; in final form, January 31, 2014
A novel series of uracil derivatives with a 3,5-dimethylbenzyl group at the C-6 position were synthesized and evaluated as non-nucleoside HIV-1 reverse transcriptase inhibitors. Purity of the compounds has been confirmed by TLC. Structures of these compounds were established on the basis of elemental analyses and spectral studies. Some of the tested compounds showed moderate to potent activities against wild-type HIV-1, and N-1 alkylated derivatives were highly active.
Keywords: HIV-1 RT, S-DABOs analogues, HEPTs analogues, Anti HIV-1 activity DOI: 10.7868/S0132342314040058
INTRODUCTION
Since discovery of the human immunodeficiency virus (HIV) as the cause of the acquired immunodeficiency syndrome (AIDS) [1], many efforts have been undertaken to design and synthesize potential agents against HIV. The non-nucleoside reverse transcriptase inhibitors (NNRTIs) serve as an increasingly important role in the therapy of HIV infection, due to their unique antiviral potency, high specificity and low toxicity, such as Nevirapine and Efavirenz [2—4]. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are a structurally diverse group of compounds that bind to the viral enzyme reverse transcriptase (RT), where the NNRTIs [5—7] interact with a specific allosteric non-substrate binding pocket site. NNRTIs are highly hydrophobic and chemically diverse compounds that comprise over 50 different members. Among them, 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT) was the first NNRTIs be identified [8, 9]. Di-hydro-alkoxy benzyl-oxopyrimidines (DABOs) are structurally modified HEPT with improved inhibitory activity and pharmacokinetic properties [10—13]. Both HEPTs and DABOs are the derivatives of 4-pyri-midinone series and have an aromatic ring at C-6 position of the pyrimidine ring, which played an important role in their anti-HIV activities [14, 15]. Molecu-
# Corresponding author (e-mail: nagykhalifa@hotmail.com).
lar modeling studies have suggested that the C-2 side chain of DABOs, a major contributor to the anti-HIV-1 activity, was locked in the same region of the binding site of the N1-substituted HEPTs [16]. As an extension ofour research, we planned to investigate the antiviral properties of new compounds of 5-allyl-6-(3,5-dimethyl benzyl)-3,4-dihydro-2-mercapto- 4- oxopyrimidines, which bearing an alkyl groups directly linked to the N-1 and S2 atoms [17].
RESULTS AND DISCUSSION
The synthesis of the newly designed compounds was described in Schemes 1 and 2. Treatment of 3,5-dimeth-ylbenzyl cyanide with 3—5 molar excess of a-bromoester in the presence of activated zinc dust in refluxing tetrahy-drofuran obtained the P-ketoester in 83% yield, followed by treatment with allyl iodide to give ethyl-2-(2-(3,5-dimethylphenyl)acetyl)pent-4-enoate (1) (Scheme 1). The latter compound 1 was converted by reaction with thiourea and sodium in boiling ethanol to give the key intermediate 2-thiouracil 2 in 71% yield. Next, selective S-alkylation of 2 with appropriate substituted alkyl and cycloalkyl halides in dry DMF in the presence of K2CO3 at room temperature afforded the designed compounds 3a—k in 55—67% yields. The sulfide 3g was oxidized to the corresponding sulfone 4 on reaction with m-chloroperbenzoic acid (Scheme 1). Reac-
tion of thiouracil 2 with 2-bromoacetaldehyde acetal in dry DMF at 65°C in the presence ofK2CO3 gave S-alky-lated thiouracil 5. The latter compound was treated with N,O-bis(trimethylsilyl)acetamide (BSA) and trimethylsi-lyltrifluoromethanesulfonate (TMS triflate) as a catalyst
to give N-1 and N-3 cyclized products 6 and 7, respectively. The N-1 regioisomer 6 was isolated as the main product in 76% yield whereas the N-3 regioisomer 7 was isolated as minor product in 11% yield (Scheme 2).
Ov
Ov
O.
BrCH2CH(OCH2CH3)2 DMF/K2CO3/65°C
S N
1. H2NC(S)NH2 Na/EtOH
2. HCl
m-CPBA dry CH2Cl2
-O,
Ov
HN
„A
R-X/rt
DMF/K2CO3
R.
HN
^ N
3a—g
a, R = CH2CH=CH2
b, R = (CH2)3Br
c, R = (CH2)3OH
d, R = CH2COOCH3
e, R = cyclopentyl
f, R = butyrolactone
g, R = cyclohexyl
Scheme 1.
O
HN S^ ~N
5
1. BSA/MeCN
2. TMS triflate
-30-0°C
+
/n
o-X 6
Scheme 2.
2
2
The reaction can easily followed by TLC and the N-1 and N-3 alkylated products could be identified by their Rf values (Rf N-3 > Rf N-1). Compound 6 was easily distinguished from its corresponding regioiso-mers 7 by 1H nuclear Overhauser effects (NOE). The formed thiouracil 2 was desulfurized using chloroace-tic acid to give the uracil derivative 8 in yield of 73%. Compound 8 was silylated by heating at reflux in 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and underwent alkylation by the treatment with diethoxymethane,
dimethoxymethane, and methylthiomethylacetae in the presence of trimethylsilyltrifluoromethanesulfonate (TMS triflate) to afford the acyclic nucleosides 9a— c in 55—63% yield. Also, compound 8 can react with 2-acetoxyethylacetoxymethyl ether in the presence of N,O-bis(trimethylsilyl)acetamide (BSA) and stannic chloride to afford the acyclic nucleoside 10 in 51% yield. Subsequen deprotection of 10 by the treatment with sodium methoxide in methanol gave the free acyclic nucleoside 11 in 73% yield (Scheme 3).
O
HN
A
1. HMDS/(NH4)2SO4
2. TMS triflate/MeCN (CH3CH2O)2CH2 or (CH3O)2CH2 or CH3SCH2OAc
1. AcOCH2CH2OCH2OAc, BSA
2. SnCl4
O
HN
Л
R
X
J
9a—c
a, R = CH3, X = O
b, R = H, X = O
c, R = H, X = S
HN O^N
J
MeONa
О
HN O^N
J
О
OAc
10
Scheme 3.
OH
11
8
Antiviral Activity
The anti-HIV activities and cytotoxicities of the synthesized S-DABOs and HEPT analogues are summarized in table. The test for activity against HIV-1 was performed in MT-4 cell cultures infected with either wild type HIV-1 virus. The expression of HIV-1 was quantified by two different methods, either the HIV-1 antigen detection assay ELISA [18] or indirectly by the MTT assay [19]. In general, the introduction of an allyl group instead of an isopropyl group at C-5 and two methyl groups at position C-3, C-5 of the phenyl ring instead of phenyl moiety did not improve the activity against HIV-1 compared to MKC-442. Also, the activity of compounds 3a, 3f and 3g including S-DABOs derivatives in the side chains at C-2 is to be expected when compared to those previously reported for S-DABOs. The N-1 alkylation of the uracil 9a leads to higher activities against HIV-1, but does not show improved activity.
Virus and cells
The HIV-1 strains HTLV-IIIB and NNRTI resistant strain N 119 were propagated in H9 cells at 37°C, 5% CO2 using RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (growth medium). The culture supernatant was filtered (0.45 nm), aliquoted and stored at —80°C until use. Both HIV-1 strains were obtained from the NIH AIDS Research and Reference program.
Inhibition of HIV-1 replication
Compounds were examined for possible antiviral activity against both strains of HIV-1 using MT4 cells as target cells. MT4 cells were incubated with virus (0.005 MOI) and growth medium containing the test dilutions of compounds for six days in parallel with virus-infected and uninfected control cultures without compound added. Expression of HIV in the cultures was quantitated by the HIV-1 antigen detection assay
Cytotoxicity and anti-HIV-1 activity of newly synthesized compounds in MT-4 cells
Compound IC-50a/^M CC-50b/^M
3a 2.7 >100
3f 2.6 >100
3g 0.49 >100
4 3.9 >100
5 0.55 >100
6 35 >100
9a 0.32 >100
MKC-442 0.005 141
AZT 0.04 52
1 50% inhibitory concentration; b 50% Cytotoxic concentration.
ELISA [18] or indirectly quantified using the MTT assay [19]. Compounds mediating less than 30% reduction of HIV expression were considered without biological activity. Compounds were tested in parallel for cytotoxic effect in uninfected MT4 cultures containing the test dilutions of compound as described above. A 30% inhibition of cell growth relative to control cultures was considered significant. The 50% inhibitory concentrations (IC-50) and the 50% cytotoxic concentrations (CC-50) were determined by interpolation from the plots of percent inhibition vs. concentration of compound.
EXPERIMENTAL
All melting points were uncorrected and determined on a Buchi melting point apparatus. NMR spectra were recorded on a varian Gemini 2000 NMR spectrometer at 300 MHz for 1H and 75 MHz for 13C with TMS as internal standard. EI mass spectra were recorded on Finnigan MAT SSQ 710, FAB mass spectra on a Kratos MSSORF instrument spectrometer at University of Southern Denmark, Denmark. Elemental analyses were performed at Atlantic Microlab, Inc., Atlanta, Georgia, USA; the found values agreed favourably with the calculated ones and the results were within ±0.3 from the theoretical values. The progress of reactions was monitored by TLC (analytical silica gel plates 60 F254). Merck silica gel (0.040—0.063 mm) was used for column chromatography.
5-Allyl 6-(3,5-dimethylbenzyl)-2,3-dihydro-2-thioxo-pyrimidin-4(1H)-one (2). A mixture of P-ketoester (1) (4.0 mmol) and thiourea (60 mmol) in sodium ethox-ide solution (sodium 2 g in 45 mL absolute EtOH) was heated under reflux overnight. After cooling, the reaction mixture was evaporated under reduced pressure, the residue was dissolved in water and neutralized with HCl. The preci
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