Synthesis and anti-HIV-1 activity of new MKC-442 analogues with an alkynyl-substituted 6-benzyl group

Article abstract of DOI:10.1002/ardp.200600163

Synthesis and antiviral activities are reported of a series of 6-(3-alkynyl benzyl)-substituted analogues of MKC-442 (6-benzyl-1-(ethoxymethyl)-5- isopropyluracil), a highly potent agent against HIV. The 3-alkynyl group is assumed to give a better stackin

Full text of DOI:10.1002/ardp.200600163

Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
Y. L. Aly et al.
225
Full Paper
Synthesis and Anti-HIV-1 Activity of New MKC-442 Analogues
with an Alkynyl-Substituted 6-Benzyl Group*
Youssef L. Aly1,2, Erik B. Pedersen1, Paolo La Colla3, and Roberta Loddo3
1 Nucleic Acid Center**, Department of Physics and Chemistry, University of Southern Denmark, Odense M,
Denmark
2 Chemistry Department, Faculty of Education Kafr El-Sheikh branch, Tanta University, Kafr El-Sheikh, Egypt
(on leave)
3 Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e
Biotecnologie Microbiche, Universita di Cagliari, Monserrato, Italy
Synthesis and antiviral activities are reported of a series of 6-(3-alkynyl benzyl)-substituted analo-
gues of MKC-442 (6-benzyl-1-(ethoxymethyl)-5-isopropyluracil), a highly potent agent against HIV.
The 3-alkynyl group is assumed to give a better stacking of the substituted benzyl group to
reverse transcriptase (RT) and this was believed to improve antiviral activity against HIV-1. The
bromo derivatives, 5-alkyl-6-(3-bromo-benzyl)-1-ethoxymethyl derivatives 7a, b and 5-alkyl-6-(3-
bromobenzyl)-1-allyloxymethyl derivatives 9a, b, showed activity against HIV on the same level
as their corresponding analogues 10a–d with a 3-trimethylsilylalkynylbenzyl substituent and
their desilylated analogues 11a–d. However, they all showed activity against HIV-1 wild type in
the range of more than 10fold lower than the one of MKC-442. Moderate activity against Y181C
and Y181C + K103N mutated strains was also observed and, in some cases, they were marginally
better than those found for MKC-442. A few amino-DABO and S-DABO analogues were also syn-
thesized but they were found to be inactive against HIV.
Keywords: Human immunodeficiency virus (HIV-1) / MKC-442 / Non-nucleoside reverse transcriptase inhibitors
(NNRTI) / Stacking / Sonogashira coupling /
Received: October 4, 2006; accepted: February 6, 2007
DOI 10.1002/ardp.200600163
highly specific as their binding site is a hydrophobic
pocket located approximately 10 ꢀ from the polymerase
active site [3]. Several RT inhibitors have been developed
and approved by FDA (US Food and Drugs Administra-
tion) and are currently in clinical use. In particular, the
non-nucleoside RT inhibitors [4–6] are highly effective
drugs with few side effects.
6-Benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442
or emivirine, Fig. 1) [7] showed high activity against HIV-1
and was chosen as a candidate for clinical trials with
AIDS patients [8]. Unfortunately, it was reported that
MKC-442 triggers the liver enzyme cytochrome P450,
Introduction
The reverse transcriptase (RT) of the human immunodefi-
ciency virus type 1 (HIV-1) is a key enzyme, which plays
an essential and multifunctional role in the replication
cycle. This enzyme is responsible for the conversion of a
single-stranded RNA genome of HIV-1 into a double-
stranded DNA chain that subsequently is incorporated
into the DNA of the infected host cell. Non-nucleoside
inhibitors of HIV-1 reverse transcriptase (NNRTIs) [1] inhi-
bit the enzyme by occupation of an induced allosteric
binding site very close to the active site [2], they are
Correspondence: Erik B. Pedersen, Nucleic Acid Center, Department of
Physics and Chemistry, University of Southern Denmark, Campusvej 55,
DK-5230 Odense M, Denmark.
E-mail: EBP@Chem.sdu.dk
Fax: +45 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.
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Y. L. Aly et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
As an attempt to optimize the MKC-442 lead structure
[10–15], we have therefore introduced alkynyl groups at
the m-position of the aromatic ring hoping to obtain bet-
ter stacking MKC-442 analogues with improved activity
against HIV.
Figure 1. Chemical structure of MKC-442.
Results and discussion
Chemistry
leading to drug interactions between MKC-442 and pro- For the synthesis of m-alkynyl MKC-442 analogous 11a–d,
tease inhibitors [9].
the key intermediates ethyl 4-(m-bromophenyl)acetoace-
The inhibitor MKC-442 is held in place in the hydro- tates 3a, b were synthesized in 72–78% yield by reaction
of m-bromophenylacetonitrile 1 with a zinc organometal-
phobic pocket by hydrogen bonds and hydrophobic
bonds to the nearby amino acids. Although m-methyl lic reagent prepared from ethyl 2-bromopropionate or
groups on the benzyl substituent of MKC-442 can make ethyl 2-bromobutyrate in anhydrous THF. In this reac-
favourable van-der-Waals contacts with Trp229 of the tion, the pyridinones 4a, b were obtained as by-products
in 2–3% yield. The acetoacetates 3a, b were then used as
binding pocket, they are metabolically unfavourable,
being open to electrophilic attack [3]. Instead, we consid- starting materials for the synthesis of uracil rings in a
ered m-ethynyl as an interesting possibility due to its bet- ring-closure reaction with thiourea and sodium ethoxide
in boiling ethanol. The so formed 6-(3-bromoben-
ter stacking properties. One could think improved stack-
ing properties of the inhibitor to be important for inhibi- zyl)thiouracils 5a, b were desulfurized with aqueous
tion of those mutated HIV that have a reduced number of chloroacetic acid [16, 17] to give the corresponding uracil
aromatic rings in the binding pocket of RT, due to repla- derivatives 6a, b in an overall yield of 51–56% for the two
steps from 3a, b. Compounds 6a, b were silylated using
N,O-bis-(trimethylsilyl)acetamide (BSA) in acetonitrile
cement of Tyr181, Tyr188 or Phe227 with non-aromatic
amino acids.
Scheme 1. Synthesis route of compounds 3–9.
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Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
MKC-442 Analogues with an Alkynyl-Substituted 6-Benzyl
227
Scheme 2. Synthesis route of compounds 10–14.
and subsequently N-1 alkylated using chloromethylethyl inhibitor to this type of mutated RT. Also, we found it
ether [15] to give the non-nucleoside analogues 7a, b in worthy to consider the effect of aminopropynyl substitu-
46–61% yield and the N-1, N-3 bis-alkylated by-products ents where additional chances were anticipated for an
8a, b in 6–8% yield. The uracils 6a, b were also silylated extra hydrogen bonding between their protonated forms
and reacted with bis-(allyloxy)methane in the presence of and RT at physiological conditions. As the Sonogashira
trimethylsilyl trifluoromethanesulfonate (TMS-triflate) coupling can be done in a secondary amine as a solvent,
as a Lewis-acid catalyst [18] to give the non-nucleoside it was considered obvious to form propargylamines in
analogues 9a, b in 35–46% yield (Scheme 1).
situ prior to the Sonogashira coupling. Reaction of 7a, b
Coupling of the bromo derivatives 7a, b and 9a, b with with propargyl chloride in the presence of the secondary
trimethylsilylacetylene (TMSA) in the presence of bis(tri- amines diisopropylamine, morpholine and pyrrolidine
phenylphosphine)palladium(II)dichloride and CuI in dii- afforded the propargylamine products 14a–f in 36–70%
sopropylamine (DIPA) as solvent under nitrogen using yield (Scheme 2).
the condition of Price and Tour [19] afforded the corre-
sponding trimethylsilylethynyl derivatives 10a–d in 42–
For the synthesis of amino-DABOs 15a, b, the b-ketoe-
sters 3a, b were reacted with 1,1-dimethylguanidine sul-
48% yield. Desilylation of 10a–d was achieved using phate and EtONa in EtOH to afford 15a, b in 38–42%
potassium hydroxide and methanol to afford m-alkynyl- yield. The reaction of thiouracil derivative 5a, b with
benzyl analogues 11a–d in 90–96% yield (Scheme 2). CH3I in DMF afforded the corresponding S-DABOs 16a, b
Coupling of compounds 7a, b with propargyl alcohol in 62–68% yield. Unfortunately, the Sonogashira cou-
using the above condition for the Sonogashira coupling pling failed on both amino-DABOs 15a, b and S-DABOs
afforded 12a, b in 43–48% yield. Compounds 12a, b were 16a, b. Coupling of compounds 11a, b with the aryl
oxidized using o-iodoxybenzoic acid (IBX) in ethyl acetate iodides 3-iodoanisole and 3-iodopyridine in the presence
to afford the aldehyde derivatives 13a, b in 28–33% yield. of bis(triphenylphosphine)palladium(II)dichloride and
These aldehydes were also considered interesting target CuI in triethylamine as solvent under nitrogen afforded
compounds because a chemical reaction to form a thio- the coupling products 17a–d in 33–57% yield and the
hemiacetal could be anticipated to take place between homo-coupling product 18 as by-product in 3% yield
the aldehyde group of the inhibitor and the mercapto (Scheme 3). The compounds 17a–d were synthesized in
group in cysteine of the Y181C RT mutation. This would order to further increase the staking ability of the
be an alternative way to ensure strong binding of the benzylic part of the MKC-442 analogue and to explore
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Y. L. Aly et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
Table 1. Antiviral activity of compounds 4–17 against HIV-1 in
MT-4 cellsa).
Com-
CC50
EC50 (lM)c)
Y181C
pound (lM)b)
No
Wild-type EFVR
K103N+-
Y181C
4a
4b
5a
5b
6a
6b
7a
7b
9a
A100
A100
A100
A100
A100
A100
A100
A100
A100
58 l 9
A100
36 l 5
41 l 8
30 l 5
A100
A100
A100
26 l 6
A100
A100
21 l 7
A100
A100
46 l 2
A100
A100
A100
A100
A100
A100
A100
32 l 2
43 l 5
65 l 5
42 l 1
A100
A100
A100
67 l 12
A100
41 l 5
0.5 l 0.2 A100
0.05 l 0.01 A100
0.5 l 0.02 88 l 12
0.02 l 0.05 17 l 2
A100
A100
A100
A100
A100
A100
A100
A100
A100
A100
A100
A100
16 l 1
5 l 1
12 l 1
2.7 l 0.3 12 l 2
51 l 5
A36
15 l 4
9 l 1
35 l 4
50 l 4
11 l 0.5
A26
A100
A100
A21
9 l 0.5
A100
A46
A100
A100
A100
A100
A100
A100
A100
15 l 5
90 l 10
9b
10a
10b
10c
10d
11a
11b
11c
11d
12a
12b
13a
13b
14a
14b
14c
14c
14e
14f
15b
16a
16b
17a
17b
17c
17d
4 l 2
1.3 l 0.5
A100
A36
A100
A36
0.5 l 0.05 A41
A41
0.4 l 0.05 A30
A30
2 l 0.5
A100
A100
A100
42 l 4
A26
1.9 l 0.1 A100
0.3 l 0.05 A100
4 l 0.05
A26
1.9 l 0.01 A100
A100
A100
A21
62 l 7
A100
A46
0.4 l 0.05 A100
A21
A21
Scheme 3. Synthesis route of compounds 15–18.
0.1 l 0.01 A100
14 l 3
0.6 l 0.3
56 l 13
56 l 13
75 l 25
6 l 1
A100
A46
A100
A100
A100
A100
A100
A100
A100
A32
A100
A100
A100
A100
88 l 5
95 l 5
90 l 10
A32
A100
A100
A100
A100
85 l 15
A100
A100
A32
how this could influence the activity against HIV-1, the
risk being a too bulky substituent.
Biological screening
9 l 1
6 l 3
The cytotoxicity and the anti-HIV activity of the synthe-
sized alkynyl MKC-442 analogues are reported in Table 1.
In general, the introduction of an alkynyl group in the
benzylic part at the 3-position did not improve the activ-
ity against HIV-1 compared to MKC-442. In fact, com-
pounds 10a–d, 11a–d and 12a, b showed more than
10fold lower activity against the wild type HIV-1 than
MKC-442. When compared with MKC-442, it was there-
fore surprising to find better activity for some of these
compounds against the mutant Y181C than for MKC-442.
When considering the bromo derivative 7b as the refer-
ence compound, the activity against wild type HIV-1 was
also lower in all cases when replacing the bromo substi-
tuent with substituents comprising acetylene. However,
for the activity against the mutant Y181C, it was retained
for the acetylene derivatives 10c, d, 11c and 13b. Com-
11 l 1
8 l 0.2
7 l 3
2 l 0.5
10 l 0.5
A43
A43
A43
A65
A65
A65
A42
A42
A42
MKC-442 A100
0.03 l 0.005 86 l 14
24 l 3
A100
a)
Data represent mean values of at least two separate experi-
ments.
b)
c)
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. The symbol (A) indicates that CC50 was
not reached at the highest concentration tested. For descrip-
tion of assay see the Section 3 (Experimental).
pound 7b in turn was more potent against this mutant ficient prerequisite for activity against the HIV mutants.
than MKC-442. An extra aminomethyl group or an aryl The examples of amino-DABOs 15a, b and S-DABOs 16a, b
group on the ethynyl substituent was detrimental to the with a 3-bromo substituent in the benzylic part were
activity against mutated virus. Furthermore, the activity devoid of activity against HIV-1. One can conclude that
against wild type HIV-1 was also reduced. The lower activ- the activity against HIV can not be increased by acetylene
ity of the compounds 17a–d against wild type and the substitution in order to improve stacking properties of
mutants indicates that the stacking principle is not a suf- the benzylic part of the drug. It has previously been
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Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
MKC-442 Analogues with an Alkynyl-Substituted 6-Benzyl
229
attempted to replace the benzylic part with a 1-naphthyl- Ethyl 4-(3-bromophenyl)-2-methyl-3-oxo-butyrate 3a
Yield 15.4 g (78%) as an oil. 1H-NMR (CDCl3): d (ppm) = 1.24–1.34
(m, 6H, 26CH3), 3.59 (q, 1H, J = 7.2 Hz, CH), 3.80 (s, 2H, ArCH2),
4.16 (q, 2H, J = 7.2 Hz, CH2O), 7.11–7.21 (m, 4H, aryl). 13C-NMR
(CDCl3): d (ppm) = 12.72 (CH3), 14.01 (CH3), 47.73 (CH2Ar), 52.12
(COCHCO), 61.50 (OCH2CH3), 122.55, 128.22, 130.07, 130.25,
132.55, 135.58 (aryl), 170.16 (C=O), 202.45 (C=O).
methyl substituent, but when using the same N-1 and 5-
substituents as in the present investigation, this was also
found a disadvantage when compared with the activity
against MKC-442 [20–22].
This work received funding from the European Community's
Sixth Framework Programme under contract number LSHP-
CT-2004-503162 (Selection and development of microbicides for
mucosal use to prevent sexual HIV transmission/acquisition).
Ethyl 4-(3-bromophenyl)-2-ethyl-3-oxo-butyrate 3b
Yield 14.8 g (72%) as an oil. 1H-NMR (CDCl3): d (ppm) = 0.88 (t, 3H, J
= 7.4 Hz, CH3CH2), 1.25 (t, 3H, J = 7.1 Hz, CH3CH2O), 1.89 (quint,
2H, J = 7.6 Hz, CH2CH3), 3.44 (t, 1H, J = 7.3 Hz, CH), 3.79 (s. 2H,
ArCH2), 4.15 (q, 2H, J = 7.1 Hz, CH2O), 7.10–7.41 (m, 4H, Harom).13C-
NMR (CDCl3): d (ppm) = 11.76 (CH3), 14.05 (CH3), 21.48 (CH2CH3),
48.15 (CH2Ar), 59.84 (COCHCO), 61.39 (OCH2CH3), 122.52, 128.26,
130.05, 130.24, 132.58, 135.46 (aryl), 169.40 (C=O), 201.85 (C=O).
HRMS-MALDI: m/z = 335.0250 [M+Na+] (C14H17BrNaO3); requires
335.0253.
Experimental
Chemistry
NMR spectra were recorded on a Varian Gemini 2000 NMR spec-
1
trometer (Varian, Palo Alto, CA, USA) at 300 MHz for H and
6-(3-Bromobenzyl)-4-hydroxy-3,5-dimethylpyridin-2
75 MHz for 13C with TMS as an internal standard. Chemical shifts
are reported in parts per million (d), and signals are expressed as
s (singlet), d (doublet), t (triplet), q (quartet) or m (multiplet).
MALDI spectra were recorded on an IonSpec Fourier Transform
Ion Cyclotron Resonance Mass Spectrometer (IonSpec Corpora-
tion, Lake Forest, CA, USA). Melting points were determined on a
Bꢁchi melting point apparatus (Bꢁchi Labortechnik, Flawil,
Switzerland). Thin-layer chromatography (TLC) was performed
on silica gel DC-alufolio 60 F254 plates from Merck (Merck, Darm-
stadt, Germany). The silica gel (0.040–0.063 mm) used for col-
umn chromatography was purchased from Merck. Solvents used
for column chromatography were distilled prior to use, while
reagents were used as purchased.
(1H)-one 4a
Yield 0.49 g (2%); m. p. 250–2528C. 1H-NMR (DMSO): d (ppm) =
1.87, 1.88 (26s, 6H, 26CH3), 3.39 (s, 1H, OH), 3.85 (s, 2H, ArCH2),
7.19–7.43 (m, 4H, aryl), 11.26 (s, 1H, NH). 13C-NMR (DMSO): d
(ppm) = 8.93 (CH3), 10.43 (CH3), 34.80 (CH2Ar), 104.77 (C-3), 105.17
(C-5), 121.64, 127.09, 129.18, 130.58, 130.72, 139.28 (aryl), 140.84
(C-6), 162.29 (C-4), 163.51 (C=O).
6-(3-Bromobenzyl)-3,5-diethyl-4-hydroxypyridin-2
(1H)-one 4b
1
Yield 0.7 g (3%); m. p. 225–2278C; H-NMR (DMSO): d (ppm) =
0.82–0.91 (m, 6H, 26CH3), 2.18–2.21 (m, 4H, 26CH2), 3.88 (s,
2H, ArCH2), 3.96 (br s, 1H, OH), 7.28–7.52 (m, 4H, aryl), 11.18 (br
s, 1H, NH). 13C-NMR (DMSO): d (ppm) = 13.11 (CH3), 14.23 (CH3),
16.21 (CH2CH3), 17.85 (CH2CH3), 34.92 (CH2Ar), 105.93 (C-3),
106.55 (C-5), 121.51, 127.27, 129.23, 130.68, 138.32, 140.06,
(aryl), 141.49 (C-6), 161.26 (C-4), 163.26 (C=O).
General procedure for preparation of compounds
3a, 3b and 4a, 4b
Zink dust (25–30 g) was activated by stirring with hydrochloric
acid (4 M, 100 mL) for 5 min. The zinc dust was filtered and
washed sequentially with H2O (100 mL), ethanol (100 mL) and
anhydrous ether (100 mL). The zinc dust was dried by evapora-
tion under reduced pressure at 808C for 5 h, and kept in vacuo
overnight. The active zinc was suspended in anhydrous THF
(150 mL) and heated to reflux. A few drops of ethyl 2-bromopro-
pionate 2a or ethyl 2-bromobutyrate 2b were added and the mix-
ture was refluxed until a green colour appeared. m-Bromo-
phenylacetonitrile (1, 66 mmol) was added in one portion and
2a or 2b (130 mmol) in 10 mL THF was added dropwise. After
complete addition the mixture was refluxed for 5 h. After cool-
ing to room temperature and dilution with THF (300 mL), the
mixture was quenched by addition of saturated aqueous K2CO3
(100 mL). The mixture was stirred for 1 h and then the THF layer
was decanted off, and the aqueous phase was washed with THF
(36100 mL). The combined THF fractions were stirred with 10%
aqueous hydrochloric acid (90 mL) for 45 min, and the solution
was evaporated under reduced pressure and dichloromethane
(300 mL) was added. The organic phase was washed with satur-
ated aqueous NaHCO3 (26100 mL), dried over anhydrous mag-
nesium sulphate, and evaporated under reduced pressure. The
residue was chromatographed on a silica gel column with petro-
leum ether (60–808C)/ethyl acetate (v/v = 80:20) to give 3a, b and
4a, b.
General procedure for preparation of 5,6-substituted
2-thioxo-2,3-dihydro-1H-pyrimidin-4-one 5a, b
Sodium (25.1 g, 1.1 mol) was dissolved in anhydrous ethanol
(500 mL). Thiourea (58.23 g, 0.77 mol) was added and the mix-
ture was heated to reflux. Compound 3a or 3b (51 mmol) was
added dropwise, and the mixture was refluxed for 8 h. The sol-
vent was evaporated in vacuo and the residue was dissolved in
water (400 mL). The 2-thiouracil 5a or 5b was precipitated by
neutralization with concentrated hydrochloric acid, and fil-
tered off, washed with water and chromatographed on a silica
gel column with petroleum ether (60–808C)/ethyl acetate (v/v
1:1) to give 5a and 5b.
6-(3-Bromobenzyl)-5-methyl-2-thioxo-2,3-dihydro-1H-
pyrimidin-4-one 5a
1
Yield 9.8 g (62%); m. p. 222–2248C. H-NMR (CDCl3): d (ppm) =
1.80 (s, 3H, CH3), 3.86 (s, 2H, CH2Ar), 7.28–7.50 (m, 4H, aryl),
12.26 (s, 1H, NH), 12.44 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) =
9.84 (CH3), 34.33 (CH2Ar), 111.53 (C-5), 121.75, 127.14, 129.66,
130.78, 131.06, 139.01 (aryl), 148.69 (C-6), 161.82 (C-4), 174.03 (C-
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Y. L. Aly et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
2). HRMS-MALDI: m/z = 310.9849 [M+H+] (C12H11BrN2OS); requires
310.9848.
6.9 Hz, OCH2CH3), 4.14 (s, 2H, CH2Ar), 5.14 (s, 2H, NCH2O), 7.03–
7.42 (m, 4H, aryl), 9.76 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) =
10.90 (CH3), 14.95 (CH2CH3), 33.55 (CH2Ar), 64.99 (OCH2CH3),
72.81 (NCH2O), 111.16 (C-5), 123.34, 125.87, 130.30, 130.52,
130.70, 137.07 (aryl), 148.75 (C-6), 151.74 (C-2), 163.74 (C-4).
HRMS-MALDI: m/z = 375.0310 [M+Na+] (C15H17BrN2NaO3); requires
375.0315.
6-(3-Bromobenzyl)-5-ethyl-2-thioxo-2,3-dihydro-1H-
pyrimidin-4-one 5b
Yield 9.6 g (58%); solid; m. p. 201–2038C. 1H-NMR (CDCl3): d
(ppm) = 0.82 (t, 3H, J = 7.2 Hz, CH3CH2), 2.25–2.32 (m, 2H,
CH2CH3), 3.86 (s, 2H, CH2Ar), 7.23–7.49 (m, 4H, aryl), 12.24 (s, 1H,
NH), 12.44 (s, 1H, 1NH). 13C-NMR (CDCl3): d (ppm) = 12.84 (CH3),
17.68 (CH2CH3), 33.96 (CH2Ar), 117.32 (C-5), 121.71, 127.05,
129.61, 130.73, 130.97, 139.45 (aryl), 148.34 (C-6), 161.33 (C-4),
174.17 (C-2). HRMS-MALDI: m/z = 324.9997 [M+H+] (C13H13BrN2OS);
requires 325.0005.
6-(3-Bromobenzyl)-1-ethoxymethyl-5-ethyl-1H-pyrimidin-
2,4-dione 7b
Yield 2.2 g (61%); semisolid. 1H-NMR (CDCl3): d (ppm) = 1.04–1.20
(m, 6H, 26CH3CH2), 2.44 (q, 2H, J = 7.3 Hz, CH2CH3), 3.60 (q, 2H, J
= 7.1 Hz, OCH2CH3), 4.14 (s, 2H, CH2Ar), 5.11 (s, 2H, NCH2O),
7.03–7.42 (m, 4H, aryl), 9.74 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm)
= 13.70 (CH3), 14.97 (CH3), 19.15 (CH2CH3), 32.96 (CH2Ar), 65.05
(OCH2), 72.75 (NCH2O), 117.20 (C-5), 123.32, 125.84, 130.34,
130.51, 130.67, 137.61 (aryl), 148.13 (C-6), 151.88 (C-2), 163.28 (C-
4). HRMS-MALDI: m/z = 367.0642 [M+H+] (C16H19BrN2O3); requires
367.0657.
General procedure for preparation of 5,6-substituted
1H-pyrimidine-2,4-diones 6a, b
Compound 5a or 5b (0.1 mol) was suspended in aqueous chloroa-
cetic acid (10%, 300 mL) and refluxed overnight. The mixture
was cooled to room temperature, and the precipitated uracil
was filtered off, washed with water, and dried in vacuo to give
compounds 6a and 6b.
6-(3-Bromobenzyl)-1,3-bis-(ethoxymethyl)-5-methyl-1H-
pyrimidin-2,4-dione 8a
6-(3-Bromobenzyl)-5-methyl-1H-pyrimidin-2,4-dione 6a
Yield 0.24 g (6%); semisolid. 1H-NMR (CDCl3): d (ppm) = 1.15–1.25
(m, 6H, 26CH3), 2.02 (s, 3H, CH3 at C-5), 3.57–3.74 (m, 4H,
26CH2CH3), 4.14 (s, 2H, CH2Ar), 5.16 (s, 2H, NCH2O), 5.47 (s, 2H,
NCH2O), 7.18–7.42 (m, 4H, aryl). 13C-NMR (CDCl3): d (ppm) = 11.51
(CH3 at C-5), 14.96 (CH2CH3), 15.15 (CH2CH3), 33.59 (CH2Ar), 65.06,
65.90 (26OCH2CH3), 71.23 (N3CH2O), 73.58 (N1CH2O), 110.52 (C-
5), 123.31, 125.89, 130.30, 130.49, 130.67, 137.14 (aryl), 147.33
(C-6), 152.41 (C-2), 163.02 (C-4). HRMS-MALDI: m/z = 433.0721
[M+Na+] (C18H23BrN2NaO4); requires 433.0733.
1
Yield 26.8 g (91%); m. p. 272–2748C. H-NMR (CDCl3): d (ppm) =
1.77 (s, 3H, CH3), 3.77 (s, 2H, CH2Ar), 7.28–7.51 (m, 4H, aryl),
10.79 (s, 1H, NH), 11.06 (s, 1H, 1NH). 13C-NMR (CDCl3): d (ppm) =
9.66 (CH3), 34.79 (CH2Ar), 105.36 (C-5), 121.74, 127.17, 129.59,
130.73, 130.95, 139.20 (aryl), 148.17 (C-6), 150.82 (C-2), 164.82 (C-
4). HRMS-MALDI: m/z = 316.9893 [M+Na+] (C12H11BrN2NaO2);
requires 316.9896.
6-(3-Bromobenzyl)-5-ethyl-1H-pyrimidin-2,4-dione 6b
1
Yield 21.7 g (88%); m. p. 242–2448C. H-NMR (CDCl3): d (ppm) =
6-(3-Bromobenzyl)-1,3-bis-(ethoxymethyl)-5-ethyl-1H-
pyrimidin-2,4-dione 8b
0.84 (t, 3H, J = 7.2 Hz, CH3CH2), 2.25 (q, 2H, J = 7.2 Hz, CH2CH3),
3.77 (s, 2H, CH2Ar), 7.26–7.51 (m, 4H, aryl), 10.75 (s, 1H, NH),
11.05 (s, 1H, 1NH). 13C-NMR (CDCl3): d (ppm) = 13.48 (CH3), 17.56
(CH2CH3), 34.41 (CH2Ar), 111.55 (C-5), 121.71, 127.12, 129.56,
130.70, 130.95, 139.63 (aryl), 147.83 (C-6), 150.85 (C-2), 164.39 (C-
4). HRMS-MALDI: m/z = 309.0237 [M+H+] (C13H13BrN2O2); requires
309.0233.
Yield 0.33 g (8%); oil. 1H-NMR (CDCl3): d (ppm) = 1.06 (t, 3H, J =
7.2 Hz, CH3CH2 at C-5), 1.15–1.28 (m, 6H, 26CH3CH2 at N1and
N3), 2.47 (q, 2H, J = 7.4 Hz, CH2CH3 at C-5), 3.57–3.74 (m, 4H,
26OCH2CH3), 4.13 (s, 2H, CH2Ar), 5.13 (s, 2H, N1CH2O), 5.47 (s,
2H, N3CH2O), 7.04–7.42 (m, 4H, aryl). 13C-NMR (CDCl3): d (ppm) =
13.65, 14.98, 15.17 (36CH3), 19.73 (CH2CH3 at C-5), 33.04 (CH2Ar),
65.12, 65.95 (26OCH2), 71.16, 73.53 (26NCH2O), 116.46 (C-5),
123.30, 125.87, 130.35, 130.50, 130.66, 137.62 (aryl), 146.84 (C-6),
152.47 (C-2), 162.56 (C-4).
General procedure for the synthesis of 7a, b and 8a, b
N,O-Bis-(trimethylsilyl)acetamide (BSA, 6.2 mL, 2.5 mmol) was
dissolved in anhydrous chloroform (30 mL) under N2. Com-
pound 6a or 6b (10 mmol) was added and after 10 min the mix-
ture become a clear solution. Chloromethylethyl ether (1.5 mL,
15 mmol) was added and the reaction mixture was stirred over-
night at room temperature under N2, quenched with 25 mL ice
cold saturated aqueous NaHCO3, and extracted with dichloro-
methane (2650 mL). The combined organic phases were dried
over anhydrous magnesium sulphate, and evaporated under
reduced pressure. The product was chromatographed on a col-
umn of silica gel with ethyl acetate/petroleum ether (60–808C)
(v/v = 1:1) to give compounds 7a, b and 8a, b.
General procedure for the synthesis of compounds 9a, 9b
Compound 6a or 6b (3 mmol) was dissolved in anhydrous aceto-
nitrile (30 mL) under N2 and BSA (2.6 mL, 10.5 mmol) was added.
The reaction mixture became clear after 15 min and after cool-
ing to –508C trimethylsilyl trifluromethansulfonate (TMS tri-
flate) (0.54 mL, 3 mmol) was added followed by dropwise addi-
tion of bis-(allyloxy)methane. The reaction mixture was stirred
at room temperature for 30 h and quenched with a cold solution
of saturated NaHCO3 (5 mL). The solvent was evaporated under
reduced pressure and the residue was extracted with diethyl
ether (3650 mL), dried over anhydrous magnesium sulphate,
and evaporated under reduced pressure. The product was chro-
matographed on a silica gel with ethyl acetate/petroleum ether
(60–808C) (v/v = 1:1) to give 9a and 9b.
6-(3-Brombenzyl)-1-ethoxymethyl-5-methyl-1H-
pyrimidin-2,4-dione 7a
Yield 1.6 g (46%); m. p. 102–1048C. 1H-NMR (CDCl3): d (ppm) =
1.16 (t, 3H, J = 7.1 Hz, CH3CH2), 2.03 (s, 3H, CH3), 3.59 (q, 2H, J =
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
MKC-442 Analogues with an Alkynyl-Substituted 6-Benzyl
231
130.41, 131.02, 134.81 (aryl), 133.44 (CH=CH2), 148.95 (C-6)
151.81 (C-2), 163.77 (C-4). HRMS- MALDI: m/z = 405.1585 [M+Na+]
(C21H26N2NaO3Si); requires 405.1605.
1-Allyloxymethyl-6-(3-bromobenzyl)-5-methyl-1H-
pyrimidin-2,4-dione 9a
Yield 0.5 g (46%); oil. 1H-NMR (CDCl3): d (ppm) = 2.01 (s, 3H, CH3),
4.11 (d, 2H, J = 6.3 Hz, OCH2CH), 4.15 (s, 2H, ArCH2), 5.17 (s, 2H,
NCH2O), 5.26–5.32 (m, 2H, CH2=CH), 5.78–5.91 (m, 1H, CH=CH2),
7.03–7.43 (m, 4H, aryl), 9.83 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm)
= 10.92 (CH3), 33.59 (CH2Ar), 70.47 (OCH2CH), 72.57 (NCH2O),
111.28 (C-5), 117.92 (CH2=CH), 123.37, 125.86, 130.31, 130.56,
130.73, 136.98 (aryl), 133.36 (CH=CH2), 148.57 (C-6), 151.77 (C-2),
1-Ethoxymethyl-5-ethyl-6-[3-(trimethylsilylethynyl)-
benzyl]-1H-pyrimidin-2,4-dione 10c
Yield 2.1 g (48%); brown foam. 1H-NMR (CDCl3): d (ppm) = 0.18 (s,
9H, (CH3)3Si), 0.96–1.21 (m, 6H, 26CH3CH2), 2.37 (q, 2H, J =
7.2 Hz, CH2CH3), 3.55 (q, 2H, J = 7.1 Hz, OCH2CH3), 4.05 (s, 2H,
CH2Ar), 5.02 (s, 2H, NCH2O), 6.98–7.31 (m, 4H, aryl), 9.64 (s, 1H,
NH). 13C-NMR (CDCl3): d (ppm) = 0.13 ((CH3)3Si), 13.69 (CH3), 14.99
(CH3), 19.14 (CH2CH3), 33.05 (CH2Ar), 65.04 (OCH2), 72.74 (NCH2O),
95.19, 104.18 (acetylene), 117.09 (C-5), 124.14, 127.41, 129.05,
130.47, 130.95, 135.40 (aryl), 148.51 (C-6), 151.93 (C-2), 163.39 (C-
4). HRMS-MALDI: m/z = 407.1761 [M+Na+] (C21H28N2NaO3Si);
requires 407.1747.
163.70 (C-4). HRMS-MALDI: m/z
=
387.0307 [M+Na+]
(C16H17BrN2NaO3); requires 387.0315.
1-Allyloxymethyl-6-(3-bromobenzyl)-5-ethyl-1H-
pyrimidin-2,4-dione 9b
Yield 0.4 g (35%); oil. 1H-NMR (CDCl3): d (ppm) = 1.06 (m, 3H, CH3),
2.44 (q, 2H, J = 7.6 Hz, CH2CH3), 4.12 (d, 2H, J = 5.6 Hz, OCH2), 4.14
(s, 2H, CH2Ph), 5.13 (s, 2H, NCH2O), 5.26–5.32 (m, 2H, CH2=CH),
5.80–5.89 (m, 1H, CH=CH2), 7.04–7.43 (m, 4H, aryl), 9.83 (s, 1H,
NH). 13C- NMR (CDCl3): d (ppm) = 13.70 (CH3), 19.13 (CH2CH3),
33.00 (ArCH2), 70.53 (OCH2), 72.51 (NCH2O), 117.28 (C-5), 117.88
(CH2 =CH), 123.33, 125.83, 130.34, 130.53, 130.69, 137.48 (aryl),
133.41 (CH=CH2), 148.01 (C-6), 151.82 (C-2), 163.27 (C-4). HRMS-
MALDI: m/z = 407.0456 [M+Na+] (C17H19BrN2NaO3); requires
401.0471.
1-Allyloxymethyl-5-ethyl-6-[3-
(trimethylsilylethynyl)benzyl]-1H-pyrimidin-2,4-dione 10d
1
Yield 2.5 g (46%); foam. H-NMR (CDCl3): d (ppm) = 0.25 (s, 9H,
(CH3)3Si), 1.06 (t, 3H, J = 7.6 Hz, CH3CH2), 2.45 (q, 2H, J = 7.6 Hz,
CH2CH3), 4.10 (d, 2H, J = 6.1 Hz, OCH2CH), 4.13 (s, 2H, ArCH2), 5.12
(s, 2H, NCH2O), 5.17–5.32 (m, 2H, CH2=CH), 5.79–5.92 (m, 1H,
CH=CH2), 7.05–7.39 (m, 4H, aryl), 9.74 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 0.13 ((CH3)3Si), 13.70 (CH3), 19.14 (CH2CH3),
33.11 (CH2Ar), 70.55 (OCH2CH), 72.53 (NCH2O), 95.25, 104.15
(acetylene), 117.18 (C-5), 117.82 (CH2=CH), 124.17, 127.42, 129.09,
130.48, 131.00, 135.30 (aryl), 133.49 (CH=CH2), 148.40 (C-6)
151.92 (C-2), 163.36 (C-4). HRMS-MALDI: m/z = 419.1757 [M+Na+]
(C22H28N2NaO3Si); requires 419.1761.
General procedure for the synthesis of compounds 10a–
d
Compounds 7a, b or 9a, b (14.24 mmol) were dissolved in diiso-
propylamine (DIPA, 50 mL) under N2 and bis(triphenylphosphi-
ne)palladium(II)dichloride (0.3 g, 0.42 mmol), CuI (0.16 g,
0.85 mmol) were added. The reaction mixture was stirred for
General procedure for the deprotection of compounds
15 min
and
trimethylsilylacetylene
(TMSA;
2.21 mL,
15.66 mmol) was added. The reaction mixture was heated at
708C for 15 h, cooled and filtered. The solvent was evaporated
under reduced pressure and the residue was chromatographed
on a silica gel with ethylacetate: petroleum ether (60–808C) (v/v
= 1:1) to give 10a–d.
10a–d and synthesis of compounds 11a–d
Compounds 10a–d (10 mmol) were dissolved in methanol
(50 mL) and KOH (1 M, 10 mL) were added. The reaction mixture
was stirred for 5 h. The solvent was evaporated under reduced
pressure and the residue was extracted with diethyl ether to give
11a–d.
1-Ethoxymethyl-5-methyl -6-[3-(trimethylsilylethynyl)-
1-Ethoxymethyl-6-(3-ethynylbenzyl)-5-methyl-1H-
pyrimidin-2,4-dione 11a
benzyl]-1H-pyrimidin-2,4-dione 10a
Yield 2.2 g (46%); brown foam. 1H-NMR (CDCl3): d (ppm) = 0.25 (s,
9H, (CH3)3Si), 1.17 (t, 3H, J = 7.2 Hz, CH3CH2), 2.01 (s, 3H, CH3), 3.60
(q, 2H, J = 7.1 Hz, OCH2CH3), 4.13 (s, 2H, CH2Ar), 5.13 (s, 2H,
NCH2O), 7.06–7.39 (m, 4H, aryl), 9.69 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 0.12 ((CH3)3Si), 10.91 (CH3), 14.98 (CH2CH3),
33.65 (CH2Ar), 64.99 (OCH2CH3), 72.80 (NCH2O), 95.22, 104.17
(acetylene), 111.09 (C-5), 124.17, 127.49, 129.08, 130.41, 130.98,
134.89 (aryl), 149.06 (C-6), 151.82 (C-2), 163.80 (C-4). MS (EI): m/z =
371 [M+H+] (22); 326 (100).
Yield 2.8 g (96%); brown foam. 1H-NMR (CDCl3): d (ppm) = 1.17 (t,
3H, J = 7.0 Hz, CH3CH2), 2.01 (s, 3H, CH3), 3.11 (s, 1H, ethynyl),
3.62 (q, 2H, J = 7.1 Hz, OCH2CH3), 4.14 (s, 2H, CH2Ar), 5.14 (s, 2H,
NCH2O), 7.10–7.41 (m, 4H, aryl), 9.83 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 10.89 (CH3), 14.95 (CH3CH2), 33.62 (CH2Ar),
64.95 (OCH2CH3), 72.77 (NCH2O), 78.06, 82.92 (acetylene), 111.11
(C-5), 123.13, 127.78, 129.19, 130.75, 131.02, 135.10 (aryl), 148.92
(C-6), 151.82 (C-2), 163.77 (C-4). HRMS-MALDI: m/z = 321.1207
[M+Na+] (C17H18N2NaO3); requires 321.1210.
1-Allyloxymethyl-5-methyl-6-[3-(trimethylsilylethynyl)-
1-Allyloxymethyl-6-(3-ethynylbenzyl)-5-methyl-1H-
benzyl]-1H-pyrimidin-2,4-dione 10b
1
pyrimidin-2,4-dione 11b
Yield 2.1 g (42%); oil. H-NMR (CDCl3): d (ppm) = 0.25 (s, 9H,
(CH3)3Si), 2.01 (s, 3H, CH3), 4.10 (d, 2H, J = 6.2 Hz, OCH2CH), 4.13 (s,
2H, ArCH2), 5.15 (s, 2H, NCH2O), 5.20–5.32 (m, 2H, CH2=CH),
5.79–5.92 (m, 1H, CH=CH2), 7.06–7.39 (m, 4H, aryl), 9.71 (s, 1H,
NH). 13C-NMR (CDCl3): d (ppm) = 0.13 ((CH3)3Si), 10.93 (CH3), 33.70
(CH2Ar), 70.48 (OCH2CH), 72.58 (NCH2O), 95.26, 104.15 (acety-
lene), 111.18 (C-5), 117.86 (CH2=CH), 124.21, 127.49, 129.13,
Yield 2.8 g (90%); oil. 1H-NMR (CDCl3): d (ppm) = 2.01 (s, 3H, CH3),
3.11 (s, 1H, ethynyl), 4.10 (d, 2H, J = 5.7 Hz, OCH2CH), 4.15 (s, 2H,
ArCH2), 5.16 (s, 2H, NCH2O), 5.21–5.32 (m, 2H, CH2=CH), 5.78–
5.91 (m, 1H, CH=CH2), 7.10–7.41 (m, 4H, aryl), 9.84 (s, 1H, NH).
13C-NMR (CDCl3): d (ppm) = 10.92 (CH3), 33.68 (CH2Ar), 70.45
(OCH2CH), 72.55 (NCH2O), 78.10, 82.89 (acetylene), 111.20 (C-5),
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

232
Y. L. Aly et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
117.85 (CH2=CH), 123.17, 127.77, 129.23, 130.74, 131.07, 134.99
(aryl), 133.40 (CH=CH2), 148.81 (C-6) 151.81 (C-2), 163.75 (C-4).
HRMS-MALDI: m/z = 333.1209 [M+Na+] (C18H18N2NaO3); requires
333.1210.
CH2OH), 5.09 (s, 2H, NCH2O), 7.09–7.35 (m, 4H, aryl), 8.18 (s, 1H,
NH). 13C-NMR (CDCl3): d (ppm) = 13.68 (CH3), 14.95 (CH3), 19.12
(CH2CH3), 33.04 (CH2Ar), 51.34 (CH2OH), 65.09 (OCH2CH3), 72.72
(NCH2O), 84.75, 88.29 (acetylene), 117.05 (C-5), 123.65, 127.52,
129.14, 130.18, 130.54, 135.50 (aryl), 148.52 (C-6), 151.84 (C-2),
163.30 (C-4). HRMS-MALDI: m/z
=
365.1472 [M+Na+]
1-Ethoxymethyl-5-ethyl-6-(3-ethynylbenzyl)-1H-
(C19H22N2NaO4); requires 365.1464.
pyrimidin-2,4-dione 11c
1
Yield 2.8 g (91%); brown foam. H-NMR (CDCl3): d (ppm) = 1.04–
General procedure for the synthesis of compounds 13a, b
Compounds 12a, b (2 mmol) were dissolved in ethylacetate
(30 mL). Iodoxybenzoic acid (IBX) (1.68 g, 6 mmol) was added.
The reaction mixture was heated at 808C over night. The solvent
was evaporated under reduced pressure, and the residue was
chromatographed on a silica gel with ethyl acetate/petroleum
ether (60–808C) (v/v = 7 : 3) to give 13a, b.
1.20 (m, 6H, 26CH3CH2), 2.45 (q, 2H, J = 7.1 Hz, CH2CH3), 3.11 (s,
1H, ethynyl), 3.60 (q, 2H, J = 7.0 Hz, OCH2CH3), 4.14 (s, 2H, CH2Ar),
5.11 (s, 2H, NCH2O), 7.10–7.41 (m, 4H, aryl), 9.87 (s, 1H, NH). 13
C
NMR (CDCl3): d (ppm) = 13.67, 14.94 (26CH3), 19.11 (CH2CH3),
33.03 (CH2Ar), 64.99 (OCH2), 72.70 (NCH2O), 78.03, 82.90 (acety-
lene), 117.11 (C-5), 123.08, 127.71, 129.14, 130.76, 130.98, 135.59
(aryl), 148.35 (C-6), 151.93 (C-2), 163.38 (C-4). HRMS-MALDI: m/z =
335.1365 [M+Na+] (C18H20N2NaO3); requires 335.1366.
1-Ethoxymethyl-5-methyl-6-[3-(3-oxopropynyl)benzyl]-
1H-pyrimidin-2,4-dione 13a
1-Allyloxymethyl-5-ethyl-6-(3-ethynylbenzyl)-1H-
Yield 0.24 g (33%); foam. 1H-NMR (CDCl3): d (ppm) = 1.16 (t, 3H, J =
7.0 Hz, CH3CH2), 2.01 (s, 3H, CH3), 3.60 (q, 2H, J = 6.9 Hz,
OCH2CH3), 4.18 (s, 2H, CH2Ar), 5.14 (s, 2H, NCH2O), 7.26–7.54 (m,
4H, aryl), 9.42 (s, 1H, CHO), 9.82 (s, 1H, NH). 13C-NMR (CDCl3): d
(ppm) = 10.91 (CH3), 14.92 (CH3CH2), 33.58 (CH2Ar), 64.99
(OCH2CH3), 72.81 (NCH2O), 88.60, 93.86 (acetylene), 111.30 (C-5),
120.49, 129.63, 130.28, 131.68, 132.09, 135.81 (aryl), 148.48 (C-6),
151.76 (C-2), 163.66 (C-4), 176.55 (CHO). MS (EI): m/z = 327 [M+H+]
(22); 59 (100) (C3H7O).
pyrimidin-2,4-dione 11d
Yield 3.1 g (96%); foam. 1H-NMR (CDCl3): d (ppm) = 1.06 (t, 3H, J =
7.4 Hz, CH3CH2), 2.46 (q, 2H, J = 7.4 Hz, CH2CH3), 3.16 (s, 1H, ethy-
nyl), 4.11 (d, 2H, J = 5.7 Hz, OCH2CH), 4.16 (s, 2H, ArCH2), 5.12 (s,
2H, NCH2O), 5.19–5.32 (m, 2H, CH2=CH), 5.79–5.92 (m, 1H,
CH=CH2), 7.12–7.41 (m, 4H, aryl), 10.60 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 13.49 (CH3), 18.87 (CH2CH3), 32.88 (CH2Ar),
70.22 (OCH2CH), 72.25 (NCH2O), 78.05, 82.71 (acetylene), 117.00
(C-5), 117.53 (CH2=CH), 122.88, 127.53, 128.95, 130.57, 130.76,
135.39 (aryl), 133.30 (CH=CH2), 148.02 (C-6), 151.95 (C-2), 163.46
(C-4). HRMS-MALDI: m/z = 347.1365 [M+Na+] (C19H20N2NaO3);
requires 347.1366.
1-Ethoxymethyl-5-ethyl-6-[3-(3-oxopropynyl)benzyl]-1H-
pyrimidin-2,4-dione 13b
Yield 0.19 g (28%); foam. 1H-NMR (CDCl3): d (ppm) = 1.04–1.25 (m,
6H, 26CH3CH2), 2.47 (q, 2H, J = 7.5 Hz, CH2CH3), 3.59 (q, 2H, J =
6.5 Hz, OCH2CH3), 4.18 (s, 2H, CH2Ar), 5.11 (s, 2H, NCH2O), 7.27–
7.54 (m, 4H, aryl), 9.42 (s, 1H, CHO), 9.72 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 13.69 (CH3), 14.92 (CH3), 19.15 (CH2CH3), 32.99
(CH2Ar), 65.05 (OCH2CH3), 72.76 (NCH2O), 88.59, 93.88 (acetylene),
117.28 (C-5), 120.45, 129.60, 130.25, 131,70, 132.10, 136.31 (aryl),
147.94 (C-6), 151.81 (C-2), 163.22 (C-4), 176.59 (CHO). HRMS-
General procedure for the synthesis of compounds 12a, b
Compounds 7a, b (14.24 mmol) were dissolved in diisopropyla-
mine (DIPA, 50 mL) under N2 and bis(triphenylphosphine)palla-
dium(II)dichloride (0.3 g, 0.42 mmol), CuI (0.16 g, 0.85 mmol)
were added. The reaction mixture was stirred for 15 min and
propargyl alcohol (2.3 g, 42 mmol) was added. The reaction mix-
ture was heated at 708C for 36 h, cooled and filtered. The solvent
was evaporated under reduced pressure and the residue was
chromatographed on a silica gel with chloroform/methanol (v/v
= 9 : 1) to give 12a, b.
MALDI: m/z
363.1472.
=
363.1465 [M+Na+] (C19H20N2NaO4); requires
General procedure for the synthesis of compounds 14a–f
Compounds 6a, b (14.24 mmol) were dissolved in a secondary
amine (50 mL) (diisopropylmine, morpholine or pyrrolidene)
under N2 and bis(triphenylphosphine)palladium(II)dichloride
(0.3 g, 0.42 mmol), CuI (0.16 g, 0.85 mmol) were added. The reac-
tion mixture was stirred for 15 min and cooled to 08C. Propargyl
chloride (2.3 g, 42 mmol) was added dropwise. The reaction mix-
ture was heated at 708C for 48 h. The solvent was evaporated
under reduced pressure and the residue was washed with water
and extracted with chloroform. The organic layer was dried over
anhydrous magnesium sulphate, and evaporated under reduced
pressure. The product was chromatographed on a silica gel with
chloroform/methanol (v/v = 95 : 5) to give 14a–f.
1-Ethoxymethyl-6-[3-(hydroxypropynyl)benzyl]-5-methyl-
1H-pyrimidin-2,4-dione 12a
Yield 2.0 g (43%); foam. 1H-NMR (CDCl3): d (ppm) = 1.16 (t, 3H, J =
7.1 Hz, CH3CH2), 1.99 (s, 3H, CH3), 3.59 (q, 2H, J = 7.3 Hz,
OCH2CH3), 3.77 (s, 1H, OH), 4.12 (s, 2H, CH2Ar), 4.49 (s, 2H,
CH2OH), 5.12 (s, 2H, NCH2O), 7.08–7.35 (m, 4H, aryl), 9.64 (s, 1H,
NH). 13C-NMR (CDCl3): d (ppm) = 10.88 (CH3), 14.95 (CH3CH2), 33.63
(CH2Ar), 51.35 (CH2OH), 64.96 (OCH2CH3), 72.78 (NCH2O), 84.78,
88.29 (acetylene), 111.08 (C-5), 123.67, 127.59, 129.17, 130.15,
130.54, 135.00 (aryl), 149.09 (C-6), 151.79 (C-2), 163.79 (C-4). MS
(EI): m/z = 328 [M+H+] (15); 203 (100).
1-Ethoxymethyl-5-ethyl-6-[3-(hydroxypropynyl)benzyl]-
6-[3-(Diisopropylaminopropynyl)benzyl]-1-ethoxymethyl-
1H-pyrimidin-2,4-dione 12b
5-methyl-1H-pyrimidin-2,4-dione 14a
1
Yield 2.3 g (48%); foam. 1H-NMR (CDCl3): d (ppm) = 1.03–1.29 (m,
6H, 26CH3CH2), 2.43 (q, 2H, J = 7.4 Hz, CH2CH3), 3.59 (q, 2H, J =
7.1 Hz, OCH2CH3), 4.12 (s, 2H, CH2Ar), 4.12 (s, 1H, OH), 4.49 (s, 2H,
Yield 3.57 g (64%); oil. H-NMR (CDCl3): d (ppm) = 1.10–1.17 (m,
15H, 56CH3), 2.01 (s, 3H, CH3 at C-5), 2.95 (s, 2H, NCH2C), 3.20–
3.29 (m, 2H, 26CH[Pri]), 3.62 (q, J = 7.1 Hz 2H, OCH2CH3), 4.12 (s,
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
MKC-442 Analogues with an Alkynyl-Substituted 6-Benzyl
233
2H, CH2Ar), 5.13 (s, 2H, NCH2O), 7.01–7.32 (m, 4H, aryl), 9.69 (s,
1H, NH). 13C-NMR (CDCl3): d (ppm) = 10.88 (CH3 at C-5), 14.96
(CH3CH2), 20.56 (46CH3[Pri]), 33.64 (CH2Ar), 34.76 (CH[Pri]2), 48.49
(CH2N), 64.91 (OCH2CH3), 72.74 (NCH2O), 82.70, 90.07 (acetylene),
110.98 (C-5), 124.79, 126.61, 129.04, 130.05, 130.38, 134.83 (aryl),
149.06 (C-6), 151.75 (C-2), 163.67 (C-4). MS (EI): m/z = 397 [M+] (24);
396 [M-H] (100).
126.96, 129.04, 130.24, 130.62, 134.93 (aryl), 148.89 (C-6), 151.82
(C-2), 163.78 (C-4). HRMS-MALDI: m/z
(C22H27N3O3); requires 382.2125.
=
382.2128 [M+H+]
1-Ethoxymethyl-5-ethyl-6-[3-(pyrrolidinopropynyl)benzyl]-
1H-pyrimidin-2,4-dione 14f
1
Yield 3,76 g (67%); oil. H-NMR (CDCl3): d (ppm) = 0.95–1.10 (m,
6-[3-(Diisopropylaminopropynyl)benzyl]-1-ethoxymethyl-
6H, 26CH3CH2), 1.76–1.78 (m, 4H, pyrrolidino), 2.36 (q, 2H, J =
7.5 Hz, CH2CH3 at C-5), 2.66 (s, 2H, NCH2), 3.48–3.58 (m, 6H,
OCH2CH3, pyrrolidino), 4.03 (s, 2H, CH2Ar), 5.02 (s, 2H, NCH2O),
6.98–7.26 (m, 4H, aryl), 10.88 (s, 1H, NH). 13C-NMR (CDCl3):
d (ppm) = 13.53 (CH3), 14.80 (CH3), 18.93 (CH2CH3), 23.61 (pyrroli-
dino), 33.88 (CH2Ar), 43.23 (CH2N), 52.11 (pyrrolidino), 64.69
(OCH2CH3), 72.45 (NCH2O), 83.71, 85.93 (acetylene), 116.81 (C-5),
124.02, 126.74, 128.82, 130.16, 130.41, 135.38 (aryl), 148.05 (C-6),
152.05 (C-2), 163.60 (C-4).
5-ethyl-1H-pyrimidin-2,4-dione 14b
1
Yield 2.6 g (43%); oil. H-NMR (CDCl3): d (ppm) = 1.06–1.18 (m,
18H, 66CH3), 2.44 (q, 2H, J = 7.5 Hz, CH2CH3), 3.20–3.29 (m, 2H,
26CH[Pri]), 3.62 (q, 2H, J = 7.0 Hz, OCH2CH3), 3.64 (s, 2H, NCH2C),
4.11 (s, 2H, CH2Ar), 5.10 (s, 2H, NCH2O), 7.04–7.31 (m, 4H, aryl),
9.45 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 13.70 (CH3CH2), 14.99
(CH3CH2), 19.16 (CH2CH3), 20.59 (46CH3[Pri]), 33.09 (CH2Ar), 34.79
(CH[Pri]2), 48.51 (CH2N), 64.99 (OCH2CH3), 72.72 (NCH2O), 82.71,
90.13 (acetylene), 117.00 (C-5), 124.79, 126.60, 129.04, 130.11,
130.38, 135.33 (aryl), 148.56 (C-6), 151.80 (C-2), 163.17 (C-4).
HRMS-MALDI: m/z = 424.2580 [M+H+] (C25H35N3O3); requires
424.2595.
General procedure for the synthesis of compounds 15a, b
The b-keto esters 3a, b (3.15 mmol) were dissolved in EtONa (Na,
0.14 g, 6.3 mmol in 50 mL anhydrous ethanol). N,N-Dimethyl-
guanidine sulphate (1.17 g, 4.3 mmol) was added and the reac-
tion mixture was refluxed for 36 h, cooled, filtered, and evapo-
rated to the half volume in vacuo, poured into water and
extracted with chloroform. The organic layer was washed with
water (2650 mL), dried over anhydrous magnesium sulphate
and evaporated in vacuo. The product was chromatographed on
a silica gel with chloroform/methanol (v/v = 9:1) to give 15a, b.
1-Ethoxymethyl-5-methyl-6-[3-
(morpholinopropynyl)benzyl]-1H-pyrimidin-2,4-dione 14c
1
Yield 2.02 g (36%); oil. H-NMR (CDCl3): d (ppm) = 1.20 (t, 3H, J =
7.0 Hz, CH3CH2), 2.00 (s, 3H, CH3 at C-5), 2.66 (t, 4H, J = 4.6 Hz,
morph), 3.51 (s, 2H, NCH2), 3.59–3.62 (m, 4H, morph), 3.78–3.82
(m, 2H, OCH2CH3), 4.13 (s, 2H, CH2Ar), 5.12 (s, 2H, NCH2O), 7.06–
7.36 (m, 4H, aryl), 9.54 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) =
10.89 (CH3 at C-5), 14.97 (CH3CH2), 33.64 (CH2Ar), 47.95 (CH2N),
52.40 (morph), 64.94 (OCH2CH3), 66.78 (morph), 72.77 (NCH2O),
84.83, 85.02 (acetylene), 111.01 (C-5), 124.00, 127.21, 129.14,
130.24, 130.71, 134.97 (aryl), 148.97 (C-6), 151.69 (C-2), 163.57 (C-
4). HRMS-MALDI: m/z = 398.2067 [M+H+] (C22H27N3O4); requires
398.2074.
6-(3-Bromobenzyl)-2-dimethylamino-5-methyl-1H-
pyrimidine-4-one 15a
1
Yield 0.42 g (42%); m. p. 180–1828C. H-NMR (CDCl3): d (ppm) =
1.85 (s, 3H, CH3), 2.98 (s, 6H, 26NCH3), 3.74 (s, 2H, CH2Ar), 7.23–
7.47 (m, 4H, aryl), 10.83 (s, H, NH). 13C-NMR (CDCl3): d (ppm) =
10.02 (CH3), 36.78 (26NCH3), 39.98 (CH2Ar), 105.20 (C-5), 121.31,
127.70, 128.79, 130.21, 131.31, 141.47 (aryl), 152.72 (C-6), 161.15
1-Ethoxymethyl-5-ethyl-6-[3-(morpholinopropynyl)-
benzyl]-1H-pyrimidin-2,4-dione 14d
(C-4), 164.54 (C-2). HRMS-MALDI: m/z
=
322.0550 [M+H+]
Yield 2.63 g (45%); oil. 1H-NMR (CDCl3): d (ppm) = 0.95–1.16 (m,
6H, 26CH3), 2.37 (q, 2H, J = 7.4 Hz, CH2CH3), 2.54 (t, 4H, J = 4.7 Hz,
morph), 3.42 (s, 2H, NCH2), 3.56–3.61 (m, 4H, morph), 3.67 (q,
2H, J = 7.1 Hz, OCH2CH3), 4.03 (s, 2H, CH2Ar), 5.00 (s, 2H, NCH2O),
7.01–7.27 (m, 4H, aryl), 7.98 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm)
= 13.68 (CH3), 14.13 (CH3), 19.13 (CH2CH3), 33.04 (CH2Ar), 47.95
(CCH2N), 52.40 (morph), 64.96 (OCH2CH3), 66.76 (morph), 72.96
(NCH2O), 84.82, 85.05 (acetylene), 116.97 (C-5), 123.95, 127.15,
129.09, 130.27, 130.67, 135.48 (aryl), 148.39 (C-6), 151.73 (C-2),
(C14H16BrN3O); requires 322.0550.
6-(3-Bromobenzyl)-2-dimethylamino-5-ethyl-1H-
pyrimidine-4-one 15b
Yield 0.40 g (38%); m. p. 153–1558C. H-NMR (CDCl3): d (ppm) =
0.99 (t, 3H, J = 7.1 Hz, CH3CH2), 2.43 (q, 2H, J = 7.3 Hz, CH2CH3),
3.11 (s, 6H, 26NCH3), 3.77 (s, 2H, CH2Ar), 7.09–7.47 (m, 4H, aryl),
8.47 (s, H, NH). 13C-NMR (CDCl3): d (ppm) = 13.78 (CH3CH2), 18.42
(CH2CH3), 37.42 (26NCH3), 40.35 (CH2Ar), 112.69 (C-5), 122.14,
127.57, 129.13, 129.13, 132.00, 141.38 (aryl), 152.10 (C-6), 162.58
1
163.10 (C-4). HRMS-MALDI: m/z
=
434.2056 [M+Na+]
(C23H29N3NaO4); requires 434.2050.
(C-4), 165.94 (C-2). HRMS-MALDI: m/z
=
358.0514 [M+Na+]
1-Ethoxymethyl-5-methyl-6-[3-pyrrolidinopropynyl)-
(C15H18BrN3NaO); requires 358.0525.
benzyl]-1H-pyrimidin-2,4-dione 14e
1
Yield 3.8 g (70%); oil. H-NMR (CDCl3): d (ppm) = 1.17 (t, 3H, J =
General procedure for the synthesis of compounds 16a, b
Compounds 5a, b (2 mmol) were dissolved in anhydrous DMF
(30 mL) under N2 and CH3I (0.56 g, 0.26 mL, 4 mmol) was added.
The reaction mixture was stirred at room temperature for 48 h.
The reaction mixture was poured on cold water and the solid
formed was filtered off. The product was chromatographed on a
silica gel with chloroform/methanol (v/v = 9:1) to give 16a, b.
7.0 Hz, CH3CH2), 1.82–1.88 (m, 4H, pyrrolidino), 2.05 (s, 3H, CH3
at C-5), 2.71 (s, 2H, NCH2), 3.39–3.48 (m, 4H, pyrrolidino), 3.59 (q,
2H, J = 6.9 Hz, OCH2CH3), 4.16 (s, 2H, CH2Ar), 5.13 (s, 2H, NCH2O),
7.07–7.32 (m, 4H, aryl), 10.05 (s, 1H, NH). 13C-NMR (CDCl3): d
(ppm) = 10.86 (CH3 at C-5), 14.94 (CH3CH2), 23.72 (pyrrolidino),
33.62 (CH2Ar), 43.63 (CH2N), 52.57 (pyrrolidino), 64.87 (OCH2CH3),
72.70 (NCH2O), 83.70, 86.27 (acetylene), 110.96 (C-5), 124.25,
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234
Y. L. Aly et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
6-(3-Bromobenzyl)-5-methyl-2-(methylthio)-1H-
1-Ethoxymethyl-5-methyl-6-[3-(pyridin-3-ylethynyl)-
pyrimidine-4-one 16a
benzyl]-1H-pyrimidin-2,4-dione 17c
1
Yield 0.40 g (62%); m. p. 174–1768C. H-NMR (CDCl3): d (ppm) =
Yield 2.66 g (52%); foam. 1H-NMR (CDCl3): d (ppm) = 1.16–1.18 (t,
3H, J = 7.00 Hz, CH3CH2), 2.03 (s, 3H, CH3), 3.62 (m, 2H, J = 6.9 Hz,
OCH2CH3), 4.18 (s, 2H, CH2Ar), 5.18 (s, 2H, NCH2O), 7.14–7.48 (m,
4H, aryl), 7.82–8.81 (m, 4H, Hpyr), 10.63 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 10.81 (CH3 at C-5), 14.87 (CH3CH2), 33.57
(CH2Ar), 64.83 (OCH2CH3), 72.66 (NCH2O), 85.54, 91.84 (acetylene),
111.05 (C-5), 122.97, 123.42, 127.76, 129.20, 130.06, 130.48,
135.22, 138.47, 148.43 (Carom and Cpyr), 148.75 (C-6), 151.93 (C-2),
2.08 (s, 3H, CH3), 2.50 (s, 3H, SCH3), 3.87 (s, 2H, CH2Ar), 7.11–7.42
(m, 4H, aryl), 12.63, (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 10.63
(CH3), 13.26 (SCH3), 40.52 (CH2Ar), 116.29 (C-5), 122.36, 127.53,
129.56, 129.87, 132.04, 140.10 (aryl), 157.20 (C-6), 161.25 (C-4),
165.29 (C-2). HRMS-MALDI: m/z = 325.0004 [M+H+] (C13H13BrN2OS);
requires 325.0005.
163.97 (C-4). HRMS-MALDI: m/z
=
398.1294 [M+Na+]
(C24H24N2NaO4); requires 398.1277.
6-(3-Bromobenzyl)-5-ethyl-2-(methylthio)-1H-pyrimidine-
4-one 16b
1
Yield 0.46 g (68%); m. p. 174–1768C. H-NMR (CDCl3): d (ppm) =
1-Ethoxymethyl-5-ethyl-6-[3-(pyridin-3-ylethynyl)benzyl]-
1H-pyrimidin-2,4-dione 17d
0.83 (t, 3H, J = 7.5 Hz, CH3CH2), 2.32 (s, 3H, SCH3), 2.37 (q, 2H, J =
7.5 Hz, CH2CH3), 3.77 (s, 2H, CH2Ar), 7.13–7.44 (m, 4H, aryl),
12.47 (s, 1H, NH). 13C-NMR (CDCl3): d (ppm) = 12.56 (CH3CH2),
13.15 (SCH3), 18.03 (CH2CH3), 39.43 (CH2Ar), 116.05 (C-5), 121.31,
127.33, 129.01, 130.27, 131.73, 141.16 (aryl), 156.81 (C-6), 161.13
Yield 3.2 g (57%); foam. 1H-NMR (CDCl3): d (ppm) = 1.04–1.19 (m,
6H, 26CH3CH2), 2.45 (q, 2H, J = 7.1 Hz, CH2CH3), 3.60 (q, 2H, J =
6.9 Hz, OCH2CH3), 4.15 (s, 2H, CH2Ar), 5.12 (s, 2H, NCH2O), 7.14–
7.34 (m, 4H, Harom), 7.64–8.02 (m, 4H, Hpyr), 10.27 (s, 1H, NH). 13C-
NMR (CDCl3): d (ppm) = 13.56, 14.80 (26CH3CH2), 32.89 (CH2Ar),
64.78 (OCH2CH3), 72.52 (NCH2O), 74.28, 81.10 (acetylene), 116.97
(C-5), 122.40, 128.18, 128.34, 129.14, 131.01, 131.12, 131.69,
131.72, 131.78, 131.91, 135.80 (Carom and Cpyr), 147.95 (C-6),
151.83 (C-2), 163.27 (C-4).
(C-4), 165.16 (C-2). HRMS-MALDI: m/z
(C14H15BrN2OS); requires 339.0161.
=
339.0166 [M+H+]
General procedure for the synthesis of compounds 17a–
d and 18
Compounds 11a, b (14.24 mmol) were dissolved in triethylamine
(50 mL) under N2 and bis(triphenylphosphine)palladium(II)-
dichloride (0.3 g, 0.42 mmol), CuI (0.16 g, 0.85 mmol) and iodo
derivatives (3-iodoanisole, 3-iodopyridine, 42 mmol) were added.
The reaction mixture was stirred at room temperature for 15 h.
The solvent was evaporated under reduced pressure and the resi-
due was chromatographed on a silica gel with chloroform/
methanol (v/v = 95:5) to give 17a-d and 18.
1,4-Bis-[3-(3-Ethoxymethyl-5-methyl-2,6-dioxo-1,2,3,6-
tetrahydropyrimidin-4-ylmethyl)phenyl]-butadiyne 18
Yield 0.25 g (8%); brown foam. 1H-NMR (CDCl3): d (ppm) = 1.16 (t,
6H, J = 7.1 Hz, 26CH3CH2), 2.01 (s, 6H, 26CH3), 3.60 (q, 4H, J =
7.1 Hz, 26OCH2CH3), 4.15 (s, 4H, 26CH2Ar), 5.14 (s, 4H,
26NCH2O), 7.13–7.54 (m, 8H, aryl), 9.95 (s, 2H, 26NH). 13C-NMR
(CDCl3): d (ppm) = 10.89 (s, 26CH3), 14.92 (s, 26CH3CH2), 33.61 (s,
26CH2Ar), 64.93 (s, 26OCH2CH3), 72.76 (s, 26NCH2O), 74.45,
81.23 (acetylene), 111.15 (s, 26C-5), 122.64, 129.31, 131.16,
131.29, 131.94, 135.34 (aryl), 148.74 (26C-6), 151.80 (26C-2),
1-Ethoxymethyl-5-methyl-6-[3-(3-methoxyphenyl-
ethynyl)benzyl]-1H-pyrimidin-2,4-dione 17a
163.75 (26C-4). HRMS- MALDI: m/z
=
617.2344 [M+Na+]
Yield 2.12 g (37%); foam. 1H-NMR (CDCl3): d (ppm) = 1.18 (t, 3H, J =
7.1 Hz, CH3CH2), 2.03 (s, 3H, CH3), 3.61 (q, 2H, J = 6.8 Hz,
OCH2CH3), 3.82 (s, 3H, OCH3), 4.16 (s, 2H, CH2Ar), 5.16 (s, 2H,
NCH2O), 6.88–7.46 (m, 8H, aryl), 9.78 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 10.92 (CH3 at C-5), 14.97 (CH3CH2), 33.69
(CH2Ar), 55.25 (OCH3), 64.97 (OCH2CH3), 72.79 (NCH2O), 88.41,
90.10 (acetylene), 111.09 (C-5), 115.25, 116.18, 123.76, 124.18,
124.23, 127.31, 129.21, 129.37, 130.09, 130.57, 135.06, 149.07
(aryl), 151.82 (C-6), 159.28, 163.80 (26CO). HRMS-MALDI: m/z =
427.1624 [M+Na+] (C24H24N2NaO4); requires 427.1628.
(C34H34N4NaO6); requires 617.2371.
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 strepto-
mycin. 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 4.56106 50% cell culture
infectious dose (CCID50)/mL. The K103R + V179D + P225H mutant
(EFVR) was derived from an IIIB strain passage in MT-4 cells in the
presence of efavirenz (up to 2 lM). The Y181C mutant (NIH N119)
was derived from an AZT-sensitive clinical isolate passage initi-
ally 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). K103R + V179D + P225H (EFVR), Y181C, and
1-Ethoxymethyl-5-ethyl-6-[3-(3-methoxyphenyl-
ethynyl)benzyl]-1H-pyrimidin-2,4-dione 17b
Yield 1.96 g (33%); foam. 1H-NMR (CDCl3): d (ppm) = 1.14–1.20 (m,
6H, 26CH3CH2), 2.44 (q, 2H, J = 7.4 Hz, CH2CH3), 3.59 (q, 2H, J =
7.2 Hz, OCH2CH3), 4.15 (s, 3H, OCH3), 4.16 (s, 2H, CH2Ar), 5.10 (s,
2H, NCH2O), 7.13–7.71 (m, 8H, aryl), 9.52 (s, 1H, NH). 13C-NMR
(CDCl3): d (ppm) = 13.71 (CH3), 14.95 (CH3), 19.16 (CH2CH3), 33.05
(CH2Ar), 59.63 (OCH3), 65.00 (OCH2CH3), 72.72 (NCH2O), 74.44,
81.25 (acetylene), 117.12 (C-5), 122.63, 128.26, 128.33, 128.49,
129.31, 131.15, 131.32, 131.83, 131.95, 132.09, 135.86, 147.32
(aryl), 148.16 (C-6), 151.75 (C-2), 163.11 (C-4).
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Arch. Pharm. Chem. Life Sci. 2007, 340, 225–235
MKC-442 Analogues with an Alkynyl-Substituted 6-Benzyl
235
K103N + Y181C stock solutions had titers of 4.06107, 1.26108,
and 2.16107 CCID50/mL, respectively.
[6] N. Sluis-Cremer, A. N. Temiz, I. Bahar, Curr. HIV Res. 2004,
2, 323–332.
[7] H. Tanaka, H. Takashima, M. Ubasawa, K. Sekiya, et al., J.
Med. Chem. 1995, 38, 2860–2865.
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.
[8] M. Baba, H. Tanaka, T. Miyasaka, S. Yuasa, et al., Nucleo-
sides Nucleotides, 1995, 14, 575–583.
[9] G. M. Szczech, P. Furman, G. R. Painter, D. W. Barry, et
al., Antimicrob. Agents Chemother. 2000, 44, 123–130.
[10] O. S. Pedersen, E. B. Pedersen, Synthesis 2000, 4, 479–495.
Anti-HIV assays
[11] O. S. Pedersen, E. B. Pedersen, Antivir Chem Chemother.
1999, 10, 285–314.
The activity of test compounds against multiplication of wild
type HIV-1, K103R + V179D + P225H (EFVR), Y181C, and K103N +
Y181C in acutely infected cells was based on inhibition of virus-
induced cytopathicity in MT-4 cells. Briefly, an amount of 50 lL
of culture medium containing 16104 cells was added to each
well of flat-bottom microtiter trays containing 50 lL of culture
medium with or without various concentrations of test com-
pounds. Then an amount of 20 lL of HIV suspensions (contain-
ing the appropriate amount of CCID50 to cause complete cyto-
pathicity at day 4) was added. After incubation at 378C, cell viabi-
lity was determined by the 3-(4,5-dimethylthiazol-1-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) method [23]. 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.
[12] L. Petersen, E. B. Pedersen, C. Nielsen, Synthesis 2001, 4,
559–564.
[13] L. Petersen, T. H. Hansen, N. M. Khalifa, P. T. Jørgensen, et
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