Synthesis and anti-HIV-1 activity of new fluoro-HEPT analogues: An investigation on fluoro versus hydroxy substituents

Article abstract of DOI:10.1002/ardp.201000187

Coupling of 6-benzyl-5-hydroxymethyluracil (1) with formaldehyde acetals followed by fluorination using (diethylamino)sulfur trifluoride (DAST) afforded 1-alkenyloxymethyl and 1-propargyloxymethyl 5-fluoromethyl-6-benzyluracils 3a-c. 6-(3,5-Dimethylbenzyl)-5-ethyl-1-[(2-fluoroethoxy)methyl]pyrimidine-2,4(1H,3H)- dione (6) was synthesized by fluorination of the corresponding hydroxy derivative 5. Sonogoshira reaction was performed on 6-(3,5-dimethylbenzyl)-5- ethyl-1-(4-iodobenzyl)uracil (7) with propargyl alcohol to afford 8 which was fluorinated to give the fluoro propargyl derivative 9. Compound 7 was synthesized by N1-alkylation of the corresponding uracil. Significant activity was found against HIV-1 except for compounds with 5-hydroxymethyl and 5-fluoromethyl substituents. Copyright

Full text of DOI:10.1002/ardp.201000187

366
Arch. Pharm. Chem. Life Sci. 2011, 11, 366–371
Full Paper
Synthesis and Anti-HIV-1 Activity of New Fluoro-HEPT
Analogues: An Investigation on Fluoro versus Hydroxy
Substituents*
Yasser M. Loksha1, Erik B. Pedersen1, Roberta Loddo2, and Paolo La Colla2
1 Nucleic Acid Centerꢀꢀ, Department of Physics and Chemistry, University of Southern Denmark, Odense M,
Denmark
2 Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e
Biotecnologie Microbiche, Universita di Cagliari, Cittadella Universitaria, Monserrato, Italy
Coupling of 6-benzyl-5-hydroxymethyluracil (1) with formaldehyde acetals followed by fluorination
using (diethylamino)sulfur trifluoride (DAST) afforded 1-alkenyloxymethyl and 1-propargyloxymethyl
5-fluoromethyl-6-benzyluracils 3a–c. 6-(3,5-Dimethylbenzyl)-5-ethyl-1-[(2-fluoroethoxy)methyl]pyrimidine-
2,4(1H,3H)-dione (6) was synthesized by fluorination of the corresponding hydroxy derivative 5.
Sonogoshira reaction was performed on 6-(3,5-dimethylbenzyl)-5-ethyl-1-(4-iodobenzyl)uracil (7)
with propargyl alcohol to afford 8 which was fluorinated to give the fluoro propargyl derivative
9. Compound 7 was synthesized by N1-alkylation of the corresponding uracil. Significant activity was
found against HIV-1 except for compounds with 5-hydroxymethyl and 5-fluoromethyl substituents.
Keywords: Anti-HIV-1 activity / DAST / MKC-442 analogues / Non-nucleoside reverse transcriptase inhibitors /
Sonogashira cross-coupling
Received: June 24, 2010; Revised: July 16, 2010; Accepted: July 19, 2010
DOI 10.1002/ardp.201000187
Introduction
1-[(2-Hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT)
derivatives (Fig. 1) are the first non-nucleoside reverse tran-
scriptase inhibitor analogs shown to have potent anti-HIV
activity [3, 4]. Since the discovery of HEPT and MKC-442
(a HEPT analogue, Fig. 1) [5–7], different types of HEPT and
MKC-442 analogues, have been synthesized in order to
improve their activity against HIV-1 wild type and its resis-
tant mutants [8–14]. In this paper, some fluoro HEPT
analogues were synthesized by the reaction of DAST with
the appropriate hydroxy compounds. All of the synthesized
compounds were tested for their activity against HIV-1 wild
type and mutant strains resistant against NNRTIs. We con-
sidered it interesting to compare equally bulky fluoro and
hydroxy derivatives because of their different hydrophilic
properties.
The enzyme HIV reverse transcriptase (RT) is responsible for
the conversion of single stranded RNA viral genome into a
double-stranded DNA copy. RT inhibitors are classified as
nucleoside and non-nucleoside. Nucleoside reverse transcrip-
tase inhibitors (NRTIs) were the first anti-HIV drugs discov-
ered by Mitsuya et al. [1, 2]. They block HIV RT by chain
termination. Non-nucleoside reverse transcriptase inhibitors
(NNRTIs) represent another important group of HIV-1 RT
inhibitors with proven efficacy in the clinic. Only compounds
˚
that bind to a specific pocket situated ꢁ10 A from the
enzyme’s polymerase active site are considered NNRTIs.
Correspondence: Erik B. Pedersen, Nucleic Acid Center, Department of
Physics and Chemistry, University of Southern Denmark, Campusvej 55,
DK-5230 Odense M, Denmark.
Results and discussion
E-mail: EBP@ifk.sdu.dk
Chemistry
Fax: þ45 66158780
ꢀ The information in this document is provided as is and no guarantee or
warranty is given that the information is fit for any particular purpose. The
user thereof uses the information as its sole risk and liability.
ꢀꢀ A research center funded by The Danish National Research Foundation
for studies on nucleic acid chemical biology.
5-Hydroxmethyl-6-benzyluracil (1) [15] was silylated with N,O-
bis(trimethylsilyl)acetamide (BSA) and treated with formal-
dehyde acetals [9] in dry acetonitrile in the presence of
trimethylsilyl trifluoromethanesulfonate (TMS-triflate) as a
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2011, 11, 366–371
Synthesis and Anti-HIV-1 Activity of New Fluoro-HEPT Analogues
367
O
O
N
O
N
O
N
HN
HN
(a)
HN
HN
O
N
H
O
O
S
O
O
5
4
O
O
HO
HO
(c)
(b)
HEPT
MKC-442 (HEPT analogue)
O
N
O
Figure 1. The structures of HEPT and MKC-442.
HN
HN
O
O
N
Lewis acid catalyst [16] at ꢂ508C to afford 6-benzyl-5-hydroxy-
methyl-1-alkenyloxymethyl (2a,b) and propargyloxymethyl-
uracil (2c). Fluorination of compounds 2a–c was carried
out with diethylaminosulfur trifluoride (DAST) in methylene
chloride to furnish the fluoro derivatives 3a–c (Scheme 1).
1H-NMR of compounds 3a–c showed the disappearance of
the signal corresponding to the -OH group and the
–CH2F appeared as doublets at 5.34 ppm with coupling con-
stants of JH,F ¼ 48.9 Hz.
O
7
6
F
I
(d)
O
O
N
(e)
HN
HN
O
N
O
6-(3,5-Dimethylbenzyl)-5-ethyl-1-[(2-hydroxyethoxy)methyl]-
pyrimidine-2,4(1H,3H)-dione (5) was prepared in 92% yield by
silylation of 6-(3,5-dimethylbenzyl)-5-ethyluracil (4) using BSA
followed by coupling with 1,3-dioxolane in the presence of
TMS-triflate in dry acetonitrile. Compound 5 was fluorinated
with DAST in dry methylene chloride to furnish 6-(3,5-dime-
thylbenzyl)-5-ethyl-1-[(2-fluoroethoxy)methyl]pyrimidine-2,4-
(1H,3H)-dione (6) in 70% yield. The silylated compound of 4
was refluxed with 4-iodobenzyl bromide in acetonitrile for
90 h to give 6-(3,5-dimethylbenzyl)-5-ethyl-1-(4-iodobenzyl)-
pyrimidine-2,4(1H,3H)-dione (7) in 45% yield. The coupling
at N1 of uracil ring to afford compound 7 was confirmed by
8
9
OH
F
Scheme 2. Reagents and conditions: (a) i) BSA, CH3CN,
ii) TMS-triflate, –508C, 1,3-dioxane; (b) DAST, 08C, CH2Cl2;
(c) i) BSA, CH3CN, RT, ii) p-iodobenzyl bromide, reflux;
(d) propargyl alcohol, Et3N, CuI, PdCl2(PPh3)2; (e) DAST, 08C,
CH2Cl2.
nuclear Overhauser effects (NOE) which is based on through
space transfer of spin polarization from one spin population
to another via cross-relaxation in NMR. On irradiation of
CH2-C6 at 3.75 ppm, 3.43% NOE was detected with CH2N at
4.85 ppm which also showed 2.57% NOE with CH2-C6 by
irradiation. Sonogashira cross-coupling [17–21] was applied
on compound 7 and propargyl alcohol using copper(I) iodide
and PdCl2(PPh3)3 as catalysts to afford compound 8 in 73%
yield. Fluorination of the hydroxyl compound 8 by DAST in
dry methylene chloride furnishes the fluoromethyl deriva-
tive 9 in 75% yield (Scheme 2). According to 1H-NMR the
–CH2F appeared as a doublet at 5.18 ppm with a coupling
constant of JH,F ¼ 47.4 Hz.
O
N
OH
O
OH
HN
(a)
HN
O
O
N
H
O
2a-c
1
R
(b)
O
N
F
a, R = -CH=CH2
Antiviral activity
-C=CH2
CH3
b, R =
HN
The HIV-1 strain HTLV-IIIB in MT-4 cells was used in our assay
to investigate the anti-HIV-1 activity of MKC-442 analogues
synthesized in the present study. The results are summarized
in Table 1.
O
c, R =
-C CH
O
3a-c
R
Neither HEPT analogues with 5-hydroxymethyl groups (2a–
c) nor 5-fluoromethyl derivatives (3a–c) showed any signifi-
cant activities against HIV-1. Compounds 5–9 showed higher
Scheme 1. Reagents and conditions: (a) i) BSA, CH3CN,
ii) TMS-triflate, –508C, (RCH2O)2CH2; (b) DAST, 08C, CH2Cl2.
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368
Y. M. Loksha et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 366–371
Table 1. Cytotoxicity and anti-HIV-1 activity of compounds 2a–c, 3a-c, 5–9, and reference compounds MKC-442 and efavirenz (EFV).
Compound CC50 (mM)b
SIc
EC50 (mM)a
Y181C
Wild type
EFVR
K103N þ Y181C
2a
2b
2c
3a
3b
3c
5
6
7
8
9
>100
>100
>100
>100
>100
>100
>100
>100
43 ꢃ 3
2 ꢃ 0.1
2 ꢃ 0.1
>100
30
>16
5
>11
>16
5
>10
>5000
>16666
717
500
100
6 ꢃ 1
>20
>100
>20
>100
>100
>20
>100
22 ꢃ 2
20 ꢃ 5
>43
>2
>100
>20
>100
>100
>20
>100
>20
>100
>100
>20
>100
30 ꢃ 8
4 ꢃ 2
>43
>2
>2
9 ꢃ 0.9
6 ꢃ 1
ꢄ20
10 ꢃ 2
>100
3 ꢃ 1
0.02 ꢃ0.001
0.006 ꢃ 0.0005
0.06 ꢃ 0.01
0.004 ꢃ 0.0005
0.02 ꢃ 0.005
0.03
0.5 ꢃ 0.2
3 ꢃ 1
0.1 ꢃ 0.04
0.6 ꢃ 0.05
20
>2
100
3
MKC-442
EFV
>3333
15000
>100
0.3
0.002
0.008
a
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
b
method. The symbol (>) indicates that the CC50 was not reached at the highest concentration tested
c
Selectivity index for wild type HIV-1: Ratio CC50/EC50. EC50 and CC50 are expressed as the mean values of at least two separate
experiments.
H, N) were carried out at Chemical Laboratory II at University of
activity against HIV-1 mutants than MKC-442, especially
Copenhagen, Denmark, their results were found to be in good
against the Y181C mutant. With a terminal fluorine in the
agreement (ꢃ0.4%) with the calculated values.
ethoxymethyl substituent, compound
6 showed better
activity than the corresponding hydroxy compound 5 against
HIV-1 wild type and its mutants and it was also the compound
with the highest selectivity index (SI > 16666) in the present
investigation. Surprisingly the compounds 7–9, being 1-
benzyl derivatives, showed high activity against wild type
HIV-1 although they do not have the ether linkage like the
lead structure of HEPT (Fig. 1). However, when the hydroxyl
group in compound 8 was substituted with fluorine (com-
pound 9), the efficacy against wild type HIV-1 was decreased
General procedure for preparation of 1-substituted
6-benzyl-5-hydroxymethyluracil 2a–c
N,O-Bis-(trimethylsilyl)acetamide (BSA, 1.6 mL, 6.6 mmol) was
added to a stirred solution of compound 1 (464 mg, 2 mmol)
[15] in dry methylene chloride (100 mL) under nitrogen. After
15 min the reaction was cooled to ꢂ508C. TMS-triflate (1.08 mL,
6 mmol) was added to the reaction mixture followed by the
addition of the appropriate acetal (4 mmol). The mixture was
left overnight with stirring at room temperature. The reaction
was quenched by addition of 1 mL saturated aqueous solution of
sodium carbonate and water (10 mL). The two layers were sep-
arated and the methylene chloride phase was dried over anhy-
drous magnesium sulfate and evaporated under reduced
pressure. The product was chromatographed on a silica gel
column using petroleum ether (60–808C)/EtOAc (v/v, 1:1) as elu-
ent to afford compounds 2a–c.
by
5 fold and against the Y181 mutant by 6 fold.
Unfortunately, the compounds 7–9 showed a higher cyto-
toxic effect which reduced the selectivity index to the range
of 100–717. The influence of hydrophilic properties on the
efficacy against HIV-1 of fluoro and hydroxy derivatives fol-
lows the same trend as previously predicted by molecular
modeling [22].
1-[(Allyloxy)methyl]-6-benzyl-5-(hydroxymethyl)
pyrimidine-2,4(1H,3H)-dione 2a
White solid; yield: 49%; m.p.: 96–988C; H-NMR (CDCl3) d [ppm]:
3.50 (bs, 1H, OH), 4.10 (dt, 2H, J ¼ 5.6, 1.5 Hz, OCH CH ), 4.28
–
(s, 2H, CH2Ph), 4.54 (s, 2H, CH2OH), 5.18–5.22 (m, 3H, OCH2N and
Experimental
1
–
2
Chemistry
–
–
NMR spectra were recorded on a Varian Gemini 2000 spec-
trometer at 300 MHz for 1H and 75 MHz for 13C with TMS as
an internal standard. EI mass spectra were recorded on a
Finnigan MAT SSQ 710. MALDI spectra were recorded on a 4.7
T Ultima Fourier transform Mass spectrometer (IonSpec, Irvine,
CA). Melting points were determined in a Bu¨chi melting point
apparatus. The silica gel (0.040–0.063 mm) used for column
chromatography was purchased from Merck. Microanalyses (C,
HCH CH), 5.29 (dq, 1H, J ¼ 17.2, 1.5 Hz, HCH CH), 5.78–5.91 (m,
–
–
1H, CH2 CH), 7.14–7.36 (m, 5H, Harom), 10.15 (s, 1H, NH); 13C-NMR
–
–
–
(CDCl ) d [ppm]: 33.52 (CH Ph), 56.59 (CH OH), 70.58 (OCH -C ),
–
72.54 (OCH2N), 114.25 (C5), 117.93 (CH2 CH), 127.46, 127.55,
3
2
2
2
–
–
–
129.28, 133.375 (Carom), 134.72 (CH2 CH), 151.79 (C6), 152.57
–
(C2), 164.25 (C4); EI-MS: m/z ¼ 302 [Mþ] (14%), 41 (100%);
HRMS-MALDI: m/z ¼ 325.1164 (C16H18NaN2O4 [M þ Naþ]),
requires 325.1159.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 366–371
Synthesis and Anti-HIV-1 Activity of New Fluoro-HEPT Analogues
369
JH,F ¼ 48.9 Hz, CH2-F), 7.14–7.38 (m, 5H, Harom), 9.63 (s, 1H,
NH); 13C-NMR (CDCl3) d [ppm]: 19.41 (CH3), 33.84 (CH2Ph),
6-Benzyl-5-(hydroxymethyl)-1-{[(2-methylprop-2-enyl)oxy]
methyl}pyrimidine-2,4(1H,3H)-dione 2b
–
72.69 (OCH N), 73.76 (OCH -C ), 75.31 (d, J ¼ 165.0 Hz, CH -F),
White solid; yield: 51%; m.p.: 110–1128C; 1H-NMR (CDCl3) d [ppm]:
–
2
2
2
–
–
110.16 (d, J ¼ 17.5 Hz, C5), 112.82 (CH2 C), 127.47, 127.65,
–
1.71 (s, 3H, CH ), 3.52 (bs, 1H, OH), 4.01 (s, 2H, OCH -C ), 4.29 (s,
–
–
2H, CH2Ph), 4.53 (s, 2H, CH2OH), 4.88, 4.96 (2s, 2H, CH2 ), 5.18
3
2
–
129.38, 134.17 (Carom), 140.92 (CH2 C), 151.45 (C6), 156.60
–
–
(C2), 162.46 (C4); EI-MS: m/z ¼ 318 [Mþ] (6%), 247 (100%). Anal.
calcd. for C17H19FN2O3ꢅ0.25 H2O (322.85): C, 63.25; H, 5.85; N,
8.56. Found: C, 63.66; H, 5.89; N, 8.60.
(s, 2H, OCH2N), 7.15–7.36 (m, 5H, Harom), 10.21 (s, 1H, NH); 13C-
NMR (CDCl3) d [ppm]: 19.44 (CH3), 33.48 (CH2Ph), 56.55 (CH2OH),
–
–
72.68 (OCH -C ), 73.54 (OCH N), 112.67 (CH C), 114.23 (C5),
–
–
2
2
2
–
127.44, 127.55, 129.28, 134.74 (Carom), 141.01 (CH2 C), 151.77
–
(C6), 152.59 (C2), 164.28 (C4); EI-MS: m/z ¼ [MHþ] (2%), 227 (100%).
Anal. calcd. for C17H20N2O4 (316.14): C, 64.54; H, 6.37; N, 8.86.
Found: C, 64.30; H, 6.43; N, 8.73.
6-Benzyl-5-(fluoromethyl)-1-[(prop-2-ynyloxy)methyl]
pyrimidine-2,4(1H,3H)-dione 3c
White solid; yield: 76%; m.p.: 124–1268C; 1H-NMR (CDCl3) d [ppm]:
2.48 (t, 1H, J ¼ 2.4 Hz, CHC), 4.29 (d, 2H, J ¼ 2.4 Hz, CHC-CH2),
4.30 (s, 2H, CH2Ph), 5.25 (s, 2H, OCH2N), 5.34 (d, 2H,
JH,F ¼ 48.9 Hz, CH2-F), 7.16–7.38 (m, 5H, Harom), 9.85 (s, 1H,
NH); 13C-NMR (CDCl3) d [ppm]: 33.86 (CH2Ph), 57.41 (CHC-CH2),
72.50 (OCH2N), 75.27 (d, J ¼ 166.3 Hz, CH2-F), 75.10 (CHC), 78.75
(CHC), 110.35 (d, J ¼ 17.3 Hz, C5), 127.69, 127.49, 129.38, 134.07
(Carom), 151.61 (C6), 156.45 (C2), 162.51 (C4); EI-MS: m/z ¼ 302
[Mþ] (22%), 39 (100%). Anal. calcd. for C16H15FN2O3 (302.3): C,
63.57; H, 5.00; N, 9.27. Found: C, 63.32; H, 5.11; N, 8.89.
6-Benzyl-5-(hydroxymethyl)-1-[(prop-2-ynyloxy)methyl]
pyrimidine-2,4(1H,3H)-dione 2c
White solid; yield: 50%; m.p.: 130–1328C; 1H-NMR (CDCl3) d [ppm]:
2.48 (t, 1H, J ¼ 2.4 Hz, CHC), 3.50 (bs, 1H, OH), 4.26 (s, 2H, CH2Ph),
4.27 (s, 2H, OCH2-CCH), 4.54 (s, 2H, CH2OH), 5.23 (s, 2H, OCH2N),
7.15–7.34 (m, 5H, Harom), 10.14 (s, 1H, NH); 13C-NMR (CDCl3) d
[ppm]: 33.50 (CH2Ph), 56.50 (CH2OH), 57.26 (OCH2-C), 72.44 (CHC),
75.06 (OCH2N), 78.86 (CHC), 114.39 (C5), 127.50, 127.57, 129.29,
134.61 (Carom), 151.82 (C6), 152.47 (C2), 164.15 (C4); EI-MS:
m/z ¼ 300 [Mþ] (24%), 39 (100%). Anal. calcd. for C16H16N2O4
(300.31): C, 63.99; H, 5.37; N, 9.33. Found: C, 63.88; H, 5.44; N,
9.11.
6-(3,5-Dimethylbenzyl)-5-ethyl-1-[(2-hydroxyethoxy)
methyl]pyrimidine-2,4(1H,3H)-dione 5
Compound 4 (0.51 g, 2 mmol) [23] was stirred in dry acetonitrile
(20 mL) under nitrogen and BSA (1.4 mL, 6.6 mmol) was added.
The mixture became clear after stirring at room temperature
for 10 min. The reaction mixture was cooled to ꢂ508C and TMS-
triflate (1.1 mL, 6 mmol) was added followed by dropwise
addition of the appropriate acetal (0.3 mL, 4 mmol). The
mixture was stirred at room temperature for 3 h, quenched with
ice-cold saturated solution of sodium bicarbonate (1 mL), and
evaporated under reduced pressure. Water (50 mL) was added to
the residual material and the solid product formed was filtered
off, washed with water, and dried to afford 0.61 g (92%)
compound 5.
General procedure for the preparation of 1-substituted
6-benzyl-5-fluoromethyluracil 3a–c
To a stirred solution of compound 2a–c (1.3 mmol) in dry methy-
lene chloride (2 mL) was added diethylaminosulfur trifluoride
(DAST, 0.26 mL in 0.2 mL methylene chloride, 2 mmol) dropwise
at ꢂ58C. The reaction mixture was allowed to reach room
temperature gradually and stirred for 3 h and quenched with
5% aqueous sodium bicarbonate (0.2 mL) followed by addition of
water (5 mL) and methylene chloride (5 mL). The two layers were
separated and the organic layer was dried (sodium sulfate) and
evaporated under reduced pressure. The residual oily product
was purified by a silica gel column chromatography using ether
as eluent to afford compounds 3a–c.
M.p.: 146–1488C; 1H-NMR (CDCl3) d [ppm]: 1.07 (t, 3H,
J ¼ 7.2 Hz, CH3CH2), 2.28 (s, 6H, (CH3)2Ar), 2.47 (q, 2H,
J ¼ 7.2 Hz, CH3CH2), 3.68–376 (m, 4H, CH2CH2OH), 4.06 (s, 1H,
CH2-C6), 5.18 (s, 1H, NCH2O), 6.70 (s, 2H, Harom), 6.90 (s, 1H, Harom);
13C-NMR (CDCl3) d [ppm]: 13.68 (CH3CH2), 19.17 (CH3CH2), 21.24
((CH3)2Ar), 33.33 (CH2-C6), 61.16 (CH2OH), 70.69 (CH2CH2OH),
73.13 (NCH2O), 116.97 (C5), 124.96, 128.99, 134.79, 138.90
(Carom), 149.11 (C6), 152.21 (C2), 163.40 (C4); EI-MS: m/z ¼ 332
[Mþ] (5%), 258 (100%). Anal. calcd. for C18H24N2O4 ꢅ 0.25 H2O
(336.91): C, 64.17; H, 7.33; N, 8.31. Found: C, 64.10; H, 7.31; N, 8.17.
1-[(Allyloxy)methyl]-6-benzyl-5-(fluoromethyl)pyrimidine-
2,4(1H,3H)-dione 3a
White solid; yield: 79%; m.p.: 100–1028C; 1H-NMR (CDCl3) d [ppm]:
–
4.12 (dt, 2H, J ¼ 5.7, 1.5 Hz, OCH -CH ), 4.32 (s, 2H, CH Ph),
–
2
–
–
2
5.19–5.23 (m, 3H, OCH N and HCH CH), 5.30 (dq, 2H, J ¼ 17.2,
2
–
–
1.5 Hz, HCH CH), 5.34 (d, 2H, J ¼ 48.9 Hz, CH F), 5.80–5.93 (m,
2
1H, CH2 CH), 7.15–7.39 (m, 5H, Harom), 9.85 (s, 1H, NH); 13C-NMR
–
–
6-(3,5-Dimethylbenzyl)-5-ethyl-1-[(2-fluoroethoxy)methyl]
pyrimidine-2,4(1H,3H)-dione 6
(CDCl3) d [ppm]: 33.87 (CH2Ph), 70.76 (OCH2-CH), 72.53 (OCH2N),
75.31 (d, J ¼ 165.3 Hz, CH2-F), 110.20 (d, J ¼ 17.3 Hz, C5),
To a stirred solution of compound 5 (166 mg, 0.5 mmol) in dry
methylene chloride (5 mL) was added DAST (0.1 mL in 1 mL
methylene chloride, 0.75 mmol) dropwise at ꢂ58C. The reaction
mixture was allowed to reach room temperature and stirred for
3 h and quenched with 5% aqueous sodium bicarbonate (0.5 mL)
followed by addition of water (15 mL). The mixture was extracted
with methylene chloride (15 mL). The organic phase was dried
(sodium sulfate) and the solvent was evaporated under reduced
pressure. The residual material was purified by silica gel column
chromatography using EtOAc/CH2Cl2 (v/v, 1:1) as eluent to afford
117 mg (70%) of compound 6.
–
118.08 (CH2 CH), 127.47, 127.64, 129.37, 134.18 (Carom), 133.27
–
–
(CH2 CH), 151.56 (C6), 156.62 (C2), 162.57 (C4); EI-MS: m/z ¼ 304
–
[Mþ] (11%), 246 (100%). Anal. calcd. for C16H17FN2O3ꢅ0.25 H2O
(308.83): C, 62.23; H, 5.55; N, 9.07. Found: C, 62.61; H, 5.57; N, 9.03.
6-Benzyl-5-(fluoromethyl)-1-{[(2-methylprop-2-enyl)
oxy]methyl}pyrimidine-2,4(1H,3H)-dione 3b
1
White solid; yield: 50%; m.p.: 92–948C; H-NMR (CDCl3) d [ppm]:
–
1.72 (s, 3H, CH ), 4.03 (s, 2H, OCH -C ), 4.32 (s, 2H, CH Ph), 4.90,
–
–
3
2
2
4.97 (2s, 2H, CH2 C), 5.19 (s, 2H, OCH2N), 5.34 (d, 2H,
–
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370
Y. M. Loksha et al.
Arch. Pharm. Chem. Life Sci. 2011, 11, 366–371
M.p.: 134–1368C; 1H-NMR (CDCl3) d [ppm]: 1.08 (t, 3H,
J ¼ 7.5 Hz, CH3CH2), 2.29 (s, 6H, (CH3)2Ar), 2.48 (q, 2H,
J ¼ 7.5 Hz, CH3CH2), 3.86 (dt, 2H, J ¼ 29.7, 4.1 Hz, OCH2CH2F),
4.08 (s, 2H, CH2Ar), 4.52 (dt, 2H, J ¼ 47.7, 4.1 Hz, CH2CH2F), 5.19
(s, 2H, NCH2O), 6.71 (s, 2H, Harom), 6.90 (s, 1H, Harom), 9.64 (bs, 1H,
NH); 13C-NMR (CDCl3) d [ppm]: 13.71 (CH3CH2), 19.17 (CH3 CH2),
21.25 ((CH3)2Ar), 33.23 (CH2Ar), 68.79 (d, J ¼ 19.5 Hz, CH2CH2F),
73.12 (NCH2O), 82.42 (d, J ¼ 169.9 Hz, CH2F), 116.99 (C5), 125.00,
129.00, 134.77, 138.91 (Carom), 149.20 (C6), 152.14 (C2), 163.40
(C4); EI-MS: m/z ¼ 334 [Mþ] (30%), 255 (100%). Anal. calcd.
for C18H23FN2O3 (334.39): C, 64.65; H, 6.93; N, 8.38. Found: C,
64.78; H, 6.93; N, 8.06.
163.28 (C4); HRMS-MALDI: m/z ¼ 425.1822 (C25H26N2NaO3
(M þ Naþ]), requires 425.1836.
6-(3,5-Dimethylbenzyl)-5-ethyl-1-[4-(3-fluoroprop-1-ynyl)
benzyl]pyrimidine-2,4(1H,3H)-dione 9
To a stirred solution of compound 8 (160 mg, 0.37 mmol) in dry
methylene chloride (5 mL) was added DAST (0.075 mL in 1 mL
methylene chloride, 0.56 mmol) dropwise at ꢂ58C. The mixture
was allowed to reach room temperature gradually and stirred for
3 h. The reaction mixture was quenched with 5% aqueous
sodium bicarbonate (0.5 mL) followed by addition of water
(15 mL) and extracted with methylene chloride (15 mL). The
methylene chloride phase was dried (sodium sulfate) and evap-
orated under reduced pressure. The residual was purified by a
silica gel column chromatography using CHCl3/MeOH (v/v, 25:1)
as eluent to afford 112 mg (75%) of compound 9.
6-(3,5-Dimethylbenzyl)-5-ethyl-1-(4-iodobenzyl)
pyrimidine-2,4(1H,3H)-dione 7
Compound 4 (0.58 g, 2 mmol) in dry acetonitrile (20 mL) was
silylated under nitrogen by dropwise addition of BSA (1.6 mL,
6.6 mmol) and stirred for 15 min. 4-Iodobenzyl bromide (0.77 g,
2.6 mmol) was added in one portion to the mixture which was
refluxed for 90 h. The mixture was cooled to room temperature
and the solvent was removed under reduced pressure. The residual
material was chromatographed on a silica gel column using CHCl3/
MeOH (v/v, 25:1) as eluent to afford 425 mg (45%) of compound 7.
M.p.: 192–1948C; 1H-NMR (CDCl3) d [ppm]: 1.05 (t, 3H,
J ¼ 7.4 Hz, CH3CH2), 2.30 (s, 6H, (CH3)2Ar), 2.46 (q, 2H,
J ¼ 7.4 Hz, CH3CH2), 3.75 (s, 2H, CH2-C6), 4.85 (s, 2H, CH2-N),
6.64 (s, 2H, Harom), 6.86 (d, 2H, J ¼ 8.3 Hz, Harom), 6.92 (s,
1H, Harom), 7.66 (d, 2H, J ¼ 8.3 Hz, Harom), 9.63 (bs, 1H, NH);
13C-NMR (CDCl3) d [ppm]: 13.90 (CH3CH2), 19.35 (CH3 CH2),
21.30 ((CH3)2Ar), 34.18 (CH2-C6), 46.60 (CH2-N), 116.74 (C5),
93.01, 124.82, 127.87, 129.17, 134.37, 136.37, 138.03, 139.11
(Carom), 149.38 (C6), 151.84 (C2), 163.27 (C4); EI-MS: m/z ¼ 474
[Mþ] (55%), 217 (100%). Anal. calcd. for C22H23IN2O2 ꢅ 0.5 H2O
(483.35): C, 54.67; H, 5.00; N, 5.80. Found: C, 54.36; H, 4.60; N, 5.53.
M.p.: 83–858C; 1H-NMR (CDCl3) d [ppm]: 1.05 (t, 3H,
J ¼ 7.4 Hz, CH3CH2), 2.30 (s, 6H, (CH3)2Ar), 2.47 (q, 2H,
J ¼ 7.4 Hz, CH3CH2), 3.74 (s, 2H, CH2-C6), 4.91 (s, 2H, CH2-N),
5.18 (d, 2H, JHF ¼ 47.4 Hz, CH2F), 6.65 (s, 2H, Harom), 6.92 (s,
1H, Harom), 7.08 (d, 2H, J ¼ 8.1 Hz, Harom), 7.45 (d, 2H,
J ¼ 8.1 Hz, Harom), 9.57 (bs, 1H, NH); 13C-NMR (CDCl3) d [ppm]:
13.91 (CH3CH2), 19.37 (CH3 CH2), 21.31 ((CH3)2Ar), 34.20 (CH2-C6),
46.83 (CH2-N), 71.05 (d, J ¼ 165.4 Hz, CH2F), 83.05 (d, J ¼ 21.5 Hz,
CC–CH2F), 88.82 (d, J ¼ 12.1 Hz, CC–CH2F), 116.74 (C5), 121.20,
124.84, 125.91, 129.19, 132.47, 134.39, 137.71, 139.12 (Carom),
149.44 (C6), 151.84 (C2), 163.27 (C4); HRMS-MALDI: m/z ¼
405.1968 (C25H26FN2O2 [M þ Hþ]), requires 405.1973.
Antiviral assay procedures
Compounds were solubilized in DMSO at 200 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 mg/mL strep-
tomycin. 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 ꢆ 106 50%
cell culture infectious dose (CCID50)/mL. The K103R þ
V179D þ P225H mutant (EFVR) was derived from an IIIB strain
passage in C8166 cells in the presence of efavirenz (up to 2 mM).
The Y181C mutant (NIH N119) was derived from an AZT-sensitive
clinical isolate passage initially in CEM and then in MT-4 cells in
the presence of nevirapine (10 mM). The K103N þ Y181C (NIH
A17) was derived from the IIIB strain passaged in H9 cells in the
presence of BI-RG 587 (1 mM). K103R þ V179D þ P225H (EFVR),
Y181C, and K103N þ Y181C stock solutions had titers of
3.0 ꢆ 105, 1.3 ꢆ 106, and 2.5 ꢆ 105 CCID50/mL, respectively.
6-(3,5-Dimethylbenzyl)-5-ethyl-1-[4-(3-hydroxyprop-1-
ynyl)benzyl]pyrimidine-2,4(1H,3H)-dione 8
Using a balloon with a long needle immersed in the solution, a
stream of argon was flushed through a solution of compound 7
(354 mg, 0.7 mmol) and propargyl alcohol (0.42 mL, 7 mmol) in
dry triethylamine (15 mL). The reaction mixture was transferred
by a syringe with a long needle to another flask containing
copper(I) iodide (8 mg, 6 mol%) and PdCl2[P(Ph)3]2 (15 mg,
3 mol%) under argon at room temperature. The mixture was
stirred for 3 h, and then coevaporated with chloroform
(3 ꢆ 30 mL) to remove triethylamine. Water (20 mL) was added
to the residue and the mixture was extracted with chloroform
(30 mL). The chloroform phase was dried (sodium sulfate) and
evaporated under reduced pressure. The residual material was
purified by a silica gel column using CHCl3/MeOH (v/v, 25:1) as
eluent to afford 205 mg (73%) of compound 8.
M.p.: 93–958C; 1H-NMR (CDCl3) d [ppm]: 1.05 (t, 3H,
J ¼ 7.4 Hz, CH3CH2), 2.26 (s, 6H, (CH3)2Ar), 2.46 (q, 2H,
J ¼ 7.4 Hz, CH3CH2), 3.74 (s, 2H, CH2Ar), 4.50 (s, 2H, CH2OH),
4.90 (s, 2H, CH2N), 6.65 (s, 2H, Harom), 6.92 (s, 1H, Harom), 7.04
(d, 2H, J ¼ 8.3 Hz, Harom), 7.40 (d, 2H, J ¼ 8.3 Hz, Harom), 9.53 (bs,
1H, NH); 13C-NMR (CDCl3) d [ppm]: 13.89 (CH3CH2), 19.36 (CH3
CH2), 34.16 (CH2Ar), 46.82 (CH2OH), 51.52 (CH2N), 84.98 (CC-CH2),
87.82 (CC-CH2), 116.68 (C5), 122.04, 124.83, 125.83, 129.17,
132.28, 134.40, 136.97, 139.10 (Carom), 149.54 (C6), 151.82 (C2),
HIV titration
Titration of HIV was performed in C8166 cells by the standard
limiting dilution method (dilution 1:2, four replica wells per
dilution) in 96-well plates. The infectious virus titer was deter-
mined by light microscope scoring of syncytia after 4 days of
incubation. Virus titers were expressed as CCID50/mL.
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Arch. Pharm. Chem. Life Sci. 2011, 11, 366–371
Synthesis and Anti-HIV-1 Activity of New Fluoro-HEPT Analogues
371
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