Synthesis of novel fluoro analogues of MKC442 as microbicides

Article abstract of DOI:10.1021/jm500139a

Novel analogues of MKC442 (6-benzyl-1-(ethoxymethyl)-5-isopropylpyrimidine- 2,4(1H,3H)-dione) were synthesized by reaction of 6-[(3,5-dimethylphenyl) fluoromethyl]-5-ethyluracil (5) with ethoxymethyl chloride and formaldehyde acetals. The Sonogashira reaction was carried out on the N1-(p-iodobenzyl)oxy] methyl derivative of compound 5 using propagyl alcohol to afford compound 12 (YML220). The latter compound was selected for further studies since it showed the most potent and selective activity in vitro against wild-type HIV-1 and non-nucleoside reverse transcriptase inhibitor-, nucleoside reverse transcriptase inhibitor-, and protease inhibitor-resistant mutants and a wide range of HIV-1 clinical isolates. 12 also showed microbicidal activity in long-term assays with heavily infected MT-4 cells.

Full text of DOI:10.1021/jm500139a

Article
pubs.acs.org/jmc
Synthesis of Novel Fluoro Analogues of MKC442 as Microbicides
Yasser M. Loksha,†,§ Erik B. Pedersen,*,† Roberta Loddo,‡ Giuseppina Sanna,‡ Gabriella Collu,‡
Gabriele Giliberti,‡ and Paolo La Colla*,‡
†Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230
Odense M, Denmark
‡Dipartimento di Scienze Biomediche, Universita
̀
di Cagliari, Cittadella Universitaria, 09042 Monserrato, Cagliari, Italy
§Faculty of Pharmacy and Pharmaceutical Industries, Sinai University, Al-Arish, North Sinai, Egypt
S
* Supporting Information
ABSTRACT: Novel analogues of MKC442 (6-benzyl-1-
(ethoxymethyl)-5-isopropylpyrimidine-2,4(1H,3H)-dione) were
synthesized by reaction of 6-[(3,5-dimethylphenyl)-
fluoromethyl]-5-ethyluracil (5) with ethoxymethyl chloride
and formaldehyde acetals. The Sonogashira reaction was carried
out on the N1-(p-iodobenzyl)oxy]methyl derivative of com-
pound 5 using propagyl alcohol to afford compound 12
(YML220). The latter compound was selected for further
studies since it showed the most potent and selective activity in
vitro against wild-type HIV-1 and non-nucleoside reverse
transcriptase inhibitor-, nucleoside reverse transcriptase inhibitor-, and protease inhibitor-resistant mutants and a wide range
of HIV-1 clinical isolates. 12 also showed microbicidal activity in long-term assays with heavily infected MT-4 cells.
1), which showed favorable pharmacokinetics when delivered
through gels or vaginal rings18,19 and is now being tested in
phase III clinical trials,20 and MC122021 (Chart 1), which
showed promising potential as a microbicide in rhesus
monkeys, no matter whether delivered through liposomes or
vaginal rings.22−24
The structural similarities among MKC442, TNK651,
SJ3366, TMC120, and MC1220 prompted the present study
aimed at the synthesis and biological evaluation of a series of
fluoro analogues of MKC442. Among them, 12 (YML220)
(Scheme 3) emerged as the most potent derivative in cell-based
assays against the HIV-1 wt and variants carrying clinically
relevant NRTI, NNRTI, and protease inhibitor (PRI)
mutations. Viruses resistant to 12, MC1220, TMC120, and
TMC125 were then selected in vitro, and their mutations and
cross-resistance patterns were defined. Finally, 12 and
structurally related NNRTIs were comparatively evaluated for
microbicidal activity in long-term assays and efficacy against
clinical isolates belonging to various HIV-1 clades.
INTRODUCTION
■
MKC442 (emivirine) (Chart 1) is one of the first-generation
non-nucleoside reverse transcriptase inhibitors (NNRTIs),1−4
endowed with potent and selective activity against the HIV-1
wild type (wt) but not against NNRTI-resistant mutants.
Second-generation NNRTIs were characterized by a much
greater resilience to the presence of single-point resistance
mutations; among them are DMP266 (efavirenz)5 (Chart 1)
approved for the treatment of HIV/AIDS in combination
therapy and TMC125 (etravirine)6 (Chart 1).
Several attempts have been made to increase the activity of
MKC442 against NNRTI-resistant mutants.7−11 Hopkins et
al.12 synthesized TNK651 (Chart 1), and then El-Brollosy et
al.13 synthesized a series of hybrid analogues of MKC442 and
TNK651. Some of these compounds showed activity in the
picomolar range against the HIV-1 wt and in the
submicromolar range against clinically relevant Y181C and
K103N resistant mutants (MKC442-resistant mutants in-
cluded). Wamberg et al.14 synthesized hybrid analogues of
MKC442 and SJ3366 (Chart 1) with the aim of investigating
whether the substitution of the 6-aryl ketone in SJ336615 with a
6-arylvinyl group and that of an ethoxymethyl group with
various (allyloxy)methyl moieties could lead to improved
activity against HIV-1. The new derivatives turned out active
against wt HIV-1 in the potency range of DMP266 and
moderately active against Y181C and Y181C + K103N resistant
mutants.
CHEMISTRY
■
Two different routes were explored for the synthesis of 6-
[fluoro(3,5-dimethylphenyl)methyl]-5-ethylpyrimidine-2,4-
(1H,3H)-dione (5). In the first one, the protected 5-aroyluracil
125 was reduced by sodium borohydride in methanol to the
hydroxy compound 2, which was fluorinated by
(diethylamino)sulfur trifluoride (DAST) to give the fluoro
In the meantime, the NNRTIs emerged as microbicides with
the best potential for the prevention of transmission of HIV-1
infections. Among them were TMC120 (dapivirine)16,17 (Chart
Received: January 27, 2014
© XXXX American Chemical Society
A
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considered as a novel one-pot synthesis of 6-benzoyluracil
derivatives without separation of the intermediates. Reduction
of compound 8 to the hydroxy derivative 4a and subsequent
fluorination by DAST afforded the desired compound 5
(Scheme 2).
Chart 1. Highly Potent NNRTIs
Scheme 2
derivative 3. Deprotection of compound 3, through acidic
hydrolysis using 4 M HCl and ethanol, furnished two different
compounds, 4a,b, in which the fluoro atom was substituted
with a hydroxyl and with an ethoxy group, respectively.
However, the desired compound 5 was not obtained (Scheme
1).
Compound 5 was silylated with N,O-bis(trimethylsilyl)-
acetamide and alkylated by treatment with either ethoxymethyl
chloride or a bis(allyloxy)methane,13 in the presence of
trimethylsilyl trifluoromethanesulfonate (TMS triflate) as a
Lewis acid catalyst,28 to give the MKC442 analogues 9a−c
(Scheme 2).
Scheme 1
Danel et al.29 synthesized bis[(4-iodobenzyl)oxy]methane
(10) according to a previously published method for the
preparation of formaldehyde acetals13,30 (Scheme 3) in which
methylene bromide, p-iodobenzyl alcohol, KOH, and tetrabu-
tylammonium bromide were refluxed in benzene, and the
reaction product was isolated by column chromatography in
71% yield. We synthesized the acetal 10 by coupling 1 equiv of
methylene bromide with 2 equiv of p-iodobenzyl alcohol using
sodium hydride in DMF, and the reaction product was isolated
as a solid compound in 96% yield and used without any further
purification (Scheme 3).
Compound 11a (analogue of TNK-651)12 was synthesized
by alkylation with the acetal 10 using the same procedure
described for compounds 9b,c. The N3 coupling product was
also observed (compound 11b). The assignement of N1 versus
N3 coupling product was based on comparison with 13C NMR
spectra of the corresponding N-methyluracils.31 In support of
the assignment, it was found that the enantiotopic protons of
the methylene group at N1 in compound 11a close to the chiral
In the second route, 5-ethyl-2,4,6-trichloropyrimidine (6)26
was reacted with 3,5-dimethylbenzyl cyanide using sodium
hydride in DMF to give compound 7 as previously described by
Loksha.27 Compound 7 was refluxed in methanolic sodium
methoxide to replace the chlorine atoms with methoxy groups
and then oxidized by a stream of oxygen to achieve the carbonyl
group. This was followed by evaporization of methanol and
refluxing the residual material in 4 M HCl to give the (3,5-
dimethylbenzoyl)uracil derivative 8. This method can be
1
center were found at different H NMR chemical shifts (5.00
and 5.38 ppm) as an AB system, whereas those far away from
the chiral center at N3 in compound 11b were recorded as a
singlet at 5.45 ppm.
B
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that of TMC120, but over 10-fold superior to that of MC1220,
against the NNRTI-resistant mutants.
Scheme 3
The above results suggest that the presence of a conjugated
system, such as the benzene ring in ether linkage at N1 with the
uracil ring, leads to an increased potency against HIV-1
mutants, possibly due to stacking with the aromatic parts of the
amino acid residues present in the mutated reverse tran-
scriptase. This is in agreement with the previously published
work on TNK-651.12 The triple bond on the benzene ring can
also contribute to stacking, not to mention the importance of
the hydroxy group in establishing hydrogen bonding between
12 and an acceptor residue in the non-nucleoside binding site.
In fact, when the hydroxyl group was oxidized into a carbonyl
group, the corresponding compound 13 showed a significantly
lower potency than 12 against both wt HIV-1 and NNRTI-
resistant mutants. In all cases, with respect to the compounds
synthesized by El-Brollosy et al.13 and Danel et al.,38 the
presence of a fluoro atom at the methylene group of 6-
substituents leads to an increase of potency against HIV-1-
resistant mutants.
The comparative genomic analysis of resistant mutants and
parental wt HIV-1 (subcultured for the same time period in the
absence of drugs) shows (Table 2) that no mutations occur in
the p10, p15, and p31 genes of the latter, whereas different
mutation patterns characterize the RT (p66/p51) gene of the
former. These findings are consistent with the capability of the
above NNRTIs to inhibit the recombinant reverse transcriptase
(rRT) of the HIV-1 wt in enzyme assays (results not shown).
In particular, mutants resistant to 12 and MC1220 share the
mutation Y181C (commonly related to failure of a combination
antiretroviral therapy including nevirapine39 or other NNRTI)
associated, in the former mutant, with V106I and, in the latter,
with L100I and V179D. It is worth noting that V106I is a
polymorphism occurring with similar frequency in untreated
and NNRTI-treated patients (HIV Drug Resistance Database,
Stanford University, http://hivdb.stanford.edu/cgi-bin/
MutPrevBySubtypeRx.cgi); however, when in combination
with V179D, V106I is believed to be responsible for NNRTI
resistance.40 The L100I mutation has been described, together
with another mutation, in patients subjected to a DMP266
treatment and, as a single mutation, in patients subjected to
nevirapine or delavirdine treatments.41 V179D has been
described in clinical isolates less commonly than other
mutations.42,43 Both L100I and V179D have also been
described in variants selected by treatments with novel
NNRTIs, such as capravirine42 or benzophenones.44,45
The susceptibility of the above-mentioned mutants to 12,
NVP, MC1220, DMP266, TMC120, TMC125, AZT, and SQV
was comparatively evaluated in MT-4 cells. As reported in
Table 3, all resistant mutants show full susceptibility to AZT
and SQV, and with the exception of the TMC125-resistant
mutant, they show a significant susceptibility to DMP266.
Finally, all of them cross-resist to the other NNRTIs tested. A
notable exception is represented by the MC1220- and
TMC120-resistant mutants, which share the 100I mutation
and, interestingly, retain a significant susceptibility to 12.
As HIV-1 strains have diverged widely in different regions of
the world, the antiretroviral efficacy of 12 and reference
inhibitors was evaluated in dendritic cells infected with HIV-1
clinical isolates belonging to the subtypes predominant in
various continents and including wt strains and variants
carrying clinically relevant NRTI mutations. As shown in
Table 4, the efficacy of reference compounds in inhibiting the
Sonogashira cross-coupling32−35 was applied on compound
11a and propagyl alcohol, using copper(I) iodide and
PdCl2(PPh3)3 as catalysts, to afford compound 12. The
alcoholic part of compound 12 was oxidized to the aldehyde
derivative 13 by treatment with 2-iodoxybenzoic acid (IBX)36,37
in ethyl acetate (Scheme 3).
ANTIVIRAL ACTIVITY
■
Cytotoxicity and antiviral activity of the newly synthesized
compounds were determined in MT-4 cell-based assays. The
antiretroviral spectrum was determined using HIV-1 laboratory
strains, both the wt and variants carrying clinically relevant
NRTI, NNRTI, and PRI mutations and one or more of the
following reference inhibitors: MKC442, nevirapine (NVP),
MC1220, DMP266, TMC120, TMC125, zidovudine (AZT),
and SQV.
Test compounds (Table 1) showed cytotoxicity in the range
of 27−100 μM, whereas TMC120, TMC125, and SQV were
the most cytotoxic (CC50 = 2, 11, and 15, respectively)
reference inhibitors. Among the newly synthesized derivatives,
12 was by far the most potent against both wt and mutant HIV-
1; in this respect, it turned out to be even significantly more
potent than MKC442 and NVP. Interestingly, the potency of
12 turned out to be comparable to that of MC1220, DMP266,
TMC120, and TMC125 against wt HIV-1 and comparable to
C
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Table 1. Cytotoxicity and Antiviral Activity of Test and Reference Compounds against the HIV-1 wt and Variants Carrying
a
Clinically Relevant NRTI, NNRTI, and PRI Mutations
b
c
CC50
EC50
DMP266R (100I, 103R, 179D,
225H)
N119
(181C)
A17 (103N,
181C)
AZTR (67N, 70R, 215F, MDR (41L, 74V, 106A,
MT-4 HIV-1IIIB
219Q)
215Y)
SQVR
Test Compounds
d
9a
85
64
0.005
0.004
0.008
0.4
6
0.3
2
0.04
0.05
0.07
0.3
ND
9
9b
2
0.2
1.6
7
ND
3.4
4.2
3.8
1.2
23
9c
40
4
0.4
11a
11b
12
43
1.4
>100
0.1
2.4
0.2
2
1.5
>100
37
9
>100
0.04
0.4
>100
0.3
14
11
>100
0.02
2.3
0.006
0.08
0.01
0.1
0.02
5.2
13
27
Reference Compounds
MKC442
NVP
>100
>100
≥100
40
0.03
100
>10
2
>10
>10
0.8
>100
>10
>20
0.08
0.05
0.01
0.01
0.01
0.08
1.5
0.06
0.3
0.3
0.06
3
MC1220
DMP266
TMC120
TMC125
AZT
0.002
0.002
0.003
0.002
0.01
0.004
0.002
0.001
0.001
0.2
0.01
0.004
0.002
0.001
0.2
0.01
0.004
0.003
0.003
0.01
0.09
6
0.01
0.04
0.01
0.02
0.02
2
0.2
0.3
0.01
0.01
11
50
SQV
15
0.02
0.01
0.02
a
b
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%. Compound
c
concentration (μM) required to reduce the proliferation of mock-infected MT-4 cells by 50% as determined by the MTT method. Compound
concentration (μM) required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity as determined by the MTT method.
d
Not determined.
various clinical isolates was fairly comparable. The uracil-based
compound YLM-220 was 10-fold less potent than MC1220
against subtypes A (MP582), CRF01 (MP0044), and CRF02
(MP810). It proved as potent as MC1220 and TMC120 in
inhibiting subtypes B (W5269), C (MP1308), F1 (MP411),
and G (MP1033) and equipotent with TMC120, but 10-fold
more potent than MC1220, against subtype D (MP634).
Interestingly, all compounds were very effective (0.0001 μM)
against the NRTI-resistant subtype C (MP1315).
Table 2. Mutations in the RT Gene of HIV-1 Variants
Selected in Vitro for Resistance to 12 and the Reference
Inhibitors MC1220, TMC120, and TMC125
a
selecting
drug
mutations in the RT
gene
selecting
drug
mutations in the RT
gene
12
V106I, Y181C
TMC120
TMC125
L100I, E138G
MC1220
L100I, V179D, Y181C
L109M, E138 K,
G190E
a
Resistant variants were selected by serial passages of HIV-1IIIB in the
We previously described46 long-term assays (40 days)
suitable to evaluate the efficacy of NNRTIs in irreversibly
knocking out (KO) in vitro the HIV multiplication under a
variety of treatment conditions. Here these assays were used to
comparatively determine the maximum nontoxic dose
(MNTD) of a continuous treatment with 12, MC1220, and
TMC120, as well as their lowest concentration capable of
irreversibly inhibiting the HIV-1 multiplication following a
chronic treatment or short-term (4 h) treatments carried out
either immediately after or immediately before the HIV-1
infection of MT-4 cells with a high multiplicity of infection
(MOI) (5−10 CCID50 (50% cell culture infectious dose)/cell).
As far as the MNTDs are concerned (Table 5), the
concentrations of 12 and MC1220 allowing full exponential
growth of MT-4 cells during a treatment protracted for 40 days
(the cells were resuspended at 105 cells/mL every 4 days in new
drug-containing medium) are 30 and 10 μM, respectively,
whereas the TMC120 MNTD is considerably lower (0.3 μM).
On the other hand, following a chronic treatment, the most
effective compounds (listed in order of decreasing KO
potency) are MC1220 (<0.1 μM), TMC120 (0.25 μM), and
12 (1.5 μM). Interestingly, MC1220 shows the same KO
potency (<0.1 μM) even when the treatment is limited to the
first 2 h postinfection (pi), whereas its KO concentration is 7.5
μM when the treatment is limited to the first hour pi. Vice
versa, the KO concentrations of 12 and TMC120 are higher
presence of the drugs, whose concentrations were stepwise doubled up
to 128 times the initial EC50 values. Aliquots of virus solutions (5 ×
105 CCID50/mL) were subjected to RNA extraction, RT-PCR, and
sequencing of the pol gene to identify the mutation pattern responsible
for drug resistance.
Table 3. Cross-Resistance Profiles of Selected Resistant
a
Mutants (12R, MC1220R, TMC120R, TMC125R)
b
EC50
12R
MC1220R
TMC120R
(100I,
138G)
TMC125R
(109M,
138K, 190E)
(106I, (100I, 179D,
compd
12
HIV-1IIIB 181C)
181C)
0.006
0.3
2
0.04
4
0.01
1
0.9
>20
3
NVP
>20
0.7
MC1220
DMP266
TMC120
TMC125
AZT
0.002
0.002
0.003
0.002
0.01
3
2
0.06
0.1
0.1
0.09
>20
0.07
0.01
0.02
7
0.1
>20
0.2
0.008
0.01
0.1
0.03
0.009
0.01
0.01
0.02
SQV
0.02
a
Data represent mean values for three independent determinations.
b
Variation among duplicate samples was less than 15%. Compound
concentration (μM) required to achieve 50% protection of MT-4 cells
from HIV-1-induced cytopathogenicity as determined by the MTT
method.
D
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Table 4. Comparative Activity of 12 and Reference Compounds against Clinical Isolates Belonging to Different HIV-1
a
Subtypes
b
c
EC50
EC99
B
A
C
C (NRTIR)
MP1315
CRF01
CRF02
MP810
D
F1
G
IIIB
W5269
0.01
MP582
MP1308
MP0044
MP634
0.01
MP411
MP1033
Test Compound
0.0001
12
0.006
0.01
0.01
0.01
0.1
0.01
0.01
Reference Compounds
≤0.0001
DMP266
MC1220
TMC120
0.002
0.002
0.003
0.01
0.01
ND
ND
0.01
0.01
0.01
0.01
0.001
0.01
0.01
0.01
0.01
0.01
0.1
ND
0.01
0.01
0.01
0.01
0.01
0.001
ND
≤0.0001
0.0001
0.01
a
b
Data represent mean values for three independent determinations. Variation among duplicate samples was less than 15%. Compound
concentration (μM) required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity as determined by MTT assay.
c
Compound concentration (μM) required to achieve 99% inhibition of p24 in supernatants of HIV-1-infected dendritic cell cultures.
Table 5. Maximum Nontoxic Dose (MNTD) and Lowest
Knocking Out (KO) Concentration of 12, MC1220, and
TMC120 in Long-Term (40 days) Assays with MT-4 Cells
nately, the latter favorable properties are counteracted by the
lower potency of 12 (when compared to other NNRTIs)
against NRTI- and PRI-resistant mutants.
a
Compound 12 proves active against a wide panel of clinical
isolates belonging to the B, C, D, and F subtypes and
recombinant forms and, like the other NNRTIs tested, shows
huge potency against the C NRTI-resistant clinical isolate. At
the moment, the dramatic activity of the NNRTIs against the
clade C HIV-1 variant carrying NRTI-resistant mutations
remains unclear, especially as far as 12 is concerned; in fact, it
proved to be the NNRTI with the lowest potency against AZTR
and MDR strains.
Like other NNRTI-resistant mutants, 12-resistant variants
show full susceptibility to AZT and SQV, maintain some
susceptibility to DMP266, and show cross-resistance to the
other members of the NNRTI class.
The microbicidal potential of NNRTIs derives from their
capability, unique among antiretroviral agents, to knock out the
HIV multiplication. In this respect, also 12 has some potential:
in fact, following a chronic treatment, its KO concentration is
comparable to that of TMC120 and MC1220. However,
following short-term (4 h) treatments either before or after
infection, 12 behaves like TMC120, proving totally ineffective.
Under these conditions MC1220 shows a KO concentration of
2 μM, thus proving to be the sole microbicide with some
potential for treatments to be started immediately after an
infection is believed to have occurred.
b
MNTD
c
(μM)
KO (μM)
chronic
treatment
chronic
treatment
4 h
treatment pi
4 h treatment
d
compd
12
bi
10
30
0.3
0.5−2.5
<0.1
>20
<5
>20
<5
MC1220
TMC120
0.1−0.5
>20
>20
a
Data represent mean values for three independent determinations.
b
Variation among duplicate samples was less than 15%. Maximum
compound concentration (μM) capable of allowing the exponential
proliferation of mock-infected MT-4 cells as determined by the MTT
c
method. Lowest compound concentration (μM) required to
irreversibly knock out the HIV-1 multiplication as determined by
d
the MTT method. Before infection.
than 20 μM. Following a 4 h treatment, no matter whether
carried out immediately after or before infection, the sole
compound effective in knocking out the HIV-1 multiplcation is
MC1220 (<5 μM), whereas 12 and TMC120 fail even at
concentrations as high as 20 μM. Overall, the above long-term
experiments suggest that MC1220 is not only the least
cytotoxic, but also the most potent compound in knocking
out the HIV-1 multiplication under any circumstances.
Nevertheless, unlike short-lasting gels that must be applied
soon before sexual intercourse, vaginal rings might provide
continuous and sustained protection that affords coital
independence and minimizes the risk of nonadherence to
protocols. In conclusion, this work favorably supports the
continued development of antiretroviral compounds as topical
microbicides against HIV-1 transmission, even though surpris-
ingly few have been reported so far.
DISCUSSION AND CONCLUSIONS
■
The attempt to improve the anti-HIV-1 potency and spectrum
of MKC442-type NNRTIs was successful. The new fluoro
derivatives were synthesized in a few steps from 5-ethyl-2,4,6-
trichloropyrimidine with good overall yields and costs
comparable to those of other NNRTI microbicides (i.e.,
TMC120 and MC1220). Among the title compounds, 12
emerged as the most potent derivative, and structure−activity
relationship (SAR) studies point to the importance of the
benzene ring conjugated in ether linkage at N1 with the uracil
ring, to the presence of both a triple bond and a hydroxy group
on the benzene ring, and, finally, to the fluorine substitution on
the benzylic carbon.
In short-term experiments, the potency of 12 against the
HIV-1 wt and variants bearing 103 and 181 resistance
mutations is comparable to those of second-generation
NNRTIs. Notewhorthy, 12, followed by TMC125, proves to
be the most active NNRTI against the MC1220- and TMC120-
resistant mutants, which share the L100I mutation. Unfortu-
EXPERIMENTAL SECTION
■
General Procedures. NMR spectra were recorded on a Varian
Gemini 2000 NMR spectrometer at 300 MHz for 1H and 75 MHz for
13C with TMS as the internal standard. MALDI mass spectra were
recorded on a 4.7 T Ultima (IonSpec, Irvine, CA) Fourier transform
ion cyclotron resonance (FTICR) mass spectrometer. Melting points
were determined on a Buchi melting point apparatus. Elemental
̈
analyses were performed at the H.C. Ørsted Institute, University of
Copenhagen. Silica gel (0.040−0.063 mm) used for column
chromatography and analytical silica gel TLC plates 60 F254 were
purchased from Merck. Solvents for chromatography were purchased
E
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Journal of Medicinal Chemistry
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as HPLC grade or distilled prior to use. Reactions were in general
carried out under an argon atmosphere. CH3CN was dried over 3 Å
molecular sieves. The purity of the synthesized compounds was
determined to be ≥95% by elemental analysis (C, H, N) and/or by 1H
150.60 (C2), 164.31 (C6); EI MS m/z 302 (82, M+), 241 (100). Anal.
Calcd for C17H22N2O3 (302.37): C, 67.53; H, 7.33; N, 9.26. Found: C,
67.52; H, 7.39; N, 9.06.
2-(2, 6-Dichloro-5-ethylpyrimidin-4-yl)-2-(3, 5-
dimethylphenyl)acetonitrile (7). Sodium hydride (3.24 g, 0.135
mol, 55% suspension in paraffin oil) was added portionwise at 0 °C to
a solution of 5-ethyl-2,4,6-trichloropyrimidine (9.36 g, 0.045 mol) and
3,5-dimethylbenzyl cyanide (6.53 g, 0.045 mol) in dry DMF (75 mL)
using a CaCl2 drying tube. The reaction mixture was stirred and left to
reach room temperature gradually for 6 h (TLC; PE/E, 5:1). The
mixture was poured into ice-cold water (150 mL) with stirring, and
extracted with ether (3 × 50 mL). The ether phases were dried using
MgSO4 and evaporated under reduced pressure. The residual material
was treated with methanol (20 mL) and left at −5 °C overnight. The
solid product formed was filtered off, washed with cold methanol, and
1
NMR. H NMR spectra are included in the Supporting Information.
(3,5-Dimethylphenyl)(5-ethyl-2,6-dimethoxypyrimidin-4-yl)-
methanol (2). Using an ice cooling bath, sodium borohydride (170
mg, 4.5 mmol) was added portionwise to a stirred solution of
compound 1 (1.2 g, 4 mmol) in methanol (20 mL). The reaction
mixture was left to reach room temperature gradually with stirring for
1 h. Acetic acid (1 mL) was added followed by addition of water (40
mL). The solid product formed was filtered off and dried to afford 0.81
g of the pure compound 2: yield 67%; mp 85−87 °C; 1H NMR
(CDCl3) δ 0.80 (t, 3H, J = 7.5 Hz, CH3CH2), 2.27 [s, 6H, (CH3)2Ar),
2.36−2.43 (m, 2H, CH3CH2), 3.99, 4.06 (2 s, 6H, 2 OCH3), 5.13 (br
s, 1H, OH), 5.65 (s, 1H, CHOH), 6.89 (s, 3H, Harom); 13C NMR
(CDCl3) δ 12.43 (CH3CH2), 17.39 (CH3CH2), 21.23 [(CH3)2Ar],
54.21, 54.73 (2 OCH3), 71.60 (CHOH), 113.09 (C5), 125.10, 129.61,
138.12, 142.04 (Carom), 162.38 (C6), 166.08 (C2), 170.23 (C4); EI
MS m/z 302 (86, M+), 183 (100); HRMS (MALDI) m/z calcd for
C17H22N2NaO3 (MNa+) 325.1523, found 325.1512.
4-[(3,5-Dimethylphenyl)fluoromethyl]-5-ethyl-2,6-dime-
thoxypyrimidine (3). A solution of DAST (0.8 mL, 6 mmol) in
methylene chloride (2 mL) was added dropwise at −5 °C to a solution
of compound 2 (1.51 g, 5 mmol) in dichloromethane (10 mL) under
nitrogen. The solution was stirred and left to reach room temperature
gradually during 4 h. The reaction was quenched by addition of 2 mL
of a saturated solution of sodium carbonate with stirring. The solvents
were evaporated under reduced pressure; water (20 mL) was added to
the residual material, the resulting mixture stirred for 1 h, and the
precipitate then filtered off and dried. The precipitate was purified by
silica gel column chromatography using petroleum ether/ether (PE/E)
(2:1, v/v) as an eluent to afford 1.2 g of compound 3 (79%) as an oil:
1H NMR (CDCl3) δ 0.99 (t, 3H, J = 7.5 Hz, CH3CH2), 2.30 [s, 6H,
(CH3)2Ar], 2.51−2.60 (m, 2H, CH3CH2), 3.98, 3.99 (2s, 6H,
2OCH3), 6.44 (d, JH,F = 47.4 Hz, CHF), 6.95 (s, 1H, Harom), 7.05
(s, 2H, Harom); 13C NMR (CDCl3) δ 13.56 (CH3CH2), 17.58 (d, J =
2.3 Hz, CH3CH2), 21.19 [(CH3)2Ar], 54.05, 54.51 (2OCH3), 92.65
(d, J = 177.0 Hz, CHF), 114.77 (C5), 124.00 (d, J = 5.9 Hz, Carom),
130.11 (d, J = 1.5 Hz, Carom), 137.52 (d, J = 21.3 Hz, Carom), 137.90
(Carom), 162.90 (C2), 163.14 (d, J = 21.3 Hz, C4), 170.56 (C6);
HRMS (MALDI) m/z calcd for C17H21FNaN2O2 (MNa+) 327.1479,
found 327.1480.
Synthesis of Compounds 4a,b from Compound 3. Com-
pound 3 (1.1 g, 3.6 mmol) was refluxed in a mixture of 4 M HCl (30
mL) and ethanol (20 mL) for 16 h. The solvents were evaporated
under reduced pressure until 10 mL, and the solid product formed was
filtered off, washed with water, and dried. TLC showed the existence
of two products which were separated by silica gel column
chromatography using CH2Cl2/EtOAc (3:1, v/v) as the eluent to
furnish compounds 4a and 4b.
1
dried to afford 11.7 g of compound 7 (81%): mp 108−110 °C. H
NMR (CDCl3) δ 1.01 (t, 3H, J = 7.5 Hz, CH3CH2), 2.31 [s, 6H,
(CH3)2Ar], 2.75 (q, 2H, J = 7.5 Hz, CH3CH2), 5.34 (s, 1H, CHCN),
6.98 (s, 3H, Harom); 13C NMR (CDCl3) δ 11.91 (CH3CH2), 21.21
(CH3CH2), 21.68 [(CH3)2Ar], 42.58 (CHCN), 116.94 (CN), 125.43,
130.86, 131.66, 139.45 (Carom), 132.04 (C5), 157.57 (C2), 163.98
(C4), 164.58 (C6).
6-(3,5-Dimethylbenzoyl)-5-ethylpyrimidine-2,4(1H,3H)-
dione (8). Sodium (2.9 g, 0.13 mol) was dissolved in anhydrous
methanol (100 mL). Compound 7 (9.7 g, 0.032 mol) was added, and
the mixture was refluxed for 20 h. A stream of oxygen was pumped
through the solution at room temperature for 2 h (TLC), and the
solvent was concentrated to 20 mL under reduced pressure. HCl (4
M) (100 mL) was added, and the reaction mixture was refluxed for 24
h and cooled. The solid product formed was filtered off, washed with
water, and dried to afford 7.6 g of compound 5 (86%): mp 266−268
°C (lit.21 mp 249−250 °C).
Synthesis of 4a from Compound 8. Sodium borohydride (1.36
g, 0.036 mol) was added portionwise at 0 °C to a stirred solution of
compound 8 (7.48 g, 0.0285 mol) in methanol (75 mL). The reaction
mixture was stirred and left to reach room temperature for 2 h. Water
(100 mL) was added to the reaction mixture, and the solid product
formed was filtered off, washed with water, and dried to afford 7.6 g of
compound 4a (98%), which was identical to compound 4a synthesized
from compound 3.
6-[(3,5-Dimethylphenyl)fluoromethyl]-5-ethylpyrimidine-
2,4(1H,3H)-dione (5). A solution of DAST (6 mL, 0.038 mol) in 10
mL of CH2Cl2 was added dropwise at −5 °C to a solution of
compound 4a (6.85 g, 0.025 mol.) in dichloromethane (60 mL) under
nitrogen. The solution was stirred and left to reach room temperature
for 4 h. The reaction was quenched by addition of 10 mL of a
saturated solution of sodium carbonate with stirring. The solvents
were evaporated under reduced pressure, water (100 mL) was added
to the residual material, and the resulting mixture was stirred for 1 h.
Filtration afforded 6.8 g (99%) of the crude compound 5, which can be
used directly (TLC) or purified by silica gel column chromatography
using CH2Cl2/EtOAc (1:1, v/v) as the eluent to afford 5.5 g of
Data for 6-[(3,5-dimethylphenyl)hydroxymethyl]-5-ethylpyrimi-
1
1
compound 5 (82%): yield 82%; mp 168−170 °C; H NMR (DMSO-
dine-2,4(1H,3H)-dione (4a): yield 19%; mp 230−232 °C; H NMR
d6) δ 0.81 (t, 3H, J = 7.1 Hz, CH3CH2), 2.23−2.35 [m, 8H, CH3CH2
(DMSO-d6) δ 0.80 (t, 3H, J = 7.3 Hz, CH3CH2), 2.23−2.33 [m, 8H,
CH3CH2, (CH3)2Ar], 5.62 (s, 1H, CH), 6.25 (br s, 1H, OH), 6.93 (s,
1H, Harom), 7.07 (s, 2H, Harom), 10.01 (s, 1H, NH), 11.03 (s, 1H,
NH); 13C NMR (DMSO-d6) δ 13.14 (CH3CH2), 16.97 (CH3CH2),
68.68 (CH), 109.63 (C5), 124.05, 129.04, 137.25, 140.72 (Carom),
150.54 (C6), 150.98 (C2), 164.55 (C4); EI MS m/z 274 (100, M+).
Anal. Calcd for C15H18N2O3 (274.32): C, 65.68; H, 6.61; N, 10.21.
Found: C, 65.66; H, 6.53; N, 10.08.
and (CH3)2Ar], 6.59 (d, 1H, JH,F = 45.3 Hz, CHF), 7.06 (s, 1H,
Harom), 7.12 (s. 2H, Harom), 10.80 (s, 1H, NH), 11.20 (s, 1H, NH); 13
C
NMR (DMSO-d6) δ 13.21 (CH3CH2), 17.08 (CH3CH2), 88.08 (d, J =
174.0 Hz, CH-F), 111.23 (d, J = 2.9 Hz, C5), 124.23 (d, J = 5.6 Hz,
Carom), 130.68 (d, J = 2.4 Hz, Carom), 135.64 (d, J = 21.3 Hz, Carom),
137.88 (Carom), 145.58 (d, J = 21.3 Hz, C4), 150.58 (C2), 164.13
(C6); EI MS m/z 276 (100, M+).
Data for 6-[(3,5-dimethylphenyl)ethoxymethyl]-5-ethylpyrimi-
6-[(3,5-Dimethylphenyl)fluoromethyl]-1-(ethoxymethyl)-5-
ethylpyrimidine-2,4(1H,3H)-dione (9a). Compound 5 (0.14 g, 0.5
mmol) was stirred in dry CH2Cl2 (10 mL) under N2, and N,O-
bis(trimethylsilyl)acetamide (BSA) (0.27 mL, 1.1 mmol) was added.
After complete silylation (clear solution, after 10 min), ethoxymethyl
chloride (0.071 mL, 1.5 mmol) was added dropwise to the mixture at
room temperature and the resulting mixture stirred for 3 h. The
reaction was quenched by addition of 5% aqueous sodium bicarbonate
solution (1 mL) with stirring, and then water (10 mL) and methylene
1
dine-2,4(1H,3H)-dione (4b): yield 10%; mp 162−164 °C; H NMR
(DMSO-d6) δ 0.83 (t, 3H, J = 7.3 Hz, CH3CH2), 1.21 (t, 3H, J = 7.0
Hz, CH3CH2O), 2.27−2.33 (m, 8H, CH2CH3 and (CH3)2Ar), 3.46−
3.52 (m, 2H, OCH2CH3), 5.40 (s, 1H, CH), 6.96 (s, 1H, Harom), 7.08
(s, 2H, Harom), 10.26 (s, 1H, NH), 11.07 (s, 1H, NH); 13C NMR
(DMSO-d6) δ 13.14 (CH3CH2), 14.85 (CH3CH2O), 16.92
(CH3CH2), 20.87 [(CH3)2Ar)], 64.34 (CH3CH2O), 76.40 (CH),
111.08 (C5), 124.20, 129.39, 137.35, 138.22 (Carom), 148.63 (C4),
F
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chloride (10 mL) were added to the reaction mixture. The two layers
were separated, and the organic layer was dried using sodium sulfate.
The solvent was removed under reduced pressure, and the residual
material was purified by silica gel column chromatography using
EtOAc/CH2Cl2 (1:1, v/v) as the eluent to afford 140 mg of
compound 9a: yield 84%; mp 196−198 °C; 1H NMR (CDCl3) δ 0.96
(t, 3H, J = 7.5 Hz, CH3CH2), 1.05 (t, 3H, J = 6.9 Hz, CH3CH2O), 2.26
[s, 6H, (CH3)2Ar], 2.32−2.42 (m, 2H, CH3CH2), 3.42−3.58 (m, 2H,
CH3CH2O), 4.89 (d, J = 11.1 Hz, OCH2aN, 5.30 (d, J = 10.8 Hz,
OCH2bN), 6.71 (d, 1H, J = 47.1 Hz, CHF), 6.86 (s, 2H, Harom), 6.96
(s, 1H, Harom), 9.57 (s, 1H, NH); 13C NMR (CDCl3) δ 13.65
(CH3CH2), 14.83 (CH3CH2O), 19.24 (d, J = 4.2 Hz, CH3CH2), 21.31
[(CH3)2Ar], 64.98 (CH3CH2O), 73.19 (OCH2N), 88.43 (d, J = 176.5
Hz, CHF), 118.75 (C5), 123.68 (d, J = 4.8 Hz, Carom), 131.09 (d, J =
2.5 Hz, Carom), 135.00 (d, J = 20.5 Hz, Carom), 138.78 (Carom), 146.59
(d, J = 20.0 Hz, C6), 151.53 (C2), 163.45 (C4); EI MS m/z 334 (3,
M+), 59 (100). Anal. Calcd for C18H23FN2O3·0.25H2O (334.39): C,
63.80; H, 6.84; N, 8.27. Found: C, 63.64; H, 6.96; N, 8.27.
Synthesis of Compounds 9b,c. Compound 4a (140 mg, 0.5
mmol) was stirred in dry CH3CN (10 mL) under N2, and BSA (0.27
mL, 1.1 mmol) was added. The mixture became clear after being
stirred at room temperature for 10 min. The reaction mixture was
cooled to −50 °C, and TMS triflate (0.09 mL, 0.5 mmol) was added
followed by dropwise addition of the appropriate acetal (1 mmol). The
reaction mixture was stirred at room temperature for 3 h, quenched
with an ice-cold saturated solution of NaHCO3 (1 mL), and
evaporated under reduced pressure. The residue was extracted with
Et2O (3 × 20 mL), and the combined organic fractions were dried
(MgSO4) and evaporated under reduced pressure. The residue was
chromatographed on a silica gel column using EtOAc/CH2Cl2 (1:1) as
the eluent to afford compounds 9b and 9c.
water with stirring. The solid product formed was filtered off, washed
with water, and dried to give 9.26 g of the acetal 10: yield 96%; mp
78−80 °C (lit.26 mp 78−80 °C).
Synthesis of Compounds 11a,b. BSA (9.7 mL, 0.04 mol) was
added to a stirred solution of compound 4 (5g, 0.018 mol) in dry
CH3CN (100 mL) under nitrogen after 15 min. The mixture was
cooled to −50 °C. TMS triflate (9.8 mL, 0.054 mol) was added to the
reaction mixture followed by the addition of the acetal 10 (14 g, 0.03
mol). The reaction was stirred and left to reach room temperature
overnight. The reaction was quenched by addition of 10 mL of a
saturated aqueous solution of sodium carbonate. The solvents were
removed under reduced pressure. Then water (30 mL) and methylene
chloride (30 mL) were added to the residual material, and the two
layers were separated. The CH2Cl2 layer was dried over magnesium
sulfate and evaporated under reduced pressure. The residual material
was chromatographed on a silica gel column using CH2Cl2/EtOAc
(10:1, v/v) as the eluent to afford compounds 11a and 11b.
Data for 6-[(3,5-dimethylphenyl)fluoromethyl]-5-ethyl-1-[[(4-
iodobenzyl)oxy]methyl]pyrimidine-2,4(1H,3H)-dione (11a): yield
42%; semisolid; 1H NMR (CDCl3) δ 1.05 (t, 3H, J = 7.2 Hz,
CH3CH2), 2.45 [s, 6H, (CH3)2Ar], 2.46 (q, 2H, J = 7.2 Hz, CH3CH2),
4.53 (s, 2H, CH2Ar), 5.00 (d, J = 10.8 Hz, OCH2aN), 5.38 (d, J = 10.8
Hz, OCH2bN), 6.77 (d, JH,F = 46.8 Hz, CHF), 6.88 (s, 2H, Harom), 6.98
(d, 2H, J = 8.1 Hz, Harom), 7.01 (s, 1H, Harom), 7.62 (d, 2H, J = 8.1,
Harom), 9.38 (br s, 1H, NH); 13C NMR (CDCl3) δ 13.79 (CH3CH2),
19.19 (CH3CH2), 21.33 [(CH3)2Ar], 70.92 (CH2Ar), 73.18
(NCH2O), 88.39 (d, J = 177.6 Hz, CHF), 118.90 (C5), 123.51 (d,
J = 4.8 Hz, Carom), 131.19 (d, J = 1.9 Hz, Carom), 134.76 (d, J = 20.7
Hz, Carom), 93.42, 129.50, 137.44, 137.55, 138.93 (Carom), 146.19 (d, J
= 13.6 Hz, C6), 151.48 (C2), 163.12 (C4); HRMS (MALDI) m/z
calcd for C23H24FIN2NaO3 (MNa+) 545.0708, found 545.0681.
Data for 6-[(3,5-dimethylphenyl)fluoromethyl]-5-ethyl-3-[[(4-
iodobenzyl)oxy]methyl]pyrimidine-2,4(1H,3H)-dione (11b): yield
Data for 1-[(allyloxy)methyl]-6-[(3,5-dimethylphenyl)-
fluoromethyl]-5-ethylpyrimidine-2,4(1H,3H)-dione (9b): yield 80%;
mp 106−108 °C; 1H NMR (CDCl3) δ 0.97 (t, 3H, J = 7.2 Hz,
CH3CH2), 2.26 [s, 6H, (CH3)2Ar], 2.35−2.44 (m, 2H, CH3CH2),
3.98−4.02 (m, 2H, OCH2CHCH2), 4.89 (d, 1H, J = 11.1 Hz,
OCH2aN), 5.07−5.20 (m, 2H, CH2CH), 5.30 (d, 1H, J = 11.1 Hz,
OCH2bN), 5.66−5.79 (m, 1H, CH2CH), 6.71 (d, 1H, JH,F = 46.8
Hz, CHF), 6.85, (s, 2H, Harom), 6.96 (s, 1H, Harom), 9.28 (s, 1H, NH);
13C NMR (CDCl3) δ 13.74 (d, J = 2.4 Hz, CH3CH2), 19.21 (d, J = 4.2
1
16%; mp 114−116 °C; H NMR (CDCl3) δ 0.94 (t, 3H, J = 7.4
Hz, CH3CH2), 2.29−2.36 [m, 8H, CH3CH2 and (CH3)2Ar], 4.64 (s,
2H, CH2Ar), 5.45 (s, 2H, NCH2), 6.35 (d, JHF = 46.4 Hz, CHF), 6.98
(s, 2H, Harom), 7.06 (s, 1H, Harom), 7.10 (d, 2H, J = 8.0 Hz, Harom),
7.61 (d, 2H, J = 8.0, Harom), 8.44 (br s, 1H, NH); 13C NMR (CDCl3) δ
12.99 (CH3CH2), 18.30 (CH3CH2), 21.26 [(CH3)2Ar], 70.21
(CH2Ar), 71.62 (NCH2O), 88.11 (d, J = 176.8 Hz, CHF), 112.66
(C5), 124.36 (J = 5.7 Hz, Carom), 134.46 (d, J = 21.1 Hz, Carom),
131.95 (d, J = 2.9 Hz, Carom), 93.14, 129.47, 137.33, 137.58,
139.133(Carom), 143.53 (d, J = 20.4 Hz, C6), 150.98 (C2), 163.30
(C4); HRMS (MALDI) m/z calcd for C23H24FIN2NaO3 (MNa+)
545.0708, found 545.0681.
Hz, CH3CH2), 70.55 (OCH2CC), 72.98 (NCH2O), 88.45 (d, J =
177.0 Hz, CHF), 117.73 (CH2CH), 118.82 (C5), 123.65 (d, J = 4.9
Hz), 131.16 (d, J = 2.0 Hz, Carom), 133.44 (Carom), 134.89 (d, J = 20.8
Hz, Carom), 138.87 (CH2CH), 146.44 (d, J = 19.3 Hz, C6), 151.45
(C2), 163.24 (C4); HRMS (MALDI) m/z calcd for C19H23FNaN2O3
(MNa+) 369.1585, found 369.1583. Anal. Calcd for C19H23FN2O3
(346.4): C, 65.88; H, 6.69; N, 8.09. Found: C, 65.72; H, 6.75; N, 7.97.
Data for 6-[(3,5-dimethylphenyl)fluoromethyl]-5-ethyl-1-[[(2-
methylallyl)oxy]methyl]pyrimidine-2,4(1H,3H)-dione (9c): yield
6-[(3,5-Dimethylphenyl)fluoromethyl]-5-ethyl-1-{[[4-(3-hy-
droxyprop-1-ynyl)benzyl]oxy]methyl}pyrimidine-2,4(1H,3H)-
dione (12). By a balloon with a long needle immersed in the solution,
a stream of argon was flushed through a solution of compound 11a
(2.15 g, 0.004 mol) and propagyl alcohol (2.3 mL, 0.04 mol) in dry
triethylamine (30 mL) for 0.5 h. Then the reaction mixture was
transferred by a syringe with a long needle to another flask containing
copper(I) iodide (45 mg, 5% mol) and PdCl2(PPh3)3 (85 mg, 3%
mol) under argon. The reaction mixture was stirred under argon for 3
h. The triethylamine was evaporated under reduced pressure and
coevaporated with chloroform (3 × 30 mL). The residual material was
purified by silica gel column chromatography using CH2Cl2/MeOH
(10:1, v/v) to afford 1.3 g of compound 12 (72%): mp 110−112 °C;
1H NMR (CDCl3) δ 0.98 (t, 3H, J = 7.2 Hz, CH3CH2), 2.23 [s, 6H,
(CH3)2Ar], 2.40 (q, 2H, J = 7.2 Hz, CH3CH2), 4.42 (s, 2H, CH2OH),
4.52 (s, 2H, CH2Ar), 4.94 (d, 1H, J = 11.1 Hz, OCH2aN), 5.35 (d, 1H,
J = 11.1 Hz, OCH2bN), 6.71 (d, 1H, J = 46.8 Hz, CHF), 6.82 (s, 2H,
Harom), 6.94 (s, 1H, Harom), 7.11 (d, 2H, J = 8.1 Hz, Harom), 7.30 (d,
2H, J = 8.1 Hz, Harom), 9.36 (br s, 1H, NH); 13C NMR (CDCl3) δ
13.79 (CH3CH2), 19.18 (CH3CH2), 21.30 [(CH3)2Ar], 51.55
(CH2OH), 71.19 (CH2Ar), 73.24 (NCH2O), 85.34 (CCCH2),
87.48 (CCCH2), 88.40 (d, J = 178.1 Hz, CH2F), 118.89 (C5),
123.54 (J = 4.8 Hz, Carom), 131.19 (d, J = 2.1 Hz, Carom), 134.75 (d, J =
20.7 Hz, Carom), 122.05, 127.47, 131.66, 137.54, 138.93 (Carom), 146.17
1
83%; mp 136−138 °C; H NMR (CDCl3) δ 1.04 (t, 3H, J = 9.8
Hz, CH3CH2), 1.68 (s, 3H, CH3CC), 2.33 [s, 6H, (CH3)2Ar],
2.42−2.51 (m, 2H, CH3CH2), 3.99 (s, 2H, OCH2CC), 4.86, 4.92 (2
s, 2H, CH2C), 4.94 (d, 1H, J = 12.0 Hz, OCH2aN), 5.40 (d, 1H, J =
10.8 Hz, OCH2bN), 6.81 (d, 1H, JH,F = 47.1 Hz, CHF), 6.93 (s, 2H,
Harom), 7.03 (s, 1H, Harom), 9.53 (s, 1H, NH); 13C NMR (CDCl3) δ
13.69 (d, J = 2.1 Hz, CH3CH2), 19.19 (d, J = 3.8 Hz, CH3CH2), 19.33
(CH3CC), 21.31 [(CH3)2Ar], 73.09 (OCH2CC), 73.58
(OCH2N), 88.42 (d, J = 176.8 Hz, CHF), 112.57 (CH2C),
118.81 (C5), 123.64 (d, J = 4.7 Hz, Carom), 131.14 (d, J = 2.1 Hz,
Carom), 134.92 (d, J = 20.3 Hz, Carom), 141.08 (Carom), 138.85 (CH2
C), 146.48 (d, J = 19.7 Hz, C6), 151.47 (C2), 163.37 (C4); HRMS
(MALDI) m/z calcd for C20H25FNaN2O3 (MNa+) 383.1741, found
383.1751. Anal. Calcd for C20H25FN2O3 (360.42): C, 66.65; H, 6.99;
N, 7.77. Found: C, 66,68; H, 7.04; N, 7.65.
Bis[(4-iodobenzyl)oxy]methane (10). To a solution of 4-
idodobenzyl alcohol (9.4 g, 0.04 mol) and methylene bromide (1.4
mL, 0.02 mol) in dry DMF (25 mL) in a CaCl2 drying tube was added
portionwise sodium hydride (1.92 g of 55% suspension in paraffin oil,
0.044 mol) at 0 °C. The mixture was stirred and left to reach room
temperature for 3 h. The reaction mixture was poured into ice-cold
G
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(d, J = 19.5 Hz, C6), 151.47 (C2), 163.19 (C4); HRMS (MALDI) m/
z calcd for C26H27FN2NaO4 (MNa+) 473.1847, found 473.1855.
3-{4-[((6-((3,5-Dimethylphenyl)fluoromethyl)-5-ethyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)methyl]-
phenyl}propiolaldehyde (13). A mixture of compound 12 (100 mg,
0.22 mmol) and IBX (186 mg, 0.66 mmol) in ethyl acetate (3 mL) was
heated at 80 °C for 12 h. The reaction mixture was left to reach room
temperature and then evaporated under reduced pressure. The crude
material was purified by silica gel column chromatography using
EtOAc/CH2Cl2 (1:1, v/v) as the eluent to afford 70 mg of compound
13: yield 70%; semisolid; 1H NMR (CDCl3) δ 1.06 (t, 3H, J = 7.4 Hz,
CH3CH2), 2.30 [s, 6H, (CH3)2Ar], 2.49 (q, 2H, J = 7.4 Hz, CH3CH2),
4.63 (s, 2H, CH2Ar), 5.07 (d, 1H, J = 11.1 Hz, OCH2aN), 5.41 (d, 1H,
J = 11.1 Hz, OCH2bN), 6.49 (d, 1H, J = 46.8 Hz, CHF), 6.90 (s, 2H,
Harom), 7.01 (s, 1H, Harom), 7.26 (s, 2H, J = 8.3 Hz, Harom), 7.54 (d,
2H, J = 8.3 Hz, Harom), 9.41 (s, 1H, CHO), 9.52 (br s, 1H, NH); 13C
NMR (CDCl3) δ 13.80 (CH3CH2), 19.16 (CH3CH2), 21.31
[(CH3)2Ar], 70.88 (CH2Ar), 73.35 (NCH2O), 88.38 (d, J = 178.4
Hz, CHF), 88.50 (CCCHO), 94.87 (CCCHO), 118.68, 119.02,
127.53, 131.19, 133.27, 138.97, 140.91 (Carom), 134.77 (d, J = 20.1 Hz,
Carom), 123.39 (d, J = 5.1 Hz, C5), 145.97 (d, J = 19.2 Hz, C6), 151.59
(C2), 163.13 (C4), 176.72 (CHO); HRMS (MALDI) m/z calcd for
C26H25FN2NaO4 (MNa+) 471.1691, found 471.1668.
cytopathogenicity in MT-4 cells. Briefly, 50 μL of culture medium
containing 1 × 104 cells was added to each well of flat-bottom
microtiter trays containing 50 μL of culture medium without or with
serial concentrations of the test compounds. Then 20 μL of HIV
suspensions (containing the appropriate amount of CCID50 to cause
complete cytopathogenicity at day 4) was added. After incubation at
ο
37 C, the cell viability was determined by the MTT method. The
activity of the test compounds against clinical isolates was evaluated in
ex vivo dendritic cells using the end point determination of p24
antigen (PerkinElmer).
Selection of Drug-Resistant Mutants. Drug-resistant variants
were selected by serial passages of HIV-1IIIB in the presence of
stepwise doubling drug concentrations, starting from a cell culture
infected with an MOI of 0.01 and treated with a drug concentration
equal to the EC50. Usually, the amount of virus obtained after each
passage was sufficient to determine the infection of the next cell
culture which, after infection and washing, was incubated with a
double amount of the selecting drug. Drug-resistant viruses were
selected up to drug concentrations 128-fold greater than the EC50.
Resistant virus preparations were subjected to RNA extraction, RT-
PCR, and genome sequencing to identify the mutation patterns
responsible for resistance.
Molecular Analysis of Resistant Viruses. Viral RNAs from the
wt and drug-resistant mutants were obtained using the QIAamp viral
RNA minikit (Qiagen), starting from 140 μL of cell-free viral
suspensions containing about 5 × 105 CCID50/mL, to determine the
nucleotide sequence of the pol region of the HIV-1 genome.
Reverse transcription was carried out according to the manufac-
turer’s protocol using the Superscript II enzyme (Invitrogen) and
outR2 (5′- CTTACCTCTTATGCTTGTGCTGATATTGAAAGA-
3′) as the primer. cDNAs were amplified by four PCRs using
Platinum Pfx polymerase (Invitrogen) and using the following
primers:
Antiviral Assay Procedures. The compounds were solubilized in
DMSO at 100 μM and then diluted in culture medium.
Virus and Cells. MT-4, C8166, and H9/IIIB cells were grown at
37 °C in a 5% CO2 atmosphere in RPMI-1640 medium supplemented
with 10% fetal calf serum (FCS), 100 IU/mL penicillin G, and 100 μg/
mL streptomycin. Cell cultures were checked periodically for the
absence of mycoplasma contamination with a MycoTect kit (Gibco).
Human immunodeficiency virus type 1 (HIV-1IIIB strain) was obtained
from supernatants of persistently infected H9/IIIB cells. The HIV-1
stock solutions had titers of 4.5 × 106 CCID50/mL. The K103R +
V179D + P225H mutant (DMP266R) was derived from an HIV-1IIIB
strain passaged in MT-4 cells in the presence of increasing efavirenz
concentrations (up to 2 μM). 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 μM).
The double mutant K103N + Y181C (NIH A17) was derived from the
HIV-1IIIB strain passaged in H9 cells in the presence of BI-RG 587 (1
μM). EFVR, N119, and N117 stock solutions had titers of 4.0 × 107,
1.2 × 108, and 2.1 × 107 CCID50/mL, respectively.
• INF (5′-TGAAAGATTGTACTGAGAGACAGG-3′)/MR3
(5′-CCCTTCCTTTTCCATTTCTG-3′)
• MF1 (5′-GACCTACACCTGTCAACATA-3′)/INR (5′-
TCTATTCCTACTAAAAATAGTATTTTCCTGATTCC-3′)
• MF3 (5′-ATATGCAAGAATGAGGGGTG-3′)/IR3 (5′-
GTTCAGCCTGATCTCTTACCTGTC-3′);
• IF2 (5′-GCGGGAATCAAGCAGGAATTTGGA-3′)/IR1 (5′-
TCGTAACACTAGGCAAAGGTGGCT-3′).
PCR fragments contained 664, 1762, 1133, and 917 bp, respectively.
All PCR amplifications consisted of an initial denaturation of 3 min, 34
cycles of denaturation at 94 °C for 30 s, annealing at 51.5 °C for 30 s,
extension at 68 °C for 1.5 min, and final extension at 68 °C for 5 min.
PCR fragments were purified using the QIAquick PCR purification
kit (Qiagen) and analyzed using the cycle-sequencing method
(CIBIACI service of the University of Firenze). Both DNA strands
were sequenced with specific primers. Comparative analysis of the
chromatograms allowed the identification of the mutations responsible
for resistance to the various drugs.
Long-Term Assays. In vitro long-term assays were carried out as
previously described.41 The KO concentrations of the test compounds
were determined under conditions of chronic treatments, treatments
limited to the first 4 h pi, or treatments limited to 4 h before infection
(bi).
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 a 4 day
incubation. Virus titers were expressed as CCID50 per milliliter.
Short-Term Cytotoxicity Assays. Cytotoxicity assays were
carried out in parallel with antiviral assays. Exponentially growing
cells derived from human hematological tumors [CD4+ human T-cells
containing an integrated HTLV-1 genome (MT-4)] were seeded at an
initial density of 1 × 105 cells/mL in 96-well plates in RPMI-1640
medium supplemented with 10% fetal bovine serum (FBS), 100 units/
mL penicillin G, and 100 μg/mL streptomycin. Cell cultures were then
incubated at 37 °C in a humidified, 5% CO2 atmosphere in the
absence or presence of serial dilutions of the test compounds. Cell
viability was determined after 96 h at 37 °C by the 3-(4,5-
dimethylthiazol-1-yl)-2,5-diphenyltetrazolium bromide (MTT) meth-
od.47
Long-Term Cytotoxicity Assays. MT-4 cells, resuspended in
growth medium at a density of 1 × 105 cells/mL, were incubated at 37
°C in the absence or presence of various concentrations of the test
compounds, alone or in combination. One-tenth of the culture, in the
absence or presence of the same concentrations of the test
compounds, was transplanted every 3−4 days into new flasks at low
cell density to allow continuous exponential growth. Cell viability was
determined at each passage with the MTT method.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of the compounds studied. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*Phone: +45 66502555. E-mail: erik@sdu.dk (for chemistry).
*Phone: +39 (0) 70 6754147. E-mail: placolla@unica.it (for
virology).
Anti-HIV Assays. The activity of the test compounds against the
multiplication of wt HIV-1 and EFVR, N119, and N117 variants in
acutely infected cells was based on inhibition of virus-induced
H
dx.doi.org/10.1021/jm500139a | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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E. B.; Nielsen, C. Synthesis of 6-(3,5-Dichlorobenzyl) Derivatives as
Isosteric Analogues of the HIV Drug 6-(3,5-Dimethylbenzyl)-1-
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338, 299−304.
Author Contributions
Y.M.L. and E.B.P. contributed to chemistry and R.L., G.S.,
G.G., G.C., and P.L.C. to virology.
Notes
P.L.C. is a named inventor on granted patents for MC1220
(U.S. Patents 6.635.636 and 6.545.007).
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ACKNOWLEDGMENTS
■
This work was funded by the European Community’s Sixth
Framework Programme, Contract LSHP-CT-2004-503162
(Selection and Development of Microbicides for Mucosal
Use To Prevent Sexual HIV Transmission/Acquisition).
ABBREVIATIONS USED
■
BSA, N,O-bis(trimethylsilyl)acetamide; DAST, (diethylamino)-
sulfur trifluoride; DMF, N,N-dimethylformamide; EFV, efavir-
enz, HIV, human immunodeficiency virus; HTLV, human T-
lymphotropic virus; IBX, 2-iodobenzoic acid; NEV, nevirapine;
NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI,
nucleoside reverse transcriptase inhibitor; SQV, saquinavir;
TLC, thin-layer chromatography; TMS, trifluomethanesulfo-
nate
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