Amide-containing diketoacids as HIV-1 integrase inhibitors: Synthesis, structure-activity relationship analysis, and biological activity

Article abstract of DOI:10.1016/j.bmc.2009.01.077

HIV-1 integrase, which catalyzes the integration of the viral genome into the cellular chromosome, is an essential enzyme for retroviral replication, and represents an attractive and validated target in the development of therapeutics against AIDS. In this paper, 17 amide-containing novel diketoacids were designed and synthesized, and their ability to inhibit HIV-1 integrase was tested. The structure-activity relationships were also analyzed.

Full text of DOI:10.1016/j.bmc.2009.01.077

Bioorganic & Medicinal Chemistry 17 (2009) 2913–2919
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Amide-containing diketoacids as HIV-1 integrase inhibitors: Synthesis,
structure–activity relationship analysis, and biological activity
a,
Hongcai Li a, Chao Wang a, Tino Sanchez b, Yanmei Tan a, Chunying Jiang a, Nouri Neamati b, , Guisen Zhao
*
*
a School of Pharmaceutical Sciences, Shandong University, Ji’nan, Shandong Province 250012, PR China
b Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
HIV-1 integrase, which catalyzes the integration of the viral genome into the cellular chromosome, is an
essential enzyme for retroviral replication, and represents an attractive and validated target in the devel-
opment of therapeutics against AIDS. In this paper, 17 amide-containing novel diketoacids were designed
and synthesized, and their ability to inhibit HIV-1 integrase was tested. The structure–activity relation-
ships were also analyzed.
Received 12 December 2008
Revised 30 January 2009
Accepted 31 January 2009
Available online 7 February 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HIV
Integrase inhibitors
Diketoacids
Structure–activity relationship
1. Introduction
Among the reported HIV-1 IN inhibitors, diketo-containing
compounds, mainly aryl diketoacids (ADK) and their derivatives,
Human immunodeficiency virus type-1 (HIV-1) is the etiologi-
cal agent of acquired immunodeficiency syndrome (AIDS). Three
different classes of chemotherapeutic agents are actually available
to block the replication of HIV-1, namely reverse transcriptase
inhibitors, protease inhibitors, and fusion inhibitors.1 Highly active
anti-retroviral therapy (HAART) based on the use of the above
drugs effectively inhibits the replication cycle of HIV-1. However,
HAART fails to eradicate viral replication, and the emergence of
multi-drug-resistant viral strains in infected patients can compli-
cate the response to the treatment.2,3
In addition to reverse transcriptase and protease, another target
useful for chemotherapeutic intervention is the HIV-1 integrase
(IN), an essential enzyme that catalyzes the insertion of the viral
DNA into the genome of the host cell through a complex process,
which consists of three biochemical steps: (i) cleavage of a dinucle-
otide pair from the 30-end of the viral DNA (termed ‘30-processing’,
30-P), (ii) insertion of the resulting shortened strands into the host-
cell chromosome (termed ‘strand transfer’, ST), and (iii) removal of
the two unpaired nucleotides at the 50-end of the viral DNA and
gap-filling process.4 Due to the absence of any known human
homolog,5 IN is considered as an attractive and validated target
for the development of novel anti-HIV drugs.
represent a most promising class of compounds.6 The mechanism
appears to involve the binding of the diketoacid portion to the
Mg2+ or Mn2+ co-factor located in the active site of the enzyme.7
Several IN inhibitors containing the aryl diketo moiety have been
O
OH
N
O
OH
H
N
N
N
O
H
N
F
N N
O2S
F
S-1360
O
L-870810
O
OH
F
O
O
N
H
Cl
OH
N
N
F
O
O
N
O
N
OH
H
N
N
GS-9137
OH
MK-0518
O
H
Ar
R
N
OH
O
O
* Corresponding authors. Tel.: +1 323 442 2341; fax: +1 323 442 1390 (N.N.); tel.:
+86 531 88382009; fax: +86 531 88382835 (G.Z.).
E-mail addresses: neamati@usc.edu (N. Neamati), guisenzhao@sdu.edu.cn (G.
Zhao).
amide-containing diketoacids
Figure 1. The structure of selected ADK-based clinically studied IN inhibitors and
amide-containing diketoacids.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.077

2914
H. Li et al. / Bioorg. Med. Chem. 17 (2009) 2913–2919
O
H2N
CH3
O
H
HO
Ar
AcO
Ar OH
Cl
AcO
Ar
N
a,b
CH3
O
O
O
c
1(a-b,f,j-l)
2(a-b,f,j-l)
3(a-b,f,j-l)
O
OH
O
OH
H
H
d
e
AcO Ar
HO
Ar
N
OC2H5
N
OH
O
O
O
O
4(a-b,f,j-l)
O2N
OH
HO
b
HO
a
HO
k
f
j
l
H3C
Ar
=
HO
O2N
H3CO
OH
OH
Scheme 1. Reagents and conditions: (a) Ac2O, pyridine, rt, 24 h; (b) SOCl2, 80 °C, 5 h; (c) THF, rt, <18 h; (d) (CO2C2H5)2, NaOCH3, THF, rt, <18 h, then 1 M HCl; (e) 1 M NaOH,
THF/CH3OH (1:1), rt, 1 h, then 1 M HCl.
tested in the clinic, including S-1360, L-870810, GS-9137, and MK-
0518 (Fig. 1). Especially MK-0518, also known as raltegravir, has
been approved by the FDA as the first IN inhibitor among the clin-
ical anti-HIV drugs.8 Previous studies in this field,7,9 have sug-
gested that an H-bond donating group is required for activity.
Further studies with the chemical structures of many potent IN
inhibitors have documented that an amide group is well toler-
ated.7,10–12 In fact L-870810 and MK-0518 (Fig. 1) also contain
one or two amide groups in their templates. In this paper, seven-
teen amide-containing novel diketoacids (Fig. 1) were designed
and synthesized and their inhibitory activities on HIV-1 IN were
tested, and the structure–activity relationships were analyzed.
oxide provided an intermediate as an orange oil, which was hydro-
lyzed with aqueous sodium hydroxide, and then treated with
hydrochloric acid to afford b-diketo acids (4) with various substit-
uents on the phenyl ring in good yield (Scheme 1).
Most of the compounds reported in this paper were prepared as
depicted in Scheme 2. Different from Scheme 1, the various acyl
chlorides (2) were obtained directly by treating substituted aro-
matic acids with thionyl chloride at 80 °C. Then, a similar method
was applied to the synthesis of compounds 3 and 4, as shown in
Scheme 2.
2.2. Inhibitory activity of target compounds (4) against HIV-1
integrase in vitro
2. Results and discussion
2.1. Chemistry
All synthesized compounds were tested for their ability to
inhibit 30-processing (30-P) and strand transfer (ST). The data are
summarized in Table 1. Compound 4k had the best efficacy on both
30-P and ST with an IC50 value of 8 and 2
l
M, respectively. The
other compounds showed moderate ST inhibitory potency with
IC50 values from 4 to 33 M.
The chemical synthetic routes of target compounds are pre-
sented in Schemes 1 and 2. Analogues containing hydroxyl groups
on the terminal aryl ring (4a–b, 4j–l, and 4f) were prepared as
shown in Scheme 1. Various phenolic hydroxyl groups of carbox-
ylic acids were protected with ethanoyl in the presence of pyridine,
and then reacted with thionyl chloride under reflux at 80 °C to give
the substituted acyl chloride (2). Using 3-aminoacetophenone as
raw material, compound (3) was synthesized by acylation with
the substituted acyl chloride (2) in THF at room temperature.
Treatment of compound (3) with diethyl oxalate and sodium meth-
l
The structure–activity relationships (SAR) in these novel com-
pounds were investigated by introducing substituents on the ter-
minal aryl ring, and increasing the length of the carbon chain
between the terminal benzene ring and the amide group.
We started our work focusing on extending the chain length be-
tween the terminal aromatic ring and the amide group, to enhance
the binding interaction between the compounds and IN, and thus
O
H2N
CH3
O
H
Ar OH
Ar
Cl
Ar
N
a
CH3
O
O
O
b
1(c-e,g-i,m-q) 2(c-e,g-i,m-q)
3(c-e,g-i,m-q)
O
OH
O
H
OH
O
H
d
c
Ar
Ar
N
OC2H5
N
O
OH
O
O
4(c-e,g-i,m-q)
Cl
H3CO
F
g
c
h
Ar=
d
e
Cl
OCH3
H3CO
q
p
o
m
n
i
Cl
O
H3CO
Scheme 2. Reagents and conditions: (a) SOCl2, 80 °C, 5 h; (b) THF, rt, <18 h; (c) (CO2C2H5)2, NaOCH3, THF, rt, <18 h, then 1 M HCl; (d) 1 M NaOH, THF/CH3OH (1:1), rt, 1 h, then
1 M HCl.

H. Li et al. / Bioorg. Med. Chem. 17 (2009) 2913–2919
2915
Table 1 (continued)
Table 1
Structures and HIV IN inhibitory activities of target compounds
a
a
Code
Structure
30-P IC50
M)
ST IC50
(lM)
a
a
Code
Structure
30-P IC50
M)
ST IC50
M)
(
l
(l
(l
Cl
HO
O
OH
COOH
H
O
OH
COOH
H
N
4p
92
57
9
5
18
15
2
5
N
4a
81 11
13
7
2
O
O
O
OH
COOH
H
HO
F
O
OH
COOH
H
N
4q
Cl
4b
4c
4d
4e
4f
26
22
18
30
42
6
4
6
5
8
2
2
1
N
O
O
a
IC50: Inhibitory concentration 50% (inhibition of purified integrase).
O
OH
COOH
H
N
7
O
improve their activities. As shown in Table 1, appending an ethyl-
enic linkage resulted in a two to threefold increase in both 30-P and
ST inhibitory potency (4b vs 4a and 4e vs 4o). Compound 4b
Cl
O
OH
H
4
N
COOH
showed enzyme inhibitory activities with IC50 values of 26
lM
O
and 7
l
M for 30-P and ST, respectively. Furthermore, addition of a
methylene between the terminal aromatic ring and the amide
group also provided a twofold enhancement in 30-P inhibitory
potency and a fourfold higher inhibitory activity against ST
(4h vs 4g).
H3CO
O
O
OH
H
11
11
31
7
4
1
2
N
COOH
O
Encouraged by the improved potency of compound 4b versus
4a, we continued our optimization effort by changing the phenyl
tail group with various substituents. As shown by compounds
4b–f, both electron-withdrawing and electron-donating substitu-
ents on the terminal benzene ring were tolerated. Compared to
the parent compound 4b, substitution of the 4-OH with a methoxyl
(4e) decreased IN inhibition activities, whereas fluorine (4c)
showed similar activity and chlorine (4d) enhanced the 30-P and
ST inhibitory potency. The chlorine-substituted compound 4d
was the most active among the four analogues 4b–e. Moreover,
it was found that introducing a methoxyl at the 30-position (4f)
had little impact on activity. A similar observation was made with
compounds 4j and 4l, where addition of a methoxyl at the 3-posi-
tion had minimal change in activity. Compound 4k with a 3,5-dini-
trophenyl group showed the best inhibitory profile, with IC50
HO
OH
COOH
H
N
H3CO
O
O
OH
H
N
4g
4h
4i
>100
COOH
O
O
OH
H
N
56
6
1
COOH
O
O
O
OH
COOH
H
N
>100
41 12
33
11
2
3
O
values of 8 lM and 2 l
M for 30-P and ST, respectively.
O
Utilizing the strategy of the bioisosterism principle, we replaced
the terminal benzene group with a furan ring. As exemplified by
compound 4i, replacement of the benzene ring with a furan group
displayed HIV-1 IN inhibitory activities similar to 4g.
OH
COOH
H
N
4j
OH O
Continuing SAR studies of the substituted groups with different
locations on the terminal aryl ring, we investigated compounds 4a,
4j, and 4m–q. It was noted that 3-methoxy and 3-chloro substi-
tuted compounds (4n and 4q) showed a slight improvement in
30-P inhibitory potency, but had similar levels of ST activity relative
to the compounds with 2-substitutions (4m and 4p). A methoxyl
group in the 4-position (4o) exhibited a little loss in the IN
inhibitory activities compared to 4m and 4n. However, it is worth
noting that compound 4j, with a hydroxyl substituent at C-2,
showed a twofold increase in 30-P inhibition and similar ST activity
versus the C-4 hydroxyl substituent 4a. Moreover the C-2 hydroxyl
substituent 4k and 4l also exhibited noticeable 30-P and ST inhibi-
tory potency. The results imply that another diketo moiety was
formed between the benzamide group and the C-2 hydroxyl group,
which might bind to two divalent metal ions on the active site of
IN.
NO2
O
OH
COOH
H
N
4k
4l
8
2
0.4
4
O2N
OH O
O
OH
COOH
H
N
48 22
10
13
12
19
H3C
OH O
OCH3
O
OH
COOH
H
4m
4n
4o
73
2
N
O
O
OH
COOH
H
N
69 10
1
1
H3CO
O
O
In conclusion, 17 novel amide-containing diketoacids were de-
signed and synthesized as HIV-1 IN inhibitors. The experiments
on the inhibition on HIV-1 IN indicated that (1) the chain length
between the terminal aromatic ring and the amide group had a
large influence on the IN activities. With the exception of C-2 hy-
droxy-substituted benzamide compounds (4j–l), introducing an
H3CO
O
OH
COOH
H
N
95
5
(continued on next page)

2916
H. Li et al. / Bioorg. Med. Chem. 17 (2009) 2913–2919
ethylenic linkage or methylene could enhance the IN inhibitory
activity; (2) both electron-withdrawing and electron-donating
substituents on the terminal aromatic ring were tolerated, but
mostly electron-withdrawing substituents showed better potency;
(3) the terminal aromatic group could be replaced by a furan ring,
and it showed little impact on activities; (4) changing the positions
of the substituents on the terminal aryl ring slightly affected activ-
ities, except for the hydroxy-substituted benzamide compounds.
Because a novel diketo moiety could be formed in benzamide com-
pounds with the 2-hydroxy group, improved activities were
observed.
(10 mL ꢁ 3), 5% sodium bicarbonate solution (10 mL ꢁ 3), and
saturated sodium chloride solution (10 mL ꢁ 3). The organic phase
was dried over Na2SO4 overnight. After filtering, the solvent was
evaporated under reduced pressure to obtain products 3.
3.2.1. 4-(3-Acetylphenylcarbamoyl)phenyl acetate (3a)
White powder, yield 76.2%, mp 138–139 °C, TLC Rf = 0.50
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.48 (s, 1H), 8.36 (s, 1H), 8.07 (d, J = 7.80 Hz, 1H), 8.04 (d,
J = 7.20 Hz, 2H), 7.72 (d, J = 7.80 Hz, 1H), 7.52 (t, J = 7.80 Hz, 1H),
7.32 (d, J = 7.20 Hz, 2H), 2.58 (s, 3H), 2.32 (s, 3H). MS (ESI) m/z
298.6 [M+H]+.
3. Experimental
3.2.2. 4-(3-(3-Acetylphenylamino)-3-oxoprop-1-enyl)phenyl
acetate (3b)
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification.
Melting points were determined on a Büchi capillary melting
point apparatus. Infrared (IR) spectra were measured on KBr pel-
lets, using a Nicolet Nexus 470FT-IR and were expressed in
cmꢀ1. Proton nuclear magnetic resonance (1H NMR) spectra were
White powder, yield 58.2%, mp 118–119 °C, TLC Rf = 0.42
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.47 (s, 1H), 8.26 (s, 1H), 7.98 (d, J = 7.80 Hz, 1H), 7.72 (d,
J = 7.80 Hz, 1H), 7.69 (m, 2H), 7.63 (d, J = 16.20 Hz, 1H), 7.32 (d,
J = 7.20 Hz, 2H), 7.23 (d, J = 8.40 Hz, 1H), 6.80 (d, J = 15.60 Hz,
1H), 2.56 (s, 3H), 2.31 (s, 3H). MS (ESI) m/z 324.5 [M+H]+.
recorded with
DMSO-d6 as the solvent and tetramethyl-silane (TMS) as the
internal standard. The chemical shifts were reported in
a Bruker Avance DRX600 spectrometer with
3.2.3. N-(3-Acetylphenyl)-3-(4-fluorophenyl)acrylamide (3c)
White powder, yield 52.4%, mp 158–160 °C, TLC Rf = 0.49
(petroleum/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d: 10.44 (s,
1H), 8.27 (s, 1H), 7.97 (d, J = 8.40 Hz, 1H), 7.77 (m, 1H), 7.72 (m,
2H), 7.62 (d, J = 16.20 Hz, 1H), 7.30 (t, J = 8.00 Hz, 2H), 7.25 (t,
J = 8.00 Hz, 1H), 6.78 (d, J = 15.60 Hz, 1H), 2.58 (s, 3H). MS (ESI)
m/z 284.5 [M+H]+.
d
(ppm). Mass spectra (MS) were measured with an API 4000 mass
spectrometer, and the high resolution mass spectra data were
obtained using an Accela UPLC-LTQ Orbitrap Mass Spectrometer.
Thin-layer chromatography (TLC) was performed on silica gel
GF254 plates (layer thickness, 0.2 mm) and the compounds were
visualized using UV light. Petroleum ether used for TLC had a
boiling range of 60–90 °C.
3.2.4. N-(3-Acetylphenyl)-3-(4-chlorophenyl)acrylamide (3d)
White powder, yield 55.1%, mp 114–115 °C, TLC Rf = 0.46
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.46 (s, 1H), 8.26 (s, 1H), 7.97 (d, J = 7.80 Hz, 1H), 7.70 (d,
J = 7.80 Hz, 1H), 7.67 (d, J = 9.00 Hz, 2H), 7.61 (d, J = 15.60 Hz,
1H), 7.52 (d, J = 8.40 Hz, 2H), 7.50 (d, J = 7.80 Hz, 1H), 6.83 (d,
J = 15.60 Hz, 1H), 2.58 (s, 3H). MS (ESI) m/z 300.6 [M+H]+.
3.1. General methods for preparing substituted acyl chlorides 2
Substituted aromatic acid (15 mmol), acetic anhydride
(20 mmol) and two drops of pyridine were placed in a 50 mL
three-necked flask equipped with an electromagnetic stirrer. The
reaction proceeded at room temperature with stirring for 24 h.
The solvent was evaporated under reduced pressure to obtain acyl-
ation products. Then, sulfone chloride (20 mmol) was added to the
flask and the reaction was allowed to reflux at 80 °C overnight. The
solvent was evaporated under reduced pressure at 50 °C to obtain
the products 2(a–b, f, j–l). The products were diluted with 10 mL
anhydrous tetrahydrofuran and kept on standby.
Other acyl chlorides 2(c–e, g–i, m–q) were synthesized simi-
larly by the following procedures. Sulfone chloride (20 mmol)
was added to a 50 mL one-necked flask that contained the substi-
tuted aromatic acids 1(c–e, g–i, m–q) (0.06 mol). The reaction
was left to reflux at 80 °C overnight. The solvent was evaporated
under reduced pressure at 50 °C to obtain the product, which was
then diluted with 10 mL anhydrous tetrahydrofuran and kept on
standby.
3.2.5. N-(3-Acetylphenyl)-3-(4-methoxyphenyl)acrylamide (3e)
White powder, yield 50.3%, mp 143–145 °C, TLC Rf = 0.45
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.46 (s, 1H), 8.27 (s, 1H), 7.98 (d, J = 7.80 Hz, 1H), 7.69 (d,
J = 8.40 Hz, 3H), 7.63 (d, J = 16.20 Hz, 1H), 7.50 (t, J = 7.80 Hz, 1H),
7.22 (d, J = 8.40 Hz, 2H), 6.80 (d, J = 15.60 Hz, 1H), 2.58 (s, 3H),
2.28 (s, 3H). MS (ESI) m/z 296.6 [M+H]+.
3.2.6. 4-(3-(3-Acetylphenylamino)-3-oxoprop-1-enyl)-2-
methoxyphenyl acetate (3f)
White powder, yield 51.6%, mp 160–162 °C, TLC Rf = 0.45
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.47 (s, 1H), 8.28 (s, 1H), 7.90 (m, 2H), 7.69 (d, J = 7.80 Hz, 1H),
7.63 (d, J = 16.80 Hz, 1H), 7.42 (m, 2H), 7.18 (d, J = 7.80 Hz, 1H),
6.82 (d, J = 15.60 Hz, 1H), 3.84 (s, 3H), 2.58 (s, 3H), 2.28 (s, 3H).
MS (ESI) m/z 354.4 [M+H]+.
3.2. General methods for preparing amide compounds 313
In a 100 mL three-necked flask equipped with an electromag-
netic stirrer, dropping funnel, and a reflux condenser bearing a cal-
cium chloride drying tube, was placed 3-aminoacetophenone
(20 mmol), which was dissolved in anhydrous tetrahydrofuran
(15 mL). The acyl chloride solution (compound 2) (20 mmol) was
added dropwise with stirring in an ice bath. The reaction was con-
tinued at room temperature with stirring overnight. The solution
was filtered and the filter cake was washed twice with tetrahydro-
furan. The filtrate and washings of tetrahydrofuran were mixed
and diluted with dichloromethane, and then washed with 1 M
hydrochloric acid solution (10 mL ꢁ 3), distilled water
3.2.7. N-(3-Acetylphenyl)benzamide (3g)
White powder, yield 79.8%, mp 99–101 °C, TLC Rf = 0.58 (petro-
leum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d: 10.46 (s,
1H), 8.38 (s, 1H), 8.08 (d, J = 7.80 Hz, 1H), 7.98 (d, J = 7.20 Hz,
2H), 7.72 (d, J = 7.80 Hz, 1H), 7.62 (t, J = 7.20 Hz, 1H), 7.53 (m,
3H), 2.59 (s, 3H). MS (ESI) m/z 240.4 [M+H]+.
3.2.8. N-(3-Acetylphenyl)-2-phenylacetamide (3h)
White powder, yield 64.2%, mp 125–126 °C, TLC Rf = 0.54
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.47 (s, 1H), 8.32 (s, 1H), 7.93 (d, J = 8.40 Hz, 1H), 7.74 (d,

H. Li et al. / Bioorg. Med. Chem. 17 (2009) 2913–2919
2917
J = 7.80 Hz, 1H), 7.62 (m, 1H), 7.50 (t, J = 7.80 Hz, 2H), 7.25 (t,
J = 7.20 Hz, 1H), 7.06 (t, J = 7.20 Hz, 2H), 3.69 (s, 2H), 2.47 (s, 3H).
MS (ESI) m/z 254.3 [M+H]+.
3.2.17. N-(3-Acetylphenyl)-3-chlorobenzamide (3q)
White powder, yield 65.8%, mp 138–139 °C, TLC Rf = 0.53
(petroleum ether/acetic ether 1:1, v/v). d: 10.55 (s, 1H), 8.36 (s,
1H), 8.08 (d, J = 7.80 Hz, 1H), 8.06 (s, 1H), 7.95 (d, J = 7.80 Hz,
1H), 7.74 (d, J = 7.80 Hz, 1H), 7.69 (d, J = 7.80 Hz, 1H), 7.59 (t,
J = 7.80 Hz, 1H), 7.53 (t, J = 7.80 Hz, 1H), 2.59 (s, 3H). MS (ESI) m/z
274.3 [M+H]+.
3.2.9. N-(3-Acetylphenyl)furan-2-carboxamide (3i)
White powder, yield 41.1%, mp 132–134 °C, TLC Rf = 0.43
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.40 (s, 1H), 8.34 (s, 1H), 8.05 (d, J = 7.80 Hz, 1H), 7.97 (s, 1H),
7.71 (d, J = 7.80 Hz, 1H), 7.51 (t, J = 7.80 Hz, 1H), 7.38 (s, 1H), 6.72
(s, 1H), 2.58 (s, 3H). MS (ESI) m/z 230.2 [M+H]+.
3.3. General methods for preparing amide-containing
diketoacids 414
3.2.10. 2-(3-Acetylphenylcarbamoyl)phenyl acetate (3j)
White powder, yield 62.8%, mp 82–83 °C, TLC Rf = 0.52 (petro-
leum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d: 10.55 (s,
1H), 8.31 (s, 1H), 7.97 (m, 2H), 7.75 (d, J = 7.80 Hz, 1H), 7.53 (t,
J = 7.80 Hz, 1H), 7.45 (t, J = 7.80 Hz, 1H), 6.98 (m, 2H), 2.59 (s,
3H), 2.18 (s, 3H). MS (ESI) m/z 298.6 [M+H]+.
Cleanly cut sodium (0.69 g, 0.03 mol) was placed in a 100 mL
three-necked flask, equipped with an electromagnetic stirrer, drop-
ping funnel, and a reflux condenser bearing a calcium chloride dry-
ing tube. Absolute methanol was added dropwise. After all the
sodium had dissolved, the excess methanol was removed by re-
duced pressure distillation. Anhydrous tetrahydrofuran (15 mL)
was added when the sodium methoxide was cooled to room tem-
perature. A mixture of compounds 3 (10 mmol) and diethyl oxalate
(20 mmol) was added dropwise with stirring, over an ice bath. The
reaction proceeded at room temperature with stirring overnight. A
solution of hydrochloric acid (1 M, 30 mL) and ice water was added
into the reaction solution. The solution was extracted with dichlo-
romethane (10 mL ꢁ 3). The extract liquid was washed with satu-
rated sodium chloride solution (10 mL ꢁ 3), and was then dried
over Na2SO4. After filtering, the solvent was evaporated under re-
duced pressure to obtain an orange-red oil.
The oil was dissolved in a mixed solution of methanol and tet-
rahydrofuran (1:1, 15 mL). Sodium hydroxide solution (1 M,
20 mL) was added dropwise with stirring. The reaction proceeded
at room temperature for 1 h. The reaction solution was washed
with diethyl ether (10 mL ꢁ 3), and the water phase was acidified
with hydrochloric acid solution (1 M) to pH 1–2. The resulting solid
was collected, washed by diethyl ether and acetic ether, and dried
to obtain compounds 4.
3.2.11. 2-(3-Acetylphenylcarbamoyl)-4,6-dinitrophenyl acetate
(3k)
Yellow power, yield 47.6%, mp 200–201 °C, TLC Rf = 0.43 (petro-
leum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d: 11.08 (s,
1H), 9.08 (s, 1H), 8.86 (s, 1H), 8.28 (s, 1H), 7.93 (d, J = 8.40 Hz,
1H), 7.79 (d, J = 8.40 Hz, 1H), 7.57 (t, J = 7.80 Hz, 1H), 2.60 (s, 3H),
2.16 (s, 3H). MS (ESI) m/z 388.5 [M+H]+.
3.2.12. 2-(3-Acetylphenylcarbamoyl)-6-methylphenyl acetate
(3l)
White powder, yield 51.8%, mp 124–126 °C, TLC Rf = 0.48
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.52 (s, 1H), 8.40 (s, 1H), 8.10 (d, J = 7.80 Hz, 1H), 7.97 (d,
J = 7.80 Hz, 1H), 7.88 (d, J = 8.40 Hz, 1H), 7.60 (t, J = 8.10 Hz, 1H),
7.40 (d, J = 7.20 Hz, 1H), 7.08 (t, J = 7.80 Hz, 1H), 2.59 (s, 3H), 2.21
(s, 3H), 2.12 (s, 3H). MS (ESI) m/z 312.5 [M+H]+.
3.2.13. N-(3-Acetylphenyl)-2-methoxybenzamide (3m)
White powder, yield 59.7%, mp 72–73 °C, TLC Rf = 0.52 (petro-
leum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d: 10.32 (s,
1H), 8.36 (s, 1H), 7.97 (d, J = 8.40 Hz, 1H), 7.70 (d, J = 7.80 Hz,
1H), 7.63 (d, J = 7.80 Hz, 1H), 7.51 (m, 2H), 7.19 (d, J = 8.40 Hz,
1H), 7.07 (t, J = 7.20 Hz, 1H), 3.89 (s, 3H), 2.58 (s, 3H). MS (ESI)
m/z 270.5 [M+H]+.
3.3.1. 2-Hydroxy-4-(3-(4-hydroxybenzamido)phenyl)-4-
oxobut-2-enoic acid (4a)
White powder, yield 57.1%, mp 192–193 °C. 1H NMR (DMSO-d6)
d: 10.24 (s, 1H), 10.17 (s, 1H), 8.46 (s, 1H), 8.15 (d, J = 7.84 Hz, 1H),
7.89 (d, J = 8.53 Hz, 2H), 7.77 (d, J = 7.77 Hz, 1H), 7.54 (t, J = 7.96 Hz,
1H), 7.05 (s, 1H), 6.88 (d, J = 8.53 Hz, 2H). IR (KBr, cmꢀ1): mOH
:
:
:
3387.37, 3245.76; mC@O
: 1722.99, 1631.00, 1610.39; mC@C
3.2.14. N-(3-Acetylphenyl)-3-methoxybenzamide (3n)
White powder, yield 63.7%, mp 105–106 °C, TLC Rf = 0.50
(petroleum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d:
10.43 (s, 1H), 8.37 (s, 1H), 8.08 (d, J = 8.40 Hz, 1H), 7.72 (d,
J = 7.80 Hz, 1H), 7.57 (d, J = 7.80 Hz, 1H), 7.52 (t, J = 7.80 Hz, 2H),
7.47 (t, J = 7.80 Hz, 1H), 7.18 (d, J = 8.40 Hz, 1H), 3.85 (s, 3H), 2.59
(s, 3H). MS (ESI) m/z 270.5 [M+H]+.
1653.80, 1545.37, 1511.98, 1488.29, 1439.01, 1424.80; mC–O
1256.12, 1213.92, 1175.04; c@C–H: 847.10, 776.72. MS (ESI) m/z
328.4 [M+H]+; HRMS (ESI) m/z for C17H14NO6 [M+H]+: calcd
328.0816, found 328.0821.
3.3.2. 2-Hydroxy-4-(3-((E)-3-(4-hydroxyphenyl)acrylamido)
phenyl)-4-oxobut-2-enoic acid (4b)
Yellowish powder, yield 47.6%, mp 186–188 °C. 1H NMR
(DMSO-d6) d: 14.22 (br, 1H), 10.43 (s, 1H), 9.99 (s, 1H), 8.40 (s,
1H), 8.01 (d, J = 7.80 Hz, 1H), 7.74 (d, J = 7.20 Hz, 1H), 7.53 (m,
2H), 7.48 (d, J = 8.40 Hz, 2H), 7.01 (s, 1H), 6.84 (d, J = 8.40 Hz,
3.2.15. N-(3-Acetylphenyl)-4-methoxybenzamide (3o)
White powder, yield 70.4%, mp 167–168 °C, TLC Rf = 0.49
(petroleum ether/acetic ether 3:1, v/v). 1H NMR (DMSO-d6) d:
10.29 (s, 1H), 8.36 (s, 1H), 8.08 (d, J = 8.40 Hz, 1H), 7.99 (d,
J = 8.40 Hz, 2H), 7.69 (d, J = 7.20 Hz, 1H), 7.50 (t, J = 7.80 Hz, 1H),
7.09 (d, J = 8.40 Hz, 2H), 3.85 (s, 3H), 2.59 (s, 3H). MS (ESI) m/z
270.6 [M+H]+.
2H), 6.63 (d, J = 15.60 Hz, 1H). IR (KBr, cmꢀ1):
3246.83; mC@O
1585.84, 1548.95, 1514.72; mC–O
m
OH: 3358.42,
1719.43, 1675.42, 1625.15; mC@C 1604.67,
1256.54, 1216.93, 1168.95;
:
:
:
c
@C–H: 825.14, 760.76. MS (ESI) m/z 354.4 [M+H]+; HRMS (ESI)
m/z for C19H16NO6 [M+H]+: calcd 354.0972, found 354.0975.
3.2.16. N-(3-Acetylphenyl)-2-chlorobenzamide (3p)
White powder, yield 67.4%, mp 95–97 °C, TLC Rf = 0.55 (petro-
leum ether/acetic ether 1:1, v/v). 1H NMR (DMSO-d6) d: 10.46 (s,
1H), 8.38 (s, 1H), 8.08 (d, J = 7.80 Hz, 1H), 7.98 (d, J = 7.80 Hz,
2H), 7.72 (d, J = 7.80 Hz, 1H), 7.62 (t, J = 7.20 Hz, 1H), 7.55 (m,
1H), 7.51 (s, J = 7.80 Hz, 1H), 2.59 (s, 3H). MS (ESI) m/z 274.4
[M+H]+.
3.3.3. 2-Hydroxy-4-(3-((E)-3-(4-fluorophenyl)acrylamido)phenyl)-4-
oxobut-2-enoic acid (4c)
Yellowish powder, yield 69.4%. mp 189–190 °C. 1H NMR
(DMSO-d6) d: 14.22 (br, 1H), 10.50 (s, 1H), 8.40 (s, 1H), 8.00 (d,
J = 7.80 Hz, 1H), 7.78 (d, J = 7.80 Hz, 1H), 7.64 (d, 1H), 7.57 (m,
2H), 7.55 (t, J = 7.80 Hz, 1H), 7.30 (t, J = 8.40 Hz, 2H), 7.04 (s, 1H),

2918
H. Li et al. / Bioorg. Med. Chem. 17 (2009) 2913–2919
6.76 (d, J = 15.60 Hz, 1H). IR (KBr, cmꢀ1):
m
OH: 3333.41; m@CH
:
3.3.9. 4-(3-Furan-2-carboxamido)phenyl)-2-hydroxy-4-oxobut-
2-enoic acid (4i)
3072.27; mC@O: 1731.58, 1661.62, 1627.36; mC@C: 1599.63,
1547.87, 1509.46;
m
C–O: 1287.74, 1264.71, 1228.28, 1159.64; c@C–H
:
White powder, yield 73.2%, mp 180–181 °C. 1H NMR (DMSO-d6)
d: 14.89–14.21 (br, 2H), 10.48 (s, 1H), 8.47 (s, 1H), 8.16 (d,
J = 7.80 Hz, 1H), 7.98 (s, 1H), 7.80 (d, J = 7.80 Hz, 1H), 7.56 (t,
J = 7.80 Hz, 1H), 7.41 (s, 1H), 7.08 (s, 1H), 6.74 (s, 1H). IR (KBr,
829.87, 777.74. MS (ESI) m/z 356.5 [M+H]+; HRMS (ESI) m/z for
C19H15NO5F [M+H]+: calcd 356.0929, found 356.0931.
3.3.4. 2-Hydroxy-4-(3-((E)-3-(4-
cmꢀ1):
m
OH: 3353.96, 3118.15;
m
C@O: 1742.07, 1652.59, 1614.41;
1308.86, 1264.86,
chlorophenyl)acrylamido)phenyl)-4-oxobut-2-enoic acid (4d)
White powder, yield 75.2%, mp 188–190 °C. 1H NMR (DMSO-d6)
d: 10.51 (s, 1H), 8.38 (s, 1H), 8.00 (d, J = 7.80 Hz, 1H), 7.76 (d,
J = 7.80 Hz, 1H), 7.68 (d, J = 8.40 Hz, 2H), 7.63 (d, J = 15.60 Hz,
1H), 7.54 (m, 3H), 6.98 (br, 1H), 6.83 (d, J = 16.20 Hz, 1H).
mC@C
:
1587.55, 1548.88, 1473.12; mC–O:
1228.36; c
@C–H: 783.47, 758.24. MS (ESI) m/z 302.5 [M+H]+; HRMS
(ESI) m/z for C15H12NO6 [M+H]+: calcd 302.0659, found 302.0667.
3.3.10. 2-Hydroxy-4-(3-(2-hydroxybenzamido)phenyl)-4-
oxobut-2-enoic acid (4j)
IR (KBr, cmꢀ1): mNH
3124.02, 3063.42; mC@O
1592.78, 1545.00, 1490.30; mC–O
:
3527.55; mOH
:
3414.76, 3263.68; m@CH
:
:
:
1733.01, 1657.61, 1622.60; mC@C
Yellowish powder, yield 43.6%, mp 178–180 °C. 1H NMR
(DMSO-d6) d: 11.62 (s, 1H), 10.57 (s, 1H), 8.43 (s, 1H), 8.05 (d,
J = 8.02 Hz, 1H), 7.95 (d, J = 7.74 Hz, 1H), 7.84 (d, J = 7.81 Hz, 1H),
7.58 (t, J = 7.98 Hz, 1H), 7.46 (t, J = 7.59 Hz, 1H), 7.07 (s, 1H), 7.00
:
1339.05, 1265.86, 1218.09;
c
@C–H: 819.05, 781.47. MS (ESI) m/z 372.4 [M+H]+; HRMS (ESI)
m/z for C19H15NO5Cl [M+H]+: calcd 372.0633, found 372.0631.
(d, J = 7.96 Hz, 1H), 6.97 (d, J = 7.51 Hz, 1H). IR (KBr, cmꢀ1): mOH
3404.36; m@CH: 3066.96; mC@O: 1728.95, 1640.21, 1607.10; mC@C
:
:
:
3.3.5. 2-Hydroxy-4-(3-((E)-3-(4-
methoxyphenyl)acrylamido)phenyl)-4-oxobut-2-enoic acid
(4e)
1549.25, 1491.35, 1457.00; mC–O: 1268.72, 1216.51; c@C–H
775.42, 750.97. MS (ESI) m/z 328.4 [M+H]+; HRMS (ESI) m/z for
White powder, yield 71.6%, mp 154–155 °C. 1H NMR (DMSO-d6)
d: 14.26 (br, 1H), 10.46 (s, 1H), 8.39 (s, 1H), 8.01 (d, J = 7.80 Hz, 1H),
7.74 (d, J = 7.20 Hz, 1H), 7.68 (m, 3H), 7.58 (s, 1H), 7.53 (t,
J = 7.80 Hz, 1H), 7.02 (d, J = 8.40 Hz, 2H), 6.71 (d, J = 15.60 Hz,
C17H14NO6 [M+H]+: calcd 328.0816, found 328.0822.
3.3.11. 2-Hydroxy-4-(3-(2-hydroxy-3,5-
dinitrobenzamido)phenyl)-4-oxobut-2-enoic acid (4k)
Yellow powder, yield 36.8%, mp 186–187 °C. 1H NMR (DMSO-
d6) d: 14.25 (br, 1H), 13.43 (s, 1H), 8.87 (s, 1H), 8.60 (s, 1H), 8.50
(s, 1H), 7.94 (d, J = 7.80 Hz, 1H), 7.79 (d, J = 7.80 Hz, 1H), 7.56 (t,
1H), 3.81 (s, 3H). IR (KBr, cmꢀ1):
2936.56, 2838.60; mC@O
1546.88, 1513.15, 1426.36; mC–O
m
@CH: 3005.75;
1685.74, 1624.64, 1601.33; mC@C
1256.66, 1219.25, 1173.83;
mCH: 2961.29,
:
:
:
c
@C–H: 827.02, 775.09. MS (ESI) m/z 368.4 [M+H]+; HRMS (ESI) m/
J = 7.80 Hz, 1H), 7.10 (s, 1H). IR (KBr, cmꢀ1):
3099.25; mC@O
m
OH: 3365.73; m@CH
:
z for C20H18NO6 [M+H]+: calcd 368.1129, found 368.1157.
:
1753.38, 1673.56, 1610.61; mC@C 1544.96,
:
1439.92; mC–O: 1346.20, 1272.08; c@C–H: 787.05, 740.11. MS (ESI)
3.3.6. 2-Hydroxy-4-(3-((E)-3-(4-hydroxy-3-
m/z 418.4 [M+H]+; HRMS (ESI) m/z for C17H12N3O10 [M+H]+: calcd
methoxyphenyl)acrylamido)phenyl)-4-oxobut-2-enoic acid (4f)
Yellow powder, yield 61.7%, mp 185–186 °C. 1H NMR (DMSO-
d6) d: 14.19 (br, 1H), 10.39 (s, 1H), 9.59 (s, 1H), 8.42 (s, 1H),
8.00 (d, J = 8.40 Hz, 1H), 7.76 (d, J = 7.80 Hz, 1H), 7.53 (m, 2H),
7.21 (s, 1H), 7.09 (d, J = 7.80 Hz, 1H), 7.05 (s, 1H), 6.84 (d,
J = 7.80 Hz, 1H), 6.63 (d, J = 15.00 Hz, 1H), 3.84 (s, 3H). IR (KBr,
418.0517, found 418.0525.
3.3.12. 2-Hydroxy-4-(3-(2-hydroxy-3-
methylbenzamido)phenyl)-4-oxobut-2-enoic acid (4l)
Yellowish powder, yield 59.2%, mp 186–188 °C. 1H NMR
(DMSO-d6) d: 14.20 (br, 1H), 12.36 (s, 1H), 10.62 (s, 1H), 8.40 (s,
1H), 8.10 (d, J = 7.80 Hz, 1H), 7.93 (d, J = 7.80 Hz, 1H), 7.88 (d,
J = 8.40 Hz, 1H), 7.60 (t, J = 8.10 Hz, 1H), 7.40 (d, J = 7.20 Hz, 1H),
cmꢀ1):
1547.36, 1514.52, 1430.64; mC–O
mOH: 3314.90;
m
C@O: 1735.07, 1699.98, 1589.49; mC@C
:
:
1288.13, 1265.64, 1209.22,
1175.66;
c
@C–H: 842.24, 807.91, 773.84. MS (ESI) m/z 384.6
7.08 (s, 1H), 6.90 (m, 1H), 2.21 (s, 3H). IR (KBr, cmꢀ1): mOH
:
[M+H]+; HRMS (ESI) m/z for C20H18NO7 [M+H]+: calcd
3416.18; mC@O: 1728.82, 1646.95, 1583.69; mC@C: 1547.67,
384.1078, found 384.1082.
1486.27, 1435.38; mC–O: 1295.25, 1249.97; c@C–H: 778.68, 744.30.
MS (ESI) m/z 342.3 [M+H]+; HRMS (ESI) m/z for C18H16NO6
3.3.7. 4-(3-Benzamidophenyl)-2-hydroxy-4-oxobut-2-enoic
acid (4g)
[M+H]+: calcd 342.0972, found 342.0981.
White powder, yield 62.7%, mp 188–189 °C. 1H NMR (DMSO-d6)
d: 14.25 (br, 1H), 10.51 (s, 1H), 8.49 (s, 1H), 8.17 (d, J = 7.80 Hz, 1H),
8.00 (d, J = 7.20 Hz, 2H), 7.81 (d, J = 7.80 Hz, 1H), 7.62 (t, J = 7.50 Hz,
3.3.13. 2-Hydroxy-4-(3-(2-methoxybenzamido)phenyl)-4-
oxobut-2-enoic acid (4m)
White powder, yield 60.6%, mp 173–175 °C. 1H NMR (DMSO-d6)
d: 15.00 (br, 1H), 14.19 (br, 1H), 10.36 (s, 1H), 8.48 (s, 1H), 8.04 (d,
J = 8.40 Hz, 1H), 7.79 (d, J = 7.80 Hz, 1H), 7.63 (d, J = 7.50 Hz, 1H),
7.54 (m, 2H), 7.19 (d, J = 8.40 Hz, 1H), 7.07 (m, 2H), 3.89 (s, 3H).
1H), 7.57 (m, 3H), 7.06 (s, 1H). IR (KBr, cmꢀ1):
m
NH: 3530.83; mOH
3094.66, 3067.41; mC@O 1645.72,
1547.81, 1447.65; mC–O 1298.08, 1262.03,
:
3450.33, 3251.78; m@CH
1626.87; mC@C
:
:
:
:
1201.15;
c
@C–H: 778.87, 679.31. MS (ESI) m/z 312.4 [M+H]+; HRMS
IR (KBr, cmꢀ1):
m
OH: 3371.43;
m
C@O: 1728.22, 1626.89, 1600.26;
1294.23,
(ESI) m/z for C17H14NO5 [M+H]+ calcd 312.0866, found 312.0872.
mC@C
:
1556.09, 1488.73, 1466.82, 1439.13; mC–O:
1272.81, 1246.87;
c@C–H: 780.17, 753.28. MS (ESI) m/z 342.3
3.3.8. 2-Hydroxy-4-oxo-4-(3-(2-phenylacetamido)phenyl)but-
2-enoic acid (4h)
[M+H]+; HRMS (ESI) m/z for C18H16NO6 [M+H]+: calcd: 342.0972,
found 342.0979.
Yellowish powder, yield 32.4%, mp 174–176 °C. 1H NMR
(DMSO-d6) d: 10.57 (s, 1H), 8.32 (s, 1H), 7.93 (d, J = 8.40 Hz, 1H),
7.74 (d, J = 7.80 Hz, 1H), 7.50 (t, J = 7.80 Hz, 1H), 7.34 (m, 4H),
7.25 (t, J = 6.90 Hz, 1H), 7.00 (s, 1H), 3.69 (s, 2H). IR (KBr, cmꢀ1):
3.3.14. 2-Hydroxy-4-(3-(3-methoxybenzamido)phenyl)-4-
oxobut-2-enoic acid (4n)
White powder, yield 69.1%, mp 114–115 °C. 1H NMR (DMSO-d6)
d: 15.05 (br, 1H), 14.22 (br, 1H), 10.47 (s, 1H), 8.48 (s, 1H), 8.16 (d,
J = 8.40 Hz, 1H), 7.81 (d, J = 7.80 Hz, 1H), 7.57 (t, J = 8.10 Hz, 2H),
7.52 (s, 1H), 7.47 (m, 1H), 7.19 (d, J = 8.40 Hz, 1H), 7.06 (s, 1H),
mNH: 3539.03;
m
OH: 3248.31;
m@CH: 3142.47, 3087.86, 3063.18,
3030.78; CH: 2928.19;
m
m
C@O: 1728.21, 1658.32, 1626.34; mC@C
:
:
1597.77, 1552.01, 1494.66, 1454.15; mC–O: 1288.45, 1265.15; c@C–H
777.48, 694.55. MS (ESI) m/z 326.5 [M+H]+; HRMS (ESI) m/z for
3.85 (s, 3H). IR (KBr, cmꢀ1): mOH
1649.30, 1626.58; mC@C
:
3439.39; mC@O
: 1729.32,
C18H16NO5 [M+H]+: calcd 326.0817, found 326.0823.
:
1589.66, 1544.10, 1488.08; mC–O:

H. Li et al. / Bioorg. Med. Chem. 17 (2009) 2913–2919
2919
1292.21, 1265.58, 1200.56;
c
@C–H: 778.59, 747.62, 682.41. MS (ESI)
3.4.3. Integrase assays
m/z 342.3 [M+H]+; HRMS (ESI) m/z for C18H16NO6 [M+H]+: calcd
To determine the extent of 30-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
342.0972, found 342.0978.
3.3.15. 2-Hydroxy-4-(3-(4-methoxybenzamido)phenyl)-4-
oxobut-2-enoic acid (4o)
HEPES, pH 7.5, 50 lM EDTA, 50 lM dithiothreitol, 10% glycerol
(w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine serum albumin, 10 mM
2-mercaptoethanol, 10% dimethyl sulfoxide, and 25 mM MOPS,
pH 7.2) at 30 °C for 30 min. Then, 20 nM of the 50-end 32P-labeled
linear oligonucleotide substrate was added, and incubation was
continued for an additional 1 h. Reactions were quenched by the
White powder, yield 52.8%, mp 110–112 °C. 1H NMR (DMSO-d6)
d: 15.04 (br, 1H), 14.21 (br, 1H), 10.35 (s, 1H), 8.47 (s, 1H), 8.16 (d,
J = 7.80 Hz, 1H), 7.99 (d, J = 9.00 Hz, 2H), 7.78 (d, J = 7.80 Hz, 1H),
7.55 (t, J = 7.80 Hz, 1H), 7.08 (d, J = 8.40 Hz, 2H), 7.06 (d,
J = 7.20 Hz, 1H), 3.85 (s, 3H). IR (KBr, cmꢀ1): mOH
:
3530.43,
addition of an equal volume (16
formamide, 10 mM EDTA, 0.025% xylene cyanol and 0.025% bromo-
phenol blue). An aliquot (5 L) was electrophoresed on a denatur-
lL) of loading dye (98% deionized
3269.09; mC@O
: 1686.66, 1643.08, 1604.57; mC@C: 1578.62,
1510.58, 1427.91; mC–O
:
1304.64, 1261.46, 1171.08; c@C–H
:
l
844.37, 774.75. MS (ESI) m/z 342.3 [M+H]+; HRMS (ESI) m/z for
ing 20% polyacrylamide gel (0.09 M Tris–borate, pH 8.3, 2 mM
EDTA, 20% acrylamide, 8 M urea).
C18H16NO6 [M+H]+: calcd 342.0972, found 342.0981.
Gels were dried, exposed in a PhosphorImager cassette, and
analyzed using a Typhoon 8610 Variable Mode Imager (Amersham
Biosciences) and quantitated using ImageQuant 5.2. Percent inhibi-
tion (% I) was calculated using the following equation:
3.3.16. 2-Hydroxy-4-(3-(2-chlorobenzamido)phenyl)-4-oxobut-
2-enoic acid (4p)
White powder, yield 63.4%, mp 138–140 °C. 1H NMR (DMSO-d6)
d: 14.20 (br, 1H), 10.77 (s, 1H), 8.44 (s, 1H), 8.02 (d, J = 7.80 Hz, 1H),
7.82 (d, J = 7.80 Hz, 1H), 7.64 (d, J = 7.50 Hz, 1H), 7.56 (m, 3H), 7.48
% I ¼ 100 ꢁ ½1 ꢀ ðD ꢀ CÞ=ðN ꢀ CÞꢂ
(t, J = 7.50 Hz, 1H), 7.03 (s, 1H). IR (KBr, cmꢀ1):
m
OH: 3447.02,
where C, N, and D are the fractions of 21-mer substrate converted to
19-mer (30-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
versus percent inhibition to obtain the concentration that produced
50% inhibition.
3236.54; mC@O
: 1715.43, 1659.78; mC@C: 1592.20, 1547.44,
1487.42, 1434.60; mC–O
: 1305.19, 1265.20, 1206.34; c@C–H:
777.12, 750.22. MS (ESI) m/z 346.4 [M+H]+; HRMS (ESI) m/z for
C17H13NO5Cl [M+H]+: calcd 346.0477, found 346.0475.
3.3.17. 2-Hydroxy-4-(3-(3-chlorobenzamido)phenyl)-4-oxobut-
2-enoic acid (4q)
Acknowledgments
White powder, yield 55.8%, mp 187–188 °C.1H NMR (DMSO-d6)
d: 14.92 (br, 1H), 14.22 (br, 1H), 10.60 (s, 1H), 8.47 (s, 1H), 8.16 (d,
J = 7.80 Hz, 1H), 8.06 (s, 1H), 7.96 (d, J = 7.80 Hz, 1H), 7.83 (d,
J = 7.80 Hz, 1H), 7.70 (d, J = 7.80 Hz, 1H), 7.60 (m, 2H), 7.07 (s,
This work was supported by the National Natural Science Foun-
dation of China (No. 20872082). The work in the Neamati labora-
tory was supported in part by
Foundation.
a grant from the Campbell
1H). IR (KBr, cmꢀ1):
3066.85; mC@O
m
NH: 3531.51;
mOH: 3454.57, 3270.01; m@CH:
:
1729.75, 1646.98, 1626.48; mC@C
:
1546.21,
1488.41, 1449.44; mC–O
:
1293.95, 1260.36, 1201.54; c@C–H:
References and notes
778.15, 698.12. MS (ESI) m/z 346.4 [M+H]+; HRMS (ESI) m/z for
C17H13NO5Cl [M+H]+: calcd 346.0477, found 346.0484.
1. Pommier, Y.; Johnson, A. A.; Marchand, C. Nat. Rev. Drug Discov. 2005, 4, 236.
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4. Zhao, G. S.; Wang, C.; Liu, C.; Lou, H. X. Mini-Rev. Med. Chem. 2007, 7, 707.
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3.4. Biological activity methods
3.4.1. Materials, chemicals, and enzymes
All compounds were dissolved in DMSO, and the stock solutions
were stored at ꢀ20 °C. The
c[
32P]ATP was purchased from MP Bio-
medical. The expression systems for the wild-type IN and soluble
mutant INF185KC280S were generous gifts of Dr. Robert Craigie, Lab-
oratory of Molecular Biology, NIDDK, NIH, Bethesda, MD.
7. Walker, M. A.; Johnson, T.; Naidu, B. N.; Banville, J.; Remillard, R.; Plamondon,
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3.4.2. Preparation of oligonucleotide substrates
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The oligonucleotides 21top, 50-GTGTGGAAAATCTCTAGCAGT-30
and 21bot, 50-ACT-GCTAGAGATTTTCCACAC-30 were purchased
from the Norris Cancer Center Core Facility (University of Southern
California), and purified by UV shadowing on a polyacrylamide gel.
To analyze the extent of 30-processing and strand transfer using 50-
end-labeled substrates, 21top was 50-endlabeled using T4 polynu-
cleotide kinase (Epicenter, Madison, WI) and c[
32P]ATP (MP Bio-
medical). The kinase was heat-inactivated, and 21bot was added
in 1.5-M excess. The mixture was heated to 95 °C, allowed to cool
slowly to room temperature, and run through a spin 25 mini-col-
umn (USA Scientific) to separate the annealed double-stranded oli-
gonucleotide from unincorporated material.
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