6-(1-benzyl-1 h -pyrrol-2-yl)-2,4-dioxo-5-hexenoic acids as dual inhibitors of recombinant HIV-1 integrase and ribonuclease H, synthesized by a parallel synthesis approach

Article abstract of DOI:10.1021/jm401040b

The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC₅₀ values of 3, 3, and 2.5 μM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC₅₀ value of 26 nM.

Full text of DOI:10.1021/jm401040b

Article
pubs.acs.org/jmc
6‑(1-Benzyl‑1H‑pyrrol-2-yl)-2,4-dioxo-5-hexenoic Acids as Dual
Inhibitors of Recombinant HIV‑1 Integrase and Ribonuclease H,
Synthesized by a Parallel Synthesis Approach
Roberta Costi,*,† Mathieu Metifiot,‡ Francesca Esposito,∫ Giuliana Cuzzucoli Crucitti,† Luca Pescatori,†
́
Antonella Messore,† Luigi Scipione,† Silvano Tortorella,† Luca Zinzula,∫ Ettore Novellino,§
Yves Pommier,‡ Enzo Tramontano,∫ Christophe Marchand,*,‡ and Roberto Di Santo†
†Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti, “Sapienza” Universita
̀
di Roma,
P. le Aldo Moro 5, Rome I-00185, Italy,
‡Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health,
Building 37, Room 5068, Bethesda, Maryland 20892-4255, United States
∫ Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, Cagliari I-09124, Italy
§Dipartimento di Farmacia, Universita
̀
di Napoli “Federico II”, Via D. Montesano 49, I-80131 Napoli, Italy
S
* Supporting Information
ABSTRACT: The increasing efficiency of HAART has helped to transform
HIV/AIDS into a chronic disease. Still, resistance and drug−drug interactions
warrant the development of new anti-HIV agents. We previously discovered hit 6,
active against HIV-1 replication and targeting RNase H in vitro. Because of its
diketo-acid moiety, we speculated that this chemotype could serve to develop
dual inhibitors of both RNase H and integrase. Here, we describe a new series of
1-benzyl-pyrrolyl diketohexenoic derivatives, 7a−y and 8a−y, synthesized
following a parallel solution-phase approach. Those 50 analogues have been
tested on recombinant enzymes (RNase H and integrase) and in cell-based
assays. Approximately half (22) exibited inhibition of HIV replication.
Compounds 7b, 7u, and 8g were the most active against the RNase H activity
of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 μM, respectively.
Compound 8g was also the most potent integrase inhibitor with an IC50 value of
26 nM.
Among the drugs currently used in HAART regimens,
INTRODUCTION
■
raltegravir (1, Chart 1) received approval by the U.S. Food and
Drug Administration (FDA) in 2007. 1 inhibits HIV-1 integrase
(IN), the viral coded enzyme responsible for the insertion of
the HIV retro-transcribed dsDNA into a cell host chromo-
some.6 HIV-1 IN catalyzes two enzymatic reactions with the
same active site. In the first reaction, called 3′-processing (3′-
P), IN removes the two terminal nucleotides (GT) from each
3′-end of the dsDNA. In the second reaction, named “strand
transfer” (ST), IN catalyzes a nucleophilic attack by the free 3′-
OH of the viral processed DNA onto the target chromosomal
DNA, resulting in covalent joining of the two DNA molecules.
IN belongs to the polynucleotidyl transferase family, and its
active site shares structural similarities with the HIV-1 reverse
transcriptase (RT)-associated ribonuclease H (RNase H) active
site.7 In particular, the arrangement of the active site residues
and metal ion coordination in HIV IN and HIV RNase H are
very similar.6,8 It is also important to note that (i) the RT-
After the introduction of the highly active antiretroviral therapy
(HAART), human immunodeficiency virus (HIV) infection has
been transformed into a chronic disease.1 The main reason for
this success is the use of combinatorial therapy targeting
different viral targets, which drastically reduces the selection of
drug-resistant virus strains. However, HAART exhibits several
liabilities including long-term toxicity, emergence of drug
resistant virus strains, drug−drug interactions, and a significant
cost burden associated with the use of multiple single agent
formulations.2 Therefore, the development of new anti-HIV
drugs is warranted, particularly if targeted against novel viral
functions and/or if exploring new pharmacological approaches.
One of such innovative approaches is the use of dual-action
drugs that combine two different desired pharmacological
actions at a similarly effective dose. This approach has already
been validated in the oncology arena, where dual inhibitors of
tyrosine and phosphoinositide kinases show very promising
physiological activity3 in the anti-infective field of malaria4 and
currently represents an active aspect of drug development.5
Received: July 9, 2013
Published: October 14, 2013
© 2013 American Chemical Society
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have been evaluated against both RNase H and HIV IN and for
their cytotoxicity and activity against HIV-1 infected cells.
Chart 1. Selected Inhibitors of HIV-1 RNase H Function of
RT and/or IN Enzyme
RESULT AND DISCUSSION
Chemistry. The synthesis of derivatives 7a−y and 8a−y is
reported in Scheme 1. The mono- and disubstituted-N-benzyl-
■
a
Scheme 1. Synthesis of Derivatives 7a−y and 8a−y
a
Reagents and conditions: (i) substituted benzyl bromide, NaH, room
temp; (ii) acetone, 5 N NaOH, 50 °C; (iii) diethyl oxalate, sodium
ethoxide, THFa, room temp; (iv) 1 N NaOH, 1:1 THF−methanol,
room temp.
pyrrolyl diketohexenoic acids were synthesized in a four-step
associated RNase H function is essential for HIV-1 replication,
(ii) it is a validated target for drug development, and (iii) no
drug that targets RNase H has yet been approved.8−10 Given
the structural similarities between the domains of these two
HIV-1 enzymes, several compounds initially developed as IN
inhibitors have also been screened against RNase H and vice
versa, such as diketo acid (DKA) derivatives like 4-[5-
(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid (BTDBA,
2)11 (Chart 1) and DNA aptamers.12 Recently, three kind of
structurally different compounds as dual inhibitors were
described, including tropolones (i.e., 3),13−15 madurahydrox-
ylactone derivatives like compound 4,16 and 2-hydroxyisoqui-
nolin-1,3(2H,4H)-diones (i.e., 5)17,18 (Chart 1).
We have previously reported that DKAs initially synthesized
as IN inhibitors were also able to inhibit the HIV-1 RNase H
activity of RT.19,20 In particular, the IN inhibitor 6-[1-(4-
fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic
acid ethyl ester (RDS 1643, 6, Chart 1),19,21 has been shown to
chelate Mg2+ in the RNase H catalytic site and to be inactive
against the RT-associated DNA polymerase function.19 Thus,
pursuing our decennial studies on IN inhibitors21−28 and
considering the above observations, we decided to design a
small library of analogues of compound 6, as dual IN/RNase H
inhibitors. Taking into account that the benzyl ring of DKAs
plays a crucial role in binding to both the vDNA bases and the
hydrophobic pocket within the catalytic core of IN enzyme,29
we wanted to investigate the influence of the benzyl
substituents on the anti-IN and anti-RNase H activities while
keeping unmodified both the diketohexenoic moiety and the
pyrrolyl nucleus. In this study, we describe a small library of
pyrrolyl diketohexenoic derivatives (7a−y and 8a−y), designed
by replacement of the fluorine atom in the 4-fluorobenzyl
moiety of 6 with several substituents endowed with various
electronic and steric properties to obtain dual inhibitors of IN
and RNase H. This small library of pyrrolyl derivatives was set
up by a simple and efficient parallel solution-phase synthesis
approach. The newly synthesized compounds 7a−y and 8a−y
parallel procedure (Scheme 1) using a Buchi SynCor reactor
̈
fitted with 12 or 13 tubes (50 mL). Pyrrole-2-carboxaldehyde
was alkylated with the appropriate benzyl bromide in the
presence of NaOH to afford the N-substituted-pyrrole-2-
carboxaldehydes 9a−y, which were converted to the corre-
sponding 3-buten-2-ones 10a−y by condensation with acetone
using NaOH as a base. Compounds 10a−y were then reacted
with diethyl oxalate in the presence of NaOEt as a base to
obtain hexenoic esters 7a−y that were finally hydrolyzed in
basic medium to give the corresponding acids 8a−y.
Evaluation of Biological Activities. All the newly
synthesized compounds 7a−y and 8a−y were tested in vitro
in biochemical assays both against rRT to test their ability to
reduce the activity of the RT-associated RNase H function, and
against rIN, to evaluate inhibitory activities against the ST step
of the integration reaction. Corresponding IC50 values were
calculate using a dose−response curve and are reported in
Table 1.
The IC50 values obtained for both recombinant RNase H and
IN were plotted against each other in a correlation plot where
single dots correspond to single compounds (Figure 1). As seen
in Figure 1A, this graph does not reveal any particular
correlation between RNase H and IN inhibition. The
compounds organize themselves around two perpendicular
axes crossing the IN IC50 axis (X axis) at 1 μM and the RNase
H IC50 axis (Y axis) at 10 μM (bolded crosshair in the center of
the graph, Figure 1). These two axes splice the graph into four
quarters corresponding to RNase H/IN dual inhibitors (lower
left quarter), RNase H-selective inhibitors (lower right quarter),
IN selective inhibitors (upper left quarter), and inhibitors of
lower potency (upper right quarter).
When the compounds are categorized according to their
ester (blue, Figure 1A) or acidic function (red, Figure 1B), it
appears that the acidic function is preferred for IN potency,
with a majority of the red dots (acids) in the two left quarters
and a majority of blue dots (esters) in the two right quarters of
the plot, respectively (Figure 1A). This preference of the acidic
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Table 1. Cytotoxicity, Enzymatic, and Antiviral Activities of Compounds 7a−y and 8a−y
activity in enzyme
a
assay, IC50
antiviral activity
b
c
d
e
f
g
compd
R2
R3
R4
R5
R6
X
RH
ST
EC50 HeLa
EC50 MT-4
CC50
SI
7a
7b
7c
7d
7e
7f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
15
32
0.25
1.4
1.5
2.2
41
164
>35
15
Me
H
H
Me
H
H
F
H
3.0
9.6
7.3
6.3
9.0
8
>50
37
H
8.0
2.4
H
Me
H
>111
1.0
17.4
0.31
2.2
>50
>50
42
>2
F
1.2
5.4
1.7
>161
19
H
H
11
7g
7h
7i
H
H
Cl
H
H
CN
H
H
H
H
Me
F
F
98
<0.2
0.79
26.3
17
>50
>50
>50
>50
48
>250
>63
>1
H
H
19
8.0
32
5
6
H
Cl
H
42
7j
CN
H
9.0
>2
7k
7l
H
13
7.1
7
H
CN
H
8
>111
23
>50
0.2
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
30
7m
7n
7o
7p
7q
7r
7s
7t
OMe
H
>100
6.7
>100
6
>250
OMe
H
110
12
>50
0.6
OEt
H
>83
>3
H
19
13.8
<0.2
<0.2
0.81
0.2
F
H
8
0.53
1.00
0.45
4.00
0.60
0.49
>111
1.70
8.0
>250
>250
>16
>250
>25
>12
F
H
H
H
F
F
19
9
F
H
F
H
H
H
F
19
3
7u
7v
7w
7x
7y
8a
8b
8c
8d
8e
8f
H
F
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
2
H
F
H
5
3.9
Cl
Cl
H
H
H
Cl
H
H
Me
H
H
F
Cl
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
F
9
>50
2.5
22
>20
10
H
3
H
H
15
26
4.6
17
6.4
14
2.5
9
0.09
0.17
1.3
0.35
3.7
0.83
1.6
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
42
>142
>13
>26
Me
H
H
H
1.9
4.7
H
Me
H
1.2
>50
0.35
5.1
F
0.98
0.92
0.026
0.31
4.1
>142
>9
H
H
2.6
1.7
8g
8h
8i
H
H
Cl
H
H
CN
H
H
H
H
Me
F
F
0.63
>50
>50
>50
>50
>50
<0.2
17.4
<0.2
9.8
>79
H
H
H
Cl
H
5.0
6
>50
8j
CN
H
6
8k
8l
H
5
0.75
1.7
H
CN
H
6
8m
8n
8o
8p
8q
8r
8s
8t
OMe
H
16
3.0
64
16
6
0.53
4.1
>210
>2
OMe
H
>50
>50
30
OEt
H
0.31
1.6
>250
3
H
F
H
0.059
0.042
0.052
0.15
1.60
1.2
0.52
0.5
>50
44
>128
88
F
H
H
H
F
F
10
5
F
H
2.5
>50
>50
>50
>50
>50
>50
>50
>20
>250
F
H
H
H
F
50
86
14
7
<0.2
>50
1.7
8u
8v
8w
8x
8y
1
H
F
H
H
H
Cl
H
H
F
H
>29
>3
Cl
Cl
H
H
H
Cl
Cl
H
H
H
Cl
4.9
16.2
1.4
28
30
0.17
0.97
0.007
1.9
>35
H
>50
0.024
ND
0.016
>50
>250
>15000
2
3.2
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Table 1. continued
a
b
Inhibitory concentration 50% (μM) determined from dose−response curves. Experiments performed against HIV-1 RT-associated RNase H
c
d
e
activity. Experiments performed against HIV-1 IN ST activity. Effective concentration 50% (μM) in HIV-1 infected HeLa cells. Effective
concentration 50% (μM) in HIV-1 infected MT-4 cells. Cytotoxic concentration 50% (μM). Selectivity index = CC50/EC50 in HeLa cells.
f
g
upper and two lower quarters of the graph. RNase H inhibition
may even be more prone to ester containing compounds with
two-thirds of the blue dots distributed in the two lower quarters
of the graph (Figure 1A).
We then categorized the compounds according to the type of
substitutions and found that 8 out of 11 compounds in the
lower left quarter of the graph are fluoro substituted derivatives
(green dots, Figure 1B) and that out of these compounds,
seven are difluoro substituted. Therefore, difluoro substitutions
may be a hallmark of RNase H/IN dual inhibitors. Chloro
substituents (orange dots), which distribute around the center
of the graph, do not seem to select for inhibition of one enzyme
over the other (Figure 1B). Methyl (cyan dots) and O-alkyl
substitutions (lavender dots) do not seem to favor IN
inhibition because they are in vast majority located in the
two right quarters of the plot (Figure 1B). Moreover, these
substituents do not seem to impact RNase H inhibition with an
even distribution among the upper and lower quarters of the
graph. In contrast, the cyano substituted compounds (dark-red
dots) appear to be more selective for RNase H inhibition
(lower right quarter, Figure 1B).
Finally, the compounds were categorized according to the
position of their substitutions (Figure 1C). Compounds
bearing di-ortho (2,6) substitutions (blue dots) do not inhibit
RNase H efficiently because these compounds all distribute in
the two upper quarters of the graph in contrast to compounds
bearing at least one para substituent (magenta, position 4),
which are more potent RNase H inhibitors with 90% of the
compounds in the two lower quarters of the plot (Figure 1C).
These two positions do not impact IN inhibition.
The most part of the newly synthesized pyrrolyl derivatives
7a−y and 8a−y exhibited good potency in inhibiting the ST
step of the HIV-1 IN. All the newly synthesized acids
compounds were selective inhibitors of the ST step of the
integration process catalyzed by IN, confirming that the DKA
derivatives were ST vs 3′-P selective inhibitors. In fact, the IC50
values on the 3′-P step were around 2−3 orders of magnitude
higher if compared with the ones on ST (data not shown).
The acids were more potent than the corresponding esters.
In fact, 8a−y showed IC50 values in the range 0.026−6.0 μM
for the ST step, while the corresponding esters 7a−y were less
active, showing IC50 values in the range of >0.45−111 μM for
the ST step. Among the acid derivatives, 15 compounds
(8a,b,e−h,k−m,o,q−t,y) resulted in having submicromolar
activity values when tested against the rIN ST step while four
compounds (8i,j,q,w) can be considered almost inactive (IC50
values in the range of 4−6 μM). The other six acid compounds
(8c,d,lp,u,v) were moderate IN inhibitors because their IC50
values were in the range of 1.2−1.6 μM.
Figure 1. Scatter plot for inhibition of RNase H and IN enzymes: (A)
Compounds are categorized according to their acidic or ester function.
(B) Compounds are categorized according to the nature of their
substitutions. (B) Compounds are categorized according to the
position of their substitutions. Compounds with one IC50 value
missing such as 7a have been left out of the plot and compounds with
IC50 values above 111 μM have been arbitrary positioned at the 100
μM value.
The inhibition of the rIN by the monofluoro substituted- and
difluoro substituted-N-benzyl-pyrrolyl diketohexenoic acids was
higher if compared to that of compounds bearing other
substituents (Table 1). The most potent anti-IN compound
within this series was the 4-fluorobenzyl-pyrrolyl diketohex-
enoic acid (8g), which showed an IC50 of 26 nM. Generally, the
most active compounds were fluoro derivatives 8g, 8q, 8r, and
8s (4-F, 2,3-F2, 2,4-F2, and 2,5-F2 substituted respectively) with
over the ester function is not observed for RNase H inhibition
because red dots (acidic) are equally distributed in the two
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IC50 values ranging from 26 to 59 nM. Among the ester series,
the difluoro substituted derivatives (7q,s,u,v) resulted in being
the most active compounds with IC50 values in the range of
0.49−0.60 μM.
The anti-IN activities of monosubstituted- and disubstituted-
N-benzyl-pyrrolyl diketohexenoic acids were comparable. In
fact, their IC50 values were in the range of 6−0.026 and 4.9−
0.042 μM, respectively.
The substitution of fluorine atom in para position of the
benzyl group with either electron-donor or -withdrawing
groups leads to compounds with decreased activity (8d,i,l,n,
IC50s from 1.2 to >4.1 μM). On the contrary, a good activity
persisted when the fluorine atom in the 4-position was replaced
by a hydrogen atom, such as for compound 8a (IC50 = 0.090
μM). When the fluorine atom of the benzyl ring of 8a was
shifted from para to ortho or meta position, the resulting
compounds 8e and 8f were 10-fold less active than 8a (IC50
values 0.98 and 0.92 μM, respectively).
Among the disubstituted derivatives, the difluoro compounds
8q−s (2,3-F2, 2,4-F2, and 2,5-F2, respectively), showed IC50
values (42−59 nM) comparable to that of the 4-fluoro
counterpart (8g), while 8t−v (2,6-F2, 3,4-F2, and 3,5-F2,
respectively) were 6−60 times less potent than 8g (IC50 range
0.15−1.6 μM). Similarly to 8t−v, the dimethylderivative 8p and
the dichloroderivatives 8w−y showed reduced activities (IC50
range 0.17−4.9 μM).
ones (8w). Among the disubstituted esters derivatives, the well
tolerated combination of substituents on the benzyl mojety was
the 3,4-F2 (7u), with an IC50 value of 3 μM. A comparable
activity was observed when the fluorine atom was shifted from
4 to 5 position (7v) or when the fluorine atoms were changed
with the methyl group (7p).
Cell-Based Assays. Antiviral activity was initially assessed
using HeLa-CD4-LTR-β-gal reporter cells (Table 1). In this
assay, reference compound 1 exhibited low nanomolar activity
with an EC50 value of 24 5 nM (Table 1). Among 7a−y and
8a−y, 18 derivatives (7a,e,g,h,m,o,q−t and 8a,e,g,m,o,q,r,t)
showed submicromolar EC50 values when tested against HIV-
1(IIIB) infected HeLa-CD4-LTR-β-gal cells. Selected com-
pounds were also tested for their ability to inhibit the HIV-1-
induced cytopathic effect (CPE) in MT-4 cells. From the 10
active compounds we selected (7a,c,e,f,g and 8a,b,c,f,g), all
were antiviral in the CPE assay. The inactive compound 8i was
also found inactive in this assay (Table 1). Among the acid
derivatives, the most potent compounds were 8m,o,t, with EC50
values below 0.2 μM, while the most potent ester derivatives
were 7g,m,q,r,t, with EC50 values of 0.2 μM or below.
In parallel, the newly synthesized compounds have been
tested for their cytotoxcity using HeLa-CD4-LTR-β-gal cells.
Compounds 7a−y and 8a−y exhibited limited cytotoxicity,
with CC50 values ranging from 30 to >250 μM (Table 1). This
resulted in 13 compounds with selctivity indexes (SI) above
100. The most selective compound were 7g,m,q,r,t and 8o,t,
with SI values above 250.
In general, antiviral activity data correlated with inhibition
data obtained on recombinant enzymes. Compounds harboring
an ester function were usually less active on recombinant IN
than their acid counterparts. This is may be explained by the
fact that a free carboxylic function is required for the inhibition
of IN by metal cofactor chelation. Still, some of those acid
compounds with good potency against recombinant enzymes
(e.g., 8d,h−k,u,y) lacked activity against HIV-1 replication in a
cellular context (Table 1). Neutralization of their acid function
in the corresponding ester derivatives (7d,h−k,u,y) rescued
their antiviral activity (Table 1). Similarly, our most potent
inhibitor 8g (2.5 μM and 26 nM against RNase H and IN,
respectively) is not the most potent antiviral in this series (EC50
value of 630 nM), while the ester 7g, inactive against
recombinant enzymes, is one of the most potent antiviral
(EC50 value below 200 nM). All together, our data indicate that
acid derivatives may suffer from poor cellular uptake and that
esterification of the carboxylic group may improve bioavail-
ability. Furthermore, our ester derivatives seem to be
metabolized to the more active acidic form in cells. Thus, in
our series of derivatives, ester variants may represent potential
pro-drug candidates.
In general, activities of acid 7a−y and ester 8a−y derivatives
against HIV-1 RT-associated RNase H function were
comparable. In fact, 27 compounds (7b−g,i,k,l,n,p,q,s,u−w
and 8e,g−l,n,q,s,w) were characterized by IC50 values <9.6 μM.
The most selective inhibitor of the RNase H function was
compound 7n that showed an IC50 RNase H/IC50 IN ratio of
16.
Compounds 7b,u and 8g (2-Me, 3,4-F2, and 4-F substituted,
respectively) were the most active derivatives as RNase H
inhibitors. Although 7b and 7u were ester derivatives, while 8g
was an acid compound, it is worth noting that their IC50 values
against RNase H were comparable (3, 3, and 2.5 μM,
respectively). On the contrary, a direct comparison of their
anti-IN activities showed that the acid derivative 8g was more
potent than the esters 7b and 7u (0.026, 32, and 0.6 μM,
respectively). Compounds 7u and 8g were more selective in
inhibiting the IN enzyme because their IC50 RNase H/IC50 IN
ratios were ≈10 and 100, respectively. Conversely, 7b was more
active on RNase H (IC50 IN/IC50 RNase H ratio ≈10).
Within the ortho- and meta-benzylsubstituted pyrrolyl
diketohexenoic derivative series, the inhibitory activities against
RNase H were well correlated with the nature of the
substituent. In fact, in both cases the activity decreases with
the following order: Me > Cl, CN, F > OMe, OEt, where the
derivative ester 7b (2- Me) and the acid 8c (3- Me) showed the
best activity with an IC50 value of 3 and 4.6 μM, respectively.
Among the ortho-benzyl substituted pyrrolyl diketohexenoic
acids series, fluoro and cyano groups (8e,j) are well-tolerated in
comparison with methyl, methoxy, and ethoxy groups
(8b,m,o). The same was observed when comparing the para-
benzylsubstituted esters 7g,i,l (fluoro, chloro, and cyano,
respectively) with the esters 7d,n (methyl and methoxy,
respectively).
CONCLUSION
■
Despite the major progress of HAART, limitations arising from
resistance and drug−drug interaction warrant the development
of original antiviral agents. In this study, we took advantage of
the similarities between IN and RNase H domain of RT to
design dual inhibitors. A series of pyrrolyl diketohexenoic
derivatives has been synthesized using a simple and efficient
parallel solution-phase approach. The best compounds achieved
single-digit micromolar inhibition of RNase H and low
nanomolar IC50 values against IN in recombinant assays.
Correlation analysis of the in vitro potency data highlighted key
structural requirements for the selective inhibition of one or the
The well tolerated combination of substituents on the benzyl
moiety among the disubstituted acids derivatives was observed
when the fluorine atom is present in positions 4 (8r) or 3 (8q)
or when the fluorine atoms were changed with the chlorine
8592
dx.doi.org/10.1021/jm401040b | J. Med. Chem. 2013, 56, 8588−8598

Journal of Medicinal Chemistry
Article
6-[1-(Phenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexenoic Acid
Ethyl Ester (7a). Yield 68%; 79−81 °C; ligroin. IR ν 3400 (OH),
1732 (CO ester), 1621 (CO ketone) cm−1. 1H NMR (CDCl3) δ
1.41 (t, 3H, CH2CH3), 4.38 (q, 2H, CH2CH3), 5.27 (s, 2H, CH2),
6.33−6.43 (m, 3H, pyrrole β, hexanoate C3−H and hexanoate C5−
H), 6.90−6.97 (m, 1H, pyrrole β), 7.11 (m, 1H, pyrrole α), 7.30−7.38
(m, 5H, benzene H), 7.69 (d, 1H, Jt = 15.0 Hz, hexanoate C6−H),
12−16 (sb, 1H, OH). Anal. (C19H19NO4) C, H, N.
other enzyme. Further studies taken into account these results
should allow the rational development of dual inhibitors with
increased RNase H potency.
EXPERIMENTAL SECTION
■
Chemistry. General. Melting points were determined with a Buchi
̈
530 capillary apparatus and are uncorrected. Infrared (IR) spectra were
recorded on a Perkin-Elmer Spectrum-One spectrophotometer. 1H
NMR spectra were recorded on a bruker AC 400 spectrometer. Merck
silica gel 60 F254 plates were used for analytical TLC. Developed plates
were visualized by UV light. Column chromatography was performed
on silica gel (Merck; 70−230 mesh) or aluminum oxide (Merck; 70−
230 mesh). Compounds purity were always >95% determined by high-
pressure liquid chromatography (HPLC). HPLC analysis was carried
out using Shimadzu LC-10AD VP and CTO-10AC VP. Column used
6-[1-(2-Methylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7b). Yield 89%; 104−105 °C; benzene/
cyclohexane. IR ν 3400 (OH), 1720 (CO ester), 1610 (CO
1
ketone) cm−1. H NMR (CDCl3) δ 1.43 (t, 3H, CH2CH3), 2.36 (s,
1H, CH3), 4.41 (q, 2H, CH2CH3), 5.22 (s, 2H, CH2), 6.27−6.45 (m,
3H, Jt = 15.3 Hz, hexanoate C5−H, hexanoate C3−H and pyrrole β),
6.69−6.72 (m, 3H, benzene H, pyrrole α, β), 7.16−7.31 (m, 3H,
benzene H), 7.65 (d, 1H, Jt = 15.3 Hz, hexanoate C6−H), 12−16 (sb,
1H, OH). Anal. (C20H21NO4) C, H, N.
was generally Suplex pKb-100 (250 mm × 4.6 mm, 5 μm). Buchi
̈
6-[1-(3-Methylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7c). Yield 71%; 76−78 °C; cyclohexane. IR ν
3400 (OH), 1700 (CO ester), 1590 (CO ketone) cm−1. 1H
NMR (CDCl3) δ 1.37 (t, 3H, CH2CH3), 2.32 (s, 3H, CH3), 4.33 (q,
2H, CH2CH3), 5.19 (s, 2H, CH2), 6.32−6.40 (m, 3H, pyrrole β,
hexanoate C3−H and hexanoate C5−H), 6.38−7.26 (m, 6H, benzene
H, pyrrole α and pyrrole β), 7.63 (d, 1H, Jt = 15.3 Hz, hexanoate C6−
H), 12−16 (sb, 1H, OH). Anal. (C20H21NO4) C, H, N.
SynCor was used for parallel synthesis using 50 mL test tubes. Solvents
were reagent grade and, when necessary, purified and dried by
standard methods. Organic solutions were dried over anhydrous
sodium sulfate (Merck). Concentration of solution after reactions and
extractions involved the use of a rotary evaporator operating at
reduced pressure of approximately 20 Torr. Analytical results agreed to
within 0.40% of the theoretical values. Dimethylsulfoxide-d6 99.9%
(code 44,139-2) and deuterochloroform 98.8% (code 41,675-4) of
isotopic purity (Aldrich) were used.
6-[1-(4-Methylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7d). Yield 60%; 85−86 °C; n-hexane. IR ν 3400
1
(OH), 1721 (CO ester), 1605 (CO ketone) cm−1. H NMR
General Procedure for the Synthesis of N-[(Phenyl)methyl]-
pyrrole-2-carboxaldehyde 9a−y. Twenty-five tubes (25 mL each,
divided in two sessions of 12 and 13 tubes, respectively) were charged
with a solution of pyrrole-2-carboxaldehyde (0.016 mol) in 30 mL of
(CDCl3) δ 1.41 (t, 3H, CH2CH3), 2.36 (s, 3H, CH3), 4.39 (q, 2H,
CH2CH3), 5.23 (s, 2H, CH2), 6.32−6.43 (m, 3H, Jt = 15.4 Hz, pyrrole
β, hexanoate C3−H, hexanoate C5−H), 6.90−6.96 (m, 2H, pyrrole α,
β), 7.00 (d, 2H, benzene H), 7.18 (m, 2H, benzene H), 7.69 (d, 1H, Jt
= 15.4 Hz, hexanoate C6−H), 12−16 (sb, 1H, OH). Anal.
(C20H21NO4) C, H, N.
dry DMF treated with NaOH (0.018 mol) and placed in the Buchi
̈
SynCor reactor. Then the appropriate benzyl halide (0.011 mol) was
added, and the reaction mixtures were stirred with bascular stirring in
Buchi Syncor at room temperature at 250 rpm overnight. The solution
̈
6-[1-(2-Fluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7e). Yield 87%; 103−105 °C; benzene. IR ν
3400 (OH), 1731 (CO ester), 1680 (CO ketone) cm−1. 1H
NMR (CDCl3) δ 1.44 (t, 3H, CH2CH3), 4.41 (q, 2H, CH2CH3), 5.34
(s, 2H, CH2), 6.37 (t, 1H, pyrrole β), 6.41−6.46 (m, 2H, Jt = 15.2 Hz,
hexanoate C5−H and hexanoate C3−H), 6.86−6.92 (m, 2H, benzene
H, pyrrole β), 7.00 (s, 1H, pyrrole α), 7.12−7.18 (m, 2H, benzene H),
7.36 (m, 1H benzene H), 7.73 (d, 1H, Jt = 15.2 Hz, hexanoate C6−H),
12−16 (sb, 1H, OH). Anal. (C19H18FNO4) C, H, N, F.
was diluted with water and extracted with ethyl acetate. The collected
organic extract was washed with brine (three times) and dried, and the
solvent was evaporated under reduced pressure to obtain crude 9a−y,
which was purified by column chromatography. Chemical, physical,
and analytical data of derivatives 9a−y are reported in Table 1 of the
Supporting Information, together with the spectral data of
representative compounds.
General Procedure for the Synthesis of 4-(Pyrrol-3-yl)but-3-
en-2-one 10a−y. Twenty-five tubes (25 mL each, divided in two
sessions of 12 and 13 tubes, respectively) were charged with a solution
of the appropriate aldehyde 9a−y (0.0068 mol) in 14 mL of acetone
and were treated with 9.8 mL of NaOH 5 N and stirred at 50 °C with
6-[1-(3-Fluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7f). Yield 76%; 84−86 °C; ligroin. IR ν 3400
1
(OH), 1700 (CO ester), 1600 (CO ketone) cm−1. H NMR
(CDCl3) δ 1.37 (t, 3H, CH2CH3), 4.33 (q, 2H, CH2CH3), 5.25 (s, 2H,
CH2), 6.33−6.41 (m, 3H, pyrrole β, hexanoate C3−H and hexanoate
C5−H), 6.71 (s, 1H, pyrrole β), 6.82−7.06 (m, 4H, benzene H and
pyrrole α), 7.32 (m, 1H, benzene H), 7.58 (d, 1H, Jt = 15.4 Hz,
hexanoate C6−H), 12−16 (sb, 1H, OH). Anal. (C19H18FNO4) C, H,
N, F.
bascular stirring in Buchi Syncore at room temperature at 250 rpm
̈
overnight and then treated with water. The reaction mixture was
extracted with ethyl acetate. The collected organic extract was washed
with brine (three times) and dried, and the solvent was evaporated
under reduced pressure to obtain crude derivatives 10a−y, which was
purified by column chromatography. Chemical, physical, and analytical
data of derivatives 10a−y are reported in Table 1 of the Supporting
Information, together with the spectral data of representative
compounds.
6-[1-(4-Fluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7g). Yield 78%; 96−97 °C; ligroin. IR ν 3400
1
(OH), 1698 (CO ester), 1605 (CO ketone) cm−1. H NMR
(CDCl3) δ 1.42 (t, 3H, CH2CH3), 4.39 (q, 2H, CH2CH3), 5.25 (s, 2H,
CH2), 6.36−6.44 (m, 3H, Jt = 15.5 Hz, pyrrole β, hexanoate C3−H,
hexanoate C5−H), 6.80 (m, 1H, pyrrole β), 6.96 (m, 1H, pyrrole α),
7.05−7.09 (m, 4H, benzene H), 7.69 (d, 1H, Jt = 15.5 Hz, hexanoate
C6−H), 12−16 (sb, 1H, OH). Anal. (C19H18FNO4) C, H, N, F.
6-[1-(3-Chlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7h). Yield 75%; 93−95 °C; ligroin. IR ν 3400
General Procedure for the Synthesis of Diketo Esters 7a−y.
Twenty-five tubes (25 mL each, divided in two sessions of 12 and 13
tubes, respectively) were charged with a solution of the appropriated
acetyl derivative 10a−y (0.077 mol) and diethyl oxalate (0.077 mol)
dissolved in 8 mL of dry THF and placed in the Buchi SynCor reactor.
̈
The reaction mixtures were treated, under argon stream, with NaOEt
obtained by the dissolution of Na (0.0155 mol) in 17 mL of absolute
1
(OH), 1721 (CO ester), 1574 (CO ketone) cm−1. H NMR
ethanol. The mixture was stirred with bascular stirring in Buchi
̈
(CDCl3) δ 1.37 (t, 3H, CH2CH3), 4.33 (q, 2H, CH2CH3), 5.21 (s, 2H,
CH2), 6.32−6.40 (m, 3H, pyrrole β, hexanoate C3−H and hexanoate
C5−H), 6.86−7.01 (m, 4H, benzene H and pyrrole α, β), 7.26 (m,
2H, benzene H), 7.59 (d, 1H, Jt = 15.3 Hz, hexanoate C6−H), 12−16
(sb, 1H, OH). Anal. (C19H18ClNO4) C, H, N, Cl.
SynCor at room temperature at 250 rpm for 1 h 30 min and then was
poured into n-hexane (60 mL). The collected precipitate was
vigorously stirred for 30 min in 1 N HCl (60 mL). The solid that
formed was filtered, washed with water and light petroleum ether, and
dried under IR lamp to afford the pure diketo ester 7a−y. Yield (%),
melting point (°C), recrystallization solvent, IR, 1H NMR, and
analytical data are reported for each of the following compounds.
6-[1-(4-Chlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7i). Yield 60%; 134−136 °C; ligroin. IR ν 3400
1
(OH), 1700 (CO ester), 1600 (CO ketone) cm−1. H NMR
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Journal of Medicinal Chemistry
Article
1
(CDCl3) δ 1.42 (t, 3H, CH2CH3), 4.37 (q, 2H, CH2CH3), 5.24 (s, 2H,
CH2), 6.35−6.43 (m, 3H, hexanoate C3−H, hexanoate C5−H and
pyrrole β), 6.88−7.04 (m, 4H, benzene H and pyrrole α and pyrrole
β), 7.34 (m, 2H, benzene H), 7.62 (m, 1H, Jt = 15.4 Hz, hexanoate
C6−H), 12−16 (sb, 1H, OH). Anal. (C19H18ClNO4) C, H, N, Cl.
6-[1-(2-Cyanophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7j). Yield 80%; 179−180 °C; benzene/
cyclohexane. IR ν 3400 (OH), 2227 (CN), 1746 (CO ester), 1578
(OH), 1727 (CO ester), 1611 (CO ketone) cm−1. H NMR
(DMSO-d6) δ 1.80 (t, 3H, CH2CH3), 4.77 (q, 2H, CH2CH3), 6.05 (s,
2H, CH2), 6.81−6.87 (m, 2H, pyrrole β and hexanoate C3−H), 7.11−
7.26 (m, 2H, pyrrole α and Jt = 15.9 Hz, hexanoate C5−H), 7.48 (s,
1H, pyrrole β), 7.61−7.84 (m, 3H, benzene 3H), 8.22 (d, 1H, Jt = 15.9
Hz, hexanoate C6−H), 12−16 (sb, 1H, OH). Anal. (C19H17F2NO4)
C, H, N, F.
6-[1-(2,4-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7r). Yield 82%; 119−121 °C; ligroin. IR ν
3400 (OH), 1737 (CO ester), 1601 (CO ketone) cm−1. 1H
NMR (CDCl3) δ 1.46 (t, 3H, CH2CH3), 4.39 (q, 2H, CH2CH3), 5.29
(s, 2H, CH2), 6.37 (m, 1H, pyrrole β), 6.41−6.46 (m, 2H, hexanoate
C5−H and hexanoate C3−H), 6.85−6.99 (m, 5H, pyrrole α, pyrrole β
and benzene H) 7.65 (d, 1H, Jt = 15.3 Hz, hexanoate C6−H), 12−16
(sb, 1H, OH). Anal. (C19H17F2NO4) C, H, N, F.
1
(CO ketone) cm−1. H NMR (CDCl3) δ 1.43 (t, 3H, CH2CH3),
4.40 (q, 2H, CH2CH3), 5.52 (s, 2H, CH2), 6.41−6.46 (m, 3H, pyrrole
β, hexanoate C3−H and hexanoate C5−H), 6.81 (d, 1H, benzene H),
6.94 (m, 1H, pyrrole β) 7.01 (m, 1H, pyrrole α), 7.46 (m, 1H, benzene
H), 7.56−7.60 (m, 2H, benzene H and hexanoate C6−H), 7.78 (m,
1H, benzene H), 12−16 (sb, 1H, OH). Anal. (C20H18N2O4) C, H, N.
6-[1-(3-Cyanophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7k). Yield 83%; 105−106 °C; acetone. IR ν
3400 (OH), 2227 (CN), 1746 (CO ester), 1578 (CO ketone)
6-[1-(2,5-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7s). Yield 82%; 84−85 °C; acetone. IR ν 3400
1
cm−1. H NMR (DMSO-d6) δ 1.82 (t, 3H, CH2CH3), 4.80 (q, 2H,
1
(OH), 1724 (CO ester), 1618 (CO ketone) cm−1. H NMR
CH2CH3), 6.11 (s, 2H, CH2), 6.87 (m, 1H, pyrrole β), 6.92 (s, 1H,
hexanoate C3−H), 7.15 (d, 1H, Jt = 15.5 Hz, hexanoate C5−H), 7.55
(m, 1H, pyrrole α), 7.18 (s, 1H, pyrrole β) 7.95−8.12 (m, 3H,
benzene H), 8.17−8.21 (m, 2H, Jt = 15.4 Hz,benzene H and hexanoate
C6−H), 12−16 (sb, 1H, OH). Anal. (C20H18N2O4) C, H, N.
6-[1-(4-Cyanophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7l). Yield 82%; 128−130 °C; ligroin. IR ν 3400
(OH), 2227 (CN), 1746 (CO ester), 1578 (CO ketone) cm−1.
1H NMR (DMSO-d6) δ 1.82 (t, 3H, CH2CH3), 4.79 (q, 2H,
CH2CH3), 6.11 (s, 2H, CH2), 6.87 (m, 1H, pyrrole β), 6.92 (s, 1H,
hexanoate C3−H), 7.15 (d, 1H, Jt = 15.5 Hz, hexanoate C5−H), 7.55
(m, 1H, pyrrole α), 7.18 (s, 1H, pyrrole β), 8.02 (m, 2H, benzene H),
8.17−8.21 (m, 4H, benzene H, Jt = 15.4 Hz, hexanoate C6−H), 12−
16 (sb, 1H, OH). Anal. (C20H18N2O4) C, H, N.
(DMSO-d6) δ 1.83 (t, 3H, CH2CH3), 4.80 (q, 2H, CH2CH3), 6.04 (s,
2H, CH2), 6.86 (s, 1H, pyrrole β) 6.96 (s, 1H, hexanoate C3−H),
7.01−7.19 (m, 2H, Jt = 15.3 Hz, pyrrole α and hexanoate C5−H), 7.55
(s, 1H, pyrrole β), 7.64−7.78 (m, 3H, benzene H), 8.26 (d, 1H, Jt =
15.3 Hz, hexanoate C6−H), 12−16 (sb, 1H, OH). Anal.
(C19H17F2NO4) C, H, N, F.
6-[1-(2,6-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7t). Yield 50%; 125−127 °C; ligroin. IR ν 3400
1
(OH), 1710 (CO ester), 1610 (CO ketone) cm−1. H NMR
(CDCl3) δ 1.42 (t, 3H, CH2CH3), 4.38 (q, 2H, CH2CH3), 5.27 (s, 2H,
CH2), 6.27 (m, 1H, pyrrole β), 6.40 (d, 1H, Jt = 15.2 Hz, hexanoate
C5−H), 6.48 (s, 1H, hexanoate C3−H), 6.80 (m, 1H, pyrrole β),
6.91−7.01 (m, 3H, benzene H and pyrrole α), 7.35 (m, 1H, benzene
H), 7.95 (d, 1H, Jt = 15.2 Hz, hexanoate C6−H), 12−16 (sb, 1H,
OH). Anal. (C19H17F2NO4) C, H, N, F.
6-[1-(2-Methoxyphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7m). Yield 100%; 116−118 °C; benzene/
cyclohexane. IR ν 3400 (OH), 1730 (CO ester), 1620 (CO
6-[1-(3,4-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7u). Yield 60%; 74−75 °C; acetone. IR ν 3400
1
(OH), 1737 (CO ester), 1633 (CO ketone) cm−1. H NMR
1
ketone) cm−1. H NMR (CDCl3) δ 1.37 (t, 3H, CH2CH3), 3.90 (s,
(DMSO-d6) δ 1.83 (t, 3H, CH2CH3), 4.79 (q, 2H, CH2CH3), 6.01 (s,
2H, CH2), 6.84 (m, 1H, pyrrole β) 6.93 (s, 1H, hexanoate C3−H),
7.14 (d, 1H, Jt = 15.4 Hz, hexanoate C5−H), 7.51−7.63 (m, 3H,
benzene H and pyrrole β), 7.78−7.86 (m, 2H, benzene H and pyrrole
α) 8.20 (d, 1H, Jt = 15.4 Hz, hexanoate C6−H), 12−16 (sb, 1H, OH).
Anal. (C19H17F2NO4) C, H, N, F.
3H, CH3), 4.34 (q, 2H, CH2CH3), 5.21 (s, 2H, CH2), 6.27 (t, 1H,
pyrrole β), 6.35 (d, 1H, Jt = 15.3 Hz, hexanoate C5−H), 6.37 (s, 1H,
hexanoate C3−H), 6.73 (m, 1H, pyrrole β), 6.83−6.94 (m, 5H,
benzene H, pyrrole β and pyrrole α), 7.25 (m, 1H, benzene H), 7.74
(d, 1H, Jt = 15.4 Hz, hexanoate C6−H), 12−16 (sb, 1H, OH). Anal.
(C20H21NO5) C, H, N.
6-[1-(3,5-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
6-[1-(4-Methoxyphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
enoic Acid Ethyl Ester (7v). Yield 58%; 75−78 °C; ligroin. IR ν 3400
noic Acid Ethyl Ester (7n). Yield 64%; 84−86 °C; ethanol. IR ν 3400
1
(OH), 1735 (CO ester), 1630 (CO ketone) cm−1. H NMR
1
(OH), 1701 (CO ester), 1598 (CO ketone) cm−1. H NMR
(DMSO-d6) δ 1.46 (t, 3H, CH2CH3), 4.40 (q, 2H, CH2CH3), 5.28 (s,
2H, CH2), 6.40−6.45 (m, 3H, pyrrole β, hexanoate C3−H and
hexanoate C5−H), 6.57−6.59 (m, 2H, pyrrole α and pyrrole β), 6.78
(m, 1H, benzene H), 6.92 (m, 1H, benzene H), 6.97 (m, 1H, benzene
H) 7.57 (d, 1H, Jt = 15.3 Hz, hexanoate C6−H), 12−16 (sb, 1H, OH).
Anal. (C19H17F2NO4) C, H, N, F.
(CDCl3) δ 1.41 (t, 3H, CH2CH3), 3.82 (s, 3H, CH3), 4.37 (q, 2H,
CH2CH3), 5.20 (s, 2H, CH2), 6.32−6.43 (m, 3H, Jt = 15.4 Hz, pyrrole
β, hexanoate C3−H, hexanoate C5−H), 6.86−7.08 (m, 6H, benzene
4H and pyrrole α and pyrrole β), 7.70 (d, 1H, Jt = 15.4 Hz, hexanoate
C6−H), 12−16 (sb, 1H, OH). Anal. (C20H21NO5) C, H, N.
6-[1-(2-Ethoxyphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid Ethyl Ester (7o). Yield 85%; 73−75 °C; benzene/
cyclohexane. IR ν 3400 (OH), 1731 (CO ester), 1680 (CO
6-[1-(2,4-Dichlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7w). Yield 100%; 110−113 °C; benzene/
cyclohexane. IR ν 3400 (OH), 1773 (CO ester), 1610 (CO
ketone) cm−1. 1H NMR (DMSO-d6) δ 1.30 (t, 3H, CH2CH3), 4.26 (q,
2H, CH2CH3), 5.48 (s, 2H, CH2), 6.35−6.36 (m, 2H, pyrrole β and
hexanoate C3−H), 6.52 (d, 1H, benzene H), 6.77 (m, 1H, hexanoate
C5−H), 7.02 (s, 1H, pyrrole β), 7.26 (s, 1H, pyrrole α) 7.41−7.47 (m,
3H, benzene H and hexanoate C6−H), 7.74 (d, 1H, benzene H), 12−
16 (sb, 1H, OH). Anal. (C19H17Cl2NO4) C, H, N, Cl.
1
ketone) cm−1. H NMR (CDCl3) δ 1.37 (t, 3H, CH2CH3), 1.45 (t,
3H, CH2CH3), 4.12 (q, 2H, CH2CH3), 4.35 (q, 2H, CH2CH3), 5.25 (s,
2H, CH2), 6.27 (t, 1H, pyrrole β), 6.33 (s, 1H, hexanoate C3−H),
6.35 (d, 1H, Jt = 15.3 Hz, hexanoate C5−H), 6.83−6.89 (m, 3H,
benzene H, pyrrole β), 6.97 (s, 1H, pyrrole α), 7.22 (d, 1H, benzene
H), 7.73 (d, 1H, Jt = 15.4 Hz, hexanoate C6−H), 12−16 (sb, 1H,
OH). Anal. (C21H23NO5) C, H, N.
6-[1-(3,5-Dimethylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-
hexenoic Acid Ethyl Ester (7p). Yield 46%; 99−101 °C; ligroin. IR ν
3400 (OH), 1729 (CO ester), 1616 (CO ketone) cm−1. 1H
NMR (CDCl3) δ 1.38 (t, 3H, CH2CH3), 2.27 (s, 6H, CH3), 4.34 (q,
2H, CH2CH3), 5.14 (s, 2H, CH2), 6.30 (m, 1H, pyrrole β), 6.33−6.37
(m, 2H, Jt = 15.1 Hz, hexanoate C5−H and hexanoate C3−H), 6.67
(s, 1H, benzene H), 6.84 (m, 1H, pyrrole β), 6.90−6.93 (m, 3H,
pyrrole α and benzene H), 7.65 (d, 1H, Jt = 15.1 Hz, hexanoate C6−
H), 12−16 (sb, 1H, OH). Anal. (C21H23NO4) C, H, N.
6-[1-(2,6-Dichlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7x). Yield 69%; 146−148 °C; ligroin. IR ν
3400 (OH), 1730 (CO ester), 1578 (CO ketone) cm−1. 1H
NMR (CDCl3) δ 1.45 (t, 3H, CH2CH3), 4.43 (q, 2H, CH2CH3), 5.51
(s, 2H, CH2), 6.27 (m, 1H, pyrrole β), 6.49−6.53 (m, 2H, Jt = 15.2
Hz, hexanoate C5−H and hexanoate C3−H), 6.64 (s, 1H, pyrrole β),
6.90 (m, 1H, pyrrole α), 7.36 (m, 1H, benzene H), 7.45−7.47 (d, 2H,
benzene 2H), 7.98 (d, 1H, Jt = 15.2 Hz, hexanoate C6−H), 12−16
(sb, 1H, OH). Anal. (C19H17Cl2NO4) C, H, N, Cl.
6-[1-(2,3-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7q). Yield 79%; 67−69 °C; acetone. IR ν 3400
6-[1-(3,5-Dichlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid Ethyl Ester (7y). Yield 62%; 112−113 °C; ligroin. IR ν
8594
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3400 (OH), 1723 (CO ester), 1646 (CO ketone) cm−1. 1H
NMR (CDCl3) δ 1.38 (t, 3H, CH2CH3), 4.35 (q, 2H, CH2CH3), 5.20
(s, 2H, CH2), 6.35−6.42 (m, 3H, pyrrole β, hexanoate C3−H and
hexanoate C5−H), 6.88−6.91 (m, 4H, benzene H and pyrrole β), 7.28
(m, 1H, pyrrole α), 7.54 (d, 1H, Jt = 15.3 Hz, hexanoate C6−H), 12−
16 (sb, 1H, OH). Anal. (C19H17Cl2NO4) C, H, N, Cl.
(OH), 1689 (CO acid), 1598 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.38 (s, 2H, CH2), 6.25−6.33 (m, 2H, pyrrole β and
hexanoate C3−H), 6.62 (d, 1H, Jt = 15.6 Hz, hexanoate C5−H),
6.93−7.01 (m, 3H, benzene H and pyrrole β), 7.15 (d, 2H, benzene
H), 7.26 (s, 1H, pyrrole α), 7.46 (d, 1H, Jt = 15.6 Hz, hexanoate C6−
H), 12−18 (sb, 2H, OH acid and OH enole). Anal. (C17H14FNO4) C,
H, N, F.
6-[1-(3-Chlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8h). Yield 79%; 144−145 °C; toluene/cycloexane. IR ν
3400 (OH), 1750 (CO acid), 1610 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.43 (s, 2H, CH2), 6.31 (m, 1H, pyrrole β), 6.38 (s, 1H,
hexanoate C3−H), 6.66 (d, 1H, Jt = 15.2 Hz, hexanoate C5−H),
6.97−7.40 (m, 6H, benzene H and pyrrole β, α), 7.58 (d, 1H, Jt = 15.3
Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and OH enole). Anal.
(C17H14ClNO4) C, H, N, Cl.
6-[1-(4-Chlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8i). Yield 76%; 170−172 °C; benzene/toluene. IR ν 3400
(OH), 1685 (CO acid), 1548 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.44 (s, 2H, CH2), 6.37 (m, 1H, pyrrole β), 6.48 (s, 1H,
hexanoate C3−H), 6.71 (d, 1H, Jt = 15.5 Hz, hexanoate C5−H), 7.05
(s, 1H, pyrrole β), 7.10 (m, 2H, benzene H), 7.32 (m, 1H, pyrrole α),
7.42 (m, 2H, benzene H), 7.60 (d, 1H, Jt = 15.5 Hz, hexanoate C6−
H), 12−18 (sb, 2H, OH acid and OH enole). Anal. (C17H14ClNO4)
C, H, N, Cl.
6-[1-(2-Cyanophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8j). Yield 58%; 179−180 °C; 2-propanol. IR ν 3400 (OH),
2227 (CN), 1746 (CO acid), 1578 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.71 (s, 2H, CH2), 6.42 (m, 1H, pyrrole β), 6.53 (s, 1H,
hexanoate C3−H), 6.72−6.76 (m, 2H, hexanoate C5−H and benzene
H), 7.09 (s, 1H, pyrrole β), 7.31 (s, 1H, pyrrole α), 7.53 (m, 1H,
benzene H), 7.63−7.71 (m, 2H, benzene H and hexanoate C6−H),
7.95 (m, 1H, benzene H), 12−18 (sb, 2H, OH acid and OH enole).
Anal. (C18H14N2O4) C, H, N.
General Procedure for the Synthesis of Diketo Acids 8a−y.
Twenty-five tubes (25 mL each, divided in two sessions of 12 and 13
tubes, respectively) were charged with a mixture of 1 N NaOH (9.48
mL) and the appropriated ester 7a−y (0.0028 mol) in 1:1 THF−
methanol (12 mL) was stirred with bascular stirring in Buchi Syncor at
̈
room temperature at 250 rpm for 1 h 30 min and then poured into
crushed ice. The aqueous mixture was treated with 1 N HCl until pH 3
was reached and extracted with ethyl acetate (three times). The
collected organic extract was washed with brine (three times) and
dried, and the solvent was evaporated under reduced pressure to give
the pure diketo acids 8a−y. Yield (%), melting point (°C),
recrystallization solvent, IR, 1H NMR, and analytical data are reported
for each of the following compounds.
6-[1-(Phenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexenoic Acid
(8a). Yield 68%; 146−148 °C; toluene. IR ν 3400 (OH), 1710
1
(CO acid), 1598 (CO ketone) cm−1. H NMR (DMSO-d6) δ
5.43 (s, 2H, CH2), 6.32 (m, 1H, pyrrole β), 6.45 (s, 1H, hexanoate
C3−H), 6.62 (d, 1H, Jt = 15.4 Hz, hexanoate C5−H), 7.00 (m, 1H,
pyrrole β), 7.10 (m, 1H, pyrrole α), 7.27−7.40 (m, 5H, benzene H),
7.61−7.79 (d, 1H, Jt = 15.4 Hz, hexanoate C6−H), 12−18 (sb, 2H,
OH acid and OH enole). Anal. (C17H15NO4) C, H, N.
6-[1-(2-Methylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8b). Yield 95%; 146−147 °C; benzene/toluene. IR ν 2923
(OH), 1700 (CO acid), 1580 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 2.35 (s, 3H, CH3), 5.39 (s, 2H, CH2), 6.30−6.43 (m,
2H, pyrrole β and hexanoate C3−H), 6.65 (d, 1H, Jt = 15.2 Hz,
hexanoate C5−H), 6.98 (s, 1H, pyrrole β), 7.06−7.25 (m, 5H,
benzene H and pyrrole α), 7.48 (d, 1H, Jt = 15.2 Hz, hexanoate C6−
H), 12−18 (sb, 2H, OH acid and OH enole). Anal. (C18H17NO4) C,
H, N.
6-[1-(3-Cyanophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8k). Yield 44%; 126−128 °C; 2-propanol/isopropyl ether.
IR ν 3400 (OH), 2231 (CN), 1717 (CO acid), 1582 (CO
1
ketone) cm−1. H NMR (DMSO-d6) δ 5.52 (s, 2H, CH2), 6.34 (m,
6-[1-(3-Methylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8c). Yield 95%; 121−123 °C; benzene/cyclohexane. IR ν
3400 (OH), 1700 (CO acid), 1590 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 2.49 (s, 3H, CH3), 5.33 (s, 2H, CH2), 6.28−6.33 (m,
2H, hexanoate C3−H and pyrrole β), 6.63 (d, 1H, Jt = 15.7 Hz,
hexanoate C5−H), 6.79−7.34 (m, 6H, benzene H, pyrrole α, β), 7.58
(d, 1H, Jt = 15.7 Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and
OH enole). Anal. (C18H17NO4) C, H, N.
6-[1-(4-Methylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8d). Yield 72%; 149−151 °C; benzene/toluene. IR ν 3400
(OH), 1705 (CO acid), 1575 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 2.27 (s, 3H, CH3), 5.34 (s, 2H, CH2), 6.27 (m, 1H,
pyrrole β) 6.37 (d, 1H, hexanoate C5−H), 6.55 (s, 1H, hexanoate
C3−H), 6.90 (m, 1H, pyrrole β), 7.13−7.24 (m, 5H, benzene H and
pyrrole α), 7.46 (d, 1H, Jt = 15.6 Hz, hexanoate C6−H), 12−18 (sb,
2H, OH acid and OH enole). Anal. (C18H17NO4) C, H, N.
6-[1-(2-Fluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8e). Yield 85%; 132−133 °C; benzene/toluene. IR ν 3400
(OH), 1710 (CO acid), 1590 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.46 (s, 2H, CH2), 6.32 (m, 1H, pyrrole β), 6.68 (d, 1H,
Jt = 16.0 Hz, hexanoate C5−H), 6.87−6.95 (m, 2H, hexanoate C3−H
and pyrrole β), 7.14−7.38 (m, 5H, benzene H and pyrrole α), 7.64 (m,
1H, Jt = 16.0 Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and OH
enole). Anal. (C17H14FNO4) C, H, N, F.
1H, pyrrole β), 6.46 (s, 1H, hexanoate C3−H), 6.68 (d, 1H, Jt = 15.3
Hz, hexanoate C5−H), 7.01 (s, 1H, pyrrole β), 7.34−7.37 (m, 2H,
benzene H), 7.52−7.66 (m, 3H, Jt = 15.3 Hz, benzene H, hexanoate
C6−H and pyrrole α), 7.79 (d, 1H, benzene H), 12−18 (sb, 2H, OH
acid and OH enole). Anal. (C18H14N2O4) C, H, N.
6-[1-(4-Cyanophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8l). Yield 93%; >250 °C; washed with isopropyl ether. IR ν
3400 (OH), 2227 (CN), 1746 (CO acid), 1578 (CO ketone)
cm−1. 1H NMR (DMSO-d6) δ 5.59 (s, 2H, CH2), 6.37 (m, 1H, pyrrole
β), 6.48 (s, 1H, hexanoate C3−H), 6.71 (d, 1H, Jt = 15.4 Hz,
hexanoate C5−H), 7.05 (s, 1H, pyrrole β), 7.23 (m, 2H, benzene H),
7.36 (m, 1H, pyrrole α), 7.60 (d, 1H, Jt = 15.4 Hz, hexanoate C6−H),
7.86 (m, 2H, benzene H), 12−18 (sb, 2H, OH acid and OH enole).
Anal. (C18H14N2O4) C, H, N.
6-[1-(2-Methoxyphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8m). Yield 100%; 142−143 °C; toluene. IR ν 3400 (OH),
1732 (CO acid), 1601 (CO ketone) cm−1. 1H NMR (DMSO-d6)
δ 3.85 (s, 3H, CH3), 5.21 (s, 2H, CH2), 6.25 (m, 1H, pyrrole β), 6.41
(s, 1H, hexanoate C3−H), 6.62 (d, 1H, Jt = 15.3 Hz, hexanoate C5−
H), 6.69−7.03 (m, 4H, benzene H and pyrrole β), 7.20−7.23 (m, 2H,
benzene H, pyrrole α), 7.66 (d, 1H, Jt = 15.3 Hz, hexanoate C6−H),
12−18 (sb, 2H, OH acid and OH enole). Anal. (C18H17NO5) C, H, N.
6-[1-(4-Methoxyphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8n). Yield 64%; >300 °C; washed with isopropyl ether. IR ν
3400 (OH), 1698 (CO acid), 1600 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 3.73 (s, 3H, CH3), 5.34 (s, 2H, CH2), 6.27−6.40 (m,
2H, pyrrole β and hexanoate C3−H) 6.66 (d, 1H, hexanoate C5−H),
6.89−6.93 (m, 3H, benzene H and pyrrole β), 7.07 (d, 2H, benzene
H), 7.28 (s, 1H, pyrrole α), 7.66 (d, 1H, Jt = 15.6 Hz, hexanoate C6−
H), 12−18 (sb, 2H, OH acid and OH enole). Anal. (C18H17NO5) C,
H, N.
6-[1-(3-Fluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8f). Yield 76%; 132−135 °C; toluene. IR ν 3400 (OH),
1700 (CO acid), 1600 (CO ketone) cm−1. 1H NMR (DMSO-d6)
δ 5.44 (s, 2H, CH2), 6.31 (m, 1H, pyrrole β), 6.40 (s, 1H, hexanoate
C3−H), 6.64 (d, 1H, Jt ≈ 15 Hz, hexanoate C5−H), 7.79−7.19 (m,
4H, benzene H and pyrrole β), 7.26−7.47 (m, 2H, benzene H), 7.62
(d, 1H, Jt ≈ 15 Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and
OH enole). Anal. (C17H14FNO4) C, H, N, F.
6-[1-(2-Ethoxyphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8o). Yield 80%; 159−160 °C; toluene. IR ν 2923 (OH),
1731 (CO acid), 1601 (CO ketone) cm−1. 1H NMR (DMSO-d6)
6-[1-(4-Fluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hexe-
noic Acid (8g). Yield 54%; 98−99 °C; benzene/toluene. IR ν 3400
8595
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δ 1.32−1.38 (t, 3H, CH2CH3), 4.05−4.10 (q, 2H, CH2CH3), 5.33 (s,
2H, CH2), 6.24 (t, 1H, pyrrole β), 6.42 (s, 1H, hexanoate C3−H),
6.62 (d, 1H, Jt = 15.4 Hz, hexanoate C5−H), 6.74 (d, 1H, benzene H),
6.83 (t, 1H, bezene H), 6.95 (s, 1H, pyrrole β), 6.99 (d, 1H, benzene
H), 7.20−7.23 (m, 2H, benzene H, pyrrole α), 7.66 (d, 1H, Jt = 15.4
Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and OH enole). Anal.
(C19H19NO5) C, H, N.
6-[1-(3,5-Dimethylphenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-
hexenoic Acid (8p). Yield 78%; 145−147 °C; benzene. IR ν 3400
(OH), 1713 (CO acid), 1608 (CO ketone) cm−1. 1H NMR
(DMF-d7) δ 2.18 (s, 6H, CH3), 5.29 (s, 2H, CH2), 6.25 (m, 1H,
pyrrole β), 6.44 (s, 1H, hexanoate C3−H), 6.61 (d, 1H, Jt = 15.4 Hz,
hexanoate C5−H), 6.68 (s, 1H, benzene H), 6.86 (s, 1H, pyrrole β),
6.95−6.96 (d, 1H, benzene H), 7.26 (s, 1H, pyrrole α), 7.61 (d, 1H, Jt
= 15.4 Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and OH
enole). Anal. (C19H19NO4) C, H, N.
6-[1-(2,3-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8q). Yield 47%; 147−148 °C; 2-propanol/n-hexane. IR ν
3400 (OH), 1758 (CO acid), 1630 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.54 (s, 2H, CH2), 6.35 (m, 1H, pyrrole β), 6.50 (s, 1H,
hexanoate C3−H), 6.69−6.75 (m, 2H, pyrrole β and hexanoate C5−
H), 7.03 (s, 1H, pyrrole α), 7.19−7.45 (m, 3H, benzene H), 7.72 (d,
1H, Jt = 15.3 Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and OH
enole). Anal. (C17H13F2NO4) C, H, N, F.
6-[1-(2,4-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8r). Yield 67%; 130−132 °C; ethyl acetate. IR ν 3400
(OH), 1758 (CO acid), 1630 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.46 (s, 2H, CH2), 6.31 (m, 1H, pyrrole β), 6.50 (s, 1H,
hexanoate C3−H), 6.72 (d, 1H, Jt = 15.7 Hz, and hexanoate C5−H),
6.97−7.03 (m, 2H, pyrrole β and benzene H), 7.10 (m, 1H, benzene
H) 7.28−7.37 (m, 2H, pyrrole α and benzene H), 7.69 (d, 1H, Jt =
15.7 Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and OH enole).
Anal. (C17H13F2NO4) C, H, N, F.
hexanoate C5−H), 7.07 (s, 1H, pyrrole β), 7.29 (s, 1H, pyrrole α),
7.42 (m, 1H, benzene H), 7.7 (d, 1H, Jt = 15.3 Hz, hexanoate C6−H),
7.73 (d, 1H, benzene 1H), 12−18 (sb, 2H, OH acid and OH enole).
Anal. (C17H13Cl2NO4) C, H, N, Cl.
6-[1-(2,6-Dichlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8x). Yield 84%; 132−134 °C; toluene. IR ν 3400 (OH),
1758 (CO acid), 1630 (CO ketone) cm−1. 1H NMR (DMSO-d6)
δ 5.55 (s, 2H, CH2), 6.23 (m, 1H, pyrrole β), 6.61 (s, 1H, hexanoate
C3−H), 6.67 (s, 1H, pyrrole β), 6.81 (d, 1H, Jt = 15.7 Hz, hexanoate
C5−H), 7.01 (s, 1H, pyrrole α), 7.52 (m, 1H, benzene H), 7.63−7.65
(m, 2H, benzene H), 7.94 (d, 1H, Jt = 15.7 Hz, hexanoate C6−H),
12−18 (sb, 2H, OH acid and OH enole). Anal. (C17H13Cl2NO4) C, H,
N, Cl.
6-[1-(3,5-Dichlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8y). Yield 95%; >300 °C; toluene/cyclohexane. IR ν 3400
(OH), 1743 (CO acid), 1521 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.38 (s, 2H, CH2), 6.24 (s, 1H, pyrrole β), 6.54 (m, 1H,
hexanoate C3−H), 6.33 (s, 1H, hexanoate C5−H), 7.02−7.35 (m, 5H,
pyrrole α, pyrrole β and hexanoate C6−H), 7.50 (s, 1H, benzene H),
12−18 (sb, 2H, OH acid and OH enole). Anal. (C17H13Cl2NO4) C, H,
N, Cl.
Biological Methods. HIV-1 RNase H Inhibition. The RT-
associated RNase H activity was measured in a polymerase-
independent cleavage assay in which the Poly(dC)-[3H]Poly(rG)
hybrid was used as reaction substrate as previously described.30
HIV-1 IN Inhibition. ST activity was monitored using an
electrochemiluminescent plate-based assay as previously described.27
Briefly, donor DNA obtained from BioVeris (Gaithersburg, MD) was
incubated for 30 min at 37 °C in the presence of 250 nM of
recombinant HIV-1 IN. After addition of the compounds, the
integration reaction was initiated by addition of target DNA and
carried out for 60 min at 37 °C. Finally, plates were read on the
BioVeris M-SERIES analyzer.
6-[1-(2,5-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8s). Yield 51%; 141−143 °C; toluene/n-hexane. IR ν 3400
(OH), 1747 (CO acid), 1711 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 5.50 (s, 2H, CH2), 6.35 (m, 1H, pyrrole β), 6.51 (s, 1H,
hexanoate C3−H), 6.68−6.77 (m, 2H, pyrrole β and hexanoate C5−
H), 7.04 (s, 1H, pyrrole α), 7.21−7.39 (m, 3H, benzene H), 7.72 (d,
1H, Jt = 15.2 Hz, hexanoate C6−H), 12−18 (sb, 2H, OH acid and OH
enole). Anal. (C17H13F2NO4) C, H, N, F.
6-[1-(2,6-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8t). Yield 64%; 182−183 °C; toluene. IR ν 3400 (OH),
1720 (CO acid), 1610 (CO ketone) cm−1. 1H NMR (DMSO-d6)
δ 5.40 (s, 2H, CH2), 6.22 (m, 1H, pyrrole β), 6.50 (s, 1H, hexanoate
C3−H), 6.70 (d, 1H, Jt = 15.4 Hz, hexanoate C5−H), 6.91 (m, 1H,
pyrrole β), 7.10−7.20 (m, 2H, pyrrole α and benzene H), 7.45−7.50
(m, 2H, benzene H), 7.87 (d, 1H, Jt = 15.4 Hz, hexanoate C6−H),
12−18 (sb, 2H, OH acid and OH enole). Anal. (C17H13F2NO4) C, H,
N, F.
6-[1-(3,4-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8u). Yield 100%; >270 °C; DMF/H2O. IR ν 3400 (OH),
1758 (CO acid), 1630 (CO ketone) cm−1. 1H NMR (DMSO-d6)
δ 5.38 (s, 2H, CH2), 5.80 (s, 1H, pyrrole β), 6.30 (s, 1H, hexanoate
C3−H), 6.53 (m, 1H, hexanoate C5−H), 6.80−6.90 (m, 2H, pyrrole β
and benzene H), 7.14−7.60 (m, 4H, benzene H, hexanoate C6−H and
pyrrole α), 12−18 (sb, 2H, OH acid and OH enole). Anal.
(C17H13F2NO4) C, H, N, F.
6-[1-(3,5-Difluorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8v). Yield 60%; 146−149 °C; washed with ethanol. IR ν
3400 (OH), 1712 (CO acid), 1595 (CO ketone) cm−1. 1H NMR
(DMSO-d6) δ 6.29 (s, 2H, CH2), 6.36 (s, 1H, pyrrole β), 6.47 (s, 1H,
hexanoate C3−H), 6.70−6.78 (m, 3H, benzene H and hexanoate C5−
H), 7.03 (s, 1H, pyrrole β), 7.20 (m, 1H, pyrrole α), 7.35 (s, 1H,
benzene 1H), 7.60 (d, 1H, hexanoate C6−H), 12−18 (sb, 2H, OH
acid and OH enole). Anal. (C17H13F2NO4) C, H, N, F.
HIV-1 Replication Inhibition. Compounds antiviral activity was
determined in a cell-based assay according to the procedure described
previously31 and modified as follows. HeLa-CD4-LTR-β-gal cells were
maintained in DMEM with 10% serum and 0.5 mg/mL G418. The day
prior experimentation, 96-well plates were prepared to contain 10000
cells per well in 100 μL of DMEM medium complemented with 10%
serum. On day one, each drug is serial diluted directly on cells
following a 3-fold dilution over 6 points and each well is then filled to
200 μL with either fresh medium or concentrated viral supernatant
(HIV-1(IIIB), Advanced Biotechnologies Inc.). The highest com-
pound concentration tested was 50 μM. On day two, cells are washed
three times with PBS before adding 200 μL of a solution containing 50
mM Tris-HCl pH 7.5, 100 mM β-mercaptoethanol, 0.05% Triton
X100, and 5 mM 4-methyl-umbelliferyl-β-D-galactopyranoside (4-
MUG, Sigma). On day three, sealed plates are read in a SpectraMax
GEMINI-XS (Molecular Devices) with λex/em = 360/460 nm.
Selected compounds (see Table 1) were tested for anti-HIV activity
against the replication of HIV-1 (IIIB) in MT-4 cells. MT-4 cells were
grown and maintained in RPMI 1640 medium supplemented with
10% heat-inactivated fetal calf serum, 2 mM glutamine, 0.1% sodium
bicarbonate, and 20 μg/mL gentamicin (culture medium). HIV-1
(HTLV-IIIB/LAI) was used in all experiments. Inhibition of virus-
induced cytopathic effect by the compounds was monitored by a
classical survival assay. Briefly, 50 μL of HIV-1 (100−300 CCID50
(50% cell culture infective dose)) were added to a flat-bottomed
microtiter tray with 50 μL of medium containing various
concentrations of the test compounds. MT-4 cells were added at a
final concentration of 6 × 105 cells/mL. After 5 days of incubation at
37 °C, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazoliumbromide (MTT).
Cellular Toxicity. Similarly to the antiviral assays, plates were
prepared with 10000 HeLa-CD4-LTR-β-gal cells per well and a serial
dilution of compounds in 100 μL. After 24 h of culture, 100 μL of
ATPlite reagent (Perkin-Elmer) was added to each well. After 5 min at
room temperature, plates’ luminescence was quantified using an
EnVision multilabel reader (Perkin-Elmer) according to manufac-
turer’s instructions.
6-[1-(2,4-Dichlorophenyl)methyl-1H-pyrrol-2-yl]-2,4-dioxo-5-hex-
enoic Acid (8w). Yield 68%; 145−146 °C; toluene. IR ν 3400 (OH),
1758 (CO acid), 1630 (CO ketone) cm−1. 1H NMR (DMSO-d6)
δ 5.51 (s, 2H, CH2), 6.37 (m, 1H, pyrrole β), 6.47 (s, 1H, hexanoate
C3−H), 6.56 (m, 1H, benzene H), 6.63 (d, 1H, Jt = 15.3 Hz,
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ASSOCIATED CONTENT
■
S
* Supporting Information
Spectroscopic data and analyses of compounds 7a−y, 8a−y,
9a−y, and 10a−y. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*For R.C.: phone, +39-06-49693247; fax, +39-06-49913133; E-
mail, roberta.costi@uniroma1.it.
*For C.M.: phone, +1-301-435-2463; fax, +1-301-402-0752; E-
mail, marchand@nih.gov.
Author Contributions
R.C. and M.M. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Italian MIUR for financial support PRIN 2010, the
Italian Ministry of Health for financial support AIDS 2009-2010
and Fondazione Banco di Sardegna. R.D.S. and R.C. thanks
CHAARM project for partial support. Francesca Esposito and
Luca Zinzula were supported by RAS fellowships, cofinanced
with funds of PO Sardinia FSE 2007-2013 and of LR 7/2007,
projects CRP2_683 and CRP2_682, respectively. Our studies
were also supported by the National Institutes of Health
Intramural Program, Center for Cancer Research, National
Cancer Institute and by National Institutes of Health grants
from the AIDS Intramural Targeted Program (IATAP). The
following reagent was obtained through the NIH AIDS Reagent
Program, Division of AIDS, NIAID, NIH: HeLa-CD4-LTR-β-
gal from Dr. Michael Emerman.32
203. (b) Met
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ABBREVIATIONS USED
■
HAART, highly active antiretroviral therapy; HIV, human
immunodeficiency virus; HIV-1, human immunodeficiency
virus type 1; RNase H, ribonuclease H; RT, reverse
transcriptase; IN, integrase; rRT, recombinant reverse tran-
scriptase; rIN, recombinant integrase; FDA, Food and Drug
Administration; 3′-P, 3′-processing; ST, strand transfer; GT,
guanine thymine; DKA, diketo acids; IR, infrared; HPLC, high-
performance liquid chromatography; NMR, nuclear magnetic
resonance
Mollmann, U.; Heinisch, L.; Mellors, J. W.; Le Grice, S. F.;
̈
Pommier, Y. Madurahydroxylactone derivatives as dual inhibitors of
human immunodeficiency virus type 1 integrase and RNase H.
Antimicrob. Agents Chemother. 2008, 52, 361−364.
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Mouscadet, J. F.; Calmels, C.; Andreola, M. L.; Witvrouw, M.;
Christ, F.; Debyser, Z.; Cotelle, P. Design, synthesis, and biological
evaluation of a series of 2-hydroxyquinoline-1,3(2H,4H)-diones as dual
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Cotelle, P. 2-Hydroxyisoquinoline-1,3(2H,4H)-diones as inhibitors of
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