Synthesis of novel uracil non-nucleoside derivatives as potential reverse transcriptase inhibitors of HIV-1

Article abstract of DOI:10.1002/ardp.200900139

Novel emivirine and TNK-651 analogues 5a-d were synthesized by reaction of chloromethyl ethyl ether and / or benzyl chloromethyl ether, respectively, with uracils having 5-ethyl and 6-(4-methylbenzyl) or 6-(3,4-dimethoxybenzyl) substituents. A series of new uracil non-nucleosides substituted at N-1 with cyclopropylmethyloxymethyl 9a-d, 2-phenylethyloxymethyl 9e-h, and 3-phenylprop-1-yloxymethyl 9i-l were prepared on treatment of the corresponding uracils with the appropriate acetals 8a-c. Some of the tested compounds showed good activity against HIV-1 wild type. Among them, 1-cyclopropylmethyloxymethyl- 5-ethyl-6-(3,5-dimethylbenzyl)uracil 9c and 5-ethyl-6-(3,5-dimethylbenzyl)-1-(2- phenylethyloxymethyl)uracil 9g showed inhibitory potency equally to emivirine against HIV-1 wild type. Furthermore, compounds 9c and 9g showed marginal better activity against NNRTI resistant mutants than emivirine.

Full text of DOI:10.1002/ardp.200900139

Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
N. R. El-Brollosy et al.
663
Full Paper
Synthesis of Novel Uracil Non-Nucleoside Derivatives as
Potential Reverse Transcriptase Inhibitors of HIV-1
Nasser R. El-Brollosy1, Omar. A. Al-Deeb1, Ali A. El-Emam1, Erik B. Pedersen2, Paolo La Colla3,
Gabriella Collu3, Giuseppina Sanna3, and Roberta Loddo3
1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
2 Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Odense M,
Denmark
3 Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e
Biotecnologie Microbiche, Universitꢀ di Cagliari, Monserrato, Italy
Novel emivirine and TNK-651 analogues 5a–d were synthesized by reaction of chloromethyl
ethyl ether and / or benzyl chloromethyl ether, respectively, with uracils having 5-ethyl and 6-(4-
methylbenzyl) or 6-(3,4-dimethoxybenzyl) substituents. A series of new uracil non-nucleosides
substituted at N-1 with cyclopropylmethyloxymethyl 9a–d, 2-phenylethyloxymethyl 9e–h, and
3-phenylprop-1-yloxymethyl 9i–l were prepared on treatment of the corresponding uracils with
the appropriate acetals 8a–c. Some of the tested compounds showed good activity against HIV-1
wild type. Among them, 1-cyclopropylmethyloxymethyl-5-ethyl-6-(3,5-dimethylbenzyl)uracil 9c
and 5-ethyl-6-(3,5-dimethylbenzyl)-1-(2-phenylethyloxymethyl)uracil 9g showed inhibitory
potency equally to emivirine against HIV-1 wild type. Furthermore, compounds 9c and 9g
showed marginal better activity against NNRTI resistant mutants than emivirine.
Keywords: Drug research / Emivirine analogues / HIV-1 / Non-nucleoside reverse transcriptase inhibitors / TNK-651
analogues /
Received: June 21, 2009; Accepted: August, 12, 2009
DOI 10.1002/ardp.200900139
vir) [5], which act as chain terminators to stop the attach-
ment of further nucleosides and so prevent an ongoing
viral DNA synthesis, and non-nucleoside reverse tran-
scriptase inhibitors (NNRTIs) which, in contrast to NRTIs,
are highly specific as their binding site is a hydrophobic
pocket located approximately 10 ꢀ from the polymerase
site. They bind allosterically forcing the RT-subunit into
an inactive conformation [6, 7]. Much effort has been put
into the synthesis and design of NNRTIs; more than 30
structurally different classes of NNRTIs have been
reported [8, 9], One of the first NNRTIs was 1-[(2-hydroxy-
Introduction
The reverse transcriptase (RT) enzyme is the pivot in the
retroviral cycle of human immunodeficiency virus type-1
(HIV-1). It is responsible for the conversion of a single-
stranded RNA viral genome into a double-stranded DNA
required for the human cell's genetic machinery to repli-
cate the virus [1, 2]. Two main classes of RT inhibitors,
nucleoside reverse transcriptase inhibitors (NRTIs) such
as zidovudine (AZT, Retrovir) [3], zalcitabine (ddC, Hivid)
[4], didanosine (ddI, Videx) [5], and lamivudine (3TC, Epi-
ethoxy)methyl]-6-(phenylthio)thymine
(HEPT)
[10].
Although HEPT did not show very high activity against
HIV-1, it was considered as an interesting lead compound
for the synthesis of new analogues, among them the 6-
Correspondence: Nasser R. El-Brollosy, Departement of Pharmaceuti-
cal Chemistry, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, Saudi Arabia
benzyl-1-(ethoxymethyl)-5-isopropyluracil
(Emivirine)
E-mail: brollosy@yahoo.com
Fax: +966-1-467-6220
[11], the corresponding 1-benzyloxymethyl analogues
(TNK-651) [12] and 6-(3,5-dimethylbenzyl)-1-ethoxy-
methyl-5-isopropyluracil (GCA-186) [13], which all
showed high activity against HIV-1. Emivirine was chosen
Abbreviations: N,O-bis-(trimethylsilyl)acetamide (BSA); non-nucleoside
reverse transcriptase inhibitors (NNRTIs); nucleoside reverse transcrip-
tase inhibitors (NRTIs)
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664
N. R. El-Brollosy et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
Figure 1. Structures of HEPT, Emivirine, TNK-651, GCA-186.
as a candidate for clinical trials with AIDS patients, how-
ever, the phase-III studies was halted when emivirine was
found to activate a liver enzyme Cytochrome P450 which
metabolizes protease inhibitors [14].
The mutations that appear in the presence of NNRTIs
are the Y181C, Y181C + K103N, and the triple mutant
K103R + V179D + P225H, which are critical for the bind-
ing of the NNRTIs to RT. GCA-186 differs structurally
from Emivirine only in the introduction of two methyl
substituents in the 3- and 5-position at the C-6 benzyl
group. It showed higher antiviral activity against HIV-1
wild type and greater tolerance to the presence of the
mutated HIV strains than Emivirine, due to binding of
the two methyl substituents in two hydrophobic pockets
in the NNRTI-binding site of RT as revealed by X-ray crys-
tallography [13]. In an effort to improve the activity
against HIV-1 wild type and NNRTI resistant mutants,
and as a part of our interest in the chemistry of NNRTIs
[15–25], the present study describes synthesis and antivi-
ral evaluation of novel non-nucleosides analogues of emi-
virine and GCA-186 with a N-1 cyclopropylmethoxy-
methyl substituent. Also, a series of TNK-651 analogues
substituted at N-1 with 2-phenylethyloxymethy and 3-
phenylprop-1-yloxymethyl moieties has been investi-
gated. The objective was to investigate if these substitu-
ents could lead to an improved antiviral activity against
HIV-1. In addition, the synthesis of new HEPT analogues
should also provide information regarding the ongoing
investigation of the NNRTIs binding site.
Reagents and Conditions: (a) Zn, THF; (b) K2CO3; (c) HCl, 91–93% (one-pot syn-
thesis); (d) (H2N)2CS, NaOEt, 61–64%; (e) aq. ClCH2CO2H, 81–87%; (f)
R1CH2OCH2Cl, CsI, CHCl3, 23–69%.
Scheme 1. Synthesis of compounds 2-6.
sodium ethoxide [26]. The NMR spectra of the crude com-
pounds 2a–d showed an impurity identified as another b-
keto ester resulting from self-condensation of ethyl 2-bro-
mobutyrate. On reaction with thiourea, this b-keto ester
impurity also formed a pyrimidine compound as an
impurity in the raw materials of 3a–d. However, pure
compounds 3a–d were easily obtained by recrystalliza-
tion from aqueous ethanol [27]. Desulfurization of 3a–d
was achieved by treatment with boiling aqueous chloro-
acetic acid to give the corresponding uracil derivatives
4a–d. Silylation of 5-ethyl-6-(4-methylbenzyl)uracil 4b
and 5-ethyl-6-(3,4-dimethoxybenzyl)uracil 4d with N,O-
bis-(trimethylsilyl)acetamide (BSA) in anhydrous chloro-
form followed by treatment with chloromethyl ethyl
ether and / or benzyl chloromethyl ether in the presence
of cesium iodide gave the corresponding 1-ethyloxy-
methyl 5a, b and 1-benzyloxymethyluracils 5c, d, respec-
tively. The 1,3-dialkylated uracils 6a, b were also
obtained in case of the reaction with chloromethyl ethyl
ether (Scheme 1).
Results and discussion
Chemistry
Phenylacetonitrile, 4-methylphenylacetonitrile, 3,5-
Bis-(cyclopropylmethyloxy)methane 8a, bis-(2-phenyl-
dimethylphenylacetonotrile, and / or 3,4-dimethoxyphe- ethyloxy)methane 8b [23], and bis-(3-phenylprop-1-yloxy)-
nylacetonitrile 1a–d were reacted with the zinc organo-
metallic reagent prepared from ethyl 2-bromobutyrate in
methane 8c were prepared from the corresponding alco-
hols cyclopropylcarbinol 7a, 2-phenylethanol 7b, and 3-
anhydrous THF to afford the corresponding 4-aryl-2- phenyl-1-propanol 7c, respectively, in a reaction with
ethyl-3-oxo esters 2a–d in good yields. 5-Ethyl-6-substi-
tuted-2-thiouracils 3a–d were synthesized by treating 2a–
d with thiourea in boiling ethanol in the presence of
dibromomethane using potassium hydroxide in anhy-
drous benzene in the presence of tetrabutylammonium
bromide according to the method of Nazaretyan et al.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
Novel Uracil Non-Nucleoside Derivatives
665
Table 1. Cytotoxicity and anti-HIV-1 activity of compounds 5a–d and 9a–l.
Compound
CC50 (lM)§
EC50 (lM)#
MT-4
wtIIIB
N119
A17
EFVR
(Y181C)
(K103N+Y181C)
(K103R+V179D+P225H)
5a
5b
5c
5d
9a
9b
9c
9d
>100
>100
44 € 0.5
80 € 6.5
>100
>100
51 € 3
>100
43 € 2.5
60
5.3 € 0.3
59€4.5
1.1 € 0.2
12 € 1
1.1 € 0.1
37 € 6
0.03 € 0.01
>100
>100
>100
>44
>100
>100
>44
>100
>100
>44
>80
>80
>80
>100
>100
5.1 € 0.8
>100
>43
>100
>100
>51
>100
>43
>100
>100
>51
>100
>43
9e
9f
0.4 € 0.02
>60
>60
>60
>60
9g
9h
9i
41 € 2
73
23 € 0.5
43
0.03 € 0.01
>73
0.2 € 0.01
>43
5.6 € 1.2
>73
>23
>41
>73
>23
>43
=20
>73
>23
>43
9j
>43
9k
9l
43 € 3
38
0.05 € 0.01
>38
8.8 € 1.9
>38
>43
>38
>43
>38
Emivirine
EFV
>100
38 € 1.5
0.03 € 0.005
0.002 € 0.0003
>20
0.02 € 0.005
56
0.1 € 0.03
>100
14 € 2.5
§ Compound dose required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method; # compound
dose required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity, as determined by the MTT method.
[28]. Compounds 4a–d were silylated with BSA in anhy- activity of emivirine, a well examined HEPT analogues,
drous acetonitrile and reacted with the appropriate ace- and efavirenz, the most active anti-HIV-1 drug used in
tals 8a–c, using trimethylsilyl trifluoromethanesulfonate therapy today. Results are listed in Table 1.
(TMS triflate) as a Lewis acid catalyst [29], to give the cor-
As seen from the results listed in Table 1, compounds
responding N-1 cyclopropylmethyloxymethyl, 2-phenyl- 5c, 9a, 9c, 9e, 9g, 9i, and 9k showed good activity against
ethyloxymethyl and 3-phenylprop-1-yloxymethyl-uracils HIV-1 wild type with a wide range of EC50 values of 1.1 to
9a–d, 9e–h, and 9i–l, respectively (Scheme 2).
0.03 lM. The most active compounds were 1-cyclopropyl-
The newly synthesized compounds were confirmed by methyloxymethyl-5-ethyl-6-(3,5-dimethylbenzyl)uracil 9c
comparison of similar NMR data [15–20, 22, 24]. MS(EI), and 5-ethyl-6-(3,5-dimethylbenzyl)-1-(2-phenylethyloxy-
and HRMS are in full agreement with the proposed struc- methyl)uracil 9g, which showed inhibitory potency (EC50)
tures.
equally to emivirine against HIV-1 wild type. In general,
the two methyl substituents at the 3- and 5-positions of
the benzyl moiety improved the activity against HIV-1
Biological screening
According to structure–activity relationships (SAR), stud- wild type compared with those of unsubstituted benzyl
ies of several crystal structures of the RT complex with non-nucleosides. Compounds having 1-cyclopropylme-
inhibitors such as HEPT, GCA-186, and TNK-651 indicate thyloxymethyl and 6-benzyl 9a or 6-(3,5-dimethylbenzyl)
that a methyl or isopropyl at C-5 and a benzyl or substi- 9c showed activity against HIV-1 wild type with EC50 val-
tuted benzyl, specially 3,5-dimethylbenzyl, at C-6 are the ues of 1.1 and 0.03 lM, respectively. Compound 9c was
optimal substituents of the uracil ring with respect to over 30-fold more potent than 9a. Non-nucleosides bear-
HIV-inhibition [30]. The present study describes the syn- ing 2-phenylethyloxymethyl moiety at N-1, 9g, showed
thesis of some novel uracil non-nucleosides having the inhibitory potency equal to those of emivirine, while 9e
optimal substituents at C-5 and C-6 with some new modi- was approximately 13 times less active than 9g. Com-
fications at N-1 position of the uracil ring. Compounds pounds having a 3-phenylprop-1-yloxymethyl moiety at
5a–d and 9a–l were tested for their biological activity N-1, 9i and 9k, showed good activity against HIV-1 wild
against HIV-1 wild type and strains carrying the clinically type with EC50 values of 0.2 and 0.05 lM, respectively.
relevant mutations N119 (Y181C), A17 (K103N + Y181C), Compounds 9c, 9g, and 9k showed higher activity than
and the triple mutant EFVR (K103R + V179D + P225H) in emivirine against the mutant strain N119. Compounds
MT-4 cells. Results were compared with the antiviral 5c, 9e, 9g, 9i, 9j, 9k, and 9l showed marginal higher activ-
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666
N. R. El-Brollosy et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
Resonance Mass Spectrometer (IonSpec Corporation, Lake For-
est, CA, USA). The progress of reactions was monitored by TLC
(DC-alufolio 60 F254) from Merck (Darmstadt, Germany). For col-
umn chromatography, Merck silica gel (0.040-0.063 mm) was
used.
Ethyl 4-aryl-2-ethyl-3-oxobutyrates 2b and 2d
Zinc dust (14 g) was activated by stirring with 4 M HCl (30 mL)
for 5 min. The zinc dust was filtered, washed sequentially with
H2O, EtOH, and dry Et2O; then it was dried. The active zinc was
suspended in dry THF (60 mL) and heated to reflux. Few drops of
ethyl 2-bromobutyrate were added and the mixture was refluxed
for 10 min. p-Methylphenylacetonitrile (3.94 g, 0.03 mol) and /
or 3,4-dimethoxyphenylacetonitrile (5.32 g, 0.03 mol) was added
in one portion and ethyl 2-bromobutyrate (0.06 mol) was added
dropwise. After the addition was completed, the mixture was
refluxed for 30 min, then it was diluted with THF (150 mL) and
quenched by addition of sat. aq. K2CO3 (60 mL). The mixture was
stirred for 1 h at room temperature. The THF layer was decanted
and the residue was washed with THF (3630 mL). The combined
THF fractions were stirred with 10% aq. HCl (40 mL) for 30 min.
The solution was concentrated under reduced pressure and
diluted with CH2Cl2 (100 mL). The organic phase was washed
with sat. aq. NaHCO3 (2660 mL), dried (Na2SO4), and evaporated
under reduced pressure to give the product that was used for fur-
ther synthesis without purification.
Ethyl 2-ethyl-4-(4-methylphenyl)-3-oxobutyrate 2b
Faint yellow oil; yield: 6.75 g (91%); 1H-NMR (CDCl3, 500 MHz) d:
0.97 (t, J = 7.0 Hz, 3H, CH3), 1.25 (t, J = 7.5 Hz, 3H, CH3), 1.89–1.93
(m, 2H, CH2), 2.37 (s, 3H, CH3), 3.46 (t, J = 7.5 Hz, 1H, CH), 3.76 (s,
2H, CH2), 4.12 (q, J = 7.0 Hz, 2H, CH2), 7.08, 7.13 (26d, J = 8.0 Hz,
4H, Harom); 13C-NMR (CDCl3, 125 MHz) d: 11.89 (CH3), 13.52 (CH3),
18.42 (CH2), 21.00 (CH3), 43.73 (CH2), 60.72 (CH2), 61.22 (CH),
127.76, 129.47, 130.12, 136.77 (Carom), 169.64 (CO), 202.79 (CO).
Reagents and Conditions: (a) CH2Br2, KOH, Bu4NBr, benzene, 56–63%; (b) BSA,
CH3CN, 8, TMS triflate, 44–67%.
Scheme 2. Synthesis of compounds 8 and 9.
Ethyl 2-ethyl-4-(3,4-dimethoxyphenyl)-3-oxobutyrate 2d
Faint yellow oil; yield: 8.18 g (93%); 1H-NMR (CDCl3, 500 MHz) d:
0.82 (t, J = 7.0 Hz, 3H, CH3), 1.24 (t, J = 7.5 Hz, 3H, CH3), 1.81–1.83
(m, 2H, CH2), 3.45 (t, J = 7.5 Hz, 1H, CH), 3.74 (s, 2H, CH2), 3.85,
3.86 (26s, 6H, 26OCH3), 4.12 (q, J = 7.0 Hz, 2H, CH2), 6.70–6.82
(m, 3H, Harom); 13C-NMR (CDCl3, 125 MHz) d: 13.90 (CH3), 14.05
(CH3), 18.40 (CH2), 43.71 (CH2), 55.86 (OCH3), 55.88 (OCH3), 60.69
(CH2), 61.81 (CH), 111.45, 112.78, 121.87, 125.78, 148.31, 149.10
(Carom), 169.69 (CO), 202.86 (CO).
ity than emivirine against the mutant strains A17 and
EFVR.
In summary, we synthesized novel uracil non-nucleo-
sides analogues of emivirine, GCA-186, and TNK-651 and
investigated their antiviral activity against HIV-1. Com-
pounds 9c, 9g, and 9k showed higher activity than emi-
virine against the mutant strain N119. Compounds 5c,
9e, 9g, 9i, 9j, 9k, and 9l showed marginal higher activity
than emivirine against the mutant strains A17 and EFVR.
5-Ethyl-6-substituted-2-thiouracils 3b and 3d
Na (9.98 g, 0.434 mol) was dissolved in absolute EtOH (150 mL).
Thiourea (22.84 g, 0.3 mol) was added and the mixture was
heated to reflux. Compound 2b and / or 2d (0.02 mol) was added
dropwise and the mixture was refluxed for 18 h. The solvent was
evaporated to dryness under reduced pressure and the residue
was redissolved in H2O (150 mL). The product was precipitated
by addition of conc. HCl (16 mL) and then glacial acetic acid till
pH = 4. The precipitate was filtered off, washed with H2O, dried,
and crystallized from aq. EtOH.
Experimental
Chemistry
Melting points (uncorrected) were determined on a Gallenkamp
melting point apparatus (Weiss-Gallenkamp, London, UK). NMR
spectra were recorded on a Bruker AC 500 Ultra Shield NMR
spectrometer (Bruker Bioscience, USA) at 500 MHz for 1H and
125 MHz for 13C with TMS as an internal standard. Electron
impact mass spectra were recorded on Perkin Elmer Clarus 600
GC Mass Spectrometer (Perkin Elmer, Norwalk, CT, USA). MALDI
spectra were recorded on a Fourier Transform (FT) Ion Cyclotron
5-Ethyl-6-(4-methylbenzyl)-2-thiouracil 3b
White solid; yield: 3.16 g (61%); m.p.: 203–2058C (dec.); 1H-NMR
(DMSO-d6, 500 MHz) d: 0.80 (t, J = 7.0 Hz, 3H, CH3), 2.26 (s, 3H,
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Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
Novel Uracil Non-Nucleoside Derivatives
667
CH3), 2.37 (q, J = 7.0 Hz, 2H, CH2), 3.80 (s, 2H, CH2), 7.06, 7.13
(26d, J = 8.2 Hz, 4H, Harom), 12.29, 12.38 (26s, 2H, 26NH); 13C-
NMR (DMSO-d6, 125 MHz) d: 13.97 (CH3), 18.17 (CH2), 21.04 (CH3),
34.63 (CH2), 116.51 (C-5), 128.47, 129.66, 134.14, 136.29 (Carom),
149.95 (C-6), 161.95 (C-4), 174.82 (C-2); EI MS m/z (%): 260 [M+] (36).
3H, CH3), 2.34 (s, 3H, CH3), 2.47 (q, J = 7.5 Hz, 2H, CH2), 3.58 (q, J =
7.0 Hz, 2H, CH2), 4.13 (s, 2H, CH2), 5.13 (s, 2H, CH2), 7.01, 7.03
(26d, J = 8.0 Hz, 4H, Harom), 9.68 (s, 1H, NH); 13C-NMR (CDCl3,
125 MHz) d: 13.75 (CH3), 15.03 (CH3), 19.16 (CH2), 20.94 (CH3),
33.04 (CH2), 64.97 (CH2), 72.71 (CH2), 116.79 (C-5), 127.20, 129.87,
132.16, 136.98 (Carom), 149.40 (C-6), 151.99 (C-2), 163.40 (C-4); MS
(MALDI) m/z (%): 325 [M+ + Na] (82). Anal. calcd. for C17H22N2NaO3:
325.1523. Found: 325.1514.
5-Ethyl-6-(3,4-dimethoxybenzyl)-2-thiouracil 3d
1
White solid; yield: 3.94 g (64%); m.p.: 251–2538C (dec.); H-NMR
(DMSO-d6, 500 MHz) d: 0.85 (t, J = 7.0 Hz, 3H, CH3), 2.29 (q, J =
7.0 Hz, 2H, CH2), 3.72, 3.74 (26s, 6H, 26OCH3), 3.76 (S, 2H, CH2),
6.73–6.91 (m, 3H, Harom), 12.17, 12.38 (26s, 2H, 26NH); 13C-NMR
(DMSO-d6, 125 MHz) d: 13.43 (CH3), 18.22 (CH2), 34.57 (CH2), 56.05
(OCH3), 56.07 (OCH3), 116.52 (C-5), 112.63, 113.12, 120.53, 129.47,
148.26, 150.00 (Carom), 149.25 (C-6), 161.95 (C-4), 174.69 (C-2); EI
MS m/z (%): 306 [M+] (14).
5-Ethyl-1-ethyloxymethyl-6-(3,4-dimethoxybenzyl)uracil
5b
White solid; yield: 143 mg (41%); m.p.: 89–918C; 1H-NMR (CDCl3,
500 MHz) d: 1.08 (t, J = 7.5 Hz, 3H, CH3), 1.18 (t, J = 7.0 Hz, 3H,
CH3), 2.48 (q, J = 7.5 Hz, 2H, CH2), 3.61 (q, J = 7.0 Hz, 2H, CH2), 3.86,
3.87 (26s, 6H, 26OCH3), 4.10 (s, 2H, CH2), 5.14 (s, 2H, CH2), 6.61,
6.80 (26d, J = 8.2 Hz, 2H, Harom), 6.67 (s, 1H, Harom), 9.78 (s, 1H,
NH); 13C-NMR (CDCl3, 125 MHz) d: 13.81 (CH3), 15.05 (CH3), 19.15
(CH2), 21.40 (CH3), 32.97 (CH2), 55.97 (OCH3), 56.00 (OCH3), 65.02
(CH2), 72.68 (CH2), 116.79 (C-5), 110.91, 111.87, 119.19, 127.56,
148.44, 149.68 (Carom), 149.32 (C-6), 152.01 (C-2), 163.42 (C-4); MS
(MALDI) m/z (%): 371 [M+ + Na] (39). Anal. calcd. for C18H24N2NaO5:
371.1577. Found: 371.1577.
5-Ethyl-6-substituted-uracils 4b and 4d
Compounds 3b, d (0.01 mol) were suspended in 10% aq.
ClCH2CO2H (200 mL). The suspension was refluxed for overnight
and filtered after cooling. The precipitate thus formed was
washed with H2O, cold EtOH, then Et2O and dried.
5-Ethyl-6-(4-methylbenzyl)uracil 4b
White solid; yield: 1.98 g (81%); m.p.: 256–2588C (dec.); 1H-NMR
(DMSO-d6, 500 MHz) d: 0.81 (t, J = 7.0 Hz, 3H, CH3), 2.22 (q, J =
7.0 Hz, 2H, CH2), 2.26 (s, 3H, CH3), 3.70 (s, 2H, CH2), 7.12, 7.14
(26d, J = 8.0 Hz, 4H, Harom), 10.71, 10.99 (26s, 2H, 26NH); 13C-
NMR (DMSO-d6, 125 MHz) d: 13.94 (CH3), 18.05 (CH2), 21.03 (CH3),
35.10 (CH2), 111.65 (C-5), 128.48, 129.60, 134.28, 136.20 (Carom),
149.38 (C-6), 151.40 (C-2), 164.98 (C-4); EI MS m/z (%): 244 [M+] (97).
1-Benzyloxymethyl-5-ethyl-6-(4-methylbenzyl)uracil 5c
White solid; yield: 237 mg (65%); m.p.: 94–958C; 1H-NMR (CDCl3,
500 MHz) d: 1.07 (t, J = 7.5 Hz, 3H, CH3), 2.36 (s, 3H, CH3), 2.47 (q, J
= 7.5 Hz, 2H, CH2), 4.13 (s, 2H, CH2), 4.68 (s, 2H, CH2), 5.23 (s, 2H,
CH2), 6.97–7.41 (m, 9H, Harom), 9.78 (s, 1H, NH); 13C-NMR (CDCl3,
125 MHz) d: 13.79 (CH3), 19.16 (CH2), 20.97 (CH3), 33.08 (CH2),
71.79 (CH2), 72.83 (CH2), 116.88 (C-5), 127.24, 127.82, 128.48,
129.99, 131.96, 132.05, 137.42, 141.08 (Carom), 149.24 (C-6), 152.04
(C-2), 163.37 (C-4); MS (MALDI) m/z (%): 387 [M+ + Na] (94). Anal.
calcd. for C22H24N2NaO3: 387.1679. Found: 387.1668.
5-Ethyl-6-(3,4-dimethoxybenzyl)uracil 4d
White solid; yield: 2.53 g (87%); m.p.: 211–2138C; 1H -NMR
(DMSO-d6, 500 MHz) d: 0.85 (t, J = 7.5 Hz, 3H, CH3), 2.27 (q, J =
7.5 Hz, 2H, CH2), 3.66 (s, 2H, CH2), 3.72, 3.74 (26s, 6H, 26OCH3),
6.75, 6.77 (26d, J = 8.0 Hz, 2H, Harom), 6.93 (s, 1H, Harom), 10.67,
10.97 (26s, 2H, 26NH); 13C-NMR (DMSO-d6, 125 MHz) d: 14.03
(CH3), 18.08 (CH2), 35.05 (CH2), 56.04 (OCH3), 113.09 (C-5), 111.52,
112.61, 120.58, 129.63, 149.22, 149.47 (Carom), 148.20 (C-6), 151.41
(C-2), 164.99 (C-4); EI MS m/z (%): 290 [M+] (25).
1-Benzyloxymethyl-5-ethyl-6-(3,4-dimethoxybenzyl)uracil
5d
White solid; yield: 284 mg (69%); m.p.: 131–1338C; 1H-NMR
(CDCl3, 500 MHz) d: 1.08 (t, J = 7.5 Hz, 3H, CH3), 2.47 (q, J = 7.5 Hz,
2H, CH2), 3.85, 3.87 (26s, 6H, 26OCH3), 4.11 (s, 2H, CH2), 4.68 (s,
2H, CH2), 5.25 (s, 2H, CH2), 6.57–7.38 (m, 8H, Harom), 9.51 (s, 1H,
NH); 13C-NMR (CDCl3, 125 MHz) d: 13.85 (CH3), 19.15 (CH2), 33.00
(CH2), 55.97 (OCH3), 56.00 (OCH3), 71.86 (CH2), 72.84 (CH2), 116.85
(C-5), 110.86, 111.83, 119.18, 127.38, 127.78, 128.00, 128.48,
137.39, 148.46, 149.67 (Carom)), 149.16 (C-6), 151.93 (C-2), 163.19
(C-4); MS (MALDI) m/z (%): 433 [M+ + Na] (96). Anal. calcd. for
C23H26N2NaO5: 433.1734. Found: 433.1716.
Ethyloxymethyl and benzyloxymethyluracil derivatives
5a–d and 6a, b
N,O-Bis-(trimethylsilyl)acetamide (BSA) (0.87 mL, 0.0035 mol) was
added to a suspension of 4a–d (0.001 mol) in anhydrous CHCl3
(20 mL) and the mixture was stirred at room temperature under
nitrogen. After a clear solution was obtained (10 min), chloro-
methyl ethyl ether and / or benzyl chloromethyl ether
(0.015 mol) followed by CsI (0.26 g, 0.001 mol) were added. The
reaction mixture was stirred at room temperature under nitro-
gen for 3–5 h. Saturated aq. NaHCO3 (20 mL) was added and the
mixture was extracted with CH2Cl2 (3650 mL). The organic
phase was collected, dried (MgSO4), and evaporated under
reduced pressure. The residue was chromatographed on silica
gel column using CHCl3 to give the corresponding N-1 alkylated
5a–d and 1,3-bis-alkylated uracils 6a, b.
1,3-Bis-(ethyloxymethyl)-5-ethyl-6-(4-methylbenzyl)uracil
6a
Colourless viscous oil, yield: 94 mg (26%); 1H-NMR (CDCl3,
500 MHz) d: 1.07 (t, J = 7.0 Hz, 3H, CH3), 1.19 (t, J = 7.0 Hz, 3H,
CH3), 1.24 (t, J = 7.0 Hz, 3H, CH3), 2.34 (s, 3H, CH3), 2.48 (q, J =
7.0 Hz, 2H, CH2), 3.60 (q, J = 7.0 Hz, 2H, CH2), 3.70 (q, J = 7.0 Hz,
2H, CH2), 4.12 (s, 2H, CH2), 5.14 (s, 2H, CH2), 5.48 (s, 2H, CH2), 7.01,
7.14 (26d, J = 8.0 Hz, 4H, Harom); 13C-NMR (CDCl3, 125 MHz) d:
13.70 (CH3), 15.03 (CH3), 15.20 (CH3), 19.74 (CH2), 20.94 (CH3),
33.11 (CH2), 65.04 (CH2), 65.90 (CH2), 71.14 (CH2), 73.49 (CH2),
116.08 (C-5), 127.22, 129.86, 132.18, 136.97 (Carom), 148.09 (C-6),
152.63 (C-2), 162.75 (C-4); EI MS m/z (%): 360 [M+] (9).
5-Ethyl-1-ethyloxymethyl-6-(4-methylbenzyl)uracil 5a
White solid; yield: 115 mg (38%); m.p.: 137–1388C; 1H-NMR
(CDCl3, 500 MHz) d: 1.07 (t, J = 7.5 Hz, 3H, CH3), 1.18 (t, J = 7.0 Hz,
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N. R. El-Brollosy et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
1,3-Bis-(ethyloxymethyl)-5-ethyl-6-(3,4-
dimethoxybenzyl)uracil 6b
6-Benzyl-1-cyclopropylmethyloxymethyl-5-ethyluracil 9a
White solid; yield: 189 mg (60%); m.p.: 91–928C; 1H-NMR (CDCl3,
500 MHz) d: 0.21–0.22, 0.52–0.54 (26m, 4H, 26CH2, Hcyclopropyl),
1.06 (t, J = 7.0 Hz, 3H, CH3), 1.09–1.11 (m, 1H, CH, Hcyclopropyl), 2.48
(q, J = 7.0 Hz, 2H, CH2), 3.40 (d, J = 7.0 Hz, 2H, CH2), 4.20 (s, 2H,
CH2), 5.16 (s, 2H, CH2), 7.13–7.36 (m, 5H, Harom), 9.97 (s, 1H, NH);
13C-NMR (CDCl3, 125 MHz) d: 3.03, 13.74 (Ccyclopropyl), 10.47 (CH3),
18.88 (CH2), 33.41 (CH2), 72.71 (CH2), 74.09 (CH2), 116.98 (C-5),
127.27, 127.34, 129.20, 135.33 (Carom), 149.13 (C-6), 152.04 (C-2),
163.49 (C-4); MS (MALDI) m/z (%): 337 [M+ + Na] (46). Anal. calcd.
for C18H22N2NaO3: 337.1523. Found: 337.1534.
Colourless viscous oil, yield: 93 mg (23%); 1H-NMR (CDCl3,
500 MHz) d: 1.07 (t, J = 7.5 Hz, 3H, CH3), 1.15–1.24 (m, 6H,
26CH3), 2.48 (q, J = 7.5 Hz, 2H, CH2), 3.48 (q, J = 7.0 Hz, 2H, CH2),
3.68 (q, J = 7.0 Hz, 2H, CH2), 3.84, 3.85 (s, 6H, 26OCH3), 4.08 (s,
2H, CH2), 5.15 (s, 2H, CH2), 5.47 (s, 2H, CH2), 6.59–6.81 (m, 3H,
H
arom);13C-NMR (CDCl3, 125 MHz) d: 13.75 (CH3), 15.00 (CH3), 15.05
(CH3), 19.73 (CH2), 33.03 (CH2), 55.96 (OCH3), 55.99 (OCH3), 65.09
(CH2), 65.88 (CH2), 71.13 (CH2), 73.45 (CH2), 116.06 (C-5), 110.97,
111.83, 119.17, 127.57, 148.03, 148.43 (Carom), 149.65 (C-6), 152.60
(C-2), 162.73 (C-4); EI MS m/z (%): 406 [M+] (13).
1-Cyclopropylmethyloxymethyl-5-ethyl-6-(4-
methylbenzyl)uracil 9b
Bis-(cyclopropylmethyloxy)methane 8a and bis-(3-
phenylprop-1-yloxy)methane 8c
White solid; yield: 211 mg (64%); m.p.: 126–1288C; 1H-NMR
(CDCl3, 500 MHz) d: 0.12–0.24, 0.53–0.56 (26m, 4H, 26CH2,
Cyclopropylcarbinol (7a, 7.2 g, 0.1 mol) and / or 3-phenyl-1-prop-
anol (7c, 13.6 g, 0.1 mol), dibromomethane (8.79 g, 0.0505 mol),
and tetrabutylammonium bromide (1.74 g, 0.00535 mol) were
added to potassium hydroxide (5.66 g, 0.101 mol) in anhydrous
benzene (30 mL), and the suspension was heated under reflux
for 5 h. After cooling, H2O (50 mL) was added and the resulting
solution was extracted with ether (3650 mL). The ether phase
was dried with anhydrous MgSO4 and evaporated under reduced
pressure to afford 8a and 8c as colourless oils. As determined
from NMR, 8a was contaminated with the starting material 7a
in a 5 : 2 ratio. The acetal 8a was used for further synthesis with-
out purification, while 8c was purified on a silica gel column
using petrol ether / CHCl3 (1 : 1).
H
cyclopropyl), 1.03–1.06 (m, 1H, CH, Hcyclopropyl), 1.07 (t, J = 7.5 Hz, 3H,
CH3), 2.35 (s, 3H, CH3), 2.47 (q, J = 7.5 Hz, 2H, CH2), 3.41 (d, J =
7.0 Hz, 2H, CH2), 4.16 (s, 2H, CH2), 5.16 (s, 2H, CH2), 7.02, 7.15
(26d, J = 8.0 Hz, 4H, Harom), 9.51 (s, 1H, NH); 13C-NMR (CDCl3,
125 MHz) d: 3.03, 13.76 (Ccyclopropyl), 10.47 (CH3), 19.16 (CH2), 20.95
(CH3), 33.03 (CH2), 72.70 (CH2), 74.10 (CH2), 116.80 (C-5), 127.22,
129.88, 132.13, 137.00 (Carom), 149.44 (C-6), 151.90 (C-2), 163.31 (C-
4); EI MS m/z (%): 328 [M+] (5).
1-Cyclopropylmethyloxymethyl-5-ethyl-6-(3,5-
dimethylbenzyl)uracil 9c
White solid; yield: 212 mg (62%); m.p.: 132–1338C; 1H-NMR
(CDCl3, 500 MHz) d: 0.23–0.25, 0.53–0.56 (26m, 4H, 26CH2,
Hcyclopropyl), 1.05–1.07 (m, 1H, CH, Hcyclopropyl), 1.08 (t, J = 7.5 Hz, 3H,
CH3), 2.30 (s, 6H, 26CH3), 2.48 (q, J = 7.5 Hz, 2H, CH2), 3.41 (d, J =
7.5 Hz, 2H, CH2), 4.13 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.73 (s, 2H,
Harom), 6.92 (s, 1H, Harom), 9.60 (s, 1H, NH); 13C-NMR (CDCl3,
125 MHz) d: 3.04, 13.75 (Ccyclopropyl), 10.49 (CH3), 19.17 (CH2), 21.27
(CH3), 33.23 (CH2), 72.74 (CH2), 74.10 (CH2), 116.82 (C-5), 125.05,
128.93, 135.04, 138.85 (Carom), 149.46 (C-6), 151.46 (C-2), 163.41 (C-
4); MS (MALDI) m/z (%): 365 [M+ + Na] (48). Anal. calcd. for
C20H26N2NaO3: 365.1836. Found: 365.1850.
Bis-(cyclopropylmethyloxy)methane 8a
1
Colourless oil, yield: 4.4 g (56%); H-NMR (CDCl3, 500 MHz) d:
0.55–0.58 (m, 8H, 46CH2), 1.07–1.14 (m, 2H, 26CH), 3.46 (d, J =
7.0 Hz, 4H, 26CH2), 4.83 (s, 2H, OCH2O); 13C-NMR (CDCl3,
125 MHz) d: 2.65, 10.47 (Ccycloprpyl), 72.41 (CH2), 94.64 (OCH2O); EI
MS m/z (%): 156 [M+] (12).
Bis-(3-phenylprop-1-yloxy)methane 8c
Colourless oil, yield: 8.9 g (63%); 1H-NMR (CDCl3, 500 MHz) d:
1.97–2.03 (m, 4H, 26CH2), 2.77 (t, J = 7.5 Hz, 4H, 26CH2), 3.64 (t,
J = 6.5 Hz, 4H, 26CH2), 4.78 (s, 2H, OCH2O), 7.25–7.38 (m, 10H,
Harom);13C-NMR (CDCl3, 125 MHz) d: 31.45 (CH2), 32.51 (CH2), 67.19
(CH2), 95.48 (OCH2O), 125.87, 128.40, 128.49, 141.94 (Carom); EI MS
m/z (%): 284 [M+] (9).
1-Cyclopropylmethyloxymethyl-5-ethyl-6-(3,4-
dimethoxybenzyl)uracil 9d
White solid; yield: 252 mg (67%); m.p.: 97–988C; 1H-NMR (CDCl3,
500 MHz) d: 0.22–0.24, 0.53–0.56 (26m, 4H, 26CH2, Hcyclopropyl),
1.05–1.07 (m, 1H, CH, Hcyclopropyl), 1.09 (t, J = 7.5 Hz, 3H, CH3), 2.48
(q, J = 7.5 Hz, 2H, CH2), 3.41 (d, J = 7.0 Hz, 2H, CH2), 3.87 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 4.14 (s, 2H, CH2), 5.17 (s, 2H, CH2), 6.61,
6.81 (26d, J = 8.0 Hz, 2H, Harom), 6.68 (s, 1H, Harom), 9.33 (s, 1H,
NH); 13C-NMR (CDCl3, 125 MHz) d: 3.05, 13.83 (Ccyclopropyl), 10.48
(CH3), 19.17 (CH2), 32.96 (CH2), 55.97 (OCH3), 56.00 (OCH3), 72.68
(CH2), 74.17 (CH2), 116.78 (C-5), 110.90, 111.82, 119.16, 127.48,
148.44, 149.40 (Carom), 149.66 (C-6), 151.82 (C-2), 163.23 (C-4); MS
(MALDI) m/z (%): 397 [M+ + Na] (11). Anal. calcd. for C20H26N2NaO5:
397.1734. Found: 397.1728.
1-Cyclopropylmethyloxymethyl-5-ethyluracils 9a–d
Compounds 4a–d (1 mmol) were stirred in anhydrous CH3CN
(15 mL) under nitrogen and BSA (0.87 mL, 3.5 mol) was added.
After a clear solution was obtained (10 min), the reaction mix-
ture was cooled to –508C and TMS triflate (0.18 mL, 1 mmol) was
added followed by dropwise addition of bis-(cyclopropylmethyl-
oxy)methane 8a (312 mg, 2 mmol). The mixture was stirred at
room temperature for 3–4 h. The reaction was quenched with
sat. aq. NaHCO3 solution (5 mL) and evaporated under reduced
pressure. The residue was extracted with ether (3650 mL), the
combined organic fractions were dried (MgSO4) and evaporated
under reduced pressure. The residue was chromatographed on
silica gel column with CHCl3 to afford the corresponding non-
nucleosides 9a–d.
1-(2-Phenylethyloxymethyl)uracil derivatives 9e–h
Compounds 4a–d (1 mmol) were stirred in anhydrous CH3CN
(15 mL) under nitrogen and BSA (0.87 mL, 3.5 mol) was added.
After a clear solution was obtained (10 min), the reaction mix-
ture was cooled to –508C and TMS triflate (0.18 mL, 1 mmol) was
added followed by dropwise addition of bis-(2-phenylethyloxy)-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
Novel Uracil Non-Nucleoside Derivatives
669
methane 8b (512 mg, 2 mmol). The reaction mixture was stirred
at room temperature for 3–5 h and the mixture was worked up
as described in preparation of compounds 9a–d to give the non-
nucleosides 9e–h.
1-(3-Phenylprop-1-yloxymethyl)uracil derivatives 9i–l
Compounds 4a–d (1 mmol) were stirred in anhydrous CH3CN
(15 mL) under nitrogen and BSA (0.87 mL, 3.5 mol) was added.
After a clear solution was obtained (10 min), the reaction mix-
ture was cooled to –508C and TMS triflate (0.18 mL, 1 mmol) was
added followed by dropwise addition of bis-(3-phenylprop-1-
yloxy)methane 8c (568 mg, 2 mmol). The reaction mixture was
stirred at room temperature for 4–5 h and the mixture was
worked up as described in preparation of compounds 9a–d to
give the non-nucleosides 9i–l.
6-Benzyl-5-ethyl-1-(2-phenylethyloxymethyl)uracil 9e
White solid; yield: 214 mg (59%); m.p.: 96–978C; 1H-NMR (CDCl3,
500 MHz) d: 1.04 (t, J = 7.0 Hz, 3H, CH3), 2.44 (q, J = 7.0 Hz, 2H,
CH2), 2.86 (t, J = 6.5 Hz, 2H, CH2), 3.82 (t, J = 6.5 Hz, 2H, CH2), 3.98
(s, 2H, CH2), 5.11 (s, 2H, CH2), 7.07–7.34 (m, 10H, Harom), 9.43 (s,
1H, NH); 13C-NMR (CDCl3, 125 MHz) d: 13.71 (CH3), 19.16 (CH2),
33.20 (CH2), 36.06 (CH2), 70.07 (CH2), 72.83 (CH2), 126.37, 127.26,
127.30, 128.38, 128.88, 129.20, 135.30, 138.65 (Carom), 149.08 (C-
6), 151.89 (C-2), 163.19 (C-4); MS (MALDI) m/z (%): 387 [M+ + Na]
(94). Anal. calcd. for C22H24N2NaO3: 387.1679. Found: 387.1693.
6-Benzyl-5-ethyl-1-(3-phenylprop-1-yloxymethyl)uracil 9i
Colourless viscous oil; yield: 185 mg (49%); 1H-NMR (CDCl3,
500 MHz) d: 1.10 (t, J = 7.0 Hz, 3H, CH3), 1.86 (m, 2H, CH2), 2.50 (q,
J = 7.5 Hz, 2H, CH2), 2.67 (t, J = 6.5 Hz, 2H, CH2), 3.58 (t, J = 6.0 Hz,
2H, CH2), 4.20 (s, 2H, CH2), 5.14 (s, 2H, CH2), 7.14–7.39 (m, 10H,
Harom), 9.44 (s, 1H, NH); 13C-NMR (CDCl3, 125 MHz) d: 13.77 (CH3),
19.21 (CH2), 30.99 (CH2), 32.16 (CH2), 33.44 (CH2), 68.62 (CH2),
72.99 (CH2), 116.99 (C-5), 125.93, 127.35, 128.38, 128.40, 129.26,
135.26, 141.46 (Carom), 149.09 (C-6), 151.86 (C-2), 163.23 (C-4); MS
(MALDI) m/z (%): 401 [M+ + Na] (95). Anal. calcd. for C23H26N2NaO3:
401.1836. Found: 401.1845.
5-Ethyl-6-(4-methylbenzyl)-1-(2-
phenylethyloxymethyl)uracil 9f
Colourless viscous oil; yield: 198 mg (52%); 1H-NMR (CDCl3,
500 MHz) d: 1.05 (t, J = 7.0 Hz, 3H, CH3), 2.35 (s, 3H, CH3), 2.42 (q,
J = 7.0 Hz, 2H, CH2), 2.86 (t, J = 6.5 Hz, 2H, CH2), 3.81 (t, J = 6.5 Hz,
2H, CH2), 3.94 (s, 2H, CH2), 5.11 (s, 2H, CH2), 6.95, 7.14 (26d, J =
8.0 Hz, 4H, Harom), 7.21–7.32 (m, 5H, Harom), 9.47 (s, 1H, NH); 13C-
NMR (CDCl3, 125 MHz) d: 13.73 (CH3), 19.15 (CH2), 20.95 (CH3),
32.82 (CH2), 36.07 (CH2), 70.03 (CH2), 72.80 (CH2), 116.74 (C-5),
126.44, 127.19, 128.37, 128.72, 129.86, 132.13, 136.96, 138, 65
(Carom), 149.37 (C-6), 151.91 (C-2), 163.25 (C-4); EI MS m/z (%): 378
[M+] (4).
5-Ethyl-6-(4-methylbenzyl)-1-(3-phenylprop-1-
yloxymethyl)uracil 9j
Colourless viscous oil; yield: 174 mg (44%); 1H-NMR (CDCl3,
500 MHz) d: 1.11 (t, J = 7.5 Hz, 3H, CH3), 1.88 (m, 2H, CH2), 2.37 (s,
2H, CH2), 2.51 (q, J = 7.5 Hz, 2H, CH2), 2.68 (t, J = 6.5 Hz, 2H, CH2),
3.59 (t, J = 6.0 Hz, 2H, CH2), 4.16 (s, 2H, CH2), 5.16 (s, 2H, CH2),
7.03–7.32 (m, 9H, Harom), 9.84 (s, 1H, NH); 13C-NMR (CDCl3,
125 MHz) d: 13.81 (CH3), 19.20 (CH2), 20.98 (CH3), 31.03 (CH2),
32.16 (CH2), 33.05 (CH2), 68.60 (CH2), 72.97 (CH2), 116.88 (C-5),
125.92, 127.25, 128.38, 128.41, 129.93, 132.14, 137.04, 141.50
(Carom), 149.38 (C-6), 152.02 (C-2), 163.48 (C-4); EI MS m/z (%): 392
[M+] (3).
5-Ethyl-6-(3,5-dimethylbenzyl)-1-(2-
phenylethyloxymethyl)uracil 9g
White solid; yield: 201 mg (51%); m.p.: 123–1258C; 1H-NMR
(CDCl3, 500 MHz) d: 1.06 (t, J = 7.0 Hz, 3H, CH3), 2.30 (s, 6H,
26CH3), 2.43 (q, J = 7.0 Hz, 2H, CH2), 2.87 (t, J = 6.5 Hz, 2H, CH2),
3.83 (t, J = 6.5 Hz, 2H, CH2), 3.92 (s, 2H, CH2), 5.13 (s, 2H, CH2), 6.66
(s, 2H, Harom), 6.91 (s, 1H, Harom), 7.21–7.33 (m, 5H, Harom), 9.66 (s,
1H, NH); 13C-NMR (CDCl3, 125 MHz) d: 13.72 (CH3), 19.16 (CH2),
21.27 (CH3), 33.05 (CH2), 36.09 (CH2), 70.09 (CH2), 72.85 (CH2),
116.75 (C-5), 125.02, 126.36, 128.38, 128.89, 135.06, 138.68,
138.84 (Carom), 149.40 (C-6), 152.00 (C-2), 163.39 (C-4); MS (MALDI)
m/z (%): 415 [M+ + Na] (97). Anal. calcd. for C24H28N2NaO3:
415.1992. Found: 415.2001.
5-Ethyl-6-(3,5-dimethylbenzyl)-1-(3-phenylprop-1-
yloxymethyl)uracil 9k
White solid; yield: 209 mg (51%); m.p.: 110–1128C; 1H-NMR
(CDCl3, 500 MHz) d: 1.11 (t, J = 7.5 Hz, 3H, CH3), 1.89 (m, 2H, CH2),
2.32 (s, 6H, 26CH3), 2.50 (q, J = 7.5 Hz, 2H, CH2), 2.68 (t, J = 6.5 Hz,
2H, CH2), 3.59 (t, J = 6.0 Hz, 2H, CH2), 4.12 (s, 2H, CH2), 5.16 (s, 2H,
CH2), 6.74 (s, 2H, Harom), 6.93 (s, 1H, Harom), 7.18–7.32 (m, 5H, Harom),
9.54 (s, 1H, NH); 13C-NMR (CDCl3, 125 MHz) d: 13.77 (CH3), 19.21
(CH2), 21.28 (CH3), 31.01 (CH2), 32.16 (CH2), 33.27 (CH2), 68.59
(CH2), 73.01 (CH2), 116.86 (C-5), 125.05, 125.92, 128.38, 128.40,
128.98, 135.04, 138.90, 141.48 (Carom), 149.38 (C-6), 151.95 (C-2),
163.37 (C-4); MS (MALDI) m/z (%): 429 [M+ + Na] (96). Anal. calcd.
for C25H30N2NaO3: 429.2150. Found: 429.2131.
5-Ethyl-6-(3,4-dimethoxybenzyl)-1-(2-
phenylethyloxymethyl)uracil 9h
White solid; yield: 198 mg (47%); m.p.: 109–1118C; 1H-NMR
(CDCl3, 500 MHz) d: 1.06 (t, J = 7.5 Hz, 3H, CH3), 2.44 (q, J = 7.5 Hz,
2H, CH2), 2.86 (t, J = 6.5 Hz, 2H, CH2), 3.82 (t, J = 6.5 Hz, 2H, CH2),
3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.92 (s, 2H, CH2), 5.12 (s, 2H,
CH2), 6.55, 6.79 (26d, J = 8.0 Hz, 2H, Harom), 6.59 (s, 1H, Harom),
7.20–7.31 (m, 5H, Harom), 9.31 (s, 1H, NH); 13C-NMR (CDCl3,
125 MHz) d: 13.81 (CH3), 19.15 (CH2), 32.75 (CH2), 36.08 (CH2),
55.98 (OCH3), 56.00 (OCH3), 70.11 (CH2), 72.76 (CH2), 116.71 (C-5),
110.86, 111.82, 119.14, 126.37, 127.50, 128.37, 128.87, 138.66,
148.41, 149.29 (Carom), 149.64 (C-6), 151.86 (C-2), 163.17 (C-4); MS
(MALDI) m/z (%): 447 [M+ + Na] (97). Anal. calcd. for C24H28N2NaO5:
447.1890. Found: 447.1904.
5-Ethyl-6-(3,4-dimethoxybenzyl)-1-(3-phenylprop-1-
yloxymethyl)uracil 9l
Colourless viscous oil; yield: 207 mg (47%); 1H-NMR (CDCl3,
500 MHz) d: 1.11 (t, J = 7.5 Hz, 3H, CH3), 1.88 (m, 2H, CH2), 2.51 (q,
J = 7.5 Hz, 2H, CH2), 2.67 (t, J = 6.5 Hz, 2H, CH2), 3.59 (t, J = 6.0 Hz,
2H, CH2), 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.12 (s, 2H, CH2),
5.16 (s, 2H, CH2), 6.62, 6.82 (26d, J = 8.0 Hz, 2H, Harom), 6.68 (s, 1H,
Harom), 7.16–7.30 (m, 5H, Harom), 9.55 (s 1H, NH); 13C-NMR (CDCl3,
125 MHz) d: 13.86 (CH3), 19.19 (CH2), 30.99 (CH2), 32.16 (CH2),
32.98 (CH2), 55.98 (OCH3), 56.02 (OCH3), 116.82 (C-5), 110.89,
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www.archpharm.com

670
N. R. El-Brollosy et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 663–670
111.87, 119.19, 125.93, 127.47, 128.38, 141.43, 148.47, 149.70
(Carom), 149.32 (C-6), 151.89 (C-2), 163.32 (C-4); MS (MALDI) m/z (%):
461 [M+ + Na] (93). Anal. calcd. for C25H30N2NaO5: 461.2047.
Found: 461.2041.
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Antiviral-assay procedures
Compounds were solubilized in DMSO at 100 mM and then
diluted in culture medium. Cells and viruses: MT-4, C8166, and
H9/IIIB cells were grown at 378C in a 5% CO2 atmosphere in RPMI
1640 medium supplemented with 10% fetal calf serum (FCS),
100 IU/mL penicillin G, and 100 lg/mL streptomycin. Cell cul-
tures were checked periodically for the absence of mycoplasma
contamination with a MycoTect Kit (Gibco). Human immunode-
ficiency viruses type 1 (HIV-1, IIIB strain) was obtained from
supernatants of persistently infected H9/IIIB cells. The HIV-1
stock solutions had titers of 4.56106 50% cell culture infectious
dose (CCID50)/mL. The Y181C mutant (NIH N119) was derived
from an AZT-sensitive clinical isolate passaged initially in CEM
and then in MT-4 cells in the presence of nevirapine (10 lM). The
double mutant K103N + Y181C (NIH A17) was derived from the
IIIB strain passaged in H9 cells in the presence of BI-RG 587
(1 lM). The triple mutant K103R + V179D + P225H (EFVR) was
derived from an IIIB strain passaged in MT-4 cells in the presence
of efavirenz (up to 2 lM). N119, A17, and EFVR stock solutions
had titers of 1.26108, 2.16107, and 4.06107 CCID50/mL, respec-
tively.
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HIV titration: Titration of HIV was performed in C8166 cells by
the standard limiting dilution method (dilution 1 : 2, four rep-
lica wells per dilution) in 96-well plates. The infectious-virus titer
was determined by light microscope scoring of syncytia after
four days of incubation. Virus titers were expressed as CCID50/
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[18] M. Wamberg, E. B. Pedersen, N. R. El-Brollosy, C. Nielsen,
Anti-HIV assays
Bioorg. Med. Chem. 2004, 12, 1141–1149.
The activity of test compounds against multiplication of HIV-1
wild type IIIB, N119, A17, and EFVR in acutely infected cells, was
based on inhibition of virus-induced cytopathogenicity in MT-4
cells. Briefly, an amount of 50 lL of culture medium containing
26104 cells was added to each well of flat-bottom microtiter
trays containing 50 lL of culture medium with or without vari-
ous concentrations of test compounds. Then, an amount of
20 lL of HIV suspensions, containing the appropriate amount of
CCID50 to cause complete cytopathogenicity at day 4, was added.
After incubation at 378C, cell viability was determined by the 3-
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bromide
(MTT) method [31]. The cytotoxicity of test compounds was eval-
uated in parallel with their antiviral activity and was based on
the viability of mock-infected cells, as monitored by the MTT
method.
[24] N. R. El-Brollosy, E. R. Sorensen, E. B. Pedersen, G. Sanna, et
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The financial support of the Research Center, College of Pharmacy,
King Saud University (Project # C.P.R.C.226), is greatly appreciated.
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51, 426–430.
The authors have declared no conflict of interest.
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i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com