Synthesis and anti-HIV-1 activity of novel MKC-442 analogues containing alkenyl chains or reactive functionalities in the 6-benzyl group

Article abstract of DOI:10.1007/s00706-006-0548-3

In an effort to obtain more insight into the anti-HIV efficacy of MKC-442 analogues (1-(alkoxymethyl)-6-benzyluracils), a new series of compounds was synthesized and evaluated for inhibition of HIV-1 replication. The modifications include a reactive cente

Full text of DOI:10.1007/s00706-006-0548-3

Monatshefte f€ur Chemie 137, 1557–1570 (2006)
DOI 10.1007/s00706-006-0548-3
Synthesis and Anti-HIV-1 Activity of Novel
MKC-442 Analogues Containing Alkenyl
Chains or Reactive Functionalities
in the 6-Benzyl Group
Youssef L. Aly1;#, Erik B. Pedersen1;ꢀ, Paolo La Colla2,
and Roberta Loddo2
Nucleic Acid Center^, Department of Physics and Chemistry,
1
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, Monserrato, Italy
Received May 4, 2006; accepted May 22, 2006
Published online November 17, 2006 # Springer-Verlag 2006
Summary. In an effort to obtain more insight into the anti-HIV efficacy of MKC-442 analogues
(1-(alkoxymethyl)-6-benzyluracils), a new series of compounds was synthesized and evaluated for
inhibition of HIV-1 replication. The modifications include a reactive center such as an aldehyde or an
epoxide substituted at the benzyl group. It was believed that such reactive groups could improve the
activity against HIV for the Y181C mutant by forming a covalent bond to the mercapto group in
cysteine in the hydrophobic pocket. Unfortunately, only moderate activities were found in cell-based
assays for such compounds against wild-type HIVand no activity against the Y181C mutant. However,
higher activities were found for a corresponding oxime and the precursor molecules with butenyl and
allyloxy substituents in the benzyl group. A few amino-DABO and S-DABO analogues were also
synthesized, but they were found inactive against HIV.
Keywords. Bioorganic chemistry; Drug research; HIV-1; MKC-442; Non-nucleoside.
Introduction
A major challenge facing medicinal chemistry over the next few years will be the
development of drugs with significantly improved resistance profiles for chronic
use in anti-HIV combination therapy. An important component of such regimens
ꢀ
Corresponding author. E-mail: ebp@chem.sdu.dk
#
On leave from Chemistry Department, Faculty of Education Kafr El-Sheikh branch, Tanta
University, Kafr El-Sheikh, Egypt
^
A research Center Funded by the Danish National Research Foundation for Studies on Nucleic Acid
Chemical Biology

1558
Y. L. Aly et al.
Fig. 1. Non-nucleoside reverse transcriptase inhibitors of HIV-1
will be non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1. This is
a class of structurally diverse aromatic compounds, such as nevirapine, delavirdine,
efavirenz, MKC-442 (emivirine or coactinon), HEPT (1-[(2-hydroxyethoxy)methyl]-
6-(phenylthio)thymine, 1, Fig. 1), TIBO, and thiocarboxanilides [1]. Structural studies
have revealed that NNRTIs inhibit HIV-1 reverse transcriptase (RT) by binding to
˚
an allosteric site, approximately 10 A from the polymerase active site [2–4] causing a
distortion of the catalytic aspartate triad [5].
MKC-442 (2a, Fig. 1) is an optimized structure of HEPT which was abandoned
during phase III clinical trials [6]. Also 2,6-disubstituted and 2,5,6-trisustituted-
4(3H)-pyrimidinones (amino-DABOs and S-DABOs) [7, 8] are antiviral agents in-
hibiting HIV-1 RT [9]. In an attempt to optimize the MKC-442 lead structure we
have previously introduced a vinyl group at C-5 of the uracil ring (2b, Fig. 1) instead
of the isopropyl group [10]. This compound has shown good activity against HIV-1
(IC-50 ¼ 0.07 ꢀM) being almost as active as MKC-442 (IC-50 ¼ 0.02 ꢀM).
When NNRTIs are used as drugs against HIV the major problem is the fast
development of resistance. A very common mutation when using MKC-442 is the
conversion of tyrosine at position 181 in RT into cysteine (Y181C). We have pre-
viously reported the synthesis of an MKC-442 analogue with a carbaldehyde at
position C-5 [10]. It was hoped that the drug would covalently bind to the hydro-
phobic pocket by formation of an a thiohemiacetal between the carbaldehyde and
the mercapto group in cysteine of the mutated virus. This carbaldehyde turned out
to be inactive toward the wild-type as well as the mutant. We have also attempted
to synthesize an epoxide directly attached to C-5 and also further away from the
uracil ring. However, the first compounds turned out to be too reactive to be iso-
lated, its ring being opened by the m-chlorobenzoic acid formed during epoxidation
of the corresponding alkene, the second one has been isolated [10, 11]. In this work
we have introduced the same reactive groups in the aromatic ring instead of the
uracil ring hoping for a more appropriate position of the reactive groups for reac-
tion with the mercapto group in the Y181 mutation of RT. A flexible linker to the
reactive groups from the aromatic ring may further improve the chance of a reac-
tion with the mercapto group.
Results and Discussion
Chemistry
m-(Allyloxy)benzyl chloride (3a) was synthesized from reaction of m-hydroxyben-
zyl alcohol with allyl bromide in a successive alkylation reaction and chloronation

Novel MKC-442 Analogues
1559
with thionyl chloride [12]. The corresponding m-butenylbenzyl bromide (3b) was
synthesized by reaction of m-bis(bromomethyl)benzene with allylmagnesium bro-
mide in 43% yield. Refluxing 3a, 3b with KCN in aqueous ethanol in the presence
of KI furnished the nitriles 4a, 4b in 52–73% yield. Reaction of 4a, 4b with the
zinc organometallic reagent prepared from ethyl 2-bromobutyrate in anhydrous
THF afforded the ꢁ-ketoesters 5a, 5b in 53–55% yield and the pyridinones 6a,
6b as by-products. Compounds 5a, 5b were used as starting materials for the
synthesis of uracil rings in a ring closure reaction with thiourea and NaOEt. The
formed thiouracils 7a, 7b were desulfurized with chloroacetic acid [13, 14] to give
the uracil derivatives 8a, 8b in overall yield of 37% in both cases for the two steps
from 5a, 5b. Compounds 8a, 8b were silylated using bis(trimethylsilyl)acetamide
(BSA) and subsequently N-1 alkylated using chloromethyl ethyl ether [13] to give
Scheme 1

1560
Y. L. Aly et al.
Scheme 2
the non-nucleoside analogues 9a, 9b in 53–61% yield and the N-1,N-3 bisalkylated
by-products 10a, 10b in 6–13% yield. The uracils 8a, 8b were also silylated and re-
acted with bis(allyloxy)methane in the presence of trimethylsilyl trifluoromethane-
sulfonate (TMS triflate) as a Lewis acid catalyst [15] to give the non-nucleoside
analogues 11a, 11b in 48–67% yield (Scheme 1).
The uracil derivatives 9a, 9b were reacted with ozone to cleave the double
bond and the aldehydes 12a, 12b were isolated in 18–28% yield and compound
12a was converted to the corresponding oxim 13a in 38% yield. In order to
epoxidize the double bond, the compounds 9a, 9b were reacted with m-chloro-
perbenzoic acid (MCPBA) in dry CHCl3 to give the epoxides 14a, 14b in 18–37%
yield (Scheme 2).
Alkylation of 7a with isopropyl bromide or isobutyl bromide in the presence
of K2CO3 in dry DMF gave O- and S-bisalkylated products 16a, 16b. The mono
S-alkylated derivatives were not observed. When sodium methoxide and excess
Scheme 3

Novel MKC-442 Analogues
1561
methyl iodide were used, the S-alkylated product 15 was obtained without any
bisalkylated products. For the synthesis of amino-DABO derivatives the ꢁ-ketoester
5a was reacted with dimethylguanidine sulphate in EtONa to afford compound 17
in 38% yield (Scheme 3).
Antiviral Activity
The anti-HIV activities and cytotoxicities of the synthesized MKC-442 analogues
are summarized in Table 1. In general, the introduction of an allyloxy or a butenyl
group in the benzylic part at the 3-position did not improve the activity against
HIV-1 compared to MKC-442. In fact that compounds 9a, 9b and 11a, 11b showed
a 10-fold lower activity against the wild-type HIV-1 than MKC-442. Therefore it
was surprising to find better activities for these compounds against the mutant
Y181C than the one found for MKC-442. Even against the double mutant, activity
was observed for the derivatives 9b and 11b. The compounds containing an oxim
13a, a reactive aldehyde group 12a, 12b or an reactive epoxides 14a, 14b in the
Table 1. Cytotoxicity and anti-HIV-1 activity of compounds 6–17a
Wild-type
Mutants=ꢀM
Y181C
CC-50b=ꢀM
IC-50c=ꢀM
EFVR
K103N þ Y181C
6a
6b
77
95 ꢁ 5
86
>20
>95
>20
>95
>20
>80
>20
>66
>65
>55
>20
>45
20
>20
>95
>20
>95
7a
>20
>80
>20
>80
>20
>80
7b
8a
80 ꢁ 18
74
>20
>66
>20
>66
>20
>66
8b
66 ꢁ 4
65 ꢁ 6
55 ꢁ 5
41
9a
0.5 ꢁ 0.05
0.3 ꢁ 0.1
>20
7 ꢁ 3
2 ꢁ 0.2
>20
>65
9b
10 ꢁ 1
>20
>45
10a
10b
11a
11b
12a
12b
13a
14a
14b
15
45 ꢁ 5
44 ꢁ 2
42 ꢁ 2
45 ꢁ 1
>100
>100
80 ꢁ 20
>100
80 ꢁ 20
>100
>100
>100
>100
>45
>45
0.4 ꢁ 0.005
0.2 ꢁ 0.05
8 ꢁ 1
4 ꢁ 0.8
1 ꢁ 0.2
15 ꢁ 5
51 ꢁ 8
35 ꢁ 5
>80
>44
5 ꢁ 2
>45
30 ꢁ 8
>45
>100
>100
>80
>100
>80
>100
>100
>20
100
1.9 ꢁ 0.05
0.3 ꢁ 0.1
14 ꢁ 3
9 ꢁ 1
>100
54
>80
80 ꢁ 20
>80
80 ꢁ 20
>80
>80
16a
16b
17
>100
>100
>20
>100
>100
>20
>100
>100
>20
MKC-442
0.03
20
>100
a
b
Data represent mean values of at least two separate experiments; compound dose required to
reduce the viability of mock-infected cells by 50%, as determined by the MTT method; c compound
dose required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity, as
determined by the MTT method

1562
Y. L. Aly et al.
side chain of the benzylic group showed rather moderate activities against the wild-
type virus when compared to MKC-442. Also, they do not show activity against the
mutants Y181C and K103N þ Y181C. The latter indicates that the covalent bind-
ing of the drug to cysteine in the RT hydrophobic pocket has not taken place or that
this principle is not a sufficient prerequisite for activity against the HIV mutants.
The examples of S-DABO 15 and amino-DABO 17 with an 3-allyloxy substitutent
in the benzylic part were also devoid of activity against HIV-1.
Experimental
NMR spectra were recorded on a Varian Gemini 2000 spectrometer at 300MHz for 1H and 75MHz for
1
13C NMR, internal standards used in H NMR spectra were TMS. Accurate-ion mass determinations
were performed using the 4.7T Ultima Fourier transform mass spectrometer (Ion Spec, Irvine, CA).
The (Mþ H)þ and (Mþ Na)þ ions were peak matched using ions derived from the 2,5-dihydroxy-
benzoic acid matrix. Elemental analyses were performed at H.C. Ørsted Institute, University of
Copenhagen; the found values agreed favourably with the calculated ones. Thin-layer chromatography
(TLC) analyses were carried out with Silica Gel 60 F254 TLC plates purchased from E. Merck and
were visualized in UV light (254 nm). The silica gel (0.040–0.063mm) used for column chromato-
graphy was purchased from E. Merck. Solvents used for column chromatography were distilled prior to
use, while reagents were used as purchased.
m-Butenylbenzyl bromide (3b)
To m-bis(bromomethyl)benzene (1.35 g, 5.14mmol) in dry ether (30 cm3), was added dropwise allyl-
magnesium chloride (5.14 mmol) in 20cm3 dry ether at 0ꢂC. After complete addition the reaction
mixture was refluxed for 20 h. The solvent was evaporated and the residue was chromatographed using
petroleum ether (60–80ꢂC)=ethyl acetate (1=1, v=v) to afford compound 3b. Yield 43%; oil; 1H NMR
(300 MHz, CDCl3): ꢂ ¼ 2.34–2.39 (m, 2H, CH2CH), 2.66–2.71 (m, 2H, CH2Ph), 4.45 (s, 2H, CH2Br),
5.05–5.06 (m, 2H, CH2¼CH), 5.82–5.88 (m, 1H, CH¼CH2), 7.09–7.24 (m, 4H, Harom) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 33.68 (CH2CH), 35.10 (CH2Ph), 35.24 (CH2Br), 115.07 (CH2¼CH), 137.69
(CH¼CH2), 125.84, 126.49, 128.52, 128.67, 129.04, 142.42 (Carom) ppm; MS (EI): m=z ¼ 224 (Mþ).
General Procedure for Preparation of Compounds 4a and 4b
To compound 3a or 3b (150mmol) in 100 cm3 ethanol, were added KCN (22 g, 340 mmol) and KI (5g,
30mmol) in 100 cm3 H2O. The reaction mixture was refluxed for 4 h, cooled to room temperature,
filtered, and the ethanol was evaporated. The residue was dissolved in H2O and extracted with diethyl
ether. The organic layer was dried over MgSO4 and evaporated and the residue was chromatographed
(petroleum ether (60–80ꢂC)=ethyl acetate ¼ 8=2, v=v) to afford the compounds 4a and 4b.
1-Allyloxy-3-cyanomethylbenzene (4a)
1
Yield 73%; oil; IR: ꢃꢀ¼ 2251 (CN) cmꢃ1; H NMR (300MHz, DMSO-d6): ꢂ ¼ 3.98 (s, 2H, CH2CN),
4.56 (d, J ¼ 2.1 Hz, 1H, CH2O), 5.36–5.42 (m, 2H, CH2¼CH), 6.01–6.05 (m, 1H, CH¼CH2), 6.93–
7.33 (m, 4H, Harom) ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼ 22.28 (CH2CN), 68.09 (CH2O), 117.9
(CH2¼CH), 119.06 (CN), 132.79 (CH¼CH2), 114.19, 114.37, 120.21, 130.07, 131.20, 159.01 (Carom
)
ppm; MS (EI): m=z ¼ 173 (Mþ).
1-(But-3-enyl)-3-cyanomethylbenzene (4b)
Yield 52%; oil; 1H NMR (300 MHz, CDCl3): ꢂ ¼ 2.35–2.37 (m, 2H, CH2CH), 2.68–2.73 (t,
J ¼ 7.7 Hz, 2H, CH2Ph), 3.70 (s, 2H, CH2CN), 4.99–5.00 (m, 2H, CH2¼CH), 5.78–5.84 (m, 1H,
CH¼CH2), 7.12–7.30 (m, 4H, Harom) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 23.43 (CH2CN), 35.22

Novel MKC-442 Analogues
1563
(CH2CH), 36.04 (CH2Ph), 115.14 (CH2¼CH), 117.89 (CN), 137.52 (CH¼CH2), 125.32, 127.90,
128.05, 128.95, 129.75, 142.87 (Carom) ppm; MS (EI): m=z ¼ 171 (Mþ).
General Procedure for Preparation of Compounds 5a, 5b and 6a, 6b
Zn dust (25–30g) was activated by stirring with 4 M HCl (100cm3) for 5 min. The zinc dust was
filtered off and washed sequentially with H2O, ethyl alcohol, and ether, dried by evaporation under
reduced pressure at 80ꢂC for 5 h, and kept in vacuo overnight. The active Zn was suspended in dry
THF (150 cm3) and heated to reflux. A few drops of ethyl 2-bromobutyrate were added and the mixture
was refluxed until a green colour appeared. Compound 4a or 4b (66 mmol) was added in one portion
and ethyl 2-bromobutyrate (25.3 g, 130 mmol) in 10 cm3 THF was added dropwise. After complete
addition the mixture was refluxed for 5 h. After cooling and dilution with THF (300cm3), the mixture
was quenched by addition of sat aqueous K2CO3 (100cm3). The mixture was stirred for 1 h and then
the THF layer was decanted and the residue was washed with THF (3ꢄ100 cm3). The combined THF
fractions were stirred with 10% aqueous HCl (90 cm3) for 45 min, then the solution was concentrated
under reduced pressure and CH2Cl2 (300 cm3) was added. The organic phase was washed with sat
aqueous NaHCO3 (2ꢄ100 cm3), dried over MgSO4, and evaporated under reduced pressure. The
residue was chromatographed on silica gel (petroleum ether (60–80ꢂC)=ethyl acetate ¼ 80=20, v=v)
to give 5a, 5b and 6a, 6b.
Ethyl 2-ethyl-3-oxo-4-[3-(allyloxy)phenyl]butyrate (5a)
1
Yield 55%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 0.84–0.92 (m, 3H, CH3CH2), 1.22–1.24 (m, 3H,
CH3CH2O), 1.84–1.88 (m, 2H, CH2CH3), 3.44–3.47 (m, 1H, COCHCO), 3.77 (s, 2H, PhCH2), 4.14–
4.17 (m, 2H, OCH2CH3), 4.50–4.52 (m, 2H, OCH2CH), 5.29–5.37 (m, 2H, CH2¼CH), 6.01–6.08 (m,
1H, CH¼CH2), 6.76–7.26 (m, 4H, Harom) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 11.74, 14.03 (2s,
2CH3), 21.46 (CH2CH3), 49.03 (CH2 ꢅ Ph), 59.36 (COCHCO), 61.24 (OCH2CH3), 68.67 (PhOCH2),
117.57 (CH2¼CH), 133.10 (CH¼CH2), 113.41, 116.03, 122.07, 129.57, 134.64, 158.74 (Carom),
169.52 (COCH2Ph), 202.43 (COOCH2) ppm; MS (EI): m=z ¼ 290 (Mþ).
Ethyl 2-ethyl-3-oxo-4-[3-(but-3-enyl)phenyl]butyrate (5b)
1
Yield 53%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 0.81–0.85 (m, 3H, CH3CH2), 1.22–1.26 (m, 3H,
CH3CH2O), 1.83–1.88 (m, 2H, CH2CH3), 2.34–2.39 (m, 2H, CH2CH), 2.66–2.71 (m, 2H, CH2CH2),
3.46 (t, J ¼ 7.4 Hz, 1H, COCHCO), 3.78 (s, 2H, Ph ꢅ CH2CO), 4.11–4.16 (m, 2H, OCH2CH3), 4.98–
5.00 (m, 2H, CH2¼CH), 5.79–5.88 (m, 1H, CH¼CH2), 7.02–7.26 (m, 4H, Harom) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 11.74, 14.04 (2s, 2CH3), 21.47 (CH2CH3), 35.17 (PhCH2CH2), 35.37 (CH2CH),
49.06 (COCH2Ph), 59.35 (COCHCO), 61.20 (OCH2CH3), 114.93 (CH2¼CH), 133.15 (CH¼CH2),
127.07, 127.24, 128.55, 129.68, 137.83, 142.26 (Carom), 169.54 (COCH2Ph), 202.64 (COOCH2) ppm;
MS (EI): m=z ¼ 288 (Mþ).
6-[3-(Allyloxy)benzyl]-4-hydroxy-3,5-diethylpyridin-2(1H)-one (6a, C19H23NO3)
1
Yield 2%; mp 188–190ꢂC; H NMR (300 MHz, DMSO-d6): ꢂ ¼ 0.84–0.97 (m, 6H, 2CH3), 2.36–
2.51 (m, 4H, 2CH2), 3.78 (s, 2H, PhCH2), 4.50 (d, J ¼ 5.18 Hz, 2H, OCH2), 5.21–5.47 (m, 2H,
CH2¼CH), 5.94–6.07 (m, 1H, CH¼CH2), 6.76–7.26 (m, 4H, Harom) 9.11–9.29 (br.s, 1H, OH),
11.04–11.12 (br.s, 1H, NH) ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼ 13.10, 14.30 (2s, 2CH3),
16.04, 17.97 (2CH2), 35.02 (CH2Ph), 68.04 (PhOCH2), 117.40 (CH2¼CH), 133.61 (CH¼CH2),
111.03 (C-3), 111.33 (C-5), 112.15, 114.88, 120.46, 129.43, 139.84, 139.98 (Carom), 158.18 (C-6),
161.64 (C-4), 163.22 (C¼O) ppm.
6-[3-(But-3-enyl)benzyl]-4-hydroxy-3,5-diethylpyridin-2(1H)-one (6b, C20H25NO2)
1
Yield 3%; mp 105–107ꢂC; H NMR (300 MHz, DMSO-d6): ꢂ ¼ 0.78–0.93 (m, 6H, 2CH3), 2.22–
2.58 (m, 8H, 4CH2), 3.75 (s, 2H, PhCH2), 4.93–4.99 (m, 2H, CH2¼CH), 5.74–5.82 (m, 1H,
CH¼CH2), 6.98–7.17 (m, 4H, Harom), 9.15–9.18 (br.s, 1H, OH), 11.18–11.20 (br.s, 1H, NH)

1564
Y. L. Aly et al.
ppm; 13C NMR (75 MHz, DMSO-d6): ꢂ ¼ 13.06, 14.20 (2s, 2CH3), 16.02, 17.95 (2CH2CH3), 34.39
(PhCH2CH2), 34.84 (CH2Ph), 35.10 (CH2CH), 115.10 (CH2¼CH), 137.82 (CH¼CH2), 110.93
(C-3), 111.25 (C-5), 125.51, 126.27, 128.18, 128,27, 138.32, 140.06, (Carom), 141.49 (C-4), 161.16
(C-6), 163.26 (C¼O) ppm.
General Procedure for Preparation of 5,6-Substituted 2-Thioxo-2,3-dihydro-1H-
pyrimidin-4-one 7a and 7b
Na (25.1 g, 1.1 mol) was dissolved in 500cm3 absolute ethanol. Thiourea (58.23g, 0.77mol) was
added and the mixture was heated to reflux. Compound 5a or 5b (0.015 mol) was added dropwise,
and the mixture was refluxed for 5 h. Ethanol was evaporated in vacuo and the residue was dissolved
in 400 cm3 H2O. The thiouracil 7a or 7b was precipitated by neutralization with conc HCl. The
precipitate was filtered off, washed with H2O, and chromatographed on silica gel (petroleum ether
(60–80ꢂC)=ethyl acetate ¼ 1=1, v=v) to give 7a and 7b.
6-[3-(Allyloxy)benzyl]-5-ethyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (7a)
1
Yield 41%; mp 69–71ꢂC; H NMR (300 MHz, CDCl3): ꢂ ¼ 0.81–0.86 (m, 3H, CH3CH2), 2.26–2.29
(m, 2H, CH2CH3), 3.81 (s, 2H, CH2Ph), 4.53 (d, J ¼ 4.9 Hz, 2H, OCH2), 5.35–5.40 (m, 2H, CH2¼CH),
6.00–6.18 (m, 1H, CH¼CH2), 6.78–7.27 (m, 4H, Harom), 12.21, 12.41 (2s, 2H, 2NH) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 12.82 (CH3), 17.67 (CH2CH3), 34.42 (CH2Ph), 68.06 (PhOCH2), 112.62 (C-5),
117.40 (CH2¼CH), 133.65 (CH¼CH2), 114.89, 117.05, 120.35, 129.64, 138.16, 149.02 (Carom),
158.23 (C-6), 161.38 (CO), 174.11 (C¼S) ppm; MS (MALDI): m=z ¼ 325 (Mþ Naþ).
6-[3-(But-3-enyl)benzyl]-5-ethyl-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (7b, C17H20N2OS)
Yield 41%; mp 179–181ꢂC; 1H NMR (300MHz, CDCl3): ꢂ ¼ 1.00–1.05 (t, J ¼ 7.4 Hz, 3H, CH3CH2),
2.32–2.47 (m, 4H, CH2CH3, CH2CH2), 2.65–2.70 (q, J ¼ 7.6 Hz, 2H, CH2CH), 3.84 (s, 2H, CH2Ph),
4.98–5.01 (m, 2H, CH2¼CH), 5.86–5.88 (m, 1H, CH¼CH2), 6.98–7.26 (m, 4H, Harom), 10.47, 10.51
(2s, 2H, 2NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.12 (CH3), 18.23 (CH2CH3), 35.11 (CH2CH),
35.25 (CH2CH2Ph), 35.58 (CH2Ph), 115.18 (C-5), 118.40 (CH2¼CH), 137.62 (CH¼CH2), 125.92,
127.88, 128.59, 129.14, 134.09, 142.97 (Carom), 149.15 (C-6), 161.88 (CO), 173.83 (C¼S) ppm;
HRMS (MALDI, peak matching): m=z ¼ 323.1193 (M þ Naþ) calcd 323.1189.
General Procedure for Preparation of 5,6-Substituted 1H-Pyrimidine-2,4-dione 8a and 8b
Compound 7a or 7b (0.01 mol) was suspended in 300 cm3 10% aqueous chloroacetic acid. The sus-
pension was refluxed overnight and the precipitate was filtered off after cooling. The precipitate was
washed with H2O and dried in vacuo to give compounds 8a and 8b.
6-[3-(Allyloxy)benzyl]-5-ethyl-1H-pyrimidin-2,4-dione (8a, C16H18N2O3)
Yield 91%; mp 155–157ꢂC; 1H NMR (300MHz, CDCl3): ꢂ ¼ 0.81–0.86 (m, 3H, CH3CH2), 2.24–2.26
(m, 2H, CH2CH3), 3.71 (s, 2H, CH2Ph), 4.53 (d, J ¼ 1.4 Hz, 2H, OCH2), 5.22–5.26 (m, 2H, CH2¼CH),
6.00–6.04 (m, 1H, CH¼CH2), 6.81–7.26 (m, 4H, Harom), 10.72, 11.01 (2s, 2H, 2NH) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 13.46 (CH3), 17.57 (CH2CH3), 34.92 (CH2Ph), 68.07 (PhOCH2), 111.27 (C-5),
117.39 (CH2¼CH), 133.60 (CH¼CH2), 112.63, 114.84, 120.41, 129.61, 138.33, 148.52 (Carom),
150.88 (C-6), 158.24, 164.45 (2CO) ppm; HRMS (MALDI, peak matching): m=z ¼ 309.1213
(Mþ Naþ) calcd 309.1384.
6-[3-(But-3-enyl)benzyl]-5-ethyl-1H-pyrimidin-2,4-dione (8b, C17H20N2O2)
Yield 91%; mp 182–184ꢂC; 1H NMR (300MHz, CDCl3): ꢂ ¼ 1.01–1.05 (m, 3H, CH3CH2), 2.30–2.35
(m, 2H, CH2CH3), 2.45–2.68 (m, 4H, PhCH2CH2, CH2CH), 3.76 (s, 2H, CH2Ph), 4.92–5.02 (m, 2H,
CH2¼CH), 5.75–5.84 (m, 1H, CH¼CH2), 7.04–7.25 (m, 4H, Harom), 9.88 (s, 2H, 2NH) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 13.60 (CH3), 18.15 (CH2CH3), 35.10 (CH2CH), 35.25 (CH2CH2Ph), 36.02
(CH2Ph), 113.28 (C5), 115.04 (CH2¼CH), 137.69 (CH¼CH2), 126.11, 127.58, 128.75, 128.91,

Novel MKC-442 Analogues
1565
134.89, 142.71 (Carom), 148.65 (C-6), 151.98, 164.96 (2CO) ppm; HRMS (MALDI, peak matching):
m=z ¼ 307.1418 (Mþ Na)þ calcd 307.1417.
General Procedure for Synthesis of 9a, 9b and 10a, 10b
N,O-Bis(trimethylsilyl)acetamide (BSA, 2.5 mmol, 6.2 cm3) was dissolved in 30cm3 dry CHCl3 under
nitrogen. Compound 8a or 8b (10 mmol) was added and after 10min the mixture become a clear
solution. Chloromethyl ethyl ether (15 mmol, 1.5 cm3) was added and the reaction mixture was stirred
overnight at room temperature under nitrogen, quenched with 25cm3 ice cold sat aqueous. NaHCO3,
and extracted with CH2Cl2 (2ꢄ50 cm3). The organic layer was dried over MgSO4 and evaporated
under reduced pressure and the residue was purified by column chromatography using ethyl acetate=
petroleum ether (60–80ꢂC) (1=1, v=v) to give compounds 9a, 9b and 10a, 10b.
6-[3-(Allyloxy)benzyl]-1-ethoxymethyl-5-ethyl-1H-pyrimidin-2,4-dione (9a, C19H24N2O4)
1
Yield 53%; oil; H NMR (300MHz, CDCl3): ꢂ ¼ 1.05–1.22 (m, 6H, 2CH3CH2), 2.45–2.52 (m, 2H,
CH2CH3), 3.60–3.72 (m, 2H, OCH2CH3), 4.11 (s, 2H, CH2Ph), 4.49 (d, J ¼ 1.40Hz, 2H, OCH2CH),
5.30–5.42 (m, 2H, CH¼CH2), 5.46 (s, 2H, NCH2O), 5.98–6.04 (m, 1H, CH¼CH2), 6.70–7.26 (m, 4H,
Harom), 8.75 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.72, 14.99 (2CH3), 19.09 (CH2CH3),
32.28 (CH2Ph), 64.92 (OCH2CH3), 68.73 (PhOCH2), 72.64 (NCH2O), 112.94 (C-5), 117.86
(CH2¼CH), 132.90 (CH¼CH2), 114.09, 116.87, 119.75, 130.14, 136.90, 148.90 (Carom), 151.94
(C-6), 159.18, 163.38 (2CO) ppm; MS (EI): m=z ¼ 344 (Mþ).
6-[3-(But-3-enyl)benzyl]-1-ethoxymethyl-5-ethyl-1H-pyrimidin-2,4-dione (9b, C20H26N2O3)
1
Yield 61%; oil; H NMR (300MHz, CDCl3): ꢂ ¼ 1.04–1.28 (m, 6H, 2CH3CH2), 2.23–2.35 (m, 2H,
CH2CH3), 2.47–2.71 (m, 4H, PhCH2CH2, CH2CH), 3.60–3.65 (m, 2H, OCH2CH3), 4.13 (s, 2H,
CH2Ph), 4.98–5.00 (m, 2H, CH2¼CH), 5.12 (s, 2H, NCH2O), 5.77–5.82 (m, 1H, CH¼CH2), 6.95–
7.27 (m, 4H, Harom), 9.97 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.70, 14.98 (2s, 2CH3),
19.08 (CH2CH3), 33.31 (CH2CH), 35.10 (CH2CH2Ph), 35.31 (CH2Ph), 64.90 (OCH2), 72.62 (NCH2O),
115.10 (C-5), 116.81 (CH2¼CH), 124.74, 127.33, 129.05, 135.11, 142.90 (Carom), 137.60 (CH¼CH2),
149.17 (C-6), 152.06, 163.55 (2CO) ppm; HRMS (MALDI, peak matching): m=z ¼ 365.1830
(Mþ Naþ) calcd 365.1836.
6-[3-(Allyloxy)benzyl]-1,3-bis(ethoxymethyl)-5-ethyl-1H-pyrimidin-2,4-dione (10a)
1
Yield 6%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 1.05–1.25 (m, 9H, 3CH3), 2.45–2.50 (m, 2H,
CH2CH3), 3.57–3.74 (m, 4H, 2OCH2CH3), 4.16 (s, 2H, CH2Ph), 4.50 (d, J ¼ 1.29 Hz, 2H,
OCH2CH¼CH2), 5.13 (s, 2H, N3CH2O), 5.26–5.30 (m, 2H, CH¼CH2), 5.47 (s, 2H, N1CH2O),
5.98–6.07 (m, 1H, CH¼CH2), 6.70–7.27 (m, 4H, Harom) ppm; 13C NMR (75 MHz, CDCl3):
ꢂ ¼ 13.67, 15.00, 15.15 (3CH3), 19.69 (CH2CH3), 33.36 (CH2Ph), 65.02, 65.86 (2OCH2CH3),
68.75 (PhOCH2), 71.09 (N3CH2O), 73.45 (N1CH2O), 112.84 (C-5), 117.89 (CH2¼CH), 132.89
(CH¼CH2), 114.21, 116.13, 119.79, 130.14, 136.79, 147.63 (Carom), 152.51 (C-6), 159.17, 162.66
(2CO) ppm; MS (EI): m=z ¼ 402 (Mþ).
6-[3-(But-3-enyl)benzyl]-1,3-bis(ethoxymethyl)-5-ethyl-1H-pyrimidin-2,4-dione
(10b, C23H32N2O4)
Yield 13%; oil; 1H NMR (300MHz, CDCl3): ꢂ ¼ 1.04–1.26 (m, 9H, 3CH3), 2.32–2.35 (m, 2H,
CH2CH3), 2.48–2.70 (m, 4H, PhCH2CH2, CH2CH), 3.57–3.74 (m, 4H, 2OCH2CH3), 4.12 (s, 2H,
CH2Ph), 4.97–4.98 (m, 2H, CH2¼CH), 5.13 (s, 2H, N1CH2O), 5.47 (s, 2H, N3CH2O) 5.76–5.82 (m,
1H, CH¼CH2), 6.92–7.27 (m, 4H, Harom) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.67, 15.00, 15.15
(3CH3), 19.69 (CH2CH3), 33.39 (CH2CH2Ph), 35.10 (CH2Ph), 35.31 (CH2CH), 64.98, 65.84 (2OCH2),
71.07, 73.42 (2NCH2O), 115.10 (C-5), 116.04 (CH2¼CH), 124.74, 127.34, 127.36, 129.05, 135.11,
142.89 (Carom), 137.59 (CH¼CH2), 147.89 (C-6), 152.54, 162.67 (2CO) ppm; HRMS (MALDI, peak
matching): m=z ¼ 423.2237 (Mþ Naþ) calcd 423.2254.

1566
Y. L. Aly et al.
General Procedure for Synthesis of 11a and 11b
Compound 8a or 8b (3mmol) was dissolved in 30cm3 CH3CN under nitrogen and BSA (10.5mmol,
2.6 cm3) was added. The reaction mixture became clear after 15min and after cooling to ꢃ50ꢂC TMS
triflate (3mmol, 0.54 cm3) was added followed by dropwise addition of bis(allyloxy)methane. The
reaction mixture was stirred at room temperature for 30h, quenched with a cold solution of sat aqueous
NaHCO3 (5cm3). The solvent was evaporated under reduced pressure and the residue was extracted
with diethyl ether (3ꢄ50 cm3), dried over MgSO4, evaporated under reduced pressure, and chromato-
graphed using ethyl acetate=petroleum ether (60–80ꢂC) (1=1, v=v) to give 11a and 11b.
6-[3-(Allyloxy)benzyl]-1-allyloxymethyl-5-ethyl-1H-pyrimidin-2,4-dione (11a, C20H24N2O4)
Yield 48%; semisolid; 1H NMR (300MHz, CDCl3): ꢂ ¼ 1.05–1.10 (m, 3H, CH3), 2.44–2.51 (m, 2H,
CH2CH3), 4.11 (d, J ¼ 5.2 Hz, 2H, PhOCH2), 4.13 (s, 2H, PhCH2), 4.50 (d, J ¼ 5.3 Hz, 2H, OCH2CH),
5.14 (s, 2H, NCH2O), 5.16–5.32 (m, 4H, 2CH2¼CH), 5.81–6.07 (m, 2H, 2CH¼CH2), 6.80–7.27 (m,
4H, Harom), 9.73 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.73 (CH3), 19.10 (CH2CH3),
33.34 (CH2Ph), 68.74 (PhOCH2), 70.47 (2OCH2CH), 72.48 (NCH2O), 112.99 (C-5), 117.65, 117.86
(2CH2¼CH), 114.11, 116.97, 119.76, 130.17, 136.66, 148.80 (Carom), 132.89, 133.57 (2CH¼CH2),
151.92 (C-6) 159.20, 163.33 (2CO) ppm; HRMS (MALDI, peak matching): m=z ¼ 379.1623 (M þ Naþ)
calcd 379.1628.
6-[3-(But-3-enyl)benzyl]-1-allyloxymethyl-5-ethyl-1H-pyrimidin-2,4-dione (11b, C21H26N2O)
Yield 67%; semisolid; 1H NMR (300MHz, CDCl3): ꢂ ¼ 1.04–1.98 (m, 3H, CH3), 2.30–2.35 (m, 2H,
CH2CH3), 2.47–2.52 (m, 2H, CH2CH¼CH2), 2.65–2.71 (m, 2H, PhCH2CH2), 4.11 (s, 2H, CH2Ph),
4.20 (s, 2H, NCH2O), 5.03–5.16 (m, 2H, OCH2CH), 5.26–5.33 (m, 4H, 2CH2¼CH), 5.80–5.91 (m,
2H, 2CH¼CH2), 6.92–7.27 (m, 4H, Harom), 10.01 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3):
ꢂ ¼ 13.72 (CH3), 19.09 (CH2CH3), 33.37 (PhCH2CH2), 35.10 (CH2Ph), 35.30 (CH2CH¼CH2), 70.47
(OCH2), 72.68 (NCH2O), 115.13 (C-5), 116.91, 117.62 (2CH2 ¼CH), 124.75, 127.34, 127.38, 129.09,
137.59, 142.94 (Carom), 133.59, 135.01 (2CH¼CH2), 149.09 (C-6) 152.07, 163.53 (2CO) ppm; HRMS
(MALDI, peak matching): m=z ¼ 377.1825 (M þ Naþ) calcd 377.1836.
General Procedure for Synthesis of the Aldehydes 12a and 12b
Compound 9a or 9b (0.75 mmol) was dissolved in 15 cm3 dry CH2Cl2 and cooled to ꢃ78ꢂC. O3 was
bubbled through the solution until a blue colour appeared (ca. 2.5 min). Then O2 was bubbled through
the solution until the blue colour disappeared. Dimethyl sulfide (0.28 g, 4.5 mmol) was then added at
ꢃ78ꢂC, and the solution was allowed to warm to room temperature and stirred overnight. Then the
solvent was evaporated under reduced pressure and the residue was chromatographed using methylene
chloride=ethyl acetate (1=1, v=v) to afford compounds 12a and 12b.
[3-(3-Ethoxymethyl-5-ethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-
ylmethyl)phenoxy]acetaldehyde (12a, C18H22N2O5)
1
Yield 28%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 1.04–1.25, (m, 6H, 2CH3), 2.45–2.50 (m, 2H,
CH2CH3), 3.37 (d, J ¼ 2.3 Hz, 2H, OCH2CHO), 3.59–3.62 (m, 2H, OCH2CH3), 4.14 (s, 2H, CH2Ph),
4.58 (s, 2H, NCH2O), 6.72–7.30 (m, 4H, Harom), 9.84 (s, 1H, NH) 9.85 (s, 1H, CHO) ppm; 13C NMR
(75 MHz, CDCl3): ꢂ ¼ 13.71, 14.97 (2CH3), 19.16 (CH2CH3), 33.23 (CH2Ph), 64.97 (OCH2CH3),
72.62 (NCH2O), 74.94 (PhOCH2), 112.50 (C-5), 114.34, 116.96, 120.81, 130.46, 137.32, 148.80
(Carom), 151.81 (C-6), 158.22, 163.24 (2CO), 198.54 (CHO) ppm; HRMS (MALDI, peak matching):
m=z ¼ 396.1420 (Mþ Naþ) calcd 369.1421.
3-[3-(3-Ethoxymethyl-5-ethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-ylmethyl)phenyl]propanal
(12b, C19H24N2O4)
1
Yield 18%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 1.04–1.25 (m, 6H, 2CH3CH2), 2.45–2.48 (m,
2H, CH2CH3), 2.74–2.79 (m, 2H, PhCH2CH2), 2.91–2.96 (m, 2H, CH2CHO), 3.57–3.64 (m, 2H,

Novel MKC-442 Analogues
1567
OCH2CH3), 4.13 (s, 2H, CH2Ph), 5.11 (s, 2H, NCH2O), 6.94–7.28 (m, 4H, Harom), 9.71 (s, 1H,
NH), 9.80 (s, 1H, CHO) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.70, 14.98 (2CH3), 19.12
(CH2CH3), 27.87 (PhCH2CH2), 33.28 (CH2Ph), 45.06 (CH2CHO), 64.94 (OCH2), 72.67 (NCH2O),
118.88 (C-5), 125.15, 127.20, 127.31, 129.40, 135.55, 141.49 (Carom), 149.01 (C-6), 151.94,
163.40 (2CO), 201.04 (CHO) ppm; HRMS (MALDI, peak matching): m=z ¼ 367.1627 (M þ Naþ)
calcd 367.1628.
[3-(3-Ethoxymethyl-5-ethyl-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-
ylmethyl)phenoxy]acetaldehyde oxime (13a)
Compound 12a (0.34 g, 1 mmol) was dissolved in 50 cm3 absolute ethanol. Hydroxylamine hydro-
chloride (0.06 g, 1 mmol) and triethylamine (0.20g, 2 mmol) were added. The reaction mixture was
refluxed for 2 h. The ethanol was removed in vacuo and the residue was chromatographed using
methylene chloride=ethyl acetate (1=1, v=v) to afforded compound 13a.
1
Yield 38%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 1.05–1.25, (m, 6H, 2CH3), 2.46–2.48 (m,
2H, CH2CH3), 3.59 (m, 2H, OCH2CH), 3.62–3.63 (m, 2H, OCH2CH3), 4.13 (s, 2H, CH2Ph), 4.78
(s, 2H, NCH2O), 4.88–5.10 (m, 1H, CH¼NOH), 6.68–7.57 (m, 4H, Harom), 9.74 (br.s, 1H, OH)
10.14 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.61,14.90 (2CH3), 19.01 (CH2CH3),
33.23 (CH2Ph), 61.75 (PhOCH2), 64.76 (OCH2CH3), 72.62 (NCH2O), 112.95 (C-5), 113.48,
116.91, 120.33, 130.32, 136.88, 148.78 (Carom), 146.52 (C¼NOH), 152.00 (C-6), 158.56, 163.77
(2CO) ppm.
General Procedure for Synthesis of the Epoxide 14a and 14b
Compound 9a or 9b (1.25 mmol) was dissolved in 20cm3 dry CHCl3 at room temperature. MCPBA
(235 mg, 1.5 mmol) was added and the reaction mixture was stirred at room temperature under nitrogen
for 3 days. Then 20 cm3 CHCl3 were added and the organic phase was washed with 2ꢄ20 cm3 sat
aqueous Na2S2O3, 2ꢄ20cm3, sat aqueous NaHCO3, and 2ꢄ20cm3 brine. The organic phase was
dried (MgSO4), and evaporated under reduced pressure. The products 14a and 14b were isolated after
column chromatography (ethyl acetate=petroleum ether (60–80ꢂC) (1=1, v=v).
6-[-3-(2-Oxiranylmethoxy)benzyl]-1-ethoxymethyl-5-ethyl-1H-pyrimidine-2,4-dione
(14a, C19H24N2O5)
1
Yield 18%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 0.85–0.91, 1.06–1.09 (2m, 6H, 2CH3CH2), 2.45–
2.48 (m, 2H, CH2CH3), 2.74, (m, 2H, CH2 epoxid), 3.34–3.37 (m, 2H, OCH2CH), 3.60–3.62 (m, 2H,
OCH2CH3), 3.92–3.94 (m, 1H, CH epoxide), 4.12 (s, 2H, CH2Ph), 5.11 (s, 2H, NCH2O), 6.69–7.27
(m, 4H, Harom), 9.88 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.68, 14.95 (2CH3), 19.05
(CH2CH3), 33.22 (CH2Ph), 44.51 (CH2 epoxide), 49.95 (CH epoxide), 64.89 (OCH2CH3), 68.65
(PhOCH2), 72.62 (NCH2O), 112.73 (C-5), 114.04, 116.85, 120.09, 130.17, 136.87, 148.80 (Carom),
151.93 (C-6), 159.03, 163.42 (2CO) ppm; HRMS (MALDI, peak matching): m=z ¼ 383.1567
(Mþ Naþ) calcd 383.1577.
6-[-3-(2-Oxiranylethyl)benzyl]-1-ethoxymethyl-5-ethyl-1H-pyrimidine-2,4-dione
(14b, C20H26N2O4)
1
Yield 37%; oil; H NMR (300MHz, CDCl3): ꢂ ¼ 1.04–1.20 (m, 6H, 2CH3CH2), 1.78–1.86 (m, 2H,
CH2CH3), 2.46–2.48 (m, 2H, PhCH2CH2), 2.74–2.80 (m, 4H, PhCH2CH2, CH2 epoxid), 2.93–2.95
(m, 1H, CH epoxid), 3.57–3.64 (m, 2H, OCH2CH3), 4.13 (s, 2H, CH2Ph), 5.11 (s, 2H, NCH2O), 6.95–
7.27 (m, 4H, Harom), 9.55 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.72, 15.00 (2CH3),
19.14 (CH2CH3), 32.11 (PhCH2CH2), 33.32 (CH2Ph), 34.14 (CH2CH), 47.11 (CH2 epoxid), 51.64 (CH
epoxide), 64.95 (OCH2), 72.68 (NCH2O), 116.82 (C-5), 124.93, 127.30, 129.27, 135.37, 142.41
(Carom), 149.13 (C-6) 151.90, 163.34 (2CO) ppm; HRMS (MALDI, peak matching): m=z ¼ 381.1773
(Mþ Naþ) calcd 381.1785.

1568
Y. L. Aly et al.
6-[3-(Allyloxy)benzyl]-2-methylthio-5-ethyl-1H-pyrimidine-4-one (15, C17H20N2O2S)
Compound 7a (0.6g, 2 mmol) was dissolved in 30cm3 dry DMF under nitrogen and methyl iodide
(0.56 g, 4 mmol, 0.26 cm3) was added. The reaction mixture was stirred at room temperature for 48h
and the solvent was evaporated under reduced pressure. The residue was chromatographed using ethyl
acetate=petroleum ether (60–80ꢂC) (1=1, v=v) to give 15.
Yield 35%; semisolid; 1H NMR (300MHz, CDCl3): ꢂ ¼ 1.05–1.11 (m, 3H, CH3CH2), 2.50 (s, 3H,
SCH3), 2.53–2.58 (m, 2H, CH2CH3), 3.87 (s, 2H, CH2Ph), 4.49 (d, 2H, J ¼ 5.2 Hz, OCH2), 5.28–5.35
(m, 2H, CH2¼CH), 6.00–6.08 (m, 1H, CH¼CH2), 6.74–7.20 (m, 4H, Harom), 12.64, (s, 1H, NH) ppm;
13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.15 (CH3), 13.19 (SCH3), 18.76 (CH2CH3), 40.46 (CH2Ph), 68.69
(PhOCH2), 112.38 (C-5), 117.61 (CH2¼CH), 133.27 (CH¼CH2), 115.62, 121.52, 122.17 129.20,
139.80, 157.03 (Carom), 158.56 (C-6), 161.46 (CO), 165.08 (C¼N); HRMS (MALDI, peak matching):
m=z ¼ 339.1127 (Mþ Naþ) calcd 339.1138.
General Procedure for Synthesis of Compounds 16a and 16b
Compound 7a (0.61 g, 2.03 mmol) was dissolved in 20 cm3 dry DMF. K2CO3 (0.27 g, 1.95 mmol) and
isopropyl bromide or isobutyl bromide (2.33 mmol) were added. The reaction mixture was stirred at
room temperature for 24 h, then poured on cold H2O and extracted with ethyl acetate. The organic
phase was washed with 2ꢄ50 cm3 sat aqueous NaCl, dried over MgSO4, and evaporated under reduced
pressure. The products 16a and 16b were isolated after column chromatography (chloroform=
petroleum ether (60–80ꢂC) (70=30, v=v).
4-[3-(Allyloxy)benzyl]-5-ethyl-6-isopropoxy-2-(isopropylsulfanyl)pyrimidin (16a, C22H30N2O2S)
1
Yield 72%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 0.93–0.98 (m, 3H, CH3CH2), 1.31 (d, J ¼ 7.3 Hz,
6H, 2CH3), 1.41 (d, J ¼ 7.1 Hz, 6H, 2CH3), 2.46–2.53 (m, 2H, CH2CH3), 3.88–3.90 (m, 1H, SCHPri),
3.96 (s, 2H, CH2Ph), 4.49 (d, J ¼ 1.3 Hz, 2H, PhOCH2), 5.23–541 (m, 3H, CH2¼CH, OCHPri), 5.97–
6.07 (m, 1H, CH¼CH2), 6.72–7.25 (m, 4H, Harom) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.19 (CH3
at C-5), 18.27 (CH2CH3), 21.91, 23.07 (2s, 4CH3Pri), 35.60 (SCH), 40.53 (CH2Ph), 68.69 (PhOCH2),
69.04 (OCHPri), 112.33 (C-5), 117.60 (CH2¼CH), 133.29 (CH¼CH2), 115.47, 116.69, 121.42, 129.18,
131.19, 140.15 (Carom), 158.56 (C-6), 165.33, (CO), 167.11 (C¼N) ppm; HRMS (MALDI, peak
matching): m=z ¼ 409.1923 (Mþ Naþ) calcd 409.1920.
4-[3-(Allyloxy)benzyl]-5-ethyl-6-isobutyloxy-2-(isobutylsulfanyl)pyrimidin (16b, C24H34N2O2S)
1
Yield 68%; oil; H NMR (300 MHz, CDCl3): ꢂ ¼ 0.94–0.99 (m, 3H, CH3CH2), 1.01 (d, J ¼ 3.6 Hz,
6H, 2CH3), 1.02 (d, J ¼ 6.7 Hz, 6H, 2CH3), 2.01–2.09 (m, 2H, 2CHButi), 2.51–2.59 (m, 2H, CH2CH3),
2.98 (d, J ¼ 6.7 Hz, 2H, SCH2), 3.97 (s, 2H, CH2Ph), 4.09 (d, J ¼ 6.4 Hz, 2H, OCH2) 4.47 (d,
J ¼ 5.2 Hz, 2H, PhOCH2), 5.23–5.24 (m, 2H, CH2¼CH), 5.99–6.07 (m, 1H, CH¼CH2), 6.72–7.25
(m, 4H, Harom), 12.64 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3): ꢂ ¼ 13.29 (CH3 at C-5), 18.29
(CH2CH3), 19.21, 21.95 (4CH3Buti), 27.86, 28.68 (CHButi), 39.43 (SCH2), 40.48 (CH2Ph), 68.69
(PhOCH2), 72.59 (OCH2Buti), 112.37 (C-5), 117.61 (CH2¼CH), 133.28 (CH¼CH2), 115.43, 116.49,
121.42, 129.20, 131.18, 140.19, (Carom), 158.59 (C-6), 165.25, (CO), 167.48 (C¼N) ppm; HRMS
(MALDI, peak matching): m=z ¼ 437.2224 (M þ Naþ) calcd 437.2233.
6-[3-(Allyloxy)benzyl]-2-dimethylamino-5-ethyl-1H-pyrimidine-4-one (17, C18H23N3O2)
The ꢁ-ketoester 5a was dissolved in EtONa (Na, 0.14g, 6.3 mmol in 50 cm3 abs EtOH). N,N-
Dimethylguanidine sulfate (1.17 g, 4.3 mmol) was added and the reaction mixture was refluxed for
48h, cooled, filtered, evaporated to the half volume in vacuo, poured into H2O, and extracted with
chloroform. The organic layer was washed with H2O (2ꢄ50cm3), dried over MgSO4, and evaporated
in vacuo. The residue was chromatographed using CHCl3=MeOH (9=1, v=v). Yield 38%; oil; 1H NMR
(300 MHz, CDCl3): ꢂ ¼ 0.96–1.01 (m, 3H, CH3), 2.42–2.49 (m, 2H, CH2CH3), 3.11 (s, 6H, 2NCH3),
3.78 (s, 2H, PhCH2), 4.49 (d, J ¼ 5.3Hz, 2H, OCH2CH), 5.23–5.27 (m, 2H, CH2 ¼ CH), 6.01–6.08 (m,
1H, CH ¼ CH2), 6.72–7.25 (m, 4H, Harom), 9.74 (s, 1H, NH) ppm; 13C NMR (75 MHz, CDCl3):

Novel MKC-442 Analogues
1569
ꢂ ¼ 13.72 (CH3), 18.44 (CH2CH3), 37.20 (2NCH3), 40.91 (CH2Ph), 68.65 (PhOCH2), 112.13 (C-5),
117.52 (CH2 ¼ CH), 133.35 (CH¼CH2), 112.72, 115.50, 121.51, 129.01, 140.61, 152.03 (Carom),
158.46 (C-6), 163.44 (CO), 165.94 (NC¼N) ppm; HRMS (MALDI, peak matching): m=z ¼ 314.2001
(Mþ Hþ) calcd 314.1980.
Antiviral Assay Procedures
Compounds were solubilized in DMSO at 200mM and then diluted in culture medium.
Cells and Viruses
MT-4, C8166, and H9=IIIB cells were grown at 37ꢂC in a 5% CO2 atmosphere in RPMI 1640 medium
supplemented with 10% fetal calf serum (FCS), 100 IU=cm3 penicillin G, and 100ꢀg=cm3 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) were obtained
from supernatants of persistently infected H9=IIIB cells. The HIV-1 stock solutions had titers of
4.5ꢄ106 50% cell culture infectious dose (CCID50)=cm3. The K103R-V179D-P225H mutant was
derived from a IIIB strain passaged in C8166 cells in the presence of efavirenz (up to 2ꢀM). The
Y181C mutant (NIH N119) was derived from an AZT-sensitive clinical isolate passaged initially in
CEM and then in MT-4 cells in the presence of nevirapine (10 ꢀM). The K103N þ Y181C (NIH A17)
was derived from the IIIB strain passaged in H9 cells in the presence of BI-RG 587 (1ꢀM).
K103R þV179D þ P225H (EFVR), Y181C, and K103N þ Y181C stock solutions had titers of
3.0ꢄ105, 1.3ꢄ106, and 2.5ꢄ105 CCID50=cm3, respectively.
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 determined by light
microscope scoring of syncytia after 4 days of incubation. Virus titers were expressed as CCID50=cm3.
Anti-HIV Assays
The activity of test compounds against multiplication of wild type HIV-1, Y181C, and
K103N þ Y181C in acutely infected cells was based on inhibition of virus-induced cytopathicity in
MT-4 cells. The activity of the compounds against the EFVR multiplication in acutely infected cells
was based on inhibition of p24 antigen in C8166 cells. Briefly, an amount of 50mm3 of culture
medium containing 1ꢄ104 cells was added to each well of flat-bottom microtiter trays containing
50mm3 of culture medium with or without various concentrations of test compounds. Then an amount
of 20 mm3 of HIV suspensions (containing the appropriate amount of CCID50 to cause complete
cytopathicity at day 4) was added. After incubation at 37ꢂC, cell viability was determined by the
3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT) method [16]. Alternatively, p24
levels were determined by an immunoenzymatic kit (Abbott). 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.
Acknowledgement
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) [17].
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1570
Y. L. Aly et al.: Novel MKC-442 Analogues
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[17] 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