Discovery of 3-acetyl-4-hydroxy-2-pyranone derivatives and their difluoridoborate complexes as a novel class of HIV-1 integrase Inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.08.067

HIV-1 integrase (IN) has emerged as an important therapeutic target for anti-HIV drug development. Its uniqueness to the virus and its critical role in the viral life cycle makes IN suitable for selective inhibition. The recent approval of Raltegravir (MK-0518) has created a surge in interest and great optimism in the field. In our ongoing IN drug design research, we herein report the discovery of substituted analogs of 3-acetyl-4-hydroxy-2-pyranones and their difluoridoborate complexes as novel IN inhibitors. In many of these compounds, complexation with boron difluoride increased the potency and selectivity of IN inhibition. Compound 9 was most active with an IC₅₀ value of 9 μM and 3 μM for 3'-processing and strand transfer inhibition, respectively.

Full text of DOI:10.1016/j.bmc.2008.08.067

Bioorganic & Medicinal Chemistry 16 (2008) 8988–8998
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of 3-acetyl-4-hydroxy-2-pyranone derivatives and their
difluoridoborate complexes as a novel class of HIV-1 integrase Inhibitors
Kavya Ramkumar a, Konstantin V. Tambov b, Rambabu Gundla a, Alexandr V. Manaev b,
Vladimir Yarovenko b, Valery F. Traven b, Nouri Neamati a,
*
a Department of Pharmacology & Pharmaceutical Sciences, University of Southern California, School of Pharmacy, Room 304, 1985 Zonal Avenue, Los Angeles, CA 90089, USA
b D. Mendeleev University of Chemical Technology of Russia, Miusskaja st.3, Moscow 125047, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
HIV-1 integrase (IN) has emerged as an important therapeutic target for anti-HIV drug development. Its
uniqueness to the virus and its critical role in the viral life cycle makes IN suitable for selective inhibition.
The recent approval of Raltegravir (MK-0518) has created a surge in interest and great optimism in the
field. In our ongoing IN drug design research, we herein report the discovery of substituted analogs of 3-
acetyl-4-hydroxy-2-pyranones and their difluoridoborate complexes as novel IN inhibitors. In many of
these compounds, complexation with boron difluoride increased the potency and selectivity of IN inhibi-
Received 30 May 2008
Revised 15 August 2008
Accepted 20 August 2008
Available online 30 August 2008
Keywords:
HIV integrase inhibitor
Boron
Pyranone
Docking
tion. Compound 9 was most active with an IC50 value of 9
l
M and 3
lM for 3’-processing and strand
transfer inhibition, respectively.
Ó 2008 Elsevier Ltd. All rights reserved.
GOLD
eHITS
1. Introduction
emergence of resistant IN strains necessitates continual efforts to
design structurally novel IN inhibitors.3,14 Moreover, studies with
diverse classes of inhibitors may also help better understand the
mechanisms of IN action.
In recent years, HIV-1 integrase (IN) has emerged as an impor-
tant therapeutic target for the design of anti-HIV agents. IN cata-
lyzes the insertion of HIV proviral DNA into the host genome.
This integration occurs via a multi-step process, in which the cleav-
age of a dinucleotide pair from the 3’-end of the proviral DNA (3’-
processing) and the subsequent insertion of the shortened strand
into the host genome (strand transfer) are the key catalytic func-
tions of the enzyme.1 Compounds inhibiting IN block one or both
of these steps. There is a plethora of literature describing diverse
structural classes of IN inhibitors.2–4 Among these, the b-diketo
acid class of compounds has shown selective IN inhibition and con-
siderable clinical efficacy. Previous studies from our lab have also
identified several potent and selective IN inhibitors based on b-
diketo acid and chalcone pharmacophores.5,6 Several other phar-
macophore based approaches have also resulted in diverse IN
inhibitors.7,8 Many of these compounds have been reported to sig-
nificantly suppress retroviral replication.9,10 Raltegravir (MK-
0518), a pyrimidone carboxamide, was recently approved by the
FDA as an anti-HIV drug and is the first member of the new class
of IN inhibitor drugs.11 Elvitegravir (GS-9137), another IN inhibitor
based on a dihydroquinoline carboxylic acid structure, is undergo-
ing advanced clinical evaluations.12,13 However, the propensity for
In our on-going efforts to identify structurally diverse com-
pounds with IN inhibitory activity, we discovered a novel class of
substituted analogs of 3-acetyl-4-hydroxy-pyranone and their
difluoridoborate complexes that show potent IN inhibitory effect.
Various substituted pyranones have been previously reported to
exhibit anticancer, antimicrobial, anticoagulant and HIV protease
inhibitory properties.15,16 Similarly, boron-containing compounds
have been used in boron capture therapy for cancer treatment
and as metallocarboranes for HIV protease inhibition.17,18 This is
however the first report of substituted pyranones and their difluor-
idoborate complexes exhibiting potent IN inhibitory activities. We
herein report the synthesis and IN inhibition profile of these novel
compounds as well as the results of molecular docking studies,
exploring the binding interactions of potent compounds in the IN
active site.
2. Results and discussion
2.1. Chemistry
Recently, we have found complexation with boron to greatly
increase the reactivity of acetyl(hydroxy)hetarenes toward
* Corresponding author. Tel.: +1 323 442 2341; fax: 1 323 442 1390.
E-mail address: neamati@usc.edu (N. Neamati).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.067

K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
8989
aldehydes. We prepared various
a
,b-unsaturated ketones from the
Compound 2, in which boron is complexed with the pyranone
structure containing N,N-dimethylaniline, showed moderate selec-
reaction between 3-acetyl-4-hydroxycoumarin difluoridoborate
complex and carbonyl derivatives.19–21 These products have high
electronic absorption and emission. Difluoridoborate complexes
of 2-quinoline and 2-pyranone derivatives of coumarins also
possessed high reactivity toward aldehydes. This led to easy
derivatization of the 3-acetyl-function in acetylhydroxyhetarenes.
The starting compound 3-acetyl-6-methyl-2-oxo-2H-pyran-4-yl
difluoridoborate (I) was prepared, using the method previously
described for coumarin analogs.19
Condensation of 3-acetyl-6-methyl-2-oxo-2H-pyran-4-yl diflu-
oridoborate I with triethyl orthoformate in acetic anhydride in
the presence of triethylamine formed symmetric B-complex 1
(Scheme 1). Condensation of 3-acetyl-6-methyl-2-oxo-2H-pyran-
4-yl difluoridoborate complex I with p-R-substituted benzalde-
hydes provided complexes 2, 3 and 5 by heating of the starting
compounds in Ac2O or AcOH–H2SO4 mixture (Scheme 2). Com-
pounds 4 and 6 have been prepared by the hydrolysis of 2, 3 and
5 in EtOH–H2O in the presence of Na2CO3. Condensation of I with
heterocyclic aldehydes in similar conditions provided complexes
7, 9 and 11. Compounds 8, 10 and 12 were prepared by subsequent
hydrolysis (Scheme 3) of these complexes.
tivity towards strand transfer inhibition (IC50 value of 56
1 lM).
Compounds 3 and 4 share a bromobenzene substitution at the 3-
acetyl position in the pyranone structure. While compound 4
lacked any IN inhibition at 100
lM, its boron complexed analog,
compound 3, exhibited potent inhibition of 3’-processing (IC50 va-
lue of 12
(IC50 of 4
6
2
l
M) and a 3-fold selectivity against strand transfer
lM). Compounds 5 and 6, with a fluorobenzene sub-
stitution showed similar activity profile to compounds 3 and 4.
Compound 5 inhibited 3’-processing with an IC50 of 9 M and
strand transfer with an IC50 of 16 M. Compound 6 showed a
moderate inhibition of 3’-processing (IC50 value of 64 M) but
did not inhibit strand transfer at 100 M.
3 l
4
l
l
l
Compound 8 with N,N-dimethylthiophen-2-amine substitution
inhibited 3’-processing and strand transfer with IC50 values of 19
and 11 lM, respectively. However, its boron complexed derivative,
compound 7, did not possess any IN inhibitory effect. Compounds
9 and 10 showed a 3-fold selectivity towards strand transfer inhi-
bition. Compound 9, complexed with boron, inhibited 3’-process-
ing and strand transfer with IC50 values of 9 and 3 lM, respectively.
Compounds 11 and 12 containing 3-acetyl-4-hydroxy-6-methyl-
pyranone structure with a bulky, hydrophobic 1,3,3-trimethyl-2-
methyleneindoline substitution were both inactive. Compounds
13 and 14 with a butylene bridge and N,N-dimethylaniline substitu-
tion did not show any IN inhibition, however compound 13 in the
boron complex showed weak strand transfer inhibition with an
Complexes 13 and 15 were obtained from the condensation of
3-acetyl-6-methyl-2-oxo-2H-pyran-4-yl difluoridoborate
I with
p-R-substituted aldehydes. Hydrolysis of 13 and 15 resulted in
unsaturated aldehydes 14 and 16 (Scheme 4). Due to the complex-
ation of 3-acetyl-4-hydroxy-6-methyl-2-pyranone with BF3, the 6-
methyl group was reactive toward aldehydes as well. Condensa-
tion of complexes 2, 3 and 5 with p-Me2N-benzaldehyde resulted
in complexes 17, 19 and 21. Subsequent hydrolysis gave com-
pound 18, 20 and 22 in good yields (Scheme 5). Structures of all
compounds have been confirmed by 1H NMR and mass spectra,
as well as by elemental analysis.
IC50 value of 100 lM. Compounds 15 and 16, having a similar struc-
ture with the exception of a methoxy substitution, exhibited potent
and selective strand transfer inhibition. As observed with the previ-
ous compounds, the boron complexed form of compound 15 has low
IC50 values, 11 and 4 lM for 3’-processing and strand transfer inhibi-
tion, respectively than the corresponding uncomplexed form. Fur-
ther substitutions at the 6-methyl position resulted in compounds
17–22. These compounds lacked any IN inhibitory activity.
2.2. In vitro IN inhibitory profile of novel compounds
Overall, 3-acetyl-4-hydroxy-6-methyl-pyranones with hydro-
phobic substitutions separated by alkene bridges selectively inhib-
ited IN strand transfer. Due to the shallow substrate binding site on
IN, the size and position of the substituent in these compounds ap-
pear to play a critical role in determining IN inhibitory activities.
Interestingly, complexation with boron difluoride increased the
potency and selectivity of IN inhibition. Figure 1 shows the inhibi-
tion of IN catalytic activities by active compounds. Compound 9
stands out as the most potent compound in the set with an IC50 va-
Inhibition of the IN catalytic activities were measured using an
in vitro assay specific for IN. Table 1 presents the structure of each
compound and its IC50 values for IN inhibition. Compound 1, which
has a dimeric 3-acetyl-4-hydroxy-6-methyl-pyranone structure
and is complexed with boron difluoride, inhibited 3’-processing
and strand transfer activities of IN with IC50 values of 13 8 and
7
2
lM, respectively. These results led us to further study some
key structural features by modifying compound 1.
F
F
F
F
F
F
B
B-
B
O
O
O
O
O
O
CH(OEt)3
+
NHEt3
2
NEt3, Ac2O
O
O
O
O
O
O
1
I
Scheme 1. Synthesis of compound 1.
F
F
F
F
B
CHO
B
O
O
OH
O
O
O
O
R
1.Na2CO3,EtOH,H2O
2.HCl
Ac2O, AcOH H2SO4
O
O
O
R
R
O
O
4, 6
2, 3, 5
I
Scheme 2. Synthesis of compounds 2–6. R = N(CH3)2 (2); R = Br (3,4); R = F (5,6).

8990
K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
F
F
B
F
F
O
O
B
O
O
OH
O
O
O
CHO
R
S
1.Na2CO3,EtOH,H2O
2.HCl
S
S
O
O
Ac2O, AcOH H2SO4
O
O
R
R
7, 9
8, 10
I
F
F
B
O
O
O
O
OH
OHC
N
1.Na2CO3,EtOH,H2O
2.HCl
N
N
Ac2O
O
O
O
11
12
Scheme 3. Synthesis of compounds 7–12. R = N(CH3)2 (7,8); R = Br (9, 10).
F
F
F
F
B
B
CHO
O
O
O
O
O
OH
R
1.Na2CO3,EtOH,H2O
2.HCl
O
O
Ac2O, AcOH H2SO4
R
O
O
O
O
R
I
13, 15
14, 16
Scheme 4. Synthesis of compounds 13–16. R = N(CH3)2 (13,14); R = OCH3(15,16).
F
F
B
F
F
CHO
R1
O
O
B
R1
O
O
N
R2
O
O
Ac2O
R2
O
O
R3
N
R3
17, 19, 21
R1
R3
O
O
OH
O
2, 3, 5
1.Na2CO3,EtOH,H2O
2.HCl
R2
N
18, 20, 22
Scheme 5. Synthesis of compounds 17–22. R1 = R3 = H, R2 = N(CH3)2 (17,18); R1 = R3 = H, R2 = OCH3 (19, 20), R1 = OCH3, R2 = H, R3 = Br (21, 22).
lue of 9
l
M for 3’-processing and 3
l
M for strand transfer inhibi-
scores and eHITS scores, along with selected physicochemical
properties, are given in Table 2. A general trend was observed, in
which the more active difluoridoborate complexes scored higher
than corresponding uncomplexed pyranones. Overall, there was
no significant correlation between the fitness scores and IN inhib-
itory activities of the compounds. Figure 2 depicts a comparison
between the bound conformation of representative active com-
pound 3 inside the active site of IN and its corresponding inactive
uncomplexed pyranone 4. The predicted binding areas of 3 and 4 in
the IN active site were similar to that of 5CITEP. Hydrogen bonding
interactions and the interacting amino acid residues at the active
site are given in Table 3. These compounds commonly occupy a
wide cavity surrounded by the following amino acids: K159,
K156, N155, E152, I151, P142, I141, G140, F139, N117, D116,
H114, D64 and Q62. It was observed that the active compound 3
established strong interactions with key amino acid residues D64
tion. These results were further studied and confirmed through
molecular docking studies.
2.3. Cytotoxicity and antiviral studies
CytotoxicitywasassessedbycellviabilityinMTTassay. At10 lM,
none of the compounds exhibited any significant cytotoxicity. Com-
pounds 1, 3, 9 and 15 with potent in vitro IN inhibitory effects
showed no substantial antiviral activity. Further studies are in pro-
gresstodesignmorepotentanalogswithimprovedantiviralactivity.
2.4. Computational studies
Compounds 1–22 were docked onto IN active site using eHITS
and GOLD in order to identify their binding sites. The GOLD fitness

K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
8991
Table 1
Inhibition of HIV-1 IN catalytic activities and cytotoxicity by substituted and boron complexed pyranones
Compound
Structure
Inhibition of IN catalytic activity IC50
(lM)
Cytotoxicity (l
M)c
3’-Processinga Strand transfera Selectivity indexb
F
F
F
-
F
O
B
B
B
O
O
O
1
13
8
7
56
4
2
1
2
1.9
>10
+
NHEt3
O
O
O
O
N
F
F
O
O
2
3
4
5
6
7
>100
>1.8
3.3
—
>10
>10
>10
>10
>10
>10
O
O
F
F
B
O
O
12
6
O
O
O
Br
Br
OH
>100
>100
O
F
O
F
B
O
O
9
3
16
4
>100
>100
0.6
<0.7
—
O
O
O
F
F
OH
64
O
F
O
F
B
O
O
>100
S
O
O
O
N
OH
8
9
19
4
2
11
6
2
1.8
3.2
>10
S
O
F
O
N
F
B
O
O
9
3
> 10
S
Br
O
O
(continued on next page)

8992
K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
Table 1 (continued)
Compound
Structure
Inhibition of IN catalytic activity IC50
(lM)
Cytotoxicity (l
M)c
3’-Processinga Strand transfera Selectivity indexb
OH
O
O
10
65 40
23
3
2.8
>10
S
O
F
Br
N
F
B
O
O
11
12
>100
>100
>100
>100
—
—
>10
>10
O
O
O
OH
N
O
F
O
F
B
O
O
13
14
15
16
17
>100
100
—
>10
>10
>10
>10
>10
O
O
O
N
N
OH
>100
>100
—
O
F
O
F
B
O
O
11
3
4
3
2.5
>2.6
—
O
O
O
O
O
OH
>100
39 14
O
O
F
F
B
O
O
>100
>100
N
O
O
O
N
N
OH
O
18
>100
>100
—
>10
O
N

K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
8993
Table 1 (continued)
Compound
Structure
Inhibition of IN catalytic activity IC50
(lM)
Cytotoxicity (l
M)c
3’-Processinga Strand transfera Selectivity indexb
F
F
B
O
O
19
20
21
>100
>100
—
>10
N
N
O
O
O
O
O
OH
>100
>100
>100
>100
>100
100
—
—
—
>10
>10
>10
O
F
O
F
B
O
O
O
N
N
O
O
O
Br
O
OH
22
O
O
Br
a
Results are from at least three independent experiments.
Selectivity index = (IC50(3’-processing)/IC50(strand transfer)).
Determined by MTT assay.
b
c
and E152 and Mg2+ ion that are important for IN catalytic activity.
The inactive compound 4 did not form such interactions in the ac-
tive site of IN.
4.1.1. 3-Acetyl-6-methyl-2-oxo-2H-pyran-4-yl difluoridoborate
(I)
A solution of 3-acetyl-4-hydroxy-6-methyl-2-pyranone (10 g,
0.06 mol) in dry benzene (17 mL) was treated with boron trifluor-
ide etherate (10 g, 0.07 mol) and heated for 1 h. The resultant pre-
cipitate was filtered, washed with benzene and crystallized from
benzene. Complex I, yellow crystals, yield 80%, mp 156–157 °C.
1H NMR (DMSO-d6) d, ppm: 2.39 (s, 3H, 6-CH3), 2.86 (s, 3H, 3-
CH3), 6.13 (s, 1H, H5). Found %: C 44.45; H 3.21. C8H7BF2O4. Calcu-
lated %: C 44.50; H 3.27.
3. Conclusion
We have identified a novel class of IN inhibitors with a unique
3-acetyl-4-hydroxy-pyranone scaffold. Some of the difluoridob-
orate complexes of the pyranones were found to be more potent
IN inhibitors than uncomplexed pyranones. The potent and selec-
tive inhibition profile and the lack of cytotoxicity of these novel
compounds make them suitable leads for further modifications.
Structural optimization and mechanistic studies to enhance selec-
tive IN inhibitory activity is in progress.
4.1.2. Triethylammonium 2,2-difluoro-4-[(O4’’-B)-4’-[(difluoro-
boryl)oxy]-4’-(4’’-oxo-3’’,4’’-dihydropyranone-3’’-ylidene)-2’-buten-
1’-ylidene]di-oxaboratabenzo[c]pyranone (1)
A solution of a 3-acetyl-6-methyl-2-oxo-2H-pyran-4-yl difluor-
idoborate I (2 mmol) in acetic anhydride (5 mL) was treated with
triethyl orthoformate (0.15 g, 1 mmol) and triethylamine (0.28 g,
2 mmol). The mixture was heated to 60 °C for 30 min and then
cooled and allowed to stand for 4 h. The resultant precipitate
was filtered, washed with hexane, and crystallized from glacial
acetic acid. Compound 1, yield 60%, mp 214–215 °C. 1H NMR
(DMSO-d6) d, ppm: 1.14 (t, 9H, 3CH3), 2.23 (s, 6H, CH3), 3.15 (q,
6H, 3CH2), 6.16 (s, 2H, H5), 7.43 (d, 2H, CH), 8.87 (t, 1H, CH). MS,
m/z: 441 [M+]. Found %: C 50.79; H 5.06; N 2.58. C23H27B2F4NO8.
Calculated %: C 50.87; H 5.01; N 2.58.
4. Experimental
4.1. Chemistry
Compounds 1–22 were synthesized as a continuation of our
previous studies on the synthesis, structure and reactivity of
a,b-
unsaturated ketones-3-acyl-4-hydroxycoumarin 3-acyl-4-hydro-
xy-2-quinolone derivatives and their analogs.19–21 1H NMR spectra
was recorded on a Bruker WP-200-SY instrument in CDCl3 and
DMSO-d6 with Me4Si as the internal standard. The mass spectra
were obtained on a MAT-112 mass spectrometer; the ionizing elec-
tron energy was 80 eV, the ion source temperature was equal to
250 °C, and the inlet temperature 240 °C.
4.1.3. General procedure for the preparation of 2, 7, 11 and 13
A solution of appropriate aldehyde (0.002 mol) in acetic anhy-
dride (2 mL) was added to a solution of 3-acetyl-6-methyl-2-oxo-

8994
K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
8
3
1
A
STP
21 mer
19 mer
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
9
10
15
16
B
STP
21 mer
19 mer
19 20 21 22 23
9 10 11 12 13 14 15 16 17 18
1
2
3
4
5
6
7
8
Figure 1. A representative gel showing inhibition of purified IN by selected compounds. (A) Lane 1—DNA alone; Lane 2—DNA and IN alone with no drug; Lanes 3–16—DNA
with IN and selected drug concentrations (Compounds 1 and 3–100, 33.3, 11.1, 3.7 and 1.2 M; Compound 8–100, 33.3, 11.1 and 3.7 M). (B) Lanes 1 and 13—DNA alone;
Lanes 2 and 14—DNA and IN without drug; Lanes 3–12, 15–23—DNA with IN and selected drug concentrations (Compound 15–100, 33.3, 11.1, 3.7, 1.2 and 0.4
l
l
l
M; Compound
9–100, 33.3, 11.1, 3.7 and 1.2 lM; Compounds 16 and 10–100, 33.3, 11.1 and 3.7 lM).
Table 2
Docking scores and selected physicochemical properties of substituted and boron complexed pyranone
Compound
MWa
RBb
HBAc
HBDd
ALogPe
S + logPf
PSAg
eHits_Score
GOLD_Score
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
441.89
347.12
382.95
335.15
322.04
274.24
353.15
305.35
388.98
341.18
399.20
351.40
373.16
325.36
360.12
312.32
478.30
430.50
465.25
417.45
544.15
496.35
2
3
2
3
2
3
3
4
2
3
2
3
4
5
4
5
6
7
6
7
6
7
8
5
4
4
4
4
5
5
4
4
5
5
5
5
5
5
6
6
6
6
6
6
0
0
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
0
1
4.358
3.82
4.41
2.87
3.86
2.33
3.90
2.36
4.18
2.64
4.65
3.12
4.29
2.75
4.11
2.57
5.69
4.15
5.51
3.97
6.26
4.72
2.53
3.80
4.11
3.88
3.60
3.35
3.42
2.92
3.84
3.53
5.29
4.61
4.59
3.85
4.33
3.65
6.33
5.48
6.12
5.36
6.92
6.15
91.59
50.07
46.83
63.60
46.83
63.60
78.30
95.08
75.06
91.84
50.07
66.84
50.07
66.84
56.06
72.83
53.31
70.08
59.30
76.06
59.30
76.07
ꢀ1.08
ꢀ0.28
ꢀ2.59
ꢀ1.96
ꢀ1.68
ꢀ1.74
ꢀ2.15
ꢀ2.01
ꢀ1.38
ꢀ2.12
ꢀ2.41
ꢀ1.87
ꢀ1.62
ꢀ1.68
ꢀ1.75
ꢀ1.79
ꢀ0.62
ꢀ0.04
ꢀ1.10
ꢀ1.47
ꢀ0.67
ꢀ1.92
51.99
46.91
49.89
42.13
46.55
44.45
42.36
47.72
50.76
48.39
42.10
43.37
43.43
39.44
54.98
50.12
42.35
43.74
48.97
43.74
41.48
49.18
a
Molecular weight.
b
c
d
e
f
Number of rotatable bonds.
Number of H-bond acceptors.
Number of H-bond donors.
Calculated atom-based log P.
S + logP (Simulations Plus LogP model).
2H-pyran-4-yl difluoridoborate (0.5 g, 0.002 mol) in acetic anhy-
dride (6 mL) at 60 °C. The mixture was then heated at 90 °C for
0.5 h. The reaction mixture was cooled; the precipitate formed
was filtered and washed with acetic acid, and recrystallized from
glacial acetic acid.
4.1.3.1. 3-{(2E)-3-[4-(Dimethylamino)phenyl]prop-2-enoyl}-6-
methyl-2-oxo-2H-pyran-4-yl difluoridoborate (2). Yield 84%,
mp 238–239 °C. 1H NMR (CDCl3) d, ppm: 2.34 (s, 3H, CH3), 3.17
(s, 6H, N(CH3)2), 6.07 (s, 1H, H5), 6.70 (d, 2H, Har, J = 8.8 Hz), 7.68
(d, 2H, Har, J = 8,8 Hz), 8.08 (d, 1H, CH, J = 14.9 Hz), 8.42 (d, 1H,

K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
8995
A
B
E152
E152
D64
D64
D116
D116
C
D
H67
H67
K159
K159
2.44Å
E92
T66
2.51Å
N155
T66
E92
C65
C65
N120
N155
3.01Å
N120
K156
S153
2.90Å
D64
Mg2+
K156
S153
Mg2+
D64
S119
3.29Å
S119
L63
L63
E152
I151
Q62
I151
Q62
E152
D116
D116
D117
D117
Q148
Q148
S147
S147
Figure 2. The bound conformation of compounds 3 and 4 on the catalytic core domain of HIV-1 IN. (A) and (B) shows the compounds 3 and 4 (pink), respectively overlaid on
crystal ligand 5CITEP (yellow) and binding surface is shown in green. The magenta sphere represents metal ion (Mg2+). (C) and (D) demonstrate the detailed interactions
between IN and the ligands 3 and 4, respectively.
6.01 (s, 1H, H5), 6.10 (d, 1H, CH, J = 14 Hz), 7.04–7.47 (m, 5H,
Table 3
H-bonding interactions and the active site amino acid residues interacting with
compounds 3 and 4
4Har, CH), 8.73 (t, 1H, CH). MS, m/z: 400 [M+]. Found %: C 63.15;
H 5.10; N 3.49 C21H20BF2NO4. Calculated %: C 63.18; H 5.05; N 3.51.
Compound
H-bonding
Interacting amino acid residues
interactionsa
4.1.3.4. 3-{(2E,4E)-5-[4-(Dimethylamino)phenyl]penta-2,4-die-
noyl}-6-methyl-2-oxo-2H-pyran-4-yl difluoridoborate (13). Yield
64%, mp 242–243 °C. 1H NMR (DMSO-d6) d, ppm: 2.33 (s, 3H, CH3),
3.31 (s, 6H, N(CH3)2), 6.41 (s, 1H, H5), 6.79 (d, 2H, Har, J = 8.7 Hz),
7.28 (t, 1H, CH), 7.57 (d, 1H, CH, J = 14.3 Hz), 7.65 (d, 2H, Har,
J = 8.7 Hz), 7.68 (d, 1H, CH, J = 14.3 Hz), 8.17 (t, 1H, CH). MS, m/z:
374 [M+]. Found %: C 61.19; H 4.89; N 3.65. C19H18BF2NO4. Calcu-
lated %: C 61.16; H 4.86; N 3.75.
3
4
C@Oꢁꢁ HO E152 (3.29)
C@O HN K156 (3.01)
C@O HN N155 (2.51)
C–O HN K159 (2.44)
L63, D64, C65, T66, H67, E92, D116,
Q148, I151, E152, N155, K156, K159
D64, C65, T66, H67, D116, Q148, I151,
E152, N155, K156, K159
a
Values in parentheses are H-bond distances in Å.
CH, J = 14.9 Hz). MS, m/z: 348 [M+]. Found %: C 58.79; H 4.60; N
4.00. C17H16BF2NO4. Calculated %: C 58.82; H 4.65; N 4.04.
4.1.4. General procedure for the preparation of 3, 5, 9 and 15
A solution of appropriate aldehyde (0.002 mol) in glacial acetic
acid (2 mL) and sulfuric acid (0.13 mL) was added to a solution of
3-acetyl-6-methyl-2-oxo-2H-pyran-4-yl difluoridoborate (0.5 g,
0.002 mol) in glacial acetic acid (6 mL) at 60 °C. The mixture was
then refluxed for 0.5 h. The reaction mixture was cooled; the
precipitate that formed was filtered, washed with acetic acid, and
recrystallized from glacial acetic acid.
4.1.3.2. 3-{(2E)-3-[5-(Dimethylamino)-2-thienyl]prop-2-enoyl}-6-
methyl-2-oxo-2H-pyran-4-yl difluoridoborate (7). Yield 88%,
mp 223–224 °C. 1H NMR (DMSO-d6) d, ppm: 2.26 (s, 3H, CH3),
3.38 (s, 6H, N(CH3)2), 6.22 (s, 1H, H5), 6.86 (d, 1H, Hhet
,
,
J = 5.1 Hz), 7.06 (d, 1H, CH, J = 12.9 Hz), 8.01 (d, 1H, Hhet
J = 5.1 Hz), 8.21 (d, 1H, CH, J = 12.9 Hz). MS, m/z: 354 [M+]. Found
%: C 51.04; H 4.09; N 3.96; S 9.10. C15H14BF2NO4S. Calculated %:
C 51.02; H 4.00; N 3.97; S 9.08.
4.1.4.1. 3-[(2E)-3-(4-Bromophenyl)prop-2-enoyl]-6-methyl-
2-oxo-2H-pyran-4-yl difluoridoborate (3). Yield 80%, mp 195–
196 °C. 1H NMR (CDCl3) d, ppm: 2.38 (s, 3H, CH3), 6.16 (s, 1H, H5),
7.62 (s, 4H, Har), 8.36 (s, 2H, CH). MS, m/z: 383 [M+]. Found %: C
4.1.3.3. 6-Methyl-2-oxo-3-[(2E,4E)-4-(1,3,3-trimethyl-1,3-dihy-
dro-2H-indol-2-ylidene)but-2-enoyl]-2H-pyran-4-yl
difluori-
doborate (11). Yield 76%, mp 250–251 °C. 1H NMR (CDCl3) d,
47.08;
H
2.66.
C
15H10BBrF2O4. Calculated %:
C
47.05;
ppm: 1.68 (s, 6H, (CH3)2), 2.29 (s, 3H, CH3), 3.54 (s, 3H, N(CH3)),
H 2.63.

8996
K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
4.1.4.2. 3-[(2E)-3-(4-Fluorophenyl)prop-2-enoyl]-6-methyl-2-
oxo-2H-pyran-4-yl difluoridoborate (5). Yield 65%, mp 176–
177 °C. 1H NMR (CDCl3) d, ppm: 2.40 (s, 3H, CH3), 6.16 (s, 1H,
H5), 7.76 (s, 4H, Har), 8.37 (s, 2H, CH). MS, m/z: 323 [M+]. Found
%: C 55.96; H 3.18. C15H10BF3O4. Calculated %: C 55.94; H 3.13.
tered, treated with a solution of hydrochloric acid (pH 6.5–7).
The precipitate that formed was filtered, washed with water, and
recrystallized in isopropanol.
4.1.6.1. 3-[(2E)-3-(4-Bromophenyl)prop-2-enoyl]-4-hydroxy-6-
methyl-2H-pyran-2-one (4). Yield 63%, mp 128–129 °C. 1H
NMR (CDCl3) d, ppm: 2.28 (s, 3H, CH3), 5.96 (s, 1H, H5), 7.25 (s,
4H, Har), 7.83 (d, 1H, CH, J = 15.9 Hz), 8.25 (d, 1H, CH,
J = 15.9 Hz), 17.75 (s, 1H, OH). MS, m/z: 336 [M+]. Found %: C
53.77; H 3.35. C15H11BrO4. Calculated %: C 53.76; H 3.31.
4.1.4.3. 3-[(2E)-3-(5-Bromo-2-thienyl)prop-2-enoyl]-6-methyl-
2-oxo-2H-pyran-4-yl difluoridoborate (9). Yield 88%, mp 156–
157 °C. 1H NMR (CDCl3) d, ppm: 2.40 (s, 3H, CH3), 6.13 (s, 1H, H5),
7.19 (d, 1H, Hhet, J = 5.1 Hz), 7.38 (d, 1H, Hhet, J = 5.1 Hz), 8.00 (d,
1H, CH, J = 12.9 Hz), 8.36 (d, 1H, CH, J = 12.9 Hz). MS, m/z: 389
[M+]. Found %: C 40.15; H 2.10; S 8.27. C13H8BBrF2O4S. Calculated
%: C 40.14; H 2.07; S 8.24.
4.1.6.2. 3-[(2E)-3-(4-Fluorophenyl)prop-2-enoyl]-4-hydroxy-
6-methyl-2H-pyran-2-one (6). Yield 55%, mp 114–115 °C. 1H
NMR (CDCl3) d, ppm: 2.27 (s, 3H, CH3), 5.95 (s, 1H, H5), 7.26 (s,
4H, Har), 7.95 (d, 1H, CH, J = 15.9 Hz), 8.28 (d, 1H, CH,
J = 15.9 Hz), 17.84 (s, 1H, OH). MS, m/z: 275 [M+]. Found %: C
65.73; H 4.05. C15H11FO4. Calculated %: C 65.69; H 4.04.
4.1.4.4. 3-[(2E,4E)-5-(4-Methoxyphenyl)penta-2,4-dienoyl]-6-
methyl-2-oxo-2H-pyran-4-yl difluoridoborate (15). Yield 74%,
mp 198–199 °C. 1H NMR (CDCl3) d, ppm: 2.36 (s, 3H, CH3), 3.87 (s,
3H, OCH3), 6.11 (s, 1H, H5), 6.92 (d, 2H, Har, J = 8.7 Hz), 7.02 (t, 1H,
CH), 7.20 (d, 1H, CH, J = 14.3 Hz), 7.53 (d, 2H, Har, J = 8.7 Hz), 7.80
(d, 1H, CH, J = 14.3 Hz), 8.19 (t, 1H, CH). MS, m/z: 361 [M+]. Found
%: C 60.08; H 4.16. C18H15BF2O5. Calculated %: C 60.03; H 4.20.
4.1.6.3. 3-{(2E)-3-[5-(Dimethylamino)-2-thienyl]prop-2-enoyl}-
4-hydroxy-6-methyl-2H-pyran-2-one (8). Yield 88%, mp 190–
191 °C. 1H NMR (DMSO-d6) d, ppm: 2.17 (s, 3H, CH3), 3.16 (s, 6H,
N(CH3)2), 6.04 (s, 1H, H5), 6.25 (d, 1H, Hhet, J = 4.1 Hz), 7.26 (d,
1H, CH, J = 14.3 Hz), 7.57 (d, 1H, Hhet, J = 4.1 Hz), 8.04 (d, 1H, CH,
J = 14.3 Hz), 14.00 (s, 1H, OH). MS, m/z: 306 [M+]. Found %: C
59.03; H 4.98; N 4.63; S 10.56 C15H15NO4S. Calculated %: C
59.00; H 4.95; N 4.59; S 10.50.
4.1.5. General procedure for the preparation of 17, 19 and 21
A solution of 4-dimethylaminobenzaldehyde (0.05 g, 0.4 mmol)
in acetic anhydride (2 mL) was added to a solution of appropriate
difluoridoborate (0.4 mmol) in acetic anhydride (2 mL) at 60 °C.
The mixture was refluxed for 0.5 h. The reaction mixture was
cooled; the precipitate that formed was filtered, washed with ace-
tic acid, and recrystallized from glacial acetic acid.
4.1.6.4. 3-[(2E)-3-(5-Bromo-2-thienyl)prop-2-enoyl]-4-hydro-
xy-6-methyl-2H-pyran-2-one (10). Yield 76%, mp 115–116 °C.
1H NMR (CDCl3) d, ppm: 2.27 (s, 3H, CH3), 5.94 (s, 1H, H5), 7.05
(d, 1H, Hhet, J = 4.1 Hz), 7.16 (d, 1H, Hhet, J = 4.1 Hz), 7.87 (d, 1H,
CH, J = 14.3 Hz), 7.98 (d, 1H, CH, J = 14.3 Hz), 17.80 (s, 1H, OH).
MS, m/z: 342 [M+]. Found %: C 45.81; H 2.69; S 9.42 C13H9BrO4S.
Calculated %: C 45.77; H 2.66; S 9.40.
4.1.5.1. 3-{(2E)-3-[4-(Dimethylamino)phenyl]prop-2-enoyl}-6-
{(E)-2-[4-(dimethylamino)phenyl]vinyl}-2-oxo-2H-pyran-4-yl
difluoridoborate (17). Yield 47%, mp 330–331 °C. 1H NMR
(DMSO-d6+TFA) d, ppm: 3.03 (s, 6H, N(CH3)2), 3.12 (s, 6H,
N(CH3)2), 6.37 (s, 1H, H5), 6.82 (m, 4H, Har, J = 15.8 Hz), 7.60 (m,
3H, CH, Har), 7.71 (d, 2H, Har, J = 8.7 Hz), 7.86 (d, 1H, CH,
J = 15.8 Hz), 8.00 (d, 1H, CH, J = 16.2 Hz), 8.15 (d, 1H, CH,
J = 16.2 Hz). MS, m/z: 479 [M+]. Found %: C 65.24; H 5.23; N 5.86.
4.1.6.5. 4-Hydroxy-6-methyl-3-[(2E,4E)-4-(1,3,3-trimethyl-
1,3-dihydro-2H-indol-2-ylidene)but-2-enoyl]-2H-pyran-2-one
(12). Yield 76%, mp 250–251 °C. 1H NMR (CDCl3) d, ppm: 1.66 (s,
6H, (CH3)2), 2.20 (s, 3H, CH3), 3.32 (s, 3H, N(CH3)), 5.85 (s, 1H, H5),
6.17 (d, 1H, CH, J = 14 Hz), 6.78–7.48 (m, 5H, 4Har CH), 8.39 (t, 1H,
CH), 18.82 (s, 1H, OH). MS, m/z: 352 [M+]. Found %: C 71.75; H
6.08; N 4.02 C21H21NO4. Calculated %: C 71.78; H 6.02; N 3.99.
C26H25BF2N2O4. Calculated %: C 65.29; H 5.27; N 5.86.
4.1.5.2. 6-{(E)-2-[4-(Dimethylamino)phenyl]vinyl}-3-[(2E)-3-
(4-methoxyphenyl)prop-2-enoyl]-2-oxo-2H-pyran-4-yl difluori-
doborate (19). Yield 42%, mp 295–296 °C. 1H NMR (DMSO-
d6 + TFA) d, ppm: 3.42 (s, 6H, N(CH3)2), 3.83 (s, 3H, OCH3), 6.44
(s, 1H, H5), 6.74 (d, 2H, Har, J = 8.6 Hz), 6.84 (d, 1H, CH,
J = 15.7 Hz), 7.08 (d, 2H, Har, J = 8.7 Hz), 7.60 (d, 2H, Har,
J = 8.6 Hz), 7.74 (d, 1H, CH, J = 15.7 Hz), 7.81 (d, 2H, Har,
J = 8.7 Hz), 8.17 (d, 1H, CH, J = 16 Hz), 8.28 (d, 1H, CH, J = 16 Hz).
MS, m/z: 466 [M+]. Found %: C 64.50; H 4.73; N 3.01. C25H22BF2NO5.
Calculated %: C 64.54; H 4.77; N 3.01.
4.1.6.6. 3-{(2E,4E)-5-[4-(Dimethylamino)phenyl]penta-2,4-die-
noyl}-4-hydroxy-6-methyl-2H-pyran-2-one (14). Yield 72%,
mp 204–205 °C. 1H NMR (DMSO-d6) d, ppm: 2.23 (s, 3H, CH3), 2.30
(s, 6H, N(CH3)2), 6.19 (s, 1H, H5), 6.72 (d, 2H, Har, J = 8.7 Hz.), 7.07–
7.71 (m, 6H, 2Har, 4CH). MS, m/z: 326 [M+]. Found %: C 70.10; H
5.92; N 4.31. C19H19NO4. Calculated %: C 70.14; H 5.89; N 4.30.
4.1.6.7. 4-Hydroxy-3-[(2E,4E)-5-(4-methoxyphenyl)penta-2,4-
dienoyl]-6-methyl-2H-pyran-2-one (16). Yield 74%, mp 156–
157 °C. 1H NMR (CDCl3) d, ppm: 2.28 (s, 3H, CH3), 3.84 (s, 3H,
OCH3), 5.92 (s, 1H, H5), 6.88–7.79 (m, 8H, 4Har, 4CH), 18.18 (s,
1H, OH). MS, m/z: 313 [M+]. Found %: C 69.20; H 5.11. C18H16O5.
Calculated %: C 69.22; H 5.16.
4.1.5.3. 3-[(2E)-3-(5-Bromo-2-methoxyphenyl)prop-2-enoyl]-6-
{(E)-2-[4-(dimethylamino)phenyl]vinyl}-2-oxo-2H-pyran-4-yl
difluoridoborate (21). Yield 38%, mp 345–346 °C. 1H NMR
(DMSO-d6 + TFA) d, ppm: 3.08 (s, 6H, N(CH3)2), 3.96 (s, 3H,
OCH3), 6.37 (s, 1H, H5), 6.71–6.85 (m, 3H, CH, Har), 7.10 (d, 1H,
Har J = 7.8 Hz), 7.53 (d, 1H, Har, J = 7.8 Hz), 7.63 (m, 3H, Har, CH),
,
7.73 (s, 1H, Har), 8.02 (d, 1H, CH, J = 15.8 Hz), 8.23 (d, 1H, CH,
J = 15.8 Hz). MS, m/z: 546 [M+]. Found %: C 55.21; H 3.92; N 2.58.
C25H21BBrF2NO5. Calculated %: C 55.18; H 3.89; N 2.57.
4.1.6.8. 3-{(2E)-3-[4-(Dimethylamino)phenyl]prop-2-enoyl}-6-
{(E)-2-[4-(dimethylamino)phenyl]vinyl}-4-hydroxy-2H-pyran-
2-one (18). Yield 72%, mp 256–257 °C. 1H NMR (DMSO-d6+TFU)
d, ppm: 3.51 (s, 12H, 2N(CH3)2), 6.52 (s, 1H, H5), 6.92 (d, 1H, CH,
J = 15.8 Hz), 7.67 (d, 2H, Har, J = 8.8 Hz), 7.71 (d, 2H, Har, J =
8.7 Hz), 7.75 (d, 1H, CH, J = 15.8 Hz), 7.87 (d, 2H, Har, J = 8.8 Hz),
7.99 (d, 2H, Har, J = 8.7 Hz), 8.03 (d, 1H, CH, J = 16.2 Hz), 8.44 (d,
1H, CH, J = 16.2 Hz). MS, m/z: 431 [M+]. Found %: C 72.50; H
6.01; N 6.50. C26H26N2O4. Calculated %: C 72.54; H 6.09; N 6.51.
4.1.6. General procedure for the preparation of 4, 6, 8, 10, 12, 14,
16, 18, 20 and 22
Appropriate difluoridoborate was dissolved in (10 mL) aqueous
alcoholic solution of sodium carbonate (1.60 g, 15 mmol). The mix-
ture was refluxed for 1–5 h. The reaction mixture was cooled, fil-

K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
8997
where C, N, and D are the fractions of 21-mer substrate converted
into 19-mer (product of 3’-processing) or strand-transfer products
for DNA alone, DNA plus IN without drug and with drug, respec-
tively. IC50 values were determined by plotting the logarithm of
drug concentration as a function of %I to obtain the concentration
that produced 50% inhibition.5,22
4.1.6.9. 6-{(E)-2-[4-(Dimethylamino)phenyl]vinyl}-4-hydroxy-
3-[(2E)-3-(4-methoxyphenyl)prop-2-enoyl]-2H-pyran-2-one
(20). Yield 64%, mp 240–241 °C. 1H NMR (DMSO-d6 + TFA) d,
ppm: 3.06 (s, 6H, N(CH3)2), 3.83 (s, 3H, OCH3), 6.40 (s, 1H, H5),
6.87 (d, 1H, CH, J = 15.7 Hz), 7.67 (d, 2H, Har, J = 8.6 Hz), 7.64 (d,
2H, Har, J = 8.7 Hz), 7.78 (d, 1H, CH, J = 15.7 Hz), 7.84 (d, 2H, Har,
J = 8.6 Hz), 7.95 (d, 2H, Har, J = 8.7 Hz), 8.13 (d, 1H, CH, J = 16 Hz),
8.24 (d, 1H, CH, J = 16 Hz). MS, m/z: 418 [M+]. Found %: C 71.85;
H 5.59; N 3.32. C25H23NO5. Calculated %: C 71.93; H 5.55; N 3.36.
4.2.4. Cytotoxicity assay
Human colon cancer cell line HCT-116 was maintained as a
monolayer culture and 8 ꢂ 103 cells were seeded into each well
on a 96-well tissue culture plate. After overnight attachment, com-
pounds dissolved in RPMI-1640 medium (with 10% bovine serum
4.1.6.10. 3-[(2E)-3-(5-Bromo-2-methoxyphenyl)prop-2-enoyl]-
6-{(E)-2-[4-(dimethylamino)phenyl]vinyl}-4-hydroxy-2H-
pyran-2-one (22). Yield 65%, mp 263–264 °C. 1H NMR (DMSO-
d6+TFA) d, ppm: 3.05 (s, 6H, N(CH3)2), 3.87 (s, 3H, OCH3), 6.32 (s,
1H, H5), 6.85 (d, 1H, CH, J = 16 Hz), 7.05 (m, 3H, Har), 7.55 (m, 2H,
Har, CH), 7.65 (d, 2H, Har, J = 6.8 Hz), 7.74 (s, 1H, Har), 7.97 (d, 1H,
CH, J = 16 Hz), 8.25 (d, 1H, CH, J = 16 Hz). MS, m/z: 497 [M+]. Found
%: C 60.52; H 4.50; N 2.80. C25H22BrNO5. Calculated %: C 60.50; H
4.47; N 2.82.
albumin) were added to a final concentration of 10
72 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) solution (5 mg/mL; 20 L) was added into the cell cul-
lM. After
l
ture plate and incubated with cells for 4 h. The media from each
well was removed and the cell-associated MTT crystals were dis-
solved in DMSO (150 lL/well) on a shaker at room temperature.
The absorbance intensity was measured at 570 nm against appro-
priate blank controls using a microplate reader from Molecular
devices.23
4.2. Biological activity
4.3. Computational studies
4.2.1. Materials, chemicals and enzymes
All compounds were dissolved in DMSO, and stock solutions
Compounds 1–22 were docked onto the active site of IN to
determine the biologically active conformation and possible mech-
anism of binding with IN. Docking calculations were performed
using GOLD24 and eHITS25 software packages on co-crystal struc-
tures of IN (described below).
were stored at ꢀ20 °C.
c
-[32P]-ATP was purchased either from
Amersham Biosciences or ICN. The expression system for wild-type
IN was a generous gift of Dr. Robert Craigie, Laboratory of Molecu-
lar Biology, NIDDK, NIH (Bethesda, MD).
4.2.2. Preparation of oligonucleotide substrates
4.3.1. Ligand preparation
21-mer oligonucleotides [21top (5’-GTGTGGAAAATCTCTAG
CAGT-3’) and 21bot (5’-ACTGCTAGAGATTTTCCA CAC-3’)] were
purchased from Norris Cancer Center Microsequencing Core Facil-
ity (University of Southern California) and purified by UV shadow-
ing on polyacrylamide gel. To analyze the extent of 3’-processing
and strand transfer with 5’-end labeled substrates, 21top was 5’-
end labeled by using T4 polynucleotide kinase (Epicentre, Madison,
The structures of compounds 1–22 were built and minimized
using Catalyst (Accelrys, Inc.) running on a multiprocessor Linux
machine and a 24-processor Silicon Graphics Onyx workstation.
The poling algorithm implemented within Catalyst was used to
generate conformations 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 Cata-
lyst. The lowest energy conformation of each ligand was chosen
for docking simulation.
WI) and [c-
32P]-ATP (Amersham Biosciences or ICN). The kinase
was heat-inactivated and 21bot was added in 1.5 M excess. The
mixture was heated at 95 °C, allowed to slowly cool to room tem-
perature, and purified through a spin 25 minicolumn (USA Scien-
tific, Ocala, FL) to separate annealed double-stranded
oligonucleotide from unincorporated material.
4.3.2. Protein preparation
The crystal structure of IN with its bound inhibitor 5CITEP (1-
(5-chloroindol-3-yl)-3-hydroxy-3-(2H-tetrazol-5-yl)-propenone))
from the Protein Data Bank (PDB entry 1QS4)26 was used for the
docking simulations. Some residues were unresolved in each chain
of the X-ray crystal structure. Chain A, which binds to 5-CITEP and
also orients Mg2+ chelating residues D64 and D116, was selected as
the docking target. The four unresolved residues from chain A were
modeled. Y143 and N144 were obtained from chain B of 1QS4,
while residues I141 and P142 were modeled from the IN structure
1BIS via backbone alignment. Entire modeling and minimization of
protein was done using Insight II suite of software (Accelrys, Inc).
All water molecules present in the crystal structure were removed,
but Mg2+ ion was left unchanged. Hydrogen atoms were added, the
acidic and basic residues in the active site were in their ionic form,
and the protonation state of protein at pH 7.0 was retained during
the docking studies. The ligand (5CITEP) was subsequently re-
moved to make the binding pocket available during simulations
studies.
4.2.3. In vitro IN inhibition assay
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 the reaction buffer [50 mM NaCl, 1 mM HEPES
(pH 7.5), 50 lM EDTA, 50 lM dithiothreitol, 10% glycerol (w/v),
7.5 mM MnCl2, 0.1 mg mLꢀ1 bovine serum albumin, 10 mM 2-
mercaptoethanol, 10% dimethyl sulfoxide, and 25 mM MOPS (pH
7.2)] at 30 °C for 30 min. Next, 20 nM of the 5’-end 32P-labeled lin-
ear oligonucleotide substrate was added, and incubation for an
additional 1 h. Reactions were quenched by addition of 50ꢂ of
loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xy-
lene cyanol, and 0.025% bromophenol blue). An aliquot (5 lL)
was subjected to electrophoresis on a denaturing polyacrylamide
gel (0.09 M Tris–borate, pH 8.3, 2 mM EDTA, 20% acrylamide, 8 M
urea). Gels were dried under vacuum, exposed in a PhosphorImag-
er cassette and visualized with a Typhoon 8610 Variable Mode Im-
ager (Amersham Biosciences). Quantification was done with Image
Quant 5.2 software. Percent inhibition (%I) was calculated using the
following equation:
4.3.3. GOLD v. 3.2
Docking studies of IN inhibitors were carried out using the
GOLD software package running on our multi-processor linux ma-
chine and a 24-processor Silicon Graphics Onyx workstation. A col-
lection of minimized compounds (1–22) were docked on to IN
%I ¼ 100 ꢂ ½1 ꢀ ðD ꢀ CÞ=ðN ꢀ CÞꢃ;
ð1Þ

8998
K. Ramkumar et al. / Bioorg. Med. Chem. 16 (2008) 8988–8998
active site with the GOLD. A spherical area with a radius of 20 Å
was defined and centered at the carboxylate oxygen (OD1) of
D64 in the active site. Standard set parameters of GOLD were used
throughout the simulations. For each of the 10 independent genet-
ic algorithm runs, with a selection pressure of 1.1, 100,000 opera-
tions were performed on a set of 5 islands with a population size of
100 individuals. Default operator weights were used for crossover,
mutation, and migration of 95, 95 and 10, respectively. Default cut-
offs values of 2.5 Å for hydrogen bonds and 4.0 Å for van der Waals
were employed. All other values were set to the default. Top 20
poses were saved for each ligand and best score values were used
to correlate with experimental data.
References and notes
1. Brown, P. O. In Retroviruses; Coffin, J. M., Hughes, S. H., Varmus, H. E., Eds.; Cold
Spring Harbor Laboratory Press: Cold Spring Harbor, Woodbury, NY, 1997; pp
161–203.
2. Dayam, R.; Gundla, R.; Al-Mawsawi, L. Q.; Neamati, N. Med. Res. Rev. 2008, 28,
118.
3. Nair, V.; Chi, G. Rev. Med. Virol. 2007, 17, 277.
4. Neamati, N. Expert Opin. Ther. Pat. 2002, 12, 709.
5. Dayam, R.; Sanchez, T.; Neamati, N. J. Med. Chem. 2005, 48, 8009.
6. Deng, J.; Sanchez, T.; Al-Mawsawi, L. Q.; Dayam, R.; Yunes, R. A.; Garofalo, A.;
Bolger, M. B.; Neamati, N. Bioorg. Med. Chem. 2007, 15, 4985.
7. Carlson, H. A.; Masukawa, K. M.; Rubins, K.; Bushman, F. D.; Jorgensen, W. L.;
Lins, R. D.; Briggs, J. M.; McCammon, J. A. J. Med. Chem. 2000, 43, 2100.
8. Deng, J.; Lee, K. W.; Sanchez, T.; Cui, M.; Neamati, N.; Briggs, J. M. J. Med. Chem.
2005, 48, 1496.
9. Pommier, Y.; Johnson, A. A.; Marchand, C. Nat. Rev. Drug Discov. 2004, 4, 236.
10. 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.; Danovich, R. M.;
Witmer, M. V.; Wilson, K. A. A.; Tussey, L.; Schleif, W. A.; Gabryelski, L. S.; Jin, L.;
Miller, M. D.; Casimiro, D. R.; Emini, E. A.; Shiver, J. W. Science 2004, 305, 528.
11. Cooper, D.; Gatell, J.; Rockstroh, J.; Katlama, C.; Yeni, P.; Lazzarin, A.; Chen, J.;
Isaacs, R.; Teppler, H.; Nguyen, B. Y. Abstract of papers, 14th Conference on
Retroviruses and Opportunistic Infections, Los Angeles, CA, February 25–28,
2007; 105aLB.
12. Dayam, R.; Al-Mawsawi, L. Q.; Neamati, N. Drugs R&D 2007, 8, 155.
13. Sato, M.; Motomura, T.; Aramaki, H.; Matsuda, T.; Yamashita, M.; Ito, Y.;
Kawakami, H.; Matsuzaki, Y.; Watanabe, W.; Yamataka, K.; Ikeda, S.; Kodama,
E.; Matsuoka, M.; Shinkai, H. J. Med. Chem. 2006, 49, 1506.
4.3.4. eHITs
Docking for these 22 compounds was also performed using
eHiTS docking program. All docking simulations using eHITS were
performed on parallel nodes at Sun Grid Compute Utility accessed
at http://Network.com. eHITS automatically evaluates all possible
protonation states of the receptor and ligands for each receptor-li-
gand pair. The docking method systematically covers the confor-
mational and positional search space to avoid severe steric
clashes. The top 20 conformations were saved and eHITs scores
were calculated for each of the 20 saved ligand conformations.
The highest score of each ligand was selected to correlate with
its biological activity.
14. Lataillade, M.; Kozal, M. J. AIDS Patient Care STDS 2006, 20, 489.
´
15. Bransová, J.; Brtko, J.; Uher, M.; Novotny, L. Int. J. Biochem. Cell. Biol. 1995, 27,
701.
16. Gupta, S. P.; Babu, M. S.; Kaw, N. J. Enzyme Inhib. 1999, 14, 109.
17. Barth, R. F.; Coderre, J. A.; Vicente, M. G.; Blue, T. E. Clin. Cancer Res. 2005, 11,
3987.
18. Cígler, P.; Kozísek, M.; Rezácová, P.; Brynda, J.; Otwinowski, Z.; Pokorná, J.;
Plesek, J.; Grüner, B.; Dolecková-Maresová, L.; Mása, M.; Sedlácek, J.; Bodem, J.;
Kräusslich, H. G.; Král, V.; Konvalinka, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
15394.
19. Traven, V. F.; Manaev, A. V.; Chibisova, T. A. Dyes Pigments 2003, 58, 41.
20. Manaev, A. V.; Chibisova, T. A.; Lyssenko, R. A.; Antipin, M. Yu.; Traven, V. F.
Russ. Chem. Bull. 2006, 55, 2091.
21. Manaev, A. V.; Chibisova, T. A.; Traven, V. F. Russ. Chem. Bull. 2006, 55, 2223.
22. Al-Mawsawi, L. Q.; Fikkert, V.; Dayam, R.; Witvrouw, M.; Burke, T. R., Jr.;
Borchers, C. H.; Neamati, N. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10080.
23. Burger, A. M.; Jenkins, T. C.; Double, J. A.; Bibby, M. C. Br. J. Cancer 1999, 81, 367.
24. GOLD. 1.2; CCDC: Cambridge, UK, 2002.
4.3.5. Computational ADMET analysis
Structures of all compounds were exported to ADMET Predictor
(Simulations Plus, Inc.) to calculate properties such as MW, num-
ber of rotatable bonds, number of hydrogen bond donors, number
of hydrogen bond acceptor, and S + logP. AlogP 98 and polar sur-
face area was calculated using Discovery studio (Accelrys, Inc.).
Acknowledgments
Work in Dr. Neamati’s laboratory was supported by funds
from The Campbell Foundation. Work at D. Mendeleev University
of Chemical Technology was carried out as a part of the research
project of the Russian Foundation of Basic Research, N 07-03-
00936.
25. Zsoldos, Z.; Reid, D.; Simon, A.; Sadjad, S. B.; Johnson, A. P. J. Mol. Graph. Model.
2007, 26, 198.
26. Goldgur, Y.; Craigie, R.; Cohen, G. H.; Fujiwara, T.; Yoshinaga, T.; Fujishita, T.;
Sugimoto, H.; Endo, T.; Murai, H.; Davies, D. R. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 13040.