Synthesis and Antiviral Evaluation of 1-[(2-Phenoxyethyl)oxymethyl] and 6-(3,5-Dimethoxybenzyl) Analogues of HIV Drugs Emivirine and TNK-651

Article abstract of DOI:10.1055/s-0035-1559683

Novel emivirine analogues 6a, b were synthesized by reacting chloromethyl ethyl ether with 5-ethyl/isopropyl-6-(3,5-dimethoxybenzyl)uracils 5e, f. On the other hand, A series of new TNK-651 analogues 10a-f substituted at N-1 with phenoxyethoxymethyl moiety was prepared on treatment of the corresponding uracils 5a-f with bis(phenoxyethoxy)methane (9). The newly synthesized non-nucleosides were tested for antiviral activity against wild type HIV-1 IIIB as well as the resistant strains N119 (Y181C), A17 (K103N+Y181C), and the triple mutant EFV^R (K103R+V179D+P225H) in MT-4 cells. Most of the tested compounds showed good activities. Among them 6-(3,5-dimethylbenzyl)-5-ethyl-1-[(2-phenoxyethyl)oxymethyl]uracil (10c) and 6-(3,5-dimethylbenzyl)-5-isopropyl-1-[(2-phenoxyethyl)oxymethyl]uracil (10d) that showed inhibitory potency higher than emivirine against both wild type HIV-1 and the tested mutant strains, as well as higher activity than efavirenz against EFV^R.

Full text of DOI:10.1055/s-0035-1559683

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
Synthesis and Antiviral Evaluation of 1-[(2-Phenoxyethyl)
oxymethyl] and 6-(3,5-Dimethoxybenzyl) Analogues of
HIV Drugs Emivirine and TNK-651
Authors
N. R. El-Brollosy1, R. Loddo2
Affiliations
1 Faculty of Science, Heterocyclic Research Unit, Department of Chemistry, Tanta University, Tanta, Egypt
2 Dipartimento di Scienze Biomediche, Sezione di Microbiologia e Virologia, Università di Cagliari, Cittadella Universitaria,
Monserrato (Cagliari), Italy
Key words
the resistant strains N119 (Y181C), A17
(K103N+Y181C), and the triple mutant EFVR
(K103R+V179D+P225H) in MT-4 cells. Most of
the tested compounds showed good activities.
Among them 6-(3,5-dimethylbenzyl)-5-ethyl-
1-[(2-phenoxyethyl)oxymethyl]uracil (10c) and
6-(3,5-dimethylbenzyl)-5-isopropyl-1-[(2-phe-
noxyethyl)oxymethyl]uracil (10d) that showed
inhibitory potency higher than emivirine against
both wild type HIV-1 and the tested mutant
strains, as well as higher activity than efavirenz
against EFVR.
Abstract
▼
▶
HIV-1
emivirine
TNK-651
non-nucleosides
reverse transcriptase
inhibitors
●
▶
●
Novel emivirine analogues 6a, b were synthe-
sized by reacting chloromethyl ethyl ether with
5-ethyl/isopropyl-6-(3,5-dimethoxybenzyl)ura-
cils 5e, f. On the other hand, A series of new TNK-
651 analogues 10a–f substituted at N-1 with
phenoxyethoxymethyl moiety was prepared on
treatment of the corresponding uracils 5a–f with
bis(phenoxyethoxy)methane (9). The newly syn-
thesized non-nucleosides were tested for antivi-
ral activity against wild type HIV-1 IIIB as well as
▶
●
▶
●
▶
●
▶
●
uracils
Introduction
to stop the attachment of further nucleosides
and so prevent an ongoing viral DNA synthesis;
and non-nucleoside reverse transcriptase inhibi-
tors (NNRTIs), which in contrast to NRTIs, are
highly specific as their binding site is a hydro-
phobic cavity located approximately 10Å far
from the polymerase active site. The formation of
RT–NNRTI complex results in short- and long-
range conformational changes that lock the
enzyme in an inactive form [6–8]. One of the first
▼
Acquired immunodeficiency syndrome (AIDS)
caused by human immunodeficiency virus (HIV)
is considered a pandemic disease, that is present
over a large area and is actively spreading. Since
its discovery, AIDS which is characterized by the
loss of helper T-lymphocytes and heavy damage
to the lymphatic tissues, has caused an estimated
36 million deaths worldwide and approximately
35.3 million people are living with HIV [1]. A
number of therapeutic agents with various
mechanisms of action have been developed to
combat HIV infection and replication, however,
clinical treatment of the disease often leads to
the emergence of drug-resistant strains due to
the rapid mutability of the HIV virus [2]. One of
the most promising anti-HIV agents is reverse
transcriptase inhibitors (RTIs). Reverse tran-
scriptase (RT) is a key enzyme in the HIV replica-
tion cycle and is one of the main targets in the
development of drugs for treating AIDS. Due to
its essential role in HIV-1 life cycle, RT was iden-
tified as a privileged target for anti-AIDS highly
active anti-retroviral therapy (HAART) [3–5]. 2
types of reverse transcriptase inhibitors have
been developed: nucleoside reverse transcriptase
inhibitors (NRTIs) which act as chain terminators
received 01.07.2015
accepted 31.07.2015
NNRTIs was 1-[(2-hydroxyethoxy)methyl]-6-
▶ꢀ
(phenylthio)thymine (HEPT) [9] ( Fig. 1) which
●
Bibliography
DOI http://dx.doi.org/
10.1055/s-0035-1559683
Published online: 2015
Drug Res
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 2194-9379
was considered an interesting lead compound for
the synthesis of new active analogues. Among
them the 6-benzyl-1-(ethoxymethyl)-5-isopro-
▶ꢀ
pyluracil (Emivirine) [10] ( Fig. 1), the corre-
●
sponding 1-benzyloxymethyl analogues (TNK-651)
▶ꢀ
[11] ( Fig. 1) and 6-(3,5-dimethylbenzyl)-1-
●
ethoxymethyl-5-isopropyluracil (GCA-186) [12]
▶ꢀ
(
Fig. 1), which all showed high activity against
●
Correspondence
N. R. El-Brollosy
Department of Chemistry,
Faculty of Science
Tanta University
31527, Tanta
Egypt
Tel.: +20/403/344352 518
Fax: +20/403/350 804
brollosy@science.tanta.edu.eg
HIV-1.
GCA-186 differs structurally from emivirine only
in the introduction of 2 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 2 methyl substituents in 2 hydro-
El-Brollosy NR, Loddo R. Novel Analogues of Emivirine and TNK-651… Drug Res

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
Fig. 1 Structures of HEPT, Emivirine, TNK-651
and GCA-186.
O
N
O
N
R1
HN
O
HN
O
S
O
R1
HO
O
R
Emivirine; R=CH , R1=H
TNK-651; R=Ph, R1=H
3
HEPT
GCA-186; R=R1=CH
3
phobic pockets in the NNRTI-binding site of RT [12]. In an
attempt to enhance the activity against HIV-1 wild type and
NNRTI resistant mutants, and as a part of our interest in the
chemistry of NNRTIs [13–23], the present study describes syn-
thesis and antiviral evaluation of novel emivirine analogues
with 3- and 5-dimethoxy substituents at the benzyl ring. Also, a
series of TNK-651 analogues substituted at N-1 with phenoxy-
ethoxymethyl moiety has been investigated. The objective was
to investigate if these substituents 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.
wise. After the addition was completed, the mixture was
refluxed for 30min, it was diluted with THF (150mL) and
quenched by addition of sat. aq. K2CO3 (60mL). The mixture was
stirred for 1h at room temperature. The THF layer was decanted
and the residue was washed with THF (3×30mL). The combined
THF fractions were stirred with 10% aq. HCl (40mL) for 30min.
The solution was concentrated under reduced pressure and
diluted with CH2Cl2 (100mL). The organic phase was washed
with sat. aq. NaHCO3 (2×60mL), dried (Na2SO4), and evaporated
under reduced pressure to give the product that was used for
further synthesis without purification.
Ethyl 4-(3,5-dimethoxyphenyl)-2-ethyl-3-oxobutyrate (3e)
Yellow oil; yield 8.46g (96%). 1H NMR (CDCl3): δ=0.86 (t, 3H,
J=7.0Hz, CH3), 1.27 (t, 3H, J=7.5Hz, CH3), 1.87–1.89 (m, 2H,
CH2), 3.49 (t, 1H, J=7.5Hz, CH), 3.76 (s, 2H, CH2), 3.86 (s, 6H,
2×OCH3), 4.17 (q, 2H, J=7.0Hz, CH2), 6.39 (s, 2H, Harom.), 6.48 (s,
1H, Harom.); 13C NMR (CDCl3): δ=13.57 (CH3), 14.08 (CH3), 21.54
(CH2), 43.77 (CH2), 55.63 (OCH3), 60.73 (CH2), 61.33 (CH), 99.25,
107.70, 135.37, 160.97 (Carom.), 169.63 (CO), 202.51 (CO).
Materials and Methods
▼
Melting points (°C) were measured in open glass capillaries
using a Branstead 9100 Electrothermal melting point apparatus
and are uncorrected. NMR spectra were obtained on a Bruker AC
500 Ultra Shield NMR spectrometer operating at 500.13MHz for
1H and 125.76MHz for 13C, the chemical shifts are expressed in
δ (ppm) downfield from tetramethylsilane (TMS) as internal
standard; coupling constants (J) are expressed in Hz and signals
are expressed as s (singlet), d (doublet), t (triplet), q (quartet), or
m (multiplet). Electrospray ionization mass spectra (ESI-MS)
were recorded on an Agilent 6410 Triple Quad tandem mass
spectrometer at 4.0kV for the positive ions. MALDI spectra were
recorded on a Fourier Transform (FT) Ion Cyclotron Resonance
Mass Spectrometer (IonSpec Corporation). Elemental analyses
(C, H, N) were conducted using the Elemental Analyser Vario
ELIII. The progress of reactions was monitored by TLC (DC-alufo-
lio 60 F254) from Merck, and visualization with ultraviolet light
(UV) at 365 and 254nm. For column chromatography Merck sil-
ica gel (0.040–0.063mm) was used.
Ethyl 4-(3,5-dimethoxyphenyl)-2-isopropyl-3-
oxobutyrate (3f)
Yellow oil; yield 8.65g (94%). 1H NMR (CDCl3): δ=0.83–0.85 (m,
9H, 3×CH3), 2.33–2.35 (m, 1H, CH), 3.25 (d, 1H, J=7.0Hz, CH),
3.48 (s, 2H, CH2), 3.65 (s, 6H, 2×OCH3), 4.02 (q, 2H, J=7.0Hz,
CH2), 6.25 (s, 2H, Harom.), 6.26 (s, 1H, Harom.); 13C NMR (CDCl3):
δ=13.94 (CH3), 20.27 (CH3), 20.44 (CH3), 28.32 (CH), 43.27 (CH2),
55.04 (OCH3), 61.02 (CH2), 65.97 (CH), 99.01, 107.65, 135.24,
160.85 (Carom.), 168.74 (CO), 201.86 (CO).
5-Alkyl-6-(3,5-dimethoxybenzyl)-2-thiouracils 4e and 4f
Na (9.98g, 0.434mol) was dissolved in absolute EtOH (150mL).
Thiourea (22.84g, 0.3mol) was added and the mixture was
heated to reflux. Compound 3e,f (0.02mol) was added dropwise
and the mixture was refluxed for overnight. The solvent was
evaporated under reduced pressure to dryness and the residue
was re-dissolved in H2O (150mL). The product was precipitated
by addition of conc. HCl (16mL) and then glacial acetic acid till
pH=4. The precipitate was filtered off, washed with H2O, dried
and crystallized from aq. EtOH.
Ethyl 2-alkyl-4-(3,5-dimethoxyphenyl)-3-oxobutyrates
3e and 3f
Zinc dust (14g) was activated by stirring with 4M HCl (30mL)
for 5min. The zinc dust was filtered, washed sequentially with
H2O, EtOH and dry Et2O then dried. The active zinc was sus-
pended in dry THF (60mL) and heated to reflux. Few drops of
ethyl 2-bromobutyrate (2a) or ethyl 2-bromoisovalerate (2b)
was added and the mixture was refluxed for 10min. 3,5-Dimeth-
oxyphenylacetonitrile (1c, 5.32g, 0.03mol) was added in one
portion and the bromoester 2a/2b (0.06mol) was added drop-
6-(3,5-Dimethoxybenzyl)-5-ethyl-2-thiouracil (4e)
White solid, yield 3.84g (63%); mp 189–191°C. 1H NMR (DMSO-
d6): δ=0.86 (t, 3H, J=7.0Hz, CH3), 2.30 (q, 2H, J=7.0Hz, CH2),
El-Brollosy NR, Loddo R. Novel Analogues of Emivirine and TNK-651… Drug Res

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
3.72 (s, 6H, 2×OCH3), 3.77 (s, 2H, CH2), 6.41, 6.42 (2×s, 3H,
Harom.), 12.17 (s, 1H, NH), 12.38 (s, 1H, NH); 13C NMR (DMSO-d6):
δ=13.04 (CH3), 17.36 (CH2), 34.57 (CH2), 55.10 (OCH3), 98.15,
106.49, 138.81, 160.41 (Carom.), 117.07 (C-5), 148.90 (C-6),
161.36 (C-4), 174.13 (C-2); ms (ESI) m/z (%): 307 (M+H+ , 89).
3,5-(Dimethoxybenzyl)-5-ethyl-1-ethyloxymethyluracil
(6a)
White solid, yield 148mg (43%); mp 144–145°C. 1H NMR
(CDCl3): δ=1.09 (t, 3H, J=7.5Hz, CH3), 1.20 (t, 3H, J=7.0Hz, CH3),
2.48 (q, 2H, J=7.5Hz, CH2), 3.62 (q, 2H, J=7.0Hz, CH2), 3.79 (s,
6H, 2×OCH3), 4.11 (s, 2H, CH2), 5.14 (s, 2H, CH2), 6.28 (s, 2H,
Harom.), 6.37 (s, 1H, Harom.), 9.34 (s, 1H, NH); 13C NMR (CDCl3):
δ=13.81 (CH3), 15.09 (CH3), 19.20 (CH2), 33.52 (CH2), 55.37
(OCH3), 65.05 (CH2), 72.77, CH2), 98.59, 105.65, 137.59, 161.46
(Carom.), 116.96 (C-5), 148.90 (C-6), 151.83 (C-2), 163.23 (C-4);
ms (ESI) m/z (%): 349 (M+H+ , 81).
6-(3,5-Dimethoxybenzyl)-5-isopropyl-2-thiouracil (4f)
White solid, yield 3.95g (62%); mp 231–233°C. 1H NMR (DMSO-
d6): δ=1.11 (d, 6H, J=7.0Hz, 2×CH3), 2.84 (h, 1H, J=7.0Hz, CH),
3.72 (s, 6H, OCH3), 3.80 (s, 2H, CH2), 6.40, 6.41 (2×s, 3H, Harom.),
12.12 (s, 1H, NH), 12.29 (s, 2H, NH); 13C NMR (DMSO-d6):
δ=19.58 (CH3), 26.58 (CH), 34.65 (CH2), 56.00 (OCH3), 98.20,
106.28, 138.90, 160.52 (Cacom.), 119.54 (C-5), 148.60 (C-6),
160.57 (C-4), 174.06 (C-2); ms (ESI) m/z (%): 321 (M+H+ , 76).
Anal. Calcd. For C18H24N2O5 (348.39): C 62.05, H 6.94, N 8.04%.
Found C 61.98, H 6.90, N 8.13%.
3,5-(Dimethoxybenzyl)l-1-ethyloxymethyl-5-
5-Alkyl-6-(3,5-dimethoxybenzyl)uracils 5e and 5f
Compound 4e,f (2g) was suspended in 10% aq. ClCH2CO2H
(200mL). The suspension was heated under reflux for overnight
and filtered after cooling. The precipitate thus formed was
washed with H2O, cold EtOH then Et2O, dried and crystallized
from EtOH.
isopropyluracil (6b)
White solid, yield 142mg (39%); mp 110–111°C. 1H NMR
(CDCl3): δ=1.21 (t, 3H, J=7.0Hz, CH3), 1.33 (d, 6H, J=7.0Hz,
2 × CH3), 2.88 (h, 1H, J=7.0Hz, CH), 3.63 (q, 2H, J=7.0Hz, CH2),
3.80 (s, 6H, 2×OCH3), 4.13 (s, 2H, CH2), 5.15 (s, 2H, CH2), 6.29 (s,
2H, Harom.), 6.38 (s, 1H, Harom.), 9.05 (s, 1H, NH); 13C NMR (CDCl3):
δ=15.10 (CH3), 20.47 (CH3), 28.39 (CH), 33.59(CH2), 55.38
(OCH3), 65.06 (CH2), 70.92 (CH2), 98.74, 105.56, 137.68, 161.46
(Carom.), 119.76 (C-5), 148.35 (C-6), 151.78 (C-2), 162.26 (C-4);
ms (MALDI) m/z (%): 385 (M+Na+ , 79).
6-(3,5-Dimethoxybenzyl)-5-ethyluracil (5e)
White solid, yield 1.68g (89%); mp 232–234°C. 1H NMR (DMSO-
d6): δ=0.86 (t, 3H, J=7.5Hz, CH3), 2.27 (q, 2H, J=7.5Hz, CH2),
3.67 (s, 2H, CH2), 3.73 (s, 6H, 2×OCH3), 6.40 (s, 1H, Harom.), 6.44
(s, 2H, Harom.), 10.67 (s, 1H, NH), 10.98 (s, 1H, NH); 13C NMR
(DMSO-d6): δ=13.46 (CH3), 17.59 (CH2), 35.08 (CH2), 55.09
(OCH3), 98.20, 106.47, 138.96, 160.52 (Carom.), 111.29 (C-5),
148.35 (C-6), 150.84 (C-2), 164.40 (C-4); ms (ESI) m/z (%): 291
(M+H+ , 95).
Anal. Calcd. for C19H26N 2NaO5: 385.1734. Found: 385.1740.
1,3-Bis-(ethoxymethyloxy)-6-(3,5-dimethoxybenzyl)-5-
ethyluracil (7a)
Viscous oil, yield 64mg (16%). H NMR (CDCl3): δ=1.08 (t, 3H,
1
J=7.0Hz, CH3), 1.18 (t, 3H, J=7.0Hz, CH3), 1.23 (t, 3H, J=7.0Hz,
CH3), 2.49 (q, 2H, J=7.0Hz, CH2), 3.61 (q, 2H, J=7.0Hz, CH2), 3.71
(q, 2H, J=7.0Hz, CH2), 3.78 (s, 6H, 2×OCH3), 4.09 (s, 2H, CH2),
5.16 (s, 2H, CH2), 5.48 (s, 2H, CH2), 6.28 (s, 2H, Harom.), 6.37 (s, 1H,
Harom.); 13C NMR (CDCl3): δ=13.76 (CH3), 15.09 (CH3), 15.23
(CH3), 19.76 (CH2), 33.58 (CH2), 55.36 (2×OCH3), 65.12 (CH2),
65.95 (CH2), 71.18 (CH2), 73.54 (CH2), 98.48, 105.71, 137.64,
161.45 (Carom.), 116.27 (C-5), 147.58 (C-6), 152.58 (C-2), 162.75
(C-4); ms (ESI) m/z (%): 407 (M+H+ , 69).
Anal. Calcd. For C15H18N2O4 (290.31): C 62.06, H 6.25, N 9.65%.
Found C 62.12, H 6.21, N 9.58%.
6-(3,5-Dimethoxybenzyl)-5-isopropyluracil (5f)
White solid, yield 1.65g (87%); mp 264–266°C. 1H NMR (DMSO-
d6): δ=1.11 (d, 6H, J=7.0Hz, 2×CH3), 3.33 (h, 1H, J=7.0Hz, CH),
3.69 (s, 2H, CH2), 3.72 (s, 6H, 2×OCH3), 6.39 (s, 1H, Harom.), 6.43
(s, 2H, Harom.), 10.63 (s, 1H, NH), 10.87 (s, 1H, NH); 13C NMR
(DMSO-d6): δ=20.09 (CH3), 26.39 (CH), 35.25 (CH2), 55.08
(OCH3), 98.24, 106.24, 139.07, 160.49 (Carom.), 113.89 (C-5),
147.99 (C-6), 150.83 (C-2), 163.70 (C-4); ms (ESI) m/z (%): 305
(M+H+ , 91).
1,3-Bis-(ethoxymethyloxy)-6-(3,5-dimethoxybenzyl)-5-
isopropyluracil (7b)
Viscous oil, yield 88mg (21%). 1H NMR (CDCl3): δ=1.21–1.26 (m,
6H, CH3), 1.32 (d, 6H, J=7.0Hz, 2×CH3), 2.88–2.89 (m 1H, CH),
3.63 (q, 2H, J=7.0Hz, CH2), 3.65 (q, 2H, J=7.5Hz, CH2), 3.78 (s,
6H, 2×OCH3), 4.12 (s, 2H, CH2), 5.16 (s, 2H, CH2), 5.45 (s, 2H,
CH2), 6.28 (s, 2H, Harom.), 6.37 (s, 1H, Harom.); 13C NMR (CDCl3):
δ=15.09 (CH3), 15.35 (CH3), 20.47 (CH3), 28.61 (CH), 33.66 (CH2),
55.37 (OCH3), 65.12 (CH2), 65.93 (CH2), 70.89 (CH2), 73.71 (CH2),
98.63, 105.61, 137.73, 161.44 (Carom.), 119.06 (C-5), 147.09 (C-6),
152.56 (C-2), 161.68 (C-4); ms (ESI) m/z (%): 421 (M+H+ , 73).
Anal. Calcd. For C16H20N2O4 (304.34): C 63.14, H 6.62, N 9.20%.
Found C 62.97, H 6.60, N 9.11%.
1-Ethyloxymethyl and 1,3-bis-(ethyloxymethyl)uracils
(6a,b and 7a,b)
N,O-Bis-(trimethylsilyl)acetamide (BSA) (0.87mL, 0.0035mol)
was added to a suspension of 5e,f (0.001mol) in anhydrous
CHCl3 (20mL) and the mixture was stirred at room temperature
under nitrogen. After a clear solution was obtained (20min),
chloromethyl ethyl ether (0.14g, 0.0015mol) followed by CsI
(0.26g, 0.001mol) were added. The reaction mixture was stirred
at room temperature under nitrogen for 4h. Sat. aq. NaHCO3
(20mL) was added and the mixture was extracted with CH2Cl2
(3×50mL). The organic phase was collected, dried (MgSO4) and
evaporated under reduced pressure. The residue was chromato-
graphed on silica gel column using CHCl3 to give 6a,b and 7a,b.
Bis-[(2-phenoxyethyl)oxy]methane (9)
2-Phenoxyethanol (8, 13.8g, 0.1mol), dibromomethane (8.79g,
0.0505mol), and tetrabutylammonium bromide (1.74g,
0.00535mol) were added to anhydrous benzene (30mL) con-
taining potassium hydroxide (5.66g, 0.101mol) and the suspen-
sion was heated under reflux for 5h. After cooling, H2O (50mL)
was added and the resulting solution was extracted with ether
(3×50mL). The ether phase was dried with anhydrous MgSO4
and evaporated under reduced pressure. The residue was
El-Brollosy NR, Loddo R. Novel Analogues of Emivirine and TNK-651… Drug Res

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
chromatographed on a silica gel column using pet. ether/CHCl3
(1:1) to afford 8.52g (59%) of 9 as a colourless oil.
1H NMR (CDCl3): δ=3.98 (t, 4H, J=4.5Hz, 2×CH2), 4.18 (t, 4H,
J=4.5Hz, 2×CH2), 4.91 (s, 2H, CH2), 6.96–7.34 (m, 10H, Harom.);
13C NMR (CDCl3): δ=66.31 (2×CH2), 67.23 (2×CH2), 95.75 (CH2),
114.69, 120.97, 129.47, 158.78 (Carom.).
6-(3,5-Dimethylbenzyl)-5-isopropyl-1-[(2-
phenoxyethyl)oxymethyl]uracil (10d)
Viscous oil; yield 218mg (52%). 1H NMR (CDCl3): δ=1.32 (d, 6H,
J=7.0Hz, 2×CH3), 2.30 (s, 6H, 2×CH3), 2.87–2.88 (m, 1H, CH),
4.01 (t, 2H, J=4.5Hz, CH2), 4.12 (s, 2H, CH2), 4.15 (t, 2H, J=4.5Hz,
CH2), 5.26 (s, 2H, CH2), 6.72–7.31 (m, 8H, Harom.), 9.51 (s, 1H,
NH); 13C NMR (CDCl3): δ=20.42 (CH3), 21.27 (CH3), 28.39 (CH),
66.82 (CH2), 68.09 (CH2), 73.35 (CH2), 119.74 (C-5), 114.63,
121.06, 125.04, 128.87, 129.50, 135.00,138.84, 158.57 (Carom.),
148.89 (C-6), 152.18 (C-2), 162.56 (C-4); ms (MALDI) m/z
(%)=445 (M+Na+ , 89). Anal. calcd for C25H30N2NaO4: 445.2098.
Found: 445.2103.
5-Alkyl-1-[(2-phenoxyethyl)oxymethyl]-6-substituted-
uracils (10a-f)
General Procedure.
Compound 5a–f (1mmol) was stirred in anhydrous CH3CN
(15mL) under nitrogen and BSA (0.87mL, 3.5mol) was added.
After a clear solution was obtained (10min), the reaction mix-
ture was cooled to −50°C and TMS triflate (0.18mL, 1mmol)
was added followed by dropwise addition of bis-[2-
(phenoxyethyl)oxy]methane (9) (576mg, 2mmol). The mixture
was stirred at room temperature for 3–4h. The reaction was
quenched with sat. aq. NaHCO3 solution (5mL) and evaporated
under reduced pressure. The residue was extracted with ether
(3×50mL), the combined organic fractions were dried (MgSO4)
and evaporated under reduced pressure. The residue was chro-
matographed on silica gel column with CHCl3 to afford the cor-
responding non-nucleosides 10a–f.
6-(3,5-Dimethoxybenzyl)-5-ethyl-1-[(2-phenoxyethyl)
oxymethyl]uracil (10e)
White solid; yield 269mg (61%); m.p. 136–137°C. 1H NMR
(CDCl3, 500MHz): δ=1.08 (t, 3H, J=7.0Hz, CH3), 2.48 (q, 2H,
J=7.0Hz, CH2), 3.78 (s, 6H, 2×OCH3), 3.99 (t, 2H, J=4.5Hz, CH2),
4.10–4.12 (m, 4H, 2×CH2), 5.25 (s, 2H, CH2), 6.27 (s, 2H, Harom.),
6.38 (s, 1H, Harom.), 6.90–7.31 (m, 8H, Harom.), 9.46 (s, 1H, NH). 13
C
NMR (CDCl3, 125MHz): δ=13.78 (CH3), 19.21 (CH2), 33.47 (CH2),
55.36 (OCH3), 66.82 (CH2), 68.11 (CH2), 73.18 (CH2), 117.11
(C-5), 98.68, 105.65, 114.61, 121.07, 129.50, 137.50, 158.56,
161.47 (Carom.), 148.85 (C-6), 151.99 (C-2), 163.23 (C-4); ms (ESI)
m/z (%): 441 (M+H+ , 78).
6-Benzyl-5-ethyl-1-[(2-phenoxyethyl)oxymethyl]uracil
(10a)
Anal. Calcd. For C24H28N2O6 (440.49): C 65.44, H 6.41, N 6.36%.
Found C 65.57, H 6.38, N 6.47%.
Viscous oil; yield 181mg (48%). 1H NMR (CDCl3): δ=1.07 (t, 3H,
J=7.5Hz, CH3), 2.48 (q, 2H, J=7.5Hz, CH2), 3.99 (t, 2H, J=4.5Hz,
CH2), 4.10 (t, 2H, J=4.5Hz, CH2), 4.17 (s, 2H, CH2), 5.24 (s, 2H,
CH2), 6.89–7.36 (m, 10H, Harom.), 9.56 (s, 1H, NH); 13C NMR
(CDCl3): δ=13.71 (CH3), 19.21 (CH2), 33.40 (CH2), 66.87 (CH2),
68.15 (CH2), 73.20 (CH2) 117.07 (C-5), 114.66, 121.08, 127.32,
127.35, 129.24, 129.48, 135.22, 158.58 (Carom.), 149.08 (C-6),
152.05 (C-2), 163.24 (C-4); MS (MALDI) m/z (%)=403 (M+Na+ ,
97).
6-(3,5-Dimethoxybenzyl)-5-isopropyl-1-[(2-
phenoxyethyl)oxymethyl]uracil (10f)
White solid; yield 267mg (59%); mp 107–108°C. 1H NMR
(CDCl3): δ=1.33 (d, 6H, J=7.0Hz, 2×CH3), 2.88–2.89 (m, 2H, CH),
3.78 (s, 6H, 2×OCH3), 4.01 (t, 2H, J=4.5Hz, CH2), 4.12–4.13 (m,
4H, 2×CH2), 5.25 (s, 2H, CH2), 6.27 (s, 2H, Harom.), 6.38(s, 1H,
Harom), 6.90–7.30 (m, 5H, Harom.), 9.11 (s, 1H, NH); 13C NMR
(CDCl3): δ=20.46 (CH3), 28.40 (CH), 33.54 (CH2), 55.37 (OCH3),
66.84 (CH2), 68.14 (CH2), 73.35 (CH2), 119.91 (C-5), 98.82,
105.55, 114.62, 121.07, 129.50, 137.57, 158.56, 161.46 (Carom.),
148.34 (C-6), 151.92 (C-2), 161.24 (C-4); ms (ESI) m/z (%): 455
(M+H+ , 91).
Anal. Calcd for C22H24N2NaO4: 403.1628. Found: 403.1635.
6-Benzyl-5-isopropyl-1-[(2-phenoxyethyl)oxymethyl]
uracil (10b)
Viscous oil; yield 233mg (59%). 1H NMR (CDCl3): δ=1.30 (d, 6H,
J=7.0Hz, 2×CH3), 2.87–2.88 (m, 1H, CH), 4.00 (t, 2H, J=4.5Hz,
CH2), 4.11 (s, 2H, CH2), 4.20 (t, 2H, J=4.5Hz, CH2), 5.25 (s, 2H,
CH2), 6.90–7.35 (m, 10H, Harom.), 9.11 (s, 1H, NH); 13C NMR
(CDCl3): δ=20.41 (CH3), 28.38 (CH), 33.47 (CH2), 66.84 (CH2),
68.14 (CH2), 73.65 (CH2), 119.86 (C-5), 114.62, 121.08, 127.26,
127.30, 129.22, 129.50, 135.32, 158.55 (Carom.), 148.55 (C-6),
151.98 (C-2), 162.24 (C-4); ms (MALDI) m/z (%)=417 (M+Na+ ,
67).
Anal. Calcd. For C25H30N2O6 (454.52): C 66.06, H 6.65, N 6.16%.
Found C 66.18, H 6.62, N 6.28%.
Antiviral Assay Procedures
▼
Compounds were solubilized in DMSO at 100mM and then
diluted in culture medium.
Anal. Calcd for C23H26N2NaO4: 417.1785. Found: 417.1779.
Viruses and cells
6-(3,5-Dimethylbenzyl)-5-ethyl-1-[(2-phenoxyethyl)
oxymethyl]uracil (10c)
MT-4, C8166, and H9/IIIB cells were grown at 37°C in a 5% CO2
atmosphere in RPMI 1640 medium supplemented with 10%
fetal calf serum (FCS), 100IU/mL penicillin G, and 100μg/ml
streptomycin. Cell cultures were checked periodically for the
absence of mycoplasma contamination with a MycoTect Kit
(Gibco). Human immunodeficiency 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 4.5×106 50%
cell culture infectious dose (CCID50)/mL. The Y181C mutant (NIH
N119) was derived from an AZT-sensitive clinical isolate pas-
saged initially in CEM and then in MT-4 cells in the presence of
nevirapine (10μM). The double mutant K103N+Y181C (NIH
White solid; yield 258mg (63%); mp 137–138°C. 1H NMR
(CDCl3): δ=1.08 (t, 3H, J=7.5Hz, CH3), 2.29 (s, 6H, 2×CH3), 2.48
(q, 2H, J=7.5Hz, CH2), 3.99 (t, 2H, J=4.5Hz, CH2), 4.10 (s, 2H,
CH2), 4.11 (t, 2H, J=4.5Hz, CH2), 5.25 (s, 2H, CH2), 6.71–7.32 (m,
8H, Harom.), 9.73 (s, 1H, NH); 13C NMR (CDCl3): δ=13.73 (CH3),
19.21 (CH2), 21.26 (CH3), 33.22 (CH2), 66.87 (CH2), 68.10 (CH2),
73.19 (CH2), 116.95 (C-5), 114.66, 121.07, 125.06, 128.96,
129.48, 134.97, 138.88, 158.61 (Carom.), 149.37 (C-6), 152.17
(C-2), 163.43 (C-4); ms (MALDI) m/z (%)=431 (M+Na+ , 97).
Anal. Calcd for C24H28N2NaO4: 431.1941. Found: 431.1952.
El-Brollosy NR, Loddo R. Novel Analogues of Emivirine and TNK-651… Drug Res

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
R
Br
X
i- Zn/THF
CN
+
X
O
O
R
ii- K2CO3
iii- HCl
O
O
O
X
X
1
2
3
O
X
O
X
R
i- H2NCSNH2 / NaOEt
ClCH2COOH
H2O
HN
R
HN
ii- HCl
O
N
H
X
N
H
S
X
4
5
O
O
OCH3
OCH3
R
R
i- BSA / CHCl3
O
N
HN
O
+
OCH3
O
OCH3
N
O
N
ii-
O
Cl
O
CsI
6
7
3, 4, 5
2
1
6, 7
X
R
X
R
R
a
H
a
Et
a
b
c
d
e
f
H
Et
a
Et
Pri
Pri
Pri
Et
CH3
OCH3
b
c
b
H
b
CH3
CH3
OCH3
OCH3
Pri
Et
Pri
Fig. 2 Synthesis of compounds 3–7.
A17) was derived from the IIIB strain passaged in H9 cells in the
presence of BI-RG 587 (1μM). 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 2μM).
N119, A17 and EFVR, stock solutions had titers of 1.2×108,
2.1×107 and 4.0×107 CCID50/mL, respectively.
ous concentrations of test compounds. Then, an amount of 20μL
of HIV suspensions, containing the appropriate amount of CCID50
to cause complete cytopathogenicity at day 4, was added. After
incubation at 37°C, cell viability was determined by the
3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide
(MTT) method [24]. The cytotoxicity of test compounds was
evaluated in parallel with their antiviral activity and was based
on the viability of mock-infected cells, as monitored by the MTT
method.
HIV titration
Titration of HIV was performed in C8166 cells by the standard
limiting dilution method (dilution 1:2, 4 replica wells per dilu-
tion) in 96-well plates. The infectious virus titer was determined
by light microscope scoring of syncytia after 4 days of incuba-
tion. Virus titers were expressed as CCID50/mL.
Results and Discussion
▼
Chemistry
Anti-HIV assays
Substituted acetonitriles, namely Phenylacetonitrile (1a),
3,5-dimethylphenylacetonitrile (1b), and/or 3,5-dimethoxyphe-
nylacetonitrile (1c) were reacted with the zinc organometallic
reagents prepared from ethyl 2-bromobutyrate (2a) and/or ethyl
2-bromoisovalerate (2b) in anhydrous THF to afford the corre-
sponding 4-aryl-2-ethyl-3-oxo esters 3a–f in approximately
quantitative yields. Treatment of compounds 3a–f with thiourea
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 50μL of culture medium containing
1×104 cells was added to each well of flat-bottom microtiter
trays containing 50μL of culture medium with or without vari-
El-Brollosy NR, Loddo R. Novel Analogues of Emivirine and TNK-651… Drug Res

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
O
O
O
OH
CH2Br2 , Bu4NF
KOH , C6H6
O
O
8
9
X
O
N
O
X
i- BSA / MeCN
R
R
HN
O
HN
ii- 9 / TMS triflate
O
X
N
H
O
X
O
5a-f
10
X
R
a
H
Et
Pri
Et
b
c
d
e
f
H
CH3
CH3
OCH3
OCH3
Pri
Et
Pri
Fig. 3 Synthesis of compounds 9 and 10a–f.
in boiling ethanol in the presence of sodium ethoxide followed
by acidification with HCl [25] gave the corresponding 2-thioura-
cils 4a–f in good yields. The NMR spectra of crude compounds
3a–f showed impurities identified as other β-keto esters result-
ing from self-condensation of 2-bromoesters. On reaction with
thiourea, these β-keto esters impurities also formed pyrimidines
as impurities in the raw materials of 4a–f. However, pure com-
pounds 4a–f were easily obtained by recrystallization from
aqueous ethanol [26,27]. The desired uracil stating materials
5a–f were achieved, in good yields, by desulfurization of the
corresponding thiouracils 4a–f with boiling aqueous chloro-
acetic acid. Emivirine analogues 6a, b, that were also regarded
as isosteric analogues of GCA-186 in which the methyl groups
at 3- and 5- positions of the benzyl moiety were replaced
with methoxy groups, have been synthesized. Silylation of
6-(3,5-dimethoxybenzyl)-5-ethyluracil (5e) and/or 6-(3,5-
anesulfonate (TMS triflate) as a Lewis acid catalyst [29], to give
the corresponding N-1 phenoxyethyloxymethyluracils 10a–f in
▶ꢀ
48–63% yields ( Fig. 3).
●
The newly synthesized compounds were confirmed by 1H-NMR,
13C-NMR and mass spectral data. HRMS and elemental analyses
are in full agreement with the proposed structures. The single
crystal X-ray structure of 6-(3,5-dimethylbenzyl)-5-ethyl-1-
▶ꢀ
[2-(phenoxyethyl)oxymethyl]uracil (10c)
(
Fig. 4) [30],
●
confirmed the N-1 alkylation reaction to afford the TNK-651
analogues 10a–f.
Antiviral screening
Compounds 4e, f, 5e, f, 6a, b and 10a–f were tested for antiviral
activity against HIV-1 wild type and strains carrying clinically
relevant mutations N119 (Y181C), A17 (K103N+Y181C), and the
triple mutant EFVR (K103R+V179D+P225H) in MT-4 cells.
Results were compared with the antiviral activity of emivirine, a
well examined HEPT analogues, and efavirenz, the most active
anti-HIV-1 drug used in therapy today. Results are listed
dimethoxybenzyl)-5-isopropyluracil
(5f)
with
N,O-bis-
(trimethylsilyl)acetamide (BSA) in anhydrous chloroform fol-
lowed by treatment with chloromethyl ethyl ether in the
presence of cesium iodide afforded the corresponding 1-ethyl-
oxymethyl-6-(3,5-dimethoxybenzyl)uracils 6a, b and their cor-
responding dialkylated derivatives 7a, b in 43, 39% and 16, 21%
▶ꢀ
in● Table 1.
▶ꢀ
As seen from the results listed in● Table 1, the starting materials
4e, f and 5e, f showed no activity against HIV-1. The non-
nucleosides with N-1 ethyloxymethyl moieties 6a and 6b
showed activities against HIV-1 wild type with EC50 =0.6±0.1
and 0.3±0.001μM, respectively. So, the isopropyl substituent at
C-5 of the uracil ring improved the activity compared with the
ethyl group. Compounds having 1-phenoxyethyloxymethyl and
6-(3,5-dimethoxybenzyl) substituents 10e, f showed inhibitory
potency (EC50)=0.35±0.07μM, while compounds with N-1
phenoxyethyloxymethyl and 6-benzyl or 6-(3,5-dimethylbenzyl)
substituents 10a–d showed high activities with (EC50)=0.07±
0.005−0.009±0.002μM, revealing to that the 3,5-dimethoxy
▶ꢀ
yields, respectively (● Fig. 2).
On the other hand, TNK-651 analogues with N-1 (2-phenoxye-
thyl)oxymethyl substituent 10a–f have been synthesized by
treating the uracils 5a–f with the acetal 9. 2-phenoxyethanol (8)
was treated with dibromomethane and potassium hydroxide in
anhydrous benzene in the presence of tetrabutylammonium
bromide, according to the method of Nazaretyan et al. [28], to
give bis(2-phenoxyethyl)metane (9) in 59% yield. The uracils
5a–e were silylated with BSA in anhydrous acetonitrile and
reacted with compound 9 using trimethylsilyl trifluorometh-
El-Brollosy NR, Loddo R. Novel Analogues of Emivirine and TNK-651… Drug Res

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
Fig. 4 Single crystal X-ray structure of compound 10c (Colorꢀfigureꢀavailableꢀonlineꢀonly).ꢀ
Table 1 Cytotoxicity and anti-HIV-1 activity of compounds 4e, f, 5e, f, 6a, b and 10a–f.
bEC50 [µM]
aCC50 [µM]
Compds
wtIIIB
N119 (Y181C)
A17 (K103N+Y181C)
EFVR (K103R+V179D+P225H)
MT-4
>100
>100
>100
>100
>100
>100
32±1
35
4e
4f
>100
>100
>100
>100
>100
>100
>100
>100
5e
>100
>100
>100
>100
5f
>100
>100
>100
>100
6a
0.6±0.1
30.5±4.9
28±1.4
>32
>100
>100
6b
0.3±0.001
0.07±0.005
0.06±0.001
0.009±0.002
0.01±0.0009
0.35±0.07
0.35±0.07
0.03±0.005
0.002±0.0003
90±5
>32
>100
10a
10b
10c
>32
>35
>35
18.5±2.1
11±1.4
2.3±0.2
>41
41±7.5
35
2.6±0.4
1.85±0.07
>41
6.5±1.6
13.3±0.4
>41
10d
10e
10f
41
33
>33
>33
>33
Emivirine
Efavirenz
>100
38±1.5
>20
56
>100
0.02±0.005
0.1±0.03
14±2.5
Data represent mean values for 3 independent determinations. Variation among duplicate samples was less than 15%. aCompound dose required to reduce the viability of
mock-infected cells by 50%, by as determined the MTT method. bCompound dose required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity, as
determined by the MTT method
substituents at the benzyl moiety reduced the anti-HIV activ-
ity. Compounds with 3,5-dimethyl groups at the benzyl
moiety 10c, d showed higher activities than those of unsub-
stituted benzyl 10a, b confirming that the 2 methyl groups at
3- and 5- positions of the benzyl substituent enhanced the
anti HIV-1 activity. The most active compounds are 6-
(3,5-dimethylbenzyl)-5-ethyl-1-[2-(phenoxyethyl)oxy-
methyl]uracil (10c) and 6-(3,5-dimethylbenzyl)-5-isopropyl-
1-[2-(phenoxyethyl)oxymethyl]uracil (10d) that both showed
inhibitory potency (EC50) higher than those of emevirine against
the wild type as well as the N119, A17 and EFVR mutant strains.
Also, compounds 10c,d showed higher activity than efavirenz
against the mutant strain EFVR.
El-Brollosy NR, Loddo R. Novel Analogues of Emivirine and TNK-651… Drug Res

DrugRes/2015-07-1036/17.8.2015/MPS
Original Article
13 El-Brollosy NR, Jorgensen PT, Dahan B et al. Synthesis of novel N-1
(allyloxymethyl) analogues of 6-benzyl-1-(ethoxymethyl)-5-isopro-
pyluracil (MKC-442, emivirine) with improved activity against HIV-1
and its mutants. J Med Chem 2002; 45: 5721–5726
14 El-Brollosy NR, Pedersen EB, Nielsen C. Synthesis of novel MKC-442
analogues with potent activities against HIV-1. Arch Pharm Pharm
Med Chem 2003; 336: 236–241
15 El-Essawy AA, El-Brollosy NR, Pedersen EB et al. Synthesis of new uracil
non-nucleoside derivatives as potential inhibitors of HIV-1. J Hetero-
cyclic Chem 2003; 40: 213–217
16 Wamberg M, Pedersen EB, El-Brollosy NR et al. Synthesis of 6-arylvinyl
analogues of the HIV drugs SJ-3366 and emivirine. Bioorg Med Chem
2004; 12: 1141–1149
Conclusion
▼
In summary, the present work describes synthesis and anti-
HIV-1 activity of novel emivirine analogues with 6-(3,5-dimeth-
oxybenzyl) substituents as well as new TNK-651 analogues
substituted at N-1 with (2-phenoxyethyl)oxymethyl moiety. The
most active compounds are 6-(3,5-dimethylbenzyl)-5-ethyl-
1-[(2-phenoxyethyl)oxymethyl]uracil (10c) and 6-(3,5-dime-
thyl--benzyl)-5-isopropyl-1-[(2-phenoxyethyl)oxymethyl]
uracil (10d) that showed inhibitory potency higher than emi-
virine against both wild type HIV-1 and the tested mutant
strains, as well as higher activity than efavirenz against EFVR.
17 El-Brollosy NR, Nielsen C, Pedersen EB. Synthesis of N-1-
(indanyloxymethyl) and N-1-(4-hydroxybut-2-enyloxymethyl) ana-
logues of the HIV drugs emivirine and GCA-186. Monatsh Chem 2005;
136: 1247–1254
18 Sorensen ER, El-Brollosy NR, Jorgensen PT et al. Synthesis of
6-(3,5-Dichlorobenzyl) derivatives as isosteric analogues of the HIV
Conflict of Interest
drug
6-(3,5-Dimethylbenzyl)-1-(ethoxymethyl)-5-isopropyluracil
▼
(GCA-186). Arch Pharm Chem Life Sci 2005; 338: 299–304
19 El-Brollosy NR. Synthesis of novel quinazoline non-nucleosides ana-
logues of the HIV drug emivirine. J Heterocyclic Chem 2006; 43:
1435–1440
Authors hereby declare that there are no conflicts of interest
with respect to this study.
20 El-Brollosy NR, Al-Omar MA, Al-Deeb OA et al. Synthesis of novel ura-
cil non-nucleosides analogues of 3,4-dihydro-2-alkylthio-6-benzyl-
4-oxopyrimidines and 6-benzyl-1-ethoxymethyl-5-isopropyluracil.
J Chem Res 2007; 263–267
21 El-Brollosy NR. Synthesis of new quinazoline-2,4(1H,3H)-dione non-
nucleoside analogues of the reverse transcriptase inhibitor TNK-651.
J Chem Res 2007; 358–361
22 El-Brollosy NR, Sørensen ER, Pedersen EB et al. Synthesis and antiviral
evaluation of 6-(trifluoromethylbenzyl) and 6-(fluorobenzyl) ana-
logues of HIV drugs emivirine and GCA-186. Arch Pharm Chem Life
Sci 2008; 341: 9–19
23 El-Brollosy NR, Al-Deeb OA, El-Emam AA et al. Synthesis of novel uracil
non-nucleoside derivatives as potential reverse transcriptase inhibi-
tors of HIV-1. Arch Pharm Chem Life Sci 2009; 342: 663–670
24 Pauwels R, Balzarini J, Baba M et al. Rapid and automated tetrazolium-
based colorimetric assay for the detection of anti-HIV compounds.
J Virol Methods 1988; 20: 309–321
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26 Danel K, Nielsen C, Pedersen EB. Anti-HIV active naphthyl analogues of
HEPT and DABO. Acta Chem Scand 1997; 51: 426–430
27 Attia MI, Kansoh AL, El-Brollosy NR. Antimicrobial pyrimidinones II:
synthesis and antimicrobial evaluation of certain novel 5,6-disubsti-
tuted 2-(substituted amino)alkylthiopyrimidin-4(3H)-ones. Monatsh
Chem 2014; 145: 1825–1837
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