3
062 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 19
Mazumder et al.
column; eluent MeOH:phosphoric acid, 0.5% (v/v), at 1.5 mL/
min (gradient system from 65:35 to 100:0 within 35 min).
Melting points were taken on a Buchi 530 melting point
apparatus and are uncorrected. All experiments were moni-
tored by thin layer chromatography using aluminum sheets
coated with a 0.2 mm layer of silica gel 60 F254 (Merck art.
no. 5554).
H yd r ogen a t ion of Cu r cu m in (5). Tet r a h yd r ocu r -
cu m in (8). Compound 5 (1.1 g, 0.003 mol) in EtOAc (50 mL)
was hydrogenated over 10% Pd/C (100 mg) for 2 h. The
catalyst was removed by filtration, and the filtrate was
evaporated to dryness. The residue was crystallized from
ether/hexane to give 0.9 g (81%) of 8 as colorless crystals: mp
94-95 °C (lit 11 mp 95-96 °C).
P r ep a r a tion of Ra d iola beled DNA Su bstr a tes. The
following oligonucleotides were HPLC purified by and pur-
chased from Midland Certified Reagent Co. (Midland, TX):
AE118, 5′-GTGTGGAAAATCTCTAGCAGT-3′; AE146, 5′-
GGACGCCATAGCCCCGGCGCGGTCGCTTTC-3′; AE156, 5′-
GTGTGGAAAATCTCTAGCAGGGGCTATGGCGTCC-3′;
AE117, 5′-ACTGCTAGAGATTTTCCACAC-3′; AE157, 5′-
GAAAGCGACCGCGCC-3′, AE118S, 5′-GTGTGGAAAAT-
CTCTAGCA-3′. To analyze the extents of 3′-processing or
strand transfer using 5′-end labeled substrates, AE118 was
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
1
-5. 1,7-Bis(4-m eth oxyp h en yl)-1,6-h ep ta d ien e-3,5-d ion e
(
3). 2,4-Pentanedione (1.0 g, 0.01 mol) and boric anhydride
(0.49 g, 0.007 mol) were dissolved in EtOAc (10 mL) and stirred
for 0.5 h at 40 °C. 4-Methoxybenzaldehyde (2.72 g, 0.02 mol)
and tributyl borate (4.6 g, 0.02 mol) were added, and the
reaction mixture was stirred for 0.5 h. After the dropwise
addition of a solution of n-butylamine (1.1 g, 0.015 mol) in
EtOAc (10 mL) over a period of 15 min, the mixture was stirred
for a further 24 h at 40 °C. The mixture was hydrolyzed by
the addition of 10% HCl (10 mL) and heating to 60 °C for 1 h.
The organic layer was separated, and the aqueous layer was
extracted three times with EtOAc. The combined organic
5′-end-labeled using T
4
polynucleotide kinase (Gibco BRL) and
3
2
[γ- P]ATP (DuPont-NEN). The kinase was heat-inactivated,
and AE117 was added to the same final concentration. The
mixture was heated at 95 °C, allowed to cool slowly to room
temperature, and run on a G-25 Sephadex quick spin column
(Boehringer Mannheim) to separate annealed double-stranded
oligonucleotide from unincorporated label.
layers were washed with water and dried over Na
2 4
SO .
Evaporation of the solvent left a yellow powder which was
purified by column chromatography (silica gel, cyclohexane-
EtOAc, 50:50 (v/v)) to give 3 as crystals from ether/hexane (2.2
1
g, 65%): mp 138 °C; H NMR (DMSO-d
(
6
) δ 3.81 (6 H, s), 6.10
To analyze the extent of strand transfer using the “pre-
cleaved” substrate, AE118S was 5′-end-labeled, annealed to
AE117, and column purified as above. To determine the extent
of 30-mer target strand generation during disintegration,
AE156 was 5′-end-labeled, annealed to AE157, AE146, and
AE117, and column purified as above.
1 H, s), 6.79 (2 H, d, J ) 15.8 Hz), 7.01 (4 H, d, J ) 8.5 Hz),
7
.60 (2 H, d, J ) 15.8 Hz), 7.69 (4 H, d, J ) 8.5 Hz); EIMS
•
+
m/ z 336 (M , 51). Anal. (C21
20 4
H O ) C, H.
1
,7-Dip h en yl-1,6-h ep ta d ien e-3,5-d ion e (1): crystals from
9
ether/hexane; mp 141-142 °C (lit. mp 139-140 °C).
1
2): crystals from ether/hexane; mp 224 °C (lit. mp 223-224
C).
,7-Bis(4-h yd r oxyp h e n yl)-1,6-h e p t a d ie n e -3,5-d ion e
In tegr a se P r otein s a n d Assa ys. Recombinant HIV-1
9
23
(
°
integrase was purified as described.
Integrase at a final
concentration of 200 nM was preincubated with inhibitor for
30 min at 30 °C in reaction buffer (50 mM NaCl, 1 mM
HEPES, pH 7.5, 50 µM EDTA, 50 µM dithiothreitol, 10%
1
,7-Bis(4-h yd r oxy-3-m et h oxyp h en yl)-1,6-h ep t a d ien e-
3
,5-d ion e (cu r cu m in , 5): crystals from MeOH; mp 180-182
9
°
C (lit. mp 182-183 °C).
-(4-Hyd r oxyp h en yl)-7-(4-h yd r oxy-3-m eth oxyp h en yl)-
,6-h ep ta d ien e-3,5-d ion e (4). 4 was prepared by modifica-
glycerol (wt/vol), 7.5 mM MnCl
2
, 0.1 mg/mL bovine serum
1
albumin, 10 mM 2-mercaptoethanol, 10% dimethyl sulfoxide,
3
2
1
and 25 mM MOPS, pH 7.2). Then, 20 nM of the 5′-end P-
labeled linear oligonucleotide substrate was added, and the
reaction continued for an additional 1 h. The final reaction
volume was 16 µL.
tion of the above-described method using 4-hydroxybenzalde-
9
hyde and vanillin as aldehydes. Separation of the products
4
by thin layer chromatography (silica gel 60 PF254 with CaSO ,
cyclohexane-EtOAc, 80:20 (v/v)) using the technique of radial
The disintegration reactions were performed as above with
development (Chromatotron) gave 18% of pure 4: mp 170-
the “Y”-branched oligonucleotide substrate and an integrase
9
50-212
1
71 °C (lit. mp 172-173 °C).
deletion mutant, IN
, which lacks the N-terminal zinc
Dem eth yla tion of Cu r cu m in (5). 1,7-Bis(3,4-d ih yd r ox-
finger and C-terminal DNA-binding domains.
yp h en yl)-1,6-h ep ta d ien e-3,5-d ion e (6) a n d 1-(3,4-Dih y-
d r oxyp h en yl)-7-(4-h yd r oxy-3-m et h oxyp h en yl)-1,6-h ep -
ta d ien e-3,5-d ion e (7). In a solution of 5 (5.5 g, 0.015 mol)
E lect r op h or esis a n d Qu a n t it a t ion . Reactions were
quenched by the addition of an equal volume (16 µL) of
Maxam-Gilbert 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). Gels were dried,
exposed in a Molecular Dynamics PhosphorImager cassette,
and analyzed using a Molecular Dynamics PhosphorImager
(Sunnyvale, CA). Percent inhibition was calculated using the
following equation:
2 2 3
in CH Cl (250 mL) was suspended AlCl (6.7 g, 0.05 mol), and
pyridine (16 mL, 0.2 mol) was added dropwise under stirring.
The mixture was refluxed for 24 h, cooled in ice, and acidified
with diluted HCl. The aqueous layer was extracted exhaus-
tively with EtOAc. The combined organic layers were sepa-
rated and evaporated to dryness. The residue was dissolved
in MeOH (5 mL) and separated by thin layer chromatography
(
5
(
silica gel 60 PF254 with CaSO
5:40:5 (v/v/v)) using the technique of radial development
Chromatotron) to give 3.1 g (60%) of 6 and 0.8 g (15%) of 7.
,7-Bis(3,4-d ih yd r oxyp h en yl)-1,6-h ep t a d ien e-3,5-d i-
4
, cyclohexane-EtOAc-EtOH,
%I ) 100 × [1 - (D - C)/(N - C)]
1
1
on e (6): yellow powder from CHCl
NMR (DMSO-d ) δ 6.08 (1 H, s), 6.57 (2 H, d, J ) 15.8 Hz),
.79 (2 H, d, J ) 8.2 Hz), 7.02 (2 H, dd, J ) 8.2, 1.5 Hz), 7.08
2 H, d, J ) 1.5 Hz), 7.46 (2 H, d, J ) 15.8 Hz), 8.7-9.9 (4 H,
br s, D
O exchangeable); EIMS m/ z 340 (M•+, 5.0). Anal.
) C, H.
-(3,4-Dih yd r oxyp h e n yl)-7-(4-h yd r oxy-3-m e t h oxy-
3
; mp 306-308 °C dec; H
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 integrase, and integrase
plus drug, respectively. IC50 was determined by plotting the
drug concentration versus percent inhibition and determining
the concentration which produced 50% inhibition.
6
6
(
2
(
19 16 6
C H O
1
DNA Bin d in g via Sch iff Ba se F or m a tion . This proce-
6
p h en yl)-1,6-h ep ta d ien e-3,5-d ion e (7): yellow powder from
dure was performed essentially as described previously.
1
CHCl
.06 (1 H, s), 6.58 (1 H, d, J ) 15.8 Hz), 6.75 (1 H, d, J ) 15.8
Hz), 6.79 (1 H, d, J ) 8.2 Hz), 6.82 (1 H, d, J ) 8.2 Hz), 7.02
1 H, dd, J ) 8.2, 1.8 Hz), 7.09 (1 H, d, J ) 1.8 Hz), 7.14 (1 H,
dd, J ) 8.2, 1.8 Hz), 7.32 (1 H, d, J ) 1.8 Hz), 7.47 (1 H, d, J
15.8 Hz), 7.54 (1 H, d, J ) 15.8 Hz), 9.19 (1 H, br s, D
exchangeable), 9.60-9.70 (2 H, br s, D O exchangeable);
(M ) 354.1103, obsd 354.1101.
3
; mp 165-167 °C; H NMR (DMSO-d
6
) δ 3.84 (3 H, s),
Integrase (200 nM) was preincubated with the inhibitor for
30 min at 30 °C in reaction buffer (see above). Then, the viral
DNA substrate (20 nM) containing a single enzymatically
generated abasic site was added for 2 min at room tempera-
ture. Sodium borohydride was added for an additional 5 min.
An equal volume (16 µL) of 2× SDS-PAGE buffer (100 mM
Tris, pH 6.8, 4% 2-mercaptoethanol, 4% SDS, 0.2% bromophe-
nol blue, and 20% glycerol) was added to each reaction mixture,
and the reaction mixture was heated at 95 °C for 3 min prior
6
(
)
2
O
2
•
+
HRMS m/ z calcd for C20
Anal. (C20 ) C, H.
18 6
H O
18 6
H O