Design and synthesis of novel indole β-diketo acid derivatives as HIV-1 integrase inhibitors

Article abstract of DOI:10.1021/jm049944f

Diketo acids such as S-1360 (1A) and L-731,988 (2) are potent and selective inhibitors of HIV-1 integrase (IN). A plethora of diketo acid-containing compounds have been claimed in patent literature without disclosing much biological activities and synthetic details (reviewed in Neamati, N. Exp. Opin. Ther. Pat. 2002, 12, 709-724). To establish a coherent structure - activity relationship among the substituted indole nucleus bearing a β-diketo acid moiety, a series of substituted indole-β-diketo acids (4a-f and 5a-e) were synthesized. All compounds tested showed anti-IN activity at low micromolar concentrations with varied selectivity against the strand transfer process. Three compounds, the indole-3-β-diketo acids 5a and 5c, and the parent ester 9c, have shown an antiviral activity in cell-based assays. We further confirmed a keto-enolic structure in the 2,3-position of the diketo acid moiety of a representative compound (4c) using NMR and X-ray crystallographic analysis. Using this structure as a lead for all of our computational studies, we found that the title compounds extensively interact with the essential amino acids on the active site of IN.

Full text of DOI:10.1021/jm049944f

5298
J . Med. Chem. 2004, 47, 5298-5310
Design a n d Syn th esis of Novel In d ole â-Dik eto Acid Der iva tives a s HIV-1
In tegr a se In h ibitor s
Mario Sechi,*,† Massimiliano Derudas,† Roberto Dallocchio,‡ Alessandro Dess`ı,‡ Alessia Bacchi,§ Luciano Sannia,†
Fabrizio Carta,† Michele Palomba,† Omar Ragab,| Carney Chan,| Robert Shoemaker, Shizuko Sei,#
Raveendra Dayam,| and Nouri Neamati*,|
Dipartimento Farmaco Chimico Tossicologico, Universita` di Sassari, Via Muroni 23/ A, 07100 Sassari, Italy, CNR-Istituto di
Chimica Biomolecolare, sez. di Sassari, Trav. La Crucca 3, reg. Baldinca, 07040 Li Punti, Italy, Dipartimento di Chimica
Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Universita` di Parma, Parco Area delle Scienze 17/ A,
43100 Parma, Italy, Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California,
1985 Zonal Avenue, PSC 304, Los Angeles, California 90089, and Screening Technologies Branch, DTP, DCTD, and
Laboratory of Antiviral Drug Mechanisms, SAIC-Frederick, National Cancer Institute at Frederick, Frederick, Maryland 21702
Received J anuary 19, 2004
Diketo acids such as S-1360 (1A) and L-731,988 (2) are potent and selective inhibitors of HIV-1
integrase (IN). A plethora of diketo acid-containing compounds have been claimed in patent
literature without disclosing much biological activities and synthetic details (reviewed in
Neamati, N. Exp. Opin. Ther. Pat. 2002, 12, 709-724). To establish a coherent structure-
activity relationship among the substituted indole nucleus bearing a â-diketo acid moiety, a
series of substituted indole-â-diketo acids (4a -f and 5a -e) were synthesized. All compounds
tested showed anti-IN activity at low micromolar concentrations with varied selectivity against
the strand transfer process. Three compounds, the indole-3-â-diketo acids 5a and 5c, and the
parent ester 9c, have shown an antiviral activity in cell-based assays. We further confirmed a
keto-enolic structure in the 2,3-position of the diketo acid moiety of a representative compound
(4c) using NMR and X-ray crystallographic analysis. Using this structure as a lead for all of
our computational studies, we found that the title compounds extensively interact with the
essential amino acids on the active site of IN.
In tr od u ction
Despite significant advances in antiretroviral therapy,
the search for new anti-HIV-1 agents continues in the
attempt to develop drugs capable of overcoming toxicity
and resistance.1,2 Combination therapies for the treat-
ment of HIV infection are mainly based on drugs
targeting reverse transcriptase and protease. The rapid
emergence of resistant variants, often cross-resistant to
the members of a given class, limits the efficacy of
existing antiretroviral drugs.3 In this context, new
targets have been envisaged and suitable drugs are
under development.1,2 HIV-1 integrase (IN) is one of the
targets whose inhibition might be very efficacious in
preventing viral replication. In fact, integration of viral
DNA into the host cell genome is a critical step in the
HIV life cycle. This essential process is catalyzed by the
viral enzyme IN through two different reactions: 3′-
processing and strand transfer.4-6 Initially, the enzyme
recognizes the LTR termini of the viral dsDNA, of which
it removes the last two nucleotides (GT), thus leaving
two recessed 3′-OH ends (3′-processing reaction). Then,
IN catalyzes the joining of the processed 3′-ends to the
5′-ends of the host DNA (strand transfer reaction).7 The
integration reaction is completed by the removal of
F igu r e 1. Structures of selected diketo acid-containing HIV-1
IN inhibitors.
unpaired nucleotides and gap repair by cellular enzymes
that lead to provirus formation. Because no cellular
homologue has been found in humans, IN is being
considered as a promising target for the development
of selective anti-AIDS drugs.8-10 Recently, a number of
compounds bearing a diketo acid moiety were indepen-
dently discovered by scientists from Shionogi & Co. and
Merck (for recent reviews, see refs 9 and 11) as a new
class of IN inhibitors. One of these compounds, 5CITEP
(1B; Figure 1), was subsequently cocrystallized with the
enzyme providing the first X-ray crystal structure of an
inhibitor in complex with the HIV-1 IN.12 5CITEP
belongs to the diketo acid-containing compounds, be-
cause the tetrazole group is a well-known bioisostere of
a carboxylic acid.13 Selective strand transfer inhibitors
2 and 3 constitute major leads in anti-HIV-1 IN drug
* To whom correspondence should be addressed. (M.S.) Tel: +39
079 228753. Fax: +39 079 228720. E-mail: mario.sechi@uniss.it.
(N.N.) Tel: 323-442-2341. Fax: 323-442-1390. E-mail: neamati@usc.edu.
† Universita` di Sassari.
‡ CNR-Istituto di Chimica Biomolecolare.
§ Universita` di Parma.
| University of Southern California.
Screening Technologies Branch, DTP, DCTD.
Laboratory of Antiviral Drug Mechanisms, SAIC-Frederick.
#
10.1021/jm049944f CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/16/2004

Indole-â-diketo Acid Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5299
Resu lts a n d Discu ssion
Ch em istr y. Synthetic approaches for the preparation
of target compounds 4a -f and 5a -e and their inter-
mediates are depicted in Schemes 1-5. Indol 2-(3-)-â-
diketo acids 4a -f and 5a -e were obtained by alkaline
hydrolysis from their respective â-diketo ester 7a -f and
9a -e intermediates. These intermediates were pre-
pared by Claisen condensation18,19 of 2-acetylindoles
6a -f and 3-acetylindoles 8a -e with diethyl oxalate and
sodium methoxide dissolved in CH3OH or THF (Scheme
1). Under these conditions, only methyl esters were
obtained by a trans-esterification reaction between the
methoxide and the ethyl esters. The parent compounds
6a -f and 8a -e were synthesized following different
synthetic routes. Alkylation of esters 10 and 11 with
appropriate alkyl halides in powdered KOH/DMSO20
gave the corresponding alkyl indoles 12-15. Esters 17
and 18 were synthesized from commercially available
compounds 16 under identical conditions. Acids 19a -f
were in turn obtained in good yield upon alkaline
hydrolysis. Their conversion into the desired acetyl
derivatives 6a -f (Scheme 2) was performed using
methyl lithium in diethyl ether.21 The corresponding
methyl ester 10 and the methyl dioxolindole-2-carboxy-
late 11 were prepared by cyclization of azidoesters 23
and 24 in refluxing xylene, according to the Hemets-
berger reaction.22,23 The latter were easily obtained by
Claisen condensation of aldehydes 20 and 21 with
methyl azidoacetate 22 (Scheme 3). The 3-acetyl-indoles
8a ,c,e were prepared by alkylation of available 3-acetyl-
indole 25 with the appropriate alkyl halides (Scheme
4). The intermediates 8b,d were instead obtained using
the synthetic approach of Scheme 5. Thus, the esters
13 and 14 were first treated with trifluoroacetic anhy-
dride, acetic acid, and 85% phosphoric acid to give the
3-acetyl- derivatives 26 and 27 with 80% yields.24 The
latter compounds were converted into the acids 28 and
29 by alkaline hydrolysis with powdered KOH under
refluxing ethanol. Finally, decarboxylation gave the
expected 3-acetyl-dioxole-indoles 8b,d (Scheme 5).
F igu r e 2. Design of target compounds 4a -f and 5a -e.
discovery, and in cell-based assays, they inhibit integra-
tion without affecting earlier phases of the HIV-1
replication cycle.14-16
Although this class of compounds afforded promising
leads for the development of IN inhibitors, only limited
structure-activity relationships (SAR) and detailed
synthetic studies of these inhibitors have been re-
ported.9,16,17 In this context, on the basis of the assump-
tion that the â-diketo acid moiety is necessary for the
anti-IN activity, our aim was to examine several struc-
tural and biological features of these compounds. By
combining important features of lead compounds 1B and
2, we designed a series of substituted indoles of general
structure I (Figure 2). Therefore, we made three major
modifications of I in order to better understand the role
of different substituents on activity. First, positions 2
and 3 of the diketo moiety in the indole were initially
explored to determine optimum geometry. Second, a
dioxole ring was introduced to increase the extension
of the molecular backbone. Third, N-alkylation of the
indole was introduced to modulate lipophilicity. These
structural modifications led to compounds 4a -f and
5a -e (Figure 2). Also, the inhibitory activity of the
corresponding â-diketo ester intermediates 7a -d ,f and
9b,c was evaluated in order to establish a coherent SAR
among these compounds.
Cr ysta l a n d Solu tion Str u ctu r es of Com p ou n d
4c. To determine whether the â-diketo acid moiety is
present as â-diketo or its keto-enolic tautomer in solu-
tion and in solid state, a series of NMR and X-ray
crystallographic studies were carried out. The analysis
of 13C NMR and distortionless enhancement by polar-
ization transfer (DEPT) for compound 4c showed that
the CH2 signal of the diketo moiety corresponded to CH,
an indication of the enolic form. Moreover, the 13C NMR
spectrum (chloroform-d) showed a signal at 101.9 ppm
(see Experimental Section) due to the CH enolic carbon
whereas the resonance of the corresponding CH2 carbon
of the diketo form was predicted to reside at about 56-
60 ppm upfield.25,26 The DEPT experiment supports this
observation, and only C-H signals were observed in the
aromatic region. The results of X-ray crystallographic
data of compound 4c confirm that the enolic bond is
situated in the 2,3-positions with respect to the car-
boxylic group of the â-diketo acid moiety (Figure 3,
Tables 1 and 2, and Supporting Information). These
observations are consistent with those of Bertolasi et
al. who suggested that the enolic proton preferably
resides on the carbon atom adjacent to the most elec-
tronegative group.27 The system is characterized by the
To study the nature of the drug-binding sites, we
extended our SAR studies to solve the crystal and
solution structures of a representative compound. We
also observed that selectivity against the strand transfer
process is important, but not essential, for antiviral
activity.

5300 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Sechi et al.
Sch em e 1a
a
Reagents and conditions: (i) Diethyloxalate, CH3ONa, CH3OH, reflux 4 h (THF, reflux 28 h for 9e). (ii) 2 N NaOH, CH3OH, room
temperature for 5 h, 1 N HCl.
Sch em e 2a
a
Reagents and conditions: (i) CH3I, CH3CH2Br or BnBr, KOH pellets, DMSO, room temperature for 1 h. (ii) 12% KOH, reflux for 1 h,
1 N HCl. (iii) 1.4 M CH3Li in Et2O, diethyl ether, reflux for 7 h.
Sch em e 3a
H‚‚‚O4′ ) 113(2)°]. The most relevant assembling mode
of 4c in the solid state is however represented by the
typical carboxylic acid dimeric association. This is based
on very strong H-bonds between -COOH groups that
face each other in the crystal packing, belonging to pairs
of molecules related by centers of inversion (-x + 1, -y
+ 1, -z). The C1dO1 [1.253(2) Å] and C1-O2-H [1.254-
(2) Å] bonds in the carboxylate group are equivalent due
to the remarkable strength of the H-bond [O2-H‚‚‚O1
(-x + 1, -y + 1, -z) O-H ) 1.31(5), O‚‚‚O ) 2.617(2),
H‚‚‚O ) 1.312(5), O-H‚‚‚O ) 172(4)°], with the proton
located exactly in the midpoint between the donor and
the acceptor atoms (Table 2 and Supporting Informa-
tion).
a
Reagents and conditions: (i) CH3ONa, CH3OH, -10 °C, 4 h.
(ii) Xylene, reflux for 15 min.
Sch em e 4a
Biology. Compounds 4a -f and 5a -e inhibited puri-
fied IN at a low micromolar concentration range (Table
3). All compounds were more potent in inhibiting IN-
catalyzed strand transfer processes than the 3′-process-
ing reactions, as previously reported for other deriva-
tives of this class.9,15-17 In general, the diketo acid
moiety constitutes a prominent structural feature for
the selectivity for strand transfer and that the potency
can be influenced by the aromatic portion. For example,
compounds 4a -f and 5a -e showed 5-50-fold selectivity
toward strand transfer in comparison to 3′-processing
reaction (for a representative gel, see Figure 4). Inter-
estingly, the dioxole ring as well as the position of the
diketo acid function did not lead to a significant varia-
tion of anti-IN activity. Moreover, the presence of the
alkyl substituents on the indolic nitrogen seems to not
significantly influence the potency. Nevertheless, it is
interesting to note that the best activity was displayed
a
Reagents and conditions: (i) KOH pellets, CH3I, CH3CH2Br
or BnBr, DMSO, room temperature for 1 h.
resonance-assisted intramolecular H-bond (RAHB) be-
tween the enolic -OH donor group (O3) and the ketonic
acceptor (O4). The potential delocalization of double
bond character on the keto-enolic skeleton gives par-
ticular strength to the interaction [O3‚‚‚O4 ) 2.610(2)
Å, O3-H‚‚‚O4 ) 147(3)°], as predicted by the RAHB
paradigm. The same groups are also involved in an
intermolecular H-bond involving pairs of molecules
related by a center of symmetry in the crystal packing.
Thus, the O3-H donor interacts with a O4 acceptor of
a molecule located at the (-x + 2, -y, -z) position,
forming a dimeric aggregate [H-bond geometry: O3-
H‚‚‚O4′ (-x + 2, -y, -z), O3‚‚‚O4′ ) 2.943(2) Å, O3-

Indole-â-diketo Acid Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5301
Sch em e 5a
a
Reagents and conditions: (i) (CF3CO)2O, glacial CH3COOH, 85% H3PO4, acetonitrile, room temperature for 14 h. (ii) KOH pellets,
EtOH, reflux for 2-5 h, 6 N HCl. (iii) CuCrO2, quinoline, 200 °C, 50 min.
Ta ble 2. Bond Lengths (Å) and Angles (°) for 4c
length
(Å)
bond
type
angles
(°)
bond
bond
N-C(8)
1.368(3)a
1.401(2)
1.465(2)
1.253(2) C-OH O(3)-C(2)-C(1)
1.254(2) C-OH C(3)-C(2)-C(1)
1.325(2) C-OH C(2)-C(3)-C(4)
1.241(2) CdO
1.502(3)
1.330(3) CdC
1.461(3) C-C
1.450(3)
1.373(3)
1.404(3)
1.403(3)
1.410(3)
1.408(3)
1.360(4)
1.401(4)
1.368(4)
O(1)-C(1)-C(2)
O(2)-C(1)-C(2)
O(3)-C(2)-C(3)
117.47(18)
117.87(18)
126.12(19)
112.94(17)
120.91(18)
121.85(19)
123.50(17)
119.31(18)
117.15(17)
108.32(17)
127.30(17)
124.36(17)
108.74(19)
134.2(2)
N-C(5)
N-C(13)
O(1)-C(1)
O(2)-C(1)
O(3)-C(2)
O(4)-C(4)
C(1)-C(2)
C(2)-C(3)
C(3)-C(4)
C(4)-C(5)
C(5)-C(6)
C(6)-C(7)
C(7)-C(12)
C(7)-C(8)
C(8)-C(9)
C(9)-C(10)
C(10)-C(11)
C(11)-C(12)
C(13)-C(14)
C(8)-N-C(5)
O(4)-C(4)-C(5)
O(4)-C(4)-C(3)
C(5)-C(4)-C(3)
C(6)-C(5)-N
C(6)-C(5)-C(4)
N-C(5)-C(4)
C(5)-C(6)-C(7)
C(12)-C(7)-C(6)
C(12)-C(7)-C(8)
C(6)-C(7)-C(8)
N-C(8)-C(9)
F igu r e 3. ORTEP diagram of 4c showing the atomic notation.
Ta ble 1. Crystal Data and Structural Refinement for 4c
119.7(2)
106.06(18)
129.6(2)
109.16(17)
121.2(2)
empirical formula
formula weight
temperature
C14H13NO4
259.25
293(2)K
N-C(8)-C(7)
C(9)-C(8)-C(7)
1.502(3)
107.72(15)
C(10)-C(9)-C(8)) 116.9(2)
C(9)-C(10)-C(11) 122.9(2)
C(12)-C(11)-C(10) 120.6(3)
C(11)-C(12)-C(7) 118.7(3)
wavelength
0.71069 Å
crystal system
space group
monoclinic
P21/n
C(8)-N-C(13) 123.73(16)
C(5)-N-C(13) 128.47(16)
O(1)-C(1)-O(2) 124.66(19)
unit cell dimensions (Å, °)
a ) 4.1535(6)
b ) 12.003(2)
c ) 24.963(4)
â ) 90.543(3)
1244.5(3) Å3
4
N-C(13)-C(14)
113.12(18)
a
In parentheses are the su values. Parameters relevant to the
discussion are shown in boldface.
volume
Z
calculated density
absorption coefficient
F(000)
Ta ble 3. Inhibition of HIV-1 IN Catalytic Activities and
Antiviral Activities of Indole â-diketo Derivatives
1.384 Mg/m3
0.102 mm-1
544
3′-processing strand transfer
b
c
compd IC50 (µΜ)
IC50 (µΜ)
SIa
CC50
81
>200
110
57
ND
3.8
>200
108
EC50
TId
crystal size
θ range for data collection
index ranges
0.5 × 0.4 × 0.3 mm3
1.63-23.27°
-4 e h e 4, -12 e k e 13,
-27 e l e 27
5360
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
7a
7b
7c
7d
7f
50 ( 15
11 ( 4
6 ( 2
2 ( 1
1.6 ( 1
5 ( 1
8
5
>81
>200
>110
>57
ND
>3.8
16 ( 5
10
11
20
17
50
33
31
15
12
2
reflections collected
55 ( 6
independent reflections
completeness to θ ) 23.27°
absorption correction
1793 [R(int) ) 0.0340]
20 ( 3
1 ( 0.5
2 ( 1
35 ( 5
99.8%
100 ( 10
100 ( 10
93 ( 23
45 ( 5
2 ( 1
37 ( 5 >5.4
symmetry-releted measurements
3 ( 2
>108
max - min transmission factor 1.00000-0.46069
3 ( 1
108 ( 24
>200
ND
9 ( 1 12
123 ( 9 >1.6
ND
refinement method
full-matrix least-squares on F2
3 ( 2
data/restraints/parameters
goodness-of-fit on F2
final R indices [I > 2σ(I)]
R indices (all data)
extinction coefficient
largest ∆F max/min
1793/0/225
1.017
12 ( 8
1 ( 0.5
45 ( 10
50 ( 20
ND
100 ( 10
100 ( 10
ND
106
>200
60
75
1.1
31
>106
R1 ) 0.0398, wR2 ) 0.1019
R1 ) 0.0568, wR2 ) 0.1124
0.005(2)
2
>200
ND
1.3
7
>1.6
>3.8 >200
>60
85 ( 20
100 ( 20
>100
65 ( 7
14 ( 8
60 ( 30
26 ( 5
>75
0.157/-0.164 e Å-3
>1.1
9b
9c
>31
by the N-benzyl derivatives 4e and 5e. With identical
IC50 values (1 ( 0.5 µM), these compounds were the
most potent inhibitors of the strand transfer reaction.
This may be rationalized by correlating inhibitory
potency with the size of the substituent on position 1 of
the ring; the larger the substituent, the higher the
activity issno matter whether the diketo acid is present
at position 2 or 3 of the indole. These results confirm
that the â-diketo acid group reasonably influences the
selectivity for one of the two catalytic steps and suggest
that the indole nucleus allows a consistent and selective
inhibition to be maintained. Considerable variation in
the inhibition of the 3′-processing reaction (IC50 values
>100
19 ( 4 >10.5
a
b
SI, selectivity index. CC50, cytotoxic concentration 50%.
c EC50, effective concentration 50%. TI, therapeutic index ) CC50
EC50; ND, not determined.
/
d
range from 11 to 100 µM) played a significant role in
the selectivity index (SI) results. This implies that most
of these compounds are loosely bound to the preas-
sembled IN-DNA complex, whereas they are tightly
bound to postassembled IN-DNA complexes.
Another interesting feature that could potentially play
a major role in the inhibition of IN is the ability to
chelate a divalent metal ion on the active site of IN and

5302 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Sechi et al.
F igu r e 4. Representative gel showing inhibition of purified IN by selected compounds. (A) A 21-mer blunt end oligonucleotide
corresponding to the U5 end of the HIV-1 LTR, 5′-end-labeled with 32P, is reacted with purified IN. The initial step involves
nucleolytic cleavage of two bases from the 3′-end, resulting in a 19-mer oligonucleotide. Subsequently, 3′-ends are covalently
joined at several sites to another identical oligonucleotide that serves as the target DNA. This reaction is referred to as strand
transfer, and the products formed migrate slower than the original substrate (shown in the figure as STP for strand transfer
products). (B) Lane 1, DNA alone; lanes 2, 15, and 25, IN and DNA with no drug; and lanes 3-24, IN, DNA, and selected drug
concentrations (100, 33.3, 11.1, and 3.7 µM) indicated above each lane.
possibly interfere with the target DNA. Although all
crystal structures of IN reveal a single binding site for
Mg2+, several studies suggest a plausible model in which
the IN active site binds two metal ions.28-30 This
phenomenon could perhaps explain the variability in
potency between the inhibitors. For example, we ob-
served that the indole-â-diketoesters (7a ,b,d ,f and 9b,c)
exhibit much reduced inhibitory potencies than their
respective acids and only compound 7f showed an
interesting selectivity toward the strand transfer pro-
cess (SI ) 7). These results could be explained consider-
ing the ability of both carboxylic and diketo function-
alities to potentially chelate two divalent ions where the
diketo esters bind preferably to only one. This clearly
indicates that the free carboxylic function is necessary
for a strong interaction with the catalytic residues on
the IN active site, particularly a metal ion.
be ineffective in preventing the HIV-1 cytopathic effect
in cell-based assays. A possible explanation for these
results could come from SAR studies by Wai et al.16 on
compounds exemplified by 3. The authors highlighted
the importance of relative orientations between the aryl
substituents and the diketo acid moiety and have also
stated that the intrinsic potency against IN and the
antiviral activity increases as the angle of bisection
increases from 60 to 118°. This conclusion supports our
suggestion that the indole moiety fulfills the require-
ments for 1,3-aryl disubstitution for the most active
compounds 5a ,c and 9c. The antiviral potency of 9c
could be attributed to the favorable structural combina-
tion of the aromatic framework and its â-diketo ester
moiety. However, we cannot rule out the possibility that
9c could potentially hydrolyze to a free acid upon
cellular uptake.
As far as the antiviral activity is concerned, only
indole-3-â-derivatives inhibited the HIV-1 cytopathic
effect in cell-based assays at non cytotoxic concentra-
tions. EC50 values were 37 ( 5, 9 ( 1, and 19 ( 4 µM
for 5a , 5c, and 9c, respectively. From the data presented
in Table 3, an additional observation could be made
concerning the correlation of the compound’s cytotoxicity
with their molecular size. For example, the acid 4f and
its respective ester 7f, which contain a bulky benzyl
group and a dioxole ring, were the most cytotoxic (CC50
) 3.8 and 1.1 µM) of the tested compounds. Unfortu-
nately, compounds 4a -f and 5b,d , although endowed
with selective activity against IN (Figure 4), proved to
Dock in g Stu d ies. Several computational docking
studies using the crystal structures of the IN core
domain and known inhibitors have recently been
reported.31-38 In most studies, the bound conformation
of 5CITEP in the IN-5CITEP cocrystal structure could
not be reproduced perhaps due to the crystal packing
effects. To identify the bioactive conformations of indole
â-diketo acid derivatives 4a -9c, we docked all of the
title compounds onto the IN active site using GOLD.
The core domain of IN (PDB 1BIS), wherein all of the
amino acid residues in the active site region were
resolved, was considered for docking purposes. The best
bound conformations (the bound conformation with the

Indole-â-diketo Acid Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5303
are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS) used as an internal standard. Split-
ting patterns are designated as follow: s ) singlet, d ) doublet,
t ) triplet, q ) quadruplet, m ) multiplet, bs ) broad singlet,
and dd ) double doublet. The assignment of exchangeable
protons (OH and NH) was confirmed by the addition of D2O.
Electron ionization mass spectra (70 eV) were recorded on a
Hewlett-Packard 5989 Mass Engine Spectrometer. Analytical
thin-layer chromatography (TLC) was done on Merck silica
gel F-254 plates. Pure compounds showed a single spot on TLC.
For flash chromatography, Merck Silica gel 60 was used with
a particle size 0.040-0.063 mm (230-400 mesh ASTM).
Elemental analyses were performed on a Perkin-Elmer 2400
instrument at Laboratorio di Microanalisi, Dipartimento di
Chimica, Universita` di Sassari, Italy, and the results were
within (0.4% of the theoretical values.
Gen er a l P r oced u r e for th e P r ep a r a tion of In d ole
â-d ik eto Acid s (4a -f a n d 5a -e). A solution of an appropriate
indole â-diketo ester (7a -f and 9a -e) (1 mmol) and 2 N NaOH
(4 equiv) in methanol (15 mL) was stirred at room temperature
for 5 h. After dilution with water, the mixture was acidified
with 1 N HCl. The yellow or orange precipitate that formed
was filtered off, washed with water, and recrystallized from
water/ethanol.
highest fitness score) of some of the compounds are
shown in Figure 5. The observed H-bonding and metal-
compound interactions are given in Tables 4 and 5. A
trend was observed between 3′-processing activity and
GOLD fitness scores, where potent compounds scored
favorable binding energies while some of the inactive
compounds scored low. The predicted bound conforma-
tion of the compounds was highly sensitive to the
substituents on the indole core unit. The presence of the
H-bond donor or acceptor and steric (hydrophobic)
groups significantly influenced the binding preferences
of the compounds.
Compounds with free acid adopted different bound
conformations than the esters (Figure 5). The carboxy-
late functionality of â-diketo acids formed strong H-
bonding interactions with N155 and K159 (Table 4). The
dioxole-bearing compounds adopted similar bound con-
formations, and the dioxole group formed strong H-
bonding interactions with N155 and K159. Compounds
with a benzyl substituent on indolic nitrogen adopted a
very different bound conformation than compounds with
methyl or ethyl substituents. Recurring and strong
interactions were observed between several compounds
and active site amino acid residues, such as K159, N155,
E152, I151, D116, E92, C65, H67, D64, and Q62. Some
of these amino acids are very important for the catalytic
activity of IN.39 The predicted bound conformations, and
especially their predominant interactions with active
2-Hyd r oxy-4-(1-m eth yl-1H-in d ol-2-yl)-4-oxobu t-2-en o-
ic Acid (4a ). Yield: 62%; mp 186-188 °C. IR (Nujol): v cm-1
1
1610 (CdO), 1710 (CdO acid). H NMR (CDCl3): δ 7.69 (1H,
d, Ar-H), 7.47-7.43 (3H, m, Ar-H), 7.20-7.12 (1H, m, Ar-
H), 7.07 (1H, s, HCdC), 5.10-3.90 (1H, bs, COOH), 4.12 (3H,
s, CH3). MS: m/z 245 (M+, 4), 172 (base). Anal. (C13H11NO4)
C, H, N.
2-H yd r oxy-4-(5-m et h yl-5H -[1,3]d ioxolo[4,5-f]in d ol-6-
yl)-4-oxobu t-2-en oic Acid (4b). Yield: 55%; mp 227-228 °C.
IR (Nujol): v cm-1 1660 (CdO), 1700 (CdO acid). 1H NMR
(CDCl3 + DMSO-d6): δ 7.35 (1H, s, Ar-H), 7.00 (1H, s,
HCdC), 6.97 (1H, s, Ar-H), 6.91 (1H, s, Ar-H), 6.03 (2H, s,
OCH2O), 4.06 (3H, s, CH3), 4.60-3.00 (1H, bs, COOH). MS:
m/z 289 (M+, 3), 216 (base). Anal. (C14H11NO6‚0.15H2O) C, H,
N.
4-(1-E t h yl-1H -in d ol-2-yl)-2-h yd r oxy-4-oxob u t -2-en oic
Acid (4c). Yield: 72%; mp 170-172 °C. IR (Nujol): v cm-1 1630
(CdO), 1690 (CdO acid). 1H NMR (CDCl3): δ 7.71 (1H, d, Ar-
H), 7.49 (1H, s, Ar-H), 7.45-7.32 (2H, m, Ar-H), 7.18 (1H,
d, Ar-H), 7.14 (1H, s, CHdC), 4.67 (2H, q, J 6.4 Hz, CH2),
3.80-3.10 (1H, bs, COOH), 1.42 (3H, t, J 6.4 Hz, CH3). 13C
NMR (CDCl3): δ 187.1 (CO), 164.1 (CdCOH), 162.2 (COOH),
140.0 (C2), 133.4 (C7a), 126.5 (C5), 126.1 (C4a), 123.1 (C4), 120.9
(C6), 112.7 (C3), 110.3 (C7), 101.9 (C-H), 40.2 (CH2), 15.4 (CH2).
MS: m/z 259 (M+, 8), 171 (base). Anal. (C14H13NO4) C, H, N.
4-(5-Eth yl-5H-[1,3]d ioxolo[4,5-f]in d ol-6-yl)-2-h yd r oxy-
4-oxobu t-2-en oic Acid (4d ). Yield: 38%; mp 210-211 °C. IR
(Nujol): v cm-1 1620 (CdO), 1700 (CdO acid). 1H NMR (CDCl3
+ DMSO-d6): δ 7.46 (1H, s, Ar-H), 7.15-7.04 (2H, m, Ar-H
+ CHdC), 6.96 (1H, s, ArH) 6.04 (2H, s, OCH2O), 5.20-4.30
(1H, bs, COOH), 4.57 (2H, q, J 6.6 Hz, CH2), 1.31 (3H, t, J 6.6
Hz, CH3). MS: m/z 303 (M+, 5), 188 (base). Anal. (C15H13NO6)
C, H, N.
site amino acid residues K159, N155, D64, and Mg2+
,
may explain the inhibitory profiles of the title com-
pounds.
Con clu sion . We have demonstrated that the diketo
acid functionality is important for selectivity against
strand transfer and that the aromatic ring plays a major
role in potency. Our results clearly demonstrate that
selectivity for strand transfer is not sufficient for
antiviral activity. Moreover, the high throughput assays
designed to selectively identify inhibitors of the strand
transfer step of the catalysis fail to discover potential
inhibitors of the 3′-processing step. Although diketo
acid-containing compounds represent a novel class of
compounds and are considered as a step forward in drug
design in targeting IN, many analogues showed signifi-
cant cytotoxicity and lack of antiviral activity. A pre-
requisite for all strand transfer inhibitors must be an
efficient nuclear uptake. It is, therefore, important to
bear in mind that the physicochemical properties of a
compound could potentially make a major contribution
to antiviral activity in vivo (for a recent review, see ref
40).
4-(1-Ben zyl-1H -in d ol-2-yl)-2-h yd r oxy-4-oxob u t -2-en o-
ic Acid (4e). Yield: 68%; mp 158-160 °C. IR (Nujol): v cm-1
Exp er im en ta l Section
1
1610 (CdO), 1730 (CdO acido). H NMR (CDCl3): δ 7.74 (1H,
d, Ar-H), 7.51 (1H, s, Ar-H), 7.39-7.33 (3H, m, Ar-H), 7.24-
7.18 (5H, m, Ar-H), 7.11 (1H, s, CHdC), 5.90 (2H, s, CH2),
5.40-3.80 (1H, bs, COOH). MS: m/z 321 (M+, 8), 90 (base).
Anal. (C19H15NO4) C, H, N.
4-(5-Ben zyl-5H-[1,3]d ioxolo[4,5-f]in d ol-6-yl)-2-h yd r oxy-
4-oxobu t-2-en oic Acid (4f). Yield: 39%; mp 178-182 °C. IR
(Nujol): v cm-1 1620 (CdO), 1710 (CdO acid). 1H NMR (CDCl3
+ DMSO-d6): δ 7.44 (1H, s, Ar-H), 7.25-7.21 (5H, m, Ar-
H), 7.04-6.98 (2H, m, Ar-H + CHdC), 6.80 (1H, s, Ar-H),
5.98 (2H, s, OCH2O), 5.83 (2H, s, CH2). MS: m/z 365 (M+, 6),
90 (base). Anal. (C20H15NO6‚0.27H2O) C, H, N.
Ch em istr y. Anhydrous solvents and all reagents were
purchased from Aldrich, Merck, or Carlo Erba. Anhydrous
diethyl ether was obtained by distillation from Na/benzophe-
none under nitrogen. All reactions involving air or moisture
sensitive compounds were performed under a nitrogen atmo-
sphere using oven-dried glassware and syringes to transfer
solutions. Melting points (mp) were determined using an
Electrothermal or a Ko¨fler apparatus and are uncorrected.
Infrared (IR) spectra were recorded as thin films or Nujol mulls
on NaCl plates with a Perkin-Elmer 781 IR spectrophotometer
and are expressed in v (cm-1). Nuclear magnetic resonance
(1H NMR, 13C NMR, and DEPT) spectra were determined in
CDCl3, DMSO-d6, or CDCl3/DMSO-d6 (1:3 ratio) on a Varian
XL-200 (200 MHz) as described.41,42 Chemical shifts (δ scale)
2-Hyd r oxy-4-(1-m eth yl-1H-in d ol-3-yl)-4-oxobu t-2-en o-
ic Acid (5a ). Yield: 26%; mp 177-178 °C. IR (Nujol): v cm-1
1
1660 (CdO), 1730 (CdO acid). H NMR (CDCl3 + DMSO-d6):

5304 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Sechi et al.
F igu r e 5. Predicted bound conformation of compounds 4a ,b,e (violet), 5a ,b,e (yellow), 7a ,b,f (cyan), and 9b,c (red) inside the
HIV-1 IN active site. The gray ribbon model shows the active site region while the ball-and-stick models represent prominent
amino acid residues. The Mg2+ ion is represented by the magenta sphere.

Indole-â-diketo Acid Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5305
Ta ble 4. GOLD Fitness Scores, H-Bonding Interactions, and the HIV-1 IN Active Site Amino Acid Residues Interacting with the
Indole â-Diketo Acid Derivatives 4a -9ca
compd
GOLD score
H-bonding interactions (Å)
interacting amino acid residues
K159, N155, I151, V77, V75, D64, Q62
4a
4b
36.31
46.43
-C-Od′‚ ‚ ‚HN K159 (2.38)
-OHb′‚ ‚ ‚OdC E92 (1.58)
-CdOa′‚ ‚ ‚HN H67 (3.25)
-CH2-Of′‚ ‚ ‚HN N155 (2.66)
-CH2-Oe′‚ ‚ ‚HN K159 (2.60)
-C-Od′‚ ‚ ‚HN K159 (2.30)
-OHb′‚ ‚ ‚OdC E92 (1.46)
-CdOa′‚ ‚ ‚HN H67 (2.94)
-CH2-Of′‚ ‚ ‚HN N155 (3.56)
-CH2-Oe′‚ ‚ ‚HN K159 (2.53)
-OHb′‚ ‚ ‚OdC D64 (2.61)
-OHb′‚ ‚ ‚OdC C65 (3.49)
-OHb′‚ ‚ ‚OdC D64 (3.02)
-OHb′‚ ‚ ‚OdC C65 (3.83)
-C-Od′‚ ‚ ‚HN N155 (2.18)
-C-Oc′‚ ‚ ‚HN K159 (1.69)
-OHb′‚ ‚ ‚OdC D116 (2.37)
-CH2-Of′‚ ‚ ‚HN N155 (4.05)
-CH2-Oe′‚ ‚ ‚HN K159 (2.38)
-OHb′‚ ‚ ‚OdC D64 (3.02)
-C-Od′‚ ‚ ‚HN N155 (2.01)
-C-Oc′‚ ‚ ‚HN K159 (2.11)
-OHb′‚ ‚ ‚OdC D116 (2.27)
-CH2-Of′‚ ‚ ‚HN N155 (3.82)
-CH2-Oe′‚ ‚ ‚HN K159 (2.39)
-OHb′‚ ‚ ‚OdC D64 (2.09)
-C-Od′‚ ‚ ‚HN N155 (2.17)
-C-Oc′‚ ‚ ‚HN K159 (1.92)
K159, K156, N155, E92, H67, T66, D64
4c
4d
39.74
46.97
K159, N155, I151, V77, V75, D64, Q62
K159, N155, K156, N155, E92, H67, T66, D64
4e
4f
52.75
50.02
42.46
52.11
H114, D116,C65, D64, Q62
P142, I141, F139, D116, H114, C65, D64, Q62
K159, N155, E152, I151, T66, D64, Q62
K159, K156, N155, D116, E92, H67, T66, D64
5a
5b
5c
5d
5e
44.44
52.72
53.04
K159, N155, E152, I151, T66, D64, Q62
K159, K156, N155, D116, E92, H67, T66, D64
K159, N155, I151, V77, V75, D64, Q62
7a
7b
40.89
47.52
K159, N155, F121, N120, E92, H67, T66, D64
K159, K156, N155, E92, H67, T66, C65, D64
-OHb′‚ ‚ ‚OdC C65 (1.67)
-CH2-Of′‚ ‚ ‚HN N155 (1.68)
-CH2-Oe′‚ ‚ ‚HN K159 (2.94)
-OHb′‚ ‚ ‚OdC C65 (1.58)
-CH2-Of′‚ ‚ ‚HN N155 (3.25)
-CH2-Oe′‚ ‚ ‚HN K159 (1.73)
-CH2-Oe′‚ ‚ ‚HN K159 (1.79)
-OHb′‚ ‚ ‚OdC D116 (2.16)
-CH2-Of′‚ ‚ ‚HN N155 (3.22)
-CH2-Oe′‚ ‚ ‚HN K159 (2.18)
-OHb′‚ ‚ ‚OdC I151 (2.56)
-C-Oc′‚ ‚ ‚HN Q62 (3.55)
7d
46.06
K159, K156, N155, E92, H67, T66, C65, D64
7f
9b
52.64
52.78
K159, K156, N155, E152, I151, E92, H67, T66, C65, D64, Q62
K159, K156, N155, N120, D116 E92, H67, T66, C65, D64
9c
41.07
N155, E152, I151, Q148, P142, I141, D116, H114, D64, Q62
a
The superscript letter indicates the position of the H-bond donor or acceptor atom of compounds 4a -9c. Compounds 4a -5e were
modeled in their monoanionic carboxylate form.
δ 8.31-8.26 (1H, m, Ar-H), 7.95 (1H, s, Ar-H), 7.32-7.45
(3H, m, Ar-H + HCdC), 6.94 (1H, s, Ar-H), 3.89 (3H, s,
NCH3), 2.45-1.50 (1H, bs, COOH). MS: m/z 245 (M+, 8), 157
(base). Anal. (C13H11NO4) C, H, N.
4-(5-Eth yl-5H-[1,3]d ioxolo[4,5-f]in d ol-7-yl)-2-h yd r oxy-
4-oxobu t-2-en oic Acid (5d ). Yield: 32%; mp 200-201 °C.
IR (Nujol): v cm-1 1620 (CdO), 1700 (CdO acid). 1H NMR
(CDCl3 + DMSO-d6): δ 7.88 (1H, s, Ar-H), 7.75 (1H, d,
HCdC), 7.49 (1H, s, Ar-H), 6.88 (1H, s, Ar-H), 6.00 (2H, s,
OCH2O), 4.18 (2H, q, J 7.2 Hz, CH2), 4.20-3.00 (1H, bs,
COOH), 1.50 (3H, t, J 7.2 Hz, CH3). MS: m/z 303 (M+, 4), 215
(base). Anal. (C15H13NO6‚0.05H2O) C, H, N.
2-H yd r oxy-4-(5-m et h yl-5H -[1,3]d ioxolo[4,5-f]in d ol-7-
yl)-4-oxobu t-2-en oic Acid (5b). Yield: 34%; mp 193-195 °C.
IR (Nujol): v cm-1 1620 (CdO), 1750 (CdO acid). 1H NMR
(CDCl3 + DMSO-d6): δ 7.76 (1H, s, Ar-H), 7.64 (1H, s, Ar-
H), 7.46 (1H, s, Ar-H), 6.81 (1H, s, HCdC), 5.99 (2H, s,
OCH2O), 3.79 (3H, s, NCH3), 3.65-3.15 (1H, bs, COOH). MS:
m/z 289 (M+, 6), 201 (base). Anal. (C14H11NO6) C, H, N.
4-(1-E t h yl-1H -in d ol-3-yl)-2-h yd r oxy-4-oxob u t -2-en oic
Acid (5c). Yield: 56%; mp 145-146 °C. IR (Nujol): v cm-1 1650
4-(1-Ben zyl-1H -in d ol-3-yl)-2-h yd r oxy-4-oxob u t -2-en o-
ic Acid (5e). Yield: 69%; mp 181-182 °C. IR (Nujol): v cm-1
1
1620 (CdO), 1700 (CdO acid). H NMR (CDCl3 + DMSO-d6):
δ 8.39 (1H, d, Ar-H), 8.11 (1H, s, Ar-H), 7.49-7.21 (6H, m,
Ar-H + HCdC), 7.22 (1H, s, Ar-H), 6.90 (1H, s, HCdC), 6.89
(1H, s, Ar-H), 5.42 (2H, s, CH2), 3.98-3.75 (1H, bs, COOH).
MS: m/z 321 (M+, 9), 90 (base). Anal. (C19H15NO4) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion s of In d ole
â-Dik eto Ester s (7a -f a n d 9a -e). A solution of an appropri-
ate alkylacetylindole (6a -f and 8a -e) (5.4 mmol) and diethyl-
1
(CdO), 1720 (CdO acid). H NMR (CDCl3): δ 8.32-8.28 (1H,
m, Ar-H), 8.00 (1H, s, Ar-H), 7.55-7.31 (3H, m, Ar-H +
HCdC), 6.94 (1H, s, Ar-H), 4.26 (2H, q, J 7.0 Hz, CH2), 4.20-
3.90 (1H, bs, COOH), 1.57 (3H, t, J 7.0 Hz, CH3). MS: m/z
259 (M+, 3), 171 (base). Anal. (C14H13NO4) C, H, N.

5306 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Sechi et al.
Ta ble 5. Interactions of Indole â-Diketo Acids with Mg2+ Iona
compd Mg2+ interactions (Å) compd Mg2+ interactions (Å)
s, NCH3), 2.10-1.55 (1H, bs, COOH). MS: m/z 259 (M+, 3),
157 (base). Anal. (C14H13NO4) C, H, N.
Meth yl 2-Hyd r oxy-4-(5-m eth yl-5H-[1,3]d ioxolo[4,5-f]in -
d ol-7-yl)-4-oxo-bu t-2-en oa te (9b). Yield: 75%; mp 165-164
°C. IR (Nujol): v cm-1 1640 (CdO), 1735 (CdO ester). 1H NMR
(CDCl3): δ 7.81 (1H, s, Ar-H), 7.69 (1H, s, Ar-H), 7.55 (1H,
s, Ar-H), 6.79 (1H, s, CHdC), 6.01 (2H, s, OCH2O), 3.93 (3H,
s, OCH3), 3.80 (3H, s, NCH3). MS: m/z 303 (M+, 7), 201 (base).
Anal. (C15H13NO6) C, H, N.
Meth yl 4-(1-Eth yl-1H-in d ol-3-yl)-2-h yd r oxy-4-oxo-bu t-
2-en oa te (9c). Yield: 59%; mp 159-161 °C. IR (Nujol): v cm-1
1610 (CdO), 1740 (CdO ester). 1H NMR (CDCl3): δ 8.41-8.36
(1H, m, Ar-H), 7.93 (1H, s, Ar-H), 7.58-7.28 (3H, m, Ar-
H), 6.87 (1H, s, HCdC), 4.28 (2H, q, J 7.4 Hz, NCH2), 3.94
(3H, s, OCH3), 1.57 (3H, t, J 7.4 Hz, CH3). MS: m/z 273 (M+,
7), 171 (base). Anal. (C15H15NO4) C, H, N.
4a
4b
4c
4d
4e
5e
7a
-C-Oc′‚ ‚ ‚Mg2+ (1.60)
HOb′‚ ‚ ‚Mg2+ (1.93)
-C-Od′‚ ‚ ‚Mg2+ (2.22)
HOb′‚ ‚ ‚Mg2+ (1.94)
-C-Od′‚ ‚ ‚Mg2+ (2.00)
HOb′‚ ‚ ‚Mg2+ (1.98)
-C-Od′‚ ‚ ‚Mg2+ (1.91)
-CdOa′‚ ‚ ‚Mg2+ (2.50)
HOb′‚ ‚ ‚Mg2+ (2.40)
-CdOa′‚ ‚ ‚Mg2+ (1.50)
HOb′‚ ‚ ‚Mg2+ (1.64)
-C-Oc′‚ ‚ ‚Mg2+ (1.61)
HOb′‚ ‚ ‚Mg2+ (2.17)
-C-Oc′‚ ‚ ‚Mg2+ (1.68)
HOb′‚ ‚ ‚Mg2+ (1.93)
-C-Oc′‚ ‚ ‚Mg2+ (2.18)
7b
7d
7f
4f
5a
5b
HOb′‚ ‚ ‚Mg2+ (1.97)
9b
9c
-C-Oc′‚ ‚ ‚Mg2+ (1.61)
5c
5d
HOb′‚ ‚ ‚Mg2+ (2.27)
-C-Oc′‚ ‚ ‚Mg2+ (1.64)
Meth yl 4-(5-Eth yl-5H-[1,3]d ioxolo[4,5-f]in d ol-7yl)-2-h y-
d r oxy-4-oxo-bu t-2-en oa te (9d ). Yield: 32%; mp 154-155 °C.
IR (Nujol): v cm-1 1630 (CdO), 1710 (CdO ester). 1H NMR
(CDCl3): δ 7.81 (1H, s, Ar-H), 7.76 (1H, s, Ar-H), 6.82 (1H,
s, HCdC), 6.79 (1H, s, Ar-H), 6.01 (2H, s, OCH2O), 4.13 (2H,
q, J 7.0 Hz, CH2), 3.93 (3H, s, OCH3), 1.53 (3H, t, J 7.0 Hz,
CH3). MS: m/z 317 (M+, 4), 215 (base). Anal. (C16H15NO6‚
0.70H2O) C, H, N.
a
The superscript letter indicates the atom on a compound that
is involved in a coordinate bonding interaction with the Mg2+ ion.
See Table 4 for 4a -9c.
oxalate (1.3 equiv) in methanol (30 mL) or THF (30 mL) was
added to a solution of sodium methoxide (3.2 equiv) (generated
from sodium in methanol). The mixture was refluxed under a
nitrogen atmosphere for 4 h (or 24 h for compound 9e). After
dilution with water, the solution was acidified with 1 N HCl.
The yellow precipitate formed was filtered and purified by
flash chromatography over silica gel using petroleum ether and
ethyl acetate as eluent.
Meth yl 2-Hyd r oxy-4-(1-m eth yl-1H-in d ol-2-yl)-4-oxobu t-
2-en oa te (7a ). Yield: 55%; mp 97-98 °C. IR (Nujol): v cm-1
1630 (CdO), 1740 (CdO ester). 1H NMR (CDCl3): δ 7.70 (1H,
d, Ar-H), 7.48-7-56 (4H, m, Ar-H), 7.07 (1H, s, HCdC), 4.13
(3H, s, OCH3), 3.94 (3H, s, NCH3). MS: m/z 259 (M+, 9), 172
(base). Anal. (C14H13NO4) C, H, N.
Meth yl 2-Hyd r oxy-4-(5-m eth yl-5H-[1,3]d ioxolo[4,5-f]in -
d ol-6-yl)-4-oxobu t-2-en oa te (7b). Yield: 46%; mp 180 °C
(dec). IR (Nujol): v cm-1 1630 (CdO), 1730 (CdO ester). 1H
NMR (CDCl3): δ 7.29 (1H, s, Ar-H), 6.98 (2H, m, Ar-H +
HCdC), 6.78 (1H, s, Ar-H), 6.01 (2H, s, OCH2O), 4.06 (3H, s,
OCH3), 3.93 (3H, s, NCH3). MS: m/z 303 (M+, 6), 216 (base).
Anal. (C15H13NO6) C, H, N.
Met h yl 4-(1-et h yl-1H -in d ol-2-yl)-2-h yd r oxy-4-oxob u t -
2-en oa te (7c). Yield: 51%; mp 103 °C (dec). IR (Nujol): v cm-1
1615 (CdO), 1720 (CdO ester). 1H NMR (CDCl3 + DMSO-d6):
δ 7.71 (1H, d, Ar-H), 7.50-7.39 (3H, m, Ar-H), 7.23-7.15
(1H, m, Ar-H), 7.09 (1H, s, CHdH), 4.65 (2H, q, J 7.2 Hz,
CH2), 3.95 (1H, s, OCH3), 1.42 (3H, t, J 7.2 Hz, CH3). MS: m/z
273 (M+, 171), 4 (base). Anal. (C15H15NO4) C, H, N.
Met h yl 4-(5-E t h yl-5H -[1,3]d ioxolo[4,5-f]in d ol-6-yl)-2-
h yd r oxy-4-oxo-bu t-2-en oa te (7d ). Yield: 42%; mp 170-172
°C. IR (Nujol): v cm-1 1630 (CdO), 1730 (CdO ester). 1H NMR
(CDCl3 + DMSO-d6): δ 7.33 (1H, s, Ar-H), 6.97 (2H, s, Ar-H
+ CHdC), 6.85 (1H, s, Ar-H), 6.02 (2H, s, OCH2O), 4.56 (2H,
q, J 7.0 Hz, CH2), 3.92 (3H, s, OCH3), 1.38 (3H, t, J 7.0 Hz,
CH3). MS: m/z 317 (M+, 7), 230 (base). Anal. (C16H15NO6) C,
H, N.
Meth yl 4-(1-Ben zyl-1H-in dol-2-yl)-2-h ydr oxy-4-oxo-bu t-
2-en oa te (7e). Yield: 71%; mp 167 °C (dec). IR (Nujol): v cm-1
1640 (CdO), 1710 (CdO ester). 1H NMR (CDCl3): δ 7.62 (1H,
d, Ar-H), 7.28-6.89 (9H, m, Ar-H + HCdC), 6.71 (1H, s, Ar-
H), 5.83 (2H, s, CH2), 3.67 (3H, s, OCH3). MS: m/z 335 (M+,
7), 90 (base). Anal. (C20H17NO4‚0.50H2O) C, H, N.
Meth yl 4-(5-Ben zyl-5H-[1,3]d ioxolo[4,5-f]in d ol-6-yl)-2-
h yd r oxy-4-oxo-bu t-2-en oa te (7f). Yield: 72%; mp 131 °C
(dec). IR (Nujol): v cm-1 1630 (CdO), 1750 (CdO ester). 1H
NMR (CDCl3 + DMSO-d6): δ 7.33-6.98 (7H, m, Ar-H), 6.81
(1H, s, HCdC), 6.70 (1H, s, Ar-H), 5.95 (2H, s, OCH2O), 5.83
(2H, s, CH2), 3.82 (3H, s, CH3). MS: m/z 379 (M+, 5), 90 (base).
Anal. (C21H17NO6) C, H, N.
Met h yl 2-H yd r oxy-4-(1-m et h yl-1H -in d ol-3-yl)-4-oxo-
bu t-2-en oa te (9a ). Yield: 81%; mp 174-175 °C. IR (Nujol):
v cm-1 1610 (CdO), 1745 (CdO ester). 1H NMR (CDCl3): δ
8.42-8.34 (1H, m, Ar-H), 7.86 (1H, s, Ar-H), 7.42-7.30 (3H,
m, Ar-H), 6.85 (1H, s, HCdC), 3.93 (3H, s, OCH3), 3.88 (3H,
Meth yl 4-(1-Ben zyl-1H-in dol-3-yl)-2-h ydr oxy-4-oxo-bu t-
2-en oa te (9e). Yield: 37%; mp 178-179 °C. IR (Nujol): v cm-1
1610 (CdO), 1730 (CdO ester). 1H NMR (CDCl3): δ 8.49-8.42
(1H, m, Ar-H), 7.91 (1H, s, Ar-H), 7.40-7.32 (8H, m, Ar-
H), 6.84 (1H, s, HCdC), 5.39 (2H, s, CH2), 3.92 (3H, s, OCH3).
MS: m/z 335 (M+, 5), 90 (base). Anal. (C20H17NO4) C, H, N.
P r ep a r a tion of N-Alk ylin d oles (8a ,c,e 12-15, 17, a n d
18). Anhydrous DMSO (20 mL) was added to KOH (4 equiv,
crushed pellets), and the mixture was stirred at room tem-
perature for 5 min. An appropriate indole (10, 11, 16, and 25)
(10 mmol) was then added, and the mixture was stirred at
room temperature for 45-60 min. An appropriate alkyl halide
(2 equiv) was added, and the mixture was stirred at room
temperature for 45-60 min. The progress of reaction was
monitored by TLC throughout the course of the experiment.
Then, water was added and the white precipitate that formed
was filtered and washed with water. The compounds that did
not precipitate were extracted from the aqueous solution with
diethyl ether. The organic layer was washed with water and
dried over Na2SO4, and the solvent was removed in vacuo to
give the desired final product as a solid or oil.
1-(1-Meth yl-1H-in d ol-3-yl)eth a n on e (8a ).43 Yield: 57%;
mp 107-108 °C. IR (Nujol): v cm-1 1640 (CdO). 1H NMR
(CDCl3): δ 8.45-8.35 (1H, m, Ar-H), 7.69 (1H, s, Ar-H),
7.42-7.28 (3H, m, Ar-H), 3.85 (3H, s, NCH3), 2.51 (3H, s,
CH3). MS: m/z 173 (M+, 100), 173 (base).
1-(1-Eth yl-1H-in d ol-3-yl)eth a n on e (8c).44 Yield: 69%; mp
1
87-89 °C. IR (Nujol): v cm-1 1630 (CdO). H NMR (CDCl3):
δ 8.45-8.35 (1H, m, Ar-H), 7.78 (1H, s, Ar-H), 7.45-7.25
(3H, m, Ar-H), 4.25 (2H, q, J 7.2 Hz, CH2), 1.57 (3H, t, J 7.2
Hz, CH3). MS: m/z 187 (M+, 62), 172 (base).
1-(1-Ben zyl-1H-in d ol-3-yl)eth a n on e (8e).43 Yield: 67%;
mp 109-110 °C. IR (Nujol): v cm-1 1630 (CdO). 1H NMR
(CDCl3): δ 8.43-8.37 (1H, m, Ar-H), 7.77 (1H, s, Ar-H),
7.40-7.16 (8H, m, Ar-H), 5.35 (2H, s, CH2), 2.52 (3H, s, CH3).
MS: m/z 249 (M+, 43), 91 (base).
Meth yl 1-Meth yl-1H-in d ole-2-ca r boxyla te (12).45 Yield:
1
93%; mp 99-101 °C (dec). IR (Nujol): v cm-1 1710 (CdO). H
NMR (CDCl3): δ 7.72 (1H, d, Ar-H), 7.42-7.35 (3H, m, Ar-
H), 7.20-7.10 (1H, m, Ar-H), 4.06 (3H, s, OCH3), 3.90 (3H, s,
OCH3). MS: m/z 189 (M+, 100), 189 (base).
Meth yl 5-Meth yl-5H-[1,3]d ioxolo[4,5-f]in d ole-6-ca r box-
yla te (13). Yield: 89%; mp 166 °C. IR (Nujol): v cm-1 1700
1
(CdO). H NMR (CDCl3): δ 7.13 (1H, s, Ar-H), 6.95 (1H, s,
Ar-H), 6.75 (1H, s, Ar-H), 5.95 (2H, s, OCH2O), 3.96 (3H, s,
OCH3), 3.85 (3H, s, NCH3). MS: m/z 233 (M+, 100), 233 (base).
Meth yl 5-Eth yl-5H-[1,3]d ioxolo[4,5-f]in d ole-6-ca r box-
yla te (14). Yield: 91%; mp 158-159 °C. IR (Nujol): v cm-1
1680 (CdO). 1H NMR (CDCl3): δ 7.14 (1H, s, Ar-H), 6.96 (1H,
s, Ar-H), 6.78 (1H, s, Ar-H), 5.95 (2H, s, OCH2O), 4.52 (2H,

Indole-â-diketo Acid Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5307
q, J 7.4 Hz, CH2), 3.86 (3H, s, OCH3), 1.35 (3H, t, J 7.4 Hz,
CH3). MS: m/z 247 (M+, 100), 247 (base).
1-(5-Met h yl-5H-[1,3]d ioxolo[4,5-f]in d ol-6-yl)et h a n on e
(6b). Yield: 42%; mp 151-152 °C. IR (Nujol): v cm-1 1650 (Cd
1
O). H NMR (CDCl3): δ 7.13 (1H, s, Ar-H), 6.96 (1H, s, Ar-
Meth yl 5-Ben zyl-5H-[1,3]d ioxolo[4,5-f]in d ole-6-ca r box-
yla te (15). Yield: 82%; mp 142-144 °C. IR (Nujol): v cm-1 1700
H), 6.74 (1H, s, Ar-H), 5.97 (2H, s, OCH2O), 3.98 (3H, s,
NCH3), 2.52 (3H, s, CH3). MS: m/z 217 (M+, 65), 202 (base).
1-(1-Eth yl-1H-in d ol-2-yl)eth a n on e (6c).50 Yield: 65%; mp
120-122 °C. IR (Nujol): v cm-1 1660 (CdO). 1H NMR
(CDCl3): δ 7.69 (1H, d, Ar-H), 7.41-7.05 (4H, m, Ar-H), 4.61
(2H, q, J 7.2 Hz, CH2), 2.62 (3H, s, CH3), 1.35 (3H, t, J 7.2 Hz,
CH3). MS: m/z 187 (M+, 86), 172 (base).
1-(5-Eth yl-5H-[1,3]dioxolo[4,5-f]in dol-6-yl)eth an on e (6d).
Yield: 48%; mp 132-135 °C. IR (Nujol): v cm-1 1650 (CdO).
1H NMR (CDCl3): δ 7.15 (1H, s, Ar-H), 6.97 (1H, s, Ar-H),
6.78 (1H, s, Ar-H), 5.97 (2H, s, OCH2O), 4.51 (2H, q, J 7.4
Hz, CH2), 2.54 (3H, s, CH3), 1.32 (3H, t, J 7.4 Hz, CH3). MS:
m/z 231 (M+, 100), 231 (base).
1
(CdO). H NMR (CDCl3): δ 7.24 (1H, s, Ar-H), 7.21 (1H, s,
Ar-H), 7.05-6.95 (5H, m, Ar-H), 6.71 (1H, s, Ar-H), 5.91
(2H, s, OCH2O), 5.75 (2H, s, CH2), 3.82 (3H, s, OCH3). MS:
m/z 309 (M+, 76), 91 (base).
Eth yl 1-Eth yl-1H-in dole-2-car boxylate (17).46 Yield: 87%;
colorless oil. IR (Nujol): v cm-1 1710 (CdO). 1H NMR (CDCl3):
δ 7.68 (1H, d, Ar-H), 7.48-7.31 (3H, m, Ar-H), 7.13 (1H, t,
Ar-H), 4.62 (2H, q, J 7.0 Hz, CH2), 4.37 (2H, q, J 7.0 Hz,
NCH2), 1.48 (6H, m, CH3). MS: m/z 217 (M+, 100), 217 (base).
Eth yl 1-Ben zyl-1H-in dole-2-car boxylate (18). Yield: 91%;
mp 55-56 °C. IR (Nujol): v cm-1 1720 (CdO). 1H NMR
(CDCl3): δ 7.70 (1H, d, Ar-H), 7.42-7.05 (9H, m, Ar-H), 5.84
(2H, s, CH2), 4.31 (2H, q, J 6.8 Hz, CH2), 1.34 (3H, t, J 6.8 Hz,
CH3). MS: m/z 279 (M+, 57), 91 (base).
1-(1-Ben zyl-1H-in d ol-2-yl)eth a n on e (6e).24 Yield: 51%;
mp 126-128 °C. IR (Nujol): v cm-1 1650 (CdO). 1H NMR
(CDCl3): δ 7.72 (1H, d, Ar-H), 7.41-7.05 (9H, m, Ar-H), 5.87
(2H, s, CH2), 2.61 (3H, s, CH3). MS: m/z 249 (M+, 35), 91 (base).
1-(5-B e n zy l-5H -[1,3]d io x o lo -[4,5-f]in d o l-6-y l)e t h a -
n on e (6f). Yield: 39%; mp 141-142 °C. IR (Nujol): v cm-1 1650
P r ep a r a tion of N-Alk yl-in d ol-2-ca r boxylic Acid (19a -
f). A suspension of an appropriate ester (12-15, 17, 18) (5
mmol) in 12% aqueous KOH solution (50 mL) was stirred at
reflux for 1 h. The clear solution was poured into ice water
and then acidified with 2 N HCl. A white precipitate was
filtered off and washed with water.
1
(CdO). H NMR (CDCl3): δ 7.30-7.15 (4H, m, Ar-H), 7.08-
6.98 (3H, m, Ar-H), 6.71 (1H, s, Ar-H), 5.94 (2H, s, OCH2O),
5.77 (2H, s, CH2), 2.53 (3H, s, CH3). MS: m/z 293 (M+, 61), 91
(base).
1-Meth yl-1H-in dol-2-car boxylic Acid (19a).45 Yield: 92%;
mp 210-211 °C. IR (Nujol): v cm-1 1670 (CdO). 1H NMR
(CDCl3 + DMSO-d6): δ 7.66 (1H, d, Ar-H), 7.50-7.05 (1H,
bs, COOH), 7.42-7.30 (3H, m, Ar-H), 7.15-7.05 (1H, m, Ar-
H), 4.08 (3H, s, CH3). MS: m/z 175 (M+, 100), 175 (base).
5-Meth yl-5H-[1,3]dioxolo[4,5-f]in dole-6-car boxylic Acid
(19b). Yield: 89%; mp 245-247 °C. IR (Nujol): v cm-1 1690
P r ep a r a tion of Meth yl Azid oa ceta te (22).42 A solution
of methyl bromoacetate (85 mmol) and sodium azide (90 mmol,
1.06 equiv) in DMF (20 mL) was stirred at room temperature
for 2.5 h. After a white solid formed, an equivalent quantity
of water was added and the resulting solution was extracted
three times with diethyl ether. The organic layer was washed
six times with water and dried over Na2SO4. The solvent was
removed under reduced pressure to obtain a yellow oil. Yield:
94%. IR (film): v cm-1 2120 (N3), 1750 (CdO). 1H NMR
(CDCl3): δ 3.90 (3H, s, OCH3), 3.81 (2H, s, CH2). MS: m/z 115
(M+, 17), 59 (base).
1
(CdO). H NMR (CDCl3 + DMSO-d6): δ 7.07 (1H, s, Ar-H),
6.96 (1H, s, Ar-H), 6.91 (1H, s, Ar-H), 5.97 (2H, s, OCH2O),
4.50-2.80 (1H, bs, COOH), 3.97 (3H, s, CH3). MS: m/z 219
(M+, 100), 219 (base).
1-Eth yl-1H-in d ole-2-ca r boxylic Acid (19c).47 Yield: 95%;
mp 177-179 °C. IR (Nujol): v cm-1 1680 (CdO). 1H NMR
(CDCl3): δ 8.10-6.90 (1H, bs, COOH), 7.71 (1H, d, Ar-H),
7.51-7.32 (3H, m, Ar-H), 7.21-7.10 (1H, m, Ar-H), 4.64 (2H,
q, J 7.0 Hz, CH2), 1.42 (3H, t, J 7.0 Hz, CH3). MS: m/z 189
(M+, 75), 174 (base).
Gen er a l P r oced u r e for th e P r ep a r a tion of Azid ocin -
n a m a tes (23 a n d 24).42 To a solution of an appropriate
aldehyde (20 and 21) (10 mmol) and 22 (34 mmol) in methanol
(9 mL) a cold solution of sodium methoxide (30 mmol) in
methanol (5 mL) was added. The yellow slurry was stirred at
-15°C for 4 h, and then, ice cold water (36 mL) was added.
The precipitate formed was filtered off and washed with water
to give yellow crystals.
Meth yl 2-Azid o-3-p h en yla cr yla te (23).23 Yield: 52%; mp
39-41 °C. IR (Nujol): v cm-1 2120 (N3), 1700 (CdO). 1H NMR
(CDCl3): δ 7.80 (2H, d, J 7.2 Hz, Ar-H), 7.43-7.28 (3H, m, J
7.2 Hz, Ar-H), 6.91 (1H, s, CHdC), 3.90 (3H, s, OCH3). MS:
m/z 203 (M+, 100), 203 (base).
5-Eth yl-5H-[1,3]d ioxolo[4,5-f]in d ole-6-ca r boxylic Acid
(19d ). Yield: 89%; mp 200 °C (dec). IR (Nujol): v cm-1 1660
1
(CdO). H NMR (CDCl3 + DMSO-d6): δ 7.11 (1H, s, Ar-H),
6.95 (1H, s, Ar-H), 6.86 (1H, s, Ar-H), 5.97 (2H, s, OCH2O),
5.20-4.30 (1H, bs, COOH), 4.56 (2H, q, J 6.8 Hz, CH2), 1.34
(3H, t, J 6.8 Hz, CH3). MS: m/z 233 (M+, 8), 189 (base).
1-Ben zyl-1H-in dole-2-car boxylic Acid (19e).48 Yield: 93%;
mp 194-196 °C. IR (Nujol): v cm-1 1660 (CdO). 1H NMR
(CDCl3 + DMSO-d6): δ 8.40-6.90 (1H, bs, COOH), 7.68 (1H,
d, Ar-H), 7.42-7.00 (9H, m, Ar-H), 5.86 (2H, s, CH2). MS:
m/z 251 (M+, 29), 91 (base).
Meh tyl 2-Azid o-3-(1,3-ben zod ioxol-5-yl)-a cr yla te (24).
Yield: 91%; mp 61-62 °C (from EtOH/H2O). IR (Nujol): v cm-1
1
2210 (N3), 1710 (CdO). H NMR (CDCl3): δ 7.57 (1H, s, Ar-
5-Ben zyl-5H-[1,3]dioxolo[4,5-f]in dole-6-car boxylic Acid
(19f). Yield: 84%; mp 224-226 °C. IR (Nujol): v cm-1 1660 (Cd
H), 7.27 (1H, s, Ar-H), 7.17 (1H, d, J 7.8 Hz, Ar-H), 6.84
(1H, s, CHdC), 6.82 (1H, d, J 7.8 Hz, Ar-H) 6.01 (2H, s,
OCH2O), 3.90 (3H, s, OCH3). MS: m/z 247 (M+, 100), 203
(base).
1
O). H NMR (CDCl3): δ 7.70-6.10 (1H, bs, COOH), 7.23 (1H,
s, Ar-H), 7.21 (1H, s, Ar-H), 7.18-6.98 (5H, m, Ar-H), 6.73
(1H, s, Ar-H), 5.93 (2H, s, OCH2O), 5.79 (2H, s, CH2). MS:
m/z 295 (M+, 8), 91 (base).
Gen er a l P r oced u r es for th e P r ep a r a tion of Ester s (10
a n d 11). A solution of the azidocinnamates (23 and 24) (4.04
mmol) in xylene (25 mL) was heated under reflux for 15 min.
After the solution was cooled, the pale yellow precipitate
formed was filtered and washed with petroleum ether.
Meth yl 1H-In d ole-2-ca r boxyla te (10).51 Yield: 67%; mp
145-147 °C (from EtOH/H2O). IR (Nujol): v cm-1 3320 (NH),
1680 (CdO). 1H NMR (CDCl3): δ 9.12 (1H, bs, NH), 7.69 (1H,
d, J 8.2 Hz, Ar-H), 7.41 (1H, t, J 8.0 Hz, Ar-H), 7.32 (1H, t,
J 8.2 Hz, Ar-H), 7.22 (1H, s, Ar-H), 7.15 (1H, t, J 8.0 Hz,
Ar-H), 3.95 (3H, s, OCH3). MS: m/z 175 (M+, 77), 143 (base).
Meth yl 5H-[1,3]Dioxolo[4,5-f]in d ole-6-ca r boxyla te (11).
Yield: 79%; mp 194-195 °C (from EtOH-H2O). IR (Nujol): v
Syn th esis of 2-Acetylin d oles (6a -f). Under a nitrogen
atmosphere, CH3Li (1.4 M in diethyl ether, 48 mmol) was
slowly added to a suspension of an appropriate acid (19a -f)
(24 mmol) in anhydrous diethyl ether (30 mL) and cooled to 0
°C, and the mixture was refluxed for 1 h. Additional CH3Li
(1.4 M in diethyl ether, 48 mmol) was added at room temper-
ature, and the mixture was refluxed for 4 h. The reaction was
quenched by the addition of saturated aqueous NH4Cl, diluted,
and extracted with diethyl ether. The organic layer was
washed with water, dried over Na2SO4, and evaporated in
vacuo to give the desired product as an oil or a solid.
1-(1-Meth yl-1H-in d ol-2-yl)eth a n on e (6a ).49 Yield: 55%;
mp 68-70 °C. IR (Nujol): v cm-1 1665 (CdO). 1H NMR
(CDCl3): δ 7.69 (1H, d, Ar-H), 7.37-7.14 (4H, m, Ar-H), 4.06
(3H, s, CH3), 2.61 (3H, s, CH3). MS: m/z 173 (M+, 100), 173
(base).
1
cm-1 3319 (NH), 1690 (CdO). H NMR (CDCl3): δ 11.22 (1H,
bs, NH), 7.01 (1H, s, Ar-H), 6.93 (1H, s, Ar-H), 6.90 (1H, s,
Ar-H), 5.93 (2H, s, OCH2O), 3.88 (3H, s, OCH3). MS: m/z 219
(M+, 99), 187 (base).

5308 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Sechi et al.
P r ep a r a tion of Meth yl 7-Acetyl-5-m eth yl(eth yl)-5H-
[1,3]d ioxolo[4,5-f]in d ole-6-ca r boxyla te (26 a n d 27). A
solution of TFAA (24 mmol, 2.8 equiv for 26; 11 mmol, 2.8
equiv for 27), glacial acetic acid (2.8 equiv), and 85% H3PO4
(0.32 equiv) in acetonitrile (20 mL for 26; 10 mL for 27) was
stirred at room temperature for 5 min under a nitrogen
atmosphere. Then, a solution of an appropriate indole (13 and
14) (8.7 mmol, 1 equiv) in acetonitrile (20 mL for 26; 10 mL
for 27) was added, and the mixture was stirred at room
temperature for 14 h. The reaction was quenched by addition
of ice water, and the violet precipitate formed was filtered,
washed with water, and chromatographed over silica gel using
petroleum ether and ethyl acetate as the eluent.
Meth yl 7-Acetyl-5-m eth yl-5H-[1,3]d ioxolo[4,5-f]in d ole-
6-ca r boxyla te (26). Yield: 81%; mp 126-128 °C. IR (Nujol):
v cm-1 1640 (CdO), 1730 (CdO ester). 1H NMR (CDCl3): δ
7.40 (1H, s, Ar-H), 6.79 (1H, s, Ar-H), 6.01 (2H, s, OCH2O),
3.97 (3H, s, OCH3), 3.85 (3H, s, NCH3), 2.52 (3H, s, CH3). MS:
m/z 275 (M+, 71), 260 (base).
Meth yl 7-Acetyl-5-eth yl-5H-[1,3]d ioxolo[4,5-f]in d ole-6-
ca r boxyla te (27). Yield: 78%; mp 148-150 °C. IR (Nujol): v
cm-1 1660 (CdO), 1700 (CdO ester). 1H NMR (CDCl3): δ 7.40
(1H, s, Ar-H), 6.79 (1H, s, Ar-H), 6.00 (2H, s, OCH2O), 4.31
(2H, q, J 7.2 Hz, CH2), 3.97 (3H, s, CH3), 2.52 (3H, s, CH3),
1.41 (3H, t, J 7.2 Hz, CH3). MS: m/z 289 (M+, 71), 274 (base).
P r epar ation of 7-Acetyl-5-m eth yl(eth yl)-5H-[1,3]dioxolo-
[4,5-f]in d ole-6-ca r boxylic Acid (28 a n d 29). A solution of
an appropriate ester (26 and 27) (1.3 mmol) and KOH pellets
(3.6 equiv) in ethanol (40 mL) was stirred under reflux for 3
h. Then, water was added and the solution was acidified with
6 N HCl. The white precipitate that formed was filtered,
washed with water, and collected.
were carried out with the SAINT program (SAX, area detector
integration).52 The final reflections, corrected for absorption,
were obtained by SADABS.53 The phase problem was solved
by direct methods, using SIR 97.54 Full-matrix least-squares
refinements were carried out with SHELXS 97.55 Anisotropic
thermal displacement parameters were refined for all non-H-
atoms; all H-atoms were distinguished by Fourier maps and
refined isotropically. Detailed analyses are listed in Tables 2
and 3 and the Supporting Information.
Biologica l Ma ter ia ls, Ch em ica ls, a n d En zym es. All
compounds were dissolved in DMSO, and the stock solutions
were stored at -20 °C. The γ[32P]ATP was purchased from
either Amersham Biosciences or ICN. The expression systems
for the wild-type IN and soluble mutant INF185KC280S were
generous gifts of Dr. Robert Craigie, Laboratory of Molecular
Biology, NIDDK, NIH, Bethesda, MD.
P r ep a r a tion of Oligon u cleotid e Su bstr a tes. The oligo-
nucleotides 21top, 5′-GTGTGGAAAATCTCTAGCAGT-3′, and
21bot, 5′-ACTGCTAGAGATTTTCCACAC-3′, were purchased
from Norris Cancer Center Microsequencing Core Facility
(University of Southern California) and purified by UV shad-
owing on polyacrylamide gel. To analyze the extent of 3′-
processing and strand transfer using 5′-end-labeled substrates,
21top was 5′-end-labeled using T4 polynucleotide kinase
(Epicenter, Madison, WI) and γ [32P]ATP (Amersham Bio-
sciences or ICN). The kinase was heat-inactivated, and 21bot
was added in 1.5 molar excess. The mixture was heated at 95
°C, allowed to cool slowly to room temperature, and run
through a spin 25 minicolumn (USA Scientific) to separate
annealed double-stranded oligonucleotide from unincorporated
material.
IN Assa ys. To determine the extent of 3′-processing and
strand transfer, wild-type IN was preincubated at a final
concentration of 200 nM with the inhibitor in reaction buffer
[50 mM NaCl, 1 mM HEPES, pH 7.5, 50 µM EDTA, 50 µM
dithiothreitol, 10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL
bovine serum albumin, 10 mM 2-mercaptoethanol, 10% DMSO,
and 25 mM MOPS, pH 7.2] at 30 °C for 30 min. Then, 20 nM
the 5′-end 32P-labeled linear oligonucleotide substrate was
added, and incubation was continued for an additional 1 h.
Reactions were quenched by the addition of an equal volume
(16 µL) of loading dye (98% deionized formamide, 10 mM
EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue).
An aliquot (5 µL) was electrophoresed on a denaturing 20%
polyacrylamide gel (0.09 M tris-borate, pH 8.3, 2 mM EDTA,
20% acrylamide, and 8 M urea).
7-Acet yl-5-m et h yl-5H -[1,3]d ioxolo[4,5-f]in d ole-6-ca r -
boxylic Acid (28). Yield: 71%; mp 258-260 °C. IR (Nujol): v
1
cm-1 1710 (CdO acid). H NMR (CDCl3 + DMSO-d6): δ 7.38
(1H, s, Ar-H), 7.08 (1H, s, Ar-H), 6.03 (2H, s, OCH2O), 3.88
(3H, s, NCH3), 3.77-3.45 (1H, bs, COOH), 2.55 (3H, s, CH3).
MS: m/z 261 (M+, 5), 202 (base).
7-Acetyl-5-eth yl-5H-[1,3]d ioxolo[4,5-f]in d ole-6-ca r box-
ylic Acid (29). Yield: 91%; mp 221-223 °C. IR (Nujol): v cm-1
1700 (CdO acid). 1H NMR (CDCl3): δ 7.35 (1H, s, Ar-H), 6.98
(1H, s, Ar-H), 6.10 (2H, s, OCH2O), 4.77 (2H, q, J 7.2 Hz,
NCH2), 4.15-3.65 (1H, bs, COOH), 2.87 (3H, s, CH3), 1.48 (3H,
t, J 7.2 Hz, CH3). MS: m/z 275 (M+, 7), 216 (base).
P r ep a r a tion of 1-(5-Meth yl(eth yl)-5H-[1,3]d ioxolo[4,5-
f]in d ole-7-yl)eth a n on e (8b,d ). A mixture of copper chromite
(0.3 equiv for 8b; 0.37 equiv for 8d ) and an appropriate acid
(28 or 29) (4.4 mmol for 28; 1.5 mmol for 29) in quinoline (12
mL for 28; 5 mL for 29) was heated in an oil bath at 200 °C
and stirred under a nitrogen atmosphere for 50 min. Then,
after the mixture was cooled at 0 °C, water was added and
the solution was extracted with ethyl acetate. The organic
layer was acidified with 1 N HCl, washed with water, then
with a solution of NaHCO3, again with water, and eventually
dried over MgSO4. The solvent was removed in vacuo to give
a solid that was triturated with petroleum ether and ethyl
acetate and chromatographed over silica gel using hexane and
ethyl acetate as eluent.
The gels were dried, exposed in a PhosphorImager cassette,
analyzed using a Typhoon 8610 Variable Mode Imager (Am-
ersham Biosciences), and quantitated using ImageQuant 5.2.
The percent inhibition (% I) was calculated using the following
equation:
% I ) 100 × [1 - (D - C)/(N - C)]
where C, N, and D are the fractions of 21-mer substrate
converted to 19-mer (3′-processing product) or strand transfer
products for DNA alone, DNA plus IN, and IN plus drug,
respectively. The IC50 values were determined by plotting the
logarithm of drug concentration vs percent inhibition to obtain
the concentration that produced 50% inhibition.
1-(5-Me t h yl-5H -[1,3]d ioxolo[4,5-f]in d ole -7-yl)e t h a -
n on e (8b). Yield: 46%; mp 142-145 °C. IR (Nujol): v cm-1
1635 (CdO). 1H NMR (CDCl3): δ 7.81 (1H, s, Ar-H), 7.55 (1H,
s, Ar-H), 6.76 (1H, s, Ar-H), 5.98 (2H, s, OCH2O), 3.76 (3H,
s, NCH3), 2.46 (3H, s, CH3). MS: m/z 217 (M+, 70), 202 (base).
1-(5-E t h yl-5H -[1,3]d ioxolo[4,5-f]in d ole-7-yl)et h a n on e
(8d ). Yield: 61%; mp 169-171 °C. IR (Nujol): v cm-1 1630 (Cd
An ti-HIV Assa ys in Cu ltu r ed Cells. The anti-HIV activity
was evaluated in human T-cell line CEM-SS infected with
HIV-1 as described by Weislow et al.56 In brief, the cells were
plated in 96 well plates at 5 × 103 cells/well and infected with
HIV-1RF (MOI ) 0.3). Serial dilutions of compounds were then
immediately added to the cells in a final volume of 200 µL. In
each experiment, AZT and dextran sulfate were included as
control compounds for anti-HIV activity. The cells were
maintained at 37 °C with 5% CO2-containing humidified air
for 6 days. The cell viability was quantified by absorbance at
450 nm after 4 h of incubation with 2,3-bis[2-methoxy-4-nitro-
5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium hy-
droxide (XTT) at 0.2 mg/mL. The antiviral activity was graded
based on the degree of anti-HIV protection as active (80-100%
protection), moderate (50-79% protection), and inactive (0-
1
O). H NMR (CDCl3 + DMSO-d6): δ 7.82 (1H, s, Ar-H), 7.61
(1H, s, Ar-H), 6.79 (1H, s, Ar-H), 5.98 (2H, s, OCH2O), 4.12
(2H, q, J 7.4 Hz, NCH2), 2.47 (3H, s, CH3), 1.52 (3H, t, J 7.4
Hz, CH3). MS: m/z 231 (M+, 71), 260 (base).
X-r a y Cr ysta llogr a p h y. Single crystal X-ray diffraction
analysis was carried out at room temperature with a computer-
controlled Siemens SMART AXS 100 diffractometer with area
detector (CCD) using Mo KR (λ ) 0.7107 Å). No crystal decay
was observed. The reduction and integration of the intensities

Indole-â-diketo Acid Inhibitors of HIV-1 Integrase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5309
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49% protection). The toxicity of the compounds was determined
simultaneously on the same plate in uninfected CEM-SS cells.
Molecu la r Mod elin g. The structures of all of the com-
pounds (Table 3) were built and minimized using Catalyst
(Accelrys, Inc.) running on a multiprocessor Linux PC in
parallel with a 24 processor Silicon Graphics Onyx workstation
as previously described.30,57 All compounds were modeled in
their 2,3-enol-tautomeric form based on the solution and the
crystal structure of 4c (Figure 3). Compounds 4a -f and 5a -e
were modeled in their monoanionic carboxylate form. The
poling algorithm implemented within Catalyst was used to
generate conformations for all of the compounds.58-60
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For each compound, all feasible unique conformations were
generated over a 20 kcal/mol range of energies using the best
flexible conformation generation method in Catalyst. The
subunit B of the core domain X-ray structure of IN (PDB 1BIS)
in which all of the active site amino acid residues were resolved
was chosen for docking purposes.61 A Mg2+ ion was placed in
the active site between carboxylate oxygen atoms of amino acid
residues D64 and D116 considering the geometry of the Mg2+
ion that was present in the subunit A of IN in PDB 1BIS and
subunit A in the IN-5CITEP complex crystal structure (PDB
1QS4). All of the water molecules present in the protein were
removed, and hydrogen atoms were added to the protein
considering appropriate ionization states for both the acidic
and the basic amino acid residues. Docking was performed
using version 1.2 of the GOLD: Genetic Optimization for
Ligand Docking (Cambridge Crystallographic Data Centre)
software package essentially as described.30,62-64 In brief, a 20
Å radius active site was defined considering the carboxylate
oxygen atom (OD1) of amino acid residue D64 as the center of
the active site. All conformers of the compounds were docked
into the active site of the IN. On the basis of the GOLD fitness
score, for each molecule, a bound conformation with high
fitness score was considered as the best bound conformation.
All docking runs were carried out using standard default
settings with a population size of 100, a maximum number of
100000 operations, and a mutation and crossover rate of 95.
The fitness function that was implemented in GOLD consisted
basically of H-bonding, complex energy, and ligand internal
energy terms.
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Ack n ow led gm en t. This work is dedicated to the
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