Synthesis and antiviral activity of new dimeric inhibitors against HIV-1

Article abstract of DOI:10.1016/j.bmc.2007.09.015

This paper describes the synthesis and the antiviral activities of dimeric compounds derived from homo and asymmetric combinations of N-1 propynyloxymethyl analogues 1a,b of MKC-442, an N-1 4-iodobenzyloxymethyl analogue of TNK-651 5, potent contraceptive

Full text of DOI:10.1016/j.bmc.2007.09.015

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 511–517
Synthesis and antiviral activity of new dimeric
inhibitors against HIV-1
Krzysztof Danel,a,ꢀ Louise M. Larsen,a Erik B. Pedersen,a,* Giuseppina Sanna,b
Paolo La Collab and Roberta Loddob
aNucleic Acid Centerꢁ, Department of Physics and Chemistry, University of Southern Denmark,
Campusvej 55, DK-5230 Odense M, Denmark
bDipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale e Biotecnologie Microbiche,
Universita´ di Cagliari, Cittadella Universitaria, 09042 Monserrato, Italy
Received 3 May 2007; revised 31 August 2007; accepted 11 September 2007
Available online 14 September 2007
Abstract—This paper describes the synthesis and the antiviral activities of dimeric compounds derived from homo and asymmetric
combinations of N-1 propynyloxymethyl analogues 1a,b of MKC-442, an N-1 4-iodobenzyloxymethyl analogue of TNK-651 5,
potent contraceptive norgestrel and AZT. They were obtained by Sonogashira reaction, ‘click’ chemistry or Pd-catalyzed oxidative
coupling. The iodo precursor 5 turned out as a potent compound against wild type and mutated HIV-1 virus. All dimeric com-
pounds showed lower activity against HIV-1 than MKC-442, except the asymmetric dimer of AZT and 1a which showed an activity
comparable to MKC-442.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
tential NNRTIs for a long time.7–15 Recently, we have
synthesized 1a which is a very potent inhibitor of
HIV-1 (Scheme 1).13 This compound is among the first
examples containing a terminal triple bond besides
40-E-dC5 targeting RT. In order to find new MKC-442
The spread of HIV-1 virus is still a serious problem.
Currently there are a lot of possible therapeutical regi-
mens available in hands of clinicians; however, only
four, that is, nucleoside reverse transcriptase inhibitors
(NRTIs), non-nucleoside reverse transcriptase inhibitors
(NNRTIs), protease inhibitors and fusion inhibitors are
the best developed types of inhibitors so far.1–6 This
gives a total number of 21 drugs licensed for clinical
use. The efforts to synthesize new inhibitors are under-
taken both in academic and in industrial research cen-
ters all over the world. In our research group, we have
been interested in different HEPT (1-[(2-hydroxyeth-
oxy)methyl]-6-(phenylthio)thymine) derivatives as po-
Keywords: HIV-1; NNRTI; Synthesis; AZT; TNK-651; MKC-442;
AZT; Sonogashira reaction; Click chemistry; NRTI; Nucleobase; Re-
verse transcriptase; Acetylene; D-norgestrel; Mutant; Virus; Drug-res-
istant; Efavirenz; Anti-viral activity; Uracil.
*
Corresponding author. Tel.: +45 6550 2555; fax: +45 6615 8780;
e-mail: ebp@ifk.sdu.dk
ꢀ
On leave from Department of Chemistry, Agricultural University,
Balicka St. 122, 31-149 Krakow, Poland.
ꢁ
Scheme 1. Reagents and condition: (a) PdCl2(PPh3)2, CuI, Et3N,
toluene, O2, 74–90%.
A research center founded by The Danish National Research
Foundation for studies on nucleic acid chemical biology.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.015

512
K. Danel et al. / Bioorg. Med. Chem. 16 (2008) 511–517
analogues with activity against HIV, it was therefore lo-
gic to perform arylation reactions on the terminal triple
bond of 1a and Sonogashira cross-coupling reaction.
This idea was also based on the reports of using the
alkyne moiety in related drug discovery.16–22 When
starting to make Sonogashira couplings on 1a,b, we
obtained the corresponding dimers 2a,b through homo
coupling of the acetylene bonds. These dimers were
found active against HIV-1 and we realized that such
dimeric molecules could be considered as a new type
of double drugs against HIV-1 different from previous
diacetylenes that have been reported in the literature
to link nucleobases.23–25 Therefore we decided to focus
on the reactions on the acetylene bond in order to ex-
plore the potential of such double drugs. Our rationale
is supported in relevant literature,26,27 where small mol-
ecules can interfere with the HIV-1 RT dimerization
process. We also decided to combine the commercially
available NRTI-drug AZT with 1a by a standard click
chemistry.28 The result is a hybrid between an NNRTI
and an NRTI. Similar approaches have been reported
recently.29–31
sponding diynes 2a,b in 74–90% yield (Scheme 1).
The compounds 2a,b represent an idea of using a rigid
linker which in turn can contribute with stacking prop-
erties upon binding of the drug to its target. In order
to find the scope and limitation of this idea, we decided
to replace one of the acetylene bonds with a para
substituted benzene ring and also to further elongate
the rigid and stacking linker by proper combinations
of acetylene bonds and para substituted benzene rings.
Therefore the iodine substituted analogue 5 of the
extremely potent anti-HIV-1 compound TNK-65133
was prepared by a Vorbruggen reaction34 between
¨
uracil 48 and acetal 3 (Scheme 3). The latter compound
was conveniently prepared according to a published
method for preparation of formaldehyde acetals
(Scheme 2).13,35
A subsequent Sonogashira coupling of 1a and 5, under
the exclusion of O2, furnished 6 in 42% yield. On the
other hand, it was easy to elongate the rigid linker by
an additional acetylene bond by reaction of 1a with dii-
odobenzene which gave the main product 7 together
with a negligible amount of 8.
2. Chemistry
It was also possible to use the acetylene bond of
compound 1a to synthesize asymmetric double drugs
by taking advantage of click chemistry in a reaction with
AZT (Scheme 4). The regioselectivity of triazole 9 was
established by comparing the NMR spectral data with
related compounds already published.29,30 In order to
lower the drug burden and to secure the administration
of the HIV drug, the double drug idea36 was further ex-
tended to the synthesis of potential double functional
drugs by a Sonogashira coupling reaction between the
aryl iodide 5 and the contraceptive10 (ꢀ)-D-norgestrel37
to obtain 11, accompanied by a small amount of the
homocoupled product 12. The latter compound was also
prepared independently by an oxidative coupling reac-
tion (Scheme 4).
By changing the procedure of Sonogashira couplings
on 1a,b13 into a procedure of oxidative coupling using
the procedure of Ito et al.,32 we obtained the corre-
Scheme 2. Reagents and conditions: (a) KOH, CH2Br2, NBu4Br,
benzene, 85 ꢁC, 71%.
Scheme 3. Reagents and conditions: (a) MeCN, BSA, TMS triflate, 3, ꢀ50 ꢁC–rt, 81%; (b) PdCl2(PPh3)2, CuI, Et3N, 81% (6), 48% (7) and 21% (8).

K. Danel et al. / Bioorg. Med. Chem. 16 (2008) 511–517
513
K103N + Y181C mutations, which is comparable to
that found for compound 1a. However, it is interesting
to note that compound 5 showed a 20-fold higher activ-
ity than 1a against the EFVR strain. The importance of
iodine substitution has recently been reported in an
investigation of three potent pyridinones where proper
iodine substitution resulted in inhibition of drug-resis-
tant variants, including Y181C and K103N RT.38 It
should be noticed that the activity of another iodo ana-
logue 8 comprising an acetylene bond elongation of the
N-1 substituent is considerably lower, which may be due
to the rigid nature of the triple bond or to the length of
the N-1 acyclic substituent. Also the dimer type com-
pounds (2a,b, 6 and 7) with combinations of acetylene
bonds and benzene rings in the interlinking chain re-
sulted in compounds with moderate activity against
HIV-1. Compound 9, representing an attempt to synthe-
size an asymmetric double drug by click chemistry in a
reaction between 1a and AZT, showed an activity
against wild type HIV-1 and mutant strains which is
comparable to those found for MKC-442. The low
activity against the Y181C mutated strain makes it unli-
kely that the nucleoside part of 9 functions as a nucleo-
side reverse transcriptase inhibitor. In another attempt
we intended to construct a bifunctional molecule with
both contraceptive and anti-HIV-1 activity, the ratio-
nale being to reduce the drug burden for women by
combining one of the HIV-1 drugs from the combina-
tion therapy with a contraceptive agent.
Compound 11 from coupling of 5 with commercially
available 10 is an obvious possibility to test this idea.
Unfortunately, the activity against HIV-1 dropped dra-
matically when compared with compound 5 and, there-
fore, it was not interesting to investigate compound 11
as a contraceptive agent.
4. Conclusion
In summary, we have described the synthesis of new di-
meric series analogues of MKC-442, TNK-651 and
AZT. However, the dimeric compounds only showed
moderate activity against HIV. Instead, in this study
we discovered the potent inhibitor 5, with a promising
activity against HIV-1 resistant mutants.
Scheme 4. Reagents and conditions: (a) t-BuOH/H2O, CuSO4Æ5H2O,
1 M sodium ascorbate, 69%; (b) PdCl2(PPh3)2, CuI, Et3N, 85% (11);
(c) PdCl2(PPh3)2, CuI, Et3N, toluene, O2, 96%.
5. Experimental
5.1. Chemistry
3. Antiviral activity
The HIV-1 strain HTLV-IIIB in MT-4 cells was used in
our assay to investigate the anti-HIV-1 activity of
MKC-442 analogues synthesized in the present study.
The results are summarized in Table 1 together with
those of MKC-442 and efavirenz. The monomer 1a
has been investigated before13 and in the present series
of compounds this is the most active compound against
HIV-1 wild type. Among the new monomeric com-
pounds, the TNK-651 analogue 5, containing an iodine
atom in the para position of the benzyl goup in the N-1
substituent, has an activity against HIV-1 wild type and
the drug-resistant strains carrying Y181C and
NMR spectra were recorded on a Varian Gemini 2000
NMR spectrometer at 300 MHz for 1H and 75 MHz
for 13C with TMS as internal standard. MALDI mass
spectra were recorded on a 4.7 T Ultima (IonSpec, Ir-
vine, CA) Fourier Transform Ion Cyclotron Resonance
(FTICR) Mass Spectrometer. Melting points were deter-
mined on a Buchi melting point apparatus. Elemental
¨
analyses were performed at H.C. Ørsted Institute, Uni-
versity 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.

514
K. Danel et al. / Bioorg. Med. Chem. 16 (2008) 511–517
Table 1. Cytotoxicity and anti-HIV-1 activity of MKC-442 dimeric analogues 2a–b, 6, 11, 12, 7 and 9, monomers 1a, 1b, 5, and 8, and the reference
compounds MKC-442 and efavirenz (EFV)
SIc
Wild type
EC50 (lM)
Y181C
K103N + Y181C
b
a
Compound
CC50 (lM)
EFVR
Dimers
2a
2b
6
9
18
0.5
>8
>9
>8
>9
>8
4
>9
>8
8
16
1
53
0.3
0.4
>28
2.8
0.08
>16
>100
>28
>85
26
>16
>100
>28
>85
27
11
12
7
>100
28
85
>250
1
30
2.8
>28
>85
8
9
>100
>1250
Monomers
1a
>100
33
19
>33333
82
0.003
0.4
16
0.2
1.2
1b
5
>33
0.8
>44
100
3
>33
0.1
>33
0.9
2714
220
0.007
0.2
8
44
>100
30
10
20
>44
>100
0.3
MKC-442
EFV
>3333
15000
0.03
0.002
0.008
a Compound dose required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
b Compound dose required to achieve 50% protection of MT-4 cells from HIV-1-induced cytopathogenicity, as determined by the MTT method. The
symbol (>) indicates that the CC50 was not reached at the highest concentration tested.
c Selectivity index: Ratio CC50/EC50. EC50 and CC50 are expressed as the mean values of at least two separate experiments.
Solvents for chromatography were bought as HPLC
grade or distilled prior to use. Reactions were in general
carried out under argon atmosphere. CH3CN was dried
over 3 A molecular sieves. Compound 1a,b was pre-
pared according to earlier works.8,13
6H, phenyl), 9.94 (s, 2H, NH). 13C NMR (CDCl3): d
13.8 (CH2CH3), 19.1 (CH2CH3), 19.6 (2-CH3), 30.6
(CH2Ph), 58.6 (OCH2C„), 69.4 (CH2C„C–), 73.8
(NCH2O), 76.8 (CH2C„C–), 117.9 (C-5), 125.7, 126.8,
127.3, 130.6, 133.3, 136.0 (aryl), 148.8 (C-2), 153.0 (C-
6), 163.1 (C-4). HRMS (MALDI) m/z Calcd for
C36H38N4O6Na+ (MNa+) 645.2683 Found 645.2685.
˚
5.1.1. 6-(3,5-Dimethylbenzyl)-1-[6-[6-(3,5-dimethylbenzyl)-
5-ethyl-2,4-dioxo-1,2,3,4-tetrahydro-1-pyrimidinylmethoxy]-
2,4-hexadiynyloxymethyl]-5-ethyl-1,2,3,4-tetrahydro-2,4-
5.1.3. 1-Iodo-4-(4-iodobenzyloxymethoxymethyl)-benzene
(3). A mixture of KOH (1.33 g, 23.7 mmol), Bu4NBr
(0.41 g, 1.27 mmol), CH2Br2 (2.01 g, 0.81 mL, 11.6
mmol) and 4-iodobenzylalcohol (5.50 g, 23.5 mmol) in
benzene (8 mL) was heated at 85–87 ꢁC (oil bath tem-
perature) for 2 days. After cooling to room temperature,
H2O (50 mL) was added and the mixture was extracted
with Et2O (3 · 50 mL). The organic layer was dried with
MgSO4. After removing the volatiles, the residue was
purified by silica gel column chromatography using
CH2Cl2 as eluent to give compound 3 as a white crystal-
line solid. Yield: 4.02 g (71%); Rf 0.91 (25% EtOAc/
pyrimidinedione (2a).
A solution of 1a (191 mg,
0.585 mmol), PdCl2 (PPh3)2 (12 mg, 0.017 mmol), CuI
(3.0 mg, 0.016 mmol), triethylamine (3.0 mL) in toluene
(10 mL) was stirred at room temperature overnight un-
der O2 atmosphere. After evaporation of solvents, the
residue was purified by column chromatography on sil-
ica gel with CHCl3 to afford the compound 2a: yellow
foam. Yield: 140 mg (74%); Rf 0.31 (3% MeOH/CHCl3).
1H NMR (CDCl3) d 1.07 (t, 6H, J = 7.4 Hz, CH2CH3),
2.28 (s, 12H, CH3), 2.48 (q, 4H, J = 7.4 Hz, CH2CH3),
4.06 (s, 4H, CH2Ph), 4.39 (s, 4H,OCH2C„), 5.28 (s,
4H, NCH2O), 6.71 (s, 4H, aryl),6.90 (s, 2H, aryl), 9.89
(s, 2H, NH). 13C NMR (CDCl3) d 13.9 (CH2CH3),
19.2 (CH2CH3), 21.3 (CH3), 33.1 (CH2Ph), 58.6
(OCH2C„), 69.4 (CH2C„C–), 73.6 (NCH2O), 76.8
(CH2C„C–), 117.6 (C-5), 125.1, 129.0,134.9, 138.9
(aryl), 148.7 (C-2), 153.0 (C-6), 163.2 (C-4). HRMS
(MALDI) m/z Calcd for C38H42N4O6Na+ (MNa+)
673.2996, Found 673.2997.
1
petroleum ether); mp 78–80 ꢁC. H NMR (CDCl3): d
4.56 (s, 4H, PhCH2O), 4.80 (s, 2H, OCH2O), 7.05 (d,
4H, J = 8.3 Hz, aryl), 7.66 (d, 4H, J = 8.3 Hz, aryl).
13C NMR (CDCl3): d 68.9 (PhCH2O), 93.2 (C–I), 94.1
(OCH2O), 129.7, 137.4, 137.4 (aryl). HRMS (MALDI)
m/z Calcd for C15H14I2O2Na+ (MNa+) 502.8975 Found,
502.8966.
5.1.4. 6-(3,5-Dimethylbenzyl)-5-ethyl-1-(4-iodobenzyl-oxym-
ethyl)-1H-pyrimidine- 2,4-dione (5). A suspension of uracil
48 (1.08 g, 4.16 mmol) in dry MeCN (80 mL) was treated
with N,O-bis-(trimethylsilyl)acetamidate (BSA) (1.86 g,
2.23 mL, 9.13 mmol) under a nitrogen atmosphere.
When the mixture became clear, it was cooled to
ꢀ50 ꢁC, and TMS triflate (2.84 g, 2.32 mL, 12.8 mmol)
was added dropwise, followed by the acetal 3 (3.30 g,
6.87 mmol). The mixture was kept at ꢀ50 ꢁC for
15 min and then allowed to warm to room temperature.
5.1.2. 5-Ethyl-1-[6-[5-ethyl-6-(2-methylbenzyl)-2,4-dioxo-
1, 2,3,4-tetrahydro-1-pyrimidinylmethoxy]-2,4-hexadiynyl-
oxymethyl]-6-(2-methylbenzyl)-1,2,3,4-tetrahydro-2,4-pyri-
midinedione (2b). It was prepared analogously to 2a:
yield 140 mg (90%); Rf 0.29 (3% MeOH/CHCl3). 1H
NMR (CDCl3) d 1.06 (t, 6H, J = 7.4 Hz, CH2CH3),
2.37 (s, 6H, 2-CH3), 2.43 (q, 4H, J = 7.4 Hz, CH2CH3),
4.03 (s, 4H, CH2Ph), 4.39 (s, 4H, OCH2C„), 5.18 (s,4H,
NCH2O), 6.86 (d, 2H, J = 7.0 Hz, aryl), 7.11–7.24 (m,

K. Danel et al. / Bioorg. Med. Chem. 16 (2008) 511–517
515
Stirring was continued until uracil 4 was consumed as
judged from TLC. A cold saturated solution of NaH-
CO3 was added. The mixture was evaporated until dry-
ness and further extracted with Et2O (6 · 50 mL). The
etheral fractions were combined, dried, and evaporated.
The residue was purified by silica column chromatogra-
phy (chloroform) and recrystallized from petroleum
ether/ethyl acetate to deliver a pure compound. Yield:
1.69 g (81%); white solid; Rf 0.43 (3% MeOH/CHCl3);
mp 175ꢀ177 ꢁC. 1H NMR (CDCl3): d 1.06 (t, 3H,
J = 7.5 Hz, CH2CH3), 2.27 (s, 6H, 2 · CH3), 2.46 (q,
2H, J = 7.5 Hz, CH2CH3), 4.04 (s, 2H, CH2Ph), 4.58
(s, 2H, OCH2), 5.18 (s, 2H, NCH2O), 6.64 (s, 2H,aryl),
6.88 (s, 1H, aryl), 7.06 (d, 2H, J = 8.4 Hz, aryl), 7.66
(d, 2H, J = 8.4 Hz, aryl), 9.41 (s, 1H, NH). 13C NMR
(CDCl3): d 13.8 (CH2CH3), 19.2 (CH2CH3), 21.3
(2 · CH3), 33.3 (CH2Ph), 71.0 (OCH2), 72.7 (NCH2O),
93.5 (C–I), 116.9 (C-5), 124.9, 129.0, 129.7, 134.7,
137.0, 137.5, 138.9 (aryl), 149.1 (C-2), 152.0 (C-6),
163.2 (C-4). HRMS (MALDI) m/z calcd for C23H25I-
N2O3Na+ (MNa+) 527.0802 Found 527.0976. Anal.
Calcd for C23H25IN2O3Æ0.25H2O: C, 54.29; H, 5.05; N,
5.50. Found: C, 54.09; H, 4.78; N, 5.40.
(28 mg, 0.147 mmol), and Et3N (10 mL) was stirred over-
night under argon. After work up as described for com-
pound 6, the residue was subjected to silica gel column
chromatography (CHCl3) to give two fractions. The slow
eluted fraction was the target compound 7. Yield: 87 mg
(48%); white solid; Rf 0.15 (3% MeOH/CHCl3); mp
180ꢀ182 ꢁC. 1H NMR (DMSO-d6): d 0.84 (t, 6H,
J = 7.2 Hz, CH2CH3),2.18 (s, 12H, 4 · CH3), 2.28 (q,
4H, J = 7.2 Hz,CH2CH3),4.04 (s, 4H, CH2Ph), 4.50 (s,
4H, OCH2C„), 5.15 (s, 4H, NCH2O), 6.76 (s, 4H, aryl),
6.86 (s, 2H, aryl), 7.36 (s, 4H, aryl), 11.50 (s, 2H,NH). 13C
NMR (DMSO-d6): d 13.4 (CH2CH3), 18.5(CH2CH3),
20.7 (2 · CH3), 32.7(CH2Ph),56.6 (OCH2C„), 72.1
(NCH2O), 85.0 (OCH2C„C), 87.5 (OCH2C„C), 115.5
(C-5), 121.9, 124.8, 128.3, 131.5, 135.7, 137.9 (aryl),
148.1 (C-2), 151.4 (C-6), 162.9 (C-4). HRMS (MALDI)
m/z Calcd for C44H46N4O6Na+ (MNa+) 749.3310 Found,
749.3314. Anal. Calcd for C44H46N4O6Æ2H2O: C, 69.65;
H, 6.13; N, 7.34. Found: C, 69.27; H, 6.08; N, 7.34.
5.1.7. 6-(3,5-Dimethylbenzyl)-5-ethyl-1-[3-(4-iodophenyl)
prop-2-ynyloxymethyl]-1H-pyrimidine-2,4-dione (8). Iso-
lated as the fast eluted fraction. Yield: 30 mg (21%);
brown foam; Rf 0.36 (3% MeOH/CHCl3). 1H NMR
(CDCl3): d 1.01 (t, 3H, J = 7.5 Hz, CH2CH3), 2.25 (s,
6H, 2 · CH3), 2.44 (q, 2H, J = 7.5 Hz, CH2CH3), 4.09
(s, 2H, CH2Ph), 4.49 (s, 2H, OCH2C„), 5.25 (s, 2H,
OCH2N), 6.69 (s, 2H, aryl), 6.89 (s, 1H, aryl), 7.10 (d,
2H, J = 9.0 Hz, aryl), 7.63 (d, 2H, J = 9.0 Hz, aryl), 9.42
(s, 1H, NH). 13C NMR (CDCl3): d 13.7 (CH2CH3), 19.2
(CH2CH3), 21.2 (2 · CH3), 33.3 (CH2Ph), 58.0
(OCH2C„),72.6 (NCH2O), 85.6 (OCH2C„), 85.8
(OCH2C„C), 94.7 (C–I), 116.9 (C-5), 121.6, 125.0,
129.0, 133.3, 134.7, 137.5, 138.9 (aryl), 149.1 (C-2),
152.0 (C-6), 163.2 (C-4); HRMS (MALDI) m/z calcd for
C25H25IN2O3Na+ (MNa+), 551.0802 Found, 551.0791.
5.1.5. 6-(3,5-Dimethylbenzyl)-1-[4-[3-[6-(3,5-dimethylben-
zyl)-5-ethyl-2,4-dioxo-1,2,3,4-tetrahydro-1-pyrimidinyl-
methoxy]-1-propynyl]benzyloxymethyl]-5-ethyl-1,2,3,4-
tetrahydro-2,4-pyrimidinedione (6). A mixture of 1a
(163 mg, 0.5 mmol), 5 (252 mg, 0.5 mmol), PdCl2(PPh3)2
(22 mg, 0.031 mmol), and CuI (12 mg, 0.063 mmol) was
suspended in dry Et3N (30 mL) under argon. The mix-
ture was stirred at room temperature for 24 h. The sol-
vent was removed in vacuo and CHCl3 (40 mL) was
added. The organic phase was washed with H2O and
dried over Na2SO4. After removing the volatiles, the res-
idue was purified by silica gel column chromatography
(chloroform). Yield: 148 mg (42%); white foam; Rf
0.37 (3% MeOH/CHCl3); mp 113ꢀ115 ꢁC. 1H NMR
(CDCl3): d 1.08 (t, 3H, J = 7.4 Hz, CH2CH3), 1.18 (t,
3H, J = 7.4 Hz, CH2CH3), 2.27 (s, 6H, 2 · CH3), 2.28
(s,6H, 2 · CH3), 2.42 (q, 2H, J = 7.4 Hz, CH2CH3),
2.55 (q, 2H, J = 7.4 Hz, CH2CH3), 4.09 (s, 2H, CH2Ph),
4.16 (s, 2H, CH2Ph), 4.54 (s, 2H, OCH2C„), 4.69 (s,
2H,OCH2), 5.30 (br s, 4H, NCH2O), 6.68 (s, 2H, aryl),
6.74 (s, 2H, aryl), 6.89 (s, 2H, aryl), 7.15 (d, 2H,
J = 8.6 Hz, aryl), 7.18 (d, 2H, J = 8.6 Hz, aryl), 10.16
(s, 1H, NH), 10.56 (s, 1H, NH).13C NMR (CDCl3): d
14.1 (2 · CH2CH3), 19.0 (CH2CH3), 19.2 (CH2CH3),
21.3 (4 · CH3), 33.1 (CH2Ph), 33.4 (CH2Ph), 59.4
(OCH2C„), 73.1 (NCH2O), 74.0 (OCH2), 73.6
(NCH2O), 85.2 (CH2C„C), 85.4 (CH2C„C), 116.4
(C-5), 116.7 (C-5), 121.4, 125.1, 126.9, 129.0, 131.0,
134.8, 134.9, 138.5, 138.9 (aryl), 149.1 (C-2), 149.8 (C-
2), 151.1 (C-6), 151.9 (C-6), 164.4 (C-4), 164.9 (C-4).
HRMS (MALDI) m/z Calcd for C42H46N4O6Na+
(MNa+) 725.3310 Found, 725.3301.
5.1.8. 6-(3,5-Dimethylbenzyl)-5-ethyl-1-[1-[(2S,3S,5R)-2-
hydroxymethyl-5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydro-
1-pyrimidinyl)tetrahydro-3-furanyl]-1H-1,2,3-triazol-4-
ylmethoxymethyl]-1,2,3,4-tetrahydro-2,4-pyrimidinedione
(9). AZT (133.5 mg, 0.50 mmol) and 1a (163 mg,
0.50 mmol) were suspended in a 1:1 mixture of H2O
and tert-butyl alcohol (3 mL). Sodium ascorbate
(0.3 mmol, 300 lL of freshly prepared 1 M solution in
H2O) was added, followed by copper (II) sulfate penta-
hydrate (7.5 mg, 0.03 mmol, in 100 lL of H2O). The het-
erogeneous mixture was stirred vigorously overnight.
When TLC analysis indicated complete consumption
of the reactants, the reaction mixture was diluted with
H2O (50 mL), and the white precipitate was collected
by filtration. After washing with cold H2O
(2 · 25 mL), it was dried under vacuum to afford
206 mg (69%) of pure product as a white powder; Rf
0.10 (5% MeOH/CHCl3); mp 132–134 ꢁC. 1H NMR
(DMSO-d6): d 0.90 (t, 3H, J = 7.0 Hz, CH2CH3), 1.82
(s,3H, CH3), 2.22 (s, 6H, 2 · CH3), 2.28 (q,
2H,J = 7.0 Hz, CH2CH3), 2.63–2.73 (m, 2H, H-20),
3.60ꢀ3.64 (m, 2H, H-50), 3.98 (s, 2H, CH2Ph), 4.18–
4.21 (m,1H, H-30), 4.63 (s, 2H, OCH2), 5.09 (s, 2H,
NCH2O), 5.29–5.35 (m, 2H, H-40,OH), 6.42 (t, 1H,
J = 6 Hz, H-10), 6.73 (s, 2H, aryl), 6.87 (s, 1H, aryl),
7.82 (s, 1H, C-6), 8.24 (s, 1H, CH, triazole), 11.36 (s,
5.1.6. 6-(3,5-Dimethylbenzyl)-1-[3-[4-[3-[6-(3,5-dimethyl-
benzyl)-5-ethyl-2,4-dioxo-1,2,3,4-tetrahydro-1-pyrimidi-
nylmethoxy]-1-propynyl]phenyl]-2-propynyloxymethyl]-5-
ethyl-1,2,3,4-tetrahydro-2,4-pyrimidinedione (7). A mixture
of 1a (196 mg, 0.60 mmol), diiobenzene (88 mg,
0.27 mmol), PdCl2(PPh3)2 (52 mg, 0.074 mmol), CuI

516
K. Danel et al. / Bioorg. Med. Chem. 16 (2008) 511–517
1H, NH), 11.47 (s, 1H, NH). 13C NMR (DMSO-d6): d
12.2 (CH3), 13.5 (CH2CH3), 18.5 (CH2CH3), 20.8
(CH3Ph), 32.8 (C-20), 37.1 (CH2Ph), 59.1 (C-30), 60.6
(OCH2), 61.7 (C-50), 72.0 (NCH2O), 83.8 (C-40), 84.4
(C-10), 109.6 (C-5), 115.4 (C-5), 123.5, 124.9, 128.2,
135.8, 136.2, 138.0, 143.4, 148.2, 150.4, 151.5, 163.0,
163.6 (aryl, triazole, uracils). HRMS (MALDI) m/z
Calcd for C29H35N7O7Na+ (MNa+) 616.2490 Found,
616.2460.
5.2.1. 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 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 4.5 · 106 50% cell culture infec-
tious dose (CCID50)/mL. The K103R + V179D +
P225H mutant (EFVꢂ) was derived from an IIIB strain
passaged 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 passaged initially
in CEM and then in MT-4 cells in the presence of nevi-
rapine (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). EFVR,
N119, and N117 stock solutions had titers of 4.0 · 107,
1.2 · 108, and 2.1 · 107 CCID50/mL, respectively.
5.1.9. 6-(3,5-Dimethylbenzyl)-5-ethyl-1-[4-(13-ethyl-17-hyd-
roxy-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahy-
dro-1H-cyclopenta[a]phenanthren-17-ylethynyl)benzyloxym-
ethyl]uracil (11). Compound 5 (173 mg, 0.34 mmol),
D-(ꢀ)-norgestrel 10 (107 mg, 0.34 mg), PdCl2(PPh3)2
(22 mg, 0.031 mmol), and CuI (12 mg, 0.063 mmol) were
stirred in Et3N (20 mL) at rt for 2 days. The mixture was
evaporated to dryness, and CHCl3 (15 mL) was added.
The organic phase was washed with H2O (20 mL), dried
and evaporated. The residue was purified by silica gel
column chromatography (chloroform). A negligible
amount of the homocoupled product 12 was also
formed. Yield 200 mg (85%); yellow foam; Rf 0.29 (3%
5.2.2. 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 incuba-
tion. Virus titers were expressed as CCID50/mL.
1
MeOH/CHCl3); mp 178ꢀ180 ꢁC. H NMR (CDCl3): d
0.82–2.53 (m, 36H), 4.03 (s, 2H, CH2Ph), 4.62 (s, 2H,
OCH2), 5.17 (s, 2H, NCH2O), 5.83 (s, 1H, CH@),
6.63 (s, 2H, aryl), 6.88 (s, 1H, aryl), 7.25 (d, 2H,
J = 8.4 Hz, aryl), 7.37 (d, 2H, J = 8.4 Hz, aryl), 8.95 (s,
1H, NH).13C NMR (CDCl3): d 9.6, 13.7, 18.9, 19.0,
21.3, 22.5, 26.1, 26.2, 28.8, 30.7, 33.2, 35.5, 36.5, 39.4,
40.9, 42.4, 48.5, 49.0, 51.1 (alifatic), 71.2 (OCH2), 72.7
(NCH2O), 81.7, 85.7 (C-17, ꢀC„), 93.4 (ꢀC„C),
116.8 (C-5), 122.5, 124.6, 124.9, 127.6, 129.0, 131.6,
134.7, 137.5, 138.9 (aryl and ꢀCH@), 149.1 (C-6),
151.8 (C-2), 163.0 (C-4), 166.6 (>C–), 199.9 (C@O).
HRMS (MALDI) m/z Calcd for C44H52N2O5Na+
(MNa+) 711.3768 Found, 711.3742.
5.2.3. Anti-HIV assays. The activity of test compounds
against multiplication of wild type HIV-1, EFVR,
N119, and N117 in acutely infected cells was based on
inhibition of virus-induced cytopathogenicity in MT-4
cells. Briefly, an amount of 50 lL of culture medium
containing 1 · 104 cells was added to each well of flat-
bottom microtiter trays containing 50 lL of culture
medium with or without various concentrations of test
compounds. Then an amount of 20 lL of HIV suspen-
sions (containing the appropriate amount of CCID50
to cause complete cytopathogenicity at day 4) was
added. After incubation at 37 ꢁC, cell viability was
determined by the 3-(4,5-dimethylthiazol-1-yl)-2,5-
diphenyltetrazolium bromide (MTT) method.39 Alterna-
tively, p24 levels were determined by an immunoenzy-
matic 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.
5.1.10. 15-Ethyl-14-[4-(15-ethyl-14-hydroxy-5-oxotetracy-
clo[8.7.0.02,7.011,15]heptadec-6-en-14-yl)-1,3-butadiynyl]-14-
hydroxytetracyclo.[8.7.0.02,7011,?]-heptadec-6-en-5-one (12).
D-(ꢀ)-norgestrel 10 (78 mg, 0.25 mmol), PdCl2(PPh3)2
(6.2 mg, 0.0088 mmol), CuI (1.5 mg, 0.0081 mmol),
Et3N (2 mL) and toluene (6 mL) were stirred under O2
atmosphere overnight. The volatiles were removed in va-
cuo and the residue was further purified by silica gel col-
umn chromatography (chloroform). Yield: 75 mg (96%);
yellowish solid; Rf 0.30 (3% MeOH/CHCl3); mp 215–
1
217 ꢁC. H NMR (CDCl3): d 0.86–2.51 (m, 50H), 5.83
(s, 2H, @CH). 13C NMR (CDCl3): d 9.5, 18.9, 22.5,
26.1, 26.5, 28.8, 30.6, 35.4, 36.5, 39.5, 40.9, 42.4, 48.7,
48.7, 51.1 (aliphatic), 70.3 (–C„ C–C„C–), 82.1, 83.6
(C-17, -C„C–C„C-), 124.6 (@CH), 166.4 (@C<),
199.9 (C@O). HRMS (MALDI) m/z Calcd for
C42H54O4Na+ (MNa+) 645.3914 Found, 645.3892.
Anal. Calcd for C42H54O4Æ 3H2O: C, 74.46; H, 7.98.
Found: C, 74.90; H, 8.13.
Acknowledgments
This work received funding from the European Commu-
nity’s Sixth Framework Programme under contract
number LSHP-CT-2004-503162 (Selection and develop-
ment of microbicides for mucosal use to prevent sexual
HIV transmission/acquisition).40
5.2. Antiviral assay procedures
References and notes
Compounds were solubilized in DMSO at 100 mM and
then diluted in culture medium.
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