Synthesis, Biological Activity, and ADME Properties of Novel S-DABOs/N-DABOs as HIV Reverse Transcriptase Inhibitors

Article abstract of DOI:10.1002/cmdc.201200056

Previous studies aimed at exploring the SAR of C2-functionalized S-DABOs demonstrated that the substituent at this position plays a key role in the inhibition of both wild-type RT and drug-resistant enzymes, particularly the K103N mutant form. The introdu

Full text of DOI:10.1002/cmdc.201200056

MED
DOI: 10.1002/cmdc.201200056
Synthesis, Biological Activity, and ADME Properties of
Novel S-DABOs/N-DABOs as HIV Reverse Transcriptase
Inhibitors
Marco Radi,[a] Mafalda Pagano,[a] Luigi Franchi,[a] Daniele Castagnolo,[a] Silvia Schenone,[b]
Gianni Casaluce,[a] Claudio Zamperini,[a] Elena Dreassi,[a] Giovanni Maga,[c] Alberta Samuele,[c]
Encarna Gonzalo,[d] Bonaventura Clotet,[d] Josꢀ A. Estꢀ,[d] and Maurizio Botta*[a, e]
Previous studies aimed at exploring the SAR of C2-functional-
ized S-DABOs demonstrated that the substituent at this posi-
tion plays a key role in the inhibition of both wild-type RT and
drug-resistant enzymes, particularly the K103N mutant form.
The introduction of a cyclopropyl group led us to the discov-
ery of a potent inhibitor with picomolar activity against wild-
type RT and nanomolar activity against many key mutant
forms such as K103N. Despite its excellent antiviral profile, this
compound suffers from a suboptimal ADME profile typical of
many S-DABO analogues, but it could, however, represent
a promising candidate as an anti-HIV microbicide. In the pres-
ent work, a new series of S-DABO/N-DABO derivatives were
synthesized to obtain additional SAR information on the C2-
position and in particular to improve ADME properties while
maintaining a good activity profile against HIV-1 RT. In vitro
ADME properties (PAMPA permeation, water solubility, and
metabolic stability) were also experimentally evaluated for the
most interesting compounds to obtain a reliable indication of
their plasma levels after oral administration.
Introduction
Among the various anti-HIV agents, non-nucleoside reverse
transcriptase inhibitors (NNRTIs) are now an established part of
the therapy for treating HIV infection.[1,2] In addition, increased
attention has been focused on the possibility of using NNRTIs
as HIV microbicides to prevent or decrease HIV infections in
women.[3–5] Microbicides in fact represent one of the most
promising strategies for combating the HIV/AIDS epidemic in
the absence of an effective vaccine. It is generally accepted
that a microbicide should act prior to the integration of provi-
ral DNA into the host cell DNA; therefore, the two major com-
pound classes that can be used for this are entry inhibitors
and RT inhibitors. Some NNRTIs exhibit high-affinity binding
and slow dissociation from RT,[6–9] a characteristic that is well-
suited to a potential microbicide. Examples of NNRTIs in pre-
clinical and clinical evaluation as microbicides include dapivir-
ine (TMC 120), MIV 150, UC 781, and dihydroalkylthiobenzyl-
oxopyrimidines (DABOs).[10–13] In this context, DABOs have
been the object of many studies aimed at the identification of
novel analogues with potent inhibitory activity toward wild-
type HIV-1 and especially drug-resistant mutants.[14] In fact,
given the rapid selection for mutant strains, new drugs with
activity against drug-resistant viruses are currently needed.
Over the years, many derivatives characterized by various C2
side chains (S-DABOs and N-DABOs, Figure 1) with high activity
profiles have been developed.[15–19] Structure–activity relation-
ship (SAR) studies of S-DABOs along with molecular modeling
investigations into their putative binding mode have identified
a key hydrogen bond between the NH group at position 3 of
the pyrimidinone and Lys101 (Figure 1, zone C) and profitable
hydrophobic interactions between the right portion of the
molecule and a large pocket in RT defined by the residues in
zones A and B, as illustrated in Figure 1.[20,21]
The substituent at position C2, which is characteristic for
each DABO family, establishes a series of interactions within
the HIV-1 RT non-nucleoside inhibitor binding pocket (NNBP)
which are essential for the antiviral activity of these derivatives.
Although computational studies have allowed rationalization
of the activity profiles of many compounds, these simulations
[a] Dr. M. Radi,+ Dr. M. Pagano, Dr. L. Franchi, Dr. D. Castagnolo,
Dr. G. Casaluce, Dr. C. Zamperini, Dr. E. Dreassi, Prof. M. Botta
Dipartimento Farmaco Chimico Tecnologico
University of Siena, Via Alcide de Gasperi 2, 53100 Siena (Italy)
E-mail: botta.maurizio@gmail.com
[b] Prof. S. Schenone
Dipartimento di Scienze Farmaceutiche
University of Genoa, Viale Benedetto XV 3, 16132 Genova (Italy)
[c] Dr. G. Maga, Dr. A. Samuele
Istituto di Genetica Molecolare
IGM-CNR, Via Abbiategrasso 207, 27100 Pavia (Italy)
[d] Dr. E. Gonzalo, Dr. B. Clotet, Prof. J. A. Estꢀ
Retrovirology Laboratory irsiCaixa
Hospital Universitari Germans Trias i Pujol
Universitat Autonoma de Barcelona, 08916 Badalona (Spain)
[e] Prof. M. Botta
Sbarro Institute for Cancer Research and Molecular Medicine
Center for Biotechnology, College of Science and Technology
Temple University, BioLife Science Building, Suite 333
1900 North 12th Street, Philadelphia, PA 19122 (USA)
[+] Current address: Dipartimento Farmaceutico, Universitꢁ degli Studi di
Parma, V.le G.P. Usberti 27/A, 43124 Parma (Italy)
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M. Botta et al.
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Figure 1. Structures of S-DABO, N-DABO, and hit compounds 1–3. Interaction of a generic C2-para-methoxyarylalkyl DABO within the NNBP of HIV-1 RT and
general structure (I) of the newly designed derivatives.
cannot explain the explicit role played by the substituent at
position C2, owing to the considerable conformational flexibili-
ty of RT, which has complicated traditional structure-based
drug design strategies for the discovery of novel NNRTIs.[20]
In a previous study dealing with the development of novel
S-DABOs, we reported that the introduction of a para-methoxy
arylalkyl moiety at C2 significantly increases the antiviral activi-
ty due to a profitable hydrophobic interaction with a large
zone of the allosteric pocket (Figure 1, zone D).[21] In particular,
the methoxy substituent that forms a hydrogen bond with
N103 is able to disrupt the interaction of this residue with
Y188 which in turn stabilizes the closed-pocket form of the
K103N mutant, imparting resistance to NNRTIs (an analogous
hydrogen bond contact is not present in the wild-type RT
As a continuation of our research on the identification of
DABO drug candidates for the treatment or prevention of
HIV,[25] we report herein the synthesis of two series of novel
DABO derivatives (S-DABOs and N-DABOs) bearing different
linkers at position C2. We first focused on the synthesis of
novel S-DABO derivatives by introducing various hydrophobic
spacers at C2 and linkers containing hydrogen bond donor/ac-
ceptor moieties in order to evaluate the possibility of establish-
ing additional profitable interactions with the NNBP. We next
planned the synthesis of N-DABO derivatives with an accepta-
ble ADME profile in terms of improved water solubility, perme-
ation, and low first-pass metabolism.
structure). In this manner, the C2 chain of the S-DABOs is able Results and Discussion
to penetrate into the NNBP, pushing residues V179 and Y181
S-DABOs
away from Y188, which is known to be one of the major hur-
dles hampering the formation of the NNBP. Compound 1, bear-
ing a para-methoxybenzylthio chain at C2, was the first S-
DABO derivative reported to be endowed with strong activity
toward the K103N mutant due to its ability to open the NNBP.
Subsequently, several analogues were synthesized, such as
compound 2,[22] with a cinnamylthio moiety, and its cycloprop-
yl bioisostere 3,[23] which emerged as the most potent deriva-
tive (Figure 1). The latter compound, in fact, showed picomolar
activity against wild-type RT and nanomolar activity against
many key mutant viral strains. Kinetic studies also demonstrat-
ed that compound 3 dissociates very slowly, if at all, from the
RT surface with a dissociation rate constant significantly lower
than values obtained with the same technique for delavirdine,
efavirenz, and nevirapine. Despite their excellent activity pro-
files, these compounds, as is the case for most of the DABO
derivatives, suffer from poor absorption, distribution, metabo-
lism, and excretion (ADME) properties. In their review, Sweeney
and Klumpp[24] reported the importance of pharmacokinetic
factors in the success of first-line anti-HIV therapies based on
NNRTIs. A head-to-head comparison between nevirapine and
efavirenz highlighted that even if the latter possesses higher in
vitro antiviral activity, its poor pharmacokinetic profile makes it
clinically similar to the less active nevirapine.
The initial series of novel S-DABO derivatives to be synthesized
was characterized by the presence of a methyl group at C5
and C6’ (R1 =R2 =CH3) and various linkers (W) at C2. First, lead
compound 2 was modified by introducing an additional
methyl group on the double bond of the linker to increase the
lipophilicity and to further investigate the role played by this
substituent on the left portion of the molecule (compound 7).
We then focused on the terminal alkyne derivative 10 to be
used as a key intermediate for the synthesis of new analogues.
The triple bond can be easily manipulated and converted into
the triazole derivatives 14 and 15 by 1,3-dipolar cycloaddition,
or in the case of diene 13, by enyne cross-metathesis reaction.
This latter compound was designed with the intent of explor-
ing possible hydrophobic interactions between the diene fea-
ture and zone D of HIV RT, similar to those established by the
cyclopropyl moiety of lead compound 3. Moreover, the triple
bond was reduced to cis alkene 20 to compare its activity with
the corresponding trans analogue 2. Finally, we decided to in-
troduce an oxygenated moiety within the linker to evaluate
the possibility of establishing hydrogen bond interactions
within zone D of the NNBP. The other pharmacophoric groups
of the S-DABO leads 1–3 were left unchanged, with the excep-
tion of the methyl group at C6’, which was removed from
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some derivatives to compare the activity of the diastereomeric
mixtures with the corresponding racemates.
The first target compound 7 was prepared as shown in
Scheme 1. The trans-4-methoxycinnamaldehyde was allowed
to react with methylmagnesium bromide to afford the 1,2-ad-
Scheme 2. Reagents and conditions: a) K2CO3, DMF, 08C, 1 h; b) BzCl, py, r.t.,
2 h.
Scheme 1. Reagents and conditions: a) MeMgBr, THF, 08C, 1.5 h; b) 5, P(CH3)3,
DIAD, DMF, MW, 408C, 30 min.
out as shown in Scheme 4. Propargyl alcohol 16 was allowed
to react via Sonogashira coupling with the 4-iodoanisole to
give compound 17, which was further reduced with Lindlar
catalyst to give cis-alkene 18. This latter compound was finally
converted into the corresponding bromide and reacted with
thiouracil 6 to give desired pyrimidinone 20. The synthesis of
oxygenated derivatives 25–33 is illustrated in Scheme 5 and
was performed by starting from derivatives with a propan-3-
one spacer as versatile intermediates for further functionaliza-
tion. The synthesis of key intermediates 25 and 26 was ach-
ieved by hetero-Michael condensation[27] between 1-(4-me-
thoxyphenyl)prop-2-en-1-one 23 and pyrimidinones 6 and 24.
The synthesis of intermediate 25 was planned in order to
obtain a series of non-diastereomeric derivatives 27, 29, 31
and non-chiral 33.
dition product alcohol 5, which in turn reacted with 6-(1-(2,6-
difluorophenyl)ethyl)-5-methyl-3,4-dihydro-2-thioxopyrimidin-
4(3H)-one 6 via microwave-assisted Mitsunobu reaction, lead-
ing to the desired compound 7 (Scheme 1). The key intermedi-
ate alkyne 10 was then synthesized from compound 6, which
was allowed to react with propargyl bromide in the presence
of potassium carbonate in anhydrous DMF at 08C to obtain
the desired compound 9. Microwave-assisted conditions were
not used in the latter alkylation to avoid formation of an unde-
sired bicyclic side product.[26] The C4-carbonyl group was then
protected as a benzoyl derivative (compound 10) to avoid any
side cyclization reaction during subsequent synthetic steps
(Scheme 2). Sonogashira coupling of thiouracil 10 with the 4-
iodoanisole followed by work-up deprotection with an aque-
ous mixture of ammonium chloride and ammonium hydroxide
led to compound 12 (Scheme 3). Alternatively, intermediate 11
was isolated by a neutral work-up and allowed to react with
ethylene in the presence of Grubbs’ second-generation catalyst
to give, after deprotection, diene
Starting from 4-methoxybenzaldehyde 21, a Grignard reac-
tion was performed with vinylmagnesium bromide to obtain
compound 22, which was then oxidized with manganese diox-
ide to give the desired 1-(4-methoxyphenyl)prop-2-en-1-one
23 (Scheme 5). The hetero-Michael condensation was finally
13. Finally, copper-catalyzed
azide–alkyne cycloaddition reac-
tions using a microwave-assisted
protocol developed in our labo-
ratories afforded derivatives 14
and 15. Compound 10 reacted
with the para-methoxyphenyl
azide and, after in situ deprotec-
tion, gave compound 14. Reac-
tion of 10 with sodium azide
and para-methoxybenzyl chlo-
ride gave compound 15, which
differs from the previous 14 by
the presence of an extra methyl-
ene bridge between the triazole
Scheme 3. Reagents and conditions: a) 4-iodoanisole, CuI, PdCl2(PPh3)2, Et3N, DMF, r.t., 10 min; b) NH4Cl/NH4OH aq.
solution, 30 min; c) 1. (for n=0): 4-OMePhN3, sodium ascorbate/Cu(SO)4, H2O/tBuOH, MW, 1208C, 10 min; (for
n=1): NaN3, BnCl, sodium ascorbate/Cu(SO)4, H2O/tBuOH, MW, 1258C, 10 min; 2. NH4Cl/NH4OH aq. solution,
ring and the para-methoxyphen-
yl moiety. The synthesis of cis-
alkene derivative 20 was carried 30 min; d) 1. CH2=CH2 1 atm, Grubbs 2, toluene, 808C, 48 h; 2. NH4Cl/NH4OH aq. solution, 30 min.
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N-DABOs
With the aim of obtaining improved RT inhibitors endowed
with an acceptable ADME profile, we next focused our atten-
tion on the synthesis of N-DABO analogues of lead compounds
1–3. Docking studies previously performed by us on this com-
pound class revealed a further anchor point represented by
the hydrogen bond between the NH function at C2 and the
carbonyl backbone of Lys101, which could compensate for the
lack of hydrophobic interactions within the active site. Previ-
ously reported N-DABO derivatives were characterized primari-
ly by alkyl or cycloalkyl substituents on the nitrogen atom at
C2 and, despite the large amount of data on wild-type RT, very
little information was reported for drug-resistant mutants such
as K103N.[28] Considering the important role played by the 4-
methoxyphenyl substituent in opening the NNBP of the K103N
mutant in the case of the S-DABO series, we planned to syn-
thesize new N-DABOs characterized by a 4-methoxybenzyl and
4-methoxyphenylcyclopropyl C2 linker bound to a primary or
secondary amino group. In fact, while the NH group at C2
gives an additional hydrogen bond interaction with Lys101,
further functionalization of this nitrogen atom could give addi-
tional insight into the structure–activity relationships for this
class of derivatives. The classical approaches for obtaining N-
DABOs are a direct Pinner pyrimidine synthesis or a C2-nucleo-
philic substitution on DABOs or S-DABOs using the appropriate
amines. The drawbacks of these procedures are long reaction
times, high temperatures, and the use of nucleophiles in great
excess; these limit their applicability with amines that are not
readily accessible. We recently reported a versatile microwave-
assisted domino Michael addition/cyclocondensation protocol
for the preparation of pyrimidin-4(3H)-one derivatives for use
in the synthesis of novel N-DABOs.[29] Following this new ap-
proach, we synthesized alkynone
Scheme 4. Reagents and conditions: a) 4-iodoanisole, CuI, PdCl2(PPh3)2, Et3N,
DMF, r.t., 5 min; b) H2, Lindlar catalyst, quinoline, EtOAc, r.t., 48 h; c) PBr3,
Et2O, 08C, 2 h; d) 19, K2CO3, DMF, r.t., 3 h.
performed in anhydrous DMF using trifluoromethanesulfonic
acid as catalyst to obtain the desired compounds in good
yields. The ketone moiety of the side chain was manipulated
to give secondary and tertiary alcohol derivatives 27–32. Re-
duction with sodium borohydride led to compounds 27 and
28. Grignard reaction with methyl- and ethylmagnesium bro-
mide gave compounds 29, 30 and 31, 32, respectively. Finally,
we decided to treat compound 25 with hydroxyamine hydro-
chloride in ethanol to furnish the corresponding oxime 33
with the aim of evaluating the interactions of the oxime group
with the RT binding site.
38 as a key intermediate for our
Michael addition/cyclocondensa-
tion protocol. Wittig olefination
of the commercially available 1-
(2,6-difluorophenyl)ethanone 34
with
methoxymethyltriphenyl-
phosphonium chloride and n-bu-
tyllithium at ꢀ788C in THF gave
the corresponding enol ether 35,
which, after cleavage with boron
tribromide followed by hydroly-
sis, gave aldehyde 36 in good
yield (Scheme 6). Alkynone 38
was then synthesized through
a
one-pot/two-step sequence
with an initial condensation of
aldehyde 36 with the Ohira–
Bestmann reagent[30] to obtain
alkyne 37, which was submitted
directly to carbonylation to
afford the key intermediate 38.
The substituted guanidines
39a–d required for the synthesis
Scheme 5. Reagents and conditions: a) vinylmagnesium bromide, THF, 08C, 1 h; b) MnO2, Et2O, r.t., 16 h; c) 23, tri-
fluoromethanesulfonic acid, DMF, r.t., 1 h; d) NaBH4, MeOH, 08C!r.t., 15 min; e) R2-MgBr, THF, 08C!r.t., 1 h;
f) NH2OH·HCl, NaOAc, EtOH, r.t., 16 h.
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ported in Table 1. Among the novel S-DABOs, compound 7 re-
tained an activity similar to or better than that of the leads 1–
3. Interestingly, derivatives 29–33, while modestly active
against wt RT and the K103N and Y118L mutants, showed very
potent inhibitory activities against the replication of the Y18C
mutant virus. The tertiary hydroxy moiety or oxime group on
the linker of these compounds may play a role in the interac-
tions with key residues of the latter mutant. On the other
hand, compounds characterized by a secondary hydroxy group
(27 and 28) did not show any selectivity for a specific RT
mutant, and derivative 28 proved to be the most interesting
derivative, endowed with good fold resistance. With the only
exception of compound 40a, the novel N-DABO derivatives
(Entries 16–19) showed similar or even better activity profiles
than leads 1–3. More importantly, subsequent in vitro ADME
studies showed improved properties for the synthesized N-
DABOs over the corresponding S-DABOs (Table 2). In fact, the
importance of optimizing ADME properties for potential drug
candidates is widely recognized; the success of a potential
drug is determined not only by good efficacy and specificity,
but also by acceptable ADME and toxicity properties. Early as-
sessment and optimization allow earlier corrections of property
limitations, thereby decreasing the risk of drug failure in ad-
vanced phases.[32] In this regard, a few in vitro experiments can
be easily conducted to quickly establish the absorption and
stability of drug candidates in the early phase: aqueous solu-
bility, parallel artificial membrane permeability assay
(PAMPA),[33] and human liver microsome (HLM) stability deter-
mination. These experiments have proven to be reliable indica-
tors of plasma exposure levels after oral administration. Ac-
cordingly, some of the most promising compounds previously
identified were submitted to a thorough ADME study to identi-
fy promising drug candidates endowed with better aqueous
solubility, passive permeation, and low phase 1 metabolism
(Table 2).
Scheme 6. Reagents and conditions: a) (methoxymethyl)triphenylphosphoni-
um chloride, nBuLi, THF, ꢀ788C!r.t; b) BBr3, CH2Cl2, ꢀ788C, 2 h; c) dimethyl
1-diazo-2-oxopropylphosphonate, K2CO3, MeOH, r.t., 12 h; d) PdCl2, CuCl2,
CO, NaOAc, MeOH, r.t., 2 h.
of the desired N-DABO derivatives 40a,c and 41b,d (Scheme 7)
were synthesized in a few steps and in high yields by starting
from commercially available compounds. Guanidines reacted
under Michael addition/cyclization conditions with 38 to give
the desired N-DABO derivatives 40a,c and 41b,d. Compounds
40a,c were obtained as single products in good yields, where-
as derivatives 41b,d were isolated together with their re-
gioisomers 42b,d.
Passive membrane permeability was initially evaluated in
a PAMPA by using a validated protocol we recently applied to
a family of pyrazolo[3,4-d]pyrimidines.[34] The aqueous solubility
was evaluated by following the method developed by
Avdeef,[35] and the results are expressed in mgmLꢀ1. Metabolic
stability was finally evaluated by incubating the aforemen-
tioned compounds with 5 mL pooled HLM fractions for 1 h at
378C in order to simulate phase 1 metabolism. The parent
drugs and metabolites were subsequently determined by LC–
MS analysis (see Experimental Section). N-DABO derivatives
40a and 41b showed values of membrane permeation and
metabolic stability similar to that of S-DABO lead 1, yet also
showed much improved aqueous solubility. In contrast, S-
DABO derivative 27 did not show an improved ADME profile
with respect to that of lead compound 1.
Scheme 7. Reagents and conditions: a) Na2CO3, tBuOH, 1208C, MW, 20 min
(for R2 =4-methoxybenzyl) or 40 min (for R2 =4-methoxyphenylcyclopropyl).
Biological assays and in vitro ADME
The synthesized compounds were evaluated in enzyme assays
for their ability to inhibit either wild-type (wt) or mutated RTs,
as well as in assays with MT-4 cells to evaluate their cytotoxici-
ty and anti-HIV activity as described previously,[31] in compari-
son with nevirapine (NVP) and efavirenz (EFV), used as refer-
ence drugs. In particular, the following mutants were used:
K103N and Y181I for enzymatic tests, K103N, Y181C, and
Y188L for tests on cell lines. The results of these assays are re-
Conclusions
In summary, we explored the effects of various linkers at posi-
tion C2 of S- and N-DABO NNRTIs, derived from leads 1–3, on
their antiviral activity and in vitro ADME properties. Several
compounds showed nanomolar inhibitory potency against
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Table 1. Anti HIV-1 activity and cytotoxicity of the new S-/N-DABO derivatives.
Entry Compd
X
W
R1
R2
IC50 [mm][a,b]
K103N
EC50 [mm][a,c]
K103N Y181C
CC50 [mm][d]
wt
Y181I
0.8
ꢁ0.1
0.47
NL4-3 wt
Y188L
0.02
0.04
ꢁ0.003 ꢁ0.01
0.06 0.36
ꢁ0.006 ꢁ0.06
0.0006
ꢁ0.0001
0.01
ꢁ0.001
0.52
0.16
0.045
ꢁ0.007
0.4
ꢁ0.05
37.3
ꢁ0.9
1
2
7
X
S
CH3 CH3
CH3 CH3
ꢁ0.05
0.1
ꢁ0.06
0.02
12
>25
ꢁ0.07
ꢁ0.01
ꢁ0.003
0.14
ꢁ0.02
0.08
ꢁ0.01
0.9
ꢁ0.1
0.085
ꢁ0.005
15.4
ꢁ0.5
3
13
S
CH3 CH3
ND[e]
ND
>15.4
0.32
ꢁ0.02
1.11
ꢁ0.01
0.12
ꢁ0.02
0.16
ꢁ0.03
13.2
ꢁ0.5
4.23
ꢁ0.03
0.098
ꢁ0.008
3.72
ꢁ0.02
4
5
14
15
S
S
CH3 CH3
CH3 CH3
ND
ND
ND
ND
>25
>25
>25
0.76
>25
>25
>25
>25
0.13
ꢁ0.01
0.65
ꢁ0.05
6
7
20
25
S
S
CH3 CH3 >4
ND
ND
>25
0.13
ꢁ0.01
0.022
ꢁ0.002
0.67
ꢁ0.07
13.3
ꢁ0.5
13.4
ꢁ0.5
11.3
ꢁ0.5
CH3
H
ND
ND
ND
ND
ND
ND
ꢁ0.06
8
26
27
28
29
30
S
S
S
S
S
CH3 CH3 >4
>13.42
>13.42
>13.42
>13.42
2.18
ꢁ0.08
1.09
ꢁ0.09
0.44
ꢁ0.04
0.6
ꢁ0.05
9
CH3
CH3 CH3
CH3
CH3 CH3
H
>11.28
>11.28
0.08
0.18
ꢁ0.01
1
0.36
ꢁ0.06
0.12
0.03
ꢁ0.003
17.2
10
11
12
ꢁ0.01
0.48
ꢁ0.08
0.17
ꢁ0.05
0.22
ꢁ0.02
ꢁ0.05
ꢁ0.02
0.28
ꢁ0.08
0.015
ꢁ0.005
0.007
ꢁ0.001
ꢁ0.5
3.2
ꢁ0.2
3.1
H
ND
ND
>3.2
>3.2
>3.2
0.3
ꢁ0.03
0.4
ꢁ0.04
>3.1
ꢁ0.1
ND
ND
ND
>3.1
ꢁ0.1
13
14
31
32
S
S
CH3
H
ND
>25
>25
>25
0.079
ꢁ0.009
4.45
ꢁ0.05
CH3 CH3 >4
ND
ND
ND
>4.45
>4.45
>4.45
>25
0.76
0.45
ꢁ0.05
0.002
ꢁ0.0002
0.003
ꢁ0.0004
0.003
ꢁ0.0005
0.001
ꢁ0.0001
0.00003
ꢁ0.00001
0.0002
0.0015
ꢁ0.0001
1.61
ꢁ0.02
>5
10.27
ꢁ0.03
>5
15
16
33
S
CH3
H
H
ND
>10.27
ꢁ0.06
0.006
ꢁ0.001
0.010
40a
NCH3
CH2
CH2
CH2
CH3
1.055
0.366
ꢁ0.06
0.666
ꢁ0.006
ND
0.003
ꢁ0.0003
0.07
ꢁ0.009
2.23
ꢁ0.03
0.13
ꢁ0.01
0.55
0.01
ꢁ0.001
2.95
ꢁ0.05
>5
0.885
17
18
40c
41b
NCH3
NH
H
H
CH3
CH3
>5
>5
ꢁ0.004 ꢁ0.007
0.057
ꢁ0.007
0.01
5
0.06
ꢁ0.01
0.10
ꢁ0.001
0.30
0.88
0.313
ꢁ0.005
19
22
41d
1
NH
S
H
CH3
>5
>5
ꢁ0.001 ꢁ0.08
CH3 CH3
0.003 45
>20
0.95
>12.43
ꢁ0.05
0.26
ꢁ0.03
23
2
S
CH3 CH3
CH3 CH3
0.292
0.04
3.7
>20
0.13
0.07
>11.7
ꢁ0.00004
0.0006
24
25
3
S
3.00
8.00
ꢁ0.1
0.40ꢁ0.01
6.00
20.0
ꢁ0.9
0.10ꢁ0.1
0.52
0.16
0.045
37.3
NVP
NH2
–
–
–
–
0.40
ꢁ0.05
0.04
ꢁ0.06
0.47
>5
>5
>5
>5
ꢁ0.07
26
EFV
–
–
–
0.0004
ꢁ0.0002
0.01
ꢁ0.003
0.56
ꢁ0.0005
0.45
ꢁ0.01
>1
[a] Data represent the mean of at least two experiments. [b] IC50: inhibitory concentration 50; concentration required to inhibit 50% of the enzyme.
[c] EC50: effective concentration 50; concentration required to inhibit 50% of HIV-induced cell death, evaluated by the MTT method in MT-4 cells. [d] CC50
:
cytotoxic concentration 50; concentration required to induce 50% death of uninfected cells evaluated by the MTT method in MT-4 cells. [e] ND: not deter-
mined.
888
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ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 883 – 896

S-/N-DABOs as HIV RT Inhibitors
MED
Table 2. ADME properties of S-/N-DABO derivatives.
[c]
[d]
Compd
QP-logP[a]
QP-logS[b]
QP-Caco [nmsꢀ1
]
Papp ꢂ10ꢀ6 [cmsꢀ1
]
Aq. Sol. [mgmLꢀ1
]
logS
Met. Stab. [%][e]
Memb. Ret. [%][f]
27
40a
41b
1
4.44
4.98
4.33
4.8
ꢀ6.9
ꢀ6.1
ꢀ6.1
ꢀ6.3
859
2485
1119
1845
0.97
11.6
6.72
10.5
0.011
0.11
2.42
ꢀ7.6
ꢀ6.5
ꢀ5.18
ꢀ7.5
99
99
98
99
0
32
6.1
1.7
<0.01
[a] Predicted octanol/water partition coefficient; range of recommended values: (ꢀ2.0)–(+6.5). [b] Predicted aqueous solubility; range of recommended
values: (ꢀ6.5)–(+0.5). [c] Predicted apparent Caco-2 cell permeability; range of recommended values: <25: poor, >500: great. [d] PAMPA: see Experimen-
tal Section for details. [e] Metabolic stability expressed as a percentage of unmodified parent drug after 60 min incubation with HLM protein. [f] Membrane
retention expressed as percentage of compound retained within the phospholipid layer (50% DMSO).
Microwave irradiation experiments: Microwave irradiation experi-
wild-type HIV-1 and relevant drug-resistant mutants. Even if
more detailed investigations are required to clearly understand
the role of the C2 linker on the anti-HIV profile of S-DABO de-
rivatives, the data reported herein give new insight into the
chemical space that could be further explored around the C2
position of the S-DABO family. In addition, these preliminary re-
sults suggest that the conversion of the S-DABO derivatives
into the corresponding N-DABOs may lead to new derivatives
characterized by improved ADME profiles (especially aqueous
solubility) for this potent family of antiviral agents, and will aid
in the further rational design of more effective NNRTIs.
ments were conducted with a CEM Discover Synthesis Unit (CEM
Corp., Matthews, NC, USA). The instrument consists of a continuous
focused microwave power delivery system with operator-selectable
power output from 0 to 300 W. The temperature of the vessel con-
tents was monitored with a calibrated infrared temperature control
unit mounted under the reaction vessel. All experiments were per-
formed with a stirring option, whereby the contents of the vessel
are stirred by means of a rotating magnetic plate located below
the floor of the microwave cavity and a Teflon-coated magnetic stir
bar in the vessel.
ADME assays
Chemicals: All solvents, reagents, and l-a-phosphatidylcholine
Experimental Section
were from Sigma–Aldrich Srl (Milan, Italy). Dodecane was pur-
chased from Fluka (Milan, Italy). Pooled HLM fractions (20 mgmLꢀ1
)
General chemistry
from human male donors were obtained from BD Gentest-Biosci-
ences (San Jose, CA, USA). Milli-Q purified water (Millipore, Milford,
MA, USA) was used. Hydrophobic filter plates (MultiScreen-IP, Clear
Plates, 1pore =0.45 mm), 96-well microplates, and 96-well UV-trans-
parent microplates were obtained from Millipore (Bedford, MA,
USA).
Materials and methods: All commercially available reagents were
purchased from Sigma–Aldrich and were used as received. Solvents
were reagent grade and, when necessary, purified and dried by
standard methods. Anhydrous reactions were run under positive
pressure of dry N2 or Ar gas. IR spectra were recorded on a Perki-
nElmer BX FTIR system, using KBr pellets. Thin-layer chromatogra-
phy (TLC) was carried out using Merck TLC silica gel 60 F254 plates.
Flash chromatographic purifications were performed on columns
packed with Merck silica gel 60, 23–400 mesh. 1H NMR and
13C NMR spectra were recorded at 400 MHz on a Bruker Avance
DPX400, at 300 MHz on a Varian VXR-300, and at 200 MHz on
a Bruker AC200F spectrometer. Chemical shifts are reported relative
to tetramethylsilane at 0.00 ppm. Elemental analyses (C, H, N) were
performed in house using a PerkinElmer Elemental Analyzer 240C
and data are within 0.4% of the theoretical values. Melting points
were taken with a Gallenkamp melting point apparatus and are un-
corrected. MS data were obtained with an Agilent 1100 LC/MSD VL
system (G1946C) at a flow rate of 0.4 mLminꢀ1 using a binary sol-
vent system of 95:5 MeOH/H2O. UV detection was monitored at
254 nm. MS data were acquired in positive and negative mode,
scanning over the mass range 50–1500. The following ion source
parameters were used: drying gas flow, 9 mLminꢀ1; nebulizer pres-
sure, 40 psig; drying gas temperature, 3508C. All target com-
pounds possessed a purity of ꢂ95% as verified by elemental anal-
yses by comparison with the theoretical values.
Parallel artificial membrane permeability assay (PAMPA): Donor
solution (0.5 mm) was prepared by diluting 1 mm compound stock
solution in DMSO with phosphate buffer (0.025m, pH 7.4). Filters
were coated with 5 mL 1% (w/v) solution of phosphatidylcholine in
dodecane. Donor solution (150 mL) was added to each well of the
filter plate. To each well of the acceptor plate were added 300 mL
solution (50% DMSO in phosphate buffer). All compounds were
tested in three different plates in different days. The sandwich was
incubated for 5 h at room temperature under gentle shaking. After
the incubation time, the sandwich plates were separated and sam-
ples were taken from both receiver and donor sides and analyzed
by LC with UV detection. LC analyses were conducted by HPLC
(PerkinElmer series 200) using a Polaris C18 column (150ꢂ4.6 mm,
5 mm particle size) at a flow rate of 0.8 mLminꢀ1 with a mobile
phase composed of CH3CN/0.1% HCOOH(aq) (60:40 v/v) for each
compound. Permeability (Papp) for PAMPA was calculated according
to Equation (1), obtained from the equation of Wohnsland and
Faller[36] and Sugano et al.[37] with some modification, in order to
obtain permeability values in cmsꢀ1
:
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www.chemmedchem.org
889

M. Botta et al.
MED
VDVA
Papp
¼
ꢀ lnð1 ꢀ rÞ
ð1Þ
Table 3. Chromatographic and MS parameters of selected compounds.
ðVD þ VAÞAt
Compd
tR [min][a]
Monitored
Collision
Capillary
Transition [m/z]
Energy [eV]
Voltage [V]
27
40a
41b
1
5.9
5.9
5.3
6.4
433!147
386!121
372!121
403!121
ꢀ15
34
50
69
40
for which VA is the volume in the acceptor well, VD is the volume in
the donor well (cm3), A is the “effective area” of the membrane
(cm2), t is the incubation time (s), and r is the ratio between drug
concentration in the acceptor and equilibrium concentration of the
drug in the total volume (VD +VA). Drug concentration is estimated
by using peak area integration.
ꢀ19.0
ꢀ17.0
ꢀ17.0
[a] Retention time.
Microsomal stability assays: Each compound in DMSO solution
was incubated at 378C for 60 min in 125 mm phosphate buffer
(pH 7.4) and 5 mL HLM protein (0.2 mgmLꢀ1) in the presence of
a NADPH-generating system at a final volume of 0.5 mL (final com-
pound concentration: 50 mm); DMSO did not exceed 2% (final so-
lution). The reaction was stopped by cooling in ice and adding
1.0 mL CH3CN. The reaction mixtures were then centrifuged
(10 min, 48C, 10000 rpm), and the parent drug and metabolites
were subsequently determined by LC–MS. Chromatographic analy-
ses were performed with an Agilent 1100 LC/MSD VL system
(G1946C; Agilent Technologies, Palo Alto, CA, USA) consisting of
a vacuum solvent degassing unit, a binary high-pressure gradient
pump, an 1100 series UV detector, and an 1100 MSD model VL
benchtop mass spectrometer. Chromatographic separation was
carried out with a Varian Polaris 5 C18 A column (150–4.6 mm, 5 mm
particle size) and gradient elution [eluent A: CH3CN; eluent B:
HCOOH(aq) (0.1%)]. The analysis started with 2% eluent A, which
was rapidly increased to 70% over 10 min, then slowly increased
to 98% over 15 min. The flow rate was 1.0 mLminꢀ1, and injection
volume was 20 mL. The mass spectra detection (MSD) single-quad-
rupole instrument was equipped with orthogonal spray API-ES
(Agilent Technologies). N2 was used as both nebulizing and drying
gas (pressure: 40 psi, flow rate: 9 mLminꢀ1, capillary voltage:
3000 V, fragmentor voltage: 70 V, vaporization temperature:
3508C). UV detection was set at l 254 nm. LC–ESIMS determination
was performed by operating the MSD in the positive ion mode.
Spectra were acquired over the scan range m/z 100–1500 using
a step size of 0.1 u. The percentage of non-metabolized compound
was calculated by comparison with reference solutions.
a given compound was made by comparison with the appropriate
calibration curves generated with standard solutions in MeOH.
Biological methods
Chemicals: [3H]dTTP (40 Cimmolꢀ1) was obtained from Amersham,
and unlabeled dNTPs were purchased from Boehringer. GF/C filters
were supplied by Whatman. All other reagents were of analytical
grade and were purchased from Merck or Fluka.
Nucleic acid substrates: The homopolymer poly(rA) (Pharmacia)
was mixed at weight ratios in nucleotides of 10:1 to the oligomer
oligo(dT)12–18 (Pharmacia) in 20 mm Tris·HCl (pH 8.0) containing
20 mm KCl and 1 mm EDTA, heated at 658C for 5 min, and then
slowly cooled to room temperature.
Expression and purification of recombinant HIV-1 RT forms: Re-
combinant heterodimeric wild-type RT, Leu100Ile, Val179Asp,
Lys103Asn, and Tyr181Ile were expressed and purified to >95%
purity (as determined by SDS-PAGE) as described.[25]
HIV-1 RT RNA-dependent DNA polymerase activity assays: RNA-
dependent DNA polymerase activity was assayed as follows: a final
volume of 25 mL contained buffer A (50 mm Tris·HCl pH 7.5, 1 mm
DTT, 0.2 mgmLꢀ1 BSA, 4% glycerol), 10 mm MgCl2, 0.5 mg poly(rA)/
oligo(dT) 10:1 (0.3 mm 3’-OH ends), 10 mm [3H]dTTP (1 Cimmolꢀ1),
and 5–10 nm RT. Reactions were incubated for 10 min at 378C. Ali-
quots (20 mL) were then spotted onto GF/C glass fiber filters, which
were immediately immersed in ice-cold 5% trichloroacetic acid
(TCA). Filters were washed twice in ice-cold 5% TCA and once in
EtOH for 5 min, dried, and the acid-precipitable radioactivity was
measured by scintillation counting.
Water solubility assays: Each solid compound (1 mg) was added
to 1 mL H2O. Samples were shaken in a shaker bath at 208C for
24–36 h. The suspensions were filtered through a 0.45 mm nylon
filter (Acrodisc), and the dissolved compound analyzed by LC–MS–
MS. Determinations were performed in triplicate for each com-
pound. An LC–MS system was used for quantification and consist-
ed of a Varian apparatus including a vacuum solvent degassing
unit, two pumps (212-LC), a triple quadrupole MSD (Model 320-LC)
mass spectrometer with ES interface and Varian MS Workstation
System Control Ver. 6.9 software. Chromatographic separation was
carried out with a Pursuit C18 column (50ꢂ2.0 mm) (Varian) with
3 mm particle size and gradient elution [eluent A: CH3CN; eluent B:
HCOOH(aq) (0.1%)]; Analysis started with 0% eluent A, which was
increased linearly to 70% over 10 min, then slowly increased to
98% over 15 min. The flow rate was 0.2 mLminꢀ1, and injection
volume was 5 mL. The instrument was operated in positive mode
and parameters were as follows: detector 1850 V, drying gas pres-
sure 25.0 psi, desolvation temperature 300.08C, nebulizing gas
45.0 psi, needle 5000 V, and shield 600 V. N2 was used as nebulizer
and drying gas. Collision-induced dissociation was performed with
Ar as the collision gas at a pressure of 1.8 mTorr in the collision
cell. The transitions, capillary voltage, and collision energy used for
each compound are summarized in Table 3. Quantification of
Inhibition assays: Reactions were performed under the conditions
described for the HIV-1 RT RNA-dependent DNA polymerase activi-
ty assay. Incorporation of radioactive dTTP into poly(rA)/oligo(dT)
was monitored in the presence of increasing amounts of inhibitor
as indicated in the Figure legends. Data were analyzed according
to Equation (2):
v ¼ Vmax=ð1 þ I=IC50Þ
ð2Þ
in which v is the apparent velocity of the reaction, Vmax is the ap-
parent maximum reaction rate (in absence of inhibitor), I is the in-
hibitor concentration, and IC50 is the concentration required to in-
hibit the enzymatic activity by 50%. Experiments were performed
in triplicate, and mean values were fitted to the equation with
GraphPad Prism 3.0 software.
Chemical synthesis
(E)-4-(4-Methoxyphenyl)but-3-en-2-ol (5): To a solution of trans-4-
methoxycinnamaldehyde (1 g, 6.173 mmol) in 10 mL THF, MeMgBr
890
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ChemMedChem 2012, 7, 883 – 896

S-/N-DABOs as HIV RT Inhibitors
MED
(3.0m in THF; 3.09 mL, 9.259 mmol) was added dropwise at 08C,
and the mixture was stirred at this temperature for 1.5 h. The mix-
ture was then diluted with Et2O and quenched with a small
amount of saturated NH4Cl(aq). The resulting suspension was filtered
through Celite, and to the filtered solution H2O was added fol-
lowed by two extractions with Et2O. The organic layers were col-
lected, dried over Na2SO4, and concentrated under reduced pres-
sure. The crude product was purified by flash chromatography
(Et2O/petroleum ether (PE), 40:60) to obtain pure product (990 mg,
C23H18F2N2O2S: C 65.08, H 4.27, N 6.60, found: C 65.21, H 4.34, N
6.69.
6-(1-(2,6-Difluorophenyl)ethyl)-2-(3-(4-methoxyphenyl)prop-2-
ynylthio)-5-methylpyrimidin-4-yl benzoate (11) and 6-(1-(2,6-di-
fluorophenyl)ethyl)-2-(3-(4-methoxyphenyl)prop-2-ynylthio)-5-
methylpyrimidin-4(3H)-one (12): To a solution of compound 10
(54.3 mg,0.128 mmol) in dry DMF, 4-iodoanisole
9 (30 mg,
0.128 mmol), Et3N (36 mL, 0.256 mmol), PdCl2(PPh3) (9 mg,
0.0128 mmol), and CuI (7.3 mg, 0.038 mmol) were added. The reac-
tion mixture was stirred for 10 min at room temperature and then
extracted with EtOAc. The combined extract was dried over anhy-
drous Na2SO4 and concentrated under reduced pressure to give 11
(68 mg, yield >99%); mp: 231–2338C; 1H NMR (400 MHz, CDCl3):
d=7.75 (d, J=8 Hz, 2H), 7.49–7.44 (m, 3H), 7.25–7.20 (m, 2H),
7.09–7.06 (m, 1H), 6.76–6.73 (m, 4H), 4.58–4.56 (m, 1H), 4.15–4.00
(m, 2H), 3.73 (s, 3H), 1.95 (s, 3H), 1.63 ppm (d, 3H); MS (ESI) m/z:
531 [M+H]+; Anal. calcd for C30H24F2N2O3S: 67.91, H 4.56, N 5.28,
found: C 67.87, H 4.50, N 5.19. Compound 11 was then dissolved
in EtOAc, a solution of NH4Cl/NH4OH (10:1) was added, and the re-
sulting mixture was stirred for 30 min. The organic phase was then
separated, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. Purification by flash chromatography (eluent:
PE/EtOAc 4:1) give the final product 12 (54.5 mg, yield >99%);
1
yield 90%) as a yellow solid. H NMR (400 MHz, CDCl3): d=7.29 (d,
J=7.2 Hz, 1H), 6.82 (d, J=7.3 Hz, 1H), 6.49 (d, Jtrans =15.9 Hz, 1H),
5.59–6.18 (dd, 1H, J=7.3 Hz, Jtrans =15.9 Hz, 1H), 4.32–4.52 (m, 1H),
3.78 (s, 3H), 1.33 ppm (d, 3H, J=7.2 Hz); Anal. calcd for C11H14O2: C
74.13, H 7.92, found: C 74.25, H 7.98.
2-((E)-4-(4-Methoxyphenyl)but-3-en-2-ylthio)-6-(1-(2,6-difluoro-
phenyl)ethyl)-5-methylpyrimidin-4(3H)-one (7): 6-(1-(2,6-Difluoro-
phenyl)ethyl)-5-methyl-3,4-dihydro-2-thioxopyrimidin-4(3H)-one
6
(40 mg, 0.141 mmol) and compound 5 (38 mg, 0.212 mmol) were
suspended in the minimal amount of dry DMF (1 mL) in the pres-
ence of trimethylphosphine (1m solution in toluene, 212 mL,
212 mmol). The reaction mixture was cooled in an ice bath, and
DIAD was added (42 mL, 0.212 mmol). The mixture was microwave
irradiated at 408C for 30 min and was then diluted with H2O (2 mL)
and extracted with Et2O (3ꢂ10 mL). Organic phases were collected,
dried over anhydrous Na2SO4, and evaporated to dryness. The resi-
due was purified on silica gel by flash chromatography to obtain
1
mp: 220–2228C; H NMR (400 MHz, CDCl3): d=12.38 (bs, 1H), 7.25–
7.20 (m, 2H), 7.09–7.06 (m, 1H), 6.76–6.73 (m, 4H), 4.58–4.56 (m,
1H), 4.15–4.00 (m, 2H), 3.73 (s, 3H), 1.95 (s, 3H), 1.63 ppm (d, 3H);
13C NMR (100 MHz, CDCl3): d=163.42, 162.51 (dd, J1 =246 Hz, J2 =
9 Hz, 2C), 154.75, 133.24, 128.18 (d, J=9 Hz, 1C), 114.91, 113.83,
111.62, 111.36 (d, J=25 Hz, 2C), 83.07, 82.36, 55.27, 34.05, 20.47,
17.66, 9.84 ppm; MS (ESI) m/z: 427 [M+H]+, 449 [M+Na]+; Anal.
calcd for C23H20F2N2O2S: C 64.77, H 4.73, N 6.57, found: C 64.92, H
4.69, N 6.48.
compound
7 (18 mg, yield 28%) as a
white solid. 1H NMR
(400 MHz, CDCl3): d=7.24–7.03 (m, 3H), 6.79–6.72 (m, 4H), 6.42
(dd, Jtrans =15.9 Hz, 1H), 6.14 (dd, J=7.32 Hz, Jtrans =15.9 Hz, 1H),
4.66–4.61 (q, J=7.8 Hz, 1H), 4.54–4.52 (q, J=7.8 Hz, 1H), 3.74 (s,
3H), 1.93 (s, 3H), 1.63–1.61 (d, J=7.1 Hz, 3H), 1.48 ppm (dd, J=
6.7 Hz, 3H); MS (ESI) m/z: 443 [M+H]+; Anal. calcd for
C24H24F2N2O2S: C 65.14, H 5.47, N 6.33, found: C 65.37, H 5.53, N
6.76.
6-(1-(2,6-Difluorophenyl)ethyl)-2-(3-(4-methoxyphenyl)-2-methyl-
enebut-3-enylthio)-5-methylpyrimidin-4(3H)-one (13): A solution
of compound 11 (80 mg, 0.1509 mmol) in toluene and ruthenium
carbene complex (Grubbs 2; 10 mol%) was stirred at 808C under
ethylene gas (1 atm) for 48 h. The solvent was removed under re-
duced pressure, and then the crude product was dissolved in
EtOAc and a solution of NH4Cl/NH4OH (10:1) was added. The mix-
ture was stirred for an additional 30 min and then the organic
layer was separated, dried over Na2SO4, concentrated under re-
duced pressure, and purified by flash chromatography (eluent:
Hex/Et2O 1:1) to give the desired product 13 (10 mg, yield 15%);
mp: 227–2298C; 1H NMR (400 MHz, CDCl3): d=7.14 (d, J=8 Hz,
2H), 7.10–7.06 (m, 1H), 6.79–6.73 (m, 4H), 5.26 (s, 1H), 5.19 (s, 1H)
5.12 (s, 1H), 5.02 (s, 1H), 4.53–4.50 (m, 1H), 4.11–4.06 (m, 2H), 3.74
(s, 3H), 1.92 (s, 3H), 1.61 ppm (d, 3H); 13C NMR (100 MHz, CDCl3):
d=164.14 (dd, J1 =245 Hz, J2 =9 Hz, 2C), 159.81, 155.29, 147.92,
143.32, 132.96, 129.39, 128.16 (d, J=9 Hz, 1C), 119.41, 116.13,
114.29, 113.52, 111.63, 111.38 (d, J=26 Hz, 2C), 55.27, 34.06, 29.70,
17.68, 9.89 ppm; MS (ESI) m/z: 455 [M+H]+, 477 [M+Na]+; Anal.
calcd for C25H24F2N2O2S: C 66.06, H 5.32, N 6.16, found: C 66.11, H
5.27, N 6.19.
6-(1-(2,6-Difluorophenyl)ethyl)-5-methyl-2-(prop-2-ynylthio)pyri-
midin-4(3H)-one (9): Compound 6 (400 mg, 1.41 mmol) was sus-
pended at 08C under Ar in a minimal amount of dry DMF, then
K2CO3 (147 mg, 1.41 mmol) and propargyl bromide 15 (139 mL,
1.56 mmol) were added, and the mixture was stirred at 08C for 3 h.
H2O was added to the reaction mixture, which was then extracted
with EtOAc, dried over Na2SO4, concentrated under reduced pres-
sure, and purified by flash chromatography (eluent: PE/EtOAc 2:1)
to give the desired product 9 (382 mg, yield 84%); mp: 223–
1
2258C; H NMR (400 MHz, CDCl3): d=7.09–7.05 (m, 1H), 6.83–6.79
(m, 2H), 4.57–4.51 (m, 1H), 3.91–3.87 (m, 2H), 2.10 (s, 1H), 1.97 (s,
3H), 1.60 ppm (d, 3H); MS (ESI) m/z: 321 [M+H]+, 343 [M+Na]+;
Anal. calcd for C16H14F2N2OS: C 59.99, H 4.40, N 8.74, found: C
60.02, H 4.55, N 8.89.
6-(1-(2,6-Difluorophenyl)ethyl)-5-methyl-2-(prop-2-ynylthio)pyri-
midin-4-yl benzoate (10): To a solution of compound 9 (378 mg,
1.18 mmol) in pyridine (19 mL), benzoyl chloride 19 (685 mL,
6 mmol) was added, and the mixture was stirred at room tempera-
ture for 2 h. The reaction mixture was quenched with saturated
NaHCO3(aq) (50 mL), extracted with EtOAc (3ꢂ50 mL), and acidified
with 1n HCl. The combined extract was dried over anhydrous
Na2SO4 and concentrated under reduced pressure to give the de-
sired product 10 (451 mg, yield 90%); mp: 234–2368C; 1H NMR
(400 MHz, CDCl3): d=8.09 (d, J=8 Hz, 2H), 7.60–7.56 (m, 1H),
7.45–7.41 (m, 2H), 7.11–7.09 (m, 1H), 6.81–6.77 (m, 2H), 4.68–4.63
(m, 1H), 3.85–3.80 (m, 2H), 2.07 (s, 1H), 1.93 (s, 3H), 1.70 ppm (d,
3H); MS (ESI) m/z: 425 [M+H]+, 447 [M+Na]+; Anal. calcd for
6-(1-(2,6-Difluorophenyl)ethyl)-2-((1-(4-methoxyphenyl)-1H-
1,2,3-triazol-4-yl)methylthio)-5-methylpyrimidin-4(3H)-one (14):
A
mixture of 4-iodoanisole (468 mg, 2 mmol) NaN3 (156 mg,
2.4 mmol), CuI (38 mg, 0.2 mmol) l-proline (48 mg, 0.4 mmol), and
a solution of NaOH 20% in DMSO (4 mL) was heated at 608C
under Ar for 18 h. The cooled mixture was partitioned between
EtOAc and H2O. The organic layer was separated, and the aqueous
layer extracted with EtOAc twice. The combined organic layers
were separated, washed with brine, dried (Na2SO4), filtered, and
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891

M. Botta et al.
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evaporated to give the crude 4-methoxyphenylazide. Then com-
pound 10 (51.6 mg, 0.12 mmol) and freshly synthesized para-me-
thoxyphenylazide (18 mg, 0.12 mmol) were suspended in a 1:1
mixture of H2O and tBuOH (1.5 mL each) in a 10 mL glass vial
equipped with a small magnetic stir bar. To this was added sodium
ascorbate (0.1 equiv) and CuSO4·5H2O (0.01 equiv). The mixture
was then irradiated for 10 min at 1258C using an irradiation power
of 100 W. After completion of the reaction a solution of NH4Cl/
NH4OH (10:1) was added, stirring was continued for 30 min, fol-
lowed by extraction with EtOAc, drying over Na2SO4, concentration
under reduced pressure, and purification by flash chromatography
(eluent: EtOAc) to give the desired product 14 (47 mg, yield 82%);
mp: 241–2438C; 1H NMR (400 MHz, CDCl3): d=7.49–7.46 (m, 1H),
7.10–7.04 (m, 1H), 6.77–6.73 (m, 3H), 6.62–6.60 (m, 2H), 6.49 (s,
1H), 4.44–4.42 (m, 1H), 3.96 (bs, 2H), 3.69 (s, 3H), 1.86 (s, 3H),
1.54 ppm (d, 3H); 13C NMR (100 MHz, CDCl3): d=162.63, 161.13
(dd, J1 =245 Hz, J2 =9 Hz, 2C), 160.16, 140.25, 138.20, 128.27 (d, J=
8 Hz, 1C), 128.07, 116.37, 111.67, 111.41 (d, J=26 Hz, 2C), 104.65,
55.31, 33.69, 31.28, 17.61, 10.17 ppm; MS (ESI) m/z: 470 [M+H]+;
Anal. calcd for C23H21F2N5O2S: C 58.84, H 4.51, N 14.92, found: C
58.92, H 4.59, N 15.01.
flash chromatography (eluent: Hex/Et2O 1:1) to give the desired
product 18 (57.4 mg, yield 25%); mp: 112–1148C; 1H NMR
(400 MHz, CDCl3): d=7.09 (d, J=8 Hz, 2H), 6.81 (d, J=8 Hz, 2H),
6.43 (d, Jcis =12 Hz, 1H), 5.74–5.68 (m, Jcis =12 Hz, 1H), 4.36 (d, 2H),
3.71 ppm (s, 3H); MS (ESI) m/z: 163 [MꢀH]ꢀ; Anal. calcd for
C10H12O2: C 73.15, H 7.37, found: C 73.21, H 7.42.
(Z)-1-(3-Bromoprop-1-enyl)-4-methoxybenzene (19): To a solution
of compound 18 (28.4 mg, 0.173 mmol) in Et2O, PBr3 (16 mL,
0.173 mmol) was added at 08C under Ar. The reaction mixture was
stirred at 08C for 1 h, then quenched with H2O (5 mL) and a saturat-
ed solution of NaHCO3, and extracted with EtOAc (3ꢂ50 mL). The
combined extract was dried over anhydrous Na2SO4 and concen-
trated under reduced pressure to give the desired product 19
(39 mg, yield >99%); mp: 116–1188C; 1H NMR (400 MHz, CDCl3):
d=7.09 (d, J=8 Hz, 2H), 6.81 (d, J=8 Hz, 2H), 6.38 (d, Jcis =12 Hz,
1H), 5.70–5.63 (m, Jcis =12 Hz, 1H), 3.98 (d, 2H), 3.70 ppm (s, 3H);
MS (ESI) m/z: 226 [MꢀH]ꢀ; Anal. calcd for C10H11OBr: C 52.89, H
4.88, found: C 52.80, H 4.79.
(Z)-6-(1-(2,6-Difluorophenyl)ethyl)-2-(3-(4-methoxyphenyl)allyl-
thio)-5-methylpyrimidin-4(3H)-one (20): Compound 6 (33.4 mg,
0.12 mmol) was suspended under Ar in a minimal amount of dry
DMF. Then K2CO3 (16.3 mg, 0.130 mmol) and compound 19
(29.6 mg, 1.56 mmol) were added, and the resulting mixture was
stirred for 3 h at room temperature. H2O was added to the reaction
mixture, the organic layer was separated, and the aqueous phase
was extracted with EtOAc (3ꢂ50 mL), dried over Na2SO4, concen-
trated under reduced pressure, and purified by flash chromatogra-
phy (eluent: PE/Hex 4:1) to give the desired product 20 (23 mg,
yield 45%); mp: 220–2228C; 1H NMR (400 MHz, CDCl3): d=11.79
(bs, 1H), 7.15 (d, J=8 Hz, 2H), 7.07–7.04 (m, 1H), 6.77–6.73 (m,
4H), 6.41 (d, Jcis =12 Hz, 1H), 6.03–5.97 (m, Jcis =12 Hz, 1H), 4.57–
4.52 (m, 1H), 3.97–3.91 (m, 2H), 3.75 (s, 3H), 1.95 (s, 3H), 1.62 ppm
(d, 3H); 13C NMR (100 MHz, CDCl3): d=163.71, 162.80 (dd, J1 =
246 Hz, J2 =9 Hz, 2C), 155.58, 132.58, 130.12, 128.17 (d, J=9 Hz,
1C), 127.61, 122.28, 113.92, 111.63 (d, J=25 Hz, 2C), 55.29, 33.24,
29.70, 17.74, 9.84 ppm; MS (ESI) m/z: 427 [MꢀH]ꢀ; Anal. calcd for
C23H22F2N2O2S: C 64.47, H 5.18, N 6.54, found: C 64.39, H 5.09, N
6.43.
6-(1-(2,6-Difluorophenyl)ethyl)-2-((1-(4-methoxybenzyl)-1H-1,2,3-
triazol-4-yl)methylthio)-5-methylpyrimidin-4(3H)-one (15): para-
Methoxybenzyl chloride (17.4 mL, 0.13 mmol), compound 10
(54 mg, 0.13 mmol) and NaN3 (9.22 mg, 0.14 mmol) were suspend-
ed in a 1:1 mixture of H2O and tBuOH (1.5 mL each) in a 10 mL
glass vial equipped with a small magnetic stir bar. To this were
added sodium ascorbate (0.1 equiv) and CuSO4·5H2O (0.01 equiv),
and the vial was microwave irradiated (100 W) for 10 min at 1258C.
After completion of the reaction, a solution of NH4Cl/NH4OH (10:1)
was added, and stirring was continued for 30 min. The mixture was
then extracted with EtOAc, dried over Na2SO4, concentrated under
reduced pressure, and purified by flash chromatography (eluent:
PE/EtOAc 3:1) to give the desired product 15 (34.5 mg, yield 56%);
mp: 217–2198C; 1H NMR (400 MHz, CDCl3): d=7.20–7.18 (m, 1H),
7.09–7.04 (m, 2H), 6.77–6.73 (m, 3H), 6.49 (s, 1H), 5.05 (bs, 2H),
4.45–4.42 (m, 2H), 3.97 (s, 3H), 1.86 (s, 3H), 1.54 ppm (d, 3H,);
13C NMR (100 MHz, CDCl3): d=162.54, 161.13 (dd, J1 =245 Hz, J2 =
9 Hz, 2C), 160.08, 140.24, 128.27 (d, J=8 Hz, 1C), 128.15, 116.31,
111.65, 111.40 (d, J=26 Hz, 2C), 104.64, 65.84, 34.12, 33.68, 31.27,
17.58, 10.14 ppm; MS (ESI) m/z: 484 [M+H]+; Anal. calcd for
C24H23F2N5O2S: C 59.61, H 4.79, N 14.48, found: C 59.73, H 4.82, N
14.55.
1-(4-Methoxyphenyl)prop-2-en-1-ol (22): A solution of para-anis-
aldehyde (15 mmol) in dry THF (50 mL) at ꢀ158C was treated with
vinylmagnesium bromide (45 mmol). The resulting mixture was al-
lowed to warm to room temperature and stirred for 1 h. Saturated
NH4Cl(aq) (50 mL) was added, and the aqueous layer was extracted
with EtOAc (3ꢂ50 mL). The organic layers were recollected,
washed with brine (1ꢂ10 mL), dried over Na2SO4, and evaporated
under reduced pressure. The residue was purified by flash chroma-
tography (CH2Cl2) to furnish compound 22 (1.603 g, yield 65%) as
3-(4-Methoxyphenyl)prop-2-yn-1-ol (17): To a solution of 4-iodo-
anisole (1 g, 4.27 mmol) in dry DMF, propargyl alcohol (746 mL,
12.81 mmol), Et3N (1.18 mL, 8.54 mmol), PdCl2(PPh3) (300 mg,
0.42 mmol) and CuI (246.5 mg, 1.29 mmol) were added. The reac-
tion mixture was stirred at room temperature for 5 min before
quenching with H2O (50 mL) and extraction with EtOAc (3ꢂ50 mL).
The combined extract was dried over anhydrous Na2SO4, concen-
trated under reduced pressure, and purified by flash chromatogra-
phy (eluent: PE/EtOAc 3:1) to give the desired product 17 (700 mg,
1
a yellow oil. TLC Rf (CH2Cl2) 0.53; H NMR (CDCl3): d=7.26 (d, J=
8.5 Hz, 2H), 6.86 (d, J=8.5 Hz, 2H), 6.01 (sept, J1 =16.6 Hz, J2 =
10.6 Hz, J3 =8.6 Hz, 1H), 5.29 (d, J=16.6 Hz, 1H), 5.15 (d, J=
10.6 Hz, 1H), 5.10 (d, J=8.6 Hz, 1H), 3.77 ppm (s, 3H); Anal. calcd
for C10H12O2: C 73.15, H 7.37, found: C 73.21, H 7.42.
1
yield 92%); mp: 165–1678C; H NMR (400 MHz, CDCl3): d=7.30 (d,
J=8 Hz, 2H), 6.76 (d, J=8 Hz, 2H), 4.41 (s, 2H), 3.70 (s, 3H),
3.33 ppm (bs, 1H); MS (ESI) m/z 161 [MꢀH]ꢀ; Anal. calcd for
C10H10O2: C 74.06, H 6.21, found: C 74.11, H 6.37.
1-(4-Methoxyphenyl)prop-2-en-1-one (23):
A solution of 22
(16 mmol) in dry Et2O (130 mL) was treated at room temperature
with MnO2 (160 mmol). The resulting mixture was stirred overnight
at the same temperature. The black suspension was filtered on
a Celite pad and evaporated under reduced pressure. The residue
was purified by flash chromatography (CH2Cl2/MeOH 9:1) to furnish
compound 23 (2.072 g, yield 80%) as a yellow oil. TLC Rf (CH2Cl2/
PE 1:1) 0.66; 1H NMR (CDCl3): d=7.80 (d, J=7.3 Hz, 2H), 7.00 (q,
J1 =16.5 Hz, J2 =10.3 Hz, 1H), 6.76 (d, J=7.3 Hz, 2H), 6.26 (d, J=
(Z)-3-(4-Methoxyphenyl)prop-2-en-1-ol (18): To a solution of com-
pound 17 (227 mg, 1.401 mmol) in EtOAc, quinoline (109 mL,
0.924 mmol) and Lindlar catalyst (227 mg) were added, and the
mixture was stirred under H2 for 48 h. The mixture was then fil-
tered on a Celite pad and washed with a solution of 1n HCl, dried
over Na2SO4, concentrated under reduced pressure, and purified by
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S-/N-DABOs as HIV RT Inhibitors
MED
16.5 Hz, 1H), 5.67 (d, J=10.3 Hz, 1H), 3.66 ppm (s, 3H); Anal. calcd
for C10H10O2: C 74.06, H 6.21, found: C 74.11, H 6.27.
for C22H22F2N2O3S: C 61.10, H 5.13, N 6.48, found: C 61.17, H 5.24, N
6.52.
6-(1-(2,6-Difluorophenyl)ethyl)-2-(3-(4-methoxyphenyl)-3-oxo-
propylthio)-5-methylpyrimidin-4(3H)-one (25): A solution of 6
(0.584 mmol) in dry DMF (8 mL) under Ar was treated with trifluor-
omethanesulfonic acid (0.117 mmol). After 15 min 23 (1.753 mmol)
was added, and the solution stirred at room temperature for 1 h.
EtOAc (12 mL) and saturated NaHCO3(aq) (5 mL) were then added,
and the organic phase was washed with distilled H2O (2ꢂ10 mL)
and brine (2ꢂ10 mL), dried over Na2SO4, and evaporated under re-
duced pressure. The residue was purified by flash chromatography
(EtOAc/PE 1:3) to furnish compound 25 (126 mg, yield 50%) as
a white solid; mp: 1688C; TLC Rf (EtOAc/PE 2:3) 0.27; IR (CHCl3):
6-(1-(2,6-Difluorophenyl)ethyl)-2-(3-hydroxy-3-(4-methoxyphe-
nyl)propylthio)-5-methylpyrimidin-4(3H)-one (28): A solution of
26 (0.046 mmol) in dry MeOH (1 mL) at 08C under Ar was treated
with NaBH4 (0.027 mmol) portionwise until TLC indicated complete
consumption of starting material. The solvent was removed under
reduced pressure. The residue was diluted with EtOAc (5 mL), and
the organic layer was washed with distilled H2O (2ꢂ5 mL) and
brine (2ꢂ5 mL), dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by TLC (EtOAc/PE 1:1) to furnish
compound 28 (20 mg, yield >99%) as a white solid. TLC Rf
(EtOAc/PE 2:3) 0.30; 1H NMR (CDCl3): d=7.25 (d, J=7.49 Hz, 2H),
7.11 (m, 1H), 6.86 (d, J=7.49 Hz, 2H), 6.76 (t, J=8.26 Hz, 2H), 4.77
(m, 1H), 4.56 (m, 1H), 3.79 (s, 3H), 3.33–3.15 (m, 2H diast.), 2.20–
1.99 (m, 2H diast.), 1.91 (d, 3H, diast.), 1.63 ppm (d, 3H, diast.);
13C NMR (CDCl3): d=163.37, 163.00, 161.28 (dd, J1 =246.11 Hz, J2 =
9.19 Hz, 2C), 158.16, 155.68, 136.13, 128.48 (d, J=9.19 Hz), 127.29
(2C), 114.70 (d, J=24.28 Hz), 114.09 (2C), 111.34 (d, J=24.28 Hz,
2C), 71.90, 55.43, 38.67, 31.14, 27.44, 26.98, 10.57 ppm; MS: m/z
447 [M+1]+, 469 [M+Na]+; Anal. calcd for C23H24F2N2O3S: C 61.87,
H 5.42, N 6.27, found: C 61.93, H 5.50, N 6.31.
1
n˜1600, 1665 cmꢀ1; H NMR ([D6]DMSO): d=7.76 (d, J=8.62 Hz, 2H),
7.14 (dd, J1 =7.70 Hz, J2 =6.65 Hz, 1H), 7.02 (d, J=8.62 Hz, 2H),
6.90 (t, J=7.70 Hz, 2H), 3.87 (s, 2H), 3.84 (s, 3H), 3.01 (q, J1 =
10.85 Hz, J2 =4.90 Hz, 4H), 2.01 ppm (s, 3H); 13C NMR ([D6]DMSO):
d=196.32, 163.40, 163.24, 161.29 (dd, J1 =245.21 Hz, J2 =8.98 Hz,
2C), 158.26, 152.27, 130.15 (2C), 129.42, 128.91 (d, J=8.98 Hz),
128.76, 114.12, 114.03 (2C), 111.14 (d, J=24.54 Hz, 2C), 55.72,
37.60, 26.83, 24.42, 10.20 ppm; MS: m/z 431 [M+H]+, 453 [M+
Na]+; Anal. calcd for C22H20F2N2O3S: C 61.38, H 4.68, N 6.51, found:
C 61.29, H 4.53, N 6.47.
6-(2,6-Difluorobenzyl)-2-(3-hydroxy-3-(4-methoxyphenyl)butyl-
thio)-5-methylpyrimidin-4(3H)-one (29) and 6-(1-(2,6-difluoro-
phenyl)ethyl)-2-(3-hydroxy-3-(4-methoxyphenyl)butylthio)-5-
methylpyrimidin-4(3H)-one (30): A solution of 25 or 26 (1 equiv)
in dry THF (1 mL) at 08C under Ar was treated with MeMgBr
(3 equiv). After 1 h, EtOAc (5 mL) and a saturated solution of NH4Cl
(5 mL) were added. The organic layer was washed with distilled
H2O (2ꢂ5 mL), dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by TLC (EtOAc/PE 1:1) to furnish
compound 29 (yield 83%) as a white solid. 1H NMR (CDCl3): d=
7.30 (d, J=8.83 Hz, 2H), 7.17 (m, 1H), 6.85 (d, J=8.83 Hz, 2H), 6.83
(m, 2H), 3.93 (s, 3H), 3.79 (s, 3H), 3.02–2.72 (dq, J1 =72.01 Hz, J2 =
7.23 Hz, 2H diast.), 2.12 (s, 3H), 1.94 (t, J=7.23 Hz, 2H), 1.12 ppm
(s, 3H); 13C NMR (CDCl3): d=163.95, 162.17, 161.60 (dd, J1 =
247.55 Hz, J2 =8.99 Hz, 2C), 157.36, 155.12, 142.63, 127.05 (d, J=
8.99 Hz), 126.77 (2C), 113.63 (2C), 113.44 (d, J=24.48 Hz), 110.88
(d, J=23.96 Hz, 2C), 75.01, 55.19, 34.13, 29.53, 26.13, 10.04 ppm;
MS: m/z 429 [MꢀH2O]+, 469 [M+Na]+; Anal. calcd for
C23H24F2N2O3S: C 61.87, H 5.42, N 6.27, found: C 61.92, H 5.49, N
6-(2,6-Difluorobenzyl)-2-(3-(4-methoxyphenyl)-3-oxopropylthio)-
5-methylpyrimidin-4(3H)-one (26): A solution of 24 (0.553 mmol)
in dry DMF (8 mL) under Ar was treated with trifluoromethanesul-
fonic acid (0.111 mmol). After 15 min 23 (1.753 mmol) was added,
and the solution was stirred at room temperature for 1 h. EtOAc
(12 mL) and saturated NaHCO3(aq) (5 mL) were then added, and the
organic phase was washed with distilled H2O (2ꢂ10 mL) and brine
(2ꢂ10 mL), dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by flash chromatography
(EtOAc/PE 1:3) to furnish compound 26 (167 mg, yield 68%) as
a white solid; mp: 1808C; TLC Rf (EtOAc/PE 2:3) 0.23; IR (CHCl3):
n˜1600, 1665 cmꢀ1 1H NMR (CDCl3): d=7.85 (d, J=8.78 Hz, 2H),
;
7.04 (m, 1H), 6.91 (d, J=8.78 Hz, 2H), 6.75 (t, J=8.37 Hz, 2H), 4.59
(q, J=7.23 Hz, 1H), 3.87 (s, 3H), 3.55 (q, J=6.60 Hz, 2H), 3.29 (q,
J=6.60 Hz, 2H), 2.02 (s, 3H), 1.64 ppm (d, J=7.23 Hz, 2H); 13C NMR
(CDCl3): d=196.59, 165.02, 163.78, 161.75 (dd, J1 =247.66 Hz, J2 =
9.27 Hz, 2C), 156.07, 130.24 (2C), 129.73, 128.22, 128.12 (d, J=
9.27 Hz), 113.70 (3C), 111.45 (d, J=26.12 Hz, 2C), 55.48, 38.29,
34.13, 24.89, 17.65, 9.82 ppm; MS: m/z 467 [M+Na]+; Anal. calcd
for C23H23F2N2O3S: C 62.15, H 4.99, N 6.30, found: C 62.21, H 5.03, N
6.37.
1
6.34. Compound 30 (yield 95%), white solid; H NMR (CDCl3): d=
7.28 (d, J=8.54 Hz, 2H), 7.08 (q, J=6.83 Hz, 1H), 6.82 (d, J=
8.29 Hz, 2H), 6.76 (t, J=8.29 Hz, 2H), 4.45 (q, J=7.07 Hz,1H), 3.70
(s, 3H), 3.17–2.87 (m, 2H diast.), 2.14 (m, 2H), 1.88 (d, J=3.90 Hz,
2H diast.), 1.55 (t, J=6.58 Hz, 3H diast.), 1.50 ppm (d, J=2.92 Hz,
3H diast.); 13C NMR (CDCl3): d=164.79, 163.00, 161.28 (dd, J1 =
248.03 Hz, J2 =9.73 Hz, 2C), 158.38, 155.76, 141.32, 128.15 (d, J=
9.73 Hz), 125.94 (2C), 114.12 (2C), 113.64 (d, J=23.96 Hz), 111.58 (d,
J=23.96 Hz, 2C), 74.19, 55.26, 43.70, 34.00, 30.30, 25.53, 17.72,
9.76 ppm; MS: m/z 461 [M+1]+, 483 [M+Na]+; Anal. calcd for
C24H26F2N2O3S: C 62.59, H 5.69, N 6.08, found: C 62.64, H 5.73, N
6.11.
6-(2,6-Difluorobenzyl)-2-(3-(hydroxyimino)-3-(4-methoxyphenyl)-
propylthio)-5-methylpyrimidin-4(3H)-one (27): A solution of 25
(0.046 mmol) in dry MeOH (1 mL) at 08C under Ar was treated with
NaBH4 (0.027 mmol) portionwise until TLC indicated complete con-
sumption of starting material. The solvent was removed under re-
duced pressure. The residue was diluted with EtOAc (5 mL), and
the organic layer was washed with distilled H2O (2ꢂ5 mL) and
brine (2ꢂ5 mL), dried over Na2SO4, and evaporated under reduced
pressure. The residue was purified by TLC (EtOAc/PE 1:1) to furnish
compound 27 (20 mg, yield 100%) as a white solid. TLC Rf (EtOAc/
6-(2,6-Difluorobenzyl)-2-(3-hydroxy-3-(4-methoxyphenyl)pentyl-
1
PE 2:3) 0.30; H NMR (CDCl3): d=7.22 (d, J=8.45 Hz, 2H), 7.13 (m,
thio)-5-methylpyrimidin-4(3H)-one (31):
A
solution of 25
1H), 6.87 (d, J=8.45 Hz, 2H), 6.81 (t, J=8.45 Hz, 2H), 4.63 (q, J=
4.25 Hz, 1H), 3.93 (s, 2H), 3.80 (s, 3H), 3.14–2.98 (m, 2H diast.), 2.14
(s, 3H), 1.99–1.77 ppm (m, 2H, diast.); 13C NMR ([D6]DMSO): d=
163.03, 162.87, 161.16 (dd, J1 =245.68 Hz, J2 =8.52 Hz, 2C), 159.81,
158.16, 137.43, 128.65 (d, J=8.52 Hz), 126.85 (2C), 114.04 (d, J=
24.10 Hz), 113.86 (2C), 110.09 (d, J=24.10 Hz, 2C), 70.31, 54.97,
38.22, 26.76, 26.53, 9.98 ppm; MS: m/z 455 [M+Na]+; Anal. calcd
(0.046 mmol) in dry THF (1 mL) at 08C under Ar was treated with
EtMgBr (0.139 mmol). After 1 h, EtOAc (5 mL) and saturated
NH4Cl(aq) (5 mL) were added. The organic layer was washed with
distilled H2O (2ꢂ5 mL) and brine (2ꢂ5 mL), dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by
TLC (EtOAc/PE 1:1) to furnish compound 31 (19 mg, yield 92%) as
1
a white solid. TLC Rf (EtOAc/PE 1:1) 0.23; H NMR (CDCl3): d=7.20
ChemMedChem 2012, 7, 883 – 896
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893

M. Botta et al.
MED
(d, J=8.69 Hz, 2H), 7.10 (q, J=7.47 Hz, 1H), 6.79 (m, 4H), 3.87 (s,
2H), 3.74 (s, 3H), 2.90–2.62 (m, 2H diast.), 2.05 (s, 3H), 1.99 (m, 2H
diast.), 1.65 (m, 2H diast.), 0.66 ppm (t, J=7.47 Hz, 3H); 13C NMR
(CDCl3): d=164.14, 162.82, 160.44 (dd, J1 =244.92 Hz, J2 =9.07 Hz,
2C), 159.63, 156.17, 136.57, 128.12 (d, J=9.07 Hz), 126.44 (2C),
113.38 (d, J=24.22 Hz), 111.47 (2C), 110.80 (d, J=24.22 Hz, 2C),
76.22, 55.13, 41.92, 35.62, 27.59, 25.10, 10.24, 7.56 ppm; MS: m/z
461 [M+1]+, 483 [M+Na]+; Anal. calcd for C24H26F2N2O3S: C 62.59,
H 5.69, N 6.08, found: C 62.52, H 5.62, N 5.99.
1C), 102.28, 59.84, 13.86 ppm; Anal. calcd for C10H10F2O: C 65.21, H
5.47, found: C 65.33, H 5.52
2-(2,6-Difluorophenyl)propanal (36): Compound 35 (45 mg,
0.244 mmol) was dissolved in CH2Cl2, and the solution was cooled
to ꢀ788C. Then BBr3 (1m in CH2Cl2; 244 mL, 0.244 mmol) was
added dropwise, and the mixture was stirred at ꢀ788C for 2 h. H2O
was then added, the organic phase separated, and the aqueous
layer extracted twice with CH2Cl2. The organic phases were collect-
ed, washed with NaHCO3 and brine, dried over Na2SO4, and con-
centrated to obtain 36 in sufficient purity for use in the next step
6-(1-(2,6-Difluorophenyl)ethyl)-2-(3-hydroxy-3-(4-methoxyphe-
nyl)pentylthio)-5-methylpyrimidin-4(3H)-one (32): A solution of
26 (0.046 mmol) in dry THF (1 mL) at 08C under Ar was treated
with EtMgBr (0.139 mmol). After 1 h, EtOAc (5 mL) and saturated
NH4Cl(aq) (5 mL) were added. The organic layer was washed with
distilled H2O (2ꢂ5 mL) and brine (2ꢂ5 mL), dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified by
TLC (EtOAc/PE 1:1) to furnish compound 32 (20 mg, yield 94%) as
1
without purification (yield: quant.); H NMR (400 MHz, CDCl3): d=
9.68 (s, 1H), 7.26–7.17 (m, 1H), 6.87–6.83 (m, 2H), 3.84–3.79 (q, J=
7.4 Hz, 1H), 1.42–1.40 ppm (d, J=7.3 Hz, 3H); MS (ESI) m/z: 171
[M+H]+; Anal. calcd for C9H8F2O: C 65.21, H 5.47, found: C 65.29,
H 5.42.
Methyl 4-(2,6-difluorophenyl)pent-2-ynoate (38): To a solution of
36 (41 mg, 0.241 mmol) in MeOH (5 mL), K2CO3 (67 mg,
0.482 mmol) and dimethyl-1-diazo-2-oxopropylphosphonate (43 mL,
0.289 mmol) were added, and the mixture was stirred at room
temperature overnight under Ar. The mixture was then diluted
with 15 mL MeOH, and NaOAc (19 mg, 0.234 mmol), PdCl2 (0.5 mg,
0.03 mmol), and CuCl2 (32 mg, 0.234 mmol) were added. The mix-
ture was stirred under CO for 2 h (the color of the mixture turned
from green to grey). Then mixture was filtered on Celite and dried
by rotary evaporation. The crude product was purified by flash
chromatography (EtOAc/Hex 90:10) to obtain compound 38
1
a white solid. TLC Rf (EtOAc/PE 1:1) 0.24; H NMR (CDCl3): d=7.28
(d, J=8.52 Hz, 2H), 7.13 (m, 1H), 6.86 (dd, J1 =8.97 Hz, J2 =2.91 Hz,
2H) 6.84 (d, J=8.52 Hz, 2H), 4.54 (q, J=7.17 Hz, 1H), 3.79 (ds, J=
3.78 Hz, 3H), 3.24–2.82 (m, 2H diast.), 2.19 (m, 2H), 1.93 (ds, J=
9.19 Hz, 3H), 1.84 (m, 2H diast.), 1.60 (q, J=5.35 Hz, 3H), 0.74 ppm
(t, J=5.35 Hz, 3H); 13C NMR (CDCl3): d=164.32, 162.94, 161.22 (dd,
J1 =244.92 Hz, J2 =9.07 Hz, 2C), 158.29, 155.70, 136.73, 128.11 (d,
J=9.07 Hz), 126.42 (2C), 113.48 (d, J=24.22 Hz), 111.58 (2C), 111.32
(d, J=24.22 Hz, 2C), 55.14, 42.43, 42.23, 35.60, 33.90, 30.82, 25.24,
17.62, 9.70, 7.56 ppm; MS: m/z 475 [M+1]+, 497 [M+Na]+; Anal.
calcd for C25H28F2N2O3S: C 63.27, H 5.95, N 5.90, found: C 63.31, H
5.99, N 5.97.
1
(13 mg, yield 34%) as a colorless oil. H NMR (400 MHz, CDCl3): d=
7.18–7.10 (m, 1H), 6.82–6.78 (m, 2H), 4.24–4.19 (q, J=7.2 Hz, 1H),
3.67 (s, 3H), 1.53–1.51 ppm (d, J=7.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3): d=161.91 (dd, J1 =245 Hz, J2 =9 Hz, 2C), 154.08, 129.15,
117.18, 112.44 (d, J=27 Hz, 2C), 89.08, 72.31, 52.58, 20.94,
20.02 ppm; MS (ESI) m/z: 225 [M+H]+; Anal. calcd for C12H10F2O2:
C 64.28, H 4.50, found: C 64.31, H 4.62.
6-(2,6-Difluorobenzyl)-2-(3-(hydroxyimino)-3-(4-methoxyphenyl)-
propylthio)-5-methylpyrimidin-4(3H)-one (33): A solution of 25
(0.032 mmol) in dry EtOH (2 mL) was treated with NH2OH·HCl
(0.098 mmol) and NaOAc (0.098 mmol). The mixture was held at
reflux overnight. Et2O (5 mL) and saturated NaHCO3(aq) (5 mL) were
added, and the organic layer was washed with distilled H2O (2ꢂ
5 mL) and brine (2ꢂ5 mL), dried over Na2SO4, and evaporated
under reduced pressure to give compound 33 as a white solid resi-
due (15 mg, 100%). TLC Rf (EtOAc/PE 1:1) 0.21; 1H NMR
([D6]acetone): d=7.41 (d, J=8.35 Hz, 2H), 7.21 (m, 1H), 6.88 (t, J=
7.50 Hz, 2H), 6.79 (d, J=8.35 Hz, 2H), 3.94 (s, 2H), 3.73 (s, 3H), 3.12
(t, J=7.03 Hz, 2H), 2.86 (t, J=7.03 Hz, 2H), 2.02 ppm (s, 3H); MS:
m/z 468 [M+Na]+; Anal. calcd for C23H23F2N3O3S: C 60.12, H 5.05, N
9.14, found: C 60.21, H 5.17, N 9.19.
General procedure for the synthesis of 40a,c 41b,d and 42b,d:
In a microwave sealed tube, the appropriate substituted guanidine
hydrochloride (1.2 equiv), compound 38 (1 equiv) and NaHCO3
(2.4 equiv) in tBuOH (3 mL) were irradiated for 20 min (for R2 =4-
methoxybenzyl) or 40 min (for R2 =4-methoxyphenylcyclopropyl)
in a self-tunable CEM microwave synthesizer at 1208C and finally
allowed to cool to room temperature. The reaction mixture was di-
rectly evaporated to dryness to give a solid crude material. The res-
idue was purified by flash chromatography (CH2Cl2/MeOH) to
obtain final products.
1,3-Difluoro-2-(1-methoxyprop-1-en-2-yl)benzene (35): To a sus-
pension of methoxymethyl(triphenyl)phosphonium chloride
(988 mg, 2.88 mmol) in THF (20 mL), a solution of nBuLi (2m in
hexane; 1.44 mL, 2.88 mmol) was added dropwise at ꢀ788C under
Ar. The resulting orange solution was stirred for 30 min at ꢀ788C
and then at room temperature for 30 min until a red color ap-
peared. The solution was then cooled again to ꢀ788C, and a solu-
tion of 1-(2,6-difluorophenyl)ethanone (250 mL, 1.921) previously
dissolved in THF (5 mL) was added dropwise. The mixture was
stirred at room temperature for 18 h and was then quenched with
H2O, the organic phase separated, and the aqueous layer extracted
twice with Et2O. The organic phases were collected, washed with
brine, dried over Na2SO4, and concentrated by rotary evaporation.
The crude product was purified by flash chromatography (pen-
tane/Et2O 95:5) to obtain compound 35 (276 mg, yield 78%) as
a colorless oil. 1H NMR (400 MHz, CDCl3): d=7.09–7.04 (m, 1H),
6.82–6.75 (t, 2H), 6.08 (s, 1H), 3.64 (s, 3H), 1.88 ppm (s, 3H);
13C NMR (100 MHz, CDCl3): d=161.08 (dd, J1 =245 Hz, J2 =9 Hz,
2C), 137.34, 127.48 111.97 (d, J=27 Hz, 2C), 111.95 (d, J=10 Hz,
2-(N-(4-Methoxybenzyl)-N-methylamino)-6-(1-(2,6-difluorophen-
yl)ethyl)pyrimidin-4(3H)-one (40a): White solid (yield 41%);
1H NMR (400 MHz, CDCl3): d=11.14 (bs, NH), 7.09–7.05 (m, 1H),
7.00–6.98 (d, 2H, J=8.4 Hz), 6.77–6.69 (m, 4H), 5.59 (s, 1H), 4.67–
4.44 (dd, 2H, J=93.9 Hz, 14.8 Hz), 4.24–4.19 (q, 1H, J=7.2 Hz), 3.71
(s, 3H), 2.95 (s, 3H), 1.56–1.55 ppm (d, 3H, J=7.2 Hz); 13C NMR
(100 MHz, CDCl3): d=171.41, 166.48, 162.90, 160.60, 159.01, 153.82,
129.30, 127.87, 119.94, 113.87, 111.37, 97.71, 55.25, 52.23, 36.24,
34.63, 16.73 ppm; MS (ESI) m/z: 386 [M+H]+; Anal. calcd for
C21H21F2N3O2: C 65.44, H 5.49, N 10.90, found: C 65.52, H 5.55, N
11.05.
2-(4-Methoxybenzylamino)-6-(1-(2,6-difluorophenyl)ethyl)pyrimi-
din-4(3H)-one (41b): White solid (yield 23%); 1H NMR (400 MHz,
CDCl3): d=11.98 (bs, NH), 7.13–7.03 (m, 3H), 6.79–6.75 (t, 2H, J=
8.3 Hz), 6.71–6.69 (d, 2H, J=8.5 Hz), 6.38 (bs, NH), 5.49 (s, 1H),
4.38–4.13 (m, 3H), 3.71 (s, 3H), 1.53–1.52 ppm (d, 3H, J=7.1 Hz);
13C NMR (100 MHz, CDCl3): d=172.6, 166.2, 162.8, 160.4, 158.8,
153.6, 130.7, 129.3, 128.0, 119.7, 113.8, 111.5, 98.5, 55.2, 44.1, 29.7,
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S-/N-DABOs as HIV RT Inhibitors
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16.7 ppm; MS (ESI) m/z: 372 [M+H]+; Anal. calcd for C20H19F2N3O2:
C 64.68, H 5.16, N 11.31, found: C 64.71, H 5.19, N 11.42.
[1] J. Adams, N. Patel, N. Mankaryous, M. Tadros, C. D. Miller, Ann. Pharmac-
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3-(4-Methoxybenzyl)-2-amino-6-(1-(2,6-difluorophenyl)ethyl)pyri-
midin-4(3H)-one (42b): White solid (yield 23%); H NMR (400 MHz,
[3] R. W. Buckheit, Jr., K. M. Watson, K. M. Morrow, A. S. Ham, Antiviral Res.
2010, 85, 142–158.
1
[4] T. Mertenskoetter, P. E. Kaptur, Eur. J. Med. Res. 2011, 16, 1–6.
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2116.
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350.
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mirri, G. Maga, Antiviral Res. 2010, 86, 268–275.
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li, R. Silvestri, G. Maga, Antiviral Res. 2009, 81, 47–55.
CD3OD): d=7.15–7.07 (m, 1H), 6.76–6.69 (m, 6H), 6.09 (s, 1H),
5.06–5.83 (dd, 2H, J=92.2 Hz, 18.1 Hz), 4.42–4.37 (q, 1H, J=
6.3 Hz), 3.68 (s, 3H) 1.51–1.49 ppm (d, 3H, J=6.5 Hz); 13C NMR
(100 MHz, CD3OD): d=173.02, 161.9, 159.5, 159.2, 158.0, 155.9,
129.8, 125.7, 125.4, 116.9, 113.9, 111.8, 105.8, 54.4, 47.7, 30.8,
17.9 ppm; MS (ESI) m/z: 372 [M+H]+; Anal. calcd for C20H19F2N3O2:
C 64.68, H 5.16, N 11.31, found: C 64.73, H 5.19, N 11.45.
2-(N-((2-(4-Methoxyphenyl)cyclopropyl)methyl)-N-methylamino)-
6-(1-(2,6-difluorophenyl)ethyl)pyrimidin-4(3H)-one (40c): Color-
less oil (yield 10%); 1H NMR (400 MHz, CDCl3): d=7.07–7.00 (m,
1H), 6.85–6.81 (m, 2H), 6.76–6.71 (m, 4H), 5.60 (s, 1H), 4.21–4.16
(q, 1H, J=7.3 Hz), 3.71 (s, 3H), 3.58–3.33 (m, 2H), 3.05 (s, 3H),
1.69–1.59 (m, 1H), 1.56–1.54 (d, 3H, J=7.3 Hz), 1.27–1.06 (m, 1H),
0.84–0.63 ppm (m, 2H); 13C NMR (100 MHz, CDCl3): d=171.42,
166.16, 163.11, 160.58, 157.92, 153.55, 134.35, 127.87, 126.92,
119.93, 113.80, 111.33, 97.48, 55.32, 53.16, 35.25, 29.70, 22.33, 21.04,
16.78, 14.05 ppm; MS (ESI) m/z: 426 [M+H]+; Anal. calcd for
C24H25F2N3O2: C 67.75, H 5.92, N 9.88, found: C 67.81, H 5.97, N
9.93.
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Chemother. 2009, 53, 487–495.
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10.1002/med.20241.
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2007/043094, 2007.
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A. Tafi, G. Casaluce, G. Giorgi, M. Armand-Ugon, E. Gonzalez, J. A. Estꢀ,
M. Baltzinger, G. Bec, P. Dumas, E. Ennifar, M. Botta, J. Med. Chem. 2009,
52, 840–851.
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muca, R. Ragno, A. Samuele, S. Zanoli, M. Armand-Ugon, I. Clotet-
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Mai, J. Med. Chem. 2008, 51, 4641–4652.
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8000–8008.
2-((2-(4-Methoxyphenyl)cyclopropyl)methylamino)-6-(1-(2,6-di-
fluorophenyl)ethyl)pyrimidin-4(3H)-one (41d): Colorless oil (yield
28%); 1H NMR (400 MHz, CDCl3): d=11.80 (bs, NH), 7.19–7.00 (m,
1H), 6.88–6.85 (m, 2H), 6.75–6.71 (m, 4H), 5.96 (bs, NH), 5.56 (s,
1H), 4.24–4.15 (q, 1H, J=7.1 Hz), 3.71 (s, 3H), 3.42–3.29 (m, 1H),
3.20–3.06 (m, 1H), 1.69–1.61 (m, 1H), 1.55–1.53 (d, 3H, J=7.1 Hz),
1.24–1.09 (m, 1H), 0.76–0.64 ppm (m, 2H); 13C NMR (100 MHz,
CDCl3): d=172.65, 166.17, 162.91, 160.36, 157.73, 153.91, 134.46,
128.02, 127.12, 113.75, 111.40, 98.42, 55.31, 44.84, 36.39, 22.04,
21.23, 16.86, 13.80 ppm; MS (ESI) m/z: 412 [M+H]+; Anal. calcd for
C22H21F2N3O2: C 66.49, H 5.33, N 10.57, found: C 66.54, H 5.41, N
10.59.
2-Amino-6-(1-(2,6-difluorophenyl)ethyl)-3-((2-(4-methoxyphen-
yl)cyclopropyl)methyl)pyrimidin-4(3H)-one (42d): White solid
(yield 15%); 1H NMR (400 MHz, CD3OD): d=7.38–7–11 (m, 1H),
7.00–6.79 (m, 4H), 6.72–6.70 (d, 2H, J=8.3 Hz), 5.97 (s, 1H), 4.49–
4.45 (q, 1H, J=7 Hz), 3.97–3.88 (m, 1H), 3.71–3.61 (m, 4H), 1.94–
1.69 (m, 2H), 1.59–1.54 (m, 3H), 1.29–0.75 ppm (m, 3H); 13C NMR
(100 MHz, CD3OD): d=172.99, 168.41, 161.90, 158.20, 155.53,
133.06, 130.03, 126.71, 114.21, 113.50, 112.02, 105.73, 54.30, 47.01,
31.13, 21.71, 20.51, 17.90, 12.25 ppm; MS (ESI) m/z: 412 [M+H]+;
Anal. calcd for C22H21F2N3O2: C 66.49, H 5.33, N 10.57, found: C
66.53, H 5.41, N 10.62.
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Acknowledgements
This work was supported by the European Union (“CHAARM” Col-
laborative Project, grant number HEALTHF3-2009-242135 and
“THINC” Collaborative Project, grant number HEALTH-2007-2.3.2-
1) to M.B., the National AIDS Program Grant 40H26 to G.M., and
MICINN project SAF2010-216117-C02 to B.C.
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Keywords: ADME · antiviral agents · lead optimization ·
NNRTIs · reverse transcriptase
ChemMedChem 2012, 7, 883 – 896
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Received: January 18, 2012
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Published online on March 14, 2012
896
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