Diketo acids derivatives as dual inhibitors of human immunodeficiency virus type 1 integrase and the reverse transcriptase RNase H Domain

Article abstract of DOI:10.2174/092986711796504619

The HIV-1 integrase (IN) and reverse transcriptase (RT) are essential enzymes in the virus cycle. RT is crucial for the retrotranscription of the RNA viral genome, while IN is involved in the insertion in host chromosome of the proviral double strand DNA produced by RT. This enzyme has two associated functions: the RNA- and DNA-dependent DNA polymerase (RDDP and DDDP) and the ribonuclease H (RNase H). The RNase H function catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex replication intermediate. Since the discovery that catalytic cores of both HIV-1 RNase H and IN are folded in a very similar way, have very similar active site geometries, and show the same DDE triad absolutely required for catalytic activity, some researches were devoted to study IN and RNase H dual inhibitor. Our decennial interest in design and synthesis of IN inhibitors led us to study the activity of our compounds also on RNase H activity. The results of the activities showed by pyrrolyl and quinolonyl diketo acids are reported and discussed.

Full text of DOI:10.2174/092986711796504619

Current Medicinal Chemistry, 2011, 18, 3335-3342
3335
Diketo Acids Derivatives as Dual Inhibitors of Human Immunodeficiency Virus Type
1 Integrase and the Reverse Transcriptase RNase H Domain
R. Di Santo*
Pasteur Institute - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” University of
Rome, Italy
Abstract: The HIV-1 integrase (IN) and reverse transcriptase (RT) are essential enzymes in the virus cycle. RT is crucial for the retro-
transcription of the RNA viral genome, while IN is involved in the insertion in host chromosome of the proviral double strand DNA pro-
duced by RT. This enzyme has two associated functions: the RNA- and DNA-dependent DNA polymerase (RDDP and DDDP) and the
ribonuclease H (RNase H). The RNase H function catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex
replication intermediate. Since the discovery that catalytic cores of both HIV-1 RNase H and IN are folded in a very similar way, have
very similar active site geometries, and show the same DDE triad absolutely required for catalytic activity, some researches were devoted
to study IN and RNase H dual inhibitor. Our decennial interest in design and synthesis of IN inhibitors led us to study the activity of our
compounds also on RNase H activity. The results of the activities showed by pyrrolyl and quinolonyl diketo acids are reported and dis-
cussed.
Keywords: HIV-1, integrase, reverse transcriptase, ribonuclease H, RNase H, RNase H inhibitor, IN inhibitor, dual inhibitors, diketo acids,
pyrrole derivatives, quinolinone drivatives.
Human immunodeficiency virus type 1 (HIV-1) integrase (IN)
is an enzyme essential for viral replication that mediates the inser-
tion of viral DNA within the host cell genome via a multistep proc-
ess occurring in the following biochemical stages: (i) assembly of a
stable DNA-enzyme complex with specific DNA sequences at the
end of the HIV-1 long terminal repeat regions, (ii) 3ꢀ-processing
that consists of the endonucleolytic cleavage of the 3ꢀ-ends of the
viral DNA, (iii) transport through the nuclear envelope of the mul-
timeric complex, called pre-integration complex (PIC), including
viral proteins, IN and the processed DNA, (iv) strand transfer,
which consists of the ligation of the viral 3ꢀ-OH DNA ends (gener-
ated by 3ꢀ-processing) to the 5ꢀ-DNA phosphate of a host chromo-
some, (v) removal of the two unpaired nucleotides at the 3ꢀ-ends of
the viral DNA, and (vi) gap filling, probably accomplished by cel-
lular enzymes, Fig. (1). In the past decade, IN has emerged as an
attractive target; in fact, it is essential for retroviral replication, and
no host-cell equivalent of IN is known, so that IN inhibitors should
not interfere with normal cellular processes, and therefore have a
high therapeutic index. HIV IN is a 32-kDa protein comprising
three structural domains: the amino-terminal domain, the carboxy-
terminal domain and the catalytic core domain that contains a triad
of invariant carboxylate residues required for catalysis: D64, D116,
and E152, called DDE motif. A Mg2+ ion is coordinated by D64
and D116 along with water molecules, whereas E152 that lies close
to D64 does not participate in metal binding [1, 2].
into later stage trials, the latter being the first drug approved as IN
inhibitor in 2007, Fig. (2).
It is generally believed that IN inhibitors such as DKA (A), or
their bioisoster dihydroxypyrimidine carboxamide class (B), includ-
ing raltegravir (1) bind these two metal ions in the active site while
the hydrophobic aryl group participates in a specific interaction
with an adjacent hydrophobic pocket or surface, Fig. (3) [22, 23].
Reverse transcriptase (RT) is another enzyme that is essential in
the early steps of the HIV-1 life cycle for the conversion of the viral
single-stranded RNA genome into a double-stranded DNA, which
is subsequently translocated into the cell nucleus and integrated into
the cell host DNA [24, 25]. RT is a multifunctional enzyme that
possesses several distinct associated activities including RNA- and
DNA-dependent DNA polymerase (RDDP and DDDP, respec-
tively), ribonuclease H (RNase H), strand transfer, strand displace-
ment synthesis and nucleotide excision, Fig. (4) [24-26].
RNase H activity, which degrades the RNA of the RNA-DNA
hybrid molecules, is required at several steps during the reverse
transcription process, and a functional RNase H is essential for
retroviral replication. HIV IN and RNase H belong to a broader
class of nucleotide-related enzymes including nucleases, polym-
erases, and polynucleotidyl transferases [3, 27].
Phosphoryl transfer is a common chemical transformation,
which is essential for a number of basic cellular functions such as
DNA replication and transcription, signal transduction and metabo-
lism. HIV-1 RNase H catalyzes phosphoryl transfer through nu-
cleophilic substitution reactions on phosphate esters, requiring the
concerted action of a general base activating the nucleophile and a
general acid protonating the leaving group. In particular, catalysis
occurs by deprotonation of a water molecule to form a nucleophilic
hydroxide group that attacks the scissile phosphate group on RNA
[28, 29]. In order to accomplish this reaction, the HIV-1 RT-
associated RNase H function requires either Mg2+ or Mn2+, thus
being classified as a metal dependent transposases, Fig. (5) [30].
However, differently from the other RNase Hs such as the E. coli
RNase H that needs a single divalent cation in the active site [31],
the structure of the HIV-1 RNase H domain has been proposed to
need two divalent cations that, consistently with the phosphoryl
transfer geometry, seem to be coordinated by the active site car-
boxylates D443, E478, D498 and D549, Fig. (5) [29]. These resi-
dues compose a three-amino-acid DDE motif, highly conserved in
many HIV-1 strains in the core domain of the RNase H active site:
mutations in any of the D443, D498 and E478 residues abolish
enzyme activity [32, 33].
Whereas structural studies of IN reveal a single binding site for
Mg2+, the number of metal ions present and required in the active
site during the process remains controversial. Since a second metal
has been observed in an avian sarcoma virus IN crystal structure
[3], and because of the two-metal structure for polynucleotide trans-
ferases [4, 5], it has been proposed that a second metal (Mg2+ or
Mn2+) can be coordinated between D116 and E152 once HIV-1 IN
binds its DNA substrate(s) [6, 7]. It is therefore likely that the
metal(s) coordinate(s) IN and the phosphodiester backbone of the
DNA substrate(s) during the 3ꢀ-processing and strand-transfer steps.
A great number of HIV-1 IN inhibitors with metal binding
properties have been described, including diketo acids (DKAs) and
their bioisosters, and numerous reviews have been published [8-18].
Among them, two compounds, Elvitegravir [19] and Raltegravir
[20, 21], demonstrated promising clinical trial results and advanced
*Address correspondence to this author at the Pasteur Institute - Fondazione Cenci
Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, “Sapienza” University
of Rome, P.le Aldo Moro 5, I-00185 Rome, Italy; Tel: +39-06-49913150; Fax: +39-06-
49913133; E-mail roberto.disanto@uniroma1.it
0929-8673/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

3336 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
R. Di Santo
INTEGRATION PROCESS
CAGT
ACTG
Cytoplasm
Viral dsDNA
GTCA
TGAC
ACTG
OH
GTCA
CA
Viral dsDNA
IN, proteins
3’-PROCESSING
PIC
HO
AC
ACTG
OH
GTCA
CA
Nucleus
Viral dsDNA
HO
AC
STRAND TRANSFER
Human DNA
CA
TG
AC
GT
CA
GT
TG
AC
Proviral DNA
integrated in
human DNA
5’-END JOINING
Fig. (1). Steps of integration process performed by HIV-1 IN.
F
O
O
O
Cl
OH
F
HO
O
N
N
N
H
N
H
N
O
N
O
N
OH
O
1
2
Fig. (2). Chemical structures of a HIV-1 IN inhibitors Raltegravir (1) and Elvitegravir (2).
H
N
opment of several classes of RT inhibitors (RTI), which have been
discovered and approved for the treatment of HIV-1 infected pa-
tients [37, 38]. However, although RT is a multifunctional enzyme,
all approved RTI actually target, only the RT-associated polym-
erase function. Inhibitors of RT-associated RNase H function were
less studied if compared with both RT-associated polymerase func-
tion and IN inhibitors. However, in the last five years more atten-
tion has been given to the HIV-1 RT-associated RNase H activity as
drug target. As a result, several compound screenings were per-
formed by different teams and diverse classes of inhibitors were
found to be able to inhibit the HIV-1 RNase H activity. Among
them, natural products, hydrazones, naphthalene sulfonic acids,
DKAs, N-hydroxyimides, hydroxypyrimidine carboxylates, nitro-
furan-2-carboxylic acid carbamoylmethyl ester derivatives, viny-
logous ureas, and thiocarbamates and triazoles, were reported, the
most part of which having metal-chelating ability [39].
O
O
O
Mg2+
Mg2+
N
O
O
R
Mg2+
Mg2+
Ar
O
N
H
O
A
B
Fig. (3). Metal binding ability of DKA (A) and dihydroxypyrimidine
carboxamide (B).
Surprisingly, determination of the three-dimensional structure
of the catalytic core of HIV-1 RNase H led to the finding that HIV-
1 RNase H and HIV-1 IN are folded in a very similar way and have
very similar active site geometries. Catalytic cores of both HIV-1
RNase H and IN share a similar ꢀꢁ-fold containing a central five-
stranded mixed ꢁ-sheet surrounded by ꢀ-helices on both sides in
the same topological order [34-36]. Moreover, RNase H and IN
have remarkably similar active sites with the same DDE triad abso-
lutely required for catalytic activity.
Within the above cited class of RNase H inhibitors, some
DKAs inhibitors of HIV-1 IN have shown anti-RNase H activities
[40, 41] whereas DNA aptamers first described as inhibitors of
RNase H have been reported to inhibit HIV-1 integrase [42]. Re-
cently, tropolones [43-45] and madurahydroxylactone derivatives
[46] have been reported to inhibit both enzymes.
The pivotal role of RT in the HIV-1 life cycle has made this en-
zyme a key target in the AIDS chemotherapy leading to the devel-

Diketo Acids Derivatives as Dual Inhibitors of Human Immunodeficiency Virus
Current Medicinal Chemistry, 2011 Vol. 18, No. 22
3337
RNA (+)
R
U5
PBS
GAG
POL
POL
ENV
ENV
PPT
U3
R
RNase H
RNA (+)
R
R
U5
U5
PBS
GAG
ENV
PPT
U3
R
DNA (-)
strand transfer
RNA (+)
PBS
GAG
POL
PPT
U3
R
R
U5
DNA (-)
RNase H
RNase H
U3 R
RNA (+)
U5
PBS
PBS
GAG
GAG
POL
POL
ENV
ENV
PPT
PPT
U3
R
DNA (-)
DNA (-)
PPT
PPT
PBS
GAG
POL
ENV
U3
R
R
U5
U5
RNase H
DNA (+)
PBS
PPT
PPT
U3
U3
R
R
U5
U5
PBS
GAG
POL
ENV
DNA (-)
RNase H
DNA (+)
U3
U3
R
R
U5
U5
PBS
PBS
GAG
POL
ENV
PPT
DNA (-)
strand transfer
DNA (+)
U3
R
U5
PBS
PBS
GAG
POL
ENV
PPT
U3
R
U5
DNA (-)
DNA (+)
U3
U3
R
R
U5
U5
PBS
PBS
GAG
GAG
POL
POL
ENV
ENV
PPT
PPT
U3
U3
R
R
U5
U5
DNA (-)
ꢀ
Fig. (4). HIV-1 RNA genome conversion into DNA by RT.
Base
HO
O
O
O
_
Glu478
O
OH
O
P
M2+
O-
O
Asp498
_
Base
O
O
O
O-
H2O
M2+
H
O
OH
_
O
P
Asp443
O-
O
O
O
O
_
O
Asp549
Fig. (5). Schematic representation of the two-metal ion mechanism of catalysis for the RNase H activity of HIV-1 reverse transcriptase.
Since the hypermutability of the HIV-1 genome easily results in
drug-resistant strains, in time this leads to an overall reduction of
drug efficacy. Therefore, the identification of new RTI that target
other essential RT-associated functions such as the RNase H activ-
ity could be an attractive approach to enhance the anti-HIV-1 drug
armamentarium effectiveness [47]. Further, the simultaneous inhi-
bition of HIV-1 IN, RT polymerase, and RT RNase H activities by
metal-chelating compounds appears as a novel and attractive thera-
peutic, way which could contribute to overcome the problems oc-
curring during combination therapy.

3338 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
R. Di Santo
OH
OH
OH
O
OH
O
N
HO
HO
OH
OH
O
O
3
F
O
4
OH
O
N
O
O
5b
F
Fig. (6). Design of dikeyo hexenoic acids (i.e. 5b) and reference structures 3 and 4. The cinnamoyl and the benzyl portions are highlighted with dashed and
full line, respectively, while diketo portion and carboxylic function are pointed out with bold bonds.
Our efforts to find HIV-1 IN inhibitors, started in the last part
of 90s, time in which we designed and synthesized conformation-
ally restrained cinnamoyl derivatives belonging to the polyhydroxy-
late aromatic compound class [48,49]. Then, after the demonstra-
tion that polyhydroylated aromatic derivatives were inhibitors of
HIV-1 entry in cell-based assays [50] and did not target the IN in
vivo, we decided to focus our attention on the aryl diketoacid com-
pounds, which were proven to be effective HIV-1 IN inhibitors both
in cell-based assays and in vivo [51, 52].
the carboxylic function found both in DKAs (i.e. 3) and in our de-
rivatives (i.e. 4); iv) an benzyl portion typical for both DKAs and
cinnamoyl derivatives (i.e. 1-benzylpyrrole or trihydroxyben-
zylidene group, respectively), Fig. (6). The newly designed N-
benzylpyrrolyl diketohexenoic acids (5) were a promising tool to
explore how the elongation of the diketobutanoic chain would af-
fect anti-IN activity [53-55].
Compound 5b and 75 congeners were synthesized and tested
against both recombinant IN in enzyme assays and HIV-1 infected
cells in cell-based assays. The synthetic pathway to obtain deriva-
tives 5 is reported in Scheme 1.
Starting form the structure of the i) pyrrole derivative L-
731,988 (3) reported by Hazuda as anti-IN agent [51] and ii) poly-
hydroxy cinnamoyl derivatives (such as 4) that we previously iden-
tified as potent IN inhibitors in enzyme assays [48, 49], we de-
signed new pyrrolyl diketohexenoic acid derivatives as potential IN
inhibitors (i.e. 5b), Fig. (6). Pyrrolyl diketohexenoic acids (i.e. 5b)
are characterized by i) the cinnamoyl moiety of polyhydroxy aro-
matic derivatives, in which the phenyl ring is replaced by the bioi-
sosteric pyrrole moiety; ii) the diketo acid group of DKA series; iii)
Data of cytotoxicity and antiviral activities in enzyme (IN) and
cell-based assays of selected pyrrolyl diketohexenoic acids 5 and a
few related congeners 6 and 7 are reported in Table 1, using 3 as the
reference compound.
In general, compounds 5-7 were found to be potent ST inhibi-
tors, with activities within the range 0.026-110 ꢀM. These DKA
O
CH3
N
N
c
O
a
b
N
H
H
O
H
R
R
COOEt
COOH
N
N
d
O
OH
O
OH
R
R
5
Scheme 1. Synthetic pathway to obtain pyrrolyl diketohexenoic acids 5.
(a) Benzylhalide, K2CO3, DMF, 18 h, 90°C; (b) 2-propanone, 5N NaOH, 24 h, 25 °C: (c) diethyl oxalate, NaOEt, THF, 1h, 45 min, 25 °C; (d) 1N NaOH, THF,
MeOH, 1.5 h, 25 °C.

Diketo Acids Derivatives as Dual Inhibitors of Human Immunodeficiency Virus
Current Medicinal Chemistry, 2011 Vol. 18, No. 22 3339
Table 1. Cytotoxicity and Antiviral Activities in Enzyme (IN - Related to ST, and RNase H) and Cell-Based Assays of Pyrrolyl Diketo Acids 5-7
COOX
O
R
O
OH
N
O
OH
COOX
COOX
R
N
N
F
F
5
7
6
IC50a (ꢁM)
Compd
R
X
ST
RH
EC50b (ꢁM)
CC50c (ꢁM)
SId
5a
5b
5c
5d
5e
5f
H
Et
H
15
0.09
8.0
-
0.25
0.35
3
>50
>50
>50
>50
32
>200
>143
>17
>38
>2
H
15
9.8
5.0
32
6
2-Cl
Et
H
2-Cl
4.0
1.3
2-CN
2-CN
2-F
Et
H
9.00
0.26
0.98
0.98
32
17
>50
0.31
0.35
1.4
>50
>50
>50
>50
>50
33
-
5g
5h
5i
Et
H
6.3
6.4
3.0
26
>161
>143
>36
>14
55
2-F
2-Me
2-Me
2-OEt
2-OEt
2-OMe
2-OMe
Et
H
5j
0.17
12
3.7
5k
5l
Et
H
>100
64
0.6
0.31
23
>0.2
0.2
33
>165
195
>250
5m
5n
Et
H
> 100
16
39
0.53
0.2
>50
5o
5p
3-Cl
3-Cl
Et
H
6
19
0.79
>50
7.1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>63
-
0,31
-
9
5q
3-CN
3-CN
3-F
Et
H
5
>7
-
5r
0.75
11
5
>50
2.2
5s
Et
H
9.0
14
>23
>10
>21
>26
>143
>38
>2
-
5t
3-F
0.92
8.0
5.1
5u
3-Me
3-Me
3-OMe
3-OMe
4-Cl
Et
H
9.6
4.6
20
2.4
5v
1.3
1.9
5w
5x
Et
H
60
0.35
1.3
0.49
42
>100
8.0
5.0
8
5y
Et
H
26.3
>50
>50
>50
<0.2
0.63
17.4
>50
>50
17.4
0.66
0.63
1.4
5z
4-Cl
ꢀ4.1
>111
1.70
2.3
5aa
5ab
5ac
5ad
5ae
5af
5ag
5ah
6a
4-CN
4-CN
4-F
Et
H
-
6
-
Et
H
8.0
2.5
7.3
17
>250
>79
>3
-
4-F
0.026
ꢀ111
1.2
4-Me
4-Me
4-OMe
4-OMe
=O
Et
H
Et
H
110
4.1
6.7
3.0
7.5
2.0
7.0
-
>3
H
0.232
0.204
0.73
0.057
1.9
6b
NH2
H
7
-
H
3
1.5
54
36
BTDBA
3.2
>50
aCompound concentration required to reduce rIN strand transfer (ST) and rRNase H activity (RH) by 50%. b Compound concentration required to achieve 50% protection of MT-4/KB
cells from the HIV-1 induced cytopathicity, as determined by MTT method. cCytotoxicity: compound dose required to reduce the viability of mock-infected cells by 50%. dSelectivity
index: CC50/EC50 ratio.

3340 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
R. Di Santo
Table 2.
Cytotoxicity and Antiviral Activities in Enzyme (IN - Related to ST and RNase H) and Cell-Based Assays of Quinolonyl Diketo Acids 8
and 9
O
O
OH
OH
O
O
O
OH
COOX
XOOC
COOX
R
N
N
8
9
F
F
Compd
R
X
IC50a (ꢀM) ST
inhib %b (10 ꢀM) RH
EC50c (ꢀM)
CC50d (ꢀM)
SIe
8a
8b
8c
8d
8e
8f
7-Pyf
7-Pyf
6-F
Et
H
2.3
0.028
0.58
0.030
1.3
55.6
70.7
21.5
61.6
23.9
6
4.1
0.17
>50
>50
>50
>50
ntg
>200
>200
>200
>200
>200
>200
ntg
>49
>1176
Et
H
-
-
-
-
6-F
7-F
Et
H
7-F
0.020
21
8g
8h
8i
6-Cl
6-Cl
7-Cl
7-Cl
Et
H
24.5
-10.3
-19.5
-10.1
11.8
80.4
0.028
14
46.1
ntg
>200
ntg
>4
Et
H
8j
0.019
0.34
0.012
>50
0.91
4.29
>200
191
-
9a
Et
210
9b
H
>200
>47
c
aCompound concentration required to reduce rIN strand transfer (ST) by 50%. bPercentage of inhibition of RNase H (RH) activity at 10 ꢀM. Compound concentration required to
reduce the exponential growth of MT-4/KB cells by 50%. dCytotoxicity: compound dose required to reduce the viability of mock-infected cells by 50%. eSelectivity index: CC50/EC50
ratio. fPy = pyrrolidin-1-yl. gnt = not tested.
derivatives were also endowed of antiretroviral activity against
HIV-1 infected cells (EC50 from 0.2 to 26.3 ꢀM), and good selectiv-
ity indexes (up to >250), with few exceptions (5f,p,r,z,aa,ab,af,ag).
However, an accurate analysis of the data led us to think that further
targets could be involved in the inhibition of the life cycle of HIV-1
in infected cells, since no direct correlation could be found between
the anti-IN data and the antiviral activities. In particular, a number
of compounds 5-7 were more potent in cell-based than in enzyme
assays against rIN (see compounds 5a,c,d,g-i,k,m-o,s,u,w,y,ac,ae
and 7). First we thought that RNase H function of the RT could be
inhibited besides IN, based on i) the above cited similarities of the
active sites of IN and RNase H [34-36], and ii) the reports on the
us to pursue the research in this field and actually, new compounds
as RNase H inhibitors are in development that will be publish soon.
In parallel to the development of pyrrolyl diketo acids, we de-
signed also quinolonyl diketo acids [56-62] characterized by one (8)
[56] or two (9) DKA chains [57], as IN inhibitors. The monofunc-
tional derivatives 8 were selective ST inhibitor, while the bifunc-
tional compounds 9 were active also against 3’-P. Thus IN inhibi-
tors 8 and 9 were useful tools to study the mechanism of action of
IN. In particular, we demonstrated through cross-linking experi-
ments between IN and its DNA substrate that derivatives 9 bind to
both DNA acceptor and donor sites [63].
Based on the results obtained in the enzyme assays against
RNase H with pyrrolyl diketo acids 5-7, we decided to test also our
quinolonyl diketo acids against the RNase H function of the RT. A
first group of quinolonyl DKAs were tested against rRNase H at 10
ꢀM. Compound 8 and 9 in general showed good activity against the
RNase H function with % that ranged from 6 to 80.4. In general,
both monofuntional and bifunctional derivatives were good RNase
H inhibitors (Table 2). Interestingly in a few cases (see 8h-j) we
found increased activity of RNase H activity. This issue will be
further studied and discussed in the future.
anti-RNase
H activity of 4-[5-(benzoylamino)thien-2-yl]-2,4-
dioxobutanoic acid (BTDBA), a DKA discovered by Merck [40].
Thus, we decided to test all pyrrolyl diketo acids synthesized in our
laboratories, against RNase H in enzyme assays. The results of this
screening are reported for selected derivatives 5-7 in Table 1, using
BTDBA as the reference compound. Very interestingly, the major-
ity of tested compounds were active in inhibiting the RNase H ac-
tivity of RT. The data of activity were within the range 2-64 ꢀM,
and only a few compounds (5a,k,m,x) were inactive in these as-
says. So we decided to study more deeply the properties of 5ac, one
of the most potent derivatives against RNase H. Compound 5ac
(RDS 1643) selectively inhibits the RNase H function of the HIV-1
RT, without affecting neither its DNA polymerase associated activ-
ity, nor the RNase H activity associated to the Avian Myeloblasto-
sis Virus (AMV) RT or the E. coli RNase H, while it barely af-
fected the HIV-1 IN activity [41]. We also performed a partial
characterization of the interaction between RDS 1643 and the HIV-
1 RT by time of addition, kinetic and drug association studies. Fi-
nally, we demonstrated that RDS 1643 inhibits in cell-based assays
the replication of wild type and HIV-1 strains resistant to RTIs
currently used in clinical practice [41]. This interesting results led
The data in our hand led us to conclude that both classes of
compounds pyrrolyl and quinolonyl DKAs, are dual inhibitors of
IN and RNase H function of the RT. Further studies will be devoted
to develop potent agents active against both enzymes (dual inhibi-
tors). These dual inhibitors could be excellent antiretroviral agents
useful to overcome the resistance of the virus.
ACKNOWLEDGEMENTS
Thanks are due to Yves Pommier, Christophe Marchand, Enzo
Tramontano, Stuart Le Grice for biological data. The research lead-

Diketo Acids Derivatives as Dual Inhibitors of Human Immunodeficiency Virus
Current Medicinal Chemistry, 2011 Vol. 18, No. 22 3341
[19]
[20]
[21]
Dejesus, E.; Berger, D.; Markowitz, M.; Cohen, C.; Hawkins, T.; Ruane, P.;
Elion, R.; Farthing, C.; Zhong, C.L.; Cheng, A.K.; McColl, D.; Kearney,
B.P. Antiviral activity, pharmacokinetics, and dose response of the HIV-1 in-
tegrase inhibitor GS-9137 (JTK-303) in treatment-naive and treatment-
experienced patients. J. Acquired Immune Defic. Syndr., 2006, 43, 1-5.
Grinsztejn, B.; Nguyen, B.Y.; Katlama, C.; Gatell, J.M.; Lazzarin, A.; Vitte-
coq, D.; Gonzalez, C.J.; Chen, J.; Harvey, C.M.; Isaacs, R.D. Safety and effi-
cacy of the HIV-1 integrase inhibitor raltegravir (MK-0518) in treatment-
experienced patients with multidrug-resistant virus: A phase II randomised
controlled trial. Lancet, 2007, 369, 1261-1269.
Markowitz, M.; Morales-Ramires, J.O.; Nguyen, B.Y.; Kovacs, C.M.; Steig-
bigel, R.T.; Cooper, D.A.; Liporace, R.; Schwartz, R.; Isaacs, R.; Gilde, L.R.;
Wenning, L.; Zhao, J.; Teppler, H. Antiretroviral activity, pharmacokinetics,
and tolerability of MK-0518, a novel inhibitor of HIV-1 integrase, dosed as
monotherapy for 10 days in treatment-naive HIV-1-infected individuals. J.
Acquired Immune Defic. Syndr., 2006, 43, 509-515.
Bacchi, A.; Biemmi, M.; Carcelli, M.; Carta, F.; Compari, C.; Fisicaro, E.;
Rogolino, D.; Sechi, M.; Sippel, M.; Sotriffer, C.A.; Sanchez, T.W.; Nea-
mati, N. From ligand to complexes. Part 2. Remarks on human immu-
nodeficiency virus type 1 integrase inhibition by ꢀ-diketo acid metal
complexes. J. Med. Chem., 2008, 51, 7253-7264.
Sechi, M.; Bacchi, A.; Carcelli, M.; Compari, C.; Duce, E.; Fisicaro, E.;
Rogolino, D.; Gates, P.; Deruda, M.; Al-Mawsawi, L.Q.; Neamati, N. From
ligand to complexes: inhibition of human immunodeficiency virus type 1 in-
tegrase by beta-diketo acid metal complexes. J. Med. Chem., 2006, 49, 4248-
4260.
ing to these results has received funding from the European Com-
munity’s Seventh Framework programme (FP7) under grant agree-
ment CHAARM no. 242135 and by Italian PRIN 2008 prot.
2008CE75SA.
ABBREVIATIONS
HIV-1
IN
PIC
=
=
=
=
=
=
=
=
=
=
Human immunodeficiency virus type 1
integrase
pre-integration complex
reverse transcriptase
RNA-dependent DNA polymerase
DNA-dependent DNA polymerase
ribonuclease H
RT
RDDP
DDDP
RNase H
DKA
RTI
[22]
[23]
[24]
diketo acid
RT inhibitors
BTDBA
4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic
acid
Telesnitsky, A.; Goff, S.P. in Retroviruses, Coffin, J.M.; Hughes, H; Var-
mus, H.E. Eds., Cold Spring Harbor Laboratory Press: Cold Spring Harbor,
NY, 1997, pp. 121-160.
AMV
=
Avian Myeloblastosis Virus
[25]
[26]
Hughes, S.H.; Arnold, E.; Hostomsky, Z. In Ribonucleases H, Crouch, R.J;
Toulmé, J.J. Eds. Les Editions Inserm: Paris, 1998; pp.195-224.
Nikolenko, G.N.; Palmer, S.; Maldarelli, M.; Mellors, J.W.; Coffin, J.M.;
Pathak, V.K. Mechanism for nucleoside analog-mediated abrogation of HIV-
1 replication: Balance between RNase H activity and nucleotide excision.
Proc. Natl. Acad. Sci. USA, 2005, 102, 2093-2098.
Haren, L.; Ton-Hoang, B.; Chandler, M. Integrating DNA: Transposases and
retroviral integrases. Annu. Rev. Microbiol., 1999, 53, 245-281.
Klumpp, K.; Hang, J.Q.; Rajendran, S.; Yang, Y.; Derosier, A.; Wong Kai,
P.; Overton, H.; Parkes, K.E.; Cammack, N.; Martin, J.A. Two-metal ion
mechanism of RNA cleavage by HIV RNase H and mechanism-based design
of selective HIV RNase H inhibitors. Nucleic Acids Res., 2003, 31, 6852-
6859.
Klumpp, K.; Mirzadegan, T. Recent progress in the design of small molecule
inhibitors of HIV RNase H. Curr. Pharm. Drug, 2006, 12, 1909-1922.
Keck, J.L.; Goedken, E.R.; Marqusee, S. Activation/attenuation model for
RNase H. A one-metal mechanism with second-metal inhibition. J. Biol.
Chem., 1998, 273, 34128-34133.
Katayanagi, K.; Okumura, M.; Morikawa, K. Crystal structure of Es-
cherichia coli RNase HI in complex with Mg2+ at 2.8 A resolution: proof for
a single Mg(2+)-binding site. Proteins, 1993, 17, 337-347.
Mizrahi, V.; Usdin, M.; Harington, A.; Dudding, L. Site-directed mutagene-
sis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of
HIV-1 reverse transcriptase. Nucleic Acids Res., 1990, 18, 5359-5363.
Mizrahi, V.; Brooksbank, R.; Nkabinde, N. Mutagenesis of the conserved
aspartic acid 443, glutamic acid 478, asparagine 494, and aspartic acid 498
residues in the ribonuclease H domain of p66/p51 human immunodeficiency
virus type I reverse transcriptase. Expression and biochemical analysis. J.
Biol. Chem., 1994, 269, 19245-19249.
Andréola, M.L. Closely related antiretroviral agents as inhibitors of two
HIV-1 enzymes, ribonuclease H and integrase: “Killing two birds with one
stone”. Curr. Pharm. Des., 2004, 10, 3713-3723.
Yang, W.; Hendrickson, W.A.; Crouch, R.J.; Satow, Y. Structure of ribonu-
clease H phased at 2 Å resolution by MAD analysis of the selenomethionyl
protein. Science, 1990, 249, 1398-1405.
Davies, J.F., II; Hostomska, Z.; Hostomsky, Z.; Jordan, S.R.; Matthews, D.A.
Crystal structure of the ribonuclease H domain of HIV-1 reverse tran-
scriptase. Science, 1991, 252, 88-95.
REFERENCES
[1]
[2]
[3]
Goldgur, Y.; Dyda, F.; Hickman, A.B.; Jenkins, T.M.; Craigie, R.; Davies,
D.R. Three new structures of the core domain of HIV-1 integrase: an active
site that binds magnesium. Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 9150-
9154.
[27]
[28]
Maignan, S.; Guilloteau, J.P.; Zhou-Liu, Q.; Clément-Mella, C.; Mikol, V.
Crystal structures of the catalytic domain of HIV-1 integrase free and com-
plexed with its metal cofactor: High level of similarity of the active site with
other viral integrases. J. Mol. Biol., 1998, 282, 359-368.
Bujacz, G.; Alexandratos, J.; Wlodawer, A.; Merkel, G.; Andrake, M.; Katz,
R.A.; Skalka, A.M. Binding of different divalent cations to the active site of
avian sarcoma virus integrase and their effects on enzymatic activity. J. Biol.
Chem., 1997, 272, 18161-18168.
Yang, W.; Steitz, T.A. Recombining the structures of HIV integrase, RuvC
and RNase H. Structure, 1995, 3, 131-134.
[29]
[30]
[4]
[5]
Beese, L.S.; Steitz, T.A. Structural basis for the 3’-5’exonuclease activity of
Escherichia coli DNA polymerase I: a two metal ion mechanism. EMBO J.,
1991, 10, 25-33.
[31]
[32]
[33]
[6]
Grobler, J.A.; Stillmock, K.; Hu B.; Witmer, M.; Felock, P.; Espeseth, A.S.;
Wolfe, A.; Egbertson, M.; Bourgeois, M.; Melamed, J.; Wai, J.S.; Young, S.;
Vacca. J.; Hazuda, D.J. Diketo acid inhibitor mechanism and HIV-1 inte-
grase: implications for metal binding in the active site of phosphotransferase
enzymes. Proc. Natl. Acad. Sci. USA, 2002, 99, 6661-6666.
Marchand, C.; Johnson, A.A.; Karki, R.G.; Pais, G.C.; Zhang, X.; Cowan-
sage, K.; Patel, T.A.; Nicklaus, M.C.; Burke, T.R., Jr.; Pommier, Y. Metal-
dependent inhibition of HIV-1 integrase by ꢀ-diketo acids and resistance
of the soluble double-mutant (F185K/C280S). Mol. Pharmacol., 2003, 64,
600-609.
[7]
[34]
[35]
[36]
[8]
[9]
Pommier, Y.; Johnson, A.A.; Marchand, C. Integrase inhibitors to treat
HIV/AIDS. Nat. Rev. Drug Discovery, 2005, 4, 236-248.
Johnson, A.A.; Marchand, C.; Pommier, Y. HIV-1 integrase inhibitors: A
decade of research and two drugs in clinical trial. Curr. Top. Med. Chem.,
2004, 4, 1059-1077.
[10]
[11]
[12]
[13]
Dayam, R.; Deng, J.; Neamati, N. HIV-1 integrase inhibitors: 2003-2004
update. Med. Res. Rev., 2006, 26, 271-309.
Makhija, M.T. Designing HIV integrase inhibitors: Shooting the last arrow.
Curr. Med. Chem., 2006, 13, 2429-2441.
[37]
[38]
[39]
Jochmans, D. Novel HIV-1 reverse transcriptase inhibitors. Virus Res., 2008,
134, 171-185.
De Clercq, E. Anti-HIV drugs: 25 compounds approved within 25 years after
the discovery of HIV. Int. J. Antimicrob. Agents, 2009, 33, 307-320.
Tramontano, E.; Di Santo, R. HIV-1 RT-associated RNase H function inhibi-
tors: recent advances in drug development. Curr. Med. Chem., 2010, 17,
2837-2853.
Lataillade, M.; Kozal, M.J. The hunt for HIV-1 integrase inhibitors. AIDS
Patient Care STDS, 2006, 20, 489-501.
Maurin, C.; Bailly, F.; Cotelle, P. Structure-activity relationships of HIV-1
integrase inhibitors: enzyme-ligand interactions. Curr. Med. Chem., 2003,
10, 1795-1810.
[14]
[15]
[16]
[17]
Cotelle, P. Patented HIV-1 integrase inhibitors (1998-2005). Recent Patents
Anti-Infect. Drug Discovery, 2006, 1, 1-15.
[40]
Shaw-Reid, C.A.; Munshi, V.; Graham, P.; Wolfe, A.; Witmer, M.; Dan-
zeisen, R.; Olsen, D.B.; Carroll, S.S.; Embrey, M.; Wai, J.S.; Miller, M.D.;
Cole, J.L.; Hazuda, D.J. Inhibition of HIV-1 ribonuclease H by a novel
diketo acid, 4-[5-(benzoylamino)thien-2-yl]-2,4-dioxobutanoic acid. J. Biol.
Chem., 2003, 278, 2777-2780.
Dayam, R.; Neamati, N. Small-molecule HIV-1 integrase inhibitors: the
2001-2002 update. Curr. Pharm. Des., 2003, 9, 1789-1802.
Neamati, N. Patented small molecules inhibitors of HIV-1 integrase: a 10-
year saga. Expert Opin. Ther. Patents, 2002, 12, 709-724.
Witvrouw, M.; Van Maele, B.; Vercammen, J.; Hantson, A.; Engelborghs,
Y.; De Clercq, E.; Pannecouque, C.; Debyser, Z. Novel inhibitors of HIV-1
integration. Curr. Drug. Metab., 2004, 5, 291-304.
[41]
Tramontano, E.; Esposito, F.; Badas, R.; Di Santo, R.; Costi, R.; La Colla, P.
6-[1-(4-Fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic
acid
ethyl ester a novel diketo acid derivative which selectively inhibits the HIV-1
viral replication in cell culture and the ribonuclease H activity in vitro. Anti-
viral Res., 2005, 65, 117-124.
[18]
Young, S.D. Inhibition of HIV-1 integrase by small molecules: the potential
for a new class of AIDS chemotherapeutics. Curr. Opin. Drug Discov. De-
vel., 2001, 4, 402-410.

3342 Current Medicinal Chemistry, 2011 Vol. 18, No. 22
R. Di Santo
[42]
[43]
de Soultrait, V.; Lozach, P.; Altmeyer, R.; Tarrago-Litvak, L.; Litvak, S.;
Andréola, M.L. DNA aptamers derivated from HIV-1 RNase H inhibitors are
strong anti-integrase agents. J. Mol. Biol., 2002, 324, 195-203.
[54]
[55]
Di Santo, R.; Costi, R.; Artico, M.; Ragno, R.; Greco, G.; Novellino, E.;
Marchand, C.; Pommier, Y. Design, synthesis and biological evaluation of
heteroaryl diketohexenoic and diketobutanoic acids as HIV-1 integrase in-
hibitors endowed with antiretroviral activity. Il Farmaco, 2005, 60, 409-417.
Costi, R.; Di Santo R.; Artico, M.; Roux, A.; Ragno, R.; Massa, S.; Tramon-
tano, E.; La Colla, M.; Loddo, R.; Marongiu, M.E.; Pani, A.; La Colla, P. 6-
Aryl-2,4-dioxo-5-hexenoic acids, novel integrase inhibitors active against
HIV-1 multiplication in cell-based assays. Bioorg. Med. Chem. Lett., 2004,
14, 1745-1749.
Di Santo, R.; Costi, R.; Roux, A.; Artico, M.; Lavecchia, A.; Marinelli, L.;
Novellino, E.; Palmisano, L.; Andreotti, M; Amici, R.; Galluzzo, C.M., Nen-
cioni, L.; Palamara, A.T.; Pommier, Y.; Marchand, C. Novel bifunctional
quinolonyl diketo acid derivatives as HIV-1 integrase inhibitors: design, syn-
thesis, biological activities and mechanism of action. J. Med. Chem., 2006,
49, 1939-1945.
Di Santo, R.; Costi R.; Roux, A.; Miele, G.; Cuzzucoli Crucitti, G.; Iacovo,
A.; Rosi, F.; Lavecchia, A.; Marinelli, L.; Di Giovanni, C.; Novellino, E.;
Palmisano, L.; Andreotti, M.; Amici, R.; Galluzzo, C.M.; Nencioni, L.;
Palamara, A.T.; Pommier, Y.; Marchand, C. Novel quinolinonyl diketo acid
derivatives as HIV-1 integrase inhibitors: design, synthesis and biological ac-
tivities. J. Med. Chem., 2008, 51, 4744-4750.
Bona, R.; Andreotti, M.; Buffa, V.; Leone, P.; Galluzzo, C.M.; Amici, R.;
Palmisano, L.; Mancini, M.G.; Michelini, Z.; Di Santo, R.; Costi, R.; Roux,
A.; Pommier, Y.; Marchand, C.; Vella, S.; Cara, A. Development of a human
immunodeficiency virus vector-based, single-cycle assay for evaluation of
anti-integrase compounds. Antimicrobial Ag. Chemother., 2006, 50, 3407-
3417.
Cianfriglia, M.; Dupuis, M.L.; Molinari, A.; Verdoliva, A.; Costi, R.; Gal-
luzzo, C.M.; Andreotti, M.; Cara, A.; Di Santo, R.; Palmisano, L. HIV-1 in-
tegrase inhibitors are substrates for the multidrug transporter MDR1-P-
glycoprotein. Retrovirology, 2007, 4, 17.
Terrazas-Aranda, K.; Van Herrewege, Y.; Hazuda, D.; Lewi, P.; Costi, R.; Di
Santo, R.; Cara, A.; Vanham, G. Human immunodeficiency virus type 1
(HIV-1) integration: a potential target for microbicides to prevent cell-free or
cell-associated HIV-1 infection. Antimicrob. Agents Chemother., 2008, 52,
2544-2554.
Jegede, O.; Babu, J.; Di Santo, R.; McColl, D. J.; Weber, J.; Quiñones-
Mateu, M.E. HIV type 1 integrase inhibitors: from basic research to clinical
implications. AIDS Rev., 2008, 10, 172-189.
Michelini, Z.; Galluzzo, C.M.; Negri, D.R.; Leone, P.R.; Amici, R.; Bona,
R.; Summa, V.; Di Santo, R.; Costi, R.; Pommier, Y.; Marchand, C.;
Palmisano, L.; Vella, S.; Cara, A. Evaluation of HIV-1 integrase inhibitors
on human primari macrophages using a luciferase-based single-cycle pheno-
typic assay. J. Virol. Methods, 2010, 168, 272-276.
Didierjean, J.; Isel, C.; Querré, F.; Mouscadet, J.F.; Aubertin, A.M.; Valnot,
J.Y.; Piettre, S.R.; Marquet, R. Inhibition of human immunodeficiency virus
type 1 reverse transcriptase, RNase H, and integrase activities by hydroxy-
tropolones. Antimicrob. Agents Chemother., 2005, 49, 4884-4894.
Budihas, S.; Gorshkova, I.; Gaidamakov, S.; Wamiru, A.; Bona, M.; Parniak,
M.; Crouch, R.; McMahon, J.; Beutler, J.; Le Grice, S. Selective inhibition of
HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxy-
lated tropolones. Nucleic Acids Res., 2005, 33, 1249-1256.
Semenova, E.A.; Johnson, A.A.; Marchand, C.; Davis, D.A.; Yarchoan, R.;
Pommier, Y. Preferential inhibition of the magnesiumdependent strand trans-
fer reaction of HIV-1 integrase by ꢀ-hydroxytropolones. Mol. Pharmacol.,
2006, 69, 1454-1460.
Marchand, C.; Beutler, J.A.; Wamiru, A.; Budihas, S.; Mollmann, U.;
Heinisch, L.; Mellors, J.W.; Le Grice, S.F.; Pommier, Y. Madurahydroxylac-
tone derivatives as dual inhibitors of human immunodeficiency virus type 1
integrase and RNase H. Antimicrob. Agents Chemother., 2008, 52, 361-364.
Tramontano, E. HIV-1 RNase H: recent progress in an exciting, yet little
explored, drug target Mini-Rev. Med. Chem., 2006, 6, 727-737.
Artico, M.; Di Santo, R.; Costi, R.; Novellino, E.; Greco, G.; Massa, S.;
Tramontano, E.; Marongiu, M.E.; De Montis, A.; La Colla, P. Geometrically
and conformationally restrained cinnamoyl-compounds as inhibitors of HIV-
1 integrase: synthesis, biological evaluation and molecular modeling. J. Med.
Chem., 1998, 41, 3948-3960.
Costi, R.; Di Santo R.; Artico, M.; Massa, S.; Ragno, R.; Loddo, R.; La
Colla, M.; Tramomtano, E.; La Colla, P.; Pani, A. 2,6-Bis(3,4,5-
trihydroxybenzylidene) derivatives of cyclohexanone: novel potent HIV-1
integrase inhibitors that prevent HIV-1 multiplication in cell-based assays.
Bioorg. Med. Chem., 2004, 12, 199-215.
Pluymers, W.; Neamati, N.; Pannecouque, C.; Fikkert, V.; Marchand, C.;
Burke, T.R., Jr.; Pommier, Y.; Schols, D.; De Clercq, E.; Debyser, Z.;
Witvrouw, M. Viral entry as the primary target for the anti-HIV activity of
chicoric acid and its tetra-acetyl esters. Mol. Pharmacol., 2000, 58, 641-648.
Hazuda, D.J.; Felock, P.; Witmer, M.V.; Wolfe, A.; Stillmock, K.; Grobler,
J.A.; Espeseth, A.; Gabryelski L.; Schleif, W.A.; Blau, C.; Miller, M.D. In-
hibitors of strand transfer that prevent integration and inhibit HIV-1 replica-
tion in cells. Science, 2000, 287, 646-650.
Hazuda, D.J.; Young, S.D.; Guare, J.P.; Anthony, N.J.; Gomez, R.P.; Wai,
J.S.; Vacca, J.P.; Handt, L.; Motzel, S.L.; Klein, H.J.; Dornadula, G.; Dano-
vich, R.M.; Witmer, M.V.; Wilson, K.A.; Tussey, L.; Schleif, W.A.;
Gabryelski, L.S.; Jin, L.; Miller, M.D.; Casimiro, D.R.; Emini, E.A.; Shiver,
J.W. Integrase inhibitors and cellular immunity suppress retroviral replica-
tion in rehesus macaques. Science, 2004, 305, 528-532.
Di Santo, R.; Costi, R.; Artico, M.; Tramontano, E.; La Colla, P.; Pani, A.
HIV-1 integrase inhibitors that block HIV-1 replication in infected cells.
Planning synthetic derivatives from natural products. Pure Appl. Chem.,
2003, 75, 195-206.
[44]
[45]
[56]
[57]
[58]
[46]
[47]
[48]
[49]
[59]
[60]
[50]
[51]
[52]
[61]
[62]
[63]
Johnson, A.A.; Marchand, C.; Patil, S.S.; Costi, R.; Di Santo, R.; Burke,
T.R., Jr.; Pommier, Y. Probing HIV-1 integrase inhibitor binding sites with
position-specific integrase-DNA cross-linking assays. Mol. Pharmacol.,
2007, 71(3), 893-901.
[53]
Received: February 26, 2011
Revised: May 29, 2011
Accepted: May 30, 2011