Design and synthesis of dual inhibitors of HIV reverse transcriptase and integrase: Introducing a diketoacid functionality into delavirdine

Article abstract of DOI:10.1016/j.bmc.2008.02.007

Cost and toxicity problems associated with highly active antiretroviral therapy (HAART) in HIV/AIDS treatment could be alleviated by using designed multiple ligands (DMLs). Dual inhibitors of HIV reverse transcriptase (RT) and integrase (IN) were rationally designed by introducing a diketoacid (DKA) functionality into the tolerant C-5 site of RT inhibitor delavirdine. The resulting compounds all demonstrate good activity against both RT and IN in enzymatic assays and HIV in cell-based assay, whereas their C-7 regioisomers are all inactive in these assays. Balanced activities were observed with C-3 halogenated inhibitors.

Full text of DOI:10.1016/j.bmc.2008.02.007

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3587–3595
Design and synthesis of dual inhibitors of HIV reverse
transcriptase and integrase: Introducing a diketoacid
functionality into delavirdine
Zhengqiang Wang* and Robert Vince*
Center for Drug Design, Academic Health Center, University of Minnesota, 516 Delaware Street SE, Minneapolis, MN 55455, USA
Received 8 January 2008; revised 1 February 2008; accepted 5 February 2008
Available online 8 February 2008
Abstract—Cost and toxicity problems associated with highly active antiretroviral therapy (HAART) in HIV/AIDS treatment could
be alleviated by using designed multiple ligands (DMLs). Dual inhibitors of HIV reverse transcriptase (RT) and integrase (IN) were
rationally designed by introducing a diketoacid (DKA) functionality into the tolerant C-5 site of RT inhibitor delavirdine. The
resulting compounds all demonstrate good activity against both RT and IN in enzymatic assays and HIV in cell-based assay,
whereas their C-7 regioisomers are all inactive in these assays. Balanced activities were observed with C-3 halogenated inhibitors.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
HAART as the standard AIDS chemotherapy are still
compromised by major issues that originate from using
multiple drugs, such as high cost and toxicity.11 Side ef-
fects can be particularly problematic as they normally
lead to poor patient adherence, and as a result, patients
experience viral rebound, and even worse, multi-drug
resistance (MDR).12 We envision that these issues could
be alleviated by using designed multiple ligands
(DMLs)13–15 which are single structures engaging multi-
ple biological targets. These multifunctional compounds
should work to contain much of the resistance as
HAART does, albeit at a lower cost and possibly with
less side effects. Identifying multiple ligands through ra-
tional design has been a subject of growing interest in
medicinal chemistry.14
Human immunodeficiency virus (HIV) is the causative
agent of acquired immunodeficiency syndrome
(AIDS).1,2 Despite FDA approval of over 30 antiviral
regimens for the treatment of AIDS,3 HIV continues to
be a devastating pandemic that claimed 2.1 million lives
in 2007 alone.4 The increasing mortality of HIV/AIDS is
largely due to the lack of vaccines5 and the persistence of
viral reservoirs,6 which render this disease unpreventable
and incurable. These complications underscore the
imperativeness of chemotherapeutic agents as they pro-
vide the only relief for HIV/AIDS patients. Unfortu-
nately, clinical usage of singly dosed antivirals is
limited by the rapid selection of resistant viral strains. In-
stead, cocktails combining mechanistically distinct drugs
are administered to form highly active antiretroviral
therapy (HAART),7 which can effectively suppress viral
replication to undetectable level. Inhibitors of reverse
transcriptase (RT) and protease (PR) constitute the cor-
nerstone of HAART, which is enhanced by recent FDA-
approved anti-HIV drugs that target new molecular
mechanisms of HIV replication, such as entry inhibitors
enfuvirtide8 and maraviroc,9 and integrase (IN) inhibitor
raltegravir (MK-0518).10 Nevertheless, the benefits of
2. Design of RT/IN dual inhibitors
HIV RT and IN are two virally encoded enzymes that
function sequentially in two closely related steps of HIV
replication. Interestingly, RT has two distinct active sites
for polymerase and RNase H activities, respectively,
whereas IN uses the same binding site for two catalytic
activities: 30 processing and strand transfer. It could be
more amenable to target RNase H and IN as these two
share a common binding mechanism. However, targeting
dissimilar binding sites could be essential to avoiding
quick emergence of drug resistance. Therefore, our design
is focused on the polymerase active site of RT along with
Keywords: HIV; Dual inhibitors; Reverse transcriptase; Integrase.
*
Corresponding authors. Tel.: +1 612 625 8253; fax: +1 612 625
8252; e-mail addresses: vince001@umn.edu; wangx472@umn.edu
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.007

3588
Z. Wang, R. Vince / Bioorg. Med. Chem. 16 (2008) 3587–3595
IN. Strategically, our design generally involves the incor-
poration of an IN pharmacophore to a known potent RT
inhibitor as illustrated by the design of the first rationally
designed RT/ IN inhibitor 1 (Fig. 1).16 Apparently the key
to this strategy would be to identify a tolerant region in
the RT structure. Crystal structure of TNK-651, a RT
inhibitor of the 1-[(2-hydroxyethoxy)methyl]-6-(phenyl-
thio)thymine (HEPT) family,17 reveals that the N-1
pendant of the pyrimidine core extends from the non-
nucleoside RT inhibitor (NNRTI) binding pocket to the
protein/solvent interface, which presents an attractive site
for incorporating a moiety to generate anti-IN activity
(Fig. 1).
HN
HN
N
N
N
N
H
N
H
N
N
O
N
O
O
O
O
S
N
HO
H
OH O
3a, RT/ IN dual inhibitor
2, Delavirdine, RT inhibitor
Figure 2. The design of RT/IN dual inhibitor 3a: replacing the
methylsulfonamide group of RT inhibitor 2 with a DKA functionality.
The synthesis starts with the preparation of 6a (Scheme
2). Unfortunately, the direct Friedel–Crafts acylation of
ethyl indole-2-carboxylate (7) yielded a mixture of three
regioisomers (Scheme 2a).25 Clearly acylation at C-3 po-
sition of the indole ring is largely favored to give 8
(62%), and the desired 5-acyl compound 6a was pro-
duced as a minor product (15%) only, which was found
inseparable from 8. Remarkably, 7-acyl product 6b is
well separated from the inseparable mixture of 6a and
8 and has a significantly different solubility in organic
solvents, presumably due to an internal H-bonding in
6b (Scheme 2a). Nevertheless, the direct Friedel–Crafts
acylation provides a viable synthesis for isomeric 7-acyl
indole-2-carboxylate 6b instead of the desired com-
pound 6a.
Similarly, crystallographic study has shown that the
methylsulfonamide group at the C-5 position of delavir-
dine (2),18 an RT inhibitor of the bis(heteroaryl)pipera-
zine (BHAP) family, is situated in an open area where
structural modification could be tolerated. Therefore,
replacing this sulfonamide group with a DKA could
generate inhibitory activity against IN without signifi-
cantly affecting the RT binding (Fig. 2). Notably, known
pharmacophore model19,20 indicates that, besides a
DKA type of metal-chelating functionality, IN binding
also requires a hydrophobic aromatic ring directly con-
nected to the chelator, which can be satisfied by the in-
dole ring of 3a in our design.
Other FDA-approved RT inhibitors nevirapine21 and
efavirenz22 are almost entirely enclosed by the NNRTI
binding pocket, which is largely hydrophobic and there-
fore an inappropriate environment for the polar metal-
chelating pharmacophores of IN inhibitors.
The alternative Fisher indole synthesis was then pursued
for the synthesis of 6a (Scheme 2b). In this event, aniline
9 was diazotized and reacted with enolized malonate 10
to produce the requisite hydrazone 11 in excellent yield.
The subsequent Fisher indole synthesis26 was effected by
microwaving the hydrazone in trifluoroacetic acid
(TFA) at 140 ꢁC for 15 min. Gratifyingly, the micro-
wave reaction gave a much higher yield (45%) than the
same reaction with conventional heating (4%).25 An-
other strategy to get around the regioselectivity issue
in the acylation reaction is to block the most reactive
C-3 site. We chose to halogenate this site because (a)
the dehalogenation could be viable; (b) halogens are bio-
isosteric to hydrogen; and (c) these halogens could be
used as handles for further functionalization to study
the SAR at this site. As expected, the bromination
(12a) and chlorination (12b) occurred exclusively at C-
3 position (Scheme 2c), and the subsequent acylation
at C-5 and C-7 positions yielded products (6c, 6d and
6e, 6f) that are easily separated.
3. Chemistry
Our synthetic strategy for inhibitor 3a is outlined in
Scheme 1. We envision that inhibitor 3a can be synthet-
ically accessed from acyl intermediate 4, which can be
prepared from piperazine 5 and indole ester 6. Direct
acylation of commercial ester 7 could give 6, which
might be complicated by the regioselectivity issue. Alter-
natively, indole ester 6 can be synthesized via Fisher in-
dole synthesis23 from hydrazone 11, which can be
prepared via a classic Japp–Klingeman reaction24 be-
tween aniline 9 and malonate 10.
To complete the synthesis (Scheme 3), ethyl 5-acyl-in-
dole-2-carboxylates 6a–f were saponified and coupled
with piperazine compound 5 27 to produce intermediates
4a–f, which were further elaborated to produce inhibi-
tors 3a–f through a condensation with ethyl oxalate
and a subsequent saponification.
RT Pharmacophore
O
HN
O
N
O
O
IN Pharmacophore
4. Results and discussion
OH
O
OH
1
Results of anti-RT, anti-IN, and anti-HIV assays of syn-
thesized RT/IN inhibitors are summarized in Table 1.
Remarkably, the substitution pattern appears to affect
both RT binding and IN binding dramatically. As
Figure 1. A rationally designed RT/IN dual inhibitor featuring a
HEPT pharmacophore for anti-RT activity and a DKA pharmaco-
phore for IN activity.

Z. Wang, R. Vince / Bioorg. Med. Chem. 16 (2008) 3587–3595
3589
HN
HN
N
N
N
N
HN
H
N
H
N
N
O
N
O
HN
N
O
N
+
H
N
HO
O
OEt
O
5
OH
O
3a
O
4
7
direct
acylation
O
H
OEt
O
O
O
N
Fisher indole
synthesis
O
+
Japp-Klingeman
OEt
N
N
OEt
NH2
H
O
11
6
10
9
Scheme 1. Synthetic plan for dual inhibitor 3a.
H
N
H
O
OEt
OEt
O
N
H
H
N
a
OEt
O
N
+
OEt
O
O
+
(a)
O
O
inseparable
6a
8
7
6b
O
O
O
O
H
N
OEt
O
OEt
O
c
(b)
(c)
10
N
N
H
OEt
NH2
b
O
9
7
11
6a
H
N
H
H
OEt
O
OEt
O
N
OEt
O
N
d or e
f
R
X
X
6c, X = Br, R = 5-Ac
6d, X = Br, R = 7-Ac
6e, X = Cl, R = 5-Ac
6f, X = Cl, R = 7-Ac
12a, X = Br
12b, X = Cl
Scheme 2. Preparation of acyl indole-2-carboxylates 6a–f. Reagents and conditions: (a) AcCl, AlCl3, CH2ClCH2Cl, rt, 1 h, 100%, 6a/8/6b = 15:62:23;
(b) NaNO2, HCl; 10, KOH, 91%; (c) microwave, 140 ꢁC, 15 min, 45%; (d) NBS, DMF, rt, 96% (12a); (e) NCS, DMF, rt, 95% (12b); (f) AcCl, AlCl3,
CH2ClCH2Cl, rt, 1 h; for 12a: 73%, 6c/6d = 1.5:1; for 12b: 75%, 6e/6f = 1.6:1.
clearly shown in Table 1, compounds with DKA substi-
tution at C-7 position (3b, 3d, 3f) are virtually inactive
against both enzymes, whereas compounds with C-5
DKA functionality (3a, 3c, 3e) show sub- or low micro-
molar activity against RT and IN in enzymatic assays as
well as HIV in cell-based assay. Apparently C-7 substi-
tution results in angular compounds with nearly closed
conformations which might not favor binding to IN,
while RT modeling28 shows that the DKA functionality
of compounds with C-7 substitution has unfavorable
electrostatic interactions with the backbone carbonyls
of K103 and K104. By contrast, inhibitors with C-5 sub-
stitution have a pseudo-linear conformation resembling
delavirdine, which apparently benefits both RT and IN
binding. This observed overwhelming preference of C-
5 substitution over C-7 proves the importance of identi-
fying tolerant regions in the design of dual inhibitors
with two distinct pharmacophores.
Significantly, it was also observed that introducing a hal-
ogen (Cl or Br) atom at C-3 position of the indole ring
reduces the anti-RT potency while slightly enhancing
the anti-IN activity, reflecting the dissimilarity of these
two binding sites. As a result, balanced binding affinity
against both RT and IN is achieved with inhibitors 3c
and 3e (Table 1), particularly the latter (IC50: 1.1 lM
against RT, 4.7 lM against IN and 0.98 lM against
HIV-1). By contrast, inhibitor 3a lacks the halogen sub-
stituent at C-3 and its activities against RT and IN are
separated by 3 orders of magnitude, which is similar to
inhibitor 1. Importantly, the major thrust for developing
dual inhibitors active against two dissimilar targets is to

3590
Z. Wang, R. Vince / Bioorg. Med. Chem. 16 (2008) 3587–3595
HN
N
HN
N
N
HN
OH
N
H
N
N
H
N
H
N
O
OR
O
O
5
a
O
O
b
O
X
X
X
6a-f
4a-f
13a-f
a, X = H, sub at 5
b, X = H, sub at 7
c, X = Br, sub at 5
d, X = Br, sub at 7
e, X = Cl, sub at 5
f, X = Cl, sub at 7
HN
HN
N
N
N
N
c
7
H
N
H
N
d
N
O
N
O
O
O
HO
EtO
HO
5
3
X
X
HO
O
O
14a-f
3a-f
Scheme 3. Synthesis of inhibitors 3a–f. Reagents and conditions: (a) NaOH (aq), EtOH, reflux, 3 h, 44%; (b) 5, CDI, DMF, rt, 62%; (c) Na/EtOH,
diethyl oxalate, rt, 3 h; (d) NaOH (aq), EtOH/CHCl3, rt, 1 h, 41% over 2 steps.
sults do demonstrate that activities can be balanced via
functionalizing the initial lead compound.
Table 1. Anti-RT, anti-IN and anti-HIV assay results for inhibitors
3a–fa
In conclusion, we have designed a scaffold for dual inhib-
itors of HIV reverse transcriptase (RT) and integrase (IN)
by introducing a diketoacid (DKA) functionality into the
tolerant C-5 site of RT inhibitor delavirdine. Assay results
show that C-5 substituted inhibitors 3a, 3c, and 3e all
demonstrate good activity against both RT and IN in
enzymatic assays and HIV in cell-based assay, whereas
their C-7 regioisomers are all inactive. We have also dem-
onstrated that balanced activities can be achieved via
functionalizing the initial lead compound. Inhibitor 3e
demonstrates the best balance against RT (IC50:
1.1 lM), IN (IC50: 4.7 lM), and HIV (EC50: 0.98 lM).
Inhibitor
X
Sub
RT IC50
(lM)
IN IC50
(lM)b
HIV EC50
(lM)
1c
2
—
—
H
—
—
5
0.057
0.036
0.0059
>100
0.12
2.4
>100
11
0.033
0.021
0.52
>10
3a
3b
3c
3d
3e
3f
H
7
>100
3.9
75
Br
Br
Cl
Cl
5
0.79
>10
5. Experimental
5.1. Chemistry
7
>100
1.1
>100
5
4.7
>100
0.98
>10
7
a IC50
concentration.
:
50% inhibitory concentration; EC50
:
50% effective
5.1.1. General procedures. All commercial chemicals
were used as supplied unless otherwise indicated. Dry
solvents (THF, Et2O, CH2Cl2, and DMF) were dis-
pensed under argon from an anhydrous solvent system
with two packed columns of neutral alumina or molec-
ular sieves. Anhydrous ethanol was purchased from Sig-
ma–Aldrich. Flash chromatography was performed with
Silia-P flash silica gel (silicycle, 230–400 mesh) with indi-
cated mobile phase. All reactions were performed under
inert atmosphere of ultra-pure argon with oven-dried
b Average activity against 30 processing and strand transfer.
c Data taken from Ref. 16.
achieve the potential advantage of avoiding selection of
resistance mutations. If the activities are largely unbal-
anced, as in the case of inhibitor 3a, the selection of resis-
tant mutation at the more potent site (RT) would almost
certainly lead to treatment failure, as the activity against
the less potent site (IN) would not be sufficient to sup-
press viral replication. In this scenario, an even worse
consequence of resistance mutations at both RT and
IN sites is highly possible. Therefore, achieving balanced
binding affinity against dissimilar targets is essential,
which, unfortunately, remains the biggest challenge in
designing multiple ligands.14 The examples of balanced
activities with 3c and 3e are not ideal as they reflect a loss
of anti-RT activity compared to 3a. However, these re-
1
glassware. H and 13C NMR spectra were recorded on
a Varian 600 MHz spectrometer. High resolution mass
data were acquired on an Agilent TOF II TOS/MS spec-
trometer capable of ESI and APCI ion sources. Micro-
wave reactions were conducted with
Chemistry Emrys Optimizer Microwave Reactor.
a Personal
5.1.1.1. (E)-Ethyl 2-(2-(4-acetylphenyl)hydrazono)pro-
panoate (11). This compound was prepared according to

Z. Wang, R. Vince / Bioorg. Med. Chem. 16 (2008) 3587–3595
3591
a known procedure.25 The crude hydrazone was ob-
tained in 91% yield, which was recrystallized from tolu-
ene to give pure hydrazone 11 (65%) as a purple solid:
1H NMR (600 MHz, CDCl3) d 8.19 (s, 1H), 7.89 (d,
J = 8.4 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 4.31 (q,
J = 7.2 Hz, 2H), 2.53(s, 3H), 2.12 (s, 3H), 1.36 (t,
J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) d 197.1,
165.2, 147.5, 135.4, 131.2, 130.6, 113.7, 61.7, 26.5,
14.5, 10.9.
5.1.2.1. Ethyl 7-acyl-3-1H-indole-2-carboxylate (6b).
Yield (23%); H NMR (600 MHz, CDCl3) d 10.75 (s,
1
1H), 7.87 (d, J = 7.8 Hz, 1H), 7.83 (d, J = 7.2 Hz, 1H),
7.21 (d, J = 1.8 Hz, 1H), 7.16 (t, J = 7.2 Hz, 1H ), 4.39
(q, J = 7.2 Hz, 2H), 2.65 (s, 3H), 1.40 (t, J = 7.2 Hz,
3H); 13C NMR (150 MHz, CDCl3) d 199.8, 161.5,
135.4, 129.5, 129.1, 129.0, 128.2, 121.2, 120.1, 108.5,
61.3, 26.6, 14.6.
5.1.2.2. Ethyl 5-acyl-3-bromo-1H-indole-2-carboxylate
(6c). Yield (50%); 1H NMR (600 MHz, CDCl3) d
9.51(br s, 1H), 8.28 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H),
7.44 (d, J = 8.4 Hz, 1H), 4.47 (q, J = 7.2 Hz, 2H), 2.69
(s, 3H), 1.44 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
CDCl3) d 197.8, 160.9, 137.9, 131.6, 127.7, 126.4,
126.0, 123.9, 112.5, 100.1, 62.1, 26.8, 14.5.
5.1.1.2. Ethyl 5-acyl-1H-indole-2-carboxylate (6a).
Hydrazone 11 (1.00 g · 5, 4.00 mmol · 5) was placed
into 5 reaction tubes. To each tube was added 6.0 mL
of TFA. The resulting dark purple suspension was
microwaved (140 ꢁC) for 15 min. TFA was then re-
moved and the residue was subjected to flash chroma-
tography (silica gel, hexanes/EtOAc = 4:1) to afford
1
compounds 6a (2.05 g, 45%) as a light purple solid: H
5.1.2.3. Ethyl 7-acyl-3-bromo-1H-indole-2-carboxylate
1
NMR (600 MHz, CDCl3) d 9.69 (s, 1H), 8.34 (s, 1H),
7.97 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.31
(s, 1H), 4.43 (q, J = 7.8 Hz, 2H), 2.65 (s, 3H), 1.42 (t,
J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) d 198.3,
162.0, 139.5, 131.0, 129.4, 127.1, 125.3, 125.2, 112.3,
110.3, 61.6, 26.8, 14.6.
(6d). Yield (33%); H NMR (600 MHz, CDCl3) d 10.79
(br s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.86 (d, J = 7.8 Hz,
1H), 7.24 (d, J = 7.2 Hz, 1H), 4.44 (q, J = 7.2 Hz, 2H),
2.68 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H); 13C NMR
(150 MHz, CDCl3) d 199.8, 160.3, 134.0, 129.4, 129.3,
127.8, 125.8, 121.1, 120.7, 98.6, 61.7, 26.8, 14.6.
5.1.1.3. Ethyl 3-bromo-1H-indole-2-carboxylate (12a).
solution of N-bromosuccinimide (NBS, 3.92 g,
5.1.2.4. Ethyl 5-acyl-3-chloro-1H-indole-2-carboxylate
(6e). Yield (45%); H NMR (600 MHz, CD3OD) d 8.24
1
A
22.0 mmol) in DMF (15.0 mL) was added dropwise to
an ice-cooled solution of ethyl 1H-indole-2-carboxylate
(3.78 g, 20.0 mmol) in DMF (10.0 mL). Upon comple-
tion of addition (ca. 20 min), the resulting mixture was
allowed to warm to room temperature and stirred for
an additional 2 h. This mixture was then poured into
300 mL of ice water and the resulting precipitate was
collected by filtration, washed with water and dried un-
der vacuum to afford compound 12a (5.25 g, 98%) as a
(s, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.41 (d, J = 8.4 Hz,
1H), 4.39 (q, J = 7.2 Hz, 2H), 2.63 (s, 3H), 1.40 (t,
J = 7.2 Hz, 3H); 13C NMR (150 MHz, CD3OD) d
199.1, 160.8, 137.9, 130.4, 125.5, 125.4, 124.3, 122.5,
113.5, 112.8, 61.4, 26.2, 14.0.
5.1.2.5. Ethyl 7-acyl-3-chloro-1H-indole-2-carboxylate
1
(6f). Yield (45%); H NMR (600 MHz, CDCl3) d 10.60
(br s, 1H), 7.85 (d, J = 7.2 Hz, 2H), 7.19 (t, J = 7.8 Hz,
1H), 4.43 (q, J = 7.2 Hz, 2H), 2.65 (s, 3H), 1.42 (t,
J = 7.2 Hz, 3H); 13C NMR (150 MHz, CDCl3) d 199.8,
160.2, 133.2, 129.3, 127.5, 126.5, 124.0, 121.0, 120.5,
112.7, 61.6, 26.7, 14.6.
1
yellow solid: H NMR (600 MHz, CDCl3) d 9.30 (br s,
1H), 7.66 (d, J = 8.4 Hz, 1H), 7.40–7.34 (m, 2H),
7.21(t, J = 7.8 Hz, 1H), 4.45 (q, J = 6.6 Hz, 2H), 1.44
(t, J = 6.6 Hz, 3H).
5.1.1.4. Ethyl 3-chloro-1H-indole-2-carboxylate (12b).
This compound was prepared using NCS following a similar
procedure described for the preparation of 12a: yield (95%);
1H NMR (600 MHz, CDCl3) d 9.25 (br s, 1H), 7.71 (d,
J = 8.4 Hz, 1H), 7.39-7.35 (m, 2H), 7.21(t, J = 7.2 Hz,
1H), 4.47 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H); 13C
NMR (150 MHz, CDCl3) d 161.5, 135.1, 126.8, 126.4,
122.6, 121.5, 120.4, 112.6, 112.3, 61.7, 14.6.
5.1.3. General procedure for the hydrolysis of indole ester
6a–f. A solution of ester (3.20 mmol) in EtOH/CH2Cl2
(1:1, 10.0 mL) was treated with 1 N NaOH (13.0 mL,
13.0 mmol). The resulting mixture was stirred for 5 h.
After separation, the aqueous was extracted with 20%
MeOH in CH2Cl2 (15.0 mL · 2) and then acidified with
1 N HCl to pH 4. The precipitate was collected by filtra-
tion, washed thoroughly with water, and dried under
vacuum to give acids 13a–f.
5.1.2. General procedure for the Friedel–Crafts acyla-
tion25. A suspension of AlCl3 (30.0 mmol) in dichloro-
ethane (DCE, 100 mL) was cooled to 0 ꢁC. To this
was added acetyl chloride (30.0 mmol) and the mixture
was stirred for 5 min. A solution of an indole-2-carbox-
ylate in 20.0 mL of DCE was then added dropwise. The
resulting mixture was stirred at room temperature for
2 h and then poured into 100 mL of ice water. The aque-
ous was extracted with EtOAc (100 mL · 2) and the
combined organics were dried over Na2SO4 and concen-
trated under reduced pressure. The residue was sub-
jected to flash chromatography (silica gel, hexanes/
EtOAc, 6:1) to afford desired acyl compounds.
5.1.3.1. 5-Acyl-1H-indole-2-carboxylic acid (13a).
Yield (44%,); H NMR (600 MHz, DMSO-d6) d 13.06
(s, 1H), 10.09 (s, 1H), 8.38 (s, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.46 (d, J = 8.4 Hz, 1H), 7.23 (s, 1H), 2.57 (s,
3H), 1.42 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
DMSO-d6) d 198.0, 163.1, 140.2, 131.0, 130.5, 127.0,
125.5, 124.4, 113.1, 109.7, 27.2.
1
5.1.3.2. 7-Acyl-1H-indole-2-carboxylic acid (13b).
1
Yield (37%,); H NMR (600 MHz, DMSO-d6) d 13.37
(s, 1H), 10.65 (s, 1H), 8.03 (d, J = 7.2 Hz, 1H), 8.00 (d,
J = 7.8 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.22 (d,

3592
Z. Wang, R. Vince / Bioorg. Med. Chem. 16 (2008) 3587–3595
J = 2.4 Hz, 1H ), 2.66 (s, 3H); 13C NMR (150 MHz,
DMSO-d6) d 200.7, 162.8, 135.1, 130.3, 129.6, 129.2,
129.1, 121.3, 120.9, 108.4, 27.2.
J = 1.8 Hz, 1H), 6.92–6.89 (m, 2H), 6.83 (d,
J = 7.8 Hz, 1H), 4.18 (s, 1H), 4.09 (br s, 4H), 3.54 (m,
1H), 3.18 (br s, 4H), 2.61 (s, 3H), 1.23 (d, J = 6.6 Hz,
6H); 13C NMR (150 MHz, DMSO-d6) d 198.2, 162.8,
150.0, 138.9, 136.7, 135.1, 131.1, 130.6, 127.0, 124.5,
124.3, 120.8, 117.0, 112.2, 107.0, 49.4, 44.0, 36.7, 31.7,
26.8, 23.1.
5.1.3.3. 5-Acyl-3-bromo-1H-indole-2-carboxylic acid
1
(13c). Yield (66%); H NMR (600 MHz, DMSO-d6) d
13.45 (br s, 1H), 12.51 (s, 1H), 8.14 (s, 1H), 7.89 (d,
J = 9.0 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 2.61 (s, 3H);
13C NMR (150 MHz, DMSO-d6) d 197.8, 161.9, 138.7,
131.1, 127.5, 127.2, 125.6, 122.9, 113.9, 97.8, 27.4.
5.1.4.3. 1-(3-Bromo-2-(4-(3-(isopropylamino)pyridin-2-
(4c).
yl)piperazine-1-carbonyl)-1H-indol-5-yl)ethanone
1
Yield (92%); H NMR (600 MHz, CDCl3) d 11.21 (s,
1H), 8.13 (s, 1H), 7.85 (d, J = 9.0 Hz, 1H), 7.63 (d,
J = 4.8 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 6.89 (dd,
J = 7.8 Hz, 4.8 Hz, 1H), 6.80 (d, J = 7.8 Hz, 1H), 4.15
(d, J = 6.6 Hz, 1H), 3.85 (br s, 4H), 3.51 (m, 1H), 3.14
(t, J = 4.8 Hz, 4H), 2.62 (s, 3H), 1.19 (d, J = 6.0 Hz,
6H); 13C NMR (150 MHz, CDCl3) d 197.9, 162.8,
149.9, 138.4, 136.7, 135.0, 131.0, 130.3, 126.6, 124.5,
122.3, 120.9, 117.0, 112.5, 93.4, 43.9, 36.7, 31.6, 26.8,
23.1; HRMS (ESI+) calcd for C23H27N5O2Br [M+H]+
484.1353, found 484.1338 (E = ꢀ3.2 ppm).
5.1.3.4. 7-Acyl-3-bromo-1H-indole-2-carboxylic acid
(13d). Yield (66%,); H NMR (600 MHz, DMSO-d6) d
10.78 (s, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.85 (d,
J = 8.4 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 2.66 (s, 3H);
13C NMR (150 MHz, DMSO-d6) d 200.4, 162.1, 134.0,
129.6, 129.4, 127.8, 126.2, 120.7, 112.5, 98.5, 26.4.
1
5.1.3.5. 5-Acyl-3-chloro-1H-indole-2-carboxylic acid
1
(13e). Yield (90%,); H NMR (600 MHz, DMSO-d6) d
13.64 (br s, 1H), 12.38 (s, 1H), 8.23 (s, 1H), 7.89 (d,
J = 8.4 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 2.61 (s, 3H);
13C NMR (150 MHz, DMSO-d6) d 197.8, 161.9, 137.8,
130.9, 125.7, 125.6, 125.3, 122.0, 113.8, 111.5, 27.3.
5.1.4.4. 1-(3-Bromo-2-(4-(3-(isopropylamino)pyridin-2-
(4d).
yl)piperazine-1-carbonyl)-1H-indol-7-yl)ethanone
1
Yield (44%); H NMR (600 MHz, CDCl3) d 10.80 (s,
1H), 7.84 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H),
7.63 (m, 1H), 7.25–7.20 (m, 1H), 6.91–6.88 (m, 1H),
6.81 (d, J = 7.8 Hz, 1H), 3.84 (br s, 4H), 3.51 (m, 1H),
3.15 (br s, 4H), 3.11 (m, 1H), 2.66 (s, 3H), 1.20 (d,
J = 6.0 Hz, 6H); 13C NMR (150 MHz, CDCl3) d 200.0,
162.3, 150.0, 136.7, 135.0, 134.1, 130.2, 128.4, 127.8,
126.6, 124.7, 121.0, 120.8, 117.1, 112.3, 92.5, 50.7,
44.0, 26.7, 23.1, 23.0.
5.1.3.6. 7-Acyl-3-chloro-1H-indole-2-carboxylic acid
(13f). Yield (52%,); H NMR (600 MHz, DMSO-d6) d
10.59 (br s, 1H), 7.81 (d, J = 7.2 Hz, 2H), 7.16 (t,
J = 7.8 Hz, 1H), 2.63 (s, 3H); 13C NMR (150 MHz,
CDCl3) d 200.3, 162.5, 133.1, 129.3, 127.4, 126.3,
123.9, 121.0, 120.3, 112.5, 26.3.
1
5.1.4. General procedure for the coupling of acids 13a–f
with amine 5. To a solution of acid (2.13 mmol) in
8.00 mL of DMF was added carbonyl diimidazole
(CDI, 0.42 g, 2.56 mmol) at 0 ꢁC. After the mixture
was stirred for 30 min, a solution of amine 5 (0.56 g,
2.56 mmol) in 2.00 mL of DMF was added and the
resulting mixture was stirred for 18 h. The precipitate
was filtered via a pad of celite. The filtrate was diluted
with CH2Cl2, washed with a saturated NaHCO3 aque-
ous solution and brine, and dried over Na2SO4. After
the solvent was removed, the residue was subjected to
flash chromatography (silica gel, hexanes/EtOAc, 1:1)
to afford compounds 4a–f.
5.1.4.5. 1-(3-Chloro-2-(4-(3-(isopropylamino)pyridin-
2-yl)piperazine-1-carbonyl)-1H-indol-5-yl)ethanone (4e).
1
Yield (55%); H NMR (600 MHz, DMSO-d6) d 12.37
(s, 1H), 8.20(s, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.53 (d,
J = 4.2 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 6.91–6.87
(m, 2H), 4.50 (d, J = 8.4 Hz, 1H), 3.85 (br s, 2H), 3.65
(br s, 2H), 3.56 (m, 1H), 3.00 (br s, 4H), 2.62 (s, 3H),
1.14 (d, J = 6.6 Hz, 6H); 13C NMR (150 MHz,
DMSO-d6) d 197.8, 161.0, 150.2, 137.6, 136.6, 134.6,
130.8, 129.7, 124.5, 124.3, 120.9, 117.3, 113.3, 104.8,
79.9, 43.6, 36.5, 31.4, 27.4, 22.9.
5.1.4.1. 1-(2-(4-(3-(Isopropylamino)pyridin-2-yl)piper-
azine-1-carbonyl)-1H-indol-5-yl)ethanone (4a). Yield
(62%); H NMR (600 MHz, CDCl3) d 11.02 (s, 1H),
5.1.4.6. 1-(3-Chloro-2-(4-(3-(isopropylamino)pyridin-
2-yl)piperazine-1-carbonyl)-1H-indol-7-yl)ethanone (4f).
Yield (67%); H NMR (600 MHz, CDCl3) d 10.64 (s,
1
1
8.28(s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.66 (d,
J = 4.2 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 6.90–6.89
(m, 2H), 4.44 (d, J = 7.8 Hz, 1H), 3.90 (br s, 4H), 3.56
(m, 1H), 3.02 (d, J = 4.8 Hz, 4H), 2.86 (s, 3H), 1.16 (d,
J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) d 200.3,
163.0, 150.4, 136.6, 134.7, 133.8, 132.3, 129.3, 128.9,
128.0, 121.2, 120.7, 120.5, 117.3, 104.9, 71.0, 43.6,
36.4, 31.4, 27.3, 23.0.
1H), 7.82 (t, J = 7.8 Hz, 2H), 7.65 (d, J = 4.8 Hz, 1H),
7.20 (t, J = 7.8 Hz, 1H), 6.87 (m, 1H), 6.79 (d,
J = 7.8 Hz, 1H), 4.12 (d, J = 4.8 Hz, 1H), 3.84 (br s,
4H), 3.50 (m, 1H), 3.15 (m, 4H), 2.64 (s, 3H), 1.20 (d,
J = 6.0 Hz, 6H); 13C NMR (150 MHz, CDCl3) d 199.9,
161.7, 150.1, 136.6, 135.1, 133.5, 128.1, 127.8, 126.8,
125.6, 121.0, 120.6, 120.5, 116.9, 106.2, 49.8, 44.0,
26.7, 23.1.
5.1.4.2. 1-(2-(4-(3-(Isopropylamino)pyridin-2-yl)piper-
azine-1-carbonyl)-1H-indol-7-yl)ethanone (4b). Yield
(58%); 1H NMR (600 MHz, DMSO-d6) d 10.97 (s,
1H), 7.98 (d, J = 7.8 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H),
7.55–7.54 (m, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.98 (d,
5.1.5. General procedure for the synthesis of final acid
compounds 3a–f from 4a–f. Sodium (4.38 mmol) was
added to 3.00 mL of anhydrous EtOH at room temper-
ature under argon. The mixture was stirred until a clear
solution was obtained. To this was added diethyl oxalate

Z. Wang, R. Vince / Bioorg. Med. Chem. 16 (2008) 3587–3595
3593
(0.237 mL, 1.75 mmol) and a suspension of compound
4a–f (0.88 mmol) in 2 mL of anhydrous EtOH. The solu-
tion turned yellow immediately and precipitate formed.
This mixture was stirred for 4 h then quenched with a
saturated aqueous solution of NH4Cl (5.0 mL), ex-
tracted with 20% MeOH in CH2Cl2 (10.0 mL · 3). The
combined organics were washed with brine (15.0 mL)
and dried over Na2SO4. After the solvent was removed,
the residue was directly taken to next step, except for the
intermediate from 4c, which was purified by flash chro-
matography (silica gel, 5% MeOH in CH2Cl2) to afford
compound 14c.
5.1.5.4. (Z)-4-(3-Bromo-2-(4-(3-(isopropylamino)pyri-
din-2-yl)piperazine-1-carbonyl)-1H-indol-5-yl)-2-hydro-
xy-4-oxobut-2-enoic acid (3c). Yield (49% from 14c); H
1
NMR (600 MHz, CD3OD/CDCl3) d 8.23 (s, 1H), 7.91
(d, J = 9.0 Hz, 1H), 7.58 (d, J = 3.6 Hz, 1H), 7.48 (d,
J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.07–7.01 (m 2H), 3.84
(br s, 4H), 3.57 (m, 1H), 3.19 (s, 4H), 1.22 (d,
J = 6.6 Hz, 6H); 13C NMR (150 MHz, CDCl3) d 191.5,
168.4, 164.4, 163.5, 162.6, 148.2, 139.0, 137.9, 131.9,
130.5, 128.5, 126.8, 123.9, 121.7, 121.4, 119.2, 113.0,
98.3, 93.0, 44.1, 36.7, 29.7, 22.4; HRMS (ESIꢀ) calcd
for C25H25N5O5Br [MꢀH]ꢀ 554.1039. Found: 554.1076.
The intermediate (14c and the crude residue for those
from 4a, 4b, 4d–f, 0.45 mmol) was dissolved into
EtOH/CH2Cl2 (1:1, 2 mL) and treated with 1N NaOH
(2 mL). The resulting mixture was stirred for 2 h and
then thoroughly extracted with 20% MeOH in CH2Cl2.
The aqueous phase was then acidified with 1 N HCl to
pH 4. The precipitate was collected by filtration,
washed with water, and dried under vacuum to give
acids 3a–f.
5.1.5.5. (Z)-4-(3-Bromo-2-(4-(3-(isopropylamino)pyri-
din-2-yl)piperazine-1-carbonyl)-1H-indol-7-yl)-2-hydro-
xy-4-oxobut-2-enoic acid (3d). Yield (47% over two
1
steps); H NMR (600 MHz, CD3OD/CDCl3) d 7.83 (d,
J = 7.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.30 (t,
J = 7.8 Hz, 1H), 7.25 (s, 1H), 7.14–7.11 (m, 1H), 7.06
(d, J = 7.8 Hz, 1H), 3.84 (br s, 4H), 3.47 (m, 1H), 3.16
(br s, 4H), 3.13 (m, 1H), 1.24 (d, J = 6.0 Hz, 6H); 13C
NMR (150 MHz, CDCl3) d 191.7, 166.3, 163.0, 162.8,
148.7, 136.9, 135.2, 134.1, 130.4, 128.6, 127.9, 126.6,
124.4, 121.3, 120.9, 117.5, 112.7, 92.8, 44.2, 26.7, 23.4,
23.1; HRMS (ESIꢀ) calcd for C25H25N5O5Br [MꢀH]ꢀ
554.1039. Found: 554.1028.
5.1.5.1. (Z)-Ethyl-4-(3-bromo-2-(4-(3-(isopropylami-
no)pyridin-2-yl)piperazine-1-carbonyl)-1H-indol-5-yl)-2-
hydroxy-4-oxobut-2-enoate (14c). Yield (45%); 1H NMR
(600 MHz, CDCl3) d 11.43 (s, 1H), 7.96 (s, 1H), 7.83 (d,
J = 7.8 Hz, 1H), 7.62 (d, J = 3.6 Hz, 1H), 7.35 (d,
J = 7.2 Hz, 1H), 7.10 (s, 1H), 6.90 (dd, J = 7.8 Hz,
4.8 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 4.36 (q,
J = 7.2 Hz, 2H), 3.65 (q, J = 7.2 Hz, 1H), 3.50 (m,
1H), 3.85 (br s, 4H), 3.13 (s, 4H), 1.37 (t, J = 7.2 Hz,
3H), 1.18 (d, J = 6.0 Hz, 6H); 13C NMR (150 MHz,
CDCl3) d 191.6, 168.3, 163.0, 162.8, 149.8, 139.0,
136.8, 134.8, 130.6, 128.4, 126.9, 124.0, 121.9, 121.0,
117.2, 113.0, 98.3, 93.5, 62.7, 50.6, 44.0, 36.8, 31.7,
18.5, 14.3.
5.1.5.6. (Z)-4-(3-Chloro-2-(4-(3-(isopropylamino)pyri-
din-2-yl)piperazine-1-carbonyl)-1H-indol-5-yl)-2-hydro-
xy-4-oxobut-2-enoic acid (3e). Yield (34% over two
1
steps); H NMR (600 MHz, CD3OD/CDCl3) d 8.28 (s,
1H), 7.90 (d, J = 9.0 Hz, 1H), 7.57 (d, J = 3.6 Hz, 1H),
7.48 (d, J = 9.0 Hz, 1H), 7.07–7.02 (m 2H), 3.86 (br s,
4H), 3.58 (m, 1H), 3.19 (s, 4H), 1.23 (d, J = 6.6 Hz,
6H); 13C NMR (150 MHz, CD3OD) d 191.2, 169.2,
164.7, 162.1 (2), 148.4, 138.4, 137.9, 132.0, 128.3,
125.1, 123.9, 121.3, 120.7, 119.1, 113.0, 106.9, 44.0,
22.2; HRMS (ESI+) calcd. for C25H27N5O5Cl [M+H]+
512.1701. Found: 512.1708.
5.1.5.2. (Z)-4-(2-(4-(3-(Isopropylamino)pyridin-2-yl)piper-
azine-1-carbonyl)-1H-indol-5-yl)-2-hydroxy-4-oxobut-2-
enoic acid (3a). Yield (41% over two steps); H NMR
1
5.1.5.7. (Z)-4-(3-Chloro-2-(4-(3-(isopropylamino)pyri-
din-2-yl)piperazine-1-carbonyl)-1H-indol-7-yl)-2-hydro-
xy-4-oxobut-2-enoic acid (3f). Yield (35% over two steps);
1H NMR (600 MHz, CD3OD/CDCl3) d 7.93 (d,
J = 7.2 Hz, 1H), 7.82 (t, J = 7.2 Hz, 2H), 7.78 (d,
J = 4.8 Hz, 1H), 7.74 (d, J = 4.8 Hz, 1H), 7.16 (d,
J = 6.6 Hz, 1H), 7.03–7.00 (m, 1H), 4.15 (d, J = 7.2 Hz,
1H), 3.89 (br s, 4H), 3.22 (br s, 4H), 3.17 (m, 1H), 1.24
(d, J = 6.0 Hz, 6H); 13C NMR (150 MHz, CDCl3) d
191.9, 166.4, 163.1, 162.8, 148.9, 137.1, 135.3 134.2,
130.3, 128.6, 127.8, 126.7, 124.5, 121.1, 120.8, 117.8,
112.8, 93.0, 44.4, 27.1, 23.6, 23.2; HRMS (ESI+) calcd
for C25H27N5O5Cl [M+H]+ 512.1701. Found: 512.1702.
(600 MHz, CD3OD/CDCl3) d 8.28 (s, 1H), 7.78 (d,
J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H),
7.26 (d, J = 7.8 Hz, 1H), 7.21–7.18 (m, 1H), 6.93 (s,
1H), 4.04 (br s, 4H), 3.63 (m, 1H), 3.29 (s, 4H), 1.24
(d, J = 6.0 Hz, 6H); 13C NMR (150 MHz, CD3OD) d
191.4, 169.1, 164.7, 162.9, 146.5, 139.5, 138.8, 131.4,
127.7, 127.6, 127.2, 123.9, 123.1, 121.2, 121.1, 112.3,
106.8, 97.5, 48.7, 44.1, 21.2; HRMS (ESIꢀ) calcd for
C25H25N5O5 [MꢀH]ꢀ 476.1934. Found: 476.1938.
5.1.5.3. (Z)-4-(2-(4-(3-(Isopropylamino)pyridin-2-yl)piper-
azine-1-carbonyl)-1H-indol-7-yl)-2-hydroxy-4-oxobut-2-
enoic acid (3b). Yield (24% over two steps); H NMR
1
(600 MHz, CD3OD/CDCl3) d 8.15 (d, J = 7.8 Hz, 1H),
8.01 (d, J = 7.8 Hz, 1H), 7.58–7.53 (m, 3H), 6.97–6.86
(m, 2H), 6.68 (s, 1H), 3.95 (br s, 4H), 3.56 (m, 1H),
3.14 (s, 4H), 1.22 (d, J = 6.0 Hz, 6H); 13C NMR
(150 MHz, CD3OD) d 197.8, 167.0, 163.1, 150.0,
138.8, 137.3, 135.9, 134.3, 133.9, 128.9, 128.0, 125.5,
123.8, 122.2, 121.0, 117.5, 114.2, 111.6, 43.8, 42.5,
38.5, 22.5; HRMS (ESIꢀ) calcd for C25H25N5O5
[MꢀH]ꢀ 476.1934. Found: 476.1925.
5.2. Biology
5.2.1. RT assay. This assay was performed using the
Quan-T-RT Assay System kit from Amersham Biosci-
ences (TRK 1022). Experiments were done using
0.65 mL snap cap tubes with a SPA (scintillation prox-
imity assay) bead, where a short poly (A) tail and a oligo
(T) primer have been coupled via a biotin linkage to a
resin bead containing scintillation cocktail. This resin

3594
Z. Wang, R. Vince / Bioorg. Med. Chem. 16 (2008) 3587–3595
was incubated with a drug compound, the [3H]-TTP tra-
cer and the reaction was initiated by the addition of the
RT (RT) enzyme from Ambion: the RNA Company
(AM2045). The [3H]TTP was incorporated into the poly
(A) tail by RT enzyme but in the presence of the drug
candidate this incorporation should be inhibited. This
reaction was incubated for 3 h at 37 ꢁC and quenched
by the addition of the stop buffer. These tubes were then
transferred to 20 mL scintillation tubes (glass) and
counted for 1 min per sample using a beta counter.
Acknowledgments
This research was supported by the Center for Drug De-
sign at the University of Minnesota. We thank Dr. Eric
Bennett for discussion, Dr. Christine Salomon for IN as-
say and Christine Dreis for RT assay. We also thank
Roger Ptak at Southern Research Institute for cell-
based assay, Dr. Robert Craigie of NIH for the integr-
ase plasmid and Dr. William Shannon for consultation
and discussion.
5.2.2. IN assay. IN was expressed using Escherichia coli
BL21(DE3) and standard IPTG induction.29 In a typical
assay, DNA substrate 50-biotin ATGTGGAAAA
TCTCTAGCAGT and 30-cy5 ACTGCTAGAGATTTT
CCACAT (IDT) were annealed in DNA annealing buf-
fer (50 mM Tris, pH 7.5, 10 mM MgCl2) by heating to
95 ꢁC and allowed to cool over 10 min in a thermocy-
cler. For each inhibitor concentration 12.5 lL of
600 nM IN in reaction buffer (10 mM HEPES, pH 7.5,
10 mM MnCl2, 1 mM DTT, 10% glycerol, 0.1 mg/ml
BSA 0.05% Brij-35) was added to each well in a 96-well
plate. 0.5 lL of inhibitor or DMSO was added to each
sample and allowed to equilibrate with the enzyme for
10 min at room temperature. After the incubation,
12.5 lL of 50 nM DNA substrate in reaction buffer
was added to each well to initiate the reaction. The reac-
tion was incubated at 37 ꢁC for 2 h, after which 75 lL of
4· binding buffer (20 mM Tris, pH 8.0, 1.6 M NaCl,
40 mM EDTA, 0.4 mg/ml salmon sperm DNA) was
added and the entire mixture was transferred to a
StreptaWell High bind plate (Roche). The DNA was al-
lowed to bind to the plate for 1 h at room temperature
with mild agitation. Non-specific DNA was removed
from the plate by washing 3 · 5 min with 200 lL dena-
turing buffer (30 mM NaOH, 0.2 M NaCl, 1 mM
EDTA). The plate was washed briefly with 200 lL of
TE. To remove the DNA for fluorescence detection
100 lL of formamide was added to each well and incu-
bated for 15 min at room temp with mild agitation.
Ninety microliters of the formamide solution was trans-
ferred into a black 384-well plate and read on a molec-
ular devices analyst using the continuous lamp
excitation 620 nm, emission 665 nm.
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